New hydrazides derivatives of isoniazid against Mycobacterium tuberculosis: Higher potency and lower hepatocytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2018.01.071

Tuberculosis (TB) is one of the leading causes of death worldwide. The emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) and TB-HIV co-infection are major public health challenges. The anti-TB drugs of first choice were developed more than 4 decades ago and present several adverse effects, making the treatment of TB even more complicated and the development of new chemotherapeutics for this disease imperative. In this work, we synthesized two series of new acylhydrazides and evaluated their activity against different strains of Mtb. Derivatives of isoniazid (INH) showed important anti-Mtb activity, some being more potent than all anti-TB drugs of first choice. Moreover, three compounds proved to be more potent than INH against resistant Mtb. The Ames test showed favorable results for two of these substances compared to INH, one of which presented expressly lower toxicity to HepG2 cells than that of INH. This result shows that this compound has the potential to overcome one of the main adverse effects of this drug.

Full text of DOI:10.1016/j.ejmech.2018.01.071

Accepted Manuscript
New hydrazides derivatives of isoniazid against Mycobacterium tuberculosis: Higher
potency and lower hepatocytotoxicity
Frederico Silva Castelo-Branco, Evanoel Crizanto de Lima, Jorge Luiz de Oliveira
Domingos, Angelo C. Pinto, Maria Cristina S. Lourenço, Karen Machado Gomes,
Mariana Marques Costa-Lima, Carlos Fernando Araujo-Lima, Claudia Alessandra
Fortes Aiub, Israel Felzenszwalb, Thadeu Estevam M.M. Costa, Carmen Penido,
Maria G. Henriques, Nubia Boechat
PII:
S0223-5234(18)30084-9
10.1016/j.ejmech.2018.01.071
EJMECH 10148
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 October 2017
Revised Date: 9 January 2018
Accepted Date: 21 January 2018
Please cite this article as: F.S. Castelo-Branco, E.C. de Lima, J.L. de Oliveira Domingos, A.C. Pinto,
M.C.S. Lourenço, K.M. Gomes, M.M. Costa-Lima, C.F. Araujo-Lima, C.A.F. Aiub, I. Felzenszwalb,
T.E.M.M. Costa, C. Penido, M.G. Henriques, N. Boechat, New hydrazides derivatives of isoniazid
against Mycobacterium tuberculosis: Higher potency and lower hepatocytotoxicity, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.071.
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ACCEPTED MANUSCRIPT
New Hydrazides Derivatives of Isoniazid Against Mycobacterium
tuberculosis: Higher Potency and Lower Hepatocytotoxicity
a,b
c
Frederico Silva Castelo-Branco , Evanoel Crizanto de Lima , Jorge Luiz de Oliveira
b
e
d
e
Domingos , Angelo C. Pinto , Maria Cristina S. Lourenço , Karen Machado Gomes ,
f
f, ,h
Mariana Marques Costa-Lima , Carlos Fernando Araujo-Lima g , Claudia Alessandra
f
g
i,j
i,j
Fortes Aiub , Israel Felzenszwalb , Thadeu Estevam M. M. Costa , Carmen Penido , Maria
i,j
a*
G. Henriques , and Nubia Boechat
a
Fundacao Oswaldo Cruz, Instituto de Tecnologia em Fármacos, Farmanguinhos-Fiocruz,
Departamento de Sintese Farmacos, Manguinhos, 21041-250 Rio de Janeiro, RJ, Brazil.
b
Programa de Pos-Graduacao em Quimica da Universidade Federal do Rio de Janeiro,
Instituto de Química, Departamento de Quimica Organica, 21941-909 Rio de Janeiro, RJ,
Brazil.
c
Universidade Federal do Rio de Janeiro, Instituto de Quimica, Campus Macae, 27930-560
Macae, RJ, Brazil.
d
Universidade do Estado do Rio de Janeiro, Instituto de Quimica, Departamento de
Quimica Organica, 20550-900, Rio de Janeiro, RJ, Brazil.
e
Fundacao Oswaldo Cruz, Instituto Nacional de Infectologia Evandro Chagas, Laboratório
de Bacteriologia e Bioensaios em Micobacterias, 21045-900, Rio de Janeiro, RJ, Brazil.
f
Laboratorio de Genotoxicidade, Departamento de Genetica e Biologia Molecular,
Universidade Federal do Estado do Rio de Janeiro, 20211-010, Rio de Janeiro, RJ, Brazil.

ACCEPTED MANUSCRIPT
g
Laboratorio de Mutagenese Ambiental, Departamento de Biofisica e Biometria,
Universidade do Estado do Rio de Janeiro, 20551-030, Rio de Janeiro, RJ, Brazil.
h
Programa de Pos-graduacao em Biociencias da Universidade do Estado do Rio de Janeiro,
Instituto de Biologia, 20551-030, Rio de Janeiro, RJ, Brazil.
i
Fundacao Oswaldo Cruz, Instituto de Tecnologia em Farmacos, Farmanguinhos-Fiocruz,
Laboratorio de Farmacologia Aplicada, Manguinhos, 21041-250, Rio de Janeiro, RJ,
Brazil.
j
Fundacao Oswaldo Cruz, Centro de Desenvolvimento Tecnologico em Saúde,
CDTS/INCT-IDN-Fiocruz 21040-361, Rio de Janeiro, RJ, Brazil
ABSTRACT: Tuberculosis (TB) is one of the leading causes of death worldwide. The
emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) and TB-
HIV co-infection are major public health challenges. The anti-TB drugs of first choice were
developed more than 4 decades ago and present several adverse effects, making the
treatment of TB even more complicated and the development of new chemotherapeutics for
this disease imperative. In this work, we synthesized two series of new acylhydrazides and
evaluated their activity against different strains of Mtb. Derivatives of isoniazid (INH)
showed important anti-Mtb activity, some being more potent than all anti-TB drugs of first
choice. Moreover, three compounds proved to be more potent than INH against resistant
Mtb. The Ames test showed favorable results for two of these substances compared to INH,
one of which presented expressly lower toxicity to HepG2 cells than that of INH. This

