Discovery of imidazo[2,1- b ]thiazole HCV NS4B inhibitors exhibiting synergistic effect with other direct-acting antiviral agents

Article abstract of DOI:10.1021/jm501934n

The design, synthesis, and SAR studies of novel inhibitors of HCV NS4B based on the imidazo[2,1-b]thiazole scaffold were described. Optimization of potency with respect to genotype 1b resulted in the discovery of two potent leads 26f (EC₅₀ = 16

Full text of DOI:10.1021/jm501934n

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Article
Discovery of imidazo[2,1-b]thiazole HCV NS4B inhibitors exhibiting
synergistic effect with other direct-acting antiviral agents
Ning-Yu Wang, Ying Xu, Wei-Qiong Zuo, Kun-Jie Xiao, Li Liu, Xiu-Xiu Zeng, Xin-Yu You, Li-
Dan Zhang, Chao Gao, Zhi-Hao Liu, Ting-Hong Ye, Yong Xia, Ying Xiong, Xue-Jiao Song,
Qian Lei, Cui-Ting Peng, Hong Tang, Sheng-Yong Yang, Yu-Quan Wei, and Luo-Ting Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501934n • Publication Date (Web): 24 Feb 2015
Downloaded from http://pubs.acs.org on February 27, 2015
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Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
Xiong, Ying; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
Song, Xue-Jiao; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
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Lei, Qian; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
Peng, Cui-Ting; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School; Sichuan University, School of
Chemical Engineering
Tang, Hong; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
Yang, Sheng-Yong; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
Wei, Yu-Quan; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
Yu, Luo-Ting; Sichuan University, State Key Laboratory of
Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School
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Discovery of imidazo[2,1ꢀb]thiazole HCV NS4B
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inhibitors exhibiting synergistic effect with other directꢀ
acting antiviral agents
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§
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§ ‡
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NingꢀYu Wang§, †, Ying Xu , †, WeiꢀQiong Zuo , KunꢀJie Xiao , Li Liu , XiuꢀXiu Zeng ‡, XinꢀYu You
,
,
§
‡
§ ‡
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§
,
,
, LiꢀDan Zhang , Chao Gao , ZhiꢀHao Liu , TingꢀHong Ye , Yong Xia , Ying Xiong , XueꢀJiao
Song§, Qian Lei§, CuiꢀTing Peng§ ‡, Hong Tang§, ShengꢀYong Yang§, YuꢀQuan Wei§, and LuoꢀTing
,
§
Yu*
§State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
‡ Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan
University, Chengdu, Sichuan 610065, China.
†These authors contributed equally.
Corresponding Author: *To whom correspondence should be addressed.
L.T.Y.: Tel: +86 8550 3817; Fax: +86 8550 3817; Eꢀmail: yuluot@scu.edu.cn.
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ABSTRACT
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The design, synthesis and SARs studies of novel inhibitors of HCV NS4B which based on
imidazo[2,1ꢀb]thiazole scaffold were described. Optimization of potency with respect to genotype 1b
resulted in the discovery of two potent leads 26f (EC50=16 nM) and 28g (EC50= 31 nM). The resistance
profile studies revealed that 26f and 28g targeted at HCV NS4B, more precisely the second amphipathic
α helix of NS4B (4BAH2). Crossꢀresistance between our 4BAH2 inhibitors and other directꢀacting
antiviral agents targeting at NS3/4A, NS5A and NS5B was not observed. For the first time, the
synergism of a series of combinations based on 4BAH2 inhibitor was evaluated. The results
demonstrated that our 4BAH2 inhibitor 26f was synergistic with NS3/4A inhibitor Simeprevir, NS5A
inhibitor Daclatasvir and NS5B inhibitor Sofosbuvir, and it could also reduce the dose of these drugs at
almost all effect levels. Our study suggested that favorable effects could be achieved by combination
4BAH2 inhibitors like 26f with these approved drugs and new allꢀoral antiviral combinations based on
4BAH2 inhibitors were worth developing to supplement or even replace current treatment regiments for
curing HCV infection.
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Introduction
Approximately 150 million people worldwide are tortured by chronic Hepatitis C (CHC) and its
complications, which place tremendous economic burden on both the HCVꢀinfected subjects and the
healthcare system.1, 2 Despite considerable effort on the understanding of Hepatitis C, there is still no
preventive vaccine available to date.3 Current standard of care (SoC) for HCVꢀinfected subjects
involves a combination of pegylated interferon alpha (PEG IFNꢀα), ribavirin (RBV) and one of the five
approved directꢀacting antivirals (DAAs), including Boceprevir,4 Telaprevir,5 Simeprevir,6 Sofosbuvir7
and Daclatasvir (Figure 1).8 Although these triple therapies can achieve distinct improvement in the
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cure rate compared with previous SoC just consisting of PEG IFNꢀα and RBV, the adverse events
associated with interferon and RBV cannot be eliminated, and the treatment durations of these new
regiments are still lengthy.9,10 There is still a long way to go to achieve the final destination of
eradication of hepatitis C virus globally.
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The high mutation rate of HCV provides it the ability to generate drug resistance to almost all DAAs
after a shortꢀterm treatment when a single drug is used exclusively, which makes interferon
indispensable for traditional treatments. Interferonꢀsparing combination regimen, which comprises two
or more DAAs, could effectively decrease the generation of resistant mutation and achieve high
sustained virologic response (SVR) rates, especially when the combination treatments contain an
antiviral agent with high resistance barrier such as nucleoside NS5B polymerase inhibitors or the
secondꢀgeneration NS3/4A protease inhibitors.11 Such allꢀoral treatments (AOTs) have open a new era
for HCV treatment as the approval of Gilead's twoꢀagent combo Harvoni12 and AbbVie's fourꢀagent
combo Viekira Pak13. Both of Harvoni and Viekira Pak can achieve SVR in 95%+ of difficultꢀtoꢀtreat
genotype 1ꢀinfected subjects with duration as short as 8ꢀ12 weeks while avoid of the uncomfortable
adverse events produced by IFN. These impressive results have spurred us to develop more novel and
safe DAAs to supplement current treatment and further shorten the treatment duration to less than 8
weeks.
The HCV genome encodes a polyprotein precursor of about 3010 amino acids, which is processed
to yield mature structural (Core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A,
and NS5B) proteins in the host cells. Almost all current clinical candidates and approved DAA drugs
addressing HCV infection target at one of the three nonstructural proteins, including the NS3/4A
protease, NS5A, and the RNAꢀdependent RNA polymerase NS5B. Compared to these nonstructural
proteins, the nonstructural protein NS4B is a less well characterized 27 kDa protein which plays several
essential roles in HCV replication.14 And its inhibitors were not well developed or reported until
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recently. NS4B could specifically recognize and bind to the 3’ terminus of the negative viral strand, and
the RNA binding function could be blocked by clemizole (1a), an H1 histamine receptor antagonist.15
This compound, which was the first reported NS4B inhibitor, was found to substantially inhibit HCV
replication and be highly synergistic with HCV protease inhibitors in vitro.16 However, the clinical
efficiency of clemizole is unavailable at present yet. The second amphipathic α helix of NS4B (4BAH2)
could induce vesicle aggregation which is essential for the membranous web formation.17 It is reported
recently that small molecule like anguizole (1b) that disrupts the membrane association of 4BAH2 could
alter the subcellular distribution pattern of the NS4B protein and thus inhibits the replication of HCV.18
In addition to anguizole, a series of compounds with various chemotypes have been reported to target at
NS4B.19ꢀ24 Because all of these inhibitors shared similar key resistant mutants with anguizole,
specifically H94N/R and V105L/M (genotype 1b),18 to discriminate these NS4B inhibitors from RNA
binding inhibitors like Clemizole, we thus termed this kind of NS4B inhibitors as 4BAH2 inhibitor in
this study. Some of these 4BAH2 inhibitors exhibit high potency against genotype 1 in vitro and in
PXB® mice,20 the clinical efficiency of these compounds is yet to be verified. More importantly,
expected as a part of future oral drug cocktail, the combination effects of this kind of NS4B (4BAH2)
inhibitor with other DAAs are also required to be evaluated to provide us the feasible evidence of
developing new allꢀoral antiꢀHCV therapies based on these 4BAH2 inhibitor.
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Our research group was actively seeking new antiꢀHCV agents.25 Herein we describe our efforts to
develop novel 4BAH2 inhibitor candidates. A series of imidazo[2,1ꢀb]thiazole derivatives were
designed and synthesized based on the imidazo[1,2ꢀa]pyridine NS4B inhibitors (1c) reported by
GlaxoSmithKline.22 Extensive structure−activity relationship (SAR) studies were performed leading to
the discovery of several nanomolar HCV inhibitors and the resistance profile as well as crossꢀresistance
of the most potent compounds 26f and 28g were then evaluated. Finally, the most potent compound 26f
was further selected to evaluate the synergism of drugꢀdrug interaction as potential combination partner
with other DAAs, including the NS3/4A inhibitor Simeprevir, NS5A inhibitor Daclatasvir, NS5B
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inhibitor Sofosbuvir as well as the NS4B RNA binding inhibitor Clemizole, which provide the first
evidence of the feasibility of developing new allꢀoral antiꢀHCV therapies based on 4BAH2 inhibitors.
