Results and discussion
CHAPTER 5
RESULTS AND DISCUSSION
5.1
CHEMISTRY
The most common method for the synthesis of substituted azetidine-2-
one is the Staudinger-keteneimine cyclo-addition, which involves the reaction
of imines with acid chloride in the presence of a tertiary base. Likewise 4thiazolidinones are derivatives of thiazolidine with a carbonyl group at the 4position. Several protocols for the synthesis of 4-thiazolidinones are available
in the literature. Essentially they are three-component reactions involving an
amine, a carbonyl compound, and a mercapto-acid. The process can be either
a one-pot three-component condensation or a two-step process. The synthesis
of compounds was achieved through the versatile and efficient synthetic route
outlined in Scheme 29. It is apparent from the scheme that the new
heterocyclic compounds contain azetidine-2-one and thiazolidine-4-one
moieties. Reaction of substituted arylidene hydrazide (Schiff base) with
thioglycollic acid and chloroacetyl chloride seemed to be a convenient route
to fulfill this aim. The yield of the compounds was not optimized.
Intermediate quinoxaline-2,3-dione (1a ) was synthesized based on reported
procedure. Compound 1a was added to a mixture of aniline and ethanol and
few drops of glacial acetic acid were added to yield 5.1g of 3-((phenylimino)3,4-dihydroquinoxaline)-one (1b) (yield 85 %). The compound 1b and ophenylenediamine
react
to
yield
N-((E)-3-(phenylimino)-3,4-dihydro
qunoxalin-2(1H)-ylidene)benzene-1,2-diamine(1c). Compound 1c, underwent
Schiff reaction with the required aromatic aldehyde (1d-m). The
cyclocondensation of Schiff base(s) 1n-w with chloroacetyl chloride and
triethylamine resulted in the formation of azetidinones (2n-w) whereas
68
Results and discussion
cyclocondensation
of
Schiff
bases
with
thioglycolic
acid
yielded
thiazolidinones (3n-w).
Several protocols for the synthesis of mannich bases are reported in
literature [Willard M, 1977, Garazd YL, 2003, Nabel AN 2005, Chipeleme A
2007]. We have carried out the synthesis of quinoxaline -2, 3-dione 1 by the
most common reaction between o-phenylenediamine and oxalic acid under
solvent free conditions. The intermediate 2a was synthesized by a simple
condensation reaction of compound 1 with aniline while the replacement of
aniline with p–chloroaniline yielded compound 2b. The synthesis of title
compounds 4a-f was achieved through nucleophilic addition of 2a with
formaldehyde and substituted piperazines.
Where as, the nucleophilic
addition of 2b with formaldehyde and piperazines yielded the title
compounds 5a-f. A simple and efficient method has been developed for the
synthesis
of
series
of
N-Mannich
bases
of
(E)-3-(phenylimino/4-
chlorophenylimino)-2,3-dihydro-1-[(N-substitutedpiperazinyl)methyl]quinoxaline-2-(1H)-one 4a-f and 5a-f. The requisite 2a and 2b were obtained by
reaction between quinoxaline-2,3-dione 1 and aniline / p-chloroaniline. These
compounds underwent N-mannich reaction with various substituted
piperazines to yield title compounds 4a-f and 5a-f. The synthetic route is
outlined in Scheme 30.Structures of these compounds were confirmed by
spectral studies (IR, 1H NMR, 13C NMR and Mass) and elemental analysis.
5.1.1 4-(substituted phenyl)-1-(2(3-(phenylimino)-3,4-dihydroquinoxalin
-2-(1H)- ylidene)amino)phenyl)azetidin-2-one (2n-w).
β-Lactam formation is characterized by the disappearance of the
The IR spectra of the compounds 2n-w
showed strong absorption band in the range of 1753-1648 cm-1 characteristic
of the β-lactam carbonyl group, which are lower than so far reported values
(1800 cm-1) of the ketones, aldehydes and other amides. This may be due to
the conjugation of unpaired electrons of the ring nitrogen with carbonyl group
in the β-lactam of azetidinone resulting in increased single bond character and
69
Results and discussion
lowering of carbonyl single absorption frequency. The absorption peaks due
to C-Cl appeared in the range of 670-765 cm-1 in the compounds 2n-w. The
methoxy group in compounds 2p and 2w showed a peak at 1229 cm-1. The
strong absorption band due to hydroxyl group was observed at 3610, 3396,
3389 cm-1 in compounds 2n, 2o and 2w respectively. Steric hindrance
prevents hydrogen bonding and no bonded hydroxyl band is observed. The
aromatic methyl group in compound 2s showed a strong peak at 2850. The
absorption peak at 1290 cm-1 in compound 2t confirmed the presence of
dimethyl amino group. The presence of nitro group on compound 2u is
confirmed by the peak at 1536 cm-1.
