Identification of benzothiazones containing a hexahydropyrrolo[3,4-: C] pyrrol moiety as antitubercular agents against MDR-MTB

Article abstract of DOI:10.1039/d0ra00750a

IMB1603, a spiro-benzothiazone compound discovered by our lab, displayed potent anti-MTB activity in vitro and in vivo. In this study, we reported a series of new BTZs containing the hexahydropyrrolo[3,4-c]pyrrol moiety based on the structure of IMB1603. Among them, BTZs 11 and 24 displayed potent anti-MTB (MIC < 0.035 μM) and MDR-MTB (MIC, 0.053-0.102 μM) activity, good solubility (1.82-1.85 μg mL^-1), and low cytotoxicity (CC₅₀ > 200 μM), suggesting BTZs 11 and 24 may serve as promising candidates for further study. The molecular docking study of 11 toward DprE was also investigated, and revealed that 11 mimicked the binding pattern of PBTZ169 in the active site of DprE1.

Full text of DOI:10.1039/d0ra00750a

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Identification of benzothiazones containing
a hexahydropyrrolo[3,4-c]pyrrol moiety as
antitubercular agents against MDR-MTB†
Cite this: RSC Adv., 2020, 10, 14410
Xican Ma,‡ab Bing Han,‡ab Aoyu Wang,c Lu Yang,b Menghao Huang,d
d
a
c
and Kai Lvb
*
*
Kushan Chowdhury,
Jian Gu, Kai Zhang
IMB1603, a spiro-benzothiazone compound discovered by our lab, displayed potent anti-MTB activity in
vitro and in vivo. In this study, we reported a series of new BTZs containing the hexahydropyrrolo[3,4-c]
pyrrol moiety based on the structure of IMB1603. Among them, BTZs 11 and 24 displayed potent anti-
MTB (MIC < 0.035 mM) and MDR-MTB (MIC, 0.053–0.102 mM) activity, good solubility (1.82–1.85 mg
mLꢀ1), and low cytotoxicity (CC50 > 200 mM), suggesting BTZs 11 and 24 may serve as promising
candidates for further study. The molecular docking study of 11 toward DprE was also investigated, and
revealed that 11 mimicked the binding pattern of PBTZ169 in the active site of DprE1.
Received 24th January 2020
Accepted 23rd March 2020
DOI: 10.1039/d0ra00750a
rsc.li/rsc-advances
Tuberculosis (TB) is a chronic infectious disease caused mainly throughout the world for the scientists and researchers working
by Mycobacterium tuberculosis (MTB).1 The World Health Orga- in the eld of anti-TB.17–21
nization (WHO) estimated that approximately 10 million people
According to the binding pharmacophore revealed by the
were infected and 1.5 million died from TB worldwide in 2018.2 crystal structures of BTZ043/PBTZ169 complexed with DprE1,
The current therapy for TB infected patients requires a combi- the BTZ core interacted with the active site cavity, whereas the
nation of four front-line drugs for 6–9 months and does not cyclohexane or spirocyclic moiety was located at the protein
favor patient compliance. Even worse, the prevalence of surface (Fig. 1).13,16 More precisely, the CF3 group was well
multidrug-resistant (MDR) TB and extensively drug-resistant placed in a small pocket and interacted with Asn392; the nitro
(XDR) TB has exacerbated the situation.3–5 Although new group of PBTZ169 or BTZ043 was converted to nitroso which
drugs with novel mechanisms such as bedaquiline, delamanid covalently binds to Cys387 or Cys394 residue of DprE1 enzyme,
and pretomanid have been approved in recent years for the leading to irreversible inactivation of the enzyme.13,16 It
treatment of MDR-TB,6,7 some adverse events or warnings have appeared that the BTZ core was crucial for the activity, but the
been noted and limited their use in the clinic.8 Therefore, there spirocyclic or cyclohexane at the C-2 position might be open for
is still an urgent unmet medical need for safer and more structure modication.
effective agents for the treatment of TB.
