Eur J Med Chem (1990) 25,765-774

0 Elsevier, Paris
765
Original article
Inotropic, vasodilator and low Km, CAMP-selective,
cGMP-inhibited phosphodiesterase (PDE III) inhibitory activities
of 4a-methyl-4,4a-dihydro-SH-indeno[l,2-c]pyridazin-3(2H)-ones
and 4a-methyl-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3(2~)-ones
SJ Bakewell, WJ Coates, MB Comer, ML Reeves, BH Warrington*
Smith Kline & French Research Ltd, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK
(Received 15 December 1990; accepted 21 May 1990)
Summary - Novel 7-substituted-4,4a-dihydro-4a-methyl-5H-indeno[l,2-c]pyridazin-3[2K]-ones and S-substituted-4a-methyl-
benzo[h]cinnolin3[2H]-ones have been synthesized and their PDE III inhibitory, inotropic and vasodilator potencies compared with
those of their normethyl analogues and their bicyclic 4,5-dihydro-6-phenylpyridazinone analogues. The structure-activity relation-
ships of the tricyclic pyridazinones differ from those of bicyclic pyridazinones mainly in respect of the effect of introducing the
methyl group into the pyridazinone ring. Whilst in the 4,5-dihydro-6-phenylpyridazin-3(2H)-ones, introduction of a 5-methyl group
has been widely reported to lead to compounds of significantly greater potency, the novel tricyclic 4a-methylpyridazinones showed
similar levels of inotropic, vasodilator and PDE III inhibitory potency to their normethyl analogues. Possible reasons for this differ-
ence in behaviour are discussed.
Resume - Activite inotropique, vasodilatatrice et inhibitrice K,,,-bas, CAMP-selective, cGMP-inhibe phosphodiesterase
(PDE III) de 4,4a-dihydro-4a-methyl SH-indeno[l,2-clpyridazin-3(2H)-ones et 4a-methyl-4,4a,5,6-tetrahydro benzo[h]cinno-
iin-3(2H)-ones. De nouvelles 4,4a-dihydro-4a-methyl-SH-indeno[l,Z-c]pyridazin-3(2H)ones suhstituees en 7 et 4a-methyl henzo(hl-
cinnolin-3(2H)ones substituees en 8 ant ete synthetisees et leurs proprietes PDE III inhibitrice, inotropique et vasodilatatrice compa-
r&es aver celles de leurs analogues normethyles et 4,5-dihydro-6-phenylpyridazinone. Les relations structut-e-activite des
pyridazinones tricycliques d@rent de celles des hicycliques, principalement dans leffet dintroduction du groupe methyle sur la pyri-
dazinone. Tandis que dans les 4,5-dihydro-6-phenylpyridazin3(2H)-ones, 1Tntroduction dun methyle en 5 a ete largement decrite
pour mener a des composes de puissance significativement plus grande, les nouvelles 4a-methylpyridazinones tricycliques montrent
des valeurs dactivite inotropique, vasodilatrice et PDE III inhibitrice similaires a celles de leurs analogues normethyles. Les raisons
possibles de cette dt@rence de comportement sent discutees.
benzocinnolinones / indenopyridazinones / inotropic activity / phosphodiesterase inhibitory activity / pyridazinones / vaso-
dilator activity
Introduction
The inotropic, antihypertensive and platelet anti-
aggregatory properties of 6-aryl-4,5dihydropyri-
dazin-3(2H)-ones 1 and their rigid tricyclic analogues
2, 3 have been widely reported [l-16]. It is probable
that these pharmacological effects arise largely from
their ability to inhibit the low K,,,, CAMP-selective,
*Correspondence and reprints
Abbreviations: PDE, phosphodiesterase; CAMP, adenosine-
3,5-cyclic monophosphate; cGMP, guanosine-3,5-cyclic
monophosphate
cGMP-inhibited phosphodiesterase (PDE III)** [7, 13,
171. The potency of 6-aryl-4,5-dihydro-Smethylpyri-
dazinones 4 as modulators of the above pharrnaco-
logical effects, and as inhibitors PDE III, normally
exceeds that of their normethyl homologues 1 [l-8].
To explore the effect of introducing an analogous
**In an alternative classification system, this enzyme would be
described as type IV [17]. However, the present classification
allows distinction between the low K, CAMP-snecific. cGMP-
, . , 1
inhibited phosphodiesterase and the CAMP-specific enzyme
which is inhibited by rolipram (4-[3-(cyclopentyloxy)~4-
methoxyphenyll-2-pyrrolidinone) see [31]
766
1: RI= H 2: R =H 3: RI= H
4: Rt = CH,
Structures 14: R* represents a wide range of substituents
containing electronegative centres l-3 bond lengths remo-
ved from the phenyl ring eg CH,CONH-, imidazol-1-yl,
C=N, etc.
methyl group into the tricyclic pyridazinones 2 and 3,
the novel 4a-methyl-4,4a-dihydro-SH-indeno[ 1,2-c]-
pyridazinones (table I; 5-9) and 4a-methylbenzo[h]-
cinnolinones (table I; 10-14) have been synthesized
and their PDE III inhibitory, inotropic and vasodilator
potencies compared with those of their normethyl and
4,5-dihydro-6-phenylpyridazinone analogues (table I;
1516,17-21 and 22-28 respectively).
Chemistry
The 4,4a-dihydrobenzocycloalkanopyridazin-3(2H)-
ones 10, 14, 16, 17, 20 and 21 (table I) were derived
from known benzocycloalkan-l-ones as shown in
scheme 1. In a manner akin to that described by Sircar
et al [lo] 6-acetamido-1 -tetralone [ 141 was converted
by way of the Mannich base 29 and the quaternary
salt 30 into the nitrile 31. 6-Methoxy-1-tetralone and
5-acetamido-1-indanone [14] were similarly convert-
ed into the nitriles 32 and 33 respectively, but the
intermediacy of a quatemary salt was found to be
unnecessary as the Mannich bases 34 and 35 could be
transformed directly by treatment with cyanide.
