Synthesis and biological activity of novel amino acid-(N'-benzoyl) hydrazide and amino acid-(N'-nicotinoyl) hydrazide derivatives

Article abstract of DOI:10.3390/10091218

The coupling reaction of benzoic acid and nicotinic acid hydrazides with N-protected L-amino acids including valine, leucine, phenylalanine, glutamic acid and tyrosine is reported. The target compounds, N-Boc-amino acid-(N'-benzoyl)- and N-Boc-amino acid-(N'-nicotinoyl) hydrazides 5a-5e and 6a-6e were prepared in very high yields and purity using N-[(dimethylamino)-1H- 1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide (HATU) as coupling reagent. The antimicrobial activity of the Cu and Cd complexes of the designed compounds was tested. The products were deprotected affording the corresponding amino acid-(N'-benzoyl) hydrazide hydrochloride salts (7a-7e) and amino acid-(N'-nicotinoyl) hydrazide hydrochloride salts (8a-8e). These compounds and their Cu and Cd complexes were also tested for their antimicrobial activity. Several compounds showed comparable activity to that of ampicillin against S. aureus and E. coli.

Full text of DOI:10.3390/10091218

Molecules 2005, 10, 1218-1228
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis and Biological Activity of Novel Amino Acid-
(N'-Benzoyl) Hydrazide and Amino Acid-(N'-Nicotinoyl)
Hydrazide Derivatives.
Sherine N. Khattab
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
Alexandria 21321, Egypt. E-mail: ShKh2@link.net
Received: 9 November 2004; in revised form: 12 July 2005 / Accepted: 13 July 2005 / Published:
30 September 2005
Abstract: The coupling reaction of benzoic acid and nicotinic acid hydrazides with N-
protected L-amino acids including valine, leucine, phenylalanine, glutamic acid and
tyrosine is reported. The target compounds, N-Boc-amino acid-(N`-benzoyl)- and N-
Boc-amino acid-(N`-nicotinoyl) hydrazides 5a-5e and 6a-6e were prepared in very high
yields and purity using N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-
methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide (HATU) as coupling
reagent. The antimicrobial activity of the Cu and Cd complexes of the designed
compounds was tested. The products were deprotected affording the corresponding
amino acid-(N`-benzoyl) hydrazide hydrochloride salts (7a-7e) and amino acid-(N`-
nicotinoyl) hydrazide hydrochloride salts (8a-8e). These compounds and their Cu and Cd
complexes were also tested for their antimicrobial activity. Several compounds showed
comparable activity to that of ampicillin against S. aureus and E. coli.
Keywords: Benzoic acid hydrazide, nicotinic acid hydrazide, amino acid hydrazide
derivatives, Copper and Cadmium complexes, antimicrobial agents.

