Syntheses of 2,4,6-trisubstituted pyrimidine derivatives as a new class of antifilarial topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.10.046

A series of 21 compounds of trisubstituted pyrimidine derivatives have been synthesized and evaluated for their in vitro topoisomerase II inhibitory activity against filarial parasite Setaria cervi. Out of these, five compounds (12, 13, 14, 25 and 28) exhibited 70-80% inhibition at 10 μg/mL and three compounds (13, 14 and 28) have shown 40-60% inhibition at 5 μg/mL concentration. A series of 21 compounds of trisubstituted pyrimidine derivatives have been synthesized and evaluated for their in vitro topoisomerase II inhibitory activity against filarial parasite Setaria cervi. Out of these, seven compounds (8, 11-14, 25 and 28) have shown 60-80% inhibition at 40 and 20 μg/mL concentration. Five compounds (12, 13, 14, 25 and 28) exhibited 70-80% inhibition at 10 μg/mL concentration and three compounds (13, 14 and 28) have shown 40-60% inhibition at 5 μg/mL concentration. All the above mentioned compounds have shown better topo II inhibitory activity than standard antifilarial drug (DEC) and enzyme topo II inhibitors (Novobiocin, Nalidixic acid).

Full text of DOI:10.1016/j.bmcl.2004.10.046

Bioorganic & Medicinal Chemistry Letters 15 (2005) 47–50
Syntheses of 2,4,6-trisubstituted pyrimidine derivatives as a
new class of antifilarial topoisomerase II inhibitorsq
Sanjay Babu Katiyar,a Iti Bansal,b J. K. Saxenab and P. M. S. Chauhana,*
aMedicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226001, India
bBiochemistry Division, Central Drug Research Institute, Lucknow 226001, India
Received 26 July 2004; accepted 13 October 2004
Available online 5 November 2004
Abstract—A series of 21 compounds of trisubstituted pyrimidine derivatives have been synthesized and evaluated for their in vitro
topoisomerase II inhibitory activity against filarial parasite Setaria cervi. Out of these, seven compounds (8, 11–14, 25 and 28) have
shown 60–80% inhibition at 40 and 20lg/mL concentration. Five compounds (12, 13, 14, 25 and 28) exhibited 70–80% inhibition at
10lg/mL concentration and three compounds (13, 14 and 28) have shown 40–60% inhibition at 5lg/mL concentration. All the
above mentioned compounds have shown better topo II inhibitory activity than standard antifilarial drug (DEC) and enzyme topo
II inhibitors (Novobiocin, Nalidixic acid).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Previously in our laboratory some prototype molecules
have been synthesized, which have shown adulticidal
and antiinflammatory activities with additional DNA
topoisomerase II inhibitory activity. These compounds
possessed both macrofilaricidal and microfilaricidal
actions combined with sterilizing effect due to DNA
topoisomerase II inhibitory activity.4,5 Earlier we have
also reported some pyrido-indole and quinolone
derivatives as novel antifilarial agents, some of them
also exhibited topo II inhibitory activity.6,7 In this
manuscript we are reporting some novel trisubstituted
pyrimidine derivatives as antifilarial topoisomerase II
inhibitors.
The lymphatic filariasis is a major vector born disease of
the developing world and around 120 million persons
are infected worldwide.1 Within last 10years, though
significant progress has been made in the treatment
and control strategies by introducing new diagnostic
and monitoring tools, single annual or biannual dose
therapy, combination therapy with diethylcarbamazine
(DEC), ivermectin and albendazole,1 lymphatic filariasis
still continues to be a very severe problem due to lack of
definite actions on adult worms. Among the various en-
zymes identified for drug development against parasitic
diseases DNA topoisomerases have been chosen as a no-
vel target for antifilarial drug development. DNA topo-
isomerases are the enzymes required for the replication,
transcription and recombination of DNA. These en-
zymes play crucial roles in the organization of DNA
within the cell nucleus as well as in its structure and
function. The presence of ATP-dependent DNA topo-
isomerase II activity in the filarial parasites has been
demonstrated in our laboratory.2,3
2. Chemistry
Synthesis of compound 1 was carried out according to
the literature procedure8–10 with slight modifications.
Syntheses of targeted compounds 8–28 is shown in
Scheme 1.
Compounds 2, 3 and 4 were synthesized by reacting
compound 1 with guanidine hydrochloride and different
imidines such as N-imidino morpholine hydrochloride
and 4-amino benzimidine dihydrochloride, respec-
tively,10–13 in presence of NaH in dry DMF. Com-
pounds 2, 3 and 4 were oxidized to corresponding
sulfones 5, 6 and 7 in presence of m-chloroperoxy
Keywords: Pyrimidine; Antifilarial; Topoisomerase II.
q CDRI Communication no 6606.
