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Substitution of terminal amide with 1H-1,2,3-triazole: Identification of unexpected class of potent antibacterial agents
Bioorganic & Medicinal Chemistry Letters 28 (2018) 884–891
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Substitution of terminal amide with 1H-1,2,3-triazole: Identification of unexpected class of potent antibacterial agents Fangchao Bi a, Shengli Ji b, Henrietta Venter c, Jingru Liu a, Susan J. Semple c, Shutao Ma a,⇑ a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China b ReaLi Tide Biological Technology (Weihai) Co. Ltd., East Longhai Road & South Yangguang Road, Nanhai New District, Weihai 264207, China c School of Pharmacy & Medical Sciences, Sansom Institute for Health Research, University of South Australia, GPO Box 2471, Adelaide 5001, Australia
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Article history: Received 28 October 2017 Revised 25 December 2017 Accepted 1 February 2018 Available online 2 February 2018 Keywords: 1H-1,2,3-Triazole Terminal amide Mimic Antibacterial agents In silico prediction
a b s t r a c t 3-Methoxybenzamide (3-MBA) derivatives have been identified as novel class of potent antibacterial agents targeting the bacterial cell division protein FtsZ. As one of isosteres for the amide group, 1,2,3-triazole can mimic the topological and electronic features of the amide, which has gained increasing attention in drug discovery. Based on these considerations, we prepared a series of 1H-1,2,3-triazolecontaining 3-MBA analogues via isosteric replacement of the terminal amide with triazole, which had increased antibacterial activity. This study demonstrated the possibility of developing the 1H-1,2, 3-triazole group as a terminal amide-mimetic element which was capable of both keeping and modulating amide-related bioactivity. Surprisingly, a different action mode of these new 1H-1,2,3-triazolecontaining analogues was observed, which could open new opportunities for the development of antibacterial agents. Ó 2018 Elsevier Ltd. All rights reserved.
Infections caused by multidrug-resistant (MDR) bacteria are becoming a serious and global threat to public health.1,2 Furthermore, the number of new therapeutic agents approved or under development is limited in recent years.3,4 Therefore, there is an urgent need for new antibiotics for fighting against the rampant multidrug-resistant bacteria. The validation of bacterial cell division protein FtsZ as a novel antibacterial drug target has been confirmed by various research groups, and different smallmolecule inhibitors targeting FtsZ offer promising candidates for new antibacterial agents development.5–12 Among those FtsZtargeting compounds, 3-methoxybenzamide (3-MBA) 1 was identified as a promising lead.13 In order to improve its potency and optimize drug-like properties, a series of modifications have led to a potent 3-MBA derivative PC190723 (2), and other synthetic analogues (Fig. 1).13–17 The crystal structure of S. aureus FtsZ-PC190723 complex and docking models have shown that the benzamide group plays an important role for antibacterial activity.17–19 However, little successful modifications on the amide group are achieved through introducing small polar substituents or substituting other close groups for the amide function.15,20 Nevertheless, considering its
⇑ Corresponding author. E-mail address: [email protected] (S. Ma). https://doi.org/10.1016/j.bmcl.2018.02.001 0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
key function for antibacterial activity, it is meaningful to further investigate this crucial amide group. Over the past few years, 1,2,3-trizole has gained increasing attention in all aspects of drug discovery since the introduction of the click chemistry by Sharpless and co-workers.21 Remarkably, the 1,2,3-triazole serves as a functional moiety that can mimic the topological and electronic properties of an amide bond (Fig. 2).22–24 What’s more, unlike amides, the triazole ring is not sensitive to hydrolytic cleavage or redox modification.22,25 From the present research, several examples of amide-to-triazole point mutation have been reported and exerted positive results (Fig. 3).26–28 These findings have initially confirmed that the triazole ring is an effective amide surrogate capable of both sustaining and modulating amide-related bioactivities. Furthermore, to our knowledge, the existent amidomimetic analogues are all 1,4-substituted triazoles, and no information has been reported on the terminal amide isosteric exchange with triazole. Based on the above considerations and the peculiar structure of benzamide antibacterial agents, we designed and synthesized a series of 1,2,3-triazole-substituted 3-MBA analogues, and evaluated their preliminary antibacterial activity. The brief design route of this program is shown in Fig. 4. Replacing the amide moiety with 1H-1,2,3-triazole ring led to series A. Further investigation was transferred to non-fluorinated series B. Next, we changed the 1,2,3-triazole into 1,2,4-triazole or tetrazole,
F. Bi et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 884–891
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Fig. 2. Topological and electronic similarities of amide and 1,2,3-triazole groups.
