Synthesis, characterization and biological evaluation of C5′-N-cyclopropylcarboxamido-C6-amino-C2-alkynylated purine nucleoside analogues

Article abstract of DOI:10.1080/15257770.2017.1375117

In an effort to develop potent antibacterial and anticancer agents, a series of C5′-N-cyclopropylcarboxamido-C6-amino-C2-alkynylated purine nucleoside analogues 11a-g were synthesized through a Sonogashira cross-coupling reaction. The nine-step synthesis is easy to perform, and employs commercially available reagents. 2-Iodo-5′-N-cyclopropylcarboxamidoadenosine (9) was used as the starting intermediate for the synthesis of title derivatives 11a-g. Synthetic intermediates (2–9) and final products (11a-g) were appropriately characterized by IR, ¹H NMR, ¹³C NMR and mass spectroscopy. The synthesized purine nucleoside analogues (11a-g) were evaluated for their in vitro antibacterial activity against two gram-positive and two gram-negative bacteria. They were then tested for cytotoxicity against MDA-MB-231 and Caco-2 cancer cell lines to determine their anti-cancer activity. Among the tested compounds, compounds 11c and 11g showed most potent antibacterial activity against S.aureus and P.aeruginosa bacterial strains. Compounds 11b and 11e displayed considerable IC₅₀^s of 7.9 and 6.8 μg/mL, respectively, vs MDA-MB-231 cell lines of 7.5 and 8.3 μg/mL, respectively, against the Caco-2 cell lines.

Full text of DOI:10.1080/15257770.2017.1375117

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis, characterization and biological
evaluation of C5′-N-cyclopropylcarboxamido-
C6-amino-C2-alkynylated purine nucleoside
analogues
Arasavelli Ananda Mohan, Ganapavarapu Veera Raghava Sharma & Siddaiah
Vidavalur
To cite this article: Arasavelli Ananda Mohan, Ganapavarapu Veera Raghava Sharma &
Siddaiah Vidavalur (2017) Synthesis, characterization and biological evaluation of C5′-N-
cyclopropylcarboxamido-C6-amino-C2-alkynylated purine nucleoside analogues, Nucleosides,
Nucleotides and Nucleic Acids, 36:10, 637-651, DOI: 10.1080/15257770.2017.1375117
To link to this article: https://doi.org/10.1080/15257770.2017.1375117
Published online: 29 Nov 2017.
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Date: 29 December 2017, At: 17:52

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
https://doi.org/./..
Synthesis, characterization and biological evaluation of
Cꢀ-N-cyclopropylcarboxamido-C-amino-C-alkynylated
purine nucleoside analogues
Arasavelli Ananda Mohana,b, Ganapavarapu Veera Raghava Sharmac, and
Siddaiah Vidavalurd
aDepartment of Organic Chemistry, Dr. B.R. Ambedkar University, Srikakulam, Etcherla, Andhra Pradesh,
India; bDepartment of Chemistry, Jawaharlal Nehru Technological University Kakinada, Kakinada, Andhra
Pradesh, India; cDepartment of Chemistry, GIT, GITAM University, Visakhapatnam, Andhra Pradesh, India;
dDepartment of Organic Chemistry & FDW, Andhra University, Visakhapatnam, Andhra Pradesh, India
ABSTRACT
ARTICLE HISTORY
Received  March 
Revised  August 
Accepted  August 
In an effort to develop potent antibacterial and anticancer
agents, a series of C5ꢀ-N-cyclopropylcarboxamido-C6-amino-C2-
alkynylated purine nucleoside analogues 11a-g were synthesized
through a Sonogashira cross-coupling reaction. The nine-step
synthesis is easy to perform, and employs commercially avail-
able reagents. 2-Iodo-5ꢀ-N-cyclopropylcarboxamidoadenosine (9)
was used as the starting intermediate for the synthesis of title
derivatives 11a-g. Synthetic intermediates (2–9) and final prod-
KEYWORDS
Sonogashira coupling; purine
nucleosides; antibacterial
activity; cytotoxicity;
MDA-MB-; Caco-
1
ucts (11a-g) were appropriately characterized by IR, H NMR, 13C
NMR and mass spectroscopy. The synthesized purine nucleoside
analogues (11a-g) were evaluated for their in vitro antibacterial
activity against two gram-positive and two gram-negative bac-
teria. They were then tested for cytotoxicity against MDA-MB-
231 and Caco-2 cancer cell lines to determine their anti-cancer
activity. Among the tested compounds, compounds 11c and 11g
showed most potent antibacterial activity against S.aureus and
P.aeruginosa bacterial strains. Compounds 11b and 11e displayed
considerable IC50s of 7.9 and 6.8 µg/mL, respectively, vs MDA-MB-
231 cell lines of 7.5 and 8.3 µg/mL, respectively, against the Caco-2
cell lines.
Introduction
Over the past years, many structurally modified organic compounds or drugs have
been synthesized for the treatment of cancer, as well as bacterial and viral diseases
and have become available for investigation in studies or clinical trials. Among
them, the class of antineoplastic drugs belonging to the group of purine nucle-
oside analogues is important.[1] Currently, a good number of cytotoxic nucleo-
side analogues have been approved by the Food and Drugs Administration (FDA)
CONTACT Arasavelli Ananda Mohan
anandamohan.organic@gmail.com
Dr. B. R. Ambedkar University, Srikakulam, Etcherla-Mandal, Andhra Pradesh  , India.
Department of Organic Chemistry,
©  Taylor & Francis Group, LLC

