Identification of Novel Benzoxa-[2,1,3]-diazole Substituted Amino Acid Hydrazides as Potential Anti-Tubercular Agents

Article abstract of DOI:10.3390/molecules24040811

Discovery and development of new therapeutic options for the treatment of Mycobacterium tuberculosis (Mtb) infection are desperately needed to tackle the continuing global burden of this disease and the efficacy and cost limitations associated with current medicines. Herein, we report the synthesis of a series of novel benzoxa-[2,1,3]-diazole substituted amino acid hydrazides in a two-step synthesis and evaluate their inhibitory activity against Mtb and selected bacterial strains of clinical importance utilising an end point-determined REMA assay. Alongside this, their potential for undesired cytotoxicity against mammalian cells was assessed employing standard MTT assay methodologies. It has been demonstrated using modification at three sites (the hydrazine, amino acid, and the benzodiazole) it is possible to change both the antibacterial activity and cytotoxicity of these molecules whilst not affecting their microbial selectivity, making them attractive architectures for further exploitation as novel antibacterial agents.

Full text of DOI:10.3390/molecules24040811

molecules
Article
Identification of Novel Benzoxa-[2,1,3]-diazole
Substituted Amino Acid Hydrazides as Potential
Anti-Tubercular Agents
Alistair K. Brown 1,2 , Ahmed K. B. Aljohani 1, Jason H. Gill 2,3 , Patrick G. Steel 4 and
Jonathan D. Sellars 1,2,5,
*
1
Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University,
Catherine Cookson Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK;
alistair.brown2@newcastle.ac.uk (A.K.B.); a.aljohani2@newcastle.ac.uk (A.K.B.A.)
School of Pharmacy, Faculty of Medical Sciences, King George VI Building,
Newcastle upon Tyne NE1 7RU, UK
2
3
4
5
0
Northern Institute for Cancer Research, Paul O Gorman Building, Faculty of Medical Sciences,
Newcastle University, Newcastle upon Tyne NE2 4HH, UK; jason.gill@newcastle.ac.uk
Department of Chemistry, Durham University, Lower Mountjoy, Stockton Road, Durham DH1 3LE, UK;
p.g.steel@durham.ac.uk
Chemistry, School of Natural and Environmental Sciences, Newcastle University,
Newcastle upon Tyne NE1 7RU, UK
*
Correspondence: jon.sellars@newcastle.ac.uk; Tel.: +44-(191)-2082357
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Peter J. Rutledge
Received: 29 January 2019; Accepted: 20 February 2019; Published: 23 February 2019
Abstract: Discovery and development of new therapeutic options for the treatment of
Mycobacterium tuberculosis (Mtb) infection are desperately needed to tackle the continuing global
burden of this disease and the efficacy and cost limitations associated with current medicines. Herein,
we report the synthesis of a series of novel benzoxa-[2,1,3]-diazole substituted amino acid hydrazides
in a two-step synthesis and evaluate their inhibitory activity against Mtb and selected bacterial strains
of clinical importance utilising an end point-determined REMA assay. Alongside this, their potential
for undesired cytotoxicity against mammalian cells was assessed employing standard MTT assay
methodologies. It has been demonstrated using modification at three sites (the hydrazine, amino
acid, and the benzodiazole) it is possible to change both the antibacterial activity and cytotoxicity of
these molecules whilst not affecting their microbial selectivity, making them attractive architectures
for further exploitation as novel antibacterial agents.
Keywords: benzoxa-[2,1,3]-diazole; antibacterial; Mycobacterium tuberculosis; amino acid; hydrazide
1. Introduction
Tuberculosis (TB) remains the predominant bacterial infectious disease globally, with approximately
10.4 million new cases and 1.7 million deaths recorded in 2016 [
over 20% of the population is latently infected with Mycobacterium tuberculosis (Mtb), the causative
agent of TB [ ]. Current treatment regimens of TB require patients to administer drug combinations
for an extended period of time, at least six months, which is costly, prone to differential response
rates, and exhibits significant problems with patient compliance and adherence [ ]. The current
1]. Additionally, estimates suggest that
2
3
treatment regimen involves combinations of rifampicin (RIF), isoniazid (INH), ethambutol (EMB), and
pyrazinamide. However, resistance to these drug therapies has exacerbated the management issues
of TB. In 2016 alone, almost 500,000 new cases of multidrug-resistant TB (resistant to both RIF and
INH) and over 100,000 cases of RIF-resistant TB were identified [1]. Consequently, the identification of
Molecules 2019, 24, 811; doi:10.3390/molecules24040811
www.mdpi.com/journal/molecules

Molecules 2019, 24, 811
2 of 14
novel therapeutic options is an absolute necessity for the management of TB in the future [
4
]. In this
context, in recent years several new drugs and/or regimens have been reported for TB, including
bedaquiline [ ], delamanid [ ], clofazimine [ ], bedazuiline-protomanid-linezolid [ ]. Worryingly,
resistance to several of these approaches has already been reported, primarily because of similar
drug resistance mechanisms and target pathways [ 10]. Consequently, the development of new drug
5
6
7
8
9
,
therapies for TB requires the discovery of new drug targets and novel structures which circumvent
resistance mechanisms, whilst also enabling shorter treatment regimens.
Benzoxa-[2,1,3]-diazole, benzothia-[2,1,3]-diazole and benzotriazoles are a class of heterocycle
which exhibit widespread therapeutic opportunities [11]. In addition, no studies have evaluated these
compounds or their derivatives against Mtb. Inspired by virtual screening studies which identified
simple substituted nitro benzoxa-[2,1,3]-diazoles as potential inhibitors of Mtb ATP phosphoribosyl
transferase (HisG) and APS reductase (APSR) [12
,13], we were attracted to this scaffold as a new
starting point for the development of anti-TB drugs.
In this report we describe a series of novel benzoxa-[2,1,3]-diazole substituted amino acid
hydrazides as selective drugs for the treatment of TB, highlighting the importance of the
benzo-[2,1,3]-diazole, amino acid (AA) and the substituted aryl hydrazine (R1), towards Mtb selectivity,
potency, efficacy, and avoidance of toxicity against mammalian cells (Figure 1).
Figure 1. Inhibitors of M. tuberculosis based on the benzoxa-[2,1,3]-diazole framework highlighting the
key modifications.
2. Results and Discussion
In the context of a study to identify novel antibacterial agents designed to overcome antimicrobial
resistance, a small library of diverse bioactive compounds had previously been synthesised within
our team. Using the Resazurin Microtiter Assay (REMA) [14
–16], these compounds were screened
for antibacterial activity at a fixed concentration (128 g/mL) against a range of drug-susceptible
µ
bacteria including Gram-positive, Gram-negative and mycolata bacteria (Supporting Information,
Table S1) which revealed that many possessed little utility, even at these high concentrations. However,
benzo-[2,1,3]-diazole architectures
1–12 were shown to possess antibacterial activity, including activity
against mycolata bacteria and Mtb (Figure 2).

Molecules 2019, 24, 811
3 of 14
Figure 2. Benzodiazole structures from the initial 128
Gram-negative and mycolata bacteria.
µg/mL screen against a range of Gram-positive,
To gain an improved understanding of the antibacterial potency and scope of these compounds,
a dose-range REMA assay was performed (128–0.125 µg/mL, converted to µM if active) (Table 1).
Table 1. Selective antibacterial activity of benzodiazole compounds present in the compound library,
expressed as mean inhibitory concentration (MIC) (µM). (-) = No activity in the REMA assay.
MIC (µM)
Compound
S. aureus
E. coli
S. equi subsp. equi
S. agalactiae
S. pyogenes
R. equi
M. tuberculosis mc27000
1
-
11.9
-
-
-
11.9
128.9
17.16
0.59
80.7
109.7
23.0
-
2
0.7
29.8
5.6
0.9
-
-
3
3.4
3.4
1.7
20.3
-
5.1
4
7.1
-
0.9
2.4
-
9.5
5
-
-
-
-
-
-
6
-
-
-
-
-
-
7
-
-
-
-
-
-
-
8
-
-
-
-
-
-
9
10
-
-
-
-
-
3.6
-
31.1
36.7
148.4
44.0
0.91
0.03
9.79
-
-
-
-
-
11
-
-
27.6
-
-
10.4
11.6
-
12
-
-
-
-
-
Isoniazid
Rifampin
Ethambutol
-
0.12
-
-
7.78
-
-
0.03
-
-
0.97
-
-
0.49
-
7.78
-

