(Chlorosulfonyl)benzenesulfonyl Fluorides - Versatile Building Blocks for Combinatorial Chemistry: Design, Synthesis and Evaluation of a Covalent Inhibitor Library

Article abstract of DOI:10.1021/acscombsci.8b00130

Multigram synthesis of (chlorosulfonyl)benzenesulfonyl fluorides is described. Selective modification of these building blocks at the sulfonyl chloride function under parallel synthesis conditions is achieved. It is shown that the reaction scope includes the use of (hetero)aromatic and electron-poor aliphatic amines (e.g., amino nitriles). Utility of the method is demonstrated by preparation of the sulfonyl fluoride library for potential use as covalent fragments, which is demonstrated by a combination of in silico and in vitro screening against trypsin as a model enzyme. As a result, several inhibitors were identified with activity on par with that of the known inhibitor.

Full text of DOI:10.1021/acscombsci.8b00130

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Article
(Chlorosulfonyl)benzenesulfonyl fluorides – versatile
building blocks for combinatorial chemistry. Design,
synthesis and evaluation of a covalent inhibitor library
Kateryna Tolmachova, Yurii S. Moroz, Angelika Konovets, Maxim Platonov, Oleksandr
Vasylchenko, Petro Borysko, Sergey Zozulya, Anastasia Gryniukova, Andrey V. Bogolubsky,
Sergey Pipko, Pavel K. Mykhailiuk, Volodymyr Brovarets, and Oleksandr O. Grygorenko
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00130 • Publication Date (Web): 24 Oct 2018
Downloaded from http://pubs.acs.org on October 27, 2018
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(Chlorosulfonyl)benzenesulfonyl fluorides –
versatile building blocks for combinatorial
chemistry. Design, synthesis and evaluation of
a covalent inhibitor library
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Kateryna A. Tolmachova,a,b Yurii S. Moroz,a,e Angelika Konovets,a,c Maxim O. Platonov,c
Oleksandr V. Vasylchenko,c Petro Borysko,d Sergey Zozulya,d Anastasia Gryniukova,d
Andrey V. Bogolubsky,c Sergey Pipko,c Pavel K. Mykhailiuk,a Volodymyr S. Brovarets,b
Oleksandr O. Grygorenkoa,c*
a National Taras Shevchenko University of Kyiv, Volodymyrska Street 60, Kyiv 01601,
Ukraine
b Institute of Bioorganic Chemistry & Petrochemistry, NAS of Ukraine
Murmanska Street 1, Kyiv 02660, Ukraine
c Enamine Ltd., Chervonotkatska Street 78, Kyiv 02094, Ukraine, www.enamine.net
d Bienta/Enamine Ltd., Chervonotkatska Street 78, Kyiv 02094, Ukraine, www.bienta.net
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eChemspace, Ilukstes iela 38‐5, Riga, LV‐1082, Latvia, www.chem-space.com
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E-mail: gregor@univ.kiev.ua
Keywords: Sulfonyl halides; Parallel synthesis; Chemoselectivity; Covalent fragments;
Sulfonamides; Serine protease inhibitors
R1
N
S
R2
O
O
O
Cl
O
N
O
S
S
S
S
O
O
R1R2NH
O
O
N
N
O
S
S
O
F
F
F
O
Trypsin inhibitor
IC50 = 84 M
R1 = hetaryl, EWGCH(R3)
R2, R3 = H, alkyl
Abstract. Multigram synthesis of (chlorosulfonyl)benzenesulfonyl fluorides is
described. Selective modification of these building blocks at the sulfonyl chloride
function under parallel synthesis conditions is achieved. It is shown that the reaction
scope includes the use of (hetero)aromatic and electron-poor aliphatic amines (e.g.
amino nitriles). Utility of the method is demonstrated by preparation of the sulfonyl
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fluoride library for potential use as covalent fragments, which is demonstrated by a
combination of in silico and in vitro screening against trypsin as a model enzyme. As a
result, several inhibitors were identified with activity on the par to that of the known
inhibitor.
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Introduction.
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Sulfonyl fluorides have gained much interest in recent years primarily due to their
reasonable reactivity towards nucleophilic attack. In organic synthesis, the sulfur (VI) –
fluoride exchange (SufFEx) reaction was named as “another good reaction for click
chemistry” due to its high efficiency and tolerance to a range of functional groups.1 In
medicinal chemistry, sulfonyl fluorides have been considered as “privileged warheads”
for design of covalent modifiers, which is again due to their controllable reactivity in the
biological systems.2 The best known representatives of this class are benzyl sulfonyl
fluoride (PMSF) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF),
widely used in biochemical studies to inhibit serine proteases in an irreversible manner
(Figure 1).3 These compounds are also examples of covalent fragments, which have
attracted much attention in recent years.4
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SO2F
Cl
SO2F
+H3N
PMSF
AEBSF
Figure 1. Known sulfonyl fluorides – covalent serine protease inhibitors
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Either the SufFEx methodology or a design of covalent inhibitors can benefit from
functionalized sulfonyl fluorides, especially if the use of a parallel synthesis
methodology is intended. Recent examples of building blocks illustrating this idea
include sulfonyl fluorides bearing the moieties of heteroaliphatic amines,5 chalcones,6
aldehydes,7 carboxylic acids,8 organoboronates,9 as well as benzyl10,11 aryl,9 or vinyl12
bromides. Meanwhile, it is widely accepted that sulfonyl fluorides and sulfonyl chlorides
differ significantly in their reactivity.13 Therefore, chlorosulfonyl-substituted sulfonyl
fluorides might be envisaged as useful building blocks for the parallel synthesis of
libraries of sulfonyl fluorides bearing a sulfonamide moiety.
