Discovery of Potent Dual-Tailed Benzenesulfonamide Inhibitors of Human Carbonic Anhydrases Implicated in Glaucoma and in Vivo Profiling of Their Intraocular Pressure-Lowering Action

Article abstract of DOI:10.1021/acs.jmedchem.9b02090

The design of three dual-tailed sulfonamide series 11a-11g, 14a-14h, and 16a-16e as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors are presented. All compounds were evaluated for inhibitory action against pharmacologically relevant human CA isoforms I, II, IV, and VII. Compounds 11a-11g emerged as potent CA inhibitors against the four tested isoforms with a significant selectivity to CA II, which is implicated in glaucoma (K_i in the range 0.36-6.9 nM). X-ray crystallographic analysis of three compounds (11a, 11d, and 11g) bound to CA II showed the validity of the adopted drug design strategy as specific moieties within the ligand structure interacted directly with the hydrophobic and hydrophilic halves of the CA II active site. Compounds 11b-11d and 11g were evaluated for their intraocular pressure-lowering effects in a rabbit model of glaucoma. 11b and 11d showed significant efficacy when compared to the clinically used drug dorzolamide.

Full text of DOI:10.1021/acs.jmedchem.9b02090

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Article
Discovery of potent dual-tailed benzenesulfonamide inhibitors
of human carbonic anhydrases im-plicated in glaucoma and
in vivo profiling of their intraocular pressure lowering action.
Mohamed Fares, Wagdy M. Eldehna, Silvia Bua, Cecilia Lanzi, Laura Lucarini, Emanuela Masini,
Thomas S. Peat, Hatem A. Abdel-Aziz, Alessio Nocentini, Paul A. Keller, and Claudiu Trandafir Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02090 • Publication Date (Web): 07 Feb 2020
Downloaded from pubs.acs.org on February 8, 2020
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Discovery of potent dual-tailed benzenesulfonamide inhibitors of human carbonic anhydrases
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implicated in glaucoma and in vivo profiling of their intraocular pressure lowering action.
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Mohamed Fares , Wagdy M. Eldehna , Silvia Bua , Cecilia Lanzi , Laura Lucarini , Emanuela
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1,*
Masini , Thomas S. Peat , Hatem A. Abdel-Aziz , Alessio Nocentini , Paul A. Keller , Claudiu T.
Supuran4,*
1
School of Chemistry & Molecular Bioscience, Molecular Horizons, University of Wollongong, and Illawarra Health & Medical Research Institute,
Wollongong, NSW 2522, Australia.
2
School of Chemistry, the University of Sydney, NSW 2006, Australia.
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt.
4
University of Florence, Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, via Ugo Schiff 6, 50019, Sesto Fiorentino,
Florence, Italy.
5
University of Florence, Department of NEUROFARBA, Section of Pharmacology and Toxicology, viale Gaetano Pieraccini 6, 50100, Florence, Italy.
6
CSIRO, Biomedical Manufacturing Program, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Department of Applied Organic Chemistry, National Research Center, Dokki, Giza 12622, Egypt
ABSTRACT: The design of three dual-tailed sulfonamide series 11a-g, 14a-h and 16a-e as carbonic
anhydrase (CA, EC 4.2.1.1) inhibitors are presented. All compounds were evaluated for inhibitory
action against pharmacologically relevant human (h) CAs isoforms I, II, IV, and VII. Compounds 11a-g
emerged as potent CA inhibitors against the four tested isoforms with a significant selectivity to CA II,
which is implicated in glaucoma (K in the range 0.36 - 6.9 nM). X-ray crystallographic analysis of three
i
compounds (11a, 11d and 11g) bound to CA II showed the validity of the adopted drug design strategy
as specific moieties within in the ligand structure interacted directly with the hydrophobic and
hydrophilic halves of the CA II active site. Compounds 11b-d and 11g were evaluated for their
intraocular pressure (IOP) lowering effects in a rabbit model of the glaucoma. 11b and 11d showed
significant efficacy when compared to the clinically used drug dorzolamide.
Keywords: glaucoma; metalloenzyme; sulfonamide; tail; inhibition, selectivity.
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1
. INTRODUCTION
Glaucoma is an optic neuropathy disease that affects more than 64 million people worldwide and is
implicated with 5 million instances of blindness which accounts for 12% of blindness, globally.1, 2
Despite profound advances in the area, the understanding of the primary causes of glaucoma is still not
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complete. Glaucoma is categorized into two major classes according to the anatomic configuration of
the aqueous humor, namely open angle glaucoma (OAG), which is prevalent in Europe and Africa and
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angle-closure glaucoma (CAG), which mainly affects Asian countries. Risk factors of OAG are age-
related and are associated with high intra ocular pressure (IOP) and thin central cornea.3
Current approaches used in the clinic to manage OAG mainly include IOP lowering drugs and surgery.
There are fundamentally five drug classes used to lower IOP, namely, carbonic anhydrase inhibitors,
cholinomimetics, sympathomimetic, β-blockers and prostaglandin analogues.4 Limitations in
determining IOP reduction in vitro make common drug discovery approaches such as cell-based assay
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or high throughput screening not useful to assess the drop in IOP. Whilst some promising candidates
are under clinical trials as anti-glaucoma agents, the development of new, safe and effective glaucoma
drugs is in high demand.1
Carbonic anhydrases (CA, EC 4.2.1.1) constitute a superfamily of ubiquitous metalloenzymes which
principally act as catalysts in the CO hydration reaction. The 15 isoforms identified in human belong
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to the α-class of the 8 genetically diverse families of CA discovered to date (α-, β-, γ-, δ-, ζ-, η-, θ- and
ι-CAs). These 15 isozymes differ in multiple factors including catalytic activity, structural fold, tissue
and subcellular localization and response to different kinds of modulators.5-10 Abnormal expression of
hCAs isoforms such as II and IV were detected in glaucoma.7, 10, 11
Great effort has been made in the design and synthesis of selective CA inhibitors (CAI) with anti-
glaucoma activity. Primary sulfonamides such as acetazolamide AAZ (Figure 1) have been clinically
used for almost 70 years as systemic anti-glaucoma agents despite a range of side effects due to the
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lack of enzymatic selectivity. The second generation antiglaucoma drugs, such as dorzolamide DRZ
and brinzolamide BRZ (Figure 1), act topically thus leading to a significant side effects reduction.12
They contain an amino group which can be converted to a HCl salt, conferring better water solubility.
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O O
S
O O
S
H
S
S
O
N
N
S
SO NH2
SO NH2
2
2
SO NH
2
2
O
N
N
HN
HN
1
2
3
AAZ
DRZ
BRZ
H NO S
2
2
H NO S
2
2
H NO S
O
2
2
N
Ar
S
O O
O O
S
N
H
N
N
NH2
H
H
R
5
4
6
ts-SA
SA
SO-SA
R
H NO S
2
2
O
N
H
CN
7
CA-SA
Figure 1. Structures of clinically used CAIs and other inhibitors derived from sulfanilamide (SA).
