Phosphorylated ganciclovir derivatives: Design, synthesis and in vitro and in vivo immunomodulatory activity

Article abstract of DOI:10.1007/s00044-013-0819-6

Synthesis of a series of novel phosphorylated ganciclovir derivatives with bioactive amines/aminoacid esters as substituents was accomplished. These compounds were structurally characterized by IR, NMR (¹H, ¹³C, ³¹P), mass spectra and CHN analysis. The compounds (5a-m) have been evaluated for its in vitro and in vivo immunomodulatory activities. The amino acid ester-substituted ganciclovir derivatives, especially the 5g and 5i, increased the intracellular killing activity of the stimulated neutrophils. The in vivo experiment results show that the administration of compounds 5g and 5i (8 mg/kg body weight) to diet-induced immune impaired obese rats ameliorated the significant increase in immune cell counts (lymphocytes, neutrophils and monocytes). In addition, TNF-α, IL-6 and IL-1 secretions were considerably restored to normal by the compounds 5g and 5i with regulation in the release of C-reactive protein suggesting their potentiating effect on immune system dysfunction. These in vitro and in vivo results suggest that the phosphorylated ganciclovir derivatives 5g and 5i are strong immunomodulators. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0819-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2242–2251
DOI 10.1007/s00044-013-0819-6
ORIGINAL RESEARCH
Phosphorylated ganciclovir derivatives: design, synthesis
and in vitro and in vivo immunomodulatory activity
•
Kuruva Chandra Sekhar Devineni Subba Rao
•
•
Kalla Chandra Mouli Tartte Vijaya
•
Chamarthi Naga Raju
Received: 13 July 2013 / Accepted: 1 October 2013 / Published online: 15 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Synthesis of a series of novel phosphorylated
ganciclovir derivatives with bioactive amines/aminoacid
esters as substituents was accomplished. These compounds
were structurally characterized by IR, NMR (1H, 13C, 31P),
mass spectra and CHN analysis. The compounds (5a–
m) have been evaluated for its in vitro and in vivo
immunomodulatory activities. The amino acid ester-
substituted ganciclovir derivatives, especially the 5g and
5i, increased the intracellular killing activity of the stimu-
lated neutrophils. The in vivo experiment results show that
the administration of compounds 5g and 5i (8 mg/kg body
weight) to diet-induced immune impaired obese rats ame-
liorated the significant increase in immune cell counts
(lymphocytes, neutrophils and monocytes). In addition,
TNF-a, IL-6 and IL-1 secretions were considerably restored
to normal by the compounds 5g and 5i with regulation in the
release of C-reactive protein suggesting their potentiating
effect on immune system dysfunction. These in vitro and
in vivo results suggest that the phosphorylated ganciclovir
derivatives 5g and 5i are strong immunomodulators.
In vitro nitroblue tetrazolium test ꢀ Immune cell counts ꢀ
Immune modulating biofactors
Introduction
Ganciclovir is a guanine containing acyclic nucleoside pro-
drug (Cytovene, RxList) that is activated by phosphoryla-
tion (Nichols and Boeckh, 2000). This undergoes trip-
hosphorylation to become active, with the initial
monophosphorylation catalyzed by UL97-encoded kinase
and subsequently by cellular kinases (Raymund, 2011).
Ganciclovir triphosphate inhibits viral deoxyribonucleic
acid (DNA) synthesis through competitive incorporation
during viral DNA synthesis, thereby leading to DNA chain
termination (Karen, 2006). It has long been used as anti-
viral medication with the success in treating cytomegalo-
virus, human herpes virus infections, even it is also
effective against pneumonia, encephalitis and gastrointes-
tinal diseases (Meyers, 1991; Jeffery et al., 2011).
Viral infections such as herpes, cytomegali and human
immunodeficiency viruses have substantial impact on host
immune responses (Chereshnev et al., 2013; Brennan, 2001;
Lorenzo et al., 2013). Following infection, viruses infiltrate
the cell and produce immediate-early antigens that regulate
DNA production. During the ensuing, the virus also produces
late antigens that direct nucleocapsid protein production. It
also causes up-regulation of (interleukin) IL-2, the master
regulatory molecules of immune response and down regu-
lation of major histocompatibility complex (MHC)-1 mole-
cules on the surface of infected cells to evade host immune
recognition (Brennan, 2001). The initial promise of antiviral
agents has been offset by the development of antiviral
resistance in specific populations, such as immune compro-
mised hosts (McHutchison et al., 1998).
Keywords Phosphorylated ganciclovir derivatives ꢀ
In vivo immunomodulatory activity ꢀ
K. C. Sekhar ꢀ D. S. Rao ꢀ C. Naga Raju (&)
Department of Chemistry, Sri Venkateswara University,
Tirupati 517502, Andhra Pradesh, India
e-mail: rajuchamarthi10@gmail.com
K. C. Mouli
Department of Biotechnology, Sri Venkateswara University,
Tirupati 517502, Andhra Pradesh, India
T. Vijaya
Department of Botany, Sri Venkateswara University,
Tirupati 517502, Andhra Pradesh, India
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Med Chem Res (2014) 23:2242–2251
2243
In addition, for many viruses effective antivirals are not
available. Augmenting the host’s natural immune response
to viruses by the supplementation of immune modulating
molecules is, however, an alternative approach in the
antiviral therapy (Dockrell and Kinghorn, 2001). Hence, it
is imperative for the treatment of active viral diseases with
a combination of immunomodulation and antiviral therapy
(Blair and Emily, 2008).
tetramethylguanidine (TMG) (0.002 mol) as a base. After
completion of the addition, the reaction mixture was stirred
for 2 h at 0 °C to offord an intermediate, 2-amino-9-([2-
(chloromethyl)-2-oxo-1,3,2k5-dioxaphosphinan-5-yl]oxym-
ethyl)-6,9-dihydro-1H-6-purinone (3). The reaction pro-
gress was monitored by thin layer chromatography (TLC)
using methanol:ethyl acetate (1:1). After completion of the
reaction, it was filtered to remove TMGꢀHCl. Ethane-1,2-
diamine (4a) (0.001 mol) in dry THF (10 mL) was added
dropwise to the filtrate (3) at -10 °C and stirring was
continued for 4 h at 50 °C. The progress of the reaction
was monitored by TLC using methanol:ethyl acetate (1:1).
