Quinones

BIOCHEMISTRY RESEARCH TRENDS
QUINONES

OCCURRENCE, MEDICINAL
USES AND PHYSIOLOGICAL
IMPORTANCE

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QUINONES

OCCURRENCE, MEDICINAL
USES AND PHYSIOLOGICAL
IMPORTANCE

ERVIN R. PRICE
AND
SMITH C. JOHNSON
EDITORS

New York

Copyright 2013 by Nova Science Publishers, Inc.

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Published by Nova Science Publishers, Inc. New York
ISBN: 978-1-62618-324-7 (eBook)

CONTENTS

Preface vii
Chapter 1 Dihydropyrimidinone Derivatives: Redox
Reactivity, Pharmacological Relevance
and Medicinal Applications 1
Marta Pineiro, Bruno F. O. Nascimento
and Antnio M. dA. Rocha Gonsalves
Chapter 2 Biological Implications of Benzoquinones 57
Jisook Kim
Chapter 3 Quinone Monoacetal Compounds in Application
to Controlled Reactions with Nucleophiles 85
Toshifumi Dohi and Yasuyuki Kita
Chapter 4 Catecholquinones as Substrates of the NRH:
Quinone Oxidoreductase 2 in the Brain
and Retina 141
Lucia de Fatima Sobral Sampaio
Chapter 5 Plasma Membrane CoQ, Porin, and Redox
Control of Autism 157
Brian F. Teske, I. L. Sun, Anna Gvozdjakova,
Hans Low and Frederick L. Crane
Index 173

PREFACE

In this book, the authors present current research in the study of the
occurrence, medicinal uses and physiological importance of quinones. Topics
discussed in this compilation include the biological implications of
benzoquinones; dihydropyrimidinone derivates and their redox reactivity and
pharmacological relevance; quinone monoacetal compounds in the application
of controlled reactions with nucleophiles; quinone oxidoreductase 2 in the
brain and retina; and plasma membrane CoQ, porin, and redox control of
autism.
Chapter 1 Biginelli and Biginelli-like dihydropyrimidines constitute an
ubiquitously recognized class of nitrogen-containing compounds. The present
chapter intends to assess the currently available scientific literature on the
developments specifically regarding the important oxidation/reduction
reactivity of dihydropyrimidinones, their thione analogs and other derivatives.
Accounts dealing with the pharmacological properties and uses in medicine of
dihydropyrimidine scaffolds will also be reviewed.
Chapter 2 Benzoquinones (BQs) represent the simplest form of
quinones, containing two carbonyl groups on a six-membered ring. They are
ubiquitously found in diverse organisms as free quinones, protein cofactors, or
an integral part of the mitochondrial electron transport chain (ETC). In
addition, many BQs are identified as environmental toxins generated from
industrial processes as the metabolites of polycyclic aromatic hydrocarbons,
contributing to bioaccumulation. To date, animal and epidemiological studies
revealed that the quinone derivatives of benzene metabolites serves as a source
of inducing abnormal cell behavior, leading to cancer or triggering immune
response. Whether occurring endogenously in living organisms or
exogenously in the environment, there is a universal understanding on the role
of BQs as potential toxins, except some limited cases like protein-bound BQs
Ervin R. Price and Smith C. Johnson viii
or electron carriers in the ETC. Studies done at a molecular-level approach
revealed that BQs exhibit both genotoxicity and non-genotoxicity/epigenetic
toxicity, targeting both cellular DNA and proteins. The mechanism of their
action is thought to occur via the combination of oxidative damage intervened
by redox-cycling, adduct formation with DNA and proteins, and protein cross-
linking/protein conformation change.
Chapter 3 A summary of the preparation, synthetic utility, and
application of quinone monoacetals is presented with focus on the following
points. Quinone monoacetals (QMAs), the oxidized compounds of phenols as
well as the desymmetrized alternative of quinones, have attracted considerable
interest due to their broad utilities in organic transformations as intermediates
and important building blocks for the synthesis of natural products. Recently,
increasing interest in the development and utilization of QMAs has been
occurring due to their unique bifunctionalities of both ,-unsaturated
carbonyl and allylacetal moieties. The varied reactivities in nucleophilic attack
on QMA carbons can occur, for instance, addition to the carbonyl carbon and
conjugated addition to the enone moiety. In contrast to these established
addition chemistries, the reports of the utility of QMAs in substitution
reactions are quite limited. This chapter principally deals with the progress in
the emerging theme of the selectivity during the reactions of QMAs toward
nucleophiles, especially with emphasis on the latter topic, the section of which
starts for i) efficient prearation of QMAs, ii) general guideline for the
reactivities of QMAs toward nucleophiles, and iii) newly developed methods
for the regioselective introduction of aromatic or alkene nucleophiles by
controlled coupling strategies using specific acid catalysts. In particular, the
authors new strategies can now provide attractive synthetic routes to the
valuable oxygenated biaryls, terphenyls, dihydrobenzofurans, and other related
functionalized compounds. Several important results, such as the syntheses of
key modules of natural products and preparation of regio-controlled phenol
oligomers, are also discussed for the promising expansion of these future
applications.
Chapter 4 Quinones are highly toxic products of the degradation of
many compounds surging from live organisms. Certain of these highly toxic
products are substrates of the NRH: quinone oxidoreductase 2 (NQO2). This
flavoenzime has a ping-pong bi bi catalytic mechanism, where the coenzyme
FAD is reduced by rare cosubstrates, such as N-rybosil-hidronicotinamide
(NRH) and N-metyl-hidronicotinamide (NMH). The NQO2 detoxifying
activity occurs synchronically with the activation of the anti-cancer protein
p53, which is primarily activated in response to xenobiotic and radiation. It is
Preface ix
not clear if the over activation of the NQO2 produces ionic reactive
compounds that are capable of activating p53, or if the xenobiotic presence is
capable of triggering a NQO2-p53 binding, which, in turn, activates protein
p53. Among the diversity of putative NQO2 substrates, the authors highlighted
catechol quinones produced from catecholamines in the brain and retina. The
catecholamines participation in neurological diseases, as well the NQO2
influence in the neurological diseases physiopathology, is incontestable.
Accordingly, in this chapter, the authors aim to discuss the implication of the
characteristic catechol quinone reductase NQO2 function in the catechol
quinones metabolism, which takes place similarly in neurons from the brain
and retina, associating with NQO2 cancer-preventing activity and with those
neurological diseases related to catecholamine metabolisms dysfunctions.
Chapter 5 Autism is a neurological condition starting in childhood that is
characterized by behavioral and intellectual problems. Its occurrence is
increasing and although there are some treatments, they are of limited effect or
have undesirable side effects. A recent study showed that autistic children had
increased serum levels of auto-antibodies to Voltage Dependent Anion
Channel (VDAC). Interestingly, in addition to the membrane transport
function of VDAC a second function was recently described by A. Lawens
group at Monash University in Melbourne. This group showed that VDAC
was also a trans-PM NADH dehydrogenase. The VDAC autoantibody detected
in autistic children inhibits the dual transport and dehydrogenase functionality
of VDAC. In this report the authors implicate Coenzyme Q as an important co-
factor for redox control of PM pores including VDAC. The authors show that
the PM redox function is dependent on Coenzyme Q and propose that this
novel function for CoQ has therapeutic implications for treatment of autism
disorders. More broadly, the Coenzyme Q requirement for the PM redox
function of porin in diverse species including bacteria, plants, and mammals
suggests a mechanistically conserved feature of pore redox control.

In: Quinones ISBN: 978-1-62618-323-0
Editors: E. R. Price and S. C. Johnson 2013 Nova Science Publishers, Inc.

Chapter 1

DIHYDROPYRIMIDINONE DERIVATIVES:
REDOX REACTIVITY, PHARMACOLOGICAL
RELEVANCE AND MEDICINAL APPLICATIONS

Marta Pineiro, Bruno F. O. Nascimento
and Antnio M. dA. Rocha Gonsalves
Department of Chemistry, University of Coimbra,
Coimbra, Portugal

ABSTRACT

Biginelli and Biginelli-like dihydropyrimidines constitute an
ubiquitously recognized class of nitrogen-containing compounds. The
present chapter intends to assess the currently available scientific
literature on the developments specifically regarding the important
oxidation/reduction reactivity of dihydropyrimidinones, their thione
analogs and other derivatives. Accounts dealing with the pharmacological
properties and uses in medicine of dihydropyrimidine scaffolds will also
be reviewed.

INTRODUCTION

Pietro Biginelli reported, in 1893, the synthesis of 4-phenyl-5-
ethylcarboxylate-6-methyl-3,4-dihydropyrimidin-2(1H)-one (4) in a one-pot,
M. Pineiro, B. F. O. Nascimento and A. M. dA. Rocha Gonsalves 2
three-component acid catalysed cyclocondensation reaction of ethyl
acetoacetate (1), benzaldehyde (2) and urea (3) [1], (Scheme 1). Although the
reaction did not attract too much attention until the end of the twentirth-
century, the increasing importance of the multicomponent reactions in organic
and medicinal chemistry [2] jointly with the apparent similarity of
dihydropyrimidinones with the dihydropyridine calcium channel modulators
of the Hantzsch type [3] significantly increased the interest in the development
of the synthesis and study of the pharmacological properties of this interesting
heterocycle scaffold.

Scheme 1.
The Biginelli reaction was extended to the synthesis of an immense
variety of compounds through variation at all three components; using
different aldehydes from aromatic to aliphatic, diverse -ketoesters and
thiourea instead of urea, thousands of compounds were synthetised.
The synthetic methods and the mechanistic aspects related to these
compounds, as well as their structure functionalization [4] have already been
extensively and critically evaluated. [5] The products of these reactions are
3,4-dihydropyrimidi-2(1H)-ones or 3,4-dihydropyrimidin-2(1H)-thiones which
are the reduce form of pyrimidin-2(1H)-ones and the oxidized form of
tetrahydropyrimidin-2(1H)-ones, Figure 1, or their corresponding thione
derivatives.
The exploration of the oxidation and reduction reactions that allow the
transformation of one derivative in to other and the pharmacological properties
of the reduced and oxidized derivatives is the scope of this review. The interest
in these particular aspects is based on the following reasons: 1) The studies on
the activity or inactivity of Hantzschs compounds as calcium channel
modulators were related to their redox properties.
N
H
NH
EtO
2
C
O
EtO
2
C
O
O H
H
2
N O
NH
2
+
HCl(cat)
EtOH,
1
2
3
4
Dihydropyrimidinone Derivatives 3

Figure 1. Structures of pyrimidines and pyrimidinones and related hydroderivatives.
In fact, the short plasma half-life of nifedipine (Adalat
) was related to
metabolic oxidation to pyridines [6] and the substitution of the sp
2
carbon for
nitrogen (from dihydropyridines to dihydropyrimidinones) appears to prevent
both chemical and biological oxidation to inactive aromatic products [7] 2)
while the oxidation of dehydropyrimidines of the Hantzsch type to aromatic
pyrimidines is an easy process [8] dehydrogenation/aromatization of the
Biginelli compounds is more difficult and one of the most studied reactions for
these compounds. 3) the treatment of ones and thiones is similar but
pyrimidin-2(1H)-thiones are scarcer compounds and so, they deserve a
differential treatment 4) despite their many biological activities only the
application as calcium channel modulators, selective
1a
adrenoreceptor
antagonist and mitotic kinesis inhibitors was properly reviewed, 5)
additionally, their antioxidant activity was recently reported.

1. OXIDATIONS

1.1. Dihydropyrimidinones

The first report on the oxidation of 3,4-dihydropyrimidin-2(1H)-ones is an
isolated work: in 1964 Akira Takamizawa and Kentaro Hirai published the
studies on the pyrimidine derivatives.[9] The synthesis of 4,6-unsubstituted
3,4-dihydropyrimidin-2(1H)-one 6 was achieved from the condensation of
ethyl-3-ethoxy-2-methoxymethyleneproprionate (5) with urea. Dehydro-
N
N
pyrimidine
N
NH
1,6-dihydropyrimidine
N
H
NH
1,2,3,4-tetrahydropyrimidine
N
H
NH
hexahydropyrimidine
N
NH
1,6-dihydropyrimidin-2-ol
OH N
NH
1,4,5,6-tetrahydropyrimidin-2-ol
OH N
N
OH
pyrimidin-2-ol
N
H
NH
3,4-dihydropyrimidin-2(1H)-one
O N
H
NH
tetrahydropyrimidin-2(1H)-one
O
N
H
N
O
pyrimidin-2(1H)-one
N
H
NH
hexahydropyrimidin-2-ol
OH
+2H
-2H
+2H
-2H
+2H
-2H
+2H
-2H
+2H
-2H
+2H
-2H
+2H
-2H
+
2
H
-
2
H
+
2
H
-
2
H
genation of 6 by the action of bromine in acetic acid under reflux during 1
hour yields pyrimidi-2(1H)-one 7 in 56% yield (Scheme 2).

Scheme 2.
It was only in the 90s of the past century that this reaction began to be
explored. The oxidation of 1N-methyl-3,4-dihydropyrimidin-2(1H)-ones on
melting PCl
5
or on boiling in a solution of PCl
5
in POCl
3
affords different
products depending on the substituent at C-6. The oxidation of 6-unsubstituted
derivative 8a yields 1N-methylpyrimidin-2(1H)-one 9a in 35% and increasing
the reaction time 2-chloropyrimidine 10a was obtained exclusively. The
analogous dihydropyrimidin-2(1H)-one with a phenyl group at the 6 position
oxidized to a mixture of pyrimidin-2(1H)-one 9b, the demethylation product
11b and the corresponding chloropyrimidine 10b, in moderated yields. The
reaction of 1N-methyl-3,4-dihydropyrimidi-2(1H)-ones, with a methyl group
at the C-6, with PCl
5
do not afford any oxidation product, the reaction product
being a mixture of chlorinated compounds 12-14(Scheme 3).[10]

Scheme 3.
N
H
NH
O
O
O
O
O
+
H
2
N NH
2
O
EtO
O
EtOH, H
+
reflux, 8h
Br
2,
AcOH
reflux, 1h
N
H
N
O
EtO
O
5
3
6 7 56%
N
NH
O
EtO
O
Me
R
8a R =H
8b R =Ph
8c R =Me
N
N
O
EtO
O
Me
R
9a R =H 35%
9b R =Ph 30%
N
N
Cl
EtO
O
R
10a R =H
10b R =Ph 20%
N
H
N
O
EtO
O
R
11b R =Ph 15%
+ +
PCl
5
/POCl
3
R =H
or R =Ph
R =Me
N
NH
O
EtO
O
Me
Cl
2
HC
N
NH
O
EtO
O
Me
ClHC
Cl
N
NH
O
EtO
O
Me
Cl
2
C
Cl
12 25%
13 20%
14 19%
When Biginelli compounds 4 and 8c with a methyl substituent at the C-6
are oxidized with SeO
2
in refluxing dioxane the product of the
dyhydrogenation was obtained but with further oxidation at the methyl group
affording as main product the carboxylic acid derivatives 15a and 15b in 50
and 53% yield respectively, (Scheme 4). The oxidation of 5-carbamoyl-3,4-
dihydropyrimidin-2(1H)-one 16 originates the corresponding oxidized form
with a carboxylic acid at C-6 17 which through intramolecular condensation
affords 1H-pyrrolo[3,4-d]pyrimidine-2,5,7(6H)-trione 18 in 36% yield
(Scheme 5).[11]
5-Carbonitrile-3,4-dihydropyrimidin-2(1H)-one 19 and 1,2,3,9b-
tetrahydro-5H-indeno[1,2-d]pyrimidines 21a,b were effectively oxidized to the
corresponding dehydrogenated products 20 and 22a,b with palladium on
charcoal in diphenyl ether at high temperature but this methodology is
inapplicable for the oxidation of 3,4-dihydropyrimidin-2(1H)-ones with an
ester derivative at C-5 (Scheme 6).[12]

Scheme 4.

Scheme 5.
N
NH
O
EtO
O
R
Me
4 R =H
8a R =Me
SeO
2,
dioxane
reflux, 5h
N
N
O
EtO
O
R
HOOC
15a R =H
15b R =Me
N
NH
O
H
2
N
O
CH
3
Me
SeO
2,
dioxane
reflux, 5h N
N
O
CH
3
N
N
O
H
2
N
O
CH
3
HOOC
HN
O
O
16
17
18 36%

Scheme 6.
In 1997 Kappe and co-workers reported the oxidation of 2-methoxy-1,4-
dihydropyrimidines 23a-h to 2-methoxy-pyrimidines 24a-h with different
oxidants: cerium ammonium nitrate (CAN) in water, manganese oxide,
chloranil and 2,6-dichloro-3,5-dicyanobenzoquinone (DDQ) using, in all of
them, CH
2
Cl
2
as solvent. The best results were obtained using DDQ at room
temperature with reaction times from 8 to 18 hours and yields up to 70%. Two
of the resulting 2-methoxy-pyrimidines, 24b and 24c, were converted by O-
demethylation with pyridinium hydrochloride under reflux for 30 minutes into
the corresponding pyrimidinones 25b and 25c in moderate yields.[13]

Scheme 7.
Following this work, Kappe and co-workers presented the first study that
could be considered a methodology for the oxidation of 6-methyl-3,4-
N
H
NH
O Me
NC
Pd/C(10%), Diphenyl ether
230 C, 2 h
19
20 81%
N
H
N
O Me
NC
N
NH
O
Pd/C(10%), Diphenyl ether
210 C, 1 h
21a R =H
21b R =Me
22a R =H 68%
22b R =Me 75%
O
R
N
NH
O
O
R
N
H
N
OMe
R
Me
23a R =2-thienyl
23b R =Ph
23c R =i-propyl
23d R =n-propyl
23e R =2-furyl
23f R =2-ClC
6
H
4

23g R =2-MeOC
6
H
4
23h R =CH(Et)
2
O
EtO
DDQ, CH
2
Cl
2
r.t. 8-18 h
N
N
OMe
R
Me
O
EtO
24a R =2-thienyl 50%
24b R =Ph 55%
24c R =i-propyl 60%
24d R =n-propyl 55%
24e R =2-furyl 65%
24f R =2-ClC
6
H
4
60%
24g R =2-MeOC
6
H
4
70%
24h R =CH(Et)
2
40%
Py, HCl
N
H
N
O
R
Me
O
EtO
25b R =Ph 45%
25c R =i-propyl 40%
reflux, 30 min.
dihydropyrimidin-2(1H)-ones. In this work eight dihydropyrimidin-2(1H)-
ones were successfully oxidized to the corresponding 6-methlylpyrimidin-
2(1H)-ones 27a-g with nitric acid at low temperature in moderate to good
yields (29-77%) without any reference to the oxidation of the methyl group at
C-6. Per-nitrated compound 28 was obtained in good yield using higher
temperature (Scheme 8).[14] This approach was later used for the synthesis of
6-methyl-pyrimidin-2(1H)-one-5-carboxylates 30a and 30b in 80 and 75%
yield, respectively (Scheme 9).[15] These compounds were reduced through
nucleophilic addition, as referred in the reduction section (Scheme 28).
Dehydrogenated product 25b was later used as reagent for the synthesis of
multifunctionalized pyrimidines trhough the phosphonium based reagent
bromo-tris-pyrrolidino phosphoniumhexafluorophosphate (PyBroP) mediated
coupling with C, N, O and S nucleophiles.[16]

Scheme 8.

Scheme 9.
Ceric ammonium nitrate (CAN) has been explored for the
dehydrogenation of 3,4-dihydropyrimidin-2(1H)-ones. Shamumugan and
Perumal obtained pyrimidin-2(1H)-ones 32 or tetrahydropyrimidin-2,4-
(1H,3H)-diones 33 regioselectively by varying the reaction conditions.
Pyrimidin-2(1H)-ones 32 were obtained in high yields using CAN and
N
NH
O
R
2
Me
O
R
3
R
1
4 R
1
=H; R
2
=Ph; R
3
=OEt
26a R
1
=H; R
2
=Me; R
3
=OEt
26b R
1
=H; R
2
=4-NO
2
C
6
H
4
; R
3
=OEt
26c R
1
=H; R
2
=3-MeOC
6
H
4
; R
3
=OMe
26d R
1
=H; R
2
=2-CF
3
C
6
H
4
; R
3
=OEt
26e R
1
=Me; R
2
=Ph; R
3
=OEt
26f R
1
=Me; R
2
=3-NO
2
C
6
H
4
; R
3
=OBn
26g R
1
=H; R
2
=Ph; R
3
=NEt
HNO
3
(50-60%)
0 C, 2-30 min
N
N
O
R
2
Me
O
R
3
R
1
25b R
1
=H; R
2
=Ph; R
3
=OEt 77%
27a R
1
=H; R
2
=Me; R
3
=OEt 54%
27b R
1
=H; R
2
=4-NO
2
C
6
H
4
; R
3
=OEt 59%
27c R
1
=H; R
2
=3-MeOC
6
H
4
; R
3
=OMe 76%
27d R
1
=H; R
2
=2-CF
3
C
6
H
4
; R
3
=OEt 29%
27e R
1
=Me; R
2
=Ph; R
3
=OEt 65%
27f R
1
=Me; R
2
=3-NO
2
C
6
H
4
; R
3
=OBn 76%
27g R
1
=H; R
2
=Ph; R
3
=NEt 61%
N
NH
O
Ph O
EtO
N
O
2
N
OH
28
N
NH
O Me
O
EtO
R
HNO
3
(40%)
0 C to r.t., 30 min
N
N
O Me
O
EtO
R
29a R =Me
29b R =H
30a R =Me 80%
30b R =H 75%
NaHCO
3
as buffering agent, suspended in acetone in ice-cooled conditions,
followed by stirring the reaction mixture at ambient temperature for 1 h. Using
CAN as oxidant and acetic acid as solvent, reaction conditions widely
employed for the oxidation of dihydropyridines,[17] 3,4-dihydropyrimidin-
2(1H)-ones with a methyl group at C-6 are regioselectively converted into the
corresponding tetrahydropyrimidin-2,4-(1H,3H)-diones in moderate yields
(Scheme 10). The scope of the reaction was assessed through the synthesis of a
large set of structurally diverse pyrimidines (Figure 2).[18]

Scheme 10.

Figure 2. Diverse pyrimidinones obtained by oxidation of the corresponding dehydro
compound with CAN/ NaHCO
3
.
More recently, the combination of CAN and HCl was used for the rapid
and efficient dehydrogenation of 3,4-dihydropyrimidin-2(1H)-ones at room
temperature in high yields, being the pure reaction product easily separated by
precipitation from CH
2
Cl
2
/hexane (Scheme 11).[19]
N
NH
O
R
1
Me
O
R
2
H
CAN (3 Equiv.)
NaHCO
3
(5 Equiv.)
Aq. Acetone
Argon, -5 C to r.t., 1h
N
N
O
R
1
Me
O
R
2
H
CAN (5 Equiv.)
AcOH
Argon, 80 C, 1-2h
N
NH
O
R
O
O
EtO
H
4 R
1
=Ph; R
1
=OEt
31a R
1
=4-biphenyl; R
2
=OEt
31b R
1
=2-ClC
6
H
4
; R
2
=OEt
31c R
1
=2-NO
2
C
6
H
4
; R
2
=OEt
31d R
1
=3-HOC
6
H
4
; R
2
=OEt
31e R
1
=4-MeOC
6
H
4
; R
2
=OEt
31f R
1
=4-MeOC
6
H
4
; R
2
=OMe
25a R
1
=Ph; R
2
=OEt 83%
32a R
1
=4-biphenyl; R
2
=OEt 79%
32b R
1
=2-ClC
6
H
4
; R
2
=OEt 85%
32c R
1
=2-NO
2
C
6
H
4
; R
2
=OEt 80%
32d R
1
=3-HOC
6
H
4
; R
2
=OEt 81%
32e R
1
=4-MeOC
6
H
4
; R
2
=OEt 81%
32f R
1
=4-MeOC
6
H
4
; R
2
=OMe 83%
33a R

=Ph 61%
33b R

=2-NO
2
C
6
H
4
55%
33c R

=4-MeOC
6
H
4
68%
N
H
N
O Me
O
EtO
N
H
N
O
O
EtO
OMe
N
H
N
O F
3
C
O
EtO
OMe
N
H
N
O
O
EtO
Br
N
H
N
O Me
O
EtO
N N
Ph
Br
N
N
O Me
O
EtO
Me
N
N
O Me
O
EtO
Me
27e 85%
N
N
O Me
O
EtO
Me
39 69 %
OMe
N
H
N
O
O
N
H
N
O
O
N
HN
N
NH
O
O
OEt
O
EtO
O O
O
Me Me
34 82%
35 71% 36 80% 37 76% 38 80%
40 65% 41 61%
42 71% 43 60%

Scheme 11.
Yamamoto and co-workers used tert-butylhydroperoxide (TBHP) and
CuCl
2
for the catalytic dehydrogenation of 3,4-dihydropyrimidin-2(1H)-ones
46 to the more oxidized pyrimidin-2-ol derivatives 47.

Scheme 12.
The authors evaluated several copper, palladium, ruthenium and iron salts in
different solvents concluding that the optimal reaction conditions for the
aromatization/dehydrogenation reaction was 1 mol% of CuCl
2
, 2 to 2.5
equivalents of TBHP in CH
2
Cl
2
and 0.1 to 0.3 equivalent of K
2
CO
3
, which led
to a significant rate acceleration at 40 C for 15 to 24 h (Scheme 12).[20] This
methodology was used for the synthesis of C-2 substituted pyrimidines by
N
NH
O
R
1
Me
O
R
2
H
CAN (2 Equiv.)
50% aq. HCl
AcOH, r.t., 20 min
N
N
O
R
1
Me
O
R
2
H
4 R
1
=Ph; R
2
=OEt
31b R
1
=2-ClC
6
H
4
; R
2
=OEt
31e R
1
=4-MeOC
6
H
4
; R
2
=OEt
44a R
1
=3-MeOC
6
H
4
; R
2
=OEt
23g R
1
=2-MeOC
6
H
4
; R
2
=OEt
44c R
1
=4-MeC
6
H
4
; R
2
=OEt
44d R
1
=3-MeC
6
H
4
; R
2
=OEt
44e R
1
=4-ClC
6
H
4
; R
2
=OEt
44f R
1
=3-NO
2
C
6
H
4
; R
2
=OEt
44g R
1
=2-MeO-5-BrC
6
H
3
; R
2
=OEt
44h R
1
=4-ClC
6
H
4
; R
2
=CH
3

44i R
1
=4-MeC
6
H
4
; R
2
=CH
3
25a R
1
=Ph; R
2
=OEt 85%
31b R
1
=2-ClC
6
H
4
; R
2
=OEt 80%
32e R
1
=4-MeOC
6
H
4
; R
2
=OEt 82%
45a R
1
=3-MeOC
6
H
4
; R
2
=OEt 75%
45b R
1
=2-MeOC
6
H
4
; R
2
=OEt 72%
45c R
1
=4-MeC
6
H
4
; R
2
=OEt 80%
45d R
1
=3-MeC
6
H
4
; R
2
=OEt 78%
45e R
1
=4-ClC
6
H
4
; R
2
=OEt 83%
45f R
1
=3-NO
2
C
6
H
4
; R
2
=OEt 76%
45g R
1
=2-MeO-5-BrC
6
H
3
; R
2
=OEt 70%
45h R
1
=4-ClC
6
H
4
; R
2
=CH
3
78%
45i R
1
=4-MeC
6
H
4
; R
2
=CH
3
76%
N
NH
O
R
1
R
2
O
MeO
H
1mol% CuCl
2
TBHP (2-2.5 Equiv.)
K
2
CO
3
(0.1 Equiv.)
CH
2
Cl
2
, 40C, 15-24h
N
N
OH
R
1
R
2
O
MeO
46a R
1
=Ph; R
2
=Me
46b R
1
=4-FC
6
H
4
; R
2
=Me
46c R
1
=4-ClC
6
H
4
; R
2
=Me
46d R
1
=4-MeC
6
H
4
; R
2
=Me
46e R
1
=cyclopropyl; R
2
=Me
46f R
1
=Ph; R
2
=i-Pr
46g R
1
= 4-FC
6
H
4
; R
2
=i-Pr
46h R
1
= i-Pr; R
2
=Ph
31a R
1
=4-MeOC
6
H
4
; R
2
=Me
47a R
1
=Ph; R
2
=Me 80%
47b R
1
=4-FC
6
H
4
; R
2
=Me 93%
47c R
1
=4-ClC
6
H
4
; R
2
=Me 85%
47d R
1
=4-MeC
6
H
4
; R
2
=Me 84%
47e R
1
=cyclopropyl; R
2
=Me 97%
47f R
1
=Ph; R
2
=i-Pr 93%
47g R
1
= 4-FC
6
H
4
; R
2
=i-Pr 97%
47h R
1
= i-Pr; R
2
=Ph 77%
47i R
1
=4-MeOC
6
H
4
; R
2
=Me 83%
sequential oxidation, esterification and cross-coupling with N, S and O
nucleophiles in PEG-400 at room temperature.[21]
The oxidizing hypervalent iodine compound (diacetoxyiodo)benzene
(DIB, PhI(OAc)
2
) was unable to dehydrogenate 3,4-dihydropyrimidin-2(1H)-
ones by itself, but using TBHP as an additive the dehydrogenation takes place
in mild conditions to yield pyrimidin-2(1H)-ones in good yields after 3-4
hours (Scheme 13).[22]

Scheme 13.

Scheme 14.
N
NH
O
R
Me
O
EtO
H
PhI(OAc)
2
(1.1 Equiv.)
TBHP (2 Equiv.)
CH
2
Cl
2
r.t., 3-4 h
N
N
O
R
Me
O
EtO
H
4 R =Ph
26b R =4-N
2
OC
6
H
4
31e R =4-MeOC
6
H
4
44a R =3-MeOC
6
H
4
44c R =4-MeC
6
H
4
44e R =4-ClC
6
H
4
44f R =3-N
2
OC
6
H
4
48a R =3,4-(MeO)
2
C
6
H
3
48b R =3-BrC
6
H
4
48c R =(CH
3
)
2
CH
48g R =(CH
3
)
2
CHCH
2
25b R =Ph 84%
27b R =4-N
2
OC
6
H
4
79%
32e R =4-MeOC
6
H
4
83%
45a R =3-MeOC
6
H
4
79%
45c R =4-MeC
6
H
4
83%
45e R =4-ClC
6
H
4
84%
45f R =3-N
2
OC
6
H
4
83%
49a R =3,4-(MeO)
2
C
6
H
3
80%
49b R =3-BrC
6
H
4
79%
49c R =(CH
3
)
2
CH 73%
49g R =(CH
3
)
2
CHCH
2
72%
N
NH
O
R
1
Me
O
R
2
H
TBO (3.5 Equiv.)
Acetonitrile
Argon, 100 C
N
N
O
R
1
Me
O
EtO
H
50a R
1
=PhCH
2
CH
2
; R
2
=OEt
50b R
1
=4-(CH
3
)
2
NC
6
H
4
; R
2
=OEt
50c R
1
=2-thienyl; R
2
=OEt
44h R
1
=4-ClC
6
H
4
; R
2
=CH
3
44i R
1
=4-NO
2
C
6
H
4
; R
2
=CH
3
50d R
1
=Ph; R
2
=CH
3
50e R
1
=2-CH
3
OC
6
H
4
; R
2
=CH
3
50f R
1
=3-CH
3
OC
6
H
4
; R
2
=CH
3
50g R
1
=4-CH
3
OC
6
H
4
; R
2
=CH
3
50h R
1
=2-ClC
6
H
4
; R
2
=CH
3
50i R
1
=3-ClC
6
H
4
; R
2
=CH
3
50j R
1
=4-BrC
6
H
4
; R
2
=CH
3
50k R
1
=2-NO
2
C
6
H
4
; R
2
=CH
3
50l R
1
=3-NO
2
C
6
H
4
; R
2
=CH
3
50m R
1
=4-CH
3
C
6
H
4
; R
2
=CH
3
50n R
1
=4-(CH
3
)
2
NC
6
H
4
; R
2
=CH
3
50o R
1
=2-thienyl; R
2
=CH
3
R
2
=OEt
51a R
1
=PhCH
2
CH
2
87%
51b R
1
=4-(CH
3
)
2
NC
6
H
4
89%
51c R
1
=2-thienyl 86%
N
N
O
R
1
Me
O
H
3
C
H
TBO (3.5 Equiv.)
Acetonitrile
Argon, 100 C
45h R
1
=4-ClC
6
H
4
88%
45i R
1
=4-NO
2
C
6
H
4
50%
52d R
1
=Ph 86%
52e R
1
=2-CH
3
OC
6
H
4
82%
52f R
1
=3-CH
3
OC
6
H
4
70%
52g R
1
=4-CH
3
OC
6
H
4
86%
52h R
1
=2-ClC
6
H
4
80%
52i R
1
=3-ClC
6
H
4
78%
52j R
1
=4-BrC
6
H
4
85%
52k R
1
=2-NO
2
C
6
H
4
60%
52l R
1
=3-NO
2
C
6
H
4
70%
52m R
1
=4-CH
3
C
6
H
4
83%
52n R
1
=4-(CH
3
)
2
NC
6
H
4
86%
52o R
1
=2-thienyl 80%
R
2
=CH
3

Scheme 15.
A very similar set of 5-carboethoxy-3,4-dihydopyrimidin-2(1H)-ones and
the analogous 5-acetyl derivatives were oxidized to the corresponding
pyrimidin-2(1H)-ones, also in high yields, using benzoyl peroxide (BPO)
without any other additive in acetonitrile under an argon atmosphere at 100 C.
The reaction time varied from 1 to 8 hours for the 5-carboethoxy derivatives
and from 3 to 10 hours for the 5-acetyl analogues. Besides compounds 4, 23g,
26b, 31a,b, 44a,c,e and 48a three more 5-carboethoxy derivatives 50a-c and
the twelve 5-acetyl derivatives 44h,i and 50d-o were successfully oxidized to
the corresponding pyrimidin-2(1H)-ones (Scheme 14).[23]
Potassium persulfate, K
2
S
2
O
8
, in aqueous aceotinitrile was used for the
first time as oxidant for 3,4-dihydopyrimidin-2(1H)-ones mixed with
hexahydrate Co(II) nitrate. In 2007, Shanmugan and Perumal reported the
oxidation-dealkylation of 6-methyl and bromomethyl dihydropyrimidin-
2(1H)-ones to the corresponding demethylated products 11b and 53 when the
reaction solution was heated at 80 C for 3 hours. The oxidation of the 4-alkyl
derivatives in acetonitrile at reflux afford 2,6-dioxo-1,2,5,6-
tetrahydropyrimidine-5-carboxylate (54). The oxidation reaction of less
sensitive substituents afford the pyrimidin-2(1H)-ones 36, 37, 41 and 42 in
moderate yields (Scheme 15).[24]
Memmarian and Farhadi used potassium persulfate, K
2
S
2
O
8
, in aqueous
acetotinitrile without additives to successfully oxidize ten 6-methyl-5-
carboxyethyl-3,4-dihydropyrimidin-2(1H)-ones to the corresponding
pyrimidin-2(1H)-ones without demethylation. Using acetonitrile/water under
reflux for 15 to 110 minutes, the corresponding dehydrogenated derivatives
N
H
NH
O
R
1
R
2
O
EtO
N
H
N
O
R
1
H
O
EtO

53 R
1
=2-NO
2
C
6
H
4
78%
11b R
1
=Ph 65%
R
1
=Ph or 2-NO
2
C
6
H
4
R
2
=CH
3
or CH
2
Br
Co(NO
3
)
2
.6H
2
O/K
2
S
2
O
8
Aq. CH
3
CN, 80C N
H
N
O O
O
EtO
54 73%
Co
2+
/S
2
O
8
2-
CH
3
CN
Reflux
R
1
=C
2
H
5
ou (CH
3
)
2
CH
R
2
=CH
3
N
H
N
O
O
EtO
OMe
N
H
N
O F
3
C
O
EtO
OMe
36 65% 37 52%
N
H
N
O
O
N
H
N
O
O
41 45% 42 77%
were obtained in 60-85 % yields.[25] The reaction times were reduced to 5-27
minutes and the yields improved to 90-95% performing the reaction at 70 C
under ultrasound irradiation.[26] Using the same procedure 6-methyl-5-acetyl-
3,4-dihydropyrimidin-2(1H)-ones were dehydrogenated in 10 to 40 minutes to
yield the corresponding pyrimidin-2(1H)-ones in 85-97% yield.[27] Using
only water as solvent and under microwave irradiation the same set of
compounds was obtained in shorter reaction times and yields between 87 to
95% (Scheme 16).[28]
The photocatalytic system anatase TiO
2
/O
2
[29]

and anatase

TiO
2
nanoparticles [30] was successfully used for the oxidation of 3,4-
dihydropyrimidin-2(1H)-ones in high yields in 2-4 hours, using 40 mg of
catalyst per mmol of heterocycle. The electron withdrawing or donor character
of the substituent on the phenyl group influenced the reaction rate (Scheme
17). The use of light and chloroform without any additional catalyst was also
reported as a method for the dehydrogenation of 3,4-dihydropyrimidin-2(1H)-
ones.

