Discovery of thienothiazolocarboxamide analogues as novel anti-tubercular agent

Article abstract of DOI:10.1016/j.bmc.2020.115797

In order to identify anti-tubercular agents with a novel scaffold, commercial libraries of small organic compounds were screened against a fluorescent strain of Mycobacterium tuberculosis H37Rv, using a dual phenotypic assay. Compounds were assessed again

Full text of DOI:10.1016/j.bmc.2020.115797

Journal Pre-proofs
Discovery of Thienothiazolocarboxamide Analogues as Novel Anti-tubercular
Agent
Guanghai Jin, Young Mi Kim, Aram Lee, Junghwan Choi, Sunhee Kang,
Mooyoung Seo, Jeong Jea Seo, Sumi Lee, Juhee Kang, Jaeseung Kim,
Sinyoung Park, Minjeong Woo, Virginia Carla, Honggun Lee, Jinyeong Heo,
David Shum, Kaapjoo Park, Vincent Delorme, Inhee Choi
PII:
S0968-0896(20)30627-1
https://doi.org/10.1016/j.bmc.2020.115797
BMC 115797
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 August 2020
28 September 2020
28 September 2020
Please cite this article as: G. Jin, Y. Mi Kim, A. Lee, J. Choi, S. Kang, M. Seo, J. Jea Seo, S. Lee, J. Kang, J.
Kim, S. Park, M. Woo, V. Carla, H. Lee, J. Heo, D. Shum, K. Park, V. Delorme, I. Choi, Discovery of
Thienothiazolocarboxamide Analogues as Novel Anti-tubercular Agent, Bioorganic & Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.bmc.2020.115797
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Discovery of Thienothiazolocarboxamide Analogues as Novel Anti-tubercular Agent
a
a
a
a
a
Guanghai Jin , Young Mi Kim , Aram Lee , Junghwan Choi , Sunhee Kang , Mooyoung
Seo a, Jeong Jea Seo a, Sumi Lee a, Juhee Kang a, Jaeseung Kim a, Sinyoung Park b, Minjeong
c
c
d
d
d
a
Woo , Virginia Carla , Honggun Lee , Jinyeong Heo , David Shum , Kaapjoo Park , Vincent
c
a
Delorme , Inhee Choi
a Medicinal Chemistry, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu, Seongnam-si,
Gyeonggi-do, 13488 Rep. of Korea
b Animal Facility and Lab Support, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu,
Seongnam-si, Gyeonggi-do, 13488 Rep. of Korea
c Tuberculosis Research Lab, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu,
Seongnam-si, Gyeonggi-do, 13488 Rep. of Korea
d Screening Development Platform, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu,
Seongnam-si, Gyeonggi-do, 13488 Rep. of Korea
Corresponding author: Inhee Choi, inhee.choi@ip-korea.org
Highlights
 Thienothiazolocarboxamide analogues were prepared and evaluated as anti-tubercular
agents.
 Compound 42 showed activities against M. tuberculosis H37Rv replicating outside and
inside macrophages (extracellular IC50 = 0.76 μM, intracellular IC50 = 0.19 μM).
 Compound 42 showed favorable in vivo PK profile and efficacy in a murine model of
tuberculosis which proposed it as a potential lead compound for further evaluation.
1

ABSTRACT
In order to identify anti-tubercular agents with a novel scaffold, commercial libraries of small
organic compounds were screened against a fluorescent strain of Mycobacterium tuberculosis
H37Rv, using a dual phenotypic assay. Compounds were assessed against bacteria replicating
in broth medium, as well as inside macrophages, and thienothiazolocarboxamide (TTCA)
scaffold was identified as hit in both assays, with submicromolar inhibitory concentrations.
Derivatives of TTCA were further synthesized and evaluated for their inhibitory effects on
M. tuberculosis H37Rv. In the present study we report the structure-activity relationship of
these TTCA derivatives. Compounds 28, 32 and 42 displayed good anti-tubercular activities,
as well as favorable ADME and PK properties. Compound 42 exhibited excellent oral
bioavailability in mice with high distribution to lungs, within 1 hour. It was found to be
efficacious in a dose dependent manner in a murine model of M. tuberculosis infection. Hence,
compound 42 is now under evaluation as a potential lead candidate for treatment of tuberculosis.
Graphical abstract
Keywords: Tuberculosis (TB); Thienothiazolocarboxamide (TTCA); Structure-activity
relationship (SAR); Structure-property relationship (SPR); Pharmacokinetics (PK); in vivo
efficacy.
2

1. Introduction
Tuberculosis (TB), mostly caused by Mycobacterium tuberculosis in humans, remains one
of the top 10 causes of death worldwide and is the leading cause of death from a single
infectious agent [1]. TB typically affects the lungs, but may also affect other parts of the body.
According to the WHO report published in 2019, 10 million people fell ill with TB in 2018,
while nearly 1.5 million people died from the disease, including about 250,000 patients co-
infected with the human immunodeficiency virus (HIV) [2]. The current standard regimen
against TB involves a combination of four drugs, isoniazid (INH), rifampicin (RIF),
pyrazinamide (PZA) and ethambutol (EMB) [3]. Treatment of an infected individual requires
at least six months of chemotherapy. The emergence of drug-resistant strains, including
multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains, threatens the goal of
TB elimination worldwide [4]. During the last decade, several compounds reached pre-clinical
and clinical trials, and three new drugs ‒ bedaquiline, delamanid, and pretomanid ‒ have
recently been approved [5–7]. However, treatment for drug-resistant TB consists in lengthy
regimens with drugs that are more expensive and more toxic [2]. Therefore, there is an urgent
need for new therapeutic strategies that could not only shorten the treatment regimen but also
help control the spread of drug-resistant forms of the disease.
In order to search for novel chemotypes to be developed as potential anti-tubercular agents,
a library of 30,270 small molecules was subjected to high-throughput screening using a
conventional phenotypic approach featuring M. tuberculosis strain H37Rv, constitutively
expressing a green fluorescent protein (GFP), replicating in broth medium (extracellular assay).
In parallel, compounds were also tested against the same mycobacterial strain replicating inside
macrophages, or intracellular assay [8,9]. Among the hit compounds for this dual screening,
compound 1, containing thienothiazolocarboxamide (TTCA) scaffold (Fig 1A), exhibited
submicromolar inhibitory concentrations with IC50 values less than 1 μM in both extracellular
and intracellular assays. This promising scaffold, which is structurally different from known
anti-tubercular drugs (Fig 1B), was further investigated through chemical modifications
focused on R1 and R2 position. Compounds from this TTCA series showed higher potency
against M. tuberculosis replicating within macrophages, as compared to broth culture medium.
Herein, we describe the synthesis, structure-activity relationship (SAR) and structure-property
relationship (SPR) analysis of these TTCA compounds, along with their in vitro biological
3

evaluation. Selected compounds with good pharmacokinetics (PK) profiles were tested for in
vivo efficacy in a murine mode of TB.
A.
O
S
N
S
O
S
N
S
N
R2
O
NH
R1 NH
TTCA
1
B.
First line
O
O
NH2
N
H
N
NH2
N
N
Isoniazid
(INH, H)
Rifampicin
(RIF, R)
Pyrazinamide
(PZA, Z)
Ethambutol
(EMB, E)
Second line
O
O
O
S
O
F
NH
N
O
N
F
OH
OH
OH
NH2
HO
N
N
N
H2N
N
O
O
p-aminosalicylic
acid
Ethionamide
(ETH or Eto)
Moxifloxacin
(MXF, Mox or Mfx)
Ofloxacin (Ofx)
(PAS)
Recently approved
N
O
F
F
O
F
F
F
O
F
N
O
OH
N
O
N+
Br
O
-O
N
N
N+
O
O
-O
N
O
N
O
Bedaquiline
(BDQ)
Delamanid
Pretomanid
4

