This document summarizes research on the synthesis and evaluation of novel imidazopyridine and pyrrolopyridine compounds against tuberculosis and other infectious diseases. Specifically, it discusses the synthesis of various imidazopyridine and pyrrolopyridine derivatives and their antimycobacterial and antimicrobial activities based on literature reports. It provides background on the need to develop new drugs to address resistance to existing antibiotics and antifungals. Examples are given of imidazopyridine and pyrrolopyridine compounds that have shown potent activity against Mycobacterium tuberculosis or other pathogens.

1. Synthesis and applications of some novel imidazopyridine
and pyrrolopyridine compounds
By
Gilish Jose
USN: 12PPCH0004
Department of Chemistry
Jain University, Bangalore
Under the supervision of
Dr. Suresha Kumara T.H.
Associate Professor
U.B.D.T. College of Engineering (A constituent college of V.T.U)
Davangere, Karnataka

2. CONTENTS
 Chapter 1: Introduction
 Chapter 2: Synthesis, crystal structure, molecular docking and antimycobacterial
evaluation of imidazo[1,2-a]pyridine-2-carboxamide derivatives
 Chapter 3: Synthesis, crystal structure, molecular docking and antimicrobial evaluation of
new polyfunctional imidazo[4,5-c]pyridine derivatives
 Chapter 4: Synthesis, crystal structure, molecular docking, antimicrobial and
antimycobacterial evaluation of new pyrrolo[3,2-c]pyridine derivatives
 Chapter 5: Summary of findings
 List of publications
 Conferences and Presentations
2

3. CHAPTER-1: Introduction
Background of the research
 Tuberculosis (TB) is a highly dangerous bacterial infectious disease caused by an airborne bacterium known as
bacillus Mycobacterium tuberculosis [1].
 In recent years, the resistance to first-line TB drugs (isoniazid, ethambutol, pyrazinamide and rifampicin) has
amplified the new forms of TB such as multiple drug-resistant TB (MDR-TB) and extensively drug-resistant TB
(XDR-TB) [2,3].
 Infections caused by bacteria and fungi remain a major health concern due to the development of resistance to
currently using antibiotics (chlorampenicol, streptomycin, tetracyclin, azythromycin, sulfadiazin, pencillin –V,
cefadroxil etc) and antifungals (fluconazole, amphotericin B, abafungin, sulconazole etc.) [4-6].
1. WHO. 2016. ‘Global Tuberculosis report 2016’.
2. de Souza MVN. 2006. ‘Promising drugs against tuberculosis’. Recent Patents on Anti-Infective Drug Discovery. 1:1: 33-44.
3. Goldman R, Plumley KV, Laughon BE. 2007. ‘The evolution of extensively drug resistant tuberculosis (XDR-TB): History, status and issues for global control’. Infectious
Disorders - Drug Targets. 7:2: 73-91.
4. Chu DTW, Plattner JJ, Katz L. 1996. ‘New directions in antibacterial research’, Journal of Medicinal Chemistry. 39:20: 3853-3874.
5. Davies J. 1996. ‘Bacteria on the rampage’, Nature. 383: 219-220.
6. Hitchock CA. 1993. ‘Resistance of Candida albicans to azole antifungal agents’, Biochemical Society Transactions. 21: 1039-1047. 33

4.  To address these issues, there is an emerging need to develop more potent drugs to treat these lethal diseases.
 In this direction, a variety of nitrogen heterocycles are being designed, synthesized and evaluated against these
lethal microbes.
 Among them, pyridine-containing heterocyclic scaffolds such as Imidazopyridine and Pyrrolopyridine derivatives
are very important, versatile motifs since several of its derivatives have been found to be medicinally useful [7-11].
N
N
H
N
N N
Imidazo[1,2-a]pyridine
N N
H
N
N
N
Imidazo[1,5-a]pyridine Imidazo[4,5-b]pyridine Imidazo[4,5-c]pyridine
N
N
H
N
N
H
N N
H
N N
H
Pyrrolo[2,3-b]pyridinePyrrolo[2,3-c]pyridinePyrrolo[3,2-c]pyridine Pyrrolo[3,2-b]pyridine
Fig. 1.1: Biologically important imidazopyridine and pyrrolopyridine core moieties
Background of the research ...
7. Krenitsky TA, Rideout JL, Chao EY, Koszalka GW, Gurney F, Crouch RC, Cohn NK, Wolberg G, Vinegar R. 1986. ‘Imidazo[4,5-c]pyridines (3-deazapurines) and their nucleosides
as immunosuppressive and antiinflammatory agents’, Journal of Medicinal Chemistry. 29:1: 138-143.
8. Ramasamy K, Imamura N, Hanna NB, Finch RA, Avery TL, Robins RK, Revankar GR. 1990. ‘Synthesis and antitumor evaluation in mice of certain 7-deazapurine (pyrrolo[2,3-
d]pyrimidine) and 3-deazapurine (imidazo[4,5-c]pyridine) nucleosides structurally related to sulfenosine, sulfinosine, and sulfonosine’, Journal of Medicinal Chemistry. 33:4: 1220-
1225.
9. Robertson DW, Beedle EE, Krushinski JH, Pollock GD, Wilson H, Wyss VL, Hayes JS. 1985. ‘Structure-activity relationships of arylimidazopyridine cardiotonics: discovery and
inotropic activity of 2-[2-methoxy-4-(methylsulfinyl)phenyl]-1H-imidazo[4,5-c]pyridine’, Journal of Medicinal Chemistry. 28:6: 717-727.
10. Temple Jr. C 1990. ‘Antimitotic agents. Synthesis of imidazo[4,5-c]pyridin-6-ylcarbamates and imidazo[4,5-b]pyridin-5-ylcarbamates’, Journal of Medicinal Chemistry. 33:2: 656-
661.
11. Temple Jr. C, Rose JD, Comber RN, Rener GA. 1987. ‘Synthesis of potential anticancer agents: imidazo[4,5-c]pyridines and imidazo[4,5-b]pyridines’, Journal of Medicinal
Chemistry. 30:10: 1746-1751.
4

5. Fig 1.2: Specific examples of imidazopyridine based antimycobacterial and antimicrobial agents [12-18]
N N
NH2
O 1, X = OCH3, Mtb MIC = 0.5 µg/mL
2, X = O
F
F
, Mtb MIC = 0.5 µg/mL
3, X = O
O
, Mtb MIC = 0.25 µg/mL
4, X = O
F , Mtb MIC = 0.25 µg/mLN
X
N N
H
N
O
N N
H
N
O
O
Cl
N N
H
N
O
O
F
N N
H
N
O
O
F
5 6
7 8
5-8, Mtb MIC = <0.006µM
N N
H
N
O
9, Mtb MIC50 = 2.03 µM
N N
H
N
O
10, Mtb MIC50 = 2.03 µM
N N
H
N
O
O
O
F
F
F
O
11, Mtb MIC50 = 0.013 µM
N N
H
N
O
N
O
F F
F
Q203
12, Mtb MIC50 = 0.0027 µM
Cl
N N
H
N
R
Br
R = F
O
ClF
F
F
F
F
13 14 15
16 17 18
N N
N
HN
N N
N
Cl
N N
H
N N
Cl
N N
H
N N
Cl
Br
19 20 21
N N
F
R
R =
O
N
N
N
N
N
22 23
2524
Literature survey
12. Ramachandran S, Panda M, Mukherjee K, Choudhury NR, Tantry SJ, Kedari CK, Ramachandran V, Sharma S, Ramya VK, Guptha S, Sambandamurthy VK, 2013. ‘Synthesis and
structure activity relationship of imidazo[1,2-a]pyridine-8-carboxamides as a novel antimycobacterial lead series’, Bioorganic & Medicinal Chemistry Letters. 23:17: 4996-5001.
13. Moraski GC, Markley LD, Cramer J, Hipskind PA, Boshoff H, Bailey MA, Alling T, Ollinger J, Parish T, Miller MJ. 2013. ‘Advancement of imidazo[1,2-a]pyridines with improved
pharmacokinetics and nM activity vs. Mycobacterium tuberculosis’, ACS Medicinal Chemistry Letters. 4:7: 675-679.
14. No Z, Kim J, Brodin P, Seo MJ, Kim YM, Cechetto J, Jeon H, Genovesio A, Lee S, Kang S, Ewann FA, Nam JY, Christophe T, Fenistein DPC, Jamung H, Jiyeon J. 2011. ‘Anti-
infective compounds’. WO 2011/113606 A1.
15. Pethe K, Bifani P, Jang J, Kang S, Park S, Ahn S, Jiricek J, Jung J, Jeon HK, Cechetto J, Christophe T, Lee H, Kempf M, Jackson M, Lenaerts AJ, Pham H, Jones V, Seo MJ, Kim YM,
Seo M, Seo JJ, Park D, Ko Y, Choi I, Kim R. 2013. ‘Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis’, Nature Medicine. 19: 1157-1160.
16. Lavanya P, Suresh M, Kotaiah Y, Harikrishna N, Venkata Rao C. 2011. ‘Synthesis, antibacterial, antifungal and antioxidant activity studies on 6-bromo-2-substituted phenyl-1H-
imidazo [4,5-b] pyridine’. Asian Journal of Pharmaceutical and Clinical Research. 4:4: 69-73.
17. Mallemula VR, Sanghai NN, Himabindu V and Chakravarthy AK. 2015. ‘Synthesis and characterization of antibacterial 2-(pyridin-3-yl)-1H-benzo[d]imidazoles and 2-(pyridin-3-yl)-
3H-imidazo[4,5-b]pyridine derivatives’, Research on Chemical Intermediates. 41:4: 2125-2138.
18. Cheng CC, Shipps Jr. GW, Yang Z, Sun B, Kawahata N, Soucy KA, Soriano A, Orth P, Xiao L, Mann P, Black T. 2009. ‘Discovery and optimization of antibacterial AccC inhibitors’,
Bioorganic & Medicinal Chemistry Letters. 19:23: 6507-6514.
5

