Discovery and potency optimization of 2-amino-5-arylmethyl-1,3-thiazole derivatives as potential therapeutic agents for prostate cancer

Article abstract of DOI:10.1002/ardp.200800201

A new chemical series was identified via high-throughput screening as having antiproliferative activity on DU-145 human prostate carcinoma cell line (hit compound potency - 2.9 μM). Medicinal chemistry optimization of two peripheral diversity vectors of t

Full text of DOI:10.1002/ardp.200800201

420
Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
Full Paper
Discovery and Potency Optimization of 2-Amino-5-arylmethyl-
1,3-thiazole Derivatives as Potential Therapeutic Agents for
Prostate Cancer
Mikhail Krasavin1, Ruben Karapetian2, Igor Konstantinov1, Yuri Gezentsvey1, Konstantin
Bukhryakov1, Elena Godovykh2, Olga Soldatkina2, Yan Lavrovsky2, Andrei V. Sosnov1,
and Andrei A. Gakh3
1 Department of Medicinal Chemistry, Chemical Diversity Research Institute, Khimki, Moscow Reg., Russia
2 Department of Lead Discovery, Chemical Diversity Research Institute, Khimki, Moscow Reg., Russia
3 Oak Ridge National Laboratory, Oak Ridge, TN, USA
A new chemical series was identified via high-throughput screening as having antiproliferative
activity on DU-145 human prostate carcinoma cell line (hit compound potency – 2.9 lM). Medic-
inal chemistry optimization of two peripheral diversity vectors of the hit molecule, independ-
,
ently, led to SAR generalizations and identification of the best’ moieties. The latter were merged
in a single compound that exhibited an over 100-fold better potency than the hit compound.
For the most potent compounds it was confirmed that the observed antiproliferative potency
was not associated with the compounds' non-specific cytotoxicity.
Keywords: Antiproliferative activity / Cytotoxicity / Drug-likeness / Prostate cancer / Small molecules /
Received: November 15, 2008; accepted: February 12, 2009
DOI 10.1002/ardp.200800201
molecules as well as antibodies targeted at disrupting
Introduction
vital signaling pathways in cancerous cells have a poten-
tial to provide a new basis for innovative treatment of
prostate cancer (and other proliferative disorders) in the
years to come [4].
Prostate cancer is the number one cancer diagnosed in
men today. While it occurs to certain extent throughout
the world (least commonly in Eastern / Southern Asia), it
is viewed as the major public health threat in Western
Europe and, especially, the United States [1]. In the US
alone, it has been estimated [2] that 186,295 new cases of
prostate cancer (mostly – among men over fifty) were
diagnosed in 2008, accounting for 25% of all cancers
diagnosed in men that year and 10% of the total cancer-
related mortality. Appropriate diet (including dietary
supplements) and exercise are currently the common
themes for prostate cancer prevention while classical
treatments are limited to surgery, radiation therapy, and
hormone therapy. Chemotherapy of late-stage prostate
cancer is still largely experimental; however, it may lead
to increased survival in the future [3]. Specifically, small
The present study was part of an ongoing effort in our
laboratories to find novel antiproliferative agents as
potential treatments for cancer. It was aimed at identify-
ing new small heterocyclic molecules in the Chemical
Diversity Research Institute collection [5] that would be
specifically inhibitory to DU-145 human prostate carci-
noma cell line (a ,classical' cell line of prostate cancer) [6]
while exhibiting no non-specific (general) cytotoxicity.
High-throughput screening of a highly diverse set of over
7000 compounds comprising over 200 chemical classes
led to several confirmed hit classes. Among these, two
closely related thiazole compounds, 1 and 2 (Fig. 1), that
exhibited inhibition of DU-145 cell proliferation in dose-
response manner, attracted our attention due to their
drug-likeness [7], structural simplicity, presence in their
structure of two distinct types of peripheral appendages
(thus allowing for informative SAR exploration), and syn-
theses reported in the literature for related structures.
Correspondence: Mikhail Krasavin, Department of Medicinal Chemistry
– Chemical Diversity Research Institute, Rabochaya St. 2a, Khimki, Mos-
cow Reg., 141400, Russia.
E-mail: myk@chemdiv.com
Fax: +7 495 626 9780
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
Discovery and Potency Optimization of 2-Amino-5-arylmethyl-1,3-thiazoles
421
Table 1. Structures and DU-145 proliferation inhibition data of
compounds 3a–m.
Figure 1. Structures of the hit compounds identified in DU-145
cell proliferation HTS.