After completion of the reaction, it was filtered to remove
TMGꢀHCl, and the filtrate was concentrated in a rota-
evaporator to obtain crude product. The residue was dissolved
in chloroform (10 mL) and washed with 1 M hydrochloric
acid solution (2 9 15 mL), saturated sodium bicarbonate
(2 9 10 mL), and then water (3 9 15 mL). Then the organic
phase was dried over 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 of 2-amino-9-[(2-[(2-aminoethyl)amino]methyl-
2-oxo-1,3,2k5-dioxaphosphinan-5-yl)oxy]methyl-6,9-dihydro-
1H-6-purinone with high yield, 79 %.
The previous findings in our laboratory on biological
evaluation of phosphorylated drugs displayed broad spec-
trum of potential pharmacological activities (Rao et al.,
2010; Rao et al., 2011). Considering the above, the primary
effort in the present study, therefore, is to synthesize the
phosphorylated derivatives of ganciclovir and further to
screen them for the in vitro and in vivo immune modulating
potentials.
Materials and methods
Chemistry
Chemicals were procured from Sigma-Aldrich and Merck
and used as such without further purification. All solvents
used for spectroscopic and other physical studies were
reagent grade and were further purified by standard meth-
ods (Armarego and Perrin, 1997). Melting points (mp)
were determined using a calibrated thermometer by Guna
Digital Melting Point apparatus and expressed in degree
centigrade (°C) and are uncorrected. Infrared spectra (IR)
were obtained on a Perkin-Elmer Model 281-B spectro-
photometer. Samples were analyzed as potassium bromide
(KBr) disks. Absorptions were reported in wave numbers
The same experimental procedure was adopted for the
preparation of the remaining title compounds 5b–
l (Scheme 1) using various amines (4b–f) and amino acid
esters (4g–l).
Spectroscopic analysis
2-Amino-9-[(2-[(2-aminoethyl)amino]methyl-2-oxo-
1,3,2k5-dioxaphosphinan-5-yl)oxy]methyl-6,9-dihydro-1H-
6-purinone (5a)
1
(cm-1). H and 13C NMR spectra were recorded as solu-
tions in DMSO-d6 on a Bruker AMX 400 MHz spec-
1
trometer operating at 400 MHz for H, 100 MHz for 13C
Yield 79 %; white solid, mp 235–237 °C; IR (KBr): m 3,196,
ꢀ
1
and 161.9 MHz for 31P NMR. The H and 13C chemical
3,137, 3,097, 3,057, 1,250, 1,021, 1,025, 728 cm-1; 1H NMR
(400 MHz, DMSO-d6): d 10.65 (br, s, 1H, H-3, NH), 7.79 (s,
1H, H-8), 6.50 (s, 2H, H-17, NH2), 5.42 (s, 2H, H-10),
4.48–2.47 (14H, H-12 to H-16 and H-10-40); 13C NMR
(100 MHz, DMSO-d6): d 157.3 (C-4), 154.2 (C-2), 151.6
(C-6), 137.7 (C-8), 116.8 (C-5), 73.7 (C-13), 72.0 (C-12), 69.6
(C-10), 50.1 (C-20), 43.4 (C-16), 40.5 (C-30); 31P
NMR(161.9 MHz, DMSO-d6): d -10.9; LC MS (%): m/z
374.3 (65 %), 254.4 (100 %); Anal. Calcd. for C12H20N7O5P:
C 38.61; H 5.40; N 26.26; Found: C 38.51; H 5.28; N 26.19.
shifts were expressed in parts per million (ppm) with ref-
erence to tetramethylsilane (TMS) and 31P chemical shifts
to 85 % H3PO4. LCMS mass spectra were recorded on a
Jeol SX 102 DA/600 mass spectrometer.
Synthesis of 2-amino-9-[(2-[(2-aminoethyl)amino]methyl-
2-oxo-1,3,2k5-dioxaphosphinan-5-yl)oxy]methyl-6,9-
dihydro-1H-6-purinone (5a)
2-Amino-9-((1,3-dihydroxypropan-2-yloxy)methyl)-1H-
purin-6(9H)-one [ganciclovir (1)] (0.001 mol) was dis-
solved in the mixture of solvents tetrahydrofuran (THF)
(10 mL) and pyridine (5 mL). To this stirred solution of
Ganciclovir, chloromethylphosphonic dichloride (2)
(0.001 mol) in 5 mL of THF was added dropwise over a
period of 15 min at -10 °C in the presence of 1,1,3,3-
2-Amino-9-[(2-[(2-hydroxyethyl)amino]methyl-2-oxo-
1,3,2k5-dioxaphosphinan-5-yl)oxy]methyl-6,9-dihydro-1H-
6-purinone (5b)
ꢀ
Yield 75 %; white solid, mp 229–231 °C; IR (KBr): m
3,436, 3,155, 3,075, 3,023, 1,245, 1,018, 1,022, 771 cm-1
;
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Med Chem Res (2014) 23:2242–2251
Scheme 1 Synthesis of
phosphorylated derivatives of
ganciclovir
1H NMR (400 MHz, DMSO-d6): d 10.63 (br, s, 1H, H-3,
NH), 7.72 (s, 1H, H-8), 6.55 (s, 2H, H-17, NH2), 5.49 (s,
2H, H-10), 4.45-2.91 (13H, H-12 to H-16 and H-10-40); 13C
NMR (100 MHz, DMSO-d6): d 157.4 (C-4), 154.6 (C-2),
151.2 (C-6), 137.5 (C-8), 116.8 (C-5), 73.9 (C-13), 72.6 (C-
12), 69.4 (C-10), 59.4 (C-30), 52.9 (C-20), 43.9 (C-16); 31P
NMR(161.9 MHz, DMSO-d6): d -11.0; LC MS (%): m/z
375.1 (72 %), 254.4 (100 %); Anal. Calcd. for C12H19N6
O6P: C 38.51; H 5.12; N 22.45; Found: C 38.42; H 5.04; N
22.36.
