Synthesis and biological evaluation of 2-thiopyrimidine derivatives

Article abstract of DOI:10.1016/j.bmc.2005.02.047

Various 2-thiopyrimidine derivatives have been synthesized by an efficient, one-pot reaction of functionalized amines with either 4-isothiocyanato-4- methyl-2-pentanone or 3-isothiocyanatobutanal. All the synthesized compounds were fully characterized by

Full text of DOI:10.1016/j.bmc.2005.02.047

Bioorganic & Medicinal Chemistry 13 (2005) 3185–3195
Synthesis and biological evaluation of 2-thiopyrimidine derivatives
Sham M. Sondhi,a,* Rajendra N. Goyal,a Anand M. Lahoti,a Nirupma Singh,a
Rakesh Shuklab and Ram Raghubirb
aDepartment of Chemistry, Indian Institute of Technology Roorkee (IITR), Roorkee 247 667, India
bDepartment of Pharmacology, Central Drug Research Institute (CDRI), Lucknow 226 001, India
Received 1 February 2005; revised 19 February 2005; accepted 19 February 2005
Available online 19 March 2005
Abstract—Various 2-thiopyrimidine derivatives have been synthesized by an efficient, one-pot reaction of functionalized amines with
either 4-isothiocyanato-4-methyl-2-pentanone or 3-isothiocyanatobutanal. All the synthesized compounds were fully characterized
1
by elemental analysis (CHN), FT-IR, H NMR, and mass spectral data. One of the compounds, 7,7,8a-trimethyl-hexahydro-thia-
zolo[3,2-c]pyrimidine-5-thione (17) showed good anti-inflammatory (37.4% at 100 mg/kg p.o.) and analgesic activity (75% at 100 mg/
kg p.o.). 7-(1-Mercapto-3,3,4a-trimethyl-4,4a,5,9b-tetrahydro-3H-pyrido[4,3-b]indol-7-yl)-3,3,4a-trimethyl-3,4,4a,5-tetrahydrobenzo[4,5]-
imidazo[1,2-c]pyrimidine-1-thiol (3) showed moderate activity against CDK-1(IC 50 = 5 lM). The other compounds showed
moderate anti-inflammatory (5–20%), analgesic (25–75%) and protein kinase (CDK-5, GSK-3) inhibitory activities (IC50
10 lM).
>
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, two PCT international applications12 revealed
that, 2-TP derivatives possess potent activity against
inflammation and immune disorders. Thus, search for
novel, potent and selective 2-TP derivatives is desirable
in order to substitute drugs having major side effects
such as peptic-ulcer formation and gastro-intestinal
damage.13 During the past few years our laboratory
has been actively involved in the synthesis of a variety
of 2-TP derivatives14 by a simple but efficient, one-pot
reaction of functionalized amines with the well-known
precursor b-isothiocyanatoketone.15 Various mono-,
di-, tri- and tetra-cyclic, di/tetrahydro-2-TP derivatives
have been synthesized and evaluated for anti-inflamma-
tory and analgesic activity (both in vivo and in vitro).16
Some of them have shown moderate to good activity for
inflammation.17 Recently we have extended our scope of
research to in vitro screening of 2-TP derivatives for ki-
nase (CDK-1, CDK-5, and GSK-3) inhibition.18 In con-
tinuation of our search for potent bioactive molecules, it
was considered worthwhile to synthesize a variety of di/
tetrahydro 2-TP derivatives. The synthesis19 and biolog-
ical studies of various 2-TP derivatives have been dis-
cussed in this paper.
Pyrimidine derivatives have been very well known in
medicinal chemistry for their therapeutic applications.1
One possible reason for their activity is presence of a
pyrimidine base in thymine, cytosine and uracil, which
are essential building blocks of nucleic acids,2 DNA
and RNA. One important class of pyrimidine is 2-thio-
pyrimidine (2-TP) and its derivatives, which are also well
known as 2-mercaptopyrimidine compounds.3 In 2-TP
ring sulfur atom serves as an interesting replacement
for the existing oxygen atom bonded to C-2 in uridine
base.4 Considering this assumption 2-TPs have attracted
substantial interest of synthetic-biochemists.5 The Euro-
pean patent6 revealed the application of 2-TP derivatives
in preparation of cardiotonic drugs. Studies by Pathak
et al. evaluated primary activity of 2-TP derivatives
against Mycobacterium tuberculosis7 (Mtb). 2-TPs also
serve as important precursors for asymmetric synthesis
of allylic sulfides/sulfonates.8 Thus synthesis,9 biologi-
cal10 as well as analytical studies11 of 2-TP derivatives
have been topics of interest for chemists.
2. Chemistry
Keywords: Synthesis; 2-Thiopyrimidine derivatives; b-Isothiocyana-
toketone; Anti-inflammatory; Analgesic; Kinase inhibition.
*
Various 2-thiopyrimidine derivatives (3, 4, 6, 7a, 9a–c,
11a,b, 13a,b, 15, 17, 19, and 21) have been synthesized
Corresponding author. Tel.: +91 1332 285811; fax: +91 1332
273560; e-mail: sondifcy@iitr.ernet.in
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.047

3186
S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
in reasonable yields by the one-pot reaction of either 4-
isothiocyanato-4-methyl-2-pentanone20 (2a) or 3-isothio-
cyanatobutanal20 (2b) with commercially available
functionalized amines (1a,b, 5a,b, 8, 10, 12, 14, 16, 18,
and 20) in absolute methanol. Temperature and pH con-
ditions play an important role in these reactions.
distinguish dihydro-2-TP (3) from tetrahydro-2-TP (4)
derivatives. Compound 4 is supposed to be symmetrical
since a singlet at d 4.85, was integrated for two identical
olefinic protons on either sides of biphenyl ring.
General mechanism21 for condensation of the function-
alized amine with b-keto/aldo-isothiocynate, is pre-
sented in Figure 1. At an elevated temperature the
initial attack of para –NH2 group of compound 1a on
the isothiocyanate group of 2a can generate an interme-
diate species (i), which has an unreacted ketone (or alde-
hyde) group. This reactive intermediate exists in a
favorable cyclic six-member structure (i) due to the pres-
ence of geminal dimethyl groups. Furthermore intramo-
lecular nucleophilic attack of the –NH group over the
3. Results and discussion
Two moles of 4-isothiocyanato-4-methyl-2-pentanone
(2a), on condensation with 3,30-diaminobenzidine (1a)
in absolute methanol (pH ꢀ 4–5) under refluxing condi-
tions gave tetrahydro 2-thiopyrimidobenzimidazole
derivative 3 with 77% yield (Scheme 1). The structure
of 3 was fully supported by elemental analysis, FT-IR,
1H NMR and mass spectral studies. Infra-Red spectra
of 3 showed characteristic absorption bands near 1201
and 3150 cmÀ1 for the stretching frequency of C@S
H
N
R1
R2
R1
R2
S
NH2
1
N
and N–H group, respectively. H NMR spectra of 3 in
+
NH
XH
CDCl3 + 2 drops of DMSO-d6, clearly showed two clo-
sely packed doublets (d 2.24 and 2.55 with Jab = 13.5 Hz
each) for the anisotropic protons (2· –HaCHbC*CH3).
