Design, synthesis and anti colon cancer activity evaluation of phosphorylated derivatives of Lamivudine (3TC)

Article abstract of DOI:10.2174/157018011793663921

The novel phosphorylated derivatives of Lamivudine (5a-5l) as potential anti colon cancer agents are synthesized. These title compounds are designed based on the basic pyrimidine derivative lamivudine as a starting compound and reacted with various phosph

Full text of DOI:10.2174/157018011793663921

Letters in Drug Design & Discovery, 2011, 8, 59-64
59
Design, Synthesis and Anti Colon Cancer Activity Evaluation of Phos-
phorylated Derivatives of Lamivudine (3TC)
Valasani Koteswara Raoa, Sanapalli S. Reddya, Balam S. Krishnaa, Cirandur S. Reddya,
Nimmanapalli P. Reddyb, Tamatam C. M. Reddyb Chamarthi N. Raju*,a and S. K. Ghoshc
aDepartment of Chemistry, Sri Venkateswara University, Tirupati – 517 502, India
bSchool of life Sciences, University of Hyderabad, Hyderabad- 500 046, India
cBioorganic Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
Received April 19, 2010: Revised July 25, 2010: Accepted September 08, 2010
Abstract: The novel phosphorylated derivatives of Lamivudine (5a-5l) as potential anti colon cancer agents are synthe-
sized. These title compounds are designed based on the basic pyrimidine derivative lamivudine as a starting compound
and reacted with various phosphorodichloridates followed by the introduction of bioactive groups at the phosphorus. Their
structures were characterized by IR, 1H, 13C, 31P NMR and mass spectral analyses. All the compounds were evaluated for
their anti colon cancer activity against COLO-205 cell lines in vitro studies. Among them 5a and 5b emerged as lead
compounds with 0.003 μM and 0.0001 μM values.
Keywords: Anti colon cancer activity, COLO- 205 cell lines, MTT assay, Phosphorylated derivatives of lamivudine.
INTRODUCTION
phate competes with endogenous 2ꢀ-deoxycytidine–5ꢀ-
triphosphate for binding on HIV-I reverse transcriptase. As
lamivudine- triphosphate does not have a hydroxyl group at
any position analogous to the 3ꢀ- hydroxyl group of the natu-
ral 2ꢀ-deoxy nucleosides, its use as a substrate by HIV-I re-
verse transcriptase effectively terminates chain extension
beyond its incorporation into the HIV-DNA genome [8].
Nucleoside analogs, such as lamivudine, zidovudine, zal-
citabine and didanosine are an important class of anticancer
and antiviral agents [1, 2]. Lamivudine (3TC), the negative
enatiomer of (-) 2-deoxy-3ꢀ-thiacytidine is a dideoxy nucleo-
side which was disclosed by Soudeyns et al. a structurally
novel potent anti-HIV analog BCH -189 (2'-3'-dideoxy-3'-
thiacytidine) [3], in which the methylene group at the 3ꢀ-
position of the ribose ring is replaced by a sulfur atom.
Lamivudine is a dideoxynucleoside analog reverse tran-
scriptase inhibitor that is used in combination with other
nucleoside reverse transcriptase inhibitor, non-nucleoside
reverse transcriptase inhibitor, and protease inhibitors in the
treatment of HIV-1 infection as a component of currently
recommended highly active antiretroviral therapy [4].
Among D-dideoxynucleoside analogs, 2ꢀ-3ꢀ didehydro-2ꢀ, 3ꢀ
dideoxythymidine and ddC (2'-3'-dideoxycytidine) are a few
examples of anti-HIV drugs and D-dideoxynucleoside ana-
logs like ꢁ-D- arabinofuranosylcytosine and gemcitabine are
anticancer drugs [5].
Lamivudine has been proved in clinical use for the treat-
ment of HIV and hepatitis-B [9], L-FMAU [1-(2-fluoro-5-
methyl-beta,L-arabinofuranosyl) uracil] [10] and 2ꢀ, 3ꢀ-
dideoxy-2ꢀ, 3ꢀ-didehydro-ꢁ-L-5ꢀ-fluorodeoxycytidine, which
are in phase II clinical trials as anti hepatitis-B virus agents,
and ꢁ-L (-)-dioxolanecytidine [11, 12] which is in the phase
II clinical trials as an anti cancer agent. This compound is in
the advanced stage of clinical evaluation for the treatment of
both diseases. Clinical trials also demonstrated that 3'-azido-
3'-deoxythymidine has a significant effect in controlling the
progression of AIDS in patients [13]. Critical clinical evalua-
tion of the therapeutic potential of nucleoside analogs re-
quires a large amount of material be readily accessible which
translates into efficient synthetic protocols for preparation of
derivatives with bio-active groups.
