Studying synergism of methyl linked cyclohexyl thiophenes with triazole: Synthesis and their cdk5/p25 inhibition activity

Article abstract of DOI:10.1016/j.ejmech.2011.02.059

A novel clubbed triazolyl thiophene series of cdk5/p25 inhibitors, potentially useful for the treatment of Alzheimer's disease, is disclosed. Evaluation of the SAR of substitution within these series has allowed the identification of a range of compounds

Full text of DOI:10.1016/j.ejmech.2011.02.059

European Journal of Medicinal Chemistry 46 (2011) 2066e2074
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Studying synergism of methyl linked cyclohexyl thiophenes with triazole:
Synthesis and their cdk5/p25 inhibition activity
,
Mahendra Shiradkar a, John Thomas b, Vanita Kanase c, Rajendra Dighe d
*
a Dr. Reddy’s Laboratories, Ameerpet, Hyderabad, India
b Inogent Laboratories Limited, Hyderabad, India
c Natco Pharma Limited, Hyderabad, India
d Dr. Reddy’s Laboratories, Bachupally, Hyderabad, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel clubbed triazolyl thiophene series of cdk5/p25 inhibitors, potentially useful for the treatment of
Alzheimer’s disease, is disclosed. Evaluation of the SAR of substitution within these series has allowed
the identification of a range of compounds which significantly reduce brain cdk5/p25 and thus have
potential as possible treatments for Alzheimer’s disease.
Received 16 November 2010
Received in revised form
23 February 2011
Accepted 24 February 2011
Available online 3 March 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Thiophene
Triazole
cdk5/p25
Alzheimer’s disease
1. Introduction
Based on this hypothesis, we embarked on a cdk5/p25 inhibitor
discovery program to find an orally bioavailable, high potency
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder accompanied by memory decline, cognitive impairment,
and visual-spatial disorientation, for which no effective treatment
exists today. Postmortem brain analysis of AD patients reveals
extensive formation of neurofibrillary tau protein tangles and
amyloid plaques. The serine/threonine kinase cdk5 along with its
cofactor p25 [1] (or the longer cofactor, p35) has been supposed to
hyperphosphorylate tau [2], leading to the formation of paired
helical filaments and deposition of cytotoxic neurofibrillary tangles
[3] and thus responsible to neurodegenerative disorders such as
Alzheimer’s disease, Parkinson’s disease, stroke, or Huntington’s
disease [4]. Cdk5 also phosphorylates Dopamine and Cyclic AMP-
Regulated Phosphorprotein (DARPP-32) at threonine 75 and is thus
indicated in having a role in dopaminergic neurotransmission [5].
Inhibition of the anomalous cdk5/p25 complex is, therefore,
a viable target for treating Alzheimer’s disease by preventing tau
hyperphosphorylation and neurofibrillary tangle formation. Liter-
ature survey reveals that thiophene derivatives [6] as the potential
inhibitors of cdk5/p25 for the treatment of Alzheimer’s disease and
other neurodegenerative disorders [7e13].
compound/s. Screening of an in-house database provided several
hits with modest cdk5/p25 inhibitory activity, one of which was the
clubbed triazolyl thiophene (IC50 ¼ 46 ꢀ 2 nM).
In recent years, environmentally benign synthetic methods have
received considerable attention and solvent-free protocols are
reported. A fast, highly efficient and eco-friendly solvent-free
chemical transformation, for the synthesis of title compounds,
under microwave irradiation, using acidic alumina is designed.
2. Result and discussion
2.1. Chemistry
Compounds 1AeD, 2AeD, 3AeL, 4AeL, 5AeL, 6AeL, 7AeL,
8AAe8AJ, 8Me8Z, 9AeL and 10AeL were synthesized using
reported methods [4,8,9,14e27]. Compounds 1AeD was converted
to thiocarbazate salts by treatment with carbon disulphide and
potassium hydroxide, which on treatment with hydrazine hydrate
gave 2AeD. Compounds 2AeD was treated with 4echlorobenzoyl
chloride at 0 ꢁC to yield 3AeL (Scheme 1). The transformed
compounds 3AeL on treatment with diiodomethane in the pres-
ence of strong alkali i.e. sodium hydroxide gave 4AeL (Scheme 2).
Title compounds 3AeL were treated with chloroacetonitrile, which
* Corresponding author. Tel.: þ91 9850661065.
E-mail address: digherd@gmail.com (R. Dighe).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.059

M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
2067
O
X
O
C H
2
O
KOH/CS
NHNH
5
2
2
X
X
X
CN
NH NH H O
COOEt
COOEt
2
2
2
R COCl
1
Step-4
O
Step-2
Step-3
S, ( Et ) NH, EtOH
2
R
R
1
1
S
S
S
NH
2
Step-1
NHCOR
N
2
NH
2
S
H
X
N
X
SH
SH
R COCl
2
X
N
SK
NH NH H O
2
2 2
N
H
N
N
N
1
X = -CH2-
N
Step-6
Step-5
O
R
R
S
R
S
1
1
S
3
2
1
Scheme 1. Synthesis of lead compound/s 3 [4,8,9,14e27].
on neutralization with sodium carbonate gave a precipitates of
compounds 5AeL (Scheme 3). Compounds 3AeL, when treated
with methyl bromoacetate in basic condition produced 6AeL.
Chemical transformation of 6AeL to 7AeL was achieved by treat-
ment it with hydrazine hydrate (Scheme 4). While compounds
7AeL, on treatment with appropriate acid chlorides, furnished
8AeL. Schiff bases, the condensation products of 9AeL, were
synthesized by treating 7AeL with benzaldehyde and confirmed by
absence of triplet of NH of hydrate. Compounds 7AeL were con-
verted to thiocarbazate salts by treatment with carbon disulphide
and potassium hydroxide, which on treatment with hydrazine
hydrate gave 10AeL (Scheme 5). The NMR spectra confirmed
formation of triazole derivative from hydrazide, which shows
presence of sulfhydryl proton at 3.1.
s-Alkylation with acetonitrile provided the first analogs 5A and
5I that demonstrated with excellent activity, while others exhibited
moderate to poor activity. Thus it was decided to modify the
structure as SH group. In order to optimize the sulfhydryl compo-
nent, compounds 6AeL were synthesized and investigated, which
revealed loss of activity. A further modification of compounds 6AeL
produced compounds 7AeL. The results of the cdk5/p25 inhibitory
activity are quite interesting as 7AeD have shown impressive
percentage of inhibition. Compounds 7AeL was selected for further
studies as it has a free amino group, which opened an area for
further modification at this point. Compounds 8AeAJ was obtained
by treatment with acid chlorides which ultimately showed
decreased activity. Furthermore, compounds 7AeL were converted
to Schiff bases with benzaldehyde, and on investigation, 9AeE have
shown promising activity while other remained inactive.
Compounds 10AeL were found to be inactive.
2.2. Cyclic-dependent kinase 5/p25 inhibiting activity
Attention was then turned to optimization of the 9AeE in order
to gain the selectivity over cdk2. On comparing, 9E afforded
improved cdk5 potency that is >17.5 -fold selectivity versus cdk2.
The compound 9A was equally selective versus cdk2 and had
slightly improved cdk5 IC50. Other derivatives were had noticeably
decreased cdk5 activity.
Kinase inhibition was measured by use of scintillation proximity
assays (SPA). The results of the assays are reported in Tables 1e4.
During the preliminary screening compound 2A has emerged as hit
cdk5/p25 (IC50 ¼ 043 ꢀ 02 nM), with good potency and more
opportunities for chemical transformation for the optimization.
Testing of 2A against other cdk’s revealed that 2A was essentially
equipotent at inhibiting cdk2/cyclin E (IC50 ¼ 52 ꢀ 5 nM), a cancer
target. Thus with an objective to improve cdk5 potency and mini-
mize cdk2 activity, certain chemical modification have been per-
formed. Variation of amine side chain of 2A with MAOS allowed us
to rapidly explore the first arm of the parmacophore. As a first step
towards lead optimization amino group was protected to the cor-
responding compounds 3AeL however, all of these modifications
were resulted in a substantial decrease in activity. The next struc-
tural modification made was a dimeric product of 4AeL but these
changes were also resulted in a substantial loss of biological
activity.
