Identification of benzothiazinones containing an oxime functional moiety as new anti-tuberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2019.111595

A series of benzothiazinones (BTZs) containing an oxime moiety, based on the structure of ZR-10 discovered in our lab, were designed and synthesized. Most of the compounds with alkoxyimino groups attached to the piperazine or cyclohexyl ring of PBTZ169, e

Full text of DOI:10.1016/j.ejmech.2019.111595

Journal Pre-proof
Identification of benzothiazinones containing an oxime functional moiety as new anti-
tuberculosis agents
Apeng Wang, Kai Lv, Zeyu Tao, Jian Gu, Lei Fu, Mingliang Liu, Baojie Wan, Scott G.
Franzblau, Chao Ma, Xican Ma, Bing Han, Aoyu Wang, Shijie Xu, Yu Lu
PII:
S0223-5234(19)30729-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111595
EJMECH 111595
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2019
Revised Date: 4 August 2019
Accepted Date: 5 August 2019
Please cite this article as: A. Wang, K. Lv, Z. Tao, J. Gu, L. Fu, M. Liu, B. Wan, S.G. Franzblau, C. Ma,
X. Ma, B. Han, A. Wang, S. Xu, Y. Lu, Identification of benzothiazinones containing an oxime functional
moiety as new anti-tuberculosis agents, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111595.
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Graphic Abstract

Identification of Benzothiazinones Containing an Oxime Functional
Moiety as New Anti-tuberculosis Agents
Apeng Wang a,1, Kai Lv a,1, Zeyu Tao a, Jian Gu b, Lei Fu c, Mingliang Liu a, *, Baojie
Wand, Scott G. Franzblau d, Chao Ma a, Xican Ma a,b, Bing Han a,b, Aoyu Wang a,e,
Shijie Xu a, Yu Luc, *
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
b College of Pharmacy, Southwest Minzu University, Chengdu, 610041, China
cBeijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology,
Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital
College of Pharmacy, Medical University, Beijing 100149, China
dInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois, Chicago, Illinois
60612, USA
e Hebei Medical University, Shijiazhuang, 050017, China
1These authors contributed equally to this work.
*Corresponding author: lmllyx@126.com, 86-010-63030965 (M.L. Liu);
luyu4876@hotmail.com, 86-010-89509357 (Y. Lu)
ABSTRACT: A series of benzothiazinones (BTZs) containing an oxime moiety,
based on the structure of ZR-10 discovered in our lab, were designed and synthesized.
Most of the compounds with alkoxyimino groups attached to the piperazine or
cyclohexyl ring of PBTZ169, exhibit excellent in vitro activity against both
drug-sensitive and clinically isolated multidrug-resistant Mycobacterium tuberculosis
(MTB) strains (MIC: < 0.016 -0.037 µg/mL) and low cell cytotoxicity. Two close
PBTZ169-analogues 3a and 3b with proper ADME/T and PK properties show potent
in vivo efficacy in an acute mouse model of tuberculosis. Compound 3a is under
evaluation as a potential clinical candidate for treatment of tuberculosis.
KEYWORDS: Benzothiazinones; Oximes; Antimycobacterial activity; Synthesis

1.Introduction
Tuberculosis (TB) affects one-third of the world's population, with approximately
10.0 million new TB cases and 1.6 million TB-related deaths estimated by the World
Health Organization (WHO) in 2017 [1]. This disease is mainly caused by
Mycobacterium tuberculosis (MTB), currently treated with a standard therapy
combination of four drugs, during a six months course that does not favour patient
compliance. Recently, resistance to this therapy has risen, creating the new labels
multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB, which pose a
serious global health threat [2-5]. Although bedaquiline and delamanid were approved
for the treatment of MDR-TB, some adverse events have been noted [6]. Hence, the
development of more effective and safer anti-TB agents with novel mechanisms
cannot be overemphasized [7].
As a novel class of anti-TB agents targeting decaprenyl phosphoryl-β-D-ribose
2ʹ-epimerase (DprE1), benzothiazinones (BTZs), have garnered great interest recently.
Two candidates BTZ043 and PBTZ169 (Figure 1), are in Phase I [8] and II [9] clinical
trials at present, respectively, for the treatment of both drug-susceptible TB and
MDR-TB. Structure-activity relationship (SAR) studies of BTZs demonstrate that the
crucial moieties for potent activity are the S atom and carbonyl group on the
thiazinone ring, a strong electron-withdrawing one (CF3, CN, etc.) at C-6 position,
and more importantly the NO2 group at C-8 position [10, 11]. Many series of new BTZs
were reported to have potent antimycobacterial activity [12-23]. Some BTZs containing
oxime-functionalized N-heterocycles exampled by ZR-10 (Fig. 1), were identified as
new anti-TB agents in our lab [14], highlighting the importance of oximes with respect
to antimycobacterial activity of these compounds. Further, the oximino group is
frequently employed in the most popular antibiotics - third /fouth generation
cephalosporins and some quinolones (gemifloxacin, zabofloxacin). Thus, we intended
to design and synthesize a series of novel BTZ derivatives, wherein oximes are
attached to nitrogen heterocycles through a methylene or an ethylene, or the

cyclohexyl ring of PBTZ169 is replaced with a symmetric cyclic ketoxime moiety in
this study (Fig. 1). Our primary objective was to find new BTZ derivatives with
potent antimycobacterial activity and facilitate the further development of these
compounds.
Figure 1. Design of new BTZ derivatives
2. Results and discussion
2.1. Chemistry
The syntheses of target compounds 1-2 and 3-4 are shown in Schemes 1 and 2,
respectively.
Nucleophilic
substitution
of
N-Boc-piperazine
5
with
1-bromopropan-2-one and Michael addition with but-3-en-2-one gave ketones 6a and
6b, respectively. Oximation of the ketones 6a and 6b with O-methyl/ ethyl/
benzylhydroxylamine, and then removal of the Boc protecting group with

trifluoracetic acid (TFA) yielded side chain compounds 7a-f. Coupling of BTZ core
compound 11 with 7a-f in the presence of Et3N (TEA) afforded the target compounds
1a-f. Compounds 2a-e were conveniently obtained from 8 in a similar manner as
compounds 1a-f (Scheme 1).
Reagents and conditions: a) 1-bromopropan-2-one or but-3-en-2-one, K2CO3, CH3CN, rt, 3-4h, 52%
- 61%; b) R1ONH2, Pyridine, MeOH, rt, 5-7h; c) TFA, DCM, rt, 3-4 h, 48%-72%, two steps; d)
Et3N, MeOH, 40℃, 1-3 h, 27%-45%.
Scheme 1. Synthesis of compounds 1a-f and 2a-e
Oximation of 4-(hydroxymethyl)cyclohexanone 12 with O-methylhydroxylamine
(MeONH2) or O- ethylhydroxylamine (EtONH2) gave oximes 13a and 13b,
respectively. Tosylation of the alcohols 13a and 13b with p-toluenesulfonyl chloride
(TsCl) in the presence of pyridine yielded 14a and 14b, respectively. Nucleophilic
substitution of the esters 14a and 14b with N-Boc-piperazine derivatives, and then
removal of the Boc protecting group with TFA afforded side chain compounds 15a-o.
Coupling of 11 with 15a-o in the presence of Et3N afforded the target compounds
3a-o. Compounds 4a-f were furnished as a similar procedure for compounds 3a-o

using 3-(hydroxymethyl)cyclobutanone 16 as the starting material (Scheme 2).
Reagents and conditions: a) MeOH, MeONH2 or EtONH2, Pyridine, rt, 5-7h; b) Pyridine, TsCl, rt,
overnight, 56%-64%; c) N-Boc-piperazine derivatives, K2CO3, KI, 18-crown-6, CH3CN, 80℃,
overnight; d) TFA, DCM, rt, 3-4h, 35%-42%, two steps; e) Et3N, MeOH, 40℃, 1-3 h, 25%-41%.
Scheme 2. Synthesis of compounds 3a-o and 4a-f
2.2. In-vitro anti-MTB activity
The target compounds 1-4 were initially screened for in vitro activity against MTB
[24]
H37Rv ATCC 27294 strain using the Microplate Alamar Blue Assay (MABA)
.
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 isoniazid
(INH), rifampicin (RFP) and PBTZ169 for comparison are presented in Table 1.
The data reveal that with a few exceptions (2c-e), all the BTZ derivatives show
potent in vitro activity against this strain (MIC: < 0.1 µg/mL). 19 compounds were
found to have the same excellent potency (MIC: <0.016 µg/mL) as PBTZ169, while
being more active than RFP and INH (MIC: 0.031-0.078 µg/mL). Overall, the
potency of the derivatives is related to the whole structures of the side chains.
Moreover, exception for 1f, 2e and 3l, all of them have lower ClogP values (3.25 –

4.96) than PBTZ169 (5.10), indicating that they should have slightly less lipophilicity
than PBTZ169. However, no clear correlation between ClogP and antimycobacterial
activity was found in this study.
We are pleased to find that the first designed series 1a-f exhibit roughly equal MIC
values of <0.016 – 0.037 µg/mL to PBTZ169, suggesting that oxime-functionalities
on the terminal nitrogen of the piperazine ring are well acceptable, and the carbon
chain length (n) and the size of the alkyl group (R1) on the oxime moiety have little
effects on antimycobacterial activity. However, replacement of the piperazine ring
with 2,8-diazaspiro[4.5]decanes leads to decreased activity (1 vs 2), probably due to
the rigid structures of the spiro-heterocycles.
In further modifications, oximes were directly introduced on the para-position of
the cyclohexane ring of PBTZ169 (CLogP: 5.10), the corresponding compounds 3a
(CLogP: 3.70) and 3b (CLogP: 4.23) with less lipophilicity remain excellent potency
(MIC: <0.016 µg/mL). The effect of R2 group (s) on the piperazine ring of 3a, b was
also investigated. All of the methylated derivatives 3c-o show potent activity (MIC:
<0.016 – 0.031 µg/mL) comparable to 3a, b, indicating that the position, number and
chirality of the methyl group(s) have hardly influence on antimycobacterial activity.
Yet surprisingly, exception for 4b (3b vs 4b), replacement of the
cyclohexanone-oximes with symmetric cyclobutanone-oximes leads to decreased
activity (3a vs 4a, 3n vs 4c, 3o vs 4d, 3k vs 4e, 3l vs 4f), maybe due to the ring tension
of the cyclobutanone-oximes.
Table 1. Structures and activity of compounds 1-4 against MTB H37Rv
6
NOR1
Me
7
( )n
1
N
( )n
N
N
N
W =
3
NOR1
4
9
10
Me
NO2
2a-e
1a-f
S
W
2
6
3
N
5
2
3
N
F3C
N
N
N
O
6
5
R2
R2
NOR1
NOR1
3a-o
4a-f

