Synthesis and biological evaluation of novel 4-(hetero) aryl-2-piperazino quinazolines as anti-leishmanial and anti-proliferative agents

Article abstract of DOI:10.1016/j.bmcl.2009.03.020

A series of new class of 4-(hetero)aryl-2-piperazino quinazolines were synthesized and assessed for in vitro activity against extracellular promastigotes and intracellular amastigotes of Leishmania donovani. Among the compounds evaluated, compound 4bb and 4cb showed the selectivity index (SI) value > 8.03 and 4.21, respectively, which is promising as compared with sodium stilbogluconate (SSG) and pentamidine with the SI of 6.38 and 2.07, respectively. The synthesized compounds were also tested for anti-proliferation activity in a panel of mammalian cell lines. Compound 4aa which is quite inactive and 4ab which is hardly selective in anti-leishmanial assay are found to have significant activity in anti-proliferative assay.

Full text of DOI:10.1016/j.bmcl.2009.03.020

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2542–2545
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel 4-(hetero) aryl-2-piperazino
quinazolines as anti-leishmanial and anti-proliferative agents q
Shailesh Kumar a, Nishi Shakya b, Suman Gupta b, Jayanta Sarkar c, Devi Prasad Sahu a,
*
a Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow, India
b Division of Parasitology, Central Drug Research Institute, Lucknow, India
c Drug Target Discovery and Development Division, Central Drug Research Institute, Lucknow, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new class of 4-(hetero)aryl-2-piperazino quinazolines were synthesized and assessed for
in vitro activity against extracellular promastigotes and intracellular amastigotes of Leishmania donovani.
Among the compounds evaluated, compound 4bb and 4cb showed the selectivity index (SI) value > 8.03
and 4.21, respectively, which is promising as compared with sodium stilbogluconate (SSG) and pentam-
idine with the SI of 6.38 and 2.07, respectively. The synthesized compounds were also tested for anti-pro-
liferation activity in a panel of mammalian cell lines. Compound 4aa which is quite inactive and 4ab
which is hardly selective in anti-leishmanial assay are found to have significant activity in anti-prolifer-
ative assay.
Received 12 December 2008
Revised 5 March 2009
Accepted 9 March 2009
Available online 13 March 2009
Keywords:
2,4-Disubstituted quinazolines
Bis-heteroaryls
Antileishmanial agents
Antiproliferative agents
Ó 2009 Elsevier Ltd. All rights reserved.
Diseases caused by protozoal parasites such as leishmaniasis
have an overwhelming impact on public health throughout the
world, particularly in the tropics and subtropics. Leishmaniasis is
caused by protozoan parasites of the genus Leishmania presenting
several forms of the disease such as cutaneous (CL), mucocutane-
ous (MCL) and visceral leishmaniasis (VL). The later form caused
by the parasite Leishmania donovani can be fatal if untreated. The
disease is transmitted in vertebrate after inoculation of promastig-
otes by the bite of the infected female phlebotomine sandfly.1 Sub-
sequently, the promastigotes are quickly phagocytized by the
macrophages of the host and further changes to the amastigotes
form. The clinical manifestation of the disease is a consequence
of the multiplication of the amastigotes inside the macrophages.
According to the World Health Organization’s recent reports 88
countries are affected by leishmaniasis, comprehending 12 million
infected people worldwide, with approximately 350 million people
at risk. The incidence is increasing worldwide, with 1–2 million
new cases registered annually.2 For many decades, the first choice
of drug treatment for leishmaniasis has been the pentavalent anti-
monials, sodium stilbogluconate (PentostamÒ) and meglumine
antimonate (GlucantimeÒ) despite the fact that they exhibit renal
and cardiac toxicity. Alternative drugs pentamidine and amphoter-
icin B, have not experienced widespread use due to toxicity and
cost.3 Miltefosine, originally developed as an anticancer agent,
has been found to be highly effective against leishmaniasis but
possess severe gastrointestinal problems.4 As the leishmaniasis
chemotherapy is still inefficient, therefore the immediate action
is to develop new agents, more potent and selective for treating
this increasing parasitosis. A great number of natural and synthetic
compounds comprising divergent chemical structures have been
tested in the past few years in antileishmanial assays and among
these, quinazoline class of compounds have also been reported to
possess antiprotozoal activities through DHFR inhibition.5
Biaryls and biheteroaryls are significant building blocks in a
large number of natural products6 and pharmocophores in a vari-
ety of biologically active compounds.7 Naturally occurring napthyl-
isoquinoline alkaloids ancistrotanzanine B and ancistroealaine A
are potent antileishmanial agents.8 These important biheteroaryls
were earlier synthesized by metal catalyzed reactions which re-
quire multistep reactions,9 involving toxic or expensive catalysts.
