Synthesis and biochemical evaluation of triazole/tetrazole-containing sulfonamides against thrombin and related serine proteases

Article abstract of DOI:10.1016/j.bmcl.2011.07.023

A small library of 25 triazole/tetrazole-based sulfonamides have been synthesized and further evaluated for their inhibitory activity against thrombin, trypsin, tryptase and chymase. In general, the triazole-based sulfonamides inhibited thrombin more efficiently than the tetrazole counterparts. Particularly, compound 26 showed strong thrombin inhibition (K_i = 880 nM) and significant selectivity against other human related serine proteases like trypsin (K_i = 729 μM). Thrombin binding affinity of the same compound was determined by ITC and demonstrated that the binding of this new triazole-based scaffold is enthalpically driven, making it a good candidate for further development.

Full text of DOI:10.1016/j.bmcl.2011.07.023

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5305–5309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biochemical evaluation of triazole/tetrazole-containing
sulfonamides against thrombin and related serine proteases
Rogelio Siles a, Yuko Kawasaki a, Patrick Ross a, Ernesto Freire a,b,
⇑
a Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
b Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of 25 triazole/tetrazole-based sulfonamides have been synthesized and further evaluated
for their inhibitory activity against thrombin, trypsin, tryptase and chymase. In general, the triazole-
based sulfonamides inhibited thrombin more efficiently than the tetrazole counterparts. Particularly,
compound 26 showed strong thrombin inhibition (Ki = 880 nM) and significant selectivity against other
Received 29 May 2011
Revised 30 June 2011
Accepted 6 July 2011
Available online 14 July 2011
human related serine proteases like trypsin (Ki = 729 lM). Thrombin binding affinity of the same com-
pound was determined by ITC and demonstrated that the binding of this new triazole-based scaffold is
enthalpically driven, making it a good candidate for further development.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Thrombosis or thrombin inhibition
Sulfonamide synthesis
Isothermal titration calorimetry
Trypsin selectivity
Triazole and tetrazole scaffolds
Even though much work has been done to prevent and treat
vascular diseases, they are still the leading cause of death world-
wide.1 The national vital statistical reports published recently2
showed that in 2009, cardiovascular diseases were the major rea-
son of death in the US (almost 32% of the total number of deaths
reported in that year) and cerebrovascular ailments along with
strokes occupied the fourth place in the list of mortality. For those
reasons, the development of new, safe and effective antithrombotic
drugs continues to be a major priority in the pharmaceutical
industry.
Traditional therapy for the treatment of thrombosis (cerebral,
coronary and deep venous) and thromboembolic disorders (pul-
monary embolism) has included the use of parenteral anticoagu-
lants like unfractionated heparins (UFH), low-molecular-weight
heparins (LMWH), factor Xa (FXa) inhibitors (fondaparinux), vita-
min K antagonists (warfarin, acenocoumarol, phenprocoumon)
and thrombin inhibitors (hirudin, desirudin, argatroban and biva-
lirudin).3–7 Small molecular weight thrombin inhibitors with high
selectivity and good bioavailability have been difficult to obtain.7,8
Thrombin, a trypsin-like serine protease, is an established target
for the design of antithrombotic drugs since it plays an important
role in the formation of blood clots by converting fibrinogento fibrin,
activating other coagulation factors (V, VIII, XI, and XIII) present in
the coagulation cascade and stimulating platelet aggregation.7–13
Structurally, thrombin has three domains9 (the active site and
exosites 1 and 2). Thrombin’s active site is primarily constituted
by a catalytic triad (Ser195, His57, and Asp102), a basic-recognition
S1 pocket (Asp189 is the most important residue), a hydrophobic
proximal S2 pocket (Tyr60A and Trp60D are the main residues)
and a bigger lipohilic distal S3 pocket (Leu 99, Ile174, and
Trp215).7,9–12 Additionally, subsites S10, S20 and S30 and the oxyan-
ion hole have also been considered in the design of stronger and
more selective inhibitors.10,14
The development of highly potent and selective direct thrombin
inhibitors (DTIs) is an active area of research.15,16 We have identi-
fied a series of heterocyclic sulfonamides that inhibits the activity
of thrombin with high potency and good selectivity to related ser-
ine proteases. These inhibitors do not contain the traditional highly
basic functionalities (guanidine, amidine, amino, imidazole,
hydroxyguanidine)8,17,18 used to interact with the Asp189 present
in the specific S1 pocket of serine proteases. Herein, we report the
chemical synthesis of 25 triazole/tetrazole-based sulfonamides
and their biochemical evaluation against thrombin, trypsin, tryp-
tase and chymase.
