Synthesis and evaluation of new carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.03.061

A series of new sulfamide derivatives have been synthesized, their structures were confirmed by ¹H NMR and ESI-MS. Some target compounds were assessed by the tool of Dock6, and inhibition effects of all the new compounds on carbonic anhydrase II have been investigated. In addition, some compounds have been investigated for their antihypoxic effects in mice. Results indicated that nine target compounds exhibit as effectively as acetazolamide and 10 compounds have more potent inhibition effects on carbonic anhydrase II than acetazolamide. Three of them (I-8, I-18 and I′-3) can prolong markedly the survival time of mice in hypoxia, which are worth carrying out further studies.

Full text of DOI:10.1016/j.bmc.2011.03.061

Bioorganic & Medicinal Chemistry 19 (2011) 3221–3228
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of new carbonic anhydrase inhibitors
Zhonghai Xiao a, , Ruifeng Duan a, , Wenyu Cui b, Yanfang Zhang b, Shouguo Zhang c, Fangjin Chen d,
a,b,e,
Yankun Zhang a, Jiaying Liu a, Dongxiang Zhang a, Yuan Meng e, Lin Wang c, , Hai Wang
⇑
⇑
a Department of Environmental Medicine, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Tianjin 300050, China
b Department of Cardiovascular Pharmacology, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
c Department of Medicinal Chemistry, Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China
d Department of Spinal Cord Injury Repair, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China
e Thadweik Academy of Medicine, Beijing 100039, China
a r t i c l e i n f o
a b s t r a c t
A series of new sulfamide derivatives have been synthesized, their structures were confirmed by 1H NMR
and ESI-MS. Some target compounds were assessed by the tool of Dock6, and inhibition effects of all the
new compounds on carbonic anhydrase II have been investigated. In addition, some compounds have
been investigated for their antihypoxic effects in mice. Results indicated that nine target compounds
exhibit as effectively as acetazolamide and 10 compounds have more potent inhibition effects on car-
bonic anhydrase II than acetazolamide. Three of them (I-8, I-18 and I0-3) can prolong markedly the sur-
vival time of mice in hypoxia, which are worth carrying out further studies.
Article history:
Received 20 January 2011
Revised 25 March 2011
Accepted 26 March 2011
Available online 2 April 2011
Keywords:
Synthesis
Acetazolamide
Ó 2011 Elsevier Ltd. All rights reserved.
Human carbonic anhydrase II
Acute mountain sickness
Hypoxia
1. Introduction
1,3,4-thiadiazole and sulfamide group as pharmacophore, two ser-
ies of new derivatives have been synthesized and evaluated. The
Acute mountain sickness (AMS) is a common disease at high
altitude, its disease rate reaches up to 60% among Chinese people
and about 42% at abroad.1,2 The clinical symptoms are headache,
cardiopalmus, dyspnea, anorexia, nausea, acratia, cyanosis and oe-
dema et al. Prevention and treatment of AMS is an important topic.
One of the carbonic anhydrase (CA) inhibitors-acetazolamide (AZA)
is regarded as an efficient drug to prevent and cure AMS. In Amer-
ica, AZA is the unique medicine to the indication, which was ap-
proved by FDA in 1994.3
But the common dose is much higher (500 g everyday) and the
rate of significant side-effects of AZA for preventing and treating
AMS is about 64.1%,4 the generalization and application of AZA
are confined greatly. Therefore, it is important to find a new kind
of safe and effective medicine to prevent and cure AMS. It has been
discovered that 1,3,4-thiadiazole and sulfamide group in the
molecular structure of AZA (Fig. 1) are the chief functional groups
of the sulfamide CA II inhibitors.5 To find more potent and less
side-effects drug than AZA, with AZA as a lead compound and
lipophilic groups were introduced manually in order to increase
compounds’ inhibition effects and selectivity. Assisted by com-
puter-aided drug design, the interactions between 10 compounds
and CA II were assessed by flex-docking method. The inhibition ef-
fects on CA II of 36 new compounds were investigated, and the
antihypoxic effects in mice of 10 compounds were assessed.
2. Results and discussion
2.1. Chemistry
With the 2-(p-toluenesulfonamido)-l,3,4-thiadiazole-5-sulfon-
amide (3I) or 2-benzene-sulfonamido-l,3,4-thiadiazole-5-sulfon-
amide (3I0) as a scaffold; we have synthesized a new class of AZA
derivatives of such system to be tested against the cytosolic iso-
zymes as well as the AMS-associated CA II.6,7
O
O
S
N
N
⇑
Corresponding authors. Tel./fax: +86 856 10 6693 2239 (L.W.); tel.: +86 10 6818
6828; fax: +86 10 6693 1557 (H.W.).
H
N
H3C
NH2
S
E-mail addresses: zhonghai.x@163.com (Z. Xiao), wanglin07@sina.com
(L. Wang), wh9588@yahoo.com.cn (H. Wang).
O
These authors contributed equally to this work.
Figure 1. Structure of acetazolamide.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.061

3222
Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
O
S
The target compounds, 2-[(p-toluenesulfonamido)-N-yl]-l,3,4-
thiadiazole-5-sulfonamides I-1–I-19, were obtained by refluxing
O
O
S
N
N
N
N
N
N
H
N
a
c
b
H2N
NH2
H3C
NH2
NH2
S
S
2-(p-toluenesulfonamido)-l,3,4-thiadiazole-5-sulfonamide
(3I)
O
O
with different halogenated hydrocarbons in alkaline medium fol-
lowed by acidification. Similarly, the derivatives 2-(benzenesulfon-
2
1
amido-N-yl)-l,3,4-thiadiazole-5-sulfonamides
I0-1–I0-15
were
O
O
S
O
S
O
S
N
N
H
N
prepared by refluxing 2-benzene-sulfonamido-l,3,4-thiadiazole-5-
sulfonamide (3I0) with different halogenated hydrocarbons in alka-
line medium followed by acidification. The target compounds II-1
and II0-1 were prepared by refluxing 3I and 3I0 with a large excess
of different halogenated hydrocarbons and alkali followed by
acidification.
R1
S
R1
N
NH2
S
S
O
O
O
R2
O
3I or 3I'
I-1~I-19; I'-1~I'-15
Scheme 1. Synthesis routes for target compounds I-1–I-19 and I0-1–I0-15. Reagents
and conditions: (a) HCl, ethanol, reflux 4 h; (b) p-toluenesulfonyl chloride, acetone,
2.5 mol Lꢀ1 NaOH aq, 5 mol Lꢀ1 NaOH aq, stir 30 min at 0–5 °C, HCl, Et2O;
benzenesulfonyl chloride, acetone, 2.5 mol Lꢀ1 NaOH aq, 5 mol Lꢀ1 NaOH aq, stir
30 min at room temperature, HCl, Et2O; (c) 3I or 3I’(1 mol), R2X (X = Br or I, 1 mol),
KOH (1 mol), DMF, 60–80 °C (2–4 h).
2.2. Carbonic anhydrase inhibition
2.2.1. In vitro inhibition
Derivatives I-1–I-19, I0-1–I0-15, II-1 and II0-1 were investigated
for their inhibitory effects on hCA II (Table 1). The reported inhib-
itory data for the potent CA inhibitor AZA was also mentioned for
comparison. Because the inhibition rate of AZA was about 50%
O
S
O
S
O
S
O
S
N
N
N
N
R2
R2
H
N
d
R1
N
N
NH2
R1
S
S
O
R2
O
O
O
when the concentration of AZA was 0.3 l
mol Lꢀ1 in the conditions
3I or 3I'
II-1; II'-1
of the experiments, we use the same concentration for all com-
pounds to compare their inhibition effects on hCA II.
Scheme 2. Synthesis routes for target compounds II-1 and II0-1. Reagents and
conditions: (d) 3I or 3I’ (1 mol), R2X (X = Br or I, 3 mol), KOH (3 mol), DMF, 60 °C
(2 h).
