Synthesis, molecular docking analysis and biological evaluations of saccharide-modified thiadiazole sulfonamide derivatives

Article abstract of DOI:10.3390/ijms22115482

A series of saccharide-modified thiadiazole sulfonamide derivatives has been designed and synthesized by the “tail approach” and evaluated for inhibitory activity against carbonic anhydrases II, IX, and XII. Most of the compounds showed high topological polar surface area (TPSA) values and excellent enzyme inhibitory activity. The impacts of some compounds on the viability of HT-29, MDA-MB-231, and MG-63 human cancer cell lines were examined under both normoxic and hypoxic conditions, and they showed certain inhibitory effects on cell viability. Moreover, it was found that the series of compounds had the ability to raise the pH of the tumor cell microenvironment. All the results proved that saccharide-modified thiadiazole sulfonamides have important research prospects for the development of CA IX inhibitors.

Full text of DOI:10.3390/ijms22115482

International Journal of
Molecular Sciences
Article
Synthesis, Molecular Docking Analysis and Biological
Evaluations of Saccharide-Modified Thiadiazole
Sulfonamide Derivatives
Zuo-Peng Zhang, Ye Zhong, Zhen-Bin Han, Lin Zhou, Hua-Sheng Su, Jian Wang, Yang Liu *
and Mao-Sheng Cheng *
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China; zzp2354172@gmail.com (Z.-P.Z.);
zymc666@gmail.com (Y.Z.); wuwudiyi@gmail.com (Z.-B.H.); zevangeline101@gmail.com (L.Z.);
huashengsu375@gmail.com (H.-S.S.); 102030118@syphu.edu.cn (J.W.)
* Correspondence: y.liu@syphu.edu.cn (Y.L.); mscheng@syphu.edu.cn (M.-S.C.)
Abstract: A series of saccharide-modified thiadiazole sulfonamide derivatives has been designed and
synthesized by the “tail approach” and evaluated for inhibitory activity against carbonic anhydrases
II, IX, and XII. Most of the compounds showed high topological polar surface area (TPSA) values
and excellent enzyme inhibitory activity. The impacts of some compounds on the viability of HT-29,
MDA-MB-231, and MG-63 human cancer cell lines were examined under both normoxic and hypoxic
conditions, and they showed certain inhibitory effects on cell viability. Moreover, it was found that
the series of compounds had the ability to raise the pH of the tumor cell microenvironment. All the
results proved that saccharide-modified thiadiazole sulfonamides have important research prospects
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
for the development of CA IX inhibitors.
Citation: Zhang, Z.-P.; Zhong, Y.;
Han, Z.-B.; Zhou, L.; Su, H.-S.; Wang,
Keywords: saccharide; tail approach; pH of the tumor cell microenvironment; CA IX inhibitors
J.; Liu, Y.; Cheng, M.-S. Synthesis,
Molecular Docking Analysis and
Biological Evaluations of
Saccharide-Modified Thiadiazole
Sulfonamide Derivatives. Int. J. Mol.
Sci. 2021, 22, 5482. https://doi.org/
10.3390/ijms22115482
1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are lyases that are widely distributed in all
organisms from archaea to higher animals. According to the different coding genes, CAs
can be divided into eight families:
genetically different CA families, human carbonic anhydrases (hCAs) belong to the
family [ ]. hCAs are a class of zinc metalloenzymes with 15 subtypes. In the human body,
their main physiological roles are as catalysts for the reversible interconversion of carbon
dioxide and bicarbonate [ ]. CA IX is a special isoform of the hCAs family. Its expression
in normal tissues is limited to gastrointestinal epithelial cells but it is overexpressed in many
solid tumors, contributing to the growth advantage of cancer cells [ 11]. Therefore, CA IX
is considered an important target for tumor treatment, and hCA modulators are considered
promising drugs in clinical applications [12]. At present, CA IX-targeted drugs have been
studied in clinical research. The CA IX inhibitor SLC-0111 combined with gemcitabine for
the treatment of metastatic pancreatic ductal cancer in patients overexpressing the CA IX
isoform has entered clinical phase II [13–15].
α-, β-, γ-, δ-, ζ-, η-, θ-, and ι-CAs. Among the eight
Academic Editor: Claudiu T. Supuran
α
-
1–4
Received: 12 April 2021
Accepted: 20 May 2021
Published: 22 May 2021
5–7
8–
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
X-ray crystallography studies showed that the catalytic domains of the hCA family are
highly similar, which causes great difficulties for the search for selective CA inhibitors [6].
Copyright:
© 2021 by the authors.
To alleviate the clinical side effects, carbonic anhydrase inhibitors such as brinzolamide, a
drug for glaucoma, are administered locally [16]. Local administration is difficult for CA
IX inhibitors, and the resulting side effects seriously hinder the development of selective
CA IX inhibitors. Until the “tail approach” was proposed, it provided a favorable weapon
for selective CA IX inhibitors [17]. The “tail approach” is a simple and effective method
that refers to appending a “tail” with a wide spectrum of chemical functionalities to the
Zn2+ binding group through a simple chemical reaction to change the mode of action of
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the inhibitor in different isoforms of CAs. A proper “tail” structure is of great significance
for the research on selective CA IX inhibitors. One of the characteristics of CA IX is that its
subcellular localization is outside the cell membrane, while its common off-target isoform
CA II is located in the cytoplasm (Figure 1). The “tail” modification of appropriate frag-
ments can improve the polarity of inhibitors to reduce cell membrane permeability, which
is conducive to obtaining CA IX selectivity. Saccharides have the characteristics of low
toxicity, high polarity, and high solubility. In the area of medicinal chemistry, glycol deriva-
tives and glycosyl modification compounds have shown a wide range of pharmacological
activities [18
have achieved remarkable success, and various molecules of saccharide-modified CA IX in-
hibitors have been reported [23 25]. The high polarity of saccharide-modified compounds
–22]. In the research on CA IX inhibitors, saccharide-modified compounds
–
puts them at a disadvantage when they penetrate the cell membrane, which is conducive to
the selectivity. At the same time, the hydrogen bonding between the saccharide fragment
and the residues in the hydrophilic pocket at the rim of the enzymatic cavity is beneficial
to improvement of the activity.
Figure 1. Subcellular distribution of hCAs.
To investigate the application of saccharide modifications in CA IX inhibitors, we de-
signed and synthesized sixteen novel compounds in this work by linking glucose fragments
and thiadiazole sulfonamides. These compounds were then tested for their inhibitory ef-
fects on the tumor-associated proteins hCA IX and XII and off-target hCA II subtypes.
Their inhibitory activities were comparable to that of the positive control AZM in the low
nanomolar region. The study of molecular docking studies and the summary of preliminary
structure–activity relationships have provided a basis for further research.
2. Results and Discussion
2.1. Chemistry
The general synthetic route for the synthesis of the target saccharide-modified thiadi-
azole sulfonamide derivatives is shown in Scheme 1. We adopted a convergent strategy
to complete the syntheses of compounds, which mainly include two parts: saccharide
fragments and small molecule fragments. In the saccharide fragment, glucose was chosen
as the starting material, and intermediate
4 was synthesized from glucose by benzoylation
(
2
), bromination ( ) and azide substitution. After hydrogen reduction with Pd/C as the
3
catalyst, the product underwent a one pot reaction with succinic anhydride or glutaric
anhydride to generate saccharide intermediates 5a and 5b. In the small molecule fragment,
different substituted aminobenzoic acids 6a
acid chlorides after protection of the amino groups. Acid chlorides 8a
intermediate 10 to give the corresponding intermediates 11a
deprotected to obtain small molecule intermediates 12a–h.
–
h
were converted into the corresponding
reacted with
. Intermediates 11a were
–
h
–h
–h