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Although examples of such building blocks are known in the literature for more than
nine decades (compounds 1–7, Figure 2),14–17 only a few isolated examples that
demonstrate their selective modification at the sulfonyl chloride moiety have been
reported to date.15–20 In this work, we describe synthesis of all three isomeric
(chlorosulfonyl)benzenesulfonyl fluorides 1–3, as well as demonstrate their utility for the
preparation of a library of sulfonamide-containing sulfonyl fluorides 8 (Scheme 1). In
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addition to that, their potential for the covalent inhibition of serine proteases was
evaluated using the classical target of this class – trypsin – as a model enzyme.
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SO2F
SO2F
ClO2S
X
SO2F
SO2Cl
ClO2S
1
2
4
5
3
, X = H
, X = Me
, X = OH
SO2F
SO2F
ClO2S
ClO2S
6
7
Figure 2. Known examples of chlorosulfonyl-substituted sulfonyl fluorides
H
SO2F
SO2F
N
R1
R2
R2
O
S
9
N
R1
ClO2S
O
8
1, o-isomer
2, m-isomer
3, p-isomer
Scheme 1.
Results and discussion.
It should be noted that although all three sulfonyl fluorides 1–3 have been mentioned
in the literature, their syntheses were not described in the corresponding papers;
preparation of the compound 2 was mentioned only in a patent.21 We have prepared all
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three building blocks 1–3 using a modification of the above-mentioned method (Scheme
2).21 The synthesis commenced from the commercially available nitrobenzenesulfonyl
chlorides 10–12, which reacted with KF – Et3N to give the corresponding sulfonyl
fluorides 13–15 (82–93% yield). Catalytic hydrogenation of 13–15 led to the formation of
anilines 16–18 (40–95% yield), which were subjected to the diazotation, followed by the
Sandmeyer reaction with in situ generated SO2 to give the target compounds 1–3 in 60–
91% yield.
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SO2F
SO2Cl
SO2F
SO2F
1. NaNO2, HCl
H2
KF
Et3N
O2N
2. SOCl2, H2O,
CuCl
Pd-C
ClO2S
O2N
10
H2N
13
14
15
16
17
18
1
2
3
, o-
, m-
, p-
, o-, 82%
, m-, 89%
, p-, 93%
, o-, 40%
, m-, 79%
, p-, 95%
, o-, 60%
, m-, 85%
, p-, 91%
11
12
Scheme 2. Synthesis of the building blocks 1–3
The next part of our work included design and synthesis of the library 8{1–3,9} based
on the building blocks 1–3. Initially, validation of the common sulfonamide synthesis
protocol was performed for the sulfonyl halides 1–3. A sulfonamide set of 150
compounds was enumerated from 1–3 and nucleophiles 9{1–120}, followed by random
selection (with at least one product per reagent included). It was found that reaction of
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1–3 with most primary and secondary (hetero)aromatic amines 9{1–102} (R1 = (het)aryl;
R2 = H, alkyl, Figure 3) in the presence of pyridine as a base and CH2Cl2 as the solvent
occurred slowly but more or less cleanly at rt; typically, the conversion was completed
after 48 h. The products 8{1–3,1–102} were obtained with 61% synthesis success rate
and 45% average yield (Table 1). Increasing the temperature to 40 C led to the loss of
chemoselectivity, so that mixtures containing mono- and bis-sulfonamides, as well as
unreacted starting material were formed.