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Chemistry
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The target compounds 26aꢀ26k, 27a-27f, 28a-28g for this study were prepared by coupling two crucial
building blocks, the carboxylic acid segment A and the amine segment B, with EDCI and HOBt in
dichloromethane (Scheme 1). Unless commercially available, the preparation of the important
intermediates 2ꢀaminothiazoles for the synthesis of carboxylic acid segment A are illustrated in Scheme
2. Reacting 1ꢀcyclopropylethanone 2 with bromine in methanol furnished αꢀbromoketone 3, which was
cyclized with thiocarbamide in ethanol to afford 4ꢀcyclopropylthiazolꢀ2ꢀamine 8f. Starting from the
common substrate 4ꢀmethylpentanꢀ2ꢀone, compound 8d and 8g were prepared after two collective steps
with a ratio of 1:7 due to lack of chemoselectivity for bromination. For the synthesis of 5ꢀ
isopropylthiazolꢀ2ꢀamine 8c, bromination and cyclization were completed one pot due to the instability
of 2ꢀbromoꢀ3ꢀmethylbutyraldehyde under room temperature.26 Cyclization of various 2ꢀaminothiazoles
8a-g with ethyl bromopyruvate followed by chlorination of the resultant intermediates 9a-g furnished
imidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylic acid esters 10a-i, which went through LiOHꢀmediated ester
hydrolysis to produce carboxylic acids 11a-i (Scheme 3).
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The tricyclic carboxylic acids were prepared from cyclic ketone or commercially available 2ꢀ
benzothiazolamines through the sequence depicted in Scheme 4. Bromination of cyclopentanone 12a
through a literature procedure27 followed by cyclization of the resultant 2ꢀbromocyclopentanone
produced 5,6ꢀdihydroꢀ4Hꢀcyclopenta[d]thiazolꢀ2ꢀamine 14a, The corresponding tricyclic carboxylic
acid 16a was then obtained from 14a following our standard cyclizationꢀchlorinationꢀhydrolysis
sequence. This method could also be applied to the synthesis of tricyclic carboxylic acids 16b-f
(Experimental Section). Commercially available 2ꢀbenzothiazolamines 17a-e proceeded standard
cyclizationꢀchlorinationꢀhydrolysis steps to afford corresponding tricyclic carboxylic acids 18a-e.
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The amine building block B was synthesized as outlined in Scheme 5. Treatment glycine with 4ꢀ
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nitrophenylsulfonamido)acetic acid 22, which was condensed with various amines and cyclized with
1,2ꢀdibromoethane to give the piperazinone intermediates 24a-c.28 Subsequently deprotection of 24a-c
by thiols like methyl 3ꢀmercaptopropionate produced corresponding amines 25a-c.
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Results and Discussion
Potency of the compounds described in this study were determined using GT 1b subgenomic replicon
assay.29 Starting from compound 26a, we first evaluated a number of simple substituents on the thiazole
ring, and the results were summarized in Table 1. The size of the substituent has a substantial impact on
replicon activity. Compound 26a without substituent on the thiazole ring (EC50>10 ꢁM) exhibited no
inhibitory activity against Gtꢀ1b replicon and a small methyl substituted at C2ꢀ (26b) or C3ꢀposition
(26e) just provided weak inhibitory activity, whereas a much bulkier group like isopropyl (26c,
EC50=1.32 ꢁM) or cyclopropyl (26f, EC50=0.016 ꢁM) provided significant increase in replicon activity.
However, the introduction of isobutyl at C3ꢀposition (26g, EC50=0.74 ꢁM) led to a 46ꢀfold decrease in
potency compared with its cyclopropyl counterpart 26f, suggesting a bulky substituent over 4 carbon
atoms at C3ꢀposition was unfavourable for the potency. Compound with two substituents on the thiazole
ring (26d) was less active than its single substituent counterpart (26c), which can be further confirmed
by comparing the potency of 26f and 26g with their C2ꢀposition chloroꢀsubstituted counterparts 26h
(EC50=8.6 ꢁM) and 26i (EC50=6.7 ꢁM). As the best substituent on thiazole scaffold was found to be
cyclopropyl at C3ꢀposition, variants of R3 in the context of 3ꢀcyclopropylimidazo[2,1ꢀb]thiazole were
subsequently explored. Unfortunately, when cyclopentyl was replaced by hydrophilic transꢀ4ꢀ
hydroxycyclohexyl in 26j (EC50=0.89 ꢁΜ), a substantial loss of potency was observed, and attempt to
increase potency by introduction of unsaturated fiveꢀmembered ring (26k, EC50=0.079 ꢁΜ) was also
unsuccessful.
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Our initial effort have revealed that the imidazo[2,1ꢀb]thiazole core could be a favorable scaffold for
HCV inhibitor, and a single substituent at C3ꢀposition of thiazole ring with a size of 3 carbon atoms was
optimal. We next questioned if a ring fused to the imidazo[2,1ꢀb]thiazole scaffold was tolerant. A series
of tricyclic analogues were thus synthesized and evaluated. As shown in Table 2, the introduction of
various saturated rings fused to the imidazo[2,1ꢀb]thiazole resulted in a series of less potent HCV
inhibitors and the potency of the analogues declined as the size of the fused rings increasing (27a,
EC50=0.13 ꢁM, 27b, EC50=0.19 ꢁM, 27c, EC50=0.94 ꢁM), consistant with the SAR observed in the
bicyclic series. Among the sixꢀmembered fused ring analogues, compound without substituent on the
fused ring (27b) proved to be the most potent. The introduction of a single methyl provided a slightly
decrease in potency, while the gemꢀdimethyl variant showed a 6ꢀfold loss of potency. The replacement
of the hydrophobic cyclohexane ring in 27b with a relative hydrophilic cycloketone ring resulted in an
inactive analogue (27f), suggesting hydrophobic interactions between this region and the target existed.
A series of rigid benzo[d]imidazo[2,1ꢀb]thiazole derivatives were thus synthesized. When the saturated
rings fused on the imidazo[2,1ꢀb]thiazole scaffold were replaced by a benzene ring in 28a (EC50=0.092
ꢁM), a slight improvement in potency was observed, whereas the introduction of substituents into
benzene rings (28b-28e) were detrimental to the potency, regardless of their electronic and lipophilic
effects, which might attribute to their size exceeding the optimal range, consistent with the trends in
27a-f. The influence of R3 on the potency was also investigated. The replacement of cyclopentyl in 28a
by transꢀ4ꢀhydroxycyclohexyl in 28f (EC50=0.33 ꢁM) led to a 3ꢀfold decrease in potency, while the
introduction of unsaturation to the fiveꢀmembered ring (28g, EC50=0.031 ꢁM) provided 3ꢀfold increase
in potency. 28g combined with 26f outstanding from the above series of imidazo[2,1ꢀb]thiazole
derivatives were selected for further biologic evaluation.
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In order to verify that the synthesized imidazo[2,1ꢀb]thiazole HCV inhibitors indeed target at
NS4B, sideꢀbyꢀside transient transfection GT 1b assays with wild type (WT) and a panel of
representative resistant replicons of NS4B were performed on 26f and 28g, with the results shown in
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Table 3. Three of the five strains bearing NS4B mutations (H94R, F98C, V105M),18,22 which have
emerged as a hallmark of many 4BAH2 inhibitors such as anguizole and 1c, showed significant
resistance to 26f and 28g. Unexpectedly, the other two strains bearing NS4B mutations (H3R and
Q26R) mapped to Nꢀterminal region of NS4B, which were reported resistant to Bristol Myers Squibb’s
undisclosed NS4B inhibitor,14 showed resistance to 26f and 28g as well. To preclude the possibility that
our specific imidazo[2,1ꢀb]thiazole scaffold endow their resistance to these resistant strains, the
imidazo[1,2ꢀa]pyridine 4BAH2 inhibitor 1c was also evaluated for its potency against these two
resistant strains. The results in Table 3 showed that 1c exhibited significant shift in potency on both
strains. Although other 4BAH2 inhibitors were not evaluated in this assay, our study proved that Nꢀ
terminal region of NS4B was also important for the potency of some 4BAH2 inhibitor. The observation
of cross resistance between our imidazo[2,1ꢀb]thiazole HCV inhibitors and the reported 4BAH2
inhibitor 1c had confirmed that the imidazo[2,1ꢀb]thiazole derivatives in our study were NS4B
inhibitors, more precisely 4BAH2 inhibitors.
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We further investigated whether there was crossꢀresistance between our 4BAH2 inhibitors and
other DAAs, including NS3/4A inhibitors, NS5A inhibitors, nucleoside and nonꢀnucleoside NS5B
inhibitors. The potency of 26f and 28g against a panel of HCV replicons containing representative
mutations resistant to NS3/4A inhibitors (A156T),30 NS5A inhibitors (Y93H),31,32 nucleoside (S282T)33
and nonꢀnucleoside (M423I, P495A, Y448H)34 NS5B inhibitors were tested, and all of these replicons
were equally sensitive to 26f and 28g compared with the wild type 1b replicon (Table 3), suggesting
that there was no crossꢀresistance between our 4BAH2 inhibitors and other DAAs. It also implied the
potential of the combination of our 4BAH2 inhibitors with other DAAs to develop novel antiviral
therapies.
Combination therapy is indispensable for chronic hepatitis C individual, and it has become
increasingly clear that traditional interferonꢀbased SoC will soon be replaced by allꢀoral therapies.
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Previous studies have developed a series of allꢀoral antiꢀHCV regimens and the efficiency of which
have been verified in clinic.35ꢀ39 However, to our knowledge there is no report addressed on the
possibility of developing novel allꢀoral antiꢀHCV regimens based on 4BAH2 inhibitor currently, even if
in vitro. To investigate the drugꢀdrug interaction between our 4BAH2 inhibitors and other DAAs, we
next assessed the additive, antagonistic, or synergistic properties of combination therapies based on our
4BAH2 inhibitor 26f in the HCV subgenomic GT 1b replicon system.
