chapter iv synthesis and biolgical evaluation of schiff...
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54
CHAPTER –IV
Synthesis And Biolgical Evaluation of
Schiff Bases Bearing Pyrimidine Moiety.
55
INTRODUCTION
Pyrimidines:
Pyrimidine is a heterocyclic aromatic organic compound similar to
benzene and pyridine, containing two nitrogen atoms at positions 1
and 3 of the six-member ring. It is isomeric with two other forms
of diazine. Pyrimidine is a colourless compound, melting point
(22.5o C), boiling point (124oC). Pyrimidines are aromatic, basic and
water soluble compounds and pyrimidine is a much weaker base
than pyridine.
Three pyrimidine derivatives that are integral part of DNA
and RNA well as to structure of medicinally active agents.
Thymine may also referred as 5-methyl uracil. The substituted
pyrimidines are complex molecules because of the nature of the
substituents. Uracil and thymine may be considered to contain the
neutral urea unit or the acidic imide moiety.
The metabolism of these unique pyrimidines is important from the
standpoint of both biochemical utilization of these compounds and
drug metabolism of pyrimidine derivatives. Uracil is converted into
a useful compound uridylic acid needed for synthesis of RNA. In a
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similar manner cytosine is conjugated with PRPP to yield cytidine-
5- monophosphate or cytidylic acid. Thymine is metabolized by
conjugation via salvage pathway with PRPP to the thymine ribosyl
–5-phosphate. This form of thymidylic acid can be utilized in
specific RNA molecules.
Two special pyrimidines are barbituric acid and substituted
barbiturates. The substituted barbiturates represent a special
class of compounds. Which have been used for their sedative
hypnotic action.
Barbituric acid is a fairly strong acid with a Pka of 4.12, but
upon substitution at the 5 positions, the Pka rises dramatically,
the 5,5- disubstituted barbiturates react with sodium hydroxide to
form a salt that is quite water-soluble.
Properties of Pyrimidines
Aromaticity1:
A new σ-п separatability criterion is used to divide the total
energy of planner ring system into two parts. A σ part and п part.
The behavior of these parts under distortions towards resonance
structure is investigated. The Callen showed that the σ energy
tends towards bond equalization. The total structure depends on
the relative dominance of “σ vs п” electron tends. The properties of
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benzene referred to as aromaticity as due to a forced п-electrons
delocalization.
Oxidation: All the three peracids2 (U, T, C) giving N- oxides but
care must be taken with pyrimidines3 due to the relative instability
of the products under the acidic conditions. Pyrazines form N, N1 –
dionides most easily, but pyridazine4 requires forcing conditions5.
Alkylation:
The pyrimidines react with alkyl halides to give mono
quaternary salts, through somewhat less readily than comparable
to pyridines.
Pyrimidines of Pharmacological interest:
Certain pyrimidine derivatives are also known to display
antimalarial6, antifilarial7, and anti-leishmanial8 activities. The
biodynamic property of this ring system prompted us to design
pyrimidine derivatives stimulating pharmacophores and
substituents responsible for diverse pharmacological activities.
Nandeeshaiah et al.,9 reported the synthesis of (1) which
showed the blood platelet disaggregating property.
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SeNN
N
NHR
R=H
(1)
Hansa et al.,10 have reported the synthesis of (2) which
showed potential anti-microbial activity.
(2)
Upadhyay et al.,11 have reported the synthesis of (3)
pyrimidine and azo pyrimidines as biodynamic agents.
(3)
Heidal et al.,12 prepared 5-Fluorouracil (4) and
demonstrated its significant tumour inhibiting activity.
(4)
Parmar et al.,13 have reported the synthesis of thiazolidinones
(5) from hydrazino pyrimidine as potential anti-microbial agent.
(5)
Sondhi et al.,14 have reported anti-inflammatory and
analgesic activity of synthesized pyrimidine derivatives (6,7).
