hERG optimizations of IMB1603, discovery of alternative benzothiazinones as new antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2019.06.053

IMB1603, a new benzothiazinone lead discovered by our lab, exhibited potent anti-MTB activity in vitro and in vivo, but significant hERG binding potency (IR > 90% at 10 μM). Thus, we embarked on a lead optimization program with the goal of identifying alternative leads that could reduce the hERG liability without sacrificing antimycobacterial potency. Compounds 2c and 4c were identified to maintain the anti-MTB activity (MICs <0.035–0.078 μM), and had lower hERG binding affinity (IR < 50% at 10 μM). Both of them were also found to have acceptable safety and pharmacokinetic properties. Studies to determine the in vivo efficacy of 2c and 4c are currently underway.

Full text of DOI:10.1016/j.ejmech.2019.06.053

Accepted Manuscript
hERG optimizations of IMB1603, discovery of alternative benzothiazinones as new
antitubercular agents
Kai Lv, Apeng Wang, Zeyu Tao, Lei Fu, Hongtao Liu, Bin Wang, Chao Ma, Hongjian
Wang, Xican Ma, Bing Han, Auyu Wang, Kai Zhang, Mingliang Liu, Yu Lu
PII:
S0223-5234(19)30580-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.053
EJMECH 11456
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2019
Revised Date: 23 May 2019
Accepted Date: 18 June 2019
Please cite this article as: K. Lv, A. Wang, Z. Tao, L. Fu, H. Liu, B. Wang, C. Ma, H. Wang, X. Ma,
B. Han, A. Wang, K. Zhang, M. Liu, Y. Lu, hERG optimizations of IMB1603, discovery of alternative
benzothiazinones as new antitubercular agents, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.06.053.
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ACCEPTED MANUSCRIPT
Graphic Abstract
IMB1603, a new benzothiazinone lead discovered by our lab, exhibited potent anti-TB
activity in vitro and in vivo, but significant hERG binding potency (IR > 90% at 10 µM).
Thus, we embarked on a hERG optimization program. Multi-series of compounds were
designed, synthesized, and evaluated. Among them, compounds 2c and 4c were found to
have acceptable hERG binding potency, potent anti-TB activity in vitro, acceptable safety,
and pharmacokinetic properties. Currently, compounds 2c and 4c were selected as new
leads and subjected for in vivo assessment.

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hERG optimizations of IMB1603, discovery of alternative
benzothiazinones as new antitubercular agents
Kai Lv,a, † Apeng Wang,a, † Zeyu Tao,a Lei Fu,b Hongtao Liu,a, c Bin Wang,b Chao Ma,a
Hongjian Wang,a Xican Ma,a Bing Han,a Auyu Wang,d Kai Zhang,d Mingliang Liu,a, *
Yu Lub, *
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100050, China
bBeijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of
Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing
Chest Hospital, Capital Medical University, Beijing 100149, China
cDepartment of Pharmacy, Hebei General Hospital, Hebei, Shijiazhuang 050051, China
d
Department of Pharmaceutical Chemistry, School of Pharmacy, Hebei Medical
University, Shijiazhuang, 050017, P. R. China
† These 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
IMB1603, a new benzothiazinone lead discovered by our lab, exhibited potent anti-MTB
activity in vitro and in vivo, but significant hERG binding potency (IR > 90% at 10 µM).
Thus, we embarked on a lead optimization program with the goal of identifying
alternative leads that could reduce the hERG liability without sacrificing
antimycobacterial potency. Compounds 2c and 4c were identified to maintain the anti-
MTB activity (MICs <0.035-0.078 µM), and had lower hERG binding affinity (IR < 50%
at 10 µM). Both of them were also found to have acceptable safety and pharmacokinetic
properties. Studies to determine the in vivo efficacy of 2c and 4c are currently underway.
KEYWORDS: Antitubercular agents, benzothiazinones, structure-activity relationships,
hERG
1. Introduction
Tuberculosis (TB) is an old disease which has existed for millennia. The TB is
caused by Mycobacterium tuberculosis (MTB), which can spread when people who are

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sick with TB expel bacterial into the air, such as coughing.1 According to World Health
Organization (WHO) statistics, TB has been the top 10 leading cause of death in the past
decades. Thus, WHO has declared TB a global health emergency since 1993, published a
global TB report every year since 1997, and started End TB Strategy at the World Health
Assembly in 2014. But nevertheless, TB still caused about 1.6 million deaths including
1.3 million HIV-negative people in 2017.2 At the current control efforts, the goal of
ending the TB epidemic by 2030 will not be achieved.3 In addition, the emergence of
multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB have intensified
the situation.4-6 The treatment of MDR-TB is extremely difficult, requiring daily
administration of a combination of 5−7 drugs for a period of 18−24 months.7 Some of
these existing drugs show significant toxicities. Even now XDR-TB has no guidelines or
evidence that may guide its treatment.8 Although Bedaquiline and Delamanib were
approved for the treatment of MDR-TB in recent years, both of them were limited for use
in the clinical due to pronounced issues in terms of hERG toxicity concerns and ADME
issues.4 Therefore, there is an urgent need for the discovery of new drugs with novel
mechanisms of action for the treatment of TB.
Figure 1. Structures of BTZ043, PBTZ169 and IMB1603
Benzothiazinones (BTZs), a novel class of TB agents targeting decaprenyl
phosphoryl-β-D-ribose 2ʹ-epimerase (DprE1), were reported to have strong inhibitory
potency against drug-sensitive MTB, MDR-MTB and XDR-MTB strains.9-14 BTZ043
and PBTZ169 (
now known as Macozinone)15, the most advanced candidates (Figure 1)
from this series, are in Phase I16 and Phase II17 clinical trials at present, respectively, for
the treatment of both drug-susceptible TB and MDR-TB. However, PBTZ169 also have
limitations such as its low solubility.
The co-crystal structures of the DprE1 with BTZ043 / PBTZ169 complexes and the
structure-activity relationship (SAR) of this series suggested that the crucial moieties for
potent activity are the S atom and carbonyl group on the thiazinone ring, a strong

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electron-withdrawing one (CF3, CN, etc.) at C-6 position, and more importantly the NO2
group at C-8 position.10-11 On the other hand, it should be prudent to continue populating
the pipeline of antimycobacterial leads due to the high failure rate in new drug
development.18-19 Based on it, we have previously focused on structural modification of
the side chain at C-2 position of the BTZ core to found more effective candidates.20-22
Among of the derivatives, IMB1603 (Figure 1) containing a C-2 nitrogen spiro-
heterocycle moiety displayed excellent in vitro activity against MTB H37Rv and clinical
isolated MDR-MTB strains (MIC: < 0.035 µM), acceptable pharmacokinetic property,
greater aqueous solubility than PBTZ169.20 More importantly, IMB1603 showed strong
efficacy in a mouse model of acute MTB H37Rv infection (25 mg/kg, P.O., once daily
for 5 days a week for 3 weeks in BALB/c mice), producing 3.20 logs of colony forming
unit (CFU) reduction in the lung (no published). However, our subsequent investigations
revealed that IMB1603 turned out to inhibit in vitro hERG ion channels in mammalian
cell lines, with inhibition rate (IR) of >90% at 10 µM, predicting potential
electrocardiogram (QT) prolongation risk. Consequently, we embarked on a lead
optimization program to identify novel analogues with reduced hERG cardiac toxicity but
not at the expense of antimycobacterial potency to make critical compound progression
decisions.
2. Results and Discussion
Many efficient strategies to combat the affinity of drugs to hERG have been proposed,
but in fact making even subtle changes in the structures of small molecules have been
found to cause profound impact on the hERG activity.23-24 Herein, we described SAR of
three moieties (benzene ring, linker, N-heterocycle) on the C-2 side chain of the BTZ
core toward the discovery of the promising leads 2c and 4c. We believe that the SAR
developed could also be applicable to other compound series.
2.1. Substitutions on Benzene Ring
We first examined the influence of benzene substitutions, such as halogen (F, Br) and /
or strong electron-withdrawing groups (CF3, CN, NO2) in IMB1603 derivatives 1a-i
which were easily synthesized according previous reported procedure.20 As shown in
Table 1, all of them displayed the same excellent in vitro antimycobacterial activity (MIC
< 0.035 µM) as PBTZ169. Compared with mono substituted compounds (1a-e, IR >