Compound
R1
R2
IC50 (lM)a)
3a
NA
Herein, we report on the syntheses of several dozen
analogs of the hit 2-acylamino-5-arylmethyl-1,3-thiazole
compounds 1 and 2, leading to the initial SAR generaliza-
tions for this chemical class, significant improvement of
the inhibitory potency, and to confirmation that even for
the most potent compounds synthesized in this work,
their inhibition of DU-145 proliferation was due to a spe-
cific effect of these compounds on the cancerous cells
rather than general cytotoxicity.
3b
3c
14.3 l 8.7
5.2 l 0.8
3d
3e
0.9 l 0.3
1.7 l 0.1
Results and discussion
To confirm that the observed activity of 1 and 2 was
indeed associated with the 2-acylamino-5-arylmethyl-1,3-
thiazole chemotype and to obtain initial SAR clues for
further potency optimization, we selected and screened
13 close analogs 3a–m from our screening collection [5].
The results are summarized in the Table 1.
A number of important observations emerged from
this preliminary SAR information. Firstly, the antiproli-
ferative potency of the compounds tested is quite sensi-
tive to small structural variations both in the benzyl and
the acylamino portions. For example, the potency of 3g
drops over ten-fold when just one chlorine atom is
removed from the benzyl group (as in 3f). Secondly, there
seems to a preference for a rigid two-atom linker (as in 3i,
3j, 3l, or 3m) between the (hetero)aromatic group and the
carbonyl of the acylamino portion over a flexible one. At
the same time, there is a clear preference for para-substi-
tution in the benzyl portion (compare 3b and 3h), with
the most potent compound so far (3k) having a para-flu-
oro substituent.
3f
11.9 l 6.1
1.2 l 0.3
3g
3h
3i
1.00 l 0.05
0.79 l 0.16
0.90 l 0.08
0.62 l 0.08
1.7 l 0.3
3j
3k
3l
We decided to undertake further SAR exploration of
the series in the following two experimental cohorts: i)
the benzyl portion will be kept intact while acylamino
group variations are explored; ii) the acyl group will be
kept unchanged while more analogs with variations in
the benzyl portion are prepared.
3m
0.9 l 0.2
According to the literature analogy [8, 9], the 2-amino-
5-benzyl-1,3-thiazole precursors 4 substituted at the ben-
zyl portion, can be synthesized in two steps from readily
available anilines 5 as shown in Scheme 1. The respective
diazonium salts 6 were reacted with acrolein in the pres-
a)
IC50 have been evaluated for standard error. The values
reported are the mean of two determinations, standard error
provided; NA: not active.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

422
M. Krasavin et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
Scheme 1. Preparation of 2-amino-5-benzyl-1,3-thiazoles 4a–j.
ence of cupric chloride in biphasic toluene-water
medium to provide respective 3-aryl-2-chloropropanals 7.
These, after reaction workup and isolation of the crude
material, were reacted, without further purification,
with thiourea in refluxing ethanol to provide the requi-
site 2-amino-5-benzyl-1,3-thiazole core building blocks 4
in moderate to good yields. Thus, we introduced two
notable improvements to the original procedure [8, 9],
namely, the use of the biphasic toluene-water reaction
medium throughout the syntheses, and avoidance of the
purification of intermediates 7 by distillation. These,
however, lead to virtually no yield improvement com-
pared to the originally described procedure but rather
simplified the syntheses operationally. Having synthe-
sized the core 2-aminothiazoles 4, we proceeded to pre-
Scheme 2. Acylation of 2-amino-5-benzyl-1,3-thiazoles 4.
pare derivatives of them in SAR-informative manner, as response experiments to determine their IC50 values
outlined above.
For the acylamino portion variations, we chose 4-fluo-
(Table 2).
For the optimization of the benzyl group, we chose to
robenzyl-substituted core 4a, based on high potency of keep the (3-chromanoyl)amino portion unchanged,
the compound 3k (IC50 = 0.62 lM) in the initial screening based on the high potency of compound 3i (IC50
=
set. In terms of further acylation, we found 4a to be 0.79 lM) in the initial screening set. The core thiazole-2-
rather non-reactive even toward acyl chlorides amines 4a–j were efficiently acylated with 3-chromanoic
(Scheme 2, method A). Additionally, for a wider variety of acid using method B (Scheme 2) and their activity was
acyl groups, it would be desirable if the acylation could measured in DU-145 proliferation-inhibition assay to
be achieved using carboxylic acids directly. We discov- directly determine their IC50 values (unlike for the acyla-
ered that a range of carboxylic acids were effective acylat- mino portion optimization, we were limited here by the
ing agents toward 4a when activated with 1,19-carbonyl- availability of core amines 4). The results of these find-
bis(3-methyl-1H-imidazol-3-ium triflate) 8 that was pre- ings are summarized in the Table 3.
