Summary.
The introduction of antimicrobial agents into general clinical use represents one
of the landmark medical advances of modern medicine. In the last half of the
20th century, a number of new antimicrobials came into clinical use, presenting
clinicians with an array of choices when treating many types of infectious
diseases. However, the issue of antimicrobial resistance that has been a concern
ever since the beginning of the antimicrobial era, has taken on more importance
recently. Clinicians are witnessing increasing rates of in vitro resistance among
previously susceptible organisms and the emergence of intrinsically resistant
organisms as pathogens in immuno-compromised hosts. The spread of resistance
has in turn limited the treatment options for some serious and life-threatening
diseases. To curtail the development and spread of antimicrobial resistance we
require both the preservation of current antimicrobials through their appropriate
use, as well as the discovery and development of new agents.
There is clearly a need for new antimicrobials to combat disease due to resistant
pathogens in serious and life-threatening diseases. Improved clinical trial design
requires the use of better tools in the drug development process to evaluate
efficacy and safety. While there remain significant challenges in the discovery
and development of new antimicrobials, there are still advantages for those
willing to continue to advance the science and public health in this field. After
detailed survey of literature, a wide variety of antimicrobial and antitubercular
like quinolines10-12, thiazoles18, imidazoles19, piperazines20 etc. have been
1
Summary.
prepared in our laboratory. Keeping the above consideration in view, the present
research investigation was undertaken by me under the supervision of Dr. Abha
Bishnoi, which aimed at the synthesis and pharmacological evaluation of newer
compounds with the expectation to arrive at a biologically active agent with a
more specific action, greater potency, profound safety and with none of the
undesirable side effects.
The present researches include the synthesis and pharmacological screening of
studies of the following types of compounds:
1. Pyrazoline Derivatives:
Type
I:
2-(4-aryl-2-thiazolyl)-7-benzylidene-3,3a,4,5,6,7-hexahydro-3-
phenyl-2H-indazoles.
R
O
NH2
N
N
X
R
R
3(a-d)
R
4(a-f)
2
Summary.
R
R1
N
N
N
X
R
5(a-l)
The requisite starting material 2,6-bis(aryl methylidene)cyclohexanones (3a-d)
were prepared by the Claisen condensation of cyclohexanone with different
aldehydes according to the known procedure35-40. Reaction of 1 with substituted
hydrazines under acidic conditions yielded 7-benzylidene – 3, 3a, 4,5, 6, 7
hexahydro-3-phenyl-2-thiocarmoyl-2H-indazoles
(4a-d).
The
cyclo
condensation reaction of (4a-d) with substituted acetophenones in presence of
iodine gave the corresponding 2-(4-aryl-2-thiazoyl)-7-benzylidene-3,3a,4,5,6,7hexahydro-3 phenyl-2H-indazole compounds (5a-j).
The new series of arylthiazolyl pyrazoline derivatives were evaluated for
antimicrobial activity. It was found that among the arylthiazolyl pyrazolines
synthesized, compounds 5j, 5g and 5l possess pronounced antimicrobial activity
against S. aureus, B. subtilis and C. albicans in comparison with the other
compounds. Interestingly all the three compounds bear a 4-chlorophenyl group.
Compound 5g showed significant growth inhibitory action against all tested
3
Summary.
strains. The above observation indicate that the presence of 4-chlorophenyl
group enhances the antimicrobial activity. Compounds 5e, 5g and 5l were most
active against S. aureus with an MIC of 12.5 μg/ml which is quite close to the
standard drug Ampicillin. Compounds 5b and 5j showed good activity against
B.subtilis with a MIC value of 12.5 μg/ml but none of them could compete with
that of the standard drug Erythromycin. Compounds 5g, 5l, 5j (MIC 12.5 μg/ml)
and 5l (MIC 6.25 μg/ml) of this series showed excellent activity activity against
C. albicans which is quite comparable with that of Fluconazole.
Type II: 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-N-hydroxy3,3a,4,5,6,7-hexahydro-2H-indazole-2-carbothioamide/ 3-substitutedphenylN-hydroxy-5-methyl-7-(4-substituted benzylidene)-4,6-diphenyl-3,3a,4,5,6,7hexahydro-2H-pyrazolo[4,3-c]pyridine-2-carbothioamide.
O
CHR'
R'HC
NHOH
NHNH2 .HCl
S
7
N
CH3
8a,b
4
Summary.
R
CHR'
NHOH
N
S
NHOH
N
N
H3 C
R
N
N
S
R'
10(a-b)
9(a-d)
The requisite starting material N-hydroxy thiosemicarbazide (7) were prepared
from hydroxylamine hydrochloride and hydrazine hydrate in carbon disulphide
in the presence of KOH. This solution was then stirred at 800C for 1 hour.