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90%), disubstituted ones (1f-i) exhibited decreased binding potency against hERG
channel, with IR values of < 90% which were still not acceptable. Subsequently, two
compounds 1j and 1k with bulkier naphthalene rings were also investigated.
Interestingly, β-naphthalene compound (1j) with potent antimycobacterial activity (MIC
< 0.035 µM), displayed a relatively lower level of hERG inhibition (IR = 78%) than all of
substituted benzene ones with an exception of 1g.
Table 1. Activity against MTB H37Rv and hERG inhibition of BTZs with a substituted
benzene moiety
W
MIC
hERG
Compd.
(µM)
<0.035
(IR at 10 µM)
95.7±1.3%
1a
(IMB1603)
1b
<0.035
94.9±1.5%
<0.035
<0.035
<0.035
<0.035
90.2±1.8%
94.1±2.2%
90.6±3.4%
88.5±3.5%
1c
1d
1e
1f
<0.035
<0.035
68.6±4.8%
83.3±2.3%
1g
1h
<0.035
88.6±3.6%
1i

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0.055
NT
1j
<0.035
78.1±5.2%
41.6±5.3%
1k
<0.035
0.269
0.164
PBTZ169
INH
RFP
INH, isoniazid; RFP, rifampicin.
2.2. Benzene Ring Modifications.
Among the most successful reported approaches for diminishing binding to the hERG
channel is reducing the lipophilicity, as measured by cLog P.23, 25 We probed the
consequences of replacing the benzene moiety in the lead IMB1603 with other
heteroaromatic rings (1l-s, cLog P: 2.32 – 4.65). With a few exceptions, these analogues
were found to have much less antimycobacterial activity than IMB1603. For the top 3
most active compounds 1m, 1q and 1s, their antimycobacterial activity and hERG
affinity decreased as cLog P values reduced. For example, thiophene derivative 1m (cLog
P = 4.28) maintaining the same potency to IMB1603 (MIC: < 0.035 µM), turned out to
be a strong hERG inhibitor at 10 µM (IR = 96.2%). Oxazole derivative 1m with the
lowest CLog P of 2.32 displayed moderate hERG inhibition (IR = 53.2%) (Table 2). It
suggested that increasement of the polarity could decrease hERG inhibition, but this also
results in reduced antimycobacterial activity possible due to lower membrane
permeability and is illustrative of the challenges of multidimensional optimization.
Table 2. Activity against MTB H37Rv and hERG inhibition of BTZs with a modified
benzene ring
Compd.
W
MIC
hERG
(IR at 10
µM)
cLog P
(µM)

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<0.035
95.7±1.3%
4.78
4.65
1a
(IMB1603)
1l
1.478
NT
<0.035
0.479
96.2±0.8%
NT
4.28
3.71
1m
1n
0.477
0.235
NT
NT
3.14
3.14
1o
1p
0.122
1.576
0.248
<0.035
74.1±1.5%
NT
3.14
3.24
2.32
5.10
1q
1r
53.2±2.4%
41.6±5.3%
1s
PBTZ169
2.3. Linker Modifications.
The effect of the methylene linker between the benzene and spiro-heterocycle in
IMB1603 was subsequently investigated. As shown in Table 3, the length of the linker
had little impact on antimycobacterial activity, and hERG inhibition slightly decreased as
the carbon chain extended (1a vs 2a vs 2b). Surprisingly, compound 2c by removal of the
methylene linker, maintained excellent antimycobacterial potency (MIC: < 0.035 µM)
and was virtually inactive in hERG inhibition assay at 10 µM with IR value of 23% lower
than PBTZ169 (IR = 41%). The significantly decreased hERG binding affinity of 2c
might attribute by the removal of the basic center, or the rigid structure of the C-2 side
chain. Based on the above data, compound 2c merited further study.
At the same time, an alkyl group was tried to introduced on the methylene linker of
IMB1603 to explore the impact of the steric hindrance. However, the synthesis of these
alkyl derivatives has low yields, and the products were very hard to purification. Thus, an

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alkyl group was introduced to IMB1604 (3a) and IMB1605 (3b) which has the similar
structure feature and pharmacological properties to IMB1603. As shown in Table 3,
replacement of a hydrogen atom on the methylene of 3a with methyl remained the same
antimycobacterial potency (MIC: < 0.035 µM), and led to reduced hERG inhibition (3a
vs 3c), although changes of the halogen atom at para-position of the benzene ring had
little effect on hERG activity (3c vs 3d vs 3e). The same trend was also observed by
comparison between 3b and 3f. Introduction of ethyl seemed to be preferred over methyl
(3f vs 3g). Our results suggested that a bulky alkyl on the methylene linker might be
favorable for reduction of hERG potency. Currently, more analogues with other bigger
substituents were under investigation.
Table 3. Activity against MTB H37Rv and hERG inhibition of linker modified BTZs
MIC
hERG
W
Compd.
(µM)
(IR at 10 µM)
<0.035
0.055
95.7±1.3%
92.7±0.6%
1a (IMB1603)
2a
<0.035
<0.035
85.8±2.6%
23.0±3.3%
2b
2c
<0.035
<0.035
97.0±1.6%
94.6±2.5%
3a (IMB1604)
3b (IMB1605)
<0.035
0.107
77.2±3.0%
74.0±4.2%
3c
3d

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<0.035
<0.035
<0.035
73.4±4.6%
3e
3f
72.9±4.0%
69.7±2.1%
3g
2.4. N-Heterocycle Modifications.
In order to get more candidates with diversity structure features, changes were
subsequently focused on the spiro-heterocycle of IMB1603 while the para-fluorobenzene
moiety was maintained. As shown in table 4, compounds 4b-d exhibited good
antimycobacterial activity (MIC: < 0.035 – 0.078 µM). To our delight, compound 4c
containing an azabicyclo[2.2.2]octane ring showed significantly decreased hERG affinity
with IR of 34.9% at 10 µM, even lower than that of PBTZ169, indicating its potential for
further development.
Table 4. Activity against MTB H37Rv and hERG inhibition of N-heterocycle modified
BTZs
MIC
Herg
Compd. Q
(µM)
(IR at 10 µM)
0.248
NT
4a
4b
<0.035 94.3±0.6%

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0.078
0.054
34.9±2.9%
95.6±0.5%
4c
N
4d
N
N
2.5. Additional assessments of selected compounds
On the bases of the above studies, compounds 2c and 4c with potent antimycobacterial
activity (MIC: <0.035-0.078 µM) and poor hERG inhibition (IR:23.0-34.9%) at 10 µM
were evaluated against two clinical isolated MTB-MDR (16833 and 16995) strains
resistant to both INH and RFP. As shown in Table 5, both of them exhibited strong
activity (MIC: < 0.035-0.062 µM) similar to that against MTB H37Rv, suggesting their
promising potential for both drug-sensitive and resistant MTB strains (Tables 3-5).
The above 2 compounds were tested for in vivo tolerability by recording the number
of survivors after a single oral dose in ICR mice of 500 mg/kg, followed by a 7-day
observation. In general, compounds 2c and 4c showed good tolerance in mice at 500
mg/kg (Table 6).
Table 5. Anti-MDR-MTB activity and acute toxicity of selected compounds
MICa (µM)
Acute
Toxicityb
Compd.
MDR-MTB16883 MDR-MTB16995
<0.035
0.062
<0.035
<0.035
<0.035
>40
5/5
5/5
5/5
-
2c
4c
PBTZ169 <0.035
>40
INH
>40
>40
-
RFP
aMDR-MTB 16883 and MDR-MTB 16995 were isolated from patients in Beijing Chest
Hospital. bNo. of animals that survived / total no. of animals.
Compounds 2c and 4c with low oral acute lethal toxicity were evaluated for their in
vivo PK profiles in ICR mice after a single oral administration of 25 mg/kg. As shown in