638
A. A. MOHAN ET AL.
Figure . Structures of nucleoside analogues: Cladribine, Fludarabine, PHPNECA and ATL-e.
and the European Medicines Agency (EMA). Cladribine 2-chloro-9-(2ꢀ-deoxy-
β-D-ribofuranosyl)-adenine and fludarabine 2-fluoro-9-(β-D-arabinofuranosyl)-
adenine (Figure 1) are examples of these analogues and are used for the treating
hematological malignancies.[2,3]
Some recent reports have demonstrated that modified purine nucleo-
sides show cytotoxic activity against different cancer cell lines and antibacte-
rial activity against various bacterial strains.[4,5] Particularly 2-alkynyl-5ꢀ-N-
ethylcarboxamidoadenosine and 2-alkynyl-5ꢀ-N-cyclopropyl carboxamidoadeno-
sine derivatives are a class of analogues with promising properties, and many
of them are agonists of the A2A adenosine receptor (A2AAR).[6,7] 2-(R,S)-
Phenylhydroxypropynyladenosine-C5ꢀ-N-ethylcarboxamide (PHPNECA) and
apadenoson (ATL-146e) (Figure 1) are examples of potent agonists of A2AARs.[8,9]
The synthesis of 2-alkynyl-5ꢀ-N-ethylcarboxamido adenosine and 2-alkynyl-5ꢀ-
N-cyclopropylcarboxamido adenosine derivatives and the evaluation of their bio-
logical activities have been well documented in the literature.[10,11] These deriva-
tives are typically prepared from 2-iodo-5ꢀ-ethyl or cyclopropyl carboxamidoadeno-
sine through the Sanogashira cross-coupling reaction with the appropriate terminal
alkyne.[12]
Sonogashira coupling is the Pd-catalyzed cross-coupling of the sp2 carbon of vinyl
or aryl halide with the terminal sp carbon of alkyne in the presence of cuprous iodide
(CuI).[13] Because of its broad synthetic potential, the Sonogashira cross-coupling
reaction has become an essential step in the synthesis of conjugated enynes or aryl
alkynes. The 2-iodo-5ꢀ-N-ethyl or cyclopropyl carboxamidoadenosine derivative
was previously synthesized from 2-iodoadenosine in four steps, which was conse-
quently prepared from guanosine in four steps.[14,15]
Pioneering work by Robert et al. described the synthesis and biological activity
of 2-(3-(1-methoxycarbonyl)piperidene-4-yl)propyn-1-yl)-5ꢀ-N-ethylcarboxami-
doadenosine and 2-(3-(1-methoxycarbonyl)piperidene-4-yl)propyn-1-yl)-5ꢀ-N-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
639
cyclopropylcarboxamidoadenosine derivatives.[16] However, 2-(3-aromatic/satu-
rated heterocyclic substituted)propyn-1-yl–5ꢀ-N-cyclopropylcarboxamido adeno-
sine derivatives remain unexplored.
Considering the above data, in continuation of ongoing research for the devel-
opment of biologically active compounds, the titled compounds 11a-g were synthe-
sized and evaluated for their in vitro anti-bacterial and anti-cancer activities.
Experimental
Chemistry
All reagents and solvents were used as received from commercial sources unless oth-
erwise mentioned. All the reactions were monitored by thin-layer chromatography
analysis using silica gel plates (Kieselgel 60F254 Merck). Silica coated plates 60 F254
for TLC supplied by MERCK, compounds were visualized by UV light (254 nm)
after spraying with 10% sulfuric acid/methanol solution. Melting points were deter-
mined in open capillary tubes on a Boetius melting point apparatus and were uncor-
rected. The FT IR spectra as wave number (ν cm−1) were recorded using KBr pellets
in BRUKER spectrophotometer. 1H NMR and 13C NMR spectra were obtained at
room temperature on Varian Gemini spectrometer at 400 MHz (1H) and 100 MHz
(13C). Chemical shifts are expressed as relative ppm from the solvent. Mass mea-
surements were recorded on JEOL Sx102 mass spectrometer coupled with liquid
chromatography system in Electrospray Time of Flight (TOF) mode. The elemen-
tal analysis was performed on Perkin Elmer CHN elemental analyzer. The purity
of compounds was determined using reversed-phase (RP) WATERS HPLC system
equipped with symmetry C18 analytical column (4.6× 75 mm). Peaks were detected
by UV absorption (254 nm) using a diode array detector. Column chromatography
was used for purification of crude compounds over silica gel MERCK 60.
Biological assay
Antibacterial activities
A collection of four bacterial organisms including two gram-positive and two gram-
negative organisms were used for this study. Staphylococcus aureus (MTCC 3160)
(Microbial Type Culture Collection and Gene Bank), Bacillus alcalophilus (MTCC
1485), Escherichia coli (MTCC 1563) and Pseudomonas aeruginosa (MTCC 6642)
were obtained from Institute of Microbial Technology, Chandigarh, India. The title
analogues 11a-g was tested for antibacterial activity in vitro by agar well diffusion
[17]
method
was carried out using two gram-positive viz., Staphylococcus aureus,
Bacillus alcalophilus, and two gram-negative viz., Escherichia coli, Pseudomonas
aeruginosa with norfloxacin as a standard reference. The bacterial strains were reac-
tivated from stock culture by incubating the inoculated broths at 37°C for 24 h
in an incubator. Final inoculums containing 1X106 c.f.u/mL colonies were added

640
A. A. MOHAN ET AL.
aseptically to Mueller-Hinton agar medium and poured into Sterile Petri dishes.
100 µL of each test extract from stock concentrations (1 mg/mL) were added to
the wells (8 mm) punched on an agar surface. The reference standard was main-
tained with norfloxacin. Plates were incubated in a bacterial incubator for overnight
at 37°C. The activity was evaluated by measuring the diameter of inhibition zone
(mm).
Anti-cancer activities
Polystyrene disposables for cell cultivation were produced by Corning (Thomas Sci-
entific). Media, supplements and other reagents for cell culture maintenance were
obtained from Sigma-Aldrich (USA). The chemicals used were of analytical or
HPLC grade. MDA-MB-231 and Caco-2 cell lines were procured from NCCS, Pune,
India. Both cell lines were verified to be mycoplasma-free at the time of purchase,
and no further testing specifically for myco plasma occurred. The human breast can-
cer cell line MDA-MB-231 and human colon cancer cell line Caco-2 were routinely
grown in polystyrene tissue culture dishes (100 × 20 mm, Becton Dickinson Lab-
ware) with Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 5%
fetal bovine serum, 2 mmol L-glutamine, 100 units/mL penicillin and 100 units/mL
streptomycin. Cells were incubated in humidified atmosphere containing 5% CO2
at 37°C. The cell line was passaged twice weekly for maintaining sub-confluent state.
The MTT (3-(4,5-dimet hylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colori-
metric assay was used to determine growth inhibition.[18] The principle of the assay
lies in the fact that the MTT is reduced to formazan ((E,Z)-5-(4,5-demethylthiazol-
2-yl)-1,3-diphenylformazan) crystals. DXN (Doxoru bicin) a known anti-cancer
drug was used as reference compound in the assay. The treated and untreated cells
were washed with PBS (Phosphate Buffer solution) and MTT was added and incu-
bated at 37°C for 5 h. The MTT was then removed and the formazan crystals were
dissolved by adding DMSO (Dimethyl Sulphoxide). The absorbance at 540 nm rep-
resents the viable cells. The absorbance was recorded using multi scan spectrum.
The absorbance of the untreated cells was defined as 100% viable cells. The % of
viable cells were plotted (Y-axis) against concentration (X-axis). The IC50 values can
be interpolated from the graph.
General procedure for the synthesis of modified purine nucleosides (a-g)
The C5ꢀ-N-cyclopropylcarboxamido-C6-amino-C2-alkynylated purine nucleoside
derivatives 11a-g were synthesized from 2-iodo-5ꢀ-N-cyclopropylcarboxami-
doadenosine (9) and different terminal alkynes (10a-g) through Sanogashira
cross-coupling reaction, 2-iodo-5ꢀ-N-cyclopropylcarboxamidoadenosine (9) was
obtained from commercially available guanosine in eight steps (Scheme 1) by using
a known method.[19]
ꢀ,ꢀ,ꢀ-Tri-O-acetylguanosine ()
White solid, 62.3 g, 70% yield, mp 168°C, Rf value: 0.2 (ethyl acetate/methanol 50:1).
1H NMR (400 MHz, d6-DMSO): δ = 10.8 (s, 1H, NH), 7.93 (s, 1H, =CHN), 6.52

