Diaryltriazenes as antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA) and Mycobacterium smegmatis

Article abstract of DOI:10.1016/j.ejmech.2016.12.060

Diaryltriazene derivatives were synthesized and evaluated for their antimicrobial properties. Initial experiments showed some of these compounds to have activity against both methicillin-resistant strains of Staphylococus aureus (MRSA) and Mycobacterium s

Full text of DOI:10.1016/j.ejmech.2016.12.060

Accepted Manuscript
Diaryltriazenes as antibacterial agents against methicillin resistant Staphylococcus
aureus (MRSA) and Mycobacterium smegmatis
Jure Vajs, Conor Proud, Anamaria Brozovic, Martin Gazvoda, Adrian Lloyd, David I.
Roper, Maja Osmak, Janez Košmrlj, Christopher G. Dowson
PII:
S0223-5234(16)31066-2
10.1016/j.ejmech.2016.12.060
EJMECH 9151
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 November 2016
Revised Date: 28 December 2016
Accepted Date: 30 December 2016
Please cite this article as: J. Vajs, C. Proud, A. Brozovic, M. Gazvoda, A. Lloyd, D.I. Roper, M.
Osmak, J. Košmrlj, C.G. Dowson, Diaryltriazenes as antibacterial agents against methicillin resistant
Staphylococcus aureus (MRSA) and Mycobacterium smegmatis, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.060.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Diaryltriazenes as Antibacterial Agents Against
Methicillin Resistant Staphylococcus aureus
(MRSA) and Mycobacterium smegmatis
Jure Vajs,a Conor Proud,b Anamaria Brozovic,c Martin Gazvoda,a Adrian Lloyd,b
David I. Roper,b Maja Osmak,c, * Janez Košmrlj a, * and Christopher G. Dowson b, *
aFaculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113,
SI-1000 Ljubljana, Slovenia
bSchool of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL,
United Kingdom
cDivision of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54,
HR-10000 Zagreb, Croatia
KEYWORDS: Antibacterial agent, Methicillin resistant Staphylococcus aureus (MRSA),
Mycobacterium smegmatis, Antibiotic resistance, Triazene
ABSTRACT: Diaryltriazene derivatives were synthesized and evaluated for their antimicrobial
properties. Initial experiments showed some of these compounds to have activity against both
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methicillin-resistant strains of Staphylococus aureus (MRSA) and Mycobacterium smegmatis,
with MICs of 0.02 and 0.03 µg/mL respectively. Those compounds with potent anti-
staphylococcal and anti-mycobacterial activity were not found to act as growth inhibitors of
mammalian cell lines or yeast. Furthermore, we demonstrated that one of the most active anti-
MRSA diaryltriazene derivatives was subject to very low frequencies of resistance at <10–9.
Whole genome sequencing of resistant isolates identified mutations in the enzyme that lysylates
phospholipids. This could result in the modification of phospholipid metabolism and
consequently the characteristics of the staphylococcal cell membrane, ultimately modifying the
sensitivity of these pathogens to triazene challenge. Our work has therefore extended the
potential range of triazenes, which could yield novel antimicrobials with low levels of resistance.
1. Introduction
Historically the discovery of sulphonamides and β-lactam antibiotics triggered the next 50
years of research, during which the majority of antimicrobial agents in use today were
discovered [1]. However, the emergence of multidrug-resistance among bacteria including
Staphylococcus aureus (S. aureus) and other members of the ESKAPE pathogens, and latterly
extreme drug resistance in these and Mycobacterium tuberculosis (M. tuberculosis; XDRTB),
has challenged the effectiveness of antibiotics in the advent of modern medicine. As such,
antibiotic resistance has become one of the most serious health care problems in the world [2].
This has been exacerbated by a collapse in antibiotic discovery by global pharma R&D, and
resulted in a lack of new antibiotics coming to the market [3,4]. Resistance arises either by
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alterations to the antibiotic target, by the acquisition of antibiotic modifying enzymes, or by
reducing the local concentration of antibiotic via changes in cell wall permeability or efflux.
Considering the above, there is an urgent need to develop new effective antibacterial agents
that circumvent the emergence of resistance [5]. Based on our recent research [6] we were
prompted to further explore the antimicrobial activity of 1,3-diaryltriazenes. Triazenes possess a
number of biological properties, including antifungal [7], anorexic [8], and anticancer activity
[9,10]. The mechanism of action of alkyltriazenes is generally connected with the formation of
reactive diazonium species that are able to alkylate DNA [11,12].
Here we describe the preparation and testing of 46 compounds from the 1,3-diaryltriazene
family, including 5 compounds containing an isoniazid-like (INH) structural element, and 31
compounds containing a fluorine atom, with the potential to be more biologically active than
their non-fluorinated analogues [13]. As a result, several triazenes were identified as possesing
potent antimicrobial activity towards methicillin resistant S. aureus (MRSA) and M. Smegmatis-
a model mycobacterial representative of M. tuberculosis.
2. Result and discussion
2.1. Chemistry
A series of 46 triazene derivatives 1–19 were prepared according to Scheme 1, and their
structures are depicted in Figure 1. These include 1,3-diaryltriazenes (1), their N-acyl substituted
derivatives 2–15, including the carbamate 4e, and triazenide salts 16–19.
The selection of substituents at the triazene 1,3-diphenyl rings was mainly focused on strongly
electro-withdrawing groups, in most cases possessing both –I and –R effects, whereas the
electron-donating groups were not considered. This selection was stimulated by our recent
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observation on strong in vitro activity of electron-deficient triazenes against M. tuberculosis [6].
To effectively participate in modulating the electron density, ortho- and/or para- substitution
patterns relative to the triazene group were predominantly selected.
Some 1,3-diaryltriazenes were modified by N-acylation. Nicotinoyl (14), and isonicotinoyl
(15) derivatives were tentatively considered as potential multi-target hybrid compounds of 1,3-
diaryltriazenes with isoniazid (INH) structures, potentially releasing the isonicotinoyl radical and
1,3-diaryltriazenes. INH is a highly specific first-line TB prodrug, which after activation couples
isonicotinoyl radicals to NADPH and binds irreversibly to enoyl-AcpM reductase, blocking cell
wall synthesis [14]. In addition to nicotinoyl (14) and isonicotinoyl (15) derivatives, some other
acyl analogues 2–13 were considered. Since alkyltriazenes are well known as DNA alkylating
agents [11,12], these derivatives were not investigated for genotoxicity reasons.