Molecules 2019, 24, 811
4 of 14
The results of the endpoint REMA assay revealed a mix of activity against the organisms tested,
with simple substituted benzodiazole compounds providing broad spectrum activity. Whilst the
nitrobenzoxa-[2,1,3]-diazole showed the highest levels of activity against Mtb, the lack of selectivity
was a cause for concern. Replacing the nitro group with a sulphonamido amino acid moiety greatly
improved specificity for mycolata bacteria, with substituted benzoxa-[2,1,3]-diazole showing much
higher activity than , suggesting poor cell wall penetration of is due to the carboxylic acid moiety.
Notwithstanding this, replacement of the benzoxa-[2,1,3]-diazole with benzothia-[2,1,3]-diazoles and
led to a complete loss of activity suggesting that the benzoxa-[2,1,3]-diazole plays a crucial role in
these compounds antibacterial activity.
Further analysis of the results revealed that conversion of the ester to an aryl hydrazide
1–4
3
6
5
5
7
8
9–12
provided compounds more consistent activity across a range of structures. Consequently, substituted
benzoxa-[2,1,3]-diazoles were chosen as the partner to amino acid hydrazides for further investigation,
via a SAR study to further understand the importance of the amino acid (AA) and the hydrazine (R1)
on anti-mycobacterial activity.
2.1. Chemical Synthesis of Benzoxa-[2,1,3]-diazole Amino Acid Hydrazides
To undertake this investigation, a two-step synthesis was engaged starting from N-Boc amino acids
(Scheme 1). DCC coupling with a monosubstituted hydrazine produced the intermediate protected
amino acid hydrazine which, following deprotection and condensation with benzoxa-[2,1,3]-diazole
sulphonyl chloride afforded the desired products in moderate to good overall yields following flash
chromatography purification (Table 2). Interestingly, several of the compounds exhibited restricted
rotation as demonstrated by VT-1H-NMR spectroscopy (Supporting Information, Figure S2).
Scheme 1. Synthesis of amino acid hydrazides and the desired benzoxa-[2,1,3]-diazole amino
acid hydrazides.
2.2. Structure–Activity Relationships
Following the synthetic approach, Boc amino acid hydrazides 13a
–22a and benzoxa-[2,1,3]-diazole
amino acid hydrazide 10 14b 22b were screened in the same way against the same range of
9,
,
–
drug-susceptible bacteria as described above. Importantly, in line with the preliminary screening data
these compounds showed high selectivity for mycolata bacteria (Supporting Information, Table S3).
Focusing on the Mtb response, initially exploring the role of the amino acid, fixing the hydrazide and
increasing the bulk of the amino acid substituent 13a
of this component (Table 2). Subsequently, fixing the amino acid to glycine, we then evaluated the
role of the hydrazine component (18a 22a). Introduction of an unsubstituted aromatic hydrazine 18a
alongside halogenated hydrazines 19a 22a did not provide any significant enhancement in activity
although a marked increase in cytotoxicity was observed. For both series, enhanced antibacterial
–17a resulted in diminished antibacterial activity
–
–
activity was restored on coupling to the benzoxa-[2,1,3]-diazole
increased cytotoxicity, as noted for this subunit [17].
9, 10, 14b–22b albeit at the cost of

Molecules 2019, 24, 811
5 of 14
Table 2. Results of synthesis of amino acid hydrazides and benzoxa-[2,1,3]-diazole amino acid
hydrazides and the antibacterial activity and cytotoxicity.
Amino Acid
(AA)
Hydrazine
(R1)
M. tuberculosis
Mammalian
Cell Toxicity
Compound
Yield
Rotamer Ratio
mc27000 (µM)
13a
9
47%
7%
-
7:1
-
12.01
31.13
117.24
60.85
214.26
16.33
151.14
59.27
46.06
17.25
90.46
36.67
112.95
20.00
106.75
19.22
213.50
19.22
106.75
19.22
0%
>90%
0%
Gly
Phg
Pro
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
H
14a
14b
15a
15b
16a
16b
17a
17b
18a
10
95%
63%
85%
85%
83%
85%
93%
60%
76%
60%
78%
57%
96%
43%
80%
22%
95%
29%
-
>90%
0%
-
>90%
<10%
>90%
0%
-
Phe
Ala
Gly
Gly
Gly
Gly
Gly
-
-
-
>90%
57%
0%
5:1
4:1
4:1
4:1
4:1
6:1
4:1
5:1
3:1
4:1
19a
19b
20a
20b
21a
21b
22a
22b
59%
60%
43%
80%
55%
45%
<20%
70%
4-F
4-Cl
3-Cl
2-Cl
3. Discussion
Worryingly, as drug-resistant bacterial infections are on the rise and with the recent removal of
antibiotic drug discovery programmes, there will be a significant demand for new chemical entities to
address this condition. This study has identified that benzoxa-[2,1,3]-diazole substituted amino acid
hydrazides have considerable potential as selective and potent agents against Mtb.
Throughout this study, the benzoxa-[2,1,3]-diazole core appears to be essential for activity. Whilst,
as observed in some examples, the use of this unit is commonly associated with cytotoxicity this can
be effectively modulated through the addition of the amino acid hydrazine. For example, we observed
that conjugation with the amino acid hydrazides 19a or 21a provides a reduction in cytotoxicity (19b
,
21b, Table 2).
Excitingly, this modulation led to the use of a simple unsubstituted hydrazide 18a which, although
in isolation showed significant cytotoxicity, when conjugated with the benzoxadiazole 10 provides a
compound with good level of activity against Mtb and no observable cytotoxicity.