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Scheme 1. The general synthesis route of compounds 14a–p. Reagents and conditions: (i) BzCl,
Scheme 1. The general synthesis route of compounds 14a–p. Reagents and conditions: (i) BzCl,
4-dimethylaminopyridine (DMAP), Py, r.t.; (ii) HBr-HOAc, dichloromethane (DCM), 0 ◦C -r.t.;
◦
(iii) NaN3, N, N-Dimethylformamide (DMF), 50 C; (iv) Pd/C, H2, succinic anhydride/glutaric
anhydride, r.t.; (v) Tetrahydrofuran (THF)/CHCl3, Fmoc-Cl, 60 ◦C; (vi) SOCl2, reflux; (vii)
EtOH, HCl, reflux; (viii) acetone, Py, 0 ◦C-r.t.; (ix) diethylamine, DMF, r.t.; (x) N-ethyl-N0-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI), Py, r.t.; (xi) MeOH/Et3N/H2O, r.t.
In the presence of EDCI, saccharide intermediates (5a
termediates (12a ) were condensed to obtain intermediates 13a
compounds 14a were obtained by deprotection (some compounds contained a few
/5b) and small molecule in-
–
h
–p. Finally, the target
–
p
amounts of stereoisomers).
2.2. Carbonic Anhydrase Inhibition
The inhibitory activities of the novel saccharide-modified thiadiazole sulfonamide
derivatives 14a–p and small molecule intermediates 12a–h toward three physiologically
relevant hCA isoforms, hCA II (common off-target cytoplasmic isoform), hCA IX, and
XII (tumor-associated membrane-bound isoforms), were evaluated by monitoring the
hydrolysis of 4-nitrophenyl acetate (4-NPA). AZM was chosen as the positive control drug.
The inhibition data are summarized in Table 1.
Table 1. The IC50 against relevant hCA isoforms and the topological polar surface area (TPSA) of the
intermediates 12a–h and compounds 14a–p (Comp. 12a–h and 14a–p).
hCA II
(IC50, nM)
hCA IX
(IC50, nM)
hCA XII
(IC50, nM)
TPSA (Å2)
Comp.
R
12a
12b
12c
12d
12e
12f
R = H
-
R = 3-F
R = 3-Cl
R = 3-Br
R = 3-NO2
R = 3-CH3
R = 3-OCH3
109.9 ± 6.9
79.1 ± 5.4
44.6 ± 8.3
63.8 ± 6.3
41.3 ± 6.2
36.5 ± 7.6
65.9 ± 8.3
55.1 ± 11.5
43.8 ± 6.6
36.4 ± 8.3
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
140.3
140.3
140.3
140.3
140.3
183.1
140.3
149.2
242.4 ± 9.4
401.1 ± 12.3
543.2 ± 10.3
701.3 ± 13.2
119.7 ± 14.8
147.2 ± 15.3
12g
12h
14a
14b
14c
14d
n = 3, R = H
n = 3
n = 3, R = 3-F
n = 3, R = 3-Cl
140.9 ± 12.9
80.3 ± 11.5
215.0 ± 9.5
765.4 ± 15.1
29.0 ± 4.2
29.0 ± 1.5
76.6 ± 6.2
279.8 ± 10.2
140.8 ± 5.5
56.0 ± 8.5
81.8 ± 11.1
n.t.
266.2
266.2
266.2
266.2

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Table 1. Cont.
hCA II
(IC50, nM)
hCA IX
(IC50, nM)
hCA XII
(IC50, nM)
TPSA (Å2)
Comp.
R
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
AZM
n = 3, R = 3-Br
n = 3, R = 3-NO2
n = 3, R = 3-CH3
n = 3, R = 3-OCH3
n = 2, R = H
n = 2
n = 2, R = 3-F
n = 2, R = 3-Cl
n = 2, R = 3-Br
n = 2, R = 3-NO2
n = 2, R = 3-CH3
n = 2, R = 3-OCH3
885.6 ± 13.7
995.3 ± 10.6
165.2 ± 9.1
271.7 ± 9.0
197.4 ± 7.9
260.3 ± 12.6
474.4 ± 7.1
612.0 ± 8.5
823.7 ± 11.1
1403.1 ± 24.6
269.8 ± 13.9
332.1 ± 16.8
16.7 ± 4.7
1082.0 ± 8.5
117.1 ± 6.9
53.4 ± 6.2
n.t.
n.t.
70.5 ± 10.9
84.9 ± 7.6
52.8 ± 11.4
40.6 ± 6.9
n.t.
266.2
309.0
266.2
275.1
266.2
266.2
266.2
266.2
266.2
309.0
266.2
275.1
113.8
68.8 ± 10.2
54.9 ± 6.2
69.4 ± 5.7
141.1 ± 8.1
255.9 ± 7.9
865.0 ± 10.7
290.9 ± 10.9
135.0 ± 7.0
47.9 ± 7.1
n.t.
n.t.
n.t.
n.t.
42.3 ± 9.4
50.2 ± 12.8
38.2 ± 10.3
Referring to the data in Table 1, the structure–activity relationships (SAR) of interme-
diates 12a–h and compounds 14a–p are preliminarily summarized below.
(i). The IC50 values of intermediates 12a–h against hCA II and IX were 79.1–701.3 and
36.4–65.9 nM, respectively. Among them, 12b showed the strongest inhibitory activity
against hCA II, with an IC50 of 79.1 nM. The substitution of halogen or electron
withdrawing groups led to a decrease in hCA II inhibitory activity. Intermediate 12h
showed the strongest inhibitory activity with hCA IX, with an IC50 of 36.4 nM. The
effect of different substitutions on hCA IX inhibitory activity was not obvious. In the
in vitro enzyme inhibitory activity test, 12a–h generally showed selectivity for hCA
IX, and 12f exhibited the best selectivity for hCA IX; its inhibitory activity for hCA IX
was 10 times higher than that for hCA II;
(ii). The IC50 values of saccharide-modified compounds 14a–p against hCA II and IX were
80.3–1403.1 and 29.0–1082.0 nM, respectively; 14b exhibited the strongest inhibitory
activity against hCA II, with an IC50 of 80.3 nM, while 14a and 14b had the best
inhibitory activity against hCA IX, with an IC50 of 29.0 nM. Compared with 12a
–h, the
TPSA values of compounds 14a were significantly increased, which was beneficial
–
p
to improving the selectivity through the differences in subcellular localization of
isoforms. However, the inhibitory activity against hCA II and IX decreased to varying
degrees, and the inhibitory activity against hCA II decreased significantly;
(iii). Among the compounds 14a–p, 14a–b and 14i–j, which did not feature substitution
on the benzene ring, were the compounds with the best hCA II inhibitory activities
when the aliphatic chain lengths were n = 2 and 3, respectively. Whether the benzene
ring was substituted by an electron-donating group or electron-donating group, the
inhibitory activity of the compounds against hCA II decreased, especially for the
substituents Cl, Br and NO2. The activity data showed that for hCA II, the inhibitory
activities of compounds 14a
than those of compounds 14i–p with n = 2;
(iv). Compared with hCA II, compounds 14a generally had higher inhibitory activity
against hCA IX, among which 14a 14g–j and 14p exhibited more desirable IC50
–h with aliphatic chain length n = 3 were generally higher
–
p
–
c,
values of less than 100 nM. When the benzene ring was substituted by F or an electron
donating group, the inhibitory activity of the compounds toward hCA IX did not
change significantly, but when the substitution was for Cl, Br, or -NO2, the inhibitory
activity was obviously reduced. Similar to hCA II, the inhibitory activities toward
hCA IX for the compounds with aliphatic chain length n = 3 were higher than those
of the compounds with n = 2;
(v). Compounds 14a
–c, 14g–j, and 14p with good inhibitory activity with hCA IX were
tested for their inhibitory activities with hCA XII. The values of IC50 ranged from