*
Corresponding author. Tel.: +91 522 2212411x4332; fax: +91 522
2623405; e-mail addresses: drpmschauhan@rediffmail.com;
premsc58@hotmail.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.046

48
S. B. Katiyar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 47–50
CH3
S
O
2-Acetyl thiophene
a
SCH3
SCH3
d
b
SCH3
S
S
S
S
SCH3
O
N
N
N
N
1
c
NH2
2
SCH3
NH2
N
N
4
N
O
3
e
e
e
SO2CH3
S
SO2CH3
SO2CH3
S
S
N
N
N
N
N
N
N
NH2
5
O
NH2
f
6
7
f
f
R'''
S
R''
R'
N
S
N
N
S
N
N
N
N
NH2
O
20-24
NH2
25-28
8-19
Scheme 1. Reagents and conditions: (a) CS2, NaOH(s), MeI, THF, 0°C–rt; (b) guanidineÆHCl, NaH, DMF, 110°C; (c) N-imidino morpholineÆHCl,
NaH, DMF, 110°C; (d) 4-aminobenzimidineÆ2HCl, NaH, DMF, 110°C; (e) m-CPBA, DCM, 0°C–rt; (f) different amines, THF, 100°C, in closed
steel vessel.
benzoic acid (2.5equiv). The sulfones 5, 6 and 7 were
subjected to nucleophilic substitution with various
amines in closed steel vessel to yield targeted compounds
8–28. All the synthesized compounds (Table 1) were well
characterized by spectroscopic data such as mass, IR,
NMR and elemental analysis.15
occasional shaking and centrifuged at 10,000g for
30min and 1,05,000g for 1h.
Topoisomerase activity was estimated by monitoring the
relaxation of supercoiled pBR322 DNA as reported pre-
viously (Pandya et al., 1999). Assay mixture (20lL) con-
tained 50mM Tris–HCl, (pH7.5); 50mM KCl; 1mM
MgCl2; 1mM ATP; 0.1mM EDTA; 0.5mmM DTT;
30lg/mL BSA; 0.25lg pBR322 DNA and enzyme pro-
tein. The reaction mixture was incubated at 37°C for
30min and 5lL stop buffer (0.25%, bromophenol blue,
1M sucrose, 1mM EDTA, 0.5% SDS) was added. The
entire reaction mixture was loaded on 1% agarose gel
and electrophoresed in 40mM Tris–acetate buffer,
pH8.3, 1mM EDTA at 20V for 20h. The gel was
stained with ethidium bromide (0.5lg/mL) and photo-
graphed in GDS 7500 UVP (Ultra Violet Products,
UK) transilluminator. The effect of inhibitors on the en-
zyme activity was measured by incubating enzyme with
inhibitor for 10min at 37°C and starting the reaction
with addition of pBR322 DNA. The percent inhibition
was measured by micro densitometry of the gel with
GEL BASE/GEL BLOT PRO GEL analysis software program
(Ultra Violet Products, UK).
3. Biological activity
3.1. Material and methods
DNA topoisomerase was partially purified from the
filarial parasites Setaria cervi according to slightly modi-
fied method of Pandaya et al.14 Filarial parasites were
homogenized in nuclei isolation buffer (NIB) (2.5mM
potassium phosphate buffer, pH7.0; 2mM MgCl2;
0.1mM EDTA; 1mM EGTA; 1mM DTT and 1mM
PMSF) and centrifuged at 3000g for 10min at 4°C.
The pellet was washed with NIB and resuspended in
NIB containing 4mM EDTA; 0.35% (v/v) Triton x-
100 and 0.375M NaCl. The suspension was gently agi-
tated for 15min on ice and polyethylene glycol (9%, w/v)
was added. The mixture was kept on ice for 1h with

S. B. Katiyar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 47–50
49
Table 1.
Compound no. R0
Compound no. R0
Compound no. R00
Compound no. R000
8
NH–(CH2)2–CH3 14
20
21
22
25
26
27
N
N
N
N
N
CH3
Ph
N
N
N
N
CH3
Ph
N
N
N
N
N
CH3
Ph
9
NH–(CH2)3–CH3 15
NH–(CH2)5–CH3 16
CH3
CH3
NH-(CH2)3-N
10
O
O
11
12
13
NH–(CH2)2–OH 17
23
24
28
HN
HN
HN
NH-(CH2)2
NH-(CH2)3
N
O
N
NH-(CH2)3
N
O
N
18
19
––
––
NH-(CH2)2
NH-(CH2)3
N
O
O
––
N
Table 2. Topoisomerase II inhibitory activity against filarial parasite Setaria cervi
S. no.
Compounds
% Inhibition at different concentrations
40lg/mL
20lg/mL
10lg/mL
5lg/mL
1
2
3
4
5
6
7
8
9
10
8
11
60
60
80
80
80
80
70
45
80
80
60
60
80
80
80
80
70
10
20
40
NI
NI
80
80
80
80
70
NI
10
20
NI
NI
NI
60
12
13
14
25
60
25
28
DEC (antifilarial)
40
NI
NI
NI
Novobiocin (topo II inhibitor)
Nalidixic acid (topo II inhibitor)
NI = no inhibition.