Molecules 2019, 24, 811
6 of 14
4. Materials and Methods
4.1. Chemistry
4.1.1. Synthesis of Hydrazides—General Procedure
A solution of N-Boc amino acid (0.25 g, 1.43 mmol, 1 equiv.), HOBt (2 equiv.) and DCC (1.2 equiv.)
◦
was dissolved in THF (7.5 mL), cooled to 0 C and stirred for 15 min. The solution was treated
with N-Aryl/Alkyl hydrazine * (1.2 equiv.) before warming to room temperature and stirring for
a further 1.5 h. The mixture was then poured into sat. aq. NH4Cl (20 mL) before separating and
extracting the aqueous layer with EtOAc (40 mL). The organic layer was further washed with sat.
aq. NaHCO3 (20 mL) and then brine (20 mL). The combined organic layers were dried over MgSO4,
filtered, concentrated and dried in vacuo. Flash chromatography (DCM/EtOH/NH3 [600:8:1], [400:8:1],
[200:8:1]) afforded the desired N-Boc amino acid hydrazides.
* When the hydrazine hydrochlorides were used, Et3N (1.2 equiv.) is added to neutralise the salt
tert-Butyl 2-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)ethylcarbamate, 13a
Following the general procedure outlined, N-Boc-L-Glycine (0.50 g, 2.9 mmol) and 4-trifluorophenyl
hydrazine (0.61 g, 3.5 MMOL) was transformed following flash chromatography into the title
◦
compound which was isolated as a white solid (0.45 g, 47%); m.p. 185–187 C;
υ
(ATR) 3370
max
(NH), 3278 (NH), 3234 (NH), 3108, 3058, 2996, 1649 (C=O), 1615, 1518 (C=O), 1330, 1245, 1156, 1107,
1052, 828, 559 cm−1
;
δH (700 MHz, DMSO-d6) 9.78 (1H, s, Ar-NHNH), 8.32 (1H, s, Ar-NHNH), 7.40
(2H, d, J 9, Ar-H), 7.07 (1H, t, J 6, BocNH), 6.78 (2H, d, J 9, Ar-H), 3.60 (2H, d, J 6, NHCH2), 1.37 (9H, s,
(CH3)3COC(O)NH); δC (176 MHz, DMSO-d6) 170.2 (CONH), 156.6 ((CH3)3COC(O)NH), 153.1 (Ar-C),
126.7 (Ar-C), 126.4 (Ar-C), 118.82 (Ar-C), 118.63 (Ar-C), 112.1 (Ar-C), 78.8 (NHCH2), 42.7 ((CH3)3CO),
28.8 ((CH3)3CO); δF (658 MHz, DMSO-d6) -59.30; m/z (ES+) 356 (MNa+), 689 (2M + Na+); HRMS (ES+)
Found MH+, 334.13722 (C14H19F3N3O3 requires 334.13730).
tert-Butyl (S)-(2-oxo-1-phenyl-2-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)carbamate, 14a
Following the general procedure outlined, N-Boc-L-phenylglycine (0.250 g, 0.99 mmol) and
4-trifluorophenyl hydrazine (0.30 g, 1.75 mmol) was transformed following flash chromatography
into the title compound as a brown solid (0.386 g, 95%); Rf 0.44 (DCM/EtOH/NH3 [200:8:1]); m.p.
138–143 ◦C;
1067, 834, 696, 558, 488 cm
υ
max (ATR) 3328 (N-H), 2982, 2930, 2851, 1678 (C=O), 1655 (C=O), 1524, 1322, 1158, 1113,
−1
; δH (400 MHz, CDCl3) 8.00 (1H, bs, NH), 7.46–7.37 (7H, m, Ar-H), 6.68 (2H,
d, J 8, Ar-H), 6.24 (1H, bs, NH), 5.69 (1H, d, J 7, NHCHCO), 5.35 (1H, bs, NH), 1.42 (9H, s, C(CH3)3);
δC (176 MHz, CDCl3) 157.0 (NHCHCO), 150.3 (NHCOO), 129.3 (Ar-C), 128.9 (Ar-C), 128.4 (Ar-C),
127.2 (Ar-C), 127.1 (Ar-C), 126.5 (Ar-C), 125.1 (Ar-C), 123.6 (CF3), 112.7 (Ar-C), 80.3 (C(CH3)3), 59.4
(NHCHCO), 28.3 (C(CH3)3); δF (376 MHz, CDCl3) -61.60 (3F, s, CF3); m/z (ES+) 410 (MH+), 432 (MNa+),
841 (2M + Na+); HRMS (ES+) Found MH+, 410.1689 (C20H23F3N3O3 requires 410.1686).
tert-Butyl (S)-2-(2-(4-(trifluoromethyl)phenyl)hydrazine-1-carbonyl)pyrrolidine-1-carboxylate, 15a
Following the general procedure outlined, N-Boc-L-proline (0.250 g, 1.16 mmol) and 4-trifluorophenyl
hydrazine (0.30 g, 1.75 mmol) was transformed following flash chromatography into the title compound
as a golden-brown solid (0.366 g, 85%); Rf 0.38 (DCM/EtOH/NH3 [200:8:1]); m.p. 116–120 ◦C;
υ
max
(ATR) 3265 (N-H), 2979, 2932, 2851, 1672 (C=O), 1618, 1399, 1324, 1107, 1065, 833, 591 cm−1
;
δH
(400 MHz, CDCl3) 8.91 (1H, bs, NH), 7.45 (2H, d, J 8, Ar-H), 6.87 (2H, d, J 8, Ar-H), 6.34 (1H, bs,
NH), 4.45–4.34 (1H, m, NCHCO), 3.52–3.35 (2H, m, N(CH2)3), 2.06–1.87 (4H, m, N(CH2)3), 1.53 (9H,
s, C(CH3)3); δC (176 MHz, CDCl3) 172.2 (NCHCO), 156.1 (NCOO), 150.8 (Ar-C), 126.4 (Ar-C), 125.2
(Ar-C), 123.7 (CF3), 112.7 (Ar-C), 81.0 (C(CH3)3), 58.4 (NCHCO), 47.2 (NCH2(CH2)2), 28.4 (C(CH3)3),
25.5, (NCH2(CH2)2), 24.8 (NCH2(CH2)2); m/z (ES+) 374 (MH+), 396 (MNa+), 769 (2M + Na+); HRMS
(ES+) Found MNa+, 396.1497 (C17H22F3N3O3Na requires 396.1505).

Molecules 2019, 24, 811
7 of 14
tert-Butyl (S)-(1-oxo-3-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)carbamate, 16a
Following the general procedure outlined, N-Boc-L-phenylalanine (0.250 g, 0.94 mmol) and
4-trifluorophenyl hydrazine (0.30 g, 1.75 mmol) was transformed following flash chromatography
into the title compound as a golden-brown solid (0.330 g, 83%); Rf 0.39 (DCM/EtOH/NH3 [200:8:1]);
m.p. 157–159 ◦C;
1156, 1103, 1067, 835, 716, 515 cm−1
υ
max (ATR) 3324 (N-H), 3274 (N-H), 2931, 2851, 1686 (C=O), 1660 (C=O), 1520, 1328,
δH (400 MHz, CDCl3) 7.93 (1H, bs, NH), 7.39 (2H, d, J 8, Ar-H),
;
7.36–7.32 (3H, m, Ar-H), 7.26–7.22 (2H, m, Ar-H), 6.61 (2H, d, J 8, Ar-H), 6.18 (1H, bs, NH), 5.09 (1H, bd,
J 7, NH), 4.46 (1H, q, J 8, CH2CHCO), 3.12 (2H, d, J 8, CH2CHCO), 1.47 (9H, s, C(CH3)3); δC (176 MHz,
CDCl3) 171.6 (NHCHCO), 150.3 (NHCOO), 135.96 (Ar-C), 129.3 (Ar-C), 128.9 (Ar-C), 127.2 (Ar-C),
126.4 (Ar-C), 126.4 (Ar-C), 125.2 (Ar-C), 123.6 (CF3), 112.7 (Ar-C), 80.9 (C(CH3)3), 54.5 (NHCHCO),
33.5 (CH2CHCO), 28.3 (C(CH3)3); δF (376 MHz, CDCl3) -61.56 (3F, s, CF3); m/z (ES+) 424 (MH+), 446
(MNa+), 869 (2M + Na+); HRMS (ES+) Found MH+, 424.1847 (C21H25F3N3O3 requires 424.1843).
tert-Butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)carbamate, 17a
Following the general procedure outlined, N-Boc-L-alanine (0.250 g, 1.32 mmol) and 4-trifluorophenyl
hydrazine (0.30 g, 1.75 mmol) was transformed following flash chromatography into the title compound
◦
as a dark brown solid (0.425 g, 93%); Rf 0.37 (DCM/EtOH/NH3 [200:8:1]); m.p. 126–131 C;
υ
max
−1
(ATR) 3321 (N-H), 2929, 2851, 1683 (C=O), 1664 (C=O), 1617, 1524, 1322, 1157, 1101, 1066, 830, 641 cm
;
δH (400 MHz, CDCl3) 8.50 (1H, bs, NH), 7.46 (2H, d, J 8, Ar-H), 6.87 (2H, d, J 8, Ar-H), 6.34 (1H, bs,
NH), 5.07 (1H, bd, J 6, NH), 4.40 (1H, m, CH3CH), 1.50 (9H, s, C(CH3)3), 1.43 (3H, d, J 7, CH3CH);
δC (176 MHz, CDCl3) 171.7 (NHCHCO), 158.7 (NHCOO), 129.3 (Ar-C), 128.9 (Ar-C), 127.2 (Ar-C),
126.4 (CF3), 112.8 (Ar-C), 80.9 (C(CH3)3), 54.6 (NHCHCO), 33.7 (CHCH3), 28.3 (C(CH3)3); δF (376 MHz,
CDCl3) -61.55 (3F, s, CF3); m/z (ES+) 348 (MH+), 370 (MNa+), 717 (2M + Na+); HRMS (ES+) Found
MNa+, 370.1352 (C15H20F3N3O3Na requires 370.1349).
tert-Butyl 2-oxo-2-(2-phenylhydrazinyl)ethylcarbamate, 18a
Following the general procedure outlined, N-Boc-L-Glycine (0.50 g, 2.9 mmol) and phenyl hydrazine
(0.30 mL, 3.1 mmol) was transformed following flash chromatography into the title compound as a
white solid (0.30 g, 39%); Rf 0.1 (n-hexane/EtOAc 7:3) as a mixture of rotamers in the ratio [5:1] by
NMR @ 25 ◦C; m.p. 120–122 ◦C;
(C=O), 1519, 1488, 1418, 1375, 1260, 1192, 1106, 1036, 874, 760 cm
υ
(ATR) 3348 (NH), 3274 (NH), 2922, 2852, 1746, 1652 (C=O), 1640
max
−1
; δH (700 MHz, DMSO-d6) 9.59 (1H,
s, NHNHPh), 9.08 (0.2H, s, NHNHPh), 7.85 (0.22H, s, NHNHPh), 7.65 (1H, s, NHNHPh), 7.17 (0.45H, t,
J 8, Ar-H), 7.08 (2H, t, J 8, Ar-H), 7.02 (1H, t, J 5, BocNH), 6.74 (0.23H, t, J 8, Ar-H), 6.71 –6.63 (3H, m,
Ar-H), 6.61 (0.23H, m, Ar-H), 3.68 (0.34H, d, J 6, BocNHCH2), 3.58 (2H, d, J 5, BocNHCH2), 1.37 (9H, s,
(CH3)3COC(O)NH), 1.34 (1.33H, s, (CH3)3COC(O)NH); δC (176 MHz, DMSO-d6) 174.2 (CONH), 169.9
(CONH), 156.5 ((CH3)3COC(O)NH), 149.9 (Ar-C), 149.1 (Ar-C), 129.3 (Ar-C), 119.0 (Ar-C), 112.8 (Ar-C),
112.6 (Ar-C), 78.7 (NHCH2), 78.5 (NHCH2), 42.7 ((CH3)3CO), 31.9 ((CH3)3CO), 28.8 ((CH3)3CO), 22.8
((CH3)3CO); m/z (ES+) 266 (MH+), 531 (2M + H+); HRMS (ES+) Found MH+, 266.24985 (C13H20O3N3
requires 266.14992).
tert-Butyl (2-(2-(4-fluorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 19a
Following the general procedure outlined, N-Boc-L-Glycine (0.25 g, 1.45 mmol) and 4-fluorophenyl
hydrazine (0.20 g, 1.57 mmol) was transformed following flash chromatography (DCM/EtOH/NH3
[600:8:1], [400:8:1], [200:8:1]) into the title compound as a light brown solid (0.32 g, 78%) as a mixture of
rotamers [4:1] by NMR @ 25 ◦C; Rf 0.32 (DCM/EtOH/NH3 [200:8:1]); m.p. 127–129 ◦C;
3364 (NH), 3269 (NH), 3132, 2984, 1652 (C=O), 1505, 1393, 1370, 1269, 1225, 1224, 1162, 1029, 1007 cm
υ
(ATR)
max
−1
;
NMR data given for major rotamer δH (700 MHz, CDCl3) 8.35 (1H, bs, (CH3)3COC=ONH), 6.89 (2H, t,
J 17, Ar-H), 6.75 (2H, dd, J 9, 4, Ar-H), 5.30 1H, bs, NHC=O), 4.07 (1H, bs, NH), 3.85 (2H, d, J 6, CH2NH),
1.45 (9H, s, (CH3)3COC=ONH); δC (176 MHz, CDCl3) 170.4 (C=O), 157.4 (C=O), 158.6 (Ar-C) 143.7
δ