The synthesis of the triazole-containing inhibitors which is de-
picted in Scheme 2 involved seven steps while the preparation of
the tetrazole-based analogs was achieved in just five steps. The ini-
tial step in the synthesis of the triazole sulfonamides comprised
the formation of aromatic azides that were prepared by any of
the three methods shown in Scheme 1.
Azides 1–3 were prepared by the reaction of their bromide
⇑
Corresponding author. Tel.: +1 410 516 7743; fax: +1 410 516 6469.
E-mail address: ef@jhu.edu (E. Freire).
precursors with sodium azide in ethanol.19 On the other hand,
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.023

5306
R. Siles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5305–5309
reaction conditions, once the azide was formed it was gradually
transformed into the respective amine as reported previously,20
accounting for the moderate yields obtained as shown in Scheme
1. Finally, an alternative procedure used to convert an alcohol to
the corresponding azide21 consisted in using tosylimidazole (TsIm),
with catalytic amount of TBAI for the preparation of 3-(azido-
methyl)pyridine 8. Alkylation of 3-aminophenol was performed
first by protecting the amino group with (Boc)2O and then alkylat-
ing the resulting Boc-phenol with propargyl bromide in the pres-
ence of potassium carbonate,22,23 finally obtaining the Boc-
protected alkyne 9 with 79% yield. Formation of triazole-contaning
anilines 10–17 was successfully achieved by using in sequence first
a Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition24 between al-
kyne 9 and azides 1–8 and then deprotecting the resulting Boc-tri-
azoles with TFA.22
Synthesis of tetrazole 25 involved a 3-step route in which, 3-
nitrobenzonitrile initially reacted with sodium azide and ZnBr2 to
form its corresponding nitrotetrazole25 which was subsequently
alkylated26 by 3-chlorobenzylbromide and afterwards reduced
with Zn/AcOH27 to form the desired 3-(2-(3-chlorobenzyl)-2H-tet-
razol-5-yl)aniline. Preparation of sulfonyl chlorides 18–24 was
performed by heating the corresponding 3-substituted styrenes
with sulfuryl chloride in DMF solution.28 The final step in the syn-
thesis of triazoles 26–42 and tetrazoles 43–50 consisted of the
reaction of anilines 10–17 and 25 with sulfonyl chlorides 18–24
using water as the solvent and Na2CO3 as the base.29 We observed
that when THF was added to this reaction as a co-solvent in a 1:1
Scheme 1. Synthesis of azides. Reagents and conditions: (a) NaN3, EtOH, rt, 24 h;
(b) NaN3, PPh3 (1 equiv), CCl4–DMF, 80–90 °C, 4 h; (c) NaN3, TsIm, TBAI, TEA, DMF,
100 °C, 21 h.