3. Conclusion
In conclusion, a novel series of clubbed triazolyl thiophene
derivatives that inhibit cdk5/p25 has been discovered. It was found
that the potency of the screening hit 2A could be enhanced first by
structural transformation to a 2-position of thiophene core and
amino and sulfhydryl groups in triazole core and subsequently by
the introduction of appropriate constituents on both the hetero-
cyclic rings leading to the most promising compounds 9A and 9B.
Finally it can be concluded that an ideal cdk5/p25 inhibitor with
minimal toxicity and potential activity can be designed using above
said compounds as lead molecules. The said inhibitor can be
synthesized using MAOS so as to get the benefits of this novel
technique.
Scheme 2. Synthesis of Bis derivatives 4.
Scheme 3. Synthesis of cyano analogs 5.

2068
M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
Scheme 4. Synthesis of hydrazides 7.
4. Experimental protocols
4.1.2. Preparation of N-[3-(4-Amino-5-mercapto-4H-[1,2,4]triazol-
3-ylmethyl)-4,5,6,7-tetrahydro-benzo(b)thiophen-2-yl]-2-chloro-
acetamide (2A), N-[3-(4-Amino-5-mercapto-4H-[1,2,4]triazol-3-
ylmethyl)-4,5,6,7-tetrahydro-benzo(b)thiophen-2-yl]-2-acetamide
(2B), N-[3-(4-Amino-5-mercapto-4H-[1,2,4]triazol-3-ylmethyl)-
4,5,6,7-tetrahydro-benzo(b)thiophen-2-yl]-2- benzamide (2C),
N-[3-(4-Amino-5-mercapto-4H-[1,2,4]triazol-3-ylmethyl)-4,5,6,7-
tetrahydrobenzo(b)thiophen-2-yl]-2- propionic acid (2D)
Above titled compound were prepared using cited methods
[31,32,35].
4.1. Chemistry
The melting points were recorded on electrothermal apparatus
and are uncorrected. 1H NMR spectra on a Bruker Avance 300 MHz
instrument using CDCl3 as solvent using TMS as internal standard;
the chemical shifts (d) are reported in ppm and coupling constants
(J) are given in Hertz. Signal multiplicities are represented by s, d, t,
ds, dd, m, and br s. Mass spectra were recorded on a Finnigan LCQ
mass spectrometer. Microwave irradiation was carried out in Raga
Scientific Microwave Systems, Model RG31L at 2450 MHz.
Elemental analysis was performed on a Heracus CHN-Rapid Ana-
lyser. Analysis indicated by the symbols of the elements of func-
tions was within ꢀ0.4% of the theoretical values. The purity of the
compounds was checked on silica gel coated Al plates (Merck).
4.1.2.1. N-[3-(4-Amino-5-mercapto-4H-[1,2,4]triazol-3-ylmethyl)-
4,5,6,7-tetrahydro-benzo (b) thiophen-2-yl]-2-chloro-acetamide (2A).
Yield 76%; mp 254e256 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.53e1.68
(m, 4H, cyclohexane), 2.02 (s, 2H, NH2), 2.20e2.42 (t, 4H, cyclohexane
CH2, J ¼ 4.9 Hz), 3.2 (s, 1H, SH), 3.76 (s, 2H, CH2), 4.15 (s, 2H, CH2Cl),
8.02 (s,1H, NH); MS m/z (%) 358 (Mþ,100), 323 (67.8), 290 (19.2), 248
(44.8),192 (10.1),177 (23.6); Anal. Calcd. for C13H16ClN5OS2: C, 43.63;
H, 4.51; N, 19.57. Found: C, 43.76; H, 4.47; N, 19.72.
4.1.1. Preparation of N0-[2-(2-chloro-acetylamino)-4,5,6,7-tetrahydro-
benzo[b]thiophen-3 yl]-hydrazinecarbodithioic potassium salt (1A),
N0-[2-(2-Acetylamino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-
acetyl]-hydrazinecarbodithioic potassium salt (1B), N0-[2-(2-benzoyl-
amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-acetyl]-hydrazine-
carbodithioic potassium salt (1C), 3-{3-[2-(N0-dithiocarboxy-
hydrazino)-2-oxo-ethyl]-4,5,6,7-tetrahydro-benzo[b]thiophen-2-
ylamino}-propionic potassium salt (1D)
4.1.3. General preparation of 7-chloro-hepta-2,4,6-triynoic acid -
{3-[2-(2-substituted-amino) 4, 5, 6, 7-tetrahydro-benzo[b]
thiophen-3-ylmethyl]-5-mercapto-[1,2,4] triazol-4-yl}-amide
[30,31,35]
The triazole (2) (1 mmol) in 20 ml of 10% NaOH was treated drop
wise with an equimolar amount of the 4-chlorobenzoyl chloride at
Above titled compound were prepared as per the reported
method [28e33,35].
0
ꢁC, which was stirred for 30e45 min. At the end of stirring,
precipitate was observed. It was then filtered, washed thoroughly
with water and crystallized.
4.1.1.1. N0-[2-(2-Chloro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thi-
ophen-3-yl]-hydrazine carbodithioic potassium salt (1A). Yield 86%;
mp 213e215 ꢁC; 1H NMR (300 MHz, CDCl3):
d
1.43e1.59 (m, 4H,
4.1.3.1. 7-Chloro-hepta-2,4,6-triynoic acid -{3-[2-(2-chloro-acetyla-
mino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3-ylmethyl]-5-mercapto-
[1,2,4]triazol-4-yl}-amide (3A). Yield 71%; mp 284e286 ꢁC; 1H NMR
cyclohexane), 2.45e2.51 (t, 4H, cyclohexane CH2, J ¼ 4.4 Hz), 3.41 (s,
2H, CH2), 4.19 (s, 2H, CH2Cl), 4.25e4.50 (dd, 2H, JNHeNH ¼ 4.21,
JNHeNH ¼ 4.52), 8.03 (s, 2H, NH); MS m/z (%) 415 (Mþ, 55), 376 (32.4),
341 (53.7), 250 (100), 208 (12.8), 165 (7.2), 151 (31.6), 99 (27), 85
(15.6); Anal. Calcd. for C13H15ClKN3O2S3:C, 37.53; H, 3.63; N, 10.10.
Found: C, 37.41; H, 3.52; N, 10.05.
(300 MHz, CDCl3):
d 1.14e1.61 (m, 4H, cyclohexane), 2.25e2.58 (t,
4H, cyclohexane CH2, J ¼ 4.8 Hz), 3.1 (s, 1H, SH), 3.72 (s, 2H, CH2),
4.15 (s, 2H, CH2Cl), 7.12e7.36 (m, 4H, ArH), 8.06 (s, 2H, NH); MS m/z
(%) 496 (Mþ,100), 494 (80), 457 (52), 373 (33.8), 296 (17), 268 (9.8),
Scheme 5. Synthesis of triazole-amides 8, Schiff bases 9 and Triazolo-s-triazole 10.

M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
2069
Table 1
cdk5 IC50 values of the compound 1Ae5L.
Sr. No.
R1
R2
R3
Cdk5 IC50 (nM)a
Sr. No.
R1
R2
R3
Cdk5 IC50 (nM)a
1A
1B
1C
1D
2A
2B
2C
2D
3A
3B
3C
3D
3E
3F
3G
3H
3I
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
247 ꢀ 37
346 ꢀ 41
373 ꢀ 39
397 ꢀ 35
043 ꢀ 02
386 ꢀ 85
621 ꢀ 23
268 ꢀ 31
343 ꢀ 12
460 ꢀ 71
183 ꢀ 11
457 ꢀ 43
751 ꢀ 74
572 ꢀ 63
891 ꢀ 112
674 ꢀ 67
689 ꢀ 82
584 ꢀ 47
618 ꢀ 46
433 ꢀ 24
340 ꢀ 22
367 ꢀ 21
4C
4D
4E
4F
4G
4H
4I
NHCOC6H5
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
eCH3
eCH3
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
627 ꢀ 39
164 ꢀ 11
669 ꢀ 42
646 ꢀ 83
695 ꢀ 47
577 ꢀ 38
849 ꢀ 85
839 ꢀ 56
766 ꢀ 81
734 ꢀ 36
058 ꢀ 12
415 ꢀ 75
551 ꢀ 72
422 ꢀ 12
657 ꢀ 64
393 ꢀ 61
486 ꢀ 78
4874 ꢀ 86
061 ꢀ 06
458 ꢀ 84
564 ꢀ 64
641 ꢀ 74
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
e
e
NHCOC6H5
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
4J
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
eCH3
eCH3
eCH3
e4-ClC6H4
e4-ClC6H4
4K
4L
5A
5B
5C
5D
5E
5F
5G
5H
5I
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
eCH3
NHCOC6H5
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
3J
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
3K
3L
4A
4B
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
5J
5K
5L
eCH3
eCH3
eCH3
a
Inhibitory concentration; The IC50 for Roscovitine (Positive control) was observed as 280 ꢀ 41.