Like other dideoxynucleosides, the antiviral activity of
lamivudine is due to its active 5ꢀ-triphosphate anabolite [6].
Lamivudine is anabolized intracellularly by a step wise proc-
ess, first to the monophosphate form by deoxycytidine
kinase, then to the diphosphate form by cytidine monophos-
phate kinase, and deoxycytidine monophosphate kinase, and
finally to the active triphosphate form by pyrimidine nucleo-
side diphosphate kinase [6]. In vitro studies have shown
lamivudine- diphosphate to be the predominant anabolite;
thus, the rate-limiting step in lamivudine phosphorylation is
from lamivudine- diphosphate to lamivudine- triphosphate
[7]. To exert its antiretroviral effect, lamivudine- triphos-
Lamivudine is the derivative of pyrimidine, and although
originally developed as an antiviral for human immuno defi-
ciency virus (HIV) was found to selectively repress COLO-
205 cell line and is also effective for repression of necroin-
flammation, liver fibrosis, seroconversion of serum HBV-
DNA and HBeAg (hepatitis B “e” antigen), and normaliza-
tion of serum ALT (alanine amino transferase) [14]. Further,
pharmacokinetic properties and cellular permeability of a
drug can be modulated by derivatization of bioreversible
forms of this drug.
The current work describes such an effort in which phos-
phorylated derivatives of lamivudine are synthesized and
screening for anti colon cancer activity against COLO -205
*Address correspondence to this author at the Department of Chemistry, Sri
Venkateswara University, Tirupati – 517 502, India; Tel: +91877-2249666 –
479; Fax: +91877- 2225211; E-mail: rajuchamarthi10@gmail.com
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

60 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 1
Rao et al.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl]methyl(2-dihydrogenphosphato)(4-nitrophenyl)
phosphate (5a)
cell lines in in vitro was envisaged. The anti-cancer activity
of D-deoxy nucleoside analongs led us to develop the syn-
thesis of new phosphorylated derivatives of lamivudine.
Substituents were selected with different electronic and
solubility characteristics with the aim of investigating the
substituent effect at the phosphorus atom.
Yield 72%; m.p. 171-173 ºC; IR (ꢀmax, cm-1, KBr): 3415
1
(O-H), 3295 (N-H), 1228 (P=O); H NMR (400 MHz,
DMSO-d6 , ꢀ , ppm): 3.10 (dd, 1H, J = 4.1, 11.9 Hz), 3.43
(dd, 1H, J = 5.2, 11.5 Hz), 3.76-3.80 (m, 2H), 5.19-5.21 (m,
1H), 5.80 (d, 1H, J = 7.6 Hz), 6.20-6.23 (m, lH), 7.30-7.42
(m, 2H), 7.72-7.92 (br m, 2H), 7.98 (d,1H, J = 7.6 Hz), 8.10-
8.22 (m, 2H), 9.6 (br s, 2H, P-OH); 13C NMR (100 MHz,
MATERIALS AND METHODS
Chemicals were procured from Sigma-Aldrich, Merck
and Lancaster, and used as such without further purification.
