Design, synthesis and biological evaluation of carbohydrate-based sulphonamide derivatives as topical antiglaucoma agents through selective inhibition of carbonic anhydrase II

Article abstract of DOI:10.1080/14756366.2019.1705293

A series of new carbohydrate-based sulphonamide derivatives were designed, synthesised by employing the so-call ‘sugar-tail’ approach. The compounds were evaluated in vitro against a panel of CAs. Compared to their parent compound p-sulfamoylbenzoic acid, these compounds showed nearly 100-fold improvement in their binding affinities against hCA II in vitro. All of compounds showed great water solubility and the pH value of their water solutions of compounds is 7.0. Such properties are advantageous to make them much less irritating to the eye when applied topical glaucomatous drugs, compared to the relatively highly acidic dorzolamide preparations (pH 5.5). Notably, compounds 7d, 7 g, 7 h demonstrated to topically lower intraocular pressure (IOP) in glaucomatous animals better than brinzolamide when applied as a 1% solution directly into the eye. Low cytotoxicity on human cornea epithelial cell was observed in the tested concentrations by the MTT assay.

Full text of DOI:10.1080/14756366.2019.1705293

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation of
carbohydrate-based sulphonamide derivatives
as topical antiglaucoma agents through selective
inhibition of carbonic anhydrase II
Zhuang Hou, Chuanchao Li, Yichuang Liu, Miao Zhang, Yitong Wang,
Zhanfang Fan, Chun Guo, Bin Lin & Yang Liu
To cite this article: Zhuang Hou, Chuanchao Li, Yichuang Liu, Miao Zhang, Yitong Wang,
Zhanfang Fan, Chun Guo, Bin Lin & Yang Liu (2020) Design, synthesis and biological evaluation
of carbohydrate-based sulphonamide derivatives as topical antiglaucoma agents through selective
inhibition of carbonic anhydrase II, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
383-390, DOI: 10.1080/14756366.2019.1705293
To link to this article: https://doi.org/10.1080/14756366.2019.1705293
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 383–390
https://doi.org/10.1080/14756366.2019.1705293
RESEARCH PAPER
Design, synthesis and biological evaluation of carbohydrate-based sulphonamide
derivatives as topical antiglaucoma agents through selective inhibition of carbonic
anhydrase II
Zhuang Hou , Chuanchao Li, Yichuang Liu, Miao Zhang, Yitong Wang, Zhanfang Fan, Chun Guo, Bin Lin and
Yang Liu
Key Laboratory of Structure-Based Drugs Design and Discovery (Ministry of Education), School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Shenyang, China
ABSTRACT
ARTICLE HISTORY
Received 10 October 2019
Revised 3 December 2019
Accepted 5 December 2019
A series of new carbohydrate-based sulphonamide derivatives were designed, synthesised by employing
the so-call ‘sugar-tail’ approach. The compounds were evaluated in vitro against a panel of CAs. Compared
to their parent compound p-sulfamoylbenzoic acid, these compounds showed nearly 100-fold improve-
ment in their binding affinities against hCA II in vitro. All of compounds showed great water solubility and
the pH value of their water solutions of compounds is 7.0. Such properties are advantageous to make
them much less irritating to the eye when applied topical glaucomatous drugs, compared to the relatively
highly acidic dorzolamide preparations (pH 5.5). Notably, compounds 7d, 7 g, 7 h demonstrated to topic-
ally lower intraocular pressure (IOP) in glaucomatous animals better than brinzolamide when applied as a
1% solution directly into the eye. Low cytotoxicity on human cornea epithelial cell was observed in the
tested concentrations by the MTT assay.
KEYWORDS
Carbonic anhydrase II
inhibitors; sugar tail;
hydrophilic half;
antiglaucoma
Introduction
or reddening of the eye, blurred vision, pruritus, and other local side
effects after the topical administration (Figure 1)13,14
.
Glaucoma is an ophthalmologic disease featured by a high intraocu-
lar pressure (IOP), ultimately resulting in the irreversible loss of per-
ipheral vision, and eventually into blindness1. Clinically, there are
many treatments available for glaucoma such as surgery, laser, and
pharmacologic therapies. For the majority of glaucoma patients, low-
ering IOP by pharmacologic therapy is the preferred choice. It is well-
known that the systemic administration of inhibitors of carbonic
anhydrase (CA) II, an enzyme present in ciliary epithelial cells located
in the ciliary body, reduces aqueous humour secretion and lowers
the IOP2. Carbonic anhydrase inhibitors (CAIs) such as acetazolamide
(AAZ), methazolamide (MZA), ethoxzolamide (EZA), and dichlorophe-
namide (DCP) can be used as pressure-lowering systemic drugs in
the treatment of glaucoma3–5. However, they may cause undesired
side effects such as fatigue, augmented diuresis, or paresthesias due
to inhibition of the carbonic anhydrase isoforms present in many
other tissues and organs than the eye6,7. To avoid the above-men-
tioned side effects, it is better for the inhibitors to have topical activ-
ity therefore can be applied directly into the eye. Currently, two
topically effective CAIs have been developed: dorzolamide (DZA) and
brinzolamide (BRZ)8,9. Both drugs can be applied topically as water
To reduce this side effect, many efforts were made to design
topical antiglaucoma sulphonamides with more balanced pKas.
