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A Dissertation for the Degree of Doctor of Philosophy in Pharmacy

Synthesis and structure-activity

relationship (SAR) studies of novel side

chain analogues of Q203 as

antitubercular agents

결핵 후보물질 개발을 위한 새로운 Q203

유도체 합성 및 구조-활성 상관관계 연구

August 2017

Graduate School of Pharmacy

Seoul National University

Pharmaceutical chemistry Major

Sunhee Kang

i

Abstract

Synthesis and structure-activity relationship (SAR) studies of

novel side chain analogues of Q203 as antitubercular agents

A clinical unmet need to respond to spread of multi-drug resistance (MDR) and

extensively drug resistance (XDR) is rapidly increasing and novel drug which has

novel mode of action (MOA) is urgently needed. Q203 is new TB drug candidate

in clinical trials having imidazo[1,2-a]pyridine-3-carboxamide (IPA) scaffold. In

this study, a set of new analogues of Q203 which were varied side chain region was

synthesized and evaluated their anti-tubercular activities. According to our

structure-activity relationship studies, many analogues showed potent anti-

tubercular activities against H37Rv-GFP replicating liquid broth culture medium as

well as within the macrophage in spite of conformational changes. On the other

hand, reduced lipophilicity of the side chain region led to decreased anti-tubercular

activity. The small set of compounds, 8, 16, 21 and 42 exibited excellent in vivo

pharmacokinetic values with high drug exposure level after oral administration.

The representative compounds, 21 and 42 reduce significant bacterial burden in the

lung and spleen in the infected mouse model, which suggests that they are

promising clinical candidates as new anti-tubercular agent.

Key word: Tuberculosis, multi-drug resistance (MDR), extensively drug resistance

(XDR), Q203, imidazo[1,2-a]pyridine-3-carboxamide (IPA), structure-

activity relationship (SAR)

Student Number: 2011-30497

ii

Table of Contents

I. Introduction ---------------------------------- 1

II. Results and Discussion ---------------------------------- 3

III. Conclusions ---------------------------------- 20

IV. Experimental ---------------------------------- 21

V. References ---------------------------------- 51

VI. Acknowledgements ---------------------------------- 56

Appendix I ---------------------------------- 57

Appendix II ---------------------------------- 66

Abstract in Korean ---------------------------------- 68

1

I. Introduction

Tuberculosis (TB) is an infectious disease that threat global health with high

motality rate. According to the latest estimates, 9.6 million new TB cases were

reported and 1.5 million people died in 2014.1 In addition, high prevalence of

multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB and co-

infection with human immunodeficiency virus (HIV) make treatment difficult and

mortality rate high. The effective regimen involving a combination of drugs,

isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) has

being applied for drug susceptible TB patients but the cure rates of MDR/XDR TB

patients are unacceptably low.2-3 Encouraged two drugs, bedaquiline and delamanid

were recently approved for the treatment of drug resistant TB patients by the

United States Food and Drug Administration and European Medicines Agency

respectively (Figure 1).4-7 However, because of the continuing emergence of drug-

resistant TB and the lack of new chemical entities in clinical trials, development of

additional drug candidates are clearly needed.

Q203 (Figure 1), which belong to imidazo[1,2-a]pyridine-3-carboxamide (IPA)

scaffold, was developed by our group and it is in currently clinical trials.8-10 It has

novel mode of action which blocks ATP production within M. tuberculosis by

targeting QcrB, which encodes the b subunit of the electron transport compiles

2

ubiquinol-cytochrome c reductase.8,11 According to our previous SAR study of

Q203, the imidazo[1,2-a]pyridine-3-carboxamides are critical and the linearly

extended lipophilic side chain led to unprecedented anti-tubercular activity.9 In this

study, a series of new IPA analogues, which was varied chain lengths and

flexibility of side chain were designed and synthesized and the study suggests

promising new clinical candidates that have comparable potency as well as in vivo

pharmacokinetic properties and effecacy.

Figure 1. Structure of bedaquiline, delamanid and Q203

3

II. Results and Discussion

II-1. Chemistry

The synthetic procedures for the preparation of IPA analogues are described in

Scheme 1-4. The appropriately substituted imidazo [1,2-a]pyridine-3-carboxylic

acids (47a-e) were prepared from unsubstituted -keto ester, 43 (Scheme 1).

Commercially available ethyl-3-oxopentanoate (43) was treated with N-

bromosuccinimide (NBS) and over 2 equivalent of ammonium acetate in

diethylether to obtain 2-brominated product (44).9, 12 Compound 44 was condensed

with adequate aminopyridines, 45a-e in ethanol at reflux temperature9, 13 and the

resulting intermediate ester 46a-e were saponified using lithium hydroxide14 to

give imidazo [1,2-a]pyridine carboxylic acid, 47a-e.

Scheme 1. General synthetic scheme of 2-ethylimidazo[1,2-a]pyridine-3-carboxylic

acidsa

aReagents and conditions: (i) NBS, NH4OAc (over 2.0 eq.), Et2O, 6h; (ii) 44, EtOH,

reflux, overnight; (iii) LiOH, EtOH/H2O (3:1, v/v), overnight

4

The synthetic route for the benzylamine counterpars is shown in Scheme 2-3. The

benzylamines that possess various linkers between two aryl rings were prepared as

shown in Scheme 2. The benzyloxy analogues, 48a and 48b, were synthesized

from 4-hydroxybenzonitrile and adequate benzylbromide via SN2 reaction using

sodium hydride. The methylamine analogue 50 was synthesized by reductive

amination with 4-aminobenzonitrile and 4-trifluoromethoxybenzaldehyde using

sodium triacetoxyborohydride. Compounds 52a and 52b, which have an oxygen or

a nitrogen atom between two phenyl rings, were prepared from 4-

fluorobenzonitrile and adequate aniline or phenol by heating in the presence of

potassium carbonate. The N-methyl analogue 54 was obtained by methylation from

52b using sodium hydride. For the preparation of benzylamines which possess a

cyclic amine between two aryl moieties, a mixture of 4-fluorobenzonitrile and

adequate cyclic amine was heated with potassium carbonate to obtain nitrile

intermediate 56a-d. Compounds 58a-d and 60a-b, which possess ether linker next

to cyclic amine were synthesized via Mitsunobu reaction from 56a or 56c with

adequate phenol in the presence of DIAD and PPh3. Piperazine containing

analogue 63 was synthesized from 56d through deprotection of tert-buthyloxy

carbonyl group and then reductive amination with 4-fluorobenzaldehyde using

sodium triacetoxyborohydride was followed. Most benzonitrile intermediated were

converted to benzylamines, 49a-b, 51, 53a-b, 55, 57, 59a-d, 61a-b and 64, using

5

lithium aluminum hydride (LAH) in thetrahydrofuran14, whereas compound 65 was

synthesized by reduction using Raney-Ni at H2 atmosphere.

The benzylamines containing simple fused ring were obtained from commercial

source or synthesized from commercially available benzonitrile by reduction using

lithium aluminum hydride (LAH) and 2-substituted benzo[d]oxazole containing

benzylamines were prepared as shown in Scheme 3. Cyclohexanecarboxylic acid

66 was activated by oxalyl chloride then coupling reaction with 3-amino-4-

hydroxybenzonitrile 69 was accomplished. The resulting amide intermediate was

condensed in the presence of pyridinium p-toluene sulfonate at reflux temperature

to give compound 67.15 In the similar way, the compounds 75a and 75b was

synthesized from 73a and 73b respectively via activation using thionyl chloride,

amide coulpling with 69 and condensation. On the other hand, the compound 71

which possess methyl morpholine moiety at 2-position was introduced

chloromethyl group from 69 with 2-chloro-1,1,1-trimethoxyethane by heating then,

morpholine was employed by SN2 reaction. The synthesized benzonitrile

intermediate 68 and 72 was subsequently reduced to benzylamines through mild

nickel borohydride recution16 but 75a and 75b was converted to benzylamines

using lithium aluminum hydride.

6

Scheme 2. Synthesis of benzylamine counterparts for IPAs listed in Table 1a

aReagents and conditions: (i) NaH, DMF, 0°C - room temperature, 4 h; (ii) LAH, T

HF, 0°C – reflux, 2 h; (iii) NaBH(OAc)3, MC, overnight; (iv) adequate amine, aniline or

phenol, K2CO3, DMF or DMSO, 120–150°C, 6 h; (v) NaH, iodomethane, 0°C - room

temperature, 2h; (vi) corresponding phenol, DIAD, PPh3, MC, overnight; (vii) TFA,

MC, rt, 4 h; (viii) 4-trifluoromethoxybenzaldedyde, NaBH(OAc)3, MC, overnight; (viiii)

Raney-Ni, H2, MeOH, 2 h

7

Scheme 3. Synthetic scheme of 2-substituted benzo[d]oxazole-5-yl-methanaminea

aReagents and conditions: (i) oxalyl chloride, CHCl3, rt, 1 h; (ii) 69, 1,4-dioxane, reflux,

overnight; (iii) pyridinium p-toluene sulfonate, xylene, reflux, overnight; (iv) NaBH4, NiCl2,

EtOH, 0°C, rt, 1 h; (v) 2-chloro-1,1,1-trimethoxyethane, EtOH, reflux, 5 h; (vi) morpholine,

DMF, rt, overnight; (vii) thionyl chloride, 69, MC, rt, 1 h; (viii) LAH, THF, reflux, 3 h

The synthetic route for target IPA analogues (4-42) is depicted in Scheme 4. For

the preparation of compounds 4-22 and 25-42, EDC mediated amide coupling was

accomplished with precosor acids and corresponding amines in the final step.

However, in the case of carbonyl analogues, 23 and 24, amide coupling was

conducted with 47c and 65 then, the deprotection of the tert-butyloxy carbonyl

group and reaction with corresponding acyl chloride was followed.

8

Scheme 4. Synthesis of target IPA derivatives (4-42)a

aReagents and conditions: (i) corresponding amine, EDC, HOBt, TEA, DMF, 80°C,

2–4 h; (ii) TFA, MC, 6 h; (iii) corresponding acyl chloride, TEA, MC, 1.5 h

II-2. Structure-activity relationship (SAR)

The structure-activity relationship (SAR) was investigated in two types of

analogues that have i) various linker between two aryl rings and ii) fused ring in

the side chain region. All synthesized compounds were evaluated for their anti-

tubercular activity against H37Rv-GFP replicating liquid broth culture medium

(extracellular MIC80) and results are summarized in Table 1. Our initial study was

focused on the analogues that have various linkers, such as ether, methyl ether,

amine, methyl amine, cyclic amine containing ethers and carbolyl linker between

two aryl moieties (Table 1). Compounds 4-9 have a methyl ether linker (-OCH2-);

thus, they are conformationally more flexible than Q203. First, we introduced

various halogen atoms at 6 or 7 as R1 because a substitution at 5 or 8 was less

9

potent (data not shown). The results showed that the small highly electronegative

fluorine group (5) was approximately 3-fold less potent and the sterically more

hindered bromo group (6) led to more seriously decreased activity compared to

compound 4 (extracellular MIC80 = 84 nM and 432 nM, respectively). However,

the chlorine analogues showed increased potency over a hydrogen atom (7 and 8,

extracellular MIC80 = 15 nM for both) whether it was placed at posion 6 or 7.

