Synthesis, characterization, and biologic activity of new acyl hydrazides and 1,3,4-oxadiazole derivatives

molecules

Article

Synthesis, Characterization, and Biologic Activity of

New Acyl Hydrazides and

1

,3,4-Oxadiazole Derivatives

1

, ,†

2,3,†

, Petre Ionita ¹ , Arnaud Tatibouët ,

4

Irina Zarafu * , Lilia Matei

, Coralia Bleotu

1

3,5

6

Anca Păun , Ioana Nicolau , Anamaria Hanganu

Roxana Maria Nemes

, Carmen Limban , Diana Camelia Nuta ,

7

2,†

8,9,

,

, Carmen Cristina Diaconu

and Cristiana Radulescu

*

1

Faculty of Chemistry, University of Bucharest, 050663 Bucharest, Romania;

petre.ionita@chimie.unibuc.ro (P.I.); anca.paun@chimie.unibuc.ro (A.P.);

ioana.nicolau@chimie.unibuc.ro (I.N.)

2

3

4

“

Stefan S Nicolau” Institute of Virology, Romanian Academy, 030304 Bucharest, Romania;

lilia.matei@virology.ro (L.M.); coralia.bleotu@virology.ro (C.B.); carmen.diaconu@virology.ro (C.C.D.)

Research Institute of the University of Bucharest (ICUB), Life, Environmental and Earth Sciences Division,

University of Bucharest, 060023 Bucharest, Romania; anamaria.hanganu@unibuc.ro

Institute of Organic and Analytical Chemistry ICOA-UMR7311, University of Orleans,

4

5067 Orleans, France; arnaud.tatibouet@univ-orleans.fr

5

6

Institute of Organic Chemistry “C.D. Nenitescu” of the Romanian Academy, 060023 Bucharest, Romania

Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, 020021 Bucharest, Romania;

carmen.limban@umfcd.ro (C.L.); diana.nuta@umfcd.ro (D.C.N.)

National Institute of Pneumology Marius Nasta, 050152 Bucharest, Romania; roxanamarianemes@gmail.com

Faculty of Sciences and Arts, “Valahia” University of Targoviste, 130004 Targoviste, Romania

Institute of Multidisciplinary Research for Science and Technology, Valahia University of Targoviste,

7

8

9

1

3004 Targoviste, Romania

*

†

Correspondence: irina.zarafu@chimie.unibuc.ro (I.Z.); cristiana.radulescu@valahia.ro (C.R.)

These authors contributed equally to this work.

Received: 19 June 2020; Accepted: 21 July 2020; Published: 21 July 2020

Abstract: Starting from isoniazid and carboxylic acids as precursors, thirteen new hydrazides

and 1,3,4-oxadiazoles of 2-(4-substituted-phenoxymethyl)-benzoic acids were synthesized and

characterized by appropriate means. Their biological properties were evaluated in terms of apoptosis,

cell cycle blocking, and drug metabolism gene expression on HCT-8 and HT-29 cell lines. In vitro

antimicrobial tests were performed by the microplate Alamar Blue assay for the anti-mycobacterial

activities and an adapted agar disk diﬀusion technique for other non-tubercular bacterial strains. The

best antibacterial activity (anti-Mycobacterium tuberculosis eﬀects) was proved by

9. Compounds 7, 8,

and determined blocking of G1 phase. Compound proved to be toxic, inducing apoptosis in 54%

9

7

of cells after 72 h, an eﬀect that can be predicted by the increased expression of mRNA caspases 3

and 7 after 24 h. The inﬂuence of compounds on gene expression of enzymes implicated in drug

metabolism indicates that synthesized compounds could be metabolized via other pathways than

NAT2, spanning adverse eﬀects of isoniazid. Compound

9 had the best antibacterial activity, being

used as a disinfectant agent. Compounds , and , seemed to have antitumor potential. Further

7,

8

9

studies on the action mechanism of these compounds on the cell cycle may bring new information

regarding their biological activity.

Keywords: isoniazid; oxadiazole; synthesis; antibacterial activity; apoptosis; cell cycle; drug

metabolism genes expression

Molecules 2020, 25, 3308; doi:10.3390 /molecules25143308

www.mdpi.com /journal /molecules

Molecules 2020, 25, 3308

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1

. Introduction

Hydrazides represent a class of organic compounds containing a nitrogen–nitrogen covalent bond

with at least one acyl substituent [ ]. They are used as starting materials for the synthesis of surfactants

and of various heterocycles as of 1,2,4-triazoles [ ], 1,3,4-thiadiazoles [ ], or 1,3,4-oxadiazoles [ ] with

pharmacological activity [ ]. Hydrazides are well known for their antitumor [ ], anti-inﬂammatory [ ],

analgesic [ ], antibacterial [ ], and antiviral [10] activities, while 1,3,4-oxadiazoles are known for

antibacterial, antifungal, and anti-inﬂammatory activities [11,12].

Is well known that isoniazid (INH) is used as valuable drug for anti-tuberculosis treatments

mostly against the active metabolize and multiply of Mycobacterium tuberculosis bacteria [13 14], but less

1

2

3

3,4

5

6

7

8

7,9

,

eﬀective than dormant ones [15]. The hydrazide of isonicotinic acid is considered a prodrug capable of

crossing the M. tuberculosis cell wall through passive diﬀusion [16]. After reaching the inside of the

cell [17], KatG (mycobacterial catalase-oxidase) is activating it. The active forms of INH shown diﬀerent

eﬀects on the cell wall mycolic acids synthesis [18], on nucleic acids replication/transcription [19], and

on bacterial respiratory metabolism, as well [20]. All this time, it never been completely discovered the

complex mechanism of action of isoniazid as prodrug [21,22].

In terms of toxicity eﬀect over the human body, the INH behavior is diﬀerent for each

patient. The isoniazid is degradaded mainly by acetylation with N-acetyltransferase (NAT),

which conduct to N1-acetyl-N2-isonicotinylhydrazine (acetylisoniazid) metabolite, and further, by

metabolization process, result the mono-acetylhydrazine and the nontoxic di-acetylhydrazine [23–25].

The polymorphisms of genes that codify NATs, also inﬂuence the metabolism of INH. On the other

hand, diﬀerent degrees of activity for NATs and an individual acetylation proﬁle are determined by

the presence of various polymorphisms [26]. On this basis, the humans may be classiﬁed in three

classes such as slow, intermediate, and fast acetylators [27]. Therefore, a fast acetylation of INH lead

to a lowering concentration of active drug [14], being responsible for applied therapy eﬀectiveness,

mostly to some patients which required single weekly doses [28]. On the other hand, for slow

acetylators the risk for hepatotoxic eﬀects occurrence is possible due to high concentrations of isoniazid

metabolites [24,29,30]. Literature [22,31,32] revealed that the any change added to isoniazid molecule

such as the inclusion of a functional group to the hydrazine structure could block the acetylation of the

drug by NATs, therefore inﬂuencing its activity and toxicity.

It had been previously shown that isoniazid derivatives exhibited a good anti-mycobacterial

activity, some of them being more active than isoniazid or other anti-tubercular drugs used in the

current therapy regimens [33–38]. Various studies have postulated that the majority of isoniazid

derivatives are activated before reaching the intracellular medium of M. tuberculosis [39]. On the other

hand, based on QSAR study [40], the Ventura group presumed that isoniazid derivatives activation

involve the formation of electrophilic intermediate species (hydrazyl radical or ion) that are transformed

+

in acyl radicals which can couple with NADH/NAD , the formed adducts inhibiting enoyl-acyl carrier

protein reductase (InhA) activity [41].

To the best of our knowledge, the synthesis of new isoniazid derivatives based clinical drugs

is important for the therapeutic ﬁeld. These observations encouraged the authors to continue the

previous research (i.e., synthesis, characterization, and pharmacological evaluation of new heterocyclic

compounds), where hydrazides/indazoles/oxadiazoles linked with other heterocycles proved as

eﬀective antimicrobial agents [42–48]. The purpose of current study was to characterize the new

synthesized isoniazid derivatives and to preliminary evaluate their biological activity to identify their

usefulness as drugs in medical ﬁeld.

2

. Results and Discussion

2

.1. Synthesis and Structural Characterization

The benzoic acid hydrazides were prepared following the method described in the general

experimental procedure. The carboxylic acids were obtained by a method reported earlier by Limban

Molecules 2020, 25, 3308

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group [49], by reﬂuxing the phthalide with potassium p-substituted phenolates in xylene. The obtained

corresponding acid was treated with thionyl chloride in 1,2-dichloroethane under reﬂux. The crude acid

chloride thus obtained was added to isoniazid dissolved in dichloromethane and the reaction mixture

was stirred at room temperature. Product formation was monitored by thin-layer chromatography on

silica gel, and the obtained products were separated and puriﬁed on chromatographic columns. In the

case of 2-(4-methyl/methoxy/ethyl-phenoxymethyl)-benzoic acid derivatives, the presence of two new

compounds in each reaction was observed.

