Synthesis, structure-activity relationship and antiviral activity of 3′-N,N-dimethylamino-2′,3′-dideoxythymidine and its prodrugs

Article abstract of DOI:10.1016/j.ejmech.2010.05.028

A probable NRTI molecule, viz. 3′-N,N-dimethylamino-2′, 3′-dideoxythymidine (4) and its 5′-O-carboxyl ester prodrugs - 5′-(N-α-BOC-l-phenylalanyl)-3′-N,N-dimethylamino-2′, 3′-dideoxythymidine (5), 5′-l-phenylalanyl-3′-N,N- dimethylamino-2′,3′-dideoxythymidine (6) and 5′-decanoyl- 3′-N,N-dimethylamino-2′,3′-dideoxythymidine (7) have been synthesized and screened against HIV, HSV-1 and 2, parainfluenza-3, vesicular stomatitis and several other viruses. The compound 6 showed good antiviral activity with EC₅₀ value 0.03 μM (SI = 8) against VSV in Hela and HEL cell lines. However, the lead compound 4 and its derivatives 5, 6 and 7 showed no remarkable activity against HIV-1 and other viruses. Molecular docking studies with HIV-1 RT using DS 2.5 and pymol softwares have shown marked differences in the interaction patterns between the lead compound 4 and AZT. Synthesis, molecular modeling and antiviral/anti-HIV activity of 3′-N,N-dimethylamino-2′,3′-dideoxythymidine and its prodrugs have been described.

Full text of DOI:10.1016/j.ejmech.2010.05.028

European Journal of Medicinal Chemistry 45 (2010) 3787e3793
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structureeactivity relationship and antiviral activity of 30-N,
N-dimethylamino-20,30-dideoxythymidine and its prodrugsq
,
a
a
a
b
b
Ramendra K. Singh a , Dipti Yadav , Diwakar Rai , Garima Kumari , C. Pannecouque , Erik De Clercq
*
a Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad 211002, India
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
A probable NRTI molecule, viz. 30-N,N-dimethylamino-20,30-dideoxythymidine (4) and its 50-O-carboxyl ester
prodrugs e 50-(N- -phenylalanyl)-30-N,N-dimethylamino-20,30-dideoxythymidine (5), 50-
-BOC- -phenyl-
alanyl-30-N,N-dimethylamino-20,30-dideoxythymidine 50-decanoyl-30-N,N-dimethylamino-
Article history:
Received 4 March 2010
Received in revised form
11 May 2010
Accepted 12 May 2010
Available online 20 May 2010
a
L
L
(6)
and
20,30-dideoxythymidine (7) have been synthesized and screened against HIV, HSV-1 and 2, parainfluenza-3,
vesicularstomatitisand severalotherviruses. Thecompound6 showedgood antiviralactivity withEC50 value
0.03
m
M (SI ¼ 8) against VSV in Hela and HEL cell lines. However, the lead compound 4 and its derivatives 5, 6
and 7 showed no remarkable activity against HIV-1 and other viruses. Molecular docking studies with HIV-1
RT using DS 2.5 and pymol softwares have shown marked differences in the interactionpatterns between the
lead compound 4 and AZT.
Keywords:
Dideoxythymidine
NRTIs
Ó 2010 Elsevier Masson SAS. All rights reserved.
VSV
HIV-1
HIV-1 RT
MTT assay
1. Introduction
toxicity by interacting with human DNA polymerase g [10], present
in mitochondria, which results in mitochondrial dysfunctions. Thus
NRTIs, show many side effects [11e13]. In addition to this, four more
significant obstacles currently limiting the clinical application of
nucleoside analogues are oral absorption, cellular uptake, short half-
life and viral resistance [14]. So, there is a need for new nucleoside
mimics and their prodrugs to overcome the limitations. We have
already reported several nucleosidic [15,16] and non-nucleosidic
molecules [17] with significant antiviral activities.
The NRTIs, by virtue of acting as chain terminators of (ꢀ) strand
DNA synthesis during reverse transcription, have been developed
as potential drugs against HIV/AIDS. In order to explore such new
inhibitors, we report here the synthesis of 30-N,N-dimethylamino-
20,30-dideoxythymidine (DMAT) and its 50-O-ester prodrugs with
amino and fatty acids. The lead molecule, DMAT, was designed
keeping AZT, an approved NRTI drug against HIV/AIDS, in mind,
where azido group has been replaced by dimethylamino function,
Fig. 1. Thus, this molecule being a dideoxynucleoside is expected to
act as NRTI against HIV-1 RT enzyme. Similarly, the prodrugs,
having biodegradable ester linkages, are supposed to enhance the
cellular uptake due to their high lipophilic nature.