Molecules 2005, 10
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Introduction:
Amino acids have proven to play a significant role in the synthesis of novel drug candidates with
the use of non-proteinogenic and unnatural amino acids [1-8]. Recently a series of novel diflunisal
hydrazide-hydrazone derivatives have demonstrated significant antimicrobial activity [9]. These
synthesized compounds and additional hydrazide derivatives were screened for their wide range of
biological activities, antimycobacterial activity against Mycobacterium tuberculosis, antimicrobial
activities against various bacteria (Escherichia coli and Bacillus subtilis), fungi, and yeast species [10-
13]. In addition, nicotinamide and nicotinic acid have been in use for 65 years due to their unusual
antimicrobial spectrum. Many drugs possess modified pharmacological and toxicological properties
when administered in form of metallic complexes [14-16]. In view of this it was interesting to
synthesize several new compounds to evaluate the effect of amino acids on the bioactivity of both
benzoic acid hydrazide and nicotinic acid hydrazide. It was also of interest to study the effect of metals
such as Cu and Cd on the antibacterial activity of these compounds, thus both hydrazide ligands and
complexes were tested for their antibacterial and antifungal activity.
Results and Discussion
Chemistry
For the synthesis of the desired compounds, the sequence of reactions shown in Scheme 1 was
followed. The reaction of benzoic acid hydrazide (3) with different N-Boc-L-amino acids in
dimethylformamide in the presence of triethylamine (Et3N) as a base and N-[(dimethylamino)-1H-
1,2,3-triazolo[4,5-b]pyridin-1-yl-methyl-ene]-N-methylmethanaminium hexafluorophsphate N-oxide
(HATU) as coupling reagent at 0°C gave the desired compounds 5a-5e. The structures of compounds
1
5a-5e were established by means of IR, H-NMR, 13C-NMR, and elemental analyses. The infrared
spectra of these compounds showed the absorption peaks of the urethane and the amide carbonyls in
the 1715-1727 cm-1 and 1663-1693 cm-1 ranges, respectively, and the absorption peak of the N-H in
1
the 3226-3336 cm-1 range. The H-NMR showed the presence of three exchangeable signals for the
NH and the 13C-NMR detected the presence of one urethane carbonyl and two amide carbonyls in the
153-173 ppm range. Similarly, nicotinic acid hydrazide (4) was allowed to react under the same
conditions with the same N-Boc-L-amino acids to give the desired compounds 6a-6e. The structures of
compounds 6a-6e were also established by means of IR, 1H-NMR, 13C-NMR, and elemental analyses.
Compounds 5 and 6 were subjected to N-deprotection by passing HCl gas into a solution of these
compounds in methylene chloride/ether. The latter series upon deprotection of Boc group afforded the
new series of compounds 7 and 8 (Table 1, Table 2).
Compounds 5, 6, 7 and 8 were allowed to undergo complexation with Cu and Cd. All complexes
were obtained by reacting one equivalent of the ligands 5, 6, 7 or 8 once with 2 equivalents of
Cu(NO3)2 and once with 2 equivalents of Cd(CH3COO)2 in methanol.

Molecules 2005, 10
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The Cu and Cd complexes were later decomposed and their Cu and Cd contents were analyzed by
atomic absorption to determine the ratio of complex formation of Cu and Cd to ligand. The atomic
absorption analysis showed the formation of complexes Cu:L and Cd:L in the ratio (1:1).
Scheme 1
O
O
OH
NH
1) CH3-OH, H2SO4
2) NH2-NH2, Et-OH
NH2
X
X
X = CH; 3
X = N; 4
X = CH; 1
X = N; 2
R
HATU,Et3N
OH
Boc-NH
DMF
O
O
O
O
NH
HN
NH
HN
O
HCl gas
X
X
R
NH2
R
NH-Boc
HCl
X = CH; 7
X = N; 8
X = CH; 5
X = N; 6
a, R = CH(CH3)2; b, R = CH2-CH2-COOBn; c, R = CH2-CH(CH3)2;
d, R = CH2-Ph; e, R = CH2-[4-(OBn)C6H4].
Table 1. Yields, Color, Mps., and Elemental Analyses of Compounds 7 and 8.
Cmpd Yield
(%)
Color
mp
Elemental Analysis (Found)
(°C)
C
H
N
7a
7b
7c
7d
7e
75
84
93
83
89
77
81
78
89
96
White
White
White
White
White
White
White
White
White
White
244 53.04(52.89) 6.68 (6.83)
155 58.24(58.39) 5.66 (5.89)
190 54.64(54.91) 7.05 (7.32)
238 60.09(59.84) 5.67 (5.92)
233 64.86(64.58) 5.68 (5.89)
290 --------------- --------------- ---------------
267 --------------- --------------- ---------------
253 --------------- --------------- ---------------
215 --------------- --------------- ---------------
197 --------------- --------------- ---------------
15.46(15.62)
10.72(10.96)
14.70(14.95)
13.14(13.32)
9.87 (9.59)
8a*
8b*
8c*
8d*
8e*
* = Elemental analysis was unreliable due to their hygroscopic nature.

