Synthesis and biological evaluation of modified purine homo-N-nucleosides containing pyrazole or 2-pyrazoline moiety

Article abstract of DOI:10.3109/14756366.2012.755623

9-Substituted (pyrazol-5-yl)methyl- or (2-pyrazolin-5-yl)methyl-9H-purines were synthesized from 9-allyl-6-chloro-9H-purine through the 1,3-dipolar cycloaddition reaction with nitrile imines, prepared in situ from the corresponding hydrazone and NBS/Et3N under MW or from hydrazinoylchloride and Et3N under reflux. The coupling of new 6-chloropurines with amines in H2O under microwaves resulted quantitatively to modified pyrazol-5-yl- or 2-pyrazolin-5-yl adenine homo-N-nucleosides. The new compounds were tested in vitro for their ability to: (i) interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH), (ii) inhibit lipid peroxidation, (iii) inhibit the activity of soybean lipoxygenase, (iv) inhibit in vitro thrombin and for (v) their antiproliferative and cytotoxic activity. Pyrazolines were found to be more potent in vitro. Compound 7a exhibited satisfactory combined antioxidant and anti-lipid peroxidation activity, inhibition of lipoxygenase (89%) and thrombin inhibitory ability, whereas compound 7b exhibited high lipoxygenase inhibitory activity in combination to significant anti-thrombin activity. No compound exhibited a significant cytotoxic activity, while all showed moderate antiproliferative activity.

Full text of DOI:10.3109/14756366.2012.755623

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(1): 109–117
2014 Informa UK Ltd. DOI: 10.3109/14756366.2012.755623
!
Synthesis and biological evaluation of modified purine
homo-N-nucleosides containing pyrazole or 2-pyrazoline moiety
Andreas Thalassitis1, Anna-Maria Katsori2, Konstantinos Dimas3, Dimitra J. Hadjipavlou-Litina2, Fokion Pyleris1,
Nikolaos Sakellaridis3, and Konstantinos E. Litinas1
2
1Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece, Department of
3
Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece, and Department of Pharmacology,
Faculty of Medicine, Thessaly University, Biopolis Larissa, Greece
Abstract
Keywords
9-Substituted (pyrazol-5-yl)methyl- or (2-pyrazolin-5-yl)methyl-9H-purines were synthesized
from 9-allyl-6-chloro-9H-purine through the 1,3-dipolar cycloaddition reaction with nitrile
imines, prepared in situ from the corresponding hydrazone and NBS/Et3N under MW or from
hydrazinoylchloride and Et3N under reflux. The coupling of new 6-chloropurines with amines in
H2O under microwaves resulted quantitatively to modified pyrazol-5-yl- or 2-pyrazolin-5-yl
adenine homo-N-nucleosides. The new compounds were tested in vitro for their ability to:
(i) interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH), (ii) inhibit lipid peroxidation, (iii) inhibit
the activity of soybean lipoxygenase, (iv) inhibit in vitro thrombin and for (v) their
antiproliferative and cytotoxic activity. Pyrazolines were found to be more potent in vitro.
Compound 7a exhibited satisfactory combined antioxidant and anti-lipid peroxidation activity,
inhibition of lipoxygenase (89%) and thrombin inhibitory ability, whereas compound 7b
exhibited high lipoxygenase inhibitory activity in combination to significant anti-thrombin
activity. No compound exhibited a significant cytotoxic activity, while all showed moderate
antiproliferative activity.
9-Allylpurines, 1,3-dipolar cycloaddition
reaction, homo-N-nucleosides, lipid
peroxidation inhibitors, nitrile imines,
pyrazole, pyrazoline, thrombin inhibitors
History
Received 19 November 2012
Revised 1 December 2012
Accepted 1 December 2012
Published online 23 January 2013
Introduction
cycloaddition reactions of mesityl nitrile oxide with 9-allyl
derivatives of 6-chloropurine, 6-piperidinylpurine, 6-morpholi-
nylpurine, 6-pyrrolidinyl purine and 6-N,N-dibenzoyladenine
inhibited thrombin and lipid peroxidation. The majority of these
compounds showed significant lipoxygenase inhibitory activity
too. Most of the LO inhibitors are antioxidants or free radical
scavengers, since lipoxygenation occurs via a carbon-centered
radical11.
It is generally accepted that there is a close association
between cancer and chronic inflammation12–14. Epidemiological
studies have also shown that chronic inflammation preexists in
some types of cancer. It is therefore evident that the use of multi-
target ligands, that interact with multiple targets, could be
valuable for the treatment of the abovementioned pathophysiolo-
gical conditions15. It has already been proven for a number of
commercially available nonsteroidal anti-inflammatory drugs
(NSAIDs), e.g. aspirin, that they possess a combination of these
properties.
Pyrazoline16–18 and pyrazole19 derivatives are important
biological agents. In particular, pyrazoline derivatives have
antimicrobial, anticancer, antiviral, anti-inflammatory20, antide-
pressant21, anticonvulsant22, antiamoebic23 activity. The pyrazole
derivatives present19 analgesic, hypoglycemic, antibacterial, anti-
inflammatory, insecticidal, anti-influenza virus24, antimicrobial25
activity. Pyrazolines are prepared mainly by cyclization of
chalcones with hydrazines16,20,23, from 1,3-dipolar cycloaddition
reactions of nitrile imines to alkenes26 by heating27 or under
microwaves28 or from the cyclocondensation of hydrazines with
1,3-dihalides under MW irradiation29. Pyrazoles are synthesized
Nucleosides represent a class of compounds that possess very
interesting biological activities1,2. The adenosine generated at
inflamed site is receiving increasing interest as an endogenous
anti-inflammatory agent and presents potential pharmacological
uses as anti-inflammatory agent3,4. The homo-N-nucleosides with
a CH2-group between adenine and carbocyclic or heterocyclic
ring possess higher conformational flexibility5,6 to combine with
the bases of DNA/RNA by a lowering of the electrostatic
repulsion6.
Oxidation is an important process which produces free radicals
in living systems. Persistently high levels of reactive oxygen
species (ROS) are believed to produce pathological conditions.
ROS, like superoxide radical anion, hydrogen peroxide and
hydroxyl radical, are produced during the inflammation process
by phagocytic leukocytes at the inflamed site and they are
involved in the biosynthesis of prostaglandins and in the
cycloxygenase (COX)- and lipoxygenase (LO)-mediated conver-
sion of arachidonic acid into proinflammatory intermediates7,8
.