59
( 6 ) ( 7 )
RIVEW OF LITERATURE
Rajesh et al.,15 synthesized some 2- amino- 4, 6-
diarylsubstituted pyrimidines (8) and then screened them for
antibacterial and herbicidal activity.
( 8 )
Laura et al., 16 synthesized some new amino derivatives of 1,
2, 3- triazole [4, 5- d] pyrimidines (9) and then screened them for
their affinity towards A1 and A2A adenosine receptors.
( 9 )
Erric et al., 17 synthesized analogues of 4- benzylamino-2-
7H- pyrrolo [2, 3- d] pyrimidines (10) and then screened them for
their anxiolytic activity.
(10)
Silvana et al., 18 synthesized novel purine and pyrimidines
nucleoside analogues containing 2, 3- epoxypropyl, 3-amino- 2-
hydroxyl or 2, 3- epoxypropyl ether moieties (11) and then
screened them for their anti-tumour and antiviral activity.
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(11)
Munchhof et al., 19 synthesized thieno pyrimidines and
thieno pyridines (12) and then screened them for anticancer
activity.
(12)
Nakashima et al., 20 synthesized fused pyrimidine
derivatives (13), and screened them for blood oxygen partial
pressure amelioration.
(13)
John et al., 21 synthesized thieno [3, 2- d] pyrimidines and
furo [3, 2- d] pyrimidines (14) and then screened them for
purinergic receptor antagonists.
(14)
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SCHEME-II
CHO
FH3C
O
O
O
CH3
Piperidine
CH3COOH
F
COOCH3
H2N
S
HN
CH3
2
H2SO4
F
HN
N
O
OCH3
CH3
CH3
SH3C
F
N
N
O
OCH3
CH3
CH3
SH3C
mCPBA
MDC
F
N
N
O
OCH3
CH3
CH3
SH3C
O O
Methanol
CH3NH2
F
N
N
O
OCH3
CH3
CH3
NH
H3C
F
N
N
O
OCH3
CH3
CH3
NH3C
NaH \ DMF
CH3SO2Cl
SH3C
O
O
DIBAL-H
Toluene
F
N
NCH3
CH3
NH3C
SH3C
O
O
OH
F
N
N
CHO
CH3
CH3
NH3C
SH3C
O
O
DDQ
Toluene
Toluene
MnO2
F
N
NCH3
CH3
NH3C
SH3C
O
O
N
R-NH2
CH3
HMPA
R
(SA-1)
(SA-2)(SA-3)(SA-4)
(SA-5) (SA-6)(SA-7I)
(SA-8a-j)
CH3COOH
p- FluroBenzaldehyde
Methyl isobutyrylacetate
O
62
EXPERIMENTAL
Procedure for the preparation of Compound SA-1:
P-fluorobenzaldahyde (5.94g, 0.1 mol), methylisobutyryl acetate
(6.33g, 0.1 mol), piperidine (0.42g, 0.01mol) and acetic acid (0.2ml)
was placed in a round bottom flask fitted with a condenser and the
mixture was heated at 85-90o C for 3hrs. Cooled the reaction
mixture and added chloroform (100ml). The separated chloroform
layer was washed with 0.1N HCl (50ml), 5 % aq. Sodium
bicarbonate (50ml) and finally with water (100ml). The solvent was
evaporated completely. The crude mass (12g) was purified by
Column chromatography. (Yield-70%, b.p- 320oC).
