Synthesis of Hydrazones from Amino Acids and their Antimicrobial and Cytotoxic Activities

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Article

Synthesis of Hydrazones from Amino Acids and their Antimicrobial and Cytotoxic

Activities

a

b

c

*

Obaid-ur-Rahman Abid , Ghamama Khatoon, Muhammad Arfan, Imran Sajid,

Peter Langer,^dWajid Rehman,^a,eFazal Rahim,^aMuhammad Yasir,^aMuhammad Waqar^a

and Kashif Syed Haleem^f

^aDepartment of Chemistry, Hazara University, Mansehra, Pakistan

^bDepartment of Chemistry, School of Natural Sciences, National University of Sciences and Technology, Islamabad,

Pakistan

^cDepartment of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan

^dInstitut für Chemie, Universität Rostock, Rostock, Germany

^eTechnical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190 China

^fDepartment of Microbiology, Hazara University, Mansehra, Pakistan

(Received: December 10, 2016; Accepted: June 7, 2017; DOI: 10.1002/jccs.201600859)

Hydrazones 6a–6n were synthesized from different amino acids with various aldehydes under

reﬂux in methanol/ethanol. The structures of synthesized compounds were ascertained by elemen-

tal analysis and spectroscopic techniques. A comparative study of the antimicrobial activity and

cytotoxicity was carried out of the N-protected amino acids, their esters, hydrazides, and the

respective hydrazones, providing good results in cytotoxicity studies.

Keywords: Hydrazones; Cytotoxicity; Amino acids; Antimicrobial.

INTRODUCTION

Hydrazones are potential candidates for drug

synthesis is the best strategy for this purpose. Generally,

benzamide derivatives are known for their medicinal

importance such as a muscle relaxant as well as antemetic,

antipsychotic, and antiarrahythmic agents.^24,25Recently,

hydrazone derivatives of benzoyl-protected glycine have

been reported for their anticonvulsant activities.²⁶

Determination of the biological activity of synthetic

and biological compounds has played a great role in drug

design during the last few years. Antimicrobial activity

screening is important in drug development, which in turn

is helpful in curing human and animal diseases. Keeping

in mind the signiﬁcance of hydrazones and their amino

acid derivatives in relation to their biological activities,

this paper reports the synthesis of hydrazones from

N-benzoylated amino acids and different substituted alde-

hydes and their biological evaluation.

development because of their reactive azomethine

group (−CO–NH–N CH–).¹Many researchers in the

organic chemistry and medicinal ﬁelds have focused on

the study of such molecules for years because of their

important applications. Hyrdrazones have been found

to exhibit diverse biological properties including

antiviral,²antifungal,³antibacterial,⁴antimalarial,⁵

antitubercular,^6,7

inﬂammatory,⁸

antiplatelet,⁹

anticonvulsant,¹⁰antitumor,¹¹and anticancer activ-

ities.^12,13Among the signiﬁcant pharmacophores in case

of antiinﬂammatory and antiplatelet drug structures are

hydrazine and acyl hydrazine moieties.^9,14Hydrazones

with the cyanoacetyl group have been reported for their

antitumor activities.^15–17

Amino acids have been used in the synthesis of Schiff

bases and their complexes. Such moieties have shown

promising results in their application studies such as ﬂuo-

rescent properties¹⁸and biological activities over a wide

range.^19–23Hydrazones from N-protected amino acids are

less reported. Protection via sulfonamide or benzamide

RESULTS AND DISCUSSION

Chemistry

Amino acids (1a–1c) were converted to N-

benzoylated amino acids by stirring their solutions in

NaOH (10%) with benzoyl chloride for 3–4 h. A solid

*Corresponding author. Email: obaid_chem@yahoo.com

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1

Article

Abid et al.

material appeared in solution, to which ice and hydro-

chloric acid (dil + cold) were added till the pH reached

3. After ﬁltration and washing with cold distilled water,

the benzoyl-protected amino acid was dried. IR spectro-

scopic data indicated the completion of protection of

the amino group by the disappearance of two separate

bands for the NH₂asymmetric and symmetric stretch-

ing vibrations, whereas only one band for NH stretch-

ing appeared in the range 3365–3296 cm⁻¹. Appearance

of amidic carbonyl bands in the range 1692–1646 cm⁻¹

and aromatic C C bands in the range 1578–1542 cm⁻¹

also indicated the successful protection.

Each protected amino acid (2a–2c) was dissolved

separately in dry methanol, a few drops of sulfuric acid

were added, and reaction was reﬂuxed for 10–19 h. The

solvent was removed using a rotary evaporator. The

remaining material was then worked up. IR spectro-

scopic data indicated the formation of the ester by the

disappearance of broad band due to the OH group cen-

tered near 3000 cm⁻¹. The appearance of ester carbonyl

band in the range 1742–1718 cm⁻¹indicated the suc-

cessful esteriﬁcation.

indicated the synthesis of the hydrazide. Each hydrazide

(4a–4c) was dissolved in 5 mL of ethanol by stirring,

and an equivalent mmol of the aldehyde and few drops

of acetic acid were added. The solution was reﬂuxed for

3–9 h, and then the product formed was ﬁltered by add-

ing cold distilled water and dried. Products (6a–6n)

were characterized by elemental analysis and spectro-

scopic data. The results of elemental analysis were in

good agreement with the proposed stoichiometry. IR

spectroscopic data indicated the formation of imines by

the disappearance of two separate bands for the NH₂

asymmetric and symmetric stretching vibrations. The

appearance of the imine (C N) band in the range

1651–1589 cm⁻¹also indicated the synthesis of imines.

