Synthesis of novel triazolothione, thiadiazole, triazole-functionalized furo/thieno[2,3-b]pyridine derivatives and their antimicrobial activity

Article abstract of DOI:10.1080/00397911.2017.1354379

A series of novel triazolothione, thiadiazole, triazole-functionalized furo/thieno[2,3-b]pyridine derivatives 9a–l, respectively, were prepared starting from 2 (1H) pyridone 3 through selective O/S-alkylation followed by Thorpe–Ziegler cyclization to form furo/thieno[2,3-b]pyridine derivatives 6. Compounds 6 on reaction with hydrazine hydrate resulted carbohydrazide derivatives 7 and further reacted with diverse substituted phenyl isothiocyanates to form phenyl hydrazine carbothiamide derivatives 8. Each compound 8 is independently reacted in presence of NaOH, H₂SO₄, and N₂H₄.H₂O to form triazolothione, thiadiazole, triazole-functionalized furo/thieno[2,3-b]pyridine derivatives 9a–l, respectively. All the products 9a–l were screened against Gram +ve, Gram –ve bacteria and fungal strains. Compounds 9c–h showed high activity against Bacillus subtilis microbial-type culture collection (MTCC) 121 at <8.0 micromolar concentration. Promising compounds further screened for minimum bactericidal concentration against B. subtilis MTCC 121 using ciprofloxacin as standard and found to show very good activity. These compounds also screened for biofilm inhibition activity against B. subtilis MTCC 121 using erythromycin as standard and confirmed the high activity.

Full text of DOI:10.1080/00397911.2017.1354379

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis of novel triazolothione, thiadiazole,
triazole functionalized furo/thieno [2,3-b] pyridine
derivatives and their antimicrobial activity
G. Santhosh Kumar, Y. Poornachandra, K. Ratnakar Reddy, C. Ganesh Kumar
& B. Narsaiah
To cite this article: G. Santhosh Kumar, Y. Poornachandra, K. Ratnakar Reddy, C. Ganesh Kumar
& B. Narsaiah (2017): Synthesis of novel triazolothione, thiadiazole, triazole functionalized furo/
thieno [2,3-b] pyridine derivatives and their antimicrobial activity, Synthetic Communications
To link to this article: http://dx.doi.org/10.1080/00397911.2017.1354379
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Date: 21 July 2017, At: 05:18

Synthesis of novel triazolothione, thiadiazole, triazole
functionalized furo/thieno [2,3-b] pyridine derivatives and
their antimicrobial activity
G. Santhosh Kumar
Fluoro Organic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
India
Y. Poornachandra
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Tarnaka, Hyderabad, India
K. Ratnakar Reddy
Fluoro Organic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
India
C. Ganesh Kumar
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Tarnaka, Hyderabad, India
B. Narsaiah*
1

Fluoro Organic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
India
Address correspondence to B. Narsaiah. Tel: +91-40-27193630; Fax: +91-40-27160387. E-mail:
narsaiahbanda84@gmail.com
ABSTRACT
A series of novel triazolothione, thiadiazole, triazole functionalized furo/thieno [2,3-b]
pyridine derivatives 9a-l respectively were prepared starting from 2 (1H) pyridone 3 via selective
O/S-alkylation followed by Thorpe-Ziegler cyclization to form furo/thieno [2,3-b] pyridine
derivatives 6. Compounds 6 on reaction with hydrazine hydrate resulted carbohydrazide
derivatives 7 further reacted with diverse substituted phenyl isothiocyanates to form phenyl
hydrazine carbothiamide derivatives 8. Each compound 8 is independently reacted in presence of
NaOH, H2SO4 and N2H4.H2O to form triazolothione, thiadiazole, triazole functionalized
furo/thieno [2,3-b] pyridine derivatives 9a-l respectively. All the products 9a-l were screened
against Gram +ve, Gram –ve bacteria and fungal strains. Compounds 9c-h showed high activity
against Bacillus Subtilis MTCC121 at < 8.0 micro molar concentration. Promising compounds
further screened for minimum bactericidal concentration against Bacillus Subtilis MTCC 121
using ciprofloxacin as standard and found to show very good activity. These compounds also
screened for bio-film inhibition activity against Bacillus Subtilis MTCC 121 using Erythromycin
as standard and confirmed the high activity.
Graphical Abstract
2