Benzothiazinones (BTZs), a novel class of TB agents targeting
decaprenylphosphoryl-b-D-ribose
20-epimerase
(DprE1),9,10
exhibited exceptional inhibitory activity against MTB and MDR-
MTB strains.11–13 PBTZ169 (macozinone) and BTZ043, the most
advanced BTZ candidates, is currently in phase II14 and phase I
clinical trial15 for the treatment of both drug-susceptible TB and
MDR-TB.16 BTZ scaffold has become a research hot spot
aCollege of Pharmacy, Southwest Minzu University, Chengdu, 610041, China. E-mail:
gujiancd@163.com
bInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking
Union Medical College, Beijing, 100050, China
cDepartment of Pharmaceutical Chemistry, School of Pharmacy, Hebei Medical
University, Shijiazhuang, 050017, PR China. E-mail: zhk810728@163.com
dDepartment of Biochemistry and Molecular Biology, Indiana University School of
Medicine, Indianapolis, IN 46202, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra00750a
‡ These authors contributed equally to this work.
Fig. 1 Design of new BTZs.
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Table 2 The structure and anti-MTB activity of new BTZs series 2
Compd
12
Ar or R0
MIC (mM)
0.796
29
1.667
30
31
32
1.849
1.981
0.463
Scheme
1
Reagents and conditions: (a): (i)
X
¼
H, ArCHO,
Na(OAc)3BH, AcOH, CH2Cl2, RT, 3–4 h, 71–85%; OR (ii) X ¼ Me,
Ti(OiPr)4, acetophenones, MeOH, NaCNBH3, 40 ꢁC, 5–7 h, 30–55%;
(b) TFA, DCM, rt, 3–4 h; (c) (i) Et3N, MeOH, 40 ꢁC, 1–1.5 h, 22–41%, two
steps; (ii) TFA, DCM, 70% (Boc deprotection in TFA was only needed for
compound 36).
33
34
0.899
Based on the binding characteristics and reported structure–
activity relationship (SAR), our group focused on the discovery
of alternative moieties at the C-2 position of BTZ core.22–25
Recently, we identied IMB1603 with a spiro-heterocyclic
segment as a potent anti-TB lead by combining the structure
feature of PBTZ169 and BTZ043.23 In this study, replacement of
the spiro-heterocyclic group of IMB1603 with hexahydropyrrolo
[3,4-c]pyrrol gave a new series of BTZs (Fig. 1). The anti-TB
activity, solubility, and toxicity of these new BTZs were evalu-
ated, aiming to identify alternative groups at position 2 of BTZs
27.350
35
36
PBTZ169
Boc
H
1.720
>40
<0.035
and nd optimized potent anti-TB drug candidates with
improved drug-like properties through SAR study.
The synthesis of target compounds 1–43 was shown in
Scheme 1. Reductive amination of hexahydropyrrolo[3,4-c]pyr-
rol A in the presence of aryl aldehydes or acetophenones gave
Table 1 The structure and anti-MTB activity of new BTZs series 1
Table 3 The structure and anti-MTB activity of new BTZs series 3
Compd
R
MIC (mM)
Compd
R
MIC (mM)
1
2
3
4
5
6
7
8
0.462
0.244
1.654
0.953
0.616
0.451
0.112
1.832
0.218
1.926
<0.035
0.796
0.222
0.217
0.203
0.223
17
18
19
20
21
22
23
24
25
26
27
28
PBTZ169
INH
RFP
m-NO2
m-CF3
m-MeO
o-F
p-F, m-F
p-Cl, m-Cl
p-F, m-Cl
p-Cl, m-F
p-F, o-Cl
p-F, o-F
p-Cl, o-Cl
p-F, o-Br
0.414
0.101
3.671
3.595
0.111
0.077
0.104
<0.035
0.888
0.232
0.566
0.783
<0.035
0.262
0.166
p-F
p-Cl
p-Br
p-CN
p-NO2
p-CF3
p-MeO
p-tBu
p-Me
p-CF3O
H
Compd
R00
MIC (mM)
9
37
38
39
40
41
42
43
PBTZ169
p-CF3
p-OCF3
0.202
0.209
0.110
0.219
0.108
0.205
0.189
<0.035
10