The newly synthesized N,N’-diacylhydrazines (1–6) were diluted in toluene and treated with

phosphoryl chloride. The reaction mixture was reﬂuxed for 6 h and then stirred overnight at room

temperature. Product formation was monitored by thin layer chromatography on silica gel. Toluene

and residual phosphoryl chloride were removed under reduced pressure and the reaction products

were taken up in dichloromethane and washed with 5% NaOH solution and with saturated aqueous

NaCl solution. The organic layer was dried over anhydrous sodium sulfate and the solvent distilled

under reduced pressure. The newly synthesized 1,3,4-oxadiazoles were recrystallized using ethanol.

The synthesis reactions of new compounds are presented in Figure 1.

Compound

R

Compound

R

1

2

3

4

5

6

H

F

Cl

CH

OCH

7

8

9

10

11

12

CH

OCH

3

C2H

5

3

H

F

Cl

3

C2

H

5

1

3

CH

3

R

O

POCl₃

HCl

N

NH

-

H

R

N

O

-

3

^H^P^O4

N

O

R

N

1

0-13

COCl

1-6

+

-HCl

O

R

H

O

N

NH₂

O

N

H

N

7-9

O

R

Figure 1. Synthesis of the new isoniazid derivatives.

The synthesis yields (attempts to optimize were not made), the melting points, and the R values

f

of the new compounds obtained are presented in Table 1.

Molecules 2020, 25, 3308

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Table 1. Results obtained in the synthesis of the compounds.

◦

R_f^a^,^b^,^c

Compound

Yield [%]

m.p. [ C]

INH

-

171–173

128–129

168–169

145–146

173–175

108–111

133–135

Semisolid

210–211

298–299

341–342

249–250

0.03 ^a

b

1

2

3

4

5

6

7

8

9

52

40

25

58

54

33

5

2

6

62

65

68

73

0.50

0.57

0.56

0.17

0.13

0.17

0.60

0.52

0.63

0.72

0.79

0.75

b

a

b

10

11

12

13

b

c

0.60

a

AcOEt:CH2Cl2 = 1:1, ^bAcOEt:MeOH = 9:1, ^cCH2Cl2:MeOH = 5:1.

1

The chemical structure of the newly synthesized compounds was conﬁrmed by H-NMR,

13

1

C-NMR, IR, and by elemental analysis. The H-NMR spectra of compounds

1–

6

showed two

singlets at 10.11–11.20 ppm and 9.72–10.55 ppm, which represent the two hydrazide protons (NH-NH,

1

deuterable), while the H-NMR spectra of compounds

7–

9

showed only one singlet at 8.53–11.97 ppm,

corresponding to the NH–N proton. This indicates that compounds

1

–6

are N,N’-disubstituted, while

compounds 7–9 are N,N,N’-trisubstituted.

−

1

−1

FTIR spectra showed the C=O peak at 1690–1669 cm and the C–N peak at 1609–1592 cm , and,

−

1

also, in the case of compounds

1

–

9, the N–H stretching vibration at 3271–3191 cm . The stretching

−1

vibrations of the aromatic rings (C=C–C) are situated at 1608–1443 cm . For all compounds it can

−1

distinguish the 1,2- and 1,4-disubstitution at 748–735 cm and 841–811 cm , respectively. The

complete interpretation of H-, ¹³C-NMR and IR spectra and the elemental analyses of new compounds

are presented in the experimental part.

1

All compounds were subjected to MS analysis, the signals of molecular ions [M + H] corresponding

to the calculated molecular weight and structure of these compounds.

2

.2. Biological Evaluation

2

.2.1. Antimicrobial Activity

Taking into account that isoniazid derivatives are known for their anti-tubercular activity [30,32,36],

the newly synthesized derivatives of isoniazid were tested for their in vitro activity against a clinical

strain of M. tuberculosis. The tested compounds were quantitatively assayed using serial binary

dilutions ranging from 25 µg/mL to 0.012 µg/mL. None of the new synthesized compounds proved to

have anti-tubercular activity, apart from compound 9 that inhibited the growth of M. tuberculosis at a

concentration of 6.25 µg/mL (Table 2).

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Table 2. The anti-tubercular evaluation of the new compounds.

Compound

MIC ^a[µg/mL]

INH

0.098

>25

25

1

2

3

4

5

6

7

8

9

25

6.25

>25

1

0

1

2

3

a

Anti-mycobacterial activity (minimum inhibitory concentration (MIC)) against a clinical strain of M. tuberculosis.

The minimal inhibitory concentration (MIC) of the compound

of those reported for other isoniazid derivatives obtained by combination with acid chlorides [33

A very active derivative against M. tuberculosis is 1-isonicotinyl-2-nonanoyl hydrazine, superior to

isoniazid with a very low minimal inhibitory concentration value of 0.025 g/mL [38]. Other compounds

from this class exhibited MIC values ranging between 0.39 and 71.6 g/mL [33 37]. Regarding the

9

was moderate, which is in the range

–38].

µ

–

isoniazid-derived 1,3,4-oxadiazoles reported in literature, few of them proved anti-tuberculosis

activity [50].

In order to establish if these new synthesized compounds have antibacterial activity against

other microbial species and to determine the spectrum of their activity, the novel derivatives

were tested on other, non-tuberculosis, bacterial strains isolated from clinical specimens, both

Gram-positive (e.g., Staphylococcus hominis, Staphylococcus aureus, and coagulase-negative staphylococci)

and Gram-negative (e.g., Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter koseri,

Morganella morganni, Acinetobacter baumanii, and Pseudomonas aeruginosa), using an adapted diﬀusion

technique. The most active derivative proved to be compound

the majority of tested strains, followed by the compounds and

compounds , and 10 13. On the other hand, the N,N,N’-triacylhydrazines

antibacterial activity than their corresponding N,N’-diacylhydrazines , as well as the rest of tested

9

7

, which inhibited the growth of

. The less active derivatives were

exhibited a better

6

1,

3

–

7–9

4–6

compounds (Table 3). According to previously reported data compounds with similar structures

proved inhibitory activity on the growth of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas

aeruginosa, and Escherichia coli [33–37].

Molecules 2020, 25, 3308

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2

.2.2. The Inﬂuence of the New Isoniazid Derivatives on the Expression Levels of Some Genes

Implicated in Drug Metabolism

The main metabolic pathway of isoniazid and similar compounds in human body involves

its N-acetylation and the increased expression of NAT1 and NAT2 genes, which is suggested to be

associated with a better rate of metabolization. In this context, the inﬂuence of newly synthesized

substances on NAT1 and NAT2 expression in eukaryotic cells was evaluated in two cell lines—HCT-8

and HT-29—at a concentration of 50

NAT2*5 genotype, while the HCT-8 cell line presents the NAT2*6 genotype [51]. Isoniazid decreased

the expression of NAT1 and NAT2 in both cell lines, as well as compounds 10, and 11. NAT1

and NAT2 gene expressions were increased by compounds and in both HT-29 and HCT-8 cell

lines. Compounds and increased the gene expression of NAT1 in both used cell lines, compound

increased NAT1 expression only in HT-29, and compounds and increased it only in the HCT-8

cell line. Regarding NAT2 gene expression, it was increased by compounds and in both tested cell

lines, by compounds and 12 in HT-29 cell line, and by compound in HCT-8 cells. Generally, the

NAT2 gene expression was more inﬂuenced by tested compounds in HCT-8 cell line, than in HT-29 cell

line, with the exception of compounds 11 (Figure 2). Compounds could be used to potentiate

isoniazid metabolism in slow acetylators, and further studies should be done in this direction.

µg/mL. As we previously reported, the HT-29 cell line presents

2, 4,

6

9

3

5

1

7

8

7

8

5

3

9–

6–9

Figure 2. The inﬂuence of the new isoniazid derivatives 1–13 on the expression of NAT1 and NAT2

genes in HT-29 and HCT-8 cell lines.

In order to identify how new synthesized compounds may be degraded and eliminated from the

body, the expression of some CYP450 isoenzymes, involved in the metabolism of pharmacological

substances, was evaluated in HCT-8 and HT-29 cell lines (Figure 3). The obtained results shown that

isoniazid decreased the expression of CYP1A1 and CYP2C19 genes in both cell lines, but increased

the expression of CYP3A4 in HCT-8 cells. All tested compounds increased CYP1A1 expression in

both cell lines, except for compound 3. Regarding the expression of CYP2C19, it was increased by

Molecules 2020, 25, 3308

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compound

synthesized 1,3,4-oxadiazoles 10

by compounds and in HT-29 and in HCT-8 cells, and by compounds 1, 2, and 4

4

in both used cell lines; by compounds

1,

5,

6

,

8, and

9

in HCT-8 cells; and by new

,

11

,

12, and 13 in HT-29 cells. CYP3A4 gene expression was increased

only in HT-29 cells.