Cpds.a R1 n / R2
ClogPb MIC
(µg/mL)
Cpds.a
R1
Et
n / R2
ClogPb MIC (µg/mL)
Me
Et
1
1
1
2
2
2
3.25
3.78
4.96
3.42
3.95
5.15
0.037
(R)-2-Me 4.75
<0.016
<0.016
<0.016
<0.016
<0.016
1a
1b
1c
1d
1e
1f
3h
3i
<0.016
<0.016
<0.016
<0.016
Me (S)-2-Me 4.22
Et (S)-2-Me 4.75
Me 3,5-di-Me 4.74
Et 3,5-di-Me 5.27
Me 2,5-di-Me 4.74
Bn
Me
Et
3j
3k
3l
Bn
3m
<0.016
0.085
0.031
Me
1
3.53
Me 3-Me
4.22
2a
3n
<0.016
Et
1
4.06
3.70
4.23
5.43
3.70
4.23
4.22
4.75
4.22
4.75
4.22
0.029
Et
3-Me
H
4.75
2.98
3.51
3.50
4.02
<0.016
0.023
<0.016
0.056
0.052
0.066
0.056
0.078
0.031
<0.016
2b
2c
2d
2e
3a
3b
3c
3d
3e
3f
3o
Me
Et
2
0.469
Me
Et
4a
2
0.241
H
4b
Bn
Me
Et
2
0.119
Me 3-Me
Et 3-Me
4c
H
H
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
0.023
4d
Me 3,5-di-Me 4.01
Et 3,5-di-Me 4.54
4e
Me (S)-3-Me
Et (S)-3-Me
Me (R)-3-Me
Et (R)-3-Me
Me (R)-2-Me
4f
RFP
INH
PBTZ169
5.10
3g
a Cpds are Compounds; b ClogP is caculated in ChemOffice 2017.
Encouraged by their potent activity (MIC: < 0.016 µg/mL) against drug sensitive
MTB H37Rv strain, 19 compounds were evaluated against two clinical isolated
MTB-MDR strains (16995 and 16833) resistant to both INH and RFP. As shown in
Table 2, all of them exhibit comparable activity (MIC: < 0.016 - 0.017 µg/mL) to
PBTZ169, suggesting their promising potential for both drug-sensitive and resistant
MTB strains (Tables 1 and 2).
2.3. Cell cytotoxicity

The above 19 compounds were tested for mammalian cell cytotoxicity using Vero
cells measured as a concentration inhibiting 50% growth (CC50) as compared to a
no-treatment control, and the results are reported in Table 2. Exception for 3l, all the
BTZ derivatives show low cytotoxicity (CC50: >64 µg/mL).
Table 2. Anti-MDR-MTB activity and cytotoxicity of selected compounds
Cpds.
MIC (µg/mL)
Vero cells
CC50b (µg/mL)
Compd.
MIC (µg/mL)
Vero cells
CC50b (µg/mL)
MDR-1a MDR-2a
MDR-1a MDR-2a
1b
0.010
<0.016
<0.016
0.017
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
3h
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
>40
>64
>64
>64
>64
60.45
>64
>64
>64
>64
NT
1c
1d
1e
1f
0.010
3i
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
3j
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
3k
3l
3a
3b
3c
3d
3e
3f
3n
3o
4b
PBTZ169 <0.016
RFP
INH
>40
>40
>40
NT
a MDR-1 is MDR-MTB 16995 and MDR-2 is MDR-MTB 16833, which were isolated from
patients in Beijing Chest Hospital; NT, not tested. b The 50% cytotoxic concentration. NT: not
tested.
2.4. In vivo pharmacokinetic profiles
Among of the above compounds with potent activity against all the tested strains
and low cytotoxicity, two closest PBTZ169-analogues 3a and 3b were further
evaluated for their in vivo pharmacokinetic (PK) profiles in ICR mice after a single
oral administration of 25 mg/kg. Although shorter T1/2 (1.65 - 2.29 h), As shown in

Table 3, 3a and 3b display excellent drug exposures with Cmax (8757 - 15221 ng·mL-1)
and AUC0-∞ (27527 - 40601 h·ng·mL-1) which are significantly higher than PBTZ169.
Table 3. In vivo PK profiles of compounds 3a and 3b
Cpds.
PBTZ169
2.78±1.85
0.25±0
3a
3b
T1/2 (h)
1.65±0.9
0.25±0
2.29±1.04
0.25±0
Tmax (h)
Cmax (ng·mL-1)
AUC0-∞ (h·ng·mL-1)
MRT (h)
8573±2232
27527±3581
2.46±0.5
15221±2009
40601±6976
2.30±0.18
912±156
1657±711
3.17±1.42
2.5. In vivo efficacy
The preliminary in vivo efficacy evaluation of compounds 3a and 3b along with
PBTZ169 and INH was conducted in a murine model of acute infection with MTB
H37Rv strain. All of them were given orally at the same dose of 25 mg/kg, once daily
for 5 days a week for a total of 3 weeks in BALB/c mice, as described in published
protocols [24]. As shown in Table 4, compounds 3a and 3b appear highly active after 3
weeks of treatment, which results in a significant reduction of MTB colony-forming
unit (CFU) in lungs by 4.04 and 4.44 Logs, respectively, as compared to the untreated
control group. To our satisfaction, both of 3a and 3b demonstrate slightly stronger in
vivo efficacy compared to PBTZ169, although poorer than INH (25 mg/kg).
Compared to the previous compounds [12-23], the identification of 3a and 3b with
potent in vivo efficacy was a progress in the modification of BTZs derivatives.
Table 4. In vivo efficacy of 3a and 3b against MTB H37Rv in a BALB/c mouse
model
Cpds.
Log10 CFU/lung
6.44±0.34
Untreated

3a
2.40±0.20
2.00±0.07
2.65±0.54
1.76±0.49
3b
PBTZ169
INH
2.6. Further Appraisal
Based on the potent in vitro activity and in vivo efficacy described above,
additional studies were also conducted focusing on compounds 3a and 3b. As
presented in Table 5, 3a and 3b are not active against MTB H37Rv strain under
LORA condition (MIC
>
32 µg/mL), although they show slightly
higher inhibition ratios (86% and 79%, respectively) than PBTZ169 (78%) at the
same concentration of 32 µg/mL. Compounds 3a and 3b (IC50: 7.80 µM and >10µM,
respectively) are less active than PBTZ169 (IC50: 3.77 µM) against cytochrome P450
enzyme 2C19, and they do not, like PBTZ169, inhibit other four enzymes (1A2, 2D6,
3A4, 2C9) with IC50s > 10 µM, suggesting a very low potential for drug-drug
interactions. Low inhibition of hERG (IC50 > 30 µM) of 3a and 3b suggest a low risk
of blocking the cardiac potassium channel and causing QT prolongation. Compounds
3a and 3b, like PBTZ169, have not mutate effects on TA100 and TA102 strains, in
the presence or absence of metabolic activation (S9 fraction), in the salmonella
typhimurium reverse mutation assay (mini-Ames). A preliminary in vivo tolerability
study was carried out in SD rats with a single dose at 2 g/kg. All animals survived
after oral administration followed by a 7-day observation.
Table 5. Additional Comparative Data for 3a and 3b
Cpds.
3a
3b
PBTZ169
78
LORAa
% Inhb
MIC (µg/mL)
1A2
86
79
>32
>10
>10
>32
>10
>10
>32
CYP inhibition
>10
IC50 (µM)
2D6
>10

3A4
2C9
>10
>10
>10
>10
>10
>10
2C19
7.80
>10
3.77
hERG IC50 (µM)
47.98±5.51
No
38.92±12.78
No
101.7±24.09
No
Mutagenic effect (Ames test)
Acute Toxicity Studyc
10/10d
10/10d
10/10d
aLow-oxygen-recovery assay. bThe inhibition ratio at 32 µg/mL. cSingle oral dose in SD rats. dNo.
of animals that survived/total no. of animals after 7 days.
The PK profiles of compound 3a was also further studied in SD rats, following a
single intravenous dose (2.5 mg/kg) and oral multi-dose administrations (25, 50, 200
mg/kg, n = 6, bisexual each half). As shown in Table 6, 3a shows excellent oral PK
profiles, and its major parameters (Cmax, AUC0-∞, F%) are significantly greater than
PBTZ169 at the same doses. Thus far, all the above results support compound 3a to
be worth of further investigation.
Table 6. In vivo PK profiles of compound 3a
Cpds.
route
Dose
T1/2
(h)
Tmax
(h)
Cmax
AUC0-∞
(h·ng·mL-1)
5721±1402
26779±12301
F
(mg/kg)
(ng·mL-1)
2591±1322
(%)
3a
IV
2.5
25
1.77±0.43
-
PO
3.20±0.71 1.25±0.61 6227±2122
46.1±19.4
40.4±12.5
50
4.13±1.27 1.67±0.52 10085±4509 45909±15262
200
2.5
25
6.73±1.41 2.83±1.33 18247±4848 128143±66556 30.3±14.0
PBTZ169 IV
1.64±0.48
-
1445±234
1543±376
1604±942
3117±842
4725±967
PO
4.23±2.58 0.91±0.64 463±258
4.86±0.74 1.33±0.75 694±104
5.76±2.03 2.50±1.76 804±442
10.7±6.63
10.2±2.99
3.82±0.73
50
200

3. Conclusion
In summary, we designed and synthesized a series of novel BTZ derivatives
containing an oxime functional moiety as new anti-TB agents based on the structure
feature of ZR-10 discovered in our lab. With a few exceptions (2c-e), all of them
exhibit considerable activity against MTB H37Rv strain (MIC: < 0.1 µg/mL). All of
those compounds (1a-f, 3a-o), wherein oximes are attached to the piperazine or
cyclohexyl ring of PBTZ169, show comparable activity to PBTZ169 against MTB
H37Rv and MDR-MTB strains (MIC: < 0.016 – 0.037 µg/mL). Two close
PBTZ169-analogues 3a and 3b with proper ADME/T and PK properties were
identified to have potent in vivo efficacy in an acute mouse model of tuberculosis
which is a breakthrough respect to the previous benzothiazinones [12-23]. Compound 3a
with significantly greater major parameters than PBTZ169 at the same doses after oral
multi-dose administrations (25, 50, 200 mg/kg), has been selected as a potential
clinical development candidate for treatment of TB. Further evaluation including
combination study and deep understanding of druggability of compound 3a are
ongoing to fully assess its potential as a new agent to treat MDR-TB.
Experimental protocols
4.1. Chemistry
All commercially available solvents and reagents were obtained from
commercial suppliers and used without further purification. Melting points were
1
13
determined in open capillaries and uncorrected. H NMR and C NMR spectra were
determined on a Varian Mercury-600/500/400 spectrometer in DMSO-d6 or CDCl3
using tetra-methylsilane (TMS) as an internal standard. Electrospray ionization (ESI)
mass spectra and high-resolution mass spectra (HRMS) were 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 synthesis procedure of compounds 6a-b and 9a-b.
To a stirring solution of tert-butyl piperazine-1-carboxylate 5 (3 mmol) or
tert-butyl 2,8-diazaspiro[4.5]decane-8-carboxylate 8 or in CH3CN (50 mL) was added

