Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitubercular evaluation of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives Cui-Ting Peng a, , Chao Gao b, , Ning-Yu Wang b, Xin-Yu You a,b, Li-Dan Zhang a,b, Yong-Xia Zhu b, Ying Xv b, Wei-Qiong Zuo b, Kai Ran b, Hong-Xia Deng b, Qian Lei b, Kun-Jie Xiao b, Luo-Ting Yu a,b,⇑ a b
Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, China State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 1 December 2014 Revised 21 January 2015 Accepted 24 February 2015 Available online xxxx Keywords: Tuberculosis Benzothiazinones Structure–activity relationships (SAR) Aqueous solubility
a b s t r a c t Tuberculosis (TB) remains a major human health problem. New therapeutic antitubercular agents are urgent needed to control the global tuberculosis pandemic. We synthesized a new series of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives and evaluated their anti-mycobacterial activities against Mycobacterium tuberculosis H37Ra as well as their druggabilities. The results showed that most of these derivatives, especially the compounds with simple alkyl side chains, exhibited good antitubercular activities and favorable aqueous solubilities with no obvious cytotoxicity. It suggested that the 4-carbonyl piperazine substituents in benzothiazinone scaffold were well tolerated, in which the compound 8h, with an antitubercular activity of MIC 0.008 lM, exhibited an excellent aqueous solubility of 104 lg/mL, which was 100-fold better than the potent DprE1 inhibitor Comp.1 (BTZ038), also more soluble than PBTZ169. Ó 2015 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) remains a major human health problem. Despite the availability of current effective four-drug combination regimen and other second-line anti-TB drugs,1 there were still about 1.3 million people died of the disease in 2012.2 In the past 5–6 years, there have been some new therapeutic antitubercular agents in preclinical or clinical trials after several decades of near inactivity to fill the drug pipeline.3–7 However, the drug pipeline for the early clinical phase remains inadequate3 and challenges still exists for novel drugs’ widescale use due to their safety risks or unsatisfactory pharmacokinetics.8 Therefore, there is still a great need for more advanced candidates with novel mechanisms, extraordinary potency, favourable pharmacokinetics and good safety profiles. Decaprenylphosphoryl-b-D-ribose20 -epimerase (DprE1), a critical enzyme involved in the biosynthesis of mycobacterial cell wall, is a flavoprotein that in concert with decaprenylphosphoryl-2-keto-ribose reductase (DprE2) to catalyze the epimerization of decaprenylphosphoryl-b-D-ribose (DPR) to decaprenylphosphorylb-D-arabinofuranose (DPA), the sole precursor for the synthesis of cell wall arabinans.9 These years, DprE1 is emerging as a potential target for developing potent and safer antitubercular agents,10
⇑ Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail address: [email protected] (L.-T. Yu). These authors contributed equally to this work.
such as Nitrobenzothiazinones (BTZ043),9 Dinitrobenzamides,11 Benzoquinoxalines compound VI-937612 and Nitro-triazole inhibitor 377790 (Fig. 1).13 Benzothiazinone compound BTZ043, with minimum inhibitory concentration (MIC) against Mycobacterium tuberculosis of 2.3 nM, had exceptional cellular activity and potent activities against drug-susceptible, MDR and XDR clinical isolates of M. Tuberculosis.10 However, the efficacy of BTZ043 in the mouse model of TB was relatively lower because of its poor aqueous solubility and low bioavailability.14 Recently, some novel DprE1 inhibitors have been developed,14–18 such as PBTZ16914, TCA115 and Azaindoles (Fig. 1),16 which also showed good effectiveness in the in vivo animal models of TB. In our previous study,19 the benzothiazinone compound with a 4-carbonyl substituted piperazine group exhibited good potency against M. tuberculosis. In this Letter, considering that 4-carbonyl substituted piperazine group might facilitate the solubility profile of the molecule, a new series of 4-carbonyl piperazine substituted benzothiazinone derivatives were designed and synthesized. The anti-mycobacterial activity, solubility and cytotoxicity of these compounds were evaluated, aiming to find optimized potent antitubercular inhibitors with improved druggability through the structure–activity study. As outlined in Scheme 1, the designed compounds were synthesized in five steps with yields of 40–65%. The compound 4 was prepared from the start material 2-chloro-3-nitro-5-(trifluoromethyl)
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Please cite this article in press as: Peng, C.-T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.02.061
2
C.-T. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
NO2
O O2N
N H
O2 N
N
O Br
O
N
N
NO2
377790
VI-9376
DNB1
N N
O
O N H
S
N
O O
N
O
O
N
NH N
F3 C
S
O
N H
Azaindole
TCA1
N
F
N
S
N
NO2
N
PBTZ169
O F3 C
N S
N
O
NO2 O
*
(RS) BTZ038 (1) (S) BTZ043 Figure 1. Known DprE1 inhibitors.
