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119
SYNTHESIS, CHARACTERIZATION AND
BIOLOGICAL EVALUATION OF SOME
NEW 2 - AMINO - 4, 6 - DIARYL
PYRIMIDINES
CHAPTER - 2
120
2.1 LITERATURE REVIEW
Pyrimidine was first isolated by Gabriel and Colman in 1899.
The chemistry of pyrimidine1 and its derivatives have been studied
since the past century due to their diverse pharmacological properties.
Pyrimidine (50) and purine (51), the two nitrogen containing
heterocyclic aromatic compounds are the parents of the “bases” that
constitute a key structural unit of nucleic acids, even though
pyrimidine itself does not exist in nature. Both pyrimidine and purine
are planar and this flat shape is very important when we consider the
structure of nucleic acids.
N
N
1
2
3
4
5
6
N
N
1
2
3
N
N
H
4
5
9
8
7
6
(50) (51)
In terms of their chemistry, pyrimidine and purine resemble
pyridine. They are weak bases and relatively unreactive towards
electrophilic aromatic substitution. There is an important structural
difference between pyrimidine derivatives that bear –OH groups and
those with –NH2 groups. The structure of a pyrimidine that bears an
amino group follows directly from the structure of the parent ring
system as seen in the case of cytosine. An equilibrium exists in the
aminopyrimidines between the amino (52) and imino forms (53).
121
N
N
NH
H
N
N
NH2
(52) (53)
However, the corresponding pyrimidine having a hydroxyl group
(54) resemble an enol, but exist instead in its keto form (55), contrary
to the stable isomers with the hydroxyl groups on benzene- like rings.
This is because the keto form of the pyrimidine is also aromatic and
stable owing to amide resonance2 as shown in Scheme 12.
N
N
HO N
N
O
H
(54) (55)
N
N
H
O N
N
O
H
Scheme 12. Resonance in keto form of 4-hydroxypyrimidine
122
GENERAL METHODS OF SYNTHESIS OF PYRIMIDINES
The general methods employed in the synthesis of pyrimidines
are briefly reviewed below:
1. 1, 4-dihydro-4-phenyl-2, 6-dimethyl-3, 5-diacetylpyridines were
converted into chalcones by Claisen-Schmidt condensation with
aldehydes. The resulted chalcones were cyclized with guanidine to
give aminopyrimidines3 (Scheme 13).
NH3C CH3
CH3H3C
C6H5O O
H
2ArCHO
NH3C CH3
C6H5O O
H
ArAr
NH2H2N
NH
NH3C CH3
C6H5
H
NN NN
NH2 NH2
ArAr
Scheme 13
2. 1, 3-diaryl-propenones react with guanidine by refluxing them
together in a basic alcoholic medium to give dihydropyrimidines,
which on oxidation with H2O2 yield 4, 6-diaryl-2-
aminopyrimidines4 (Scheme 14).
123
Ar1 Ar2
O
+
NH2H2N
NH
N NH
Ar1
NH2
Ar2
N N
Ar1
NH2
Ar2H2O2
Scheme 14
3. Guanidine reacts with -ketoesters, -diketones, cyanoacetic esters
and , -unsaturated carbonyl compounds to give 2–amino
pyrimidines usually in good yields5 (Scheme 15).
H2N CNH
NH2
CH2
COCH3
COOC2H5
N
N
OH
H2N CH3
Scheme 15
4. Urea reacts with , -unsaturated esters to form dihydrouracil or
uracil6 (Scheme 16).
CO
NH2
NH2+ CH
CHCH3
COOC2H5
C
HN CH2
CHCH3
N
H
O
O
N
NHO
OH
CH3
Scheme 16
5. Ureidoethylenes undergo ready cyclization to pyrimidines in the
presence of a basic catalyst7 (Scheme 17). The required
ureidoethylenes can be prepared from N, N′-dicarbamylformamidines
by reaction with cyanoethylacetate. N, N′-dicarbamylformamidines
can be obtained by the reaction of urea with ethyl orthoformate.
124
CONH2
NH2
C2H5OCHO H2NCNCHNHCNH2
O O
H2CCOOC2H5
CN
N
CNH2
CC
H
H
O
COOC2H5
CN
NaOC2H5N
NO
H
H
NH
COOC2H5
2 H
Scheme 17
125
SPECTRAL FEATURES OF PYRIMIDINES:
UV SPECTRA OF PYRIMIDINES:
Two bands, one at 243 nm and the other at 298 nm are
generally observed in the UV spectrum of pyrimidines. When an
electron releasing substituent is present, a bathochromic shift of the
second band (n-*) usually occur while the electron withdrawing
substituent produces an opposite effect.
The more intense band at 243 nm is due to -* transition
which undergoes a bathochromic shift by both type of substituents
with an increase in intensity. Two bands, one in between 330-350 nm
and the other in between 250-260 nm are usually observed in the
case of 3, 5-diaryl pyrimidines8-10 , whereas 4,6-diarylpyrimidines
exhibit characteristic UV maxima at 254 nm for -* transition and at
350 nm for n-* transition.
IR SPECTRA OF PYRIMIDINES:
The 2-aminopyrimidine system can readily be characterized by
the appearance of absorption bands11 in between 1650 - 1630 cm-1
(C=N) , 1584-1570 cm1 (C=C) and three sharp bands in the region
3490-3460 cm-1, 3352-3350 cm-1and 3200-3100 cm-1 (NH2 free and
hydrogen bonded).The C-H stretching modes occur in between 3100-
3050 cm-1. The characteristic ring-breathing mode occurs in between
1020-990 cm-1 and another characteristic band near 800 cm-1.
NMR SPECTRA OF PYRIMIDINES:
The spectrum of 2-aminopyrimidine consists of a doublet
centered at 5.7 ppm (equivalent 4 & 6 hydrogens) and a triplet (5-
126
hydrogen) centered at 4.1 ppm superimposed on a broad peak (NH2).
The doublet and triplet splitting are both 48 Hz, and the relative areas
are estimated to be 2:1. The spectrum may be interpreted as
indicating the predominance of the amino form of the molecule in
DMSO solution. But the unsymmetrical imino form cannot be
completely ruled out from the NMR spectrum; however, the amino
structure is considered to be the prevalent species.
In the 1H NMR spectra, the relative deshielding of 4 protons is in
the order C2 - H > C4 - H = C6-H > C5-H as expected. Thus the C2-H
resonates at 8.78 and C5-H at 7.46 12, 13. The 2-amino substituted
pyrimidines show a doublet and triplet typical of A2X system with the
C4-H / C6-H signal broadened by coupling to the adjacent nitrogen14.
The 2-amino 4, 6-diarylpyrimidines show the C5-H proton as a singlet
around 7.2 – 7.47 and a broad signal in between 5.47 –5.80 due
to the amino protons, which disappears when the CDCl3 solution is
shaken with D2O15.
MASS SPECTRA OF PYRIMIDINES:
The mass spectra of pyrimidines are generally simple. The
dominant fragmentation mode of pyrimidines involves sequential loss
of two HCN molecules to give ionized acetylene at m/z 26, as the base
peak16. A similar sequential loss of two molecules of HCN 17, 18 was
observed in the fragmentation of 2-aminopyrimidines.
127
THERAPEUTIC POTENTIAL OF PYRIMIDINES:
A literature survey revealed that various substituted
pyrimidines are known to possess antimicrobial, anti-inflammatory,
anticancer, antiviral, antitubercular, antimalarial and other
miscellaneous activities. Given below is a brief account of various
modifications reported on pyrimidine nucleus, which showed a variety
of biological and pharmacological activities.
