Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

SYNTHESIS OF PHOSPHORYLATED DERIVATIVES OF RIBAVIRIN AND EVALUATION

OF THEIR CYTOTOXICITY AGAINST LUNG CANCER CELL LINES (NCI-69)
A. Janardhan Rao1,C. Naga Raju1,K. Hema Kumar2
1

Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India.

Aptus Biosciences Private Limited, Yenugonda, Mahabubnagar - 509 002, India.

ARTICLE INFO
Article history
Received 12/12/2014
Available online
30/12/2014

Keywords
Chlorophenyldichlorophosphate,
Monochlorides.
Cytotoxicity.

ABSTRACT
Synthesis of phosphorylated derivatives of ribavirin was accomplished in two steps. In the
first step, 4-chlorophenyl dichlorophosphate was reacted with various amines and alcohols
in equimolar quantities in the presence of triethylamine. These intermediate monochlorides
were further treated with ribavirine (RBV) using Et 3N as a base at 0-30 C in pyridine and
THF. All the title compounds were purified by column chromatography and characterized
by spectral data. Based on the importance of nucleoside in anticancer activity, their
cytotoxicity was evaluated against lung cancer cell lines (NCI-69). Majority of the title
compounds exhibited high activity. These results will be supported further for development
of phosphorylated nucleosides.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as A. Janardhan Rao et al. Synthesis of Phosphorylated Derivatives of Ribavirin and evaluation of
their cytotoxicity against lung cancer cell lines (NCI-69. Indo American Journal of Pharm Research.2014:4(12).

5934

Corresponding author
C. Naga Raju
Department of Chemistry,
Sri Venkateswara University, Tirupati 517 502, India,
rajuchamarthi10@gmail.com,
+919703193375.

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ISSN NO: 2231-6876

INTRODUCTION
In the search for more effective antiviral and antitumor agents, various modifications of nucleotides have been proposed.
Nucleoside analogs (eg. 5-fluoro uracil) are used in anti-cancer, anti-(retro) viral and immunosuppressive therapies. The modified
nucleosides are phosphorylated to nucleotide mono-, di-, and triphosphates intracellularly. The modified triphosphate form inhibits
human and viral polymerases and reverse transcriptases and it is incorporated into nucleic acids [1,2]. The latest antiviral drugs are in
use are emtricitamine and tenofavir. Modified nucleoside triphosphates have received much attention in search for potential
therapeutic and diagnostic agents [3]. Modified nucleosides such as lamivudine (3TC), stavudine (d 4T), zidovudine (AZT) and
ribavirine (RBV) are pro-drugs that need to be phosphorylated to their active 5-triphosphate (TP) form in order to compete with their
natural deoxyribonucleoside triphosphate (dNTP) counterpart for incorporation into the synthesized DNA [4-7].
Ribavirin, a synthetic nonspecific nucleoside analogue [8] and synthesis of 1--D-ribofuranosyl-1, 2, 4-triazole-3carboxamide (ribavirin) was first synthesized by Witkowski [9] and co-workers. Ribavirin is a unique in that as a nucleoside it readily
undergoes facilitated transport into cells. It is then phosphorylated to 1--D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide 5-phosphate
by deoxyadenosine kinase and/or adenosine kinase. Ribavirin 5-diphosphate and the 5-triphosphate have been identified in tissues
from rats given labeled ribavirin orally [10]. The absence of any phosphorylated derivatives in rat serum and urine and the presence of
these metabolites in all tissues examined suggest that phosphorylation occurs within the cell and that the 5-phosphorylated
nucleotides of ribavirin remain within the cell until enzymatic dephosphorylation takes place to the nucleoside which is a major
urinary product [11]. The previous reports from our laboratory and literature reports demonstrate that phosphorylation of drugs will
increase their biological property [12-14].Phosphorylated of nucleosides exposed better antiviral and anticancer results than their
mother nucleosides [22].
Based on these results, in our effort to discover and develop apoptosis inducers as potential new anticancer agents, we
successfully synthesized novel Phosphorylated derivatives of ribavirin and found that these compounds could act as a good antilung
cancer activity.
MATERIALS AND METHODS
Chemicals were procured from Sigma-Aldrich, Merck and Lancaster, and were used as such without further purification. All
solvents used for spectroscopic and other physical studies were reagent grade and were further purified by literature methods [15].
Melting points were determined using a calibrated thermometer by Guna Digital Melting Point apparatus. They are expressed in
degrees centigrade. IR spectra were obtained on a Perkin-Elmer Model 281-B spectrophotometer as KBr disks, absorptions were
reported in cm-1. 1H, 13C and 31P NMR spectra were recorded as solutions in MeOD on a Bruker AVANCE III 500 MHz spectrometer
operating at 500 MHz for 1H, 125 MHz for 13C and 202.4 MHz for 31P NMR. The 1H, 13C and 31P chemical shifts were expressed in
ppm with reference to tetramethylsilane and 85 % H 3PO4 respectively. Mass spectra were recorded on a JEOL GCMATE II Mass
spectrometer. Elemental analyses were performed by Central Drug Research Institute, Lucknow, India.