Overall, all compounds that have halogen substituents on R1 showed good potency

except for the 6-bromo compound. It suggested that the steric factor seems to be

more important than the electronic factor. In terms of changing R3 group from a

trifluoromethoxy group to a fluorine group had no influence on activity (9,

extracellular MIC80 = 20 nM). On the other hand, compound 10, which has an alkyl

amine linker (-NHCH2-), exhibited approximately 16-fold decreased potency

compared to compound 8 in spite of similar linker length and conformation.

Compound 11-13 have single atom (such as –O-, -NH- and –NMe-) between two

aryl moieties; thus comparably short side chain. Compound 11 having oxygen liker

instead of methyl ether showed a 4-fold decreased potency (extracellular MIC80 =

64 nM) compared to compound 8. In addition, the presence of a hydrogen bond

donor led to significant reduction in potency (12, extracellular MIC80 = 839 nM).

Interestingly, when a hydrogen atom on the nitrogen was replaced with a methyl

group, the activity strikingly recovered to 40 nM (13). Then, SARs were explored

10

for a set of analogues which have more extended linker length by the cyclic amine

moiety (14-24). The cyclic amine was varied to piperidine and piperazine and

flexible and rotatable linkers were introduced between the second cyclic amine and

the last phenyl ring. The analogues that have ether linker next to the pipeidine ring

displayed excellent potency with MIC80 values of 3-26 nM, regardless of the R3

substituent at the end (14-18). Also, substitution on R1 did not give a big difference

in potency (18, extracellular MIC80 = 21 nM) compared to compound 17. Potency

was retained by analogue 19, which possesses an alkyl linker next to piperiding

ring (extracellular MIC80 = 22 nM) compared to the compounds which have an

ether bridge. Compounds 20-21 have flexibly extended linker between piperidine

ring and last phenyl ring. Compound 20 showed tolerable potency but the

compound 21 displayed superior potency (extracellular MIC80 = 37 and less than 1

nM, repectively), despite their difference in side chain conformation compared to a

set of analogues 14-18. A series of analogues containing a piperazine in the middle

of the side chain was synthesized to give more polarity (22-24). Compound 22,

which has a benzyl group next to the piperazine, displayed good potency with a

MIC80 values of 15 nM whereas, the carbonyl containing analogues, 23 and 24,

showed over a 10-fold reduction in potency compared to compound 20 and 22,

despite having similar chain lengths. These results suggest that a rigid linker would

11

restrict side chain reorientation for target binding, and that, more importantly, the

decreased lipophilicity in the linker region may adversely affect activity.

Next, structure-activity relationships (SARs) were studied for a set of compounds

that have a fused ring moiety as the side chain and their anti-tubercular activity was

summarized in Table 2. All of them were simpler compared to Q203 that had an

extended lipophilic side chain. The study began with naphthyl group as R2 to

examine a simple hydrophobic side chain effect (25 and 26). They showed the

desired anti-tubercular activity without extended side chain (extracellular MIC80 =

31 and 125 nM, respectively). With a promising result, we introduced small fused

ring as a side chain to find compounds that have potent antitubercular activity (27-

34). The naphthyl group was replaced with indol which have hydrogen bonding

donor, activity was sharply decreased (27, extracellular MIC80 = 2220 nM).

However, a hydrogen atom was replaced with methyl group (28), the activity was

increased again (extracellular MIC80 = 500 nM). Especially the regio-isomer

compound 29 displayed the same potent anti-tubercular activity as compound 25. It

suggests that the position of nitrogen is more favorable when placed in the para

direction from the benzylic linker, as shown in Q203. In addition,

benzo[d][1,3]dioxole analogue 30 showed also favorable anti-tubercular activity

with MIC80 values of 82 nM. On the other hand, more polar fused ring was

introduced in the side chain such as benzo[b][1,4]oxazin-3-one and benzimidazole,

12

they lost anti-tubercular activity. In the case of 2-methyl benzo[d]oxazole

analogues 33 and 34, they displayed approximately 6- and 12-fold reduced potency

compared to compounds 25 and 26. Although several analogues were obtained that

showed good anti-tubercular activity without extended lipophilic side chain, but

they were hardly metabolized in human and rat liver microsomes (t1/2 < 10 min).

However, benzo[d]oxazole analogue 33 and 34 exibited improved metabolic

stability in human (t1/2 > 30 and 120 min) despite of comparably low anti-tubercular

activity (extracellular MIC80 = 203 and 1530 nM, respectively). We postulated that

they could be a good starting point to make derivatives for improvement of anti-

tubercular activity and metabolic stability. We therefore designed and synthesized

an additional set of benzo[d]oxazole containing analogues which introduced

cyclohexyl, methyl morpholine and phenyl at 2-position of benzo[d]oxazole

instead of methyl. Because the extended side chain conferred improvement of

potency in our previous studies. Compounds 35 and 36 having cyclohexyl group at

2-position of benxo[d]oxazole showed improved anti-tubercular activity

(extracellular MIC80 = 82 nM for both). Whereas, introducing of polar methyl

morpholine led to strikingly decreased potency (compound 37 and 38, extracellular

MIC80 = 2670 and 13700 nM, respectively). Notably, analogues 39-40, which have

lipophilic phenyl ring at 2-position of benzo[d]oxazole displayed superior anti-

tubercular activity with MIC 80 values of 9-27 nM.

13

Table 1. Extracellular activity of IPA analogues against M. tuberculosis H37Rva,b

aExtracellular MIC80 = inhibitory activity against M. tuberculosis H37Rv replicating in

liquid broth culture medium. MIC80 is the minimum concentration required to inhibit

growth by 80% and indicates an average value of two independent measurements. bAnti-

tubercular activity of Q203 were adapted from ref 9.

14

Table 2. Extracellular activity of fused ring IPA analogues against M. tuberculosis

H37Rva,b

aExtracellular MIC80 = inhibitory activity against M. tuberculosis H37Rv replicating in

liquid broth culture medium. MIC80 is the minimum concentration required to inhibit

growth by 80% and indicates an average value of two independent measurements. bAnti-

tubercular activity of Q203 were adapted from ref 9.

15

II-3. Anti-tubercular activity against M. tuberculosis inside macrophage

(Intracellular activity)

The in vitro activity against M. tuberculosis replicating inside macrophages8,17-18

(intracellular MIC80) was also investigated for a set of compounds that displayed

good extracellular potency (Table 3). The assay has several advantages, (i)

evaluation of anti-tubercular activity in the physiologically relevant condition, (ii)

selection of non-cytotoxic compounds during the assay, (iii) exclusion of poor

substrates for macrophage-induced efflux mechanism or compounds which have

low cell-permeability.8, 19 Compounds 5, 8, 11, 13, 15-16 and 21-22 selected from

series of analogues which have flexible and ratatable linkers, displayed good

intracellular activity as well. In particular, compounds 8, 15-16 and 21 showed

excellent potency with a single nano molar range (intracellular MIC80 = less than 1

nM-9 nM). However, compound 22 exibited quite decreased intracellular potency

(intracellular MIC80 = 82 nM) despite having same extracellular activity as

compound 8. It seems that decreased lipophilicity by piperazine group might have

influenced cell permeability in the macrophage infection assay system. Compounds

33, 35-36 and 39-42 were selected from fused ring analogues for evaluation of

intracellular activity. Among them, compounds 39, 41 and 42 which were

introduced phenyl group at 2-position of banzo[d]oxazole, displayed excellent

extracellular activity as well as intracellular activity (less than 1 nM to 9 nM).

16

Table 3. Anti-tubercular activity against M. tuberculosis H37Rv-GFP replicating

inside macrophages (Intracellular activity)a,b,c

Antimycobacterial activity against

H37Rv-GFP

Microsomal stability

t1/2, min

Compd Extracellular

MIC80 (nM) a

Intracellular

MIC80 (nM) b Human Rat

5 84 28 - -

8 15 <1 >120 46.9

11 64 27 - -

13 40 23 - -

15 3 <1 24.7 53.5

16 26 9 42.5 >120

21 <1 <1 78.0 61.5

22 15 82 - -

33 203 82 - -

35 82 82 - -

36 82 82 - -

39 9 3 25.6 54.3

40 82 741 - -

41 27 9 >120 >120c

42 9 <1 >120 >120c

Q203c 4 1.43

INH 490 200

aIntracellular MIC80 = inhibitory activity against M. tuberculosis H37Rv replicating inside

macrophage. MIC80 is the minimum concentration required to inhibit growth by 80% and

indicates an average value of two independent measurements. bAnti-tubercular activity of

Q203 were adapted from ref 9.

17

II-4. In vivo pharmacokinetics (PK) evaluation

Fitst, microsomal stability of compounds 8, 15-21, 39, 41 and 42 was investigated

in human and rat microsomes to select candidates for in vivo PK and the results are

summarized in Table 3. Most of compounds displayed good microsomal stability

(t1/2 > 30 min) in human and rat microsomes. However, compounds 15 and 39

showed comparably low stability in human liver microsome.

Based on the promising antitubercular activity and microsomal stability, the in

vivo pharmacokinetic properties of compounds 8, 16, 21 and 42 were evaluated in

mice after intravenous (i.v.) and oral (p.o.) administration of 2 and 10 mg/kg,

respectively. As shown in Table 4, all compounds exhibited long half-life (t1/2) and

low systemic clearance after intravenous administration. After oral administration,

all compounds reached a maximum concentration in plasma within 2 h and showed

high drug exposure level in the plasma (AUC0-inf, 11500, 48900, 49700 and 116400

ng.h/mL, respectively) with long half-life (t1/2, 11.9, 20.0, 9.83 and 36.3 h,

respectively). Overall, they achieved comparably superior bioavailability (53.4,

65.6, 66.3 and 40.1 %) as Q203. Especially, 40% bioavailability of 42 was

arithmetically lower than 90% of Q203 but it doesn’t actually mean lower

bioavailability than Q203 because it showed enoughly good drug exposure level at

24h with much higher concentration than EC50 values (Appendix, Fig. A1).

18

Table 4. Pharmacokinetic values of 8, 16, 21 and 42a

aThe PK values for compound Q203 were taken from reference 9 and presented for

comparison.

II-5. In vivo efficacy in chronic mouse model

On the basis of the promising in vivo PK properties, in vivo efficacy was

evaluated in an established mice model8-9, 20 for the representative compounds 21

and 42 which were selected from flexible linker and fused ring analogues

respectively. The study involved 6-8-wk-old BALB/c mice infected with 2 × 102 to

2 × 103 CFU of M. tuberculosis H37Rv via an intranasal route, with treatment

initiated after 21 days of infection. Compounds 21, 42 and reference drug (INH)

were administrated by oral gavage for 28 days, five times per week. Bacterial load

in the lung and spleen of infected mice was determined by colony forming unit

(CFU) enumeration and the results are summarized in Table 5. Compound 21

exhibited significant anti-tubercular efficacy, with a 1.42 and 2.62 log reduction of

19

the bacterial burden in the lung and spleen, respectively, of infected mice at a dose

of 50 mg/kg. On the other hand, compound 42 was less efficacious than compound

21 with a 1.04 and 0.76 log reduction of the bacterial burden in the lung and spleen

of the infected mice even at a higher dose of 100 mg/kg. The results revealed that

compound 21 was comparably potent in vivo as the reference drug, INH. In

addition we observed a reduced number of inflammatory lesions in the lung and

spleen compared with the untreated group and the INH treated group (data not

shown) and any clinical symptoms including death were not observed during the

study.