The design and development of selective CAI is challenging. Many strategies have been adopted
including the widely used ring and tail approaches.13-15 CA inhibitors such as AAZ (1), DRZ (2) and
BRZ (3) are examples of the ring approach, in which a primary sulfonamide group is attached to varied
1
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types of main aromatic rings (Figure 1). In the tail approach, a variety of molecular tails are attached
to the scaffold bearing the zinc binding group to confer better physicochemical properties, such as
increased water solubility, or to tune the inhibition profiles against the target CAs by interaction with
the most diverse section of the CA active site, the medium-outer portion.16-19 Sulfanilamide (4, Figure
1
), clinically used as antibacterial, have been the lead CA inhibitor in many drug-design strategies
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whereby a variety of tail functionalities were used, such as tosylureido (ts-SA 5), cyanoamide (CA-SA
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), or dual-tailed arylsulfone (SO-SA 6) (Figure 1).18, 20
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. RESULTS AND DISCUSSION
.1. Drug Design and Chemistry
In the current study, a new class of 3-oxo-N-(4-sulfamoylphenyl)butanamides was proposed based on
the dual tail approach (Figure 2). Considering the benzenesulfonamide as zinc-binding motif (orange in
Figure 2), aryl groups were included in the derivatives tails to interact with the CA hydrophobic region
(red in Figure 2), whereas H-bonds acceptor moieties were included for the interactions with Gln and
Asn residues present in the hydrophilic half of the active site (blue in Figure 2).
Figure 2: Design of dual-tailed sulfonamides targeting hCAs. The molecular surface of hydrophobic
and hydrophilic halves of the CA active site are colored in red and blue, respectively. The Zn(II) ion is
represented as an orange sphere (pdb 1CA2).
The synthesized compounds were thus investigated for their inhibitory effect against the cytosolic
isoforms hCA I, II and VII and the transmembrane hCA IV. The best inhibitors in terms of potency and
selectivity against the CAs implicated in glaucoma were assessed in vivo for their IOP lowering action
using DRZ as a standard. Evidence for the mode of binding of these dual tailed sulfonamides to hCA II
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9
was achieved at the molecular level by X-ray crystallographic analysis with three of the most potent
CA II inhibitors.
The key intermediate 9 was synthesized by reacting sulfanilamide 4 with ethyl acetoacetate 8 (Scheme
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). Knoevenagel condensation of acetoacetanilide 9 with the aldehydes 10a-g in the presence of
catalytic piperidine and acetic acid afforded 11a-g derivatives in yields of 30-62% (Scheme 1).
1
13
In a typical example, analysis of the H NMR and C NMR spectra of 11d revealed the disappearance
of the resonances at 3.60 ppm and 52.8 ppm, respectively, assigned to the methylene group in 9,
1
indicating the formation of arylidene 11d. The H NMR spectra showed two singlets at 2.37 and 3.70
ppm, assigned to CH and OCH , respectively. The arylidene H was assigned to the singlet at δ 7.61.
3
3
The protons ortho to the methoxy group in the aryl substituent were assigned the resonance at 6.83
ppm, while the protons meta to the methoxy group were assigned to the resonance at 7.49 ppm. The p-
sulfamoyl aryl substituents were assigned to the two doublets at 7.70 ppm (meta to the sulfamoyl
group) and 7.77 ppm (ortho to the sulfamoyl group). The halogenated derivative 15 was synthesized by
2
2
chlorination of the key intermediate 9 using sulfuryl chloride (Scheme 1). Access to derivatives of the
type 14a-h and 16a-e was accomplished via classic Japp-Klingemann coupling reaction of 3-oxo-N-(4-
sulfamoylphenyl)butanamide 9 and 2-chloro-3-oxo-N-(4-sulfamoylphenyl)butanamide 15, respectively
with different diazonium salts 13a-h and 13a-e in the presence of sodium acetate trihydrate and ethanol
23
1
(
Scheme 1). Analysis of the H NMR spectra of 14a-h and 16a-e showed the disappearance of the
resonances at ~3.60 and 5.42 ppm assigned to the methylene and methane in 9, respectively. It should
be mentioned that derivatives 16a-e may react nonspecifically with nucleophiles present in
biomolecules due to the presence of an activated chlorine atom, although we did not see this behavior
with CAs.
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H NO S
2
2
O
+
O
O
NH2
4
8
a
59%
0
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0
Ar-CHO
10a-g
H NO S
2
2
O
O
N
H
b
9
e
42%
O
1
9-62%
c
Ar-N +Cl-
H NO S
2 2
Ar-NH2
2
O
H NO S
2
2
12a-h
13a-h
O
O
d
29-59%
O
N
H
Cl
N
H
H NO S
2
2
O
15
Ar
1
1a-g
N
Ar-N +Cl-
H
d
2
N
Ar
1
0,11
Ar
-C H
4-ClC H4
N
H
13a-f
18-40%
a
b
c
d
e
f
6
5
14a-h
6
H NO S
2
2
6
2-ClC H4
O
N
6
4-OMeC H4
12-14,16 Ar
Cl
3-NitroC6H4
a
b
c
d
e
f
-C H
6 5
2-Cl-4-FC H3
H
N
6
4-FC6H4
4-ClC6H4
4-BrC6H4
4-OCH3C6H4
4-CNC6H4
-NO C H
Ar
g
-2-thienyl
N
H
16a-e
3
3
g
h
2 6 4
,4-diOCH C H
6 3
3
Scheme 1: Reagents and conditions: (a) neat reaction, reflux, 15 min; (b) MS 4 Å, piperidine, acetic
acid, ethanol, RT, 8 h; (c) NaNO /HCl, 0 °C; (d) sodium acetate trihydrate, ethanol, 0 °C; (e) SO Cl , 0
2
2
2
°C, 1 h then RT for 4 h.
.2. Carbonic Anhydrase Inhibition
2
Compounds 9, 11a-g, 14a-g, 15 and 16a-e were evaluated for their inhibition against the cytosolic CA
2
4
I, II and VII and the membrane-bound CA IV using a stopped-flow CO hydrase assay method. The
2
clinically used acetazolamide (AAZ) was used as the standard in the kinetic evaluation.
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Table 1: Inhibition data of compounds 9, 11a-g, 14a-h and 16a-e against isoforms hCA I, II, IV and
2
4
VII by a stopped-flow CO hydrase assay.
2
H NO S
H NO S
2
2
H NO S
2 2
2
2
O
O
O
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
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9
0
1
2
3
4
5
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7
8
9
0
N
Ar
N
N
H
N
N
H
H
H
R
Ar
9
14a-h; R = COCH3
11a-g
16a-e; R = Cl
a
K (nM)
i
Cmp
9
R
Ar
CA I
69.2±5.5
68.7±3.9
41.1±1.8 0.61±0.02
7.3±0.61 0.36±0.03
15.3±1.2 0.45±0.02
75.5±5.0
52.7±3.9
49.1±4.8
82.2±6.4
2669±231 8.1±0.65
2642±114 5.2±0.20
>10000
512.9±38
703.7±49 273.1±13
CA II
39.5±1.7
4.4±0.16
CA IV
CA VII
H
-
-
-
-
-
-
-
-
885.3±41
957.5±53
145.0±7.0
382.8±19
608.5±26
387.0±24
222.5±15
810.4±63
>10000
>10000
>10000
>10000
>10000
273.1±15
44.4±1.9
4.3±0.14
2.9±0.09
3.1±0.12
46.3±3.0
23.5±1.6
5.3±0.24
647.0±33
3323±159
1690±73
>10000
761.5±54
3589.4±238
792.4±55
4.4±0.31
68.1±4.5
46.6±3.0
6.1±0.52
1034.1±76
55.2±3.1
2.5±0.14
3.5±0.13
2.8±0.11
1
1a
1b
1c
1d
11e
1f
1g
C H
6
5
1
4-ClC H
6
4
1
2-ClC H
6
4
1
4-OCH C H
3 6 4
3-NO C H
4
6.9±0.11
5.6±0.25
2.9±0.10
4.3±.31
2
6
1
2-Cl-4-F-C H
6
4
1
1
1
2-thienyl
C H
4a COCH3
4b COCH3
14c COCH3
4d COCH3
4e COCH3
4f COCH3 4-OCH C H
6
5
4-FC H
6
4
4-ClC H
6
4
1
1
1
4-BrC H
370.5±16
52.2±1.4
6
4
4-CNC H
6
4
>10000
>10000
3
6
4
1
4g COCH3
14h COCH 3,4-diOCH C H
3
3-NO C H
750.6±62
59.3±2.8
227.1±11.6 40.6±2.1
295.6±14.5 34.8±1.1
44.1±2.3
925.7±57 262.1±17
71.4±3.9
5.0±0.14
2
6
4
1477±96
7364±124
7380±210
906.5±64
>10000
3
3
6
16a
6b
6c
6d
6e
Cl
Cl
Cl
Cl
Cl
C H
6
5
1
4-FC H
6 4
1
4-ClC H
3.0±0.24
6
4
1
4-BrC H
6 4
1
4-OCH C H
3
86.1±3.3 21.1±0.93 6415.6±320
3
6
AAZ
DRZ
BRZ
-
-
-
250±13
>50000
730±24
25.0±1.1
9±0.51
3±0.12
74.0±4.1
8500±245
3950±341
a Mean ± SD, from three replicates, by a stopped flow technique.