1-bromopropan-2-one or but-3-en-2-one (4 mmol) and K2CO3 (5 mmol) at room
temperature. The mixture was stirred for 3-4 hours at room temperature and filtered.
The filtrate was diluted by H2O (250 mL) and extracted by DCM (100 mL × 3). The
combined organic layer was washed by brine, dried over anhydrous MgSO4, filtered,
and concentrated. The residue was purified over silica gel column (DCM: MeOH = 40:
1) to yield oils compounds 6a-b or 9a-b (yield, 52%-61%).
4.2.1.1. tert-butyl 4-(2-oxopropyl)piperazine-1-carboxylate 6a
1
Yellow oil; yield: 61%; H NMR (500 Mz, CDCl3): δ 3.37 (t,
J
= 5.1 Hz, 4H, H-2,
H-6), 3.11 (s, 2H, -CH2C(O)-), 2.33 (t,
1.35 (s, 9H, 1-NBoc).
J = 5.1 Hz, 4H, H-3, H-5), 2.05 (s, 3H, Me),
4.2.1.2. tert-butyl 4-(3-oxobutyl)piperazine-1-carboxylate 6b
1
Yellow oil; yield: 57%; H NMR (500 Mz, CDCl3): δ 3.38 (brs, 4H, H-2, H-6), 2.64
(d,
J = 13.3 Hz, 4H, -CH2-CH2-C(O)-), 2.37 (brs, 4H, H-3, H-5), 2.14 (s, 3H, Me),
1.42 (s, 9H, 1-NBoc).
4.2.2. General synthesis procedure of compounds for 1a-f and 2a-e
To a stirred solution of 6a-b or 9a-b (0.5 mmol) in MeOH (15 mL) was added
pyridine (2 mL) and RONH2·HCl (0.6 mmol) at room temperature. The mixture was
stirred for 5-7 hours and then concentrated. The residue was dissolved with DCM and
washed by brine. The organic phase was added TFA (3 mL) at room temperature. The
mixture was stirred for 3-4 hours and concentrated to afford the crude products 7a-f or
10a-e which was used directly in the next step without further purification. To a
stirred solution of above crude products 7a-f or 10a-e in anhydrous MeOH (10 mL)
was added BTZ core compound 11 (0.3 mmol) and Et3N (0.6 mmol) at room
temperature. The mixture was stirred overnight at 40 oC and concentrated. The residue
was purified by silica gel column (DCM: MeOH = 20: 1) to yield the yellow oils or
yellow solids, which were further treated by n-hexane to give compounds 1a-f and
2a-e (yield, 27%-45%).
4.2.2.1. 2-(4-(2-(methoxyimino)propyl)piperazin-1-yl)-8-nitro-6-(trifluoromethyl)
-4H-benzo[e][1,3]thiazin-4-one (1a)
Yellow solid; yield: 43%; mp: 105-107 oC; 1H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H,
benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5), 4.17-4.06 (m, 4H,
piperazine(H-2, H-6)), 3.87 (s, 3H, CH3, -NOMe), 3.06 (s, 2H, CH2, -CH2C(NOMe)),
2.60 (brs, 4H, piperazine (H-3, H-5)), 1.91 (s, 3H, Me, -C(NOMe)CH3). 13C NMR
(100 Mz, CDCl3) δ 166.8, 162.6, 144.2, 134.1, 133.8 (q,
J
= 3.3 Hz), 130.0, 127.0,
126.4 (q, = 3.3 Hz), 122.7 (q,
J
J = 271.4 Hz), 52.8, 52.4, 46.5, 30.5, 29.6, 27.6, 8.9.
MS-ESI (m/z): 446.1 (M+H)+. HRMS-ESI (m/z): Calcd. for C17H19F3N5O4S (M+H)+:
446.1104; Found: 446.1108.

4.2.2.2.
2-(4-(2-(ethoxyimino)propyl)piperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benz
o[e][1,3]thiazin-4-one (1b)
Yellow solid; yield, 40%; mp: 112-114 oC; 1H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H,
benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5), 4.11(q,
J = 7.5 Hz, 2H,
-NOCH2-), 4.18-4.06 (m, 4H, piperazine (H-2, H-6)), 3.06 (s, 2H, CH2,
-CH2C(NOMe)), 2.59 (t,
-C(NOMe)CH3), 1.25 (t,
J
J
= 5.0 Hz, 4H, piperazine (H-3, H-5)), 1.92 (s, 3H, Me,
= 7.5 Hz, 3H, Me, -NOCH2CH3). 13C NMR (150 Mz,
CDCl3) δ 166.6, 162.3, 154.0, 144.1, 133.5 (q,
J
= 3.6 Hz), 130.0 (q,
J = 35.4 Hz),
127.0, 126.1 (q, = 3.6 Hz), 122.5 (q,
J
J = 273.3 Hz), 69.4, 62.0, 52.6, 14.8,
13.3.MS-ESI (m/z): 460.1 (M+H)+. HRMS-ESI (m/z): Calcd. for C18H21F3N5O4S
(M+H)+: 460.1261; Found: 460.1269.
4.2.2.3.
2-(4-(2-((benzyloxy)imino)propyl)piperazin-1-yl)-8-nitro-6-(trifluorome-thyl)-4H
-benzo[e][1,3]thiazin-4-one (1c)
Yellow solid; yield: 27%; mp: 101-103 oC; 1H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H,
benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5), 7.35-7.26 (m, 5H,
-NOCH2Ph), 5.11 (s, 2H, -NOCH2Ph), 4.10-3.88 (m, 4H, piperazine(H-2, H-6)), 3.06
(brs, 2H, CH2, -CH2C(NOMe)), 2.54 (brs, 4H, piperazine (H-3, H-5)), 1.95 (s, 3H, Me,
-C(NOMe)CH3). 13C NMR (100 Mz, CDCl3) δ 166.6, 162.3, 155.0, 144.0, 138.1, 134.
1, 133.5 (q, J = 3.6 Hz), 129.9 (q, J = 35.5 Hz), 128.5, 128.1, 127.9, 126.9, 126.2 (q,
J
= 3.6 Hz), 122.5 (q, J = 271.6 Hz), 75.8, 61.8, 52.5, 29.8, 13.6. MS-ESI (m/z): 522.1
(M+H)+. HRMS-ESI (m/z): Calcd. for C23H23F3N5O4S (M+H)+: 522.1417; Found:
522.1423.
4.2.2.4.
2-(4-(3-(methoxyimino)butyl)piperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benz
o[e][1,3]thiazin-4-one (1d)
Yellow solid; yield: 45%; mp: 131-133 oC; 1H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H,
benzothiazinone H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.00-3.81 (m, 4H,
piperazine(H-2, H-6)), 3.82 (s, 1.74H, OMe), 3.81 (s, 1.08H, OMe), 2.63-2.52 (m, 7H,
piperazine(H-3, H-5), -CH2CHHC(NOMe)), 2.39 (t,
-CHHC(NOMe)), 1.89 (s, 1.22 H, Me), 1.85 (s, 1.74 H, Me). 13C NMR (100 Mz,
CDCl3) δ 166.5, 162.2, 156.1, 155.6, 144.0, 134.2, 133.5 (q, = 3.4 Hz), 123.0 (q,
35.0 Hz), 126.8, 126.2 (q, = 3.4 Hz), 122.5 (q, = 271.8 Hz), 61.4, 54.5, 53.7, 52.5,
J
= 7.25 Hz, 1H,
J
J =
J
J

33.4, 26.9, 20.3, 14.2. MS-ESI (m/z): 460.1 (M+H)+. HRMS-ESI (m/z): Calcd. for
C18H21F3N5O4S (M+H)+: 460.1261; Found: 460.1271.
4.2.2.5.
2-(4-(3-(ethoxyimino)butyl)piperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[
e][1,3]thiazin-4-one (1e)
Yellow solid; yield: 45%; mp: 115-117 oC; 1H NMR (500 Mz, CDCl3): δ 9.07 (s, 1H,
benzothiazinone H-7), 8.74 (s, 1H, benzothiazinone H-5), 4.12-3.89 (m, 6H,
piperazine(H-2, H-6), -NOCH2-), 2.54-2.63 (m, 7H, piperazine(H-3, H-5),
-CH2CHHC(NOMe)), 2.40-2.37 (m,1H, -CHHC(NOMe)), 1.85 (s, 3H, Me), 1.23 (t,
= 7.0 Hz, 3H, Me, -NOCH2CH3). 13C NMR (100 Mz, CDCl3) δ 166.5, 162.2, 155.2,
144.0, 134.1, 133.5 (q, = 3.4 Hz), 129.7 (q, = 35.0 Hz) 126.8, 126.1 (q, = 3.4 Hz),
122.5 (q, = 271.8 Hz), 69.0, 54.5, 53.7, 52.4, 33.5, 26.9, 20.3, 14.8, 14.3.; MS-ESI
J
J
J
J
J
(m/z): 474.1 (M+H)+. HRMS-ESI (m/z): Calcd. for C19H23F3N5O4S (M+H)+:
474.1417; Found: 474.1423.
4.2.2.6. 2-(4-(3-((benzyloxy)imino)butyl)piperazin-1-yl)-8-nitro-6-(trifluoro-
methyl)-4H-benzo[e][1,3]thiazin-4-one (1f)
Yellow solid; yield: 37%; mp: 110-112 oC; 1H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H,
benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5), 7.36-7.26 (m, 5H,
-NOCH2Ph), 5.07 (s, 1.16H, -CHHPh), 5.06 (s, 0.79H, -CHHPh), 4.08-3.77 (m, 4H,
piperazine(H-2, H-6)), 2.65-2.56 (m, 7H, piperazine(H-3,H-5), -CH2CHHC(NOBn)),
13
2.42-2.39 (m,1H, -CHHC(NOBn)), 1.91 (s, 1.63H, Me), 1.90 (s, 1.51H, Me). C
NMR (100 Mz, CDCl3) δ 166.6, 162.2, 156.6, 156.2, 144.0, 138.5, 138.2, 134.2,
133.5 (q,
126.2 (q,
J
= 3.4 Hz), 129.5 (q,
= 3.4 Hz), 122.4 (q,
J
= 35.5Hz), 128.5, 128.4, 128.3, 127.9, 127.8, 126.8,
= 271.8 Hz), 75.5, 54.1, 53.7, 52.4, 46.2, 33.4, 29.8,
J
J
27.0, 20.3, 14.6, 8.7. MS-ESI (m/z): 536.1 (M+H)+. HRMS-ESI (m/z): Calcd. for
C24H25F3N5O4S (M+H)+: 536.1574; Found: 536.1578.
4.2.2.7. 2-(2-(2-(methoxyimino)propyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-ni-
tro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (2a)
o
1
Yellow solid; yield: 35%; mp: 113-115 C; H NMR (500 Mz, CDCl3) δ 9.10 (d, J =
1.0 Hz, 1H, benzothiazinone H-7), 8.75 (d, J = 1.0 Hz, 1H, benzothiazinone H-5),
3.99-3.81 (m, 7H, diazaspiro[4.5]decan (H-7, H-9), NOMe), 3.14 (s, 2H, -CH2
C(NOMe)), 2.68 (brs, 2H, diazaspiro[4.5]decan (H-1)), 2.48 (brs, 2H,
diazaspiro[4.5]decan (H-3)), 1.90 (s, 3H, Me), 1.79 (brs, 6H, diazaspiro[4.5]decan

