Fragment-based drug discovery of 2-thiazolidinones as BRD4 inhibitors: 2. Structure-based optimization

Article abstract of DOI:10.1021/jm501504k

The signal transduction of acetylated histone can be processed through a recognition module, bromodomain. Several inhibitors targeting BRD4, one of the bromodomain members, are in clinical trials as anticancer drugs. Hereby, we report our efforts on discovery and optimization of a new series of 2-thiazolidinones as BRD4 inhibitors along our previous study. In this work, guided by crystal structure analysis, we reversed the sulfonamide group and identified a new binding mode. A structure-activity relationship study on this new series led to several potent BRD4 inhibitors with IC₅₀ of about 0.05-0.1 μM in FP binding assay and GI₅₀ of 0.1-0.3 μM in cell based assays. To complete the lead-like assessment of this series, we further checked its effects on BRD4 downstream protein c-Myc, investigated its selectivity among five different bromodomain proteins, as well as the metabolic stability test, and reinforced the utility of 2-thiazolidinone scaffold as BET bromodomain inhibitors in novel anticancer drug development.

Full text of DOI:10.1021/jm501504k

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Article
Fragment-Based Drug Discovery of 2-Thiazolidinones
as BRD4 inhibitors: 2. Structure-based Optimization
Lele Zhao, Yingqing Wang, Danyan Cao, Tiantian Chen, Qi Wang, Yanlian Li,
Yechun Xu, Xin Wang, Danqi Chen, Lin Chen, Yue-Lei Chen, Guangxin Xia,
Zhe Shi, Yu-Chih Liu, Yijyun Lin, Ze-Hong Miao, Jingkang Shen, and Bing Xiong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501504k • Publication Date (Web): 05 Jan 2015
Downloaded from http://pubs.acs.org on January 12, 2015
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FragmentꢀBased Drug Discovery of 2ꢀThiazolidinones as BRD4
inhibitors: 2. Structureꢀbased Optimization
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Lele Zhao, Yingqing Wang, Danyan Cao, Tiantian Chen, Qi Wang, Yanlian Li, Yechun
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Xu, Naixia Zhang, Xin Wang, Danqi Chen, Lin Chen, YueꢀLei Chen, Guangxin Xia, Zhe
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Shi, YuꢀChih Liu, Yijyun Lin, Zehong Miao, Jingkang Shen, Bing Xiong
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Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
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Division of Antiꢀtumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
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Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
201203, China
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Drug discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
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Central research Institute of Shanghai pharmaceutical Holding Co. 898 Halei Road, Shanghai
201203, China
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Shanghai ChemPartner Co., Ltd, 998 Halei Road, Shanghai 201203, China
#
these authors contributed equally
*
Corresponding authors. (B. X.) Tel: +86 21 50806600 ext. 5412 fax: +86 21 50807088. Email:
bxiong@simm.ac.cn; (Z. M.) Tel: +86 21 50806600 ext. 2302. Email:zhmiao@simm.ac.cn; (J. S.)
Tel: +86 21 50806600 ext. 54120. Email: jkshen@simm.ac.cn
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Abstract
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The signal transduction of acetylated histone can be processed through a recognition
module—bromodomain. Several inhibitors targeting BRD4, one of the bromodomain
members, are in clinical trials as anticancer drugs. Hereby, we report our efforts on
discovery, optimization of a new series of 2ꢀthiazolidinones as BRD4 inhibitors along
our previous study. In this work, guided by crystal structure analysis, we reversed the
sulfonamide group and identified a new binding mode. SAR study on this new series
led to several potent BRD4 inhibitors with IC50 about 0.05ꢀ0.1 ꢁM in FP binding
assay and GI 0.1ꢀ0.3 ꢁM in cell based assays. To complete the leadꢀlike assessment
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of this series, we further checked its effects on BRD4 downstream protein cꢀMyc,
investigated its selectivity among five different bromodomain proteins, as well as the
metabolic stability test, and reinforced the utility of 2ꢀthiazolidinone scaffold as BET
bromodomain inhibitors in novel antiꢀcancer drug development.
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Keywords: SBDD; fragment; crystallography; 2ꢀthiazolidinone; BRD4;
bromodomain
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Introduction
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Lysine acetylation has been recognized as an important step of postꢀtranslational
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modifications in epigenetics. Studies have shown that histone acetyltransferases
(HATs) can add an acetyl group to the εꢀnitrogen atom of lysine residues in histones
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to exert its “writer” function in histone modification. This important mark can be
erased by another type of enzymes called histone deactylases (HDACs, or the
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, 6
“eraser”).
For signal transduction involving acetylated histones, a “reader”, as a
recognition module binding to the acetylated histones is required. Currently,
bromodomain was known as an important module to recognize and bind to acetylated
7, 8
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histones.
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The bromodomain was identified to function as lysine actylation reader in the early
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1990s in the brahma gene from Drosophila Melanogaster. After analysis of human
genome, it was found that 46 different nuclear and cytoplasmic proteins contain 61
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bromodomains. These proteins include HATs and HATꢀassociated proteins, histone
methyltransferases, helicases, ATPꢀdependent chromatin remodeling complexes,
transcriptional coꢀactivators, TBPꢀassociated factors, and nuclear scaffolding proteins.
Among them, BET (bromodomain and extraꢀterminal) proteins have received
increasing interest as it was associated with the cancer development in multiple
1
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myeloma and medulloblastoma. Besides, BRD4, an important member of BET
subfamily, has recently been identified as a component of a recurrent chromosomeal
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translocation in a very aggressive form of human squamous carcinoma. This
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translocation expresses the tandom bromodomains of BRD4 combining with the NUT
(nuclear protein in testis) protein to form NUT midline carcinoma (NMC). Previous in
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vivo studies showed that the bromodomain inhibitors suppressed tumor growth in
several mouse models, such as acute myeloid leukaemia (AML) and melanoma, and
underscored the broad utility and therapeutic potential of the inhibitors of human
bromodomain proteins.