ACCEPTED MANUSCRIPT
result shows that this compound has the potential to overcome one of the main adverse
effects of this drug.
Keywords: tuberculosis; hydrazide, isoniazid; activity; hepatotoxicity; cytotoxicity.
1
. Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a condition
that afflicts all continents on Earth.[1] Its incidence and prevalence have higher levels in
Asia, Africa and South America, where it especially affects underdeveloped or developing
1
countries. It is estimated that one third of the world's population has a latent infection of
Mtb, of which approximately 10% will develop TB.[1] In 2015, approximately 10 million
new cases of TB were reported, killing 1.8 million individuals.[1] Since 1993, the World
Health Organization (WHO) declared TB a global emergency due to the increasing
multidrug resistance of Mtb strains to the first-line drugs and due to the worrying situation
of co-infection with Mtb and human immunodeficiency virus (HIV), leading to the
comorbidity of TB-AIDS, which is the leading cause of death in AIDS patients. [1]
The first-line drugs currently available for TB chemotherapy, namely, rifampicin
(RIF), isoniazid (INH), ethambutol (EMB) and pyrazinamide (PZA), were developed more
than 40 years ago.[2,3] Such drugs have a number of side effects, and the limitations of
their application and the emergence of drug resistance have led to an urgent need for new
TB drug development. Recently, we updated the portfolio of new drug candidates for TB
chemotherapy along with the relationship between these substances’ structure and activity
and the synthetic methodologies for producing them.[4]

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INH, an inhibitor of mycolic acid synthesis of Mtb, is widely used in anti-TB
chemotherapy due to its high activity and low cost.[5] However, the process of INH N-
acetylation promoted by the enzyme N-acetyltransferase (NAT) generates the metabolite N-
acetylisoniazid, which is devoid of antimycobacterial activity.[6] The NAT enzyme is
present in both humans and mycobacteria. In humans classified as "fast acetylators", a large
amount of INH is inactivated, limiting its application to these groups.[7] Additionally, in
Mtb, this enzyme is linked to a mechanism of resistance to INH.[8] For this reason, the
addition of protecting groups (PG) to NH is used to avoid N-acetylation of INH.[9,10] As
2
an example of this strategy, our group has shown that hydrazones derived from isoniazid
and 1,2,3-triazoles present important activity against the etiological agent of TB at low
micromolar scale.[11] Hydrazone and hydrazides have been widely exploited in medicinal
chemistry.[12-16] More specifically, several studies have shown that hydrazide derivatives
present important activity against M. tuberculosis, such moiety being a key group for the
antimycobacterial activity.[17-20] In fact, isoniazid itself is a hydrazide.
In an unpublished screening of a library of compounds from our group, we have
identified that the gem-difluorinated derivative of thiosemicarbazide 1 showed low anti-Mtb
activity with a minimum inhibitory concentration (MIC) of 297 µM (Figure 1). This
compound is structurally related to thioacetazone, an anti-TB drug. The inclusion of
fluorine in the structure of this compound was based on the fact that this element can lead
to significant changes in physical, chemical and biological properties of the compound, as it
is able to increase the lipophilicity of the molecule and be a binding site for electrostatic
interactions in the target, and it may also significantly alter the reactivity of neighboring
groups through its strong electron-withdrawing inductive effect.[21]

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Based on these facts, this work is aimed at the synthesis and biological evaluation of
two novel series of hydrazide derivatives (Figure 1). Our plan was based on using INH and
the gem-difluorinated thiosemicarbazide (1) as scaffolds (Figure 1). Although 1 has shown
low antimycobacterial activity, it can be used as a starting point for the optimization of its
biological activity by modifying its thiosemicarbazide unit. The hydrazides of series A (2-
8
) were planned by keeping the 2,2-difluoro-2-phenylacetamide scaffold of 1 and
substituting it with different hydrazines in order to verify the importance of such subunits
in the antimycobacterial activity. In series B (9-17), the planned derivatives present the
INH moiety protected by 2,2-difluoro-2-phenylacetamide or 2-oxo-2-phenylacetamide
moieties aiming to observe the influence of these groups on the activity of the INH portion,
as well as to verify the importance of the inclusion of the fluorine atoms in comparison to
an additional carbonyl.

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O
NH
O
S
F
F
H N
2
H
N
N
Cl
H
H N
2
N
N
H
O
HN
INH
N
1
S
O
HN
NH2
MIC = 297 M
Thiacetazone
Series A
Series B
F
F
H
O
F
F
N
H
N
NH2
R
N
H
O
NH
O
N
NH
2
O
O
9, R=H;
1
1
1
0, R=Me;
1, R=F;
2, R=Cl;
F
F
F
F
H
N
N
13, R=OMe
O
NH
3
O
O
O
H
N
R
N
H
NH
NH2
F
O
N
H
N
NH
N
H
14, R=H
5, R=Me
O
O
1
NH
4
O
O
O
F
H
N
H
N
R
N
N
H
X
H
O
N
O
NH
NH
5
, R=H/X=CH
, R=Me/X=CH
, R=H/X=N
6
7
8
R
O
16, R=CH2Cl
17, R=Ph
O
, R=Me/X=N
Figure 1. Design of the novel hydrazides of series A and B. Series A was planned to
contain fragments of thiacetazone and gem-difluorinated thiosemicarbazide (1) in red and

ACCEPTED MANUSCRIPT
green. Series B contains fragments of isoniazid (INH) in blue and thiacetazone and
compound 1 in red.
2
. Results and Discussion
2
.1. Chemistry
2
2
The final derivatives 2,4-13 were prepared in a 3-step route starting (step a) with
the acetylation of isatins (18a-e) with acetic anhydride under reflux for 4 hours providing 1-
acetylisatins (19a-e) in good yields (Scheme 1). The acetylated isatins were characterized
by melting point and mass spectrometry, and the data are consistent with the literature.[23]
The 1-acetylisatins were deoxofluorinated (step b) using sulfur diethylaminosulfur
trifluoride (DAST) in anhydrous dichloromethane under an argon atmosphere for 16 hours
at room temperature, furnishing 1-acetyl-3,3-difluoroindolin-2-ones (20a-e), which were
purified by sublimation and obtained in yields of 50-88%. The mass spectrometry of
compounds 20a-e showed compatible molecular ions and fragmentations for these
1
13
structures. The intermediates 20d,e are novel and were fully characterized by IR, H, C
1
9
and F NMR. The use of DAST promoted, as expected, the selective fluorination of the
ketone carbonyl due to its higher electrophilicity. The difluorohydrazides of interest (4-13)
were obtained through reactions (step c) between 1-acetyl-3,3-difluoroindolin-2-ones (20a-
e) and either aminoguanidine to yield 4, the corresponding hydrazines to yield 5-8, or
isoniazid (INH) to yield 9-13 (step c). Reactions were performed in acetonitrile (ACN)
under reflux for 12 hours, and total conversion of substrate was observed, giving yields in
the range of 50-80% (Scheme 1). The preparation of compound 2 was carried out in ACN
at 0 °C for 40 minutes to give 45% yield (step d). The low temperature applied specifically