All solvents used for spectroscopic and other physical stud-
ies were reagent grade and were further purified by literature
methods [15]. Melting points (m.p.) were determined using a
calibrated thermometer by Guna Digital Melting Point appa-
ratus. They are expressed in degrees centigrade (°C) and are
uncorrected. Infrared Spectra (IR) were obtained on a
Nicolet 380 FT-IR spectrophotometer. Samples were ana-
lyzed as potassium bromide (KBr) discs. Absorptions are
reported in wave numbers (cm-1). 1H, 13C NMR and P31NMR
spectra were recorded as solutions in DMSO-d6 on a Bruker
DMSO-d6 ꢀ , ppm): 38.5, 67.4, 86.9, 87.5, 96.5, 120.6,
,
125.6, 141.1, 142.2, 152.8, 159.6, 164.7; 31P (161.9 MHz,
DMSO-d6 ꢀ , ppm): -7.16, -21.30, LCMS (m/z,%): 510
,
+
•
[MH , 100], 359 (28), 344 (31), 285 (19); Anal. Calcd.:
C14H16N4O11P2S: C 32.95, H 3.16, N 10.98; Found: C 32.62,
H 3.14, N 10.95.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl]methyl(2-hydroxyethyloxy)(4-nitrophenyl)phos-
phate (5b)
Yield: 75%; m.p.:126-128 ºC; IR (ꢀmax, cm-1, KBr): 3403
(O-H), 3278 (N-H), 1235 (P=O); 1H NMR (400 MHz,
DMSO-d6 , ꢀ, ppm): 3.14 (dd, 1H, J = 4.2, 11.2 Hz), 3.44
(dd, 1H, J = 5.2 , 11.2 Hz), 3.78-3.84 (m, 2H), 4.10-4.14 (m,
2H), 4.30-4.33 (m, 1H), 4.52 (t, 1H, J = 5.2 Hz), 5.16-5.20
(m, 1H), 5.80 (d, 1H, J = 7.6 Hz), 6.22-6.28 (m, 1H), 7.35-
7.48 (m, 2H), 7.42 (d, 1H, J = 5.3 Hz), 7.81-7.86 (br m, 2H),
7.92 (d, 1H, J = 7.5 Hz), 8.23-8.35 (m, 2H) ; 13C NMR (100
MHz, DMSO-d6 , ꢀ , ppm): 37.6, 62.3, 67.6, 68.8, 75.2, 84.5,
96.3, 121.2, 125.3, 141.3, 143.2, 152.5, 159.2, 164.5; 31P
(161.9 MHz, DMSO-d6 , ꢀ , ppm): -4.50; LCMS (m/z,%):
1
AMX 400 MHz spectrometer operating at 400 MHz for H,
1
100 MHz for 13C and 161.9 MHz for 31P NMR. The H and
13C chemical shifts are expressed in parts per million (ppm)
with reference to tetramethylsilane (TMS) and 31P chemical
shifts to 85% H3PO4. LC mass spectra were recorded on a
Jeol SX 102 DA / 600 Mass spectrometer. Elemental analy-
ses were performed by Central Drug Research Institute,
Lucknow, INDIA. The following abbreviations were used
while presenting the NMR data s = singlet, d = doublet, t =
triplet and m = multiplet.
+
•
474 [MH , 27], 387 (15), 314 (20), 277 (48), 197 (10), 183
(30), 100 (41); Anal. Calcd.: C16H19N4O9PS: C 40.51, H
4.04, N 11.81; Found: C 40.30, H 3.99, N 11.72.
SYNTHESIS
To the stirred solution of lamivudine (228.8 mg, 1.00
mmol) in dry tetrahydrofuran (20 mL) and pyridine (20 mL),
was added a solution of 4-nitrophenyl phosphorodichloridate
(137.8 mg, 1.00 mmol) in THF (20 mL) at -10 ºC in the
presence of TEA (101.2 mg, 1.00 mmol) over a period of 15
min. The reaction mixture was further stirred at 0 ºC and
continued stirring for 2 h, progress of the reaction was moni-
tored by TLC, (ethylacetate: hexane 5:5). After completion
of the reaction, it was filtered to remove triethylamine hy-
drochloride. The filtrate containing the intermediate 3a was
further reacted with mono potassium dihydrogen phosphate
(136.0 mg, 1.00 mmol) in THF (10 mL) and pyridine (10
mL) at 70-80 ºC for 10 hrs. After completion of the reaction,
NaCl was removed by filtration and the solvent was removed
in a rota-evaporator to obtain crude product. It was purified
by column chromatography on silica gel using acetone:
methanol (9:1) as eluent to afford the pure compound 5a, [5-
(4-amino-2-oxo-1, 2-dihydro-1-pyrimidinyl)-1,3-oxathiolan-