This pattern can be observed only when both (para
and meta) amino groups of the same benzene ring of
1a are involved in the cyclization with 1mol of 2a result-
ing in pyrimidobenzimidazole ring structure. The other
signals observed in the NMR of 3 were as follows; gem-
inal dimethyl groups as two closely packed singlet at d
1.34 (2· 3H) and 1.38 (2· 3H), protons of methyl groups
present on asymmetric carbon atoms (C*) as singlet at d
1.52 (2· 3H), aromatic protons in the region d 6.62–7.05
(2· 3H). On D2O exchange broad peaks for labile pro-
tons of –NH groups disappeared from the original posi-
tion of d 8.15 (2· –HN–), 8.65 (–HN–C@S), and 9.03
(–HN–C@S), which further supports the structure as-
signed for 3. Mass spectra (EI-MS) of 3 gave molecular
ion peak at m/z 492 (15%) (calcd molar mass for
C26H32N6S2; 492.213). Reaction of benzidine (1b) with
2a in absolute methanol at rt as well as under refluxing
XH
S
O
R3
R3
O
X= NH, S or O
R1=R2=R3=CH3
or
(i)
R1=CH3, R2=R3= H
H
N
R1
R2
H
N
R1
S
S
R2
H
N
R3
X
N
HO
XH
R3
OH
H
(iii)
-H2O
(ii)
-H2O
S
H
N
R1
R2
S
NH
R1
R2
N
N
R3
X
R3
1
XH
condition (pH ꢀ 4–5) gave 4 with 60% yield. The H
(iv)
(v)
NMR of 4 showed a sharp singlet at d 4.85 for olefin
protons. This singlet is characteristic in order to
Figure 1.
H2N
NH2
+
N
S
O
R
R
1a; R = NH2
1b; R = H
2a
CH3OH
r. t. / 8h
pH 5 / Reflux,/ 4h
S
S
N
S
S
HN
NH
HN
NH
N
N
N
N
N
H
H
4
3
Scheme 1.

S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
3187
neighboring ketone (or aldehyde) group can yield a alco-
hol (ii). Finally meta –NH2 group of 1a attacks over the
reactive species (ii), to yield a 2-thiopyrimidobezimida-
zole ring (iv) by elimination of the water molecule. It
was expected that the loss of the water molecule in such
a reaction was accelerated in the presence of the protic
catalyst (like H2SO4). An exact similar mechanism was
expected on the other side of biphenyl ring of 1a to yield
3. Synthesis of various pyrimidobezimidazole ring com-
pounds by a similar mechanism has been well described
in the literature.22 At rt an intermediate similar to iii was
formed by an intra-molecular nucleophilic attack of
para –NH2 group of 1b over 2a. This intermediate spe-
cies (iii) rapidly looses a water molecule to give a 2-thio-
pyrimidine ring (v) on either sides of biphenyl ring. The
mechanism in Figure 1 suggest only probable sequence
of steps involved in the formation of tetrahydro-2-thio-
pyrimidine (iv) and dihydro-2-thiopyrimidine (v)
analogues.
looks like a stereoisomer, however we were unable to
separate its different isomers. The EI-MS spectra of 6
showed a molecular ion peak at m/z 233, 40% (calcd
mass for C12H15N3S 233.099) and the fragmented cat-
ions observed in the mass spectra were presented in
the Experimental section. The structure assigned for 7a
was also fully in agreement with the observed spectral
data. Formation of 7b by the reaction of 5b with 2b
was also expected23 but when the reaction was carried
out in absolute methanol under refluxing condition
with/without adjusting pH ꢀ 4–5, in TLC only a single
spot was observed (after 6 h) at Rf 0.6 (CHCl3–CH3OH,
9.5:0.5) and its characterization further confirmed it as
7a (20%) rather than 7b.
Reaction of the hydrochloride salt of aminoacetonitrile
(8) with 2a and 2b under different experimental condi-
tions is presented in Scheme 3. Reaction of 8 with 2a,
after a 6 h reflux gave a white solid.24 Its purification
and characterization allowed its identification as 9a
rather than 9. The IR (KBr) spectra of 9a showed no
peaks in the region 2225–2260 cmÀ1 (C„N stretching),
however sharp peaks at 1735.9 and 1212.2 cmÀ1(C@O
and C@S stretching, respectively) were observed. This
suggested that the cyanide group of 8 underwent hydro-
lysis in presence of an acid catalyst (HCl) and finally a
methyl ester derivative 9a was formed in presence of
alcohol (methanol). The 1H NMR of 9a showed a char-
acteristic signal singlet d 3.78 (H3CO–C@O) and 4.79
(H2C–COOMe) further confirms the presence of methyl
ester in 9a. The FAB-MS spectra for 9a showed a base
MH+ peak m/z 229 (calcd mass for C10H16N2SO2
228.093) and also a peak at m/z 213 (40%) for a stable
(4,4,6-trimethyl-2-thioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
acetyl cation. For the synthesis of 9, it was considered
necessary to quench the acid formed during the reac-
tion of 8 with 2a. Accidentally when we used 1.3 equiv
(with respect to HCl) of sodium carbonate in the
Scheme 2 shows formation of 1,4-dihydro-2-thiopyrimi-
dine derivatives 6 and 7a by rt stirring of 3-isothiocya-
natobutanal (2b) with 2-aminobenzylamine (5a) and
2-aminobenzyl-alkanol (5b), respectively. The mecha-
nism followed in the synthesis of 6 was very similar to
that explained for iv in Figure 1. It is interesting to note
that, the reactive aldehyde group of 2b is supposed to
participate only in the second step and this behavior
of intramolecular addition of various diamines with 2b
has been reported in our earlier communication.16 IR
analysis of 6 showed characteristic bands at 3339.7,
3222.6 (N–H stretch), 1559.3, 1489.6 (C@C) and
1
1210.3 cmÀ1(C@S) and its H NMR spectra in CDCl3
showed a characteristic septet d 3.92, integrating for a
single proton at C12. The J values (4 and 6.6 Hz) for
*
*
the proton (at C12) indicated, its coupling with methyl
hydrogen and hydrogen atoms at C11. The different
peaks observed in the NMR revealed that compound 6
XH
N
+
NH2
S
O
H
5a; X=NH
2b
r . t. / 6h
CH3OH
5b; X= O
15
S
6
3
10
1
2
9
OH
O
6
3
10
5
4
9
N
NH 13
1
2
8
7
N
14
5
4
14
13
N
8
12
11
N
H
7
16
11
S
N
H
15 S
N
H
16
12
6
7b
7a
Scheme 2.

3188
S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
S
O
HN
N
O
9a
S
a
c
N
N
NH
N
O
R1
b
R2
CH3OH
Reflux
NH2 .HCl
9b
N
+
S
CN
S
O
R3
HN
HN
N
C
8
2a; R1=R2=R3=CH3
or
2b; R1=CH3, R2=R3= H
9
S
a; 6 h
O
N
b; Na2CO3 (1.3 eqi) / 8 h
c; 8 h
O
O
9c
Scheme 3.
reaction, 9b was isolated. The IR analysis of 9b neither
showed C„N nor C@O stretching vibrations, however
a peak at 1191.5 cmÀ1 was clearly observed. The 1H
NMR of 9b showed a characteristic double doublet for
anisotropic protons with J = 18.0 and 17.8 Hz, but no
characteristic singlet was observed for the olefin proton.
Thus it was established that a tetrahydro-2-thiopyrimi-
dine derivative 9b was formed during the reaction. This
could be possible only when tertiary –OH group (rather
than elimination of water) attacks over the neighboring
cyanide group to give a five member ring condensed
with 2-thiopyrimidine ring. A characteristic singlet ob-
served at d 3.16 ppm was attributed to S–CH3 protons
and this S-methylation is due to the formation of methyl
carbonium ion, in presence of excess base (Na2CO3).
