Lead Development of Thiazolylsulfonamides with Carbonic Anhydrase Inhibitory Action

Article abstract of DOI:10.1021/acs.jmedchem.7b00183

A series of congeners structurally related to pritelivir, N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide, a helicase-primase inhibitor for the treatment of herpes simplex virus infections, was prepared. The syn

Full text of DOI:10.1021/acs.jmedchem.7b00183

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Article
Lead development of thiazolyl sulfonamides
with carbonic anhydrase inhibitory action
Fabrizio Carta, Alexander Birkmann, Tamara Pfaff, helmut buschmann, wilfried
schwab, Holger Zimmermann, alfonso maresca, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00183 • Publication Date (Web): 08 Mar 2017
Downloaded from http://pubs.acs.org on March 9, 2017
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Lead development of thiazolyl sulfonamides with carbonic anhydrase inhibitory action
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Fabrizio Carta,1* Alexander Birkmann,2 Tamara Pfaff,2 Helmut Buschmann,2 Wilfried
Schwab,2 Holger Zimmermann,2 Alfonso Maresca,1 and Claudiu T. Supuran1 *
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1 Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
2 AiCuris AntiꢀInfective Cures GmbH, FriedrichꢀEbertꢀStr. 475, 42117 Wuppertal, Germany.
Abstract: A series of congeners structurally related to pritelivir, Nꢀ[5ꢀ(aminosulfonyl)ꢀ4ꢀmethylꢀ
1,3ꢀthiazolꢀ2ꢀyl]ꢀNꢀmethylꢀ2ꢀ[4ꢀ(2ꢀpyridinyl) phenyl]acetamide, a helicaseꢀprimase inhibitor for the
treatment of herpes simplex virus infections, was prepared. The synthesized primary and secondary
sulfonamides were investigated as inhibitors of six physiologically and pharmacologically relevant
human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic enzymes hCA I and II, the
mitochondrial ones hCA VA and VB, and the transꢀmembrane, tumor associated hCA IX and XII.
Low nanomolar inhibition KI values were detected for all of them, with a very interesting and well
defined structureꢀactivity relationship. As many CAs are involved in serious pathologies, among
which cancer, obesity, epilepsy, glaucoma, etc., sulfonamide inhibitors as those reported here may
be of interest as drug candidates. Furthermore, pritelivir itself is an effective inhibitor of some CAs,
also inhibiting whole blood enzymes from several mammalian species, which may be a favorable
pharmacokinetic feature of the drug which can be transported throughout the body bound to blood
CA I and II.
Key words: Carbonic anhydrase; Sulfonamide; Thiazole; Pritelivir
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Introduction.
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Pritelivir,
(Nꢀ[5ꢀ(aminosulfonyl)ꢀ4ꢀmethylꢀ1,3ꢀthiazolꢀ2ꢀyl]ꢀNꢀmethylꢀ2ꢀ[4ꢀ(2ꢀpyridinyl)
9
phenyl]acetamide), previously known as BAY 57ꢀ1293 and AIC316 (Fig. 1),1 is an antiviral agent
in Phase II clinical development, useful for the treatment of herpes simplex virus (HSV)
infections.1,2,3 Its mechanism of action is totally different from that of other antiꢀherpetic agents
such as acyclovir, penciclovir, and other nucleoside analogs (which inhibit the herpesviral DNA
polymerase after becoming activated by the viral thymidine kinase), as pritelivir acts as a helicaseꢀ
primase inhibitor that does not need to become activated.1ꢀ3 The Phase I and II clinical trials were
promising, with the compound being well tolerated and effective showing superiority over placebo
and the nucleoside analog valacyclovir.2,3
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Fig. 1: Chemical structures of the antiꢀHSV helicaseꢀprimase inhibitor pritelivir and herpesviral
DNA polymerase inhibitors acyclovir and penciclovir.
One of the interesting features of pritelivir is that the compound incorporates a primary
sulfonamide moiety, not present in any other antiviral agent. However, this functionality is well
known for its affinity for the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1),4ꢀ10 with many
aromatic, heterocylic, aliphatic and sugar derivatives incorporating this moiety, acting as highly
effective CA inhibitors (CAIs).11ꢀ15 In line with this, initial activity on carbonic anhydrase in the
micromolar range has been reported previously for pritelivir by means of a carbonic anhydrase–
catalyzed CO2 hydration assay.1
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There are six genetic families encoding CAs in virtually all organisms known to date, the αꢀ,
βꢀ, γꢀ, δꢀ, ζꢀ and ηꢀCAs.6ꢀ9 All CAs known so far are metal ionꢀdependent enzymes, with a metalꢀ
hydroxide species within the enzyme cavity acting as a nucleophile in the catalytic cycle, and a
second step (usually rateꢀdetermining) involving a proton transfer reaction from a water molecule
coordinated to the active site metal ion to the environment, for regenerating the nucleophile.4,7ꢀ10
Metal ions employed at the active site of the different CAs include Zn(II) (in all classes), Cd(II) (in
ζꢀCAs), Co(II) (in the δ class) or Fe(II) (for γꢀCAs, in anaerobic conditions).4 This pingꢀpong
mechanism makes some of the members of the CA superfamily among the most effective enzymes
known in nature, with kcat/KM values close to the limit of the diffusionꢀcontrolled processes.10,11
The CAs possess crucial physiologic functions, as the reaction products obtained from the
hydration of CO2 are either involved in pH regulation (bicarbonate and protons), but also in many
biosynthetic processes (lipogenesis, ureagenesis, gluconeogenesis) or in other important phenomena
such as for example chemosensing,4 sexual development (in pathogenic fungi),8 pH and CO2ꢀ
sensing, pathogenicity, and survival in ambient air of many bacteria, fungi and/or protozoa.7ꢀ10
In humans, 15 αꢀCAs isoforms are known to date, CA I ꢀ CA VA, CA VB, CA VI ꢀ CA
XIV, with 12 of them being catalytically active and three (CA VIII, X and XI) devoid of activity but
still playing significant functions in tumorigenesis and other physiologic as well as pathologic
processes.4
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As CO2, bicarbonate and protons are simple molecules/ions involved in a host of
physiologic processes, some of which briefly mentioned above, their upꢀ or downꢀregulation is
associated with a range of diseases.12ꢀ15 Indeed, the CAIs of the primary sulfonamide type (but
many other chemotypes were reported, such as the coumarins,16 sulfocoumarins,17 mono and
dithiocarbamates,18 etc.) are clinically used for decades as diuretics,4,11 antiepileptics,12 antiꢀobesity
agents,13 antiglaucoma drugs,4,15 or more recently as antitumorꢀagents, with one such compound in
clinical development for the treatment of hypoxic, metastatic tumors.4,19 Although many new
chemotypes with CA inhibitory properties and with various mechanisms of actions were reported in
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the last 10 years,16ꢀ18 the sulfonamides remain the most investigated class of such compounds with
many interesting representatives being reported constantly.19,20
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One of the main hurdles connected with the use of CAIs in the treatment of diverse
conditions as those mentioned above, is related to the offꢀtarget inhibition of isoforms other than the
desired one.4 In fact the various pharmacological applications of the CAIs are due to the high
number of isoforms and their involvement in different pathologies.11ꢀ15
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Results and Discussion
Compound design and synthesis. Many sulfonamide CAIs incorporate an elongated scaffold
which contains a fiveꢀmembered heterocyclic ring on which the SO2NH2 zinc binding group moiety
and a tail are attached on the two sides of the cycle, in such a way that the tail extends as much as
possible within the enzyme active site and makes interactions with amino acid residues at the
entrance of the cavity, which are the least conserved residues among the many mammalian isoforms
known.4ꢀ11,21 Most of the times the fiveꢀmembered heterocyclic ring was an 1,3,4ꢀthiadiazole,21a,b
a
thiophene,21c and rarely a thiazole nucleus.21d This type of scaffold leads to a multitude of contacts
between the inhibitor and the enzyme, as shown schematically in Fig. 2 for 5ꢀ[1ꢀ(naphthalenꢀ1ꢀyl)ꢀ
1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl]thiopheneꢀ2ꢀsulfonamide bound to human (h) isoform hCA II, as determined
by Xꢀray crystallography.21c The sulfamoyl zinc binding group (ZBG) is observed bound to the
catalytically crucial Zn(II) ion whereas the organic scaffold of the inhibitor is in contact with many
amino acid residues involved in inhibitor binding, such as Phe131, Val135, Leu204, Pro202,
extending throughout the cavity. 21c
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Fig. 2. Binding of a thiopheneꢀ2ꢀsulfonamide derivative to hCA II as determined by Xꢀray
crystallography (PDB file 4BF6). 21c
Such a binding for sulfonamide CAIs with elongated scaffolds affords not only the
possibility to obtain compounds with very high affinities for the enzyme (usually low nanomolar or
subnanomolar), but also many cases of isoformꢀselective inhibitors were detected in this way.19ꢀ21
Considering that pritelivir (Fig. 1) also has this type of scaffold, with the thiazole ring substituted
with the sulfonamide ZBG and the (2ꢀpyridinyl) phenyl]acetamide fragment at positions 5 and 2
respectively, we decided to first confirm the CA inhibitory properties of this primary sulfonamide in
our laboratory, and thereafter to use the molecule as a lead for obtaining new sulfonamide CAIs.