Initial studies of the effects of adenosine on human neutrophiles9
indicated that adenosine inhibits stimulated Oꢀ2 or H2O2
generation. From our recent research10, it was found that some
new homo-N-nucleosides, derived from the 1,3-dipolar
Address for correspondence: Konstantinos E. Litinas, Laboratory of
Organic Chemistry, Aristotle University of Thessaloniki, Thessaloniki
54124, Greece. Tel: þ302310997864. Fax: þ302310997679. E-mail:
klitinas@chem.auth.gr

110 A. Thalassitis et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 109–117
Cl
N
(iii)
N
for 3b
H
N
N
N
N
Me
Cl
N
Cl
N
Cl
PhCH=NNHAr
N
N
N
N
N
N
N
N
2a,b
N
Ar
+
NH
+
(i)
N
N
N
N
N
N
N
N
Ph
Ph
N
3a,b
1
4
N
Cl
2,3a: Ar=Ph
AH
6a-d
AH
6a-c
b: Ar=4-Me-C6H4
5
(ii)
(ii)
A
Ar
A
CH3
A
N
Ph
Ph
7a:
b:
N
N
N
O
N
A
N
N
N
Ar
N
6,8a:
b:
O
N
N
N
N
N
N
N
N
N
Ph
c:
Ph
Ph
7a-g
Ph
d:
e:
Boc-N
O
N
8a-c
c:
4-Me-C6H4
N
N
N
d:
Boc-N
N
4-Me-C6H4
4-Me-C6H4
f:
g:
Scheme 1. Reagents and conditions: (i) 2, NBS, benzene, stirring 15 min r.t.; 1, Et3N, MW, 80 ꢁC, 80 min. (ii) H2O, MW, 100 ꢁC, 1 min. (iii) DDQ, dry
toluene, reflux, 48 h.
mainly by the reaction of hydrazines with b-difunctional
O
compounds30, from tosylhydrazones of a,b-unsaturated ketones
under MW irradiation31 and by 1,3-dipolar cycloaddition reac-
tions of tosylhydrazone salts32,33 or nitrile imines25 with alkynes.
N
N
N
In continuation to our work in the field of modified homo-
N-nucleosides, we present here the reactions of 9-allyl-6-
chloropurine with nitrile imines under MW irradiation and the
replacement of chlorine by amines in the above products
(Schemes 1 and 2). The biological evaluation of the new
2-pyrazolines and pyrazoles as lipoxygenase and lipid peroxida-
tion inhibitors, cytotoxic and antiproliferative agents and
simultaneously as thrombin inhibitors is also studied.
N
N
1
10
(i)
Ph-C=NNH-Ph
3a
7a
(i)
Cl
9
Scheme 2. Reagents and conditions: (i) CHCl3, Et3N, reflux, 7 h.
Materials and methods
Melting points were determined on a Kofler hot-stage apparatus
(Arthur H. Thomas, Co, Philadelphia, PA) and are uncorrected. IR
spectra were obtained with a Perkin-Elmer (Waltham, MA) 1310
spectrophotometer as Nujol mulls. NMR spectra were recorded on
a Bruker AM 300 (Bruker A.G., Karlsruhe, Germany) (300 MHz
and 75 MHz for 1H and 13C, respectively) using CDCl3 as solvent
and TMS as an internal standard. J values are reported in Hz.
Mass spectra were determined on a Shimadzu (Manchester, UK)
LCMS-2010 EV system under electrospray ionization (ESI)
conditions. Microanalyses were performed on a Perkin-Elmer
2400-II Element analyzer. For in vitro determination a UV-Vis
Perkin-Elmer Lambda Spectrophotometer was used. The
absorbance for cytotoxic activity assay was measured at 530 nm,
in an EL-311 BIOTEK microelisa reader (BioTek, Winooski,
VT). The MW experiments were performed in a Biotage (Initiator
2.0) scientific MW oven. All the reagents used were commercially
available by Merck A.G. (Darmstadt, Germany). 1,1-Diphenyl-2-
picrylhydrazyl (DPPH), nordihydroguaiaretic acid (NDGA) were
purchased from the Aldrich Chemical Co. (Milwaukee, WI.
Soybean lipoxygenase, linoleic acid sodium salt, nicotinamido-
adenine-dinucleotide (NADH), nitrotetrazolium blue (NBT),
porcine heme and indomethacin were obtained from Sigma
Chemical, Co. (St. Louis, MO). Silica gel No. 60, Merck A.G. was

DOI: 10.3109/14756366.2012.755623
Synthesis and biological evaluation of modified purine homo-N-nucleosides 111
used for column chromatography. A549 (non-small cell lung (CDCl3) ꢀ 33.0, 47.5, 49.2, 125.5, 125.7, 128.7, 130.2, 145.4,
cancer), PC3 (prostate cancer), MB435 (melanoma), CAKI and 152.3; MS (ESI): 471/473/475 [M þ H]þ. Anal. Calcd for
SN12C (renal cancer) were obtained from the National Cancer C18H16Cl2N12: C, 45.87; H, 3.42; N, 35.66. Found: C, 45.98; H,
Institute, NIH (Bethesda, MD).
3.57; N, 35.41. The unchanged starting material (41%) was eluted
as the last one.
Synthesis
General procedure for 1,3-dipolar cycloaddition reaction with
diphenylnitrile imine (from hydrazonoyl chloride) under reflux
General procedure for 1,3-dipolar cycloaddition reaction with
nitrile imines (from hydrazones) under MW irradiation
In a mixture of purine (1) (65 mg, 0.336 mmol) and hydrazonoyl
chloride (9) (0.125 g, 0.544 mmol) in CHCl3 (7 mL) Et3N (58 mg,
0.08 mL, 0.58 mmol) was added and refluxed for 7 h. After
cooling and evaporation, the residue was separated by column
chromatography [ethyl acetate/hexane (1:2)] to give from the
faster moving band the derivative (3a) (67 mg, 51% yield)
followed by the unreacted purine (1) (23 mg, 36%).
A mixture of phenylhydrazone (2a) (0.441 g, 2.25 mmol) and
NBS (0.4 g, 2.25 mmol) in benzene (15 mL) was stirred in an MW
vial at r. t. for 15 min. The purine (1) (0.146 g, 0.75 mmol) and
Et3N (0.225 g, 0.31 mL, 2.25 mmol) were then added and the
mixture was irradiated under MW at 80 ꢁC for 80 min. The
resulted solution after cooling was evaporated and separated by
column chromatography [ethyl acetate:hexane (1:4)] to give after
the elution of unreacted hydrazone (2a) and the product (3a)
(0.155 g, 53% yield). The not consumed starting purine (1)
(63 mg, 43%) was eluted next.