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Table 6, both of them displayed good drug exposures, although shorter T1/2 (1.25-4.79 h)
than PBTZ169 (6.33 h). In particular, the AUC0-t of 2c were significantly higher than
PBTZ169. Currently, these two compounds were all subjected to in vivo assessment.
Table 6. In vivo PK profiles of selected compounds in mice at 25 mg/kg
Compd.
T1/2
(h)
Tmax
(h)
Cmax
(ng·mL-1)
AUC0-t
(h·ng·mL-1)
4.79±1.19 0.50±0
1.25±0.04 0.25±0
6.33±1.07 0.33±0
1613±231
5033±311
2c
3040±1480 3550±1450
1746±281 2475±702
4c
PBTZ169
3. Conclusion
In summary, we reported on optimization of lead compound IMB1603 discovered in
our lab, including SAR studies of three moieties (benzene ring, linker, and N-heterocycle)
on the C-2 side chain of the BTZ core, to identify alternatives with reduced hERG cardiac
toxicity while keeping potent anti-TB activity. The optimization process led to
compounds 2c and 4c which showed potent antimycobacterial activity ((MIC: <0.035-
0.078 µM) against MTB H37Rv as well as two MDR-MTB clinical isolates, acceptable
hERG affinity (IR: 23.0-34.9%) at 10 µM. In addition, these two compounds also
displayed acceptable safety and good PK properties. Studies to determine the in vivo
efficacy of 2c and 4c are currently underway.
4. Chemistry
The synthesis of these molecules was carried out as shown in various schemes 1-2. A
general synthetic route for compounds 1-3 was shown in scheme 1. Introduction of
diverse W groups to 2,8-diazaspiro[4.5]decane 8 by previous reported reductive
amination with aldehydes, Buchwald-Hartwig amination with 1-Bromo-4-Flurobenzene,
nucleophilic substitution with bromides, or Ti(OiPr) mediated reductive amination with
ketone gave compounds 9-11, which upon removal of Boc protecting group afforded the
desired side chain intermediates 12-14. Coupling compounds 12-14 with benzothiazinone
15 yielded the targets 1-3.

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o
Reagents and conditions: a): i) ArCHO, NaCNBH3, AcOH, rt-50 C (for 9a-s); OR iii)
K2CO3, CH3CN, 1-(2-Bromoethyl)-4-fluorobenzene or 1-(3-bromopropyl)-4-
fluorobenzene (for 10a-b); OR ii) Pd(OAc)2, BINAP, 1-bromo-4-fluorobenzene, t-
BuONa, anhydrous toluene (for 10c); OR iv）Ti(OiPr)4, ketone, MeOH, NaCNBH3 (for
3c-g); b): DCM, TFA; c): Et3N, MeOH.
Scheme 1. Synthesis of compounds 1a-s, 2a-c and 3c-g
The synthesis of compounds 4a-d was shown in scheme 2. Reductive amination of
commercially available compounds 16a-d with 4-fluorobenzaldehyde afforded
compounds 17a-d. Removal of Boc-protecting group followed by coupling with
compound 15 gave target compounds 4a-d.
Reagents and conditions: a) 4-fluorobenzaldehyde OR cyclohexanecarbaldehyde,
NaCNBH3, AcOH, rt-50 oC; b) DCM, TFA; c) 15, Et3N, MeOH.
Scheme 2. Synthesis of compounds 4a-d
5. Experiment section

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MIC determination. MICs against replicating M. tuberculosis were determined by the
microplate Alamar blue assay (MABA). PBTZ169 (synthesized by our lab), 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 fold 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.
Inhibition evaluation on hERG K+ channel. The electrophysiology recording of hERG
channel current was carried out following the standard protocol as described previously.30
HEK 293 cells were stably transfected with human Ether-a-gogo related gene (hERG)
channel. The voltage-gated hERG potassium channel current was recorded at room
o
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.
Acute toxicity determination of compounds 2c and 4c. The toxicity study was carried
out using female ICR mice weighting 18-21 g each. The animals were randomly

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distributed to control group and treated groups, containing five 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 (0.5 mL),
whereas each treated group received orally the agents suspended in 5% CMC solution
(500 mg/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.
Pharmacokinetic Profiles determination of compounds 2c and 4c. SPF female ICR
mice weighing 18–22ꢀg were used in the pharmacokinetic study. The ICR mice were
fasted overnight before dosing. Every treatment group contain 3 rats. 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 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.
General Chemical Methods. All commercially available solvents and reagents were
used without further purification. All moisture sensitive reactions were carried out under
1
Argon atmosphere in commercially available anhydrous solvents. H NMR spectra were
determined on a Varian Mercury-400 or Bruker 500 M spectrometer in MeOD, CDCl3, or
DMSO-d6 using tetramethylsilane as an internal standard. Electrospray ionization (ESI)
mass spectra was obtained on an Agilent 1260-6420 Mass spectrum instruments. The
reagents were all of analytical grade or chemically pure. TLC was performed on silica gel
plates (Merck, ART5554 60F254).

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Purity was determined by HPLC, and all target compounds were confirmed to
have >95% purity.
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.
General synthesis procedure of compounds 1b-s. To a stirring solution of 8 (0.3 mmol)
in MeOH (5 mL) was added the corresponding aldehyde (0.4 mmol) and NaCNBH3 (0.5
mmol) at room temperature. The mixture was adjusted to pH 6-7 by acetic acid, stirred
overnight at room temperature, and quenched by 1 M NaOH solution (5 mL). The
mixture was diluted by H2O (15 mL), and extracted by DCM (10 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 = 20 : 1) to
yield oils 9b-s (70-93%).
To a stirred solution of 9b-s (0.2 mmol) in DCM (5 mL) was added TFA (1 mL) at
room temperature. The mixture was stirred for 2 hours and concentrated to afford the
crude product 12b-s which was used directly in the next step without further purification.
To a stirred solution of above crude 12b-s in anhydrous MeOH (10 mL) was added BTZ
core compound 15 (0.2 mmol) and Et3N (0.6 mmol) at room temperature. The mixture
o
was stirred overnight at 40 C, and concentrated. The residue was purified by column
chromatography over silica gel (DCM : MeOH = 20 : 1) to yield the yellow solids 1b-s.
2-(2-(2-fluorobenzyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-(trifluoromethyl)-4H-
benzo[e][1,3]thiazin-4-one (1b) According to above general procedure, employing 2-
flurobenzaldehyde afforded compound 1b as a yellow solid (30% for two steps), mp:
o
1
115-117 C; HPLC purity: 95.3%, retention time 9.02 min; H NMR (500 MHz, CDCl3)
δ 9.14 (s, 1H), 8.78 (s, 1H), 7.43-7.40 (m, 1H), 7.30-7.28 (m, 1H), 7.17-7.15 (m, 1H),
13
7.10-7.06 (m, 1H), 4.22-3.70 (m, 6H), 2.74 (brs, 2H), 2.54 (brs, 2H), 1.79 (brs, 6H); C
NMR (125 MHz, DMSO-d6) δ 165.54, 161.28, 160.93 (d, J = 244.2 Hz), 144.75, 134.80,
131.73 (q, J = 3.5 Hz), 131.50 (d, J = 4.5 Hz), 129.30 (d, J = 8.1 Hz), 127.75 (q, J = 35.5