Scheme 16.

N
NH
O
R
1
Me
O
R
2
H
K
2
S
2
O
8,
CH
3
CN/H
2
O
Reflux
15-110 min
4 R
1
=Ph
23g R
1
=2-MeOC
6
H
4
26b R
1
=4-N
2
OC
6
H
4
31b R
1
=2-ClC
6
H
4
31e R
1
=4-MeOC
6
H
4
44a R
1
=3-MeOC
6
H
4
44c R
1
=4-MeC
6
H
4
50a R
1
=PhCH
2
CH
2
55a R
1
=3-ClC
6
H
4
55b R
1
=2-BrC
6
H
4
60-85%
K
2
S
2
O
8,
CH
3
CN/H
2
O
70 C, ultrasound,
5-27 min
N
N
O
R
1
Me
O
EtO
H
90-95%
K
2
S
2
O
8,
H
2
O
MW, 1-3 min
87-95%
R
2
=OEt
R
2
=Me
K
2
S
2
O
8,
H
2
O
MW, 4-3 min
87-95%
N
N
O
R
1
Me
O
Me
H
70 C, ultrasound,
10-40 min
K
2
S
2
O
8,
CH
3
CN/H
2
O
85-97%
56
57

Scheme 17.

Scheme 18.

Scheme 19.
The irradiation with a 400 W high-pressure mercury lamp of a chloroform
solution saturated with Argon for 10 to 26 hours (depending on the nature of
N
H
N
O
R
Me
O
EtO
N
H
NH
O
R
Me
O
EtO
TiO
2
/O
2,
h, pH =7
CH
3
CN, r. t. 2 - 4 h
4 R =Ph
26b R =4-N
2
OC
6
H
4
31c R =2-N
2
OC
6
H
4
31e R =4-MeOC
6
H
4
44c R =4-MeC
6
H
4
44e R =4-ClC
6
H
4
58 R =2,6-Cl
2
C
6
H
4
25b R =Ph 90%
27b R =4-N
2
OC
6
H
4
85%
32c R =2-N
2
OC
6
H
4
80%
32e R =4-MeOC
6
H
4
85%
45c R =4-MeC
6
H
4
95%
45e R =4-ClC
6
H
4
95%
59 R =2,6-Cl
2
C
6
H
4
96%
N
NH
O
R
Me
O
EtO
H
4 R =Ph
23g R =2-MeOC
6
H
4
26b R =4-N
2
OC
6
H
4
31b R =2-ClC
6
H
4
31e R =4-MeOC
6
H
4
44a R =3-MeOC
6
H
4
44c R =4-MeC
6
H
4
50a R =PhCH
2
CH
2
55a R =3-ClC
6
H
4
55b R =2-BrC
6
H
4
N
NH
O
R
Me
O
EtO
H
25b R =Ph 93%
24g R =2-MeOC
6
H
4
96%
27b R =4-N
2
OC
6
H
4
95%
32b R =2-ClC
6
H
4
92%
32e R =4-MeOC
6
H
4
94%
45a R =3-MeOC
6
H
4
92%
45c R =4-MeC
6
H
4
95%
51a R =PhCH
2
CH
2
92%
56a R =3-ClC
6
H
4
92%
56b R =2-BrC
6
H
4
96%
h >280 nm, CHCl
3,
Ar
r.t. 10-26 h
N
NH
O
R
1
R
2
O
EtO
H
4 R
1
=Ph ; R
2
=CH
3
31e R
1
=4-MeOC
6
H
4
; R
2
=CH
3
44c R
1
=4-MeC
6
H
4
; R
2
=CH
3
44e R
1
=4-ClC
6
H
4
; R
2
=CH
3
61a R
1
=4-BrC
6
H
4
; R
2
=CH
3
61b R
1
=Ph; R
2
=Ph
61c R
1
=4-CH
3
C
6
H
4
; R
2
=Ph
61d R
1
=4-ClC
6
H
4
; R
2
=Ph
N
N
R
1
R
2
O
EtO
H
60, h >400 nm, K
2
CO
3,
CCl
4
CH
3
CN/H
2
O, r.t. 10-26 h
O
25b R
1
=Ph ; R
2
=CH
3
82%
32e R
1
=4-MeOC
6
H
4
; R
2
=CH
3
91%
45c R
1
=4-MeC
6
H
4
; R
2
=CH
3
88%
45e R
1
=4-ClC
6
H
4
; R
2
=CH
3
86%
62a R
1
=4-BrC
6
H
4
; R
2
=CH
3
84%
62b R
1
=Ph; R
2
=Ph 84%
62c R
1
=4-CH
3
C
6
H
4
; R
2
=Ph 91%
62d R
1
=4-ClC
6
H
4
; R
2
=Ph 90%
N
N
Re
CO
Br
CO
CO
60
the substituent at the phenyl group at position 4) afforded the oxidation
product in high yields, (Scheme 18).[31]
With visible light irradiation of rhenium(I) complex 60, a photochemical
conversion of 3,4-dihydropyrimidin-2(1H)-ones with methyl and phenyl
substituents at position 6 to pyrimidin-2(1H)-ones at room temperature has
been achieved with good yields in acetonitrile-water solution containing CCl
4

and K
2
CO
3
(Scheme 19).[32]
Nitrosonium cation (NO
+
) was used as an oxidant for 3,4-
dihydropyrimidin-2(1H)-ones with aryl and alkyl substituents at position 4.
The reaction of 3,4-dihydropyrimidin-2(1H)-ones with NO
+
BF4
-
in
acetonitrile at room temperature for reaction times under 3 hours yields
pyrimidin-2(1H)-ones in quantitative yield with the only exception of DHPM
23g, whose oxidation only affords 24g in 62% yield (Scheme 20).[33]

Scheme 20.
5-carboxamide-3,4-dihydropyrimidin-2(1H)-ones 65 were oxidized to the
corresponding dehydro derivatives 66 using tetrabutylammonium
peroxydisulfate (TBAPS) in acetonitrile at 85 C. The use of ultrasound
irradiation allows the decrease of the reaction temperature in 10 C and a slight
improvement of yields. The desired oxidation products were obatined in yields
up to 80% (Scheme 21).[34]
The oxidation of 3,4-dihydropyrimidine-2(1H)-thiones to pyrimidine-
2(1H)-thiones was found to be an exceedingly more complex task than the
oxidation of the corresponding oxo analogues. In 1990 Atwal and co-workers
observed that the slow oxidation of thioheterocycles 67 with DDQ in dioxane
at room temperature afforded pyrimidinone 68 (Scheme 22).[7]

N
H
N
O
R
2
O R
1
Me
NO
+
BF
4
-
(1.2 Equiv.), CH
3
CN
r. t. 0.7-3.0 h
N
H
NH
O
R
2
O R
1
Me
4 R
1
=Ph; R
2
=OEt
23g R
1
=4-N
2
OC
6
H
4
; R
2
=OEt
31e R
1
=4-MeOC
6
H
4
; R
2
=OEt
44c R
1
=4-MeC
6
H
4
; R
2
=OEt
44e R
1
=4-ClC
6
H
4
; R
2
=OEt
63a R
1
=2-MeC
6
H
4
; R
2
=OEt
63b R
1
=i-Bu; R
2
=OEt
63c R
1
=n-Pr; R
2
=OEt
63d R
1
=i-Hex; R
2
=OEt
44i R
1
=4-MeC
6
H
4
; R
2
=CH
3
46a R
1
=4-MeC
6
H
4
; R
2
=CH
3
4 R
1
=Ph; R
2
=OEt >99%
23g R
1
=4-N
2
OC
6
H
4
; R
2
=OEt 62%
31e R
1
=4-MeOC
6
H
4
; R
2
=OEt >99%
44c R
1
=4-MeC
6
H
4
; R
2
=OEt >99%
44e R
1
=4-ClC
6
H
4
; R
2
=OEt >99%
63a R
1
=2-MeC
6
H
4
; R
2
=OEt >99%
63b R
1
=i-Bu; R
2
=OEt >99%
63c R
1
=n-Pr; R
2
=OEt >99%
63d R
1
=i-Hex; R
2
=OEt >99%
44i R
1
=4-MeC
6
H
4
; R
2
=CH
3
>99%
46a R
1
=4-MeC
6
H
4
; R
2
=CH
3
>99%

Scheme 21.

Scheme 22.
Twenty years later, Shin reported the synthesis of 2-unsubstituted
dehydropyrimidines 70 with moderate yields through desulfurative oxidation
of 3,4-dihydropyrimidine-2(1H)-thiones using H
2
O
2
in the presence of a
catalytic amount of vanadyl sulfate, or in slightly higher yields using Oxone.
In both cases, the major side product of this reactions was found to be 3,4-
dihydropyrimidin-2(1H)-ones.

N
H
N
O
R
2
HN
O R
1
Me
TBAPS, CH
3
CN
74C, ultrasound, 3-10 min.
N
H
NH
O
R
2
HN
O R
1
Me
65a R
1
=Ph; R
2
=Ph
65b R
1
=Ph; R
2
=CH
2
C
6
H
5
65c R
1
=Ph; R
2
=4-FC
6
H
4
65d R
1
=Ph; R
2
=2-ClC
6
H
4
65e R
1
=Ph; R
2
=4-ClC
6
H
4
65f R
1
=Ph; R
2
=4-BrC
6
H
4
65g R
1
=2-ClC
6
H
4
; R
2
=2-ClC
6
H
4
65h R
1
=3-ClC
6
H
4
; R
2
=2-ClC
6
H
4
65i R
1
=4-ClC
6
H
4
; R
2
=2-ClC
6
H
4
65j R
1
=2-BrC
6
H
4
; R
2
=2-ClC
6
H
4
65k R
1
=4-BrC
6
H
4
; R
2
=2-ClC
6
H
4
65l R
1
=3-N
2
OC
6
H
4
; R
2
=2-ClC
6
H
4
65m R
1
=4-N
2
OC
6
H
4
; R
2
=2-ClC
6
H
4
65n R
1
=3-CH
3
OC
6
H
4
; R
2
=2-ClC
6
H
4
65o R
1
=4-CH
3
OC
6
H
4
; R
2
=2-ClC
6
H
4
65p R
1
=4-CH
3
C
6
H
4
; R
2
=2-ClC
6
H
4
65q R
1
=CH
3
CHC
6
H
5
; R
2
=2-ClC
6
H
4
66a R
1
=Ph; R
2
=Ph 90%
66b R
1
=Ph; R
2
=CH
2
C
6
H
5
90%
66c R
1
=Ph; R
2
=4-FC
6
H
4
90%
66d R
1
=Ph; R
2
=2-ClC
6
H
4
91%
66e R
1
=Ph; R
2
=4-ClC
6
H
4
89%
66f R
1
=Ph; R
2
=4-BrC
6
H
4
88%
66g R
1
=2-ClC
6
H
4
; R
2
=2-ClC
6
H
4
92%
66h R
1
=3-ClC
6
H
4
; R
2
=2-ClC
6
H
4
91%
66i R
1
=4-ClC
6
H
4
; R
2
=2-ClC
6
H
4
92%
66j R
1
=2-BrC
6
H
4
; R
2
=2-ClC
6
H
4
90%
66k R
1
=4-BrC
6
H
4
; R
2
=2-ClC
6
H
4
90%
66l R
1
=3-N
2
OC
6
H
4
; R
2
=2-ClC
6
H
4
84%
66m R
1
=4-N
2
OC
6
H
4
; R
2
=2-ClC
6
H
4
85%
66n R
1
=3-CH
3
OC
6
H
4
; R
2
=2-ClC
6
H
4
91%
66o R
1
=4-CH
3
OC
6
H
4
; R
2
=2-ClC
6
H
4
93%
66p R
1
=4-CH
3
C
6
H
4
; R
2
=2-ClC
6
H
4
89%
66q R
1
=CH
3
CHC
6
H
5
; R
2
=2-ClC
6
H
4
80%
N
H
N
S
EtO
O
Me
NO
2
CO
2
Et
DDQ, Dioxane
r.t. 72 h
N
H
N
O
EtO
O
Me
NO
2
CO
2
Et
67 68

Scheme 23.

Scheme 24.

Scheme 25.
Further oxidation of the reaction products with KMnO
4
in acetone at room
temperature yield 2-unsubstituted pyrimidines 71. Direct oxidation of 3,4-
N
H
NH
X
EtO
O
Me
R
H
2
O
2
(30%, 3.5 Equiv.)
N
H
N EtO
O
Me
R
69a X =S; R =Ph
69b X =S; R =4-C
2
H
5
C
6
H
4
69c X =S; R =n-C
5
H
11
69d X =S; R =PhCH
2
CH
2
69e X =S; R =4-FC
6
H
4
4 X =O; R =Ph
69f X =O; R =4-C
2
H
5
C
6
H
4
69g X =O; R =n-C
5
H
11
50a X =O; R =PhCH
2
CH
2
46b X =O; R =4-FC
6
H
4
VOSO
4
.xH
2
O
KMnO
4
(1 Equiv.), acetone
r. t. 2 h
N
N EtO
O
Me
R
71a R =Ph 73%
71b R =4-C
2
H
5
C
6
H
4
73%
71c R =n-C
5
H
11
81%
71d R =PhCH
2
CH
2
87%
71e R =4-FC
6
H
4
76%
72a R =Ph 81% 81%
72b R =4-C
2
H
5
C
6
H
4
44% 62%
72c R =n-C
5
H
11
73% 79%
72d R =PhCH
2
CH
2
35% 63%
72e R =4-FC
6
H
4
65% 79%
N
N EtO
O
Me
R
OH
KMnO
4
(2.5 Equiv.)
acetone, r.t.
70a R =Ph 48%
70b R =4-C
2
H
5
C
6
H
4
43%
70c R =n-C
5
H
11
35%
70d R =PhCH
2
CH
2
73%
70e R =4-FC
6
H
4
50%
From X =S From X =O
N
H
NH
X
EtO
O
Ph
R
73a X =S; R =Ph
73b X =S; R =1-Naphthyl
8b X =O; R =Ph
73c X =O; R =1-Naphthyl
air
100 wt% activated carbon
Xylene, 140 C 41-45 h
N
N
S
EtO
O
Ph
R
S
N
N
Ph
R
O
OEt
74a R =Ph 85%
74b R =1-Naphthyl 91%
N
H
N
S
EtO
O
Ph
R
NaBH
4
MeOH/tBuOH
(1/10)
20 C, overnight
75a R =Ph 79%-82%
75b R =1-Naphthyl 79%-82%
X =S
X =O
Lawersson's Reagent (0.5 Equiv.)
Benzene, Reflux, 4h
HN SMe
NH
2
+
76
R O
EtO
2
C
NaHCO
3
DMF
77
N
H
N
R
EtO
2
C
SMe
78a R=Me
78b R =Ph
Mn(OAc)
3
N
N
R
EtO
2
C
SMe
R =Me
R =Me
MnO
2,
CH
2
Cl
2
MW, 100C, 10 min
or 25 C, 10-18 h
(82%)
79a R=Me 98%
79b R =Ph 99%
60, K
2
CO
3
h >400 nm, 12h
dihydropyrimidin-2(1H)-ones and thioneswith 2.5 equivalents of KMnO
4
in
acetone at room temperature affords 2-hydroxypyrimidines 72 in high yields
(Scheme 23).[35] These two works pointed out one of the major problems for
the preparation of pyrimidine-2(1H)-thiones: the competition with
desulfurization in reactions with oxygen reactants, i.e., oxidants.
Hayashi achieved the synthesis of 6-aryl-pyrimidine-2(1H)-thiones 75
through two different strategies, the reduction of bis(2-pyrimidyl)disulfides 74,
prepared from the oxidation of the corresponding 3,4-dihydropyrimidine-
2(1H)-thiones 73a,b with air and activated carbon, and NaBH
4
, and through
the substitution of oxygen of DHPMs 8b and 73c for sulfur using Lawerssons
reagent in benzene at reflux for 4 hours (Scheme 24).
The Atwal modification of the Biginelli reaction allows the synthesis of 2-
methylthio-1,4-dihydropyrimidines. [36] There are a few examples in the
literature where these dehydroderivatives were oxidized to 2-
methylthiopyrimidines. 2-methylthio-1,4-dihydropyrimidine 78a was oxidized
using manganic acetate under conventional heating [37], through
phtotochemical oxidation with visible light irradiation of rhenium(I) complex
60 [32], or with manganese oxide under microwave irradiation (Scheme
25).[38]
Yamamoto extended the methodology for the oxidation of 3,4-
dihydropyrimidine-2(1H)-thiones to oxidize a set of four 2-methylthio-1,4-
dihydropyrimidines using TBHP, CuCl
2
and K
2
CO
3
in dichloromethane. The
oxidation of the 6-i-propylderivatives 80c,d yields the 6-unsubstituted
pyrimidines 82, as major products (Scheme 26).[20] The dehydrogenation of
methylthio derivative 81c to pyrimidine without dealkylation was performed
using DDQ as oxidant in dicloromethane at room temperature for 30
minutes.[39]

Scheme 26.

1mol% CuCl
2
TBHP (2-2.5 Equiv.)
K
2
CO
3
(0.1 Equiv.)
CH
2
Cl
2
, 40C, 15-24h N
H
N
R
1
EtO
2
C
SMe
R
2
N
N
R
2
R
1
EtO
2
C
SMe
78a R
1
=Me; R
2
=Ph
80b R
1
=4-FC
6
H
4
; R
2
=i-Pr
80c R
1
=i-Pr; R
2
=4-FC
6
H
4
80c R
1
=i-Pr; R
2
=Ph
81a R
1
=Me; R
2
=Ph 72%
81b R
1
=4-FC
6
H
4
; R
2
=i-Pr 68%
81c R
1
=i-Pr; R
2
=4-FC
6
H
4
21%
81c R
1
=i-Pr; R
2
=Ph 25%
+
N
N
R
EtO
2
C
SMe
82a R =4-FC
6
H
4
68%
82b R =Ph 46%
2. REDUCTION

Biginelli himself reported the first reduction of dihydropyrimidines. The
treatment of 4-phenyl-5-carboethoxy-6-methyl-tetrahydropyrimidin-2(1H)-one
(4) with Na amalgam yielded two products. He showed that the one with
higher melting point contained two additional hydrogen atoms and identified it
as the hexahydropyrimidine derivative.[1] Forty years later Folkers studied the
hydrogenation of dihydropyrimidines. His first observation was the reduction
of the pyrimidine nucleus jointly with the phenyl substituent at position four of
dihydropyrimidine 4 to yield 64.7% of tetrahydroderivative 84, when treated
with Adams platinium catalyst and hydrogen at three atmospheres in glacial
acetic acid for 24 hours.[40] The selective hydrogenation of the pyrimidine
ring was achieved using copper, barium and chromium oxides as catalysts,
yielding the tetrahydropyrimidin-2(1H)-one 83, while the reduction of the
phenyl ring was performed using Ni at 145 C. The same catalyst at higher
temperatures, afforded the complete hydrogenation product, the ciclohexyl-
tetrahydropyrimidin-2(1H)-one 84.[41] The reduction of compound 4 using
copper, barium and chromium oxides as catalysts with more drastic conditions
affords a combination of hydrogenation and hydrogenolysis products, among
which it was possible to identify 2-benzyl-butan-1-ol (86) (Scheme 27).[42]
6-Methyl-5-carboxyehtyl-pyrimidin-2(1H)-ones 30a and 30b were
reduced to 6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-carboxylates by
nucleophilic addition of C-nucleophiles in anhydrous THF, under nitrogen
atmosphere at low temperature, and subsequent treatment with aqueous NH
4
Cl
(Scheme 28).[15] The regioselective reaction of 5-unsubstituted pyrimidin-
2(1H)-ones and thiones with organometallic compounds such as Grignard and
organolithium reagents was reported for the preparation of
dihydropyrimidinones and thiones. In fact, the reaction of N-phenyl-4,6-
dimethyl derivatives 87 with alkyl Grignards occurs at the C-6 in preference to
the C-4 of the pyrimidine ring, while alkyl-lithium reagents predominantly
attack the 4-position selectively affording dihydroderivatives 89 (Scheme
29).[43]


Scheme 27.

Scheme 28.
Treatment of pyrimidine-2(1H)-thione 87b, and the resulting
dihydropyrimidine-2(1H)-thiones 88b and 89b with Raney Nickel affords the
corresponding products of reductive desulfurization, 90, 91 and 92,
respectively.
The treatment of 4,6-dimethyl-1-phenylpyrimidine-2(1H)-thione (87b)
with Raney nickel for 3 hours at room temperature under hydrogen
atmosphere gave 1,2-dihydropyrimidine 90 in moderate yield.
Dihydropyrimidine-2(1H)-thiones 88b and 89b, warmed with Raney nickel in
methanol at 50 C for 1 h and then refluxed for 2 hours, afforded the isomeric
products 1,4-dihydropyrimidine 91 and 1,6-dihydropyrimidine 92, respectively
(Scheme 29).[44]
Dihydropyrimidine-2(1H)-thiones of the Biginelli type 93a-g were also
used for the synthesis of 4-aryl derivatives of 1-sustituted 1,4-dihydro-
pyrimidine-4-carboxylic acid.
N
H
NH
O
EtO
O
Me
4
N
H
NH
O
EtO
O
Me
H
2
, CuBaCrO
200 C, 1.8 h
(38.6%)
83
N
H
NH
O
EtO
O
Me
84
H
2
, Ni
145 C, 5.3 h
H
2
, Ni
175 C, 3 h (70.2%)
N
H
NH
O
EtO
O
Me
H
2
, Ni
145 C, 5.3 h
(37%)
H
2
, CuBaCrO
200 C, 2.1 h
(63%)
85
(37.7%)
H
2
, CuBaCrO
250 C, 7.25 h
HO
86 28.6%
EtOH
N
N
O Me
O
EtO
R
28a R =Me
28b R =H
i) C-nuclephile (Nu)
anydrous THF/N
2
, -78C
ii) Satd NH
4
Cl N
NH
O Me
O
EtO
R
Nu Nu =
O
OEt
O
Nu =
O
OMe
O
Nu =
Nu =
Nu =
Nu =
Nu =
29a R =Me 73%
29b R =H 57%
30 R =H 35%
31 R =Me 75%
32 R =H 50%
33 R =Me 96%
34a R =Me 77%
34b R =H 52%
35a R =Me 92%
26a R =H 51%
-CH
3
-SO
2
Ph
-COPh
-CH
2
CN
-COCH
3
The reduction with Raney nickel in acetone gave 1,4-dihydropyrimdines
in moderate yields, with the exception of the brominated derivative, which
undergoes desulfurative reduction with debromination affording 94a as the
only reaction product. The reaction of nitro derivative 93c and methoxy
derivative 93d with Raney nickel in refluxing methanol yielded compounds 95
and 96, respectively (Scheme 30).[45]

Scheme 29.

Scheme 30.
N
N
X
1) RMgBr or RLi,
ether
2) H
2
O r.t. 5-6 h
N
NH
X N
NH
X
87a X =O
87b X =S
88a X =O
88b X =S
89a X =O
89b X =S
+
RMgBr RLi
X =O
R =Me 29 95:5 65 15:85
R =Et 20 50:50 42 10:90
R=iPr 19 0:100 12 0:100
R =tBu 59 80:20 - -
X =S
R =Me 62 70:30 43 35:65
R =Et 83 60:40 42 0:100
R=iPr 48 75:25 10 0:100
R =tBu 75 30:70 - -
Yield(%) 88:89 ratio Yield(%) 88:89 ratio
Ph Ph Ph
X =S
N
N
Ph
Raney Nickel, H
2
MeOH, 3 h, r.t.
90
Raney Nickel
1)MeOH, 1 h ,50 C
2) Reflux, 2h
X =S
Raney Nickel
1)MeOH, 1 h ,50 C
2) Reflux, 2h
X =S
N
N
Ph
91
R
R
N
N
Ph
92
R
R
N
NH
S
R
2
R
Me
R
1
93a R =Ph; R
1
=Ph; R
2
=CO
2
Et
93b R =4-BrC
6
H
4
; R
1
=Ph; R
2
=CO
2
Et
93c R =4-NO
2
C
6
H
4
; R
1
=Ph; R
2
=CO
2
Et
93d R =4-CH
3
OC
6
H
4
; R
1
=Ph; R
2
=CO
2
Et
93e R =Ph, R
1
=Ph; R
2
=CONH
2
93f R =4-CH
3
OC
6
H
4
; R
1
=Ph; R
2
=CN
93g R =Ph; R
1
=CH
3
; R
2
=CO
2
Et
N
N
R
2
R
Me
R
1
95 61%
94a R =Ph; R
1
=Ph; R
2
=CO
2
Et 67%
94b R =4-BrC
6
H
4
; R
1
=Ph; R
2
=CO
2
Et --
94c R =4-NO
2
C
6
H
4
; R
1
=Ph; R
2
=CO
2
E --
94d R =4-CH
3
OC
6
H
4
; R
1
=Ph; R
2
=CO
2
Et 37% and 96
94e R =Ph, R
1
=Ph; R
2
=CONH
2
65%
94f R = 4-CH
3
OC
6
H
4
; R
1
=Ph; R
2
=CN 55%
94g R =Ph; R
1
=CH
3
; R
2
=CO
2
Et 56%
Raney Nickel, acetone
Reflux, 2 h
N
NH
EtO
2
C
Me
Ph
NO
2
N
NH
EtO
2
C
Me
Ph
OCH
3
OH
96 50%
The reductive desulfurization of the methyl ester of 97 and the thiomethyl
derivative 98, obtained by alkylation with CH
3
I and NaH in
hexamethylphosphoramide (HMP) and DMF, afforded pyrimidines 99a and
99b in 50 and 44% yields, respectively (Scheme 31).[46]
Deprotection and reduction reactions of dihydropyrimidin-2(1H)-ones
were used as models for investigating the scope of continuous flow
hydrogenation reactions using either Pd/C or Raney nickel as heterogeneous
catalysts in a compact, continuous flow H-cube,[47] suitable for high pressure
heterogeneous hydrogenation and feasible for high-throughput processing.
Reductive desulfurization of a 0.012 M acetonitrile solution of thione 93g to
yield 95% of 1,4-dihydropyrimidine 94g was achieved through continuous
flow hydrogenation at 40 C and a minimum hydrogen pressure of 1-2 bar.
Using a typical 1 mL/min flow rate complete consumption of the starting
material was obtained after one cycle within 30 minutes (Scheme 32).[48]

Scheme 31.

Scheme 32.

3. BIOLOGICAL ACTIVITY

The interest of the scientific community in dihydropyrimidinone scaffolds
originated because of their resemblance to Hantzsch-type dihydropyridines,
which at the time were well known as calcium channel modulators and
selective
1a
adrenoreceptor antagonists. In the early studies,
dihydropyrimidinones were considered simple aza analogues of Hantzschs
N
N
S Me
CH
3
CH
3
O
MeO
Reflux, 1 h
N
N
Me
CH
3
R
O
MeO
Reflux, 1 h
N
NH
Me
CH
3
O
MeO
SCH
3
97 99a R =CH
3
50%
99b R =H 44%
98
N
NH
S Me
CH
3
O
MeO
Raney Nickel, H
2
(1-2 bars)
acetonitrile, 40 C, 30 min
N
N
Me
CH
3
O
EtO
93g
94g 95%
Cont. flow (1 mL/min)
compounds that could be the solution for the improvement of the
pharmacological profile of these compounds. However, the progress with this
compounds as calcium channel modulators and selective
1a
adrenoreceptor
antagonists, as well as the new discoveries on pharmacological functions
associated to dihydropyrimidinones and thiones made this an interesting class
of compounds in their own right. The pharmacological research of 3,4-
dihydropyrimidine-2(1H)-ones has been previously reviewed with focus on
their function as calcium channel modulators, selective
1a
adrenoreceptor
antagonists, melanin-concentrating hormone receptor 1 antagonist, and as
enzyme (ROCK1 and Hsp70 ATPase) and mitotic kinesis inhibitors.[4b, 5e,
49] Moreover, 3,4-dihydropyrimidine-2(1H)-ones have shown to possess a
very diverse pharmacological profile and their capabilities as antimalarial,
antiviral, antibacterial and antioxidant among others will be the focused in this
review.

3.1. Antioxidant Activity

Antioxidant activity of 3,4-dihydropyrimidi-2(1H)-ones was referred in
the study of Stradyn and co-workers about the electrochemical reduction of
hydrogenated 2-pyrimidones. The authors claim the electrochemical
oxidation of hydrogenated 2-pyrimidones and hydrogenated 2-pyridones
proceeds at comparatively similar potential ranges, while derivatives of 1,2-
and 1,4-dihydropyridine are oxidized much more readilyThis finding is in
accord with the antioxidant properties of these compounds.[9, 50]
The firsts studies on the antioxidant capability of 3,4-dihydropyrimidi-
2(1H)-ones date from 2006. Stefani and co-workers synthetized
dihydropyrimidinones 100-103 and evaluated their antioxidant activity.
Compounds 101a and 101b exhibited a strong activity against lipid
peroxidation induced by Fe + EDTA, while compounds 101a and 103a were
the most potent in reducing reactive oxygen species (ROS) levels, Figure 3.
The diverse set of compounds in Figure 4, including 3,4-dihydropyrimidi-
2(1H)-thione 106 and more oxidized heterocycle 107, were tested for their
antioxidative capability in cumene oxidation. The antioxidative activity was
evaluated through the study of the reaction of these compounds with
cumylperoxy radicals and cumyl hydroperoxide. The capability to terminate
oxidation chains by reaction with cumylperoxy radicals was evaluated using as
example the cumene oxidation with azobis(isobutyronitrile) as initiator. The
experiments showed that one molecule of dihydropyrimidinone decomposed
up to several thousands of cumyl hydroperoxide molecules. No significant
differences related to the structural differences of these compounds were
reported.[51]

Figure 3.

Figure 4.

Figure 5.
Recently, Gangwar and Kasana [51] estimated the radical scavenging
activity and reducing power activity of 3,4-dihydropyrimidi-2(1H)-ones with
ortho and para substituents at the phenyl ring, Figure 5. The most active
compounds are the ones with the hydroxyl group at the phenyl ring. The
compounds with the hydroxyl group at the para position, 108b and 108h are
N
H
NH
O
O
O
R
100a R =H
100b R =NO
2
N
H
NH
O
O
O
R
N
Ph
Ph
101a R =H
101b R =NO
2
N
H
NH
O
O
O
R
Ph
Ph
102a R =H
102b R =NO
2
N
H
NH
O
O
O
R
103a R =H
103b R =NO
2
Ph
N
H
NH
O
EtO
O
26a
N
H
NH
O
EtO
O OH
Br
N
H
NH
S
EtO
O OH
N
H
NH
O
EtO
O
N
N
S
EtO
O
O
105 106
107
104
N
H
NH
O
R
3
O
O
R
1
R
2
4 R
3
=Et; R
1
=R
2
=H
23g R
3
=Et; R
1
=H; R
2
=NO
2
44f R
3
=Et; R
1
=NO
2
; R
2
=H
31e R
3
=Et; R
1
=H; R
2
=OCH
3
108a R
3
=Et; R
1
=OCH
3
; R
2
=OH DPPH (IC
50
) =3.65
108b R
3
=Et; R
1
=H; R
2
=OH DPPH (IC
50
) =2.71
108c R
3
=Me; R
1
=R
2
=H
108d R
3
=Me; R
1
=H; R
2
=NO
2
108e R
3
=Me; R
1
=NO
2
; R
2
=H
108f R
3
=Me; R
1
=H; R
2
=OCH
3
108g R
3
=Me; R
1
=OCH
3
; R
2
=OH DPPH (IC
50
) =5.02
108h R
3
=Me; R
1
=H; R
2
=OH DPPH (IC
50
) =2.18
the most active exhibiting 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging activity comparable to the standard gallic acid, catechin and
butylates hydroxyl toluene (BHT) which have IC
50
values of 2.06, 2.12 and
2.18 g/mL. The compounds with the hydroxyl group at the ortho position
108a and 108g present mild reducing power activity.

Figure 6.
The antioxidant capabilities of several pyrimido[5,4-c]quinolone-2,5-
diones 109 and 2-thioxopyrimido[5,4-c]quinolone-5-ones 110 were estimated
using DPPH method and benzoic acid hydroxylation method [52] for
measuring the hydroxyl radical scavenging activity. The scavenging effects on
DPPH radicals increased in the compounds with a thiourea moiety. In fact,
heterocycles 110a-d, Figure 6, present values at 250 M, comparable with the
values of standard ascorbic acid at 25 M. Hydroxyl radical scavenging
activity for all compounds increased with concentration, compounds 110b and
110d presenting the best results with values at 200 M, comparable with the
standard quercetol at 100 M.[53]
Dimethyl adducts of 6-oxo-4-substituted aryl-2-sulfanyl-1,6-
dihydropyrimidines-5-carbonitrile 111 were screened for their in vitro
antioxidant activity by various methods such as scavenging of hydrogen
peroxide, scavenging of nitric oxide radical, lipid peroxidation inhibitory
activity and reducing power determination.
All the compounds tested showed moderate antioxidant activity compared
with ascorbic acid; compounds 111i and 111j are the more potent, which may
be due to the hydroxyl group present on the benzene ring in the structure,
Figure 7.[54]
In an attempt to correlate the ulcerogenic activity with lipid peroxidation,
Amir and co-workers measured lipid peroxidation activity in the gastric
N
NH
O
HN
O
N
NH
S
HN
O
R
1
R
2
110a R
1
=R
2
=H
110b R
1
=Cl; R
2
=H
110c R
1
=H; R
2
=F
110d R
1
=H; R
2
=Cl
R
109 R =H, F or Cl
mucosa [55] of four indole derivatives of 3,4-dihydropyrimidi-2(1H)-ones and
thiones 112.
The studies showed that these compounds have inhibited induction of
gastric mucosal lesions and that the compounds showing lower ulcerogenic
activity also showed a reduction in lipid peroxidation. In all the compounds,
the values of lipid peroxidation, measured as nmol of malondialdehyde per
100 mg of gastric mucosa tissue, were lower than the standard drug Ibuprofen
(6.15 nmol/100mg), Figure 8.[56]

Figure 7.