In Schemes 1 and 2 the synthetic strategy to obtain a series of thiazolylꢀ5ꢀsulfonamides
(including pritelivir) is shown.
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Scheme 1. Synthesis of sulfonamides 4a-h and 6.
2ꢀChlorothiazoles 1a,b were transformed to the corresponding sulfonyl chlorides 2a,b. Reaction of
2a,b with ammonia or primary amines led to sulfonamides 3a-e, which were thereafter reacted
again with amines in order to obtain the 2ꢀaminoꢀthiazoleꢀ5ꢀsulfonamide derivatives 4a-h
,
incorporating a series of different substituents at the 2ꢀaminoꢀ, thiazoleꢀ4ꢀposition and
N
ꢀ
sulfonamide fragments of the molecule, in order to generate chemical diversity (Scheme 1).
Derivative 4a, incorporating a 4ꢀmethyl moiety at the thiazole ring was converted into the
corresponding 4ꢀhyroxymethylen derivative
6
via monobromination with
Nꢀbromosuccinimide,
followed by displacement of the bromide with water
5
,
thus leading to the desired 2ꢀaminomethylꢀ
5ꢀsulfonamideꢀthiazole
6
incorporating the hydroxymethylene group at position 4 (Scheme 1).
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Scheme 2. Synthesis of sulfonamides 8a-t. i) H2SO4 2% aq.; ii) 1.25 M HCl/MeOH.
In order to obtain the pritelivirꢀlike compounds, the intermediates 4a-h obtained as shown in
Scheme 1, were coupled with 4ꢀsubstitutedꢀphenylacetic acids 7a-i in the presence of carbodiimides
leading to sulfonamides 8a-t (derivative 8b is pritelivir). The chemical diversity was achieved by
varying the nature of the aromatic/heterocyclic moieties which substitute the phenylacetic ring
7 at
position 4, with phenyl, 2ꢀpyridyl, pyridinium, pyrazyl, pyrazoyl and substitutedꢀphenyl groups
included in the study (Scheme 2).1b, 23ꢀ24 The 4ꢀethylꢀ (8s) and 4ꢀhydroxymethyl (8t) analogs of
pritelivir (which has a 4ꢀmethyl such moiety) were also prepared as depicted in Scheme 2.
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All compounds were properly characterized by spectroscopic methods which confirmed
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their structure (1HꢀNMR, CꢀNMR, MS, and IR). Purity was controlled by HPLC. All compounds
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reported here were > 98% pure.
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Carbonic anhydrase inhibition. Sulfonamide reported here were tested in vitro for their inhibition
profiles against six physiologically relevant hCA enzymes,25 the cytosolic isoforms I and II, the
mitochondrial ones hCA VA and VB, and the transꢀmembrane, tumor associated hCA IX and XII
(Table 1).
Table 1. CA inhibition data against isoforms human carbonic anhydrases (hCA) I, II, VA, VB, IX
and XII with compounds 8a-8t and acetazolamide (AAZ) as standard, by a stoppedꢀflow CO2
hydrase assay.25
_______________________________________________________________________________
Compound
KI (nM)*
hCA VA
hCA I
hCA II
hCA VB
hCA IX
hCA XII
______________________________________________________________________________
8a
8b
8c
8d
8e
8f
26.9
0.9
67.6
474.0
816.0
354.0
32.0
61.7
29.0
61.2
53.5
58.9
55.1
389.0
251.0
78.0
58.0
45.6
47.0
67.8
62.0
49.1
0.9
4.9
323.0
262.0
436.0
378.0
29.3
12.8
14.0
15.6
248.0
203
81.0
464.0
261.0
35.0
69.1
22.0
1.0
77.2
61.3
36.7
77.0
61.5
59.0
3.1
8g
8h
8i
264.0
55.4
79.0
74.8
4.9
56.1
10.1
0.9
46.5
5.5
8j
37.0
104
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8k
8l
58.5
47.8
47.4
54.1
44.2
543.0
73.0
26.6
48.4
64.7
250.0
180
32.9
81.5
74.3
341
82.0
122
94.5
1.0
560
441
51.4
21.8
40.8
25.6
24.7
14.0
41.0
44.5
28.1
42.9
25.4
75.1
4.2
37.4
75.4
32.8
64.8
73.0
88.1
98.1
60.9
51.1
63.1
56.7
92.3
79.4
37.8
33.0
34.9
58.2
23.8
66.0
54.2
8m
8n
8o
57.3
52.8
67.0
60.0
31.6
67.6
48.3
43.1
5.6
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8p
8q
8r
8s
8t
10.0
12.1
AAZ
_______________________________________________________________________________
* Mean from 3 different assays, errors were within ± 10 % of the reported values, by a stoppedꢀ
flow, CO2 hydrase assay.25
The following structureꢀactivity relationship (SAR) can be drawn by considering data of Table 1. It
should be stressed from the beginning that most of these derivatives are primary sulfonamides (8a-
8l, 8s and 8t), four derivatives incorporate the SO2NHMe moiety (8m-8p) whereas 8q and 8r
possess bulkier substituents at the sulfamoyl nitrogen (see Scheme 2):
(i) the slow cytosolic isoform hCA I, widely present in the blood, gastroꢀintestinal tract and many
other tissues in humans,4ꢀ11 was effectively inhibited by sulfonamides
8 investigated here, with KIs
ranging between 26.6 and 543 nM (Table 1). Most of these sulfonamides were thus more effective
hCA I inhibitors compared to acetazolamide (AAZ, 5ꢀacetamidoꢀ1,3,4ꢀthiadiazoleꢀsulfonamide), a
clinically used drug (KI of 250 nM).4 The nature of the Het moiety present in the final part of the
tail seems to be the most important factor influencing activity. For example, the phenylꢀsubstituted
compound 8a is a 12ꢀtimes more effective hCA I inhibitor compared to pritelivir 8b which has a 2ꢀ
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pyridyl moiety instead of the phenyl one. Thus, the replacement of one CH group by an N atom
leads to important changes in the inhibitory activity, a phenomenon already documented by us for
many classes of CAIs by means of kinetic and Xꢀray crystallographic studies.15ꢀ21 Compact R2
moieties (Me and cyclopropyl) led to better hCA I inhibition in compounds incorporating them
(e.g., 8h-8l) compared to the derivative incorporating a bulkier such group (8g). There are no major
differences of activity between primary and secondary sulfonamides, and also the nature of the
group in position 4 of the thiazole (methyl, ethyl or hydroxyethyl) does not influence much the
inhibitory power of these sulfonamides (Table 1).