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Figure 1. Structures of representative inhibitors of BET bromodomains: (+)ꢀJQ1 (1) , IꢀBET762
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(2) , IꢀBET151 (3), Pfiꢀ1 (4) , 5 , 6 and our previously reported BET bromodomain ligands
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With the importance of BRD4 in cancer development, many approaches were
applied to discover novel inhibitors against BET family. Despite many fragment hits
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were disclosed in recent years, there are only a few chemical scaffolds (Figure 1)
became later potent inhibitors against bromodomain of BET subfamily, especially
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BRD4.
A molecule, (+)ꢀJQ1, containing triazolothienodiazepine core scaffold,
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developed by Qi et al., was identified as a selective inhibitor of BET subfamily, and
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demonstrated its antitumor efficacy in the xenograft model of NMC. Specificity of
inhibitor JQ1 against other bromodomain proteins was also investigated in thermal
shift assay, a method using the change in protein melting temperature to account for
the inhibitor binding affinity. From the assay, it was found that (+)ꢀJQ1 only
showed the affinity to BET subfamily of bromodomains as it increased the melting
temperature of bromodomains of BRD2, BRD3 and BRD4 more than 6 ℃.
Meanwhile, Nicodeme et al. described that a similar molecule IꢀBET762 could
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suppress the inflammatory gene expression by inhibiting the BET family of proteins.
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Later, this compound was further extended to antitumor therapy and successfully
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entered into clinical trials in 2012. Besides these similar scaffolds, several novel series
of potent compounds were developed in pharmaceutical and academic research group.
3,5ꢀDimethylꢀisoxazoleꢀcontaining IꢀBET151 was first discovered in GSK through
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fragmentꢀbased approach, then optimized to a potent and selective BET inhibitor, and
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demonstrated its in vivo efficacy murine model of MLLꢀAF9 leukaemia.
In 2012,
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Pfizer identified a novel scaffold 3,4ꢀdihydroꢀ3ꢀmethylꢀ2(1H)ꢀquinazolinone through
fragmentꢀbased drug discovery. This scaffold was further optimized to sulfonamide
derivatives with IC50 around 0.1ꢀ0.5 ꢁM in a peptide displacement biochemical assay.
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The selectivity of IBETꢀ762 and IBETꢀ151 were reported by Dawson et al., also
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showing they are selective BET subfamily bormodomain inhibitors in a fluorescence
anisotropy (FP) assay: IBETꢀ762 has IC50 values of 0.79 ꢁM, 0.4ꢁM and 0.63ꢁM to
BRD2, BRD3 and BRD4 respectively; and IBETꢀ151 has IC50 of 0.5, 0.25 and 0.79
ꢁM to BRD2, BRD3 and BRD4. Other interesting scaffolds, including
3,5ꢀDimethylisoxazole and [1,2,4]triazolo[4,3ꢀa]phthalazine, were also developed as
potent BET bromodomain inhibitors in Structural Genomics Consortium. Similar to
IꢀBET151, Compound 5 utilizes the isoxazole group to form hydrogen bonding
interaction with the conserved residue Asparagine (N140 in BRD4 protein).
Compound 6 containing a novel [1,2,4]triazolo[4,3ꢀa]phthalazine scaffold was
developed by Brennan et al. as a BRD4 inhibitor, but also shows weak binding to
other bromodomains, including ATAD2, TIF1α and PCAF.
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Previously, our BRD4 inhibitor discovery by computational docking and Xꢀray
crystallography study revealed that 2ꢀthiazolidinones 7, 8 and 9 could mimic the
acetylated lysine moiety to bind to the first bromodomain of BRD4 protein
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(BRD4(I)). Initial optimization and leadꢀlike assessment demonstrated the potential
of this new scaffold. Herein, further SAR study of this new series of compounds will
be reported. Guided by coꢀcrystal structures, we identified a new binding mode of
these 2ꢀthiazolidinone derivatives. With this interesting finding, we further explored
the SAR and obtained several potent BRD4 inhibitors, which could serve as a base for
further drug development.
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The syntheses of novel 2ꢀthiazolidinone derivatives were illustrated in schemes 1ꢀ4.
3’ꢀSulfonamide derivatives 12aꢀj were prepared using the synthetic route in Scheme 1.
Benzenesulfonamide derivatives 10aꢀj were obtained from commercially available
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sulfonyl chloride and different amines. Treatment of 10aꢀj with CuBr gave the
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desired 2ꢀbromoacetophenones 11aꢀj. KSCN was added to obtain the crude
2ꢀthiocyanatoethanone products which were used without further purification. The
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thiazolꢀ2(3H)ꢀone products 12aꢀj were obtained by reaction with 50% H SO in acetic
acid (v/v) at 100 °C for one hour.
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Scheme 1. Synthesis of the 3'ꢀSulfoamide Substituted 2ꢀThiazolidinone Derivatives 12aꢀj.
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Reagents and conditions: a) R NH
2
, pyridine, CH
2
Cl
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acetone, rt , then 50% H SO solution (v/v) in acetic acid, 100 °C.
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In Scheme 2, 4’ꢀsubstitutedꢀ3’ꢀnitroacetophenones 13aꢀd were reduced with iron
power to afford amine derivatives 14aꢀd. Conversion of 14aꢀd to sulfonyl chlorides
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15aꢀd followed the procedure described by R. V. Hoffman. Subsequent reactions
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which were similar to scheme 1 furnished compounds 18aꢀd.
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Scheme 2. Synthesis of the 3',4'ꢀ Substituted 2ꢀThiazolidinones Derivatives 18aꢀd. Reagents and
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conditions: a) Fe, NH Cl, EtOH, 80 °C; b) NaNO , HCl (conc.), acetic acid, H O, ꢀ10 °C , then
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In Scheme 3, treatment of 19 with fuming nitric acid afforded template 20, which
was used directly without deacetylation. The 2ꢀthiazolidinone scaffold 25 was
obtained through similar procedures described in scheme 1 and 2. The 4’ꢀamine
derivative 25 was then reacted with different acyl chlorides to afford 4’ꢀamide
products 26a and 26b.
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Scheme 3. Synthesis of the 3',4'ꢀ Substituted 2ꢀThiazolidinones Derivatives 25, 26a and 26b.