ACCEPTED MANUSCRIPT
Hz), 126.56, 126.50, 125.83 (d, J = 15.1 Hz), 124.66 (d, J = 2.8 Hz), 123.01 (q, J = 274.1
Hz), 115.60 (d, J = 21.8 Hz), 65.35, 53.12, 52.24, 45.13, 40.84, 37.37, 36.12; HRMS-ESI
(m/z): Calcd. For C24H23F4N4O3S (M+H)+: 523.1422; Found: 523.1422.
8-nitro-2-(2-(4-(trifluoromethoxy)benzyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1c) According to above general
procedure, employing 3-(trifluromethoxy)benzaldehyde afforded compound 1c as a
o
yellow solid (35% for two steps), mp: 119-121 C; HPLC purity: 97.5%, retention time
1
10.51 min; H NMR (500 MHz, CDCl3) δ9.14 (s, 1H), 8.79 (s, 1H), 7.39 (brs, 2H), 7.22
(d, J = 7.70 Hz, 2H), 4.30-3.85 (m, 4H), 3.64 (s, 2H), 2.70 (brs, 2H), 2.49 (brs, 2H), 1.80
(s, 6H);13C NMR (125 MHz, DMSO-d6) δ165.66, 161.36, 147.62, 144.83, 139.16, 134.84,
131.73 (q, J = 3.5 Hz), 130.44, 127.75 (q, J = 35.5 Hz), 126.56, 123.01 (q, J = 274.1 Hz),
121.12, 120.57 (q, J = 252.4 Hz), 65.40, 58.94, 53.25, 45.10, 40.84, 36.12; HRMS-ESI
(m/z): Calcd. For C25H23F6N4O4S (M+H)+: 589.1339; Found: 589.1328.
8-nitro-2-(2-(4-nitrobenzyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-(trifluoromethyl)-4H-
benzo[e][1,3]thiazin-4-one (1d) According to above general procedure, employing 4-
nitrobenzaldehyde afforded compound 1d as a yellow solid (42% for two steps), mp:
1
136-138 oC; HPLC purity: 99.8%, retention time 9.01 min; H NMR (500 MHz, DMSO-
d6) δ 8.86 (s, 1H), 8.81 (s, 1H), 8.22 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 4.07
(brs, 4H), 3.74 (s, 2H), 2.62 (t, J = 6.3 Hz, 2H), 2.48 (s, 2H), 1.74 (brs, 6H); 13C NMR
(100 MHz, DMSO-d6) δ 165.70, 161.39, 148.05, 146.92, 144.89, 134.86, 131.75 (q, J =
3.5 Hz), 129.72, 127.84 (q, J = 35.1 Hz), 126.64, 126.59, 123.88, 122.53 (q, J = 272.3
Hz), 65.35, 59.02, 53.30, 45.13, 37.15, 36.21; HRMS-ESI (m/z): Calcd. For
C24H23F3N5O5S (M+H)+: 550.1367; Found: 550.1369.
8-nitro-2-(2-(4-nitrilebenzyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-(trifluoromethyl)-4H-
benzo[e][1,3]thiazin-4-one (1e) According to above general procedure, employing 4-
formylbenzonitrile afforded compound 1e as a yellow solid (33% for two steps), mp:
o
1
161-162 C; HPLC purity: 96.4%, retention time 8.90 min; H NMR (500 MHz, CDCl3)
δ 9.13 (s, 1H), 8.79 (s, 1H), 7.64 (d, J = 7.60 Hz, 2H), 7.48 (d, J = 7.60 Hz, 2H), 4.30-
13
3.90 (m, 4H), 3.70 (s, 2H), 2.69 (brs, 2H), 2.50 (brs, 2H), 1.80 (brs, 6H); C NMR (125
MHz, DMSO-d6) δ 165.61, 161.33, 145.67, 144.81, 134.82, 132.65, 131.73 (q, J = 3.5

ACCEPTED MANUSCRIPT
Hz), 129.57, 127.84 (q, J = 35.5 Hz), 126.59, 126.54, 123.11 (q, J = 274.1 Hz), 119.37,
65.33, 59.27, 53.27, 45.13, 40.90, 36.99, 36.13; HRMS-ESI (m/z): Calcd. For
C25H23F3N5O3S (M+H)+: 530.1468; Found: 530.1465.
2-(2-(2,3-difluorobenzyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (1f) According to above general procedure, employing
2,3-difluorobenzaldehyde afforded compound 1f as a yellow solid (41% for two steps),
o
1
mp: 99-101 C; HPLC purity: 99.6%, retention time 9.15 min; H NMR (500 MHz,
CDCl3) δ 9.14 (s, 1H), 8.79 (s, 1H), 7.20-7.10 (m, 3H), 4.24-3.88 (m, 4H), 3.76 (s, 2H),
2.75 (brs, 2H), 2.56 (brs, 2H), 1.83 (brs, 6H);13C NMR (125 MHz, DMSO-d6) δ 165.61,
161.33, 150.12 (dd, J = 150.1 Hz, 13.1 Hz), 148.46 (dd, J = 251.1 Hz, 12.8 Hz), 144.83,
134.82, 132.65, 131.73 (q, J = 3.5 Hz), 128.68 (d, J = 10.7 Hz), 127.84 (q, J = 35.5 Hz),
126.59, 126.53, 124.93, 123.13 (q, J = 274.1 Hz), 116.34 (d, J = 16.8 Hz), 65.26, 53.07,
52.00, 45.05, 40.84, 37.06, 36.10; HRMS-ESI (m/z): Calcd. For C24H22F5N4O3S (M+H)+:
541.1327; Found: 541.1326.
2-(2-(2,4-bis(trifluoromethyl)benzyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1g) According to above general
procedure, employing 2,4-bis(trifluoromethyl)benzaldehyde afforded compound 1c as a
o
yellow solid (45% for two steps), mp: 137-138 C; HPLC purity: 95.8%, retention time
1
10.95 min; H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.79 (s, 1H), 7.98 (d, J = 7.2 Hz,
1H), 7.92 (s, 1H), 7.83 (d, J = 7.2 Hz, 1H), 4.24-3.88 (m, 4H), 3.88 (s, 2H), 2.75 (brs, 2H),
2.56 (brs, 2H), 1.83 (brs, 6H);13C NMR (100 MHz, CDCl3) δ 166.56, 161.75, 143.94,
142.66, 134.21, 133.40 (q, J = 3.5 Hz), 130.68, 129.62 (q, J = 35.3 Hz ), 128.66, 126.73,
126.05 (q, J = 3.5 Hz), 123.76, 123.09, 121.05, 65.34, 55.28, 53.25, 44.67, 40.92, 37.24,
36.24; HRMS-ESI (m/z): Calcd. For C26H22F9N4O3S (M+H)+: 641.1263; Found:
641.1250.
2-(2-(3,5-bis(trifluoromethyl)benzyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1h) According to above general
procedure, employing 3,5-bis(trifluoromethyl)benzaldehydeafforded compound 1h as a
o
yellow solid (37% for two steps), mp: 148-149 C; HPLC purity: 97.3%, retention time
11.25 min; 1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.79 (s, 1H), 7.85-7.80 (brs, 3H),

