Design, synthesis and antitubercular evaluation of benzothiazinones containing a piperidine moiety

Article abstract of DOI:10.1016/j.ejmech.2018.03.060

We herein report the design and synthesis of benzothiazinones containing a piperidine moiety as new antitubercular agents based on the structure feature of IMB-ZR-1 discovered in our lab. Some of them were found to have good in vitro activity (MIC < 1 μg/

Full text of DOI:10.1016/j.ejmech.2018.03.060

Accepted Manuscript
Design, synthesis and antitubercular evaluation of benzothiazinones containing a
piperidine moiety
Kai Lv, Zeyu Tao, Qian Liu, Lu Yang, Bin Wang, Shuo Wu, Apeng Wang, Menghao
Huang, Mingliang Liu, Yu Lu
PII:
S0223-5234(18)30303-9
10.1016/j.ejmech.2018.03.060
EJMECH 10325
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 January 2018
Revised Date: 27 February 2018
Accepted Date: 20 March 2018
Please cite this article as: K. Lv, Z. Tao, Q. Liu, L. Yang, B. Wang, S. Wu, A. Wang, M. Huang, M. Liu, Y.
Lu, Design, synthesis and antitubercular evaluation of benzothiazinones containing a piperidine moiety,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.060.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphic abstracts:
New benzothiazinones bearing a piperidine moiety were designed and synthesized as
antitubercular agents based on the structure feature of IMB-ZR-1 discovered in our lab.
Compound 2i were found to display potent anti-TB activity (MIC < 0.016 µg/mL) against
drug-sensitive/resistant MTB strains.

ACCEPTED MANUSCRIPT
Design, synthesis and antitubercular evaluation of benzothiazinones containing a
piperidine moiety
Kai Lv,a，1 Zeyu Tao,a，1 Qian Liu,a, b Lu Yang,a Bin Wang,c Shuo Wu,a Apeng Wang,a
c
*
Menghao Huang,d Mingliang Liu,a, * Yu Lu
，
a
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China
b Institute of Molecular Design and Synthesis, School of Pharmaceutical Science & Technology, Tianjin University, 92
Weijin Road, Nankai District, Tianjin, 300072, P. R. China.
c
Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing
Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing
101149, China
d Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine,
Indiana 46202, USA
1 These authors contributed equally to the work.
*Corresponding author: lmllyx@126.com, 86-010-63030965 (M.L. Liu); luyu4876@hotmail.com, 86-010-89509357
(Y. Lu)
ABSTRACT
We herein report the design and synthesis of benzothiazinones containing a piperidine moiety as new antitubercular
agents based on the structure feature of IMB-ZR-1 discovered in our lab. Some of them were found to have good in
vitro activity (MIC < 1 µg/mL) against drug-susceptible Mycobacterium tuberculosis H37RV strain. After two set of
modifications, compound 2i were found to display comparable in vitro anti-TB activity (MIC < 0.016 µg/mL) to
PBTZ169 against drug-sensitive and resistant mycobacterium tuberculosis strains. Compound 2i also showed
acceptable PK profiles. Studies to determine PK profiles in lung and in vivo efficacy of 2i are currently under way.
KEYWORDS: benzothiazionones, PBTZ169, antimycobacterial activity, synthesis
1. Introduction
Tuberculosis (TB) is caused mainly by mycobacterium tuberculosis (MTB), has existed for millennia and
remains a major global health problem.[1] The World Health Organization (WHO) estimated that approximately 10.4
million people were infected and 1.3 million died from TB worldwide in 2016.[2] The current guidelines for treatment
of drug-susceptible TB infection recommends a combination of four front-line drugs rifampin, isoniazid, pyrazinamide
and ethambutol for 6-9 months, often leading to significant side effects and poor patient compliance. In addition, the
spread of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB has rendered these front-line drugs
ineffective. [3] Therefore, there is an urgent need for discovery of new drugs with new mechanisms of action. Although
Bedaquiline (inhibition of mitochondrial ATP synthase) and Delamanid (inhibition mycolic acid biosynthesis) were
approved for the treatment of MDR-TB, over a huge gap of over 40 years, [4-5] both of them have pronounced issues,
including hERG toxicity concerns, as well as multiple ADME issues due to their high lipophilicity. [6]
Benzothiazinones (BTZs), a novel class of TB agents were reported to have strong inhibitory potency against
drug-sensitive TB, MDR-TB and XDR-TB strains targeting on decaprenyl phosphoryl-β-D-ribose 2ʹ-epimerase
(DprE1). [7-12] The most advanced compound from this series (PBTZ169, Figure 1), developed by the Swiss Federal

ACCEPTED MANUSCRIPT
Institute of Technology and Innovative Medicines, has progressed into Phase II clinical trials in Russian Federation for
the treatment of both drug-susceptible TB and MDR-TB.[2]
<INSERT FIGURE 1 HERE>
The structure-activity relationship (SAR) and mechanistic studies of BTZs suggest that the NO2 group at position
8 and the sulfur atom at position 1 are critical for activity, that the -CF3 at position 6 plays an important role in
maintaining activity. [7, 13-20] In our previous study, we focused on the modification at position 2, some of the
resulting compounds were found to have improved activity and pharmacokinetic properties. [11, 12] Among them,
IMB-ZR-1 with the piperidine ring displayed potent in vitro anti-TB activity against the tested MDR-TB strains (MIC
= 0.016 µg/mL). [12] Inspired by the above research results, we designed and synthesized two series of new BTZs 1-2
with piperidine moiety as ring A (Figure 1.). Shorten the linker between the two cycles of IMB-ZR-1 by removing the
methylene group gave the series 1; shifting the nitrogen atom in the linker to ring B provided the series 2. In order to
explore SAR of these BTZs, the R group on series 1 could be methyl or hydrogen; the W moiety of series 2 could be
piperidine, pyrrolidine, azepane, 4-methylpiperazine, diethylamino, or substituted piperidine, et al. The
antimycobacterial activity of all the target compounds were evaluated against drug-susceptible TB strain. The potent
compound 2i was further evaluated for antimycobacterial activity against MDR-TB strains, and in vivo PK properties,
aiming to identify alternative group at position 2 of BTZs and find more effective DprE1 inhibitors as anti-TB
candidates.
<INSERT SCHEME 1 HERE>
2. Results and Discussion
2.1 Chemistry
Detailed synthetic pathways to target compounds 1a-b and 2a-k are outlined in scheme 1. Reductive amination
of ketone 3 and N-methylcyclohexylamine or cyclohexylamine by NaCNBH3 in MeOH afforded compounds 4a-b. Boc
deprotection by TFA in DCM gave the intermediates 5a-b. Coupling of 5a-b and 6 which was prepared according
literature procedure [8] furnished the BTZs 1a-b. Condensation of the acid 7 with amines in the presence of EDC and
HOBt yielded compounds 8a-k. Removal of the Boc group followed by amide reduction with LiAlH4 formed
intermediates 9a-k. Installation of 9a-k to 6 in the same manner as the preparation of 1a-b gave targets 2a-k.
2.2 Anti-TB activity
The two series 1a-b and 2a-e bearing different kinds of amines to ensure the structure diversity were first
synthesized (Table 1). They were preliminarily screened for in vitro activity against MTB H37Rv ATCC27395 strain,
using the Microplate Alamar Blue Assay (MABA). [21] The minimum inhibitory concentration (MIC) is defined as the
lowest concentration effecting a reduction in fluorescence of >90% relative to the mean of replicate bacterium-only
controls. The MIC values of the compounds along with PBTZ169, isoniazid (INH), and rifampicin (RFP) for
comparison were obtained from three independent experiments and presented in µg/mL in Table 1.
<INSERT TABLE 1 HERE>
The data revealed that these two BTZ derivatives 1a-b and 2a-e shown considerable anti-TB activity against this
strain (MIC < 10 µg/mL), although they were less active than PBTZ169, INH and RFP. Among them, compounds 1a-b
and 2a displayed better activity (MIC < 1 µg/mL) than other BTZs 2b-e. Removal of the methyl group on 1a didn’t
influence the potency (1a vs 1b). Decrease or increase the ring size of the piperidine group on 2a resulted in a loss of
anti-TB activity (2a vs 2b vs 2c). Replacement of the piperidine group with N-methyl piperazine or diethyl amine also
leaded to a decreased potency (2a vs 2d vs 2e). Specifically, compound 2d shown more than 16-fold higher MIC value