The ubiquitous CA I was inhibited efficiently by compounds 9 and 11a-g with inhibition constants (Ki
values) spanning a narrow range between 7.3 and 75.5 nM with the 2-chlorophenyl derivative 11c
showing the best activity with a K value of 7.3 nM against CA I. An inhibition decline was observed
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for compounds of subset 14 and 16, with the exception of 14a, 14h, 16c and 16e whose K values
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spanned the range 44.1-86.1 nM. In contrast, the other derivatives of the two subsets showed inhibition
of CA I in the high nanomolar to low micromolar range (227.1-2669 nM) up to no inhibition observed
below 10 μM for 14d. Thus, incorporation of halo-substituted phenyl moieties in the scaffolds 14 and
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6 produced a detrimental effect on the inhibition of CA I.
The target CA II was potently inhibited by compounds 11a-g with low to sub nanomolar K values
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0.36-6.9 nM) whereas 9 showed a K of 39.5 nM. Of note is the subnanomolar CA II inhibition by
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1b-d, significantly lower than that of AAZ, which endow them with significant potential for the
treatment of a wealth of diseases implicating CA II abnormal levels or activities. More complex SAR
were instead produced with the inhibition data against CA II of compounds from subsets 14 and 16.
While the absence of, or presence of specific substituents certain substituents (e.g. o-chloro, p-chloro or
p-methoxy) on the outer phenyl ring enabled the compounds to hold a low nanomolar efficacy (Ki
values in the range 3.0-8.1 nM for 14a-c, h and 16c), some others led to a drop of inhibition
effectiveness culminating with K values of 370.5 and 262.1 nM arising from the incorporation of a
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para-bromo substituent in the 14 and 16 scaffolds.
The membrane bound CA IV was the least inhibited isoform here screened by compounds 9, 11a-g,
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4a-h and 16a-e. Whereas compounds 9 and 11a-g retained a submicromolar inhibition with K values
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spanning 145.0 - 885.3 nM, derivatives from subsets 14 and 16 reported some micromolar K values
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against CA IV (0.91-7.38 μM), but mostly did not produced inhibition below 10 μM (14a-f, 16d).
The cytosolic isoform CA VII, generally defined as a brain-associated isozyme, was efficiently
inhibited by compounds 11a-g, though by an order of magnitude lower than CA II with K values
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spanning 2.9 - 46.6 nM, with 11b-d being again the most potent inhibitors (K values of 4.3, 2.9 and 3.1
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nM, respectively). Again, a drop in inhibition was observed with compounds from subset 14, where Ki
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values were in the range 647.0 - >10000 nM, with the exception of the 3,4-dimethoxyphenyl derivative
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4h, which held 4.4 nM K . In contrast, most compounds bearing a chloro substituent in place of the
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acetyl group from subset 16 showed an effective CA VII inhibition in the range 6.1-68.1 nM, except
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6d, the p-Br-phenyl derivative, that displayed a K of 1034.1 nM.
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.3. X-Ray crystallography
To gain insights into the ligand-target interactions driving such intense hCA II inhibition, the X-ray co-
crystallographic analysis of the adducts of hCA II with 11a, 11d and 11g was achieved at resolutions of
1.67, 1.32 and 1.48 Å, respectively (Figure 3 and ESI: Table S2, Figure S22-25). The sulfonamide
coordination to the Zn(II) is present in all adducts, as well as the two H-bond networks involving the
-
SO NH group and Thr199. The phenyl ring bearing the sulfonamide moiety of 11a, 11d and 11g forms
2
hydrophobic interactions with Val121, Val143 and Leu198. As originally designed, the carbonyl group
of the amide and acetyl moieties are oriented towards the hydrophilic half of the hCA II active site
where they establish H-bond networks. In detail, the amidic oxo group of 11a, 11d and 11g H-bonded
to the Gln92 side chain NH . In contrast, the oxo-acetyl group of 11a and 11g are directly H-bonded to
2
the Asn67 side chain NH and by a water-bridge with Asn62, whereas the hCA II/11d adduct possesses
2
a sole water-bridged H-bond between the acetyl group and Asn67. The outer aromatic ring of 11a, 11d
and 11g were bound within the hydrophobic pocket of the hCA II active site by π-alkyl interactions
with Leu198, Pro202, Leu204, Val135 and Phe131, as hypothesized in the drug design phase.
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Figure 3: Active site view of the hCA II adduct with A) 11a (pdb 6UFB), B) 11d (pdb 6UFC) and C)
1g (pdb 6UFD). Hydrogen bonds are represented as black dashed lines.
1
2.4. Antiglaucoma Activity (IOP Lowering Activity)
The IOP lowering properties of the most potent CA II inhibitors, 11b-d and 11g, were assessed in a
rabbit model of glaucoma (Figure 4). DZA hydrochloride 1% was used as the standard, and control
experiments utilised the vehicle (0.9% NaCl + 0.1% DMSO + 0.1% EtOH). A 1% solution of the
compounds were administered as eye drops to rabbits with high IOP, induced by the injection of 0.05
mL of hypertonic saline solution (5% NaCl in distilled water) into the vitreous of both eyes. At 60 min
post-instillation compounds 11b and 11d decreased IOP by 9.0 and 10.0 mmHg, while the standard
DRZ was less effective with an IOP reduction of 8.5 mmHg (Figure 4). In contrast, 11c and 11g
produced an IOP reduction of 6.4 and 7.5 mmHg, respectively. At the 120 min mark after the
administration, 11b and 11d triggered an even more marked IOP drop to 12.8 and 12.3 mmHg. DRZ
retained a comparable action, with the IOP reduction settling at 9.0 mmHg. Derivative 11g showed an
enhanced efficacy after 120 min post-instillation, with a reduction of 8.9 mmHg, compared to DRZ. In
contrast, 11c reported a rapid reduction of efficacy (only 3.6 mmHg reduction) 120 min after post-
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administration. Oddly, 11c uniquely protracted its action after 240 min post-instillation at 2.0 mmHg,
whereas the IOP lowering activity of 11b and 11d settled at 1.3 and 1.5 mmHg, respectively.
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Figure 4. Drop of intraocular pressure (ΔIOP, mmHg) versus time (min) in hypertonic saline-induced
ocular hypertension in rabbits, treated with 50 μL of 1% solution of compounds 11b-d and 11g and
DRZ as the standard. Data are analyzed with 2way Anova followed by Bonferroni multiple comparison
test. * p<0.05 11b, 11g, DRZ vs vehicle at 60' and DRZ vs vehicle at 120'; ** p<0.01 11d and vs
vehicle at 60' and 11g vs vehicle at 120'; **** p<0.001 11b and 11d vs vehicle at 120'.