Procedure for the preparation of Compound SA-2:
Compound SA-1 (10g, 1mol), S-methyl urea hydrogen sulphate
(7.93g, 0.8mol) and hexamethyl phosphoramide (15ml) was placed
in a round bottom flask and the mixture were heated at 120o-
125oC for 24hrs. Cooled the reaction mixture to 80o C and added
toluene (50ml), 5% aq.Sodium bicarbonate (50ml). The mixture
was stirred for 30 min at 80-85o C. Cooled the reaction mixture to
25 o C and separated the toluene layer. It was washed with water
(50ml). The solvent was evaporated under reduced pressure, to get
the crude mass. Fresh toluene (50ml) was added to the crude mass
in a round bottom flask to which added a solution of 2,3-Dichloro-
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5,6-dicyno-1,4-benzoquinone(DDQ) (4.7g, 0.5mol) in toluene(50ml)
slowly for 15min at 25-30o C. The reaction mixture was stirred for
2hrs. Filtered the 2,3-Dichloro-5,6-dicyno-1,4-hydroquinone
(DDHQ) and washed the DDHQ with toluene (10ml) and filtrate
was washed with 5 % aq. NaHCO3 (4×50ml) and water (100ml). The
solvent was evaporated to get the solid. The solid was recrystallised
from ethanol. (m.p.- 85oC, yield - 76%.)
Procedure for the preparation of Compound SA-3:
In to a clean dry round bottomed flask introduced Compound
SA-2 (10g,0.1mol) in Dichloromethane(100ml) to which was added
a solution of m-choloroperbenzoic acid (13.5g, 0.25mol) in
dichloromethane (50ml) for 30min at 0-5oC. Slowly warmed the
mixture to 25-30oC and stirred for 2hrs at same temperature (m-
chlorobenzoic acid precipitated out). Filtered the m-chlorobenzoic
acid and the filtrate was washed with 5 % aq. NaHCO3 (2×50ml)
and with water (100ml). The solvent was evaporated to get the
crude mass from which pure solid was obtained by the addition of
hexane (50ml). The separated solid was recrystallised with ethanol.
(m.p.- 120 oC, yield-68%.).
64
Procedure for preparation of Compound SA-4:
The compound SA-3 (10g) in menthol (50ml) was placed in a
round bottom flask and cooled the solution to 0-5oC. Added a
solution of 25 % methylamine in methanol (100ml) at 0-5o C for
1hr. and it was refluxed for 2hrs. The solvent was evaporated to get
the solid and the solid was recrystallised with ethanol. (m.p- 95oC,
yield- 80 %.).
Procedure for the preparation of Compound SA-5:
The compound SA-4 (10g, 0.1mol) in DMF (50ml) was placed
in a round bottom flask and cooled the solution to 0-5oC. Added
60 % Sodium hydride (1.5g, 0.12mol) under nitrogen. Stirred the
reaction mixture for 30 min at 0-5o C, added a solution of methane
sulfonyl chloride (3.76g, 0.1mol) in DMF (25ml) for 10min at 0-5
oC. Stirred the mixture for 1hr. Then the reaction mixture was
added to ice cold water with stirring. Filtered the separated crude
solid and washed with 5 % aq. NaHCO3 (50ml) and water (50ml).
The product was recrystallised from ethanol. (m.p.- 109oC, yield -
50%.).
Procedure for the preparation of Compound SA-6:
The compound SA-5 (10g, 0.1mol) was dissolved in toluene (150ml)
in a clean dry round bottom flask. Cooled the reaction mixture to -
65
10 oC under nitrogen atmosphere. Slowly added a solution of 25 %
DIBAL-H in toluene (41.5ml, 0.25mol) for 30min at -10 oC. Added
water (100ml) and stirred for 30min and filtered the reaction
mixture, and washed with hot toluene (50ml). The water was
separated from filtrate. The toluene was evaporated completely to
get solid. The solid was recrystallised with ethanol. (mp.- 79 oC ,
yield-72%.).
Procedure for the preparation of Compound SA-7:
The compound SA-6 (10g) in toluene (100ml) and active Mno2 (20g)
were placed in a round bottom flask and stirred for 15 hrs at 25-30
oC. Filtered the Mno2 and washed with hot toluene (50ml). Then
the solvent was evaporated completely to get the solid. The solid
was recrystallised with ethanol. (m.p.- 62 oC, yield- 65 %.).
