Journal Pre-proofs

Benzoxepinones: A new isoform-selective class of tumor associated carbonic

anhydrase inhibitors

Aiga Grandane, Alessio Nocentini, Thomas Werner, Raivis Zalubovskis,

Claudiu T. Supuran

PII: S0968-0896(20)30317-5
DOI: https://doi.org/10.1016/j.bmc.2020.115496
Reference: BMC 115496

To appear in: Bioorganic & Medicinal Chemistry

Received Date: 18 February 2020

Revised Date: 31 March 2020
Accepted Date: 5 April 2020

Please cite this article as: A. Grandane, A. Nocentini, T. Werner, R. Zalubovskis, C.T. Supuran, Benzoxepinones:
A new isoform-selective class of tumor associated carbonic anhydrase inhibitors, Bioorganic & Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115496

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Benzoxepinones: A new isoform-selective class of tumor associated carbonic
anhydrase inhibitors
Aiga Grandanea,*, Alessio Nocentinib, Thomas Wernerc,*, Raivis Zalubovskisa,d, Claudiu T.
Supuranb,*

aLatvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia

bUniversità degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical
Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
cLeibniz Institute for Catalysis, Albert-Einstein-Straße 29a, 18059 Rostock, Germany
dInstitute of Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry,
Riga Technical University, 3/7 Paula Valdena Str., Riga LV-1048, Latvia

Abstract
Benzoxepinones (“homocoumarins”) are identified as a new class of selective inhibitors for
tumor associated human carbonic anhydrases (hCA, EC 4.2.1.1) isoforms IX and XII. Similar to
coumarins, they do not inhibit or poorly inhibit cytosolic human (h) isoforms hCA I and II, but act as
nanomolar inhibitors of the trans-membrane, tumor associated isoforms hCA IX and XII.

Keywords: Carbonic anhydrase; Benzoxepinone; Selective inhibitor; Tumor; Catalytic Wittig

reaction

1. Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes which catalyze a

reversible hydration of carbon dioxide to a bicarbonate anion and a proton, controling the optimal pH
in cells as a main function.1 15 α-CA isoforms have been identified in humans (h). Notably, hCA IX
and XII are overexpressed in hypoxic cancer cells, therefore this two hCA isoforms are defined as
drug targets.2 So far, there are several selective classes of hCA IX and XII inhibitors known. In this
regard sulfonamides incorporating triazine moieties,3 coumarins,4 thiocoumarins,4b,5 2-
thioxocoumarins,5 selenium or tellurium heterocoumarins,6 sulfocoumarins7 and lately sulfonamide
derivatives were reported, where selective inhibition of CAs was achieved by tail approach.8
Recently, 3H-1,2-benzoxathiepine 2,2-dioxides (Fig. 1) were reported as a new class of isoform-
selective tumor associated hCA IX and XII inhibitors.9
 Benzoxepinones can be considered as bioisosteres of 3H-1,2-benzoxathiepine 2,2-dioxides
where sulfonyl group was replaced by carbonyl functional group. Their straightforward synthetic
methodology has been reported recently by one of our groups.10 However, their CA inhibitory
properties were not explored. Here we report CA inhibition data of benzoxepinones for
transmembrane, tumor associated hCA IX and XII, as well as for the off-targets, widely spread
cytosolic isoforms hCA I and II.

S O
O O O
O
Coumarin Sulfocoumarin

O S O O
O O
3H-1,2-benzoxathiepine 2,2-dioxide Benzoxepinone

Fig. 1 Structure of CA inhibitors.

2. Results and discussion

2.1. Chemistry

Benzoxepinones 4a-l were synthesized according literature method described previously.10

The key intermediates 2-formylphenyl fumarates 3 were prepared in a two step procedure, where first
commercially available mono-methyl fumarate (1) was converted to acid chloride 2 by treatment with
presence of SOCl2 (Table 1). Subsequent acylation of 2-hydroxybenzaldehyde derivatives with 2 gave
esters 3a-l in good yields (61–99%, Table 1, entries 1–12). The series of target compounds
benzoxepinones 4 were obtained from appropriate 2-formylphenyl fumarates 3 by intramolecular
base-free catalytic Wittig reaction (Table 1). A commercially available methyl-1-phenyl-2-
phospholen 1-oxide (5, 5 mol%) was used as a pre-catalyst in the presence of benzoic acid (5 mol%)
as co-catalyst and trimethoxy silane as a reducing agent. The desired benzoxepinones 4a-l containing
unsubstituted core (entry 1) and electron-donating substituents (entries 2–7), as well as electron-
withdrawing substituents (entries 8–12) were isolated in a moderate yields (27–44%).

Table 1. Synthesis of benzoxepinones 4 via intramolecular catalytic Wittig reactions.