4. Results and discussion
or 4-amino phenyl group (25 and 28) at position-2. The
introduction of morpholino group at position-2 exerts
no inhibitory effect on topo II enzyme of filarial parasite
Setaria cervi.
The effects of synthesized compounds are shown in Ta-
ble 2. All the synthesized compounds were evaluated for
their in vitro topoisomerase II inhibitory activity against
filarial parasite Setaria cervi. Out of the 21 screened
compounds, 7 compounds (8, 11, 12, 13, 14, 25 and
28) exhibited 60–80% inhibition at 40 and 20lg/mL con-
centration while the standard antifilarial drug (DEC)
and topo II inhibitors (Novobiocin, Nalidixic acid) have
shown 45–80% inhibition at a dose 40lg/mL and 10–
40% inhibition at a dose 20lg/mL concentration. At
10lg/mL concentration five compounds (12, 13, 14, 25
and 28) exhibited 70–80% inhibition while the antifilar-
ial drug (DEC) has shown no inhibition and topo II
inhibitors, Novobiocin, Nalidixic acid, exhibited 10%
and 20% inhibition, respectively. Four compounds (13,
14, 25 and 28) have shown 25–60% inhibition, where
as standard drugs (DEC, Novobiocin and Nalidixic
acid) showed no inhibition at 5lg/mL concentration.
Structure activity relationship of all the 21 screened
compounds have given clear indication that amino
group and 4-aminophenyl group at position-2 are very
crucial in exerting topo II inhibitory activity against
filarial parasite Setaria cervi. Most of the active com-
pounds (8, 11, 12, 13 and 14) either having amino group
5. Conclusion
In conclusion, we have synthesized and identified new
trisubstituted pyrimidine derivatives as antifilarial topo-
isomerase II inhibitors. Some of these compounds
exhibited significant topo II inhibitory activity as com-
pared to standard antifilarial drug (DEC) and topo II
inhibitors (Novobiocin, Nalidixic acid). Activity results
indicates that compounds (8 11–14, 25 and 28) can be
utilized as lead molecules for further investigations
and optimizing purpose for antifilarial chemotherapy.
Acknowledgements
Thanks to SAIF, Lucknow for providing spectroscopic
and analytical data, S.B.K. also thankful to CSIR,
New Delhi, for the award of Senior Research Fellow-
ship. Technical assistance of Ms. Reema Ray Sonker
is greatly acknowledged.

50
S. B. Katiyar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 47–50
phy to afford compound 15 (1.20g, 71.43%). By similar
References and notes
procedure described for compound 15, compounds 8–28
were synthesized. Compound 15: mp 139–141°C; m/z
(FAB-MS): 338 (M+1); IR (KBr): 3335, 3079, 2965,
2844, 1573, 1465, 1349cmꢀ1; 1H NMR (200MHz, CDCl3):
d 7.61 (d, 1H, J = 3.55Hz, thiophene-H), 7.39 (d, 1H,
J = 4.84Hz, thiophene-H), 7.31 (d, 2H, J = 7.89Hz, Ar–
H), 7.09 (dd, 1H, J = 4.82, 3.52Hz, thiophene–H), 6.98–
6.86 (m, 3H, Ar–H), 6.35 (s, 1H, py–H), 4.77 (br s, 2H,
NH2), 3.81 (t, 4H, J = 5.08Hz, NCH2), 3.26 (t, 4H,
J = 5.10Hz, NCH2); 13C NMR (50MHz, CDCl3): d
165.21, 164.25, 163.10, 149.34, 143.25, 129.64, 129.01,
128.17, 125.98, 120.68, 116.84, 89.10, 49.55, 44.35. Anal.
Calcd for C18H19N5S: C 64.07, H 5.68, N 20.75. Found: C
64.23, H 5.75, N 20.89. Compound 21: mp 139–141°C; m/z
(FAB-MS): 408 (M+1); IR (KBr): 3087, 2935, 2865, 1600,
1. Chauhan, P. M. S. Drugs of the Future 2000, 25, 481.
2. Saxena, J. K.; Srivastava, A. K.; Murthy, P. K.; Chatter-
jee, R. K.; Ghatak, S.; Walter, R. D. Tropenmed.
Parasitol. 1984, 35, 174.
3. Pandya, U.; Saxena, J. K.; Kaul, S. M.; Murthy, P. K.;
Chatterjee, R. K.; Tripathi, R. P.; Bhaduri, A. P.; Shukla,
O. P. J. Helminthol. 1997, 71, 325.
4. Bhattacharya, S.; Katiyar, D.; Bajpai, P.; Tripathi, R. P.;
Saxena, J. K. Parasitol. Res. 2004, 92, 177.
