Exploration of novel 2-alkylimino-1,3-thiazolines: T-type calcium channel inhibitory activity

Article abstract of DOI:10.1021/cc100041m

We have developed combinatorial libraries of new 2-alkylimino-1,3- thiazolines with four diversity points, consisting of more than 500 compounds, in a parallel synthetic fashion. The synthetic strategy was based on the construction of a large library aimed at the discovery of new compounds with T-type calcium channel inhibitory activity through structure modifications of hit compound 2. The syntheses of the compounds of Chemset A with four diversity points were accomplished by the condensation of thioureas 5 with α-haloketones 6{1-66} having two diversity points each. A library of phthalimidyl 1,3-thiazolines 24 was synthesized to provide Chemset B, which allowed the introduction of other diversity points through the nucleophilic character of the amino nitrogen. A sublibrary, Chemset C, was constructed from the libraries of Chemset A and Chemset B by functionalization of the C-4 position of the 1,3-thiazoline ring. The products containing ester or acid groups at the C-4 position of the 1,3-thiazoline ring were used in amide synthesis to give a new sublibrary within Chemset C. Deprotection of the phthalimidyl moiety of 24 followed by the reaction with benzoyl chloride gave the corresponding sublibrary in Chemset C. Another sublibrary which includes secondary amino derivatives was obtained by reduction of the amide moiety or reductive amination of 23 with phenyl aldehyde. The selected compounds from the generated libraries were evaluated with respect to inhibition of T-type calcium channels, where some of them have exhibited promising activity.

Full text of DOI:10.1021/cc100041m

518
J. Comb. Chem. 2010, 12, 518–530
Exploration of Novel 2-Alkylimino-1,3-thiazolines: T-Type Calcium
Channel Inhibitory Activity
Minsoo Han, Kee Dal Nam, Dongyun Shin, Nakcheol Jeong,† and Hoh-Gyu Hahn*
Organic Chemistry Laboratory, Korea Institute of Science and Technology, Seoul 136-791, Korea
ReceiVed March 10, 2010
We have developed combinatorial libraries of new 2-alkylimino-1,3-thiazolines with four diversity points,
consisting of more than 500 compounds, in a parallel synthetic fashion. The synthetic strategy was
based on the construction of a large library aimed at the discovery of new compounds with T-type
calcium channel inhibitory activity through structure modifications of hit compound 2. The syntheses
of the compounds of Chemset A with four diversity points were accomplished by the condensation of
thioureas 5 with R-haloketones 6{1-66} having two diversity points each. A library of phthalimidyl
1,3-thiazolines 24 was synthesized to provide Chemset B, which allowed the introduction of other
diversity points through the nucleophilic character of the amino nitrogen. A sublibrary, Chemset C,
was constructed from the libraries of Chemset A and Chemset B by functionalization of the C-4 position
of the 1,3-thiazoline ring. The products containing ester or acid groups at the C-4 position of the 1,3-
thiazoline ring were used in amide synthesis to give a new sublibrary within Chemset C. Deprotection
of the phthalimidyl moiety of 24 followed by the reaction with benzoyl chloride gave the corresponding
sublibrary in Chemset C. Another sublibrary which includes secondary amino derivatives was obtained
by reduction of the amide moiety or reductive amination of 23 with phenyl aldehyde. The selected
compounds from the generated libraries were evaluated with respect to inhibition of T-type calcium
channels, where some of them have exhibited promising activity.
amiloride,8 diphenylpiperidine derivatives,9 succinimide de-
Introduction
rivatives,10 fluoxetine, NNC 55-0396,11 3,4-dihydroquinazo-
lines,12 and dioxoquinazoline carboxamides, were reported,
there is no subtype-specific drug yet available. The develop-
ment of an antagonist that acts selectively on T-type calcium
channels would be a key in understanding its function in
many physiological processes.13
Combinatorial chemistry serves as a powerful tool in lead
discovery and lead optimization by allowing rapid generation
of potential candidates for screening. Introduction of high-
throughput biological screening has increased the demand
for the preparation of large numbers of compounds. Among
the broad range of templates, heterocyclic scaffolds represent
the most promising molecules as leading structures for the
discovery of novel synthetic drugs. In particular, 2-imino-
1,3-thiazoline derivatives have been the focus of great interest
because of their remarkable biological properties. For
instance, ꢀ-turn mimic 2-acylimino-1,3-thiazolines exhibited
GPIIb/IIIa antagonist activities1 and 3-hydroxy-1,3-thiazo-
lines exhibited PHD2 antagonistic activitiy.2 This scaffold
has received more attention recently as a potent CB2
cannabinoid receptor agonist3,4 and KCNQ2/Q3 agonist for
treatment of neuropathic pain.5
To identify new small molecule blockers against T-type
calcium channels, we screened a limited set of selected
compounds from our in-house indigenous chemical library.
The compounds were evaluated against HEK293 cells which
stably express both T-type calcium channel CaV3.1 with an
a1G subunit and potassium channel Kir2.1. Primary screening
was conducted with an FDSS6000 assay system which was
developed for high-throughput screening. From the primary
screening, we identified 2-cyclohexylimino-1,3-thiazolines
2 which showed 54% inhibitory activity at a concentration
of 10 µM, while the phenylimino-1,3-thiazolines 1 showed
much weaker activity (-8 to 36%) at the same concentration
(Figure 1).14 With these initial data, we decided to synthesize
analogues of compound 2 for lead optimization.
Calcium channels play a number of critical roles in nervous
system function, including controlling pacemaker activities
in the heart, sleep, hormone secretion, mechanosensation,
and epilepsy.6 Low-voltage-activated calcium channels were
classified as T-type, defined by activation at low membrane
potentials. While several classes of T-type calcium channel
blockers, including mibefradil (withdrawn from the market
because of the toxicity induced by drug-drug interaction),7
* To whom correspondence should be addressed. Fax: +82-2-958-5189.
E-mail: hghahn@kist.re.kr.
Figure 1. T-Type Ca2+ channel inhibitory activity of 2-imino-1,3-
thiazolines.
† Current address: Korea University, Seoul 136-701, Korea.
10.1021/cc100041m  2010 American Chemical Society
Published on Web 05/07/2010