6. N N
H
O
HN
N
N
N
N
F
F F
S. aureus MIC (µM): 3.13
MRSA MIC (µM): 1.56
N N
H
O
HN
N
N
N
F
F F
S. aureus MIC (µM): 0.40
MRSA MIC (µM): 1.10
N
NH
O
O
N N
H
O
HN
N
N
N
F
F F
S. aureus MIC (µM): 0.78
MRSA MIC (µM): 1.56
N
NH
O
O
O
N N
H
O
HN
N
N
N
F
F F
S. aureus MIC (µM): 0.098
MRSA MIC (µM): 0.2
N
NH
O
O
O
26
27
28 29
Literature survey ...
N N
H
R
R = o-MeOC6H4,
p-MeOC6H4,
m-ClC6H4
N N
H
R
R = o-MeOC6H4,
p-MeOC6H4,
m-ClC6H4
C6H5
N N
H
R
R = H, o-Me-p-MeOC6H3
Cl
30 31 32
N
N
O
N
H
OH
N
N
N
Mtb MIC (µM): <0.39
N
N
O
N
H
F
N
N
O
Mtb MIC (µM): <0.39
N
N
O
N
H
F
N
N
O
Mtb MIC (µM): 0.78
N
N
O
N
H
F
N
N
O
Mtb MIC (µM): 0.78
F
O
N
NO
O
N
H
OH
N
N
N
Mtb MIC (µM): <0.39
33 34 35
36 37
Fig 1.3: Specific examples of pyrrolopyridine based antimycobacterial and antimicrobial agents [19-24]
19. Manchester JI, Dussault DD, Rose JA, Sjodin PAB, Nickelsen MU, Ioannidis G, Bist S, Fleming P, Hull KG. 2012. ‘Discovery of a novel azaindole class of antibacterial agents
targeting the ATPase domains of DNA gyrase and Topoisomerase IV’. Bioorganic Medicinal Chemistry Letters. 22:15: 5150-5166.
20. Paget SD, Boggs CM, Foleno BD, Goldschmidt RM, Hlasta DJ, Weidner-Wells MA, Werblood HM, Bush K, Macielag MJ. 2006. ‘Antibacterial activity of pyrrolopyridine-substituted
oxazolidinones: synthesis and in vitro SAR of various C-5 acetamide replacements’. Bioorganic & Medicinal Chemistry Letters. 16:17: 4537-4542.
21. Leboho TC, Vuuren SF, Michael JP, Koning CB. 2014. ‘The acid-catalysed synthesis of 7-azaindoles from 3-alkynyl-2-aminopyridines and their antimicrobial activity’. Organic &
Biomolecular Chemistry. 12:2: 307-315.
22. Shirude PS, Shandil R, Sadler C, Naik M, Hosagrahara V, Hameed S, Shinde V, Bathula C, Humnabadkar V, Kumar N, Reddy J, Panduga V, Sharma S, Ambady A, Hegde N,
Whiteaker J, McLaughlin RE, Gardner H, Madhavapeddi P, Ramachandran V, Kaur P, Narayan A, Guptha S, Awasthy D, Narayan C, Mahadevaswamy J, Vishwas KG, Ahuja V,
Srivastava A, Prabhakar KR, Bharath S, Kale R, Ramaiah M, Choudhury NR, Sambandamurthy VK, Solapure S, Iyer PS, Narayanan S, Chatterji M. 2013. ‘Azaindoles: Noncovalent
DprE1 inhibitors from scaffold morphing efforts kill Mycobacterium tuberculosis and are efficacious in vivo’. Journal of Medicinal Chemistry. 56:23: 9701-9708.
23. Shirude PS, Shandil RK, Manjunatha MR, Sadler C, Panda M, Panduga V, Reddy J, Saralaya R, Nanduri R, Ambady A, Ravishankar S, Sambandamurthy VK, Humnabadkar V, Jena
LK, Suresh RS, Srivastava A, Prabhakar KR, Whiteaker J, McLaughlin RE, Sharma S, Cooper CB, Mdluli K, Butler S, Iyer PS, Narayanan S, Chatterji M. 2014. ‘Lead optimization of
1,4-azaindoles as antimycobacterial agents’. Journal of Medicinal Chemistry. 57:13: 5728-5737.
24. Chatterji M, Shandil R, Manjunatha MR, Solapure S, Ramachandran V, Kumar N, Saralaya R, Panduga V, Reddy J, Prabhakar KR, Sharma S, Sadler C, Cooper CB, Mdluli K, Iyer
PS, Narayanan S, Shirude PS. 2014. ‘1,4-Azaindole, a potential drug candidate for treatment of tuberculosis’. Antimicrobial Agents and Chemotherapy. 58:9: 5325-5331.
6

7. Methodology
 A novel series of imidazopyridine and pyrrolopyridine derivatives were designed and synthesized.
 The chemical structures of the compounds were characterized by 1H NMR, 13C NMR, IR, LC/MS, elemental
analysis and X-ray crystallography.
 The in vitro antimicrobial activities of the compounds were evaluated against various bacterial and fungal strains.
 Also, evaluated in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC 27294)
using Microplate alalmar blue assay (MABA).
 The safety profile of the compounds were accessed by testing in vitro cytotoxicity against Human Embryonic
Kidney Cell-line 293T (HEK-293T) cells at 50 µg/mL concentration by (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay.
 The structure biological activity relationship of the compounds were explained by molecular docking study with
target enzymes.
7

8. Scheme 2.1: Synthesis of imidazo[1,2-a]pyridine-2-carboxamide derivatives (5a-q)
25. Lombardino JG. 1965. ‘Preparation and new reactions of imidazo[1,2-a]pyridines’, The Journal of Organic Chemistry. 30:7: 2403-2407
Chapter-2: Synthesis, crystal structure, molecular docking and antimycobacterial evaluation
of imidazo[1,2-a]pyridine-2-carboxamide derivatives
1. Synthesis
8