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40.6 to 140.8 nM. All tested compounds showed strong hCA XII inhibitory activity, of
which that of 14j was the most prominent.
In summary, this series of compounds showed significant inhibition against the tumor-
related isoforms hCA IX and XII, with high TPSA and potential selective qualifications,
which are worthy of further study.
2.3. Studies on Docking into the Active Site of hCA IX
We firstly re-docked the native ligands into CA IX (PDB ID: 5FL4) with optimized
docking parameters, and successfully produced the binding modes observed in the crystal
structures, with RMSD values of 0.619 Å for the docked and experimental poses. As
compounds 14a, 14e, and 14i were CA IX inhibitors; their binding mode to the active site
of CA IX was investigated by a docking simulation using Autodock 4(Zn). The results are
shown in Figure 2.
Figure 2. The modes of binding for compounds in hCA IX (
compound 14i (blue).
A,D); compound 14a (grey) (B,E); compound 14e (pink) (C,F);
By analyzing the docking results of 14a, the oxygen atom of benzamide formed two
hydrogen bonds interaction with Gln71 and Gln92 in CA IX (Figure 2A,D). The sulfonamide
group was positioned in the bottom of the cavity, interacting strongly H-bonding inter-
actions with His119 and Thr200, respectively. The ring of benzamide formed strong π–π
stacking interaction with His68. Meanwhile, the region of the thiazole sulfonamide formed
hydrophobic interactions with His94, His96, Leu199, and Trp210. In the docking results of
14e (Figure 2B,E), the oxygen atom of benzamide formed two hydrogen bonds interaction
with Gln71 in CA IX. The thiazolesulfonamide group was positioned in the bottom of the
cavity, having strong H-bonding interactions with His119, Thr200, and Thr201, respectively.
Meanwhile, the thiazolesulfonamide group also formed hydrophobic interactions with
His94, His96 and Leu199. In the docking results of 14i (Figure 2C,F), the oxygen atom
of hydroxyl formed hydrogen bonds interaction with Asp131. The oxygen atom of ben-

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zamide interacted H-bonding interactions with Gln71 and Gln92. The thiadiazole group
locating in the middle of the cavity formed hydrogen bonds interaction with Gln92 and
Thr201. The sulfonamide group was positioned in the bottom of the cavity, interacting
strongly H-bonding interactions with Thr200. The ring of thiadiazole formed strong π–π
stacking interaction with His94. At the same time, the region of the thiazolesulfonamide
formed hydrophobic interactions with His94, His96, Val121, and Leu199. Importantly, the
sulfonamide group formed a coordination bond with the zinc atom, which was reflected in
the three compounds playing a crucial role in the activity. Afterwards, in order to verify
the accuracy of the docking, we conducted dynamic simulations on them.
2.4. Molecular Dynamics Simulations
The 100 ns Molecular dynamics (MD) simulations were performed with three selected
CA IX-compound complexes (14a, 14e, and 14i) to examine their interactions between recep-
tors and ligands and elucidate the contribution of key residues during the binding process.
As shown in Figure 3, the RMSD and RMSF values of three complexes were monitored
during the whole MD simulations. In Figure 3A, the CA IX-compound 14a complex reached
equilibrium after 65 ns, where the protein RMSD value fluctuated around 5.5–7.0 Å. The
CA IX-compound 14i complex reached equilibrium after 60 ns, where the protein RMSD
value fluctuated around 6.0–7.0 Å. Throughout the MD simulation, 14e showed obvious
fluctuations and did not reach equilibrium, indicating that 14e had poor binding affinity
and the result was consistent with enzyme inhibitory activity. The RMSF plots were
displayed in Figure 3B, the fluctuations of majority residues were less than 2 Å, and several
regions with huge fluctuations located in the active site.
Figure 3. The Root-mean-square deviation (RMSD) and Root-mean-square fluctuations (RMSF) of
CA IX protein during the whole MD simulations. (
(grey), CA IX-compound 14e complex (green) and CA IX-compound 14i complex (blue). (
A
) RMSD plots of CA IX-compound 14a complex
) RMSF
B
plots of CA IX-compound 14a (grey), CA IX-compound 14e complex (orange) and CA IX-compound
14i complex (blue).
As shown in Figure 4, the histogram showed the type and ratio of interactions between
receptors and ligands, including hydrogen bonds, hydrophobic contacts, ionic interactions,
and water bridges. In Figure 4A, compound 14a formed hydrogen bonds with residues

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Arg64, Gln71, Gln92, Ala128, Arg129, Val130, Asp131, Glu173, Thr200, and Thr201, and
formed water bridges with residues Arg64, Gln71, Gln92, Ala128, Arg129, Val130, Asp131,
and Pro202. The hydrophobic interactions were with residues Val130 and Leu199. Among
them, 14a formed strong hydrogen bonds with Thr200, which accounted for 99% of the
simulation time, which played a crucial role in activities. In Figure 4B, compound 14e
formed hydrogen bonds with residues Tyr11, Arg64, Gln71, Gln92, Glu173, Thr200, and
Thr201, and formed water bridges with residues Tyr11, Arg64, Asn66, Asn68, Gln71, Gln92,
Glu173, and Thr201. The hydrophobic interactions were with residues Trp9 and His94.
Among them, 14e formed strong hydrogen bonds with Thr200, which accounted for 99%
of the simulation time. In addition, water bridges were also significant for the interactions,
such as the carbonyl oxygen formed water bridges with Tyr11, Arg64, Asn66, and Gln71,
which accounted for 40%, 40%, 35% and 42% of the simulation time, respectively. In
Figure 4C, compound 14i formed hydrogen bonds with residues Arg64, Asn66, Gln71,
Gln92, Val130, Asp131, Glu173, Thr200, and Thr201, and formed water bridges with
residues Tyr11, Arg64, Asn66, Asn68, Gln71, Gln92, Val130, Asp131, and Glu173. The
hydrophobic interactions were with residues Trp9, His94, and Val130. Among them, 14i
formed strong hydrogen bonds with Thr200, which accounted for 99% of the simulation
time. Meanwhile, residue Tyr 11 formed water bridge with 14i accounting for 50% of the
simulation time.
Figure 4. Protein-ligand contacts during the whole MD simulations. (
A) Interaction fraction of CA IX-compound 14a
complex. ( ) Interaction fraction of CA IX-compound 14e complex. ( ) Interaction fraction of CA IX-compound 14i complex.
B
C
The results of MD simulations indicated 14a and 14i were more stable during MD
simulations and revealing that 14a and 14i might have stronger interactions with CA IX. In
addition, compound 14e had the largest RMSD fluctuation, this might be due to its weak

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contact with CA IX which made the complex unstable, and could explain its lowest activity
among three compounds.
2.5. In Vitro Cytotoxicity Studies on Cancer Cells
To evaluate the ability of novel saccharide-modified compounds to inhibit the viability
of tumor cells, several compounds that exhibited the best inhibitory profiles were chosen to
evaluate their effects against human colon cancer HT-29 cells and breast adenocarcinoma
MDA-MB-231 cells via CCK-8 assay. The results are shown in Figure 5.
Figure 5. Effects of compounds 14a
, 14b, 14h, 14p, and AZM on the cell viability of HT-29 and MDA-MB-231 under
normoxic (A,C) and hypoxic (B,D) conditions.
As shown in Figure 5, compounds 14a, 14b, 14h, 14p, and AZM were evaluated for
their effects against two cancer cell lines that overexpress CA IX under normoxic and
hypoxic conditions. Based on the results, we summarize the following conclusions:
(i). The inhibitory activities of compounds 14a, 14b, 14h, and 14p on the two tumor cell
lines were better than those of the positive control drug AZM;
(ii). Under hypoxic conditions, the inhibitory activities of the tested compounds and AZM
on the tumor cell lines were all higher than those under normoxia;
(iii). Compared with the breast adenocarcinoma MDA-MB-231, the tested compounds
generally showed higher inhibitory efficacy with the colon cancer cell line HT-29.
Then, considering the high inhibitory activity of 14a on the two isoforms of CA IX and
XII, we evaluated the inhibitory activity of 14a on human osteosarcoma MG-63 featuring
high expression of CA IX and XII. The results are shown in Figure 6.

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Figure 6. Effects of compounds 14a and AZM on the cell viability of MG-63 under normoxic and
hypoxic conditions.
Similar to the previous conclusions, the effect of 14a in inhibiting the vitality of MG-63
cells under hypoxic conditions was more obvious than that under normoxic conditions.
Under hypoxic conditions, the administration of 800 µM 14a reduced the viability of MG-63
cells by approximately 30%, and the effect was significantly better than that of the positive
control AZM.
Although these novel saccharide-modified CA IX inhibitors showed promising iso-
form inhibitory activities, their inhibitory effect on the cell viability of tumor-related cells
was not obvious. This is a common problem in research on CA IX inhibitors [26,27]. This
might indicate that CA IX inhibitors are not effective enough to be used alone in cancer
therapy [25].
2.6. Extracellular pH Measurement in the Presence of Compound 14b, 14h, and 14p
The high expression of CA IX plays an important role in the maintenance of the
tumor cell microenvironment, and inhibiting its activity might have a potential impact on
the pH of the tumor microenvironment. To evaluate the effect of compounds on the pH
of the microenvironment, the pH values under normoxic and hypoxic conditions were
determined in the presence of compounds 14b, 14h, and 14p. The results showed that 14b,
14h, and 14p increased tumor pH to varying degrees at concentrations of 0.1 and 1 mM
(Figure 7). The tested compounds had no obvious effect on cells under normal conditions,
but they significantly improved extracellular acidosis induced by hypoxia. In addition, this
improvement in pH was dose-dependent.