2-yl]methyl(2-dihydrogenphosphato) (4-nitrophenyl)phos-
phate. The same experimental procedure was adopted for the
preparation of the remaining title compounds 5b-5l. Analyti-
cal and spectral data of the newly synthesized compounds
corroborated well with the proposed structures. The synthetic
protocol for all the compounds 5a–5l is presented in Scheme
1 and their spectral data are given below.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl] methyl(2-aminoethylamido) (4-nitrophenyl)
phosphate (5c)
Yield: 69%; m.p.: 182-184 ºC; IR (ꢀmax, cm-1, KBr): 3292
1
(N-H), 1229 (P=O); H NMR (400 MHz, DMSO-d6 ꢀ ,
,
ppm): 3.11 (dd, 1H, J = 4.2, 11.5 Hz), 3.40 (dd, 1H, J = 5.2
Hz, 5.4 Hz), 3.56-3.61 (m, 4H), 3.80-3.85 (m, 2H), 4.34-4.42
(m, 1H), 5.19-5.21 (m, 1H), 5.26 (br s, 2H), 5.80 (d, 1H, J =
7.4 Hz), 6.31-6.38 (m, 1H), 7.28-7.37 (m, 2H), 7.82-7.88 (br
m, 2H), 7.96 (d, 1H, J = 7.4 Hz), 8. 10-8.16 (m, 2H); 13C
NMR (100 MHz, DMSO-d6 , ꢀ , ppm): 38.2, 40.2, 45.1, 3,
67.2, 86.5, 87.5, 95.5, 121.7, 126.3, 142.2, 143.5, 152.3,
158.4, 164.5; 31P (161.9 MHz, DMSO-d6 , ꢀ , ppm): -18.20;
+
•
LCMS (m/z,%): 472 [MH ]; Anal. Calcd.: C16H21N6O7PS:
C 40.68, H 4.48, N 17.79; Found: C 40.52, H 4.44, N 17.67.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl]methyl(4-methylpiperazino)(4-nitrophenyl)
phosphate (5d)
Yield: 72%; m.p.: 116-118 ºC; IR (ꢀmax, cm-1, KBr): 3302
1
(N-H), 1218 (P=O); H NMR (400 MHz, DMSO-d6 ꢀ,
,
ppm): 2.27 (s, 3H), 2.41-2.47 (m, 4H), 2.81-2.85 (m, 4H),
3.12 (dd, 1H, J = 4.1, 11.1 Hz), 3.43 (dd, 1H, J = 5.2 Hz, 4.1

Design, Synthesis and Anti Colon Cancer Activity Evaluation
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 1 61
Hz), 3.84-3.89 (m, 2H), 5.18-5.20 (m, 1H), 5.81 (d, 1H, J =
7.1 Hz), 6.21-6.26 (m, 1H), 7.22-7.38 (m, 2H), 7.88-7.94 (br
m, 2H), 7.98 (d, 1H, J = 7.6 Hz), 8.13-8.18 (m, 2H); 13C
NMR (100 MHz, DMSO-d6 , ꢀ , ppm): 38.5, 43.1, 44.1, 56.2,
67.4, 86.0, 87.2, 96.6, 120.6, 125.5, 142.6, 143.5, 152.5,
159.4, 164.1; 31P (161.9 MHz, DMSO-d6 , ꢀ , ppm): -7.11;
[5-(4–Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl] methyl(4-chlorophenyl)(4-methylpiperazino)
phosphate (5h)
Yield: 74%; m.p.: 181-183 ºC; IR (ꢀmax, cm-1, KBr): 3286
1
(N-H), 1218 (P=O); H NMR (400 MHz, DMSO-d6 ꢀ ,
,
ppm): 2.27(s, 3H), 2.41-2.43 (m, 4H), 2.81-2.86 (m, 4H),
3.10 (dd, 1H, J = 4.5, 11.3 Hz), 3.42 (dd, 1H, J = 5.2, 11.5
Hz), 3.80-3.84(m, 2H), 4.21-4.24 (m, 1H), 5.19-5.22 (m,
1H), 5.80 (d, 1H J = 7.5 Hz), 6.02-6.13 (m, 2H), 6.20-6.24
(m, 1H), 7.21-7.36 (m, 2H), 7.88-7.94 (br m, 2H) 7.98 (d,
1H, J = 7.6 Hz), ; 13C NMR (100 MHz, DMSO-d6 , ꢀ , ppm):
38.5, 43.1, 44.1, 56.2, 67.4, 86.9, 87.5, 95.5, 120.6, 125.5,
141.1, 152.8, 142.2, 159.2, 164.7; 31P (161.9 MHz, DMSO-
+
•
LCMS (m/z,%): 512 [MH ]; Anal. Calcd.: C19H25N6O7PS:
C 44.53, H 4.92, N 16.40; Found: C 44.29, H 4.87, N 16.24.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl] methyl (4-chlorophenyl) (2-dihydrogenphos-
phato)phosphate (5e)
Yield: 71%; m.p.: 126-128 ºC; IR (ꢀmax, cm-1, KBr): 3408
(O-H), 3312 (N-H), 1224 (P=O); 1H NMR (400 MHz,
DMSO-d6 , ꢀ , ppm): 3.14 (dd, 1H, J = 4.1, 11.4 Hz), 3.40
(dd, 1H, J = 5.2, 11.9 Hz), 3.80-3.85 (m, 2H), 5.20-5.24 (m,
1H), 5.80 (d, 1H, J = 7.4 Hz), 6.25-6.31 (m, 1H), 6.32-6.48
(m, 2H), 7.20-7.35 (m, 2H), 7.85-7.89 (br m, 2H), 7.99 (d,
1H, J = 7.5 Hz), 10.2 (br s, 2H) ; 13C NMR (100 MHz,
+
•
d6 ꢀ , ppm): -7.11; LCMS (m/z,%): 501 [MH ]; Anal.