9. Scheme 2.2: Synthesis of imidazo[1,2-a]pyridine-2-carboxamide derivatives (8a-k)
9

10. Structures of newly synthesized imidazo[1,2-a]pyridine-2-carboxamide derivatives (5a-q & 8a-k)
10

11. N N
N
N
HN
O
F
5a
C19H16FN5O
Mol. Wt.: 349.36
N N
N
N
HN
O
F
5a
C19H16FN5O
Mol. Wt.: 349.36
2. Characterization
The chemical structures of the compounds were characterized by 1H NMR, 13C NMR, HRMS, LC/MS and elemental analysis.
Fig. 2.1: 1H NMR & 13C NMR spectrum of compound 5a
1H NMR (400 MHz, DMSO-D6): δ 8.93 (t, J = 6.0 Hz, 1H, CO–NH–CH2), 8.81 (s, 1H, H-5), 8.27 (s, 1H, H-3), 8.15 (s, 1H, H-3"), 7.86 (s, 1H,
H-5"), 7.57 (d, J = 10.4 Hz, 2H, H-2', H-6'), 7.38-7.35 (m, 2H, Ar–H), 7.12 (t, J = 8.8 Hz, 2H, H-3', H-5'), 4.44 (d, J = 6.4 Hz, 2H, CO–NH–
CH2–Ar), 3.87 (s, 3H, N–CH3)
13C NMR (100 MHz, DMSO-D6): δ 162.3 (CO–NH), 162.1 (C-4'), 159.8 (C-9), 142.8 (C-5), 139.7 (C-3"), 136.0 (C-1'), 135.9 (C-2), 129.4 (C-
2'), 129.3 (C-6'), 128.1 (C-7), 125.8 (C-6), 121.9 (C-5"), 118.8 (C-3), 117.3 (C-8), 114.9 (C-3'), 114.7 (C-5'), 114.6 (C-4"), 41.3 (CO–NH–CH2–
Ar), 38.7 (N–CH3)
11

12. 1H NMR (400 MHz, DMSO-D6): δ 10.06 (s, 1H, CO-NH), 8.95 (s, 1H, H-5), 8.46 (s, 1H, H-3), 7.90 (d, J = 3.2 Hz, 1H, Ar-H), 7.71-7.69 (m,
3H, Ar-H), 7.64-7.62 (m, 3H, Ar-H), 7.34-7.32 (m, 2H, Ar-H), 6.99 (d, J = 6.8 Hz, 1H, Ar-H), 2.87-2.81 (m, 8H, Al-H), 2.59-2.58 (m, 1H, Al-
H), 2.36-2.33 (m, 4H, Al-H), 2.24 (s, 3H, Ar-CH3), 2.22 (s, 3H, N-CH3), 0.59-0.56 (m, 2H, Al-H), 0.34-0.31 (m, 2H, Al-H)
13C NMR (100 MHz, DMSO-D6): δ 172.3 (CO-NH), 160.3 (CO-NH), 147.2 (C-9), 143.1 (C-5), 141.4 (Ar-C), 140.1 (Ar-C), 133.7 (C-2),
131.9 (Ar-C), 129.0 (C-7), 126.5 (2C, Ar-C), 126.4 (3C, Ar-C), 126.0 (C-6), 124.4 (Ar-C), 122.7 (Ar-C), 118.8 (Ar-C), 118.4 (Ar-C), 117.2 (C-
3), 115.6 (C-8), 55.1 (2C, Al-C), 51.4 (2C, Al-C), 45.7 (N-CH3), 36.6 (Al-C), 30.6 (Al-C), 22.2 (CH-NH), 17.6 (Ar-CH3), 5.6 (2C, CH2-CH2)
Fig. 2.2: 1H NMR & 13C NMR spectrum of compound 8a
12

13. Fig. 2.3: Molecular structure of 5f [C18H15N5O2] showing the atom labeling scheme with two molecules in the
asymmetric unit and 30% probability displacement ellipsoids.
Crystal data for 5f, [C18H15N5O2] (M = 333.35 g/mol):
monoclinic, space group P21/c (no. 14), a = 31.097(6) Å, b =
9.3652(19) Å, c = 10.816(2) Å, β = 90.084(11)°, V = 3149.9(11)
Å3, Z = 4, T = 296.15 K, μ(MoKα) = 0.090 mm-1, Dcalc = 1.383
g/cm3, 35922 reflections measured (2.62° ≤ 2Θ ≤ 50°), 5523
unique (Rint = 0.1491, Rsigma = 0.1080) which were used in all
calculations. The final R1 was 0.0908 (>2sigma(I)) and wR2 was
0.2817 (all data).
CCDC 1007948 contains the supplementary crystallographic
data for 5f.
3. X-ray crystallographic analysis
Crystal structure determination of [5f]
13

14. Fig. 2.4: Packing diagram of 5f [C18H15N5O2] viewed along the b axis. Dashed lines indicate N---H…O hydrogen
bond, which link the molecules into a two-dimensional network along (010). H atoms not involved in hydrogen
bonding have been deleted for clarity. 14

15. Crystal data for 8c, C33H40N6O2 (M = 552.71 g/mol): triclinic, space group P-1 (no. 2), a = 9.4760(13) Å, b =
10.5481(13) Å, c = 15.497(2) Å, α = 78.340(8)°, β = 86.762(8)°, γ = 88.796(8)°, V = 1514.5(4) Å3, Z = 2, T =
296.15 K, μ(MoKα) = 0.080 mm-1, Dcalc = 1.214 g/cm3, 14895 reflections measured (3.94° ≤ 2Θ ≤ 50°), 4935
unique (Rint = 0.0637, Rsigma = 0.0777) which were used in all calculations. The final R1 was 0.0933 (>2sigma(I))
and wR2 was 0.2927 (all data).
Fig. 2.5: Molecular structure of 8c (C33H40N6O2) showing the atom labelling scheme and 30% probability
displacement ellipsoids
Crystal structure determination of [8c]
15

16. Fig. 2.6: Packing diagram of 8c (C33H40N6O2) viewed along the b axis. Dashed lines indicate N---H…O hydrogen
bond, which link the molecules into a two-dimensional network along (010). H atoms not involved in hydrogen
bonding have been deleted for clarity
16

17. 3. Biology
Antimycobacterial activity
•All the final compounds (5a-q & 8a-k) were screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv
(ATCC 27294) using Microplate Alamar Blue assay (MABA) [26] at PH 7.4 with drug concentrations ranging from 25 µg/mL to 0.78 µg/mL
in duplicates.
Fig. 2.7: Antimycobacterial activity of imidazo[1,2-a]pyridine derivatives (5a-q & 8a-k)
•Amongst the screened compounds, compounds 5j, 5l, 5q, 8c, 8d, 8h, 8i, 8j and 8k have displayed MIC values ≥6.25 µg/mL.
26. Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM, Gilman RH. 1998. ‘Rapid, low-technology MIC
determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay’, Journal of Clinical Microbiology. 36:2: 362-366. 17

18. Table 2.1: Cytotoxicity of compounds 5j, 5l, 5q, 8c, 8d, 8h, 8i, 8j and 8k
Compound Cytotoxicity at
50 µg/mL
5j 26.12
5l 25.56
5q 30.06
8c 28.06
8d 26.1
8h 24.8
8i 25.56
8j 22.16
8k 29.12
Cytotoxicity
The safety profile of the active compounds was also accessed by testing in vitro cytotoxicity against human embryonic kidney cell-line
293T (HEK-293T) cells at 50 µg/mL concentration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
The entire tested compounds demonstrated a good safety profile with very low toxicity towards the HEK cells indicating the suitability
of these compounds for further drug development.
18

