Preliminary SAR and biological evaluation of antitubercular triazolothiadiazine derivatives against drug-susceptible and drug-resistant Mtb strains

Article abstract of DOI:10.1016/j.bmc.2016.10.027

Following up the SAR study of triazolothiadiazoles for their antitubercular activities targeting Mt SD in our previous study, on the principle of scaffold hopping, the C3 and C6 positions of triazolothiadiazine were examined systematically to define a preliminary structure–activity relationship (SAR) with respect to biological activity. This study herein highlights the potential of two highly potent advanced leads 6c-3, 6g-3 and several other compounds with comparable potencies as promising new candidates for the treatment of TB (6c-3, MIC-H37Rv?=?0.25?μg/mL; MIC-MDRTB?=?2.0?μg/mL; MIC-RDRTB?=?0.25?μg/mL; Mt SD-IC₅₀?=?86.39?μg/mL; and 6g-3, MIC-H37Rv?=?1.0?μg/mL; MIC-MDRTB?=?4.0?μg/mL; MIC-RDRTB?=?2.0?μg/mL; Mt SD-IC₅₀?=?73.57?μg/mL). Compounds 6c-3 and 6g-3 possessed a para-nitro phenyl at the 6 position showed low Vero and HepG2 cells toxicity, turning out to be two excellent lead candidates for preclinical trials. In addition, in vitro Mt SD inhibitory assay indicates that Mt SD is at least one of the targets for their antitubercular activity. Thus, they may turn out to be promising multidrug-resistance-reversing agents.

Full text of DOI:10.1016/j.bmc.2016.10.027

Accepted Manuscript
Preliminary SAR and biological evaluation of antitubercular triazolothiadiazine
derivatives against drug-susceptible and drug-resistant Mtb strains
Ziqiang Li, Xiaoguang Bai, Qi Deng, Guoning Zhang, Lei Zhou, Yishuang Liu,
Juxian Wang, Yucheng Wang
PII:
S0968-0896(16)31038-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.027
BMC 13354
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
19 September 2016
15 October 2016
20 October 2016
Please cite this article as: Li, Z., Bai, X., Deng, Q., Zhang, G., Zhou, L., Liu, Y., Wang, J., Wang, Y., Preliminary
SAR and biological evaluation of antitubercular triazolothiadiazine derivatives against drug-susceptible and drug-
resistant Mtb strains, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.10.027
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Preliminary SAR and biological evaluation of antitubercular
triazolothiadiazine derivatives against drug-susceptible and drug-
resistant Mtb strains
Ziqiang Li,a,† Xiaoguang Bai,b,† Qi Deng, b Guoning Zhang, b Lei Zhou, b Yishuang Liu, b Juxian
Wangb, and Yucheng Wangb,*
a School of Pharmacy, Jinzhou Medical University, No. 40, Section 3, Songpo Road, Linghe District, Jinzhou
121001, China
b Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College,
Beijing 100050, China.
* Corresponding authors. Tel: +86-10-63165263. E-mail addresses: wyc9999@gmail.com (Y. Wang),
imbjxwang@163.com (J. Wang).
† Equal contributing authors.
1

ABSTRACT
Following up the SAR study of triazolothiadiazoles for their antitubercular activities targeting Mt
SD in our previous study, on the principle of scaffold hopping, the C3 and C6 positions of
triazolothiadiazine were examined systematically to define a preliminary structure−activity
relationship (SAR) with respect to biological activity. This study herein highlights the potential
of two highly potent advanced leads 6c-3, 6g-3 and several other compounds with comparable
potencies as promising new candidates for the treatment of TB (6c-3, MIC-H37Rv = 0.25
μg/mL; MIC-MDRTB = 2.0 μg/mL; MIC-RDRTB = 0.25 μg/mL; Mt SD-IC50 = 86.39 μg/mL;
and 6g-3, MIC-H37Rv = 1.0 μg/mL; MIC-MDRTB = 4.0 μg/mL; MIC-RDRTB = 2.0 μg/mL; Mt
SD-IC50 = 73.57 μg/mL). Compounds 6c-3 and 6g-3 possessed a para-nitro phenyl at the 6
position showed low Vero and HepG2 cells toxicity, turning out to be two excellent lead
candidates for preclinical trials. In addition, In vitro Mt SD inhibitory assay indicates that Mt SD
is at least one of the targets for their antitubercular activity. Thus, they may turn out to be
promising multidrug-resistance-reversing agents.
Keywords:
Shikimate dehydrogenase
Antitubercular agent
3,6-Disubstituted triazolothiadiazine
SAR
2

1. Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a leading cause of
morbidity and mortality due to contagious and insidious disease worldwide. Despite its known
etiology, TB still remains one of the most threatening health problems globally, claiming
approximately 9.6 million cases in 2014, of which 480,000 were multi-drug-resistant (MDR). 1
The only FDA-approved TB drug, Bedaquiline, has been recently announced for the treatment of
MDR-TB since the 1960s. 2 Unfortunately, like other known second-line antitubercular drugs, it
has been reported to possess unwanted side effects, in particular its ability to cause QT (the time
interval measured from the beginning of the QRS complex to the end of the T wave in an
electrocardiogram) prolongation could lead to an abnormal and potentially fatal heart rhythm. 3
The rise of MDR and extremely drug-resistant (XDR) strains of M. tuberculosis4 has prompted a
search for new tuberculosis drug candidates and new druggable targets. Consequently, in the
development of novel antitubercular chemotherapeutics, the lack of cross-resistance with
existing drugs remains pivotal, along with good candidate potency and little or no toxicity. 5
In order to discover novel nontoxic antitubercular drugs with least chances of pre-existing
resistance, it is imperative to choose such a novel target which is not only essential for the
survival but is also exclusive to Mtb. The shikimate pathway is one such target which is found in
many human pathogens, including Mycobacterium tuberculosis, for the production of
chorismate, a biosynthetic precursor for aromatic amino acids and other aromatic compounds.
Shikimate dehydrogenase (SD), the fourth enzyme of this pathway is involved in the conversion
of 3-dehydroshikimate to shikimate using NADPH as the cosubstrate. Since, it is proven to be
necessary for the survival of Mtb and is absent in mammals. 6 M. tuberculosis shikimate
dehydrogenase (Mt SD) is therefore a potential target for the treatment of human disease,
including attractive target for new TB drug and vaccine development 7. The novel mode of action
3

