Design, synthesis, and evaluation of substituted 2-acylamide-1,3-benzo[d]zole analogues as agents against MDR- and XDR-MTB

Article abstract of DOI:10.1016/j.ejmech.2020.112898

N-(5-Chlorobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamideox-amide has been identified as a potent inhibitor of Mtb H37Rv, with a minimum inhibitory concentration (MIC) of 0.42 μM. In this study, a series of substituted 2-acylamide-1,3-zole analogues were designed and synthesized, and their anti-Mtb activities were analyzed. In total, 17 compounds were found to be potent anti-Mtb agents, especially against the MDR- and XDR-MTB strains, with MIC values < 10 μM. These analogues can inhibit both drug-sensitive and drug-resistant Mtb. Four representative compounds were selected for further profiling, and the results indicate that compound 18 is acceptably safe and has favorable pharmacokinetic (PK) properties. In addition, this compound displays potent activity against Gram-positive bacteria, with MIC values in the range of 1.48–11.86 μM. The data obtained herein suggest that promising anti-Mtb candidates may be developed via structural modification, and that further research is needed to explore other compounds.

Full text of DOI:10.1016/j.ejmech.2020.112898

Journal Pre-proof
Design, Synthesis, and Evaluation of Substituted 2-acylamide-1,3-benzo[d]zole
Analogues as Agents against MDR- and XDR-MTB
Dongsheng Li, Chao Liu, Xinhai Jiang, Yuan Lin, Jing Zhang, Yan Li, Xuefu You, Wei
Jiang, Minghua Chen, Yanni Xu, Shuyi Si
PII:
S0223-5234(20)30870-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112898
EJMECH 112898
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 June 2020
Revised Date: 11 September 2020
Accepted Date: 27 September 2020
Please cite this article as: D. Li, C. Liu, X. Jiang, Y. Lin, J. Zhang, Y. Li, X. You, W. Jiang, M. Chen, Y.
Xu, S. Si, Design, Synthesis, and Evaluation of Substituted 2-acylamide-1,3-benzo[d]zole Analogues as
Agents against MDR- and XDR-MTB, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
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Design, Synthesis, and Evaluation of Substituted 2-acylamide-1,3-benzo[d]zole
Analogues as Agents against MDR- and XDR-MTB
Dongsheng Li§, Chao Liu§, Xinhai Jiang, Yuan Lin, Jing Zhang, Yan Li, Xuefu You, Wei Jiang, Minghua
Chen*, Yanni Xu*, Shuyi Si*
Beijing Key Laboratory of Antimicrobial Agents, and National Center for New Microbial Drug Screening, Institute of Medicinal Biotechnology, Chinese
Academy of Medical Sciences & Peking Union Medical College, Tiantanxili No 1, Beijing 100050, P. R. China
ABSTRACT
N-(5-Chlorobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamideox-amide has been identified as a potent inhibitor of Mtb
H37Rv, with a minimum inhibitory concentration (MIC) of 0.42 ꢀM. In this study, a series of substituted 2-acylamide-1,3-zole
analogues were designed and synthesized, and their anti-Mtb activities were analyzed. In total, 17 compounds were found to be potent
anti-Mtb agents, especially against the MDR- and XDR-MTB strains, with MIC values < 10 ꢀM. These analogues can inhibit both
drug-sensitive and drug-resistant Mtb. Four representative compounds were selected for further profiling, and the results indicate that
compound 18 is acceptably safe and has favorable pharmacokinetic (PK) properties. In addition, this compound displays potent activity
against Gram-positive bacteria, with MIC values in the range of 1.48–11.86 ꢀM. The data obtained herein suggest that promising
anti-Mtb candidates may be developed via structural modification, and that further research is needed to explore other compounds.
KEYWORDS
Substituted 2-acylamide-1,3-zole, Anti-tuberculosis agent, MDR-MTB, XDR-MTB
§These authors contributed equally to this work.
*Correspondence:
Minghua Chen, E-mail: mingsunlight@sina.com;
Yanni Xu, E-mail: xuyanniwendeng@hotmail.com;
Shuyi Si, E-mail: sisyimb@hotmail.com, Tel:+86 10 63180604, Fax: +86 10 63180604.

1. Introduction
Tuberculosis (TB), an airborne contagious disease caused by mycobacterium tuberculosis (Mtb) bacteria, is considered to be one of
the most serious health hazards in the world, and it is ranked as the top infectious killer on a global level [1]. According to the World
Health organization (WHO), an estimated 10.0 million people contracted TB worldwide in 2018. Of the infected patients, an estimated
1.45 million die each year, including 1.2 million HIV-negative and 0.25 million HIV-positive patients [2]. The first-line drugs currently
used in combination anti-Mtb chemotherapy (rifampin, isoniazid, pyrazinamide, and ethambutol) constitute the most effective weapons
against drug-sensitive TB, as they target different physiological phases (replicating and non-replicating [3]) affected by Mtb in human
lungs. However, these drugs are limited by the need for prolonged directly observed therapy (DOT) treatment (at least 6 months) and
follow-up support [3−5]. Moreover, the emergence of multi-drug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains
of Mtb have limited the effectiveness of the available drugs and exacerbated the TB epidemic [6−8]. The risk of disease reactivation in
asymptomatically infected individuals or those harboring latent Mtb constitutes an additional challenge associated with TB, particularly
if these individuals are co-infected with HIV, have diabetes, or are subject to anti-tumor necrosis factor therapy [6,9].
Most first-line and second-line anti-Mtb drugs were discovered in the 1950s and 1960s [9]. In fact, only two drugs (bedaquiline and
delamanid) were approved by the US Food and Drug Administration (FDA) for MDR-TB treatment during the last four decades.
Although many new/repurposed drugs and combination therapies, such as FS-1, PA-824, TBA-7371, linezolid, and nitrazoxanide, are
available at the Clinical Trials website, new chemicals that simplify and shorten treatment, target MDR-MTB or XDR-MTB strains,
require low dosing frequency and can be co-administered with HIV medications [6,10,11] still need to be developed in order to control
the TB epidemic.
Whole-cell-based high-throughput screening (HTS) constitutes a promising method for the synthesis of new anti-Mtb compounds
[12−14].
Previously,
we
had
shown
that
the
IMB-T097
molecule,
N-(5-chlorobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamideox-amide (Figure 1) prepared using whole-cell-based HTS
is a potent inhibitor of Mtb H37Rv, with an MIC of 0.42 ꢀM [15]. Herein, we establish a robust method for the synthesis of drug-like
molecules based on the preliminary structure−activity relationship (SAR) analysis of IMB-T097. Considering that fused or non-fused
2-aminothiazole is a frequent emergency segment in antibacterial agents [16-20], and that non-fused 2-amino oxazol analogues have
weak anti-Mtb H37Rv activity (the data are not shown in this study), only fused oxazole derivatives are explored.
Figure 1. Hit molecule: IMB-T097
2. Chemistry
Figure 2 depicts the systematic structural modifications of IMB-T097 implemented in this study. To examine the effect of electrical
properties and steric hindrance on anti-Mtb activity, a series of derivatives with different R2 groups was synthesized. Another series of
IMB-T097 analogues was prepared by replacing the 1,2,3-thiadiazole ring with different five- or six-membered rings (R1), including
thiophene, furan, and benzene. Also, the benzoxazole group was replaced with benzothiazole, benzimidazole, or
oxazolo[4,5-b]pyridine. Among the synthesized derivatives, compound 6 that has a large hydrocarbon group (t-Bu) at C-5 position
showed excellent anti-Mtb activity (MIC: ~0.40 ꢀM). Therefore, other compounds comprising large monosubstituted groups or fused
cycles at C-4 and/or C-5 positions of the 2-amine benzo[d]oxazol were also explored.