Figure 8.
Thirty-two dihydropyrimidin-2(1H)-ones and thione analogs of compound
27g, Scheme 8, were screened for their possible antioxidant activity by DPPH.
Among the thirty-two title compounds only compounds with 3-nitro phenyl
moiety at the position 4 of dihydropyrimidine ring showed good antioxidant
activity with IC
50
values of 58 and 63 mg concentrations, respectively.[57]

3.2. Antibacterial and Antifungal and Antimycobacterial
(Antitubercular) Activity

The antibacterial and antifungal activity of 6-oxo-4-substituted aryl-2-
sulfanyl-1,6-dihydropyrimidine-5-carbonitrile 111a-j, Figure 7, were tested
against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis,
N
N
S
NC
R
O
111a R =4-CH
3
OC
6
H
4
111b R =2-FC
6
H
4
111c R =2-furyl
111d R =2-thienyl
111e R =2-N(CH
3
)
2
C
6
H
4
111f R =4-ClC
6
H
4
111g R =2-ClC
6
H
4
111h R =2,3-(CH
3
O)C
6
H
3

111i R =2-HOC
6
H
4
111j R =2,3-(HO)C
6
H
3
IC
50
(g/mL)
58 39 44 52
53 37 41 48
47 33 26 31
NO H
2
O
2
lipid peroxidation reducing power
ascorbic acid
N
H
NH
X R
112a X =O; R =4-ClC
6
H
4
0.33 4.20
112b X =O; R =4-CH
3
C
6
H
4
0.75 4.34
112c X =S; R =4-ClC
6
H
4
0.41 4.21
112d X =S; R =4-CH
3
C
6
H
4
0.54 4.36
NH
Ulcerogenic activity MDA
(severity index) nmol/100mg
Staphylococcus aureus and fungi Candida albicans and Aspergillus falvus.
Fluorinated derivative 111b was the most potent against B. subtilis with
similar antibacterial activity to the standard drug Streptomycin, closely
followed by the chlorinated derivative 111g. Furyl derivatives 111c and 111e
exhibited potent antifungal activity against C. albicans with better values than
the standard drug Amphotericin-B. [54]
Fused 3,4-dihydropyrimidin-2(1H)-ones and thiones were tested against
standard bacteria and fungi. Ghorab and co-workers [58] synthetized a series
of derivatives of 4-(4-fluorophenyl)-2-thioxo-1,2,3,4,7,8-
hexahydroquinazolin-5(6H)-one (113) and tested their antifungal activity
against four species of fungi namely, Aspergillus ochraceus, Aspergillus
falvus, Penicillium chrysogenum and Candida albicans using the hole cup-
plate agar diffusion method.[59] Compounds 114a and 115a were found to be
the most active, identical to fungicide Trosyd (Tioconazole), against
Aspergillus ochraceus and Aspergillus falvus with values of minimum
inhibitory concentration (MIC) of 40 g/mL. All compounds 114-117 posses
high activity, similar to Trosyd against Penicillium chrysogenum, while only
compound 114b exibit a activity comparable with the reference fungicide
against Candida albicans. The in vitro antibacterial activity of compounds
113b-i against standard strains of Staphylococcus aureus, Escherichia coli and
Pseudomonas aeruginosa was evaluated by the cup-plate agar diffusion
method [60] and Broth Microdilution MIC method [61]. At 50 g/mL
concentration, and using Norfloxacin as the standard drug, all compounds
showed antibacterial activity against gram-negative and gram-positive
bacteria. [62]
in the search for new 2-thiouracil-5-sulphonamide derivatives with
antibacterial and antifungal activity, Fathalla and co-workers synthetized
pyrimidin-2(1H)-ones 118a,b and thiones 118c,d and tested against Bacillus
subtilis, Escherichia coli, Candida albicans, Staphylococcus aureus, Sarcina,
Pseudomonas aeroginosa, and Mycobacterium phlei. The four compounds
showed very different bactericidal activity, while pyrimidine-2(1H)-thione
118c was active against Bacillus subtilis (MIC = 1.25 g/mL), Escherichia
coli (MIC = 412 g/mL) and Staphylococcus aureus (MIC = 125 g/mL).
Thione 188d showed activity against Bacillus subtilis and Staphylococcus
aureus. Pyrimidin-2(1H)-one 118b was weakly active against Staphylococcus
aureus and heterocycle 118a was inactive against all the bacteria strains tested,
Figure 10.[62]


Figure 9.

Figure 10.

Figure 11.
N
H
NH
X
R
3
O 113a X =S; R
1
=R
2
=H, R
3
=4-FC
6
H
4
113b X =S; R
1
=R
2
=CH
3
, R
3
=Ph
113c X =S; R
1
=R
2
=CH
3
, R
3
=4-ClC
6
H
4
113d X =S; R
1
=R
2
=CH
3
, R
3
=5-benzo(1,3)dioxole
113e X =S; R
1
=R
2
=CH
3
, R
3
=6-(2-chloro-3-methyl)quinoline
113f X =O; R
1
=R
2
=CH
3
, R
3
=Ph
113g X =O; R
1
=R
2
=CH
3
, R
3
=4-ClC
6
H
4
113h X =O; R
1
=R
2
=CH
3
, R
3
=5-benzo(1,3)dioxole
113i X =O; R
1
=R
2
=CH
3
, R
3
=6-(2-chloro-3-methyl)quinoline
N
H
N
S
O
F
R
1
R
2
114a R
1
=Ph; R
2
=H
114b R
1
=NH
2
; R
2
=CN
N
H
N
S
O
F
N
N
S
NH
2
N
H
N
S
O
F
NH
N
O
N
H
N
S
O
F
NH
S
HN
O
O
115a R =H
115b R =Me
R
Ph
116
117
R
1
R
2
N
H
NH
X
R
118a X =O; R = 4-NO
2
C
6
H
4
118b X =O; R = 4-ClC
6
H
4
118c X =S; R = 4-NO
2
C
6
H
4
118d X =S; R = 4-ClC
6
H
4
N
H
S
O O
HN
N
H
O
S
N
N
S
119a R = 4-SCH
3
C
6
H
4
119b R = 4-CH
3
OC
6
H
4
119c R = 4-ClC
6
H
4
119d R = 4-FC
6
H
4
119e R = 4-HOC
6
H
4
119f R = 2,4-Cl
2
C
6
H
3
119g R = 4-HO,3-CH
3
OC
6
H
3
119h R = 4-F,3-PhOC
6
H
3
119i R = 4,3-OCH
2
OC
6
H
3
SMe
EtO
2
C
O
R
N
N
S
SMe
EtO
2
C
O O
R
120a R = 4-NO
2
C
6
H
4
120d R = 2,4-Cl
2
C
6
H
3
120b R = 4-ClC
6
H
4
120c R = 4-BrC
6
H
4
18 23 20 18 12 18 19 20
Standard 19 25 20 18 22 18 20 20
S.aureus P.aeruginosa K.pneumoniae E.coli A.fumigatus A.flavus C.albicans P. marneffei
Thiazolo[2,3-b]pyrimidinones 119 and 120 were screened for their
antibacterial and antifungal activities against Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa and Klebsiella pneumonia and for the
antifungal assays against Aspergillus flavus, Aspergillus fumigatus, Candida
albicans and Penicillium marneffei. All compounds have exhibited moderated
to excellent growth inhibition of bacteria and fungi, especially compound 120c
with a 4-bromophenylfuranyl moiety, which showed MIC values similar to the
standard drug ciclopiroxolamine, Figure 11.[63]
Fused 2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones
with isoxazole substituents 121 were evaluated for in vitro antibacterial and
antifungal activity against various Gram-positive (Bacillus subtilis, Bacillus
sphaericus, Staphylococcus aureus), Gram-negative bacteria (Pseudomonas
Aeruginosa, Klebsiella aerogenes, and Chromobacterium violaceum), and
fungal strains (Aspergillus niger, Chrysosporium tropicum, Rhizopus oryzae,
Fusarium moniliformae and Curvularia lunata) using both dilution method
[64]

and agar cup bioassay method.[65]
All the compounds present bactericidal activity comparable to the
reference compound, Ciprofloxacin. As observed for thiazolo[2,3-
b]pyrimidinones, halogenated compounds 121d (MIC = 6-9 g/mL), and 121h
(MIC = 8-10 g/mL), are highly active when compared to unsubstituted
compound 121a (MIC = 13-20 g/mL). Identically, all the tested compounds
present antifungal activity against the five fungi used, in this case the
compounds with methyl 121b or methoxy 121c substituents on the para
position of the benzene ring are the most toxic, with higher antigungal activity
than the standard Clotrimazole.[66]
The minimum inhibitory concentration of biphenyl dihydropyrimidines
122a-h against Staphylococcus aureus, Escherichia coli, and Pseudomonas
aeruginosa was determined by standard agar dilution. The compounds in the
series presented moderate antibacterial activity when compared to
Ciprofloxacin, Sparfloxacin, and Trovafloxacin, Figure 13.[67]

Figure 12.
N
N
S
R
O
N
N
O
O
N
121a R =Ph
121b R =4-CH
3
C
6
H
4
121c R =4-CH
3
OC
6
H
4
121d R =4-BrC
6
H
4
121e R =C
6
H
5
CH
2
121f R =4-ClC
6
H
4
121g R =3-CH
3
OC
6
H
4
121h R =2-ClC
6
H
4

Figure 13.

Figure 14.
Benzylthio substituents of biphenyl dihydropyrimidines 123 were
screened for their antibacterial activity against the same bacteria strains and
Streptococcus pyogenes and Klebsiella pneumoniae bacterial strains. The
investigation of antibacterial screening data revealed that all the tested
compounds showed moderate to good bacterial inhibition. Compounds 123a,
123b, 123e, 123f, 123i and 123j showed very good activity against all the
bacterial strains compared with Ciprofloxacin. Newly prepared compounds
were screened for their antifungal activity against. The antifungal screening
against Aspergillus flavus, Aspergillus fumigatus, Candida albicans,
Penicillium marneffei and Trichophyton mentagrophytes showed moderate to
good activity. Compounds 123a, 123c, 123f, 123i and 123j emerged as very
active against all the fungal strains, Figure 14.[68]

N
H
NH
X
EtO
2
C
122a X =O; R =3-CO
2
Et
122b X =S; R =3-CO
2
Et
122c X =O; R =2-CH
2
CO
2
Et
122d X =S; R =2-CH
2
CO
2
Et
122e X =O; R =4-CH
2
CO
2
Et
122f X =S; R =4-CH
2
CO
2
Et
122g X =O; R =3-OCH
2
CO
2
Et
122h X =S; R =3-OCH
2
CO
2
Et
R
N
H
NH
S
EtO
2
C
123a R =Ph
123b R =4-BrC
6
H
4
123c R =4-(CH
2
CH
3
)C
6
H
4
123d R =4-CH
3
C
6
H
4
123e R =4-OCH
3
C
6
H
4
123f R =4-N(CH
3
)
2
C
6
H
4
123g R =4-ClC
6
H
4
123h R =4-CH(CH
3
)
2
C
6
H
4
123i R =4-C(CH
3
)
3
C
6
H
4
123j R =4-NO
2
C
6
H
4
CO
2
Et
R

Scheme 34.
Using 6-amino-2-thioxo-2,3-dihydro-2(1H)-pyrimidinone (124) as starting
material Sayed and co-workers synthetized new pyrimidine derivatives and
determined their in vitro antibacterial and antifungal activity against
Escherichia coli, Bacillus subtilis and Candida albicans. Only compounds
125a, 125a-c and 127a, are active antibacterials, comparable with standard
drug Ampicillin and compound 126c was the only one active as antifungal
agent; the authors note that cyclisation at the 5,6 position of the
dihydropyrimidine ring may eliminate or decrease the antimicrobial activity
(Scheme 34).[69] The pharmacophoric activity of pyrimidones and
thiobarbituric acid prompted the design and synthesis of 2,4,7-
tri(substituted)phenyl-2,4,8,10-tetraza-3,9-dithioxo-5-oxo-bicyclo [4.4.0] dec-
1(6)-enes (129a-d) and 2,4,7-tri(substituted)phenyl-2,4,8,10-tetraza-3-thioxo-
5,9-dioxo-bicyclo[4.4.0]dec-1(6)-enes (129e-h). All the synthetized
compounds were screened for their antibacterial activity against
Staphyllococcus aureus, Corynebacterium diphtheria, Proteus aeruginosa and
Escherichia coli bacterial strains by the disc diffusion method, showing
activity comparable with the standard drug Ampiciline trihydrate, Figure
15.[70] 4-aryl-5-isopropoxycarbonyl-6-methyl-3,4-dihydropyrimidin-2(1H)-
ones 130ag and 4-phenyl-5-isopropoxycarbonyl-6-methyl-3,4-
dihydropyrimidin-2(1H)-thione (129h) were screened for their antibacterial
activity against Staphylococcus aureus, Escherichia coli, Klebsiella
pneumoniae, Pseudomonas aeruginosa and Salmonella typhi and antifungal
activity against Candida albicans, Aspergillus flavus, Rhizopus and Mucor.

N
H
NH
S
O
H
2
N
N
H
NH
S
O
N
N
H
R
124
125a R =Ph
125b R =2-CH
3
C
6
H
4
125c R =3-CH
3
C
6
H
4
N
H
NH
S
O
N
H
126a R =4-BrC
6
H
4
126b R =3,4,5(OCH
3
)C
6
H
2
126c R =3-indolyl
126d R =4-N(CH
3
)
2
C
6
H
4
126d R =4-NO
2
C
6
H
4
HN
N
H
O
S
R
N
H
NH
S
O
N R
127a R =Ph
127b R =3-indolyl
N
H
NH
S
O
N
128a R =4-OCH
3
C
6
H
4
128b R =4-N(CH
3
)
2
C
6
H
4
R
NC
H
2
N

Figure 15.
Compounds 130b, 130c, exhibited in vitro antibacterial activity against
Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa and
antifungal activity comparable to Amphotericin B. Compound 130f with a
nitro group at the para position of the 4-aryl group and 130g with a fluorine at
the para position of the 4-aryl group showed more activity than the standard
drugs.
The thione derivative is not more active than the reference drug but is two
times more active than the corresponding 3,4-dihydropyrimidin-2(1H)-one
derivative against Candida albinas and Pseudomonas aeruginosa and four
times more active against Mucor and Staphylococcus aureus, Figure 16.[71]
Dimers of 3,4-dihydropyrimidine-2(1H)-thiones 131-134 with flexible
alkyl chain spacers of varying length and conformationally restricted spacers
with unsaturation (cis and trans) have been designed and synthesized. All
compounds were tested for antibacterial activity against Gram-positive
bacteria Staphylococcus aureus, Staphylococcus epidermidis and Bacillus
subtilis and Gram-negative bacteria such as Escherichia coli, Klebsiella
pneumoniae, and Pseudomonas aeruginosa.
The inhibitory zones (in mm) have been determined by using agar well
method. The best results were obtained with compound 133c, having moderate
activity against S. aureus and P. aeroginosa and compounds 131e and 13d also
with moderate activity against S. epidermidis. The antifungal activity was
evaluated against Candida albicans which have been found to be resistant to
all the dimers without showing zones of inhibition, Figure 17.[72]

N
N
S
O
HN
N
H
129a X =S; R
1
=Ph; R
2
=4-OCH
3
C
6
H
4
129b X =S; R
1
=2-CH
3
C
6
H
4
; R
2
=4-ClC
6
H
4
129c X =S; R
1
=2-NO
2
C
6
H
4
; R
2
=Ph
129d X =S; R
1
=2-ClC
6
H
4
; R
2
=2-OHC
6
H
4
129e X =O; R
1
=Ph; R
2
=2-OHC
6
H
4
129f X =O; R
1
=2-CH
3
C
6
H
4
; R
2
=4-OCH
3
C
6
H
4
129g X =O; R
1
=4-ClC
6
H
4
; R
2
=4-OCH
3
C
6
H
4
129h X =O; R
1
= 2-ClC
6
H
4
; R
2
=2-OHC
6
H
4
R
1
R
1
X
R
2

Figure 16.

Figure 17.
Pyrazolinyl derivatives of 3,4-dihydropyrimidin-2(1H)-ones 135 were
tested in vitro for their antibacterial activity against Staphylococcus aureus,
Staphylococcus albus, Escherichia coli, Klebsiella pneumoniae, and Proteus
vulgaris at a 250 mg/mL. Comparing with the standard drug Ciproflaxange at
50 mg/mL only compound 135e, the derivative with a chlorine atom on the
phenyl ring, shows good activity while all the other compounds shows only
moderate activity, Figure 18.[73] 3,4-Dihydropyrimidin-2(1H)-ones with an
isatin moiety 136 were tested as antibiotics against, Escherichia coli,
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia,
Staphylococcus Typhi, Staphylococcus pyogenusm and Bacillus subtilis using
Gentamycin, Ampicillin, Chloramphenicol, Ciprofloxacin and Norfloxacin as
standard antibacterial agents.

N
H
NH
X
R
Me
130a X =O; R =Ph
130b X =O; R = 4-ClC
6
H
4
130c X =O; R = 2-ClC
6
H
4
130d X =O; R = 4-CH
3
C
6
H
4
130e X =O; R = 4-OCH
3
C
6
H
4
130c X =O; R = 4-NO
2
C
6
H
4
130c X =O; R = 4-FC
6
H
4
130c X =S; R = Ph
O
HO
HN
NH
S
Me
131 R =CO
2
Et; R
1
=H
132 R =Methylproline; R =H
133 R =CO
2
Et; R
1
=OCH
3
134 R =Methylproline; R
1
=OCH
3
O
R
R
1
O NH
HN
S
Me O
R
R
1
O
Spacer
Spacer
a b c d e
(CH
2
)
3
(CH
2
)
4
(CH
2
)
5
trans-CH=CH cis-CH=CH

Figure 18.

Figure 19.
Antifungal activity was screened against the three fungal species Candida
albicans, Aspergillus niger and Aspergillus clavatus. Nystatin and Griseofulvin
was used as a standard antifungal agent. Chlorinated 136f and the
corresponding thione 136l were the most active compounds in both
pharmacological activities with values of antibacterial activity comparable to
Ampicillin and Chloramphenicol and antifungal activity comparable to
Nyastin, Figure 19.[74]
4H-Pyrimido[2,1-b]benzothiazole derivatives of curcumin 137a-h were
evaluated for their antibacterial activity against gram-positive and gram-
negative bacteria Staphylococcus aureus, Pseudomonas aeruginosa,
Salmonella typhi, Escherichia coli, Bacillus cereus and Providencia rettgeri
and antifungal activity against fungi Aspergillus niger, Aspergillus fumigates
and Aspergillus flavus. All the compounds show better activity than curcumin
against the strains of bacteria used. Hydroxyl compound 137f was the most
N
H
NH
O
N
135a R =Ph
135b R = 4-CH
3
C
6
H
4
135c R = 3,4-(CH
3
CH
2
)C
6
H
3
135d R = 2,4,6-(CH
3
)
3
C
6
H
2
135e R = 4-ClC
6
H
4
135f R = 4-N(CH
3
)
2
C
6
H
4
N
OH
R
N
H
NH
X Me
136a X =O; R =H
136b X =S; R =H
136c X =O; R =Br
136d X =S; R =Br
136e X =O; R =NO
2
136f X =S; R =NO
2
136g X =O; R =F
136h X =S; R =F
136i X =O; R =I MIC (g/mL)
136j X =S; R =I E.coli P.aeruginosa K.pneumoniae S. Typhi S.aureus S. pyogenusm B. subtilis
136k X =O; R =Cl 100 100 62.5 100 62.5 100 62.5
136l X =S; R =Cl 62.5 100 100 62.5 100 62.5 100
Ampicillin 100 100 100 100 250 100 100
Chloramphenicol 50 50 50 50 50 50 50
O
S
N
N
N
NH
O
R
active compound, especially against S. aureus, with the same value of MIC
95

(1.25 g/mL) as Ciproflaxacin used as standard drug whereas nitro compound
137d exhibited significant activity against A. niger and A. fumigates, Figure
20.[68]
Kumar and co-workers synthetized dihydropyrimidin-2(1H)-ones having a
5-chloro-pyrazole unit at position 4 of the dihydropyrimidinone ring 138a-g.
The compounds were tested for their antibacterial activity against Proteaus
valgarin, Pseudomonas aeruginosa and Escherichia coli. All compounds
showed low or moderate activity against the different types of bacteria when
compared with Gentamicin, Figure 21.[75]

Figure 20.
The substitution of the pyrazole moiety for imidazoyl or thiophenyl does
not bring significant improvement in the antebacterial activity. A series of
twenty two 3,4-dihydropyrimidin-2(1H)-ones and thiones with imidazoyl and
thiophenyl substituents at position 4of the hydropyrimidine ring were screened
for antibacterial and antifungal activity against Gram-positive bacteria, namely
Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis, and
three Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa, and
Salmonella enteritidis. No significant antibacterial activity was observed
except for weak inhibitory activity (MIC = 128 g/ml) exerted by 139b and
139l against Staphylococcus aureus and Pseudomonas aeruginosa. The
studied compounds were also screened for their antifungal activity against one
fungus, Candida albicans and one mold, Aspergillus niger. Most of the
compounds had better antifungal than antibacterial activity. Moderate
antifungal agents, 139d , 139h , 139i , 139j , 139n , and 139l were determined
among the studied compounds. No antifungal activity was observed in thienyl
N
N
S
R
H
3
CO
HO
O
H
3
CO
OH
137a R =Ph
137b R =2-OHC
6
H
4
137c R =4-ClC
6
H
4
137d R =4-NO
2
C
6
H
4
137e R =3-OCH
3
-4-OHC
6
H
3
137f R =4-OHC
6
H
4
137g R =4-CH
3
C
6
H
4
137h R =2,6-Cl
2
C
6
H
3
containing hydropyrimidines, Figure 22.[76] Hussein and co-workers
synthesized and assayed the inhibitory effects on the catalytic activity of the
Imipenemase-1 (IMP-1), a metallo--lactamase from Pseudomonas
aeruginosa and Klebsiella pneumonia, of ten 3,4-dihydropyrimidine-2(1H)-
thiones 141-146. Compounds 141a and 145 are the strongest inhibitors for
IMP-1 with inhibition constants of 20 M comparable with the known MBL
inhibitor of metallo--lactamase, L-captopril K
ic
= 12.5 M, Figure 23.[77]

Figure 21.

Figure 22.
4,6-Diaryl-pyrimidinethiones 146 and the thiosubstituted derivatives 1,6-
Dihydropyrimidin-2-ylthiobutanenitriles 147 were screened for their anti-
bacterial activity against Staphylococcus aureus, Staphylococcus pyogenes,
Pseudomonas aeruginosa and Escherichia coli. Compounds 147 were tested
for antifungal activity as primary screening in six sets against Candida
N
NH
O
R
1
138a R =H; R
1
=CO
2
Et
138b R =H; R
1
=CO
2
Me
138c R =H; R
1
=CO
2
Me
138d R =CH
3
; R
1
=CO
2
Et
138e R =CH
3
; R
1
=CO
2
Me
138f R =Ph; R
1
=CO
2
Et
138a R =Ph R
1
=CO
2
Me
Me
R
N N
Cl
Ph
Me
N
H
NH
O
R
1
139a R =CH
2
Ph; R
1
=CONH(2-ClC
6
H
4
)
139b R =CH
2
Ph; R
1
=CONH(3-ClC
6
H
4
)
139c R =CH
2
Ph; R
1
=CONH(4-ClC
6
H
4
)
139d R =CH
2
Ph; R
1
=CONH(2-Pyridyl)
139e R =CH
2
Ph; R
1
=CONH(3-Pyridyl)
139f R =NHPh; R
1
=CONH(2-ClC
6
H
4
)
139g R =NHPh; R
1
=CONH(3-ClC
6
H
4
)
139h R =NHPh; R
1
=CONH(4-ClC
6
H
4
)
139i R =NHPh; R
1
=CONH(2-Pyridyl)
139j R =NHPh; R
1
=CONH(3-Pyridyl)
139k R =NHPh; R
1
=CO
2
Me
139l R =NHPh; R
1
=CO
2
Et
139m R =CH
2
Ph; R
1
=CO
2
Me
139n R =CH
2
Ph; R
1
=CO
2
Et
Me
N
N
SMe
R
N
H
NH
X
R
Me
S
140a X =O; R =CONH(2-ClC
6
H
4
)
140b X =O; R =CONH(3-ClC
6
H
4
)
140c X =O; R =CONH(4-ClC
6
H
4
)
140d X =O; R =CONH(2-Pyridyl)
140e X =O; R =CONH(3-Pyridyl)
140f X =S; R =CO
2
CH(CH
3
)
2
140g X =S; R =CO
2
(CH
2
)
3
CH
3
140h X =S; R =CO
2
CH
2
CH(CH
3
)
2
albicans, Aspergillus niger and Aspergillus clavatus at various concentrations
of 1000, 500 and 250 g/mL.

Figure 23.
For all the strains, the more oxidized heterocycle 147 was more active
than the corresponding thio unsubtituted compounds 146, probably obtained as
3,4-dihydropyrimidine-2(1H)-thione derivatives. Compound 146 showed a
poor inhibitory effect against E. coli (MIC = 250 g/ml) while heterocycles
147e and 147g possess excellent activity against E. coli with MIC values of 25
g/ml, four times more potent than the reference compound Ampicillin (MIC
= 100 g/ml). Compounds 147a and 147k showed good activity against
Pseudomonas aeruginosa with MIC values of 50 and 25 g/ml, respectively
and compound 147j was five times more potent than the standard drug against
A. aureus. For the antifungal activity compounds 147d and 147g were very
active against C. albicans, with MIC values ten and five times lower than
Griseofulvin, respectively. Compound 147f and 147j are 10 time more active
than Griseofulvin against A. niger and 147k showed a good activtity against A.
clavatus (Scheme 35).[78]

N
H
NH
S Me
R
141a R =Ph; R
1
=CH
3
141b R =4-OCH
3
C
6
H
4
; R
1
=CH
3
141c R =styryl; R
1
=CH
3
141d R =4-OCH
3
C
6
H
4
; R
1
=OEt
141e R =styryl; R
1
=OEt
O
R
1
N
NH
S Me
O
R
1
OH
O
OMe
142
N
H
NH
S Me
OMe
143
Me
NC
NC
N
H
NH
S
OMe
144
Me
O OMe
N
H
NH
S
OMe
145
Me
N
N
OMe
N
H
NH
S
OMe
146
Me
N
H
O
S
N
H
O
O
N
N

Scheme 35.

Figure 24.
Twenty-one thiourea analogs of 3,4-dihydropyrimidine-2(1H)-thiones 148
were tested for their antibacterial activity against Escherichia coli, Staphy-
lococcus aureus, Bacillus subtilis and Salmonella typhimurium. Compounds
bearing fluorine and chlorine atoms at the ortho position of the phenyl ring,
148a and 148b, respectively are the most potent compounds showing better
activity than Ciprofloxacin. Derivatives 148c,d and 148i,k with
trifluoromethyl, trifluoromethoxyl and methoxyl substituents at ortho and para
position of the phenyl ring also present very good activity, Figure 24.[79]
A set of 3,4-dihydropyrimidin-2(1H)-ones with a phenyl carbamoyl group
at 5 position were tested for their antitubercular activity against
Mycobacterium Tuberculosis H
37
Rv. The twenty nine compounds tested
showed low to moderate activity at 6.25g/mL concentration, the best results
were obtained with 149a and 149b with a 2,3-dimethylphenyl and 3,4-
dimethyl carbamoyl side chain, respectively, which showed 65% and 63%
inhibition, Figure 25.[80]
N
NH
S
R
CN
H
2
N
147a R =Ph
147b R =4-ClC
6
H
4
147c R =2-OHC
6
H
4
147d R =3-OHC
6
H
4
147e R =4-OHC
6
H
4
147f R =4-NO
2
C
6
H
4
147g R =4-OH,3-OCH
3
C
6
H
3
147h R =2-CH
3
C
6
H
4
147i R =3-CH
3
C
6
H
4
147j R =4-CH
3
C
6
H
4
147k R =4-OCH
3
C
6
H
4
147l R =4-N(CH
3
)
2
C
6
H
4
CN
Pyridine
N
NH
SH
R
H
2
N
146a-l
N
H
NH
S Me
148a R =2-FC
6
H
4
148b R =2-ClC
6
H
4
148c R =2-CF
3
C
6
H
4
148d R =2-OCF
3
C
6
H
4
148e R =2-Cl,6-CH
3
C
6
H
3
148f R =2-Cl,6-CF
3
C
6
H
3
148g R =2-Cl,5-CF
3
C
6
H
3
148h R =2-Cl,4-CF
3
C
6
H
3
148i R =3-CF
3
C
6
H
4
148j R =4-CF
3
C
6
H
4
148k R =4-OCH
3
C
6
H
4
NH
S
N
H
R
EtO
2
C
Twenty-five 3,4-dihydropyrimidin-2(1H)-ones, thiones 150 and
thiomethyl derivatives 151 bearing the pyrazole ring at position 4 of the ring
were screened for their in vitro antimycobacterial activity at 6.25 g/mL
against Mycobacterium Tuberculosis H
37
Rv, compounds exhibiting more than
90% of inhibition were selected for the evaluation of the minimal inhibitory
concentration, two compounds (150a and 150d) inhibited MTB with MIC of
<1 g/mL and three compounds (150j, the carboxymethyl derivative of 150e,
and 151e) with MIC of <2 g/mL. Among all the compounds, 150a and 150d,
having 4-F and 4-NO
2
substituents on the phenyl ring of the pyrazolyl
substituent were the most potent compounds (MIC of 0.02 g/mL) of the
series. These compounds are better than the existing drug Isoniazid (MIC =
0.03 g/mL) in the in vitro studies, Figure 26.[81]

Figure 25.

Figure 26.
Similar structures, pirazolinyl 3,4-dihydropyrimidin-2(1H)-ones and
thiones with pirazolinyl subtituents 135a-l, Figure 19, were tested as
antibiotics against Mycobacterium Tuberculosis H
37
Rv. In the same manner as
the activity against bacteria and fungi, the best results were obtained with the
N
H
NH
O Me
149a
N
H
O
O
N
H
NH
O Me
N
H
O
NO
2
149b
N
H
NH
X Me
150a X =O; R =4-FC
6
H
4
150b X =O; R =4-ClC
6
H
4
150c X =O; R =4-BrC
6
H
4
150d X =O; R =4-NO
2
C
6
H
4
150e X =O; R =4-CH
3
C
6
H
4
150f X =S; R =4-FC
6
H
4
150g X =S; R =4-ClC
6
H
4
150h X =S; R =4-BrC
6
H
4
150i X =S; R =4-NO
2
C
6
H
4
150j X =S; R =4-CH
3
C
6
H
4
N N
R
EtO
O
N
NH
SMe Me
N N
R
EtO
O
151a R =4-FC
6
H
4
151b R =4-ClC
6
H
4
151c R =4-BrC
6
H
4
151d R =4-NO
2
C
6
H
4
151e R =4-CH
3
C
6
H
4
chlorinated compounds 135k and 135l with MIC values of 100 g/mL and
62.5 g/mL, respectively, which means a good activity when compared with
MIC value of 40 g/mL for Rifampicin and moderate to low activity when
compared with Isoniazid (MIC = 0.20 g/mL).[74] The antitubercular activity
of 3,4-dihydropyrimidin-2(1H)-ones and thiones linked to an oxadiazol and
indol ring 152 were studied against Mycobacterium Tuberculosis H
37
Rv. At
the primary screening using constant concentration (6.25 g/mL) compounds
152c, 152f, 152i and 152l showed inhibition activity of 99%, the same level as
standard drugs Isoniazid, Refampin, Ethambutol and Pyrazinamide. In the
second screening compounds 152c and 152l displayed complete inhibition at
MIC values of 3.10 and 3.12 g/mL, respectively, showing better activity than
Pyrazinamide and comparable activity to Ethambutol, Figure 27.[82]

Figure 27.
Thirty-six 3,4-dihydropyrimidin-2(1H)-ones with a chromone moiety at
C-3 were screened for their in vitro antibactierial activity against
Mycobacterium Tuberculosis H
37
Rv. Six compounds 153a, 153b, 153c, 153d,
154 and 155 showed excellent activity with MIC 3.397.38 M and were more
potent than standard drugs Ethambutol (MIC of 7.63 M) and Ciprofloxacin
(MIC of 9.44 M). Compound 153d was found to be the most active with MIC
3.39 M. It is interesting to note that, in general, the compounds having the
trifluoromethyl group has improved activity compared to methyl, phenyl and
chloromethyl substitutions, Figure 28.[73] Fused N-substituted pyrido-3,4-
dihydropyrimidine-2(1H)-thiones with aryl substituents at position 6 of the
hydropyrimidinethione ring totaling thirty-six compounds, were screened for
their antimicobacterial activity against Mycobacterium Tuberculosis H
37
Rv. In
general, N-benzylpyrido-3,4-dihydropyrimidine-2(1H)-thiones 157 showed
better activity than the N-methyl derivative 156 and compounds with
halogenated aryl ring exhibit enhanced activity. Among them heterocycles 156
and 157a-c are more active than Ethambutol (MIC = 7.6 g/mL).
N
H
NH
X Me
152a X =O; R =H
152b X =O; R =Br
152c X =O; R =NO
2
152d X =O; R =F
152e X =O; R =I
152f X =O; R =Cl
152g X =S; R =H
152h X =S; R =Br
152i X =S; R =NO
2
152j X =S; R =F
152k X =S; R =I
152l X =S; R =Cl
N
N
O
N
HN
O
R
Dichlorinated compound 157a and fluorinated derivative 157c are more active
than Ciprofloxacin (MIC = 4.7 g/mL) with MIC values of 2.8 g/mL and
3.42.8 g/mL, respectively, Figure 29.[83]

Figure 28.

Figure 29.

Figure 30.

N
H
NH
O R
O
EtO
2
C
O
R
1
R
2
R
2
153a R =CF
3
; R
1
=CH
3
; R
2
=H; R
3
=H
153b R =CH
3
; R
1
=H; R
2
=NO
2
; R
3
=H
153c R =CH
3
; R
1
=H; R
2
=H; R
3
=NO
2
153d R =CF
3
; R
1
=H; R
2
=Ph; R
3
=H
N
H
NH
O F
3
C
O
EtO
2
C
O
O
154
N
H
NH
O F
3
C
O
EtO
2
C
O
O
155
N
NH
S
156
Cl
Cl
HN
Cl
Cl
N
NH
S
Cl
HN
Cl
Cl
Cl
N
NH
S
HN
N
NH
S
HN
F
F F
F
157a
157b 157c
N
NH
SH
R
N
N
H
N
S
158a R =Ph
158b R =2-OHC
6
H
4
158c R =4-OHC
6
H
4
158d R =4-ClC
6
H
4
158e R =4-ClC
6
H
4
158e R =2-NO
2
C
6
H
4
158e R =4-NO
2
C
6
H
4
158e R =4-OCH
3
C
6
H
4
Pyrazolo[3,4-d ]tetrahydropyrimidine thiols containing a phenothiazine
nucleus 158 were preliminary in vitro screening for their anti-tubercular
activity. The activity is considerably affected by substitutions at the phenyl
ring of the pyrazolo[3,4-d]pyrimidine nucleus. It has been observed that
compounds 158b , 158d , and 158f having methoxy, chloro, and nitro groups
at position 2, showed excellent anti-tubercular activity with percentage
inhibitions of 93, 91, and 96, respectively, at a MIC of 6.25 g/mL, Figure
30.[84]

3.3. Antiparasitic Activity: Antimalarial, Antifilarial and
Anthelmintic Activity

3,4-dihydropyrimidin-2(1H)-ones and thiones with oxadiazol and indol
moieties, 152, Figure 27, were screened for antimalarial activity against
Plasmodium falciparum 3D7 Chloroquine-sensitive strain using Chloroquine
as reference drug. Compounds 152c, 152d, 152g, 152i, 152j, and 152l have
remarkable antimalarial activity with MIC values in the range of 0.035-5.0
g/mL. Nitrated compound 152c and chlorinated compound 152l with MIC
values of 0.177 g/mL and 0.035 g/mL, respectively, displayed excellent
activity when compared with the standard drug (MIC = 0.125 g/mL).[82]
To assess whether pyrimidinones inhibit Plasmodium falciparum growth,
the Apicomplexan parasite that is responsible for the most lethal forms of
human malaria, Brodsky and co-workers examined 157 compounds of this
class. N-substituted-6-methyl-3,4-dihydropyrimidin-2(1H)-ones 159-167,
Figure 31, inhibit hypoxanthine uptake into P. falciparum-infected red blood
cells inhibiting P. falciparum replication with IC
50
values between 30-1.6
M.[85]
The well-known antimalarial activity of styryl ketones (chalcones)
motivated the synthesis of thiazolopyrimidyl derivatives bearing E-chalcones
as substituents 168, which were evaluated for their antimalarial activity against
two different strains of Plasmodium falciparum i.e. 3D7 (chloroquine
sensitive) and K1 (chloroquine resistant). All the compounds except
compounds 168b, 168e, 168h, and 168u, are potent antimalarials with IC
50
< 2
g/mL against either of the two Plasmodium strains. Chloroquine was taken as
standard, whose IC
50
value was found to be 0.006 and 0.6 g/mL against 3D7
and K1 strains, respectively. Compound 168h and chlorinated compounds
168d and 168p were the most active against K1 strain, Figure 32.[86]


Figure 31.