against Mtb would ensure that the compounds targeting SD will be least prone to developing
resistance. Consequently, we set out to devise SD inhibitors that could be used as selective
antitubercular drugs. We previously developed a high-throughput screening (HTS) model
targeting Mt SD to identify competitive Mt SD inhibitors. 8 One 3,6-disubstituted
triazolothiadiazole (1, MIC-H37Rv = 8.0 μg/mL; MIC-MDRTB (Isoniazid and Rifampin
resistant strains) = 8.0 μg/mL; MIC-RDRTB (Rifampin resistant strains) = 8.0 μg/mL; Mt SD-
IC50 = 23.00 μg/mL) (Fig. 1) has been identified as a promising lead for antitubercular drug
development. We have previously reported the structure-activity relationships study of 3,6-
disubstituted triazolothiadiazoles, which gave a good number of hit compounds through
screening against Mtb H37Rv strain at 8.0 μg/mL. Moreover, selected lead compounds from
these hits were found to exhibit excellent MIC values in the range of 0.25−8.0 μg/mL against
drug sensitive strains (H37Rv) as well as MDRTB and RDRTB. These lead compounds exhibit
potential inhibitory activity on Mt SD. Subsequently, triazolothiadiazoles 2 and 3 (Fig. 1) were
found to be bactericidal and also active against Mt SD.9
The triazolothiadiazine core is a well-known privileged structure in medicinal chemistry as it
is a versatile heterocycle, possessing a wide spectrum of biological activities including
antimicrobial, 10-15 antitumor, 16-19 antioxidant, 10 anti-HIV, 18 antiviral, 20 analgesic 21, acetyl- and
butyryl-cholinesterases inhibitory 19,22 , alkaline phosphatase inhibitory 19, monoamine oxidase
inhibitory 22, 15-lipo-oxygenase inhibitory 23, and SIRT1 inhibitory 24 activities. We were
interested in the triazolothiadiazine class of compounds because triazolothiadiazines were
previously identified as having activity against M. tuberculosis 14, 25. Suresh Kumar et al. reported
clubbed isopropylthiazole triazolothiadiazines with activity against M. tuberculosis and pursued
antitubercular screening so as to obtain a potent and low-toxicity lead 4 14 (Fig. 1). Kincaid et al.
identified triazolothiadiazines with activity against M. tuberculosis by means of virtual screening
4

for UDP-galactopyranose mutase ligands. 25 The triazolothiadiazines reported in these studies
represent a variety of pharmacophores with having extensive variation at the C-3 and C-6
positions for modulation of activity. Therefore, it is an important pharmacophore for the
discovery of new drugs. However, against Mt SD, no compound has emerged as a promising lead
in the past years besides triazolothiadiazoles previously reported by us 9.
Fig. 1. Lead compounds from triazolothiadiazines.
The scaffold hopping approach is one of the strategies included within the rational design
protocol for identification of new biologically relevant small molecules. Since we have
previously studied the SAR study of 3,6-disubstituted triazolothiadiazoles 9, in our continuous
efforts to discover novel antitubercular scaffolds, scaffold hopping was used to find other
antitubercular inhibitors against Mt SD. With this in mind, a series of 3,6-disubstituted
triazolothiadiazines that maintain structural elements of the parent compound 1 have been
synthesized whereby the triazolothiadiazole is replaced with the triazolothiadiazine. Considering
above facts and the principles of group replacement and bioisosterism, we set out for
optimization of the triazolothiadiazine through systematic structural modifications at the 3 and 6
5

positions as shown in Fig. 2 to evaluate their steric and electronic effects on the
antimycobacterial activity and to deduce SAR. As a result, we have discussed the effect of
unsubstitution and different substitution of aromatic and hetero-aromatic groups at the 3 and 6
positions on the potency against Mtb. Moreover, to explore the chemical space around the C3
aryl moiety of the triazolothiadiazines, a methylene spacer is inserted between carbon atom at the
3 position and the sterically encumbered β-naphthalene ring. In detail, we conducted an
exploratory study to understand the preliminary SAR of a triazolothiadiazine series regarding
antitubercular activity, which has led to the identification of two highly potent lead compounds
6c-3 (MIC-H37Rv = 0.25 μg/mL), 6g-3 (MIC-H37Rv = 1.0 μg/mL) and others with comparable
potencies. Additionally, the several potent compounds were also screened for their in vitro Mt
SD inhibition and were tested to assess their apparent cytotoxicity toward Vero and HepG2 cells.
The results of this study are summarized in this paper.
Fig. 2. Optimization of 3,6-disubstituted triazolothiadiazines.
6

2. Chemical synthesis
Compounds for the optimization library of 3,6-disubstituted triazolothiadiazines (27
compounds in total) were synthesized according to Scheme 1. The commercially available
appropriate aromatic acid was firstly activated by 1,1'-carbonyldiimidazole and then
hydrazinolysized to give corresponding aroyl hydrazides 3a−h in 79−95% yields in one-pot,
which then reacted with carbon disulfide, in presence of potassium hydroxide in ethanol to afford
the respective potassium dithiocarbazinate 4a−h, which later cyclized to desired 4-amino-3-
mercapto-1,2,4-triazole 5a−h by reacting with hydrazine hydrate (80%) in 60−70% yields. The
resulting triazoles 5a−h were further converted to corresponding triazolothiadiazines 6 in one
pot-reaction by condensation with the appropriate phenacyl bromides in the presence of absolute
ethanol through microwave irradiation (MWI) in 90−99% yields.
7

Scheme 1. Synthesis of 3,6-disubstituted triazolothiadiazines. Reagents and conditions: (a)1,1'-
carbonyldiimidazole, THF, 2 h, room temperature (rt); (b) N2H4H2O, rt, overnight; (c) CS2, KOH,
EtOH, rt, 8 h; (d) N2H4H2O, rt, H2O, 2 h, rt/5 h, reflux; (e) ArCOCH2Br, EtOH, MWI, 30 min, 95
°C
8