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H
N
O
N
N
N
N
N
O
N
N
N
N
N
O
N
N
N
H2N
CN
NH2
H2N
H2N
NH2
NH2
H
7
9
{1}
9
{2}
9
{3}
9
{4}
9
{5}
9
{6}
9
{7}
9
{8}
8
9
H
N
H
N
CN
NH2
Cl
O
10
11
12
13
14
15
16
17
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22
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N
N
N
N
H2N
N
H2N
N
H2N
N
H2N
H
N
O
O
9
9
9
9
9
9
9
9
{16}
{9}
{10}
{11}
{12}
{13}
{14}
{15}
NH2
NH2
NH2
NH2
H2N
N
N
N
O
S
O
H2N
H2N
H2N
HN
N
N
N
N
S
N
N
N
N
9
9
9
9
9
9
{22}
9
{23}
9
{24}
{17}
{18}
{19}
{20}
{21}
HCl
H
H2N
N
N
N
N
N
O
N
O
N
O
N
N
N
N
N
N
O
N
N
N
NH2
NH2
H2N
NH2
H2N
S
H
9
{25}
9
9
9
9
9
9
9
{32}
{26}
{27}
{28}
{29}
{30}
{31}
O
H
O
N
HCl
H2N
HCl
HCl
2HCl
HCl
N
HCl
NH2
N
OPh
O
N
H2N
N
N
H2N
HCl
O
N
N
N
O
N
H
HCl
H2N
NH2
9
{40}
S
NH2
{39}
NH2
9
{33}
9
9
9
9
9
9
{34}
{35}
{36}
{37}
{38}
O
HCl
HCl
HCl
O
O
NH2
NH2
H2N
HO
NH2
NH2
NH2
H2N
N
N
N
NH
N
N
N
N
N
N
N
H
N
N
H2N
N
9
9
9
9
9
{45}
9
{46}
9
{47}
9
{48}
{41}
{42}
{43}
{44}
NH
O
NH
HN
O
NH
O
NH
S
O
S
N
S
O
O
H2N
H2N
S
N
N
NH2
{54}
O
N
S
9
9
{50}
9
{51}
9
9
9
9
9
{56}
{49}
{52}
{53}
{55}
H2N
O
NH2
O
O
N
N
N
H
NH2
CN
S
O
F
N
HN
N
NH2
NH2
N
H2N
H2N
N
HN
N
N
N
O
O
O
H2N
F
H
9
{57}
9
9
9
9
9
9
{63}
9
{64}
{58}
{59}
{60}
{61}
{62}
O
NH2
NH
NH2
NH2
NH2
N
NH2
O
N
HN
N
N
N
NH
H2N
HN
N
N
CF3
CF3
H2N
F
H2N
CN
Cl
O
9
9
9
{67}
9
9
9
9
9
{72}
{65}
{66}
{68}
{69}
{70}
{71}
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NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Br
F3C
HO
N
N
N
N
N
N
N
Cl
Cl
F
9
{73}
9
{74}
9
{75}
9
{76}
9
{77}
9
{78}
9
{79}
9
{80}
9
{81}
9
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F3C
NH2
N
NH2
NH2
NH2
NH2
NH2
NH2
Br
F
N
N
N
N
N
HN
N
H2N
Br
N
N
N
N
9
9
{83}
9
9
9
9
{87}
9
9
{82}
{84}
{85}
{86}
{88}
{89}
H
N
H2N
N
Cl
NH2
C
l
N
NH2
NH2
NH2
N
NH2
N
N
N
N
H2N
N
Br
N
H
S
NH
N
N
N
N
NH2
9
9
{91}
9
{92}
9
9
9
{95}
9
{96}
9
{97}
{90}
{93}
{94}
Cl
NH2
HCl
HO
NH2
2HCl
NH2
NH2
NH2
S
N
N
N
N
S
NH2
N
NH2
N
N
NH2
N
H2N
N
N
N
O
N
H
9
9
9
9
9
9
9
9
{105}
{98}
{99}
{100}
{101}
{102}
{103}
{104}
HCl
HCl
H2N
NH2
H2N
O
F
HCl
NH2
HCl
HCl
NH2
HN
F
NH2
NH
NH
NH2
H2N
O
NC
O
F
NH2
CN
O
N
NC
F
F
NH2
9
9
9
9
9
9
9
9
9
{114}
{106}
{107}
{108}
{109}
{110}
{111}
{112}
{113}
O
H2N
HCl
H2N CN
HCl
N
S
H
HCl
NH2
N
N
CN
NC
H
N
N
N
N
CN
N
N
CN
NH2
HO
H
H
9
9
9
9
9
9
9
9
{122}
{115}
{116}
{117}
{118}
{119}
{120}
{121}
O
O
HCl
O
H2N
HN
HCl
H
N
N
Br
H2N
O
NH2
O
N
N
N
N
O
NH2
N
N
H
S
H2N
N
H2N
NH2
9
{130}
O
N
NH2
9
9
9
9
9
9
9
{129}
{123}
{124}
{125}
{126}
{127}
{128}
NH2
H
NH2
O
S
H2N
F
NH2
NH2
H2N CN
N
N
O
N
N
N
O
N
N
H
NH2
N
N
N
CF3
N
O
9
9
9
9
9
9
9
9
{138}
{131}
{132}
{133}
{134}
{135}
{136}
{137}
Figure 3. Structures of nucleophilic reagents 9{1–138}
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Table 1. Parallel synthesis of sulfonyl fluorides 8
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#
Product
Yield
(%)
#
Product
Yield
(%)
#
Product
Yield
(%)
#
Product
Yield
(%)
1
2
3
4
5
6
7
8
9
8{1,8}
0
4
6
4
7
4
8
4
9
5
0
5
1
5
2
5
3
5
4
5
5
8{2,46}
8{2,48}
8{2,62}
8{2,66}
8{2,70}
8{2,71}
8{2,72}
8{2,80}
8{2,82}
8{2,85}
99
32
43
61
25
99
0
91 8{3,23}
92 8{3,25}
93 8{3,26}
94 8{3,27}
95 8{3,28}
96 8{3,29}
97 8{3,31}
98 8{3,32}
99 8{3,33}
8
13 8{3,85}
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6
8{1,19}
8{1,24}
8{1,40}
8{1,45}
8{1,46}
8{1,50}
8{1,51}
8{1,51}
8{1,52}
0
0
13 8{3,85}
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2
0
13 8{3,86}
8
0
0
13 8{3,86}
9
9
0
28
33
43
30
99
17
14 8{3,87}
75
10
0
0
24
0
14 8{3,88}
1
14 8{3,89}
2
11
11
0
0
14 8{3,90}
10
9
3
6
14 8{3,91}
4
1
0
99
10 8{3,34}
14 8{3,92}
0
0
5
1
1
8{1,53}
8{1,56}
0
0
5
6
8{2,87}
8{2,91}
0
7
10 8{3,35}
0
14 8{3,95}
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6
1
2
5
7
10 8{3,36}
56
14 8{3,96}