The results of in vitro inhibition evaluation for all compounds
are shown in Table 1: (i) hCA II was inhibited by compounds
I-1–I-19, I0-1–I0-15 and AZA with inhibition rate in the range of
5.75–65.85%. The inhibition rate of AZA was 55.35%. The inhibition
effects of ten compounds (I-5, I-7, I-12, I-17, I-18, I0-1, I0-2, I0-3, I0-9
and I0-11) was higher than that of AZA (P <0.05), which showed
higher activities with inhibition rate of 56.22–65.85%. The inhibi-
tion effects of nine compounds (I-3, I-4, I-8, I0-4, I0-8, I0-10, I0-12,
I0-13 and I0-14) equaled to that of AZA (P >0.05), which showed
moderate activities with inhibition rate of 52.66–56.04%, while
other derivatives have less inhibition effects than AZA. (ii) Deriva-
tives II-1 and II0-1 showed inhibition rate of ꢀ1.75% and 0.66%,
respectively. Compounds II-1 and II0-1 had no activities.
The synthesis of target compounds I-1–I-19 and I0-1–I0-15 is
outlined in Scheme 1 and that of II-1 and II0-1 is outlined in
Scheme 2. The starting compound, 2-amino-l,3,4-thiadiazole-5-
sulfonamide (2)8,9 was prepared according to the method reported
by Arslan and Clapp et al. through the reaction between AZA and
concentrated HCl in ethanol.
The preparation of the key intermediate, 2-(p-toluenesulfonam-
ido)-l,3,4-thiadiazole-5-sulfonamide (3I),10,11 was achieved
through the reaction by stirring the 2-amino-l,3,4-thiadiazole-5-
sulfonamide (2) with p-toluenesulfonyl chloride in alkaline
medium for 30 min at 0–5 °C followed by acidification. 2-Benzene-
sulfonamido-l,3,4-thiadiazole-5-sulfonamide (3I0)10,11 was achieved
through the reaction by stirring the 2-amino-l,3,4-thiadiazole-5-
sulfonamide (2) with benzenesulfonyl chloride in alkaline medium
for 30 min at room temperature followed by acidification.
2.2.2. In vivo inhibition
Derivatives I-2, I-7, I-8, I-12, I-17, I-18, I0-1, I0-2, I0-3 and I0-8
were investigated for their antihypoxic effects on hypoxic
Table 1
Inhibition data for derivatives I-1–I-19 and I0-1–I0-15 and II-1 and II0-1 investigated in the present paper and standard sulfonamide CAIs (AZA) on hCA II (x ꢁ s)
Compd
R1
R2
Inhibition rate/%
Compd
R1
R2
Inhibition rate/%
AZA
I-1
I-2
I-3
I-4
I-5
I-6
I-7
I-8
CH3-CO–
H
55.35 2.67
51.52 1.54
51.83 1.00
56.04 1.35#
52.66 1.82#
56.22 0.95*
38.75 1.93
60.17 1.32***
54.78 1.09#
50.18 3.15
12.79 13.49
38.90 2.30
65.85 1.80***
5.75 3.55
AZA
I-19
I0-1
CH3-CO–
4-CH3-Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
Ph-SO2–
4-CH3-Ph-SO2–
Ph-SO2–
H
55.35 2.67
48.76 2.36
60.83 0.95***
60.57 3.56**
58.61 0.97***
55.35 3.03#
20.86 16.45
48.22 4.10
54.62 2.87#
55.47 3.68#
57.82 0.98*
53.55 3.15#
58.19 0.93**
54.57 0.47#
55.41 1.36#
45.75 10.83
50.52 0.94
ꢀ1.75 1.66
0.66 1.75
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
4-CH3-Ph-SO2–
n-C4H9–
Ph-CH2–
n-C3H7–
CH3–
2-CH3O-Ph-(C3H6)–
C2H5–
n-C4H9–
I0-2
I0-3
n-C3H7–
C2H5–
I0-4
Ph-CH2-CH2–
4-Cl-Ph-CH2–
2,4-Cl-Ph-CH2–
CH3–
n-C5H11
i-C4H9–
n-C12H25
i-C3H7–
i-C4H9–
n-C5H11
4-Cl-Ph-CH2–
2,4-Cl-Ph-CH2–
Ph-CH2-CH2–
4-Br-Ph-CH2–
4-CH3O-Ph-CH2–
4-Br-Ph-CH2-CH2–
3,4,5-CH3O-Ph-CH2–
4-CH3-Ph-CH2–
4-F-Ph-CH2–
–
I0-5
I0-6
I0-7
I-9
–
I0-8
–
I-10
I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
I0-9
I0-10
I0-11
I0-12
I0-13
I0-14
I0-15
II-1
II0-1
n-C12H25–
Ph-CH2–
4-CH3O-Ph-CH2–
i-C3H7–
4-Br-Ph-CH2-CH2–
3,4,5-CH3O-Ph-CH2–
CH3–
47.19 1.95
34.77 7.67
49.44 1.01
62.30 0.49***
64.88 1.25***
Ph-CH2–
*
**
P <0.05.
P <0.01.
P <0.001.
***
#
P >0.05 versus AZA.

Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
3223
Table 2
Table 3
Survival time of mice for some derivatives investigated in the present paper
Grid energy of binding and other interaction energies between ligands and CA II (kcal/
mol)
Compd
Time (s)
Compd
Time (s)
Compound
Egrid
Evdw
Eele
D
Blank control
Positive control
I-2
I-7
I-8
I-12
2199 217
2726 530
2582 426
2426 385
3486 489
2441 358
I-17
I-18
I0-1
I0-2
I0-3
I0-8
2512 302
2860 392
2716 444
2977 322
3135 592
2716 369
DD
D
DD,**
Az
3I
ꢀ38.29
ꢀ43.95
ꢀ56.26
ꢀ52.77
ꢀ49.29
ꢀ50.84
ꢀ52.39
ꢀ46.57
ꢀ50.14
ꢀ50.18
ꢀ48.03
ꢀ51.53
ꢀ39.66
ꢀ23.81
ꢀ28.44
ꢀ41.38
ꢀ36.29
ꢀ38.15
ꢀ38.63
ꢀ39.62
ꢀ29.58
ꢀ37.61
ꢀ34.56
ꢀ30.40
ꢀ36.15
ꢀ32.50
ꢀ14.48
ꢀ15.51
ꢀ14.88
ꢀ16.48
ꢀ11.14
ꢀ12.21
ꢀ12.77
ꢀ16.99
ꢀ12.53
ꢀ15.62
ꢀ17.63
ꢀ15.38
ꢀ7.16
DD
I-1
I-2
I-3
I-4
I-7
3I0
I0-1
I0-3
I0-7
I0-11
I0-13
DD,**
DD,**
DD
**
P <0.01 versus positive control.
P <0.05.
P <0.01 versus blank control.
D
DD
tolerance in mice. The reported data for AZA was also mentioned
for comparison.
The following should be noted regarding data presented in
Table 2: The tolerance time of the blank control group was 2199 s,
and that of the positive control group was 2726 s. The survival time
of the target compound group (I-2, I-8, I-17, I-18, I0-2, I0-3 and I0-8)
and the positive control group (AZA) was all higher than that of the
blank control group. The survival time of the target compound
group (I-8, I-18 and I0-3) was higher than that of the positive
control group markedly, and these three compounds were worth
carrying out further study.
flex-docking procedures were performed for each ligand and CA
II. The grid energy of ligand–enzyme complex (Egrid), the interac-
tion electrostatic (Eele), and van der Waals (Evdw) energies between
the ligand and the enzyme (Evdw) were calculated (Table 3).15
The energy data showed a rough correlation between the bind-
ing grid energy (Egrid) values of target compounds and their inhib-
itory effects on hCA II. With the highest activity, the compound I-1
showed the highest Egrid values of ꢀ56.26 kcal/mol. Regarding the
electrostatic and van der Waals energy values, all derivatives
showed high Evdw values indicating the importance of this types
of interactions for enzyme binding.