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Figure 7. Extracellular pH measurement of MDA-MB-231 (
A
) and HT-29 (
B
) under normoxic and
hypoxic conditions.
3. Materials and Methods
3.1. Chemistry
All the reagents (Energy Chemical, Shanghai, China) were used without further
purification unless otherwise specified. Solvents were dried and redistilled prior to use
in the usual manner. Analytical TLC was per-formed using silica gel HF254 (Qingdao
Haiyang Chemical, Qingdao, Shandong, China). Preparative column chromatography was
performed with silica gel H. Melting points were obtained on a Büchi melting point B-540
1
apparatus (Büchi Labortechnik, Flawil, Switzerland). H and 13C NMR spectra (details
of raw data for compounds, see Figures S1–S54) were recorded on a Bruker ARX 600 or
400 MHz spectrometer (Bruker, Zurich, Switzerland) HRMS were obtained on an Agilent
ESI-QTOF instrument (Agilent, Santa Clara, CA, USA).
3.1.1. Synthesis of Intermediate 2–4
Intermediates 2–4 were prepared according to the literature procedures [28]. Interme-
diate 4 [α]2D0 +22.7 (c 1.0, acetone).
3.1.2. General Procedure of (((2R,3R,4S,5R,6R)-3,4,5-Tris(benzoyloxy)-6-
((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)amino) Acid (5a and 5b)
Intermediate
4 (1.50 g, 2.4 mmol), corresponding acid anhydride (2.4 mmol),
4-dimethylaminopyridine (15.00 mg, 0.12 mmol) and Pd/C (0.15 g) were dispersed in
45 mL THF. Then, in the presence of hydrogen, the reaction was stirred at room tempera-
ture for 6 h. After the reaction was completed, the solution was concentrated under reduced
pressure. Purification by column chromatography (75: 1, dichloromethane-methanol) to
obtain intermediate 5a and 5b.
4-Oxo-4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-
pyran-2-yl)amino)butanoic Acid (5a)
◦
20
1
Yield 64.1%. m.p. 88.2–90.0 C, [
α
]
+39.7 (c 1.0, acetone), H-NMR (DMSO-d6,
600 MHz
)
δ
12.12 (s, 1H), 8.93 (d, J = 9.5 HzD, 1H), 7.97 (d, J = 7.2 Hz, 2H), 7.84 (d, J = 7.3 Hz
,
2H), 7.79 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.69 (t, J = 7.2 Hz, 1H), 7.61 (dd, J =
16.8, 4.1 Hz, 2H), 7.55 (q, J = 7.3 Hz, 3H), 7.46 (dd, J = 9.4, 6.1 Hz, 4H), 7.41 (t, J = 7.7 Hz,
2H), 6.06 (t, J = 10.1 Hz, 1H), 5.81 (t, J = 9.3 Hz, 1H), 5.60 (t, J = 9.7 Hz, 1H), 5.35 (t, J =
9.4 Hz, 1H), 4.61 (d, J = 9.8 Hz, 1H), 4.52 (d, J = 11.1 Hz, 1H), 4.46 (dd, J = 12.4, 3.5 Hz, 1H),
2.29 (tdd, J = 23.4, 20.7, 7.6 Hz, 4H) ppm. 13C-NMR (DMSO-d6, 151 MHz)
δ 173.92, 173.08,

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172.26, 165.80, 165.54, 165.25, 134.21, 133.99, 129.64, 129.25, 129.03, 77.75, 74.40, 72.73, 72.16,
69.39, 62.92, 30.47, 29.14 ppm.
5-Oxo-5-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-
pyran-2-yl)amino)pentanoic Acid (5b)
20
Yield 70.0%.m.p. 94.4–96.8 ◦C, [
α
]
+51.0 (c 1.0, acetone), 1H-NMR (DMSO-d6, 600
MHz)
δ
12.03 (s, 2H), 8.87 (d, J = 9.5 Hz,D1H), 7.97 (dd, J = 8.2, 1.2 Hz, 2H), 7.84 (dd, J = 8.3,
1.1 Hz, 2H), 7.79 (dd, J = 8.3, 1.2 Hz, 2H), 7.72 (dd, J = 8.2, 1.1 Hz, 2H), 7.70–7.65 (m, 1H),
7.61 (td, J = 7.4, 1.6 Hz, 2H), 7.55 (q, J = 8.0 Hz, 3H), 7.46 (dd, J = 14.0, 7.6 Hz, 4H), 7.41 (t, J
= 7.8 Hz, 2H), 6.06 (t, J = 9.5 Hz, 1H), 5.81 (t, J = 9.4 Hz, 1H), 5.59 (t, J = 9.7 Hz, 1H), 5.36
(t, J = 9.4 Hz, 1H), 4.64–4.57 (m, 1H), 4.52 (dd, J = 12.4, 2.4 Hz, 1H), 4.45 (dd, J = 12.5, 3.8
Hz, 1H), 2.25 (t, J = 7.4 Hz, 3H), 2.17–2.02 (m, 4H) ppm. 13C-NMR (DMSO-d6, 151 MHz)
δ
174.56, 172.88, 165.79, 165.53, 165.23, 134.50, 134.11, 133.99, 129.81, 129.65, 129.48, 129.15,
129.02, 77.66, 74.36, 72.77, 72.14, 69.43, 62.95, 60.23, 34.83, 33.22, 20.75 ppm.
3.1.3. General Procedure of 4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)benzoic Acid
Derivatives (7a–h)
To a solution of different aminobenzoic acid (1.0 mmol) in THF (20 mL), Fmoc-Cl
(
3.88 g, 1.5 mmol) in CHCl3 was added by drop. After the addition was complete, reacted
◦
at 60 C for 12 h. Once the reaction was complete, the reaction solution was cooled to
room temperature, 20 mL of ether was added, filtered, and the solid was collected to
obtain intermediate 7a–h with a yield of 77.2–86.5%. The products were directly used
without purification.
3.1.4. General Procedure of (9H-Fluoren-9-yl)methyl (4-(chlorocarbonyl)phenyl)carbamate
Derivatives (8a–h)
Intermediate 7a–h was dispersed in 20 mL of SOCl2, and the reaction was refluxed for
5 h until the reaction solution was clear. Then, the reaction solution was cooled to room
temperature, and the solvent was removed under reduced pressure to obtain intermediate
8a–h with a yield of 88.2–93.8%. The products were directly used without purification.
3.1.5. Synthesis of 5-Amino-1,3,4-thiadiazole-2-sulfonamide (10)
Acetazolamide (5.00 g, 22.5 mmol) was dispersed in 50 mL of EtOH, 8 mL of concen-
trated hydrochloric acid was added, and the reaction was refluxed for 8 h until the reaction
solution was clear. After cooling to room temperature, the solvent was removed under
reduced pressure. Slowly added sodium bicarbonate solution until the pH of solution was
neutral. Filtrated, collected solids to obtain 3.77 g intermediates 10 with a yield of 93.0%.
1H-NMR (600 MHz, DMSO-d6)
δ 172.13, 158.34.
δ
8.06 (s, 2H), 7.81 (s, 2H). 13C-NMR (151 MHz, DMSO-d6)
3.1.6. General Procedure of (9H-Fluoren-9-yl)methyl
(4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)carbamate Derivatives (11a–h)
Intermediates 10 (1.80 g, 1.0 mmol) was dispersed in 30 mL of acetone, 0.24 mL
pyridine was added, then intermediate 7a
–h was added gradually. The mixture was stirred
at room temperature for 8 h. After the reaction was completed, filtered and collected the
solid to obtain intermediate 11a–h with a yield of 52.6–73.9%. The products were directly
used without purification.
3.1.7. General Procedure of 4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide
Derivatives (12a–h)
To a solution of intermediates 11a–h, 5 mL of diethylamine was added, and the reaction
was stirred at room temperature for 5 h. After the reaction was completed, the solution
was concentrated under reduced pressure. Then, purification by column chromatography
(25:1, dichloromethane-methanol) to obtain intermediates 12a–h.