9-[(1,3-Diphenyl-4,5-dihydro-1H-pyrazol-5-yl)methyl]-6-mor-
polinyl-9H-purine (7a). (from the reaction of allylpurine (10))
(37% yield). Orange solid, m.p. 129–131 ꢁC (DCM), IR (Nujol):
1
3070, 1640, 1580, 1495 cmꢀ1; H-NMR (CDCl3) ꢀ 3.25 (dd, 1H,
6-Chloro-9-[(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)methyl]-
9H-purine (3a). Beige solid, m.p. 168–170 ꢁC (DCM); IR (Nujol):
;
3050, 1630, 1585 cmꢀ1 1H-NMR (CDCl3) ꢀ 3.26 (dd, 1H,
J1 ¼ 4.9 Hz, J2 ¼ 17.3 Hz), 3.36 (dd, 1H, J1 ¼ 10.8 Hz,
J2 ¼ 17.3 Hz), 3.77 (t, 4H, J ¼ 4.9 Hz), 4.19–4.28 (m, 4H), 4.30
(dd, 1H, J1 ¼ 5.7 Hz, J2 ¼ 14.3 Hz), 4.61 (dd, 1H, J1 ¼ 3.3 Hz,
J2 ¼ 14.3 Hz), 4.93–5.04 (m, 1H), 6.91 (tt, 1H, J1 ¼ 1.4 Hz,
J2 ¼ 7.0 Hz), 7.25–7.38 (m, 7H), 7.50 (s, 1H), 7.53 (dd, 2H,
J1 ¼ 1.7 Hz, J2 ¼ 7.9 Hz), 8.36 (s, 1H); 13C-NMR (CDCl3) ꢀ 36.3,
44.0, 45.7, 58.7, 67.0, 113.4, 119.8, 119.9, 125.6, 128.4, 128.8,
129.6, 132.1, 139.1, 143.8, 147.8, 151.3, 152.2, 153.8; MS (ESI):
440 [M þ H]þ., 462 [M þ Na]þ; 478 [M þ K]þ. Anal. Calcd for
C25H25N7O: C, 68.32; H, 5.73; N, 22.31. Found: C, 68.34; H,
5.80; N, 22.09. The unreacted purine (10) (61%) eluted next.
J1 ¼ 4.2 Hz, J2 ¼ 17.2 Hz), 3.43 (dd, 1H, J1 ¼ 11.1 Hz,
J2 ¼ 17.2 Hz), 4.43 (dd, 1H, J1 ¼ 5.7 Hz, J2 ¼ 14.4 Hz), 4.67 (dd,
1H, J1 ¼ 3.0 Hz, J2 ¼ 14.4 Hz), 4.97–5.05 (m, 1H), 6.93 (t, 1H,
J¼ 7.2 Hz), 7.24–7.38 (m, 7H), 7.45–7.50 (m, 2H), 7.84 (s, 1H),
8.71 (s, 1H); 13C-NMR (CDCl3) ꢀ 36.7, 44.8, 58.5, 113.4, 120.2,
125.5, 126.3, 128.5, 129.1, 129.7, 131.6, 143.6, 145.7, 147.8, 151.3,
152.0, 152.2; MS (ESI): 389/391 [MþH]þ., 411/413 [MþNa]þ;
427/429 [MþK]þ. Anal. Calcd for C21H17ClN6: C, 64.31; H, 2.65;
N, 20.35. Found: C, 64.05; H, 2.68; N, 20.55.
Oxidation of 6-chloro-9-{[1-(4-methylphenyl)-3-phenyl-4,5-
dihydro-1H-pyrazol-5-yl]methyl}-9H-purine (3b) to 6-chloro-
9-{[1-(4-methylphenyl)-3-phenyl-1H-pyrazol-5-yl]methyl}-9H-
purine (4). A solution of compound 3b (50 mg, 0.12 mmol) in dry
toluene (5 mL) was treated with DDQ (39 mg, 0.17 mmol) under
reflux for 48 h [after 24 h more DDQ (10 mg, 0.044 mmol, total
49 mg, 0.214 mmol) was added]. The solvent was evaporated and
the residue was separated by column chromatography [ethyl
acetate:hexane (1:2)] to give compound 4 (47 mg, 94% yield).
6-Chloro-9-{[1-(4-methylphenyl)-3-phenyl-4,5-dihydro-1H-
pyrazol-5-yl]methyl}-9H-purine (3b). (from the reaction of 4-
methyphenylhydrazone (2b) after MW irradiation for 90 min;
eluted first) (48% yield). Beige solid, m.p. 76–78 ꢁC(DCM-
hexane), IR (Nujol): 3040, 1580, 1545, 1495 cmꢀ1 1H-NMR
;
(CDCl3) ꢀ 2.31 (s, 3H), 3.18 (dd, 1H, J1 ¼ 4.5 Hz, J2 ¼ 17.2 Hz),
3.35 (dd, 1H, J1 ¼ 11.1 Hz, J2 ¼ 17.2 Hz), 4.37 (dd, 1H,
J1 ¼ 5.7 Hz, J2 ¼ 14.4 Hz), 4.61 (dd, 1H, J1 ¼ 3.1 Hz,
J2 ¼ 14.4 Hz), 4.85–4.98 (m, 1H), 7.10–7.14 (m, 4H), 7.24–
7.31 (m, 3H), 7.44–7.49 (m, 2H), 7.84 (s, 1H), 8.69 (s, 1H);
13C-NMR (CDCl3) ꢀ 20.5, 36.7, 44.9, 58.9, 113.7, 125.5, 125.7,
128.4, 128.7, 128.9, 129.7, 131.7, 130.2, 141.5, 145.9, 147.4,
151.9, 152.2; MS (ESI): 403/405 [M þ H]þ., 425/427
[M þ Na]þ. Anal. Calcd for C22H19ClN6: C, 65.59; H, 4.75;
N, 20.86. Found: C, 65.51; H, 4.40; N, 20.68.
General procedure for the amination of 6-chloropurine deriva-
tives under MW irradiation
In an MW vial the chloropurine (3a) (20 mg, 0.051 mmol) was
added in H2O (1 mL) along with morpholine (6a) (0.009 mL,
9 mg, 0.103 mmol) and the mixture was irradiated at 100 ꢁC for
1 min. Extraction with DCM (3 ꢂ 20 mL), drying with anhydrous
Na2SO4 and evaporation of the solvent resulted to 9-[(1,3-
Diphenyl-4,5-dihydro-1H-pyrazol-5-yl)methyl]-6-morpolinyl-9H-
purine (7a) (94% yield).
6-Chloro-9-{[1-(4-methylphenyl)-3-phenyl-1H-pyrazol-5-yl]
methyl}-9H-purine (4). (from the above reaction; eluted after
compound 3b) (3% yield). Light brown solid, m.p. 79–81 ꢁC
;
(dec.), IR (Nujol): 3030, 1590, 1560, 1510 cmꢀ1 1H-NMR
(CDCl3) ꢀ 2.40 (s, 3H), 5.52 (s, 2H), 6.73 (s, 1H), 7.20–7.56 (m,
7H), 7.78–7.82 (m, 2H), 7.79 (s, 1H), 8.72 (s, 1H); 13C-NMR
(CDCl3) ꢀ 21.1, 39.0, 105.4, 125.6, 125.7, 125.9, 128.3, 128.4,
128.6, 128.7, 130.2, 137.3, 139.5, 144.3, 152.0, 152.1; MS
(ESI): 401/403 [M þ H]þ.439/441 [M þ K]þ. Anal. Calcd for
C22H17ClN6: C, 65.92; H, 4.27; N, 20.96. Found: C, 65.83; H,
4.37; N, 20.74.
9-[(1,3-Diphenyl-4,5-dihydro-1H-pyrazol-5-yl)methyl]-6-
piperidinyl-9H-purine (7b). (97% yield). Orange solid, m.p.