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(ii) The physiologically dominant isoform hCA II, widely spread all over the body and a drug target
for diuretic and antiglaucoma agents,4,11,15 was also potently inhibited by most sulfonamides
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investigated here, with KIs ranging between 0.9 and 341 nM (Table 1). Several compounds were
much more effective than AAZ as hCA II inhibitors (e.g., 8a, 8i, 8s, KIs ranging between 0.9 and
4.9 nM versus 12.1 nM for AAZ) whereas pritelivir 8b and many of its congeners (8c, 8d, 8l, 8t
)
showed comparable (KIs of 10.0 – 32.9 nM) or slightly weaker (8g, 8h, 8m, 8n, 8p, 8r) activity
compared to the standard drug (Table 1). SAR is rather similar with what stressed above for hCA I
inhibition, with the nature of the Het fragment of the molecule being the most important structural
feature influencing activity. Again phenyl or substituted phenyl (8a and 8i) seem to be more
effective than the heterocyclyl such moieties, except for 8s, case in which the 4ꢀEt instead of 4ꢀMe
leads to 12.8 times better activity of 8s compared to pritelivir 8b (the two compounds differ only by
one CH2 group).
(iii) The mitochondrial isoform hCA VA was also effectively inhibited by most sulfonamides
reported here, which showed KIs ranging between 29.0 and 816 nM (Table 1). It should be
mentioned that hCA VA (and probably also the second mitochondrial isoform hCA VB) are drug
targets for antiobesity agents.13 The only compound in clinical use for treating obesity based on
CAIs is topiramate, an antiepileptic for which this second use was approved recently.13 Several
sulfonamides reported here, such as 8a, 8e, 8f, 8g, -8j, 8l, 8n, 8o, 8s and 8t showed better or
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comparable hCA VA inhibitory activity to AAZ, with KIs ranging between 29.0 and 64.8 nM, being
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thus of considerable interest as antiobesity drug candidates. Pritelivir and some of its congeners
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(8b-d, 8k) showed weaker hCA VA inhibitory activity, with KIs ranging between 354 and 816 nM
9
(Table 1). SAR is more complicated for the inhibition of this isoform, and it is interesting to note
that the most effective inhibitor was 8g which incorporates a bulky R2 moiety, which was
detrimental to inhibition of isoforms hCA I and II. Other structural aspects which lead to effective
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inhibition (e.g., the nature of the Het and R1 moietiess from inhibitors
discussed above for their inhibition of hCA I and II.
8) are similar to what
(iv) The second mitochondrial isoform, hCA VB, was more sensitive to inhibition with
sulfonamides compared to hCA VA, showing KIs in the range of 23.8 ꢀ 441 nM. Many of these
8
sulfonamides were better or comparable hCA VB inhibitors to AAZ, among which 8a, 8e-8g, 8j,
8l, 8o-8s (KIs ranging between 23.8 and 58.2 nM). Pritelivir 8b as well as its congeners 8c and 8k
were the least effective hCA VB inhibitors with inhibition constants of 251ꢀ441 nM (Table 1).
(v) The tumor associated isoform hCA IX, a validated antitumor drug target4,14 was effectively
inhibited by sulfonamides 8, with KIs ranging between 0.9 and 464 nM (Table 1). Only two
compounds (8c and 8d) showed a KI > 100 nM whereas the remaining ones were highly effective
hCA IX inhibitors (pritelivir is one of the least effective in the group of good inhibitors, with a KI of
81.0 nM, see Table 1). Again the most important structural feature influencing activity is the nature
of the Het moiety at the terminal part of the tail, with pyridyl (as sulfate, thus probably pyridinium)
and
Nꢀmethylpyridinium (in 8c and 8d) leading to the least effective inhibitors. The best
substitution patterns in this part of the molecule include the Ph (8a, 8h) 3ꢀfluorophenyl (8j
)
moieties. Again primary and secondary sulfonamides showed similar inhibitory properties, and the
nature of the group in position 4 of the thiazole ring was not very influential for the inhibitory
activity (Table 1).
(vi) Powerful inhibitory activity was registered also against hCA XII, a second transmembrane
isoform investigated here (target for antiglaucoma and anticancer agents)4,14,15 with sulfonamides 8
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showing KIs ranging between 3.1 and 77.2 nM. Pritelivir 8b was the least effective hCA XII
inhibitor in the series, but several of its congeners (8a, 8h, 8j, 8l) has a KI of 3.1 – 5.5 nM being
highly effective inhibitors of this isoform. SAR is similar to what discussed above for hCA IX
inhibition, but it is interesting to note that the most effective hCA XII inhibitor possesses the
cyclopropyl moiety as R2 group (Table 1 and Scheme 2).
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Since mammalian blood is very rich in CAs (mainly isoforms I and II),4b we also
investigated the inhibition of whole blood CAs from three species, mouse, rat and human with the
antiviral drug pritelivir (Table 2).
Table 2. IC50 for the inhibition of mouse, rat and human whole blood with pritelivir 8b
.
___________________________________________
Species
IC50 (nM)*
____________________________________________
Mouse
Rat
134
115
65.3
Human
____________________________________________
* Mean from 3 different assays, errors within ± 10 % of the reported values, stoppedꢀflow, CO2
hydrase assay.25
From data of Table 2 it can be seen that there are no substantial differences of inhibitory
power of pritelivir against the blood CA of the different mammals included in the investigation (all
in the same order of magnitude), with the human enzymes being the most inhibited ones (IC50 of
65.3 nM), followed by the rodent blood enzymes (see Table 2).
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Conclusions. A series of thiazoleꢀ5ꢀsulfonamide derivatives was prepared by an original procedure.
4
5
6
These compounds are congeners of the helicaseꢀprimase inhibitor pritelivir,
Nꢀ[5ꢀ(aminosulfonyl)ꢀ
7
8
9
4ꢀmethylꢀ1,3ꢀthiazolꢀ2ꢀyl]ꢀNꢀmethylꢀ2ꢀ[4ꢀ(2ꢀpyridinyl) phenyl]acetamide, currently in Phase II
clinical development. The synthesized primary and secondary sulfonamides were investigated as
inhibitors of six physiologically and pharmacologically relevant hCA isoforms, the cytosolic
isoforms I and II, the mitochondrial ones hCA VA and VB, and the transꢀmembrane, tumor
associated hCA IX and XII. Low nanomolar inhibitors were detected for all of them, with a very
interesting and well defined structureꢀactivity relationship, typical for all these different isoforms.
As many CAs are involved in serious pathologies, among which cancer, obesity, epilepsy,
glaucoma, etc., sulfonamide inhibitors as those reported here may be of interest as drug candidates
for all these pathologies. Furthermore, we could confirm that pritelivir itself is an effective inhibitor
of some CA isoforms in vitro, whereas our IC50 values were reproducibly lower than those
previously reported (Kleymann 2002). However, there are considerable differences between the test
system that was used by Kleymann et al. and our assay leading to this discrepancy, first of all by
monitoring the dehydratase but not the hydratase enzymatic reaction, and second because of our
automated (stoppedꢀflow) versus manual measurements.
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Since pritelivir is in Phase II clinical development, a variety of in vivo data and data from
treatment in humans is available. In animal studies only the development of urinary bladder
hyperplasia in rats has been attributed to the CA inhibitory effect of pritelivir so far.2 This
proliferation of the transitional cells is a transient, rodentꢀspecific effect of CA inhibition which is
wellꢀknown and generally considered as not relevant for humans. Neither adverse findings or
beneficial pharmacological effects that could be related to CA inhibition in humans has been
reported for healthy subjects or patients treated with pritelivir as of today. However, these studies
were not designed to show effects on diseases or conditions targeted by CA inhibitors such as
cancer or obesity.
