Chapter-5
Results and Discussion
The present work is divided into two parts:
SECTION A: Synthetic work
SECTION B: Pharmacological investigation of quinazoline derivatives for diuretic and
antihypertensive activity
SECTION A: SYNTHETIC WORK
Total 74new substituted quinazoline derivatives were synthesized by following different
schemes(Scheme I &II). The purity of compounds was checked by single-spot TLC using
Toluene:Ethyl acetate: Formic acid (5:4:1) and Benzene: Acetone (9:1) solvent systems and
spotslocated under iodine vapors/UV light. The structures of the synthesized compounds
wereestablished on the basis of modern analytical techniques; IR, 1H-NMR,
13
C-NMR,
Massspectral data and elemental analysis.
Scheme I:Total 38 Compounds were prepared from Scheme-I; N-(substituted-4-oxo-2substituted-phenylquinazolin-3-(4H)-yl) substituted benzene sulfonamide (1-38)
The title compounds were synthesized in four steps as outline in Scheme-I, illustrated with
the synthesis of compound, N-(2-(2-chlorophenyl)-7-nitro-4-oxoquinazolin-3(4H)-yl)-4methylbenzenesulfonamide(20). In the first step, 2-amino-4-nitrobenzoic acid was treated
with 2-chlorobenzoyl chloride to yield 2-(2-chlorobenzamido)-4-nitrobenzoic acid. It showed
sharp melting point, 220°C. It was further identified on the basis of IR and 1H-NMR spectra.
IR spectrum of the compound showed characteristic bands at 1706 and 1132cm-1 for carbonyl
and C-N group respectively. The
Ph.D Thesis
1
H-NMRspectrumsthere is appeared a multipletat
179Jamia Hamdard
Chapter-5
Results and Discussion
fromδ8.06-8.08 and 8.17-8.22 for aromatic protons.The characteristic peak of broad singlet at
δ 10.82 showed the presence of NH group and disappeared by the addition of D2O
asconfirmation for this groupand there is a singlet at δ 12.82 showed the presence of COOH
group.
2-(2-chlorobenzamido)-4-nitrobenzoic acidwas cyclized on acetylation to yield 2-(2chlorophenyl)-7-nitro-4H-benzo[d][1,3]oxazin-4-one. It showed different Rf value and
melting point while comparing with starting compound.The benzoxazine derivative was
further identified on the basis of IR and 1H-NMR spectral data.The appeared peak in IR
spectra at around1690,1115and 1250cm-1 for carbonyl, C_N and C-O-C moiety. The 1H-NMR
spectrum there appeared a multiplet at around δ8.02-8.04 andδ8.10-8.13 which confirmed the
presence of aromatic protons.
The above benzoxazine derivativewas reacted with hydrazine hydrate to get compound, 3amino-2-(2-chlorophenyl)-7-nitroquinazolin-4(3H)-one. It was structurally elucidated on the
basis of IR, 1H-NMR and Mass spectral data. The appeared peak in IR spectrum at around
3023 cm-1 for CH stretching. It was also confirmed the presence of carbonyl functional group
Ph.D Thesis
180Jamia Hamdard
Chapter-5
Results and Discussion
with the band at 1695 cm-1, the band at 1498 cm-1 also confirmed the double bond. The 1HNMR showed the aromatic protons as multiplet around δ7.90-7.94 andδ8.08-8.10.
The
characteristic singlet peak atδ5.22 showed the presence of NH2. This peak disappeared by the
addition of D2O asconfirms for this group. The mass spectrum showed the peak at m/z:
316.03(M+).
The
titled
compound,N-(2-(2-chlorophenyl)-7-nitro-4-oxoquinazolin-3(4H)-yl)-4-
methylbenzenesulfonamide
20
was
synthesized
by
the
reaction
chlorophenyl)-7-nitroquinazolin-4(3H)-onewith4-toluene-1-sulfonyl
of3-amino-2-(2chloride(Hinsberg
Reaction). It was structurally elucidated on the basis of IR, IH-NMR,13C-NMR and Mass
spectral data.
The IR spectra of the mentioned molecule showed characteristic absorption bands at 3311
cm-1 (N–H), 3018 cm-1 (Ar–CH), 2880 cm-1 (C–H), 2174 cm-1 (C–N), 1733cm-1 (CO),
1634cm-1 (C=N), 1592, 1465 cm-1 (C=C), 1372cm-1 (NO2), 1287, 1195cm-1(SO2). The 1HNMR spectra showed two doublet at δ7.42, J=6.4 Hz and 8.38, J=3.2 Hz confirming the
proton positions 5 and 8 in quinazolinone ring, one doublet at δ8.32, J=8.8Hz confirmed
aromatic proton at position of 2 in phenyl ring. Multiplets at δ 7.40–7.30 and δ 7.76– 8.58
indicated three protons of phenyl ring and four protons of another phenyl ring, respectively.