3. CONCLUSIONS
Three series of dual-tailed sulfonamide CA inhibitors 11a-g, 14a-h and 16a-e were designed,
synthesized and evaluated for their inhibitory profiles against four carbonic anhydrase isoforms: hCA I,
II, IV, and VII. The tested compounds showed selectivity to CA II and compounds 11a-g emerged as
the most potent CA II inhibitors with low to sub nanomolar K values (0.36-6.9 nM). To further
i
understand and rationalize the strong inhibitory profile of the compounds against CA II, X-ray
crystallographic studies were performed which showed defined moieties within the ligand structures
specifically interact with the hydrophobic and hydrophilic halves of the CA II active site. As CA II up-
regulation is implicated with glaucoma, the most active CA II inhibitors 11b-d and 11g (K values of
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.36-2.9 nM) were evaluated for their IOP lowering action against DRZ as the standard. Compound
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1g showed a comparable IOP lowering effect to DRZ (IOP reduction = 8.5 mmHg), while compounds
1b and 11d were more potent than DRZ with IOP reduction of 12.8 and 12.3 mmHg, respectively.
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Therefore, this study presents compounds 11b and 11d as potential promising candidates for the
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development of therapeutic anti-glaucoma strategies.
4
4
. EXPERIMENTAL PROTOCOLS
.1. Chemistry
Melting points were measured with a Gallenkamp (Griffin) apparatus and are uncorrected. Reactions
were monitored by TLC analysis using silica gel GF/UV 254 and visualized with UV light. Flash
chromatography was performed using Merck silica gel 60 (70–230 mesh) under positive air pressure.
Infrared (IR) spectra were obtained on a Shimadzu IRAfinity-1 FTIR spectrometer. NMR spectra were
1
recorded on Bruker 400 and 500 MHz FT-NMR spectrometers. H spectra were run at 400 and 500
1
3
MHz, while C spectra were run at 100 and 125 MHz. Chemical shifts (δ ) are reported relative to
H
TMS as the internal standard and coupling constant (J) values are reported in Hertz. The abbreviations
used are as follows: s, singlet; d, doublet; t, triplet; m, multiplet. All reagents and solvents were purified
and dried by standard techniques. The purity of tested compounds was greater than 95% as determined
by HPLC analysis using a Shimadzu CLASS-VP LC10 analytical HPLC system equipped with a Photo
Diode Array Detector and autosampler (see Supporting Information). Low resolution mass
spectrometry (MS) was performed on a Shimadzu LC-2010 Electrospray Ionization (ESI) Mass
Spectrometer. Samples were prepared using HPLC grade methanol. The principal ion peaks m/z values
are reported with their relative intensities in parentheses. High resolution mass spectra (HRMS) were
obtained using a Waters Quadrupole-Time of Flight (Q-Tof) mass spectrometer via ESI with Leucine-
Enkephalin as internal standard. All compounds were screened for PAINS alerts using the SwissADME
server (www.swissadme.ch). Derivatives 14a-h showed alerts due to imine functionality, however,
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these compounds are not the major lead outcomes from this study and are not taken further. The
activity results can still be used in the context of SAR as an enzyme assay was used, and thus binding
results are still valid. It should be noted that drugs developed in this area are for topical use, and not
systemic use.
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-Oxo-N-(4-sulfamoylphenyl)butanamide (9) 21, 25
3
Sulfanilamide 4 (3.44 g, 20 mmol) was added to a boiling ethyl acetoacetate 8 (7.77 mL, 61 mmol) and
the mixture was heated at reflux for 20 minutes. After cooling, HCl 10% (10 mL) was added and the
mixture was stirred for 30 minutes, the solid material filtered out and crystalized from aqueous ethanol
26
to give 9 (3.02 g, 59%) as white crystals; m.p. 180 °C (Lit. 183-186 °C). The product contains about
1
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0% of enol form as analysed by H NMR data. H NMR (500 MHz, DMSO-d ) δ: 2.21 (s, 3H, CH ),
6 3
3.60 (s, 2H, CH ), 7.25 (br. s, 2H, NH ), 7.71 (d, J = 9.0 Hz, 2H, ArH), 7.76 (d, J = 9.0 Hz, 2H, ArH),
2
2
1
0.43 (s, 1H, NH). Enol form signals: 1.93 (s, 3H, CH ), 5.23 (s, 1H, =CH), 10.26 (s, 1H, NH), 13.59
3
1
3
(
br. s, 1H, OH); C NMR (126 MHz, DMSO-d ): 30.7 (CH ), 52.8 (CH ), 119.1, 127.2, 139.0, 142.2,
6 3 2
-
1
2
66.1, 203.1; MS (ESI-) 255 (100%, M-H) , 291 (34%, M+Cl); HRMS (ESI-) calcd for C H N O S:
10 11 2 4
55.0440, found 255.0437.
General procedure A for preparation of 2-arylidene-3-oxo-N-(4-sulfamoylphenyl)butanamides
11a-g): A suspension of 3-oxo-N-(4-sulfamoylphenyl)butanamide 9 (256 mg, 1 mmol), the
(
appropriate aldehyde 10a-g (1 mmol) piperidine (10 µl, 0.1 mmol) and acetic acid (6 µl, 0.1 mmol) in
absolute ethanol (10 mL) and was stirred for 6-8 h at room temperature. The mixture was slowly
poured into brine (20 mL) and extracted with dichloromethane (3 × 10 mL), dried (anhyd. MgSO ), the
4
solvent removed under reduced pressure, and the resulting residue purified via flash chromatography
(petroleum ether: ethyl acetate = 5:5) to give 11a-g.
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(
E)-2-Benzylidene-3-oxo-N-(4-sulfamoylphenyl)butanamide (11a): Using the general procedure A and
benzaldehyde 10a (106 mg, 1 mmol), compound 11a (153 mg, 45%) was isolated as a white solid, m.p.
1
172 °C. H NMR (500 MHz, DMSO-d ) δ: 2.47 (s, 3H, CH ), 7.30 (br. s, 2H, NH ), 7.41 (m, 3H, ArH),
6
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.42 (d, J = 7.0 Hz, 2H, ArH), 7.76 (s, 1H, arylidineH), 7.76-7.79 (m, 4H, ArH), 10.76 (s, 1H, NH); C
NMR (126 MHz, DMSO-d ): 26.9 (CH ), 119.6, 127.5, 129.7, 130.4, 131.4, 133.8, 138.1, 139.6, 140.9,
6
3
-
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42.3, 166.8 (CONH), 197.1 (COCH ); MS (ESI-) 343 (100%, M-H) , 379 (25%, M+Cl); HRMS (ESI-
3
) calcd for C H N O S: 343.0753, found 343.0757.
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(E)-2-(4-Chlorobenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (11b): Using the general
procedure and 4-chlorobenzaldehyde 10b (140 mg, 1 mmol), compound 11b (204 mg, 54%) was
1
isolated as a white solid, m.p. 183 °C. H NMR (500 MHz, CD OD) δ: 2.46 (s, 3H, CH ), 7.31 (br. s,
3
3
2H, NH ), 7.52 (d, J = 8.4 Hz, 2H, ArH), 7.64 (d, J = 8.4 Hz, 2H, ArH), 7.76-7.80 (m, 5H,
2
13
4
1
ArH+arylidineH), 10.77 (s, 1H, NH); C NMR (126 MHz, DMSO-d ): 26.5 (CH ), 119.2, 127.0,
6
3
3
5
29.3, 131.5, 132.3, 135.5, 138.1, 139.0, 139.2, 141.7, 166.0, 196.5; MS (ESI-) 377 (100%, M ( Cl)-
-
37
-
35
H) , 379 (34%, M ( Cl)-H) ; HRMS (ESI-) calcd for C H ClN O S: 377.0363, found 377.0367.