,
Calcd.: C19H25ClN5O5PS: C 45.47, H 5.02, N 13.95; Found:
C 45.26, H 4.97, N 13.82.
[5-(4-Amino-2-oxo-1, 2-dihydro-1-pyrimidinyl)-1,3-oxa-
thiolan-2-yl] methylbis(2-chloroethyl)amino(2-dihydro-
genphosphato)phosphate (5i)
DMSO-d6 ꢀ , ppm): 38.5, 67.4, 86.9, 87.5, 96.5, 120.6,
,
126.6, 128.4, 142.5, 148.3, 158.3, 164.7; 31P (161.9 MHz,
Yield: 71%; m.p.: 161-163 ºC; IR (ꢀmax, cm-1, KBr): 3388
(O-H), 3292 (N-H), 1221 (P=O); 1H NMR (400 MHz,
DMSO-d6 , ꢀ , ppm): 3.10 (dd, 1H, J = 4.5, 11.9 Hz), 3.42
(dd, 1H, J = 5.2, 11.5 Hz), 3.26 (t, 4H, J = 13.6 Hz), 3.62 (t,
4H, J = 13.6 Hz), 3.81-3.85 (m, 2H), 5.18-5.21 (m, 1H), 5.80
(d, 1H, J = 7.6 Hz), 6.20-6.24 (m, 1H), 7.86-7.95 (br m, 2H),
7.96 (d, 1H, J = 7.4 Hz), 11.8 (br s, 2H); 13C NMR (100
MHz, DMSO-d6 , ꢀ , ppm): 38.5, 43.2, 44.5, 64.2, 86.9, 87.5,
96.5, 142.2, 158.6, 164.9; 31P (161.9 MHz, DMSO-d6 , ꢀ ,
DMSO-d6 ꢀ , ppm): -7.40, -25.31; LCMS (m/z,%): 499
,
+
•
[MH ]; Anal. Calcd.: C14H16ClN3O9P2S: C 33.65, H 3.23, N
8.41; Found.: C 33.42, H 3.20, N 8.33.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl] methyl (4-chlorophenyl)(2-hydroxyethyloxy)
phosphate (5f)
Yield: 71%; m.p.: 126-128 ºC; IR (ꢀmax, cm-1, KBr): 3408
(O-H), 3312 (N-H), 1224 (P=O); 1H NMR (400 Hz, DMSO-
d6 , ꢀ , ppm): 3.10 (dd, 1H, J = 4.1 Hz, 11.1 Hz), 3.41 (dd,
1H, J = 5.2 Hz, 11.6 Hz), 3.82-3.87 (m, 2H), 4.12-4.16 (m,
2H), 4.36-4.40 (m, 1H), 5.21-5.24 (m, 1H), 5. 63 (s, 1H),
5.80 (d, 1H, J = 7.1 Hz), 6.20-6.24 (m, 1H), 6.17-6.23 (m,
1H), 6.35-6.46 (m, 2H), 7.22-7.30 (m, 1H), 7.87-7.92 (br m,
2H) 7.95 (d, 1H, J = 7.1 Hz); 13C NMR (100 MHz, DMSO-
d6 , ꢀ , ppm): 38.5, 61.2, 67.4, 68.2, 86.9, 87.5, 96.5, 120.6,
126.6, 129.5, 142.1, 148.3, 159.6, 164.7; 31P (161.9 MHz,
+
•
ppm): -4.11, -17.21; LCMS (m/z,%): 512 [MH ]; Anal.
Calcd.: C12H20Cl2N4O8P2S: C 28.08, H 3.93, N 10.92;
Found: C 27.86, H 3.90, N 10.82.