3. Results and discussions
3.1. Evaluation of antitubercular activity and SAR study
The library of 3,6-disubstituted triazolothiadiazines 6a-g (27 compounds) was subjected to
evaluated for their activity against Mtb H37Rv, MDRTB and RDRTB by the BacT/ALERT 3D
liquid culture technology26 using Rifampin (RFP) and Isoniazid (INH) as the standard drugs to
determine the MIC. Results are summarized in Table 1 and Table 2. Compounds were measured
at a concentration of 8.0 μg/mL (MIC) for the preliminary assessment of the activity against Mtb
H37Rv, wherein 17 compounds are inactive at 8.0 μg/mL concentration (Table 1), 4 compounds
is comparable in activity to the lead compound 1 (Table 1), whereas 6 compounds are found to
inhibit the growth of Mtb H37Rv, MDRTB and RDRTB at variable concentrations (Table 2).
Some of the most representative compounds are also tested toward Vero and HepG2 cells to
ascertain the cytotoxicity profile. To further analyze the biological profile of these promising
compounds, in vitro Mt SD inhibitory assay has been carried out as well. These modifications
lead to a variable range of activities and allow us to construct a plausible SAR as will be
described herein.
The activity of selected 6 derivatives was assessed for their ability to inhibit the growth of the
acquired clinical MDRTB and RDRTB strains from Jiangsu province hospital, China (Table 2).
To our delight, these triazolothiadiazines showed potent activity MIC = 0.25 ~ 4 μg/mL against
two tested drug-resistant strains (Table2). Control data for INH, RFP and the lead compound 1 is
also reported (Table 2) for comparison.
As Table 1 and Table 2 shows, the substituents at both 3- and 6-position are significantly
influential on antibacterial activity. Disappointingly, it is very clear that neither the weak
electron-withdrawing chloro group nor the strong electron-donating methoxy group at the para-
position of the phenyl ring attached at 3-position of conjugated skeleton is favor of the potency
9

of compounds (6b-1−4, 6d-1−4). On the contrary, bromo substituent (electron-withdrawing
group) attached at para position of the phenyl ring at the C-3 position results in a sharp increase
in potency, leading to the discovery of two highly active lead compounds 6c-1 and 6c-3 (MIC-
H37Rv = 0.25 μg/mL), which are 32-fold more potent than the lead compound 1. It could be seen
that 6c-1 and 6c-3 were significantly more potent than other triazolothiadiazines. This increase in
activity is not surprising in light of triazolothiadiazoles previously reported. 9 On the other hand,
the nature of the phenyl ring substituent at the C-6 position exhibits notable effects on the
potency. It is evident that most of the hybrid compounds bearing 4-nitrophenyl at the C-6
position are significantly active against H37Rv, MDRTB and RDRTB (6c-3, 6e-3, 6f-3, 6g-3).
The introduction of electron-withdrawing fluoro group at para-position of phenyl ring attached to
C-6 of heteroaromatic core leads to reduced activity as compared to the lead compound 1 (MIC >
8.0 μg/mL).
In the 3-phenyl series of compounds (6a-1−4), the activity requirement for the substitution
pattern of the 6-phenyl moiety is less well defined and merits further investigation. The
compounds with electron-withdrawing fluoro, nitro and electron-donating methoxy at the para-
position of the phenyl ring attached at 6-position do not show appreciable antitubercular activity
(MIC > 8.0 μg/mL) whereas compound 6a-1 incorporating unsubstituent phenyl ring attached at
6-position shows better potency than the parent compound 1 (MIC-H37Rv = 2.0 μg/mL; MIC-
MDRTB = 4.0 μg/mL; MIC-RDRTB = 2.0 μg/mL). Thus, the fact that any activity at all was
observed is somewhat intriguing to us and suggests compounds with further improvements in
potency may be obtainable.
In the 3-(4-bromophenyl) series of compounds (6c-1−4), quite surprisingly, almost all
compounds (barring 6c-2) examined possess good to excellent antitubercular activities. It is
obvious that unsubstituent and 4-bromo of the benzene ring at the 6 position are beneficial to
10

increase the potency, highlighting 6c-1 and 6c-3 as the most potent compounds in the series with
MIC value of 0.25 μg/mL which is 32-fold more active as compared to the parent compound 1.
Meanwhile, 6c-4 is comparable in activity to the lead compound 1. The results clearly suggest
that 4-bromophenyl group to the 3 position is crucial for biological activity. The most potent
compound in this series, at present, is 6c-3 (MIC-MDRTB = 2.0 μg/mL; MIC-RDRTB = 0.25
μg/mL). This result indicates that 4-bromo attached to the benzene ring at the 3 position is
important for growth inhibition activity as even compound 6c-2 with an 4-fluorophenyl group at
the 6 position showed an MIC-H37Rv > 8.0 μg/mL.
On the basis of above data, we next turn our attention to inserting a methylene spacer
between carbon atom at the 3 position and the sterically encumbered β-naphthalene ring in order
to increase molecule flexibility. Unfortunately, unsubstituent, small electron-donating methoxy
and weak electron-withdrawing fluoro at the para-position of the phenyl ring attached at 6-
position have a negative impact on activity (MIC-H37Rv > 8.0 μg/mL). However, compound 6e-
3 with 4-nitrophenyl at the C-6 position, is the most active compound of the series (MIC-
MDRTB = 4.0 μg/mL; MIC-RDRTB = 4.0 μg/mL). It suggests that a sterically hindered group at
the para position of phenyl ring at the C-3 position of triazolothiadiazine moiety has a slight
impact on activity.
On the basis of the principle of bioisosterism, aiming to improve the water solubility by
introduction of ionizable nitrogen groups, 6f-1−3 have been synthesized whereby the benzene
ring at the 3 position is replaced with a pyridine ring. Fortunately, 6f-3 incorporating 4-
methoxyphenyl attached at 6-position is emerged as an efficient candidate (MIC-MDRTB = 4.0
μg/mL; MIC-RDRTB = 2.0 μg/mL). Further attempts to introduce fluoro attached at meta
position of the pyridine ring (6g-1−4). Similar results have been also obtained. The modifications
11

lead to the identification of an active compound 6g-3 (MIC-MDRTB = 4.0 μg/mL; MIC-RDRTB
= 2.0 μg/mL) in the series.
Assessment of the collocation of these data within the SAR is difficult, but an analysis of
antimycobacterial data clearly shows that antitubercular activity of the newly synthesized
heterocyclic compounds, containing 1,2,4-triazole moiety fused with 1,3,4-thiadiazine ring
depends on the substituents rather than the basic skeleton of the molecule. In general, both
electronic and steric factors actively modulate the antitubercular activity and the presented
triazolothiadiazines are proved to be better antitubercular candidates. And then these data are an
important observation to follow up a rational design of more potent antitubercular candidates.
Table 1 Antibacterial activity of triazolothiadiazines 6 against Mtb H37Rv strain (MIC, μg/mL)
Mtb
Mtb
R1
R2
Compound
R1
R2
H37Rv
H37Rv
Compound
MIC
<8
MIC
>8
6a-1
6a-2
6d-3
6d-4
>8
>8
>8
>8
6a-3
6a-4
6b-1
6e-1
6e-2
6e-3
>8
>8
>8
<8
12