Fig. 1. A. Generic structure of the thienothiazolocarboxamide (TTCA) scaffold (left) and
chemical structure of initial hit compound 1 (right). B. Structures of representative first and
second line as well as recently approved anti-tubercular drugs.
2. Chemistry
Analogues of the hit compound 1 with modified linkers were synthesized as outlined in
Scheme 1. The commercially available 2,4-dichlorothiazole-5-carbaldehyde was alkylated
with piperidine in the presence of trimethylamine (TEA) in acetonitrile to afford compound 2.
Ethyl thioglycolate was alkylated with 2 followed by cyclization in the presence of TEA in
DMSO and heating. The key intermediate 3 was finally obtained after reaction with sodium
ethoxide. The ester 3 was further coupled with p-anisidine in the presence of
trimethylaluminum in toluene to provide hit compound 1. N-methylated compound 4 was
obtained by treating compound 1 with sodium hydride and iodomethane. Ester reduction of
compound 3 using lithium aluminium hydride afforded the primary alcohol 5, which was in
turn oxidized by Dess-Martin periodinane to afford aldehyde 6. Reductive amination using
NaBH(OAc)2 with p-anisidine finally afforded the amine linker compound 7. Compound 8 was
prepared from the ester 3 by coupling with 4-methoxybenzylamine. Hydrolysis of ester
intermediate 3 gave the carboxylic acid 9. A Curtius rearrangement was used to directly convert
9 into carbamate 10, involving diphenylphosphoryl azide (DPPA) and TEA in tert-butanol.
Deprotection with Conc. HCl afforded amine 11, which was coupled with 4-methylbenzoic
acid in the presence of EDCi, HOBt and TEA to obtain the reversed amide linker compound
12. Additionally, the ester intermediate 3 was coupled with 3,5-dimethoxyaniline or 4-
chloroaniline to give compounds 13 and 14, respectively. Finally, compound 1 was further
demethylated by BBr3 in THF to yield compound 15.
As shown in Scheme 2, compounds 18-23 were synthesized using methods similar to those
described in Scheme 1. Commercially available amines were alkylated with 2,4-
dichlorothiazole-5-carbaldehyde to give compounds 16a-e, which then afforded 17a-e through
alkylation and cyclization. Amines selected included N-boc-piperazine, morpholine, 1-
methylpiperazine, 4-difluoropiperidine and dimethylamine. Compounds 17a-e were further
coupled with p-anisidine to provide compounds 18, 19, 21, 22 and 23. Compound 20 was
prepared by de-protection of compound 18.
5

The synthesis of amine 25 and TTCA analogues 28-35 is described in Scheme 3. N-boc-
piperidine-4-carboxylic acid was coupled with dimethylamine in the presence of EDCi, HOBt
and TEA to produce amide 24, subsequently de-protected in TFA to give amine 25. Using the
same synthetic route as described in Scheme 2, 2,4-dichlorothiazole-5-carbaldehyde was
coupled with various amines (including 25) to provide intermediate aldehydes 26a-g, which
were converted into esters 27a-g. The 2-methylbenzo[d]oxazol-5-amine was coupled with 17b,
27a-g to provide the desired compounds 28-35.
Synthesis of the biaryl amines 37a-h and TTCA analogues 38-45 is described in Scheme
4. Suzuki coupling of substituted pyridines 36a-f and 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline using Pd(dppf)Cl2 and sodium carbonate gave the desired amines
37a-f. In addition, 37g and 37h were synthesized from 4-fluorophenylboronic acid by Suzuki
coupling with 3-amino-6-bromopyridine (36g) and 2-amino-5-bromopyridine (36h),
respectively. The ester 27g was coupled with amines 37a-h using trimethylaluminum in toluene
to yield compounds 38-45.
6

Scheme 1. Scheme for the synthesis of TTCA analogues with modified linkers.
O
O
O
S
N
S
S
N
S
O
S
N
S
(xiii)
(xi)
O
(xii)
S
N
S
H2N
HCl
N
HN
N
N
HN
N
HO
9
10
11
12
(x)
Cl
O
O
N
S
4
N
S
Cl
O
O
R1 NH
S
N
S
N
(vi)
N
O
(iv)
S
N
(ii), (iii)
(i)
N
N
Cl
N
N
H
O
H
S
H2N R1
S
S
EtO
1: R1 = 4-methoxyphenyl
8: R1 = 4-methoxybenzyl
13: R1 = 3,5-dimethoxyphenyl
14: R1 = 4-chlorophenyl
15: R1 = 4-hydroxyphenyl
2
3
(v)
(vii)
S
N
S
S
N
S
O
H
S
N
S
N
(ix)
(viii)
N
N
O
NH
HO
7
5
6
Reagents and conditions: (i) piperidine, TEA, CH3CN, rt, 2h; (ii) ethyl thioglycolate, TEA, DMSO, 120°C, 3h; (iii) NaOEt, EtOH, rt, 2h; (iv)
amine, AlMe3, toluene, 100°C, overnight; (v) BBr3, THF, 0°C; (vi) NaH, MeI, 0°C to rt, 2h; (vii) LAH, THF, 0°C, 2h; (viii) Dess-Martin
periodinane, MC, rt, 1h; (ix) p-anisidine, NaBH(OAc)2, MC, rt, overnight; (x) LiOH, MeOH/H2O (3:1, v/v), 50°C, 3h; (xi) DPPA, TEA, t-
BuOH, reflux, 95°C, 4h; (xii) Conc. HCl, 1,4-dioxane, rt, 2h; (xiii) 4-methoxybenzoic acid, EDCi, HOBt, TEA, DMF, rt, overnight.
7

Scheme 2. Scheme for the synthesis of TTCA derivatives 18-23.
O
S
N
S
Cl
Cl
O
N
S
N
S
R2
N
(i)
(ii), (iii)
(iv)
R2
Cl
O
NH
R2
H
H
S
EtO
S
O
O
18: R = -boc-piperazine
N
16a: R = -boc-piperazine
17a: R = -boc-piperazine
N
N
2
2
2
(v)
19: R2 = morpholine
20: R2 = piperazine
16b: R2 = morpholine
16c: R2 = 1-methylpiperazine
17b: R2 = morpholine
17c: R2 = 1-methylpiperazine
21: R2 = 1-methylpiperazine
22: R2 = 4-difluoropiperidine
23: R2 = dimethylamine
16d: R2 = 4-difluoropiperidine
16e: R2 = dimethylamine
17d: R2 = 4-difluoropiperidine
17e: R2 = dimethylamine
Reagents and conditions: (i) N-boc-piperazine or morpholine or 1-methylpiperazine or 4-difluoropiperidine or dimethylamine, TEA, CH3CN,
rt, 2h; (ii) ethylthioglycolate, TEA, DMSO, 120°C, 3h; (iii) NaOEt, EtOH, rt, 2h; (iv) p-anisidine, AlMe3, toluene, 100°C, overnight; (v) TFA,
dichloromethane, 4h.
8