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4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12a)
Yield 64.1%, 1H-NMR (600 MHz, DMSO-d6)
δ13.30 (s), 8.34 (s), 7.31 (d, J = 7.7 Hz),
7.26 (s), 7.20 (t, J = 7.8 Hz), 6.85 (dd, J = 7.9, 1.4 Hz), 5.46 (s). 13C-NMR (151 MHz, DMSO-d6)
δ 165.61, 164.02, 161.51, 148.48, 130.86, 128.58, 118.01, 114.82, 112.86.
3-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12b)
Yield 59.7%, 1H-NMR (600 MHz, DMSO-d6)
δ13.30 (s), 8.34 (s), 7.31 (d, J = 7.7 Hz),
7.26 (s), 7.20 (t, J = 7.8 Hz), 6.85 (dd, J = 7.9, 1.4 Hz), 5.46 (s). 13C-NMR (151 MHz, DMSO-d6)
δ 165.61, 164.02, 161.51, 148.48, 130.86, 128.58, 118.01, 114.82, 112.86.
4-Amino-3-fluoro-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12c)
1
Yield 59.7%, H-NMR (600 MHz, DMSO-d6)
δ 13.06 (s), 8.31 (s), 7.88 (dd, J = 12.7,
1.8 Hz), 7.80 (dd, J = 8.5, 1.9 Hz), 6.83 (t, J = 8.7 Hz), 6.23 (s). 13C-NMR (151 MHz, DMSO-d6)
δ 164.77, 164.63, 163.05, 150.38, 148.80, 142.64, 142.56, 127.00, 115.84, 115.71, 115.23, 115.20.
4-Amino-3-chloro-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12d)
Yield 48.1%, 1H-NMR (400 MHz, DMSO-d6)
δ13.09 (s, 1H), 8.29 (s, 2H), 8.13 (s, 1H),
7.88 (dd, J = 8.6, 2.1 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.37 (s, 2H). 13C-NMR (151 MHz,
DMSO-d6) δ 164.86, 164.38, 162.89, 150.03, 130.61, 129.51, 118.28, 116.63, 114.66.
4-Amino-3-bromo-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12e)
Yield 37.0%, 1H-NMR (600 MHz, DMSO-d6)
δ13.10 (s), 8.45–8.24 (m), 7.90 (dd, J = 8.6,
2.1 Hz), 6.85 (d, J = 8.6 Hz), 6.32 (s). 13C-NMR (151 MHz, DMSO-d6)
δ 164.69, 164.39, 163.15,
151.01, 133.85, 129.99, 114.55, 106.53.
4-Amino-3-nitro-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12f)
Yield 42.4%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.49 (s), 8.99 (d, J = 2.1 Hz), 8.35 (s),
8.13–8.02 (m), 7.11 (d, J = 9.0 Hz). 13C-NMR (151 MHz, DMSO-d6)
δ 165.04, 164.18, 162.77,
149.40, 135.84, 134.93, 130.18, 128.47, 119.81.
4-Amino-3-methyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12g)
1
Yield 57.3%, H-NMR (400 MHz, DMSO-d6)
δ 12.86 (s, 1H), 8.27 (s, 2H), 7.85 (d, J
= 1.5 Hz, 1H), 7.80 (dd, J = 8.5, 2.1 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 5.90 (s, 2H), 2.11 (s,
3H). 13C-NMR (151 MHz, DMSO-d6)
δ 165.23, 164.68, 162.98, 152.64, 131.56, 128.78, 120.51,
116.93, 113.17, 17.83.
4-Amino-3-methoxy-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (12h)
Yield 63.0%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.02 (s), 8.26 (s), 7.64 (d, J = 10.2 Hz),
6.69 (d, J = 8.0 Hz), 5.77 (s), 3.87 (s). 13C-NMR (151 MHz, DMSO-d6)
δ 165.61, 164.29, 163.75,
145.58, 124.17, 112.55, 110.64, 55.99.
3.1.8. General Procedure of
(2R,3R,4S,5R,6R)-2-((Benzoyloxy)methyl)-6-(4-oxo-4-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-
yl)carbamoyl)phenyl)amino)butanamido)tetrahydro-2H-pyran-3,4,5-triyl Tribenzoate
Derivatives (13a–p)
Intermediate 5a (b) (1.0 mmol) and EDCI (0.29 g, 1.5 mmol) were dissolved in 25 mL
of anhydrous pyridine. After stirring for 1 h, corresponding intermediate 12a–h (1.0 mmol)
was added and reacted at room temperature for 8 h. After the reaction completed, the
solution was diluted with ethyl acetate, then the mixture was washed with water and
saturated sodium chloride solution successively. The solution was dried over Na2SO4,
filtered, and concentrated to obtain the intermediate 13a–h. The products were directly
used without purification.

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3.1.9. General Procedure of
N1-(4-((5-Sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-((2R,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)glutaramide Derivatives (14a–p)
Dissolved 13a–p in 22 mL of a mixed solution of methanol/triethylamine/water
(8:2:1) and reacted at room temperature for 12 h. After the reaction completed, the solvent
was removed under reduced pressure, and the obtained solid was separated by column
chromatography (dichloromethane: methanol = 8:1) to obtain compound 14a–p, some
compounds contained a small amount of stereoisomer that were difficult to separate.
N1-(4-((5-Sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-((2R,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)glutaramide (14a)
Two-step yield 12.2%, 1H-NMR (600 MHz, DMSO-d6)
δ 10.05 (s, 1H), 8.35 (d, J = 9.1
Hz, 1H), 8.24 (s, 1H), 8.07 (d, J = 8.7 Hz, 2H), 7.74 (s, 2H), 7.64 (d, J = 8.6 Hz, 2H), 4.72 (t, J
= 9.1 Hz, 1H), 3.64 (dd, J = 11.7, 1.8 Hz, 1H), 3.41 (dd, J = 11.8, 5.4 Hz, 1H), 3.17 (t, J = 8.8
Hz, 1H), 3.12–2.99 (m, 3H), 2.36 (dd, J = 13.9, 6.6 Hz, 2H), 2.18 (ddq, J = 22.5, 15.0, 7.5 Hz
,
2H), 1.93–1.72 (m, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
172.84, 171.62, 170.62, 159.85,
141.55, 132.95, 129.54, 129.22, 118.37, 79.82, 78.87, 77.72, 72.69, 70.30, 61.29, 36.17, 34.99,
21.22.; HRMS (ESI): Calcd. for [M − H]− C20H25N6O10S2: 573.1152, Found 573.1143.
N1-(3-((5-Sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-((2R,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)glutaramide (14b)
Two-step yield 10.9%, 1H-NMR (600 MHz, DMSO-d6)
δ13.48 (s, 1H), 10.12 (s, 1H), 8.35
(d, J = 2.0 Hz, 2H), 8.19 (s, 2H), 7.83 (d, J = 7.9 Hz, 2H), 7.45 (t, J = 7.9 Hz, 1H), 4.96 (d, J =
4.1 Hz, 1H), 4.91–4.81 (m, 2H), 4.72 (t, J = 9.1 Hz, 1H), 4.49 (t, J = 5.1 Hz, 1H), 3.64 (dd, J
= 11.0, 3.9 Hz, 1H), 3.44–3.38 (m, 1H), 3.17 (td, J = 8.7, 3.5 Hz, 1H), 3.11–3.01 (m, 3H), 2.37
(t, J = 7.4 Hz, 2H), 2.27–2.07 (m, 2H), 1.88–1.78 (m, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
174.66, 172.79, 171.57, 163.54, 139.62, 129.09, 123.39, 122.84, 119.84, 119.74, 79.81, 78.91, 77.78,
72.74, 70.31, 61.29, 36.12, 35.00, 21.25; HRMS (ESI): Calcd. for [M
573.1152, Found 573.1139.
−
H]− C20H25N6O10S2:
N1-(2-Fluoro-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)glutaramide (14c)
Two-step yield 9.2%, 1H-NMR (600 MHz, DMSO-d6)
δ 9.77 (s, 1H), 8.36 (d, J = 9.1 Hz,
1H), 8.25 (s, 1H), 8.05–7.97 (m, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 12.1 Hz, 1H), 7.70 (s,
2H), 4.71 (dt, J = 13.4, 9.1 Hz, 2H), 3.63 (d, J = 14.1 Hz, 2H), 3.44–3.37 (m, 2H), 3.22–3.13 (m,
2H), 3.12–3.00 (m, 4H), 2.44 (t, J = 7.1 Hz, 2H), 2.24–2.16 (m, 2H), 1.82 (dt, J = 14.7, 7.3 Hz,
2H). 13C-NMR (151 MHz, DMSO-d6)
δ172.52, 171.47, 169.51, 166.33, 159.28, 153.37, 151.75,
135.45, 127.76, 124.13, 122.47, 114.77, 114.64−, 79.24, 78.25, 77.11, 72.09, 69.71, 60.71, 35.10,
34.43, 20.74; HRMS (ESI): Calcd. for [M
−
H] C20H24FN6O10S2: 591.1058, Found 591.1050.
N1-(2-Chloro-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)glutaramide (14d)
Two-step yield 7.7%, 1H-NMR (600 MHz, DMSO-d6)
δ 12.58 (s, 1H), 8.23 (d, J = 9.1 Hz,
1H), 8.10 (s, 1H), 7.85 (dd, J = 8.6, 1.8 Hz, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.29 (s, 2H), 4.88 (d, J
= 41.0 Hz, 3H), 4.70 (t, J = 9.1 Hz, 1H), 4.54 (s, 1H), 3.62 (d, J = 11.7 Hz, 1H), 3.40 (d, J = 11.4
Hz, 3H), 3.16 (t, J = 8.7 Hz, 1H), 3.11–2.99 (m, 4H), 2.07 (dq, J = 14.6, 7.4 Hz, 2H), 2.00 (t, J =
7.3 Hz, 2H), 1.63 (p, J = 7.3 Hz, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
179.27, 173.47, 167.81,
164.19, 161.93, 149.53, 130.40, 129.26, 118.94, 116.62, 114.60, 79.83, 78.80, 77.63, 72.58, 70.24,