Table 5. In vivo efficacy of 21 and 42

Compd.

Dose

(mg/kg)

CFU

(Log10)/lung

CFU

(Log10)/spleen

21 50 4.77 ± 0.30 2.71 ± 0.30

42 100 5.15 ± 0.45 4.57 ± 0.40

INH 25 4.30 ± 0.22 2.66 ± 0.16

Untreated 6.19 ± 0.45 5.33 ± 0.20

20

III. Conclusion

A series of new IPA analogues which have flexible linkers between two aryl

moieties and fused rings as side chain has been synthesized. Through a set of

analogues that have flexible and rotatable linkers between two aryl rings, we

understood the role of the right hand side chain that lipophilicity is more important

than linearity of the side chain orientation. In addition, lineary extended side chain

of Q203 was replaced with a set of shorter fused ring moieties to reduce size of the

side chain and lipophilicity as well as low metabolic stability caused by simple

fused ring was overcome by introducing phenyl group at 2-position of

benxo[d]oxazole ring with activity improvement. Compounds, 8, 16, 21 and 42,

displayed potent anti-tubercular activities against H37Rv-GFP replicating not only

liquid broth culture medium but also whthin the macrophage. In the followed in

vivo studies, they all exhibited excellent in vivo PK properties with high drug

exposure levels in the plasma and good bioavailabilites. The representative

compounds 21 and 42 indicate significant bacterial burden reduction in infected

mice. Especially, compound 21 was comparably potent in vivo as the reference

drug. Based on the promising pharmacokinetic properties and efficacy results in

vivo, compound 21 could therefore be a promising candidate as anti-TB drug which

has comparable potency and in vivo properties with Q203.

21

IV. Experimental

IV-1. General.

All reactions were carried out under an argon atmosphere in oven-dried glassware

with magnetic stirring and the reaction solvents were purified by passage through a

bed of activated alumina. Purification of reaction products was carried out by flash

chromatography using silica gel 60 (Merck, 230-400 mesh). Analytical thin layer

chromatography was performed on 0.25 mm silica gel 60-F254 plates (Merck).

Visualization was accomplished with 254 nm of UV light and PMA or potassium

permanganate staining followed by heating. Melting points (mp) were measured on

an electro thermal melting point apparatus, M-565 (BÜ CHI). 1H-NMR (at 400

MHz) and 13C-NMR (at 100 MHz) spectra were reported on a Varian 400 MHz

spectrometer. 1H-NMR spectra (CDCl3 at 7.26 ppm) and 13C-NMR spectra (CDCl3

at 77.2 ppm) were recorded in ppm using solvent as an internal standard. Data are

reported as (ap = apparent, s = singlet, d= doublet, t = triplet, q = quartet, m =

multiplet, br = broad; coupling constant(s) in Hz; integration). LC/MS data were

obtained using a Waters 2695 LC and Micromass ZQ spectrometer. The purity of

all biologically tested compounds was ≥95 % by HPLC. Yields refer to purified

products and are not optimized.

22

IV-2. Experimental procedure and spectroscopic data analysis

IV-2-1. General procedure for preparation of actid counterparts, 47a-e

Procedure for the preparation of 44. To a stirred solution of ethyl 3-

oxopentanoate (43, 13.8 mmol) in Et2O (70 mL) were added ammonium acetate

(41.4 mmol) and N-bromosuccinimide (13.8 mmol) and the reaction mixture was

stirred at room temperature for 6 hours. The reaction mixture was diluted with Et2O

(20 mL) and washed with water (50 mL × 2). The organic phase was dried over

anhydrous MgSO4 and concentrated in vacuo to give ethyl 2-bromo-3-

oxopentanoate (44) as clear oil that was used for next reaction without further

purification.

General procedures for the preparation of 46a-e. To a solution of ethyl 2-

bromo-3-oxopentanoate (44, 12.9 mmol) in EtOH (25 mL) was added 2-amino-5-

chloropyridine (12.9 mmol). The mixture was stirred at reflux temperature for

overnight. After cooling, the reaction mixture was concentrated. The resulting dark

residue was dissolved in EtOAc (20 mL) and washed with water (20 mL). The

organic phase was washed with brine (20 mL), dried MgSO4 and concentrated in

vacuo. The crude residue was purified by flash column chromatography (n-hexane:

EtOAc = 4:1) to give 46c as a pale yellow solid. In a similar manner, the

compounds 46a-b and 46d-e were synthesized according to this procedure.

23

General procedure for the preparation of 47a-e. The resulting intermediate

ester (46c, 4.3 mmol) was dissolved in EtOH (30mL) and an aqueous solution of

lithium hydroxide (13.0 mmol in 10 mL of water) was added. The mixture was

stirred at room temperature for overnight. The organic solvent was evaporated and

1N HCl was added until pH was reached to 4. The residual pale solid was collected

by filtration, washed with water and dried to give 47c as a white solid.

In similar manner, 47a-b and 47d-e were synthesized according to this procedure

and 1H NMR data of carboxylic acid 47d and intermediate ester 46a-c and 46e are

as below.

Ethyl 2-ethylimidazo[1,2-a]pyridine-3-carboxylate (46a). 1H NMR (400 MHz,

CDCl3) δ 1.36 (t, J = 7.6 Hz, 3H), 1.43 (t, J = 7.2 Hz, 3H), 3.12 (q, J = 7.6 Hz, 2H),

4.43 (q, J = 7.2 Hz, 2H), 6.95 – 6.98 (m, 1H), 7.35 – 7.39 (m, 1H), 7.63 – 7.65 (m,

1H), 9.31 – 9.33 (m, 1H).

Ethyl 2-ethyl-6-fluoroimidazo[1,2-a]pyridine-3-carboxylate (46b). 1H NMR (400

MHz, CDCl3) δ 1.35 (t, J = 7.6 Hz, 3H), 1.44 (t, J = 7.6 Hz, 3H), 3.12 (q, J = 7.6

Hz, 2H), 4.44 (q, J = 7.2 Hz, 2H), 7.28 -7.31 (m, 1H), 7.58 -7.62 (m, 1H), 9.31 –

9.33 (m, 1H).

Ethyl 6-chloro-2-ethylimidazo[1,2-a]pyridine-3-carboxylate (46c). 1H NMR (400

MHz, CDCl3) δ 1.35 (t, J = 7.6 Hz, 3H), 1.44 (t, J = 7.2 Hz, 3H), 3.11 (q, J = 7.6

24

Hz, 2H), 4.44 (q, J = 7.2 Hz, 2H), 7.35 (dd, J = 9.6, 2.0 Hz, 1H), 7.58 (d, J = 9.6

Hz, 1H), 9.42 (d, J = 2.0 Hz, 1H).

Ethyl 7-chloro-2-ethylimidazo[1,2-a]pyridine-3-carboxylate (46e). 1H NMR (400

MHz, CDCl3) δ 1.34 (t, J = 7.6 Hz, 3H), 1.43 (t, J = 7.2 Hz, 3H), 3.09 (q, J = 7.6

Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 6.95 (dd, J = 7.6, 2.0 Hz, 1H), 7.62 (d, J = 2.0

Hz, 1H), 9.26 (d, J = 7.6 Hz, 1H).

Ethyl 6-bromo-2-ethylimidazo[1,2-a]pyridine-3-carboxylic acid (47d). 1H NMR

(400 MHz, DMSO-d6) δ 1.24 (t, J = 7.6 Hz, 3H), 3.02 (q, J= 7.6 Hz, 2H), 7.63-7.69

(m, 2H), 9.41 (s, 1H), 13.3 (brs, 1H).

IV-2-2. Procedure for preparation of benzylamine counterparts

General procedure for preparation of 48a-b. To a stirred solution of 4-

hydroxybenzonitrile (1.68 mmol) in DMF (5 mL) was added NaH (60% dispersion

in paraffin, 2.01 mmol) under ice-bath. After 10min, 4-fluorobenzyl bromide (2.01

mmol) was added then the resulting solution was further stirred for 4 hours at room

temperature. The mixture was quenched with water (10 mL) and extracted with

EtOAc (10 mL × 2). The organic phase was washed with brine (10 mL), dried over

MgSO4 and concentrated in vacuo to give 48a as a white solid. In a similar manner,

48b was synthesized according to procedure above.

25

General procedure for preparation of 62. To a stirred solution of 56d (4.05

mmol) in methylene chloride (10 mL) was added trifluoroacetic acid (1 mL) and

the resulting solution was stirred for 2 hours. After reaction colpletion, the residue

was concentrated to give 62 and it was used for next reaction witout further

purification.

General procedure for preparation of 50 and 63. To a stirred solution of 4-

aminobenzonitrile (4.23 mmol) and 4-(trifluoromehtoxy)benzaldehyde (4.65

mmol) in methylene chloride (10 mL) was added sodium triacetoxyborohydride

(6.34 mmol) and the resulting mixture was stirred for overnight. The mixture was

diluted with methylene chloride (10 mL), washed with saturated Na2CO3 (aq. 10

mL) and brine (10 mL), dried over MgSO4 and concentrated in vacuo. The crude

residue was purified by flash column chromatography (n-hexane:EtOAc = 7:1) to

give 50 as a pale yellow solid (85%). In a similar manner, compound 63 was

synthesized from 62 according to this procedure.

General procedure for preparation of 52a-b and 56a-d. A mixture of 4-

fluorobenzonitrile (2.48 mmol), 4-(trifluoromethoxy)phenol (2.73 mmol) and

K2CO3 (7.44 mmol) in DMF or DMSO (3 mL) was heated to 150℃ for 6 hours.

After the cooling, the mixture was poured to the water. The resulting solid was

filtered, washed with water and dried to give 52a.

In a similar manner, 52b and 56a-d were synthesized according to this procedure.

26

Procedure for preparation of 54. To a stirred solution of 52b (1.27 mmol) in

anhydrous DMF (5 mL) was added NaH (60% dispersion in paraffin, 1.91 mmol)

under ice-bath. After 20 min, iodomethane (1.91 mmol) was added and the reaction

mixture was allowed to ambient temperature. After 2 hours of stirring, the mixture

was quenched with water (5 mL) and concentrated. The resulting residue was

diluted with EtOAc (20 mL) and washed with water (15 mL) and brine (15 mL).

The organic phase was dried over MgSO4 and concentrated in vacuo. The crude

residue was purified by flash column chromatography (n-hexane:EtOAc = 2:1) to

give 54.

General procedure for preparation of 58a-d and 60a-b. To a stirred solution

of 56a (1.38 mmol), triphenylphosphine (1.94 mmol) and 4-chlorophenol (1.38

mmol) in methylene chloride (10 mL) was added diisopropyl azodicarboxylate

(1.66 mmol) slowly and the resulting mixture was stirred for overnight. The

reaction mixture was diluted with methylene chloride (10 mL) and washed with

water (10 mL) and brine (10 mL). The resulting organic phase was dried over

MgSO4 and concentrated in vacuo. The crude residue was purified by flash column

chromatography (n-hexane:EtOAc = 7:1) to give 58b as a white solid. In a similar

manner, compound 58a, 58c-f and 60a-b were synthesized according to this

procedure.