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The Combination Index (CI) is considered to be the golden standard to define the synergism of
drugꢀdrug interaction.40ꢀ42 CI values=1 always represents additive effect, while CI values <1 and >1
mean synergistic and antagonistic interactions, respectively. Thus, a low CI value stands for strong
synergism, and vice versa. The DoseꢀReduction Index (DRI) is another important parameter to assess
the synergism of drugꢀdrug interaction. It is a measurement of how many fold the dosage of each drug
in a combination may be reduced at a given effect level compared with the dosage of each drug used
alone.41 DRI<1 indicates unfavorable doseꢀreduction, whereas DRI>1 indicates favorable doseꢀ
reduction, which is expected in drug combination therapies. Three representative marketed DAAs, the
NS3/4A inhibitor Simeprevir, the NS5A inhibitor Daclatasvir, the NS5B inhibitor Sofosbuvir as well as
the NS4B RNA binding inhibitor Clemizole were selected to combine with 26f to evaluate the CI and
DRI in a broad range of concentrations.
The combination of 26f and Simeprevir resulted in greater inhibition than either compound alone at
almost all tested concentrations (Table S2), while no obvious cytotoxicity was observed at all these
concentrations. Synergistic interactions were observed in a broad range of concentrations, and the
synergistic interactions strengthened gradually as the concentration of 26f increasing (Figure 3a), while
no obvious trend was observed as the concentration of Simeprevir increasing. Similar synergistic
interactions and safety profiles were also observed in the Daclatasvirꢀ26f and Sofosbuvirꢀ26f
combinations, In both assays the synergistic interactions were reinforced as the concentration of 26f
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increasing (Figure 3b, c). Because synergism at high effect levels is much more therapeutically relevant
than at low effect levels for infectious diseases like hepatitis C,41 our study had demonstrated the
advantages of combination therapies based on 4BAH2 inhibitors like 26f, especially when it combined
with an inhibitor with high resistance barrier such as Sofosbuvir.
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Recent study in cancer therapy have demonstrated that two drugs that hit the same biological target
through different mechanism of actions can potently synergize to give enhanced anticancer effect.43 We
hypothesized that this combination effect could be applied to antiviral therapy. The potency and
cytotoxicity of our 4BAH2 inhibitor 26f in combination with Clemizole, a NS4B RNA binding inhibitor,
were thus tested. The combination of 26f and Clemizole resulted in additive to synergistic effects at low
concentration combinations, while antagonistic interactions were observed at high concentrations of
either compound (Figure 3d). Reasonable explanation may be that the NS4B is a membrane protein,
and small molecular NS4B inhibitor like Clemizole or 26f could disrupt the normal transmembrane
structure and thus weaken the potency of another NS4B inhibitor, which in turn resulted in the
antagonistic effects at high concentrations of either compound. Moreover, cytotoxicity was also
observed under high concentrations of Clemizole, consistant with previous study which reported this
compound had a narrow safety window for HCV therapy.16 Anyway, our study had shown that two
NS4B inhibitors with different mechanism of actions could, at least at low concentration combinations,
synergistically inhibit the replication of HCV.
The DRIꢀeffect (DRIꢀFa) Plot (Figure 4) for above combination therapies showed that 26f could
reduce the dose of the other drugs from 1 to 7 ꢀfold in almost all Fa range, including the NS4B RNA
binding inhibitors Clemizole. And all of the four drugs could also reduce the dose of 26f in almost all Fa
range. This result further supported the advantages of combination 4BAH2 inhibitor 26f with other
DAAs for HCV therapy.
Over all, the combination therapy studies have provided us the first proof that 4BAH2 inhibitors
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such as 26f could combine with other DAAs in prevent HCV infection, which are worth developing to
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Conclusion
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In summary, we described the design, synthesis, SAR studies of a novel class of NS4B (4BAH2)
inhibitors based on imidazo[2,1ꢀb]thiazole scaffold. Extensive SAR studies were performed at C2ꢀ and
C3ꢀpositions on imidazo[2,1ꢀb]thiazole core to discover that a single substituent on the thiazole ring
with a size of 3 carbon atoms (26f) was optimal. A series of tricyclic analogues which introduction of a
saturated or an unsaturated ring fused to the imidazo[2,1ꢀb]thiazole scaffold were also synthesized and
evaluated to uncover that a simple benzene ring fused to the imidazo[2,1ꢀb]thiazole scaffold (28g) was
optimal. 26f (EC50=16 nM) and 28g (EC50=31 nM) outstanding as their good potency against HCV
genotype 1b replicon, were selected for further resistance profile and combination therapy studies.
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The resistance profile studies confirmed that 26f and 28g targeted at NS4B. And there was no
crossꢀresistance between our 4BAH2 inhibitors and other DAAs targeting at NS3/4A, NS5A and NS5B.
For the first time, the synergism of a series of combinations based on our 4BAH2 inhibitor 26f was
evaluated to discover that 26f could be synergistic with NS3/4A inhibitor Simeprevir, NS5A inhibitor
Daclatasvir and NS5B inhibitor Sofosbuvir to inhibit the replication of GT 1b replicon, and it could also
reduce the dose of these drugs in almost all Fa range, suggesting favorable effects could be achieved by
combination 4BAH2 inhibitors like 26f with these approved drugs and new allꢀoral antiꢀHCV
combinations based on 4BAH2 inhibitors are worth developing to supplement or even supplant current
treatment strategies.
Combination 26f with NS4B RNA binding inhibitor Clemizole aimed at determining whether two
drugs that hit the same biological target through different mechanism of actions could potently
synergize to inhibit viral replication revealed that 26f could be additive/synergistic with Clemizole at
low concentration combinations. Although no favorable effect was observed, our study implied that two
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drugs acting on the same biological target through different mechanism of actions might potently
synergize to inhibit the replication of HCV or other viruses.
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Experimental Section
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Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used
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without further purification. The H and C NMR spectra were recorded on a Bruker AVANCEш 400
spectrometer (Bruker Company, Germany) at 25 ℃ using DMSOꢀd6 or CDCl3 as the solvent. Chemical
shifts (δ) were reported in ppm relative to Me4Si (internal standard), and coupling constants (J) were
reported in Hz, and peak multiplicity are reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br s (broad singlet). Mass Spectra (MS) were performed on a Waters QꢀTOF Premier
mass spectrometer (Micromass, Manchester, UK). Thin layer chromatography (TLC) was performed on
0.20 mm Silica Gel Fꢀ254 plates (Qingdao Haiyang Chemical, China), and column chromatography
were performed using Silica gel 60 of 300ꢀ400 mesh (Qingdao Haiyang Chemical, China). The purity of
all the title compounds was determined on an UltiMate 3000 (Dionex, USA) HPLC system, and were
of >95% purity. HPLC conditions to assess the purity of the final compounds were as follows: column,
Atlantis dC18, 4.6 mm × 150 mm, 5 ꢁm; mobile phase, methanol(55%)/water(45%) or
methanol(65%)/water(35%); flow rate, 1.0 mL/min; UV wavelength, 190 − 400 nm; temperature, 35 ℃;
injection volume, 10 ꢁ L.
5ꢀIsopropylthiazolꢀ2ꢀamine (8c).26 A solution of bromine (1.0 mL, 20 mmol) in dichloromethane/
dioxane (V/V = 4/1, 12 mL) was slowly added to a cooled solution of isovaleraldehyde (1.721 g, 20
mmol) in dichloromethane/dioxane (V/V = 4/1, 36 mL). The mixture was stirred at 10 ℃ for 2 h and
was slowly added to a suspension of thiourea (1.523 g, 20 mmol) in dichloromethane/dioxane (V/V =
4/1, 30 mL). Ethanol (6 mL) and triethylamine (2.424 g, 24 mmol) were added to the reaction mixture
with vigorous stirring for 20 h at room temperature. The resulting mixture was diluted with water (100
mL), alkalized to pH = 12 with 12 N aqueous NaOH and stirred at room temperature for another 1 h.
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Then it was extracted with dichloromethane, and the organic layer was washed with brine, dried over
anhydrous MgSO4, and concentrated under vacuum to dryness. The residue was purified by column
chromatography with ethyl acetate/ petroleum ether to afford 5ꢀisopropylthiazolꢀ2ꢀamine 8c (1.202 g,
42.26% yeild) as a white solid. 1H NMR (400 MHz, CDCl3) δ 6.73 (s, 1H), 4.84 (s, 2H), 2.99 (d, J = 6.8
Hz, 1H), 1.25 (t, J = 8.3 Hz, 6H).
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4ꢀIsobutylthiazolꢀ2ꢀamine (8g) and 4ꢀisopropylꢀ5ꢀmethylthiazolꢀ2ꢀamine (8d). To a solution of 4ꢀ
methylpentanꢀ2ꢀone (2.003 g, 20 mmol) in methanol at 0 ℃ was slowly added bromine (1.0 mL, 20
mmol) dropwise. The mixture was stirred at room temperature for 2 h, diluted with water and stirred for
another 30 min. Then the mixture was partitioned between Et2O/water and the organic layer was washed
with brine, dried over anhydrous MgSO4, and concentrated under vacuum to afford a mixture of 3ꢀ
bromoꢀ4ꢀmethylpentanꢀ2ꢀone (5) and 1ꢀbromoꢀ4ꢀmethylpentanꢀ2ꢀone (6), which was used in the next
step without further purification.
The mixture of 5 and 6 from above step and thiourea (1.523 g, 20 mmol) were dissolved in ethanol
and heated under reflux for 6 h. Then the reaction mixture was cooled to room temperature and
concentrated under vacuum to dryness. The residue was dissolved in water, alkalized to pH = 12, and
extracted with ethyl acetate, The organic layer was washed with brine, dried over anhydrous MgSO4,
and concentrated under vacuum. The crude intermediate was purified by column chromatography with
ethyl acetate/ petroleum ether to afford 8d and 8g. 4ꢀIsopropylꢀ5ꢀmethylthiazolꢀ2ꢀamine (8d), 0.384 g,
12.29% yeild. 1H NMR (400 MHz, CDCl3) δ 4.68 (br s, 2H), 3.07 (dt, J = 13.7, 6.8 Hz, 1H), 2.13 (s,
1
3H), 1.20 (d, J = 6.8 Hz, 6H). 4ꢀIsobutylthiazolꢀ2ꢀamine (8g), 2.563g, 82.02% yeild. H NMR (400
MHz, CDCl3) δ 6.07 (br s, 1H), 5.07 (s, 2H), 2.37 (d, J = 7.1 Hz, 2H), 1.98 (dd, J = 13.4, 6.7 Hz, 1H),
0.91 (d, J = 6.6 Hz, 6H).