Novel 2-Alkylimino-1,3-thiazolines
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 519
Therefore, we elaborated a strategy for the construction of a
focused chemical library of alkyl (or cycloalkyl) substituents
at the imino nitrogen (R3) on the 2-imino-1,3-thiazolines.
To increase the chemical diversity at the 2-cyclohexylimino-
1,3-thiazoline nucleus using combinatorial strategies, we
planned to modify the four points (I-IV) shown in Figure
2. It seems that the spacer from R1 to the core of 2-imino-
1,3-thiazoline plays an important role in the activity.
The synthetic strategy for the preparation of the core
structure 2-alkylimino-1,3-thiazolines with four diversity
points was based on condensations of R-haloketones with
thioureas (Figure 3).14 Reactions of thioureas 3 and R-ha-
loketones 4 with two diversity points in each afforded
2-imino-1,3-thiazolines bearing four diversity points (I-IV)
in one molecule.
Figure 2. Fragment analysis of the structure of 2-cyclohexylimino-
1,3-thiazoline and targeted libraries of the analogues of the scaffold
2-imino-1,3-thiazoline.
Figure 3. 2-Imino-1,3-thiazoline core with four diversity points.
We hypothesized that our previously described construc-
tion of the 2-alkylimino-1,3-thiazoline core structure14 should
readily afford a library of Chemset A and Chemset B (Figure
4). Also, numerous analogues of the libraries should be
readily prepared through various coupling reactions with a
suitable building block collection and subsequent function-
alization at the C-4 position of the 1,3-thiazoline core to give
sublibrary C. In the case of Chemset B, the nucleophilic
character of the nitrogen of the 4-amino moiety would allow
the diversity of the library to afford an additional sublibrary
within Chemset C.
Syntheses of Building Blocks 3{1-20} and 4{1-66}. The
building blocks thioureas 3 and R-haloketones 4 used in this
study are listed in Figures 5 and 6, respectively. The building
blocks thioureas 3 were easily prepared in quantitative yields
by the reaction of the commercially available primary amines
with isothiocyanates in refluxing ethanol.
The other building blocks, γ-chloroacetoacetanilide de-
rivatives 4{1-47}, were prepared by the previously reported
method.14 The preparation of R-haloketones is summarized
in Scheme 1. New γ-chloroacetoacetamides 4{48-61} were
prepared in 25-82% yields (see the Supporting Information
Figure 4. Chemset A and Chemset B as key compounds for lead
optimization and Chemset C sublibraries thereof.
Results and Discussion
Compound Design and Strategy. Considering the struc-
tures and activities of compounds 1 and 2 (see Figure 1),
one could speculate that the substituent at the 2-imino moiety
would play a critical role in their biological activities.
Figure 5. Thiourea building blocks 3{1-20} used in this study.

520 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Han et al.
Figure 6. γ-Chloroacetoacetamides and R-haloketone building blocks 4{1-66} used in this study.
for the yields and melting points) by dropwise addition of
either aliphatic amines or benzylamine derivatives to a
-78 °C solution of γ-chloroacetoacetyl chloride (1.1 molar
equiv) in methylene chloride in the presence of triethylamine
(1.1 molar equiv) (Scheme 1). These solid products were
isolated by simple filtration and were used for the subsequent

Novel 2-Alkylimino-1,3-thiazolines
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 521
Scheme 1. Preparation of the Building Blocks R-Haloketones
4 Used in This Study
Scheme 2. Synthetic Route of 2-Imino-1,3-thiazolines and
Regiochemistry
reactions without further purification. The structures of the
new derivatives 4{47-61} were confirmed by their 1H NMR
spectroscopy. Building blocks 4{62,64} are commercially
available, and building blocks 4{63,65,66} were prepared
by bromination of the corresponding ketones (see the
Experimental Section for the reaction conditions).15
Syntheses of Chemset A. A synthetic route to 2-alky-
limino-1,3-thiazoline-4-acetamide derivatives 9 by a parallel
combinatorial method is shown in Scheme 2. The reaction
of the prepared building block γ-chloroacetoacetamides
4{1-61} with thioureas 3{1-20} in refluxing ethanol gave
the corresponding 2-alkylimino-1,3-thiazoline-4-acetamides
in quantitative yields. With regard to the regiochemistry of
the product, there are two regioisomers (9 or 10) depending
on the spatial arrangement of the two substituents (R2 and
R3) at the 1,3-thiazoline scaffold. The Hantzsch thiazole
synthesis from the condensation of R-haloketones with mono-
N-substituted thioamides produced 2-aminothiazoles and/or
2-iminothiazolines depending on the acidity of the reaction
medium.16 In case of the reaction of R-haloketones with N,N-
disubstituted thioureas to form 2-imino-1,3-thiazoline, the
regiochemistry of the product is likely dependent on the
relative bulkiness of the two substituents at thiourea nitro-
gens. Presumably, the steric repulsion between the bulky
amide group at C-4 and the substituent at C-3 of the
intermediate 1,3-thiazolidine would be a critical factor that
affects the regiochemistry of the product. Thus, the reaction
of 4 with thioureas 3 provides initially either imino sulfide
5 or 6, by attack of the sulfur in thiourea on the carbon
neighboring chlorine. Intermediates 5-8 are in equilibrium
as shown in Scheme 2. When R2 is smaller than R3, 7 is a
more favorable conformation than 8, because of the possible
steric repulsion between the bulky amide group at C-4
(R1NHCOCH2) and R3 of the 1,3-thiazolidine scaffold in
conformation 8. Accordingly, we concluded that the struc-
tures of the resulting 2-alkylimino-1,3-thiazoline derivatives
would contain the bulkier group at the imino nitrogen and
the smaller group at the C-3 nitrogen. For example, the
reaction of γ-chloroacetoacetanilide(p-phenoxy) 4{12} with
N-cyclopropyl-N′-adamantylthiourea 3{16} afforded the cor-
responding 2-imino-1,3-thiazoline 9{16,12} in which the
bulky adamantyl group was substituted at the imino nitrogen
and the smaller cyclopropyl group was substituted at C-3 of
the 1,3-thiazolidine scaffold. Further proof of the structure
was established by means of an X-ray crystallographic
analysis (Figure 7) of compound 9{16,12} (see the Support-
ing Information for X-ray crystallographic analysis of
additional compound 9{7,3}).
After the completion of the reaction, the desired products,
2-alkylimino-1,3-thiazolines, were isolated by filtration from
the reaction mixture. The obtained solids were the corre-
sponding hydrogen chloride salts of 2-alkylimino-1,3-thia-
zolines, which could be used directly for T-type calcium
channel inhibitory activity screening. We have constructed
a library of 308 compounds in this manner using Carousel
Reaction Stations in a parallel synthetic fashion without
isolating intermediates 7. The isolated yields ranged from 8
to 99% for the final step.
Figure 7. ORTEP plots of 2-adamantylimino-1,3-thiazoline
9{16,12}.

