New one-pot synthesis of anti-tuberculosis compounds inspired on isoniazid

Article abstract of DOI:10.1016/j.ejmech.2020.112699

A library of thirty N-substituted tosyl N’-acryl-hydrazones was prepared with p-toluenesulfonyl hydrazide, methyl propiolate and different aldehydes in a one-pot synthesis via an aza-Michael reaction. The scope of the reaction was studied, including aliphatic, isoprenylic, aromatic and carbocyclic aldehydes. The prepared collection was tested against Mycobacterium tuberculosis H37Rv. Nine analogs of the collection showed Minimum Inhibitory Concentration ≤10 μM, of which the most active members (MIC of 1.25 μM) were exclusively E isomers. In order to validate the mechanism of action of the most active acrylates, we tested their activity on a M. tuberculosis InhA over-expressing strain obtaining MIC that consistently doubled those obtained on the wild type strain. Additionally, the binding mode of those analogs on M. tuberculosis InhA was investigated by docking simulations. The results displayed a hydrogen bond interaction between the sulfonamide and Ile194 and the carbonyl of the methyl ester with Tyr 158 (both critical residues in the interaction with the fatty acyl chain substrate), where the main differences on the binding mode relays on the hydrophobicity of the nitrogen substituent. Additionally, chemoinformatic analysis was performed to evaluate in silico possible cytotoxicity risk and ADME-Tox profile. Based on their simple preparation and interesting antimycobacterial activity profile, the newly prepared aza-acrylates are promising candidates for antitubercular drug development.

Full text of DOI:10.1016/j.ejmech.2020.112699

European Journal of Medicinal Chemistry 208 (2020) 112699
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
New one-pot synthesis of anti-tuberculosis compounds inspired on
isoniazid
,
, d
Diego G. Ghiano a, Alejandro Recio-Balsells a, Ana Bortolotti b 1, Lucas A. Defelipe c
,
Adrian Turjanski , Hector R. Morbidoni e, Guillermo R. Labadie a
c
,
d
b,
, f, *
ꢀ
ꢀ
a Instituto de Química Rosario, UNR, CONICET, Suipacha 531, S2002LRK, Rosario, Argentina
b
ꢀ
Laboratorio de Microbiología Molecular, Facultad de Ciencias Medicas, Universidad Nacional de Rosario, Santa Fe 3100, S2002KTR, Rosario, Argentina
c
ꢀ
IQUIBICEN-CONICET, Ciudad Universitaria, Pabellon 2, C1428EHA, Ciudad de Buenos Aires, Argentina
d
ꢀ
ꢀ
Departamento de Química Biologica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellon 2, C1428EHA,
Ciudad de Buenos Aires, Argentina
e Consejo de Investigaciones Científicas, Universidad Nacional de Rosario, Argentina
f
ꢀ
ꢀ
Departamento de Química Organica, Facultad de Ciencias Bioquímicas y Farmaceuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK,
Rosario, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of thirty N-substituted tosyl N’-acryl-hydrazones was prepared with p-toluenesulfonyl hydra-
zide, methyl propiolate and different aldehydes in a one-pot synthesis via an aza-Michael reaction. The
scope of the reaction was studied, including aliphatic, isoprenylic, aromatic and carbocyclic aldehydes.
The prepared collection was tested against Mycobacterium tuberculosis H37Rv. Nine analogs of the
Received 10 February 2020
Received in revised form
22 July 2020
Accepted 25 July 2020
Available online 22 August 2020
collection showed Minimum Inhibitory Concentration ꢀ10
mM, of which the most active members (MIC
of 1.25 M) were exclusively E isomers. In order to validate the mechanism of action of the most active
m
acrylates, we tested their activity on a M. tuberculosis InhA over-expressing strain obtaining MIC that
consistently doubled those obtained on the wild type strain.
Keywords:
Aza-Michael
Tuberculosis
InhA
One-pot
Docking
Additionally, the binding mode of those analogs on M. tuberculosis InhA was investigated by docking
simulations. The results displayed a hydrogen bond interaction between the sulfonamide and Ile194 and
the carbonyl of the methyl ester with Tyr 158 (both critical residues in the interaction with the fatty acyl
chain substrate), where the main differences on the binding mode relays on the hydrophobicity of the
nitrogen substituent. Additionally, chemoinformatic analysis was performed to evaluate in silico possible
cytotoxicity risk and ADME-Tox profile.
Chemoinformatics
Based on their simple preparation and interesting antimycobacterial activity profile, the newly pre-
pared aza-acrylates are promising candidates for antitubercular drug development.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
positive people) [1]. In addition, multidrug-resistant tuberculosis
(MDR-TB, resistant to Isoniazid and Rifampicin) is becoming more
Tuberculosis (TB) remains a major infectious disease and is a
great cause of death throughout the world. The World Health Or-
ganization (WHO) estimated 10.0 million new TB cases and 1.6
million deaths in 2017 (including 300.000 deaths caused in HIV-
common [2] with 458.000 new cases in the World in 2017 [1]. It is
estimated that about 8.5% of these cases were caused by
extensively-drug-resistant M. tuberculosis (XDR-TB), showing
additional resistance to at least a fluoroquinolone and one inject-
able drug (amikacin, kanamycin or capreomycin) [1,3]. For patients
affected by XDR-TB, the therapeutic efficacy of standard treatments
is quite limited. Additionally, the emergence of “Totally drug-
resistant” M. tuberculosis strains that have a low rate of successful
treatment is becoming a health threat in certain regions of the
planet [4].
* Corresponding author. Instituto de Química Rosario, UNR, CONICET, Suipacha
531, S2002LRK, Rosario, Argentina.
E-mail address: labadie@iquir-conicet.gov.ar (G.R. Labadie).
Current address: Departamento de Microbiología, Facultad de Ciencias Bio-
1
ꢀ
The common treatment for TB consists of a cocktail of four
químicas y Farmaceuticas, Suipacha 531, Universidad Nacional de Rosario, Argen-
tine National Research Council (CONICET), Rosario, Argentina.
https://doi.org/10.1016/j.ejmech.2020.112699
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
drugs: isoniazid, rifampicin, ethambutol and pyrazinamide over a
6- to 9-month period. Also, anti-tuberculosis drugs induce hepa-
totoxicity (ATDH), causing substantial morbidity and mortality and
diminishing treatment effectiveness. Side effects are the main
reasons for the non-adherence to the current treatment, reducing
the effectiveness and eventually contributing to failure, relapse, or
the emergence of drug-resistance [5].
Therefore, there is an urgent need to develop new drugs. New
candidates need to be able to shorten and simplify the actual
treatment, to improve tolerance, be active against resistant strains
(MDR-TB, XDR-TB) and also be compatible with anti-HIV therapies
in TB-HIV patients [6].
One of the most effective and specific antituberculosis drugs is
Isoniazid (INH). It has been a key chemotherapeutic agent since it
was introduced in 1952 [7]. INH is active only on growing tubercle
bacilli, not being effective against non-replicating bacilli or under
anaerobic conditions [8,9].
INH is a prodrug that is activated by the mycobacterial catalase-
peroxidase enzyme KatG [10], producing an isonicotinoyl acyl
radical which reacts with NAD/NADH species to give the INH-NADH
adduct (Scheme 1). This adduct inhibits the enoyl ACP (Acyl Carrier
Protein) reductase (InhA), thus blocking the mycolic acid biosyn-
thesis and affecting the integrity of the cell wall, leading to cell
death [11]. Mutations in several genes (katG, ahpC, kasA, inhA) have
been described as mediating resistance to INH, however it has been
clearly shown that the essential InhA encoded by inhA is the true
target for the activated INH. Of outmost importance, mutations
abrogating the activity of KatG ewhich are vastly predominant
worldwide in clinical strains-prevent the activation of the prodrug
into active forms, rendering INH totally inactive [10]. Therefore,
new compounds targeting InhA without the need of an activation
step are desperately needed to replace INH and combat multidrug-
resistant strains of M. tuberculosis [12]. Despite its proven applica-
bility and effectivity, INH has yet another major drawback as it
produces adverse reactions that include hepatotoxicity and
neurotoxicity [13].
compounds structurally related to isoniazid. A detailed look on the
reported compounds showed that only few examples of N’-
substituted derivatives have been reported and their biological
activity has not been explored.
On the other side, tosylhydrazones have shown antibacterial
activity and have also been present in analogs with antinociceptive
activity [22]. Also, there are tosylhydrazones reported as inhibitors
of monoamine oxidase A, a target for anti depressives [23,24], and
as acetylcholinesterase inhibitors, a target for Alzheimer’s disease
treatment [25]. (Fig. 2).
In the present study, we explore the N-substituted tosylhy-
drazones scaffold (Fig. 2, Compound E), structurally related to
isoniazid, looking for the design of more effective compounds that
would overcome drug resistance and reduce or eliminate adverse
effects.
The proposed structure has a new substituent on the secondary
nitrogen. This modification will introduce stereoelectronic differ-
ences that could contribute to sustain and improve the anti-
mycobacterial activity overcoming known resistant mechanisms
related to activation of INH [20].
2. Results and discussions
2.1. Chemistry
Synthetically N-substituted tosylhydrazones scaffold can be
prepared by different strategies, but a critical point was the nature
of the secondary nitrogen substituent. In order to improve the ac-
tivity, we looked for potential moieties that contribute to the ac-
tivity as pharmacophores. We found that hetero-substituted E-
ethyl acrylate
E (Scheme 2) have shown moderate anti-
mycobacterial activity [26]. Therefore, combining tosylhydrazones
with hetero-substituted E acrylates could improve the activity as a
hybrid drug [27]. That compound also has the advantage that it can
be prepared by an aza-Michael reaction between methyl propiolate
1 and tosylhydrazone anion 2 as is shown in Scheme 2.
In recent years, compounds with structural features based on
INH with antituberculosis activity have been developed (Fig. 1)
[14e16]. Among those, there are new analogs which introduced
substituents over the terminal nitrogen, keeping the isonicotinic
portion without modifications (Fig. 1, Compound A) [17]. Another
approach exchanges the isonicotinic portion with different aro-
matics substituents (Fig. 1, Compound B) [18]. The hydrazone has
also been exchanged by a hydrazine that displayed high MIC on
M. tuberculosis H37Rv, but its IC90 on anaerobic and aerobic con-
dition was between 3 and 4 times more active, respectively (Fig. 1,
Compound C) [19,20]. Alternatively, N-arylsulfonyl derivatives have
Sulfonyl hydrazines are widely used as intermediates for het-
erocyclic synthesis, such as indazole [28], 1,2,4-triazines [29],
1,2,4,5-tetrazines [30] and H-pyrazolo [5,1a] isoquinolines [31]. The
synthesis of the N-substituted acrylates by aza-Michel reaction has
been recently explored. One example of that approach involved the
reaction of aromatic benzyl hydrazones with ethyl propiolate under
basic conditions (DABCO) [32]. Another reported procedure used
triphenylphosphine to capture the 1,3-zwitterionic intermediate
generated by the reaction of sulfonyl hydrazones with ethyl pro-
piolate [33]. Both reactions were conducted at room temperature
and were regioselective, generating only the E adduct with mod-
erate to high yields. Similar compounds can also be prepared by
Mitsunobu reactions [34] or nucleophilic substitution of alkylha-
lides [35].
been prepared as metallo-b-lactamases inhibitors to overcome the
resistance mechanisms generated by some bacterial species (Fig. 1.
Compound D) [21]. Those results encouraged us to develop new
Scheme 1. Isoniazid activation by KatG enzyme.