1

In the H NMR spectra, the presence of NH and imine

CH protons in the range 12.20–7.93 ppm and the rele-

vant aromatic protons of the protecting group and the

aldehyde group conﬁrmed the synthesis of the desired

products. Electrospray ionization mass spectrometry

(EI-MS) results showed the molecular ion peaks in only

two cases, whereas the fragment with m/z 105 (benzoyl

cation) mostly appeared as a prominent fragment and

as base peak in six cases. The conﬁrmation of target

molecules was ﬁnally achieved through high-resolution

mass spectrometry (HRMS) (Scheme 1 and Table 1).

Each ester (3a–3c) was separately dissolved in eth-

anol, and then hydrazine hydrate was added and

reﬂuxed for 3–11 h. The solvent was removed, and the

product was ﬁltered, washed with hexane, and dried. IR

spectroscopic data indicated the formation of hydra-

zides by appearance of two separate bands for NH₂

Biological activity

asymmetric (3485–3458 cm⁻¹

)

and symmetric

Antimicrobial activity

The newly synthesized com-

(3298–3269 cm⁻¹) stretching vibrations in addition to

the NH band. Disappearance of the ester carbonyl

band and the appearance of a broad band for two the

amidic carbonyls in the range 1678–1649 cm⁻¹also

pounds were investigated for their antimicrobial activity

against a panel of Gram-positive and Gram-negative

pathogenic bacteria and fungi. The test strains included

Staphylococcus aureus, MRSA (methicillin-resistant

S. aureus), Escherichia coli, Klebsiella sp., Actinobacter

sp., and Candida albicans. In this analysis, the protected

amino acids, esters, hydrazides, and hydrazones with

diverse aldehydes were studied comparatively. Each of

the compounds exhibited a different of antimicrobial

response against the different tested strains. The com-

pound 6k exhibited good antimicrobial activity against

Gram-negative bacteria such as E. coli and against

C. albicans with inhibitory zones of 20 and 19 mm,

respectively. Compounds 2a, 2c, 3a, 4b, 4c, 6a, 6b, and

6c showed moderate to low antimicrobial activity

against Gram-positive bacteria, and inhibitory zones in

the range of 7 to 16 mm were observed against

S. aureus and MRSA. Compounds 3b, 3c, 4a, and 6i,

O

H

R

H R

H

R

(a)

(b)

N

COOH

N

COOMe

(3a-c)

H₂N

COOH

H

(1a-c)

(2a-c)

R=H, CH₃, CH₂CH(CH₃)₂

O

(c)

O

R¹

H

R

H

R

NHNH₂

(d)

NHN

N

C

N

C

H

R¹CHO

O

(5a-h)

(4a-c)

(6a-n)

Scheme 1. Synthesis of Schiff bases (a) PhCOCl,

MeOH (b) dry MeOH, conc. H₂SO₄,

reﬂux (c) MeOH, hydrazine hydrate

80%, reﬂux (d) MeOH, R¹CHO, reﬂux.

2

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JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Hydrazones from Amino Acids

Table 1. Structures of the synthesized compounds

Table 1. Continued

R¹

Sample code

R

H

R¹

Sample code

R

6a

6n

CH₃

6b

6c

H

however, were found to be inactive against all the tested

strains (Table 2).The results of this antimicrobial

screening indicate that N-protected amino acids 2a, 2c,

the ester 3a, the hydrazide 4c, and the hydrazones 6a,

6b are promising candidates against Gram-positive bac-

teria. The synthesized hydrazones such as 6f, 6h, 6j, 6k,

and 6m exhibit a broad spectrum of activities against

both Gram-positive and Gram-negative bacteria and

also against the fungal test strain, so these compounds

can be the lead candidates for further biological investi-

gation and structural modiﬁcations. It is to be noted

that the hydrazones were found to be more active than

the protected amino acids as well as their esters and

hydrazides. The active compounds were also tested for

their minimum inhibitory concentrations (MICs)

against S. aureus and E. coli. In the case of Gram-

positive S. aureus, most of the compounds exhibited

MIC in the range 32–128 μg/mL, while in case of

Gram-negative E. coli, MIC in the range 16–256 μg/mL

was exhibited by compounds 6f and 6k, respectively

(Table 2).