O F3 C
OH
F3 C
(a)
Cl
Cl
F3 C
NCS
(b) Cl
Cl
NO2
NO2
NO2
2 F3 C
F3 C N
S
(d) NO2
NH
5
N
(e) or (f) N
4
O
3
O (c)
O
O
S
N
NO2
N
6a-6o 7a-7g 8a-8h
R O
Scheme 1. Reagents and conditions: (a) (COCl)2, CH2Cl2, DMF (cat), room temp (rt), 2 h; (b) NH4NCS, CH2Cl2, PEG-400 (cat), rt, 2–4 h; (c) 1-Boc-piperazine, rt, 30 min; (d) TFA, 0 °C to rt, 3 h; (e) R-COCl, 0 °C, 10 min to 2 h; (f) R-COOH, HATU, DIPEA, rt, overnight.
benzoic acid (commercially purchased) in two steps as our previously described, and PEG-400 acted as the phase-transfer catalyst.19 Next, through the nucleophilic reaction and cyclization between 1-Boc-piperazine and the isothiocyanato group of compound 4, then deprotection of Boc in trifluoroacetic acid yielded the intermediate compound 5. To get our target molecules, compound 5 was condensed with the corresponding carboxylic acids or acyl chlorides, respectively. Compound 5 reacted with carboxylic acids catalyzed by the combination of HATU and DIPEA at room temperature, or directly condensed with acyl chlorides for 10 min to 2 h to obtain the target molecules (Table 1). The structures of these new compounds were determined by 1H NMR, 13C NMR and MS (electrospray ionization (ESI)). And details of the general synthetic route of Comp.1 and PBTZ169 can be found in previous studys14,19 and the Supplementary information. The physico-chemical and biological data for these new benzothiazinones were provided in Table 1, in which compounds
bearing different kinds of substituent groups to ensure side chain flexibility and structure diversity were described. The MIC against M. tuberculosis H37Ra was conducted to evaluate their biological activity in vitro. Our previous study19 showed that the volume and lipophilicity of the substituents were important in maintaining activity. So we first explored the effect of various substituted phenyl rings. In general, different para-substituents (including electron-withdrawing and donating substituents) in phenyl rings did not significantly impact the anti-mycobacterial activities. These compounds showed moderate MIC against H37Ra (ranging from 0.016 to 0.13 lM). Yet, most of them displayed slightly improved solubilities, among which the compound 6c, with a para-trifluoromethyl substituent, showed the better solubility of 7.53 lg/mL. Meanwhile, the different anti-mycobacterial activities between 6e, 6f and 6g revealed that the ortho-and meta-substituted phenyl derivatives displayed higher potency than the para-substituted compounds, which was further validated by comparison of 6k, 6l with 6m. The compound 6k showed the MIC of 0.008 lM, 2 and 4-fold more potent than that of 6l (0.016 lM) and 6m (0.031 lM). More importantly, all ortho-substituted compounds (6b, 6e, 6k and 6n) exhibited better aqueous solubility than their meta- and para-substituted analogues, which may partly result from the decreasing of molecular planarity induced by the orthosubstituents on the benzene ring. And the formation of intermolecular hydrogen bond may also contributed to the increased solubility of 6e, 6k and 6n. The replacement of phenyl group by benzyl led to 10-fold improvement in aqueous solubility without loss of potency (6d vs 6g), which could be explained by the increasing of molecular flexibility. Then, we replaced the phenyl ring by different cycloalkyl groups varied from cyclopropyl to adamantyl (7a–7g). From comparison of the ring size, the cyclopentyl turned out to be the optimum substituents since compound 7a and 7b showed the MIC of 0.13 and 0.031 lM, respectively, while 7c exhibited improved potency of 0.016 lM. And the corresponding C log P values increased from
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C.-T. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 1 SAR evaluation and cytotoxicity of the benzothiazinone derivatives
O F3 C
N S NO2
N N
R O
a b c
Compd
R
MIC (lM)a H37Ra
C log Pb
Solubility (lg/mL)c
Cytotoxicity (lM) Vero
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 7a 7b 7c 7d 7e 7f 7g 8a 8b 8c 8d 8e 8f 8g 8h Comp.1 PBTZ169
4-Nitrophenyl 2-Nitrophenyl 4-(Trifluoromethyl)phenyl 4-Methoxybenzyl 2-Methoxyphenyl 3-Methoxyphenyl 4-Methoxyphenyl 4-Methylphenyl 4-Ethylphenyl 4-tert-Butylphenyl 2-Fluorophenyl 3-Fluorophenyl 4-Fluorophenyl 2-Chlorophenyl 4-Chlorophenyl Cyclopropyl Cyclobutyl Cyclopentyl Cyclohexyl Cyclohexylmethyl 4-(tert-Butyl)cyclohexyl Adamantyl Methyl Ethyl Propyl Butyl (Methylthio) methyl tert-Butyl 2-Methylpropyl 1-Methylpropyl