ACCEPTED MANUSCRIPT
in this reaction aimed to avoid the formation of the corresponding acid from heterocyclic
ring opening by water, since the hydrazine hydrate was used. Hydrazone 3 was obtained
from 2 by reacting it with cyclohexanone in CH
2 2
Cl and anhydrous sodium sulfate (step d).
The presence of the difluoromethylene group in the compounds’ structures could be easily
1
9
observed in F NMR by the singlet between -100 and -106 ppm. Compounds (2-13) are all
1
13
novel and were further fully characterized by H, C, mass spectrometry and elemental
analysis, and the data are compatible with their structures.
Scheme 1. Synthesis of the new α,α-difluorohydrazides 2-13. (a) Acetic anhydride, reflux,
4
h, 72-89%; (b) DAST, CH Cl , 16 h, r.t., 50-88%; (c) hydrazides, INH or
2 2

ACCEPTED MANUSCRIPT
o
aminoguanidine, ACN, reflux, 12 h, 50-80%; (d) hydrazine, ACN, 0 C, 40 min., 48% (d)
cyclohexanone, CH Cl , Na SO , r.t., 18 h, 80%.
2
2
2
4
The synthesis of compounds 14 and 15 (Scheme 2) started with optimization of the
isatin acetylation step, in which catalytic amounts of sulfuric acid were used. With this
method (step a’), we describe for the first time a significant reduction in the reaction time
from 4 hours to only 5 minutes, with high yield (95%). The next step b’ consisted in the
opening of 1-acetylisatins with INH, providing the products of interest at 56-68% yield.
The other final compounds 16 and 17 were obtained by the reaction of isatin with
chloroacetyl (step c’) and benzoyl chlorides (step d), respectively, followed by the opening
reaction with INH (step b’). The ring opening reaction's selectivity was ensured by the
addition of water as a co-solvent so that the equilibrium of hydrazone formation at the keto-
carbonyl remains unfavorable. This route proved to be efficient, inexpensive and easily
applied on a future industrial scale.
Scheme 2. Synthesis of hydrazides 14-17. (a’) acetic anhydride, H SO , reflux, 5 min,
2
4
9
5%; (b’) INH, ACN, H O, r.t. 16 h, 56-68%. (c’) chloroacetyl chloride, reflux, 16 h, 94%;
2
3
(d) benzoyl chloride, Et N, ACN, reflux, 16 h, 94%.

ACCEPTED MANUSCRIPT
To be used as a negative control in the antimycobacterial evaluation, a natural
inactive metabolite of INH, N-acetylisoniazid (Ac-INH), was synthesized by reacting INH
2
4
with acetic anhydride as previously described.
2
.2. Antimycobacterial evaluation
Substances from series A and B had their antimycobacterial activity evaluated by
Microplate Alamar Blue Assay (MABA)[25] against the Mycobacterium tuberculosis strain
H37Rv ATCC 27294 (American Type Culture Collection, Rockville, Md), which is
susceptible to all first-line anti-TB drugs. Isoniazid (INH), rifampicin (RIF), ethambutol
(EMB) and pyrazinamide (PZA) were used as positive controls, with MICs of 0.88 µM,
1
.22 µM, 15.90 µM and 813.00 µM, respectively (Table 1). The natural metabolite of INH
N-acetylisoniazid (Ac-INH) showed no inhibitory activity at concentrations up to 558.66
µM, confirming its inactivity towards Mtb.
The inactivity of all the derivatives from series A (2-8) highlights the importance of
the INH moiety for the antimycobacterial activity, as all INH derivatives found in series B
proved to be important inhibitors of M. tuberculosis. Considering the difluorinated
compounds of series B, 9 and 10 were the most potent, with MIC of 3.59 µM and 6.91 µM,
respectively, and although they are less active than INH, they are significantly more potent
than EBM and PZA (Table 1). Compounds 11 and 12 showed MIC of 16.39 µM and 17.08
µM, respectively, and were more active than 13, which showed MIC of 33.07 µM (Table
1
). It is important to notice that substituents in the phenyl ring at position 4 have negative

ACCEPTED MANUSCRIPT
influences on the activity, and these influences are not related to its electronic nature
because derivatives with electron donors and electron withdrawing groups were all less
potent than the one not substituted, 9.
Considering the results for compounds 9-13, derivatives 14-17 were proposed in
order to evaluate the importance of the difluoromethylene group in activity by replacing it
with a carbonyl group. Moreover, the conjugation of a carbonyl with the phenyl ring could
change the conformation of these molecules, in which relative planarity could be achieved.
Compounds 14 and 15 have, respectively, H and Me groups at position 4, aiming to
correlate them with the difluorinated compounds 9 and 10 that presented higher activity.
Two other compounds, 16 and 17, were also proposed in order to observe the influence on
activity of an electron withdrawing group (CH Cl) and a bulky electron donor (phenyl) in
2
the amide region. Compounds 14-16 showed remarkable anti-Mtb activity, being
approximately twice as potent as INH and RIF and 30-fold and 1,800-fold more potent than
EMB and PZA, respectively. Using substances 9 and 14 as examples, it is clear that
substitution of the difluoromethylene with a carbonyl is beneficial to the anti-Mtb activity,
in this case with an 8-fold increase in potency. Substance 17 was significantly less potent
than the others, and this result may be related to a negative influence of the bulky group
attached to the side amide moiety.
As the cell wall of Mtb is a highly lipophilic structure, compounds with higher LogP
are supposed to cross it more easily.[18] For such reason, the LogP of all compounds were
calculated in order to see if there was any correlation with the antimycobacterial activity
(Table 1). Although compounds 9-17 are all more lipophilic than INH and this may
facilitate their diffusion through the Mtb, it was not possible to notice a better activity of the

ACCEPTED MANUSCRIPT
more lipophilic substances of this series compared to those of lower CLogP. This suggests
that the antimycobacterial activity of these substances is more related to structural aspects
than to this physicochemical factor and that even the less lipophilic substances in this series
are able to diffuse through the cell wall of this mycobacteria. The lack of correlation
between higher lipophilicity and higher antimycobacterial activity has been also reported
elsewhere.[24,26-28] On the other hand, in addition to being more potent in vitro than INH,
the substances 14-16 may also have improved pharmacokinetic profiles due to its increased
lipophilicity, which can increase its volume of distribution and its half-life, which is very
short for INH, especially in fast acetylators pacients.[29]
Table 1. Antimycobacterial evaluation of the new N-acylhydrazides from series A and B
against Mtb H37Rv.
MIC
MIC
(µM)
a
a
Entry
2
Structure
CLogP Entry
Structure
CLogP
(µM)
>411.52 1.44
>309.60 3.25
>350.88 0.85
>313.48 2.68
13
14
15
16
33.07 1.49
3
4
5
0.47
0.45
0.43
0.70
0.84
0.63
6
>300.30 2.95
17
8.04
1.58