Reaction of
2,6-bis(aryl methylidene)cyclohexanones 3(a-d) and 3,5-
bis(substituted benzylidene)-1-methyl-2,6-diphenylpiperidin-4-one 8(a,b) with
N-hydroxy thiosemicarbazide under acidic conditions yielded 7-(4-substituted
benzylidene)-3-(4-substituted
phenyl)-N-hydroxy-3,3a,4,5,6,7-hexahydro-2H-
indazole-2-carbothioamide 9(a-d) and 3-substitutedphenyl-N-hydroxy-5-methyl7-(4-substituted
benzylidene)-4,6-diphenyl-3,3a,4,5,6,7-hexahydro-2H-
pyrazolo[4,3-c]pyridine-2-carbothioamide 10(a, b).
The results of antimicrobial assay reveal that the compounds showed significant
zone of inhibition of the fungi and two strains of bacteria i.e. B. subtilis and S.
aureus. Therefore, these strains were taken up for the calculation of MIC. It is
shown that compound 10b is the most active one among the series shown to
5
Summary.
inhibit all the three strains to a considerable extent. All compounds showed
average antimicrobial activity, as shown by the zone of inhibition and MIC
values, against S. aureus. Compound 10b showed maximum zone of
inhibition(12mm). Against B. subtilis, only compound 10b showed some
significant antimicrobial activity as revealed by the MIC value. Other
compounds of the series were found to be inactive. None of them would prove a
promising antibacterial agent against these two strains. As an antifungal agent
also, compound 10b was very promising and proved to be a potent antimicrobial
agent on the whole.
Primary screening of this series of compounds was also conducted at three
concentrations (3.12μg/ ml , 6.25μg/ ml and 12.5μg/ ml) against M. tuberculosis
using the Microplate Alamar Blue Assay(MABA)67. Compounds demonstrating
at least 90% inhibition in the primary screen were tested at lower concentrations
against M. tuberculosis to determine the MIC using MABA. Interesting results
were obtained from these assays. The in vitro antimycobacterial activities of
these compounds 9(a-d) and 10(a-b) were found to be inferior to that of
Isoniazid against M. tuberculosis. Further, the compounds 9(a-d) and 10(a-b)
had either little or no activity (2-13% inhibition).
Type
III:
N-(4-(1-acetyl-5-(4-substituted
pyrazol-3-yl)phenyl)-3-(4-substituted
phenyl)-4,5-dihydro-1Hphenyl)-5-(2-(4-substituted
6
Summary.
phenyl)-1-phenyl-2,3-dihydro-1H-pyrazol-4-yl)cyclopent-2enecarbothioamide.
R1
O
N N
R2
N
N
Ac
H 2NHNCHN
S
R3
16(a-c)
13(a-c)
R2
N N
Ac
N HN
N
S
R3
R1
N N
17(a-i)
The final compounds 17(a-i) were furnished by refluxing 13(a-c) with 16(a-c) in
ethanol with traces of acetic acid on a water bath at 950C.
The antimicrobial activity results were significant for only gram positive bacteria
S. aureus (ATCC9144) and fungi C. albicans (ATCC24433). The activity was
excellent for 17a against Candida sp. with greatest Zone of Inhibition of 30mm.
All other compounds also displayed an above average or good activity against
7
Summary.
the fungus. 17a and 17g showed significant activity against the bacteria S.
aureus.
2. Quinoline Derivatives
Type IV: 7-(4-substituted phenyl)-5,6-diphenylbenzo[h][1,2,4]triazolo[3,4a][2,6]naphthyridine-3(2H)-thione / one.
X
O
O
HN
N
Ph
N
Ph
Ph
Ph
N
N
R
21 (a-c)
R
22 (a-f)
Compound 21(a-c) were prepared by the reaction of isatin with substituted
acetophenones and then cyclization with benzoin in presence of PPA.
Compounds 21(a-c) were then treated with semicarbazide and thiosemicarbazide
to give the final compounds 22(a-f).
All synthesized compounds were evaluated for their biological activity. The
complete class of compounds showed significant antibacterial and antifungal
activity. Compound 22d showed maximum antibacterial zone of inhibition
against gram negative E. coli whereas compound 22b and 22f showed maximum
8
Summary.
activity against gram positive S. aureus and B. subtilis respectively. Among
antifungals compound 22e displayed excellent activity against C. albicans.
These compounds can make for good antimicrobial agents and may be
antimalarial too.
3. Benzimidazole & Triazine Derivatives
Type
V:
1-(4-substituted
benzo[d]imidazol-2-yl)hydrazines
phenyl)-3-(substituted)propan-1-(1Hand
1-(4-substituted
phenyl)-3-
substituted propan-1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)hydrazines.