Complete hydrolysis of nitrile 32 and partial hydro-
lysis of the nitriles 31 and 33 gave the methoxy acid
36 and the acetamido carboxamides 37 and 38 re-
spectively which were then cyclocondensed with
hydrazine to give the known compounds 21 [ 1],20 [9]
and 16 [9]. An alternative method for the conversion
of 6-acetamido-1-tetralone into 20 has been described
by Cignarella et al [9]. Complete hydrolysis [lo] of
compound 31 gave a solution containing 6-amino-l-
oxo- 1,2,3,4-tetrahydronaphthyl-2-acetic acid, which
was diazotized and treated with cyanide to give the
cyan0 acid 39 as a precursor in an alternative route to
17 [9]. Compounds 36 and 39 were alkylated under
basic conditions to provide the corresponding
2-methylated esters 40 and 41 which were hydrolysed
to give the acids 42 and 43 required for the syntheses
of 14 and 10 respectively.
The precursors for 4a-methylindenopyridazinones 5
and 9 were derived as shown in scheme 2. Hydroxy-
methylation of 4-bromo- and 4-methoxypropiophe-
none gave mixtures of the corresponding a-hydroxy-
methyl, a-methoxymethyl, and a-vinyl derivatives
which, without separation, were treated with sulfuric
acid to give the 2-methyl indanones 44 and 45. The
bromoindanone 44 was converted into the cyano inda-
F2
tjHCOCH3
CHZCN
\
C$CONH~
11: n-2; R2=NHCOCHs
2: n=2; R2=OCHs
f
2: n-l; R2=NHCOCHa
I
e [R~=NHCOCHJ]
\
3: n-2
3: n=l
&i.$02H
/
&!$02H
h/
/
36: R2=OCH3
39: R2=CN
R2 R2
&OfiH, i - .&02H j 1og
3
40: R2=OCHj
jj: R2=CN
42: R2=OCH3
3: R2=CN
Scheme 1. Reagents. a: HCHO/Me,NH.HCl.b: MeI/Me,-
CO. c: KCN/MeOH. d: cone H,SOdice. e: 10% H,SO,,
lOOC/3 h. f: 5 M HCl. lOOT/5 h. g: NaNOJKCN-CuCN.
h: NaH/MeI. i: 2 M HCl/AcOH/refl 3 h. j: N,H,.H,O/aq
AcOH.
767
Table I. Structures, properties and biological data of dihydropyridazinones in this study.
Compounds 5-21 Compounds 22-28
0
n Rl Rz
5
6
7
a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1
1
I
1
1
2
2
2
2
2
1
1
2
2
2
2
2
CH3
7-CN
CH3
7-CONH2
CH3
7-NH2
CH3
I-NHcoCH3
CH3
7-OCH3
CH3 S-CN
CH3
8-CONH2
-3
E-NH2
CH3
8-NHCOCH3
CH3
8.oCH3
H 7-NH2
H 7-NHCOCH3
H 8.CN
H S-NH2
H s-NH2
H 8-NHcoCH3
H 8-OCH3
CH3
4-CN
CH3
4-CONH2
CH3.
4m2
CH3
4-NHCOCH3
H 4-NH2
H 4.NHCOCH3
H 4.OCH3
-
Exemp. Yield
Methodb (%F mp W)
Inhlhition Annesthetized Cd
of PDE III Inotropic Vasodilntor
Lit. mp
C-3
Ref Formuis*
IC50d
EDsorb
%P
(PW W-P) ~mk)
13
77
43
75
23
73
85
47
42
91
33
51
24
63
57
12
54
3oi
7d
25k
50
sd
73m
75
254
3067
222
315-6
1801
302(d)
267-8
268
305
215
244-S
>300
292-S
>300
289
292
198
193-6
214-6
272-6
234.5
249-52
252-4
1.50-l
aq EtOH
aq EtOH
aq EtOH
AcOH/EtOH
EtOH
DMF/EtOH
EtOH
aq EtOH
aq EtOH
aq EtOH
1 -PIOH
aq AcOH
DMF/EtOH
aq AcOH
aq AcOH
aq AcOH
aq EtOH
sublimed
MeOH
EtOH/Et20
aq DMSO
EtOHiEt20
H20
EtOH
242(d) 10
>300 10
300 9
277-80 9
288-9 10
199-20 1
194.6 1
216-8 2
195-T 1
235.6 1
251-2 12
252 12
150-l 33
Cl3Hl lN30
C13H13N302
C12H13N30
C14H15N302
C13H14N202
C14H,3N30.0.1DMF
C14H15N302.0.5H20
C13H15N30.0.5EtOH
ClSH17N302
C14H16N202
CIlHllN30
C13H13N302
Cl3Hl lN30
C13H13N302.0.65H20
C12H13N30
C14H15N302
C13H14N202
Cl2Hl lN30
C12H13N302
CllH13N30.HCI
C13H15N302
CIOHl lN30.HCl
C,2H,3N302.0.25H20
Cl lHIZN202
9.43 (0.77)
8.38 (2.45) 0.4
30.0 (appmx)
10.3 (1.7)
19.5 (19.5)
9.9 (1.18)
5.4 (0.57)
7.5 (3.39)
3.8 (0.34)
9.4 (1.42)
56.1 (9.0)
5.81 (0.18)
8.85 (1.09)
8.30 (0.88)
34.5 (3.52)
9.22 (0.93)
12.1 (2.13)
2.04(0.11)
2.23 (0.28)
37 (12.9)
0.9 (0.16)
61.7 (19.5)
7.13 (1.7)
12.7 (6.1)
0.18
1.4
1.6 1.0
0.77 0.53
0.2 0.2
1.8 1.57
0.8 1.1
1.2
4.6
0.2
A.2
0.07
1.7
0.03
4.43
0.19
-
0.25
0.26
0.3
3.4
1.3
0.42
M.2
0.10
0.5
0.08
1.4
0.20
acompounds were selected for evaluation in vivo on the basis of their PDE III affinity and ability to cause dose-dependent
contraction of isolated guinea pig ventricular strip (data not shown). Compound 28 was too poorly soluble to test in vivo.