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(E)-2-(2-Chlorobenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (11c): Using the general
procedure and 2-chlorobenzaldehyde 10c (140 mg, 1 mmol), compound 11c (136 mg, 36%) was
1
isolated as a white solid, m.p. 192 °C. H NMR (500 MHz, CD OD) δ: 2.50 (s, 3H, CH ), 7.24 (td, J =
3
3
7.7, 1.3 Hz, 1H, ArH), 7.37, (td, J = 7.8, 1.7 Hz, 1H, ArH), 7.51 (dd, J = 8.1, 1.3 Hz, 1H, ArH), 7.65
(dd, J = 7.9, 1.7 Hz, 1H, ArH) , 7.69 (d, J = 8.8 Hz, 2H, ArH), 7.83 (d, J = 8.8 Hz, 2H, ArH), 8.01 (s,
1
3
1
H, arylidineH); C NMR (126 MHz, CD OD): 25.2 (CH ), 119.4, 126.9, 127.0, 129.1, 129.8, 131.5,
3 3
3
5
-
131.6, 134.8, 136.9, 139.1, 141.5, 166.4, 196.1; MS (ESI-): 377 (100%, M ( Cl)-H) , 379 (35%, M
(37
Cl)-H), (ESI+): 401 (M+Na); HRMS (ESI-) calcd for C H ClN O S: 377.0363, found 377.0356.
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(E)-2-(4-Methoxybenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (11d): Using the general
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procedure and 4-methoxybenzaldehyde 10d (136 mg, 1 mmol), compound 11d (217 mg, 58%) was
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isolated as a white solid, m.p. 162 °C. H NMR (DMSO-d ) δ: H NMR (500 MHz, CD OD) δ 2.37 (s,
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H, CH ), 3.70 (s, 3H, OCH ), 6.83 (d, J = 9.0 Hz, 2H, ArH), 7.49 (d, J = 9.0 Hz, 2H, ArH), 7.61 (s,
3 3
1
3
H, arylidineH), 7.70 (d, J = 9.0 Hz, 2H, ArH), 7.77 (d, J = 9.0 Hz, 2H, ArH); C NMR (126 MHz,
CD OD): 24.8 (CH ), 54.6 (OCH ), 114.2, 119.3, 125.3, 126.9, 131.9, 134.8, 139.0, 141.4, 141.8,
3
3
3
-
162.2, 168.0, 196.8; MS (ESI-) 373 (100%, M-H) , (ESI+): 397 (M+Na); HRMS (ESI-) calcd for
C H N O S: 373.0858, found 373.0840.
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(E)-2-(3-Nitrobenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (11e): Using the general
procedure and 3-nitrobenzaldehyde 10e (151 mg, 1 mmol), compound 11e (74 mg, 19%) was isolated
1
1
as a white solid, m.p. 196 °C. H NMR (DMSO-d ) δ: H NMR (400 MHz, DMSO) δ 2.50 (s, CH ,
6
3
3
H), 7.30 (br. s, 2H, NH ), 7.72-7.81 (m, 5H, ArH), 7.97 (s, 1H, arylidineH), 8.03 (d, J = 8.5 Hz, 1H,
2
1
3
ArH), 8.26 (d, J = 8.5 Hz, 1H, ArH), 8.56 (s, 1H, ArH), 10.83 (s, 1H, NH); C NMR (101 MHz,
DMSO) δ: 26.8 (CH ), 119.7, 124.5, 125.3, 127.3, 131.1, 135.5, 135.9, 138.4, 139.7, 139.8, 141.7,
3
-
1
48.5, 165.8, 196.9; MS (ESI-) 388 (100%, M-H) , (ESI+): 412 (M+Na); HRMS (ESI-) calcd for
C H N O S: 388.0603, found 388.0605.
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14
3
6
(E)-2-(2-Chloro-4-fluorobenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (11f): Using the
general procedure and 2-chloro-4-fluorobenzaldehyde 10f (158 mg, 1 mmol), compound 11f (99 mg,
1
2
7
5%) was isolated as a yellow solid, m.p. 152 °C. H NMR (400 MHz, CD OD) δ: 2.50 (s, 1H, CH ), δ
3
3
.06 (td, J = 8.5, 2.6 Hz, 1H, ArH), 7.37 (dd, J = 8.5, 2.6 Hz, 1H, ArH), 7.69-7.71 (m, 3H, ArH), 7.84
1
3
(d, J = 9.0 Hz, 2H, ArH), 7.95 (s, 1H, arylidineH); C NMR (101 MHz, CD OD) δ: 25.2 (CH ), 114.3
3
3
(
d, J2 28), 117.0 (d, J2 31), 119.4, 126.9, 130.7 (d, J3 11), 135.8, 136.0 (d, J3 14), 139.0,
C−F C−F C−F C−F
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39.0, 139.1, 141.4, 162.2 (d, J1C−F 315), 166.3, 195.9; MS (ESI-:) 395 (100%, M-H) , 397 (30%, M
(37
Cl)-H), (ESI+): 319 (M+Na); HRMS (ESI-) calcd for C H ClFN O S: 359.0269, found 395.0271.
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13
2
4
(
E)-3-Oxo-N-(4-sulfamoylphenyl)-2-(thiophen-2-ylmethylene)butanamide (11g): Using the general
0
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9
0
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3
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8
9
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9
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3
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7
8
9
0
procedure and thiophene-2-carbaldehyde 10g (112 mg, 1 mmol), compound 11g (217 mg, 62%) was
1
isolated as a yellow solid, m.p. 185 °C. H NMR (500 MHz, DMSO-d ) δ: 2.43 (s, 1H, CH ), 7.19 (dd,
6
3
J = 5.0, 3.7 Hz, 1H, ArH), 7.30 (br. s, 2H, NH ), 7.64 (dd, J = 3.7, 1.3 Hz, 1H, ArH), 7.83-7.85 (m, 5H,
2
1
3
ArH), 8.01 (s, 1H, arylidineH), 10.84 (s, 1H, NH); C NMR (126 MHz, DMSO-d ): 26.5 (CH ), 119.5,
6
3
1
27.3, 128.6, 133.7, 133.9, 134.7, 136.0, 137.0, 139.4, 142.4, 166.0, 196.1; MS (ESI-) 349 (100%, M-
-
H) , (ESI+): 373 (M+Na); HRMS (ESI-) calcd for C H N O S : 349.0317, found 349.0311.
1
5
13
2
4 2
General procedure
B
for preparation of (E)-3-oxo-2-(2-arylhydrazineylidene)-N-(4-
sulfamoylphenyl)butanamides (14a-h): The diazonium salt 13a-h was prepared by slowly adding a
cold sodium nitrite solution (0.7 g, 10 mmol) in deionized water (5 mL) to a cold solution of the
appropriate arylamine 12a-h (10 mmol) dissolved in 6 M hydrochloric acid (6 mL). To a cold solution
of 3-oxo-N-(4-sulfamoylphenyl)butanamide 9 (256 mg, 1 mmol) and sodium acetate trihydrate (408
mg, 3 mmol) in ethanol (10 mL), the appropriate diazonium chloride solution 13a-h (15 mmol) was
slowly added over a period 30 min at 0-5 °C. The reaction mixture was stirred for further 3 h at 0-5 °C
and 3 h at room temperature. The resulting solid was filtered, washed with water (2 x 10 mL), dried and
recrystallized from EtOH/DMF to afford 14a-h.