 O
R1

R2 OH O CO2Me
R3 R1 R1
O O O
(i) (ii) (iii)
HO CO2Me Cl CO2Me R 2
O CO2Me R2 O
R 3
R3 O
1 2, 99% 3 4

Entry R1 R2 R3 Product 3 Yield 3/ % Product 4 Yield 4/ %

1 H H H 3a 97 4a 36
2 H H OMe 3b 87 4b 29
3 H OMe H 3c 68 4c 44
4 OMe H H 3d 81 4d 35
5 H Me H 3e 86 4e 36
6 Me H H 3f 96 4f 40
7 t-Bu H H 3g 90 4g 34
8 H NEt2 H 3h 73 4h* 27
9 H benzo 3i 61 4i 43
10 H Cl H 3j 69 4j 32
11 Cl H H 3k 64 4k 42
12 CO2Me H H 3l 99 4l 31

Reagents and conditions: (i) SOCl2, DMF (cat.), CH2Cl2, reflux, 3 h; (ii) NEt3, CH2Cl2, RT, 16 h; (iii)
methyl-1-phenyl-2-phospholen 1-oxide (5, 5 mol%), benzoic acid (5 mol%), (MeO)3SiH, toluene,
100 °C, 16 h.

*R2=NEt2·HCl

A plausible mechanism of the catalytic Wittig reaction starts with a reduction of the oxidized
form of the catalyst methyl-1-phenyl-2-phospholen 1-oxide (5) forming phospholene 6 (step A,
Scheme 2).10, 11 The next step is a Michael addition of 6 to the α,β-unsaturated carbonyl compound 3
forming enolate 7 (step B). Subsequently ylide 8 is formed by a protonation/deprotonation sequence
(step C). Then an intramolecular Wittig reaction between aldehyde moiety and ylide leads to
formation of benzoxepinone 4 and 5 the oxidized form of the catalyst which can enter another
catalytic cycle (step D).
 CO2Me
O Ph
R 2 HSi(OMe)3
P
O
O
4 O
Me (MeO)3Si Si(OMe)3
D 5 A
Me Ph
O P

O P
R Ph Me
O CO2Me 6 O

8 O
R
C B
Me O CO2Me
O 3
O P
R Ph
O CO2Me
7

Scheme 2. Plausible mechanism for intramolecular base-free catalytic Wittig reaction.

2.2. CA inhibition

Benzoxepinones 4a-l were screened for the inhibition of four human CA isoforms - the
cytosolic, widespread hCA I and hCA II (off-targets in this case) as well as transmembrane tumor-
associated hCA IX and hCA XII which are anticancer drug targets.12–15 Inhibition data of
benzoxepinones 4a–4l (as well as the sulfonamide acetazolamide AAZ, as standard) against hCA I,
II, IX and XII, after 6 h of incubation period of the enzyme and inhibitor solutions have been collected
(Table 1).16 It should be noted that assaying the inhibition with the 15 min incubation period (as for
the sulfonamides)17 leads to very weak inhibition (data not shown). This was also observed in the
case with the coumarins.12,18–21 Hence, a 6 h incubation period has been used for assaying all
benzoxepinones as CA inhibitors.
Data presented in Table 1 represent the following SAR: the cytosolic isoforms hCA I and II
were generally not inhibited by the investigated benzoxepinones which in most cases showed
inhibition constants >10 µm. Benzoxepinones 4d, 4g, and 4k containing 7-methoxy, 7-tetrt-butyl or
7-chloro substituents demonstrated weak inhibitory activities of hCA II (KI=67–86 µm).
Acetazolamide, the sulfonamide CA inhibitor in clinical use, shows a low nanomolar inhibitory
activity of hCA II, and also significantly inhibits hCA I (KI=250 nM).
The transmembrane, tumor associated hCA IX was effectively inhibited by benzoxepinones
with inhibition constants in the range of 20.0–302.7 nM (Table 1). The weakest hCA IX inhibitors
were benzoxepinones 4e and 4i containing 8-methyl substituent or naphthyl substituent as well as
unsubstituted benzoxepinone 4a (KI in the range of 171.1–302.7 nM). Slightly better inhibition was
observed for 8-substituted benzoxepinones 4c, 4h, and 4j containing methoxy, diethylammonium
chloride or chloro functional groups (KI in the range of 78.0–93.4 nM), as well as for 7-methyl
substitued benzoxepinone 4f with KI=71.7 nM. Highly effective hCA IX inhibitors were
benzoxepinones 4b, 4d, 4g, and 4l (KI in the range of 37.3–46.3 nM). They incorporate 9-methoxy
substituent or substituents in 7th position like methoxy, tert-butyl or acyl functional groups. The most
effective hCA IX inhibitor in benzoxepinone series reported here was 7-chloro substituted compound
4k with KI=20.0 nM. Notably, this inhibitory activity is comparable with acetazolamide (AAZ, KI=25
nM).
The second transmembrane isoform, hCA XII, was also effectively inhibited by
benzoxepinones reported here with KIs in the range of 12.9–466.3 nM (Table 1). The weakest
hCA XII inhibitors were benzoxepinones 4c, 4e, 4h, 4i, and 4a (KI in the range of 184.1–466.3 nM).
They incorporated 8-methoxy, 8-methyl, 8-diethylammonium chloride substituents, naphthalene
moiety as well as unsubstituted benzoxepinone. Slightly more effective hCA XII inhibitor was 8-
chloro substituted benzoxepinone 4j (KI= 68.7 nM). High inhibitory activities were demonstrated by
benzoxepinones 4b, 4d, and 4f (KI in the range of 34.7–48.0 nM) containing 9-methoxy, 7-methoxy,
and 7-methyl substituents, respectively. Gratifyingly, excellent inhibitory activities were observed
for benzoxepinones 4g, 4k, 4l, and 4k. Benzoxepinones containing 7-tert-butyl (4g) and 7-acyl (4l)
moieties showed KI in the range between 26.8 and 25.1 nM, respectively. Furthermore, the most
effective hCA XII inhibitor by benzoxepinones reported here was 7-chloro substituted compound 4k
(KI=12.9 nM). It should be also noted that acetazolamide is a very effective hCA XII inhibitor, but
as mentioned earlier, it is a promiscuous CA inhibitor, significantly inhibiting most of the 15 hCA
isoforms.1a,22,12 Although the CA inhibitory mechanism of benzoxepinones is currently unknown, we
postulate that it could be similar to coumarins, i.e. lactone ring opening by hydrolysis to the
corresponding 4-phenyl-3-butenoic acids which thereafter anchor to the zinc-coordinated water
molecule within the enzyme active site.23