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sec-Butyl ((2R,3S,4R,5R)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-dihydroxy tetra- hydrofuran-2-yl)methyl 4-chlorophenyl

phosphate (35)
A solution of 2-butanol (0.002 mole), in dry THF (10 mL) and triethylamine(0.002 mole), was added drop wise over a period
of 15 minutes to a stirred solution of 4-chlorophenyl phosphorodichloridate (1) (0.002 mole), in 10 mL of THF at -10-0C. After
stirring for 2 hours, formation of intermediate monochloride (14) was ascertained by TLC eluted in 7:3 mixture of ethyl acetate and
hexane.. To this ribavirin (26) (0.002 mole) in 10 mL of pyridine and triethylamine (0.002 mole), was added drop wise at 0 C. After
completion of the addition, the reaction temperature was slowly raised to 30 C and continued stirring for 8 h. The progress of the
reaction was monitored by TLC eluted in 7:3 mixtures of ethyl acetate and methanol. The reaction mixture was filtered to remove
triethylamine hydrochloride and the solvent was removed in a rotaevaporator. The residue was dissolved in chloroform (10 mL) and
washed with 1M hydrochloric acid solution (2x15 mL), saturated sodium bicarbonate solution (2x10 mL), and then water (3x15 mL).
Then the organic phase was dried (MgSO4) and distilled under vacuum, and the residue was purified by column chromatography on
silica gel by eluting with 5% methanol in chloroform to afford pure product 27. The same procedure was adopted for the preparation
of other compounds28-38.

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O
Cl

O
Cl

R-H (2-13)
-10 0C, THF, TEA

Cl

(1)

Cl

(14-25)

Cl
Et3N.HCl
+
R

O
N

Cl

Cl
R

HO

Cl

THF, TEA,

N
O

R
OH

HO
(14-25)
R

OH
(26)

(27-38)

Compound

Compound

N
O
O

29

NH

N
H

33

N
H

N
H

36

32

Me
N

35

31

27

28

Pyridine 40 oC
HO

Compound

NH2

NH2

37
N

O
30

NH
N

34
N

N
H

38

Scheme-3

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(2 R , 3 S, 4 R, 5 R ) - 5- ( 3 carbamoyl 1 H - 1, 2, 4 triazol 1 yl )- 3, 4 dihydroxytetrahydrofuran 2 yl ) methyl 4chlorophenyl sec-butylphosphoramidate. (28)

MF: C18H25ClN5O7P; Yield: 58%; viscous liquid. Anal. Calcd : C, 44.13; H, 5.14; N, 14.30. Found: C, 44.09; H, 5.10, N, 14.36. IR
(KBr): 3370-3425(-OH,-NH2, NH),1684 (C=O), 1236(P=O) cm-1.1H-NMR (500 MHz, MeOD): d 8.81(s, 1H,H-5), 7.29-7.31(m, 2H,
NH2), 7.23 (d, 2H, J = 8.5Hz, Ar-H), 7.17(d, 2H, J = 9Hz, Ar-H), 6.76 ( d, 1H, J = 9Hz, H-1' ), 5.97(d, 1H, J = 3.5 Hz, OH),
5.93(d, 1H, J = 4 Hz, OH), 4.53-4.51(m, 1H, H-2' ), 4.35-4.37(m, 1H, H-3' ), 4.13-4.15 (m, 1H, H-4'), 3.83-3.85(dd, 1H, H-5') 3.723.76(dd, 1H, H-5'), 3.56-3.61(m, 2H, Bu-CH, Bu-NH), 1.51-1.57(m, 2H, Bu-CH2), 1.24-1.27(d, 3H, Bu-CH3), 0.92-0.95(t, 3H, BuCH3). 31P-NMR (202 MHz, MeOD): -4.92.

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Spectral data for the synthesized compounds