HIV-1 reverse transcriptase (HIV-1 RT), a primary target for
developing anti-HIV drugs, is the enzyme responsible for gener-
ating linear dsDNA from ssRNA e the genetic material of HIV-1. This
dsDNA acts as a provirus and leads to viral expression through
integration in the host genome, transcription and translation
processes.
There are several drugs that inhibit HIV-1 RT and they are clas-
sified as nucleos(t)ide RT inhibitors (N(t)RTIs) and non-nucleoside
RT inhibitors (NNRTIs). The NRTIs have emerged as important
therapeutic agents for the development of anti-HIV drugs [1e4]. The
NRTIs after intracellular phosphorylation to their active triphos-
phate form by cellular kinases [5], act as competitive inhibitors with
respect to the dNTP substrates. These molecules when incorporated
by HIV-1 RT in the growing template/primer, cause chain termina-
tion [6,7] since they lack 30-OH group, which is necessary for chain
elongation and thus, they inhibit HIV-1 RT [8,9]. Despite their
tremendous utility, nucleoside analogues may cause serious cellular
q
Work presented in Nucleic Acids Symposium (Series No. 52) held in Japan (Ref.
All these molecules have been evaluated for their activity
against various viruses, like HIV-1, vesicular stomatitis, HSV-1 & 2,
reovirus, parainfluenza virus and many others.
[37]).
* Corresponding author. Tel./fax: þ91 532 2461005.
E-mail address: singhramk@rediffmail.com (R.K. Singh).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.028

3788
R.K. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3787e3793
O
N
compounds was able to inhibit cytopathic effects of Influenza A or B
virus at subtoxic concentrations or the highest concentration
tested.
O
N
HN
O
HN
O
O
It was observed that compounds 6 and 7 only, showed antiviral
properties against VSV (EC50 0.03) and FCV (EC50 0.05), respec-
tively. However, the antiviral potency of the prodrug 6 was still
significantly lower than that of established antiviral drugs e (S)-
DHPA, BVDU and ribavirine against VSV as was evident from its
CC50 and EC50 values shown in Table 1.
Since nucleosides exhibit only modest bioavailability [21], we
prepared the prodrug molecules using amino and fatty acids to
improve their bioavailability [22,23] and chemical stability. This
was done to ensure enhanced cellular uptake and sustained release
of drug molecule without compromising its properties [24e27].
The lead compound 4 and its prodrug 5 did not show any
antiviral activity, whereas the prodrug 7 showed activity, albeit, too
little against FCV and FHV. However, the prodrug 6 showed good
activity against VSV with SI value equal to 8. The enhanced reac-
tivity of free amino acid ester prodrug may be attributed to its
electronic activation by the positively charged amino terminus at
O
R1O
R
2O
N
N
H3C
+N
N-
CH3
DMAT :
prodrugs:
AZT :
R
R
1 = H
R2 = H
R2 = phenylalanyl
2 = decanoyl
prodrugs:
1 = tyrosynyl, lysinyl,
phenylalanyl, valyl etc.
R1 = decanoyl, myristoyl, pamitoyl etc.
R
Fig. 1. Structures of AZT and 30-N,N-dimethylamino-20,30-dideoxythymidine and their
prodrugs.
2. Chemistry
A new thymidine analogue, viz. 30-N,N-dimethylamino-20,30-
dideoxythymidine has been synthesized from thymidine as shown
in Scheme 1. 50-O-(4,40-Dimethoxytrityl)-30-mesylthymidine,
obtained after 50-OH selective protection with DMTCl followed by
mesylation with MsCl, when reacted with potassium phthalimide
witnessed the introduction of nucleophile (phthalimide ion) into
the sugar moiety [18]. During conversion of 1e2, the elimination of
C30-mesyloxy group was effected through intramolecular
displacement by attack of carbonyl group of thymidine at C2, which
resulted in formation of 2,30-anhydronucleoside intermediate. This
result was well in accordance with the literature data. It is sug-
gested that under this condition, phthalimide ion is introduced into
the ‘down’ (ribo) configuration [19]. The 30-phthalimide derivative
2 was treated with methanolic methylamine, which through
methanolysis and aminolysis resulted in formation of compound 3
[20]. Reaction of 3 with formic acid and aqueous formaldehyde
(40%) yielded 4. In this reaction, formaldehyde used was the source
of methyl groups, while formic acid supplied the hydrogens
involved in the reduction. This treatment also removed the DMT
group from compound 3. As expected, the UV absorption spectra of
all intermediates were similar to that of thymidine, while dissimilar
Rf values showed that all these changes took place in the sugar
moiety and not in the aglycon part [19].