A series of novel triazolothione, thiadiazole, triazole functionalized furo/thieno [2,3-b] pyridine
derivatives 9a-l respectively were prepared. All the products 9a-l were screened against Gram
+ve, Gram –ve bacteria and fungal strains. Compounds 9c-h showed promising activity against
Bacillus subtilis MTCC121 at < 8.0 micro molar concentration, promising compounds further
screened for minimum bactericidal concentration against Bacillus Subtilis MTCC 121 using
ciprofloxacin as standard and found to show very good activity. These compounds also screened
for bio-film inhibition activity against Bacillus Subtilis MTCC 121 using Erythromycin as
standard and confirmed the high activity.
KEY WORDS: 2(1H) pyridone, antimicrobial activity, O/S-alkylation, thiadiazole, thorpe-
ziegler cyclization, triazole, triazolothione
1. Introduction
Among all classes of biologically active compounds, the 5,6 fused ring systems such as
furopyridines and thienopyridines attracts important position in the development of new
3

pharmaceutical agents which are structurally analogues to indoles and pyrrolopyridines, and play
a significant role in promoting activity.[1], [2] Pyridyl, furopyridine and thienopyridine ring system
also considered as a prominent scaffold present in many of bioactive molecules, played a vital
role in the development of different medicinal agents.[3] However, this core structures have
shown activity against HIV,[4] CNS disorders,[5] skin diseases[6] and hyperglycemia.[7] Azoles,
especially the bio-isosteric 1,2,4-triazolothione, 1,2,4-triazoles and 1,3,4-thiadiazoles, are
important molecules known to interact with particular receptors on enzyme receptor sites.[8]
During recent years, there has been intense investigation of different classes of thiadiazole
compounds and many of which known to possess interesting biological activity such as
antimicrobial,[9–11] antituberculosis,[12] antiinflammatory,[13–15] anticonvulsant,[16], [17] antihyper
tensive,[18], [19] local anesthetic,[20] anticancer,[21], [22] and hypoglycemic activities.[23] Recently, it
was found that the trifluoromethyl group[24], [25] at a strategic position of an organic molecule,
dramatically alters the properties of molecule in terms of lipid solubility, oxidative thermal
stability, permeability and oral bio-availability. Moreover, in our earlier reports, we have
reported the synthesis of different oxadiazole derivatives and found that the combination of these
derivatives with furo/thieno [2,3-b] pyridine fused ring exhibited promising antimicrobial
activities. Encouraged by these reports, some azole derivatives bearing a furo/thieno [2,3-b]
pyridine moiety were synthesized with an aim to achieve the compounds having better
antimicrobial activity. Keeping in view of earlier findings on the importance of azoles, here we
have synthesized a series of novel 1,2,4-triazolothione, 1,3,4-thiadiazole and 1,2,4-triazole
functionalized furo/thieno [2,3-b] pyridine derivatives, screened for antimicrobial activity and
compounds 9c-h which showed promising activity have been identified.
2. Chemistry
4

The 3-cyano-4-trifluoromethyl-6-(thiophen-2-yl)-pyridine 2(1H) one/ pyridine 2(1H)
thione[26]
3 was reacted with 2-bromoethyl acetate and obtained selectively 2-O/S-ethylacetoxy-3-
cyano-4-trifluoromethyl-6-(thiophen-2-yl) pyridine 5 and 6. Compound 5 was cyclized in DMF
using potassium carbonate as base and obtained furo/thieno pyridine derivatives 6. The sequence
of reactions is mainly an abstraction of proton from active methylene followed by cyclization
onto nitrile carbon. This type of cyclization is also known as Thorpe-Ziegler cyclization.
Compound 6 on reaction with hydrazine hydrate followed by reaction with diverse substituted
phenylisocyanate in the presence of ethanol as a solvent resulted phenylhydrazinecarbothio
amide derivatives 7. Compound 7 was on reaction with phenylisothiocyante to form thiourea
containing derivatives 8 on further reaction under different conditions with different reagents
obtained different azole substituted furo/thieno [2,3-b] pyridine derivatives 9. The synthetic
pathway leading to the title compounds is given in Scheme 1, Scheme 2 and products are
tabulated in Table 1.
3. Results & Discussion:
Antimicrobial activity &structure activity relationship
Compounds 9a-l were screened for antimicrobial activity against seven bacterial
organisms such as Micrococcus luteus MTCC 2470, Staphylococcus aureus MTCC96,
Staphylococcus aureus MLS-16 MTCC 2940, Bacillus subtilis MTCC 121, Escherichia coli
MTCC 739, Pseudomonas aeruginosa MTCC 2453, Klebsiellaplanticola MTCC 530 and one
fungal strain Candida albicans MTCC 3017. Out of twelve compounds screened, six compounds
9c, 9d, 9e, 9f, 9g and 9h showed promising activity against Gram +ve Bacillus subtilis MTCC
5