sec-Butyl ((2R,3S,4R,5R)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-dihydroxy tetra- hydrofuran-2-yl)methyl 4-chlorophenyl
phosphate (27)
MF: C18H24ClN4O8P; yield: 61%; viscous liquid. Anal. Calcd: C, 44.05; H, 4.93; N, 11.41.Found C, 44.09; H, 4.98, N, 11.37. IR
(KBr): 3350-3420 (-OH, -NH2), 1685 (C=O), 1231(P=O) cm-1. 1H-NMR (500 MHz, MeOD): d 8.83(s, 1H,H-5), 7.28-7.31(m, 2H,
NH2), 7.22 (d, 2H, J = 8.5Hz, Ar-H), 7.15(d, 2H, J = 9Hz, Ar-H), 6.78 ( d, 1H, J = 9Hz, H-1' ), 5.99(d, 1H, J = 3.5 Hz, OH),
5.95(d, 1H, J = 3.5 Hz, OH), 4.54-4.52(m, 1H, H-2' ), 4.37-4.39(m, 1H, H-3' ), 4.14-4.15 (m, 1H, H-4'), 3.84-3.87(dd, 1H, H-5') 3.713.75(dd, 1H, H-5'), 3.66-3.68(m, 1H, Bu-CH), 1.5-1.56(m, 2H, Bu-CH2), 1.25-1.28(d, 3H, Bu-CH3), 0.9-0.93(t, 3H, Bu-CH3). 13CNMR (125 MHz, MeOD): d161.2(-C=O), 156.16(C-3), 155.34(C-1" ), 151(C-5), 144(C-3" & C-5" ), 128(C-4" ),127.9(C-2" & C-6" ),
115.6(C-1' ), 85( C-4' ), 74.4 (C-aliphtic), 74.1 (C-2' ), 69.5 (C-3' ), 60.7(C-5' ), 29.6(C-aliphtic), 18.8(C-aliphtic), 7.7(C-aliphtic).31PNMR (202 MHz, MeOD): -5.78, -4.59. GCMS (m/z; % ): (491.5, 30%) [MH]+.

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(2 R, 3 S, 4 R, 5R ) 5 - ( 3 carbamoyl 1 H - 1, 2, 4 triazol 1 yl ) - 3, 4 dihydroxytetrahydrofuran 2 yl ) methyl 4chlorophenyl 4-methylpiperazin-1-ylphosphonate (29)