N

6d

H

MeO

OMe

6e

6f

H

Me₂N

H₃CO

H

6g

CH₂CH(CH₃)₂

6h

CH₂CH(CH₃)₂

OH

6i

6j

CH₂CH(CH₃)₂

Cytotoxicity (% larval mortality)

The synthesized com-

pounds were subjected to cytotoxicity screening by the

microwell cytotoxicity assay against the brine shrimp

Artimia salina. Most of the compounds exhibited mod-

erate to high cytotoxicity. The compounds 6a and 6b

were found to be less cytotoxic, with larval mortality in

the range 20–30%; however, most of the compounds

showed 50–70% larval mortality. Compounds 4b, 6f, 6j,

and 6k showed the highest percentage of larval mortal-

ity (70–85%) and hence were the most cytotoxic com-

pounds. Owing to the antimicrobial response of these

compounds, the broad spectrum activity of the com-

pounds like 6f, 6h, 6j, 6k, and 6m can be due to their

high nonspeciﬁc cytotoxicity. The high cytotoxicity of

the compound 6k might be due to the presence of two

methoxy groups, which are the activating groups.

N

6k

CH₂CH(CH₃)₂

MeO

OMe

6l

CH₂CH(CH₃)₂

Me₂N

6m

OH

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Abid et al.

Table 2. Antimicrobial activity of the synthesized compounds

Antimicrobial activity against MDR bacteria and fungi

Minimum inhibitory concentrations

(MICs) μg/mL

(zone of inhibition in mm)

Sample codes S. aureus MRSA E. coli Kleb Acineto Candida albicans

S. aureus

E. coli

2a

2b

2c

3a

3b

3c

4a

4b

4c

6a

6b

6c

6d

6e

6f

14

—

12

10

—

09

12

10

07

—

10

—

16

—

12

—

18

10

12

11

—

13

16

14

11

12

—

10

—

19

—

16

20

10

14

—

10

09

—

10

07

—

09

—

08

—

12

10

—

08

—

10

—

11

—

10

09

—

14

—

12

10

13

—

18

—

07

10

32

—

256

—

32

—

32

16

—

64

—

128

64

128

—

128

—

128

—

6g

6h

6i

6j

6k

6l

—

32

—

6m

6n

64

—

Kleb, Klebsella sp.; Acineto, Acinetobacter sp.

However, these compounds can be the promising candi-

dates for antitumor or anticancer activity and should be

investigated further for these kinds of biological activ-

ities (Table 3).

CONCLUSIONS

The hydrazones 6a–6n synthesized from three

amino acids were characterized and evaluated for their

antimicrobial activity and cytotoxicity. Compounds 6f,

6h, 6j, 6k, and 6m exhibited a broad spectrum of antibac-

terial activity and can be the lead candidates for further

biological investigations and structural modiﬁcations.

Cytotoxicity studies revealed that compounds 4b, 6f, 6j,

and 6k were the most cytotoxic. And these compounds

can be promising candidates for antitumor or anticancer

activity and should be investigated further for such bio-

logical activities. In general, hydrazones were found to be

more active than their precursors.

Table 3. Cytotoxicity results of synthesized compounds

Cytotoxicity

(% mortality)

Cytotoxicity

(n% mortality)

Samples

2a

2b

2c

3a

3b

3c

4a

4b

4c

6a

6b

6c

50.0

—

6d

6e

6f

6g

6h

6i

6j

6k

6l

—

63.8

81.0

—

58.9

64.1

—

38.6

—

76.1

85.0

65.2

55.0

65.8

—

EXPERIMENTAL

71.2

64.1

26.3

21.6

—

Different chemicals/reagents were used for the syn-

thesis of the desired products, which included amino

acids, benzoyl chloride, hydrochloric acid, sulfuric acid,

methanol (dry), magnesium sulfate(anhydrous), ethanol,

6m

6n

—

4

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Hydrazones from Amino Acids

acetic acid, hydrazine hydrate (80%), distilled water, hex-

ane, dichloromethane, ethyl acetate, acetone, chloroform,

and different aldehydes. All the chemicals used were of

analytical grade and were purchased from Merck, Riedel-

deHaen, Scharlau, Aldrich, or Fluka.

ﬁltered. The solvent was evaporated to obtain the

product.

Procedure for the synthesis of hydrazides

Six millimoles of the ester was dissolved in 4 mL

methanol by stirring in a round-bottom ﬂask, and then

1.5 mL of hydrazine hydrate was added and reﬂuxed

for 3–11 h in a reﬂux condenser. The solvent was

removed, and the product was ﬁltered, washed with

n-hexane, and dried.

All synthesized compounds were characterized by

their physical constants as well as spectroscopic data

1

such as IR, H NMR, GC-MS, HRMS, and elemental

analysis. Thin-layer chromatography (TLC) was per-

formed on precoated silica gel GF-254 aluminum plates

(Kieselgel 60, 20 × 20, 0.2–0.5 mm thick; E. Merck,

Germany). Detection under UV light illumination was

done with 254 and/or 366 nm. Melting points were

determined on a Sanyo Gallenkamp digital melting

point apparatus (MPD 350) in open capillaries. ¹H

NMR was carried out on a Bruker ARX 300 spectrome-

ter. Mass Spectroscopy was carried out on AMD

MS40, AMD 402 (AMD Intectra), Varian MAT CH

7, MAT 731, and HRMS on Finnigan MAT 95 or Var-

ian MAT 311, Bruker FTCIR, AMD 402 (AMD Intec-

tra) instruments. IRs spectra were recorded on a Bruker

IFS 66 (FT IR), Nicolet 205 FT IR, Nicolet Protege

460, or Nicolet 360 Smart Orbit (ATR) instrument

using KBr and ATR. The elemental analyzer used was

LECO CHNS-932 or Thermoquest Flash EA 1112.