280 (14.7), 289 (29.8), 246 (9.3), 220 (10.8), 164 (10.1), 98 (15.1), 85
(5.3); Anal. Calcd. for C20H19Cl2N5O2S2: C, 48.39; H, 3.86; N, 14.11.
Found: C, 48.55; H, 3.98; N, 14.03.
7.11e7.68 (m, 5H, ArH), 8.05 (s, 2H, NH); MS m/z (%) 462 (Mþ, 68),
427 (100), 426 (57), 378 (43.6), 301 (14), 285 (10.1), 246 (28.3), 220
(34.6), 164 (20.3), 98 (9), 85 (12.3); Anal. Calcd. for C20H20ClN5O2S2:
C, 52.00; H, 4.36; N, 15.16. Found: C, 52.13; H, 4.50; N, 15.20.
4.1.3.2. N-{3-[2-(2-Chloro-acetylamino)-4, 5, 6, 7-tetrahydro-benzo
[b]thiophen-3-ylmethyl]-5-mercapto-[1,2,4] triazol-4-yl}-benzamide
(3E). Yield 69%; mp 275e277 ꢁC; 1H NMR (300 MHz, CDCl3):
4.1.3.3. N-[3-(4-Acetylamino-5-mercapto-4H-[1,2,4] triazol-3-ylme-
thyl)-4, 5, 6, 7-tetrahydro-benzo[b]thiophen-2-yl]-2-chloro-acet-
amide (3I). Yield 77%; mp 282e284 ꢁC; 1H NMR (300 MHz, CDCl3):
d
1.42e1.67 (m, 4H, cyclohexane), 2.42e2.69 (t, 4H, cyclohexane
CH2, J ¼ 4.4 Hz), 3.1 (s, 1H, SH), 3.84 (s, 2H, CH2), 4.08 (s, 2H, CH2Cl),
d 1.38e1.61 (m, 4H, cyclohexane), 2.08 (s, 3H, CH3), 2.48e2.56 (t,
Table 2
cdk5 IC50 values of the compound 6Ae8Z.
Sr. No.
R1
R2
R3
Cdk5 IC50 (nM)a
Sr. No.
R1
R2
R3
Cdk5 IC50 (nM)a
6A
6B
6C
6D
6E
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
eCH3
eCH3
eCH3
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
eCH3
eCH3
eCH3
e4-ClC6H4
eCH3
eCH3
eCH3
eCH3
eCH3
e
443 ꢀ 106
389 ꢀ 35
293 ꢀ 38
541 ꢀ 18
681 ꢀ 47
578 ꢀ 74
353 ꢀ 71
641 ꢀ 119
695 ꢀ 112
389 ꢀ 97
567 ꢀ 103
733 ꢀ 137
039 ꢀ 02
064 ꢀ 02
236 ꢀ 34
201 ꢀ 31
037 ꢀ 01
192 ꢀ 47
264 ꢀ 72
455 ꢀ 79
061 ꢀ 01
249 ꢀ 36
180 ꢀ 27
931 ꢀ 24
468 ꢀ 64
639 ꢀ 67
366 ꢀ 48
363 ꢀ 34
436 ꢀ 46
411 ꢀ 83
8AF
8AG
8AH
8AI
8AJ
8B
8C
8D
8E
8F
8G
8H
8I
8J
8K
8L
8M
8N
8O
8P
8Q
8R
8S
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH3
eCH3
eCH3
eCH3
eCH3
eC6H5
CH2Cl
CH2Cl
CH2Cl
CH2Cl
eCH3
eCH3
eCH3
eC6H5
eC6H5
eC6H5
eC6H5
CH2Cl
CH2Cl
CH2Cl
CH2Cl
eCH3
eCH3
eCH3
eCH3
eC6H5
eC6H5
eC6H5
eC6H5
CH2Cl
CH2Cl
CH2Cl
CH2Cl
eCH3
eCH3
476 ꢀ 68
452 ꢀ 67
474 ꢀ 74
358 ꢀ 72
265 ꢀ 38
641 ꢀ 49
318 ꢀ 30
352 ꢀ 28
617 ꢀ 23
525 ꢀ 47
483 ꢀ 41
271 ꢀ 17
229 ꢀ 67
260 ꢀ 38
340 ꢀ 22
185 ꢀ 39
350 ꢀ 61
548 ꢀ 46
564 ꢀ 77
467 ꢀ 48
468 ꢀ 92
413 ꢀ 45
466 ꢀ 73
434 ꢀ 80
532 ꢀ 57
643 ꢀ 74
547 ꢀ 74
362 ꢀ 35
454 ꢀ 49
327 ꢀ 48
e
e
e
e
eCH3
6F
e
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eC6H5
eC6H5
eC6H5
eC6H5
eC6H5
6G
6H
6I
6J
6K
6L
7A
7B
7C
7D
7E
NHCOC6H5
e
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
e
NHCOC6H5
e
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
e
NHCOC6H5
e
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
7F
e
7G
7H
7I
7J
7K
7L
8A
8AA
8AB
8AC
8AD
8AE
NHCOC6H5
e
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
e
NHCOC6H5
e
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e
8T
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
eCH3
eCH3
eCH3
eC6H5
eC6H5
eC6H5
8U
8V
8W
8X
8Y
8Z
eC6H5
eC6H5
eC6H5
eCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
eCH3
a
Inhibitory concentration; The IC50 for Roscovitine (Positive control) was observed as 280 ꢀ 41.

2070
M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
Table 3
cdk5 IC50 values of the compound 9Ae10L.
Sr. No.
R1
R2
R3
Cdk5 IC50 (nM)a
Sr. No.
R1
R2
R3
Cdk5 IC50 (nM)a
9A
9B
9C
9D
9E
9F
9G
9H
9I
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
e
e
e
e
e
e
e
e
e
e
e
e
035 ꢀ 01
213 ꢀ 76
217 ꢀ 23
258 ꢀ 37
032 ꢀ 1
10A
10B
10C
10D
10E
10F
10G
10H
10I
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
e4-ClC6H4
eC6H5
eC6H5
eC6H5
eC6H5
eCH3
e
e
e
e
e
e
e
e
e
e
e
e
2860 ꢀ 116
6380 ꢀ 104
3280 ꢀ 217
5850 ꢀ 158
6185 ꢀ 144
6437 ꢀ 149
4587 ꢀ 179
4985 ꢀ 146
4035 ꢀ 186
5146 ꢀ 230
6142 ꢀ 201
6212 ꢀ 251
385 ꢀ 28
435 ꢀ 52
267 ꢀ 52
336 ꢀ 48
453 ꢀ 75
467 ꢀ 44
535 ꢀ 52
NHCOC6H5
NHCOC6H5
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
NHCH2CH2COOH
NHCOCH2Cl
NHCOCH3
NHCOC6H5
NHCH2CH2COOH
9J
9K
9L
eCH3
eCH3
eCH3
10J
10K
10L
eCH3
eCH3
eCH3
a
Inhibitory concentration; The IC50 for Roscovitine (Positive control) was observed as 280 ꢀ 41.
4H, cyclohexane CH2, J ¼ 4.6 Hz), 3.01 (s, 1H, SH), 3.78 (s, 2H, CH2),
4.23 (s, 2H, CH2Cl), 8.08 (s, 2H, NH); MS m/z (%) 400 (Mþ, 71), 365
(73), 316 (54.6), 301 (34.2), 273 (100), 272 (10.4), 246 (25.9), 218
(18.5), 164 (5.8), 98 (42); Anal. Calcd. for C15H18ClN5O2S2: C, 45.05;
H, 4.54; N, 17.51. Found: C, 45.16; H, 4.34; N, 17.69.