ACCEPTED MANUSCRIPT
than that of 2a. Consequently, this set of modification suggested that the cyclohexylamino (1a-b) and
piperidin-1-ylmethyl (2a) moiety were more suitable for the Q fragment (Table 1) than other moieties. Considering
compound 2a displayed best activity (MIC = 0.481 µg/mL) among these two series, we intend to make further
optimization based on 2a.
<INSERT TABLE 2 HERE>
Next, an additional set of substituted piperidine analogues as W moiety listed in Table 2 were designed and
synthesized. Our results indicated that some of them (2g-i) exhibited good activity (MIC < 1 µg/mL). Among them,
compound 2i (MIC < 0.016 µg/mL) is more active than INH (MIC = 0.037 µg/mL) and RFP (MIC = 0.084 µg/mL),
and comparable to PBTZ169 (MIC < 0.016 µg/mL). Exploration of SAR was first conducted by introduction of methyl
group to ortho-, meta-, and para-positions of piperazine ring. Introduction of methyl group to ortho-position provided
decreased potency (2a vs 2f), whereas the presence of meta- or para-methyl group afforded a slightly increased or
maintained anti-TB activity (2a vs 2g and 2h). The installation of hydroxy or ketal groups at the para-position was
detrimental to the potency (2a vs 2j and 2k). Surprisingly, the introduction of trifluoro group at the para position caused
a dramatical improvement in potency (2a vs 2i).
Inspired by the strong anti-TB potency against drug sensitive strain, compound 2i was further evaluated against two
clinical isolated MDR-TB strains (16995 and 16833) resistant to both INH and RFP (MIC > 40 µg/mL). As shown in
table 3, compound 2i (MIC < 0.016 µg/mL) displayed comparable anti-MDR-TB activity to PBTZ169 (MIC < 0.016
µg/mL), suggesting its promising potential for both drug-sensitive and resistant TB strains.
<INSERT TABLE 3 HERE>
2.3 Pharmacokinetics
We subsequently investigated the in vivo PK profiles of compound 2i in mice after a single oral administration of 50
mg/kg. As shown in Table 4, compound 2i showed acceptable PK properties although the AUC of 2i (2489 h·ng/mL) is
less than half of PBTZ169 (5478 h·ng/mL). The Tmax of 2i (0.25 h) is shorter than PBTZ169 (0.83 h), indicating that
2i was absorbed faster than PBTZ169. The Cmax is comparable to PBTZ169. We speculated that the lower AUC of 2i
probably due to extensive tissue distribution or excretion. Currently, the PK profiles of 2i in lung is under testing,
which will give the lung distribution properties of 2i.
<INSERT TABLE 4 HERE>
3. Conclusion
In summary, two series of BTZ derivatives with piperidine ring were designed as new anti-TB agents through
modifications of IMB-ZR-1. Most of them exhibited considerable in vitro inhibitory (MIC < 10 µg/mL) activity against
drug sensitive strain. Compound 2i with 4-trifuoromethyl piperidine group as the W moiety of series 2 displayed
comparable in vitro anti-TB activity to PBTZ169 against drug-sensitive and resistant TB strains. Compound 2i also
showed acceptable PK profiles, although its AUC is less than half of PBTZ169. Studies to determine further PK
profiles in lung and in vivo efficacy of 2i are currently under way.
4. Experimental protocols
4.1. Chemistry
1
Melting points were determined in open capillaries and are uncorrected. H NMR spectra were determined on a
Varian Mercury-400 spectrometer in DMSO-d6 or CDCl3 using tetramethylsilane as an internal standard. Electrospray
ionization (ESI) mass spectra was obtained on an MDSSCIEX Q-Tap mass spectrometer. The reagents were all of
analytical grade or chemically pure. TLC was performed on silica gel plates (Merck, ART5554 60F254).
4.2. Synthesis
4.2.1. General procedure for the preparation of compound 4a-b