The one broad singlet corresponding to NH was obtained at δ 10.23 that was D2O
exchangeable. The chemical shift of 13C-NMR spectra at δ20.8could be account for CH3. The
signals of
13
Ph.D Thesis
C-NMR spectra which appeared at around δ 158.5 and δ 160.8accounted for
181Jamia Hamdard
Chapter-5
Results and Discussion
carbon of N-C=Nand CO. Whereas some
13
C-NMR spectra appeared at around δ122.2(2C),
128 (2C), 132.4, 138.2(Ar-C), 122.2, 128, 130, 131.8, 132.7, 134.2 (Ar-C), 120.6, 125.8,
128.3, 130.2, 152.6(2C)(Ar-C)could be accounted for aromatic carbon. The mass spectrum
showed the presence of molecular ion peak at m/z471/472/473(M+1/+2/+3) in accordance to
the molecular formula C21H15ClN4O5S.
Scheme II:Total 36 compounds were prepared from Scheme-II; 7-substituted-3-(4-(3-(4substitutedphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-substituted quinazolin-4(3H)-one
(39-74).
The titled compounds were synthesized in four steps as outline in scheme-II, illustrated with
the synthesis of compound,7-chloro-2-(2-chlorophenyl)-3-(4-(3-phenyl-4,5-dihydroisoxazol5-yl)phenyl)quinazolin-4(3H)-one(44). 2-amino-4-chlorobenzoic acid was treated with 2chlorobenzoyl chloride to yield 4-chloro-2-(2-chlorobenzamido)benzoic acid. It showed
sharp melting point, 270°C. It was further identified on the basis of IR and 1HNMR spectra.
IR spectrum showed characteristic bands at 1696 and 1112 for carbonyl and C-N group
respectively. The 1H-NMR spectrums showed the aromatic protons as multiplet at aroundδ
8.02-8.04 and 8.07-8.10. The characteristic peak of broad singlet at δ9.34 showed the
presence of NH, this peakdisappeared by addition of D2O as confirms for this group and 1HNMR spectra, there appeared asinglet at δ 12.82 indicate the presence of COOH.
Ph.D Thesis
182Jamia Hamdard
Chapter-5
Results and Discussion
2-(2-chlorobenzamido)-4-nitrobenzoic acid was cyclized on acetylation to yield 2-(2chlorophenyl)-7-nitro-4H-benzo[d][1,3]oxazin-4-one. It showed different Rf value and
melting point while comparing with starting compound.The benzoxazine derivative was
further identified on the basis of IR and 1H-NMR spectral data. IR spectrum showed
characteristic band at 1685,1608and 1240 for carbonyl, C=N and COC moiety. In 1HNMRspectra, there appeared a multiplet around δ7.90-7.94 and 8.02-8.04 indicative of
aromatic protons.
Cl
NH2
O
O
Cl
Cl
N
Cl
N
N
O
O
C
O
CH3
CH3
3-(4-acetylphenyl)-7-chloro-2-(2-chlorophenyl)
quinazolin-4(3H)-one
The benzoxazine derivative was further treated with p-aminoacetophenone to yield 3-(4acetylphenyl)-7-chloro-2-(2-chlorophenyl)quinazolin-4(3H)-one.
It
was
structurally
elucidated on the basis of IR, 1H-NMR and Mass spectral data. IR spectrum showed the band
at 3024 for CH stretching. It also confirmed the presence of carbonyl functional group with
the band at 1698, 1708 cm-1, the band at 1465 cm-1 also confirmed the double bond. The 1HNMR showed a multiplet around δ 8.00-8.03, 8.06-8.10 and 8.10-8.12 account for aromatic
Ph.D Thesis
183Jamia Hamdard
Chapter-5
Results and Discussion
protons. The characterstic singlet peak atδ2.16 showed the presence of CH3. The mass
spectrum showed the peak at m/z: 409.04(M+1).