MF: C19H26ClN6O7P; Yield: 63%, semisolid.Anal. Calcd: C, 44.15; H, 5.07; N, 16.26. Found C, 44.11; H, 5.02, N, 16.31. IR
(KBr):3325-3410(-OH, NH2), 1686 (C=O), 1213(P=O) cm-1. 1H-NMR (500 MHz, MeOD): d 8.79(s, 1H,H-5), 7.27-7.3(m, 2H, NH2),
7.25 (d, 2H, J = 9Hz, Ar-H), 7.16(d, 2H, J = 8.5Hz, Ar-H), 6.75 ( d, 1H, J = 9.1Hz, H-1' ), 5.95(d, 1H, J = 4 Hz, OH), 5.91(d, 1H,
J = 3.5 Hz, OH), 4.51-4.49(m, 1H, H-2' ), 4.33-4.35(m, 1H, H-3' ), 4.11-4.14 (m, 1H, H-4'), 3.82-3.84(dd, 1H, H-5'), 3.73-3.76(dd,
1H, H-5'), 2.67(t, 4H, CH2N-), 2.45(t, 4H, CH2N), 2.29(s, 3H, N-Me). 31P-NMR (202 MHz, MeOD): -5.52.
(2R,3S,4R,5R)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl) methyl 4-chlorophenyl piperazin-1ylphosphonate (30)
MF: C18H24ClN6O7P;Yield: 56%; viscous liquid. IR (KBr):3345-3415 (-OH, -NH, NH2),1687 (C=O), 1227(P=O) cm-1. Anal. Calcd :
C, 42.99; H, 4.81; N, 16.71. Found C, 42.95; H, 4.77, N, 16.77. 1H-NMR (500 MHz, MeOD): d 8.78(s, 1H,H-5), 7.29-7.32(m, 2H,
NH2), 7.22 (d, 2H, J = 8.5Hz, Ar-H), 7.15(d, 2H, J = 9Hz, Ar-H), 6.74-6.76 ( d, 1H, J = 9Hz, H-1' ), 5.95(d, 1H, J = 3.5 Hz, OH),
5.91(d, 1H, , J = 2.5 Hz, OH), 4.51-4.49(m, 1H, H-2' ), 4.33-4.35(m, 1H, H-3' ), 4.11-4.14 (m, 1H, H-4'), 3.82-3.84(dd, 1H, H-5'),
3.73-3.76(dd, 1H, H-5'), 2.67(t, 4H, CH2N-), 2.45(t, 4H, CH2N). 31P-NMR (202 MHz, MeOD): -7.59.
(2R,3S,4R,5R)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl) methyl 4-chlorophenyl piperidin-1ylphosphonate (31)
MF: C19H25ClN5O7P; Yield: 62%, Viscous liquid. IR (KBr):3335-3410(-OH, NH2),1685 (C=O), 1219(P=O) cm-1. 1H-NMR (500
MHz, MeOD): d 8.83(s, 1H,H-5), 7.29-7.31(m, 2H, NH2), 7.23 (d, 2H, J = 8.5Hz, Ar-H), 7.17(d, 2H, J = 9Hz, Ar-H), 6.77-6.79 (
d, 1H, J = 9Hz, H-1' ), 5.98 (d, 1H, J = 3.5 Hz, OH), 5.95(m, 1H, J = 4 Hz, OH), 4.52-4.54(m, 1H, H-2' ), 4.37-4.39(m, 1H, H-3' ),
4.14-4.15 (m, 1H, H-4'), 3.84-3.87(dd, 1H, H-5'), 3.72-3.75(dd, 1H, H-5'), 3.17-3.13(m, 4H, CH2N-),1.46-1.56(m, 6H, (CH2 )3 ). 13CNMR (125 MHz, MeOD): d161.5(-C=O), 157.12(C-3), 155.9(C-1" ), 151.7(C-5), 144.8(C-3" & C-5" ), 127.6(C-4" ),128(C-2" & C-6"
), 113.9(C-1' ), 87( C-4' ), 74.1 (C-2' ), 69.5 (C-3' ), 60.7(C-5' ), 49.1 ( pip-C-2, C-6), 25.8( pip-C-3, C-5), 23.5(pipC-4). 31P-NMR (202
MHz, , MeOD): -4.54. GCMS (m/z; % ): (501.5, 30%) [MH]+.
(2R, 3S, 4R, 5R) -5 - (3- carbamoyl 1 H -1, 2, 4-triazol 1 - yl) -3, 4- dihydroxytetrahydrofuran 2 - yl) methyl 4-chlorophenyl
morpholinophosphonate (32)
MF: C18H23ClN5O8P; Yield: 61%, Viscous liquid. Anal. Calcd : C, 42.91; H, 4.60; N, 13.90. Found C, 42.88; H, 4.56, N, 13.96.IR
(KBr): 3362-3430(-OH, NH2),1687 (C=O), 1227(P=O) cm-1.1H-NMR (500 MHz, MeOD): d 8.86(s, 1H,H-5), 7.28-7.32(m, 2H, NH2),
7.24 (d, 2H, J = 8.5Hz, Ar-H), 7.15(d, 2H, J = 9Hz, Ar-H), 6.77 ( d, 1H, J = 9Hz ,H-1' ), 5.97 (m, 1H, J = 3.5 Hz , OH), 5.935.94(d, 1H, J = 3.5 Hz, OH), 4.51-4.53(d, 1H, H-2' ), 4.35-4.37(m, 1H, H-3' ), 4.12-4.13 (m, 1H, H-4'), 3.84-3.87(m, 2H, H-5'), 3.66(t,
4H, CH2-O), 3.6 (t, 4H, CH2N). 31P-NMR (202 MHz, MeOD): 12.5.
(2 R, 3 S, 4 R, 5 R ) 5 - ( 3 carbamoyl 1 H - 1, 2, 4 - triazol 1 - yl ) - 3, 4 dihydroxytetrahydrofuran 2 yl ) methyl 4chlorophenyl 1-phenylethylphosphoramidate(33)
MF: C22H25ClN5O7P; Yield: 58%, viscous liquid.Anal. Calcd: C, 49.12; H, 4.68; N, 13.02. Found C, 49.15; H, 4.73, N, 12.97. IR
(KBr):3360- 3410(-OH, NH, NH2),1683 (C=O), 1230(P=O) cm-1.1H-NMR (500 MHz, MeOD): d 8.84(s, 1H,H-5), 7.3-7.33 (m, 2H,
NH2), 7.17-7.26(m, 5H, Ar-H), 7.09(d, 2H, J = 8Hz, Ar-H), 7.02(d, 2H, J = 9 Hz, Ar-H), 6.75 ( d, 1H, J = 9.1Hz, H-1' ), 5.96 (d, 1H,
J = 3.5 Hz , OH), 5.93(d, 1H, J = 4 Hz, OH), 4.87(s,1H, Ar-CH-NH ), 4.51-4.53(m, 1H, H-2' ), 4.35-4.37(m, 1H, H-3' ), 4.12-4.13
(m, 1H, H-4'), 3.98-4.04 (m, 1H, Ar-CH-NH ), 3.84-3.87(m, 2H, H-5'), 1.64-1.66 (d, 3H, J = 8Hz , -CH3). 31P-NMR (202 MHz, ,
MeOD): 18.7.

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(2 R, 3 S, 4 R, 5 R ) 5 - ( 3 - carbamoyl 1 H - 1, 2 , 4 triazol 1 yl ) - 3, 4 dihydroxytetrahydrofuran 2 yl ) methyl 4chlorophenyl thiophen-2-ylmethylphosphoramidate(35)