Bynthesized by the method of the compound shown. Compounds 2228 were prepared by the literature methods indicated
under Ref and are not described in the experimental section. CNO attempt was made to optimise yields. dAl1 compounds were
analysed for C, H and N within 0.4% of the calculated values. eIC& value is the concentration required to inhibit enzyme acti-
vity by 50%. Values are means + SEM (n = 3-5, except for compound 7). fED,, values are doses producing 50% increase in
dLVPldt,,,, from control levels. sED,, values are doses producing 15% decrease in autoperfused hindquarters perfusion pres-
sure. Walculated from the dose-response curves and expressed as arithmetic means of 2 or more separate experiments.
iFrom 24. jFrom 22. kOveral1 from 4-acetamidopropiophenone. From 27. W-(4-AcetamidophenyI)-4-oxobutanoic acid.
From 4-(4-methoxyphenyl)-4-oxobutanoic acid. OFree base
768
R2 F2
0
0 +
&OCHCH3
LH20R
@=Br,OCH3;
0
0
bOT=CH,
CH3
R=H,CH3
.fe-- 39
CH2C02C2H5
44
45: R2=OCHs
46: Rz=CN
Rz=OCH3,CN
e
1.2
Scheme 2. Reagents. a: HCHO/MeOH.b: cone H,SO,.
c: CuCN/quinoline. d: NaH/BrCH,CO,Et. e: N2H4.H20.
none 46 by treatment with cyanide. Alkylation of 45
and 46 by ethyl bromoacetate gave the crude inda-
none-2-acetic esters which were treated with hydra-
zine to give compounds 9 and 5.
The remainder of the tricyclic compounds shown in
table I were prepared by derivatization of the hydrazine
cyclocondensation products 5, 10,16, 17,20 as shown
in a generalized form in scheme 3. Hydrolysis of
cyan0 compounds 5,lO and 17 gave the carboxamides
6, 11 and 18 respectively. The amines 7 and 12 were
obtained by Hoffmann rearrangement of the corres-
ponding carboxamides 6 and 11. The amines 15 [lo],
19 [9] were prepared by hydrazinolysis of the amides
16 and 20 respectively. Acetylation of the amines 7
and 12 afforded the acetamido derivatives 8 and 13.
The bicyclic compounds 22-28 were prepared by the
literature methods indicated in table I.
In the cyclocondensations with hydrazine leading to
pyridazinones described above, it was of interest to
note that reactions involving esters proceeded more
slowly and in a poorer yield than those in which an
acid or an amide was the substrate. A possible expla-
nation is that reactions involving acid or amide sub-
strates proceed by way of a lactone or lactam interme-
diate (eg 47).
Biological evaluation
Compounds 5-28 were assayed for inhibition of PDE
III derived from cat heart. Their inhibitory potencies,
expressed as the concentration required to cause 50%
inhibition of the enzyme, are shown in table I.
Selected compounds (table I; 6, 8, 9, 11, 13-16,
18-21, 23-27), were studied in ganglion-blocked,
h-blocked anaesthetised cats. Following a bolus intra-
venous injection of the test compound, the maximum
value of the first derivative of left ventricular press-
ure, dLVPldt,,,, was measured as an index of con-
tractility and inotropic activity was quantitatively
assessed as the dose (ED,,) required to cause a 50%
increase in this parameter. During the anaesthetised
cat experiments, autoperfused hindquarters perfusion
pressure, heart rate and left intraventricular pressure
were also monitored. Changes in heart rate and left
intraventricular pressure were minimal in these exper-
iments and values are not given. Vasodilator activity
was assessed by the dose (ED,5) required to cause a
15% fall in perfusion pressure.
Results and discussion
The pharmacological properties of 6-aryl-4,5dihydro-
pyridazinones 1 have long been recognised. Recently,
interest has focused on the inotropic and vasodilator
properties of bicyclic and tricyclic compounds l-4 in
dogs [5-10,131 and substantial evidence [ 18-261 has
been amassed that their pharmacological effects are
mediated, at least in part, by inhibition of PDE III, the
low K,,,, CAMP-selective phosphodiesterase which is
potently inhibited by cGMP. Correlation of the in vivo
CN
$ONH2
0
I
NaOBr From 5, 11
g,~.~.g 1.12. II, 19
Scheme 3. RI and R are as shown in table I.
inotropic potency and in vitro PDE III inhibitory acti-
vity of a range of arylpyridazinones 1 with different
phenyl ring 4-substituents [7], and of a more structu-
rally diverse series of inotropes [ 131, has also been
demonstrated. Earlier studies were concerned mainly
with the blood pressure lowering [l-4] and platelet
anti-aggregatory [4, 111 effects of the bicyclic com-
pounds 1 and 4 in rodents. The structure-activity re-
lationships elucidated [ 1, 21 for these effects were
remarkably similar to those shown for inotropic activ-
ity and PDE III inhibition, suggesting that antihyper-
tensive and anti-aggregatory effects are also mediated
to a large extent by CAMP, but these last 2 effects are
the predominant biological effects in rodent models
[6, 141. This conclusion would also be consistent with
the known consequences of elevating CAMP levels
[27, 281. Several structure-related patterns can be
identified in the results in table I for PDE III inhibi-
tion by compounds 5-28, which reflect previously
reported structure-activity relationships. For example,
as reported [lo] for inotropic activity in dogs for a
different range of phenyl sustituents R2, tricyclic pyri-
dazinones 2 and 3 here show levels of PDE III inhi-
bition similar to those of analogously substituted bi-
cyclic compounds 1 (compare the amino compounds
15, 19, acetamido compounds 16, 20 and methoxy
compound 21 with their bicyclic analogues 26-28). In
addition, within the series (table I) 5-9, 15-16, 17-21,
22-25, 26-28, which differ only in respect of R2,
compounds in which the phenyl ring substituent R2 is
acetamido, carboxamido or cyan0 show greater inhibi-
tory potency than the amino compound and reflect the
similar ranking of phenyl substituents with respect to
potency found for antihypertensive [ 1,2], platelet
aggregation inhibitory [4] and inotropic [lo] effects.