pared in situ by treatment of CDI (1,19-carbonylbis-1H-imi-
At this point, it was clear that 2-(2-furyl)ethenoyl group
dazole) with methyl triflate [10] under microwave irradi- gave the best results among the variety of the acyl groups
ation (Scheme 2, method B). We synthesized a series of screened (9e, IC50 = 0.25 lM) while 4-methoxybenzyl
over 30 N-acyl derivatives of 4a using method B and group (10b, IC50 = 0.059 lM) provided the best inhibitory
screened them for DU-145 proliferation inhibition at potency over other nine substituted benzyl groups
10 lM concentration. Eight compounds 9a–h displaying screened. To test if these SAR findings would have a con-
over 50% inhibition at 10 lM were selected for dose- vergent effect on the potency, compound 11 incorporat-
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
Table 2. Optimization of the acylamino portion.
Discovery and Potency Optimization of 2-Amino-5-arylmethyl-1,3-thiazoles
423
Table 3. Optimization of the benzyl portion.
Compound
R
Yield (%)
67
IC50 (lM)a)
1.8 l 0.6
0.7 l 0.1
Compound
Starting amine Yield (%)
IC50 (lM)a)
10a
10b
10c
10d
10e
10f
10g
10h
10i
4a
4b
4c
4d
4e
4f
45
56
75
64
77
42
50
55
67
71
0.52 l 0.13
9a
9b
0.059 l 0.009
0.095 l 0.001
0.26 l 0.02
NA
0.35 l 0.04
0.40 l 0.01
0.48 l 0.02
0.7 l 0.1
93
4g
4h
4i
9c
72
88
0.75 l 0.35
1.7 l 0.3
10j
4j
NA
a)
§ IC50 have been evaluated for standard error. The values
reported are the mean of two determinations, standard error
provided; NA: not active.
9d
9e
9f
65
95
92
63
0.25 l 0.01
3.55 l 0.05
7.6 l 0.5
gate if the antiproliferative activity of the compounds
was due to some non-specific cytotoxicity. To this end,
the cytotoxicity of the most potent compounds (9e, 10b
and 11 with IC50 values of 0.25, 0.059, and 0.015 lM,
respectively) was measured on HepG2 and DU-145 cell
lines [11] using a known cytotoxic digitonin, as positive
control. As it is evident from Fig. 3, none of these com-
pounds exhibited over 20% cytotoxicity at concentra-
tions as high as 50 lM, while digitonin itself caused 100%
cell death in nanomolar concentration range. Thus, the
observed antiproliferative activity of the most potent
compounds in the series is not due to non-specific cyto-
toxicity but must be rather exerted via a specific cellular
target and is selective of the abnormally dividing human
prostate carcinoma cells.
9g
9h
partial
a)
IC50 have been evaluated for standard error. The values
reported are the mean of two determinations, standard error
provided.
Conclusion
In the present work, we have undertaken SAR-guided
optimization of the inhibitory potency of the new class
of antiproliferative chemical series, 2-acylamino-5-aryl-
methyl-1,3-thiazoles. The optimization was undertaken
by selecting two best moieties for the benzyl and the acyl-
amino portion from the early screening set and optimiz-
ing the two portions independently. This two-vector SAR
exploration led to identification of the new moieties
which, when combined in the same compound 11, led to
significant improvement of the DU-145 proliferation-
inhibitory potency (two orders of magnitude starting
from the initial hit compounds 1 and 2). We also demon-
strated that the observed antiproliferative activity of the
Figure 2. The ”best” SAR-merging compound 11.
ing both of the ,best' groups was synthesized (Fig. 2). Its
IC50 measured at 15 nM in the DU-145 proliferation-inhib-
ition assay, thus confirming the additive effects of the
two portions of the molecule on the potency and the full
applicability of the independent two-vector optimization
strategy undertaken here.