Procedure for the synthesis of hydrazones

One millimole of hydrazide was dissolved in 5 mL

of ethanol by stirring in a round-bottom ﬂask, and an

equivalent mmol of aldehydes and few drops of acetic

acid were added. The solution was reﬂuxed for 3–9 h,

and the product was ﬁltered, washed by cold distilled

water, and dried. Characteristic data for all the synthe-

sized compounds 6a–6n are given in detail below.

N⁰-(Anthracene-10-ylmethylene)-2-benzamidoacetohydra-

zide 6a

Yellow powder; Yield: 89%; m.p: 202–205^ꢀC; IR

(cm⁻¹): 3352 (NH), 1680 (C O), 1643(C N), 1537

(C C); Elemental analysis: calcd. for C₂₄H₁₉N₃O₂: C,

75.57; H, 5.02; N, 11.02, Found: C, 75.69; H, 4.91; N,

1

11.18; H NMR (DMSO-d₆, 300 MHz); δ 11.72 (s, 1H,

NH), 8.77–8.74 (m, 2H, NH/CH), 8.63(d, 2H,

J = 6.6 Hz, Ar–H), 8.17 (d, 2H, J = 8.4 Hz, Ar–H),

7.98–7.92 (m, 2H, Ar–H), 7.69–7.48 (m, 8H, Ar–H),

4.49 (d, 2H, J = 6.0 Hz, CH₂); MS (GC, 70 eV): m/z

(%): 204(16), 203(100), 202(10), 176(9), 175(8), 150(5),

101(7), 88(11), 75(6); HRMS Pos (ESI) calcd. for

C₂₄H₁₉N₃O₂[M + H,]⁺382.155, Found 382.15492 and

[M + Na]⁺calcd. 404.13695, Found 404.1369 and the

exact ion mass is 381.14773.

Procedure for the N-protection of amino acids

Ten millimoles of the amino acid was dissolved

2.5 mL of NaOH (10%). Then, 1.5 mL of benzoyl chlo-

ride was added, and the solution was stirred for 3–4

h. A solid material was obtained, which was further

acidiﬁed with hydrochloric acid (dilute + cold) to pH 3.

After ﬁltration and washing by cold distilled water, the

benzoyl-protected amino acid was dried and then

weighed.

N⁰-(p-Biphenylmethylene)-2-benzamidoacetohydrazide 6b

Light brown powder; Yield: 86%; m.p:

238–239^ꢀC; IR (cm⁻¹): 3315 (NH), 1687 (C O),1634

(C N), 1547 (C C); Elemental analysis: calcd. for

C₂₂H₁₉N₃O₂: C, 73.93; H, 5.36; N, 11.76. Found: C,

73.87; H, 5.19; N, 11.94. ¹H NMR (DMSO-d₆,

Procedure for the esteriﬁcation of N-protected amino

acids

The protected amino acid (8.0 mmol) was dis-

solved in 5 mL of dry methanol in a round-bottom

ﬂask, and a few drops of sulfuric acid were added. The

solution was stirred and reﬂuxed for 10–19 h. After

completion of the reaction, the solvent was evaporated

and ethyl acetate was added, and the solution was

washed with NaHCO₃solution and distilled water.

Then MgSO₄was added and the organic layer was

300 MHz);

δ 11.58 (s, 1H, NH), 8.71 (t, 1H,

J = 6.6 Hz, NH), 8.06 (s, 1H, CH), 7.92(d, 2H,

J = 6.3 Hz, Ar–H), 7.82–7.77 (m, 4H, Ar–H), 7.73 (d,

2H, J = 8.7 Hz, Ar–H), 7.57–7.47 (m, 5H, Ar–H), 7.41

(d, 1H, J = 6.3 Hz, Ar–H) 4.46 (d, 2H, J = 6.0 Hz,

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Abid et al.

CH₂); MS (GC, 70 eV): m/z (%): 358 (M + 1⁺,1),

357 (M⁺, 5), 223(10), 197(15), 196(83), 195(13), 179

(13), 167(12), 165(27), 152(16), 135(13), 134(27), 105

(100), 77(52), 51(11); HRMS (EI) calcd. for

C₂₂H₁₉N₃O₂[M⁺]: 357.14718, Found: 357.1483.