121. Structure verses activity relationship revealed that, specific variation in structure showed
enhanced activity. If we consider compound 9a-d series of 1,2,4-triazolothione derivatives,
compounds 9a and 9b (furopyridine derivatives) showed moderate activity MIC 15.6 µg/mL and
compounds 9c and 9d (thienopyridine derivatives) showed promising activity MIC 7.8 µg/mL. It
may be due to the presence of sulphur in place of oxygen. Compounds 9e-l (1,3,4-thiadiazole
derivatives) plays a crucial role to promote antibacterial activity against Gram +ve Bacillus
subtilis MTCC 121. Among all the derivatives, compound 9h showed high activity i.e. MIC is
3.9 µg/mL. It may be attributed to the presence of thiophene and methoxyphenyl group.
However, compounds 9a-b and 9i-l could not show activity upto the concentration of 125 µg/mL
against all the organisms. The details of activity data is outlined in Tables 2–4.
Experimental section
Melting points were recorded on Casia-Siamia (VMP-AM) melting point apparatus and
are uncorrected. IR spectra were recorded on a Perkin-Elmer FT-IR 240-C spectrophotometer
1
using KBr optics. H NMR spectra were recorded on Bruker AV 300MHz in CDCl3 & DMSO-
d6 using TMS as internal standard. Electron impact (EI) and chemical ionization mass spectra
were recorded on a VG 7070 H instrument at 70 eV. All high-resolution spectra were recorded
on QSTARXL hybrid MS/MS system (Applied Biosystems, USA) under Electrospray
ionization.All the reactions were monitored by thin layer chromatography (TLC) on pre-coated
silica gel 60 F254 (mesh); spots were visualized with UV light. Merck silica gel (60-120 mesh)
was used for column chromatography. CHN analysis was recorded on a Vario EL analyser.
Antibacterial assay
6

The antimicrobial activity of the synthesized compounds was determined using well
diffusion method[27] against different pathogenic bacterial and Candida strains procured from the
Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-Institute of Microbial
Technology, Chandigarh, India. The pathogenic reference strains were seeded on the surface of
the media petri plates, containing Muller-Hinton agar with 0.1 ml of previously prepared
microbial suspensions individually containing 1.5 × 108cfu ml-1 (equal to 0.5 McFarland). Wells
of 6.0 mm diameter were prepared in the media plates using a cork borer and the synthesized
compounds dissolved in 10% DMSO at a dose range of 125-0.97 µg were added in each well
under sterile conditions in a laminar air flow chamber. Standard antibiotic solutions of neomycin
(bacterial strains) and miconazole (Candida strains) at a dose range of 125-0.97 µg well-1, served
as positive controls, while the well containing DMSO served as negative control. The plates were
incubated for 24 h at 30°C and the well containing the least concentration showing the inhibition
zone is considered as the minimum inhibitory concentration. All experiments were carried out in
duplicates and mean values are represented.
Minimum bactericidal concentration (MBC) assay
Bactericidal assay[28] (NCCLS, 2000) was performed in sterile 2.0 mL micro fuge tubes
against a panel of pathogenic bacterial strains, including Micrococcus luteus MTCC 2470,
Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS-16 MTCC 2940, Bacillus
subtilis MTCC 121, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 2453 and
Klebsiella planticola MTCC 530, cultured overnight in Mueller Hinton broth. Serial dilutions of
test compounds were prepared in Mueller Hinton broth with different concentrations ranging
from 0 to150 µg mL-1. To the test compounds, 100 µL of overnight cultured bacterial
suspensions were added to reach a final concentration of 1.5 × 108cfu mL-1 (equal to 0.5
7