3

9

(

A)

(

B)

Figure 3. The inﬂuence of the new isoniazid derivatives

1–13 on the expression of CYP1A1, CYP2C19,

and CYP3A4 genes in the eukaryotic cells; (A) HT-29 cells; (B) HCT-8 cells

Isoniazid inhibits CYP2C19 and CYP3A4 activities and mechanistically inactivates CYP1A2,

CYP2A6, CYP2C19, and CYP3A4 in human liver microsomes [52,53]. El Sayed’s group analyzed the

Molecules 2020, 25, 3308

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inﬂuence of some isoniazid derivatives on the expression of mRNA and activity of enzymes implicated

in drug metabolism in mice [54]. Although the analyzed compounds determined some changes in

mRNA expressions, their inﬂuence on enzymes activity was minor [54]. The increased expression

levels of CYP1A1 induced by the new synthesized isoniazid derivatives could be explained by the

induction of their metabolism. CYP450 enzymes are implicated in phase I of xenobiotic and drug

metabolism, and CYP1A1 catalyzes the hydroxylation and other oxidative transformations of aromatic

substances [53

,55]. Regarding diﬀerent proﬁles of CYP2C19 expression in the two cell lines used, this

could account for the existence of a polymorphic locus.

2

.2.3. Cytotoxicity and Eﬀects on Cell Cycle

The obtained compounds have also been tested to establish their cytotoxicity and the apoptotic

eﬀect on HT-29 cell line. The eﬀect of the treatment with 50

was evaluated by ﬂow cytometry using FITC-labeled annexin-V and propidium iodide staining that

discriminate between apoptotic and intact cells. The most toxic compound proved to be compound

followed by compounds and . The treatment of HT-29 cells with 50 g/mL of compound has no

µg/mL of substances for 24, 48, and 72 h

7,

8

9

µ

7

toxic eﬀect after 24 h, but after 48 and 72 h it induced necrosis in 28.6% and 54%, respectively, of the

eukaryotic cells (Table 4).

Kumar’s group evaluated a series of N’-(E)-(substituted-benzylidene)-isonicotinohydrazide

derivatives regarding their anticancer activity against several types of tumors and determined that

isonicotinoyl hydrazides display moderate to potent anticancer activity [56]. Later, Rodrigues et al.

evaluated thirty-two N’-[(E)-(disubstituted-phenyl)-methylidene]-isonicotino-hydrazides for their

activity against four human cancer cell lines and established that the biological activity of this

compounds is determined by the number, the positions, and the types of substituents attached to the

aromatic ring. The best results were proved by disubstituted derivatives, and the presence of hydroxyl

groups on the benzene ring plays an important role in the anticancer activity of this series, especially

when it is located in o-position. Hydroxyl groups located in o-position in hydrazone systems are good

ligands for metals, the action mechanism of this class could possibly be based on the formation of

complexes that are likely to inactivate enzymes involved in abnormal cell division [57].

In current case, the most cytotoxic activity had been proved by a compound containing a methyl

group in p-position of a benzene ring (compound 7). This eﬀect could be probably explained by its

metabolism, the tolyl moiety being oxidized to the corresponding alcohol and carboxylic acid and to

o-cresyl [58], the presence of hydroxyl groups on the benzene ring in o-position playing an important

role in the anticancer activity [57]. The metabolic pathway of compound

oxidation of ethyl group resulting the corresponding alcohols and acids, which are less toxic [59]. In the

case of compound , the major metabolic pathway could involve O-demethylation of this compound,

9 possibly involves the

8

similar to other compounds containing p-methoxyphenyl moiety [60], with isoniazid derivatives

containing a hydroxyl moiety in the p-position of a benzene ring being less toxic [57].

The cytotoxicity of the newly synthesized compounds was also studied at the molecular level by

analyzing the expression of genes implicated in apoptosis. From all tested substances, compounds

determined an increase in the expression levels of caspases 3, 7, 8, and 9 genes. Compounds 10 and 13

slightly increased the gene expression of caspases 3, 7, and 9, and compound 12 increased caspase 3 and 9

gene expression, while compounds , and increased only the expression of caspase 9. Regarding the

proapoptotic Bax (Bcl-2-associated X protein) and antiapoptotic Bcl-2 (B-cell lymphoma 2), their gene

expression was increased by compounds 10, and 12. Compound 13 increased Bcl-2 and decreased Bax

3 and

7

2

,

8

9

2,

gene expressions, while other tested compounds decreased both genes expressions. Still, the Bax/Bcl-2

ratio was positive accounting for an induction of apoptotic program. Regarding MCL1 (Myeloid Cell

Leukemia Sequence 1), compounds

compounds increased it (Figure 4).

1, 2, 4, and 5 decreased expression of this gene, while the other

Molecules 2020, 25, 3308

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Table 4. Apoptosis induction by the new isoniazid derivatives in eukaryotic cells.

Necrosis

% of Total Cells]

Late Apoptosis

[% of Total Cells]

Early Apoptosis

[% of Total Cells]

Viable Cells

[% of Total Cells]

Compound

[

7

2 h

HT-29

INH

1.46

5.25

0.854

0.493

0.382

11.1

12.2

5.82

20.7

8.38

10.8

0.766

1.43

0.553

0.658

0.206

3.09

0.503

0.937

0.304

1.85

3.28

2.7

33.3

7.05

2.8

0.317

0.301

0.82

0.239

0.057

0.915

0.278

0.305

0.294

2.15

1.56

1.01

2.93

1.99

98.3

90.7

98.4

98.3

99.0

84.9

83

1

2

3

4

5

6

7

8

9

90.5

43

82.6

85.2

98.7

97.8

98.4

98.9

1.22

10

11

12

13

0.196

0.457

0.23

0.194

4

8 h

HT-29

INH

1.27

5.96

2.35

2.37

0.706

4.32

5.19

7.28

16.2

6.64

10.1

0.599

3.15

0.734

0.732

0.264

2.63

0.325

0.221

0.121

1.5

4.35

2.6

12.4

2.52

0.13

0.661

0.337

0.431

0.538

0.703

1.17

0.346

0.736

0.614

0.708

0.435

0.337

0.527

0.131

98.3

90.8

97.0

98.6

93.5

89.3

89.8

70.7

90.2

84.6

98.6

95.7

98.4

99.0

1

2

3

4

5

6

7

8

9

4.52

10

11

12

13

0.359

0.834

0.381

0.174

2

4 h

HT-29

INH

0.68

3.68

0.612

0.637

1.1

0.032

1.85

0.404

1.06

98.9

93.4

98.9

98.7

98.1

92.6

93

92.8

91.5

92.1

92.4

98.9

98.5

97.9

98.3

1

2

3

4

5

6

7

8

9

0.411

0.505

0.794

0.964

0.132

1.2

1.14

1.71

1.5

0.169

0.127

0.816

0.23

0.124

0.204

0.019

0.832

0.237

1.14

0.87

1.95

1.32

5.58

6.6

4.88

6.51

4.22

4.82

0.835

1.16

1.15

1.4

10

11

12

13

0.129

0.164

0.11

0.058

Molecules 2020, 25, 3308

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(

A)

(

B)

Figure 4. The inﬂuence of the new isoniazid derivatives on the expression of genes implicated in

apoptosis induction in HCT-8 cells; (A) pro-apoptotic genes; (B) anti-apoptotic genes.

The activation of the eﬀector caspase 3 can be induced by the extrinsic pathway via tumor necrosis

factor (TNF) family receptors (e.g., Fas), FADD (Fas-activated death domain protein), and caspase

8

, or by the intrinsic pathway via the mitochondrial release of cytochrome c and Apaf-1–mediated

processing of caspase 9 [61]. In the case of compound , the increased expression of caspase 9 could be

associated with activation of intrinsic apoptotic pathway. Although the phenotypic changes induced

by the treatment of compound with 50 g/mL for 24 h are minor, the increased expression of mRNA

caspase 3 and 7 predicts the apoptotic phenomena occurring at 48 and 72 h. In the case of compounds

, and 13, although all evaluated caspases and Bax gene expressions were increased, the expression of

7

µ

3

,

1

2

antiapoptotic Bcl-2 and MCL1 genes was also high, and no apoptotic eﬀect was observed at 48 or 72 h.

The inﬂuence of the new compounds on the cell cycle was determined on HCT-8 cells treated for

24 h with 50

µg/mL of the tested compounds. Compounds 4–9 and 13, containing methyl, ethyl, or

methoxy moieties, increased the G0/G1 phase and decreased the S phase, while the other compounds

Molecules 2020, 25, 3308

13 of 27

decreased the G0/G1 phase and increased the S phase. Compounds

4–6

slightly increased the G2/M

phase, while the rest of compounds decreased this phase. N,N,N’-Triacylhydrazines

7–9 exhibited a

stronger inﬂuence on the cell cycle than the other tested compounds (Figure 5). The evaluation of the

treatment at 48 h showed a decrease of S and G2/M phases in the case of N,N,N’-triacylhydrazines

and 8, and in addition, the appearance of the subG0 peak associated with apoptosis (Figure 6).

7

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

1

2

3

4

5

6

7

8

9

10 11 12 13 INH

G2/M 10.2210.1110.5215.1115.6212.21 10.2 10.97 6.49 10.8610.36 9.92 10.6215.0113.83

44.3143.2946.4425.2527.3532.8311.3312.51 4.18 42.0940.5843.2936.9638.4839.43

M

S

G0/G1 41.7142.9541.1163.1556.06 47.9 74.9472.2388.8542.9745.0444.5849.6944.1246.43

Figure 5. The inﬂuence of the new isoniazid derivatives on cell cycle in eukaryotic cells after a

2

4 h treatment.