Figure 2. Systematic modification and SAR of IMB-T097
The synthetic route used to prepare the target compounds is outlined in Scheme 1. The derivatives in which X is an oxygen atom
were prepared by reacting different 2-amino phenols (1a−11a, 25a, 27a−29a) with cyanogen bromide in methanol. The resulting
2-amine benzo[d]oxazols were obtained at relatively high yields (75%−93%). Compounds 25c and 26c were synthesized via
2-aminobenzo[d]oxazol-4-ol substitution of compound 25b at C-4 position in the presence of a halide and a base.
Benzo[d]thiazol-2-amine 12b and 1H-benzo[d]imidazol-2-amine 13b were directly purchased from a commercial supplier. As for the
substituted 2-acylamide-1,3-benzo[d]zole analogues (1
with amide (1b 11b, 25c 26c, 27b 29b) using the EDCI-HoBt-TEA system and the DMF solvent. The yields of these analogues
ranged between 22 and 75%. The compounds comprising an unsaturated cycle at C-4 or C-5 positions (compounds 18 24) were
−17, 25−29), they were prepared by condensation reaction of carboxylic acid
−
−
−
−
synthesized via the Suzuki-Miyaura cross coupling reaction of 4-Br/5-Br substituted compounds 4 or 7 with the corresponding boric
acid. Considering that pyridin-2-ylboronic acid is an unstable agent that cannot be readily used in the laboratory, compound 19 was
prepared via the Stille coupling reaction with an alternative agent, 2-(tributylstannyl)pyridine. Finally, compound 24 was obtained with
19% yield by reacting compound 4 with morpholine (Buchwald−Hartwig cross coupling reaction).

Scheme 1. General scheme of the synthesis of Disubstituted Azole Analogues 1
−29. Reagents and conditions: (a) BrCN, MeOH, 0°C – rt, overnight; (b) copper,
Cs2CO3, DMF, 110°C, 2 h or K2CO3, ACN, reflux, 8 h; (c) carboxylic acid, EDCI, HoBt, TEA, DMF, 0°C – rt, 16 h; (d) Suzuki
Pd(PPh3)4, K3PO4, DMF-H2O, microwave, 150°C, 30 min; Stille reaction: 2-(tributylstannyl)pyridine, Pd2(dba)3, K3PO4, anhydrous DMF, reflux, 5 h;
−
Miyaura reaction: boronic acid,
Buchwald-Hartwig reaction: XPhos, Pd(PPh3)4, t-BuOK, DME, 110°C, 2 h.
3. Results and discussion
3.1. In vitro anti-Mtb activity and structure activity relationship (SAR)
In vitro analyses of the activities of all synthesized 2-acylamide-1,3-benzo[d]zole analogues against M. tuberculosis H37Rv, the MDR
strain (FJ05120), and the XDR strain (FJ05195) were conducted in 7H9 medium using the Micro plate Alamar Blue Assay (MABA).
The isoniazid (INH), rifampicin (RIF), streptomycin (SM), and ethambutol (EMB) first-line drugs were used as standards (Table 1).
Seventeen of the 29 target compounds prepared herein showed high anti-H37Rv potency, as well as excellent activity against the
MDR- and XDR-MTB strains (MIC < 10 ꢀM). In fact, many of these compounds were found to be more effective than the standard
drugs. Specifically, strong anti-Mtb activity (MIC < 10 ꢀM) was observed for the compounds having electron-withdrawing or
electron-donating groups on the aryl ring (1-10). Notably, the anti-Mtb activity of the compound comprising a 5-tert-butyl substituent
(compound 6, MIC ~0.40 ꢀM) was found to be 16 times less than that of the compound with a 6-tert-butyl substituent (compound 9,
MIC 3.16−6.32 ꢀM), which indicates that the exotonier of C-5 is more suitable for holding large groups.
When the benzene ring of compound 2 was replaced with pyridine (compound 11), the MIC of anti-Mtb activity was increased from
0.48 to 7.66 ꢀM, possibly due to the low cLogP value of 11. Compared with the oxazole analogues, the thiazole and imidazole
compounds (12 and 13) showed lower anti-Mtb activity (MIC 28.95−57.90 ꢀM), indicating that oxazole is a more suitable segment in
the scaffold. Similarly, the substitution of the 1,2,3-thiadiazole ring with other aromatic rings diminished anti-Mtb activity, especially
for compound 17 (MIC 268.63−537.25 ꢀM).
Table 1
In vitro anti-MTB evaluation of all the synthesized compounds and four controls against H37Rv, MDR-MTB (FJ05120), and
XDR-MTB (FJ05195) and cell cytotoxicity in HEK-293.
Strain range of anti-MTB MIC
MTT IC50
(ꢀM)
(ꢀM)
Compd
ClogPa
MDRb
FJ05120
HRc
XDRc
H37Rv
FJ05195
HEK-293
HRSCOc
1
2
3
4
5
6
1.60
0.84
1.03
1.75
1.34
2.67
0.42
0.48
0.45
2.95
1.82
0.40
1.70
0.48
0.45
1.47
7.29
0.40
1.70
0.48
1.80
1.47
7.29
0.40
6.65
19.56
42.03
45.01
21.93
6.73

7
8
1.75
1.60
2.67
0.89
-0.17
1.53
0.99
1.93
2.50
3.25
2.75
1.45
1.45
1.24
1.24
2.70
0.85
0.71
1.40
0.49
2.84
0.89
0.89
1.47
0.42
0.74
0.42
0.74
0.85
10.59
7.14
2.42
NDd
15.18
>100
>100
>100
81.94
ND
9
3.16
6.32
6.32
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
INH
RIF
SM
EMB
3.14
1.57
3.14
7.66
3.83
7.66
28.95
30.85
17.53
16.38
14.35
268.63
0.74
57.90
30.85
70.11
32.75
14.35
537.25
0.37
57.90
30.85
35.06
32.75
14.35
537.25
0.37
>100
>100
ND
23.71
2.96
23.71
1.48
47.43
2.96
54.93
ND
1.48
0.74
1.48
1.27
0.63
0.63
ND
1.47
0.73
1.47
ND
11.58
5.44
23.16
2.72
23.16
5.44
18.32
61.82
>100
80.86
ND
2.82
5.64
2.82
103.12
102.79
102.79
<3.65
<0.61
<0.69
<2.45
103.12
6.42
103.12
25.70
25.70
29.17
>311.08
87.83
39.16
25.70
7.29
59.03
--
>311.08
<0.69
4.89
--
--
--
aClogP values were calculated using ChemDraw version 12.0, Perkin–Elmer.
bMDR-MTB = multidrug resistant Mtb; XDR-MTB = extensively drug resistant Mtb.
cDrugs abbreviated as H = isoniazid, R = rifampicin, S = streptomycin, C = capreomycin, O = ofloxacin.
dNot determined.
All of the derivatives with substituents at C-4 (compounds 19, 25, and 26) or C-5 (compounds 18, and 20-24) presented potent
anti-Mtb activity (MIC 0.37−23.16 ꢀM). Except for the compound substituted with an alkyl heterocycle (compound 24, MIC
11.58−23.16 ꢀM), the C-5-substitued derivatives (compounds 18, and 20−23) showed particularly high anti-Mtb activity (MIC
0.37−2.96 ꢀM), with pyridin-2-yl substitution (compound 18) yielding the best effectiveness (MIC 0.37 ꢀM). The obtained results
confirm that C-4 and C-5 are capable of accommodating large groups, resulting in broader exposure to the target. Inspired by the
effectiveness of the C-4 and C-5 substituted derivatives, three other analogues with fused aromatic rings at these positions (compounds
27-29) were further synthesized. Unfortunately, these compounds showed relatively low anti-Mtb activity, which is probably due to
their rigid characteristics.
3.2. Cell cytotoxicity and acute toxicity
The above 29 compounds were tested for mammalian cell cytotoxicity using human embryonic kidney cells (HEK-293) measured as
IC50 value as compared to control, and the results are reported in Table 1. The result reflects that compounds 1
−11 (except compound 10)
exhibit a certain degree of cytotoxicity (IC50, 2.42 45.01 ꢀM). Meanwhile, the compounds 18, 20, 25, 26 and 29, which have different
−
aryl groups at C-4 or/and C-5 site, show lower cytotoxicity (IC50 ≥ 54.93 ꢀM).
Considering the potent anti-Mtb activities, compounds 1, 6, 18, and 26, which represent different design strategies were selected for