Table 2: Inhibition data of human CA isoforms hCA I, II, IX and XII by compounds 4a-l and the
standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
CO2Me O
R O N N
S NH2
N S
H O
O
4a-l O AAZ

KI* (nM)

Cmpd R hCAI hCA II hCA IX hCA XII

4a H >100000 >100000 254.0 352.6

4b 9-OMe >100000 >100000 46.3 39.8
4c 8-OMe >100000 >100000 83.8 156.2
4d 7-OMe >100000 66818 40.2 34.7
4e 8-Me >100000 >100000 171.1 184.1
4f 7-Me >100000 >100000 71.7 48.0
4g 7-tBu >100000 85680 37.3 26.8
4h 8-NEt2·HCl >100000 >100000 93.4 156.1
4i 8,9-benzo >100000 >100000 302.7 466.3
4j 8-Cl >100000 >100000 78.0 68.7
4k 7-Cl >100000 76195 20.0 12.9
4l 7-COOMe >100000 >100000 39.3 25.1
AAZ - 250 12 25 5.7

*Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5–10 % of the
reported values).

3. Conclusions

In conclusion, we report here benzoxepinones as a new class of tumor associated carbonic

anhydrases IX and XII isoform-selective inhibitors. Benzoxepinones were obtained in a
straightforward synthesis from 2-hydroxybenzaldehydes using an intramolecular base-free catalytic
Wittig reaction. The title compounds were investigated for the inhibition of four hCA isoforms with
medicinal chemistry applications, the cytosolic hCA I and II, and the transmembrane, tumor-
associated hCA IX and XII. Benzoxepinones generally do not inhibit the ubiquitous, off-target
cytosolic isoforms hCA I and II, but showed selective inhibition of the two transmembrane hCAs,
with KI ranging between 20.0 and 302.7 nM against hCA IX, and between 12.9 and 466.3 nM against
hCA XII. As hCA IX and XII are validated anti-tumor targets, such isoform-selective inhibitors as
the benzoxepinones reported here, may be useful for identifying suitable drug candidates for clinical
trials.

4. Experimental
4.1. Chemistry
All reagents were purchased from commercial sources and used as received without further
purification. Thin layer chromatography was performed on Merck TLC-plates with fluorescence
indication (silica type 60, F254), spots were visualized using UV-light or KMnO4 stains. Flash
chromatography was performed using silica with a grain size of 40–63 µm from Macherey-Nagel.
Deuterated chloroform was purchased from Deutero. NMR spectra were recorded on Bruker 300
Fourier, Bruker AV 300 and Bruker AV 400 spectrometers. The chemical shifts () for 1H in CDCl3
are given in parts per million (ppm) and referenced to 7.26, respectively. Coupling constants are
expressed in Hertz (Hz). The following abbreviations are used: s= singlet, d= doublet, t= triplet, q=
quadruplet, m= multiplet.