Scheme 3. Scheme for the synthesis of TTCA derivatives 28-35.
O
O
N
O
N
(i)
(ii)
TFA
HN
Boc
N
Boc
N
OH
24
25
O
O
S
N
S
Cl
O
Cl
O
R2
S
N
S
N
N
(iv), (v)
(vi)
(iii)
R2
NH
O
Cl
R2
H
H
EtO
S
S
N
28: R2 = morpholine
17b: R2 = morpholine
16b: R2 = morpholine
29: R2 = 4-hydroxypiperidine
27a: R2 = 4-hydroxypiperidine
26a: R2 = 4-hydroxypiperidine
30: R2 = 4-(hydroxymethyl)piperidine
31: R2 = 2,6-dimethylmorpholine
32: R2 = 4-(dimethylcarbamoyl)piperidine
33: R2 = 2-(methylamino)ethan-1-ol
34: R2 = 3-(methylamino)propan-1-ol
35: R2 = 2-morpholinoethanamine
27b: R2 = 4-(hydroxymethyl)piperidine
27c: R2 = 2,6-dimethylmorpholine
27d: R2 = 4-(dimethylcarbamoyl)piperidine
27e: R2 = 2-(methylamino)ethan-1-ol
27f: R2 = 3-(methylamino)propan-1-ol
27g: R2 = 2-morpholinoethanamine
26b: R2 = 4-(hydroxymethyl)piperidine
26c: R2 = 2,6-dimethylmorpholine
26d: R2 = 4-(dimethylcarbamoyl)piperidine
26e: R2 = 2-(methylamino)ethan-1-ol
26f: R2 = 3-(methylamino)propan-1-ol
26g: R2 = 2-morpholinoethanamine
Reagents and conditions: (i) dimethylamine, EDCi, HOBt, TEA, dichloromethane, rt, overnight; (ii) TFA, dichloromethane, 4h; (iii) TEA,
CH3CN, rt, 2h; (iv) ethylthioglycolate, TEA, DMSO, 120°C, 3h; (v) NaOEt, EtOH, rt, 2h; (vi) 2-methylbenzo[d]oxazol-5-amine, AlMe3, toluene,
100°C, overnight.
9

Scheme 4. Scheme for the synthesis of biaryl amines 37a-h and TTCA analogues 38-45.
(i)
O
O
X
Br
X
NH2
+
B
NH2
Y
Z
Y Z
36a X = Me, Y = CH, Z = N
36b X = OMe, Y = CH, Z = N
36c X = CF3, Y = CH, Z = N
36d X = Cl, Y = CH, Z = N
36e X = F, Y = CH, Z = N
36f X = F, Y = N, Z = CH
37a X = Me, Y = CH, Z = N
37b X = OMe, Y = CH, Z = N
37c X = CF3, Y = CH, Z = N
37d X = Cl, Y = CH, Z = N
37e X = F, Y = CH, Z = N
37f X = F, Y = N, Z = CH
(i)
OH
Br
NH2
F
NH2
+
F
B
OH
X
Y
X Y
36g X = N, Y = CH
36h X = CH, Y = N
37g X = N, Y = CH
37h X = CH, Y = N
O
S
N
S
O
S
N
S
(ii)
NH
38: R1 =
39: R1 =
NH
R1 NH
N
N
EtO
37a-37h
N
N
O
O
O
27g
CF3
40: R1 =
N
Cl
F
41: R1 =
42: R1 =
43: R1 =
44: R1 =
45: R1 =
N
N
F
N
F
N
F
N
Reagents and conditions: (i) Pd(dppf)Cl2, Na2CO3, DME/H2O (3:1, v/v), 150°C, 1h; (ii) AlMe3,
toluene, 100°C, overnight.
10

3. Results and discussion
The in vitro activity of synthetic TTCA analogues was evaluated against M. tuberculosis
H37Rv expressing GFP (H37Rv-GFP) replicating in liquid broth culture medium (extracellular
IC50), but also inside macrophages (intracellular IC50) using already established assays [7,8].
The initial structure-activity relationship (SAR) studies evaluated a set of analogues that
contained replacement of the amide linker in an attempt to affect potency of hit compound 1
(Table 1). Replacement of hydrogen with methyl in the amide linker yielded compound 4,
which showed no activity against H37Rv-GFP replicating outside and inside macrophages.
Extension of the length of the amide linker by one carbon towards the phenyl ring (8) did not
yield any activities as well. Finally, compounds with an amine (7) or a reversed amide (12)
linker showed no potency in any of the two assays. Therefore, this set of linker modifications
revealed that the carboxamide linked to a phenyl ring was a critical feature to maintain anti-
mycobacterial activity.
Table 1. Anti-tubercular activity of TTCA analogues with linker modification.
S
N
S
O
X
N
Anti-tubercular activity
Compound
1
X
Extracellular
IC50 (µM)
Intracellular
IC50 (µM)
O
O
0.83
0.17
N
H
4
7
8
> 20
> 20
> 20
> 20
> 20
> 20
N
N
H
O
N
H
O
12
> 20
0.40
> 20
0.35
N
H
INH
11

RIF
0.01
0.06
Next, modifications were focused on R1 and R2 positions, as shown in Table 2. In position
R1, the anti-tubercular activity was lost when substituting the phenyl ring with two methoxy
groups in meta position (13), and it showed poor metabolic stability. This suggested that the
para position on the phenyl ring might be important for the compound potency. A chloro
substitution in para position (14) showed similar potency and metabolic stability compared
with compound 1. Replacement of methoxy with hydroxyl, as in compound 15, reduced the
activity against H37Rv-GFP replicating outside and inside macrophages by approximately 7
and 14 fold, respectively. Replacement of the piperidine ring at R2 position with morpholine
gave compound 19, which showed similar potency and increased metabolic stability as
compared to compound 1. With a piperazine substituent (20), IC50 values for both assays were
increased. Substitution of the piperazine with a methyl in -NH position, as in compound 21,
restored the overall potency while decreasing the metabolic stability. Compound 22, presenting
a 4-difluoropiperidine substituent at position R2, showed activities and metabolic stabilities
very similar to that of compound 1. Opening the ring at position R2, as in the dimethylamine
substituted compound 23, slightly decreased the potency but reduced the metabolic stability.
Overall, R2 substitutions were found to affect mostly the metabolic stability, while R1 position
was more critical for the activity.
Table 2. Biological evaluation of TTCA derivatives.
O
S
N
S
R2
R1 NH
Metabolic stability
Phase I (min)
Anti-tubercular activity
Compound
R1
R2
Extracellular
IC50 (µM)
Intracellular
IC50 (µM)
Human
Mouse
O
N
1
0.83
0.17
> 20
> 60
13.9
O
O
N
13
> 20
24.5
33.6
12