further profiling. As shown in Table 2, compounds 1 and 6 yield similar IC50 values (6.65 and 6.73 ꢀM, respectively), with the latter
showing a higher selectivity index value due to its stronger anti-Mtb activity (MIC of 6 is 4 times less than that of 1). As for compounds
18 and 26, they both present low cytotoxicity (IC50 > 100 ꢀM) and high selectivity index. In vivo acute toxicity study was implemented
by recording the number of survivors after an oral single dose in mice of 1000 mg/kg following a 7 days observation. As shown in Table
2, compound 1 shows some oral acute lethal toxicity, while compounds 6 and 18 show very low oral acute lethal toxicity. In general,
derivatives 1, 6, and 18 are safe for use in mice.
Table 2
The cytotoxicity and acute toxicity of compounds 1, 6, 18, and 26.
IC50 (ꢀM)
HEK-293 cell lines
P. O. dose
(mg/kg)
no. of animals that survived/
total no. of animals
compd
Selectivity Index (SI) a
6.65
6.73
4
1000
1000
1000
N/Ab
4/6
6/6
1
6
17
>100
>270
6/6
N/Ab
18
>100
>18
26
aSelectivity Index: IC50 (ꢀM)/MIC (ꢀM); bnot tested, since 26 shows poor PK property
3.3. In vivo pharmacokinetics (PK)
The in vivo pharmacokinetic (PK) profiles of compounds 6, 18, and 26 were tested in rats after oral administration of a single 40
mg/kg dose. Blood samples were collected at times ranging between 0 and 8 h post-ingestion. As shown in Table 3, the Cmax, AUC(0-t)
,
MRT, and Tmax values of compound 26 are 6.93 ± 1.44 ng/mL, 37.97 ± 10.21 h•ng/mL, 4.12 ± 0.38 h, and 2.83 h, respectively.
Comparatively, the PK properties of compound 6 are relatively better, with Cmax = 6836.55 ± 740.10 ng/mL, AUC(0-t) = 24802.17 ±
2888.66 h•ng/mL, and MRT = 5.26 ± 0.35. However, the best properties were observed for compound 18 whose Cmax, AUC(0-t), and
MRT values are 9048.41 ± 1473.34 ng/mL, 27794.11 ± 4255.06 h•ng/mL, and 5.59 ± 0.12 h, respectively. Since the analyzed blood
samples were collected during the limited period of 8 h only, the T1/2, Vd/F, and CL/F values of 6 and 18 could not be obtained. Overall,
the PK results suggest that substitution at C-5 position favors the PK profiles of the benzoxazole scaffold.
Table 3
Pharmacokinetic parameters of compounds 6, 18, and 26 in rats administered with single 40 mg/kg oral doses (Mean ± SD, n = 3).
compd
6
Tmax (h)
≥8
Cmax (ng/mL)
6836.55 ± 740.10
9048.41 ± 1473.34
6.93 ± 1.44
AUC(0-t) (h·ng/mL)
24802.17 ± 2888.66
27794.11 ± 4255.06
37.97 ± 10.21
MRT (h)
5.26 ± 0.35
5.59 ± 0.12
4.12 ± 0.38
≥8
18
2.83
26
3.4. Broad-spectrum panel study
Ten representative bacterial strains (Five Gram-positive: S. aureus 29213, S. aureus 33591, S. epidermidis 12228, E. faecalis 29212,
and E. faecium 700221; and five Gram-negative: E. coli 25922, K. pneumonia 700603, K. pneumonia 7, P. aeruginosa 27853, A.
calcoacetious 19606) were used to study the broad-spectrum panels of compounds 6 and 18. The results summarized in Table 4 indicate
that both compounds exhibit more potent activity against the Gram-positive bacterial strains (MIC 1.48−12.64 ꢀM) than against the
Gram-negative ones (MIC > 379.42 ꢀM).
Table 4
Broad-spectrum panels of compounds 6 and 18.
MIC (ꢀM)
Species
6
18
S. aureusATCC29213a
S. aureusATCC33591b
S. epidermidisATCC12228c
E. faecalisATCC29212d
6.32
6.32
6.32
12.64
5.93
1.48
2.96
11.86

E. faeciumATCC700221 e
E. coli ATCC25922f
12.64
5.93
> 404.58
> 404.58
> 404.58
> 404.58
> 404.58
> 379.42
> 379.42
> 379.42
> 379.42
> 379.42
K. pneumoniaATCC700603g
K. pneumonia 7f
P. aeruginosaATCC27853
A. calcoacetiousATCC19606
b
c
aMethicillin sensitive Staphylococcus aureus, MSSA; Methicillin resistant Staphylococcus aureus, MRSA; Methicillin sensitive Staphylococcus epidermidis,
MSSE; dVancomycin susceptible Enterococcus, VSE; eVancomycin resistant Enterococcus, VRE; Extended-Spectrum β-Lactamases negative, ESBLs (-);
f
gExtended-Spectrum β-Lactamases positive, ESBLs (+).
4. Conclusion
A series of substituted 2-acylamide-1,3-benzo[d]zole analogues were designed and synthesized, and their activities against H37Rv,
MDR-MTB, and XDR-MTB were assessed. The obtained results indicate that most of the synthesized compounds exhibit strong
anti-Mtb activity. The mechanism of this activity is probably novel, considering that the analogues can inhibit both, drug sensitive and
drug resistant Mtb. Based on preliminary structure−activity relationship (SAR) assessments, large-group-substitution at positions C-4
and C-5 of IMB-T097 might lead to higher activity due to greater exposure to the target. Of the 29 compounds prepared herein, four
were assessed in terms of their potential to be used as drugs. Of these four compounds, 18 displayed the lowest cytotoxicity and acute
toxicity, as well as the best pharmacokinetic (PK) properties. Broad-spectrum panel studies revealed that compound 18 is also a potent
anti-Gram-positive agent. In order to develop a promising novel anti-Mtb candidate for TB treatment, especially MDR-TB and
XDR-TB, a further SAR exploration of compound 18 and corresponding ADMET research are in progress and the study results will be
communicated in due time.
5. Experimental
5.1. General information
All of the reagents and solvents were purchased from commercial suppliers and used without further purification. The reactions were
monitored using thin-layer chromatography (TLC) analysis performed on commercial silica-gel plates (GF254), or using LC-MS
analysis on an Agilent 1100 HPLC system. 1H NMR (nuclear magnetic resonance) and 13C NMR spectra were recorded in
DMSO-d6/CDCl3/CD3OD solvent on a Bruker spectrometer (400 MHz/100 MHz, and 600 MHz/150 MHz), using the tetramethylsilane
(TMS) peak as an internal reference. ESI-HRMS data were measured on Thermo LTQ XL.
5.2. Synthesis of compounds
5.2.1 Ethyl 4-methyl-1,2,3-thiadiazole-5-carboxylatete
Methyl hydrazinecarboxylate (5.40 g, 0.06 mol) was dissolved in 30 mL anhydrous EtOH, and then a solution of ethyl acetoacetate
(7.81 g, 0.06 mol) in 20 mL anhydrous EtOH was slowly added. The mixture was stirred and reacted at room temperature for 6 h before
evaporating the solvent under reduced pressure. The yellow solid (hydrazone) obtained at the end was used in the next step without
further purification.
A solution of the crude hydrazone dissolved in 50 mL dichloromethane was added dropwise to 15 mL of SOCl2 at temperatures < 5°C.
The mixture was stirred and reacted overnight at room temperature, and then the solvent and excess SOCl2 were evaporated under
reduced pressure. The residue was purified on a silica chromatography column using PE/EA (1/1, v/v) as mobile phase. The yield of the
yellow oil (5.37 g) collected at the end was found to be 52%, and based on MS-ESI (mass spectrometry-electron spray ionization)
analysis, its m/z ratio is 173.06 [M+H]+.
5.2.2 4-methyl-1,2,3-thiadiazole-5-carboxylic acid
In
a 100 mL flask, 20 mL NaOH(aq) (3 M) were added to 10 mL EtOH and 5.14 g (0.03 mol) ethyl
4-methyl-1,2,3-thiadiazole-5-carboxylatet. The mixture was stirred at room temperature for 3 h, and then EtOH was evaporated and the
residue was dispersed in 40 mL of water. The pH of the solution was adjusted to ~3 using HCl (conic.). The precipitated solid was
collected and washed with ester to give the title compound (white solid, 3.67 g) at 85% yield without any further purification. 1H NMR
(400 MHz, CD3OD): δ 2.85 (s, 3H). 13C NMR (100 MHz, CD3OD): δ 161.74, 160.48, 140.82, 12.29. MS-ESI (m/z): 145.0 [M+H]+.