Methyl 3-(chloroformyl)acrylate (2).10 Monomethyl fumarate (1, 5.00 g, 38.4 mmol) was dissolved
in CH2Cl2 (125 mL). After DMF (2 drops) was added the solution was cooled to 0 ºC and thionyl
chloride (3.1 mL, 42.3 mmol) was added dropwise. The resulting mixture was refluxed for 3 h. The
solvent was distilled off, afford the title compound as a yellow oil (5.6 g, 37.7 mmol, 99%). 1H NMR
(300 MHz, CDCl3) δ: 3.86 (s, 3H), 6.96 (d, J = 15.5 Hz, 1H), 7.03 (d, J = 15.5 Hz, 1H).

4.1.1. General procedure 1 (GP 1) for the synthesis of fumarates 3a-l.

Appropriate benzaldehyde derivative (1 equiv) was dissolved in CH2Cl2 (5 mL·mmol–1) and methyl
3-(chloroformyl)acrylate (2, 1.1–2.0 equiv) was added dropwise. The solution was cooled to 0 °C and
NEt3 (1.6–2.0 equiv) was added dropwise. The resulting suspension was stirred at room temperature
for 16 h. The mixture was quenched with H2O (2.5 mL·mmol–1) and washed with saturated aq.
KHCO3 solution (2  2.5 mL·mmol–1). The organic layer was dried over Na2SO4. Volatiles were
removed under vacuum and the crude product was purified by column chromatography on silica gel
(cyclohexane:EtOAc).

2-Formylphenyl methyl fumarate (3a).10, 24 Obtained according GP1 from 2-hydroxybenzaldehyde

(500 mg, 0.44 mL, 4.09 mmol), methyl 3-(chloroformyl)acrylate (2, 668 mg, 4.50 mmol) and NEt3
(662 mg, 0.91 mL, 6.54 mmol). The crude product was purified by column chromatography on silica
gel (cyclohexane:EtOAc= 20:1) to yield a yellow oil (925 mg, 3.95 mmol, 97 %). Rf (SiO2,
cyclohexane:EtOAc= 3:1)= 0.32; 1H NMR (400 MHz, CDCl3, δ): 3.87 (s, 3H), 7.08 (d, 1H, J = 15.8
Hz), 7.13 (d, 1H, J =15.8 Hz), 7.23–7.26 (m, 1H), 7.43–7.48 (m, 1H), 7.65–7.70 (m, 1H), 7.92 (dd,
1H, J = 7.7, 1.7 Hz), 10.10 (s, 1H).

2-Formyl-6-methoxyphenyl methyl fumarate (3b).10 Obtained according GP1 from 2-hydroxy-4-

methoxybenzaldehyde (400 mg, 2.63 mmol), methyl 3-(chloroformyl)acrylate (2, 430 mg, 2.89
mmol) and NEt3 (426 mg, 0.59 mL, 4.21 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 5:1) to yield a colorless solid (605 mg, 2.29
mmol, 87%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.18; 1H NMR (300 MHz, CDCl3, δ): 3.86 (s,
3H), 3.37 (s, 3H), 7.08 (d, 1H, J = 15.8 Hz), 7.15 (d, 1H, J = 15.8 Hz), 7.22–7.26 (m, 1H), 7.34–7.41
(m, 1H), 7.46–7.50 (m, 1H), 10.11 (s, 1H).

2-Formyl-5-methoxyphenyl methyl fumarate (3c).10 Obtained according GP1 from 2-hydroxy-3-

methoxybenzaldehyde (400 mg, 2.63 mmol), methyl 3-(chloroformyl)acrylate (2, 430 mg, 2.89
mmol) and NEt3 (426 mg, 0.59 mL, 4.21 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 5:1) to yield a colorless solid (472 mg, 1.79
mmol, 68%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.18; 1H NMR (300 MHz, CDCl3, δ): 3.86 (s,
3H), 3.89 (s, 3H), 6.72 (d, 1H, J = 2.4 Hz), 6.93 (dd, 1H, J = 8.7, 2.4 Hz), 7.06 (d, 1H, J = 15.8 Hz),
7.12 (d, 1H, J = 15.8 Hz), 7.83 (d, 1H, J = 8.7 Hz), 9.93 (s, 1H).