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Finally, we could show that pritelivir interacts with whole blood enzymes from several
mammalian species, which may be a favorable pharmacokinetic feature of a drug which can be
transported throughout the body bound to blood enzymes such as CA I and II. In fact, pritelivir has
a long halfꢀlife in the body of up to 80 hours allowing even once weekly dosing for suppression of
genital herpes.3
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In summary, by investigating a series of primary and secondary sulfonamides, we could
identify several compounds with oneꢀdigit nanomolar or even subꢀnanomolar activity on certain CA
isoenzymes. A potential use of these potent inhibitors for CA associated conditions and diseases
remains to be investigated.
Experimental protocols
Chemistry
General. Anhydrous solvents and all reagents were purchased from SigmaꢀAldrich, Alfa Aesar and
TCI. All reactions involving airꢀ or moistureꢀsensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic
resonance (1HꢀNMR, 13CꢀNMR, 19FꢀNMR) spectra were recorded using a Bruker Advance III 400
MHz spectrometer in DMSOꢀ
coupling constants ( ) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; brs, broad singlet; dd, doublet of doublets. The
assignment of exchangeable protons (O and N ) was confirmed by the addition of D2O.
d6. Chemical shifts are reported in parts per million (ppm) and the
J
H
H
Analytical thinꢀlayer chromatography (TLC) was carried out on Merck silica gel Fꢀ254 plates. Flash
chromatography purifications were performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM) as the
stationary phase and ethyl acetate/nꢀhexane were used as eluents. Melting points (m.p.) were
measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are
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uncorrected. HPLC was performed by using a Waters 2690 separation module coupled with a
photodiode array detector (PDA Waters 996) and as column a NovaꢀPak C18 4 ꢁm 3.9 mm × 150
mm (Waters), silicaꢀbased reverse phase column. Sample was dissolved in acetonitrile 10%, and an
injection volume of 45 ꢁL was used. The mobile phase, at a flow rate of 1 mL/min, was a gradient
of water + trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile + TFA 0.1% (B), with steps as
follows: (A%:B%), 0−10 min 90:10, 10−25 min gradient to 60:40, 26:28 min isocratic 20:80,
29−35 min isocratic 90:10. TFA 0.1% in water as well in acetonitrile was used as counterion. All
compounds reported here showed more than 96% HPLC purity. The solvents used in MS measures
were acetone, acetonitrile (Chromasolv grade), purchased from SigmaꢀAldrich (Milan ꢀ Italy), and
mQ water 18 Mꢂ, obtained from Millipore's Simplicity system (MilanꢀItaly). The mass spectra
were obtained using a Varian 1200L triple quadrupole system (Palo Alto, CA, USA) equipped by
Electrospray Source (ESI) operating in both positive and negative ions. Stock solutions of analytes
were prepared in acetone at 1.0 mg mLꢀ1 and stored at 4°C. Working solutions of each analyte were
freshly prepared by diluting stock solutions in a mixture of mQ H2O/ACN 1/1 (v/v) up to a
concentration of 1.0 ꢁg mLꢀ1 The mass spectra of each analyte were acquired by introducing, via
syringe pump at 10 /L minꢀ1, of the working solution. Rawꢀdata were collected and processed by
Varian Workstation Vers. 6.8 software.
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Synthesis of 2ꢀchloroꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonyl chloride (2a) and 2ꢀchloroꢀ4ꢀethylthiazoleꢀ5ꢀ
sulfonyl chloride (2b). 1b, 22
2ꢀChloroꢀ4ꢀmethylthiazole (1a) or 2ꢀchloroꢀ4ꢀethylthiazole (1b) (1.0 eq) was added dropꢀwise to a
solution of thionyl chloride (2.5 eq) and chlorosulfonic acid (5.0 eq). The reaction mixture was
stirred at 120 °C for 48 hrs, cooled down , quenched with slush and extracted with DCM (3 x 20
ml). The combined organic layers were dried over Na2SO4, filteredꢀoff, and concentrated under
vacuo to give a residue that was purified by fractional distillation (2a, 17 mbar, 85ꢀ95°C; 2b, 0.9
mbar, 59ꢀ64 °C).
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2ꢀChloroꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonyl chloride (2a); m/z (ESI positive) 231.90 [M+H]+.
2ꢀChloroꢀ4ꢀethylthiazoleꢀ5ꢀsulfonyl chloride (2b); m/z (ESI positive) 247.12 [M+H]+.
Experimental data in agreement with reported data 1b, 22
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22
Synthesis of 2ꢀchloroꢀ4ꢀalkylthiazoleꢀ5ꢀsulfonyl amides 3a-e
.
2ꢀChloroꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonyl chloride (2a) or 2ꢀchloroꢀ4ꢀethylthiazoleꢀ5ꢀsulfonyl chloride
2b) (1.0 eq.) was treated with the proper amine (1.0 eq) in THF and stirred until consumption of
(
the starting material occurred (TLC monitoring). Then the solvents were removed under vacuo and
the obtained residue was immediately used for the next step without further purification.
2ꢀChloroꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonamide 3a
;
m/z (ESI positive) 212.95 [M+H]+.22
,4ꢀdimethylthiazoleꢀ5ꢀsulfonamide 3b;
m/z (ESI positive) 226.96 [M+H]+.
2ꢀChloroꢀ
2ꢀChloroꢀ
[M+H]+.
N
N
ꢀ(2ꢀhydroxyethyl)ꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonamide 3c
; m/z (ESI positive) 256.97
2ꢀChloroꢀ
N
ꢀcyclopropylꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonamide 3d
m/z (ESI positive) 240.98 [M+H]+.
;
m/z (ESI positive) 252.98 [M+H]+.
2ꢀChloroꢀ4ꢀethylꢀNꢀmethylthiazoleꢀ5ꢀsulfonamide 3e;
Synthesis of 4ꢀalkylꢀ2ꢀ(alkylamino)thiazoleꢀ5ꢀsulfonamido derivatives 4a-h 22
.
2ꢀChloroꢀ4ꢀalkylthiazoleꢀ5ꢀsulfonyl amides 3a-e (1.0 eq) were dissolved in acetonitrile and treated
with the appropriate amine (3.3 eq) at 50 °C until consumption of the starting material (TLC
monitoring). The reaction solution was cooled down to r.t. the solvent was removed under vacuo to
give a residue that was treated with H2O. The solid formed was collected by filtration and dried
under vacuo to afford the titled compounds which didn’t require further purification.
4ꢀMethylꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (4a); m.p. 192 °C.22; m/z (ESI positive) 208.01
[M+H]+.
2ꢀ((2ꢀ(Dimethylamino)ethyl)amino)ꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonamide (4b); m/z (ESI positive)
265.07 [M+H]+.
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2ꢀ(Cyclopropylamino)ꢀ4ꢀmethylthiazoleꢀ5ꢀsulfonamide (4c); m/z (ESI positive) 234.03 [M+H]+.
4
5
6
N
,4ꢀDimethylꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (4d); m/z (ESI positive) 222.03 [M+H]+.
7
8
9
2ꢀ((2ꢀ(Dimethylamino)ethyl)amino)ꢀ
279.09 [M+H]+.
N,4ꢀdimethylthiazoleꢀ5ꢀsulfonamide (4e); m/z (ESI positive)
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N
ꢀCyclopropylꢀ4ꢀmethylꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (4f); m/z (ESI positive) 248.04
[M+H]+.
N
ꢀ(2ꢀHydroxyethyl)ꢀ4ꢀmethylꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (4g); m/z (ESI positive)
252.04 [M+H]+.
4ꢀEthylꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (4h); m/z (ESI positive) 222.03 [M+H]+.
Synthesis of 4ꢀ(hydroxymethyl)ꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (6).