Molecules 2019, 24, 811
8 of 14
(Ar-CNH), 115.7 (Ar-C), 114.9 (Ar-C), 81.0 ((CH3)3COC=ONH) 43.5 (CH2), 28.7 ((CH3)3COC=ONH);
δF (376MHz, CDCl3) -123.19 (Ar-F); m/z (ES+) 284 (MH+), 306 (MNa+), 589 (2M + Na+); HRMS (ES+)
Found MH+, 284.1412 (C13H19N3O3F requires 284.1410).
tert-Butyl (2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 20a
Following the general procedure outlined, N-Boc-L-Glycine (0.25 g, 1.45 mmol) and 4-chlorophenyl
hydrazine (0.22 g, 1.57 mmol) was transformed following flash chromatography into the title compound
◦
as a brown solid (0.41 g, 96%) as a mixture of rotamers [4:1] by NMR @ 25 C; Rf 0.2 (n-hexane/EtOAc
◦
[1:1]); m.p. 118–120 C;
υ
(ATR) 3365, 3275 (NH), 3122, 3044, 2929, 2851, 1697, 1650 (C=O),
max
1525, 1491, 1441, 1391, 1369, 1311, 1268, 1241, 1158, 1091, 1053 cm−1; NMR data given for major
rotamer δH (700 MHz, CDCl3) 8.07 (1H, bs, C=ONH), 7.17 (2H, d, J 9, Ar-H), 6.76 (2H, d, J 9, Ar-H),
5.16 (1H, bs, NH), 3.87 (2H, d, J 7, CH2NH), 3.45 (1H, bs, NH), 1.47 (9H, s, (CH3)3COC=ONH); δC
(176 MHz, CDCl3) 169.0 (C=O), 156.5 (C=O), 146.2 (Ar-CCl), 129.1 (Ar-C), 126.2 (Ar-CNH), 114.8 (Ar-C),
80.4 ((CH3)3COC=ONH), 43.8 (CH2NH), 28.3 ((CH3)3COC=ONH); m/z (ES+) 300 ([35Cl]MH+), 302
([37Cl]MH+), 322 ([35Cl]MNa+), 324 ([37Cl]MNa+), 623 ([35,35Cl]2M+Na+), 625 ([35,37Cl]2M + Na+), 627
([37,37Cl]2M + Na+); HRMS (ES+) Found [35Cl]MH+, 300.1134 (C13H19N3O335Cl requires 300.1115).
tert-Butyl (2-(2-(3-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 21a
Following the general procedure outlined, N-Boc-L-Glycine (0.25 g, 1.45 mmol) and 3-chlorophenyl
hydrazine hydrochloride (0.28 g, 1.57 mmol) was transformed following flash chromatography into
the title compound as a pale brown solid (0.34 g, 80%) as a mixture of rotamers [4:1] by NMR
◦
◦
@ 25 C; Rf 0.38 (DCM/EtOH/NH3 [200:8:1]); m.p. 119.5–121.7 C;
υ
(ATR) 3343 (NH), 3269
max
(NH), 3077, 2980, 2932, 1659 (C=O), 1597, 1512, 1490, 1368, 1242, 1230, 1156, 1046, 1030 cm−1; NMR
data given for major rotamers δH (700 MHz, CDCl3) 8.09 (1H, bs, NH), 7.12 (1H, t, J 8, Ar-H), 6.85
(1H, ddd, J 8, 2, 1, Ar-H), 6.81 (1H, dd, J 2, Ar-H), 6.70 (1H, ddd, J 8, 2, 1, Ar-H), 5.17 (1H, bs,
NH), 3.88 (2H, d, J 6, CH2NH), 1.47 (9H, s, (CH3)3COC=ONH); δC (176 MHz, CDCl3) 170.0 (C=O),
157.5 (C=O), 148.9(Ar-C), 135.1 (Ar-CNH), 130.2 (Ar-C), 121.3 (Ar-C), 113.4 (Ar-C) 111.8 (Ar-C), 81.2
((CH3)3COC=ONH), 28.3 ((CH3)3COC=ONH); m/z (ES+) 300 ([35Cl]MH+), 302 ([37Cl]MH+), 322
([35Cl]MNa+), 324 ([37Cl]MNa+), 623 ([35,35Cl]2M + Na+), 625 ([35,37Cl]2M + Na+), 627 ([37,37Cl]2M +
Na+); HRMS (ES+) Found [35Cl]MH+, 300.1119 (C13H19N3O335Cl requires 300.1115).
tert-Butyl (2-(2-(2-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 22a
Following the general procedure outlined, N-Boc-L-Glycine (0.25 g, 1.45 mmol) and 2-chlorophenyl
hydrazine hydrochloride (0.28 g, 1.57 mmol) was transformed following flash chromatography into
◦
the title compound as a light brown gum (0.41 g, 95%) as a mixture of rotamers [3:1] by NMR @ 25 C;
Rf 0.50 (DCM/EtOH/NH3 [200:8:1]);
υ
(ATR) 3262 (NH), 3074, 2960, 2928, 2851, 2118, 1668 (C=O),
max
1593, 1536, 1498, 1456, 1393, 1365, 1281, 1254, 1226, 1171, 1053, 1035 cm−1; NMR data given for major
rotamer δH (700 MHz, CDCl3) 8.23 (1H, bs, NH), 7.31 (1H, d, J 8, Ar-H), 7.14 (1H, t, J 8, Ar-H), 6.88
(1H, dd, J 8, J 1, Ar-H), 6.84 (1H, t, J 8, Ar-H), 6.40 (1H, bs, NH), 3.90 (2H, d, J 6, CH2NH), 3.47 (1H, bs,
NH), 1.47 (9H, s, (CH3)3COC=ONH); δC (176 MHz, CDCl3) 169.7 (C=O), 156.9 (C=O), 143.5 (Ar-CCl),
129.5 (Ar-C), 127.6 (Ar-C), 121.4 (Ar-C), 119.5 (Ar-CNH), 113.5 (Ar-C), 80.9 ((CH3)3COC=ONH), 43.5
(CH2NH), 28.2 ((CH3)3COC=ONH); m/z (ES+) 300 ([35Cl]MH+), 302 ([37Cl]MH+), 322 ([35Cl]MNa+),
324 ([37Cl]MNa+), 623 ([35,35Cl]2M + Na+), 625 ([35,37Cl]2M + Na+), 627 ([37,37Cl]2M + Na+); HRMS
(ES+) Found [35Cl]MH+, 300.1108 (C13H19N3O335Cl requires 300.1115).
4.1.2. Synthesis of Benzoxa-[2,1,3]-Diazole Peptidomimetics—General Procedure
N-Boc amino acid hydrazides (1.20 equiv.) were dissolved in 4 M HCl solution in dioxane (3 mL)
and stirred for 30 min at room temperature. The solvent was then removed in vacuo, the resulting
solid was suspended in THF (3 mL) and Et3N (3 equiv.) was added. The solution was cooled to
0 ◦C, before treating with 7-chlorobenzoxa-[2,1,3]-diazole-4-sulphonyl chloride (50 mg, 0.20 mmols,