(H-4, H-6, H-10)); 13C NMR (125 Mz, CDCl3) δ166.5, 161.7, 144.0, 134.2, 133.4,
128.8 (q, = 34.5 Hz), 126.7, 126.0, 122.4 (q, = 272.4 Hz), 61.5, 59.4, 58.5, 53.2,
J
J
44.7, 40.9, 37.1, 36.2, 18.4, 12.9; MS-ESI (m/z): 500.1 (M+H)+.
4.2.2.8. 2-(2-(2-(ethoxyimino)propyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (2b)
o
Yellow solid; yield: 27%; mp: 109-111 C; 1H NMR (500 Mz, CDCl3) δ 9.15 (s, 1H,
benzothiazinone H-7), 8.80 (s, 1H, benzothiazinone H-5), 4.16-3.89 (m, 6H,
diazaspiro[4.5]decan (H-7, H-9), -NOCH2-), 3.16 (s, 2H, -CH2 C(NOEt)), 2.71 (s, 2H,
diazaspiro[4.5]decan (H-1)), 2.51 (s, 2H, diazaspiro[4.5]decan (H-3)), 1.94 (s, 3H,
Me), 1.79 (brs, 6H, diazaspiro[4.5]decan (H-4, H-6, H-10)), 1.24 (t,
J = 7.0 Hz, 3H,
Me, -NOCH2CH3); 13C NMR (125 Mz, DMSO-d6) δ165.7, 161.4, 144.9, 134.9, 131.7,
127.8 (q,
J = 34.5 Hz), 126.6, 123.1 (q, J = 272.4 Hz), 68.6, 65.3, 59.3, 53.3, 40.9,
37.1, 36.1, 15.1, 13.3; MS-ESI (m/z): 514.1 (M+H)+.
4.2.2.9. 2-(2-(3-(methoxyimino)butyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (2c)
o
1
Yellow solid; yield: 38%; mp: 113-115 C; H NMR (500 Mz, CDCl3) δ 9.10 (d, J =
1.5 Hz, 1H, benzothiazinone H-7), 8.75 (d, J = 1.5 Hz, 1H, benzothiazinone H-5),
3.94-3.82 (m, 7H, diazaspiro[4.5]decan (H-7, H-9), -NOCH3), 2.65-2.58 (m, 4H,
-NCH2CH2C(NOMe)), 2.52-2.50 (m, 2H, diazaspiro[4.5]decan (H-1)), 2.36 (t, J = 7.6
Hz, 2H, diazaspiro[4.5]decan (H-3)), 1.88 (s, 3H, Me), 1.79-1.73 (m, 6H,
13
diazaspiro[4.5]decan (H-4, H-6, H-10)); C NMR (100 Mz, CDCl3) δ 166.5, 161.7,
156.6, 156.1, 144.0, 134.3, 131.7 (q,
J
= 3.5 Hz), 130.1, 127.7 (q,
J = 35.5 Hz), 126.6,
123.1 (q,
J = 273.2 Hz), 65.2, 61.2, 53.4, 44.7, 40.6, 37.5, 36.3, 35.0, 14.1; MS-ESI
(m/z): 514.1 (M+H)+.
4.2.2.10. 2-(2-(3-(ethoxyimino)butyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (2d)
1
Orange oil; yield: 43%; H NMR (500 Mz, CDCl3) δ 9.14 (s, 1H, benzothiazinone
H-7), 8.80 (s, 1H, benzothiazinone H-5), 4.12-3.85 (m, 6H, diazaspiro[4.5]decan (H-7,
H-9), -NOCH2-), 2.81-2.50 (m, 8H, -NCH2CH2C(NOEt), diazaspiro[4.5]decan (H-1,
H-3)), 1.93-1.80 (m, 9H, diazaspiro[4.5]decan (H-4, H-6, H-10), Me), 1.30-1.28 (m,
3H, NOCH2CH3); 13C NMR (100 Mz, CDCl3) δ 165.6, 161.3, 156.4, 155.9, 144.8,
134.8, 131.7 (q, J = 3.5 Hz), 130.1, 127.7 (q, J = 35.5 Hz), 126.6, 123.1 (q, J = 273.2
Hz), 68.3, 68.3, 53.2, 52.8, 45.2, 43.4, 37.2, 36.2, 34.8, 15.1, 15.1; MS-ESI (m/z):
527.1 (M+H)+.

4.2.2.11.
2-(2-(3-((benzyloxy)imino)butyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-(trifluo
romethyl)-4H-benzo[e][1,3]thiazin-4-one (2e)
1
Orange oil; yield: 45%; H NMR (500 Mz, CDCl3) δ 9.15 (s, 1H, benzothiazinone
H-7), 8.80 (s, 1H, benzothiazinone H-5), 7.40-7.31 (m, 5H, NOCH2Ph), 5.11 (s, 2H,
NOCH2Ph), 4.16-3.83 (m, 4H, diazaspiro[4.5]decan (H-7, H-9)), 2.72-2.42 (m, 8H,
-NCH2CH2C(NOBn), diazaspiro[4.5]decan (H-1, H-3)), 1.94 (s, 3H, Me), 1.75 (brs,
6H, diazaspiro[4.5]decan (H-4, H-6, H-10)); 13C NMR (125 Mz, DMSO-d6) δ 165.7,
161.3, 156.9, 144.8, 139.0, 134.8, 131.7, 128.7, 128.0, 127,9, 127.8 (q,
J
= 34.5 Hz),
126.6, 123.1 (q,
J = 272.4 Hz), 74.8, 65.0, 53.2, 52.5, 51.7, 45.1, 40.7, 36.0, 34.6, 14.4;
MS-ESI (m/z): 590.2 (M+H)+.
4.2.3. General synthesis procedure of compounds 13a-b, 17a-b.
To a stirred solution of compound 12 or 16 (1.0 mmol) in MeOH (20 mL) was
added pyridine (10 mL) and MeONH2·HCl or EtOHNH2.HCl (2.5 mmol) at room
temperature. The mixture was stirred for 5-7 hours and washed by brine, then
concentrated for next step without purification.
To a stirred solution of the above compounds (1.0 mmol) in pyridine (20 mL)
was added TsCl (285.7 mg, 1.5 mmol) at room temperature. The mixture was stirred
overnight and concentrated. The residue was purified over silica gel column (EtOAc:
n-hexane = 1: 5) to give compounds 13a-b, 17a-b in a yield of 56%-64%.
4.2.3.1. (4-(methoxyimino)cyclohexyl)methyl 4-methylbenzenesulfonate (13a)
1
Yellow oil; yield: 64%; H NMR (500 Mz, CDCl3): δ 7.77 (d,
J
= 8.0 Hz, 2H,
= 6.0 Hz, 2H,
-S(O)2Ph(H)), 7.34 (d,
-S(O)2OCH2), 3.79 (s, 3H, -OMe), 3.17 (d,
-PhMe), 2.37 (d, = 14.0 Hz, 1H, H-5), 2.05 (dt,
J
= 8.0 Hz, 2H, -MePh(H)), 3.86 (d,
J
J
= 14.5 Hz, 1H, H-3), 2.45 (s, 3H,
= 4.0 Hz, = 13.5 Hz, 1H, H-3),
= 4.0 Hz, = 13.5 Hz, 1H, H-2),
J
J
J
1.94-1.83 (m, 3H, H-5, H-2, H-6), 1.73 (dt,
1.20-1.06 (m, 2H, H-6, H-1).
J
J
4.2.3.2. (4-(ethoxyimino)cyclohexyl)methyl 4-methylbenzenesulfonate (13b)
1
Yellow oil; yield: 61%; H NMR (500 Mz, CDCl3): δ 7.75 (d,
J
= 6.0 Hz, 2H,
= 5.0 Hz, 2H, -NOCH2 ),
= 14.0 Hz, 1H, H-3), 2.42 (s, 3H, -PhMe), 2.34
= 13.0 Hz, 1H, H-3), 1.90-1.81 (m, 3H, H-5, H-2,
= 13.0 Hz, 1H, H-2), 1.19 (t, = 7.00 Hz, 3H,-NOCH2CH3), 1.15-1.06
(m, 2H, H-6, H-1).
-S(O)2Ph(H)), 7.32 (d,
3.83 (brs, 2H, -S(O)2OCH2), 3.17 (d,
(d, = 14.0 Hz, 1H, H-5), 2.03 (t,
H-6), 1.71 (t,
J = 6.0 Hz, 2H, -MePh(H)), 4.00 (d, J
J
J
J
J
J
4.2.3.3. (3-(methoxyimino)cyclobutyl)methyl 4-methylbenzenesulfonate (17a)