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
641
Scheme . Synthesis of modified purine nucleoside derivatives 11a-g.
(s, 2H, NH2), 6.05 (t, 1H, J = 7.1 Hz, H-2R), 5.8 (t, 1H, J = 6.3 Hz, H-5R), 5.5 (d,
1H, J = 6.5 Hz, H-3S), 4.83 (d, 1H, J = 5.5 Hz, H-4S), 4.25 (d, 2H), 2.16 (s, 3H), 2.10
(s, 6H). 13C NMR (100 MHz, d6-DMSO): δ = 176.3 (CO2CH3), 164.7, 160.0, 143.5,
142.1 (C-purine), 138.7 (CH-purine), 60.0 (CH2), 26.7, 21.4, 15.6 (CH3). IR (KBr):
ν = 3350.97 (N-H str) 3162.18 (Ar =CH str), 2928.48, 2892.48 (C-H str), 1631.76
(CONH str), 1615.73 (COOMe str), 1554.96 (Ar C=C str), 1336.56 (C-N str), 1118
(C-O-C str). Anal. Calcd. for C16H19N5O8: C, 46.95; H, 4.68; N, 17.11. Found: C,
47.10; H, 4.62; N, 17.02. LC-MS (TOF) m/z: 410.0 [M+1]+.
-Amino--chloro--(,,-tri-O-acetyl-β-D-ribofuranosyl)purine ()
Light yellow solid, 37.1 g, 64.1% yield, mp 189°C, Rf value: 0.4 (CHCl3/CH3OH 4:1).
1H NMR (400 MHz, d6-DMSO): δ = 7.94 (s, 1H, =CHN), 6.08 (t, 1H, J = 7.5 Hz,
H-5R), 6.02 (d, 1H, J = 6.9 Hz, H-3S), 6.85 (t, 1H, J = 6.5 Hz, H-2R), 5.13 (s, 2H,
NH2) 4.33 (d, 1H, J = 4.9 Hz, H-4S), 4.25 (d, 2H), 2.25 (s, 3H), 2.20 (s, 6H). 13C NMR
(100 MHz, d6-DMSO): δ = 174.2 (CO2CH3), 162.3, 153.4, 140.0, 135.0 (C-purine),
144.5 (CH-purine), 60.9 (CH2), 33.4, 29.6, 23.2 (CH3). IR (KBr): ν = 3441.82 (OH
str), 3280.98 (N-H str) 3163.09 (N=CH str), 2927.26 (C-H str), 2851.87 (C-H str),
1511.37, 1422.10 (Ar C=C str), 1362.44 (C-N str), 1165.40 (C-O-C str), 724.18 (C-
Cl). Anal. Calcd. for C16H18ClN5O7: C, 44.92; H, 4.24; N, 16.37. Found: C, 44.75; H,
4.19; N, 16.39. LC-MS (TOF) m/z: 427.41[M]+ and 429.38 [M+2]+.