27

Procedure for preparation of 67. To a stirred solution of cyclohexylcarboxylic

acid (3.8 mmol) in CHCl3 (15 mL) was treated oxalyl chloride (11.4 mmol) and the

mixture was stirred for an hour at room temperature. The solvent was evaporated

under reduced pressure then the resulting residue was dissolved in 1,4-dioxane (20

mL). 3-Amino-4-hydroxybenzonitrile (4.2 mmol) was added and the reaction

mixture was refluxed for overnight. Again, the organic solvent was evaporated.

Xylene (20 mL) and pyridinium p-toluene sulfonate (4.2 mmol) were added and

the reaction mixture was refluxed for overnight. After reaction completion, the

mixture was diluted with EtOAc and washed with water and brine. The organic

phase was dried over MgSO4 and concentrated in vacuo. The crude residue was

washed with n-hexane to give benzonitrile intermediate 67 as white solid. 1H NMR

(400 MHz, CDCl3) δ 1.37 – 1.48 (m, 3H), 1.64 – 1.77 (m, 3H), 1.84 – 1.89 (m, 2H),

2.14 – 2.18 (m, 2H), 2.95 – 3.00 (m, 1H), 7.54 – 7.60 (m, 2H), 7.98 (d, J = 0.8 Hz,

1H); LCMS (ESI) m/z 227 [M + H]+.

Procedure for preparation of 70. A mixture of 3-amino-4-hydroxybenzonitrile

(2.98 mmol) and 2-chloro-1,1,1-trimethoxyethane (3.28 mmol) in ethanol (15 mL)

was stirred and refluxed for 5h. The reaction mixture was cooled to room

temperature then evaporated. Cold diethyl ether was poured to the crude residue

and the generating insoluble solid was filtered off. The resulting filtrate was

concentrated and the crude residue was purified by flash column chromatography

28

(n-hexane:EtOAc = 5:1 ratio) to give compound 70 (86%, white solid). 1H NMR

(400 MHz, CDCl3) δ 4.77 (s, 2H), 7.66 – 7.71 (m, 2H), 8.08 – 8.01 (m, 1H); LCMS

(ESI) m/z 193 [M + H]+.

Procedure for preparation of 71. To a stirred solution of compound 70 (1.04

mmol) in N,N-dimethylformamide (4 mL) was added morpholine (1mL) and the

reaction mixture was stirred for overnight at room temperature. The mixture was

concentrated and the crude residue was purified by flash column chromatography

(n-hexane:EtOAc = 3:2 ratio) to give compound 71 (71%, white solid). 1H NMR

(400 MHz, CDCl3) δ 2.64 – 2.67 (m, 4H), 3.76 – 3.78 (m, 4H), 3.89 (s, 2H), 7.62 –

7.67 (m, 2H), 8.04 (d, J = 0.8 Hz, 1H); LCMS (ESI) m/z 244 [M + H]+.

General procedure for preparation of 75a-b. To a stirred solution of 4-

trifluoromethoxybenzoic acid (73b, 4.4 mmol) in dry methylene chloride (5 mL)

was added thionyl chloride (5.5 mmol) slowly and the mixture was stirred for an

hour. The mixture was concentrated and the residue was dissolved in 1,4-dioxane

(10 mL). 3-Amino-4-hydroxybenzonitrile (3.7 mmol) was added and the reaction

mixture was stirred at refluxed temperature for overnight. Again, the mixture was

concentrated. Xylene (10 mL) and pyridinium p-toluene sulfonate (4.4 mmol) were

added and the reaction mixture was refluxed for overnight. After reaction

completion, the mixture was diluted with EtOAc and washed with water and brine.

The organic phase was dried over MgSO4 and concentrated in vacuo. The crude

29

residue was purified by recrystallization with diethyl ether to give compound 75b

(88%, white solid). 1H NMR (400 MHz, DMSO-d6) δ 7.61 (d, J = 8.8 Hz, 2H), 7.90

(dd, J = 8.0, 0.8 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.31 – 8.33 (m, 2H), 8.40 – 8.41

(m, 1H); LCMS (ESI) m/z 305 [M + H]+.

In a similar manner, the compound 75a was synthesized according to this

procedure.

IV-2-3. Reduction of benzonitrile

Method A (For 68 and 72): To a stirred solution of benzonitrile (0.69 mmol)

and nickel chloride hexahydrate (1.05 mmol) in ethanol (20 mL) was added sodium

borohydride (2.10 mmol) under ice-bath. The reaction was allowed to room

temperature and further stirred for 1.5 h. Water (5 mL) was added to the mixture

and stirred for 5 min. The resulting insoluble black residue was filtered off using

cellite and washed with methylene chloride. The filtrate was concentrated to give

crude product and it was used for next reaction without further purification.

Method B (For 49a-b, 51, 53a-b, 55, 57, 59a-d, 61a-b, 64 and 76a-b): To a

stirred solution of benzonitrile (0.43 mmol) in tetrahydrofuran (5 mL) was added

lithium aluminum hydride (1.30 mmol) and the mixture was refluxed for 3h. The

reaction was quenched with 1N aqueous NaOH under ice-bath and insoluble

residue was filtered off using cellite. The resulting filtrate was extracted with

30

EtOAc (2 times), washed with brine, dried over MgSO4 and concentrated in vacuo.

The crude product was used for next reaction without further purification.

Method C (For 65): To a stirred solution of benzonitrile (1.55 mmol) in methanol

(10 mL) was added a portion of Raney-Ni and the mixture was stirred for 4h under

H2 atmosphere. The reaction mixture was filtered by cellite and the filtrate was

concentrated. The crude residue was purified by flash column chromatography

(methylene chloride:MeOH = 20:1).

IV-2-4. Procedures for the preparation of target imidazo[1,2-a]pyridine-3-

carboxamides

General procedure for the preparation of 4-22, 77 and 25-42. To a stirred

solution of appropriate ethylimidazo[1,2-a]pyridine-3-carboxylic acid (47a-e, 2.83

mmol) in anhydrous DMF (10 mL) was added 1-(3-dimethylaminopropyl)-3-

ethylcarbodiimide hydrochloride (3.84 mmol), 1-hydroxybenzotriazole (1.54

mmol), triethylamine (5.12 mmol) and corresponding benzylamines (2.56 mmol) at

room temperature, then the resulting solution was heated to 70℃ with stirring.

After 2 hours, the reaction mixture was cooled to room temperature and evaporated.

Water (50 mL) was added into the crude residue, the resulting solid was collected

by filtration, the filter cake was washed with water (50 mL) and dried to afford

crude product. The resulting crude compound was purified by flash column

31

chromatography (n-hexane:EtOAc:methylene chloride = 1:1:1), then recrystallized

from EtOAc to give target compound.

Procedure for the preparaion of 23 and 24. To a stirred solution of 77 (14.6

mmol) in methylene chloride (30 mL) was added trifluoroacetic acid (10 mL) and

the resulting mixture was stirred at room temperature for 4h. The reaction mixture

was diluted with water (40 mL) and basified with aqueous 1N NaOH. Then the

mixture was extracted with EtOAc (100 mL × 3) and the combined extract was

washed with water (100 mL) and brine (100 mL). The resulting organic phase was

dried over anhydrous Na2SO4 and concentrated to give intermediate amine with

98% yield. The intermediate amine (2.48 mmol) was dissolved with

tetrahydrofuran (5 mL) and triethylamine (7.4 mmol) was added. The reaction

mixture was cooled under ice-bath and benzoyl chloride (2.98 mmol) was added

slowly. The resulting solution was allowed to room temperature and further stirred

for 30min. The mixture was diluted with water (100 mL) and extracted with EtOAc

(50 mL × 3). The resulting organic phase was washed with brine (20 mL), dried

over Na2SO4 and concentrated. The crude residue was purified by flash column

chromatography (PE:EA = 4:1 to 1:2) to give 23. In a similar manner, compound

24 was synthesized according to this procedure.

32

2-Ethyl-N-(4-((4-(trifluoromethoxy)benzyl)oxy)benzyl)imidazo[1,2-a]pyridine-3-

carboxamide (4). White solid; mp = 138.7 ℃; 1H NMR (400 MHz, CDCl3) δ 1.36

(t, J = 7.6 Hz, 3H), 2.95 (q, J = 7.6 Hz, 2H), 4.61 (d, J = 5.2 Hz, 2H), 5.03 (s, 2H),

6.15 (brt, J = 5.6 Hz, 1H), 6.86 (ddd, J = 1.2, 7.2, 7.2 Hz, 1H), 6.93 (d, J = 8.4 Hz,

2H), 7.21 (d, J = 8.0 Hz, 2H), 7.26 – 7.30 (m, 3H), 7.43 (d, J = 8.8 Hz, 2H), 7.56 (d,

J = 9.2 Hz, 1H), 9.34 (d, J = 7.2 Hz, 1H); LCMS (ESI) m/z 470 [M + H]+.

2-Ethyl-6-fluoro-N-(4-((4-(trifluoromethoxy)benzyl)oxy)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (5). White solid; mp = 157.4 ℃; 1H NMR (400 MHz,

CDCl3) δ 1.39 (t, J = 7.2 Hz, 3H), 2.96 (q, J = 7.2 Hz, 2H), 4.63 (d, J = 5.6 Hz, 2H),

5.06 (s, 2H), 6.08 (brt, J = 5.2 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 7.22 – 7.26 (m,

3H), 7.31 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.56 (dd, J = 5.2, 9.6 Hz,

1H), 9.43 – 9.45 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 13.38, 23.81, 43.21,

69.36, 115.39, 115.82, 115.98, 116.94, 117.03, 118.63, 118.88, 119.37, 121.34,

121.92, 124.42, 129.02, 129.31, 130.89, 135.76, 143.89, 149.09, 151.82, 152.48,

154.83, 158.29, 161.34; LCMS (ESI) m/z 488 [M + H]+.

6-Bomo-2-ethyl-N-(4-((4-(trifluoromethoxy)benzyl)oxy)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (6). Pale yellow solid; mp = 189.7 ℃; 1H NMR (400

MHz, CDCl3) δ 1.38 (t, J = 7.6 Hz, 3H), 2.95 (q, J = 7.6 Hz, 2H), 4.63 (d, J = 5.6

Hz, 2H), 5.05 (s, 2H), 6.08 (brt, J = 5.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 2H), 7.22 (d,

33

J = 8.4 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.38 (dd, J = 1.6, 9.2 Hz, 1H), 7.44 –

7.49 (m, 3H), 9.61 (d, J = 1.6 Hz, 1H); LCMS (ESI) m/z 548 [M + H]+.

6-Chloro-2-ethyl-N-(4-((4-(trifluoromethoxy)benzyl)oxy)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (7). White solid; mp = 168.5 ℃; 1H NMR (400 MHz,

CDCl3) δ 1.39 (t, J = 7.6 Hz, 3H), 2.96 (q, J = 7.6 Hz, 2H), 4.64 (d, J = 5.6 Hz, 2H),

5.06 (s, 2H), 6.05 (brs, 1H), 6.97 (d, J = 8.4 Hz, 2H), 7.20 - 7.33 (m, 5H), 7.46 (d, J

= 8.8 Hz, 2H), 7.54 (d, J = 9.2 Hz, 1H), 9.53 (d, J = 1.2 Hz, 1H); LCMS (ESI) m/z

504 [M + H]+.