1,3-Diaryltriazenes 1a–h were prepared by treating the appropriately substituted anilines with
either sodium nitrite in hydrochloric acid, isoamyl nitrite, or sodium hexanitrocobaltate(III). An
acylation of the selected 1,3-diaryltriazenes 1 into N-acyl-1,3-diaryltriazenes 2–15 was achieved
by using the appropriate acyl chloride in the presence of triethylamine as a base. Acyl chlorides
of nine different aliphatic, aromatic, and heteroaromatic carboxylic acids were selected,
including acetyl, chloroacetyl, methoxyacetyl, acetoxyacetyl, benzoyl, fluorobenzoyl,
(trifluoromethyl)benzoyl, pentafluorobenzoyl, nicotinoyl, and isonicotinoyl chlorides. In
addition, four different triazenide salts were prepared from diaryltriazene 1a. The reaction with
methyl
or
ethyl
propiolate
in
the
presence
of
triethylamine
yielded
alkoxycarbonylvinyltriethylammonium salts 16a and 17a, whereas trituration with triethylamine
and potassium hydroxide gave triazenide salts 18a and 19a respectively (Scheme 1).
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Scheme 1. Synthesis of compounds 1–19a
aReagents: a) NaNO2/HCl [15]; b) isoamyl nitrite [15]; c) Na3Co(NO2)6 [16]; d) Et3N, ClCOR
[15]; e) Et3N, HC CCO2CH3 or HC CCO2C2H5 [17]; f) Et3N [17]; g) KOH [17]. For a key of
substituents, see Figure 1.
≡
≡
Figure 1. The structures of 1,3-diaryltriazenes 1, N-acylated 1,3-diaryltriazenes 2–15 and
triazenide salts 16–19.
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2. 2. Antimicrobical properties
Initial susceptibilities to compounds 1–19 were determined for M. smegmatis MC2 155,
MRSA USA300 (as a representative of Gram-positive pathogen), E. coli DH10β, as a
representative Gram-negative bacterium, and Saccharomyces cerevisiae (S. cervisiae) wild strain
as a representative eukaryote. The results are presented in Table 1. None of the examined
compounds 1–19 however were found to be toxic towards S. cerevisiae.
Table 1. The initial MIC (minimal inhibitory concentrations) and MBC (minimal bactericidal
concentration) screenings of the compounds 1–19
M. smegmatis MC2 155
MRSA USA300
E. coli DH10β
S. cerevisiae wild strain
Compd
1a
1b
1c
MICa
>128
/
MBCa
MICa
>128
>128
>128
>128
2
MBCa
MICa
>128
/
MBCa
MICa
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
MBCa
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
>128
/
/
/
>128
/
/
/
/
/
1d
1e
>128
>128
8
/
4
>128
>128
>128
>128
64
/
/
>128
1
/
/
1f
128
1
/
1g
1h
2a
2e
>128
16
/
>128
0.25
4
/
/
128
0.25
>128
>128
128
128
>128
/
16
>128
4
64
128
>128
32
>128
8
32
32
2i
/
>128
2
/
64
2j
3a
3b
3d
3k
>128
2
128
/
/
/
/
/
>128
>128
>128
>128
>128
/
/
/
/
>128
64
128
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128
>128
32
>128
32
32
64
128
64
/
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
4e
/
8
16
64
5k
>128
0.5
0.5
>128
32
6a
128
>128
>128
>128
32
>128
32
32
32
128
128
64
64
64
128
/
6e
/
>128
>128
>128
8
/
128
64
7a
/
/
7e
/
/
64
7k
>128
>128
>128
128
/
8
32
8e
128
16
8
8
32
8k
8
8
64
9e
16
0.25
>128
>128
16
0.5
>128
64
10a
10e
10k
11e
12k
13a
14e
14i
14j
14k
14l
15e
15i
15j
15k
15l
16a
17a
18a
19a
>128
>128
>128
>128
32
/
128
128
64
64
>128
32
>128
16
32
64
16
/
/
/
128
64
/
16
/
>128
2
/
64
>128
128
/
2
128
32
64
32
32
>128
>128
64
64
64
128
8
/
64
128
32
128
>128
>128
/
32
16
64
4
4
32
128
>128
>128
128
64
16
16
16
32
32
>128
16
/
128
32
128
32
32
32
32
/
>128
>128
16
32
2
2
32
4
0.0156
0.03125
0.0156
0.0156
0.0156
0.03125
0.03125
0.0156
>128
>128
/
4
32
/
0.03125
0.03125
0.5
0.5
/
/
/
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a In µg/mL.
The most potent diaryltriazene compounds identified against MRSA USA300 (16a–19a
inclusive, which had MIC values of <0.02 µg/mL) compared favorably to reported
susceptibilities for this organism: gentamycin 0.5–8 µg/mL, kanamycin >64 µg/mL, tobramycin
0.5–2 µg/mL, rifampin <0.125–0.5 µg/mL, ciprofloxacin 0.125–>64 µg/mL, levofloxacin 0.25–
32 µg/mL, gatifloxacin 0.5–4 µg/mL, vancomycin 0.25–2 µg/mL, teichoplanin <0.125–0.25
µg/mL, clindamycin <0.125–>64 µg/mL, erythromycin 8–>64 µg/mL, linezolid 2–4 µg/mL,
oxacillin 16–32 µg/mL, chloroamphenicol 4–32 µg/mL, mupirocin 0.5–>64 µg/mL, fusidic acid
<0.125–0.5 µg/mL, tetracycline <0.125–4 µg/mL, minocycline, and doxycycline both <0.125–
0.5 µg/mL [18].
Similarly, the diaryltriazenes we identified with potent antibacterial activity against M.
smegmatis compared favorably in potency to established antibiotics that this organism is resistant
to. For example, MIC values for β-lactams (amoxicillin 8–16 µg/mL, ampicillin 128 µg/mL,
carbenicillin 512 µg/mL, oxacillin 32 µg/mL cefoxitin 2–4 µg/mL and ceftriaxone 32 µg/mL)
[19] and also for vancomycin (45 µg/mL) and D-cycloserine (105 µg/mL) [20] are considerably
greater than the MICs for 18a and 19a.