19. 4. Molecular docking
Docking with M. tuberculosis InhA
To explain the structure-anti TB activity relationship of the active compounds (5j, 5l and 5q), the binding affinity of the compounds were
studied against the 2-trans-enoyl-ACP reductase from M. tuberculosis (M. tuberculosis InhA ), an enzyme essential for cell wall biogenesis in
M. tuberculosis [27].
Ligand Binding energy (kJ/mol) Bonding
5j -9.00
5j::DRG1:NAR:InhA:B:Met98:HN
5j::DRG1:OAM:InhA:B:Tyr158:HH
5l -8.85
5l::DRG1:HBC:InhA:B:His265:O
5l::DRG1:NAM:InhA:B:Arg225:HH11
5q -10.03
5q::DRG1:HAL:InhA:B:Gly3:O
5q::DRG1:OAM:InhA:B:Trp249:HE1
Table 2.2: Molecular docking of imidazo[1,2-a]pyridine-2-carboxamides (5j, 5l and 5q) with M. tuberculosis InhA
•The ligands 5j, 5l & 5q were formed hydrophobic interactions with different amino acid chains with an added stability of two H-bonds.
•The high affinity of ligands with in the receptor active site of M. tuberculosis InhA revealed that the in vitro antimycobacterial activity (Mtb
MIC, 6.25 µg/mL) maybe due to the inhibition of the enzyme.
Fig. 2.8: Molecular interactions between ligands (5j, 5l, and 5q) and M. tuberculosis InhA
27. Pauli I, dos Santos RN, Rostirolla DC, Martinelli LK, Ducati RG, Timmers LFSM, Basso LA, Santos DS, Guido RVC, Andricopulo AD, de Souza ON. 2013. ‘Discovery of
New Inhibitors of Mycobacterium tuberculosis InhA Enzyme Using Virtual Screening and a 3D-Pharmacophore-Based Approach’ J. Chem. Inf. Model., 53 (9) 2390–2401
19

20. Docking with M. tuberculosis glutamine synthetase
To explain the structure-anti TB activity relationship of the active compounds (8c, 8d, 8h, 8i, 8j and 8k), the binding affinity of the
compounds were studied against M. tuberculosis glutamine synthetase, a potential target to develop antimycobacterial drug [28].
Ligands Binding affinity (kJ/mol) Bond length (Å) Bonding Hydrophobic interactions
8c
-6.1
3.08 8c:O2::His237:ND1
Asn92, Thr77, Asn233, Asp240, Leu244, Arg79, Lys4, Glu16, Asp153.23 8c:N6::Asp241:OD1
2.91 His237:O::Asp241:N
8d -5.1 3.03 8d:N6::Asn208:O Asp8, Leu12, Gly210, Lys4, Ala78, Ser209, Ile80, Arg79, Glu16
8h -6.1 2.90 8h:O2::Ile80:N Ser209, Arg79, Asn208, Leu12, Ala78, Lys4, Asp8
8i -5.5 - - Ala78, Asn208, Glu16, Leu12, Lys11, Lys4, Asp8
8j -4.8 - - Asn208, Ile80, Gly210, Ser209, Arg79, Ala78, Leu12, Lys4, Asp8
8k -5.8 - - Arg79, Gly210, Ser209, Ala78, Leu12, Lys4, Asp8
Table 2.3: Molecular docking of imidazo[1,2-a]pyridine-2-carboxamides (8c, 8d, 8h, 8i, 8j and 8k) with M. tuberculosis glutamine
synthetase
•The ligands 8c, 8d, and 8h were established hydrophobic contacts with an added stability of one or more H-bonds with amino acid units
in the receptor active site of M. tuberculosis glutamine synthetase.
•The ligands 8i, 8j and 8k have formed only hydrophobic contacts with different amino acid residues without any H-bond.
•The high affinity of imidazo[1,2-a]pyridine-2-carboxamides (8c, 8d, 8h, 8i, 8j and 8k) within the binding pocket of M. tuberculosis
glutamine synthetase revealed that the in vitro antimycobacterial activity maybe due to the inhibition of the enzyme.
28. Ponnan P, Gupta S, Chopra M, Tandon R, Baghel AS, Gupta G, Prasad AK, Rastogi RC, Bose M, Raj HG. 2013. ‘2D-QSAR, docking studies, and in silico ADMET prediction of
polyphenolic acetates as substrates for protein acetyltransferase function of glutamine synthetase of Mycobacterium tuberculosis’, ISRN Structural Biology. 2013: 1-12.
20

21. Fig. 2.9: Ligplot images of protein-ligand (8c, 8d, 8h, 8i, 8j and 18k) interactions with M. tuberculosis glutamine synthetase
21

22. 29. Bavetsias V, Atrash B, Naud SGV, Sheldrake PW, Blagg J. 2012. ‘Pyrrolopyridineamino derivatives as mps1 inhibitors’. WO 2012/123745 A1.
Scheme 3.1: Synthesis of imidazo[4,5-c]pyridine derivatives (15a-l)
Scheme 3.1 
Chapter-3: Synthesis, crystal structure, molecular docking and antimicrobial evaluation of
imidazo[4,5-c]pyridine derivatives
1. Synthesis
22

23. Scheme 3.1: Synthesis of imidazo[4,5-c]pyridine derivatives (15a-l)
23

24. Structures of newly synthesized imidazo[4,5-c]pyridine derivatives (15a-l)
24

25. Fig. 3.1: 1H NMR & 13C NMR spectrum of compound 15a
1H NMR (400 MHz, CDCl3): δ 10.06 (s, 1H, NH), 7.91 (s, 1H, H-6), 7.39 (t, J = 2.4 Hz, 1H, Ar-H), 7.37-7.36 (m, 1H, Ar-H), 7.34 (t, J = 6.4 Hz, 1H, Ar-H), 7.30
(d, J = 1.2 Hz, 1H, Ar-H), 7.28 (d, J = 1.6 Hz, 1H, Ar-H), 7.24-7.22 (m, 1H, Ar-H), 7.15 (d, J = 7.6 Hz, 1H, Ar-H), 7.08 (d, J = 9.2 Hz, 1H, Ar-H), 7.00-6.95 (dt, J
= 8.4, 2.4 Hz, 1H, Ar-H), 4.76 (d, J = 12.0 Hz, 1H, Al-H), 4.42 (d, J = 10.4 Hz, 1H, Al-H), 4.28 (s, 2H, S-CH2), 3.95 (q, J = 9.2 Hz, 1H, Al-H), 3.69 (t, J = 10.0 Hz,
1H, Al-H), 2.89-2.83 (m, 1H, Al-H), 2.12-2.08 (m, 1H, Al-H), 1.98-1.92 (m, 1H, Al-H), 1.91-1.82 (m, 1H, Al-H), 1.66-1.60 (m, 1H, Al-H)
13C NMR (100 MHz, CDCl3): δ 164.8 (Ar-C-F), 162.4 (C-4), 150.2 (C-2), 148.8 (C-6), 138.9 (Ar-C), 138.4 (Ar-C), 138.3 (Ar-C), 134.7 (Ar-C), 131.3 (Ar-C),
130.6 (Ar-C), 129.7 (C-7a), 128.1 (Ar-C), 127.8 (C-3a), 123.4 (C-7), 121.4 (CN), 114.7 (Ar-C), 114.5 (Ar-C), 113.4 (Ar-C), 49.1 (Al-C), 47.1 (Al-C), 32.3 (S-CH2),
28.9 (Al-C), 27.9 (Al-C), 23.9 (Al-C).
2. Characterization
The chemical structures of the compounds were characterized by 1H NMR, 13C NMR, IR, HRMS, LC/MS and elemental analysis.
25

26. Crystal data for C25H22N5OF (M =427.47): monoclinic, space
group P21/a (no. 14), a = 10.5330(5) Å, b = 16.8672(7) Å, c =
12.9704(6) Å, β = 111.127(6)°, V = 2149.46(18) Å3, Z = 4, T =
173(2) K, μ(CuKα) = 0.731 mm-1, Dcalc = 1.321 g/mm3, 13948
reflections measured (8.996 ≤ 2Θ ≤ 145.056), 4227 unique
(Rint = 0.0398) which were used in all calculations. The final
R1 was 0.0739 (I > 2σ(I)) and wR2 was 0.2092 (all data)
Fig. 3.2: ORTEP drawing of 15c shows the labelling scheme and 30% probability displacement ellipsoids.
3. X-ray crystallographic analysis
Crystal structure determination of [15c]
26

27. Fig. 3.3: Molecular packing for 15c viewed along the b axis. Dashed lines indicate N---H…N hydrogen bonds linking
the molecules into chains along [010]. Hydrogen atoms not involved in H-bonding have been removed for clarity.
27