Fig. 1. Chemical structures of some FtsZ inhibitors and their MICs against S. aureus.
leading to series C and series D. Further exploration was focused on B6 by changing the 1H-1,2,3-triazole and introducing other heteroatoms into this five-membered ring, producing E1, F1 and G1. The alkoxy substituent attached to the ortho- or para-position of the benzene ring gave B6-o and B6-p. Methylation of the 1H-1,2,3-triazole ring obtained three N-methylated analogues, B6-1m, B6-2m, B6-3m. The brief design route of this program is shown in Fig. 4. The chemistry and preliminary bioactivity evaluation of these novel derivatives are presented as follows. The synthetic routes of the above proposed compounds were illustrated in Schemes 1–4. The synthetic route of the series A was outlined in Scheme 1. Commercially available 2,4-difluorophenol 7 was protected by benzyl group, and then treated with DMF in the presence of n-BuLi, which was followed by deprotection of the benzyl group, to give the key intermediate 9. Alkylation of 9 with different alkyl chloride or bromide afforded aldehyde product 10. Conversion of the aldehyde group of 10 to 1H-1,2,3-triazole ring was carried out using two step reactions of treatment with nitromethane and p-TsOH-mediated cycloaddition.29 As for the synthesis of compound A7, at first we tried the alkylation of 9 with bromomethyl thiazole, but this reaction did not work as shown in Scheme 1a, which was consistent with the report previously.30 Secondly, an alternative route II was that conversion of aldehyde group of 9 to glycol acetal 9a was followed by
alkylation of 9b. Unfortunately, the attempt to convert 9b to the desired compound 10 by acid-catalyzed hydrolysis failed because multiple byproducts were observed. Finally, the third route III was explored by conversion of 9 to nitroalkene 9c and then alkylation of 11 gave A7 according to the general route. The synthesis of series B was achieved in similar method to that of series A. The synthetic route of the series C and D was outlined in Scheme 2. Bis-benzylation of 3-hydroxybenzoic acid was followed by hydrolysis to produce 3-hydroxy-protected benzoic acid 16. Benzoic acid 16 was subjected to reduction and then chlorination to benzyl chloride 18. Substitution reaction of 18 with 1,2,4-triazole or tetrazole was followed by deprotection of benzyl group to generate two key intermediates 20 and 22, respectively. Alkylation of 20 or 22 with different haloalkanes afforded the target compounds. The synthesis of the series E, F and G was illustrated in Scheme 3. Treatment of intermediate 16 with oxalyl chloride produced the corresponding acid chloride 23, which was further converted in the presence of hydrazine or ammonium carbonate to hydrazide 24 and amide 29, respectively. Cyclization of 24 using trimethyl orthoformate or triphosgene gave 25 or 27, respectively. The series E and F were prepared after deprotection and alkylation of hydroxy group. Cyclization of amide 29 to 31 was carried out in two steps of treating with DMF-DMA and hydroxylamine hydrochloride.31 Finally, through similar procedures mentioned above, the series G was obtained.
Fig. 3. Structural modifications through changing amide group to 1,2,3-triazole ring.
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Fig. 4. Brief design process of isosteric modification on benzamide derivatives.
Methylation of the 1H-1,2,3-triazole led to three N-methylated compounds B6-1m, B6-2m and B6-3m using the reported synthetic method.32 The substituent position of methyl group was identified by 1H NMR spectra, and hydrogen atoms of the methyl group at different positions had distinctive chemical shifts, which was corresponding to the published data.32–35 Two isomers B6-o and B6-p of B6 were also prepared by the same procedures as those of series B (Scheme 4). According to the standard broth microdilution method recommended by NCCLS,36 all the newly synthesized compounds were determined for their in intro antibacterial activity (Supplementary data, Antibacterial Testing). The tested strains included four Gram-positive strains which were B. subtilis ATCC9372, S. aureus ATCC25923, S. aureus ATCC43300, S. aureus PR and two
Gram-negative strains which were E. coli ATCC25922, K. pneumoniae ATCC700603. The antibacterial activity of seven series of compounds are shown in Tables 1–3, and expressed as minimum inhibitory concentration (MIC) in units of lg/mL. As shown in Table 1, these novel difluoro-triazole analogues didn’t show desired results. On the other hand, compound A6 bearing a long alkyl chain exhibited good activity (4, 8, 8, 4 and 8 lg/mL) against four Gram-positive and one negative strains respectively. For the non-fluorinated series B, inspiring results were found as shown in Table 2, triazole analogue B6 was more potent (2, 2, 4, 4 and 4 lg/mL) than its amide precursor 6 (16, 16, 16, 16 and 8 lg/mL) against five bacteria including two drug-resistant and one Gram-negative strains. Furthermore, when compared with
Scheme 1. Synthetic routes of series A and B. Regents and conditions: (i) benzyl chloride, K2CO3, NaI, MeCN, 45 °C, 99%; (ii) n-BuLi, DMF, THF, 50 °C to r.t., 82%; (iii) 10% Pd/ C, ammonium formate, EtOH, 65 °C, 96%; (iv) haloalkanes, K2CO3, DMF, 25–45 °C, 83–91%; (v) nitromethane, NH4OAc, AcOH, reflux; (vi) NaN3, p-TsOH, DMF, 60 °C, 53–75% for two steps.
F. Bi et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 884–891
Scheme 1a. The exploration of three possible routes for the synthesis of A7.