physiological pH, which derives support from the fact that L-amino
acid esters enhance nucleoside absorption via human peptide
transporters (hPEPT1) of active transport system [28,29]. Further-
more, since bone marrow progenitor cells lack this active transport
system for amino acid, prodrug 6 is expected to show reduced
toxicity at bone marrow level. Therefore, compound 6 has potential
to be developed as an effective antiviral agent against VSV.
The lead compound 4, being a 20,30-dideoxynucleoside, was
expected to act as chain terminator and thus, interfere with viral
cell metabolism and its prodrugs were designed and synthesized to
enhance its cellular uptake. However, this molecule showed almost
no anti-HIV activity and rather proved cytotoxic. This might be
O
N
O
N
O
N
HN
HN
HN
O
O
O
i
OH
ii
DMTO
DMTO
O
O
O
O
N
H2N
iii
HO
3
1
2
O
O
O
N
3. Results and discussion
HN
HN
O
O
C
O
N
H3C
O
3.1. Antiviral activity
H
N
iv
O
OH
CH
H3C
C O
O
H3C
H2C
All compounds 4e7 have been screened for their antiviral
potential against HIV and several other viruses and the results are
shown in Table 1. The lead compound DMAT and its prodrugs were
found inactive against HIV-1 ROD and HIV-1 IIIB strains and showed
toxicity. Compounds 4 and 5 have showed similar values of CC50 and
EC50 against all viruses studied and thus no activity was observed,
4
N
5
N
H3C
H3C
CH3
CH3
vi
v
O
N
O
N
while 50-
potentially active at an EC50 of 0.03
L-phenylalanyl derivative 6, having free amino function was
HN
HN
mM against VSV in HeLa and HEL
O
C
O
O
O
cell cultures. The EC50 value of compound 6 has been found to be
eightfold lower than its CC50 value (SI ¼ 8). Besides this, it has also
H2N CH
H2C
C
O
CH3(CH2)7CH2
O
O
O
showed moderate activity at an EC50 of around 0.05 mM against HSV-
N
6
N
H3C
H3C
1 & 2, coxsackie B4, and RSV in HeLa cell culture. At the same EC50
value, it was found active against VV, TK HSV, in HEL cell lines; PIV-3,
RV-1, SV and PTV in Vero cell culture and ECV (FIPV) and FHV in CRFK
cell culture. The 50-decanoyl derivative 7 has shown some activity
against FCV (FIPV) and FHV in CRFK cell culture.
7
CH3
CH3
i. DMTCl, MsCl, potassium phthalimide, DMAP, TEA, Pyr; ii. methylamine in
methanol, HCl, 105°C; iii. HCOOH/ HCHO (40%), 70°C; iv. BOC-phenylalanine, DCC,
DMAP, pyr, r.t.; v. TFA/ DCM; Et3N/DCM, r.t.; vi. C9H19COCl, DMAP, pyr, r.t.
All compounds were also tested against influenza A H1N1/H3N2
subtype and Influenza B in MDCK cell lines. However, none of these
Scheme 1. Synthesis of DMAT (4) and its prodrugs (5e7).

Table 1
Antiviral and cytotoxic effects of compounds 4e7.