In 1H NMR spectrum of azetidinones 2n-w the aromatic multiplets
appeared in the range of δ 7.0-8.9 ppm. β-Lactam formation is characterized
by the disappearance of the N=CH proton of the Schiff’s base and appearance
of the H3 and H4 proton of the β -lactam ring. A review of the literature
revealed that the protons of the β -lactam at H3 and H4 can vary in position
due to surrounding factors and substituents. The CH-Cl of Azetidinone was
confirmed by singlet at δ 4.6-5.4 ppm. The NH proton of title compounds
appeared in the range of δ 3.8-4.4 ppm. The deviation of the linear hydrogenbond leads to downfield shift and the shielding effect. The hydroxyl group
was confirmed by singlet at δ 7.7, 7.2 and 4.9 ppm in compounds 2n, 2o and
2w. The compounds 2p and 2w showed a singlet at δ 3.9 and 3.7 ppm
respectively due to methoxy group. The singlet at 2.3 ppm was attributed to
methyl group in compound 2s. The dimethyl amino group,s proton gives a
broad peak at δ 2.9 ppm in compound 2t which has a character typical of the
group with hindered rotation. In summary the 1H NMR values correlated well
with expected structures of title compounds 2n-w.
The
13
C NMR is evaluated for compounds 2n. The 12 aromatic
carbons showed multiplet in the region δ128-139 ppm for compounds 2n.
The carbon of CH-Cl showed a singlet at δ 55.32. The CH2 of azetidinones in
70
Results and discussion
compounds 2n was found in δ 55.37 ppm. The carbonyl carbon in both the
compounds was seen at δ 163.8. The amide carbon exhibited singlet in δ
169.5 and 173.52 ppm. The molecular weight of the synthesized compounds
2n-w was confirmed by mass spectra. The molecular ion peaks obtained were
in good agreement with the molecular weight of the compounds. Elemental
analysis was carried out for carbon, hydrogen and nitrogen. The results
obtained were within 0.4 % of the theoretical value.
To conclude the IR, 1HNMR,
13
CNMR, elemental analysis and are in
good correlation with expected structures. This confirmed the structures of
synthesised compounds.
5.1.2 2-(substitutedphenyl)-3-(2-(3-(phenylimino)-3,4-dihydroquinoxalin
-2-1H)-ylidene)amino)phenyl)thiazolidin-4-one (3n-w)
The IR is of value in this study because cyclization of the Schiff’s
bases to the corresponding β-lactams results in certain features that are very
easily and clearly characterized by IR, such as the carbonyl absorption of the
β -lactam, clearly indicated the formation of β -lactam .The –HC=N–
stretching mode of the Schiff’s base showed an absorption at 1660-1640 cm-1,
usually conjugation with an aromatic ring produces enhanced absorption near
1625 cm-1 as shown in compounds 1n-w. But this peak disappeared in the title
compounds 2n-w and 3n-w. The thiazolidinones were mainly observed by
presence of strong absorption band in the range of 1610-1660 cm-1 which is
characteristic of five membered lactones. The absorption band in all the
compounds 3n-w which lies in the range of 613-720 cm-1 confirmed the
presence of C-S-C bond formation. The aromatic multiplets were present in
2850-3086 cm-1. The NH peak of quinoxaline is observed at 3106-3615 cm-1.
The phenolic OH is found in the range of 3367, 3349 and 3375 cm-1 in
compounds 3n, 3o and 3w respectively. Steric hindrance here prevents
hydrogen bonding and no bonded hydroxyl band is observed. The aromatic
nitro peaks were seen at 1536, cm-1in compound 3u. The absorptions in the
71
Results and discussion
751in the compounds 3r and 3q is attributed to aromatic chloro group. The
peak due to methoxy group is seen at 1278, and 1245cm-1 in compounds 3p
and 3w.
In 1 H NMR the aromatic multiplets were observed at δ 6.3-8.9 ppm in
all the titled compounds 3n-w. All the thiazolidinones showed a singlet at δ
3.3-3.7 due to presence of –CH proton of thiazolidinone, which is flanked by
nitrogen and sulfur from two sides and with benzene on third side. The CH2S
signal is present in all the compounds in the range of δ 4.6-5.1 ppm. The CHN proton is found in the range of δ 6.0-6.6 ppm. The amino proton of NH is
observed at δ 4.1- 4.7 ppm. The deviation of the linear hydrogen-bond leads
to downfield shift and the shielding effect. The more acidic phenolic hydroxyl
group of 3n, 3o and 3w generated a lower-field resonance signal which is
observed in all the compounds in the range δ 2.2-2.3 ppm. The methoxy
proton is observed at δ 3.3, 3.4 3.1 and 3.0 ppm in compounds 3p and 3w
respectively. This deshielding of the protons of the methoxy methyl group due
to carbon bonding to an electronegative oxygen atom causes a down field
shift. The compound 3t showed a singlet at δ 2.9 ppm due to 6 proton of
dimethylamino group which has a character typical of the group with
hindered rotation. The
13
C NMR is evaluated for compounds 3n.The 12
aromatic carbons showed multiplet in the region δ110-140 ppm for compound
3n. The carbon of CH-S showed a singlet at δ 55.32 ppm in both the
compounds. The CH2 of thiazolidinones in compounds 3n were found in δ
162.56. The carbonyl carbon in both the compounds was seen at δ 181.5. The
amide carbon exhibited singlet in δ 166.26. Elemental analysis was carried
out for carbon, hydrogen and nitrogen. The results obtained were within 0.4
% of the theoretical value. The molecular weight of the compounds was
confirmed by mass spectra. The fragmentation patterns of the compounds
were also studied.
72
Results and discussion
5.1.2 3-(phenylimino)-2,3-dihydro-1-[(N-substitutedpiperazinyl)methyl]
quinoxaline-2-(1H)-one 4a-f and 5a-f
The formation of mannich base is confirmed by the appearance of NCH2 bond. The absorption peak at 1105-1459 cm-1 is due the presence of CH2
group. The IR spectra of compounds showed absorption bands due to
stretching vibrations of N-H, C=O and C-N at 3686-3771cm-1,1590-1698 cm-1
and 1527-1614cm-1 respectively while the vibrational frequencies of the
COOH and C-F peaks were observed at 1266-1403cm-1 and 1201-1266 cm-1.