2-Formyl-4-methoxyphenyl methyl fumarate (3d).10 Obtained according GP1 from 2-hydroxy-5-

methoxybenzaldehyde (400 mg, 2.63 mmol), methyl 3-(chloroformyl)acrylate (2, 430 mg, 2.89
mmol) and NEt3 (426 mg, 0.59 mL, 4.21 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 5:1) and recrystallized from EtOH to yield a light
yellow solid (560 mg, 2.12 mmol, 81%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.29; 1H NMR (300
MHz, CDCl3, δ): 3.86 (s, 3H), 3.87 (s, 3H), 7.06 (d, 1H, J = 15.8 Hz), 7.12 (d, 1H, J = 15.8 Hz), 7.13–
7.21 (m, 2H), 7.36–7.38 (m, 1H), 10.07 (s, 1H).

2-Formyl-5-methylphenyl methyl fumarate (3e).10 Obtained according GP1 from 2-hydroxy-4-

methylbenzaldehyde, methyl 3-(chloroformyl)acrylate (2, 480 mg, 3.23 mmol) and NEt3 (476 mg,
0.66 mL, 4.70 mmol). The crude product was purified by column chromatography on silica gel
(cyclohexane:EtOAc= 5:1) to yield a yellow oil (625 mg, 2.52 mmol, 86%). Rf (SiO2,
cyclohexane:EtOAc= 3:1)= 0.34; 1H NMR (300 MHz, CDCl3, δ): 2.44 (s, 3H), 3.86 (s, 3H), 7.02–
7.04 (m, 1H), 7.06 (d, 1H, J = 15.8 Hz), 7.12 (d, 1H, J = 15.8 Hz), 7.21–7.26 (m, 1H), 7.75–7.82 (m,
1H), 10.02 (s, 1H).

2-Formyl-4-methylphenyl methyl fumarate (3f).10 Obtained according GP1 from 4-methyl-2-

hydroxybenzaldehyde (500 mg, 3.67 mmol), methyl 3-(chloroformyl)acrylate (2, 818 mg, 5.51
mmol) and NEt3 (594 mg, 0.82 mL, 5.87 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 3:1) to yield a yellow oil (875 mg, 3.52 mmol,
96%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.42; 1H NMR (300 MHz, CDCl3, δ): 2.43 (s, 3H), 3.85
(s, 3H), 7.06 (d, 1H, J = 15.8 Hz), 7.11 (d, 1H, J = 15.8 Hz), 7.12 (d, 1H, J = 8.2 Hz), 7.43–7.48 (m,
1H), 7.68–7.70 (m, 1H), 10.05 (s, 1H).

4-(tert-Butyl)-2-formylphenyl methyl fumarate (3g).10 Obtained according GP1 from 5-tert-butyl-2-

hydroxybenzaldehyde (450 mg, 2.52 mmol), methyl 3-(chloroformyl)acrylate (2, 561 mg, 3.78
mmol) and NEt3 (581 mg, 0.63 mL, 4.54 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 20:1) to yield a yellow oil (661 mg, 2.46 mmol,
90%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.45; 1H NMR (300 MHz, CDCl3, δ): 1.36 (s, 9H), 3.86
(s, 3H), 7.07 (d, 1H, J = 15.8 Hz), 7.13 (d, 1H, J = 15.8 Hz), 7.16 (d, 1H, J = 8.6 Hz), 7.68 (dd, 1H,
J = 8.6, 2.6 Hz), 7.90 (d, 1H, J = 2.6 Hz), 10.09 (s, 1H).

5-(Diethylamino)-2-formylphenyl methyl fumarate (3h).10 Obtained according GP1 from 4-

diethylamino-2-hydroxybenzaldehyde (500 mg, 2.59 mmol), methyl 3-(chloroformyl)acrylate (2, 576
mg, 3.88 mmol) and NEt3 (459 mg, 0.58 mL, 4.14 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 3:1) to yield a green oil (580 mg, 1.90 mmol,
73%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.21; 1H NMR (300 MHz, CDCl3, δ): 1.21 (t, 6H, J = 7.2
Hz), 3.42 (q, 4H, J = 7.2 Hz), 3.85 (s, 3H), 6.31 (d, 1H, J = 2.5 Hz), 6.57 (dd, 1H, J = 8.9, 2.5 Hz),
7.06 (d, 1H, J = 15.8 Hz), 7.12 (d, 1H, J = 15.8 Hz), 7.66 (d, 1H, J = 8.9 Hz), 9.73 (s, 1H).

2-Formylnaphthalen-1-yl methyl fumarate (3i).10 Obtained according GP1 from 1-hydroxy-2-

naphthylaldehyde (400 mg, 2.32 mmol), methyl 3-(chloroformyl)acrylate (2, 520 mg, 3.48 mmol)
and NEt3 (376 mg, 0.52 mL, 3.71 mmol). The crude product was purified by column chromatography
on silica gel (cyclohexane:EtOAc= 20:1) and recrystallized from EtOH to yield a colorless solid (400
mg, 1.41 mmol, 61%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.37; 1H NMR (300 MHz, CDCl3, δ):
3.90 (s, 3H), 7.22 (d, 1H, J = 15.8 Hz), 7.30 (d, 1H, J = 15.8 Hz), 7.50–7.71 (m, 2H), 7.85–7.97 (m,
4H), 10.25 (s, 1H).