26
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8{1,57}
8{1,73}
8{1,76}
8{1,77}
8{1,85}
8{1,98}
8{1,99}
8{1,102}
8{1,116}
8{1,118}
0
0
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0
0
0
0
0
0
0
5
8
8{2,93}
8{2,94}
8{2,97}
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10 8{3,37}
23
19
0
14 8{3,97}
99
99
99
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8
1
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10 8{3,39}
14 8{3,98}
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5
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0
99
10 8{3,41}
15 8{3,98}
5
0
1
6
6
1
8{2,106} 23
8{2,108} 43
8{2,109} 16
8{2,110} 43
8{2,112} 99
8{2,113} 54
8{2,114} 44
8{2,115} 37
10 8{3,44}
0
15 8{3,99}
6
1
1
7
6
2
10 8{3,45}
99
99
99
26
0
15 8{3,100} 33
7
2
1
8
6
3
10 8{3,46}
15 8{3,101}
0
8
3
1
9
6
4
10 8{3,47}
15 8{3,104} 24
9
4
2
0
6
5
11 8{3,48}
15 8{3,105}
0
0
5
2
1
6
6
11 8{3,49}
15 8{3,107} 32
1
6
2
2
6
7
11 8{3,54}
0
15 8{3,108} 36
2
7
2
3
8{1,122} 13
8{1,123} 60
8{1,133} 74
8{1,137} 10
6
8
11 8{3,55}
0
15 8{3,108} 38
3
8
2
4
6
9
8{2,119}
8{2,134}
8{3,1}
0
5
0
11 8{3,56}
35
0
15 8{3,109} 30
4
9
2
5
7
0
11 8{3,58}
16 8{3,110} 20
5
0
2
7
11 8{3,59}
0
16 8{3,111} 38
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8{1,138} 37
8{1,140} 38
7
2
8{3,2}
8{3,3}
8{3,4}
8{3,5}
8{3,6}
8{3,7}
8{3,8}
8{3,9}
8{3,10}
8{3,12}
8{3,13}
8{3,14}
8{3,15}
0
11 8{3,60}
43
0
16 8{3,112} 53
7
2
2
8
7
3
66
68
26
0
11 8{3,61}
16 8{3,114} 31
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3
2
9
8{2,3}
59
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0
7
4
11 8{3,63}
0
16 8{3,117}
0
0
0
9
4
3
0
8{2,4}
7
5
12 8{3,64}
0
16 8{3,119}
0
5
3
1
8{2,8}
7
6
12 8{3,65}
0
16 8{3,120}
1
6
3
2
8{2,15}
8{2,18}
8{2,19}
8{2,22}
8{2,23}
8{2,28}
8{2,29}
8{2,30}
35
48
56
12
8
7
7
14
46
0
12 8{3,67}
0
16 8{3,121} 75
2
7
3
3
7
8
12 8{3,68}
99
28
46
52
55
55
99
16 8{3,124} 99
3
8
3
4
7
9
12 8{3,69}
16 8{3,125} 99
4
9
3
5
8
0
12
0
12 8{3,74}
17 8{3,126}
8
5
0
3
6
8
1
12 8{3,75}
17 8{3,127} 42
6
1
3
7
15
27
57
8
2
99
99
23
12 8{3,76}
17 8{3,128} 72
7
2
3
8
8
3
12 8{3,76}
17 8{3,129} 99
8
3
3
9
8
4
12 8{3,77}
17 8{3,130} 61
9
4
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0
8{2,32}
8{2,34}
8{2,36}
8{2,38}
8{2,42}
8{2,43}
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35
59
97
51
99
8
5
8{3,16}
8{3,17}
8{3,18}
8{3,19}
8{3,20}
8{3,21}
0
13 8{3,78}
3
17 8{3,131} 23
0
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1
8
6
23
30
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13 8{3,79}
38
34
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17 8{3,132}
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13 8{3,81}
17 8{3,135} 10
2
7
4
3
8
8
13 8{3,82}
17 8{3,136} 74
3
8
4
4
8
9
13 8{3,83}
0
17 8{3,137} 46
4
9
4
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9
0
30
13 8{3,84}
27
5
Low chemoselectivity was also observed with primary amines 9{103–106}, including
sterically hindered tert-butylamine 9{103}, even if the reaction was performed at room
temperature (although we managed to isolate the library members 8{2,104} and
8{2,106}, both in 23% yield, after chromatographic purification). Nevertheless, the
method could be extended to less nucleophilic aliphatic amines bearing electron-
withdrawing groups (e.g. -alkyl -amino nitriles 9{107–109} or fluorinated amines
9{110,111}, as well as acyl hydrazides 9{112–114} and phenols 9{115}, although in
these cases, isolated yields were slightly lower (average value is 38%). The procedure
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did not work with the parent N-(cyanomethyl)amines 9{116–118} and ,-dialkyl -
amino nitriles 9{119,120}: mixtures of unidentified products were obtained in these
experiments.