(

A)

Figure 6. The inﬂuence of compounds

A) compound 7; (B) compound 8; (C) control cells (HT 29).

(B)

(C)

7

and 8 on the cell cycle in eukaryotic cells after a 48-h treatment:

(

At molecular level, the cell cycle is regulated by cyclin-dependent kinases (CDKs) that are

activated by cyclins. In order to determine the inﬂuence of the tested compounds on the expression

of diﬀerent factors implicated in the regulation of the cell cycle, the expression of cyclin A, cyclin B,

CDK1 (Cyclin-dependent kinase 1), and CDC20 (cell division cycle protein 20) genes was analyzed

(

Figure 7). Compounds

induced the expression of the cyclin B gene. Compounds

gene expression and induced the expression of the CDK1 gene. Compound

inﬂuence on the expression of genes implicated in the cell cycle by slightly increasing the expression of

cyclin A, cyclin B, and CDK1 genes and slightly reducing the expression of CDC20. Compounds

, and 11 reduced the expression levels of cyclin A, cyclin B, CDK1, and CDC20 genes, indicating

4

and

5

slightly reduced cyclin A, CDK1, and CDC20 gene expression and

and 10 reduced cyclin A, cyclin B, and CDC20

proved to have a weak

2

6

1, 7,

8, 9

Molecules 2020, 25, 3308

14 of 27

an inhibition of cell cycle progression, which is in agreement with the decrease of S and G2 phases

demonstrated by ﬂow cytometry. Compounds 12, and 13 increased the evaluated genes expressions.

3,

The inﬂuence of these compounds on the gene expression of cyclin B and CDK1 could explain no

apoptotic eﬀect observed disregard increased levels of expression of caspases. CDK1/cyclin B complex

could phosphorylate procaspase 8, inhibiting its processing and, thus, the activation of apoptosis

through extrinsic pathway [62], and, also, could phosphorylate caspase 9 at an inhibitory site [63],

consequently inhibiting the intrinsic apoptotic pathway. On the other hand, CDK1/cyclin B could

also target p53, its Ser-315 phosphorylation enhancing ATP generation and membrane potential in

mitochondria [64].

Figure 7. The inﬂuence of the new isoniazid derivatives

CDK1, and CDC20 genes in the eukaryotic cells.

1–13 on the expression of cyclin A, cyclin B,

The blocking in the G1 phase observed after 24 h of treatment with some isoniazid derivatives

was previously reported in the literature [65]. The inﬂuence of these compounds on the cell cycle was

explained by their activity as Fe-chelators, determining cyclin D proteolysis and cyclin E accumulation,

and a decrease of CDK2 (Cyclin-dependent kinase 2) level [65,66].

In the current study, compounds 7–9 reduced the expression levels of genes implicated in S, G2,

and M phases of cell cycle and caused cell cycle blocking in G0/G1 phase. This eﬀect can also be

explained by their metabolism (discussed above), with presumed metabolites being capable of forming

complexes that could inactivate enzymes involved in abnormal cell division [57].

The inﬂuence of compounds

7–9 on cell cycle indicates their antitumor potential and further

studies on their mechanism of action should/will be done.

Molecules 2020, 25, 3308

15 of 27

3

. Materials and Methods

3

.1. Chemicals and Analytical Techniques

All chemicals and reagents were used as purchased from commercial suppliers (MilliporeSigma,

Burlington, MA, USA and Chimopar, Bucharest, Romania, respectively) and were of the highest

available purity. The solvents were used as absolute solvents, as well.

Analytical thin-layer chromatography (TLC) was performed on aluminium TLC plates that were

silica gel-coated with ﬂuorescent indicator F254, with size 20

×

20 cm, (Merck KGaA, Darmstadt,

Germany). The spots were visualized using a ﬁxed wavelength (254 nm) UV light.

1

13

The H( C)-NMR spectra, in deuterated solvents DMSO-d /CDCl (isotopic purity 99.9%), were

6

3

recorded on Varian Inova-400 NMR spectrometer (Agilent, Santa Clara, CA, USA, former Varian Inc.,

1

13

Palo Alto, CA, USA) of 400 MHz ( H)and 100 MHz ( C), equipped with a Varian broadband direct

(

1

H/X) probe. The standard for chemical shift is tetramethylsilane (TMS) and J-values were calculated

[ppm] are referenced to protonated solvent signals as internal standard

in Hz. Chemical shifts

δ

1

13

1

13

DMSO-d₆:

Signal multiplicities are abbreviated as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), dt

doublet of triplet), q (quadruplet), and m (multiplet) with the preﬁx b in case of broad signals [67 70].

Molecular identiﬁcation of organic functional groups in solid sample was performed by Fourier

δ = 2.50 ppm ( H), 39.52 ppm ( C), and CDCl : δ = 7.26 ppm ( H), 77.16 ppm ( C) [63–66].

3

(

–

transform infrared spectroscopy using a Vertex 80v spectrometer (Bruker, Ettlingen, Germany),

equipped with attenuated total reﬂectance (ATR) accessory. FTIR spectra were recorded in the range of

−

1

−1

4

000 to 400 cm , with 0.2 cm spectral resolution, 0.1% T accuracy, and 32 scans/spectra.

Melting points were determined on a melting point meter Krüss M5000 (A. Krüss Optronic,

◦

Hamburg, Germany), up to 400 C, and are given uncorrected.

Mass spectra were recorded on a Maxis Bruker 4G spectrometer (Bruker Corporation, Billerica,

MA, USA) with an electrospray ionization source (ESI). For the biological approach, the synthetized

compounds were initially dissolved in DMSO (i.e., 1 mg/mL) to prepare a concentrated stock solution.

Finally, additional dilutions (e.g., 1

µg/mL in methanol) were prepared in culture medium. The

scanning range of molecular ions (m/z) was 50 to 1250.

3

.2. Synthesis and Characterization of Isoniazid Derivatives

3

.2.1. N,N’-diacylhydrazines and N,N,N’-triacylhydrazines

To 5 mmol of 2-(4-substituted-phenoxymethyl)-benzoic acid dissolved in 25 mL of

1

,2-dichloroethan was added 10 mmol of thionyl chloride. The mixture was reﬂuxed for 3 h. The

excess of thionyl chloride and 1,2-dichloroethan was removed under reduced pressure. A quantity of

mmol of isoniazid dissolved in 30 mL of dichloromethane was added to the crude acid chloride and

5

the mixture was stirred overnight at room temperature. The next day, a double volume of water was

added to the reaction mixture and the solid formed was separated by ﬁltration and washed twice with

dichloromethane. After ﬁltration, the aqueous phase was extracted three times with dichloromethane

and the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under

reduced pressure and the products (Figure 8) were separated and puriﬁed on chromatographic columns

ﬁlled with silica gel using dichloromethane: ethyl acetate in gradient for elution.

Molecules 2020, 25, 3308

16 of 27

3

''

4''

5''

6''

O

2

''

7'''

2''' 3'''

4

''

5''

1''

2''

1'''

3

4 5

NH NH

6'

5'

4'''

1

''

O

R 8'''

6''

3''

3

2

4'

5

1' N

7'''

O

6

''' 5'''

4

6

'

5'

O

NH N

2'

3'

1

2

4'

2'''

1'''

1'N

O

O1

3

'''

6

'''

2

'

3'

4

''' 5'''

R

O

R

8

'''

(

A)

R

H

F

(B)

Compound

R

Compound

R

1

2

3

4

5

6

CH

3

7

8

9

CH

3

OCH

3

OCH

3

Cl

C

2

H

5

C2H

5

Figure 8. General formula of newly synthesized N,N’-diacylhydrazines (compounds

N,N,N’-triacylhydrazines (compounds 7–9) (B).

1–6) (A) and

Compound 1

N-(2-Phenoxymethyl-benzoyl)-N’-(isonicotinoyl)-hydrazine

Yield: 52%

M.p.: 128–129 C

◦

Elemental analysis: C H N O , M = 347; calcd: C = 69.16%, H = 4.9%, N = 12.1%; found:

20

17

3

C = 69.14%, H = 4.93%, N = 12.08%.

1

H-NMR (400 MHz, DMSO-d ,

δ

ppm, J Hz): 10.93 (s, 1H, H-3), 10.52 (s, 1H, H-4), 8.89 (d, 2H,

6

0

00

0

00

H-2 , H-6 , 5.9), 7.96 (d, 1H, H-3 , 7.4), 7.81(d, 2H, H-3 , H-5 , 6.0), 7.63 (t, 1H, H-5 , 7.3), 7.54 (d, 1H,

0

00

000

H-6 ,7.8), 7.30 (t, 1H, H-4 , 7.4), 7.34 (t, 2H, H-3 , H-5 , 8.0), 7.03-6.99 (m, 3H, H-2 , H-4 , H-6 ),

000

5

.21 (s, 2H, H-7 ).