Compound
Virus
Cell
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
t
HeLa
HeLa
HeLa
HEL
HEL
HEL
HEL
HEL
Vero
Vero
Vero
Vero
Vero
CRFK
CRFK
MDCK
MDCK
MDCK
MTT
MTT
4
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
EC50
CC50
>0.37
>0.37
>0.20
>0.20
0.03
0.24
>0.23
>0.23
146
>250
10
>0.37
>0.37
>0.20
>0.20
>0.05
0.24
>0.23
>0.23
>250
>250
30
>0.37
>0.37
>0.20
>0.20
>0.05
0.24
>0.23
>0.23
146
>250
30
>0.37
>0.37
>0.20
>0.20
>0.05
0.24
>0.23
>0.23
e
>0.37
>0.37
>0.20
>0.20
>0.05
0.24
>0.23
>0.23
e
>0.37
>0.37
>0.20
>0.20
>0.05
0.24
>0.23
>0.23
e
>0.37
>0.37
>0.20
>0.20
0.03
0.24
>0.23
>0.23
e
>0.37
>0.37
>0.20
>0.20
>0.05
0.24
>0.23
>0.23
e
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.23
>0.23
>250
>250
95
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.23
>0.23
>250
>250
250
>250
e
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.23
>0.23
>250
>250
111
>250
e
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.23
>0.23
>250
>250
>250
>250
e
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.23
>0.23
>250
>250
50
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.05
>0.23
e
>0.37
>0.37
>0.20
>0.20
>0.05
>0.24
>0.05
>0.23
e
>0.37
e
>0.37
e
>0.37
e
>0.46
e
>0.46
e
5
>0.20
e
>0.20
e
>0.20
e
>0.16
e
>0.16
e
6
>0.24
e
>0.24
e
>0.24
e
>0.18
e
>0.18
e
7
>0.23
e
>0.23
e
>0.23
e
>0.15
e
>0.15
e
(S)-DHPA
Ribavirin
Brivudine
Ganciclovir
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
10
50
>250
50
>250
0.03
>100
e
85
>250
2
>250
>100
>100
e
>250
>250
>250
>250
>100
>100
e
125
>250
50
>250
0.03
>100
e
e
e
7
9
9
e
e
>250
e
>250
e
>250
e
>250
0.08
>250
0.03
>100
e
>250
e
>250
e
e
e
>100
e
>100
e
>100
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
>100
>100
e
2.6
>100
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Oseltamivir-
carboxylate
Amantidin
e
e
e
e
e
e
e
e
0.07
>100
45
>100
20
1.7
>100
4
>100
0.8
>100
4
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
>100
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
>100
>100
e
e
Rimantidin
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
>100
e
e
a ¼ Vesicular stomatitis virus; b ¼ coxsackie virus B4; c ¼ respiratory syncytial virus; d ¼ herpes simplex virus-1(KOS); e ¼ herpes simplex virus-2 (G); f ¼ vaccinia virus; g ¼ vesicular stomatitis virus; h ¼ herpes simplex virus-1
TK-KOS ACV; i ¼ para-influenza-3 virus; j ¼ reovirus-1; k ¼ sindbis virus; l ¼ coxsakie virus B4; m ¼ punta toro virus; n ¼ feline corona virus (FIPV); o ¼ feline herpes virus; p ¼ influenza A H1N1 subtype; q ¼ influenza A H3N2
subtype; r ¼ influenza B; s ¼ HIV-1 ROD; t ¼ HIV IIIB.
EC50 ¼ compound concentration (in
m
M) required to reduce virus yield by 50%, CC50 ¼ compound concentration (in
mM) required to reduce cell viability by 50%.

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R.K. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3787e3793
Table 2
Drug characteristics of thymidine, AZT and DMAT.
Molecules M.W.
Mol. vol. No. of
No. of
TPSA
log P
H-acceptors H-donors
Thymidine 242.231 207.189
7
9
7
3
2
2
104.557 ꢀ1.433
134.084 ꢀ0.099
87.567 ꢀ0.771
AZT
267.245 224.063
269.301 245.078
DMAT
expected because of introduction of dimethylamino group at 30-C of
ribose moiety, which did not show any interaction with HIV RT
during molecular docking experiments. The lead molecule 4 was
designed because of its similarity with AZT, but it completely failed
in fulfilling the objectives. It was surprising to see that replacement
of azido group by dimethylamino group turned the molecule highly
toxic.
3.2. Molecular docking
The fact that the lead molecule DMAT (and its prodrugs) being
a dideoxynucleoside and having structural features similar to AZT,
did not show any activity against HIV and rather proved toxic,
prompted us to study its interaction with HIV-1 RT.1 We performed
elaborate molecular computational studies using the software
Discovery Studio (DS) 2.5. This molecule showed some striking
differences with AZT e the approved anti-HIV drug as shown in
Table 2. The physiochemical studies suggested that both the
molecules e AZT and DMAT, followed Lipinski0s rule of five but they
have marked differences in their molecular volume, total polar
surface area (TPSA) and log P values. Similarly, the docking exper-
iment also showed quite different patterns of their interaction with
HIV-1 RT (Figs. 2e4). Although DMAT formed one more H-bond
with HIV-1 RT than AZT, the crucial interactions as shown by AZT
were missing in the case of DMAT. The azido function on AZT
formed three H-bonds e one with Arg72 and two with Asp113 (the
amino acid constituting the dNTP-binding site), whereas the
dimethylamino function on DMAT formed no H-bonds at all.