ACCEPTED MANUSCRIPT
To a stirred solution of tert-butyl 4-oxopiperidine-1-carboxylate (200 mg, 0.67 mmol) in anhydrous MeOH (5 mL)
was added N-methylcyclohexanamine/cyclohexanamine, NaCNBH3 (166 mg, 2.68 mmol) at room temperature. The
mixture was adjusted to pH 6 by acetic acid, and stirred for 3 hours. The mixture quenched by H2O (20 mL), and
extracted by DCM. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The residue was
purified by chromatography on a silica gel column to yield compound 4a-b.
4.2.1.1. tert-butyl 4-(cyclohexyl(methyl)amino)piperidine-1-carboxylate 4a
Following the general procedure, employing N-methylcyclohexanamine yielded compound 4a as a colorless oil in
40% yield, 1HNMR (500 MHz, CDCl3) δ 4.30 (2H, brs), 3.34 (1H, brs), 3.17 (1H, brs), 2.73 (2H, brs), 2.65 (3H, s),
2.20 (2H, m), 1.96 (2H, m), 1.73 (2H, m), 1.58 (2H, brs), 1.42 (9H, s), 1.25 (7H, brs); MS-ESI (m/z): 319 (M + Na)+.
4.2.1.2. tert-butyl 4-(cyclohexylamino)piperidine-1-carboxylate 4b
Following the general procedure, employing cyclohexanamine yielded compound 4b as a colorless oil in 50% yield,
MS-ESI (m/z): 283 (M + H)+.
4.2.2. General procedure for the procedure of compound 5a-b
To a stirred solution of 4a-b (0.25 mmol) in DCM (5 mL) was added TFA, stirred for 1 hour, and concentrated. The
residue was diluted by DCM, concentrated again for three times to gave the crude 5a-b which was used directly for the
next step.
4.2.3 General procedure for the preparation of compound 8a-k
To a stirred solution of compound 7 (200 mg, 0.87 mmol) in DCM (10 mL) was added amines (1.0 mmol), EDC
(186 mg, 1.2 mmol), and HOBt (153 mg, 1.2 mmol) at room temperature. The mixture was stirred for 3 hrs, quenched
by H2O (15 mL), and extracted by DCM (15 mL × 3). The organic layer was dried over anhydrous MgSO4, filtered,
and concentrated. The residue was purified by chromatography on a silica gel column to afford compound 8a-l as a
colorless oil.
4.2.3.1. tert-butyl 4-(piperidine-1-carbonyl)piperidine-1-carboxylate 8a
Following the general procedure, employing piperidine as the amine yielded compound 8a as a colorless oil in 85%
yield, 1H NMR (500 MHz, CDCl3) δ 4.14 (brs, 2H), 3.55 (brs,2H), 3.43 (brs, 2H), 2.76 (brs, 2H), 2.62 (m, 1H), 1.66 (m,
6H), 1.55 (brs, 4H), 1.45 (s, 9H); MS-ESI (m/z): 297 (M + H)+.
4.2.3.2. tert-butyl 4-(pyrrolidine-1-carbonyl)piperidine-1-carboxylate 8b
Following the general procedure, employing piperidine as the amine yielded compound 8b as a colorless oil in 90%
yield, which was used directly for the next step.
4.2.3.3. tert-butyl 4-(azepane-1-carbonyl)piperidine-1-carboxylate 8c
Following the general procedure, employing azepane as the amine yielded compound 8c as a colorless oil in 94%
yield, 1H NMR (500 MHz, CDCl3) δ 4.15(brs, 2H), 3.51(brs,2H), 3.47(t, J = 5.9 Hz, 2H), 2.75(brs, 2H), 2.60(m, 1H),
2.01(m, 2H), 1.73(m, 6H), 1.57(brs, 4H), 1.46(s, 9H); MS-ESI (m/z): 333 (M + Na)+.
4.2.3.4. tert-butyl 4-(4-methylpiperazine-1-carbonyl)piperidine-1-carboxylate 8d
Following the general procedure, employing azepane as the amine yielded compound 8d as a colorless oil in 75%
yield, 1H NMR (500 MHz, CDCl3) δ 4.14 (brs, 2H), 3.65 (brs, 2H), 3.53 (brs, 2H), 2.75 (brs, 2H), 2.6 1(m, 1H), 2.41 (d,
J = 13.4 Hz, 4H), 2.32 (s, 3H), 1.67 (m, 4H), 1.45 (s, 9H); MS-ESI (m/z): 312 (M + H)+, 334 (M + Na)+.
4.2.3.5. tert-butyl 4-(diethylcarbamoyl)piperidine-1-carboxylate 8e
Following the general procedure, employing diethylamine as the amine yielded compound 8e as a colorless oil in
80% yield, 1H NMR (500 MHz, CDCl3) δ 4.15 (brs, 2H), 3.34 (q, J = 9.3 Hz, 4H), 2.74 (brs, 2H), 2.54 (t, J = 11.0 HZ,
1H), 1.75 (q, J = 10.1 Hz, 2H), 1.6 3(m, 2H), 1.45 (s, 9H), 1.20 (t, J = 6.9 HZ, 3H), 1.09 (t, J = 6.9 HZ, 3H); MS-ESI
(m/z): (M + H)+; MS-ESI (m/z): 285 (M + H)+.