Having improved the antiproliferative potency of the
series by two orders of magnitude, we set off to investi-
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

424
M. Krasavin et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
and were not corrected. Analytical thin-layer chromatography
was carried out on EM Separations Technology (Gibbstown, NJ,
USA) F254 silica gel plates. Compounds were visualized with short-
wavelength UV light. 1H-NMR and 13C-NMR spectra were recorded
on Bruker DPX-300 spectrometers (Bruker Bioscience, USA) in
DMSO-d6 using TMS as an internal standard. LCMS analyses were
obtained on a PE SCIEX API 150EX mass spectrometer (Perkin-
Elmer, Norwalk, CT, USA) following separation on a Shimadzu
LC-10AD liquid chromatography system (Shimadzu, Tokyo,
Japan) equipped with Shimadzu SP D-10A UV-Vis detector
(254 nm) and Sedex 75 ELSD detector. Elemental analyses were
obtainedatResearchInstituteforChemicalCrop Protection(Mos-
cow, Russia) using Carlo Erba Strumentazione 1106 analyzer
(Carlo Erba, Milan, Italy). All solvents and reagents were obtained
from commercialsources andused withoutpurification.
The prostate cancer Du-145 and hepatocellular carcinoma
HepG2 cell lines were purchased from the American Type Cell
Collection (HTB-81 and HB-8065 respectively). Du-145 was cul-
tured in RPMI-1640 complemented with 10% fetal bovine serum
and 2 mM L-Glutamine, HepG2 in DMEM complemented with
10% fetal bovine serum and 2 mM L-Glutamine. All the cell lines
were cultured at 378C in a 5% CO2. All screening manipulations
were done using Biomek FX and Biomek 2000 Laboratory Auto-
mated Workstations (Beckman Coulter Inc., Fullerton, CA, USA).
Fluorescence was measured on Wallac 1420 multilabel counter
(Perkin-Elmer).
Chemistry
General procedure for the synthesis of 5-arylmethyl-1,3-
thiazole-2-amines 4a–j
A three-necked round-bottomed flask equipped with an addition
funnel, septum, and a gas outlet was charged with cuprous
chloride dihydrate (1 g, 5.8 mmol). It was suspended in 5 mL of
toluene and acrolein (1.35 mL, 20 mmol) was added via syringe.
Vigorous stirring was initiated and an ice-cold aqueous solution
of the respective arenediazonium chloride 6 (prepared by diazo-
tization of 20 mmol of the corresponding aniline 5) was placed
in the addition funnel and added to the reaction mixture at
such a rate as to keep the exothermic reaction temperature
around ambient. After the addition was complete, the stirring
was continued for another 3 h. At that point the toluene layer
was separated and the aqueous layer was extracted with ether
(2625 mL). The combined organic extracts were dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The pres-
ence of the target 3-aryl-2-chloropropanals 7 was established by
characteristic C-2 proton triplets around 4.5 ppm in the 1H-NMR
spectrum of the crude products (CDCl3).
Figure 3. Cytotoxicity of the most active compounds prepared
in this work and digitonin measured on HepG2 (A) and DU-145
(B) cells.
compounds was not due to non-specific cytotoxicity. The
compound 11 resulting from the present work thus rep-
resents a promising new lead for development of novel
therapeutic agents for treatment of prostate cancer. The
specific cellular mechanism of action of this compound
remains to be investigated.
This research was supported by the Global IPP program
through the International Science and Technology Center
(ISTC). Oak Ridge National Laboratory is managed and oper-
ated by UT-Battelle, LLC, under U.S. Department of Energy con-
tract DE-AC05-00OR22725. This paper is a contribution from
the Discovery Chemistry Project.
This material was dissolved in ethanol (20 mL) and thiourea
(1.6 g, 0.21 mmol) was added. The homogeneous mixture was
brought to reflux and kept at that temperature for 2 h. Upon
cooling to rt, water (100 mL) was added to the reaction mixture
and it was then neutralize with 28% aq. NH4OH. The resulting
precipitate was removed by filtration, air-dried, and crystallized
from chloroform.
The authors have declared no conflict of interest.
1
Compounds 4a–b, 4d–h had their H-NMR data (DMSO-d6)
consistent with the previously reported [8, 9] data. The identity
of these compounds was further confirmed by LCMS analyses.
Data for new compounds are provided below.
Experimental
5-(4-Isopropylbenzyl)-1,3-thiazol-2-amine 4c
All reactions were run in oven-dried glassware in atmosphere of
nitrogen. Melting points were measured with a Bꢀchi -520 melt-
ing point apparatus (Bꢀchi Labortechnik, Flawil, Switzerland)
LCMS [M + H+] 233. 1H-NMR (300 MHz, DMSO-d6) d: 7.12 (q, J = 8.2
Hz, 4H), 6.73 (s, 1H), 6.64 (s, 2H), 3.8 (s, 2H), 2.81 (ddd, J = 13.8, 7.1,
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
Discovery and Potency Optimization of 2-Amino-5-arylmethyl-1,3-thiazoles
425
7.0 Hz, 1H), 1.24 (d, J = 6.9 Hz, 6 H);13C-NMR (75 MHz, DMSO-d6) d:
167.4, 146.8, 136.8, 135.0, 127.8, 127.5, 126.2, 33.3, 32.5, 23.64.