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Therefore, the developed procedure for the synthesis of SO2F-containing
sulfonamides 8{1–3,9} can be effective for (hetero)aromatic, as well as aliphatic amines
with low nucleophilicity, hydrazides, and phenols. The method tolerates azole, azinone,
and carboxamide N–H bonds, hydroxyl, nitrile, and aromatic bromide functions, as well
as 2- or 4-chloropyridine moieties. 92 of 150 library members 8{1–3,9} were prepared in
the parallel synthesis during the validation step (61% synthesis success rate, 43%
average yield), although in most cases, chromatographic purification of the products
was necessary. Analysis of unsuccessful results obtained with aromatic amines did not
reveal any additional regularities; in our opinion, the reasons behind these failures might
be related to isolation and/or purification issues and not chemical problems.
The following order of efficiency in the synthesis of the library 8 was observed for the
sulfonyl halides 1–3 according to the synthesis success rate: 2 (87%) > 3 (61%) > 1
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(14%). Poor results obtained with o-isomer 1 might be attributed to the limited stability of
the final products 8{1,9} towards hydrolysis (possibly due to the participation of the o-
sulfonamide moiety) since in most cases, the corresponding sulfonic acid was observed
as the main component in the crude products according to LCMS data. These
conclusions are supported by the results obtained from stability of the compounds 8 in
DMSO solutions: whereas in the case of the products obtained from the sulfonyl halides
2 and 3, the compounds were stable at rt for several days, the compounds 8{1,9}
showed considerable degradation at these conditions after overnight storage.
Therefore, it is preferable to use their freshly prepared solutions.
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Further extension of the library 8 was aimed at demonstrating its potential to inhibit
serine proteases using the classical target of this class – trypsin – as a model enzyme.
The library enumeration followed the principles of the REAL concept published
elsewhere.22 In particular, virtual coupling of sulfonyl halides 1–3 and 1224
(hetero)aromatic or fluorinated aliphatic amines, or -aminonitriles 9 with in-house
validated reactivity in the sulfonamide bond formation, afforded 31224 = 3672
synthesizable library members. These were subjected to covalent docking into the
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trypsin S1 pocket that is quite deep and planar so that comfortable accommodation of
aromatic cores is possible.23 The catalytically active Ser195 was responsible for the
covalent bond formation; in addition to that, several hydrogen bond donors/acceptors
were also present in the binding site, including side chains of Asp189, Ser190, Gln192,
as well as backbone NH groups of Ser195, Gly193 and Gly216.
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As a result of the docking procedure, 100 compounds were selected as virtual hits
according to the following criteria: the built-in QXP scoring function,24 the number of
hydrogen bonds (was exposed to a minimum of four hydrogen bonds), the protein-
ligand contact surface area and the distances from ligand to the key points of the
corresponding pharmacophore model (Figure 4). The model was based on the limited
ligand mobility due to the covalent bond with Ser195 and the structural features of the
entire series of compounds. Formation of hydrogen bonds between oxygen atoms of the
two sulfo groups and Gly193, as well as Gln192 and/or Gly216, respectively, was
desirable. An additional hydrogen bond with Gly219 might be also present for the
compounds bearing a sulfonamide NH group. This interaction fixed the (het)aryl
fragment L2 in the S1 sub-pocket; filling the sub-pocket and approaching a part of the
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ligand to Asp 189 was considered as one of the selection criteria. A separate filter
removed ligands strongly exposed to the solution. Further processing of the complexes
was carried out by visual inspection according to the assumed pharmacophore
interaction model.
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Figure 4. Pharmacophore model of ligand binding in the S1 pocket of trypsin (red and
blue spheres show hydrogen bond donors and acceptors, respectively; L1, L2 denote
aromatic/hydrophobic moieties
62 of 100 sulfonyl fluorides from the resulting set were synthesized using the
developed procedure (Table S1). These products were subjected to in vitro screening
against trypsin, including protein thermal shift assay (TSA),25–27 followed by BApNA (N-
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benzoyl-DL-arginine p-nitroanilide) enzymatic digestion assay28 (Table 2). It is
interesting to note that most of the identified hits demonstrated negative thermal shift.
This indicates destabilization of the folded protein in the presence of the ligand,29 and is
sometimes perceived as an undesirable feature of the TSA hits.30,31 Nevertheless, three
of these compounds had IC50 values lower than the standard (PMSF) in the enzymatic
digestion assay, the compound 8{1,122} being five-fold more active than PMSF (Figure
5); other showed activity comparable to that of PMSF. It should be noted that all the
identified hits were rather polar (cLogP = 0.77–2.6032), which might be useful to satisfy
the hydrogen-bonding potential of the compounds in the protein binding site. In
particular, the ligand-lipophilicity efficiency (defined as LLE = pIC50 – cLogP33) for the
most active compound 8{1,122} (3.28) was improved as compared to that of PMSF
(1.93). These data demonstrate that the library 8{1–3,9} can be a promising tool for
discovery of covalent modifiers.