642
A. A. MOHAN ET AL.
-Chloro--iodo--(,,-tri-O-acetyl-β-D-ribofuranosyl)-H-purine ()
White solid, 23 g, 44% yield, mp 196°C, Rf value: 0.6 (CHCl3/CH3OH 4:1). 1H NMR
(400 MHz, d6-DMSO): δ = 7.94 (s, 1H, =CHN), 6.85 (t, 1H, J = 7.5 Hz, H-2R), 6.08
(t, 1H, J = 6.9 Hz, H-5R), 6.02 (d, 1H, J = 6.5 Hz, H-3S), 4.33 (d, 1H, J = 4.8 Hz,
H-4S), 4.25 (d, 2H), 2.25 (s, 3H), 2.20 (s, 6H). 13C NMR (100 MHz, d6-DMSO): δ
= 170.6 (CO2CH3), 156.9 (CH-purine), 138.5, 133.6, 132.5, 127.9 (C-purine), 55.7
(CH2), 29.8, 24.6, 23.0 (CH3). IR (KBr): ν = 3436.54 (OH str), 3285.54 (N-H str),
3143.62 (N=CH str), 2927.10 (C-H str), 2850.73 (C-H str), 1522.19, 1440.78 (Ar
C=C str), 1327.80 (C-N str), 1142.61 (C-O-C str), 873.86 (C-Cl), 730.13 (C-I). Anal.
Calcd. for C16H16ClIN4O7: C, 35.67; H, 2.99; N, 10.40. Found: C, 35.76; H, 3.02;
+
N, 10.50. HRMS (TOF) m/z found 539.0037 [cacld for C16H16ClIN4O7 539.6862
(M+H)+].
-Iodoadenosine ()
White solid, 13.5 g, 80% yield, mp 162°C, Rf value: 0.5 (20% Methanol in chloro-
form). 1H NMR (400 MHz, d6-DMSO): δ = 8.28 (s, 2H, NH), 7.82 (s, 1H, =CHN),
7.20 (broad singlet, 1H, OH), 7.65 (broad singlet, 1H, OH), 5.83 (t, 1H, J = 7.0 Hz,
H-2R), 5.48 (t, 1H, H-5R), 5.23 (d, 1H, J = 5.5 Hz, H-3S), 5.12 (d, 1H, J = 5.8 Hz,
H-4S), 4.05 (d, 2H, J = 3.5 Hz), 1.80 (s, 1H). 13C NMR (100 MHz, d6-DMSO): δ
= 145.8 (CH-purine), 135.2, 132.3, 131.7, 128.7 (C-purine), 60.1 (CH2). IR (KBr):
ν = 3452.86 (OH str), 3225.34 (N-H str), 3156.24 (N=CH str), 2926.59 (C-H str),
2851.91 (C-H str), 1594.33, 1546.19 (Ar C=C str), 1441.26 (C-N str), 1065.38 (C-
O-C str), 789.72 (C-I). Anal. Calcd. for C10H12IN5O4: C, 30.55; H, 3.08; N, 17.81.
Found: C, 30.58; H, 3.12; N, 17.78. HRMS (TOF) m/z found 395.3415 [calcd for
C10H12IN5O4 395.1537 (M+2)+].
-Iodo-ꢀ,ꢀ-O-isopropylideneadenosine ()
White solid, 10.4 g, 70% yield, mp 174°C, Rf value: 0.5 (ethyl acetate/methanol 20:1).
1H NMR (400 MHz, d6-DMSO): δ = 7.75 (s, 1H, =CHN), 7.28 (s, 2H, NH), 5.80 (t,
1H, J = 7.0 Hz, H-2R), 5.41 (t, 1H, J = 6.2 Hz, H-5R), 5.20 (d, 1H, J = 7.0 Hz, H-3S),
4.52 (d, 1H, H-4S), 4.05 (d, 1H), 3.85 (d, 1H, J = 5.2 Hz), 2.05 (s, 1H, OH), 1.6 (s, 3H,
CH3), 1.4 (s, 3H, CH3). 13C NMR (100 MHz, d6-DMSO): δ = 145.8 (CH-purine),
135.2, 132.2, 128.7, 128.2 (C-purine), 126.5 (C(CH3)2), 73.0 (CH2), 24.9, 23.0 (CH3).
IR (KBr): ν = 3426.78 (OH str), 3220.52 (N-H str), 3146.94 (N=CH str), 2925.14
(C-H str), 2849.61 (C-H str), 1592.32, 1501.06 (Ar C=C str), 1368.91 (C-N str),
1142.94 (C-O-C str), 706.19 (C-I). Anal. Calcd. for C13H16IN5O4: C, 36.04; H, 3.72;
N, 16.17; Found: C, 36.14; H, 3.80; N, 16.11. LC-MS (TOF) m/z: 433.43 [M]+.
-Iodo-ꢀ,ꢀ-O-isopropylidene-ꢀ-carboxylicacid adenosine ()
White solid, 8.4 g, 78.6% yield, mp 191°C, Rf value: 0.2 (20% Methanol in chloro-
form). 1H NMR (400 MHz, d6-DMSO): δ = 7.75 (s, 1H, =CHN), 7.28 (s, 2H, NH),
5.80 (t, 1H, J = 7.0 Hz, H-2R), 5.41 (t, 1H, J = 6.8 Hz, H-5R), 5.20 (d, 1H, J = 5.5 Hz,
H-3S), 4.52 (d, 1H, H-4S), 4.05 (d, 1H), 1.6 (s, 3H, CH3), 1.4 (s, 3H, CH3). 13C NMR
(100 MHz, d6-DMSO): δ = 176.6 (COOH), 138.9 (CH-purine), 131.2, 130.3, 110.5,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