4.1.4.3. N,N0-(Methylenebis{sulfanedial-5{3-[(2-chloro-acetylamino)-
4,5,6,7-tetrahydro- benzo [b]thiophen-3-ylmethyl]}-4H-[1,2,4]triazole-
3,4-dial})di-acetamide (4I). Yield 89%; mp 259e261 ꢁC; 1H NMR
(300 MHz, CDCl3):
d 1.24e1.59 (m, 8H, cyclohexane), 2.08 (s, 6H,
COCH3), 2.44e2.56 (t, 8H, cyclohexane CH2, J ¼ 4.3 Hz), 3.61 (s, 4H,
CH2), 4.14 (s, 4H, CH2Cl), 4.51 (s, 4H, SCH2), 7.98 (s, 4H, NH); Anal.
Calcd. for C31H36Cl2N10O4S4: C, 45.86; H, 4.47; N, 17.25. Found: C,
45.78; H, 4.59; N, 17.18.
4.1.4. General preparation of N,N0-(methylenebis{sulfanedial-5{3-
[(2- substituted-amino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
ylmethyl]}-4H-[1,2,4]triazole-3,4-dial})di-4-chloro benzamide [7,35]
The triazole (3) (1 mmol), diiodomethane (1.5 mmol) and 5.6 g
(1 mmol) potassium hydroxide were dissolved in 20 ml of
dichloromethane. To the said mixture acidic alumina (20 g) was
added. Dichloromethane was evaporated in vacuos, and mixture
was kept inside the alumina bath and irradiated for 5e6 min at the
power level of 300 W. The mixture was cooled. The solid thus
separated was dissolved in hot ethanol and filtered. After cooling,
the filtrate gave the product as white.
4.1.5. General preparation of 7-chloro-hepta-2, 4, 6-triynoic acid {3-
[2-(2- substituted-amino)-4, 5, 6, 7-tetrahydro-benzo[b]thiophen-3-
ylmethyl]-5-cyanomethylsulfanyl-[1,2,4] triazol-4-yl}-amide [34,35]
The triazole (3) (1 mmol) was mixed with 1.2 ml (2 mmol) of
chloroacetonitrile and dissolved in 25 ml of water. Neutralization
with sodium carbonate gave a precipitate, which was filtered,
washed with cold water (2 ꢂ 20 ml), and crystallized.
4.1.5.1. 7-Chloro-hepta-2, 4, 6-triynoic acid {3-[2-(2-chloro-acetyla-
mino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3-ylmethyl]-5-cyanome-
thylsulfanyl-[1,2,4] triazol-4-yl}-amide (5A). Yield 86%; mp
4.1.4.1. N,N0-(Methylenebis {sulfanedial-5{3-[(2-chloroacetylamino)-
4, 5, 6, 7-tetrahydro-benzo[b]thiophen-3-ylmethyl]}-4H-[1,2,4] tri-
azole-3,4-dial}) di-4-chlorobenzamide (4A). Yield 84%; mp
245e247 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.25e1.49 (m, 4H,
266e268 ꢁC; 1H NMR (300 MHz, CDCl3):
d
1.31e1.60 (m, 8H,
cyclohexane), 2.51e2.68 (t, 4H, cyclohexane CH2, J ¼ 4.8 Hz), 3.81 (s,
2H, CH2), 4.12 (s, 2H, CH2CN), 4.42 (s, 2H, CH2Cl), 7.17e7.41 (m, 4H,
ArH), 8.0 (s, 2H, NH); MS m/z (%) 535 (Mþ, 100), 480 (21.9), 421
(20.6), 324 (34.2), 310 (40.8), 254 (17.9), 83 (28.9), 69 (16.1), 55
(11.3); Anal. Calcd. for C22H20Cl2N6O2S2: C, 49.35; H, 3.76; N, 15.69.
Found: C, 49.23; H, 3.64; N, 15.81.
cyclohexane), 2.02e2.24 (t, 8H, cyclohexane CH2, J ¼ 4.5 Hz), 3.81 (s,
4H, CH2), 4.18 (s, 4H, CH2Cl), 4.62 (s, 2H, CH2), 7.15e7.36 (m, 8H,
ArH), 8.04 (s, 4H, NH); Anal. Calcd. for C41H38Cl4N10O4S4: C, 49.00;
H, 3.81; N, 13.94. Found: C, 49.11; H, 3.75; N, 13.99.
4.1.4.2. N,N0-(Methylenebis{sulfanedial-5{3-[(2-chloro-acetylamino)-
4,5,6,7-tetrahydro
azole-3,4-dial})di-benzamide (4E). Yield 79%; mp 278e280 ꢁC; 1H
NMR (300 MHz, CDCl3): 1.20e1.51 (m, 8H, cyclohexane), 2.21e2.39
(t, 8H, cyclohexane CH2, J ¼ 4.7 Hz), 3.61 (s, 4H, CH2), 4.15 (s, 4H,
CH2Cl), 4.35 (s, 4H, SCH2), 7.18e7.55 (m, 10H, ArH), 8.05 (s, 4H, NH);
Anal. Calcd. for C41H40Cl2N10O4S4: C, 52.61; H, 4.31; N, 14.96. Found:
C, 52.58; H, 4.41; N, 14.82.
e
benzo [b]thiophen-3-ylmethyl]}-4H-[1,2,4]tri-
4.1.5.2. N-{3-[2-(2-Chloro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]
thiophen-3-ylmethyl]-5-cyanomethylsulfanyl-[1,2,4]
benzamide (5E). Yield 82%; mp 268e270 ꢁC; 1H NMR (300 MHz,
CDCl3): 1.38e1.55 (m, 4H, cyclohexane), 2.44e2.62 (t, 4H, cyclo-
triazol-4-yl}-
d
d
hexane CH2, J ¼ 4.4 Hz), 3.68 (s, 2H, CH2), 4.09 (s, 2H, CH2CN), 4.22
(s, 2H, CH2Cl), 7.13e7.56 (m, 5H, ArH), 8.04 (s, 2H, NH); MS m/z (%)
501 (Mþ, 54), 484 (36), 425 (100), 324 (9.3), 310 (12), 254 (28), 161
(32), 83 (40), 69 (7.9), 55 (14.6); Anal. Calcd. for C22H21ClN6O2S2: C,
52.74; H, 4.22; N, 16.77. Found: C, 52.67; H, 4.31; N, 16.68.
Table 4
Selectivity ratio of most active compounds.
4.1.5.3. N-[3-(4-Acetylamino-5-cyanomethylsulfanyl-4H-[1,2,4] triazol-
3-ylmethyl)-4, 5, 6, 7-tetrahydro-benzo[b]thiophen-2-yl]-2-chloro-
acetamide (5I). Yield 81%; mp 271e273 ꢁC; 1H NMR (300 MHz,
Compound
Cdk5 IC50 (nm)a
Cdk2 IC50 (nm)a
Select k2/k5
2A
5A
5I
7A
7B
7E
7I
43 ꢀ 2
58 ꢀ 12
61 ꢀ 6
39 ꢀ 2
64 ꢀ 2
37 ꢀ 1
61 ꢀ 1
35 ꢀ 1
32 ꢀ 1
52 ꢀ 5
1136 ꢀ 154
1319 ꢀ 89
799 ꢀ 58
587 ꢀ 48
487 ꢀ 68
5986 ꢀ 132
48 ꢀ 7
1.2
19.6
21.6
20.5
9.2
13.2
98.1
1.4
CDCl3):
d 1.34e1.52 (m, 4H, cyclohexane), 2.06 (s, 3H, COCH3),
2.44e2.62 (t, 4H, cyclohexane CH2, J ¼ 4.2 Hz), 3.72 (s, 2H, CH2), 3.81
(s, 2H, CH2CN), 4.22 (s, 2H, CH2Cl), 8.04 (s, 2H, NH); MS m/z (%) 439
(Mþ, 49), 422 (79), 363 (19), 328 (62.3), 287 (53.4), 272 (100), 258
(19.5), 216 (12.4), 160 (6.8), 83 (29), 69 (9.7), 55 (13); Anal. Calcd. for
C17H19ClN6O2S2: C, 46.52; H, 4.36; N,19.15. Found: C, 46.58; H, 4.28; N,
19.07.
9A
9E
560 ꢀ 21
17.5
a
Inhibitory concentration.