Cl
N
N
14
15
1.1
4.8
0.11
2.75
> 60
–
22.5
–
HO
O
O
N
O
19
20
1.1
0.21
2.1
57.6
–
> 60
–
N
NH
13.7
N
N
N
O
O
O
21
22
23
1.3
1.1
1.6
0.11
0.33
0.27
16.2
> 60
23.6
1.4
F
F
35.9
13.7
N
INH
RIF
0.40
0.01
0.35
0.06
Overall increase in activity was obtained when R1 was changed from 4-methoxy to
benzoxazole (Table 3). Various substitutions at position R2 were explored with several
saturated ring and aliphatic chain substituents. Compound 28, displaying a morpholine
substituent at position R2, exhibited good metabolic stability with high potency. With a
hydroxyl group in para position of the piperidine ring (29), the activity was not enhanced in
both extracellular and intracellular assays, while high metabolic stabilities were maintained.
Moving the hydroxyl further away from the piperidine ring using a one carbon linker (30) did
not improve extracellular and intracellular activities, as compared to 28. Substitution of the
morpholine ring with two methyl (31) reduced the anti-mycobacterial activity and was not
favorable for the stability in mouse liver microsome. Presenting a dimethylamine carbonyl
group substituted at para position of the piperidine ring (32) resulted in similar extracellular
13

and intracellular activity as compared to compound 28, solubility and metabolic stability were
similarly maintained as well.
Table 3. Biological evaluation of TTCA derivatives.
O
S
N
S
R2
NH
O
N
Solubility
(ug/mL)
Metabolic stability
Phase I (min)
Anti-tubercular activity
Extracellular Intracellular
Compound
R2
pH
7.4
pH
2.1
Human
Mouse
IC50 (µM)
IC50 (µM)
N
O
28
0.42
0.20
0.82
5.3
> 60
> 60
N
N
OH
29
30
31
0.64
0.50
1.1
0.46
0.23
0.25
2.4
0.87
1.1
9.7
7.9
1.9
> 60
–
> 60
–
OH
N
O
> 60
27.2
O
N
N
N
32
33
34
0.50
0.88
1.0
0.51
1.3
1.2
4.5
1.3
4.0
> 60
> 60
> 60
> 60
37.7
> 60
20.6
10.6
OH
N
0.70
OH
14

NH
N
35
1.35
0.47
2.1
37.1
> 60
> 60
O
INH
RIF
0.40
0.01
0.35
0.06
In order to improve the solubility, the polar substituents and linear alkyl chain were
presented. Therefore, aliphatic linkers with hydroxyl group were prepared and evaluated.
Compound 33, mimicking an opened morpholine ring through a two carbon linker, exhibited
enhanced solubility but slightly decreased potency and metabolic stability in mouse liver
microsome. A three carbon linker (34) showed high metabolic stability with slightly increased
solubility, but no enhancement of the potency. Spacing the morpholine from the core by a two
carbon linker (35) resulted in a moderate increase in solubility with high metabolic stability,
but a slight decrease in activity. These results revealed that compounds with aliphatic linker
formation exhibited better physicochemical properties as compared to the saturated ring
formation at position R2.
In an effort to enhance the potency outside and inside macrophages, the R2 position was
fixed with the ethyl-morpholine substituent, while R1 position was further varied with biaryl
substituents presenting both pyridine and phenyl rings (Table 4). The methyl pyridine
compound 38 showed moderate anti-mycobacterial activity and increased solubility but lower
metabolic stability. Additionally, methoxy, trifluoromethyl and chloro pyridine rings led to
higher extracellular activities (39-41). However, substitution with 5-fluoro pyridine
(compound 42) showed strong activity against H37Rv-GFP replicating outside as well as inside
macrophages (extracellular IC50 = 0.76 μM, intracellular IC50 = 0.19 μM), with enhanced
solubility and good metabolic stability. Maintaining this fluoro substitution, different positions
of the nitrogen atom on the biaryl rings were also investigated (43-45). The 6-fluoro pyridine
compound 43 showed the highest potency against extracellular and intracellular mycobacteria
but showed poor solubility and decreased metabolic stability. Lastly, when switching the
position of phenyl and pyridine rings (44 and 45), around 3 to 5 fold reduction of both
extracellular and intracellular anti-tubercular activities was observed. Compound 42 has also
15

shown promising inhibition against tested ten MDR and one XDR clinical isolates with MIC
less than 2 to 4 uM (Supplementary Table 1). We have successfully synthesized TTCA
derivatives with anti-mycobacterial activities which are comparable or similar to the known
drug, INH, used as the control with RIF.
Table 4. Biological evaluation of TTCA derivatives.
O
S
N
S
NH
R1 NH
N
O
Solubility
(ug/mL)
Metabolic stability
Phase I (min)
Anti-tubercular activity
Compound
R1
Extracellular
IC50 (µM)
Intracellular
IC50 (µM)
pH 7.4
pH 2.1
Human
Mouse
38
39
40
41
1.35
1.35
4.5
0.31
0.19
0.21
0.21
1.3
0.20
9.8
52.3
20.9
27.1
N
N
O
>50
44.3
43.4
<30
–
<30
–
CF3
N
Cl
3.2
0.20
–
–
N
F
F
42
43
0.76
0.61
0.19
0.10
0.76
1.1
43.6
1.5
> 60
25.1
45.5
45.6
N
N
F
44
45
4.6
4.7
0.37
0.36
0.57
0.56
>50
–
–
–
–
N
F
44.2
N
INH
RIF
0.40
0.01
0.35
0.06
16

3.1 In vitro ADME evaluation
On the basis of the SAR study, we selected compounds 28, 32 and 42 for in vitro ADME
evaluation, as shown in Table 5. In the drug discovery process, metabolic stability, plasma
stability and plasma protein binding are important parameters to consider before initiating
further in vivo PK studies. Generally, results from Phase I metabolic stability studies highlights
susceptibility to oxidation/reduction, while results from Phase II indicates glucuronidation. The
metabolic stability of the three compounds was studied in microsomes from human and mouse.
All three exhibited good stability for both phase I and II. These compounds also showed good
plasma stability for both human and mouse. Compounds 28 and 42 showed high rates of
binding to plasma proteins, but these were more moderate for compound 32. Drug-drug
interaction is another critical factor to consider for an anti-tubercular agent, due to combination
therapy with other medications for treatment. Therefore, we also evaluated the cytochrome
P450 inhibition with 5 different isozymes for compounds 32 and 42, as shown in Table 6.
Compound 32 did not show inhibition tested in 5 CYP isozymes. However, compound 42 was
found to be non-inhibitor for 1A2 and 2C19, and it inhibited moderately in rest of the isoforms.
All selected compounds were stable in the plasma, and free unbound fraction of compounds
suggested that these compounds are suitable for further PK studies.
Table 5. ADME evaluation of selected compounds 28, 32 and 42.
Metabolic stability
Phase I (t1/2, min)
Metabolic stability
Phase II (t1/2, min)
Plasma protein
binding (%)
Plasma Stability
(at 4h, %)/ (at 2h, %)*
Compound
Human
>60
Mouse
>60
Human
>60
Mouse
>60
Human
Mouse Human
Mouse
99.9
28
86.8
74.7
98.8
32
42
>60
>60
>60
99.7
>60
124
>60
86.1*
98.2*
95.7*
95.1
99.8
96.4
99.8
45.5
>100*
17