643
109.0 (C-purine), 124.9 (C(CH3)2), 31.0, 26.6 (CH3). IR (KBr): ν = 3433.03 (OH
str), 3374.50 (N-H str), 3170.57 (N=CH str), 2927.27 (C-H str), 2851.61 (C-H str),
1615.13 (C=O str), 1492.67 (Ar C=C str), 1442.59 (C-N str), 1165.25 (C-O-C str),
790.99 (C-I str). Anal. Calcd. for C13H14IN5O5: C, 34.92; H, 3.16; N, 15.66; Found:
C, 35.01; H, 3.13; N, 15.59. LC-MS (TOF) m/z: 448.27 [M+1]+.
-Iodo-ꢀ,ꢀ-O-isopropylidene-ꢀ-N-cyclopropylcarboxamidoadenosine ()
White solid, 6.4 g, 70.3% yield, mp 178°C, Rf value: 0.4 (ethylacetate/n-hexanes 1:1).
1H NMR (400 MHz, d6-DMSO): δ = 7.75 (s, 1H, =CHN), 7.28 (s, 1H, CONH),
6.25 (t, 1H, J = 7.2 Hz, H-3R), 6.02 (t, 1H, J = 7.0 Hz, H-6R), 5.82 (s, 2H, NH2),
5.18 (d, 1H, H-6S), 4.62 (d, 1H, J = 4.9 Hz, H-4S), 2.65 (d, 1H, CHNH), 1.75 (s,
3H, CH3), 1.42 (s, 3H, CH3), 0.65 (t, 2H, CH2-CH2), 0.2 (t, 2H, CH2-CH2). 13C
NMR (100 MHz, d6-DMSO): δ = 170.1 (CONH), 142.0, 137.6, 132.6, 125.1 (C-
purine), 139.4 (CH-purine), 29.4, 26.6 (CH3), 24.0 (CH-cyclopropyl). IR (KBr): ν
= 3430.35 (N-H str), 3112.26 (N=CH str), 2926.94, (C-H str), 2852.82, (C-H str),
1600.30 (CONH str), 1503.55 (Ar C=C str), 1439.67 (C-N str), 1155.55 (C-O-C str),
792.46 (C-I str). Anal. Calcd. for C16H19IN6O4: C, 39.52; H, 3.94; N, 17.28. Found: C,
39.59; H, 4.01; N, 17.19. HRMS (TOF) m/z found 487.1712 [calcd for C16H19IN6O4
487.2723 (M+1)+].
-Iodo-ꢀ-N-cyclopropylcarboxamidoadenosine ()
White solid, 4.3 g, 73.4% yield, mp169°C, Rf value: 0.4 (15% Methanol in chlo-
1
roform). H NMR (400 MHz, d6-DMSO): δ = 8.24 (s, 1H, =CHN), 8.2 (s, 1H,
CONH), 7.82 (s, 2H, NH2), 5.95 (t, 1H, J = 7.0 Hz, H-2S), 5.82 (t, 1H, J = 6.9 Hz, H-
5R), 5.18 (m, 1H, H-3R), 4.62 (d, 1H, J = 5.3 Hz, H-4S), 4.25 (s, 1H, OH), 4.2 (s, 1H,
OH), 2.6 (m, 1H, CH), 0.65 (d, 4H, CH2-CH2). 13C NMR (100 MHz, d6-DMSO): δ
= 171.3 (CONH), 160.9, 135.2, 128.6, 126.52 (C-purine), 145.7 (CH-purine), 25.0
(CH-cyclopropane). IR (KBr): ν = 3424.41 (O-H str), 3346.79 (N-H str), 3181.25
(N=CH str), 2927.89 (C-H str), 2853.50 (C-H str), 1616.76 (CONH str), 1544.19,
1442.91 (Ar C=C str), 1408.21(C-N str), 1165.21 (C-O-C str), 794.88 (C-I str). Anal.
Calcd. for C13H15IN6O4: C, 34.99; H, 3.39; N, 18.83. Found: C, 35.01; H, 3.32; N,
18.89. LC-MS (TOF) m/z: 447.26 [M+1]+.
General procedure for Sonogashira cross-coupling
2-Iodo-5ꢀ-N-cyclopropylcarboxamidoadenosine (9) (2 g, 4.4 mmol) was dissolved
in a mixture of 13.2 mL of dry N,N-dimethylformamide (DMF) and 20 mL of dry
triethyl amine, and 5.0 mmol of the desired terminal alkyne derivative[20,21] (10a-
g) was added to this mixture. The reaction mixture was bubbled with nitrogen
for 15 minutes, and subsequently CuI (152 mg, 0.4 equivalents) and Pd(PPh3)2Cl2
(0.2 equivalents) were added under nitrogen atmosphere. The reaction mixture was
stirred for 24 h at room temperature. The solvents were removed under reduced
pressure to obtain an oily alkynylated purine derivative. The oily residue was dis-
solved in 120 mL of AcOEt and then washed with water (9 ×10 mL) and brine

