Synthesis of new pyrrolo[2,3-d]pyrimidine derivatives and evaluation of their activities against human colon cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2009.12.050

New pyrrolo[2,3-d]Pyrimidines with heteroaryl substitution at 5th position through sulfur linker were synthesized incorporating putative pharmacophoric moieties like benzimidazole and benzothiazole as heteroaryl groups. Cytotoxic effect of all the compounds was carried out on HCT116 colon cancer cell lines. Compounds 6c and 6h with nitrobenzimidazole and pyrimidyl heterocycles attached at 5th position via sulfur were the most potent of all with IC50 values ≈17.6?μM. Among the four compounds tested for apoptosis induction activity, 6c induced apoptosis in a dose dependent manner.

Full text of DOI:10.1016/j.ejmech.2009.12.050

European Journal of Medicinal Chemistry 45 (2010) 1453–1458
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new pyrrolo[2,3-d]pyrimidine derivatives and evaluation of their
activities against human colon cancer cell lines
*
Saritha Jyostna Tangeda, Achaiah Garlapati
University College of Pharmaceutical Sciences, Kakatiya University Warangal 506 009, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New pyrrolo[2,3-d]Pyrimidines with heteroaryl substitution at 5th position through sulfur linker were
synthesized incorporating putative pharmacophoric moieties like benzimidazole and benzothiazole as
heteroaryl groups. Cytotoxic effect of all the compounds was carried out on HCT116 colon cancer cell
lines. Compounds 6c and 6h with nitrobenzimidazole and pyrimidyl heterocycles attached at 5th
Received 5 February 2009
Received in revised form
14 December 2009
Accepted 23 December 2009
Available online 22 January 2010
position via sulfur were the most potent of all with IC50 values z17.6
tested for apoptosis induction activity, 6c induced apoptosis in a dose dependent manner.
Ó 2010 Elsevier Masson SAS. All rights reserved.
mM. Among the four compounds
Keywords:
Pyrrolo(2,3-d)pyrimidine
Cytotoxicity
Apoptosis
1. Introduction
Over the past years benzimidazole derivatives are one of the
most extensively studied classes of heterocyclic compounds. Mole-
cules with benzimidazole nucleus have been reported to posses
anticancer, anti-infective, antibacterial and antifungal properties
due to their structural similarity with naturally occurring purines as
they can easily interact with biomolecules of the living systems
[12,13]. FB642 is a member of the benzimidazole carbamate
exhibited in vitro anti-tumor activity against both the murine B16
TS (Thymidylate Synthase) plays an important role in the
biosynthesis of nucleic acid precursors [1,2]. TS which catalyses the
conversion of deoxyuridylate (dump) to deoxy thymidylate (dTMP),
has been recognized as an attractive target for the development of
anti-tumor agents [3]. 5-Fluorouracil, the first known TS inhibitor,
has been used clinically as an anti-tumor agent. The emergence of
resistance as well as the insensitivity of certain tumor types to 5-FU
has prompted the design of novel folate analogues as potential TS
inhibitors and anti-tumor agents [4]. Fig. 1 illustrates important
examples of clinically used TS inhibitors such as Raltitrexed [5] and
Pemetrexed [6].
Classical antifolates containing
molecule have short-comings such as drug resistance due to
decreased folyl poly- -glutamate synthetase (FPGS) and impair-
ment of reduced folate carrier (RFC) system required to enter into
the cell [7]. The problems associated with classical antifolates are
overcome by non-classical lipophilic analogues by deleting (or)
melanoma (IC50 ¼ 8.5
mM) and human HT-29 colon carcinoma
(IC50 ¼ 9.5 M) cell lines [14,15]. Similarly benzothiazole constitutes
m
an important Scaffold of drugs possessing several pharmacological
functions, rendering this molecule and its derivatives powerful anti-
tumor agents [16,17]. DF203 is identified as anticancer agent with
remarkable differential activity in vitro and in vivo [18,19].