ACCEPTED MANUSCRIPT
4.2.3.6. tert-butyl 4-(2-methylpiperidine-1-carbonyl)piperidine-1-carboxylate 8f
Following the general procedure, employing 2-methylpiperidine as the amine yielded compound 8f as a colorless
oil in 87% yield, 1H NMR (500 MHz, CDCl3) δ 4.92 (brs, 1H), 4.49 (m, 1H), 4.15 (brs, 3H), 3.66 (m, 1H), 3.13 (m,
1H), 2.76 (brs, 2H), 2.59 (brs, 1H), 1.63 (m, 8H), 1.45 (s, 9H), 1.27 (brs, 3H); MS-ESI (m/z): 333 (M + Na)+.
4.2.3.7. tert-butyl 4-(3-methylpiperidine-1-carbonyl)piperidine-1-carboxylate 8g
Following the general procedure, employing 3-methylpiperidine as the amine yielded compound 8g as a colorless
oil in 87% yield, 1H NMR (500 MHz, CDCl3)δ4.43 (m, 1H), 4.14 (brs, 2H), 3.80 (d, J = 11.8 Hz, 1H), 3.71 (d, J = 13.4
Hz, 1H), 2.98 (m, 1H), 2.76 (brs, 2H), 2.63 (m, 1H), 2.24 (t, J = 11.2 Hz, 1H), 1.83 (d, J=12.5 Hz, 1H), 1.68 (m, 5H),
1.45 (brs, 9H), 1.25 (m, 1H), 1.15 (m, 1H), 0.92 (d, J = 10.7 Hz, 3H); MS-ESI (m/z): 333 (M + Na)+.
4.2.3.8. tert-butyl 4-(4-methylpiperidine-1-carbonyl)piperidine-1-carboxylate 8h
Following the general procedure, employing 4-methylpiperidine as the amine yielded compound 8h as a colorless
oil in 87% yield, 1H NMR (500 MHz, CDCl3) δ 4.57 (brs, 1H), 4.14 (brs, 2H), 3.85 (brs, 1H), 3.01 (brs, 1H), 2.76 (m,
2H), 2.62 (m, 1H), 2.54 (m, 1H), 1.66 (m, 7H), 1.45 (s, 9H), 1.08 (m, 2H), 0.95 (d, J = 6.5 Hz, 2H).
4.2.3.9. tert-butyl 4-(4-(trifluoromethyl)piperidine-1-carbonyl)piperidine-1-carboxylate 8i
Following the general procedure, employing 4-trifluoromethylpiperidine as the amine yielded compound 8i as a
colorless oil in 68% yield, 1H NMR (500 MHz, CDCl3) δ 4.75 (d, J = 11.2 Hz, 1H), 4.14 (brs, 2H), 4.01 (d, J = 11.4 Hz,
1H), 3.05 (m, 1H), 2.77 (brs, 2H), 2.61 (m, 1H), 2.53 (m, 1H), 2.28 (m, 1H), 1.94 (m, 2H), 1.72 (m, 2H), 1.66 (brs, 2H),
1.45 (brs, 11H); MS-ESI (m/z): 365 (M + H)+.
4.2.3.10. tert-butyl 4-(4-hydroxypiperidine-1-carbonyl)piperidine-1-carboxylate 8j
Following the general procedure, employing 4- hydroxypiperidine as the amine yielded compound 8j as a
colorless oil in 68% yield, 1H NMR (500 MHz, CDCl3) δ 4.08 (m, 2H), 3.95 (brs,2H), 3.76 (brs, 1H), 3.23 (m, 2H),
2.76 (brs, 2H), 2.63 (brs, 1H), 2.07 (m, 2H), 1.89 (brs, 2H), 1.69 (brs, 2H), 1.50 (m, 2H), 1.45 (brs, 9H); MS-ESI (m/z):
335 (M + Na)+.
4.2.3.11. tert-butyl 4-(1,4-dioxa-8-azaspiro[4.5]decane-8-carbonyl)piperidine-1-carboxylate 8k
Following the general procedure, employing 4- hydroxypiperidine as the amine yielded compound 8k as a colorless
oil in 75% yield, 1H NMR (500 MHz, CDCl3) δ 4.14 (d, J = 11.6 Hz, 2H), 3.98 (s, 4H), 3.68 (brs, 2H), 3.56 (brs, 2H),
2.76 (t, J = 12.2 Hz, 2H), 2.64 (m, 1H), 1.69 (m, 8H), 1.45 (s, 9H); MS-ESI (m/z): 355 (M + H)+.
4.2.4. General procedure for the preparation of 9a-k
To a stirred solution of 8a-k (0.25 mmol) in DCM (5 mL) was added TFA, and stirred for 1 hour. The mixture was
concentrated, and diluted by DCM, concentrated again for three times to fully remove the TFA. The residue was
dissolved in THF. To the mixture was added LiAlH4 (0.5 mL,1 M solution in THF) at 0 oC, and stirred for 30 mins. The
mixture was slowly quenched by MeOH (0.5 mL), and NaOH solution (0.2 mL, 1 M). The mixture was filtered through
celite, washed by MeOH, and concentrated. The residue was concentrated and purified by chromatography on a silica
gel column (DCM : MeOH : NH3·H2O = 5 : 3 : 1) to afford crude 9a-k as brown oils，which were used directly for the
next step.
4.2.5. General procedure for the preparation of 1a-b and 2a-k
To a stirred solution of 5a-b or 9a-k (0.1 mmol) in anhydrous MeOH (5 mL) was added compound 6 (32 mg, 0.1
o
mmol) and Et3N (28 µL, 0.2 mmol) at room temperature under argon. The mixture was heated to 40 C, stirred for 3
hours, and concentrated. The mixture was diluted by DCM (15 mL) and washed by H2O. The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated. The residue was purified by chromatography on a silica gel column