522 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Han et al.
Scheme 3. Preparation of 2-Imino-1,3-thiazoline Derivatives 11-13
Along with the syntheses of 2-alkylimino-1,3-thiazoline-
4-acetamides 9, we prepared analogues 11-13 from the
reaction of thioureas 5 with building blocks 6{63-65} by
the same method described above to give the corresponding
2-alkylimino-1,3-thiazolines 11-13 (Scheme 3).
Table 1. Yields, Purities, and Molecular Weights of
Representative 2-Alkylimino-1,3-thiazoline-4-acetamides 9 and
Their Analogues
entry
compound
yielda
MW (found)b
purityc
1
2
3
4
5
6
7
8
9{1,30}
9{2,7}
9{3,12}
9{4,26}
9{5,6}
80
57
80
74
80
87
84
66
86
78
76
19
74
83
69
93
96
97
466.13857 (MH+)
400.23989 (MH+)
450.21934 (MH+)
420.18538 (MH+)
426.25656 (MH+)
434.22686 (MH+)
380.14093 (MH+)
316.15252 (MH+)
436.20458 (MH+)
332.14342 (MH+)
438.25544 (MH+)
424.24122 (MH+)
450.25715 (MH+)
462.22103 (MH+)
402.25659 (MH+)
466.28870 (MH+)
363.21128d [(M - Br)+]
293.13328d [(M - Br)+]
89
86
85
50
100
61
100
70
88
62
98
92
96
93
87
98
88
92
Figure 8 provides representative structures of the prepared
2-alkylimino-1,3-thiazolines 9 and 11-13, and Table 1 lists
yields and HRMS data. The acid or ester functions in
molecules 11-13 were further used for preparation of an
amide library to increase the chemical diversity of the
synthesized libraries. We synthesized nine compounds as key
intermediates, which were used for the construction of
additional sublibraries within Chemset C through various
coupling reactions and subsequent functionalization at the
C-4 position of the 1,3-thiazoline core. Figure 7 provides
representative structures of the prepared 2-alkylimino-1,3-
thiazolines of Chemset A, and Table 1 lists the yields and
MS data.
9{7,6}
9{9,12}
9{10,1}
9{11,12}
9{9,56}
9{12,7}
9{13,6}
9{16,8}
9{20,12}
9{12,48}
9{16,49}
9{14,62}
12{12,64}
9
10
11
12
13
14
15
16
17
19
a Yields (percent) were calculated on the basis of the weight of the solid
obtained by filtration of the reaction mixture. b Molecular ions were
determined by mass spectrometry (ESI) after removal of the HCl salt by the
treatment of triethylamine. c Purities (percent) were determined by HPLC
at 210 nm. d Molecular ions were determined by mass spectrometry (ESI)
without removal of the HX salt (X ) Cl or Br).
Syntheses of Chemset B. To increase the diversity,
we have synthesized 4-aminomethyl-1,3-thiazoline 15
(see Scheme 4), a key intermediate for the preparation of
new 2-imino-1,3-thiazoline derivatives, by using the
nucleophilic character of its 4-amino moiety. The reaction
Figure 8. Representative structures of the prepared 2-alkylimino-1,3-thiazolines of Chemset A.