The structure-activity relationship (SAR) for compounds 1–19 was determined for MRSA
USA300 strain. The MIC values, spanning from >128 µg/mL to 0.0156 µg/mL, are highly
dependent on structure. Briefly, the comparison of antibacterial activities for compounds 2a–j,
3a and 3k, having different R2 groups (H, F, Cl, Br, CF3) while keeping R4 (NO2) unchanged,
reveals a decreasing trend in MIC values according to the R2 group, so that H < Br < F < Cl <
CF3 (Table 1, Figure 2A). A comparison of the MIC values for the compounds 3a, 3b, and 3d,
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having a fixed CF3 group at R2, indicates that the NO2 group at the position 4 of the 1,3-diphenyl
rings is a crucial one, and substituting this group for Cl or CF3 deactivates this compound (Figure
2B). The above results suggest that the 4-nitro-2-(trifluoromethyl)phenyl pattern may be the key
structural element in identifying the “hit” diaryltriazene compound (R2 = CF3, R4 = NO2, Figure
2C), at least within the derivatives under this investigation that possess electron-deficient 1,3-
diaryl substituents.
Figure 2. A) The general structure of diaryltriazenes with a variable group R2; B) The general
structure of diaryltriazenes with a variable group R4; C) The structure of the parent fragment
crucial for the antibacterial activity; D) The effect of “N1 substitution pattern” on antibacterial
activity of 1,3-bis(4-nitro-2-(trifluoromethyl)phenyl)triazenes.
Also evident from Table 1 is the influence of the N-acyl group in N-acyl-1,3-diaryltriazenes on
MIC value. Higher activities were observed for the N-acyl groups that had a more electron
deficient carbonyl group (for example, comparing MICs between 2a and 3a; Table 1, Figure
2D). The trend suggests that the liberation of a triazenide anion, presumably through the reaction
of N-acyl-1,3-diaryltriazenes with intracellular nucleophilic reagents, may play a role in the
compounds antibacterial activity. This is in line with the triazenide salts 16–19 being the most
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potent killers of strain MRSA USA300. The selection of the cation in the triazenide salts 16–19
appears not to be important in the activity against MRSA USA300, although this was not found
for M. smegmatis (comparing 16a and 17a with 18a and 19a).
Interestingly, some of the examined compounds, notably 14j, showed a significant activity
against the wild type E. coli strain (Table 1). This observation, although not investigated further
here, opens the possibility of discovering novel diaryltriazene derivatives, possessing greater
potencies against Gram-negative organisms. This could be a particularly important observation
given the clinical concern currently associated with multi-drug resistant Gram-negative
infections.
As non-specific bactericidal activity usually corresponds to a lack of prokaryotic specificity
and therefore potential toxicity in mammals [21], we decided to focus on identifying potentially
membrane-damaging diaryltriazenes.
The BacLight™ assay is a particularly useful probe for determining the effect of compounds
on bacterial membrane integrity [22]. Ten of the most active diaryltriazenes (1g, 1e, 2a, 3a, 6a,
10a, 16a, 17a, 18a, and 19a) having MICs against MRSA USA300 of less than 2 µg/mL, were
selected for testing using this assay. The results of these assays on the cell membrane of this
strain are summarized in Table 2. Here it appears that all the tested compounds, with the
exception of compounds 1, are not membrane damaging.
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Table 2. MRSA USA300 membrane integrity
MRSA USA300
Compd c (µg/mL) cells with intact membrane (%)a
8
2
2
8
2
2
2
2
2
2
64.6±9.4
31.2±3.2
103.0±5.8
100.0±2.4
95.8±2.3
103.8±4.7
105.5±1.3
107.3±3.5
104.3±6.5
107.6±9.6
1e
1g
2a
3a
6a
10a
16a
17a
18a
19a
a The compounds upon which the amount of MRSA USA300 with intact membrane was 95%
and 110% are not considered to be membrane damaging. Data are shown as mean±SD obtained
from at least three independent experiments.
Nine of the above compounds (1g, 2a, 3a, 6a, 10a, 16a, 17a, 18a, and 19a), active against
both, MRSA USA300 and also M. smegmatis MC2 155, were selected for antimicrobial activity
evaluation against a variety of Gram-positive and Gram-negative bacteria. The results are
summarized in Table 3. All selected compounds were water soluble small molecules, stable in
solid state as well as in solution with common organic solvents and water. As such, they could be
relatively easily modified for further optimization [23].
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Table 3. Susceptibility (MIC, MBC) of the selected diaryltriazenes to the different Gram-
positive and Gram-negative bacteria
Organism
Compd
1g
1
2a
3a
6a
0.5
1
10a
0.5
4
16a
17a
0.0156 0.0156
0.25 0.25
0.0156 0.0156
18a
19a
MICa
MBCa
MICa
MBCa
0.25
0.5
0.03
0.5
0.0156
0.25
0.0156
0.5
MRSA wild
strain 1653
1
0.5
1
0.06
0.5
0.03125 0.5
0.125
0.5
1
0.0156
0.125
0.0156
0.125
S. pneumoniae R6
1
0.125
0.125
0.0312
5
0.0312
5
MICa
1
0.125 0.03125 0.5
2
0.0156
2
0.0156
B. subtilis ATCC
6051
MBCa
MICa
MBCa
MICa
MBCa
MICa
MBCa
MICa
MBCa
MICa
MBCa
MICa
MBCa
MICa
MBCa
32
2
4
4
4
1
16
>64
2
2
2
>128 >128 0.5
>128
>128 >128 >128
0.5
1
0.5
Vancomycin
resistant E.
faecalis 501
>128 >128 >128
>128 >128 >128
/
/
>128
>128
>128
>128
>128
>128
P. aeruginosa
PA0001
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
>128
/
>128
/
>128
>128
>128
P. aeruginosa
ATCC 27853
/
/
/
>128 >128 >128
>128 >128 >128
>128
>128
>128
K. pneumoniae
ATCC 13882
/
/
/
/
/
/
/
/
/
>128 >128 >128
>128 >128 >128
>128
>128
>128
K. pneumoniae
ATCC 700603
/
/
/
/
/
/
/
/
/
>128 >128 >128
>128 >128 >128
>128
>128
>128
A. baumannii
ATCC 19606
/
/
/
/
/
/
/
/
/
>128 >128 >128
>128 >128 >128
>128
/
>128
/
>128
/
A. baumannii
BAA1605
/
/
/
/
/
/
a In µg/mL.