ACCEPTED MANUSCRIPT
7
8
9
>312.40 1.71
>299.40 2.22
Ac-INH
>558.66 -0.58
0.88 -0,71
INH
RIF
3.59
6.91
1.41
1.54
1.22
3.85
1
0
1
2
EMB
PZA
15.90 -0.12
813.00 -0.71
1
1
16.39 2.14
17.08 1.69
a
CLogP calculated by ALOGPS 2.1.[30]
The difluorinated derivatives of INH 9-12 were also screened against resistant
strains of Mtb (Table 2). In the case of the wild-type clinical isolate INH-resistant strain
WTINHR-Mtb, KatG mutant), compounds 9-12 were all equipotent and showed moderate
(
activity with MICs ranging from 130.9 µM to 143.7 µM. The weaker activity of such
compounds against this strain compared with the INH-susceptible strain shows that the
mode of action of these substances may be the same as that of INH. The 2-oxo-2-
phenyacetamide derivative of INH 14 was also elected for evaluation against this strain and
showed MIC of 19.2 µM, which is far lower than those of the difluorinated derivatives 9-
1
2. Moreover, it is important to notice that substance 14 proved to be at least 4-fold more
potent than INH against this strain, which shows that it is somehow able to partially bypass
the mechanism of INH resistance of this mycobacteria.

ACCEPTED MANUSCRIPT
Considering the Mtb strain SR 2571/0215, which also shows resistance to INH due
to KatG mutation, it is possible to observe that the gem-difluorinated substances 9 and 10
were weakly active. However, it is noteworthy that derivatives 14-16 had better activity
than INH, specially in the cases of 14 and 15, which proved to be 3-fold more potent than
that this reference drug.
Similar results were found when these substances were evaluated against Mtb strain
T113/09, which exhibits resistance to INH (KatG mutation) and RIF (rpoB mutation), in
which it is possible note again that derivatives 14-16 hava a higher potency than that of
INH.
Considering the improved activities of the acylhydrazides 14-16 in comparison with
INH against both susceptible and resistant strains, it is possible to infer that their activities
not only depend on the INH moiety, otherwise, there would be no improvement in
antimycobacterial activity. Another point that should be emphasized is that the activity of
these substances may not be associated with its hydrolysis followed by INH release, since
gem-difluorinated derivatives 9-13 must be more susceptible to this type of reaction, as the
carbonyl that links the INH moiety is more electrophilic due to the higher electron
withdrawing effect of the α-carbonyl fluorine atoms. In fact, unlike hydrazones, hydrazides
have good stability in aqueous media [31], being some acylhydrazide derivatives of INH
not susceptible to hydrolysis, even in gastric juice, as shown by Giannola et al.[32]
Nevertheless, the inactive acetylated INH metabolite, Ac-INH, is itself an acylhydrazide
and its formation is a therapeutic issue due to the stability of the bond between the acyl
group and the INH moiety, since if it were easily hydrolyzed, the inactivation process
would not be relevant.

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Table 2. Antimycobacterial evaluation of derivatives of INH (9-12, 14-16) against resistant
Mtb strains.
a
b
c
Compd.
9
WTINHR-Mtb
143.7
138.1
136.6
130.9
19.2
INHR-Mtb
287.4
>276.2
NT
INH/RIFR-Mtb
287.4
276.2
NT
1
1
1
1
1
1
0
1
2
4
5
6
NT
NT
38.3
38.3
36.7
34.7
58.4
NT
36.7
NT
69.4
INH
>80
116.8
a. INH-resistant Mtb (wild-type clinical isolate, KatG mutation); b. INH-resistant Mtb (strain SR 2571/0215,
KatG mutation); c. INH and RIF-resistant Mtb (strain T113/09, KatG and rpoB mutation). NT. not tested.
2
.3. Cytotoxicity evaluation on macrophages
Compounds 14-17 were evaluated for their cytotoxicity against macrophages of the
J774A.1 lineage (Table 3). Considering that macrophages are the main target cells in Mtb
infections, this evaluation is of particular interest, since the developed substances should be
toxic only to the mycobacteria and not to the host cell. The results are expressed in terms of
the cell viability promoted by the substances at a given concentration. All substances
showed high cell viability at concentrations up to 100 µM. However, at the higher
concentration, derivatives 14 and 15 were superior to the others, presenting a percentage of

ACCEPTED MANUSCRIPT
viable cells above 93%. Indeed, the mean cell viability promoted by these derivatives was
also slightly superior to that of INH. It is important to note the high selectivity of these
substances over Mtb, since they were able to maintain high viability of macrophages even
at concentrations 200 times higher than their MICs for Mtb.
Table 3. Cell viability assay of compounds 14-17.
Cell viability (%±SEM)
Macrophage J774A.1
Entry
1
nM
10nM
98.7±0.6
100.0
100nM
97.7±1.0
99.5±0.5
97.9±1.8
99.8±0.1
97.3±1.3
1µM
10µM
100µM
93.3±0.7
94.0±3.2
63.3±2.1
86.5±1.7
90.3±1.2
14
15
16
17
99.6±0.4
100.0
99.5±0.4
99.2±0.8
96.5±3.1
98.2±1.8
98.7±0.8
97.1±1.3
98.2±1.8
98.8±1.1
98.0±2.0
98.5±1.2
99.0±0.9
100.0
98.5±0.8
99.4±0.4
98.2±0.9
INH
99.5±0.4
2
.4. Hepatotoxic potential on HepG2 cells
One of the main adverse effects of INH is its considerable hepatotoxicity; in severe
cases INH can cause liver failure.[33,34] This serious side-effect is believed to be caused
by different hydrazine and acetylhydrazine INH metabolites or direct oxidation of
2
INH.[35,36] The high reactivity of the INH terminal NH seems to be involved in these
processes. For this reason, modifications to this functional group may change the
hepatotoxicity profile of INH derivatives. For this reason, we decided to investigate the
hepatotoxicity of the new compounds using HepG2 cells, which represent a fast,
inexpensive, and easily executed model to predict this outcome (OECD).[37] This model

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has been largely used through the last decade to detect drug genetic toxicity[38] and predict
its human hepatotoxicity.[39] Drug-induced hepatotoxicity is mainly divided into hepatic
steatosis, cholestasis, or necrosis. In this sense, Van-Summeren et al. (2011)[40] described,
through proteomic analysis, that HepG2 cells are a better model to detect hepatotoxicity by
cholestasis, the type of liver injury induced by the chronic use of INH.[41]
Thus, the hepatocytotoxicity of substances 14-17 was performed on HepG2
hepatocarcinoma cells at times of 3 and 24 hours, and the results express the hepatotoxic
potential of these compounds (Table 4). Although substance 14 showed higher
hepatocytotoxicity (31.5±9.2 µM) in 24 h than INH (105.0±21.2 µM), an important result
was achieved with derivative 15, the LC of which was higher than 200 µM, even after 24
5
0
hours. This concentration is at least twice as high as that presented by INH, and it suggests
that 15 has lower hepatotoxic potential in vivo than the drug that generated it, presenting a
selectivity index (SI) higher than 444, which is at least 4-fold superior to that of INH.
Table 4. Hepatocytotoxicity evaluation of compounds 14-17.
HepG2
a
LC (µM±SD)
5
0
Compound
SI
3
h
24h
31.5±9.2
>200
1
1
1
1
4
5
6
7
>200
>200
>200
>200
>200
67
>444
256
>25
119
110.0±14.1
>200
INH
105.0±21.2
a
selectivity index calculated by dividing HepG2 LC (24h) by MIC against Mtb H37Rv.
50