Antimicrobial activity:
The finding that 2,4-diaminopyrimidines inhibit the growth of
microorganisms by interfering with their utilization of folic acid led to
an intensive search for antiinfective agents in this class of heterocyclic
compounds. Trimethoprim developed as an antimalarial drug had
unique broad spectrum antimicrobial action. The pioneering work of
Hitchings19 led to the combination of trimethoprim with sulfa drug,
sulfamethoxazole constituting an important advance in the
development of clinically effective antimicrobial agents. Chemical
modification of trimethoprim led to potent antibacterial compound
tetroxoprim (56)
H3CO
H3COH2CH2CO
OCH3
CH2
N
NH2N NH2
(56)
128
Patel et al.20 synthesized some new 2-amino-4-
substitutedphenyl-6-(8-quinolinol-5-yl) pyrimidines (57) which
showed moderate to potent antimicrobial activity.
N N
NH2
R
NOH
(57)
Mishra et al.21 reported the synthesis of a series of pyrimidine-2-one
derivatives (58) which showed antimicrobial activity relative to
norfloxacin against Gram-positive and Gram-negative bacteria using
serial dilution technique.
N
N
R
O
H
R2
N
NN
R1
CH3
H
(58)
Bodke et al.22 reported the synthesis of some new benzofuro [3, 2-d]
pyrimidines (59). These compounds were screened for antibacterial
and antifungal activity.
129
O
NNH
OH
O
N
(59)
Murthy et al.23 synthesized some new chromanopyrimidines
(60). They were tested against Gram -positive bacteria B. subtillis and
B. pumilus and Gram -negative bacteria E. coli and P. vulgaris.
O
N N
OHMe
Me
NH2
R
(60)
Kudari et al. 24 reported the synthesis and antimicrobial
properties of bis- 2-thioxo-1,2,3,4,5,6-hexahydropyrimidine 4,6-diones
(61).
NN
OO
S
C CH2O O CH2C
OO
N N
OO
S
RR
(61)
130
Anticancer activity:
Some novel substituted pyrimidines (62) bearing a benzofuran
substituent were synthesized and evaluated for antitumor activity by
Babu et al 25, of which compounds with R2=SH and R1=4-MeOC6H4
showed significant antitumor activity.
O N
N
R1
R2
(62)
Fathalla et al. 26 synthesized a series of some new pyrimidine
derivatives ( 63 & 64) and evaluated them for antitumor activity. The
results indicated that some of these compounds showed antitumor
activity against liver cancer (HEPG 2) tumor cell line tested, but with
varying intensities in comparison to the known anticancer drugs, 5-
fluoro uracil and doxorubicin.
N
NS
H
R
CN
NH NH2
N
N
CN
H
S
OCH3
N
O
(63) (64)
131
Wang et al. 27 synthesized some 2-anilino-4-(1H-pyrrol-3-yl)
pyrimidines (65) inhibiting CDK2 & CDK4.
N
N
N
H
Me
NO2
NH
MeMe
Me
(65)
Fahmy et al.28 synthesized new fluorinatedthiazolo [4,5-
d]pyrimidines (66-68) which showed anticancer activity.
N
N
N
SS
Me
F
ON
F
F
N
N
N
SS
Me
F
NHF
N
N
N
SS
Me
F
Cl
(66) (67) (68)
Kaldirkyan et al.29 synthesized disubstituted 5-(3-methyl-4-
alkoxy- benzyl) pyrimidines (69), which were screened for their
anticancer activity.
R= Me, Et, Pr, Me2CH
(69)
N
N
CH2
Me
OR
OHHS
OH
132
Grigoryan et al.30 synthesized some novel 2, 5-substituted
pyrimidines (70). These compounds were tested for their antitumor
activity.
N
N
OH
OHS R
(70)
Chan et al.31 synthesized new diaminopyrimidine (71) with ω-
carboxyalkoxy or ω-carboxy-1-alkynyl substitution and these
compounds possessed significant and selective inhibition of DHFR.
N
N N
NH2
H2N
Me OMe
C
C
COOH
(71)
Antimalarial acitivity:
Vishwanadhan et al.32 reported the synthesis of some novel 5-
substituted amino-2, 4-diamino-8-chloropyrimido-[4, 5-b] quinolines
(72). These molecules were evaluated for blood schizonticidal activity
in mice infected by Plasmodium berghei. Some of these compounds
had significant curative potential when compared with chloroquine.
133
N N
N
NH
O2S
NH
R
NH2
NH2Cl
(72)
Chauhan et al.33 synthesized a series of 2, 4, 6-trisubstituted
pyrimidines ( 73) and evaluated them for their in vitro antimalarial
activity against Plasmodium falciparum. Of the 18 compounds
synthesized, 14 compounds showed MIC in the range of 0.25- 2
μg/mL and were several fold more active than pyrimethamine.
N
N
N
NH
R1
R
(73)
134
Antiviral activity:
Chern et al.34 synthesized some fused pyrimidines (74)
possessing specificity against human enteroviruses.
N
N
NN
NNPh
Ph
Ph
(74)
Sheriff et al.35 synthesized 2-(benzoxazol-2-yl-amino)-3H-4-
oxopyrimidines (75) and screened them for in vitro anti-HIV activity.
N
O
N
HN
N
H
O
H
HO
(75)
Novikov et al.36 synthesized new 1-[{2-(phenoxy) ethoxy}methyl]-
uracil derivatives (76). These compounds showed viral inhibition
properties against HIV-1.
N
N
Br
O
O Me
OO
R
R = Cl or Me
(76)
135
Zhou et al.37 synthesized some novel (Z) - and (E)-[2-fluoro-2-
(hydroxy methyl) cyclopropylidienemethyl] pyrimidines (77) which are
methylenecyclopropane analogues of nucleosides.
N
N
NN
NH2
F
HO
(77)
Anti-inflammatory activity:
Pirisino et al.38 have synthesized a new 2- phenylpyrazolo-4-
ethyl-4, 7-dihydro [1,5-a]-pyrimidine-7-one (78) and was evaluated
for anti-inflammatory activity by carrageenan-induced paw oedema
and cotton pellet-induced granuloma methods. This compound was
found to possess the activity similar to indomethacin, phenylbutazone
and isoxicam.
N
N
N
C2H5
O
(78)
Cenieola et al.39 evaluated some imidazolo [1, 2-c] pyrimidines
(79) for anti-inflammatory activity by carrageenan-induced paw
oedema method and found to show activity comparable to
indomethacin. These compounds were devoid of ulcerogenic property.
136
N
N
H
R1
CH3
R2
N
R1= Cl, OCH3, CH3
(79) R2=COOH, CH2COOH
Nargund et al.40 reported the synthesis of few substituted 2-
mercapto-3-(N-alkyl) pyrimido [5, 4-c] cinnolin-4- ones (80) and
screened for anti-inflammatory activity by carrageenan-induced rat
paw oedema method. Some of these compounds showed significant
reduction in paw oedema when compared to phenylbutazone.
NN
NN
S
R1
O
R
H
R= H, CH3
(80) R1= H, OCH3, CH3, Cl
Several pyrazolo [3, 4-d] pyrimidine derivatives were synthesized
as potential inhibitors of adenosine kinase by Cottam et al.41. One of
the compounds (81), was found to display good anti-inflammatory
activity at a dose of 30 mg/kg when evaluated in vivo in rat pleurisy
inflammation model.
137
N
N N
N
I
O
HO
OH
CH2OH
NH2
(81)
Bruni et al.42 synthesized a series of pyrazolo [1,5-a] pyrimidin-
7-one (82), which when evaluated for anti-inflammatory activity by
carrageenan-induced rat paw oedema method, significant activity was
observed, when phenyl group at 2- position was replaced by 2-theinyl
or 2-pyridinyl group. Compounds with alkyl and saturated ring
systems in the place of R at 2-position showed reduction in activity.
N
NN
R
CH3
O
(82)
Vidal et al.43 have studied the effects of some hexahydroimidazo
[1, 2-c] pyrimidine derivatives (83) on leukocyte function in vitro and
screened for anti-inflammatory activity in two models of inflammation.