Synthesis of (E)-7-chloro-2-(2-chlorophenyl)-3-(4-cinnamoylphenyl)quinazolin-4(3H)-one
was carried out by Claisen–Schmidt method, in which 3-(4-acetylphenyl)-7-chloro-2-(2chlorophenyl)quinazolin-4(3H)-one treated with benzaldehyde. It was structurally elucidated
on the basis of IR, 1H-NMR and Mass spectral data. IR spectra appeared at around3010 cm-1
for CH stretching. It also confirmed the presence of carbonyl functional group with the band
at 1680, 1710 cm-1, the band at 1460 cm-1account for the double bond. The 1H-NMR spectra
there appeared a multiplet around δ 8.02-8.06, 8.08-8.10, 8.12-8.14 and 8.17-8.22 indicative
of aromatic protons. The characteristicdoublet peak atδ6.62 (1H, J=7.8Hz, Ha) and δ7.10
(1H, J= 8.2Hz, Hb)showed the presence of CH=CH. The mass spectrum showed the peak at
m/z : 497.07(M+1).
Ph.D Thesis
184Jamia Hamdard
Chapter-5
Results and Discussion
(E)-7-chloro-2-(2-chlorophenyl)-3-(4-cinnamoylphenyl)quinazolin-4(3H)-one was cyclized
by using hydroxyl amine hydrochloride to form of 7-chloro-2-(2-chlorophenyl)-3-(4-(3phenyl-4, 5-dihydroisoxazol-5-yl) phenyl)quinazolin-4(3H)-one.The yield, melting point,
molecular formula and molecular weight are providedin experimental part.
IR spectrum of 44showed the characteristic absorption bands at 3034 cm-1 (Ar-CH), 1710 cm1
(CO), 1660 cm-1 (C=N), 1598, 1480 cm-1 (C=C), 1224 cm-1 (C-O-N), 1176 cm-1 (C-N), 610
cm-1 (C-Cl) respectively. The aromatic protons in 1H-NMR spectra appeared in theform of
doublet at aroundδ 8.14, J=0.6Hz. The characteristic peak of 1H-NMR spectra appeared as
doublet at around δ 3.94indicate the presence of CH2and double doublet at around δ5.86
indicate for CH in isoxazole. The aromatic protons in 1H-NMR spectra appeared in the
multiplets at around δ 8.06-8.03, 8.08-8.06 and 8.22-8.20 respectively. The isoxazole carbons
were observed in
13
C-NMR spectra at around δ 45.7, 88.5 and 154.7. The signals of
13
C-
NMR spectra which appeared at around δ 155.9 and δ 162.4 could be assigned to the carbons
of N-C=N and CO. The signals of aromatic carbons were observed at around δ 128.2 (2C),
130.8(2C), 131.4, 132.3 (Ar-C), 120.6, 124.8, 130.2, 132.3, 134.5, (Ar-C), 125.84(2C),
127.8(2C), 132.4, 137.6 (Ar-C) and signals 118.3,120.6,128.2, 130.8, 138.2, 151.3 of
quinazoline moiety respectively.The mass spectrum shows the presence of molecular ion
peak at m/z 512.38/513 (M+1/+2) accordance to the molecular formula, C29H19Cl2N3O2.
Ph.D Thesis
185Jamia Hamdard
Chapter-5
Results and Discussion
SECTION B: PHARMACOLOGICAL INVESTIGATION OF QUINAZOLINE
DERIVATIVES FOR DIURETIC AND ANTIHYPERTENSIVE ACTVITY
DIURETIC ACTIVITY
The final compounds in Scheme-I (1–38) were evaluated for diuretic activityby the Lipschitz
et al. method. The Diuretic activity at a dose of 2mg afteroral administration of test drug and
1mg of standard drugmetolazone (9.15±0.175** total urine output in mL)were also
observed.Compound no.10, 12, 20,21, 28 and 29showed 7.57±0.148**, 7.24±0.153**,
7.86±0.019**, 8.05±0.028**, 10.58±0.244** and 11.46±0.257** mL cumulative urine
output,
among
which
N-[7-chloro-2-(4-methoxyphenyl)-4-oxoquinazolin-3(4H)-yl]-4-
nitrobenzenesulfonamide (29) was highly substantial (11.46±0.257)(p<0.01), i.e. increased by
>200% with respect to control.Diuretic action of compound no.29 was found to be 1.99 (1.25
timesmore potent than metolazone at a double dose). The Lipschitzvalue (the ratio T/U, in
which T is the response of the testcompound and U is that of urea treatment, indices of 1.0
andhigher are regarded as a positive effect in terms of diureticactivity) shows that compound
no.29was 1.41 times potent than urea.
Further, compound no. 29 showed a substantial increase in the Na+excretion (p<0.01), i.e.