A6, this defluorine analogue B6 also showed enhanced antibacterial activity. As shown in Table 3, tetrazole analogues D1 and D2 lost their activity. In the 1,2,4-triazole analogues, only C6 exhibited modest antibacterial activity (16, 16, 32, 32 and 16 lg/mL) against five tested strains. However, two oxadiazole derivatives E1 and G1 whose polar NAH of 1H-1,2,3-triazole was replaced by O, didn’t exhibit notable activity. While the 1,3,4-oxadiazol-2(3H)-one derivative F1 reserving a polar NAH bond was found to possess potent activity (4, 4, 8, 8 and 4 lg/mL) against five tested strains. This difference implied that the polar hydrogen on nitrogen atom could play an improvement role in the antibacterial activity through forming hydrogen bond or other biological functions. As we expected, three methylated analogues B6-(1m, 2m and 3m) showed a remarkable decreased activity (32 lg/mL). As shown in Table 2, the ortho-substituted triazole analogue B6o was slightly less effective than the meta-substituted analogue B6, and the para-substituted B6-p had a comparable efficacy to B6. This result indicated that the relative location between 1H-1,2,3triazole and alkoxy chain on the benzene ring could have some
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impact on the antibacterial activity, reflecting an order of meta- para- > ortho-position. B7 with the linear alkoxy group possessed similar potency to B6 with the branched alkoxy, while B8 with short-chained alkoxy group was much less active than B7 with long-chained alkoxy group. As the antibacterial activity revealed, in these diverse series, the analogues bearing the long alkoxy side chain showed the most potent activity. In contrast, the analogues with the rigid substituted benzene or heterocycle ring were not remarkable in the antibacterial activity. This indicated that the long alkoxy side chain could interact with the hydrophobic region of FtsZ that was space-limited. Thus, the long flexible alkoxy group could fit comfortably into the critical hydrophobic binding site, while the rigid phenyl or heterocyclic side chain was somewhat restricted. Slightly confusingly, among these outstanding compounds B6, B6- (o and p), B7, C6 and F1, none of them led to distinct filamentation of B. substilis or ballooning of S. aureus as cell division inhibition indicating their on-target activity of FtsZ, typical morphological change before and after treatment was recorded on Fig. S1. This unintentional result, together with the different impact on the activity of benzamide and triazole derivatives caused by fluorine atoms, indicated that these novel triazole-substituted 3-MBA analogues might not target FtsZ as their amide precursors. PharmMapper server was used to investigate the potential target of the triazole-containing analogues.37 B6 was chosen as a representative compound and the 3D Mol2 file was uploaded to PharmMapper (http://59.78.96.61/pharmmapper/). Among the top 300 targets obtained, 8 bacterial-related proteins were picked out, the PDB codes of which were 1hzp, 1xnz, 1zow, 1bsj, 2jff, 3il7, 4alm and 4cuz, respectively. After further analyzing the results of the virtual screening including the pharmacophore models and molecular features of the above targets, which were provided by PharmMapper, we speculated that peptide deformylase (PDF) could be the potential target of B6 for antibacterial activity. This protein target was appeared as a PDB code 1bsj among the results. The pharmacophore model of 1bsj and molecular features of B6 were shown in Fig. 5. PDF plays a key role in prokaryotic protein synthesis. In prokaryotes, protein synthesis begins with a formylated methionine (fMet).38 After translation initiation, the formyl group of the nascent polypeptide is removed by PDF, which is a necessary step to obtain the final protein and is essential for bacterial survival.39
Scheme 2. Synthetic routes of series C and D. Regents and conditions: (i) a) benzyl chloride, K2CO3, DMF, 45 °C, b) NaOH, H2O, EtOH, 60 °C, 89% for two steps; (ii) LiAlH4, THF, 0 °C to r.t.; (iii) SOCl2, DCM, 0 °C, 85%; (iv) 1,2,4-triazole or tetrazole, NaH, DMF, r.t.; (v) 10% Pd/C, ammonium formate, EtOH, 65 °C, 65–70%; (vi) haloalkanes, K2CO3, DMF, 25–45 °C, 80–88%.
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Scheme 3. Synthetic routes of series E, F and G. Regents and conditions: (i) a) benzyl chloride, K2CO3, DMF, 45 °C, b) NaOH, H2O, EtOH, 60 °C, 98% for two steps; (ii) oxalyl chloride, DCM, DMF, r.t., 100%; (iii) a) MeOH, b) N2H4H2O, 95% for two steps; (iv) trimethyl orthoformate, NH4Cl, EtOH, reflux, 90%; (v) 10% Pd/C, ammonium formate, EtOH, 65 °C; (vi) haloalkanes, K2CO3, DMF, 25–45 °C. (vii) triphosgene, DIPEA, DCM, r.t., 85%; (viii) (NH4)2CO3, DCM, r.t., 95%; (ix) DMF-DMA, toluene, 90 °C, 84%; (x) a) NH2OHHCl, NaOH, AcOH, H2O, r.t.; b) AcOH, 1,4-dioxane, 90 °C, 72% for two steps.
Scheme 4. Synthetic routes of five derivatives of B6. Regents and conditions: (i) CH3I, K2CO3, DMF, r.t, 23% for B6-1m, 52% for B6-2m, 13% for B6-3m; (ii) haloalkanes, K2CO3, DMF, 40 °C; (iii) nitromethane, NH4OAc, AcOH, reflux; (iv) NaN3, p-TsOH, DMF, 60 °C.