>8
>8
6b-2
6e-4
6b-3
6b-4
6c-1
8
6f-1
6f-2
6f-3
8
>8
<8
>8
<8
6c-2
6c-3
6c-4
>8
<8
8
6g-1
6g-2
6g-3
6g-4
>8
>8
<8
8
6d-1
6d-2
>8
>8
Table 2 Antibacterial (MIC) activity against H37Rv, MDRTB and RDRTB and inhibition of Mt
SD activity of 6 promising triazolothiadiazines
compound
Mtb H37Rv Mtb MDRTB Mtb RDRTB
Inhibition of Mt
SD IC50(µg/mL)
MIC(µg/mL) MIC(µg/mL)
8.0 8.0
MIC(µg/mL)
8.0
23.00±1.18
1
13

2.0
4.0
2.0
2.0
4.0
4.0
4.0
≥16.0
4.0
2.0
1.0
0.25
4.0
2.0
2.0
16.0
1.0
28.38±1.22
252.5±2.26
86.39±1.21
11.68±1.08
20.99±1.23
73.57±1.62
6a-1
6c-1
6c-3
6e-3
6f-3
0.25
0.25
4.0
2.0
1.0
6g-3
RFPa
INHb
0.25
0.12
IC50 values are indicated as means ± SD of three independent experiments; RFPa, Rifampin;
INHb, Isoniazid.
3.2. In vitro Mt SD inhibitory activity
In order to further demonstrate that this series of compounds actually target Mt SD, the 6
potent compounds were selected for further evaluation of their in vitro Mt SD inhibitory activity
(Table 2). As a consequence, the results account for the extent of inhibitory effect on Mt SD
correlates to the extent of antitubercular activity. The Mt SD inhibitory assay reveals that most of
the triazolothiadiazines (barring 6c-1) possess at least a low to moderate inhibitory property.
However, a weak correlation between inhibition of mycobacterial growth and Mt SD inhibitory
activity is observed. For example, 6c-1 is very efficient in killing mycobacterial cells on a par
with the control drug INH, but it displays almost no effect on Mt SD. Within this context the
potent derivatives, 6c-3, 6f-3 and 6g-3 reveal potential inhibitory activity on Mt SD (6c-3, Mt
14

SD-IC50 = 86.39 μg/mL; 6f-3, Mt SD-IC50 = 20.99 μg/mL; 6g-3, Mt SD-IC50 = 73.57 μg/mL).
These findings confirm that specific endogenous targets for these triazolothiadiazines are still
elusive; however, the results indicate that they may have pleiotropic modes of action and
potential to reverse antitubercular resistance. As TB treatment regimens always comprise of a
combination of complementary drugs, this off-target effect of the triazolothiadiazines could have
a positive impact on the effectiveness of drug treatment regimens. In fact, the potent
mycobacterial growth inhibitors showing moderate Mt SD inhibitory properties may also prove
to be prospective leads in a multidrug therapy owing to synergistic combinations, should that
arise. Thus, 6c-3 and 6g-3 could be used in combination with standard antitubercular drugs such
isoniazid or rifampin to reverse multidrug resistance in tuberculosis.
3.3. Cytotoxicity
The 4 potent derivatives synthesized were tested to assess their apparent cytotoxicity toward
Vero and HepG2 cells (Table 3). In general, the selectivity index (SI), that in this case is the ratio
between IC50 toward Vero or HepG2 cells and the MIC toward Mtb H37Rv, for a compound to
be considered a valuable lead has usually to be > 10. As Table 3 shows, we are pleased to notice
that most compounds are apparently not toxic (SI>10), while maintaining good activity
compared with the hit compound 1. Unfortunately, modifications of 6e-3 leading to an
enhancement of activity, also results in a counterproductive improvement of cytotoxicity.
Summarizing, the preliminary investigation around the SAR for these antitubercular
triazolothiadiazines leads to the synthesis of more active compounds, meanwhile, devoid of
apparent cytotoxicity. Thus, 6c-3 and 6g-3 are emerged as advanced lead compounds for further
preclinical drug development.
Table 3 Cytotoxicity (IC50 in μM) and SI of 4 promising triazolothiadiazines against Vero, and
HepG2 cell lines
15

compound
Vero
Vero
SI*
HepG2
HepG2
SI*
Mtb H37Rv
Cytotoxicity
Cytotoxicity
IC50(µM)
MIC(µM)
IC50(µM)
24.54
0.67
0.60
9.95
2.80
71.05±2.53
58.50±1.11
13.92±1.18
69.71±1.23
354.57±1.56
2.90
46.58±2.28
234.66±1.56
10.41±1.66
72.57±1.21
165.78±1.58
1.90
1
87.31
23.21
7.01
350.25
17.34
7.29
6c-1
6c-3
6e-3
6g-3
126.63
59.21
SI* is the ratio of cytotoxicity (IC50 in μM) to in vitro activity against M. tuberculosis H37Rv
(ATCC 25618 strain) expressed as MIC in μM.
16

4. Conclusions
In conclusion, following up the SAR study of triazolothiadiazoles for their antitubercular
activities targeting Mt SD in our previous study 9, on the principle of scaffold hopping, the
preliminary SAR study on triazolothiadiazines for their antitubercular activities against Mtb
H37Rv, MDRTB and RDRTB strains were performed, building upon the identification of
promising early lead compounds 1 obtained from an high-throughput screening campaign.
Similarly, the purpose of this SAR study was to optimize the aromatic or hetero-aromatic
substituents attached at 3-position and 6-position of triazolothiadiazine through systematic
modifications to determine its potential for progression as a drug candidate. It has been found
that the nature of the substitute group on the para site of 3-phenyl exerts noteworthy effects on
the antitubercular activity. For example, electron-donating methoxy and weak electron-
withdrawing chloro groups at the para-position of the phenyl ring attached at 3-position are
detrimental to the activity. On the contrary, bromo substituent (electron-withdrawing group) at
this position dramatically increases the potency. This breakthrough finding in this SAR study has
led to the discovery of 6c-1 and 6c-3 with exceptional potency (MIC-H37Rv = 0.25 μg/mL),
which possess a p-bromophenyl substituent attached at 3-position along with a phenyl or a p-
nitrophenyl attached at 6-position of triazolothiadiazine core. Also, 4 other compounds were
found to possess comparable potencies (MIC-H37Rv ≤4.0 μg/mL), including 6g-3 (MIC-H37Rv
= 1.0 μg/mL), bearing a p-nitrophenyl group at the 6 position and a 3-fluoropyridyl group at the
3 position. These advanced lead compounds do not show appreciable cytotoxicity against Vero
and HepG2 cells (SI > 10). The advanced leads 6c-3 and 6g-3 exhibit the similar potencies
against drug-resistant Mtb clinical isolates, as anticipated. Other important findings in this SAR
study include the fact that besides 6a-3 and 6d-3, most compounds bearing 4-nitrophenyl moiety
at the 6 position prove to be very potent against Mtb H37Rv, MDRTB and RDRTB strains
17