5-Chloro-2-formylphenyl methyl fumarate (3j).10 Obtained according GP1 from 4-chloro-2-

hydroxybenzaldehyde (400 mg, 2.55 mmol), methyl 3-(chloroformyl)acrylate (2, 568 mg, 3.83
mmol) and NEt3 (476 mg, 0.57 mL, 4.08 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 5:1) to yield a colorless solid (474 mg, 1.91
mmol, 69%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.42; 1H NMR (300 MHz, CDCl3, δ): 3.87 (s,
3H), 7.07–7.11 (m, 2H), 7.30 (d, 1H, J = 1.9 Hz), 7.43 (dd, 1H J = 8.3, 1.9 Hz), 7.85 (d, 1H, J=8.3
Hz), 10.05 (s, 1H).
4-Chloro-2-formylphenyl methyl fumarate (3k).10 Obtained according GP1 from 5-chloro-2-
hydroxybenzaldehyde (500 mg, 3.19 mmol), methyl 3-(chloroformyl)acrylate (2, 712 mg, 4.79
mmol) and NEt3 (581 mg, 0.80 mL, 5.74 mmol). The crude product was purified by column
chromatography on silica gel (cyclohexane:EtOAc= 5:1) and recrystallized from EtOH to yield a
colorless solid (551 mg, 2.06 mmol, 64%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.37; 1H NMR (300
MHz, CDCl3, δ): 3.86 (s, 3H), 7.07–7.12 (m, 2H), 7.22 (d, 1H, J = 8.7 Hz), 7.61 (dd, 1H, J = 8.7, 2.6
Hz), 7.87 (d, 1H, J = 2.6 Hz), 10.05 (s, 1H).

2-Formyl-4-(methoxycarbonyl)phenyl methyl fumarate (3l).10 Obtained according GP1 from methyl-

3-formyl-4-hydroxybenzoate (400 mg, 2.22 mmol), methyl 3-(chloroformyl)acrylate (2, 494 mg, 3.33
mmol) and NEt3 (0.359 g, 0.50 mL, 3.55 mmol) as a light brown solid (3l, 646 mg, 2.21 mmol, 99%).
1H NMR (300 MHz, CDCl3, δ): 3.86 (s, 3H), 3.96 (s, 3H), 7.05–7.16 (m, 2H), 7.35 (d, 1H, J = 8.5
Hz), 8.32 (dd, 1H, J = 8.5, 2.2 Hz), 8.57 (d, 1H, J = 2.2 Hz), 10.11 (s, 1H).

4.1.2. General procedure 2 (GP 2) for the synthesis of benzoxepinones 4a-l. Fumarate derivative 3a-l
(1 equiv) was dissolved in anhydrous toluene (2 mL·mmol–1). To the solution 3-methyl-1-phenyl-2-
phospholen 1-oxide (5, 0.05 equiv.), benzoic acid (0.05 equiv) and (MeO)3SiH (3 equiv) were added
under argon. The resulting solution was stirred at 100 °C for 16 h. After cooling to room temperature
all volatiles were removed. The crude product was purified by column chromatography on silica gel
(cyclohexane:EtOAc gradient).

Methyl 2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4a).10, 25 Obtained according GP2 from

fumarate derivative 3a (300 mg, 1.28 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 12 mg,
0.064 mmol), benzoic acid (8 mg, 0.06 mmol) and (MeO)3SiH (469 mg, 0.48 mL, 3.74 mmol) as a
colorless solid (101 mg, 0.463 mmol, 36%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.34; 1H NMR
(400 MHz, CDCl3, δ): 3.44 (s, 2H), 3.88 (s, 3H), 7.26–7.31 (m, 2H), 7.41–7.51 (m, 2H), 7.87 (s, 1H).

Methyl 9-methoxy-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4b).10 Obtained according GP2

from fumarate derivative 3b (160 mg, 0.606 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 6
mg, 0.03 mmol), benzoic acid (4 mg, 0.03 mmol) and (MeO)3SiH (222 mg, 0.23 mL, 1.82 mmol) as
a colorless solid (44 mg, 0.178 mmol, 29%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.21; 1H NMR
(300 MHz, CDCl3, δ): 3.44 (s, 2H), 3.87 (s, 3H), 3.91 (s, 3H), 6.96–7.00 (m, 1H), 7.02–7.07 (m, 1H),
7.18–7.24 (m, 1H), 7.86 (s. 1H).
Methyl 8-methoxy-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4c).10 Obtained according GP2
from fumarate derivative 3c (200 mg, 0.757 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5,
7 mg, 0.04 mmol), benzoic acid (5 mg, 0.04 mmol) and (MeO)3SiH (278 mg, 0.29 mL, 2.27 mmol)
as a colorless solid (83 mg, 334 mmol, 44%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.27; 1H NMR
(400 MHz, CDCl3, δ): 3.43 (s, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 6.78 (d, 1H, J = 2.5 Hz), 6.85 (dd, 1H,
J = 8.7, 2.5 Hz), 7.32 (d, 1H, J = 8.7 Hz), 7.82 (s, 1H).