4ꢀCyclopropylthiazolꢀ2ꢀamine (8f). Compound 8f was prepared from 1ꢀcyclopropylethanone (1.681 g,
20 mmol) by following the procedures described for the preparation of 8d and 8g. 1.900 g, 67.86% yield
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1
as a white powder. H NMR (400 MHz, DMSOꢀd6) δ 6.63 (br s, 2H), 5.99 (s, 1H), 1.78ꢀ1.69 (m, 1H),
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0.80ꢀ0.69 (m, 4H).
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Ethyl imidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9a). To a solution of 2ꢀamino thiazole (0.500 g, 5 mmol)
in tetrahydrofuran was added ethyl bromopyruvate (90%, 1.625 g, 7.5 mmol) dropwise and the reaction
was stirred at room temperature for 8 h. Then the precipitate was filtered and washed with THF. The
filter cake was then dissolved in ethanol and heated under reflux for 8 h. Then the resulting reaction
mixture was cooled to room temperature and concentrated under vacuum to dryness. The residue was
diluted with water and the precipitate was filtered, washed with water, and dried in vacuum to afford
intermediate 9a (0.375 g, 38.27% yield) as a white solid. 1H NMR (400 MHz, DMSOꢀd6) δ 8.42 (s, 1H),
7.95 (d, J = 4.5 Hz, 1H), 7.45 (d, J = 4.5 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).
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General Procedures of Method A for the Synthesis of 9b-9g. The intermediate 2ꢀaminothiazoles
(8b-8g) (1.0 equiv) and ethyl bromopyruvate (1.0 equiv) was dissolved in butanone and heated under
reflux for 12 h, then another portion of ethyl bromopyruvate (1.0 equiv) was added to the reaction
mixture and it was heated under reflux for another 12 h. The resulting reaction mixture was cooled to
room temperature and concentrated under vacuum to dryness. The residue was extracted with ethyl
acetate and water, and the organic layer was washed with brine, dried over anhydrous MgSO4, and
concentrated under vacuum. The crude intermediate was purified by column chromatography with
dichloromethane/methanol to afford 9bꢀ9g.
Ethyl 2ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9b), 16.86% yield, 1H NMR (400 MHz, CDCl3)
δ 8.13 (s, 1H), 7.51 (s, 1H), 4.23 (t, J = 8.0 Hz, 2H), 2.44 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H).
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Ethyl 2ꢀisopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9c), 30.04% yield, H NMR (400 MHz,
DMSOꢀd6) δ 8.28 (s, 1H), 7.72 (s, 1H), 4.25 (d, J = 7.1 Hz, 2H), 3.18ꢀ3.12 (m, 1H), 1.30ꢀ1.25 (m, 9H).
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Ethyl 2ꢀisopropylꢀ3ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9d), 22.09% yield, H NMR (400
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MHz, DMSOꢀd6) δ 8.01 (s, 1H), 4.22 (q, J = 7.0 Hz, 2H), 3.19ꢀ3.14 (m, 1H), 1.30ꢀ1.24 (m, 9H).
MS(ESI) m/z: 251.0 [MꢀH]ꢀ.
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Ethyl 3ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9e), 41.33% yield, 1H NMR (400 MHz, CDCl3)
δ 7.99 (s, 1H), 6.83 (s, 1H), 4.39 (q, J = 7.1 Hz, 2H), 2.48 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). MS(ESI)
m/z: 211.0 [M+H]+.
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Ethyl 3ꢀcyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9f), 37.89% yield, H NMR (400 MHz,
CDCl3) δ 8.17 (s, 1H), 6.49 (s, 1H), 4.42 (q, J = 6.9 Hz, 2H), 2.15 (s, 1H), 1.43 (t, J = 7.0 Hz, 3H), 1.11ꢀ
1.01 (m, 2H), 0.85ꢀ0.79 (m, 2H). MS(ESI) m/z: 237.0 [M+H]+.
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Ethyl 3ꢀisobutylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (9g), 17.74% yield, H NMR (400 MHz,
CDCl3) δ 8.05 (s, 1H), 6.77 (s, 1H), 4.40 (q, J = 6.9 Hz, 2H), 2.56 (d, J = 7.1 Hz, 2H), 2.05ꢀ1.95 (m,
1H), 1.40 (t, J = 7.0 Hz, 3H), 0.93 (d, J = 6.6 Hz, 6H). MS(ESI) m/z: 253.1 [M+H]+.
General Procedures of Method B for the Synthesis of 10a ꢀ 10g. To a solution of 9a - 9g (1.0 equiv,
0.2 mol/L) in N,Nꢀdimethylformamide at room temperature was slowly added Nꢀchlorosuccinimide
(1.05 equiv), and the mixture was stirred for 3ꢀ6 h until the substrate had been completely consumed,
then the reaction mixture was diluted with water and stirred for another 30 min. The precipitate was
filtered, washed with cold water and dried in vacuum to afford intermediate 10aꢀ10g.
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Ethyl 5ꢀchloroꢀimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10a), 87.14% yield. H NMR (400 MHz,
CDCl3) δ 7.52 (d, J = 3.3 Hz, 1H), 7.21 (s, 1H), 4.46 (q, J = 7.0 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H).
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Ethyl 5ꢀchloroꢀ2ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10b), 84.45% yield. H NMR (400
MHz, CDCl3) δ 7.29 (s, 1H), 4.25 (t, J = 8.0 Hz, 2H), 2.45 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H).
Ethyl 5ꢀchloroꢀ2ꢀisopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10c), 78.01% yield. 1H NMR (400
MHz, CDCl3) δ 7.25 (s, 1H), 4.33 (d, J = 7.1 Hz, 2H), 3.24ꢀ3.17 (m, 1H), 1.45ꢀ1.35 (m, 9H).
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Ethyl 5ꢀchloroꢀ2ꢀisopropylꢀ3ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10d), 100% yield. H
NMR (400 MHz, CDCl3) δ 4.22 (q, J = 7.0 Hz, 2H), 3.18ꢀ3.12 (m, 1H), 1.33 (t, J = 7.1 Hz, 3H), 1.24 (d,
J = 13.2 Hz, 6H).
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Ethyl 5ꢀchloroꢀ3ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10e), 52.11% yield. H NMR (400
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MHz, CDCl3) δ 6.62 (s, 1H), 4.35 (q, J = 7.0 Hz, 2H), 2.59 (s, 3H), 1.36 (t, J = 7.0 Hz, 3H).
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Ethyl 5ꢀchloroꢀ3ꢀcyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10f), 77.43% yield. H NMR
(400 MHz, CDCl3) δ 6.47 (s, 1H), 4.43 (q, J = 6.9 Hz, 2H), 2.25 (s, 1H), 1.44 (t, J = 7.0 Hz, 3H), 1.13ꢀ
1.03 (m, 2H), 0.89ꢀ0.81 (m, 2H).
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Ethyl 5ꢀchloroꢀ3ꢀisobutylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10g), 58.58% yield. H NMR (400
MHz, CDCl3) δ 6.60 (s, 1H), 4.40 (q, J = 6.9 Hz, 2H), 2.81 (d, J = 7.1 Hz, 2H), 2.07ꢀ1.96 (m, 1H), 1.43
(t, J = 7.0 Hz, 3H), 0.95 (d, J = 6.6 Hz, 6H).
General Procedures for the Synthesis of 10h and 10i. To a solution of 9f or 9g (1.0 equiv, 0.2
mol/L) in DMF at room temperature was slowly added Nꢀchlorosuccinimide (1.05 equiv), and the
mixture was stirred for 3ꢀ6 h. Then another portion of Nꢀchlorosuccinimide (1.05 equiv) was added and
stirred for 12 h. The reaction mixture was then diluted with water and stirred for another 30 min. The
precipitate was filtered, washed with cold water and dried in vacuum to afford dualꢀchlorinated
intermediates 10hꢀ10i.
Ethyl 2,5ꢀdichloroꢀ3ꢀcyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10h), 69.53% yield. 1H NMR
(400 MHz, CDCl3) δ 4.45 (q, J = 7.0 Hz, 2H), 2.05 (s, 1H), 1.45 (t, J = 7.0 Hz, 3H), 1.15ꢀ1.07 (m, 2H),
0.91ꢀ0.83 (m, 2H). MS(ESI) m/z: 305.0 [M+H]+.
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Ethyl 2,5ꢀdichloroꢀ3ꢀisobutylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarboxylate (10i), 43.11% yield. H NMR
(400 MHz, CDCl3) δ 4.43 (q, J = 7.0 Hz, 2H), 2.85 (d, J = 7.0 Hz, 2H), 2.09ꢀ1.98 (m, 1H), 1.45 (t, J =
7.0 Hz, 3H), 0.97 (d, J = 6.6 Hz, 6H) MS(ESI) m/z: 320.0 [M+H]+.
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General Procedures of Method C for the Synthesis of 11aꢀ11i. 1N aqueous LiOH (5.0 equiv) was
added to a solution of 10aꢀ10i in THF/methanol (v/v=1:1, 0.1 mol/L), the reaction mixture was stirred at
room temperature for 3ꢀ12 h until the substrate had been completely consumed. Then the reaction
mixture was concentrated under vacuum and the residue was dissolved in water and acidified to pH=3
with 4N aqueous HCl. The precipitate was filtered, washed with cold water, and dried in vacuum to
afford 11aꢀ11i, which was used in the next step without further purification.