D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
3
Fig. 1. Antitubercular INH related compounds.
Fig. 2. Bioactive tosyl hydrazones.

4
D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
Scheme 4. Study of the stability of the tosylhidrazone anion by Kong et al.
Scheme 2. Proposed merged scaffold and synthesis.
Hydrazones are known to be toxic and potentially explosive,
being a major drawback when used in any synthetic scheme,
particularly when being the starting material. That becomes
particularly important for industrial applications where scaling up
the synthesis is needed, handling grams to kilograms of diazo
compounds [36]. That instability requires special care and conve-
nient handling. As an alternative procedure to deal with those
disadvantages, we have proposed a new alternative method
involving a one-pot synthesis, without hydrazone intermediates
isolation, to generate the acrylate derivates in a simple and safe
manner. Therefore, it was necessary to find an alternative method
that would allow us to prepare the hydrazone with a concomitant
Michael reaction, without isolating the condensation product. Prof.
Aggarwal’s group has a long history of using diazo compounds as
valuable synthetic intermediates [37]. Between those applications,
they have reported a synthetic procedure to prepare pyrazoles. This
reaction starts with the condensation of tosylhydrazine and benz-
aldehyde, followed by addition of an aqueous solution of sodium
hydroxide. The benzaldehyde tosylhydrazone sodium salt I, formed
the diazo intermediate II that reacts with an internal alkyne in a
[3 þ 2] cycloaddition (Route A, Scheme 3).
Scheme 5. Synthesis of tosylhydrazone acrylates.
effect of PPh3 as a stabilizing agent of the tosylhydrazone anion [35]
(Scheme 4). Without the addition of PPh3, the tosylhydrazone anion
decomposes in less than 3 h. Therefore, there is an open window of
stability to add the Michael acceptor, that will afterwards react to
produce the adduct (see Scheme 5).
We started the optimization process of the reaction using
benzaldehyde as a model substrate. Initially, the hydrazone for-
mation was attempted by the reaction of the aldehyde with tosyl-
hydrazine in anhydrous acetonitrile at room temperature over
several hours. To favor the formation of the reaction intermediate, it
was necessary to run the reaction under reflux to promote the
dehydration, adding molecular sieves to remove the water. Under
Our goal was to find conditions to capture the tosylhydrazone
anion I with a Michael acceptor (Route B, Scheme 3) before spon-
taneously losing a sulfinate, generating the corresponding diazo
intermediate. Thus, the aza-Michael reaction will be competing
with a 1,3-dipolar cycloaddition. Previously, Kong et al. have stud-
ied this competing process in different conditions, analyzing the
Scheme 3. Different possible pathway of the reaction.

D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
5
that condition, it was possible to produce the intermediate, that
30 compounds.
was then treated with an aqueous solution of sodium hydroxide,
leading to a solution of benzaldehyde tosylhydrazone sodium salt
in 30 min. Finally, we proceeded to add methyl propionate and
allow it to react overnight at room temperature. On our first at-
tempts, only the Z isomer at low yields was obtained. To improve
the yield, we decided to raise the temperature of this step, running
the reaction at reflux. Under this condition the conversion was
complete, obtaining a mixture of regioisomers in good yield and
reducing the reaction time from 12 to 4 h.
Having optimized the reaction procedure, we studied the scope
of the reaction with a diverse selection of aldehydes in order to
prepare a collection of analogs. First, different substituted benzal-
dehydes were evaluated, generating derivatives 2 to 7 (Table 1)
without substantial differences with the model substrate. 2-
Naphthaldehyde was also used in order to have a higher steric
demand; and the effects of a heteroatom on the aromatic ring was
explored including furfural and pyrrole-2-carboxaldehyde. Re-
actions provided the expected products 8, 9 and 10 with variable
2.2. Biological evaluation
2.2.1. In vitro activity against M. tuberculosis
All of the synthesized N-acrylates were assayed in vitro against
M. tuberculosis H37Rv obtaining the MIC by serial microdilution
colorimetric assay using MTT as viability indicator [38,39]. The re-
sults are presented in Table 2 and Fig. 3. At the higher concentration
tested (20
tubercular activity. The remaining thirteen derivatives (43% of the
collection) showed MIC ranging from 20 to 1.25 M. Of those, an-
alogs 3E, 8Z, 10Z and 14Z had a MIC ¼ 20 M, three (11Z, 12E and
15E) displayed a MIC ¼ 10 M, and another three derivatives (10E,
11E and 14E) showed a MIC of 5.0 M. Finally, the most active
M. The structure-
mM), seventeen of the analogs did not show anti-
m
m
m
m
analogs were 1E, 2E and 8E with MIC ¼ 1.25
m
activity relationship analysis indicates the importance of the
regiochemistry of the acrylate on activity profile. Out of the thirteen
active members of the collection, nine analogs were E isomers, and
more importantly, were exclusively the compounds that displayed
yields. Finally, the reactivity of
a,b-unsaturated aldehydes was
assayed obtaining the derivatives 11e15 in good yields. However,
when pentaldehyde was used, no product was observed (Table 1).
All the isomers were successfully separated by flash column and
characterized by 1H and 13C NMR, and mass spectrometry. The
yields were calculated including both isolated isomers. The E:Z ratio
was calculated by 1H NMR signals and was near 40:60 for all the
reactions. Following this methodology, it was possible to synthesize
the best (below 10 mM) MIC.
A detailed analysis of the effect of the substituent shown evi-
dence of steric and electronic effect have an impact on the activity
profile of the compounds. The E isomer of the simplest aromatic
derivative (1E) that holds a phenyl group was one of the most active
compounds. The analogs with substituted phenyl rings shown that
the ortho derivatives were more active than the para substituted
ones. The substitution of the para position seems to be sensitive to
the nature of the group. Activating substituent 2E (2-OMe)
conserved the potency display by the non-substituted derivative
1E, meanwhile, a compound containing a strong deactivating
methyltrifluoro group (3E), was inactive. The para substituted de-
rivatives were all inactive, not being possible to infer any effect of
activating and deactivating groups of the aromatic ring. On the
other side, fused aromatic ring derivatives, like the naphthyl analog
8E, were as active as analog 1E, demonstrating that the size of the
ring does not affect the potency when the regiochemistry is
conserved. Only the furanyl analog 10 was active out of the two
heterocyclic derivatives prepared. Derivatives 11 to 15 included
different conjugated double bonds of different size and length. The
activity of this series increases as the size of the substituent de-
creases, being derivatives 11E and 14E clear examples of that
behavior.
Table 1
N-Tosylacrylate synthesized.
Substituent family
Aromatic
R
Compounds
Yield %
phenyl
1E
2E
3E
4E
5E
6E
7E
8E
9E
1Z
2Z
3Z
4Z
5Z
6Z
7Z
8Z
9Z
68
26
58
90
50
87
67
27
71
2-OMe-Ph
2-CF3-Ph
binaphtyl
4-NO2-Ph
4-Br-Ph
Recently, Ghiya et al. have prepared and evaluated a library of
N’-substituted-4-methylbenzenesulfonohydrazides
mycobacterial, antibacterial and antifungal activities [40]. Those
compounds were prepared from substituted benzaldehyde (Cl, Br,
OMe) and p-toluensulphonyl hydrazine being similar to our pro-
posed scaffold but lacking the N-methyl acrylates group. The
for
anti-
3,4-diMeO -Ph
2-napthyl
Heterocyclic
10E
10Z
42
Table 2
Antimycobacterial activity compounds in MtH37Rv.
a
,
b
-unsaturated
prenyl
11E
12E
11Z
12Z
52
33
Compound
MIC
m
M
Compound
MIC
m
M
Compound
MIC mM
1Z
1E
2Z
2E
3Z
3E
4Z
4E
5Z
5E
INH
20
6Z
6E
7Z
7E
8Z
8E
9Z
9E
>20
>20
>20
>20
20
1.25
>20
>20
20
11Z
11E
12Z
12E
13Z
13E
14Z
14E
15Z
15E
10
1.25
>20
1.25
>20
20
>20
>20
>20
>20
0.36
5.0
>20
10
>20
>20
20
5.0
>20
10
13E
13Z
85
14E
15E
14Z
15Z
35
63
10Z
10E
Rif
5.0
0.30
Saturated
pentyl
Not observed
INH ¼ isoniazid Rif: Rifampycin