Figure 32.
N
NH
O Me
BnO
O
NO
2
N
O
N
O
O
HN
159
N
NH
O Me
BnO
O
N
O
O
HN
160
N
NH
O Me
BnO
O
OCH
3
161
N
NH
O Me
EtO
O
163
NO
2
OH
O
N
NH
O Me
BnO
O
NO
2
N
O
N
O
O
HN
162
N
NH
O Me
BnO
O
164
NH
O
N
Me
CO
2
Et
N
NH
O Me
EtO
O
167
NH
O
N
Me
CO
2
Et
NO
2
N
NH
O Me
EtO
O
Bn
O
S
O O
NO
2
165
N
H
NH
O
EtO
O
166
N
N
S
O R
R
1
R
1
168a R =Ph; R
1
=Ph
168b R =Ph; R
1
=4-ClC
6
H
4
168c R =Ph; R
1
=3-ClC
6
H
4
168d R =Ph; R
1
=2-ClC
6
H
4
IC
50
=0.36 g/mL
168e R =Ph; R
1
=4-BrC
6
H
4
168f R =Ph; R
1
=4-FC
6
H
4

168g R =Ph; R
1
=4-OCH
3
C
6
H
4
168h R =Ph; R
1
=C
6
H
4
-4-OBn
168i R =Ph; R
1
=3,4,5-(OCH
3
)
3
C
6
H
3
IC
50
=0.30 g/mL
168j R =4-OCH
3
C
6
H
4
; R
1
=Ph
168k R =4-OCH
3
C
6
H
4
; R
1
=4-ClC
6
H
4
168l R =4-OCH
3
C
6
H
4
; R
1
=4-FC
6
H
4
168m R =4-OCH
3
C
6
H
4
; R
1
=2-thiophenyl
168n R =3,4,5-(OCH
3
)
3
C
6
H
2
; R
1
=3,4,5-(OCH
3
)
3
C
6
H
2
168o R =3,4,5-(OCH
3
)
3
C
6
H
2
; R
1
=4-OCH
3
C
6
H
4
168p R =4-ClC
6
H
4
; R
1
=Ph
168q R =4-ClC
6
H
4
; R
1
=2-ClC
6
H
4
IC
50
=0.27 g/mL
168r R =4-ClC
6
H
4
; R
1
=3-ClC
6
H
4
168s R =4-ClC
6
H
4
; R
1
=4-ClC
6
H
4
168t R =4-ClC
6
H
4
; R
1
=4-BrC
6
H
4
168u R =4-ClC
6
H
4
; R
1
=C
6
H
4
-4-OBn
S-Alkyl-1,4-dihydropyrimidinthiones 169a-n were evaluated in vitro for
their macrofiliracudal activity against Brugia malayi one of the three parasites
causing lymphatic filariasis. Compounds 169c, 169h and 169k affected both
motility (irreversible loss at 100 M) and inhibition of growth ( > 50%).
Compound 169k was tested in vivo to study its adulticidal activity against
filarial worms in B. malayijird model displaying about 46% adulticidal
activity and 34% of the sterilized female worms were recovered, very good
results compared with the standard antifilarial drug Diethylcarbamazine
citrate, which did not show any noticeable microfilaricidal activity and about
only 10% adulticidal activity when compared to untreated controls. Figure
33.[87]

Figure 33.
6-Oxo-4-substituted aryl-2-sulfanyl-1,6-dihydropyrimidines-5-carbonitrile
derivatives 111 were screened for anthelmintic activity using earthworms,
Perituma posthuma. The compounds were evaluated by the time taken for
complete paralysis and death of earthworms, comparing the mean lethal time
with the standard drug Albendazole. Compounds 111e, 111d and 111i exhibits
less time for death than the standard and compound 111j showed anthelmintic
activity comparable with the standard.[54]

3.4. Insecticide

The mosquito larvicidal activity of 2-thioxo-2,3,6,10b-tetrahydro-1H-
pyrimido[5,4-c]quinolin-5-ones 121, screened for bactericidal and antifungal
activity, was also tested. The LC
50
values for the measurement of toxicity and
activity against fourth instar larvae of Culex quinquefasciatus showed that
N
H
NH
S
R
EtO
O
R
1
169a R =1-naphthyl; R
1
=benzyl
169b R =1-naphthyl; R
1
=n-pentyl
169c R =1-naphthyl; R
1
=n-butyl
169d R =1-naphthyl; R
1
=n-tetradecyl
169e R =3-NO
2
C
6
H
4
; R
1
=benzyl
169f R =3-NO
2
C
6
H
4
; R
1
=n-pentyl
169g R =3-NO
2
C
6
H
4
; R
1
=n-butyl
169h R =4-OCH
3
C
6
H
4
; R
1
= benzyl
169i R =4-OCH
3
C
6
H
4
; R
1
=n-pentyl
169j R =4-OCH
3
C
6
H
4
; R
1
=n-tetradecyl
169k R =4-FC
6
H
4
; R
1
=n-pentyl
169l R =4-FC
6
H
4
; R
1
=n-tetradecyl
169m R =4-ClC
6
H
4
; R
1
=n-pentyl
169n R =Thiophenyl; R
1
=benzyl
compounds 121b, 121d and 121h are the most toxic to larvae with values of
0.85, 0.88 and 0.98, respectively, Figure 12.[66]

3.5. Anti-Inflammatory Activity

The anti-inflammatory activity of 3,4-dihydropyrimidin-2(1H)-ones and
thiones 112a-d, Figure 8, was evaluated by the carrageenan induced paw
edema method. The compounds were tested in vivo and compared with the
standard drug Ibuprofen in the same oral dose. After 4 h, dihydro-
pyrimidinones showed higher activity than the corresponding thiones, the best
result being obtained with compound 112a, that showed a maximum inhibition
of 70.7% comparable with Ibuprofen (86.4%).[56]
Compounds 124c, 125c, 126b and 127b previously referred in Figure 15,
were investigated in comparison with ibuprofen as standard anti-inflammatory
agents. Compounds 125c and 126b show higher activity than ibuprofen at 3
and 4h after administration, while compounds 124c and 127b are less active;
this could be related to the cyclisation at the 5,6 position of dihydropyrimidine
ring that also decreases or eliminates the activity as antimicrobial.[69]
Thiourea analogs of 3,4-dihydropyrimidine-2(1H)-thiones 148, Figure 24,
screened for their antibacterial activity, were also evaluated for their anti-
inflammatory activity against the proinflammatory cytokines TNF- and IL-6
target using Dexamethasone as standard. Compounds 148d, 148e-i and 148l
were found to exhibit either higher or comparable anti-inflammatory activity
than the standard. Compound 148g, bearing a chlorine atom and a
trifluoromethyl group on the phenyl ring presents a percentage of inhibition of
IL-6 of 96% at 10M and the difluorinated derivative 148l showed a
percentage of inhibition of IL-6 of 90%.[79]
The anti-inflammatory activity of [4,6-(4-substituted aryl)-2-thioxo-3,4-
dyhydro-pyrimidin-5-yl]-acetic acid 170 was evaluated according to the
method of Winter et al.[56] in albino rats employing the carangeenan induced
rat paw edema test. All the compounds showed tendency to cause a fall in
edema and showed anti-inflammatory activity when compared with sodium
Diclofenac. The anti-inflammatory activity data shows that the presence of
methoxy at the para position of the phenyl group, compounds 170c, 170i and
170o, increase the activity of the compounds, Figure 34.[56, 88]
Shinde and co-workers also evaluated the anti-inflammatory activity of
propanoic acid analogs to 169. The new compounds were found to be of short
term action but with anti-inflammatory effects comparable with Diclofenac.
Compounds 171a and 171b were the most effective at 1 h from administration,
Figure 35.[89]

Figure 34.

Figure 35.
Antagonists of the TRPA1 receptor could find possible therapeutic use as
antinociceptive or anti-inflammatory agents[90] In a high throughput screen
aimed at the identification of TRPA1 antagonists, 4-phenyl-2-thioxo-1,2,3,4
tetrahydroindeno[1,2-d]pyrimidin-5-one (172a) was identified as a TRPA1
receptor antagonist, with a potency exceeding the previously described
antagonists 173 and 174 (HC-030031)[91]. Gijsen and co-workers synthetized
a series of derivatives 172 with different substituents on the phenyl ring at C-4
of the dihydropyrimidinethione ring and tested their activity on both the
human and rat TRPA1 channel.
In general, a comparable activity was observed for the human and rat
channel, for most compounds a slightly higher potency for rat TRPA1.
Especially metamethoxy substituted 172b displayed a more than 10-fold
enhanced potency, and additional analogs similar to 172b were investigated.
172c was the most active compound obtained, with IC
50
of 0.050 M. The
separation of the racemates shows than only the dextrarotatory enantiomer is
active, with IC
50
of 0.013 M, Figure 36.[92]
N
H
NH
S
R
1
170a R =H; R
1
=Ph
170b R =H; R
1
=4-ClC
6
H
4
170c R =H; R
1
=4-OCH
3
C
6
H
4
170d R =H; R
1
=2-thiophenyl
170e R =H; R
1
=furfuryl
170f R =H; R
1
=3-nicotinyl
HO O
R
170m R =CH
3
; R
1
=Ph
170n R =CH
3
; R
1
=4-ClC
6
H
4
170o R =CH
3
; R
1
=4-OCH
3
C
6
H
4
170p R =CH
3
; R
1
=2-thiophenyl
170q R =CH
3
; R
1
=furfuryl
170r R =CH
3
; R
1
=3-nicotinyl
170g R =Cl; R
1
=Ph
170h R =Cl; R
1
=4-ClC
6
H
4
170i R =Cl; R
1
=4-OCH
3
C
6
H
4
170j R =Cl; R
1
=2-thiophenyl
170k R =Cl; R
1
=furfuryl
170l R =Cl; R
1
=3-nicotinyl
N
H
NH
S
R
1
171a R =CH
3
; R
1
=2-thiophenyl
171b R =H; R
1
=2-OHC
6
H
4
R
O
HO
The effect of 3,4-dihydropyrimidinone derivatives as potential anti-
neuroinflammatory substances using LPS-stimulated BV-2 microglial cells
was examined and compounds 175a,b and 176 have shown inhibitory effects
on NO production in BV-2 cells.
Compound 175b showed stronger inhibitory activity on NO production
than the others in LPS-stimulated BV-2 cells. The studies on the regulation of
microglial cell activation in activated BV-2 cells show that compound 175b
suppressed pro-inflammatory factors in LPS-activated microglial BV-2 cells.
In addition, compound 175b prevented LPS-induced microglial activation
mediated neurotoxicity in N2a cells suggesting that compound 175b has
potential neuroprotective effects in inflammatory-related brain damage
induced by microglial cell activation, Figure 37.[93]

Figure 36.

Figure 37.

Figure 38.
N
H
NH
S
172a R =H IC
50
=1.8M
172b R =OCH
3
172c R =O(CH
2
)
3
OCH
3
O
R
N
H
NH
S
OCH
3
EtO
2
C
173 R =H IC
50
>10M 174 R =H IC
50
>10M
N
N
O
N
N
O
N
H
O
N
H
NH
X Me
175a X =O
175b X =S
EtO
O
O
N
H
NH
O Me
EtO
O OH
Cl
176
HN NH
X
Me
R
1
O
O
O
OR
OR
OR
OR
177a X =O; R =Ac; R
1
=Me
177b X =S; R =Ac; R
1
=Me
177c X =O; R =Ac; R
1
=OMe
177b X =S; R =Ac; R
1
=OMe
177d X =O; R =Ac; R
1
=OEt
177e X =S; R =Ac; R
1
=OEt
177f X =O; R =H; R
1
=Me
177g X =S; R =H; R
1
=Me
177h X =O; R =H; R
1
=OMe
177i X =S; R =H; R
1
=OMe
177j X =O; R =H; R
1
=OEt
177k X =S; R =H; R
1
=OEt
3.6. Sedative-Hypnotic Activity

The sedative-hypnotic activities of 3,4-dihydropyrimidin-2(1H)-ones and
thiones with allopyranoside substituent at the para position of the phenyl ring,
177, were evaluated in vivo by recording the number of spontaneous
locomotions in mice using an actophotometer.[94] The higher sedative-
hypnotic activities of derivatives 177a , 177c-f suggested that the introduction
of the acetyl group in the sugar moiety led to an increase in the sedative-
hypnotic effect, Figure 38.[95]

3.7. Antagonist of Hsp70 - Alzheimer Disease

Using a newly described high-throughput screening (HTS) method, a
library of 2800 known bioactive compounds were evaluated and five active
compounds belonging to at least three chemical scaffolds were identified as
activators and inhibitors of the ATPase activity of Hsp70.
Among them, the dihydropyrimidines 178 and 179 were found to increase
Hsp70 function by 45% with EC
50
values of 120150 M.[96] 3,4-
dihydropyrimidin-2(1H)-one 178 was tested in vitro as antagonist of Hsp70
and proved to be capable of compensating for insufficient chaperone levels
and promoting anti-aggregation activity. in vitro activity as antagonist of
Hsp70 might activate in vivo endogenous Hsp70 and combat
neurodegenerative diseases without concomitant involvement of stress
response, compound 178 opens a possibility for therapy of neurodegenerative
diseases such as Alzheimer, Figure 39.[97]

3.8. Calcitonin Mimetics - Hypercalcemia Associated with
Pagets Disease and Osteoporosis

Mathews and co-workers identified a novel series of dihydropyrimidines
with capability to act as calcitonin mimetic Calcitonin, which plays an
important role in inhibiting bone resorption through the mediation of
osteoclasts.
By inhibiting bone resorption and promoting renal calcium excretion,
calcitonin has therapeutic applications in a variety of clinical disorders,
including hypercalcemia associated with Pagets disease and osteoporosis.[98]
Several analogues of compound 180 inhibited parathyroid hormone stimulated
bone resorption in a bone organ culture assay in a dose-dependent manner:
compound 180 was efficacious in a Weanling rat model when administered
subcutaneously.

Figure 39.

Figure 40.
While it was possible to significantly improve the oral bioavailability and
potency, while retaining efficacy in the mouse calvaria model, preliminary oral
evaluation of 181 in the Weanling rat study showed no in vivo efficacy. These
compounds may serve as a template for future small molecule calcitonin
mimetic ligands, Figure 40.[99]

3.9. Inhibitors of Fatty Acid Transpoter FATP4 Obesity

Several potent, cell permeable 4-aryl-dihydropyrimidinones have been
identified as inhibitors of fatty acid transport protein 4 (FATP4). Since FATP4
inhibition would result in the accumulation of free fatty acids rather than
triglycerides blocking the absorption of triglycerides this could result in a
treatment of obesity.
In a previous screening 3,4-dihydropyrimidin-2(1H)-one 182 was found to
have an IC
50
value of 1.2 M for the inhibition of FATP4 and showed
N
NH
O Me
EtO
2
C
178
Br
O
O
N
NH
O Me
BnO
2
C
Cl
OH
O
Cl
179
N
H
NH
O
180 EC
50
=5.8 M
O
O
H
3
CO N
H
NH
O
181 EC
50
=1.3 M
O
O
H
N
O
N
selectivity over FATP2 and FATP5. Using this heterocycle as lead compound,
a series of 23 compounds with ester groups at position 5 were synthetized and
evaluated, cyclopentyl ester 183 being the most active with IC
50
of 0.25 M.
The evaluation of the two isomers shows that only isomer S is active for the
inhibition of FATP4. When the optimal cyclopentyl ester group was retained,
several compounds with different aryl groups at position 4 were synthetized
and evaluated but no significant potency enhancements were observed
compared to the nitro compound. The most potent compound was 184, with a
trifluoromethyl subtituent. Replacement of the substituted phenyl group with
heteroaromatics did not result in any significant activity and the replacement
of an oxygen atom by a sulfur atom in the pyrimidinone ring resulted in a loss
in potency. As in the case of 183 the evaluation of the enantiomers showed
that 184-S is more active than R isomer with IC
50
values of 0.6 M and > 30
M respectively, Figure 41.[100]

Figure 41.

Figure 42.

N
H
NH
O
183
O
O
NO
2
N
H
NH
O
182
N
H
O
NO
2
EtS
N
H
NH
O
O
O
CF
3
184
N
H
NH
O
185a R =3-OHC
6
H
4
EC
50
=0.087 M
185b R =4-OHC
6
H
4
EC
50
=0.031 M
185c R =4-NHCOCH
3
C
6
H
4
EC
50
=0.063 M
185d R =4-FC
6
H
4
EC
50
=0.024 M
O
O R
3.10. Anti-HIV Agents

Compounds 168 with a chalcone thiazole-pyrimidyl moiety, Figure 32,
were evaluated for their Human immunodeficiency virus (HIV) reverse
transcriptase inhibitory activity. Among all the compounds screened,
heterocycles 168d and 168h show very good activities with percentages of
inhibition of 73.44 and 66.92%, respectively.[86]
In a high-throughput screening campaign for the discovery of novel
antiretrovirals No and co-workers identified a series of compounds containing
a dihydropyrimidinone scaffold that exhibited inhibitory activities against HIV
replication at low micromolar concentrations. From this starting point, they
synthetized and evaluated a library of 33 compounds as HIV-1 replication
inhibitors in vitro. Compounds 185a-d showed EC
50
values under 0.1 M.
Chiral separation of the enantiomers showed that the S configuration on the C-
4 is a crucial factor for antiviral activity: only the S isomer of compound 185
exhibited antiviral activity with EC
50
value of 0.038 M, Figure 42.[101]

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In: Quinones ISBN: 978-1-62618-323-0

Chapter 2

BIOLOGICAL IMPLICATIONS
OF BENZOQUINONES

Jisook Kim

Department of Chemistry,
University of Tennessee at Chattanooga, Chattanooga, TN, US

ABSTRACT

Benzoquinones (BQs) represent the simplest form of quinones,
containing two carbonyl groups on a six-membered ring. They are
ubiquitously found in diverse organisms as free quinones, protein
cofactors, or an integral part of the mitochondrial electron transport chain
(ETC). In addition, many BQs are identified as environmental toxins
generated from industrial processes as the metabolites of polycyclic
aromatic hydrocarbons, contributing to bioaccumulation. To date, animal
and epidemiological studies revealed that the quinone derivatives of
benzene metabolites serves as a source of inducing abnormal cell
behavior, leading to cancer or triggering immune response. Whether
occurring endogenously in living organisms or exogenously in the
environment, there is a universal understanding on the role of BQs as
potential toxins, except some limited cases like protein-bound BQs or
electron carriers in the ETC. Studies done at a molecular-level approach
revealed that BQs exhibit both genotoxicity and non-
genotoxicity/epigenetic toxicity, targeting both cellular DNA and

Email: jisook-kim@utc.edu
58
prot
com
form
con

1.

1.1. Fr

Fre
[1] and
as a qui
Both oB
carbony
to Mich
A w
convers
such as
the plan
proper
severe
distingu
bond in
Und
which i
through
as stim
differen

Figure 1
acids.
teins. The me
mbination of o
mation with D
nformation chan
. ENDOGEN
ree Benzoqu
e benzoquino
p-benzoquino
inone form or
BQ and pBQ h
yl groups. Due
hael addition a
well-known ca
sion of urushio
poison oak, p
nts and transf
manner. The
allergic con
uished by the
the alkyl grou
der a mildly o
is known to
h Michael add
mulating antig
nt pathways oc
. Structure of ur
J
echanism of th
oxidative dama
DNA and pro
nge.
NOUSLY O
uinones: o-B
ones such as
one (1,4-benzo
r a reduced fo
have two doub
e to their ,-c
as well we carb
ase for oBQ a
ol to oBQ. W
poison ivy, an
ferred to the
exposure of
ntact dermatit
different carb
ups at the R po
oxidizing cond
form a hapt
ition [2-5]. Th
gens which c
ccurring in an
rushiol and its c
isook Kim
heir action is
age intervened
oteins, and pr
CCURRING
Benzoquinon
o-benzoquino
oquinone, pBQ
orm mostly in
ble bonds in th
conjugated sys
bonyl condens
associated wit
When a person
nd poison sum
skin unless p
urushiol-conta
tis [1]. Sev
bon chain leng
osition (Figure
dition, urushio
tenyl protein
hen, the oBQ-
an trigger an
antigen presen
conversion to o
thought to o
d by redox-cy
rotein cross-li
G BENZOQU
ne and p-Be
one (1,2-Benz
Q) families ar
n plants and in
he six-membe
stem, they bot
sation.
th skin outbre
is in contact
mac, urushiol
prompt washi
aining plants
veral differen
gth and the n
e 1).
ol is converted
or ahapten m
-conjugated p
n immune re
nting cell (Fig
oBQ.Nu, nucleo
occur via the
ycling, adduct
inking/protein
UINONES
enzoquinon
zoquinone, oB
re available eit
nsects (Figure
ered ring and t
th are suscepti
eaks involves
with toxic pla
is released fr
ing is done i
to skin leads
nt urushiols
number of dou
d readily to o
modified prot
roteins can se
esponse in th
gure 2).
ophilic amino
e
BQ)
ther
e 1).
two
ible
the
ants
rom
in a
s to
are
uble
oBQ
tein
erve
hree

Biological Implications of Benzoquinones 59

Figure 2. Proposed mechanism of immune response in an antigen presenting cell
(APC, APC in gray color) upon exposure to oBQ (Q) after oxidation of urushiol.
In an endogenous major histocompatibility complex (MHC) class I
activation pathway, the oBQ-conjugated proteins are generated endogenously
and broken down into the antigenic peptides through the action of cytosolic
proteasomes. Then, the antigenic peptides are transported to the endoplasmic
reticulum where they form a complex with MHC class I molecules.
Eventually, the complexes are brought to the cellular surface of the antigen
presenting cell by the MHC class I molecules and recognized by CD8
+
T cells
during antigen-specific activation. In an exogenous MHC class I activation
pathway as a cross-presentation mechanism, apathogen containing the oBQ-
conjugated proteins is taken by host cells which become antigen presenting
cells and this results in expression of MHC class I molecules which are
recognized by CD8
+
T cells. In an endogenous MHC class II activation
pathway, exogenously occurring oBQ-conjugated proteins are endocytosed to
the target cells and broken down into oBQ-labeled peptide in endosome or
lysosome. This action leads to MHC class II activation and eventually the
activation of CD4
+
T cells. In an exogenous MHC class II activation pathway,
a pathogen containing the oBQ-conjugated proteins can be taken up by the
target cells and the pathogen can be removed through endocytic pathway
leading to MHC class II activation.
To note, only a handful of published studies were dedicated to elucidating
the exact mechanism of the action of oBQ, whether it occurs through the MHC
class I pathway or the MHC class II pathway.
Jisook Kim 60

Figure 3. Model haptenyl analogs for urushiol-protein reactions via oBQ.
To summarize, it is generally understood that oBQ-induced allergic
dermatitis is a T-cell mediated-response, and the activations of both CD4
+
and
CD8
+
T cells were observed upon presentation of urushiol to human T cells [1,
6, 7]. In addition, the chemical model study suggests that the hapten formation
of oBQ with a protein occurs mostly likely at the C-5 or C-6 position of oBQ
(Figure 3) [5].
Free pBQs exist in various structures in many organisms such as plants,
fungi, bacteria, and insects, however found in part as intermediate metabolites
or their reduced form [8]. Thompson published a thorough work on naturally
occurring quinones including pBQs, discussing well-above 300 types of pBQs
isolated in many organisms and cultures [8]. Among many interesting topics
related to the identified pBQs, the spray mechanism of bombardier beetles
certainly grabbed attention of many, primarily due to the outcome of the
insects action to the stored grains in warehouses [9-11]. When exposed to a
threat, many insects such as bombardier beetles spray a solution of quinols,
hydrogen peroxide, and oxygen in high pressure, which is utilized as a
chemical and thermal weapon and discharged at 100C explosively, against
their predators [9, 10]. Then, the quinols are oxidized to pBQs upon ejection of
the spraying solution. To the benefit of the quinoid solution-discharging
insects, this action serves as a defensive mechanism. However, the downside is
the infestation of the stored grains such as flour and seeds by the beetles,
therefore with high in the level of pBQs. The structures of pBQs from pBQ-
releasing insects are shown in Figure 4 [8, 10]. When Swiss albino mice were
exposed to flour infested with beetles, biscuits with the flour, and, 1,4-
benzoquinone, respectively, it was shown that the mice developed tumors in
the organs such as the liver and the spleen [11]. In this mice research, elMofty
and coworkers concluded that 1,4-benzoquinone by itself or the cocktail of
substituted pBQs resulted in the tumori-genesis of the mice.

Figure 4
pBQ, 1,4
benzoqu

1.2. Be

Sub
quinone
amine o
globifor
(ECAO
member
residue,
from bo

Figure 5
Bio
. Structures of r
4-benzoquinone
uinone; D, 2-me
enzoquinone
bstituted BQs
e containing c
oxidase (BPA
rmis amine o
) [12, 13]. T
red quinone f
, called TOPA
oth eukaryotes
. Structure of T
ological Impli
representative p
e; B, 2-methyl-1
thyl-3-methoxy
es as Protei
are found as
copper amine
AO), pea seed
oxidase (AGA
The name TO
form of an act
A quinone or
s and prokaryo
TOPA quinone.
cations of Ben
pBQs found in p
1,4-benzoquino
y-1,4-benzoquin
n Cofactors
s the covalent
e oxidases (C
dling amine o
AO), and esc
OPA originat
tive-site based
TPQ (Figure
otes [14].
nzoquinones
pBQ-releasing i
one; C, 2-metho
none.
s
tly-bound cof
CAOs) such a
oxidase (PSA
cherichia coli
ted by abbre
d 2,4,5-trihydr
5) which is p

insects [8]. A,
oxy-1,4-
factors of TO
as bovine plas
O), arthrobac
i amine oxid
eviating the s
roxyphenylala
present in CA
61
OPA
sma
cter
dase
six-
aine
AOs

Jisook Kim 62
Table 1. Class of amine oxidases by enzyme source

Enzyme class Enzyme source
Flavin-depedent Monoamine Oxidases Beef Liver
Human Placenta
Rat Liver
Copper-containing Amine Oxidases Porcine Plasma
Horse Plasma
Sheep Plasma
Pea Seedling
Soybean Seedling
Chick Pea Seedling
Pig Kidney
Lysyl Oxidases Beef Aorta
Human Placenta
Semicarbazide-sensitive Amine Oxidases Porcine Aorta
Beef Aorta
Rat Aorta

CAOs serve an important purpose in nutrient catabolism in prokaryotic
organisms, while the role of higher organisms appears to be more complicated
[15, 16]. It seems there are at least two different types of mammalian copper
amine oxidases: the cellular amine oxidases that regulate histamine and
polyamine levels, and the serum proteins that control the level of circulating
biogenic amines such as dopamine and phenethylamine [16]. CAOs belong to
the family of amine oxidases along with flavin-dependent mitochondrial
monoamine oxidases, quinone-dependent CAOs which include the lysyl
oxidases, and semicarbazide-sensitive amine oxidases (Table 1) [15].
The important reaction CAOs catalyze involves two-electron oxidation of
an aliphatic amine at the expense of reducing O
2
to H
2
O
2
, and this is initiated
through the binding of the amine to the benzoquinone-based cofactor. A
primary amine (RCH
2
NH
2
) may be dehydrogenated to an imine which then
hydrolyzes to an aldehyde, and NH
3
or an aldehyde may be formed directly
(equation 1).

RCH
2
NH
2
+ H
2
O + O
2
RCHO + NH
3
+ H
2
O
2
(equation 1)

Klinman and coworkers released the comparison studies of the quinone-
containing peptide sequences along with those of the enzymes expressed from
the coding genes and identified the TOPA quinone (TPQ) formed from a
precursor active-site tyrosyl residue, through a Cu
2+
-dependent auto-oxidation
[17]. It turns out TPQ exists in a highly conserved sequence, asparagine-
tyrosine (TOPA quinone)-aspartate/glutamate-tyrosine [18]. There is
considerable structural homology among the various copper containing amine
oxidases, as confirmed by the recently reported crystal structures for PSAO
[19], AGAO [20], and ECAO [21]. The amino-acid sequences near the active
site for these amine oxidases are shown in Table 2.
Although the copper is an absolute prerequisite for biogenesis of the TPQ
cofactor, the latter being initiated by coordination to copper of the precursor
tyrosine phenolate, the role of copper in the catalytic cycle of amine
deamination has been a subject debate for a while. Based on the combined
evidence from the known enzyme structures, the TPQ side chain is flexible
enough to permit the aromatic group to rotate about the C-C bond, so that
the TPQ backbone can move between bonding and non-bonding positions with
respect to the copper atom. With regard to the details of copper coordination,
in the three enzymes with known structures of ECAO, PSAO and AGAO, the
copper has square pyramidal coordination, with three histidines and two water
molecules, one as an equatorial ligand and the other as an axial ligand (see
Figure 6 and Table 3) [16-19, 21-23].

Table 2. Sequence of copper amine oxidases near the active-site. Amino
acids are represented in one-letter abbreviation

Sequence Enzyme
LVFRSVSTMLNYDYVWDMVFYPNGAIEVKLHAT BPAO
LIVRTIVTVGNYDNVIDWEFKASGSIKPSIALS PSAO
MVISFFTTIGNYDYGFYWYLYLDGTEFEAKAT AGAO
LVVRWISTVGNYDYIFDWIFHENGTICIDAGAT ECAO

Table 3. Conserved residues in the active site of each amine oxidase.

Enzyme BPAO PSAO AGAO ECAO
Cofactor TPQ 470 TPQ 387 TPQ 382 TPQ 466

Histidine
ligands
to copper

H 442
H 519
H 521

H 442
H 444
H 603

H 431
H 433
H 592

H 524
H 526
H 689

Active site base Asp Asp 300 Asp 298 Asp 383
64
Figure 6
Asp, asp
Als
the opp
general
21-23].

1.3. Be
Transp

BQ
[25] pla
transpor
group a
CoQ an
a repea
skeleton
(UbQ).
differs.
CoQ ha
revealed
. Representativ
parate; Tyr, tyro
so the catalyti
posite side of
base catalyst
enzoquinone
port Chain
Q derivatives s
ay an importa
rt chain (ETC
at the C-3 pos
nd PQ are quin
ated isoprene
n. It should b
Depending on
For instance,
as 6 units. PQ
d that the num
J
e active site of
osine; His, histid
c base is beli
the TPQ from
in the abstrac
es as Electr
such as coenzy
ant role as ele
C) [24, 26-28]
sition, while P
none derivativ
unit, attache
be noted that
n the organism
, mammals C
is commonly
mber of the isop
isook Kim
CAOs (adapted
dine.
eved to be a
m the copper
ction of a pro
on Carriers
yme Q (CoQ)
ctron carriers
]. As shown i
PQ lacks the m
ves containing
ed on a 6-m
CoQ is com
m, the number
CoQ has 10 r
found in plan
prene unit var
d from reference
conserved asp
r center and p
oton from the
s in the Elec
) [24] and pla
in the mitoch
in Figure7, Co
methyl group.
g a long hydro
membered ben
mmonly know
r of the repea
repeated units
nt organism, an
ries even amon

es [12, 21, 109]
partate residue
plays a role a
substrate [16-
ctron
astoquinone (P
hondrial elect
oQ has a met
. Otherwise, b
ophobic tail, w
nzoquinone r
wn as ubiquin
ated isoprene u
s while bacte
nd a recent stu
ng plants [25]
]).
e at
as a
-19,
PQ)
tron
thyl
both
with
ring
none
unit
erial
udy
.
Figure 7
oxidized
Fig
quinone
oxidized
frame.
semiqui
reduced
intercon
to serv
reductio
example
oxidore
transfer
oxidore
carrier
membra
action o
electron
electron
Sin
nature,
mobile
cellular
[25, 28
features
chain to
isopente
polypre
polypre
hydroxy
tyrosine
Bio
.Structures and
d quinone form;
ure 7 shows
es exist in thre
d, it has two c
As Q accept
inone form (Q
d to become qu
nversion betw
ve as reversib
on potential o
e, UbQ can
ductase) as w
r the electr
ductase) in th
in the ETC
ane, however a
of CoQ in m
ns from the p
ns to the cytoc
ce their long
however the
in lipophilic m
membrane fo
, 30, 32]. In
s due to the fa
o the quinone
enyl pyropho
nyl pyrophos
nyl-4-hydroxy
y benzoic aci
e or phenylalan
ological Impli
redox reaction
QH, semiquin
the scheme o
ee oxidation s
carbonyl group
ts one electr
QH). Then, a
uinol (QH
2
) af
een Q and QH
ble electron
of a species t
accept elect
ell as the com
rons to the
e mitochondri
of chloroplas
also found in
mitochondria, P
photosystem
hrome bf com
isoprene chai
quinol form b
membranes; th
or CoQ, the thy
addition, bio
act that both c
backbone. Bio
osphate (Isop
sphate (Polyp
y benzoic acid
id which is
nine, forming
cations of Ben
of CoQ (R = C
none form; QH
2
of the quinon
states whether
ps and two do
ron and a pr
at the subsequ
fter accepting
H
2
is reversibl
acceptors an
they couple w
trons from t
mplex II (succin
e complex
ial ETC [29]. P
sts and is loc
the chloroplas
PQ picks up
II, becoming
mplex [31].
in makes both
being less hyd
he inner mitoc
ylakoid and th
ogenesis of C
ontain the iso
osynthesis of C
petenyl PP),
prenyl PP). T
d transferase, p
a derivative
CoQ [24]. Bi
nzoquinones
CH
3
group) and
2
, reduced quino
nes redox rea
r CoQ or PQ.
ouble bonds in
roton, it is c
uent step, the
an electron an
le, allowing b
nd donors de
with on a giv
the complex
nate-UbQ oxi
III (UbQH
2
PQ serves as a
cated mostly
st envelope [3
protons from
g plastoquinol
h CoQ and PQ
drophobic, the
chondrial mem
he chloroplast
CoQ and PQ
oprene unit att
CoQ includes
farnesylpyro
Then, through
polyprenyl PP
or metabolic
iosynthesis of
PQ (R = H). Q,
one form.
action where
When Q is fu
n a benzoquin
converted into
e semiquinone
nd a proton. T
both CoQ and
epending on
ven reaction.
I (NADH-U
idoreductase)
2
-cytochrome
a mobile elect
at the thylak
0]. Similar to
m the stroma
l, and transpo
Q hydrophobic
ey are found
mbrane and ot
t envelope for
shares comm
tached as the s
s the formation
ophosphate,
h the action
P couples with
intermediate
f PQ includes a
65

,
the
ully
none
o a
e is
This
PQ
the
For
UbQ
and
C
tron
koid
the
and
orts
c in
and
ther
PQ
mon
side
n of
and
of
h 4-
e of
also
Jisook Kim 66
the formation of isopentenyl PP, and eventually the formation of the conjugate
of polyprenyl PP and homogentisate which is a derivative of phenylalanine
[25, 30].