L-glutamic acid moiety in the
In spite of the above and although many new pyrrolo[2,3-
d]pyrimidine derivatives have been synthesized as potential
anti-tumor agents, there is scarce literature data on pyrrolo(2,3-
d)pyrimidines possessing benzimidazole and benzothiazole as
substituent. Therefore it is thought worthwhile to synthesize pyr-
rolo[2,3-d]pyrimidine derivatives bearing various heterocyclic nuclei
such as benzothiazole, benzimidazole etc and evaluate them for their
anti-tumor activity. The newly proposed synthesized compounds,
characterized by spectral and analytical properties, are evaluated for
anti-tumor against human colon cancer cell lines (HCT 116) (Fig. 2).
g
modifying L-glutamic acid component from the folate analogues,
which would not be dependent on FPGS for their potency and could
passively diffuse into cells [8,9]. Nolatrexed is the first non-classical
TS inhibitor to reach clinical trials. Gangjee et al. reported the
synthesis of 2-amino-4-oxo-5-thiopyridyl-6-methyl pyrrolo(2,3-
d)pyrimidine (1) as a potent TS inhibitor (IC50 ¼ 0.34
mM) [10,11].
2. Chemistry
2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimi-
dine (5) the key intermediate for the synthesis of title compounds
* Corresponding author. Tel.: þ91 9440601054; fax: þ91 870 2453508.
E-mail address: achaiah_g@yahoo.co.in (A. Garlapati).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.050

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S.J. Tangeda, A. Garlapati / European Journal of Medicinal Chemistry 45 (2010) 1453–1458
Fig. 1. Examples of some potential TS inhibitors.
6a–j (Scheme 1) was prepared by the addition of chloroacetone (4)
to a solution of 2,6-diamino-4-hydroxy pyrimidine (3) in sodium
acetate and water as reported earlier [11]. Various heteroaryl
moieties like benzimidazole, benzothiazole, pyridyl, substituted
oxadiazoles etc. were linked via sulfur to the 5th position of pyr-
rolopyrimidines in a one step oxidative addition of corresponding
mercapto heteroaryl compounds in presence of iodine [20].
Completion of the reaction was monitored by TLC. The title
compounds 6a–j were obtained in good yields (32–67%) [21].
trypan blue exclusion method [21]. The inhibitory potencies (IC50
are listed in Table 1. Compound 6c and 6h were equipotent and the
most active of series with IC50 close to 17.60 M. The compound
with 5-nitrobenzimidazole (6c) exhibited more than 2 times
potency when compared to the one without 5-nitro substitution
)
m
(6b; IC50 ¼ 38.04
mM). However, introduction of electron with-
drawing chloro substituent at 5th position of benzimidazole
nucleus did not result in any significant increase in potency (6d;
IC50 ¼ 36.23
potency (6c; IC50 ¼ 45.27
releasing methyl group at 5th position resulted in a little increase in
M). Compound 6a and 6i showed lower
mM). On contrary 5-bromo substitution decreased the
mM). However substitution with electron
3. Biological studies
potency (6f; IC50 ¼ 34.56
m
All the synthesized compounds (6a–j) were evaluated for
cytotoxic activity against the colon cancer HCT 116 cells using
activity which indicates phenyl substituted oxadiazole and pyridyl
moieties did not contribute much to the activity. However,

S.J. Tangeda, A. Garlapati / European Journal of Medicinal Chemistry 45 (2010) 1453–1458
1455
Fig. 2. (A) A cytotoxic effect of compounds 6a–j. (B) Apoptosis of HCT 116 cells upon treatment with 6c, 6d, 6f and 6g. Mean of three independent experiments; Significance levels;
*p < 0.005; **p < 0.0005.
introduction of another nitrogen into the pyridine ring (6i) to result
2-pyrimidinyl compound (6h) greatly enhanced the potency
Among all the compounds, 6c with nitrobenzimidazole
heterocycle exhibited maximum activity. Other electron with-
drawing groups like chloro on benzimidazole did not result in
such increase in potency. Benzothiazole containing compound, 6g
exhibited much greater potency when compared to the benz-
imidazole counter part (6b) which further suggests that nitro
substitution on benzothiazole might result in further improve-
ment in potency. The results suggested that compounds 6a–j
suppressed cell proliferation and a few of them induced apoptosis
in human colon cancer cell lines. It has been well established that
excessive proliferation and lack of apoptosis leads to tumor
growth and above study clearly suggests that compound 6c can
be a potential candidate for further studies as an anti-tumor
agent.