1
Yellow oil; yield: 60%; H NMR (500 Mz, CDCl3): δ 7.78 (d,
J = 7.0 Hz, 2H,
S(O)2Ph(H)), 7.35 (d, = 8.0 Hz, 2H, -MePh(H)), 4.05 (brs, 2H, -S(O)2OCH2), 3.79 (s,
J
3H, OMe), 3.00-2.94 (m, 2H, H-2, H-4), 2.69-2.67 (m, 1H, H-2), 2.62-2.53 (m, 2H,
H-4, H-1), 2.45 (s, 3H, -PhMe).
4.2.3.4. (3-(ethoxyimino)cyclobutyl)methyl 4-methylbenzenesulfonate (17b)
1
Yellow oil; yield: 56%; H NMR (500 Mz, CDCl3): δ 7.77 (d,
J = 7.0 Hz, 2H,
S(O)2Ph(H)), 7.34 (d,
NOCH2), 3.01-2.91 (m, 2H, H-2, H-4), 2.69-2.64 (m, 1H, H-2), 2.59-2.54 (m, 2H,
H-4, H-1), 2.44 (s, 3H, -PhMe), 1.21 (t, = 7.0 Hz, 3H, -NOCH2CH3).
J = 7.0 Hz, 2H, -MePh(H)), 4.05-4.01 (m, 4H, -S(O)2OCH2,
J
4.2.4. General synthesis procedure of compounds 3a-o, 4a-f.
To a stirred solution of compound 13a-b, 17a-b (1.0 mmol) in CH3CN (20 mL)
were added N-Boc-piperazine (180.9 mg, 0.9 mmol), K2CO3 (205.9 mg, 1.5 mmol),
KI (166.0 mg, 1.0 mmol) and 18-crown-6 (26.4 mg, 0.1 mmol) at room temperature.
The mixture was stirred overnight at 80oC and filtered. The filtrate was diluted by
DCM and washed by brine. The organic layer was concentrated for next step. To a
stirred solution of the above compounds in DCM (20 mL) was added TFA (3 mL) at
room temperature. The mixture was stirred for 3-4 hours and concentrated to afford
the crude products 15a-o and 19a-f in a yield of 35%-42%. To a stirred solution of
above crudes in anhydrous MeOH (10 mL) was added BTZ core compound 11 (403.2
mg, 1.0 mmol) and Et3N (0.2 mL, 1.5 mmol|) at room temperature. The mixture was
stirred for 1-3 hours at 40 oC and concentrated. The residue was purified by silica gel
column (DCM: MeOH = 20: 1) to give 1a-f and 2a-e (25%-41% for two steps),
4.2.4.1. 2-(4-((4-(methoxyimino)cyclohexyl)methyl)piperazin-1-yl)-8-nitro-6-(tri-
fluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3a)
1
Yellow solid; yield: 41%; mp: 192-193 ; HPLC purity: 98.68%; H NMR (500 Mz,
CDCl3): δ 9.10 (s, 1H, benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5),
4.14-3.82 (m, 4H, piperazine(H-2, H-6)), 3.82 (s, 3H, NOMe), 3.19 (d,
1H, N-CHHCH-), 2.56 (brs, 4H, piperazine(H-3, H-5) ), 2.42 (d, = 14.0 Hz, 1H,
N-CHHCH-), 2.23 (d, = 7.5 Hz, 2H, cyclohexane (H-3)), 2.10 (dt, = 4.5 Hz,
13.5 Hz, 1H, cyclohexane (H-1)), 2.00 (d, = 12.0 Hz, 1H, cyclohexane (H-5)), 1.94
(d, = 11.0 Hz, 1H, cyclohexane (H-5)), 1.83-1.69 (m, 2H, cyclohexane (H-2)),
1.20-1.04 (m, 2H, cyclohexane (H-6)). 13C NMR (125 Mz, CDCl3) δ 166.5, 162.1,
J = 14.5 Hz,
J
J
J
J =
J
J
159.6, 144.0, 134.2, 133.4, 129.7 (q,
J = 35.3 Hz), 126.8, 126.1, 122.5 (q, J = 274.7
Hz), 63.8, 61.1, 53.2, 46.7, 34.4, 31.3, 31.2, 30.1, 24.1. MS-ESI (m/z): 500.1 (M+H)+.
HRMS-ESI (m/z): Calcd. for C21H25F3N5O4S (M+H)+: 500.1574; Found: 500.1578.
4.2.4.2. 2-(4-((4-(ethoxyimino)cyclohexyl)methyl)piperazin-1-yl)-8-nitro-6-(tri-

fluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3b)
1
Yellow solid; yield: 40%; mp: 197-199 ; HPLC purity: 97.78%; H NMR (500 Mz,
CDCl3): δ 9.10 (s, 1H, benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5),
4.27-3.81 (m, 4H piperazine(H-2, H-6)), 4.06 (q,
= 14.5 Hz, 1H, N-CHHCH-), 2.56 (brs, 4H, piperazine(H-3, H-5)), 2.42 (d,
Hz, 1H, N-CHHCH-), 2.24 (d, = 6.5 Hz, 2H, cyclohexane (H-3)), 2.11 (t,
J
= 6.5 Hz, 2H, NOCH2-), 3.22 (d,
J
= 14.0
= 13.0
J
J
J
Hz, 1H, cyclohexane (H-1)), 2.04-1.93 (m, 2H, cyclohexane (H-5)), 1.83-1.66 (m, 2H,
cyclohexane (H-2)), 1.25 (t,
J = 7.0 Hz, 3H, NOCH2CH3), 1.20-1.09 (m, 2H,
13
cyclohexane (H-6)). C NMR (100 Mz, CDCl3) δ 166.6, 162.2, 159.3, 144.0, 134.2,
133.5 (q, J = 4.0 Hz), 129.8 (q, J = 35.3 Hz), 126.8, 126.1 (q, J = 3.0 Hz), 122.5 (q, J
= 273.7 Hz), 68.8, 63.8, 53.1, 46.8, 34.4, 31.3, 30.1, 24.2, 14.7. MS-ESI (m/z): 514.1
(M+H)+. HRMS-ESI (m/z): Calcd. for C22H27F3N5O4S (M+H)+: 514.1730; Found:
514.1738.
4.2.4.3.
(S)-2-(4-((4-(methoxyimino)cyclohexyl)methyl)-3-methylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3c)
1
Yellow oil; yield: 31%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.71-4.36 (m, 1H, piperazine(H-2)),
4.10-3.90 (m, 1H, piperazine(H-6)), 3.81 (s, 3H, NOMe), 3.81-3.58 (m, 1H,
piperazine(H-2)), 3.48-3.23 (m, 1H, piperazine(H-6)), 3.19 (d, J = 14.5 Hz, 1H,
N-CHHCH-), 3.02-2.84 (m, 1H, piperazine(H-3)), 2.70-2.40 (m, 2H,
piperazine(H-5) ), 2.39 (d, J = 14.5 Hz, 1H, N-CHHCH-), 2.35 (brs, 1H, cyclohexane
(H-1)), 2.14-2.07 (m, 3H, cyclohexane (H-3, H-5)), 1.92 (d, J = 12.0 Hz, 1H,
cyclohexane (H-5)), 1.78 (dt, J = 5.5 Hz, J = 14.0 Hz, 1H, cyclohexane (H-2)), 1.68
(brs, 1H, cyclohexane (H-6)), 1.20-1.00 (m, 5H, cyclohexane (H-2, H-6), Me). 13C
NMR (100 Mz, CDCl3) δ 166.6, 162.1, 159.8, 144.0, 134.2, 133.5 (q, J = 3.0 Hz),
129.8 (q, J = 35.3 Hz), 126.9, 126.1 (q, J = 4.0 Hz), 122.5 (q, J = 273.7 Hz), 61.2,
58.9, 55.4, 35.1, 31.2, 30.3, 24.2. MS-ESI (m/z): 514.1 (M+H)+. HRMS-ESI (m/z):
Calcd. for C22H27F3N5O4S (M+H)+: 514.1730; Found: 514.1738.
4.2.4.4.
(S)-2-(4-((4-(ethoxyimino)cyclohexyl)methyl)-3-methylpiperazin-1-yl)-8-nitro-6-(t
rifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3d)
1
Yellow oil; yield: 35%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.70-4.43 (m, 1H, piperazine(H-2)), 4.05 (q,
J = 6.4 Hz, 2H, -NOCH2-), 4.07-3.87 (m, 1H, piperazine(H-6)), 3.80-3.60 (m, 1H,
piperazine(H-2)), 3.46-3.28 (m, 1H, piperazine(H-6)), 3.22 (d, J = 14.0 Hz, 1H,
N-CHHCH-), 3.06-2.89 (m, 1H, piperazine(H-3)), 2.70-2.45 (m, 2H, piperazine(H-5)),
2.43-2.31 (m, 2H, N-CHHCH-, cyclohexane (H-1)), 2.11-2.00 (m, 3H, cyclohexane
(H-3, H-5)), 1.87-1.77 (m, 2H, cyclohexane (H-5, H-2)), 1.70 (brs, 1H, cyclohexane