Table 1 Series A analogues with their in vitro antibacterial activity. Minimum inhibitory concentration/MIC (lg/mL)
Comp
a b
B. subtilis ATCC9372
S. aureus ATCC25923
S. aureus PRa
S. aureus ATCC43300b
E. coli ATCC25922
K. pneumoniae ATCC700603
A1 A2 A3 A4 A5 A6 A7 A8 A9
32 32 >64 16 >64 4 >64 >64 >64
32 32 >64 16 64 8 >64 >64 >64
32 32 >64 16 64 8 >64 >64 >64
16 16 >64 8 >64 4 >64 >64 >64
>64 >64 >64 >64 >64 >64 >64 >64 >64
>64 64 >64 >64 >64 8 >64 >64 >64
2 5
0.25 2
0.5 4
0.5 8
1 4
>64 >64
0.25 4
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized. S. aureus ATCC43300: methicillin-resistant strain.
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F. Bi et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 884–891 Table 2 Series B and B6 analogues with their in vitro antibacterial activity. Minimum inhibitory concentration/MIC (lg/mL)
Comp
a b
B. subtilis ATCC9372
S. aureus ATCC25923
S. aureus PRa
S. aureus ATCC43300b
E. coli ATCC25922
K. pneumoniae ATCC700603
B1 B2 B3 B4 B5 B6 B7 B8 B9
>64 >64 32 >64 16 2 2 >64 >64
>64 32 32 >64 16 2 4 >64 >64
>64 >64 32 >64 32 4 4 >64 >64
>64 >64 16 >64 16 4 4 >64 >64
>64 >64 >64 >64 >64 >64 >64 >64 >64
64 >64 32 >64 16 4 4 >64 >64
B6-o B6-p B6-1m B6-2m B6-3m
8 2 32 >64 64
8 4 64 >64 64
8 4 >64 >64 >64
8 4 32 >64 >64
>64 >64 >64 >64 >64
8 4 4 >64 16
6
16
16
16
16
>64
8
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized. S. aureus ATCC43300: methicillin-resistant strain.
Table 3 Series C to G analogues with their in vitro antibacterial activity. Minimum inhibitory concentration/MIC (lg/mL)
Comp
a b
B. subtilis ATCC9372
S. aureus ATCC25923
S. aureus PRa
S. aureus ATCC43300b
E. coli ATCC25922
K. pneumoniae ATCC700603
C1 C2 C3 C4 C5 C6
>64 >64 >64 >64 >64 16
>64 >64 >64 >64 >64 16
>64 >64 >64 >64 >64 32
>64 >64 >64 >64 >64 32
>64 >64 >64 >64 >64 >64
>64 >64 64 64 >64 16
D1 D2
>64 >64
>64 >64
>64 >64
>64 >64
>64 >64
>64 8
E1
16
32
>64
>64
>64
16
G1
>64
>64
>64
>64
>64
>64
F1 F2
4 >64
4 >64
8 >64
8 >64
>64 >64
4 >64
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized. S. aureus ATCC43300: methicillin-resistant strain.
Subsequently, LigsiteCSC webserver was used to search the binding pocket of protein PDF. LigsiteCSC calculates the potential binding affinity of a protein based on the notion of surface-solventsurface events and the degree of conservation of the involved surface residues.40 The crystal structure of the PDF (PDB code: 1bsj) was submitted to LigsiteCSC (http://projects.biotec.tu-dresden.de/ pocket/), and potential binding sites were obtained. Next, molecular modeling study was performed to analyze the interaction between B6 and PDF using AutoDock. The docking mode of B6 in the possible PDF binding pocket indicated that the 1H-1,2,3-triazole and the ether oxygen could form hydrogen bonds with the
surrounding amino acid residues; the branched-alkyl chain extended to a hydrophobic region. MOE calculation also showed similar ligand interactions in the active sites (Fig. 6). According to the above results, we deduced that this novel class of 1H1,2,3-triazole-substituted 3-MBA analogues probably inhibited PDF to exhibit antibacterial activity. In spite of these multi-level in silico evidences, further systematic experiments should be carried out to elaborate this speculation. In summary, we have identified several interesting new class of potential antibacterial agents, and our results highlighted that the bioisostere 1H-1,2,3-triazole group substituting for the amide
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Fig. 5. The pharmacophore models of B6 binding with 1bsj. Big sphere indicates pharmacophore model of 1bsj, small sphere indicates molecular features of B6, and the coincidence degree reflects binding affinity of B6 and target.
Fig. 6. (A) The docking model (PDB ID: 1bsj) of B6 in the possible PDF binding pocket, performed by AutoDock. The hydrogen bonds were shown as yellow dot lines. (B) MOE cartoon of B6 ligand interactions in active site.
group of 3-MBA derivatives increased the antibacterial activity. This research also provided a novel direction for further modification of the terminal amide moiety of benzamide antibacterial agents and afforded some new scaffolds for antibiotics discovery. On the other hand, the docking results did not necessarily correlate well with the biological activity, and the accurate target and SARs of these novel class of 1H-1,2,3-triazole-substituted analogues needed further research.
research and development project of Shandong Province (2017CXGC1401), Major Project of Science and Technology of Shandong Province (2015ZDJS04001) and the Project-sponsored by SRF for ROCS, SEM. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2018.02.001.