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Reagents and conditions: a) Fuming nitric acid, acetic acid, 0 °C; b) NaNO , HCl (conc.), acetic
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2 2 2 2
acid, H O, ꢀ10 °C , then SO , CuCl, acetic acid, 10 °C; c) Cyclopentylamine, pyridine, CH Cl , rt;
1
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2 2 4
d) CuBr , EtOAc, 80 °C; e) KSCN, acetone, rt , then 50% H SO solution (v/v), 100 °C; f) Fe,
2
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4
NH Cl, EtOH, 80 °C; g) R 'COCl, pyridine, THF, rt.
1
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In Scheme 4, 28a and 28b were synthesized with good overall yields using a
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reported sequence starting from 27a and 27b and dibromoisocyanuric acid. Using
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Cuꢀcatalyzed CꢀN coupling reaction, we converted bromoꢀsubstituted 28a and 28b to
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corresponding amineꢀsubstituted 34a and 34b. Sulfonamide and thiazolꢀ2(3H)ꢀone
ring construction using previously described conditions led to target compounds 33a,
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33b, 39aꢀ42a and 39bꢀ42b. Compound 43 was obtained by reductive amination
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from 39b.
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Scheme 4. Synthesis of the 3',5'ꢀ or 3',4',5'ꢀ Substituted 2ꢀThiazolidinones Derivatives 33a, 33b,
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39aꢀ42a, 39bꢀ42b and 43. Reagents and conditions: a) Dibromoisocyanuric acid, H
2
SO
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(conc.), rt,
CO
then NaI, Na SO acetic acid, rt; b) tertꢀbutyl carbamate, CuI,
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,
K
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,
2 2 2
N,N'ꢀdimethylethylenediamine, toluene, 110 °C, then TFA, CH Cl , 0 °C; c) NaNO , HCl (conc.),
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acid, 100 °C; g) Fe, NH Cl, EtOH, 80 °C; h) R 'COCl, pyridine, THF, rt; i) 37% formaldehyde
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
Results and Discussion
Initial Optimization.
6
7
8
9
0
1
2
In our previous paper, 2ꢀthiazolidinone scaffold with sulfonylaminophenyl
1
9
substitution was reported. These compounds displayed encouraging potency in the
fluorescence anisotropy binding assay, with IC50 around 1 ꢁM, but showed weak
proliferation inhibition activity in human colon cancer HTꢀ29 cell line (the most
potent compound with GI50=37.3±15.5 ꢁM in HTꢀ29 cells). Therefore, it would be
worthwhile to find new 2ꢀthiazolidinone bromodomain inhibitors with potency for
both BRD4(I) binding and cellular based assays.
1
1
1
13
14
Figure 2. Initial optimization of 2ꢀthiazolidinone BRD4 inhibitors with reversing the sulfonamide
substituents
1
5
1
1
6
7
Lately, we continued the structural modification on 2ꢀthiazolidinone by replacing
the sulfonylaminophenyl substitution with amino sulfonyl phenyl group (Figure 2).
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9
1
1
2
3
4
5
6
7
8
9
0
1
2
Various types of R ꢀsubstituents were tested (Table 1, 12aꢀ12j) and significantly
improved BRD4(I) activity was obtained when being compared with the fragment hit
7. The cyclopropyl derivative 12b had an encouraging ligand efficiency of 0.404 ꢁM,
while cyclopentyl derivative 12a and cyclohexylꢀcontaining 12c showed marginally
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
1
increased activity but with a trend of decreasing ligand efficiency. For R ,
introduction of aromatic rings including phenyl and isoxazole groups decreased the
1
BRD4(I) potency dramatically (compounds 12d, 12e and 12f). Larger R groups
(12gꢀ12i) were not tolerated at all. This was different from the SAR study of the
previous work, implying that the reversed sulfonamide substituents may interact with
distinct binding modes.
1
1
1
1
Table 1. Effects of R ꢀsubstituted 2ꢀthiazolidinones on inhibition of BRD4(I) by fluorescence
a
anisotropy assay
1
3
1
1
Compound
number
R
BRD4 (I)
IC50 (ꢁM)
0.058±0.01
Ligand
efficiency
0.319
Compound
number
12d
R
BRD4 (I)
IC50 (ꢁM)
>10
Ligand
efficiency
ꢀ
(
+)ꢀJQ1
ꢀ
ꢀ
ꢀ
ꢀ
IꢀBET 151
0.10±0.01
>100
0.309
ꢀ
12e
12f
12g
8.33±0.71
7.34±1.65
>10
0.300
0.332
0.299
7
8
4.1±0.5
0.352
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b
1
2a
2b
1.40±0.08
2.26±0.08
2.06±0.02
0.379
0.404
0.351
12h
12i
12j
>10
0.304
0.311
0.339
1
9.31±0.20
5.82±1.12
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2c
a
1
2
3
The IC50 in the table was calculated from two independent experiments. The fluorescent
compound used in the assay was JQ1ꢀFITC. (The synthesis route was provided in our previous
1
9
b
28
work ). Ligand efficiency (LE) was calculated as 1.37 (pIC50/number of heavy atoms).
4
5
6
7
Figure 3. Coꢀcrystal structures of BRD4(I) bound with 2ꢀthiazolidinone derivatives. The ligands
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7
8
9
1
2
3
4
5
6
7
8
9
0
and interacted important residues were shown in stick model, while the protein was shown in
ribbon model. (A) 12aꢀBRD4 (I) (pdb entry: 4QR3); (B) superimposed the complexes of 2ꢀBRD4
(I) (pdb entry: 4HXR) and 12aꢀBRD4 (I); (C) 18cꢀBRD4 (I) (pdb entry: 4QR4); (D) 40aꢀBRD4
(I) (pdb entry: 4QR5).
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
To decipher the interaction of this series of 2ꢀthiazolidinone derivatives, we
performed crystallization experiment and solved the coꢀcrystal structure of 12a bound
to BRD4(I). As shown in Figure 3AꢀB, compound 12a had a different binding mode
compared to the previous Nꢀphenyl sulfonamide 8, as the cyclopentyl of 12a pointed
to LEU94 and ASN140 residues, which were on the opposite side of WPF shelf. The
sulfonamide group formed a hydrogen bonding network through three water
molecules, which may account for the increase of the binding affinity. From previous
1
11
29
1
2
SAR studies, it was found that, for many known BRD4 inhibitors, the WPF shelf
13
was considered to be the major binding subsite of bromodomain module, however,
this was not the case for this series of BRD4 inhibitors.