Molecules 2019, 24, 811
9 of 14
1 equiv.) before warming to room temperature and stirring for 2 h. The mixture was then poured
into sat. aq. NH4Cl (20 mL). The aqueous layer was separated and extracted with EtOAc (20 mL).
The combined organic layers were dried over MgSO4, filtered, concentrated and dried in vacuo. Flash
chromatography (DCM/EtOH/NH3 [600:8:1], [400:8:1], [200:8:1]) afforded the desired sulphonamides.
7-Chloro-N-(2-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)ethyl)benzo[c][1,2,5]oxadiazole-4-
sulfonamide, 9
Following the general procedure outlined, tert-Butyl 2-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)
ethylcarbamate, 13a (79 mg, 0.24 mmol) was transformed following flash chromatography into the
title compound as a pale brown solid (6 mg, 7%); Rf 0.6 (DCM/MeOH 9:1) as a mixture of rotamers in
the ratio [7:1] by NMR @ 25 ◦C; m.p. 211–213 ◦C;
1418, 1326, 1155, 1107, 1062, 952, 834, 632 cm−1
υ
max (ATR) 332, 3158, 1685 (C=O), 1614, 1524, 1473,
;
δH (500 MHz, DMSO-d6) 9.88 (1H, s, Ar-NHNH), 9.31
(0.13H, s, Ar-NHNH), 8.73 (1H, t, J 6, SO2NH), 8.46 (0.14H, s, Ar-NHNH), 8.41 (0.14H, t, J 6, SO2NH),
8.26 (1H, s, Ar-NHNH), 8.02 (1H, d, J 8, Ar-H), 7.95 (0.16H, d, J 7, Ar-H), 7.91 (0.17H, d, J 7, Ar-H), 7.85
(1H, d, J 8, Ar-H), 7.57 (0.34H, d, J 8, Ar-H), 7.43 (2H, d, J 8, Ar-H), 6.79 (0.32H, d, J 8, Ar-H), 6.68 (2H,
d, J 8, Ar-H), 3.94 (0.25H, d, J 6, NHCH2), 3.88 (2H, d, J 6, NHCH2); δC (126 MHz, DMSO-d6) 168.4
(C=O), 152.6 (Ar-C), 149.4 (Ar-C), 146.2 (Ar-C), 134.5 (Ar-C), 131.5 (Ar-C), 129.1 (Ar-C), 126.7 (Ar-C),
−
125.6 (Ar-C), 112.1 (Ar-C), 44.3 (NHCH2); δF (376 MHz, DMSO) -59.71, -59.89; m/z (ES ) 448 ([35Cl]M-),
450 ([37Cl]M−), 897 ([35Cl,35Cl]2M−), 899 ([35Cl,37Cl]2M−), 900 ([37Cl,37Cl]2M−); HRMS (ES−) Found
[35Cl]M−, 448.00931 (C15H1035ClF3N5O4S requires 448.00996).
(S)-7-Chloro-N-(2-oxo-1-phenyl-2-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)benzo[c][1,2,5]
oxadiazole-4-sulfonamide, 14b
Following the general procedure outlined, tert-butyl (S)-(2-oxo-1-phenyl-2-(2-(4-(trifluoromethyl)
phenyl)hydrazineyl)ethyl)carbamate, 14a (97 mg, 0.24 mmol) was transformed following flash
chromatography into the title compound as a pale yellow solid (66 mg, 63%); Rf 0.46 (DCM/EtOH/NH3
[200:8:1]); m.p. 183–187 ◦C;
υ
(ATR) 3275 (N-H), 2930, 1693 (C=O), 1618, 1521, 1325, 1161, 1105,
max
1067, 840, 800, 636, 619, 590, 570, 530, 504, 491 cm−1
; δH (700 MHz, DMSO-d6) 10.20 (1H, s, NH), 9.35
(1H, bs, NH), 8.36 (1H, s, NH), 7.97 (1H, d, J 7, Ar-H), 7.82 (1H, d, J 7, Ar-H), 7.32–7.27 (4H, m, Ar-H),
7.18–7.15 (3H, m, Ar-H), 6.44 (2H, d, J 8, Ar-H), 5.30 (1H, s, NHCHCO); δC (176 MHz, DMSO-d6) 168.9
(NHCHCO), 152.3 (Ar-C), 148.9 (Ar-CNO), 145.3 (Ar-CNO), 137.1 (Ar-C), 134.9 (Ar-C), 131.1 (Ar-C),
128.9 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 127.9 (Ar-C),127.7 (Ar-C), 126.4 (Ar-C), 125.6 (Ar-C), 124.5 (CF3),
111.6 (Ar-C), 59.0 (NHCHCO); m/z (ES+) 526 ([35Cl] MH+), 528 ([37Cl] MH+), 548 ([35Cl] MNa+), 550
([37Cl] MNa+), 1073 ([35,35Cl] 2M + Na+), 1075 ([35,37Cl] 2M + Na+), 1077 ([37,37Cl] 2M+Na+); HRMS
(ES+) Found [35Cl]MNa+, 526.0565 (C21H16F3N5O4S35Cl requires 526.0558).
(S)-1-((7-Chlorobenzo[c][1,2,5]oxadiazol-4-yl)sulfonyl)-N0-(4-(trifluoromethyl)phenyl)pyrrolidine-2-
carbohydrazide, 15b
Following the general procedure outlined, tert-butyl (S)-2-(2-(4-(trifluoromethyl)phenyl)hydrazine-1-
carbonyl)pyrrolidine-1-carboxylate, 15a (89 mg, 0.24 mmol) was transformed following flash
chromatography into the title compound as a yellow solid (82 mg, 85%); Rf 0.46 (DCM/EtOH/NH3
◦
[200:8:1]); m.p. 166–170 C;
1066, 946, 836, 616 cm
υ
max (ATR) 3324 (N-H), 2929, 2851, 1682 (C=O), 1621, 1574, 1325, 1146, 1102,
−1
; δH (700 MHz, DMSO-d6) 10.09 (1H, s, NH), 8.39 (1H, bs, NH), 8.08 (1H, d, J 7,
Ar-H), 7.86 (1H, d, J 7, Ar-H), 7.44 (2H, d, J 9, Ar-H), 6.82 (2H, d, J 9, Ar-H), 4.43 (1H, dd, J 9, 4, NCHCO),
3.61–3.57 (1H, ddd, J 10, 7, 5, NCH2(CH2)2), 3.47–3.41 (1H, dt, J 10, 7, NCH2(CH2)2), 1.26–0.96 (4H,
m, NCH2(CH2)2); δC (176 MHz, DMSO-d6) 171.4 (NHCHCO), 152.8 (Ar-C), 149.5 (Ar-CNO), 146.3
(Ar-CNO), 136.8 (Ar-C), 131.2 (Ar-C), 127.7 (Ar-C), 126.5 (Ar-C), 126.3 (Ar-C), 125.5 (Ar-C), 124.7 (CF3),
111.9 (Ar-C), 60.7 (NCHCO), 49.3 (NCH2(CH2)2), 33.9 (NCH2(CH2)2), 24.9 (NCH2(CH2)2); m/z (ES+)
490 ([35Cl] MH+), 492 ([37Cl] MH+); HRMS (ES+) Found [35Cl]MH+, 490.0555 (C18H16F3N5O4S35Cl
requires 490.0558).