Formation of S-methylated derivative of 2-TP have been
well reported during acidic/basic reaction conditions.17
The structure assigned for 9b was fully supported by
its FAB-MS spectra. Reaction of 8 with 2b in refluxing
methanol gave 9c in very low yield (10%). In the forma-
tion of 9c it was expected that the secondary alcohol
generated during the formation of tetrahydro-2-thiopyr-
imidine ring underwent O-methylation25 (instead of
elimination of a water molecule) due to an acid catalyst
(HCl) and –C„N group underwent hydrolysis and
esterification (in presence of HCl) to yield 9c. 1H
NMR spectra of 9c in CDCl3+2 drops of DMSO-d6
showed a characteristic singlet d 3.37 (O–CH3) with a
multiplet in the region d 1.69 and 2.11 ppm, which sup-
ports presence of two asymmetric centers in the vicinity
of –CH2.
aza-fluorene-4-thione (11b), respectively (Scheme 4a).
Formation of 11a from 10 was concluded in the presence
of an acid catalyst (pH 4–5) in refluxing methanol.
Reaction of 10 with 2b at rt (10 days) gave 11b and its
characterization confirmed it as tetrahydro-2-thiopyrim-
idine derivative (iv) rather than dihydro-2-TP derivative
(v).
Reaction of 4-aminobutyric acid (12) with 2a was ex-
pected to give 13 (Scheme 4b) however 13a was isolated
under the investigational conditions. A compound 13b
was obtained by a reaction of 12 with 2b at rt after an
interval of 15 days. The typical nature of the dehydra-
tion of alcohol (ii) (Fig. 1) was not seen in 13b, it may
be due to the stability of the secondary alcohol at rt. Full
spectral data for 13a and 13b was presented in the
Experimental section.
Compound 17 (Scheme 5) was formed in 59% yield by a
reaction of cysteaminiumchloride (16) with 2a in pres-
ence of Na2CO3 in refluxing methanol. Compounds
15, 19, and 21 were isolated in reasonable yields
(ꢀ60%) by stirring of 4-aminobenzonitrile (14), 2,2,6,6-
tetraethyl-piperidin-4-ylamine (18) and dapsone (20),
respectively, with 2a, in absolute methanol at rt. Com-
pound 21 was separated out of the reaction mixture
immediately after 2 h. This typical tendency of dihy-
dro-2-TP derivative to separate out the reaction mixture
is well documented in the literature.26
3.1. Pharmacology
2-Amino-3-hydroxy-pyridine (10) on reaction with iso-
thiocyanate 2a and 2b gave a 1-(3-hydroxypyridin-2-
yl)-4,4,6-trimethyl-3,4-dihydropyrimidine-2(1H)-thione
(11a) and 2-methyl-1,2,3,9a-tetrahydro-9-oxa-3,4a,5-tri-
The compounds 3, 4, 6, 7a, 9a–c, 11a,b, 13a,b, 15, 17, 19,
and 21 have been tested for (i) anti-inflammatory activ-
ity in the carrageenin-induced paw oedema model at
100 and 50 mg/kg p.o. (ii) Analgesic activity in the

S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
3189
H
N
S
N
N
N
a
OH
R1
R2
N
NH2
OH
CH3OH
11a
N
+
S
S
O
R3
b
NH
10
2a; R1=R2=R3=CH3
N
or
2b; R1=CH3, R2=R3= H
O
a; pH 5/ Reflux/ 14 h
11b
b; r. t. / 10 days
(a)
S
HO
N
NH
O
13a
S
c
R1
R2
CH3OH
N
NH
N
HO
+
NH2
S
O
R3
O
O
O
d
13
12
2a; R1=R2=R3=CH3
or
S
2b; R1=CH3, R2=R3= H
HO
N
NH
O
HO
c: pH 5/ Reflux/ 12 h
d; r. t. / 15 days
13b
(b)
Scheme 4.
phenylquinone writhing assay at 100 and 50 mg/kg p.o.
and (iii) cyclin-dependent kinase (CDK-1and 5) and
glycogen synthase kinase (GSK-3) inhibitory activity.
Results of these biological activities are summarized in
Table 1.
efficient, one-pot reaction. All these compounds have
been synthesized in reasonable yield. Screening of these
compounds for anti-inflammatory, analgesic, and pro-
tein kinase (CDK-1, CDK-5, and GSK-3) inhibitory
activities revealed that compound 3 showed some activ-
ity against CDK-1and compound 17 possess good anti-
inflammatory as well as analgesic activities.
It is clear from Table 1, compound 3 is having some
activity against CDK-1and CDK-5 and the IC 50 values
were 5 and 25 lM, respectively. The other compounds
showed moderate activities against CDK-5 and GSK-3
with IC50 > 1 0lM. Compound 17 is having good anti-
inflammatory (37.4% at 100 mg/kg p.o.) and analgesic
activities (75% at 100 mg/kg p.o.) compared to the stan-
dard drug Ibuprofen. Compounds 4, 7a, 9a, 9b, 11a,
13a, 15, 19, and 21 showed moderate to good anti-
inflammatory activity. Compounds 7a, 11a also showed
good analgesic activity. Other compounds showed mod-
erate analgesic activity.
5. Experimental
Melting points were uncorrected and obtained on a cap-
illary apparatus. Infra-Red spectra were recorded using
a Perkin Elmer 1600 FT Spectrophotometer and only
characteristic peaks are reported. 1H NMR spectra were
measured in appropriate deuteriated solvent (CDCl3,
DMSO-d6, D2O) with TMS as internal standard by
using
Bruker
AC-300F,
NMR
Spectrometer
(300 MHz). Chemical shifts (d) are reported in parts
per million (ppm) of the applied field and coupling con-
stants (J) are expressed in Hertz (Hz). The EI-MS
spectra were recorded on VG-70-S mass spectrometer.
FAB-MS spectra were reordered on Jeol SX-102
(FAB) mass spectrometer. Elemental analyses were
4. Conclusions
Various 2-TP derivatives (3, 4, 6, 7a, 9a–c, 11a,b, 13a,b,
15, 17, 19, and 21) have been synthesized by a simple,

3190
S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
S
NH
CH3OH
+
NC
NH2
2a
2a
NC
N
r. t. / 6 h
14
15
S
CH3OH/ Na2CO3(1 eqiv)
Reflux/ 10 h
NH2 .HCl
N
NH
+
HS
S
S
16
17
NH
CH3OH
HN
N
2a
+
NH2
HN
r. t. / 12 h
18
19
O
O
S
+
N
S
H2N
NH2
CH3OH
O
O
2a
20
r. t. / 2 h
O
S
S
S
HN
N
N
NH
21
Scheme 5.
Table 1. Anti-inflammatory, analgesic, and kinase (CDK-1, CDK-5 and GSK-3) inhibitory activity data of various 2-TP derivatives
Compound
Anti-inflammatory (%)
100 mg/kg p.o. 50 mg/kg p.o.
Analgesic (%)
100 mg/kg p.o. 50 mg/kg p.o.
Protein kinase IC50 (lM)
CDK-1
CDK-5
GSK-3
3
0.0
11.2
0.0
—
—
25.0
25.0
50.0
75.0
25.0
50.0
0.0
—
5.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
25.0
>10
>10
>10
>10
>10
>10
>10
>1
>100
>10
>10
>10
>10
>10
>10
>10
4
0.0
6
7a
—
—
25.0
50.0
—
14.7
11.2
10.3
0.0
9a
9b
—
—
25.0
—
9c
11a
—
—
5.2
75.0
50.0
—
11b
13a
—
—
—
0
>
16.9
—
—
50.0
25.0
10.0
25.0
50.0
20.0
20.0
75.0
—
>10
>10
>10
>10
>10
>10
—
>10
>10
>10
>10
>10
>10
—
13b
15
18.3a
—
—
23.7
37.4
—
50.0
75.0
17
19
—
19.4a
18.7a
—
—
—
21
Ibuprofen
Phenylbutazone
—
56.0
58.4
100
—
36.4
—
—
a ALD50 = > 1000 mg/kg p.o.
performed using a Vario EL III elementar analyzer.