The following notes were observed regarding the docked struc-
tures (Fig. 2): (i) The amino group of target compounds replace the
hydroxyl ion/water molecule coordinated to zinc atom in the na-
tive enzyme. The zinc ion remains in its stable tetrahedral geome-
try being coordinated, in addition to the amino group, with
imidazole nitrogens of His94, His96 and His119. (ii) The Zn–N bond
distance is 1.99 Å, which is comparable with the reported data
(1.95–2.10 Å).16 Although there is no clear relationship between
the Zn–N bond distance of the docked compounds and their activ-
ities, it was noted that compound I-1 showed the shorter bond dis-
tance consistent with its higher inhibitory activity.
2.3. Structure–activity relationships
The structure–activity relationships of derivatives I-1–I-19,
I0-1–I0-15, II-1 and II0-1 were analyzed as shown that:
ꢂ When R1 is the same, R2 different:
(1)
For compounds group I: When R2 is an aryl, the carbon
chains connected with nitrogen are much shorter and
the para orientation substituents are much smaller, the
activities are much stronger; there is no activity if the car-
bon chains are too long or the para orientation substitu-
ents are too bulky; when R2 is an alkyl and the lengths
of carbon chains are 2–4, the activities are much better
than others, the excess long carbon chains are disadvanta-
geous to the compounds’ activities.
For compounds group I0: The most compounds have no
activity when R2 is an aryl. The compounds’ activities
are strong when R2 is an alkyl, the lengths of carbon
chains don’t influence the activities.
3. Conclusion
(2)
The synthesis of a new series of 1,3,4-thiadiazole-2-sulfamide
derivatives was reported here. The compounds have been tested
for their inhibitory effects on hCA II. The results revealed that some
derivatives were very effective inhibitors for hCA II. Some com-
pounds have been investigated for their antihypoxic effects, the
survival time of mice administered some derivatives was much
longer apparently than that of AZA. In addition, the binding mode
of the tested compounds inside the hCA II active site was predicted
using a docking technique initially. And structure–activity rela-
tionship study is useful theoretical and experimental evidence to
optimize the compound’s structure and find new drugs for the pre-
vention and treatment of AMS.
ꢂ
When R2 is the same, R1 different:
(1) When R2 is an alkyl, compounds group I and I0 all have activ-
ities, the activities of compounds group I0 are stronger than
that of I, which is consistent with that of CADD.
(2) When R2 is an aryl, the activities of compounds group I is
stronger than that of I0, and three compounds’ activities
are stronger than that of AZA.
ꢂ The compound II-1 and II0-1 have no activity. All these results
demonstrate the sulfamide group R-SO2NH2 is an essential
group for inhibition of enzyme activity.
4. Experimental
4.1. General
2.4. Docking studies
We tried to predict such activity by docking the synthesized
compound 3I, 3I0, I-1, I-3, I-4, I-5, I-9, I0-1, I0-2, I0-3, I0-8, I0-9 and
AZA into hCA II active site using the tool of Dock6.12,13
The X-ray crystallographic structure of human carbonic anhy-
drase II complexed with brinzolamide (hCA II PDB: 1A42)14 was
used as the model to discover the inhibitor of catalytic site. The
Melting points were recorded in open capillaries with electro-
thermal melting point apparatus (RY-1, China) and are
uncorrected. 1H NMR (400 MHz) spectra were recorded on ‘JNM-
ECA-400’ (Japan) with DMSO-d6 or CDCl3 as a solvent, TMS as an
internal reference and the chemical shifts are expressed in parts

3224
Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
Figure 2. 3D docked structure of I-1 (ball and stick) at hCA II active site. The purple ball is represented catalytically critical Zn(II), and the Zn–N (–SO2NH2) bond distance is
1.99 Å. The figure has been generated by using ‘Linux’ operating system installed on the Xeon with a 2.6 MHz processor and 1G RAM. The X-ray crystallographic structure of
hCA II complexed with brinzolamide (hCA II PDB: 1A42) was obtained from the Protein Data Bank.
per million (d, ppm). Mass spectra (MS) were performed on ‘LCQ
Advantage MAX 10 spectrometer’ (Finnigan, America). Elemental
analyses were performed on ‘Carlo Erbal 106’ (Italy). All reactions
were monitored by thin-layer chromatography (TLC) using Silica
Gel 60 GF245 precoated sheets 20 ꢃ 20 cm, layer thickness
0.2 mm (Qing Dao Hai Yang, China) and were visualized by UV-
lamp at wavelength (k) 254 nm. All chemicals and solvents were
analytical reagents, and the solvents were distilled before use.
chloride was replaced by benzenesulfonyl chloride (2.10 g,
0.012 mol). The crude product was recrystallized from 60 mL of
hot water to give the white floccular crystal. Mp: 230–232 °C
(0.27 g, 15.5%). Anal. Calcd for C8H8N4O4S3: C, 29.99; H, 2.52; N,
17.49. Found: C, 30.10; H, 2.36; N, 18.02. ESI-MS, m/z: [MꢀH]ꢀ
(calcd) 318.80 (318.97).
4.2.4. General procedure for the synthesis of 2-[(p-toluen-
esulfonamido)-N-yl]-l,3,4-thiadiazole-5-sulfonamides (I-1–I-
19)
4.2. Chemistry
2-(p-Toluenesulfonamido)-l,3,4-thiadiazole-5-sulfonamide (3I)
(0.16 g, 0.5 mmol) was dissolved in N,N-dimethylformamide
(DMF, 1 mL) with stirring at room temperature. Potassium hydrox-
ide (0.03 g, 0.5 mmol) was suspended in DMF (1 mL) then added to
the above solution, potassium hydroxide was dissolved slowly.
Then a mixture of halogenated hydrocarbons (0.6 mmol) and
DMF (1 mL) was added to the reaction mixture and heated to
60–80 °C in an oil-bath for 1–3 h (controlled by TLC). The solvent
was evaporated to dryness in vacuum, the residue was flaxen solid.
The compound I-1–I-19 was obtained by chromatographic
fractionation.
4.2.1. Synthesis of 2-amino-l,3,4-thiadiazole-5-sulfonamide
(2)8,9
A mixture of AZA (12.15 g, 0.055 mol), 12 mL of concentrated
HC1 and 180 mL of anhydrous ethanol was heated in 80 °C oil bath
for 4 h. After a part of ethanol had evaporated, the obtained sus-
pension was allowed to cool slowly. The solid was filtered and
crystallized from water to give the analytical sample (86.0%);
mp: 216–219 °C; 1H NMR (DMSO-d6) d: 7.81 (s, 2H, H2N-thiadia-
zole), 8.05 (s, 2H, –SO2NH2); ESI-MS, m/z: [M+H]+ (calcd) 180.90
(180.98), [MꢀH]ꢀ (calcd) 178.88 (178.98).
4.2.2. Synthesis of 2-(p-toluenesulfonamido)-l,3,4-thiadiazole-
5-sulfonamide (3I)10,11
4.2.4.1. 2-[(p-Toluenesulfonamido)-N-(n-butyl)]-l,3,4-thiadia-
zole-5-sulfonamide (I-1).