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We were unable to detect activity of the selected compounds in Gram-negative strains, i.e. E.
coli (Table 1), P. aeruginosa, K. pneumonia, A. baumannii (Table 3). This may have been due to
a lack of penetration of the tested compounds through the outer membranes of these organisms,
or possibly due to efficient efflux functioning. To decipher between these two possibilities we
performed MIC evaluations of the selected compounds using an E. coli membrane permeable
strain of D22, and an E. coli strain of D22 knockout of the membrane pump N43 CGSC 5583.
The results, presented in Table 4, suggest that the inactivity of the tested compounds in the case
of Gram negative-bacteria, represented by the E. coli DH10β strain, was indeed related to the
low permeability of the Gram-negative outer membrane to these compounds. The aberrant
behaviour of compounds 6a and 10a could have arisen from their structure. These compounds
are unique amongst those tested from Table 3, in their possession of a (substituted) N-benzoyl
group. Although this may well be an issue that uniquely impinges upon the ability of 6a and 10a
to penetrate cells, the explanation of the actions of 6a and 10a may also reside with the E. coli
strain D22 used in these experiments. D22 contains a mutation in the LpxC gene which encodes
UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, an enzyme involved in
the synthesis of lipid A [24,25]. Lipid A is a lipopolysaccharide appended to the outer membrane
of E. coli. Perturbations to the synthesis of lipid A, caused by LpxC mutations, would therefore
likely impact upon the ability of hydrophobic compounds such as 6a and 10a to penetrate the
cell.
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Table 4. MIC and MBC evaluation for E. coli membrane permeable strain D22, and an E. coli
strain containing a knockout of the membrane pump N43 CGSC 5583
E. coli membrane E.
coli
knockout
of
permeable strain membrane pump N43 CGSC
D22
5583
MICa
>128
>128
>128
>128
>128
>128
>128
>128
>128
Compd MICa
MBCa
>128
>128
16
MBCa
2
/
/
/
/
/
/
/
/
/
1g
2a
1
0.5
>128
>128
0.25
0.5
0.5
0.5
3a
6a
/
/
10a
16a
17a
18a
19a
>128
>128
>128
>128
a in µg/mL.
Critically, resistance to methicillin and vancomycin underpins medically intractable infections
caused by MRSA and vancomycin resistant enterococci (VRE). The acute diaryltriazene
sensitivity of MRSA USA300, a highly resistant clinical MRSA strain, therefore prompted us to
investigate the possibility that our antimicrobial diaryltriazene compounds might sensitise these
pathogens to β-lactams and vancomycin, which would ultimately be of clinical interest.
However, no synergism [26] between the compound 16a, one of the most promising new
diaryltriazene compounds, was detected when tested with methicillin against MRSA, nor with
vancomycin against Vancomycin resistant E. faecalis (VRE). This finding led us to conclude that
the mode of action of the diaryltriazene 16a was either unrelated to the action of methicillin and
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vancomycin (bacterial peptidoglycan biosynthesis) or did not impact on the resistance
mechanisms employed by MRSA and VRE towards β-lactams and glycopeptides.
In order to better characterise the mode of action of the compound 16a, MRSA strain USA300
was grown continuously in the presence of this active compound, at progressively inhibitory
concentrations (2-, 4-, 6- and 8-times it’s MIC value), allowing resistance to develop. Following
incubation three 16a-resistant MRSA USA300 isolates were obtained, and for all cultures, the
mutation rates when exposed to the different concentrations of compound 16a was calculated
(Table 5).
Table 5. Mutation rate assaya and resistant strains isolated
Diaryltriazene 16a
16a-resistant
Concentration
(µg/mL)
isolate name
Mutation rate MIC (µg/mL)
3.96×10–6
0.03125
Isolate R1
Isolate R2
Isolate R3
/
0.03125
0.0625
0.09375
0.125
8×10–9
0.03125
8×10–9
0.0625
< 8×10–9
/
a Mutation rate assays were performed using MRSA USA300 strain.
MRSA USA300 isolates resistant to the compound 16a, and sensitive parental strains were
subjected to whole genome sequencing in order to identify the underlying genetic
polymorphisms present in resistant isolates. Resistant isolates (identified as Isolates R1, R2, and
R3) were compared to the parental (control) strain, allowing the genetic polymorphisms
differentiating these isolates to be identified (Figure 3).
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Isolate R1 contained a single 47 base pair deletion at the position 128454 of the genome,
corresponding to the position of a pseudogene in the reference, which was potentially reactivated
by this deletion. The gene was identified as Staphylococcal protein A (SpA), a known virulence
protein. The deletion occurred at amino acid (AA) position 390 of the 522AA protein, causing
the protein to shift back into frame. A comparison with the NCBI blast database revealed that
this was a common deletion found among S. aureus isolates. Cell wall associated SpA functions
to bind the Fcγ domain of human immunoglobulin [27]. This molecule consists of a hydrophobic
C-terminal, thought to be embedded within the cell membrane, flanked at the N-terminal end by
a charged tail [28,29]. The mechanism by which this protein reduces its sensitivity to the
diaryltriazene 16a therefore remains unclear; its surface location could however affect the
interaction or permeability of the cell to this compound.
Isolate R2 carried a single non-synonymous single nucleotide polymorphism (SNP) at position
1396319 when compared to the control strain. This guanine to thymine SNP altered the amino
acid sequence of fmtC, a lysylphosphatidylglycerol synthetase, an 843 amino acid protein, from
Glu307, to an ATG “stop” codon. This gene is not essential in S. aureus. The function of FmtC
appears to be to impart a positive charge on the negatively charged phospholipid membrane
bilayer [30]. This is achieved through the addition of a lysine residue, through the positive
charge of its epsilon amino group. This altering of the surface charge as a route to antibiotic
resistance is widely documented, particularly for the pore-forming peptides such as nisin [31,32].