the synthesis of azides 4–7 was accomplished by the reaction of
equimolar amounts of triphenylphosphine and their alcohol pre-
cursors with sodium azide using carbon tetrachloride and dimeth-
ylformamide as solvents. We could observe that under these
Scheme 2. Synthesis of triazole and tetrazole-based sulfonamides. Reagents and conditions: (a) (Boc)2O, THF, 61 °C, 24 h, 83% yield; (b) K2CO3, NaI, DMF, propargyl bromide,
68 °C, 45 h, 98% yield; (c) Na(ascb), CuSO4ꢀ5H2O, H2O–EtOH (1:1), rt, 24 h, 65–99% yield; (d) TFA, DCM, rt ꢁ2 h, >93% yield; (e) SO2Cl2, DMF, 60–80 °C, 3–7 h; (f) NaN3, ZnBr2,
H2O, reflux, 31 h, 94% yield; (g) TEA, 3-chlorobenzylbromide, AN, rt, 23 h, 34% yield; (h) Zn, AcOH, rt, 2.5 h, 97% yield; (i) 1 M Na2CO3, THF–H2O, rt, 1.5–5 h, % yields are shown
in Tables 1 and 2.

R. Siles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5305–5309
5307
Table 1
Inhibition of thrombin and related serine proteases by triazole-based sulfonamides
Compound
Yieldd (%)
R2
R1
Ki (l
M)
Ratiob
vi/v0 (10 l
M)a
Thrombin
Trypsin
729
Thrombin
0.06
Tryptase
Chymase
26
27
28
29
30
70
84
72
49
58
Cl
0.88
1.71
1.97
1.75
2.27
828
71
0.95
0.98
0.89
0.99
0.88
0.95
NO2
CF3
NH2
F
122
0.06
1.0
127
64
0.09
0.97
71
41
0.11
0.99
119
48
0.12
0.95
31
56
NO2
6.89
nd
—
0.18
0.98
1.00
32
33
52
63
NO2
Cl
4.60
5.43
187
nd
41
—
0.19
0.19
1.0
0.95
1.00
0.98
34
35
56
76
Cl
Cl
ndc
nd
nd
nd
—
—
0.24
0.27
0.99
0.98
1.00
0.96
36
37
53
78
Cl
Cl
8.91
nd
nd
nd
—
—
0.37
0.41
0.97
0.95
1.00
0.99
38
32
Cl
13.7
nd
—
0.50
0.95
0.97
39
40
41
42
41
47
60
64
CF3
NO2
F
nd
nd
nd
nd
nd
—
—
—
—
0.58
0.70
0.71
0.76
0.96
1.0
1.00
1.00
0.99
0.98
62.5
nd
1.0
Cl
39.2
0.99
a
b
c
Residual enzyme activity determined in the presence of 10
Ratio of Ki trypsin/Ki thrombin.
Not determined.
lM inhibitor.
d
% Yield of the last step shown in Scheme 2.
ratio, the yields were considerably increased. The percentage yields
of these reactions are shown in Tables 1 and 2 for triazoles and
tetrazoles, respectively.
removed to be transformed into analogs 35 (vi/v0 = 0.27) and 37
(vi/v0 = 0.41), the thrombin inhibitory potency drops approxi-
mately five and seven times respectively. Similarly, when the thio-
The inhibitory potency and selectivity of the compounds were
first evaluated by measuring the enzymatic activity (vi/v0) of
thrombin, tryptase and chymase in the absence (v0) and in the
phene ring of substituent R1 of compound 26 (Ki = 0.88
lM) is
removed to become analog 36 (Ki = 8.91 M), the thrombin inhib-
l
itory potency activity drops by a factor of ten. These data suggests
that both rings, the benzene and thiophene, are important struc-
tural features for thrombin inhibition. We also noticed that the
presence of a nitrogen atom in substituent R1 reduces the inhibi-
tory capacity against thrombin. For example, compounds 39–42,
which all contain a pyridine ring in R1, exhibited residual activities
presence (vi) of 10 lM inhibitor. The residual enzymatic activity
is expressed as the ratio (vi/v0). The inhibition constants (Ki) for
thrombin and trypsin were determined for some selected com-
pounds (compounds 26–33, 36, 38, 40, and 42) and are reported
in Tables 1 and 2. According to the enzyme inhibition studies in
the triazole-based series (Table 1), compounds containing a ben-
zene-fused heterocyclic systems (see substituent R1 of compounds
26–34) are the most active against thrombin. We noticed that if the
benzene ring of substituent R1 of compound 26 (vi/v0 = 0.06) is
higher than 0.58. In particular, when compound 36 (Ki = 8.91
lM)
is transformed into its pyridine analog 42 (Ki = 39.2 M), the inhib-