Molecules 2005, 10
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Table 2. 1H-NMR Data of Compounds 7 and 8.
Compound
7a
1H-NMR (DMSO-d6): (δ) ppm
0.89, 0.97 (2d, 6H, 2 CH3), 1.92 (m, 1H, CH), 4.89 (d, 1H, CH), 7.40-7.88
(m, 5H, aromatic), 8.38 (br.s, 2H, 2 NH), 10.64 (br.s, 2H, 2 NH).
1.99 (m, 2H, CH2), 2.63, 2.75 (2m, 2H, CH2-CO), 4.55 (m, 1H, CH), 5.14
m, 2H, CH2-O), 7.21-7.90 (m, 10H, aromatic), 8.36 (br.s, 2H, 2 NH),
10.66 (br.s, 2H, 2 NH).
7b
7c
0.91 (dd, 6H, 2 CH3), 1.63 (m, 2H, CH2), 1.81 (m, 1H, CH), 3.83 (m, 1H,
CH), 7.46-7.87 (m, 5H, aromatic), 8.42 (br.s, 2H, 2 NH), 10.61 (br.s, 2H,
2 NH).
7d
7e
8a
3.07 (2m, 2H, CH2), 4.16 (m, 1H, CH), 7.22-7.89 (m, 10H, aromatic),
8.34 (br.s, 2H, 2 NH), 10.65 (br.s, 2H, 2 NH).
3.08 (m, 2H, CH2), 4.29 (m, 1H, CH), 5.11 (br.s, 2H, CH2), 7.24-7.96 (m,
14H, aromatic), 8.45 (br.s, 2H, 2 NH), 10.59 (br.s, 2H, 2 NH).
δ 0.87, 0.96 (2d, 6H, 2 CH3), 1.96 (m, 1H, CH), 4.01 (m, 1H, CH), 7.79
(d, 2H, aromatic), 8.40 (br.s, 2H, 2 NH), 8.78 (d, 2H, aromatic), 10.62
(br.s, 2H, 2 NH).
8b
8c
8d
8e
2.03 (m, 2H, CH2), 2.35 (m, 2H, CH2-CO), 4.09 (m, 1H, CH), 5.14 m, 2H,
CH2-O), 7.33-7.82 (m, 7H, aromatic), 8.44 (br.s, 2H, 2 NH), 8.77 (d, 2H,
aromatic), 10.66 ( br.s, 2H, 2 NH).
δ 0.92 (d, 6H, 2 CH3), 1.59 (m, 2H, CH2), 1.79 (m, 1H, CH), 4.07 (m, 1H,
CH), 7.79 (d, 2H, aromatic), 8.39 (br.s, 2H, 2 NH), 8.75 (d, 2H, aromatic),
10.71 (br.s, 2H, 2 NH).
3.09 (m, 2H, CH2), 4.33 (m, 1H, CH), 7.25-7.39 (m, 5H, aromatic), 7.85
(d, 2H, aromatic), 8.43 (br.s, 2H, 2 NH), 8.79 (d, 2H, aromatic), 10.79
(br.s, 2H, 2 NH).
3.04 (m, 2H, CH2), 4.27 (m, 1H, CH), 5.14 (br.s, 2H, CH2), 7.24-7.47 (m,
9H, aromatic), 7.82 (d, 2H, aromatic), 8.45 (br.s, 2H, 2 NH), 8.79 (d, 2H,
aromatic), 10.62 (m, 2H, 2 NH);
Biological Test Results
The synthesized compounds 5, 6, 7 and 8 and their Cu and Cd complexes have been evaluated for
their antimicrobial activity. The microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and
Sabouraud Liquid Medium (Oxoid) were used for the determination of antibacterial and antifungal
activity [17]. The minimal inhibitory concentration (MIC) values listed in Table 3 show that all the test
compounds have lower antifungal activity than clotrimazole (Canesten®, Bayer).