(IC50 ¼ 17.60
Compound 6g with benzothiazole linked to pyrrolopyrimidine
M) when compared to its
mM).
exhibited greater potency (IC50 ¼ 26.27
m
benzimidazole containing compound (6b). Compound 6j with N-
methyl imidazole substitution exhibited lower potency
(IC50 ¼ 48.90
mM) indicating the importance of benzo fusion to
imidazole for activity. Among all the compounds synthesized, four
have been selected for their effect on apoptosis by EtBr/AO assay
[22]. 6c, 6d, 6f and 6g induced apoptosis in HCT116 cells in a dose
dependent manner (6c, 28.33% in 5
mmol/L, 49.33% in 10
mmol/L,
65% in 20 mol/L, 85% in 50 mol/L and 94% in a 100 m
m
m
mol/L) versus
untreated cells.
4. Conclusions
5. Experimental protocols
The synthetic methodology adopted resulted in sulfanyl heter-
oaryl linked pyrrolopyrimidines in overall high yields. The
compounds thus obtained were evaluated for cytotoxic activity
against colon cancer cell lines to obtain the IC50 values.
5.1. Chemistry
Melting points were determined on Shital scientific melting
point apparatus and are uncorrected. 1H NMR spectra were

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5.1.3. 2-Amino-6-methyl-5-[20-(benzimidazolyl)sulfanyl]
3,4-dihydro-4-oxo-7H-pyrrolo [2,3-d] pyrimidine 6b
Compound 6b (synthesized as described for 6a); yield 53%; pale
white solid; mp 280–284 ꢀC (dec); 1H NMR (300 MHz, DMSO-d6)
d
2.22 (3H, s, CH3), 6.15 (2H, s, NH2), 7.05–7.41 (4H, m, H ar), 10.33
Table 1
Cytotoxicity IC50 values in
m
M for the tested compounds against HCT116 Cancer cell
lines.
Compound
Ar HCT 116 (colon)
N
N
6a
88.00
38.04
O
N
Scheme 1. Reagents and conditions (a) NaOAc/H2O, 80 ꢀC, 4–5 h (b) ArSH, I2, EtOH/
H2O (3:2), 90 ꢀC, upto 3 h.
6b
N
H
determined on a Bruker AVANCE-300 spectrometer. All NMR
spectra were measured in DMSO-d6 solutions using tetra methyl
silane as an internal standard and 1H chemical shifts are
reported as ppm. Mass spectra were recorded by using electro-
spray ionization technique (ESI) on the VG170708H mass spec-
trometer. Solvents were dried by conventional methods.
Silicagel 60–120 mesh (Merck) was used as an adsorbent for
column chromatography. TLC was performed on 5–10 cm
aluminum plates coated with silicagel 60F-254 (Merck) in an
appropriate solvent.
NO2
Cl
N
6c
17.61
36.23
45.27
N
H
N
6d
N
H
5.1.1. 2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo
[2,3-d]-pyrimidine 5
A suspension of 2,6-diamino-4-hydroxy pyrimidine (1.26 g,
10 mmol) in 25 mL of water containing sodiumacetate (0.82 g,
10 mmol) was heated to 80 ꢀC until it formed a clear solution.
Chloroacetone (0.79 mL,10 mmol) was added to this solution in one
lot, following which a precipitate began to form within 30 min. The
reaction mixture was heated with stirring at 80 ꢀC for an additional
4 h, cooled and filtered. The crude residue thus obtained was
recrystallised with water to get 5 as a off white solid, yield 68%, mp
Br
N
6e
N
H
CH3
N
250 ꢀC (dec); 1H NMR (300 MHz, DMSO-d6)
d 2.14 (3H, s, CH3), 5.84
(1H, s, 5-CH), 5.96 (2H, s, NH2), 10.13 (1H, s, NH), 10.79 (1H, s, NH);
Anal. C7H8N4O (C, H, N).
6f
34.56
26.27
N
H
5.1.2. 2-Amino-6-methyl-5-[20-(50-phenyl-10,30,40-
oxadizolyl)sulfanyl]-3,4-dihydro-4-oxo-7H-pyrrolo [2,3-d]
pyrimidine 6a
S
To a solution of 5 (0.164 g, mmol) in a mixture of ethanol,
water (3:2; 40 ml) was added 2-mercapto-5-phenyl-1,3,4-oxa-
diazole (0.356 g, 2 mmol) and the reaction miture was heated to
90 ꢀC for 30 min. Then I2 (0.500 g, 2 mmol) was added and the
heating continued with stirring until starting material dis-
appeared as monitored by TLC.. To this mixture was added excess
sodium thiosulfate and filtered. The residue was washed well
with water and air dried. The crude solid obtained was purified
through column chromatography on silicagel using 1–8% meth-
anol in dichloromethane. Fractions containing the desired spot
were pooled and evaporated to dryness to give 6a as a pale
yellow solid (32%); mp 200–204 ꢀC (dec); 1H NMR (300 MHz,
6g
N
H
N
N
6h
6i
17.60
50.00
N
DMSO-d6)
d 2.26 (3H, s, CH3), 6.17 (2H, s, NH2), 7.54 (3H, m, H ar),
7.83 (2H, d, H ar), 10.32 (1H, s, NH), 11.57 (1H, s, NH); Anal.