68–70 ꢁC (DCM), IR (Nujol): 3050, 1635, 1570, 1495 cmꢀ1
;
1H-NMR (CDCl3) ꢀ 1.58–1.80 (m, 6H), 3.25 (dd, 1H,
J1 ¼ 4.9 Hz, J2 ¼ 17.3 Hz), 3.36 (dd, 1H, J1 ¼ 10.8 Hz,
J2 ¼ 17.3 Hz), 4.10–4.24 (m, 4H), 4.27 (dd, 1H, J1 ¼ 5.9 Hz,
J2 ¼ 14.3 Hz), 4.60 (dd, 1H, J1 ¼ 3.4 Hz, J2 ¼ 14.3 Hz), 4.93–
5.05 (m, 1H), 6.90 (t, 1H, J ¼ 6.9 Hz), 7.24–7.43 (m, 7H), 7.51
(s, 1H), 7.55 (dd, 2H, J1 ¼ 1.7 Hz, J2 ¼ 7.7 Hz), 8.34 (s, 1H);
13C-NMR (CDCl3) ꢀ 24.7, 26.1, 36.4, 44.1, 46.6, 58.8, 113.5,
119.7, 119.9, 125.7, 128.5, 128.8, 129.6, 132.2, 138.6, 143.9,
147.9, 150.6, 151.1, 152.0; MS (ESI): 438 [M þ H]þ, 460
[M þ Na]þ; 476 [M þ K]þ. Anal. Calcd for C26H27N7: C,
71.37; H, 6.22; N, 22.41. Found: C, 71.32; H, 6.35; N, 22.16.
5,50-Bis[(6-chloro-9H-purin-9-yl)methyl]-4,40,5,50-tetrahydro-
1H,10H-3,30-bipyrazole (5). (from the above reaction; eluted after
compound 4) (6% yield). Oil, IR (Nujol): 3230, 3060, 3020, 1585,
1
1560, 1490 cmꢀ1; H-NMR (CDCl3) ꢀ 3.73 (dd, 1H, J1 ¼ 8.6 Hz,
J2 ¼ 11.1 Hz), 3.88 (dd, 1H, J1 ¼ 4.1 Hz, J2 ¼ 11.1 Hz), 4.59 (dd,
1H, J1 ¼ 8.3 Hz, J2 ¼ 14.4 Hz), 4.67–4.78 (m, 1H), 5.03 (dd, 1H,
J1 ¼ 3.8 Hz, J2 ¼ 14.4 Hz), 8.25 (s, 1H), 8.77 (s, 1H); 13C-NMR

112 A. Thalassitis et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 109–117
9-[(1,3-Diphenyl-4,5-dihydro-1H-pyrazol-5-yl)methyl]-6-pyr-
rolidinyl-9H-purine (7c). (97% yield). Yellow solid, m.p. 98– (m, 1H), 7.14 (d, 2H, J ¼ 8.6 Hz), 7.18 (d, 2H, J ¼ 8.6 Hz), 7.23–
J2 ¼ 14.3 Hz), 4.56 (dd, 1H, J1 ¼ 3.3 Hz, J2 ¼ 14.3 Hz), 4.86–4.97
100 ꢁC (DCM), IR (Nujol): 3060, 1640, 1585 cmꢀ1
;
1H-NMR 7.36 (m, 3H), 7.49 (s, 1H), 7.53 (dd, 1H, J1 ¼ 1.6 Hz, J2 ¼ 7.8 Hz),
(CDCl3) ꢀ 1.90–2.11 (m, 4H), 3.24 (dd, 1H, J1 ¼ 5.0 Hz, 8.34 (s, 1H); 13C-NMR (CDCl3) ꢀ 20.5, 29.7, 36.4, 43.9, 47.9,
J2 ¼ 17.3 Hz), 3.35 (dd, 1H, J1 ¼ 10.8 Hz, J2 ¼ 17.3 Hz), 3.60– 59.2, 113.8, 120.1, 125.6, 128.4, 128.5, 129.2, 130.0, 132.4,
4.23 (m, 4H), 4.27 (dd, 1H, J1 ¼ 6.1 Hz, J2 ¼ 14.3 Hz), 4.60 (dd, 139.2, 141.9, 147.4, 150.6, 152.9, 153.1; MS (ESI): 438
1H, J1 ¼ 3.3 Hz, J2 ¼ 14.3 Hz), 4.92–5.04 (m, 1H), 6.90 (tt, 1H, [M þ H]þ., 460 [M þ Na]þ. Anal. Calcd for C26H27N7: C,
J1 ¼ 1.7 Hz, J2 ¼ 6.7 Hz), 7.24–7.39 (m, 7H), 7.50 (s, 1H), 7.55 71.37; H, 6.22; N, 22.41. Found: C, 71.66; H, 5.94; N, 22.32.
(dd, 2H, J1 ¼ 1.8 Hz, J2 ¼ 7.8 Hz), 8.36 (s, 1H); 13C-NMR
9-{[1-(4-Methylphenyl)-3-phenyl-1H-pyrazol-5-yl] methyl}-6-
(CDCl3) ꢀ 29.7, 36.4, 44.0, 48.1, 58.8, 113.5, 119.8, 120.1,
morpholinyl-9H-purine (8a). (94% yield). Light brown solid,
125.7, 128.4, 128.7, 129.6, 132.2, 139.3, 143.9, 147.8, 150.6,
m.p. 68–70 ꢁC (DCM-hexane), IR (Nujol): 3020, 1595,
152.6, 154.3; MS (ESI): 424 [M þ H]þ, 446 [M þ Na]þ; 462
1520 cmꢀ1 1H-NMR (CDCl3) ꢀ 2.41 (s, 3H), 3.82 (t, 2H,
;
[M þ K]þ. Anal. Calcd for C25H25N7: C, 70.90; H, 5.95; N, 23.15.
J ¼ 4.8 Hz), 4.28 (t, 2H, J ¼ 4.8 Hz), 5.41 (s, 2H), 6.61 (s, 1H),
7.24–7.47 (m, 7H), 7.49 (s, 1H), 7.79 (dd, 2H, J1 ¼ 1.5 Hz,
J2 ¼ 6.8 Hz), 8.34 (s, 1H); 13C-NMR (CDCl3) ꢀ 21.2, 43.5, 45.7,
67.1, 105.0, 113.8, 125.5, 125.8, 128.6, 130.0, 132.7, 136.5,
137.8, 138.6, 138.9, 139.0, 141.0, 152.0, 152.5, 152.6; MS (ESI):
452 [M þ H]þ., 474 [M þ Na]þ. Anal. Calcd for C26H25N7O: C,
69.16; H, 5.58; N, 21.71. Found: C, 69.41; H, 5.83; N, 21.48.