Table 6. CYP inhibition profile for compounds 32 and 42.
CYP Inhibition (IC50, µM)
Compound
3A4
2D6
1A2
2C9
2C19
>50
32
42
>50
>50
>50
>50
6.95
6.85
>100
7.41
>100
3.2 Toxicity evaluation
Cytotoxicity of compounds 32 and 42 was evaluated against a panel of four human-cancer
cell lines, as seen in Table 7. Tested compounds were found to be non-cytotoxic to all these
cells, at concentrations up to 50 μM. As bedaquiline and delamanid were reported to have
potential cardiotoxicity issues [10,11], we used the hERG potassium channel patch-clamp
assay to test compounds 32 and 42. Results suggested almost no risk for cardiotoxicity. In
addition, compounds 32 and 42 showed no genetic toxicity in a mini-Ames mutagenicity test.
Table 7. Cytotoxicity and mutagenicity evaluations for compounds 32 and 42.
Cytotoxicity (IC50, µM)
HEK-
Mini-Ames
hERG
(µM)
Compound
S9(-)
TA98/TA100
S9(+)
A549
>50
>50
HepG2 Jurkat
293
TA98/TA100
32
42
>50
>50
>50
>50
>50
>30
negative/negative negative/negative
negative/negative negative/negative
>50
27.6
3.3 In vivo pharmacokinetic (PK) studies
Based on their acceptable in vitro drug-like ADME properties, we evaluated compounds
28, 32 and 42 for in vivo PK in mice, after intravenous (i.v.) and oral (p.o.) administration
(Table 8). Compound 28 exhibited short half-life and high clearance with low volume of
distribution. In contrast, the two other compounds displayed moderate volume of distributions
and low clearance. Compounds 32 and 42 showed highest AUC with low clearance and
18

excellent bioavailability. Additionally, compound 42 (T1/2 = 2.3 h) had longer half-life than
compound 32 (T1/2 = 0.99 h). The distribution of compounds 32 and 42 in the target organ ‒
the lungs ‒ was assessed at Tmax 1 hour, as shown in Table 9. Compounds 32 and 42 showed
good distribution to the lung. Especially, compound 42 showed good distribution to the lung
as compared to plasma, with a ratio of 4.8.
Table 8. In vivo pharmacokinetic evaluation of compounds 28, 32 and 42.
Pharmacokinetics (i.v.)
Pharmacokinetics (p.o.)
Compound
t1/2
(h)
Cl
Vdss
(mL/kg)
Dose
(mg/kg)
t1/2
(h)
Cmax
(ng/mL)
AUClast
(ng·h/mL)
F
(%)
Dose
(mg/kg)
(mL/min/kg)
28
32
42
0.33
0.92
1.2
121
11.8
14.9
626
701
2
2
3
ND
0.99
2.3
313
7010
2937
ND
ND
71.2
102
10
10
10
10005
10369
1520
ND: PK parameters were not calculated due to the limited number of data points.
Table 9. Plasma and lung distribution for compound 32 and 42.
Pharmacokinetics (p.o.)
Compound
Plasma conc. Lung conc. Lung/Plasma
Dose
(mg/kg)
Tmax
(h)
(ng/mL)
(ng/mL)
ratio
32
42
4400
2986
0.7
1
1
10
10
3380
16180
4.8
3.4 In vivo efficacy
Based on its desirable in vivo PK profile, we conducted an in vivo efficacy study in mice
for compounds 32 and 42 (Fig. 2). Compound treatment was initiated three weeks after
infection of BALB/c female mice with 300 to 600 CFU (colony forming units) of M.
tuberculosis H37Rv by the intranasal route (5 mice per groups). Compounds 32, 42 and
19

positive control isoniazid (INH) were administered by oral gavage for 28 days, five times per
week. Bacterial loads were enumerated in the lung of infected mice at the end of the 28 days
of treatment. Compound 32 was used at 25, 50 and 100 mg/kg, compound 42 was used at 20,
50 and 100 mg/kg dose and INH was used as the positive control at a 25 mg/kg dose.
Compound 32 showed no effect at any dose. In contrast, compound 42 displayed a decrease in
CFU that was proportional to the administered dose. It had a better activity against M.
tuberculosis at 50 mg/kg compared to the control INH, but was not effective at 20 mg/kg. At
100 mg/kg, compound 42 showed very high reduction of the bacterial burden in the lungs of
infected mice (4 to 5 logs reduction) and the number of CFU fell below the detection limit (2.3
logs).
A.
B.
20

Fig 2. A. In vivo efficacy of compound 32; B. In vivo efficacy of compound 42. Groups of 5
Balb/C mice were infected intranasaly with ~300 to 600 colony forming units (CFU) of
M. tuberculosis H37Rv. A control group was sacrificed one day after infection (D1) to
ascertain the required amounts of CFU were administered. Another control group was
sacrificed 3 weeks post-infection (D21) to validate the progression of the infection. The
remaining mice were treated from D21 by daily gavage (5 times a week), for a period of four
weeks, with TTCA compounds or the control drug isoniazid (INH). A control group was left
untreated and mice received only the vehicle used for TTCA compound (NT). Animals were
sacrificed one day after the treatment completion and lungs were collected. One lobe was
homogenized and serial-diluted in PBS to enumerate the CFU. The limit of detection (LOD,
below which no CFU could be counted) was approximately 200 CFU per mice (2.3 logs).
Statistical significance for the number of CFU in treated against untreated mice (NT) was
calculated using a Bonferroni’s multiple comparison test. Adjusted P values are indicated on
the graph. ****: < 0.0001; ns: not significant.
4. Conclusions
In summary, a new series of thienothiazolocarboxamide analogues were designed and
synthesized as potential anti-tuberculosis agents. Preliminary SAR and SPR studies on the
scaffold revealed that the carboxamide linker with a phenyl group was critical for the anti-
mycobacterial activity. In addition, biaryl substituents at position R1 led to increased anti-
21

tubercular potency, while varying the substituents at R2 position allowed tuning of the
physicochemical properties with minor influence on the activity. The compound 42 showed
good in vitro activity and drug-like ADME properties. It also demonstrated absence of toxicity
in various human-cancer cells, low risk of cardiotoxicity and no genotoxicity. In addition,
compound 42 exhibited a favorable in vivo pharmacokinetic profile, with an oral bioavailability
over 100% in mice (10 mg/kg). Compound 42 was efficacious in a murine model of TB, in a
dose-dependent manner. Hence, 42 was selected as a new lead compound for further evaluation
as a potential clinical candidate to combat tuberculosis.
5. Experimental section
5.1 Chemistry
5.1.1 General information
All commercially available chemicals were of reagent grade and were used without further
purification. Solvents were dried with standard procedures. All reactions were carried out under
argon atmosphere in flame-dried glassware with magnetic stirring. The proton nuclear
magnetic resonance (1H-NMR) spectra and carbon nuclear magnetic resonance (13C-NMR)
spectra were determined on a Varian (400 MHz, 100 MHz) or Bruker (400 MHz, 100 MHz)
spectrometer. Melting points (mp) were measured on an electro thermal melting point
apparatus, M-565 (BÜCHI). LC/MS data were obtained using a Waters ZQ2000 platform. The
purity of all biologically tested compounds were determined as ≥ 90 % by HPLC. All of the
reactions were purified by flash column chromatography using silica gel 60 (Merck, 230-400
mesh). Additionally, thin-layer chromatography was performed on 0.25 mm silica gel 60-F254
plates (Merck).
5.1.2. Procedure for the synthesis of compound 2.
To a stirred solution of 2,4-dichlorothiazole-5-carbaldehyde (1.0 equiv.) in CH3CN (10
mL) was added piperidine (1.2 equiv.) and triethylamine (3.0 equiv.) and the mixture was
stirred for 4 hours at room temperature. After the reaction was completed, as determined by
TLC, the reaction mixture was poured to the cold water and then extracted with EtOAc (20
mL), washed with sat. NaHCO3 (10 mL), dried over MgSO4 and concentrated. The crude
22