13

000 0 0

C-NMR (100 MHz, DMSO-d , δ ppm): 167.3 (C-5), 164.5 (C-2), 159.0 (C-1 ), 149.7 (C-2 , C-6 ),

6

0 00 00 00 000 000 00 00

40.8 (C-4 ), 136.5 (C-1 ), 133.6 (C-2 ), 132.3 (C-5 ), 129.7 (C-3 , C-5 ), 128.5 (C-4 , 127.7 (C-3 ),

24.6 (C-6 ), 121.7 (C-3 , C-5 ), 121.0 (C-4 ), 114.3 (C-2 , C-6 ), 68.5 (C-7 ).

FT-IR (ATR in solid,

140; 815; 742.

HRMS m/z 348.1346 [M + H] (calcd for C H N O : 348.1342).

1

0

000

−

ν

cm ¹): 3199; 3030; 2915; 2468; 1672; 1607; 1588; 1547; 1510; 1455; 1282; 1240;

1

+

20

18

3

R (silicagel, AcOEt:MeOH = 9:1): 0.50.

f

Compound 2

N-[2-(p-Fluoro-phenoxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine Yield: 40%

◦

M.p.: 168–169 C

Elemental analysis: C H FN O , M = 365; calcd: C = 65.75%, H = 4.38%, N = 11.51%; found:

20

16

3

C = 65.73%, H = 4.39%, N = 11.53%.

1

H-NMR (400 MHz, DMSO-d ,

δ

0

ppm, J Hz): 10.98 (s, 1H, H-3), 10.56 (s, 1H, H-4), 8.87 (d, 2H,

6

0

00

H-2 , H-6 , 6.0), 7.95 (d, 2H, H-3 , H-5 , 5.92), 7.66 (m, 2H, H-3 , H-6 ), 7.57 (t, 1H, H-5 , 7.18), 7.51 (t,

H, H-4 , 7.41), 7.04 (d, 2H, H-2 , H-6 , 8.4), 6.9 (d, 2H, H-3 , H-5 , 8.4), 5.35 (s, 2H, H-7 ).

0

000

1

Molecules 2020, 25, 3308

17 of 27

1

3

000

C-NMR (100 MHz, DMSO-d ,

δ

ppm): 167.9 (C-5), 164.2 (C-2), 162.1 (C-4 ), 156.4 (C-1 ),

6

0

00

1

(

50.0 (C-2 , C-6 ), 140.8 (C-4 ), 136.5 (C-1 ), 133.4 (C-2 ), 131.1 (C-5 ), 129.5 (C-4 ), 127.5 (C-3 ), 127.3

00 0 0 000 000 000 000 000

C-6 ), 122.2 (C-3 , C-5 ), 116.2 (C-3 , C-5 ), 114.5 (C-2 , C-6 ), 66.5 (C-7 ).

−

1

FT-IR (ATR in solid,

28; 739.

HRMS m/z 366.1248 [M + H] (calcd for C H FN O : 366.1248).

ν cm ): 3242; 3037; 2923; 2451; 1689; 1598; 1581; 1524; 1443; 1237; 1134; 1114;

8

+

20

17

3

R (silicagel, AcOEt:MeOH = 9:1): 0.57.

f

Compound 3

N-[2-(p-Chloro-phenoxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine Yield: 25%

◦

M.p.: 145–146 C

Elemental analysis: C H ClN O , M = 381.5; calcd: C = 62.91%, H = 4.19%, N = 11.01%; found:

20

16

3

C = 62.89%, H = 4.16%, N = 11.03%.

1

H-NMR (400 MHz, DMSO-d ,

δ ppm, J Hz): 11.2 (s, 1H, H-3), 10.96 (s, 1H, H-4), 9.12 (d, 2H,

6

0

00

H-2 , H-6 , 5.95), 8.34 (d, 2H, H-3 , H-5 , 5.8), 7.99 (m, 2H, H-3 , H-6 ), 7.81 (t, 1H, H-5 , 7.0), 7.76 (t,

00 000 000 000 000 000

1

H, H-4 , 7.32), 7.5 (d, 2H, H-3 , H-5 , 8.0), 6.70 (d, 2H, H-2 , H-6 , 8.1), 5.74 (s, 2H, H-7 ).

13

000 0 0

C-NMR (100 MHz, DMSO-d , δ ppm): 167.9 (C-5), 164.4 (C-2), 156.6 (C-1 ), 150.1 (C-2 , C-6 ),

6

0 00 00 00 000 00 000 000

40.7 (C-4 ), 136.6 (C-1 ), 133.7 (C-2 ), 131.4 (C-5 ), 125.1 (C-4 ), 129.9 (C-4 ), 129.2 (C-3 , C-5 ),

28.2 (C-3 ), 127.8 (C-6 ), 122.2 (C-3 , C-5 ), 115.4 (C-2 , C-6 ), 67.1 (C-7 ).

FT-IR (ATR in solid,

41; 713.

HRMS m/z 382.0954 [M + H] (calcd for C H ClN O : 382.0952).

1

0

00

0

000

−1

ν cm ): 3215; 3037; 2968; 1690; 1598; 1573; 1510; 1452; 1247; 1218; 1139; 826;

7

+

20

17

3

R (silicagel, AcOEt:MeOH = 9:1): 0.56.

f

Compound 4

N-[2-(p-Tolyloxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine Yield: 58%

◦

M.p.: 173–175 C

Elemental analysis: C H N O , M = 361; calcd: C = 69.81%, H = 5.26%, N = 11.63%; found:

21

19

3

C = 69.83%, H = 5.25%, N = 11.65%.

1

H-NMR (400 MHz, DMSO-d ,

δ

0

000

ppm, J Hz): 10.96 (s, 1H, H-3), 10.55 (s, 1H, H-4), 8.84 (d, 2H,

6

0

00

H-2 , H-6 , 6.05), 7.91 (d, 2H, H-3 , H-5 , 5.86), 7.62 (m, 2H, H-3 , H-6 ), 7.55 (t, 1H, H-5 , 7.22), 7.46 (t,

H, H-4 , 7.42), 7.08 (d, 2H, H-3 , H-5 , 8.2), 6.90 (d, 2H, H-2 , H-6 , 8.39), 5.30 (s, 2H, H-7 ), 2.22

0

000

1

(

000

s, 3H, H-8 ).

13

000 0 0

C-NMR (100 MHz, DMSO-d , δ ppm): 167.5 (C-5), 163.9 (C-2), 156.1 (C-1 ), 149.6 (C-2 , C-6 ),

6

0 00 00 00 000 00 000 000

40.2 (C-4 ), 136.0 (C-1 ), 133.0 (C-2 ), 130.5 (C-5 ), 129.8 (C-4 ), 129.5 (C-4 ), 127.8 (C-3 , C-5 ),

27.6 (C-3 ), 127.5 (C-6 ), 121.8 (C-3 , C-5 ), 114.6 (C-2 , C-6 ), 66.5 (C-7 ), 20.0 (C-8 ).

FT-IR (ATR in solid,

140; 811; 739.

MS m/z 362.1023 [M + H] (calcd for C H N O : 362.4018).

1

0

00

0

000

−

1

ν cm ): 3191; 3027; 2919; 2461; 1669; 1607; 1590; 1551; 1510; 1455; 1291; 1244;

1

+

21

20

3

R (silicagel, AcOEt:CH Cl = 1:1):0.16.

f

2

Compound 5

N-[2-(p-Methoxy-phenoxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine Yield: 54%

◦

M.p.: 108–111 C

Elemental analysis: C H N O , M = 377; calcd: C = 66.84%, H = 5.04%, N = 11.17%; found:

21

19

3

4

C = 66.82%, H = 5.02%, N = 11.17%.

1

0

H-NMR (400 MHz, CDCl ,

δ

ppm, J Hz): 10.11 (s, 1H, H-3), 9.72 (s, 1H, H-4), 8.63 (d, 2H, H-2 ,

3

0

00

0

00

H-6 , 5.5), 7.74 (d, 1H, H-3 , 7.62), 7.62 (d, 2H, H-3 , H-5 , 5.67), 7.52 (m, 2H, H-5 , H-6 ), 7.42 (t, 1H,

Molecules 2020, 25, 3308

18 of 27

0

000

H-4 , 6.64), 6.93 (d, 2H, H-3 , H-5 , 8.98), 6.78 (d, 2H, H-2 , H-6 , 8.98), 5.22 (s, 2H, H-7 ), 3.73 (s,

000

3

H, H-8 ).

1

3

000 000

C-NMR (100 MHz, CDCl , δ ppm): 166.5 (C-5), 162.8 (C-2), 154.7 (C-1 ), 152.2 (C-4 ), 150.6

3

0 0 0 00 00 00 00 00

C-2 , C-6 ), 138.5 (C-4 ), 135.7 (C-1 ), 132.1 (C-2 ), 131.8 (C-3 ), 130.4 (C-5 ), 129.0 (C-4 ), 128.7

(

0

000

C-6 ), 121.0 (C-3 , C-5 ), 116.61 (C-3 , C-5 ), 114.82 (C-2 , C-5 ), 69.55 (C-7 ), 55.72 (C-8 ).