The compound having the fluorine in the place of R showed significant
inhibition of paw swelling with reduced PGE2 levels in paw
homogenates.
138
O
O N
N
N
N SO2 CH3
OH
H3C
H3C
R
(83) R= H, p-Br, p- Cl, o- Cl
Bruni et al.44 reported the synthesis of some new 2, 5-
cycloamino-5H-benzopyrano[4,3-d] pyrimidines (84), which showed
anti-inflammatory activity at 100 mg/kg dose level.
O
NR2
N N
NR1
(84)
NR1= NR2= pyrrolo, piperidino, morpholino
Ferri et al.45 synthesized some 2-tosylamino and 2-
tosyliminopyrimidine derivatives (85 & 86) and studied their
interference with some leukocyte functions and 5-lipooxygenase ( 5-
LO) activity. The study demonstrated that all the compounds
inhibited cell free 5-LO activity and reduced activation of neutrophils,
which may have relevance for the modulation of the inflammatory
response.
139
N
N NTs
CH2 CONHR
(85)
N
N N Ts
CH2
H
CONHR
(86)
Ihsan et al.46 synthesized some new 1,2,4-triazolo[1,5-c]-
pyrimidines (87-89), having anti-inflammatory activity against
carrageen-induced rat paw oedema.
N N
R1
R2
N
N
Ph N N
N
N
CN
Ph
OMe
O
O
N
N
N
NN
CN
Ph
Br
(87) (88) (89)
Kandeel et al.47 synthesized some thienopyrimidines (90)
exhibiting anti- inflammatory activity.
N
N
N
NS
R2
Me
Me
R2= H, Me, SH, Ph, 2-Br C6H4,
4- Br C6H4, 4-Me C6H4
(90)
140
Carmen et al.48 synthesized pyrazolo [1, 5-a] pyrimidines (91)
possessing significant and selective COX-2 inhibitory activity.
N N
NR1
R2
R3
SO2Me
F
(91)
Miscellaneous activities:
Pandey et al.49 synthesized some novel terpenylpyrimidines (92
& 93) having antileishmanial activity.
Me Me N N
R1
NH2
Me
Me Me N N
R2
NH2
Me
(92) (93)
Joubran et al.50 synthesized new arylpropanolamines contaning
dipyrrolidinyl pyrimidines (94 & 95). These compounds showed
antioxidant activity and proved as neuroprotective agents.
NH
N
N
N
N
OH
R
NH N
N
N
N
OH
R
(94) (95)
141
Therapeutically important drugs51,52 containing pyrimidine
moiety along with their structures are given below:
DRUG ACTIVITY
N
O
I
O
HO
HO
O
NH
Idoxuridine (96)
Antiviral
N
O
CF3
O
HO
HO
O
NH
Triflouridine (97)
Antiviral
N
O
CH3
O
N3
HO
O
N
H
Zidovudine (98)
Antiviral(AIDS)
142
OHO
N
N NH2O
Zalcitabine (99)
Antiviral(AIDS)
N
N
CH3
O
O
H
OHO
Stavudine (100)
Antiviral(AIDS)
N
NO
S
OHO
NH2
Lamivudine (101)
Antiviral(AIDS)
N
N
F
H
H
O
O
5-fluorouracil (102)
Antiviral(AIDS) and
Anticancer
143
N
N
NH2
OO
HO
HOHO
Cytarabine (103)
Antiviral(AIDS)
N
NO
NH
O
HO
HO
Acitabine (104)
Antiviral(AIDS)
HCl
N
N
NN
O
N
O
Busiprone (105)
Antidepressant
CH3CH2
N
N
H
H
O
O
Primidone (106)
Anticonvulsant
144
N
NN
N N
N
CH2CH2OH
CH2CH2OH
N
N
HOH2CH2C
HOH2CH2C
Dipyridamole (107)
Vasodilator
N
N
N
NH2
H2N
O
Minoxidil (108)
Antihypertensive
CH2NCH2CH2N(CH3)2
N NCH3O
Thonzylamine (109)
Antihistaminic
145
O
NH
N
N
N
H
H
Alniditan (110)
Antihistaminic
N
N
H2N CH2
NH2OCH3
OCH3
OCH3
Trimethoprim (111)
Antiinfective
N
N
H2N
NH2
CH2CH3
Cl
Pyrimethamine (112)
Antimalarial
H2N S
O
O
NH N
N
H3CO OCH3
Sulfadoxine (113)
Antimalarial
146
N
NO
H
F
NH2
Flucytosine (114)
Antifungal
H2N S
O
O
N
N
N
CH3
CH3
H
Sulfamethazine (115)
Antibacterial
H2N S
O
O
N
N
N
H
Sulfadiazine (116)
Antibacterial
147
2.2 EXPERIMENTAL WORK
Aims and Objectives:
It could be seen from the literature that pyrimidines and their
derivatives were found to possess different biological activities. The
pyrimidines synthesized earlier in our laboratories also possessed
significant anti-inflammatory, anticancer and antimicrobial activities.
In continuation of our earlier work on pyrimidines, it is thought of
synthesizing some more new pyrimidines from the chalcones
synthesized in chapter-1, in order to consolidate the results in the
substituted pyrimidine series.
1. To synthesize and purify the 2, 4, 6-trisubstituted pyrimidines
from the chalcones obtained from 3'-methyl-4'-
hydroxyacetophenone.
2. To characterize the compounds using spectral (IR, 1H NMR and
Mass) methods and Elemental analysis. The data related to
structural characterization are given individually.
3. To screen the synthesized pyrimidines for their toxicity and
possible biological activities like anti-inflammatory,
antibacterial, antifungal and anticancer.
4. To identify the active compounds for further exploitation.
148
Materials and methods:
The same chemicals, solvents, procedures and instruments that
were mentioned in chapter-1 also used here. Guanidine hydrochloride
was obtained from the local supplier and the chalcones used were
obtained from 3'-methyl-4'-hydroxyacetophenone as described in
chapter-1.
General procedure for the synthesis of 2, 4, 6-trisubstituted
pyrimidines 53-57
The condensation of the chalcones with Guanidine
hydrochloride in an alkaline medium viz., in potassium hydroxide in
the presence of ethanol, at reflux temperatures (2 to 6 hours) resulted
in the formation of corresponding pyrimidines (Scheme 18).
Completion of the reaction was established by TLC using silica gel-G.
After completion of the reaction, the reaction mixture was poured onto
crushed ice with constant stirring. The solid that separated was
filtered, dried and purified by column chromatography on silica gel,
using a mixture of ethyl acetate and hexane as the mobile phase. The
purified pyrimidine derivatives were obtained as light to bright yellow
fine powders.