5.2.3 General Procedure for the Synthesis of benzoxazol-2-amino compounds (1b–12b, 25b, 27b-29b)
BrCN (2.03 g, 17.6 mmol, 1.1eq) was slowly added to a solution of 2-aminophenol (16.0 mmol, 1.0 eq) in 30 mL MeOH. The
reaction mixture was stirred overnight at room temperature. Excess BrCN was quenched with saturated Na2CO3 in a fume hood, until
the pH value reached ~7. Then, MeOH was evaporated and EtOAc (30 mL) was added. The organic phase was washed with water (20
mL × 2) and brine (20 mL), and then it was dried over anhydrous Na2SO4, filtered, and evaporated to give the title compound without
further purification.
5.2.3.1
8.0 Hz, 1H), 7.23 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.0, 2.0 Hz, 1H). MS-ESI (m/z): 169.0 [M+H]+.
5-Chlorobenzo[d]oxazol-2-amine (1b). Pale solid, 91% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.61 (brs, 2H), 7.32 (d, J =
5.2.3.2
benzo[d]oxazol-2-amine (2b). White solid, 83% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.16 (t, J = 8.0 Hz, 2H), 7.36 (d, J
= 8.0 Hz, 2H), 9.58 (s, 2H). MS-ESI (m/z): 135.1 [M+H]+.
5.2.3.3
5-fluorobenzo[d]oxazol-2-amine (3b). Pale solid, 86% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.64 (brs, 2H), 6.76–7.31 (m,
3H). MS-ESI (m/z): 153.0 [M+H]+.
5.2.3.4
5-bromobenzo[d]oxazol-2-amine (4b). Beige solid, 87% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.61 (brs, 2H), 7.36 (d, J =
2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.4, 2.0 Hz, 1H). MS-ESI (m/z): 213.2 [M+H]+.
1
5.2.3.5
5-methylbenzo[d]oxazol-2-amine (5b). Pale solid, 93% yield. H NMR (400 MHz, DMSO-d6): δ 7.28 (brs, 2H), 7.18 (d, J =
8.0 Hz, 1H), 7.01 (d, J = 2.0 Hz, 1H), 6.77 (dd, J = 8.0, 2.0 Hz, 1H), 2.31 (s, 3H). MS-ESI (m/z): 148.9 [M+H]+.
1
5.2.3.6
5-(tert-butyl)benzo[d]oxazol-2-amine (6b). Yellow solid, 88% yield. H NMR (400 MHz, DMSO-d6): δ 7.54 (brs, 2H), 7.20
(d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 6.65 (dd, J = 8.0, 2.0 Hz, 1H), 1.30 (s, 9H). MS-ESI (m/z): 191.1 [M+H]+.
5.2.3.7
4-bromobenzo[d]oxazol-2-amine (7b). Beige solid, 81% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.25 (brs, 2H), 7.44 (d, J =
8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H). MS-ESI (m/z): 213.2 [M+H]+.
1
5.2.3.8
6-chlorobenzo[d]oxazol-2-amine (8b). Brown solid, 80% yield. H NMR (400 MHz, DMSO-d6): δ 7.61 (brs, 2H), 7.50 (s,
1H), 7.13–7.19 (m, 2H). MS-ESI (m/z): 169.0 [M+H]+.
5.2.3.9
6-(tert-butyl)benzo[d]oxazol-2-amine (9b). Pale solid, 75% yield. 1H NMR (400 MHz, DMSO-d6): δ 9.66 (brs, 2H), 7.62 (d,
J= 1.2 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H) 1.27 (s, 9H). MS-ESI (m/z): 191.2 [M+H]+.
5.2.3.10 methyl 2-aminobenzo[d]oxazole-5-carboxylate (10b). Slightly pink solid, 80% yield.1H NMR (400 MHz, DMSO-d6): δ 7.69
(d, J = 1.6 Hz, 2H), 7.61–7.63 (m, 3H), 7.40 (d, J = 8.4 Hz, 1H), 3.80 (s, 3H). MS-ESI (m/z): 193.10 [M+H]+.
1
5.2.3.11 Oxazolo[4,5-b]pyridin-2-amine (11b). Black solid, 78% yield. H NMR (400 MHz, DMSO-d6): δ 8.02 (dd, J = 5.2, 1.4 Hz,
1H), 7.60 (dd, J = 7.8, 1.4 Hz, 1H), 6.90 (dd, J = 7.8, 5.2 Hz, 1H). MS-ESI (m/z): 135.1 [M+H]+.
1
5.2.3.12 2-aminobenzo[d]oxazol-4-ol (25b). Black solid, 77% yield. H NMR (400 MHz, DMSO-d6): δ 10.23 (brs, 1H), 8.65 (brs,
2H), 8.92–8.95 (m, 2H), 6.72 (m, 1H). MS-ESI (m/z): 151.2 [M+H]+.
1
5.2.3.13 naphtho[1,2-d]oxazol-2-amine (27b). Brown solid, 83% yield. H NMR (400 MHz, DMSO-d6): δ 8.11 (d, J = 8.0 Hz, 1H),
7.95 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.51–7.56 (m, 2H), 7.46–7.47 (m, 2H), 7.42–7.44 (m, 2H). MS-ESI (m/z): 185.1
[M+H] +.
1
5.2.3.14 Oxazolo[5,4-h]quinolin-2-amine (28b). Pale solid, 83% yield. H NMR (400 MHz, DMSO-d6): δ 9.03 (dd, J = 8.8 Hz, 1H),
8.94 (dd, J = 8.8 Hz, 1H), 8.12 (brs, 2H), 7.99 (dd, J = 8.0, 4.0 Hz, 1H), 7.88 (dd, J = 8.0, 4.0 Hz, 1H), 7.72–7.82 (m, 1H). MS-ESI
(m/z): 186.1 [M+H]+.
5.2.3.15 oxazolo[4,5-f]quinolin-2-amine (29b). Slightly yellow solid, 66% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.82 (dd, J = 4.0,
1.2 Hz, 1H), 8.47 (dd, J = 8.4, 1.2 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H),7.59 (brs, 2H), 7.49 (dd, J = 8.4, 4.0 Hz,