Quenching of the membranes negative charge through the lysine addition is therefore likely to
reduce a repulsion of the negatively charged diaryltriazenes, facilitating their penetration. A loss
of FmtC function or efficacy could therefore affect the interaction between negatively charged
diaryltriazene and the negatively charged membrane leading to resistance.
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Isolate R3 carried two mutations relative to the control, a non-synonymous SNP at position
2496871, and a single thymine insertion at position 2496195, in an intergenic region. The
guanine to thymine SNP resulted in an arginine to leucine substitution at the AA-position 197 of
the 210 AA DNA-binding helix-turn-helix (HTH) domain of a possible transcriptional regulator.
Using the Basic Local Alignment Search Tool (BLAST) this gene most closely resembled a
Tetracycline Repressor (TetR) family transcriptional regulator- so named owing to their
involvement with tetracycline resistance in S. aureus [33].
The thymine insertion at position 2496195 occurred in an intergenic region prior to the
possible transcriptional regulator described above, on the forward strand, and upstream from a
hypothetical membrane protein on the reverse strand. Using the software PEPPER [34], this
region was predicted to contain a promoter sequences in the forward direction [33]. This
suggested that expression levels for the predicted transcriptional regulator could have been
affected. However, it remains unclear as to how mutations in a gene associated with tetracycline
resistance impact upon diaryltriazene resistance.
Therefore, no single target could be identified for the activity of compound 16a. Instead,
changes in MIC appear to have resulted from independent mechanisms unique to each mutant
isolate.
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Figure 3. Mutant isolates of MRSA USA300 compared to the control. The figure illustrates the
genomic locations of the different mutations relative to the control. The outer scale bar indicates
the number of base pairs from the replication origin, and can be used to compare the approximate
genomic locations for each of the mutations identified. Green indicates the location of genes on
the forward and reverse strands.
To assess the specificity and translational benefit that might arise from antimicrobials based
upon the diaryltriazene compounds used in this research we assessed the impact of the most
promising triazenide salt 16a, as well as 17a, 18a, and 19a on human keratinocytes. The results
(Figure 4) show that keratinocytes survived significantly higher concentrations of triazenide salts
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compared to those concentrations which inhibited the growth of MRSA USA300, M. smegmatis
(Table 1), MRSA wild strain, S. pneumoniae, Bacillus subtilis or Vancomycin resistant E.
faecalis (Table 3). This data clearly indicates a marked selectivity of these diaryltriazenes
towards bacterial growth inhibition.
120
100
80
16a
60
17a
40
20
0
18a
19a
0.025
0.1
0.5
2
10
25
Concentration (µg/mL)
Figure 4. Survival of healthy human keratinocytes treated with compounds 16a, 17a, 18a and
19a.
The SAR conducted suggested a strong role for the nitro group on antibacterial efficacy.
Although nitroarenes are less preferred scaffolds in drug discovery due to their potential
mutagenicity, certain drugs do contain this moiety [35]. These include chloramphenicol and
metronidazole, which are on the WHO’s Model List of Essential Medicines [36]. Despite their
potential threat, recent endeavors have shown promise in mitigating the mutagenic nature of
nitroarenes [37].
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It is interesting to note that the structures of 1,3-diaryl substituents in the triazenes identified in
this work have a similarity to benzothiazinones (BTZ043) and related compounds [14,35,38].
For antitubercular activity, it was found that these compounds need to be functionalized with a
nitro group as well as a meta- electron-withdrawing group, typically either a trifluoromethyl or
another nitro group. The nitroaromatic group was postulated to undergo bioreduction yielding a
nitrosoarene, which then reacts with an active site cysteine residue in the DprE1 enzyme to form
a semimercaptal adduct, inactivating this enzyme [14,35,38]. Recent chemical studies on
BTZ043 confirmed the postulated reactivity of this enzyme, indicating that thiolates and other
nucleophiles induce nonenzymatic reduction of the nitro groups to the corresponding nitroso
intermediates [39]. We conducted NMR experiments where equimolar amounts of reduced
glutathione (GSH) and triazenide salt 19a was incubated at room temperature in a mixture of
DMSO-d6 and phosphate buffer of pH 7.4 (1:1, v/v). The reaction mixture was monitored by 1H
NMR spectroscopy for 24 h indicating no changes in the aromatic region of the spectra. This
suggests that the molecular mechanism of action of 1,3-diarlytriazenes under this investigation
may be different to that of BTZ043.
The biological activity of alkyltriazenes has been associated with reactive diazonium
degradation products involved [11,12]. In contrast, diaryltriazenes 1a and 19a under this
investigation were found to be stable in phosphate buffer solutions at pH 7.4 with no detectable
decomposition, as indicated by TLC and NMR analyses. Thus, the molecular mechanism of
antibacterial activity in 1,3-diaryltriazenes has yet to be explored, and will be the subject of
forthcoming investigations.
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3. Conclusions
Here we have described the synthesis of a library of 1,3-diaryltriazenes, their N-acyl
derivatives, and their salts. Structure-activity relationships (SAR) revealed that the antimicrobial
properties of these compounds depended on the type of substituent group attached to the two
constituent benzene rings. Specifically, a trifluoromethyl group at position 2, and a nitro
substituent at position 4 relative to the triazene was found to be critical to its activity.
Regarding the biological activities of the new compounds, the most important result from our
study was that the selected compounds 16a, 17a, 18a and 19a were extremely active against one
of the most clinically important bacterial species, MRSA. These compounds were not found to
be membrane damaging to MRSA USA300. The molecular basis of the triazene activity against
S. aureus remained unclear, appearing to have more than one mechanism of killing based on the
observed mutations identified among resistant isolates. Where a mechanism could be
characterized, it appeared that changes in the phospholipid composition of the staphylococcal
cell membrane were ultimately responsible. In addition, the selected compounds were found to
be very effective against other Gram-positive bacteria, such as S. pneumoniae, B. subtilis,
Vancomycin resistant E. faecalis, and M. smegmatis. Promising activities of a number of the
triazene family compounds towards E. coli were also found. The majority of the tested
compounds were however inactive against Gram-negative organisms, with membrane
penetration found to be limiting in E. coli.
These compounds were also found to be inactive against lower eukaryotes (yeast), and more
importantly, exhibited only a low cytotoxicity against human keratinocytes (as compared to the
cytotoxicity observed towards certain pathogenic bacteria). These characteristics are promising
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with respect to the potential clinical utility of these triazenes for use as novel antimicrobials. Our
data suggest that 1,3-diaryltriazenes could represent a new class of antibacterial drug.