Structureꢀactivity Relationships.
1
4
15
2
1
6
Introducing different substituents (Table 2) in R position generally gave 2ꢀ10 folds
18, 30
17
improved activity compared with compound 12a, and increased cellular activity.
1
8
The electronic nature of the substituents on this position was not critical, for either
electron donating (18a, 18b and 25) or electron withdrawing (18c) groups displayed
similar BRD4(I) affinity. Larger amide substituents such as 26a and 26b were
tolerable but not optimal, as indicated by their decreased activity and ligand efficiency
when being compared with 25. As studied in several cancer cells, including acute
19
2
0
21
2
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
myloid leukemia and multiple myeloma, BRD4ꢀdependent transcriptional regulation
was related to the MYC pathway activation, and upregulated MYC is a critical driver
for many types of cancers. We also investigated their cellular activities in three
different cell lines. In our previous study, we found that human colon cancer HTꢀ29
cell, harboring evaluated expression of cꢀMyc protein, was the sensitive cell line
among a panel of screened solidꢀtumor cancer cells. The reported sensitive cell lines
MV4;11 and MM.1S were also utilized to test the cellular proliferation inhibition
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
effects. In general, our compounds exhibited encouraging potency in all of the three
tested cell lines, especially in MV4;11 cell line. The results from cellular
antiꢀproliferation assay together with the protein binding activity suggested that
further exploration could be conducted to find useful BRD4 inhibitors.
1
11
1
2
To support further optimizations, we solved the complex structure of 18c with
BRD4(I) (Figure 3C). Similar to compound 12a, the Nꢀcyclopentyl group of
compound 18c situated at the same area as that of compound 12a, and this further
confirmed the unusual binding mode of this series of compounds. By detailed analysis
of the orientation of phenyl ring in the binding site, we speculated that the substitution
at another meta position will extend to the subpocket behind the WPF shelf, which
was also a unique subpocket revealed in our previous study.
13
1
4
15
1
6
17
1
8
2
19
Table 2. Effects of R ꢀsubstituted 2ꢀthiazolidinones on inhibition of BRD4(I) in FA assay, and
2
0
antiproliferation effects against cell lines HTꢀ29, MV4;11 and MM.1S.
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2
3
4
5
6
7
8
9
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
1
Compound
number
BRD4 (I)
HTꢀ29
MV4;11
MM.1S
Ligand
2
R
a
a
b
b
IC50 (ꢁM)
IC50 (ꢁM)
IC50 (ꢁM)
IC50 (ꢁM)
efficiency
(
+)ꢀJQ1
ꢀ
ꢀ
0.058±0.01
0.104
0.945
0.023
0.119
0.459
0.547
0.341
0.109
0.299
1.899
2.415
0.319
0.309
IꢀBET 151
0.10±0.01
0.22±0.01
0.76±0.08
c
1
8a
Me
3.95±2.71
3.93±0.19
0.411
18b
OMe
0.362
18c
18d
25
Cl
F
0.25±0.00
0.50±0.01
0.12±0.01
0.41±0.15
0.80±0.05
23.67±1.71
35.16±4.25
3.28±2.12
24.64±5.58
12.99±5.62
3.369
NT
0.408
0.389
0.428
0.321
d
NT
NH
2
0.289
0.681
NT
0.793
2.310
NT
26a
26b
0.307
a
b
2
3
The IC50 in the table was calculated from two independent experimental measurements. The
c
IC in the table was calculated from one experimental measurement. Ligand efficiency (LE) was
50
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
d
1
2
3
4
5
6
7
8
9
0
calculated as 1.37 (pIC50/number of heavy atoms). NT = not tested.
3
Inspired by the crystal structure of 18cꢀBRD4(I), we continued to explore the R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
position “directing” toward the loop behind the WPF shelf. For comparison, we
2
synthesized the unsubstituted and R ꢀmethyl substituted products, as listed in Table 3.
Similar to the previous study, the substitution at this niche can significantly increase
the binding activity towards BRD4(I).
3
2
Significant BRD4(I) activity was observed if R was Br and R was kept as methyl
(33b). This compound also showed excellent activity in MV4;11 cancer cell
proliferation inhibition test, with an IC50 0.14 ꢁM and ligand efficiency of 0.427.
Changing to amine (39b) or aminomethyl group (43) decreased the activity about 2ꢀ4
folds in BRD4(I) binding assay, while increasing the activity in cellular
antiꢀproliferation assays. These three compounds displayed potencies comparable to
IꢀBET151 both in FA and cell based assays. Comparing the methyl substituted and
1
11
1
2
13
1
4
2
15
unsubstituted compounds 33a, 33b and 39aꢀ39b, it was found that when R was
1
6
methyl, the inhibitors have better cellular activities. This may be a result of better cell
permeability of compounds 33b and 39b, as the methyl substitution has larger
hydrophobic tendency.
17
1
8
3
19
To further explore the SAR of R position, we expanded the small substitutions to
2
0
larger amide groups (40bꢀ42b). An interesting phenomenon was observed when
2
21
comparing R ꢀmethyl substituted 40b and 42b with the unsubstituted analogs 40a and
2
2
2
42a: the unsubstituted analogs were 4ꢀ6 folds more active than the R ꢀmethyl
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5
6
7
8
9
1
2
3
4
5
6
7
8
9
0
substituted compounds.
3
To investigate whether R binds to the predicted subpocket, we solved the crystal
3
structure of 40a with BRD4(I) (Figure 3D). As speculated, the amide group of R of
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
40a forms a hydrogen bond with residue GLN85 and several indirect hydrogen bonds
to residues PRO82 and GLN85 through one water molecule. Compound 40a utilized
the sulfonamide to form a hydrogen bonding network with water molecules around
the ASN140 and ILE146. By comparing with the 18c, it was found that the
Nꢀcyclopentane group of sulfonamide rotated about 180 degree, which may explain
2
why the R ꢀmethyl substituted analogs were less active because the hydrogen atoms
1
of methyl group may have a close contact with the hydrogen atom at nitrogen of
sulfonamide group, therefore disturbing the binding conformation.