1H). MS-ESI (m/z): 186.1 [M+H]+.
5.2.4 4-(pyridin-2-ylmethoxy)benzo[d]oxazol-2-amine (25c)
2-(Bromomethyl)pyridine hydrobromide (835 mg, 3.3 mmol) and potassium carbonate (911 mg, 6.6 mmol) were added to a solution
of 2-aminobenzo[d]oxazol-4-ol 25b (450 mg, 3.0 mmol) in 15 mL acetonitrile. The mixture was stirred at 70°C for 4 h and the
reaction progress was monitored by LCMS. Solid impurities were removed by filtration, and the filtrate was evaporated under vacuum.
The product solution was reconstituted in 20 mL DCM, washed with water and brine, and then dried over MgSO4. Subsequently, the
solution was loaded on a silica chromatography column and eluted with DCM/MeOH (20/1, v/v) to afford the pure brown solid product
1
25c (410 mg, 57% yield). H NMR (400 MHz, DMSO-d6): δ 8.75 (brs, 2H), 8.56–8.67 (m, 2H), 8.61 (d, J = 4.8 Hz, 1H), 8.35 (d, J =
2.8 Hz, 1H), 8.22 (m, 1H), 7.89 (m, 1H), 6.59 (t, J = 8.0 Hz, 1H), 5.25 (s, 2H). MS-ESI (m/z):242.0 [M+H]+.
5.2.5 4-(pyrimidin-2-yloxy)benzo[d]oxazol-2-amine (26c)
A mixture of 2-aminobenzo[d]oxazol-4-ol 25b (650 mg, 4.33 mmol), 2-iodopyrimidine (893 mg, 4.33 mmol), copper (273 mg, 4.33
mmol), and cesium carbonate (2.11 g, 6.50 mmol) in 10 mL DMF was reacted in a microwave at 100°C for 30 min. Thereafter, the
reaction mixture was filtered by cilte and washed with DCM. The filtrate was purified by column chromatography with DCM/MeOH
(95/5, v/v) mobile phase. The yellow solid product collected at the end (210 mg) was labeled 26c, and the reaction yield was determined
to be 21%. 1H NMR (400 MHz, DMSO-d6): δ 8.57 (d, J = 4.4 Hz, 2H), 7.44 (s, 1H), 7.19–7.24 (m, 2H), 6.94–6.99 (m, 2H). MS-ESI
(m/z): 229.2 [M+H]+.
5.2.6 General Procedure for the Synthesis of compounds 1–17 and 25–29
TEA (2.0 eq), EDCI (1.5 eq), and HoBt (1.5 eq) were added to a solution of 4-methyl-1,2,3-thiadiazole-5-carboxylic acid (10 mmol,
1.0 eq) in 15 mL DMF kept at 0°C. The mixture was stirred at this temperature for 1 h before adding the amine (1.0 eq). The resulting
solution was then stirred and allowed to react for 16 h at room temperature. The progress of the reaction was monitored using LCMS. At
the end of the 16 h, the mixture was poured into 30 mL DCM-THF (5/1, v/v) and washed with water (30 mL). The organic phase was
collected, and the solvent was slowly evaporated in a rotary evaporator under vacuum. When lots of solid material appeared, the
evaporation process was ceased, and the solid was filtered. The crude material was triturated with acetonitrile or methanol to give the
desired product with high purity (> 95%).
5.2.6.1 N-(5-chlorobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (1, IMB-T097). Slightly yellow solid, 41% yield.
1H NMR (600 MHz, DMSO-d6): δ 13.33 (brs, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.36 (dd, J = 8.4, 2.4 Hz, 1H), 2.92 (s, 3H).
13C NMR (150 MHz, DMSO-d6): δ 160.1, 159.8, 146.6, 142.6, 129.3, 124.3, 113.1, 112.1, 13.5. HRMS-ESI (m/z): calculated for
C11H8N4O2ClS [M+H]+ 295.00510; found 295.00520.
5.2.6.2 N-(benzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (2). White solid, 71% yield. 1H NMR (600 MHz, DMSO-d6):
δ 13.33 (brs, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.36 (dt, J = 7.8, 1.2 Hz, 1H), 7.32 (dt, J = 7.8, 1.2 Hz, 1H), 2.93 (s,
3H). 13C NMR (150 MHz, DMSO-d6): δ 167.3, 159.9, 147.4, 143.3, 129.7, 125.4, 124.5, 113.0, 110.7, 13.5. HRMS-ESI (m/z):
calculated for C11H9O2N4S [M+H]+ 261.04407; found 261.04517.
1
5.2.6.3 N-(5-fluorobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (3). White solid, 65% yield. H NMR (600 MHz,
DMSO-d6): δ 13.34 (brs, 1H), 7.65 (dd, J = 9.0, 4.2 Hz, 1H), 7.26 (brd, J = 6.6 Hz, 1H), 7.17 (ddd, J = 9.0, 9.0, 3.0 Hz, 1H), 2.90 (s,
3H). 13C NMR (150 MHz, DMSO-d6): δ 160.2, 160.0, 158.6, 146.8, 140.0, 111.8, 111.7, 111.3, 111.1, 100.9, 13.5. HRMS-ESI (m/z):
calculated for C11H8O2N4FS [M+H]+ 279.03465; found 279.03418.
1
5.2.6.4 N-(5-bromobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (4). White solid, 46% yield. H NMR (600 MHz,
DMSO-d6): δ 13.33 (brs, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.50 (dd, J = 8.4, 2.4 Hz, 1H), 2.93 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): δ 160.0, 127.0, 116.9, 112.5, 13.5. HRMS-ESI (m/z): calculated for C11H6O2N4BrS [M-H]‒ 336.93894; found
336.93886.
1
5.2.6.5 4-methyl-N-(5-methylbenzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (5). White solid, 56% yield. H NMR (600 MHz,