644
A. A. MOHAN ET AL.
Table . Structures of the modified purine nucleosides 11a-g synthesized from compound 9 and 10a-
g.
Entry
9
10a-g
11a-g
11a
11b
11c
11d
11e
11f
11g
(15 mL). The organic layer was dried over MgSO4 and the solvents were evaporated
under vacuum to obtain desired alkynylated derivatives 11a-g. Table 1 presents the
detailed structures of the corresponding reactants and products. The pure com-
pounds were obtained through silica gel column chromatography or reverse-phase
HPLC (gradient: acetonitrile and formic acid), and their corresponding structures
were confirmed by spectral data analysis.
-(-(-Methoxycarbonyl)
piperidene--yl)propyn--yl)-ꢀ-N-cyclopropylcarboxamidoadenosine (a)
Light brown solid, 1.9 g, 85% yield, mp131.6–134.9°C, Rf value: 0.5 (CHCl3/CH3OH
4:1). 1H NMR (400 MHz, d6-DMSO): δ = 8.25 (s, 1H, =CHN), 8.23 (s, 1H, CONH),

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
645
7.42 (s, 2H, NH2), 5.95 (t, 1H, J = 7.1 Hz, H-2R), 5.62 (t, 1H, J = 6.6 Hz, H-5S), 5.18
(d, 1H, J = 5.5 Hz, H-3S), 4.62 (d, 1H, J = 5.0 Hz, 4R), 4.25 (s, 1H, OH), 4.2 (m,
1H, CH), 4.1 (broad singlet, 1H, OH), 3.6 (s, 3H, OCH3), 3.1 (t, 1H, piperidine), 2.8
(m, 2H, piperidine), 2.2 (d, 2H), 1.8 (m, 2H, piperidine), 1.2 (m, 4H, piperidine),
0.45–0.55 (d, 4H, CH2-CH2). 13C NMR (100 MHz, d6-DMSO): δ = 170.0 (CONH),
162.5 (CH-purine), 132.5, 126.8, 126.0, 122.4 (C-purine), 88.3, 82.9 (C-acetylene),
48.2, 44.5, 38.8 (CH2-piperidine), 29.4(CH-piperidine), 26.5 (CH2-cyclo propane).
IR (KBr): ν = 3422.07 (O-H str), 3263.73 (N-H str), 3120.87 (N=CH str), 2923.19,
2852.63 (C-H str), 1673.53 (CONH str), 1641.96 (COOMe str), 1593.11, 1489.76
(Ar C=C str), 1203.51 (C-N str), 1126.79, 1048.93. (C-O-C str). Anal. Calcd. for
C23H29N7O6: C, 55.30; H, 5.85; N, 19.63. Found: C, 55.16; H, 5.92; N, 19.66. LC-MS
(TOF) m/z: 500.02 [M+1]+ and 521.99 [M+Na]+.
-(-(,-Dimethoxyphenoxy)propyn--yl)-ꢀ-N-cyclopropylcarboxamidoadenosine
(b)
White solid, 1.6 g, 86% yield, mp 126.6–128.5°C, Rf value: 0.4 (CHCl3/CH3OH 4:1).
1H NMR (400 MHz, d6-DMSO): δ = 7.91 (s, 1H, =CHN), 7.38 (m, 2H, ArH), 7.28
(s, 1H, CONH), 7.10 (s, 1H, ArH), 7.02 (s, 2H, NH2), 5.26 (t, 1H, J = 6.8 Hz, H-2R),
4.68 (t, 1H, J = 5.5 Hz, H-5S), 4.20 (d, 1H, J = 5.0 Hz, H-3S), 4.04 (d, 1H, J = 5.8 Hz,
4R), 3.84 (s, 1H, OH), 3.68 (broad singlet, 1H, OH), 3.02 (s, 6H, Ar-OCH3), 2.3 (m,
1H, CH), 2.1 (s, 2H, CH2), 1.7 (t, J = 3.5 Hz, 2H, cyclopropyl), 1.1 (t, 2H, cyclo-
propyl). 13C NMR (100 MHz, d6-DMSO): δ = 174.9 (CO NH), 164.6, 131.3, 130.5,
126.8 (C-purine), 141.8 (CH-purine), 120.7, 116.8, 108.7(C-benzene), 86.4, 84.5 (C-
acetylene), 71.2, 61.4 (CH-tetrahydrofuran), 54.5 (CH3), 24.8 (CH-cyclopropane).
IR (KBr): ν = 3442.5 (OH str), 3057.1 (N-H str), 2927.4, 2846.0 (C-H str), 2805.3,
1622.1 (CONH str), 1520.2, 1478.6(Ar C=C str), 1441.7 (OCH3 bending), 1371.5,
1203.3 (C-N str), 1041.0 (C-O-C str). Anal. Cald. for C24H26N6O7: C, 56.47; H, 5.12,
N, 16.44. Found: C, 56.36; H, 5.03; N, 16.32. LC-MS (TOF) m/z: 511.6 [M+1]+.
-(-(o-Tolyloxy)propyn--yl)- ꢀ-N-cyclopropyl carboxamidoadenosine (c)
White solid, 1.1 g, 81% yield, mp 125.8°C, Rf value: 0.6 (CHCl3/CH3OH 4:1). 1H
NMR (400 MHz, d6-DMSO): δ = 8.82 (s, 1H, =CHN), 8.45 (s, 1H, CONH), 8.28
(d, 1H, ArH), 7.45 (dd, 2H, J = 7.5, 9.0 Hz, ArH), 7.02 (d, 1H, ArH), 5.94 (t, 1H, J
= 7.0 Hz, H-2R), 5.64 (s, 2H, NH2), 4.64 (t, 1H, J = 5.5 Hz, H-5S), 4.18 (d, 1H, J =
5.0 Hz, H-3S), 4.04 (d, 1H, J = 4.5 Hz, 4R), 3.84 (s, 1H, OH), 3.68 (broad singlet,
1H, OH), 3.60 (s, 3H, Ar-OCH3), 2.62 (m, 1H, CH), 1.8 (s, 2H, CH2), 0.90 (t, 2H,
cyclopropyl), 0.72 (t, 2H, cyclopropyl). 13C NMR (100 MHz, d6-DMSO): δ = 175.0
(CONH), 168.5, 146.5, 144.6, 131.3 (C-purine), 136.5 (CH-purine), 130.8, 127.2,
116.8, 107.7 (C-benzene), 87.4, 86.5 (C-Acetylene), 26.9 (CH-cyclopropane), 16.8
(CH3). IR (KBr): ν = 3417.9 (O-H str), 3057.4 (N-H str), 2927.8, 2846.7 (C-H str),
1621.7 (CONH str), 1520.2, 1478.4 (Ar C=C), 1204.0 (C-N str), 1139.2, 1100.7 (C-
O-C str). Anal. Cald. for C23H24N6O5: C, 59.48; H, 5.21, N, 18.09. Found: C, 59.36;
H, 5.13; N, 18.12. LC-MS (TOF) m/z: 465.7 [M+1]+.