Novel 2-Alkylimino-1,3-thiazolines
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 523
Scheme 4. Synthetic Route of 4-Aminomethyl-2-imino-1,3-thiazolines 15
Table 2. Yields, Purities, and Molecular Weights of
Representative Phthalimidyl 2-Imino-1,3-thiazolines 14
compound by TLC (8:2 methanol/chloroform), and its
structure was confirmed by the appearance of a broad
singlet peak at 1.37 ppm with the disappearance of the
multiplet peak at 7.70-7.90 ppm, which corresponds to
the phthalimidyl protons, in the 1H NMR spectrum. Since
4-aminomethyl-1,3-thiazoline 15 was unstable at room
temperature, it was used for the subsequent reaction
without purification. Figure 9 provides representative
structures of the prepared phthalimidyl 1,3-thiazolines,
Chemset B, and Table 2 lists the yields and HRMS data.
entry
compound
yielda
MW (found)b
purityc
1
2
3
4
5
6
7
8
14{1,66}
14{2,66}
14{3,66}
14{8,66}
14{11,66}
14{18,66}
14{19,66}
14{20,66}
14{12,66}
14{13,66}
14{14,66}
14{16,66}
90
94
83
78
87
82
78
71
94
81
79
79
356.14416 [(M - Br)+]
370.15966 [(M - Br)+]
384.17491 [(M - Br)+]
382.15853d (MH+)
84
89
82
93
76
87
90
81
85
87
88
87
370.15900 [(M - Br)+]
384.17586 [(M - Br)+]
398.19069 [(M - Br)+]
e
-
9
400.17383 [(M - Br)+]
422.18996 [(M - Br)+]
436.20564 [(M - Br)+]
434.18997 [(M - Br)+]
10
11
12
Syntheses of Sublibrary Chemset C. Lead optimization
involves structural modifications of a hit compound to lead
compound that has demonstrated desired biological or
pharmacological activities, often using an in vivo and in
vitro assay system. We synthesized 11-13 as shown in
Scheme 3 along with the library of 300 compounds of
2-alkylimino-1,3-thiazoline-4-acetamides 9. The acid or
ester functions in molecules 11-13 were used for the
preparation of an amide library to increase the chemical
diversity of the synthesized libraries. Intermediate ethyl
ester 11 and methyl ester 13 were transformed into the
corresponding acids 16 and 17, respectively, via hydrolysis
with an aqueous potassium hydroxide solution at ambient
temperature in ∼70% yield (Scheme 5). An interesting
phenomenon was observed for hydrolysis of 11. Refluxing
of 11 in an aqueous potassium hydroxide solution gave
4,5-dimethyl-2-cyclohexylimino-1,3-thiazoline, presum-
ably resulting from further decarboxylation of 16 (see the
a Yields (percent) were calculated on the basis of the weight of
the solid obtained by filtration of the reaction mixture. b Molecular
ions were determined by mass spectrometry (ESI) without removal of
the HX salt (X ) Cl or Br). c Purities (percent) were determined by
HPLC at 210 nm. d The molecular ion was determined by mass
spectrometry (ESI) after removal of the HBr salt by the treatment of
triethylamine. e Molecular ion could not be obtained.
of 4{66} with thiourea 3 in refluxing ethanol afforded
2-imino-1,3-thiazoline hydrogen bromide salt 14 in quan-
titative yield. We have synthesized a library of 12
compounds of phthalimidyl 1,3-thiazolines 14, which were
precursors of 15.17 Treatment of 14 with triethylamine in
benzene followed by addition of hydrazine hydrate (or
methylamine or ethanolamine) in ethanol at room tem-
perature gave 4-aminomethyl-1,3-thiazoline 15 in moder-
ate yields (42-65%). Product 15 was confirmed as a single
Figure 9. Representative structures of the prepared phthalimidyl 2-imino-1,3-thiazolines 14 of Chemset B.

524 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Han et al.
Scheme 5. Preparation of Amide Compounds for Increasing the Chemical Diversity of the Library
Supporting Information). Successful results were obtained
when the hydrolysis reaction conducted for a short reaction
time at room temperature (2 h) afforded 16 and 17 in
65-75% yield. The amide coupling of carboxylic acids
12, 16, and 17 with either anilines or benzylamines was
performed in the presence of 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (EDCI) as a con-
densing agent and N,N-dimethylaminopyridine (DMAP)
in a methylene chloride solution at 0 °C to give corre-
sponding amides 19.18 The used aniline or benzylamine
18 building blocks in these reactions are shown in Figure
10.
We have constructed a sublibrary of 80 compounds in
this manner using Carousel Reaction Stations in a parallel
synthetic fashion. The structures of compounds 19 were
confirmed by 1H NMR spectroscopy. The yields obtained
ranged from 15 to 99% for the final step. Figure 11
provides representative structures of the prepared 2-alky-
limino-1,3-thiazolines 19, and Table 3 lists yields and
HRMS data.
The next approach to increasing the diversity of the library
involved reduction of the amide moiety of prepared com-
pounds 9 and 19. Generally, hydrogen bonds affect
drug-target interactions in biological systems.19 Nitrogen
and oxygen are the most common atoms involved as
hydrogen bond acceptors. The nitrogen atom of an aliphatic
tertiary amine is a better hydrogen bond acceptor than that
of an amide.20 In the latter cases, the lone pair of the nitrogen
can interact with neighboring π systems to form various
resonance structures, resulting in a nitrogen that is less likely
to participate in hydrogen bonding. For this reason, we
transformed the amide moiety to the secondary amine by
reduction (Scheme 6). Reduction of compounds 9 and 19
with lithium aluminum hydride (LAH) in refluxing tetrahy-
drofuran afforded corresponding secondary amine 20 smoothly
in moderate to high yield (11-82%). We have constructed
a sublibrary of 76 compounds in this manner using Carousel
Reaction Stations in a parallel synthetic fashion. Figure 11
provides representative structures of the prepared 2-alky-
Figure 10. Building blocks anilines or benzylamines 18{1-39} used in the reactions.