The PDE III inhibitory activity of the 4a-methyl-
benzocinnolinones 10-14 was relatively insensitive to
changes in the phenyl substituent R2 and rank order
could not be reliably established.
As expected [ 1,2,4-6,8] the bicyclic dihydro-5-
methylpyridazinones 24,25 showed significantly low-
er IC,, values than the dihydropyridazinones 26, 27.
However, most of the 4a-methylpyridazinones in
table I showed levels of PDE III inhibitory potency
similar to those of their normethyl analogues. Only for
the compound pair 12 and 19 was a significant but
marginal enhancement observed for a 4a-methyl com-
pound.
Of the compounds tested (6,8,9,11,13-16, N-21,
23-27), all except 21 showed evidence of sustained
inotropic activity. The structure-related trends seen in
the IC,, values for PDE III inhibition were also
present in the ED,, data for inotropic activity. Thus,
analogous tricyclic and bicyclic compounds showed
approximately equivalent inotropic potency (compare
8, 13, 16, 20 with 27 and compare 12, 15, 19 with 26)
and ranking of phenyl substituents with respect to
inotropic potency for PDE III inhibition. The enhanc-
ed inotropic potency of 5-methyl compounds in the
bicyclic series 24, 25 relative to their not-methyl
analogues 26, 27 was well defined, but there was no
evidence for a substantial difference in the inotropic
potency of 4a-methyl tricyclic compounds and their
not-methyl analogues. Overall, the ED,, values shown
in table I could be correlated with IC,, according to
equation 1 providing further support for the mediation
of inotropic activity by PDE III.
Log IC,, = 1.15 + 0.628 Log ED,, (1)
t-2 = 0.63, s = 0.3147, F = 23.7 , n = 16
The ED,, values for vasodilation correlated poorly
with the IC,, values for PDE III inhibition as shown in
equation 2, but a moderate level of correlation was
observed between ED,, values and ED,, values for
inotropic activity as shown in equation 3, indicating
association of these two properties.
Log ICsO = 1.20 + 0.636 Log ED,,
r2 = 0.34, s = 0.42, F = 7.35, rz = 16
(2)
Log EDso = 0.122 + 1.15 Log ED,,
(3)
r2 = 0.70, s = 0.36, F = 32.5, n = 16
Visual inspection of the results showed again that
analogous compounds in the tricyclic and bicyclic
series were approximately equiactive and the 6-aryl-
4,5-dihydro-5-methylpyridazinones 24 and 25 were
more potent than 26 and 27 respectively, but the other
structure-related trends seen for I& and EDso data
were barely discernable in the ECS values, nor were
any alternative structure-related trends apparent.
From the above results it can be seen that the struc-
ture-activity relationships of the tricyclic pyridazin-
ones 2 and 3 differ from those of bicyclic pyridazin-
ones 1 mainly in the effect of introducing a methyl
group at RI. Whilst in the bicyclic series 1 this struc-
tural change has been widely reported to lead to com-
pounds of significantly greater potency, as exemplifi-
ed here in the enhanced inotropic, vasodilator and
PDE III inhibitory effects of 24 and 25 over 26 and 27
respectively, the novel tricyclic 4a-methylpyridazin-
ones showed similar levels of potency to their nor-
methyl analogues for these 3 activities. The reasons
for this difference in behaviour are not clear. X-ray
[8], molecular modelling [8, 29, 301 and UV studies
have shown that the preferred conformation of the
aryldihydropyridazinones 1 and 4 is one in which the
alignment of the phenyl and pyridazinone rings is
essentially coplanar and that in 4 the methyl group at
770
Ri adopts a pseudo-axial position. In addition, the
inotropic and PDE III inhibitory activity of non-co-
planar pyridazinones generally has been found to be
reduced relative to comparable coplanar molecules
[5,7]. It is probable, therefore, that the coplanar
4a-methylindenopyridazinones and 4a-methylbenzo-
cinnolinones mimic in a rigid fashion the biologically
effective conformation (and 5-methyl group align-
ment) of the more active series 4.
Structure 47: X = NH or 0; n = 0,l or 2.
Conclusions
The PDE III inhibitory activity of 6-aryl-4,5-dihydro-
pyridazin-3(2H)-ones is enhanced about lo-fold by
the introduction of a methyl group at the Sposition,
but reduced when a larger substituent such as an ethyl
group is introduced [7], and activity is retained mainly
by the 5-R-enantiomer [29]. These observations have
given rise to the proposition that, in addition to the
PDE III binding interactions of the pyridazinone ring
amide and the phenyl substituent made by 6-aryl-
pyridazinone based PDE III inhibitors generally, a
5-pseudo-axial methyl group of 6-aryl-4,5-dihydro-5-
methylpyridazin-3(2H)-ones is able to make a unique,
affinity-enhancing, regiospecific interaction with a
hydrophobic pocket in the CAMP-ribose-accepting
region in the PDE III binding site [8, 29, 301. In
contrast, this work shows that the analogous introduc-
tion of a 4a-methyl group into the tricyclic com-
pounds 2 and 3 at Ri does not lead generally to any
significantly increased PDE III inhibitory potency.