4. Experimental
4.1. Chemistry
Starting materials and solvents for the synthesis of the examined compounds were used as
obtained, and without further purification, from Aldrich, Fluka, Alfa Aesar and Maybridge
Chemical Company Ltd). Melting points were determined on a Kofler micro hot stage and were
uncorrected. NMR spectra were recorded with a Bruker Avance III 500 MHz instrument
operating at 500 MHz (1H), 126 MHz (13C) at 296 K. Proton spectra were referenced to Si(CH3)4
as the internal standard (
δ
= 0.00 ppm) and carbon chemical shifts were given against the central
= 77.0 ppm). Chemical shifts were given on the scale
line of the solvent signal (CDCl3 at
δ
δ
(parts per million) and coupling constants (J) were given in Hertz. IR spectra were obtained with
a Bruker ALPHA Platinum ATR spectrometer on a solid sample support (ATR). High-resolution
mass spectrometry (HRMS) analysis was performed using an Agilent 6224 Accurate Mass TOF
LC/MS spectrometer. Elemental analyses (C, H, N) were performed with Perkin Elmer 2400
Series II CHNS/O Analyser. The progress of all reactions was monitored on Fluka silica-gel
TLC-plates (with fluorescence indicator UV254), using ethyl acetate/petroleum ether as the
solvent system. A column chromatography was performed using Merck silica gel 60 (35−70 µm)
with solvent mixtures as specified in the corresponding experiments. All the compounds tested
1
were of
≥
95% purity, as verified by H NMR and 13C NMR spectroscopy as well as with
elemental microanalysis. The values obtained for C, H, N analyses were within
calculated values.
±0.40% of the
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The compounds 1a, 1c–1h, 2, 3a, 4–13 and 18 were prepared according to known procedures
[15,16,40]. Precursor 1,3-diaryltriazenes, i.e., 1,3-bis(2-fluoro-4-nitrophenyl)triazene, 1,3-bis(2-
methyl-4-nitrophenyl)triazene, 1,3-bis(2-bromo-4-nitrophenyl)triazene, and 1,3-bis(4-nitro-3-
(trifluoromethyl)phenyl)triazene, that were used for the acylation shown in Scheme 1 into 2i, 2j,
3k, 5k, 7k, 8k, 10k, 12k, 14i–l, 15i–l, were synthesized as described in the literature [6].
4.1.1. Preparation of 1,3-bis(2,4-bis(trifluoromethyl)phenyl)triazene (1b). An aqueous solution
of sodium nitrite (0.035 g, 0.5 mmol/10 mL) was added dropwise to the solution of 2,4-
bis(trifluoromethyl)aniline (0.229 g, 1 mmol) in 5% aqueous HCl (2.5 mL) at 0 °C. The reaction
mixture was stirred at room temperature for 20 h. Then, the solid material was filtered and
washed with water.
Pale yellow solid; yield 75%, mp 91–93 °C (MeOH/H2O); IR: 3343, 3101, 1632, 1594, 1527,
1
1487, 1455, 1426, 1347, 1306 cm–1; H NMR (500 MHz, DMSO-d6):
δ
(ppm) 7.89–7.95 (m,
+
2H), 8.08–8.18 (m, 4H), 13.25 (s, 1H); HRMS (ESI+) m/z for C16H8F12N3 [M + H]+: calcd
470.0521, found 470.0517. Anal. calcd for C16H7F12N3: C, 40.95; H, 1.50; N, 8.96; found C,
41.02; H, 1.37; N, 9.08.
4.1.2. General procedures for the synthesis of 3-acyl-1,3-diaryltriazenes 2–15
Method A. (3b, 3d, 3k) Triethylamine (0.279 mL, 2 mmol) was added to a stirred suspension
of the appropriate triazene 1 (1 mmol) in acetonitrile (10 mL) at room temperature, followed by
the addition of chloroacetyl chloride (0.155 mL, 2 mmol). After additional stirring for 10 min,
the reaction mixture was evaporated to dryness under reduced pressure. Then, water (5 mL) was
added and the water phase was washed with dichloromethane (3 × 10 mL). Combined organic
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phases were first washed with water (20 mL) and then dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure to dryness yielding the crude products 3. Thereafter, products
were recrystallized using the appropriate solvents.
Method B. (14, 15) Triethylamine (0.558 mL, 4 mmol) was added to a stirred suspension of the
appropriate triazene 1 (1 mmol) in dry acetonitrile (20 mL) at room temperature, followed by the
addition of nicotinoyl chloride (isonicotinoyl chloride in cases of 15). After additional stirring for
10–20 min, the reaction mixture was evaporated to dryness under reduced pressure, treated with
methanol, filtered off and washed with saturated NaHCO3 solution and methanol yielding the
crude products 14 and 15. Thereafter, all products were recrystallized using the appropriate
solvents.
4.1.3. General procedures for the synthesis of triazenide salts 16–19
Method C. (16, 17) Triethylamine (0.558 mL, 4 mmol) was added to a stirred suspension of
1,3-bis(4-nitro-2-(trifluoromethyl)phenyl)triazene (1a) (0.424 g, 0.5 mmol) in acetonitrile (7 mL)
at room temperature followed by the addition of methyl propiolate (ethyl propiolate in the case
of 17a). After additional stirring for 1 h, the reaction mixture was evaporated to dryness under
reduced pressure, treated with diethyl ether and filtered off, yielding the crude products 16a and
17a. Thereafter, both products were recrystallized using the appropriate solvents.
Method D. (19) A solution of KOH (0.224 g, 4 mmol) in water (20 mL) was added to a stirred
solution of 1,3-bis(4-nitro-2-(trifluoromethyl)phenyl)triazene in acetonitrile (10 mL) at room
temperature. After additional stirring for 20 min, acetonitrile was evaporated under reduced
pressure, and the precipitate was filtered off, yielding 19a. A crude product was recrystallized
from acetonitrile/water mixture.