6
D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
Fig. 3. Minimal Inhibitory Concentrations of the compounds prepared against M. tuberculosis.
activity of that library on M. tuberculosis H37Rv ranged from
Me85.8 M (for the 2,4-dimethoxy analog); that is
and M. bovis BCG strains expressing M. tuberculosis or M. smegmatis
InhA [46]. As expected, the ratio is 20 times higher for INH and
ethionamide respectively.
As it can be seen in Table 3 and Fig. 4, the MIC on the InhA-OE
strain doubled the observed MIC for the wild type strain for the
six compounds selected. Of note, the 2-fold difference was
observed on a number of experimental repetitions. Those results
indicate that InhA could be the target (or one of the targets) of these
compounds.
MIC ¼ 411.1
m
m
four times less active than almost half of our library and 69 less
active than compounds 1E, 2E and 8E, the most active members of
the collection. Therefore, the importance of the addition of a
Michael acceptor on the structure is clearly relevant to improve the
biological activity.
A comparison with the similar scaffold previously mentioned
shown that the most active analogs 1E, 2E and 8E are 6- and 20-fold
more active than compounds B (MIC 7.8
m
M) and C (MIC 25
m
M) but
To gain insight into the binding mode, the previously selected
analogs (1E, 2E, 8E, 10E, 11E and 14E) were docked on the crys-
tallized enoyl-acyl carrier protein reductase protein (PDB ID 4TRO).
The results showed that all the compounds had the same binding
mode. They display a hydrogen bond interaction between the sul-
fonamide oxygen and Ile194 amide nitrogen that are at, on average,
2.15 Å and an average hydrogen bond angle of 159ꢁ (Fig. 5, Table 4).
The distance between Tyr158 and the ester group is, on average,
1.78 Å and an average hydrogen bond angle of 152ꢁ (Fig. 5, Table 4),
being another hydrogen bond interaction that contributes to the
binding of the compounds.
This critical interaction of Tyr158 has also been found on the
different crystal structures. Such an example is given by the crystal
structure of InhA bound to a carbonyl arylamide inhibitor. In this
structure, the oxygen of the amide carbonyl group is hydrogen-
bonded to the hydroxyl group of Tyr158, very close to what was
observed between the carbonyl group of the ester and the same
residue [47]. Other examples included the alkyl diphenyl ethers,
where the oxygen at the 4-hydroxy group of the inhibitor make
hydrogen bond with the hydroxy group of the tyrosine [45], with 4-
hydroxy-2-pyridone that displayed similar hydrogen-bonding
pattern [43]. More recently, the crystal structure of diazaborines,
a cofactor-independent class of inhibitors of enoyl fatty acyl re-
ductases including InhA, showed hydrogen bonds and charge-
charge interaction between the diazaborine head and Tyr158 [48].
did not reach the potency of INH and compound A. It is also
important to compare the activity of those compounds with the
parent drug INH. Analogs 1E, 2E and 8E have a MIC that is 3.5 times
higher than INH. Alternatively, we can also compare the activity of
those analogs with rifampicin. This antimycobacterial polyketide is
a key component of anti-tuberculosis therapy that targets the DNA-
dependent RNA polymerase. The MIC of the aza-acrylates 1E, 2E
and 8E are 4.2 times higher than that of the antimycobacterial
polyketide. That comparison suggest that further improvement is
necessary on the proposed structures to reach rifampicin potency.
Nevertheless, when compared to rifampicin, the aza-acrylates are
synthetic small molecules that can be easily prepared, being
excellent candidates for further development.
2.2.2. In vitro cytotoxicity assay on Vero cells
All the analogs were tested for cytotoxicity on VERO (monkey
kidney fibroblast) cells by Neutral Red assay, lacking cytotoxicity up
to the maximum concentration of 4.75
this information, the most active compounds 1E, 2E and 8E have
low cytotoxicity, with IC50 > 13.3 M, > 12.2 M and >11.6 M,
mg/mL. Taking into account
m
m
m
respectively. Those values correspond to selectivity index of 10.6,
9.76 and 9.28, which indicates that are good candidates to advance
to in vivo studies [41].
2.2.3. In vitro activity against MtInhA overexpressing strain
Having in mind that the proposed scaffold was inspired by INH,
we wanted to determine if our set of active compounds shared the
same mechanism of action. To do that, we set to validate InhA as the
main target of our compounds, towards that end we compared the
activity of the most active compounds of our library on the wild-
type H37Rv and on an InhA overexpressing M. tuberculosis strain
(InhA-OE) [42]. In the former, an increase on the MIC will be sug-
gestive of the role of this enzyme as an in vivo target as has been
reported by several authors. For example, 4-hydroxy-2-pyridones
have a ratio InhA-OE/H37Rv between 1.7 and 2.2 [43], meanwhile
alkyl diphenyl ethers ranged between 9 and 12 [44,45], and thia-
diazoles varied between 4-fold or more on M. smegmatis mc2155
Table 3
Compared activity against Mtb H37Rv and Mtb OE InhA.
Compound
MIC (mM)
H37Rv
InhA-OE
1E
2E
8E
10E
11E
14E
INH
1.25
1.25
1.25
5.0
5.0
5.0
2.5
5.0
2.5
10
10
10
0.36
>7.3

D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
7
Table 4
MIC and Autodock4 Energy Score for receptor 4TRO with computed cLogP for each
compound.
Compound
MIC
m
M
Autodock4 Energy (kcal/mol)
clogP
1E
2E
8E
10E
11E
14E
1.25
1.25
1.25
5.00
5.00
5.00
ꢂ9.03
ꢂ8.41
ꢂ9.83
ꢂ7.96
ꢂ7.98
ꢂ7.99
3.17
3.18
4.36
2.43
3.25
2.99
2.2.3.1. ADME-tox calculations and molecular properties calculations.
An adequate balance between the partition coefficient and the
solubility is necessary for oral drug good bioavailability. One-third
of new chemical entities have poor pharmacokinetic properties
and have not been able to reach the clinical trial phase. The
experimentally expensive process to determine ADME-Tox prop-
erties of drug candidates can be accelerated by using in silico
methods on early steps of the development pipeline. To that pur-
pose, computational studies of all the synthesized compounds were
performed to predict their physicochemical parameters, ADME
properties, Lipinski’s rule of five (Ro5), toxicity liabilities and drug-
likeness. The rule of five proposed by Lipinski et al. [49] is a widely
used filter which may indicate if a compound presents good
theoretical oral bioavailability (logP, MW, nON, and nNHOH). Veber
expanded with other parameters (ROT and PSA) that contribute to a
better prediction of oral absorption and bioavailability [50].
Because the analysis cannot differentiate between E and Z
regioisomers, only 15 calculations were necessary. To complete the
analyses, the data were compared with first-line drugs used for TB
treatment (isoniazideINHe, rifampicin-RIF-, pyrazinamide-PZA-,
ethambutol-EMB- and streptomycin-SM-).
The results presented in Table 5 show that all the compounds
have MW < 500, log P < 5 and the number of hydrogen bond ac-
ceptors and donors also fulfill the Ro5. The prepared compounds
also obey Veber parameters [50]. In summary, all the compounds
that form the collection seem to be good candidates in term of
satisfactory membrane permeability and oral bioavailability and,
thus, might be considered as drug-like molecules. As was expected,
two of the first-line drugs (rifampicin and streptomycin), violate
some of the rules, demonstrating that Ro5 may not be considered
strictly [51].
Fig. 4. Compared activity against Mtb H37Rv and Mtb OE InhA.
A detailed Autodock4 energy profile of the docking studies of
the selected analogs is presented in Table 4. When the calculated
score energy is compared with the MTb MIC, a similar tendency
was observed. Nevertheless, comparison of the most active analogs
displaying equal MIC values (1.25 mM) showed that the simplest
analog 8E seems to form the most stable complex, followed by 1E
and 2E.
We can classify the compounds in two different groups when
looking at hydrogen bond distances and angles to Ile194. 1E, 2E and
8E have tighter interactions (2.07 A and 157ꢁ) while 10E, 11E and
14E have higher H-bond distances (2.24 A and 160.5ꢁ). On the other
hand, the H-bond distances and angles are quite similar between all
the derivatives analyzed here.
The main difference in the observed activity could be explained
by the type of hydrophobic R group. Aromatic interactions between
aromatic R substituents and Phe97 could be involved in the
observed better MIC and the computed Autodock4 Energy (Fig. 5,
dashed lines). Nevertheless, we cannot discard that the change in
hydrophobicity could also influence the ability of the compounds to
penetrate the cell wall and thus, explain the different MIC between
the compounds. The change in hydrophobicity can be seen in the
computed clogP for the compounds (Table 4). The computed clogP
of the compounds correlates quite well with the lipophilicity of
them.
The Osiris platform [52] predicted the overall toxicity based on
the presence of fragments responsible for irritant, mutagenic,
tumorigenic, or reproductive effects. The calculations shown in
Table 6 revealed that all compounds, except for compound 5, pre-
sent a low toxicity risk. When compared to the first line drug INH, it
Fig. 5. Docking poses of analogs 1E, 2E, 8E on InhA PDB ID 4TRO. Protein backbone is depicted in New cartoon representation in transparent background. Key residues are shown as
sticks while compounds are shown with Ball and Sticks representation. Color code of atoms: Carbon (Cyan), Nitrogen (Blue), Oxygen (Red) and Sulphur (Yellow). Point lines depict
Hydrogen Bonds while dashed lines indicate Pi-Pi interaction between aromatic centers. (For interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)