Novel 2-Alkylimino-1,3-thiazolines
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 525
Figure 11. Representative structures of the prepared 2-alkylimino-1,3-thiazolines of 2-alkylimino-1,3-thiazolines 19 and 20.
Table 3. Yields, Purities, and Molecular Weights of
Representative 2-Alkylimino-1,3-thiazolines 19
Scheme 6. Reduction of the Amide Moiety to Amine,
Resulting in an Increase in Diversity
entry
compound
n
yielda
MW (found)b
purityc
1
2
3
4
5
6
7
8
9
19{10,63,5}
19{16,63,13}
19{16,63,14}
19{12,64,24}
19{12,64,27}
19{11,64,16}
19{11,64,17}
19{16,65,12}
19{20,65,12}
1
1
1
0
0
0
0
2
2
66
60
51
32
42
83
49
28
21
344.18017 (MH+)
548.21405 (MH+)
528.26872 (MH+)
396.21038 (MH+)
450.18335 (MH+)
398.08522d [(M - Br)+]
393.13904d [(M - Br)+]
514.25467 (MH+)
476.23763 (MH+)
82
97
90
98
95
98
75
88
89
the preparation of amides and secondary amines through
coupling with acyl chlorides and reductive amination (Scheme
7).21
a Yields (percent) were calculated on the basis of the weight of the
solid obtained by filtration of the reaction mixture. b Molecular ions
were determined by mass spectrometry (ESI) after removal of the HCl
salt by the treatment of triethylamine.
c Purities (percent) were
Treatment of 15 with acyl chlorides 21{1-11} gave the
corresponding 1,3-thiazolines 22. Since the structures of these
compounds have NH and CO moieties in positions analogous
to the positions of those in 9 (see Scheme 1), the comparison
of the biological activities of both compounds 9 and 22 would
contribute to the elucidation of the structure-activity rela-
tionship of these series. To further increase the versatility
using intermediate 15, another reaction was employed
through reductive amination, to generate secondary amines
24 by the reaction with phenyl aldehydes 23{1,2} in the
presence of sodium borohydride in methanol. The acyl
determined by HPLC at 210 nm. d Molecular ions were determined by
mass spectrometry (ESI) without removal of the HX salt (X ) Cl or
Br).
limino-1,3-thiazolines-4-ethylamine 20, and Table 4 lists
yields and HRMS data.
The third synthetic approach for expanding the library was
the functionalization of the side chains starting with 2-ami-
nomethyl-1,3-thiazolines 15. After deprotection of the ph-
thalimimdyl moiety, the amino function of 15 was used for

526 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Han et al.
Table 4. Yields, Purities, and Molecular Weights of
Representative 2-Alkylimino-1,3-thiazolines 20
molecule library. The patch-clamp assay is more accurate
and sensitive but measures Ca2+ current one by one. All the
synthesized compounds were first screened by the FDSS6000
assay, and selected compounds with potent activity were
further screened by the patch-clamp assay, yielding IC50
values.22
entry
compound
n
yielda
MW (found)b
purityc
1
2
3
4
5
6
7
20{20,12}
20{16,12}
20{12,48}
20{18,49}
20{16,63,12}
20{12,64,16}
20{16,65,12}
1
1
1
1
1
0
2
78
59
32
39
93
75
43
448.24123 (MH+)
486.25703 (MH+)
388.27852 (MH+)
402.29360 (MH+)
500.27359 (MH+)
382.23022 (MH+)
500.27316 (MH+)
99
99
d
-
-
d
Primary screening results of the FDSS6000 assay system
and IC50 values from the patch-clamp assay of a portion of
the synthesized compounds are summarized in Table 6.
In summary, combinatorial libraries of new 2-alkylimino-
1,3-thiazolines with four diversity points consisting of more
than 500 compounds were constructed in a parallel synthetic
fashion. The synthetic strategy was a lead optimization
method of compounds with T-type calcium channel inhibi-
tory activity, designed through structural modifications of
hit compound 2. The compounds of Chemset A were
synthesized by the condensation of thioureas 5 with R-ha-
loketones 6{1-66}. The libraries included intermediates
4-aminomethyl-1,3-thiazoline 15 and 1,3-thiazolinecarboxylic
acids 12, 16, and 17, which gave sublibrary C through
various coupling reactions with suitable building block
collection and subsequent functionalization at the C-4
position of the 1,3-thiazoline core. The selected compounds
from the generated library were evaluated against a T-type
calcium channel, and some of them have exhibited promising
inhibitory activity.
88
67
98
a Yields (percent) were calculated on the basis of the weight of the
solid obtained by filtration of the reaction mixture. b Molecular ions
(MH+) were determined by mass spectrometry (ESI) after removal of
the HCl salt by the treatment of triethylamine. c Purities (percent) were
determined by HPLC at 210 nm. d The purity could not be determined
due to a lack of the sample.
Scheme 7. Preparation of Sublibraries 22 and 24 from
Aminomethyl-1,3-thiazolines
chloride and phenyl aldehyde building blocks used in this
study are shown in Figure 12.
We constructed a library of 35 compounds in this manner
using Carousel Reaction Stations in a parallel synthetic
fashion. Figure 13 provides representative structures of the
prepared 2-imino-1,3-thiazolines, and Table 5 lists yields and
MS data.
Experimental Section
Synthesis of Thiourea 3 (general procedure). To a
solution of isothiocyanate (1.5 mmol) in ethanol (5 mL) was
added amine (1.5 mmol), and the mixture was stirred at room
temperature for 12 h. The reaction mixture was cooled to
room temperature, and the precipitates were filtered, washed
with cold ethyl ether, and then dried in air to afford the
corresponding alkylthiourea.
Synthesis of γ-Chloroacetoacetanilide 4{1-61} (gen-
eral procedure). To a solution of distilled diketene (49
mmol) in dried methylene chloride (50 mL) at -78 °C
under a dry ice/acetone cooling bath was added slowly a
solution of chlorine in dried methylene chloride (100 mL).
The reaction mixture was stirred for 30 min at the same
Biological Screening
The biological activities of the synthesized compounds
were evaluated against HEK293 cells which stably express
both T-type calcium channel Cav3.1 with an a1G subunit and
potassium channel Kir2.1. The percent inhibition of the Ca2+
current was measured at certain molar concentrations of the
synthesized compounds. For this purpose, two assay methods
were employed: FDSS6000 assay and patch-clamp assay
using a single cell. The FDSS6000 assay is developed for
high-throughput screening and applied to the whole small
Figure 12. Building blocks acyl chlorides 21{1-11} and phenyl aldehydes 23{1,2} used in the reactions.