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61.21, 38.60, 35.41, 21.94; HRMS (ESI): Calcd. for [M
Found 607.0756.
−
H]− C20H24ClN6O10S2: 607.0762,
N1-(2-Bromo-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)glutaramide (14e)
Two-step yield 5.9%, 1H-NMR (600 MHz, DMSO-d6)
δ 8.32 (d, J = 9.0 Hz), 8.20 (d, J =
1.6 Hz), 7.86 (dd, J = 8.5, 1.7 Hz), 6.81 (d, J = 8.5 Hz), 5.99 (s), 4.69 (t, J = 9.0 Hz), 3.62 (d,
J = 11.7 Hz), 3.41 (dd, J = 11.8, 5.0 Hz), 3.17 (t, J = 8.6 Hz), 3.07 (dt, J = 18.0, 9.3 Hz), 2.06
(dd, J = 15.2, 7.4 Hz), 2.00 (t, J = 7.2 Hz), 1.70–1.52 (m). 13C-NMR (151 MHz, DMSO-d6)
δ
179.22, 173.53, 173.37, 167.99, 149.56, 133.48, 129.60, 122.88, 114.47, 106.58, 79.95, 78.85, 77.57,
−
72.51, 70.28, 61.27, 38.45, 35.33, 22.12; HRMS (ESI): Calcd. for [M
651.0257, Found 651.0250.
−
H] C20H24BrN6O10S2:
N1-(2-Nitro-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)glutaramide (14f)
Two-step yield 8.3%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.55 (d, J = 81.3 Hz, 1H), 8.99
(dd, J = 6.6, 2.1 Hz, 1H), 8.42–8.29 (m, 2H), 8.10 (d, J = 9.0 Hz, 3H), 7.14 (d, J = 9.0 Hz, 1H),
4.87 (s, 1H), 4.67 (t, J = 9.0 Hz, 1H), 3.65–3.57 (m, 1H), 3.40 (dd, J = 11.8, 4.9 Hz, 2H), 3.16 (t,
J = 8.6 Hz, 1H), 3.11–2.99 (m, 2H), 2.35 (t, J = 7.1 Hz, 2H), 2.10 (ddt, J = 30.3, 15.2, 7.5 Hz,
2H), 1.76–1.60 (m, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
172.68 (s), 164.31 (s), 163.86 (s),
149.25 (s), 134.91 (s), 130.11 (s), 128.51 (s), 119.73 (s), 117.40 (s), 79.81 (s), 78.80 (s), 77.61 (s),
72.57 (s), 70.22 (s), 61.20 (s), 35.44 (s), 34.49 (s), 20.00 (s); HRMS (ESI): Calcd. for [M
C20H24N7O12S2: 618.1003, Found 618.0984.
−
H]−
N1-(2-Methyl-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)glutaramide (14g)
Two-step yield 10.4%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.21 (s, 1H), 9.37 (s, 1H), 8.35
(d, J = 9.1 Hz, 1H), 8.15 (s, 2H), 8.03 (d, J = 1.3 Hz, 1H), 7.96 (dd, J = 8.4, 1.6 Hz, 1H), 7.70
(dd, J = 23.7, 8.3 Hz, 1H), 4.97 (d, J = 0.5 Hz, 1H), 4.87 (dd, J = 11.1, 4.8 Hz, 2H), 4.73 (t, J =
9.1 Hz, 1H), 4.49 (t, J = 5.1 Hz, 1H), 3.64 (dd, J = 11.5, 2.4 Hz, 1H), 3.45–3.38 (m, 1H), 3.18 (t, J
= 8.4 Hz, 1H), 3.13–2.98 (m, 3H), 2.43 (t, J = 7.1 Hz, 2H), 2.30 (s, 3H), 2.27–2.11 (m, 2H), 1.84
(p, J = 7.3 Hz, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
172.91, 171.77, 167.19, 165.52, 163.65,
140.71, 131.17, 130.78, 126.93, 126.07, 123.97, 79.80, 78.86, 77.71, 72.70, 70.30, 61.29, 35.71,
35.03, 21.43, 18.41; HRMS (ESI): Calcd. for [M
587.1293.
−
H]− C21H27N6O10S2: 587.1308, Found
N1-(2-Methoxy-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N5-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)glutaramide (14h)
Two-step yield 11.9%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.44 (s, 1H), 9.38 (s, 1H), 8.36
(s, 2H), 8.34 (d, J = 9.1 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H), 7.78 (dd, J
= 8.5, 1.8 Hz, 1H), 4.90 (s, 1H), 4.72 (t, J = 9.1 Hz, 1H), 3.97 (s, 3H), 3.64 (dd, J = 11.7, 1.7
Hz, 1H), 3.41 (dd, J = 11.8, 5.4 Hz, 1H), 3.18 (t, J = 8.8 Hz, 1H), 3.14–2.95 (m, 3H), 2.54–2.51
(m, 2H), 2.29–2.01 (m, 2H), 1.91–1.71 (m, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
172.88,
172.33, 165.03, 162.63, 148.93, 132.83, 125.69, 122.29, 120.46, 111.13, 79.81, 78.87, 77.73, 72.71,
70.32, 61.30, 56.59, 36.05, 34.99, 21.31; HRMS (ESI): Calcd. for [M
603.1257, Found 603.1232.
−
H]− C21H27N6O11S2:

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N1-(4-((5-Sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-((2R,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)succinimide (14i)
Two-step yield 9.9%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.36 (s, 1H), 10.39 (s, 1H), 8.44
(d, J = 9.1 Hz, 1H), 8.35 (s, 2H), 8.13 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 4.87 (s, 1H),
4.71 (t, J = 9.1 Hz, 1H), 3.67–3.59 (m, 1H), 3.41 (dd, J = 11.8, 5.1 Hz, 1H), 3.35 (s, 1H), 3.17 (t, J
= 8.6 Hz, 1H), 3.11–3.01 (m, 3H), 2.65–2.59 (m, 2H), 2.56–2.51 (m, 2H). 13C-NMR (151 MHz,
DMSO-d6)
δ 174.26, 172.21, 172.13, 171.89, 171.38, 142.90, 131.11, 129.90, 118.41, 79.98, 78.91,
77.74, 72.84, 70.26, 61.21, 31.74, 30.59; HRMS (ESI): Calcd. for [M
559.0995, Found 559.0921.
−
H]− C19H23N6O10S2:
N1-(3-((5-Sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-((2R,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)succinimide (14j)
Two-step yield 9.0%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.55 (s, 1H), 10.24 (s, 1H), 8.44
(d, J = 9.1 Hz, 1H), 8.35 (d, J = 18.7 Hz, 3H), 7.94–7.74 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 4.98 (d,
J = 4.5 Hz, 1H), 4.87 (dd, J = 11.3, 5.2 Hz, 2H), 4.71 (t, J = 9.1 Hz, 1H), 4.51 (t, J = 5.7 Hz, 1H),
3.63 (dd, J = 11.5, 4.0 Hz, 1H), 3.48–3.37 (m, 1H), 3.17 (td, J = 8.6, 4.4 Hz, 1H), 3.12–2.99 (m,
3H), 2.66–2.56 (m, 2H), 2.50–2.44 (m, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
172.22, 171.32,
166.16, 165.06, 162.59, 140.06, 131.96, 129.65, 124.00, 123.20, 119.52, 79.97, 78.91, 77.73, 72.82,
70.26, 61.22, 31.58, 30.56; HRMS (ESI): Calcd. for [M
Found 559.0979.
−
H]− C19H23N6O10S2: 559.0995,
N1-(2-Fluoro-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)succinimide (14k)
Two-step yield 8.5%, 1H-NMR (600 MHz, DMSO-d6)
δ 9.95 (s, 1H), 8.43 (d, J = 9.1 Hz,
1H), 8.12 (t, J = 7.9 Hz, 1H), 7.99–7.88 (m, 4H), 4.97 (s, 1H), 4.86 (dd, J = 9.3, 5.2 Hz, 3H),
4.75–4.65 (m, 2H), 4.51 (t, J = 6.0 Hz, 2H), 3.63 (dd, J = 10.3, 5.0 Hz, 2H), 3.45–3.38 (m, 2H),
3.21–3.13 (m, 2H), 3.11–2.99 (m, 5H), 2.72–2.62 (m, 2H), 2.46–2.32 (m, 2H).
N1-(2-Chloro-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)succinimide (14l)
Two-step yield 7.6%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.26 (s, 1H), 8.42 (d, J = 9.0 Hz,
1H), 8.14 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 8.6, 2.0 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.43 (s,
2H), 4.65 (t, J = 9.0 Hz, 1H), 3.60 (d, J = 11.3 Hz, 1H), 3.40 (dd, J = 11.8, 4.5 Hz, 1H), 3.25–3.10
(m, 1H), 3.03 (dd, J = 16.3, 7.9 Hz, 3H), 2.61–2.53 (m, 2H), 2.44–2.27 (m, 2H). 13C-NMR
(151 MHz, DMSO-d6) δ 172.22, 171.62, 164.47, 164.04, 161.07, 150.01, 130.64, 129.55, 117.99,
116.64, 114.65, 79.87, 78.77, 77.58, 72.69, 70.14, 61.10, 30.92, 29.27; HRMS (ESI): Calcd. for
[M − H]− C19H22ClN6O10S2: 593.0606, Found 577.0600.
N1-(2-Bromo-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)succinimide (14m)
Two-step yield 6.6%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.25 (s, 1H), 8.39 (d, J = 9.1 Hz,
1H), 8.29 (d, J = 2.0 Hz, 1H), 7.90 (dd, J = 8.6, 2.0 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.37 (s,
2H), 4.66 (t, J = 9.1 Hz, 1H), 3.61 (d, J = 10.5 Hz, 1H), 3.39 (dd, J = 11.8, 4.9 Hz, 2H), 3.15
(dd, J = 15.2, 6.6 Hz, 1H), 3.10–2.95 (m, 3H), 2.56–2.51 (m, 2H), 2.35 (td, J = 16.8, 9.6 Hz,
2H). 13C-NMR (151 MHz, DMSO-d6)
δ 172.29, 171.61, 164.30, 164.01, 161.17, 151.06, 133.90,
130.05, 118.50, 114.53, 106.46, 79.86, 78.78, 77.61, 72.71, 70.16, 61.11, 30.97, 29.30; HRMS
(ESI): Calcd. for [M − H]− C19H22BrN6O10S2: 637.0100, Found 637.0061.

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N1-(2-Nitro-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)succinimide (14n)
Two-step yield 8.1%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.64 (s, 1H), 9.00 (d, J = 2.1
Hz, 1H), 8.41 (d, J = 9.0 Hz, 1H), 8.09 (dd, J = 8.9, 2.2 Hz, 3H), 7.13 (d, J = 9.0 Hz, 1H),
4.65 (t, J = 9.0 Hz, 1H), 3.60 (d, J = 10.5 Hz, 2H), 3.39 (dd, J = 11.8, 4.7 Hz, 2H), 3.21–3.11
(m, 1H), 3.11–2.97 (m, 3H), 2.58–2.51 (m, 3H), 2.43–2.28 (m, 2H). 13C-NMR (151 MHz,
DMSO-d6)
δ
172.29, 171.53, 164.30, 163.98, 161.29, 149.24, 134.95, 130.13, 128.51, 119.7−1,
117.39, 79.88, 78.82, 77.64, 72.73, 70.17, 61.12, 30.99, 29.32; HRMS (ESI): Calcd. for [M
C19H22N7O12S2: 604.0846, Found 604.0834.
−
H]
N1-(2-Methy-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)succinimide (14o)
Two-step yield 10.2%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.36 (s, 1H), 9.50 (s, 1H), 8.45
(d, J = 9.0 Hz, 1H), 8.36 (s, 1H), 8.04 (s, 1H), 7.91 (dd, J = 83.8, 8.5 Hz, 2H), 4.72 (t, J = 9.0 Hz,
1H), 3.64 (d, J = 11.0 Hz, 1H), 3.42 (dd, J = 11.7, 5.1 Hz, 1H), 3.22–3.15 (m, 3H), 3.12–3.02
(m, 4H), 2.75 (dd, J = 11.7, 4.4 Hz, 2H), 2.68–2.61 (m, 2H), 2.33 (s, 3H). 13C-NMR (151 MHz,
DMSO-d6)
δ 172.46, 171.49, 165.56, 164.88, 163.45, 162.71, 141.64, 131.29, 130.59, 127.10,
123.54, 79.95, 78.85, 77.66, 72.80, 70.24, 61.22, 31.39, 30.81, 18.35; HRMS (ESI): Calcd. for [M
− H]− C20H25N6O10S2: 573.1152, Found 573.1126.
N1-(2-Methoxy-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-N4-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)succinimide (14p)
Two-step yield 10.2%, 1H-NMR (600 MHz, DMSO-d6)
δ 13.43 (s, 1H), 9.48 (s, 1H), 8.42
(d, J = 9.1 Hz, 1H), 8.36 (s, 2H), 8.30 (d, J = 8.3 Hz, 1H), 7.85 (s, 1H), 7.78 (d, J = 9.9 Hz,
1H), 4.85 (s, 1H), 4.71 (t, J = 9.1 Hz, 1H), 3.97 (s, 3H), 3.63 (d, J = 10.5 Hz, 1H), 3.60 (s, 1H),
3.41 (dd, J = 11.8, 5.1 Hz, 2H), 3.17 (t, J = 8.7 Hz, 1H), 3.12–3.01 (m, 3H), 2.77 (t, J = 6.6 Hz,
1H), 2.75–2.64 (m, 2H), 2.49–2.41 (m, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ
172.14, 171.94,
165.16, 162.75, 148.81, 133.08, 132.92, 122.37, 120.28, 111.19, 80.04, 79.02, 78.00, 73.02, 70.44,
61.36, 56.61, 31.84, 30.75; HRMS (ESI): Calcd. for [M
Found 589.1077.
−
H]− C20H25N6O11S2: 589.1101,
3.1.9.17. hCA Enzyme Inhibition Assays
Carbonic anhydrase can catalyze the hydrolysis of 4-nitrophenyl acetate (4-NPA).
According to the method reported in the literature, the reaction rate was monitored by
measuring the change of absorbance at 405 nM [8,29,30]. The experimental buffer consisted
of 15 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH = 7.40),
0.01% tetraethylene glycol monododecyl ether (Brij), and 100 mmol/L NaCl. Recombinant
human carbonic anhydrase II, IX and XII were commercially available (Sino Biological
Inc, Beijing, China) and prepared in buffer with a concentration of 3.3, 10.0, and 10.0
ng/µL, respectively. Then, 18 µL of enzyme solution was transferred into 384-well plates
in duplicate. Stock solutions of the inhibitors (10.0 mmol/L) were prepared using DMSO
as solvent, and then diluted 1:3 with DMSO. The test concentration included 600, 200,
66.667, 22.222, 7.407, 2.469, 0.823, 0.274, 0.091 µM, a total of nine concentrations. Then
2.00 µL of each inhibitor was added to each well. All compounds were allowed to incubate
with the enzyme for 15 min at 25 ◦C to form the Enzyme-Inhibitor (E-I) complex. After
that, substrate 4-NPA (1.00 mmol/L, 20.00
added into the E-I complex solution and incubated for 60, 90, 90 min at 25 C, respectively.
The absorbance of each compound was measured with Envision 2104 plate reader (Perkin
µ
L, Sigma-Aldrich, St. Louis, MI, USA) was
◦