MF: C19H21ClN5O7PS;Yield: 57%, viscous liquid. IR (KBr): 3345-3410(-OH,-NH, NH2),1683 (C=O), 1227(P=O) cm-1. 1H-NMR
(500 MHz, MeOD): d 8.86(s, 1H, H-5), 7.53(bs, 2H, NH2), 7.28-7.37 (m, 5H, Thio-H, Ar-H), 7.23(d, 2H, J = 9Hz, Ar-H), 6.28 ( d,
1H, J = 9Hz,H-1' ), 5.95 (d, 1H, OH), 5.54(d, 1H, J = 3.5 Hz, OH), 4.47-4.49(m, 1H, H-2' ), 4.32-4.34(m, 1H, H-3' ), 4.15-4.17 (m,
1H, H-4'), 3.85-3.83(dd, 1H, H-5'), 3.72-3.74(dd, 1H, H-5'), 3.15-3.18(m, 3H, Thio-CH2, Thio-NH). 13C-NMR (125 MHz, MeOD):
d161.9(-C=O), 156.9(C-3), 145.1.(Thio-C-2), 144.5(C-1" ), 143(C-5), 142.4(Thio -C-5), 128.8 (C-3" & C-5" ), 127.6 (C-4" ),

5937

(2 R, 3 S, 4 R, 5 R ) 5 - ( 3 carbamoyl 1 H - 1, 2, 4 triazol 1 yl ) - 3, 4 dihydroxytetrahydrofuran 2 yl ) methyl 4chlorophenyl pyridin-3-ylmethylphosphoramidate(34)

MF: C20H22ClN6O7P; Yield: 61%,viscous liquid.Anal. Calcd: C, 45.77; H, 4.22; N, 16.01. Found C, 45.82; H, 4.26, N, 15.97. IR
(KBr):3320-3410(-OH, NH, NH2),1687 (C=O), 1215(P=O) cm-1. 1H-NMR (500 MHz, MeOD): d 8.9(s, 1H, H-5), 8.57 (s, 1H, Py-H),
7.93-8.14 (m, 3H, Py-H ), 7.29-7.31(m, 2H, NH2), 7.18-7.26(m, 4H, Ar-H), 6.73 ( d, 1H, J = 9Hz, H-1' ), 5.97 (d, 1H, J = 3.5 Hz,
OH), 5.94(d, 1H, J = 3.5 Hz , OH), 5.3 (s, 1H, PyCH2-NH) 4.49-4.51(m, 1H, H-2' ), 4.33-4.35(m, 1H, H-3' ), 4.13-4.15 (m, 1H, H-4'),
4.03-4.07(m, 2H, Py-CH2), 3.85-3.88 (m, 2H, H-5'). 13C-NMR (125 MHz, MeOD): d163.3(-C=O), 156.2(C-3), 152.2(Py-C-2),
149.4(Py-C-6), 149.1(C-1" ), 144(C-5), 137.9(Py-C-3), 137.3(Py-C-4), 129.2(C-3" & C-5"), 128.8(C-4" ), 127.5 (C-2" & C-6" ),
124.2(Py-C-5), 96.1.(C-1' ), 87.5( C-4' ), 75.5 (C-2' ), 70.6 (C-3' ), 63.5(C-5' ), 38.5(Py-CH2). 31P-NMR (202 MHz, MeOD): 19.5.