The chalcones that were used in the synthesis of pyrimidines:
1. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-pyridinyl)-2-propen-1-one (B1)
2. 1-(3'–methyl -4'-hydroxyphenyl)-3-(3''-pyridinyl)-2-propen-1-one (B2)
3. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-pyridinyl)-2-propen-1-one (B3)
4. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-furyl)-2-propen-1-one (B4)
149
5. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-pyrrolyl)-2-propen-1-one (B5)
6. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-thienyl)-2-propen-1-one (B6)
7. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-indolyl)-2-propen-1-one (B7)
8. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-quinolinyl)-2-propen-1-one (B8)
9. 1-(3–methyl -4'-hydroxyphenyl)-3-(9''-anthracenyl)-2-propen-1-one
(B9)
10. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-fluorophenyl)-2-propen-1-one
(B10)
11. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-chlorophenyl)-2-propen-1-one
(B11)
12. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-bromophenyl)-2-propen-1-one
(B12)
13. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-methylphenyl)-2-propen-1-
one (B13)
14. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-methoxyphenyl)-2-propen-1-
one (B14)
15. 1-(3'–methyl -4'-hydroxyphenyl)-3-(3'', 4'', 5''- trimethoxyphenyl)-2-
propen-1- one (B15)
Chalcone Guanidine 2, 4, 6 – trisubstituted
(as hydrochloride salt) pyrimidine
Scheme 18
H3C
HO
C
O
C C Ar
HH
+ H2N C NH2
NH
KOH
NN
NH2
H3C
HO
Ar
12
3
4
56
1' 2'
3'
4'5'
6'
Ethanol
2 - 4 hours
Reflux
150
Where Ar =
N
N
N
B1 B2 B3
O
N
H
S
B4 B5 B6
N
H
N
B7 B8 B9
F
Cl
Br
B10 B11 B12
CH3
OCH3
OCH3
OCH3
OCH3
B13 B14 B15
151
Procedure:
Synthesis of 2-amino-4-(3'–methyl-4'-hydroxyphenyl)-6-(2''-
pyridinyl) pyrimidine (B1P1):
1-(3'–methyl-4'-hydroxyphenyl)-3-(2''-pyridinyl)-2-propen-1-one
(B1)(0.001 mol ) was condensed with guanidine hydrochloride (0.001
mol ) in the presence of potassium hydroxide (0.002 mol ) in absolute
ethanol (5 ml) at reflux temperature on a water bath for 6 hours. The
solvent was evaporated in vacuo and crushed ice was added to the
residue while mixing thoroughly, whereupon a bright yellow solid
separated out. This solid was filtered under vacuum, dried and
purified by column chromatography to give pure pale yellow solid.
The compound B1P1 was analyzed for molecular formula as
C16H14N4O, m.p. 1570C, well supported by a [M+H]+ ion at m/z 279 in
its positive mode electrospray ionization mass spectrum (Fig. 21). IR
(KBr disc, cm-1) spectrum (Fig. 19) showed the characteristic bands at
3361 (NH2), 3499 (O-H), 1602 (C=N) and 1573 (C=C) and 1508
(CH=CH).
The 1H NMR spectrum (400 MHz, CDCl3, Fig. 20) of compound
B1P1 showed the characteristic C-5-H of the pyrimidine around δ 8.28
as singlet and C-2-NH2 at δ 5.40 as singlet. The spectrum also
accounted for the other aromatic protons of the hetero aromatic and
phenyl rings in between δ 7.20 - 8.68. The spectrum also showed an
aromatic methyl group as a singlet at δ 2.32 integrating for three
protons.
152
The results of Elemental analysis were also in agreement
with those of the calculated values.
Based on the above spectral data and elemental analysis, the
structure of the compound B1P1 was confirmed as 2-amino-4-(3' –
methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine.
By adopting the above synthetic procedure, pyrimidines of 3'-
methyl-4'-hydroxyacetophenone chalcones (chapter-1) were
synthesized, the physical and spectral characteristics of these
pyrimidines (B1P1-B15P15) were presented separately in detail.
153
The new 2, 4, 6 - trisubstituted pyrimidines thus synthesized:
1. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl)
pyrimidine (B1P1)
2. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(3''-pyridinyl)
pyrimidine (B2P2)
3. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-pyridinyl)
pyrimidine (B3P3)
4. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-furyl)
pyrimidine (B4P4)
5. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyrrolyl)
pyrimidine (B5P5)
6. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-thienyl)
pyrimidine (B6P6)
7. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-indolyl)
pyrimidine (B7P7)
8. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-quinolinyl)
pyrimidine (B8P8)
9. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(9''-anthracenyl)
pyrimidine (B9P9)
10. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-fluorophenyl)
pyrimidine (B10P10)
154
11. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-chlorophenyl)
pyrimidine (B11P11)
12. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-bromophenyl)
pyrimidine (B12P12)
13. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methylphenyl)
pyrimidine (B13P13)
14. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methoxyphenyl)
pyrimidine (B14P14)
15. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(3'',4'',5''-
trimethoxyphenyl) pyrimidine (B15P15)
155
Characterization of the new 2, 4, 6-trisubstituted pyrimidines
Table 8. Physical characterization data of 2, 4, 6-trisubstituted
pyrimidines
N N
NH2
Ar
HO
H3C
Compound
Ar
Molecular
Formula
Relative
Molecular
Mass
( RMM)
Melting
Point
( oC)
Yield
(%)
B1P1
N
C16H14 N4O
278
157
76
B2P2
N
C16H14 N4O
278
160
74
B3P3
N
C16H14 N4O
278
149
75
B4P4 O
C15H13 N3O2
267
173
70
B5P5 N
H
C15H14 N4O
266
159
72
156
B6P6 S
C15H13 N3SO
283
162
78
B7P7 N
H
C19H16 N4O
316
195
68
B8P8
N
C20H16 N4O
328
183
70
B9P9
C25H19 N3O
377
170
66
B10P10
F
C17H14 N3FO
256
260
71
B11P11
Cl
C17H14 N3ClO
311.5
145
73
B12P12
Br
C17H14 N3BrO
356
252
70
B13P13
CH3
C18H17 N3O
291
110
66
157
B14P14 OCH3
C18H17 N3O2
307
155
68
B15P15
OCH3
OCH3
OCH3
C20H21 N3O4
367
165
72
158
Table 9. Elemental analysis data of 2, 4, 6-trisubstituted
pyrimidines
Compound ( % Calculated)
C H N
( % Found)
C H N
B1P1 69.05 5.07 20.13 69.02 5.04 20.12
B2P2 69.05 5.07 20.13 69.01 5.02 20.10
B3P3 69.05 5.07 20.13 69.01 5.00 20.07
B4P4 67.40 4.90 15.70 67.00 4.87 15.20
B5P5 67.16 5.97 20.89 67.11 5.92 20.84
B6P6 63.58 4.62 14.83 63.55 4.58 14.80
B7P7 72.13 5.10 17.17 72.10 5.06 17.13
B8P8 73.15 4.91 17.06 73.14 4.86 17.02
B9P9 79.55 5.07 11.13 79.50 5.00 11.15
B10P10 69.15 5.09 14.23 69.13 5.05 14.25
B11P11 65.49 4.53 13.48 65.44 4.50 13.45
B12P12 57.32 3.96 11.80 57.30 3.92 11.75
B13P13 74.20 5.88 14.42 74.17 5.84 14.47
B14P14 70.35 5.54 13.68 70.30 5.51 13.63
B15P15 65.38 5.76 11.44 65.36 5.72 11.40
159
Table 10. IR spectral data of 2, 4, 6-trisubstituted
pyrimidines
Compound Position of absorption band ( cm-1)
B1P1 3361 (NH2), 3499 (O-H), 1602 (C=N),
1573 (C=C Quadrant of Ar), 1508 (CH=CH)
B2P2 3362 (NH2), 3497 (O-H), 1600 (C=N),
1575 (C=C Quadrant of Ar),1505 (CH=CH)
B3P3 3364 (NH2), 3496 (O-H), 1602 (C=N),
1572 (C=C Quadrant of Ar), 1504 (CH=CH)
B4P4 3360 (NH2), 3498 (O-H), 1602 (C=N),
1573 (C=C Quadrant of Ar), 1504 (CH=CH), 1200 (-C-O-)
B5P5 3365 (NH2), 3499 (O-H), 1605 (C=N),
1574 (C=C Quadrant of Ar), 1505 (CH=CH)
B6P6 3361 (NH2), 3497 (O-H), 1602 (C=N),
1572 (C=C Quadrant of Ar), 1504 (CH=CH), 704 (C-S)