ACCEPTED MANUSCRIPT
4.24-3.76 (m, 6H), 2.72 (brs, 2H), 2.52 (brs, 2H), 1.82 (brs, 6H); HRMS-ESI (m/z):
Calcd. For C26H22F9N4O3S (M+H)+: 641.1263; Found: 641.1234.
2-(2-(3-bromo-4-nitrobenzyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1i) According to above general
procedure, employing 3-bromo-4-nitrobenzaldehyde afforded compound 1i as a yellow
o
solid (41% for two steps), mp: 150-152 C; HPLC purity: 98.9%, retention time 9.71
min; 1H NMR (500 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.80 (s, 1H), 8.02 (d, J = 8.2 Hz, 1H),
7.86 (s, 1H), 7.59 (d, J = 8.2 Hz, 1H), 4.07-3.86 (m, 4H), 3.71 (s, 2H), 2.63 (t, J = 6.3 Hz,
2H), 2.48 (s, 2H), 1.74 (brs, 6H); 13C NMR (125 MHz, DMSO-d6) δ 165.68, 161.38,
148.60, 146.96, 144.88, 134.86, 134.30, 131.75 (q, J = 3.5 Hz), 128.98, 127.84 (q, J =
35.5 Hz), 126.59, 126.15, 123.11 (q, J = 274.1 Hz), 113.56, 65.22, 58.24, 53.21, 45.18,
40.96, 36.96, 36.17; HRMS-ESI (m/z): Calcd. For C24H22BrF3N5O5S (M+H)+: 628.0472;
Found: 628.0443.
2-(2-(naphthalen-1-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1j) According to above general
procedure, employing 1-naphthaldehyde afforded compound 1j as a yellow solid (44%
for two steps), mp: 195-197 oC; HPLC purity: 95.6%, retention time 10.33 min; 1H NMR
(500 MHz, CDCl3) δ 9.13 (s, 1H), 8.77 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 7.6
Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.57-7.52 (m, 2H), 7.47-7.43 (m, 2H), 4.08-3.82 (m,
6H), 2.76 (brs, 2H), 2.54 (brs, 2H), 1.79 (brs, 6H); 13C NMR (125 MHz, DMSO-d6)
δ166.48, 161.58, 134.25, 133.77, 133.29 (q, J = 3.5 Hz), 132.22, 129.49 (q, J = 35.5 Hz),
128.41, 127.92, 126.70, 126.47, 125.90 (q, J = 3.5 Hz), 125.72, 125.67, 125.19, 124.46,
122.40 (q, J =272.4 Hz), 65.24, 58.33, 53.27, 44.96, 40.72, 37.49, 36.19; HRMS-ESI
(m/z): Calcd. For C28H26F3N4O3S (M+H)+: 555.1672; Found: 555.1665.
2-(2-(naphthalen-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1k) According to above general
procedure, employing 2-naphthaldehyde afforded compound 1k as a yellow solid (53%
for two steps), mp: 170-172 oC; HPLC purity: 98.6%, retention time 10.46 min; 1H NMR
(500 MHz, CDCl3) δ 9.12 (s, 1H), 8.78 (s, 1H), 7.86 (brs, 4H), 7.52 (brs, 3H), 4.01-3.78
(m, 6H), 2.78 (brs, 2H), 2.55 (brs, 2H), 1.82 (brs, 6H). 13C NMR (125 MHz, CDCl3)

ACCEPTED MANUSCRIPT
δ166.48, 161.63, 143.88, 136.58, 134.24, 133.34, 133.26 (q, J = 3.5 Hz), 132.72, 129.56
(q, J = 35.5 Hz), 127.96, 127.66, 127.64, 126.95, 126.72, 126.01, 125.92 (q, J = 4.0 Hz),
125.62, 122.40 (q, J =272.4 Hz), 65.24, 60.35, 53.37, 44.99, 40.72, 37.30, 36.40; HRMS-
ESI (m/z): Calcd. For C28H26F3N4O3S (M+H)+: 555.1672; Found: 555.1654.
2-(2-((5-methoxy-1H-indol-2-yl)methyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1l) According to above general
procedure, employing 5-methoxy-1H-indole-3-carbaldehyde afforded compound 1l as a
o
yellow solid (50% for two steps), mp: 199-201 C; HPLC purity: 95.1%, retention time
9.51 min; 1H NMR (500 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.86 (s, 1H), 8.81 (s, 1H), 7.24
(d, J = 8.5 Hz, 1H), 7.19 (s, 1H), 7.14 (s, 1H), 6.76 (d, J = 8.2 Hz, 1H), 4.08-3.72 (m, 4H),
3.78 (s, 3H), 3.71 (s, 2H), 2.60 (brs, 2H), 2.46 (brs, 2H), 1.69 (brs, 6H); HRMS-ESI (m/z):
Calcd. For C27H27F3N5O4S (M+H)+: 574.1730; Found: 574.1728.
8-nitro-2-(2-(thiophen-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1m) According to above general
procedure, employing thiophene-2-carbaldehyde afforded compound 1m as a yellow
o
solid (46% for two steps), mp: 143-145 C; HPLC purity: 96.9%, retention time 8.75
1
min; H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.80 (s, 1H), 7.28-7.26 (m, 1H), 6.99-
6.96 (m, 2H), 4.10-3.88 (m, 6H), 2.77 (brs, 2H), 2.55 (brs, 2H), 1.82 (brs, 6H);13C NMR
(125 MHz, DMSO-d6) δ 165.67, 161.37, 144.88, 143.23, 134.86, 131.72 (q, J = 3.5 Hz),
127.80 (q, J = 35.5 Hz), 126.95, 126.60, 126.56, 123.15 (q, J = 274.1 Hz), 65.03, 54.19,
53.02, 45.15, 40.85, 36.97, 36.12; HRMS-ESI (m/z): Calcd. For C22H22F3N4O3S2 (M+H)+:
511.1080; Found: 511.1058.
2-(2-((1-methyl-1H-pyrrol-2-yl)methyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1n) According to above general
procedure, employing 1-methyl-1H-pyrrole-2-carbaldehyde afforded compound 1n as a
o
yellow solid (38% for two steps), mp: 160-162 C; HPLC purity: 95.5%, retention time
8.84 min; 1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.78 (s, 1H), 6.62 (s, 1H), 6.06 (brs,
1H), 6.02 (brs, 1H), 4.23-3.82 (m, 4H), 3.69 (s, 3H), 3.59 (brs, 2H), 2.65 (brs, 2H), 2.45
(brs, 2H), 1.76 (brs, 6H);13C NMR (125 MHz, DMSO-d6) δ 165.63, 161.34, 144.84,
134.85, 131.73 (q, J = 3.5 Hz), 129.95, 127.75 (q, J = 35.5 Hz), 126.57, 123.04 (q, J =

ACCEPTED MANUSCRIPT
272.4 Hz), 122.59, 108.33, 106.22, 65.46, 53.10, 51.41, 45.13, 37.42, 35.99, 33.80;
HRMS-ESI (m/z): Calcd. For C23H25F3N5O3S (M+H)+: 508.1625; Found: 508.1649.
8-nitro-2-(2-(pyridin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (1o) According to above general procedure, employing
picolinaldehyde afforded compound 1o as a yellow solid (37% for two steps), mp: 120-
122 oC; HPLC purity: 98.8%, retention time 8.37 min; 1H NMR (500 MHz, DMSO-d6) δ
8.83 (s, 1H), 8.77 (s, 1H), 8.47 (d, J = 4.3 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.42 (d, J =
7.8 Hz, 1H), 7.25-7.23 (m, 1H), 3.83 (brs, 4H), 3.69 (s, 2H), 2.62 (t, J = 6.7 Hz, 2H), 2.48
13
(brs, 2H), 1.80-1.68 (m, 6H); C NMR (100 MHz, DMSO-d6) δ 165.67, 161.36, 159.46,
149.17, 144.88, 137.00, 134.86, 131.74 (q, J = 3.5 Hz), 127.75 (q, J = 35.5 Hz), 126.59,
123.04 (q, J = 272.4 Hz), 122.80, 122.50, 108.33, 106.22, 65.54, 61.68, 53.40, 45.20,
40.90, 37.42, 37.05, 36.27; HRMS-ESI (m/z): Calcd. For C23H23F3N5O3S (M+H)+:
506.1468; Found: 506.1451.
8-nitro-2-(2-(pyridin-3-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (1p) According to above general procedure, employing
nicotinaldehyde afforded compound 1p as a yellow solid (39% for two steps), mp: 154-
o
1
156 C; HPLC purity: 97.5%, retention time 7.53 min; H NMR (500 MHz, CDCl3) δ
9.14 (s, 1H), 8.78 (s, 1H), 8.59-8.56 (m, 2H), 7.73 (s, 1H), 7.30 (brs, 1H), 4.19-3.88 (m,
13
4H), 3.69 (brs, 2H), 2.72 (brs, 2H), 2.52 (brs, 2H), 1.81 (brs, 6H); C NMR (100 MHz,
CDCl3) δ 166.56, 161.74, 150.01, 148.79, 143.94, 136.38, 134.23, 133.33 (q, J = 3.0 Hz),
129.63 (q, J = 35.5 Hz), 126.73, 125.96 (q, 3.6 Hz), 123.48, 122.52 (q, J = 273.2 Hz),
65.14, 57.37, 53.23, 44.7, 40.77, 37.31, 36.17; HRMS-ESI (m/z): Calcd. For
C23H23F3N5O3S (M+H)+: 506.1468; Found: 506.1457.
8-nitro-2-(2-(pyridin-4-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (1q) According to above general procedure, employing
isonicotinaldehyde afforded compound 1q as a yellow solid (48% for two steps), mp:
1
137-139 oC; HPLC purity: 99.0%, retention time 7.32 min; H NMR (500 MHz, DMSO-
d6) δ 8.86 (s, 1H), 8.81 (s, 1H), 8.53 (d, J = 5.1 Hz, 2H), 7.36 (d, J = 5.1 Hz, 2H), 4.07-
3.85 (m, 4H), 3.63 (s, 2H), 2.62 (t, J = 6.3 Hz, 2H), 2.46 (s, 2H), 1.72 (brs, 6H); 13C
NMR (100 MHz, DMSO-d6) δ 165.65, 161.36, 150.00, 148.70, 144.86, 134.85, 131.73 (q,