−

1

FT-IR (ATR in solid,

30; 743.

MS m/z 378.0935 [M + H] (calcd for C H N O : 378.4012).

ν cm ): 3199; 3066; 2993; 1683; 1592; 1576; 1550; 1452; 1242; 1213; 1140; 1038;

8

+

21

20

3

4

R (silicagel, AcOEt:CH Cl = 1:1): 0.13.

f

2

Compound 6

N-[2-(p-Ethyl-phenoxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine Yield: 33%

◦

M.p.: 133–135 C

Elemental analysis: C H N O , M = 375; calcd: C = 70.40%, H = 5.60%, N = 11.20%; found:

22

21

3

C = 70.36%, H = 5.62%, N = 11.23%.

1

H-NMR (400 MHz, DMSO-d ,

δ

ppm, J Hz): 10.90 (s, 1H, H-3), 10.53 (s, 1H, H-4), 8.78 (d, 2H,

6

0

00

H-2 , H-6 , 3.91), 7.83 (d, 2H, H-3 , H-5 , 4.30), 7.60 (m, 2H, H-3 , H-6 ), 7.53 (t, 1H, H-5 ,7.23), 7.43

00 000 000 000 000 000

(

2

t, 1H, H-4 , 7.22), 7.09 (d, 2H, H-3 , H-5 , 8.01), 6.90 (d, 2H, H-2 , H-6 , 8.01), 5.29 (s, 2H, H-7 ),

000 000

.50 (m, 2H, overlapping with DMSO-d6 signal, H-8 ), 1.11 (t, 3H, H-9 , 7.42).

1

3

000 0 0

C-NMR (100 MHz, DMSO-d , δ ppm): 167.6 (C-5), 164.2 (C-2), 156.3 (C-1 ), 150.5 (C-2 , C-6 ),

6

0 00 000 00 00 00 000 000

39.4 (C-4 ), 136.1 (C-1 ), 136.0 (C-4 ), 133.1 (C-2 ), 130.6 (C-5 ), 128.7 (C-4 ), 127.9 (C-3 , C-5 ),

27.6 (C-3 ), 127.6 (C-6 ), 121.4 (C-3 , C-5 ), 114.7 (C-2 , C-2 ), 66.5 (C-7 ), 27.3 (C-8 ), 15.9 (C-9 ).

FT-IR (ATR in solid,

27; 736.

MS m/z 376.0836 [M + H] (calcd for C H N O : 376.3283).

1

0

00

0

000

−

1

ν cm ): 3265; 3030; 2961; 2926; 2870; 1690; 1608; 1581; 1509; 1453; 1218; 1140;

8

+

22

3

R (silicagel, AcOEt:CH Cl = 1:1): 0.17.

f

2

Compound 7

N,N-di[2-(p-Tolyloxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine

Yield: 5%

M.p.: semisolid

Elemental analysis: C H N O , M = 585; calcd: C = 73.85%, H = 5.30%, N = 7.18%; found:

36

31

3

5

C = 73.82%, H = 5.28%, N = 7.19%.

1

0 0

δ ppm, J Hz): 11.85 (s, 1H, H-3), 8.77 (d, 2H, H-2 , H-6 , 5.86), 7.71

H-NMR (400 MHz, DMSO-d ,

6

0

00

(

d, 2H, H-3 , 7.42), 7.57 (m, 4H, H-3 , H-5 , H-6 ), 7.50 (t, 2H, H-5 , 7.20), 7.38 (t, 2H, H-4 , 6.83), 7.04

d, 4H, H-5 , H-3 , 8.59), 6.87 (d, 4H, H-2 , H-6 , 8.40), 5.20 (s, 4H, H-7 ), 2.21 (s, 6H, H-8 ).

0

00

000

1

3

000 0 0

C-NMR (400 MHz, DMSO-d , δ ppm): 169.8(C-5), 164.6 (C-2), 155.9 (C-1 ), 150.5 (C-2 , C-6 ),

6

0 00 00 00 000 00 0 0

1

(

38.2 (C-4 ), 136.1 (C-1 ), 132.6 (C-2 ), 131.3 (C-5 ), 129.7 (C-4 ), 129.5 (C-4 ), 127.9 (C-3 , C-5 ), 127.4

0

00

000

C-3 ), 127.2 (C-6 ), 121.0 (C-3 , C-5 ), 114.6 (C-2 , C-6 ), 66.4 (C-7 ), 20.0 (C-8 ).

−

1

FT-IR (ATR in solid, ν cm ): 3271; 3030; 2923; 1689; 1609; 1583; 1508; 1217; 1140; 811; 739.

MS m/z 586.2167 [M + H] (calcd for C H N O : 586.5563).

+

36

32

3

5

R (silicagel, AcOEt:CH Cl = 1:1): 0.60.

f

2

Compound 8

N,N-di[2-(p-Methoxy-phenoxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine

Yield: 2%

M.p.: semisolid

Molecules 2020, 25, 3308

19 of 27

Elemental analysis: C H N O , M = 617; calcd: C = 70.02%, H = 5.02%, N = 6.81%; found:

36

31

3

7

C = 70.05%, H = 5.00%, N = 6.79%.

1

0 0

δ ppm, J Hz): 8.62 (d, 2H, H-2 , H-6 , 5.47), 8.53 (s, 1H, H-3), 7.69 (d,

H-NMR (400 MHz, CDCl ,

3

00 00 00 00

H, H-3 , 7.81), 7.55 (d, 2H, H-6 , 7.62), 7.45 (t, 2H, H-5 , 7.48), 7.33 (t, 2H, H-4 , 7.61), 7.15 (d, 2H,

H-3 , H-5 , 5.86), 6.88 (d, 4H, H-3 , H-5 , 9.18), 6.78 (d, 4H, H-2 , H-6 , 8.98), 5.19 (s, 4H, H-7 ),

1

0

000

3

.72 (s, 6H, H-8 ).

13

000 000

C-NMR (100 MHz, CDCl , δ ppm): 170.4 (C-5), 164.4 (C-2), 154.4 (C-1 ), 152.4 (C-4 ), 150.5

3

0 0 0 00 00 00 00 00 00

(

1

C-2 , C-6 ), 138.7(C-4 ), 135.5 (C-1 ), 133.5 (C-2 ), 131.4 (C-5 ), 131.1 (C-4 ), 128.7 (C-3 ), 126.7 (C-6 ),

0 0 000 000 000 000 000 000

20.8 (C-3 , C-5 ), 116.8 (C-3 , C-5 ), 114.9 (C-2 , C-6 ), 68.6 (C-7 ), 55.7 (C-8 ).

−

1

FT-IR (ATR in solid, ν cm ): 3267; 3065; 2932; 1688; 1556; 1503; 1463; 1207; 1139; 1032; 821; 737.

MS m/z 618.2467 [M + H] (calcd for C H N O : 618.5551).

+

36

32

3

7

R (silicagel, AcOEt:CH Cl = 1:1): 0.52.

f

2

Compound 9

N,N-di[2-(p-Ethyl-phenoxymethyl)-benzoyl]-N’-(isonicotinoyl)-hydrazine

Yield: 6%

M.p.: semisolid

Elemental analysis: C H N O , M = 613; calcd: C = 74.39%, H = 5.71%, N = 6.85%; found:

38

35

3

5

C = 74.37%, H = 5.69%, N = 6.87%.

1

0 0

δ ppm, J Hz): 11.97 (s, 1H, H-3), 8.77 (d, 2H, H-2 , H-6 , 4.69), 7.73 (d, 2H,

H-NMR (DMSO,

0

00

0

00

H-3 , 7.62), 7.58 (m, 4H, H-3 , H-5 , H-6 ), 7.50 (t, 2H, H-5 , 7.42), 7.37 (t, 2H, H-4 , 7.03), 7.07 (d, 4H,

000

H-3 , H-5 , 8.01), 6.90 (d, 4H, H-2 , H-6 , 8.20), 5.21 (s, 4H, H-7 ), 2.50 (m, 4H, overlapping with

0

00

DMSO-d6 signal, H-8 ), 1.12 (t, 6H, H-9 , 7.43).

13

000 0 0 0

C-NMR (DMSO, δ ppm): 169.9 (C-5), 164.7 (C-2), 156.1 (C-1 ), 150.5 (C-2 , C-6 ), 138.5 (C-4 ),

00 000 00 00 00 000 000 00

36.1 (C-1 ), 132.7 (C-4 ), 131.2 (C-2 ), 129.6 (C-5 ), 128.6 (C-4 ), 128.0 (C-3 , C-5 ), 127.4 (C-3 ),

27.2 (C-6 ), 121.1 (C-3 , C-5 ), 114.6 (C-2 , C-6 ), 66.4 (C-7 ), 27.3 (C-8 ), 15.8 (C-9 ).

FT-IR (ATR in solid,

31; 748.

MS m/z 614.0954 [M + H] (calcd for C H N O : 614.3095).