Alternately, anhydrous AlCl3 mediated heteroarylation of arenes
and heteroarenes could be a cheap, convenient and straightfor-
ward synthetic methodology to access the biheteroaryls. In our
ongoing program devoted to the synthesis of bioactive molecules,
we had focused on quinazolines and chalcones derivatives. Based
on these observations we hypothesized and synthesized some
substituted biheteroaryl compounds containing quinazoline motif
in search of potential antileishmanial agents. In the continuation
of our studies,10 herein we described AlCl3 mediated synthesis of
some novel 4-(hetero) aryl-2-piperazino quinazolines and their
in vitro evaluation against promastigotes and amastigotes of Leish-
mania donovani and for anti-proliferative activity against various
q
CDRI Communication No. 7663.
* Corresponding author. Tel.: +91 522 2612415x4378; fax: +91 522 2623405.
E-mail addresses: dpsahuin@yahoo.com, dp_sahu@cdri.res.in (D.P. Sahu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.020

S. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2542–2545
2543
Table 1
cancer cell lines. 2-chloro-4-(hetero)aryl quinazolines, 2-chloro-4-
(1H-indol-3-yl)-quinazoline (3a), 2-chloro-4-(2,4,6-trimethoxy-
phenyl)-quinazoline (3b), 2-chloro-4-(2,4-dimethoxy-phenyl)-qui-
nazoline (3c) and 2-chloro-4-(1-methyl-1H-indol-3-yl)-quinazo-
line (3d) were prepared by a novel method of AlCl3 mediated
selective arylation by different arenes and heteroarenes at position
4 of 2,4-dichloroquinazoline 1.
When 1 equiv of arene/heteroarene was reacted with 1 equiv of
2,4-dichloroquinazoline 211 in the presence of 1.2 equiv of anhy-
drous aluminum chloride using dichloroethane as solvent 2-
chloro-4-(hetero)aryl quinazolines 312 were formed exclusively
in excellent yields (Scheme 1). To synthesized 2,4-disubstituted
quinazolines compounds (4aa–db),13 2-chloro-4-(hetero)aryl
quinazolines were further reacted with different cyclic amines in
1,4-dioxane in presence of triethylamine to afford the desired com-
pounds (Table 1). Reactions in dioxane proceeds much more
smoothly and gave the desired product in quantitative yield unlike
previously reported methods.14
Compound
Ar(Het)–
Indole
R–
4aa
N
N
N
N
N
N
NH
4ab
4ac
4ad
4ae
Indole
Indole
Indole
Indole
NCH3
O
N
N
F
Antileishmanial activity. The L. donovani promastigotes (MHOM/
IN/Dd8; originally obtained from Imperial college, London) were
transfected with firefly luciferase gene and the transfectants were
maintained in medium 199 (Sigma chemical Co., USA) supple-
mented with 10% fetal calf serum (GIBCO) and 1% penicillin
4af
Indole
N
4ag
4ba
4bb
4ca
4cb
4da
4db
Indole
N
N
N
N
N
N
N
N
(50 U/ml), streptomycin (50 lg/mL) solution (Sigma) under pres-
sure of G418 (Sigma).15 The compounds were assessed in vitro
against transgenic L. donovani promastigotes and intracellular
amastigotes as described by Ashutosh et al.15 at various concentra-
tions using sodium stilbogluconate and pentamidine as a reference
drugs. Cytotoxicity responses were assessed by MTT assay using
mouse macrophage cell line (J-774-A-1).16 IC50 of antileishmanial
activity was calculated by Probit analysis.17
2,4,6-Tri OCH3–C6H2
2,4,6-Tri OCH3–C6H2
2,4-DiOCH3–C6H3
2,4-DiOCH3–C6H3
1-CH3–indole
NH
NCH3
NH
Anti-proliferative activity. The anti-proliferative activity of the
compounds were determined in twofold serial dilutions against
four human cancer cell lines, DU145 (Prostate carcinoma), MCF-7
(Breast adenocarcinoma), KB (Oral epidermal carcinoma) and
C33A (Cervical carcinoma) and a non cancerous VERO mammalian
NCH3
NH
cell line upto a highest concentration of 50
lg/mL in an MTT
assay.18
1-CH3–indole
NCH3
Results and discussion. The in vitro biological activities of 2,4-
disubstituted quinazolines have shown encouraging results. IC50
and SI values of synthesized quinazolines against promastigotes
and intracellular amastigotes have been displayed in Table 2.