The indenopyridazinones 2 and benzocinnolinones
3 show the overall planar topography associated with
high PDE III inhibitory potency in bicyclic 6-aryl-
pyridazinone derivatives. In addition, analogously
substituted bicyclic pyridazinones 1 and tricyclic pyri-
dazinones 2 and 3 show comparable levels of PDE III
inhibitory potency, which is modulated by changes of
phenyl substituent R* in a similar manner in all series.
Therefore, except in respect of the methyl group inter-
actions, the PDE III binding interactions of bicyclic
and tricyclic compounds are probably essentially
similar and the lack of a significant PDE III inhibitory
potency enhancing effect for the introduction of a
4a-methyl group into tricyclic pyridazinones is not the
result of a gross alteration in binding characteristics
between the 2 series. However, the high specificity
shown by 5-substituted 6-aryl-4,5-dihydro-pyridazin-
ones for the binding of a suitably oriented methyl
group only indicates that the requirements for access
to the hydrophobic pocket are highly critical and it is
possible that a small realignment of the position of the
potential binding groups of an inhibitor in the PDE III
binding site might be sufficient to deny efficient
access.
In tricyclic PDE III inhibitors, realignment is likely
to be caused by steric factors associated with the
central ring as this is the main feature distinguishing
the bicyclic and tricyclic series. Clearly, in 4,4a-di-
hydro-4a-methylindenopyridazinones, the strain im-
posed by a central 5-membered ring distorts overall
molecular topography and the relative positions of the
pyridazinone ring amide, the 4u-methyl group and the
phenyl ring substituent will differ from those of an
analogously substituted 6-aryl-4,5-dihydropyridazin-
one in a planar conformation. However, the central
ring in 4u-methylbenzocinnolinones is essentially un-
strained and the relative positions of the potential
enzyme binding moieties closely mimic those of a
coplanar bicyclic pyridazinone. Despite this close
correspondence, of the 4u-methyl-substituted benzo-
cinnolinones 10-14, only 12 shows significantly
greater PDE III inhibitory potency than its nor-methyl
homologue 19 and the degree of affinity enhancement
is much less than that seen between analogous bi-
cyclic homologues. An alternative and more robust
explanation, which can account for the absence of the
potency enhancing effect of a 4u-methyl group in both
the indenopyridazinone and benzocinnolinone series,
is that the central ring structure of a tricyclic molecule
encounters steric hindrance by a feature of the PDE III
binding site sufficient to cause a small realignment of
the bound molecule which is great enough to prevent
efficient access of the methyl group to the binding
pocket but too small to significantly alter the strength
of other binding interactions. Another more subtle
possibility is that although the 5-methyl group of
6-aryl-4,5-dihydropyridazinones contributes to affin-
ity by being regiospecifically accommodated in a
binding pocket, the greater part of its affinity-enhanc-
ing properties derive from an additional role in
damping or restricting the conformational freedom of
the 4,5-bond. Clearly, in conformationally restricted
tricyclic analogues, a 4u-methyl group would not
show this additional effect.
Experimental protocols
Melting points were determined on a Biichi capillary melting
point apparatus and are uncorrected. Analytical samples were
homogeneous by TLC performed on silica gel plates with
chloroform and methanol (9/l) as eluant. NMR spectra were
determined on Bruker AM 250 or AM 360 spectrometers and
IR spectra were recorded on a Perkin-Elmer Model 298 ins-
trument. All structural assignments are consistent with IR and
NMR spectra. Analyses indicated by the symbols of the
elements were within * 0.4% of the theoretical value.
7-Acetamido-4a-dihydro-4a-methyl-SH-indeno[l,2-c]pyrid-
azin-3(2H)-one 16
Aqueous formaldehyde solution (37% w/v; 5.1 ml; 63 mmol),
dimethvlammonium chloride (8.0 a: 98 mmol) and cone HCI
(2 drops) were stirred at room temperature for5 min and then
added to acetic anhydride (35 ml). After 20 min vigorous
boiling occurred. 5-Acetamido- 1 indanone [ 131 ( 11.5 g;
61 mmol) was then added and the mixture was stirred at 100C
for 1 h. Concentration gave a crude product which was treated
with acetone (100 ml) for 3 min then the mixture was re-
concentrated. Water (150 ml) was added to the residue and
unreacted ketone was extracted with dichloromethane. The
aqueous layer was basified with dimethylamine and extracted
with dichloromethane (100 ml). The organic layer was evapor-
ated and the residue was partitioned between 2 M HCI and
dichloromethane. Concentration of the aqueous layer gave
needles of 5-acetamido-2-dimethylaminomethyl-1-indanone
hydrochloride 35 (7.6 g; 44%), mp 182-3C (from 2 M HCI),
anal ClaHIRNT07 (C, H, N). Compound 35 (6.68 g; 24 mmol)
in methanol (50 ml) and water (25 ml) was added to a stirred
solution of KCN (7.9 P: 122 mmol) in methanol (80 ml) and
water (10 ml) at 6OC~Conc HCI was added to pH 7 and the
mixture stirred under reflux for 30 min. A further portion of
KCN (3.0 g; 46 mmol) was added followed by cone HCl to
restore pH 7. After 20 min the methanol was removed under
reduced pressure and cooling the deposited 5-acetamido-2-
cyanomethyl-1-indanone 33 (5.0 g; 93%), mp 182C remelts
209C (from I-propanol), anal C,3H,ZN202 (C, H, N). Com-
pound 33 (5.0 g; 22 mmol) was added in portions over 5 min
to a stirred mixture of cone H2S04 (10.0 ml) and glacial acetic
acid (10 ml). The solution was heated to 50C for 15 min then
poured onto ice (100 g). The mixture was stirred for 1 h and
the solution slowly precipitated 5-acetamido-l-oxo-2-indanyl-
acetamide 38 (3.16 a: 58%). mu 2368C (from water). anal
C,,H,,N,03 (C: H, NTA mixture of 38 (3.15 g; 13 mmol) and
hydrazine hydrate (1.56 ml; 31 mmol) in 50% aqueous AcOH
(28 ml) was stirred under reflux for 30 min. Cooling and fihra-
tion gave 16 (1.6 g; 5 l%), mp > 300C (from aq acetic acid).