NMR (400 MHz, DMSO-d6): d 10.66 (br, s, 1H, H-3, NH),
7.72 (s, 1H, H-8), 6.54 (s, 2H, H-17, NH2), 5.46 (s, 2H,
H-10), 4.44–3.11 (12H, H-12 to H-16 and H-20-40); 13C
NMR (100 MHz, DMSO-d6): d 157.7 (C-4), 154.6 (C-2),
151.4 (C-6), 137.8 (C-8), 116.5 (C-5), 73.3 (C-13), 72.7 (C-
12), 70.7 (C-20), 69.1 (C-10), 63.9 (C-16), 62.4 (C-30); 31P
NMR(161.9 MHz, DMSO-d6): d -14.4; LC MS (%): m/z
376.4 (68 %), 254.4 (100 %); Anal. Calcd. for
C12H18N5O7P: C 38.41; H 4.83; N 18.66; Found: C 38.32;
H 4.73; N 18.57.
2-Amino-9-([2-([1-(hydroxymethyl)propyl]aminomethyl)-
2-oxo-1,3,2k5-dioxaphosphinan-5-yl]oxymethyl)-6,9-
dihydro-1H-6-purinone (5c)
2-Amino-9-[(2-[(2-hydroxyethyl)sulfanyl]methyl-2-oxo-
1,3,2k5-dioxaphosphinan-5-yl)oxy]methyl-6,9-dihydro-1H-
6-purinone (5e)
ꢀ
ꢀ
Yield 68 %; white solid, mp 243–245 °C; IR (KBr): m
Yield 80 %; white solid, mp 246-248 °C; IR (KBr): m
1
3,445, 3,148, 3,082, 3,049, 1,257, 1,016, 1,024, 742 cm-1
;
3,420, 3,091, 3,045, 1,248, 1,014, 1,028, 752 cm-1; H
1H NMR (400 MHz, DMSO-d6): d 10.63 (br, s, 1H, H-3,
NH), 7.76 (s, 1H, H-8), 6.58 (s, 2H, H-17, NH2), 5.49 (s,
2H, H-10), 4.49–1.02 (17H, H-12 to H-16 and H-10-60); 13C
NMR (100 MHz, DMSO-d6): d 157.4 (C-4), 154.9 (C-2),
151.7 (C-6), 137.5 (C-8), 116.2 (C-5), 73.7 (C-13), 72.4 (C-
12), 69.8 (C-10), 60.7 (C-30), 60.4 (C-20), 42.3 (C-16), 24.3
(C-50), 11.5 (C-60); 31P NMR(161.9 MHz, DMSO-d6): d -
18.7; LC MS (%): m/z 403.7 (90 %), 254.4 (100 %); Anal.
Calcd. for C14H23N6O6P: C 41.79; H 5.76; N 20.89; Found:
C 41.71; H 5.64; N 20.77.
NMR (400 MHz, DMSO-d6): d 10.62 (br, s, 1H, H-3, NH),
7.74 (s, 1H, H-8), 6.54 (s, 2H, H-17, NH2), 5.44 (s, 2H,
H-10), 4.49–2.39 (12H, H-12 to H-16 and H-20-40); 13C
NMR (100 MHz, DMSO-d6): d 157.5 (C-4), 154.3 (C-2),
151.7 (C-6), 137.4 (C-8), 116.9 (C-5), 73.2 (C-13), 72.9 (C-
12), 69.7 (C-10), 57.6 (C-30), 38.6 (C-20), 32.9 (C-16); 31P
NMR(161.9 MHz, DMSO-d6): d -13.6; LC MS (%): m/z
392.3 (82 %), 254.4 (100 %); Anal. Calcd. for C12H18N5
O6PS:C 36.83; H 4.64; N 17.90; Found: C 36.71; H 4.50; N
17.74.
2-Amino-9-[(2-[(2-hydroxyethoxy)methyl]-2-oxo-1,3,2k5-
dioxaphosphinan-5-yloxy)methyl]-6,9-dihydro-1H-6-
purinone (5d)
2-Amino-9-[(2-[(2-aminoethyl)sulfanyl]methyl-2-oxo-
1,3,2k5-dioxaphosphinan-5-yl)oxy]methyl-6,9-dihydro-1H-
6-purinone (5f)
ꢀ
ꢀ
Yield 82 %; white solid, mp 268–270 °C; IR (KBr): m
Yield 73 %; white solid, mp 251–253 °C; IR (KBr): m 3,218,
1
3,467, 3,104, 3,042, 1,256, 1,013, 1,019, 768 cm-1; H
3,063, 3,027, 1,232, 1,017, 1,021, 772 cm-1 1H NMR
;
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Med Chem Res (2014) 23:2242–2251
2245
(400 MHz, DMSO-d6): d 10.61 (br, s, 1H, H-3, NH), 7.70 (s,
1H, H-8), 6.57 (s, 2H, H-17, NH2), 5.49 (s, 2H, H-10), 4.43–2.32
(13H,H-12toH-16andH-20-40); 13C NMR (100 MHz, DMSO-
d6): d 157.2 (C-4), 154.7 (C-2), 151.4 (C-6), 137.5 (C-8), 116.7
(C-5), 73.4 (C-13), 72.3 (C-12), 69.7 (C-10), 41.4 (C-30), 39.2
(C-20), 32.4 (C-16); 31P NMR(161.9 MHz, DMSO-d6):
d -15.7; LC MS (%): m/z 391.5 (75 %), 254.4 (100 %); Anal.