Yield, 51.3%; mp: 136–138 °C; 1H
With stirring at 0–5 °C, 2 mL 5 mol Lꢀ1 sodium hydroxide
(0.01 mol) and a solution of p-toluenesulfonyl chloride (2.28 g,
0.012 mol) in 3 mL of acetone were added simultaneously to a
solution of 2-amino-l,3,4-thiadiazole-5-sulfonamide (1.80 g,
0.01 mol) in 4 mL 2.5 mol Lꢀ1 sodium hydroxide (0.01 mol). The
reaction mixture, which became quite warm due to exothermic
heat of reaction, was recooled to 0–5 °C and stirred for an addi-
tional 30 min. The solution was then extracted with 15 mL of ether
and the aqueous phase was separated, treated with activated char-
coal, filtered and acidified with concentrated hydrochloric acid to
pH 3. On cooling and stirring, the product was separated slowly
from this solution as a finely divided white solid, the crude product
was recrystallized from 80 mL of hot water to give the white floc-
cular crystal. Mp: 254–255 °C (0.70 g, 10.6%). 1H NMR (DMSO-d6)
d: 2.38(s, 3H, –CH3), 7.38–7.40 (d, 2H; J = 8.1 Hz; H-3, 5 of phenyl
ring), 7.71–7.73 (d, 2H; J = 8.4 Hz; H-2, 6 of phenyl ring), 8.50 (s,
2H, –SO2NH2), 13.40 (s, 1H, HN-thiadiazole). Anal. Calcd for
C9H10N4O4S3: C, 32.33; H, 3.01; N, 16.75. Found: C, 32.52; H,
2.86; N, 17.04. ESI-MS, m/z: [MꢀH]ꢀ (calcd) 332.81 (332.99).
NMR (CDCl3) d: 0.85–0.89 (t, 3H; J = 7.3 Hz; CH3–CH2–CH2–CH2–
N—), 1.23–1.29 (m, 2H; J = 7.6 Hz; CH3–CH2–CH2–CH2–N—), 1.70–
1.75 (m, 2H; J = 7.6 Hz; CH3–CH2–CH2–CH2–N—), 2.42 (s, 3H,
CH3–Ph-SO2–), 4.13–4.17 (t, 2H; J = 7.3 Hz; CH3–CH2–CH2–CH2–
N—), 5.69 (s, 2H, –SO2–NH2), 7.29–7.31 (d, 2H; J = 7.9 Hz; H-3, 5
of phenyl ring), 7.76–7.78 (d, 2H; J = 8.4 Hz; H-2, 6 of phenyl ring).
ESI-MS, m/z: [M+H]+ (calcd) 391.04 (391.06), [MꢀH]ꢀ (calcd)
388.97 (389.04).
4.2.4.2. 2-[(p-Toluenesulfonamido)-N-benzyl]-l,3,4-thiadiazole-
5-sulfonamide (I-2).
Yield, 70.8%; mp: 179–181 °C; 1H NMR
(DMSO-d6) d: 2.38 (s, 3H, CH3-Ph-SO2-), 5.34 (s, 2H, Ph-CH2-N—),
7.19–7.22 (m, 2H; J = 7.6 Hz; Ph-CH2-N—), 7.29–7.32 (m, 3H;
J = 6.8 Hz; Ph-CH2-N—), 7.36–7.38 (d, 2H; J = 8.9 Hz; CH3-Ph-SO2-),
7.68–7.70 (d, 2H; J = 8.4 Hz; CH3-Ph-SO2-), 8.61 (s, 2H, –SO2–
NH2). ESI-MS, m/z: [M+H]+ (calcd) 424.94 (425.04), [MꢀH]ꢀ (calcd)
422.93 (423.03). Anal. Calcd for C16H16N4O4S3: C, 45.27; H, 3.80; N,
13.20. Found: C, 45.54; H, 3.50; N, 13.24.
4.2.4.3. 2-[(p-Toluenesulfonamido)-N-(n-propyl)]-l,3,4-thiadia-
4.2.3. Synthesis of 2-benzenesulfonamido-l,3,4-thiadiazole-5-
sulfonamide (3I0)10,11
The procedure was same to the above except that a reaction
temperature of room temperature was used and p-toluenesulfonyl
zole-5-sulfonamide (I-3).
Yield, 71.8%; mp: 161–163 °C; 1H
NMR (CDCl3) d: 0.86–0.90 (t, 3H; J = 7.4 Hz; CH3–CH2–CH2–N—),
1.77–1.80 (m, 2H; J = 7.4 Hz; CH3–CH2–CH2–N—), 2.42 (s, 3H, CH3-
Ph-SO2-), 4.10–4.14 (t, 2H; J = 7.4 Hz; CH3–CH2–CH2–N—), 5.69 (s,

Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
3225
2H, –SO2–NH2), 7.29–7.31 (d, 2H; J = 8.1 Hz; H-3, 5 of phenyl ring),
2H; J = 8.1 Hz; H-3,
5
of CH3-Ph-SO2-), 7.65–7.67 (d, 2H;
7.76–7.78 (d, 2H; J = 8.1 Hz; H-2, 6 of phenyl ring). ESI-MS, m/z:
J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2-), 8.59 (s, 2H, –SO2–NH2). ESI-
MS, m/z: [M+H]+ (calcd) 458.93 (459.00), [MꢀH]ꢀ (calcd) 456.91
(456.99).
[M+H]+ (calcd) 377.01 (377.04), [MꢀH]ꢀ (calcd) 374.94 (375.03).
4.2.4.4. 2-[(p-Toluenesulfonamido)-N-methyl]-l,3,4-thiadiazole-
5-sulfonamide (I-4).
Yield, 69.0%; mp: 205–208 °C; 1H NMR
4.2.4.11. 2-[(p-Toluenesulfonamido)-N-(2,4-dichlorobenzyl)]-
(DMSO-d6) d: 2.38 (s, 3H, CH3-Ph-SO2-), 3.71 (s, 3H, CH3-N—),
7.39–7.41 (d, 2H; J = 8.4 Hz; H-3, 5 of phenyl ring), 7.74–7.76 (d,
2H; J = 8.1 Hz; H-2, 6 of phenyl ring), 8.59 (s, 2H, –SO2–NH2). ESI-
MS, m/z: [M+H]+ (calcd) 348.97 (349.01), [MꢀH]ꢀ (calcd) 346.91
(346.99).
l,3,4-thiadiazole-5-sulfonamide (I-11).
Yield, 69.0%; mp:
125–126 °C; 1H NMR (DMSO-d6) d: 2.39 (s, 3H, CH3-Ph-SO2-),
5.39 (s, 2H, (Cl)2-Ph-CH2-), 7.30–7.32 (d, 2H, H-5, 6 of (Cl)2-Ph-
CH2-), 7.36–7.66 (m, 4H, CH3-Ph-SO2-), 7.95 (s, 1H, H-3 of (Cl)2-
Ph-CH2-), 8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd)
492.86 (492.96), [MꢀH]ꢀ (calcd) 490.87 (490.95).
4.2.4.5.
2-[(p-Toluenesulfonamido)-N-ethyl]-l,3,4-thiadia-
Yield, 60.8%; mp: 200–203 °C; 1H
zole-5-sulfonamide (I-5).
4.2.4.12. 2-[(p-Toluenesulfonamido)-N-phenethyl]-l,3,4-thiadi-
NMR (DMSO-d6) d: 1.24–1.28 (t, 3H; J = 7.3 Hz; CH3-CH2-N—),
2.38 (s, 3H, CH3-Ph-SO2-), 4.10–4.16 (q, 2H; J = 7.3 Hz; CH3–
CH2–N—), 7.39–7.41 (d, 2H; J = 8.1 Hz; H-3, 5 of phenyl ring),
7.74–7.76 (d, 2H; J = 8.1 Hz; H-2, 6 of phenyl ring), 8.59 (s, 2H,
–SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 363.02 (363.03), [MꢀH]ꢀ
(calcd) 360.92 (361.01).
azole-5-sulfonamide (I-12).
Yield, 87.6%; mp: 148–150 °C;
1H NMR (DMSO-d6) d: 2.40 (s, 3H, CH3-Ph-SO2-), 3.00–3.03 (t,
2H; J = 7.0 Hz; Ph-CH2-CH2-N—), 4.36–4.39 (t, 2H; J = 7.0 Hz; Ph-
CH2-CH2-N—), 7.04–7.16 (m, 5H, Ph-CH2-CH2-N—), 7.38–7.40 (d,
2H; J = 8.1 Hz; H-3,
5 of CH3-Ph-SO2-), 7.62–7.64 (d, 2H;
J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2-), 8.61 (s, 2H, –SO2–NH2). ESI-
MS, m/z: [M+H]+ (calcd) 439.02 (439.06), [MꢀH]ꢀ (calcd) 436.94
(437.04).