28. 4. Antimicrobial activity
All the newly synthesized compounds were evaluated in vitro for their antimicrobial activity against bacterial strains Staphylococcus aureus
(NCIM-5345), Bacillus cereus (NCIM-2155), Escherichia coli (NCIM-2931) & Pseudomonas aeruginosa (MTCC-4727) and fungal stains
Candida albicans (MTCC-183) & Saccharomyces cerevisiae (NCIM-3044) using Agar disc diffusion method [30].
30. Arthington-Skaggs BA, Motley M, Warnock DW, Morrison CJ. 2000. ‘Comparative evaluation of pasco and national committee for clinical laboratory standards M27-A broth
microdilution methods for antifungal drug susceptibility testing of yeasts’, Journal of Clinical Microbiology. 38:6: 2254-2260.
31. Swenson JM, Killgore GE, Tenover FC. 2004. ‘Antimicrobial susceptibility testing of acinetobacter spp. by NCCLS broth microdilution and disk diffusion methods’. Journal of
Clinical Microbiology. 42:11: 5102-5108.
Compound
Antibacterial activity (Zone of inhibition
in mm)
Antifungal activity (Zone
of inhibition in mm)
E.
coli
P.
aeruginosa
B. cereus S. aureus C. albicans S. cerevisiae
15a - 11 - - - -
15b - - - - - -
15c - - 6 - - 19
15d - - 13 - - -
15e - 13 - - 15 -
15f 12.5 - - - - -
15g 16 - - - - 15
15h - - - - - -
15i - - - - - -
15j - - - - - -
15k 12 - - 10 22 -
15l 9 8 - 23 - -
Streptomycin 21.4 20.9 20.2 23.8 -
Fluconazole - - - - 25.0 24.0
Table 3.1: Antimicrobial activity of compounds (15a-l)
 Five compounds (15c, 15e, 15g, 15k and 15l) from the above result were tested for antimicrobial activity (MIC) against respective
microorganisms using Broth microdilution method and their IC50 values were calculated [31].
28

29. Compound Microorganism MIC (µg/mL) IC50 (µg/mL)
15c Saccaromyces cerevisiae 384 192 ± 0.05
15e Candida albicans 404.2 206.4 ± 0.08
15g
Escherichia coli 197.6 74.48 ± 1.19
Saccaromyces cerevisiae 296.4 98.8 ± 1.03
15k Candida albicans 195.4 73.7 ± 0.06
15l Staphylococcus aureus 200 100 ± 1.03
Table 3.2. Antimicrobial activity MIC and IC50 of compounds (15c, 15e, 15g, 15k and 15l)
Compound 15g
Compound 15k
Compound 15l
Gram-negative antibacterial activity against E. coli
Antifungal activity against C. albicans
Gram-positive antibacterial activity against S. aureus
29

30. 5. Molecular docking
The structure-antimicrobial activity relationship of these derivatives was explained by molecular docking with target
enzyme Glucosamine-6-phosphate synthase (GlcN-6-P synthase), the target enzyme for the antimicrobial agents [32].
32. Milewski S. 2002. ‘Glucosamine-6-phosphate synthase-the multi-facets enzyme’, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1597:2:
173-192.
Ligand
Docking energy
(kcal/mol)
H-Bonding Bond length (Å)
15c -13.18 His77:HE2:::15c:NAC 2.13
15e -12.54 His77:HE2:::15e:NAC 2.15
15g -11.89 His77:HE2:::15g:NAC 1.98
15k -11.85
His77:HE2:::15k:NAP 1.96
Gly99:HN:::15k:NAT 1.72
15l -11.21 - -
Table 3.3: Molecular docking of pyrrolo[3,2-c]pyridine Mannich bases (15c, 15e, 15g, 15k and 15l) with GlcN-6-P synthase.
30

31. Fig. 3.4: Intermolecular interactions of ligands with amino acid residues in GlcN-6-P synthase
31
•The high affinity of ligands (15c, 15e, 15g, 15k & 15l) with in the binding pocket of Glucosamine-6-phophate
synthase may be due to the inhibition of enzyme

32. Scheme 4.1: Synthesis of pyrrolo[3,2-c]pyridine derivatives (18a-v)
29. Bavetsias V, Atrash B, Naud SGV, Sheldrake PW, Blagg J. 2012. ‘Pyrrolopyridineamino derivatives as mps1 inhibitors’. WO 2012/123745 A1.
Chapter-4: Synthesis, crystal structure, molecular docking, antimicrobial and antimycobacterial
evaluation of new pyrrolo[3,2-c]pyridine Mannich bases
1. Synthesis
32

33. Structures of newly synthesized pyrrolo[3,2-c]pyridine derivatives (18a-v)
Fig. 4.1: Structures of pyrrolo[3,2-c]pyridine derivatives (18a-v)
33

34. 2. Characterization
The chemical structures of the compounds were characterized by 1H NMR, 13C NMR, LC/MS and elemental analysis.
Fig. 4.2: 1H NMR & 13C NMR spectrum of compound 18a
N
Cl
N
H
N
N
N
Cl
N
H
N
N
1H NMR (400 MHz, DMSO-D6): δ 11.63 (s, 1H, NH), 7.84 (d, J = 5.6 Hz, 1H, H-6), 7.25 (d, J = 5.6 Hz, 1H, H-7), 3.66 (s, 2H, Ar-CH2-N), 2.72 (d, J = 7.6
Hz, 2H, Al-H), 2.56-2.53 (m, 1H, Al-H), 2.45-2.25 (m, 9H, Al-H), 1.70-1.57 (m, 4H, Al-H), 1.53-1.44 (m, 2H, Al-H), 1.28-1.17 (m, 2H, Al-H), 0.90 (d, J =
6.4 Hz, 6H, (CH3)2)
13C NMR (100 MHz, DMSO-D6): δ 142.4 (C-6), 141.1 (C-4), 140.7 (C-2), 138.1 (C-7a), 121.7 (C-3a), 106.9 (C-7), 106.2 (C-3), 53.5 (2C, Al-C), 52.6 (2C,
Al-C), 50.6 (Al-C), 48.0 (Ar-CH2-N), 38.9 (2C, Al-C), 31.7 (Ar-CH2), 31.2 (Al-C), 24.4 (2C, Al-C), 18.1 (2C, (CH3)2)
34

35. Fig. 4.3: Molecular structure of C13H15N2Cl (17a) showing the atom labeling scheme and 30% probability
displacement ellipsoids.
Crystal data for 17a, C13H15N2Cl (M = 234.72): monoclinic, space group P21/c, a = 9.9763(6) Å, b = 9.6777(6) Å, c
= 13.3002(9) Å, β = 106.459(7)°, V = 1231.47(14) Å3, Z = 4, T = 173(2) K, μ(CuKα) = 2.522 mm-1, Dcalc = 1.266
g/mm3, 7124 reflections measured (9.244 ≤ 2Θ ≤ 145.144), 2404 unique (Rint = 0.0381) which were used in all
calculations. The final R1 was 0.0420 (I > 2σ(I)) and wR2 was 0.1254 (all data).
CCDC 992274 contains the supplementary crystallographic data for C13H15N2Cl.
3. X-ray crystallographic analysis
Crystal structure determination of [17a]
35

36. Fig. 4.4: Packing diagram of C13H15N2Cl (17a) viewed along the a axis. Dashed lines indicate N---H…N hydrogen
bonds, which link the molecules into chains along [001]. H atoms not involved in hydrogen bonding have been
deleted for clarity.
36

37. Fig. 4.5: Molecular structure of C21H24N3OCl, CH3OH (18c) showing the atom labeling scheme with two molecules in
the asymmetric unit and 30% probability displacement ellipsoids.
Crystal data for 18c, C21H24N3OCl, CH3OH, (M = 401.92): triclinic, space group P-1, a = 10.1478(7) Å, b = 12.0945(8)
Å, c = 18.3244(10) Å, β = 90.766(5)°, V = 2147.1(2) Å3, Z = 4, T = 173(2) K, μ(CuKα) = 1.744 mm-1, Dcalc = 1.243
g/mm3, 14238 reflections measured (7.656 ≤ 2Θ ≤ 145.27), 8297 unique (Rint = 0.0330) which were used in all
calculations. The final R1 was 0.0578 (I > 2σ(I)) and wR2 was 0.1773 (all data).
CCDC 992275 contains the supplementary crystallographic data for C21H24N3OCl, CH3OH.
Crystal structure determination of [18c]
37