M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
2071
4.1.6. General preparation of [5-[2-(2-substituted-amino)-4, 5, 6, 7-
tetrahydro-benzo[b] thiophen-3-ylmethyl]-4-(7-chloro-hepta-2, 4,
6-triynoylamino)-4H-[1,2,4] triazol-3-ylsulfanyl]-acetic acid methyl
ester [7,32e35]
A solution of triazole (3) (1 mmol), 0.4 g (1 mmol) of sodium
hydroxide and methyl bromoacetate 1.53 g (1 mmol) was prepared.
To this, acidic alumina was added in 1:5 equivalent of triazole. The
reaction mixture was mixed, and mixture was kept inside the
alumina bath and irradiated for 4e5 min at the power level of
300 W. The mixture was cooled and poured on ice. The solid thus
separated was extracted with hot ethanol, filtered. After cooling,
filtrate gave almost pure product.
cyclohexane CH2, J ¼ 4.4 Hz), 3.71 (s, 2H, CH2), 3.74 (s, 2H, SCH2), 4.03
(s, 2H, CH2Cl), 4.07e4.15 (t, 1H, NH, J ¼ 4.3 Hz), 7.22e7.48 (m, 4H,
ArH), 8.2 (broad, 2H, NH); MS m/z (%) 568 (Mþ, 65), 531 (69), 488
(145), 438 (61), 380 (78), 363 (8.4), 286 (100), 258 (13), 242 (18), 210
(51), 155 (33), 123 (25), 89 (49); Anal. Calcd. for C22H23Cl2N7O3S2: C,
46.48; H, 4.08; N, 17.25. Found: C, 46.38; H, 4.11; N, 17.13.
4.1.7.2. N-{3-[2-(2-Chloro-acetylamino)-4, 5, 6, 7-tetrahydro-benzo
[b] thiophen-3-ylmethyl] -5-hydrazinocarbonylmethylsulfanyl-[1,2,4]
triazol-4-yl}-benzamide (7E). Yield 79%; mp above 300 ꢁC; 1H NMR
(300 MHz, CDCl3):
d 1.38e1.61 (m, 4H, cyclohexane), 2.06 (d, 2H,
NH2, J ¼ 6.4 Hz), 2.45e2.62 (t, 4H, cyclohexane CH2, J ¼ 4.2 Hz), 3.67
(s, 2H, CH2), 3.78 (s, 2H, SCH2), 4.07 (s, 2H, CH2Cl), 4.10e4.24 (t, 1H,
NH, J ¼ 4.7 Hz), 7.35e7.66 (m, 5H, ArH), 8.06 (broad, 2H, NH); MS m/
z (%) 534 (Mþ, 38.2), 498 (100), 455 (10.4), 439 (17.8), 380 (11.7), 363
(29), 286 (46.4), 258 (31.2), 210 (57), 155 (20.8), 123 (7.7), 89 (10.9);
Anal. Calcd. for C22H24ClN7O3S2: C, 49.48; H, 4.53; N, 18.36. Found:
C, 49.51; H, 4.39; N, 18.17.
4.1.6.1. [5-[2-(2-Chloro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]
thiophen-3-ylmethyl]-4-(7-chloro-hepta-2,4,6-triynoylamino)-4H-
[1,2,4] triazol-3-ylsulfanyl]-acetic acid methyl ester (6A). Yield 82%;
mp 279e281 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.41e1.68 (m, 4H,
cyclohexane), 2.40e2.61 (t, 4H, cyclohexane CH2, J ¼ 4.1 Hz), 3.56 (s,
3H, OCH3), 3.8 (s, 2H, CH2), 3.9 (s, 2H, SCH2), 4.28 (s, 2H, CH2Cl),
7.20e7.46 (m, 4H, ArH), 8.09 (broad, 2H, NH); MS m/z (%) 568
(Mþ,100), 509 (14), 459 (12.3), 437 (11.3), 423 (7.2), 366 (7.7), 329
(79), 328 (10), 314 (19.5), 286 (7.9), 271 (31.6), 222 (10.5), 219 (5.7);
Anal. Calcd. for C23H23Cl2N5O4S2: C, 48.59; H, 4.08; N, 12.32. Found:
C, 48.47; H, 4.22; N, 12.16.
4.1.7.3. N-[3- (4-Acetylamino-5-hydrazinocarbonylmethylsulfanyl-4H
-[1,2,4] triazol-3-ylmethyl)-4,5,6,7-tetrahydro-benzo [b] thiophen-2-
yl]-2-chloro-acetamide (7I). Yield 67%; mp 249e251 ꢁC; 1H NMR
(300 MHz, CDCl3):
d 1.24e1.48 (m, 4H, cyclohexane), 1.86 (d, 2H,
NH2, J ¼ 6.3 Hz), 2.08 (s, 3H, COCH3), 2.43e2.54 (t, 4H, cyclohexane
CH2, J ¼ 4.9 Hz), 3.43 (s, 2H, CH2), 3.60 (s, 2H, SCH2), 4.08 (s, 2H,
CH2Cl), 4.12e4.28 (t,1H, NH, J ¼ 4.0 Hz), 8.09 (broad, 2H, NH); MS m/
z (%) 472 (Mþ, 93.3), 436 (24), 393 (21), 378 (12.27), 320 (5.86), 303
(17), 271 (100), 229 (15.2), 212 (8.4), 156 (34), 123 (20), 69 (70); Anal.
Calcd. for C17H22ClN7O3S2: C, 43.26; H, 4.70; N, 20.77. Found: C,
43.20; H, 4.57; N, 20.81.
4.1.6.2. {4-Benzoylamino -5-[2-(2-chloro-acetylamino)-4, 5, 6,
-tetrahydro-benzo [b] thiophen-3-ylmethyl]-4H-[1,2,4]triazol -3
7
-ylsulfanyl}-acetic acid methyl ester (6E). Yield 75%; mp
253e255 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.61e1.84 (m, 4H,
cyclohexane), 2.38e2.51 (t, 4H, cyclohexane CH2, J ¼ 4.8 Hz), 3.53 (s,
3H, OCH3), 3.61 (s, 2H, CH2), 3.83 (s, 2H, SCH2), 4.02 (s, 2H, CH2Cl),
6.73e7.22 (m, 5H, ArH), 8.01 (broad, 2H, NH); MS m/z (%) 534 (Mþ,
100), 476 (21.9), 462 (24.6), 440 (34.2), 426 (39.8), 369 (17.9), 332
(11.3), 288 (5.8), 273 (4), 219 (3), 83 (5.9); Anal. Calcd. for
C23H24ClN5O4S2: C, 51.73; H, 4.53; N, 13.11. Found: C, 51.63; H, 4.44;
N, 13.01.
4.1.8. General preparation of 7-chloro-hepta-2, 4, 6-triynoic acid
-{3-(2- substituted -amino-4,5,6,7-tetrahydro-benzo[b]thiophen-
3ylmethyl)-5-[2-(N0-acetyl-hydrazino)-2-oxo-ethylsulfanyl] -[1,2,4]
triazol-4-yl}-amide [30,31,35]
To a solution of (7) (1 mmol) in dichloromethane (excess
amount), appropriate acid chloride (1 mmol) was added drop-wise
with constant vigorous stirring. After 25 min of stirring, acidic
alumina (10 g) was added. Dichloromethane then was evaporated
in vacuos, and mixture was kept inside the alumina bath and
irradiated for 5e6 min at the power level of 300 W. The mixture
was cooled and the product was extracted with ether. Ether was
distilled off and product thus obtained was crystallized from n-
hexane-carbon tetrachloride mixture.
4.1.6.3. {4-Acetylamino-5-[2-(2-chloro-acetylamino)-4, 5, 6, 7-tetra-
hydro-benzo [b] thiophen-3-ylmethyl]-4H-[1,2,4] triazol-3 -ylsul-
fanyl}- acetic acid methyl ester (6I). Yield 78%; mp 265e267 ꢁC; 1H
NMR (300 MHz, CDCl3):
d 1.34e1.59 (m, 4H, cyclohexane), 2.01 (s,
3H, CH3), 2.36e2.52 (t, 4H, cyclohexane CH2, J ¼ 4.1 Hz), 3.63 (s, 3H,
OCH3), 3.69 (s, 2H, CH2), 3.85 (s, 2H, SCH2), 4.20 (s, 2H, CH2Cl), 8.02
(broad, 2H, NH); MS m/z (%) 472 (Mþ, 92), 414 (67), 401 (37.6), 366
(12.8), 319 (6.3), 291 (27.1), 275 (4.9), 219 (33.2), 161 (100), 95
(55.4), 83 (16.7); Anal. Calcd. for C18H22ClN5O4S2: C, 45.81; H, 4.70;
N, 14.84. Found: C, 45.75; H, 4.53; N, 14.66.