Found: C, 70.71; H, 6.12; N, 22.93.
tert-Butyl 4-{9-[(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-
yl)methyl]-9H-purin-6-yl}piperazine-1-carboxylate (7d). (87%
yield). Beige solid, m.p. 130–132 ꢁC (DCM), IR (Nujol): 3040,
1
1665, 1640, 1585 cmꢀ1; H-NMR (CDCl3) ꢀ 1.49 (s, 9H), 3.27
(dd, 1H, J1 ¼ 5.7 Hz, J2 ¼ 17.1 Hz), 3.38 (dd, 1H, J1 ¼ 10.8 Hz,
J2 ¼ 17.1 Hz), 3.49 (t, 1H, J ¼ 4.8 Hz), 4.15–4.26 (m, 4H), 4.30
(dd, 1H, J1 ¼ 5.6 Hz, J2 ¼ 14.3 Hz), 4.60 (dd, 1H, J1 ¼ 2.9 Hz,
J2 ¼ 14.3 Hz), 4.93–5.03 (m, 1H), 6.90 (t, 1H, J ¼ 6.8 Hz), 7.23–
7.39 (m, 7H), 7.50 (s, 1H), 7.52 (dd, 2H, J1 ¼ 1.7 Hz, J2 ¼ 7.9 Hz),
8.35 (s, 1H); 13C-NMR (CDCl3) ꢀ 29.7, 36.4, 43.9, 44.0, 45.0,
58.7, 79.7, 113.4, 119.8, 125.6, 126.3, 128.4, 128.8, 129.6, 132.1,
139.1, 143.8, 147.8, 151.4, 152.4, 153.9, 154.8; MS (ESI): 539
[M þ H]þ., 561 [M þ Na]þ; 577 [M þ K]þ. Anal. Calcd for
C30H34N8O2: C, 66.89; H, 6.36; N, 20.80. Found: C, 67.11; H,
6.12; N, 20.96.
9-{[1-(4-Methylphenyl)-3-phenyl-1H-pyrazol-5-yl]methyl}-6-
piperidinyl-9H-purine (8b) (95% yield). Light brown solid, m.p.
68–70 ꢁC (DCM-hexane), IR (Nujol): 3040, 1580, 1560,
;
1495 cmꢀ1 1H-NMR (CDCl3) ꢀ 1.60–1.79 (m, 6H), 2.43 (s,
3H), 4.14–4.31 (m, 4H), 5.40 (s, 2H), 6.61 (s, 1H), 7.22–7.43 (m,
7H), 7.48 (s, 1H), 7.79 (dd, 2H, J1 ¼ 1.6 Hz, J2 ¼ 8.4 Hz), 8.31 (s,
1H); 13C-NMR (CDCl3) ꢀ 21.2, 24.6, 26.2, 38.6, 46.4, 105.0,
113.8, 125.5, 125.8, 125.9, 128.1, 128.6, 130.1, 136.6, 137.2,
138.7, 139.1, 141.3, 152.0, 152.6, 152.8; MS (ESI): 450
[M þ H]þ. Anal. Calcd for C27H27N7: C, 72.14; H, 6.05; N,
21.81. Found: C, 72.11; H, 6.29; N, 21.54.
9-{[1-(4-Methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-
yl]methyl}-6-morpholinyl-9H-purine (7e). (95% yield). Beige
solid, m.p. 167–169 ꢁC (DCM-hexane), IR (Nujol): 3040, 1580,
9-{[1-(4-Methylphenyl)-3-phenyl-1H-pyrazol-5-yl]methyl}-6-
pyrrolidinyl-9H-purine (8c) (97% yield). Light brown solid, m.p.
68–70 ꢁC (DCM-hexane), IR (Nujol): 3020, 1580, 1510 cmꢀ1; 1H-
NMR (CDCl3) ꢀ 1.94–2.11 (m, 4H), 2.41 (s, 3H),), 3.70–4.11 (m,
4H), 5.39 (s, 2H), 6.59 (s, 1H), 7.24–7.47 (m, 7H), 7.48 (s, 1H),
7.79 (d, 2H, J ¼ 7.0 Hz), 8.33 (s, 1H); 13C-NMR (CDCl3) ꢀ 21.2,
38.6, 48.2, 49.2, 105.0, 114.0, 125.5, 125.8, 126.0, 128.1, 128.6,
130.1, 136.6, 138.0, 138.8, 139.0, 141.0, 151.9, 153.1, 153.2; MS
(ESI): 436 [M þ H]þ. Anal. Calcd for C26H25N7: C, 71.70; H,
5.79; N, 22.51. Found: C, 71.71; H, 5.50; N, 22.60.
1545 cmꢀ1
;
1H-NMR (CDCl3) ꢀ 3.20 (dd, 1H, J1 ¼ 5.1 Hz,
J2 ¼ 17.2 Hz), 3.31 (dd, 1H, J1 ¼ 10.8 Hz, J2 ¼ 17.2 Hz), 3.76 (t,
4H, J ¼ 4.8 Hz), 4.22 (t, 4H, J ¼ 4.8 Hz), 4.36 (dd, 1H,
J1 ¼ 5.6 Hz, J2 ¼ 14.3 Hz), 4.57 (dd, 1H, J1 ¼ 3.3 Hz,
J2 ¼ 14.3 Hz), 4.85–4.97 (m, 1H), 7.12 (d, 2H, J ¼ 8.7 Hz), 7.17
(d, 2H, J ¼ 8.7 Hz), 7.24–7.32 (m, 3H), 7.49 (s, 1H), 7.48–7.54
(m, 2H), 8.34 (s, 1H); 13C-NMR (CDCl3) ꢀ 20.5, 36.4, 44.0, 45.7,
59.2, 67.0, 113.7, 119.8, 125.6, 128.4, 128.6, 129.3, 130.1, 132.3,
139.1, 141.8, 147.4, 151.5, 152.4, 153.9; MS (ESI): 454
[M þ H]þ., 476 [M þ Na]þ. Anal. Calcd for C26H27N7O: C,
68.85; H, 6.00; N, 21.62. Found: C, 68.56; H, 5.72; N, 21.85.
Biological assay
In vitro experiments
9-{[1-(4-Methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-
yl]methyl}-6-piperidinyl-9H-purine (7f). (96% yield). Beige
solid m.p. 144–146 ꢁC (DCM-hexane), IR (Nujol): 3040, 1570,
1540 cmꢀ1; 1H-NMR (CDCl3) ꢀ 1.55–1.77 (m, 6H), 3.20 (dd, 1H,
J1 ¼ 5.2 Hz, J2 ¼ 17.2 Hz), 3.31 (dd, 1H, J1 ¼ 10.8 Hz,
J2 ¼ 17.2 Hz), 4.11–4.22 (m, 4H), 4.23 (dd, 1H, J1 ¼ 4.9 Hz,
J2 ¼ 14.2 Hz), 4.56 (dd, 1H, J1 ¼ 3.3 Hz, J2 ¼ 14.2 Hz), 4.85–4.97
(m, 1H), 7.13 (d, 2H, J ¼ 8.5 Hz), 7.18 (d, 2H, J ¼ 8.5 Hz), 7.23–
7.34 (m, 3H), 7.49 (s, 1H), 7.53 (dd, 1H, J1 ¼ 1.5 Hz, J2 ¼ 7.8 Hz),
8.32 (s, 1H); 13C-NMR (CDCl3) ꢀ 20.5, 24.8, 26.0, 36.4, 44.5,
46.4, 59.1, 113.7, 119.7, 125.6, 128.4, 128.6, 129.2, 130.0, 132.3,
138.5, 141.9, 147.4, 151.3, 152.6, 153.9; MS (ESI): 452 [M þ H]þ,
474 [M þ Na]þ; 490 [M þ K]þ. Anal. Calcd for C27H29N7: C,
71.81; H, 6.47; N, 21.71. Found: C, 72.05; H, 6.34; N, 21.62.