4.2.4.6. 2-[(p-Toluenesulfonamido)-N-(n-lauryl)]-l,3,4-thiadia-
zole-5-sulfonamide (I-6).
Yield, 26.1%; mp: 96–99 °C; 1H
NMR (CDCl3) d: 0.86–0.90 (t, 3H; J = 6.8 Hz; CH3-(CH2)9-CH2-CH2-
N—), 1.20–1.30 (m, 18H, CH3-(CH2)9-CH2-CH2-N—), 1.72–1.75 (m,
2H; J = 6.8 Hz; CH3-(CH2)9-CH2-CH2-N—), 2.42 (s, 3H, CH3-Ph-SO2-
), 4.12–4.16 (t, 2H; J = 7.3 Hz; CH3-(CH2)9-CH2-CH2-N—), 5.81 (s,
2H, –SO2–NH2), 7.28–7.30 (d, 2H; J = 8.1 Hz; H-3, 5 of phenyl ring),
7.75–7.77 (d, 2H; J = 8.1 Hz; H-2, 6 of phenyl ring). ESI-MS, m/z:
[M+H]+ (calcd) 503.15 (503.18), [MꢀH]ꢀ (calcd) 501.05 (501.17).
4.2.4.13. 2-[(p-Toluenesulfonamido)-N-(4-bromobenzyl)]-l,3,4-
thiadiazole-5-sulfonamide (I-13).
Yield, 80.3%; mp: 235–
238 °C; 1H NMR (DMSO-d6) d: 2.39 (s, 3H, CH3-Ph-SO2-), 5.32 (s,
2H, Br-Ph-CH2-N—), 7.15–7.37 (m, 4H, Br-Ph-CH2-N—), 7.48–7.66
(m, 4H, CH3-Ph-SO2-), 8.59 (s, 2H, –SO2–NH2). ESI-MS, m/z:
[M+H]+ (calcd) 502.85 (502.95), [MꢀH]ꢀ (calcd) 500.85 (500.94).
4.2.4.14.
2-[(p-Toluenesulfonamido)-N-(4-methoxylbenzyl)]-
Yield, 66.7%; mp:
4.2.4.7. 2-[(p-Toluenesulfonamido)-N-(i-propyl)]-l,3,4-thiadia-
l,3,4-thiadiazole-5-sulfonamide (I-14).
zole-5-sulfonamide (I-7).
Yield, 45.2%; mp: 186–188 °C; 1H
225–227 °C; 1H NMR (DMSO-d6) d: 2.39 (s, 3H, CH3-Ph-SO2-),
3.72 (s, 3H, CH3O-Ph-CH2-N—), 5.26 (s, 2H, CH3O-Ph-CH2-N—),
6.83–6.85 (d, 2H; J = 8.7 Hz; H-3, 5 of CH3O-Ph-SO2-), 7.15–7.17
(d, 2H; J = 8.7 Hz; H-2, 6 of CH3O-Ph-SO2-), 7.37–7.39 (d, 2H;
J = 8.4 Hz; H-3, 5 of CH3-Ph-SO2-), 7.70–7.71 (d, 2H; J = 8.1 Hz; H-
2, 6 of CH3-Ph-SO2-), 8.59 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+
(calcd) 454.95 (455.05), [MꢀH]ꢀ (calcd) 452.95 (453.04).
NMR (DMSO-d6) d: 1.32–1.33 (d, 6H; J = 6.7 Hz; (CH3)2-CH-N—),
2.38 (s, 3H, CH3-Ph-SO2-), 4.77–4.81 (m, 1H; J = 6.6 Hz; (CH3)2-
CH-N—), 7.39–7.41 (d, 2H; J = 8.1 Hz; H-3, 5 of phenyl ring),
7.74–7.76 (d, 2H; J = 8.4 Hz; H-2, 6 of phenyl ring), 8.57 (s, 2H, –
SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 376.98 (377.04), [MꢀH]ꢀ
(calcd) 374.93 (375.03).
4.2.4.8.
zole-5-sulfonamide (I-8).
2-[(p-Toluenesulfonamido)-N-(i-butyl)]-l,3,4-thiadia-
Yield, 51.3%; mp: 182–184 °C; 1H
4.2.4.15. 2-[(p-Toluenesulfonamido)-N-(4-bromophenethyl)]-
l,3,4-thiadiazole-5-sulfonamide (I-15).
Yield, 65.9%; mp:
NMR (DMSO-d6) d: 0.79–0.81 (d, 6H; J = 6.7 Hz; (CH3)2-CH-CH2-
N—), 2.03–2.10 (m, 1H; J = 6.9 Hz; (CH3)2-CH-CH2-N—), 2.38 (s, 3H,
CH3-Ph-SO2-), 3.95–3.97 (d, 2H; J = 7.0 Hz; (CH3)2-CH-CH2-N—),
7.38–7.40 (d, 2H; J = 8.1 Hz; H-3, 5 of phenyl ring), 7.72–7.74 (d,
2H; J = 8.4 Hz; H-2, 6 of phenyl ring), 8.59 (s, 2H, –SO2–NH2). ESI-
MS, m/z: [M+H]+ (calcd) 391.02 (391.06), [MꢀH]ꢀ (calcd) 388.94
(389.04).
195–198 °C; 1H NMR (DMSO-d6) d: 2.41 (s, 3H, CH3-Ph-SO2-),
2.99–3.03 (t, 2H; J = 6.8 Hz; Br-Ph-CH2–CH2–N—), 4.38–4.41 (t,
2H; J = 6.8 Hz; Br-Ph-CH2–CH2–N—), 6.99–7.01 (d, 2H; J = 8.4 Hz;
H-3, 5 of Br-Ph-CH2–CH2–N—), 7.27–7.29 (d, 2H; J = 8.4 Hz; H-2, 6
of Br-Ph-CH2–CH2–N—), 7.39–7.41 (d, 2H; J = 8.1 Hz; H-3, 5 of
CH3-Ph-SO2–), 7.61–7.63 (d, 2H; J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2-
), 8.61 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd)
516.90(516.97), [MꢀH]ꢀ (calcd) 514.88(514.95).
4.2.4.9. 2-[(p-Toluenesulfonamido)-N-(n-pentyl)]-l,3,4-thiadia-
zole-5-sulfonamide (I-9).
Yield, 59.4%; mp: 149–152 °C; 1H
4.2.4.16. 2-[(p-Toluenesulfonamido)-N-(3,4,5-trimethoxylben-
NMR (DMSO-d6) d: 0.73–0.77 (t, 3H; J = 7.3 Hz; CH3–CH2–CH2–
CH2–CH2–N—), 1.08–1.22 (m, 4H, CH3–CH2–CH2–CH2–CH2–N—),
1.64–1.70 (m, 2H; J = 7.2 Hz; CH3–CH2–CH2–CH2–CH2–N—), 2.38
(s, 3H, CH3-Ph-SO2-), 4.09–4.13 (t, 2H; J = 6.9 Hz; CH3–CH2–CH2–
CH2–CH2–N—), 7.38–7.40 (d, 2H; J = 7.8 Hz; H-3, 5 of phenyl ring),
7.72–7.74 (d, 2H; J = 8.4 Hz; H-2, 6 of phenyl ring), 8.58(s, 2H, –
SO2-NH2). ESI-MS, m/z: [M+H]+ (calcd) 405.04 (405.07), [MꢀH]ꢀ
(calcd) 402.96 (403.06).
zyl)]-l,3,4-thiadiazole-5-sulfonamide (I-16).