Similarly, the H-bond formed by phosphate moiety of AZT (and TTP
as well) with Val111 was missing in the case of DMAT. However, the
aglycan part in both the molecules formed two H-bonds with Arg72
through oxo group and “O” atom in the sugar ring. The stability of
DMATTPeRT complex was also less than that of AZTTPeRT or
TTPeRT complexes as is evident from their minimization energies.
A comparative account of interaction of TTP, AZTTP and DMATTP
and stability of their respective complexes with HIV-1 RT has been
shown in Table 3. Thus, the physiochemical and docking studies
suggested the possible reasons for inactivity of the lead molecule
and its prodrugs.
Fig. 2. Interaction of TTP with HIV-1 RT at dNTP-binding site.
not sufficient enough criterion and rather it must possess the
characteristic features too for requisite interactions at the dNTP-
binding site of HIV-1 RT enzyme shall definitely help in designing
new NRTI molecules.
5. Experimental
5.1. Chemistry
Chemicals were obtained from E. Merck India Ltd, India and
SigmaeAldrich Chemical Company, USA. Melting points were
determined on electrothermal apparatus and are uncorrected.
4. Conclusion
In conclusion, we have synthesized 30-N,N-dimethylamino-
20,30-dideoxythymidine 4 and its 50-O-carboxyl ester prodrug
derivatives 5e7 and evaluated their antiviral activity against HIV-1
(IIIB and ROD strains), HSV-1/2, vesicular stomatitis, parainfluenza-
3 virus and many others. It was observed that compounds 6 and 7
only, showed moderate antiviral properties against VSV (EC50 0.03)
and FCV (EC50 0.05), respectively. Further mechanistic studies on
molecules 6 and 7 are in progress against vesicular stomatitis, feline
corona and feline herpes viruses.
The inference drawn from the present studies that for a nucle-
oside analogue to be an effective NRTI, being a dideoxy derivative is
1
This facility was not available in the beginning.
Fig. 3. Interaction of AZTTP with HIV-1 RT at dNTP-binding site.

R.K. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3787e3793
3791
evaporator to obtain the crude product. Silica gel column purifi-
cation with a linear gradient up to 10% ethyl acetate in hexane,
afforded the pure compound, 50-O-(4,40-dimethoxytrityl)-30-
mesyl-20,30-dideoxythymidine, which was refluxed for 12 h with
potassium phthalimide (9.0 g) in dimethylformamide (450 ml). The
solvent was removed under reduced pressure and the residue
extracted with ethyl acetate. The ethyl acetate layer was finally
washed with water (3 ꢁ10 ml), dried over anhydrous Na2SO4 and
concentrated to get the title compound in crude form (4.72 g, 70%).
5.1.2. 50-O-(4,40-Dimethoxytrityl)-30-amino-20,30-dideoxythymidine
(3)
The compound 2 (4.34 g) was stirred with methylamine (16 ml)
in methanol at 105 ꢂC for 20 h. The reaction mixture was evapo-
rated to an oily residue, washed with water, dissolved in ethanol
(100 ml) and treated with hydrochloric acid to neutralize the
residual methylamine. The acidic solution was concentrated under
reduced pressure, which resulted in crystallization. The crystals
were separated and the residual water and acids were removed by
co-evaporation with benzene under reduced pressure from the
mother liquor, which was further subjected to crystallization. The
crystalline residue was triturated with ethanol and ether. The title
compound was obtained as colorless crystals (3.04 g, 87%).
5.1.3. 30-N,N-Dimethylamino-20,30-dideoxythymidine (4)
Fig. 4. Interaction of DMATTP with HIV-1 RT at dNTP-binding site.