7-Chloro-2-ethyl-N-(4-((4-(trifluoromethoxy)benzyl)oxy)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (8). 1H NMR (400 MHz, CDCl3) δ 1.39 (t, J = 7.6 Hz,

3H), 2.95 (q, J = 7.6 Hz, 2H), 4.63 (d, J = 5.6 Hz, 2H), 5.06 (s, 2H), 6.03 (brs, 1H),

6.90 (dd, J = 7.6, 2.0 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H),

7.31 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 2.0 Hz, 1H), 9.36 (d,

J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 13.52, 22.25, 42.28, 68.63,

114.27, 115.08, 115.56, 116.03, 121.46, 128.48, 129.14, 129.86, 131.79, 132.22,

137.14, 145.14, 148.21, 151.39, 157.51, 160.91; LCMS (ESI) m/z 504 [M + H]+.

7-Chloro-2-ethyl-N-(4-((4-fluorobenzyl)oxy)benzyl)imidazo[1,2-a]pyridine-3-

carboxamide (9). Pale yellow solid; mp = 181.0 ℃; 1H NMR (400 MHz, CDCl3) δ

1.38 (t, J = 7.6 Hz, 3H), 2.95 (q, J = 7.6 Hz, 2H), 4.62 (d, J = 5.6 Hz, 2H), 5.02 (s,

2H), 6.02 (brs, 1H), 6.90 (dd, J = 7.6, 2.4 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 7.07

34

(dd, J = 8.8, 8.8 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.40 (dd, J = 5.6, 8.8 Hz, 2H),

7.58 (d, J = 1.6 Hz, 1H), 9.36 (d, J = 7.6 Hz, 1H); LCMS (ESI) m/z 438 [M + H]+.

7-Chloro-2-ethyl-N-(4-((4-(trifluoromethoxy)benzyl)amino)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (10). White solid; mp = 169.6 ℃; 1H NMR (400 MHz,

CDCl3) δ 1.36 (t, J = 7.6 Hz, 3H), 2.93 (q, J = 7.6 Hz, 2H), 4.18 (brs, 1H), 4.34 (s,

2H), 4.55 (d, J = 5.2 Hz, 2H), 6.00 (brs, 1H), 6.60 (d, J = 8.4 Hz, 2H), 6.87 (d, J =

7.6 Hz, 1H), 7.17 (d, J = 8.0 Hz, 4H), 7.38 (d, J = 8.0 Hz, 2H), 7.56 (s, 1H), 9.33 (d,

J = 7.2 Hz, 1H); LCMS (ESI) m/z 503 [M + H]+.

7-Chloro-2-ethyl-N-(4-(4-(trifluoromethoxy)phenoxy)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (11). White solid; mp = 141 - 142 ℃; 1H NMR (400

MHz, CDCl3) δ 1.41 (t, J = 7.6 Hz, 3H), 2.98 (q, J = 7.6 Hz, 2H), 4.68 (d, J = 5.6

Hz, 2H), 6.10 (brs, 1H), 6.91 (dd, J = 2.0, 7.6 Hz, 1H), 6.98 – 7.02 (m, 4H), 7.18 (d,

J = 8.8 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 2.0 Hz, 1H), 9.37 (d, J = 7.2

Hz, 1H) ); LCMS (ESI) m/z 490 [M + H]+.

7-Chloro-2-ethyl-N-(4-((4-(trifluoromethoxy)phenyl)amino)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (12). Pale yellow solid; mp = 200 ℃; 1H NMR (400

MHz, CDCl3) δ 1.40 (t, J = 7.6 Hz, 3H), 2.98 (q, J = 7.6 Hz, 2H), 4.63 (d, J = 5.6

Hz, 2H), 5.73 (s, 1H), 6.05 (brs, 1H), 6.91 (dd, J = 2.0, 7.6 Hz, 1H), 7.03 - 7.07 (m,

4H), 7.12 (d, J = 9.2 Hz, 2H), 7.29 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 1.6 Hz, 1H),

9.36 (d, J = 7.6 Hz, 1H); LCMS (ESI) m/z 489 [M + H]+.

35

7-Chloro-2-ethyl-N-(4-(methyl(4-

(trifluoromethoxy)phenyl)amino)benzyl)imidazo[1,2-a]pyridine-3-carboxamide

(13). White solid; mp = 134 ℃; 1H NMR (400 MHz, DMSO-d6) δ 1.26 (t, J = 7.6

Hz, 3H), 2.98 (q, J = 7.6 Hz, 2H), 3.24 (s, 3H), 4.50 (d, J = 6.0 Hz, 2H), 6.92 (d, J

= 8.8 Hz, 2H), 7.08 - 7.10 (m, 3H), 7.19 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.8 Hz,

2H), 7.78 (s, 1H), 8.44 (brs, 1H), 8.97 (d, J = 7.2 Hz, 1H); LCMS (ESI) m/z 503

[M + H]+.

6-Chloro-2-ethyl-N-(4-(4-(4-fluorophenoxy)piperidin-1-yl)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (14). Pale yellow solid; mp = 148.4 ℃; 1H NMR (400

MHz, CDCl3) δ 1.39 (t, J = 7.6 Hz, 3H), 1.91 - 1.94 (m, 2H), 2.06 - 2.11 (m, 2H),

2.96 (q, J = 7.6 Hz, 2H), 3.08 - 3.14 (m, 2H), 3.47 - 3.54 (m, 2H), 4.37 - 4.39 (m,

1H), 4.61 (d, J = 5.6 Hz, 2H), 6.01 (brs, 1H), 6.86 - 6.89 (m, 2H), 6.95 - 7.00 (m,

4H), 7.26 - 7.30 (m, 3H), 7.53 (d, J = 8.8 Hz, 1H), 9.53 (d, J = 1.6 Hz, 1H); LCMS

(ESI) m/z 507 [M + H]+.

6-Chloro-N-(4-(4-(4-chlorophenoxy)piperidin-1-yl)benzyl)-2-ethylimidazo[1,2-

a]pyridine-3-carboxamide (15). Pale yellow solid; mp = 165.3 ℃; 1H NMR

(400MHz, DMSO-d6) δ 0.81 (t, J = 7.6 Hz, 3H), 1.19 – 1.28 (m, 2H), 1.54 – 1.57

(m, 2H), 2.53 (q, J = 7.6 Hz, 2H), 2.55 – 2.59 (m, 2H), 3.00 – 3.06 (m, 2H), 3.98 (d,

J = 6.0 Hz, 2H), 4.05 – 4.11 (m, 1H), 6.48 (d, J = 8.8 Hz, 2H), 6.53 – 6.56 (m, 2H),

6.78 (d, J = 8.4 Hz, 2H), 6.83 – 6.87 (m, 2H), 6.98 (dd, J = 9.6, 2.4 Hz, 1H), 7.20

36

(d, J = 9.6 Hz, 1H), 7.93 (brt, J = 6.0 Hz, 1H), 8.62 (d, J = 2.4 Hz, 1H); 13C NMR

(100 MHz, DMSO-d6) δ 13.51, 22.31, 30.34, 42.38, 46.48, 72.92, 116.17, 116.30,

117.60, 118.05, 120.08, 124.67, 125.24, 127.41, 128.73, 129.73, 129.79, 143.70,

150.20, 151.41, 156.32, 160.88; LCMS (ESI) m/z 523 [M + H]+.

6-Chloro-2-ethyl-N-(4-(4-(4-(trifluoromethyl)phenoxy)piperidin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (16). White solid; 1H NMR (400

MHz, CDCl3) δ 1.39 (t, J = 7.6 Hz, 3H), 1.94 - 1.99 (m, 2H), 2.10 - 2.15 (m, 2H),

2.96 (q, J = 7.6 Hz, 2H), 3.12 - 3.18 (m, 2H), 3.47 - 3.53 (m, 2H), 4.53 - 4.57 (m,

1H), 4.61 (d, J = 5.2 Hz, 2H), 6.02 (brs, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.98 (d, J =

8.4 Hz, 2H), 7.27 - 7.31 (m, 3H), 7.51 - 7.55 (m, 3H), 9.53 (d, J = 2.0 Hz, 1H); 13C

NMR (100 MHz, DMSO-d6) δ 13.49, 22.31, 30.25, 42.38, 46.45, 72.86, 116.19,

116.29, 116.42, 117.59, 120.08, 120.96, 121.28, 121.59, 121.91, 123.65, 125.24,

126.34, 127.34, 127.38, 127.42, 128.74, 129.85, 143.70, 150.17, 151.42, 160.45,

160.88; LCMS (electrospray) m/z 557 [M + H]+.

6-Chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (17). 1H NMR (400 MHz,

CDCl3) δ 1.38 (t, J = 7.6 Hz, 3H), 1.89 - 1.97 (m, 2H), 2.07 - 2.12 (m, 2H), 2.95 (q,

J = 7.6 Hz, 2H), 3.09 - 3.15 (m, 2H), 3.48 - 3.53 (m, 2H), 4.42 - 4.47 (m, 1H), 4.60

(d, J = 5.6 Hz, 2H), 6.00 (brs, 1H), 6.88 – 6.93 (m, 3H), 6.96 (d, J = 8.4 Hz, 2H),

37

7.14 (d, J = 8.8 Hz, 1H), 7.26 - 7.31 (m, 2H), 7.58 (d, J = 2.0 Hz, 1H), 9.36 (d, J =

7.6 Hz, 1H); LCMS (electrospray) m/z 573 [M + H]+.

7-Chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (18). Pale yellow solid; mp =

141.3 ℃; 1H NMR (400 MHz, CDCl3) δ 1.39 (t, J = 7.6 Hz, 3H), 1.89 -1.97 (m,

2H), 2.07 - 2.12 (m, 2H), 2.96 (q, J = 7.6 Hz, 2H), 3.09 - 3.16 (m, 2H), 3.47 - 3.53

(m, 2H), 4.42 - 4.47 (m, 1H), 4.61 (d, J = 5.6 Hz, 2H), 6.01 (brs, 1H), 6.91 (dd, J =

2.0, 6.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8Hz, 2H), 7.26 - 7.30 (m,

3H), 7.53 (d, J = 9.6 Hz, 1H), 9.53 (s, 1H); LCMS (ESI) m/z 573 [M + H]+.

N-(4-(4-benzylpiperidin-1-yl)benzyl)-7-chloro-2-ethylimidazo[1,2-a]pyridine-3-

carboxamide (19). White solid; mp = 103.4 ℃; 1H NMR (400 MHz, CDCl3) δ

1.35 (t, J = 7.6 Hz, 3H), 1.37 - 1.44 (m, 2H), 1.63 - 1.70 (m, 1H), 1.72 - 1.76 (m,

2H), 2.57 (d, J = 6.8 Hz, 2H), 2.61 - 2.67 (m, 2H), 2.92 (q, J = 7.6 Hz, 2H), 3.63 -

3.66 (m, 2H), 4.57 (d, J = 5.2 Hz, 2H), 6.08 (brs, 1H), 6.84 - 6.87 (m, 1H), 6.90 (d,

J = 8.0 Hz, 2H), 7.15 (d, J = 7.2 Hz, 2H), 7.19 - 7.30 (m, 5H), 7.55 (d, J = 1.6 Hz,

1H), 9.29 - 9.32 (m, 1H); LCMS (ESI) m/z 487 [M + H]+.