(H-2)), 1.24 (t, J = 7.0 Hz, 3H, -NOCH2CH3-), 1.16-1.06 (m, 5H, cyclohexane (H-6),
Me). 13C NMR (100 Mz, CDCl3) δ 166.6, 162.1, 159.4, 144.0, 134.2, 133.5 (q, J = 3.0
Hz), 129.8 (q, J = 35.3 Hz), 126.9, 126.1 (q, J = 4.0 Hz), 122.5 (q, J = 273.7 Hz), 68.8,
58.9, 55.5, 35.2, 31.3, 30.4, 24.3, 14.7. MS-ESI (m/z): 528.2 (M+H)+. HRMS-ESI
(m/z): Calcd. for C23H29F3N5O4S (M+H)+: 528.1887; Found: 528.1891.
4.2.4.5.
(R)-2-(4-((4-(methoxyimino)cyclohexyl)methyl)-3-methylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3e)
1
Yellow oil; yield: 32%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.65-4.43 (m, 1H, piperazine(H-2)),
4.09-3.87 (m, 1H, piperazine(H-6)), 3.82 (s, 3H, NOMe), 3.82-3.58 (m, 1H,
piperazine(H-2)), 3.46-3.30 (m, 1H, piperazine(H-6)), 3.18 (d, J = 14.5 Hz, 1H,
N-CHHCH- ), 3.02-2.93 (m, 1H, piperazine(H-3)), 2.68-2.48 (m, 2H,
piperazine(H-5)), 2.43-2.35 (m, 2H, N-CHHCH-, cyclohexane (H-1)), 2.14-2.00 (m,
3H, cyclohexane (H-3, H-5)), 1.92 (d, J = 12.0 Hz, 1H, cyclohexane (H-5)), 1.77 (dt,
J = 5.0 Hz, J = 14.0 Hz, 1H, cyclohexane (H-2)), 1.70 (brs, 1H, cyclohexane (H-2)),
1.19-0.99 (m, 5H, cyclohexane (H-6), Me). 13C NMR (100 Mz, CDCl3) δ 166.6, 162.1,
159.8, 144.0, 134.2, 133.5 (q, J = 3.0 Hz), 129.8 (q, J = 35.3 Hz), 126.9, 126.1 (q, J =
3.0 Hz), 122.5 (q, J = 273.7 Hz), 61.2, 59.0, 55.5, 35.1, 31.6, 31.4, 30.1, 24.1.
MS-ESI (m/z): 514.2 (M+H)+.
4.2.4.6.
(R)-2-(4-((4-(ethoxyimino)cyclohexyl)methyl)-3-methylpiperazin-1-yl)-8-nitro-6-(
trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3f)
1
Yellow oil; yield: 36%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.74 (s, 1H, benzothiazinone H-5), 4.68-4.41 (m, 1H, piperazine(H-2)), 4.05 (q,
J = 7.0 Hz, 2H, NOCH2-), 4.07-3.86 (m, 1H, piperazine(H-6)), 3.82-3.58 (m, 1H,
piperazine(H-2)), 3.50-3.29 (m, 1H, piperazine(H-6)), 3.22 (d, J = 15.0 Hz, 1H,
N-CHHCH-), 3.05-2.84 (m, 1H, piperazine(H-3)), 2.66-2.48 (m, 2H, piperazine(H-5)),
2.43-2.31 (m, 2H, N-CHHCH-, cyclohexane (H-1)), 2.14-2.00 (m, 3H, cyclohexane
(H-3, H-5)), 1.92 (d, J = 12.0 Hz, 1H, cyclohexane (H-5)), 1.77 (dt, J = 5.0 Hz, J =
14.0 Hz, 2H, cyclohexane (H-2)), 1.24 (t, J = 6.5 Hz, 3H, NOCH2CH3), 1.09-1.01 (m,
5H, cyclohexane (H-6), Me). 13C NMR (100 Mz, CDCl3) δ 166.6, 162.1, 159.5, 144.0,
134.2, 133.5 (q, J = 3.0 Hz), 129.8 (q, J = 35.3 Hz), 126.9, 126.1 (q, J = 3.0 Hz),
122.5 (q, J = 273.7 Hz), 68.8, 59.0, 55.5, 35.2, 31.6, 31.4, 30.1, 24.2, 14.7. MS-ESI
(m/z): 528.2 (M+H)+.
4.2.4.7.
(R)-2-(4-((4-(methoxyimino)cyclohexyl)methyl)-2-methylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3g)

1
Yellow oil; yield: 25%; H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H, benzothiazinone
H-7), 8.76 (s, 1H, benzothiazinone H-5), 5.60-4.11 (m, 1H, piperazine(H-2)), 3.81 (s,
3H，NOMe), 3.62-3.31 (m, 1H, piperazine(H-6)), 3.20 (d, J = 14.5 Hz, 1H,
N-CHHCH-), 2.94 (d, J = 10.0 Hz, 1H, piperazine(H-6)), 2.79 (d, J = 10.5 Hz, 1H,
piperazine(H-3)), 2.42 (d, J = 13.5 Hz, 1H, N-CHHCH-), 2.30-2.07 (m, 5H,
piperazine(H-3, H-5), cyclohexane (H-1, H-3, H-5) ), 2.03-1.94 (m, 2H, cyclohexane
(H-3, H-5)), 1.83-1.71 (m, 2H, cyclohexane (H-2, H-6)), 1.49-1.43 (m, 3H, Me),
1.20-1.06 (m, 2H, cyclohexane (H-2, H-6)). 13C NMR (100 Mz, CDCl3) δ 166.6,
162.1, 159.8, 144.0, 134.2, 133.5 (q, J = 4.0 Hz), 129.8 (q, J = 36.4 Hz), 126.9, 126.1
(q, J = 4.0 Hz), 122.5 (q, J = 274.7 Hz), 61.2, 59.0, 55.5, 35.1, 31.5, 30.1, 24.1.
MS-ESI (m/z): 514.2 (M+H)+.
4.2.4.8.
(R)-2-(4-((4-(ethoxyimino)cyclohexyl)methyl)-2-methylpiperazin-1-yl)-8-nitro-6-(
trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3h)
1
Yellow oil; yield: 27%; H NMR(500 Mz, CDCl3): δ 9.10 (s, 1H, benzothiazinone
H-7), 8.76 (s, 1H, benzothiazinone H-5), 5.45-4.45 (m, 1H, piperazine(H-2)), 4.06 (q,
J = 7.5 Hz, 2H, NOCH2-), 3.76-3.28 (m, 1H, piperazine(H-6)), 3.23 (d, J = 14.5 Hz,
1H, N-CHHCH-), 2.94 (d, J = 9.5 Hz, 1H, piperazine(H-6)), 2.79 (d, J = 10.5 Hz, 1H,
piperazine(H-3)), 2.42 (d, J = 14.5 Hz, 1H, N-CHHCH-), 2.30-2.07 (m, 5H,
piperazine(H-3, H-5), cyclohexane (H-1, H-3, H-5)), 2.03-1.90 (m, 2H, cyclohexane
(H-3, H-5)), 1.84-1.72 (m, 2H, cyclohexane (H-2, H-6)), 1.50-1.38 (m, 3H, Me), 1.25
(t, J = 7.5 Hz, 3H, NOCH2CH3), 1.20-1.07 (m, 2H, cyclohexane (H-2, H-6)). 13C
NMR (100 Mz, CDCl3) δ 166.6, 162.0, 159.5, 144.1, 134.4, 133.5 (q, J = 3.0 Hz),
127.0, 126.1 (q, J = 3.0 Hz), 68.9, 63.7, 34.6, 31.4, 31.2, 30.2, 30.0, 29.9, 24.3, 14.7.
MS-ESI (m/z): 528.2 (M+H)+.
4.2.4.9.
(S)-2-(4-((4-(methoxyimino)cyclohexyl)methyl)-2-methylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3i)
1
Yellow oil; yield: 29%; H NMR(500 Mz, CDCl3): δ 9.10 (s, 1H, benzothiazinone
H-7), 8.76 (s, 1H, benzothiazinone H-5), 5.52-4.08 (m, 1H, piperazine(H-2)), 3.83 (s,
3H, NOMe), 3.67-3.27 (m, 1H, piperazine(H-6)), 3.20 (d, J = 14.5 Hz, 1H,
N-CHHCH-), 2.94 (d, J = 10.5 Hz, 1H, piperazine(H-6)), 2.79 (d, J = 10.5 Hz, 1H,
piperazine(H-3)), 2.42 (d, J = 14.0 Hz, 1H, N-CHHCH-), 2.28-2.08 (m, 5H,
piperazine(H-3, H-5), cyclohexane (H-1, H-3, H-5)), 2.03-1.94 (m, 2H, cyclohexane
(H-3, H-5)), 1.84-1.73 (m, 2H, cyclohexane (H-2, H-6)), 1.50-1.31 (m, 3H, Me),
1.30-1.16 (m, 2H, cyclohexane (H-2, H-6)). 13C NMR (100 Mz, CDCl3) δ 166.6,
162.0, 159.8, 144.1, 134.4, 133.5 (q, J = 3.0 Hz), 129.8 (q, J = 35.3 Hz), 127.0, 126.1
(q, J = 4.0 Hz), 122.5 (q, J = 273.7 Hz) , 63.7, 61.2, 57.8, 53.6, 34.5, 34.5, 31.3, 31.2,
30.2, 30.0, 29.8, 29.5, 24.2. MS-ESI (m/z): 514.2 (M+H)+.

4.2.4.10.
(S)-2-(4-((4-(ethoxyimino)cyclohexyl)methyl)-2-methylpiperazin-1-yl)-8-nitro-6-(t
rifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3j)
1
Yellow oil; yield: 27%; H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H, benzothiazinone
H-7), 8.76 (s, 1H, benzothiazinone H-5), 5.57-4.16 (m, 1H, piperazine(H-2)), 4.06 (q,
J = 7.0 Hz, 2H, NOCH2-), 3.72-3.30 (m, 1H, piperazine(H-6)), 3.23 (d, J = 14.5 Hz,
1H, N-CHHCH-), 2.94 (d, J = 10.0 Hz, 1H, piperazine(H-6)), 2.79 (d, J = 10.5 Hz,
1H, piperazine(H-3)), 2.42 (d, J = 14.0 Hz, 1H, N-CHHCH-), 2.30-2.08 (m, 5H,
piperazine(H-3, H-5), cyclohexane (H-1, H-3, H-5)), 2.03-1.93 (m, 2H, cyclohexane
(H-3, H-5)), 1.84-1.63 (m, 2H, cyclohexane (H-2, H-6)), 1.50-1.41 (m, 3H, Me), 1.25
(t, J = 7.0 Hz, 3H, NOCH2CH3), 1.20-1.02 (m, 2H, cyclohexane (H-2, H-6)). 13C
NMR (100 Mz, CDCl3) δ 166.6, 162.0, 159.4, 144.10, 134.4, 133.5 (q, J = 3.0 Hz),
129.8 (q, J = 36.4 Hz), 127.0, 126.1 (q, J = 4.0 Hz), 122.5 (q, J = 273.7 Hz), 68.8,
63.7, 53.6, 34.6, 31.4, 31.3, 31.2, 30.2, 30.0, 29.8, 24.3, 14.7. MS-ESI (m/z): 528.2
(M+H)+.
4.2.4.11.
2-(4-((4-(methoxyimino)cyclohexyl)methyl)-3,5-dimethylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3k)
1
Yellow oil; yield: 34%; H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H, benzothiazinone
H-7), 8.76 (s, 1H, benzothiazinone H-5), 4.84 (d, J = 10.0 Hz, 1H, piperazine(H-2)),
4.00 (d, J = 10.0 Hz, 1H, piperazine(H-6)), 3.81 (s, 3H, NOMe), 3.26-3.15 (m, 2H,
piperazine(H-2), N-CHHCH- ), 2.97 (brs, 1H, piperazine(H-6)), 2.70-2.55 (m, 2H,
piperazine(H-3, H-5)), 2.45-2.35 (m, 3H, N-CHHCH-, cyclohexane (H-3, H-5)),
2.11-1.95 (m, 3H, cyclohexane (H-1, H-3, H-5)), 1.73 (dt, J = 5.0 Hz, J = 14.0 Hz, 2H,
cyclohexane (H-2, H-6)), 1.20-0.98 (m, 8H, cyclohexane (H-2, H-6), Me, Me). 13C
NMR (100 Mz, CDCl3) δ 166.6, 161.7, 159.7, 144.0, 134.2, 133.6 (q, J = 3.0 Hz),
129.8 (q, J = 36.4 Hz), 126.9, 126.1 (q, J = 4.0 Hz), 122.5 (q, J = 273.7 Hz), 61.2,
56.6, 52.3, 51.6, 38.2, 31.8, 31.6, 30.6, 24.4, 19.3, 19.0. MS-ESI (m/z): 528.2
(M+H)+.
4.2.4.12.
2-(4-((4-(ethoxyimino)cyclohexyl)methyl)-3,5-dimethylpiperazin-1-yl)-8-nitro-6-(
trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3l)
1
Yellow oil; yield: 36%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.84 (brs, 1H, piperazine(H-2)), 4.05 (q, J =
7.0 Hz, 2H, NOCH2-), 4.04-3.96 (m, 1H, piperazine(H-6)), 3.27 (d, J = 14.5 Hz, 1H,
N-CHHCH-), 3.21 (brs, 1H, piperazine(H-2)), 2.99 (brs, 1H, piperazine(H-6)), 2.67
(brs, 2H, piperazine(H-3, H-5)), 2.43-2.34 (m, 3H, N-CHHCH-, cyclohexane (H-3,
H-5)), 2.10-1.96 (m, 3H, cyclohexane (H-1, H-3, H-5)), 1.76-1.69 (m, 2H,
cyclohexane (H-2, H-6)), 1.20-0.98 (m, 11H, cyclohexane (H-2, H-6), Me, Me,
13
NOCH2CH3). C NMR (150 Mz, CDCl3) δ 166.6, 161.7, 159.4, 144.0, 134.2, 133.5