50-O-(4,40-Dimethoxytrityl)-30-amino-20,30-dideoxythymidine
(3.04 g, 5 mmol) dissolved in a mixture of 98e100% formic acid
(6 ml) and 40% aqueous formaldehyde (6 ml) was refluxed at 70 ꢂC
for 20 min to give light orangish syrup. The reaction mixture was
concentrated under reduced pressure and partitioned between
water and ethyl acetate. Compound 4 (1.15 g, 77%) obtained in
water fraction was crystallized with aqueous ethanol: mp
180e180.5 ꢂC; Rf: 0.17 (DCM:MeOH 9.5:0.5); UV(EtOH) lmax
Silica gel for TLC and column chromatography (60e120 mesh) was
obtained from E. Merck India Ltd. UV measurements were carried
out on Hitachi 220S spectrophotometer. HPLC analyses were per-
formed using 250 ꢁ 4.6 mm2 C18 column and solvent system
MeOH:H2O (6:4) at a flow rate of 1 ml/min on 6 AD Binary Gradient
Shimadzu HPLC system. 1H NMR spectra were recorded on DRX
300 MHz instrument using DMSO-d6 as solvent and TMS as an
internal standard. 13C NMR spectra were recorded in CDCl3 on
a Varian XL-300 spectrometer operating at 75 MHz. Mass spectra
were obtained using a Thermofinnigan TRACE-DSQ Electrospray
Ionization (ESI) mass spectrometer. Elemental analyses were
carried out on a PerkineElmer 240-C analyzer. All solvents were
dried and distilled prior to use.
265 nm; C18 HPLC (265 nm) tR ¼ 1.47 min; 1H NMR (DMSO);
d 1.95
(s, 3H, 5-CH3); 2.15 (m, 2H, H-20); 2.27 (s, 6HeN (CH3)2) ; 2.97
(m,1H, H-30); 3.64 (m, 2H, H-50); 4.08 (m,1H, H40); 5.87 (m, 1H,
H-10); 7.54 (s, 1H, H-6);10.02 (s, 1H, NH, Thy); 13C NMR (CDCl3):
d
15.7, 31.5, 39.8, 55.7, 63.9, 77.2, 78.6, 109.3, 135.8, 152.6, 164.5; ESI-
MS m/z 269 (Mþ). Anal. calcd for C12H19N3O4: C, 53.50; H, 7.06; N,
15.60; found: C, 53.10; H, 6.92; N, 15.50.
5.1.1. 50-O-(4,40-Dimethoxytrityl)-30-phthalimidothymidine (2)
Thymidine (2.415 g, 10 mmol) was suspended in dry pyridine
(w100 ml) in a 250 mL round bottomed flask and added DMAP
(61 mg, 0.05 equivalent), TEA (1.9 ml, 1.4 equivalent) and DMTCl
(4.1 g, 1.2 equivalent) and stirred the reaction mixture for 2.5 h. TLC
at this point showed complete disappearance of thymidine. The
contents were cooled to 0 ꢂC and methanesulfonyl chloride
(1.23 ml, 10.89 mmol) was added dropwise. Stirring was continued
further for 2 h and the reaction mixture was allowed to warm up to
25 ꢂC. After stirring the reaction mixture for an additional 30 min,
the contents were poured into crushed ice/water and the viscous
mass so obtained was extracted in chloroform (50 ml) and washed
with water (2 ꢁ 10 ml). The organic phase was dried over anhy-
5.1.4. 50-(N-
dideoxythymidine (5)
a-BOC-L
-phenylalanyl)-30-N,N-dimethylamino-20,30-
Dicyclohexylcarbodiimide (515 mg, 2.5 mmol) was added to
a
stirred solution consisting of 30-N,N-dimethylamino-20,30-
dideoxythymidine (269 mg,1 mmol), DMAP (183.25 mg,1.5 mmol,)
and N- -t-boc- -phenylalanine (397.95 mg, 1.5 mmol) in pyridine
a
L
(10 ml) under anhydrous condition. The progress of reaction was
monitored by TLC. After the reaction was complete (70e72 h), the
separated DCU was filtered off. The filtrate was evaporated to
dryness under reduced pressure and the title compound was
purified by column chromatography over silica gel using a mixture
of ethyl acetate/hexane (8.5:1.5) as eluent. Yield: (269 mg, 62%); mp
97 ꢂC; Rf : 0.46 (DCM:MeOH 9.5:0.5); UV (EtOAc) lmax 265 nm. C18
HPLC (265 nm) tR ¼ 2.24 min; 1H NMR (DMSO-d6);
d
1.23 (s, 9H, t-
drous sodium sulfate, filtered and evaporated on
a rotary
Table 3
Interaction of Thymidine, AZT and DMAT triphosphates with HIV-1 RT.