In 1H NMR studies the chemical shift and multiplicity patterns correlated well
with the proposed structures. Thus the 1H NMR showed a singlet at δ 5.18
ppm corresponding to formation of N-CH2-N while that of the NH-Ar signal
appeared at δ 4.62 ppm. The presence of COOH was confirmed by a sharp
singlet at δ 14.22. We confirmed the aromatic protons by the appearance of
multiplets at δ 7.04- 8.49 ppm whereas the multiplets at δ 4.27-4.43 ppm
confirmed the presence of 4 hydrogens of piperazine.
The
13
C NMR of 4a revealed 32 carbon atoms with C=O having
highest signal at δ 200.57 ppm while two CH2 of cyclopropane appeared at δ
5.84 and δ 5.31 ppm. The remaining carbons showed signals ranging from δ
196.82 to δ 36.43. The mass spectrum of 4a showed molecular ion peak at
m/z 580.22 (25%) which was in agreement with molecular mass of compound
C32H29FN6O4 while the base peak was observed at 106 (100%). Other peaks
were found at 330.12 (28%), 236.08 (15%), 161 (11%) and 131.03(5%).
5.2
BIOLOGICAL ACTIVITY
5.2.1 Antitubercular Activity
All the synthesized compounds exhibited an interesting activity profile
against the tested mycobacterial strain. The results revealed that the activity is
considerably affected by various substituents on the aromatic ring of either 2azetidinone or 4-thiazolidinone nucleus. It is fascinating to observe that the
introduction of a hydroxyl, methoxy, chloro, dimethylamino and nitro group
73
Results and discussion
on aromatic ring (2o, 3o, 2p, 3p, 2q, 3q, 2t, 3t, 2r, 3r, 2u and 3u), resulted in
compounds with an enhanced antitubercular activity (MIC values ranging
from 0.67 – 3.70 µg/ml). Amongst them, compounds 2t, 3t, 2r, 3r and 2u
(MIC values ranging from 0.67 – 0.97 µg/ml) exhibited a significant activity
when compared with first-line drug isoniazid (MIC = 0.47g/ml). This
antitubercular activity may be attributable to the introduction of electron
withdrawing group on the aromatic ring. However substitution by electron
releasing methoxy group also resulted in compounds with moderate
antitubercular activity (2p and 3p). It is interesting to note that the
introduction of an electron releasing hydroxyl group in the p-position of
phenyl ring resulted in respectable activity (2o and 3o). On the other hand,
substitution of hydroxyl group in the o-position is found to have complete loss
of activity (2n and 3n). The replacement of hydroxyl moiety by electron
releasing methyl group in the compounds 2s and 3s showed mild activity. The
electron withdrawing substituted compounds (chloro, dimethylamino and
nitro) were found to be more active than electron releasing methoxy, hydroxyl
and methyl moiety in case of antitubercular activity. Literature survey reveals
that electron withdrawing or donating groups amend the lipophilicity of the
test compounds, which in turn alters permeability across the bacterial cell
membrane. Further, compounds 2v and 3v, substituted with a five membered
furan structure did not show any considerable activity. Similarly, introduction
of methoxy and hydroxyl groups in compounds 2w and 3w leads to devoid of
the activity. It was also studied the influence of the 2-azetidinone nucleus in
compounds 2n-w and 4-thiazolidinone nucleus in compounds 3n–w on the
biological activity. It was observed that the replacement in the core nucleus
did not alter the antitubercular activity to a greater extent. The antitubercular
activities of 2n-w and 3n-w are shown in Table 5.1 and 5.2 respectively.
Figures 5.1 and 5.2 indicate the graphical representation of these results.
74
Results and discussion
In Mannich base series 4a-f and 5a-f the compounds with quinolone
substitution exhibited better antitubercular activity. The compounds 4a, 4b
and 4c with ciprofloxacin, norfloxacin and sparfloxacin with aniline
substitution exhibited percentage inhibition of 100, 95 and 89 respectively.
The similar effect was observed in compounds 5a, 5b and 5c with percentage
inhibition of 100, 96 and 95. The order of reactivity is 4a = 5a > 5b> 4b = 5c
> 4c. The other compounds were inactive. In contrast to azetidinones and
thiazolidinone series, the electrostatic field is not affecting the antitubercular
activity of mannich base series. The least active compound 4f is having
morpholine substitution. The antitubercular activities of 4a-f and 5a-f are
shown in Table 5.3. Figure 5.3 indicates the graphical representation of these
results.