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General Procedures of Method D for the Synthesis of 14aꢀ14f.27 NBS (1.05 equiv) was added
slowly to a solution of 12a-12f (1.0 equiv, 0.2 mol/L) in DMSO at room temperature and the mixture
was stirred for 10ꢀ30 min. After completion of the reaction as monitored by TLC, the reaction mixture
was quenched with ammonium chloride solution and extracted with ethyl acetate. The organic phase
was washed with brine, dried over anhydrous MgSO4, and concentrated under vacuum to afford the αꢀ
brominated cyclic ketones 13a-13f which were used in the next step without further purification.
A solution of αꢀbrominated cyclic ketones 13a-13f and thiourea (1.523g, 20mmol) in ethanol was
heated under reflux for 3ꢀ6 h until the substrate was consumed as monitored by TLC. The reaction
mixture was cooled to room temperature and concentrated under vacuum to dryness. The residue was
dissolved in water and alkalized to pH = 12 to allow the product to deposit. The product was collected
by filtration, washed with cold water and dried under vacuum to afford the desired thiazolꢀ2ꢀamines
14a-14f.
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5,6ꢀDihydroꢀ4Hꢀcyclopenta[d]thiazolꢀ2ꢀamine (14a). H NMR (400 MHz, CDCl3) δ 4.84 (br s, 2H),
2.75 – 2.63 (m, 2H), 2.58 (dd, J = 10.1, 4.4 Hz, 2H), 2.35 – 2.23 (m, 2H).
4,5,6,7ꢀTetrahydrobenzo[d]thiazolꢀ2ꢀamine (14b), 74.68% yield, 1H NMR (400 MHz, CDCl3) δ 4.79
(br s, 2H), 2.59ꢀ2.52 (m, 4H), 1.89 – 1.66 (m, 4H).
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5,6,7,8ꢀTetrahydroꢀ4Hꢀcyclohepta[d]thiazolꢀ2ꢀamine (14c), 39.43% yield, H NMR (400 MHz,
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CDCl3) δ 4.98 (s, 2H), 2.72 – 2.63 (m, 2H), 2.63 – 2.53 (m, 2H), 1.77 (d, J = 3.7 Hz, 2H), 1.73 – 1.60
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(m, 4H).
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2ꢀAminoꢀ5,6ꢀdihydrobenzo[d]thiazolꢀ7(4H)ꢀone (14d), 23.27% yield, 1H NMR (400 MHz, DMSO) δ
8.08 (s, 2H), 2.67 (t, J = 6.1 Hz, 2H), 2.36 (t, J = 6.4 Hz, 2H), 2.03 – 1.93 (m, 2H).
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6ꢀMethylꢀ4,5,6,7ꢀtetrahydrobenzo[d]thiazolꢀ2ꢀamine (14e), 65.32% yield, H NMR (400 MHz,
CDCl3) δ 4.81 (br s, 2H), 2.75 – 2.63 (m, 2H), 2.38 – 2.27 (m, 1H), 2.08ꢀ2.01 (m, 2H), 1.69 – 1.57 (m,
2H), 1.10 (d, J = 9.2 Hz, 3H).
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6,6ꢀDimethylꢀ4,5,6,7ꢀtetrahydrobenzo[d]thiazolꢀ2ꢀamine (14f), 58.93% yield, H NMR (400 MHz,
CDCl3) δ 4.82 (br s, 2H), 2.92 (t, J = 7.6 Hz, 2H), 2.38 (s, 2H), 1.76 (t, J = 7.6 Hz, 2H), 1.02 (s, 6H).
General Procedure for the Synthesis of 15aꢀ15f. The tricyclic imidazo[2,1ꢀb]thiazole intermediates
15a-15f were prepared from 14a-14f and ethyl bromopyruvate by following the procedures described in
Method A.
Ethyl 3ꢀchloroꢀ6,7ꢀdihydroꢀ5Hꢀcyclopenta[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarboxylate (15a), 32.29%
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yield, H NMR (400 MHz, CDCl3) δ 7.94(s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 2.93 – 2.80 (m, 2H), 2.61ꢀ
2.55 (m, 2H), 2.35 – 2.28 (m, 2H), 1.40(t, J = 7.2 Hz, 3H). MS(ESI) m/z: 237.1 [M+H]+.
Ethyl 3ꢀchloroꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarboxylate (15b), 40.36% yield,
1H NMR (400 MHz, CDCl3) δ 7.91(s, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.69 (s, 2H), 2.61 (s, 2H), 1.95(s,
4H), 1.42(t, J = 7.2 Hz, 3H). MS(ESI) m/z: 251.1 [M+H]+.
Ethyl 3ꢀchloroꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀcyclohepta[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarboxylate (15c),
18.35% yield, 1H NMR (400 MHz, CDCl3) δ 7.93(s, 1H), 4.41 (q, J = 7.2 Hz, 2H), 2.75 – 2.52 (m, 4H),
1.79ꢀ1.70 (m, 2H), 1.69 – 1.58 (m, 4H), 1.43(t, J = 7.2 Hz, 3H). MS(ESI) m/z: 265.2 [M+H]+.
Ethyl 3ꢀchloroꢀ7ꢀmethylꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarboxylate (15d),
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35.38% yield, 1H NMR (400 MHz, CDCl3) δ 7.91 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.81 – 2.62 (m, 2H),
2.41 – 2.28 (m, 1H), 2.08ꢀ2.01 (m, 2H), 1.68 – 1.55 (m, 2H), 1.40 (t, J = 7.0 Hz, 3H), 1.12 (d, J = 9.2
Hz, 3H). MS(ESI) m/z: 265.1 [M+H]+.
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Ethyl 3ꢀchloroꢀ7,7ꢀdimethylꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarboxylate (15e),
37.77% yield, 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 4.42 (q, J = 7.1 Hz, 2H), 2.96 (t, J = 7.6 Hz,
2H), 2.41 (s, 2H), 1.77 (t, J = 7.6 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H), 1.07 (s, 6H). MS(ESI) m/z: 279.2
[M+H]+.
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Ethyl
3ꢀchloroꢀ8ꢀoxoꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarboxylate
(15f),
19.13% yield, 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 4.42 (d, J = 7.1 Hz, 2H), 3.03 (d, J = 6.1 Hz,
2H), 2.72 (d, J = 6.7 Hz, 2H), 2.38 (s, 2H), 1.42 (t, J = 7.0 Hz, 3H). MS(ESI) m/z: 265.1 [M+H]+.
General Procedure for the Synthesis of 16aꢀ16f. The intermediates 16a-16f were prepared from
15a-15f by following two successive steps as described in method B and Method C.
General Procedure for the Synthesis of 18aꢀ18e. The intermediates 18a-18e were prepared from
17a-17e by following three successive steps as described in method A, B and C.
Transꢀ4ꢀ((triisopropylsilyl)oxy)cyclohexanamine trifluoroacetate (20). To a solution of tertꢀbutyl
(transꢀ4ꢀhydroxycyclohexyl)carbamate (4.305 g, 20 mmol) in DMF was added triisopropylchlorosilane
(5.794 g, 30 mmol) and imidazole (3.401 g, 50 mmol) at room temperature and stirred for 12h. Then the
reaction mixture was extracted with ethyl acetate/water, the organic layer was washed successively with
distilled water and brine, dried over anhydrous MgSO4, and concentrated under vacuum to afford tertꢀ
butyl (transꢀ4ꢀ((triisopropylsilyl)oxy)cyclohexyl)carbamate (19) as a white solid (6.233 g, 83.86%),
which was used in the next step without further purification.
Compound 19 (5.574 g, 15.0 mmol) was dissolved in trifluoroacetic acid/dichloromethane (V/V =
1/4), and the mixture was stirred under room temperature for 1h. Then the solvent was removed under
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vacuum to afford 20 as a white solid. 5.945 g, 100% yield. MS(ESI) m/z: 272.2 [M+H]+.
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2ꢀ(4ꢀNitrophenylsulfonamido)acetic acid (22). To a solution of Glycine (10.0 g, 133 mmol) in 1 N
aqueous sodium hydroxide (140 mL, 140 mmol) at 0 ℃ was slowly added 4ꢀnitrobenzenesulfonyl
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chloride (29.5 g, 133 mmol). The reaction mixture was warmed to room temperature after stirring for
30min, and was alkalized to pH> 9 by 1 N sodium hydroxide solution. 2 h later, another portion of 1 N
aqueous sodium hydroxide (150 mL, 150 mmol) was added and the mixture was extracted with ethyl
acetate (100 mL × 3), the insoluble material in the aqueous phase was filtered and the filtrate was adjust
to pH=9 with 6 N aqueous HCl and stirred for 30 min. The precipitate was collected by filtration to
obtain 22 (19.73 g, 56.88 %) as a white powder. MS(ESI) m/z: 261.1 [M+H]+.
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General Procedure for the Synthesis of 23a-23c.To a solution of 22 (1.0 equiv) and various amines
(1.0 equiv) in DMF was successively added ethyl diisopropylamine (3.0 equiv) and HATU (1.5 equiv).
The mixture was stirred at room temperature for 12 h. Then water was added to the reaction mixture to
allow the product to deposit. The precipitate was collected by filtration to obtain 23a-23c.
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Nꢀcyclopentylꢀ2ꢀ(4ꢀnitrophenylsulfonamido)acetamide (23a): 73.12% yield, H NMR (400 MHz,
DMSOꢀd6) δ 8.49 (d, J = 8.8 Hz, 2H), 8.39 (s, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 6.8 Hz, 1H),
3.86 – 3.77 (m, 1H), 3.51 (s, 2H), 1.72 – 1.27 (m, 8H), MS(ESI) m/z: 350.1 [M+Na]+.