C15H12N6O2S (C, H, N).

S.J. Tangeda, A. Garlapati / European Journal of Medicinal Chemistry 45 (2010) 1453–1458
1457
Table 1 (continued)
d6) d 2.13 (3H, s, CH3), 6.08 (2H, s, NH2), 7.11 (1H, t, H ar), 8.50 (2H, d,
H ar), 10.20 (1H, bs, NH), 11.30 (1H, s, NH); MS (ESI) m/z 275.2
Compound
Ar
HCT 116 (colon)
[M þ 1]þ; Anal. C11H10N6OS (C, H, N).
N
5.1.10. 2-Amino-6-methyl-5-[20-(pyridinyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-i] pyrimidine 6i
N
CH3
6j
48.90
Compound 6i (synthesized as described for 6a); yield 48%;
yellow solid; mp 250–256 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
d
2.16 (3H, s, CH3), 6.12 (2H, s, NH2), 6.67–8.30 (4H, m, H ar), 10.24
(1H, s, NH), 11.45 (1H, s, NH); Anal. C12H11N5OS (C, H, N).
O
5.1.11. 2-Amino-6-methyl-5-[20-(10-methylimidazolyl)sulfanyl]-3,4-
dihydro-4-oxo-7H-pyrrolo-[2,3-d] pyrimidine 6j
H
N
F
5-Fluorouracil
3.03
Compound 6j (synthesized as described for 6a); yield 51%;
white solid; mp 220–226 ꢀC dec. 1H NMR (300 MHz, DMSO-d6)
O
N
H
d
2.23 (3H, s, CH3), 3.73 (3H, s, CH3) 6.07 (2H, s, NH2), 6.80 (1H, s, H
ar), 7.12 (1H, s, H ar), 10.16 (1H, s, NH), 11.23 (1H, s, NH); Anal.
C11H12N6OS (C, H, N).
(1H, s, NH), 11.50 (1H, s, NH), 11.77 (1H, s, NH); MS (ESI) m/z 313
[M þ 1]þ; Anal. C14H12N6OS (C, H, N).
5.2. Evaluation of cell cytotoxicity
5.2.1. Cell culture
Human colon cancer HCT116 cells, purchased from American
Type Culture Collection (Manassas, VA) were used and maintained
5.1.4. 2-Amino-6-methyl-5-[20-(50-nitrobenzimidazolyl)sulfanyl]-
3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d] pyrimidine 6c
Compound 6c (synthesized as described for 6a); yield 52%;
in McCoy’s 5A medium containing
with 10% heat-inactivated fetal bovine serum, 100 units/ml peni-
cillin and 100
g/mL streptomycin at 37 ꢀC and 5% CO2 atmosphere
L-glutamine and supplemented
yellow solid; mp 280–284 ꢀC (dec); 1H NMR (300 MHz, DMSO-d6)
d
2.23 (3H, s, CH3), 6.18 (2H, s, NH2), 7.50 (1H, d, H ar), 8.03 (1H, m, H
m
ar), 8.20 (1H, d, H ar), 10.50 (1H, s, NH), 11.60 (1H, s, NH), 11.61 (1H,
s, NH); Anal. C14H11N7OS (C, H, N).
and sub-cultured after trypsinization (0.5% trypsin/2.6 mM EDTA).
For all experiments, cells were seeded at 1 ꢁ 106 cells in culture
dishes (100 mm) and grown to 60–70% confluence. To study the
growth arrest and apoptosis, various doses of test compounds
5.1.5. 2-Amino-6-methyl-5-[20-(50-chlorobenzimidazolyl)sulfanyl]-
3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d] pyrimidine 6d
ranging from 2.5
mM to 100 mM were used.