75
Results and discussion
Table 5.1 Antitubercular activity of azetidinones 2n-w
Compound
MIC in µg/ml
2n
50.00 ±1.02
2o
3.00 ±0.34
2p
1.53 ±0.42
2q
1.40 ±0.33
2r
0.89 ±0.21
2s
20.6 ±1.23
2t
0.76 ±0.11
2u
0.97 ±0.21
2v
10.1 ±01.24
2w
11.8± 2.11
Standard
0.46 ±0.13
Fig. 5.1 Antitubercular activity of azetidinones 2n-w
76
Results and discussion
Table 5.2 Antitubercular activity of Thiazolidinones 3n-w
Compound
MIC in µg/ml
3n
13.8 ±0.12
3o
3.70 ±0.21
3p
1.38 ±0.16
3q
1.38 ±0.18
3r
0.76 ±0.12
3s
19.0 ±1.08
3t
0.67 ±0.12
3u
1.77 ±0.21
3v
36.00 ±2.11
3w
35 ±3.12
Standard
0.46 ±0.13
Fig. 5.2 Antitubercular activity of thiazolidinones 3n-w
77
Results and discussion
Table 5.3 Antitubercular activity of Mannich bases
Compound
MIC in µg/ml
4a
0.99 ±0.12
4b
0.95 ±0.18
4c
0.92 ±0.14
4d
40.82 ±1.22
4e
36.24 ±1.87
4f
24.76 ±1.23
5a
0.67 ±0.13
5b
0.76 ±0.19
5c
0.65 ±0.16
5d
30.56 ±1.56
5e
28.95 ±1.43
5f
40.32 ±1.22
Standard
0.46 ±0.13
Fig. 5.3 Antitubercular activity of mannich bases 4a-f and 5a-f
78
Results and discussion
5.2.2 Leptospirocidal Screening
In the first phase of screening for biological activity, the in vitro
leptospirocidal activity for all the synthesized compounds was performed. By
using microtitreplate based microbial assay, the IC50 values were determined.
(IC50 is defined as the concentration of drug at which 50% inhibition of
microorganism occurs). The results of in vitro screening revealed that
leptospirocidal activity is considerably affected by various substitutions on
the piperazine ring. The compounds 4a-c and 5a-c showed enhanced activity.
[IC
50
values ranging from 26 µg/ml-30 µg/ml]. Among them the IC50 values
of the compounds 5a-c [IC50 values 26 µg/ml] were similar to that of
reference drug benzyl penicillin [IC50 values 26 µg/ml]. Simple substitutions
by piperazine and methyl piperazine (4d, 4e and 5d, 5e) do not produce
significant activity. Morpholine substitution (4f and 5f) also resulted in less
active compounds. The leptospirocidal activities of 2n-w, 3n-w and 4a-f and
5a-f are shown in Table 5.4, 5.5 and 5.6 respectively. Figures 5.4 and 5.5 and
5.6 indicate the graphical representation of these results.
79
Results and discussion
Table 5.4 In vitro Leptospirocidal activity of azetidinones 2n-w
Compound
IC 50 (µg/ml)
2n
50±1.22
2o
48±1.34
2p
42±1.21
2q
35±1.87
2r
38±1.56
2s
38±1.11
2t
41±1.12
2u
35±1.32
2v
50±2.11
2w
50±1.23
Standard
26±0.88
Fig. 5.4 Leptospirocidal activity of azetidinones 2n-w
80
Results and discussion
Table 5.5 In vitro Leptospirocidal activity of thiazolidinones 3n-w
Compound
IC 50 (µg/ml)
3n
45±1.21
3o
42±1.11
3p
50±1.32
3q
30±1.41
3r
30±1.56
3s
47±1.65
3t
38±1.67
3u
30±1.32
3v
42±1.24
3w
42±1.26
Standard
26±0.88
Fig. 5.5 Leptospirocidal activity of thiazolidinones 3n-w
81
Results and discussion
Table 5.6 In vitro Leptospirocidal activity of Mannich bases 4a-f and 5a-f
Compound
IC 50 (µg/ml)
4a
30±1.11
4b
28±1.21
4c
28±1.10
4d
48±1.32
4e
46±1.75
4f
42±1.54
5a
26±0.98
5b
26±1.01
5c
26±1.01
5d
42±1.25
5e
50±1.76
5f
50±1.43
Standard
26±0.88
Fig.5.6 Leptospirocidal activity of mannich bases 4a-f and 5a-f
82
Results and discussion
5.2.2.1Acute oral toxicity studies (AOT)
The acute oral toxicity studies were carried out according to OECD
guidelines 423 in healthy female albino mice. No behavioral changes or
mortality were observed up to the dose of 300mg/kg. The AOT lies in range
of 300-2000 mg/kg.
5. 2.2.2 In vivo Leptospirocidal activity
The results of in vitro study prompted us to carry out the in vivo
studies. The compounds 4a-c which showed maximum activity in in vitro
testing was selected for in vivo testing. On day 3, group V and VI receiving
compound 5b at two different doses (250 mg/kg and 500 mg/kg) showed an
antibody titre value of 1:20, which remained consistent for 7 days on par with
reference drug benzyl penicillin. The results are shown in Table 5.7
Table 5.7 Results of MAT test
Antibody titre values
Day 0
Day 3
Day 5
Day7
N
N
N
N
Groups
Dose
(mg/kg)
Normal control
-
Infected control
-
N
1:40
1:60
1:20
III
250
N
1:40
1:40
1:80
IV
500
N
1:40
1:40
1:80
V
250
N
1:20
1:20
1:20
VI
500
N
1:20
1:20
1:20
VII
250
N
1:20
1:40
1:40
VIII
500
N
1:20
1:40
1:80
Benzylpenicillin
327.5
N
1:20
1:20
1:20
83
Results and discussion
The in vivo studies were performed by MAT test. During MAT test the
compounds 5a, 5b and 5c showed an antibody value similar to that of
reference drug benzyl penicillin. This may be attributed to the presence of
ciprofloxacin, norfloxacin and sparfloxacin in their structure. The fusion of
quinolone antibiotics to quinoxaline as mannich bases resulted in active
compounds.