0.063 0.13 0.031 0.13 0.063 0.031 0.13 0.031 0.031 0.016 0.008 0.016 0.031 0.016 0.016 0.13 0.031 0.016 0.031 0.016 0.004 0.031 >0.13 0.016 0.13 0.031 0.13 0.031 0.031 0.008 0.004 125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 120.1 88.7 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
Minimum inhibitory concentration against M. tuberculosis H37Ra (ATCC 25177). Calculated with ChemDraw Ultra 11.0. Aqueous solubility at room temperature.
2.18 (7a) to 3.62 (7d). Conversely, It provided a much larger decrease in aqueous solubilities (7a afforded the best solubility of 52.86 lg/mL, whereas 7c and 7d reduced to 5.34 and 3.24 lg/ mL). Meanwhile, introduction of cyclohexylmethyl (7e) resulted in slightly improved potency while a much lower solubility of 0.12 lg/mL and the higher C log P of 4.24, compared with compound 7d. Similarly, compound 7f, with a tert-butyl introduced in the 4-position of cyclohexyl, exhibited enhanced activity with a MIC of 0.004 lM, equivalent to that of Comp.1 but a reduced solubility and increased C log P value (5.47). In these cases, we can see the apparently negative correlation between the C log P and aqueous solubility. In order to get some more simplified compounds with relatively hydrophilic profile simultaneously, we introduced some simple alkyl chains attached to the scaffold. Firstly, although the methyl substituted compound 8a showed a decreased antitubercular activity, it was rather hydrophilic (C log P = 1.59) with a solubility of 83.86 lg/mL, which guided us to conduct in-depth SAR study. Encouragingly, with alkyl chain extended and branched, most of the analogues displayed increased anti-mycobacterial activities with favorable aqueous solubilities. The compound 8b and 8c were more potent than 8a although they exhibited lower aqueous solubility, while the compound 8d displayed a relatively increased solubility of 42.5 lg/mL compared with 8b and 8c. When
introduced a sulfur atom into the alkyl chain (compound 8e), the aqueous solubility was also improved without effect on the biological activity. Also, the branched alkyl groups were well tolerated for the antitubercular activity as it can be concluded from compound 8f, 8g and 8h. Generally, introduction of steric hindrance to reduce freedom to rotate is often important to improve activity, thus resulted in the different activity and solubility of 8g and 8h. Compound 8h, with a less flexible side chain attached when methyl substituted in the first carbon atom of the propyl, exhibited a increased activity (MIC = 0.008 lM) compared with 8g. Notably, compound 8h showed an excellent aqueous solubility of 104 lg/ mL, which was 100-fold more soluble than Comp.1 (0.94 lg/mL) and also was more soluble than PBTZ169 (9.07 lg/mL). To evaluate the safety profile, all these compounds were screened the cytotoxicity against Vero cells by MTT assay in an extensive concentration range (Table 1). Most of them showed an IC50 >125 lM, indicating their nontoxicity toward the normal cells and the good safety profile in vitro. In conclusion, some benzothiazinone compounds have potential antitubercular activities which target the attractive cellular enzyme DprE1, but their aqueous solubilities are almost poor, which would likely limit the oral bioavailabilities at higher doses in clinical trials. In order to improve the solubility and other druggabilities of the benzothiazinone compounds with retained antitubercular activity,
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C.-T. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