Yield, 85.6%;
mp: 82–85 °C; 1H NMR (DMSO-d6) d: 2.38 (s, 3H, CH3-Ph-SO2-),
3.57 (s, 9H, (CH3O)3-Ph-CH2-N—), 5.26 (s, 2H, (CH3O)3-Ph-CH2-
N—), 6.51 (s, 2H, H-2, 6 of (CH3O)3-Ph-CH2-N—), 7.36–7.38 (d, 2H;
J = 8.1 Hz; H-3, 5 of CH3-Ph-SO2-), 7.72–7.74 (d, 2H; J = 8.4 Hz; H-
2,
6 of CH3-Ph-SO2-), 8.61 (s, 2H, –SO2–NH2). ESI-MS, m/z:
[M+Na]+ (calcd) 536.96 (537.05), [MꢀH]ꢀ (calcd) 512.97 (513.06).
4.2.4.17. 2-[(p-Toluenesulfonamido)-N-(4-methylbenzyl)]-l,3,4-
4.2.4.10. 2-[(p-Toluenesulfonamido)-N-(4-chlorobenzyl)]-l,3,4-
thiadiazole-5-sulfonamide (I-17).
Yield, 77.1%; mp: 192–
thiadiazole-5-sulfonamide (I-10).
Yield, 44.8%; mp: 220–
195 °C; 1H NMR (DMSO-d6) d: 2.26 (s, 3H, CH3-Ph-CH2-N—), 2.39
(s, 3H, CH3-Ph-SO2-), 5.29 (s, 2H, CH3-Ph-CH2-N—), 7.09 (s, 4H,
CH3-Ph-CH2-N—), 7.36–7.38 (d, 2H; J = 8.1 Hz; H-3, 5 of CH3-Ph-
SO2-), 7.68–7.70 (d, 2H; J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2-), 8.59
224 °C; 1H NMR (DMSO-d6) d: 2.39 (s, 3H, CH3-Ph-SO2-), 5.34 (s,
2H, Cl-Ph-CH2-), 7.22–7.24 (d, 2H; J = 8.7 Hz; H-3, 5 of Cl-Ph-CH2-
), 7.34–7.36 (d, 2H; J = 8.7 Hz; H-2, 6 of Cl-Ph-CH2-), 7.35–7.37 (d,

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Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
(s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 438.92 (439.06),
[MꢀH]ꢀ (calcd) 436.95 (437.04).
2H; J = 6.8 Hz; CH3–CH2–CH2–N—), 7.39–7.87 (m, 5H, Ph-SO2-),
8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 363.00
(363.03), [MꢀH]ꢀ (calcd) 360.92 (361.01).
4.2.4.18. 2-[(p-Toluenesulfonamido)-N-(4-fluorobenzyl)]-l,3,4-
4.2.5.4.
2-(Benzenesulfonamido-N-phenethyl)-l,3,4-thiadia-
Yield, 78.1%; mp: 152–154 °C; 1H
thiadiazole-5-sulfonamide (I-18).
Yield, 58.8%; mp: 199–
zole-5-sulfonamide (I0-4).
201 °C; 1H NMR (DMSO-d6) d: 2.38 (s, 3H, CH3-Ph-SO2-), 5.33 (s,
2H, F-Ph-CH2-N—), 7.11–7.16 (t, 2H; J = 8.8 Hz; H-3, 5 of F-Ph-
CH2-N—), 7.26–7.29 (q, 2H, H-2, 6 of F-Ph-CH2-N—), 7.36–7.38 (d,
NMR (DMSO-d6) d: 3.00–3.04 (t, 2H; J = 7.0 Hz; Ph-CH2–CH2–N—),
4.37–4.41 (t, 2H; J = 7.2 Hz; Ph-CH2–CH2–N—), 7.02–7.15 (m, 5H,
Ph-CH2–CH2–N—), 7.57–7.76 (m, 5H, Ph-SO2–), 8.61 (s, 2H, –SO2–
NH2). ESI-MS, m/z: [M+H]+ (calcd) 425.01 (425.04), [MꢀH]ꢀ (calcd)
422.94 (423.03).
2H; J = 8.1 Hz; H-3,
5 of CH3-Ph-SO2-), 7.68–7.70 (d, 2H;
J = 7.9 Hz; H-2, 6 of CH3-Ph-SO2-), 8.60 (s, 2H, –SO2–NH2). ESI-MS,
m/z: [M+H]+ (calcd) 442.97 (443.03), [MꢀH]ꢀ (calcd) 440.94
(441.02).
4.2.5.5. 2-[Benzenesulfonamido-N-(4-chlorobenzyl)]-l,3,4-thia-
diazole-5-sulfonamide (I0-5).
Yield, 74.6%; mp: 198–199 °C;
1H NMR (DMSO-d6) d: 5.36 (s, 2H, Cl-Ph-CH2–N—), 7.23–7.25 (m,
2H; J = 9.0 Hz; H-3, of Cl-Ph-CH2–N—), 7.34–7.36 (m, 2H;
4.2.4.19. 2-[(p-Toluenesulfonamido)-N-(2-methoxylphenylpro-
pyl)]-l,3,4-thiadiazole-5-sulfonamide (I-19).
Yield, 55.6%;
mp: 111–113 °C; 1H NMR (DMSO-d6) d: 1.91–1.95 (m, 2H;
J = 7.4 Hz; CH3O-Ph-CH2–CH2–CH2–N—), 2.36 (s, 3H, CH3-Ph-SO2-),
3.71 (s, 3H, CH3O-Ph-CH2–CH2–CH2–N—), 4.09–4.13 (t, 2H;
J = 7.0 Hz; CH3O-Ph-CH2–CH2–CH2–N—), 6.78–7.17 (m, 4H, CH3O-
Ph-CH2–CH2–CH2–N—), 7.37–7.39 (d, 2H; J = 8.1 Hz; H-3, 5 of
CH3-Ph-SO2-), 7.72–7.74 (d, 2H; J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2-
), 8.58 (s, 2H, –SO2–NH2). 1H NMR (CDCl3) d: 2.04–2.11 (m, 2H;
J = 7.3 Hz; CH3O-Ph-CH2–CH2–CH2–N—), 2.40 (s, 3H, CH3-Ph-SO2-),
2.57–2.61 (t, 2H; J = 7.4 Hz; CH3O-Ph-CH2–CH2–CH2–N—), 4.15–
4.18 (t, 2H; J = 7.1 Hz; CH3O-Ph-CH2–CH2–CH2–N—), 5.61 (s, 2H, –
SO2–NH2), 6.80–7.20 (m, 4H, CH3O-Ph-CH2–CH2–CH2–N—), 7.26–
7.28 (d, 2H; J = 7.5 Hz; H-3, 5 of CH3-Ph-SO2–), 7.75–7.77 (d, 2H;
J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2–). ESI-MS, m/z: [M+H]+ (calcd)
483.07 (483.08), [MꢀH]ꢀ (calcd) 481.02 (481.07).
5
J = 9.0 Hz; H-2, 6 of Cl-Ph-CH2–N—), 7.55–7.80 (m, 5H, Ph-SO2-),
8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 444.88
(444.99), [MꢀH]ꢀ (calcd) 442.87 (442.97).
4.2.5.6. 2-[Benzenesulfonamido-N-(2,4-dichlorobenzyl)]-l,3,4-
thiadiazole-5-sulfonamide (I0-6).
Yield, 50.6%; mp: 125–
128 °C; 1H NMR (DMSO-d6) d: 5.40 (s, 2H, (Cl)2-Ph-CH2–N—),
7.32–7.56 (m, 3H, (Cl)2-Ph-CH2–N—), 7.58–7.79 (m, 5H, Ph-SO2–),
8.59 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 478.84
(478.95), [MꢀH]ꢀ (calcd) 476.86 (476.93).
4.2.5.7. 2-(Benzenesulfonamido-N-methyl)-l,3,4-thiadiazole-5-
sulfonamide (I0-7).
Yield, 89.5%; mp: 194–196 °C; 1H NMR
(DMSO-d6) d: 3.72 (s, 3H, CH3-N—), 7.58–7.89 (m, 5H, Ph-SO2–),
8.61 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 334.95
(334.99), [MꢀH]ꢀ (calcd) 332.90 (332.98).