4.1.8.1. 7-Chloro-hepta-2, 4, 6-triynoicacid{3-[2-(N0-acetyl-hydrazino)
-2-oxo-ethylsulfanyl]-5-[2-(2-chloro-acetylamino)-4,5,6,7-tetrahydro-
benzo[b]thiophen-3-ylmethyl]-[1,2,4]triazol-4-yl}-amide (8A). Yield
4.1.7. General preparation of 7-chloro-hepta-2, 4, 6-triynoic acid-
{3-[2-(2- substituted-amino)-4, 5, 6, 7-tetrahydro-benzo[b]thio-
phen-3-ylmethyl]-5-hydrazinocarbonyl methylsulfanyl-[1,2,4]
triazol-4-yl}-amide [31,32,35]
A solution of (6) (1 mmol) with 5 ml (1 mmol) hydrazine hydrate
(98%) was prepared in 10 ml ethanol. To this acidic alumina (10 g) was
added. Ethanol then was evaporated in vacuos, and mixture was kept
inside the alumina bath and irradiated for 5e6 min at the power level
of300 W. The mixture wascooled and theproduct wasextracted with
ether. Ether was distilled off and product thus obtained was crystal-
lized from n-hexane carbon tetrachloride mixture.
82%; mp 254e256 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.40e1.66 (m, 4H,
cyclohexane), 2.08 (s, 3H, COCH3), 2.54e2.68 (t, 4H, cyclohexane CH2,
J ¼ 4.5 Hz), 3.72 (s, 2H, CH2), 3.90 (s, 2H, SCH2), 4.08 (s, 2H, CH2Cl),
4.24e4.64 (dd, 2H, JNHeNH ¼ 4.13, JNHeNH ¼ 4.63), 7.20e7.36 (m, 4H,
ArH), 8.04 (s, 2H, NH); MS m/z (%) 610 (Mþ, 79), 575(26), 540 (52), 489
(63), 461(78),438(32), 395(41), 376(4.8), 326(19), 240(18), 223(7.6),
210 (100), 171 (8.3), 144 (3.3); Anal. Calcd. for C24H25Cl2N7O4S2: C,
47.21; H, 4.13; N, 16.06. Found: C, 47.09; H, 4.03; N, 16.17.
4.1.8.2. N- (3-{4- Acetylamino-5-[2-(N0 -benzoyl-hydrazino)-2-oxo-
ethylsulfanyl]-4H -[1,2,4]triazol-3-ylmethyl}-4,5,6,7-tetrahydro-ben-
zo[b]thiophen-2-yl)-2-chloro-acetamide (8AC). Yield 71%; mp
4.1.7.1. 7-Chloro-hepta-2, 4, 6-triynoicacid-{3 -[2 -(2 -chloro-acety-
lamino)-4,5, 6,7- tetrahydro-benzo[b]thiophen-3-ylmethyl]-5-hydra-
zinocarbonylmethylsulfanyl-[1,2,4] triazol-4-yl}-amide (7A). Yield
226e228 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.37e1.63 (m, 4H,
cyclohexane), 2.02 (s, 3H, COCH3), 2.48e2.62 (t, 4H, cyclohexane
CH2, J ¼ 4.1 Hz), 3.69 (s, 2H, CH2), 3.78 (s, 2H, SCH2), 4.11 (s, 2H,
CH2Cl), 4.22e4.64 (dd, 2H, JNHeNH ¼ 4.28, JNHeNH ¼ 4.59), 7.20e7.69
(m, 5H, ArH), 8.05 (s, 2H, NH); MS m/z (%) 576 (Mþ, 100), 541 (56.2),
83%; mp 230e232 ꢁC; 1H NMR (300 MHz, CDCl3):
d
1.41e1.62 (m, 4H,
cyclohexane), 2.14 (d, 2H, NH2, J ¼ 6.5 Hz), 2.45e2.64 (t, 4H,

2072
M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
496 (30.8), 481 (29.5), 427 (10), 379 (15.7), 325 (13.3), 287 (37), 241
(23.5), 167 (76), 124 (22), 109 (7), 98 (5.5); Anal. Calcd. for
C24H26ClN7O4S2: C, 50.04; H, 4.55; N, s17.02. Found: C, 50.13; H,
4.43; N, 17.14.
4.1.8.8. N-(3-[2-(2-Chloro-acetylamino)- 4, 5, 6, 7-tetrahydro-benzo
[b] thiophen -3-ylmethyl]-5-{2-[N0-(2-chloro-acetyl)-hydrazino]-2-
oxo-ethylsulfanyl}-[1,2,4]triazol-4-yl)-benzamide (8U). Yield 82%;
mp 235e237 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.40e1.64 (m, 4H,
cyclohexane), 2.56e2.73 (t, 4H, cyclohexane CH2, J ¼ 4.6 Hz), 3.78
(s, 2H, CH2), 3.85 (s, 2H, SCH2), 4.11 (s, 4H, CH2Cl), 4.21e4.48 (dd,
2H, JNHeNH ¼ 4.52, JNHeNH ¼ 4.74), 6.90e7.32 (m, 5H, ArH), 8.05 (s,
2H, NH); MS m/z (%) 610 (Mþ, 100), 575 (85), 533 (49), 472 (14), 431
(29), 402 (11), 353 (7.8), 310 (67), 281 (31), 234 (24), 178 (19), 134
(6.1), 98 (3.4); Anal. Calcd. for C24H25Cl2N7O4S2: C, 47.21; H, 4.13; N,
16.06. Found: C, 47.08; H, 4.24; N, 16.18
4.1.8.3. N-[3-(4-Acetylamino-5-{2-[N0-(2-chloro-acetyl)-hydrazino]-
2-oxo-ethylsulfanyl}-4H-[1,2,4]triazol-3-ylmethyl)-4,5,6,7-tetrahydro-
benzo[b]thiophen-2-yl]-2-chloro-acetamide (8AG). Yield 84%; mp
247e249 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.36e1.51 (m, 4H,
cyclohexane), 2.02 (s, 3H, COCH3), 2.41e2.63 (t, 4H, cyclohexane
CH2, J ¼ 4.4 Hz), 3.68 (s, 2H, CH2), 3.76 (s, 2H, SCH2), 4.17 (s, 4H,
CH2Cl), 4.25e4.69 (dd, 2H, JNHeNH ¼ 4.33, JNHeNH ¼ 4.63), 8.07 (s, 2H,
NH); MS m/z (%) 548 (Mþ, 14.1), 513 (38), 498 (15.7), 458 (17.3), 423
(100), 377 (23.5), 309 (3.9), 280 (13.2), 229 (5.8),177 (11),161 (29.6).;
Anal. Calcd. for C19H23Cl2N7O4S2: C, 41.61; H, 4.23; N,17.88. Found: C,
41.66; H, 4.13; N, 17.80.
4.1.8.9. N-(3-{4-Acetylamino-5-[2-(N0-acetyl-hydrazino)-2-oxo-ethy-
lsulfanyl]-4H-[1,2,4] triazol-3-ylmethyl}-4,5,6,7-tetrahydro-benzo[b]
thiophen-2-yl)-2-chloro-acetamide (8Y). Yield 81%; mp 251e253 ꢁC;
1H NMR (300 MHz, CDCl3):
d 1.39e1.65 (m, 4H, cyclohexane), 2.04 (s,
6H, COCH3), 2.47e2.61 (t, 4H, cyclohexane CH2, J ¼ 4.4 Hz), 3.68 (s,
2H, CH2), 3.72 (s, 2H, SCH2), 4.09 (s, 4H, CH2Cl), 4.18e4.49 (dd, 2H,
JNHeNH ¼ 4.26, JNHeNH ¼ 4.50), 8.03 (s, 2H, NH); MS m/z (%) 514 (Mþ,
100), 479 (62), 438 (15.8), 376 (52), 335 (71), 279 (8.7), 223 (24), 177
(27); Anal. Calcd. for C19H24ClN7O4S2: C, 44.40; H, 4.71; N, 19.07.