Amongst the 4-indolyl quinazolines 4ab–4ag, 4ab with N-methyl
piperazine as substituent at 2-position of quinazoline motiff exhib-
ited least IC50 value of 0.68 lg/mL in the series. Surprisingly,
replacement of N-methyl piperazine with more lipophilic piperi-
dine moiety as in 4ac led to decrease in anti-promastigotic activity
O
O
Cl
N
O
ii
OH
i
NH
N
+
H2N
NH2
NH2
N
H
O
Cl
2
1
iii
R
N
N
Cl
iv
N
N
Ar(Het)
Ar(Het)
3
4
Ar(Het) = 3-indolyl (3a)
Ar(Het) = 2,4,6-triOCH3-C6H2 (3b)
Ar(Het) = 2,4-diOCH3-C6H3 (3c)
Ar(Het) = 1-methyl-3-indolyl (3d)
Scheme 1. Reaction conditions: (i) 135–140 °C, 3 h; (ii) POCl3, N,N-dimethyl aniline, reflux, 5 h; (iii) Ar(Het)-H, AlCl3, dry DCE, N2, 75–80 °C, 3 h; 3a (80.3%), 3b (85.0%); 2 h,
3c (83.0%), 3d (80.6%); (iv) R–H (1.2 mmol), 3a–d (1 mmol), TEA (2 mmol), dioxane (5 mL), 90–95 °C.

2544
S. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2542–2545
Table 2
at position 4 together with piperazine or 4-methyl-piperazine at
In vitro anti-leishmanial activity of compounds 4aa–db
position 2 of quinazoline enhances the anti-proliferative activity.
Compound 4aa which is quite inactive and 4ab which is hardly
selective in anti-leishmanial assay are found to have significant
IC50 values in anti-proliferative assay. However, the replacement
of indole moiety with an aryl ring in form of 2,3-dimethoxy ben-
zene (4cb) and 2,3,5-trimethoxy benzene (4bb) together with N-
methyl group remarkably enhances the antileishmanial activity.
In conclusion, 4-(hetero) aryl-2-substituted piperazino quina-
zolines were synthesized for the first time and their in vitro antile-
ishmanial and antiproliferative activities were evaluated. Most of
the synthesized compounds exhibited moderated to good activity
Compounds
In-vitro screening
Anti-amastigote
promastigote activity (MQ/amast.
Cytotoxicity Selectivity
CC50
g/mL)
indexa
(SI)
Anti-
(
l
activity IC50
g/mL)
model) IC50 (lg/mL)
(
l
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ba
4bb
4ca
0.56
0.68
>10
Toxic
4.17
ND
3.11
7.27
—
—
1.74
—
1.80
1.72
2.66
2.37
0.25
6.20
0.624
3.79
0.28
0.62
940
25.14
12.32
8.02
50.07
23.41
1.35
5.67
—
1.99
1.90
0.17
1.19
—
4.73
Toxic
12.45
5.11
Acknowledgments
>100
3.98
18.00
16.01
1.03
297.38
>8.03
0.78
4.21
1.28
—
S.K. and Nishi are grateful to the University Grant Commission,
New Delhi and CSIR, New Delhi for the financial support and to the
Sophisticated Analytical Instrument Facility, CDRI, Lucknow for
providing spectroscopic data. Technical support of Mr. A.P. Diwedi
is duly acknowledged.