One successful approach for designing topical CAIs was termed
“water-solubilizing tail approach”, which was a special version of
‘sugar-tail approach’. It has been applied for the synthesis of
many classes of topical antiglaucoma sulphonamide CAIs15–19. In
this study, we employed this approach for designing and obtain-
ing water-soluble, high-affinity sulphonamide CA inhibitors, which
have great water solubility without the need to form hydrochlor-
ide salts. Herein, we report our work for obtaining carbohydrate-
based CA II inhibitors with great water solubility in the neutral pH
range, and potent and long-lasting in vivo properties as IOP lower-
ing agents in glaucoma animal models.
Results and discussion
Sugar-tail approach to compound design
The main goal of this study was to obtain highly potent, water
soluble CA inhibitors with potentially few side effects owing to
the neutral pH of the eye drop solution when these agents were
used in the treatment of glaucoma. Sugar-tail approach was
employed for the following reasons (Figure 2): (1) It was expected
that compounds designed by this approach could form more
solutions/suspensions and produce a prolonged reduction of IOP10,11
.
They showed fewer side effects as compared to the systemically
applied sulphonamides. Clinically, they are used as hydrochloride
salts in need of good water solubility12. However, in many cases it
will lead to the lowering of the pH in such solutions. Consequently, interactions with the CA active site for the hydrophilic glycosyl
the acidic solution will result in frequent stinging sensations, burning moiety to interact with the hydrophilic half of the active site. (2)
CONTACT Yang Liu
y.liu@syphu.edu.cn; Bin Lin
randybinlin@gmail.com Key Laboratory of Structure-Based Drugs Design and Discovery (Ministry of
Education), School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, 110016, China
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

384
Z. HOU ET AL.
Figure 1. Clinically used sulphonamides AAZ, MZA, EZA, DCP, DZA and BRZ.
Figure 2. Structures of the sugar-tail compounds 7a–7h.
Due to the highly hydrophilic nature of sugar moieties, good Intermediate 4 was formed by the reaction of a glycosyl bromide
water solubility could be achieved by incorporating them. (3) The with an excess of sodium azide. Next, the intermediate 4 was
pH of the water solutions of these compounds was in the neutral reacted with hydrogen under the catalysis of Pd/C to form an inter-
mediate 5. The condensation reaction of amino sugar with p-sul-
phonamide benzoic acid forms an intermediate 6 with an amide
bond as a linker. Finally, deacetylation with a catalytic amount of
NaOCH3 in CH3OH gave the target analogue 720. All compounds
were extensively characterised (Supplementary Material).
The different sugar derivatives were prepared by the same
method. Direct condensation of monosaccharides, such as: glu-
cose derivative 7a, galactose derivative 7b, xylcose derivative 7c,
arabinesose derivative 7d, ribose derivative 7e, rhamnose deriva-
tive 7f, glucuronic acid derivative 7g, manose derivative 7h.
pH range therefore there was no need to form hydrochloride salts,
making them much less irritating to the eyes. Target compounds
were designed by attaching aminosaccharide group as the hydro-
philic fragment via an amide bond linker to a p-sulfamoylbenzoic
acid as the zinc binding group (ZBG). Different types of aminosac-
charides were chosen to provide insight into the structure-activity
relationship (SAR) (Figure 2). Sugars used in this study were glu-
cose, galactose, arabinose, xylose, ribose, rhamnose, glucuronic
acid and mannose.
Chemistry
Biological activity
The general synthetic strategy for the synthesis of the target com-
pounds is shown in Scheme 1. Monosaccharide was selected as the CA inhibition studies
starting material, and acetylation with acetic anhydride to form
To determine the enzymatic inhibitory activity, all the newly syn-
intermediate 2 in pyridine. The terminal glycosyl bromide 3 was thesised sugar derivatives 7a–7h were evaluated by enzyme
prepared by the reaction of 2 with HBr-AcOH in good yield. assays in vitro on three physiologically relevant CA isoforms

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
385
Scheme 1. Synthesis of representative compound 7a. Reagents and Conditions: (a) Ac2O, pyridine, 5 h; (b) HBr-AcOH, CH2Cl2, r.t., 6 h, 62%; (c) NaN3, DMF, r.t. 5 h, 85%;
(d) Pd/C, EA, 2 h, 89%; (e) EDCI, DCM, r.t., 2 h, 74%; (f) NaOMe, MeOH, r.t., 85%.
Table 2. The solubility and pH of the solutions.
Table 1. The inhibitory activities of the compounds against hCA II and hCA I.