38. Fig. 4.6: Packing diagram of C21H24N3OCl, CH3OH (18c) viewed along the b axis. Dashed lines indicate O---H…N,
O---H…O, N---H…O hydrogen bonds and weak C---H...N intermolecular interactions, which link the molecules into a
two-dimensional network along (010). H atoms not involved in hydrogen bonding have been deleted for clarity. 38

39. Comp
d.
Gram-negative bacteria Gram-positive bacteria Fungus
Pseudomonasspp.
E.coli
P.aeruginosa
K.pneumoniae
S.aureus
B.flexus
C.sporogenes
S.mutans
Scopulariopsisspp.
A.tereus
18a – – – – +++ +++ +++ +++ – –
18b – – – – ++ ++ ++ ++ – –
18c – – – – ++ ++ ++ ++ – –
18d – – – – ++++ ++++ ++++ ++++ – –
18e – – – – +++++ +++++ ++++ ++++ – –
18f – – – – ++++ ++++ +++++ ++++ – –
18g – – – – ++ ++ ++ ++ – –
18h – – – – ++ ++ ++ ++ – –
18i – – – – ++ ++ ++ ++ – –
18j – – – – – – – – – –
18k – – – – ++ ++ ++ ++ – –
18l – – – – ++ ++ ++ ++ – –
18m – – – – ++ ++ ++ ++ – –
18n – – – – – – – – – –
18o – – – – – – – – – –
18p – – – – ++++ ++++ ++++ ++++ – –
18q – – – – – – – – – –
18r – – – – +++++ +++++ +++++ +++++ – –
18s – – – – +++ +++ +++ +++ – –
18t – – – – ++++ ++++ ++++ ++++ – –
18u – – – – +++ +++ +++ +++ – –
18v – – – – +++ +++ +++ +++ – –
Strept
omyci
n
+++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++ – –
Nysta
tin
– – – – – – – – +++++ +++++
Zone of inhibition in mm; +++++ = 32 mm, ++++ = 20 mm, +++ = 15 mm, ++ = 10 mm, hyphen denotes
no activity
4. Biology
Antimicrobial activity
•All the newly synthesized pyrrolo[3,2-c]pyridine Mannich bases (7a-v) were evaluated in vitro for their antibacterial
action against bacteria strains Staphylococcus aureus (NCIM 5345), Bacillus flexus (MTCC 2909), Clostridium
sporogenes (NICM 2559), Streptococcus mutans (MTCC 890), Pseudomonas spp. (NCIM 2872), Escherichia coli
(NCIM 2931), Pseudomonas aeruginosa (MTCC 4727) and Klebsiella pneumoniae (PTCC 1053) and fungal strains
Scopulariopsis sp. (MTCC 3553) and Aspergillus terreus (MTCC 1782) by Agar well diffusion method [30].
•Amoxicillin was used as antibacterial reference and nystatin was used as the antifungal reference.
30. Arthington-Skaggs BA, Motley M, Warnock DW, Morrison CJ. 2000. ‘Comparative evaluation of pasco and national committee for clinical laboratory standards M27-A broth
microdilution methods for antifungal drug susceptibility testing of yeasts’, Journal of Clinical Microbiology. 38:6: 2254-2260.
39

40. Comp
d.
Gram-negative bacteria Gram-positive bacteria Fungus
Pseudomonasspp.
E.coli
P.aeruginosa
K.pneumoniae
S.aureus
B.flexus
C.sporogenes
S.mutans
Scopulariopsisspp.
A.tereus
18a – – – – +++ +++ +++ +++ – –
18b – – – – ++ ++ ++ ++ – –
18c – – – – ++ ++ ++ ++ – –
18d – – – – ++++ ++++ ++++ ++++ – –
18e – – – – +++++ +++++ ++++ ++++ – –
18f – – – – ++++ ++++ +++++ ++++ – –
18g – – – – ++ ++ ++ ++ – –
18h – – – – ++ ++ ++ ++ – –
18i – – – – ++ ++ ++ ++ – –
18j – – – – – – – – – –
18k – – – – ++ ++ ++ ++ – –
18l – – – – ++ ++ ++ ++ – –
18m – – – – ++ ++ ++ ++ – –
18n – – – – – – – – – –
18o – – – – – – – – – –
18p – – – – ++++ ++++ ++++ ++++ – –
18q – – – – – – – – – –
18r – – – – +++++ +++++ +++++ +++++ – –
18s – – – – +++ +++ +++ +++ – –
18t – – – – ++++ ++++ ++++ ++++ – –
18u – – – – +++ +++ +++ +++ – –
18v – – – – +++ +++ +++ +++ – –
Strept
omyci
n
+++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++ – –
Nysta
tin
– – – – – – – – +++++ +++++
Zone of inhibition in mm; +++++ = 32 mm, ++++ = 20 mm, +++ = 15 mm, ++ = 10 mm, hyphen denotes
no activity
Compd.
Gram-negative bacteria Gram-positive bacteria Fungus
Pseudomonas
spp.
E. coli
P.
aeruginosa
K.
pneumoniae
S.
aureus
B. flexus
C.
sporogenes
S. mutans
Scopulariopsis
spp.
A.
tereus
18a – – – – +++ +++ +++ +++ – –
18b – – – – ++ ++ ++ ++ – –
18c – – – – ++ ++ ++ ++ – –
18d – – – – ++++ ++++ ++++ ++++ – –
18e – – – – +++++ +++++ ++++ ++++ – –
18f – – – – ++++ ++++ +++++ ++++ – –
18g – – – – ++ ++ ++ ++ – –
18h – – – – ++ ++ ++ ++ – –
18i – – – – ++ ++ ++ ++ – –
18j – – – – – – – – – –
18k – – – – ++ ++ ++ ++ – –
18l – – – – ++ ++ ++ ++ – –
18m – – – – ++ ++ ++ ++ – –
18n – – – – – – – – – –
18o – – – – – – – – – –
18p – – – – ++++ ++++ ++++ ++++ – –
18q – – – – – – – – – –
18r – – – – +++++ +++++ +++++ +++++ – –
18s – – – – +++ +++ +++ +++ – –
18t – – – – ++++ ++++ ++++ ++++ – –
18u – – – – +++ +++ +++ +++ – –
18v – – – – +++ +++ +++ +++ – –
Streptomycin +++++
++++
+
+++++ +++++ +++++ +++++ +++++ +++++ – –
Nystatin – – – – – – – – +++++ +++++
Zone of inhibition in mm; +++++ = 32 mm, ++++ = 20 mm, +++ = 15 mm, ++ = 10 mm, hyphen denotes no activity
Table 4.1: Antimicrobial activity screening of pyrrolo[3,2-c]pyridine derivatives 18a-v
The inhibition zone diameter data analysis indicated that compounds except 18j, 18n, 18o and 18q were showed only
Gram-positive antibacterial activity and no activity against Gram-negative bacteria strains and fungal strains .
40