4ꢀMethylꢀ2ꢀ(methylamino)thiazoleꢀ5ꢀsulfonamide (4a) (1.0 eq) was dissolved in MeOAc and
treated with NBS (1.0 eq) and AIBN cat. at r.t. for 3 hrs. Then the solvents were removed under
vacuo and the residue was purified by silica gel column chromatography eluting with 30% ethyl
acetate in
(methylamino)thiazoleꢀ5ꢀsulfonamide (
at 100°C. The solvents were removed in vacuo and the obtained residue was triturated from DCM
to afford the title compound ( ) in 98% yield; δH (400 MHz, DMSOꢀ 6) 3.10 (3H, d, 6.2, ꢀC 3),
4.72 (2H, d, 6.4, C 2), 4.80 (2H, t, 6.4, exchange with D2O, O ), 7.20 (1H, brs, exchange with
n
ꢀhexane followed by trituration in DCM to afford 4ꢀ(bromomethyl)ꢀ2ꢀ
5
) which was treated with a 1/1 solution of H2O/1,4ꢀdioxane
6
d
J
N
H
J
H
J
H
D2O, N
H
), 7.56 (2H, s, exchange with D2O, SO2N
H2); δC (100 MHz, DMSOꢀ
d
6) 29.8, 58.6, 110.1,
148.2, 165.0; m/z (ESI positive) 224.01 [M+H]+.
1b
Synthesis of
The proper acid 7a-i (1.0 eq.) was dissolved in dry DMF an treated with 1ꢀhydroxyꢀ1
benzotriazole (HOBT; 1.0 eq) for 10 minutes at r.t., followed by addition of the corresponding 4ꢀ
NꢀalkylꢀNꢀ(4ꢀalkylꢀ5ꢀsulfamoylꢀthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀarylꢀ2ꢀylꢀphenyl)ꢀacetamides 8a-t.
H
ꢀ
alkylꢀ2ꢀ(alkylamino)thiazoleꢀ5ꢀsulfonamido derivatives 4a-h,
6
(1.1 eq) and
Nꢀ(3ꢀ
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dimethylaminopropyl)ꢀ
N
′ꢀethylcarbodiimide hydrochloride (EDCI.HCl; 1.1 eq). The reaction
4
5
6
7
8
mixture was stirred at r.t. under a nitrogen atmosphere for until consumption of the starting
material. Then the solvent was removed under vacuo and the obtained residue was triturated from
DCM or H2O to afford the titled compounds in the pure form.
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2ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ
obtained according to the previously reported general procedure, by using 4a and 7a, in 83 % yield;
m.p. 193 °C; δH (400 MHz, DMSOꢀ 6) 2.48 (3H, s, C 3), 3.71 (3H, s, NꢀC 3), 4.23 (2H, s, C 2),
7.38 (1H, m, ArꢀH), 7.48 (2H, d, 8.4, Arꢀ ), 7.50 (2H, d, 8.4, Arꢀ ), 7.65 (2H, s, exchange with
D2O, SO2N 2), 7.80 (2H, d, 8.4, Arꢀ ); δC (100 MHz, DMSOꢀ 6) 16.5, 35.0, 42.3, 127.5, 128.0,
N
ꢀmethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)acetamide
(
8a
)
was
d
H
H
H
J
H
J
H
H
J
H
d
128.5, 129.0, 130.6, 134.2, 139.8, 140.1, 148.0, 148.1, 160.2, 171.0; m/z (ESI positive) 402.09
[M+H]+.
N
ꢀMethylꢀ
obtained according to the previously reported general procedure, by using 4a and 7b, in 74 % yield;
m.p. 190 °C; δH (400 MHz, DMSOꢀ 6) 2.48 (3H, s, C 3), 3.71 (3H, s, NꢀC 3), 4.23 (2H, s, C 2),
7.32 (1H, m, ArꢀH), 7.63 (2H, d, 8.4, Arꢀ ), 7.67 (2H, s, exchange with D2O, SO2N 2), 7.85 (2H,
appt, 8.8 , Arꢀ ), 8.00 (1H, d, 8.8, Arꢀ ), 8.10 (2H, d, 8.4, Arꢀ ), 8.66 (1H, d, 8.8, Arꢀ ); δC
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylꢀthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀpyridinꢀ2ꢀylꢀphenyl)ꢀacetamide
(
8b
)
was
d
H
H
H
J
H
H
J
H
J
H
J
H
J
H
(100 MHz, DMSOꢀ
d6) 16.6, 34.7, 42.0, 120.6, 123.0, 127.0, 128.7, 130.6, 135.4, 137.7, 137.8,
148.6, 150.0, 156.2, 158.9, 172.3; m/z (ESI positive) 403.08 [M+H]+.
Bis [
N
ꢀMethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylꢀthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀpyridinꢀ2ꢀylꢀphenyl)ꢀacetamide] sulfate
salt (8c
)
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N
ꢀmethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylꢀthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀpyridinꢀ2ꢀylꢀphenyl)ꢀacetamide (8b) (1.0 eq)
was treated at 0° C with a 2% w/w aqueous solution of H2SO4 (0.6 eq). The precipitate formed was
collected by filtration, washed with H2O and dried under vacuo to afford the titled compound in
90% yield. δH (400 MHz, DMSOꢀ
7.32 (1H, m, ArꢀH), 7.63 (2H, d, 8.4, Arꢀ
appt, 8.8 , Arꢀ ), 7.99 (1H, d, 8.8, Arꢀ
(100 MHz, DMSOꢀ
d
6) 2.48 (3H, s, C
), 7.67 (2H, s, exchange with D2O, SO2N
), 8.14 (2H, d, 8.4, Arꢀ ), 8.68 (1H, d,
H
3), 3.71 (3H, s, NꢀC
H
3), 4.23 (2H, s, C
2), 7.85 (2H,
8.8, Arꢀ ); δC
H2),
J
H
H
J
H
J
H
J
H
J
H
d
6) 16.6, 34.7, 42.0, 120.6, 123.0, 127.0, 128.7, 130.6, 135.4, 137.7, 137.8,
ꢀ +
148.6, 150.0, 156.2, 158.9, 172.3; m/z (ESI positive) 403.08 [MꢀHSO4 ] .
1ꢀMethylꢀ2ꢀ(4ꢀ(2ꢀ(methyl(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)amino)ꢀ2ꢀoxoethyl)phenyl)pyridinꢀ1ꢀ
ium acetate salt (8d) was obtained according to the previously reported general procedure, by using
4a and 7c, in 89% yield; δH (400 MHz, DMSOꢀ
d
6) 2.15 (3H, s, C
2), 7.63 (2H, d,
), 7.82 (1H, appt,
), 9.30 (1H, d, 8.8, Arꢀ ); δC (100 MHz, DMSOꢀd6)
H
3ꢀCO2), 2.25 (3H, s, C
8.4, Arꢀ ), 7.59 (2H, s,
8.8, Arꢀ ), 8.38 (1H,
H3), 3.52
(3H, s, NꢀC
H
3), 4.14 (3H, s, N+ꢀC
H
3), 4.26 (2H, s, C
H
J
H
exchange with D2O, SO2N
H
2), 7.80 (2H, d,
8.8, Arꢀ
J
8.4 , Arꢀ
H
J
H
appt, 8.8, Arꢀ ), 8.52 (1H, d,
J
H
J
H
J
H
17.0, 22.0, 23.1, 33.9, 39.8, 42.0, 121.0, 123.0, 125.1, 128.7, 130.0, 132.1, 136.5, 143.2, 146.0,
ꢀ +
148.8, 150.4, 159.0, 171.9, 172.3, 175.0; m/z (ESI positive) 417.10 [MꢀCH3CO2 ] .