The hematological and biochemical parameters were estimated in both
normal and pathological conditions. Various hematological parameters like
ESR, WBC count, RBC count and platelet count were performed. During
infection RBC and platelet counts were decreased while ESR and WBC count
were elevated. The administration of test compounds increased the RBC and
platelet counts and reduced the WBC and ESR counts. This result was
comparable to the reference drug benzyl penicillin and was displayed in
Table 5.8.
Table 5.8 Results of hematological test
RBC
WBC
ESR
Platelets
6
3
3
3
(1×10 /mm ) (1×10 /mm ) (mm/hr) (1×106/mm3)
Normal control 12.83 ± 1.02 11.0 ± 0.97 5.10 ± 0.23 4.17 ± 0.19
Group
5.30 ± 0.39
23.10 ± 1.27 43.20 ± 1.27
III
8.02 ± 0.12c
19.23 ± 1.05 20.18 ± 1.37c 1.58 ± 0.69
IV
8.47 ± 0.29c
18.79 ± 1.28c 19.59 ± 1.20c 1.96 ± 0.56c
V
10.96 ± 0.24a 14.02 ± 1.75b 11.06 ± 0.86b 2.89 ± 0.49b
VI
11.17 ± 0.79a 13.59 ± 0.92b 10.23 ± 1.06b 3.09 ± 0.87b
VII
10.51 ± 0.32b 16.44 ± 0.99c 15.01 ± 1.25b 2.04 ± 0.76c
VIII
10.86 ± 0.98b 15.96 ± 1.10b 14.79 ± 0.68b 2.21 ± 0.57c
Infected control
0.92 ± 0.05
Benzylpenicillin 11.49 ± 0.91a 11.23 ± 0.79a 7.82 ± 0.59a
Values are mean ± S.E. of 6 animals in each group;
4.02 ± 0.68a
a
p<0.0001 vs
control, b p <0.01 vs control, c p <0.05 vs control
84
Results and discussion
The infection with leptospirosis caused excessive hemolysis which
reduced RBC and platelet counts. On the other hand, WBC count and ESR
were increased in leptospirosis. Administration of test compounds decreased
the elevated levels of WBC and ESR. In contrast there was an increase in
RBC and platelet counts after administrations of test compounds.
Biochemical parameters estimated were serum total cholesterol, serum
HDL-cholesterol, serum creatinine, serum urea, serum glutamate pyruvate
transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT),
total bilirubin and triglycerides. During the infection, all these parameters
were elevated in serum and were decreased after the administration of the test
drugs. The increase in serum creatinine level during leptospirosis was due to
stimulation of the enzyme nitric oxide synthase which produced more nitric
oxide which lead to increase in serum creatinine level while the infection
caused renal damage due to which serum urea level was increased.
Stimulation of serum lipase and triglyceride synthase in leptospirosis elevated
the triglyceride level. The increase in bilurubin level was due to excessive
hemolysis [Elves, A.P., 2006, Robinson CR 1956]. Administration of test
compounds decreased the elevated levels of biochemical parameters which is
displayed in Table 5.9.
85
Results and discussion
Table 5.9 Results of biochemical parameters
Group
Total cholesterol
(mg/dL)
TG
(mg/dL)
HDL
(mg/dL)
Creatinine
(mg/dL)
Urea
(mg/dL)
SGPT
(mg/dL)
SGOT
(mg/dL)
Bilirubin
(mg/dL)
Normal
control
99.83 ± 8.18
76.86 ± 1.25
109.85 ± 9.73
0.81 ± 0.21
18.04 ± 1.92
72.43 ± 8.61
209.07 ± 7.71
1.07 ± 0.19
Infected
control
214.54 ± 3.81
173.37 ± 3.41
41.46 ± 1.22
4.16 ± 0.76
45.45 ± 1.64
135.13 ± 9.20
343.38 ± 6.67
23.72 ± 4.32
III
158.32 ± 3.62c
131.34 ± 5.68c
65.23 ± 0.43c
3.48 ± 0.35
38.99 ± 1.03
125.46 ± 3.31
278.72 ± 12.04c
16.78 ± 0.56c
IV
151.37 ± 12.07b
119.85 ± 8.90a
77.63 ± 1.26b
3.36 ± 0.16
37.04 ± 1.10
122.83 ± 3 .65
266.24 ± 10.56 b
14.72 ± 1.45c
V
145.25 ± 8.82c
112.91 ± 3.25a
75.87 ± 2.87c
2.59 ± 0.27c
31.39 ± 1.04c
109.91 ± 6.29b
260.89 ± 11.03b
2.89 ± 0.15a
VI
120.59 ± 5.18a
103.47 ± 3.81a
83.40 ± 0.47a
1.30 ± 0.21a
23.58 ± 2.06b
102.45 ± 4.00b
229.02 ± 8.04a
3.39 ± 0.24a
VII
105.16 ± 7.71a
95.43 ± 6.02a
98.62 ± 0.94a
1.47 ± 0.26a
17.42 ± 1.33a
80.72 ± 4.19a
210.37 ± 13.47a
1.44 ± 0.28a
VIII
133.63 ± 13.92b
110.78 ± 2.26b 80.75 ± 7.05b
3.01 ± 0.43c
36.36 ± 1.46c
115.91 ± 6.93c
268.30 ± 11.33c
11.88 ± 0.90b
Benzyl
penicillin
108.66 ± 6.92a
90.22 ± 7.56a
1.50 ± 0.15a
20.02 ± 3.06a
80.06 ±5.68a
222.19 ± 5.39a
3.438 ± 0.36a
91.02 ± 4.05a
Values are mean ± S.E. of 6 animals in each group;
a
p<0.0001 vs control,
b
p <0.01 vs control,
c
p <0.05 vs control
86
Results and discussion
During the estimation of transpeptidase the synthetic compounds 4a,
4b and 4c the amount of aniline released by N-(DL-glutamyl) aniline, which
is a synthetic substrate for the enzyme transpeptidase. Enzyme inhibition
studies revealed that compound 4b released the least amount of aniline from
the synthetic substrate, N-(DL-glutamyl) aniline which was very similar to
that of reference drug. It was very clear from the result that the compounds
were having leptospirocidal activity due to the inhibition of the enzyme
transpeptidase.