8
D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
Table 5
In silico parameters of acrylates compounds and first-line drugs.
Compound
MW
logP
n ON
nNHOH
TPSA
ROT
Viol.
1e15
INH
EMB
RIF
SM
PZA
Lipinski [49]
Veber [50]
347e436
137
204
822
581
0.91e3.79
ꢂ0.97
0.35
0e1
3
4
6
16
2
84.42e144.1
68.01
64.51
216.6
336.4
7e9
1
9
5
11
1
0
0
0
3
3
0
4
4
16
19
4
2.10
ꢂ7.86
ꢂ0.71
ꢀ5
123
<500
68.88
<5
<10
ꢀ140
ꢀ10
the calculated for INH. The lowest MIC obtained in our series is 4
times higher than that of INH. However, the drug-score value and
low toxicity point out that these NCE are interesting candidates for
antimycobacterial drug development.
Table 6
In silico toxicity risks and drugs-score of acrylates compounds and first line drugs.
Compound
Toxicity risksa
Drug-score
Mb
Tc
Id
Ree
Drug likeness
Drug-score
As InhA is an important target for new antimycobacterial drug
development, a wide range of inhibitors has been previously re-
ported. A search on the ChEMBL database, a chemical database of
bioactive molecules maintained by the European Bioinformatics
Institute (EMBL-EBI) [53], provided 322 compounds with reported
activity on the enzyme. In Fig. 6 we can observe that the physico-
chemical parameters (logP, MW and TPSA) of the most active
compounds of the library shown in Table 7 are in the same range of
the wide collection of InhA inhibitors reported.
To define the explored chemical space between the reported
InhA inhibitors (the 322 InhA inhibitors found on ChEMBL) and our
collection, a similarity search was performed using the web-based
platform chemmine tools [54] using a Tanimoto coefficient> 0.7. The
results were plotted in a three-dimensional space group according
to the different clusters found. The 30 synthesized analogs were
also included in the same analysis to identify their similarity with
the reported inhibitors. Fig. 7 shows the different clusters found,
where the aza-acrylate are grouped (orange) in a different cluster.
The analysis performed demonstrates that the prepared collection
has low similarity with previously reported compounds and is a
new chemotype that is located in an unexplored section of the
chemical space of the InhA inhibitors.
1e15
INH
EMB
RIF
SM
PZA
-
-
-
-
ꢂ16.4 to ꢂ4.23
ꢂ5.06
0.14e0.45
0.06
0.56
0.2
0.32
þ
þ
þ
þ
-
þ
-
-
-
2.38
-
-
þ
-
-
þ
þ
-
þ
10.12
0.83
ꢂ0.68
þ
-
0.14
a
Ranked according to: (ꢂ) not toxic, ( ) slightly toxic, (þ) highly toxic.
b
c
M, mutagenic;
T, tumorigenic;
I, irritant;
Re, reproductive effective.
d
e
has high toxicity risks for all the indicators, and the other five drugs
presented at least one alert.
Comparing the drug-likeness of the compounds against first-
line drugs, positive drug-likeness value (0.1e10) points out that a
molecule contains predominantly fragments which are frequently
present in commercial drugs; we found lower values of drug-
likeness for the compounds with the exception analog 7 which is
better than isoniazid.
The drug-score combines drug-likeness, lipophilicity, solubility,
molecular weight, and toxicity risks all in one value, and this may
be used to judge the compound overall potential as a drug. As seen
from results collected in Table 7, the most active compounds
showed low to moderate drug-score (0.14e0.45) that revealed their
potential as safe lead compounds. Most of the compounds have
higher values than first-line drugs except for ethambutol.
A rapid comparison between the most active compounds of the
series against INH (Table 7) shows a clear contrast. While the aza-
acrylates do not have toxicity risks, INH showed high toxicity
risks as observed in patients under treatment with INH. The logP
values of the compounds are in the accepted range, while the
negative value for INH confirms the low solubility in water. PSA
values suggest that both the new compounds as well as INH have
good permeability of biological membranes. Because drug-score
calculations are performed considering the toxicity and physical
properties, the drug-score is about 5 times higher in our series than
3. Conclusions
In summary, we have prepared fifteen E,Z pairs of N-substituted
tosyl N’-acryl hydrazine acrylates with good yields. This collection
was assayed against M. tuberculosis, nine of then having a MIC of
10 mM or below. The most active compounds 1E, 2E and 8E are E
isomers holding bulky aromatic (phenyl and naphthyl) sub-
stituents. As pointed out, the acrylate group is critical for the
antimycobacterial activity, identifying a potent novel scaffold
(Fig. 8). We have tested the compounds against an InhA over-
expressing strain, obtaining MIC differences suggesting that the
compounds could potentially inhibit the enzyme. Docking studies,
performed to rationalize how the compounds interact with the
enzyme, found that hydrogen bond interactions with Tyr 158 and
Table 7
In silico parameters and biological activities of acrylates hits and INH.
Compd
Toxicity risks
MW
logP
PSA
Drug-score
MICa
m
M
CYTb
m
M
SIc
1E
2E
8E
INH
- - - -
- - - -
- - - -
þþþþ
358
388
408
137
3.17
3.18
4.36
ꢂ1.02
76.05
85.28
76.05
68.01
0.43
0.41
0.33
0.06
1.25
1.25
1.25
0.37
>13.3
>12.2
>11.6
10.6
9.76
9.28
a
Activity against Mtb HR37Rv.
Citotoxicity against Vero cells.
Selectivity index.
b
c

D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
9
Fig. 6. Distribution of physicochemical parameters of the chemical library.
Fig. 8. SAR analysis of the aza acrylates chemical library.
mechanism of action to completely validate InhA as the main
molecular target.
Fig. 7. Analysis of molecular similarity of known InhA inhibitors and the prepared
collection (highlighted in orange). (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)
4. Material and methods
Ile194 were critical for binding InhA, in agreement with results by
Parikh [55,56] and Khan [57]. A complete chemoinformatics anal-
ysis of the collection was also performed, suggesting that the
compounds have no toxicity risk and comply with Lipinski’s Ro5. A
similarity search of the prepared collection with known InhA in-
hibitors revealed that the new INH inspired analogs are located in
an unexplored region of the chemical space. Isolation and charac-
terization of M. tuberculosis mutants resistant to compounds 1E, 2E
and 8E will shed light on whether they bind InhA and help un-
derstanding the protein-compound molecular interactions in vivo.
Thus, we consider that the aza-acrylate is a promising scaffold for
antitubercular drug discovery that prompt further studies on their
4.1. General
All reactions were performed under nitrogen atmosphere using
oven-dried glassware and standard syringe/septa techniques. The
reaction progress was monitored on GP TLC plates. Spots were
visualized under 254 nm UV light and/or by dipping the TLC plate
into a solution of 2 mL anisaldehyde and 10 mL glacial acetic acid
and followed by heating with a heat gun. Column chromatography
was performed with silica gel 60 (230e400 mesh) under a low
pressure of nitrogen, using increasing EtOAc-hexane gradients as a
solvent. All the solvents (hexane, ethyl acetate, acetonitrile) were
distilled before use.