S-Acetamido-4,4a,5,6-tetrahydrobenzo~]cinnolin-3(2H)-one 20
Compound 20 was prepared from 6-acetamido-l-tetralone [ 141
in a manner similar to that described for the preparation of 16,
except that the Mannich base 29 was not isolated but treated
with an excess of Mel in acetone to give (6-acetamido-l-oxo-
1,2,3,4-tetrahydronaphth-2-yl)trimethylammonium iodide 30
(60%; mp 168-70C (from acetone), anal C,6H2,N,01 (C, H,
N)). Compound 30 was further converted as described for
intermediate 29 to give (6-acetamido- 1 -oxo-tetrahydronaphth-
2-yl)acetonitrile 31 (61%; mp 189-90C (from aq acetone),
anal Ci4Hi4N202 (C, H, N)), (6-acetamido-1-oxo-1,2,3,4-tetra-
hydronaphth-2-yl)acetamide 37 (98%; mp (from water), anal
C,,Hr6N,03 (C, H, N)) and compound 20 (12%; mp 292C
(from aq ethanol)).
8-Methoxy-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3(2H)-one 21
In a manner similar to the preparation of 16, 6-methoxy-l-
tetralone was converted by way of 2-dimethylaminomethyl-6-
771
methoxy-1-oxo-1,2,3,4-tetrahydronaphthalene 34 (90%; mp
170C (from aq acetone), anal C,,H,9N0,.HCl (C, H, N, Cl-)),
into (6-methoxv-l-oxo-tetrahvdronauhth-2-vl)acetonitrile 32
(90%; mp 90C*(from methanol-hex&e), anal C,,H,,NO, (C,
H, N)). Treatment of the nitrile 32 with an excess of 5 M HCl
for 6 h and collection of the precipitate gave the carboxylic
acid 36 (76%; mp 16556C (by reprecipitation from
NaHCO,). Compound 36 (2.0 g; 8.5 mmol) and hydrazine
hydrate 2.0 ml; 40 mmol) were stirred and heated under reflux
in 50% aqueous acetic acid (50 ml) for 2 h. Cooling and
collection of the precipitate gave 21 (54%; mp 198C (from aq
ethanol)).
8-Cyano-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3(2H)-one[9] 17
Compound 31 (17.0 g; 65 mmol) in 10% H$O, (300 ml) was
heated under reflux for 3 h to give a solution of 6-amino-l-
oxo-1,2,3,4-tetrahydronaphthyl acetic acid. NaNO, (5.0 g;
73 mmol) in water (20 ml) was added dropwise at &5C and
the resulting mixture was added to a solution of CuCN (15.0 g)
and KCN (25.0 g) in water (150 ml) at 40C. When effer-
vescence had ceased the reaction mixture was extracted with
chloroform containing about 20% methanol. Evaporation of the
filtered extract gave 6-cyano-1-0x0-1,2,3,4-tetrahydronaphth-
2-yl acetic acid 39 (9.1 g; 61%), mp 205-10C (by reprecipita-
tion from NaHCO?), anal C,,H,,N03 (C, H, N)). Treatment of
39 (5.0 g; 22 mmol) with hydrazine hydrate (1.0 g; 20 mmol)
in refluxing 50% aqueous acetic acid gave compound 17
(1.2 g; 24%) , mp 292-5C (from DMF-ethanol)).
8-Cyano-4a-methyl-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3-
(2H)-one 10
Compound 39 (8.0 g; 35 mmol) was added to a stirred sus-
uension of NaH (1.8 P: 75 mmol) in drv DMF (100 ml) at
20-3OC. When effervescence had ceased, Me1 (26 g;
141 mmol) was added and stirring was continued for a further
2 h. Water was added cautiously to the cooled reaction mixture
to destroy the excess of NaH, then the reaction mixture was
partitioned between water (200 ml) and dichloromethane
(300 ml). The organic layer was evaporated and the residue
fractionated on a silica column using dichloromethane-methan-
01 mixtures of increasing polarity as eluant. Evaporation of
the appropriate fraction and trituration of the residue with
pentane gave methyl (6-cyano-2-methyl-1-oxo-1,2,3,4-tetra-
hydronaphth-2-yl)acetate 41 (4.0 g; 44%), mp 117C (from
ether-hexane), anal C,,H,,N03 (C, H, N). A suspension of
compound 41 (4.0 g) in 2 M HCl (100 ml) and glacial acetic
acid (20 ml) was refluxed for 3 h, concentrated, then extracted
with chloroform. Back extraction with a solution of NaHCO,
and acidification (HCI) of the aqueous layer gave 6-cyano-
2-methyl-l-oxo-l,2,3,4-tetrahydronaphth-2-yl)acetic acid 43
(3.53 g; 93%), mp 209-11C (by reprecipitation from
NaHCO,), anal C,4H13N03 (C, H, N). Compound 43 (3.5 g;
14 mmol) and hydrazine hydrate (2.0 ml; 40 mmol) were stirr-
ed and refluxedin 30 % aqueous acetic acid for 2 h. Cooling
gave a ureciuitate of comuound 10 (2.5 g: 73%: mu 302C
Tfrom DMF-ethanol)). 1
\ 01 , 1
S-Methoxy-4a-methyl-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3-
(2H)-one 14
As in the preparation of 10, compound 36 was transformed to
the ester 40, which was not purified, but hydrolysed to give
(6-methoxy-1-oxo-tetrahydronaphth-2-yl)acetic acid 42 (32%;
772
mp 139-4OC (from 1-propanol), anal C14H1604 (C, H)).