l
itory activity drops almost four times. The adverse presence of
nitrogen atom in substituent R1 can also be illustrated by

5308
R. Siles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5305–5309
Table 2
Inhibition of thrombin and related serine proteases by tretrazole-based sulfonamides
a
Compound Yieldc
(%)
R4
vi/v0 (10
lM)
Thrombin Tryptase Chymase
43
44
29
56
0.23b
0.34
1.0
0.99
0.91
0.97
45
46
34
66
0.54
0.55
1.0
0.94
0.95
CH3
0.97
Figure 1. An overlay of compounds 26 (green), 38 (blue) and 50 (red). This image
was generated using MOE.30
47
48
49
50
44
81
63
61
0.68
0.81
0.93
0.95
1.0
1.0
1.0
1.0
0.97
0.95
1.00
26, 38, and 50 (Fig. 1) was performed.30 By comparing the
structures of compounds 38 (blue) and 50 (red), we noticed that
the main difference is found in the dissimilar orientation that the
3-chlorobenzyl moiety has in both structures. We think that the
orientation observed in compound 38 allows this group to ap-
proach closer and more effectively to the S3 pocket of thrombin
giving account for the higher inhibitory activity. Comparison of
the conformations of compounds 26 (green) and 38 (blue) provides
additional proof that the inhibitory activity increases with the
incorporation of more hydrophobic groups into the S3 pocket.
For instance, the presence of an extra thiophene group in com-
0.97
a
b
c
Residual enzyme activity determined in the presence of 10
Ki value for this compound is 5.20 M.
% Yield of the last step shown in Scheme 2.
l
M inhibitor.
l
pound 26 (Ki = 0.88
over that of compound 38 (Ki = 13.7
l
M) increases the potency almost 15 times
M). In addition, the triazoles
l
comparing compound 26 with compounds 33 and 34. In the former
case, the replacement of the benzothiophene ring of compound 26
(Ki = 0.88 lM) by a benzothiazole in compound 33 (Ki = 5.43 lM),
show better selectivity than the tetrazoles.
In order to evaluate the thermodynamic profile of the top com-
pound (compound 26), its binding to thrombin was measured by
isothermal titration calorimetry (ITC) (Fig. 2). As shown in the fig-
ure, the binding of compound 26 to thrombin is enthalpically dri-
ven suggesting that the compound establishes strong interactions
with thrombin.31,32 The binding is characterized by a Gibbs energy
reduces the inhibitory activity almost six times while in the second
case, substitution of the same ring by an indole moiety present in
compound 34, diminishes the inhibition four times. Similar results
are found by comparing compound 27 with compounds 31 and 32
The syntheses of compounds 26–30 were carried out in order to
identify the nature of substituent R2 needed to improve thrombin
inhibition. The thrombin Ki values of compounds 26–30 reported
in Table 1 indicate that R2 decreases the enzyme activity in the fol-
lowing order: Cl > NO2 > NH2 > CF3 > F. As we can see from these
values, compound 26 is the best compound in this series not only
because it exhibits the best Ki value against thrombin
(
D
G) of ꢂ8.3 kcal/mol, a binding enthalpy (
D
H) of ꢂ9.1 kcal/mol
and an entropic contribution (ꢂT
D
S) of 0.8 kcal/mol. The thermo-
dynamic profile of compound 26 provides a good optimization
(Ki = 0.88
against trypsin (Ki = 729
l
M) but also because it shows remarkable selectivity
M, Ki ratio = 828) and the other human
l
related proteases like tryptase (vi/v0 = 0.95) and chymase (vi/
v0 = 0.98) which sets it apart from its analogs 27–30. In fact, it is
worth mentioning that many of the triazole compounds reported
in this manuscript showed excellent selectivity against tryptase
and chymase.