6-Chloro-2-ethyl-N-(4-(4-((4-(trifluoromethoxy)phenoxy)methyl)piperidin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (20). Pale yellow solid; mp =

183.6 ℃; 1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 1.50 - 1.54 (m,

2H), 1.93 - 1.95 (m, 3H), 2.72 - 2.78 (m, 2H), 2.94 (q, J = 7.6 Hz, 2H), 3.71 - 3.74

38

(m, 2H), 3.82 (d, J = 6.0 Hz, 2H), 4.59 (d, J = 5.6 Hz, 2H), 6.06 (brt, J = 5.6 Hz,

1H), 6.86 (d, J = 9.2 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H),

7.24 - 7.28 (m, 3H), 7.51 (d, J = 9.6 Hz, 1H), 9.50 (d, J = 1.2 Hz, 1H); LCMS

(ESI) m/z 587 [M + H]+.

7-Chloro-2-ethyl-N-(4(4-((4-fluorophenoxy)methyl)piperidin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (21). White solid; mp =

186.5 ℃; 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J = 7.2 Hz, 3H), 1.34 – 1.42

(m, 2H), 1.82 – 1.87 (m, 3H), 2.62 – 2.67 (m, 2H), 2.96 (q, J = 7.2 Hz, 2H), 3.67 –

3.70 (m, 2H), 3.83 (d, J = 5.6 Hz, 2H), 4.42 (d, J = 6.0 Hz, 2H), 6.90 – 6.95 (m,

4H), 7.06 – 7.11 (m, 3H), 7.21 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 1.6 Hz, 1H), 8.38

(brt, J = 6.0 Hz, 1H), 8.94 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ

13.55, 22.27, 28.63, 35.76, 42.39, 49.12, 72.95, 114.26, 115.55, 115.59, 116.06,

116.10, 116.14, 116.27, 116.33, 128.45, 128.48, 128.67, 129.70, 131.76, 145.13,

150.82, 151.34, 155.50, 155.52, 155.68, 158.02, 160.88; LCMS (ESI) m/z 587 [M

+ H]+.

6-Chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)benzyl)piperazin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (22). White solid; mp = 138.1 -

138.7 ℃; 1H NMR (400 MHz, CDCl3) δ 1.38 (t, J = 7.6 Hz, 3H), 2.58 - 2.61 (m,

4H), 2.95 (q, J = 7.6 Hz, 2H), 3.19 - 2.61 (m, 4H), 3.55 (s, 2H), 4.60 (d, J = 5.6 Hz,

2H), 6.00 (brs, 1H), 6.92 (d, J = 8.8 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 7.26 - 7.30

39

(m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 9.2 Hz, 1H), 9.52 (d, J = 2.0 Hz,

1H); LCMS (ESI) m/z 572 [M + H]+.

6-Chloro-2-ethyl-N-(4-(4-(2-(4-fluorophenyl)acetyl)piperazin-1-

yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide (23). White solid; 1H NMR

(300MHz, DMSO-d6) δ 1.24 (t, J = 7.5 Hz, 3H), 2.91 - 3.12 (m, 6H), 3.51 - 3.65

(m, 4H), 3.74 (s, 2H), 4.42 - 4.44 (m, 2H), 6.92 (d, J = 8.7 Hz, 2H), 7.10 (t, J = 8.8

Hz, 2H), 7.19 - 7.31 (m, 4H), 7.69 (dd, J = 9.6, 1.8 Hz, 1H), 7.78 (d, J = 9.6 Hz,

1H), 8.66 (t, J = 5.7 Hz, 1H), 9.10 (s, 1H); LCMS (ESI) m/z 534 [M + H]+.

6-chloro-2-ethyl-N-(4-(4-(4-fluorobenzoyl)piperazin-1-yl)benzyl)imidazo[1,2-

a]pyridine-3-carboxamide (24). White solid; 1H NMR (300 MHz, DMSO-d6) δ

1.25 (t, J = 7.5 Hz, 3H), 3.00 (q, J = 7.5 Hz, 2H), 3.08 - 3.28 (m, 4H), 3.31 - 3.91

(m, 4H), 4.43 (d, J = 5.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 7.20 - 7.33 (m, 4H),

7.49 (dd, J = 8.4, 5.4 Hz, 2H), 7.70 (dd, J = 9.2, 1.8 Hz, 1H), 7.80 (d, J = 9.2 Hz,

1H), 8.70 (t, J = 5.7 Hz, 1H), 9.10 (s, 1H); LCMS (ESI) m/z 520 [M + H]+.

6-Chloro-2-ethyl-N-(naphthalen-2-ylmethyl)imidazo[1,2-a]pyridine-3-

carboxamide (25). White solid; mp = 194.5 ℃; 1H NMR (400 MHz, CDCl3) δ

1.40 (t, J = 7.6 Hz, 3H), 2.98 (q, J = 7.6 Hz, 2H), 4.87 (d, J = 5.6 Hz, 2H), 6.19

(brs, 1H), 7.29 – 7.32 (m, 1H), 7.47 – 7.56 (m, 4H), 7.82 – 7.88 (m, 4H), 9.56 –

9.57 (m, 1H); 13C NMR (100 MHz, DMSO-d6) 13C NMR (100 MHz, DMSO-d6) δ

13.58, 22.38, 43.07, 116.23, 117.64, 120.14, 125.38, 125.95, 126.12, 126.37,

40

126.65, 127.56, 127.99, 128.40, 132.56, 133.35, 137.43, 143.81, 151.64, 161.10;

LCMS (ESI) m/z 364 [M + H]+; HRESIMS calcd for C21H18ClN3O [M + H]+

364.1211, found 364.1227.

7-Chloro-2-ethyl-N-(naphthalen-2-ylmethyl)imidazo[1,2-a]pyridine-3-

carboxamide (26). White solid; mp = 181.5 ℃; 1H NMR (400 MHz, MeOH-d4) δ

1.32 (t, J = 7.6 Hz, 3H), 3.02 (q, J = 7.6 Hz, 2H), 4.78 (s, 2H), 7.06 (dd, J = 6.8,

1.6 Hz, 1H), 7.44 – 7.59 (m, 4H), 7.82 – 7.87 (m, 4H), 8.96 (d, J = 7.6 Hz, 1H); 13C

NMR (100 MHz, DMSO-d6) δ 13.62, 22.34, 43.06, 114.34, 115.59, 116.03, 125.94,

126.12, 126.37, 126.65, 127.98, 128.39, 128.57, 131.88, 132.56, 133.35, 137.47,

145.25, 151.58, 161.11; LCMS (ESI) m/z 364 [M + H]+; HRESIMS calcd for

C21H18ClN3O [M + H]+ 364.1211, found 364.1237.

N-((1H-indol-6-yl)methyl)-6-chloro-2-ethylimidazo[1,2-a]pyridine-3-

carboxamide (27). Pale yellow solid; mp = 123.1 ℃; 1H NMR (400 MHz, DMSO-

d6) δ 1.26 (t, J = 7.6 Hz, 3H), 2.99 (q, J = 7.6 Hz, 2H), 4.61 (d, J = 6.0 Hz, 2H),

6.37 – 6.39 (m, 1H), 7.03 (dd, J = 8.0, 1.2 Hz, 1H), 7.29 – 7.31 (m, 1H), 7.39 (s,

1H), 7.45 (dd, J = 9.2, 2.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 9.2 Hz,

1H), 8.48 – 8.49 (m, 1H), 9.08 (d, J = 2.0 Hz, 1H), 11.03 (s, 1H); 13C NMR (100

MHz, DMSO-d6) δ 13.55, 22.31, 43.40, 101.28, 110.72, 116.36, 117.65, 119.37,

120.12, 120.27, 125.24, 125.70, 127.05, 127.44, 132.47, 136.42, 143.72, 151.42,

41

160.88; LCMS (ESI) m/z 353 [M + H]+; HRESIMS calcd for C19H17ClN4O [M +

H]+ 353.1164, found 353.1171.

6-Chloro-2-ethyl-N-((1-methyl-1H-indol-6-yl)methyl)imidazo[1,2-a]pyridine-3-

carboxamide (28). White solid; mp = 149.5 ℃; 1H NMR (400 MHz, CDCl3) δ

1.36 (t, J = 7.6 Hz, 3H), 2.95 (q, J = 7.6 Hz, 2H), 3.80 (s, 3H), 4.82 (d, J = 5.6 Hz,

2H), 6.13 (brs, 1H), 6.49 (d, J = 3.2 Hz, 1H), 7.08 (d, J = 2.8 Hz, 1H), 7.12 (dd, J =

8.0, 1.2 Hz, 1H), 7.30 (dd, J = 9.6, 2.0 Hz, 1H), 7.34 (s, 1H), 7.55 (d, J = 9.6 Hz,

1H), 7.63 (d, J = 8.0 Hz, 1H), 9.54 (d, J = 1.2 Hz, 1H); 13C NMR (100 MHz,

DMSO-d6) δ 13.62, 22.31, 32.80, 43.47, 100.56, 108.90, 116.43, 117.65, 119.41,

120.08, 120.68, 125.25, 127.50, 130.14, 132.69, 136.84, 143.69, 151.43, 160.88;

LCMS (ESI) m/z 367 [M + H]+; HRESIMS calcd for C20H19ClN4O [M + H]+

367.1320, found 367.1338.

6-Chloro-2-ethyl-N-((1-methyl-1H-indol-5-yl)methyl)imidazo[1,2-a]pyridine-3-

carboxamide (29). White solid; mp = 201.2 ℃; 1H NMR (400 MHz, CDCl3) δ

1.37 (t, J = 7.6 Hz, 3H), 2.94 (q, J = 7.6 Hz, 2H), 3.81 (s, 3H), 4.78 (d, J = 5.6 Hz,

2H), 6.07 (m, 1H), 6.48 (d, J = 3.2 Hz, 1H), 7.09 (d, J = 2.8 Hz, 1H), 7.24 – 7.26

(m, 1H), 7.29 (dd, J = 9.6, 2.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 9.6

Hz, 1H), 7.63 (s, 1H), 9.54 (d, J = 1.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ

13.55, 22.29, 32.94, 43.34, 100.59, 109.99, 116.40, 117.63, 119.68, 120.07, 121.56,

125.24, 127.41, 128.39, 130.16, 130.34, 136.06, 143.69, 151.38, 160.82; LCMS

42

(ESI) m/z 367 [M + H]+; HRESIMS calcd for C20H19ClN4O [M + H]+ 367.1320,

found 367.1333.

N-(benzo[d][1,3]dioxol-5-ylmethyl)-6-chloro-2-ethylimidazo[1,2-a]pyridine-3-

carboxamide (30). White solid; mp = 179.1 ℃; 1H NMR (400 MHz, CDCl3) δ

1.31 (t, J = 7.6 Hz, 3H), 2.90 (q, J = 7.6 Hz, 2H), 4.51 (d, J = 5.6 Hz, 2H), 5.89 (s,

2H), 6.38 (brs, 1H), 6.71 – 6.80 (m, 3H), 7.23 (dd, J = 9.6, 2.0 Hz, 1H), 7.43 (d, J =

9.6 Hz, 1H), 9.34 (d, J = 2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 13.27, 23.21,

43.50, 101.24, 108.32, 108.52, 116.63, 121.11, 121.70, 126.10, 128.48, 131.86,

144.35, 147.20, 148.12, 151.31, 161.15; LCMS (ESI) m/z 358 [M + H]+;

HRESIMS calcd C18H16ClN3O3 [M + H]+ 358.0953, found 358.0967.