Treatment of 42 with hydrazine hydrate gave compound 14
(91%; mp 215C (from aq ethanol)).
7-Cyano-4,4a-dihydro-4a-methyl-SH-indeno[l,2-c]pyridazin-
3(2H)-one 5
Anhydrous potassium carbonate (5.45 g; 0.04 mol), para-
formaldehyde (14.1 g; 0.47 mol) and 4-bromopropiophenone
(100 g; 0.47 mol) in methanol (400 ml) were stirred at room
temperature for 3 h to give a clear solution. Further para-
formaldehvde (20.8 P: 0.69 mol) was added and the mixture
was allowed to Stan; overnight. Dilution with water (2 l),
extraction with chloroform and evaporation of the extract gave
an oil containing 4-bromo-1-(2-hydroxymethyl)propionyl-
benzene, 4-bromo-1-(2-methoxymethyl)propionylbenzene and
l-(4-bromophenyl)prop-2-ene-l-one. This mixture was added
with cooling to cone H,S04 (300 ml), stirred at 0C for 4 h,
allowed to stand at room temperature overnight, then poured
into water (2 1). Extraction with chloroform and evaporation of
the extract gave 112 g of a resin which was extracted with hot
hexane (300 ml). The hot extract was charcoaled, allowed to
cool, filtered through diatomaceous earth and evaporated to
give 48 g of an oil. Distillation gave a fraction boiling
165-70C which was triturated with pentane to give 5-bromo-
2-methylindan-l-one 44 (22.0 g; 21%, mp 55C anal
&,H,BrG (C, H)). A mixture of 44 (2.0 g; 9 mmol) and CuCN
(4.0 g; 44 mmol) in quinoline (50 ml) was stirred at 200C for
2 h then cooled. treated with chloroform (100 ml) and filtered
to remove copper salts. The filtrate was washed with 6 M HCl
(3 x 100 ml) and evaporated to a brown gum which was crys-
tallized from aqueous ethanol to give 5-cyano-2-methylindan-
l-one 46 (1.45 g; 95%) mp 90-1C anal C,,H,NO (C, H).
Compound 46 (7.89 g; 46 mmol) was added during 5 min to a
stirred suspension of NaH (1.38 g; 76 mmol) in dry DMF
(38 ml). After 20 min, when hydrogen evolution had ceased,
ethyl bromoacetate (7.0 g; 42 mmol) was added dropwise
durmg 5 min. Stirring was-continued for a further 20 min; then
the mixture was poured into ice-water (200 ml) and acidified
with cone HCl to pH 5.0. The suspension was extracted with
chloroform and the extract evaporated at lOOC/2.0 mm to
leave 7.3 g of a brown oil containing about 30% of the starting
indanone and 70% ethvl (5-cvano-2-methvl-1-oxoindan-2-v&
acetate. Without furthe; purification, the mixture was dissol;ed
in hot 50% aqueous acetic acid (50 ml), treated with hydrazine
hydrate (5.0 ml; 100 mmol) and stirred under reflux for 24 h.
Partial evaporation gave a syrup containing an orange solid
which was treated with ethanol to give a precipitate of 5 (1.3 g;
13% (from 46)), mp 254C (from aq ethanol).
4,4a-Dihydro-7-methoxy-4a-methyl-5H-indeno[l,2-c]pyri-
dazin-3(2H)-one 9
In a similar manner to the preparation of 5, 4-methoxypropio-
phenone was converted into 5-methoxy-2-methylindan- 1 -one
45 (18.7%; mp 66-8C (from hexane) anal C,,H,,02 (C, H)),
which in turn was converted into 9 (23%; mp 160-1C (from
ethanol)).
7-Carhoxamido-4,4a-dihydro-4a-methyl-5H-indeno[l,2-c]-
pyridazine-(2H)-one 6
Finely divided 5 (4.1 g) was sprinkled into rapidly stirred cone
H,SO, (50 ml) at 40C. After all solid had dissolved the
mixture was stirred for 5 min then poured onto ice (2.5 kg).
Filtration gave 6 (3.4 g; 77%), mp 3067C (from aq ethanol).
7-Amino-4,4a-dihydro-4a-methyl-SH-indeno[l,2-c]pyridazin-
3(2H)-one 7
Bromine (1.57 g; 9.8 mmol) was added to a solution of NaOH
(1.5 g) in water (10 ml) at 0C. Compound 6 (1.31 g;
5.4 mmol)) was added followed, after 1 min, by further NaOH
(1.1 g) in water (10 ml). The mixture was heated rapidly to
80C for 2 min, then cooled and acidified with cone HCl. The
filtered solution was basified (NaOH soln) to give a gum
containing 7 and ring brominated by-products. The gum was
dissolved in ethanol (50 ml). treated with 2 M NaOH 12.5 ml)
and hydrogenated at40 psiin the presence of 10% palladium
on carbon until no further uptake occurred. Concentration of
the filtered solution and addition of water gave 7 (0.5 g; 43%),
mp 222C (from aq ethanol).
7-Amino-4,4a-dihydro-SH-indeno[l,2-c]pyridazine-3(2H)-one
[IO] I5
Compound 8 (1.50 g) was suspended in hydrazine hydrate
(30 ml) and heated under reflux with ethanol (5 ml) for 2 h.
The solution was filtered through diatomaceous earth, allowed
to cool, then extracted with chloroform. The combined extracts
were dried (MgSO,) and concentrated. Recrystallization of the
crude product gave 15 (0.41 g; 33%), mp 244-5C (from
1 -propanol).