C₂₈H₂₇N₃O₂: C, 76.86; H, 6.22; N, 9.60, Found: C,

76.69; H, 6.17; N, 9.81; ¹H NMR (DMSO-d₆,

300 MHz); δ 12.11 (s, 1H, NH), 9.69 (s, 1H, NH), 8.79

(s, 1H, CH), 8.75 (d, 2H, J = 6.6 Hz, Ar–H), 8.18 (d,

2H, J = 6.3 Hz, Ar–H), 8.05 (d, 2H, J = 6.3 Hz, Ar–

H), 7.70–7.58 (m, 8H, Ar–H), 4.44 (dd, 1H,

J = 8.0,7.5 Hz, CH), 1.88–1.79 (m, 3H, CH/CH₂), 0.92

(d, 3H, J = 6.3 Hz, CH₃), 0.85 (d, 3H, J = 6.3 Hz,

CH₃); MS (GC, 70 eV): m/z (%): 204(19), 203(100), 202

(12), 176(13), 174(8), 88(10), 75(8), 61(6); HRMS Pos

(ESI) calcd. for C₂₈H₂₇N₃O₂, [M + H]⁺calcd.

438.13354, found 438.13364 and [M + Na]⁺calcd.

460.11548, found 460.11583; exact ion mass 437.12626.

2-Benzamido-N⁰-(quinolin-2-ylmethylene)acetohydrazide 6c

Light brown powder; Yield: 93%; m.p:

226–229^ꢀC; IR (cm⁻¹): 3391 (NH), 1697 (C O),1641

(C N), 1538 (C C); Elemental analysis: calcd. for

C₁₉H₁₆N₄O₂: C, 68.66; H, 4.85; N, 16.86. Found: C,

68.83; H, 4.77; N, 16.72; ¹H NMR (DMSO-d₆,

300 MHz);

δ 11.90 (s, 1H, NH), 8.78 (t, 1H,

J = 6.6 Hz, NH), 8.42 (d, 1H, J = 8.7 Hz, Ar–H), 8.21

(s, 1H, CH), 8.14 (d, 1H, J = 8.7 Hz, Ar–H), 8.05–8.0

(m, 2H, Ar), 7.93 (d, 2H, J = 6.6 Hz, Ar–H), 7.80 (dt,

1H, J = 6.6, 2.7 Hz, Ar–H), 7.64 (dt, 1H, J = 6.6,

2.7 Hz, Ar–H), 7.57–7.50 (m, 3H, Ar–H), 4.53 (d, 2H,

J = 6.0 Hz, CH₂); MS (GC, 70 eV): m/z (%): 290(5),

198(54), 170(62), 142(68), 115(82), 105(100), 77(71),

51(18); HRMS Pos (ESI) calcd. for C₁₉H₁₆N₄O₂

[M + H]⁺calcd. 333.1346, Found: 333.13527 and

[M + Na]⁺calcd. 355.11655, Found 355.11638; exact

ion mass 332.12733.

N⁰-(2,4-Dihydroxybenzylidene)-2-benzamido-4-methylpe-

ntanehydrazide 6h

Dark brown powder; Yield: 72%; m.p:

204–209^ꢀC; IR (cm⁻¹): 3318 (NH), 1688 (C O), 1651

(C N), 1561 (C C); Elemental analysis: calcd. for

C₂₀H₂₃N₃O₄: C, 65.03; H, 6.28; N, 11.37, Found: C,

65.16; H, 6.45; N, 11.20; ¹H NMR (DMSO-d₆,

300 MHz); δ 11.60 (s, 1H, NH), 9.33 (bs, 2H, OH),

8.54 (d, 1H, J = 8.7 Hz, NH), 8.27 (s, 1H, CH), 7.90

(d, 2H, J = 6.6 Hz, Ar–H), 7.57 (d, 1H, J = 6.6 Hz,

Ar–H), 7.53 (d, 2H, J = 6.3 Hz, Ar–H), 7.26 (d, 1H,

J = 3.0 Hz, Ar–H), 6.94 (dd, 1H, J = 6.3,3.0 Hz, Ar–

H), 6.79 (d, 1H, J = 8.7 Hz, Ar–H), 4.51 (dd, 1H,

J = 8.0, 7.5 Hz, CH), 1.94–1.88 (m, 3H, CH/CH₂),

0.91 (d, 3H, J = 6.3 Hz, CH₃), 0.87 (d, 3H, J = 6.3 Hz,

CH₃); MS (GC, 70 eV): m/z (%): 256(22), 135(24), 122

(32), 105(100), 77(47), 51(14), 29(11); HRMS Pos (ESI)

calcd. for C₂₀H₂₃N₃O₄[M + H]⁺calcd. 370.17613,

Found 370.17713; [M + Na]⁺calcd. 392.15808, Found

392.15769, exact ion mass 369.16886.

N⁰-(3,5-Dimethoxybenzylidene)-2-benzamidoacetohydraz-

ide 6d

Floral white powder; Yield: 91%; m.p: 174–175^ꢀC;

IR (cm⁻¹): 3255 (NH), 1687 (C O),1631 (C N), 1573

(C C); Elemental analysis: calcd. for C₁₈H₁₉N₃O₄: C,

63.33; H, 5.61; N, 12.31, Found: C, 63.47; H, 5.53; N,

1

12.44; H NMR (DMSO-d₆, 300 MHz); δ 11.51 (s, 1H,

NH), 8.70 (t, 1H, J = 6.6 Hz, NH), 7.93 (s, 1H, CH),

7.90 (d, 2H, J = 6.6 Hz, Ar–H), 7.56–7.49 (m, 3H, Ar),

6.86 (t, 2H, J = 3.0 Hz, Ar–H), 6.55(t, 1H, J = 3.0 Hz,

Ar–H), 4.42 (d, 2H, J = 6.3 Hz, CH₂), 3.78 (s, 6H,

2OCH₃); MS (GC, 70 eV): m/z (%): 341 (M⁺, 1), 207

(26),180(18), 178(11), 153(17), 135(11), 134(24),

105 (100), 77 (67), 51 (14); HRMS Pos (ESI) calcd. for

C₁₈H₁₉N₃O₄[M + H]⁺; calcd. 342.14483, Found

342.14496, and [M + Na]⁺calcd. 364.12678, Found

364.12699; exact ion mass 341.13756.