Novel 2-Alkylimino-1,3-thiazolines
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 527
Figure 13. Representative structures of the prepared 2-alkylimino-1,3-thiazolines of 2-alkylimino-1,3-thiazolines 22 and 24.
Table 5. Yields, Purities, and Molecular Weights of
Representative 2-Alkylimino-1,3-thiazolines 21 and 23
Synthesis of Ethyl 4-Bromo-3-oxopentanoate 4{63}.
To a stirred and heated (55 °C) solution of ethyl
3-oxopentanoate (23.0 g, 0.16 mol) dissolved in benzene
(500 mL) under an oil bath was added a solution of Br2
(8.24 mL, 0.16 mol) dissolved in benzene (200 mL) over
40 min, and stirring was continued for 30 min. The solvent
was removed by evaporation to afford 4-bromo-3-oxo-
entry
compound
yielda
MW (found)b
purityc
1
2
3
4
5
6
7
8
22{1,66,8}
22{2,66,4}
47
35
43
57
62
63
62
49
59
64
21
29
364.12450 (MH+)
412.10069 (MH+)
372.21219 (MH+)
402.25788 (MH+)
376.18616 (MH+)
372.21120 (MH+)
412.20572 (MH+)
440.23710 (MH+)
428.21689 (MH+)
438.22170 (MH+)
73
94
86
86
78
95
81
90
100
95
22{3,66,10}
22{11,66,2}
22{18,66,11}
22{19,66,7}
22{12,66,5}
22{13,66,6}
22{14,66,9}
22{16,66,5}
24{11,66,2}
24{16,66,2}
1
pentanoate 4{63} (light brown oil, 72% yield): H NMR
(400 MHz, CDCl3) δ 4.55 (q, 1H, J ) 6.8 Hz, CHBr),
4.20 (q, 2H, J ) 7.1 Hz, CH2CH3), 3.74 (q, 2H, J ) 6.1
Hz, CH2), 1.76 (d, 3H, J ) 6.8 Hz, BrCHCH3), 1.27 (t,
3H, J ) 7.1 Hz, CH2CH3).
9
10
11
12
d
e
-
-
d
e
-
-
Synthesis of Methyl 5-Bromo-4-oxopentanoate 4{65}.
To a stirred and heated (70 °C) solution of 4-oxopentanoic
acid (8.1 g, 70 mmol) dissolved in methanol (90 mL) was
added a solution of Br2 (3.6 mL, 70 mmol) dissolved in
methanol (20 mL) over 10 min, and stirring was continued
for 2 h. The reaction mixture was cooled to room temper-
ature, and the solvent was removed under reduced pressure.
The residue was diluted with methylene chloride (50 mL)
and washed with a saturated NaHCO3 solution three times
and then with water. The organic layer was dried with
MgSO4. The solvent was removed to afford methyl
5-bromo-4-oxopentanoate 4{65} (light brown oil, 83%
yield): 1H NMR (400 MHz, CDCl3) δ 3.97 (s, 2H, CH2Br),
3.68 (s, 3H, OCH3), 2.97-2.94 (t, 2H, J ) 6.6 Hz,
COCH2CH2CO2CH3), 2.67-2.65 (t, 2H, J ) 6.2 Hz,
COCH2CH2CO2CH3).
a Yields (percent) were calculated on the basis of the weight of
the solid obtained by filtration of the reaction mixture. b Molecular
ions (MH+) were determined by mass spectrometry (ESI) after
removal of the HCl salt by the treatment of triethylamine. c Purities
(percent) were determined by HPLC at 210 nm. d Molecular ion
could not be obtained. e The purity could not be determined due to a
lack of the sample.
temperature. To the reaction mixture was added dropwise
a mixture of triethylamine (0.75 mL, 54 mmol) and the
appropriate amine or aniline (49 mmol) dissolved in
methylene chloride (100 mL) cooled to -78 °C under an
acetone/dry ice cooling bath. The rection mixture was
stirred for 1 h at the same temperature and allowed to
warm to room temperature. The mixture was then
washed with a 0.1 N HCl solution, a saturated aqueous
NaHCO3 solution, and then H2O. The organic layer was
dried (MgSO4) and evaporated under reduced pressure.
The resulting solid was collected by filtration, washed with
cold ethyl ether, and then dried in air to afford γ-chlo-
roacetoacetanilide 4{1-61} (25-82% yield).
Synthesis of r-Bromo-r′-phthaimidyl Acetone 4{66}.
To a mechanically stirred solution of chloroacetone (10.0
mL, 0.126 mol) dissolved in acetone (150 mL) was added
phthalimide potassuium salt (25.5 g, 0.138 mol), and the