In the in vitro assays each experiment was performed at least in
triplicate and the standard deviation of absorbance was less than
10% of the mean.
Determination of the reducing activity of the stable radical DPPH
To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol
an equal volume of the compounds dissolved in DMSO was
added34. The mixture was shaken vigorously and allowed to stand
for 20 min or 60 min; absorbance at 517 nm was determined
spectrophotometrically and the percentage of activity was
calculated. All tests were undertaken on three replicates and the
results were averaged (Table 1).
Soybean lipoxygenase inhibition study in vitro
9-{[1-(4-Methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-
yl]methyl}-6-pyrrolidinyl-9H-purine (7 g). (98% yield). Beige
solid m.p. 169–171 ꢁC (DCM-hexane), IR (Nujol): 3020, 1580,
1550 cmꢀ1; 1H-NMR (CDCl3) ꢀ 1.90–2.10 (m, 4H), 3.19 (dd, 1H,
J1 ¼ 5.2 Hz, J2 ¼ 17.2 Hz), 3.31 (dd, 1H, J1 ¼ 10.9 Hz,
J2 ¼ 17.2 Hz), 3.60–4.15 (m, 4H), 4.24 (dd, 1H, J1 ¼ 5.9 Hz,
The tested compounds dissolved in DMSO were incubated at
room temperature with sodium linoleate (0.1 mL) and 0.2 mL of
enzyme solution (1/9 ꢂ 10ꢀ4 w/v in saline)34. The conversion of
sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was
recorded and compared with the appropriate standard inhibitor.

DOI: 10.3109/14756366.2012.755623
Synthesis and biological evaluation of modified purine homo-N-nucleosides 113
Table 1. Interaction-reducing activity with DPPH (RA%); inhibition of lipid peroxidation (AAPH%); in vitro inhibition of soybean lipoxygenase (LO);
in vitro inhibition of thrombin (% Thr).
Compounds
Clog P39
RA% 0.05 mM 20 min ꢃ SD
AAPH% 0.1 mM ꢃ SD
LO (%) 0.1 mM ꢃ SD
Thr (%) 0.1 mM ꢃ SD
3a
3b
4
5
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
4.28
4.78
4.42
na
19 ꢃ 0.1
20 ꢃ 0.8
62 ꢃ 2.1
66 ꢃ 1.5
22 ꢃ 1.1
24 ꢃ 1.0
13 ꢃ 0.6
17 ꢃ 0.1
43 ꢃ 1.2
20 ꢃ 1.5
43 ꢃ 0.9
74 ꢃ 1.6
79 ꢃ 2.1
44 ꢃ 1.2
81 ꢃ 1.7
–
94 ꢃ 2.1
58 ꢃ 1.7
5 ꢃ 0.3
82 ꢃ 1.9
100 ꢃ 1.5
72 ꢃ 2.0
73 ꢃ 1.4
54 ꢃ 1.0
5.8 ꢃ 0.1
91.5 ꢃ 2.0
49 ꢃ 1.8
99 ꢃ 2.2
96 ꢃ 2.0
87 ꢃ 1.5
–
97 ꢃ 1.9
34 ꢃ 1.0
44 ꢃ 0.8
92 ꢃ 3.8
89 ꢃ 2.4
80 ꢃ 1.8
37 ꢃ 0.9
74 ꢃ 1.6
30 ꢃ 1.1
41 ꢃ 0.8
12 ꢃ 0.08
No
18 ꢃ 0.1
nt
nt
nt
3.80
5.19
4.63
5.30
4.30
5.68
5.13
3.94
5.32
4.77
–
75 ꢃ 2.6
73 ꢃ 1.8
19 ꢃ 0.1
48 ꢃ 0.6
nt
nt
nt
nt
nt
nt
nt
15 ꢃ 0.1
40 ꢃ 1.3
84 ꢃ 2.1
–
NDGA
Trolox
NAPAP
–
–
64 ꢃ 3
–
–
–
100 ꢃ 4
na: not available; nt: not tested; ꢃSD ꢄ 10%. NDGA: nordihydroguaiaretic acid; NAPAP: Na-(2-naphthyl-sulfonyl-glycyl)-D,L-p-amidinophenylalanyl-
piperidine; each experiment was performed at least in triplicate.
Inhibition of linoleic acid lipid peroxidation
was always greater than 97%. For the SRB assay, cells were
seeded into 96-well plates in 100 mL of medium at a density of
5000–15 000 cells per well, depending on the cell line, and
subsequently, the plates were incubated at standard conditions for
24 h to allow the cells to resume exponential growth prior to
addition of the compounds. In order to measure the starting cell
population, cells in one plate were fixed in situ with TCA 50%
(w/v) followed by SRB staining as described37. To determine
cytotoxic activity, all compounds were dissolved in DMSO and
then directly added in the cultures at four 10-fold dilutions (from
100 to 0.1 mM) and incubation continued for an additional period
of 48 h. The assay was terminated by the addition of cold TCA
50% (w/v) followed by SRB staining and the absorbance was
measured at 530 nm, in an EL-311 BIOTEK microelisa reader
Production of conjugated diene hydroperoxide by oxidation of
linoleic acid in an aqueous dispersion is monitored at 234 nm34.
AAPH is used as a free radical initiator. This assay can be used to
follow oxidative changes and to understand the contribution of
each tested compound.
Azo compounds generating free radicals through spontaneous
thermal decomposition are useful for in vitro studies of free
radical production. The water soluble azo compound AAPH has
been extensively used as a clean and controllable source of
thermally produced alkylperoxyl free radicals. Ten microliters of
the 16 mM linoleic acid dispersion was added to the UV cuvette
containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4
prethermostated at 37 ꢁC. The oxidation reaction was initiated at
37 ꢁC under air by the addition of 50 mL of 40 mM AAPH
solution. Oxidation was carried out in the presence of aliquots
(10 mL) in the assay without antioxidant, lipid oxidation was
measured in the presence of the same level of DMSO. The rate of
(BioTek, Winooski, VT), in order to determine the three
38
parameters GI50, TGI and LC50
.
Determination of lipophilicity as clog P
oxidation at 37 ꢁC was monitored by recording the increase in Lipophilicity was theoretically calculated as clog P values in
absorption at 234 nm caused by conjugated diene hydroperoxides. n-octanol buffer by CLOGP Programme of Biobyte Corp39.
