Substitution of terminal amide with 1H-1,2,3-triazole: Identification of unexpected class of potent antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2018.02.001

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-triazole-containing 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-triazole-containing analogues was observed, which could open new opportunities for the development of antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2018.02.001

Accepted Manuscript
Substitution of terminal amide with 1H-1,2,3-triazole: identification of unex-
pected class of potent antibacterial agents
Fangchao Bi, Shengli Ji, Henrietta Venter, Jingru Liu, Susan J. Semple, Shutao
Ma
PII:
S0960-894X(18)30082-9
https://doi.org/10.1016/j.bmcl.2018.02.001
BMCL 25597
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
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25 December 2017
1 February 2018
Please cite this article as: Bi, F., Ji, S., Venter, H., Liu, J., Semple, S.J., Ma, S., Substitution of terminal amide with
1H-1,2,3-triazole: identification of unexpected class of potent antibacterial agents, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.02.001
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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
* Corresponding author: Shutao Ma
Tel/Fax: +86 531 88382009; E-mail: mashutao@sdu.edu.cn
1

Abstract
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-triazole-containing 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-triazole-containing
analogues was observed, which could open new opportunities for the development of
antibacterial agents.
Keywords: 1H-1,2,3-Triazole; Terminal amide; Mimic; Antibacterial agents; In silico
prediction
2

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 small-molecule inhibitors targeting FtsZ offer
promising candidates for new antibacterial agents development.5-12 Among those
FtsZ-targeting 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
Fig. 1. Chemical structures of some FtsZ inhibitors and their MICs against S. aureus.
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 key function for antibacterial activity, it is meaningful to further
investigate this crucial amide group.
Fig. 2. Topological and electronic similarities of amide and 1,2,3-triazole groups.
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
3

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.
Fig. 3. Structural modifications through changing amide group to 1,2,3-triazole ring.
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, 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.
4

Fig. 4. Brief design process of isosteric modification on benzamide derivatives.
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.
5

Scheme 1. Synthetic routes of series A and B. Regents and conditions: (i) benzyl chloride, K2CO3, NaI,
MeCN, 45℃, 99%; (ii) n-BuLi, DMF, THF, -50℃ to r.t., 82%; (iii) 10% Pd/C, ammonium formate,
EtOH, 65℃, 96%; (iv) haloalkanes, K2CO3, DMF, 25–45℃, 83–91%; (v) nitromethane, NH4OAc,
AcOH, reflux; (vi) NaN3, p-TsOH, DMF, 60℃, 53–75% for two steps.
Scheme 1a. The exploration of three possible routes for the synthesis of A7.
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.
6

Scheme 2. Synthetic routes of series C and D. Regents and conditions: (i) a) benzyl chloride, K2CO3,
DMF, 45℃, b) NaOH, H2O, EtOH, 60℃, 89% for two steps; (ii) LiAlH4, THF, 0℃ to r.t.; (iii) SOCl2,
DCM, 0℃, 85%; (iv) 1,2,4-triazole or tetrazole, NaH, DMF, r.t.; (v) 10% Pd/C, ammonium formate,
EtOH, 65℃, 65–70%; (vi) haloalkanes, K2CO3, DMF, 25–45℃, 80–88%.
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.
Scheme 3. Synthetic routes of series E, F and G. Regents and conditions: (i) a) benzyl chloride, K2CO3,
DMF, 45℃, b) NaOH, H2O, EtOH, 60℃, 98% for two steps; (ii) oxalyl chloride, DCM, DMF, r.t., 100%;
(iii) a) MeOH, b) N2H4·H2O, 95% for two steps; (iv) trimethyl orthoformate, NH4Cl, EtOH, reflux,
90%; (v) 10% Pd/C, ammonium formate, EtOH, 65℃; (vi) haloalkanes, K2CO3, DMF, 25–45℃. (vii)
triphosgene, DIPEA, DCM, r.t., 85%; (viii) (NH4)2·CO3, DCM, r.t., 95%; (ix) DMF-DMA, toluene,
90℃, 84%; (x) a) NH2OH·HCl, NaOH, AcOH, H2O, r.t.; b) AcOH, 1,4-dioxane, 90℃, 72% for two
steps.
7