528 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Han et al.
Table 6. Primary Screening Results and IC50 Values of a Portion of the Synthesized Compounds
b
entry
compound
n
A
B
C
D
% inhibitiona
IC50 (µM)
1
2
3
4
5
6
7
8
9{1,5}
9{3,9}
9{8,7}
9{18,12}
9{20,12}
9{11,7}
9{12,8}
9{12,12}
9{12,5}
9{12,2}
9{12,4}
9{16,8}
9{16,7}
9{16,12}
9{14,2}
9{14,6}
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
C6H4(4-CH3)
C6H4(4-CF3)
C6H4(4-C4H9)
C6H4(4-OC6H5)
C6H4(4-OC6H5)
C6H4(4-C4H9)
C6H4[4- CH(CH3)2]
C6H4(4-OC6H5)
C6H4(4-CH3)
C6H4(4-F)
C6H4(4-Br)
C6H4[4-CH(CH3)2]
C6H4(4-C4H9)
C6H4(4-OC6H5)
C6H4(4-F)
methyl
cyclohexyl
cyclohexyl
cyclohexyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
46
68
62
82
81
75
73
77
61
74
69
75
82
83
73
76
75
76
51
60
72
78
63
73
68
60
25
64
65
70
65
73
69
77
77
55
63
55
78
53
-
n-propyl
cyclopropyl
ethyl
cyclopropyl
methyl
methyl
methyl
methyl
methyl
-
-
cycloheptyl
cycloheptyl
cycloheptyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
-
-
-
0.05 ( 0.01
0.92 ( 0.08
1.17 ( 0.08
0.77 ( 0.03
0.61 ( 0.07
0.31 ( 0.02
0.19 ( 0.02
0.43 ( 0.07
0.60 ( 0.04
0.30 ( 0.01
0.35 ( 0.01
-
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
methyl
cyclopropyl
cyclopropyl
cyclopropyl
n-propyl
n-propyl
ethyl
cyclopropyl
methyl
methyl
methyl
methyl
methyl
cyclopropyl
cyclopropyl
cyclopropyl
methyl
methyl
methyl
C6H4(4-C2H5)
C6H4(4-C2H5)
9{13,6}
H
19{16,63,12}
19{12,63,14}
19{12,63,13}
19{12,64,35}
19{12,64,28}
19{12,64,27}
19{16,65,12}
19{16,65,14}
19{16,65,13}
20{12,8}
20{12,12}
20{12,5}
20{12,2}
20{12,4}
20{16,8}
20{16,7}
20{16,12}
20{14,2}
20{14,6}
20{13,6}
22{12,66,5}
22{13,66,4}
22{16,66,5}
C6H4(4-OC6H5)
C6H4[4-OC6H4(4-CH3)]
C6H4[4-OC6H4(4-Cl)]
CH2C6H4(4-CF3)
CH2C6H4(3-Cl)
CH2C6H4(2-CF3)
CH2C6H4(4-OC6H5)
C6H4[4-OC6H4(4-CH3)]
C6H4[4-OC6H4(4-Cl)]
C6H4[4-CH(CH3)2]
C6H4(4-OC6H5)
C6H4(4-CH3)
C6H4(4-F)
C6H4(4-Br)
C6H4[4-CH(CH3)2]
C6H4(4-C4H9)
C6H4(4-OC6H5)
C6H4(4-F)
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-
-
0.14 ( 0.02
0.16 ( 0.02
0.37 ( 0.04
-
-
-
0.72 ( 0.07
0.60 ( 0.04
0.21 ( 0.05
1.09 ( 0.81
0.38 ( 0.11
0.18 ( 0.06
1.53 ( 0.83
0.31 ( 0.09
0.43 ( 0.08
1.43 ( 0.38
2.00 ( 0.67
-
methyl
methyl
cyclopropyl
cyclopropyl
cyclopropyl
n-propyl
n-propyl
ethyl
methyl
ethyl
cyclopropyl
C6H4(4-C2H5)
C6H4(4-C2H5)
C6H4(4-OCH3)
C6H3(3,5-di-Cl)
C6H4(4-OCH3)
H
H
H
H
-
-
H
a Percent inhibition values were obtained with the FDSS6000 assay system. b IC50 values were determined from the dose-response curve, obtained
by the patch-clamp method.
1
mixture was heated to reflux over 20 h. The reaction mixture
was cooled to room temperature, and the solvent was
removed by evaporation. The residue was diluted with
methylene chloride (200 mL), washed with water, and then
dried with MgSO4. The solvent was removed in vacuo, and
the resulting solid was filtered, washed with cold ethyl ether,
and then dried in air to afford R-phthaimidyl acetone as a
white solid. To a stirred and heated (70 °C) solution of
R-phthaimidyl acetone (1.01 g, 5 mmol) in acetic acid (20
mL) was added a solution of Br2 (0.3 mL, 6 mmol) dissolved
in acetic acid (5 mL) over 10 min. After the reaction mixture
had been stirred for 1 h and cooled to room temperature,
the solvent was removed under reduced pressure. The residue
was diluted with methylene chloride (50 mL), washed with
a saturated aqueous NaHCO3 solution five times and water,
and then dried with MgSO4. The solvent was removed by
evaporation, and the resulting solid was collected by filtra-
tion, washed with isopropyl ether, and then dried in air to
afford 2-(3-bromo-2-oxopropyl)isoindoline-1,3-dione 4{66}
(white solid, 54% yield): H NMR (300 MHz, CDCl3) δ
7.90-7.74 (m, 4H, ArH), 4.78 (s, 2H, NCH2), 4.01 (s, 3H,
CH2Br).
Synthesis of 2-Alkylimino-1,3-thiazoline 9 (general
procedure). The syntheses of 2-alkylimino-1,3-thiazoline 9
was accomplished using a parallel synthesizer Carousel 12-
place reaction station (Radleys Discovery Technologies). To
a solution of alkylthiourea 3 (0.01 mol) in ethanol (5 mL)
was added γ-chloroacetoacetanilide 4{1-62} (0.01 mol). The
reaction mixture was heated to reflux for 12 h. The reaction
mixture was cooled to room temperature, and the precipitates
were filtered, washed with cold ethyl ether (or isopropyl
ether), and then dried in air to afford 9 (solid, 8-99% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline Ester 11 (gen-
eral procedure). To a solution of ethyl 4-bromo-3-oxopen-
tanoate 4{63} (1.1 g, 5 mmol) dissolved in ethanol (40 mL)
was added alkylthiourea 3 (5 mmol), and the mixture was
heated to reflux over 5 h. The reaction mixture was cooled
to room temperature, and the solvent was removed under