ACCEPTED MANUSCRIPT
J = 3.5 Hz), 127.75 (q, J = 35.5 Hz), 126.58, 123.78, 123.08 (q, J = 272.4 Hz), 65.36,
58.59, 53.34, 45.17, 37.01, 36.19; HRMS-ESI (m/z): Calcd. For C23H23F3N5O3S (M+H)+:
506.1468; Found: 506.1486.
2-(2-((1l2-pyrrol-2-yl)methyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (1r) According to above general
procedure, employing 1H-pyrrole-2-carbaldehyde afforded compound 1r as a yellow
o
solid (52% for two steps), mp: 94-96 C; HPLC purity: 95.1%, retention time 8.90 min;
1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.79 (s, 1H), 6.82 (s, 1H), 6.16 (s, 1H), 6.09
(s, 1H), 4.17-3.90 (m, 4H), 3.75 (brs, 2H), 2.80 (brs, 2H), 2.59 (brs, 2H), 1.84 (brs,
6H);13C NMR (100 MHz, CDCl3) δ 166.56, 161.84, 143.94, 134.16, 133.41 (q, J = 3.5
Hz), 129.65 (q, J = 35.5 Hz), 126.70, 126.03 (q, J = 3.2 Hz), 122.54 (q, J = 272.3 Hz),
118.42, 107.97, 64.46, 58.49, 52.89, 44.58, 40.81, 37.11, 35.98; HRMS-ESI (m/z): Calcd.
For C22H23F3N5O3S (M+H)+: 494.1468; Found: 494.1492.
8-nitro-2-(2-(oxazol-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (1s) According to above general procedure, employing
oxazole-5-carbaldehyde afforded compound 1s as a yellow solid (42% for two steps),
HPLC purity: 95.5%, retention time 7.90 min; 1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H),
8.79 (s, 1H), 7.75 (s, 1H), 7.19 (s, 1H), 4.17-3.90 (m, 4H), 3.12-2.96 (m, 4H), 1.84 (brs,
6H); ); 13C NMR (125 MHz, DMSO-d6) δ 165.67, 161.84, 161.37, 144.87, 140.26,
134.85, 131.73 (q, J = 3.5 Hz), 127.80 (q, J = 35.5 Hz), 127.31, 126.60, 126.56, 123.15 (q,
J = 274.1 Hz), 64.91, 52.81, 51.17, 44.41, 40.92, 37.14, 36.24; HRMS-ESI (m/z): Calcd.
For C21H21F3N5O4S (M+H)+: 496.1261; Found: 496.1266.
General synthesis procedure of compounds 2a-b. To a stirring solution of 8 (0.5 mmol)
in CH3CN (10 mL) was added 1-(2-Bromoethyl)-4-fluorobenzene or 1-(3-bromopropyl)-
4-fluorobenzene (0.5 mmol) and K2CO3 (1.0 mmol) at room temperature. The mixture
was stirred overnight at room temperature and filtered. The filtrate was diluted by H2O
(25 mL), and extracted by DCM (20 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 = 20 : 1) to yield oils 10a-b (yield, 65-
89%).

ACCEPTED MANUSCRIPT
To a stirred solution of 10a-b (0.2 mmol) in DCM (5 mL) was added TFA (1 mL) at
room temperature. The mixture was stirred for 2 hours and concentrated to afford the
crude product 13a-b which was used directly in the next step without further purification.
To a stirred solution of above crude 13a-b in anhydrous MeOH (10 mL) was added BTZ
core compound 15 (0.2 mmol) and Et3N (0.6 mmol) at room temperature. The mixture
o
was stirred overnight at 40 C, and concentrated. The residue was purified by column
chromatography over silica gel (DCM : MeOH = 20 : 1) to yield the yellow solids, which
were further treated by n-hexane to give compounds 2a-b.
2-(2-(4-fluorophenethyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (2a) According to above general procedure, employing
1-(2-Bromoethyl)-4-fluorobenzene afforded compound 2a as a yellow solid (38% for two
o
1
steps), mp: 174-175 C; HPLC purity: 96.8%, retention time 9.63 min; H NMR (500
MHz, DMSO-d6) δ 8.85 (s, 1H), 8.79 (s, 1H), 7.30 (brs, 2H), 7.15 (brs, 2H), 4.07-3.87 (m,
4H), 3.33 (s, 4H), 2.94 (brs, 4H), 1.76 (brs, 6H); HRMS-ESI (m/z): Calcd. For
C25H25F4N4O3S (M+H)+: 537.1578; Found: 537.1596.
2-(2-(3-(4-fluorophenyl)propyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (2b) According to above general
procedure, employing 1-(3-bromopropyl)-4-fluorobenzene afforded compound 2b as a
yellow solid (45% for two steps), HPLC purity: 95.4%, retention time 9.95 min; 1H
NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 8.79 (s, 1H), 7.18 (brs, 2H), 7.03-7.01 (m, 2H),
4.20-3.84 (m, 6H), 3.07-2.65 (m, 4H), 2.34-2.10 (m, 4H), 1.81 (brs, 6H); HRMS-ESI
(m/z): Calcd. For C26H27F4N4O3S (M+H)+: 551.1735; Found: 551.1739.
2-(2-(4-fluorophenyl)-2,8-diazaspiro[4.5]decan-8-yl)-8-nitro-6-(trifluoromethyl)-4H-
benzo[e][1,3]thiazin-4-one (2c) To a stirring solution of 8 (60 mg, 0.3 mmol) in
anhydrous toluene (5 mL) was added 1-bromo-4-fluorobenzene (45 µL, 0.4 mmol),
BINAP (19 mg, 0.03 mmol), t-BuONa (58 mg, 0.6 mmol), and Pd(OAc)2 (7 mg, 0.03
mmol) at room temperature. The mixture was stirred overnight at room temperature and
filtered. The filtrate was diluted by H2O (15 mL), and extracted by DCM (10 mL × 3).
The combined organic layer was washed by brine, dried over anhydrous MgSO4, filtered,