646
A. A. MOHAN ET AL.
-(-(,,-Trimethoxyphenoxy) propyn--yl)-
ꢀ-N-cyclopropylcarboxamidoadenosine (d)
White solid, 0.9 g, 76% yield, mp 142.6°C, Rf value: 0.2 (ethylacetate/n-hexane 1:1).
1H NMR (400 MHz, d6-DMSO): δ = 8.45 (s, 1H, CONH), 8.02 (s, 1H, =CHN), 7.35
(s, 2H, NH2), 7.02 (s, 1H, ArH), 6.76 (s, 1H, ArH), 5.36 (t, 1H, J = 7.5 Hz, H-2R),
5.13 (t, 1H, J = 6.8 Hz, H-5S), 4.90 (d, 1H, J = 5.5 Hz, H-3S), 4.44 (d, 1H, J = 5.0 Hz,
4R), 4.22 (s, 1H, OH), 4.08 (s, 1H, OH), 3.16 (m, 1H, CH), 3.02 (s, 9H, Ar-OCH3),
2.07 (t, 2H, J = 1.5 Hz, cyclopropyl), 1.91 (t, 2H, J = 2.1 Hz, cyclopropyl), 1.2 (s, 2H,
CH2). 13C NMR (100 MHz, d6-DMSO): δ = 174.9 (CO NH), 167.5, 144.6, 136.2,
135.7 (C-purine), 142.5 (CH-purine), 122.2, 121.7, 119.5, 116.7, 106.9 (C-benzene),
87.9, 84.6 (C-acetylene), 61.6, 54.1 (CH3), 26.4 (CH-cyclopopane). IR (KBr): ν =
3417.9 (O-H str), 3057.7 (N-H str), 2926.8, 2849.2 (C-H str), 1739.3 (CONH str),
1622.3, 1520.7 (Ar C=C str), 1479.7 (OCH3 str), 1202.8 (C-N str), 1102.6, 1045.8
(C-O-C str). Anal. Cald. for C25H28N6O8: C, 55.55; H, 5.22, N, 15.55. Found: C,
55.42; H, 5.13; N, 15.52. LC-MS (TOF) m/z: 541.281 [M+1]+.
-(-(p-Tolyloxy)propyn--yl)-ꢀ-N-cyclopropyl carboxamidoadenosine (e)
White solid, 0.7g, 85% yield, mp141.5°C, Rf value: 0.4 (CHCl3/CH3OH 4:1). H
1
NMR (400 MHz, d6-DMSO): δ = 8.21 (s, 1H, =CHN), 7.64 (s, 1H, CONH), 7.26
(d, 2H, J = 7.5 Hz, ArH), 7.08 (d, 2H, J = 7.6 Hz, ArH), 6.70 (s, 2H, NH2), 5.04 (t,
1H, J = 5.5 Hz, H-2R), 4.74 (t, 1H, H-5S), 4.46 (d, 1H, H-3S), 4.24 (d, 1H, 4R), 4.12
(s, 1H, OH), 3.98 (s, 1H, OH), 3.60 (s, 3H, CH3), 2.62 (m, 1H, CH), 1.8 (s, 2H, CH2),
0.90 (t, 2H, J = 1.3 Hz, cyclopropyl), 0.72 (t, 2H, J = 1.0 Hz, cyclopropyl). 13C NMR
(100 MHz, d6-DMSO): δ = 174.9 (CONH), 145.4, 134.8, 129.2, 126.7 (C-purine),
137.2 (CH-purine), 121.5, 119.2, 116.7, 108.6 (C-benzene), 86.7, 84.5 (C-acetylene),
54.8 (CH2), 21.5 (CH3). IR (KBr): ν = 3415.3 (O-H str), 3057.4 (N-H), 2927.3 (C-H
str), 1739.3 (CONH str), 1621.8 (Ar C=C str), 1371.8, 1295.6 (C-O str), 1253.2 (C-
N str), 1138.8, 1007.5 (C-O-C str). Anal. Cald. for C23H24N6O5: C, 59.48; H, 5.21,
N, 18.09. Found: C, 59.39; H, 5.18; N, 18.06. HRMS (TOF) m/z found 465.13176
[calcd for C23H24N6O5 465.18864 (M+1)+].
-(-(-Chlorophenoxy)propyn--yl)-ꢀ-N-cyclopropylcarboxamidoadenosine (f)
White solid, 0.6 g, 76.9% yield, mp148.2°C, Rf value: 0.3 (ethylacetate/n-hexanes
1:1). 1H NMR (400 MHz, d6-DMSO): δ = 8.24 (s, 1H, =CHN), 7.86 (s, 1H, CONH),
7.42 (d, 2H, J = 7.4 Hz, ArH), 7.20 (s, 2H, NH2), 7.10 (d, 2H, J = 7.0 Hz, ArH), 5.26
(t, 1H, J = 5.5 Hz, H-2R), 5.04 (t, 1H, J = 5.5 Hz, H-5S), 4.62 (d, 1H, H-3S), 4.22
(d, 1H, 4R), 4.14 (s, 1H, OH), 4.03 (s, 1H, OH), 2.89 (m, 1H, CH), 2.7 (s, 2H, CH2),
2.69 (t, 2H, J = 2.1 Hz, cyclopropyl), 2.42 (t, 2H, J = 1.6 Hz, cyclopropyl). 13C NMR
(100 MHz, d6-DMSO): δ = 172.8 (CONH), 147.1, 131.0, 130.3, 128.2 (C-purine),
136.2 (CH-purine), 128.7, 127.8, 125.0, 122.1 (C-benzene), 85.1, 87.5 (C-acetylene),
26.7 (CH-cyclopropane). IR (KBr): ν = 3422.5 (O-H str), 3057.1 (N-H str), 2927.4,
2846.0 (C-H str), 1739.0 (CONH str), 1622.1 (Ar C=C str), 1371.5, 1295.4 (C-O
str), 1253.4 (C-N str), 1138.3, 1041.0 (C-O-C str), 750.2 (C-Cl). Anal. Cald. for

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
647
C22H21ClN6O5: C, 54.49; H, 4.37, N, 17.33. Found: C, 54.36; H, 4.33; N, 17.29.
HRMS (TOF) m/z found 485.15143 [calcd for C22H21ClN6O5 485.13402 (M+1)+].
-(-(-Nitrophenoxy)propyn--yl)-ꢀ-N-cyclopropylcarboxamidoadenosine (g)
Light yellow solid, 0.5 g, 85% yield, mp153.2°C, Rf value: 0.4 (CHCl3/CH3OH 4:1).
1H NMR (400 MHz, d6-DMSO): δ = 8.84 (s, 1H, =CHN), 8.46 (s, 1H, CONH),
8.22 (d, 1H, J = 7.5 Hz, ArH), 7.42 (d, 2H, J = 7.0 Hz, ArH), 6.12 (d, 1H, J = 7.0 Hz,
ArH), 5.16 (s, 2H, NH2), 4.64 (s, 1H, OH), 4.26 (t, 1H, H-2R), 4.2 (t, 1H, H-5S),
4.03 (broad singlet, 1H, OH), 3.62 (d, 1H, H-3S), 2.90 (d, 1H, 4R), 2.81 (m, 1H,
CH), 2.4 (s, 2H, CH2), 1.81 (t, 2H, J = 1.6 Hz, cyclopropyl), 0.86 (t, 2H, J = 1.2 Hz,
cyclopropyl). 13C NMR (100 MHz, d6-DMSO): δ = 172.9 (CONH), 148.1, 133.2,
129.0, 127.4 (C-purine), 140.2 (CH-purine), 124.2, 122.8, 177.7, 108.9 (C-benzene),
90.2, 88.4 (C-acetylene), 27.4 (CH-cyclopropane). IR (KBr): ν = 3448.7 (O-H str),
3347.5 (N-H str), 2941.8, 2840.2 (C-H str), 1587.3 (CONH str), 1488.5 (Ar C=C str),
1393.8 (N-O str), 1262.9 (C-N str). Anal. Cald. for C22H21N7O7: C, 53.33; H, 4.27,
N, 19.79. Found: C, 53.28; H, 4.19; N, 19.72. LC-MS (TOF) m/z: 496.17 [M+1]+.
Results and discussion
Synthesis and characterization
Scheme 1 shows the synthesis of C2, C6 and C5ꢀ modified purine nucleoside deriva-
tives from commercially available guanosine. In a one-pot reaction (step1), the
acetyl group was selectively introduced on the C2ꢀ, C3ꢀ and C5ꢀ hydroxyl groups
by using acetic anhydride. The amino group of guanine has low nucleophilicity;
therefore, it does not react with acetic anhydride. Acetylated derivative 2 was puri-
fied through reverse-phase HPLC (0%−50% gradient: acetonitrile and ammonium
acetate ). In step2, the carbonyl group in the nucleobase was converted to chloro
derivative 3 by reacting with POCl3 and N,N-dimethyl aniline in an acetonitrile
solvent. The resulting light yellow oily residue was purified through silica gel col-
umn chromatography with ethylacetate/methanol (v/v, 10/1), and the purified prod-
uct was used in the next step. The C2 amino group was directly converted to iodo
derivative 4 by reacting with t-butyl nitrite and di-iodomethane (step-3). Notably,
the expected nucleophilic aromatic substitution with NH3 at the C6 position of the
purine in compound 4 occurred easily, and this substitution was accompanied by
the cleavage of the acetate protecting groups in compound 5. The resulting residue
was recrystallized with hot isopropanol and purified through reverse-phase HPLC
(gradient: acetonitrile and ammonium acetate in water). The regioselective protec-
tion of the C2ꢀ and C3ꢀ diol moieties of compound 5 (step5) with 2,2-dimethoxy
propane in PTSA yielded compound 6, which was subsequently oxidized (step6)
with KMnO4. The C5ꢀ hydroxyl group in the sugar moiety was oxidized to carboxylic
acid derivative 7. After workup, the resulting pure crude mixture was used in the
next step without further purification. In step7, commercially available HOBt was
used to convert the C5ꢀ carboxylic group to amide derivative 8 in DMF solvent. The