a series of 4-carbonyl piperazine substituted 1, 3-benzothiazin-4one derivatives were synthesized in five steps with good yields. In general, these new compounds exhibited good anti-microbial activity against Mtb H37Ra and did not show obvious cytotoxicity toward the Vero cell. In our investigation, the various phenyl substituted compounds showed similarly good antitubercular activities and slightly improved solubilities. The cycloalkyl compounds showed better activities and the size of the groups played a critical role on the biological activity and solubility, among which compound 7f, with a 4-tert-butylcyclohexyl attached, exhibited a MIC of 0.004 lM (equivalent to that of Comp.1), but its aqueous solubility was still poor. The simplified alkyl chain substituted compounds, remarkably, displayed markedly increased aqueous solubilities and maintained potent antitubercular activities, in which compound 8h exhibited excellent activity with MIC of 0.008 lM and good solubility of 104 lg/mL, 100-fold more soluble than that of Comp.1 and 10-fold than that of PBTZ169. The results suggested that the introduction of a 4-carbonyl piperazine were well tolerated and contributed greatly to the enhanced solubilities, which paved the way to find more optimized compounds with improved druggability targeting the cellular enzyme DprE1. Acknowledgments This work was supported by The National Science and Technology Major Project of China (2012ZX09103101-021). We thank Shanghai Chempartner for in vitro antitubercular screening and Sichuan Long March Pharmaceutical Co., Ltd for their technical and financial support. We also thank Lei Li of State Key Laboratory of Biotherapy (Sichuan University) for NMR measurements.
6. 7.
8. 9.
10.
11.
12.
13.
14.
15.
16.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.02. 061.
17.
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Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitubercular evaluation of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives Cui-Ting Peng a, , Chao Gao b, , Ning-Yu Wang b, Xin-Yu You a,b, Li-Dan Zhang a,b, Yong-Xia Zhu b, Ying Xv b, Wei-Qiong Zuo b, Kai Ran b, Hong-Xia Deng b, Qian Lei b, Kun-Jie Xiao b, Luo-Ting Yu a,b,⇑ a b
Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, China State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 1 December 2014 Revised 21 January 2015 Accepted 24 February 2015 Available online xxxx Keywords: Tuberculosis Benzothiazinones Structure–activity relationships (SAR) Aqueous solubility
a b s t r a c t Tuberculosis (TB) remains a major human health problem. New therapeutic antitubercular agents are urgent needed to control the global tuberculosis pandemic. We synthesized a new series of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives and evaluated their anti-mycobacterial activities against Mycobacterium tuberculosis H37Ra as well as their druggabilities. The results showed that most of these derivatives, especially the compounds with simple alkyl side chains, exhibited good antitubercular activities and favorable aqueous solubilities with no obvious cytotoxicity. It suggested that the 4-carbonyl piperazine substituents in benzothiazinone scaffold were well tolerated, in which the compound 8h, with an antitubercular activity of MIC 0.008 lM, exhibited an excellent aqueous solubility of 104 lg/mL, which was 100-fold better than the potent DprE1 inhibitor Comp.1 (BTZ038), also more soluble than PBTZ169. Ó 2015 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) remains a major human health problem. Despite the availability of current effective four-drug combination regimen and other second-line anti-TB drugs,1 there were still about 1.3 million people died of the disease in 2012.2 In the past 5–6 years, there have been some new therapeutic antitubercular agents in preclinical or clinical trials after several decades of near inactivity to fill the drug pipeline.3–7 However, the drug pipeline for the early clinical phase remains inadequate3 and challenges still exists for novel drugs’ widescale use due to their safety risks or unsatisfactory pharmacokinetics.8 Therefore, there is still a great need for more advanced candidates with novel mechanisms, extraordinary potency, favourable pharmacokinetics and good safety profiles. Decaprenylphosphoryl-b-D-ribose20 -epimerase (DprE1), a critical enzyme involved in the biosynthesis of mycobacterial cell wall, is a flavoprotein that in concert with decaprenylphosphoryl-2-keto-ribose reductase (DprE2) to catalyze the epimerization of decaprenylphosphoryl-b-D-ribose (DPR) to decaprenylphosphorylb-D-arabinofuranose (DPA), the sole precursor for the synthesis of cell wall arabinans.9 These years, DprE1 is emerging as a potential target for developing potent and safer antitubercular agents,10
⇑ Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail address: [email protected] (L.-T. Yu). These authors contributed equally to this work.