B7P7
3365 (NH2), 3495 (O-H), 1604 (C=N),
1575 (C=C Quadrant of Ar), 1506 (CH=CH)
B8P8 3361 (NH2), 3496 (O-H), 1603 (C=N),
1570 (C=C Quadrant of Ar),1503 (CH=CH)
B9P9 3363 (NH2), 3495 (O-H), 1601 (C=N),
1573 (C=C Quadrant of Ar), 1504 (CH=CH)
B10P10 3361 (NH2), 3498 (O-H), 1605 (C=N),
1574 (C=C Quadrant of Ar), 1505 (CH=CH), 1120 (C-F)
160
B11P11 3360 (NH2), 3496 (O-H), 1600 (C=N),
1572 (C=C Quadrant of Ar), 1505 (CH=CH), 853 (C-Cl)
B12P12 3362 (NH2), 3499 (O-H), 1604 (C=N),
1575 (C=C Quadrant of Ar), 1506 (CH=CH), 1020 (C-Br)
B13P13 3360 (NH2), 3497 (O-H), 1603 (C=N),
1572 (C=C Quadrant of Ar), 1505 (CH=CH)
B14P14 3361 (NH2), 3495 (O-H), 1602 (C=N),
1571 (C=C Quadrant of Ar), 1507 (CH=CH), 1070 (-O-CH3)
B15P15 3365 (NH2), 3498 (O-H), 1604 (C=N),
1570 (C=C Quadrant of Ar), 1504 (CH=CH), 1072 (-O-CH3)
161
Table 11. 1 H NMR spectral data of 2, 4, 6-trisubstituted pyrimidines
Compound Chemical shift ( δ ) in ppm
B1P1 8.28 (1H,s, C-5-H)
5.40 (2H,s, C-2-NH2)
7.87 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
7.90 (1H,d,J=8.8Hz,C-6'-H)
8.57 (1H,d,J=8Hz,C-3''-H)
7.67 (1H,t,J=8Hz,C-4''-H)
7.47 (1H,m ,J=7.5Hz,C-5''-H)
8.68 (1H,d,J=7.8Hz, C-6''-H)
B2P2 7.71 (1H,s, C-5-H)
5.30 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
9.19 (1H,s,C-2''-H)
8.31 (1H,d,J=8.2Hz,C-4''-H)
7.42 (1H,m ,J=8.2Hz,C-5''-H)
8.69 (1H,d,J=7.8Hz, C-6''-H)
B3P3 7.86 (1H,s, C-5-H)
5.30 (2H,s, C-2-NH2)
7.83 (1H,s,C-2'-H)
2.33 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
8.75 (1H,d, J=8.1Hz ,C-2''-H)
7.93 (1H,d,J=8.1Hz,C-3''-H)
8.09 (1H,d ,J=8.1Hz,C-5''-H)
8.61 (1H,d,J=8.2Hz, C-6''-H)
162
B4P4 8.00 (1H,s, C-5-H)
5.25 (2H,s, C-2-NH2)
7.83 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
7.87 (1H,d,J=8.8Hz,C-6'-H)
7.00 (1H,d, J=6.8Hz ,C-3''-H)
7.41 (1H,d,J=6.7Hz,C-4''-H)
7.70 (1H,d,J=6.7Hz,C-5''-H)
B5P5 7.80 (1H,s, C-5-H)
5.30 (2H,s, C-2-NH2)
7.77 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
8.00 (1H,d,J=8.8Hz,C-6'-H)
6.87 (1H,d, J=6.7Hz ,C-3''-H)
6.18 (1H,m,J=6.8Hz,C-4''-H)
6.69 (1H, d, J=6.7Hz,C-5''-H)
B6P6 7.40 (1H,s, C-5-H)
5.18 (2H,s, C-2-NH2)
7.82 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
7.90 (1H,d,J=8.8Hz,C-6'-H)
7.89 (1H,d, J=6.6Hz ,C-3''-H)
7.21 (1H,m,J=6.7Hz,C-4''-H)
7.57 (1H, d, J=6.6Hz,C-5''-H)
163
B7P7
7.38 (1H,s, C-5-H)
5.44 (2H,s, C-2-NH2)
7.86 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.15 (1H,d,J=8.8Hz,C-5'-H)
7.91 (1H,d,J=8.8Hz,C-6'-H)
8.21 (1H,m,C-2''-H)
8.60 (1H,d,J=7.7Hz,C-4''-H)
7.05 (1H,t, J=7.7Hz,C-5''-H)
7.22 (1H,t, J=7.9Hz,,C-6''-H)
7.44 (1H,d, J=7.9Hz,C-7''-H)
B8P8 8.31 (1H,s, C-5-H)
5.35 (2H,s, C-2-NH2)
7.87 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.20 (1H,d,J=8.8Hz,C-5'-H)
7.90 (1H,d,J=8.8Hz,C-6'-H)
8.50 (1H,d, J=8.1Hz, C-3''-H)
8.30 (1H,d,J=8.1Hz,C-4''-H)
7.85(1H,d, J=7.9Hz,C-5''-H)
7.55 (1H,t, J=7.9Hz,C-6''-H)
7.73 (1H,t, J=7.9Hz,C-7''-H)
8.26 (1H,d,J=7.9Hz, C-8''-H)
B9P9 7.82 (1H,s, C-5-H)
5.02 (2H,s, C-2-NH2)
7.90 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.10 (1H,d,J=8.8Hz,C-5'-H)
7.91 (1H,d,J=8.8Hz,C-6'-H)
7.81 (1H,d, J=7.8Hz ,C-1''-H)
7.18 (1H,m, J=8.1Hz ,C-2''-H)
164
7.48 (1H,m, J=8.1Hz ,C-3''-H)
8.23 (1H,d,J=8.1Hz,C-4''-H)
8.23 (1H,d, J=8.1Hz,C-5''-H)
7.48 (1H,m, J=8.1Hz,C-6''-H)
7.18 (1H,m, J=7.8Hz,C-7''-H)
7.82 (1H,d,J=7.8Hz, C-8''-H)
8.70 (1H,s, C-10''-H)
B10P10 7.84 (1H,s, C-5-H)
5.45 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
6.89 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
8.10 (1H,d,J=8.6Hz,C-2''-H)
7.29 (1H,d,J=8.6Hz,C-3''-H)
7.29 (1H,d, J=8.6Hz,C-5''-H)
7.99 (1H,d, J=8.6Hz,C-6''-H)
B11P11
7.81 (1H,s, C-5-H)
5.42 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.15 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
7.85 (1H,d,J=8.4Hz,C-2''-H)
7.60 (1H,d,J=8.4Hz,C-3''-H)
7.60 (1H,d, J=8.4Hz,C-5''-H)
7.99 (1H,d, J=8.4Hz,C-6''-H)
165
B12P12 7.79 (1H,s, C-5-H)
5.30 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.30 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
8.12 (1H,d,J=8.2Hz,C-2''-H)
7.53 (1H,d,J=8.2Hz,C-3''-H)
7.53 (1H,d, J=8.2Hz,C-5''-H)
8.12 (1H,d, J=8.2Hz,C-6''-H)
B13P13
7.72 (1H,s, C-5-H)
5.43 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
6.89 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
8.05 (1H,d,J=8.1Hz,C-2''-H)
7.36 (1H,d,J=8.1Hz,C-3''-H)
2.41 (3H,s, C-4''-CH3)
7.36 (1H,d, J=8.1Hz,C-5''-H)
8.05 (1H,d, J=8.1Hz,C-6''-H)
166
B14P14
7.75 (1H,s, C-5-H)
5.45 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.10 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
8.10 (1H,d,J=7.9Hz,C-2''-H)
7.05 (1H,d,J=7.9Hz,C-3''-H)
3.80 (3H,s, C-4''-OCH3)
7.05 (1H,d, J=7.9Hz,C-5''-H)
7.99 (1H,d, J=7.9Hz,C-6''-H)
B15P15
7.57 (1H,s, C-5-H)
5.25 (2H,s, C-2-NH2)
7.84 (1H,s,C-2'-H)
2.32 (3H,s,C-3'-CH3)
7.15 (1H,d,J=8.8Hz,C-5'-H)
7.94 (1H,d,J=8.8Hz,C-6'-H)
7.61 (1H,s,C-2''-H)
3.93 (3H,s,C-3''-OCH3)
3.94 (3H,s, C-4''-OCH3)
3.93 (3H,s, C-5''-OCH3)
7.61 (1H,s, C-6''-H)
167
Fig.19. IR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine (B1P1)
168
Fig.20. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine (B1P1)
169
Fig.21. Mass spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine (B1P1)
170
Fig.22. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(3''-pyridinyl) pyrimidine (B2P2)
171
Fig.23. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-pyridinyl) pyrimidine (B3P3)
172
Fig.24. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-furyl) pyrimidine (B4P4)
173
Fig.25. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyrrolyl) pyrimidine (B5P5)
174
Fig.26. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-thienyl) pyrimidine (B6P6)
175
Fig.27. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-indolyl) pyrimidine (B7P7)
176
Fig.28. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-quinolinyl) pyrimidine (B8P8)
177
Fig.29. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(9''-anthracenyl) pyrimidine (B9P9)
178
Fig.30. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-fluorophenyl) pyrimidine (B10P10)
179
Fig.31. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-chlorophenyl) pyrimidine (B11P11)
180
Fig.32. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-bromophenyl) pyrimidine (B12P12)
181
Fig.33. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methylphenyl) pyrimidine (B13P13)
182
Fig.34. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methoxyphenyl) pyrimidine (B14P14)
183
Fig.35. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(3'', 4’, 5’’-trimethoxyphenyl) pyrimidine (B15P15)
175
2.3 BIOLOGICAL EVALUATION
PRESENT WORK
A number of 2, 4, 6-trisubstituted pyrimidines were reported to
possess diverse biological activities like antimicrobial, antidepressant,
analgesic, anti-inflammatory, anticancer, antiviral, antileishmanial,
antitubercular, anti-HIV and antimalarial. In view of the varied
biological and pharmacological importance of pyrimidine derivatives, it
is felt worthwhile to evaluate them for possible activities. These
compounds therefore were screened for anti-inflammatory,
antibacterial, antifungal and anticancer activities.