4.10±0.012, which was greater thanstandards, i.e. urea 2.49±0.023 and metolazone
(3.58±0.018). The diuretic action of compounds showing significant activitywas (single drug
or more) further analysed at dose equivalent to metolazone (1mg/kgbody weight). Among the
tested compounds, compound no.29 wasfound to be almost equally potent to metolazone.
However, Na+ and K+ excretion potential was higher than metolazone shown in Tables 1and
2, respectively. Compound no. 29 was also found to possesssignificant kaliuretic property
(p<0.01), i.e. 1.84±0.008 withregards to the Na+/ K+ ratio.
Ph.D Thesis
186Jamia Hamdard
Chapter-5
Results and Discussion
From the biological data of all the tested compounds obtained, an attempt was made to
establish relationship between structure of the compound and its diuretic activity. The SAR is
as followsThe presence of sulphonamide group like –SO2NH– at the 3rdposition in quinazolinone seems
to
be
essential
for
diureticactivity
similar
to
metolazone.
SAR
showed
that
compoundsbearing electron-withdrawing groups (like NO2) were more activethan the
compounds having electron-releasing groups (CH3).
The final compounds in Scheme-II (39–74) were also evaluated for diuretic activityby the
Lipschitz et al. method. The results at a dose of 2mg afteroral administration of test drug and
1mg of standard drugmetolazone (9.15±0.175** total urine output in mL)were also
observed.Compound no.46, 48, 60,61,62, 64and 68showed 6.33±0.063**, 6.76±0.052**,
6.33±0.079**, 6.84±0.039**, 7.73±0.047**, 6.33±0.047** and 6.30±0.051**mLcumulative
urine
output,
among
which
7-chloro-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-
yl)phenyl)-2-(4-methoxyphenyl)quinazolin-4(3H)-one
(62)
was
highly
substantial
(7.73±0.047)(p<0.01), i.e. significant to control.Diuretic action of compound no.62 was
found to be 1.62. The Lipschitzvalue (the ratio T/U, in which T is the response of the
testcompound and U is that of urea treatment, indices of 1.0 andhigher are regarded as a
positive effect in terms of diureticactivity) shows that compound no.62was 1.09 times potent
than urea.
Further, compound no.62 showed a substantial increase in the Na+excretion (p<0.01), i.e.
2.23±0.115, which was near aboutstandards, i.e. urea 2.49±0.023 and metolazone
(2.24±0.027). However, Na+ and K+ excretion potential was near about metolazone shown in
Tables 3and 4, respectively. Compound no.62 was also found to possesssignificant kaliuretic
property (p<0.01), i.e. 1.10±0.008 withregards to the Na+/ K+ ratio.
Ph.D Thesis
187Jamia Hamdard
Chapter-5
Results and Discussion
From the biological data of all the tested compounds obtained, an attempt was made to
establish relationship between structure of the compound and its diuretic activity. The SAR is
as followsQuinazoline moiety is essential for the activity. It was found that substitution of electron
withdrawing group in aromatic moiety lead to increase the activity. While compound with
methoxy group on para position of the phenyl ring emerges out to be most potent.
ANTIHYPERTENSIVE ACTVITY
In the present experiment, the normal Albino rats (body weight 200-250g) were used for the
screening of synthetic compounds. The rats were given Methylprednisolone acetate 20mg/kg
body weight for two weeks separately for induction of hypertension.
Systolic and mean arterial blood pressure was measured inconscious rats using CODA NonInvasive
Blood
PressureRecorder
by
the
Tail–Cuff
method
(Kent
Scientific
Corporation,Torrington, CT). The restrainers carrying the rat were placed in theBP
instrument with tail protruding out. The tail was gently placedin contact with a transducer
membrane, which was connected tothe digital BP display panel. The instrument was then
turned onand allowed to stabilize until steady pulse rate was observed.Once the ‘‘pulse level
ready’’ signal appeared, the BP recordingbutton was pressed and the mean arterial BP was
recorded. Albinorats (body weight 200–250 g) were used in study. Rats weregrouped,
comprising of six animals in each group. Each compound(5 mg/kg body weight) was injected
intraperitoneally aftersuspending in 1% carboxymethyl cellulose (CMC) solution. Theblood
pressure was recorded at various time intervals. The maximum reduction in MABP was
observed after 6h in all tested compounds. Therefore results of % reduction in MABP were
calculated in Table 5.