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4.1.4. 1-(1,3-Bis(2,4-bis(trifluoromethyl)phenyl)triaz-2-en-1-yl)-2-chloroethanone (3b)
Method A. Pale yellow solid; yield 91%; mp 85–87 °C (petroleum ether); IR: 1744, 1626,
1
1596, 1492, 1345, 1304, 1274, 1232, 1160, 1120 cm–1; H NMR (500 MHz, CDCl3):
δ (ppm)
4.73–4.90 (m, 2H), 7.35–7.42 (m, 1H), 7.71–7.77 (m, 1H), 7.88–7.98 (m, 2H), 7.98–8.03 (m,
1H), 8.07–8.12 (m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm) 41.7, 118.7, 121.9 (q, J = 273
Hz), 122.2 (q, J = 273 Hz), 122.8 (q, J = 273 Hz), 122.9 (q, J = 273 Hz), 124.3–124.5 (m),
124.8–125.0 (m), 127.6 (q, J = 32 Hz), 130.0 (q, J = 32 Hz), 131.9 (q, J = 32 Hz), 132.2, 133.2
–
(q, J = 32 Hz), 136.1, 147.1, 167.8; HRMS (ESI–) m/z for C16H6F12N3 [M – COCH2Cl]–: calcd
468.0376, found 468.0380. Anal. calcd for C18H8ClF12N3O: C, 39.62; H, 1.48; N, 7.70; found: C,
39.76; H, 1.43; N, 7.60.
4.1.5 1-(1,3-Bis(4-chloro-2-(trifluoromethyl)phenyl)triaz-2-en-1-yl)-2-chloroethanone (3d)
Method A. Yellow solid; yield 96%; mp 83–85 °C (diisopropyl ether); IR: 3109, 3015, 2960,
2122, 1733, 1714, 1599, 1501, 1481, 1413 cm–1; 1H NMR (500 MHz, CDCl3):
δ (ppm) 4.74 (d, J
= 13.9 Hz, 1H), 4.82 (d, J = 13.9 Hz, 1H), 7.11–7.16 (m, 1H), 7.57–7.63 (m, 2H), 7.65–7.71 (m,
2H), 7.79–7.83 (m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm) 41.9, 119.1, 121.8 (q, J = 274
Hz), 122.2 (q, J = 274 Hz), 127.2 (q, J = 5 Hz), 127.9 (q, J = 5 Hz), 128.6 (q, J = 32 Hz), 130.4
(q, J = 32 Hz), 131.2 (q, J = 2 Hz), 132.6, 132.8, 133.2, 136.1, 136.8, 143.1, 167.8; HRMS
–
(ESI–) m/z for C14H6Cl2F6N3 [M – COCH2Cl]–: calcd 399.9848, found 399.9852. Anal. calcd
for C16H8Cl3F6N3O: C, 40.15; H, 1.68; N, 8.78; found: C, 39.91; H, 1.60; N, 8.57.
4.1.6. 1-(1,3-Bis(2-bromo-4-nitrophenyl)triaz-2-en-1-yl)-2-chloroetanone (3k)
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Method A. Yellow solid; yield 91%; mp 151–153 °C (dichloromethane/diisopropyl ether); IR:
3101, 3076, 3002, 2958, 2112, 1991, 1922, 1720, 1594, 1580 cm–1; 1H NMR (500 MHz, CDCl3):
δ
(ppm) 4.76–4.90 (m, 2H), 7.44–7.49 (m, 1H), 7.59–7.65 (m, 1H), 8.23–8.29 (m, 1H), 8.33–
8.38 (m, 1H), 8.50–8.54 (m, 1H), 8.60–8.65 (m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm)
41.8, 119.8, 122.3, 123.5, 123.5, 124.1, 128.7, 129.2, 131.6, 140.4, 148.2, 148.9, 149.8, 167.2;
–
HRMS (ESI–) m/z for C12H6Br2N5O4 [M – COCH2Cl]–: calcd 441.8792, found 441.8792. Anal.
calcd for C14H8Br2ClN5O5: C, 32.24; H, 1.55; N, 13.43; found: C, 32.31; H, 1.46; N, 13.32.
4.1.7. (1,3-Bis(2-chloro-4-nitrophenyl)triaz-2-en-1-yl)(pyridin-3-yl)methanone (14e)
Method B, reaction time: 20 min. Pale yellow solid; yield 66%; mp 128–132 °C
(MeOH/acetone); IR: 3098, 1703, 1584, 1528, 1474, 1351, 1226, 1144, 1134, 1120, 1047 cm–1;
1H NMR (500 MHz, CDCl3):
δ (ppm) 7.36–7.43 (m, 1H), 7.50–7.56 (m, 1H), 7.58–7.65 (m, 1H),
8.07–8.13 (m, 1H), 8.19–8.25 (m, 1H), 8.29–8.34 (m, 1H), 8.34–8.38 (m, 1H), 8.48–8.53 (m,
1H), 8.84–8.89 (m, 1H), 9.10–9.16 (m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm) 119.7, 122.8,
122.9, 123.3, 125.6, 126.2, 128.9, 131.8, 133.2, 134.4, 137.6, 139.1, 148.1, 148.5, 149.0, 150.7,
153.0, 168.6; HRMS for C18H11Cl2N6O5 [M + H]+: calcd 461.0162, found 461.0164. Anal. calcd
for C18H10Cl2N6O5 × 0.5 H2O: C, 46.56; H, 2.53; N, 17.61; found: C, 46.41; H, 2.70; N, 17.78.
4.1.8. (1,3-Bis(2-fluoro-4-nitrophenyl)triaz-2-en-1-yl)(pyridin-3-yl)methanone (14i)
Method B, reaction time: 10 min. Brown solid; yield 76%; mp 126–128 °C (MeOH); IR: 3091,
1699, 1586, 1526, 1483, 1417, 1346, 1308, 1261, 1241, 1216 cm–1; 1H NMR (500 MHz, CDCl3):
δ
(ppm) 7.42–7.48 (m, 1H), 7.50–7.55 (m, 1H), 7.59–7.64 (m, 1H), 8.00–8.07 (m, 2H), 8.16–
13
8.22 (m, 2H), 8.24–8.30 (m, 1H), 8.83–8.88 (m, 1H), 9.09–9.13 (m, 1H); C NMR (126 MHz,
26

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CDCl3):
δ
(ppm) 112.7 (d, J = 25 Hz), 113.4 (d, J = 25 Hz), 120.0 (d, J = 15 Hz), 120.0 (d, J =
15 Hz), 121.5, 123.3, 128.5 (d, J = 15 Hz), 128.8, 131.5, 137.8, 140.1 (d, J = 8 Hz), 148.5 (d, J
= 8 Hz), 149.6 (d, J = 8 Hz), 150.8, 153.0, 156.5 (d, J = 260 Hz), 157.4 (d, J = 260 Hz), 168.5;
+
HRMS (ESI+) m/z for C18H11F2N6O5 [M + H]+: calcd 429.0754, found 429.0747. Anal. calcd
for C18H10F2N6O5: C, 50.48; H, 2.35; N, 19.62; found: C, 50.30; H, 2.22; N, 19.42.