Molecule
Total number
of H-bonds
Bonds formed
with PO4 unit
Bonds formed
with C]O on base
Bonds formed
with sugar moiety
Bonds formed with
30 functional group
Minimization
energy (kcal/mol)
TTP
AZTTP
DMATTP
11
13
14
8 Val110, 111
8 Val111,
10 Asp113,
Glu44, Lys46
1 Arg72
1 Arg72
1 Arg72
1 Arg72
1 Arg72
1 Arg72
1 (OH) Asp113
3 (N3) Arg72, Asp113
0 (Ne(CH3)2)
ꢀ61.20318
ꢀ63.93809
ꢀ53.81129

3792
R.K. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3787e3793
boc); 1.95 (s, 3H, 5-CH3); 2.13 (m, 2H, H-20); 2.26 (s, 6H, eN (CH3)2);
2.95 (m,1H, H-30); 3.18 (d, 2H, CH2eamino acid); 3.86 (t, 1H,
CHeamino acid); 4.20 (m, 2H, H-50); 4.58 (m,1H, H-40); 5.85 (m, 1H,
H-10); 7.26e7.08 (m, 5H, Ar); 7.55 (s, 1H, H-6); 10.05 (s, 1H, NH,
adsorption to the cells at 37 ꢂC, the residual virus was replaced by
cell culture medium (Eagle minimum essential medium supple-
mented with 3% fetal calf serum) and various concentrations of the
test compounds. Virus cytopathogenicity was recorded as it
reached completion in the untreated virus-infected cell cultures, i.
e., at 1e2 days for vesicular stomatitis virus; 2 days for coxsackie;
2e3 days for vaccinia, herpes simplex type 1 and 2 and sindbis; 4
days for respiratory syncytial virus and 6e7 days for reo and par-
ainfluenza viruses [33,34]. The antiviral activity of compounds is
Thy); 13C NMR (CDCl3):
d 15.8, 31.6, 33.3, 39.2, 39.7, 55.8, 60.6, 67.2,
74.6, 78.5, 109, 127.8, 128.1, 125.4, 134.8, 140.9, 127.7, 152.2,172; ESI-
MS m/z (Mþ) 488. Anal. calcd for C25H36N4O6: C, 61.46; H, 7.43; N,
11.47; found: C, 61.15; H, 7.30; N, 11.38.
5.1.5. 50-
dideoxythymidine (6)
Compound (150 mg, 0.30 mmol) was added slowly to
L
-Phenylalanyl-30-N,N-dimethylamino-20,30-
expressed as EC50 (the concentration (
induced cytopathogenicity by 50%).
mM) required to inhibit virus-
5
a mixture of 30% TFA and methylene chloride (10 ml). The solution
was stirred under anhydrous condition for 30 min. The solvent was
removed under vacuum and the residue treated with triethylamine
in methylene chloride to afford the title molecule. The product was
purified by silica gel column chromatography using ethyl acetate/
methanol (4:1) as eluent. Yield: (80 mg, 63%) mp 219e220 ꢂC; Rf :
0.29 (DCM:MeOH 9.5:0.5) UV (EtOAc) lmax 265 nm. 1H NMR
5.2.3. Cytotoxicity assays
Cytotoxicity of all compounds was assessed on the basis of two
parameters: (i) alteration of normal cell morphology, and (ii)
inhibition of macromolecule (DNA, RNA and protein) synthesis.
Cytotoxicity (CC50) of compounds was examined by trypan blue
exclusion test. To evaluate cytotoxicity, uninfected confluent cell
cultures treated with various concentrations of test compounds
were incubated in parallel with virus-infected cell cultures
prepared in plastic trays containing 24 wells (16 mm diameter;
Falcon plastics). After 2 days of incubation at 37 ꢂC in a CO2 incu-
bator, when the cell cultures were confluent, culture medium was
removed from each well and 1 ml of maintenance medium con-
taining serial concentrations of the test compounds was added. For
cell control, 1 ml of maintenance medium without compound was
added. All cultures were incubated at 37 ꢂC, and after 2 and 7 days
of incubation, compounds were withdrawn and the viability of the
cells was determined by the trypan blue exclusion method.
(DMSO-d6): d
1.94 (s, 3H, 5-CH3); 2.15 (m, 2H, H-20); 2.26 (s, 6H, eN
(CH3)2); 2.95 (m,1H, H-30); 3.17 (d, 2H, CH2eamino acid); 3.85 (t,1H,
CHeamino acid); 4.22 (m, 2H, H-50); 4.56 (m,1H, H-40); 4.65 (s, 2H,
eNH2); 5.87 (m, 1H, H-10); 7.22e7.18 (m, 5H, Ar); 7.54 (s, 1H, H-
6);10.03 (s, 1H, NH, Thy); 13C NMR (CDCl3):
d 15.2, 31.5, 39.4, 39.7,
55.2, 60.1, 67.2, 74.1, 78.9, 109.2, 125.7, 127.0, 128.7, 134.1, 140.9,
152.3,172.8, ESI-MS m/z (Mþ) 416. Anal. Calcd for C42 H41 N7 O12: C,
60.35; H, 4.91; N, 11.73 found: C, 60.14; H, 4.70; N, 11.61.