Ph.D Thesis
188Jamia Hamdard
Chapter-5
Results and Discussion
The statistical analysis was performed using GRAPHPADINSTAT 3 software (Graph Pad
Software Inc, San Diego, CA).Data obtained from animal experiments were expressed
asarithmetic mean±SEM. The comparison between variousgroups was performed by one-way
analysis of variance(ANOVA), and the effects in treatment groups were comparedwith toxic
control or control group by the Dunnet multiplecomparison test. P<0.05 was considered to be
significant[*p<0.05; **<0.01].
All the compounds in vivo showed comparable antihypertensive activity against standard
drugs prazosin (45.16% reduction in MABP) and diazoxide(44.66% reduction in MABP). On
the basis of activity reported from Scheme-I, The results werecompared with standard drug,
prazosin and diazoxide shownin Table 5. All compounds exhibited good decrease in the
systolicblood pressure up to 6 h after the administration of testcompounds and a moderate
rise afterwards, which may be dueto the excretion of compounds from body after 6 h. The %
reductionin MABPof compound no.10, 12, 20, 21 and 28 were found to show36.16, 36.66,
36.16, 38.08 and39.83.Whereas compound no.29(46.26 %MABP) showed excellent
antihypertensive activityas compared to standard drugs prazosin and diazoxide. Rest ofthe
compounds showed moderate decrease in hypertension.
From the biological data of all the tested compounds obtained, an attempt was made to
establish relationship between structure of the compound and its antihypertensive activity.
The SAR is as followsThe structure–activity relationship of quinazolinone derivativesshowed that, substitution of
phenyl rings had agreat influence on the antihypertensive activity of the compounds.These
variations might be through the stereolectronic propertiesof the substituent. In general,
substitution of electronwithdrawinglike NO2 and Cl in the phenyl ring attachedwith
quinazolinone moiety increases the antihypertensive activityirrespective of their positions, for
example compound no. 28 and 29showed excellent antihypertensive activities. On the other
hand, theelectron-releasing group like CH3 in the phenyl ring causesdecrease in the activity,
Ph.D Thesis
189Jamia Hamdard
Chapter-5
Results and Discussion
as shown in compound no.27. However,unsubstituted phenyl ring leads to loss in activity, as
shownin compound no.1, thus indicating the significance of electronwithdrawinggroups in
the quinazolinone series.
On the basis of activity reported from Scheme-II, The results were compared with standard
drug, prazosin and diazoxide shown in Table 6.All compounds exhibited good decrease in the
systolicblood pressure up to 6 h after the administration of testcompounds and a moderate
rise afterwards, which may be dueto the excretion of compounds from body after 6 h. The %
reduction in MABP of titledcompound no.46, 48, 60, 61, 64 and 68 were found to show31.33,
32.58, 33.75, 41.66, 31.16 and 36.58. Whereas compound no.62 (44.33 % reduction in
MABP) showed significant antihypertensive activityas compared to standard drugs prazosin
and diazoxide. Rest ofthe compounds showed moderate decrease in hypertension.
From the biological data of all the tested compounds obtained, an attempt was made to
establish relationship between structure of the compound and its antihypertensive activity.
The SAR is as followsIn general replacement of piperazin moiety at position 2 with substituted phenyl ring and
incorporation of substituted isoxazole ring at position 3 of quinazoline moiety produce lead to
more effective compound in comparision
to clinically used drugs such as
prazosin,
doxazosin etc.
Introduction of electron releasing group at para position of phenyl ring at position 2 along
with electron withdrawing group at phenyl ring of quinazoline ring and isoxazole linked
phenyl ring yielded significally active compounds with prolonged duration of action
(compound no.61&62). However substitution –OCH3 group produces more potent compound
of the series (compound no.62). This effect is attributed due to specific effect of –OCH3
group which gives off a negative inductive effect while still producing a more dominant
positive mesomeric effect.
The percentage reduction in mean arterial blood pressure (MABP) of compounds was shown
in Graph 1-8 at dose 5mg/kg.
Ph.D Thesis
190Jamia Hamdard
Chapter-5
5
Results and Discussion
Graph 1. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-I
Graph 2.. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-I
Ph.D Thesis
191Jamia Hamdard
Chapter-5
5
Results and Discussion
Graph 3.. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-I
Graph 4.. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-I
Ph.D Thesis
192Jamia Hamdard
Chapter-5
Results and Discussion
Graph 5. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-II
Graph 6. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-II
Ph.D Thesis
193Jamia Hamdard
Chapter-5
Results and Discussion
Graph 7. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-II
Graph 8. Percentage reduction in Mean arterial blood pressure (MABP) at dose 5mg/kg
in Scheme-II
Ph.D Thesis
194Jamia Hamdard