residue was purified by flash column chromatography (methylene chloride:MeOH = 50:1) to
give the desired compound 2.
5.1.3. Procedure for the synthesis of compound 3.
A mixture of 2 (1.0 equiv.), ethylthioglycolate (1.2 equiv.) and triethylamine (2.0 equiv.)
in DMSO (10 mL) was heated at 120 ℃ for 3 hours. The reaction mixture was diluted with
EtOAc (30 mL), washed with water (10 mL), dried over MgSO4 and concentrated. The
resulting residue was dissolved in EtOH (20 mL) and a sodium ethoxide solution (21% in EtOH,
2.0 equiv.) was added. The reaction mixture was stirred for one hour at room temperature. After
completion, the reaction was quenched with water (5 mL) and the organic solvent was
evaporated. The reaction mixture was extracted with EtOAc (20 mL), washed with water, dried
over MgSO4 and concentrated. The crude residue was purified by flash column
chromatography (n-hexane:EtOAc = 7:3) to give 3.
5.1.4. General procedure for the preparation of compounds 1, 8, 13 and 14.
To a cooled (ice bath), stirred solution of 3 (1.0 equiv.) and the desired amine (1.0 equiv.)
in toluene was added trimethylaluminum (2.0 M in toluene, 1.0 equiv.). The resulting mixture
was allowed to reach room temperature and then heated to 100 ℃ overnight. After reaction
completion, the reaction mixture was cooled down to room temperature, washed with
CH2Cl2/MeOH (20:1, v/v) and the solvent evaporated. The crude was purified by flash column
chromatography (methylene chloride:MeOH = 20:1) to obtain compounds 1, 8, 13 and 14.
5.1.4.1. N-(4-Methoxyphenyl)-2-(piperidin-1-yl)thieno[2,3-d]thiazole-5-carboxamide (1).
Ivory solid; mp = 235 °C; 1H-NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.08 (s, 1H), 7.59
(d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.73 (s, 3H), 3.52 - 3.56 (m, 4H), 1.61 - 1.65 (m,
6H); LCMS (electrospray) m/z 373 [M + H]+.
5.1.4.2. N-(4-methoxybenzyl)-2-(piperidin-1-yl)thieno[2,3-d]thiazole-5-carboxamide (8).
Yellow solid; mp = 142 °C; 1H-NMR (400 MHz, DMSO-d6) δ 8.84 (t, J = 8.0 Hz, 1H), 7.87
(s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.36 (d, J = 8.8 Hz, 2H), 3.72 (s,
13
3H), 3.49 - 3.55 (m, 4H), 1.60 - 1.65 (m, 6H); C-NMR (100 MHz, DMSO-d6) δ 173.35,
160.87, 157.64, 156.95, 130.97, 130.86, 128.05, 121.20, 119.20, 113.10, 54.47, 48.49, 41.38,
24.09, 22.90; LCMS (electrospray) m/z 388 [M + H]+.
23

5.1.4.3. N-(3,5-Dimethoxyphenyl)-2-(piperidin-1-yl)thieno[2,3-d]thiazole-5-carboxamide (13).
Yellow solid; mp = 148 °C; 1H-NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.11 (s, 1H), 6.99
(s, 1H), 6.98 (s, 1H), 6.23 (s, 1H), 3.73 (s, 6H), 3.52 - 3.56 (m, 4H), 1.61 - 1.65 (m, 6H); 13C-
NMR (100 MHz, DMSO-d6) δ 178.68, 159.81, 159.68, 158.07, 140.05, 130.56, 121.38, 120.49,
97.65, 94.89, 54.50, 48.58, 24.12, 22.89; LCMS (electrospray) m/z 404 [M + H]+.
5.1.4.4. N-(4-chlorophenyl)-2-(piperidin-1-yl)thieno[2,3-d]thiazole-5-carboxamide (14). Pale
yellow solid; mp = 275.2 °C; 1H-NMR (400 MHz, acetone-d6) δ 9.48 (brs, 1H), 7.96 (s, 1H),
7.79 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 3.58 - 3.63 (m, 4H), 1.68 - 1.74 (m, 6H);
13C-NMR (100 MHz, DMSO-d6) δ 173.75, 159.72, 158.18, 137.37, 130.12, 127.97, 126.38,
121.43, 120.93, 120.78, 48.57, 24.12, 22.88; LCMS (electrospray) m/z 378, 380 [M + H]+ (Cl-
isotope pattern).
5.1.5. Procedure for the synthesis of compound 4.
To a stirred solution of 1 (1.0 equiv.) in N,N-dimethylformamide (0.5 mL) was added
sodium hydride (1.2 equiv.) at 0 °C. After 20 min of stirring, iodomethane (2.0 equiv.) was
added to the solution and the mixture was further stirred at room temperature for 2 hours. After
reaction completion, the reaction mixture was poured in water and extracted with methylene
chloride (10 mL × 3). The organic layer was washed with brine (5 mL), dried over anhydrous
NaSO4 and concentrated in vacuo. The resulting crude residue was purified by flash column
chromatography (methylene chloride:MeOH = 20:1) to give 4.
5.1.5.1.
N-(4-Methoxyphenyl)-N-methyl-2-(piperidin-1-yl)thieno[2,3-d]thiazole-5-
carboxamide (4). Ivory solid; mp = 195 °C; lH-NMR (400 MHz, acetone-d6) δ 7.29 (d, J = 7.6
Hz, 2H), 7.02 (d, J = 7.6 Hz, 2H), 6.72 (s, 1H), 3.85 (s, 3H), 3.46 - 3.52 (m, 4H), 3.18 (s, 3H),
l.61 - 1.69 (m, 6H); 13C-NMR (100 MHz, DMSO-d6) δ 174.62, 162.23, 159.39, 159.26, 136.89,
129.94, 129.61, 124.26, 121.86, 115.46, 55.88, 49.57, 25.11, 23.94; LCMS (electrospray) m/z
388 [M + H]+.
5.1.6. Procedure for the synthesis of compound 5.
To a stirred solution of 3 (3.4 mmol) in tetrahydrofuran (20 mL) was added lithium
aluminum hydride (5.1 mmol) at 0 °C and the reaction mixture was stirred for 2 hours while
maintaining the temperature below 4 °C. The reaction was quenched by addition of water with
stirring and the insoluble solid was filtered off. The filtrate was extracted with methylene
24