4.2.5. General procedure for the synthesis of 2-(benzenesulfo-
namido-N-yl)-l,3,4-thiadiazole-5-sulfonamides (I0-1–I0-15)
0.16 g of 2-benzenesulfonamido-l,3,4-thiadiazole-5-sulfon-
amide (3I0) (0.5 mmol) was dissolved into DMF (1 mL) with stirring
at room temperature. The suspending liquid of 0.03 g of potassium
hydroxide (0.5 mmol) and 1 mL of DMF was added to the above
solutions, potassium hydroxide was dissolved slowly. Then a mix-
ture of halogenated hydrocarbons (0.6 mmol) and 1 mL of DMF
was added to the reaction mixture and heated to 60–80 °C in an
oil-bath for 1–4 h (monitored by TLC). The solvent was evaporated
to dryness in vacuum, the residue was flaxen solid. The compound
I0-1–I0-15 was obtained by chromatographic fractionation.
4.2.5.8. 2-[Benzenesulfonamido-N-(n-pentyl)]-l,3,4-thiadiazole-
5-sulfonamide (I0-8).
Yield, 66.7%; mp: 137–139 °C; 1H NMR
(DMSO-d6) d: 0.72–0.76 (t, 3H, J = 7.2 Hz; CH3–(CH2)2–CH2–CH2–
N—), 1.09–1.23 (m, 4H, J = 7.4 Hz; CH3–(CH2)2–CH2–CH2–N—),
1.63–1.70 (m, 2H, J = 7.2 Hz; CH3–(CH2)2–CH2–CH2–N—), 4.11–
4.14 (t, 2H, J = 6.8 Hz; CH3–(CH2)2–CH2–CH2–N—), 7.58–7.87 (m,
5H, Ph-SO2–), 8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd)
391.01 (391.06), [MꢀH]ꢀ (calcd) 388.94 (389.04).
4.2.5.9. 2-[Benzenesulfonamido-N-(i-butyl)]-l,3,4-thiadiazole-5-
4.2.5.1.
2-(Benzenesulfonamido-N-ethyl)-l,3,4-thiadiazole-5-
Yield, 74.6%; mp: 152–155 °C; 1H NMR
sulfonamide (I0-9).
Yield, 58.2%; mp: 125–130 °C; 1H NMR
sulfonamide (I0-1).
(DMSO-d6) d: 0.78–0.80 (d, 6H, J = 6.7 Hz; (CH3)2–CH–CH2–N—),
2.02–2.09 (m, 1H, J = 6.8 Hz; (CH3)2–CH–CH2–N—), 3.96–3.98 (d,
2H, J = 7.3 Hz; (CH3)2–CH–CH2–N—), 7.58–7.87 (m, 5H, Ph-SO2-),
8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd) 376.99
(377.04), [MꢀH]ꢀ (calcd) 374.92 (375.03).
(DMSO-d6) d: 1.25–1.28 (t, 3H; J = 7.2 Hz; CH3–CH2–N—), 4.12–
4.18 (q, 2H; J = 7.3 Hz; CH3–CH2–N—), 7.58–7.88 (m, 5H, Ph-SO2-),
8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd)
349.01(349.01), [MꢀH]ꢀ (calcd) 346.93 (346.99). Anal. Calcd for
C10H12N4O4S3: C, 34.47; H, 3.47; N, 16.08. Found: C, 35.37; H,
3.44; N, 16.26.
4.2.5.10.
2-[Benzenesulfonamido-N-(n-Lauryl)]-l,3,4-thiadia-
Yield, 41.0%; mp: 92–94 °C; 1H
zole-5-sulfonamide (I0-10).
4.2.5.2. 2-[Benzenesulfonamido-N-(n-butyl)]-l,3,4-thiadiazole-
NMR (DMSO-d6) d: 0.84–0.87 (t, 3H, J = 6.8 Hz; CH3–(CH2)9–CH2–
CH2–N—), 1.13–1.27 (m, 18H, CH3–(CH2)9–CH2–CH2–N—), 1.64–
1.67 (m, 2H, J = 6.6 Hz; CH3–(CH2)9–CH2–CH2–N—), 4.10–4.14 (t,
2H, J = 6.8 Hz; CH3–(CH2)9–CH2–CH2–N—), 7.57–7.86 (m, 5H, Ph-
SO2–), 8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: Target [M+H]+ (calcd)
489.12 (489.17), [MꢀH]ꢀ (calcd) 487.00 (487.15).
5-sulfonamide (I0-2).
Yield, 83.9%; mp: 178–181 °C; 1H
NMR (DMSO-d6) d: 0.76–0.80 (t, 3H; J = 7.3 Hz; CH3–CH2–CH2–
CH2–N—), 1.16–1.17 (m, 2H; J = 7.3 Hz; CH3–CH2–CH2–CH2–N—),
1.63–1.67 (m, 2H; J = 7.2 Hz; CH3–CH2–CH2–CH2–N—), 4.11–4.15
(t, 2H; J = 6.9 Hz; CH3–CH2–CH2–CH2–N—), 7.58–7.87 (m, 5H, Ph-
SO2–), 8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd)
377.03 (377.04), [MꢀH]ꢀ (calcd) 374.95 (375.03).
4.2.5.11. 2-(Benzenesulfonamido-N-benzyl)-l,3,4-thiadiazole-5-
sulfonamide (I0-11).
Yield, 50.2%; mp: 147–150 °C; 1H NMR
4.2.5.3. 2-[Benzenesulfonamido-N-(n-propyl)]-l,3,4-thiadiazole-
(DMSO-d6) d: 5.36 (s, 2H, Ph-CH2–N—), 7.19–7.32 (m, 5H, Ph-CH2–
N—), 7.56–7.82 (m, 5H, Ph-SO2–), 8.61 (s, 2H, –SO2–NH2). ESI-MS,
m/z: [M+H]+ (calcd) 411.01 (411.03), [MꢀH]ꢀ (calcd) 408.92
(409.01).
5-sulfonamide (I0-3).
Yield, 68.8%; mp: 150–152 °C; 1H NMR
(DMSO-d6) d: 0.78–0.80 (t, 3H; J = 3.6 Hz; CH3–CH2–CH2–N—),
1.67–1.73 (m, 2H; J = 7.2 Hz; CH3–CH2–CH2–N—), 4.08–4.12 (t,

Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
3227
4.2.5.12. 2-[Benzenesulfonamido-N-(4-methoxylbenzyl)]-l,3,4-
4.3. Carbonic anhydrase inhibition assay
4.3.1. Isozyme preparation
Human CA II cDNAs were expressed in Escherichia coli strain
BL21 (DE3) from the plasmids pET-28b/hCA II. A practical method
for screening hCA II inhibitors was successfully constituted by re-
combinant hCA II protein expressed in E. coli as the source of
hCA II enzyme.17,18
thiadiazole-5-sulfonamide (I0-12).
Yield, 63.6%; mp: 188–
191 °C; 1H NMR (DMSO-d6) d: 3.72 (s, 3H, CH3O-Ph-CH2–N—),
5.27 (s, 2H, CH3O-Ph-CH2–N—), 6.82–6.84 (d, 2H, J = 8.7 Hz; H-3, 5
of CH3O-Ph-CH2–N—), 7.15–7.18 (d, 2H, J = 8.7 Hz; H-2,
6 of
CH3O-Ph-CH2–N—), 7.57–7.83 (m, 5H, Ph-SO2–), 8.60 (s, 2H, –SO2–
NH2). ESI-MS, m/z: [M+H]+ (calcd) 440.73 (441.04), [MꢀH]ꢀ (calcd)
438.93 (439.02).
4.2.5.13.
2-[Benzenesulfonamido-N-(i-propyl)]-l,3,4-thiadia-
zole-5-sulfonamide (I0-13).