EXPERIMENTAL METHODS
ACUTE TOXICITY
The same protocols and procedures that have been followed in
Chapter-1 are used to study acute toxicity of 2, 4, 6-trisubstituted
pyrimidines.
All the pyrimidines employed in the pharmacological screening
have been found to be free form toxicity as well as toxic symptoms
even at a high dose of 1000 mg/kg body weight and hence they are
considered safe.
2.3.1 ANTI-INFLAMMATORY ACTIVITY
The same protocols and procedures that have been followed in
Chapter-1 are used to study anti-inflammatory activity of pyrimidines
(B1P1-B15P15). The results are presented in Table 12.
176
Table12. Anti-inflammatory activity of 2, 4, 6-trisubstituted
pyrimidines
Compound
Ar
% inhibition ± SEM at
various time intervals
0.5 h 1.0h 2.0h 3.0h 4.0h 6.0h
B1P1 2''-pyridinyl 7±1 8±1 38±1 45±1 77±1 80±1
B2P2 3''-pyridinyl 7±1 8±1 37±1 47±1 76±1 79±1
B3P3 4''-pyridinyl 6±1 7±1 36±1 47±1 75±1 79±1
B4P4 2''-furyl 6±1 7±1 35±1 46±1 74±1 77±1
B5P5 2''-pyrrolyl 15±1 16±1 54±1 65±1 95±1 97±1
B6P6 2''-thienyl 8±1 10±1 40±1 51±1 81±1 82±1
B7P7 2''-indolyl 15±1 17±1 55±1 67±1 96±1 98±1
B8P8 2''-quinolinyl 8±1 9±1 40±1 49±1 78±1 81±1
B9P9 9''-anthracenyl 11±1 14±1 46±1 55±1 87±1 91±1
B10P10 4''-fluorophenyl 10±1 13±1 45±1 56±1 84±1 89±1
B11P11 4''-chlorophenyl 9±1 12±1 44±1 55±1 82±1 86±1
B12P12 4''-bromophenyl 8±1 10±1 42±1 53±1 80±1 83±1
B13P13 4''-methylphenyl 11±1 14±1 50±1 60±1 91±1 95±1
B14P14 4''-methoxyphenyl 12±1 14±1 48±1 59±1 90±1 93±1
B15P15 3'',4'',5''-
trimethoxyphenyl
14±1 15±1 52±1 63±1 94±1 96±1
Aceclofenac (standard) 21±1 23±1 56±1 67±1 96±1 99±1
All values are represented as mean±SEM (n=6). *P<0.01 compared to
reference standard Aceclofenac. Student’s t-test. Dosage: Aceclofenac-
2 mg/kg and test compounds-10 mg/kg body weight of rat.
177
DISCUSSION ON THE RESULTS:
The anti-inflammatory activity of all the 2, 4, 6-trisubstituted
pyrimidines have been evaluated by using carrageenan-induced rat
paw oedema method. The results of this activity shown in Table 12.
From the results, it is evident that a number of these
pyrimidines possessed some degree of anti-inflammatory activity,
when compared to the standard drug, aceclofenac, but not at an
identical dose level, since the compounds were tested at 10 mg/kg,
whereas the drug tested at 2 mg/kg body weight dose levels. In
particular, compound B7P7 and B5P5 possessed maximum activity
Compounds B15P15, B13P13 and B14P14 carrying the electron releasing
substituents on the aromatic ring also enhanced the activity.
This suggest that pyrimidines having a number of these
substituents at different positions of the aromatic ring can be
synthesized with a hope to get promising leads in this series of
compounds. Another interesting observation emerged out of this study
is that the anti-inflammatory activity of the pyrimidines was more
than that of chalcones from which they were obtained. All these
observations were in agreement with the reports cited in the literature.
178
2.3.2 ANTIBACTERIAL ACTIVITY
Since pyrimidines have been reported to possess antibacterial
activity and hence the new 2, 4, 6-trisubstituted pyrimidines prepared
in the present work were tested for their antibacterial activity. The
same protocols and procedures that have been followed in Chapter-1
are used to study antibacterial activity of newly synthesized
pyrimidines (B1P1-B15P15). The results are presented in Table 13.
179
Table 13. Antibacterial activity of 2, 4, 6 – trisubstituted
pyrimidines
Compound
Ar
Zone of inhibition in mm
Quantity in µg/ml
B.pumilis B.subtilis E. coli P.vulgaris
50 100 50 100 50 100 50 100
B1P1 2''-pyridinyl 04 06 05 07 05 09 09 10
B2P2 3''-pyridinyl 04 05 05 06 05 07 08 09
B3P3 4''-pyridinyl 05 06 05 07 06 08 08 10
B4P4 2''-furyl 12 14 13 17 15 17 13 15
B5P5 2''-pyrrolyl 05 08 06 09 07 11 09 12
B6P6 2''-thienyl 05 07 05 08 06 10 10 11
B7P7 2''-indolyl 09 12 10 13 13 16 10 12
B8P8 2''-quinolinyl 13 15 14 17 16 18 14 16
B9P9 9''-anthracenyl 04 05 04 05 05 06 07 08
B10P10 4''-fluorophenyl 12 15 13 16 16 17 12 15
B11P11 4''-chlorophenyl 11 14 12 16 15 16 12 14
B12P12 4''-bromophenyl 10 14 12 15 14 16 11 13
B13P13 4''-methylphenyl 06 09 08 11 09 14 09 11
B14P14 4''-methoxyphenyl 05 09 07 10 08 13 09 10
B15P15 3'',4'',5''-trimethoxyphenyl
07 10 08 12 10 15 08 12
Benzyl penicillin(standard) 15 18 16 19 18 22 17 20
Control (DMSO) - - - - - - - -
180
DISCUSSION ON THE RESULTS:
All the pyrimidine derivatives (B1P1-B15P15), have been evaluated
for their antibacterial activity against, Bacillus pumilis, Bacillus
subtilis (Gram-positive) and Escherichia coli, Proteus vulgaris (Gram-
negative) using cup-plate method. The results of this evaluation have
been compared by taking benzyl penicillin as reference standard.