5.1.6. 50-Decanoyl-30-N,N-dimethylamino-20,30-dideoxythymidine
(7)
Decanoyl chloride (0.20 ml, 1 mmol) was added dropwise to an
ice-cold stirred solution consisting of the lead compound 4
(269 mg, 1 mmol), DMAP (0.32 g, 2.6 mmol) in pyridine (25 ml) and
the reaction mixture stirred overnight at room temperature under
anhydrous condition. Reaction mixture was concentrated under
reduced pressure and partitioned between ethyl acetate and water.
The organic fraction was concentrated to a residue and the product
was purified by column chromatography over silica gel using ethyl
acetate/hexane (9.5:0.5) as eluent. Yield: (200 mg, 59%) mp 160 ꢂC;
Rf : 0.84 (DCM:MeOH 9.5:0.5); UV (EtOAc) lmax 265 nm, C18 HPLC
5.2.4. Anti-HIV assay
Antiviral screening against HIV-1 (IIIB and ROD strains) was
monitored by the efficiency of test compounds to inhibit syncytia
formation after HIV infection of MT-4 cells following the MTT method
[34e36]. The activity of compounds against HIV-1 was monitored by
inhibition of HIV-1-induced cytopathogenecity in MT-4 cells. Briefly,
MT-4 cells (3 ꢁ 104 cells per well in 96 well plate) were cultured in
microdilution trays in presence of various concentrations of test
compounds added immediately after infection with 50% cell culture
infective doses of HIV-1. After 5 days of incubation at 37 ꢂC, the
number of viable cells was determined by the MTT (30-[4,5-dime-
thylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) method.
(265 nm) tR ¼ 2.84 min; 1H NMR (DMSO-d6)
d 0.97 (t, 3H); 1.29 (m,
10H); 1.33 (q, 2H); 1.68 (m, 2H,); 1.93 (s, 3H, 5-CH3); 2.13 (m, 2H, H-
20); 2.25 (t, 2H,); 2.27 (s, 6H, eN (CH3)2); 2.97(m,1H, H-30); 4.21 (m,
2H, H-50); 4.57 (m,1H, H-40); 5.85 (m, 1H, H-10); 7.57 (s, 1H, H-6);
5.2.5. Molecular modeling study
10.04 (s, 1H, NH, Thy); 13C NMR (CDCl3):
d
14.0, 15.8, 23.8, 25.4, 29.7,
Molecular docking of compound 4 and its prodrugs 5, 6 and 7 into
the dNTP-binding site of HIV-1 RT was carried out using DS 2.5
software and PDB code 3E01 of HIV-1 RT. CDOCKER was used and
flexible docking was done. Stimulated annealing was done to get the
energy-minimized structures of the molecules. The energy-mini-
mized forms were then docked into HIV-1 RT. Visualization was
done using PyMol software. All molecular modeling studies were
performed on an Intel Pentium 2.99 GHz processor, 1.99 GB RAM
with Windows XP professional version 2002 operating system.
30.6, 31.5, 33.6, 39.4, 67.2, 74.9, 109.73, 134.8, 152.5, 164.4, 172.0;
ESI-MS m/z 423 (Mþ) Anal. calcd for C22H37N3O5: C, 62.33; H, 8.73;
N, 9.91; found: C, 62.19; H, 8.51; N, 9.52.
5.2. Biological evaluation
5.2.1. Antiviral assays
Antiviral assays were based on inhibition of virus-induced
cytopathogenicity in various cell cultures following the established
procedures [29e32]. The assays were performed against several
viruses, viz. HIV, herpes simplex virus type 1 (strain KOS), herpes
simplex virus type 2 (strain G), cytomegalovirus (CMV), sindbis virus
(SV), parainfluenza virus type-3 (PIV-3) and reovirus type-3, vesic-
ular stomatitis virus (VSV), coxsackie virus (Coxs V) and respiratory
syncytial virus (RSV) using CRFK, HEL, HeLa, Vero and CD-4 cell lines.
Acknowledgements
Financial assistance from the Department of Biotechnology (DBT)
and Indian Council of Medical Research (ICMR), New Delhi, Govern-
ment of India for carrying out this work is sincerely acknowledged.
5.2.2. Virus cytopathogenecity
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