Procedure for the preparation of Compound SA-8a-j:
The compound SA-7 (0.1mol) and substituted amines (0.1mol) in
glacial acetic acid (25ml) were placed in a round bottom flask fitted
with a condenser and refluxed for 30 hrs. The mixture was added
to ice cold water with stirring, filtered the separated solid and
washed with water until the filtrate is neutral. The product was
recrystallised from ethanol and (Yields were in the range 60-70 %).
66
BIOLOGICAL ACTIVITY
All the synthesized compounds were evaluated for their
antibacterial, anti inflammatory activity by following the standard
procedure.
ANTIBACTERIAL ACTIVITY:
All the compounds synthesized in the present investigation
were screened for their anti-bacterial activity by subjecting the
compounds to standard procedures. Antibacterial activities were
tested on nutrient agar medium against streptococci and
pseudomonas aureus which are representative types of gram
positive and gram negative organisms respectively. The
antibacterial activity of the compounds was assessed by disc-
diffusion method22.
67
TABLE-6: Antibacterial activity of compounds SA-8a-j.
Sl. No
Name of the compounds
Mean zone of inhibition (in mm)
Streptococci Pseudomonas aureus
50g 100g 50g 100g
01 Streptomycin 21 25 20 23
02 SA-8a 14 19 14 19
03 SA-8b 11 20 15 20
04 SA-8c 13 19 15 19
05 SA-8d 15 18 19 21
06 SA-8e 16 17 16 21
07 SA-8f 15 20 14 16
08 SA–8g 16 21 15 17
09 SA–8h 15 21 11 16
10 SA–8i 15 20 12 17
11 SA–8j 16 21 13 18
Average triplicate ± Standard deviation
Note:- “ – “denote no activity, 06 – 07mm poor activity, 08 –
10mm moderate activity, 11-12mm good activity.
68
ANTI INFLAMMATORY ACTIVITY:
Determination of acute toxicity (LD50):
The acute toxicity of synthesized compounds was
determined by using albino mice of either sex (20-30 gm) those
maintained under standard husbandry conditions. The animals
were fasted overnight prior to the experiment and fixed dose
(OECD guideline No. 420) method of CPCSEA was adopted for
toxicity studies. 1/5th of the lethal dose was taken as effective
dose ED50 (Therapeutic dose).
Anti inflammatory activity:
Inflammation is a normal protective response to tissue injury
caused by physical trauma, noxious chemicals or microbiologic
agents. Inflammation is body‟s response for tissue repair 23.
Inflammation is triggered by the release of chemical mediators
from the injured tissues and migrating cells. The specific chemical
mediators vary with the type of inflammatory process and include
amines such as histamine, serotonin, and lipids such as
prostaglandins and small peptides such as kinins 24. The acute
inflammatory response has 3 main functions.
The affected area is occupied by a transient material
called the acute inflammatory exudates. The exudates
69
carry proteins, fluid and cells from local blood vessels
into the damaged area to mediate local defenses.
If an infective causative agent (e.g. bacteria) is present in
the damaged area, it can be destroyed and eliminated by
components of the exudates.
The damaged tissue can be broken down and partially
liquefied, and the debris removed from the site of
damage.
Mechanism of inflammation:
In and around the inflamed tissue, there is an accumulation of
oedema fluid in the interstitial compartment which comes from
blood plasma by its escape through the endothelial wall of
peripheral vascular bed. In initial stage the escape of fluid i.e. due
to vasodilatation and consequent elevation in hydrostatic pressure,
the characteristic inflammatory oedema, and exudates appear by
increased vascular permeability of micro circulation. Inflammatory
diseases including different types of rheumatic diseases are a
major cause of morbidity of the working force throughout the
world. Many drugs produced a dramatic symptomatic improvement
in rheumatic processes, but all of them shared the common
undesirable effect i.e., gastrointestinal irritation 25.