(
E)-3-Oxo-2-(2-phenylhydrazineylidene)-N-(4-sulfamoylphenyl)butanamide (14a): Using general
procedure B and aniline 12a (93 mg, 1 mmol), compound 14a (165 mg, 46%) was isolated as yellow
1
solid, m.p. 215 °C. H NMR (500 MHz, DMSO-d ) δ: 2.46 (s, 3H, CH ), 7.16 (t, J = 7.5 Hz, 1H, ArH),
6
3
7.28 (br. s, 2H, NH ), 7.41 (t, J = 7.5 Hz, 2H, ArH), 7.54 (d, J = 7.5 Hz, 2H, ArH), 7.80 (d, J = 9.0 Hz,
2
13
2
H, ArH), 7.83 (d, J = 9.0 Hz, 2H, ArH), 11.44 (s, 1H, CONH), 13.77 (s, 1H, NNH); C NMR (126
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MHz, DMSO-d ): 26.3, 116.4, 120.3, 125.5, 127.3, 127.4, 130.0, 139.9, 140.7, 142.3, 162.5, 198.8; MS
6
4
5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
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-
(
ESI-): 359 (100%, M-H) ; HRMS (ESI-) calcd for C H N O S: 359.0809, found 359.0823.
1
6
15
4
4
E)-2-(2-(4-Fluorophenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (14b): Using
general procedure B and 4-fluoroaniline 12b (111 mg, 1 mmol), compound 14b (185 mg, 49%) was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
isolated as a greenish solid, m.p. 248 °C. H NMR (400 MHz, DMSO-d ) δ: 2.50 (s, 3H, CH ), 7.25-
6
3
7
.30 (m, 4H, NH + 2ArH), 7.59-7.63 (m, 2H, H9, ArH), 7.82 (d, J = 11.0 Hz, 2H, ArH), 7.85 (d, J =
2
1
3
11.0 Hz, 2H, ArH), 11.39 (s, 1H, CONH), 13.65 (s, 1H, NNH); C NMR (101 MHz, DMSO-d ): 26.3,
6
2
3
1
1
2
3
1
6
.
7
(
d
,
J
C
−
F
2
9
H
z
)
,
1
1
8
.
2
(
d
,
J
C
−
F
1
0
H
z
)
,
1
2
0
.
3
,
1
2
7
.
3
,
1
2
7
.
7
,
1
3
9
.
0
,
1
3
9
.
9
,
1
4
0
.
7
,
1
5
8
.
7
(
d
,
J
C
−
F
-
40 Hz), 162.4, 198.7; MS (ESI-) 377 (100%, M-H) ; HRMS (ESI-) calcd for C H FN O S:
1
6
14
4
4
77.0714, found 377.0729.
(E)-2-(2-(4-Chlorophenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (14c): Using
general procedure B and 4-chloroaniline 12c (127 mg, 1 mmol), compound 14c (217 mg, 55%) was
1
isolated as a yellow solid, m.p. 260 °C. H NMR (500 MHz, DMSO-d ) δ: 2.49 (s, 3H, CH ), 7.32 (br.
6
3
s, 2H, NH ), 7.44 (d, J = 8.5 Hz, 2H, ArH), 7.56 (d, J = 8.5 Hz, 2H, ArH), 7.80 (d, J = 9.0 Hz, 2H,
2
1
3
ArH), 7.83 (d, J = 9.0 Hz, 2H, ArH), 11.34 (s, 1H, CONH), 13.51 (s, 1H, NNH); C NMR (126 MHz,
DMSO-d ): 26.2, 117.9, 120.3, 127.2, 128.8, 128.9, 129.8, 139.9, 140.7, 141.4, 162.1, 198.4; MS (ESI-
6
3
5
-
37
-
35
)
393 (100%, M ( Cl)-H) , 395 (30%, M ( Cl)-H) ; HRMS (ESI-) calcd for C H ClN O S:
16 14 4 4
393.0419, found 393.0435.
(
E)-2-(2-(4-Bromophenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (14d): Using
general procedure B and 4-bromoaniline 12d (171 mg, 1 mmol), compound 14d (258 mg, 59%) was
1
isolated as a yellow solid, m.p. >260 °C. H NMR (500 MHz, DMSO-d ) δ: 2.50 (s, 3H, CH ), 7.52 (d,
6
3
J = 9.0 Hz, 2H, ArH), 7.59 (d, J = 9.0 Hz, 2H, ArH), 7.82 (d, J = 9.0 Hz, 2H, ArH), 7.85 (d, J = 9.0 Hz,
13
2
H, ArH), 11.32 (s, 1H, CONH), 13.39 (s, 1H, NNH); C NMR (126 MHz, DMSO-d ): 26.4, 117.3,
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79
1
18.5, 120.5, 127.5, 129.3, 133.0, 140.1, 141.0, 142.1, 162.4, 198.7; MS (ESI-) 437 (100%, M ( Br)-
-
81
-
79
H) , 339 (100%, M ( Br)-H) ; HRMS (ESI-) calcd for C H BrN O S: 436.9919, found 436.9925.
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14
4
4
(
E)-2-(2-(4-Cyanophenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (14e): Using
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general procedure B and 4-aminobenzonitrile 12e (118 mg, 1 mmol), compound 14e (119 mg, 31%)
1
1
was isolated as a yellow solid, m.p. >260 °C. H NMR (DMSO-d ) δ: H NMR (500 MHz, DMSO-d )
6
6
δ: 2.50 (s, 3H, CH ), 7.32 (br. s, 2H, NH ), 7.65 (d, J = 9.0 Hz, 2H, ArH), 7.82 (d, J = 9.0 Hz, 2H,
3
2
1
3
ArH), 7.85 (d, J = 9.0 Hz, 2H, ArH), 11.15 (s, 1H, CONH), 12.85 (s, 1H, NNH); C NMR (126 MHz,
DMSO-d ) δ: 26.3, 106.1, 116.6, 119.8, 120.5, 127.5, 133.2, 134.5, 140.2, 141.1, 146.6, 161.9, 198.1;
6
-
MS (ESI-): 384 (100%, M-H) ; HRMS (ESI-) calcd for C H N O S: 384.0761, found 384.0776.
1
7
14
5
4
(
E)-2-(2-(4-Methoxyphenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (14f): Using
general procedure B and 4-methoxyaniline 12f (123 mg, 1 mmol), compound 14f (168 mg, 43%) was
1
isolated as a yellow solid, m.p. 255 °C. H NMR (500 MHz, DMSO-d ) δ: 2.50 (s, 3H, CH ), 3.78 (s,
6
3
3
7
2
H, OCH ), 7.02 (d, J = 8.5 Hz, 2H, ArH), 7.34 (br. s, 2H, NH ), 7.53 (d, J = 8.5 Hz, 2H, ArH), 7.83-
3
2
1
3
.88 (m, 4H, ArH), 11.57 (s, 1H, CONH), 14.07 (s, 1H, NNH); C NMR (126 MHz, DMSO-d ) δ:
6
5.6, 55.2, 114.7, 117.4, 119.7, 125.1, 126.7, 135.1, 139.2, 140.1, 157.1, 162.2, 198.1; MS (ESI-): 389
-
(100%, M-H) ; HRMS (ESI-) calcd for C H N O S: 389.0914, found 389.0933.
17
17
4
5
(
E)-2-(2-(3-Nitrophenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide (14g): Using
general procedure B and 3-nitroaniline 12g (138 mg, 1 mmol), compound 14g (117 mg, 29%) was
1
isolated as a yellow solid, m.p. 248 °C. H NMR (500 MHz, DMSO-d ) δ: 2.48 (s, 3H, CH ), 7.31 (br.