whereas the introduction of 4-methoxyphenyl attached at 3-position abolishs the activity (MIC >
8.0 μg/mL). In vitro Mt SD inhibitory activity by the most potent compounds, demonstrates the
extent of inhibitory effect on Mt SD correlates to the extent of antitubercular activity. In general,
compounds 6c-3 and 6g-3 show a better drug profile in terms of cytotoxicity toward Vero and
HepG2 cells and potency against MDRTB and RDRTB clinical isolates, turning out to be two
excellent lead candidates for preclinical trials. The pharmacokinetics parameters as well as in
vivo efficacy of 6c-3 and 6g-3 will be evaluated in due course in order to prove their efficacy and
to progress toward preclinical trials.
18

5. Experiments
Methods and materials
Methods. 1H and 13C NMR spectra were recorded on Bruker or Varian 400 or 500 MHz NMR
spectrometers operating at the frequencies indicated. Chemical shifts (δ) are in ppm, referenced
to tetramethylsilane. Coupling constants (J) are reported in hertz and rounded to 0.5 Hz. Splitting
patterns are abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m),
broad (br) or some combination of them. Melting points were measured on a Mettler Toledo
capillary melting point apparatus and are uncorrected. Reactions were monitored by thin-layer
chromatography (TLC, silica gel 60 F254) and spots were observed by UV-lamp. APCI high-
resolution mass spectra (HRMS) was recorded on an Autospec Ultima-TOF spectrometer.
MB/BacT ALERT 3D system, which includes a computerized database management system.
Carbon dioxide released into the medium by actively growing mycobacteria is detected through a
gas-permeable sensor containing a colorimetric indicator embedded at the bottom of culture
vials. EnsprireTM enzyme-labelling measuring instrument (Perkin Elmer) was used in vitro Mt
SD inhibitory activity and cell cytotoxicity experiments.
Materials. Modified Middlebrook 7H9 broth, MB reconstituting fluid, and the buffers were
purchased from Biomerieux. RFP and INH were purchased from National Institute for Food and
Drug Control.
5.1. Synthetic procedure and analytical data of aryl or hetero-aryl acid hydrazides 3a-g,
potassium dithiocarbazinate 4a-g and 4-amino-5-substituted -3-mercapto-1,2,4-triazoles 5a-
g
19

Hydrazides 3a-g, potassium dithiocarbazinate 4a-g and 1,2,4-triazoles 5a-g were synthesized
as described in the literature. 9
5.2. Synthetic procedure and analytical data of 3,6-disubstituted triazolothiadiazines (6a-g)
5.2.1. 3,6-biphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6a-1). A mixture of 5a (0.05 g,
0.26 mmol) and 2-bromoacetophenone (0.057 g, 0.29 mmol) were dissolved in absolute alcohol
(2.5 mL), and the resulting mixture was heated at 95 °C in the microwave for 30 min. The
mixture was allowed to stand overnight, separated solid was filtered, washed thoroughly with
cold alcohol and ether, and dried to obtain 6a-1 (0.071 g, 93% yield) as a white solid: mp 226 –
227 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.04 – 7.99 (m, 4H), 7.64 – 7.55 (m, 6H), 4.46 (s, 2H);
13C NMR (101 MHz, DMSO-d6) δ156.05, 151.65, 142.61, 133.47, 131.97, 130.24, 129.15,
128.78, 127.93, 127.56, 125.97, 22.76. HRMS (APCI) m/z calcd. for C16H12N4SH+: 293.0855.
Found: 293.0846.
The same procedure was followed for the synthesis of 6a-2−4, 6b-1−4, 6c-1−4, 6d-1−4, 6e-
1−4, 6f-1−3 and 6g-1−4.
5.2.2. 6-(4-Fluorophenyl)-3-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6a-2). White
solid (95% yield); mp 244 – 245 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.12 – 8.06 (m, 2H), 8.05
– 7.96 (m, 2H), 7.62 – 7.54 (m, 3H), 7.49 – 7.40 (m, 2H), 4.45 (s, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 165.33, 154.82, 151.45, 142.16, 130.03, 129.96, 128.50, 127.73, 125.83, 116.11,
115.89, 22.66. HRMS (APCI) m/z calcd. for C16H11FN4SH+: 311.0761. Found: 311.0763.
5.2.3. 6-(4-Nitrophenyl)-3-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6a-3). Light
yellow powder (91% yield); mp 247 – 248 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.41 (dd, J =
6.8, 2.0 Hz, 2H), 8.25 (dd, J = 6.8, 2.0 Hz, 2H), 8.01 – 7.98 (m, 2H), 7.62 – 7.55 (m, 3H), 4.52
20