Molecules 2005, 10
Table 3. Minimal inhibitory concentration (MIC) of test compounds in µg/mL.
1222
Test compound
E. coli S. aureus C. albicans
Test compound
(Cu:L),(1:1) of 6e
(Cd:L),(1:1) of 5a
E. coli S. aureus C. albicans
ampicillin
25
-------
>200
>200
100
50
12.5
--------
>200
>200
100
---------
12.5
100
100
12.5
>200
25
200
clotrimazole
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
5a
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100
(Cd:L),(1:1) of 5b >200
(Cd:L),(1:1) of 5c >200
(Cd:L),(1:1) of 5d >200
(Cd:L),(1:1) of 5e >200
(Cd:L),(1:1) of 6a >200
(Cd:L),(1:1) of 6b >200
5b
>200
25
5c
5d
>200
>200
>200
>200
>200
>200
>200
100
>200
>200
100
5e
>200
>200
100
50
6a
6b
(Cd:L),(1:1) of 6c
(Cd:L),(1:1) of 6d >200
(Cd:L),(1:1) of 6e
100
100
>200
>200
>200
>200
>200
100
6c
6d
100
50
6e
(Cu:L),(1:1) of 7a >200
(Cu:L),(1:1) of 7b >200
(Cu:L),(1:1) of 7c >200
(Cu:L),(1:1) of 7d >200
(Cu:L),(1:1) of 7e >200
(Cu:L),(1:1) of 8a >200
(Cu:L),(1:1) of 8b >200
(Cu:L),(1:1) of 8c >200
7a
100
25
7b
>200
50
7c
100
100
200
25
50
7d
>200
50
50
7e
>200
>200
>200
>200
100
8a
>200
100
8b
100
50
8c
>200
100
(Cu:L),(1:1) of 8d
(Cu:L),(1:1) of 8e >200
(Cd:L),(1:1) of 7a
100
(Cd:L),(1:1) of 7b >200
100
8d
8e
100
50
100
>200
>200
12.5
>200
>200
>200
>200
>200
>200
>200
(Cu:L),(1:1) of 5a
200
100
(Cu:L),(1:1) of 5b >200
(Cu:L),(1:1) of 5c
100
(Cu:L),(1:1) of 5d >200
(Cu:L),(1:1) of 5e
200
>200
50
(Cd:L),(1:1) of 7c
(Cd:L),(1:1) of 7d
100
>200
>200
>200
>200
>200
>200
>200
50
50
(Cd:L),(1:1) of 7e >200
(Cd:L),(1:1) of 8a >200
25
(Cu:L),(1:1) of 6a >200
(Cu:L),(1:1) of 6b >200
(Cu:L),(1:1) of 6c >200
100
(Cd:L),(1:1) of 8b
(Cd:L),(1:1) of 8c >200
(Cd:L),(1:1) of 8d
100
(Cd:L),(1:1) of 8e >200
100
>200
>200
100
(Cu:L),(1:1) of 6d
200
The test compounds are more active against S. aureus and E. coli. Compounds (Cu:L),(1:1) of 6e
and (Cd:L),(1:1) of 7c have antimicrobial activity against S. aureus comparable to that of ampicillin,
while the activity of compounds (Cu:L),(1:1) of 5e, (Cd:L),(1:1) of 5b and (Cd:L),(1:1) of 5d is
about 50% of that of ampicillin. Compounds 7b and 8a have antimicrobial activity against E. coli

Molecules 2005, 10
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comparable to that of ampicillin, while the activity of compounds 5d, 6c, 6e, 8c, 8e, (Cd:L),(1:1) of
7d is about 50% of that of ampicillin.
Conclusions
The present work describes the synthesis of amino acid derivatives and its metal complexes which
showed interesting results comparable to ampicillin. The uncomplexed compounds showed higher
antimicrobial activity against E. coli compared to their respective complexes. While the complexes
showed higher antimicrobial activity against S. aureus compared to their respective ligands. These
results prompted us to further pursue in SAR as our future plane.
Acknowledgements
Author is very grateful to Dr. Adnan A. Bekhit, Department of Pharmaceutical Chemistry, and Dr.
Elsayed Aboulmagd, Department of Microbiology, Faculty of Pharmacy, University of Alexandria,
Egypt; for performing the antimicrobial evaluation.
Experimental
General
Melting points were determined on a Mel-Temp apparatus and are uncorrected. 1H-NMR and 13C-
NMR spectra were recorded on a Bruker 300 MHz spectrometer with chemical shift values reported in
δ units (ppm) relative to an internal standard (tetramethylsilane). Infrared data were obtained on a
Perkin-Elmer 1600 series Fourier transform instrument as KBr pellets. Amino acids are abbreviated
and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (J.
Biol. Chem., 1972, 247, 977). The abbreviations: HATU (N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-
b]pyridin-1-ylmethylene]-N-methyl-methanaminium hexafluorophosphate N-oxide); Boc (t-butyloxy-
carbonyl); Et3N (triethylamine) and DMF (N,N-dimethylformamide) were used throughout the
manuscript. Follow up of the reactions and checking the homogeneity of the compounds were made by
TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by
exposure to UV-lamp at λ 254 nm for few seconds. Elemental analyses were performed in the
Chemistry Department, Faculty of Science, Cairo, University.
Chemistry
General Procedure for the Reaction of N-Boc-L-Amino Acids with Benzoic Acid Hydrazide.
A mixture of N-Boc-L-amino acid (1 mmol), HATU (0.38 g, 1 mmol) and Et3N (0.28 mL, 2 mmol)
was stirred at 0°C for 3 minutes in DMF (2 mL). Then benzoic acid hydrazide (3, 0.122 g, 1 mmol)