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Nꢀ(cyclopentꢀ3ꢀenꢀ1ꢀyl)ꢀ2ꢀ(4ꢀnitrophenylsulfonamido)acetamide (23b): 78.51% yield, H NMR (400
MHz, DMSOꢀd6) δ 8.52 – 8.24 (m, 4H), 8.03 (d, J = 8.4 Hz, 2H), 5.66 (s, 2H), 4.25 – 4.03 (m, 1H), 3.52
(d, J = 5.9 Hz, 2H), 2.55 – 2.45 (m, 2H), 2.01 (dd, J = 15.4, 3.5 Hz, 2H). MS(ESI) m/z: 326.1 [M+H]+.
2ꢀ(4ꢀNitrophenylsulfonamido)ꢀNꢀ(4ꢀtransꢀ((triisopropylsilyl)oxy)cyclohexyl)acetamide (23c): 53.13%
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yield, H NMR (400 MHz, DMSO) δ 8.45ꢀ8.35 (m, 3H), 8.02 (d, J = 8.9 Hz, 2H), 7.74 (d, J = 7.6 Hz,
1H), 3.50 (d, J = 5.0 Hz, 2H), 3.38ꢀ3.21 (m, 2H), 1.73 (d, J = 9.9 Hz, 2H), 1.59 (d, J = 10.3 Hz, 2H),
1.19 – 0.99 (m, 22H), MS(ESI) m/z: 514.1 [M+H]+.
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General Procedure for the Synthesis of 24a-24c.To a solution of 23a-23c (1.0 equiv) in dry DMF
was added potassium carbonate (10.0 equiv). The mixture was stirred at 60 ℃ for 30 min and 1,2ꢀ
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dibromoethane (10.0 equiv) was added. After 24h, the solvent was distilled off under vacuum and the
residue was suspended in water and stirred for another 30 min. The precipitates were collected by
filtration to obtain the crude intermediates, which were recrystallized from ethanol to get 24a-24c.
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1ꢀCyclopentylꢀ4ꢀ((4ꢀnitrophenyl)sulfonyl)piperazinꢀ2ꢀone (24a): 86.68% yield, H NMR (400 MHz,
DMSOꢀd6) δ 8.43 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 4.66ꢀ4.60 (m, 1H), 3.67 (s, 2H), 3.37ꢀ
3.26 (m, 4H), 1.59 – 1.39 (m, 8H). MS(ESI) m/z: 354.1 [M+H]+.
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1ꢀ(Cyclopentꢀ3ꢀenꢀ1ꢀyl)ꢀ4ꢀ((4ꢀnitrophenyl)sulfonyl)piperazinꢀ2ꢀone (24b): 48.71% yield, H NMR
(400 MHz, DMSOꢀd6) δ 8.44 (d, J = 8.4 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 5.77 (s, 2H), 4.25 – 4.03 (m,
1H), 3.68 (s, 2H), 3.39 (d, J = 5.0 Hz, 2H), 3.28 (d, J = 5.0 Hz, 2H), 2.75ꢀ2.65 (m, 2H), 2.06 (dd, J =
15.4, 3.7 Hz, 2H). MS(ESI) m/z: 352.1 [M+H]+.
4ꢀ((4ꢀNitrophenyl)sulfonyl)ꢀ1ꢀtransꢀ(4ꢀ((triisopropylsilyl)oxy)cyclohexyl)piperazinꢀ2ꢀone
(24c):
42.80% yield, 1H NMR (400 MHz, DMSOꢀd6) δ 8.44 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 4.14ꢀ
4.04 (m, 1H), 3.65 (s, 2H), 3.65ꢀ3.55 (m, 1H), 3.32 (d, J = 5.0 Hz, 2H), 3.26 (d, J = 5.0 Hz, 2H), 1.86 (d,
J = 11.3 Hz, 2H), 1.57 – 1.00 (m, 6H), 1.00 (s, 18H). MS(ESI) m/z: 540.3 [M+H]+.
General Procedure for the Synthesis of 25a-25c. To a suspension of lithium hydroxide (3.0 equiv)
in dry acetonitrile/DMSO (49:1 v/v) at 50 ℃ was added 3ꢀmercaptopropionate (2.5 equiv) with
vigorous stirring. After 30 min, a solution of compound 24a-24c (1.0 equiv) in dry acetonitrile/dimethyl
sulfoxide (49:1 v/v) was slowly added to the reaction mixture, which was left to stir for another 1.5ꢀ3.0
h. After completion of the reaction as monitored by TLC, the solvent was removed under vacuum and
the residue was dissolved in dichloromethane. This organic layer was extracted with 1 N aqueous HCl,
and the aqueous phase was alkalized to pH=12 with aqueous KOH (10% m/m). Then the aqueous phase
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was extracted with dichloromethane and the combined organic phase was dried over anhydrous MgSO4
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and concentrated under vacuum to afford 25a-25c.
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1ꢀCyclopentylpiperazinꢀ2ꢀone (25a): 70.5 % yield, 1H NMR (400 MHz, CDCl3) δ 5.03ꢀ4.94 (m, 1H),
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3.55 (s, 2H), 3.23 (s, 2H), 3.07 (s, 2H), 1.85ꢀ1.45 (m, 8H).
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1ꢀ(Cyclopentꢀ3ꢀenꢀ1ꢀyl)piperazinꢀ2ꢀone (25b): 48.57% yield, H NMR (400 MHz, CDCl3) δ 5.75 (s,
2H), 5.34 ꢀ 5.20 (m, 1H), 3.59 (br s, 2H), 3.38 – 3.26 (m, 2H), 3.20 – 3.05 (m, 2H), 2.59 (dd, J = 16.0
Hz, 8.8Hz , 2H), 2.19 (dd, J = 16.1 Hz, 8.8Hz , 2H).
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1ꢀ(Transꢀ4ꢀ((triisopropylsilyl)oxy)cyclohexyl)piperazinꢀ2ꢀone (25c): 42.19% yield, H NMR (400
MHz, CDCl3) δ 4.64ꢀ4.47 (m, 1H), 3.67ꢀ3.50 (m, 3H), 3.26 (s, 2H), 3.11ꢀ2.98 (m, 2H), 1.86 ꢀ 1.07 (m,
8H), 0.98 (s, 18H).
General Procedures of Method E for the Synthesis of 26aꢀ26k, 27a-27f, 28a-28g. To a suspension
of acids (1.0 equiv) and amines (1.2 equiv) in dichloromethane was added EDCI (1.5 equiv) and HOBt
(1.2 equiv). The mixture was stirred at room temperature for 3ꢀ12 h until the starting materials were
consumed. Then water was added to the reaction mixture and the organic phase was washed
successively with 1 N aqueous HCl, saturated potassium carbonate solution and brine, dried over
anhydrous MgSO4, filtered and concentrated under vacuum. The residue was purified by column
chromatography to afford the title compound.
4ꢀ(5ꢀChloroimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone (26a). 26a was
obtained from 11a (50 mg, 0.25 mmol) and 25a (50 mg, 0.30 mmol) by following the procedures
described in Method E: 56 mg, 64% yield as a pale solid. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 3.4
Hz, 1H), 7.04 (d, J = 4.6 Hz, 1H), 4.95 (d, J = 9.0 Hz, 1H), 4.74 (s, 1H), 4.39 (s, 1H), 4.28 (s, 1H), 3.95
(s, 1H), 3.40 (s, 2H), 1.96 – 1.40 (m, 8H).13C NMR (100 MHz, CDCl3) δ 165.35, 146.51, 134.98,
120.76, 116.54, 115.63, 54.13, 50.92, 47.26, 40.03, 28.04, 24.12. MS(ESI) m/z: 391.1 [M + K] +.
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4ꢀ(5ꢀChloroꢀ2ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone (26b). 26b
was obtained from 11b (64 mg, 0.30 mmol) and 25a (60 mg, 0.36 mmol) by following the procedures
described in Method E: 71 mg, 65% yield as a white solid.1H NMR (400 MHz, CDCl3) δ 7.20 (s, 1H),
4.87 (s, 1H), 4.62 (s, 1H), 4.33 (s, 1H), 4.15 (s, 1H), 3.87 (s, 1H), 3.33 (s, 2H), 2.43 (s, 3H), 1.79 (s, 2H),
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1.72 – 1.37 (m, 6H). C NMR (100 MHz, CDCl3) δ 165.25, 161.33, 145.49, 131.23, 108.22, 54.21,
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50.85, 47.11, 40.24, 28.11, 24.12, 14.30. MS(ESI) m/z: 389.2 [M + Na] +.
4ꢀ(5ꢀChloroꢀ2ꢀisopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone (26c). 26c
was obtained from 11c (118 mg, 0.48 mmol) and 25a (98 mg, 0.58 mmol) by following the procedures
described in Method E: 136 mg, 72% yield as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.14 (s, 1H),
4.96 (s, 1H), 4.75 (s, 1H), 4.38 (s, 1H), 4.29 (s, 1H), 3.94 (s, 1H), 3.38 (s, 2H), 3.20 – 3.04 (m, 1H), 1.86
ꢀ 1.40 (m, 8H), 1.38 (d, J = 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 165.27, 161.09, 145.64, 132.84,
130.94, 128.85, 105.65, 54.11, 50.91, 47.25, 40.25, 29.31, 28.05, 24.11, 23.47. MS(ESI) m/z: 395.1 [M
+ H] +.