(s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 154.39, 151.88, 149.16, 142.47, 139.41, 130.40,
128.97, 128.84, 128.03, 125.73, 124.16, 22.89. HRMS (APCI) m/z calcd. for C16H11N5O2SH+:
338.0706. Found: 338.0713.
5.2.4. 6-(4-Methoxyphenyl)-3-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6a-4).
White solid (96% yield); mp 240 – 241 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.07 – 7.94 (m,
4H), 7.65 – 7.52 (m, 3H), 7.14 (d, J = 8.8 Hz, 2H), 4.43 (s, 2H), 3.86 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 162.35, 155.71, 151.37, 142.71, 130.26, 129.48, 128.77, 127.91, 125.91,
125.44, 114.61, 55.59, 22.53. HRMS (APCI) m/z calcd. for C17H14N4OSH+: 323.0961. Found:
323.0975.
5.2.5 3-(4-Chlorophenyl)-6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6b-1). White
solid (97% yield); mp 273 – 274 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.03 (m, 4H), 7.68 (d, J =
8.4 Hz, 2H), 7.65 – 7.56 (m, 3H), 4.46 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.26,
150.75, 142.90, 135.04, 133.37, 132.06, 129.60, 129.17, 128.99, 127.64, 124.80, 22.78. HRMS
(APCI) m/z calcd. for C16H11ClN4SH+: 327.0466. Found: 327.0469.
5.2.6. 3-(4-Chlorophenyl)-6-(4-fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6b-
2). White solid (95% yield); mp 284 – 285 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.09 (dd, J =
8.8, 5.2 Hz, 2H), 8.03 (dd, J = 6.4, 2.0 Hz, 2H), 7.67 (dd, J = 6.8, 2.0 Hz, 2H), 7.46 – 7.42 (m,
2H), 4.45 (s, 2H). HRMS (APCI) m/z calcd. for C16H10ClFN4SH+: 345.0371. Found: 345.0368.
5.2.7 3-(4-Chlorophenyl)-6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6b-
3). Light yellow powder (99% yield); mp 259 – 260 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.40
(dd, J = 6.8, 2.0 Hz, 2H), 8.26 (dd, J = 6.8, 2.0 Hz, 2H), 8.02 (dd, J = 6.4, 2.0 Hz, 2H), 7.68 (dd,
J = 6.8, 2.0 Hz, 2H), 4.53 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 154.58, 150.98, 149.19,
21

142.76, 139.28, 135.21, 129.73, 129.06, 129.04, 124.53, 124.14, 22.93. HRMS (APCI) m/z
calcd. for C16H10ClN5O2SH+: 372.0316. Found: 372.0301.
5.2.8. 3-(4-Chlorophenyl)-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6b-4). White solid (90% yield); mp 247 – 248 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.05 (dd, J
= 6.8, 2.0 Hz, 2H), 8.00 (dd, J = 6.8, 2.0 Hz, 2H), 7.67 (dd, J = 6.8, 2.0 Hz, 2H), 7.13 (dd, J =
7.2, 2.0 Hz, 2H), 4.42 (s, 2H), 3.86 (s, 3H). HRMS (APCI) m/z calcd. for C17H13ClN4OSH+:
357.0571. Found: 357.0561.
5.2.9. 3-(4-Bromophenyl)-6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6c-1). White
solid (98% yield); mp 266 – 267 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.04 – 8.00 (m, 2H), 7.97
(dd, J = 6.8, 2.0 Hz, 2H), 7.81(dd, J = 6.8, 2.0 Hz, 2H), 7.65 – 7.56 (m, 3H), 4.46 (s, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 156.25, 150.83, 142.93, 133.35, 132.05, 131.90, 129.78, 129.16,
127.64, 125.14, 123.86, 22.76. HRMS (APCI) m/z calcd. for C16H11BrN4SH+: 372.9940. Found:
372.9936.
5.2.10. 3-(4-Bromophenyl)-6-(4-fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6c-2). White solid (97% yield); mp 269 – 270 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.09 (dd, J
= 8.8, 5.2 Hz, 2H), 7.95 (dd, J = 6.8, 2.0 Hz, 2H), 7.81 (dd, J = 6.8, 2.0 Hz, 2H), 7.44 (t, J = 8.8
Hz, 2H), 4.45 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 155.28, 150.84, 142.84, 131.89,
130.38, 130.29, 129.81, 125.10, 123.86, 116.38, 116.16, 22.76. HRMS (APCI) m/z calcd. For
C16H10BrFN4SH+: 390.9846. Found: 390.9852.
5.2.11. 3-(4-Bromophenyl)-6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6c-
3). Light yellow powder (96% yield); mp 254 – 255 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.40
(dd, J = 7.2, 2.0 Hz, 2H), 8.26 (dd, J = 6.8, 2.0 Hz, 2H), 7.95 (dd, J = 6.8, 2.0 Hz, 2H), 7.81 (dd,
22

J = 6.8, 2.0 Hz, 2H), 4.52 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 154.58, 151.09, 149.20,
142.80, 139.28, 131.96, 129.92, 129.07, 124.89, 124.15, 124.05, 22.91. HRMS (APCI) m/z
calcd. for C16H10BrN5O2SH+: 417.9792. Found: 417.9794.
5.2.12. 3-(4-Bromophenyl)-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6c-4). White solid (99% yield); mp 253 – 254 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.02 – 7.95
(m, 4H), 7.81 (dd, J = 6.8, 2.0 Hz, 2H), 7.13 (dd, J = 6.8, 2.0 Hz, 2H), 4.42 (s, 2H), 3.86 (s, 3H).
13C NMR (101 MHz, DMSO-d6)) δ 162.36, 155.74, 150.63, 142.90, 131.86, 129.72, 129.55,
125.37, 125.26, 123.74, 114.59, 55.58, 22.51. HRMS (APCI) m/z calcd. for C17H13BrN4OSH+:
403.0046. Found: 403.0044.
5.2.13. 3-(4-Methoxyphenyl)-6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6d-1).
White solid (91% yield); mp 202 – 203 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.03 – 8.00 (m,
2H), 7.98 (dd, J = 7.0, 2.0 Hz, 2H), 7.64 – 7.57 (m, 3H), 7.14 (dd, J = 7.0, 2.0 Hz, 2H), 4.44 (s,
2H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.73, 155.85, 151.48, 142.01, 133.55,
131.91, 129.46, 129.14, 127.53, 118.36, 114.26, 55.36, 22.72. HRMS (APCI) m/z calcd. for
C17H14N4OSH+: 323.0961. Found: 323.0982.
5.2.14. 6-(4-Fluorophenyl)-3-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6d-2). White solid (93% yield); mp 242 – 243 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.08 (dd, J
= 9.0, 5.0 Hz, 2H), 7.96 (dd, J = 6.5, 2.0 Hz, 2H), 7.43 (dd, J = 9.0, 2.0 Hz, 2H), 7.14 (dd, J =
7.0, 2.0 Hz, 2H), 4.43 (s, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.74, 154.90,
151.48, 141.93, 130.25, 130.16, 129.48, 118.33, 116.37, 116.16, 114.27, 55.36, 22.72. HRMS
(APCI) m/z calcd. for C17H13FN4OSH+: 341.0867. Found: 341.0865.
23