Calcd. for C12H19N6O5PS: C 36.92; H 4.91; N 21.53; Found:C
36.84; H 4.86; N 21.40.
H-10), 4.52-1.02 (19H, H-12 to H-16 and H-10-70); 13C
NMR (100 MHz, DMSO-d6): d 170.7 (C-30), 157.5 (C-4),
154.2 (C-2), 151.7 (C-6), 137.4 (C-8), 116.7 (C-5), 73.7 (C-
13), 72.9 (C-12), 69.1 (C-10), 62.4 (C-20), 51.3 (C-50), 41.4
(C-16), 33.4 (C-60), 20.2 (C-70); 31P NMR(161.9 MHz,
DMSO-d6): d -19.8; LC MS (%): m/z 445.2 (73 %), 254.4
(100 %); Anal. Calcd. for C16H25N6O7P: C 43.24; H 5.67;
N 18.91; Found: C 43.20; H 5.59; N 18.82.
Ethyl 2-[(5-[(2-amino-6-oxo-6,9-dihydro-1H-9-
purinyl)methoxy]-2-oxo-1,3,2k5-dioxaphosphinan-2-
ylmethyl)amino]-4-methylpentanoate (5j)
Ethyl 2-amino-3-[(5-[(2-amino-6-oxo-6,9-dihydro-1H-9-
purinyl) me thoxy]-2-oxo-1,3,2k5-dioxabphosphinan-2-
ylmethyl) sulfanyl] propanoate (5g)
Yield 58 %; white solid, mp 275–277 °C; IR (KBr): mꢀ 3,134,
3,112, 3,065, 1,253, 1,027, 1,023, 761 cm-1 1H NMR
;
ꢀ
Yield 65 %; white solid, mp 296–298 °C; IR (KBr): m
1
3,162, 3,106, 3,068, 1,245, 1,023, 1,029, 765 cm-1; H
(400 MHz, DMSO-d6): d 10.49 (br, s, 1H, H-3, NH), 7.63 (s,
1H, H-8), 6.44 (s, 2H, H-17, NH2), 5.53 (s, 2H, H-10), 4.52–1.02
(23H,H-12toH-16andH-10-90); 13C NMR (100 MHz, DMSO-
d6): d 165.2 (C-30), 157.3 (C-4), 154.8 (C-2), 151.3 (C-6), 137.8
(C-8), 116.1 (C-5), 73.4 (C-13), 72.8 (C-12), 69.7 (C-10), 58.3
(C-50), 59.8 (C-20), 39.4 (C-70), 24.4 (C-80), 23.2 (C-90), 41.1 (C-
16), 15.7 (C-60); 31P NMR(161.9 MHz, DMSO-d6): d -18.9;
LC MS (%): m/z 473.4 (58 %), 254.4 (100 %); Anal. Calcd. for
C18H29N6O7P: C 45.76; H 6.19; N 17.79; Found: C 45.67; H
6.11; N 17.72.
NMR (400 MHz, DMSO-d6): d 10.32 (br, s, 1H, H-3, NH),
8.66 (s, 2H, H-80, NH2), 7.68 (s, 1H, H-8), 6.43 (s, 2H,
H-17, NH2), 5.49 (s, 2H, H-10), 4.48-1.32 (15H, H-12 to
H-16 & H-20-70); 13C NMR (100 MHz, DMSO-d6): d 168.3
(C-40), 157.5 (C-4), 154.4 (C-2), 151.9 (C-6), 137.2 (C-8),
116.1 (C-5), 73.6 (C-13), 72.3 (C-12), 69.7 (C-10), 62.1 (C-
60), 54.4 (C-30), 36.5 (C-20), 31.9 (C-16), 15.8 (C-70); 31P
NMR(161.9 MHz, DMSO-d6): d -23.3; LC MS (%): m/z
463.6 (88 %), 254.4 (100 %); Anal. Calcd. for
C15H23N6O7PS: C 38.96; H 5.01; N 18.17; Found: C 38.87;
H 4.93; N 18.11.