IC50 (lM)a,b
Selectivity ratio
hCA I hCA II hCA IX hCA I/hCA II hCA IX/hCA II
Compounds
Solubility (%)
pH
AAZ
DZA
BRZ
7a
0.21
0.67
0.05
1.0
6.5
5.5
5.5
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
Compounds
p-Sulfamoylbenzoic acid 120
15
0.27
24
0.69
8
1.6
2.5
4.5
8.2
9.0
10.9
10.9
17
7a
7b
7c
7d
7e
7f
7g
7h
AAZ
12
10
7
4
2.3
2.5
1.8
3.6
0.3
44.4
47.6
77.7
66.6
28.8
35.7
36.0
0.21
0.09
0.06
0.08
0.07
0.05
0.009
0.012
0.95
0.74
0.54
0.87
0.76
0.85
0.65
0.030
7b
2.5
7c
1.5
7d
7e
1.0
2.5
7f
1.0
7g
1.0
400
25
7.2
2.5
7h
2.0
aHuman recombinant enzymes, by the esterase assay (4-nitrophenylacetate
as substrate).
sulfamoylbenzoic acid (IC50 ¼ 24 lM) for hCA IX inhibition.
This is also presumably due to the matching the hydrophilic
half of the active site with the hydrophilic glucosyl moiety, in
addition to the sulphonamide as the zinc binding group.
bRelative errors were in the range of 5–10% of the reported values, calculated
from three independent assays.
including CA I, CA II and CA IX (Table 1). AAZ was chosen as the iv. Then, the selectivity of compounds 7a–7h was explored for
CA I, CA II and CA IX. The parent fragment p-sulfamoylben-
zoic acid displayed weak selectivity for hCA II with respect to
isozymes hCA I (8-fold) and hCA IX (1.6-fold). The compounds
7a–7h displayed high selectivity for the hCA II isozyme com-
control compound.
Inhibition data for sugar derivatives 7a–7h reported here, p-sul-
famoylbenzoic acid and clinically used agent AAZ are reported in
Table 1. The following structure-activity relationship (SAR) were
summarised based on the CA inhibition data with these sugar
derivatives investigated here:
pared with both hCA
I (400-fold) and IX (17-fold).
Collectively, these results showed it was possible to generate
hCA II selective inhibitors by tethering a sugar tail onto the
CA anchor pharmacophore over the physiologically abundant
hCA I and IX isozymes. These results also indicated that
selective inhibition of carbonic anhydrase II of sugar-deriva-
tives may reduce the side effects of current clinical drugs,
and is expected to be a good candidate for treatment of
antiglaucoma.
i. The ubiquitously isoform hCA I in the body was weakly
inhibited by the parent compound p-sulfamoylbenzoic acid
(IC50 of 120 lM), as well as by sugar derivatives 7a–7h (IC50
1.8–12 lM). As CA I is considered an off-target CA isozyme,
this weak CA I inhibition is desirable for the compounds
designed to target the glaucoma-associated enzymes.
ii. Against the physiologically dominant isoform hCA II, these
sugar compounds showed IC50 values of 0.009–0.27 lM
(Table 1). Compared to the parent compound, p-sulfamoyl-
benzoic acid, which has an IC50 value of 15 lM against hCA
II, the sugar derivatives 7a–7h are at least two orders of
magnitude more potent. Compound 7h, possessing manose
fragment in its structure, was the best hCA II inhibitor (IC50
¼ 0.009 nM). The combination of both designed by the
sugar-tail approach increased the inhibitory activity by two
orders of magnitude, presumably due to simultaneously
binding to the zinc ion and occupying the hydrophilic sub-
pocket of the active site. Such modes of binding were further
analysed and supported in detail in the molecular docking
analysis section.
Solubility and pH assay
Generally, in order to have noticeable pharmacological effects,
the majority of topical antiglaucoma drugs need to possess
acceptable solubility. For example, dorzolamide must be dis-
solved as hydrochloride salt and as a consequence, the rather
acidic solution (pH ¼ 5.5) causing eye irritation. Therefore,
some important physicochemical properties of new sulphona-
mides were investigated in detail (Table 2). Firstly, we explored
the solubility of compounds 7a–7h in water. Compounds
7a–7h showed excellent water solubility which would be suit-
able for developing topical IOP lowering agents. In contrast,
clinically used carbonic anhydrase inhibitors such as AAZ, DZA
and BRZ possess lower water solubility. Next, we explored the
pH values of compounds 7a–7h in water. It was shown that
all the solutions had pH of 7 while the solutions of AAZ, DZA,
iii. The SAR for the inhibition of the tumor-associated isoform
hCA IX was similar to hCA II: Notably, compounds 7a–7h
(IC50
¼
0.54–0.95 lM) were also much better than p- and BRZ had pH lower than 7 (Table 2). Thus, it was easy to

386
Z. HOU ET AL.
0
60
120
180
240
300
360
0
-1
-2
-3
-4
-5
-6
-7
-8
-9
BRZ
7d
7g
7h
Vehicle
Time（min）
Figure 3. Effect of topically administered sulphonamide CA inhibitors (1% water solutions/suspensions) on the IOP of hypertensive rabbits. Errors were in the range of
3–5% of the reported values.
formulate them as eye-drops at neutral pH values. This also
showed that due to the presence of the glycosyl tail in deriva-
tives 7a–7h their water solubility was drastically increased while
the pH of their solutions stayed at neutral pH, which consti-
tuted a very desirable pharmacological feature for topically act-
ing antiglaucoma drugs.