Starting materials were procured from Aldrich (USA)
and Spectrochemꢁ (India) and were used as received.
Solvents were purified by standard procedures. All the

S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
3191
reactions were monitored over silica gel-G (Merck) TLC
plates and spots were visualized by iodine vapors or by
irradiation with ultraviolet light (254 nm). Silica gel
(60–120 mesh) used for column chromatography was
obtained from Qualigensꢁ (India).
5.1.4.1. 7-(1-Mercapto-3,3,4a-trimethyl-4,4a,5,9b-tet-
rahydro-3H-pyrido[4,3-b]indol-7-yl)-3,3,4a-trimethyl-3,4,
4a,5-tetrahydro-benzo[4,5]imidazo[1,2-c]pyrimidine-1-thiol
(3). Solvent of crystallization, methanol; Violet solid,
mp 213–215 ꢂC (0.760 g, 77%); IR (KBr m cmÀ1
)
3202.3 (N–H), 1613.1, 1494.5 (C@C), 1201.1 (C@S);
1H NMR (CDCl3+DMSO-d6 d); 1.34 (s, 6H, 2·
–CH3), 1.38 (s, 6H, 2· –CH3), 1.52 (s, 6H, 2· –CH3),
2.24 (d, 2H, J = 13.5 Hz, –CH2), 2.55 (d, 2H,
J = 13.47 Hz, –CH2), 6.30 (d, 2H, J = 8.1Hz, Ar–H),
6.62 (d, 1H, J = 7.9 Hz, Ar–H), 6.80 (d+s, 2H, Ar–H),
7.05 (q, 1H, Ar–H), 8.15 (d, 2H, exchanges with D2O,
2· –HN–), 8.65 (d, 1H, exchanges with D2O,
–HN–C@S), 9.03 (s, 1H, exchanges with D2O, –HN–
5.1. Chemistry
5.1.1. General procedure for rt reaction of 2a. Function-
alized amine (2 mmol) was dissolved in absolute metha-
nol (15 mL). To this was added 4-isothiocyanato-4-
methyl-2-pentanone (2a) (0.314 g, 2 mmol).27 The result-
ing solution was stirred at rt for an appropriate time and
the reaction was monitored at different time intervals.
After completion of the reaction, methanol was allowed
to evaporate at rt in a beaker and the resulting sticky
mass was crystallized by using a appropriate solvent or
it was subjected to column chromatography.
C@S); EI-MS m/z (% abundance): 492 (M+, 15%), 460
SH
N
N
(M+À2CH4, 12.1%), 232 (
, 42.3%), 188 (
,
N
N
N
H
N
H
100%); Anal. Calcd for C26H32N6S2: C, 63.38; H, 6.55;
N, 17.06. Found: C, 63.43; H, 6.60; N, 17.00.
5.1.4.2. 4,40-Bis(4,4,6-trimethyl-3,4-dihydro-1H-pyrim-
idine-2-thioxo) biphenyl (4). Solvent of elution, EtOAc–
hexane (4:6); White solid, mp 273–275 ꢂC (0.552 g,
60%); IR (KBr m cmÀ1) 3253.3 (N–H), 1643.1, 1514.6
(C@C), 1201.9 (C@S); 1H NMR (DMSO-d6 d);
1.37 (s, 12H, 4· –CH3), 1.51 (s, 6H, 2· –CH3), 4.86 (s,
2· 1H, olefinic), 7.27 (d, 4H, J = 8.1Hz, Ar–H), 7.67
(d, 4H, J = 8.2 Hz, Ar–H), 8.41(br s, 2H, exchanges
with D2O, 2· –HN–C@S); FAB-MS m/z (% abun-
5.1.2. General procedure for pH adjusted reaction of 2a.
Functionalized amine (2 mmol) was dissolved in abso-
lute methanol (25 mL) by heating and cooled to rt. To
this was added 4-isothiocyanato-4-methyl-2-pentanone
(2a) (0.314 g, 2 mmol).27 The pH of the resulting solu-
tion was adjusted to ꢀ4–5 by adding few drops of
10% sulfuric acid in methanol. If any turbidity (salt)
was observed it was filtered off and the clear solution
was kept for reflux for an appropriate time. Reac-
tion was monitored at different time intervals and after
completion, methanol was distilled off under reduced
pressure. The resulting mixture was washed with 10%
sodium bicarbonate solution28 (2 · 5 mL) and further
with cold water (2 · 5 mL) and then crystallized or sub-
jected to column chromatography.
+
dance): 463 (MH+, 20%), 431(MH À2CH4, 50%), 415
(m/z 431–CH , 40%); Anal. Calcd for C26H30N4S2: C,
4
67.50; H, 6.54; N, 12.11. Found: C, 67.41; H, 6.45; N,
12.18.
5.1.4.3. 3-Methyl-2,3,4,4a,9,10-hexahydro-2,9a,10-tri-
aza-anthracene-1-thione (6). Solvent of crystallization,
methanol; White solid, mp 180–182 ꢂC (0.115 g, 25%);
IR (KBr m cmÀ1): 3222.3 (N–H), 1559, 1489 (C@C),
1215.1 (C@S); 1H NMR (CDCl3 + DMSO-d6 d)
1.20–1.27 (dd, 3H, J = 6.5 and 6.6 Hz, –CH3), 1.73–
1.81 (dd, 2H, J = 3.8 and 6.6 Hz, H2C11), 3.92 (septet,
1H, J = 4 and 6.6 Hz, H*C12), 4.27 (m, 1H, HC8),
4.60–4.67 (m, 2H, H2C10), 6.57–7.10 (m, 4H, Ar–H)
5.46 and 7.33 (br s, 2· 1H, exchanges with D2O, HN7
and HN13); EI-MS m/z (% abundance): 233 (M+,
45%), 232 (M+ÀH, 3.8%), 200 (M+ÀSH, 15%), 132
5.1.3. General procedure for rt reaction of 2b. Function-
alized amine (2 mmol) was dissolved in absolute metha-
nol (15 mL). 3-Isothiocyanatobutanal (2b) (0.258 g,
2 mmol) was also dissolved separately in absolute meth-
anol (10 mL). Any insoluble polymeric material was fil-
tered off and a clear solution was added to the amine
solution. The resulting mixture was stirred at rt. The
reaction was monitored at different time intervals and
after completion, the contents were transferred to a bea-
ker and methanol was allowed to evaporate at rt. The
resulting sticky mass was crystallized or subjected to col-
umn chromatography.
N
(
, 27%), 105 (
, 100%); Anal. Calcd for
N
H
NH
C12H15N3S: C, 61.77; H, 6.48; N, 18.01. Found: C,
61.82; H, 6.55; N, 17.97.
5.1.4. General procedure for Na2CO3 added reactions.
Functionalized amine hydrochloride (2 mmol) was dis-
solved in absolute methanol (25 mL). The insoluble
material was filtered off and to the clear solution 4-isothio-
cyanato-4-methyl-2-pentanone (2a) (0.314 g, 2 mmol)
was added. To this reaction mixture a fine powder of so-
dium carbonate (0.106 g, 1 mmol) was added and kept
for reflux with constant stirring. The reaction was moni-
tored at different time intervals and after completion,
methanol was distilled off under reduced pressure. The
resulting solid was washed with cold water (2 · 5 mL)
and then crystallized or subjected to column
chromatography.