11
1
2
2
3
13
14
Table 3. Effects of R , R ꢀsubstituted 2ꢀthiazolidinones on inhibition of BRD4(I) in FA assay, and
antiproliferation effects against cell lines HTꢀ29, MV4;11 and MM.1S.
1
5
Compound
number
BRD4 (I)
HTꢀ29
MV4;11
MM.1S
Ligand
2
3
R
R
ꢀ
a
a
b
b
IC50 (ꢁM)
IC50 (ꢁM)
50
IC (ꢁM)
IC50 (ꢁM)
efficiency
(
+)ꢀJQ1
ꢀ
0.058±0.01
0.104
0.023
0.109
0.319
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
IꢀBET 151
ꢀ
ꢀ
0.10±0.01
0.30±0.01
0.06±0.01
0.46±0.01
0.14±0.02
0.06±0.01
0.22±0.08
0.17±0.02
0.93±0.07
0.11±0.01
0.79±0.00
0.25±0.01
0.945
0.119
0.299
NT
0.309
d
c
3
3a
H
Br
57.10±12.69
5.46±1.84
6.15±0.90
0.86±0.42
5.69±0.48
28.48±2.02
13.86±1.15
23.48±10.38
25.22±6.08
20.09±4.15
0.85±.855
NT
0.403
0.427
0.392
0.405
0.364
0.323
0.341
0.293
0.326
0.276
0.374
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
33b
Me
H
Br
0.141
0.689
0.184
0.228
0.450
0.791
NT
0.999
3.580
0.570
1.055
1.755
2.533
NT
39a
39b
40a
40b
NH
NH
2
2
Me
H
Me
H
41a
H
N
41b
42a
42b
Me
H
*
O
0.272
0.602
0.198
3.305
4.095
0.505
Me
Me
43
NHMe
a
b
1
2
3
4
The IC50 in the table was calculated from two independent experimental measurements. The
c
IC50 in the table was calculated from one experimental measurement. Ligand efficiency (LE) was
d
calculated as 1.37 (pIC50/number of heavy atoms). NT = not tested.
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2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
0
Bromodomain selectivity profile
Bromodomain is the conserved module in evolution, and they share a common 3D
structure pattern of one short helix (helix Z) and three long helices(Helix AꢀC). The
acetylated lysine can bind to the top of the bromodomains by forming several
hydrogen bonds with the conserved residue asparagine (ASN140 in BRD4). As
selectivity is critical for the success of drug discovery, we selected six compounds
from this series to profile the binding specificity in five representative bromodomain
modules, including BRD4(I), BRD2(I), EP300, BRG1 and ATAD2 (detailed
sequence information can be found in supporting materials). After cloned and
expressed these modules, we utilized the thermal shift assay to test the binding
tendency of inhibitors, which is to measure the thermal stability of a target protein and
a subsequent increase in protein melting temperature upon binding of a ligand to the
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
1
11
1
2
31
13
protein. From the Table 4, it was clearly shown that compounds of this series are
generally selective inhibitors for BET subfamily, showing good binding affinity to
BRD2(I) and BRD4(I). And similar to (+)ꢀJQ1, our compounds showed better binding
affinity to BRD4(I) rather than BRD2(I). Interestingly, although compounds of this
series showed little affinity for bromodomains of EP300 and BRG1, they displayed
certain binding activity towards bromodomain of ATAD2, implying that it is a
promising starting point for developing ATAD2 inhibitors.
1
4
15
1
6
17
1
8
19
2
0
21
Table 4. Thermal shift analysis of selected compounds against the bromodomains from BRD4(I),
BRD2(I), EP300, BRG1 and ATAD2.
2
2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound
number
BRD4(I)
BRD2(I)
EP300
BRG1
ATAD2
a
∆T (°C)
∆T (°C)
∆Tm (°C)
∆Tm (°C)
∆T (°C)
m
m
m
(
+)ꢀJQ1
9.58±0.20
6.30±0.15
5.19±0.50
7.76±0.26
9.22±0.58
7.25±0.25
8.48±0.20
5.47±0.19
3.47±0.25
2.82±0.21
4.06±0.17
5.92±0.22
5.04±0.16
5.63±0.20
ꢀ1.24±0.41
ꢀ0.17±0.65
0.49±0.23
ꢀ0.08±0.28
ꢀ1.01±0.38
0.19±0.25
ꢀ0.54±0.31
0.14±0.29
0.69±0.25
0.41±0.46
0.39±0.25
0.94±0.26
0.57±0.39
1.03±0.47
1.04±0.27
1.44±0.54
1.36±0.30
3.28±0.62
3.42±0.08
2.70±0.46
3.55±0.53
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8a
1
8bꢀ
2
5
33b
3
9b
40a
a
1
2
3
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5
6
7
8
9
0
1
2
3
Heat map shows relative ꢁT
m
: red indicates large ꢁT
m
m
, and green indicates small ꢁT . The data
are calculated from four time measurements.
Effects on cꢀMyc protein and mRNA Expression
Delmore et al. discovered the BET bromodomain proteins as regulatory factors for
13
oncogene cꢀMyc. And the BRD4 inhibitor (+)ꢀJQ1 induces an antiproliferative
effect associated with the downꢀregulation of cꢀMyc transcription. To assess the
2ꢀthiazolidinone BRD4 inhibitors, we performed the Western blotting experiment and
quantitative realꢀtime PCR (RTꢀqPCR) to study the cellular effect related to cꢀMyc.