Yield, 67.3%; mp: 163–166 °C;
4.3.2. Evaluation of carbonic anhydrase inhibitory activity
1H NMR (DMSO-d6) d: 1.32–1.34 (d, 6H, J = 6.4 Hz; (CH3)2–CH–
N—), 4.77–4.84 (m, 1H, J = 6.6 Hz; (CH3)2–CH–N—), 7.59–7.88 (m,
5H, Ph-SO2–), 8.60 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+ (calcd)
362.98 (363.03), [MꢀH]ꢀ (calcd) 360.94 (361.01).
The dosage of AZA was to be a standard. When the inhibition
rate of AZA was 50%, the density of AZA was 0.3 l
mol Lꢀ1, so we
use the same density for all compounds to compare their inhibi-
tion effects on CA II. Derivatives I-1–I-19, I0-1–I0-15, II-1 and II0-1
were investigated for their inhibitory effects on hCA II. The hCA
II activity was detected under pH 7.6 and 25 °C by its esterase
activity which can decompose p-nitrophenyl acetate (PNPA) to
increase the photoabsorption at 348 nm by UV-240 ultraviolet
4.2.5.14. 2-[Benzenesulfonamido-N-(4-bromophenethyl)]-l,3,4-
thiadiazole-5-sulfonamide (I0-14).
Yield, 71.7%; mp: 193–
195 °C; 1H NMR (DMSO-d6) d: 2.99–3.03 (t, 2H, J = 6.8 Hz; Br-Ph-
CH2–CH2–N—), 4.38–4.42 (t, 2H, J = 6.8 Hz; Br-Ph-CH2–CH2–N—),
6.99–7.01 (d, 2H, J = 8.1 Hz; H-3, 5 of Br-Ph-CH2–CH2–N—), 7.29–
7.31 (m, 2H, J = 8.4 Hz; H-2, 6 of Br-Ph-CH2–CH2–N—), 7.58–7.76
(m, 5H, Ph-SO2–), 8.63 (s, 2H, –SO2–NH2). ESI-MS, m/z: [M+H]+
(calcd) 502.90 (502.95), [MꢀH]ꢀ (calcd) 500.85 (500.94).
2ꢀ
spectrophotometry (SHIMADZU, Japan).19,20 2.2 mL Tris–SO4
buffer solution, 0.1 mL compound solution, 0.6 mL substrate
solution and 0.1 mL enzyme solution were added to reaction sys-
tem one by one, the bulk volume of reaction system was 3.0 mL.
At first, the absorbance value (A) was corrected to ‘zero’ by dis-
tilled water, then the absorbance value was recorded every
0.5 min within 3.0 min. The curve was made by absorbance va-
lue and time, the increase value of absorbance value in 1 min
4.2.5.15. 2-[Benzenesulfonamido-N-(3,4,5-trimethoxylbenzyl)]-
l,3,4-thiadiazole-5-sulfonamide (I0-15).
Yield, 69.1%; mp:
84–88 °C; 1H NMR (DMSO-d6) d: 3.58 (s, 9H, (CH3O)3-Ph-CH2–
N—), 5.27 (s, 2H, (CH3O)3-Ph-CH2-N—), 6.53 (s, 2H, H-2, 6 of
(CH3O)3-Ph-CH2-N—), 7.55–7.86 (m, 5H, Ph-SO2-), 8.61 (s, 2H, –
SO2-NH2). ESI-MS, m/z: [M+Na]+ (calcd) 522.97 (523.04), [MꢀH]ꢀ
(calcd) 498.94 (499.04).
(DA/min) was computed by linear part of the curve. The molar
extinction coefficient of paranitrophenol was 5.0 ꢃ 106 under
pH 7.6 and 25 °C. Three formulas were as followed:
(1)
D
A348nm/min =
D
Asample/min ꢀ
D
Acompare germ/min.
(2) Specific activity (U/mg) = (
D
A348nm/min)/(5.0 ꢃ mg enzyme/
mL solution).
4.2.6. Synthesis of 2-[(p-toluenesulfonamido)-N-methyl]-5-
[N,N-dimethylsulfonamide]-l,3,4-thiadiazole (II-1)
(3) Inhibition rate (%) = [1 ꢀ (sample + compound) specific
activity/(sample) specific activity] ꢃ 100.
2-(p-Toluenesulfonamido)-l,3,4-thiadiazole-5-sulfonamide (3I)
(0.16 g, 0.5 mmol) was dissolved in 1 mL DMF with stirring at room
temperature. Potassium hydroxide (0.10 g, 1.5 mmol) was sus-
pended in DMF (1 mL) then added to the above solution, potassium
hydroxide was dissolved slowly. Then a mixture of methyl iodide
(1.8 mmol) and DMF (1 mL) was added to the reaction mixture
for 1 h at room temperature (monitored by TLC). The solvent was
evaporated to dryness in vacuum, the residue was flaxen solid.
The compound II-1 was obtained by chromatographic fraction-
ation. Yield, 74.5%; mp: 172–175 °C; 1H NMR (DMSO-d6) d: 2.38
(s, 3H, CH3-Ph-SO2-), 2.90 (s, 6H, (CH3)2-N-), 3.72 (s, 3H, CH3-N—),
7.39–7.41 (d, 2H; J = 8.1 Hz; H-3, 5 of CH3-Ph-SO2-), 7.75–7.77 (d,
2H; J = 8.1 Hz; H-2, 6 of CH3-Ph-SO2-). ESI-MS, m/z: [M+H]+ (calcd)
377.03 (377.04).
4.3.3. Evaluation of some compounds’ antihypoxic effects in
mice
AZA was a positive control drug, the antihypoxic effects of
some compounds in mice were evaluated.21 Kunming mice were
randomly divided into blank control group, positive control
group and target compound group. In dose of 80 mg/kg, target
compounds, AZA and physiological saline were administered to
the target compound group, positive control group and blank
control group for five days, respectively. The mice were fasted
for 12 h before experiment, and were dealed with intragastric
administration after 1 h experimentized. After putting the mice
into sealed bottles (250 mL) with 10 g of Soda Lime, the survival
time of the mice was recorded.
4.2.7. Synthesis of 2-(benzenesulfonamido-N-benzyl)-5-[N,N-
dibenzylsulfonamido]-l,3,4-thiadiazole (II0-1)
2-Benzenesulfonamido-l,3,4-thiadiazole-5-sulfonamide
(3I0)
4.4. Docking studies
(0.16 g, 0.5 mmol) was dissolved into DMF (1 mL) with stirring at
room temperature. Potassium hydroxide (0.10 g, 1.5 mmol) was
suspended in DMF (1 mL) then added to the above solutions, potas-
sium hydroxide was dissolved slowly. Then a mixture of benzyl
bromide (0.31 g, 0.0018 mol) and DMF (1 mL) was added to the
reaction mixture and heated to 80 °C in an oil-bath for 4 h (moni-
tored by TLC). The solvent was evaporated to dryness in vacuum,
the residue was flaxen solid. The compound II-1 was obtained by
chromatographic fractionation. Yield, 77.4%; mp: 130–132 °C; 1H
NMR (DMSO-d6) d: 4.49 (s, 4H, (Ph-CH2)2-N-), 5.31 (s, 2H, Ph-
CH2-N—), 7.17–7.25 (m, 12H; J = 7.5 Hz; 3Ph-CH2-), 7.32–7.35
(m, 3H; J = 8.7 Hz; 3Ph-CH2-), 7.59–7.80 (m, 5H, Ph-SO2-). ESI-MS,
m/z: [M+H]+ (calcd) 591.04 (591.12).
The molecular modeling calculations and docking studies were
performed using Dock6. The program operated under ‘Linux’ oper-
ating system installed on the Xeon with a 2.6 MHz processor and
1G RAM.
The X-ray crystallographic structure of hCA II complexed with
brinzolamide (1A42) was obtained from the Protein Data Bank.
The enzyme was prepared for docking studies where: (i) The ligand
molecule was removed from the enzyme active site. (ii) Hydrogen
atoms were added to the structure with their standard geometry.
(iii) Virtual screen was used to discover the inhibitor of hCA II.
The ligand were minimize by MMFF94 force field.22

3228
Z. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 3221–3228
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