10
D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
1H and 13C NMR spectra were measured on a 300 MHz Bruker
Avance II using CDCl3 as a solvent. Chemical shifts were reported in
ppm downfield from tetramethylsilane ( ) as the internal standards
and coupling constants are in hertz (Hz). Assignments of proton
resonances were confirmed by correlated spectroscopy (Hetero-
nuclear Single Quantum Coherence, HSQC) high-resolution mass
spectra (HRMS) were recorded on a Bruker MicroTOF II spray
source. All the melting points were determined in open Pyrex
capillaries with an Electrothermal 9000 melting point apparatus
and are uncorrected.
4.2.1.4. Methyl (E)-3-(2-((E)-2-methoxybenzylidene)-1-
tosylhydrazinyl) acrylate (2E). White solid. Mp: 154.8e155.8 ꢁC.
d
1H NMR (300 MHz, CDCl3):
d
9.08 (s, 1H, C4eH), 8.12 (d, J ¼ 13.5 Hz,
1H, C3eH), 7.90 (d, J ¼ 7.5 Hz, 1H, Harom), 7.67 (d, J ¼ 8.6 Hz, 2H,
C5eH), 7.50 (d, J ¼ 8.2 Hz, 1H, Harom), 7.32 (d, J ¼ 7.9 Hz, 2H, C6eH),
6.97 (t, J ¼ 8.0 Hz, 2H, Harom), 5.32 (d, J ¼ 13.5 Hz, 1H, C2eH), 3.89 (s,
3H, OCH3), 3.69 (s, 3H, OCH3), 2.43 (s, 3H, Ts-CH3). 13C NMR
(75 MHz, CDCl3):
d 167.4 (C), 166.1 (CH), 159.6 (C), 145.1 (C), 141.9
(CH), 134.4 (CH), 133.8 (C), 129.9 (CH), 128.0 (CH), 127.4 (CH), 120.8
(CH), 111.3 (CH), 101.5 (CH), 55.7 (CH3), 51.4 (CH3), 21.7 (CH3). IR
(KBr) y: 1714, 1624, 1597, 1487, 1436, 1371, 1288, 1253, 1170, 1145,
1087, 1045, 756, 705, 659 cmꢂ1.ESI-HRMS Calcd for (M þ Hþ)
4.2. Synthesis
C
19H21N2O5S 389.1166, found 389.1166.
4.2.1. General procedure for the preparation of acrylates
4.2.1.5. Methyl (Z)-3-(1-tosyl-2-((E)-2-(trifluoromethyl)benzylidene)
hydrazinyl) acrylate (3Z). Colorless oil. 1H NMR(300 MHz, CDCl3):
The p-toluene sulfonyl hydrazide (279 mg, 1.5 mmol) was added
to a solution of aldehyde (1.5 mmol) in dry acetonitrile and the
mixture was stirred at 90 ꢁC for 6 h. A solution of 5 M NaOH
(1.5 mmol) was added and the mixture was stirred for another
30 min at RT. Then the propiolate (7.5 mmol) was added dropwise,
and the mixture was stirred at 90 ꢁC. The volatiles were evaporated
under reduced pressure, and the residue was dissolved in a 1:1
mixture of brine-ethylacetate (70 mL). The organic layer was
separated and dried over Na2SO4. After filtration and removal of the
solvent under reduced pressure, the crude material was purified by
flash chromatography over silica gel using an increasing AcOEt/
hexane gradient.
d
8.23 (s,1H, C4eH), 8.07 (d, J ¼ 7.8 Hz,1H, Harom), 7.81 (d, J ¼ 8.4 Hz,
2H, C5eH), 7.67 (d, J ¼ 7.8 Hz, 1H, Harom), 7.57- 7.49 (m, 2H, Harom),
7.32 (d, J ¼ 8.0 Hz, 2H, C6eH), 6.39 (d, J ¼ 8.8 Hz, 1H, C3eH), 5.91 (d,
J ¼ 8.8 Hz, 1H, C2eH), 3.66 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3).13
C
NMR (75 MHz, CDCl3):
d 164.8 (C), 146.8 (CH), 145.0 (C), 134.2 (C),
132.1 (CHx2), 131.3 (CH); 130.3 (CH), 129.8 (CH), 128.3 (CH), 127.5
(CH), 125.9 (CH), 117.8 (CH), 51.8 (CH3), 21.6 (CH3). IR (film) y: 1714,
1624, 1597, 1438, 1315, 1166, 1122, 1033, 769, 671 cmꢂ1. ESI-HRMS
Calcd for (M þ Hþ) C19H17N2NaO4S 449.0753, found 449.0753.
4.2.1.6. Methyl (E)-3-(1-tosyl-2-((E)-2-(trifluoromethyl)benzylidene)
hydrazinyl) acrylate (3E). Colorless oil. 1H NMR (300 MHz, CDCl3):
4.2.1.1. Methyl (Z)-3-(2-((E)-benzylidene)-1-tosylhydrazinyl) acrylate
d
9.05 (s,1H, C4eH), 8.17 (d, J ¼ 13.5 Hz,1H, C3eH), 8.14-8.11 (m,1H,
(1Z). Colourless oil. 1H NMR (300 MHz, CDCl3):
d 8.24 (s,1H, C4eH),
H
arom), 7.80-7.74 (m, 1H, Harom), 7.69 (d, J ¼ 13.5 Hz, 2H, C5eH),
7.74 (d, J ¼ 8.3 Hz, 2H, Harom), 7.65 (d, J ¼ 8.1, 2H, C5eH), 7.43-7.40
(m, 3H, Harom), 7.31 (d, J ¼ 8.1 Hz, 2H, C6eH), 6.53 (d, J ¼ 9.1 Hz, 1H,
C3eH), 5.65 (d, J ¼ 9.1 Hz, 1H, C2eH), 3.57 (s, 3H, OCH3), 2.42 (s, 3H,
7.62-7.58 (m, 2H, Harom), 7.32 (d, J ¼ 8.1 Hz, 2H, C6eH), 5.52 (d, J ¼
13.5 Hz, 1H, C2eH), 3.73 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR
(75 MHz, CDCl3):
d 167.2 (C), 160.1 (CH), 145.7 (C), 143.7 (C), 141.4
Ts-CH3). 13C NMR (75 MHz CDCl3):
d 165.5 (CH), 158.3 (C), 144.9 (C),
(CH), 133.6 (C), 132.2 (CHx2), 131.7 (C), 130.1 (CH), 128.4 (C), 128.1
(CH), 127.9 (CH), 126.2 (CH), 102.4 (CH), 51.5 (CH3), 21.7 (CH3). IR
134.2 (CH), 133.3 (C), 133.1 (CH), 131.5 (C), 130.0 (CH), 129.7 (CH),
128.8 (CH), 128.3 (CH), 113.3 (CH), 51.7 (CH3), 21.6 (CH3). IR (film)
y:
(film)
y: 1714, 1624, 1597, 1438, 1315, 1166, 1122, 1033, 769,
3061, 1712, 1625, 1604, 1435, 1170, 1143, 1087, 813, 704, 657,
574 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C18H19N2O4S 359.1060,
found 359.1060.
671 cmꢂ1.ESI-HRMS Calcd for (M þ Hþ) C19H17N2NaO4S 449.0753,
found 449.0753.
4.2.1.7. Methyl (Z)-3-(2-((E)-[1,10-biphenyl]-4-ylmethylene)-1-
tosylhydrazinyl) acrylate (4Z). Colourless oil. 1H NMR (300 MHz,
4.2.1.2. Methyl (E)-3-(2-((E)-benzylidene)-1-tosylhydrazinyl) acry-
late (1E). Colourless oil. 1H NMR (300 MHz, CDCl3):
d 8.69 (s, 1H,
CDCl3):
d 8.30 (s, 1H, C4eH), 7.76-7.72 (m, 4H, Harom), 7.65-7.60 (m,
C4eH), 8.12 (d, J ¼ 13.6 Hz,1H, C3eH), 7.77 (d, J ¼ 7.5 Hz, 2H, Harom),
7.65 (d, J ¼ 8.5 Hz, 2H, C5eH), 7.53 (d, J ¼ 7.3 Hz, 1H, Harom), 7.46 (d,
J ¼ 7.5 Hz, 2H, Harom), 7.31 (d, J ¼ 8.2 Hz, 2H, C6eH), 5.37 (d,
4H, Harom), 7.49-7.43 (m, 2H, Harom), 7.40-7.38 (m, 1H, Harom), 7.31
(d, J ¼ 8.1 Hz, 2H, C6eH), 6.57 (d, J ¼ 9.2 Hz, 1H, C3eH), 5.65 (d,
J ¼ 9.2 Hz, 1H, C2eH), 3.59 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3). 13
C
J ¼ 13.6 Hz, 1H, C2eH), 3.70 (s, 3H, OCH3), 2.43 (s, 3H, TsCH3). 13
C
NMR (75 MHz, CDCl3):
d 165.5 (C), 158.4 (CH), 144.9 (C), 144.2 (C),
NMR (75 MHz, CDCl3):
d 169.4 (CH), 167.3 (C), 145.3 (C), 141.8 (CH),
140.0 (C), 134.2 (C), 133.4 (CH), 132.0 (C), 129.7 (CH), 129.0 (CH),
128.9 (CH), 128.7 (CH), 128.3 (CH), 127.5 (CH), 127.1 (CH), 112.8 (CH),
133.7 (C), 132.8 (CH), 132.3 (C), 129.9 (CH), 129.1 (CH), 129.0 (CH),
127.9 (CH), 102.0 (CH), 51.4 (CH3), 21.7 (CH3). IR (film) y: 3061, 1712,
51.7 (CH3), 21.7 (CH3). IR (film) y: 3062, 3030, 2993, 2951, 2920,
1625, 1604, 1435, 1170, 1143, 1087, 813, 704, 657, 574 cmꢂ1. ESI-
2848, 1714, 1623, 1600, 1371, 1170, 1145, 1087 cmꢂ1. ESI-HRMS Calcd
HRMS Calcd for (M þ Hþ) C18H19N2O4S 359.1060, found 359.1060.
for (M þ Hþ) C24H22N2NaO4S 457.1192, found 457.1193.
4.2.1.3. Methyl (Z)-3-(2-((E)-2-methoxybenzylidene)-1-
4.2.1.8. Methyl (E)-3-(2-((E)-[1,10-biphenyl]-4-ylmethylene)-1-
tosylhydrazinyl)acrylate (4E). White solid. Mp 107e109 ꢁC.NMR
tosylhydrazinyl) acrylate (2Z). Colorless oil. 1H NMR (300 MHz,
CDCl3):
d
8.59 (s, 1H, C4eH), 7.73 (m, 3H, Harom-C5-H), 7.39 (m, 1H,
(300 MHz, CDCl3):
d
8.73 (s, 1H, C4eH), 8.13 (d, J ¼ 13.5, 1H, C3eH),
H
arom), 7.29 (d, J ¼ 8.1 Hz, 2H, C6eH), 6.95 (d, J ¼ 7.6 Hz, 1H, Harom),
7.85 (d, J ¼ 8.31, 2H, Harom), 7.72-7.60 (m, 6H, Harom-C5-H), 7.48 (t,
J ¼ 7.3 Hz, 2H), 7.42 (d, J ¼ 7.1 Hz, 1H), 7.33 (d, J ¼ 8.1, 2H, C6eH),
5.39 (d, J ¼ 13.5 Hz, 1H, C2eH), 3.71 (s, 3H, OCH3), 2.44 (s, 3H, Ts-
CH3). 13C NMR (75 MHz, CDCl3): 169.0 (CH), 167.3 (C), 145.6 (C),
145.3(C), 141.8 (CH), 139.8 (C), 133.7 (C), 131.1(C), 130.0 (CH), 129.6
(CH), 129.0 (CH), 128.3 (CH), 128.0 (CH), 127.6 (CH), 127.2 (CH), 101.9
6.89 (d, J ¼ 7.9 Hz, 1H, Harom), 6.46 (d, J ¼ 9.2 Hz, 1H, C3eH), 5.65 (d,
J ¼ 9.2 Hz, 1H, C2eH), 3.85 (s, 3H, OCH3), 3.60 (s, 3H, OCH3), 2.41 (s,
3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
d 165.6 (C), 158.8 (C), 153.6
(CH), 144.7 (C), 134.4 (C), 133.4 (CH), 132.8 (CH), 129.6 (CH), 128.3
(CH); 126.8 (CH), 121.6 (C), 120.7 (CH), 113.5 (CH), 111.1 (CH), 55.6
(CH3), 51.6 (CH3), 21.6 (CH3). IR (film)
y
: 1714,1624, 1597, 1487,1436,
(CH), 51.42 (CH3), 21.7 (CH3).IR (KBr) y: 3062, 3030, 2993, 2951,
1371, 1288, 1253, 1170, 1145, 1087, 1045, 756, 705, 659 cmꢂ1. ESI-
2920, 2848, 1714, 1623, 1600, 1371, 1170,1145, 1087 cmꢂ1. ESI-HRMS
HRMS Calcd for (M þ Hþ) C19H21N2O5S 389.1166, found 389.1166.
Calcd for (M þ Hþ) C24H22N2NaO4S 457.1192, found 457.1193.