ACCEPTED MANUSCRIPT
2
.5. Mutagenic evaluation
The mutagenicity and carcinogenicity of hydrazines and, in particular, metabolite-
induced mutagenicity of isoniazid have been reported.[42,43] For this reason, compounds
1
4 and 15 were selected for further investigation of whether they present mutagenic
properties through the Salmonella reverse mutation assay (Ames Test)[44] following the
Organization for Economic Co-operation and Development (OECD) test guideline 471
(Table 5). Considering the TA100 strain, none of the tested substances showed
mutagenicity even with exogenous metabolism from the S9 mix. However, under this
condition, INH and 15 were cytotoxic at concentrations of 500 and 5,000 µM, respectively,
masking an eventual positive mutagenic result. Regarding the TA98 strain, in the non-
metabolized assays, 14 did not present mutagenicity at the concentrations evaluated, while
1
5 was mutagenic only at the highest concentration of 5,000 µM, which is more than 10-
fold greater than the anti-TB MIC of this substance. Under the same conditions, INH
presented mutagenicity at concentrations higher than 50 µM, evidencing the lower
mutagenic potential of the derivatives developed in comparison with the drug they derived
from. In the assay with exogenous metabolism for strain TA98, derivatives 14 and 15
showed potential mutagenicity only at concentrations ≥ 500 µM.

ACCEPTED MANUSCRIPT
+
Table 5. Mean values ±SD (IF) of revertant His colonies of Salmonella enterica serovar
Typhimurium strains used in Salmonella/microsome assay after incubation with the
compounds.
-S9
+S9
Entry
µM
TA98
TA100
TA98
TA100
0
30 ± 3 (1.0)
30 ± 2 (1.0)
31 ± 6 (1.1)
37 ± 2 (1.2)
38 ± 2 (1.3)
40 ± 4 (1.3)
29 ± 4 (1.0)
36 ± 3 (1.2)
45 ± 2 (1.6)
52 ± 5 (1.8)
56 ± 1 (1.9)
99 ± 7 (1.0)
99 ± 3 (1.0)
128 ± 3 (1.3)
129 ± 6 (1.3)
131 ± 9 (1.3)
141 ± 10 (1.4)
131 ± 1 (1.0)
159 ± 12 (1.2)
162 ± 13 (1.2)
173 ± 17 (1.4)
190 ± 2 (1.4)
43 ± 4 (1.0)
50 ± 7 (1.1)
50 ± 1 (1.1)
70 ± 2 (1.6)
85 ± 19 (2.0)
111 ± 9 (2.5)
38 ± 3 (1.0)
38 ± 3 (1.0)
65 ± 5 (1.7)
69 ± 4 (1.8)
129 ± 20 (3.4)
153 ± 4 (4.0)
38 ± 3 (1.0)
51 ± 2 (1.3)
51 ± 2 (1.3)
57 ± 5 (1.5)
60 ± 3 (1.6)
Cytotoxic
125 ± 1 (1.0)
0.5
135 ± 13 (1.1)
135 ± 4 (1.1)
152 ± 6 (1.2)
153 ± 1 (1.2)
158 ± 9 (1.3)
119 ± 20 (1.0)
126 ± 1 (1.1)
174 ± 4 (1.5)
Cytotoxic
-
5
1
1
4
5
5
0
5
5
00
,000
0
0.5
5
5
0
5
5
00
,000
100 ± 11 (3.4) 207 ± 9 (1.6)
-
0
29 ± 4 (1.0)
44 ± 5 (1.5)
47 ± 5 (1.6)
59 ± 1 (2.0)
106 ± 8 (3.7)
131 ± 1 (1.0)
144 ± 14 (1.1)
153 ± 13 (1.2)
166 ± 6 (1.3)
207 ± 4 (1.6)
119 ± 20 (1.0)
163 ± 8 (1.4)
166 ± 16 (1.4)
Cytotoxic
-
0.5
5
INH
5
0
5
5
00
,000
116 ± 8 (4.0) 251 ± 15 (1.9)
-
SD: standard deviation; -S9: absence of metabolic activation; +S9: presence of metabolic
activation.Bold denotes IF > 2 and p > 0.05 in one-way ANOVA;Positive controls without
S9: 4NQO (1.0 µg/pl.) for TA98 120±10 revertants; AS (1.0 µg/pl.) for TA100 607±56
revertants; With S9: 2AA (1.0 µg/pl.) for TA98 305±1 revertants; B[a]P (50 µg/pl.) for
TA100 1436±40 revertants.

ACCEPTED MANUSCRIPT
2
.6. Structure-activity relationship (SAR)
Considering the antimycobacterial activity against the strain H37Rv of Mtb, it is
possible to highlight that the presence of the isonicotyl moiety is essential for the activity,
since all INH-derivatives were active, in contrast to the loss of activity of the derivatives
that lacks it. In addition, the change from the difluoromethyl group to a carbonyl group
increases by up to 15-fold the activity, as seen in the comparison of substances 10 (MIC =
6
.91 µM) and 15 (MIC = 0.45 µM). In the amide moiety, the acyl group is able to
significantly influence the activity, once the phenylacetic group present in substance 17
MIC = 8.04 µM) decreases the activity by about 18-fold when compared to the acetyl
(
group (14, MIC = 0.47 µM and 15, MIC = 0.45 µM) and chloroacetyl (16, MIC = 0.43
µM). In regard to hepatocytotoxicity, it is important to note that the inclusion of a methyl in
the benzene ring in substance 15 (LC > 200 µM) leads to HepG2 cells toxicity at least 6-
5
0
fold lower than when not substituted in the same position (14, LC = 31.5 µM). Figure 2
5
0
presents a summary of the SAR of these series of substances.
Figure 2. Structure-activity relationship.