4cb
4da
4db
Sodium
stilbogluconate
(SSG)
4.28
12.50
Toxic
53.62
6.38
Pentamidine
0.643
12.11
25.15
2.07
References and notes
ND: not determined.
a
Selectivityindex(SI)definedbytheratioCC50 (J-774 A-1 cells)/IC50 (Leishmania amastigotes)
.
1. (a) Desjeux, P. World Health Stat. Q 1992, 45, 267; (b) Desjeux, P. Trans. R. Soc.
Trop. Med. Hyg. 2001, 9, 239; (c) Guerin, P. J.; Olliaro, P.; Sundar, S.; Boelaert, M.;
Croft, S. L.; Desjeux, P.; Wasunna, M. K.; Bryceson, A. D. Lancet Infect. Dis. 2002,
2, 494.
2. (a) Tropical Disease Research: Progress 1999–2000; World Health Organization:
Geneva, 2001.; (b) Gelb, M. H.; Hol, W. G. J. Science 2002, 297, 343; (c) Trouiller,
P.; Torreele, E.; Olliaro, P.; White, N.; Foster, S.; Wirth, D.; Pécoul, B. Trop. Med.
Int. Health 2001, 6, 945.
3. Mcgregor, A. Lancet 1998, 351, 575.
4. Sangraula, H.; Sharma, K. K.; Rijal, S.; Dwivedi, S.; Koirala, S. J. Assoc. Physians
India. 2003, 51, 686.
5. Berman, J. D.; King, M.; Edwards, N. Antimicrob. Agents Chemother. 1989, 33,
1860.
6. Bringmann, G.; Ochse, M.; Schupp, O.; Tasler, S. Progress in the Chemistry of
Organic Natural Products; Springer: Wien, 2001. pp. 82.
7. (a) Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893; (b)
Wang, G. T.; Wang, S.; Gentles, R.; Sowin, T.; Leitza, S.; Reilly, E. B.; von Geldern,
T. W. Bioorg. Med. Chem. Lett. 2005, 15, 195.
8. Bringmann, G.; Hamm, A.; Schraut, M. Org. Lett. 2003, 5, 16, 2805.
9. (a) Liu, W. J.; Xie, Y. X.; Liang, Y.; Li, J. H. Synthesis 2006, 860; (b) Marion, N.;
Navarro, O.; Mei, J.; Stevens, E. D.; Scott, N. M.; Nolan, S. P. J. Am. Chem. Soc.
2006, 128, 4101; (c) Lützen, A.; Hapke, M. Eur. J. Org. Chem. 2002, 2292; (e) Mee,
S. P. H.; Lee, V.; Baldwin, J. E. Angew. Chem. 2004, 116, 1152.
with IC50 value more than 10 lg/mL. With 4-(2,4,6-trimethoxy-
phenyl), 4-(2,4-dimethoxyphenyl) and 4-(1-methyl indole) quina-
zolines reverse trend were exhibited in the anti-promastigote
activity with variation of substituents at 2-position as 4-methyl
piperazinyl derivatives (4bb, 4cb and 4db) have lower anti-pro-
mastigote activity compared to piperazinyl derivative (4ba, 4ca
and 4da), but no similar trends were exhibited in their anti-amas-
tigote activities with variation of substituent in general at 2-posi-
tion of 4-aryl quinazolines. The compounds 4af and 4db
exhibited least CC50 values at 1.35
tively. 4bb with least cytotoxicity (CC50 value above 100
l
g/mL and 1.03
l
g/mL respec-
g/mL)
l
has selectivity index above 8.03 which is comparable with that
of sodium stilbogluconate. From the IC50 and SI values for intracel-
lular amastigotes of the test derivatives indicate that two
compounds,
2-(4-methyl-piperazin-1-yl)-4-(2,4,6-trimethoxy-
10. Diwedi, A. P.; Kumar, S.; Varshney, V.; Singh, A. B.; Srivastava, A. K.; Sahu, D. P.
Bioorg. Med. Chem. Lett. 2008, 18, 2301.