Molecules 2019, 24, 811
10 of 14
(S)-7-Chloro-N-(1-oxo-3-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)benzo[c]
[1,2,5]oxadiazole-4-sulfonamide, 16b
Following the general procedure outlined tert-butyl (S)-(1-oxo-3-phenyl-1-(2-(4-(trifluoromethyl)
phenyl)hydrazineyl)propan-2-yl)carbamate, 16a (100 mg, 0.24 mmol) was transformed following
flash chromatography into the title compound which was isolated as a brown solid (91 mg, 85%); Rf
◦
0.41 (DCM/EtOH/NH3 [200:8:1]); m.p. 80–84 C; υm−a1x (ATR) 3321 (N-H), 2932, 2854, 1677 (C=O), 1617,
1323, 1156, 1109, 1065, 834, 700, 632, 618, 577, 488 cm
; δH (700 MHz, DMSO-d6) 10.13 (1H, s, NH), 9.06
(1H, d, J 9, NH), 8.43 (1H, bs, NH), 7.76 (1H, d, J 7, Ar-H), 7.71 (1H, d, J 7, Ar-H), 7.41 (2H, d, J 9, Ar-H),
7.00–6.96 (2H, m, Ar-H), 6.84–6.79 (3H, m, Ar-H), 6.70 (2H, d, J 9, Ar-H), 4.16–4.12 (1H, m, CH2CHCO),
2.87 (1H, dd, J 14, 4, CH2CHCO), 2.68 (1H, dd, J 14, 11, CH2CHCO); δC (176 MHz, DMSO-d6) 170.9
(NHCHCO), 152.6 (Ar-C), 148.9 (Ar-CNO), 144.7 (Ar-CNO), 136.9 (Ar-C), 133.9 (Ar-C), 130.7 (Ar-C),
129.5 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 126.5 (Ar-C), 126.2 (Ar-C), 126.2 (Ar-C), 125.5 (Ar-C), 124.6 (CF3),
111.8 (Ar-C), 57.4 (NHCHCO), 38.0 (CH2CHCO); m/z (ES+) 540 ([35Cl] MH+), 542 ([37Cl] MH+), 562
([35Cl] MNa+), 564 ([37Cl] MNa+), 1101 ([35,35Cl] 2M + Na+), 1103 ([35,37Cl] 2M + Na+), 1105 ([37,37Cl]
2M + Na+). HRMS (ES+) Found [35Cl]MH+, 540.0642 (C22H1835ClF3N5O4S requires 540.0715).
(S)-7-Chloro-N-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)benzo[c][1,2,5]
oxadiazole-4-sulfonamide, 17b
Following the general procedure outlined, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)
hydrazineyl)propan-2-yl)carbamate, 17a (82 mg, 0.24 mmol) was transformed following flash
chromatography into the title compound as a pale yellow solid (55 mg, 60%); Rf 0.34 (DCM/EtOH/NH3
[200:8:1]); m.p. 127–131 ◦C;
υ
(ATR) 3388 (N-H), 3315 (N-H), 3274, 2929, 2851, 1667 (C=O), 1619,
max
1323, 1159, 1110, 1067, 949, 836, 633, 597, 579, 511 cm−1
; δH (700 MHz, DMSO-d6) 9.90 (1H, bs, NH),
8.80 (1H, d, J 8, NH), 8.24 (1H, bs, NH), 8.00 (1H, d, J 7, Ar-H), 7.82 (1H, d, J 7, Ar-H), 7.39 (2H, d, J 9,
Ar-H), 6.61 (2H, d, J 9, Ar-H), 4.20 – 4.15 (1H, m, CH3CHCO), 1.26 (3H, d, J 7, CH3CHCO); δC (176 MHz,
DMSO-d6) 171.4 (NHCHCO), 152.5 (Ar-C), 149.2 (Ar-CNO), 145.6 (Ar-CNO), 134.4 (Ar-C), 131.2 (Ar-C),
128.9 (Ar-C), 128.3 (Ar-C), 126.5 (Ar-C), 125.4 (Ar-C), 124.6 (CF3), 111.7 (Ar-C), 51.2 (NHCHCO), 19.8
(CHCH3); m/z (ES+) 464 ([35Cl] MH+), 466 ([37Cl] MH+); HRMS (ES+) Found [35Cl]MH+, 464.0400
(C16H14F3N5O4S35Cl requires 464.0402).
7-Chloro-N-(2-oxo-2-(2-phenylhydrazinyl)ethyl)benzo[c][1,2,5]oxadiazole-4-sulfonamide, 10
Following the general procedure outlined, tert-butyl 2-oxo-2-(2-phenylhydrazinyl)ethylcarbamate, 18a
(64 mg, 0.24 mmol) was transformed following flash chromatography into the title compound as a
yellow solid (41 mg, 55%); Rf 0.4 (DCM/MeOH 9:1) as a mixture of rotomers in the ratio [4:1] by NMR
@ 25 ◦C; m.p. 209–211 ◦C;
υ
(ATR) 3262 (NH), 1738, 1647 (C=O), 1606, 1530, 1494, 1410, 1346, 1190,
max
1157, 947, 834, 747 cm−1
; δH (500 MHz, DMSO-d6) 9.70 (1H, s, Ar-NHNH), 9.13 (0.24H, s, Ar-NHNH),
8.68 (1H, t, J 6, SO2NH), 8.35 (0.24H, t, J 6, SO2NH), 8.00 (1H, d, J 7, Ar-H), 7.94 (0.29H, d, J 7, Ar-H),
7.90 (0.27H, d, J 7, Ar-H), 7.84 (1H, d, J 7, Ar-H), 7.60 (1H, s, Ar-NHNH), 7.22 (0.55H, t, J 7, Ar-H),
7.10 (2H, t, J 7, Ar-H), 6.80 (0.25H, t, J 7, Ar-H), 6.71–6.66 (1.6H, m, Ar-H), 6.58 (2H, d, J 7, Ar-H), 3.96
(2H, d, J 6, NHCH2), 3.85 (2H, d, J 6, NHCH2); δC (126 MHz, DMSO-d6) 168.2 (C=O), 149.47 (Ar-C),
149.43 (Ar-C), 146.1 (Ar-C), 134.4 (Ar-C), 131.5 (Ar-C), 129.8 (Ar-C), 129.3 (Ar-C), 125.5 (Ar-C), 119.2
(Ar-C), 112.7 (Ar-C), 44.4 (NHCH2); m/z (ES+) 382 ([35Cl]MH+), 384 ([37Cl]MH+); HRMS (ES+) Found
[35Cl]MH+, 382.03702 (C14H1335ClN5O4S requires 382.03713).
7-Chloro-N-(2-(2-(4-fluorophenyl)hydrazineyl)-2-oxoethyl)benzo[c][1,2,5]oxadiazole-4-sulfonamide,
19b
Following the general procedure outlined, tert-butyl (2-(2-(4-fluorophenyl)hydrazineyl)-2-oxoethyl)
carbamate, 19a (68 mg, 0.24 mmol) was transformed following flash chromatography into the title
◦
compound as a brown solid (46 mg, 57%) as a◦mixture of rotamers [4:1] by NMR @ 25 C; Rf 0.48
(DCM/EtOH/NH3 [200:8:1]); m.p. 180–185 C;
υ
(ATR) 3364, 3245 (NH), 2918, 2850, 1673
max