such as Nitrobenzothiazinones (BTZ043),9 Dinitrobenzamides,11 Benzoquinoxalines compound VI-937612 and Nitro-triazole inhibitor 377790 (Fig. 1).13 Benzothiazinone compound BTZ043, with minimum inhibitory concentration (MIC) against Mycobacterium tuberculosis of 2.3 nM, had exceptional cellular activity and potent activities against drug-susceptible, MDR and XDR clinical isolates of M. Tuberculosis.10 However, the efficacy of BTZ043 in the mouse model of TB was relatively lower because of its poor aqueous solubility and low bioavailability.14 Recently, some novel DprE1 inhibitors have been developed,14–18 such as PBTZ16914, TCA115 and Azaindoles (Fig. 1),16 which also showed good effectiveness in the in vivo animal models of TB. In our previous study,19 the benzothiazinone compound with a 4-carbonyl substituted piperazine group exhibited good potency against M. tuberculosis. In this Letter, considering that 4-carbonyl substituted piperazine group might facilitate the solubility profile of the molecule, a new series of 4-carbonyl piperazine substituted benzothiazinone derivatives were designed and synthesized. The anti-mycobacterial activity, solubility and cytotoxicity of these compounds were evaluated, aiming to find optimized potent antitubercular inhibitors with improved druggability through the structure–activity study. As outlined in Scheme 1, the designed compounds were synthesized in five steps with yields of 40–65%. The compound 4 was prepared from the start material 2-chloro-3-nitro-5-(trifluoromethyl)
http://dx.doi.org/10.1016/j.bmcl.2015.02.061 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
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2
C.-T. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
NO2
O O2N
N H
O2 N
N
O Br
O
N
N
NO2
377790
VI-9376
DNB1
N N
O
O N H
S
N
O O
N
O
O
N
NH N
F3 C
S
O
N H
Azaindole
TCA1
N
F
N
S
N
NO2
N
PBTZ169
O F3 C
N S
N
O
NO2 O
*
(RS) BTZ038 (1) (S) BTZ043 Figure 1. Known DprE1 inhibitors.
O F3 C
OH
F3 C
(a)
Cl
Cl
F3 C
NCS
(b) Cl
Cl
NO2
NO2
NO2
2 F3 C
F3 C N
S
(d) NO2
NH
5
N
(e) or (f) N
4
O
3
O (c)
O
O
S
N
NO2
N
6a-6o 7a-7g 8a-8h
R O
Scheme 1. Reagents and conditions: (a) (COCl)2, CH2Cl2, DMF (cat), room temp (rt), 2 h; (b) NH4NCS, CH2Cl2, PEG-400 (cat), rt, 2–4 h; (c) 1-Boc-piperazine, rt, 30 min; (d) TFA, 0 °C to rt, 3 h; (e) R-COCl, 0 °C, 10 min to 2 h; (f) R-COOH, HATU, DIPEA, rt, overnight.