Novel 2-Alkylimino-1,3-thiazolines
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 529
reduced pressure; the resulting solid was filtered, washed with
cold ethyl ether (or isopropyl ether), and then dried in air to
afford 11 (solid, 70-98% yield).
and the corresponding amine or aniline 18 (2 mmol) was
added to the reaction mixture while it was being stirred. The
reaction mixture was allowed to warm to room temperature
over 5-10 h. The reaction mixture was diluted with
methylene chloride, washed sequentially with 0.1 N HCl, a
saturated aqueous NaHCO3 solution, and water, and then
dried with anhydrous MgSO4. The solvent was removed by
evaporation, and the residue was purified by flash chroma-
tography on silica gel to yield the corresponding 19 (solid,
2-99% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline-4-ethylamine
20 (general procedure). To a solution of 9 (or 19) (0.4
mmol) in tetrahydrofuran (10 mL) was added LiAlH4 (76
mg, 2 mmol). The reaction mixture was heated to reflux over
7-20 h. The reaction progress was monitored by TLC.
Glauber’s salt (67 mg, 0.2 mmol) was added to destroy
excess LiAlH4 while the mixture was being stirred for 1 h
at room temperature. The precipitates were removed by
filtration through Celite. The solvent was removed by
evaporation, and the residue was separated by flash chro-
matography using silica gel to give 20 (solid, 6-93% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline Hydrochloride
22 (general procedure). To a solution of 15 (0.3 mmol)
dissolved in methylene chloride (10 mL) was added acyl
chloride 21 (0.3 mmol). The reaction mixture was stirred at
room temperature for 5 h. The precipitates were filtered,
washed with methylene chloride, and then dried in air to
afford 22. To obtain the free amine of 22, the reaction
mixture was treated with triethylamine (42 µL, 0.3 mol) at
room temperature while being stirred for 30 min. The solvent
was removed by evaporation, and the residue was purified
by flash chromatography using silica gel and a mixture of
ethyl acetate and n-hexane (1:1) to afford the free amine of
22 (23-83% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline Acid 12 (gen-
eral procedure). To a solution of ethyl 3-bromopyruvic acid
4{64} (1.01 g, 6 mmol) dissolved in ethanol (10 mL) was
added alkylthiourea 3 (6 mmol), and the mixture was heated
to reflux over 2 h. The reaction mixture was cooled to room
temperature, and the solvent was removed by evaporation;
the resulting solid was filtered, washed with cold ethyl ether
(or isopropyl ether), and then dried in air to afford 12 (solid,
85-99% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline Ester 13 (gen-
eral procedure). To a solution of methyl 5-bromo-4-
oxopentanoate 4{65} (1.7 g, 8 mmol) dissolved in ethanol
(20 mL) was added alkylthiourea 3 (8 mmol), and the mixture
was heated to reflux over 3 h. The reaction mixture was
cooled to room temperature, and the solvent was removed
by evaporation; the resulting solid was filtered, washed with
cold ethyl ether (or isopropyl ether), and then dried in air to
afford 13 (solid, 70-98% yield).
Synthesis of Phthalimidyl 1,3-Thiazoline 14 (general
procedure). To a solution of 4{66} (5.6 g, 20 mmol)
dissolved in ethanol (60 mL) was added alkylthiourea 3 (20
mmol), and the mixture was heated to reflux over 2 h. The
reaction mixture was cooled to room temperature, and the
solvent was removed by evaporation; the resulting solid was
filtered, washed with cold ethyl ether, and then dried in air
to afford 14 (solid, 76-93% yield).
Synthesis of 4-Aminomethyl-2-imino-1,3-thiazoline 15
(general procedure). To a solution of 14 (4 mmol) dissolved
in benzene (30 mL) was added triethylamine (0.61 mL, 4
mmol), and the reaction mixture was stirred for 2 h at room
temperature. The produced precipitates were filtered out, and
the solvent was removed under reduced pressure. The residue
was diluted with ethanol (50 mL) and added hydrazine
monohydrate (0.39 mL, 8 mmol) and the mixture stirred
at ambient temperature for 10 h. The resulting solid was then
filtered off and washed with methylene chloride. The filtrate
was concentrated in vacuo to yield 15 (oil, 42-65% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline Acids 16 and
17 (general procedure). To a solution of 11 (or 13) (5
mmol) dissolved in methanol (3.3 mL/g) was added KOH
(15 mmol). The reaction mixture was stirred for 2 h, and
the solvent was removed under reduced pressure. The
reaction mixture was poured onto water (30 mL/g) and
stirred for 1 h. The mixture was washed with methylene
chloride, and the aqueous layer was acidified (pH 2-3)
by addition of concentrated HCl. The reaction mixture
was evaporated, and the produced residue was filtered off
using acetone and methylene chloride. The filtrate was
dried with anhydrous MgSO4, and the solvent was
removed by evaporation to afford the corresponding 16
(or 17) (solid, 65-75% yield).
Synthesis of 2-Alkylimino-1,3-thiazoline 24 (general
procedure). A solution of 15 (0.5 mol) in methanol (5 mL)
and aldehyde 23 (0.5 mol) was stirred at room temperature
for 5 h. To the reaction mixture was added NaBH4 (38 mg,
1 mol), and stirring was continued at the same temperature
for 10 h. To the resulting reaction mixture was added a 1 N
NaOH aqueous solution to destroy excess NaBH4. This
mixture was extracted with methylene chloride, and the
organic extract was dried with anhydrous MgSO4. The
solvent was removed by evaporation, and the crude product
was purified by flash chromatography on silica gel to yield
24 (15-36% yield).
Acknowledgment. We thank Prof. H. Mah, Prof. S. H.
Cheon, and Mr. I. M. El-Deeb for assistance in preparation
of the manuscript.
Supporting Information Available. Yields, melting points,
and 1H NMR data for all the compounds, mass and HPLC data
for the representative compounds, and X-ray crystallographic
data for 9{7,3} and 9{16,12} (CIF). This material is available
free of charge via the Internet at http://pubs.acs.org.
Synthesis of 2-Alkylimino-1,3-thiazoline 19 (general
procedure). To a suspension of 12 (or 16 or 17) (1 mmol)
dissolved in methylene chloride (10 mL) at 0 °C were added
DMAP (0.25 g, 2 mmol) and EDCI (0.20 g, 1 mmol)
sequentially. The resulting mixture was stirred for 30 min,
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