Inhibition of thrombin
Results and discussion
As a substrate, tosyl-Gly-Pro-Arg-pNA was used at 1 mM final Synthesis
concentration35. Compounds were dissolved at a final concentra-
The reaction of 9-allyl-6-chloropurine10 (1) with the diphenylni-
tion of 100 mL in a Tris-buffer (0.05 M Tris, 0.154 M NaCl, ethanol
5%, pH 8.0). Three minutes after the addition of bovine thrombin
(2.5 unit/mg), the reaction was ended by adding 0.1 mL acetic acid
50%. The absorption of the released p-nitroaniline was measured at
405 nm. NAPAP: Na-(2-naphthyl-sulfonyl-glycyl)-D,L-p-amidi-
nophenylalanyl-piperidine) was used as a reference compound.
trile imine, generated in situ from the hydrazone (2a) with NBS in
the presence of Et3N, in benzene40 under MW irradiation at 80 ꢁC
for 80 min resulted in 1,3-dipolar cycloaddition product (3a)
(53%) (Scheme 1) with 43% of the starting purine remaining
unchanged. The regioselectivity of this reaction for the formation
of 5-substituted 2-pyrazoline is quite similar to that expected from
the literature27. The same product (3a) (51%) is isolated also from
the reaction of purine (1) with the diphenylnitrile imine prepared
In vitro cytotoxic and antiproliferative activity of the compounds
A549 (Non-small cell lung cancer), PC3 (prostate cancer), in situ from the treatment of hydrazonoyl chloride41 (9) with Et3N
MB435 (melanoma), CAKI and SN12C (renal cancer) were under reflux (Scheme 2), while 36% of the starting purine
adapted to propagate in RPMI 1640 medium supplemented with remained unchanged.
5% heat-inactivated fetal calf serum, 2 mM L-glutamine and
antibiotics. The cultures were grown in a 37 ꢁC humidified (6a), piperidine (6b), pyrrolidine (6c) or N-Boc-piperazine (6d)
incubator with 5% CO2-atmosphere
under microwave (MW) irradiation at 100ꢁC in H2O for 1 min
The amination of compound 3a (Scheme 1) with morpholine
In vitro cytotoxic activity of all congeners was determined by afforded almost quantitatively the adenine derivatives (7a–d). This
the SRB assay36. Cell viability was assessed at the beginning of procedure seems to be very efficient and eco friendly without the
each experiment by the trypan blue dye exclusion method, and use of any other reagent except H2O. The morpholinyl-substituted

114 A. Thalassitis et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 109–117
compound 7a (37%) is isolated also [accompanied by the scavenging activity45. These advantages made the DPPH
unreacted purine (10) (61%)] from the 1,3-dipolar cycloaddition method interesting for the testing of our compounds.
reaction of 9-allyl-6-morpholin-4-yl-9H-purine10 (10) with the
The use of the free radical reactions initiator AAPH is
diphenylnitrile imine, received in situ under reflux from the recommended as more appropriate for measuring radical-scaven-
hydrazonoyl chloride (9) (Scheme 2), after treatment with Et3N. ging activity in vitro, because the activity of the peroxyl radicals
The reaction of 9-allylpurine (1) with the nitrile imine, produced by the action of AAPH shows a greater similarity to
generated in situ from the p-tolylhydrazone (2b) with NBS in cellular activities such as lipid peroxidation46.
the presence of Et3N, in benzene under microwaves at 80 ꢁC for
The interaction/reducing activity (RA) of the examined
90 min gave the pyrazoline derivative (3b) (48% yield) followed by compounds with the stable free radical DPPH is shown in
its oxidation product the pyrazole derivative (4) (3%), the Table 1. This interaction, indicating their radical scavenging
bipyrazoline derivative (5) (6%) and the unchanged starting ability in an iron-free system, was measured at 50 mM (20 min).
material (41%). The derivative (5) shows the expected molecular The new compounds 3a–b, 7a–d, 7f present mild reducing
ion in MS spectrum (471/473/475 [M þ H]þ), the expected pattern activities (13–24%) after 20 min, whereas 7e, 7g, 8c present
1
for 2-pyrazolin-5-yl methylene group in the H-NMR spectrum 43–44% and compounds 4, 5, 8a and 8b (62–79%). The presence
[3.73 (dd, 1H, J1 ¼ 8.6 Hz, J2 ¼ 11.1 Hz), 3.88 (dd, 1H, J1 ¼ of a pyrazole ring (compound 4) leads to a higher interaction
4.1 Hz, J2 ¼ 11.1 Hz), 4.59 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 14.4 Hz), value, compared to the corresponding pyrazoline analogue
4.67–4.78 (m, 1H), 5.03 (dd, 1H, J1 ¼ 3.8 Hz, J2 ¼ 14.4 Hz)], while (4 ¼ 62%, whereas 3b ¼ 20%). Compound 5 shows similar
there are no other aromatic protons except the protons of the purine antioxidant activity with 4. Comparing 7a to 8a and 7b to 8b, it
skeleton. There is also absorption for N–H group in the IR seems that the pyrazoles (8a–8b) are more potent that the
spectrum (3230 cmꢀ1). Oxidation of pyrazoline (3b) with 2,3- corresponding pyrazolines (7a–7b).
dichloro-4,5-dicyano-o-benzoquinone (DDQ) in toluene under
reflux produced pyrazole (4) in 94% yield. Derivative 3b after one the reduction of the DPPH radical by an electron transfer (ET)
month exposure to the air oxidized to pyrazole (4). from the antioxidant. Particularly effective such antioxidants are
In the DPPH assay, the dominant chemical reaction involved is
The aminations of pyrazolines (3b) and pyrazoles (4) the phenoxide anions from phenolic compounds like catechol and
(Scheme 1) with morpholine (6a), piperidine (6b) or pyrrolidine derivatives, such as nordihydroguaiaretic acid (NDGA). Herein,
(6c) under microwave irradiation at 100 ꢁC in H2O for only 1 min the most potent antioxidants 8a and 8b did not contain such
afforded almost quantitatively the homo-N-nucleosides containing structural characteristics. Their interaction values did present big
pyrazoline (7e–g) and pyrazole (8a–c) moieties, respectively.
differences although in 8a, substituent A is a morpholinyl group
and in 8b A is a piperidinyl moiety. In general, the replacement of
Cl by an amine (a substituent) did not lead the antioxidant activity
in an increase.
Biological studies
Oxidation is an important process, which produces reactive
oxygen species ROS. Antioxidants are defined as substances that
even at low concentration significantly delay or prevent oxidation
of easy oxidizable substrates and there is an increased interest of
using antioxidants for medical purposes in recent years. Free
radicals are formed in both physiological and pathological
conditions in mammalian tissues. The uncontrolled production
of free radicals is considered as an important factor in the tissue
damage induced by several pathophysiologies. It is well known
that free radicals play an important role in the inflammatory
process. Lipid peroxidation mediated by free radicals is
considered to be the major mechanism of cell membrane
destruction and cell damage inducing alterations of ion transport
and inhibition of metabolic processes42. Phospholipids containing
polyunsaturated fatty acids are predominantly susceptible to
peroxidation. ROS are produced during the inflammation process
by phagocytic leukocytes at the inflamed site and they are
involved in the biosynthesis of prostaglandins and in the
cycloxygenase- and lipoxygenase-mediated conversion of arachi-
donic acid into proinflammatory intermediates7,8. LO products
are regulators of platelet [Ca2þ] mobilization and aggregation in
response to some agonists. LO inhibitors may work in part by
modifying platelet cyclic AMP metabolism.