The antibacterial activity data of pyrimidine derivatives (B1P1-
B15P15, Table 13) indicated that the compounds have some degree of
inhibitory activity on all the bacteria at both 50 µg (0.05 ml) and 100
µg (0.1ml) dose levels, when compared with the reference standard.
From the results, it is evident that the compounds B8P8, B4P4
and B10P10 exhibited significant antibacterial activity, at a
concentration of 0.1 ml dose level and is comparable to that of
standard drug, benzyl penicillin at a concentration of 100 µg/ml.
Compounds B11P11, B12P12 and B7P7 showed moderate antibacterial
activity. The results are consistent with the biological activity of
existing drugs. Further studies have to be conducted to explore the
mechanism of action of these compounds.
181
2.3.3 ANTIFUNGAL ACTIVITY
The same protocols and procedures that have been followed in
Chapter-1 are used for antifungal activity of pyrimidines (B1P1-B15P15)
and the results are given in Table 14.
182
Table 14. Antifungal activity of 2, 4, 6 – trisubstituted
pyrimidines
Compound
Ar
Zone of inhibition in mm
Quantity in µg/ml
A.niger P.crysogenium
50 100 50 100
B1P1 2''-pyridinyl 04 05 05 07
B2P2 3''-pyridinyl 04 06 05 06
B3P3 4''-pyridinyl 04 05 06 07
B4P4 2''-furyl 09 12 08 10
B5P5 2''-pyrrolyl 05 07 05 06
B6P6 2''-thienyl 05 06 05 07
B7P7 2''-indolyl 08 11 07 09
B8P8 2''-quinolinyl 10 12 09 11
B9P9 9''-anthracenyl 04 05 05 06
B10P10 4''-fluorophenyl 13 16 12 14
B11P11 4''-chlorophenyl 12 15 12 13
B12P12 4''-bromophenyl 11 14 11 12
B13P13 4''-methylphenyl 06 09 06 07
B14P14 4''-methoxyphenyl 05 08 06 07
B15P15 3'',4'',5''-trimethoxyphenyl
07 10 07 08
Fluconazole (standard) 17 21 15 18
Control (DMSO) - - - -
183
DISCUSSION ON THE RESULTS:
The antifungal activity of substituted pyrimidines obtained in
the present study was evaluated against A. niger and P.crysogenium
employing fluconazole as the standard drug and using cup-plate
method.
A close examination of Table 14, pertaining to the antifungal
data of pyrimidine derivatives (B1P1-B15P15) revealed that all the
compounds in this series have been found to be effective against all
fungi at both 50 µg (0.05 ml and 100 µg (0.1 ml dose levels, when
compared with reference standard fluconazole.
Among the compounds tested, compounds B10P10, B11P11 and
B12P12 were found to be more potent than other compounds. This
clearly revealed the contribution of electron withdrawing groups (like
halogens) on the aromatic ring in enhancing the antifungal activity.
Literature reports were also in agreement with these observations.
184
2.3.4 ANTICANCER ACTIVITY
A number of pyrimidines synthesized earlier in our laboratory,
when screened for anticancer activity, found to be active on only
prostate cancer cell lines and infact some of them were found to
possess significant activity. Moreover, several reports on the
usefulness of pyrimidines as anticancer agents were available in
literature.
The synthesized pyrimidines have been screened for anticancer
activity on prostate cancer cell lines (DU-145) using MTT based
cytotoxicity assay described by Mosmann58 in 1983.
Chemicals/Biochemicals used in the present study:
(a) Media: DMEM (Dulbecco’s Modified Eagles Medium)
(b) 10% Fetal bovine serum (FBS)
(c) MTTreagent:
[3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide]
(d) Cell lines: DU-145 cell lines, were obtained from the National
Centre for Cell Science ( NCCS), Pune ( India)
Procedure:
This method is based on a colorimetric assay which takes
into account the ability of a mitochondrial dehydrogenase
enzyme from viable cells to cleave the tetrazolium rings of the
pale yellow MTT and form dark blue formazan crystals which is
largely impermeable to cell membranes, thus resulting in its
accumulation within healthy cells. The level of formazan created
185
is a reflection of the number of surviving cells and shows a
proportionality relationship between them.
The required cell proliferation assay kit was
obtained from Roche Applied Sciences, Germany. The procedure
consists of seeding an equal number of DU-145 cells in each
well of a 96- well microplate and incubating at 37○C in the
presence of 5% CO2. Various concentrations of the test
substances were added to the cells. For every 24 hours the
culture medium was renewed with the test substances. 0.5%
DMSO was added into the vehicle control culture wells. After 72
hours treatment, 5 µl of MTT reagent (R&D Systems, USA) along
with 45 µl of phenol red and FBS free DMEM (Sigma Life
Science, USA) was added to each well and incubated for 4 hours
at 37○C in presence of 5% CO2. Then 50 µl of solublization
buffer (R&D Systems, USA) was added to each well to solubilize
the coloured formazan crystals produced by the reduction of
MTT. After 24 hours, the optical density was measured at 550
nm using a microplate reader (BioRad, USA). The results (mean
O.D.± SD) obtained from quadruplicate wells were used in
calculation to determine the IC50 of the test compounds.
186
The percent inhibition is then calculated from the
formula:
% inhibition = Control O.D. – Sample O.D. × 100
Control O.D.
The IC50 values of the newly synthesized pyrimidine derivatives
were shown in Table 15.
Table 15. Anticancer activity of selective 2, 4, 6 – trisubstituted
Pyrimidines on DU-145 cell lines
S.No.
Compound
IC50 for cell proliferation (50µg/ml)
1 B5P5 10.56
2 B7P7 24.79
3 B8P8 32.18
4 B10P10 185.23
5 B11P11 55.46
6 B12P12 42.17
187
DISCUSSION ON THE RESULTS:
The above IC50 values for pyrimidines revealed that they did not
have any significant anticancer activity against the cell lines (DU-145)
tested. Of all the compounds B10P10 showed maximum activity. The
fluorine substituent present on the phenyl ring in B10P10 contributed
favorably to the observed anticancer activity, which is consistent with
the literature reports. In fact, a number of anticancer drugs currently
used in therapy possessed one or more fluorine substituents in their
structures. Pyrimidines having number of substituents at different
positions of the phenyl ring can be synthesized as the resulting
compounds are likely to possess significant activity. A QSAR study on
a large set of compounds need to be carried out in order to arrive at
the structural requirements and contributing physico-chemical
properties for the anticancer activity of 2,4,6-trisubstituted
pyrimidines. These compounds also need to be tested on other cancer
cell lines in order to predict their activity and therapeutic usefulness.
188
2.4 REFERENCES
1. Kidwai, M., Saxena, S., Rastogi, S. and Venkataramanan, R.,
Current Med. Chem. Anti-infective agents, 2, 269 (2003).
2. Carey, F.A., in: Organic Chemistry, The McGraw- Hill companies
(7th Indian Edition), New delhi, 1164 ( 2008).
3. Thore, S.N. et al., Asian J. Chem., 19, 4429 (2007).
4. Varga, L., Nagy, T., Kovesdi, I., Benet, B. J., Dorman, G., Urge, L.
and Darvis, F., Tetrahedron, 59, 655 (2003).
5. Kenner, G.W. and Todd, A., in: Hetrocyclic compounds, Chapman
and Hall, New York, 242 (1957).
6. Phillippi, E., Hendgen, F. and Hernler, F., Monatsch. Chemie., 69,
270 (1936).
7. Whitehead, C. and Traverso, J., J.Am.Chem. Soc., 75, 671 (1953).
8. Armarego, W.L.F., in: Physical Methods in Hetrocyclic Chemistry,
Academic Press, New York, 90 (1971).