Ethyl 2-[(5-[(2-amino-6-oxo-6,9-dihydro-1H-9-
purinyl)methoxy]-2-oxo-1,3,2k5-dioxaphosphinan-2-
ylmethyl)amino]-2-phenylacetate (5k)
Ethyl 2-[(5-[(2-amino-6-oxo-6,9-dihydro-1H-9-
purinyl)methoxy]-2-oxo-1,3,2k5-dioxaphosphinan-2-
ylmethyl)amino]-3-hydroxypropanoate (5h)
ꢀ
Yield 67 %; white solid, mp 241–243 °C; IR (KBr): m
1
3,157, 3,125, 3,078, 1,246, 1,049, 1,023, 757 cm-1; H
ꢀ
Yield 70 %; white solid, mp 232–234 °C; IR (KBr): m
NMR (400 MHz, DMSO-d6): d 10.62 (br, s, 1H, H-3, NH),
7.70 (s, 1H, H-8), 7.05-7.21 (m, 5H, Ar’-H), 6.48 (s, 2H,
H-17, NH2), 5.50 (s, 2H, H-10), 4.84 (s, NH, H-10),
4.52–1.31 (13H, H-12 to H-16 and H-10-60);13C NMR
(100 MHz, DMSO-d6): d 168.7 (C-30), 157.2 (C-4), 154.9
(C-2), 151.3 (C-6), 137.2 (C-8), 132.2 (C-Ph), 127.4 (C-
Ph), 126.9 (C-Ph), 125.4 (C-Ph), 116.6 (C-5), 73.1 (C-13),
72.8 (C-12), 69.7 (C-10), 64.8 (C-20), 59.2 (C-50), 40.4 (C-
16), 15.5 (C-60); 31P NMR(161.9 MHz, DMSO-d6): d -
24.7; LC MS (%): m/z 493.5 (88 %), 254.4 (100 %); Anal.
Calcd. for C20H25N6O7P: C 48.78; H 5.12; N 17.07; Found:
C 48.70; H 5.05; N 17.00.
3,445, 3,155, 3,092, 3,076, 1,252, 1,028, 1,021, 772 cm-1
;
1H NMR (400 MHz, DMSO-d6): d 10.37 (br, s, 1H, H-3,
NH), 7.62 (s, 1H, H-8), 6.41 (s, 2H, H-17, NH2), 5.47 (s,
2H, H-10), 4.48–1.35 (17H, H-12 to H-16 and H-10-80); 13C
NMR (100 MHz, DMSO-d6): d 166.7 (C-30), 157.2 (C-4),
154.8 (C-2), 151.4 (C-6), 137.4 (C-8), 116.4 (C-5), 74.5 (C-
20), 73.8 (C-13), 72.1 (C-12), 69.9 (C-10), 61.1 (C-50), 60.9
(C-70), 41.8 (C-16), 15.4 (C-60); 31P NMR(161.9 MHz,
DMSO-d6): d -20.6; LC MS (%): m/z 447.13 (79 %),
254.4 (100 %); Anal. Calcd. for C15H23N6O8P: C 40.36; H
5.19; N 18.83; Found: C 40.28; H 5.13; N 18.77.
Methyl 2-[(5-[(2-amino-6-oxo-6,9-dihydro-1H-9-
purinyl)methoxy]-2-oxo-1,3,2k5-dioxaphosphinan-2-
ylmethyl)amino]-3-methylbutanoate (5i)
Ethyl 2-[(5-[(2-amino-6-oxo-6,9-dihydro-1H-9-purinyl)
methoxy]-2-oxo-1,3, 2k5dioxaphosphinan-2-ylmethyl)
amino] 2-(4-hydroxy phenyl) acetate (5l)
ꢀ
ꢀ
Yield 63 %; white solid, mp 264–267 °C; IR (KBr): m
Yield 72 %; white solid, mp 282–284 °C; IR (KBr): m
1
3,144, 3,118, 3,063, 1,248, 1,036, 1,018, 775 cm-1; H
3,585, 3,168, 3,115, 3,063, 1,283, 1,037, 1,026, 745 cm-1
;
NMR (400 MHz, DMSO-d6): d 10.42 (br, s, 1H, H-3, NH),
7.66 (s, 1H, H-8), 6.45 (s, 2H, H-17, NH2), 5.51 (s, 2H,
NMR (400 MHz, DMSO-d6): d 10.37 (br, s, 1H, H-3, NH),
7.74 (s, 1H, H-8), 6.82-7.08 (m, 4H, Ar’-H), 6.58 1H (s, 2H,
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Med Chem Res (2014) 23:2242–2251
H-17, NH2), 5.62 (s, 1H, Ph-OH), 5.45 (s, 2H, H-10), 4.65-
1.38 (14H, H-12 to H-16 and H-10-30); 13C NMR
(100 MHz, DMSO-d6): d 169.2 (C-30), 157.4 (C-4), 156.4
(C-Ph-OH), 154.6 (C-2), 151.2 (C-6), 137.5 (C-8), 127.5
(C-Ph), 128.1 (C-Ph), 115.2 (C-Ph), 116.8 (C-5), 73.9 (C-
13), 72.6 (C-12), 69.4 (C-10), 64.2 (C-20), 59.7 (C-50), 40.4
(C-16), 15.1 (C-60); 31P NMR(161.9 MHz, DMSO-d6): d -
21.4; LC MS (%): m/z 509.9 (76 %), 254.4 (100 %); Anal.
Calcd. for C20H25N6O8P: C 47.25; H 4.96; N 16.53; Found:
C 47.15; H 4.84; N 16.44.
Bangalore, India. Animals were housed at five rats per cage in
an air-conditioned room at 23 1 °C, 55–60 % relative
humidity, and a 12 h light/dark cycle (07:00 lights on, 19:00
lights off), and were given a laboratory regular rodent diet for
1 week. The high-fat diet (HFD) with energy density
466.78 kcal/100 g purchased primarily from National Insti-
tute of Nutrition (NIN), Hyderabad, India, was used to induce
obesity with altered immunity. The caloric density of the
normal diet (ND) was 292 kcal/100 g and obtained from
HindustanLeverLimited, Bombay.Allofanimalexperiments
were carried out according to the guidelines of the Sri
Venkateswara University’s Institutional Animal Care and Use
Committee (No./02(i)/a/CPCSCA/IAEC/SVU/TV). For
treatments with compounds 5a–m, after acclimatization for
1 week, rats were randomlydivided intofifteen groups(n = 5
per group), and respectively fed a normal diet (ND) ? vehi-
cle, a high-fat diet (HFD) ? vehicle, HFD ? 5m (ganciclo-
vir): 8 mg/kg bodyweight (b.w), HFD ? 5a: 8 mg/kg b.w,
HFD ? 5b: 8 mg/kg b.w, HFD ? 5c: 8 mg/kg b.w, HFD ?