Molecules 2019, 24, 811
11 of 14
(C=O), 1507, 1453, 1414, 1367, 1346 (S=O), 1216, 1157 (S=O), 1108, 1042 cm−1; NMR data given
for major rotamer δH (700 MHz, DMSO-d6) 9.69 (1H, bs, Ar-NH), 8.63 (1H, t, J 9, CH2NH), 7.98
(1H, d, J 7, Ar-H), 7.81 (1H, d, J 7, Ar-H), 6.91 (2H, m, Ar-H), 6.56 (2H, m, Ar-H), 3.81 (2H, d, J 6,
CH2NH); δF (376 MHz, DMSO) -126.45 (Ar-F); δC (176 MHz, DMSO-d6) 167.9 (C=O), 155.6 (Ar-C),
149.1 (Ar-C), 145.8 (Ar-C), 145.7 (Ar-C), 134.1 (Ar-C), 131.2 (Ar-C), 129.0 (Ar-C), 125.3 (Ar-C), 115.5
(Ar-C), 113.8 (Ar-C), 44.1 (CH2NH); m/z (ES+) 400 ([35Cl]MH+), 402 ([37Cl]MH+), 422 ([35Cl]MNa+),
424 ([37Cl]MNa+), 799 ([35,35Cl]2M + H+), 803 ([35,37Cl]2M + H+), 804 ([37,37Cl]2M + H+), 821
([35,35Cl]2M + Na+), 823 ([35,37Cl]2M + Na+), 825 ([37,37Cl]2M + Na+); HRMS (ES+) Found [35Cl]MH+,
400.0271 (C14H12N5O4FS35Cl requires 400.0283).
7-Chloro-N-(2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoethyl)benzo[c][1,2,5]oxadiazole-4-sulfonamide,
20b
Following the general procedure outlined, tert-butyl (2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoethyl)
carbamate, 20a (72 mg, 0.24 mmol) was transformed following flash chromatography into the title
◦
compound as a brown solid (35 mg, 43%) as a mixture of rotamers [6:1] by NMR @ 25 C; Rf 0.37
(DCM/EtOH/NH3 [200:8:1]); m.p. 174–177 ◦C;
υ
(ATR) 3326, 3277 (NH), 3151, 3932, 2856, 1668
max
(C=O), 1523, 1491, 1325 (S=O), 1158 (S=O), 1107, 1067, 1043 cm−1; NMR data given for major rotamer
δH (700 MHz, DMSO-d6) 9.72 (1H, bs, Ar-NH), 8.62 (1H, t, J 9, NHCH2), 7.98 (1H, d, J 7, Ar-H), 7.81 (1H,
d, J 7, Ar-H), 7.72 (1H, bs, NH), 7.10 (2H, d, J 7, Ar-H), 6.55 (2H, d, J 7, Ar-H), 3.81 (2H, d, J 6, CH2NH);
δC (176 MHz, DMSO-d6) 168.0 (C=O), 149.1 (Ar-C), 148.3 (Ar-C), 145.8 (Ar-C), 134.2 (Ar-C), 131.3
(Ar-C), 128.9 (Ar-C), 128.7 (Ar-C), 125.3 (Ar-C), 122.3 (Ar-C), 114.0 (Ar-C), 44.1 (CH2NH); m/z (ES+) 416
([35,35Cl]MH+), 418 ([35,37Cl]MH+), 420 ([37,37Cl]MH+), 438 ([35,35Cl]MNa+), 440 ([35,37Cl]MNa+), 442
([37,37Cl]MNa+), 853 ([35,35,35,35Cl]2M + Na+), 855 ([35,35,35,37Cl]2M + Na+), 857 ([35,35,37,37Cl]2M + Na+),
859 ([35,37,37,37Cl]2M + Na+), 861 ([37,37,37,37Cl]2M + Na+); HRMS (ES+) Found [35,35Cl]MH+, 415.998
(C14H12N5O4S35Cl2 requires 415.9987).
7-Chloro-N-(2-(2-(3-chlorophenyl)hydrazineyl)-2-oxoethyl)benzo[c][1,2,5]oxadiazole-4-sulfonamide,
21b
Following the general procedure outlined, tert-butyl (2-(2-(3-chlorophenyl)hydrazineyl)-2-oxoethyl)
carbamate, 21a (72 mg, 0.24 mmol) was transformed following flash chromatography into the title
compound as a pale yellow solid (29 mg, 22%) as a mixture of rotamers [5:1] by NMR @ 25 ◦C; Rf 0.29
(DCM/EtOH/NH3 [200:8:1]); m.p. 211–213 ◦C;
υ
(ATR) 3332, 3266, 3163 (NH), 2927, 2852, 1688
max
(C=O), 1597, 1524, 1469, 1425, 1371 (S=O), 1339, 1159 (S=O), 1073, 1043 cm−1; NMR data given for
major rotamer δH (700 MHz, DMSO-d6) 9.75 (1H, bs, Ar-CNH), 8.66 (1H, t, J 6, CH2NH), 8.10 (1H, s,
NH), 7.98 (1H, d, J 7, Ar-H), 7.82 (1H, d, J 7, Ar-H), 7.08 (1H, t, J 7, Ar-H), 6.68 (1H, ddd, J 8, 2, 1, Ar-H),
6.61 (1H, t, J 4, Ar-H), 6.52 (1H, ddd, J 8, 2, 1, Ar-H), 3.82 (2H, d, J 7, CH2NH); δC (176MHz, DMSO-d6)
168.13 (C=O), 150.9 (Ar-C), 149.2 (Ar-C), 145.8 (Ar-C), 134.1 (Ar-C), 131.2 (Ar-C), 130.7 (Ar-C), 128.9
(Ar-C), 125.3 (Ar-C), 118.4 (Ar-C), 114.0 (Ar-C), 111.8 (Ar-C), 111.1 (Ar-C), 44.2 (CH2NH); m/z (ES+) 416
([35,35Cl]MH+), 418 ([35,37Cl]MH+), 420 ([37,37Cl]MH+), 471 ([35,35Cl]MNa + MeOH), 473 ([35,37Cl]MNa
+ MeOH), 475 ([37,37Cl]MNa + MeOH); HRMS (ES+) Found [35,35Cl]MH+, 415.9981 (C14H12N5O4S35Cl2
requires 415.9987).
7-Chloro-N-(2-(2-(2-chlorophenyl)hydrazineyl)-2-oxoethyl)benzo[c][1,2,5]oxadiazole-4-sulfonamide,
22b
Following the general procedure outlined, tert-butyl (2-(2-(2-chlorophenyl)hydrazineyl)-2-oxoethyl)
carbamate, 22a (72 mg, 0.24 mmol) was transformed following flash chromatography into the title
◦
compound as a yellow solid (24 mg, 29%) as a mixture of rotamers [4:1] by NMR @ 25 C; Rf 0.16
(DCM/EtOH/NH3 [600:8:1]); m.p. 173–175 ◦C;
υmax (ATR) 3324 (NH), 2928, 2851, 1669 (C=O), 1626,
1574, 1526, 1343, 1311, 1243, 1159 (S=O), 1088, 1042 cm−1; NMR data given for major rotamer δH
(700 MHz, DMSO-d6) 9.85 (1H, bs, Ar-NH), 8.69 (1H, t, J 6, CH2NH), 8.00 (1H, d, J 7, Ar-H), 7.84 (1H, d,
J 7, Ar-H), 7.27 (1H, s, C=ONH), 7.21 (1H, dd, J 8, 2, Ar-H), 7.10 (1H, dt, J 8, 7, Ar-H), 6.71 (1H, m, Ar-H),

Molecules 2019, 24, 811
12 of 14
6.58 (1H, dd, J 8, 1, Ar-H), 3.85 (2H, d, J 6, CH2NH); δC (176 MHz, DMSO-d6) 168.1 (C=O), 149.2 (Ar-C),
145.8 (Ar-C), 144.5 (Ar-C), 134.2 (Ar-C), 131.2 (Ar-C), 129.5 (Ar-C), 128.9 (Ar-C), 125.3 (Ar-C), 120.6
(Ar-C), 117.5 (Ar-C), 113.2 (Ar-C), 44.1 (CH2NH); m/z (ES+) 416 ([35,35Cl]MH+), 418 ([35,37Cl]MH+), 420
([37,37Cl]MH+), 471 ([35,35Cl]MNa + MeOH), 473 ([35,37Cl]MNa + MeOH), 475 ([37,37Cl]MNa + MeOH);
HRMS (ES+) Found [35,35Cl]MH+, 415.9972 (C14H12N5O4S35Cl2 requires 415.9987).
4.2. Biological Assessment
Bacterial strains and growth media used in this study (Supporting Information, Table S4).
4.2.1. Bacterial Growth Inhibition Assays
The minimum inhibitory concentration of the compounds against all strains using stand REMA
assay protocols [14]. Briefly, 100
plate (Corning Incorporated, Corning, NY, USA). The wells in rows A to H in columns 1 received
94.88 L of growth medium (7H9 media was supplemented with 0.2% casamino acids, 24 g/mL
µL of relevant growth media was added to all wells of a sterile 96-well
µ
µ
pantothenate and 10% OADC, Beckton Dickinson, Sparks, MD, USA). Compounds were added to
rows A1-H1 (quadruplet per compound) followed by 1:2 serial dilutions across the plate to column 11
were 100
µL of excess medium was discarded from the wells in column 11. The bacterial cultures
at 0.5 McFarland standard diluted 1:25 was added to the wells in rows A to H in columns 1 to 11
(100 µL), where the wells in column 12 served as drug-free controls (positive and negative). The plates
were sealed with parafilmTM and incubated at 37 ◦C, unless 30 ◦C was stated as the optimum for the
organism. Freshly prepared filter sterilised resazurin (0.2% w/v, Sigma Aldrich, Dorset, UK) was filter
sterilised and 10 µ
L added to all wells and re-incubated at 37 ◦C or 30 ◦C for 24 h or until the positive
and negative controls showed a clear result.
4.2.2. Mammalian Cytotoxicity Determination Using the MTT Assay
In vitro chemosensitivity of Human NCI-H460 lung carcinoma cells to the agents were determined
using the MTT assay, described elsewhere [18]. Cells were exposed to the amino acid hydrazides or
benzoxa-[2,1,3]-diazole amino acid hydrazides (10 µM), or solvent (dimethyl sulphoxide; DMSO) in
quadruplicate. Solvent concentrations did not exceed 0.1% and were not cytotoxic. Chemosensitivity
and cell survival was assessed following 96 h compound exposure, with cytotoxicity relative to vehicle
control subsequently determined.
5. Conclusions
As shown by the present study, this interplay between cytotoxicity and antibacterial activity can
be readily manipulated through the substitution patterns on each component, aromatic hydrazides, the
size of the amino acid side chain and the benzoxa-[2,1,3]-diazole (Figure 3). The ease of manipulation
makes this an attractive template and a full examination of all these parameters is the subject of
ongoing efforts which will be reported in due course.
Figure 3. SAR profile of benzoxa-[2,1,3]-diazole substituted amino acid hydrazides.