5.1.4.4. 2-Methyl-1,2,3,10a-tetrahydro-9H-10-oxa-3,4a-
diaza-phenanthrene-4-thione (7a). Solvent of elution,
EtOAc–CHCl3 (1:9); White solid, mp 193–195 ꢂC
(0.210 g, 45%); IR (KBr m cmÀ1) 3184.0 (N–H),
1
1525.9, 1439.4 (C@C), 1212.2 (C@S); H NMR (CDCl3
d) 1.28–1.31 (d, J = 6.3 Hz, –CH3) 1.94–2.04 (m,
1H, J = 2.0, 3.5, 4.0, 6.0 Hz, HC14), 2.19–2.26 (m,
1H, J = 2.0, 3.5, 4.0, 6.0 Hz, HC14), 3.83–3.91(m, 1H,
J = 2.4, 4.0, 6.5 Hz, HC13), 4.91–4.93 (t, 1H, J =
3.3 Hz, HC8), 5.06 (s, 2H, H2C10), 6.8 (br s, 1H,
exchanges with D2O, –HN–C@S), 7.01–7.04 (d, 1H,
J = 6.0 Hz, Ar–H), 7.18–7.30 (dd, 2H, J = 6.0 and

3192
S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
6.1 Hz, Ar–H), 8.21–8.23 (d, 1H, J = 6.0 Hz, Ar–H); EI-
MS m/z (% abundance): 234 (M+, 100%), 201 (M+ÀSH,
, 20%), 133
6.98–7.03 (t, 1H, Ar, J = 7.0 and 8.0 Hz), 7.36–7.38 (d,
1H, Ar, J = 8.0 Hz), 7.64–7.66 (d, 1H, Ar, J = 7.0 Hz),
9.83 (br s, 1H, –HN–C@S, exchanges with D2O),
12.05 (very br s, 1H, OH-Ar, exchanges with D2O);
EI-MS m/z (% abundance): Does not show M+ ion peak
30%), 175 (M+ÀHSCN, 10%), 147 (
O
N
O
(
, 60%); Anal. Calcd for C12H14N2SO: C,
N
but 190 (M+ÀHSCN, 13.7%), 175 (
, 100%);
61.51; H, 6.02; N, 11.96. Found: C, 61.55; H, 5.95; N,
12.01.
N
OH
Anal. Calcd for C12H15N3OS: C, 57.81; H, 6.06; N,
16.85. Found: C, 57.88; H, 6.10; N, 16.79.
5.1.4.5. (4,4,6-Trimethyl-2-thioxo-3,4-dihydro-2H-pyrim-
idin-1-yl)-acetic acid methyl ester (9a). Solvent of
crystallization, methanol; White solid, mp 158–160 ꢂC
(0.185 g, 47%); IR (KBr m cmÀ1) 3198.5 (N–H), 1735.9
(C@O), 1694.6 (C@N), 1540.3 (C@C), 1222.2 (C@S);
1H NMR (CDCl3 d) 1.31 (2s, 6H, 2· –CH3), 1.87 (d,
3H, J = 1.2 Hz, –CH3), 3.78 (s, 3H, H3OC–C@O), 4.79
(d, 1H, J = 1.1 Hz, olefinic), 4.85 (s, 2H, H2C–N–),
6.95 (br s, 1H, exchanges with D2O, HN–C@S); FAB-
MS m/z (% abundance): 229 (MH+, 100%), 213
(M+ÀCH3, 40%), 195 (M+ÀSH, 10%); Anal. Calcd for
C10H16N2O2S: C, 52.61; H, 7.06; N, 12.27. Found: C,
52.55; H, 7.10; N, 12.30.
5.1.4.9.
2-Methyl-1,2,3,9a-tetrahydro-9-oxa-3,4a,5-
triaza-fluorene-4-thione (11b). Solvent of crystallization,
methanol; Pale yellow solid, mp 180–182 ꢂC (0.050 g,
10%); IR (KBr m cmÀ1): 3216.7 (N–H), 1653.0, 1557.9
1
(C@C), 1208.7 (C@S); H NMR (CDCl3 + DMSO-d6
d): 1.39–1.41 (d, J = 6.0 Hz, –CH3), 1.92–2.05
(dd, J = 3.8 and 12.0 Hz, 1H, HaCHb–HC*CH3), 2.61–
2.67 (2t, 1H, J = 3.9 and 12.0 Hz each, HbCHa–
HC*CH3), 3.75–3.79 (t, 1H, J = 6.0 Hz, HC*CH3),
5.98–6.03 (dd, 1H, J = 3.7 and 3.8 Hz, O–C*H–N),
6.93–6.96 (dd, 1H, J = 5.1and 6.9 Hz, Ar–H), 7.07–
7.09 (d, 1H, J = 6.7 Hz, Ar–H), 7.92 (br s, 1H,
exchanges with D2O, HN–C@S), 8.03–8.05 (d, 1H,
J = 5.1Hz, Ar–H); FAB-MS m/z (% abundance): 222
(MH+, 32%), 221(M +, 10%), 206 (M+ÀCH3, 5%); Anal.
Calcd for C10H11N3OS: C, 54.28; H, 5.01; N, 18.99.
Found: C, 54.15; H, 5.10; N, 19.01.
5.1.4.6. 7,7,8a-Trimethyl-5-methylsulfanyl-8,8a-dihy-
dro-7H-oxazolo[3,2-c]pyrimidin-2-ylideneamine (9b). Sol-
vent of elution, EtOAc–CHCl3 (5:5); Green solid, mp
175–177 ꢂC (0.136 g, 30%); IR (KBr m cmÀ1) 3295.7
1
(N–H), 1634.0 (C@N), 1191.5 (C–S); H NMR (CDCl3
d): 1.35 (s, 3H, –CH3), 1.44 (s, 3H, –CH3), 1.66 (s, 3H,
–CH3), 1.85 (d, 1H, Jab = 18.0 Hz, HaCHb–C*),
2.26 (d, 1H, Jba = 18.0 Hz, HbCHa–C*), 3.16 (s, 3H,
H3C–S–), 4.55 (d, 1H, Jcd = 17.4 Hz, HcCHd–N), 5.18
(d, 1H, Jdc = 17.7 Hz, HdCHc–N–), 8.14 (br s, 1H, ex-
changes with D2O, HN–C@S); FAB-MS m/z (% abun-
dance): 228 (MH+, 100%), 212 (M+ÀCH3, 10%), 113
5.1.4.10. 4-(4,4,6-Trimethyl-2-thioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-butyricacid (13a). Solvent of crystalliza-
tion, methanol; White solid, mp 208–210 ꢂC (0.330 g,
68%); IR (KBr m cmÀ1): 3259.0 (N–H), 1706.8 (C@O),
1643.9, 1538.3 (C@C), 1205.3 (C@S); 1H NMR
(CDCl3+DMSO-d6 d): 1.22–1.23 (2s, 6H, 2·
–CH3), 1.89–2.03 (s+m, 5H, –CH3+–CH2), 2.31–2.38
(dd, 2H, J = 6.0 Hz each, –CH2), 4.23–4.29 (m, 2H,
–CH2), 4.68 (s, 1H, olefinic), 7.62 (br s, 1H, exchanges
with D2O, HN–C@S), 12.05 (very br s, 1H, exchanges
with D2O, –COOH); EI-MS m/z (% abundance): 242
(M+, 48%), 227 (M+ÀCH3, 61.8%), 209 (M+ÀSH,
2.5%), 198 (M+ÀCO2, 1.5%), 183 (M+ÀHSCN, 5.0%),
N
NH
(
, 80%); Anal. Calcd for C10H17N3OS: C, 52.84;
O
H, 7.54; N, 18.48. Found: C, 52.90; H, 7.60; N, 18.45.