Methyl 7-methoxy-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4d).10 Obtained according GP2

from fumarate derivative 3d (300 mg, 1.14 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 11
mg, 0.06 mmol), benzoic acid (7 mg, 0.06 mmol) and (MeO)3SiH (416 mg, 0.43 mL, 3.41 mmol) as
a colorless solid (98 mg, 0.40 mmol, 35%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.29; 1H NMR (300
MHz, CDCl3, δ): 3.43 (s, 2H), 3.83 (s, 3H), 3.87 (s, 3H), 6.85 (d, 1H, J = 3.1 Hz), 7.01 (dd, 1H, J =
9.0, 3.1 Hz), 7.20 (d, 1H, J = 9.20 Hz), 7.82 (s, 1H).

Methyl 8-methyl-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4e).10 Obtained according GP2

from fumarate derivative 3e (300 mg, 1.21 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 12
mg, 0.06 mmol), benzoic acid (7 mg, 0.06 mmol) and (MeO)3SiH (444 mg, 0.46 mL, 3.63 mmol) as
a colorless solid (101 mg, 435 mmol, 36%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.32; 1H NMR
(400 MHz, CDCl3, δ): 2.42 (s, 3H), 3.42 (s, 2H), 3.87 (s, 3H), 7.07–7.11 (m, 2H), 7.28–7.31 (m, 1H),
7.84 (s, 1H).

Methyl 7-methyl-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4f).10 Obtained according GP2

from fumarate derivative 3f (300 mg, 1.21 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 12
mg, 0.06 mmol), benzoic acid (7 mg, 0.06 mmol) and (MeO)3SiH (444 mg, 0.46 mL, 3.63 mmol) as
a colorless solid (112 mg, 482 mmol, 40%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.39; 1H NMR
(300 MHz, CDCl3, δ): 2.38 (s, 3H), 3.42 (s, 2H), 3.87 (s, 3H), 7.16 (d, 1H, J = 8.5 Hz), 7.19–7.21 (m,
1H), 7.25–7.29 (m, 1H), 7.82 (s, 1H).

Methyl 7-(tert-butyl)-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4g).10 Obtained according

GP2 from fumarate derivative 3g (300 mg, 1.03 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5,
10 mg, 0.05 mmol), benzoic acid (6 mg, 0.05 mmol) and (MeO)3SiH (378 mg, 0.39 mL, 3.09 mmol)
as a colorless solid (96 mg, 0.35 mmol, 34%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.45; 1H NMR
(300 MHz, CDCl3, δ): 1.34 (s, 9H), 3.44 (s, 2H), 3.88 (s, 3H), 7.20 (d, 1H, J = 8.6 Hz), 7.38 (d, 1H,
J = 2.5 Hz), 7.50 (dd, 1H, J = 8.6, 2.5 Hz), 7.88 (s, 1H).

N,N-Diethyl-4-(methoxycarbonyl)-2-oxo-2,3-dihydrobenzo[b]oxepin-8-ammonium chloride (4h).10

Obtained according GP2 from fumarate derivative 3h (300 mg, 0.983 mmol), 3-methyl-1-phenyl-2-
phospholen 1-oxide (5, 9 mg, 0.05 mmol), benzoic acid (6 mg, 0.05 mmol) and (MeO)3SiH (360 mg,
0.38 mL, 2.95 mmol). Purification by column chromatography on silica gel (cyclohexane:EtOAc
gradient) gave a yellow oil (98 mg, 0.34 mmol). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.37. The crude
was dissolved in Et2O (1.5 mL) and 2M HCl solution in Et2O (0.20 mL, 0.406 mmol) was added.
Precipitates were collected by filtration yielding a colorless solid (85 mg, 0.26 mmol, 27%). 1H NMR
(300 MHz, CDCl3, δ): 1.28 (t, 6H, J = 7.2 Hz), 3.47 (s, 2H), 3.52 (q, 4H, J = 7.2 Hz), 3.88 (s, 3H),
7.45–7.50 (m, 1H), 7.54 (d, 1H, J=8.5 Hz), 7.67–7.75 (m, 1H), 7.85 (s, 1H).