Found: C, 44.34; H, 4.87; N, 19.28.
4.1.8.4. 7-Chlorohepta-2, 4, 6-triynoic acid {3- [2- (N0-benzoyl-hydra-
zino)- 2-oxo-ethylsulfanyl]-5-[2-(2-chloro-acetylamino)-4,5,6,7-tetra-
hydro-benzo[b]thiophen-3-ylmethyl]-[1,2,4]triazol-4-yl}-amide (8E).
Yield 71%; mp 231e233 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.65e1.64
(m, 4H, cyclohexane), 2.51e2.64 (t, 4H, cyclohexane CH2, J ¼ 4.2 Hz),
3.70 (s, 2H, CH2), 3.79 (s, 2H, SCH2), 4.23 (s, 2H, CH2Cl), 4.34e4.62 (dd,
2H, JNHeNH ¼ 4.23, JNHeNH ¼ 4.76), 6.90e7.38 (m, 9H, ArH), 8.03 (s, 2H,
NH); MS m/z (%) 672 (Mþ, 100), 614 (11.1), 579 (13.2), 534 (32), 480
(3.6), 432 (3.4), 379 (8.2), 325 (8.1), 287 (11.9), 241 (15.4), 167 (23.8),
124 (54.2), 109 (25.7), 98 (22); Anal. Calcd. for C29H27Cl2N7O4S2: C,
51.79; H, 4.05; N, 14.58. Found: C, 51.72; H, 4.10; N, 14.42.
4.1.9. General preparation of 7-chloro-hepta-2, 4, 6-triynoic acid -
{3-(benzylidene-hydrazinocarbonylm ethylsulfanyl)-5-[2-(2-
substituted -amino)- 4, 5, 6, 7-tetrahydro-benzo[b]thiophen-
3ylmethyl]- [1,2,4] triazol-4-yl}-amide [31,32,35]
A solution of (7) (1 mmol) with benzaldehyde (1 mmol) was
prepared in 10 ml ethanol. To this acidic alumina (10 g) was added.
Ethanol then was evaporated in vacuos, and mixture was kept
inside the alumina bath and irradiated for 1 min at the power level
of 300 W. The mixture was cooled and poured on ice. The solid thus
separated was filtered and extracted with ether. Ether was distilled
off and product thus obtained was crystallized from hot ethanol.
4.1.8.5. 7-Chloro-hepta- 2,4,6-triynoic acid (3-[2-(2-chloro-acetyla-
mino)-4,5, 6, 7-tetrahydro -benzo[b]thiophen-3-ylmethyl]-5-{2-[N0
-(2-chloro-acetyl)-hydrazino]-2-oxo-ethylsulfanyl}-[1,2,4]triazol-4-yl)-
amide (8I). Yield 74%; mp 290e292 ꢁC; 1H NMR (300 MHz, CDCl3):
d
1.33e1.55 (m, 4H, cyclohexane), 2.44e2.62 (t, 4H, cyclohexane CH2,
J ¼ 4.8 Hz), 3.76 (s, 2H, CH2), 3.78 (s, 2H, SCH2), 4.13 (s, 4H, CH2Cl),
4.26e4.64 (dd, 2H, JNHeNH ¼ 4.43, JNHeNH ¼ 4.68), 7.17e7.40 (m, 4H,
ArH), 8.02 (s, 2H, NH); MS m/z (%) 644 (Mþ, 35.5), 609 (43), 574
(27.7), 559 (5.5), 519 (40), 471 (26.8), 436 (10.5), 408 (13.2), 390 (8.7),
338 (4.7), 310 (100), 280 (9.1), 229 (20.2), 177 (33.3), 161 (8.8); Anal.
Calcd. for C24H24Cl3N7O4S2: C, 44.69; H, 3.75; N, 15.20. Found: C,
44.79; H, 3.63; N, 15.18.
4.1.9.1. 7-Chloro-hepta-2, 4, 6-triynoic acid-{3-(benzylidene-hydra-
zinocarbonylm ethylsulfanyl)-5-[2-(2-chloro-acetylamino)-4,5,6,7-
tetrahydro-benzo[b]thiophen-3ylmethyl]-[1,2,4] triazol-4-yl}-amide
(9A). Yield 86%; mp decomposed around 226e228 ꢁC; 1H NMR
(300 MHz, CDCl3):
d 1.38e1.61 (m, 4H, cyclohexane), 2.51e2.62 (t,
4H, cyclohexane CH2, J ¼ 4.4 Hz), 3.76 (s, 2H, CH2), 3.94 (s, 2H,
SCH2), 4.24 (s, 2H, CH2Cl), 6.91e7.40 (m, 9H, ArH), 8.06 (s, 3H, NH),
8.24 (s, 1H, N]CH); MS m/z (%) 656 (Mþ, 69), 615 (19), 570 (37), 532
(21), 468 (58), 399 (24), 348 (35), 287 (100), 210 (18.4), 157 (11.4),
103 (12.3); Anal. Calcd. for C29H27Cl2N7O3S2: C, 53.05; H, 4.14; N,
14.93. Found: C, 53.14; H, 4.03; N, 14.87.
4.1.8.6. N-{3-[2-(N0-Acetyl-hydrazino)-2-oxo-ethylsulfanyl]-5-[2-(2-
chloro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3-ylmeth-
yl]-[1,2,4]triazol-4-yl}-benzamide (8M). Yield 57%; mp 170e172 ꢁC;
1H NMR (300 MHz, CDCl3):
d 1.31e1.59 (m, 4H, cyclohexane), 2.06
(s, 3H, COCH3), 2.45e2.64 (t, 4H, cyclohexane CH2, J ¼ 4.7 Hz), 3.71
(s, 2H, CH2), 3.74 (s, 2H, SCH2), 4.19 (s, 4H, CH2Cl), 4.28e4.61 (dd,
2H, JNHeNH ¼ 4.23, JNHeNH ¼ 4.58), 7.08e7.68 (m, 5H, ArH), 8.04 (s,
2H, NH); MS m/z (%) 576 (Mþ, 13.6), 540 (13), 489 (40.9), 438 (6),
376 (51.3), 326 (100), 240 (77.4), 223 (15.9),171 (25),144 (4.4); Anal.
Calcd. for C24H26ClN7O4S2: C, 50.04; H, 4.55; N, 17.02. Found: C,
50.19; H, 4.49; N, 17.21.
4.1.9.2. N-{3-(Benzylidene-hydrazinocarbonylmethylsulfanyl)-5-[2-(2
-chloro
thyl]-[1,2,4]triazol-4-yl}-benzamide (9E). Yield 78%; mp 182e184 ꢁC;
1H NMR (300 MHz, CDCl3):
1.66e1.87 (m, 4H, cyclohexane),
-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3-ylme-
d
2.40e2.51 (t, 4H, cyclohexane CH2, J ¼ 4.9 Hz), 3.64 (s, 2H, CH2), 3.80
(s, 2H, SCH2), 4.19 (s, 2H, CH2Cl), 6.78e7.54 (m, 10H, ArH), 7.8 (d, 2H,
benzene CH), 7.96 (s, 3H, NH), 8.13 (s, 1H, N]CH); MS m/z (%) 622
(Mþ, 6.9), 573 (31), 535 (3), 500 (6), 470 (13.7), 399 (3.4), 348 (39),
319 (16), 287 (100), 224 (67.6), 210 (29),157 (5),103 (9.8); Anal. Calcd.
for C29H28ClN7O3S2: C, 55.98; H, 4.54; N, 15.76. Found: C, 55.98; H,
4.42; N, 15.69.