N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]-4,5,6,7-
tetrahydro-1-benzothiophene-3-carboxamide 9d
1
LCMS [M + H+] 373. H-NMR (300 MHz, DMSO-d6) d: 11.90 (s, 1H),
8.21 (s, 1H), 7.23 (dd, J = 6.0, 9.0 Hz, 2H), 7.08 (s, 1H), 6.85 (t, J = 9.0
Hz, 2H), 4.05 (s, 2H), 2.85 (m, 2H), 2.72 (m, 2H), 1.70–1.85 (m, 4H).
5-[2-Chloro-5-(trifluoromethyl)benzyl]-1,3-thiazol-2-
amine 4i
LCMS [M + H+] 293. 1H-NMR (300 MHz, DMSO-d6) d: 7.43–7.50 (m,
3H), 6.82 (s, 1H), 5.42 (s, 2H), 4.11 (s, 2H); 13C-NMR (75 MHz,
DMSO-d6) d: 167.7, 138.1, 137.1, 136.2, 129.7, 128.9 (q, J = 33.0
Hz), 126.7 (q, J = 3.75 Hz), 124.7 (q, J = 3.5 Hz), 123.7, 121.4 (q, J =
273.0 Hz), 30.4.
(2E)-N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]-3-(2-
furyl)acrylamide 9e
1
LCMS [M + H+] 329. H-NMR (300 MHz, DMSO-d6) d: 11.75 (s, 1H),
7.25 (m, 2H), 7.04 (s, 1H), 6.98 (t, J = 9.0 Hz, 2H), 5.87 (s, 1H), 5.78
(s, 1H), 4.02 (s, 2H), 2.87 (t, J = 9.8 Hz, 2H), 2.68 (t, J = 9.8 Hz, 2H).
5-[4-Chloro-3-(trifluoromethyl)benzyl]-1,3-thiazol-2-
amine 4j
1
LCMS [M + H+] 293. H-NMR (300 MHz, DMSO-d6) d: 7.52 (s, 1H),
4-(4-Chlorophenoxy)-N-[5-(4-fluorobenzyl)-1,3-thiazol-2-
7.46 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 6.81 (s, 1H), 5.48 (s,
2H), 4.02 (s, 2H); 13C-NMR (75 MHz, DMSO-d6) d: 167.8, 138.5,
135.8, 132.3, 131.2, 130.0, 128.0 (q, J = 30.0 Hz), 127.0 (q, J = 5.0
Hz), 125.1, 122.4 (q, J = 273.0 Hz), 32.1.
yl]butanamide 9f
LCMS [M + H+] 406. 1H-NMR (300 MHz, DMSO-d6) d: 11.87 (s, 1H),
7.25 (m, 3H), 7.16 (s, 1H), 7.08 (t, J = 6.0 Hz, 2H), 6.78 (d, J = 6.0 Hz,
2H), 4.07 (s, 2H), 3.95 (t, J = 10.2 Hz, 2H), 2.56 (t, J = 9.8Hz, 2H),
2.02 (m, 2H).
General procedure for acylation of 2-amino-5-arylmethyl-
1,3-thiazoles 4a–j
4-(4-Bromophenoxy)-N-[5-(4-fluorobenzyl)-1,3-thiazol-2-
To
a solution of CDI (1,19-carbonylbis-1H-imidazole; 1.6 g,
yl]butanamide 9g
9.9 mmol) in anhydrous nitromethane (15 mL) was added MeOTf
(3.26 g, 19.9 mmol) at 108C. This reagent {1,19-carbonylbis(3-
methyl-1H-imidazol-3-ium) triflate} was used immediately in
the acylation procedure.
1
LCMS [M + H+] 450. H-NMR (300 MHz, DMSO-d6) d: 11.72 (s, 1H),
7.42 (m, 2H), 7.03 (s, 1H), 6.98 (t, J = 9.0 Hz, 2H), 5.86 (s, 1H), 5.81
(d, J = 12.0 Hz, 1H), 5.69 (s, 1H), 4.02 (s, 2H), 2.80 (m, 2H), 2.65 (m,
2H), 1.85 (m, 1H), 1.53 (m, 1H), 1.14 (d, J = 8.6 Hz, 3H), 1.02 (m,
2H), 0.89 (d, J = 8.6 Hz, 1H), 0.82 (m, 1H), 0.55 (m, 1H), 0.44 (m,
1H).