This enzyme is required for the cross linking of
peptidoglycon which is an important step in cell wall synthesis. Hence it is
concluded that leptospirocidal activity of test compounds may be due to
inhibition of cell wall synthesis. Figure 5.7 represents the graphical results.
B- Blank, NC- Normal control, Std- Standard
Fig 5.7 Results of inhibition of enzyme transpeptidase
87
Results and discussion
5.3
QSAR STUDIES
A high correlation was seen between the anti-tubercular and anti-
leptospiral activity (r = 0.834). Compounds 4a, 4b, 4c, 5a, 5b and 5c showed
both good anti-tubercular and anti-leptospiral activity. Investigation revealed
that the presence of ciprofloxacin moiety along with quinoxaline scaffold
gives rise to the best compounds (4a, 4b, 4c, 5a, 5b and 5c). Chlorobenzene
substitution at the azetidinone and thiazolidinone ring showed good activities
against both the organisms (2r and 3r). Antitubercular activity is generally
good when substituents are electron withdrawing groups (-Cl, -NO2) and as in
2q, 2u, 3q, 3u and moderate to poor incase of electron releasing groups
including (-OH, -CH3) 2n, 2o, 2s, 3n, 3o and 3s with the exception of 2r and
3r. Both antitubercular and antileptospiral activities are poor in compounds
4d, 4e, 4f, 5d, 5e and 5f which have simple piperazine and morpholine
substitution.
Statistically significant models are obtained for the series of
compounds for their antitubercular and antileptospiral activities as shown in
Table 9.10. The square of the correlation coefficient r2 is satisfactory (r2 >
0.72). The internal predictive ability of the models is acceptable with q2 >
0.61. The external cross – validation coefficient is also considerable with
pred_r2 > 0.70. Among the many equations generated the best equations for
antitubercular and leptospirocidal activities were selected based upon
statistical parameters. They are
for tuberculosis,
BA = -2.4274 + 0.1808 * G_C_N_7- 1.020 * G_O_O_6 - 0.8166* T_O_O_3
for leptospirosis,
BA = -2.7843 + 0.1771 * chiV4PathCluster – 0.1832 * T_O_O_6
88
Results and discussion
Table 5.10
Model
2D QSAR models for the Antitubercular and Antileptospiral
activity of substituted quinoxalines, azetidinones and
thiazolidinones
Training set
r2
r2
adj
q2
F
ratio
Press Pred_r2 Ro2
Ro’2
K
K’
Z
Score
M.
tuberculosis
H37Rv
26
0.716 0.682 0.614 18.521 3.536
0.814 0.812 0.723 1.004 0.945 4.270
Leptosprira
interrogans
25
0.878 0.866 0.856 78.786 0.196
0.718 0.854 0.762 1.045 0.954 11.039
The closeness of r2 to either Ro2 or Ro’2 and values of either k or k’
near to 1 further enhances the significance of the models and proves their
proximity towards an ideal model. The correlation matrix for the observed
activity and the descriptors is shown in Tables 5.11 and 5.12. It was found
that there is no correlation between the descriptors indicating the absence of
inter-correlation which is desired while developing QSARs. In the case of the
antitubercular activity, observed activity has high correlation with G_C_N_7
and G_O_O_6 (both descriptors are opposite in their effects) and only
moderate correlation is seen for the descriptor T_O_O_3. The observed
antileptospiral activity shows very high correlation towards chiV4
Path/cluster and moderate correlation towrds T_O_O_6.
Table 5.11
Correlation matrix between the descriptors and Antitubercular
activity
G_C_N_7
G_O_O_6
T_O_O_3
G_C_N_7
G_O_O_6
1
-0.1811
1
-
Observed
Activity
-
T_O_O_3
-0.1069
-0.1276
1
-
Observed
Activity
0.5748
-0.5930
-0.3599
1
89
Results and discussion
Table 5.12 Correlation matrix between the descriptors and Antileptospiral
activity
chiV4
PathCluster
T_O_O_6
Observed
Activity
chiV4
PathCluster
1
-
-
T_O_O_6
-0.054876153
1
-
Observed
Activity
0.901058356
-0.303993647
1
The data also complies with the contributions of each descriptor on the
activity. Figures 5.8 and 5.9 show the contributions of each descriptor on the
activity.