Compounds with antioxidant properties are expected to offer
protection in inflammation and thrombosis and to lead to effective
drugs, in comparison to the well known antioxidant agent NDGA.
In our studies AAPH was used as a free radical initiator to
follow oxidative changes of linoleic acid to the conjugated diene
hydroperoxide34,46. Azo compounds generating free radicals
through spontaneous thermal decomposition are useful for free
radical production studies in vitro. All compounds showed
significant inhibition of lipid peroxidation 49–100 (Table 1)
compared to 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox), used as a standard (64%). No differences are
observed between compounds 7b (piperidinyl) and 7c (pyrrodi-
nyl) indicating that the magnitude of the ring (5- or 6-membered)
does not influence significantly the inhibition. However, 7d
presents a decrease (54%) possibly due to the presence of the
bulky Boc group, whereas 7a, the morpholinyl-substituted
molecule, highly inhibits lipid peroxidation 100%. Between the
two 3a, 3b (6-Cl substituted pyrazolines), 3a seems to be more
potent inhibitor lipid peroxidation. The presence of Ar ¼ 4-Me–
C6H4– group decreases the biological response. Minor changes
are observed within the inhibitory activities of 3a–7a with the
replacement of the 6-Cl group by the morpholinyl moiety. On the
contrary, significant high differences are observed between
the antioxidant and inhibitory values of 3b–7e.
Taking into account the multifactorial character of oxidative
stress and inflammation, we evaluated the in vitro antioxidant
activity of the synthesized molecules using two different
antioxidant assays: (a) interaction with the stable free radical
DPPH and (b) interaction with the water-soluble azo compound
AAPH.
It is worthy to note that small changes in Ar substituent are
followed by significant decrease in the anti-lipid peroxidation
behavior, e.g. 7a–7e, 7b–7f, 7c–7g. Comparing pyrazolines 7e–g
to pyrazoles 8a–c, big differences are observed within the %
inhibition values of 7e and 8a, 7g and 8c.
Both require a spectrophotometric measurement and a certain
reaction time in order to obtain reproducible results43. In its
oxidized form, the DPPH radical has an absorbance maximum
centered at about 517 nm44. The DPPH method is described as a
simple, rapid and convenient method independent of
sample polarity for screening many samples for radical
Since lipophilicity is a significant physicochemical property
determining distribution, bioavailability, metabolic activity and
elimination, we tried to theoretically calculate clog P values39 as
an expression of lipophilicity. Perusal of these values did not give
any evidence of influence on the above determined antioxidant
properties.

DOI: 10.3109/14756366.2012.755623
Synthesis and biological evaluation of modified purine homo-N-nucleosides 115
Table 2. In vitro results for cytotoxic and antiproliferative activity.
Compounds
CELL LINES
Parameters
7a
7d
7b
7c
4
7g
8b
8a
8c
A549
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
55.2
54.7
59.0
50.4
99.4
4100
35.2
80.7
4100
36.8
65.4
94.1
25.8
55.2
84.7
39.3
66.8
94.3
37.6
67.0
96.4
36.1
64.0
91.9
44.6
51.0
55.4
63.5
4100
4100
43.3
83.2
4100
64.4
4100
4100
74.8
4100
4100
56.3
104.0
4100
4100
4100
25.7
91.8
4100
75.2
4100
4100
103.8
4100
4100
55.2
4100
4100
4100
4100
41.3
86.9
4100
4100
4100
28.7
82.5
4100
64.4
4100
4100
104.7
4100
4100
78.7
4100
4100
4100
4100
40.5
92.5
4100
80.6
4100
4100
103.7
4100
4100
61.6
4100
4100
4100
4100
43.2
95.9
4100
71.2
4100
4100
4100
4100
4100
4100
4100
52.2
99.4
4100
75.6
4100
4100
4100
4100
4100
PC3
MB435
CAKI
SN12C
52.1
92.9
52.0
92.2
4100
70.7
4100
4100
51.3
90.4
4100
4100
58.9
4100
4100
35.2
81.0
4100
LC50
GI50
TGI
66.3
4100
4100
64.9
4100
4100
LC50
The in vitro anticancer activity of compounds was determined using the SRB antiproliferative assay as instructed by the NCI. The nine compounds were
tested against a panel of five human cancer cell lines derived from four different types of solid tumors. Three parameters growth inhibiting activity
(GI50), cytostatic activity (TGI) and cytotoxic activity (LC50) were evaluated for each cell line and each compound tested. Results represent mean of
three experiments each one run in triplicates with a C.V. ꢄ 15%.
Figure 1. The growth curves of cells treated with the compound 4 which was found to be the most active. Cells were exposed to various concentrations
of the agent for 48 h and the growth rates were calculated using the SRB method (see Materials and methods section). Points represent the mean of three
independent experiments each one run in triplicates ꢃ SD.
Compounds were further evaluated for inhibition of soybean However, in this data set lipophilicity does not seem to affect
lipoxygenase (LO) by the UV absorbance based enzyme assay10. the LO inhibition.
All the tested derivatives inhibit soybean LO with the exception of
It is now widely accepted that activation of the coagulation
8a, which under the experimental conditions did not present any cascade, with initiation of thrombin and fibrin deposition is a
inhibition, as well as 7g and 8b, which showed limited inhibition. consequence of inflammation. Once thought to be completely
Compounds 3b, 4, 7c, 7e, 8c follow with close inhibition values different processes, the boundaries between inflammation and
(30–44%). Slight differences are observed among the most potent coagulation are now nearly indistinguishable50. Serineprotease
derivatives 5, 7a, 7b and 7d. Compound 5 presents the higher thrombin, can elicit many inflammatory responses in micro-
activity at 92% as well as compound 3a (97%). Compound 3a vascular endothelium. Its multiple role in thrombosis makes
seems to be more potent inhibitor of LO and of lipid peroxidation. thrombin an important target for the therapeutic agents designed
No differentiation was observed between the piperidinyl-(7b) and for thrombus prevention51. LO inhibitors reduce platelet aggrega-
N-Boc-piperazinyl (7d) substituted derivatives.
Lipophilicity is as
referred47–49
physichochemical property for lipoxygenase inhibition. active, low molecular weight synthetic thrombin inhibitors have
tion induced by thrombin and U46619 and modify release of Ca2þ
an
important from intracellular stores52. The development of selective, orally

116 A. Thalassitis et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 109–117
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We would like to thank Dr A. Leo and Biobyte for free access to the
C-QSAR program.
Declaration of interest
This research has been co-financed by the European Union (European
Social Fund – ESF) and Greek national funds through the Operational
Program ‘‘Education and Lifelong Learning’’ of the National Strategic
Reference Framework (NSRF) – Research Funding Program: Heracleitus
II (A. Thalassitis) Investing in knowledge society through the European
Social Fund.
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