41. Comp
d.
Gram-negative bacteria Gram-positive bacteria Fungus
Pseudomonasspp.
E.coli
P.aeruginosa
K.pneumoniae
S.aureus
B.flexus
C.sporogenes
S.mutans
Scopulariopsisspp.
A.tereus
18a – – – – +++ +++ +++ +++ – –
18b – – – – ++ ++ ++ ++ – –
18c – – – – ++ ++ ++ ++ – –
18d – – – – ++++ ++++ ++++ ++++ – –
18e – – – – +++++ +++++ ++++ ++++ – –
18f – – – – ++++ ++++ +++++ ++++ – –
18g – – – – ++ ++ ++ ++ – –
18h – – – – ++ ++ ++ ++ – –
18i – – – – ++ ++ ++ ++ – –
18j – – – – – – – – – –
18k – – – – ++ ++ ++ ++ – –
18l – – – – ++ ++ ++ ++ – –
18m – – – – ++ ++ ++ ++ – –
18n – – – – – – – – – –
18o – – – – – – – – – –
18p – – – – ++++ ++++ ++++ ++++ – –
18q – – – – – – – – – –
18r – – – – +++++ +++++ +++++ +++++ – –
18s – – – – +++ +++ +++ +++ – –
18t – – – – ++++ ++++ ++++ ++++ – –
18u – – – – +++ +++ +++ +++ – –
18v – – – – +++ +++ +++ +++ – –
Strept
omyci
n
+++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++ – –
Nysta
tin
– – – – – – – – +++++ +++++
Zone of inhibition in mm; +++++ = 32 mm, ++++ = 20 mm, +++ = 15 mm, ++ = 10 mm, hyphen denotes
no activity
Compd. R1 NR2R3
Gram-positive bacteria
S. aureus
B.
flexus
C.
sporogenes
S.
mutans
18a cyclopentylmethyl 4-isopropylpiperazin-1-yl >50 >50 >50 >50
18b phenyl piperidin-4-ol >50 >50 >50 >50
18c phenyl 3,3-dimethylpiperidin-4-ol >50 >50 >50 >50
18d cyclohexylmethyl morpholino 35 40 40 30
18e cyclohexylmethyl 4-methylpiperazin-1-yl 14 15 13 20
18f cyclohexylmethyl N-benzylethanamino 10 9 10 10
18g phenyl 4-methylpiperazin-1-yl 40 40 30 35
18h phenyl 2-(piperazin-1-yl)ethanol >50 >50 >50 >50
18i phenyl N,N-dimethylpiperidin-4-amine >50 >50 >50 >50
18j phenyl 4-(methylsulfonyl)piperazin-1-yl >50 >50 >50 >50
18k phenyl 4-(pyrrolidin-1-yl)piperidin-1-yl >50 >50 >50 >50
18l phenyl morpholino >50 >50 >50 >50
18m phenyl piperidine-4-carboxamide >50 >50 >50 >50
18n phenyl piperidine-3-carboxamide >50 >50 >50 >50
18o cyclopentylmethyl 3-bromo-N-phenylpyridin-2-amine >50 >50 >50 >50
18p cyclohexylmethyl pyrrolidin-1-yl 25 30 35 40
18q cyclohexylmethyl 4-(methylsulfonyl)piperazin-1-yl >50 >50 >50 >50
18r cyclohexylmethyl N-methyl-2-phenylethanamine 10 8 9 10
18s cyclohexylmethyl N,N-dimethylpiperidin-4-amine 30 35 35 40
18t cyclohexylmethyl piperidine-3-carboxamide 12 15 15 14
18u cyclohexylmethyl piperidine-4-carboxamide 20 25 25 20
18v cyclohexylmethyl isoindolin-2-yl 40 35 30 40
Streptomycin 2.0 1.5 1.0 0.5
The MIC values were evaluated at concentration range 5-50 µg/mL
Table 4.2: Gram-positive antibacterial activity of pyrrolopyridine Mannich
bases
N
N
H
Cl N
R1
R2
R3
Amongst the tested compounds, 18e, 18f,
18r, 18t and 18u were exerted the best
inhibitory activity against Gram-positive
antibacterial strains.
Compounds 18d, 18g, 18p, 18s and 18v
were showed moderate antibacterial activity
•All the compounds (18a-v) were evaluated
in vitro for their Gram-positive antibacterial
activity against bacteria strains S. aureus, B.
flexus, C. sporogenes and S. mutans by using
Broth microdilution [31]
•The MIC values for the synthesized
compounds were determined with drug
concentrations ranging from 50 µg/mL to 5
µg/mL.
•Streptomycin was used as an antibacterial
reference compound for comparison.
Gram-positive antibacterial activity
31. Swenson JM, Killgore GE, Tenover FC. 2004.
‘Antimicrobial susceptibility testing of acinetobacter spp. by
NCCLS broth microdilution and disk diffusion methods’.
Journal of Clinical Microbiology. 42:11: 5102-5108.
41

42. Antimycobacterial activity
•All the final compounds were screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC 27294)
using Microplate Alamar Blue assay (MABA) [26] at PH 7.4 with drug concentrations ranging from 25 µg/mL to 0.78 µg/mL in duplicates.
•Isoniazid, ethambutol, and rifampicin were used as anti-TB reference compounds for comparing the in vitro results.
•Amongst the screened compounds, three pyrrolopyridine derivatives 18r, 18t, and 18u have displayed MIC values ≥6.25 µg/mL.
•Compared to the first-line anti-TB drug ethambutol (MIC 3.125 µg/mL), compound 18r was found to be less potent with MIC 6.25 µg/mL,
compound 18t was found more potent (MIC <0.78 µg/mL) and compound 18u was found equipotent (MIC 3.125 µg/mL).
26. Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM, Gilman RH. 1998. ‘Rapid, low-technology MIC
determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay’, Journal of Clinical Microbiology. 36:2: 362-366. 42

43. 5. Molecular docking study
Docking with GlcN-6-P synthase
To explain the antibacterial activity of pyrrolopyridine mannich bases, the binding affinity of the compounds were studied against
Glucosamine-6-phosphate synthase (GlcN-6-P synthase), the target enzyme for the antibacterial agents [32].
Table 4.4: Molecular docking of pyrrolo[3,2-c]pyridine Mannich bases (18e, 18f, 18r, 18t & 18u) with GlcN-6-P synthase.
•All the ligands except 18e have formed hydrogen (H) or halogen (X) bonds with GlcN-6-P Synthase.
•The highest docking energy was found in the analogue 18e (-10.4 kcal/mol). The compound 18e showed hydrophobic interactions with 13
different amino acid residues without any H-bond.
•The moderate docking energy was found in the two ligands 18f (-10.89 kcal/mol) and 18r (-13.83 kcal/mol).
•The least docking energy was found in the analogue 18u (-24.92 kcal/mol). The ligand 18u represented 8 hydrophobic interactions with
different amino acid chains with an added stability of one H-bond and one X-bond.
•The ligand 18t represented 11 hydrophobic interactions with different amino acid chains with an added stability of one H-bond. The chlorine
atom of 18t has formed intermolecular bonds with Asp192 in a fashion that resemble H-bonds
32. Milewski S. 2002. ‘Glucosamine-6-phosphate synthase-the multi-facets enzyme’, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1597:2: 173-
192.
Ligand
Docking energy
(kcal/mol)
Bonding
Bond length
(Å)
Hydrophobic interactions
18e -10.40 - - Ser379, Glu351, Gly378, Leu238, Pro377, Tyr25, Asp192, Val376, Arg26, Pro177, Arg173, Arg383, Ser380
18f -10.89 18f:NH::::OH:Tyr25 2.86
Ser349, Val179, Leu178, Ser191, Leu194, Leu238, Asp192, Thr395, Arg26, Ser604, Pro177, Val376, Glu351,
Phe205
18r -13.83 18r:N:::OH:Tyr25 2.79 Arg26, Asp192, Glu351, Phe205, Pro377, Val376, Gly378, Leu238, Asn375, Thr395, Gly394, Ser379, Leu23
18t -23.55 18t:N:::COOH:Asp192 3.10 Ser349, Gln348, Pro177, Phe205, Arg26, Thr395, Tyr25, Val376, Leu238, Gln193, Ser604
18u
-24.92
18u:N:::OH:Tyr25 3.33
Ser349, Leu238, Val376, Gly394, Thr395, Tyr240, Glu396, Arg2618u:Cl:::NH2:Gln193
(Halogen bonding)
3.03
43

44. Fig. 4.8: Ligplot image of protein-ligand interactions with GlcN-6-P synthase
•The docking study reveals that the high affinity of pyrrolo[3,2-c]pyridine mannich bases within the binding pocket of GlcN-6-P synthase
strongly enhanced the determined activities of these derivatives as potent antibacterial agents. 44