(q, J = 3.0 Hz), 129.8 (q, J = 34.7 Hz), 126.9, 126.2 (q, J = 3.0 Hz), 122.5 (q, J =
271.8 Hz), 68.8, 58.6, 56.6, 51.0, 38.2, 31.8, 31.6, 30.6, 24.6, 18.5, 14.7. MS-ESI
(m/z): 542.2 (M+H)+.
4.2.4.13.
2-(4-((4-(methoxyimino)cyclohexyl)methyl)-2,5-dimethylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3m)
1
Yellow oil; yield: 25%; H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H, benzothiazinone
H-7), 8.76 (s, 1H, benzothiazinone H-5), 5.42 (brs, 0.30 H, piperazine(H-2)), 4.97 (brs,
0.39 H, piperazine(H-2)), 4.50 (brs, 0.39H, piperazine(H-6)), 3.94 (brs, 0.39 H,
piperazine(H-6)), 3.82 (s, 3H, NOMe), 3.20 (d, J = 14.5 Hz, 1.50H, piperazine(H-6),
N-CHHCH-), 2.89 (brs, 1.50H, piperazine(H-6), piperazine(H-3)), 2.60 (t, J = 11.0
Hz, 1H, piperazine(H-5)), 2.42 (d, J = 14.0 Hz, 1H, N-CHHCH-), 2.32 (brs, 2H,
piperazine(H-3), cyclohexane (H-3) ), 2.15-2.05 (m, 2H, cyclohexane (H-3, H-5)),
1.91-1.65 (m, 4H, cyclohexane (H-1, H-5, H-2)), 1.52-1.39 (m, 3H, Me), 1.17-1.03 (m,
5H, cyclohexane (H-6), Me). 13C NMR (100 Mz, CDCl3) δ 166.6, 161.6, 159.8, 144.1,
134.4, 133.5 (q, J = 4.0 Hz), 129.8 (q, J = 35.3 Hz), 127.0, 126.1 (q, J = 3.0 Hz),
122.5 (q, J = 272.0 Hz), 61.2, 58.2, 56.7, 35.3, 31.6, 31.4, 31.2, 31.1, 30.3, 29.9, 29.8,
24.3, 24.1. MS-ESI (m/z): 528.2 (M+H)+.
4.2.4.14.
2-(4-((4-(methoxyimino)cyclohexyl)methyl)-3-methylpiperazin-1-yl)-8-nitro-6-(tri
fluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3n)
1
Yellow oil; yield: 33%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.67-4.48 (m, 1H, piperazine(H-2)),
4.10-3.90 (m, 1H, piperazine(H-6)), 3.82 (s, 3H, NOMe), 3.75-3.67 (m, 1H,
piperazine(H-2)), 3.48-3.29 (m, 1H, piperazine(H-6)), 3.19 (d, J = 14.5 Hz, 1H,
N-CHHCH- ), 3.02-2.86 (m, 1H, piperazine(H-3)), 2.70-2.50 (m, 2H,
piperazine(H-5)), 2.43-2.35 (m, 2H, N-CHHCH-, cyclohexane (H-3)), 2.14-2.01 (m,
3H, cyclohexane (H-1, H-3, H-5)), 1.94-1.67 (m, 3H cyclohexane (H-5, H-2, H-6)),
1.16-1.01 (m, 5H, cyclohexane (H-2, H-6), Me). 13C NMR (150 Mz, CDCl3) δ 166.6,
162.1, 159.8, 144.0, 134.2, 133.5 (q, J = 3.0 Hz), 129.8 (q, J = 34.7 Hz), 126.9, 126.2
(q, J = 3.0 Hz), 122.5 (q, J = 273.3 Hz), 61.2, 55.5, 35.2, 31.6, 31.4, 31.2, 30.3, 30.1,
24.2, 24.1. MS-ESI (m/z): 514.1 (M+H)+.
4.2.4.15. 2-(4-((4-(ethoxyimino)cyclohexyl)methyl)-3-methylpiperazin-1-yl)-8 ni-
tro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3o)
1
Yellow oil; yield: 34%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.70-4.45 (m, 1H, piperazine(H-2)), 4.05 (q,
J = 7.0 Hz, 2H, NOCH2-), 4.00-3.86 (m, 1H, piperazine(H-6)), 3.78-3.67 (m, 1H, ,
piperazine(H-2)), 3.48-3.30 (m, 1H, piperazine(H-6)), 3.19 (d, J = 15.0 Hz, 1H,
N-CHHCH-), 3.08-2.86 (m, 1H, piperazine(H-3)), 2.67-2.48 (m, 2H, piperazine(H-5)),

2.43-2.27 (m, 2H, N-CHHCH-, cyclohexane (H-3)), 2.14-2.00 (m, 3H, cyclohexane
(H-1, H-3, H-5)), 1.94-1.70 (m, 3H, cyclohexane (H-5, H-2, H-6)), 1.25 (t, J = 7.0 Hz,
3H, NOCH2CH3), 1.16-1.01 (m, 5H, , cyclohexane (H-2, H-6), Me). 13C NMR (150
Mz, CDCl3) δ 166.6, 162.1, 159.5, 144.0, 134.2, 133.5 (q, J = 2.9 Hz), 129.8 (q, J =
35.5 Hz), 126.9, 126.1 (q, J = 3.3 Hz), 122.5 (q, J = 273.3 Hz), 68.8, 59.1, 58.6, 55.5,
35.2, 31.6, 31.4, 31.3, 30.4, 30.1, 24.3, 18.6, 14.7. MS-ESI (m/z): 528.2 (M+H)+.
4.2.4.16. 2-(4-((3-(methoxyimino)cyclobutyl)methyl)piperazin-1-yl)-8-nitro-6-(tri-
fluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4a)
1
Yellow solid; yield: 33%; mp: 186-187 ; H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H
,
benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5), 4.19-3.83 (m, 4H
,
piperazine(H-2, H-6)), 3.83 (s, 3H, NOMe), 3.07-3.02 (m, 2H, -NCH2CH-), 2.60-2.54
(m, 9H, piperazine(H-3, H-5), cyclobutene (H-1, H-2, H-4)). 13C NMR (125 Mz, CDCl3)
δ 166.5, 162.2, 155.6, 144.0, 134.1, 133.4 (q, J = 4.5 Hz), 126.8, 126.2 (q, J = 3.0 Hz),
122.5 (q, J = 271.9 Hz), 63.2, 61.6, 52.7, 46.7, 36.3, 35.5, 25.9. MS-ESI (m/z): 472.2
(M+H)+.
4.2.4.17. 2-(4-((3-(ethoxyimino)cyclobutyl)methyl)piperazin-1-yl)-8-nitro-6-(tri-
fluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4b)
1
Yellow solid; yield: 31%; mp: 182-184 ; H NMR (500 Mz, CDCl3): δ 9.10 (s, 1H,
benzothiazinone H-7), 8.76 (s, 1H, benzothiazinone H-5), 4.26-3.76 (m, 4H,
piperazine(H-2, H-6)), 4.06 (q, J = 7.0 Hz, 2H, NOCH2-), 3.06-3.03 (m, 2H,
-NCH2CH-), 2.62-2.54 (m, 9H piperazine(H-3, H-5), cyclobutene (H-1, H-2, H-4)),
1.25 (t, J = 7.0 Hz, 3H, NOCH2CH3). 13C NMR (150 Mz, CDCl3) δ 166.6, 162.2,
155.3, 144.1, 133.6 (q, J = 3.0 Hz), 129.9 (q, J = 36.2 Hz), 126.9, 126.2 (q, J = 3.0
Hz), 122.5 (q, J = 273.3 Hz), 69.4, 63.4, 52.8, 36.4, 35.8, 29.8, 26.0, 14.8. MS-ESI
(m/z): 486.2 (M+H)+. HRMS-ESI (m/z): Calcd. for C20H23F3N5O4S (M+H)+:
486.1417; Found: 486.1419.
4.2.4.18.
2-(4-((3-(methoxyimino)cyclobutyl)methyl)-3-methylpiperazin-1-yl)-8-nitro-6-(tri
fluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4c)
1
Yellow oil; yield: 29%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.76-4.40 (m, 1H, piperazine(H-2)),
4.12-3.87 (m, 1H, piperazine(H-6)), 3.82 (s, 3H, NOMe), 3.82-3.57 (m, 1H,
piperazine(H-2)), 3.46-3.20 (m, 1H, piperazine(H-6)), 3.13-2.87 (m, 3H,
piperazine(H-3), NCH2CH-), 2.87-2.67 (m, 1H, cyclobutene (H-1)), 2.61-2.48 (m, 4H,
cyclobutene (H-2, H-4)), 2.42-2.35 (m, 2H, piperazine(H-5)), 1.14 (brs, 3H, Me). 13C
NMR (100 Mz, CDCl3) δ 166.6, 162.2, 155.7, 144.0, 134.1, 133.5 (q, J = 3.0 Hz),