4ꢀ(5ꢀChloroꢀ2ꢀisopropylꢀ3ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone
(26d). 26d was obtained from 11d (49 mg, 0.19 mmol) and 25a (39 mg, 0.23 mmol) by following the
procedures described in Method E: 49 mg, 63% yield as a pale solid. 1H NMR (400 MHz, CDCl3) δ4.87
(s, 1H), 4.51 (s, 1H), 4.30 (s, 1H), 4.14 – 3.94 (m, 1H), 3.85 (s, 1H), 3.34 (s, 2H), 3.18 (d, J = 5.3 Hz,
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1H), 2.54 (s, 3H), 1.79 (s, 2H), 1.72ꢀ1.36 (m, 6H), 1.23 (d, J = 13.3 Hz, 6H). C NMR (100 MHz,
CDCl3) δ 165.11, 160.47, 145.95, 136.25, 123.10, 54.15, 50.93, 47.19, 39.91, 30.03, 28.11, 24.11, 23.79,
11.95. MS(ESI) m/z: 431.2 [M + Na] +.
4ꢀ(5ꢀChloroꢀ3ꢀmethylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone (26e). 26e
was obtained from 11e (64 mg, 0.30 mmol) and 25a (60 mg, 0.36 mmol) by following the procedures
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described in Method E: 74 mg, 67% yield as a pale solid. H NMR (400 MHz, CDCl3) δ 6.52 (s, 1H),
5.32 (s, 1H), 4.89 (d, J = 8.0 Hz, 1H), 4.56 (s, 1H), 4.31 (s, 1H), 3.87 (s, 1H), 3.33 (s, 2H), 2.61 (s, 3H),
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1.80 (s, 2H), 1.70ꢀ1.32 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 165.18, 160.50, 148.07, 134.01, 130.17,
54.19, 50.90, 47.18, 39.99, 28.08, 24.11, 14.51. MS(ESI) m/z: 389.2[M + Na] +.
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(26f).
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26f was obtained from 11f (243 mg, 1.00 mmol) and 25a (201 mg, 1.20 mmol) by following the
procedures described in Method E: 355 mg, 91% yield as a pale powder. 1H NMR (400 MHz, CDCl3) δ
6.37 (d, J = 1.2 Hz, 1H), 4.87 (d, J = 7.9 Hz, 1H), 4.57 (s, 1H), 4.32 (s, 1H), 4.12 (s, 1H), 3.88 (s, 1H),
3.32 (s, 2H), 2.17 (s, 1H), 1.79 (s, 2H), 1.71ꢀ1.48 (m, 4H), 1.49 – 1.36 (m, 2H), 1.00 (q, J = 6.1 Hz, 2H),
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0.81 – 0.72 (m, 2H). C NMR (100 MHz, CDCl3) δ 165.40, 161.99, 146.63, 135.64, 130.92, 115.11,
109.16, 54.19, 50.99, 47.26, 40.09, 28.09, 24.17, 8.68, 7.28. MS(ESI) m/z: 415.2 [M + Na]+. HRMS
(ESI) m/z calcd for C18H21ClN4O2S, 393.1147; found, 393.1165.
4ꢀ(5ꢀChloroꢀ3ꢀisobutylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone (26g). 26g
was obtained from 11g (30 mg, 0.12 mmol) and 25a (25 mg, 0.15 mmol) by following the procedures
described in Method E: 47 mg, 96% yield as a pale powder. 1H NMR (400 MHz, CDCl3) δ 6.52 (s, 1H),
4.86 (d, J = 7.5 Hz, 1H), 4.54 (s, 1H), 4.32 (s, 1H), 4.07 (s, 1H), 3.87 (s, 1H), 3.33 (s, 2H), 2.78 (d, J =
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4.6 Hz, 2H), 2.12 – 1.40 (m, 9H), 0.96 (d, J = 6.2 Hz, 6H). C NMR (100 MHz, CDCl3) δ 165.15,
161.24, 145.67, 133.54, 129.78, 114.30, 110.78, 54.18, 50.88, 47.17, 39.92, 36.63, 28.08, 27.64, 24.11,
22.02. MS(ESI) m/z: 431.3 [M + Na]+ .
1ꢀCyclopentylꢀ4ꢀ(2,5ꢀdichloroꢀ3ꢀcyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)piperazinꢀ2ꢀone
(26h). 26h was obtained from 11h (90 mg, 0.33 mmol) and 25a (66 mg, 0.39 mmol) by following the
1
procedures described in Method E: 54 mg, 38% yield as a pale solid. H NMR (400 MHz, CDCl3) δ
5.06 – 4.72 (m, 1H), 4.53 (s, 1H), 4.31 (s, 1H), 4.07 (d, J = 10.0 Hz, 1H), 3.87 (s, 1H), 3.28 (s, 2H), 1.95
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– 1.71 (m, 3H), 1.69ꢀ1.50 (m, 4H), 1.49 – 1.32 (m, 2H), 1.18ꢀ1.09 (m, 2H), 1.07ꢀ0.99 (m, 2H). C
NMR (100 MHz, CDCl3) δ 165.25, 161.37, 143.16, 134.09, 130.90, 118.59, 115.34, 54.19, 50.83, 47.19,
40.01, 28.05, 24.10, 8.72, 6.49..MS(ESI) m/z: 449.2 [M + Na]+.
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1ꢀCyclopentylꢀ4ꢀ(2,5ꢀdichloroꢀ3ꢀisobutylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)piperazinꢀ2ꢀone
(26i).
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26i was obtained from 11i (65 mg, 0.22 mmol) and 25a (42 mg, 0.25 mmol) by following the
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procedures described in Method E: 51 mg, 52% yield as a pale solid. H NMR (400 MHz, CDCl3) δ
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4.96ꢀ4.83 (m, 1H), 4.54 (s, 1H), 4.32 (s, 1H), 4.08 (s, 1H), 3.87 (s, 1H), 3.32 (s, 2H), 2.84 (d, J = 4.6 Hz,
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2H), 2.10 (s, 1H), 1.80 (s, 2H), 1.64 (s, 2H), 1.57 (s, 2H), 1.43 (s, 2H), 0.97 (d, J = 5.0 Hz, 6H). C
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NMR (100 MHz, CDCl3) δ 165.25, 161.21, 143.87, 134.15, 130.76, 117.21, 114.73, 54.23, 50.89, 47.23,
40.04, 33.34, 29.08, 28.07, 24.11, 21.91. MS(ESI) m/z: 465.1[M + Na] +.
4ꢀ(5ꢀChloroꢀ3ꢀcyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀ(transꢀ4ꢀ
hydroxycyclohexyl)piperazinꢀ2ꢀone (26j). Compound 11f (73 mg, 0.30 mmol) and 25c (128 mg, 0.36
mmol) went through the procedures as described in method E affording the intermediate 4ꢀ(5ꢀchloroꢀ3ꢀ
cyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀ(transꢀ4ꢀ((triisopropylsilyl)oxy)cyclohexyl)piperazinꢀ
2ꢀone (72 mg, 0.12 mmol), which was then dissolved in the methanol solution of hydrogen chloride and
stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column
chromatography to afford 26j (49 mg, 39% yield) as a pale solid. 1H NMR (400 MHz, CDCl3) δ 6.46 (s,
1H), 4.63 (s, 1H), 4.43 (s, 2H), 4.16 (s, 1H), 3.94 (s, 1H), 3.58 (s, 1H), 3.38 (s, 2H), 2.24 (s, 1H), 2.06 ꢀ
1.21 (m, 8H), 1.09 (s, 2H), 0.85 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 165.11, 161.93, 147.92, 135.59,
129.51, 114.49, 109.03, 69.78, 51.66, 47.45, 44.38, 40.27, 34.33, 27.19, 8.65, 7.27. MS(ESI) m/z:
445.2 [M + Na] +.
4ꢀ(5ꢀChloroꢀ3ꢀcyclopropylimidazo[2,1ꢀb]thiazoleꢀ6ꢀcarbonyl)ꢀ1ꢀ(cyclopentꢀ3ꢀenꢀ1ꢀyl)piperazinꢀ2ꢀ
one (26k). 26j was obtained from 11f (45 mg, 0.20 mmol) and 25b (40 mg, 0.24 mmol) by following
the procedures described in Method E: 26 mg, 33% yield as a pale solid. 1H NMR (400 MHz, CDCl3) δ
6.48 (s, 1H), 5.75 (s, 2H), 5.45 (s, 1H), 4.62 (d, J = 20.5 Hz, 1H), 4.39 (s, 1H), 4.17 (s, 1H), 3.93 (s, 1H),
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3.33 (s, 2H), 2.78 – 2.63 (m, 2H), 2.35 – 2.16 (m, 3H), 1.14 – 1.01 (m, 2H), 0.94 – 0.77 (m, 2H). C
NMR (100 MHz, CDCl3) δ 164.86, 161.81, 147.87, 135.63, 129.43, 114.37, 109.27, 50.90, 50.51, 47.09,
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40.02, 36.37, 8.63, 7.25. MS(ESI) m/z: 413.2[M + Na] +.
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4ꢀ(3ꢀChloroꢀ6,7ꢀdihydroꢀ5Hꢀcyclopenta[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀ
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cyclopentylpiperazinꢀ2ꢀone (27a). 27a was obtained from 16a (97 mg, 0.40 mmol) and 25a (84 mg, 0.50
mmol) by following the procedures described in Method E: 106 mg, 68% yield as a pale solid. 1H NMR
(400 MHz, CDCl3) δ 4.97 (s, 1H), 4.74 (s, 1H), 4.38 (s, 1H), 4.27 (s, 1H), 3.93 (s, 1H), 3.38 (s, 2H),
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3.11 (s, 2H), 2.92 (t, J = 6.8 Hz, 2H), 2.63 – 2.51 (m, 2H), 1.95 – 1.45 (m, 8H). C NMR (100 MHz,
CDCl3) δ 165.31, 161.03, 145.79, 133.01, 127.49, 125.33, 54.10, 50.87, 47.20, 40.04, 33.09, 28.06,
26.34, 25.72, 24.13. MS(ESI) m/z: 415.2 [M + Na] +.