DMSO-d6): δ 13.29 (brs, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.13 (d, J = 8.4 Hz, 1H), 2.94 (s, 3H), 2.39 (s, 3H). 13C NMR (150
MHz, DMSO-d6): δ 167.3, 160.0, 159.8, 147.6, 141.3, 135.1, 129.6, 125.1, 112.8, 110.2, 21.0, 13.5. HRMS-ESI (m/z): calculated for
C12H11N4O2S [M+H]+ 275.05972; found 275.05963.
1
5.2.6.6 N-(5-(tert-butyl)benzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (6). Slightly brown solid, 70% yield. H NMR
(600 MHz, DMSO-d6): δ 13.21 (brs, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.46 (d, J = 1.8 Hz, 1H), 7.36 (dd, J = 9.0, 1.8 Hz, 1H), 2.92 (s, 3H),
1.30 (s, 9H). 13C NMR (150 MHz, DMSO-d6): δ 167.2, 160.1, 159.8, 148.5, 147.4, 141.2, 129.4, 121.8, 110.0, 109.5, 34.8, 31.3, 13.5.
HRMS-ESI (m/z): calculated for C15H17O2N4S [M+H]+ 317.10667; found 317.10661.
1
5.2.6.7 N-(4-bromobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (7). Pale solid, 22% yield. H NMR (400 MHz,
DMSO-d6): δ 7.71 (dd, J = 8.4, 0.8 Hz, 1H), 7.57 (d, J = 8.4, 0.8 Hz, 1H), 7.27 (t, J = 8.4 Hz, 1H), 2.85 (s, 3H).13C NMR (100 MHz,
DMSO-d6): δ 160.5, 155.4, 147.6, 127.8, 125.4, 109.9, 13.4. HRMS-ESI (m/z): calculated for C11H8O2N4BrS [M+H]+ 339.9546; found
338.9549.
1
5.2.6.8 N-(6-chlorobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (8). White solid, 58% yield. H NMR (600 MHz,
DMSO-d6): δ 13.37 (brs, 1H), 7.84 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.42 (dd, J = 8.4, 1.8 Hz, 1H), 2.93 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): δ 160.0, 159.6, 146.9, 144.1, 128.3, 125.5, 114.3, 111.3, 109.1, 13.5. HRMS-ESI (m/z): calculated for
C11H8O2N4ClS [M+H]+ 295.00510; found 295.00517.
1
5.2.6.9 N-(6-(tert-butyl)benzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (9). Yellow solid, 36% yield. H NMR (400
MHz, DMSO-d6): δ 13.24 (brs, 1H), 7.61 (s, 1H), 7.39 (m, 2H), 2.93 (s, 3H), 1.30 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 159.5,
156.0, 143.7, 122.3, 112.6, 107.5, 105.8, 34.9, 13.6, 12.2. HRMS-ESI (m/z): calculated for C15H17O2N4S [M+H]+ 317.10667, found
317.10795.
1
5.2.6.10 methyl 2-(4-methyl-1,2,3-thiadiazole-5-carboxamido)benzo[d]oxazole-5-carboxylate (10). White solid, 28% yield. H NMR
(600 MHz, DMSO-d6): δ 13.46 (brs, 1H), 7.99 (s, 1H), 7.95 (dd, J = 8.4, 1.8 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H), 2.94 (s, 3H).
13C NMR (150 MHz, DMSO-d6): δ 165.4, 160.1, 126.8, 126.0, 110.9, 52.5, 13.5. HRMS-ESI (m/z): calculated for C11H9ON4S2 [M+H]+
277.02123, found 277.02144.
1
5.2.6.11 4-methyl-N-(oxazolo[4,5-b]pyridin-2-yl)-1,2,3-thiadiazole-5-carboxamide (11). Pale solid, 22% yield. H NMR (600 MHz,
DMSO-d6): δ 8.32 (d, J = 4.2 Hz, 1H), 8.03 (dd, J = 8.4, 1.2 Hz, 1H), 7.33 (dd, J = 8.4, 5.4 Hz, 1H), 2.90 (s, 3H). 13C NMR (150 MHz,
DMSO-d6): δ 160.0, 159.0, 119.3, 118.0, 13.4. HRMS-ESI (m/z): calculated for C10H8O2N5S [M+H]+ 262.03932, found 262.03888.
5.2.6.12 N-(benzo[d]thiazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (12). Pale solid, 46% yield. 1H NMR (600 MHz,
DMSO-d6): δ 10.97 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 2.80 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ 159.3,
157.9, 143.7, 139.7, 132.0, 121.7, 118.8, 111.8, 13.2. HRMS-ESI (m/z): calculated for C11H9O2N4S [M+H]+ 261.04407, found
261.04502.
1
5.2.6.13 N-(1H-benzo[d]imidazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (13). Yellow solid, 70% yield. H NMR (400 MHz,
DMSO-d6): δ 12.70 (brs, 1H), 7.41 (m, 2H), 7.22 (m, 2H), 2.95 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 166.5, 158.8, 152.1, 149.1,
129.0, 123.0, 111.6, 13.4; HRMS-ESI (m/z): calculated for C11H10ON5S [M+H]+ 260.06006; found 260.06141.
5.2.6.14 N-(benzo[d]oxazol-2-yl)furan-2-carboxamide (14). White solid, 66% yield. 1H NMR (600 MHz, DMSO-d6): δ 12.02 (brs, 1H),
8.00 (brs, 1H), 7.62 (overlap, 1H), 7.60 (overlap, 2H), 7.33 (dt, J = 7.2, 1.2 Hz, 1H), 7.29 (dt, J = 7.2, 1.2 Hz, 1H), 6.72 (s, 1H). 13C
NMR (150 MHz, DMSO-d6): δ 154.9, 147.8, 147.3, 145.8, 140.6, 124.7, 123.8, 118.4, 117.0, 112.3, 110.2. HRMS-ESI (m/z): calculated
for C12H9O3N2 [M+H]+ 229.06077; found 229.06061.
5.2.6.15 N-(benzo[d]oxazol-2-yl)thiophene-2-carboxamide (15). Slightly yellow solid, 71% yield. 1H NMR (600 MHz, DMSO-d6): δ
12.13 (brs, 1H), 8.15 (brs, 1H), 7.95 (brs, 1H), 7.62 (overlap, 2H), 7.33 (dt, J = 7.2, 1.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.23 (brs, 1H).
13C NMR (150 MHz, DMSO-d6): δ 158.8, 155.0, 147.7, 137.7, 133.9, 131.4, 128.4, 124.8, 123.8, 118.3, 110.2. HRMS-ESI (m/z):