benzoic acid (commercially purchased) in two steps as our previously described, and PEG-400 acted as the phase-transfer catalyst.19 Next, through the nucleophilic reaction and cyclization between 1-Boc-piperazine and the isothiocyanato group of compound 4, then deprotection of Boc in trifluoroacetic acid yielded the intermediate compound 5. To get our target molecules, compound 5 was condensed with the corresponding carboxylic acids or acyl chlorides, respectively. Compound 5 reacted with carboxylic acids catalyzed by the combination of HATU and DIPEA at room temperature, or directly condensed with acyl chlorides for 10 min to 2 h to obtain the target molecules (Table 1). The structures of these new compounds were determined by 1H NMR, 13C NMR and MS (electrospray ionization (ESI)). And details of the general synthetic route of Comp.1 and PBTZ169 can be found in previous studys14,19 and the Supplementary information. The physico-chemical and biological data for these new benzothiazinones were provided in Table 1, in which compounds
bearing different kinds of substituent groups to ensure side chain flexibility and structure diversity were described. The MIC against M. tuberculosis H37Ra was conducted to evaluate their biological activity in vitro. Our previous study19 showed that the volume and lipophilicity of the substituents were important in maintaining activity. So we first explored the effect of various substituted phenyl rings. In general, different para-substituents (including electron-withdrawing and donating substituents) in phenyl rings did not significantly impact the anti-mycobacterial activities. These compounds showed moderate MIC against H37Ra (ranging from 0.016 to 0.13 lM). Yet, most of them displayed slightly improved solubilities, among which the compound 6c, with a para-trifluoromethyl substituent, showed the better solubility of 7.53 lg/mL. Meanwhile, the different anti-mycobacterial activities between 6e, 6f and 6g revealed that the ortho-and meta-substituted phenyl derivatives displayed higher potency than the para-substituted compounds, which was further validated by comparison of 6k, 6l with 6m. The compound 6k showed the MIC of 0.008 lM, 2 and 4-fold more potent than that of 6l (0.016 lM) and 6m (0.031 lM). More importantly, all ortho-substituted compounds (6b, 6e, 6k and 6n) exhibited better aqueous solubility than their meta- and para-substituted analogues, which may partly result from the decreasing of molecular planarity induced by the orthosubstituents on the benzene ring. And the formation of intermolecular hydrogen bond may also contributed to the increased solubility of 6e, 6k and 6n. The replacement of phenyl group by benzyl led to 10-fold improvement in aqueous solubility without loss of potency (6d vs 6g), which could be explained by the increasing of molecular flexibility. Then, we replaced the phenyl ring by different cycloalkyl groups varied from cyclopropyl to adamantyl (7a–7g). From comparison of the ring size, the cyclopentyl turned out to be the optimum substituents since compound 7a and 7b showed the MIC of 0.13 and 0.031 lM, respectively, while 7c exhibited improved potency of 0.016 lM. And the corresponding C log P values increased from
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C.-T. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 1 SAR evaluation and cytotoxicity of the benzothiazinone derivatives
O F3 C
N S NO2
N N
R O
a b c
Compd
R
MIC (lM)a H37Ra
C log Pb
Solubility (lg/mL)c
Cytotoxicity (lM) Vero
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 7a 7b 7c 7d 7e 7f 7g 8a 8b 8c 8d 8e 8f 8g 8h Comp.1 PBTZ169
4-Nitrophenyl 2-Nitrophenyl 4-(Trifluoromethyl)phenyl 4-Methoxybenzyl 2-Methoxyphenyl 3-Methoxyphenyl 4-Methoxyphenyl 4-Methylphenyl 4-Ethylphenyl 4-tert-Butylphenyl 2-Fluorophenyl 3-Fluorophenyl 4-Fluorophenyl 2-Chlorophenyl 4-Chlorophenyl Cyclopropyl Cyclobutyl Cyclopentyl Cyclohexyl Cyclohexylmethyl 4-(tert-Butyl)cyclohexyl Adamantyl Methyl Ethyl Propyl Butyl (Methylthio) methyl tert-Butyl 2-Methylpropyl 1-Methylpropyl
0.063 0.13 0.031 0.13 0.063 0.031 0.13 0.031 0.031 0.016 0.008 0.016 0.031 0.016 0.016 0.13 0.031 0.016 0.031 0.016 0.004 0.031 >0.13 0.016 0.13 0.031 0.13 0.031 0.031 0.008 0.004 125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 120.1 88.7 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
Minimum inhibitory concentration against M. tuberculosis H37Ra (ATCC 25177). Calculated with ChemDraw Ultra 11.0. Aqueous solubility at room temperature.