In an effort to evaluate the importance of the triazole ring for
the inhibitory potency against thrombin, we initially synthesized
eight tetrazole-based sulfonamides 43–50. Based on these preli-
minary results, we noticed that the triazole-based scaffold inhibits
thrombin much better than the tetrazole-based series. This obser-
vation is supported by comparing the inhibitory activity of com-
pound 38 (vi/v0 = 0.50) with compound 50 (vi/v0 = 0.95). As
noticed, replacement of the triazole ring in 38 by the tetrazole moi-
ety in 50 increases the thrombin inhibitory activity almost 2-fold.
To further corroborate the superiority of triazoles over tetrazoles,
an overlay of the energy-minimized conformations of compounds
Figure 2. The thermodynamic signature for the binding of compound 26 to
thrombin.

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11. Obst, U.; Betschmann, P.; Lerner, C.; Seiler, P.; Diederich, F.; Gramlich, V.;
Weber, L.; Banner, D. W.; Schönholzer, P. Helv. Chim. Acta 2000, 83, 855.
12. Di Cera, E.; Dang, Q. D.; Ayala, Y. M. Cell. Mol. Life Sci. 1997, 53, 701.
13. Squizzato, A.; Dentali, F.; Steidl, L.; Ageno, W. Intern. Emerg. Med. 2009, 4, 479.
14. Betschmann, P.; Sahli, S.; Diederich, F.; Obst, U.; Gramlich, V. Helv. Chim. Acta
2002, 85, 1210.
15. Lee, C. J.; Ansell, J. E. Direct Thrombin Inhibitors. Br. J. Clin. Pharmacol. [Online
early access]. doi:10.1111/1365-2125.2011.03916.x 2011. Published online:
January 17, 2011. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2125.
2011.03916.x/abstract (accessed May 10, 2011).
scenario since it already exhibits strong favorable enthalpy. It is
well known that the binding entropy is easier to optimize33
In summary, we discovered and synthesized a small library of
25 triazole/tetrazole-based sulfonamides which were evaluated
for their inhibitory activity against thrombin, trypsin, tryptase
and chymase. In particular, five compounds (26–30) showed out-
standing Ki values for thrombin inhibition and most importantly,
they had significant selectivity against other human related serine
proteases which make them good candidates for further
development.
16. Turpie, A. G. Curr. Opin. Drug Disc. Dev. 2009, 12, 497.
17. Young, M. B.; Barrow, J. C.; Glass, K. L.; Lundell, G. F.; Newton, C. L.; Pellicore, J.
M.; Rittle, K. E.; Selnick, H. G.; Stauffer, K. J.; Vacca, J. P.; Williams, P. D.; Bohn,
D.; Clayton, F. C.; Cook, J. J.; Krueger, J. A.; Kuo, L. C.; Lewis, S. D.; Lucas, B. J.;
McMasters, D. R.; Miller-Stein, C.; Pietrak, B. L.; Wallace, A. A.; White, R. B.;
Wong, B.; Yan, Y.; Nantermet, P. G. J. Med. Chem. 2004, 47, 2995.
18. Tucker, T. J.; Brady, S. F.; Lumma, W. C.; Lewis, S. D.; Gardell, S. J.; Naylor-Olsen,
A. M.; Yan, Y.; Sisko, J. T.; Stauffer, K. J.; Lucas, B. J.; Lynch, J. J.; Cook, J. J.;
Stranieri, M. T.; Holahan, M. A.; Lyle, E. A.; Baskin, E. P.; Chen, I. W.; Dancheck,
K. B.; Krueger, J. A.; Cooper, C. M.; Vacca, J. P. J. Med. Chem. 1998, 41, 3210.
19. Sha, C. K.; Tsou, C. P. J. Org. Chem. 1990, 55, 2446.
Acknowledgments
This work was supported by grants from the National Institutes
of Health (GM56550 and GM57144) and the National Science
Foundation (MCB0641252).