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Elmer, Waltham, MA, USA). The inhibitor AZM was used as standard to investigate the
inhibitory activity of these compounds.
3.2. Preparation for Docking Studies and Prediction by TPSA
Molecular docking calculations using the ligand molecules with CA IX (PDB code:
5FL4) [31] and were conducted with the modified version of Autodock (version Autodock
4(Zn), Scripps Research Institute, San Diego, CA, USA) [32,33]. A grid box with a grid
spacing of 0.375 Å was generated to define the binding pocket.
AutoGrid 4 was used as the auxiliary program to generate affinity grid fields. Com-
pound structures were built and minimized with the Accelrys Discovery Studio 3.0 software
package with flexible torsions assigned, and all dihedral angles were able to freely rotate.
We applied the Lamarckian genetic algorithm to determine the appropriate binding po-
sitions, orientations, and conformation of ligands. The optimized parameters were as
follows: the maximum number of energy evaluations was increased to 25,000,000 per run,
the iterations of Solis & Wets local search were 3000, the number of individuals in the
population was 300, and the number of generations was 25. Results differing by < 2 Å in a
positional root mean square deviation were clustered together. In each group, the lowest
binding energy configuration with the highest percentage frequency was selected as the
group representative. All other parameters were maintained as default.
ADME/T profile of the selected compounds was assessed using the standard de-
scriptors protocol in the DS (version 3.0, Accelrys Inc., San Diego, CA, USA) [34]. In the
ADME/T module, various parameters including TPSA were used to quantitatively predict
the properties of a set of rules that specify the ADME/T characteristics of the compounds.
3.3. Preparation for Molecular Dynamics Simulation
The 100 ns molecular dynamics (MD) simulations were carried out of CA IX-compound
14a, CA IX-compound 14e, and CA IX-compound 14i, respectively. The simulations were
performed by Desmond v3.8 module in the Schrödinger suite (version 9.6, Schrödinger
Inc., NY, USA). The system was solvated with simple point charge (SPC) water and
neutralized by adding an appropriate amount of counter ions in an orthorhombic box
(
10 Å × 10 Å × 10 Å) to form a buffer area between the protein atoms and the side of
the box. Then, the Optimized Potentials for Liquid Simulation (OPLS_2005) force field
was used to minimize the energy of the complex system, and the maximum number of
iterations in the minimization process was set to 5000, and the convergence threshold was
kept at 1.0 kcal/mol/Å. A mixed method of the steepest descent and the limited memory
Broyden-Fletcher-Goldfarb-Shanno (LBFGS) algorithms with the maximum of 5000 steps
were performed to minimize the system energy until a gradient threshold of 25 kcal/mol/Å
was reached. Before MD simulations, a 100 ps simulation was performed to relax the whole
system. The temperature was set to 300 K and maintained throughout by implementing
Nose-Hoover thermostat with the pressure set to 1.01325 bar and maintained through
Martyna-Tobias-Klein pressure bath. Finally, the 100 ns MD simulations were carried out, a
time interval of every trajectory was recorded at every 100 ps.
3.4. CCK-8 Assay In Vitro
The MDA-MB-231, HT-29 and MG-63 cell lines were provided by KeyGEN BioTECH
Ltd (Nanjing, Jiangsu, China). The cells were digested and counted to prepare a cell
suspension of 5.0
×
104 cells/mL, and then 100
µL of cell suspension was added to each
well of the 96-well cell culture plate. The plates were placed in a 37 ◦C, 5% CO2 incubator
(Corning Incorporated, Corning, New York, USA) for 24 h. The drug was diluted with
the culture medium to the required concentration, ed100 µL of the corresponding drug-
containing medium was added to each well, and a negative control group was set up at the
same time. The 96-well cell culture plates were placed into a normoxic incubator (37 ◦C,
5% CO2) and hypoxic incubator (37 ◦C, 5% CO2, 0.5% O2, 94.5% N2) for 48 h. Finally, the
96-well plate was stained with CCK-8 and the OD value determined (λ = 450 nm).

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3.5. Measurement of Extracellular pH
The measurement of the change of extracellular pH referred to the reported litera-
ture [26]. In brief, cancer cells were incubated for 12 h. After removing the medium, fresh
medium was added to the plates and then incubated for 48 h under hypoxia or normoxia.
The pH of the medium was measured immediately. The tested compounds were dissolved
in DMSO and diluted with culture medium to the desired concentration. After the addition
of tested compounds, the same procedure was repeated.
4. Conclusions
Herein, a series of saccharide-modified thiadiazole sulfonamide derivatives were
reported. Intermediates 12a–h and compounds 14a–p were assayed for their inhibitory
activities against three hCA isoforms of pharmacological relevance: tumor-associated
transmembrane isoforms hCA IX and XII and cytosolic off-target isoform hCA II. The results
indicated that all the tested compounds showed activity against hCA II, IX, and XII. Among
them, the IC50 values for the saccharide-modified derivatives with high TPSA values for
hCA II and IX ranged from 80.3–1403.1 nM and 29.0–1082.0 nM, respectively. Molecular
docking results showed that the interaction with residue Gln92 and the distance between
sulfanilamide and Zn2+ had important impacts on the activity. Further cell experiments
showed that compounds 14a, 14b, 14h, and 14p had inhibitory effects on the viability of
cancer cell lines with high expression of CA IX, and this viability inhibition was enhanced
under hypoxic conditions. Compound 14a had better inhibitory activity than the positive
control AZM on MG-63 cells with high expression of CA IX and XII. In addition, compounds
14b, 14h, and 14p significantly modified the acidic microenvironments of cancer cells.
Based on the above results and the known importance of the acidic microenvironment for
tumor growth, we believe that saccharide-modified CA IX inhibitors have prospects for
further development as combination drugs for tumor treatment.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3
390/ijms22115482/s1.
Author Contributions: M.-S.C. and Y.L. supervised the whole experiment and provided technical
guidance. Z.-P.Z. designed and synthesized all of novel compounds. Y.Z., Z.-B.H., L.Z. and H.-S.S.
assisted in the synthetic and biology research. J.W. supervised and carried out molecular modeling
experiments. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Natural Science Foundation of China, grant number
81473087, 81872752.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Yang Liu wishes to express his thanks for the support by the Program for
Liaoning Innovative Talents in University (grant number LR2017043).
Conflicts of Interest: The authors declare no conflict of interest.
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