1

0

000

−

1

ν cm ): 3231; 3022; 2958; 2923; 1674; 1628; 1599; 1554; 1507; 1476; 1244; 1134;

8

3

+

38

36

3

5

R (silicagel, AcOEt:CH Cl = 1:1): 0.63.

f

2

.2.2. 1,3,4-Oxadiazoles Synthesis

A quantity of 1 mmol N,N’-diacylhydrazine was suspended in 15 mL toluene. After 20 min of

stirring, 5 mmol phosphoryl chloride was added dropwise. The reaction mixture was reﬂuxed for

h and stirred overnight at room temperature. The solvent and residual phosphoryl chloride were

6

removed under reduced pressure. The residue was taken up in dichloromethane and washed three

times with 5% NaOH solution and three times with saturated aqueous NaCl solution. The organic

layer was dried over anhydrous sodium sulfate and distilled under reduced pressure to remove the

solvent. The ﬁnal compound (Figure 9) was recrystallized using ethanol.

Molecules 2020, 25, 3308

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4

3

N

Comp.

R

5'

3''

6'

4'

4''

5''

5

2

2''

O

1

' N

1

0

H

F

3

'

1"

2'

7'''

6''

2

'''

O

11

12

1'''

3

'''

6'''

4'''

Cl

R

5'''

8

'''

1

3

CH

3

Figure 9. General formula of newly synthesized 1,3,4-oxadiazoles (compounds 10–13).

Compound 10

2

2-[2-(Phenoxymethyl)-phenyl]-5-(pyridine-4-yl)-1,3,4-oxadiazole

Yield: 62%

◦

M.p.: 210–211 C

Elemental analysis: C H N O , M = 329; calcd: C = 72.95%, H = 4.56%, N = 12.77%; found:

20

15

3

2

C = 72.97%, H = 4.58%, N = 12.74%.

1

0 0 00

δ ppm, J Hz): 8.87 (d, 2H, H-2 , H-6 , 4.00), 8.24 (d, 1H, H-3 ,

H-NMR (400 MHz, DMSO-d6,

0 0 00 00 00

.00), 8.05 (d, 2H, H-3 , H-5 , 4.00), 7.80 (d, 1H, H-6 , 8.00), 7.72 (t, 1H, H-5 , 7.99), 7.62 (t, 1H, H-4 ,

.99), 7.29 (d, 2H, H-3 , H-5 , 8.00), 7.02-6.93 (m, 3H, H-2 , H-4 , H-6 ), 5.58 (s, 2H, H-7 ).

8

7

0

00

000

1

3

000 0 0

C-NMR (100 MHz, DMSO-d6, δ ppm): 164.8 (C-5), 162.1(C-2), 158.2(C-1 ), 149.2 (C-2 , C-6 ),

0 00 00 00 000 000 00 00

36.7 (C-4 ), 132.3 (C-5 ), 132.0 (C-2 ), 130.4 (C-4 ), 129.6 (C-3 , C-5 ), 129.2 (C-6 ), 128.6 (C-3 ),

21.4 (C-1 ),121.1 (C-4 ), 120.0 (C-3 , C5 ), 114.5 (C-2 , C-6 ), 67.6 (C-7 ).

FT-IR (ATR in solid,

24; 742.

HRMS m/z 330.1237 [M + H] (calcd for C H N O : 330.1237).

1

0

000

0

000

cm ¹): 3030; 2964; 2815; 2160; 1604; 1574; 1552; 1503; 1452; 1232; 1125; 1015;

−

ν

8

+

20

16

3

2

R (silicagel, AcOEt:MeOH = 9:1): 0.72.

f

Compound 11

2

2-[2-(p-Fluoro-phenoxymethyl)-phenyl]-5-(pyridine-4-yl)-1,3,4-oxadiazole

Yield: 65%

M.p.: 298–299 C

◦

Elemental analysis: C H FN O , M = 347; calcd: C = 69.16%, H = 4.03%, N = 12.1%; found:

20

14

3

2

C = 69.15%, H = 4.05%, N = 12.12%.

1

0 0 00

δ ppm, J Hz): 8.74 (d, 2H, H-2 , H-6 , 4.68), 8.21 (d, 1H, H-3 , 7.3),

H-NMR (400 MHz, DMSO-d6,

0 0 00 00 00

.84 (d, 2H, H-3 , H-5 , 5.7), 7.75 (d, 1H, H-6 , 7.42), 7.67 (t, 1H, H-5 , 6.6), 7.57 (t, 1H, H-4 , 7.6), 6.87

7

(

000 000 000 000 000

d, 2H, H-3 , H-5 , 8.21), 7.03 (d, 2H, H-2 , H-6 , 8.2), 5.51 (s, 2H, H-7 ).

13

000 000

δ ppm): 164.9 (C-5), 162.8 (C-2), 156.6 (C-1 ), 152.1 (C-4 ),

C-NMR (100 MHz, DMSO-d6,

0 0 0 00 00 00 00 00

51.3 (C-2 , C-6 ), 137.2 (C-4 ), 132.5 (C-5 ), 131.0 (C-2 ), 129.9 (C-1 ), 129.8 (C-3 ), 129.5 (C-6 ), 129.0

1

(

0

000

C-4 ), 120.7 (C-3 , C5 ), 115.2 (C-3 , C-5 ), 114.9 (C-2 , C-6 ), 68.3 (C-7 ).

−

1

FT-IR (ATR in solid,

41; 739.

HRMS m/z 348.1145 [M + H] (calcd for C H FN O : 348.1142).

ν cm ): 3029; 2974; 2844; 2163; 1608; 1581; 1508; 1455; 1241; 1124; 1114; 1015;

8

+

20

15

3

2

R (silicagel, AcOEt:MeOH = 9:1): 0.79.

f

Molecules 2020, 25, 3308

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Compound 12

2

2-[2-(p-Chloro-phenoxymethyl)-phenyl]-5-(pyridine-4-yl)-1,3,4-oxadiazole

Yield: 68%

M.p.: 341–342 C

◦

Elemental analysis: C H ClN O , M = 363.5; calcd: C = 66.02%, H = 3.85%, N = 11.55%; found:

20

14

3

2

C = 66.01%, H = 3.87%, N = 11.57%.

1

0 0 00

δ ppm, J Hz): 8.71 (d, 2H, H-2 , H-6 , 4.68), 8.14 (d, 1H, H-3 , 7.3),

H-NMR (400 MHz, DMSO-d6,

0 0 00 00 00

.81 (d, 2H, H-3 , H-5 , 5.82), 7.73 (d, 1H, H-6 , 7.35), 7.64 (t, 1H, H-5 , 6.65), 7.58 (t, 1H, H-4 , 7.69),

7

0

00

000

.52 (d, 2H, H-3 , H-5 , 8.39), 6.74 (d, 2H, H-2 , H-6 , 8.39), 5.49 (s, 2H, H-7 ).

13

000 0 0

C-NMR (100 MHz, DMSO-d6,

δ ppm): 164.1 (C-5), 161.9 (C-2), 155.9 (C-1 ), 150.3 (C-2 , C-6 ),

0 00 00 00 00 00 000 000

36.5 (C-4 ), 132.0 (C-5 ), 130.4 (C-2 ), 129.4 (C-1 ), 129.2 (C-3 ), 129.0 (C-6 ), 128.7 (C-3 , C-5 ),

1

0

000

0

000

28.1 (C-4 ), 124.8(C-4 ), 120.0 (C-3 , C5 ), 114.8 (C-2 , C-6 ), 67.7 (C-7 ).

−1

FT-IR (ATR in solid,

45; 713.

HRMS m/z 364.0837 [M + H] (calcd for C H ClN O : 364.2827).

ν cm ): 3030; 2991; 2843; 2159; 1604; 1582; 1551; 1510; 1455; 1218; 1125; 840;

7

+

20

15

3

2

R (silicagel, AcOEt:MeOH = 9:1): 0.75.

f

Compound 13

2

2-[2-(p-Tolyloxymethyl)-phenyl]-5-(pyridine-4-yl)-1,3,4-oxadiazole

Yield: 73%

M.p.: 249–250 C

◦

Elemental analysis: C H N O , M = 343; calcd: C = 73.47%, H = 4.96%, N = 12.24%; found:

21

17

3

2

C = 73.45%, H = 4.93%, N = 12.27%.

1

0 0 00

H-NMR (400 MHz, DMSO-d6, δ ppm, J Hz): 8.79 (d, 2H, H-2 , H-6 , 4.71), 8.22 (d, 1H, H-3 , 7.29),

0 0 00 00 00

.89 (d, 2H, H-3 , H-5 , 5.73), 7.78 (d, 1H, H-6 , 7.40), 7.70 (t, 1H, H-5 , 6.63), 7.62 (t, 1H, H-4 , 7.69),

7

0

00

000

.08 (d, 2H, H-3 , H-5 , 8.35), 6.87 (d, 2H, H-2 , H-6 , 8.44), 5.53 (s, 2H, H-7 ), 2.21 (s, 3H, H-8 ).