5.2.15. 3-(4-Methoxyphenyl)-6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6d-3). Light yellow powder (95% yield); mp 237 – 238 °C; 1H NMR (400 MHz, DMSO-d6) δ
8.41 (dd, J = 7.2, 2.0 Hz, 2H), 8.25 (dd, J = 6.8, 2.0 Hz, 2H), 7.95 (dd, J = 6.8, 2.0 Hz, 2H), 7.15
(dd, J = 6.8, 2.0 Hz, 2H), 4.51 (s, 2H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.89,
154.28, 151.71, 149.14, 141.94, 139.47, 129.64, 128.97, 124.16, 118.00, 114.35, 55.40, 22.86.
HRMS (APCI) m/z calcd. for C17H13N5O3SH+: 368.0812. Found: 368.0805.
5.2.16. 3,6-Bis(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6d-4). White
solid (91% yield); mp 228 – 229 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.03 – 7.95 (m, 4H), 7.17
– 7.11 (m, 4H), 4.41 (s, 2H), 3.86 (s, 3H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
162.32, 160.80, 155.62, 151.17, 142.20, 129.50, 129.47, 125.50, 118.15, 114.60, 114.28, 55.59,
55.39, 22.48. HRMS (APCI) m/z calcd. for C18H16N4O2SH+: 353.1067. Found: 353.1077.
5.2.17. 3-(β-naphthylmethyl)-6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6e-1).
White solid (93% yield); mp 230 – 231 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.30 (d, J = 8.0
Hz, 1H), 8.00 – 7.92 (m, 3H), 7.85 (d, J = 7.6 Hz, 1H), 7.63 – 7.53 (m, 5H), 7.52 – 7.44 (m, 2H),
4.75 (s, 2H), 4.39 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 154.76, 152.35, 140.48, 133.43,
131.96, 131.93, 131.50, 129.04, 128.49, 127.59, 127.51, 127.46, 126.25, 125.83, 125.53, 124.10,
27.76, 22.76. HRMS (APCI) m/z calcd. for C21H16N4SH+: 357.1168. Found: 357.1155.
5.2.18. 6-(4-Fluorophenyl)-3-(β-naphthylmethyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6e-2). White solid (91% yield); mp 223 – 224 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J =
8.0 Hz, 1H), 8.07 – 8.02 (m, 2H), 7.96 – 7.91 (m, 1H), 7.85 (dd, J = 7.2, 2.0 Hz, 1H), 7.59 – 7.51
(m, 2H), 7.50 – 7.39 (m, 4H), 4.74 (s, 2H), 4.38 (s, 2H). HRMS (APCI) m/z calcd. For
C21H15FN4SH+: 375.1074. Found: 375.1072.
24

5.2.19. 3-(β-Naphthylmethyl)-6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6e-3). Light yellow powder (92% yield); mp 235 – 236 °C; 1H NMR (400 MHz, DMSO-d6) δ
8.39 (dd, J = 7.2, 2.0 Hz, 2H), 8.29 (d, J = 8.0 Hz, 1H), 8.21 (dd, J = 6.8, 2.0 Hz, 2H), 7.97 –
7.93 (m, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.60 – 7.46 (m, 4H), 4.76 (s, 2H), 4.46 (s, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 152.97, 152.57, 149.12, 140.31, 139.38, 133.44, 131.81, 131.50, 128.83,
128.51, 127.65, 127.63, 126.29, 125.85, 125.58, 124.10, 124.05, 27.81, 22.82. HRMS (APCI)
m/z calcd. for C21H15N5O2SH+: 402.1019. Found: 402.1010.
5.2.20. 6-(4-Methoxyphenyl)-3-(β-naphthylmethyl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6e-4). White solid (95% yield); mp 217 – 218 °C; 1H NMR (400 MHz,
DMSO-d6) δ 8.29 (d, J = 8.0 Hz, 1H), 7.98-7.42 (m, 3H), 7.86 (d, J = 7.6 Hz, 1H), 7.61 – 7.53
(m, 2H), 7.52 – 7.44 (m, 2H), 7.11 (d, J = 8.8 Hz, 2H), 4.76 (s, 2H), 4.37 (s, 2H), 3.85 (s, 3H).
HRMS (APCI) m/z calcd. for C22H18N4OSH+: 387.1274. Found: 387.1281.
5.2.21. 6-Phenyl-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6f-1). Light
yellow powder (92% yield); mp 275 – 276 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.06 – 9.00 (m,
2H), 8.51 – 8.40 (m, 2H), 8.12 – 8.07 (m, 2H), 7.70 – 7.61 (m, 3H), 4.53 (s, 2H). 13C NMR (101
MHz, DMSO-d6) δ 157.13, 155.75, 148.37, 145.52, 145.33, 133.02, 132.44, 129.24, 127.93,
123.13, 23.00. HRMS (APCI) m/z calcd. for C15H11N5SH+: 294.0808. Found: 294.0807.
5.2.22. 6-(4-Fluorophenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6f-2).
Light yellow powder (90% yield); mp 275 – 276 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J
= 5.2 Hz, 2H), 8.39 (d, J = 4.8 Hz, 2H), 8.17 (dd, J = 8.8, 5.2 Hz, 2H), 7.47 (t, J = 8.8 Hz, 2H),
4.51 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.78, 163.28, 156.09, 148.48, 145.93, 145.12,
25

137.68, 130.71, 130.62, 129.60, 129.57, 123.02, 116.47, 116.25, 22.99. HRMS (APCI) m/z
calcd. for C15H10FN5SH+: 312.0713. Found: 312.0707.
5.2.23. 6-(4-Methoxyphenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6f-
3). Light yellow powder (99% yield); mp 276 – 277 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.02
(dd, J = 5.6, 1.6 Hz, 2H), 8.45 (d, J = 6.4 Hz, 2H), 8.08 (dd, J = 6.8, 2.0 Hz, 2H), 7.16 (dd, J =
7.2, 2.0 Hz, 2H, 4.48 (s, 2H), 3.88 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.69, 156.60,
148.14, 145.50, 145.32, 138.25, 129.93, 124.98, 123.03, 114.67, 55.67, 22.70. HRMS (APCI)
m/z calcd. for C16H13N5OSH+: 324.0914. Found: 324.0921.
5.2.24. 3-(3-Fluoropyridin-4-yl)-6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6g-1).
Light yellow powder (90% yield); mp 223 – 224 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.87 (d, J
= 2.0 Hz, 1H), 8.69 (d, J = 4.8 Hz, 1H), 7.97 – 7.94 (m, 2H), 7.89 – 7.84 (m, 1H), 7.65 – 7.60
(m, 1H), 7.59 – 7.54 (m, 2H), 4.52 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.26, 154.24,
146.32, 146.27, 143.74, 139.39, 139.16, 133.11, 132.20, 129.15, 127.61, 124.65, 121.24, 121.13,
23.15. HRMS (APCI) m/z calcd. for C15H10FN5SH+: 312.0713. Found: 312.0705.
5.2.25. 6-(4-Fluorophenyl)-3-(3-fluoropyridin-4-yl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6g-2). Light yellow powder (92% yield); mp 226 – 227 °C; 1H NMR (400
MHz, DMSO-d6) δ 8.87 (d, J = 2.0 Hz, 1H), 8.68 (d, J = 4.8 Hz, 1H), 8.03 (dd, J = 8.8, 5.2 Hz,
2H), 7.90 – 7.82 (m, 1H), 7.45 – 7.39 (m, 2H), 4.51 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ
155.27, 154.22, 146.32, 146.26, 143.63, 139.38, 139.15, 130.36, 130.27, 129.65, 124.66, 121.21,
116.41, 116.19, 23.13. HRMS (APCI) m/z calcd. for C15H9F2N5SH+: 330.0619. Found:
330.0612.
26