11
12
13
14
15
16
p-F, m-F
p-Cl, m-F
p-F, m-Cl
m-F, m-F
p-F, m-F, m-F
m-F
m-Cl
m-Br
m-CN
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Table 4 Anti-MDR-MTB activity and cytotoxicity of selected compounds
MIC (mM)
MDR-MTB1a
MDR-MTB2a
CC50 (mM)
Water solubilityc (mg mLꢀ1
)
b
Compd
7
11
18
21
22
23
24
39
0.080
0.085
0.209
0.119
0.110
0.115
0.102
0.121
0.188
<0.035
>40
0.053
0.053
0.112
0.058
0.056
0.058
0.073
0.087
0.097
<0.035
>40
166.37
>200
55.77
38.63
27.31
40.79
>200
54.98
50.68
>200
ND
2.30
1.85
2.29
3.50
1.94
2.01
1.82
2.97
2.55
0.90d
NT
41
PBTZ169
INH
RFP
>40
>40
ND
NT
a
MDR-MTB1 (MDR-MTB 16833) and MDR-MTB2 (MDR-MTB 16995) were obtained from the State Laboratory of Tuberculosis Reference of China.
The 50% cytotoxic concentration. The water solubility was tested in 0.01 M HCl solution (approximate pH 2.0). This data was from ref. 24.
b
c
d
compound B. Thereaer, deprotection of the Boc group in tri- and b-naphthalene 33 showed decreased potency, whereas
uoroacetic (TFA) gave the intermediate C. Coupling of BTZ the a-naphthalene 32 displayed a slightly increased anti-TB
core D with A or C in the presence of Et3N (TEA) afforded the activity. The indole moiety was also not favored, leading to
target compounds 1–35 and 37–43. Removing the Boc group of a dramatically decreased anti-TB activity (34, MIC ¼ 27.35
35 in TFA gave the target compound 36.
mM). In addition, BTZ 35 with Boc-group was synthesized and
The target compounds 1–43 were initially screened for in proved to display moderate anti-TB potency (MIC < 2 mM).
vitro activity against MTB H37Rv ATCC 27294 strain using the Removal of the R0 group, as in compound 36, led to a total loss
Microplate Alamar Blue Assay (MABA).26 The minimum inhibi- of anti-TB potency (MIC > 40 mM), indicating the existence of
tory concentration (MIC) was dened as the lowest concentra- N–H bond might not be tolerated at the C2-position of the
tion effecting a reduction in uorescence of >90% relative to the BTZ core.
mean of replicate bacterium-only controls. The MIC values of
the compounds along with isoniazid (INH), rifampicin (RFP) linker were investigated in this set. The R00 group of 37–41 were
and PBTZ169 were presented in Tables 1–3 for comparison. selected from the BTZs with potent anti-TB activity in Table 1
Finally, as shown in Table 3, BTZs with a methyl group at the
As shown in Table 1, BTZs 1 and 2 with cyclohexyl and p- (MIC < 0.15 mM). Compared to the corresponding BTZs in Table
uorobenzyl group from PBTZ169 and IMB1603 were initially 1, the introduction of a methyl group resulted in a decreased
synthesized and evaluated. The MIC value of BTZ 2 was lower anti-TB potency (37–41 vs. 7, 11, 21, 23–24). In addition, BTZs 42
than that of 1, and comparable to that of INH. According to our with double m-F and 43 with p-F, double m-F, were designed and
previous SAR ndings of spiro-BTZs, the substituents at the synthesized according above SAR ndings. However, the anti-
phenyl ring were crucial for anti-TB activity.23 In parallel with TB potency of BTZs 42–43 was also not as good as we ex-
the spiro-BTZ series, we synthesized compounds 3–28 with pected, both of them displayed lower anti-TB activity than 39.