Compound 6d (synthesized as described for 6a); yield 56%; off
white solid; mp 260–264 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
5.2.2. Cytotoxicity assay
d
2.22 (3H, s, CH3), 6.16 (2H, s, NH2), 7.05–7.45 (3H, m, H ar),10.31 (1H,
Trypan blue exclusion assay has the ability to stain cells with
damaged plasma membrane blue. Hence blue stained cells are
considered as died and unstained cells are viable [23]. HCT116 cells
(10,000 cells) are layered in a 6 well plate for 24 h, treated with
various concentrations (2.5–100 mM) of test compounds (6a–j) and
then incubated at 37 ꢀC. Viability assay is done for HCT116 cells after
24 h of treatment with test compounds. The floating and adherent
s, NH),11.50 (1H, s, NH),11.80 (1H, s, NH); Anal. C14H11N6OSCl(C, H, N).
5.1.6. 2-Amino-6-methyl-5-[20-(50-bromobenzimidazolyl)sulfanyl]-
3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d] pyrimidine 6e
Compound 6e (synthesized as described for 6a); yield 57%; light
brown solid; mp 270–274 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
d
2.22 (3H, s, CH3), 6.16 (2H, s, NH2), 7.19–7.59 (3H, m, H ar), 10.32
cells were collected, suspended in 25 mL of phosphate buffered saline
(1H, s, NH), 11.54 (1H, s, NH), 11.86 (1H, s, NH); Anal. C14H11N6OSBr
(C, H, N).
(PBS) and mixed with 0.4% trypan blue stain. After a few minutes,
stained and unstained cells were counted on a haemocytometer by
light microscopy. Values are expressed as % of viable cells.
5.1.7. 2-amino-6-methyl-5-[20-(50-methylbenzimidazolyl)sulfanyl]-
3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine 6f
5.2.3. Detection of apoptosis
Compound 6f (synthesized as described for 6a); yield 57%; off
Apoptosis in cells induced by test compounds is detected by
their ability to get stained with fluorescent dyes, acridine orange
and ethidium bromide. Apoptotic cells that have lost their
membrane integrity appear orange due to costain with ethidium
bromide showing condensed chromatin. Alive cells stain uniformly
white solid; mp 260–264 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
d
2.22 (3H, s, CH3), 2.33 (3H, s, CH3), 6.20 (2H, s, NH2), 6.84–7.31 (3H,
m, H ar), 10.34 (1H, s, NH), 11.50 (1H, s, NH), 11.70 (1H, s, NH); Anal.
C15H14N6OS (C, H, N).
green. HCT 116 cells were exposed to 6c, 6d, 6f and 6g (2.5–100 mM)
5.1.8. 2-Amino-6-methyl-5-[20-(benzothiazolyl)sulfanyl]-3,4-
dihydro-4-oxo-7H-pyrrolo[2,3-d] pyrimidine 6g
for 48 h 25
with 1
100
PBS) just prior to microscopy. A 10
m
L of the cell suspension (w5 ꢁ 106 per mL) was added
mL of acridine orange/ethidium bromide (one part each of
Compound 6g (synthesized as described for 6a); yield 67%; off
mg/mL acridine orange and 100
m
g/mL ethidium bromide in
white solid; mp 240–246 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
m
L aliquot of the gently mixed
d
2.25 (3H, s, CH3), 6.19 (2H, s, NH2), 7.25 (1H, t, H ar), 7.39 (1H, t, H
suspension was placed on microscope slides, covered with glass
slips and examined under an Olympus AX71 microscope (Tokyo,
Japan) connected to a digital imaging system with SPOT RT software
version 3.0. Cells were scored into following categories: C1, cells
with large, green, noncondensed nuclei as non-apoptotic, viable
cells; C2, cells with red/orange nuclei that showed signs of nuclear
bead formation as apoptotic cells and C3, cells with large red nuclei
that did not show signs of nuclear condensation or bead formation
ar), 7.76 (1H, d, H ar) 7.82 (1H, d, H ar),10.36 (1H, s, NH),11.70 (1H, s,
NH); MS (ESI) m/z 330.2 (M þ 1)þ; Anal. C14H11N5OS2 (C, H, N).
5.1.9. 2-Amino-6-methyl-5-[(20-pyrimidinyl)sulfanyl]-3,4-dihydro-
4-oxo-7H-pyrrolo[2,3-d] pyrimidine 6h
Compound 6h (synthesized as described for 6a); yield 37%;
paleyellow solid; mp 240–246 ꢀC (dec). 1H NMR (300 MHz, DMSO-

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