45. Docking with Mycobacterium tuberculosis glutamine synthetase
To explain the structure-anti TB activity relationship of the compounds (18r, 18t and 18u), the binding affinity of the compounds were
studied against M. tuberculosis glutamine synthetase, a potential target to develop an antimycobacterial drug [28].
Table 4.2: Molecular docking of pyrrolo[3,2-c]pyridine Mannich bases (18r, 18t & 18u) with M. tuberculosis glutamine synthetase
Ligand
Binding affinity
(kcal/mol)
Bonding Bond length (Å) Hydrophobic interactions
18r -6.6 18r:NH:::COOH:Ala78 3.23 Arg79, Ile80, Asn208, Lys4, Pro75, Val9, Leu12, Lys11, Asp8
18t -6.2 18t:NH2CO:::NH2:Ile80 2.99 Pro75, Ala78, Lys4, Arg79, Asp8, Leu12, Asp15
18u -6.4 18u:NH:::COOH:Ala78 2.99 Lys4, Val9, Arg79, Pro75, Ser209, Asp8, Leu12, Asn208, Ile80, Glu16, Asp15
28. Ponnan P, Gupta S, Chopra M, Tandon R, Baghel AS, Gupta G, Prasad AK, Rastogi RC, Bose M, Raj HG. 2013. ‘2D-QSAR, docking studies, and in silico ADMET prediction
of polyphenolic acetates as substrates for protein acetyltransferase function of glutamine synthetase of Mycobacterium tuberculosis’, ISRN Structural Biology. 2013: 1-12.
Fig. 4.9: Ligplot images of protein-ligand (18r, 18t and 18u) interactions with M. tuberculosis glutamine synthetase
•The ligands 18r, 18t and 18u were represented , hydrophobic interactions with different amino acid chains with an added stability of one H-bond
45

46. Fig. 4.10: 3D representation of the interaction of ligands (18r, 18t & 18u) with M. tuberculosis glutamine synthetase by using educational
version of PyMol. The ligands were represented in green colour, H-bonds with their respective distances were represented with cyan colour,
and the interacting residues were represented in ball and stick model representation.
The high affinity of pyrrolo[3,2-c]pyridine mannich bases within the binding pocket of M. tuberculosis glutamine synthetase revealed
that the in vitro antimycobacterial activity maybe due to the inhibition of the enzyme. 46

47. Chapter 5: Summary of findings
Table 5.1: New imidazopyridine based anti-tubercular agents
5j, Mtb, MIC: 6.25 µg/mL 5l, Mtb, MIC: 6.25 µg/mL 5q, Mtb, MIC: 6.25 µg/mL
8c, Mtb, MIC: 6.25 µg/mL 8d, Mtb, MIC: 6.25 µg/mL 8h, Mtb, MIC: 6.25 µg/mL
8i, Mtb, MIC: 6.25 µg/mL 8j, Mtb, MIC: 6.25 µg/mL 8k, Mtb, MIC: 3.125 µg/mL
47

48. Table 5.2: New imidazopyridine based antimicrobial agents
15c 15e 15g
15k 15l
18e 18f 18r Mtb, MIC: 6.25 µg/mL
18t Mtb, MIC: <0.78 µg/mL 18u Mtb, MIC: 3.125 µg/mL
Table 5.3: New pyrrolopyridine based antibacterial and antimycobacterial agents
48

49.  Efficiently synthesized a novel series of imidazo[4,5-c]pyridines, imidazo[1,2-a]pyridines
and pyrrolo[3,2-c]pyridines with good anti-tuberculosis, antibacterial and antifungal
activity.
 I believe that these identified hit compounds warrants further optimization and has the
potential to deliver a candidate drug for TB and antimicrobial infections.
49

50. List of publications
1. Gilish Jose, T.H. Suresha Kumara, Gopalpur Nagendrappa, H.B.V. Sowmya, Dharmarajan Sriram, Perumal
Yogeeswari, Jonnalagadda Padma Sridevi, Tayur N. Guru Row, Amar A. Hosamani, P.S. Sujan Ganapathy, N.
Chandrika, L.V. Narendra, ‘Synthesis, molecular docking and anti-mycobacterial evaluation of new imidazo[1,2-
a]pyridine-2-carboxamide derivatives’, European Journal of Medicinal Chemistry 89 (2015) 616–627 [Impact Factor:
3.902, ISSN: 0223-5234]
2. Gilish Jose, T.H. Suresha Kumara, Gopalpur Nagendrappa, H.B.V. Sowmya, Jerry P. Jasinski, Sean P. Millikan, Sunil S.
More, Bhavya Janardhan, B.G. Harish, N. Chandrika, ‘Synthesis, crystal structure, molecular docking and antimicrobial
evaluation of new pyrrolo[3,2-c]pyridine derivatives’, Journal of Molecular Structure 1081 (2015) 85–95 [Impact
Factor: 1.78, ISSN: 0022-286].
3. Gilish Jose, T.H. Suresha Kumara, Gopalpur Nagendrappa, H.B.V. Sowmya, Jerry P. Jasinski, Sean P. Millikan, N.
Chandrika, Sunil S. More, B.G. Harish, ‘New polyfunctional imidazo[4,5-c]pyridine motifs: Synthesis, crystal studies,
docking studies and antimicrobial evaluation’, European Journal of Medicinal Chemistry 77 (2014) 288–297 [Impact
Factor: 3.902, ISSN: 0223-5234]
50

51. Conferences and Presentations
1. ‘Synthesis, crystal structure, molecular docking, antimicrobial and antimycobacterial evaluation of new pyrrolo[3,2-
c]pyridine mannich bases’ in International conference on Science and Technology: Future Challenges and
Solutions (STFCS-2016) at University of Mysore, Mysore on August 8-9, 2016.
2. ‘Synthesis, crystal structure, molecular docking and anti-mycobacterial evaluation of new imidazo[1,2-a]pyridine-2-
carboxamide derivatives’, in UGC sponsored National conference on Recent Trends in Medicinal Chemistry at
Jyothi Nivas College, Bangalore on September 3-4, 2014 (Awarded best poster presentation).
3. ‘Microwave-assisted synthesis of imidazo[4,5-c]pyridines mediated by propylphosphonic anhydride’, in National
conference on Frontiers and Challenges in Biological Organometallic Compounds (FCBOM-2013) at M.S.
Ramaiah Institute of Technology, Bangalore on June 20-21, 2013.
4. ‘Versatile synthesis of novel 4-chloro-1H-pyrrolo[3,2-c]pyridine derivatives’, in National conference on Frontiers
and Challenges in Biological Organometallic Compounds (FCBOM-2013) at M.S. Ramaiah Institute of Technology,
Bangalore on June 20-21, 2013 (Awarded best poster presentation).
51

52. Acknowledgement
I would like to express my deep gratitude to Dr. Suresha Kumara TH, my research guide, for his encouragement and support throughout
my research.
My grateful thanks are also extended to Dr. Sandeep Shastri (Honorable Pro-Vice Chancellor, Jain University), Dr. Mythili P. Rao (Dean
- Languages & Research Coordinator, Jain University) and Prof. G. Nagendrappa (Department of Chemistry, Jain University).
I convey my sincere thanks to Dr. Jerry P. Jasinski (Department of Chemistry, Keene state College, Keene, USA) and Dr. T.N. Guru Row
(Solid State and Structural Chemistry Unit, IISc, Bangalore, for providing X-ray crystallographic data.
It is my pleasant duty to express my grateful thanks to Dr. D. Sriram (Department of Pharmacy, BITS-Pilani, Hyderabad) for providing
antimycobacterial activity evaluation.
I would like to express my sincere thanks to Dr. Sunil S. More and Dr. P.S. Sujan Ganapathy (Jain University, Bangalore) Dr. B.G.
Harish (VTU PG Center, VTU, Bangalore) for their support in getting docking studies and biological studies.
It is my duty to express sincere thanks to the NMR Research Center and the Inorganic and Physical Chemistry department, Indian Institute
of Science, Bangalore for providing the spectral data.
Finally, I wish like to thank my colleagues and my family for their support throughout my study.
Thank you….