ACCEPTED MANUSCRIPT
and concentrated. The residue was purified over silica gel column (EtOAc: n-hexane = 5 :
1) to yield oils 10c (58 mg, 58%).
To a stirred solution of 10c (50 mg, 0.15 mmol) in DCM (5 mL) was added TFA (2
mL) at room temperature. The mixture was stirred for 2 hours and concentrated to afford
the crude product 13c which was used directly in the next step without further
purification. To a stirred solution of above crude 13c in anhydrous MeOH (10 mL) was
added BTZ core compound 15 (48 mg, 0.15 mmol) and Et3N (0.6 mmol) at room
o
temperature. The mixture was stirred overnight at 40 C, and concentrated. The residue
purified by column chromatography over silica gel (DCM : MeOH = 20 : 1) to yield the
yellow solids, which were further treated by n-hexane to give 2c (32 mg, 43%), mp: 192-
o
1
194 C; HPLC purity: 96.3%, retention time 14.07 min; H NMR (500 MHz, CDCl3) δ
9.15 (s, 1H), 8.80 (s, 1H), 7.00 (t, J = 8.5 Hz, 2H), 6.53-6.50 (m, 2H), 4.20-3.84 (m, 4H),
3.45 (t, J = 6.7 Hz, 2H), 3.26 (brs, 2H), 2.03 (t, J = 6.7 Hz, 2H), 1.86 (brs, 4H); HRMS-
ESI (m/z): Calcd. For C23H21F4N4O3S (M+H)+: 509.1265; Found: 509.1245.
General synthesis procedure of compounds 3c-g. A mixture of compound 8 (0.3 mmol)
o
and corresponding ketones (0.4 mmol) in Ti(OPr)4 was stirred at 70 C for 8 hours and
cooled to room temperature. MeOH (5 mL) and NaCNBH3 (1.6 mmol) was added to the
o
mixture, and stirred for 5 hours at 40 C. The mixture was quenched by 1 N NaOH (10
mL), filtered by celite, and washed by MeOH. The MeOH was evaporated under vacuo.
The residue was diluted by H2O, and extracted by Et2O. 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 = 30 : 1) to yield oils 11c-g (yield,
40-70%).
To a stirred solution of 11c-g in DCM (5 mL) was added TFA (1 mL) at room
temperature. The mixture was stirred for 2 hours and concentrated to afford the crude
product 14c-g which was used directly in the next step without further purification. To a
stirred solution of above crude 14c-g in anhydrous MeOH (10 mL) was added BTZ core
compound 15 (0.3 mmol) and Et3N (0.6 mmol) at room temperature. The mixture was
o
stirred overnight at 40 C, and concentrated. The residue was purified by column

ACCEPTED MANUSCRIPT
chromatography over silica gel (DCM : MeOH = 20 : 1) to yield the yellow solids, which
were further treated by n-hexane to give 3c-g.
2-(9-(1-(4-bromophenyl)ethyl)-3,9-diazaspiro[5.5]undecan-3-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3c) According to above general
procedure, employing 1-(4-bromophenyl)ethan-1-one as ketone afforded compound 3c as
a yellow solid (38% for two steps), mp: 171-172 oC; HPLC purity: 97.7%, retention time
1
10.67 min; H NMR (500 MHz, DMSO-d6) δ 8.82 (s, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.29
(d, J = 7.9 Hz, 2H), 3.96 (brs, 2H), 3.79 (brs, 2H), 3.48 (q, J = 6.8 Hz, 1H), 2.39 (brs, 2H),
2.32 (brs, 2H), 1.52 (brs, 8H), 1.29 (d, J = 6.5 Hz, 3H);13C NMR (100 MHz, DMSO-d6) δ
165.69, 161.37, 144.90, 143.81, 134.90, 131.71, 131.4, 130.05, 126.63, 122.53 (q, J =
272.5 Hz), 120.04, 63.44, 45.76, 35.55, 30.41, 19.33; HRMS-ESI (m/z): Calcd. For
C26H27BrF3N4O3S (M+H)+: 611.0934; Found: 611.0936.
2-(9-(1-(4-fluorophenyl)ethyl)-3,9-diazaspiro[5.5]undecan-3-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3d) According to above general
procedure, employing 1-(4-fluorophenyl)ethan-1-one as ketone afforded compound 3d as
a yellow solid (35% for two steps), mp: 166-167 oC; HPLC purity: 97.5%, retention time
9.74 min; 1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.79 (s, 1H), 7.30 (brs, 2H), 7.06-
7.03 (m, 2H), 4.11 (brs, 2H), 3.83 (brs, 2H), 3.46 (q, J = 6.4 Hz, 1H), 2.50 (brs, 2H), 2.42
(brs, 2H), 1.82 (brs, 8H), 1.41 (d, J = 6.4 Hz, 3H);13C NMR (100 MHz, CDCl3) δ 166.55,
162.35 (d, J = 247.5 Hz), 161.69, 143.93, 139.66 (d, J = 3.0 Hz), 134.29, 133.35 (q, J =
3.5 Hz), 129.54 (q, J = 35.5 Hz), 128.98, 126.79, 125.96 (q, J = 3.7 Hz), 122.54 (q, J =
272.5 Hz), 115.12, 114.91, 64.17, 45.99, 43.06, 35.95, 30.25, 19.69; HRMS-ESI (m/z):
Calcd. For C26H27F4N4O3S (M+H)+: 551.1735; Found: 551.1721.
2-(9-(1-(4-chlorophenyl)ethyl)-3,9-diazaspiro[5.5]undecan-3-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (3e) According to above general
procedure, employing 1-(4-chlorophenyl)ethan-1-one as ketone afforded compound 3e as
a yellow solid (46% for two steps), mp: 178-179 oC; HPLC purity: 98.9%, retention time
1
10.42 min; H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.79 (s, 1H), 7.30-7.28 (m, 4H),
4.11 (brs, 2H), 3.83 (brs, 2H), 3.45 (q, J = 6.4 Hz, 1H), 2.50 (brs, 2H), 2.41 (brs, 2H),
1.82 (brs, 8H), 1.38 (d, J = 6.4 Hz, 3H);13C NMR (125 MHz, DMSO-d6)165.69, 161.38,

ACCEPTED MANUSCRIPT
144.91, 143.35, 134.89, 131.74, 131.56, 129.66, 128.52, 127.88, 126.65, 123.10 (q, J =
272.5 Hz), 63.38, 45.79, 35.55, 30.40, 19.32; HRMS-ESI (m/z): Calcd. For
C26H27ClF3N4O3S (M+H)+: 567.1439; Found: 567.1427.
8-nitro-6-(trifluoromethyl)-2-(9-(1-(4-(trifluoromethyl)phenyl)ethyl)-3,9-
diazaspiro[5.5]undecan-3-yl)-4H-benzo[e][1,3]thiazin-4-one (3f) According to above
general procedure, employing 1-(4-(trifluoromethyl)phenyl)ethan-1-one as ketone
o
afforded compound 3f as a yellow solid (51% for two steps), mp: 141-142 C; HPLC
purity: 97.3%, retention time 10.64 min; 1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H),
8.79 (s, 1H), 7.61 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 7.8 Hz, 2H), 4.12 (brs, 2H), 3.83 (brs,
2H), 3.50 (q, J = 6.4 Hz, 1H), 2.52 (brs, 2H), 2.41 (brs, 2H), 1.63 (brs, 8H), 1.40 (d, J =
6.4 Hz, 3H);13C NMR (100 MHz, DMSO-d6) δ 164.60, 160.30, 148.48, 143.83, 133.81,
130.67 (q, J = 3.6 Hz), 127.47, 126.71 (q, J = 35.5 Hz), 125.57, 125.51 (q, J = 3.5 Hz),
124.43, 124.41, 122.08 (q, J = 272.4 Hz), 62.68, 44.85, 42.13, 34.48, 29.31, 18.29;
HRMS-ESI (m/z): Calcd. For C27H27F6N4O3S (M+H)+: 601.1703; Found: 601.1695.
8-nitro-6-(trifluoromethyl)-2-(9-(1-(4-(trifluoromethyl)phenyl)propyl)-3,9-
diazaspiro[5.5]undecan-3-yl)-4H-benzo[e][1,3]thiazin-4-one (3g) According to above
general procedure, employing 1-(4-(trifluoromethyl)phenyl)propan-1-one as ketone
o
afforded compound 3g as a yellow solid (38% for two steps), mp: 231-233 C; HPLC
purity: 99.1%, retention time 10.89 min; 1H NMR (500 MHz, DMSO-d6) δ 8.87 (s, 1H),
8.81 (s, 1H), 7.92 (d, J = 7.5 Hz, 2H), 7.79 (d, J = 7.5 Hz, 2H), 3.98 (brs, 2H), 3.80 (brs,
2H), 3.69 (d, J = 10.5 Hz, 1H), 3.39 (brs, 2H), 3.02 (brs, 2H), 2.32 (brs, 1H), 2.08 (brs,
1H), 2.03-1.54 (m, 8H), 1.29 (t, J = 6.5 Hz, 3H); HRMS-ESI (m/z): Calcd. For
C28H29F6N4O3S (M+H)+: 615.1859; Found: 615.1846.
General synthesis procedure of compounds 4a-d. To a stirring solution of 16a-d (0.3
mmol) in MeOH (5 mL) was added the f 4-fluorobenzaldehyde (0.4 mmol) and
NaCNBH3 (0.5 mmol) at room temperature. The mixture was adjusted to pH 6-7 by
acetic acid, stirred overnight at room temperature, and quenched by 1 M NaOH solution
(5 mL). The mixture was diluted by H2O (15 mL), and extracted by DCM (10 mL × 3).
The combined organic layer was washed by brine, dried over anhydrous MgSO4, filtered,