McFarland) and incubated at 37 ºC for 24 h. After 24 h of incubation, the Minimum Bactericidal
Concentration (MBC) was determined by sampling 10 µL of suspension from the tubes onto
Mueller Hinton agar plates and were incubated for 24 h at 37 ºC to observe the growth of test
organisms. MBC are the lowest concentration of test compound required to kill a particular
bacterium strain. All the experiments were carried in duplicates.
Bio-film inhibition assay
The test compounds were screened in sterile 96 well polystyrene micro titer plates using
the modified bio-film inhibition assay,[29] against a panel of pathogenic bacterial strains
including Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS16 MTCC 2940,
Bacillus subtilis MTCC121, Pseudomonas aeruginosa MTCC 2453, and Klebsiella planticola
MTCC 530, which were cultured overnight in tryptone soy broth (supplemented with 0.5%
glucose). The test compounds of predetermined concentrations ranging from 0 to 250 µgmL-1
were mixed with the bacterial suspensions having an initial inoculum concentration of 5×105 cfu
mL-1. Aliquots of 100 µL were distributed in each well and then incubated at 37 ºC for 24 h
under static conditions. The medium was then discarded and washed with phosphate buffered
saline to remove the non-adherent bacteria. Each well of the micro titer plate was stained with
100 µL of 0.1% crystal violet solution followed by 30 min., incubation at room temperature.
Later, the crystal violet solution from the plates was discarded, thoroughly washed with distilled
water for 3 to 4 times and air dried at room temperature. The crystal violet stained bio-film was
solubilized in 95% ethanol (100 µL) and the absorbance was recorded at 540 nm using TRIAD
multimode reader (Dynex Technologies, Inc, Chantilly, VA, USA). Blank wells were employed
as background check. The inhibition data were interpreted from the dose-response curves, where
IC50 value is defined as the concentration of inhibitor required to inhibit 50% of bio-film
8

formation under the above assay conditions. All the experiments were carried out in triplicates
and the values are indicated as mean ± S.D.
4. Conclusion
In conclusion, we have successfully prepared a series of novel triazolothione, thiadiazole,
triazole functionalized furo/thieno [2,3-b] pyridine derivatives 9a-l, screened against Gram +ve,
Gram –ve bacteria and fungal strains. Compounds 9c-h showed high activity against Bacillus
subtilis MTCC121 at < 8.0 micro molar concentration. Promising compounds further screened
for minimum bactericidal concentration against Bacillus Subtilis MTCC 121 using ciprofloxacin
as standard and found to show moderate to very good activity.
General procedure for the synthesis of 3-(3-Amino-6-(thiophen-2-yl)-4-
(trifluoromethyl) furo[2,3-b]pyridin-2-yl)-4-phenyl-1H-1,2,4-triazole-5(4H)-
thione derivatives (9a-d)
2-(3-Amino-6-(thiophen-2-yl)-4-(trifluoromethyl)furo[2,3-b]pyridine-2-carbonyl)-N-
phenyl hydrazinecarbothioamide (8a) (0.01 mol) in 2 N NaOH was allowed to reflux for 4-6 h.
The resulting solution was cooled to room temperature and acidified to pH 3˗4 with 37%
hydrochloric acid. The precipitate formed was filtered and washed with distilled water, dried to
afford compound 9a.
3-(3-Amino-6-(thiophen-2-yl)-4-(trifluoromethyl)furo[2,3-b]pyridin-2-yl)-4-phenyl-1H-
1,2,4-triazole-5(4H)-thione (9a):
White solid; mp181-183oC;IR (KBr, cm-1): 3380, 3310 (-NH2) 1223 (-NHCS-); 1H NMR
(DMSO-d6, 300 MHz): δ ppm 6.22 (br, s, 2H, NH2), 7.12-7.18 (m, 2H, Ar-H), 7.41-7.48 (m, 4H,
9