A suspension or a solution of a carboxylic acid (2 mmol) in
dioxane (1.5 mL) was placed in a 10 mL microwave reactor vessel.
A solution of 1,19-carbonylbis(3-methyl-1H-imidazol-3-ium) tri-
flate (2 mmol) in nitromethane was added and the mixture was
stirred at 408C for 20 min. Then, an appropriate 2-amino-5-aryl-
methyl-1,3-thiazole (2 mmol) in dioxane (2 mL) was added. The
reaction vessel was capped and heated at 708C for 2 h in a Biot-
age InitiatorTM microwave synthesizer operating at 100 W (Biot-
age, Uppsala, Sweden). After cooling to rt, the volatiles were
removed in vacuo and the residue was purified by column chro-
matography on silica gel using an appropriate gradient of meth-
anol in dichloromethane as eluent.
N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]-3-{5-[trans-2-
methylcyclopropyl]-2-furyl}propanamide 9h
LCMS [M + H+] 385. 1H-NMR (300 MHz, DMSO-d6) d: 11.82 (s, 1H),
7.38 (d, J = 6.0 Hz, 2H), 7.28 (t, J = 9.2 Hz, 2H), 7.15 (s, 1H), 7.07 (t,
J = 9.2 Hz, 2H), 6.85 (d, J = 6.0 Hz, 2H), 4.08 (s, 2H), 3.97 (t, J = 9.6
Hz, 2H), 2.52 (t, J = 9.2Hz, 2H), 2.03 (m, 2H).
N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]chromane-3-
(2E)-N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]-3-(5-methyl-
carboxamide 10a
LCMS [M + H+] 369. 1H-NMR (300 MHz, DMSO-d6) d: 12.23 (s, 1H),
7.24 (m, 2H), 7.19 (s, 1H), 7.02–7.08 (m, 4H), 6.82 (ddd, J = 12.0,
12.0, 1.2 Hz, 1H), 6.75 (dd, J = 12.0, 1.2 Hz, 1H), 4.37 (m, 1H), 4.11
(s, 2H), 4.06 (m, 1H), 2.79–3.22 (m, 3H).
2-furyl)acrylamide 9a
LCMS [M + H+] 343. 1H-NMR (300 MHz, DMSO-d6) d: 7.27 (d, J = 16.0
Hz, 1H), 7.25 (dd, J = 7.5, 9.0 Hz, 2H), 7.07 (s, 1H), 6.96 (t, J = 9.0 Hz,
2H), 6.57 (s, 1H), 6.55 (d, J = 16.0 Hz, 1H), 6.12 (s, 1H), 4.03 (s, 2H),
2.47 (s, 3H) [amide protons in exchange with H2O].
N-[5-(4-Methoxybenzyl)-1,3-thiazol-2-yl]chromane-3-
N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]-3,4-
carboxamide 10b
dimethoxybenzamide 9b
1
LCMS [M + H+] 381. H-NMR (300 MHz, DMSO-d6) d: 12.18 (s, 1H),
LCMS [M + H+] 373. 1H-NMR (300 MHz, DMSO-d6) d: 12.10 (s, 1H),
7.72 (d, J = 9.0 Hz, 1H), 7.69 (s, 1H), 7.27 (dd, J = 6.0, 12.0 Hz, 2H),
7.15 (s, 1H), 7.03 (t, J = 12.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 1H), 4.12 (s,
2H), 3.73 (s, 3H), 3.70 (s, 3H).
7.02–7.18 (m, 5H), 6.84 (dd, J = 9.0, 4.2 Hz, 2H), 6.80 (m 1H), 6.74
(d, J = 9.0 Hz, 1H), 4.36 (m, 1H), 4.01 (s, 2H), 4.00 (m, 1H), 2.80–
3.25 (m, 3H).
(2E)-N-[5-(4-Fluorobenzyl)-1,3-thiazol-2-yl]-3-(3-
N-[5-(4-Isopropylbenzyl)-1,3-thiazol-2-yl]chromane-3-
methylphenyl)acrylamide 9c
carboxamide 10c
1
1
LCMS [M + H+] 353. H-NMR (300 MHz, DMSO-d6) d: 11.92 (s, 1H),
LCMS [M + H+] 393. H-NMR (300 MHz, DMSO-d6) d: 12.30 (s, 1H),
7.62 (d, J = 16.0 Hz, 1H), 7.36 (d, J = 6.0 Hz, 1H), 7.32 (s, 1H), 7.26 (t,
J = 6.0 Hz, 2H), 7.14 (d, J = 6.0 Hz, 1H), 7.09 (s, 1H) 7.01 (t, J = 9.0
Hz, 2H), 6.83 (d, J = 16.0 Hz, 1H), 4.04 (s, 2H), 2.38 (s, 3H).