8-Acetamido-4a-methy1-4,4a,5,6-tetrahydrobenzo[h]cinnolin-
3(2H)-one 13
A suspension of compound 12 (300 mg; 1.3 mmol) in water
(20 ml) was treated with cone HCl then 2 M NaOH to give a
turbid solution at pH 4. Acetic anhydride (1 ml; 9.6 mmol)
was added and the mixture was stirred for 2 h, cooled in ice-
water and filtered to afford 13 (150 mg; 42%), mp 305C
(from ethanol).
Inhibition of phosphodiesterases
Three peaks of cyclic nucleotide phosphodiesterase activity
[PDE (peak I), PDE (peak II) and PDE (peak III)] from cat
heart were separated by chromatography on DEAE-Sepharose
CL-6B [(diethylamino)ethyl]cellulose with a bead size of
45-165 pm) [31]. The high-speed supematant from a cat heart
homogenate tissue (2 9) in 20 ml of 20 mM PIPES Iuinera-
zine-i?-Nl-bis(2-ethane&lfonic acid)], 50 mM sodium a&ate,
pH 6.5) was applied to a 15 x 1.5 cm column of DEAE-
Sepharose equilibrated with the homogenisation buffer.
The PDE activities were eluted with a gradient of 0.05-l M
sodium acetate in 20 mM PIPES. The elution profile is shown
in figure 1. There were 3 major peaks which had the following
characteristics: PDE (peak I) had high affinity for cyclic AMP
and cyclic GMP and- was characte&ed by-an activation by
Caz+/calmodulin comnlex: PDE (ueak II) demonstrated relativ-
ely .high affinity fbr iAMP and was not affected by
Ca2+/calmodulin complex. This activity was characterised as
being potently inhibited by rolipram (4-[3-(cyclopentyloxy)-4-
methoxyphenyll-2-pyrrolidinone) [ 171; PDE (peak III) had
high affinity for CAMP. It could also hydrolyse cGMP though
the preferred substrate was CAMP. Cyclic-GMP was a potent
inhibitor of the hydrolysis of CAMP. The activity was also
insensitive to Ca2+jcalmodulin activation.
The enzvmes were assaved bv incubation at 37C for
4-30 min in 50 mM Tris, -5 mM MgCl,, pH 7.5 with 3H-
labelled cyclic nucleotide (4 x 105 disintegrations min-1) and
773
I
A
I\
AA
I
A
i
A
I
Fraction Number
Fig 1. Elution profile of cat cardiac ventricle PDE. Activity
peaks were labelled 1, II and III in order of elution from the
DEAE-Sepharose column (see Experimental protocols).
Fractions were assayed using 1 PM CAMP as the substrate.
14C-labelled nucleotide 5-monophosphate (3 x 10s disinteg-
rations mint). The assay was stopped by boiling, and the 3H-
labelled 5-monophosphate product separated from substrate on
boronate columns [32]. The reaction mixture was diluted with
0.5 ml 100 mM HEPES [N-(2-hydroxyethyl)piperazine-N-2-
ethanesulfonic acid], 100 mM NaCl, pH 8.5, and applied to the
column. The column was extensively washed with the same
buffer, and the 5-nucleotide eluted with 6 ml of 0.25 M acetic
acid. The recovery of product as judged by t4C-recovery was
approximately 80%. All assays were linear with time of incu-
bation and concentration of enzyme over the range used in
these experiments.
IC,, values (the concentration of inhibitor required for 50%
inhibition of activity) were obtained for PDE (peak III) by
incubation of the enzyme at 1 PM CAMP, and a range of inhi-
bitor concentrations from 0.1 x IC,,, to 100 x IQ,.
Anaesthetised cat screen
Cats of either sex, weighing betwen 1.9 and 2.5 kg, were
anaesthetized with sodium pentobarbitone (Sagatal) 60 mg/kg
ip. The trachea was cannulated to maintain a free passage of
air. Blood pressure was recorded from a brachial artery, us&g a
Bell and Howell Tvue 4-422 nressure transducer connected
through a pressure preamplifier io a recorder. The blood press-
ure signal was also used to trigger a heart rate meter. A
brachial vein was cannulated for administration of drugs. The
abdomen was opened and the descending aorta, caudal to the
renal arteries, was cleared of surrounding tissue, and the in-
ferior mesenteric artery was tied off. Prior to cannulation the
preparation was left for 3/4 h to allow small broken vessels to
seal and then 500 units/kg iv of heparin was administered to
prevent blood clotting in the extracorporeal system. The
descending aorta was cannulated toward the heart to receive
blood, and away from the heart toward the hindquarters to
return blood by means of a Watson-Marlow peristaltic pump.
The hindquarters were perfused at a constant flow rate, such
that the pressure closely matched the mean systemic blood
pressure, allowing calculation of hindquarter vascular resis-
tance.
Left ventricular pressure was recorded via a polythene
cannula introduced down the right carotid artery and manipu-
lated through the semilunar valves into the left ventricle. The
pressure signal was electronically differentiated to give a con-
tinuous linear record of dLVP/dt,,, which was taken as an
index of contractility.
All animals were pretreated with 5 mg/kg pempidine (a
ganglion blocking agent) to prevent reflex activity and produce
a stable preparation with a lowered heart rate, blood pressure,
and contractile state, which was sensitive to isoprenaline given
in a range of doses from 0.01 to 0.1 pg/kg iv and any cat not
responding with an increase in contractility of at least 100%
was rejected. Propranolol, a fl-adrenoreceptor antagonist, was
then given at a dose 1 mg/kg iv plus 3-4 mg/kg SC. When the
preparation was stable, the test compound was given as a bolus
intravenous injection and the time course of the response was
followed for at least 1 h.
Acknowledgments
The authors gratefully acknowledge the contributions made to
this work by JC Emmett, ST Flynn and CM Whittaker.
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