Fig 5.8 Contribution of G_C_N_7, G_O_O_6 and T_O_O_3 towards the
antitubercular activity
90
Results and discussion
Fig 5.9
Contribution of chiV4 PathCluster and T_O_O_6 towards the
leptospirocidal activity
Parity plots between the observed and the predicted activities for both
the models are shown in the figures 5.10 and 5.11. The dotted lines indicate
the 95% confidence lines. The presence of most of the points within the
confidence levels indicates that the models built are significant.
2
Predicted Activity
1
0
-1
-2
-3
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
Observed Activity
Fig 5.10
Parity plot between M. tuberculosis H37 RV activity and the
QSAR model (dotted lines show 99% confidence lines;
closed dots are training set and open dots are the test set)
91
Results and discussion
-1.2
-1.4
Predicted Activity
-1.6
-1.8
-2.0
-2.2
-2.4
-2.6
-2.8
-2.6
-2.4
-2.2
-2.0
-1.8
-1.6
-1.4
Observed Activity
Fig 5.11 Parity plot between Leptospirosis activity and the QSAR model
(dotted lines show 99% confidence lines; closed dots are
training set and open dots are the test set)
The QSAR model generated for the antitubercular activity of
substituted quinoxalines, azetidinones and thiazolidinones showed that three
descriptors involved in predicting the activity of the compounds. Descriptors
G_C_N_7 and G_O_O_6 are alignment independent geometric descriptors
which are defined as the average geometrical distance between two attributes
separated by a particular number of bonds. Thus G_C_N_7 is defined as the
average geometrical distance between carbon atoms (single, double or triple
bonded) separated from nitrogen atoms by a topological distance of seven
bonds. This descriptor has a positive contribution towards the activity.
G_C_N_7 is particularly large when there is an N, N- dimethyl aniline
substitution to the azetidinone or thiazolidinone ring as revealed by the
compounds 2t and 3t. These compounds show very good activity against M.
tuberculosis but are only moderately active against Leptospira interrogans.
Analyzing the structures of the compounds under study we can see that
G_C_N_7 is very nonspecific and cannot be used to predict the exact
structural features. However it can be inferred that presence of nitrogen
containing functional groups especially at position 14 (azetidinone ring) and
position 9 (thiazolidinone ring) increased the activity of the compounds. A
very good correlation (r = 0.85) is found with chiV4 Path/Cluster obtained for
92
Results and discussion
the antileptospiral model. The latter descriptor suggested that understanding
molecular connectivity and skeletal branching is necessary to design effective
derivatives. The most active compounds including 2t, 3t, 4a, 4b, 5a and 5b
showed a good positive correlation (r = 0.64) and the least active compounds
including 2n, 2s, 3s, 3v and 3w showed a good negative correlation (r = -0.61)
towards the descriptor G_C_N_7.
G_O_O_6 is defined as the average geometrical distance between two
oxygens (single or double bonded) separated by a topological distance of 6
bonds. This descriptor which has a negative effect on the activity is exhibited
by compounds 2n, 2v, 3n and 3v. 2n and 3n possess a hydroxyl group at the
ortho position of the substituted azetidinone and thiazolidinone respectively
while 2v and 3v contain a furan ring. Azetidinones and thiazolidinones are
both structural subunits of penicillin and as literature reports conveyed, the
spatial disposition of the electrostatic negative well created by the oxygen of
the carbonyl group is the key factor for their antibacterial activity especially
in binding and interaction with proteins such as acyl transferases [Colette
Goffin et al., 2002]. Knut Baumann explained the binding of steroids to CBG
using such alignment independent descriptors. His interpretation of the
geometric descriptors is related to change in binding constants as a result of
the differences in the geometrical distance of the particular substituent [Knut
Baumann., 2002] Comparing the above compounds revealed that, the activity
is poor at decreased geometrical distances of the oxygens, suggesting that
orientation of the oxygen associated with the hydroxyl group as well as the
furan ring influences negatively on the binding constants of the compounds
and interferes with spatial positioning of the electrostatic well (C=O group of
azetidinone ring) and thus decreasing the compounds potency.
The alignment independent topological descriptor T_O_O_3 has a
negative effect on the anti-tubercular activity. This descriptor indicates the
presence of number of oxygens separated from other oxygens by a topological
93
Results and discussion
distance of 3 bonds. Only compounds 2w and 3w showed this descriptor and
they have poor activity (high MIC values). From the structure of the
compounds it can be inferred that the presence of a methoxy group along with
the hydroxyl group contributes to decreased activity. A balance between the
lipophilic and the electronegative substituents is necessary to determine an
appreciable antimycobacterial activity. There have been instances where side
chains consisting of both hydrophilic group and a lipophilic group have
shown poor antitubercular activities than those compounds which don’t have
any substituent at that position. On analyzing the compounds we found that
compounds 2o and 3o which do not have the methoxy substitution (but have
the hydroxyl substitution) are far more active than both 2w and 3w which
have both the substitutions.
The model derived for the antileptospiral activity of substituted
quinoxalines, azetidinones and thiazolidinones yielded two informative
descriptors, namely chiV4 path/cluster and T_O_O_6. The descriptor chiV4
path/cluster or 4χvPC is a Keir’s molecular connectivity index. The χ indices are
whole molecular descriptors, where a single value is computed for the whole
molecule which represented the entire molecule [Lowell H. et al., 2001]. 1 χ
is the direct representation of the molecular structure which encodes a degree
of branching. The number associated with the index is simply a count of the
atoms or bonds, thus making
1
χ a weighted count of skeletal bonds. A
molecular connectivity index represents certain aspects of the skeletal
structure [Lowell H. et al., 2001].