129.8 (q, J = 35.3 Hz), 126.9, 126.2 (q, J = 4.0 Hz), 122.5 (q, J = 273.7 Hz), 61.7,
58.4, 55.2, 53.6, 36.3, 35.6, 35.5, 26.1, 26.1. MS-ESI (m/z): 486.2 (M+H)+.
4.2.4.19. 2-(4-((3-(ethoxyimino)cyclobutyl)methyl)-3-methylpiperazin-1-yl)-8-ni-
tro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4d)
1
Yellow oil; yield: 31%; H NMR (500 Mz, CDCl3): δ 9.08 (s, 1H, benzothiazinone
H-7), 8.74 (s, 1H, benzothiazinone H-5), 4.71-4.38 (m, 1H, piperazine(H-2)), 4.05 (q,
J = 7.0 Hz, 2H, NOCH2-), 4.05-3.82 (m, 1H, piperazine(H-6)), 3.82-3.55 (m, 1H,
piperazine(H-2)), 3.46-3.23 (m, 1H, piperazine(H-6)), 3.03-2.87 (m, 3H,
piperazine(H-3), NCH2CH-), 2.74 (brs, 1H, cyclobutene (H-1)), 2.61-2.48 (m, 4H,
cyclobutene (H-2, H-4)), 2.43-2.38 (m, 2H, piperazine(H-5)), 1.24 (t, J = 7.00 Hz, 3H,
13
NOCH2CH3), 1.13 (brs, 3H, Me). C NMR (100 Mz, CDCl3) δ 166.6, 162.2, 155.3,
144.0, 134.2, 133.6 (q, J = 3.0 Hz), 129.9 (q, J = 35.3 Hz), 126.9, 126.2 (q, J = 3.0
Hz), 122.5 (q, J = 273.7 Hz), 69.4, 58.5, 55.2, 36.3, 35.8, 29.8, 26.2, 14.8. MS-ESI
(m/z): 500.1 (M+H)+.
4.2.4.20.
2-(4-((3-(methoxyimino)cyclobutyl)methyl)-3,5-dimethylpiperazin-1-yl)-8-nitro-6
-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4e)
1
Yellow oil; yield: 27%; H NMR (500 Mz, CDCl3): δ 9.09 (s, 1H, benzothiazinone
H-7), 8.75 (s, 1H, benzothiazinone H-5), 4.85 (brs, 1H, piperazine(H-2)), 4.03 (brs,
1H, piperazine(H-6)), 3.81 (s, 3H, NOMe), 3.20 (brs, 1H, piperazine(H-2)), 3.03-2.96
(m, 3H, piperazine(H-6), NCH2CH- ), 2.82 (d, J = 4.5 Hz, 2H, cyclobutene (H-2,
H-4)), 2.74 (brs, 2H, cyclobutene (H-2, H-4)), 2.58-2.52 (m, 3H, cyclobutene (H-1),
13
piperazine(H-3, H-5)), 1.24-1.18 (m, 6H, Me). C NMR (100 Mz, CDCl3) δ 166.6,
161.8, 155.2, 144.0, 134.1, 133.5 (q, J = 3.0 Hz), 1329.8 (q, J = 35.3 Hz), 126.9,
126.1 (q, J = 4.0 Hz), 123.0 (q, J = 273.7 Hz), 61.7, 54.9, 54.0, 52.4, 51.6, 36.5, 35.8,
29.8, 26.5, 18.8, 18.6. MS-ESI (m/z): 500.2 (M+H)+.
4.2.4.21.
2-(4-((3-(ethoxyimino)cyclobutyl)methyl)-3,5-dimethylpiperazin-1-yl)-8-nitro-6-(t
rifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4f)
1
Yellow oil; yield: 30%; H NMR (500 Mz, CDCl3): δ 9.06 (s, 1H, benzothiazinone
H-7), 8.73 (s, 1H, benzothiazinone H-5), 4.84 (brs, 1H, piperazine(H-2)), 4.03 (q, J =
6.50 Hz, 3H, NOCH2-, piperazine(H-6)), 3.19 (brs, 1H, piperazine(H-2)), 3.04-2.94
(m, 3H, piperazine(H-6), NCH2CH-), 2.81 (d, J = 6.0 Hz, 2H, cyclobutene (H-2,
H-4)), 2.73 (brs, 2H, cyclobutene (H-2, H-4)), 2.56-2.52 (m, 3H, cyclobutene (H-1),
piperazine(H-3, H-5)), 1.22 (t, J = 7.0 Hz, 3H, NOCH2CH3), 1.24-1.04 (m, 6H, Me).
13C NMR (100 Mz, CDCl3) δ 166.5, 161.7, 154.8, 144.0, 134.1, 133.5 (q, J = 3.0 Hz),
129.8 (q, J = 35.3 Hz), 126.8, 126.1 (q, J = 3.0 Hz), 122.4 (q, J = 274.7 Hz), 69.3,
54.8, 54.0, 52.4, 51.6, 36.5, 35.9, 26.4, 18.8, 18.5, 14.7. MS-ESI (m/z): 514.2
(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（ATCC27294）and clinical isolate strains were 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 folds
dilutions of test compounds and positive controls 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. Inhibition evaluation on hERG K+ channel
Inhibition evaluation on hERG K+ channel. The electrophysiology recording of
hERG channel current was carried out following the standard protocol as described
previously. HEK 293 cells were stably transfected with human Ether-a-gogo related
gene (hERG) channel. The voltage-gated hERG potassium channel current was
o
recorded at room temperature (25 C) from randomly selected transfected cells using
whole-cell recording technique with an EPC10 USB (HEKA) or Multiclamp 700B
amplifier (Molecular Devices), while electrical data was digitalized by
Digidata1440A with acquisition rate of 10 kHz and signals filtered at 2.5 KHz using
Patchmaster or pClamp10 respectively. Dofetilide was included as a positive control
to ensure the accuracy and sensitivity of the test system. All experiments were
performed in 3-4 times.
4.5. Pharmacokinetic Profiles determination
Pharmacokinetic Profiles determination of compounds 3a and 3b. SPF female ICR
mice (obtained from JOINN (Suzhou) weighing 18–22ꢀg were used in the
pharmacokinetic study. The ICR mice were fasted overnight before dosing. Every
treatment group contain 3 mice. Mice were dosed with the tested compounds

suspension at 25ꢀmg/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 dissolve 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. The SD female rats obtained from JOINN
(Suzhou) weighing 180 - 220 g were also used in the pharmacokinetic study of
compound 3a as a similar procedure.
4.6. Acute toxicity determination
The toxicity study was carried out using SD rats weighting 180 - 220 g each. The
animals were randomly distributed to control group and treated groups, containing ten
animals per group. They were maintained on animal cubes, provided with water and
foods. After depriving the animals’ food overnight, the control group received 5%
CMC solution orally whereas each treated group received orally the agents suspended
in 5% CMC solution (2 g /kg). The animals were observed continuously for the first 4
hours and then each hour for the next 24 h and at 8 hourly intervals for the following
7 days after administering, to observe any death or changes.
4.7. In vivo activity determination
In vivo TB infection assay of compounds 3a, 3b and PBTZ169. SPF BALB/c mice
(female) weighing 18-20 g were used in this study. Each group was composed of 6
mice. Mice were infected via aerosol with a suspension of 5×106 CFU/mL M.
tuberculosis (H37Rv) using a Glas-Col inhalation system, to deposit 50-100 bacilli
into the lungs of each animal. The course of infection was followed by plating
homogenates of harvested organs [n = 3] on 7H11 agar plates (7H11 plates containing
10% oleic acid albumin-dextrose-catalase (OADC) enrichment and 50 µg/mL
cycloheximide, 200 U/mL polymyxin B, 50 mg/mL carbenicillin, and 20 mg/mL
trimethoprim) and determining CFU on days 3, 10, and 30 post infection. compounds
3a, 3b and PBTZ169 were dissolved or suspended in 0.5% CMC and administered by
oral gavage in a maximum volume of 200 mL such that a dose of 25 mg/kg body
weight was achieved. The control group was received only 0.5% CMC. Mice were
treated on day 10 post infection and 5 times per week. The treatment period was 3
weeks. Mice were sacrificed the day after the last day of treatment, lungs removed,
homogenized, and serially diluted in 10-fold steps in HBSS. One hundred mL were

o
spread on 7H11 agar in duplicate. The plates were incubated at 37 C for 3 weeks.
Data are expressed as the log10 (and as log10 reduction) provided by a given dose of
the compound against the growth of the organism in the untreated control group.
Mean log10 values were calculated from bacterial burden counts.
4.8. Purity determination
The purity of compounds 3a and 3b was checked by HPLC analyzing using Agilent
1260 HPLC-UV system with the Zorbax SB-C18 column (250 mm × 4.6 mm, 5 µm,
PN: 883975-902) as our previous method [22]. Compounds were dissolved in MeOH
with a concentration of 0.3 mg / mL, the mobile phase contains solvent A (methanol)
and solvent B (0.1% TFA + H2O). Gradient elution procedure was set up as: 0 min, 10%
A; 3 min, 50% A; 10 min, 100% A; 12 min, 10% A (or 12min, 50% A and then
18min, 10% A). The Ultraviolet absorption wavelength is 254 nm and the
column temperature is 25 . The injection volume is 10 µL and the flow rate is 1.3
mL/min. As the above procedure, the HPLC purity of 3a and 3b is 98.68% and
97.78%, respectively.
Funding sources
This work is supported by the CAMS Innovation Fund for Medical Science
(CAMS-2017-I2M-1-011), National Mega-project for Innovative Drugs
(2018ZX09721001-004-007; 2018ZX09711001-007-002). NSFC 81872753
Notes
The authors declare no competing financial interest.
References
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[2] A. N. Unissa, L. E. Hanna, S. Swaminathan, A Note on Derivatives of Isoniazid,
Rifampicin, and Pyrazinamide Showing Activity Against Resistant Mycobacterium
tuberculosis, Chem. Biol. Drug Des. 87 (2016) 537-550.
[3] A. Campaniço, R. Moreira, F. Lopes, Drug discovery in tuberculosis. New drug targets and
antimycobacterial agents, Eur. J. Med. Chem. 150 (2018) 525-545.
[4] R.S. Wallis, M. Maeurer, P. Mwaba, J. Chakaya, R. Rustomjee, G.B. Migliori, B. Marais,
M. Schito, G. Churchyard, S. Swaminathan, M. Hoelscher, A. Zumla,
Tuberculosis-advances in development of new drugs, treatment regimens, host-directed