9. Vanderhaeghe, H. and Claesen, M., Bull. Sci. Chim. Belg., 66, 276
(1957).
10. Simson, M.M., J. Am. Chem. Soc., 71, 1470 (1949)
11. Baddar, F.G., Al-Hajjar, F.H and El-Rayyes, N.R., J. Het. Chem.,
13, 257 (1971).
189
12. Brown, D.J., in: Comprehensive Heterocyclic Chemistry, Pergman,
3, 57 (1984).
13. Al-Hajjar, F.H. and Sabir, S.S., J. Het. Chem., 19, 1087 (1982).
14. Reddy, G.S.I., Hobgood, R.T. and Gold Stein, J.H., J. Am. Chem.
Soc., 84, 336 (1962).
15. El-Rayyes, N. R. and Al-Hajjar, F.H., J. Het. Chem., 14, 367
(1977).
16. Rice, J.M., Dudek, G.O. and Barber, M., J. Am. Chem. Soc., 87,
4569 (1965).
17. Nishiwaki, T., Tetrahedron, 22, 3117 (1966).
18. Spitller, G., in: Physical Methods in Hetrocyclic Chemistry,
Academic Press, New York, 3, 286 (1971).
19. Hitchings, G.H., Ann. N.Y. Acad. Sci., 23, 700 (1961).
20. Patel, V.G. and Patel, J.C., Ultra Scientist of Physical Sci., 16, 111
(2004).
21. Mishra, B.K., Mishra, R. and Harinarayana Moorthy, N.S., Trends
in Applied Sciences Research, 3, 203 (2008).
22. Bodke, Y. and Sangapure, S.S., J. Indian Chem. Soc., 80, 187
(2003).
23. Murthy, Y.L.N., Rani, N., Ellaiah, P. and Bhavani Devi, R., Het.
Comm., 11, 189 (2005).
190
24. Kudari, S.M., Munera, W. and Beede, S.M., Oriental J. Chem., 12,
167 (1996).
25. Babu, V., Harinadha Kumar, P., Senthil Srinivasan, K.K. and
Bhat, G. V., Indian J. Pharm. Sci., 66, 647 (2004).
26. Fathalla, O.A., Zeid, I.F., Haiba, M.E., Soliman, A.M., Abid-
Elmoez, S.I.A. and EI-Serwy, W.S., World J. Chemistry., 4, 127
(2009).
27. Wang, S. et al., Bioorg. Med. Chem. Lett., 14, 4237 (2004).
28. Fahmy, H.T.Y., Rostom Sherif, A.F., Saudi Manal, N., Zjawiony
Jordan, K. and Robins David, J., Archiv der Pharmazie., 336,
216 (2003).
29. Kaldirkyan, M.A., Grigoryan, L.A., Geboyan, V.A., Arsenyan, F.G.,
Stepayan, G.M. and Garibdzhanyan, B.T., Pharm. Chem. J., 34,
521 (2006).
30. Grigoryan, L.A., Kaldrikyan, M.A., Melik-Ogandzhanyan, R.G.,
Arsenyan, F.G., Stepayan, G.H. and Garibdzhanyan, B.G., Pharm.
Chem. J., 33, 468 (2005).
31. Chan, D.C.M., Fu, H. F., Ronalf, A., Queener, S. F. and
Rosowsky, A., J. Med. Chem., 48, 4426 (2005).
32. Vishwanadhan, C.L., Joshi, A.A. and Sachin, S.N., Bioorg. Med.
Chem. Lett., 15, 73 (2005).
191
33. Chauhan, P.M.S., Anu, A., Srivastava, K. and Puri, S.K., Bioorg.
Med. Chem., 13, 6226 (2005).
34. Chern, J.H. et al., Bioorg. Med. Chem. Lett., 14, 2519 (2004).
35. Sheriff, A.F.R., Fahmy Hesham, T.Y. and Saudi Manal, N.S.,
Scientia Pharmaceutica, 71, 57 (2003).
36. Novikov, M.S., Ozerov, A.A., Orlova, Y.A. and Buckheit, R.W.,
Chem. Het. Compounds, 41, 625 (2005).
37. Zhou, S., Kern, E. R., Gullen, E., Yung-Chi, C., Drach, J.,
Matsumi, S., Mitsuya, H. and Zem Licka J., J. Med. Chem., 47,
6964 (2004).
38. Pirisino, R., Bainchini, F., Banchelli, J., Ignesti, G. and Ramondi,
L., Pharmacol. Res. Comm., 18, 241 (1996).
39. Cenieola, M. L., Donnoli, D., Stella, L., Paola, C.D., Constantino,
M., Anignente, E., Arena, F., Luraschi, E. and Saturnino, C.,
Pharmacol. Res., 22, 80 (1990).
40. Nargund, L.V.G., Badiger, V.V. and Yarnal, S.M., J. Pharm. Sci.,
81, 365 (1992).
41. Cottam, H.B., Wasson, D.B., Shih, H.C., Raychaudhary, A.,
Pasquale, G.D. and Carson, D.A., J. Med. Chem., 36, 3424 (1993).
42. Bruni, F., Costazo, A., Selleri, S., Guerrini, G., Fantozzi, R.,
Pirisino, R. and Brunelleschi, S., J. Pharm. Sci., 82, 480 (1993).
192
43. Vidal, A., Ferrandiz, M.L., Ubeda, A., Alarcon, A.A., Arques, J.S.
and Alcarz, M.J., J. Pharm. Pharmacol., 53, 1379 (2001).
44. Bruni, O., Brullo, C., Ranise, A., Schenone, S., Bondavalls, S.,
Barvocelli, E., Ballabeni, V., Chivarani, M., Tognolini, M. and
Jmpicciatore, M., Bioorg. Med. Chem. Lett., 11, 1397 (2001).
45. Ferri, P.F., Ubeda, A., Guillen, J., Lasri, J., Rosende, M.E.G.,
Akssir, M. and Arques, J.S., Eur. J. Med. Chem., 38, 289 (2003).
46. Ihsan, A.S. et al., J. Saudi Chem. Society, 7, 207 (2003).
47. Kandeel, M.M. and Omar, A.H., Bull. Faculty Pharmacy, 41, 43
(2003).
48. Carmen, A. et al., J. Med. Chem., 44, 350 (2001).
49. Pandey, S., Suryawanshi, S.N., Suman, G. and Srivastavam,
V.M.L., Eur. J. Med. Chem., 39, 969 (2004).
50. Joubran, L., Jackson, W. R., Compi, E. M., Robinson, A. J.,
Wells, B. A., Godfreay, P. D., Callaway, J. K. and Jaraott, B.,
Austrian J. Chem., 56, 597 (2003).
51. Burger, A., in: Burger’s Medicinal Chemistry and Drug Discovery,
John- Wiley publications Inc. 5th Edition (1995).
52. Foye, W.O., Lemke, T.L. and David, W., in: Principles of Medicinal
Chemistry, B. I. Waverly pvt. Ltd, 4th edition (1995).
193
53. Reddy, C.S. and Nagaraj, A., J. Heterocycl. Chem., 44, 1181
(2007).
54. Suryawanshi, S.N., Bhat, B.A., Susmita, P., Naveen, C. and
Suman, G., Eur. J. Med. Chem., 42, 1211 (2007).
55. Chauhan, P.M.S., Naresh, S., Agarwal, A., Sanjay babu, K.,
Nishi, N. G. and Suman, G., Bioorg. Med. Chem., 14, 7706 (2006).
56. Akbar, M., Naser, F., Golnar, K. and Neda, F., Synthesis and
Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry,
37, 279 (2007).
57. Shujang, Tu, Fang, F., Chunbao, M., Hong, J., Youjian, F.,
Daqing, S. and Xiangshan, W., Tetrahedron Lett.,44, 6153 (2003).
58. Mosmann, T., J.Immunol Methods, 65, 55 (1983).