2. EXOGENOUSLY OCCURRING BENZOQUINONES

2.1. Benzoquinones and Polycyclic Aromatic Hydrocarbons

Exogenous benzoquinones are generated as metabolites of polycyclic
aromatic hydrocarbon (PAH) such as benzene, substituted benzene, and
naphthalenes [33]. PAHs are widespread environmental toxins found in the
general environment as well as in the occupational setting. Most importantly,
PAHs serve as precursors for their metabolites including PAH quinones
(Figure 8) [34-40]. Animal studies and epidemiological studies have revealed
that the benzene family (benzene and substituted benzenes) is capable of
inducing abnormal cell or tissue behavior such as cancer and neurotoxicity
[34-54]. Huff and coworkers reported the clear carcinogenic effect of benzene
toward a group of rats and mice exposed to benzene [41-44]. They reported the
high frequencies of developing multiple carcinomas such as squamous cell
carcinomas of the oral cavity and the skin, malignant lymphomas, ovarian
granulosa cell tumors, and ovarian benign tumors due to benzene
bioaccumulation. Xylene and toluene are found to result in high risk of
developing cancer and leukemia based on epidemiological studies carried out
with workers [39, 40]. p-Dichlorobenzene is used as an active component for
deodorants, pesticides, toilet bowl cleaners, and mothballs. Recently, it was
discovered that that p-dichlorobenzene was able to produce neurotoxic effects
[45] and ichthyosis-like dermatosis [46]. Matsumoto and coworkers reported
that p-dichlorobenzene exhibited carcinogenicity and chronic toxicity in mice
such as hepatocellular carcinomas, hepatoblastomas, and hepatic histiocytic
sarcomas [47, 48, 55]. In addition, Versonnen and coworkers reported that p-
dichlorobenzene is estrogenic based on a yeast estrogen screen and zebrafish
assay [49]. Very interesting studies were reported regarding butylated-
hydroxyanisol (BHA)s ability of inducing apoptosis and toxicity: BHA is
known as a commonly used food preservative and chemotherapeutic agent.
However, a number of studies discussed that BHA exhibited toxic and
carcinogenic effect by inducing papilloma and carcinoma in rats, mice,
hamsters, and pigs [50-54].

Figure 8
outcome

2.2. Be
of Ben

As
the met
play an
modific
modific
Foc
out ex
indepen

Figure 9
(adapted
1,2-benz
G, 1,2,4-

Bio
. General overv
e. Q, PAH quino
enzene Meta
nzoquinones
to how PAHs
abolites of PA
important rol
cations, oxida
cations [33].
cusing on a si
xtensive mec
ndently and sh
. A mechanistic
d from reference
zenediol (catech
-benzenetriol (2
ological Impli
view of generati
ones.
abolism Lea
s
s exhibit their
AHs in their q
e in causing to
ative damage,
mplest PAH,
chanism stud
ed light on its
c view of benze
es [44, 56, 57]).
hol); D, oBQ; E
2-hydroxyhydro
cations of Ben
ion of PAH quin
ading to the
r toxicities, it
quinone forms,
oxic abnorma
, lipid modif
benzene, Huf
dies on the
intracellular m
ene metabolism
. A, benzene ox
, pBQ; F, 1,4-b
oquinone); H, 2-
nzoquinones
nones and their
e Formation
is universally
, whether redu
al cell behavio
fications, and
ff and Snyder
e bioactivatio
metabolism [4
generating oBQ
xide; B, benzene
benzenediol (hy
-hydroxy-1,4-b
r biological
n
y understood t
uced or oxidiz
or through prot
d/or nucleic a
rs groups carr
on of benz
44, 56, 57].
Q and pBQ
e dihydrodiol; C
ydroquinone, HQ
benzoquinone.
67

that
zed,
tein
acid
ried
zene

C,
Q);
Jisook Kim 68
As shown in Figure 9, it has been suggested that the bio-activation of
benzene occurs through the action of cellular proteins such as cytochrome
P450 monooxygenase, epoxide hydrolase, dehydrogenase, and tyrosinase,
generating electrophilic metabolites, namely, benzene oxide (A), oBQ (D),
pBQ (E), and 2-hydroxy-p-benzoquinone (H) [44, 56, 57]. These electrophilic
metabolites are thought to react with many cellular proteins, lipids, and nucleic
acids.
Undoubtedly, PAH quinones are found to be the major cause for the
observed PAH toxicity and thought to exhibit their fatal activities by reacting
with cellular proteins and nucleic acids through redox-cycling and adduct
formation [33, 56, 58-72]. The biological outcomes as well as the cellular
targets of PAH quinones appear to be broad even for the BQ-based simple
PAH quinones.

2.3. Lipid Peroxidation and Benzoquinones

The potential targets for BQs are notably lipids, nucleic acids, and
proteins, however it should be noted that only few studies are available
regarding the interaction of BQs and lipids [73, 74]. In an effort to give insight
on the role of pBQ toward lipid peroxidation, Soucek and coworkers
investigated the interconversions of pBQ and hydroquinone (HQ) via the
radical form of an intermediate called semiquinone in buffered conditions or in
the microsomal system in the presence of chemically induced cytochrome
P450 (CYP2E1) [73]. The study indicated that the redox-cycling between pBQ
and HQ was affected by the presence of NADPH as well as microsomes.
Furthermore, their findings suggest that the formation of OH radicals was
facilitated by both pBQ and HQ in the presence of NADPH, however lipid
peroxidation was inhibited in the same condition.
On another note, Afanasev points out that several quinones including
pBQ promote lipid peroxidation in endothelial cells, yet inhibit lipid
peroxidation in the presence of NADPH [74]. Taken together, the effect of
pBQ on lipid peroxidation appears to be complex and requires more in-depth
investigation to elucidate the exact mechanism on how the redox-cycling of
pBQ affects lipid peroxidation.
For now, the idea that BQs redox-cycling disrupts the glutathione redox
cycle, affecting lipid peroxidation indirectly, is generally accepted (Figure 10).

Figure 1
lipid per
dismutas
glutathio
disulfide
Fig
Oxidize
exposed
HQ, int
of H
2
O
2
convert
form of
to GSH
conjuga
redox-c
leading
and GS
arylatio

2.4. Be

The
situation
minimiz
BQs. O
cellular
or prote

Bio
0. Interplay bet
oxidation. HQ,
se; GTPO, gluta
one reductase; G
e; OLs, oxidized
ure 10 presen
ed lipid mole
d to H
2
O
2
wh
tervened by th
2
also triggers
s glutathione
f GSH. The ac
H. Then, the r
ates [75] or w
ycling of BQ
to the consum
SH redox-cyc
n or oxidation
enzoquinone
e arylation of
n to cells sin
zed at the exp
On the other h
toxicity for a
ein modificatio
ological Impli
tween BQ redox
hydroquinone;
athione peroxid
GSH, glutathion
d lipid molecule
ts the complex
ecules (OLs)
hich may resu
he action of su
s the activatio
(GSH) into
ction of glutat
regenerated G
with BQ to fo
Q can couple
mption of GSH
le contributes
n of GSH by th
es and Glut
cellular GSH
nce the detrim
pense of the re
hand, a low le
cell not to be
ons by BQs or
cations of Ben
x-cycling and g
BQ, benzoquin
dase; GTST, glu
ne in a reduced
es.
xity of the rol
are generate
lt from the re
uperoxide dism
on of glutathio
glutathione d
thionereductas
GSH may reac
form BQ-GSH
with the redo
H. The interpl
s to the toxic
he BQ/HQ pai
tathione
by BQs/HQs
mental effects
eacting GSH t
evel GSH or G
able to protec
r HQs.
nzoquinones
glutathione redo
none; SOD, sup
utathione S-tran
form; GSSG, g
le of BQ on li
ed when a li
edox-cycling b
mutase (SOD)
one peroxidas
disulfide (GSS
se (GTR) redu
ct with OLs to
H conjugates.
ox-cycle of G
lay between B
city of BQs
ir.
certainly crea
s of BQs can
through adduc
GSH depletio
ct itself from o
ox-cycle affectin
peroxide
nsferase; GTR,
glutathione
ipid peroxidati
ipid molecule
between BQ
). The generat
e (GTPO) wh
SG), an oxidi
uces GSSG b
o form OL-G
In addition,
GSH and GSS
BQ redox-cycl
whether throu
ates an interest
n be avoided
ct formation w
on could resul
oxidative dam
69

ng
ion.
e is
and
tion
hich
ized
back
GSH
the
SG,
ling
ugh
ting
d or
with
lt in
mage
70
Figure 1
benzoqu
and GSH
In t
of the c
adducts
extensiv
well as
identifie
methyl-
of Ca
2+

multiply
in the
multiply
chemica
accepto
electrop
observe
cowork
benzoqu
[69]. In
thiol (SH
SH grou
formatio
and cow
1,4-ben
compreh
1. Structures of
uinone, 2-metho
H.
this regard, th
conjugated add
from the rea
ve mechanism
s in vitro co
ed mono-sub
-BQ or 2-meth
and ATP [77
y substituted g
presence of
y substituted g
al model rea
r such as BQ
philic quinone
ed both in che
ers presented
uinone (BrBQ
n their work, a
H) groups, wa
ups in the pro
on between B
workers invest
zoquinone [8
hensive and c
J
f the conjugated
oxy-1,4-benzoqu
here has been a
ducts of BQs
actions of BQ
m studies perf
nditions (Fig
stituted gluta
hoxy-BQ was
7, 78, 80]. Ho
glutathion-S-y
microsomes
glutathion-S-y
ction [79]. T
Qs or HQs an
es react with s
emical and bi
d a series o
Q) and sulfur n
a reduced form
as shown to re
otein, and this
rBQ and sulfu
tigated alkylat
81]. In a r
collective insi
isook Kim
d adducts of div
unone, 2,3,5,6-t
an interest on
and GSH. Th
Qs and GSH
formed at the
gure 11) [76-
athion-S-yl hy
incubated wi
wever, van O
yl HQ either t
[76]. Boatm
yl HQ when pB
The adduct fo
nd sulfur-cont
sulfur nucleop
ological syste
f adducts fo
nucleophile th
m of ribonucl
eact with pBQ
s finding sugg
ur nucleophile
tion of cytochr
review articl
ight on the im
verse BQs (2-me
tetrachloro-1,4-
n examining th
he structures o
have been id
e chemical mo
-80]. OBrien
ydroquinone
ith hepatocyte
Ommen and co
tri- or tetra-su
man and cowo
BQ was reacte
formation betw
taining specie
philes, and suc
ems [63, 69, 8
ormed betwee
hrough alkalin
lease A (RNas
Q, forming cov
gests the poss
e-containing p
rome C by (gl
e, Monk an
mportance of
ethyl-1,4-
-benzoquinone)
he biological r
of the conjuga
dentified throu
odel reactions
n and cowork
when either
es in the prese
oworkers isola
ubstituted addu
orkers identif
ed with GSH i
ween a Mich
es is expected
ch reactions w
81]. Hanzlik
en 2-bromo-1
ne permethylat
se), with 8 in
valent bonds w
sibility of add
proteins [63]. L
lutathionyl-S-
nd Lau offe
BQ-GSH add

)
role
ated
ugh
s as
kers
2-
ence
ated
ucts
fied
in a
hael
d as
were
and
1,4-
tion
ntact
with
duct
Lau
yl)-
ered
duct
formation [82], addressing the multiple effects of the quinone-based
polyphenolic-GSH conjugateson DNA damage, chemical-induced stress
response, neurotoxicity, carcinogenicity and hematotoxicity. In the essence,
due to their higher if not equal reactivity than their quinone precursors, BQ-
GSH conjugates creates toxic situations for cells instead of remaining as a
final product of a cells defense mechanism to remove quinones.

2.5. Nucleic Acids and Benzoquinones

The reactions of BQs and nucleic acids have been studied extensively,
however mostly focused on adduct formation. In vivo and in vitro studies
carried out with human lympocytes showed that the benzene metabolites such
as pBQ induce chromatid exchange and micronuclei formation [83,84]. In
addition, several groups reported that the adduct formation was observed when
the nucleobases or plasmids with certain sequences were treated with pBQ [58,
60, 85], tetrachlorohydroquinone [68], tert-butylhydroquinone [86],
butylatedhydroxytoluene [87], and phenylphenol [88]. tert-Butylhydroquinone
is known to be a major metabolite of butylatedhydroxyanisole and form 8-
hydroxydeoxyguanosine in calf thymus DNA [86]. Chlorinated benzoquinones
were found to be produced during drinking water disinfection processes [67,
89-91] and were found to modify DNA as well as the building blocks of DNA
[33, 67, 68]. The adducts were identified either by
32
P adduct mapping
approach or mass spec analyses, however the reported yield of such adducts
was as low as 0.1% in certain conditions [58, 60, 68, 92]. In terms of
identifying DNA nucleoside-BQ adduct structures, several model studies were
carried out at physiologically relevant conditions leading to isolation of the
following adducts; 3-hydroxy-1,N
6
-benzetheno-2-deoxyadenosine-3-
phosphate (A) from the reaction of 2-deoxyadenosine-3-phosphate and pBQ
[93], 9-hydroxy-1,N
6
-benzetheno-2-deoxyadenosine (B) from the reaction of
2-deoxyadenosine and pBQ [60], 3-hydroxy-1,N
4
-benzetheno-2-
deoxycytidine (C) from the reaction of 2-deoxycytidine and pBQ [60], and
7,8-dichlro-3-(2-deoxyribofuranos-1-yl)-3H-imidazo-[4,5:4,5]pyrimido[1,2-
]benzimidazole-6,9,11(5H)-trione (D) from the reaction of 2-
deoxyguanosine and tetrachloro-1,4-benzoquinone [68] (Figure 12). In
addition to the evidenced DNA modification via adduct formation with BQs,
DNA depurination and strand scissionwere thought as potential mechanism
leading to DNA damage, when DNA is exposed to reactive oxygen species
(ROS) generated through BQ redox-cycling [33].
Jisook Kim 72

Figure 12. Identified adducts of DNA nucleoside and BQs. BQ-contribution
highlighted in green color.

2.6. Proteins and Benzoquinones

Protein modifications induced by BQs can lead to epigenetic genotoxic
stress through post-translational modifications. As shown in the model scheme
(Figure 13) [71, 72], BQs can modify a protein in three different ways. First,
redox-cycling between BQs and the reduced form (HQs) can lead to the
formation of ROS (Figure 10) which can be fatal to biological events. The
outcome of BQ redox-cycling would be the generation of ROS such as
superoxide, depletion of cellular GSH, oxidative damage of proteins, and
disruption of GSH redox-cycle. Second, BQs can alkylate a protein via a
nucleophilic attack by the protein, undergoing adduct formation with the
reacting protein. The reaction can be initiated by N, O, and S-containing
nucleophilic amino acids such as lysine, serine, cysteine in a protein. Third,
BQs can induce protein cross-linking. In this pathway, BQs can react with a
nucleophilic lysine residue of a protein resulting in lysine oxidation, leading to
the formation of allysine (i.e., aldehyde containing lysine). The allysine can
then condense with an intact lysine residue from another protein molecule
generating intramolecular cross-linking. Repeated cross-linking may
ultimately cause the formation of oligomers and furthermore polymeric
aggregates.
Generally, it is understood that the main mechanism for BQ-induced
protein modifications occurs through adduct formation, suggested by the
studies utilizing mass spectrometric approach, target enzyme activity assays,
and
14
C labeling experiment [62-65, 81, 94-98]. For instance, McDonald and
coworkers carried out
14
C labeling experiments, exposing the blood and bone
marrow of mice and rats to benzene [64, 65].

Figure 1
Inte
from pB
from th
finding
biologic
compreh
hemoglo
destruct
structur
to pBQ
was co
Interesti
only wi
and cow
when d
cowork
glutama
suggest
pBQ ba
complex
cyclized
[81, 96-
thiol gro
of ureas
Bio
3. Proposed sch
erestingly, out
BQ adduct for
he adduct form
provides crit
cally importa
hensive under
obin. Kondro
tion induced b
ral investigatio
or bromoben
oncluded that
ingly, they we
th a reduced f
worker monito
ifferent forms
ers investigat
ate and aspart
s that the mo
ased on m/z v
x nature of p
d diquinone-ly
-98]. Krajews
oups as well a
se by a series o
ological Impli
heme for the act
t of the total h
rmation, 12%
mation induce
tical evidence
ant proteins.
rstanding on w
ova and cow
by pBQ and pH
on on the add
nzene using E
t pBQ react
ere able to de
form of RNase
ored lysine and
s of RNase w
ted cytochrom
tate were clea
odification of
value increase
pBQ-induced a
ysine adduct o
ska and cowor
as oxidations o
of quinones [9
cations of Ben
tion of BQs tow
emoglobin mo
were from cy
ed by both o
e showing the
However, t
what happened
orkers report
HQ [94]. Han
duct of ribonuc
ESI-MS appro
ted with sul
etect this predo
e, not with a n
d cysteine mod
ere used as th
me c modific
arly modified
lysine residu
e by 105. Lau
adduct format
or a Michael
rkers elaborat
of thiols being
95].
nzoquinones
ward a protein.
odification of
ysteinyl bindin
oBQ and benz
e pBQs abili
their study
d to the other
ted the time-
nzlik and cowo
clease (RNase
ach [62, 63].
lfhydryl grou
ominant cyste
native form of
dification via
heir model pro
cation and re
d by pBQ [97
ues occurred v
u and cowork
tion that can
adduct of pB
ted their work
g responsible
mice, 5.5% w
ng, and 3% w
zene oxide. T
ity in modify
does not o
r 79.5% modif
-dependent P4
orkers carried
e) upon expos
In their work
ups of cyste
eine modificat
f RNase. Hanz
adduct format
otein. Fisher
eported that f
7]. Their find
via alkylation
kers reported
involve eithe
BQ and glutam
k on arylation
for the inhibit
73

were
were
This
ying
ffer
fied
450
out
sure
k, it
ine.
tion
zlik
tion
and
few
ding
n by
the
er a
mate
n of
tion
Jisook Kim 74
With regard to the effect of BQ redox-cycling on protein modifications,
many studies focused on the role of BQ redox-cycling affecting ROS
generation and GSH redox-cycle (Figure 10) rather than looking into direct
evidence revealing protein oxidation due to increased level of ROS. Both
OBrien and Bolton discussed extensively the outcome of quinone redox-
cycling leading to cytotoxicity due to increased oxidative stress [33, 80]. What
complicates BQ redox-cycling is the presence of ascorbic acid in the system as
described in published studies [71] [99, 100]. Verrax and coworkers discussed
enhancement of quinone redox-cycling in the presence of ascorbic acid,
accompanying induction of caspase-3 independent apoptosis [100]. Roginsky
and coworkers carried out kinetic studies on redox reaction between quinones
and ascorbic acid, revealing ascorbic acid mediates quinone redox-cycling
[99]. Kim and coworkers reported the inhibition of 2-chlorobenzoquinone-
induced RNase modification in the presence of ascorbic acid and NADH,
respectively [71].
By far, BQ-induced protein cross-linking is the topic that was visited the
least, even though it was postulated as a possibility [63, 71, 72]. Recently, Kim
and coworkers demonstrated both pBQ and 2-chloro-1,4-benzoquinone
effectively induced RNase aggregation via cross-linking, utilizing SDS-PAGE,
fluorescence spectroscopy, and confocal microscopy. This finding is important
since there is a strong connection between protein aggregation and disorders
such as Parkinson's disease, Alzheimer's disease, and Huntington's disease
[101-103]. There are many factors influencing protein stability leading to
aggregation, which are pH [104, 105], pressure [106], temperature [106, 107],
mutation [108], and the presence of destabilizing chemicals [104, 105, 107].
The evidence showing BQs ability serving as protein cross-linkers offers
insight on the role of ubiquitously present quinones on protein aggregation and
related diseases, since quinone-induced protein aggregation/modification has
received very little attention.

CONCLUSION

In summary, BQs are wide-spread whether they occur endogenously or
exogenously. The types of biological/chemical events they participate are far
more complicated than one can ever imagine; namely as free quinones,
electron carriers in the mitochondrial/chloroplast ETC, TOPA quinone
cofactors in CAOs, and PAH quinones as metabolites. Considering the simple
six-membered ring structure with two carbonyl groups, however with some
variations on substituents, the outcome of their action is broad, leading to
regulation, disruption, or destruction of cellular activities. Their redox-cycling
and ability to serve as Michael acceptors present opportunities for them to
react with cellular components like GSH, nucleic acids, lipid, and proteins.

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In: Quinones ISBN: 978-1-62618-323-0

Chapter 3

QUINONE MONOACETAL COMPOUNDS
IN APPLICATION TO CONTROLLED
REACTIONS WITH NUCLEOPHILES

Toshifumi Dohi and Yasuyuki Kita

College of Pharmaceutical Sciences,
Ritsumeikan University, Kusatsu, Shiga, Japan

ABSTRACT

A summary of the preparation, synthetic utility, and application of
quinone monoacetals is presented with focus on the following points.
Quinone monoacetals (QMAs), the oxidized compounds of phenols as
well as the desymmetrized alternative of quinones, have attracted
considerable interest due to their broad utilities in organic transformations
as intermediates and important building blocks for the synthesis of
natural products. Recently, increasing interest in the development and
utilization of QMAs has been occurring due to their unique
bifunctionalities of both ,-unsaturated carbonyl and allylacetal
moieties. The varied reactivities in nucleophilic attack on QMA carbons
can occur, for instance, addition to the carbonyl carbon and conjugated
addition to the enone moiety. In contrast to these established addition
chemistries, the reports of the utility of QMAs in substitution reactions
are quite limited. This chapter principally deals with the progress in the

Tel and Fax +81(77)68798229; E-mail: kita@ph.ritsumei.ac.jp

Toshifumi Dohi and Yasuyuki Kita 86
emerging theme of the selectivity during the reactions of QMAs toward
nucleophiles, especially with emphasis on the latter topic, the section of
which starts for i) efficient prearation of QMAs, ii) general guideline for
the reactivities of QMAs toward nucleophiles, and iii) newly developed
methods for the regioselective introduction of aromatic or alkene
nucleophiles by controlled coupling strategies using specific acid
catalysts. In particular, our new strategies can now provide attractive
synthetic routes to the valuable oxygenated biaryls, terphenyls,
dihydrobenzofurans, and other related functionalized compounds. Several
important results, such as the syntheses of key modules of natural
products and preparation of regio-controlled phenol oligomers, are also
discussed for the promising expansion of these future applications.

Keywords: Quinones; Quinone Monoacetals; Selectivity; Nucleophile;
Addition; Substitution; Natural Products Syntheses

INTRODUCTION

Quinone-type compounds (Figure 1) are frequently included in
commercial and industrial chemicals of many broad and attractive
applications. They also ubiquitously exist in nature, and notably, such structures
are known to sometimes show unique biological activities. [1, 2] Besides,
these quinone compounds are important in organic chemistry as synthetic
intermediates and building blocks. Therefore, quinones and their related
molecules remain valuable structural motives of continuous interest in modern
scientific fields. [2].
In this context, the quinone monoacetal (QMA), a mono-protected
quinone compound, has attracted considerable interests due to their broad
utilities and vast synthetic potential for the substrate in many organic
transformations. They usually serve as useful desymmetrized quinones in
selective chemical transformations, and thus are called masked
benzoquinones. [3] Extensive synthetic applications of QMAs as versatile
intermediates of natural products and related molecules account for the
versatility and importance of these compounds in modern scientific fields,
which continuously encourage the development of new preparative methods
that can effectively construct a unique structural unit. Indeed, numerous
reports have appeared aiming at developing a greener system for QMA
synthesis, [3] most of which involved the oxidation of phenols and their
Quinone Monoacetal Compounds in Application 87
derivatives using metal-based oxidants [4] and other classical synthetic
systems.

[5-7].

Figure 1. Quinones and their monoacetal compounds.

Figure 2. Structural characteristics and reactivities of QMAs.
In the past few decades, these compounds can be more easily accessed
from the corresponding phenols by treatment with organic oxidants,
specifically phenyliodine(III) diacetate (PhI(OAc)
2
, PIDA) and
phenyliodine(III) bis(trifluoroacetate) (PhI(OCOCF
3
)
2
, PIFA) in suitable
alcohol solvents. [8].
In contemporary chemical synthesis, the advancements in the strategies
for the efficient and selective formations of new carbon-carbon and carbon-
heteroatom bonds enabling the construction of complex molecules from
readily available synthetic materials emerges as a universal synthetic goal. [9]
In this regard, rising interest in chemistry and utilization of QMAs has been
occurring since 1970s due to their unique bifunctional structure, bearing not
only an ,-unsaturated carbonyl unit, but also the allylacetal moiety in a one-
ring skeleton (Figure 2).
Usually, these QMAs behaved in the synthesis as masked quinone
compounds where only one carbonyl moiety of the ,-unsaturated carbonyl
unit is protected from these reactions. As a result, QMAs could potentially
participate in many types of reactions as a versatile electrophile for various
nucleophiles and dienophiles in cycloadditions. In these cases, the acetal
protection is designed to differentiate the electronic nature of the carbon-
carbon double bond of the initial quinones and define the regioselectivity to
nucleophiles and dienes. QMAs derived from p-quinones are stable and
isolatable compounds, whereas some ortho-QMAs that are obtained from o-
quinone can rapidly undergo self-condensation via the Diels-Alder pathway,
sometimes failed to be isolated based on the high reactivities of the ,-
unsaturated carbonyl and diene units. [10].
In terms of the reactions regarding QMAs with nucleophiles (Nu), some
chemo- and regio-selectivity issues arising from the presence of the carbonyl
functionality, enone unit, and allyl acetal moiety are usually considered as the
Achilles heel for their efficient utilization in organic synthesis. Meanwhile,
synthetic chemistry was used to realize such a reaction control and has attained
high levels of selectivity with a specific strategy for partitioning the desired
course of competitive reactions. [9] This typically involves strategies for
controlling the reaction pathway by an efficient reagent and/or suitable
modification of the substrate itself. Hence, QMAs with both unsaturated
carbonyl and allylacetal functional motifs are considered to be potential
organic precursors due to their inherently ambient unique reactivities if
transformations onto their own organic framework with a regioselective value
are an option by using a suitable activator for discriminating the reactive
functionalities.
Due to the uniqueness of the privileged bifunctional structure as well as its
easy and efficient preparative methods, a wide range of potential reactivities
toward nucleophilic attack on QMAs was already revealed to produce the
addition reactions toward hard and soft nucleophiles (Figure 3), for instance,
the addition to the carbonyl carbon (1,2-addition) [11-18] and conjugated
addition to the enone moiety (i.e., 1,4-addition). [19-26]

However, in sharp
contrast to the established addition chemistry of QMAs regarding the
reactivity of the enone moiety, strategies for utilizing the allylacetal
functionality as an electrophilic unit for substitution reactions are quite
limited.
Except for hydrolysis (vide infra), only a few examples of acetal
displacement were reported until recently for inducing the substitution at the
allylic position of the allylacetal moiety (in other words, -position of the
carbonyl group of QMA). [27-37].


Figure 3. Reactivities of QMAs toward nucleophiles based on the four electrophilic
carbons.
Motivated by this unexploited reactivity and synthetic potential of QMAs,
we and other groups have recently developed new research projects by
focusing the chemistry with particular aim for proving the ambient nature of
QMAs toward a diverse series of nucleophiles based on the reagent controls.
Accordingly, some of these QMAs have already proved as regiospecific
quinone equivalents even for substitution reactions, and their transformations
can dramatically expand the utilities for encompassing the obtainable
structures to a wide array of naturally occurring systems.

[38-43] Moreover,
the alternate synthetic utility of QMAs becomes a powerful tool in other
aspects as it afforded a new synthetic method for designing some useful fine
chemicals, such as biaryls for ligands in catalytic reactions, and further
elongated aromatic oligomers are valuable in materials chemistry and
nanotechnology. [34, 35].
To the best of our knowledge, there is no recent seminal account and
review that summarizes the ambient reactivities of QMAs, thus we present
in this book chapter the following subjects about the chemisty of QMAs: 1)
efficient synthesis of QMAs, 2) general guideline for the reactivities of QMAs
with nucleophiles, and finally, 3) our newly developed strategies for the
regioselective introduction of diverse aromatic or alkene nucleophiles by
controlled coupling using specific acid promoters to provide attractive
synthetic pathways to the valuable oxygenated biaryls, terphenyls,
dihydrobenzofurans, and other related functionalized compounds as well as
recent examples by other research groups. The promising applications for the
formation of the key modules of natural products using these methods are also
briefly described in the last section, 4) intramolecular reactions for the
synthesis of natural products. This chapter exclusively summarizes the
chemistry of the quionone O,O-acetals, and the reactivities of their derivatives,
ex., iminoquinone acetals, quionone O,S-acetals, etc., are only briefly
introduced along with the former main QMA compounds.
1. EFFICIENT PREPARATION OF QUINONE MONOACETALS

Mono-protected quinone acetals, QMAs, were conventionally prepared by
a large number of approaches (Figure 4). [3] The synthetic pathways are
generally classified as (i) chemical oxidation of p-alkoxy phenols, for which
the iron(III) salt was reported in 1957 by Martius et al., [4a]

and successive
works with a diversity of oxidizing reagents, such as other ferric salts, [4b-e]
lead(IV) acetate, [4b, f, g] silver oxide,

[4c] copper(II)-pyridine complex, [4h]
manganese(IV) oxide, [4i, j] thallium(III) nitrate (TNN),

[4k-m] mercury(II)
oxide in the presence of iodine, [4n] bismuth(III) acetate,

[4o] ceric(IV)
ammonium nitrate, [4p, q] calcium hypochlorite, [4r] N-bromosuccinimide
(NBS), [4q, s] 2,6-dichloro-3,5-dicyanobenzo quinone (DDQ)

[4e, i] and its
derivatives, hypobromite and bromine,

[4s, t] and periodic acid

[4u] have
appeared for this conversion, which were sometimes more efficient for the
direct conversion of non-alkoxy phenols. Similarly, QMAs can be also formed
from p-methoxyanilides or sulfonanilides. [44] Other methods include (ii)
anodic oxidations of p- or o-methoxyphenols, [5a-c] dimethoxybenzenes, [5d-
f] simple phenols, [5g] and their trimethylsilyl ether derivatives, [5h] (iii)
partial acetal formation of quinones, [6] and (iv) monohydrolysis of quinone
bisacetals. [7].

Figure 4. Classical approaches for preparation of QMAs.

Figure 5. Preparation of QMAs by hypervalent iodine oxidations (Ac = OCOCH
3
).
In the past few decades, chemical oxidation methods were frequently used
as a mild and straightforward strategy toward QMAs, but several limitations
remained problematic, such as a high loading of these toxic oxidants, narrow
substrate and reaction scope of these oxidants, and low yields of the QMA
formations.
Under the situation, hypervalent iodine reagents, specifically,
phenyliodine(III) diacetate (PIDA) and phenyliodine(III) bis(trifluoroacetate)
(PIFA), have emerged as attractive oxidants for the synthesis of QMAs. [8] In
1987, the research group of Tamura and Kita first generalized this new
practical method with PIDA and PIFA to provide a variety of p-benzoquinone
monoacetals from p-alkoxyphenols in excellent yields under very mild
conditions (Figure 5). [8a] The green chemistry concept as well as facile
handling of the hypervalent iodine reagents that have less toxicity and safer
characteristics compared to the heavy-metal oxidizers have greatly encouraged
their use in QMA synthesis in recent years, and these reagents as
environmentally friendly alternatives to the classically-used chemical oxidants
now seem to have become very popular and a promising choice to achieve the
efficient and green preparation of QMAs.

2. GENERAL GUIDELINES FOR THE REACTIVITIES
OF QUINONE MONOACETALS TOWARD NUCLEOPHILES

Intrigued by the potential of QMAs as useful building blocks by the
controlled reactions to both their enone and allylacetal fragments, we now
present a brief summary of the major developments in this area. The QMA
bifunctionalities are electronically differentiated and thus capable of
elaboration for controlling the regioslectively in a wide array of nucleophilic
attacks, which can be roughly classified into addition and substitution
reactions. The former category also includes a) addition to a carbonyl carbon
(1,2-addition) [11-18] and b) conjugated addition to an enone moiety (1,4-
addition and others), [19-26] the fundamental theories of which have been
extensively studied from 1970 to the 1990s, concluding that the nature of the
used hard and soft nucleophiles is the dominant factor that alters the
reactivities toward the enone group.

2.1. Addition Chemistry: a) 1, 2-Additions

QMAs cause additions at the remaining carbonyl carbon for common hard
nucleophiles, and behave as a useful mono-protected quinone equivalent.
Ronln and co-workers demonstrated in 1975 this type of 1,2-addition of
QMAs with an organolithium reagent to produce the corresponding p-quinol
ketal in 84% yield (Figure 6, Eq. 1). [5a] At almost the same time, Evans
group reported the 1,2-addition of lithium enolate generated by the treatment
of lithium diisopropyl amide (LDA) at -78
o
C to give the p-quinol ketal in
good yields by employing methyl 3-(3,4,5-trimethoxyphenyl)propionate, and
used it for the synthesis of phenanthrenoid compounds upon treatment of the
p-quinol ketal intermediates with acid for the sequential intramolecular
cyclization (Eq. 2). [11a-c] Nonetheless, to selectively obtain the same p-
quinol products in the reaction of quinones themselves by the same 1,2-
addition procedure is difficult. As the acetal moieties are the protecting group
of the carbonyl group, such a QMA strategy with successive deprotection of
the acetal in the products, for example Eq. 3, is accepted as the formal
selective carbonyl addition of nucleophiles toward quinone compounds. [11d,
e] In this way, the inherent regioselectivity problem of quinone compounds
can be generally alleviated by desymmetrizing the two carbonyl groups of the
quinones by protection of one acetal carbon.
A wide range of organolithium species and Grignard reagents have since
been reported to undergo similar 1,2-additions. [11f-i] The Swentons research
group simultaneously presented the 1,2-addition of a series of aryllithium
reagents to QMAs, which provided a facile route to aryl phenols by the
accompanying reductive rearomatization (Figure 7, Eq. 1). [12a, b] The aryl
phenol formation was explained to proceed via acid hydrolysis of the p-quinol
ketals to the corresponding p-quinols followed by their reduction. This tandem
addition-reduction sequence was also used for the C1-lithiated glycols in order
to obtain C-aryl glycosides (Eq. 2). [12c].
The continuous efforts of many researchers were then dedicated to further
contributions to this 1,2-addition chemistry, showing diverse collaboration of
Lewis acid catalysts with organolithium or Grignard reagents. One important
example is the RMgX/cerium chloride

combination (where R = alkyl, aryl,
etc.; X = Br, Cl) reported by Imamoto that significantly accelerated the rate of
the 1,2-addition over the competitive conjugated addition (Figure 8). [13].

Figure 6. Early reports of 1,2-additions of QMAs.

Figure 7. 1,2-addition/reductive rearomatization sequence (Ar = aryl, TBS =
Si
t
BuMe
2
).


Figure 8. Cerium-accelerated 1,2-addition.

Figure 9. Trifluoromethylation of the carbonyl group (TMS = trimethylsilyl, DMF =
N,N-dimethylformamide).