calculated for C12H9O2N2S [M+H]+ 245.03792; found 245.03760.
1
5.2.6.16 N-(benzo[d]oxazol-2-yl)-5-chlorothiophene-2-carboxamide (16). White solid, 49% yield. H NMR (600 MHz, DMSO-d6): δ
7.74 (brs, 1H), 7.59 (d, J = 7.2 Hz, 1H), 7.51 (brs, 1H), 7.33 (dt, J = 7.2, 1.2 Hz, 1H), 7.29 (dt, J = 7.2, 1.2 Hz, 1H), 7.24 (brs, 1H). 13
C
NMR (150 MHz, DMSO-d6): δ 131.2, 128.5, 125.0, 124.0, 110.3. HRMS-ESI (m/z): calculated for C12H8ClO2N2S [M+H]+ 278.99895,
found 278.99908.
1
5.2.6.17 N-(benzo[d]oxazol-2-yl)benzamide (17). White solid, 75% yield. H NMR (600 MHz, DMSO-d6): δ 12.06 (brs, 1H), 8.05
(overlap, 2H), 7.63 (brt, J = 7.2 Hz, 2H), 7.60 (overlap, 1H), 7.54 (brt, J = 7.2 Hz, 2H), 7.33 (dt, J = 7.2, 1.2 Hz, 1H), 7.30 (dt, J = 7.2,
1.2 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δ 165.0, 155.5, 147.8, 140.6, 132.6, 128.5, 124.7, 123.9, 118.3, 110.2. HRMS-ESI (m/z):
calculated for C14H11O2N2 [M+H]+ 239.08150; found 239.08170.
1
5.2.6.18 4-methyl-N-(4-(pyridin-2-ylmethoxy)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (25). Yellow solid, 47% yield. H
NMR (400 MHz, DMSO-d6): δ 10.66 (s, 1H), 8.45 (ddd, J = 4.8, 1.6, 0.8 Hz, 1H), 7.81 (dt, J = 7.6, 2.0 Hz, 1H), 7.44 (brd, J = 7.6 Hz,
1H), 7.29 (ddd, J = 6.0, 4.8, 0.8 Hz, 1H), 7.14 (dd, J = 8.4, 8.4 Hz, 1H), 7.13 (s, 1H), 6.79 (ddd, J = 8.4, 8.4, 6.0 Hz, 1H), 5.55 (s, 2H),
2.80 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 163.7, 159.3, 155.8, 154.8, 149.1, 148.4, 145.8, 144.3, 137.1, 124.6, 122.7, 121.4,
117.4, 112.8, 105.7, 102.0, 48.9, 13.4. HRMS-ESI (m/z): calculated for C17H14O3N5S [M+H]+ 368.08119, found 368.08169.
5.2.6.19 4-methyl-N-(4-(pyrimidin-2-yloxy)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (26). White solid, 29% yield. 1H
NMR (400 MHz, DMSO-d6): δ 8.63 (d, J = 4.8 Hz, 2H), 7.59 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.28 (t, J = 4.8 Hz, 1H), 7.24
(d, J = 8.0, 0.8 Hz, 1H), 2.83 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 164.3, 160.3, 160.1, 158.4, 158.0, 129.7, 124.7, 118.4, 117.2,
108.0, 105.8, 13.4. HRMS-ESI (m/z): calculated for C15H11O3N6S [M+H]+ 355.06079, found 355.06039.
1
5.2.6.20 4-methyl-N-(naphtho[1,2-d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (27). Black solid, 58% yield. H NMR (400 MHz,
DMSO-d6) δ 8.58 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.69 (dt, J = 8.0, 1.2 Hz, 1H),
7.59 (dt, J = 80, 1.2 Hz, 1H), 2.93 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.0, 130.8, 128.8, 127.2, 125.7, 125.2, 121.9, 110.7,
13.5. HRMS-ESI (m/z): calculated for C15H11O2N4S [M+H]+ 311.05972, found 311.05972.
5.2.6.21 4-methyl-N-(oxazolo[5,4-h]quinolin-2-yl)-1,2,3-thiadiazole-5-carboxamide (28). Brown solid, 35% yield. 1H NMR (400 MHz,
DMSO-d6): δ 9.02 (s, 1H), 8.55 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 7.2 Hz, 2H), 7.64 (s, 1H), 2.90 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 160.1, 150.9, 137.1, 125.9, 124.7, 121.1, 111.6, 105.7, 13.4. HRMS-ESI (m/z): calculated for C14H10O2N5S [M+H]+
312.05497, found 312.05450.
1
5.2.6.22 4-methyl-N-(oxazolo[4,5-f]quinolin-2-yl)-1,2,3-thiadiazole-5-carboxamide (29). Yellow solid, 25% yield. H NMR (400 MHz,
DMSO-d6): δ 8.83 (s, 2H), 7.90 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 6.8 Hz, 1H), 7.50 (s, 1H), 2.82 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 164.5, 157.8, 148.6, 145.4, 143.2, 120.5, 119.1, 113.4, 13.3. HRMS-ESI (m/z): calculated for C14H10O2N5S [M+H]+
312.05497, found 312.05453.
5.2.7 4-methyl-N-(5-(pyridin-2-yl)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (18)
A
mixture of N-(5-bromobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide 4 (600 mg, 1.77 mmol) and
2-(butyldipentylstannyl)pyridine (843 mg, 2.12 mmol) was dissolved in 10 mL anhydrous DMF, and then CuI (50 mg, 0.27 mmol),
Pd2(dba)3 (165 mg, 0.18 mmol), and TEA (354 mg, 3.54 mmol) were added. The solution was stirred and reacted at 90°C under nitrogen
atmosphere. After 5 h, the reaction mixture was filtered through celite, and the solvent was evaporated under vacuum. The residue was
purified by prep-TLC using THF/MeOH (20/1, v/v) as an eluent. The yield of the obtained while solid product 18 (310 mg) was found
to be 52%.1H NMR (400 MHz, DMSO-d6): δ 13.46 (brs, 1H), 8.68 (brd, J = 6.0 Hz, 1H), 8.16 (d, J = 1.2 Hz, 1H), 8.03 (dd, J = 8.4, 1.6
Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.90 (td, J = 8.0, 1.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.38 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 2.95 (s,
3H).13C NMR (100 MHz, DMSO-d6): δ 160.0, 155.0, 149.6, 137.5, 136.4, 123.2, 122.8, 120.4, 110.8, 105.8, 13.5. HRMS-ESI (m/z):
calculated for C16H12O2N5S [M+H]+ 338.07062, found 338.07050.
5.2.8 4-methyl-N-(4-(pyridin-2-yl)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (19). The method used to synthesize this

compound is the same as that used to prepare 18. 19: slightly yellow solid, 41% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.38 (ddd, J =
4.8, 2.0, 0.8 Hz, 1H), 7.76 (ddd, J = 8.0, 7.6, 2.0 Hz, 1H), 7.65 (dt, J = 8.0, 0.8 Hz, 1H), 7.60 (brd, J = 8.4 Hz, 2H), 7.50 (dd, J = 8.4, 2.0
Hz, 1H), 7.45 (ddd, J = 7.6, 4.8, 0.8 Hz, 1H), 2.93 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.0, 150.6, 141.5, 139.6, 128.2, 127.1,
123.5, 116.9, 112.6, 13.5. HRMS-ESI (m/z): calculated for C16H12O2N5S [M+H]+ 338.07062, found 338.07068.
5.2.9 4-methyl-N-(5-(pyridin-3-yl)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (20)
A
mixture of N-(5-bromobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide
4
(130 mg, 0.38 mmol),
pyridin-3-ylboronic acid (230 mg, 1.90 mmol), K3PO4 (400 mg, 1.90 mmol), and Pd(PPh3)4 (43 mg, 0.038 mmol) in 7 mL DMF/H2O
(6/1, v/v) was heated in a microwave at 150°C under nitrogen atmosphere and monitored using LCMS. After 30 min, the solvent was
evaporated under vacuum, and the residue was purified by prep-TLC using DCM/THF/MeOH (10/10/1, v/v/v) as an eluent. The yield
of the obtained yellow solid product 20 (45 mg) was found to be 35%. 1H NMR (400 MHz, DMSO-d6): δ 8.84 (d, J = 1.6 Hz, 1H), 8.52
(dd, J = 4.8, 1.6 Hz, 1H), 8.01 (brd, J = 8.0 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.44 (dd, J = 7.6, 4.8 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H),
7.35 (dd, J = 8.4, 1.6 Hz, 1H), 2.89 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 165.8, 163.7, 157.4, 147.8, 147.6, 147.4, 136.4, 134.0,
132.0, 123.8, 120.1, 114.7, 108.8, 105.8, 13. 3. HRMS-ESI (m/z): calculated for C16H12O2N5S [M+H]+ 338.07062, found 338.07100.
5.2.10
4-methyl-N-(5-(pyridin-4-yl)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (21). The method used to synthesize this
compound is the same as that used to prepare 20. 21: pale solid, 31% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.58 (d, J = 5.2 Hz, 2H),
7.82 (s, 1H), 7.65 (d, J = 5.2 Hz, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 2.89 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
δ 165.9, 163.8, 157.4, 152.1, 150.0, 148.1, 147.8, 147.7, 144.6, 132.0, 121.1, 120.0, 114.6, 109.5, 108.8, 13.3. HRMS-ESI (m/z):
calculated for C16H12O2N5S [M+H]+ 338.07062, found 338.07070.
5.2.11
methyl 4-(2-(4-methyl-1,2,3-thiadiazole-5-carboxamido)benzo[d]oxazol-5-yl)benzoate (22). The method used to synthesize
this compound is the same as that used to prepare 20. 22: white solid, 45% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.65 (m, 4H), 7.19
(m, 3H), 3.84 (s, 3H), 1.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 166.2, 163.2, 151.9, 139.6, 135.6, 129.9, 126.7, 125.3, 52.6, 13.5.
HRMS-ESI (m/z): calculated for C19H15O4N4S [M+H]+ 395.08085, found 395.08017.
5.2.12
4-methyl-N-(5-(1-methyl-1H-pyrazol-4-yl)benzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (23). The method used to
synthesize this compound is the same as that used to prepare 20. 23: yellow solid, 35% yield. 1H NMR (600 MHz, DMSO-d6): δ 8.04 (s,
1H), 7.78 (s, 1H), 7.46 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.18 (dd, J = 7.8, 1.8 Hz, 1H), 3.85 (s, 3H), 2.87 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): δ 165.1, 162.9, 157.2, 152.4, 146.2, 144.1, 135.8, 127.4, 127.3, 122.7, 117.9, 112.4, 108.4, 38.6, 13.2.
HRMS-ESI (m/z): calculated for C15H13O2N6S [M+H]+ 341.08152, found 341.08195.
5.2.13
4-methyl-N-(5-morpholinobenzo[d]oxazol-2-yl)-1,2,3-thiadiazole-5-carboxamide (24)
XPhos (76mg, 0.16 mmol) and Pd(PPh3)4 (92 mg, 0.08 mmol) were added to
a
mixture of
N-(5-bromobenzo[d]oxazol-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide 4 (260 mg, 0.77 mmol), morpholine (134 mg, 1.54 mmol),
and t-BuOK (173mg, 1.54 mmol) in 10 mL anhydrous DME. The resulting solution was reacted at 110°C under nitrogen atmosphere for
2 h and monitored using LCMS. Then, the solvent was evaporated under vacuum and the residue was purified by prep-TLC using
DCM/MeOH (20/1, v/v) as eluent. The yellow solid product obtained at the end was labeled 24 (50 mg), and its yield was determined to
be 19%. 1H NMR (400 MHz, DMSO-d6): δ 7.46 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.6 (s, 1H), 3.75 (t, J = 4.8 Hz, 4H), 3.08
(t, J = 4.8 Hz, 4H), 2.93 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.7, 149.6, 112.6, 110.8, 66.0, 49.2, 13.5. HRMS-ESI (m/z):
calculated for C15H16O3N5S [M+H]+ 346.09684, found 346.09750.
5.3. Determination of Anti-Mtb MIC values
The anti-Mtb activities of the synthesized compounds were tested against the standard Mtb strain H37Rv (ATCC 27294), as well as
the drug-resistant MDR-FJ05120 and XDR-FJ05195 strains (clinical isolates). The first-line anti-TB drugs isoniazid, rifampicin,
streptomycin, and ethambutol were used as positive controls. The MICs of M. tuberculosis replication inhibition were determined by
Microplate Alamar Blue Assay (MABA). The final concentrations of the investigated compounds varied between 0.125 and 128 mg/L.
The bacterial strains (106 cfu/mL cell concentration) were cultured with each one of the synthesized compounds (varying concentrations)