4.1.8.7. N-{3 -[2-(N0-Benzoyl-hydrazino)- 2 -oxo-ethylsulfanyl]-5-[2-
(2-chloro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3-ylmet-
hyl]-[1,2,4]triazol-4-yl}-benzamide (8Q). Yield 91%; mp 124e126 ꢁC;
1H NMR (300 MHz, CDCl3):
d 1.38e1.54 (m, 4H, cyclohexane),
2.44e2.62 (t, 4H, cyclohexane CH2, J ¼ 5.2 Hz), 3.69 (s, 2H, CH2), 3.79
(s, 2H, SCH2), 4.16 (s, 4H, CH2Cl), 4.22e4.62 (dd, 2H, JNHeNH ¼ 4.46,
JNHeNH ¼ 4.61), 7.38e7.74 (m, 10H, ArH), 8.02 (s, 2H, NH); MS m/z (%)
638 (Mþ, 100), 603 (43), 579 (36), 534 (84), 480 (54.6), 432 (9.9), 392
(12), 379 (37), 325 (40), 298 (32), 287 (26), 241 (6.3), 167 (10), 109
(5.3), 98 (14); Anal. Calcd. for C29H28ClN7O4S2: C, 54.58; H, 4.42; N,
15.36. Found: C, 54.39; H, 4.33; N, 15.43.
4.1.9.3. N-{3-[4-Acetylamino -5-(benzylidene-hydrazinocarbonylme-
thylsulfanyl)-4H-[1,2,4] triazol-3-ylmethyl]-4,5,6,7-tetrahydro-benzo
[b]thiophen-2-yl}-2-chloro-acetamide (9I). Yield 81%; mp decom-
posed around 221e223 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.42e1.60
(m, 4H, cyclohexane), 2.03 (s, 3H, COCH3), 2.44e2.60 (t, 4H, cyclo-
hexane CH2, J ¼ 4.6 Hz), 3.75 (s, 2H, CH2), 3.92 (s, 2H, SCH2), 4.26 (s,

M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
2073
2H, CH2Cl), 6.91e7.12 (m, 5H, ArH), 8.01 (s, 3H, NH), 8.12 (s, 1H, N]
CH); MS m/z (%) 560 (Mþ, 100), 525 (3.7), 474 (13.5), 432 (9), 363
(12), 322 (17), 305 (60.4), 263 (32), 231 (76), 175 (38.6); Anal. Calcd.
for C24H26ClN7O3S2: C, 51.47; H, 4.68; N, 17.51. Found: C, 51.55; H,
4.49; N, 17.60.
incorporation of [33P] from the gamma phosphate of [33P] ATP
(Amersham, cat. no. AH-9968) into biotinylated peptide substrate
PKTPKKAKKL. Reactions were carried out in a buffer containing
50 mM TriseHCI, pH 8.0; 10 mM MgCl2, 0.1 mM Na3VO4, and
1 mM DTT. The final concentration of ATP was 0.5
specific radioactivity of 4 Ci/nmol), and the final concentration
of substrate was 0.75 M. Reactions, initiated by the addition of
mM (final
m
4.1.10. General preparation of 7-chloro-hepta-2, 4, 6-triynoic acid -
{3-(4- amino-5-mercapto-4H-[1,2,4] triazol-3-ylmethylsulfanyl)-5-
[2-(2- substituted -amino)-4, 5, 6, 7-tetrahydro-benzo[b]thiophen-
3ylmethyl] e [1,2,4] triazol-4-yl}-amide [31,32,35]
m
cdk5 and activator protein p25, were carried out at room
temperature for 60 min. Reactions were stopped by addition of
0.6 volume of buffer containing (final concentrations): 2.5 mM
The (7) (1 mmol) was dissolved in alcoholic potassium
hydroxide (1 mmol) and kept for stirring. Carbon disulphide
(1.5 mmol) was added drop wise to this solution with stirring. Thick
solid mass was obtained, to which 50 ml of absolute alcohol was
added. Stirring was continued for 16 h. At the end of 16th h, dry
ether was added to the mixture. The precipitate (thiocarbazate)
obtained was taken immediately for the next step.
A solution of thiocarbazate (1 mmol) with hydrazine hydrate
(1 mmol) was prepared in 10 ml ethanol. To this acidic alumina
(10 g) was added. Ethanol then was evaporated in vacuos, and
mixture was kept inside the alumina bath and irradiated for
5e6 min at the power level of 300 W. The mixture was cooled and
poured on ice. The solid thus separated was filtered, extracted with
ether, ether was distilled off and product thus obtained was crys-
tallized from hot ethanol.
EDTA, 0.05% Triton-X 100, 100 mM ATP, and 1.25 mg/mL strepta-
vidin coated SPA beads (Amersham cat. no. RPNQ0007). Radio-
activity associated with the beads was quantified by scintillation
counting. We have also done cytotoxicity analysis of the above-
synthesized compounds, using neutral red uptake by using Vero-
C-1008 cell line [36] at various concentrations (6.25e50 mg/mL),
none of them were found toxic. Hence the activities of the above-
synthesized compounds were not due to cytotoxicity of
compounds.
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4.1.10.1. 7-Chloro-hepta-2,4,6-triynoic acid -{3-(4- amino-5-mer-
capto-4H-[1,2,4] triazol-3-ylmethylsulfanyl)-5-[2-(2-chloro-acetyla-
mino)-4,5,6,7-tetrahydro-benzo[b]thiophen-3ylmethyl]
triazol-4-yl}-amide (10A). Yield 72%; mp 232e234 ꢁC; 1H NMR
(300 MHz, CDCl3): 1.31e1.58 (m, 4H, cyclohexane), 2.04 (s, 2H,
e
[1,2,4]
d
NH2), 2.56e2.73 (t, 4H, cyclohexane CH2, J ¼ 4.3 Hz), 3.06 (s, 1H,
SH), 3.82 (s, 2H, CH2), 4.12 (s, 2H, SCH2), 4.27 (s, 2H, CH2Cl),
7.14e7.41 (m, 4H, ArH), 8.1 (s, 2H, NH); MS m/z (%) 624 (Mþ, 82), 570
(43), 517 (31), 461 (26), 384 (31), 326 (13.2), 247 (15), 226 (17), 126
(100); Anal. Calcd. for C23H23Cl2N9O2S3: C, 44.23; H, 3.71; N, 20.18.
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thylsulfanyl)-5-[2-(2-chloro-acetyl amino)-4,5,6,7-tetrahydro-benzo
[b]thiophen-3-ylmethyl]-[1,2,4]triazol-4-yl}-benzamide (10E). Yield
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80%; mp 256e258 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.43e1.72 (m,
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4H, cyclohexane), 2.03 (s, 2H, NH2), 2.36e2.62 (t, 4H, cyclohexane
CH2, J ¼ 4.2 Hz), 3.03 (s,1H, SH), 3. 86 (s, 2H, CH2), 4.07 (s, 2H, SCH2),
4.20 (s, 2H, CH2Cl), 7.06e7.62 (m, 5H, ArH), 8.08 (s, 2H, NH); MS m/z
(%) 590 (Mþ, 100), 536 (17), 483 (22), 427 (61), 350 (14), 326 (45.7),
247 (39), 226 (6.6), 126 (22.3), 89 (19.7); Anal. Calcd. for
C23H24ClN9O2S3: C, 46.81; H, 4.10; N, 21.36. Found: C, 46.93; H, 4.19;
N, 21.48.
4.1.10.3. N-{3-[4-Acetylamino -5 -(4-amino-5- mercapto-4H-[1,2,4]
triazol -3 -yl methylsulfanyl)-4H-[1,2,4]triazol-3-ylmethyl]-4,5,6,7-
tetrahydro-benzo[b]thiophen-2-yl}-2-chloro-acetamide (10I). Yield
77%; mp 269e271 ꢁC; 1H NMR (300 MHz, CDCl3):
d 1.26e1.59 (m,
4H, cyclohexane), 1.97 (s, 2H, NH2), 2.08 (s, 3H, COCH3), 2.52e2.71
(t, 4H, cyclohexane CH2, J ¼ 4.5 Hz), 3.12 (s, 1H, SH), 3.76 (s, 2H,
CH2), 4.17 (s, 2H, SCH2), 4.23 (s, 2H, CH2Cl), 8.04 (s, 2H, NH); MS
m/z (%) 528 (Mþ, 56.3), 474 (52), 421(100), 365 (10), 288 (48), 246
(93), 219 (8.1), 83 (72.3); Anal. Calcd. for C18H22ClN9O2S3: C,
40.94; H, 4.20; N, 23.87. Found: C, 40.85; H, 4.07; N, 23.98.
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4.2. Cyclin-dependent kinase 5/p25 inhibiting activity
Kinase inhibit ion was measured by use of scintillation prox-
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M. Shiradkar et al. / European Journal of Medicinal Chemistry 46 (2011) 2066e2074
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