Note References The authors declare that this study was carried out only with public funding. There is no funding or no agreement with commercial for profit firms. Acknowledgement This research was supported financially by the National Natural Science Foundation of China (81673284), Major Project of Research and development of Shandong Province (2016GSF201202), Key
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Substitution of terminal amide with 1H-1,2,3-triazole: Identification of unexpected class of potent antibacterial agents Fangchao Bi a, Shengli Ji b, Henrietta Venter c, Jingru Liu a, Susan J. Semple c, Shutao Ma a,⇑ a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China b ReaLi Tide Biological Technology (Weihai) Co. Ltd., East Longhai Road & South Yangguang Road, Nanhai New District, Weihai 264207, China c School of Pharmacy & Medical Sciences, Sansom Institute for Health Research, University of South Australia, GPO Box 2471, Adelaide 5001, Australia
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Article history: Received 28 October 2017 Revised 25 December 2017 Accepted 1 February 2018 Available online 2 February 2018 Keywords: 1H-1,2,3-Triazole Terminal amide Mimic Antibacterial agents In silico prediction
a b s t r a c t 3-Methoxybenzamide (3-MBA) derivatives have been identified as novel class of potent antibacterial agents targeting the bacterial cell division protein FtsZ. As one of isosteres for the amide group, 1,2,3-triazole can mimic the topological and electronic features of the amide, which has gained increasing attention in drug discovery. Based on these considerations, we prepared a series of 1H-1,2,3-triazolecontaining 3-MBA analogues via isosteric replacement of the terminal amide with triazole, which had increased antibacterial activity. This study demonstrated the possibility of developing the 1H-1,2, 3-triazole group as a terminal amide-mimetic element which was capable of both keeping and modulating amide-related bioactivity. Surprisingly, a different action mode of these new 1H-1,2,3-triazolecontaining analogues was observed, which could open new opportunities for the development of antibacterial agents. Ó 2018 Elsevier Ltd. All rights reserved.
Infections caused by multidrug-resistant (MDR) bacteria are becoming a serious and global threat to public health.1,2 Furthermore, the number of new therapeutic agents approved or under development is limited in recent years.3,4 Therefore, there is an urgent need for new antibiotics for fighting against the rampant multidrug-resistant bacteria. The validation of bacterial cell division protein FtsZ as a novel antibacterial drug target has been confirmed by various research groups, and different smallmolecule inhibitors targeting FtsZ offer promising candidates for new antibacterial agents development.5–12 Among those FtsZtargeting compounds, 3-methoxybenzamide (3-MBA) 1 was identified as a promising lead.13 In order to improve its potency and optimize drug-like properties, a series of modifications have led to a potent 3-MBA derivative PC190723 (2), and other synthetic analogues (Fig. 1).13–17 The crystal structure of S. aureus FtsZ-PC190723 complex and docking models have shown that the benzamide group plays an important role for antibacterial activity.17–19 However, little successful modifications on the amide group are achieved through introducing small polar substituents or substituting other close groups for the amide function.15,20 Nevertheless, considering its
⇑ Corresponding author. E-mail address: [email protected] (S. Ma). https://doi.org/10.1016/j.bmcl.2018.02.001 0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
key function for antibacterial activity, it is meaningful to further investigate this crucial amide group. Over the past few years, 1,2,3-trizole has gained increasing attention in all aspects of drug discovery since the introduction of the click chemistry by Sharpless and co-workers.21 Remarkably, the 1,2,3-triazole serves as a functional moiety that can mimic the topological and electronic properties of an amide bond (Fig. 2).22–24 What’s more, unlike amides, the triazole ring is not sensitive to hydrolytic cleavage or redox modification.22,25 From the present research, several examples of amide-to-triazole point mutation have been reported and exerted positive results (Fig. 3).26–28 These findings have initially confirmed that the triazole ring is an effective amide surrogate capable of both sustaining and modulating amide-related bioactivities. Furthermore, to our knowledge, the existent amidomimetic analogues are all 1,4-substituted triazoles, and no information has been reported on the terminal amide isosteric exchange with triazole. Based on the above considerations and the peculiar structure of benzamide antibacterial agents, we designed and synthesized a series of 1,2,3-triazole-substituted 3-MBA analogues, and evaluated their preliminary antibacterial activity. The brief design route of this program is shown in Fig. 4. Replacing the amide moiety with 1H-1,2,3-triazole ring led to series A. Further investigation was transferred to non-fluorinated series B. Next, we changed the 1,2,3-triazole into 1,2,4-triazole or tetrazole,
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Fig. 2. Topological and electronic similarities of amide and 1,2,3-triazole groups.