phenyl)-quinazoline 4bb and 4-(2,4-dimethoxy-phenyl)-2-(4-
methyl-piperazin-1-yl)-quinazoline 4cb exhibited higher activity
11. Curd, F. H. S.; Landquist, J. K.; Rose, F. L. J. Chem. Soc. 1947, 775.
12. Typical procedure for 4-(hetero)aryl quinazolines 3a–d: To the solution of 2,4-
dichloroquinazoline (1 mmol) in dichloroethane (10 mL), was added AlCl3
(1.2 mmol) under N2 atmosphere and allowed to stirred for 2–5 min.
Nucleophilic substrates, Ar(Het)H (1 mmol) were added and allowed to
stirred for 2–3 h. After completion of the reaction, the reaction mixture was
cooled at room temperature and poured into ice-cold water (100 mL) with
continuous stirring for 15–20 min. The crude products were isolated by
extracting with ethyl acetate (10 mL Â 3). The organic phase was separated
and washed with brine, dried over sodium sulphate and evaporated under
vacuum. The crude products obtained were further purified by silica gel
column chromatography using ethyl acetate–hexane mixture; Compound 3c:
Yield: 83.0%; mp: 140–142 °C; 1H NMR (300 MHz, CDCl3): d 3.71 (s, 3H), 3.90
(s, 3H), 6.61 (d, J = 2.2 Hz, 1H), 6.67 (dd, J = 8.4 and 2.3 Hz, 1H), 7.41 (d,
J = 8.4 Hz, 1H), 7.49–7.55 (m, 1H), 7.77 (dd, J = 8.0 and 0.8 Hz, 1H), 7.85–7.90
(m, 1H), 7.99 (d, J = 8.4 Hz, 1H); 13C NMR (75 MHz, CDCl3): d 55.89, 56.01,
99.23, 105.64, 123.45, 127.70, 128.03, 128.65, 132.68, 135.00, 152.52, 158.66,
163.26, 171.21; mmax (KBr, cmÀ1): 1611, 1209, 1161; FAB (m/z): 300, [M+H]+
301; HRMS-EI: found: 300.0662, calcd: 300.0665. Compound 3d: Yield: 80.3%;
mp: 142–144 °C; 1H NMR (300 MHz, CDCl3): d 3.93 (s, 3H), 7.28–7.36 (m, 2H),
7.38–7.43 (m, 1H), 7.55–7.61 (m, 1H), 7.78 (s, 1H), 7.84–7.89 (m, 1H), 7.97 (dd,
J = 8.3 and 0.7 Hz, 1H), 8.17–8.20 (m, 1H), 8.41 (dd, J = 8.6 and 0.7 Hz, 1H); 13C
NMR (75 MHz, CDCl3): d 33.68 (CH3), 109.96 (CH), 112.19 (C), 121.53 (C),
122.04 (CH), 122.12 (CH), 123.52 (CH), 127.00 (C), 127.29 (CH), 127.46 (CH),
128.05 (CH), 133.93 (CH), 134.33 (CH), 137.65 (C), 152.90 (C), 157.37 (C),
166.15 (C); mmax (KBr, cmÀ1): 1563, 1347, 735; ES–MS (m/z): [M+H]+ 294;
HRMS-EI: found: 293.0689, calcd: 293.0719.
against L. donavani (IC50 = 12.45 and 4.28
compared to reference drugs sodium stilbogluconate (IC50
53.62 g/mL) and pentamidine (IC50 = 12.11 g/mL) and thus rep-
resent the interesting leads as antileishmanial agents.
lg/mL, respectively) as
=
l
l
In anti-proliferative assay, compounds 4aa and 4ab showed the
proliferative inhibition in KB (Oral squamous cell carcinoma) cell
line. The compound 4aa showed the IC50 values of 4
8.2 g/mL in KB and C-33A respectively, and compounds 4ab
showed the IC50 values of 4.9 g/mL and 6.0 g/mL in KB and
lg/mL and
l
l
l
MCF-7, respectively. The activity profile is shown in Table 3.