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N
ꢀMethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylꢀthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀpyrazolꢀ1ꢀylꢀphenyl)ꢀacetamide
(
8e
)
was
4
5
6
7
8
obtained according to the previously reported general procedure, by using 4a and 7e, in 60% yield;
δH (400 MHz, DMSOꢀ 6) 2.46 (3H, s, C 3), 3.69 (3H, s, NꢀC 3), 4.20 (2H, s, C 2), 6.42 (1H, s,
Arꢀ ), 7.42 (2H, d, 8.4, Arꢀ ), 7.48 (1H, s, Arꢀ ), 7.58 (2H, d, 8.4, Arꢀ ), 7.63 (2H, s,
exchange with D2O, SO2N 2), 8.00 (1H, s; Arꢀ ); δC (100 MHz, DMSOꢀ 6) 15.8, 32.0, 42.0,
d
H
H
H
9
H
J
H
H
J
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
d
110.1, 112.4, 125.7, 126.8, 130.2, 131.1, 138.6, 141.0, 147.9, 159.0, 170.1; m/z (ESI positive)
391.08 [M+H]+.
N
ꢀmethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀ(pyrazinꢀ2ꢀyl)phenyl)acetamide
(8f
)
was
obtained according to the previously reported general procedure, by using 4a and 7d, in 63% yield;
m.p. 220 °C; δH (400 MHz, DMSOꢀ
7.53 (2H, s, exchange with D2O, SO2N
(1H, s, Arꢀ ), 8.80 (1H, d, 8.8, Arꢀ
d
6) 2.30 (3H, s, C
2), 7.65 (2H, d,
), 8.82 (1H, d,
H
3), 3.7 (3H, s, NꢀC
8.4, Arꢀ ), 8.10 (2H, d,
); δC (100 MHz, DMSOꢀd
H
3), 4.08 (2H, s, C
8.4, Arꢀ ), 8.76
6) 15.4,
H2),
H
J
H
J
H
H
J
H
J
8.8, Arꢀ
H
32.0, 41.9, 111.0, 126.5, 130.3, 135.8, 142.4, 143.4, 144.8, 148.0, 153.2, 160.4, 172.3; m/z (ESI
positive) 404.08 [M+H]+.
2ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ
yl)acetamide (8g) was obtained according to the previously reported general procedure, by using 4b
and 7a, in 72 % yield; δH (400 MHz, DMSOꢀ 6) 2.30 (3H, s, C 3), 2.35 (6H, s, 2 x NꢀC 3), 2.71
(2H, t, 6.4 CH2ꢀC 2ꢀN(CH3)2), 3.64 (2H, t, 6.4 C 2ꢀCH2ꢀN(CH3)2), 3.92 (2H, s, C 2), 7.40 (1H,
8.4, ArꢀH), 7.54 (2H, d, 8.4, ArꢀH), 7.68 (2H, s, exchange with D2O,
8.4, ArꢀH); δC (100 MHz, DMSOꢀ 6) 16.0, 42.4, 48.0, 48.1, 60.2, 110.4,
Nꢀ(2ꢀ(dimethylamino)ethyl)ꢀNꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀ
d
H
H
J
H
J
H
H
m, ArꢀH), 7.50 (2H, d,
SO2NH2), 7.79 (2H, d,
J
J
J
d
128.0, 128.5, 129.0, 130.6, 134.2, 139.8, 140.1, 148.0, 148.1, 160.2, 172.0; m/z (ESI positive)
459.14 [M+H]+.
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Journal of Medicinal Chemistry
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4
5
6
7
8
9
2ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ
obtained according to the previously reported general procedure, by using 4c and 7a, in 82% yield;
m.p. 164 °C; δH (400 MHz, DMSOꢀ 6) 1.06 (2H, m), 1.32 (2H, m), 2.16 (3H, s, C 3) 3.98 (2H, s,
2), 4.18 (1H, m), 7.36 (2H, d, 8.4; Arꢀ ), 7.39 (1H, appt, 8.4, ArꢀH), 7.49 (2H, d, 8.4, Arꢀ
H), 7.53 (2H, s, exchange with D2O, SO2N 2), 7.70 (2H, d, 8.4, Arꢀ ), 7.78 (2H, d, 8.4; Arꢀ );
NꢀcyclopropylꢀNꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)acetamide (8h) was
d
H
C
H
J
H
J
J
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
J
H
J
H
δC (100 MHz, DMSOꢀd6) 10.4, 15.9, 35.0, 38.4, 110.1, 127.8, 128.0, 128.3, 129.1, 130.4, 135.0,
139.9, 141.1, 148.0, 162.3, 169.4; m/z (ESI positive) 428.10 [M+H]+.
2ꢀ(2'ꢀFluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ
N
ꢀmethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)acetamide
(8i)
was obtained according to the previously reported general procedure, by using 4a and 7f, in 94 %
yield; m.p. 209 °C; δH (400 MHz, DMSOꢀ
7.27 (1H, appt, 8.7, ArꢀH), 7.38 (2H, d,
with D2O, SO2N 2), 7.63 (2H, d,
); δC (100 MHz, DMSOꢀ 6) 16.2, 32.0, 39.2, 110.4, 115.8 (d, JCꢀF 23.5), 125.0, 128.2, 129.1,
d
6) 2.20 (3H, s, C
8.4; Arꢀ ), 7.49 (1H, m, Arꢀ
8.6, Arꢀ
H
3) 3.78 (s, NꢀC
), 7.53 (2H, s, exchange
), 7.75 (2H, d, 8.6; Arꢀ
H3), 4.12 (2H, s, CH2),
J
J
H
H
H
J
8.4, ArꢀH), 7.69 (1H, appt,
J
H
J
H
d
129.3 (d, JCꢀF 23.5), 130.2, 130.9, 134.6, 135.0, 148.0, 158.9, (d, JCꢀF 247) 159.1, 171.3; m/z (ESI
positive) 420.08 [M+H]+.
2ꢀ(3'ꢀFluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ
was obtained according to the previously reported general procedure, by using 4a and 7g, in 84 %
yield; m.p. 148 °C; δH (400 MHz, DMSOꢀ 6) 2.20 (3H, s, C 3) 3.78 (s, NꢀC 3), 4.12 (2H, s, C 2),
7.21 (1H, m, Arꢀ ), 7.28 (1H, d, 8.4; Arꢀ ), 7.38 (1H, d, 8.4; Arꢀ ), 7.53 (2H, s, exchange with
D2O, SO2N 2), 7.54 (2H, m, ArꢀH), 7.65 (2H, d, 8.4, Arꢀ ); δC (100 MHz, DMSOꢀ 6) 16.0, 32.4,
40.1, 114.1 (d, JCꢀF 24.0), 116.1 (d, JCꢀF 24.0), 123.6, 127.8, 128.1, 130.2, 130.9, 134.5, 139.9,
N
ꢀmethylꢀ
N
ꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)acetamide
(8j)
d
H
H
H
H
J
H
J
H
H
J
H
d
141.2, 148.0, 159.1, 162.1 (d,
J
CꢀF 247), 171.0; m/z (ESI positive) 420.08 [M+H]+.
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7
8
9
2ꢀ(2',5'ꢀDifluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ
8k) was obtained according to the previously reported general procedure, by using 4a and 7h, in 80
% yield; m.p. 188 °C; δH (400 MHz, DMSOꢀ 6) 2.19 (3H, s, C 3) 3.80 (s, NꢀC 3), 4.12 (2H, s,
2), 7.23 (1H, m, Arꢀ ), 7.36 (1H, m; Arꢀ ), 7.37 (2H, d, 8.4; Arꢀ ), 7.50 (1H, m; Arꢀ ), 7.52
(2H, s, exchange with D2O, SO2N 2), 7.64 (2H, d, 8.4, Arꢀ ); δC (100 MHz, DMSOꢀ 6) 16.0,
32.0, 40.3, 110.1, 116.0, 116.1 (d, CꢀF 24.0), 118.0 (d, CꢀF 24.0), 128.2, 130.0, 132.9, 134.4, 135.4,
NꢀmethylꢀNꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)acetamide
(
d
H
H
C
H
H
H
J
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
J
H
d
J
J
148.0, 154.5, 157.4 (d, JCꢀF 247), 158.0 (d, JCꢀF 247), 159.1, 171.3; m/z (ESI positive) 438.07
[M+H]+.