5d:8 mg/kgb.w, HFD ? 5e:8 mg/kgb.w,HFD ? 5f:8 mg/
kg b.w, HFD ? 5g: 8 mg/kg b.w, HFD ? 5h: 8 mg/kg b.w,
HFD ? 5i: 8 mg/kg b.w, HFD ? 5j: 8 mg/kg b.w,
HFD ? 5k: 8 mg/kg b.w and HFD ? 5l: 8 mg/kg b.w. The
doses of compounds were determined by preliminary tests
using concentrations of 50, 100, and 200 mg/kg as per Orga-
nization for Economic Co-operation and Development
(OECD)-423 guidelines. The lethal dose (LD50) was deter-
mined as 80 mg/kg b.w. Hence, the experimental dose (1/10)
8 mg/kg b.w was selected for the immunomodulatory effect.
Compounds were dissolved in distilled (2 mg/mL) water for
oral administration at the desired doses in a volume of 5 mL/
kg daily at 8:00 h. All groups were treated for 8 weeks. At the
end of the treatmentperiod, blood was collected throughretro-
orbital bleeding under chloroform anesthesia. The serum
samples were prepared by centrifugation of the collected
blood samples (2,500 rpm for 15 min), then stored at -80 °C
for biochemical determinations. Total counts of immune cells
viz. lymphocytes, neutrophils, monocytes were determined
using hematology analyser. Levels of cytokines such as
TNF-a, IL-6, IL-1 and C-reactive protein (CRP) in serum
samples were determined by specific enzyme-linked immu-
nosorbent assay (ELISA) techniques according to the manu-
facturer’s instructions (Genex Bio, New Delhi, India).
In vitro immunomodulatory activity (Nitroblue
tetrazolium test)
Neutrophils were isolated from venous blood of healthy vol-
unteers. The heparinized blood (5 mL containing 100 units of
heparin) was added to 1 mL of 4.5 % dextran B in physio-
logical saline. The mixture was gently shaken and allowed to
stand for 60 min at room temperature to sediment erythro-
cytes. Neutrophils were isolated by Ficoll-Hypaque density
gradient centrifugation according to Ferrante and Thong
(Ferrante and Thong, 1980). After the removal of the residual
erythrocytes by hypotonic lysis, the neutrophils were washed
with Hank’s balanced salt (HBS) solution. The cells were
suspended at a final concentration of 5 9 106 neutrophils/mL
in HBS solution for NBT reduction test (Basaran et al., 1997).
The viability of neutrophils was tested by trypan blue exclu-
sion and was greater than 90 %. A suspension of neutrophils
(5 9 106/mL) was prepared in 0.5 mL of phosphate buffered
saline (PBS) solution in different tubes; 0.1 mL of PBS
solution (control) and 0.1 mL of endotoxin-activated plasma
(standard) were added to 1st tube and 2nd tubes, respectively,
0.1 mL of different concentrations (10, 20, 40, 100 and
1,000 lg/mL) of the test samples (5a–m) was added to the
other tubes. 0.2 mL of freshly prepared 0.15 % nitroblue tet-
razolium (NBT) solution was added to each tube and incu-
bated at 37 °C for 20 min. The tubes were centrifuged at
4009g for 3–4 min to discard the supernatant. The cells were
resuspended in a small volume of PBS solution. A thin film
was made with the drop on the slide, dried, fixed by heating,
counter stained with dilute carbol fuchsin for 15 s. The slide
waswashedunder tapwater, driedandfocused under1009 oil
immersion objectives. Two hundred neutrophils were counted
for the % of NBT positive cells containing blue granules/
lumps (Wilkinson, 1981).
The concentration of cytokines was determined spec-
trophotometrically at 450 nm. A standard curve using
cytokines standards was also constructed. The cytokine
concentrations for unknown samples were calculated
according to standard curve.
In vivo immunomodulatory activity (high-fat diet-
induced obese rats with altered immunity)
Experimental design
Statistical analysis
Male Wistar strain Albino rats weighing 110–130 g were
purchased from Sri Venkateswara Animal Agency at
All data are presented as mean S.D. Differences
between groups were analyzed using the one-way analysis
123

Med Chem Res (2014) 23:2242–2251
2247
of variance (ANOVA) with Dunnett’s t test. A value of
P \ 0.01 was considered statistically in significant using
GraphPad InStat 3 statistical analytical software.
organic phase was dried (MgSO4) and distilled under
vacuum, and the residue was purified by column chroma-
tography on silica gel eluting with 5 % methanol:chloro-
form to obtain the title compounds (5a–l).
The resulted compounds were characterized by their
spectral analysis. The IR spectra of 5a–l showed the
expected absorption bands at 3,218–3,027 and
1,283–1,232 cm-1 for the NH and P=O stretching vibra-
tions, respectively (Thomas, 1974). Aliphatic OH bands
were observed in between 3,445 and 3,425 for the com-
Results and discussion
Chemistry
Chloromethylphosphonic dichloride (2) in THF/Pyridine
was reacted at -10 °C with ganciclovir (1 or 5m) in the
presence of 1,1,3,3-tetramethylguanidine (TMG) as a base.