6
3
s, 2H, NH ), 7.65 (t, 1H, J = 8.5 Hz, 1H, ArH), 7.81 (d, J = 8.5 Hz, 2H, ArH), 7.84 (d, J = 8.5 Hz, 2H,
2
ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.95 (d, J = 8.0 Hz, 1H, ArH), 8.32 (s, 1H, ArH), 11.15 (s, 1H,
1
3
CONH), 12.92 (s, 1H, NNH); C NMR (126 MHz, DMSO-d ) δ: 25.9, 110.4, 118.5, 120.1, 121.9,
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27.2, 131.3, 132.1, 139.8, 140.8, 144.0, 149.0, 161.6, 197.7; MS (ESI-): 404 (100%, M-H) ; HRMS
(ESI-) calcd for C H N O S: 404.0659, found 404.0673.
16 14 5 6
6
7
8
9
1
1
1
1
1
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1
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1
1
2
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2
2
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(
E)-2-(2-(3,4-Dimethoxyphenyl)hydrazineylidene)-3-oxo-N-(4-sulfamoylphenyl) butanamide (14h):
0
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7
8
9
0
1
2
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8
9
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9
0
Using general procedure B and 3,4-dimethoxyaniline 12h (153 mg, 1 mmol), compound 14h (168 mg,
1
4
0%) was isolated as an orange solid, m.p. 247 °C. H NMR (500 MHz, DMSO-d ) δ: 2.52 (s, 3H,
6
CH ), 3.77 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 7.02 (d, J = 9.0 Hz, 1H, ArH), 7.12 (dd, J = 9.0, 2.5 Hz,
3
3
3
1H, ArH), 7.26 (d, J = 2.5 Hz, 1H, ArH), 7.31 (br. s, 2H, NH ), 7.82 (d, J = 9.0 Hz, 2H, ArH), 7.85 (d,
2
13
J = 9.0 Hz, 2H, ArH), 11.54 (s, 1H, CONH), 13.99 (s, 1H, NNH); C NMR (126 MHz, DMSO-d ) δ:
6
2
6.4, 56.2, 56.5, 101.4, 108.6, 113.1, 120.5, 126.1, 127.5, 136.1, 140.0, 140.9, 147.5, 150.3, 162.9,
-
1
98.8; MS (ESI-): 419 (100%, M-H) , HRMS (ESI-) calcd for C H N O S: 419.1020, found
18 19
4
6
419.1035.
2
-Chloro-3-oxo-N-(4-sulfamoylphenyl)butanamide (15): To a stirred suspension of 3-oxo-N-(4-
sulfamoylphenyl)butanamide 9 (2.56 g, 10 mmol) in toluene (30 mL) with ice cooling was added
dropwise over 1 h sulfuryl chloride (1.50 g, 11 mmol) and the reaction warmed at 40-50 °C until
completion as analysed by TLC. The toluene was evaporated, and the residue was crystallized from
ethanol to afford 15 (1.22 g, 42%) as white crystals, m.p. 140 °C. The product contains about 32% of
1
1
enol form as analysed by H NMR. H NMR (500 MHz, DMSO-d ) δ: 2.32 (s, 3H, CH ), 2.52 (s, 3H,
6
3
CH ), 5.42 (s, 1H, CH), 7.31 (br. s, 2H, NH ), 7.34 (br. s, 2H, NH ), 7.74-7.76 (m, 4H, ArH), 7.80-7.82
3
2
2
13
(m, 4H, ArH), 7.34 (br. s, 2H, NH ), 10.98 (s, 1H, NH), 10.99 (s, 1H, NH); CNMR (126 MHz,
2
DMSO-d ) δ: 24.7, 27.5, 64.2, 84.0, 120.0, 121.3, 127.1, 127.3, 140.0, 140.6, 140.7, 141.3, 162.3,
6
3
5
37
163.9, 192.3, 197.3; MS (ESI-): 289 (100%, M( Cl)-H), 391 (30%, M ( Cl)-H); HRMS (ESI-) calcd
35
for C H ClN O S: 289.0044, found 289.0058.
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General
procedure
C
for
preparation
of
(Z)-2-oxo-N-aryl-2-((4-
sulfamoylphenyl)amino)acetohydrazonoyl chloride (16a-f): To a cold solution of 2-chloro-3-oxo-N-
(4-sulfamoylphenyl)butanamide 15 (290 mg, 1 mmol) and sodium acetate trihydrate (408 mg, 3 mmol)
in ethanol (10 mL), the appropriate diazonium chloride solution 13a-f (15 mmol) was slowly added
over a period 30 min at 0-5 °C. The reaction mixture was stirred for further 3 h at 0-5 °C and 3 h at
0
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2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
room temperature. The resulting solid was filtered, washed with water (2 x 10 mL), dried (MgSO ) and
4
recrystallized from EtOH/DMF to afford 16a-f.
(Z)-2-Oxo-N-phenyl-2-((4-sulfamoylphenyl)amino)acetohydrazonoyl chloride (16a): Using general
procedure C and aniline 12a (93 mg, 1 mmol), compound 16a (151 mg, 34%) was isolated as a white
1
solid, m.p. 245 °C. H NMR (500 MHz, DMSO-d ) δ: 7.00 (t, J = 7.5 Hz, 1H, ArH), 7.29 (br. s, 2H,
6
NH ), 7.34 (t, J = 7.5, 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.82 (d, J = 9.0 Hz, 2H, ArH),
2
13
7
.92 (d, J = 9.0 Hz, 2H, ArH), 10.34 (s, 1H, NH), 10.41 (s, 1H, NH); C NMR (126 MHz, DMSO-d )
6
δ: 115.4, 118.2, 120.8, 122.6, 127.0, 129.5, 139.5, 141.8, 143.2, 158.1; MS (ESI-): 351 (100%, M
3
5
-
37
-
35
( Cl)-H) , 353 (33%, M ( Cl)-H) , HRMS (ESI-) calcd for C H ClN O S: 351.0319, found
1
4
12
4
3
3
51.0310.
(Z)-N-(4-Fluorophenyl)-2-oxo-2-((4-sulfamoylphenyl)amino)acetohydrazonoyl chloride (16b): Using
general procedure C and 4-fluoroaniline 12b (111 mg, 1 mmol), compound 16b (100 mg, 27%) was
1
isolated as a buff solid, m.p. 250 °C. H NMR (500 MHz, DMSO-d ) δ: 7.16 (t, J = 8.8 Hz, 2H, ArH
6
7
8
.28 (br. s, 2H, NH ), 7.61 (dd, J = 8.8, 5.0 Hz, 2H, ArH), 7.82 (d, J = 8.5 Hz, 2H, ArH), 7.91 (d, J =
2
1
3
.5 Hz, 2H, ArH), 10.32 (s, 1H, NH), 10.42 (s, 1H, NH); C NMR (126 MHz, DMSO-d ) δ: 116.0 (d,
6
J2 23 Hz), 116.8 (d, J3 8 Hz), 118.2, 120.8, 126.9, 139.4, 139.7, 141.7, 157.3 (d, J1C−F 238 Hz),
C−F
C−F
35
-
37
-
1
58.0; MS (ESI-): 369 (100%, M ( Cl)-H) , 371 (33%, M ( Cl)-H) ; HRMS (ESI-) calcd for
3
5
C H F ClN O S: 369.0224, found 369.0232.
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(Z)-N-(4-Chlorophenyl)-2-oxo-2-((4-sulfamoylphenyl)amino)acetohydrazonoyl chloride (16c): Using
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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general procedure C and 4-chloroaniline 12c (127 mg, 1 mmol), compound 16c (154 mg, 40%) was
1
isolated as a yellow solid, m.p. 255 °C. H NMR (500 MHz, DMSO-d ) δ: 7.29 (br. s, 2H, NH ), 7.37
6
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(d, J = 8.3 Hz, 2H, ArH), 7.62 (d, J = 8.3 Hz, 2H, ArH), 7.82 (d, J = 8.5 Hz, 2H, ArH), 7.92 (d, J = 8.5
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Hz, 2H, ArH), 10.36 (s, 1H, NH), 10.51 (s, 1H, NH); C NMR (126 MHz, (CD ) CO) δ: 123.2, 129.5,
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30.0, 131.1, 132.6, 137.0, 139.3, 143.6, 157.0, 163.2; MS (ESI-): 385 (100%, ( Cl) M-H) , HRMS
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(ESI-) calcd for C H Cl N O S: 384.9929, found 384.9935.