ACCEPTED MANUSCRIPT
(DCM : MeOH : NH3·H2O = 10 : 1 : 0.01) or prepare TLC (DCM : MeOH : NH3·H2O = 10 : 1 : 0.01) to give BTZs
1a-b or 2a-k.
4.2.5.1. 2-(4-(cyclohexyl(methyl)amino)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one 1a
Following the general procedure, employing 5a yielded compound 1a as yellow solid in 40% yield, mp:146-148 oC;
HPLC purity, 98.1%; 1H NMR (500 MHz, CDCl3) δ 9.09 (1H, s), 8.75 (1H, s), 5.24 (1H, brs), 4.43 (1H, brs), 3.18
(2H,brs), 3.05 (1H, brs), 2.68 (1H,brs), 2.35 (3H, s), 2.05 (2H, brs), 1.83 (4H, brs), 1.65 (2H, d, J = 11.7 Hz), 1.37 (2H,
m), 1.26 (2H, m), 1.12 (2H, m); ESI-MS (m/z): 471 (M + H)+.
4.2.5.2. 2-(4-(cyclohexylamino)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one 1b
Following the general procedure, employing 5b yielded compound 1b as yellow solid in 40% yield, mp:171-173 oC;
HPLC purity, 96.3%; 1H NMR (500 MHz, CDCl3) δ 9.09 (1H, s), 8.75 (1H, s), 4.99 (1H, brs), 4.34 (1H, brs), 3.48 (1H,
s), 3.38 (2H, brs), 3.11 (1H, brs), 2.62 (1H, brs), 2.09 (2H, brs), 1.91 (2H, d, J = 9.65 Hz), 1.76 (2H, d, J = 12.3 Hz),
1.63 (2H, d, J = 11.8 Hz), 1.53 (2H, brs), 1.26 (2H, m), 1.16 (2H, m); ESI-MS (m/z): 457 (M + H)+.
4.2.5.3. 8-nitro-2-(4-(piperidin-1-ylmethyl)piperidin-1-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one 2a
Following the general procedure, employing 9a yielded compound 2a as yellow solid in 40% yield, mp:165-167 oC;
HPLC purity, 99.0%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.10 (s, 1H), 8.74 (s, 1H), 5.25 (brs, 1H), 4.37 (brs, 1H),
3.29 (brs, 1H), 3.01 (brs, 1H), 2.35 (brs, 4H), 2.18-1.95 (m, 7H), 1.57-1.27 (m, 6H); 13C NMR (400 MHz, CDCl3) δ
(ppm) 166.46, 161.62, 144.03, 134.28, 133.26 (q, J = 3.5 Hz), 129.61 (q, J = 35.4 Hz), 126.88, 125.85 (q, J = 3.6 Hz),
122.44 (q, J = 272.95 Hz), 64.36, 55.20, 46.94, 33.63, 30.84, 25.63, 24.16; ESI-MS (m/z): 457 (M + H)+.
4.2.5.4. 8-nitro-2-(4-(pyrrolidin-1-ylmethyl)piperidin-1-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one 2b
Following the general procedure, employing 9b yielded compound 2b as yellow solid in 20% yield, mp:156-158 oC;
HPLC purity, 93.9%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.09 (s, 1H), 8.74 (s, 1H), 5.30-5.24 (m, 1H), 4.38 (brs, 1H),
3.91-3.05 (m, 2H), 2.71 (brs, 4H), 2.52 (brs, 3H), 2.21-2.00 (m, 4H), 1.87 (brs, 2H), 1.35-1.25 (m, 2H); 13C NMR (400
MHz, CDCl3) δ (ppm) 166.57, 161.74, 143.94, 134.30, 133.34 (q, J = 3.4 Hz), 129.60(q, J = 35.4 Hz), 126.73, 126.00
(q, J = 3.7 Hz), 122.42(q, J = 273.5 Hz), 61.72, 54.76, 46.81, 35.08, 30.67, 23.48; ESI-MS (m/z): 443 (M + H)+.
4.2.5.5. 2-(4-(azepan-1-ylmethyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one 2c
Following the general procedure, employing 9c yielded compound 2c as yellow solid in 28% yield, mp: 141-143 oC;
HPLC purity, 92.8%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.10 (s, 1H), 8.74 (s, 1H), 5.30-5.24 (m, 1H), 4.38 (brs, 1H),
3.29 (brs, 1H), 3.04 (brs, 1H), 2.67 (brs, 4H), 2.40 (brs, 2H), 2.22-1.88 (m, 5H), 1.62 (d, J = 26.0 Hz, 6H), 1.28-1.25 (m,
2H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.61, 161.62, 143.95, 134.39, 133.34(q, J = 35.3 Hz), 126.78, 125.97 (q,
J = 3.6 Hz), 122.44 (q, J = 272.4 Hz), 63.29, 55.98, 47.16, 34.81, 30.75, 27.83, 27.17; ESI-MS (m/z): 471.6 (M + H)+.
4.2.5.6.
2-(4-((4-methylpiperazin-1-yl)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]
thiazin-4-one 2d
Following the general procedure, employing 9d yielded compound 2d as yellow solid in 28% yield, mp: 143-145 oC;
1
HPLC purity, 95.1%; H NMR (500 MHz, CDCl3) δ (ppm) 9.09 (s, 1H), 8.74 (s, 1H), 5.25 (brs, 1H), 4.37 (brs, 1H),
3.28-3.20 (m, 1H), 3.11-3.00 (m, 1H), 2.53 (brs, 8H), 2.37 (s,3H), 2.24 (d, J = 6.6Hz, 2H), 1.99-1.90 (m, 3H), 1.28-1.26
(m, 2H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.57, 161.67, 143.95, 134.32, 133.35 (q, J = 3.5 Hz), 129.59 (q, J =
35.5 Hz), 126.76, 125.98 (q, J = 3.6 Hz), 122.42 (q, J = 271.6 Hz), 63.53, 54.90, 52.96, 46.96, 45.58, 33.60, 30.67;
ESI-MS (m/z): 472.6 (M + H)+.
4.2.5.7. 2-(4-((diethylamino)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one 2e
Following the general procedure, employing 9e yielded compound 2e as yellow solid in 38% yield, mp: 117-118
oC; HPLC purity, 92.9%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.07 (s, 1H), 8.72 (s, 1H), 5.28 (brs, 1H), 4.36 (brs, 1H),