70
Procedure 26, 27:
Carrageenan induced rat paw oedema model:
Albino rats of either sex weighing 150-200 gm were selected.
They were maintained on standard pellet diet and free access to
water.
The animals were divided into 12 groups each having six
animals. The various groups were treated as follows:
Group 1 - Normal Control (treated with 0.2mlof 5% gum acacia
p.o.)
Group 2 - Diclofenac (50 mg / kg, p.o.)
Group 3 - Compound SA-8a (50 mg/ kg, p.o.)
Group 4 - Compound SA-8b (50 mg/ kg, p.o.)
Group 5 - Compound SA-8c (50 mg/ kg, p.o.)
Group 6 - Compound SA-8d (50 mg/ kg, p.o.)
Group 7 - Compound SA-8e (50 mg/ kg, p.o.)
Group 8 - Compound SA-8f (50 mg/ kg, p.o.)
Group 9 - Compound SA-8g (50 mg/ kg, p.o.)
Group 10 - Compound SA-8h (50 mg/ kg, p.o.)
Group 11 - Compound SA-8i (50 mg/ kg, p.o.)
Group 12 - Compound SA-8j (50 mg/ kg, p.o.)
The normal control, diclofenac and test compounds were
administered to the rats 30 minutes before the injection of 0.1ml of
1% Carrageenan suspension in normal saline. Carrageenan
suspension was injected into the sub-planar region of the left hind
71
paw, and the right hind paw served as reference. Immediately there
after the oedema volume of the injected paws were measured
plethysmographically by mercury displacement method.
For comparison purpose, the volume of oedema at various
prefixed time intervals was measured. The difference between paw
volumes of the treated animals was measured and the mean
oedema volume was calculated.
Percentage reduction in oedema volume was calculated by using
the formula,
Vo - Vt Percentage reduction = x 100 Vo
Where, Vo = Volume of the paw of control at time„t‟.
Vt = Volume of the paw of drug treated at time„t‟.
From the data obtained, the mean oedema volume with
standard error (SEM), standard deviation (S.D.), percentage
reduction in oedema was calculated. The anti-inflammatory activity
of synthesized compound is given in the Table No-7.
72
TABLE-7:Anti-inflammatory activity of compounds SA-8a-j.
Sl.
No.
Name
Compounds
Dose
mg/kg
Mean difference in paw
volume S.E. after
3hr(ml)
Percentage of inhibition
01 Control -- 0.80 0.024* --
02 Standard
(Diclofenac Na) 50 0.16 0.01* 80.00
03 SA-8a 50 0.48 0.038* 40.00
04 SA-8b 50 0.31 0.027* 61.25
05 SA-8c 50 0.34 0.020* 58.00
06 SA-8d 50 0.50 0.033* 37.50
07 SA-8e 50 0.45 0.020* 43.75
08 SA-8f 50 0.54 0.032* 33.00
09 SA-8g 50 0.42 0.019* 47.50
10 SA-8h 50 0.29 0.026* 63.75
11 SA-8i 50 0.55 0.025* 31.25
12 SA-8j 50 0.49 0.021* 38.75
73
Result and Discussions:
All the synthesized pyrimidine derivatives were evaluated for
their antibacterial activity against the organisms Streptococci,
Pseudomonas aureus by disc diffusion method. Streptomycin
was used as a standard drug for compression. Antibacterial
evolution results indicate that the compounds are moderately
active against both the organism at the concentration 50µg/ml and
100µg/ml. Though the schiff‟s bases are known to show broad
spectrum of biological activity, perhaps the steric hindrance due to
the different crowed groups may hinder the activity of the
compounds irrespective of nature of substituents.
Among the series of the compounds SA-8a-SA-8j screened
for anti-inflammatory activity the compounds SA-8b, SA-8c and
SA-8h showed significant activity when compared with rest of the
compounds of the series. However these active compounds proved
to posse‟s moderate activity when compared with standard
Diclofenac sodium.
74
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