Topical antiglaucoma activity
The clinical typical symptom of glaucoma is IOP increase. Next, we
investigated the IOP lowering ability of the most active com-
pounds against the hCA II. An animal model of glaucoma, i.e., rab-
bits with high IOP, induced by the injection of 0.1 mL of
hypertonic saline solution (5% in distilled water) into the vitreous
of both eyes21–23
.
Figure 4. Effects of compounds 7a–7h, BRZ and AAZ, at 1% and 2% concentra-
tions, on the viability of HCEC cell line. Values are the average of three independ-
ent experiments. Relative errors are generally within 5–10%.
Three of the best in vitro CA inhibitors investigated here,
7d, 7g, 7h were formulated as 1% water solution and adminis-
tered topically to hypertensive rabbits. The variation of IOP of
hypertensive rabbits treated topically with one drop (50 lL) of
1% solution of inhibitors is shown in Figure 3. The drug BRZ
(1%, suspension) was also included in our experiments. It can
be seen that BRZ was an efficient IOP-lowering agent, with a
maximal effect (around 5 mm Hg) achieved at 1 h post-adminis-
tration, and a return to basal IOP values after 3 h. However,
the three sugar sulphanilamides investigated here were much
more effective IOP lowering agents as compared to the clinic-
ally used drug BRZ. Especially, the arabinose derivative 7d was
again maximally active after 1 h post-administration, producing
an IOP lowering of around 5 mm Hg. A potent IOP lowering of
3.5–5 mm Hg was then maintained for the next 4–5 h. The glu-
curonic acid derivative 7g was slightly more effective than 7d.
The maximal effect (6 mmHg) has been observed after 1 h post
administration, and it lasted for up to 4–5 h. The best IOP low-
ering agent was the manose derivative 7h. In this case, the
maximal IOP lowering (of around 8 mm Hg) has been achieved
Cytotoxicity of tested compounds on mammalian cells
In order to ensure selectivity of the antiglaucoma effects, the cyto-
toxicity of compounds 7a–7h were evaluated by MTT assay using
four mammalian cell lines, including HCEC (human corneal epithelial
cell), HepG2 (human liver carcinoma), HT-29 (human colorectral can-
cer) and H9C2 (normal, rat myocytes). Meanwhile, we compared the
effects of these sugar derivatives with those of the standard sul-
phonamide inhibitors AAZ and BRZ, working in the same conditions.
Compounds 7a–7h, AAZ and BRZ did not display any appreciable
toxicity to the four mammalian cell lines when tested up to 100 lM
(data not shown). However, our target compounds were designed as
a topical antiglaucoma drug, which was usually made into an eye
drop that concentration is 1–2%, so the topical concentration were
large. With this in mind, we prepared compounds 7a–7h at concen-
trations ranging from 1% to 2% and tested the cytotoxicity on HCEC
cell again. As shown in Figure 4, compounds 7a–7h had low toxicity
against the HCEC cell (74–88% cell viability) while the cell viability
after 2 h post-administration, and this very potent effect was rates of AAZ and BRZ towards HCEC cell were 80% and 40% at 1%
concentration. The increase of the concentration did significantly
affect the compound toxicity. For instance, at 2% concentration AAZ
showed a viability dropping (10%) and reduced cell viability was
observed for compounds 7a–7h (62–89% cell viability).
maintained for the next 5–6 h when pressure returned to the
basal values. Thus, the three derivatives showed much more
effective IOP lowering as compared to BRZ: both the magni-
tude of the effect as well as its duration of action were very
much augmented, which constitute very desirable properties for
topically acting antiglaucoma drugs. Moreover, no ocular dis-
comfort or eye irritation of the experimental animals have
been observed after administration of these sugar sulphona-
mide derivatives.
Molecular docking analyses
Molecular docking was employed to provide insight into the
inhibitory activities against the CA I, II and IX enzymes listed in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
387
Figure 5. The binding mode of compound 7c in the active site of CA I and II. (a) and (b) are surface representations for CA I and II, respectively. (c) and (d) are 2D
representations for CA I and II, respectively.
Table 1. The data showed that the inhibitory activities of the tar- amine N atom of compound 7c binds directly to the active-site Zn
get compounds were more of the same magnitude towards the
same enzyme. It was expected that the structural difference
among the sugars were minor and did not cause big difference in
the inhibitory activities. It was also observed that the inhibitory
activities of the target compounds were more of the same magni-
tude between CA II and CA IX while about one order of magni-
tude more potent for CA II than CA I. It was also not surprising
because sequence analysis showed that the amino acids com-
posed of the active sites were almost identical between CA II and
CA IX while there were several key differences between CA II and
CA I. Consequently, the following docking studies were performed
between compound 7c as the representative compound in the
active sites of CA II and CA I.