ACCEPTED MANUSCRIPT
3.28 (brs, 1H), 3.06-2.99 (m, 1H), 2.51 (q, J = 7.0 Hz, 4H), 2.27 (d, J = 6.5 Hz, 2H), 2.02-1.99 (m, 2H), 1.86-1.81 (m,
1H), 1.30-1.21 (m, 2H), 0.99 (t, 6H); ESI-MS (m/z): 445 (M + H)+.
4.2.5.8.
2-(4-((2-methylpiperidin-1-yl)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]
thiazin-4-one 2f
Following the general procedure, employing 9e yielded compound 2e as yellow solid in 36% yield, mp: 128-130
oC; HPLC purity, 98.1%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.09 (s, 1H), 8.74 (s, 1H), 5.29-5.25 (m, 1H), 4.37 (brs,
1H), 3.29 (brs, 1H), 3.00 (brs, 1H), 2.84 (brs, 1H), 2.54 (brs, 1H), 2.62-1.89 (m, 4H), 1.63 (brs,4H), 1.28-1.25 (m, 6H),
1.02 (s, 3H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.58, 161.57, 143.39, 133.34(q, J = 3.5 Hz), 129.54 (q, J = 35.2
Hz), 126.79, 125.96 (q, J = 3.7 Hz), 122.44 (q, J = 272.9 Hz), 59.31, 56.88, 53.12, 47.19, 34.71, 30.90, 25.93, 23.57,
19.05, 14.15; ESI-MS (m/z): 471 (M + H)+.
4.2.5.9.
2-(4-((3-methylpiperidin-1-yl)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]
thiazin-4-one 2g
Following the general procedure, employing 9g yielded compound 2g as yellow solid in 36% yield, mp: 127-128
oC; HPLC purity, 98.1%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.10 (s, 1H), 8.74 (s, 1H), 5.34-5.25 (m, 1H), 4.37 (brs,
1H), 3.30 (brs, 1H), 3.02 (brs, 1H), 2.72 (brs, 2H), 2.16 (brs, 2H), 2.04-1.85 (m, 5H), 1.70-1.55 (m, 5H), 1.26 (brs, 2H),
0.86 (d, J = 5.7 Hz, 3H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.60, 161.62, 143.95, 134.39, 133.34(q, J = 3.4 Hz),
129.56(q, J = 35.5 Hz), 126.79, 125.96 (q, J = 3.7 Hz), 122.44 (q, J = 273.8 Hz), 64.36, 62.79, 54.78, 47.12, 33.81,
32.98, 31.09, 29.35, 25.51, 19.71; ESI-MS (m/z): 471 (M + H)+.
4.2.5.10.
2-(4-((4-methylpiperidin-1-yl)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]
thiazin-4-one 2h
Following the general procedure, employing 9h yielded compound 2h as yellow solid in 36% yield, mp: 130-131
oC; HPLC purity, 97.6%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.14 (s, 1H), 8.79 (s, 1H), 5.39-5.28 (m, 1H), 4.41 (brs,
1H), 3.33 (brs, 1H), 3.06 (brs, 1H), 2.84 (brs, 1H), 2.22 (d, J = 4.0 Hz, 2H), 1.96 (brs, 5H), 1.66-1.63 (m, 3H), 1.30 (brs,
4H), 0.97 (d, J = 6.3 Hz, 3H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.58, 161.62, 143.94, 133.33 (q, J = 3.5 Hz),
129.54 (q, J = 35.4 Hz), 126.79, 125.95(q, J = 3.6 Hz), 122.44(q, J = 273.2 Hz), 63.21, 54.65, 53.46, 47.06, 34.27,
33.84, 30.78, 21.87; ESI-MS (m/z): 471 (M + H)+.
4.2.5.11. 8-nitro-6-(trifluoromethyl)-2-(4-((4-(trifluoromethyl)piperidin-1-yl)methyl)piperidin-1-yl)-4H-benzo[e][1,3]
thiazin-4-one 2i
Following the general procedure, employing 9i yielded compound 2i as yellow solid in 36% yield, mp:
158-159 oC; HPLC purity, 98.4%; 1H NMR (500 MHz, CDCl3) δ (ppm) 9.14 (s, 1H),8.79 (s, 1H),5.30 (brs, 1H), 4.43
(brs, 1H), 3.33 (brs, 1H), 2.98 (brs, 4H), 2.26 (brs, 2H), 2.01 (brs, 3H), 1.87 (brs, 4H), 1.66 (brs,2H), 1.32 (d, J = 7.2 Hz,
2H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.58, 161.70, 143.95, 134.30, 133.35 (q, J = 3.5 Hz), 129.61 (q, J = 35.5
Hz), 126.75, 126.00 (q, J = 3.6 Hz), 122.42 (q, J = 272.5 Hz),63.73, 53.08, 46.92, 40.33 (q, J = 28.4 Hz), 33.83, 30.69,
24.63; ESI-MS (m/z): 525.6 (M + H)+.
4.2.5.12. 2-(4-((4-hydroxypiperidin-1-yl)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]
thiazin-4-one 2j
Following the general procedure, employing 9j yielded compound 2j as yellow solid in 36% yield, mp: 78-80 oC;
HPLC purity, 93.4%; 1H NMR (500 MHz, CDCl3)δ(ppm) 9.09(s, 1H),8.7 5(s, 1H), 5.26 (brs, 1H), 4.39 (brs, 1H),
3.71-3.52 (m, 1H), 3.29-3.04 (m, 5H), 2.73 (brs, 2H), 2.33-2.20 (m, 4H), 2.00-1.90 (m, 3H), 1.56 (brs, 2H), 1.26 (brs,
2H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.60, 161.71, 143.95, 134.32, 133.36 (q, J = 3.3 Hz), 129.60 (q, J = 34.9
Hz), 126.75, 126.00 (q, J = 3.3 Hz), 122.42 (q, J = 273.1 Hz), 63.51, 51.60, 46.98, 34.47, 33.91, 30.79, 29.72, 29.35,
27.24; ESI-MS (m/z): 473.6 (M + H)+.

ACCEPTED MANUSCRIPT
4.2.5.13. 2-(4-((1,4-dioxa-8-azaspiro[4.5]decan-8-yl)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e]
[1,3]thiazin-4-one 2k
Following the general procedure, employing 9k yielded compound 2k as yellow solid in 36% yield, mp: 78-80 oC;
1H NMR (500 MHz, CDCl3) δ 9.09 (s, 1H), 8.75 (s, 1H), 5.34-5.25 (m, 1H), 4.41 (brs, 1H), 3.96 (brs, 4H), 3.28 (brs,
1H), 3.05 (brs, 1H), 2.59 (brs, 4H), 2.40-2.33 (m, 2H), 2.23-2.20 (m, 1H), 1.79-1.77 (brs, 4H), 1.63-1.58 (m, 2H), 1.27
(brs, 2H); 13C NMR (400 MHz, CDCl3) δ (ppm) 166.58, 161.72, 143.95, 134.30, 133.36 (q, J = 3.6 Hz), 129.60 (q, J =3
5.9 Hz), 126.75, 126.00(q, J = 3.6 Hz), , 122.42 (q, J = 272.9 Hz), 64.37, 63.13, 51.99, 46.78,34.48, 33.80, 30.84, 27.24;
ESI-MS (m/z): 515.6 (M + H)+.
4.3. MIC determination
MICs against replicating M. tuberculosis were determined by the microplate Alamar blue assay (MABA). RIF and
INH were included as positive controls. M. tuberculosis H37Rv and clinical isolate strains was grown to late log phase
(70 to 100 Klett units) in Difco Middlebrook 7H9 Broth (catalog no. 271310) supplemented with 0.2% (vol/vol)
glycerol, 0.05% Tween 80, and 10% (vol/vol) albumin-dextrosecatalase (BBL Middlebrook ADC Enrichment, catalog
no. 212352) (7H9-ADCTG). Cultures were centrifuged, washed twice, and then suspended in phosphate
phosphate-buffered saline. Suspensions were then passed through an 8 µm-pore-size filter to remove clumps, and
aliquots were frozen at -80 °C. Two fold dilutions of target compounds were prepared in 7H9-ADC-TG in a volume of
100 µl in 96-well, black, clear-bottom microplates (BD Biosciences, Franklin Lakes, NJ). M. tuberculosis (100 µl
containing 2 × 105 CFU) was added, yielding a final testing volume of 200 µl. The plates were incubated at 37°C; on
day 7 of incubation, 12.5 µl of 20% Tween 80 and 20 µl of Alamar blue were added to all wells. After incubation at
37 °C for 16 to 24 h, the fluorescence was read at an excitation of 530 nm and an emission of 590 nm. The MIC was
defined as the lowest concentration effecting a reduction in fluorescence of ≥90% relative to the mean of replicate
bacterium-only controls.
4.4. Pharmacokinetic Profiles determination
SPF female ICR mice weighing 20-25ꢀg were used in the pharmacokinetic study. The rats were fasted overnight
before dosing. Every treatment group contained 3 mice. Mice were dosed with the tested compounds suspension at
50ꢀmg/kg (p.o.). Compounds were suspended in 0.5% CMC for oral administration. Blood was collected from the
jugular vein of each animal at the following times after administration of drugs: 0.25, 0.5, 1, 2, 4, 6, 8 and 24ꢀh after a
single oral dosing. All blood samples were centrifuged at 3000ꢀr/min for 10ꢀmin to obtain serum which was then stored
at −20ꢀ°C. 150ꢀµL of the serum was added to 500ꢀµL of acetonitrile and the mixture was centrifuged at 13000ꢀr/min for
10ꢀmin to remove protein. The supernatant was dried and dissolved in 100ꢀµL of acetonitrile, the solution was
centrifuged at 13000ꢀr/min for 10ꢀmin. The supernatant was moved to a sample bottle for HPLC analysis. Total area
under the concentration time curve (AUC), the elimination half-time (t1/2), the peak concentration (Cmax) and the time to
reach peak concentration (Tmax) of samples were determined directly from the experimental data using WinNonlin
V6.2.1.
4.5 HPLC purity determination.
All samples were performed on an Agilent 1260 HPLC-UV system. Conditions (solvent A = methanol, solvent B =
0.1% TFA + H2O): Zorbax SB-C18 column (250 mm × 4.6 mm, 5 µm, PN: 883975-902). Injection volumn: 10 µL.
Flow: 1.3 mL/min. Gradient elution: 0.00 min, 10% A; 3 min, 50% A; 15 min, 100% A; 16 min, 10% A; 18 min 10%A.
UV at 254 nm.
Notes
The authors declare no competing financial interest.
Abbreviations