Figure 10. Formation of QMA imines and their applications.
Besides these organometallic reagents, the use of the trifluoromethyl anion
equivalent was reported for the 1,2-addition by Langlois and co-workers in
2000 for the trifluoromethylation of QMAs using a gaseous trifluoromethane
(HCF
3
)/tris(trimethylsilyl) amine (N(TMS)
3
)/tetramethylammonium fluoride
(Me
4
N
+
F
-
) or difluorotriphenylsilicate (Me
4
N
+
[F
2
SiPh
3
]
-
, TBAT) combination
to obtain the trifluoromethylated tertiary p-quinols (Figure 9). [14].
Heteroatom nucleophiles can also react with the carbonyl carbon of
QMAs, although it raises the problem that the conjugated addition is
sometimes accompanied by a 1,2-addition. The intramolecular amine of the in
situ generated QMAs from the bis-ketals

immediately cyclized at the carbonyl
group under weakly acidic conditions to produce the bicyclic iminoquinone
monoacetal (Figure 10, Eq. 1). [15a] The orientation for the hydrolysis of the
two acetal groups can be explained probably by a directing group effect of the
methoxy substituent (X = OMe) for facilitating the protonation of the cleaved
acetal.

Figure 11. Quinone methide formation.
Recently, it was reported that the intermolecular addition of sulfinyl
amines was successful without causing the conjugated addition, which has
been applied as a strategy for forming chiral benzoquinone imines, a versatile
asymmetric intermediate for several alkaloid syntheses, such as (-)-3-
demethoxyerythratidinone (Eq. 2). [15b] Hydrazines could also react with the
carbonyl group in an intermolecular manner to produce the corresponding
hydrazones. [15c].
Evans reported the generation of p-quinone methide ketals by
condensation of the carbonyl group of the QMAs and

N,N-dimethyl--
trimethylsilylacetamide (Figure 11). [16a] Utilizing this unique reactive
intermediate, the total synthesis of the amaryllidaceous alkaloid, cherylline,
was achieved. The carbonyl group of the QMAs can also participate in the
Wittig reactions and is valuable for various quinone methide ketal formations.

[16b-d] Pelter and co-workers intelligently applied this Wittig-type coupling
during the approach to the biomimetic synthesis of aryltetralin lignans. [16b,
c].
Unfortunately, it seems that asymmetric addition to the carbonyl group of
simple QMAs with good reagent control has never appeared in the literature.
Instead, chiral acetals were found for this application. Regarding the
asymmetric reduction of the carbonyl group, Tamura, Fujioka, and
collaborators introduced hydrobenzoins and related units as a cyclic chiral
acetal group for ortho-quinones as shown in Figure 12 (Eq. 1). [17a, b] The
effective removal of the chiral acetal moiety of the obtained hydroxy-,-
unsaturated compounds could afford the enantio-enriched cyclic allyl alcohols.
Interestingly, both enantiomers of the allyl alcohols were obtained in this
substrate control strategy by the proper choice of the additive for the
reduction; the (R)-alcohol was preferentially obtained by carbonyl reduction
using the lithium alminium hydride (LAH)/lithium bromide combination,
while the opposite (S)-product was exclusively produced by the treatment with
LAH/magnesium bromide.

Figure 12. Stereoselective 1,2-reduction of QMAs having chiral acetal groups.
In this connection, Wipf and Jung reported some unusual long-range directing
effects of the fluoroalkyl group at the chiral carbon center of the acetal group
(Eq. 2). [17c] In this prior example, the addition of the methyl Grignard
reagent to the QMAs afforded the addition products with the preference of one
isomer in a 2:1 ratio.
The addition chemistry to the carbonyl of QMAs was extensively applied
to a number of natural product syntheses. The research group of Zard reported
the total synthesis of fortucine, in which the amidyl radical initiator for
producing the key cyclization cascade was attached to the quinol architecture
by the 1,2-addition to the QMA carbonyl group (Figure 13, Eq. 1). [18a, b] A
recent report of the total synthesis of millingtonine A includes the addition of a
lithium enolate to synthesize the intermediate functionalized quinol (Eq. 2).

[18c] The Magnus group demonstrated the introduction of enediyne anions to
the carbonyl group of o-QMA during their synthesis of calicheamicinone (Eq.
3). [18d, e] A similar strategy was also reported by Danishefsky for the
chemoselective addition to produce an o-quinol derivative. [18f] The total
syntheses of hasubanonine [18g] and (-)-acutumine [18h] were accomplished
via quinol intermediates obtained by the addition of allyl Grignard and zinc
reagents to the appropriate o-QMA structures, respectively.

b) Conjugated Additions
One of the earliest examples of the conjugated addition of QMAs was the 1,4-
addition of various heteroatom nucleophiles, such as methanol, methanethiol,
and morpholine, to the enone moiety of the p-QMAs, which was reported in
1978 by Foster and Payne, producing the mono- or bis-1,4-adducts in good
yields (Figure 14). [19a] Later, an extended study was conducted by Ciufolini
et al. to confirm the controlled mono- and bis-conjugate additions for a wide
range of amine nucleophiles. [19b] Parker and Kang reported in 1980 the
reactivity of carbon nucelophiles toward QMAs, and disclosed the exclusive
addition of the malonate anion to the ,-unsaturated carbonyl moiety (Figure
15, Eq. 1). [19c] Other soft carbon nucelophiles can react in the same manner
as the enone moiety, which were frequently utilized for many natural product
syntheses, such as the podocarpate systems, [19d] daunomycinones, [19e] and
lactonamycin. [19f] The reaction of cyanide to specific QMAs directly
produced an aryl nitrile as a result of the sequential base-triggered
aromatization (Eq. 2). [19g] It was nicely displayed by Swenton and co-
workers that complexation of a bulky Lewis acid, that is, methylaluminum
bis(2,6-di-tert-butyl-4-methyl phenoxide) (MAD) reagent, to the carbonyl
group of the QMAs catalyzed conjugated addition of organolithiums to
produce conjugated additions, which could overwhelm the inherent preference
of organometallic reagents for the 1,2-additions (vide supra). [19h, i] Thus, the
conjugated addition of organolithium and Grignard reagents exclusively
occurred at the carbon-carbon double bonds by the treatment with MAD
reagent that can sterically protect the carbonyl carbon from the nucleophiles
(Eq. 3). This strategy was elegantly applied to the concise approach for the
defucogilvocarcin synthetic modules (Eq. 4). [19j, k].
In general, the enone moiety of the QMAs can enjoy the exploited
chemistry and reactivity patterns already found for the common ,-
unsaturated carbonyl compounds. Difunctionalization of the double bond of
the QMAs was thus operative, and tandem conjugated addition/electrophile
trapping is such a case which proves a similar utility of the enone moiety of
QMAs for chemical transformations. An interesting example was reported by
Semmelhack as an early study, who has developed the tandem conjugated
addition with the acylnickel carbonylate anion followed by electrophile
trapping of the resulting metal enolate of the quinone carbonyl group,
affording the acyl and alkyl-difunctionalized ,-adducts of QMAs in
excellent product yields of up to 91% (Figure 16). [20].

Figure 13. Applications for the natural product syntheses.

Figure 14. Early report of conjugated additions of QMAs.
Moreover, the rapid evolution of annulation chemistry continuously
emerged in these past 30 years based on the anionic addition-cyclization
cascades, which has allowed the synthetic utility of QMAs in modern
syntheses. [21] In order to construct polycyclic ring systems, Swentons
research group first demonstrated an annulation strategy using an enolate of
the acyclic diester (Figure 17, Eq. 1). [21a] The in situ generated sodium
enolate caused addition in a 1,4-fashion to the less-hindered enone -carbon,
and the addition intermediate subsequently induced a Dieckmann-type
condensation at the original -carbon of the QMA, affording the cyclic
compound analogous to adriamycin. Very recently, Deslongchamps
annulations were demonstrated by Petrovi and Brckner to smoothly occur
using a compound having Michael donor and accepter parts in the same
molecule, which offered versatile access to certain kinds of decalindiones (Eq.
2). [21b].
Russell and Warrener utilized cyanophthalide and the generated carbanion
for producing the formal [4+2] type condensation via the intermediate A to
construct anthraquinone structures with 13C isotope labeling (Eq. 3). [21c]
Very similar annulation processes to complete the syntheses of various
anthraquinone derivatives were used for p-QMAs by Russell, [21d, g, h]
Swenton, [21e] Monneret, [21f] Achmatowicz and Szechner, [21i] and for o-
QMAs by Mitchell and Russell. [21j, k] In addition, other cyclic C4 synthons
were adopted in this type of condensation to accommodate the desirable multi-
functionalities and substituent patterns in the tricyclic quinone products.


Figure 15. Conjugated additions using carbon nucleophiles.

Figure 16. ,-Functionalization of QMAs via conjugated addition.

Figure 17. Formal [4+2] cyclizations via conjugated addition pathways.
Swenton, [21l] Paredes, [21m-o] and Mal [21p,q] employed the Hauser-
type cyclic sulfone counterparts for the smart syntheses of daunomycin and
related compounds (Eq. 4). In this method, the naphthoquinone derivatives can
be also obtained by the appropriate choice of a cyclic C4 component. [21r].
With regard to other types of cyclizations, McDonald and Dreiding reported
in 1973 the synthesis of triasteranetrione, which is of particular interest due to
its bisectional cyclopropyl-methylene bridge, from the bicyclic acetal dione
intermediate. [22a]

Figure 18. Other type cyclizations.
It includes the important key step and early finding of the formal [3+3]
reactivity of benzoquinone mono(ethylene) acetal that behaved as the two
consecutive Michael acceptor (Figure 18, Eq. 1, path a). On the other hand,
when the -keto ester was deprotonated under electro chemical conditions, the
tandem Michael and oxo-Michael addition toward the double enone moiety
occurred to generate the alternative bicyclic product (path b). [22b] Coates and
MacManus reported the related Nenitzescu-type condensation of QMAs,
affording the [3.3.1] adducts (Eq. 2). [22c] Recently, Aub et al. clarified that
such cyclization modes would generally change depending on the substrates
and substituents, [22d] and in their later studies, the synthesis of the [3.2.1]
bicylooctanone structures toward ()-gelsemine was achieved by the
carbophilic double conjugate addition of the lithium dianion of a
nitropropionate ester (Eq. 3). [22e] The soft nucleophiles having sulfur
heteroatoms, such as 2-mercaptoethanols, reacted with the QMAs in a
conjugated addition manner to instead produce syn-2,3-disubstituted-2,3-
dihydro-1,4-benzoxathiin rings by acid treatment, [22f] where no [3+3] type
adduct was formed. The method for the [3+2] cyclopentanation at the ,-
unsaturated enone moiety was established for the synthesis of the cis-5,6
fused-ring systems using a new C3 synthon (Eq. 4). [22g] Similarly, p-
toluenesulfonyl methyl isocyanide (TosMIC) was used for the [3+2]
cycloaddition to construct the pyrrole ring. [22h].
o-QMAs contain the ,,,-unsaturated cyclic ketone moieties, and such
molecules show the reactivities of both 1,4- and 1,6-additions when subjected
to nucleophiles for conjugated addition. Indeed, some specifically substituted
o-QMAs, possessing an electron-withdrawing group at the -position, [23a]
directed the reactivity of the substrates to undergo -addition of the
nucelophiles rather than the aforementioned -addition (Figure 19, Eq. 1: in
this case, aromatization accompanied by elimination of the acetate group
under weakly basic conditions after the addition of an azide). On the other
hand, the substrate control strategy was also used for directing the addition to
the -carbon of the unsaturated carbonyl moieties, and indeed the presence of
the exo-cyclic electron-withdrawing group at the -position excluded the 1,6-
addition to furnish the bond-formation in an unusual manner at the ,-
carbons of the unsaturated carbonyl of the o-QMAs (Eq. 2). [23b] This
reaction was applied to the tandem cyclization to form highly-substituted
naphthalene molecules, by which the total syntheses of the perylenequinone
phleichrome and protein kinase C inhibitor, calphostin C, were achieved.
[23c].
In contrast to the established reduction of already mentioned carbonyl
group (vide supra), ,-reduction of the enone moiety by hydride reagent was
rarely reported. [24] Quideau et al. reported that reduction of the double bond
of the o-QMAs occurred by the action of hydride reagent, as example,
Selectride
R
, but it gave the desired cyclic ketone product in only low yields
(Figure 20). [24a]


Figure 19. 1,6- versus 1,4-additions of ortho-QMAs by substrate controls.

Figure 20. Example of 1,4-reduction.

The QMAs are susceptible to rearomatization under reductive conditions and
these hydrides promptly caused the tandem ,-reduction/aromatization (see
Figure 15, Eq. 2) due to the strong basicity of the reagents, which is the major
problem of using hydride reagent for the reduction of the QMA double bond.
Among the reactions of QMAs that are mentioned in this chapter, the
enantioselective variants of the conjugated addition of the enone moiety has
undoubtedly advanced the most in the past few decades in terms of the reagent
control. In 1997, Feringa reported the novel copper chiral phosphoramide-
catalyzed asymmetric 1,4-addition of alkylzinc reagents to p-QMAs with
remarkably high enantioselectivities (Figure 21, Eq. 1). [25a-c] To date,
related studies for validating new catalytic species are present for this type of
asymmetric conjugated additions. [25d] An elegant application of this addition
strategy has recently been conducted as a new enantioselective approach
toward naturally-occurring type biphenols. [25e]


Figure 21. Asymmetric conjugated additions by chiral transition metal catalysts.

Thus, the total synthesis of bismurrayaquinone A was achieved by the
traceless central-to-axial chirality exchange of the dearomatized
diastereomieric biphenol units, which were effectively obtained from QMAs
by cooperation of the catalytic asymmetric 1,4-addition of zinc reagents
followed by the oxidative dimerization of the formed enantio-enriched ketones
(Eq. 2). [25f].

Figure 22. Asymmetric additions using organoboron reagents.
Organoboron species can also be used as the nucleophilic partner of QMAs
in asymmetric conjugated additions. Tokunaga and Hayashi first reported the
asymmetric 1,4-addition of a significant number of organoboron reagents to
QMAs catalyzed by their originally developed chiral diene/rhodium complex,
which paved the way to a new synthetic route to the enantioenriched 2-aryl
tetralones (Figure 22, Eq. 1). [26a] Shortly thereafter, Corey et al.
independently developed the rhodium(I)-triethylamine catalyzed asymmetric
addition of potassium isopropenyl trifluoroborate to the enone moieties, and its
application to an effective enantioselective route to the core structure of
biologically active platensimycins (Eq. 2). [26b, c].

2.2. Substitution Chemistry: a) Acetal Displacement (Hydrolysis)

This section describes the substitution chemistry of QMAs. Typically,
under acidic conditions, acetal displacement by water leading to quinones
rapidly occurs in QMAs, by the so-called deprotection or hydrolysis
(Figure 23).

Figure 23. Acetal hydrolysis.
Meanwhile, an example of the reaction of other nucelophiles to replace the
acetal at the quaternary carbon atom is very rare, and has never been discussed
in the literature.

b) Substitution at the Allyl Acetal Moiety
In sharp contrast to the rich addition chemistry of QMAs in terms of the
reactivity at the enone moiety, strategies for utilizing the allylacetal
functionality for substitution reactions to the -position of the carbonyl group
(which also corresponds to the allylic position of the allyacetal moiety) are
quite limited.
The earliest report of an allylic substitution reaction for QMA with the
methyl Grignard reagent (MeMgI) was presented by Coutts and Hamblin more
than 30 years ago to give the rearomatized diphenyl phenol ether in some
extent, in which the reaction favored the unusual substitution course due to the
formation of the stable magnesium phenoxide consisting of the organometallic
reagent and the specific acetal leaving group (Figure 24, Eq. 1). [27].
The introduction of an excellent leaving group, such as the carboxyl
group, at the acetal part in some stable and/or electron-deficient QMAs should
be promising to bias the reactivity of the QMAs for substitution versus the
above-mentioned addition preference (for an example, see Eq. 2).

[28]
However, such reactive QMAs are sometimes too unstable to smoothly handle.
1,3-Rearrangement of the acetal methoxy group in a methanol solution might
occur via the allylic substitution pathway (Eq. 3). [29]

Figure 24. Rare examples of substitution reactions of QMAs.

These strategies under basic and neutral conditions, despite being useful for
directing the reaction to the substitution course, might not perfectly eliminate
the competitive addition processes and not be applicable to simple and readily
accessible QMAs as well as extended nucleophiles. Indeed, the S
N
2
displacement of the dimethyl acetals in QMAs to the ortho-ally and propargyl
phenols only occurred via the 1,2-addition of the organometallic species to the
carbonyl group followed by rearrangement (Eq. 4). [30].

c) Recent Studies under Acidic Conditions
Considering the aforementioned addition reactivities promoted under
basic conditions, [11-26] one can alternatively expect that acidic activation of
the QMAs might offer a new opportunity in substitution chemistry. However,
the limited range of usable nucelophiles under acidic conditions, which
typically have a lower nucelophilicity than the basic reagents, significantly
restricts the versatility of this alternative approach. As a result, attractive
examples for the nucleophilic substitutions of QMAs were only claimed in a
few research references until very recently. The S
N
2 reactivity promoted by
diethyaluminum chloride (EtAlCl
2
) was demonstrated by Sartori et al., who
proposed coordination of the aluminum Lewis acid to the acetal as well as
phenol nucleophile and the pseudo-intramolecular S
N
2 substitution process for
the unprecedented formation of the unsymmetrical bisphenols (Figure 25).
[31].
Swenton utilized the S
N
2 reactivity of the quinone monoacetals to design
a new [3+2] cyclization with electron-rich alkenes leading to dihydro
benzofurans. [32a] Promoted by the Brnsted acids, vinyl sulfide reacted with
several QMAs to give the products, albeit in low yields (Figure 26, Eq. 1).

Figure 25. Unique substitution based on the pseudo-intramolecular introduction of
nucleophiles.

Figure 26. Few examples of substrates for substitutions under acidic conditions.
Actually, Mohr and co-workers alternatively employed acidic clays,
specifically, montmorillonites, for chromenes with a few QMAs for the
construction of the pterocarpan-type dihydrobenzofuran structures (Eq. 2).

[32b].
Another interesting annulation is based on the generation of the
phenoxenium ion by the action of a strong acid on the QMAs. Bchi
emphatically studied this type chemistry for a long time and suggested a new
series of valuable [5+2] cycloaddition processes utilizing the phenoxenium
ions (for an example, see Figure 27). [33a-f]. The dienone core of the
generated cation effectively serves as a 5-atom component toward the cationic
cycloaddition with alkenes to provide bicyclo [3.2.1]octanoid scaffolds, which
are important in natural product syntheses, such as lignans.

Figure 27. Substitution/cyclization sequence via generation of phenoxenium ion (CSA
= (+)-10-camphorsulfonic acid).
Extended reactions were demonstrated by Grieco [33g] and other research
groups [33h-j] by treatment with various acid initiators and substrates.
Motivated by the inherent bifunctionalities of QMA as well as its
unexploited reactivities and utilizations regarding the substitution chemistry,
we postulated that there is still plenty of room for the development of new
reactions, particularly for the introduction of electron-rich arenes or alkene
nucleophiles to QMAs based on alternative reagent controls. The obtained
results are briefly described in the next section.

i) Coupling with Aromatic Nucleophiles by Sandwiched Brnsted
Acids [34]
As recently reported by Canesis group, quinone acetal-type compounds
would cause the tandem conjugated addition/aromatization by aromatic
nucelophiles in the presence of a boron Lewis acid. [45] Thus, it was generally
accepted that electron-rich aromatic compounds would not participate in the
substitution reaction except for the intra- and pseudo-intramolecular processes.

[31] To realize the intermolecular substitution, our research group engaged in
the investigation of the reactivity of quinone O,S-acetals toward aromatic
nucleophiles in the preliminary studies. [46] In the presence of trimethylsilyl
triflate (TMSOTf), the cyclic quinone O,S-acetals derived from the thiophenol
benzoic acid were activated and the reactions would mainly undergo an
unexpected S
N
2 displacement of the carboxyl group at the allyl O,S-acetal
moiety to form sulfur-containing biaryls (Figure 28).
As expected by the previous reports, [33] the experiment of the reaction
using a simple p-QMA with 1,3-dimethoxybenzene in the presence of
TMSOTf in acetonitrile resulted in only a trace amount of the biaryl formation
(Figure 29). The desired substitution reaction scarcely occurred using other
typical Lewis acids, such as boron trifluoride and metal reagents, and even
Et
2
AlCl [31] did not produce an ideal result for the formation of the oxgenated
biaryl.

Figure 28. Substitution of cyclic quinone O,S-acetals.

Figure 29. New coupling method of QMAs using montmorillonite (MT) clay catalyst
in HFIP (HFIP = hexafluoroisopropanol).
Extensive Brnsted acids having different acid strength values were also
examined but with only disappointing results.
At this stage, we hypothesized the strategy of the steric blocking of the -
position of the enone moiety and the acetal carbon of QMAs with the aid of
specifically-shaped Brnsted acids for effecting this transformation. To our
delight, the sandwiched solid acid catalyst, that is, montmorillonite K-10 (MT
K-10) clay, was found to be the most appropriate activator suitable for this
coupling substitution. [34a] On the other hand, while screening of the solvents,
we found that fluoroalcohols, [47] especially hexafluoroisopropanol (HFIP),
matches the proposed activation mode of the MT clay toward the QMA,
probably as a charge-stabilizing polar medium, which produced the biaryl
product in up to 90% yield.
The montmorillonites are known to consist of higher order 2D and 3D
clusters of silicate anions with nanospaces between their layers, in which a
number of protons (H
+
) are absorbed along with the sheet-like polyanions. The
unusual protons captured in the interlayers of the solid acids could possibly be
considered as a special Brnsted acid activator to generate charged species that
are effectively stabilized by the soft poly-anion counterparts. It is thus
assumed that the charged intermediate can react with an aromatic nucleophile
in the allylic manner at the less hindered carbon opposite to the acetal, rather
than the sterically-blocked tertiary acetal carbon. Under such reagent control,
the substitution could occur in good to high yields toward oxygenated mixed
biaryl products with a high efficiency and broad applicability (Figure 30).
The used MT clay could be recovered by simple filtration of the reaction
mixture and reused several times without any loss in activity.

Figure 30. Proposed activation mode and scope of the reactions.
Our group has succeeded in achieving a novel route for the synthesis of
the naphthobenzopyran-6-one structure, the common synthetic intermediate of
the biologidally active natural products, defucogilvocarcins, by utilizing our
reagent-controlled coupling for the naphthoquinone monoacetal with a
commercial aromatic nucleophile using MT clay followed by lactonization of
the formed biaryl (Figure 31).
The obtained products are the biaryl phenols, which can supply the
substrates for continuous arylation processes during the reaction with the
second nucleophile (Ar
2
H) after their conversion to aryl QMAs by the usual
PIDA oxidation. [8]

Figure 31. Concise approach for defucogilvocarcin synthetic module.
This repetitive oxidation/rearomatization strategy, that is, the oxidation of
phenols and the bond-forming rearomatization of QMAs, provides a concise
and expeditious route toward structurally well-defined oxygenated terphenyls
and further elongated oligomers as a result of the controlled arylation of
phenols under mild conditions (Figure 32). Hence, QMAs were first
synthesized from phenols by treatment with PhI(OAc)
2
(PIDA) in methanol,
which was then subjected to a controlled coupling with an oxygenated
aromatic nucleophile (Ar
1
H) in the presence of the MT clay. The arylaryl
bond formation selectively occurred at the original phenol ortho-position
along with the accompanying regeneration of the phenol functionality, giving
the mono-arylated phenol compound. Thus, this reaction sequence was
repetitive and further arylation of the obtained aryl phenol enabled
transformation to the highly-oxygenated m-terphenyls with introduction of the
second nucleophile (Ar
2
H) in a good reaction yield.

Figure 32. Synthesis of oxygenated terphenyls by repetitive couplings (Bz = benzoyl).
The broad scope of this new aryl-elongation strategy in terms of the starting
phenols, aromatic compounds (Ar
1
H and Ar
2
H), and functional groups as well
as the perfect levels of the regioselectivity controls are well documented in the
original paper. [34b].
The other terphenyl isomers, para-linked linear terphenyls, can also be
accessible by the modified procedure in combination with conventional
synthetic techniques and the unique recognition of the acetal group by the MT
clay catalyst. Notably, the linear terphenyl isomer was obtained using the same
starting material as that for the meta-terphenyl (Figure 33). The synthesis
started by the desymmetrization of the acetal part by replacing methanol with a
secondary alcohol in the first oxidation step of the phenol. In the resulting
QMA intermediate, the smaller methoxy group of the mixed acetal can
preferentially interact with the interlayer protons within the sandwich sheets of
the clay catalyst, thus causing selective elimination of methanol during the
arylation step. To transfer the phenol functionality to the para position, the
phenol group of the intermediate ortho-aryl phenol was first protected as a
methoxy group, and then the isopropyl group was chemoselectively removed
by a conventional procedure. Finally, the resulting meta-aryl phenol could be
transformed into the linear terphenyl using the standard reaction sequence in
the hypervalent iodine oxidant and MT clay.
In addition, the use of these synthesized compounds as coupling substrates
for our reaction could allow convergent access to a series of more elongated
and structurally defined phenol-based oligomers (Figure 34). For example, the
use of the prepared aryl phenol as the nucleophile for the arylation of the
QMA produced a two-plus-two tetrameric oligomer as a single product.
Similarly, the terphenyl reacted with the QMA to afford the quinquephenyl in
an acceptable yield.

Figure 33. Modified synthetic route to linear p-terphenyl.

Figure 34. Further oligomers.

As the oxygenated biaryls and related oligomers have important
applications and are found in nature, these methods prove useful in the
synthesis of natural products and analogues, and in the preparation of these
compounds in other scientific studies.

ii) Controlled Coupling of Alkenes by Combination of an Acid
and Hydrogen Bond Donor Solvent [35]
For the [3+2] coupling of QMAs and alkenes, Swenton and Morrow
reported acetic acid and MT clay, respectively, as a reaction initiator (see
Figure 26), [32] but this is usable only for a limited number of extremely
activated alkenes, i.e., vinyl sulfide and electron-rich chromenes. Accordingly,
we then tackled the [3+2] coupling reaction for allowing an extensive substrate
scope, and designed a suitable acid activator of this reaction (Figure 35). Since
the former solid acid, MT clay, showed a poor performance in this [3+2]
coupling, several types of Brnsted acid activators involving a series of
carboxylic acids having various pH values were thus evaluated for screening.
As a result, the strategy for the [3+2] coupling of QMAs applicable for a
series of alkene nucleophiles to provide a wide array of dihydrobenzofuran
products was realized with a new concept; we have clarified that the utilization
of an activated Brnsted acid in situ formed in equilibrium by the aid of the
hydrogen bond donor solvent, hexafluoroisopropanol (HFIP), would
effectively cause the [3+2] coupling in a concerted pseudo-S
N
2 manner. [35a]
The generation of the phenoxenium ion [33] during the mechanism can be
ruled out when considering no formation of [5+2] cyclization product (see
Figure 27).
In this reaction, only carboxylic acids with suitable acidic proton strengths
showed a good performance, among which pentafluorobenzoic acid (PFBA)
(pKa value: ca. 1.5) especially indicated the most promising result with up to
quantitative yield of the dihydrobenzofuran formation. Interestingly, neither
stronger nor weaker acids provided a comparable result.

Figure 35. Brnsted acid activation of QMAs for pseudo-S
N
2 substitution.

Figure 36. Perfluorobenzoic acid-promoted [3+2] coupling of QMAs and alkenes.
The hydrogen bond-donor solvent, HFIP, has also played an indispensable
role in this new coupling system because the consumption of the QMAs in
other solvents was very slow. Therefore, the combined use of the
stoichiometric acid and the solvent is highly important to achieve this effective
coupling. The coupling strategy has surely expanded the scope of coupling
substrates with regard to both QMAs and nucleophilic alkene partners (Figure
36). The reaction was also applicable for QMA imines to produce indolines.
Therefore, a novel regiospecific [3+2] coupling of QMAs with various
alkenes to give dihydrobenzofurans has been established on the basis of a new
reagent control strategy promoted by a specific acid promoter. However, the
used PFBA is usually considered to be a stoichiometric amount of waste
material after the reactions. To make the reaction cleaner, we have newly
developed a heterogeneous recoverable polymer alternative carrying the
specific PFBA functions in the polymer chain as our continued effort in
developing greener and more attractive syntheses. The renewed solid acid,
polystyrene-anchored perfluorobenzoic acid (PS-PFBA), involving im-
mobilized PFBA sites on the surfaces of the polystyrene bead was proven to
be an efficient recyclable catalyst for the same controlled coupling of QMAs
with a set of alkenes having an efficiency similar to the free PFBA itself
(Figure 37). [35b].

Figure 37. Reusable alternative of PFBA (PS = polystyrenes).
This reusable solid PS-PFBA was readily prepared using commercial
perfluoroterephthalic acid in one synthetic step relying on the well-established
amidation procedure in Merrifields solid-phase peptide synthesis. Various
advantages regarding the simple recovery, excellent reusability, and high
reproducibility as well as the perfect chemoselectivity control for the QMAs
were certified as the key features of the solid acid catalyst, PS-PFBA, for the
reactions.
Another significant merit of the use of PS-PFBA to be noted is the
improvement of the substrate and catalyst stoichiometry for the reactions as
well as the expanding scope of the nucelophiles, since the polymer acid was
almost inert to the alkenes as a milder acid. The results clarified the success of
the reactions using only 1.2 equiv. of the alkene nucleophiles related to the
QMAs. Utilizing these important characteristics toward nucleophiles, other
acid-sensitive carbon nucleophiles, such as enol silylethers, furanyl silylether,
as well as soft heteroatoms could be selectivity introduced to the quinone
architecture by employing the solid acid catalyst (Figure 38). Hence, the
coupling protocols utilizing the new PS-PFBA are expected to supply clean
routes to the important dihydrobenzofuran molecules and other functionalized
phenols.

Figure 38. Other tested nucleophiles.
iii) Others
In the literature, a similar S
N
2 displacement and cyclizing substitution for
the introduction of electron-rich arenes or alkene nucleophiles to QMAs have
recently appeared by other research groups using alternative reaction
promoters. Porco et al. investigated the ABCD ring construction of kibdelones,
in which the discovery of a novel arylation of QMA to produce a complex 2-
vinylbiphenyl adduct was nicely described (Figure 39, Eq. 1). [36] The unique
arylative substitution could proceed by the action of inorganic platinum(IV)
catalysts in the presence of an appropriate amount of water, whereas a number
of other screened metal salts as well as Brnsted and Lewis acids did not
similarly work so well for the reaction.
The activation mode of the metal salt still remains unclear, while based on
the requirement of water for the reaction, the authors suggested two possible
activation models by platinum(IV)-aqua complexes, that is, a) hydrogen bond
activation of the acetal, and b) dual activations of the QMA double bond and
acetal group, for the reaction process and control.
Very recently, Liu and co-workers reported ketene dithioacetals as a new
C2 synthon for the [3+2] type coupling of QMAs by the catalysis of tin(IV)
tetrachloride (Eq. 2). [37] This synthetic method could provide a facile [3+2]
coupling route to various benzofurans and is utilized for the synthesis of the
coumestan families.

Figure 39. Recent reports of new substitution reactions.
The authors have proposed two roles of the Lewis acid in coordination to
both the acetal and carbonyl groups, where the former coordination should
occur for activation of the QMA and the latter might be essential for a pseudo-
intramolecular process to facilitate transfer of the ketene dithioacetals from the
metal center.
In fact, heteroatom coordination of the nucleophiles would play a key role
in the transformation, otherwise, the selected Lewis acid seems not to exclude
the competitive [5+2] cyclizations for other simple alkenes without the
heteroatoms. [33].

3. INTRAMOLECULAR REACTIONS FOR THE SYNTHESIS
OF NATURAL PRODUCTS

In addition to the regioselective theme for the intermolecular reactions, we
finally present the chemical transformations of the intramolecular nucleophilic
reactions of QMAs regarding applications to natural product syntheses. The
selected literature reports are briefly summarized in the last section of this
chapter.
As the early reports of the intramolecular cyclization studies, Umezawa
and co-workers demonstrated the preparation of 2-hydroxyaporphine cycles by
a biaryl annulation strategy of o-quinol acetates. It was revealed that the
predicentrine and isodomesticine-type tetrahydroisoquinoline structures were
formed in low yields by the treatment with acetic anhydride and concentrated
sulfuric acid (Figure 40, Eq. 1). [38a].

Figure 40. Intramolecular cyclizations for the construction of biaryl
tetrahydroisoquinoline structure.

Figure 41. Application for -tropolone synthesis.
The cyclization was rationalized by the 1,6-conjugated addition to the 2,4-
dienone moiety of the o-QMA unit followed by the rapid aromatization with
release of the acetoxy group. The reverse electron-demanding coupling of this
type of compound was also investigated (Eq. 2). [38b] The reaction produced
the same-type product, while the mechanistic path was altered to a S
N
2
substitution in the latter case from the conjugated addition due to the structural
requirement of the used substrate. The syntheses of an extended series of
analogous alkaloids were reported in this acid-induced coupling by the same
research group. [38c-e].
Referring to the Umezawas protocol, the first fully regiocontrolled total
synthesis of the colchicine alkaloids was accomplished by Banwell utilizing
the efficient biomimetic conversion of the suitably functionalized
benzoquinone monoacetal into the -tropolone-O-Me ester as the key step
(Figure 41). [39a].

Figure 42. Synthesis of ellagitannins.
The reaction was explained by the initial generation of the phenoxenium
ion by the addition of trifluoroacetic acid followed by intramolecular capture
of the cationic intermediate by the tethered trimethoxyaromatic fragment.
Related approaches were also used for the syntheses of the tropolone
isoquinoline alkaloids, imerubrine and grandirubrine. [39b, c].
The cyclization of suitably protected glucose-derived digalloyl esters by
activation of the o-QMA unit with a Lewis acid was found to produce a small
amount of a biaryl product in the synthetic study toward the ellagitannin
natural product (Figure 42). [40] The authors considered the generation of the
phenoxenium ion species followed by the adventitious intramolecular trapping
by another galloyl group or S
N
2-type concerted substitution mechanism at the
vinylogous allyl acetate.
The total synthesis of 1-O-methyllateriflorone by Nicolaou includes the
construction of a complex spiroxalactone framework by the intramolecular
substitution of QMA (Figure 43). [41] It should be noted that the cyclization of
the corresponding quinone tentatively failed due to the unacceptably poor
nucleophilicity of the tertiary hydroxyl group during the conjugated addition
process, and thus the QMA structure specifically served as an excellent
acceptor for the desired ring closure.
Matsumoto and collaborators developed a novel approach to the
erythrinan alkaloids by utilizing o-QMAs as the synthetic modules. [42]

Figure 43. Key cyclization for lateriflorone.


Figure 44. Synthesis of erythrinan-type alkaloid (Boc = t-butoxycarbonyl).

The Lewis acid-promoted aza-spirocyclization of the suitably designed o-
QMA precursor possessing a carbamate group on the side chain proceeded
with an excellent yield using boron trifluoride as the initiator (Figure 44).
[42a] Transmission axial to the spiro center chirality should occur during the
formal S
N
2 intramolecular displacement at the allylacetal moiety, enabling
enantiopure access to the core structure of the erythrinan alkaloids. Based on
this approach, the total synthesis of the enantiopure O-methylerysodienone
was completed. [42b] More recently, the first total synthesis of (+)-11-
hydroxyerythratidine, a C-11 oxygenated erythrinan alkaloid, was reported by
the same group featuring the stereoselective construction of the C(5) spiro
center in this QMA strategy. [42c] In this case, copper(II) trifluoro-
methanesulfonate was the most effective for furnishing the desired aza-
spirocycles, and the efficiency of the product formation was optimized to a
95% yield with an excellent 14:1 diastereomeric excess.
The cationic [5+2] cycloaddition described in section 2-2-c) [33] was
applied to the intramolecular annulation for achieving the formal total
synthesis of ()-isocomene (Figure 45). [43]

Figure 45. Intramolecular [5+2] cyclization for concise synthesis of isocomene.
Trimethylsilyl triflate (TMSOTf) in a 3.0 M lithium perchlorate solution of
ethyl acetate effectively promoted the intramolecular cycloaddition process,
giving rise to the tricyclic diketone, which is subsequently converted to the
known isocomene intermediate after a few steps.