Fig. 1. Chemical structures of some FtsZ inhibitors and their MICs against S. aureus.
leading to series C and series D. Further exploration was focused on B6 by changing the 1H-1,2,3-triazole and introducing other heteroatoms into this five-membered ring, producing E1, F1 and G1. The alkoxy substituent attached to the ortho- or para-position of the benzene ring gave B6-o and B6-p. Methylation of the 1H-1,2,3-triazole ring obtained three N-methylated analogues, B6-1m, B6-2m, B6-3m. The brief design route of this program is shown in Fig. 4. The chemistry and preliminary bioactivity evaluation of these novel derivatives are presented as follows. The synthetic routes of the above proposed compounds were illustrated in Schemes 1–4. The synthetic route of the series A was outlined in Scheme 1. Commercially available 2,4-difluorophenol 7 was protected by benzyl group, and then treated with DMF in the presence of n-BuLi, which was followed by deprotection of the benzyl group, to give the key intermediate 9. Alkylation of 9 with different alkyl chloride or bromide afforded aldehyde product 10. Conversion of the aldehyde group of 10 to 1H-1,2,3-triazole ring was carried out using two step reactions of treatment with nitromethane and p-TsOH-mediated cycloaddition.29 As for the synthesis of compound A7, at first we tried the alkylation of 9 with bromomethyl thiazole, but this reaction did not work as shown in Scheme 1a, which was consistent with the report previously.30 Secondly, an alternative route II was that conversion of aldehyde group of 9 to glycol acetal 9a was followed by
alkylation of 9b. Unfortunately, the attempt to convert 9b to the desired compound 10 by acid-catalyzed hydrolysis failed because multiple byproducts were observed. Finally, the third route III was explored by conversion of 9 to nitroalkene 9c and then alkylation of 11 gave A7 according to the general route. The synthesis of series B was achieved in similar method to that of series A. The synthetic route of the series C and D was outlined in Scheme 2. Bis-benzylation of 3-hydroxybenzoic acid was followed by hydrolysis to produce 3-hydroxy-protected benzoic acid 16. Benzoic acid 16 was subjected to reduction and then chlorination to benzyl chloride 18. Substitution reaction of 18 with 1,2,4-triazole or tetrazole was followed by deprotection of benzyl group to generate two key intermediates 20 and 22, respectively. Alkylation of 20 or 22 with different haloalkanes afforded the target compounds. The synthesis of the series E, F and G was illustrated in Scheme 3. Treatment of intermediate 16 with oxalyl chloride produced the corresponding acid chloride 23, which was further converted in the presence of hydrazine or ammonium carbonate to hydrazide 24 and amide 29, respectively. Cyclization of 24 using trimethyl orthoformate or triphosgene gave 25 or 27, respectively. The series E and F were prepared after deprotection and alkylation of hydroxy group. Cyclization of amide 29 to 31 was carried out in two steps of treating with DMF-DMA and hydroxylamine hydrochloride.31 Finally, through similar procedures mentioned above, the series G was obtained.
Fig. 3. Structural modifications through changing amide group to 1,2,3-triazole ring.
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Fig. 4. Brief design process of isosteric modification on benzamide derivatives.
Methylation of the 1H-1,2,3-triazole led to three N-methylated compounds B6-1m, B6-2m and B6-3m using the reported synthetic method.32 The substituent position of methyl group was identified by 1H NMR spectra, and hydrogen atoms of the methyl group at different positions had distinctive chemical shifts, which was corresponding to the published data.32–35 Two isomers B6-o and B6-p of B6 were also prepared by the same procedures as those of series B (Scheme 4). According to the standard broth microdilution method recommended by NCCLS,36 all the newly synthesized compounds were determined for their in intro antibacterial activity (Supplementary data, Antibacterial Testing). The tested strains included four Gram-positive strains which were B. subtilis ATCC9372, S. aureus ATCC25923, S. aureus ATCC43300, S. aureus PR and two
Gram-negative strains which were E. coli ATCC25922, K. pneumoniae ATCC700603. The antibacterial activity of seven series of compounds are shown in Tables 1–3, and expressed as minimum inhibitory concentration (MIC) in units of lg/mL. As shown in Table 1, these novel difluoro-triazole analogues didn’t show desired results. On the other hand, compound A6 bearing a long alkyl chain exhibited good activity (4, 8, 8, 4 and 8 lg/mL) against four Gram-positive and one negative strains respectively. For the non-fluorinated series B, inspiring results were found as shown in Table 2, triazole analogue B6 was more potent (2, 2, 4, 4 and 4 lg/mL) than its amide precursor 6 (16, 16, 16, 16 and 8 lg/mL) against five bacteria including two drug-resistant and one Gram-negative strains. Furthermore, when compared with
Scheme 1. Synthetic routes of series A and B. Regents and conditions: (i) benzyl chloride, K2CO3, NaI, MeCN, 45 °C, 99%; (ii) n-BuLi, DMF, THF, 50 °C to r.t., 82%; (iii) 10% Pd/ C, ammonium formate, EtOH, 65 °C, 96%; (iv) haloalkanes, K2CO3, DMF, 25–45 °C, 83–91%; (v) nitromethane, NH4OAc, AcOH, reflux; (vi) NaN3, p-TsOH, DMF, 60 °C, 53–75% for two steps.
F. Bi et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 884–891
Scheme 1a. The exploration of three possible routes for the synthesis of A7.