5. Katiyar, D.; Tiwari, V. K.; Tripathi, R. P.; Reddy, V. M.;
Bhattacharya, S. M.; Saxena, J. K. Arzreimittelforschung
2003, 53, 857.
6. Srivastava, S. K.; Agarwal, A.; Chauhan, P. M. S.;
Agarwal, S. K.; Bhaduri, A. P.; Singh, S. N.; Fatima, N.;
Chatterjee, R. K. J. Med. Chem. 1999, 42, 1667.
7. Srivastava, S. K.; Chauhan, P. M. S.; Bhaduri, A. P.;
Fatima, N.; Chatterjee, R. K. J. Med. Chem. 2000, 43,
2275.
1575, 1465, 1379cmꢀ1 1H NMR (200MHz, CDCl3): d
;
7.61 (d, 1H, J = 3.24Hz, thiophene–H), 7.38 (d, 1H,
J = 4.28Hz, thiophene–H), 7.31 (d, 2H, J = 8.44Hz, Ar–
H), 7.09 (dd, 1H, J = 4.20, 3.22Hz, thiophene–H), 6.98–
6.90 (m, 3H, Ar–H), 6.29 (s, 1H, pyr–H), 3.82–3.54 (m,
8. Potts, K. T.; Cipullo, M. J.; Ralli, P.; Theodoridis, G. J.
Org. Chem. 1982, 47, 3027.
12H, OCH2, NCH2), 3.27 (t, 4H, J = 5.10Hz, NCH2); 13
C
NMR (50MHz, CDCl3): d 164.98, 164.15, 163.20, 148.56,
143.08, 129.69, 129.08, 128.32, 125.78, 120.15, 116.35,
89.21, 71.43, 59.18, 49.56, 44.24. Anal. Calcd for
C22H25N5OS: C 64.84, H 6.18, N 17.18. Found: C 64.52,
H 6.34, N 17.27. Compound 26: mp 120–123°C (dec.); m/z
(FABMS): 414 (M+1); IR (KBr): 3453, 3089, 2921, 2845,
1576, 1513, 1445, 1379cmꢀ1; 1H NMR (200MHz, CDCl3):
9. Junjappa, H.; Ila, H.; Asokan, C. V. Tetrahedron 1990, 5423.
10. Rudorf, W. D.; Schierhorn, A.; Augustin, M. Tetrahedron
1979, 35, 551.
11. Chauhan, S. M. S.; Junjappa, H. Tetrahedron 1976, 32,
1779.
12. Chauhan, S. M. S.; Junjappa, H. Synthesis 1974, 880.
13. Potts, K. T.; Cipullo, M. J.; Ralli, P.; Theodoridis, G. J.
Org. Chem. 1983, 47, 3027.
d
8.34 (d, 2H, J = 8.52Hz, Ar–H), 7.74 (d, 1H,
J = 3.64Hz, thiophene–H), 7.43 (d, 1H, J = 4.86Hz,
thiophene–H), 7.29 (d, 2H, J = 7.62Hz, Ar–H), 7.13 (dd,
1H, J = 4.82 and 3.62Hz, thiophene–H), 7.01–6.87 (m,
3H, Ar–H), 6.69 (s, 1H, pyr–H), 6.74 (d, 2H, J = 8.54Hz,
Ar–H), 3.95 (t, 4H, J = 5.10Hz, NCH2), 3.87 (br s, 2H,
NH2), 3.34 (t, 4H, J = 5.08Hz, NCH2); 13C NMR
(50MHz, CDCl3): d 164.10, 163.24, 158.70, 151.54,
149.07, 144.96, 130.24, 129.66, 129.14, 128.56, 128.22,
125.92, 120.66, 116.86, 114.85, 93.87, 49.56, 44.40. Anal.
Calcd for C24H23N5S: C 69.70, H 5.61, N 16.94. Found: C
69.35, H 5.76, N 16.83.
14. Pandya, U.; Saxena, J. K.; Kaul, S. M.; Murthy, P. K.;
Chatterjee, R. K.; Tripathi, R. P.; Bhaduri, A. P.; Shukla,
O. P. Med. Sci. Res. 1999, 27, 103.
15. General experimental procedure for the compound 15: The
solution of compound 5 (1.27g, 5mmol) and 1-phenyl
piperazine (0.81g, 5mmol) in dry THF was heated in
closed steel vessel at 100°C for 24h. The solvent was
removed under vacuum and resultant residue was dis-
solved in 100mL CHCl3. The organic phase was washed
with H2O (three times), dried over Na2SO4. The solution
was concentrated and purified with column chromatogra-