6-Chloro-2-ethyl-N-((3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-

yl)methyl)imidazo[1,2-a]pyridine-3-carboxamide (31). Pale yellow solid; mp =

228.6 ℃; 1H NMR (400 MHz, DMSO-d6) δ 1.23 (t, J = 7.6 Hz, 3H), 2.30 (q, J =

7.6 Hz, 2H), 4.57 (s, 2H), 5.32 (s, 2H), 6.95 – 7.05 (m, 4H), 7.27 (dd, J = 7.6, 2.0

Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 9.20 (d, J = 7.6 Hz, 1H), 10.76 (s, 1H); 13C NMR

(100 MHz, DMSO-d6) δ 13.51, 22.29, 42.33, 67.20, 114.32, 115.27, 115.59, 115.96,

116.38, 122.45, 127.56, 128.52, 131.87, 134.15, 142.56, 145.22, 151.51, 160.98,

165.42; LCMS (ESI) m/z 385 [M + H]+; HRESIMS calcd C19H17ClN4O3 [M + H]+

385.1062, found 386.0906.

43

N-((1H-benzo[d]imidazol-6-yl)methyl)-6-chloro-2-ethylimidazo[1,2-a]pyridine-3-

carboxamide (32). Pale yellow solid; mp = 245.8 ℃; 1H NMR (400 MHz, DMSO-

d6) δ 1.25 (t, J = 7.6 Hz, 3H), 2.98 (q, J = 15.2, 7.6 Hz, 2H), 4.63 (d, J = 5.6 Hz,

2H), 7.09 (dd, J = 7.6, 2.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.54 – 7.56 (m, 2H),

7.78 (d, J = 1.6 Hz, 1H), 8.17 (s, 1H), 8.51 – 8.53 (m, 1H), 8.96 (d, J = 7.2 Hz, 1H),

12.40 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 13.58, 22.30, 43.23, 114.30,

115.58, 115.61, 116.10, 122.05, 128.48, 131.81, 133.50, 142.59, 145.17, 151.42,

160.94; LCMS (ESI) m/z 354 [M + H]+; HRESIMS calcd C18H16ClN5O [M + H]+

354.1116, found 354.1115.

6-Chloro-2-ethyl-N-((2-methylbenzo[d]oxazol-5-yl)methyl)imidazo[1,2-

a]pyridine-3-carboxamide (33). White solid; mp = 172.5 ℃; 1H NMR (400 MHz,

CDCl3) δ 1.39 (t, J = 7.6 Hz, 3H), 2.64 (s, 3H), 2.97 (q, J = 7.6 Hz, 2H), 4.78 (d, J

= 5.6 Hz, 2H), 6.15 (brs, J = 5.6 Hz, 1H), 7.31 -7.35 (m, 2H), 7.46 (d, J = 8.4 Hz,

1H), 7.55 (d, J = 9.2 Hz, 1H), 7.65 (s, 1H), 9.54 (s, 1H); 13C NMR (100 MHz,

DMSO-d6) δ 13.54, 14.59, 22.34, 42.86, 110.48, 116.19, 117.63, 118.29, 120.14,

124.55, 125.33, 127.55, 136.17, 141.70, 143.78, 149.88, 151.58, 161.02, 164.75;

LCMS (ESI) m/z 369 [M + H]+; HRESIMS calcd for C19H16ClN4O2 [M + H]+

369.1113, found 369.1124.

7-Chloro-2-ethyl-N-((2-methylbenzo[d]oxazol-5-yl)methyl)imidazo[1,2-

a]pyridine-3-carboxamide (34). White solid; mp = 245.6 ℃; 1H NMR (400

44

MHz, CDCl3) δ 1.38 (t, J = 7.6 Hz, 3H), 2.64 (s, 3H), 2.96 (q, J = 7.6 Hz, 2H), 4.77

(d, J = 6.0 Hz, 2H), 6.14 (brt, J = 6.0 Hz, 1H), 6.90 (dd, J = 7.6, 2.4 Hz, 1H), 7.31

(dd, J = 8.4, 2.0 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.65 (d,

J = 2.0 Hz, 1H), 9.36 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 13.58,

14.58, 22.31, 42.86, 110.47, 114.35, 115.59, 115.99, 118.29, 124.54, 128.54,

131.88, 136.21, 141.70, 145.23, 149.88, 151.53, 161.02, 164.75; LCMS (ESI) m/z

369 [M + H]+; HRESIMS calcd for C19H16ClN4O2 [M + H]+ 369.1113, found

369.1124.

6-Chloro-N-((2-cyclohexylbenzo[d]oxazol-5-yl)methyl)-2-ethylimidazo[1,2-

a]pyridine-3-carboxamide (35). White solid; mp = 167.5 ℃; 1H NMR (400 MHz,

CDCl3) δ 1.30 – 1.44 (m, 6H), 1.59 – 1.74 (m, 3H), 1.84 – 1.88 (m, 2H), 2.14 –

2.17 (m, 2H), 2.96 (q, J = 7.6 Hz, 3H), 4.78 (d, J = 5.6 Hz, 2H), 6.19 (brs, 1H),

7.28 (d, J = 1.6 Hz, 1H), 7.30 – 7.34 (m, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.53 (d, J =

9.2 Hz, 1H), 7.67 (s, 1H), 9.53 (d, J = 2.4 Hz, 1H); LCMS (ESI) m/z 437 [M + H]+;

13C NMR (100 MHz, CDCl3) δ 13.07, 21.15, 22.55, 25.57, 30.44, 37.93, 43.81,

110.56, 113.96, 116.47, 117.78, 119.09, 124.31, 124.67, 125.92, 126.47, 132.37,

133.93, 141.72, 150.10, 158.95, 171.14; HRESIMS calcd for C24H25ClN4O2 [M +

H]+ 436.1666, found 436.1677.

7-Chloro-N-((2-cyclohexylbenzo[d]oxazol-5-yl)methyl)-2-ethylimidazo[1,2-

a]pyridine-3-carboxamide (36). White solid; mp = 154.6 ℃; 1H NMR (400 MHz,

45

CDCl3) δ 1.25 – 1.46 (m, 6H), 1.60 – 1.73 (m, 3H), 1.84 – 1.87 (m, 2H), 2.13 –

2.17 (m, 2H), 2.92 – 2.97 (m, 3H), 4.77 (d, J = 5.6 Hz, 2H), 6.12 (brs, 1H), 6.89 (d,

J = 7.2 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H),

7.67 (s, 1H), 9.36 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 13.56,

22.31, 25.32, 25.74, 30.38, 37.28, 42.93, 110.63, 114.33, 115.59, 115.97, 118.61,

124.64, 128.55, 131.87, 136.20, 141.40, 145.23, 149.52, 151.53, 161.03, 170.57;

LCMS (ESI) m/z 437 [M + H]+; HRESIMS calcd for C24H25ClN4O2 [M + H]+

436.1666, found 436.1684.

6-Chloro-2-ethyl-N-((2-(morpholinomethyl)benzo[d]oxazol-5-

yl)methyl)imidazo[1,2-a]pyridine-3-carboxamide (37). Pale yellow solid; mp =

93.0 ℃; 1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 2.62 - 2.64 (m,

4H), 2.94 (q, J = 7.6 Hz, 2H), 3.74 -3.76 (m, 4H), 3.85 (s, 2H), 4.78 (d, J = 5.6 Hz,

2H), 6.18 (brt, J = 5.6 Hz, 1H), 7.28 (dd, J = 9.6, 2.0 Hz, 1H), 7.37 (dd, J = 8.0, 1.2

Hz, 1H), 7.51 -7.54 (m, 2H), 7.70 (d, J = 1.2 Hz, 1H), 9.51 (d, J = 2.0 Hz, 1H); 13C

NMR (100 MHz, DMSO-d6) δ 13.53, 22.35, 42.88, 53.19, 54.99, 66.52, 110.97,

116.16, 117.63, 118.88, 120.15, 125.27, 125.36, 127.56, 136.49, 141.08, 143.80,

149.81, 151.60, 161.04, 163.95; LCMS (ESI) m/z 454 [M + H]+; HRESIMS calcd

for C23H24ClN5O3 [M + H]+ 453.1568, found 453.1579.

7-Chloro-2-ethyl-N-((2-(morpholinomethyl)benzo[d]oxazol-5-

yl)methyl)imidazo[1,2-a]pyridine-3-carboxamide (38). White solid; mp =

46

200.8 ℃; 1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 2.63 -2.65 (m,

4H), 2.94 (q, J = 7.6 Hz, 2H), 3.75 -3.77 (m, 4H), 3.86 (s, 2H), 4.79 (d, J = 6.0 Hz,

2H), 6.13 (brt, J = 6.0 Hz, 1H), 6.90 (dd, J = 7.2, 2.0 Hz, 1H), 7.37 (dd, J = 8.4, 1.6

Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H),

9.36 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 13.58, 22.32, 42.88,

53.19, 54.98, 66.52, 110.95, 114.35, 115.59, 115.95, 118.88, 125.26, 128.56,

131.89, 136.53, 141.08, 145.24, 149.79, 151.56, 161.05, 163.94; LCMS (ESI) m/z

454 [M + H]+; HRESIMS calcd for C23H24ClN5O3 [M + H]+ 453.1568, found

453.1578.

6-Chloro-2-ethyl-N-((2-(4-fluorophenyl)benzo[d]oxazol-5-yl)methyl)imidazo[1,2-

a]pyridine-3-carboxamide (39). White solid; mp = 258.0 ℃; 1H NMR (400 MHz,

DMSO-d6) δ 1.25 (t, J = 7. 6 Hz, 3H), 2.99 (q, J = 7.6 Hz, 2H), 4.65 (d, J = 5.6 Hz,

2H), 7.41 – 7.46 (m, 4H), 7.64 (d, J = 9.6 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.78 (s,

1H), 8.21 – 8.25 (m, 2H), 8.54 (t, J = 5.6 Hz, 1H), 9.08 (d, J = 2.0 Hz, 1H); 13C

NMR (100 MHz, DMSO-d6) δ 13.52, 22.38, 42.91, 111.08, 116.15, 116.90, 117.12,

117.63, 118.97, 120.15, 123.52, 123.55, 125.38, 125.58, 127.56, 130.30, 130.39,

137.01, 142.05, 143.81, 149.79, 151.63, 161.09, 162.30, 163.43, 165.93; LCMS

(ESI) m/z 449 [M + H]+; HRESIMS calcd for C24H18ClFN4O2 [M + H]+ 448.1102,

found 448.1107.

47

7-Chloro-2-ethyl-N-((2-(4-fluorophenyl)benzo[d]oxazol-5-yl)methyl)imidazo[1,2-

a]pyridine-3-carboxamide (40). White solid; mp = 261.4 ℃; 1H NMR (400 MHz,

DMSO-d6) δ 1.25 (t, J = 7. 2 Hz, 3H), 2.98 (q, J = 7.2 Hz, 2H), 4.64 (d, J = 5.6 Hz,

2H), 7.07 (d, J = 7.6 Hz, 1H), 7.42 – 7.46 (m, 3H), 7.75 (d, J = 8.4 Hz, 2H), 7.77 (s,

1H), 8.23 (d, J = 8.4 Hz, 2H), 8.55 (brs, 1H), 8.96 (d, J = 7.2 Hz, 1H); 13C NMR

(100 MHz, DMSO-d6) δ 13.58, 22.34, 42.90, 111.09, 114.37, 115.59, 115.96,

116.92, 117.14, 118.97, 125.58, 128.58, 130.32, 130.41, 131.90, 137.06, 142.05,

145.26, 149.79, 151.59, 161.10, 162.38, 163.4, 165.9; LCMS (ESI) m/z 449 [M +

H]+; HRESIMS calcd for C24H18ClFN4O2 [M + H]+ 448.1102, found 448.1108.