A6, this defluorine analogue B6 also showed enhanced antibacterial activity. As shown in Table 3, tetrazole analogues D1 and D2 lost their activity. In the 1,2,4-triazole analogues, only C6 exhibited modest antibacterial activity (16, 16, 32, 32 and 16 lg/mL) against five tested strains. However, two oxadiazole derivatives E1 and G1 whose polar NAH of 1H-1,2,3-triazole was replaced by O, didn’t exhibit notable activity. While the 1,3,4-oxadiazol-2(3H)-one derivative F1 reserving a polar NAH bond was found to possess potent activity (4, 4, 8, 8 and 4 lg/mL) against five tested strains. This difference implied that the polar hydrogen on nitrogen atom could play an improvement role in the antibacterial activity through forming hydrogen bond or other biological functions. As we expected, three methylated analogues B6-(1m, 2m and 3m) showed a remarkable decreased activity (32 lg/mL). As shown in Table 2, the ortho-substituted triazole analogue B6o was slightly less effective than the meta-substituted analogue B6, and the para-substituted B6-p had a comparable efficacy to B6. This result indicated that the relative location between 1H-1,2,3triazole and alkoxy chain on the benzene ring could have some
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impact on the antibacterial activity, reflecting an order of meta- para- > ortho-position. B7 with the linear alkoxy group possessed similar potency to B6 with the branched alkoxy, while B8 with short-chained alkoxy group was much less active than B7 with long-chained alkoxy group. As the antibacterial activity revealed, in these diverse series, the analogues bearing the long alkoxy side chain showed the most potent activity. In contrast, the analogues with the rigid substituted benzene or heterocycle ring were not remarkable in the antibacterial activity. This indicated that the long alkoxy side chain could interact with the hydrophobic region of FtsZ that was space-limited. Thus, the long flexible alkoxy group could fit comfortably into the critical hydrophobic binding site, while the rigid phenyl or heterocyclic side chain was somewhat restricted. Slightly confusingly, among these outstanding compounds B6, B6- (o and p), B7, C6 and F1, none of them led to distinct filamentation of B. substilis or ballooning of S. aureus as cell division inhibition indicating their on-target activity of FtsZ, typical morphological change before and after treatment was recorded on Fig. S1. This unintentional result, together with the different impact on the activity of benzamide and triazole derivatives caused by fluorine atoms, indicated that these novel triazole-substituted 3-MBA analogues might not target FtsZ as their amide precursors. PharmMapper server was used to investigate the potential target of the triazole-containing analogues.37 B6 was chosen as a representative compound and the 3D Mol2 file was uploaded to PharmMapper (http://59.78.96.61/pharmmapper/). Among the top 300 targets obtained, 8 bacterial-related proteins were picked out, the PDB codes of which were 1hzp, 1xnz, 1zow, 1bsj, 2jff, 3il7, 4alm and 4cuz, respectively. After further analyzing the results of the virtual screening including the pharmacophore models and molecular features of the above targets, which were provided by PharmMapper, we speculated that peptide deformylase (PDF) could be the potential target of B6 for antibacterial activity. This protein target was appeared as a PDB code 1bsj among the results. The pharmacophore model of 1bsj and molecular features of B6 were shown in Fig. 5. PDF plays a key role in prokaryotic protein synthesis. In prokaryotes, protein synthesis begins with a formylated methionine (fMet).38 After translation initiation, the formyl group of the nascent polypeptide is removed by PDF, which is a necessary step to obtain the final protein and is essential for bacterial survival.39
Scheme 2. Synthetic routes of series C and D. Regents and conditions: (i) a) benzyl chloride, K2CO3, DMF, 45 °C, b) NaOH, H2O, EtOH, 60 °C, 89% for two steps; (ii) LiAlH4, THF, 0 °C to r.t.; (iii) SOCl2, DCM, 0 °C, 85%; (iv) 1,2,4-triazole or tetrazole, NaH, DMF, r.t.; (v) 10% Pd/C, ammonium formate, EtOH, 65 °C, 65–70%; (vi) haloalkanes, K2CO3, DMF, 25–45 °C, 80–88%.
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Scheme 3. Synthetic routes of series E, F and G. Regents and conditions: (i) a) benzyl chloride, K2CO3, DMF, 45 °C, b) NaOH, H2O, EtOH, 60 °C, 98% for two steps; (ii) oxalyl chloride, DCM, DMF, r.t., 100%; (iii) a) MeOH, b) N2H4H2O, 95% for two steps; (iv) trimethyl orthoformate, NH4Cl, EtOH, reflux, 90%; (v) 10% Pd/C, ammonium formate, EtOH, 65 °C; (vi) haloalkanes, K2CO3, DMF, 25–45 °C. (vii) triphosgene, DIPEA, DCM, r.t., 85%; (viii) (NH4)2CO3, DCM, r.t., 95%; (ix) DMF-DMA, toluene, 90 °C, 84%; (x) a) NH2OHHCl, NaOH, AcOH, H2O, r.t.; b) AcOH, 1,4-dioxane, 90 °C, 72% for two steps.
Scheme 4. Synthetic routes of five derivatives of B6. Regents and conditions: (i) CH3I, K2CO3, DMF, r.t, 23% for B6-1m, 52% for B6-2m, 13% for B6-3m; (ii) haloalkanes, K2CO3, DMF, 40 °C; (iii) nitromethane, NH4OAc, AcOH, reflux; (iv) NaN3, p-TsOH, DMF, 60 °C.