[5-(4-Amino-2-oxo-1, 2-dihydro-1-pyrimidinyl)-1,3-oxa-
thiolan-2-yl] methylbis( 2-chloroethyl)amino(2-hydroxy-
ethyloxy)phosphate (5j)
Yield: 72%; m.p.: 171-173 ºC; IR (ꢀmax, cm-1, KBr): 3396
(O-H), 3295 (N-H), 1228 (P=O); 1H NMR (400 MHz,
DMSO-d6 , ꢀ , ppm): 3.10 (dd, 1H, J = 4.5, 11.3 Hz), 3.28 (t,
4H, J = 13.6 Hz), 3.42 (dd, 1H, J = 5.2, J = 11.5 Hz), 3.61 (t,
4H, J = 13.6 Hz), 3.84-3.88 (m, 2H), 4.10-4.16 (m, 2H),
4.21-4.26 (m, 1H), 4.52 (t, 2H, J =13.5 Hz), 5.17-5.20 (m,
1H), 5.80 (d, 1H, J = 7.1 Hz), 6.18-6.24 (m, 1H), 7.85-7.92
(br m, 2H) 7.98 (d, 1H, J = 7.6 Hz); 13C NMR (100 MHz,
DMSO-d6 , ꢀ , ppm): 44.2, 45.5, 62.6, 67.4, 68.8, 74.5, 86.9,
87.5, 96.5, 142.2, 158.3, 165.5; 31P (161.9 MHz, DMSO-d6 ,
+
•
DMSO-d6 ꢀ , ppm): -7.40; LCMS (m/z,%): 463 [MH ];
,
Anal. Calcd.: C16H19ClN3O7PS: C 41.43, H 4.13, N 9.06;
Found: C 41.12, H 4.09, N 8.79.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl]methyl(4-chlorophenyl)(2-aminoethyl amido)
phosphate (5g)
Yield: 80%; m.p.: 114-116 ºC; IR (ꢀmax, cm-1, KBr): 3316
+
•
ꢀ , ppm): -7.11; LCMS (m/z,%): 476 [MH ]; Anal. Calcd.:
C14H23Cl2N4O6PS: C 35.23, H 4.86, N 11.74; Found: C
34.98, H 4.82, N 11.63.
1
(N-H), 1231 (P=O); H NMR (400 MHz, DMSO-d6 ꢀ ,
,
ppm): 3.12 (dd, 1H, J = 4.1, 11.2 Hz), 3.41 (dd, 1H, J = 5.2,
11.4 Hz), 3.58-3.61 (m, 4H), 3.85-3.89 (m, 2H), 4.28-4.32
(m, 1H), 5.16-5.20 (m, 1H), 5.26 (br s, 2H), 5.80 (d, 1H, J =
7.5 Hz), 6.32-6.48 (m, 2H), 6.14-6.21 (m, 1H), 7.16-7.24 (m,
2H), 7.85-7.92 (br m, 2H), 7.98 (d, 1H, J = 7.5Hz); 13C NMR
(100 MHz, DMSO-d6 , ꢀ , ppm): 38.5, 40.2, 44.6, 67.4, 86.9,
87.5, 96.5, 120.6, 126.5, 130.2, 142.8, 147.7, 159.1, 164.7;
31P (161.9 MHz, DMSO-d6 , ꢀ , ppm): -4.10; LCMS (m/z,%):
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl] methylbis (2-chloroethyl amino) (2-amino-
ethylamido) phosphate (5k)
Yield: 78%; m.p.: 112-114 ºC; IR (ꢀmax, cm-1, KBr): 3292
1
(N-H), 1221 (P=O); H NMR (400 MHz, DMSO-d6 ꢀ ,
+
•
,
461 [MH ]; Anal. Calcd.: C16H21ClN5O5PS: C 41.61, H
4.58, N 15.16; Found: C 41.40, H 4.54, N 15.00.
ppm): 3.10 (dd, 1H, J = 4.1, 11.2 Hz), 3.28 (t, 4H, J = 13.6
Hz), 3.40 (dd, 1H, J = 5.2, 11.0 Hz), 3.48-3.51 (m, 4H), 3.60
(t, 4H, J = 13.6 Hz), 3.80-3.82 (m, 2H), 4.20-4.26 (m, 1H),

62 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 1
Rao et al.
5.19-5.21 (m, 1H), 5.28 (br s, 2H), 5.80 (d, 1H, J = 7.2 Hz),
6.20-6.23 (m, 1H), 7.85-7.94 (br m, 2H) 7.98 (d, 1H, J = 7.8
Hz), ; 13C NMR (100 MHz, DMSO-d6 , ꢀ , ppm): 36.2, 38.5,
40.2, 45.1, 67.4, 75.1, 86.9, 87.5, 96.7, 141.2, 158.6, 166.1;
31P (161.9 MHz, DMSO-d6 , ꢀ , ppm): -7.11; LCMS (m/z,%):
COLO-205 cells were collected and resuspended in fresh
culture medium with 10% FBS. Before the treatment with
test compound, cells were washed with PBS and fresh me-
dium was added.