After completion of the addition, the reaction mixture was
stirred for 2 h at 0 °C to obtain an intermediate, 2-amino-9-
([2-(chloromethyl)-2-oxo-1, 3, 2k5-dioxa phosphinan-5-
yl]oxymethyl)-6,9-dihydro-1H-6-purinone (3). It was fil-
tered to remove TMG.HCl. Various amines (4b–f) and
amino acid esters (4g–l) were reacted with (3) at -10 to
0 °C and further stirring was continued for 4–7 h by raising
the temperature to 35–50 °C. The progress of the reaction
was monitored by TLC using methanol:ethyl acetate (1:1)
as eluent. After completion of the reaction, it was filtered to
remove TMGꢀHCl, and the filtrate was concentrated in a
rota-evaporator to get crude product. It was dissolved in
chloroform (10 mL) and washed with 1 M hydrochloric
acid (2 9 15 mL), saturated sodium bicarbonate solution
(2 9 10 mL), and then water (3 9 15 mL). Then the
1
pounds 5b, 5c, 5d, 5e, 5h and 5l. H NMR signals and
proton counting of the title compounds were observed in
their expected region. All 13C signals of phosphorylated
ganciclovir derivatives were observed in their expected
regions (Prasadaraju et al., 2009). 31P NMR signals
appeared in the range of -24.7 to -10.9 ppm as expected
for the P=O group of the title compounds.
In vitro immunomodulatory activity
To investigate the potential immunomodulating activity of
the synthesized compounds, the phosphorylated derivatives
of ganciclovir were subjected to in vitro nitroblue tetra-
zolium (NBT) test and in vivo activity on high-fat diet-
induced immune impaired obese rats.
The NBT dye reduction test gives information about the
phagocytic and intracellular killing functions of neutrophils
Table 1 Effect of the phosphorylated ganciclovir derivatives on NBT-positive neutrophils (%)
Test sample
NBT-positive neutrophils (%)
10 lg/mL
20 lg/mL
40 lg/mL
100 lg/mL
1,000 lg/mL
NC
PC
5m
5a
5b
5c
5d
5e
5f
18.41 0.03
39.67 0.17*
39.43 0.18
40.12 0.31
33.38 0.16
31.37 0.21
34.31 0.20
32.25 0.26
33.77 0.13
45.03 0.24#
39.80 0.06
41.18 0.17#
39.17 0.12
40.62 0.13
39.58 0.21
19.36 0.15
49.57 0.25*
53.21 0.3
24.59 0.04
51.34 0.36*
55.89 0.25
45.35 0.12
48.54 0.16
46.76 0.12
49.85 0.33
47.93 0.14
49.32 0.12
58.04 0.32#
48.11 0.50
60.83 0.22#
49.31 0.21
46.63 0.12
49.49 0.13
25.42 0.16
54.14 0.48*
58.23 0.17
47.30 0.11
50.57 0.18
53.98 0.15
51.23 0.17
52.15 0.28
50.36 0.11
60.16 0.24#
53.09 0.15
62.45 0.15#
51.44 0.23
52.91 0.27
52.31 015
27.57 0.08
58.68 0.11*
60.11 0.22
50.46 0.43
55.25 0.31
54.32 0.14
53.02 0.20
55.35 0.16
57.91 0.21
66.82 0.17#
56.02 0.24
65.14 0.46#
58.03 0.15
60.32 0.10#
62.25 0.08#
42.51 0.11
45.98 0.53
43.15 0.27
46.44 0.31
43.73 0.11
45.64 0.32
56.34 0.15#
39.92 0.19
55.09 0.26#
40.92 0.11
43.11 0.23
42.46 0.14
5g
5h
5i
5j
5k
5l
Values are mean SD. Statistical analysis was by ANOVA followed by Dunnett’s t test
5m, ganciclovir; PBS (NC) phosphate buffer saline (normal control), EAP (PC) endotoxin-activated plasma (positive control)
* P \ 0.01 as compared to normal control
#
P \ 0.01 as compared to endotoxin-activated plasma
123

2248
Med Chem Res (2014) 23:2242–2251
which are necessary for normal microbicidal activity. The
dye is taken into neutrophils by phagocytosis and then
stimulation of the hexose monophosphate-shunt pathway
(HMP) of glucose oxidation and concomitant changes in
oxidative metabolism lead to the reduction of the dye
(Akbay et al., 2002).
pattern of purine ring substitution is absolutely critical. The
consequences of immune stimulation by guanosine analogs
include both humoral and cellular immune responses
(Jongdae et al., 2003). Our results reported in the present
study shed light on these events.
The ganciclovir and all its synthesized phosphorylated
derivatives at the concentrations of 10, 20, 40, 100 and
1,000 lg/mL increased the intracellular killing activity of
stimulated neutrophils assayed by in vitro NBT reduction
test. The results are shown in Table 1. Among them,
compounds 5g and 5i showed significant (P \ 0.01)
activity as compared to the standard ganciclovir (5m) and
endotoxin-activated plasma indicating their possible
immunomodulatory effect. 5k and 5l also showed signifi-
cant activity at high dose (1,000 lg/mL) compared with the
positive control group, but did not show a significant effect
with ganciclovir (5m). A number of derivatives of guano-
sine and analogs thereof have been studied over the years
for their ability to stimulate the immune system in mouse
and human models. The structural requirements for gua-
nosine analogs to be immunomodulatory are such that the
In vivo immunomodulatory activity
The immunomodulatory study of ganciclovir and its
phosphorylated derivatives was also performed using the
high-fat diet-induced immune impaired obese animal
model. Obesity is associated with an impaired immune
response (Alexia et al., 2007). Diet-induced obesity is a
more physiologically relevant model of human obesity
(Ikejima et al., 2005). The marked body weights of rats
reflecting obesity by high-fat diet is considerable and is
accompanied by increased immune cells viz., total lym-
phocytes, neutrophils, monocytes were evident (Sonia and
Ranjit Chandra, 2001). Furthermore, several lines of evi-
dence have supported a link between adipose tissue and
immunocompetent cells. This interaction is illustrated in
obesity, where excess adiposity and impaired immune
3
12
*
*
2.5
2
10
8
1.5
6
4
2
0
#
#
#
#
1
0.5
0
Experimental Groups
Experimental Groups
A Lymphocytes count
B Neutrophils count
0.45
0.4
*
0.35
0.3
0.25
0.2
#
#
0.15
0.1
0.05
0
Experimental Groups
C Monocytes count
Fig. 1 The levels of total immune cell counts in impaired immune obese rats by the treatment of phosphorylated derivatives of ganciclovir.