6-Chloro-2-ethyl-N-((2-(4-(trifluoromethoxy)phenyl)benzo[d]oxazol-5-

yl)methyl)imidazo[1,2-a]pyridine-3-carboxamide (41). White solid; mp =

239.7 ℃; 1H NMR (400 MHz, CDCl3) δ 1.40 (t, J = 7.6 Hz, 3H), 2.99 (q, J = 7.6

Hz, 2H), 4.82 (d, J = 6.0 Hz, 2H), 6.20 (brs, 1H), 7.31 (dd, J = 7.6, 1.6 Hz, 1H),

7.35 (d, J = 8.4 Hz, 2H), 7.42 (dd, J = 8.4, 1.6 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H),

7.59 (d, J = 8.4 Hz, 1H), 7.77 (s, 1H), 8.29 (d, J = 8.8 Hz, 2H), 9.55 (d, J = 1.6 Hz,

1H); 13C NMR (100 MHz, DMSO-d6) δ 13.53, 22.38, 42.90, 111.22, 116.20,

117.63, 119.14, 120.15, 122.11, 125.38, 125.89, 126.02, 127.58, 130.00, 137.15,

141.98, 143.82, 149.86, 151.08, 151.64, 161.10, 161.93; LCMS (ESI) m/z 515 [M

+ H]+; HRESIMS calcd for C25H18ClF3N4O3 [M + H]+ 514.1020, found 514.1023.

48

7-Chloro-2-ethyl-N-((2-(4-(trifluoromethoxy)phenyl)benzo[d]oxazol-5-

yl)methyl)imidazo[1,2-a]pyridine-3-carboxamide (42). White solid; mp =

244.6 ℃; 1H NMR (400 MHz, DMSO-d6) δ 1.27 (t, J = 7.2 Hz, 3H), 3.00 (q, J =

7.2 Hz, 2H), 4.66 (d, J = 6.0 Hz, 2H), 7.10 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 7.48 –

7.50 (m, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.77 – 7.81 (m, 3H), 8.32 (d, J = 8.4 Hz,

2H), 8.56 (t, J = 6.0 Hz, 1H), 8.98 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz,

DMSO-d6) δ 13.58, 22.34, 42.90, 111.21, 114.37, 115.60, 115.95, 119.13, 122.12,

125.88, 126.02, 128.59, 129.99, 131.92, 137.19, 141.98, 145.26, 149.86, 151.00,

151.60, 161.10, 161.93; LCMS (ESI) m/z 515 [M + H]+; HRESIMS calcd for

C25H18ClF3N4O3 [M + H]+ 514.1020, found 514.1025.

49

IV-3. Biological evaluations

Minimum inhibitory concentration determination (Extracellular MIC)

H37Rv-GFP was dispensed into 384-well plates in 7H9 medium without glycerol.

Mycobacterial growth was determined by measuring fluorescence intensity at 488

nm after 5 d of incubation. The MIC80 were determined using an eight- or ten-point

concentration curve with threefold serial dilution.

Minimum inhibitory concentration determination (Extracellular MIC)

The assay was performed as previously described.8,17-18 Briefly, Raw 264.7 cells

(American Type Culture Collection TIB-71) were infected with M. tuberculosis

H37Rv-GFP at a multiplicity of infection of 2:1 and dispensed into 384-well plates.

After 5 d of infection, macrophages were stained with Syto 60. Image acquisition

was performed on an EVOscreen Mark III platform integrated with Opera.

Bacterial load and macrophage number were quantified using proprietary image

analysis software.17-18 The MIC80 were determined using an eight- or ten-point

concentration curve with threefold serial dilution.

In vivo pharmacokinetic (PK) evaluation

BALB/c mice and Sprague Dawley rats were used for pharmacokinetic studies.

Compounds were given at a dose of 2 mg per kg body weight intravenously or 10

50

mg per kg body weight orally. Blood samples were taken through the caudal vena

cava using 1-mL syringes before perfusion. Samples were collected from three

mice or rats at 0.5, 1, 2, 6, 12, 24 and 48 h post-dose. Blood samples were

centrifuged at 3,200g for 10 min at 4 °C. Following centrifugation, plasma was

collected and frozen until further analysis. Compound concentrations were

determined by LC-MS.

In vivo efficacy in the mouse model of tuberculosis

The acute model was performed as previously described.8, 20 Briefly, mice were

infected with a high dose of M. tuberculosis H37Rv. Dosing was initiated 6 d after

infection. Drugs were administered orally for 3 d. Bacterial load in the lungs of

infected mice was determined by colony-forming unit (CFU) enumeration. For the

established mouse model, BALB/c mice were infected with 2 × 102 to 2 × 103 CFU

of M. tuberculosis H37Rv by the intranasal route. Treatment was initiated 3 weeks

after infection. Drugs were formulated in 20% TPGS and administered by oral

gavage for 28 d, five times per week. Bacterial load in the lungs of infected mice

was determined by CFU enumeration.

51

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WHO/HTM/TB/2015.22.

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tuberculosis with Extensive Resistance to Second-Line Drugs — Worldwide,

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diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.

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(6) European Medicines Agency. EPAR summary for the public;

EMA/731960/2013.

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Lenaerts, A.J.,; Pham, H.; Jones, V.; Seo, M.J.; Kim, Y.; Seo, M.; Seo, J.; Park,

D.; Ko, Y.; Choi, I.; Kim, R.; Kim, S.; Lim, S.; Yim, S.; Nam, J.; Kang, H.;

Kwon, H.; Oh, C.; Cho, Y.; Jang, Y.; Kim, J.; Chua, A.; Tan, B.H.; Nanjundappa,

M.B.; Rao, S.P.; Barnes, W.S.; Wintjens, R.; 1 Walker, J.R.; Alonso, S.; Lee, S.;

Kim, J.; Oh, S.; Oh, T.; Nehrbass, U.; Han, S-J.; No, Z.; Lee, J.; Brodin, P.; Cho,

S.; Nam, K.; Kim, Jaeseung. Discovery of Q203, a potent clinical candidate for

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(9) Kang, S.; Kim, R. Y.; Seo, M. J.; Lee, S.; Kim, Y. M.; Seo, M.; Seo, J. J.; Ko,

Y.; Choi, I.; Jang, J.; Nam, J.; Park, S.; Kang, H.; Kim, H. J.; Kim, J.; Ahn, S.;

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(10) U.S. National Institutes of Health. ClinicalTrials.gov; NCT02530710.

(11) Abrahams, K. A.; Cox, J. A. G.; Spivey, V. L.; Loman, N. J.; Patten, M.J.;

Constantinidoou,C.; Fernandex, R.; Alemparte,C.; Remuinan, M. J.; Barros, D.;

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Ballell, L.; Besra, G.S. Identification of Novel Imidazo[1,2-a]pyridine Inhibitors

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470-471.

(13) Ribeiro, I. G.; da Silva, K. C. M.; Parrini, S. C.; de Miranda, A. L. P.; Fraga, C.

A. M.; Barreiro, E. J. Synthesis and antinociceptive properties of new

structurally planned imidazo[1,2-a]pyridine 3-acylarylhydrazone derivatives.

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(14) Perner R.J.; DiDomenico S.; Koenig J.R.; Gomtsyan A.; Bayburt E.K.;

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M.; Kim, J.; Genovesio, A.; Carralot, J.P.; Ewann, F.; Kim, E.H.; Lee, S.Y.;

Kang, S.; Seo, M.J.; Park, E.J.; Skovierová, H.; Pham, H.; Riccardi, G.; Nam,

J.Y.; Marsollier, L.; Kempf, M.; Joly-Guillou, M.L.; Oh, T.; Shin, W.K.; No, Z.;

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2264.

56

VI. Acknowledgements

Reproduced from Sunhee Kang, Young Mi Kim, Heekyung Jeon, Sejin Park, Min

Jung Seo, Saeyeon Lee, Dongsik Park, Jiyeon Nam, Seokwoo Lee, Kiyean Nam,

Sanghee Kim and Jaeseung Kim. Synthesis and structure-activity relationships of

novel fused ring analogues of Q203 as antitubercular agents. Eur. J. Med. Chem.,

2017, 136, 420-427. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Reproduced from Sunhee Kang, Young Mi Kim, Ryang Yeo Kim, Min Jung Seo,

Zaesung No, Kiyean Nam, Sanghee Kim and Jaeseung Kim. Synthesis and

structure-activity studies of side chain analogues of the anti-tubercular agent,

Q203. Eur. J. Med. Chem., 2017, 125, 807-815. Copyright © 2016 Elsevier Masson

SAS. All rights reserved.

57

Appendix I

NMR Spectra of Representative

Compounds

58

59

60

61

62

63

64

65

66

Appendix II

In vivo Pharmacokinetics of 42

67

Figure A1. In vivo pharmacokinetics of compound 42

68

국 문 초 록

결핵 후보물질 개발을 위한 새로운 Q203 유도체 합성 및

구조-활성 상관관계 연구

약제내성 및 다약제내성 결핵균주의 출현에 따른 급증하는 의학적 미

충족 요구에 의해 새로운 작용기전을 지니는 결핵치료제의 개발이 시급

하다. 본 연구진은 선행된 연구에서 보고한 imidazo[1,2-a]pyridine-3-

carboxamide 골격의 유도체인 Q203의 구조로부터 side chain을 다양하게

변화시킨 화합물을 고안 및 합성하고, 액상배지 및 대식세포 내 서식하

는 결핵균에 대한 활성을 시험하였다. Q203의 side chain region에 다양하

게 변화된 linker에 따른 conformation의 변화에도 불구하고 많은 화합물

들이 우수한 항 결핵활성을 보여주었으며, side chain의 감소된 lipophilicity

는 화합물의 항 결핵활성에 부정적인 영향을 끼쳤다.

화합물의 우수한 in vitro 활성을 바탕으로 소규모 화합물들에 대한 in

vivo pharmacokinetic 시험을 하였으며, 그 결과 화합물 11, 16, 21 및 42화

합물은 우수한 in vivo pharmacokinetic 결과를 보여주었다. Mouse를 이용한

생체 내 효능 시험에서 대표 화합물 21 과 42 는 폐 및 이자에서 현저히

감소된 결핵균의 수치를 보여주어, 이들의 생체 내 효능이 입증되었다.

따라서 본 연구진은 우수한 in vivo pharmacokinetic 시험 결과와 생체 내

69

효능시험 결과를 바탕으로, 새로운 결핵 치료제 후보물질로서 화합물 21

을 제시하고자 한다.

주요어: 결핵, 다약제내성, 광범위 약제내성, imidazo[1,2-a]pyridine-3-

carboxamide, 구조-활성 상관관계

학번: 2011-30497 강선희