7.04–7.18 (m, 7H), 6.83 (t, J = 7.2 Hz, 1H), 6.74 (d, J = 7.2 Hz, 1H),
4.25 (m, 1H), 4.07 (s, 2H), 4.04 (m, 1H), 2.82–3.25 (m, 3H), 2.78 (m,
1H), 1.15 (d, J = 6.7 Hz, 6H).
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

426
M. Krasavin et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
N-[5-(4-Ethylbenzyl)-1,3-thiazol-2-yl]chromane-3-
Biology
carboxamide 10d
Biological assay to determine the inhibition of proliferation
of the DU-145 cells
LCMS [M + H+] 379. 1H-NMR (300 MHz, DMSO-d6) d: 12.21 (s, 1H),
7.02–7.19 (m, 7H), 6.82 (t, J = 7.7 Hz, 1H), 6.73 (d, J = 7.7 Hz, 1H),
4.38 (m, 1H), 4.08 (s, 2H), 4.06 (m, 1H), 2.83–3.15 (m, 3H), 2.64 (q,
J = 7.5 Hz, 2H), 1.12 (t, J = 7.5 Hz, 3H).
Du-145 cells were plated in a 384-well plate at the density of
4000 cells per well. 4 mM solutions of compounds in DMSO were
diluted 100 times with medium and added to cells to a final con-
centration of 20 lM (40 lL of cell culture plus 40 lL of com-
pound solution, final concentration of DMSO = 0.5%). Taxol at a
final concentration of 1 lM was used as positive control. Cells
were incubated with compounds for three days. Alamar Blue
was then added to the cells to a final concentration of 50 lM.
After incubation for 4–6 h at 378C, plate fluorescence was read
using fluorescence plate reader Wallac 1420 (Perkin-Elmer Life
Sciences; 530 nm excitation filter, 590 nm emission filter). Pro-
liferation inhibition was calculated using formula:
N-(5-Benzyl-1,3-thiazol-2-yl)chromane-3-carboxamide
10e
LCMS [M + H+] 351. H-NMR (300 MHz, DMSO-d6) d: 12.18 (s, 1H),
1
7.18–7.38 (m, 6H), 7.35 (m, 2H), 6.83 (t, J = 7.2 Hz, 1H), 6.72 (d, J =
7.2 Hz, 1H), 4.37 (m, 1H), 4.11 (s, 2H), 3.96 (m, 1H), 2.85–3.17 (m,
3H).
% INH = 1006((Fnegative –compound signal)/(Fnegative Fpositive))
N-[5-(4-Methylbenzyl)-1,3-thiazol-2-yl]chromane-3-
carboxamide 10f
Fnegative: DMSO was added to cells (viable cells)
1
LCMS [M + H+] 365. H-NMR (300 MHz, DMSO-d6) d: 12.15 (s, 1H),
7.04–7.40 (m, 7H), 6.82 (t, J = 7.4 Hz, 1H), 6.73 (d, J = 7.4 Hz, 1H),
4.35 (m, 1H), 4.05 (s, 2H), 4.04 (m, 1H), 2.83–3.15 (m, 3H), 2.37 (s,
3H).
F
positive: taxol (1lM) was added to cells (number of cells at the
first day of incubation).
Cytotoxicity assay
Du-145 or HepG2 cells were plated in 384-well plate at the den-
sity of 10000 or 20000 cells per well, respectively. Serial dilu-
tions (200 times) of tested compounds in DMSO were prepared.
After that, compounds were diluted 100 times in medium and
added to the cells (40 lL of cell culture plus 40 lL of compound
solution, final concentration of DMSO = 0.5%). Cells were incu-
bated with compounds overnight. Known toxic compound digi-
tonin was used as control. The next day, Alamar Blue was added
to cells to final concentration of 50 lM. After incubation for 4–6
hours at 378C, plate fluorescence was read using fluorescence
plate reader Wallac 1420 (Perkin-Elmer) (530 nm excitation fil-
ter, 590 nm emission filter). Compound cytotoxity was calcu-
lated using formula:
N-[5-(4-Chlorobenzyl)-1,3-thiazol-2-yl]chromane-3-
carboxamide 10g
1
LCMS [M + H+] 386. H-NMR (300 MHz, DMSO-d6) d: 12.23 (s, 1H),
7.06–7.32 (m, 7H), 6.80 (m, 1H), 6.65 (m, 1H), 4.40 (m, 1H), 4.10 (s,
2H), 4.02 (m, 1H), 2.85 – 3.15 (m, 3H).