Ar-H), 7.60 (dd, J = 4.90, 1H, Ar-H), 7.78 (dd, J = 3.76, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 13.28
13
(br, s, 1H, -NHCS-); C NMR (DMSO-d6, 75 MHz): δ ppm 123.0, 123.1, 123.7, 125.2, 126.0,
128.0, 129.0, 129.8, 129.9, 131.3, 133.4, 133.8, 134.6, 142.1, 146.6, 150.4, 157.0, 162.3;MS
(ESI): m/z [(M+H)+]: 460. HRMS m/z Calcd.for C20H12F3N5OS2 [(M+H)+]: 460.0469 Found:
460.0472.
General procedure for the synthesis of 2-(5-(Phenylamino)-1,3,4-thiadiazol-2-
yl)-6-(thiophen-2-yl)-4-(trifluoromethyl)furo[2,3-b]pyridin-3-amine derivatives
(9e-h)
A mixture of 0.001 mol of 2-(5-(phenylamino)-1,3,4-thiadiazol-2-yl)-6-(thiophen-2-yl)-
4-(trifluoromethyl)furo[2,3-b]pyridin-3-amine (8a) and concentrated H2SO4 (1 mL) was stirred
at room temperature for 1 h. Then the reaction mixture was poured over crushed ice. The
precipitated solid was washed with sodium carbonate solution followed by water to afford
compound (9e).
2-(5-(Phenylamino)-1,3,4-thiadiazol-2-yl)-6-(thiophen-2-yl)-4-(trifluoromethyl)furo[2,3-
b]pyridin-3-amine (9e):
o
1
Light yellow solid; mp 236-238 C; IR (KBr, cm-1): 3386, 3313 (-NH2); H NMR
(CDCl3+DMSO-d6, 300 MHz): δ ppm 6.23 (br. s, 2H, -NH2), 7.32-7.36 (m, 2H, Ar-H), 7.53 (dd,
J = 4.78, 2H, Ar-H), 7.59-7.62 (m2H, Ar-H), 7.71 (dd, J = 4.75, 1H, Ar-H), 8.86 (dd, J = 3.71,
1H, Ar-H), 8.12 (s, 1H, Ar-H), 10.14 (br. s, 1H, -NH-),;13C NMR (DMSO-d6, 75 MHz): δ ppm
121.2, 122.8, 123.9, 125.7, 127.0, 128.4, 129.1, 130.1, 131.5, 133.5, 134.9, 136.6, 139.7, 140.7,
142.6, 143.8, 152.5, 153.7; MS (ESI): m/z [(M+H)+]: 460. HRMS m/z Calcd.for C20H12F3N5OS2
[(M+H)+]: 460.0460, Found 460.0464.
10

General procedure for the synthesis of 5-(3-Amino-6-(thiophen-2-yl)-4-
(trifluoromethyl) furo[2,3-b]pyridin-2-yl)-N3-phenyl-4H-1,2,4-triazole-3,4-
diamine(9i-l)
2-(3-Amino-6-(thiophen-2-yl)-4-(trifluoromethyl)furo[2,3-b]pyridine-2-carbonyl)-N-
phenyl hydrazinecarbothioamide (8a) (0.01 mol) was mixed with N2H4.H2O (0.01 mol) and
MeOH (1mL). The solution was refluxed for 5-6 h. After cooling to room temperature, ice water
(10mL) was added to the reaction mixture, which was then neutralized with 3N HCl to form a
precipitate. The precipitate was isolated by filtration to afford the Triazole derivatives 9i-l.
5-(3-Amino-6-(thiophen-2-yl)-4-(trifluoromethyl)furo[2,3-b]pyridin-2-yl)-N3-phenyl-
4H-1,2,4-triazole-3,4-diamine (9i):
o
1
White solid; mp 222-224 C; IR (KBr, cm-1): 3482, 3420 (-NH2),3378, 3318 (-NH2); H
NMR (CDCl3+DMSO-d6, 300 MHz): δ ppm 5.21(br. s, 2H, -NH2), 6.26 (br. s, 2H, -NH2), 7.01
(br. s, 1H, -NH-), 7.29-7.33 (m, 2H, Ar-H), 7.50 (dd, J = 4.71, 1H, Ar-H), 7.58-7.62 (m, 3H, Ar-
H), 7.68(dd, J = 4.71, 1H, Ar-H), 7.79 (dd, J = 3.71, 1H, Ar-H), 7.91 (s, 1H, Ar-H);13C NMR
(DMSO-d6, 75 MHz): δ ppm 119.9, 120.8, 123.0, 123.4, 124.6, 125.6, 126.4, 127.9, 129.6,
131.4, 133.9, 134.2, 135.6, 137.3, 139.5, 141.8, 147.9, 149.8 121.7, 123.4, 123.9, 124.6, 125.1,
126.2, 126.8, 128.4, 130.3, 131.7, 132.7, 134.7, 136.9, 139.2, 140.7, 142.6, 143.7, 146.4, 149.7,
154.4;MS (ESI): m/z [(M+H)+]: 458. HRMS m/z Calcd. for C20H14F3N7OS [(M+H)+]: 458.1023,
Found 458.1026.
Acknowledgements
11