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Table 2. Results of in vitro testing of the sulfonyl fluorides 8 against trypsin
Entry No.
Compound
Tm, C IC50 (M) cLogP32
LLE
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9
O
S
F
O
O
338
224
648
795
2.23
2.60
2.09
1.37
1.24
1.05
1.10
1.73
S
1
2
3
4
–9.5
–10.6
–7.8
N
N
O
H
Z1672278953
8
{3,74}
O
S
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F
O
O
N
O
S
O
S
N
O
H
Z1672278926
8
{3,56}
O
S
F
O
N
O
S
S
N
O
H
Z1672278918
8
{3,98}
F
O
S
N
O
O
N
S
N
0.16
O
Z2216296236
8
{1,140}
O
N
S
O
O
S
1721
84
2.34
0.80
0.77
0.42
3.28
2.24
5
6
7
–3.0
–3.6
F
O
Z1737943418
8
{1,51}
O
N
S
S
O
N
N
O
S
F
O
Z2216296226
8
{1,122}
O
S
N
N
N
O
O
988
–12.0
S
O
F
Z2216296225
8
{1,123}
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O
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F
480
1.39
1.93
O
8
–0.11
PMSF
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Figure 5. Dose-response curves for the most active compounds 8{1,122} and 8{3,56}
compared to those for PMSF (red curves are the same)
Analysis of the binding mode for the two most active compounds 8{1,122} and
8{3,56} showed that in the case of 8{3,56}, the ligand fit well with the pharmacophore
model shown in Figure 4. In particular, the sulfo groups forms hydrogen bonds with
Gln192, Gly216, and Gly193; and the hydrogen bond was also present between the
sulfonamide NH and Gly219 (Figure 6). A different binding mode was observed for
8{1,122}: while the key hydrogen bonds with Gln192 and Gly193 were retained, the
sulfo group attached to Ser195 also formed hydrogen bond with catalytic His57. In
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addition to that, the N-4 atom of the thiadiazole ring formed a hydrogen bond with
Ser214 buried in the S1 sub-pocket. In both cases, the corresponding heteroaromatic
moieties filled the S1 sub-pocket of trypsin, as it was suggested by the pharmacophore
model.
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8{1,122}
8{3,56}
Figure 6. Binding poses of compounds 8{1,122} and 8{3,56} in the S1 pocket of trypsin
according to the docking results
Conclusions.
The difference in reactivity of sulfonyl halides can be used to selectively modify the
sulfonyl chloride moiety in the presence of the sulfonyl fluoride one within the same
molecule.
We
demonstrated
this
feature
for
three
isomeric
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(chlorosulfonyl)benzenesulfonyl fluorides. Therefore, these bifunctional building blocks
are convenient starting points to generate the libraries of sulfonyl fluorides. The scope
of the developed parallel synthesis procedure includes various (hetero)aromatic, as well
as electron-poor aliphatic amines (i.e. -amino nitriles or fluorinated amines). The library
of the obtained sulfonyl fluorides is a promising tool for design of covalent modifiers
targeting serine/threonine and lysine residues in the protein molecules, which is
validated by discovery of several trypsin inhibitors having two- and five-times higher
activity compared with the known protease inhibitor PMSF.
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Experimental part.
General. All chemicals and solvents were obtained from Enamine Ltd. and used
1
13
without further purification. H and C NMR spectra were acquired on Bruker Advance
DRX 400 and Bruker Avance DRX 500 spectrometers using DMSO-d6 as a solvent and
tetramethylsilane as an internal standard. Melting points were determined on a Buchi
melting point apparatus. LC-MS data were recorded on Agilent 1100 HPLC equipped
with diode-matrix and mass-selective detector Agilent LC/MSD SL, column: Zorbax SB-
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C18, 4.6 mm × 15 mm; eluent: A, acetonitrile – H2O with 0.1% of TFA (95:5), B, H2O
with 0.1% of TFA; flow rate: 1.8 mL/min. Elemental analyses were performed at the
Laboratory of Organic Analysis, Department of Chemistry, Kyiv National Taras
Shevchenko University.
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Preparative flash chromatography was performed on a Combiflash Companion
chromarograph (12 g RediSep columns, gradient Hexanes – i-PrOH as eluent).
Synthesis of sulfonyl fluorides 13–15. To a solution of sulfonyl chloride 10–12 (103 g, 0.467
mol) in CH2Cl2 (1000 mL), solutions of KF (81.3 g, 1.40 mol) in H2O (400 mL) and Et3N (78.1
mL, 0.560 mol) in CH2Cl2 (400 mL) were added. The mixture was stirred at room temperature
for 4 h, then the organic phase was separated, washed with H2O (400 mL), saturated aq NaHCO3
(400 mL) and evaporated in vacuo.