ACCEPTED MANUSCRIPT
3
. Conclusion
In this work, two series with 16 new acylhydrazides (2-17) were developed, and 2
new intermediates (20d-e) were reported. Series B showed important antimycobacterial
activity, evidencing the importance of the isoniazid moiety to their anti-Mtb activity.
Although the INH was essential, the moieties 2,2-difluoro-2-phenylacetamide and 2-oxo-2-
phenylacetamide showed influence on the activity as well. Among the synthesized
compounds, 14-16 were more potent against susceptible strain (H37Rv) than all the drugs
of first choice for the treatment of tuberculosis. Moreover, 14-15 were also more potent
than INH against resistant Mtb. The Ames test showed favorable results for substances 14
and 15 compared to INH. Even at concentrations up to 100 µM, derivatives 14 and 15
proved to be non-toxic to macrophages of the J774A.1 lineage with very high cell viability.
Compound 15 was also significantly less toxic to HepG2 cells than INH, suggesting that
this substance may have lower hepatotoxic potential than this reference drug in vivo. In
addition, such derivative may exhibit even more pronounced in vivo antimycobacterial
activity, as being a protected derivative of INH it may prevent deactivation by metabolic N-
acetylation. In this way, the preclinical development of compound 15 is promising, aiming
at the development of an alternative to INH with less toxic and more potent activity for the
treatment of tuberculosis.
4
. Experimental
4
.1. Chemistry
Reagents were purchased from Aldrich or Merck and used without further
purification. Thin-layer chromatography was performed with silica gel plates (Merck,

ACCEPTED MANUSCRIPT
Kieselgel 60 F ), and the spots were visualized using ultraviolet (UV, 254 nm). Yields are
2
54
products after respective purification. Melting points were obtained on a Buchi melting
point apparatus model B-545 and are uncorrected. Infrared spectra were obtained on a
Shimadzu IR spectrophotometer model Prestige-21 through KBr reflectance. Gas
chromatography with mass spectrometry detection (GC-MS) was performed using the
Agilent® Model 6890 Chromatograph with Agilent® Model 5973 at 70 eV. The values of
the fragmentations and molecular ion are expressed in mass / load (m / z) relation. The
column used was an Agilent® 122-5532 DB-5MS (5% diphenyl: 95%
dimethylpolysiloxane), and analyses were performed using the temperature ramp of 50 °C
to 350 °C. The electron-ionization mass spectrometry (ESI-MS, scan ES+ capillary (3.0
kV)/cone (30 V)/extractor (1 V)/RF lens (1.0 V)/source temperature (150 °C)/desolvation
temperature (300 °C)) was recorded using a Micromass/Waters Spectrometer model: ZQ-
4
000. High Resolution Mass Spectrometry (HRMS) data were obtained using an LC-MS
1
13
19
Bruker Daltonics MicroTOF (time of flight analyzer). H, C and F nuclear magnetic
resonance (NMR) spectra were obtained at 400, 100 and 376 MHz, respectively, using a
BRUKER Avance instrument equipped with a 5 mm probe. Tetramethylsilane was used as
an internal standard. The chemical shifts (δ) are reported in ppm. Coupling constants (J) are
reported in hertz and indicate apparent peak multiplicities. CHN elemental analyses were
performed on a Perkin-Elmer 2400 CHN elemental analyzer and are within 0.4% of
theoretical values. Substances 14-17 had their purity determined by HPLC-UV-DAD by the
method described herein. An Agilent 1100 Series Liquid Chromatography System was used
with a G1312A binary pump, G1322A degasser, G1313A automatic injector, G1316A
column oven and G1315B diode array detector (DAD). Acquisition of data and control
were conducted using Agilent ChemStation 1100 Series software. The column and

ACCEPTED MANUSCRIPT
precolumn used were the XDB C18 – 150 x 4,6 mm; 5 µm and XDB C18 – 12,5 x 4,6 mm;
5
µm, respectively. Mobile phase: (a) 5 mM ammonium acetate buffer; (b) ACN. Gradient
programing: from 0 to 25 min. - 10 to 90% of (b), from 25 to 30 min - 90% of (b), from 30
to 35 min - 90 to 10% of (b) and from 35 to 40 min - 10% of (b). The absorption was
determined at 225 nm. Procedure: To 1 or 2 mg of sample, 1,000 µl of methanol was added,
followed by ultrasonic bath for 2-5 min. Aliquots of 400 or 200 µL of solution were
transferred to 2.0 mL volumetric flasks and concentrated with methanol (concentration: 200
µg mL -1). Subsequently, solutions were filtered through 0.45 µm PVDF membrane and
injected (20 µL).
4
.1.1 N-acetylation of isatins
4.1.1.1. General procedure
Into a bound-bottom flask connected to a reflux condenser was added 13 mmol of the
proper indoline-2,3-dione (18a-e) and 15 equivalents of freshly distilled acetic anhydride.
The reaction medium was heated to reflux with magnetic stirring for 4 hours. Then, the
flask was cooled to room temperature and taken to the freezer (– 20 ° C) for 12 hours. The
formed solid was filtered and washed with water and hexane and allowed to air dry. The
product was recrystallized in ethyl acetate: hexane (1:1) with activated charcoal. The solid
formed was filtered and washed with cold hexane.
1
-acetylindoline-2,3-dione (19a)
Yield: 83%. Yellow solid, mp: 141-143 C (Lit.: 141 ºC)[45]. MS (EI): m/z 189 (11%), m/z
46 (100%), m/z 147 (22%), m/z 90 (34%), m/z 43 (45%)
o
1

ACCEPTED MANUSCRIPT
1
-acetyl-5-methylindoline-2,3-dione (19b)
o
Yield: 89%. Yellow solid, mp: 172-173 C. MS (EI): m/z 203 (17%), m/z 161 (52%), m/z
1
60 (100%), m/z 133 (21%), m/z 104 (30%), m/z 43 (27%).
1
-acetyl-5-chloroindoline-2,3-dione (19c)
o
Yield: 87%. Yellow, solid, mp: 208-210 C. MS (EI): m/z 223 (9%), m/z 181 (54%), m/z
1
80 (100%), m/z 153 (20%), m/z 124 (36%).
1
-acetyl-5-fluoroindoline-2,3-dione (19d)
o
Yield: 72%. Yellow solid, mp: 148-150 C. MS (EI): m/z 207 (14%), m/z 165 (50%), m/z
1
64 (100%), m/z 137 (17%), m/z 108 (65%).
1
-acetyl-5-methoxyindoline-2,3-dione (19e)
o
Yield: 80%, Yellow solid, mp: 146-148 C. MS (EI): m/z 219 (20%), m/z 177 (100%), m/z
1
76 (51%), m/z 149 (55%), m/z 120 (51%), m/z 106 (23%).
4
.1.1.2. Improved method for N-acetylation of isatins
To a flask was added 13 mmol of the corresponding isatin, 5 equivalents of acetic
anhydride and two drops of sulfuric acid. The reaction medium was maintained at reflux for
5
minutes. The flask was then cooled to room temperature and kept at -20 °C for 12 hours.
The formed solid was washed with water, having sufficient purity to be used in the next
step.
1
-acetylindoline-2,3-dione (19a)