chloride (50 mL × 2), dried over anhydrous MgSO4 and concentrated in vacuo. The crude
product was purified by flash column chromatography (n-hexane:EtOAc = 3:1) to give 5.
5.1.6.1. (2-(Piperidin-1-yl)thieno[2,3-d]thiazol-5-yl)methanol (5). 1H-NMR (400 MHz,
DMSO-d6) δ 7.02 (s, 1H), 5.39 (t, J = 5.6 Hz, 1H, OH), 4.56 (d, J = 5.6 Hz, 2H), 3.42 - 3.45
(m, 4H), 1.58 - 1.60 (m, 6H); LCMS (electrospray) m/z 255 [M + H]+.
5.1.7. Procedure for the synthesis of compound 7.
To a stirred solution of 5 (0.87 mmol) in methylene chloride (20.0 mL) was added Dess-
Martin periodinane (0.95 mmol) and the resulting mixture was stirred at room temperature for
one hour. The reaction mixture was diluted with methylene chloride (10.0 mL), washed with
brine (20 ml × 2), dried over anhydrous MgSO4 and concentrated in vacuo. The crude product
was purified by flash column chromatography (n-hexane:EtOAc = 5:1) to give 6. To the
aldehyde 6 (0.20 mmol) in methylene chloride (10.0 mL) was then added p-anisidine (0.22
mmol) and sodium triacetoxyborohydride (0.40 mmol) and the resulting mixture was stirred at
room temperature overnight. After reaction completion, the mixture was diluted with
methylene chloride (10 mL), washed with brine (10 mL × 2), dried over anhydrous MgSO4 and
concentrated in vacuo. The crude residue was purified by flash column chromatography (n-
hexane:EtOAc = 4:1) to give 7.
5.1.7.1. 4-Methoxy-N-((2-(piperidin-1-yl)thieno[2,3-d]thiazol-5-yl)methyl)aniline (7). Yellow
solid; mp = 105 °C; 1H-NMR (400 MHz, acetone-d6) δ 7.07 (s, 1H), 6.72 (d, J = 8.8 Hz, 2H),
6.69 (d, J = 8.8 Hz, 2H), 5.09 (brs, 1H), 4.47 (d, J = 5.6 Hz, 2H), 3.67 (s, 3H), 3.48 - 3.50 (m,
4H), 1.65 - 1.68 (m, 6H); LCMS (electrospray) m/z 360 [M + H]+.
5.1.8. Procedure for the synthesis of compound 10.
To a stirred suspension of ester compound 3 in MeOH (6.0 mL) was added an aqueous
solution of lithium hydroxide (6.32 mmol in 2.0 mL of water) and the mixture was heated to
50 °C for 3 hours. The organic solvent was evaporated and 1N HCl was added until the pH
reached 4. The residual white solid was collected by filtration, washed with water and dried in
vacuo to give the desired acid 9 as a white solid. To a solution of 9 (1.11 mmol) in tert-buthanol
(8 mL) was added diphenylphosphorylazide (1.34 mmol). The reaction mixture was stirred at
95 °C for 4 hours. The reaction mixture was diluted with methylene chloride (5 mL) and
25

washed with brine (5 mL). The organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo. The crude product was purified by flash column chromatography (n-
hexane: EtOAc = 10:1) to give the desired product 10.
5.1.8.1. tert-Butyl (2-(piperidin-1-yl)thieno[2,3-d]thiazol-5-yl)carbamate (10). Ivory solid; mp
= 157 °C; 1H-NMR (400 MHz, CDCl3) δ 6.62 (s, 1H), 6.61 (brs, 1H), 3.45 - 3.49 (m, 4H), 1.66
-1.71 (m, 6H), 1.50 (s, 9H); LCMS (electrospray) m/z 339 [M + H]+.
5.1.9. Procedure for the synthesis of reversed amide compound 12.
To a stirred solution of carbamate 10 (0.58 mmol) in 1,4-dioxane (4 mL) was added 35%
hydrogen chloride (0.7 mL). The reaction mixture was stirred at room temperature. After 2
hours, the reaction mixture was concentrated in vacuo and the crude product containing the
intermediate product 11 was used without further purification. To a stirred solution of 4-
methoxybenzoic acid (0.16 mmol) in anhydrous DMF (2.0 mL) were added l-(3-
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(0.24
mmol),
1-
hydroxybenzotriazole (0.082 mmol), triethylamine (0.33 mmol) and amine 11 (0.15 mmol) at
room temperature. The resulting solution was stirred at room temperature overnight. The
reaction mixture was concentrated, and the resulting crude residue purified by flash column
chromatography to obtain the desired product 12.
5.1.9.1. 4-Methoxy-N-(2-(piperidin-1-yl)thieno[2,3-d]thiazol-5-yl)benzamide (12). Green
solid; mp = 92 °C; 1H-NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1Η), 7.96 (d, J = 8.8 Hz, 2H),
7.08 (d, J = 8.8 Hz, 2H), 6.97 (s, 1H), 3.84 (s, 3H), 3.41 - 3.44 (m, 4H), 1.59 - 1.63 (m, 6H);
13C-NMR (100 MHz, DMSO-d6) δ 170.84, 161.81, 161.50, 146.68, 133.57, 128.88, 124.48,
117.88, 113.21, 102.28, 54.86, 48.56, 24.03, 23.00; LCMS (electrospray) m/z 374 [M + H]+.
5.1.10. Procedure for the synthesis of compound 15.
To a solution of compound 1 (1.0 equiv.) in methylene chloride was added
borontribromide (1.0 M in methylene chloride) at 0 °C. The reaction mixture was stirred at
room temperature for 2 hours. After reaction completion, the reaction mixture was quenched
with cold water and extracted with methylene chloride (10 mL × 2). The organic layer was
dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by
flash column chromatography (methylene chloride:MeOH = 50:1) to give 15.
26

5.1.10.1. N-(4-Hydroxyphenyl)-2-(piperidin-1-yl)thieno[2,3-d]thiazole-5-carboxamide (15).
Yellow solid; mp = 246 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.05 (s, 1H), 7.44
13
(d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H), 3.51 - 3.55 (m, 4H), 1.60 - 1.64 (m, 6H); C-
NMR (100 MHz, DMSO-d6) δ 173.47, 159.22, 157.41, 153.01, 131.16, 129.78, 121.50, 121.26,
119.81, 114.44, 48.54, 24.11, 22.89; LCMS (electrospray) m/z 360 [M + H]+.
5.1.11. General procedure for the preparation of compounds 16a-e.
To a stirred solution of 2,4-dichlorothiazole-5-carbaldehyde (1.0 equiv.) in CH3CN (10
mL) were added the desired amine (1.2 equiv.) and triethylamine (3.0 equiv.) and the mixture
was stirred for 4 hours at room temperature. After the reaction completion, the reaction mixture
was poured in cold water and extracted with EtOAc (20 mL), washed with sat. NaHCO3 (10
mL), dried over MgSO4 and concentrated. The crude residue was purified by flash column
chromatography (methylene chloride:MeOH = 50:1) to give desire compounds 16a-e.
5.1.12. General procedure for the preparation of compounds 17a-e.
A mixture of aldehyde (16a-e) (1.0 equiv.), ethylthioglycolate (1.2 equiv.) and
triethylamine (2.0 equiv.) in DMSO (10 mL) was heated to 120 ℃ for 3 hours. The reaction
mixture was diluted with EtOAc (30 mL), washed with water (10 mL), dried over MgSO4 and
concentrated. The resulting residue was dissolved in EtOH (20 mL) and sodium ethoxide
solution (21% in EtOH, 2.0 equiv.) was added. The reaction mixture was stirred for one hour
at room temperature. After completion, the reaction was quenched with water (5 mL) and the
organic solvent was evaporated. The reaction mixture was extracted with EtOAc (20 mL),
washed with water, dried over MgSO4 and concentrated. The crude residue was purified by
flash column chromatography (n-hexane:EtOAc = 7:3) to give compounds 17a-e.
5.1.13. General procedure for the preparation of compounds 18-19 and 21-23.
To a stirred solution of ester (17a-e) (1.0 equiv.) and p-anisidine (1.0 equiv.) in toluene
was added trimethylaluminum (2.0 M in toluene, 1.0 equiv.) under cooling with an ice bath.
The resulting mixture was allowed to reach room temperature and then heated at 100 ℃
overnight. After completion, the reaction mixture was cooled down to room temperature,
washed with CH2Cl2/MeOH (20:1, v/v) and the solvent evaporated. The crude was purified by
flash column chromatography (methylene chloride:MeOH = 20:1) to obtain compounds 18-19
and 21-23.
27