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123.5(C-2" & C-6" ), 116.4(Fu-C-4), 115.2 (Fu-C-3), 92.7(C-1' ), 85.8( C-4' ), 75.2 (C-2' ), 70.3 (C-3' ), 61.4(C-5' ), 35.5(Fu-CH2).
31
P-NMR (202 MHz, MeOD): -14.72. GCMS (m/z; % ): (530.5, 25%) [MH]+.
(2 R , 3 S, 4 R, 5 R )- 5 - (3 carbamoyl 1 H - 1, 2 ,4 triazol 1 - yl) -3, 4 dihydroxytetrahydrofuran 2 -yl) methyl 4chlorophenyl furan-2-ylmethylphosphoramidate(36)
MF: C19H21ClN5O8P; Yield: 51%,viscous liquid.Anal. Calcd:C, 44.41; H, 4.12; Cl, N, 13.63. Found C, 44.35; H, 4.16, N, 13.72. IR
(KBr):3330-3424(-OH, NH, NH2),1682 (C=O), 1225(P=O) cm-1.1H-NMR (500 MHz, MeOD): d 8.78(s, 1H, H-5), 7.55(bs, 2H, NH2),
7.16-7.23 (m, 7H, Fu-H, Ar-H), 6.52 ( d, 1H, J = 9.1Hz H-1' ), 5.93 (s, 1H, OH), 5.5(s, 1H, OH), 4.5(s, 1H, H-2' ), 4.36 (s, 1H, H-3' ),
4.13 (s, 1H, H-4'), 3.83(d, 1H, J = 11.5 Hz, H-5'), 3.71(d, 1H, J = 12 Hz H-5'), 3.16-3.18(m, 3H, Fu-CH2, Fu-NH). 13C-NMR (125
MHz, MeOD): d163.3(-C=O), 158.2(C-3), 148.4.(Fu-C-2), 146.4.(C-1" ), 145(C-5), 141.4(Fu-C-5), 129..9 (C-3" & C-5" ), 129.4 (C4" ), 123.4.(C-2" & C-6" ), 111.9(Fu-C-4) 111.2 (Fu-C-3), 94.(C-1' ), 87.1( C-4' ), 76.5 (C-2' ), 71.6 (C-3' ), 62.8(C-5' ), 36.8(Fu-CH2).
31
P-NMR(202 MHz, MeOD):-17.2. GCMS (m/z; % ): (514.96, 17%) [MH]+.
(2 R, 3 S, 4 R, 5 R) 5 - (3 carbamoyl 1 H - 1, 2, 4- triazol 1 - yl) - 3, 4 dihydroxytetrahydrofuran 2 yl ) methyl 4chlorophenyl pyridin-3-yl phosphate(37)
MF: C19H19ClN5O8P;Yield: 57%; viscous liquid. Anal. Calcd: C, 44.59; H, 3.74; N, 13.68. Found C, 44.65; H, 3.78, N, 13.61. IR
(KBr)3335-3410(-OH.-NH2),1685 (C=O), 1231(P=O) cm-1.1H-NMR (500 MHz, MeOD): d 8.74(s, 1H, H-5),8.11 (s, 1H, Py-H), 7.858.05 (m, 3H, Py-H ),7.28-7.31(m, 2H, NH2),7.25 (d, 2H, J = 9Hz, Ar-H), 7.18 (d, 2H, J = 9.5Hz, Ar-H), 6.71 ( d, 1H, J = 9Hz, H-1'
), 5.95 (d, 1H, J = 3.5 Hz , OH), 5.97(d, 1H, J = 4 Hz, OH),), 4.43-4.45(m, 1H, H-2' ), 4.31-4.33(m, 1H, H-3' ), 4.13-4.15 (m, 1H,
H-4'), 3.83-3.88 (m, 2H, H-5').31P-NMR (202 MHz, MeOD): 20.8.
(2 R, 3 S, 4 R, 5 R ) 5 - ( 3 carbamoyl 1 H - 1, 2, 4 triazol 1 - yl) - 3, 4 dihydroxytetrahydrofuran -2- yl ) methyl 4chlorophenyl 1-phenylethyl phosphate(38)
MF: C22H24ClN4O8P ; Yield: 62%, viscous liquid. IR (KBr):3340-3410(-OH,-NH2 ),1687(C=O), 1227(P=O) cm-1. 1H-NMR (500
MHz, MeOD): d 8.76(s, 1H, H-5), 7.29-7.32 (m, 2H, NH2), 7.15-7.24(m, 5H, Ar-H), 7.1(d, 2H, J = 8Hz, Ar-H), 7.04(d, 2H, J = 9
Hz, Ar-H) 6.72 ( d, 1H, J = 9Hz, H-1' ), 5.94 (d, 1H, J = 3.5 Hz,OH), 5.96(d, 1H, , J = 4 Hz, OH), 4.41-4.43(m, 1H, H-2' ), 4.324.34(m, 1H, H-3' ), 4.14-4.16 (m, 1H, H-4'), 4.09-4.12(m, 1H, Ar-CH), 3.81-3.86 (m, 2H, H-5'), 1.62-1.64 (d, 3H, J = 8Hz , -CH3).
31
P-NMR (202 MHz, MeOD): 23.4. GCMS (m/z; % ): (539.05, 20%) [MH]+.

Cytotoxicity against lung cancer cell lines (NCI-69)

Compounds 29, 30, 31, 32, 34, 35, 36, 37 and 38 showed increased activity with the increase in concentration and exhibited
good activity in 20 g/mL concentration (Table 1 and Table 2). All the compounds showed better activity than that of Ribavirin.
Compounds 26, 27, 28 and 33 showed significant increase in the cell viability in all concentrations when compared with Positive

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RESULTS AND DISCUSSION

Various amines and alcohols (2-13) in dry THF (10 mL) was reacted with 4-chlorophenyl phosphorodichloridate (1) in 10
mL of THF at -10 C using ET3N as base, and stirred for 2h at 0 C to obtain intermediate monochlorides (14-25). To this ribavirin
(26) in 10 mL of pyridine was added at 0 C in the presence of Et3N. After completion of the addition, the reaction temperature was
slowly raised to 30 C later the reaction mixture was slowly warmed to 40 C and continued stirring for 8-10 h to afford title
compounds (27-38).
All the compounds exhibited characteristic infrared absorption bands for P=O, C=O, OH and NH in the regions 12131231,1682-1687and 3325-3430 cm-1 respectively [17,18].The signal due to C-5 protons in triazole ring were observed as a singlet [19, 20]
in the region 8.74-8.9 and the -NH2 protons appeared as a multiplet and broad singlets [21-24] in the region 7.28-7.55ppm. The 13C
NMR spectral data for 27, 31, 34, 35 and 36 are given in the experimental section. The chemical shifts of the amide carbonyl carbon
atom resonated at 161.2-163.3 [11]. The31P NMR chemical shifts are appeared in the region -17.23 to 23.4 [25-27].