ACCEPTED MANUSCRIPT
o
Yield: 95%. Yellow solid, mp: 141-143 C (Lit.: 141 ºC)[45]. MS (EI): m/z 189 (11%), m/z
1
46 (100%), m/z 147 (22%), m/z 90 (34%), m/z 43 (45%)
1
-acetyl-5-methylindoline-2,3-dione (19b)
o
Yield: 95%. Yellow solid, mp: 172-173 C. MS (EI): m/z 203 (17%), m/z 161 (52%), m/z
1
60 (100%), m/z 133 (21%), m/z 104 (30%), m/z 43 (27%).
4
.1.2. Deoxofluorination of 1-acetylindoline-2,3-diones
General procedure
The corresponding 1-acetyl-indoline-2,3-dione (6 mmol) and a magnet were added
to a round-bottom flask and then sealed with a septum. The atmosphere of the system was
replaced by argon, and 37 equivalents of freshly distilled anhydrous dichloromethane were
added. The mixture was stirred for 5 minutes. Then, 2 equivalents of diethylaminosulfur
trifluoride (DAST) were added through a syringe. The reaction medium was stirred for 16
hours at room temperature under argon atmosphere. After this period, the reaction mixture
was poured into crushed ice. The organic phase was separated, washed 2 times with
distilled water, dried with anhydrous Na SO and concentrated under vacuum. The product
2
4
was purified by vacuum sublimation and stored in amber vials in an argon atmosphere.
1
-acetyl-3,3-difluoroindolin-2-one (20a)
Yield: 65%. Off-white solid, mp: 110-113 C (Lit.: 109-111 C)[22]. MS (EI): m/z 211
13%), m/z 169 (100%), m/z 141 (66%), m/z 114 (12%), m/z 43 (61%).
o
o
(
1
-acetyl-3,3-difluoro-5-methylindolin-2-one (20b)

ACCEPTED MANUSCRIPT
o
o
Yield: 66%. Off-white solid, mp: 71-73 C (Lit.: 72-74 C)[22]. MS (EI): m/z 225 (12%),
m/z 183 (100%), m/z 184 (10%), m/z 155 (74%), m/z 43 (35%).
1
-acetyl-5-chloro-3,3-difluoroindolin-2-one (20c)
o
o
Yield: 88%. Off-white solid, mp: 136-137 C (Lit.:134-136 C)[22]. MS (EI): m/z 245
(14%), m/z 205 (32%), m/z 203 (100%), m/z 175 (56%), m/z 43 (91%).
1
-acetyl-3,3,5-trifluoroindolin-2-one (20d)
o
–1
1
Yield: 72%. Off-white solid, mp: 73-75 C. IR νmax (cm ) 1797, 1716, 1097. H NMR
(DMSO-d , 400 MHz) δ ppm 3.34 (s, 3H, H-9), 7.57 (td, J = 9.1, 2.6 Hz, 1H, H-6), 7.90
6
1
3
(
dd, J = 7.3, 2.6 Hz, 1H, H-4), 8.25 (dd. J = 9.1, 4.4 Hz, 1H, H-7). C NMR (DMSO-d ,
6
1
00 MHz) δ ppm 26.09, 109.33 (t, J = 248 Hz), 112.22 (d, J = 25.8 Hz), 119.05 (d, J = 7.9
Hz), 120.25 (td, J = 22.6 Hz, 8.8 Hz), 121.04 (d, J = 23.0 Hz), 137.52 (td, J = 6.8, 2.5 Hz),
1
9
1
59.46 (d, J = 244.8 Hz), 164.60 (t, J = 30.5 Hz). 169.71. F NMR (DMSO-d
δ ppm – 105.0, – 114.8. MS (EI): m/z 229 (13%), m/z 187 (100%), m/z 159 (64%), m/z 132
12%).
6
, 377 MHz)
(
1
-acetyl-3,3-difluoro-5-methoxyindolin-2-one (20e)
o
–1
1
Yield: 50%. Off-white solid, mp: 158-159 C. IR νmax (cm )1799, 1707, 1080. H NMR
(
DMSO-d , 400 MHz) δ ppm 2.58 (s, 3H), 3.83 (s, 3H), 7.25 (d, J = 8.9 Hz, 1H), 7.46 (s,
6
1
3
1
1
1
H), 8.14 (d, J = 8.9 Hz, 1H). C NMR (DMSO-d , 100 MHz) δ ppm 26.00, 55.88, 109.47,
6
09.97 (t, J = 246 Hz), 118.33, 119.61 (t, J = 22Hz), 119.86, 134.25 (t, J = 7.0 Hz). 157.26,
1
9
64.89 (t, J = 30.8 Hz), 169.49. F NMR (DMSO-d , 377 MHz) δ ppm– 104.5 (CF ) ppm.
6
2
MS (EI): m/z 241 (15%), m/z 199 (100%), m/z 171 (55%), m/z 156 (38%).

ACCEPTED MANUSCRIPT
4
.1.3. Synthesis of the new α.α-difluorohydrazides 4-13.
General procedure
Into a bound-bottom flask coupled to a condenser was added 0.6 mmol of the
corresponding 1-acetyl-3,3-difluoroindolin-2-one and 3 mL of ACN. The mixture was
stirred at room temperature for 5 minutes. Then, 2 equivalents of INH or proper hydrazine
was added to the medium. The flask was then attached to a reflux condenser and the system
kept under stirring at 82 °C for 12 h. At the end of this period the formed precipitate was
vacuum filtered and washed with cold ACN and then with distilled water. The product was
air dried and stored under argon atmosphere with protection from light.
N-(2-(2-(2-carbamimidoylhydrazinyl)-1,1-difluoro-2-oxoethyl)phenyl)acetamide (4)
1
Yield: 70%. Off-white solid. H NMR (MeOD, 400 MHz) δ ppm 2.17 (s, 3H), 7.18 (t, J =
1
9
7
.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H). F
NMR (MeOD, 377 MHz) δ ppm – 103.0. ESI-MS(+): m/z 286.
N-(2-(1,1-difluoro-2-oxo-2-(2-phenylhydrazinyl)ethyl)phenyl)acetamide (5)
1
Yield: 79%. Off-white solid, mp.: 176-177 ºC. H NMR (MeOD, 400 MHz) δ ppm 2.13 (s,
3
7
H), 6.70 (d, J = 7.8 Hz, 1H), 6.81 (t, J = 7.3 Hz, 1H), 7.14 (t, J = 7.6 Hz, 2H), 7.35 (t, J =
1
3
.3 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 8.0, 1H). C
NMR (MeOD, 100 MHz) δ ppm 23.8, 112.2, 114.8, 117.3, 119.3, 125.0, 126.1 (t, J = 8.9
1
9
Hz), 126.2, 128.8, 131.7, 135.9, 148.1, 163.9 (t, J = 30.7 Hz), 168.4. F NMR (MeOD, 377
.
MHz) δ ppm – 106.2 ESI-MS(+): m/z 342. Anal. Calcd. for C H F N O : C, 60.18; H,
1
6
15
2
3
2
4
.73; N, 13.16; found: C, 60.12; H, 4.46; N, 13.11