5.1.13.1. N-(4-Methoxyphenyl)-2-morpholinothieno[2,3-d]thiazole-5-carboxamide (19). Pale
brown solid; mp = 248.8 °C; 1H-NMR (400 MHz, acetone-d6) δ 9.32 (s, 1H), 7.96 (s, 1H), 7.66
(d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.79 - 3.81 (m, 4H), 3.78 (s, 3H), 3.57 - 3.59 (m,
4H); 13C-NMR (100 MHz, DMSO-d6) δ 173.99, 159.28, 157.05, 154.88, 131.72, 131.29,
121.70, 121.22, 120.07, 113.23, 64.73, 54.58, 47.43; LCMS (electrospray) m/z 376 [M + H]+.
5.1.13.2.
N-(4-Methoxyphenyl)-2-(4-methylpiperazin-1-yl)thieno[2,3-d]thiazole-5-
1
carboxamide (21). White solid; mp = 219.8 °C; H-NMR (400 MHz, DMSO-d6) δ 10.03 (s,
1H), 8.11 (s, 1H), 7.63 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 3.73 - 3.78 (m, 4H), 3.58
(s, 3H), 3.53 (s, 3H), 2.23 - 2.78 (s, 4H); 13C-NMR (100 MHz, DMSO-d6) δ 174.74, 160.38,
158.35, 155.95, 132.52, 132.39, 122.74, 122.28, 121.12, 114.31, 55.66, 54.06, 48.39, 46.11;
LCMS (electrospray) m/z 389 [M + H]+.
5.1.13.3.
2-(4,4-Difluoropiperidin-1-yl)-N-(4-methoxyphenyl)thieno[2,3-d]thiazole-5-
carboxamide (22). Pale yellow solid; mp = 263.5 °C; 1H-NMR (400 MHz, DMSO-d6) δ 10.06
(s, 1H), 8.11 (s, 1H), 7.58 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.74 (s, 3H), 3.69 - 3.72
(m, 4H), 2.09 - 2.19 (m, 4H); 13C-NMR (100 MHz, DMSO-d6) δ 174.10, 160.33, 158.04,
155.97, 132.99, 132.36, 123.33, 122.89, 122.29, 121.13, 114.32, 55.66, 45.68 (t, J = 5.0 Hz),
33.01 (t, J = 24.0 Hz); LCMS (electrospray) m/z 410 [M + H]+.
5.1.13.4. 2-(Dimethylamino)-N-(4-methoxyphenyl)thieno[2,3-d]thiazole-5-carboxamide (23).
Pale brown solid; mp = 192.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H, ΝΗ), 8.12
(s, 1H), 7.63 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 9.2 Hz, 2H) 3.78 (s, 3H), 3.18 (s, 6H); LCMS
(electrospray) m/z 334 [M + H]+.
5.1.14. Procedure for the synthesis of compound 20.
To a stirred solution of 18 in methylene chloride (10.0 mL) was added excess
trifluoroacetic acid at room temperature. The mixture was stirred at room temperature for 2-4
hr. The solvent was evaporated in vacuo to give compound 20 without further purification.
5.1.14.1. N-(4-Methoxyphenyl)-2-(piperazin-1-yl)thieno[2,3-d]thiazole-5-carboxamide (20).
1
Pale yellow solid; H-NMR (400 MHz, acetone-d6) δ 9.32 (s, 1H), 7.96 (s, 1H), 7.67 (d, J =
8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.58 - 3.59 (m, 4H), 3.00 - 3.01 (m, 4H);
LCMS (electrospray) m/z 375 [M + H]+.
28

5.1.15. Procedure for the synthesis of compound 25.
To a stirred solution of 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (4.11 mmol)
in methylene chloride (20.0 mL) was added dimethylamine hydrochloride (4.93 mmol), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(6.17
mmol),
1-
hydroxybenzotriazole hydrate (6.17 mmol) and triethylamine (13.2 mmol) at room temperature.
The mixture was stirred at room temperature overnight. The reaction mixture was evaporated,
extracted with EtOAc (30 mL) and washed with water (20 mL). The organic layer was dried
over anhydrous MgSO4 and concentrated in vacuo. The resulting crude residue was purified by
flash column chromatography (methylene chloride:MeOH = 50:1) to give 24. To a mixture of
compound 24 (3.12 mmol) in methylene chloride (10.0 mL) was added trifluoroacetic acid (2.0
mL) at room temperature. The mixture was stirred at room temperature for 2-4 hr. The solvent
was evaporated in vacuo to give compound 25 without further purification.
5.1.16. General procedure for the preparation of aldehydes 26a-g.
To a stirred solution of 2,4-dichlorothiazole-5-carbaldehyde (1.0 equiv.) in CH3CN was
added the desired amine (1.2 equiv.), triethylamine (3.0 equiv.) and the mixture was stirred for
4 hours at room temperature. After the reaction completion, the reaction mixture was poured
in cold water and extracted with EtOAc, washed with sat. NaHCO3, dried over MgSO4 and
concentrated. The crude residue was purified by flash column chromatography (methylene
chloride:MeOH = 50:1) to give the desired compounds 26a-g.
5.1.17. General procedure for the preparation of esters 27a-g.
A mixture of aldehyde (26a-e) (1.0 equiv.), ethylthioglycolate (1.2 equiv.) and
triethylamine (2.0 equiv.) in DMSO (10 mL) was heated to 120 ℃ for 3 hours. The reaction
mixture was diluted with EtOAc (30 mL), washed with water (10 mL), dried over MgSO4 and
concentrated. The resulting residue was dissolved in EtOH (20 mL) and sodium ethoxide
solution (21% in EtOH, 2.0 equiv.) was added. The reaction mixture was stirred for one hour
at room temperature. After completion, the reaction was quenched with water (5 mL) and the
organic solvent evaporated. The reaction mixture was extracted with EtOAc (20 mL), washed
with water, dried over MgSO4 and concentrated. The crude residue was purified by flash
column chromatography (n-hexane:EtOAc = 3:1) to give compounds 27a-g.
5.1.18. General procedure for synthesis of compounds 28-35.
29