diversity R substituents at the phenyl ring. To our delight,
On the bases of above studies, BTZs 7, 11, 18, 21–24, 39, and
compounds 7, 11, 18 and 21–24 displayed increased potency 41 with potent anti-TB activity (MIC < 0.15 mM) were evaluated
(MIC < 0.15 mM). Especially, compound 11 with p-CF3O and 24 against two clinical isolated MDR-MTB (16833 and 16995)
with p-Cl, m-F were found to exhibit comparable anti-TB activity strains resistant to both INH and RFP.§ As shown in Table 4,
to PBTZ169 (MIC < 0.035 mM). It is interesting that moving the although the MIC values of these new BTZs were all lower than
para substituent to meta position led to an improved anti-TB that of PBTZ169 (MIC < 0.035 mM), most of them displayed
activity (2–7 vs. 13–19), while transferring to the ortho position considerable anti-MDR-TB activity (MIC < 0.15 mM).
seemed to result in a decreased potency (2 vs. 20). Notably, BTZs
These compounds were then tested for mammalian cell
with double substituents at the para and meta position of the cytotoxicity using Vero cells{ measured as a concentration
benzyl moiety showed better anti-TB activity (MIC < 0.15 mM)
than the corresponding mono-substituted BTZs (21–24 vs. 2–3
and 13–14), whereas introducing double substituents to the
para and ortho position of benzyls were not well tolerated (25–
28, MIC > 0.2 mM).
As a continuing SAR study, BTZs 29–34 with other aromatic
cyclic groups as replacement of the benzyl moiety were
explored. Compared to BTZ 12, the pyridyl analogues 29–31
§ The M. tuberculosis strains used in these studies included the laboratory strain
H37Rv (ATCC 27294; American Type Culture Collection, Rockville, MD) and
drug-resistant clinical isolates. All isolates were obtained from the State
Laboratory of Tuberculosis Reference of China.
{ African green monkey kidney (Vero) cells were purchased from the American
Type Culture Collection (ATCC), and were cultured in Minimum Essential
Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (GBICO) and
antibiotics (100 U per mL penicillin and 100 mg per mL streptomycin) at 37 ꢁC
in a 5% CO2 incubator.
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Conclusions
In summary, a series of new BTZs containing hexahydropyrrolo
[3,4-c]pyrrol moiety were designed and synthesized based on the
spiro-BTZ IMB1603 discovered in our lab. Many of them
exhibited potent in vitro anti-TB activity. Especially, compounds
11 and 24 were found to display excellent anti-MTB activity
against the drug-sensitive MTB strain H37Rv (MIC < 0.035 mM),
and also potent anti-MDR-MTB activity against the two drug-
resistant clinical isolates (MIC, 0.053–0.102 mM). In addition,
BTZs 11 and 24 showed low cytotoxicity (CC50 > 200 mM), and
exhibited better water solubility than PBTZ169, suggesting both
of them may serve as new and promising candidates for further
antitubercular drug discovery. The molecular docking results
suggested that 11 mimicked the binding pattern of PBTZ169 in
the active site of DprE1. Studies to determine the PK proles
and in vivo efficacy of 11 and 24 are currently under way.
Conflicts of interest
There are no conicts to declare.
Fig. 2 Overlay of the docking hydroxylamine intermediate of 11
(carbons are green) on the crystallized semimercaptal adduct (carbons Acknowledgements
are off white). (A) The overall view of the binding pattern; (B) close-up
view of the DprE1 active site.
This work is supported by the National Mega-project for Inno-
vative Drugs (2018ZX09721001-004-007; 2018ZX09711001-007-
002), National Natural Science Foundation of China (81872753).
inhibiting 50% growth (CC50) as compared to a no-treatment
control and the results were reported in Table 4. Most of the
tested targets displayed higher cytotoxicity than that of
PBTZ169. However, to our delight, compounds 11 and 24
showed low cytotoxicity (CC50 > 200 mM). Subsequently,
considering acidic gastrointestinal environments, these new
BTZs were evaluated for their water solubility at pH 2 (0.01 M
HCl solution) by using an HPLC-UV method.23 All of them dis-
played good solubility (1.82–3.50 mg mLꢀ1), and were more
water-soluble than PBTZ169 (0.90 mg mLꢀ1).
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