5.2.26. 3-(3-Fluoropyridin-4-yl)-6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6g-3). Light yellow powder (94% yield); mp 193 – 194 °C; 1H NMR (400
MHz, DMSO-d6) δ 8.87 (s, 1H), 8.69 (d, J = 4.4 Hz, 1H), 8.39 (d, J = 8.8 Hz, 2H), 8.19 (d, J =
8.8 Hz, 2H), 7.88 – 7.86 (m, 1H), 4.58 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 154.57,
154.21, 149.28, 146.34, 143.62, 139.42, 139.18, 139.01, 129.03, 124.68, 124.15, 121.03, 23.27.
HRMS (APCI) m/z calcd. for C15H9FN6O2SH+: 357.0564. Found: 357.0548.
5.2.27. 3-(3-Fluoropyridin-4-yl)-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6g-4). Light yellow powder (92% yield); mp 215 – 216 °C; 1H NMR (400
MHz, DMSO-d6) δ 8.92 (d, J = 2.0 Hz, 1H), 8.74 (dd, J = 5.2, 1.2 Hz, 1H), 7.99 (dd, J = 6.8, 2.0
Hz, 2H), 7.92 (dd, J = 6.0, 5.2 Hz, 1H), 7.17 (dd, J = 6.8, 2.0 Hz, 2H), 4.53 (s, 2H), 3.90 (s, 3H).
HRMS (APCI) m/z calcd. for C16H12FN5OS H+: 342.0819. Found: 342.0809.
5.3. Biological assays
Bacterial strains and growth
H37Rv and clinical Mtb strains MDRTB and RDRTB exhibiting resistant profiles to Isoniazid
and Rifampin resistant strains were used. For evaluation of drug sensitivity all strains were
grown in Difco 7H9 Middlebrook liquid medium (BD Biosciences, 271310) supplemented with
casein, bovine albumen and catalase at 37 °C. The growth of Mycobacterium tuberculosis (≤36
hours, subculture, then the growth was diluted 1:1 in sterile distilled water) formed the direct
growth control of approximately Mc Farland no.2 (DGC).
5.3.1. Antibacterial Activity assay
27

Antibacterial Activity assay was used as described previously 9 to assess the minimum
inhibitory concentrations of the compounds on M. tuberculosis H37Rv, and MDRTB and
RDRTB. Briefly, the assay was conducted in a The BacT/Alert MP bottle format. MIC of the
compounds was determined by MB/BacT in triplicate. All the synthesized compounds, isonizaid
and rifampcin were solubilized in DMSO to the working solution (2,560 µg/mL) and serially
diluted (2-fold dilutions). Working solution (2,560 µg/mL) was added aseptically to 0.5 mL of
reconstitution fluid (Tween 80, glycerol and amaranth), 0.5 mL DGC and MB Bact medium to
achieve the required concentration (16, 8, 4, 2, 1, 0.5, 0.25, 0.12, 0.06 µg/mL) in a final volume
of 10 mL each BacT/Alert MP bottle. To 9 mL of MB Bact medium, 0.5 mL of reconstitution
fluid and 0.5 mL DGC were added to a bottle and this was the positive control. To 9.5 mL of MB
Bact medium, 0.5 mL of reconstitution fluid was inoculated into another bottle formed the
negative control. These bottles were incubated in the system at 35 °C for 8-9 days and monitored
to detect growth.
The compound was considered as ineffective if the bottles containing it flagged positive at
the same time or before the positive control. The compound was considered effective if the bottle
containing it remained negative during the test period or flagged positive 2 days after the positive
control. If the positive control did not flag positive in 12 days the test was invalidated and had to
be repeated.
5.3.2. In vitro Mt SD inhibitory assay
Mtb SD Protein Preparation was carried out as described 27. Mt SD inhibitory assay was
carried out as described previously 9. All spectrophotometric assays were performed at 25 °C,
and the increase in NADPH was monitored at 340 nm. Briefly, the assays were conducted in a
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final volume of 100 µL, containing the following components: 100 mM Tris HCl, pH 9.0, 1 mM
shikimic acid, 0.5 mM NADP+ and 25 U/100 µL Mt SD. All of the components except for the
SD enzyme were premixed in a reservoir and dispensed. The reaction mixture was incubated at
25 °C for 5 min to reach a stable background. The SD enzyme was added in the end to trigger the
reaction as described 8. Stock solutions of all compounds were prepared in DMSO such that the
final concentration of this co-solvent was constant at 1% v/v in a final volume 100 µL for all
kinetic reactions. Negative control reactions were carried out with the same conditions as
described above but without inhibitor. The inhibitory activity of each derivative was expressed as
the percentage inhibition of Mt SD activity with respect to the negative control reaction without
inhibitor. All activity assays were performed in triplicate.
5.3.3. Cytotoxicity assay
The cytotoxicity of the compounds was tested against Vero and HepG2 cells. Vero and
HepG2 cells were grown without CO2 in L15 medium supplemented with antibiotics and heat
inactivated calf serum. Serial 2-fold dilutions of the drugs were performed in the 96-well
microplates. The Vero and HepG2 cells, in medium containing 2× Alamar Blue, were added to
the wells to a final concentration of 1.3 × 104 cells per well. The plates were incubated for 3 days
at 37 °C. The IC50 was calculated according to manufacturer directions.
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