CONCLUSION

In our long-term investigations of quinone-type compounds in terms of
their preparation, reactivity, synthetic utility, and applications toward natural
products, we have had the opportunity of understanding the reactivities and
utilization of these types of specific masked quinone acetal compounds, which
have gained much attention in the synthetic arena due to their privileged
bifunctionalities and ambident reactivity nature that are potentially useful for
organic transformations. In this chapter, we specifically summarized the
fundamental reactivities of QMAs with recent progresses in the use of diverse
promoters for controlling the additions or substitutions to the desymmetrized
quinone compounds, which can provide new strategies for efficient bond
formations with high chemo- and regio-selectivities suitable for the synthesis
of complex molecules.
The research reports including the representative results certify the QMA
motives as useful building blocks based on both the enone and allylacetal
functionalities for the synthesis by the reagent or substrate controls, in which
the substitution reactions as the emergent research theme in the last few
decades have been succinctly showcased in this review. In the latter cases,
designing the regio-controlling proton activators, such as the solid acid
montmorillonites, perfluorinated acids and polystyrene-anchored recyclable
ones developed in our studies, [34, 35] in combination of the transition state
stabilizing solvent, i.e., a perfluorinated hydrogen bond-donor alcohol, have
been actively studied to realize the ambident reactivities of QMAs for
exploiting the new reactivites and reaction controls, expanding the utility of
these quinone analogues and promising a novel and simple strategy for
achieving several types of complete regiospecific carbon-carbon bond
formations.
With the reactivity summary, the various approaches individually
introduced in this context have valuable synthetic uses capable of allowing the
smart synthesis of natural products employing QMAs as key synthetic
modules.
We hope that this review might be helpful for understanding the recent
advances and versatile methods in the use of QMAs that can serve as a
powerful and efficient synthetic tool for motivating chemical researchers by
not only advertising the use of multi-functionalized QMAs for rapid access to
a wide range of naturally-occurring systems involving aromatized or
dearomatized core structures that are derived from phenolic compounds, but
also for the further development of the controlled reactions among conjugated
additions, allylic substitution, and many types of other cyclizations. The scope
of the products in the field of QMA chemistry is becoming broader along with
the enhanced use of a wider variety of the nucleophiles for the generation of
other useful intermediates. In the next stage, further advances in the
asymmetric addition and coupling chemistry based on the ambident
reactivities of the QMA unit are expected to maintain the status in these
compounds in a new frontier for researchers including the authors and readers
of this chapter.

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In: Quinones ISBN: 978-1-62618-323-0

Chapter 4

CATECHOLQUINONES AS SUBSTRATES
OF THE NRH: QUINONE OXIDOREDUCTASE
2 IN THE BRAIN AND RETINA

Lucia de Fatima Sobral Sampaio
*

Laboratory of Biochemistry of Central Nervous System Development.
Biological Sciences Institute, Federal do Par University.
Belm, PA, Brazil

ABSTRACT

Quinones are highly toxic products of the degradation of many
compounds surging from live organisms. Certain of these highly toxic
products are substrates of the NRH: quinone oxidoreductase 2 (NQO2).
This flavoenzime has a ping-pong bi bi catalytic mechanism, where the
coenzyme FAD is reduced by rare cosubstrates, such as N-rybosil-
hidronicotinamide (NRH) and N-metyl-hidronicotinamide (NMH). The
NQO2 detoxifying activity occurs synchronically with the activation of
the anti-cancer protein p53, which is primarily activated in response to
xenobiotic and radiation. It is not clear if the over activation of the NQO2
produces ionic reactive compounds that are capable of activating p53, or
if the xenobiotic presence is capable of triggering a NQO2-p53 binding,
which, in turn, activates protein p53. Among the diversity of putative
NQO2 substrates, we highlighted catechol quinones produced from

*
Email: lsampaio@ufpa.br.
Lucia de Fatima Sobral Sampaio 142
catecholamines in the brain and retina. The catecholamines participation
in neurological diseases, as well the NQO2 influence in the neurological
diseases physiopathology, is incontestable. Accordingly, in this chapter,
we aim to discuss the implication of the characteristic catechol quinone
reductase NQO2 function in the catechol quinones metabolism, which
takes place similarly in neurons from the brain and retina, associating
with NQO2 cancer-preventing activity and with those neurological
diseases related to catecholamine metabolisms dysfunctions.

1. INTRODUCTION

1.1. NAD(P)H: Quinone Oxidoreductase 1 (NQO1) and NRH:
Quinone Oxidoreductase 2 (NQO2)

Quinones are electron carries. Ubiquinone, the quinone present in the
mitochondrial electron transport chain, is of special importance in the animals
metabolism. Xenobiotic quinones, such as vitamin K, are detoxified by phase
II detoxification enzymes.
Oxidoreductases NAD(P)H: quinone oxidoreductase 1 (NQO1) and NRH:
quinone oxidoreductase 2 (NQO2) are cytosolic flavoproteins that catalyze the
preventive two-electron reduction of quinones and quinonoid compounds to
hydroquinones. NQO1 is a well characterized enzyme with protective action
against oxidative stress and neoplasia, while NQO2 is not.
They have about 50% similarity. They are different in length;, NQO2 is a
231 aminoacid protein, while NQO1 is 43 aminoacids longer. Another great
difference is in relation to preference for cosubstrates. At NQO2 coenzyme,
FAD is reduced by product of the degradation of the dinucleotides, such as N-
rybosil-hidronicotinamide (NRH) and N-metyl-hidronicotinamide (NMH),
while NQO1 is reduced by the major electron carrier of synthesis reaction on
metabolism, nicotinamide adenine dinucleotide phosphate acid (NADPH).
Inhibitors of NQO1 are not useful to inhibit NQO2, which is inhibited for
quercetin and benzo(a)pyrene that do not inhibit NQO1 (Jaiswal, 1994; Long
and Jaiswal, 2000; Strassburg et al., 2002; Mailliet et al., 2005; Celli et al.,
2006; Calamine et al., 2008).
Physiologically, in humans, NQO2 gene expression is inhibited by the Sp3
transcription factor, which has a binding site in the NQO2 promoter (Wang
and Jaiswal, 2004; Wang et al., 2008).

Catecholquinones as Substrates of the NRH 143
1.2. Tissue Distribution of NQO2

NQO2 distribution is tissue specific. In human tissues screened to NQO1
and 2 gene expression (heart, brain, placenta, lung, liver, skeletal muscle,
kidney and pancreas), a generally higher expression of NQO1 than NQO2 was
found. Only NQO1 presence was observed in the placenta, and traces of both
quinone reductases gene expression were observed in the pancreas. A trace of
the NQO2 gene expression was observed in the brain (Jaiswal, 1994).
Quinone reductase enzymes tissue specificity is specie specific too. In the
mouse, no quinone reductase gene expression was observed in the brain,
spleen, lung or skeletal muscle. The highest NQO1 expression was observed in
the heart and kidney, while NQO2 was in the liver and tests that do not express
the NQO1 gene (Long and Jaiswal, 2000). Other pharmacological and
pharmacogenetic studies show NQO2 presence in bone marrow, blood cells
(Long et al., 2002; Kwiek et al., 2004; Iskander et al., 2005, 2006 and 2009)
and skin (Iskander et al., 2004; Ahn et al., 2007; Shen et al., 2010). A recent
immunocytochemistry study shows NQO2 presence in bovine oocytes, but
does not in blastocysts (Sampaio et al., 2012).
Immunohistochemical and pharmacological studies show NQO2 presence
in the prefrontal cortex and hippocampus in humans, and in the rat
hippocampus (Benoit et al., 2010 and Hashimoto et al., 2011), and pharma-
cological and pharmacogenetic studies are highly in favor of the enzyme
presence in the substantia nigra (Wang et al., 2008). A unique pharmacological
study shows NQO2 presence in chick neural retina (Sampaio, 2009), while its
expression in mammalian retinal pigment epithelium is very well shown
(Zmijewski et al., 2009).

1.3. NQO2 Physiology

NQO2 and NQO1 are phase II detoxifying enzymes, however the NQO2
physiology is not fully clear (Vela et al., 2005; Sampaio, 2009). The NQO2
mysteries are being disclosed in function of its importance as a
pharmacological target for cancer and neuropsychiatric therapeutic diseases.
Menadione (2-methylnaphthalene-1,4-dione) is a para-quinone precursor
of the vitamin K (Gong et al., 2008). Most of the in vitro NQO2 kinetic studies
use menadione as a substrate, which even though presenting an elevated Km
(M range) has been useful to identify NQO2 inhibitors (Celli et al., 2006;
Calamine et al., 2008). This substrate choice to in vitro assays is reinforced
because in the absence of the enzyme (NQO2-/- mutated mice) menadione
hepatic toxicity does not occur. On the other hand, these NQO2 null mice
present hyperplasia myeloid of bone marrow and increased neutrophils,
basophils, eosinophils, and platelets in the peripheral blood (Long et al., 2002),
associated with increased sensitivity to benzene toxicity (Iskander and Jaiswal,
2005), and with the potentiation of the Tumor necrosis factor induced
apoptosis in keratinocytes (Ahn et al., 2007). An in vitro study shows NQO2
as preferable catechol ortho-quinone reductase than a para-quinone reductase
(Fu et al., 2008).
Taken together, results from studies using NQO2 null mice and kinetic
assays show a difference in preference for substrates, in relation to NQO1.
Different substrates lead to a specific physiology. In this chapter the
significance of NQO2 in catechol ortho-quinone metabolism is highlighted.

2. CATECHOL QUINONES

2.1. Catechol Quinones as Product of the Catecholamines
Oxidation

Catecholamines metabolism has been reviewed since the middle of the
past century. Dopamine (4-(2-aminoethyl)benzene-1,2-diol), adrenaline ((R)-
4-(1-hydroxy-2-methylamino)ethyl) benzene-1,2-diol) and noradrenaline (4-
[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol)) are synthesized from
tyrosine. Dopamine is an intermediate step in adrenaline and noradrenaline
synthesis. All catecholamines are under synaptic release, vesicular uptake and
storage, and inactivation targeting the residue containing the amino group. The
last step occurs first by catechol O-methyl-transferase (COMT) enzymatic
methylation, then by monoamine oxidase (MAO) enzymatic oxidation (Hanna,
1965).
New studies through new approaches showed that catecholamine
metabolism is not as simple as it was shown in the past. What is important for
this chapter is the dynamic equilibrium between catecholamine leakage from
vesicles to cytoplasm and their reuptake through the vesicular transporter. In
neurons, most catecholamine metabolism is cytoplasmic. Thus, the vesicular
transporter has been pointed as more important to the modulation of the
catecholamine turn-over than neuronal activity, which comprises catecho-
lamine release (cellular exocytose answering an electrical membrane signal),
followed by uptake by the plasmatic membrane transporter (Eisenhofer et al.,
2004).
An amount of catecholamine leakage from vesicles into cytosol favors the
increase in the catechol ring redox chemistry in pH-neutral forming
catecholamine ortho-quinone. This parallel non-enzymatic catabolic pathway
is particularly studied regarding dopamine, a major transmitter found mainly
in the retina and the nigrostriatal tract, because it oxidizes more spontaneously
than cyclizes, compared with adrenaline and noradrenaline that have fast
cyclization. In these catecholamines, the cyclization occurs by intramolecular
1, 4-Michael addition. The cyclized resulting compound leukoaminochrome is
the precursor of aminochrome that suffers polymerase reaction yielding the
protective neuromelanin. This complex polymer bound to lipofuscin granules
is the responsible for the dark aspect of dopaminergic neurons. The level of the
highly toxic dopamine ortho-quinone is a function of the dopamine level, of
the integrity of the dopamine vesicular transporter and of the antioxidant agent
present in the cytosol. Then dopamine ortho-quinone cyclize to form the
dopamine chrome or it can be reduced back to dopamine by action of the
endogenous reductants, such as ascorbic acid. The ortho-quinone (or semi-
quinone) formation has an oxidative fashion, a consequence of the parallel
production of H
2
O
2
and its derivatives (
.
OH, O
2
-
). Indeed, dopamine quinone
makes covalent adducts in protein and DNA (Tse et al., 1976; Graham, 1978;
Graham et al., 1978). Taken together, dopaminergic neurons have a specific
dopamine oxidative stress.

2.2. Dopamine Quinone and Protein Adducts

Dopamine ortho-quinone binds covalently to nucleophilic suphydryl
groups on protein cysteinyl residues forming quimoprotein (protein-bound
quinone). These 5-S-cysteinyl-dopamine adducts are especially dangerous
because the protein function is lost. In thesis, all cysteinyl residues opened to
an aqueous environment can be reactive. Regarding transporters, 5-S-
cysteinyl-dopamine adducts at Cys
342
of the human dopamine transporter
hampers the vesicular uptake (Whitehead et al., 2001), which can increase
cytosolic levels of the dopamine ortho-quinone in a vicious cycle.
Exemplifying with enzymes at time, 5-S-cysteinyl-dopamine adducts occurs in
parkin. This protein takes part in the multi-protein complex E3 ubiquitin ligase
that is an enzyme related to ubiquitin-proteasome system. This system targets
protein for degradation. An absence or a mutation in parkin leads to
Parkinsons disease (Fallon et al., 2002 and 2006; LaVoie et al., 2005 and
2007; Ducan et al., 2011). Another important adduct is made in the protein -
synuclein. This protein primarily takes part in the formation of the Lewy bory,
which is found in neurons from substantia nigra in Parkinsons disease. The 5-
S--synucleinil-dopamine formed does not have the capability to form Lewy
bory, so an accumulation of protofibrils occurs (Conwey et al., 2001). This
increase in protofibril life-time can retard the appearance of -
synucleinopathies (Volpicelli-Daley et al., 2011).
The formation of dangerous dopamine ortho-quinones by enzymatic
pathways such as prostaglandin H synthase (Hastings et al., 1995),
lipoxygenase (Nelson et al., 1995), tyrosinase (Siegbahn, 2004), and xanthine
oxidase (Panoutsopoulos and Beedham, 2004) must be added to spontaneous
dopamine autoxidation, leading to increasing dopamine ortho-quinone
accumulation in the cytosol. In counterpart, in both, the formation of the
protective neuromelanin does not occur (Zeca et al., 2003).
In function of the failure of the dopaminergic system, the vesicular storage
capability is reduced in Parkinsons disease, accumulating dopamine in the
cytosol, which is a side effect of the Levodopa-based therapies (Asanuma et
al., 2008). Therefore, a coadjuvant therapeutic, including inhibitors of the
enzymatic dopamine oxidative pathways, is being developed (Asanuma et al.,
2012).

2.3. Catechol Quinone and DNA Adducts

Dopamine quinone-6-N3adenine and dopamine quinone-6-N7guanine
adducts are formed by the competitive intermolecular 1, 4-Michael addition to
nucleophiles in DNA. It occurs because lower pH induces the partial
protonation of the dopamine ortho-quinone amino group and impedes catechol
quinone cyclization to leukoaminochrome. Estrogens also are able to oxidize
forming catechol estrogen-3, 4-quinone, which also react by 1, 4-Michael
addition to form 4-hydroxi-estrone (estradiol)-1-N3adenine [4-OH-estrone
(estradiol)-1N3adenine] and 4-OH-estrone (estradiol)-1-N7guanine adducts. In
the process of the DNA repair, the purine base linked to the catechol quinone
is excised, forming a DNA mutated by apurinic sites (Cavalieri et al., 2002;
Zahid et al., 2010 and 2011).
Catechol estrogens are metabolized to catechol estrogen quinones by
phase I microsomal enzymes p450 (CYP) in the liver and kidney, with the
production of free radicals from the oxidation of estradiol, which forms
catechol estrogens, 4-hydroxyestradiol and 2-OH-estradiol, among the major
metabolites. These, in turn, are oxidized to the quinones, estradiol-3,4-quinone
and estradiol-2,3-quinone, which can react with DNA. Oxidation of estradiol
to 2-OH-estradiol is mainly catalyzed by cytochrome CYP1A1, and CYP3A4.
The catechol estrogens oxidation pathway is tissue specific. In extrahepatic
tissues, the oxidation of estradiol to 4-OH-estradiol is mainly catalyzed by
CYP1B1 as well as some CYP3As (Roy et al., 1992; Wang et al., 1994;
Hammon et al., 1997; Zang et al., 2007). Catechol estrogen quinone can also
be produced by other oxidant enzymes such prostaglandin H synthase
(Hastings et al., 1995), lipoxygenase (Nelson et al., 1995), tyrosinase
(Siegbahn, 2004), and xanthine oxidase (Panoutsopoulos and Beedham, 2004).

2.4. Some Natural Antioxidants as a Quencher of Catechol
Quinones

2.4.1. Gluthatione
The protein gluthatione makes a cysteinyl nucleophilic attack against
cytotoxic ortho-quinones, resulting in the formation 5-S-gluthationil-dopamine
adducts or gluthationil-catechol estrogen quinone adducts. This reaction can
lead to complete neutralization of the ortho-quinone, depending upon
reductive forces present in the cytosol. If an imbalance occurs in the
accumulation of the 5-S-gluthationil-quinone adducts side, the glutathione
stocks are depleted and toxic compounds rise in the cytosol (Zhou and Lim,
2010).

2.4.2. N-Acetyl-Cysteine
The aminothiol precursor of the aminoacid cysteine, N-acetyl-cysteine has
nucleophile and oxy radical scavenger activity. It reacts with estradiol-3,4-
quinone forming 4-OH-estradiol-N-acetyl-cysteine in normal mouse breast
cells (Venugopal et al., 2007), and in human breast cells (Zahid et al., 2010),
where it is able to block the development of the starting breast cancer (Zahid et
al., 2011). Likewise, N-acetylcysteine prevents the toxicity of the free
dopamine oxidation products such as H
2
O
2
and dopamine quinone. It was
shown to occur in rat brains, where this aminothiol prevents dopamine
mediated inhibition of the Na+, K+-ATPase and of the mitochondrial electron
transport chain activity, which is extremely significant for the control of the
neuropathologies such as Parkinsons disease (Bagh et al., 2008).

3. NQO2 REDUCING CATHECHOL QUINONES

3.1. Cancer

Accumulation of the catechol estrogens quinones occurs by a myriad of
mechanisms and can lead to malignancy appearing by formation of
depurinating catechol estrogen quinone-DNA adducts that are highly
genotoxic, representing the major risk factor for breast cancer in woman
(Jefcoate et al., 2000; Cavalieri and Rogan, 2011). The double reduction of the
estrogen catechol-quinones by NQO2 is able to attenuate tumorigenesis
induced by estradiol. In vitro studies show that estradiol-3,4-quinone has more
substrate selectivity to NQO2 than NQO1 in an assay where the NQO2
catalyze was inhibited by its inhibitor quercetin (resveratrol) (Gaikwad et al.,
2007 and 2009; Jamieson et al., 2007; Yu et al., 2009).
In vivo and in vitro studies show that the ability of NQO2 to make a
double reduction of the dopamine quinone hampers the formation of the
apurinic DNA, which occurs when dopamine ortho-quinone adducts are
excised in DNA repairing process. In this way, it prevents or ameliorates skin
carcinogenesis (Chakraborty et al., 1991; Gomez Sarosi et al., 2003; Iskander
et al., 2004; Hsieh et al., 2005; Gong et al., 2007).
In general, the anti-cancer double reduction of catechol quinone NQO2
activity is accompanied by stabilization of the genome guardian protein p53,
by a protein-protein binding (Gong et al., 2007). In addition, it has been
revealed that NQO2 activity produces free radicals (Reybier et al., 2011) that,
in turn, can also activate the p53 protein.

3.2. Neuropsychiatry Disorders

3.2.1. Parkinsons Disease
Parkinsons disease is the second most common neurodegenerative
disorder. It is placed in nigrostriatal tract, which is characterized by a great
number of dopaminergic neurons. Pathogenesis includes mitochondrial
dysfunction, inflammation, oxidative stress, impairment of the ubiquitin-
proteasome system, and the accumulation of the dopamine ortho-quinones. As
the accumulation of the dopamine ortho-quinones must lead to others
physiopathologic factors, the dopaminergic neuron-specific stress alone
aggravates or generates other types of Parkinsons disease (Asanuma et al.,
2004). In fact, in the idiopathic form of Parkinsons disease was not found to
be correlated with NQO1 and NQO2 gene polymorphism (Okada et al., 2005),
while a NQO2 promoter polymorphism was observed in familial and sporadic
types (Wang et al., 2008).
Depending on advancement of the parkinsonism, retrieving dopamine by
NQO2 double reduction of dopamine-quinone can lead to a vicious cycle by
failure of the dopamine storage capability, hampering the formation of
neuromelanin, and aggravating the disease.

3.2.2. NQO2 and Aging
The key symptom of pathological aging or dementia is a deficit in
acquisition and loss of the memory. The most common dementia is
Alzheimers disease. In pathological aging models using rodents, it was
observed that null NQO2 mice presented non-altered behaviors related to
anxiety, psychosis and depression, associated with an increase in learning
capability (Benoit et al., 2010). These results are in agreement with a
hypothesis of the dopamine stress oxidative taking part in Alzheimer
physiopathology, and of the harmful, but not starting participation of the
NQO2. In the absence of the power dopamine machinery, NQO2 enhances
dopamine stress oxidative, by accumulating dopamine it produces a vicious
cycle. In accordance with this possibility, another study using humans, 50
years old and over, showed a strength correlation with NQO2 polymorphism
and cognitive decline (delayed memory recall over time) (Payton et al., 2010).
Finally, the analyses of the hippocampus of the post-mortem Alzheimers
patients showed high levels of the NQO2 gene expression (Hashimoto and
Nakai, 2011).

3.2.3. Methamphetamine Psychosis and Alcoholism
The abuse of methamphetamine produces dopamine quinone, which must
be involved in its related psychosis symptoms. Investigating genetic factors
contribution for drug abuse, polymorphism was observed in the promoter
region of the NQO2 gene in patients with prolonged-type metamphetamine
psychosis and controls. No polymorphism was observed in the NQO1 gene
(Ohgake et al., 2005). A polymorphism in the promoter region of the NQO2
gene is also related to pathogenesis of alcoholism and alcohol withdrawal
symptoms associated with dopamine accumulation, such as delirium tremens,
hallucinations and seizures. Once more, no correlation with NQO1 promoter
genes polymorphism was found (Okubo et al., 2003). These are strongly
suggestive findings of the NQO2 action neutralizing dopamine quinones.

CONCLUSION

We conclude that catechol estrogen quinones and dopamine quinones are
preferential substrates of NQO2. These enzyme functions in dopamine
physiologic retrieving, preventing the formation of the dopamine ortho-
quinones-S-protein adducts. At the same time, it neutralizes catechol
estrogens, avoiding the formation of the tumorigenic depurinating DNA
adducts. We propose that in pathological conditions cursing with dopamine
stress oxidative, the NQO2 preventive action turns off by failure of the
dopaminergic machinery, increasing the occurrence of neuronal dopamine
accumulation, which enhances the dopamine oxidative stress.

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In: Quinones ISBN: 978-1-62618-323-0

Chapter 5

PLASMA MEMBRANE COQ, PORIN,
AND REDOX CONTROL OF AUTISM

Brian F. Teske
1
, I. L. Sun
2
, Anna Gvozdjakova
3
,

Hans Low
4
and Frederick L. Crane
*2

1
Department of Biochemistry and Molecular Biology, Indiana University,
School of Medicine, Indianapolis, IN, US
2
Department of Biological Science, Purdue University,
W. Lafayette, IN, US
3
School of Medicine, Comenius University, Bratislava, Slovakia
4
Department of Molecular Medicine and Surgery,
Karolinska Institute, Stockholm, Sweden

ABSTRACT

Autism is a neurological condition starting in childhood that is
characterized by behavioral and intellectual problems. Its occurrence is
increasing and although there are some treatments, they are of limited
effect or have undesirable side effects. A recent study showed that autistic
children had increased serum levels of auto-antibodies to Voltage
Dependent Anion Channel (VDAC). Interestingly, in addition to the
membrane transport function of VDAC a second function was recently
described by A. Lawens group at Monash University in Melbourne. This
group showed that VDAC was also a trans-PM NADH dehydrogenase.

*
Address Correspondence to: Frederick L. Crane, flccoq10@aol.com
Brian F. Teske, I. L. Sun, Anna Gvozdjakova et al. 158
The VDAC autoantibody detected in autistic children inhibits the dual
transport and dehydrogenase functionality of VDAC. In this report we
implicate Coenzyme Q as an important co-factor for redox control of PM
pores including VDAC. We show that the PM redox function is
dependent on Coenzyme Q and propose that this novel function for CoQ
has therapeutic implications for treatment of autism disorders. More
broadly, the Coenzyme Q requirement for the PM redox function of porin
in diverse species including bacteria, plants, and mammals suggests a
mechanistically conserved feature of pore redox control.

Keywords: Coenzyme Q10, NADH-Ferricyanide reductase, Redox, Autism,
VDAC

Abbreviations: Coenzyme Q10 (CoQ10), PM (PM), Ferricyanide
(FeCN
6
), Voltage Dependent Anion Channel (VDAC), Uncoupler Protein
(UCP), Inner Mitochondrial Membrane (IMM), para-dichloromercury benzoic
acid (pCMB).

INTRODUCTION

Diverse Role of Q10 in Biological Membranes

Coenzyme Q
10
(CoQ
10
) is well recognized for energy coupling and the
function of the electron transport chain in the inner mitochondrial membrane
(IMM). CoQ
10
is also present in other biological membranes [1, 2] where it
functions in diverse roles. For example, electron transport by CoQ
10
is thought
to be important for acidification and maintenance of the lysosomal pH gradient
required for proper function of lysosomal enzymes [3]. This proton pumping
in the lysosome has been shown to be inhibited by chloroquine which may
reflect an inhibition of coenzyme Q function in the lysosome [4]. In this
review we will focus on the role of CoQ
10
in the Plasma membrane (PM) and
provide evidence for a role between the CoQ
10
- directed trans-PM electron
transport system and the function of VDAC. We also discuss the implications
for the emerging biomedical implications of CoQ
10
in relation to the function
of VDAC and autism spectrum disorders. These new insights point to a more
general role of CoQ
10
in redox control of many types of PM pores, which
could warrant CoQ
10
supplementation for therapy or treatment of other
neurological conditions.
Plasma Membrane CoQ, Porin, and Redox Control of Autism 159
CoQ
10
exists in two pools within the cell, in lipid bilayers CoQ
10
is
thought to freely diffuse within the membrane where the reduced form,
CoQ
10
H
2
, participates in free radical scavenging. CoQ
10
can also be bound to
proteins within the cell, these proteins include CoQ
10
transport proteins and
membrane bound proteins such as pores. CoQ
10
has been shown to directly
bind to a rapidly expanding list of pore proteins, suggesting a direct yet
unknown mechanism for CoQ control of pore redox states. For example CoQ
binding has been demonstrated in V. cholera where a Gly-Gly amino acid pair
in the NADH:quinone oxidoreductase is proposed to participate in CoQ
binding and required for Na
+
pumping of this pore [5]. Following the original
recognition of CoQ in mitochondria, CoQ has been found in all membranes in
amounts sufficient to have significant function in membrane structure or redox
activity. CoQ has also been shown to be important for the function of the
uncoupler protein in the inner mitochondrial membrane. The uncoupler
proteins (UCP) facilitate diffusion of H
+
ions into the mitochondrial matrix
thus bypassing the ATPase and effectively uncoupling electron transport from
oxidative phosphorylation. The importance of CoQ for electron transport chain
uncoupling was shown [6-8], where addition of CoQ
10
was necessary for the
holoenzyme function of recombinant UCP. Given the emerging function of
PM CoQ
10
in whole cell redox control and the expanding list of CoQ
10
binding
proteins we provide evidence that suggests the CoQ
10
directed PM redox
system is required for the proper function of VDAC.

THE SENTINELS OF PLASMA MEMBRANE REDOX
CONTROL

Most cells have two systems for transfer of electrons from cytosolic
NADH through the plasma membrane to the external surface. Each of these
systems involves interaction with coenzyme Q (Figure 1). The first system to
be discovered was an NADH-oxidase which was shown to be subject to
hormonal and growth factor control and involved in proliferation of
transformed cells [9, 10]. The NADH oxidase in the plasma membrane
involves two possible dehydrogenases, NADH cyotochrome b5 reductase [11]
or DT-diaphorase encoded by the NQO1 gene located on the cytosolic side of
the membrane. Each dehydrogenase acts as a NADH coenzyme Q reductase to
reduce the coenzyme Q within the plane of the lipid bilayer. The reduced QH
2

is then oxidized by a reduced coenzyme Q oxidase on the extracellular side of
the membrane plasma. High levels of NQO1 activity cause an increase in the
CoQ
10
H
2
pools and is condisered protective due to heightened free radical
scavenging within the cell. On the contrary, mutations or lowered expression
of NQO1 is detrimental to cells [12].
The second system discovered was an NADH-ferricyanide reductase,
which transfers electrons to reduce ferricyanide outside the cell. This later
system was recently identified as the protein polymer VDAC [13, 14]. The
VDAC has no oxidase activity but it transfers electrons from inside the cell to
the external oxidant ferricyanide [13, 14]. In VDAC the only known groups
for electron transport across the membrane are two cysteine residues (-SH) on
the inside of the channel [15]. Both of these systems oxidize cytosolic NADH
to NAD
+
and consequently increase cytosolic NAD
+
concentrations (Figure 1)
[16]. Principally, VDAC was shown to selectively reduce ferricyanide, and is
therefore not classified as an NADH-oxidase [17]. Currently, ferricyanide is
the only oxidant that works as an external electron acceptor for VDAC, a
likely natural electron acceptor would be semi-dihydroascorbate (discussed
below) [18] especially in neural tissue where ascorbate concentration is found
to be high [19]. VDAC also reduces coenzyme Q, but this reaction may be
based on a functional requirement for coenzyme Q in the function of this
enzyme. In other words, coenzyme Q would act as part of the electron chain as
in the mitochondria or as part of the VDAC holoenzyme rather than as a
substrate for VDAC. Therefore the natural and final electron acceptor for
VDAC remains elusive and it is tempting to speculate that a natural redox
function for VDAC is in keeping coenzyme Q reduced in the plasma
membrane (Figure 1).
A third system for trans-PM electron transport, which involves ascorbate
recycling, has been proposed [20, 21]. Although no ascorbate is used with
isolated plasma membrane in this study, ascorbate recycling is a biologically
relevant process that should be noted in trans-plasma membrane electron
transport. In particular, an ascorbic oxidase could provide a good supply of
ascorbate free fradicals (AFR) [22-24] for the VDAC system given the high
concentrations of naturally occurring ascorbate in the brain [19, 22-25].
Experimentally the requirement for coenzyme Q involvement in these
systems is often demonstrated by depletion or inhibition of coenzyme Q
followed by a rescue effect after coenzyme Q reintroduction. For example,
extraction of coenzyme Q from membrane preparations decreases NADH-
oxidase activity, which would be restored by re-addition of coenzyme Q. This
experimental concept will be further demonstrated in the context of VDAC
[4].

Teske, B.F. et al.
Figure 1. Trans-plasma membrane electron transport. (Left to right): 1. NADH-oxygen
oxidoreductase (NADH Oxidase), 2. NADH-ferricyanide reductase, (VDAC), 3.
NADH-ascorbate free radical reductase, 4. Ascorbate/Ascorbic free radical
oxidoreductase.

REQUIREMENT OF Q IN PM MEDIATED
REDUCTION OF FECN6

When it was found that the Plasma Membrane (PM) had NADH-
ferricyanide reductase activity it was assumed to be based on the microsomal
NADH dehydrogenase (NADH cytochrome reductase). Later when the PM
NADH-oxidase was found, ferricyanide reduction was assumed to be a side
reaction from the primary dehydrogenase. When we studied the ferricyanide
reductase at Monash University, Lawen and Wolvetang found a connection
between the PM oxidase and apoptosis [26]. In the course of further study
Baker and Lawen took up purification of the NADH-ferricyanide reductase
from liver preparations. When it was purified they discovered that it was the
protein which was contained in VDAC found in many PMs . This was
surprising because at first the only apparent electron carriers in VDAC were
two internally located cysteine residues. However, in 1992 during a study of
coenzyme Q function we found that coenzyme Q was necessary for the
erythrocyte NADH-ferricyanide reductase activity in the erythrocyte PM. At
the time we did this study the ferricyanide reductase activity of the PM was
attributed to residual activity from a broken down oxidase. Ten years later,
Lawens group purified the reductase and identified it as VDAC.

PLASMA MEMBRANE COENZYME Q IS REQUIRED FOR
FULL REDOX FUNCTION OF VDAC

The evidence for coenzyme Q involvement with VDAC is exemplified
with plasma membrane preparations and E coli deficient for porin. Direct
testing with isolated recombinant VDAC enzyme will solidify the key
observations provided in this study.

Table 1. Inhibition of plasma membrane electron transport by PCMB.

PCMB
a
concentration (M) Membrane preparation Inhibition (%)
1.0 erythrocyte 100%
2.0 erythrocyte 100%
10 erythrocyte 100%
5 Rat Liver 60%
100 Ehrlich Ascites 89%
100 HeLa 55%
a
para-dichloromercury benzoic acid

The properties of the erythrocyte plasma membrane are unusual in that
there is no detectable NADH oxidase activity despite a high activity for
ferricyanide reduction. The presence of an NADH-oxidase in erythrocyte
plasma membranes would be futile and compete with the oxygen loading of
hemoglobin in this cell type. This means the erythrocyte plasma membrane
system used in this study has potential to uncouple the competing plasma
membrane redox systems found in other cells and provide a means to attribute
the NADH-ferricyanide reductase capacity of erythrocyte plasma membranes
to VDAC. An important distinguishing characteristic of electron transport in
VDAC is the extreme sensitivity to thiol inhibitors such as mercurials or lead
[27]. Thus the electron transport by ferricyanide reductase of erythrocytes is
100% inhibited by micromolar levels of (1.0 M) PCMB, whereas rat liver
plasma membrane ferricyanide reduction is only partially inhibited (15%) by a
PCMB concentration 100 times greater (100 M) (Table 1). These larger 100
M PCMB concentrations were also needed to inhibit ferricyanide reduction
in preparations of HeLa cell plasma membranes where a 55% inhibition was
noted. These results highlight the unique sensitivity of the erythrocyte plasma
membrane system used in this study when compared to plasma membrane
preparations of other cell types. The contrasting sensitivities for mercurial
compounds between the cell lines tested indicates that erythrocyte membranes
have key differences, likely attributed to VDAC exclusively when compared
with other plasma membranes which have a ancillary ferricyanide reductase
capacity and are therefore less sensitive to mercurial inhibition (Table 1).