N⁰-(p-Biphenyl)-2-benzamido-4-methylpentanehydrazide 6i

Floral white powder, Yield: 89%; m.p: 234–238^ꢀC;

IR (cm⁻¹): 3345 (NH), 1672 (C O), 1619 (C N), 1558

(C C); Elemental analysis: calcd. for C₂₆H₂₇N₃O₂: C,

75.52; H, 6.58; N, 10.16, Found: C, 75.82; H, 6.73; N,

1

10.03; H NMR (DMSO-d₆, 300 MHz);δ 11.89 (s, 1H,

NH), 8.52 (s, 1H, CH), 7.94 (d, 2H, J = 6.60 Hz, Ar–

H), 7.82–7.77 (m, 4H, Ar–H), 7.74 (d, 2H, J = 6.3 Hz,

Ar–H), 7.61–7.47(m, 5H, Ar–H), 7.42 (d, 1H,

J = 8.7 Hz, Ar–H), 4.55 (dd, 1H, J = 8.0,7.5 Hz, CH),

1.79–1.73 (m, 3H, CH/CH₂), 0.92 (d, 3H, J = 6.3 Hz,

CH₃), 0.85 (d, 3H, J = 6.3 Hz, CH₃); MS (GC, 70 eV):

N⁰-(anthracene-10-ylmethylene)-2-benzamido-4-methylpe-

ntanehydrazide 6g

Dark orange powder; Yield: 79%; m.p:

225–230^ꢀC; IR (cm⁻¹): 3298 (NH), 1663 (C O), 1624

(C N), 1549 (C C); Elemental analysis: calcd. for

6

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J. Chin. Chem. Soc. 2017

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Hydrazones from Amino Acids

m/z (%): 300(5), 180(20), 179(100), 165(11), 152(7), 105

(80), 77(40), 51(8); HRMS Pos (ESI) calcd. for

C₂₆H₂₇N₃O₂[M + H]⁺calcd. 414.13354, Found

414.13404 and [M + Na]⁺calcd. 436.11548, Found

436.11598; exact ion mass 413.12626.

N⁰-(4-(Dimethylamino)benzylidene)-2-benzamido-4-meth-

ylpentanehydrazide 6l

Orange powder; Yield: 96%;m.p: 130–135^ꢀC; IR

(cm⁻¹): 3315 (NH), 1648 (C O), 1593(C N), 1524

(C C); Elemental analysis: calcd. for C₂₂H₂₈N₄O₂: C,

69.45; H, 7.42; N, 14.73, Found: C, 69.61; H, 7.33; N,

1

14.51; H NMR (DMSO-d₆, 300 MHz); δ 11.54 (s, 1H,

2-Benzamido-4-methyl-N⁰-(quinolin-2-ylmethylene)penta-

nehydrazide 6j

NH), 8.52 (d, 1H, J = 8.7 Hz, NH), 8.32 (s, 1H, CH),

7.90 (d, 2H, J = 6.6 Hz, Ar–H), 7.58–7.49 (m, 5H, Ar–

H), 6.77 (d, 2H, J = 8.7 Hz, Ar–H), 4.56 (dd, 1H,

J = 8.0, 7.5 Hz, CH), 2.98 (s, 6H, NMe₂), 1.81–1.74

(m, 3H, CH/CH₂), 1.01 (d, 3H, J = 6.0 Hz, CH₃), 0.93

(d, 3H, J = 6.3 Hz, CH₃); MS (GC, 70 eV): m/z (%):

267(44), 162(17), 148(29), 146(100), 132(8), 118(14), 105

(34), 91(8), 77(31), 51(9); HRMS Pos (ESI) calcd. for

C₂₂H₂₈N₄O₂[M + H]⁺calcd. 381.11314, Found

381.11369 and [M + Na]⁺calcd. 413.09508, Found

413.09564; and exact ion mass 380.10586.