5938

Trypan blue assay

Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is
based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the
visualization of cell morphology. So this method is choosed for screening of Cytotoxicity evaluation of title compounds.
Trypan blue solution was mixed with cell sample solution [16]. Clean hemacytometer and coverslip with 70% isopropanol and
blot dry with kimwipe. Hemacytometer and coverslip were examined under microscope to verify that they are clean and that no cells
are adhering to the surfaces. Culture sample was mixed to resuspend cells. Remove 20 L of culture sample and dispense into a
microfuge tube.
20 uL of 0.4% Trypan blue solution was added to the same tube. The above solution was mixed by gently aspirating and dispensing
the solution with the micropipette. The sample was transferred to hemacytometer, center the coverslip on top of the hemacytometer.
The metal notches should be partially exposed. 10 L of the cell culture/Trypan blue mixture was aspirated. The micropipettes hold
straight up and dispense 10 L of the cell/Trypan blue solution into notch of the hemacytometer. The tip of the pipette should be very
close to the metal surface. The solution spread through capillary action. Cells were observed under the microscope turn on the
microscope per microscope SOP. Hemacytometer was placed on microscope stage. It was focused on the hemacytometer grid using
100X magnification (10X objective lens). Live cells are clear. Dead cells are blue.

Vol 4, Issue 12, 2014.

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control (Doxorubicin hydrochloride) i.e. less activity. Compounds 29, 30, 31, 34, 35, 36 and 37 exhibited high activity in 20 g/mL
concentration (Table 3 & Table 4). Compounds 30 and 31 exhibited potent anti-cancer activity when camped with the other
compounds including ribavirin (Table 5).
Table 1: Viable cells in triplicates.
PC
5 g/mL

Conc

NC

Rb

27

28

29

30

31

32

33

34

313 212 200 193 191 181 170 180 187 183
309 220 198 190 188 175 172 178 184 187
148
311 212 196 189 185 177 174 175 184 180
300 210 196 186 179 168 165 170 181 170
5 g/mL
303 207 193 183 173 166 162 170 178 169
307 203 190 178 170 162 166 166 181 170
151
302 201 188 174 164 154 159 165 179 166
10 g/mL 310 198 182 170 162 158 153 163 178 166
306 197 186 171 166 151 150 164 180 164
308 190 186 165 151 150 150 159 175 155
155
20 g/mL 307 193 182 166 149 144 143 160 173 158
314 190 180 160 144 140 142 157 169 150
Key: PC = Positve control (Doxorubicin hydrochloride), Conc: Concentrations, NC = Negative
Compounds 27 to 38.
1 g/mL

35

36

37

175 179 175

172 174 171
171 177 170
167 171 170
168 173 168
166 173 164
160 169 163
156 166 160
159 164 162
152 152 158
153 156 154
150 151 153
control (DMSO),

38
173
183
178
172
170
175
170
166
162
156
152
151
Rb = Ribavirin,

Table 2: Viable cells count in Mean and standard deviation.

Con
1
5
10
20

Mn
SD
Mn
SD
Mn

PC
----151.3
3.51
---

NC
311c
2
303.3c
3.51
306 c

Rb
214.6c
4.61
206.6c
3.51
198.6c

27
198 c
2
193 c
3
185.3c

28
190.6c
2.08
182.3c
4.04
171.6c

29
188c
3
174c
4.58
164b

30
177.6c
3.05
165.3b
3.05
154.3

31
172 c
2
164.3b
2.08
154

32
177.6c
2.51
168.6c
2.30
164 c

33
185 c
1.73
180 c
1.73
179 c

34
183.3c
3.51
169.6c
0.57
165.3c

35
172.6c
2.08
167 c
1
158.3a

36
176.6c
2.51
172.3c
1.15
166.3c

37
172 c
2.64
167.3c
3.05
161.6b

38
178 c
5
172.3c
2.51
166 c

SD

---

Mn

---

4
309.6c

2.08
191 c

3.05
182.6c

2.08
163.6a

2
148

3.51
144.6

4.58
145

1
158.6

1
172.3c

1.15
154.3

2.08
151.6

2.51
153

1.52
155

4
153

SD

---

3.78

1.73

3.05

3.21

3.60

5.03

4.35

1.52

3.05

4.04

1.52

2.64

2.64

2.64

Key: PC = Positve control (Doxorubicin hydrochloride), Conc: Concentrations (g/mL), NC = Negative control (DMSO), Rb =
Ribavirin, Compounds 27 to 38, Mn: mean, SD: Standard deviation.
P values are a = P<0.05, b = P<0.01, c = P<0.001.
Table 3: Dead cells in triplicates.
Conc

NC

Rb

27

28

29

31

32

33

34

124 128 119 121 122

126 127 116 124 125
129 131 113 120 123
131 137 123 127 129
133 133 127 129 131
138 137 122 130 129
140 139 135 133 133
141 141 133 131 137
143 144 137 129 137
147 149 140 135 145
145 147 144 137 144
148 146 142 134 147
Concentrations, NC = Negative