Cell proliferation was assayed by MTT (3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)
assay as described by Mosmann (1983) [16]. Cells (5ꢁ103
cells per well) were incubated in 96-well plates in the pres-
ence or absence of isolated compound for 24 hrs in a final
volume of 100 ꢂL. At the end of the treatment, 20 ꢂL of
MTT (5 mg/mL in PBS) was added to each well and incu-
bated for an additional 4 h at 37 °C. The purple-blue forma-
zan precipitate was dissolved in 100 ꢂL of DMSO and the
optical density was measured at 570 nm on Quant Bio-tek
Instrument, Inc, micro titer plate reader. Data are expressed
as mean ±SE. (n=3) % inhibition. Percentage growths in the
treated samples were calculated with respect to control. The
concentration of the compound that inhibited cell growth by
50% (IC50) was determined from cell survival plots. The
results are summarized in Table 1 and anti - proliferative
activity of active compounds against COLO- 205 cell line
with IC50 values are graphically represented in Fig. (2), and
the values are tabulated in Table 1.
+
•
474 [MH ]; Anal. Calcd.: C14H25Cl2N6O4PS: C 35.38, H
5.30, N 17.68; Found: C 35.23, H 5.25, N 17.56.
[5-(4-Amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxath-
iolan-2-yl]
methylbis(2-chloroethyl)amino(4-methylpi-
perazino)phosphate (5l)
Yield: 72%; m.p.: 171-173 ºC; IR (ꢀmax, cm-1, KBr): 3295
1
(N-H), 1228 (P=O); H NMR (400 MHz, DMSO-d6 ꢀ ,
,
ppm): 2.27 (s, 3H), 2.41-2.43 (m, 4H), 2.81-2.86 (m, 4H),
3.10 (d, 1H, J = 5.4, 11.2 Hz), 3.28 (t, 4H, J = 13.6 Hz), 3.42
(dd, 1H, J = 5.2 Hz, 11.5 Hz), 3.60 (t, 4H, J = 13.5 Hz),
3.76-3.80 (m, 2H), 5.21-5.24 (m, 1H), 5.80 (d, 1H, J = 7.6
Hz), 6.21-6.26 (m, 1H), 7.80-7.84 (br m, 2H), 7.99 (d, 1H, J
= 7.6 Hz); 13C NMR (100 MHz, DMSO-d6 , ꢀ , ppm): 38.5,
43.2, 44.5, 48.7, 56.8, 67.4, 75.4, 86.9, 87.5, 95.5, 141.5,
159.6, 166.5; 31P (161.9 MHz, DMSO-d6 , ꢀ , ppm): -7.11;
+
•
LCMS
(m/z,%):
514
[MH ];
Anal.
Calcd.:
C17H29Cl2N6O4PS. C 39.62, H 5.67, N 16.31; Found: C
39.42, H 5.62, N 16.15.
RESULTS AND DISCUSSION
In vitro Experiments
The synthesis of phosphorylated derivatives of lami-
vudine (Scheme 1) was carried out as follows. 4-Amino-1-
(2-(hydroxymethyl)-1, 3-oxathiolan-5-yl) pyrimidin-2(1H)-
one was treated with 4-nitrophenyl phosphorodichloridate in
the presence of triethylamine (TEA) in tetrahydrofuran
(THF) and pyridine at -10 ºC. The reaction progress was
monitored by thin layer chromatography (TLC) (ethylace-
tate: hexane 1:1) to form the monochloride 3a. Further, the
monochloride was reacted with ethylenediamine/ ethyle-
neglycol/ N-methylpiperazine/ mono potassium dihydrogen
phosphate to get 5a-d in high yields. Similarly the same
The newly synthesized compounds 5a-5l in the present
study were evaluated for their anti-proliferative activity
against COLO-205 cell line by the following procedure. The
COLO-205 cells were grown in suspension in RPMI 1640
medium supplemented with 10% heat inactivated fetal bo-
vine serum (FBS), 100 IU/mL penicillin, 100 mg/mL strep-
tomycin and 2 mM-glutamine. Cultures were maintained in a
humidified atmosphere with 5% CO2 at 37 °C. The cells
were sub cultured twice per week, seeding at a density of
about 2x105 cells/mL. For treatment, exponentially growing
NH2
NH2
NH2
N
N
N
O
P
Cl
Cl
N
Pyridine, THF
O
O
O
R
N
OH
+
O
0 ˚C, 10 hrs
R1-H
O
N
O
-10 ˚C, 2 hrs
O
P
Cl
O
O
P
R1
O
R
R
S
4a-l
S
1
2
S
3
5a-l
5a-d
R= OC6H4NO2
R= OC6H4Cl
R1
Compounds
R1
Compounds
5a, e & i
O
5e-h
5i-l
H
N
5c, g & k
NH2
Me
N(CH2-CH2-Cl)2
O
P
OH
R=
OH
N
N
5b, f & j
O
5d, h & l
OH
Scheme 1. Synthesis of lamivudine (3TC) derivatives 5(a-l).