D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
11
4.2.1.9. Methyl (Z)-3-(2-((E)-4-nitrobenzylidene)-1-tosylhydrazinyl)
3.88 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 2.40 (s, 3H, TsCH3). 13C NMR
(75 MHz, CDCl3): 170.2 (CH), 167.3 (C), 153.4 (C), 149.5 (C), 145.2
acrylate (5Z). White solid. Mp: 120.0e121.0 ꢁC. 1H NMR (300 MHz,
d
CDCl3):
d
8.24 (d, J ¼ 8.9 Hz, 2H, Harom), 7.98 (s, 1H, C4eH), 7.78 (t,
(C), 141.9 (CH), 133.8 (C), 129.9 (CH), 128.0 (CH), 125.4 (CH), 125.0
(C), 110.7 (CH), 109.2 (CH), 101.5 (CH), 56.1 (CH3), 56.0 (CH3), 51.4
(CH3), 21.7 (CH3). IR (film)Ʋ: 1730,1597,1514,1438,1267,1166,1024,
813, 765 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C20H22N2NaO4S
441.1091, found 441.1091.
J ¼ 8.2 Hz, 4H, Harom-C5-H), 7.33 (d, J ¼ 8.0 Hz, 2H, C6eH), 6.37 (d,
J ¼ 8.7 Hz, 1H, C3eH), 5.93 (d, J ¼ 8.7 Hz, 1H, C2eH), 3.68 (s, 3H,
OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
d 164.7 (C),
148.9 (CH), 148.7 (C), 145.3 (C), 139.1 (C), 134.1(C), 132.1 (CH), 129.8
(CH), 128.4 (CH), 128.3 (CH), 124.1 (CH), 118.2 (CH), 51.9 (CH3), 21.7
(CH3). IR (KBr)
y
: 1732, 1597, 1519, 1435, 1344, 1172, 1087, 1037, 854,
4.2.1.15. Methyl (Z)-3-(2-((E)-naphthalen-2-ylmethylene)-1-
748, 671 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C18H17N3NaO6S
tosylhydrazinyl) acrylate (8Z). Colourless oil. 1H NMR (300 MHz,
426.0730, found 426.0730.
CDCl3): d 8.43 (s, 1H, C4eH), 8.01 (s, 1H, Harom), 7.87- 7.84 (m, 4H,
H
arom), 7.76 (d, J ¼ 8.3 Hz, 2H, C5eH), 7.56-7.53 (m, 2H, Harom), 7.31
4.2.1.10. Methyl (E)-3-(2-((E)-4-nitrobenzylidene)-1-tosylhydrazinyl)
(d, J ¼ 8.1 Hz, 2H, C6eH), 6.61 (d, J ¼ 9.3 Hz, 1H, C3eH), 6.65 (d, J ¼
acrylate (5E). Colourless oil. 1H NMR (300 MHz, CDCl3):
d 8.78 (s,
9.3 Hz, 1H, C2eH), 3.56 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR
1H, C4eH), 8.29 (d, J ¼ 8.6 Hz, 2H, Harom), 8.10 (d, J ¼ 13.5 Hz, 1H,
C3eH), 7.92 (d, J ¼ 8.6 Hz, 2H, Harom), 7.66 (d, J ¼ 8.3 Hz, 2H, C5eH),
7.33 (d, J ¼ 8.6 Hz, 2H, C6eH), 5.54 (d, J ¼ 13.5 Hz,1H, C2eH), 3.74 (s,
(75 MHz, CDCl3): d 165.6 (C), 159.0 (CH), 144.9 (C), 134.9 (C), 134.2
(C), 133.4 (CH), 133.0 (C), 131.1 (CH), 130.8 (CH), 129.7 (CH), 128.8
(CH), 128.3 (CH), 127.9 (CH), 127.7 (CH), 126.8 (CH), 122.9 (CH), 112.6
3H, OCH3), 2.74 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
d
167.0
(CH), 51.7 (CH3), 21.7 (CH3). IR (film) y: 1712, 1625, 1371, 1170, 1145,
(C), 162.0 (CH), 145.9 (C), 141.2 (CH), 138.1 (C), 133.4 (C), 130.2 (CH),
129.5 (CH), 127.8 (CH), 124.2 (CH), 103.2 (CH), 51.6 (CH3), 21.7
(CH3).IR (film)Ʋ: 1732, 1597, 1519, 1435, 1344, 1172, 1087, 1037, 854,
748, 671 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C18H17N3NaO6S
426.0730, found 426.0730.
1087, 813, 704 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C22H20N2O4S
409.1217, found 409.1217.
4.2.1.16. Methyl (E)-3-(2-((E)-naphthalen-2-ylmethylene)-1-
tosylhydrazinyl) acrylate (8E). White solid. Mp ¼ 146.2e147.0 ꢁC.
1H NMR (300 MHz, CDCl3):
d
8.85 (s, 1H, C4eH), 8.16 (d, J ¼ 13.4 Hz,
4.2.1.11. Methyl (Z)-3-(2-((E)-4-bromobenzylidene)-1-
1H, C3eH), 8.14 (s, 1H, Harom), 7.96- 7.85 (m, 4H, Harom), 7.68 (d, J ¼
8.4 Hz, 2H, C5eH), 7.58 (quint, J1 ¼ 5.9 Hz, J2 ¼ 1.2 Hz, 2H, Harom),
7.32 (d, J ¼ 8.4 Hz, 2H, C6eH), 5.44 (d, J ¼ 13.5 Hz, 1H, C2eH), 3.72
tosylhydrazinyl) acrylate (6Z). White solid. Mp: 120.2e121.0 ꢁC. 1H
NMR (300 MHz, CDCl3):
d
8.11 (s, 1H, C4eH), 7.72 (d, J ¼ 8.2 Hz, 2H,
C5eH), 7.51 (m, 4H, Harom), 7.30 (d, J ¼ 8.2, 2H, C6eH), 6.48 (d, J ¼
8.3 Hz, 1H, C3eH), 5.69 (d, J ¼ 9.0 Hz, 1H, C2eH), 3.59 (s, 3H, OCH3),
(s, 3H, OCH3), 2.43 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
d 169.1
(CH), 167.3 (C), 145.3 (C), 141.8 (CHx2), 135.5 (C), 133.7 (C), 132.8
(CH), 132.6 (C), 130.0 (CH), 129.0 (CHx2), 128.4 (CH), 128.0 (CH),
2.41 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
d 165.3 (C), 155.6
(CH), 145.0 (C), 134.1 (C), 133.1 (CH), 132.1 (CH), 129.8 (CH), 129.4
(CH),128.2 (CH),125.9 (C),114.2 (CH), 51.7 (CH3), 21.6 (CH3). IR (KBr)
Ʋ: 1730, 1643, 1591, 1487, 1435, 1359, 1280, 1172, 1087, 1068, 1008,
817, 738, 667 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C18H18BrN2O4S
437.0165, found 437.0165.
127.1 (CH), 123.1 (CH), 102.0 (CH), 51.4 (CH3), 21.7 (CH3). IR (KBr) y:
1712, 1625, 1371, 1170, 1145, 1087, 813, 704 cmꢂ1. ESI-HRMS Calcd
for (M þ Hþ) C22H20N2O4S 409.1217, found 409.1217.
4.2.1.17. Methyl (E)-3-(2-((E)-(1H-pyrrol-2-yl)methylene)-1-
tosylhydrazinyl)acrylate (9E). Colourless oil. 1H NMR (300 MHz,
4.2.1.12. Methyl (E)-3-(2-((E)-4-bromobenzylidene)-1-
CDCl3):
J ¼ 8.3 Hz, 2H, C5eH), 7.30 (d, J ¼ 8.1 Hz, 2H, C6eH), 7.01 (s, 1H,
arom), 6.78 (s,1H, Harom), 6.35 (s,1H, Harom), 5.25 (d, J ¼ 13.5 Hz,1H,
C2eH), 3.69 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR (75 MHz,
CDCl3): 167.4 (C), 160.6 (CH), 145.1 (C), 142.1 (CH), 133.7 (C), 129.9
(CH), 128.0 (CH), 125.7 (C), 124.8 (CH), 120.0 (CH), 111.4 (CH), 101.0
d
8.42 (s, 1H, C4eH), 8.12 (d, J ¼ 13.5 Hz, 1H, C3eH), 7.63 (d,
tosylhydrazinyl) acrylate (6E). Colourless oil. 1H NMR(300 MHz,
CDCl3):
d
8.65 (s, 1H, C4eH), 8.10 (d, J ¼ 13.4 Hz, 1H, C3eH), 7.65-
H
7.61 (m, 6H, Harom-C5-H), 7.31 (d, J ¼ 8.0 Hz, 2H, C6eH), 5.37 (d, J ¼
13.4 Hz, 1H, C2eH), 3.71 (s, 3H, OCH3), 2.43 (s, 3H, Ts-CH3). 13C NMR
d
(75 MHz, CDCl3):
d 167.3 (CH), 167.2 (C), 145.4 (C), 141.6 (CH), 133.6
(C),132.4 (C), 131.2 (CH),130.3 (CH),130.0 (CH), 127.9 (CH),127.6 (C),
102.2 (CH), 51.5 (CH3), 21.7 (CH3). IR (film)Ʋ: 1730, 1643, 1591, 1487,
1435, 1359, 1280, 1172, 1087, 1068, 1008, 817, 738, 667 cmꢂ1. ESI-
HRMS Calcd for (M þ Hþ) C18H18BrN2O4S 437.0165, found 437.0165.
(CH), 51.4 (CH3), 21.7 (CH3). IR (film) y:1699, 1597, 1436, 1369, 1168,
1087, 1037, 813, 748 cmꢂ1. ESI- HRMS Calcd for (M þ Naþ)
C16H17N3NaO4S 370.0832, found 370.0832.
4.2.1.18. Methyl (Z)-3-(2-((E)-furan-2-ylmethylene)-1-
4.2.1.13. Methyl (Z)-3-(2-((E)-3,4-dimethoxybenzylidene)-1-
tosylhydrazinyl) acrylate (10Z). White solid. MP: 102.1e102.9 ꢁC.
tosylhydrazinyl) acrylate (7Z). Colourless oil. 1H NMR (300 MHz,
1HNMR (300 MHz, CDCl3):
d
8.23 (s, 1H, C4eH), 7.71 (d, J ¼ 8.4 Hz,
CDCl3):
d
8.34 (s, 1H, C4eH), 7.85 (d, J ¼ 8.2 Hz, 1H, Harom), 7.70 (d,
2H, C5eH), 7.55 (s, 1H, Harom), 7.31 (d, J ¼ 8.1 Hz, 2H, C6eH), 6.89 (d,
J ¼ 3.5 Hz, 1H, Harom), 6.61 (d, J ¼ 9.4 Hz, 1H, C3eH), 6.53 (q,
J1 ¼ 3.4 Hz, J2 ¼ 1.8 Hz, 1H, Harom), 5.54 (d, J ¼ 13.5 Hz, 1H, C2eH),
3.58 (s, 3H, OCH3), 2.41 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
J ¼ 8.3 Hz, 2H, C5eH), 7.29 (d, J ¼ 8.0 Hz, 2H, C6eH), 7.19 (m, 1H,
H
arom), 6.88 (d, J ¼ 8.2 Hz, 1H, Harom), 6.67 (d, J ¼ 9.3 Hz, 1H, C3eH),
5.48 (d, J ¼ 9.3 Hz, 1H, C2eH), 3.92 (s, 3H, OCH3), 3.89 (s, 3H, OCH3),
3.51 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
d
165.4 (C), 150.1 (CH), 148.5 (C), 145.9 (C), 145.0 (CH), 133.9 (C),
d
170.2 (CH), 166.2 (C), 154.5 (C), 153.4 (C), 141.9 (CH), 134.2 (C),
133.8 (CH), 129.8 (CH), 128.2 (CH), 116.7 (CH), 112.2 (CH), 111.3 (CH),
129.9 (CH), 128.0 (CHx2), 126.9 (C), 125.4 (C), 110.4 (CH), 109.0 (CH),
101.5 (CH), 56.0 (CH3x2), 51.4 (CH3), 21.7 (CH3). IR (film)Ʋ: 1730,
1597, 1514, 1438, 1267, 1166, 1024, 813, 765 cmꢂ1. ESI-HRMS Calcd
for (M þ Hþ) C20H22N2NaO4S 441.1091, found 441.1091.
51.5 (CH3), 21.6 (CH3). IR (KBr) y: 2951, 2096, 1712, 1643, 1471, 1435,
1371, 1130, 1087, 937, 661 cmꢂ1. ESI-HRMS Calc for (M þ Hþ)
C16H17N2O5S 349.0853, found 349.0853.
4.2.1.19. Methyl (E)-3-(2-((E)-furan-2-ylmethylene)-1-
4.2.1.14. Methyl (E)-3-(2-((E)-3,4-dimethoxybenzylidene)-1-
tosylhydrazinyl) acrylate (10E). Colourless oil. 1H NMR (300 MHz,
tosylhydrazinyl) acrylate (7E). Colourless oil. 1H NMR (300 MHz,
CDCl3):
d
8.52 (s, 1H, C4eH), 8.09 (d, J ¼ 13.5 Hz, 1H, C3eH), 7.65 (d,
CDCl3)
d
8.56 (s, 1H, C4eH), 8.11 (d, J ¼ 13.5 Hz, 1H, C3eH), 7.64 (d,
J ¼ 8.6 Hz, 2H, C5eH), 7.64 (s, 1H, Harom), 7.31 (d, J ¼ 8.2 Hz, 2H,
C6eH), 7.05 (d, J ¼ 3.6 Hz, 1H, Harom), 6.59 (q, J1 ¼ 13.5 Hz,
J2 ¼ 13.5 Hz, 1H, Harom), 5.38 (d, J ¼ 13.5 Hz, 1H, C2eH), 3.70 (s, 3H,
J ¼ 8.1 Hz, 2H, C5eH), 7.34- 7.28 (m, 4H, Harom-C6-H), 6.90 (d, J ¼
8.3 Hz,1H, Harom), 5.31 (d, J ¼ 13.5 Hz,1H, C2eH), 3.93 (s, 3H, OCH3),