Three potent inhibitors were selected (33b, 39b and 40a) based on the protein binding
and cellular antiproliferation assays. The results (Figure 4A) showed that at either 5
µM or 10 µM concentration, compound 39b was more active than other two
compounds (33b and 40a), and could significantly inhibit the expression of cꢀMyc
1
1
1
1
protein. As compound 39b is most active in cellular antiproliferation assay, the
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doseꢀdependent effect on cꢀMyc protein was also studied along with the effect of
IꢀBET151 and (+)ꢀJQ1. As indicated in Figure 4B and 4C, 39b showed good
inhibition to the expression of cꢀMyc, which is comparable to compounds (+)ꢀJQ1
and IꢀBET151. Similarly, as indicated by RTꢀqPCR assays, compounds 33b, 39b and
40a strongly downꢀregulated the expression of cꢀmyc mRNA (Figure 4D), three
compounds of which revealed more than 60% inhibition at 5 µM, especially
compound 39b showing about 90% inhibition. From the doseꢀdependent study on
expression of cꢀmyc mRNA, 39b showed clear effect even at concentration about
0.3µM, which is about 20% lower than (+)ꢀJQ1 and IꢀBET151. Taking together the
selectivity profiles, these data implied that the antiproliferation effects of
2ꢀthiazolidinone series of compounds may go through the BRD4ꢀdependent pathway,
as confirmed from the study of (+)ꢀJQ1.
10
11
12
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Figure 4. (AꢀC) Inhibition of the tested compounds on the expression of cꢀMyc protein. HTꢀ29
cells were treated with different compounds (5 µM) (A), 39b or JQ1 (0ꢀ5 µM) (B), and 39b or
IꢀBET 151 (0ꢀ5 µM) (C) for 24 h. Cells were collected and lyzed for Western blotting. The level of
cꢀMyc protein was detected and the expression of GAPDH protein was chosen as the loading
control. The experiments were repeated for three times. (DꢀF) Inhibition of the tested compounds
on the expression of cꢀmyc mRNA. HTꢀ29 cells were treated with compounds (5 µM) (D), 39b or
JQ1 (0ꢀ5 µM) (E), and 39b or IꢀBET 151 (0ꢀ5 µM) (F) for 24 h. Total RNA was isolated and
reverseꢀtranscribed for RTꢀqPCR analyses. The data were expressed as mean ± SD, representing
the relative levels of cꢀmyc mRNA from at least three independent experiments.
0
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1
In Vitro metabolic stability Assessment
11
To complete the drugꢀlikeness assessment of this series of BRD4 inhibitors, we
conducted in vitro metabolic stability profile of eight potent inhibitors as the
representatives of 2ꢀthiazolidinone compounds. As shown in Table 5, the results
provided that most compounds except 18a and 43 have favorable metabolic stability
profiles in the human liver microsome assay, as the clearance rates of six compounds
are below than 100 ꢁL/min/mg. 43 is the most metabolic unstable compound with the
clearance rate about 174 ꢁL/min/mg, indicating the aminomethyl group is sensitive in
human liver. In addition to the liver microsome assay, five cytochrome P450 enzymes
commonly metabolizing exogenous chemicals were used to test the direct inhibition
of these eight compounds. The evaluation showed that compounds 18a, 25 and 42a
can inhibit certain CYPs more than 50% ratios at the concentration of 10 ꢁM; while
other compounds showed satisfactory profiles in five CYPs inhibition assay. As CYP
1
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enzymes usually have different structural preference when binding to small molecules,
it is difficult to assess the metabolic stability simply from the functional group. From
the timeꢀdependent inhibition assay for five CYPs, the results showed that last four
compounds in Table 5 have less possibilities of covalent binding to these CYPs.
Nevertheless, from the metabolic stability profiling in vitro, it is promising that, with
detailed exploration of chemical space of the 2ꢀthiazolidinone series, useful inhibitors
of BRD4 (I) such as 39b can be identified as a good candidate for further drug
development.
10
11
12
13
14
15
16
17
18
19
20
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23
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25
26
27
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29
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31
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33
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37
38
39
40
41
42
43
44
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1
Table 5. In vitro liver microsome stability assay and cytochrome P450 enzymes inhibition assays
a
HLM
Stability
(ꢁl/min/mg
protein)
78
CYP DI ratio, % (compd concn of 10 ꢁM)
b
TDI
Cpd
3A4
2D6
2C9
1A2
2C19
(+)ꢀJQ1
IꢀBET
151
30/28%
21/22%
2%
18%
33%
2%
18%
19%
2C19
0
18%
18%
no inhibition
1
8a
128
56
20
84
66
62/68%
20/30%
70/82%
42/53%
42/46%
27%
no inhibition
18%
no inhibition
10%
52%
3%
45%
26%
57%
28%
3%
2C9、2C19
2D6、2C19
3A4
18b
2
5
74%
22%
47%
17%
33b
39%
no inhibition
no inhibition
2C9
3
9b
3%
no inhibiton
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
4
4
4
4
4
5
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5
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5
5
5
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4
4
0a
2a
74
64
14/21%
17/34%
41/47%
17%
44%
36%
14%
57%
29%
45%
37%
32%
46%
17%
no inhibition
no inhibition
no inhibition
4
3
174
no inhibition
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Footnote:
a
CYPs direct inhibition, sometimes referred to reversible inhibition, is assessed by measurement
of an enzyme (CYP) activity in the presence of increasing concentration of inhibitor without a
preꢀincubation step.
b
TDI stands for timeꢀdependent inhibition, which is referring to a change in enzyme inhibition
during an in vitro incubation and means an irreversible inactivation of CYPs.
7
Conclusions
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1
2
3
4
In summary, above studies disclosed the unexpected finding that reversing the
sulfonamide moiety can significantly improve the binding activity of 2ꢀthiazolidinone
derivatives. Through the detailed analysis of a coꢀcrystal structure of 12aꢀBRD4(I), a
new binding mode was revealed for this series of compounds. With the guidance of
crystal structures, we elaborated the SAR of the newly designed 2ꢀthiazolidinone
analogs, optimized the structure to reach a high potency as indicated from protein
binding assay and three cancer cell lines assays. From the selectivity profiling of eight
compounds against five bromodomain proteins, it was found that this series of
2ꢀthiazolidinones was selective BET subfamily inhibitors. Taking together with the
cellular effect study on downstream protein cꢀMyc and metabolic stability test, we
1
1
1
1
1
15
1
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regarded 39b as a promising BRD4 inhibitor, and a useful lead for further antiꢀcancer
drug development.