ACCEPTED MANUSCRIPT
TB, tuberculosis; MTB, Mycobacterium tuberculosis; MDR, multidrug-resistant; XDR, extensively drug-resistant;
WHO, World Health Organization; ATP, adenosine triphosphate; hERG, human ether-a-go-go related gene; ADME,
absorption, distribution, metabolism, excretion, toxicity; BTZs, nitrobenzothiazinones; DprE1, Decaprenyl
phosphoryl-β-D-ribose 2/-epimerase; SAR, structure-activity relationship; PK, pharmacokinetic; MeOH, methanol;
NaCNBH3, sodium cyanoborohydride; TFA, trifluoroacetic acid; DCM, methylene chloride; MIC, minimum inhibitory
concentration; HOBt, hydroxybenzotriazole; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; LiAlH4, lithium
aluminum hydride; MABA, microplate alamar blue assay; INH, isoniazid; RFP, rifampicin; T1/2, half-life; Cmax, the
maximum serum concentration; Tmax, the time at which the Cmax is observed; AUC, area under the curve; MRT,
mean residence time.
Ethical statement
All animal experiments were carried out in accordance with the guidelines of the Chinese Association for Laboratory
Animal Sciences, and approved by the institutional ethical committee (IEC) of Peking Union Medical College.
Acknowledgment
This work is supported by the National Mega-project for Innovative Drugs (2017ZX09201001; 2015ZX09102007-008;
2015ZX09304006-016), CAMS Initiative for Innovative Medicine (2016-I2M-1-010), CAMS Innovation Fund for
Medical Science (CAMS-2017-I2M-1-011), PUMC Youth Fund (2017350011) and Beijing Municipal Administration
of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201304).
References
[1] A. Nusrath Unissa, L.E. Hanna, S. Swaminathan, A note on derivatives of isoniazid, rifampicin, and pyrazinamide
showing activity against resistant mycobacterium tuberculosis, Chemical biology & drug design, 87 (2016) 537-550.
[2] Global tuberculosis report 2017. World Health Organization, http://www.who.int/tb/publications/global_report/en/.
[3] X. Li, V. Hernandez, F.L. Rock, W. Choi, Y.S.L. Mak, M. Mohan, W. Mao, Y. Zhou, E.E. Easom, J.J. Plattner, W.
Zou, E. Perez-Herran, I. Giordano, A. Mendoza-Losana, C. Alemparte, J. Rullas, I. Angulo-Barturen, S. Crouch, F.
Ortega, D. Barros, M.R.K. Alley, Discovery of a potent and specific M. tuberculosis leucyl-tRNA synthetase inhibitor:
(S)-3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)-ol (GSK656), J. Med. Chem. 60
(2017) 8011-8026.
[4] J. Cohen, Infectious disease. Approval of novel TB drug celebrated--with restraint, Science, 339 (2013) 130.
[5] N.J. Ryan, J.H. Lo, Delamanid: first global approval, Drugs, 74 (2014) 1041-1045.
[6] D.T. Hoagland, J. Liu, R.B. Lee, R.E. Lee, New agents for the treatment of drug-resistant Mycobacterium
tuberculosis, Adv. Drug Deliv. Rev. 102 (2016) 55-72.
[7] V. Makarov, G. Manina, K. Mikusova, U. Mollmann, O. Ryabova, B. Saint-Joanis, N. Dhar, M.R. Pasca, S. Buroni,
A.P. Lucarelli, A. Milano, E. De Rossi, M. Belanova, A. Bobovska, P. Dianiskova, J. Kordulakova, C. Sala, E. Fullam,
P. Schneider, J.D. McKinney, P. Brodin, T. Christophe, S. Waddell, P. Butcher, J. Albrethsen, I. Rosenkrands, R.
Brosch, V. Nandi, S. Bharath, S. Gaonkar, R.K. Shandil, V. Balasubramanian, T. Balganesh, S. Tyagi, J. Grosset, G.
Riccardi, S.T. Cole, Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis, Science, 324
(2009) 801-804.