The docking studies started with a redocking experiment by
AutoDock 4.2.6 with the AutoDock4Zn force field for AAZ with CA
I and II (PDB: 1AZM and 3HS4). The redocking results showed that
the experimental pose of AAZ was reproduced fairly well, espe-
cially with the interactions between the sulphonamide functional
group of AAZ and the zinc ion in CA II. This is the characteristic of
sulphonamides as CA inhibitors, which the compounds in Table 1
will follow. Next, the same model was applied to the representa-
atom along with the side chains of HIS94, HIS96 and HIS119 and
the sulphonamide group forms a hydrogen bond to THR199.
Furthermore, the sugar moiety of the compound preferably inter-
acted with the hydrophilic region of the CA active site (high-
lighted in red in Figure 5). It is consistent with the fact that sugars
have many hydroxyl groups in their structures therefore they are
highly hydrophilic and they tend to interact with the hydrophilic
region of the active site favourably. The major difference in the
active sites of CA I and II is the ASN 67 in CA II, which could form
hydrogen bond with the nearby hydroxyl group in the sugar while
the corresponding amino acid in CA I is HIS 67, which could not
form hydrogen bond with the same hydroxyl group because it
could not satisfy the criteria of distance and angle constraint.
Such evidence explained that why compound 7c was about one
order of magnitude more active against CA II than CA I.
Conclusion
Here, we report a novel class of water-soluble, topically very
effective and long-acting antiglaucoma sulphonamides, obtained
tive compound 7c. The results showed that the sulphonamide by attaching aminosaccharide moieties to well-known aromatic

388
Z. HOU ET AL.
sulphonamides incorporating free carboxy group. Eight of the (1.0 mol/l, 1 mL) was added. After it was stirred overnight, the mix-
novel carbohydrate-based compounds exhibited excellent CA II ture was neutralised with Dowex H þ resin to pH 7, then filtered.
inhibitory activity with 100-fold of the initial fragment p-sulfamoyl- The filtrate was concentrated and purified by a silica gel column
benzoic acid. In addition, all of the newly reported derivatives chromatography (5:1, CH2Cl2-MeOH) to afford 7a (52 mg, 47%) as
a white solid.
showed very good water solubility, in the range of 1–2.5% at neu-
tral pH values. In particular, some of the most active inhibitors
strongly lowered IOP in glaucomatous rabbits, showing a pro-
longed duration of action in comparison to brinzolamide. The
cytotoxicity on human cornea epithelial cell from novel carbohy-
drate-based compounds 7a–7h was evaluated, and low cytotox-
icity was observed. These compounds constitute valuable leading
compounds for obtaining topically acting antiglaucoma CAIs of a
new generation, with improved efficacy, longer duration of action,
and reduced side effects.
4-Aminosulfonyl-N-(b-D-glucopyranosyl) benzamide (7a)
M.p. 112.2–114.2 ꢀC; 1H NMR (400 MHz, DMSO)
d 9.04 (d,
J ¼ 8.8 Hz, 1H), 8.06 (d, J ¼ 8.4 Hz, 2H), 7.91 (d, J ¼ 8.4 Hz, 2H), 7.48
(s, 2H), 5.02 (d, J ¼ 4.5 Hz, 1H), 4.99 (d, J ¼ 5.3 Hz, 1H), 4.95 (t,
J ¼ 9.0 Hz, 1H), 4.91 (d, J ¼ 5.3 Hz, 1H), 4.51 (t, J ¼ 5.7 Hz, 1H), 3.68
(dd, J ¼ 10.3, 5.6 Hz, 1H), 3.44 (dt, J ¼ 11.5, 5.7 Hz, 1H), 3.37–3.32
(m, 1H), 3.28–3.23 (m, 1H), 3.22–3.17 (m, 1H), 3.14–3.07 (m, 1H).
13C NMR (151 MHz, DMSO) d 166.11, 146.93, 137.47, 128.74,
126.00, 80.82, 79.31, 77.93, 72.58, 70.47, 61.42. ESI-MS (m/z): 363.1
[M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ C13H19N2O8S: 363.0817,
Found 363.0858.
Experimental section
Chemistry
Reagents and starting materials in current study were of analytic
grade and used without further purification unless otherwise
specified. Analytical thin layer chromatography was performed on
silica gel HF254 plates. Preparative column chromatography was
performed using silica gel H. Melting points were determined by a
4-Aminosulfonyl-N-(b-D-galactopyranosyl) benzamide (7 b)
M.p. 115.7–117.3 ꢀC; 1H NMR (600 MHz, DMSO)
d 9.10 (d,
J ¼ 8.9 Hz, 1H), 8.08 (d, J ¼ 8.4 Hz, 2H), 7.89 (d, J ¼ 8.3 Hz, 2H), 7.49
(s, 2H), 4.92 (t, J ¼ 8.9 Hz, 1H), 4.88 (s, 2H), 4.62 (s, 1H), 4.50 (s, 1H),
3.73 (s, 1H), 3.65 (t, J ¼ 9.2 Hz, 1H), 3.52 (s, 1H), 3.46 (s, 2H), 3.40 (s,
1H). 13 C NMR (151 MHz, DMSO) d 166.12, 146.86, 137.52, 128.81,
125.92, 81.31, 77.48, 74.59, 69.83, 68.77, 61.02. ESI-MS (m/z): 363.1
[M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ C13H19N2O8S: 363.0817,
Found 363.0850.
Buchi melting point B-540 apparatus. All 1H and 13C NMR spectra
€
were recorded at 600 MHz and 150 MHz on a Bruker ARX 600 MHz
model spectrometer in DMSO-d6 with TMS as the internal stand-
ard. NMR spectra were analysed and interpreted using
MestReNova. ESI-MS spectra were recorded on an Agilent ESI-
QTOF instrument. HR-MS were obtained on a Bruker micrOTOF_Q
spectrometer.
4-Aminosulfonyl-N-(b-L-arabinopyranosyl) benzamide (7c)
M.p. 114.7–116.6 ꢀC; 1H NMR (400 MHz, DMSO)
d 9.04 (d,
J ¼ 8.7 Hz, 1H), 8.05 (d, J ¼ 8.5 Hz, 2H), 7.91 (d, J ¼ 8.5 Hz, 2H), 7.49
(s, 2H), 5.08 (d, J ¼ 4.5 Hz, 1H), 5.04 (d, J ¼ 5.3 Hz, 1H), 4.99 (d,
J ¼ 5.0 Hz, 1H), 4.86 (t, J ¼ 8.9 Hz, 1H), 3.70 (dd, J ¼ 11.1, 5.2 Hz, 1H),
3.36 (s, 1H), 3.30 (dd, J ¼ 9.4, 5.2 Hz, 1H), 3.20 (td, J ¼ 8.8, 4.5 Hz,
1H), 3.11 (t, J ¼ 10.8 Hz, 1H). 13 C NMR (151 MHz, DMSO) d 166.37,
146.96, 137.41, 128.74, 126.02, 81.62, 77.95, 72.33, 70.13, 68.01.
ESI-MS (m/z): 333.1 [M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ
C12H17N2O7S: 333.0712, Found 333.0748.
General procedure for the synthesis of compounds (7a–7g)
To a solution of 1 (5 g, 27.8 mmol) in pyridine (50 mL) was added
acetic anhydride (35 mL). The reaction mixture was stirred for 5 h
and then H2O (100 mL) was added and stirring continued for
20 min. The reaction mixture was washed with 0.1 M HCl (15 mL),
satd aq NaHCO3 (15 mL), and dried (MgSO4), Filtration, evaporation
of solvent under diminished pressure to dryness to give 2. To a
solution of 2 in dichloromethane (35 mL) was added HBr-HOAc
(35 mL). The reaction mixture was stirred for 5 h and then H2O
(50 mL) was added and stirring continued for 20 min. The reaction
mixture was washed with satd aq NaHCO3 (25 mL), and dried
(MgSO4). Filtration, evaporation of solvent under diminished pres-
sure and chromatography of the residue (EtOAc-petroleum ether,
1:8) gave 3 as a white solid (4.4 g, 65%). To a solution of 3 (4.4 g,
10.8 mmol) in DMF (30 mL) was added NaN3 (3.5 g, 53.8 mmol).
The reaction mixture was stirred for 4 h. Filtration, evaporation of
solvent under diminished pressure and chromatography of the
residue (EtOAc-petroleum ether, 1:5) gave 4 as a white solid (2.1 g,
53%). To a solution of 4 (2.1 g, 5.6 mmol) in EtOAc (20 mL) was
added Pd/C(1.05 g) and add hydrogen. The reaction mixture was
stirred for 2 h. Filtration, evaporation of solvent under diminished
pressure to dryness to give 5 (1.6 g, 85%). To a solution of com-
pound 5 (200 mg, 0.58 mmol) in dry CH2Cl2 (10 mL), p-sulfamoyl-
benzoic acid (116 mg, 0.58 mmol) and EDCI (111.2 mg,0.58 mmol)
were added at room temperature and the reaction mixture was
stirred for 2 h. The reaction mixture was washed with 0.1 M HCl
4-Aminosulfonyl-N-(b-D-xylopyranosyl) benzamide (7d)
M.p. 123.7–125.2 ꢀC; 1H NMR (400 MHz, DMSO)
d 9.06 (d,
J ¼ 8.7 Hz, 1H), 8.05 (d, J ¼ 8.4 Hz, 2H), 7.90 (d, J ¼ 8.4 Hz, 2H), 7.48
(s, 2H), 4.90 (d, J ¼ 5.1 Hz, 2H), 4.86 (d, J ¼ 8.5 Hz, 1H), 4.60 (d,
J ¼ 2.9 Hz, 1H), 3.70 (s, 1H), 3.69–3.67 (m, 1H), 3.67–3.61 (m, 1H),
3.50 (d, J ¼ 11.3 Hz, 1H), 3.45 (ddd, J ¼ 8.5, 5.2, 3.2 Hz, 1H). 13C
NMR (151 MHz, DMSO) d 166.17, 146.92, 137.50, 128.72, 126.01,
81.35, 73.90, 69.75, 68.49, 67.82. ESI-MS (m/z): 333.1 [M þ H]þ;
HRMS (ESI): Calcd for [M þ H]þ C12H17N2O7S: 333.0712,
Found 333.0748.
4-Aminosulfonyl-N-(b-D-ribofuranosyl) benzamide (7e)
M.p. 109.8–111.3 ꢀC; 1H NMR (600 MHz, DMSO)
d 8.85 (d,
J ¼ 8.1 Hz, 1H), 7.94 (s, 4H), 7.51 (s, 2H), 5.64 (s, 1H), 5.34 (dd,
J ¼ 8.1, 4.1 Hz, 1H), 5.22 (d, J ¼ 6.9 Hz, 1H), 4.93 (d, J ¼ 5.8 Hz, 1H),
3.93 (s, 1H), 3.65–3.62 (m, 1H), 3.58 (dd, J ¼ 5.3, 2.7 Hz, 2H), 3.37
(5 mL), satd aq NaHCO3 (5 mL), and dried (MgSO4), Filtration, evap- (q, J ¼ 9.0 Hz, 1H). 13C NMR (151 MHz, DMSO) d 165.76, 147.18,
oration of solvent under diminished pressure to dryness to give 6 137.51, 128.09, 126.44, 77.36, 71.73, 67.49, 67.04, 60.94. ESI-MS (m/
(162 mg, 53%). To a solution of 6 (162 mg, 0.31 mmol) in CH2Cl2- z): 333.1 [M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ C12H17N2O7S:
MeOH (1:1, 5 mL), freshly prepared NaOMe in MeOH solution 333.0712, Found 333.0737.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
389
4-Aminosulfonyl-N-(b-L-rhamnosyl) benzamide (7f)
M.p. 116.6–118.2 ꢀC; 1H NMR (600 MHz, DMSO)
MTT assay in vitro
d
8.47 (d,
Cell proliferation was evaluated using an MTT assay. Briefly,
5 ꢂ 104 cells per well were seeded into 96-well plates and grown
at 37 ꢀC for 24 h. Subsequently, compounds AAZ, BRZ and 7a–7g
were added to each well at increasing concentrations, and then
the cells were incubated for 96 h. The MTT solution was added to
each well, and the cells were incubated at 37 ꢀC for another 4 h.
Formazan crystals were dissolved in 150 mL of DMSO. Cell viability
was assessed by measuring the absorbance at 540 nm wavelength
using a microplate reader (BioTek ELx800 USA).
J ¼ 8.6 Hz, 1H), 8.03 (d, J ¼ 8.4 Hz, 2H), 7.89 (d, J ¼ 8.3 Hz, 2H), 7.50
(s, 2H), 5.23 (d, J ¼ 8.6 Hz, 1H), 5.05 (d, J ¼ 4.5 Hz, 1H), 4.81 (d,
J ¼ 5.3 Hz, 1H), 4.74 (d, J ¼ 5.9 Hz, 1H), 3.72 (s, 1H), 3.38 (ddd,
J ¼ 9.0, 5.9, 3.3 Hz, 1H), 3.25–3.20 (m, 1H), 3.20–3.15 (m, 1H), 1.17
(d, J ¼ 5.9 Hz, 3H). 13C NMR (151 MHz, DMSO) d 165.26, 147.03,
137.11, 128.74, 126.09, 78.93, 74.36, 74.02, 72.15, 71.09, 18.46. ESI-
MS (m/z): 347.1 [M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ
C13H19N2O7S: 347.0868, Found 347.0904.
Hypertensive rabbit IOP lowering studies
4-Aminosulfonyl-N-(b-D-glucuronic acid methyl ester) benza-
mide (7g)
All animal experiments were conformed to the guidelines of the
Animal Ethics Committee of Shenyang Pharmaceutical University.
Rabbits were provided by the Laboratory Animal Centre of
Shenyang Pharmaceutical University. Adult male rabbits weighing
2.0–2.5 kg were employed in our studies. Animals were anaesthe-
tised using zoletil and injected with 0.1 mL of hypertonic saline
solution (5% in distilled water) into the vitreous of both eyes. IOP
was measured using a Tono-Pen tonometer (Tono-pen Avia ton-
ometer, Reichhert Inc., Depew, NY 14043, USA) prior to hypertonic
saline injection (basal) at 1, 2, 3, 4, 5 and 6 h after drug adminis-
tration. Vehicle (phosphate buffer 7.00 plus DMSO 2%) or drugs
were instilled immediately after the injection of hypertonic saline.
Eyes were randomly assigned to different groups. Vehicle or drug
(0.50 mL) was directly instilled into the conjunctive pocket at the
desired doses (1 ꢁ 2%)21,22. Four different animals were used for
each tested compound.
M.p. 114.5–117.1 ꢀC; 1H NMR (600 MHz, DMSO)
d 8.78 (d,
J ¼ 8.6 Hz, 1H), 8.04 (d, J ¼ 8.3 Hz, 2H), 7.91 (d, J ¼ 8.4 Hz, 2H), 7.52
(s, 2H), 5.71 (dd, J ¼ 8.5, 5.3 Hz, 1H), 5.42 (d, J ¼ 5.2 Hz, 1H), 5.23 (d,
J ¼ 5.7 Hz, 1H), 5.12 (d, J ¼ 4.7 Hz, 1H), 4.06 (d, J ¼ 9.0 Hz, 1H), 3.82
(dd, J ¼ 8.5, 4.6 Hz, 1H), 3.64 (s, 3H), 3.59–3.55 (m, 1H), 3.44 (td,
J ¼ 8.6, 5.3 Hz, 1H). 13C NMR (151 MHz, DMSO) d 170.35, 167.31,
147.04, 137.45, 129.08, 125.87, 77.54, 73.51, 72.19, 72.04, 70.39,
52.24. ESI-MS (m/z): 391.1 [M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ
C14H19N2O9S: 391.0767, Found 391.0784.
4-Aminosulfonyl-N-(b-D-mannopyranosyl) benzamide (7h)
M.p. 112.6–114.4 ꢀC; 1H NMR (600 MHz, DMSO)
d 8.48 (d,
J ¼ 8.7 Hz, 1H), 8.03 (d, J ¼ 8.3 Hz, 2H), 7.90 (d, J ¼ 8.0 Hz, 2H), 7.50
(s, 2H), 5.27 (d, J ¼ 8.6 Hz, 1H), 5.05 (s, 1H), 4.77 (s, 2H), 4.48 (s,
1H), 3.71 (s, 2H), 3.45–3.41 (m, 2H), 3.17 (d, J ¼ 6.6 Hz, 2H). 13C
NMR (151 MHz, DMSO) d 165.18, 147.04, 137.09, 128.71, 126.12,
79.84, 78.90, 74.29, 71.02, 67.23, 61.76. ESI-MS (m/z): 363.1
[M þ H]þ; HRMS (ESI): Calcd for [M þ H]þ C13H19N2O8S: 363.0817,
Found 363.0840.
Molecular docking simulations
The experimental crystallographic structures of CA I and CA II
complex were retrieved from the Protein DataBank (PDB: 1AZM
and 3HS4). The molecular model of the sugar tail sulphonamide
derivative 7c were built by Discovery Studio 3.5. Both the protein
and the ligands were prepared by adding polar hydrogen atoms
and partial charges with the assistance of AutoDockTools 1.5.6.
AutoDockZN force field was used to handle the zinc ion in the
binding site. Grid points were increased to 60 at x, y, z axes. The
number of Dock runs was increased to 20. All other parameters
were kept as their default values.
CA inhibition
A multifunctional enzyme marking instrument has been used for
assaying CA isoenzyme activities according to the esterase assay
method described previously by Verpoorte et al.24. Initial rates of
4-nitrophenyl acetate hydrolysis catalysed by different CA iso-
zymes were recorded on a Cary 3 instrument at 400 nm interfaced
with a DELL-compatible PC. Solutions of substrate (4-nitrophenyl
acetate) were prepared in DMSO; the substrate concentration was
2 mM at room temperature. A molar absorption coefficient of
400 Mꢁ1 cmꢁ1 was used for the 4-nitrophenolate formed by
hydrolysis in the conditions of the experiments (pH 7.5).
Nonenzymatic hydrolysis rates were subtracted from the observed
rates. The experiments were repeated three times at each inhibitor
concentration. Stock solutions of the inhibitor (100 mM) were pre-
pared and diluted up to 0.5 nM with analytical buffer. At least
eight different inhibitor concentrations have been used, ranging
from 5 mM, 0.5 mM, 50 mM, 5 mM, 0.5 mM, 50 nM, 5 nM, 0.5 nM.
Inhibitor and enzyme solutions were preincubated together for
10 min at r.t. prior to the assay study, to allow for the formation
of the E-I complex. Enzyme concentrations were 1 ng/L for CA II25.
Isoforms hCA IX and hCA I have a low esterase activity compared
to hCA II. However, we also obtained a good test condition by
constantly adjusting the dosage of hCA IX and hCA I enzyme.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors appreciate the financial supports from the National
Natural Science Foundation of China [No. 81903463], Department
of Education of Liaoning Province [2017LQN03] and Department
of Science and Technology of Liaoning Province [20180540032].
ORCID
Zhuang Hou
Yang Liu
http://orcid.org/0000-0002-4933-4226
http://orcid.org/0000-0002-9284-1944

390
Z. HOU ET AL.
2018;28:709–12. (b) Supuran CT. Advances in structure-based
drug discovery of carbonic anhydrase inhibitors. Exp Opin
Drug Discov 2017;12:61–88.
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