2-Nitrobenzenesulfonyl fluoride (13). Yield 78.6 g, 82%. For spectral and physical data, see
ref.8,34
3-Nitrobenzenesulfonyl fluoride (14). Yield 85.3 g, 89%. For spectral and physical data, see
ref.35
4-Nitrobenzenesulfonyl fluoride (15). Yield 89.1 g, 93%. For spectral and physical data, see
ref.34,35
Synthesis of sulfonyl fluorides 16–18. Nitrobenzene derivative 16–18 (78.2 g, 0.381 mol)
was dissolved in MeOH (750 mL), and 5% Pd-C (12.5 g) was added. The resulting mixture was
hydrogenated in an autoclave at 50 C for 72 h. The catalyst was filtered off, and the combined
filtrates were evaporated in vacuo. The residue was triturated with t-BuOMe (750 mL). The
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solution was decanted from the residue and dried over Na2SO4. The solvent was removed under
reduced pressure, and the residue was dried in vacuo.
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2-Aminobenzenesulfonyl fluoride (16). Yield 26.7 g, 40%. For spectral and physical data, see
ref.36
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3-Aminobenzenesulfonyl fluoride (17). Yield 52.7 g, 79%. Colorless liquid. H NMR (400
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MHz, CDCl3) δ 7.45 – 7.31 (m, 2H), 7.22 (s, 1H), 6.98 (d, J = 6.9 Hz, 1H), 4.03 (s, 2H). C
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NMR (125 MHz, CDCl3) δ 147.6, 133.7 (d, J = 23.2 Hz), 130.5, 121.4, 117.6, 113.3. F NMR
(376 MHz, CDCl3) δ 64.6. LC/MS (EI): m/z = 176 [M+H]+. Anal. calcd. for C6H6FNO2S: C,
41.14; H, 3.45; N, 8.00; S, 18.30. Found: C, 41.03; H, 3.49; N, 7.97; S, 17.95.
4-Aminobenzenesulfonyl fluoride (18). Yield 63.4 g, 95%. For spectral and physical data, see
ref.37
Synthesis of sulfonyl fluorides 1–3. Thionyl chloride (41.0 mL, 0.564 mol) was added
dropwise to ice (250 g). The mixture was left for 48 h, then cooled to –5 C, and CuCl (0.574 g,
5.80 mmol) was added to give the solution A. Sulfonyl fluoride 16–18 (25.6 g, 0.146 mol) was
dissolved in 20% aq HCl (250 mL), the solution was cooled to –10 C, and a solution of NaNO2
(12.1 g, 0.175 mol) in H2O (29.0 mL) was added dropwise. The resulting solution was stirred at
room temperature for 20 min, cooled to –5 C, and added dropwise to a solution A. The mixture
was stirred at –5 C for 3 h and then filtered. The filtrates were extracted with CH2Cl2 (3250
mL), the combined extracts were dried over Na2SO4 and evaporated in vacuo. The residue was
recrystallized from hexanes.
2-(Chlorosulfonyl)benzenesulfonyl fluoride (1). Yield 22.6 g, 60%. Orange crystals; mp =
88–90 C. 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 7.4 Hz, 1H), 8.41 (d, J = 7.7 Hz, 1H), 8.03
13
(quint, J = 2.7 Hz, 2H). C NMR (125 MHz, CDCl3) δ 142.5, 136.3, 135.7, 133.3 (d, J = 1.9
Hz), 132.3, 131.3 (d, J = 28.6 Hz). 19F NMR (376 MHz, CDCl3) δ 66.0. GC/MS (EI): m/z = 258
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[M]+. Anal. calcd. for C6H4ClFO4S2: C, 27.86; H, 1.56; S, 24.79; Cl, 13.71. Found: C, 28.24; H,
1.65; S, 24.67; Cl, 14.02.
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3-(Chlorosulfonyl)benzenesulfonyl fluoride (2). Yield 32.1 g, 85%. Yellowish crystals; mp =
27–29 C. 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.36 (d, J = 8.1
Hz, 1H), 7.96 (t, J = 8.1 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 145.8, 135.2 (d, J = 28.1 Hz),
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134.3, 133.4, 131.7, 127.1. F NMR (470 MHz, DMSO-d6) δ 66.2. LC/MS (EI): m/z = 258
[M]+. Anal. calcd. for C6H4ClFO4S2: C, 27.86; H, 1.56; S, 24.79; Cl, 13.71. Found: C, 27.96; H,
1.78; S, 24.82; Cl, 13.47.
4-(Chlorosulfonyl)benzenesulfonyl fluoride (3). Yield 34.4 g, 91%. Yellowish powder; mp =
1
109–111 C. H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 8.4 Hz, 2H).
13C NMR (100 MHz, CDCl3) δ 149.6, 138.9 (d, J = 27.4 Hz), 130.0, 128.3. 19F NMR (376 MHz,
CDCl3) δ 65.6. LC/MS (EI): m/z = 258 [M]+. Anal. calcd. for C6H4ClFO4S2: C, 27.86; H, 1.56; S,
24.79; Cl, 13.71. Found: C, 27.95; H, 1.75; S, 24.48; Cl, 13.69.