#
Values are mean SD; *P \ 0.01 as compared to normal diet-ND (control group), P \ 0.01 compared to high-fat diet-HFD (obese group)
123

Med Chem Res (2014) 23:2242–2251
2249
10
9
8
7
6
5
4
3
2
1
0
80
*
*
70
60
50
40
30
20
10
0
#
#
#
#
Experimental Groups
Experimental Groups
α
A TNF- levels
B IL-6 levels
45
40
35
30
25
20
15
10
5
180
160
140
120
100
80
*
*
#
#
#
#
60
40
20
0
0
Experimental Groups
Experimental Groups
D CRP levels
C IL-1 levels
Fig. 2 The levels of biofactors in immune impaired obese rats upon the treatment with phosphorylated derivatives of ganciclovir. Values are
#
mean SD; *P \ 0.01 as compared to normal diet-ND (control group), P \ 0.01 as compared to high-fat diet-HFD (obese group)
function have been described in both humans and geneti-
cally obese rodents (Marti et al., 2001). The impact of
obesity on immune cells has been evaluated with results
suggesting that being overweight or obese is associated
with higher immune cell levels (Womack et al., 2007). In
the present study, oral administration of compounds 5g and
5i (8 mg/kg b.w) significantly ameliorated the counts of
total lymphocytes, neutrophils and monocytes (Fig. 1a–c)
induced by high-fat diet in immune impaired rats when
compared normal controls.
biofactors (cytokines) in obese rats. Data presented in
Fig. 2a–c) show the effects of ganciclovir and its deriva-
tives on TNF-a, IL-6 and IL-1 levels in the experimental
animals. Cytokines (TNF-a, IL-6 and IL-1) secretion was
not affected significantly by the compounds (5a–5f), while
the compounds (5g and 5i) exhibited significantly consid-
erable levels (36.5 7.5, 5.2 1.2, 122.9 13.5 and
37.1 13.2, 5.4 1.3, 123.6 12.4 pg/mL respectively)
when compared to the rats with dysfunctioned immune
system.
The development of high-fat diet-induced obesity is
associated with an altered leptin production (Lin et al.,
2000). Leptin is mainly secreted by the adipose tissue and has
a significant immune modulatory role (Laharrague et al.,
1998; Lord et al., 1998), demonstrating that mouse lym-
phocytes express the leptin receptor, and that leptin modu-
lates in these cells for increased cytokines (TNF-a, IL-6 and
IL-1) production. Considering the aforementioned issues, it
seemed plausible to assume that the increased lymphocyte
count reported here with the high-fat diet fed immune
impaired rats would affect the cytokine production.
Impaired immunity in high-fat diet-induced obese rats is
associated with increased production of the cytokines
including IL-6 (Santos-Alvarez et al., 1999). Second, IL-6
induces up-regulation of CRP (Shamsuzzaman et al.,
2004). Thus, we prompted to investigate the possibility of
the compounds 5a–m which could be potent immuno-
modulators acting via CRP. In view of the above, in this
article, we present evidence that upon the treatment with
compounds 5g and 5i to immune impaired obese rats, the
levels of CRP (21.9 0.51 and 22.3 1.37 ng/mL) were
significantly restored to normal as compared to high-fat
diet alone fed rats with immune dysfunction (Fig. 1d).
However, CRP production was not affected significantly in
In this study, we also investigated the effects of Gan-
ciclovir and its synthetic derivatives on immune-related
123

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neuroendocrine systems perspective. Int Rev Immunol 32(3):
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other immune functions altered rats with the phosphory-
lated derivatives of ganciclovir (5a–5f, 5h, 5j–5l) sug-
gesting their immune modulating potential.
Dockrell DH, Kinghorn GR (2001) Imiquimod and resiquimod as
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Conclusion
In conclusion, the data corroborate that the ganciclovir and
its phosphorylated derivatives (GPDs) can alter the intra-
cellular killing activity of the stimulated neutrophils indi-
cating their involvement in immune response. In addition,
the in vivo experiment indicated that the GPDs might be
cytokine modulators that regulate the TNF-a, IL-6 and IL-1
secretion levels, further IL-6 production also induces up-
regulation in CRP (biofactors for impaired immunity in
obesity) levels. Furthermore, the GPDs seem to augment
the immune cell proliferation, the useful indicator of an
immune response to treatment. When compared with the
title compounds, amino acid ester-substituted ganciclovir
derivatives presented the potent pharmcological activities.
These in vitro and in vivo results suggest that the GPDs
might be strong immunomodulators. Investigations of
synthetic compounds and their analogs or derivatives that
can modulate the immune system are gaining attention in
the research areas of immunopharmacology. Together,
targeting the immune response with these GPDs is highly
effective in mobilizing or restoring immunity, supporting
the potential therapeutic use of this novel immunomodu-
lators as adjuvants in the treatment of immune mediated
and viral diseases.
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Acknowledgments The authors express their grateful thanks to
BRNS (DAE) research project (2012/37C/21/BRNS/785), Mumbai,
India for providing financial assistance through the project.
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