Teske, B.F. et al.
Figure 2. Coenzyme Q is required for NADH-Ferricyanide reduction. (A) Ferricyanide
reductase activity was measured for erythrocyte PM preparations (control). Membranes
that were lipid depleted with heptane extraction (extracted) or rescued with 10M
Coenzyme Q (Extracted + Q) were also measured for Ferricyanide reduction. (B)
NADH-Ferricyanide reductase activity was measured in the presence of CoQ analogs
or in the presence the CoQ analog with Q rescue as indicated. Data is represented as
the mean values of three independent experiments.
Further evidence for coenzyme Q function in VDAC is shown by two
additional lines of investigation. The first experiment in this series exhibited a
requirement of CoQ
10
for NADH-ferricyanide reduction demonstrated after the
depletion of CoQ
10
by heptane extraction of erythrocyte membrane
preparations (Figure 2A). This lipid depletion caused an 81% decrease in
ferricyanide reduction, an activity that was restored by the addition of CoQ
10
.
Thus it appeared that coenzyme Q was required for the NADH-ferricyanide
reductase activity of VDAC as proposed [28]. The second experiment featured
a more specific line of experimentation by the addition of the Coenzyme Q
analogs, such as EthoxycoQ or DichloroQ, to erythrocyte membrane
preparations. EthoxycoQ caused a 90% inhibition of NADH-ferricyanide
reductase activity which was fully rescued with the addition of CoQ
10
(Figure
2B). A more modest, 63%, decrease in ferricyanide reduction was noted in
DichloroQ treated samples. This inhibition was similarly rescued with the
addition of Coenzyme Q further indicating a direct contribution of coenzyme
Q to the activity of NADH-ferricyanide reductase [29]. Taken together these
experiments suggested that CoQ
10
was required for VDAC mediated NADH-
ferricyanide reductase activity of human erythrocytes PMs as proposed [28].
The requirement of coenzyme Q for the function of a pore protein, such as
VDAC, is not without precedence, for example binding of coenzyme Q toUCP
in the mitochondria is required for optimal function [7, 8]. Mechanistically, the
requisite binding of coenzyme Q to VDAC may lower the effective redox
potential of coenzyme Q from 100 mV to a value closer to the redox potential
of thiol groups at approximately -225 mV. It is noted that there is also
evidence for residual, chelator-sensitive iron in the plasma membrane, which
may bridge any remaining redox potential gap [30, 31].

COQ
10
IS REQUIRED FOR NADH-FERRICYANIDE
REDUCTION IN DIVERSE SPECIES

The CoQ
10
requirement of NADH-ferricyanide reductase also extends to
E.coli. In this line of experimentation both Wild type E. coli and E. coli
deficient for CoQ (ubi F-) were analyzed. Figure 3A depicts a growth
comparison between Wild type and ubi F- mutants. Here, the E. coli strain
deficient for coenzyme Q displayed a less robust growth phenotype when
compared with the wild type strain. E coli were also tested for NADH-
ferricyanide reductase activity. The CoQ deficient ubi F- E. coli had a
diminished ability to reduce ferricyanide with the largest 3-fold difference
detected during the mid-log growth phase between 6 and 8 hours. To
determine if the defect in ferricyanide reduction was due specifically to CoQ
deficiency the Q deficient ubi F- strain was supplemented with either CoQ
1
or
CoQ
8
or no treatment (Figure 4). In this experiment the ferricyanide reductase
activity was measured with increasing concentrations of ferricyanide substrate
present 1-10 M. Importantly, the addition of CoQ
1
and to a lesser extent
CoQ
8
was able to rescue the NADH-Ferricyanide reductase activity to near
wildtype levels. Ferricyanide reductase activity was also determined for the
fepA- E. coli strain. This strain has a mutation in the outmembrane porin
OmpF and exhibited a 5-fold decrease in ferricyanide reductase activity
compared to WT control (Table 2). Collectively this data suggests that CoQ
and the PM electron transport system are required for the NADH-ferricyanide
reductase activity of VDAC in a diversity of species. This evolutionary
conservation suggests that the CoQ and VDAC connection is a key tenant for
PM electron transport and ferricyanide reduction and proposed to be a general
feature of other membrane pore proteins.

Teske, BF et al.
Figure 3. Coenzyme Q deficiency causes a reduced capacity for growth and FeCN
6

reduction in E. Coli. (A) Wildtype E.coli AN704 (wt) and E. coli deficient for
Coenzyme Q AN761 (ubi F
-
) were grown on complete media supplemented with 2,3
dihydrobenzoate and delta-aminolevulinic acid, growth was monitored at time points
indicated. (B) Wildtype E. coli and E. coli deficient for Coenzyme Q (ubi F-) were
assayed for ferricyanide reductase activity at the time points indicated.
Table 2. Ferricyanide reduction in E.coli.

Strain
growth
(klet units)
Ferricyanide reduction
(nmole/min/10
7
cells)
WT 300 0.16
fepA- 100 0.03
ubi- 225 0.05

Teske, BF et al.
Figure 4. CoQ supplementation improves Fe(CN)
6
reduction in Q deficient E. coli.
Addition of coenzyme Q rescues ferricyanide reduction in E. coli deficient for
coenzyme Q. E. Coli AN761 deficient for coenzyme Q was incubated with CoQ
1

(0.06mM), CoQ
8
(0.04 mM) or untreated. Ferricyanide reductase activity was
measured with increasing levels of K
3
Fe(CN)
6
as indicated.
VDAC AND REDOX CONTROL OF PORIN IN AUTISM

Experimental evidence is rapidly emerging linking VDAC dysregulation
to autism spectrum disorders and other neurological conditions [32]. With this
in mind the requirement of CoQ
10
for the function of VDAC may provide an
avenue of therapeutic potential for the treatment of these debilitating
conditions. The preliminary studies in this report provide an important
connection between CoQ, VDAC, and electron transport of porin. One of the
foundational studies linking VDAC to autism was a study by Gozalez-Gronow
[33]. In this study an increase in VDAC autoantibodies in autistic patients was
discovered. Other immunological links from Sun, I.L., et al. showed VDAC
electron transport is inhibited by cytokines [34]. The cytokines TNFa and IL2
that inhibit plasma membrane ferricyanide reduction [34] are increased in
autism [35, 36]. Similar to VDAC dysregulation, deficiencies and alterations
in other PM redox systems have been implicated in autistic patients. It has
been reported that in triiodothyronine (T3) has properties in stimulating the
plasma membrane oxidase which could represent an important link between
thyroid hormone deficiencies and reduced plasma membrane redox function
[37]. Deficiencies in T3 and other thyroid hormones, which are necessary for
neuronal migration and fetal brain development, have been noted in autistic
patients [38, 39]. Glutathione is low in autism, which may decrease electron
transport through VDAC. There are also documented positive effect of
hyperbaric oxygen on autism [40] which can be based on stimulation of the
plasma membrane oxidase. Serum profiles of autistic patients also indicate that
the levels of ceruloplasmin are diminished [41]. Ceruloplasmin can act as an
ascorbate oxidase and acts to stimulate the reductase cycling of the ascorbic
free radical oxidoreductase by increasing the availability of substrate.
Although this last point is still under investigation it should be mentioned that
ascorbic acid supplementation has been the subject of preliminary trials as a
therapy for autism. Since ascorbate is also involved in transmembrane
oxidation systems it should be considered along with CoQ for combinatorial
treatment of autism.

CONCLUDING REMARKS

Attack on the VDAC pore is proposed to be the underlying basis for a
specific disease such as autism whereas chronic oxidation through oxygen
radicals would be suggested to contribute to a more pleiotropic range of
neurological conditions such as diseases of aging. Therefore, attempts to relate
the antioxidant affects to damage of a specific enzyme is not very successful
and in its most unspecific form would tend to generate clusters of diseases.
Broader age related diseases would more likely be base on oxidative damage
to a specific functional system in this case the effect could be from selective
damage to a key component or deficiency of many components. Therefore
inhibition of electron transport increases apoptosis and maintenance of
electron flux through the PM transport system would encourage cell survival
[42].

METHODS

E. Coli Growth and NADH-FeCN6 Reduction

The Escherichia coli K12 strains (ilv C
-
, arg H
-
, ent A
-
, hem A
-
and leu
-
)
were provided by F. Gibson and G. B. Cox (Australian National University).
Strains were grown on media with growth supplements as described (cox gb
(1977) BBA 462 113-120). Cells were grown in a 37 degree C shaker and 1 ml
aliquots were taken at hourly intervals. Growth was determined using a Klett
colorimeter. For ferricyanide reduction experiments cell samples were
centrifuged in the cold room and the supernatant was discarded. Samples were
resuspended to a total volume of 3.0 ml in 0.1 M potassium phosphate buffer
pH 7.0. FeCN
6
reduction was measured using an Aminco DW-2a
spectrophotometer in the dual wavelength mode subtracting absorbance at 500
nm from absorbance at 420 nm. The change in rate of absorbtion was
determined for two to five minutes before addition of FeCN6 to a final
concentration of 5 M. Rates were measured at 20 degrees C. For CoQ rescue
experiments CoQ was added in ethanol.

Erythrocyte PM Preparation

Human erythrocyte PMs were prepared from blood bank erythrocytes with
final separation on dextran gradients as described [43]. PM preparations were
subject to heptane extraction in the dark for 4-6 hours at room temperature.
CoQ
10
was added to the CoQ extracted membranes in heptane, the heptane
was removed by evaporation prior to analysis of NADH-FeCN
6
activity.
NADH-FeCH
6
activity was determined spectrophotometrically by following
the decrease in absorbance at 420 nm. The assay was carried out in sodium
phosphate buffer (100 mM, pH 7.0) containing 0.17 mM NADH and 0.35 mM
K
3
Fe(CN)
6
and the reactions were carried out at room temperature. Coenzyme
Q and analogs were added from a stock solution prepared in ethanol.

ACKNOWLEDGMENTS

The authors acknowledge Eric Teske for assistance with figures. Dr I.G.
Young and Dr. Graeme Cox, Biochemistry Department, Australian National
University , Canberra for preparation of E. coli mutants

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INDEX

A
abnormal cell behavior, vii, 57, 67
access, 99, 114, 123, 125, 137, 139
acetic acid, 4, 8, 18, 45, 115
acetone, 8, 16, 20
acetonitrile, 11, 14, 21, 110
acid, 2, 5, 7, 24, 30, 45, 49, 63, 65, 74, 86,
89, 90, 92, 93, 97, 103, 108, 109, 110,
111, 115, 116, 117, 118, 120, 121, 122,
123, 124, 127, 129, 138, 139, 142, 155,
158, 162, 165, 171
acidic, 95, 106, 108, 109, 116
activated carbon, 17
active compound, 23, 33, 34, 46, 47
active site, 63, 64, 76
adamantane, 129
adduction, 83
adenine, 142
adrenaline, 144, 145
agar, 26, 28, 31
aggregation, 48, 74, 77, 84
albumin, 81
alcohol withdrawal, 149, 154
alcoholism, 149
alcohols, 96, 127, 131, 140
aldehydes, 2
alkaloids, 121, 122, 123, 139
alkene nucleophiles, viii, 86, 89, 110, 115,
118, 119
alkenes, 108, 109, 115, 116, 117, 118, 120
alkylation, 21, 70, 73
allylacetal moieties, viii, 85
amalgam, 18
amine(s), 61, 62, 63, 76, 77, 84, 94, 95, 97,
126
amino acid, 58, 72, 159
ammonium, 6, 7, 90
anatase, 12
antibiotic, 54
anti-cancer, viii, 141, 148
antigen, 58, 59
anti-inflammatory agents, 44, 46
antimalarials, 41
antioxidant, 3, 22, 24, 25, 145, 168, 170,
171, 172
antioxidative activity, 22
antiretrovirals, 50
antithyroid agents, 172
antitumor agent, 132
anxiety, 149
apoptosis, 66, 74, 79, 82, 137, 144, 150,
161, 168
Arabidopsis thaliana, 77
aromatic compounds, 110, 113, 128
aromatic hydrocarbons, 77
ascorbic acid, 24, 74, 145, 167, 170
aspartate, 63, 64, 73
asymmetric synthesis, 135
atmosphere, 11, 18, 19
atoms, 37
Index 174
ATP, 70
Autism, 157, 158, 167, 171, 172
autistic children, 157
auto-antibodies, 157, 167
autoimmunity, 153
B
Bacillus subtilis, 25, 26, 28, 30, 31, 32, 34,
37
bacteria, 26, 28, 29, 31, 33, 34, 38, 54, 60,
128, 158
bacterial strains, 29, 30
barium, 18
basicity, 104
basophils, 144
beetles, 60
behaviors, 149
benign, 66, 78
benign tumors, 66
benzene, 10, 17, 24, 28, 57, 66, 67, 68, 71,
72, 73, 78, 80, 81, 82, 83, 129, 144, 152
benzo(a)pyrene, 142, 152
benzoquinones, 58, 66, 71, 83, 86, 126
benzoyl peroxide, 11
bias, 107
bioaccumulation, 57, 66
bioassay, 28
bioavailability, 49
biochemical processes, 127
biological activities, 3, 86
biological systems, 70
biosynthesis, 77, 169
bismuth, 90
blood, 72, 80, 143, 168
bonds, 87, 127
bone marrow, 72, 80, 143, 144, 153
bone resorption, 48
brain, 46, 142, 143, 150, 151, 155, 160, 167,
170
brain damage, 46
brainstem, 171
Brazil, 141
breast cancer, 147, 148, 155
Britain, 75
bromine, 4, 90
building blocks, 71, 85, 86, 91, 124, 126
C
Ca
2+
, 70, 81, 82
calcitonin, 48, 49
calcium, 2, 3, 21, 48, 90, 127
calvaria, 49
cancer, 57, 66, 77, 78, 142, 143, 151, 156
carbon, 3, 58, 85, 87, 88, 91, 92, 94, 96, 97,
99, 100, 103, 106, 111, 118, 124, 130,
140
carbonyl groups, 57, 58, 65, 74, 92, 120
carboxyl, 107, 110
carboxylic acid, 5, 19, 115, 116
carcinogenesis, 79, 148, 152, 155
carcinogenicity, 66, 71, 78
carcinoma, 66
catabolism, 62
catalysis, 119
catalyst, 12, 18, 64, 111, 114, 117, 118, 138
catalytic activity, 35
catecholamines, ix, 142, 144, 145, 152, 155
category a, 91
cation, 14, 109
CD8+, 59, 60, 75
cDNA, 153
cell death, 84
cell lines, 163
cell surface, 171
cerium, 6, 93, 131
ceruloplasmin, 167, 172
chemical degradation, 127
chemical reactivity, 75
chemicals, 74, 78, 89
chemotherapeutic agent, 66
childhood, 157
children, 157, 171, 172
chiral catalyst, 136
chiral molecules, 135
chirality, 105, 123, 136, 139
chlorination, 83
chlorine, 32, 37, 45
chloroform, 12, 13
Index 175
chloroplast, 65, 74, 77
cholera, 159
chromatid, 71, 82
chromium, 18
clinical disorders, 48
cloning, 153
closure, 122
clusters, 111, 168
coding, 63
codon, 76
coenzyme, 64, 128, 141, 142, 158, 159, 160,
162, 163, 164, 166, 169, 170
Coenzyme Q, 77, 158, 162, 163, 164, 165,
169, 170
commercial, 86, 112, 118
community, 21
competition, 17
complexity, 69
compounds, 1, 2, 3, 4, 5, 7, 11, 12, 18, 20,
22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33,
34, 36, 37, 38, 39, 41, 44, 45, 46, 47, 49,
50, 85, 86, 87, 89, 92, 96, 97, 101, 110,
114, 115, 124, 125, 127, 129, 130, 131,
132, 141, 142, 147, 151, 152, 163
Concise, 112
condensation, 3, 5, 58, 88, 95, 99, 102, 135
configuration, 50
consensus, 76
conservation, 165
construction, 87, 109, 119, 120, 122, 123,
133
consumption, 21, 69, 117
contact dermatitis, 58
cooperation, 105
coordination, 63, 108, 120
copper, 9, 18, 61, 62, 63, 64, 76, 77, 84, 90,
104, 123
correlation, 149
covalent bond, 70, 75
cross-linking/protein, viii, 58
crystal structure, 63
culture, 48
curcumin, 33
cyanide, 97
cycles, 120
cycling, 58, 68, 69, 71, 72, 74, 75, 81, 84,
167
cysteine, 72, 73, 147, 156, 160, 161
cytochrome, 65, 68, 70, 73, 77, 79, 81, 82,
83, 147, 154, 155, 161
cytokines, 44, 167, 171
cytoplasm, 144
cytotoxicity, 74, 82
D
decomposition, 83
defects, 83
deficiency, 164, 165, 168
deficit, 149, 172
degradation, 141, 142, 145, 152
dehydrogenase, ix, 68, 128, 157, 159, 161,
171
delirium tremens, 149
dementia, 149
depression, 149
derivatives, 1, 2, 3, 5, 9, 11, 14, 18, 19, 22,
25, 26, 30, 32, 33, 35, 36, 38, 41, 44, 46,
47, 57, 64, 82, 87, 89, 90, 99, 101, 125,
126, 128, 130, 134, 137, 140, 145
dermatitis, 60
dermatosis, 66
destruction, 73, 75, 83
detectable, 162
detection, 83
detoxification, 142, 154
detoxifying activity, viii, 141
dienes, 88
differential treatment, 3
diffusion, 26, 30, 159
dihydropyrimidinone, vii, 21, 22, 34, 46, 50
dimerization, 105
dimethylformamide, 94
dipeptides, 127
diseases, 48, 74, 142, 143, 151, 168
disinfection, 71, 81, 83
disorder, 148, 172
dispersion, 77
displacement, 88, 106, 108, 110, 119, 123
distribution, 143
Index 176
diversity, 90, 141, 165
DMF, 21, 94
DNA, 57, 71, 72, 80, 81, 82, 83, 145, 146,
147, 148, 150, 151, 156, 171
DNA damage, 71, 82
DNA repair, 146, 148
donors, 65
dopamine, 62, 145, 146, 147, 148, 149, 150,
151, 152, 156
dopaminergic, 145, 146, 148, 150
double bonds, 58, 65, 97
down-regulation, 150
drinking water, 71, 83
drug abuse, 149
drugs, 31, 39, 151
E
E.coli, 164, 165, 166
earthworms, 44
edema, 44, 45
electrolyte, 130
electron(s), 12, 57, 62, 64, 65, 74, 103, 107,
108, 110, 115, 119, 121, 137, 142, 147,
150, 158, 159, 160, 161, 162, 165, 167,
168, 169, 170, 171
elongation, 113
elucidation, 83
enantiomers, 49, 50, 96, 135
enantioselective synthesis, 126
encephalopathy, 79
endonuclease, 81
endothelial cells, 68
energy, 158
environment, 57, 66, 145
enzyme, 22, 62, 63, 72, 83, 142, 143, 144,
145, 150, 154, 155, 160, 162, 168, 169
eosinophils, 144
epithelial cells, 155
epithelium, 143, 156
equilibrium, 115, 144
erythrocyte membranes, 163, 171, 172
ester, 5, 21, 49, 102, 121, 135
estrogen, 66, 146, 147, 148, 150, 154, 155,
156
ethanol, 130, 168, 169
ethers, 126, 128, 129, 130, 131, 133, 138,
140
ethyl acetate, 124, 140
ethylene, 102, 129
etiology, 151, 156
eukaryotic, 76
evaporation, 168
evidence, 63, 73, 74, 75, 158, 159, 162, 163,
164, 167
evolution, 99
excretion, 48
exposure, 58, 59, 73, 78, 171
extraction, 160, 163, 168
F
FAD, 141, 142
families, 58, 119
fatty acids, 49
fibrillation, 84
filtration, 111
flavoenzime, 141
flour, 60, 76
fluorescence, 74
fluorine, 31, 37
formation, 58, 60, 65, 68, 69, 70, 71, 72, 73,
75, 83, 84, 89, 90, 92, 95, 103, 107, 108,
110, 113, 116, 123, 132, 145, 146, 147,
148, 149, 150, 154, 156
fragments, 91, 138
free radicals, 146, 148
functionalization, 2
fungi, 26, 28, 33, 38, 60
furan, 140
G
gastric mucosa, 25
gene expression, 142, 143, 149, 155, 156
gene promoter, 155, 156
genes, 63, 149, 154
genetic factors, 149
genome, 148
Index 177
genotoxicity, 57
glucose, 122
glutamate, 63, 73
glutathione, 68, 69, 82, 147, 151, 156
glycine, 169
granules, 145
Grignard reagents, 92, 93, 97, 131, 133
growth, 28, 41, 43, 159, 164, 165, 166, 168,
170, 172
guardian, 148
H
half-life, 3
hallucinations, 149
hemoglobin, 73, 80, 81, 162
hepatocellular carcinoma, 66
hepatocytes, 70, 82
heptane, 163, 168
hexane, 8
hippocampus, 143, 149
histamine, 62
histidine, 64
HIV, 50
hormone, 22, 167
humoral immunity, 153
hydrides, 104
hydrocarbons, 78
hydrogen, 18, 19, 21, 24, 60, 115, 117, 119,
124, 138
hydrogen atoms, 18
hydrogen peroxide, 24, 60
hydrogenation, 18, 21
hydrolysis, 88, 92, 95, 106, 130, 134
hydroquinone, 67, 68, 69, 70, 81, 82, 83,
127, 128
hydroxyl, 23, 24, 83, 122
hyperactivity, 172
hypercalcemia, 48
hyperplasia, 144, 152, 153
hypothesis, 149
I
ibuprofen, 44
identification, 46, 82
idiopathic, 148
immune response, vii, 57, 58, 59
immunodeficiency, 50
in utero, 172
in vitro, 24, 26, 28, 30, 31, 32, 38, 39, 40,
43, 48, 50, 70, 71, 75, 82, 84, 143, 148,
152, 155
in vivo, 43, 44, 47, 48, 49, 79
incidence, 155
induction, 25, 74, 75, 79, 82, 152
industrial chemicals, 86
inflammation, 148
ingestion, 172
inhibition, 28, 29, 31, 35, 37, 38, 39, 43, 44,
45, 49, 50, 73, 74, 83, 147, 150, 153,
158, 160, 163, 164, 168, 171
inhibitor, 35, 77, 103, 135, 148
insects, 58, 60, 61
integrity, 145
intoxication, 79
iodine, 10, 90, 91, 114, 127, 129, 134, 137,
140
ionization, 81
ions, 109, 159
Iran, 52
Ireland, 139
iron, 9, 90, 126, 164
irradiation, 12, 13, 14, 17, 83
isolation, 71
isomers, 49, 114
isoprene, 64, 65
isotope, 99
K
K
+
, 147, 150
keratinocytes, 144, 150
ketones, 41, 105
kidney, 143, 146, 156
kinetic studies, 74, 143
Index 178
kinetics, 154
L
labeling, 72, 99
larvae, 44
lead, 49, 72, 90, 128, 144, 147, 148, 149,
162
learning behavior, 151
lesions, 25, 76
leukemia, 66
Lewis acids, 110, 119
ligand, 63, 171
lignans, 95, 109, 131
lipid peroxidation, 22, 24, 25, 68, 69, 81,
172
lipid peroxides, 155
lipids, 68
Listeria monocytogenes, 34
lithium, 18, 92, 96, 103, 124, 140
liver, 60, 77, 81, 83, 143, 146, 154, 161,
162, 169, 170
lymph node, 75
lymphocytes, 82
lysine, 72, 73, 83
lysosome, 59, 158
lysozyme, 84
M
machinery, 149, 150
magnesium, 96, 107, 136
major histocompatibility complex, 59
malaria, 41
malignancy, 148
mammals, 64, 158
manganese, 6, 17, 90
mass, 71, 72, 81, 83
media, 165, 168
mediation, 48
medicine, 1, 151
melanin, 22
melanoma, 151, 152
melatonin, 151, 155, 156
membranes, 65, 158, 159, 162, 168, 169
memory, 149
menadione, 137, 143, 153
mercury, 13, 90, 127
meta-analysis, 78
metabolism, 67, 142, 144, 151, 152, 153
metabolites, 57, 60, 66, 67, 68, 71, 74, 82,
83, 147, 154, 155
metabolized, 146
metal salts, 119
Methamphetamine, 149
methanol, 19, 20, 97, 107, 113, 114, 127,
129
methodology, 5, 6, 9, 17, 130, 138
methyl group, 4, 5, 7, 8, 64
methylation, 144
MHC, 59
mice, 47, 60, 66, 72, 73, 75, 76, 78, 79, 80,
144, 149, 153
microscopy, 74
microsomes, 68, 70, 77, 81, 83
migration, 167
mitochondria, 65, 81, 159, 160, 164, 169
models, 21, 75, 119, 149
moderate activity, 31, 32, 34, 37
modifications, 67, 69, 72, 74, 83
modules, 86, 89, 97, 122, 124
mold, 34
molecules, 23, 59, 63, 69, 86, 87, 103, 118,
124, 129
Montenegro, 132
mortality, 78
mucosa, 25
multidimensional, 138
mutant, 84, 151
mutation, 74, 82, 145, 160, 165
N
Na
+
, 147, 150, 159, 169
NAD, 142, 152, 153, 155, 160, 170
NADH, 65, 74, 157, 158, 159, 160, 161,
162, 163, 164, 168, 169, 170, 171, 172
nanoparticles, 12
nanotechnology, 89
Index 179
naphthalene, 103, 135
necrosis, 144
neolignans, 137
neuroblastoma, 152
neurodegenerative diseases, 48
neurological diseases, 142
neurons, 142, 144, 145, 146, 148, 171
neuropathologies, 147
neurotoxicity, 46, 66, 71, 79, 150
neutral, 108, 145
neutrophils, 144
NH2, 131
nickel, 19, 20, 21, 133
nicotinamide, 142
nigrostriatal, 145, 148
nitric oxide, 24, 171
nitrobenzene, 79
nitrogen, 1, 3, 18, 75
nucleic acid, 67, 68, 71, 75
nucleophiles, 7, 10, 18, 70, 75, 81, 86, 88,
89, 92, 94, 97, 100, 103, 108, 110, 115,
118, 119, 120, 125, 146
nucleophilic attack, viii, 72, 85, 88, 91, 147
nucleophilicity, 122
nucleus, 18, 40, 130
nutrient, 62
O
obesity, 49
octane, 129
OH, 68, 131, 145, 146, 147
oligomers, 72, 86, 89, 112, 114, 115, 138
oocyte, 155
opportunities, 75
oral cavity, 66
organ, 18, 48, 60, 94, 97, 107, 108
organic solvents, 78
organism, 64
osteoporosis, 48
ovarian cancer, 153
ox, 86, 89, 111, 112, 113, 115, 123, 131,
137, 138
oxidation, 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 14,
15, 16, 17, 22, 59, 62, 63, 65, 69, 72, 81,
86, 90, 91, 112, 114, 125, 126, 127, 128,
129, 130, 134, 135, 137, 144, 146, 147,
154, 155, 167
oxidation products, 14, 134, 147
oxidative damage, 58, 67, 69, 72, 168
oxidative stress, 74, 142, 145, 148, 150, 153
oxidoreductase, 65, 141, 142, 150, 151, 152,
153, 154, 155, 156, 159, 161, 167, 169,
170, 172
oximes, 135
oxygen, 17, 49, 60, 81, 130, 161, 162, 167,
172
P
palladium, 5, 9
pancreas, 143
paralysis, 44
parasite(s), 41, 43
parathyroid hormone, 48
parkinsonism, 149, 150
pathogenesis, 149, 150
pathological aging, 149
pathology, 155
pathways, 58, 75, 89, 90, 101, 146, 152, 155
peptide(s), 59, 62, 118, 127
perchlorate, 124, 140
peripheral blood, 144
permit, 63
peroxidation, 24, 25, 68
peroxide, 152
pH, 74, 115, 145, 146, 158, 168, 169
pharmaceutical, 54
pharmacological research, 22
pharmacology, 82
phenol, viii, 86, 92, 106, 108, 113, 114, 125,
126, 129, 138, 140
phenol oxidation, 138
phenolic compounds, 125, 128, 129
phenotype, 164
phenylalanine, 65, 77
phosphate, 71, 83, 142, 168, 169
phosphorylation, 159
physiology, 143, 144, 151
physiopathology, 142, 149
Index 180
plants, 58, 60, 64, 81, 158
plasma membrane, 159, 160, 161, 162, 164,
167, 169, 170, 171, 172
plasmid, 82
platelets, 144
platinum, 119
pneumonia, 28, 32, 35
poison ivy, 58, 75
polar, 111, 138
polar media, 138
poly(vinyl chloride), 78
polyamine, 62
polycyclic aromatic hydrocarbon, vii, 57,
66, 78
polymer, 117, 118, 145, 160
polymerase, 145
polymorphism, 149, 154, 156
polystyrene, 117, 124, 138
pools, 159, 160
potassium, 11, 106, 136, 168
precipitation, 8
predators, 60
prefrontal cortex, 143
pregnancy, 172
preparation, 17, 18, 85, 90, 91, 115, 120,
124, 128, 131, 134, 135, 162, 169
present value, 24
preservative, 66
prevention, 151
pro-inflammatory, 46
prokaryotes, 61
proliferation, 75, 79, 152, 159
promoter, 117, 142, 149
protection, 88, 92, 152
protein kinase C, 103, 135
protein oxidation, 74
protein-bound, 57, 145
proteins, 58, 59, 62, 68, 70, 72, 73, 75, 80,
83, 84, 159, 165, 169, 172
protons, 65, 111, 114
Pseudomonas aeruginosa, 25, 26, 28, 30, 31,
32, 33, 34, 35, 36
psychosis, 149, 154
purification, 161
pyrimidine, 3, 5, 14, 17, 18, 19, 26, 30, 40
pyrophosphate, 65
Q
quercetin, 142, 148
quinone(s), 57, 58, 61, 62, 64, 65, 67, 71,
74, 76, 77, 81, 82, 84, 85, 86, 87, 89, 90,
92, 95, 98, 99, 108, 110, 118, 122, 124,
126, 128, 129, 130, 131, 132, 133, 135,
136, 137, 138, 140, 141, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 159, 169, 170
R
race, 133
radiation, 141, 153
radicals, 22, 24, 68, 83, 168
rare cosubstrates, 141
reactants, 17
reaction rate, 12
reaction temperature, 14
reaction time, 4, 6, 11, 12, 14
reactions, 2, 3, 15, 17, 21, 60, 70, 71, 86, 87,
88, 89, 91, 95, 104, 109, 110, 112, 117,
118, 120, 125, 126, 128, 130, 134, 136,
138, 140, 169
reactive oxygen, 22, 71, 81
reactivity, 1, 71, 76, 88, 89, 97, 102, 103,
106, 107, 108, 110, 124, 129, 130, 137
reagents, 18, 90, 91, 92, 94, 97, 104, 105,
106, 108, 110, 129, 131, 133, 134, 136,
137, 140
recall, 149
receptors, 155
recognition, 114, 159
recovery, 118
recycling, 160, 171
red blood cells, 41
redox control, vii, ix, 158, 159
regeneration, 113
regioselective, viii, 18, 86, 88, 89, 120
regioselectivity, 88, 92, 113
reintroduction, 160
Index 181
relaxation, 77
relevance, vii, 78
repair, 80
replication, 41, 50
repression, 155
researchers, 92, 125
residues, 63, 73, 145, 156, 160, 161, 169
resolution, 76
response, viii, 60, 141
resveratrol, 148, 152
reticulum, 59, 169
retina, 142, 143, 145
reusability, 118
reverse transcriptase, 50
rhenium, 14, 17
Rhizopus, 28, 30
rhodium, 106, 136
rings, 103, 132, 135
risk, 66, 78, 148
rodents, 149
room temperature, 6, 8, 10, 14, 16, 17, 19,
137, 168
Rouleau, 151
routes, 86, 118, 137
ruthenium, 9
S
Salmonella, 30, 31, 33, 34, 37
salts, 9, 90, 126, 128, 140
scope, 2, 8, 21, 91, 112, 113, 115, 117, 118,
125
SDS-PAGE, 74
sedative, 47
selectivity, 49, 86, 88, 118, 130, 148
self-assembly, 84
sensitivity, 75, 144, 153, 162
sensitization, 75
serine, 72
serum, 62, 76, 84, 157, 172
side chain, 37, 63, 65, 77, 123
side effects, 157
signalling, 152
silicon, 131
silver, 90
single-nucleotide polymorphism, 153
skeletal muscle, 143
skeleton, 64, 87
skin, 58, 66, 75, 143, 148, 152, 155
Slovakia, 157
sodium, 45, 99, 169
solution, 4, 11, 13, 14, 21, 22, 60, 107, 124,
169
solvents, 9, 78, 87, 111, 117, 140
species, 22, 26, 33, 65, 70, 71, 81, 92, 104,
106, 108, 111, 122, 158, 165
spectroscopy, 74
spleen, 60, 143
squamous cell carcinoma, 66
stability, 74, 84, 136, 151
stabilization, 130, 148, 151, 152
stimulation, 167
stock, 169
stoichiometry, 80, 118
storage, 144, 146, 149
stress, 48, 71, 72, 148, 149, 150, 172
stroma, 65
structure, 2, 24, 74, 76, 77, 87, 88, 106, 112,
120, 122, 123, 132, 136, 139, 153, 159
substitution, 3, 17, 34, 85, 88, 89, 91, 106,
107, 108, 110, 111, 116, 119, 121, 122,
124, 125, 132, 138, 140
substitution reaction, 85, 88, 89, 106, 107,
110, 119, 124
substrate, 64, 77, 84, 86, 88, 91, 96, 103,
104, 115, 118, 121, 124, 143, 148, 160,
164, 167
sulfate, 15
sulfur, 17, 49, 70, 75, 81, 103, 110, 134
sulfuric acid, 120
supplementation, 158, 166, 167
survival, 150, 168
susceptibility, 54, 152, 156
symptoms, 79, 149, 154
synthesis, 1, 2, 3, 7, 8, 9, 15, 17, 19, 30, 41,
77, 80, 85, 86, 87, 88, 89, 91, 92, 95, 96,
101, 103, 105, 112, 114, 115, 118, 119,
121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 135, 136, 137,
138, 139, 140, 142, 144, 153, 169, 171
Index 182
synthetic methods, 2
T
T cell, 59, 60, 75
T lymphocytes, 75
target, 44, 59, 72, 143, 171
temperature, 5, 7, 8, 17, 18, 74, 169
terphenyls, 86, 89, 112, 113, 114, 138
testing, 162
thallium, 90, 127
therapeutic use, 46
therapy, 48, 150, 158, 167
thione analogs, 1, 25
thiophenol, 110
thymus, 71, 82
thyroid, 167
tin, 119
tissue, 25, 66, 143, 147, 153, 155, 160
TNF-, 44
toluene, 24, 66
toxic products, 141
toxicity, 44, 57, 66, 68, 69, 79, 80, 82, 91,
144, 147, 152, 153
toxicology, 77, 82
transcription, 142
transferrin, 170, 172
transformations, 2, 85, 86, 88, 89, 97, 111,
120, 124, 155
transition metal, 105
transport, 49, 57, 64, 142, 147, 150, 157,
158, 159, 160, 161, 162, 165, 167, 168,
169, 170, 171
treatment, 3, 18, 19, 49, 87, 92, 96, 97, 103,
109, 113, 120, 158, 164, 167, 172
trifluoroacetate, 87, 91
trifluoroacetic acid, 121
triglycerides, 49
triiodothyronine, 167
tumor(s), 60, 66, 151, 152, 170
tumorigenesis, 148, 156
tyrosine, 63, 64, 65, 76, 144
U
ubiquitin-proteasome system, 145, 148
ultrasound, 12, 14
urea, 2, 3
urinary bladder, 79
V
variations, 75
vector, 82
versatility, 86, 108
vitamin E, 136
vitamin K, 142, 143, 152
W
waste, 117
wastewater, 83
water, 6, 11, 14, 63, 77, 81, 106, 119
withdrawal, 79
workers, 6, 9, 14, 22, 24, 26, 30, 34, 41, 45,
46, 48, 50, 66, 78, 92, 94, 95, 97, 109,
119, 120
worms, 43
X
xanthones, 137
Y
yeast, 66
yield, 4, 5, 7, 10, 12, 14, 16, 18, 19, 21, 71,
92, 111, 113, 114, 116, 123, 130
Z
zinc, 97, 105

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