4.1.9. (1,3-Bis(4-nitrophenyl)triaz-2-en-1-yl)(pyridin-3-yl)methanone (14j)
Method B, reaction time: 10 min. Pale violet solid; yield 58%; mp 225–227 °C (MeOH); IR:
1
3054, 1689, 1610, 1584, 1519, 1470, 1416, 1330, 1300, 1209, 1170, 1132 cm–1; H NMR (500
MHz, CDCl3):
δ (ppm) 7.47–7.56 (m, 5H), 8.16–8.21 (m, 1H), 8.22–8.28 (m, 2H), 8.43–8.49 (m,
2H), 8.83–8.88 (m, 1H), 9.07–9.11 (m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm) 123.2, 123.3,
124.9, 125.0, 129.5, 130.3, 137.7, 140.6, 148.2, 148.3, 150.8, 151.6, 152.8, 169.2; HRMS (ESI+)
+
m/z for C18H13N6O5 [M + H]+: calcd 393.0942, found 393.0939. Anal. calcd for C18H12N6O5: C,
55.11; H, 3.08; N, 21.42; found: C, 54.86; H, 2.85; N, 21.33.
4.1.10. (1,3-Bis(2-bromo-4-nitrophenyl)triaz-2-en-1-yl)(pyridin-3-yl)methanone (14k)
Method B, reaction time: 20 min. Yellow-brown solid; yield 49%; mp 142–144 °C
(MeOH/acetone); IR: 3098, 1702, 1583, 1531, 1519, 1485, 1469, 1418, 1345, 1289, 1250, 1223
1
cm–1; H NMR (500 MHz, CDCl3):
δ (ppm) 7.33–7.39 (m, 1H), 7.50–7.55 (m, 1H), 7.57–7.61
(m, 1H), 8.13–8.18 (m, 1H), 8.19–8.25 (m, 1H), 8.38–8.43 (m, 1H), 8.48–8.51 (m, 1H), 8.65–
8.69 (m, 1H), 8.83–8.88 (m, 1H), 9.12–9.16 (m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm)
119.5, 122.7, 123.3, 123.5, 123.5, 124.0, 128.7, 129.0, 129.2, 131.8, 137.7, 141.1, 148.1, 148.8,
+
149.6, 150.7, 153.0, 168.1; HRMS (ESI+) m/z for C18H11Br2N6O5 [M + H]+: calcd 548.9152,
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found 548.9147. Anal. calcd for C18H10Br2N6O5: C, 39.30; H, 1.83; N, 15.28; found: C, 39.22; H,
1.73; N, 15.22.
4.1.11. (1,3-Bis(4-nitro-3-(trifluoromethyl)phenyl)triaz-2-en-1-yl)(pyridin-3-yl)methanone (14l)
Method B, reaction time: 10 min. Pale yellow solid; yield 36%; mp 131–133 °C (MeOH); IR:
1
3094, 2018, 1698, 1587, 1538, 1495, 1477, 1419, 1354, 1314, 1293, 1271 cm–1; H NMR (500
MHz, CDCl3): δ (ppm) 7.52–7.58 (m, 1H), 7.65–7.72 (m, 2H), 7.75–7.80 (m, 1H), 7.81–7.85 (m,
1H), 7.92–7.98 (m, 1H), 8.11–8.16 (m, 1H), 8.17–8.22 (m, 1H), 8.85–8.92 (m, 1H), 9.08–9.13
(m, 1H); 13C NMR (126 MHz, CDCl3):
δ
(ppm) 121.30 (q, J = 274 Hz), 121.33 (q, J = 274 Hz),
123.0 (q, J = 5 Hz), 123.3, 125.21 (q, J = 35 Hz), 125.25 (q, J = 35 Hz), 125.6, 126.6, 126.8,
128.8, 129.1 (q, J = 5 Hz), 134.1, 137.7, 138.4, 147.9, 148.3, 149.6, 150.7, 153.2, 168.89; HRMS
+
(ESI+) m/z for C20H11F6N6O5 [M + H]+: calcd 529.0690, found 529.0690. Anal. calcd for
C20H10F6N6O5: C, 45.47; H, 1.91; N, 15.91; found: C, 45.39; H, 1.91; N, 15.67.
4.1.12. (1,3-Bis(2-chloro-4-nitrophenyl)triaz-2-en-1-yl)(pyridin-4-yl)methanone (15e)
Method B, reaction time: 20 min. Pale violet solid; yield 63%; mp 140–142 °C
(MeOH/acetone); IR: 3092, 1698, 1586, 1520, 1480, 1410, 1346, 1293, 1249, 1223, 1131 cm–1;
1H NMR (500 MHz, CDCl3):
δ (ppm) 7.28–7.33 (m, 1H), 7.59–7.63 (m, 1H), 7.68–7.72 (m, 2H),
8.08–8.14 (m, 1H), 8.30–8.34 (m, 1H), 8.34–8.39 (m, 1H), 8.49–8.54 (m, 1H), 8.85–8.93 (m,
2H); 13C NMR (126 MHz, CDCl3):
δ
(ppm) 119.6, 122.8, 123.0, 123.0, 125.7, 126.3, 131.7,
133.3, 134.4, 138.7, 140.4, 148.2, 148.3, 149.1, 150.3, 168.6; HRMS for C18H11Cl2N6O5 [M +
H]+: calcd 461.0162, found 461.0165. Anal. calcd for C18H10Cl2N6O5: C, 46.87; H, 2.19; N,
18.22; found: C, 46.76; H, 2.16; N, 18.01.
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