Design, synthesis and evaluation of covalent inhibitors of DprE1 as antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2020.112773

Decaprenylphosphoryl-β-D-ribose 2′-oxidoreductase (DprE1) is a promising drug target for the development of novel anti-tubercular agents, and inhibitors of DprE1 are being investigated extensively. Among them, the 1,3-benzothiazinone compounds such as BTZ

Full text of DOI:10.1016/j.ejmech.2020.112773

European Journal of Medicinal Chemistry 208 (2020) 112773
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and evaluation of covalent inhibitors of DprE1 as
antitubercular agents
a
b
c
a
ꢀ d
Lingfeng Liu , Chengcheng Kong , Marco Fumagalli , Karin Savkova , Yiwen Xu ,
Stanislav Huszar , Jose C. Sammartino e, Dongguang Fan a, Laurent R. Chiarelli c
,
d
c
,
, ****
ꢀ
ꢀ
Katarína Mikusova ***, Zhaogang Sun b **, Chunhua Qiao a
,
,
, *
ꢁ
ꢀ d
a College of Pharmaceutical Sciences, Soochow University, 199 Renai Road, Suzhou, 215123, PR China
b Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research
Institute, Beijing Chest Hospital, Capital Medical University, 97 Ma Chang Street, Beijing, 101149, PR China
c Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100, Pavia, Italy
d
ꢀ
ꢁ
Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina, Ilkovicova 6, 842 15, Bratislava, Slovakia
e University School for Advanced Studies e IUSS Pavia, Piazza Della Vittoria 15, 27100, Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
Decaprenylphosphoryl-b-D
-ribose 20-oxidoreductase (DprE1) is a promising drug target for the devel-
Article history:
Received 24 June 2020
Received in revised form
5 August 2020
Accepted 17 August 2020
Available online 30 August 2020
opment of novel anti-tubercular agents, and inhibitors of DprE1 are being investigated extensively.
Among them, the 1,3-benzothiazinone compounds such as BTZ043, and its closer congener, PBTZ169, are
undergoing clinical studies. It has been shown that both BTZ compounds are prodrugs, the nitro group is
reduced to nitroso first, to which an adjacent Cys387 in the DprE1 binding pocket is covalently bound
and results in suicide enzyme inhibition. We figured that replacement of the nitro with an electrophilic
warhead would still achieve covalent interaction with nucleophilic Cys387, while the required reductive
activation could be circumvented. To test this hypothesis, a number of covalent inhibitors of DprE1 were
designed and prepared. The compounds inhibitory potency against DprE1 and anti-tubercular activity
were investigated, their chemical reactivity, formation of covalent adduct between the warhead and the
enzyme was demonstrated by mass spectrometry.
Keywords:
Anti-tuberculosis
DprE1
1,3-Benzothiazin-4-one scaffold
Covalent inhibitors
© 2020 Elsevier Masson SAS. All rights reserved.
Tuberculosis (TB), the disease caused primarily by Mycobacte-
rium tuberculosis bacillus, has been torturing mankind since the
ancient times. With the invention of isoniazid, pyrazinamide, rifa-
mycin and ethambutol in the 1950s and 1960s, the disease was
considered curable. Although the number of TB cases has been
decreasing globally during the last 20 years, the last report by
World Health Organization still estimates 10 million new cases and
1.45 million deaths in 2018 [1]. In addition, surveillance data
emphasize the ongoing problem of multidrug resistant TB [1],
related to poor adherence to the professional prescription, or co-
morbidities resulting in the compromised immune response. The
occurrence of drug resistance and the need to make the long and
demanding TB treatment more efficient, leads to continuous efforts
to develop novel drugs against TB.
One of the most promising targets for the development of new
antituberculosis agents is decaprenylphosphoryl-D
-ribose 20-
oxidoreductase (DprE1) [2]. DprE1 is a highly conserved FAD-
containing enzyme, which, along with its downstream NADH-
dependent reductase DprE2, catalyzes the conversion of decap-
renylphosphoryl-
-arabinofuranose (DPA). DPA is essential for the synthesis of the
mycobacterial cell wall component arabinan [3], and the sole donor
substrate for series of membrane-embedded arabinosyl-
b-D-ribofuranose (DPR) to decaprenylphosphoryl-
b-D
a
* Corresponding author.
** Corresponding author.
*** Corresponding author.
**** Corresponding author.
transferases including the ethambutol targets EmbA, EmbB, and
EmbC [4]. Recently, the periplasmic localization of DprE1 was
confirmed, which further substantiates the vulnerability of DprE1
as a promising antitubercular drug target [5].
E-mail addresses: laurent.chiarelli@unipv.it (L.R. Chiarelli), katarina.mikusova@
ꢁ
ꢀ
uniba.sk (K. Mikusova), sunzg75@163.com (Z. Sun), qiaochunhua@suda.edu.cn
(C. Qiao).
The first identified class of DprE1 inhibitors were nitro-
https://doi.org/10.1016/j.ejmech.2020.112773
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
benzothiazinones (BTZs), with compound BTZ043 (Fig. 1) [2] as the
lead. The minimal inhibitory concentration (MIC) value, for the next
generation nitro-benzothiazinone, PBTZ 169 (Fig. 1), is as low as
1 ng/mL against Mycobacterium tuberculosis [6]. This value is 20
folds lower than that of the well known first line drug isoniazid.
Structure-activity relationship study confirmed that the 8-nitro
group is critical for both enzymatic and cellular activity. Upon
binding to DprE1, the nitro group is reduced to a nitroso and that
specifically forms a covalent adduct with the Cys387 residue in the
active site of DprE1[7,8].
Recently, a boost to develop covalent drugs has occurred, ex-
amples of purposely designed covalent inhibitors are currently
approved or at advanced stage of clinical trials [9]. Compared to the
non-covalent counterparts, covalent drugs show strong and long
acting interaction property that allows better pharmacologic
response at a lower concentration [10]. To engineer a covalent drug,
the protein/inhibitor interacting pocket features the existence of a
nucleophilic residue, such as Ser/Thr, Lys, Met or Cys. Accordingly,
the inhibitor molecule bears an electrophilic warhead, or a moiety
that could be metabolically activated to the electrophilic part.
Typical warhead was observed as a Michael addition acceptor [11],
or a cyano group [12]. In DprE1, the presence of Cys387 in the
proximity of the BTZ binding site provides the opportunity to
achieve selectivity by covalent targeting with electrophilic in-
hibitors [13]. In this study, we investigated three types (A, B, and C)
of BTZ derived compounds, with nitro group replaced by electro-
philic warheads (Fig. 2). Compared to the reported prodrug
PBTZ169, our designed compounds were expected to inhibit DprE1
through formation of covalent adduct without going through the
reductive activation step.
Fig. 2. Design rationale for covalent inhibitors of DprE1.
As shown in Scheme 2, The cyano containing compounds B01,
B02 and B03 were prepared from commercial available starting
material 2,6-dichloronicotinic acid or 2,4-dichloropyrimidine-5-
carboxylic acid. The carboxylic acid was first transformed using
oxalyl chloride to acyl chloride 3a/3b, which was treated with
ammonium thiocyanate to provide isothiocyanate 4a/4b. Com-
pound B02 was successfully prepared through one more step re-
action between 4b and 1-(cyclohexylmethyl)piperazine. While
reaction of 4a with 1-(cyclohexylmethyl)piperazine gave interme-
diate 5 instead of the desired cyclization product, further treatment
of 5 with triethylamine in toluene under reflux condition provided
intermediate 6. Finally, compound B01 was prepared through re-
action of 6 with zinc cyanide using palladium catalyst.
1.2. Biological evaluation and chemical stability study
The prepared compounds were evaluated for their inhibitory
activity against both DprE1 and M. tuberculosis H37Rv (ATCC
25618). The results are shown in Table 1.
Overall, five out of seven type A compounds displayed good
DprE1 inhibitory activity, with A02, A03 and A06, in which nitro
group replaced by acrylamide, N-methacrylamide and methyl
1. Results and discussion
1.1. Chemistry
The designed type A and type C compounds were synthesized
following the synthetic route shown in Scheme 1.
propiolate exhibited IC50 less than 0.4
(methyl acrylate and N,N-dimethyl acrylamide, respectively) dis-
played similar IC50 1.25 M against DprE1. It was noted that low
activity is observed for compound A07 (no inhibitory activity at
concentration up to 20 M), considering this compound bears more
mM; and A01 and A04
PBTZ169 (1) was prepared according to the published procedure
[6]. Next, reduction of the nitro group to amino was achieved using
iron and ammonium chloride. The following Sandmeyer reaction
proceeded successfully using tert-butyl nitrite/NaNO2 in combina-
tion with copper halides, and converted the aromatic amino group
to the corresponding halides (C01, C02 and C03). Compound C03
further served as a common intermediate for the preparation of
compounds C04 and A01-A07. Specifically, reaction of C03 with
zinc cyanide using tetrakis (triphenylphosphine)palladium (0) as
catalyst gave compound C04. Reaction of C03 with methyl acrylate,
acrylamide, N-methacrylamide, N,N-dimethyl acrylamide, acrylo-
nitrile, methyl propiolate, 2-vinylpyridine provided A01-A07,
accordingly. Hydrogenation of A01, A05 and A02 using pd/C as
catalyst furnished D01, D02 and D03. All double bond configuration
was assigned as trans-based on the coupling constant in the 1HNMR
[14].
m
m
bulky pyridyl (A07), the result may suggest that the binding pocket
for the BTZ scaffold at 8-position is space restricted, consistent with
previously reported study [15]. It is surprising to notice compound
A05, with cyano group as warhead, showed dramatic loss of
enzyme inhibition activity. Subsequent chemical reactivity study
with methyl thioglycolate and stability investigation in PBS buffer
revealed A05 decomposition in the assay solution, explaining the
observed low biological activity.
Inconsistent with the enzyme activity, compounds with high
inhibitory potency against DprE1 did not display good cellular ac-
tivity. As shown in Table 1, all type A compounds displayed low
anti-Mtb H37Rv activity, with MIC values mostly higher than
The designed type B compounds were prepared following the
route shown in Scheme 2.
20 mM. This result suggested the compound poor ability to pene-
trate the mycomembrane and implement the inhibitory ability.
To validate the covalent inhibitor design rationale, we investi-
gated the compound reactivity toward a nucleophile like methyl
thioglycolate (MT) [16]. The result is shown in Table 2. Indeed,
except A03 and A07, all other tested compounds show reactivity
with electrophile, and covalent adducts with MT were detected as
subjecting to mass analysis. The most reactive A05 had a half life
0.2 h. Further stability study in pH7.4 PBS buffer recorded a half life
about 25 min, suggesting the possible decomposition process in the
assay solution, which also explains the observed lack of DprE1 and
Fig. 1. Reported 1,3-benzothiazinone type DprE1 inhibitors BTZ043 and PBTZ169.

L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
3
Scheme 1. Synthetic route for compounds A01-A07, C01eC04 and D01-D03.
Scheme 2. Synthetic route for compound B01, B02 and B03.
anti mycobacterial activity. By comparison, compound A06 (methyl
propiolate), A01 (methyl acrylate), A02 (acrylamide), A04 (N,N-
dimethyl acrylamide) displayed stability improvement, half life
between 1.0 h and 24 h in the presence of MT. Although A07 dis-
played low chemical reactivity toward MT, its weak DprE1 inhibi-
tory activity was more likely due to the intolerance of the bulky
pyridyl group in the enzyme binding pocket. Additionally, our
detected MT adduct peak for each compound correlates well with
the calculated mass (supporting information).
With the 7-cyano warhead, compounds B01 and B03 were ex-
pected to act as covalent DprE1 inhibitors. Unfortunately, both
compounds lost DprE1 activity. Compound B02, prepared as the
reaction intermediate for B03, also shown low DprE1 activity.
Taking together, the results suggest only phenyl other than other
hetero aromatic rings could be accommodated in the binding
pocket, which is in agreement with the previously reported study
[2]. Alternatively, it could be possible that 7-substitution on the
phenyl ring was deleterious for enzyme activity. With low DprE1
inhibition capability, the type B compounds demonstrated weak
(B03, MIC 6.7 mM) or no (B02, MIC>20 mM) antibacterial activity
(Table 1). We confirmed that B02 could form covalent adduct with
MT, and the half life is about 4 h.
During the process of preparation type A compounds, the
halogen substituted C01, C02 and C03 were readily obtained.
Although these compounds would not act through a covalent in-
hibition manner as PBTZ169 does, it was interesting to justify how
well the 1,3-benzothiazin-4-one scaffold exerts its enzymatic in-
hibition activity in a non-covalent binding mode. Thus, compounds
C01eC03 were examined for their DprE1 inhibition activity, and
micromolar IC50s were recorded (Table 1), which confirmed the

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L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
Table 1
Inhibition of DprE1 by prepared compounds and their activity against M. tuberculosis H37Rv strain.
Compd
A01
core
A
R(X)
IC50, DprE1 (
1.24 ( 0.30)
m
M)
MIC(mM, Mtb H37Rv)
>20.00
A02
A03
A
A
0.20 ( 0.04)
0.39 ( 0.11)
20.00 ( 1.4)
20.00 ( 1.56)
A04
A
1.25 ( 0.20)
>20.00
a
A05
A06
A
A
>20.00
0.14 ( 0.02)
10.00 ( 0.26)
a
A07
B01
B02
B03
A
B
B
B
>20.00
Y¼C,
Y¼N,
Y¼N,
7.72 ( 0.75)
>20.00
>12.00
>20.00
>20.00
6.73 ( 0.44)
C01
C02
C
C
0.92 ( 0.13)
1.84 ( 0.30)
0.03 ( 0.01)
0.16 ( 0.02)
C03
C04
C
C
2.35 ( 0.25)
1.70 ( 0.84)
0.63 ( 0.04)
0.16 ( 0.10)
D01
D02
C
C
5.40 ( 0.77)
13.2 ( 1.77)
>9.00
>9.00
D03
C
16.7 ( 1.95)
>9.00
1(PBTZ169)
0.01 ( 0.001)
0.011 ( 0.02)
“a":compound shows less than 70% inhibitory activity at 20 mM.
significance of the nitro group for DprE1 activity in PBTZ169. The
cyano is an apparent covalent warhead and drugs incorporated
cyano were discovered in several precedents [17], thus, compound
C04 was prepared and studied. It was found that C04 displayed IC50
compounds were superior to the current first-line drug isoniazid
(INH, MIC 2.5 M).
m
1.3. Type a compounds bind covalently to DprE1
1.7
m
M for the enzyme. However, all type C compounds did retain
M for C01,
M for C03. Compared to PBTZ169 (MIC
M), the antibacterial activity decrease for C01 was not as
significant M. tuberculosis potency, with MIC 0.03
0.16
0.01
m
To confirm the designed type A compounds could covalently
bind to DprE1, A01 and A02 were selected as representative and co-
incubated with the enzyme. As a control, DprE1 was incubated with
only the solvent DMSO. The mixture was then dialyzed and sub-
jected for mass analysis. As shown in Fig. 3, DprE1 protein
mM for C02 and 0.63 m
m
pronounced as the enzyme inhibition activity loss. Particularly, it
was worthy to say that the antibacterial potency of type C

L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
5
Table 2
Compound reactivity toward methyl thioglycolate (MT).
incubated with DMSO showed a MW of 50,940 Da. By contrast, the
protein showed a MW of 51,430 Da after incubation with A01, and
51,390 Da when incubated with A02, thus an increase in molecular
compounds from A-series against DprE1 enzyme, their MIC values
for M. tuberculosis H37Rv were rather high (Table 1). Therefore, we
were interested if these molecules target DprE1 in the cells. For this
experiment we chose compounds A01, A02, A03 and A06 and
examined their effects in M. tuberculosis H37Rv by metabolic la-
beling, as described [18]. The cultures were radiolabeled in three
different conditions: (i) for 24 h with [14C] acetate added along with
the drugs at 100 ꢀ MICs, (ii) for 24 h with [14C] acetate added along
with the drugs at 10 ꢀ MICs, (iii) for 24 h at 10 ꢀ MICs with [14C]
acetate added after 24 h of pre-treatment with the drug. The
weight of 490
20 or 460
20 Da, respectively, was observed,
which supports the covalent adduct formation between DprE1 and
the compounds.
Next, to confirm that the covalent bond is formed with the
Cys387, the modified protein was subjected to trypsin digestion,
and the peptide fragments were analyzed. The LC-MS profile of the
protein incubated with A01 showed a peak with a retention time of
43.27 min, that was absent in profile of the DprE1 alone (Fig. S1,
panel B, supporting information). The fragmentation of the peptide
showed a sequence compatible with 387-CVDFP-391, with the
compound forming an adduct with Cys387 (Fig. S2, top panel).
Similarly, the LC-MS profile of the protein incubated with A02
showed an additional peak with a retention time of 29.33 min
(Fig. S1, panel C, supporting information) with the peptide
compatible with the sequence 385-NICVDFPIK-393, with the com-
pound bound to the Cys387 (Fig. S2, bottom panel). Thus, the data
confirm that the compounds react with the Cys387 of DprE1,
forming a covalent adduct.
To further examine the covalent inhibitor design rationale, the
double bond in the warhead in the A-type of compounds A01, A05
and A02 was reduced to single bond and three type D compounds
(D01-D03) were obtained. Compared to the corresponding double
bond counterparts, D01 lost its inhibitory activity about 5 times,
D02 increased its inhibitory activity and D03 displayed dramatic
loss of activity against DprE1 enzyme. Our efforts to identify the
covalent adduct between D01 and DprE1 enzyme failed, which
suggests that covalent bond was not formed in this case, as
expected.
compound A01, for which MIC was found to be > 20.00 mM was
tested at 0.2 mM and 2 mM concentration. The lipids were
extracted from the radiolabeled cells and analyzed by thin-layer
chromatography (TLC). As shown in Fig. 4, addition of the control
inhibitors, BTZ043 and PBTZ169 caused accumulation of trehalose
dimycolates (TDM) and trehalose monomycolates (TMM) in com-
parison with the control cells. Inhibition of DprE1 by these drugs
results in restriction of synthesis of arabinan chains available for
the attachment of mycolic acids. Under these conditions, TMM, the
donor of mycolic acids for their deposition on arabinogalactan,
serves as an acceptor of surplus mycolic acids and TDM is produced.
Consequently, increased amounts of TMM and TDM can be
considered as a “signature” for inhibitors targeting arabinogalactan
portion of mAGP. This phenotype was clearly produced by the
control drugs BTZ043 and PBTZ169 at 100 ꢀ MIC, while the effects
of the tested compounds from A-series were not so profound. For
compound A06 we observed just overall decrease in [14C] acetate
incorporation into lipids under these conditions. However, at 10 ꢀ
MIC and longer incubation in the presence of the tested com-
pounds, we can see the expected phenotype, particularly for the
compounds A01, A02 and A06. The results of this experiment
suggest that these compounds target DprE1 in M. tuberculosis
H37Rv. Likewise, compounds C01 and C02, which were examined
by the same approach, appear to target DprE1 based on the lipid
profiles obtained from the radiolabeled cells, although covalent
binding is not expected in this case.
1.4. Metabolic labeling of M. tuberculosis indicates inhibition of
DprE1 by the target compounds in the cells
Despite promising inhibitory activities of the selected

6
L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
Fig. 3. Mass spectrometry analysis of DprE1 and DprE1-A01/DprE1-A02 complexes demonstrates the formation of a covalent adduct between the protein and the compound. A)
ESI-MS profile of DprE1; B) deconvolution of the spectrum in panel A; C) ESI-MS profile of DprE1 after incubation with A01; D) deconvolution of the spectrum in panel C. E) ESI-MS
profile of DprE1 after incubation with A02; F) deconvolution of the spectrum in panel E.
2. Conclusion
3. Experimental section
In summary, we have designed and prepared a number of co-
valent inhibitors of DprE1. Biochemical evaluation of these com-
pounds was performed. We validated the concept of selective
targeting of the cysteine of the DprE1 active site through incorpo-
ration of a warhead at the 8 position of 1,3-benzothiazin-4-one
scaffold. Compared to the reported nitro BTZ inhibitors, our
designed covalent inhibitors circumvent the required reductive
activation step. Nonetheless, delicate balance for the compound
stability and reactivity towards a nucleophile is critical for practical
application of these covalent inhibitors.
All commercially available reagents and solvents were used as
received. All reactions were monitored by thin layer chromatog-
raphy (TLC). Compounds were detected by ultraviolet light (UV)
absorption at either 254 or 365 nm. TLC was performed on silica
HSGF254 plates. All final products were characterized by 1H NMR,
13C NMR and MS analyses.1H NMR and 13C NMR spectra were
measured on an INOVA-400 MHz or Agilent DD2-600 MHz spec-
trometer and referenced to TMS. Analysis of sample purity was
performed on SHIMADZU LC-20AD high performance liquid chro-
matography (HPLC) system. HPLC conditions were as follows:

L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
7
Fig. 4. Evaluation of the effects of the prepared compounds by metabolic radiolabeling. TLC analysis of the lipids from radiolabeled M. tuberculosis H37Rv. Mycobacteria were
incubated with [14C]-acetate and BTZ043, PBTZ169 or compounds A01, A02, A03, A06, C01 and C02 at 100 ꢀ MIC (left panel) or 10 ꢀ MIC concentration for 24 h (middle panel).
Inhibitors were added along with the label (left and middle panels) or 24 h prior to label addition (right panel). TMM, trehalose monomycolates; TDM, trehalose dimycolates. PE,
phosphatidyl ethanolamine, CL e cardiolipin. TLC analysis was performed on Silica Gel plates in CHCl3/CH3OH/H2O (20:4:0.5).
solvent A: water, solvent B: MeOH, and flow rate 1.0 mL/min.
Compounds were eluted with a gradient from 10 to 100% MeOH/
water in 30 min, and purity was determined by the absorbance at
254 nm and 300 nm. All tested compounds have a purity of ꢁ98%
before sending for biological test.
45.9, 34.8, 31.7, 26.6, 25.9; HRMS (ESIþ) m/z [M þ H]þ calcd for
C
20H24F3IN3OS, 538.0631; found, 538.0610.
3.2. General procedure for the preparation of compound A01e05,
A07 via heck coupling reactions
To a solution of compound C03 (1.0 equiv) in N,N-dime-
thylformamide (DMF) under a nitrogen atmosphere was added a
series reagent containing propylene group (1.2 equiv; methyl
acrylate, acrylamide, N-methacrylamide, N,N-dimethyl acrylamide,
acrylonitrile, 2-vinylpyridine), triethylamine (2.0 equiv), palladium
acetate (0.5 equiv). The reaction mixture was heated to 85 ꢂC for
3e6 h. After being cooled to room temperature, the solvent was
evaporated under reduced pressure and diluted with ethyl acetate
(20 mL) and washed with 10 mL H2O. The solvents were removed.
The products were purified using flash chromatography using
mixtures of EtOAc and PET as eluent. The isolated yields were
56e92%.
3.1. Preparation of intermediate 2
To a solution of compound 1 (116 mg, 0.25 mmol) in EtOH/water
(3:1, 8 mL) was added NH4Cl (0.7 mg, 0.013 mmol) and Fe powder
(142 mg, 2.54 mmol). The resulting mixture was heated at reflux
and stirred for 1 h. After cooling to room temperature, the mixture
was filtered through a pad of diatomaceous earth, which was then
washed with more methanol (MeOH, 10 mL ꢀ 3). The filtrate was
concentrated under reduced pressure and the resulting aqueous
fraction was extracted with dichloromethane (DCM, 20 mL ꢀ 3).
The combined extracts were washed with brine, dried (Na2SO4),
filtered, and concentrated. The crude residue was purified by flash
column chromatography on silica gel with DCM: MeOH (90:1) to
afford intermediate 2 (88 mg, yield 82%) as a white solid: Rf ¼ 0.2,
CH2Cl2/MeOH: 90:1.
3.2.1. Methyl (E)-3-(2-(4-(cyclohexylmethyl)piperazin-1-yl)-4-oxo-
6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-8-yl)acrylate (A01)
The title compound was prepared from C03 and methyl acrylate
using the general procedure for Heck reaction (yield 92%) as a light
white solid: Rf ¼ 0.24, EtOAc-PET (1:1); 1H NMR (400 MHz, CDCl3)
3.1.1. 2-(4-(Cyclohexylmethyl)piperazin-1-yl)-8-iodo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (C03)
d
8.75 (s, 1H), 7.91, 7.89 (d, J ¼ 16.0 Hz, 1H), 6.54 (d, J ¼ 16.0 Hz, 1H),
To intermediate 2 (100 mg, 0.23 mmol) suspended in water
(20 mL) at 0e5 ꢂC was added dilute HCl (10%, 2 mL) and NaNO2
(24 mg, 0.35 mmol, 1.5 equiv.) solutions successively, and the
resulting reaction mixture was stirred for 1 h to complete the
diazotization process. Then KI (78 mg, 0.47 mmol, 2.0 equiv.) was
slowly added, and the reaction was stirred overnight at room
temperature. The mixture was filtered and washed with distilled
water (10 mL) and extracted with EtOAc (10 mL ꢀ 3), and the
combined organic layer was washed with 10% aqueous solution of
Na2SO3 (15 mL) and then dried over anhydrous Na2SO4, filtered,
and concentrated. The crude residue was purified by flash column
chromatography on silica gel with ethyl acetate (EtOAc) and pe-
troleum ether (PET), EtOAc-PET (1:1) to afford compound C03
(70 mg, yield 56%) as a yellow solid: Rf ¼ 0.2, EtOAc:PET (1:1); 1H
4.12 (br, 2H), 3.86 (s, 3H), 3.63 (br, 2H), 2.56 (s, 4H), 2.22 (d, J ¼ 4 Hz,
2H), 1.80e1.67 (m, 6H), 1.51 (s, 1H), 1.32e1.28 (m, 2H), 0.93e0.87
(m, 2H); 13C NMR (151 MHz, CDCl3)
d 167.8, 166.1, 160.2, 137.1, 135.9,
132.5, 130.0 (q, J ¼ 33.2 Hz), 128.5 (d, J ¼ 3.0 Hz), 126.3 (d,
J ¼ 3.0 Hz), 124.3, 123.9, 123.2 (q, J ¼ 273.3 Hz), 65.1, 52.9, 52.2, 46.1,
34.9, 31.7, 26.6, 26.0; HRMS (ESIþ) m/z [MþH]þ calcd for
C24H29F3N3O3S, 496.1876; found 496.1893.
3.2.2. (E)-3-(2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)acrylamide (A02)
The title compound was prepared from C03 and acrylamide
using the general procedure for Heck reaction (yield 56%) as a
brown solid: Rf ¼ 0.25, EtOAc-PET (1:1); 1H NMR (400 MHz, CD3OD)
NMR (400 MHz, CDCl3)
d
8.75 (s, 1H), 8.18 (s, 1H), 4.43 (br, 2H), 4.09
d
8.59 (s, 1H), 8.14 (s, 1H), 7.83 (d, J ¼ 15.2 Hz, 1H), 6.83 (d,
(br, 2H), 2.62 (s, 4H), 2.26 (s, 2H), 1.81e1.75 (m, 6H), 1.71 (s, 1H),
J ¼ 15.2 Hz, 1H), 4.88 (s, 2H), 4.03 (br, 4H), 2.72 (br, 4H), 2.36 (s, 2H),
1.33e1.21 (m, 2H), 0.92e0.87 (m, 2H); 13C NMR (151 MHz, CDCl3)
1.85e1.63 (m, 6H), 1.53 (br, 1H), 1.29e1.17 (m, 2H), 0.97e0.88 (m,
d
169.4, 161.7, 141.0, 138.3(d, J ¼ 3.0 Hz), 131.5 (d, J ¼ 34.7 Hz), 127.2
2H); 13C NMR (100 MHz, CDCl3)
d
167.1, 164.9, 159.5, 135.1, 134.1,
(d, J ¼ 4.5 Hz), 126.5, 125.2, 122.5 (q, J ¼ 273.3 Hz), 94.9, 65.0, 52.9,
131.8, 128.8 (q, J ¼ 33.5 Hz), 127.1 (d, J ¼ 3.3 Hz),125.0 (d, J ¼ 2.9 Hz),

8
L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
124.6, 123.2, 122.3 (q, J ¼ 271.3 Hz), 64.1, 52.0, 33.9, 30.7, 28.7, 25.7,
25.0; HRMS (ESIþ) m/z [MþH]þ calcd for C23H28F3N4O2S, 481.1880;
found, 481.1863.
(br, 1H), 1.24e1.15 (m, 2H), 0.90e0.87 (m, 2H); 13C NMR (151 MHz,
CDCl3)
J ¼ 34.4 Hz), 129.3 (d, J ¼ 3.3 Hz), 124.2, 122.8 (q, J ¼ 273.0 Hz), 117.8,
88.8, 78.9, 65.1, 53.3, 53.0, 46.3, 35.0, 31.7, 26.7, 26.0; HRMS (ESIþ)
m/z [MþNa]þ calcd for C24H26F3N3O3SNa, 516.1539; found,
516.1521.
d
167.3, 160.6, 153.3, 140.5, 133.1 (d, J ¼ 2.8 Hz), 130.1 (q,
3.2.3. (E)-3-(2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)-N-
methylacrylamide (A03)
The title compound was prepared from C03 and N-methyl
acrylamide using the general procedure for Heck reaction (yield
56%) as a brown solid: Rf ¼ 0.23, EtOAc-PET 1:1; 1H NMR (400 MHz,
3.2.7. (E)-2-(4-(Cyclohexylmethyl)piperazin-1-yl)-8-(2-(pyridin-2-
yl)vinyl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (A07)
The title compound was prepared from C03 and 2-vinylpyridine
using the general procedure for Heck reaction as a brown solid
(yield 57%): Rf ¼ 0.35, EtOAc-PET 1:1; 1H NMR (400 MHz, CDCl3)
CDCl3)
d
8.66 (s, 1H), 7.83 (d, J ¼ 14.8, 1H), 7.82 (s, 1H), 6.56 (d,
J ¼ 15.2 Hz, 1H), 6.45 (d, J ¼ 3.2 Hz, 1H), 4.08 (br, 2H), 3.76 (br, 2H),
2.98 (d, J ¼ 4.4 Hz, 3H), 2.49 (s, 4H), 2.16 (d, J ¼ 6.0 Hz, 2H),
1.78e1.69 (m, 6H), 1.48 (br, 1H), 1.31e1.19 (m, 2H), 0.91e0.85 (m,
d
8.70 (s,1H), 8.68 (d, J ¼ 4.0 Hz,1H), 8.01 (s,1H), 7.90 (d, J ¼ 16.0 Hz,
1H), 7.74 (t, J ¼ 8.0 Hz, 1H), 7.40 (d, J ¼ 4.0 Hz, 1H), 7.26 (s, 1H), 7.21
(d, J ¼ 12.0 Hz, 1H), 4.07 (br, 2H), 3.83 (br, 2H), 2.51 (s, 4H), 2.17 (d,
J ¼ 8.0 Hz, 2H), 1.79e1.70 (m, 6H), 1.49 (br, 1H), 1.33e1.25 (m, 2H),
2H); 13C NMR (151 MHz, CDCl3)
d 168.1, 164.9, 160.6, 136.0, 133.7,
133.2, 129.7 (q, J ¼ 28.2 Hz), 129.0 (q, J ¼ 277.4 Hz), 127.9 (d,
J ¼ 3.6 Hz), 126., 125.9 (d, J ¼ 2.7 Hz), 124.2, 65.1, 53.1, 53.0, 34.9,
31.7, 29.7, 26.7, 26.0; HRMS (ESIþ) m/z [MþH]þ calcd for
0.92e0.87 (m, 2H); 13C NMR (151 MHz, CDCl3)
d 168.4, 161.0, 153.8,
149.9, 136.9, 135.1, 133.9, 129.8 (q, J ¼ 33.2 Hz), 126.7, 125.5 (d,
J ¼ 3.0 Hz), 125.2, 124.0, 123.7, 123.5 (q, J ¼ 273.3 Hz), 123.4, 65.0,
52.9, 45.8, 34.8, 31.7, 26.6, 25.9; HRMS (ESIþ) m/z [MþH]þ calcd for
C
24H30F3N4O2S, 495.2036; found: 495.2000.
3.2.4. (E)-3-(2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)-N,N-
dimethylacrylamide (A04)
C27H30F3N4OS, 515.2087; found 515.2078.
3.3. General procedure for the synthesis of compound C01, C02
The title compound was prepared from C03 (20 mg, 0.04 mmol)
and N,N-dimethyl acrylamide using the general procedure for Heck
reaction as a brown solid (yield 59%): Rf ¼ 0.5, EtOAc-PET 1:1; 1H
A mixture of tert-butyl nitrite (1.25 equiv), copper (I) chloride or
copper (II) bromide (1.25 equiv), in CH3CN was heated to 60 ꢂC,
then treated with intermediate 2 (1.0 equiv) over 10 min, stirring is
continued for 2 h. The mixture was cooled to room temperature,
and diluted with ethyl acetate. The organic layer was washed with
10% hydrochloric acid and filtered, concentrated. The residue was
applied to column chromatography to afford the title compound
C01and C02.
NMR (400 MHz, CDCl3)
d
8.74 (s,1H), 7.91 (d, J ¼ 16.0 Hz,1H), 7.89 (s,
1H), 6.98 (d, J ¼ 16.0 Hz, 1H), 4.13 (br, 2H), 3.81 (br, 2H), 3.23 (s, 3H),
3.12 (s, 3H), 2.54 (br, 4H), 2.21 (br, 2H), 1.81e1.71 (m, 6H), 1.52 (br,
1H), 1.33e1.25 (m, 2H), 0.94e0.88 (m, 2H); 13C NMR (151 MHz,
CDCl3)
d
168.1, 165.0, 160.7, 135.9, 135.2, 133.6, 129.8 (d, J ¼ 34.7 Hz),
127.9 (d, J ¼ 3.0 Hz), 125.9 (d, J ¼ 3.0 Hz), 124.2, 123.5, 123.3 (q,
J ¼ 273.3 Hz), 65.1, 53.0, 46.4, 37.5, 36.1, 34.9, 31.7, 26.6, 26.0; HRMS
(ESIþ) m/z [M þ H]þ calcd forC25H32F3N4O2S, 509.2193; found
509.2207.
3.3.1. 8-Chloro-2-(4-(cyclohexylmethyl)piperazin-1-yl)-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (C01)
The title compound was prepared from intermediate 2 (100 mg,
0.24 mmol), copper (I) chloride (280 mg, 0.48 mmol, 2.0 eq) and
tert-butyl nitrite (48 mg, 0.47 mmol, 2.0 eq.) using the above pro-
cedure as a white solid (58 mg, yield 54%): Rf ¼ 0.68, EtOAc-PET 1:1;
3.2.5. (E)-3-(2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)acrylonitrile (A05)
The title compound was prepared from C03 and acrylonitrile
using the general procedure for Heck reaction as a yellow solid
(yield 76%): Rf ¼ 0.4, EtOAc-PET 1:1; 1H NMR (400 MHz, CDCl3)
1H NMR (400 MHz, CDCl3)
d 8.73 (s, 1H), 7.97 (s, 1H), 4.13 (br, 2H),
3.81 (br, 2H), 2.54 (br, 4H), 2.20 (s, 2H), 1.81e1.71 (m, 6H), 1.40 (br,
d
8.78 (s, 1H), 7.85 (s, 1H), 7.64 (d, J ¼ 16.0 Hz, 1H), 6.05 (d, J ¼ 16 Hz,
1H), 1.25e1.15 (m, 2H), 0.90e0.88 (m, 2H); 13C NMR (151 MHz,
1H), 4.12 (br, 2H), 3.80 (br, 2H), 2.54 (br, 4H), 2.19 (s, 2H), 1.80e1.70
CDCl3)
d
168.3,161.2,137.7,131.9 (d, J ¼ 1.5 Hz),130.9 (d, J ¼ 36.2 Hz),
(m, 6H), 1.50 (br, 1H), 1.32e1.15 (m, 2H), 0.90e0.84 (m, 2H); 13C
126.5 (d, J ¼ 3.0 Hz), 126.0, 122.7 (q, J ¼ 273.3 Hz), 120.4, 65.0, 52.8,
NMR (151 MHz, CDCl3)
d 167.3, 159.5, 143.1, 135.7, 131.5, 130.2 (q,
46.0, 34.7, 31.7, 26.5, 25.9; HRMS (ESIþ) m/z [MþH]þ calcd for
J ¼ 34.7 Hz),129.3 (d, J ¼ 3.0 Hz),125.8 (d, J ¼ 1.5 Hz),124.5,123.0 (q,
J ¼ 273.3 Hz), 116.5, 103.0, 65.1, 53.0, 46.3, 34.9, 31.7, 26.7, 26.0;
HRMS (ESIþ) m/z [MþH]þ calcd for C23H26F3N4OS, 463.1774; found,
463.1783.
C20H24ClF3N3OS, 446.1275; found, 446.1294.
3.3.2. 8-Bromo-2-(4-(cyclohexylmethyl)piperazin-1-yl)-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (C02)
The title compound C02 was prepared from intermediate 2
(50 mg, 0.12 mmol), copper (II) bromide (34 mg, 0.24 mmol, 2.0
equiv) and tert-butyl nitrite (15 mg, 0.14 mmol, 1.2 equiv) using the
above procedure. The product was obtained as a white solid (32 mg,
yield 52%): Rf ¼ 0.48, EtOAc-PET 1:1; 1H NMR (400 MHz, CDCl3)
3.2.6. Methyl3-(2-(4-(cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)propiolate (A06)
To a stirred solution of compound C03 (20 mg, 0.04 mmol) in
tetrahydrofuran (THF, 5 mL) was added methyl propiolate (5 mg,
0.05 mmol, 1.2 equiv), potassium carbonate (10 mg, 0.08 mmol, 2.0
equiv), Pd(PPh3)2Cl2 (2 mg, 0.2 equiv) and CuI (1 mg, 0.2 equiv.) and
the resulting mixture was stirred for 4 h at room temperature. The
reaction mixture was then diluted with water (10 mL) and extrac-
ted with DCM (10 mL ꢀ 3). The organic extracts were combined and
dried over anhydrous MgSO4 and concentrated under reduced
pressure. The crude residue was purified by flash column chro-
matography on silica gel to afford compound A06 (15 mg, yield
82%) as a brown solid: Rf ¼ 0.23, EtOAc: PET ¼ 1:5.1H NMR
d
8.72 (s, 1H), 7.97 (s, 1H), 4.15 (br, 2H), 3.85 (br, 2H), 2.59 (s, 4H),
2.24 (s, 2H), 1.77e1.71 (m, 6H), 1.53 (br, 1H), 1.41e1.25 (m, 2H), 0.88
(br, 2H); 13C NMR (151 MHz, CDCl3)
d
168.3, 161.1, 137.9, 131.8, (d,
J ¼ 3.0 Hz),130.8 (d, J ¼ 33.2 Hz),126.4 (d, J ¼ 3.0 Hz),126.1,126.0 (d,
J ¼ 273.3Hz), 120.3, 65.1, 53.0, 46.3, 34.9, 31.7, 26.7, 26.0; HRMS
(ESIþ) m/z [MþNa]þ calcd for C20H24BrF3N3OSNa, 512.0590; found,
512.0755.
3.3.3. 2- (4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazine-8-carbonitrile (C04)
(400 MHz, CDCl3)
d
8.78 (s, 1H), 7.99 (s, 1H), 4.15 (br, 2H), 3.90 (s,
3H), 3.78 (br, 2H), 2.57 (br, 4H), 2.22 (s, 2H), 1.80e1.71 (m, 6H), 1.51
To a solution of compound C03 (20 mg, 37.2
mmol) in DMF

L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
9
(10.0 mL) under nitrogen atmosphere was added zinc (II) cyanide
(3 mg, 0.02 mmol) and tetrakis (triphenylphosphine)palladium (0)
(2 mg, 10%). The reaction mixture was stirred at 90 ꢂC for 4 h and
was then diluted with toluene (10 mL) and the phases were sepa-
rated. The aqueous phase was extracted twice with toluene
(10 mL ꢀ 2). The combined organic phase was washed with brine
(10 mL) and saturated aqueous ammonium hydroxide, dried over
Na2SO4 and concentrated. The crude residue was purified by flash
column chromatography on silica gel to afford compound C04
(14 mg, yield 86%) as a white solid: Rf ¼ 0.26, EtOAc: PET (1:5); 1H
2.29 (s, 2H), 1.83e1.72 (m, 6H), 1.60 (s, 1H), 1.28e1.21 (m, 2H),
0.94e0.87 (br, 2H); 13C NMR (100 MHz, CDCl3)
172.6, 168.6, 160.9,
137.5, 135.3, 128.1, 125.9, 123.8, 65.1, 52.9, 46.1, 34.4, 31.7, 29.7, 27.7,
26.6, 26.0; HRMS (ESIþ) m/z [MþH]þ calcd for C23H30F3N4O2S,
483.2036; found, 483.2062.
d
3.5. Preparation of intermediate 5
Oxalyl chloride (165 mg, 1.3 mmol, 2.5 equiv) and catalytic
amount of DMF were added successively to the suspension of 2,6-
dichloronicotinic acid (100 mg, 0.52 mmol) in DCM (10 mL). The
reaction was stirred at room temperature for 1 h and evaporated in
vacuo. The residue was dissolved in 20 mL DCM and added to
ammonium thiocyanate (59 mg, 0.78 mmol, 1.5 equiv) dropwise
under stirring. A catalytic amount of PEG-400 was added to this
suspension, it was then stirred at room temperature for 20 min.
After consumption of the starting material, 1-(cyclohexylmethyl)
piperazine (95 mg, 0.52 mmol) was added and stirred for 1 h at
room temperature. The reaction mixture was diluted with water
(30 mL) and extracted with DCM (20 mL ꢀ 3). The combined
organic extracts were dried over anhydrous MgSO4 and concen-
trated under reduced pressure. The residue was purified by column
chromatography on silica gel with EtOAc-PET (1:3) to afford inter-
mediate 5 as a colorless liquid (142 mg, yield 64%): Rf ¼ 0.27, EtOAc:
PET (1:3). MS(þESI) m/z calcd for C18H25Cl2N4OS [MþH]þ ¼ 415.10,
found, 414.7.
NMR (400 MHz, CDCl3) d 8.91 (s, 1H), 8.08 (s, 1H), 4.15 (br, 2H), 3.78
(br, 2H), 2.54 (s, 4H), 2.19 (s, 2H), 1.80e1.71 (m, 6H), 1.67 (br, 1H),
1.28e1.18 (m, 2H), 0.90e0.87 (m, 2H); 13C NMR (151 MHz, CDCl3)
d
166.3, 159.2, 140.5, 132.7 (d, J ¼ 2.7 Hz), 131.5 (d, J ¼ 3.0 Hz), 130.7
(q, J ¼ 34.9 Hz), 124.6, 122.3 (q, J ¼ 273.8 Hz), 113.9, 110.9, 65.0, 53.1,
35.0, 31.7, 29.7, 26.7, 26.0; HRMS (CIþ) m/z [MþH]þ calcd for
C
21H24F3N4OS, 437.1617; found, 437.1614.
3.4. General procedure for the synthesis of compound D01, D02,
D03
To a solution of compound A01, or A02 or A05 in methanol was
added 20% Pd/C. The mixture was stirred under hydrogen at room
temperature. After completion, the mixture was filtered through
Celite and the filtrate was concentrated. The residue was applied to
column chromatography for purification to afford the title com-
pound D01, D02, D03.
3.6. Preparation of intermediate 6
3.4.1. Methyl-3-(2-(4-(cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)propanoate (D01)
The title compound was prepared from A01 (20 mg, 0.04 mmol)
and Pd/C (4 mg, 20%) using the general procedure for reduction
reaction (18 mg, yield 90%) as a cyan-blue solid: Rf ¼ 0.34, EtOAc-
To a solution of intermediate 5 (20 mg, 0.04 mmol) in 10 mL
toluene was added triethylamine (67 mg, 0.66 mmol, 2.0 equiv).
The reaction mixture was stirred at 100 ꢂC for 1 h and then diluted
with 10 mL H2O and the phases were separated. The aqueous phase
was extracted twice with toluene (10 mL ꢀ 2). The combined
organic phase was washed with brine and saturated aqueous
ammonium hydroxide, dried over sodium sulfate and concentrated.
The crude residue was purified by flash column chromatography on
silica gel to afford intermediate 6 as a white solid (12 mg, yield 92%).
PET 1:1; 1H NMR (400 MHz, CDCl3)
d 8.64 (s, 1H), 7.61 (s, 1H),
4.07 (br, 2H), 3.79 (br, 2H), 3.70 (s, 3H), 3.11 (t, J ¼ 8.0 Hz, 2H), 2.73
(t, J ¼ 8.0 Hz, 2H), 2.51 (s, 4H), 2.18 (d, J ¼ 8.0 Hz, 2H), 1.79e1.70 (m,
6H), 1.49 (s, 1H), 1.27e1.22 (m, 2H), 0.89e0.86 (m, 2H); 13C NMR
(151 MHz, CDCl3)
d 172.1, 168.5, 160.7, 137.1, 135.3, 129.5 (q,
J ¼ 33.2 Hz),128.1 (d, J ¼ 3.0 Hz),125.9 (d, J ¼ 3.0 Hz),123.8,123.5 (q,
J ¼ 273.3 Hz), 65.1, 53.0, 51.9, 46.2, 34.9, 33.0, 31.7, 27.7, 26.7, 26.0;
HRMS (ESIþ) m/z [MþH]þ calcd forC24H31F3N3O3S, 498.2033;
found, 498.2009.
EtOAc-PET (1:3, Rf ¼ 0.24); 1H NMR (400 MHz, CDCl3)
d 8.57 (d,
J ¼ 8.2 Hz, 1H), 7.38 (d, J ¼ 8.2 Hz, 1H), 4.12 (s, 2H), 3.69 (s, 2H), 2.51
(s, 4H), 2.17 (d, J ¼ 6.8 Hz, 2H), 1.76 (d, J ¼ 14.8 Hz, 4H), 1.69 (s, 2H),
1.48 (s, 1H), 1.28e1.21 (m, 2H), 0.93e0.85 (m, 2H).
3.4.2. 3-(2-(4-(cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)propanenitrile
(D02)
3.6.1. 2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-4H-pyrido
[3,2-e][1,3]thiazine-7-carbonitrile (B01)
To a 20 mL microwave vial containing a mixture of intermediate
6 (30 mg, 0.08 mmol), zinc cyanide (11 mg, 0.1 mmol, 1.2 equiv),
zinc powder (4 mg, 0.02 mmol, 0.2 equiv), and DPPF (9 mg,
0.02 mmol, 0.2 equiv) was added DMSO (6 mL). The suspension was
stirred with degassing under nitrogen for 5 min. Next, tris(di-
benzylideneacetone)dipalladium (0) (7 mg, 0.01 mmol, 0.1 equiv)
was added. The reaction mixture was sealed and stirred with
heating at 100 ꢂC (heat block) for 20 min. Then it was allowed to
cool to RT, diluted with 20 mL of water, and adjusted to pH~8 with
sat NaHCO3, and extracted with DCM (10 mL ꢀ 3). The organic
extracts were dried over anhydrous MgSO4 and concentrated under
reduced pressure. The residue was purified by column chroma-
tography on silica gel to afford compound B01 as a white solid
(20 mg, yield 65%): EtOAc-PET (1:3, Rf ¼ 0.23); 1H NMR (400 MHz,
The title compound was prepared from A05 (20 mg, 0.04 mmol)
and Pd/C (4 mg, 20%) using the general procedure for reduction
reaction (18 mg, yield 95%) as a cyan-blue solid: Rf ¼ 0.36, EtOAc-
PET 1:1; 1H NMR (400 MHz, CDCl3)
d 8.70 (s, 1H), 7.65 (s, 1H),
4.10 (br, 2H), 3.79 (br, 2H), 3.17 (t, J ¼ 8.0 Hz, 2H), 2.76 (t, J ¼ 8.0 Hz,
2H), 2.52 (s, 4H), 2.18 (d, J ¼ 8.0 Hz, 2H), 1.79e1.67 (m, 6H), 1.49 (br,
1H), 1.28e1.20 (m, 2H), 0.95e0.86 (m, 2H); 13C NMR (151 MHz,
CDCl3)
d
168.1, 159.9, 135.3, 134.7, 129.8 (q, J ¼ 33.2 Hz), 128.5, 126.7,
124.1, 121.3 (q, J ¼ 271.8 Hz), 117.8, 65.1, 53.0, 46.4, 35.0, 31.7, 28.4,
26.7, 26.0, 17.1; HRMS (ESIþ) m/z [MþH]þ calcd forC23H28F3N4OS,
465.1930; found, 465.1912.
3.4.3. 3-(2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-6-
(trifluoromethyl)-4H-benzo[e][1,3]thiazin-8-yl)propanamide (D03)
The title compound was prepared from A02 (20 mg, 0.04 mmol)
and Pd/C (4 mg, 20%) using the general procedure for reduction
reaction (19 mg, yield 98%) as a white solid: Rf ¼ 0.28, EtOAc-PET
CDCl3)
d
8.75 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 8.0 Hz, 1H), 4.15 (br, 2H),
3.72 (br, 2H), 2.53 (s, 4H), 2.18 (s, 2H), 1.79e1.70 (m, 6H), 1.49 (br,
1H), 1.27e1.20 (m, 2H), 0.92e0.87 (m, 2H); 13C NMR (151 MHz,
CDCl3)
d 167.6, 161.9, 156.5, 139.1, 135.7, 127.4, 122.6, 115.8, 65.0,
1:1; 1H NMR (400 MHz, CDCl3)
d
8.62 (s, 1H), 7.64 (s, 1H), 5.65 (s,
53.0, 34.9, 31.7, 29.7, 26.7, 26.0; HRMS (CIþ) m/z [MþH]þ calcd for
1H), 5.54 (s, 1H), 4.24 (br, 2H), 4.03 (br, 2H), 3.14 (s, 2H), 2.63 (s, 6H),
C19H24N5OS, 370.1696; found, 370.1700.

10
L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
3.6.2. 7-Chloro-2-(4-(cyclohexylmethyl)piperazin-1-yl)-4H-
3.7.2. MIC determination
pyrimido[5,4-e][1,3]thiazin-4-one (B02)
M. tuberculosis H37Rv (ACTT 25618) strains were subcultured on
LowensteineJensen medium at 37 ꢂC for 28 days. Bacterial sus-
pension was adjusted to a turbidity equivalent to 1.0 McFarland
standard, then further diluted 1:20 in Middle brook 7H9 broth
supplemented with 10% (vol/vol) OADC enrichment and 0.2% (vol/
vol) glycerol. MICs against replicating M. tuberculosis were deter-
mined by the microplate alamar blue assay (MABA) [19]. PBTZ 169
was used as positive control. Compound stock solutions (1.0 mg/
mL) in DMSO were prepared and serially diluted in two-fold. For
the most active compounds, the stock solution concentration was
To a solution of 2,4-dichloropyrimidine-5-carbonyl chloride
(100 mg, 0.47 mmol) in DCM (10 mL) added ammonium thiocya-
nate (54 mg, 0.71 mmol, 1.5 equiv) dropwise under stirring. A drop
of PEG-400 was added to the suspension, it was then stirred at
room temperature for 20 min. After consumption of the starting
material, 1-(cyclohexylmethyl)piperazine (86 mg, 0.47 mmol) was
added. It was then stirred for 1 h at room temperature. The reaction
mixture was then diluted with water (30 mL) and extracted with
DCM (20 mL ꢀ 3). The organic extracts were dried over anhydrous
MgSO4 and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel to afford com-
pound B02 as a white solid (159 mg, yield 89%): EtOAc-PET (1:3,
3.2
0.002
100
the 96-well plate containing the corresponding drugs, and the
plates were incubated at 37 ꢂC. On day 7, 50
L of 5% Tween 80 and
20 L of alamar blue were added to all wells. After incubation for
m
g/mL and the final testing concentration range was 2 to
g/mL, respectively.
L of bacterial culture suspension was added to the wells of
m
m
Rf ¼ 0.27); 1H NMR (400 MHz, CDCl3)
d 9.36 (s, 1H), 4.17 (br, 2H),
3.74 (br, 2H), 2.57 (s, 4H), 2.21 (s, 2H), 1.81e1.73 (m, 6H), 1.51 (br,
m
1H), 1.35e1.23 (m, 2H), 0.92e0.88 (m, 2H); 13C NMR (151 MHz,
m
CDCl3)
d
167.3, 166.6, 162.3, 161.0, 160.0, 115.9, 65.0, 53.0, 52.8, 47.0,
24 h, the colors of all wells were recorded. A color change from blue
to pink indicates bacterial growth. MIC was defined as the lowest
concentration of the drug that showed no color change, which was
the lowest concentration of drug capable of inhibiting the visible
growth of tested isolates.
46.6, 35.0, 31.6, 26.7, 26.0; HRMS (CIþ) m/z [MþH]þ calcd for
C
17H23ClN5OS, 380.1306; found, 380.1317.
3.6.3. 2-(4-(Cyclohexylmethyl)piperazin-1-yl)-4-oxo-4H-pyrimido
[5,4-e][1,3]thiazine-7-carbonitrile(B03)
To a solution of compound B02 (50 mg, 0.13 mmol) in DMSO
(5 mL) was added zinc cyanide (19 mg, 0.16 mmol, 1.2 equiv) and
3.7.3. IC50 determination
M. tuberculosis DprE1 was expressed and purified in E. coli cells,
as previously reported [20]. Enzyme activity was determined using
the Amplex Red/peroxidase coupled assay was performed accord-
DPPF (14.59 mg, 26.32 mmol, 0.2 equiv). The suspension was stirred
with degassing under nitrogen for 5 min. Next, tris(dibenzylide-
neacetone)dipalladium (0) (15 mg, 0.03 mmol, 0.2 equiv) was
added. The reaction mixture was sealed and stirred with heating at
80 ꢂC for 2 h. Then it was allowed to cool to RT, diluted with 20 mL
of water, and adjusted to pH~8 with sat NaHCO3, extracted with
DCM (10 mL ꢀ 3). The organic extracts were dried over anhydrous
MgSO4 and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel to afford com-
pound B03 (26 mg, yield 53%) as a white solid: EtOAc-PET (1:3,
ing to published procedure [21]. Briefly, DprE1 (0.5
bated at 30 ꢂC in 20 mM glycylglycine pH 8.5, containing, 0.050 mM
Amplex Red, and 0.35 M horseradish peroxidase. The reaction was
mM) was incu-
m
initiated by adding 500 mM FPR, and the formation of resorufin was
followed at 572 nm (ε ¼ 54,000 Mꢃ1 cmꢃ1). Initial inhibition assays
were carried out in the presence of 20
mM of each compound
(dissolved in DMSO). DMSO was used as negative control, PBTZ169
as positive control. Compounds with more than 70% inhibitory
activity at 20
mM were further analyzed for IC50 calculated ac-
Rf ¼ 0.28); 1H NMR (400 MHz, CDCl3)
d 9.48 (s, 1H), 4.13 (s, 2H), 3.72
cording to equation:
(s, 2H), 2.56 (s, 2H), 2.50 (s, 2H), 2.17 (d, J ¼ 6.8 Hz, 2H), 1.77e1.69
ꢀ
ꢁ
(m, 6H), 1.47 (br, 1H), 1.28e1.21 (m, 2H), 0.90e0.85 (m, 2H); 13C
½Iꢄ
A
½Iꢄ
¼ A
ꢀ
1 ꢃ
½0ꢄ
NMR (151 MHz, CDCl3) d 166.4,165.8,159.8,159.1,144.9,118.3,114.6,
½Iꢄ þ IC50
64.9, 53.0, 52.8, 47.3, 46.8, 35.0, 31.6, 26.7, 26.0; HRMS (CIþ) m/z
[MþH]þ calcd for C18H23N6OS, 371.1649; found, 371.1657.
where A[I] is the activity of the enzyme at inhibitor concentration [I]
and A[0] is the activity of the enzyme without inhibitor. Otherwise,
the activity was recorded as “a", as indicated in Table 1. All results
are mean SD of three replicates.
3.7. Reactivity of compounds with methyl thioglycolate in PBS
buffer
3.7.4. Covalent modification
To assess the compound reactivity with methyl thioglycolate,
the prepared compounds (10 mM) and methyl thioglycolate
(50 mM) was incubated at 37 ꢂC in 50 mM PBS buffer (pH 7.4) in a
DprE1 (3 mg/mL, 100
ml) was incubated with 100
m
M A01 or A02
at 30 ꢂC for 30 min. The sample was dialyzed, concentrated to 20
mL,
diluted 1:5 in methanol-water 1:1 containing 0.01% formic acid,
and directly analyzed in mass spectrometry, using an Ion Trap (LCQ
Fleet™) mass spectrometer with electrospray ionization (ESI) ion
source controlled by Xcalibur software 2.2. Mass spectra, recorded
for 2 min, were generated in positive ion mode under constant
instrumental conditions: source voltage 5.0 kV, capillary voltage
46 V, sheath gas flow 20 (arbitrary units), auxiliary gas flow 10
(arbitrary units), sweep gas flow 1 (arbitrary units), capillary tem-
perature 210 ꢂC, tube lens voltage 105 V. MS/MS spectra, obtained
by CID studies in the ion trap, were performed with an isolation
width of 3 Da m/z, the activation amplitude was 35% of ejection RF
amplitude that corresponds to 1.58 V.
total volume of 160 mL. The reaction was monitored by TLC and
analyzed by injection of aliquots to LCꢃMS/MS at predetermined
intervals within a period of 48 h. Control reaction was run in the
absence of methyl thioglycolate. The formation of methyl thio-
glycolate adduct was determined by detection of the corresponding
MS adduct peak.
3.7.1. T1/2 Determination
The above reaction was monitored by HPLC. The disappearance
of the parent compounds was monitored as % remaining relative to
time zero, and the data were fitted to first-order kinetics by plotting
the natural log of % remaining as a function of time. The pseudo-
first-order rate constant (k), which is the negative slope of the
linear fitting, was used to calculate the reaction half-life (t1/
Spectra deconvolution was performed using UniDec 3.2.0 soft-
ware [22].
To identify the site of attachment of the compound the DprE1
reacted with A01 and A02 was digested with trypsin and then
¼ 0.693/k).
2

L. Liu et al. / European Journal of Medicinal Chemistry 208 (2020) 112773
11
drug ethambutol, Science 368 (2020) 1211e1219.
peptides analyzed. Briefly, DprE1 (10 mg/mL, 100
with 300
M A01 or A02 at 30 ꢂC for 60 min. As control the protein
was incubated with DMSO, then samples were then incubated with
g trypsin (Trypsin Gold, Promega) at 37 ꢂC overnight. Peptides
analyses were carried out on an LC-MS (Thermo Finnigan, San Jose,
CA, United States) system consisting of a thermostated column
oven Surveyor autosampler controlled at 25 ꢂC; a quaternary
gradient Surveyor MS pump equipped with an UV/V is detector and
an Ion Trap (LCQ Fleet™) mass spectrometer with electrospray
ionization (ESI) ion source controlled by Xcalibur software 2.2.
Analytes were separated by RP-HPLC on a Jupiter (Phenomenex,
ml) was incubated
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E. Kilacskova, V. Mokosova, Z. Svetlíkova, M. Sarkan, J. Neres, J. Kordulakova,
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S.T. Cole, K. Mikusova, DprE1 is a vulnerable tuberculosis drug target due to its
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Torrance, CA, United States) C18 column (150 ꢀ 2 mm, 4
mm, 90 Å
particle size) using a linear gradient (2e60% solvent B in 45 min;
60e98% B in 5 min and 98% B in 10 min) in which solvent A con-
sisted of 0.1% aqueous formic acid and solvent B of acetonitrile
containing 0.1% formic acid. Flow-rate was 0.2 mL/min. Mass
spectra were generated by using the same conditions described
above.
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b W.J. Metzler, J. Yanchunas, C. Weigelt, K. Kish, H.E. Klei, D. Xie, Y. Zhang,
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Declaration of competing interest
[14] Neil E. Jacobsen, NMR spectroscopy explained, John Wiley & Sons, Inc (2007)
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgement
This work was supported by the National Natural Science
Foundation of China (8187169); Italian Ministry of Education,
University and Research (MIUR) (Dipartimenti di Eccellenza, Pro-
gram 2018e2022) to Department of Biology and Biotechnology, “L.
Spallanzani”, University of Pavia (to M.F., J.C.S., L.R.C.); and Slovak
Research and Development Agency (APVV-15-0515, to K.M.).
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T. Kucera, H. Kocova -Vlckova, S. Huszar, Z. Konyarikova, K. Konecna,
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A. Hrabalek, K. Mikusova, J. Roh, Development of 3,5-dinitrophenyl-contain-
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Appendix A. Supplementary data
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ing 1,2,4-triazoles and their trifluoromethyl analogues as highly efficient
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112773.
antitubercular agents inhibiting decaprenylphosphoryl-b
-D-ribofuranose 20-
oxidase, J. Med. Chem. 62 (2019) 8115e8139.
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