Methyl 2-oxo-2,3-dihydronaphtho[1,2-b]oxepine-4-carboxylate (4i).10 Obtained according GP2 from

fumarate derivative 3i (300 mg, 1.06 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 10 mg,
0.05 mmol), benzoic acid (6 mg, 0.05 mmol) and (MeO)3SiH (389 mg, 0.40 mL, 3.18 mmol) as a
yellow solid (122 mg, 0.455 mmol, 43%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.39; 1H NMR (300
MHz, CDCl3, δ): 3.49 (s, 2H), 3.91 (s, 3H), 7.45 (d, 1H, J = 8.5 Hz), 7.59–7.66 (m, 2H), 7.71 (d, 1H,
J = 8.5 Hz), 7.84–7.91 (m, 1H), 7.99 (s, 1H), 8.36–8.43 (m, 1H).

Methyl 8-chloro-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4j).10 Obtained according GP2

from fumarate derivative 3j (300 mg, 1.12 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 11
mg, 0.056 mmol), benzoic acid (7 mg, 0.056 mmol) and (MeO)3SiH (411 mg, 0.43 mL, 3.36 mmol)
as a colorless solid (91 mg, 0.36 mmol, 32%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.32; 1H NMR
(400 MHz, CDCl3, δ): 3.44 (s, 2H), 3.88 (s, 3H), 7.26–7.29 (m, 1H), 7.29–7.31 (m, 1H), 7.35–7.38
(m, 1H), 7.83 (s, 1H).

Methyl 7-chloro-2-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (4k).10 Obtained according GP2

from fumarate derivative 3k (300 mg, 1.12 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 11
mg, 0.056 mmol), benzoic acid (7 mg, 0.056 mmol) and (MeO)3SiH (411 mg, 0.43 mL, 3.36 mmol)
as a colorless solid (118 mg, 0.467 mmol, 42%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.37; 1H NMR
(300 MHz, CDCl3, δ): 3.44 (s, 2H), 3.89 (s, 3H), 7.20–7.25 (m, 1H), 7.39–7.45 (m, 2H), 7.79 (s, 1H).
Dimethyl 2-oxo-2,3-dihydrobenzo[b]oxepine-4,7-dicarboxylate (4l).10 Obtained according GP2 from
fumarate derivative 3l (300 mg, 1.03 mmol), 3-methyl-1-phenyl-2-phospholen 1-oxide (5, 10 mg,
0.05 mmol), benzoic acid (6 mg, 0.05 mmol) and (MeO)3SiH (378 mg, 0.39 mL, 3.09 mmol) as a
colorless solid (87 mg, 0.315 mmol, 31%). Rf (SiO2, cyclohexane:EtOAc= 3:1)= 0.24; 1H NMR (300
MHz, CDCl3, δ): 3.46 (s, 2H), 3.89 (s, 3H), 3.94 (s, 3H), 7.33 (d, 1H, J = 8.5 Hz), 7.90 (s, 1H), 8.10-
8.16 (m, 2H).

4.2. CA inhibition

An Applied Photophysics stoppedflow instrument has been used for assaying the CA catalysed CO2
hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. Saturated solutions of CO2 in water at 20°C were used (which have
a concentration of 17 mM). They were diluted with distilled water in order to arrive at CO2
concentrations as low as 1.7 mM. These were used thereafter for the determination of the kinetic
parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the
reaction have been used for determining the initial rate. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were
prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with the assay
buffer. Inhibitor and enzyme solutions were preincubated together for 15 min—6 h at room
temperature (15 min) or 4 °C (6 h) prior to assay, in order to allow for the formation of the E–I
complex. Data from Table 1 were obtained after 6 h incubation of enzyme and inhibitor, as for
coumarins reported earlier.1, 4d, 4f, 11, 17, 19a, 25 The inhibition constants were obtained by non-linear
leastsquares methods using PRISM 3, as reported earlier,2 and represent the mean from at least three
different determinations. All CA isofoms were recombinant ones obtained in-house as reported
earlier.1, 4d, 4f, 12, 18–21 26, 27

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

Acknowledgements
 This work was supported by the European Regional Development Fund (ERDF, project no.
1.1.1.2/VIAA/1/16/235). CTS thanks the Italian Ministry for University and Research (MIUR) for
the grant PRIN 2017XYBP2R.

Author Information

*E-mail addresses: aiga@osi.lv (A. Grandane); thomas.werner@catalysis.de (T. Werner);

claudiu.supuran@unifi.it (C. T. Supuran)

ORCID
Aiga Grandane: 0000-0002-9563-131X
Thomas Werner: 0000-0001-9025-3244
Raivis Zalubovskis: 0000-0002-9471-1342

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Graphical abstract