13

000 0 0

C-NMR (100 MHz, DMSO-d6,

δ ppm): 164.5 (C-5), 162.4 (C-2), 156.1 (C-1 ), 150.8 (C-2 , C-6 ),

0 00 00 000 000 00 00 00

36.8 (C-4 ), 132.2 (C-5 ), 130.4 (C-2 ), 129.9 (C-3 , C-5 ), 129.6 (C-1 ), 129.5 (C-3 ), 129.1 (C-6 ),

28.5 (C-4 ), 121.4 (C-4 ), 120.2 (C-3 , C5 ), 114.5 (C-2 , C-6 ), 67.7 (C-7 ), 20.1 (C-8 ).

FT-IR (ATR in solid,

10; 735.

HRMS m/z 344.1394 [M + H] (calcd for C H N O : 344.3465).

1

0

000

0

000

−1

ν cm ): 3028; 2924; 2870; 2158; 1608; 1587; 1550; 1509; 1457; 1240; 1125;

8

+

21

18

3

2

R (silicagel, CH Cl :MeOH = 5:1): 0.60.

f

2

3

.3. Anti-Mycobacterial Activity

The M. tuberculosis clinical strain used for anti-mycobacterial assay was maintained on Middlebrook

7

H9 broth. The anti-mycobacterial activity was evaluated by the microplate Alamar Blue assay (MABA)

as stated by Franzblau et al. [71]. We used serial dilutions of the new synthesized compounds and

isoniazid, the ﬁnal drug concentrations being between 25

M. tuberculosis inoculum and tested compounds were mixed and incubated at 37 C for eight days in

µ

g/mL to 0.012

µ

g/mL. Equal volumes of

◦

9

6-well plates covered and sealed with paraﬁlm. On day nine, a volume of 10 µL of freshly prepared

mixture (1:1) of Alamar Blue reagent (0.1 mg/mL) and tween 80 (10%) was added to each well and kept

◦

at 37 C for another 24 h. A blue color in the well was interpreted as no bacterial growth, and a pink

color was scored as growth. The MIC (Minimal Inhibition Concentration) was deﬁned as the lowest

drug concentration, which prevented a color change from blue to pink.

Molecules 2020, 25, 3308

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3

.4. Antibacterial Activity

The antimicrobial activity of the newly synthesized compounds was determined according to

Matei et al. [51], using the following strains: Acinetobacter baumanii, Citrobacter koseri, Escherichia coli,

Enterobacter cloacae, Enterococcus faecalis, Klebsiella pneumonia, Morganella morganii, Pseudomonas aeruginosa,

Peudomonas putida, Salmonella typhimurium, Serratia marcescens, Shigella sonnei, Stenotrophomonas

maltophilia, Staphylococcus hominis, Staphylococcus aureus, and coagulase-negative staphylococci, using

an adapted agar disk diﬀusion technique. Brieﬂy, bacterial suspensions of 0.5 McFarland density

obtained from 24 h cultures were seed on solid medium. Subsequent, a volume of 5 µL of tested

compounds (1 mg/mL solutions) were spotted on inoculated plates. Anti-bacterial activity was assessed

◦

after incubation for 24 h at 37 C.

3

.5. Cell Culture

Two cell lines, HCT-8 (CCL-224) and HT-29 (ATCC-HTB-38), were used to evaluate the activity of

0

new compounds on eukaryotic cells. These cells were maintained as adherent cultures in Dulbecco s

Modiﬁed Essential Medium (DMEM) (Sigma, Ronkonkoma, NY, USA) supplemented with 10%

◦

heat-inactivated fetal bovine serum (Sigma, USA) at 37 C, 5% CO , in a humid atmosphere.

2

3

.6. Apoptosis Detection

The apoptotic eﬀect of tested compounds was evaluated using Annexin V-FITC Apoptosis

Detection Kit I (BD Bioscience Pharmingen, USA) according to manufacturer protocol. In this regard,

6

HT-29 cells (3

×

10 ), were seeded in 3.5 cm diameter wells and treated with 50

µ

g/mL of tested

L of binding buﬀer and stained with 5

L propidium iodide for 10 min in dark. At least 10,000 events from each

sample were acquired using a Beckman Coulter ﬂow cytometer. The analysis was done using FlowJo v

compounds. After 24 h, the cells were resuspended in 100

Annexin V-FITC and 5

µ

µL

µ

8

.8.6 software (Ashland, OR, USA).

3

.7. Cell Cycle Analysis

6

To assess the inﬂuence of new compounds on cell cycle, a number of 3

×

10 HT-29 cells were

treated with compounds at 50

µ

g/mL concentration for 24 h. Treated cells were harvested, washed in a

◦

cold phosphate-buﬀered saline solution (PBS), then ﬁxed in cold ethanol (70%) and stored at

After 24 h, the cells were washed with PBS, resuspended in 100

A and labeled with 100 g/mL propidium iodide in the dark at room temperature for 30 min. DNA

−

20 C.

µ

l PBS, treated with 1 mg/mL RNase

µ

content of cells was quantiﬁed on a Beckman Coulter EPICS XL ﬂow cytometer and analyzed using

FlowJo 8.8.6 software (Ashland, OR, USA).

3

.8. Quantitative RT-PCR for Analysis of Genes Expression

6

A number of 3

concentration of 50

USA) as indicated in the protocol. For each sample, 2

High Capacity cDNA Reverse Transcription Kit with RNAse inhibitor (Applied Biosystem). Following,

0 ng of cDNA from each sample was used in real-time PCR reaction that was performed on an

×

10 HT-29 or HCT-8 cells treated with isoniazid and compounds

1

–

13 at ﬁnal

µ

g/mL for 24 h were used to extract total RNA with Trizol Reagent (Invitrogen,

µg of total RNA was reverse transcripted using

5

ABI 7300 Real Time PCR System. Gene expression was evaluated using pre-validated Taqman Gene

Expression Assays (Applied Biosystems) for NAT1 (Hs00265080_s1), NAT2 (Hs01854954_s1), BAX

(

Hs00180269_m1), MCL1 (Hs00172036_m1), BCL2 (Hs00153350_m1), CASP3 (Hs00234387_m1), CASP7

Hs00169152_m1), CASP8 (Hs00154256_m1), CASP9 (Hs00154261_m1), CCNB1 (Hs00259126_m1),

CDK1 (Hs00938777_m1), CCNA2 (Hs00153138_m1), CDC20 (Hs00426680_mH) as well as human beta

actin (endogenous control). Also, for CYP1A1, CYP2C19, CYP3A4 and GAPDH (endogenous control),

forward and reverse primers were used as described by Matei et al. [51]. Results were analyzed with

RQ study software (Applied Biosystems) applying ∆∆C method.

T

Molecules 2020, 25, 3308

23 of 27

4

. Conclusions

Starting from isoniazid, six new N,N’-diacylhydrazines were obtained, together with three new

N,N,N’-triacylhydrazines ones; also, the N,N’-diacylhydrazines were converted by synthesis in the

corresponding new oxadiazoles. All the compounds were characterized by IR, NMR, MS, and elemental

analysis. The newly obtained derivatives of isoniazid exhibit diﬀerent biological activities, depending

on their structure. The best antioxidant and antibacterial activities (including anti-Mycobacterium

tuberculosis eﬀects) were proved by compound

G0/G1 phase. Moreover, compound proved to be the most toxic, inducing apoptosis in 54% of cells

after 72 h, an eﬀect that can be predicted by the increased expression of mRNA caspase 3 and 7 after a

4 h treatment. The inﬂuence of tested compounds on gene expression of some enzymes implicated in

9. Compounds 7–9 determined cell cycle blocking in

7

2

drug metabolism indicate that newly synthesized compounds could be metabolized on other pathways

than NAT2, spanning adverse eﬀects of isoniazid.

In summary, compound

agent, and, along with compounds

9

had the best antibacterial activity and could be used as disinfectant

and , seemed to have antitumor potential. Further studies of

7

8

action mechanism of these compounds on cell cycle may bring some new signiﬁcant information

regarding their activity potential.

Author Contributions: Conceptualization, I.Z., L.M., P.I., C.B., and C.L.; methodology, I.Z., L.M., C.B., P.I., A.T.,

A.P., and I.N.; software, I.N. and A.H.; validation, P.I., A.T., and I.Z.; investigation, L.M., C.B., P.I., A.T., A.P., I.N.,

A.H., C.C.D., C.L., D.C.N., R.M.N., and I.Z.; resources, A.T., C.C.D. and C.L.; data curation, L.M., C.B., and I.Z.;

writing—original draft preparation, I.Z., L.M., and C.R.; writing—review and editing, I.Z., L.M., C.B., C.C.D.,

and C.R.; visualization, I.Z. and P.I.; supervision, I.Z.; project administration, C.R.; funding acquisition, C.R. All

authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by projects: CNFIS-FDI-0397 Performan ¸t ă s¸ i excelen ¸t ă în cercetarea

multidisciplinară (PerExcel) (Performance and excellence in multidisciplinary research) 2020 and Competitiveness

Operational Programme (COP) A1.1.4. ID: P_37_798 MyeloAL-EDiaProT, Contract 149/26.10.2016, (SMIS: 106774),

MyeloAL Project.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

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Synthesis, characterization, and biologic activity of new acyl hydrazides and 1,3,4-oxadiazole derivatives

DOI: 10.3390/molecules25143308

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