From the structural activity relationship it is found that the
presence of free NH group in form of 1-H-indole as the substituent
Table 3
Inhibition of proliferation of the compounds 4aa–dba
Compounds
IC50 (lg/mL)
DU145
MCF-7
C-33A
KB
Vero
4aa
4ab
12.5
20.8
14.1
6.0
8.2
11.5
4.0
4.9
8.9
22.3
13. General procedure for 2,4-disubstituted quinazolines 4aa–db: A mixture of 2-
chloro-4-(hetero)aryl quinazolines 3a–d (1 mmol), amines, RH (1.2 mmol) and
triethylamine (2 mmol) in dioxane (5 mL) was heated at 90–95 °C for 2–3 h.
After completion of the reaction, the solvent was evaporated and added water
a
Compounds 4ac–db were also tested against these cell lines but had an IC50
g/mL.
value superior to 50
l

S. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2542–2545
2545
(5 mL) and extracted with ethyl acetate (5 mL Â 3) to afford the crude product
which was further purified by silica gel column chromatography using 3–5%
methanol–chloroform mixture. Compound 4db: Yield: 96.3%; mp: 212–214 °C;
1H NMR (300 MHz, CDCl3): d 2.37 (s, 3H), 2.54–2.58 (t, 4H), 3.92 (s, 3H), 4.07–
4.10 (t, 4H), 7.14–7.19 (m, 1H), 7.24–7.29 (m, 1H), 7.32–7.37 (m, 1H), 7.42 (d,
J = 7.9 Hz, 1H,), 7.63–7.65 (m, 3H), 8.12–8.18 (m, 2H); 13C NMR (75 MHz,
CDCl3): d 33.53, 44.19, 46.49, 55.44, 109.83, 113.43, 118.43, 121.36, 122.03,
122.16, 123.08, 126.45, 127.29, 127.49, 132.66, 133.31, 137.70, 153.83, 159.11,
(2 Â CH2), 46.51 (2 Â CH2), 55.36 (NCH3), 55.66 (OCH3), 56.19 (2 Â OCH3), 91.37
(2 Â CH), 109.03 (C), 120.49 (C), 122.06 (CH), 125.97 (CH), 127.32 (CH), 133.32
(CH), 152.59 (C), 159.24 (2 Â C), 159.41 (C), 162.18 (C), 166.63 (C); mmax (KBr,
cmÀ1): 1605, 1546, 1132; ES–MS (m/z): [M+H]+ 395; HRMS-EI: found:
394.2006, calcd: 394.2005.
14. Ram, V. J.; Farhanullah; Tripathi, B. K.; Srivastava, A. K. Bioorg. Med. Chem. 2003,
11, 2439.
15. Ashutosh, G. S.; Ramesh, S. S.; Goyal, N. Antimicrob. Agetns Chemother. 2005, 49,
3776.
164.03;
m
max(KBr, cmÀ1): 1545, 754; ES–MS (m/z): [M+H]+ 358; HRMS-EI:
found: 357.1993, calcd: 357.1953; Compound 4bb: Yield: 96.6%; mp: 139–
141 °C; 1H NMR (300 MHz, CDCl3): d 2.34 (s, 3H, NCH3), 2.51 (t, 4H), 3.64 (s, 6H,
2 Â OCH3), 3.88 (s, 3H, OCH3), 4.00 (t, 4H), 6.25 (s, 2H), 7.01–7.06 (m, 1H), 7.33
(d, 1H, J = 8.5 Hz), 7.54- 7.61 (m, 2H); 13C NMR (75 MHz, CDCl3): d 44.25
16. Huber, W.; Koella, J. C. Acta Tropica. 1993, 55, 257.
17. Finney, D. J. Probit Analysis, 3rd ed.; Cambridge University Press, 1971.
18. Mosmann, T. J. Immunol Methods 1983, 6, 55.