2.18 (7a) to 3.62 (7d). Conversely, It provided a much larger decrease in aqueous solubilities (7a afforded the best solubility of 52.86 lg/mL, whereas 7c and 7d reduced to 5.34 and 3.24 lg/ mL). Meanwhile, introduction of cyclohexylmethyl (7e) resulted in slightly improved potency while a much lower solubility of 0.12 lg/mL and the higher C log P of 4.24, compared with compound 7d. Similarly, compound 7f, with a tert-butyl introduced in the 4-position of cyclohexyl, exhibited enhanced activity with a MIC of 0.004 lM, equivalent to that of Comp.1 but a reduced solubility and increased C log P value (5.47). In these cases, we can see the apparently negative correlation between the C log P and aqueous solubility. In order to get some more simplified compounds with relatively hydrophilic profile simultaneously, we introduced some simple alkyl chains attached to the scaffold. Firstly, although the methyl substituted compound 8a showed a decreased antitubercular activity, it was rather hydrophilic (C log P = 1.59) with a solubility of 83.86 lg/mL, which guided us to conduct in-depth SAR study. Encouragingly, with alkyl chain extended and branched, most of the analogues displayed increased anti-mycobacterial activities with favorable aqueous solubilities. The compound 8b and 8c were more potent than 8a although they exhibited lower aqueous solubility, while the compound 8d displayed a relatively increased solubility of 42.5 lg/mL compared with 8b and 8c. When
introduced a sulfur atom into the alkyl chain (compound 8e), the aqueous solubility was also improved without effect on the biological activity. Also, the branched alkyl groups were well tolerated for the antitubercular activity as it can be concluded from compound 8f, 8g and 8h. Generally, introduction of steric hindrance to reduce freedom to rotate is often important to improve activity, thus resulted in the different activity and solubility of 8g and 8h. Compound 8h, with a less flexible side chain attached when methyl substituted in the first carbon atom of the propyl, exhibited a increased activity (MIC = 0.008 lM) compared with 8g. Notably, compound 8h showed an excellent aqueous solubility of 104 lg/ mL, which was 100-fold more soluble than Comp.1 (0.94 lg/mL) and also was more soluble than PBTZ169 (9.07 lg/mL). To evaluate the safety profile, all these compounds were screened the cytotoxicity against Vero cells by MTT assay in an extensive concentration range (Table 1). Most of them showed an IC50 >125 lM, indicating their nontoxicity toward the normal cells and the good safety profile in vitro. In conclusion, some benzothiazinone compounds have potential antitubercular activities which target the attractive cellular enzyme DprE1, but their aqueous solubilities are almost poor, which would likely limit the oral bioavailabilities at higher doses in clinical trials. In order to improve the solubility and other druggabilities of the benzothiazinone compounds with retained antitubercular activity,
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C.-T. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
a series of 4-carbonyl piperazine substituted 1, 3-benzothiazin-4one derivatives were synthesized in five steps with good yields. In general, these new compounds exhibited good anti-microbial activity against Mtb H37Ra and did not show obvious cytotoxicity toward the Vero cell. In our investigation, the various phenyl substituted compounds showed similarly good antitubercular activities and slightly improved solubilities. The cycloalkyl compounds showed better activities and the size of the groups played a critical role on the biological activity and solubility, among which compound 7f, with a 4-tert-butylcyclohexyl attached, exhibited a MIC of 0.004 lM (equivalent to that of Comp.1), but its aqueous solubility was still poor. The simplified alkyl chain substituted compounds, remarkably, displayed markedly increased aqueous solubilities and maintained potent antitubercular activities, in which compound 8h exhibited excellent activity with MIC of 0.008 lM and good solubility of 104 lg/mL, 100-fold more soluble than that of Comp.1 and 10-fold than that of PBTZ169. The results suggested that the introduction of a 4-carbonyl piperazine were well tolerated and contributed greatly to the enhanced solubilities, which paved the way to find more optimized compounds with improved druggability targeting the cellular enzyme DprE1. Acknowledgments This work was supported by The National Science and Technology Major Project of China (2012ZX09103101-021). We thank Shanghai Chempartner for in vitro antitubercular screening and Sichuan Long March Pharmaceutical Co., Ltd for their technical and financial support. We also thank Lei Li of State Key Laboratory of Biotherapy (Sichuan University) for NMR measurements.
6. 7.
8. 9.
10.
11.
12.
13.
14.
15.
16.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.02. 061.
17.
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