35

36

37

127 121 125

129 120 127
126 119 123
131 129 129
132 130 127
135 131 129
139 131 137
137 133 138
141 135 135
148 145 141
149 147 139
147 146 140
control (DMSO),

38
121
124
120
120
126
128
131
134
135
144
142
142
Rb = Ribavirin,

Page

26 93
102 104 109
22 97
100 107 111
141
27 92
105 103 114
20 97
111 113 119
5 g/mL
18 95
109 117 120
22 97
109 110 123
139
24 99
113 112 132
10 g/mL 22 101 109 121 135
25 98
115 118 137
21 109 119 123 139
131
20 g/mL 21 102 121 126 141
23 107 123 127 144
Key: PC = Positve control (Doxorubicin hydrochloride), Conc:
Compounds 27 to 38.
1 g/mL

30

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PC
5 g/mL

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Vol 4, Issue 12, 2014.

A. Janardhan Rao et al.

ISSN NO: 2231-6876

Table 4: Dead cells count in Mean and standard deviation.

Con

PC
NC
Rb
27
28
29
30
31
32
33
34
35
36
37
Mn --25c
94c
102.3c 104.6c 111.3c 126.3b 128.6 116c 121.6c 123.3c 127.3b 120c 125c
1
SD --2.64 2.64 2.51
2.08
2.51
2.51
2.08
3
2.08
1.52
1.52
1
2
Mn 137 20c
96.3c 109.6c 113.3c 120.6c 134
135.6 124c 128.6a 129.6 132.6 130 128.3a
5
SD 5.29 2
1.15 1.15
3.51
2.08
3.60
2.30
2.64 1.52
1.15
2.08
1
1.15
Mn --23.6c 99.3c 112.3c 117c
134.6 141.3 141.3 135 131
135.6 139
133 136.6
10
SD --1.52 1.52 3.05
4.58
2.51
1.52
2.51
2
2
2.30
2
2
1.52
Mn --21.6c 106c 121c
125.3b 141.3 146.6a 147.3b 142c 135.3c 145.3c 148c
146c 140c
20
SD --1.15 3.60 2
2.08
2.51
1.52
1.52
2
1.52
1.52
1
1
1
Key: PC = Positve control (Doxorubicin hydrochloride), Conc: Concentrations (g/mL), NC = Negative control (DMSO),
Ribavirin, Compounds 27 to 38, Mn: mean, SD: Standard deviation.
Percentage of Viability was calculated by the following formula:
Viable cell count / Total cell count x 100

38
121.6c
2.08
124.6b
4.16
133.3
2.08
142.6c
1.15
Rb =

Table 5: Percentage of Viabilityin Mean and standard deviation.

Con PC
NC
Rb
27
28
29
30
31
32
1
--92.55 69.53 65.93 64.56 62.81 60.49 57.21 60.49
5
52.48 93.81 68.20 63.78 61.67 59.06 55.22 54.78 57.62
10
--92.83 66.66 62.26 59.45 54.92 52.19 52.15 54.84
20
--93.47 64.30 60.14 56.62 51.15 49.65 49.60 52.76
Key: PC = Positve control (Doxorubicin hydrochloride), Conc: Concentrations
Ribavirin, Compounds 27 to 38, Mn: mean, SD: Standard deviation.

33
34
60.33 59.78
58.32 56.68
57.74 54.93
56.01 51.50
(g/mL), NC =

35
36
37
38
57.55 59.54 57.91 59.41
55.74 56.99 56.96 58.03
53.24 55.56 54.19 55.46
50.60 51.17 52.54 51.57
Negative control (DMSO), Rb =

CONCLUSION
We have synthesized a series of novel bioactive phosphorylated derivatives of Ribavirine and their structures were established by
spectral data. Their cytotoxicity was assayed in vitro against NCI-69 lung cancer cell lines. We conclude that the compounds 29, 30,
31, 32, 34, 35, 36, 37 and 38 showed increased activity with the increase in concentration and exhibited good activity in 20 g/mL
concentrations. All the compounds showed better activity than that of Ribavirin. Compounds 26, 27, 28 and 33 showed significant
increase in the cell viability in all concentrations when compared with Positive control (Doxorubicin hydrochloride) i.e. less activity.
Compounds 29, 30, 31, 34, 35, 36 and 37 exhibited better activity in 20 g/mL concentration. Compounds 30 and 31 exhibited potent
anti-cancer activity (Table 1 to Table 5)). Their potency may be further established by in vivo study. These results will be supported
further for development of phosphorylated nucleosides.

Page

5940

ACKNOWLEDGEMENTS
The authors express their grateful thanks to BRNS (DAE) research project (2012/37C/21/BRNS/785), Mumbai, India for
providing financial assistance throughout the project.

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