Peach powder; Yield: 66%; m.p: 110–113^ꢀC; IR

(cm⁻¹): 3312 (NH), 1637 (C O), 1589 (C N), 1565

(C C); Elemental analysis: calcd. for C₂₃H₂₄N₄O₂: C,

71.11; H, 6.23; N, 14.42, Found: C, 71.29; H, 6.09; N,

1

14.49; H NMR (DMSO-d₆, 300 MHz); δ 12.20 (s, 1H,

NH), 8.69 (d, 1H, J = 8.7 Hz, NH), 8.63 (s, 1H, CH),

8.44 (d, 1H, J = 8.7 Hz, Ar–H), 8.14 (d, 1H,

J = 8.7 Hz, Ar–H), 8.07–8.01 (m, 2H, Ar–H), 7.96 (d,

2H, J = 6.3 Hz, Ar–H), 7.81 (dt, 1H, J = 8.7, 3.0 Hz,

Ar–H), 7.68–7.55 (m, 4H, Ar–H), 4.59 (dd, 1H,

J = 8.0,7.5 Hz, CH), 1.90–1.84 (m, 3H, CH/CH₂), 0.98

(d, 3H, J = 6.3 Hz, CH₃), 0.94 (d, 3H, J = 6.3 Hz,

CH₃); MS (GC, 70 eV): m/z (%): 275(2), 171(12), 170

(84), 142(59), 140(15), 115(100), 105(63), 77(97), 51(37),

50(18); HRMS Pos (ESI) calcd. for C₂₃H₂₄N₄O₂

[M + H,]⁺calcd. 389.1972, Found 389.19777;

[M + Na]⁺calcd. 411.17915, found 411.1787; exact ion

mass 388.188993.

2-Benzamido-N⁰-((1-hydroxynaphthalen-2-yl)methylene)-

4-methylpentanehydrazide 6m

Lemon yellow powder; Yield: 95%; m.p:

217–220^ꢀC; IR (cm⁻¹): 3265 (NH), 1644 (C O), 1621

(C N), 1579 (C C); Elemental analysis: calcd. for

C₂₄H₂₅N₃O₃:C, 71.44; H, 6.25; N, 10.41, Found: C,

71.33; H, 6.19; N, 10.55; ¹H NMR (DMSO-d₆,

300 MHz); δ 12.78 (s, 1H, OH), 12.20 (s, 1H, NH),

9.50 (s, 1H, CH), 8.23 (d, 1H, J = 8.7 Hz, Ar–H),

8.02–7.90 (m, 4H, Ar–H), 7.66–7.57 (m, 4H, Ar–H),

7.42 (t, 1H, J = 6.3 Hz, Ar–H), 7.25 (d, 1H,

J = 6.3 Hz, Ar–H), 4.64 (dd, 1H, J = 8.0, 7.5 Hz, CH),

1.93–1.85 (m, 3H, CH/CH₂), 0.98 (d, 3H, J = 6.0 Hz,

CH₃), 0.94 (d, 3H, J = 6.0 Hz, CH₃); MS (GC, 70 eV):

m/z (%): 217(18), 174(22),161(26), 147(30), 130(16), 105

(100), 77(37), 51(9); HRMS Pos (ESI) calcd. for

C₂₄H₂₅N₃O₃[M + H]⁺calcd. 404.10582, Found

404.10614 and [M + Na]⁺calcd. 426.15637, found

426.15685; and exact ion mass 403.06308.

N⁰-(3,5-Dimethoxybenzylidene)-2-benzamido-4-methylpe-

ntanehydrazide 6k

White powder; Yield: 83%; m.p: 190–193^ꢀC; IR

(cm⁻¹): 3346 (NH), 1681 (C O), 1645 (C N), 1547

(C C); Elemental analysis: calcd. for C₂₂H₂₇N₃O₄:C,

66.48; H, 6.85; N, 10.57, Found: C, 66.58; H, 6.71; N,

1

10.76; H NMR (DMSO-d₆, 300 MHz); δ 11.87 (s, 1H,

NH), 8.52 (d, 1H, J = 8.7 Hz, NH), 8.39 (s, 1H, CH),

7.92 (d, 2H, J = 6.3 Hz, Ar–H), 7.61–7.53 (m, 3H, Ar–

H), 6.88 (d, 2H, J = 3.0 Hz, Ar–H), 6.58 (t, 1H,

J = 3.0 Hz, Ar–H), 4.56 (dd, 1H, J = 8.0, 7.5 Hz, CH),

3.80 (s, 6H, 2OCH₃),1.84–1.76 (m, 3H, CH/CH₂), 1.02

(d, 3H, J = 6.3 Hz, CH₃), 0.94 (d, 3H, J = 6.3 Hz,

CH₃); MS (GC, 70 eV): m/z (%): 284(9), 164(12), 163

(75), 106(9), 105(100), 77(77), 76(10), 51(25), 50(9);

HRMS Pos (ESI) calcd. for C₂₂H₂₇N₃O₄[M + H]⁺

calcd. 398.20743; Found 398.2069 and [M + Na]⁺

calcd. 420.18938, Found 420.18889; exact ion mass

397.20016.

N⁰-(anthracen-10-ylmethylene)-2-benzamidopropanehydr-

azide 6n

Orange powder; Yield: 54%; m.p: 219–221^ꢀC; IR

(cm⁻¹): 3254 (NH), 1670 (C O), 1634 (C N), 1532

(C C); Elemental analysis: calcd. for C₂₅H₂₁N₃O₂:C,

75.93; H, 5.35; N, 10.63, Found: C, 75.69; H, 5.47; N,

1

10.79; H NMR (DMSO-d₆, 300 MHz); δ 11.81 (s, 1H,

NH), 8.78 (m, 1H, NH), 8.73 (s, 1H, CH), 8.67 (d, 2H,

J. Chin. Chem. Soc. 2017

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7

Article

Abid et al.