R1
N R2
R1
N R2
H
N
H
N
N
H
N
N
H
R
28(a-d)
N
H
N N
N
R
N
H
29 (a-c)
The preparation of novel 1-(4-substituted phenyl)-3-substituted propane-1-(1H
benzo[d]imidazol-2-yl)hydrazines 28(a-d) and 1-(4-substituted phenyl)-3substitutedpropane-1-(5H-[1,2,4]- triazino[5,6-b] indol-3-yl) hydrazines 29(a-c)
involves three steps; i) mannich reaction of different acetophenones and
secondary amines, ii) reduction of the keto compounds by sodium borohydride
to corresponding 3-N-substituted amino-1-phenyl-propanols and iii) the reaction
of alcohol derivatives with thionyl chloride followed by addition of 29
Summary.
hydrazinyl-1-H-benzo[d]imidazoles/
3-hydrazinyl-1-5H-[1,2,4]triazino[5,6-
b]indole in alchohol to produce 28(a-d) and 29(a-c) respectively.
Seven compounds were examined for their antifungal activity at a concentration
of 500 ppm. Biological data against Macrophomina phaseolina indicate that
benzimidazole derivatives of Mannich bases 28(a-d) exhibit slightly better
inhibition than triazine derivatives of these bases 29(a-c). It is very interesting to
observe that two compounds 28b and 29c with substituents R (=OH) and R1R2
(= morpholine group) inhibited the growth of fungus to a considerable extent.
Antifungal efficacy of 28b is also correlated with its energetically favored
conformation. It seems that the caged geometry of the compound 28b allows it
to interact in a more favorable way with the receptor site of the organism. On the
other hand, all the seven compounds were found to show random activity
towards Sclerotium rolfsii.
Most of the synthesized compounds have shown promising activity against S.
aureus while very mild activity against P. aeruginosa. Compounds 28c and 29a
exhibited moderate activity against C. albicans, E. coli and B. subtilis. It may
therefore be inferred that in order to elucidate a structure activity relationship,
synthesis and antimicrobial evaluation of more such compounds with larger
number of substituents is required.
4. Pyrimidine Derivatives:
10
Summary.
Type
VI:
4-(4-substitutedphenyl)-3-(3-substituted
phenyl)-4H-
spiro[isoxazole-5,3'-pyrido[1,2-a]pyrimidine]-2',4'-dione.
N
N
N
N
O
N
O
O
O
Ar
O
H
N
O
N
O
Ar
30
R
31 (a-c)
33 (a-f)
The cyclization of 2-amino pyridine and malonic acid (equimolar amounts) in
absolute ethanol leaded to the formation of compound 30. This cyclized product
30 underwent Claisen condensation with various substituted benzaldehydes in
presence of NaOH and ethanol to yield compounds 31(a-c). Compound 31(a-c)
was then stirred with 3-nitro benzaldoxime 32a and 4-chloro benzaldoxime 32b
on a magnetic
stirrer at 0-50C in presence of NaOCl via 1,3-dipolar
cycloaddition reaction, resulting in the formation of spiroisoxazline derivatives
33(a-f).
These spiro derivatives were evaluated for their anti-tubercular and antimicrobial
activities. The overall results were quite satisfactory. Among the six derivatives
prepared, three derivatives (33d, 33e & 33f) displayed modest in vitro activity
against Mycobacterium
tuberculosis via Microplate Alamar Blue Assay
(MABA). The other compounds showed either little or no anti-tubercular
11
Summary.
activity (0-16% inhibition only). The compounds 33e and 33f showed good
antimicrobial activity against almost all bacterial strains (gram positive as well
as gram negative) and fungi. All the other derivatives were found to be inactive.
The active compounds of this class i.e. 33e & 33f consisted of 3,4dioxomethylene phenyl group and proved quite promising in both antimicrobial
and anti-tubercular assays.
Type
VII:
3-(4-substituted
phenyl)-3H-isoxazolo[3,4-d]pyrido[1,2-
a]pyrimidin-4(3aH)-one.
Type VIII: 3-(4-substituted phenyl)-3.3a-dihydropyrazolo[3,4-d]pyrido[1,2a]pyrimidin-4(2H)-one.
N
N
N
O
O
Ar
N
O
34 (a-c)
Ar
N
N
N
H
35 (a-c)
Compounds 31(a-c) were refluxed with hydroxylamine hydrochloride and 2%
NaOH on one side and hydrazine hydrate and glacial acetic acid on the other to
12
Summary.
yield final compounds 34(a-c) and 35(a-c) respectively. These final compounds
were then tested for their antimicrobial and anti-tubercular activities.
The result was quite the same with isooxazole 34(a-c) and pyrazole derivatives
35(a-c) as the spiro derivatives. The benzo[d][1,3]dioxole substituted
compounds 34c and 35c were more active than all other substituents. These
compounds also showed marvelous zone of inhibition of 27 and 28 mm against
the fungal strain C. albicans. The increased acivity of benzo[d][1,3]dioxole
substitution may be due to its structure leading to a better fit at the receptor site.
As far as antitubercular activity is concerned, none of the substituent was found
to be significantly active.
13