20. Reddy, G. V. S.; Rao, G. V.; Subramanyam, R. V. K.; Iyengar, D. S. Synth. Commun.
2000, 30, 2233.
21. Soltani Rad, M. N.; Behrouz, S.; Khalafi-Nezhad, A. Tetrahedron Lett. 2007, 48,
3445.
Supplementary data
22. Pastine, S. J.; Youn, S. W.; Sames, D. Org. Lett. 2003, 5, 1055.
23. Brown, F. J.; Bernstein, P. R.; Cronk, L. A.; Dosset, D. L.; Hebbel, K. C.; Maduskuie,
T. P.; Shapiro, H. S.; Vacek, E. P.; Yee, Y. K. J. Med. Chem. 1989, 32, 807.
24. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
Supplementary data (detailed experimental syntheses, and bio-
chemical assay protocols) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.07.023.
25. Demko, Z. P.; Sharpless, K. B. J. Org. Chem. 2001, 66, 7945.
26. Nelson, D. W.; Gregg, R. J.; Kort, M. E.; Perez-Medrano, A.; Voight, E. A.; Wang,
Y.; Grayson, G.; Namovic, M. T.; Donnelly-Roberts, D. L.; Niforatos, W.; Honore,
P.; Jarvis, M. F.; Faltynek, C. R.; Carroll, W. A. J. Med. Chem. 2006, 49, 3659.
27. Borrel, C.; Thoret, S.; Cachet, X.; Guénard, D.; Tillequin, F.; Koch, M.; Michel, S.
Bioorg. Med. Chem. 2005, 13, 3853.
28. Mallireddigari, M. R.; Pallela, V. R.; Reddy, E. P.; Reddy, M. V. R. Synthesis 2005,
2005, 3639.
29. Deng, X.; Mani, N. S. Green Chem. 2006, 8, 835.
30. Molecular Operating Environment (MOE 2009.10). Chemical Computing Group
Inc: Montreal, 2009.
31. Freire, E. Drug Discovery Today 2008, 13, 869.
32. Freire, E.; Kawasaki, Y.; Velazquez-compoy, A.; Schon, A. In Biophysical
Approaches Determining Ligand Binding to Biomoleculecular Targets: Detection
Measurement and Modelling; Podjarny, A., Dejaegere, A., Kieffer, B., Eds.; Royal
Society of Chemistry, 2011; Vol. 22, pp 275–299.
References and notes
1. World Health Organization, The top 10 causes of death, 2011. http://
www.who.int/mediacentre/factsheets/fs310/en/index2.html. (accesed April
27, 2011).
2. Kochanek, K. D.; Xu, J.; Murphy, S. L.; Miniño, A. M. Natl. Vital Stat. Rep. 2011, 59,
1.
3. Mannucci, P. M.; Franchini, M. Ann. Med. 2011, 43, 116.
4. Douketis, J. D. Curr. Pharm. Des. 2010, 16, 3436.
5. Garcia, D. Thromb. Res. 2009, 123, S50.
6. Gomez-Outes, A.; Lecumberri, R.; Pozo, C.; Rocha, E. Curr. Vasc. Pharmacol. 2009,
7, 309.
7. Steinmetzer, T.; Stürzebecher, J. Curr. Med. Chem. 2004, 11, 2297.
8. Pfau, R. Curr. Opin. Drug Disc. Dev. 2003, 6, 437.
9. Srivastava, S.; Goswami, L. N.; Dikshit, D. K. Med. Res. Rev. 2005, 25, 66.
10. Schärer, K.; Morgenthaler, M.; Seiler, P.; Diederich, F.; Banner, D. W.; Tschopp,
T.; Obst-Sander, U. Helv. Chim. Acta 2004, 87, 2517.
33. Freire, E. Drug Discovery Today 2008, 13, 869.