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3
Experimental Section
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1
Chemistry
General:
All solvents were dried and purified prior to use: Toluene was distilled from sodium,
2
THF was distilled from potassium, and DCM was distilled from CaH . Compounds
13aꢀ13d, 19, 27a, 27b and other commercially available reagents were used as
1
received. H NMR (400 MHz) spectra were recorded by using a Varian Mercuryꢀ400
1
1
1
1
1
1
1
1
1
1
2
2
High Performance Digital FTꢀNMR spectrometer with tetramethylsilane (TMS) as an
13
internal standard. C NMR (126 MHz) spectra were recorded by using a Varian
Mercuryꢀ500 High Performance Digital FTꢀNMR spectrometer. Abbreviations for
peak patterns in NMR spectra: br = broadened, s = singlet, d = doublet, t = triplet, dd
= doublet of doublets and m = multiplet. Lowꢀresolution mass spectra were obtained
with a Finnigan LCQ Deca XP mass spectrometer using a CAPCELL PAK C18 (50
mm × 2.0 mm, 5 µM) or an Agilent ZORBAX Eclipse XDB C18 (50 mm × 2.1 m, 5
µM) in positive or negative electrospray mode. Highꢀresolution mass spectra were
recorded by using a Finnigan MATꢀ95 mass spectrometer. Purity of all compounds
was determined by analytical Gilson highꢀperformance liquid chromatography (HPLC)
using an YMC ODS3 column (50 mm × 4.6 mm, 5 µM). Conditions were as follows:
ꢀ1
CH
3
CN/H
2
O eluent at 2.5 mLmin flow [containing 0.1% trifluoroacetic acid (TFA)]
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3 3
at 35 °C, 8 min, gradient 5% CH CN to 95% CH CN, monitored by UV absorption at
214 nm and 254 nm. TLC analysis was carried out with glass precoated silica gel
GF254 plates. TLC spots were visualized under UV light. Flash column
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chromatography was performed with a Teledyne ISCO CombiFlash R system. All
f
solvents and reagents were used directly as obtained commercially unless otherwise
noted. All air and moisture sensitive reactions were carried out under an atmosphere
of dry argon with heatꢀdried glassware and standard syringe techniques. Melting
points were determined using a SGW Xꢀ4 hot stage microscope and are uncorrected.
(spectra data of the synthesized compounds were provided as supporting material)
Synthetic Procedures:
1
11
General procedure A for the reactions in Scheme 1, entries 10aꢀ12a:
1
2
3ꢀAcetylꢀNꢀcyclopentylbenzenesulfonamide
(10a).
To
a
solution
of
13
cyclopentanamine (0.675 mL, 6.86 mmol) and pyridine (0.74 mL, 9.15 mmol) in
CH Cl (20 mL) was added commercially available 3ꢀacetylbenzeneꢀ1ꢀsulfonyl
1
4
2
2
15
chloride (1 g, 4.57 mmol) and the mixture was stirred at room temperature for 1 hour.
The reaction was monitored by TLC. Upon completion, 1 N HCl (10 mL) was added
1
6
17
2 2
and the aqueous layer was extracted with CH Cl . The organic layers were combined,
1
8
washed with brine, dried with Na SO , and then removed in vacuo. The residue was
2
4
19
purified by flash column chromatography (gradient elution, gradient 5 to 30%
2
0
EtOAc/60ꢀ90 °C petroleum ether) to give the title compound 10a as a pale yellow
1
21
solid (1.07 g, 88% yield). H NMR (400 MHz, CDCl
3
) δ 8.45 (s, 1H), 8.16 (d, J = 7.8
2
2
Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 4.84 (d, J = 6.2 Hz, 1H),
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3.64 (q, J = 6.9 Hz, 1H), 2.67 (s, 3H), 1.85ꢀ1.75 (m, 2H), 1.67ꢀ1.57 (m, 2H),
–
1.55ꢀ1.46 (m, 2H), 1.41ꢀ1.31 (m, 2H); LCMS m/z (ESI, negative) found [MꢀH]
266.15; retention time 2.99 min, > 99% pure.
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
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3ꢀ(2ꢀBromoacetyl)ꢀNꢀcyclopentylbenzenesulfonamide (11a). To a solution of 10a (2
2
g, 7.48 mmol) in EtOAc (50 mL) was added CuBr (2 g, 8.98 mmol) and the mixture
was heated to reflux overnight. The mixture was cooled to room temperature and
extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with
2 4
brine (50 mL), dried (Na SO ), filtered and concentrated in vacuo to give the crude
product. The residue was purified by silica gel column chromatography (gradient
1
elution, gradient 5 to 20% EtOAc/ 60ꢀ90 °C petroleum ether) to give 11a as a pale
1
11
yellow solid (1.84 g, 71% yield). H NMR (400 MHz, CDCl
3
) δ 8.47 (t, J = 1.8 Hz,
1
2
1H), 8.20 (dd, J = 7.8, 1.8 Hz, 1H), 8.13 (dd, J = 7.8, 1.9 Hz, 1H), 7.69 (t, J = 7.8 Hz,
1H), 4.71 (d, J = 7.4 Hz, 1H), 4.47 (s, 2H), 3.72ꢀ3.61 (m, 1H), 1.84ꢀ1.77 (m, 2H),
1.68ꢀ1.61 (m, 2H), 1.56ꢀ1.46 (m, 2H), 1.41ꢀ1.32 (m, 2H); LCMS m/z (ESI, negative)
13
1
4
–
15
found [MꢀH] 345.91; retention time 3.26 min, > 98% pure.