12
D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
157.5 (CH), 147.9 (C), 147.2 (CH), 145.4 (C), 141.5 (CH), 133.6 (C), 130.0
(CH), 127.9 (CH), 119.3 (CH), 112.6 (CH), 102.0 (CH), 51.4 (CH3), 21.7
d
163.3 (C),
(CH3), 21.6 (CH3), 21.0 (CH3). IR (film)y: 1732, 1614, 1433, 1371, 1257,
1170, 1143, 1001, 734 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ)
C21H27N2O4S 403.1686, found 403.1686.
(CH3). IR (film) y: 2951, 2096, 1712, 1643, 1471, 1435, 1371, 1130,
1087, 937, 661 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C16H17N2O5S
4.2.1.25. Methyl (E)-3-(2-((E)-(6,6-dimethylbicyclo[3.1.1]hept-2-en-
349.0853, found 349.0853.
3-yl)methylene)-1-tosylhydrazinyl)acrylate (13E). White solid. Mp:
87.9e88.6 ꢁC. 1H NMR (300 MHz, CDCl3):
d 8.24 (s, 1H, C4eH), 8.06
4.2.1.20. Methyl (Z)-3-(2-((E)-3-methylbut-2-en-1-ylidene)-1-
(d, J ¼ 13.6 Hz, 1H, C3eH), 7.58 (d, J ¼ 8.5 Hz, 2H, C5eH), 7.29 (d, J ¼
8.0 Hz, 2H, C6eH), 6.39 (s, 1H, CH), 5.20 (d, J ¼ 13.6 Hz, 1H, C2eH),
3.69 (s, 3H, OCH3), 2.87 (t, J ¼ 5.1 Hz,1H, CH), 2.55-2.42 (m, 6H), 2.19
(s, 1H, CH), 1.32 (s, 3H, CH3), 1.17 (d, J ¼ 9.2 Hz, 1H, CH), 0.81 (s, 3H,
tosylhydrazinyl)acrylate (11Z). Yellow solid. Mp 90e92 ꢁC.1H NMR
(300 MHz, CDCl3):
d
8.36 (d, J ¼ 10.1, 1H, C4eH), 7.66 (d, J ¼ 8.4, 2H,
C5eH), 7.30 (d, J ¼ 8.1, 2H, C6eH), 6.61 (d, J ¼ 9.5, 1H, C3eH), 6.00
(d, J1 ¼10.5, 1H, CH), 5.41 (d, J1 ¼ 9.5, 1H, C2eH), 3.63 (s, 3H, OCH3),
2.41 (s, 3H, Ts-CH3), 1.96 (s, 3H, CH3), 1.93 (s, 3H, CH3). 13C NMR
CH3). 13C NMR (75 MHz, CDCl3):
d 170.2 (CH), 167.4 (C), 165.4 (C),
145.4 (C), 141.9 (CH), 141.9 (CH), 133.7 (C), 129.8 (CH), 128.0 (CH),
101.2 (CH), 52.6 (CH3), 40.5 (CH), 39.7 (CH), 37.7 (C), 33.1 (CH2), 30.9
(75 MHz, CDCl3):
d 165.6 (C), 162.9 (CH), 152.1 (C), 144.7 (C), 134.3
(CH), 134.1 (C), 129.8 (CH), 128.1 (CH), 121.5 (CH), 109.3 (CH), 51.3
(CH2), 25.8 (CH3), 21.7 (CH3), 21.0 (CH3). IR (KBr) y: 1732, 1614, 1433,
(CH3), 27.1 (CH3), 21.7 (CH3), 19.5 (CH3). IR (KBr)
y: 1712, 1620, 1435,
1371, 1257, 1170,1143, 1001, 734 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ)
1369, 1168, 1141, 1087, 839, 705 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ)
C21H27N2O4S 403.1686, found 403.1686.
C
16H21N2O4 337.1217, found 337.1217.
4.2.1.26. Methyl (Z)-3-(2-((1E,2E,4E)-hexa-2,4-dien-1-ylidene)-1-
4.2.1.21. Methyl (E)-3-(2-((E)-3-methylbut-2-en-1-ylidene)-1-
tosylhydrazinyl) acrylate (14Z). Colourless oil. 1H NMR (300 MHz,
tosylhydrazinyl) acrylate (11E). White solid. Mp: 123.4e124.3 ꢁC.
CDCl3):
d
7.99 (d, J ¼ 9.6 Hz, 1H, C4eH), 7.67 (d, J ¼ 8.2 Hz, 2H,
1H NMR (300 MHz, CDCl3):
d
8.55 (d, J ¼ 10.4 Hz,1H, C4eH), 8.05 (d,
C5eH), 7.29 (d, J ¼ 8.2 Hz, 2H, C6eH), 6.69 (dd, J1 ¼ 15.5 Hz,
J2 ¼ 10.3 Hz, 1H, CH), 6.53 (d, J ¼ 9.4 Hz, 1H, C3eH), 6.21 (dd,
J1 ¼ 15.5 Hz, J2 ¼ 10.3 Hz, 2H, CH), 6.03 (dd, J1 ¼ 15.0 Hz, J2 ¼ 6.6H,
1H, CH), 5.49 (d, J ¼ 9.4 Hz, 1H, C2eH), 3.64 (s, 3H, OCH3), 2.41 (s,
3H, Ts-CH3), 1.85 (d, J ¼ 6.9 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
J ¼ 13.6 Hz, 1H, C3eH), 7.64(d, J ¼ 8.3 Hz, 2H, C5eH), 7.31 (d, J ¼
8.1 Hz, 2H, C6eH), 6.15 (d, J ¼ 10.2 Hz, 1H, CH), 5.21 (d, J ¼ 13.6 Hz,
1H, C2eH), 3.68 (s, 3H, OCH3), 2.41 (s, 3H, Ts-CH3), 2.00(s, 3H, CH3),
1.98 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 169.7 (CH), 167.4 (C),
155.7 (C),145.1 (C),141.8 (CH), 133.7 (C), 129.9 (CH),127.9 (CH),121.1
(CH), 101.1 (CH), 51.3 (CH3), 27.1 (CH3), 21.7 (CH3), 19.5 (CH3). IR
d 165.4 (C), 163.5 (CH), 144.8 (CH), 144.7 (CH), 137.4 (CH), 134.1 (CH),
133.9 (C), 130.8 (C), 129.7 (CH), 128.2 (CH), 124.9 (CH), 111.1 (CH),
(KBr)
y
: 1712, 1620, 1435, 1369, 1168, 1141, 1087, 839, 705 cmꢂ1. ESI-
51.6 (CH3), 21.6 (CH3), 18.6 (CH3). IR (film) y: 1714, 1614, 1435, 1371,
HRMS Calcd for (M þ Hþ) C16H21N2O4 337.1217, found 337.1217.
1170, 1145, 1087, 813, 705 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ)
C17H21N2O4S 349.1217, found 349.1217.
4.2.1.22. Methyl (Z)-3-(2-((E)-cyclohex-1-en-1-ylmethylene)-1-
tosylhydrazinyl)acrylate (12Z). Colourless oil. 1H NMR (300 MHz,
4.2.1.27. Methyl (E)-3-(2-((1E,2E,4E)-hexa-2,4-dien-1-ylidene)-1-
CDCl3):
d
7.88 (s, 1H, C4eH), 7.67 (d, J ¼ 8.4 Hz, 2H, C5eH), 7.29 (d,
tosylhydrazinyl) acrylate (14E). Colourless oil. 1H NMR (300 MHz,
J ¼ 8.0 Hz, 2H, C6eH), 6.49 (d, J ¼ 9.2 Hz,1H, C3eH), 6.33 (s, 1H, CH),
5.47 (d, J ¼ 9.2 Hz, 1H, C2eH), 3.64 (s, 3H, OCH3), 2.42 (s, 3H, Ts-
CH3), 2.24 (m, 2H, CH2), 2.16 (m, 2H, CH2), 1.64 (m, 4H, CH2). 13C
CDCl3):
d
8.27 (d, J ¼ 9.9 Hz, 1H, C4eH), 8.06 (d, J ¼ 13.8 Hz, 1H,
C3eH), 7.63 (d, J ¼ 8.3 Hz, 2H, C5eH), 7.30 (d, J ¼ 8.3 Hz, 2H, C6eH),
6.83 (dd, J1 ¼ 15.2 Hz, J2 ¼ 10.5 Hz, 1H, CH), 6.34- 6.16 (m, 3H, CH),
5.25 (d, J ¼ 13.7 Hz, 1H, C2eH), 3.69 (s, 3H, OCH3), 2.41 (s, 3H, Ts-
NMR (75 MHz, CDCl3): d 165.6 (C), 164.9 (CH), 144.7 (C), 142.5 (CH),
135.3 (C), 134.1 (C), 134.0 (CH), 129.5 (CH), 128.3 (CH), 110.8 (CH),
CH3), 1.89 (d, J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 172.2
51.5 (CH3), 26.4 (CH2), 23.0 (CH2), 22.1 (CH2 x2), 21.6 (CH3). IR (film)
(CH), 167.4 (C), 147.9 (CH), 145.2 (C), 141.7 (CH), 139.5 (CH), 133.7 (C),
130.6 (CH), 130.0 (CH), 129.8 (CH), 124.2 (CH), 101.3 (CH), 51.4 (CH3),
y
: 1714; 1622; 1435; 1371; 1286; 1170; 1087; 813; 669 cmꢂ1. ESI-
HRMS Calcd for (M þ Hþ) C18H23N2O4S 363.1373, found 363.1373.
21.7 (CH3), 18.8 (CH3). IR (film) y: 1714, 1614, 1435, 1371, 1170, 1145,
1087, 813, 705 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C17H21N2O4S
4.2.1.23. Methyl (E)-3-(2-((E)-cyclohex-1-en-1-ylmethylene)-1-
349.1217, found 349.1217.
tosylhydrazinyl)acrylate (12E). Colourless oil. 1H NMR (300 MHz,
CDCl3):
d
8.15 (s, 1H, C4eH), 8.04 (d, J ¼ 13.4 Hz, 1H, C3eH), 7.63 (d,
4.2.1.28. Methyl (Z)-3-(2-((1E,2E)-3-(4-methoxyphenyl)allylidene)-
J ¼ 8.4 Hz, 2H, C5eH), 7.30 (d, J ¼ 8.2 Hz, 2H, C6eH), 6.47 (s,1H, CH),
5.20 (d, J ¼ 13.6 Hz, 1H, C2eH), 3.68 (s, 3H, OCH3), 2.41 (s, 3H, Ts-
CH3), 2.30-2.25 (m, 4H, CH2), 1.67 (m, 4H, CH2). 13C NMR (75 MHz,
1-tosylhydrazinyl) acrylate (15Z). White solid. Mp: 137.6e138.6 ꢁC.
1H NMR (300 MHz, CDCl3):
d
8.14 (d, J ¼ 9.4 Hz, 1H, C4eH), 7.70 (d,
J ¼ 8.3 Hz, 2H, Harom), 7.43 (d, J ¼ 8.8 Hz, 2H, C5eH), 7.31 (d, J ¼
8.3 Hz, 2H, C6eH), 7.04 (d, J ¼ 15.9 Hz, 1H, CH), 6.90 (d, J ¼ 8.8 Hz,
2H, Harom), 6.77 (dd, J1 ¼ 16.1 Hz, J2 ¼ 9.4 Hz, 1H, CH), 6.57 (d, J ¼
9.2 Hz, 1H, C3eH), 5.52 (d, J ¼ 9.2 Hz, 1H, C2eH), 3.84 (s, 3H, OCH3),
3.65 (s, 3H, OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
CDCl3):
d 173.5 (CH), 157.4 (C), 146.1 (CH), 141.9 (CH), 135.2 (C), 134.6
(C), 133.7 (C), 129.8 (CH), 127.9 (CH), 101.1 (CH), 51.3 (CH3), 26.6
(CH2), 23.0 (CH2), 21.7 (CH2 x2), 21.5 (CH3). IR (film) y: 1714; 1622;
1435; 1371; 1286; 1170; 1087; 813; 669 cmꢂ1. ESI-HRMS Calcd for
(M þ Hþ) C18H23N2O4S 363.1373, found 363.1373.
d
165.4 (C), 163.8 (CH), 161.0 (C), 144.8 (CH), 144.1 (C), 134.1 (CH),
134.0 (C), 129.7 (CH), 129.2 (CH), 128.2 (CH), 128.1 (C), 121.6 (CH),
114.4 (CH), 111.0 (CH), 55.4 (CH3), 51.6 (CH3), 21.6 (CH3). IR (KBr) y:
4.2.1.24. Methyl (Z)-3-(2-((E)-(6,6-dimethylbicyclo[3.1.1]hept-2-en-
3-yl)methylene)-1-tosylhydrazinyl)acrylate (13Z). Colourless oil. 1H
1714, 1602, 1579, 1510, 1438, 1359, 1305, 1257, 1170, 1087, 1028, 819,
738, 661, 597 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C21H23N2O5S
415.1322, found 415.1322.
NMR (300 MHz, CDCl3):
d
7.99 (s, 1H, C4eH), 7.60 (d, J ¼ 8.3 Hz, 2H,
C5eH), 7.26 (d, J ¼ 8.2 Hz, 2H, C6eH), 6.60 (d, J ¼ 9.7 Hz, 1H, C3eH),
6.23 (s, 1H, CH), 5.43 (d, J ¼ 9.3 Hz, 1H, C2eH), 3.61 (s, 3H, OCH3),
2.78 (t, J ¼ 5.5 Hz,1H, CH), 2.49-2.41 (m, 6H), 2.15 (s, 1H, CH), 1.29 (s,
3H, CH3), 1.12 (d, J ¼ 9.2 Hz, 1H, CH2), 0.76 (s, 3H, Ts-CH3). 13C NMR
4.2.1.29. Methyl (Z)-3-(2-((1E,2E)-3-(4-methoxyphenyl)allylidene)-
1-tosylhydrazinyl) acrylate (15E). Yellow oil. NMR (300 MHz,
(75 MHz, CDCl3):
d
165.5 (C), 162.9 (CH), 145.3 (C), 144.7 (C), 138.3
CDCl3):
d
8.41 (d, J ¼ 9.7 Hz, 1H, C4eH), 8.10 (d, J ¼ 13.6 Hz, 1H,
(CH), 134.3 (CH), 134.0 (C), 129.5 (CH), 128.3 (CH), 109.8 (CH), 51.3
(CH3), 40.6 (CH), 39.7 (CH), 37.6 (C), 32.8 (CH2), 30.9 (CH2), 26.0
C3eH), 7.66 (d, J ¼ 8.4 Hz, 2H, C5eH), 7.48 (d, J ¼ 8.7 Hz, 2H, Harom),
7.31 (d, J ¼ 8.2 Hz, 2H, C6eH), 7.18 (d, J ¼ 15.9 Hz, 1H, CH), 6.93 (d,