2ꢀ(3'ꢀFluoroꢀ2'ꢀmethoxyꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ
yl)acetamide (8l) was obtained according to the previously reported general procedure, by using 4a
and 7i, in 65 % yield; δH (400 MHz, DMSOꢀ 6) 2.20 (3H, s, C 3), 3.78 (s, NꢀC 3), 3.80 (3H, s, Oꢀ
3), 4.11 (2H, s, C 2), 7.21 (1H, m, Arꢀ ), 7.30 (1H, m; Arꢀ ), 7.38 (2H, d, 8.4; Arꢀ ), 7.52
(2H, s, exchange with D2O, SO2N 2), 7.63 (2H, d, 8.4, Arꢀ ), 7.74 (1H, m, Arꢀ ); δC (100 MHz,
DMSOꢀ 6) 16.0, 32.1, 40.2, 58.1, 110.1, 115.4 (d, JCꢀF 24.0), 119.3, 126.2, 127.3, 128.2, 130.1,
NꢀmethylꢀNꢀ(4ꢀmethylꢀ5ꢀsulfamoylthiazolꢀ2ꢀ
d
H
H
C
H
H
H
H
J
H
H
J
H
H
d
134.6, 136.4, 148.0 (d,
[M+H]+.
JCꢀF 24), 148.1, 152.4 (d, JCꢀF 247), 156.0, 171.4; m/z (ESI positive) 450.09
2ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ
was obtained according to the previously reported general procedure, by using 4d and 7a, in 93 %
yield; m.p. 177 °C; δH (400 MHz, DMSOꢀ 6) 2.21 (3H, s, C 3), 2.50 (3H, s, SO2NHꢀC 3), 3.80 (s,
NꢀC 3), 4.12 (2H, s, C 2), 7.41 (1H, appt, 6.84, Arꢀ ), 7.38 (2H, d, 8.4; Arꢀ ), 7.49 (2H, d,
8.4; Arꢀ ), 7.63 (2H, d, 8.4, Arꢀ ), 7.72 (2H, d, 8.4, Arꢀ ), 7.75 (1H, s, exchange with D2O,
SO2NH-); δC (100 MHz, DMSOꢀ 6) 16.4, 30.1, 32.0, 40.2, 110.4, 127.4, 127.6, 128.2, 129.0, 129.8,
NꢀmethylꢀNꢀ(4ꢀmethylꢀ5ꢀ(Nꢀmethylsulfamoyl)thiazolꢀ2ꢀyl)acetamide (8m)
d
H
H
H
H
J
H
J
H
J
H
J
H
J
H
d
134.5, 139.6, 140.1, 148.0, 159.1, 172.3; m/z (ESI positive) 416.10 [M+H]+.
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Journal of Medicinal Chemistry
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7
8
9
2ꢀ(2'ꢀFluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ
yl)acetamide (8n) was obtained according to the previously reported general procedure, by using 4d
and 7f, in 87 % yield; m.p. 182 °C; δH (400 MHz, DMSOꢀ 6) 2.20 (3H, s, C 3), 2.50 (3H, s,
2), 7.27 (1H, appt, 8.7, ArꢀH), 7.38 (2H, d, 8.4;
8.4, ArꢀH), 7.69 (1H, appt, 8.6, Arꢀ ), 7.75 (2H, d,
6) 16.2, 30.4, 32.0,
NꢀmethylꢀNꢀ(4ꢀmethylꢀ5ꢀ(Nꢀmethylsulfamoyl)thiazolꢀ2ꢀ
d
H
SO2NHꢀC
Arꢀ ), 7.49 (1H, m, Arꢀ
8.6; Arꢀ
H
3), 3.78 (s, NꢀC
H
3), 4.12 (2H, s, C
H
J
J
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
), 7.63 (2H, d,
J
J
H
J
H
), 7.76 (1H, s, exchange with D2O, SO2NH-); δC (100 MHz, DMSOꢀd
39.2, 110.4, 115.8 (d, JCꢀF 23.5), 125.0, 128.2, 129.1, 129.3 (d, JCꢀF 23.5), 130.2, 130.9, 134.6,
CꢀF 247) 159.1, 171.3; m/z (ESI positive) 434.09 [M+H]+.
135.0, 148.0, 158.9, (d,
J
N
ꢀmethylꢀNꢀ(4ꢀmethylꢀ5ꢀ(Nꢀmethylsulfamoyl)thiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀ(pyridinꢀ2ꢀyl)phenyl)acetamide
hydrochloride salt (8o
)
N
ꢀMethylꢀ
8o’) was treated with a commercially available 1.25 M hydrochloric acid solution in methanol. The
precipitate formed was collected by filtration and dried under vacuo to afford the titled compound
8o in 98 % yield; m.p. 240 °C; δH (400 MHz, DMSOꢀ 6) 2.20 (3H, s, C 3), 2.50 (3H, s, SO2NHꢀ
3), 3.78 (s, NꢀC 3), 4.15 (2H, s, C 2), 7.32 (1H, m, ArꢀH), 7.63 (2H, d,
s, exchange with D2O, SO2NH-), 7.85 (2H, appt, 8.8 , Arꢀ ), 7.99 (1H, d,
d, 8.4, Arꢀ ), 8.68 (1H, d, 8.8, Arꢀ ); δC (100 MHz, DMSOꢀ
Nꢀ(4ꢀmethylꢀ5ꢀ(Nꢀmethylsulfamoyl)thiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀ(pyridinꢀ2ꢀyl)phenyl)acetamide
(
d
H
C
H
H
H
J
J
8.4, Arꢀ
H
), 7.74 (1H,
), 8.14 (2H,
J
H
8.8, Arꢀ
H
J
H
J
H
d6) 16.6, 30.2, 34.7, 42.0, 120.6,
123.0, 127.0, 128.7, 130.6, 135.4, 137.7, 137.8, 148.6, 150.0, 156.2, 158.9, 172.2; m/z (ESI
positive) 432.12 [MHꢀClꢀ]+.
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7
8
9
2ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ
yl)acetamide (8p) was obtained according to the previously reported general procedure, by using 4e
and 7a, in 81 % yield; δH (400 MHz, DMSOꢀ 6) 2.21 (3H, s, C 3), 2.35 (6H, s, 2 x NꢀC 3), 2.50
(3H, s, SO2NHꢀC 3), 2.71 (2H, t, 6.4 CH2ꢀC 2ꢀN(CH3)2), 3.64 (2H, t, 2ꢀCH2ꢀN(CH3)2),
3.80 (s, NꢀC 3), 4.13 (2H, s, C 2), 7.41 (1H, appt, 8.4; Arꢀ ), 7.49
(2H, d, 8.4; Arꢀ ), 7.63 (2H, d, 8.4, Arꢀ ), 7.72 (2H, d,
Nꢀ(2ꢀ(dimethylamino)ethyl)ꢀNꢀ(4ꢀmethylꢀ5ꢀ(Nꢀmethylsulfamoyl)thiazolꢀ2ꢀ
d
H
H
H
J
H
J
6.4 C
H
J
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
J
6.84, Arꢀ
H
), 7.38 (2H, d,
H
J
H
J
H
J
8.4, ArꢀH), 7.74 (1H, s, exchange with
D2O, SO2NH-); δC (100 MHz, DMSOꢀ
d6) 16.4, 32.1, 42.4, 48.0, 48.1, 110.3, 127.6, 127.8, 128.3,
129.0, 130.0, 134.5, 139.8, 141.0, 148.0, 159.0, 172.2; m/z (ESI positive) 473.16 [M+H]+.