Authors are thankful to the Council of Scientific and Industrial Research (CSIR), New
Delhi, India for the financial assistance through a XII five year plan project DITSF code:CSC-
0204. Authors (G. Santhosh Kumar, Y. Poorna Chandra and K. Ratnakar Reddy) are also
thankful to CSIR for providing Senior Research Fellowship and contingency grant.
Supporting Information
Full experimental detail, 1H and 13C NMR spectra, ESI MS and HRMS. This material can
be found via the “Supplementary Content” section of this article’s webpage.
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14

Table 1. Preparation of 1,3,4-oxadiazole substituted furo/thieno [2,3-b] pyridine derivatives 9a-l.
Entry
1.
Compound
R
X
O
O
S
R′
Yield(%)
81
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
C6H5
9a
9b
9c
9d
9e
9f
2.
4-OCH3C6H4 82
C6H5 79
4-OCH3C6H4 80
C6H5 88
4-OCH3C6H4 85
C6H5 70
4-OCH3C6H4 72
C6H5 85
4-OCH3C6H4 82
C6H5 80
4-OCH3C6H4 78
3.
4.
S
5.
O
O
S
6.
7.
9g
9h
9i
8.
S
9.
O
O
S
10.
11.
12.
9j
9k
9l
S
15

Table 2. Antibacterial and antifungal activity.
En Com
try pds.
Minimum Inhibitory Concentration (MIC) (µg/ml)
Microc Staphylo Staphylo Baci Escher Pseudo Klebsiellapl Candida
occus
luteus
MTCC
2470
coccus
aureus
MTCC
96
coccus
aureus
MLS-16
MTCC
2940
llus
subti
lis
ichia
coli
monas
anticola
albicans
aerugin MTCC 530 MTCC
MTC
osa
3017
MT C 739 MTCC
CC
2453
121
1
2
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>12 >125.0 >125.0
5.0
>125.0
>125.0
>125.0
>125.0
9a
9b
>12 >125.0 >125.0
5.0
3
4
5
6
7
8
9
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
15.6 >125.0 >125.0
15.6 >125.0 >125.0
7.8 >125.0 >125.0
7.8 >125.0 >125.0
7.8 >125.0 >125.0
3.9 >125.0 >125.0
>12 >125.0 >125.0
5.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
>125.0
9c
9d
9e
9f
9g
9h
9i
10
>125.0
>125.0
>125.0
>12 >125.0 >125.0
5.0
>125.0
>125.0
9j
16

11
12
>125.0
>125.0
0.9
>125.0
>125.0
0.9
>125.0
>125.0
0.9
>12 >125.0 >125.0
>125.0
>125.0
0.9
>125.0
>125.0
–
9k
9l
5.0
>12 >125.0 >125.0
5.0
Ciprofloxa
cin
0.9
0.9
0.9
Miconazol
e
7.8
–
–
–
–
–
–
–
(Standard)
17

Table 3. Bactericidal activity results.
S. No.
Test compound
Minimum Bactericidal Concentration (µg/ml)
Bacillus subtilis MTCC 121
1
2
3
4
5
6
15.6
15.6
7.8
9c
9d
9e
9f
7.8
7.8
9g
9h
3.9
Ciprofloxacin (Standard)
1.17
18

Table 4. Bio-film inhibition assay results.
S. No.
Test compound
IC50 values in (μg/ml)
Bacillus subtilis MTCC 121
4.2  0.26
1
2
3
4
5
6
9c
9d
9e
9f
16.4 0.32
6.1 0.16
3.6 0.12
6.8 0.34
1.9 0.11
0.2 0.09
9g
9h
Erythromycin (Standard)
19

Scheme 1.[Aq]
20

Scheme 2.[Aq]
21