D.G. Ghiano et al. / European Journal of Medicinal Chemistry 208 (2020) 112699
13
J ¼ 8.80 Hz, 2H, Harom), 6.88 (dd, J1 ¼ 16.3 Hz, J2 ¼ 9.7 Hz, 1H, CH),
5.29 (d, J ¼ 13.6 Hz, 1H, C2eH), 3.86 (s, 3H, OCH3), 3.70 (s, 3H,
OCH3), 2.42 (s, 3H, Ts-CH3). 13C NMR (75 MHz, CDCl3):
172.4 (CH),
167.4 (C), 161.6 (C), 147.3 (CH), 145.2 (C), 141.8 (CH), 138.1 (C), 133.7
(C), 130.0 (CH), 129.6 (CH), 127.9 (CH), 120.7 (CH), 114.5 (CH), 101.3
4.4. Docking studies
d
The receptor structure (PDBID 4TRO) was manually checked,
and protonation states were manually assigned using Maestro [60].
Docking calculations were made with AutoDock4 using a phar-
macophoric bias [61] on an aromatic interaction between Phe 149
and various ligands computed with an in house script using PyMOL
[62] (PDB ids 2B35 2IDZ 2IE0 2IEB 2NTJ 2NV6 2PR2 2X22 2X23
4OHU 4OIM 4OXK 4OXN 4OXY 4OYR 2IDZ 4BQP 2NV6 2X22 2X23
3FNE 3FNF 3FNG 4BQP 4BQR 4COD 4TZK 4U0K 5COQ 5CP8). 100
different runs were used, and the results were clustered while
keeping the lowest energy cluster with at least 20% population.
After picking the lowest energy conformer a minimization with
AMBER 99SB þ parm@Frosst [63] for 1000 cycles using Sander 16
from AmberTools [64]. Visualization was performed with VMD [65].
(CH) 55.4 (CH3), 51.4 (CH3), 21.7 (CH3). IR (KBr) y: 1714, 1602, 1579,
1510, 1438, 1359, 1305, 1257, 1170, 1087, 1028, 819, 738, 661,
597 cmꢂ1. ESI-HRMS Calcd for (M þ Hþ) C21H23N2O5S 415.1322,
found 415.1322.
4.3. Biology
4.3.1. Bacterial strains
M. tuberculosis strain H37Rv was kindly provided by Dr. L. Bar-
ꢀ
rera (Instituto Nacional de Microbiología “C.G. Malbran”,
Argentina). M. tuberculosis strain H37Rv InhA-OE was generated in
the laboratory of one of us (HRM) [42]. All the mycobacterial strains
were routinely grown in Middlebrook 7H9 broth (Difco Labora-
tories, Detroit, MI, USA) supplemented with 1/10 v/v of ADS (50 g/L
BSA fraction V, 20 g/L dextrose and 8.1 g/L NaCl), glycerol (1% w/v).
Tween 80 was added to prevent clumping (0.05% w/v). This me-
dium was designated as 7H9-ADS-Gly for short. When needed,
solid media Middlebrook 7H11 supplemented with ADS (1/10 v/v)
and glycerol (1% v/v) was used. All strains were grown at 37 ꢁC
under gentle agitation.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
This work was supported in part by grants from National
Research Council of Argentina, CONICET (2012-14/0448); Agencia
ꢀ
ꢀ
Nacional de Promocion Científica y Tecnologica, ANPCyT- Argentina
(PICT 2011/0589 to GRL and PICT 2005/38198 to HRM), Universidad
4.3.2. In vitro compound activity against M. tuberculosis (Mtb)
strains
Stock solutions for all the tested compounds were made in
DMSO at 40 mM. Working solutions were made by dilution in the
above described 7H9 - ADS-G medium at a final concentration of
ꢀ
Nacional de Rosario (BIO503) and Fundacion Josefina Prats. AT, AB,
GRL and LAD are members of the scientific staff of CONICET-
Argentina. HRM is a career member of CIUNR-Argentina. DGG
and ARB thanks CONICET for the award of a fellowship.
400
mМ. Antimycobacterial activity was determined by two-fold
Appendix A. Supplementary data
dilution of the compounds in Middlebrook 7H9-ADS-G medium
as described previously [58]. For this purpose, 96-well plates (Fal-
con, Cat number 3072, Becton Dickinson, Lincoln Park, NJ) were
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112699.
used. The 96 well-plates received 100 mL of Middlebrook 7H9 broth
and a serial two-fold dilution of the compounds was made directly
on the plate. The initial and final drug concentrations tested were
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