4 v
χ PC
index described the skeletal branching of a compound by
summation over all sub graphs with an isobutane skeleton [Jonathan D et al.,
2008] This index is found to increase with increased branching or adjacency.
As this is descriptor is positive and contributes a very high percentage
towards the activity (77.6%) it is ideal to design future compounds that have
increased branching and adjacency. The compounds exhibiting highest
94
Results and discussion
activity including 4a, 4b, 4c, 5a, 5b and 5c are highly branched and the
branches are quite adjacent to each other. The high degree of branching is
aided by the presence of the ciprofloxacin (4a, 5a), norfloxacin (4b, 5b) and
sparfloxacin (4c, 5c) moiety. In fact it is found that the fluoroquinolone
antibiotic ciprofloxacin is active against virulent strains of Leptospira
interrogans both in vitro and in vivo [Itamar Shalit et al., 1989]. No clinical
trials have been conducted using ciprofloxacin and its derivatives but
quinolones are estimated to be useful in treating systemic leptospirosis. The
quinoxaline fused fluoroquinolones used in our study might prove to be a
potential skeleton for further investigation.
The other descriptor explaining the activity of the antileptospiral
compounds is the alignment independent topological descriptor T_O_O_6 and
is found to have a negative contribution. Compounds 2v, 3n and 3v which
have poor activity exhibit this descriptor. T_O_O_6 has a close relationship
with geometric descriptor, G_O_O_6, which describes the anti tubercular
activity of the compounds. They exhibit a very high correlation with r = 0.99.
T_O_O_6 gave the number of oxygens while G_O_O_6 defines the geometric
distance between them. Hence the effect of T_O_O_6 can be associated to the
spatial disposition of the electronegative well formed by the carbonyl oxygen
of the azetidinone and thiazolidinone rings. As described earlier the presence
of oxygen in the ortho position of compound 3n and the furan ring of
compounds 2v and 3v might disrupt the integrity of the well at the carbonyl
group and alter the binding constants. We found that the presence of oxygen
at topological distance equivalent to 6 bonds to the carbonyl oxygen of the
azetidinone and thiazolidinone ring is imparting an overall negative effect to
the compounds.
95
Results and discussion
5.4
TOXICITY PREDICTION
From the property prediction results, it was observed that the all the ten
compounds have log P values less than 5. The log P value of a compound,
which is the logarithm of its partition coefficient between n-octanol and water
log (coctanol/cwater), is a well established measure of the compound's
hydrophilicity. Low hydrophilicities and therefore high logP values cause
poor absorption or permeation. It has been shown for compounds to have a
reasonable propability of being well absorbed their log P value must not be
greater than 5.0. Optimizing compounds for high activity on a biological
target almost often goes along with increased molecular weights. However,
compounds with higher weights are less likely to be absorbed and therefore to
ever reach the place of action. More than 80 % of all traded drugs have a
molecular weight below 450. But the molecular weights of the synthesized
compounds were more than 500. But some of the macrolide antibiotics like
erythromycin and quinoxaline containing antibiotic echinomycin have
molecular weight exceeding 500. In druglikeness property a positive value for
the chemicals states that the molecule contains predominantly fragments
which are frequently present in commercial drugs. All the ten compounds had
a positive value for the drug likeness. The drug score combines druglikeness,
LogP, molecular weight and toxicity risks in one handy value than may be
used to judge the compound's overall potential to qualify for a drug. The
results are given in Tables 5.13, 5.14 and 5.15. Toxicity prediction results are
valued and color coded. Properties with high risks of undesired effects like
mutagenicity or a poor intestinal absorption are shown in red. Whereas a
green color indicates drug-conform behavior. All the compounds showed
green color for all the toxic parameters. So the synthesised compounds were
predicted to be safe.
96
Results and discussion
Table 5.13 Predicted molecular properties of azetidinones
Compound C Log P
2n
2o
2p
2q
2r
2s
2t
2u
2v
2w
3.14
3.14
3.33
4.05
4.05
3.75
3.43
3.17
2.11
3.03
Drug
Likeness
2.34
2.34
2.59
2.78
2.78
2.22
3.34
2.34
0.57
1.25
Drug
Score
0.34
0.34
0.41
0.34
0.34
0.37
0.39
0.35
0.42
0.35
Table 5.14 Predicted molecular properties of thiazolidinones
Compound
C Log P
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3.23
3.23
3.42
4.14
4.14
3.84
3.52
3.53
2.28
3.12
Drug
Likeness
2.21
2.21
2.02
3.26
3.26
0.49
0.9
2.04
0.03
1.9
Drug
Score
0.24
0.24
0.22
0.20
0.20
0.18
0.11
0.22
0.21
0.22
97
Results and discussion
Table 5.15 Predicted molecular properties of Mannich bases
Compound
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
C Log P
1.02
0.88
1.33
4.05
4.05
3.75
2.1
1.4
1.65
0.45
0.74
0.62
Drug
Likeness
7.39
6.5
5.63
2.78
2.78
2.22
0.55
2.88
1.69
5.65
9.22
4.26
Drug
Score
0.44
0.44
0.41
0.34
0.34
0.37
0.26
0.41
0.33
0.88
0.89
0.87
98