ACCEPTED MANUSCRIPT
N-(2-(1,1-difluoro-2-oxo-2-(2-phenylhydrazinyl)ethyl)-4-methylphenyl)acetamide (6)
Yield: 81%. Off-white solid, mp.: 185-186 ºC. H NMR (MeOD, 400 MHz) δ ppm 2.11 (s,
3
7
H), 2.40 (s, 3H), 6.72 (d, J = 8.0 Hz, 2H), 6.82 (t, J = 7.4 Hz, 1H), 7.15 (t, J = 8.0 Hz, 2H),
19
.37 (d, J = 8.2 Hz, 1H), 7.52 (s, 1H), 7.68 (d, J = 8.2 Hz, 1H). F NMR (MeOD, 377
MHz) δ ppm – 106.0. ESI-MS(–): m/z 332. Anal. Calcd. for C H F N O : C, 61.25; H,
1
7
17
2
3
2
5
.14; N, 12.61; found: C, 61.26; H, 4.79; N, 12.62
N-(2-(1,1-difluoro-2-oxo-2-(2-(pyridin-2-yl)hydrazinyl)ethyl)phenyl)acetamide (7)
1
Yield: 78%. H NMR (MeOD, 400 MHz) δ ppm 2.15 (s, 3H), 6.71 (d, J = 8.4 Hz), 6.81 (t,
J = 6.2 Hz), 7.41 (t, J = 7.5 Hz), 7.57-7.65 (m), 7.75 (d, J = 7.8 Hz), 8.07 (d, J = 4.7 Hz).
1
3
C NMR (MeOD, 100 MHz) δ ppm 23.6, 109.6, 113.4, 115.9, 117.1, 127.5, 127.6 (t, J =
19
4
.0 Hz), 129.2, 133.0, 136.8, 139.8, 147.7, 159.8, 166.4 (t, J = 31.2 Hz), 173.0. F NMR
(
MeOD, 377 MHz) δ ppm – 107.4. ESI-MS(+): m/z 343. Anal. Calcd. for C15H F N O :
14 2 4 2
C, 56.25; H, 4.41; N, 17.49; found: C, 56.23; H, 3.95; N, 17.22.
N-(2-(1,1-difluoro-2-oxo-2-(2-(pyridin-2-yl)hydrazinyl)ethyl)-4-
methylphenyl)acetamide (8)
1
Yield: 71%. Light brown solid, m.p: 153-154 ºC. H NMR (MeOD, 400 MHz) δ ppm 2.13
(s, 3H), 2.41 (s, 3H), 6.71 (d, J = 8.4 Hz), 6.81 (t, J = 6.4 Hz), 7.38 (d, J = 8.1 Hz), 7.47 (d,
1
3
J = 8.1 Hz), 7.56-7.61 (m), 8.07 (d, J = 4.5 Hz). C NMR (MeOD, 100 MHz) δ ppm 20.5,
2
1
3.3, 107.9, 114.5, (t, J = 252.5 Hz), 115.0, 126.5 (t, J = 7.7 Hz), 127.1, 128.6, 131.9,
1
9
33.2, 135.1, 138.0, 146.7, 158.0, 163.4 (t, J = 30.1 Hz), 169.0. F NMR (MeOD, 377
MHz) δ ppm – 107.2. ESI-MS(–): m/z 333. Anal. Calcd. for C H F N O : C, 57.48; H,
1
6
16
2
4
2
4
.82; N, 16.76; found: C, 57.40; H, 4.43; N, 16.51

ACCEPTED MANUSCRIPT
N-(2-(1,1-difluoro-2-(2-isonicotinoylhydrazinyl)-2-oxoethyl)phenyl)acetamide (9)
o
–1
1
Yield: 50%. Off-white solid, mp: 199-201 C. IR νmax (cm ) 1730, 1685, 1629, 1076. H
NMR (DMSO-d , 400 MHz) δ ppm 2.08 (s, 3H), 7.38 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6
6
Hz, 1H), 7.71 (bs, 2H), 7.79 (d, J = 4.8 Hz, 2H), 8.80 (d, J = 4.8 Hz, 2H), 9.22 (s, 1H),
1
3
13
1
1
1
1.09 (s, 1H), 11.37 (s, 1H). C NMR C NMR (DMSO-d , 100 MHz) δ ppm 23.53,
6
14.58 (t, J = 252 Hz), 121.27, 125.46, 125.88 (t, J = 24.2 Hz), 126.39 (t, J = 7.2 Hz),
1
9
27.66, 131.76, 135.91, 138.68, 150.60, 162.97 (t, J = 31.2 Hz), 164.37, 168.77. F NMR
, 377 MHz) δ ppm – 104.4. ESI-MS(–): m/z 347. Anal. Calcd. for
: C, 55.17; H, 4.05; N, 16.09. Found: C, 55.15; H, 4.25; N, 15.86.
(DMSO-d
6
C
16 14 2 4 3
H F N O
N-(2-(1,1-difluoro-2-(2-isonicotinoylhydrazinyl)-2-oxoethyl)-4-
methylphenyl)acetamide (10)
o
–1
1
Yield: 55%. Off-white solid, mp: 201-203 C. IR νmax (cm ) 1730, 1685, 1631, 1078. H
NMR (DMSO-d , 400 MHz) δ ppm 2.05 (s, 3H), 2.36 (s, 3H), 7.38 (d, J = 8 Hz, 1H), 7.52
sl, 2H), 7.79 (d, J = 2.8 Hz, 2H), 8.79 (sl, 2H), 9.16 (s, 1H), 11.10 (s, 1H), 11.35 (s, 1H).
6
(
1
3
C NMR (DMSO-d , 100 MHz) δ ppm 20.48, 23.41, 114.55 (t, J = 251 Hz), 121.30,
6
1
26.13 (t, J = 23.3 Hz), 126.62, 128.05, 132.17, 133.24, 135.10, 138.66, 150.60, 162.93 (t,
1
9
J = 30.3 Hz), 164.44, 168.84. F NMR (DMSO-d , 377 MHz) δ ppm – 100.6. ESI-MS(–):
6
m/z 361. Anal. Calcd. for C H F N O :C, 56.35; H, 4.45; N, 15.46. Found: C, 56.52; H,
1
7
16
2
4
3
4
.68; N, 15.15.
N-(4-chloro-2-(1,1-difluoro-2-(2-isonicotinoylhydrazinyl)-2-oxoethyl)phenyl)acetamide
11)
(