N
ꢀ(5ꢀ(
yl)phenyl)acetamide (8q) was obtained according to the previously reported general procedure, by
using 4f and 7b, in 77 % yield; δH (400 MHz, DMSOꢀ 6) 1.06 (2H, m), 1.32 (2H, m), 2.48 (3H, s,
3), 3.71 (3H, s, NꢀC 3), 4.20 (2H, s, C 2), 4.22 (1H, m), 7.32 (1H, m, ArꢀH), 7.63 (2H, d,
), 7.74 (1H, s, exchange with D2O, SO2NH-), 7.85 (2H, appt, 8.8 , Arꢀ ), 8.00 (1H, d,
), 8.10 (2H, d, 8.4, Arꢀ ), 8.66 (1H, d, 8.8, Arꢀ ); δC (100 MHz, DMSOꢀ
Nꢀcyclopropylsulfamoyl)ꢀ4ꢀmethylthiazolꢀ2ꢀyl)ꢀNꢀmethylꢀ2ꢀ(4ꢀ(pyridinꢀ2
d
C
H
H
H
J
J
8.4,
8.8,
Arꢀ
H
H
J
H
Arꢀ
J
H
J
H
d6) 14.2, 16.7,
20.0, 31.2, 38.0, 110.2, 121.0, 123.2, 126.4, 130.1, 134.2, 137.2, 138.0, 148.4, 150.0, 155.7, 159.0,
172.0; m/z (ESI positive) 443.11 [M+H]+.
2ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ
methylacetamide (8r) was obtained according to the previously reported general procedure, by
using 4g and 7a, in 88 % yield; m.p.170 °C; δH (400 MHz, DMSOꢀ 6) 2.21 (3H, s, C 3), 3.00 (2H,
dd, 6.4, 6.6, SO2NHꢀCH2-), 3.43 (2H, m, ꢀCH2-OH), 3.82 (s, NꢀC 3), 4.10 (2H, s, C 2), 4.45 (1H,
t, ), 7.41 (1H, appt, 6.84, Arꢀ ), 7.49
(2H, d, 8.4, Arꢀ ), 7.73 (1H, s, exchange with
D2O, SO2NH-), 7.75 (2H, d, 6) 16.2, 30.1, 38.4, 45.6, 59.9,
Nꢀ(5ꢀ(Nꢀ(2ꢀhydroxyethyl)sulfamoyl)ꢀ4ꢀmethylthiazolꢀ2ꢀyl)ꢀNꢀ
d
H
J
H
H
J
6.4, exchange with D2O, O
8.4; Arꢀ ), 7.63 (2H, d,
8.4, Arꢀ
H
), 7.38 (2H, d,
J
8.4; Arꢀ
H
J
H
J
H
J
8.4, Arꢀ ), 7.72 (2H, d,
H
J
H
J
H); δC (100 MHz, DMSOꢀ
d
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Journal of Medicinal Chemistry
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3
110.2, 127.6, 128.0, 128.2, 129.0, 130.0, 134.5, 140.0, 140.9, 148.1, 159.8, 172.2; m/z (ESI
4
5
6
positive) 446.11 [M+H]+.
7
8
9
N
ꢀ(4ꢀEthylꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)ꢀ
N
ꢀmethylꢀ2ꢀ(4ꢀ(pyridinꢀ2ꢀyl)phenyl)acetamide
(
8s
)
was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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obtained according to the previously reported general procedure, by using 4h and 7b, in 82 % yield;
δH (400 MHz, DMSOꢀ
NꢀC 3), 4.22 (2H, s, C
with D2O, SO2N
), 8.66 (1H, d,
d
6) 1.35 (3H, d,
J
6.4, CH2ꢀC
H
3), 3.00 (2H, q,
8.4, Arꢀ
8.8, Arꢀ
J
6.4, C
), 7.66 (2H, s, exchange
), 8.10 (2H, d, 8.4, Arꢀ
H2ꢀCH3), 3.71 (3H, s,
H
H
2), 7.32 (1H, m, ArꢀH), 7.63 (2H, d,
J
H
H
2), 7.85 (2H, appt,
J
8.8 , Arꢀ
H
), 8.00 (1H, d,
J
H
J
H
J
8.8, ArꢀH); δC (100 MHz, DMSOꢀd6) 14.2, 19.9, 34.7, 42.0, 120.6, 123.0, 127.0,
128.7, 130.6, 135.4, 137.7, 137.8, 148.6, 150.0, 156.2, 159.1, 172.0; m/z (ESI positive) 417.10
[M+H]+.
N
ꢀ(4ꢀ(hydroxymethyl)ꢀ5ꢀsulfamoylthiazolꢀ2ꢀyl)ꢀ
N
ꢀmethylꢀ2ꢀ(4ꢀ(pyridinꢀ2ꢀyl)phenyl)acetamide (8t
and 7b, in 64 %
2), 4.60 (2H, d, 6.2,
8.4, Arꢀ ), 7.66 (2H, s,
8.8, Arꢀ ), 8.10 (2H, d,
)
was obtained according to the previously reported general procedure, by using
6
yield; δH (400 MHz, DMSOꢀ
2OH), 4.78 (1H, t, 6.2, O
exchange with D2O, SO2N 2), 7.85 (2H, appt,
8.4, Arꢀ ), 8.66 (1H, d, 8.8, Arꢀ
d
6) 3.72 (3H, s, NꢀC
H
3), 4.20 (2H, s, C
H
J
C
H
J
H), 7.32 (1H, m, ArꢀH), 7.63 (2H, d,
J
H
H
J
8.8 , Arꢀ
H
), 8.00 (1H, d,
J
H
J
H
J
H
); δC (100 MHz, DMSOꢀ
d
6) 14.2, 19.9, 34.7, 42.0, 120.6,
123.0, 127.0, 128.7, 130.6, 135.4, 137.7, 137.8, 148.6, 150.0, 156.2, 159.1, 172.0; m/z (ESI
positive) 417.10 [M+H]+.
CA inhibition
An Applied Photophysics stoppedꢀflow instrument has been used for assaying the CA catalyzed
CO2 hydration activity.25 Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM
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Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CAꢀcatalyzed
CO2 hydration reaction for a period of 10ꢀ100 s. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least
six traces of the initial 5ꢀ10% of the reaction have been used for determining the initial velocity.
The uncatalyzed rates were determined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilledꢀdeionized water and dilutions
up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay, in order to allow for the
formation of the EꢀI complex. The inhibition constants were obtained by nonꢀlinear least squares
methods using PRISM 3 and the ChengꢀPrusoff equation, as reported earlier,26ꢀ29 and represent the
mean from at least three different determinations. All CA isofoms were recombinant ones obtained
inꢀhouse as reported earlier,26ꢀ29 and their concentrations in the assay system were in the range of
7.1 – 12.3 nM.
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Supporting Information. Supporting information is available free of charge on the ACS
Publications website: SMILES representation for compounds (CSV).
Acknowledgements. This research was financed by AiCuris Antiꢀinfective Cures GmbH. The
authors would like to gratefully acknowledge the contributions of Wolfgang Bender, Peter
Eckenberg, Rüdiger Fischer, Gabriele Handke, Martin Hendrix, Axel Jensen, and Udo Schneider to
synthesis and characterization of some of the thiazolyl sulfonamide compounds
*Corresponding Author: Fabrizio Carta (FC) Phone: +39ꢀ055ꢀ4573666; fax: +39ꢀ055ꢀ4573385;
Eꢀmail: fabrizio.carta@unifi.it; Prof. Claudiu T. Supuran (CTS) Phone: +39ꢀ055ꢀ4573729; fax:
+39ꢀ055ꢀ4573385; Eꢀmail: claudiu.supuran@unifi.it
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Nonstandard abbreviations. CA, carbonic anhydrase; CAI, CA inhibitor; KI, inhibition constant;
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