J = 6.3 Hz, Ar–H), 8.16 (d, 1H, J = 8.7 Hz, Ar–H),

8.00–7.93 (m, 2H, Ar–H), 7.67–7.48 (m, 8H, Ar–H),

4.66 (q, 1H, J = 7.8 Hz, CH), 1.52 (d, 3H, J = 8.1 Hz,

CH₃): MS (GC, 70 eV): m/z (%): 204(14), 203(100), 201

(9), 176(8), 175(10), 150(6), 88(8), 87(7), 75(7), 63(4),

50(4); HRMS Pos (ESI) calcd. for C₂₅H₂₁N₃O₂

[M + H]⁺calcd. 396.17065, found 396.17106 and

[M + Na]⁺calcd. 418.1526, found 418.15308; exact ion

mass 395.16338.

organism was considered as the MIC of the respective

sample.

The cytotoxicity of the compounds was deter-

mined by microwell cytotoxicity assay against the brine

shrimp (Artimia salina). The eggs powder of the

A. salina (0.5 g) was added to a 1-L separating funnel

ﬁlled with 500 mL of artiﬁcial seawater. The suspension

was aerated by bubbling air into the funnel and kept

for 24–48 h at room temperature. Later, the aeration

was stopped and the suspension was kept undisturbed

for 1 h to let the remaining eggs settle down. In order

to use only the active larvae, one side of the separating

funnel was covered with an aluminum foil and the other

side illuminated with a lamp to gather the phototropic

larvae at the illuminated side. Then with the help of a

micropipette, 30–40 shrimp larvae were transferred to a

deep-well microtiter plate, with each well ﬁlled with

0.2 mL of sea water. Initially, the dead larvae were

counted (value N), then 20 μg of the compounds in

DMSO was added to the designated wells, and the plate

was kept at room temperature in the dark. After 24 h,

the number A of the dead larvae in each well was

counted again under the microscope. The surviving lar-

vae were killed by the addition of 0.5 mL methanol to

each well, and subsequently the total number G of the

larvae was determined. Each test row was accompanied

by a blind sample containing pure DMSO instead of

the test compounds. As a positive control with 100%

mortality, namely actinomycin D (20 μg/mL), was used.

The percentage mortality M was calculated using the

following formula:

DETERMINATION OF ANTIMICROBIAL

ACTIVITY, MINIMUM INHIBITORY

CONCENTRATIONS (MICS), AND

CYTOTOXICITY

The antimicrobial activity of the synthesized com-

pounds was determined by the agar diffusion method

against the following test strains; S. aureus, MRSA,

E. coli, Klebsiella sp., Acintobacter sp., and C. albicans.

Luria–Bertani (LB) agar plates were prepared by pour-

ing 16 mL of molten agar in to Petri plates. After solid-

iﬁcation, the plates were overlayed with 4 mL of seed

agar having a standardized inoculum of the test strains.

The plates were left for 15 min at room temperature,

and the agar wells were made with the help of a sterile

cork borer. Then 50 μL of the samples was loaded in

each well, which were labeled accordingly. The plates

were left at room temperature for 2 h and incubated at

37^ꢀC for 18–24 h. The plates were observed for clear

zones around the wells, which indicated growth inhibi-

tion of the test organism by the compounds. The zones

of inhibition were measured in millimeters.

The minimum inhibitory concentrations (MICs) of

the compounds were determined by the broth dilution

method against the two representative Gram-positive

and Gram-negative test organisms including S. aureus

and E. coli, respectively. The pure cultures of the test

organisms were grown in the LB broth and the inocu-

lum was standardized by comparing with 0.5 McFar-

land standard. Twofold dilutions of each of the

compounds were prepared in sterile LB broth in the fol-

lowing range: 1024, 512, 256, 128, 64, 32, 16, 8, 4,

2, and 1 μg/mL. Each test tube contained 5 mL of LB

broth, which was inoculated with 50 μL of the cell sus-

pension. The tubes were incubated at 37^ꢀC for 18–24 h

and were observed for the growth of the test organism,

which was indicated by turbidity. The lowest dilution

that did not exhibit any turbidity or growth of the

M = [(A – B – N)/(G – N)] × 100

where

M = percent of the dead larvae after 24 h.

A = number of the dead larvae after 24 h.

B = average number of the dead larvae in the

blind samples after 24 h.

N = number of the dead larvae before starting of

the test.

G = total number of larvae.

ACKNOWLEDGMENTS

The authors acknowledge the support provided by

HEC, Pakistan, under its ASIP program for the analy-

sis of the products synthesized.

8

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J. Chin. Chem. Soc. 2017

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Hydrazones from Amino Acids

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Article Doi

Synthesis of Hydrazones from Amino Acids and their Antimicrobial and Cytotoxic Activities

DOI: 10.1002/jccs.201600859

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Article abstract of DOI:10.1002/jccs.201600859

Full text of DOI:10.1002/jccs.201600859

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