648
A. A. MOHAN ET AL.
Table . In vitro antibacterial activity of compounds 11a-g tested by the well-diffusion agar assay
expressed as the diameter (mm) of the inhibition zone.
Zone of Inhibition (Diameter mm) . mg/mL
Staphylococcus aureus Bacillus alcalophilus
Pseudomonas
Escherichia coli (−ve) aeruginosa (−ve)
Tested compounds
(+ve)
(+ve)
11a
—




—

—




—


—




—


—




—


11b
11c
11d
11e
11f
11g
Norfloxacin

resulting compound was purified through reverse-phase HPLC (gradient: acetoni-
trileand trifluoroacetic acid in water). The deprotection (step8) of the C2ꢀ and C3ꢀ
diol moieties was performed by treatment with formic acid to yield compound 9.
Generally, Pd-catalyzed cross-coupling reactions[22,23] are efficient for the alkyny-
lation of many iodo- or bromo- substituted nucleoside derivatives with terminal
acetylenes. Therefore, different C2 alkynylated purine nucleoside analogues 11a-
g were synthesized (step 9) by performing Sonogashira cross-coupling with corre-
sponding C2 iodo- derivative 9 and terminal alkyne derivatives 10a-g.
Reagents and conditions
(A) Ac2O, Dry DMF, Dry pyridine, 3.75 h, 75°C; (B) POCl3, N,N-Dimethyl ani-
line, Tetra methyl ammonium chloride, Acetonitrile, 16 h, 0°C to rt; (C) t-Butyl
nitrite, CH2I2, Acetonitrile, 4.5 h, 80°C; (D) Liquid NH3, 18 h, −78°C to rt; (E) p-
Toluene sulfonic acid (PTSA), 2,2-Dimethoxy propane, Acetone, 4 h, rt; (F) KMnO4,
KOH, H2O, 72 h, rt; (G) EDCl, HOBt (N-hydroxybenzo triazole), Cyclopropy-
lamine, DMF, TEA, Over night; (H) 50% Formic acid, 1.5 h, 80°C; (I) Pd(PPh3)2Cl2,
Cuprous iodide, Dry DMF, Dry acetonitrile, Dry triethylamine, 24 h, rt.
Biological evaluation
Antibacterial activity
The in vitro inhibitory effects of modified purine nucleoside analogues 11a-g
and the known antibacterial agent norfloxacin were evaluated against two gram-
positive bacteria Staphylococcus aureus and Bacillus alcalophilus, as well as two
gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, in agar well
diffusion assays. The screening results (Table 2) indicated that all synthesized com-
pounds exert variable inhibitory effects on gram-positive and gram-negative bacte-
ria.
Compounds 11a and 11f did not exert inhibitory effects on the examined bac-
terial species. Compared with norfloxacin compounds 11c and 11g showed higher
inhibitory effects on S. aureus and P. aeruginosa. Compound 11c exerted similar
inhibitory effects against E. coli as did norfloxacin and showed intermediate effects

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
649
Table . In vitro anti-cancer activity of compounds 11a-g tested by the MTT assay expressed as IC
(µg/mL) values.
IC (µg/mL)
Tested compound
MDA-MB-
Caco-
11a
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
11b
11c
11d
11e
11f
11g
Doxorubucin
against the other bacterial species. However, compound 11b and 11d did not exert
considerable inhibitory effects against the examined bacterial species.
Anticancer activity
The in vitro antiproliferative effects of modified purine nucleoside analogues 11a-
g were evaluated against human breast and colon cancer cell lines MDA-MBA-231
and Caco-2, respectively, in the MTT assay. The known anticancer drug doxorubicin
(DXN) was used as a reference. Table 3 shows the antoproliferative effects of the
modified purine nucleoside analogues as represented by the IC50 values.
Compounds 11b and 11e exerted considerable antiproliferative effects, where as
compounds 11c and 11d showed moderate antifroliferative effects against the MDA-
MB-231 cell line. Compounds 11d and 11g and DXN had similar antiproliferative
effects against the Caco-2 cell line.
Conclusion
The present study describes the convenient Sonogashira cross-coupling reaction-
based synthesis of C5ꢀ-N-cyclopropylcarboxamido-C6-amino-C2-alkynylated
purine nucleoside analogues (11a-g) by using bis(triphenylphosphine)palladium
(II)chloride and cuprous iodide. The heftiness of step 9 is demonstrated by
the synthesisof numerous derivatives (11a-g). The synthetic intermediates and
1
final derivatives were appropriately characterized through IR, H NMR, 13C
NMR, and mass spectroscopy. The antibacterial activity of the final deriva-
tives was evaluated against two gram-positive and two gram-negative bacte-
ria, and anticancer activity was examined against MDA-MB-231 and Caco-
2 cell lines. Compounds 11e and 11g had higher antibacterial activity than
did norfloxacin and compounds 11b and 11e showed considerable anticancer
activity.
Acknowledgements
We gratefully acknowledge the financial support of Dr.B.R.Ambedkar University, Srikakulam.
Arasavelli Ananda Mohan specifically thanks to Dr.G.V.R.Sharma and Dr.V. Siddaiah for provid-
ing required facilities and motivation for completion of the research work.

650
A. A. MOHAN ET AL.
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