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).
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℃; (iii)
nitromethane, NH4OAc, AcOH, reflux; (iv) NaN3, p-TsOH, DMF, 60℃.
According to the standard broth microdilution method recommended by NCCLS,33 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 Table 1–3,
and expressed as minimum inhibitory concentration (MIC) in units of μg/mL.
Table 1
Series A analogues with their in vitro antibacterial activity.
Minimum inhibitory concentration/MIC (μg/mL)
Comp
B. subtilis
S. aureus
S. aureus
PRa
32
S. aureus
ATCC43300b
E. coli
ATCC25922
>64
K. Pneumoniae
ATCC9372
ATCC25923
ATCC700603
32
32
32
32
16
16
>64
64
A1
A2
A3
A4
A5
A6
A7
A8
A9
32
>64
>64
16
>64
16
>64
16
>64
8
>64
>64
>64
>64
8
>64
>64
4
64
64
>64
4
>64
8
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.25
2
0.5
4
0.5
8
1
4
>64
>64
0.25
4
2
5
8

a S. aureus PR: penicillin-resistant strain isolated clinically, not characterized.
b S. aureus ATCC43300: methicillin-resistant strain.
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 μg/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 μg/mL) than its amide precursor 6 (16, 16, 16,
16 and 8 μg/mL) against five bacteria including two drug-resistant and one Gram-negative
strains. Furthermore, when compared with A6, this defluorine analogue B6 also showed
enhanced antibacterial activity.
Table 2
Series B and B6 analogues with their in vitro antibacterial activity.
Comp
Minimum inhibitory concentration/MIC (μg/mL)
B. subtilis
S. aureus
S. aureus
PRa
>64
>64
32
S. aureus
ATCC43300b
E. coli
ATCC25922
>64
K. Pneumoniae
ATCC9372 ATCC25923
ATCC700603
>64
>64
32
>64
32
32
>64
16
2
>64
>64
16
64
>64
32
>64
16
4
B1
B2
>64
>64
B3
>64
16
>64
32
>64
16
>64
B4
>64
B5
2
4
4
>64
B6
2
4
4
4
>64
4
B7
>64
>64
8
>64
>64
8
>64
>64
8
>64
>64
8
>64
>64
>64
8
B8
>64
B9
>64
B6-o
B6-p
B6-1m
B6-2m
B6-3m
2
4
4
4
>64
4
32
64
>64
64
>64
>64
>64
32
>64
4
>64
64
>64
>64
>64
>64
16
>64
16
16
16
16
>64
8
6
The superscripts are the same as annotated in Table 1.
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 μg/mL)
against five tested strains. However, two oxadiazole derivatives E1 and G1 whose polar N-H
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 N-H bond was found to possess
potent activity (4, 4, 8, 8 and 4 μg/mL) against five tested strains. This difference implied that
the polar hydrogen on nitrogen atom could play an improvement role in the antibacterial
9

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
μg/mL).
Table 3
Series C to G analogues with their in vitro antibacterial activity.
Minimum inhibitory concentration/MIC (μg/mL)
Comp
B. subtilis
ATCC9372
>64
S. aureus
ATCC25923
>64
S. aureus
PRa
S. aureus
ATCC43300b ATCC25922
E. coli
K. Pneumoniae
ATCC700603
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
64
C1
C2
C3
C4
C5
C6
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
16
16
16
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
D1
D2
16
32
>64
>64
>64
>64
>64
>64
16
E1
>64
>64
>64
G1
4
4
8
8
>64
>64
4
F1
F2
>64
>64
>64
>64
>64
The superscripts are the same as annotated in Table 1.
As shown in Table 2, the ortho-substituted triazole analogue B6-o 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-1,2,3-triazole and alkoxy chain on the benzene ring could have some 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
10

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.34 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).35 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.36
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. (Appear in color online only)
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-solvent-surface events and the degree of conservation of the involved surface
residues.37 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
11

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 1H-1,2,3-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.
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. (Appear in color online only)
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 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.
Note
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), the Natural Science Foundation of Shandong (ZR2015BM019), Major
Project of Research and development of Shandong Province (2016GSF201202), 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
12

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15

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, *
Highlights
> 1H-1,2,3-triazole-containing 3-MBA analogues were prepared via isosteric replacement.
> The analogues exhibited better antibacterial activity than their amide precursor.
> The 1H-1,2,3-triazole group served as a good mimic of the terminal amide group.
> Potential target for these novel analogous was predicted using computational techniques.
16

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, *
(Appear in color online only)
17