Molecules 2019, 24, 811
13 of 14
Supplementary Materials: The following are available online, Table S1: Initial screen of 99 compounds against
a range of Gram-positive, Gram-negative and mycolata bacterial in a 96-well plate REMA assay at 128 g/mL.
µ
Blue cells indicate no bacterial growth. Empty cells indicate bacterial growth, Figure S1: Several of the
amino acid hydrazides presented as a mixture of rotamers by NMR, Table S2: Antibacterial activity against
Gram-positive, -negative and mycolata bacteria and Mammalian Cell Toxicity of amino acid hydrazides and
benzoxa-[2,1,3]-diazole amino acid hydrazides, expressed as MIC (
µ
M) or percentage relative to control (100%).
- = No activity from the REMA assay at 128
µg/mL, Table S3: Bacterial strains and growth media used in the
REMA assays, 1H NMR, 13C NMR for all compounds.
Author Contributions: Conceptualization and design of research, A.K.B., J.H.G., J.D.S.; methodology, A.K.B.,
J.H.G., P.G.S., J.D.S.; experimental investigation, A.K.B., A.K.B.A., J.H.G., and J.D.S.; interpreted results of
experiments, A.K.B., J.H.G., and J.D.S.; writing—original draft, J.D.S. preparation; writing—review and editing,
A.K.B., J.H.G., and P.G.S.; project administration, J.DS.; approval of final version of the manuscript, A.K.B.,
A.K.B.A., J.H.G., P.G.S., and J.D.S.
Funding: This research received no external funding.
Acknowledgments: We would also like to thank A. M. Kenwright (University of Durham) and Corinne Wills for
assistance with NMR spectroscopy and J. A. Mosely (University of Durham) for mass spectra.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
O0Neill, J. Review on Antimicrobial Resistance, Tackling Drug-Resistant Infections Globally: Final Report and
Recommendations; Wellcome Trust and UK Government: London, UK, 2016.
Houben, R.M.; Dodd, P.J. The Global Burden of Latent Tuberculosis Infection: A Re-estimation Using
Mathematical Modelling. PLoS Med. 2016, 13, e1002152. [CrossRef] [PubMed]
World Health Organization. Guidelines on the Management of Latent Tuberculosis Infection; World Health
Organization: Geneva, Switzerland, 2015.
Byrne, A.L.; Fox, G.J.; Marais, B.J. Better than a pound of cure: Preventing the development of
multidrug-resistant tuberculosis. Future Microbiol. 2018, 13, 577–588. [CrossRef] [PubMed]
Patel, R.V.; Riyaz, S.; Park, S. Bedaquiline: A new hope to treat multi-drug resistant tuberculosis. Curr. Top.
Med. Chem. 2014, 14, 1866–1874. [CrossRef] [PubMed]
Baptista, R.; Fazakerley, D.M.; Beckmann, M.; Baillie, L.; Mur, L.A. Untargeted metabolomics reveals a new
mode of action of pretomanid (PA-824). Sci. Rep. 2018, 8, 5084. [CrossRef] [PubMed]
Lechartier, B.; Cole, S.T. Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones
against Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2015, 59, 4457–4463. [CrossRef] [PubMed]
Boeree, M.J.; Heinrich, N.; Aarnoutse, R.; Diacon, A.H.; Dawson, R.; Rehal, S.; Kibiki, G.S.; Churchyard, G.;
Sanne, I.; Ntinginya, N.E.; et al. High-dose rifampicin, moxifloxacin, and SQ109 for treating tuberculosis:
A multi-arm, multi-stage randomised controlled trial. Lancet Infect. Dis. 2017, 17, 39–49. [CrossRef]
Hu, Y.-Q.; Zhang, S.; Zhao, F.; Gao, C.; Feng, L.-S.; Lv, Z.-S.; Xu, Z.; Wu, X. Isoniazid derivatives and their
anti-tubercular activity. Eur. J. Med. Chem. 2017, 133, 255–267. [CrossRef] [PubMed]
9.
10. Rivera, B.; Castellsagué, E.; Bah, I.; van Kempen, L.C.; Foulkes, W.D. Biallelic NTHL1 Mutations in a Woman
with Multiple Primary Tumors. N. Engl. J. Med. 2015, 373, 1985–1986. [CrossRef] [PubMed]
11. Piccionello, A.; Guarcello, A. Bioactive Compounds Containing Benzoxadiazole, Benzothiadiazole,
Benzotriazole. Curr. Bioact. Compd. 2010, 6, 266–283. [CrossRef]
12. Cho, Y.; Ioerger, T.R.; Sacchettini, J.C. Discovery of Novel Nitrobenzothiazole Inhibitors for Mycobacterium
tuberculosisATP Phosphoribosyl Transferase (HisG) through Virtual Screening. J. Med. Chem. 2008, 51,
5984–5992. [CrossRef] [PubMed]
13. Cosconati, S.; Hong, J.A.; Novellino, E.; Carroll, K.S.; Goodsell, D.S.; Olson, A.J. Structure-Based Virtual
0
Screening and Biological Evaluation of Mycobacterium tuberculosisAdenosine 5 -Phosphosulfate Reductase
Inhibitors. J. Med. Chem. 2008, 51, 6627–6630. [CrossRef] [PubMed]
14. Palomino, J.-C.; Martin, A.; Camacho, M.; Guerra, H.; Swings, J.; Portaels, F. Resazurin microtiter assay plate:
Simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob.
Agents Chemother. 2002, 46, 2720–2722. [CrossRef] [PubMed]

Molecules 2019, 24, 811
14 of 14
15. Scalacci, N.; Brown, A.K.; Pavan, F.R.; Ribeiro, C.M.; Manetti, F.; Bhakta, S.; Maitra, A.; Smith, D.L.; Petricci, E.;
Castagnolo, D. Synthesis and SAR evaluation of novel thioridazine derivatives active against drug-resistant
tuberculosis. Eur. J. Med. Chem. 2017, 127, 147–158. [CrossRef] [PubMed]
16. Bhakta, S.; Scalacci, N.; Maitra, A.; Brown, A.K.; Dasugari, S.; Evangelopoulos, D.; McHugh, T.D.;
Mortazavi, P.N.; Twist, A.; Petricci, E.; et al. Design and Synthesis of 1-((1,5-Bis(4-chlorophenyl)-2-methyl-
0
1H-pyrrol-3-yl)methyl)-4-methylpiperazine (BM212) andN-Adamantan-2-yl-N -((E)-3,7-dimethylocta-2,6-
dienyl)ethane-1,2-diamine (SQ109) Pyrrole Hybrid Derivatives: Discovery of Potent Antitubercular Agents
Effective against Multidrug-Resistant Mycobacteria. J. Med. Chem. 2016, 59, 2780–2793. [PubMed]
17. Ricci, G.; Maria, F.; Antonini, G.; Turella, P.; Bullo, A.; Stella, L.; Filomeni, G.; Federici, G.; Caccuri, A.
7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases
Mechanism of Action of Potential Anticancer Drugs. J. Biol. Chem. 2005, 280, 26397–26405. [CrossRef]
[PubMed]
18. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef]
Sample Availability: Samples of all compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).