ACCEPTED MANUSCRIPT
and concentrated. The residue was purified over silica gel column (DCM : MeOH = 20 :
1) to yield oils 17a-d (yields，65-83%).
To a stirred solution of 17a-d (0.3 mmol) in DCM (10 mL) was added TFA (3 mL)
at room temperature. The mixture was stirred for 2 hours and concentrated to afford the
crude product 18a-d which was used directly in the next step without further purification.
To a stirred solution of above crude 18a-d in anhydrous MeOH (10 mL) was added BTZ
core compound 15 (0.3 mmol) and Et3N (0.6 mmol) at room temperature. The mixture
o
was stirred overnight at 40 C, and concentrated. The residue was purified by column
chromatography over silica gel (DCM : MeOH = 20 : 1) to yield the yellow solids, which
were further treated by n-hexane to give 4a-d.
2-(5-(4-fluorobenzyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4a) According to above general
procedure, employing compound 16a and 4-fluorobenzaldehyde afforded compound 4a
o
as a yellow solid (45% for two steps), mp: 175-176 C; HPLC purity: 95.2%, retention
1
time 8.96 min; H NMR (500 MHz, CDCl3) δ 9.20 (s, 1H), 8.81 (s, 1H), 7.30 (brs, 2H),
7.05-7.01 (m, , 2H), 4.23 (brs, 1H), 4.03-3.96 (m, 2H), 3.64 (m, 3H), 3.17 (brs, 1H), 3.04
(brs, 1H), 2.73-2.61 (m, 4H);13C NMR (125 MHz, DMSO-d6) δ165.03, 161.62 (d, J =
243.6 Hz), 159.43, 144.50, 135.53, 135.13, 132.10, 130.60, 130.54, 127.83 (q, J = 35.5
Hz), 126.64, 125.50 (q, J = 3.6 Hz), 123.14 (q, J = 272.3 Hz), 115.41, 115.27,59.94,
59.81, 58.03, 56.35, 53.48, 41.74; HRMS-ESI (m/z): Calcd. For C22H19F4N4O3S (M+H)+:
495.1109; Found: 495.1109.
2-(5-(4-fluorobenzyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-8-nitro-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (4b) According to above general
procedure, employing compound 16b (tert-butyl 2,5-diazabicyclo[2.2.1]heptane-2-
carboxylate) and 4-fluorobenzaldehyde afforded compound 4b as a yellow solid (47% for
two steps), mp: 240-241 oC; HPLC purity: 95.2%, retention time 8.59 min; 1H NMR (500
MHz, CDCl3) δ 9.20 (s, 1H), 8.81 (s, 1H), 5.49 (brs, 0.6H), 4.81 (brs, 0.4H), 4.22 (brs,
0.4H), 3.84-3.59 (m, 4.6H), 3.17-2.76 (m, 2H), 2.24-2.14 (m, 1H), 2.02-1.89 (m, 1H);13C
NMR (125 MHz, DMSO-d6) δ 166.19, 166.00, 162.88, 161.26, 160.59, 159.33, 143.59,
143.54, 134.43, 134.31, 133.73, 133.68, 129.88, 129.85, 129.83, 129.49, 127.05, 126.98,

ACCEPTED MANUSCRIPT
125.95 (q, J = 3.5 Hz), 125.86 (q, J = 3.5 Hz), 122.40 (q, J = 272.4 Hz), 115.40, 115.38,
115.26, 60.90, 60.24, 59.70, 59.12, 58.78, 58.74, 57.91, 57.15, 54.45, 52.56, 36.55, 34.83;
HRMS-ESI (m/z): Calcd. For C21H17F4N4O3S (M+H)+: 481.0952; Found: 481.0931.
2-(5-(4-fluorobenzyl)-2,5-diazabicyclo[2.2.2]octan-2-yl)-8-nitro-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (4c) According to above general procedure, employing
compound 16c (tert-butyl 2,5-diazabicyclo[2.2.2]octane-2-carboxylate) and 4-
fluorobenzaldehyde afforded compound 4c as a yellow solid (49% for two steps), HPLC
1
purity: 95.1%, retention time 8.20 min; H NMR (500 MHz, DMSO-d6) δ 8.86-8.80 (m,
2H), 7.38-7.35 (m, 2H), 7.15-7.12 (m, 2H), 5.01 (brs, 0.6H), 4.39 (brs, 0.4H), 4.04-3.97
(m, 1H), 3.78-3.70 (m, 3H), 3.07-3.03 (m, 1H), 2.91-2.83 (m, 2H), 2.07 (brs, 1H), 1.96
(brs, 1H), 1.86 (brs, 1H), 1.68 (brs, 1H); HRMS-ESI (m/z): Calcd. For C22H19F4N4O3S
(M+H)+: 495.1109; Found: 495.1093.
2-(4-(1-(4-fluorobenzyl)piperidin-4-yl)piperazin-1-yl)-8-nitro-6-(trifluoromethyl)-
4H-benzo[e][1,3]thiazin-4-one (4d) According to above general procedure, employing
compound 16d and 4-fluorobenzaldehyde afforded compound 4d as a yellow solid (55%
o
1
for two steps), mp: 157-158 C; HPLC purity: 95.0%, retention time 7.45 min; H NMR
(500 MHz, DMSO-d6) δ 8.84 (s, 1H), 8.78 (s, 1H), 7.31 (dd, J = 7.2 Hz, 6.5 Hz, 2H), 7.12
(t, J = 8.0 Hz, 2H), 3.93 (brs, 2H), 3.83 (brs, 2H), 3.42 (s, 2H), 2.82 (d, J = 10.4 Hz, 2H),
2.63 (brs, 4H), 2.25 (t, J = 11.1 Hz, 1H), 1.92 (brs, 2H), 1.72 (d, J = 10.5 Hz), 1.41 (dd, J
= 11.1 Hz, 10.4 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ 165.64, 162.49, 161.67 (d, J
=245.5 Hz), 144.87, 134.73, 131.79, 131.07, 127.84 (q, J = 34.5 Hz), 126.63, 126.60,
123.10 (q, J = 272.4 Hz), 125.37, 115.23, 61.54, 61.36, 52.78, 48.77, 40.53, 28.19;
HRMS-ESI (m/z): Calcd. For C25H26F4N5O3S (M+H)+: 552.1687; Found: 552.1668.
Funding Sources
This work is supported by the National Mega-project for Innovative Drugs
(2018ZX09721001-004-007; 2018ZX09711001-007-002), CAMS Innovation Fund for
Medical Science (CAMS-2018-I2M-3-004; CAMS-2017-I2M-1-011; CAMS-2016-I2M-
1-010), National Natural Science Foundation of China (81872753; 21502041), The
University Science and Technology Research Project of Hebei (YQ2014031). .

ACCEPTED MANUSCRIPT
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