Design, Synthesis and Anti Colon Cancer Activity Evaluation
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 1 63
NH2
NH2
N
N
N
O
N
O
O
O
O
O
OH
O
P
P
O
O
P
OH
OH
O
O
S
S
O
O
NO2
NO2
5a
5b
Fig. (1). The structures of the potent lamivudine (3TC) phosphorylated derivatives 5a and 5b.
above protocol was employed for the synthesis of 5e-l by
using 4-chlorophenyl phosphorodichloridate/ bis (2-
chloroethyl) phosphoramidicdichloride. The two correspond-
ing intermediate monochlorides (3b and 3c) were further
reacted with mono potassium dihydrogen phosphate, ethyl-
ene glycol, ethylenediamine and N-methyl piperazine in the
presence of TEA to obtain the target compounds in good
yields (69-80%). The purity of compounds was checked by
TLC and elemental analyses, and the compounds of this
study were identified by spectral data. Characteristic IR
stretching absorptions were observed in the regions 1218-
1235 cm-1, 3278-3316 cm-1 and 3388-3415 cm-1 for P=O, N-
parent drug (Lamivudine) for comparison are reported (Table
1). The obtained results revealed that the compounds 5a-5c
exhibited good anti colon cancer activity and compounds 5a
and 5b were identified as the most potent compounds pre-
sented in Fig. (2) (IC50 values 0.003 ꢀM and 0.0001 ꢀM re-
spectively) and exhibited more potent activity than lami-
vudine against COLO-205 cell line. In this series the most
active compounds were 5a, 5b and 5c than the reference
drug against COLO-205 cell lines. 4-Nitro phenyl phos-
phato, 2-hydroxyethyloxy and dihydrogen phosphate con-
taining derivatives in Fig. (1) (compounds 5a & 5b respec-
tively) exhibited promising activities against COLO-205 cell
line (Table 1).
1
H [17] and O-H [17] respectively. In H NMR spectra of 5a-
l, the chemical shifts of aromatic hydrogen atoms of the
phenyl ring were observed as two doublets in the region ꢀ
6.52-7.99 [18]. The N-H proton resonated as a broad mul-
tiplet at ꢀ 7.80-7.94 and P-O-H protons were observed as
singlet at ꢀ 8.10-9.60. 31P-NMR signals of 5a-l were ob-
served in the region -7.11 to -19.39 ppm [19]. 13C NMR
chemical shifts for compounds 5a-l were observed in their
expected regions [20].
All compounds were screened for their in vitro anti colon
cancer activity against COLO-205 cell lines by MTT method
and IC50 values of the synthesized compounds along with the
Table 1. In Vitro Antiproliferative Activity Values of Lami-
vudine Derivatives 5(a-e) Against COLO-205
Cells
Compound
IC50 (μM )
Fig. (2). Growth curves of COLO-205 cells against lamivudine
(3TC) phosphorylated derivatives. Reference compound is lami-
vudine. Data is expressed as mean ±SE. (n=3).
5a
0.003
0.0001
0.31
5b
5c
5d
32
CONCLUSION
5e
>100
>100
>100
>100
>100
>100
>100
>100
0.425
In summary, this work describes the preparation and in
vitro anti colon cancer evaluation of novel phosphorylated
derivatives prepared by reaction of lamivudine with phos-
phorus dichlorides. Three of these compounds (5a, 5b and
5c) were more active than the reference drug against COLO-
205 cell line, other derivatives also displayed moderate ac-
tivities against the COLO-205 cell line. It is conceivable that
these derivatives showing anti-colon cancer activity can be
further modified to exhibit better potency than the standard
drug (3TC). It seems that the anti colon cancer activity de-
5f
5g
5h
5i
5j
5k
5l
Reference (Lamivudine) [3TC]

64 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 1
Rao et al.
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