at 37°C in Middlebrook 7H9 broth (Difco) supplemented with 0.2% glycerol and 10% oleic acid-alumin-dextrose-citric acid (OADC).
The culture was arrested when the mid-log phase of growth was reached. The MIC values were then measured in sterile 96-well plates
with a final volume of 100 µL per well. These values correspond to the lowest drug concentrations for which the color of alamar blue
reagent remains unchanged [21].
5.4. Cell cytotoxicity
The cytotoxicity of all compounds was examined in HEK-297T cells. The compounds were dissolved in DMSO at a concentration of
10 mM, and then the solutions were diluted with DMEM culture medium containing 5% PBS to varying concentrations between 0.78
and 200 µM. The cells were seeded in 96-well plates at a density of 5 × 103 cells per well, and then they were incubated with the
compounds at 37°C. Forty-eight hours later, 20 µL of the Cell Counting Kit-8 (CCK-8) solution was added to each well, and the
mixtures were incubated for another 4 h. Finally, the absorbance of each well was measured at 450 nm using a Microplate reader
(EnVision, PerkinElmer). The IC50 values were calculated based on a concentration-response curve using GraphPad Prism 5.
5.5. Acute Toxicity
SPF BALAC/c female mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) weighing
between 18 and 22 g were used to evaluate the in vivo acute toxicity of compounds 1, 6, and 18. The mice were randomly divided into
groups of six, and each group was administered with one of the investigated compounds in 0.5% CMC-Na solution (single 1000 mg/kg
dose) by oral gavage. All of the mice were reared in appropriate environments where the temperature was maintained between 20 and
24oC, with mean day and night times. The number of surviving mice was monitored during the 14-day period following compound
administration. The animal care and experimental procedures performed herein are in accordance with the regulations of the
Institutional Animal Care and Use Committee of the Institute of Medicinal Biotechnology.
5.6. Determination of pharmacokinetic profiles
SPF male SD rats weighing between 180 and 220 g (Shanghai Sippr-BK laboratory animal Co., Ltd.) were fasted overnight in
metabolism cages before being administered with one of the investigated compounds. The rats were allowed free access to water, but
they were given food only after 4 h of compound administration. Each treatment group consisted of three rats, and all of them were
treated with a single dose (40 mg/kg) of the tested compounds suspended in 5% DMSO, 10% Solutol, and 85% deionized water. Blood
samples (200 µL) were collected from the jugular veins of rats at 0.25, 0.5, 1, 2, 4, 6, and 8 h after compound administration. The
samples were put on ice and centrifuged at 6800 g and 4°C for 6 min in order to obtain the plasmas after 2 h. The protein contents in 20
µL aliquots of the plasma samples were precipitated with 400 µL MeOH containing the internal standard (100 ng/mL). The mixtures
were vortexed for 1 min then centrifuged at 18000 g for 7 min. Subsequently, 200 µL of the supernatants were transferred to 96-well
plates, and 3 µL aliquots were analyzed by LC-MS/MS. The area under the concentration time curve (AUC), the peak concentration
(Cmax), the time to reach peak concentration (Tmax), and mean residence time (MRT) of each sample were determined based on the
plasma concentration data using WinNonlin.
5.7. Broad spectrum panel analysis
The anti-bacterial activities and MIC values of compounds 6 and 18 were determined for a series of Gram-positive and
Gram-negative bacterial pathogens, in accordance with the agar dilution method recommended by the CLSI. The selected strains
represent important bacteria, and they include five Gram-positive (S. aureus ATCC 29213, S. aureus ATCC 33591, S. epidermidis ATCC
12228, E. faecalis ATCC 29212, E. faecium ATCC 700221) and five Gram-negative strains(E. coli ATCC 25922, K. pneumonia
ATCC700603, K. pneumonia 7, P. aeruginosa ATCC 27853, A. calcoacetious ATCC 19606). Inoculations were adjusted using a
multipoint inoculator (Bolney, Sussex, UK) to yield cell concentrations of about 104 cfu/spot, and then the cells were incubated at 35°C
for 18 h. The MICs of compounds 6 and 18 were taken to be the lowest concentrations at which bacterial growth is inhibited.
Acknowledgments

This work was supported by the Institute of Medicinal Biotechnology, CAMS & PUMC, Central Public-Interest Scientific Institution
Basal Research Fund (No. IMBF201301), the Drug Innovation Major Project of China (Grant Nos. 2015ZX09304006-016,
2018ZX09735001-002, and 2018ZX09711001-007), the National Natural Science Foundation of China (No. 81773784), and Beijing
Nova Program (No. Z181100006218075).
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
We thank Professor Kanglin Wan’s group from the Chinese Center for Disease Control and Prevention (CCDC) and Professor Yu
Lu’s group from Beijing Chest Hospital for their assisting us in evaluating the intracellular anti-TB activity of the compounds of this
paper.
Abbreviations
EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride; HoBt, 1-hydroxybenzotriazole; TEA, triethylamine; DMF,
N,N-dimethylformamide;
XPhos,
(2-aminobiphenyl)-cyclometalated
palladium
mesylateprecatalyst
complex;
DME,
1,2-dimethoxyethane; AUC, area under curve; MRT, mean residence time.
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Highlights
ꢀ Twenty-nine 2-acylamide-1,3-benzo[d]zole analogues were synthesized.
ꢀ 17 compounds were found to be potent anti-Mtb agents, especially against the
MDR- and XDR-MTB strains, with MIC values < 10 µM.
ꢀ Compound 18, with a 5-(pyridin-2-yl) at R2, show potent anti-Mtb activities,
acceptably safe and favorable pharmacokinetic (PK) properties.
ꢀ Compound 18 is also a potent anti-Gram-positive agent.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: