Design, synthesis and antitubercular evaluation of benzothiazinones containing an oximido or amino nitrogen heterocycle moiety

Article abstract of DOI:10.1039/c6ra25712g

A series of 8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-ones (BTZs) bearing an oximido or amino nitrogen heterocycle moiety through modifications at the C-2 position of BTZ043 and BPTZ169 were designed and synthesized as new antitubercular agents. Many of the target compounds demonstrate excellent in vitro activity (MIC: <0.016-0.088 μg mL^?1) against the drug susceptive H37Rv strain and two clinically isolated multidrug-resistant Mycobacterium tuberculosis (MTB) strains. Compound 10a displays acceptable safety, aqueous solubility and pharmacokinetic properties, opening up a new possibility for further development.

Full text of DOI:10.1039/c6ra25712g

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Design, synthesis and antitubercular evaluation of
benzothiazinones containing an oximido or amino
nitrogen heterocycle moiety†
Cite this: RSC Adv., 2017, 7, 1480
a
a
b
a
a
Rui Zhang,‡ Kai Lv,‡ Bin Wang, Linhu Li, Bo Wang, Mingliang Liu, a Huiyuan Guo,a
*
a
b
*
Apeng Wang and Yu Lu
A series of 8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-ones (BTZs) bearing an oximido or amino
nitrogen heterocycle moiety through modifications at the C-2 position of BTZ043 and BPTZ169 were
designed and synthesized as new antitubercular agents. Many of the target compounds demonstrate
excellent in vitro activity (MIC: <0.016–0.088 mg mLꢀ1) against the drug susceptive H37Rv strain and two
clinically isolated multidrug-resistant Mycobacterium tuberculosis (MTB) strains. Compound 10a displays
acceptable safety, aqueous solubility and pharmacokinetic properties, opening up a new possibility for
further development.
Received 24th October 2016
Accepted 15th December 2016
DOI: 10.1039/c6ra25712g
www.rsc.org/advances
Tuberculosis (TB), one of the world's major causes of illness and positions, respectively, so there is only one possible structural
death, is caused mainly by Mycobacterium tuberculosis (MTB).1 modications at the C-2 position.
The World Health Organization (WHO) estimated that approx-
On the other hand, some of uoroquinolones (FQs), a class
imately one third of the world population is infected with MTB, of important second-line anti-TB drugs, such as ciprooxacin,
and 9.6 million people were infected and 1.5 million died from ooxacin and levooxacin, are frequently used for management
TB worldwide in 2014.2 In particular, the spread of multidrug- of TB including MDR-TB.15 Pyrrolidinyl, piperazinyl and piper-
resistant (MDR) TB and the emergence of extensively drug- idinyl are the most common groups at the C-7 position of FQs. It
resistant (XDR) TB have revitalized drug discovery efforts in is interesting that recently the discovery of gemioxacin and
search of novel drugs recently.3–5 It is encouraging that beda- IMB-070593 highlights the importance of oxime-functionalized
quiline and delamanid have been approved for the treatment of nitrogen heterocycles with respect to antibacterial activity.16–18
MDR-TB, aer a huge gap of over 40 years.6,7 However, there are
Inspired by the above research results, we intended to
only two new chemical entities, Q203 and TBA-354, in phase 1 replace the spiroketal moiety of BTZ043 with nitrogen cyclo-
clinical trials at present. Therefore, it is urgent to develop new ketone oximes and shi the terminal nitrogen on the piperazine
anti-TB drugs.8,9
ring of BPTZ169 outside the ring (Fig. 1), which were expected to
8-Nitro-6-(triuoromethyl)-1,3-benzothiazin-4-ones (BTZs), explore SAR of BTZs though variations on the sizes of the
a novel class of TB agents targeting DprE1,10–12 were reported to heterocycle and the alkyl group of the oxime. Thus, a series of
have potent activity in multiple models.13 BTZ043 (Fig. 1) and novel BTZ derivatives containing an oximido or amino nitrogen
PBTZ169 (Fig. 1) are being studied preclinically. Compared with heterocycle moiety were designed and synthesized in this study.
BTZ043, PBTZ169 is slightly more potent and not being ster- Our primary objective was to identify alternative groups at the
eoselective which makes it easier and cheaper to synthesize.14
The structure–activity relationship (SAR) studies of BTZs show
that –CF3 and –NO2 are the optimal groups at the C-6 and C-8
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking
Union Medical College, Beijing 100050, China. E-mail: lmllyx@126.com; Tel:
+86-10-63030965
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 101149, China. E-mail:
luyu4876@hotmail.com; Tel: +86-10-89509357
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra25712g
Fig. 1 Design of the new molecules.
‡ These authors contributed equally to this work.
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Scheme 1 Synthesis of target compounds 10a–i and 11a, 11b. Reagents and conditions: (i) RONH2$HCl, K2CO3, C2H5OH, rt, 40–65%; (ii) TFA,
CH2Cl2, rt, 50–52%; (iii) c-C6H11CH2Br, K2CO3, DMF, 80 ꢁC, 50–52%; (iv) SOCl2, NH3$H2O, reflux, 76%; (v) CS2, CH3I, DMSO, rt, 41%; (vi) (C2H5)3N,
C2H5OH, 60 ꢁC, 45–59%.
C-2 position of BTZs with potent antimycobacterial activity and more potent than BPTZ169, INH and RFP (MIC: 0.049–0.116
facilitate the further development of BTZs.
mg mLꢀ1). The potency of the oxime BTZ derivatives (10a–h) in
Detailed synthetic pathways to novel BTZ derivatives 10a–i this study is related to both of the sizes of the heterocycle and
and 11a, 11b are outlined in Scheme 1. 4-7-Membered nitrogen alkyl group (R). Generally, 4- and 6-membered heterocycles
cycloketones 1a–i were smoothly converted to the oximes 2a by show the best activity, followed by 7- and 5-membered ones with
reaction with O-alkylhydroxyamines in ethanol at room the same R group (CH3) successively (10a vs. 10d vs. 10e vs. 10h).
temperature. The N-Boc protecting group on 2a–i was removed Moreover, the contribution of the alkyl groups of the oxime
with triuoroacetic acid (TFA) in dichloromethane to afford the moiety to the activity is relevant to the heterocycles. The activity
nitrogen cycloketone oximes 3a–i in 40–65% yield.
of the R groups is as follows: benzyl ¼ ethyl > methyl for
Compounds 6a, 6b were easily prepared from the corre- azetidyl-based BTZs (10a vs. 10b vs. 10c), and but benzyl >
sponding tert-butyl 3-(methylamino) pyrrolidine/piperidine-1- methyl J ethyl for piperidinyl-based ones (10e vs. 10f vs. 10g).
carboxylate 4a, 4b via nucleophilic substitution reaction with Further investigation also suggests that introduction of an
(bromomethyl)cyclohexane in the presence of K2CO3 in amino group on the heterocycles is detrimental. For instance, 3-
dimethyl formamide (DMF) at 80 ꢁC, and then the resulting amino-4-(methoxyimino)piperidin-1-yl derivative (10i) displays
cyclohexylmethylates 2a, 2b were treated with TFA.
Amidation of 2-chloro-3-nitro-5-(triuoromethyl) benzoic 10e (MIC: 0.029 mg mLꢀ1).
MIC value of 0.436 mg mLꢀ1 which is 15-fold less potent than
acid 7 gave amide 8, which was condensated with carbon
On the other hand, when the nitrogen atom of the piperazine
disulde and methyl iodide yielded BTZ core compound 9.10 ring of PBTZ169 was converted to an exocyclic tertiary amino
Derivatives 10a–i and 11a, 11b were conveniently obtained from group, the resulting compound 11a demonstrates increased
9 by nucleophilic substitution with side chain compounds 3a–i activity (MIC: 0.051 mg mLꢀ1). It is clear that the piperidin-1-yl
and 6a, 6b, respectively.
group (11a) could be displaced by a pyrrolidin-1-yl moiety
The synthesized compounds 10a–i and 11a, 11b were (11b) without obviously affecting the potency. These results
preliminarily screened for in vitro activity against MTB H37Rv indicate that 4-aminopiperidine and 3-aminopyrrolidine rings
ATCC 27294 strain, using the Microplate Alamar Blue Assay are preferred over piperazine.
(MABA).19 The minimum inhibitory concentration (MIC) is
The compounds 10a–i and 11a, 11b were examined for
dened as the lowest concentration effecting a reduction in toxicity (CC50) in human lung adenoma A549 cell lines by MTT
uorescence of >90% relative to the mean of replicate assay and the results are reported in Table 1. All of them (CC50
bacterium-only controls. The minimum inhibitory concentra- 57.66–707.33 mg mLꢀ1) are more cytotoxic than PBTZ169 (CC50
:
:
tion (MIC) values of the compounds along with PBTZ169, 784.20 mg mLꢀ1), but the selectivity index (SI: 7667–>44 208) of
isoniazid (INH) and rifampicin (RFP) for comparison are pre- compounds 10b, 10c, 10g, 11a and 11b are bigger than PBTZ169
sented in Table 1.
(SI: 6760) for MTB H37Rv ATCC 27294.
The data reveal that with a few exceptions (10d, 10f, 10i), all
Encouraged by their strong potency against the drug-
of the BTZ derivatives have potent in vitro activity against this sensitive MTB H37Rv strain, these BTZ derivatives except
strain (MIC: <0.1 mg mLꢀ1), which is better than BPTZ169 (MIC: compounds 10d, 10f and 10i were further evaluated against
0.116 mg mLꢀ1). In particular, the most active compounds 10b, two clinical isolated MDR-MTB strains 16892 (resistant to
10c and 10g (MIC: <0.016 mg mLꢀ1) were found to be >3–>7 fold both of INH and RFP) and 16802 (resistant to INH, RFP,
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Table 1 Structures and in vitro activity of 10a–i and 11a, 11b against Table 2 In vitro activity against MDR-MTB strains, solubility and
MTB H37Rv ATCC 2729a metabolic stability of selected compoundsa
MIC (mg mLꢀ1
)
MDR-MTB MDR-MTB Water Solubility Metabolic stability
Compd 16892b
16802b
(mg mLꢀ1
)
t1/2 (min)
10a
10b
10c
10e
10g
10h
11a
11b
0.016
0.016
0.016
0.016
0.016
0.031
0.016
0.016
0.031
0.016
0.016
0.016
0.016
0.016
0.016
0.016
0.016
2.5
0.24
0.03
0.05
0.02
0.05
0.03
4.05
3.11
0.30
NT
>120
NT
NT
NT
NT
b
MIC
CC50
(mg mLꢀ1
Compd
W
(mg mLꢀ1
)
)
SIc
10a
10b
0.030
91.37 ꢂ 35.18
3046
NT
49.1
24.3
19.5
NT
<0.016
707.33 ꢂ 133.77 >44 208
PBTZ169 0.016
INH
RFP
10c
10d
<0.016
0.116
157.74 ꢂ 66.57
57.66 ꢂ 11.34
>9858
497
>40
>40
20
NT
NT
a
NT: not tested. b MDR-MTB 16892 and MDR-MTB 16802 were isolated
from patients in Beijing Chest Hospital.
10e
10f
10g
0.029
0.232
<0.016
102.07 ꢂ 7.13
191.29 ꢂ 33.94
3520
825
Table 3 PK Profiles of 10a, 11a and 11b dosed orally in micea at 50 mg
kgꢀ1 (n ¼ 3)
440.69 ꢂ 102.73 >27 543
Compd
Cmax
10a
11a
11b
327 ꢂ 137
PBTZ169
1512 ꢂ 696
5767 ꢂ 3190 173 ꢂ 76.8
10h
10i
0.060
0.436
58.30 ꢂ 3.78
241.49 ꢂ 47.5
972
554
(ng mLꢀ1
Tmax (h)
AUC0–N
)
0.333 ꢂ 0.144 1.000 ꢂ 0.866 0.250 ꢂ 0
0.583 ꢂ 0.382
5681 ꢂ 1756
7678 ꢂ 4395 627 ꢂ ND
646 ꢂ 460
(h ng mLꢀ1
t1/2 (h)
MRT (h)
)
1.15 ꢂ 0.540 1.76 ꢂ ND
1.85 ꢂ 0.634 2.92 ꢂ ND
1.69 ꢂ 0.483 3.31 ꢂ 0.187
2.42 ꢂ 0.171 4.48 ꢂ 0.484
11a
11b
0.051
0.088
530.97 ꢂ 180.70 10 411
a
Male CD-1 mice was acquired from WuXi AppTec (Shanghai) CO., Ltd;
ND: not determined (parameters not determined due to inadequately
dened terminal elimination phase).
67.46 ꢂ 19.96
784.20 ꢂ 185.8
7667
6760
PBTZ169
0.116
greater solubility (0.24 mg mLꢀ1) which is only slightly smaller
than PBTZ169 (0.30 mg mLꢀ1). As expected, exocyclic tertiary
amino derivatives 11a, 11b possess excellent aqueous solubility
(3.11–4.05 mg mLꢀ1) which is more than ten times that of
BPTZ169 (Table 2). Moreover, all of 10a, 11a and 11b show
better metabolic stability (t1/2: 24.3–>120 min) in human liver
microsomes compared to BPTZ169 (t1/2: 19.5 min).
INH
RFP
0.050
0.049
a
b
MTB H37Rv ATCC 2729 was acquired from ATCC. CC50: 50%
c
cytotoxic concentration. SI: selectivity index for MTB H37Rv ATCC
27294, CC50/MIC; INH: isoniazid: RFP: rifampicin.
Based on the measured activity levels against the tested
strains, solubility and metabolic stability, 10a, 11a and 11b were
streptomycin, ethambutol and levooxacin) (Table 2). The further tested for in vivo pharmacokinetic (PK) proles in mice
results indicate that all of 10a–c, 10e, 10g, 10h and 11a, 11b aer a single oral administration of 50 mg kgꢀ1. As shown in
have excellent activity against both of the two strains with Table 3, compound 10a, with a 3-(methoxyimino) azetidine
similar MIC values (0.016–0.031 mg mLꢀ1) to PBTZ169 (MIC: moiety, displays good PK properties, with Cmax of 5767 ng mLꢀ1
,
0.016 mg mLꢀ1), suggesting their promising potential for both AUC0–N of 7678 h ng mLꢀ1 and t1/2 of 1.15 h. Compounds 11a
drug-sensitive (MIC: <0.1 mg mLꢀ1) and resistant MTB strains and 11b have a relatively longer t1/2 of 1.69–1.76 h and MRT of
(Tables 1 and 2).
2.42–2.92 h, but a poor Cmax of 173–327 ng mLꢀ1 and AUC0–N of
The BTZ derivatives were initially evaluated for their water 627–646 h ng mLꢀ1. Unexpectedly, all of the three compounds
solubility which was determined by HPLC measurement of the have a shorter t1/2 of 1.15–1.76 h compared to BPTZ169 (t1/2: 3.31
concentration of a micromembrane ltered saturated solu- h). The in vitro and in vivo half-times of inconsistency may be
tion.18 Compared with other oxime derivatives (0.02–0.05 mg due to differences in species between human liver microsomes
mLꢀ1, clog P: 3.28–4.98), compound 10a (clog P: 2.83) has much and mice.
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Conclusions
A series of novel BTZ derivatives bearing an oximido or amino
nitrogen heterocycle moiety were designed as new antituber-
cular agents through modications at the C-2 position of
BTZ043 and BPTZ169. Many of them exhibit excellent in vitro
inhibitory activity against both drug-sensitive MTB strain
H37Rv and drug-resistant clinical isolates. Compound 10a
displays acceptable safety, aqueous solubility and PK proper-
ties, and it may serve as a new and promising lead compound
for further antitubercular drug discovery. Studies to determine
the in vivo efficacy of 10a are currently underway.
Ethical statement
Animals were maintained in accordance with the guidelines of
the Chinese Association for Laboratory Animal Sciences, Bei-
jing, China, and approved by the Institutional Ethical
Committee (IEC) of Peking Union Medical College.
Acknowledgements
This work is supported by the National S&T Major Special Project
on Major New Drug Innovations (2014ZX09507009-003,
2015ZX09102007-008), NSFC (81502923, 81373267, 21502237)
and Beijing Municipal Administration of Hospitals Clinical
Medicine Development of Special Funding Support (ZYLX201304).
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