ACCEPTED MANUSCRIPT
[8] V. Makarov, B. Lechartier, M. Zhang, J. Neres, A.M. van der Sar, S.A. Raadsen, R.C. Hartkoorn, O.B. Ryabova, A.
Vocat, L.A. Decosterd, N. Widmer, T. Buclin, W. Bitter, K. Andries, F. Pojer, P.J. Dyson, S.T. Cole, Towards a new
combination therapy for tuberculosis with next generation benzothiazinones, EMBO Mol. Med. 6 (2014) 372-383.
[9] C. Trefzer, M. Rengifo-Gonzalez, M.J. Hinner, P. Schneider, V. Makarov, S.T. Cole, K. Johnsson,
Benzothiazinones: prodrugs that covalently modify the decaprenylphosphoryl-beta-D-ribose 2'-epimerase DprE1 of
Mycobacterium tuberculosis, J. Am. Chem. Soc. 132 (2010) 13663-13665.
[10] T. Karoli, B. Becker, J. Zuegg, U. Mollmann, S. Ramu, J.X. Huang, M.A. Cooper, Identification of antitubercular
benzothiazinone compounds by ligand-based design, J. Med. Chem. 55 (2012) 7940-7944.
[11] K. Lv, X. You, B. Wang, Z. Wei, Y. Chai, B. Wang, A. Wang, G. Huang, M. Liu, Y. Lu, Identification of better
pharmacokinetic benzothiazinone derivatives as new antitubercular agents, ACS Med. Chem. Lett. 8 (2017) 636-641.
[12] R. Zhang, K. Lv, B. Wang, L. Li, B. Wang, M. Liu, H. Guo, A. Wang, Y. Lu, Design, synthesis and antitubercular
evaluation of benzothiazinones containing an oximido or amino nitrogen heterocycle moiety, Rsc Adv. 7 (2017)
1480-1483.
[13] J. Neres, F. Pojer, E. Molteni, L.R. Chiarelli, N. Dhar, S. Boy-Rottger, S. Buroni, E. Fullam, G. Degiacomi, A.P.
Lucarelli, R.J. Read, G. Zanoni, D.E. Edmondson, E. De Rossi, M.R. Pasca, J.D. McKinney, P.J. Dyson, G. Riccardi, A.
Mattevi, S.T. Cole, C. Binda, Structural basis for benzothiazinone-mediated killing of mycobacterium tuberculosis, Sci.
Transl. Med. 4 (2012) 150ra121.
[14] F. Kloss, V. Krchnak, A. Krchnakova, S. Schieferdecker, J. Dreisbach, V. Krone, U. Mollmann, M. Hoelscher,
M.J. Miller, In vivo dearomatization of the potent antituberculosis agent BTZ043 via Meisenheimer Complex
Formation, Angew Chem. Int. Edit. 56 (2017) 2187-2191.
[15] R. Tiwari, P.A. Miller, L.R. Chiarelli, G. Mori, M. Sarkan, I. Centarova, S.H. Cho, K. Mikusova, S.G. Franzblau,
A.G. Oliver, M.J. Miller, Design, Syntheses, and Anti-TB Activity of 1,3-Benzothiazinone Azide and Click Chemistry
Products Inspired by BTZ043, ACS Med. Chem. Lett. 7 (2016) 266-270.
[16] V. Makarov, J. Neres, R.C. Hartkoorn, O.B. Ryabova, E. Kazakova, M. Sarkan, S. Huszar, J. Piton, G.S. Kolly, A.
Vocat, T.M. Conroy, K. Mikusova, S.T. Cole, The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1
from Mycobacterium tuberculosis, Antimicrobial agents and chemotherapy, 59 (2015) 4446-4452.
[17] C.T. Peng, C. Gao, N.Y. Wang, X.Y. You, L.D. Zhang, Y.X. Zhu, Y. Xv, W.Q. Zuo, K. Ran, H.X. Deng, Q. Lei,
K.J. Xiao, L.T. Yu, Synthesis and antitubercular evaluation of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one
derivatives, Bioorganic & medicinal chemistry letters, 25 (2015) 1373-1376.
[18] R. Tiwari, U. Mollmann, S. Cho, S.G. Franzblau, P.A. Miller, M.J. Miller, Design and Syntheses of
Anti-Tuberculosis Agents Inspired by BTZ043 Using a Scaffold Simplification Strategy, ACS medicinal chemistry
letters, 5 (2014) 587-591.
[19] C. Gao, T.H. Ye, N.Y. Wang, X.X. Zeng, L.D. Zhang, Y. Xiong, X.Y. You, Y. Xia, Y. Xu, C.T. Peng, W.Q. Zuo,
Y. Wei, L.T. Yu, Synthesis and structure-activity relationships evaluation of benzothiazinone derivatives as potential
anti-tubercular agents, Bioorganic & medicinal chemistry letters, 23 (2013) 4919-4922.
[20] R. Tiwari, P.A. Miller, S. Cho, S.G. Franzblau, M.J. Miller, Syntheses and Antituberculosis Activity of
1,3-Benzothiazinone Sulfoxide and Sulfone Derived from BTZ043, ACS medicinal chemistry letters, 6 (2015)
128-133.
[21] Y. Lu, M. Zheng, B. Wang, L. Fu, W. Zhao, P. Li, J. Xu, H. Zhu, H. Jin, D. Yin, H. Huang, A.M. Upton, Z. Ma,
Clofazimine analogs with efficacy against experimental tuberculosis and reduced potential for accumulation,
Antimicrob. agents Chemother. 55 (2011) 5185-5193.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Table 1. Structures and activities of 1a-b and 2a-e
Q
NO2
S
N
N
F3C
O
Compd.
Q
MIC
0.973
1a
0.959
1b
0.481
3.578
1.320
2a
2b
2c
N
7.844
1.825
2d
2e
N
N
Me
<0.016
0.037
0.084
PBTZ169
INH
RFP
INH: isoniazid; RFP: rifampicin

ACCEPTED MANUSCRIPT
Table 2. Structures and activities of 1a-b and 2a-e
Compd.
W
MIC
0.481
2a
1.922
0.241
2f
2g
0.488
<0.016
1.961
3.709
2h
2i
2j
2k
<0.016
PBTZ169

ACCEPTED MANUSCRIPT
Table 3. Anti-MDR TB activity of compound 2i
MIC (µg/mL)
Compd.
MDR-TB 16995a
MDR-TB 16883a
2i
<0.016
<0.016
<0.016
<0.016
PBTZ169
INH
>40
>40
>40
>40
RFP
INH: isoniazid; RFP: rifampicin; a MDR-TB 16995 and MDR-TB 16883 were isolated from patients in Beijing Chest Hospital.
Table 4. PK profiles of compound 2i dosed orally in mice at 50 mg/kg (n = 3)
PK parameters
T1/2 (h)
PBTZ169
2i
3.3 ± 3.01
0.25 ± 0
2.87 ± 1.03
0.83 ± 0.29
1300 ± 422
5478 ± 1730
3.73 ± 0.94
Tmax (h)
Cmax (ng/mL)
AUC0-inf (h·ng/mL)
MRT (h)
1165 ± 223
2489 ± 1273
4.26 ± 3.74

ACCEPTED MANUSCRIPT
Figure 1. Design of new BTZs
Scheme 1. Synthesis of BTZs 1a-b and 2a-k

ACCEPTED MANUSCRIPT
Highlights:
1. Two series of BTZs 1-2 with piperidine moiety were designed and synthesized.
2. Some targets showed considerable in vitro activity (MIC<1 µg/mL) against MTB strain.
3. Compound 2i displayed potent in vitro anti-MDR-TB activity (MIC < 0.016 µg/mL).