5.1.4.7. (6-Methoxy-4-methyl-2-thioxo-tetrahydro-
pyrimidin-1-yl)-acetic acid methyl ester (9c). Solvent of
elution, EtOAc–CHCl3 (2:8); White solid, mp 198–
200 ꢂC (0.045 g, 10%); IR (KBr m cmÀ1): 3143.7 (N–
H), 1728.8 (C@O), 1227.1 (C@S), 1136.6 (C–O);
1H NMR (CDCl3 d): 1.26 (q, 3H, J = 3.2 and
6.6 Hz, –CH3), 1.69 (m, 1H, –HaCHb–C*CH3), 2.11
(m, 1H, –HbCHa–C*CH3), 2.60 (t, 1H, J = 6.5 Hz,
HC*CH3N), 3.37 (d, 3H, –OCH3), 3.75 (s, 3H, H3CO–
C@O), 4.04 (d, 1H, Jab = 17.6 Hz, HaCHb–COOCH3),
4.56 (t, 1H, HC–OCH3), 5.38 (d, 1H, Jba = 17.5 Hz,
HbCHa–COOCH3), 7.23 (br s, 1H, exchanges with
D2O, HN–C@S); FAB-MS m/z (% abundance): 232.66
(MH+, 15%); Anal. Calcd for C9H16N3O3S: C,
46.53; H, 6.94; N, 12.06. Found: C, 46.49; H, 6.90; N,
12.12.
S
S
HN
NH
HN
NH
156 (
, 6.0%), 141 (
, 100%), 87 (
,
COOH
11.6%); Anal. Calcd for C11H18N2O2S: C, 54.52; H,
7.49; N, 11.56. Found: C, 54.48; H, 7.54; N, 11.60.
5.1.4.11. 4-(6-Hydroxy-4-methyl-2-thioxo-tetrahydro-
pyrimidin-1-yl)-butyric acid (13b). Solvent of crystalliza-
tion, methanol; White solid, mp 140–142 ꢂC (0.077 g,
18%); IR (KBr m cmÀ1): 3271.0 (O–H), 1702.2 (C@O),
1
1200.0 (C@S); H NMR (CDCl3+DMSO-d6+D2O, d):
1.22–1.24 (d, 3H, J = 6.0 Hz, –CH3), 2.08–2.14
(m, 2H, –CH2), 2.29–2.34 (dd, 2H, J = 2.0 Hz each,
H2C–C*OH), 3.34–3.38 (q, 1H, J = 6.0 Hz, HC–CH3),
3.50–3.63 (m, 2H, –CH2), 4.23–4.29 (m, 2H, –CH2),
4.54–4.55 (d, 1H, J = 1.8 Hz, HC*N–OH); EI-MS m/z
(% abundance): 232 (M+, 0.7%), 214 (M+ÀH2O,
S
5.1.4.8.
1-(3-Hydroxy-pyridin-2-yl)-4,4,6-trimethyl-
3,4-dihydro-1H-pyrimidine-2-thione (11a). Solvent of
crystallization, methanol; Yellow solid, mp 175–177 ꢂC
(0.336 g, 67%); IR (KBr m cmÀ1): 3115.3 (N–H),
1643.6 (C@N), 1542.8, 1515.1 (C@C), 1203.6 (C@S);
1H NMR (CDCl3 + DMSO-d6 d): 1.77 (2s, 6H,
2· –CH3), 2.00 (s, 3H, –CH3), 4.65 (s, 1H, olefinic),
2.4%), 199 (M+ÀSH, 0.4%), 198 (HOOC
, 3.3%),
N
N
S
N
NH
196 (
, 52%), 163 (m/z 196 –SH, 100%); Anal.
O

S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
3193
Calcd for C9H16N2O3S: C, 46.53; H, 6.94; N, 12.06.
Found: C, 46.48; H, 7.02; N, 12.08.
Anal. Calcd for C26H30N4O2S3: C, 59.29; H, 5.74; N,
10.64. Found: C, 59.34; H, 5.70; N, 10.66.
5.2. Pharmacology
5.1.4.12. 4-(4,4,6-Trimethyl-2-thioxo-3,4-dihydropyr-
imidin-1(2H)-yl)benzonitrile29 (15). Solvent of elution,
EtOAc–hexane (2:8); White solid, mp 190–192 ꢂC
(0.295 g, 57%); IR (KBr m cmÀ1): 3437.3, 3169.6 (N–
H), 2229.3 (C„N), 1683.6, 1522.3 (C@C), 1213.8
(C@S); 1H NMR (CDCl3 d): 1.39 (s, 6H, 2·
–CH3), 1.52 (s, 3H, –CH3), 4.69 (s, 1H, olefinic), 6.67
(br s, 1H, exchanges with D2O, –HN–C@S), 7.36 (d,
2H, J = 6.0 Hz, Ar–H), 7.73 (d, 2H, J = 6.0 Hz, Ar–
H); EI-MS m/z (% abundance): 257 (M+, 27%), 242
(M+ÀCH3, 100%), 198 (M+ÀHSCN, 15%); Anal. Calcd
for C14H15N3S: C, 65.34; H, 5.87; N, 16.33. Found: C,
65.30; H, 5.92; N, 16.35.
5.2.1. Anti-inflammatory activity assay.30 Anti-inflam-
matory activity screening was carried out using carra-
geenin-induced paw oedema in albino rats. Oedema in
one of the hind paws was induced by injection of carra-
geenin solution (0.1mL of 1%) into planter aponeurosis.
The volume of the paw was plethysmographyically mea-
sured immediately and also 3 h after the injection of the
irritant. The difference in the volume gave the amount of
the oedema developed. Percent inhibition of the oedema
between the control group and the compound treated
groups was calculated and compared with the group
receiving a standard drug.
5.2.2. Analgesic activity assay.31 Analgesia was mea-
sured by the writhing assay using Swiss mice (15–20 g).
Female mice were screened for writhing on day one, by
injecting intraperitonially 0.2 cm3 of aqueous solution
of phenylquinone. They were kept on a flat surface and
the numbers of writhes of each mouse was recorded for
20 min. The mice showing significance (>10) were sorted
out and used for analgesic assay on the following day.
The mice consisting of 5 in each group and showing sig-
nificant writhing were given orally a 50 or 100 mg kgÀ1
p.o. dose of the test compounds 15 min prior to phenyl-
quinone challenge. Writhing was again recorded for each
mouse in a group and a percentage protection was calcu-
lated by using the following formula:
5.1.4.13. 7,7,8a-Trimethyl-hexahydro-thiazolo[3,2-c]-
pyrimidine-5-thione (17). Solvent of crystallization,
methanol; Yellow solid, mp 170–172 ꢂC (0.254 g, 59%);
IR (KBr m cmÀ1): 3191.8 (N–H), 1270.8 (C@S); 1H
NMR (CDCl3 d): 1.34 and 1.37 (2s, 6H, 2·
–CH3), 1.76 (s, 3H, –CH3), 2.31(s, 2H, H C–), 3.11–
2
3.15 (t, 2H, J = 6.6 Hz, H2C–N), 4.00–4.09 (p, 1H,
J = 6.0 and 6.6 Hz, HaHbC–S–), 4.88–4.96 (p, 1H,
J = 6.0 and 6.6 Hz, HbHaC–S–), 6.46 (br s, 1H, ex-
changes with D2O, HN–C@S); FAB-MS m/z (% abun-
dance): 217 (MH+, 100%), 183 (M+ÀSH, 10%), 100
S
(
, 13%); Anal. Calcd for C9H16N2S2: C, 49.96; H,
N
7.45; N, 12.95. Found: C, 46.85; H, 7.51; N, 13.01.
Protection ¼ 100 À ½fðNo: writhing for treated miceÞ=
ðNo: of writhing for untreated-miceÞg
ꢁ 100ꢂ
This was taken as percent analgesia response and was
averaged in each group of mice. Percent of animals
exhibiting analgesic was determined with each dose.
5.1.4.14. 4,4,6-Trimethyl-1-(2,2,6,6-tetramethyl-pip-
eridin-4-yl)-3,4-dihydro-1H-pyrimidine-2-thione (19). Sol-
vent of crystallization, methanol; Pale yellow solid, mp
195–197 ꢂC (0.390 g, 53%); IR (KBr m cmÀ1): 3357.3
(N–H), 1692.1 (C@N), 1538.2 (C@C), 1220.0 (C@S);
1H NMR (CDCl3 d): 1.44–1.49 (2s, 21H, 7·
–CH3), 2.04 (m, 2H, –CH2), 2.12 (m, 2H, –CH2), 3.35
(m, 1H, –HC–N), 4.74 (d, 1H, J = 5.9 Hz, olefinic),
7.03 (s, 1H, exchanges with D2O, –HN), 7.31(br s,
1H, exchanges with D2O, –HN–C@S); EI-MS m/z (%
abundance): Does not show M+ ion peak, 263
(M+À2 · CH4, 100%), 248 (M+ÀSH, –CH3, 25%), 204
5.2.3. Kinase inhibition activity.
5.2.3.1. Biochemical reagents. Sodium orthovanadate,
EGTA, EDTA, Mops, b-glycerophosphate, phenylphos-
phate, sodium fluoride (NaF), dithiothreitol (DTT), glu-
tathione-agarose, glutathione, bovine serum albumin
(BSA), nitrophenylphosphate, leupeptin, aprotinin, pep-
statin, soybean trypsin inhibitor, benzamidine, histone
H1(type III-S) were obtained from Sigma. The [c-32P]-
ATP (PB 168) was obtained from Amersham. The GS-
1peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE)
was synthesized by Peptide synthesis unit, Institute
of Biomolecular Sciences, University of Southampton,
UK.
N
(m/z 263 –HSCN, 39%) 107 (
, 35.4%); Anal. Calcd
for C16H29N3S: C, 65.04; H, 9.89; N, 14.22. Found: C,
65.13; H, 9.94; N, 14.25.
5.1.4.15.
4,40-Bis(4,4,6-trimethyl-3,4-dihydro-1H-
pyrimidine-2-thioxo) biphenyl sulfone (21). Solvent of
elution, EtOAc–CHCl3 (3:7); White solid, mp 228–
230 ꢂC (0.496 g, 55%); IR (KBr m cmÀ1): 3265.0 (N–
H), 1691.7, 1649.3, 1595.1, 1532.3 (C@C), 1318.7
5.2.3.2. Buffers. Homogenization of buffer: 60 mM
b-glycerophosphate, 15 mM p-nitrophenyl-phosphate,
25 mM Mops (pH 7.2), 15 mM EGTA, 15 mM MgCl2,
1mM DTT, 1mM sodium vanadate, 1mM NaF,
1mM phenylphosphate, 10 lg leupeptin/mL, 10 lg apro-
tinin/mL, 10 lg soybean trypsin inhibitor/mL and
100 lM benzamidine. Buffer A: 10 mM MgCl2, 1 mM
EGTA, 1mM DTT, 25 mM Tris–HCl (pH 7.5), 50 lg
1
(O@S@O), 1210.9 (C@S); H NMR (CDCl3+DMSO-
d6 d): 1.35–1.37 (2s, 12H, 4· –CH3), 1.45 (s, 6H,
2· –CH3), 4.85 (s, 2· 1H, olefinic), 6.69 (d, 2H,
J = 8.7 Hz, Ar–H), 7.32 (d, 2H, J = 8.4 Hz, Ar–H),
7.64 (d, 2H, J = 7.8 Hz, Ar–H), 7.9 (d, 2H, J = 7.6 Hz,
Ar–H), 8.00 (br s, 2H, exchanges with D2O, 2· –HN–
C@S); FAB-MS m/z (% abundance): 563 (MH+, 30%);

3194
S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
heparin/mL. Buffer C: homogenization buffer but 5 mM
EGTA, no NaF and no protease inhibitors. Tris-Buf-
fered-Salin-Tween-20 (TBST): 50 mM Tris (pH 7.4),
150 mM NaCl, 0.1% Tween-20. Hypotonic Lysis Buffer
(HLB): 50 mM Tris–HCl (pH 7.4), 120 mM NaCl, 10%
glycerol, 1% Nonidet-P40, 5 mM DTT, 1 mM
EGTA, 20 mM NaF, 1mM orthovanadate, 5 lM
microcystin, 100 lg/mL each of leupeptin, aprotinin
and pepstain.
References and notes
1. (a) Segal, H.; Hedgcoth, C.; Skinner, C. G. J. Med.
Pharmaceut. Chem. 1962, 5, 871; (b) Cumming, J. G.;
Mckenzie, C. L.; Bowden, S. G.; Campbell, D.; Masters,
D. J.; Breed, J.; Jewsbury, P. J. Bioorg. Med. Chem. Lett.
2004, 14, 5389.
2. Calvin, M.; Jorgenson, M. J. Bio-Organic Chemistry;
W.H. Freeman and Company: San Francisco, 1968;
p 78.
3. (a) Mathes, R. A. J. Am. Chem. Soc. 1953, 75, 1747; (b)
Chung, H.; Kweon, D.; Kang, Y.; Park, J.; Yoon, Y. J.
Heterocycl. Chem. 1999, 36, 905.
4. (a) Sierzputowska-Gracz, H.; Sochacka, E.; Malkiewicz,
A.; Kuo, K.; Gehrke, C.; Agris, P. F. J. Am. Chem. Soc.
1987, 109, 7171; (b) Sochacka, E.; Fratczak, I. Tetrahedron
Lett. 2004, 45, 6729.
5. (a) Stoyanov, S.; Petkov, I.; Antonov, L.; Stoyanova, T.;
Karagiannides, P.; Aslanidis, P. Can. J. Chem. 1990, 68,
1482; (b) Hazelton, J. C.; Iddon, B.; Suschitzky, H.;
Woolley, L. H. J. Chem. Soc., Perkin Trans. 1 1992, 6,
685.
6. Hajos, Z. G.; Kanojia, R. M.; Press, J. B. Eur. Pat. Appl.
EP 458459 A2, 1991; Chem. Abstr. 1991, 116,
83701.
7. Pathak, A. K.; Pathak, V.; Seit, L. E.; Sulng, W. J.;
Reynolds, R. C. J. Med. Chem. 2004, 41, 273.
8. Gias, H.; Jagusch, T.; Spathoff, N.; Gerhards, F.; Frank,
M.; Raabe, G. Chem. Eur. J. 2003, 9, 4202.
9. Kesavan, V.; Bonnet-Delpon, D.; Begue, J. Synthesis
(Stuttgart) 2000, 2, 223.
10. Holla, B. S.; Rao, B. S.; Sarojini, B. K.; Akberali, P. M.
Eur. J. Med. Chem. 2004, 39, 777.
11. (a) Goyal, R. N.; Singh, U. P.; Abdullah, A. A. Bioelect-
rochem., in press; (b) Tripathi, G. N. R.; Clements, M. J.
Phys. Chem. B 2003, 107, 11125.
12. (a) Belema, M.; Bunker, A.; Nguyen, V.; Beaulieu, F.;
Ouellet, C.; Marinier, A.; Roy, S.; Yung, X.; Qiu, Y.;
Zhang, Y.; Martel, A.; Zusi, C. PCT Int. Appl. WO
2003084959, 2003; Chem. Abstr. 2003, 139, 337987; (b)
Bonnert, R. V.; Cage, P. A.; Hunt, S. F.; Walters, I. J. S.;
Austin, R. P. PCT Int. Appl. WO-2003024966, 2003;
Chem. Abstr. 2003, 138, 271701.
13. Sondhi, S. M.; Singhal, N.; Johar, M.; Reddy, B. S. N.;
Lown, J. W. Curr. Med. Chem. 2002, 9, 1045.
14. (a) Sondhi, S. M.; Sharma, V. K.; Verma, R. P.; Singhal,
N.; Shukla, R.; Raghubir, R.; Dubey, M. P. Synthesis
(Stuttgart) 1999, 5, 878; (b) Sondhi, S. M.; Verma, R. P.;
Singhal, N.; Shukla, R.; Raghubir, R.; Dubey, M. P.
Indian Drugs 1999, 36, 50; (c) Sondhi, S. M.; Johar, M.;
Dastidar, S. G.; Shukla, R.; Raghubir, R.; Bharti, N.;
Azam, A. Aust. J. Chem. 2001, 54, 461.
5.2.3.3. Kinase preparations and assays. Kinases activ-
ities were assayed in Buffer A or C (unless otherwise sta-
ted), at 30 ꢂC, at final ATP concentration of 15 lM.
Blank values were subtracted and activities calculated
as p moles of phosphate were incorporated for a
10 min incubation. The activities are usually expressed
in percentage of the maximal activity (in absence of
the inhibitors). Controls were performed with appropri-
ate dilutions of dimethylsulfoxide. GSK-3 was purified
from porcine brain. It was assayed, following a 1/100
dilution in 1mg BSA/mL 10 mM DTT, with 5 lL,
40 lM GS-1peptide as a substrate, in buffer A, in pres-
ence of 15 lM [c-32P] ATP (3000 Ci/mM; 1mCi/mL) in
a final volume of 30 lL. After 30 min incubation at
30 ꢂC, 25 lL aliquots of supernatant were spotted onto
2.5 · 3 cm pieces of Whatman P81phosphocellulose
paper, and 20 s later, the filters were washed five times
(for at least 5 min each time) in a solution of 10 mL
phosphoric acid per liter of water. The wet filters were
counted in presence of 1mL ACS (Amersham) scintilla-
tion fluid. CDK-1/ cyclin B was extracted in a homo-
genization buffer from M phase starfish (Marthasterias
glacialis) oocytes and purified by affinity chromato-
graphy on P9CKShs1 sepharose beads, from which it
was eluted by free P9CKShs1 as reported in the
literature.32 The kinase activity was assayed in buffer
C, with 1 mg histone H1/mL, in presence of 15 lM
[c-32P]-ATP (3000 Ci/mmol; mCi/mL) in a final volume
of 30 lL. After 10 min incubation at 30 ꢂC, 25 lL
aliquots of supernatant were spotted onto P81phospho-
cellulose papers and treated as described above. CDK-5/
p25 was reconstituted33 by mixing equal amounts of
recombinant mammalian CDK-5 and p25 expressed in
E. coli as GST (Glutathione-S-transferase) fusion-
protein and purified by affinity chromatography on glu-
tathione-agarose.34 The activity of p25 was assayed in
buffer C as described for CDK-1/cyclin B.
15. Sondhi, S. M.; Singh, N.; Rajvanshi, S. Monatsh. Chem.
2004, 135, 119.
16. Sondhi, S. M.; Rajvanshi, S.; Johar, M.; Bharti, N.; Azam,
A.; Singh, A. K. Eur. J. Med. Chem. 2002, 37,
835.
Acknowledgements
17. Sondhi, S. M.; Rajvanshi, S.; Singh, N.; Jain, S.; Lahoti,
A. M. Cent. Eur. J. Chem. 2004, 2, 141.
18. (a) Meijer, L. Chem. Biol. 2003, 10, 1255; (b) Meijer, L.
Acc. Chem. Res. 2003, 36, 417; (c) Meijer, L.; Flajolet, M.;
Greengard, P. Trends Pharmacol. Sci. 2004, 25,
471.
19. Electroanalytical studies of some of the 2-TP derivatives
are under investigation.
20. Synthesis of 2a and 2b were carried out by procedures
reported in the literature: Mathes, R. A.; Stewart, F. D.;
Swedish, F., Jr. J. Am. Chem. Soc. 1948, 70, 1452.
We would like to thank Olivier Lozach and Laurent
Meijer (CNRS, Station Biologique de Roscoff, France)
for performing the kinase assays. One of the authors
(AML) is grateful to IITR, for awarding him an institute
fellowship, sponsored by the Ministry of Human Re-
source Department (MHRD), Govt. of India. Financial
support for synthesis work was provided by MHRD
through IITR grant code no. 106-20-77. We are thank-
ful to SAIF-PU Chandigarh and RSIC-CDRI Luc-
know, for spectral analyses.

S. M. Sondhi et al. / Bioorg. Med. Chem. 13 (2005) 3185–3195
3195
21. (a) Singh, H.; Kumar, S. J. Chem. Soc., Perkin Trans. 1
1987, 261; (b) Singh, H.; Kumar, S. Tetrahedron 1987, 43,
2177; (c) Singh, H.; Singh, S. Aust. J. Chem. 1973, 26,
2453.
27. In case of functionalized diamine/tetramine, 4 mmol of 2a
were used.
28. Washing with 10% NaHCO3 solution was excluded during
the work-up of 13a.
22. Sondhi, S. M.; Singhal, N.; Verma, R. P.; Arora, S. K.;
Shukla, R. K.; Raghubir, R. Monatsh. Chem. 2000, 131,
501.
29. Ovechkin, P. L.; Ignatova, L. A.; Gekhman, A. E.;
Unkovskii, B. V. Khim. Geterotsikl. Soedin. 1972, 7, 937,
Chem. Abstr. 1972, 77, 126534.
23. Zigeuner, G.; Linstschinger, W. B.; Fuchsgruber, A.;
Kollmann, K. Monatsh. Chem. 1976, 107, 171.
24. Synthesis of 9a by the reaction of methyl-2-aminoacetate
(glycine ester) with 2a in refluxing ethanol has been
reported in the literature: Zigeuner, G.; Kollmann, K.;
Linstschinger, W. B.; Fuchsgruber, A. Monatsh. Chem.
1976, 107, 183.
25. Formation of O-methylated derivative during the reaction
of b-isothiocyantoketone with functionalized amine was
documented in: Singh, H.; Kumar, S.; Singh, P. J. Chem.
Res. (M) 1984, 1641.
30. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp.
Biol. Med. 1962, 111, 544–547.
31. Singh, P. P.; Junnarkar, A. Y.; Rao, C. S.; Verma, R. K.;
Shridhar, D. R. Methods Find. Exp. Clin. Pharmacol.
1983, 5, 601–606.
32. (a) Lecler, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb,
J. A. J. Biol. Chem. 2001, 276, 251; (b) Leost, M.; Schultz,
C.; Link, A.; Wu, Y. Z.; Biernat, J.; Mandelkow, E. M.
Eur. J. Biochem. 2000, 267, 5983.
33. The p25 is a truncated version of p35 the 35 kDa CDK-5
activator.
26. Gakhar, H. K.; Madan, A.; Kumar, N. Indian J. Chem.
1980, 19B, 965.
34. Vectors were kindly provided by Dr. J. H. Wang, CNRS,
France.