Table 1 Series A analogues with their in vitro antibacterial activity. Minimum inhibitory concentration/MIC (lg/mL)
Comp
a b
B. subtilis ATCC9372
S. aureus ATCC25923
S. aureus PRa
S. aureus ATCC43300b
E. coli ATCC25922
K. pneumoniae ATCC700603
A1 A2 A3 A4 A5 A6 A7 A8 A9
32 32 >64 16 >64 4 >64 >64 >64
32 32 >64 16 64 8 >64 >64 >64
32 32 >64 16 64 8 >64 >64 >64
16 16 >64 8 >64 4 >64 >64 >64
>64 >64 >64 >64 >64 >64 >64 >64 >64
>64 64 >64 >64 >64 8 >64 >64 >64
2 5
0.25 2
0.5 4
0.5 8
1 4
>64 >64
0.25 4
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized. S. aureus ATCC43300: methicillin-resistant strain.
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F. Bi et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 884–891 Table 2 Series B and B6 analogues with their in vitro antibacterial activity. Minimum inhibitory concentration/MIC (lg/mL)
Comp
a b
B. subtilis ATCC9372
S. aureus ATCC25923
S. aureus PRa
S. aureus ATCC43300b
E. coli ATCC25922
K. pneumoniae ATCC700603
B1 B2 B3 B4 B5 B6 B7 B8 B9
>64 >64 32 >64 16 2 2 >64 >64
>64 32 32 >64 16 2 4 >64 >64
>64 >64 32 >64 32 4 4 >64 >64
>64 >64 16 >64 16 4 4 >64 >64
>64 >64 >64 >64 >64 >64 >64 >64 >64
64 >64 32 >64 16 4 4 >64 >64
B6-o B6-p B6-1m B6-2m B6-3m
8 2 32 >64 64
8 4 64 >64 64
8 4 >64 >64 >64
8 4 32 >64 >64
>64 >64 >64 >64 >64
8 4 4 >64 16
6
16
16
16
16
>64
8
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized. S. aureus ATCC43300: methicillin-resistant strain.
Table 3 Series C to G analogues with their in vitro antibacterial activity. Minimum inhibitory concentration/MIC (lg/mL)
Comp
a b
B. subtilis ATCC9372
S. aureus ATCC25923
S. aureus PRa
S. aureus ATCC43300b
E. coli ATCC25922
K. pneumoniae ATCC700603
C1 C2 C3 C4 C5 C6
>64 >64 >64 >64 >64 16
>64 >64 >64 >64 >64 16
>64 >64 >64 >64 >64 32
>64 >64 >64 >64 >64 32
>64 >64 >64 >64 >64 >64
>64 >64 64 64 >64 16
D1 D2
>64 >64
>64 >64
>64 >64
>64 >64
>64 >64
>64 8
E1
16
32
>64
>64
>64
16
G1
>64
>64
>64
>64
>64
>64
F1 F2
4 >64
4 >64
8 >64
8 >64
>64 >64
4 >64
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized. S. aureus ATCC43300: methicillin-resistant strain.
Subsequently, LigsiteCSC webserver was used to search the binding pocket of protein PDF. LigsiteCSC calculates the potential binding affinity of a protein based on the notion of surface-solventsurface events and the degree of conservation of the involved surface residues.40 The crystal structure of the PDF (PDB code: 1bsj) was submitted to LigsiteCSC (http://projects.biotec.tu-dresden.de/ pocket/), and potential binding sites were obtained. Next, molecular modeling study was performed to analyze the interaction between B6 and PDF using AutoDock. The docking mode of B6 in the possible PDF binding pocket indicated that the 1H-1,2,3-triazole and the ether oxygen could form hydrogen bonds with the
surrounding amino acid residues; the branched-alkyl chain extended to a hydrophobic region. MOE calculation also showed similar ligand interactions in the active sites (Fig. 6). According to the above results, we deduced that this novel class of 1H1,2,3-triazole-substituted 3-MBA analogues probably inhibited PDF to exhibit antibacterial activity. In spite of these multi-level in silico evidences, further systematic experiments should be carried out to elaborate this speculation. In summary, we have identified several interesting new class of potential antibacterial agents, and our results highlighted that the bioisostere 1H-1,2,3-triazole group substituting for the amide
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Fig. 5. The pharmacophore models of B6 binding with 1bsj. Big sphere indicates pharmacophore model of 1bsj, small sphere indicates molecular features of B6, and the coincidence degree reflects binding affinity of B6 and target.
Fig. 6. (A) The docking model (PDB ID: 1bsj) of B6 in the possible PDF binding pocket, performed by AutoDock. The hydrogen bonds were shown as yellow dot lines. (B) MOE cartoon of B6 ligand interactions in active site.
group of 3-MBA derivatives increased the antibacterial activity. This research also provided a novel direction for further modification of the terminal amide moiety of benzamide antibacterial agents and afforded some new scaffolds for antibiotics discovery. On the other hand, the docking results did not necessarily correlate well with the biological activity, and the accurate target and SARs of these novel class of 1H-1,2,3-triazole-substituted analogues needed further research.
research and development project of Shandong Province (2017CXGC1401), Major Project of Science and Technology of Shandong Province (2015ZDJS04001) and the Project-sponsored by SRF for ROCS, SEM. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2018.02.001.
Note References The authors declare that this study was carried out only with public funding. There is no funding or no agreement with commercial for profit firms. Acknowledgement This research was supported financially by the National Natural Science Foundation of China (81673284), Major Project of Research and development of Shandong Province (2016GSF201202), Key
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