Parallel synthesis of sulfonyl fluorides 8. Amine 9 (1 mmol), pyridine (0.5 mL), CHCl3
(1 mL), and sulfonyl chloride 1–3 (258 mg, 1 mmol) were placed into a vial, and the mixture was
shaken at room temperature for 48 h. Then it was evaporated in vacuo, CHCl3 (3 mL) was added,
the solution was washed with H2O (1 mL), and evaporated in vacuo. The residue was purified by
preparative flash chromatography.
3-(N-(Pyridin-4-yl)sulfamoyl)benzene-1-sulfonyl fluoride (9{2,85}). Yield 86 mg, quant.
1
Yellowish solid; mp = 207–209 C. H NMR (400 MHz, DMSO-d6) δ 12.98 (br s, 1H), 8.41 –
13
8.20 (m, 3H), 8.11 – 8.02 (m, 2H), 7.93 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.2 Hz, 2H). C NMR
(125 MHz, DMSO-d6) δ 162.5, 146.5, 139.8, 134.1, 132.6 (d, J = 24.2 Hz), 132.2, 131.4, 125.6,
115.5. 19F NMR (376 MHz, DMSO-d6) δ 66.1. LC/MS (CI): m/z = 317 [M+H]+. Anal. calcd. for
C11H9FN2O4S2: C, 41.77; H, 2.87; N, 8.86; S, 20.27. Found: C, 41.69; H, 2.92; N, 9.14; S, 20.56.
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Virtual screening. Molecular docking was performed using a flexible ligand and a fixed
receptor model. We used an algorithm of MCDOCK (Monte Carlo search) implemented
in QXP docking software, which had shown high reproducing ability of ligand
conformation with minimum RMSD in comparison to the crystallographic data, but more
time-consuming than standard methods.38 The binding site model was created on the
basis of PDB 2PLX.23 When converting the structure of the protein and creating a
binding site model, the center was exposed on Ser 190. The sulfonyl fluorides database
was prepared for the dock combinatorial library. The maximum number of MCDOCK
routine steps was set to 500, and the 10 best structures (based on built-in QXP scoring
function)24 were retained for each compound. The resulting protein-ligand complex
structures had been filtered by intrinsic Flo+ filters and multiRMSD software package.39
In vitro screening. Reference compounds – PMSF (phenylmethyl sulfonyl fluoride) and
benzamidine – were obtained from Enamine Ltd. (Kyiv, Ukraine). Stock solutions of the
tested compounds were prepared at 20 mM in DMSO and were stored at –20 °C until
use. All thermal shift assay (TSA) experiments with trypsin (Sigma, Cat. T8003) were
performed using ViiA™7 real-time PCR System equipped with 384-well heat block
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(Applied Biosystems, USA). General TSA methodology was adopted from the
literature24-26,31 and experimentally modified in order to optimize conditions for
measuring trypsin melting temperature shifts upon interaction with small molecules. To
define the optimal buffer composition for the TSA procedure, a matrix of common
biological buffers combined on a 96-well microplate, including phosphate, acetate,
TRIS, HEPES, MES at different pH and inorganic salts content, was tested in thermal
melt experiment on a 384-well microplate (each buffer composition in quadruplicate). In
addition, each buffer composition was tested at two different buffering component
concentrations, resulting in two 384-well plate buffer screening experiments and the
total of 192 different buffer compositions tested.
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Selection of the optimal buffer composition was based on balancing two criteria
including maximized melting temperature (thermal stability) of trypsin and maximized
melting temperature shift induced by the benzamidine at the concentration of 500 µM.
As a result, the buffer consisting of 20 mM Tris-HCl pH 7.0 and 1 mM CaCl2 was
selected for the screening. Trypsin was pre-mixed with SYPRO Orange dye (Thermo
Fischer Scientific, Cat. S6650, 5000 stock) to prepare a master mix at 345 µg/mL
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protein and 10 dye concentration. PMSF and Benzamidine were used as reference
compounds at the final concentrations of 200 uM and 500 uM, respectively. Tested
compounds were added to the protein-dye master mix at 200 uM (1% DMSO
concentration) and incubated at room temperature for 15 min in MicroAmp® optical 384-
well reaction plates (ThermoFisher, Cat. 4309849) sealed with optical sealing film
(ThermalSeal RT2, Excel Scientific, Cat. TS-RT2). The volumes of all reaction mixtures
were 10 µL (3.45 µg of trypsin per well). Thermal scanning was performed by raising
temperature to 34 °C at 2.5 °C/min without signal detection followed by 34 °C to 75 °C
temperature ramp at 0.1 °C/s with constant fluorescence intensity reading at 1 sec
intervals using EX470/EM623 nm filter set. Screening of compounds was carried out in
quadruplicate (n = 4). The raw data of dye fluorescence intensity change upon protein
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melt
were
exported
using
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ViiA7
RUO
software
1.2
(Applied
Biosystems/ThermoFischer Scientific). Further data visualization, curve fitting, melting
temperature calculations on the raw fluorescence data were performed using custom-
made Microsoft Excel scripts. The peak of the first derivative for the fluorescence curve
was used to define melting temperature (Tm). Averaged Tm values for the control wells
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