4ꢀ(3ꢀChloroꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ
2ꢀone (27b). 27b was obtained from 16b (128 mg, 0.50 mmol) and 25a (102 mg, 0.60 mmol) by
following the procedures described in Method E: 86 mg, 42% yield as a pale solid. 1H NMR (400 MHz,
CDCl3) δ 4.95 (s, 1H), 4.66 (s, 1H), 4.38 (s, 1H), 4.20 (s, 1H), 3.94 (s, 1H), 3.40 (s, 2H), 3.06 (s, 2H),
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2.72 (s, 2H), 2.03ꢀ1.77 (m, 6H), 1.77ꢀ1.56 (m, 4H), 1.56ꢀ1.42 (m, 2H). C NMR (100 MHz, CDCl3) δ
165.29, 161.34, 145.14, 133.14, 127.20, 125.19, 117.78, 54.12, 50.94, 47.23, 40.00, 28.05, 24.71, 24.11,
23.34, 22.31, 21.61. MS(ESI) m/z: 429.1 [M + Na] +.
4ꢀ(3ꢀChloroꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀcyclohepta[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀ
cyclopentylpiperazinꢀ2ꢀone (27c). 27c was obtained from 16c (27 mg, 0.10 mmol) and 25a (20 mg, 0.12
1
mmol) by following the procedures described in Method E: 21 mg, 50% yield as a pale solid. H NMR
(400 MHz, CDCl3) δ 4.93 (s, 1H), 4.68 (s, 1H), 4.39 (s, 1H), 4.22 (s, 1H), 3.97 (s, 1H), 3.42 (s, 2H),
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3.13 (t, J = 7.1 Hz, 2H), 2.79 (t, J =7.1 Hz, 2H), 2.03ꢀ1.57 (m, 12H), 1.55ꢀ1.40 (m, 2H). C NMR
(100 MHz, CDCl3) δ 165.31, 161.17, 145.43, 132.94, 127.27, 125.24, 54.11, 50.92, 47.25, 40.02, 29.33,
28.97, 28.43, 28.05, 26.51, 24.12, 19.79. MS(ESI) m/z: 421.2 [M + H] +.
4ꢀ(3ꢀChloroꢀ7ꢀmethylꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀ
cyclopentylpiperazinꢀ2ꢀone (27d). 27d was obtained from 16d (180 mg, 0.67 mmol) and 25a (135 mg,
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0.80 mmol) by following the procedures described in Method E: 160 mg, 57% yield as a pale solid. H
NMR (400 MHz, CDCl3) δ 4.96 (s, 1H), 4.64 (s, 1H), 4.38 (s, 1H), 4.16 (s, 1H), 3.93 (s, 1H), 3.41 (s,
2H), 3.22 (d, J = 15.1 Hz, 1H), 2.98 (s, 1H), 2.77 (d, J = 13.9 Hz, 1H), 2.44 – 2.30 (m, 1H), 2.05 (d, J =
9.1 Hz, 2H), 1.86 (s, 2H), 1.78 – 1.41 (m, 7H), 1.14 (d, J = 6.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
165.20, 160.91, 146.21, 132.62, 127.04, 125.16, 54.13, 50.90, 47.21, 39.90, 32.46, 29.57, 28.92, 28.06,
24.11, 22.99, 20.87. MS(ESI) m/z: 443.2 [M + Na] +.
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4ꢀ(3ꢀChloroꢀ7,7ꢀdimethylꢀ5,6,7,8ꢀtetrahydrobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀ
cyclopentylpiperazinꢀ2ꢀone (27e). 27e was obtained from 16e (45 mg, 0.16 mmol) and 25a (34 mg, 0.20
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mmol) by following the procedures described in Method E: 58 mg, 84% yield as a pale solid. H NMR
(400 MHz, CDCl3) δ 4.87 (s, 1H), 4.58 (s, 1H), 4.30 (s, 1H), 4.09 (d, J = 17.9 Hz, 1H), 3.86 (s, 1H),
3.33 (s, 2H), 2.98 (s, 2H), 2.42 (s, 2H), 1.78 (s, 2H), 1.71 – 1.48 (m, 6H), 1.49 – 1.31 (m, 2H), 1.03 (s,
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6H). C NMR (100 MHz, CDCl3) δ 165.28, 160.89, 145.19, 133.28, 125.91, 124.51, 54.10, 50.89,
47.19, 40.23, 38.00, 34.44, 30.61, 28.05, 27.54, 24.11, 21.09. MS(ESI) m/z: 457.2 [M + Na] +.
3ꢀChloroꢀ2ꢀ(4ꢀcyclopentylꢀ3ꢀoxopiperazineꢀ1ꢀcarbonyl)ꢀ6,7ꢀdihydrobenzo[d]imidazo[2,1ꢀb]thiazolꢀ
8(5H)ꢀone (27f). 27f was obtained from 16f (181 mg, 0.67 mmol) and 25a (135 mg, 0.80 mmol) by
following the procedures described in Method E: 52 mg, 18% yield as a pale solid. 1H NMR (400 MHz,
CDCl3) δ 5.30 (s, 1H), 4.96 (s, 1H), 4.62ꢀ4.14 (m, 2H), 4.01 – 3.58 (m, 1H), 3.43 (s, 2H), 2.68 (t, J = 6.4
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Hz, 2H), 2.29 (s, 2H), 1.97ꢀ1.40 (m, 10H). C NMR (100 MHz, CDCl3) δ 191.42, 166.37, 161.57,
144.48, 125.38, 122.13, 54.10, 50.86, 47.27, 40.14, 37.16, 28.16, 24.12, 22.19, 21.96. MS(ESI) m/z:
419.5 [M – H] ꢀ；MS(ESI) m/z: 421.3 [M + H] +.
4ꢀ(3ꢀChlorobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone (28a). 28a
was obtained from 18a (101 mg, 0.40 mmol) and 25a (81 mg, 0.48 mmol) by following the procedures
described in Method E: 70 mg, 43% yield as a pale solid. 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 7.6
Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.54 – 7.40 (m, 2H), 5.09 – 4.86 (m, 1H), 4.75 (s, 1H), 4.41 (s, 1H),
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4.26 (s, 1H), 3.96 (s, 1H), 3.39 (d, J = 15.4 Hz, 2H), 1.95 – 1.45 (m, 8H). 13C NMR (100 MHz, CDCl3)
δ 166.33, 161.34, 145.95, 135.02, 131.99, 130.41, 126.47, 126.17, 124.36, 114.26, 54.18, 50.93, 47.30,
40.15, 28.06, 24.13. MS(ESI) m/z: 403.1 [M + H] +.
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4ꢀ(3ꢀChloroꢀ7ꢀmethoxybenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone
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(28b). 28b was obtained from 18b (57 mg, 0.20 mmol) and 25a (40 mg, 0.24 mmol) by following the
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procedures described in Method E: 45 mg, 52% yield as a pale solid. H NMR (400 MHz, CDCl3) δ
8.19 (s, 1H), 7.25 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H), 4.98 (s, 1H), 4.77 (s, 1H), 4.42 (s, 1H), 4.28 (s, 1H),
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3.97 (s, 1H), 3.91 (s, 3H), 3.42 (s, 2H), 1.89 (s, 2H), 1.72 – 1.44 (m, 6H). C NMR (100 MHz, CDCl3)
δ 165.37, 160.82, 158.07, 131.89, 126.59, 125.88, 119.66, 114.97, 113.74, 108.66, 55.99, 54.15, 50.91,
47.47, 40.05, 28.08, 24.13. MS(ESI) m/z: 455.2 [M + Na] +.
1ꢀCyclopentylꢀ4ꢀ(3,7ꢀdichlorobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)piperazinꢀ2ꢀone (28c). 28c
was obtained from 18c (72 mg, 0.25 mmol) and 25a (51 mg, 0.30 mmol) by following the procedures
described in Method E: 78 mg, 71% yield as a pale solid. 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 7.6
Hz, 1H), 7.68 (s, 1H), 7.41 (d, J = 7.9 Hz, 1H), 4.87 (s, 1H), 4.66 (d, J = 14.6 Hz, 1H), 4.34 (s, 1H),
4.15 (d, J = 18.6 Hz, 1H), 3.89 (s, 1H), 3.35 (s, 2H), 1.80 (s, 2H), 1.72 – 1.50 (m, 4H), 1.46 (d, J = 14.8
Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 165.34, 160.97, 145.09, 134.98, 132.10, 130.42, 127.02,
126.80, 124.19, 115.13, 114.98, 54.33, 50.82, 47.27, 40.07, 28.08, 24.12. MS(ESI) m/z: 437.3 [M + H]
+
.
4ꢀ(7ꢀBromoꢀ3ꢀchlorobenzo[d]imidazo[2,1ꢀb]thiazoleꢀ2ꢀcarbonyl)ꢀ1ꢀcyclopentylpiperazinꢀ2ꢀone
(28d). 28d was obtained from 18d (33 mg, 0.10 mmol) and 25a (20 mg, 0.12 mmol) by following the
1
procedures described in Method E: 28 mg, 58% yield as a pale solid. H NMR (400 MHz, CDCl3) δ
8.09 (d, J = 7.4 Hz, 1H), 7.82 (s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 4.88 (s, 1H), 4.67 (s, 1H), 4.34 (s, 1H),
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4.18 (s, 1H), 3.89 (s, 1H), 3.34 (s, 2H), 1.81 (s, 2H), 1.71 – 1.50 (m, 4H), 1.44 (s, 2H). C NMR (100
MHz, CDCl3) δ 165.29, 161.02, 144.89, 135.14, 130.87, 129.77, 127.00, 124.51, 119.32, 115.27, 54.25,
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