1
6
NꢀCyclopentylꢀ3ꢀ(2ꢀoxoꢀ2,3ꢀdihydrothiazolꢀ4ꢀyl)benzenesulfonamide (12a). To a
suspension of KSCN (702 mg, 7.22 mmol) in acetone (20 mL) was added 11a (500
mg, 1.44 mmol), and the mixture was stirred at room temperature for 10 minutes. The
mixture was concentrated in vacuo, extracted with EtOAc (3 × 50 mL). The combined
17
1
8
19
2
0
2 4
organic layers were washed with brine (50 mL), dried (Na SO ), filtered and
21
concentrated. The crude white solid was dissolved in acetic acid (10 mL) and 2 mL
2
2
2
50% H SO
4
solution (v/v), and the mixture was heated to 100 °C for 1 hour. Upon
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0
completion, the mixture was cooled to room temperature. EtOAc (100 mL) was added,
the organic layer was washed with saturated aqueous NaHCO
mL), brine (50 mL), dried (Na SO ), filtered and concentrated in vacuo to give the
crude product. Purification by silica gel column chromatography (gradient elution,
3
(50 mL), water (100
2
4
0
1
2
3
4
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0
gradient 0 to 5% MeOH/CH
190ꢀ191 °C (from CH Cl
2
Cl
2
) to give 12a as a white solid (0.32 g, 69% yield). Mp
1
2
2
and MeOH); H NMR (400 MHz, DMSOꢀd
6
) δ 12.01 (s,
1H), 8.08 (d, J = 1.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H),
7.71ꢀ7.63 (m, 2H), 6.97 (s, 1H), 3.47 (p, J = 6.9 Hz, 1H), 2.09 (s, 2H), 1.66ꢀ1.49 (m,
13
4H), 1.43ꢀ1.35 (m, 2H), 1.33ꢀ1.23 (m, 2H); C NMR (126 MHz, DMSOꢀd ) δ 173.26,
6
1
142.91, 133.07, 130.86, 130.36, 128.88, 126.63, 123.36, 100.78, 54.89, 32.92 (2 × C),
11
16 2 2 3
23.30 (2 × C); HRMS (EI) m/z calcd for C14H N S O : 324.0602, found 324.0603;
1
2
retention time 2.91 min, > 99% pure.
13
Following procedure A as for compound 12a gave compounds 12bꢀ12j.
1
4
NꢀCyclopropylꢀ3ꢀ(2ꢀoxoꢀ2,3ꢀdihydrothiazolꢀ4ꢀyl)benzenesulfonamide (12b): White
1
15
solid, 73% yield; mp 204ꢀ205 °C (from CH
2
Cl
2
and MeOH); H NMR (400 MHz,
1
6
DMSOꢀd
6
) δ 12.04 (s, 1H), 8.08 (s, 1H), 7.97 (d, J = 2.6 Hz, 1H), 7.91 (d, J = 7.8 Hz,
17
1H), 7.78 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 6.98 (s, 1H), 2.16ꢀ2.12 (m, 1H),
13
1
8
0.54ꢀ0.44 (m, 2H), 0.41ꢀ0.31 (m, 2H); C NMR (126 MHz, DMSOꢀd
6
) δ 173.26,
19
141.56, 133.04, 130.96, 130.40, 129.19, 126.94, 123.60, 100.88, 24.66, 5.64 (2 × C);
2
0
12 2 2 3
HRMS (EI) m/z calcd for C12H N S O : 296.0289, found 296.0287; retention time
21
2.65 min, > 99% pure.
2
2
NꢀCyclohexylꢀ3ꢀ(2ꢀoxoꢀ2,3ꢀdihydrothiazolꢀ4ꢀyl)benzenesulfonamide (12c): White
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solid, 70% yield; mp 207ꢀ209 °C (from CH
2 2
Cl and MeOH); H NMR (400 MHz,
DMSOꢀd ) δ 12.02 (s, 1H), 8.09 (t, J = 1.6 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.78 (d,
6
J = 7.8 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 6.97 (d, J = 1.9 Hz,
10
11
12
13
14
15
16
17
18
19
20
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1H), 3.04ꢀ2.98 (m, 1H), 1.59ꢀ1.53 (m, 4H), 1.46ꢀ1.42 (m, 1H), 1.20ꢀ1.07 (m, 4H),
13
1.19ꢀ1.00 (m, 1H); C NMR (126 MHz, DMSOꢀd
6
) δ 173.26, 143.62, 133.08, 130.83,
130.37, 128.79, 126.42, 123.08, 100.78, 52.47, 33.71 (2 × C), 25.29 (2 × C), 24.79 (2
18 2 2 3
× C); HRMS (EI) m/z calcd for C15H N S O : 338.0759, found 338.0767; retention
time 3.19 min, > 99% pure.
Nꢀ(4ꢀMethoxyphenyl)ꢀ3ꢀ(2ꢀoxoꢀ2,3ꢀdihydrothiazolꢀ4ꢀyl)benzenesulfonamide (12d):
1
1
White solid, 80% yield; mp 221ꢀ222 °C (from CH
MHz, DMSOꢀd
7.66ꢀ7.55 (m, 2H), 6.99 (d, J = 8.0 Hz, 2H), 6.89 (s, 1H), 6.81 (d, J = 8.0 Hz, 2H),
2
Cl
2
and MeOH) ; H NMR (400
11
6
) δ 11.99 (s, 1H), 9.98 (s, 1H), 8.00 (s, 1H), 7.84 (d, J = 7.3 Hz, 1H),
1
2
13
13
3.66 (s, 3H); C NMR (126 MHz, DMSOꢀd
6
) δ 173.21, 157.04, 140.79, 132.89,
1
4
130.89, 130.32, 130.30, 129.35, 126.79, 123.94 (2 × C), 123.55, 114.79 (2 × C),
15
14 2 2 4
100.90, 55.60; HRMS (EI) m/z calcd for C16H N S O : 362.0395, found 362.0387;
1
6
retention time 2.98 min, > 99% pure.
17
Nꢀ(4ꢀAminophenyl)ꢀ3ꢀ(2ꢀoxoꢀ2,3ꢀdihydrothiazolꢀ4ꢀyl)benzenesulfonamid
(12e):
1
1
8
White solid, 50% yield; mp 206ꢀ207 °C (from CH
2
Cl
2
and MeOH) ; H NMR (400
19
MHz, DMSOꢀd ) δ 11.98 (brs, 1H), 9.53 (s, 1H), 7.93 (s, 1H), 7.84ꢀ7.80 (m, 1H),
6
2
0
7.61ꢀ7.56 (m, 2H), 6.85 (s, 1H), 6.69 (d, J = 8.4 Hz, 2H), 6.39 (d, J = 8.4 Hz, 2H),
13
21
6
4.99 (brs, 2H); C NMR (126 MHz, DMSOꢀd ) δ 173.22, 147.14, 141.04, 133.01,
2
2
130.75, 130.10, 129.11, 126.92, 125.43, 125.18 (2 × C), 123.62, 114.43 (2 × C),
29
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