N-[5-(3-Methylbenzyl)-1,3-thiazol-2-yl]chromane-3-
carboxamide 10h
LCMS [M + H+] 365. 1H-NMR (300 MHz, DMSO-d6) d: 12.18 (s, 1H),
7.12–7.24 (m, 7H), 6.84 (m, 1H), 6.74 (m, 1H), 4.36 (m, 1H), 4.08 (s,
2H), 4.04 (m, 1H), 2.92–3.17 (m, 3H), 2.32 (s, 3H).
%TOX = 1006((Fnegative –compound signal)/(Fnegative Fpositive))
Fnegative: DMSO was added to cells (viable cells)
N-{5-[2-Chloro-5-(trifluoromethyl)benzyl]-1,3-thiazol-2-
yl}chromane-3-carboxamide 10i
1
LCMS [M + H+] 454. H-NMR (300 MHz, DMSO-d6) d: 12.28 (s, 1H),
Fpositive: digitonin was added to cells (dead cells).
7.77 (s, 1H), 7.65 (m, 2H), 7.25 (s, 1H), 7.07 (m, 2H), 6.77 (t, J = 6.8
Hz, 1H), 6.68 (d, J = 6.8 Hz, 1H), 4.38 (m, 1H), 4.25 (s, 2H), 4.00 (m,
1H), 2.87–3.08 (m, 3H).
References
[1] D. M. Parkin, Oncogene 2004, 38, 6329–6340.
N-{5-[4-Chloro-3-(trifluoromethyl)benzyl]-1,3-thiazol-2-
[2] A. Jemal, R. Siegel, E. Ward, Y. Hao, et al., CA Cancer J. Clin.
2008, 58, 71–96 (early–2008 estimate); Recent update
“Cancer Facts and Figures 2008” is available at www.cancer.-
org.
yl}chromane-3-carboxamide 10j
1
LCMS [M + H+] 454. H-NMR (300 MHz, DMSO-d6) d: 12.25 (s, 1H),
7.70 (s, 1H), 7.58 (m, 2H), 7.25 (s, 1H), 7.05 (m, 2H), 6.80 (t, J = 9.0
Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 4.41 (m, 1H), 4.17 (s, 2H), 4.02 (m,
1H), 2.84 – 3.12 (m, 3H).
[3] C. Moon, J. C. Park, Y. K. Chae, J. H. Yun, S. Kim, Cancer Lett.
2008, 266, 116–134.
[4] F. L. Chen, A. J. Armstrong, D. J. George, Cancer J. 2008, 14,
40–45.
(2Z)-3-(2-Furyl)-N-[5-(4-methoxybenzyl)-1,3-thiazol-2-
[5] Parts of the screening collection can be accessed at
www.chemdiv.com.
yl]acrylamide 11
1
LCMS [M + H+] 341. H-NMR (300 MHz, DMSO-d6) d: 12.05 (s, 1H),
7.75 (s, 1H), 7.43 (d, J = 12.2 Hz, 1H), 7.17 (m, 3H), 6.84 (m, 3H),
6.66 (d, J = 12.2 Hz, 1H), 6.58 (s, 1H), 4.02 (s, 2H), 3.62 (s, 3H).
[6] F. Alimirah, J. Chen, Z. Basrawala, H. Xin, D. Choubey,
FEBS Lett. 2006, 580, 2294–2300.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 420–427
Discovery and Potency Optimization of 2-Amino-5-arylmethyl-1,3-thiazoles
427
[7] W. Walters, P. Ajay, M. A. Murcko, Curr. Opin. Chem. Biol.
1999, 3, 384–387.
[11] For cytotoxicity experiments, higher seeding densities
(10000 or 20000 cells per well) were used (see Experimen-
tal, section 4.2.2) to more accurately observe potentially
diminishing number of cells. This is in contrast with the
measurement of antiproliferative activity where initial
seeding density (4000 cells per well) was chosen to allow
more room for cell proliferation.
[8] N. D. Obushak, V. S. Matiichuk, N. I. Ganushchak, Russ. J.
Org. Chem. 1997, 33, 1010–1013.
[9] N. D. Obushak, V. S. Matiichuk, R. Y. Vasylyshin, Y. V. Osta-
pyuk, Russ. J. Org. Chem. 2004, 40, 383–389.
[10] A. K. Saha, H. Rapoport, P. Schultz, J. Am. Chem. Soc. 1989,
111, 4856–4859.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com