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(Z)-N-(4-Bromophenyl)-2-oxo-2-((4-sulfamoylphenyl)amino)acetohydrazonoyl chloride (16d): Using
general procedure C and 4-bromoaniline 12d (171 mg, 1 mmol), compound 16d (137 mg, 32%) was
1
isolated as a yellow solid, m.p. >260 °C. H NMR (500 MHz, DMSO-d ) δ: 7.29 (br. s, 2H, NH ), 7.50
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(d, J = 9.0 Hz, 2H, ArH), 7.56 (d, J = 9.0 Hz, 2H, ArH), 7.81 (d, J = 9.0 Hz, 2H, ArH), 7.91 (d, J = 9.0
13
Hz, 2H, ArH), 10.37 (s, 1H, NH), 10.51 (s, 1H, NH); C NMR (126 MHz, DMSO-d ) δ: 113.5, 116.8,
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18.5, 120.2, 126.3, 131.5, 138.9, 141.0, 142.0, 157.2; MS (ESI-): 429 (100%, M-H) , HRMS (ESI-)
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calcd for C H Br ClN O S: 428.9424, found 428.9428.
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(Z)-N-(4-Methoxyphenyl)-2-oxo-2-((4-sulfamoylphenyl)amino)acetohydrazonoyl chloride (16e): Using
general procedure C and 4-anisidine 12e (123 mg, 1 mmol), compound 16e (69 mg, 18%) was isolated
1
as a yellow solid, m.p. 250 °C. H NMR (500 MHz, DMSO-d ) δ: 3.72 (s, 3H, OCH ), 7.26 (br. s, 2H,
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NH ), 7.51 (d, J = 9.0 Hz, 2H, ArH), 7.78 (d, J = 9.0 Hz, 2H, ArH), 7.82 (d, J = 9.0 Hz, 2H, ArH), 7.89
2
13
(
d, J = 9.0 Hz, 2H, ArH), 10.24 (s, 1H, NH), 10.27 (s, 1H, NH); C NMR (126 MHz, DMSO-d ) δ:
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5.7, 114.8, 116.6, 118.2, 120.6, 126.4, 136.9, 139.9, 141.9, 155.3, 158.1; MS (ESI-): 381 (100%,
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( Cl) M-H) , HRMS (ESI-) calcd for C H ClN O S: 381.0424, found 381.0407.
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CA II protein was concentrated to 7 mg/mL and set up in SD-2 plates (Molecular Dimensions) with
00 nL plus 200 nL drops (protein plus reservoir solution) over 50 μL reservoirs and incubated at 8º C.
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The reservoir conditions consisted of 2.5-2.8 M ammonium sulfate with 100 mM Tris pH 8.5-9.0.
Compounds were soaked into the crystals for approximately 24 hours and subsequently glycerol (20%
final concentration) was added to cryoprotect the crystals for cryo-cooling. Data were collected at the
Australian Synchrotron MX1 beamline in Oct 2017 and at the MX2 beamline in Nov 2017. Data were
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auto-processed with XDS and then Aimless was used to scale the reflections, Phaser was used
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with model 4cq0 to phase the data and Coot was used to manually place the atoms in electron density.
AFITT (OpenEye Scientific Software) was used to place the ligands into density and generate the
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dictionary files. REFMAC was used to refine the structures. The 11d (high resolution at 1.32 Å)
structure used anisotropic refinement whereas the other structures were refined using isotropic
refinement. The models and structure factors have been deposited at the RCSB with PDB codes: 6UFB
(ligand 11a), 6UFC (ligand 11g) and 6UFD (ligand 11g).
4.3. CA Inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
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hydration activity. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and 20 mM Na SO (for
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maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO hydration
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reaction for a period of 10−100 s. The CO concentrations ranged from 1.7 to 17 mM for the
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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 velocity. 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
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done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6
hours at room temperature prior to assay in order to allow for the formation of the E−I complex. The
inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the
Cheng−Prusoff equation, as reported earlier,32, 33 and represent the mean from at least three different
determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier. 34, 35
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.4. Hypertensive Rabbit IOP Lowering Studies
The experimental procedures were carried out on New Zealand albino rabbits, following the Resolution
of the Association for Research in Vision and Ophthalmology, the Good Laboratory Practice for the
use of animals upon authorization of Italian Ministry of Health (number 1179/2015-PR), in agreement
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with the European Union Regulations (OJ of ECL 358/1, 12/12/1986). Male albino rabbits (body
weight 2-2.5 kg) were kept in individual cages, food and water was provided ad libitum. The animals
were maintained on a 12 h-12 h light/dark cycle in a temperature-controlled room (22-23 °C). Animals
were identified with a tattoo in the ear, numbered consecutively and examined before the beginning of
the study to verify the good general and ophthalmic health condition. All the compounds were
dissolved in pyrogen free sterile 0.9% NACl solution (i.e. physiologic solution) and 0.1% DMSO,
0
.1% EtOH at 1 mM concentration. Vehicle was 0.9% NaCl + 0.1% DMSO + 0.1% EtOH. The
viability of compounds was evaluated after repeated administration using the Draize Eye Test
(Wilhelmus, 2001). Topical delivery into the conjunctival cul-de-sac is the most common route of
ocular drug delivery. All compounds were given prior to saline injection and the IOP was measured at
the very beginning of the experimental session to establish basal IOP. Ocular hypertension was induced
by the injection of 0.05 mL of sterile hypertonic saline (5%) into the vitreous bilaterally with local
anaesthesia provided by one drop of 0.2% oxybuprocaine hydrochloride in each eye one minute prior.
IOP was measured using a Pneumotonometer (Reichert Inc. Depew, NY) after hypertonic saline
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injection after stabilization (normally 10 minutes) to verify the rise of IOP into the suitable
experimental range (IOP >30 and < 40 mmHg) and after 60, 90, 120, 240 minutes in all groups after
drug or vehicle treatment. One drop of 0.2% oxybuprocaine hydrochloride was instilled in each eye
immediately before each set of pressure measurements
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ASSOCIATED CONTENT
The Supporting Information is available free of charge on the ACS Publications website at DOI: NMR
spectra of compounds 9, 11a-g, 14a-h, 15, 16a-d; HPLC purity; X-ray Crystallography data 6UFB
(ligand 11a), 6UFC (ligand 11d) and 6UFD (ligand 11g). Figure 2 pdb 1CA2. Molecular formula
strings. Authors will release the atomic coordinates and experimental data upon article publication.
AUTHOR INFORMATION
Corresponding Author
*(P.A.K.) Telephone: +61 2 42214692; E-mail:
keller@uow.edu.au
*
(C.T.S.) Telephone: +39 0554573729; E-mail:
claudiu.supuran@unifi.it.
Notes
The authors declare no competing financial interest
Funding Sources
ACKNOWLEDGMENTS
MF thanks UOW for a University Postgraduate Award and International Postgraduate Tuition Award
scholarships and USyd for an internship. We thank OpenEye Scientific Software for a license to use
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Afitt, the CSIRO Collaborative Crystallization Centre (www.csiro.au/c3) for all crystallization
experiments, and the Australian Synchrotron and the beamline scientists at the MX beamlines for their
support in data collection.
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ABBREVIATIONS
CA, carbonic anhydrase; CAI, CA inhibitor; K , inhibition constant; IOP, intraocular pressure; TLC,
I
thin layer chromatography.
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