Molecules 2005, 10
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was added. The reaction mixture was stirred at 0°C for 1 hour and left overnight at room temperature.
The reaction mixture was diluted with ethyl acetate (80 mL) and the mixture was washed successively
with 5% aqueous citric acid solution (2×10 mL), saturated sodium bicarbonate solution (2×10 mL) and
saturated sodium chloride solution (2×10 mL). The organic layer was dried over anhydrous sodium
sulphate, filtered and the solvent was removed in vacuo. The crude product was crystallized from
methylene chloride-hexane. In the case of N-Boc L-(O-benzyl)-tyrosine (5e) and N-Boc-L-
phenylalanie (5d) the reaction mixture was poured into ice water, filtered, washed with 5% aqueous
citric acid solution, saturated sodium bicarbonate solution and finally with water, dried and
recrystallized from methylene chloride-hexane.
Boc-valyl(N`-benzoyl)-hydrazide (5a): White crystals, 0.32 g (95.4 % yield); mp 156-157°C; IR: 3308,
3226 (NH), 1715 (C=O, urethane), 1689 (C=O, amide), 1668 (C=O, amide) cm-1; 1H-NMR (CDCl3): δ
0.87, 0.95 (2d, 6H, 2 CH3), 1.35 (s, 9H, 3 CH3), 1.88 (m, 1H, CH), 4.93 (d, 1H, CH), 7.20-7.77 (m,
13
6H, 1 NH, aromatic), 9.17, 9.34 (2 br.s, 2H, 2 NH); C-NMR (CDCl3): δ 19.61, 19.70, 26.40, 28.62,
50.49, 80.69, 127.40, 129.00, 129.71, 131.79, 132.70, 136.58, 156.00, 164.62, 169.01; Anal. Calcd for
C17H25N3O4: C, 60.88; H, 7.51; N, 12.53. Found: C, 60.72; H, 7.68; N, 12.81.
Boc-(o-benzyl)-glutamyl(N`-benzoyl)-hydrazide (5b): White powder, 0.38 g (80 % yield); mp 126°C;
IR: 3336, 3253 (NH), 1753 (C=O, ester), 1727 (C=O, urethane), 1686 (C=O, amide), 1663 (C=O,
amide) cm-1; 1H-NMR (CDCl3): δ 1.37 (s, 9H, 3 CH3), 1.90, 2.05 (2m, 2H, CH2), 2.60, 2.71 (2m, 2H,
CH2-CO), 4.50 (m, 1H, CH), 5.15 m, 2H, CH2-O), 7.20-7.81 (m, 11H, 1 NH, aromatic), 9.17, 9.34 (2
br.s, 2H, 2 NH); 13C-NMR (CDCl3): δ 24.96, 28.16, 28.67, 49.70, 66.94, 80.72, 127.79, 128.65,
128.94, 128.95, 129.02, 131.87, 132.69, 136.10, 136.16, 153.64, 165.20, 169.82, 173.20; Anal. Calcd
for C24H29N3O6: C, 63.28; H, 6.42; N, 9.22. Found: C, 63.02; H, 6.21; N, 8.96.
Boc-leucyl(N`-benzoyl)-hydrazide (5c): White crystals, 0.26 g (74 % yield); mp 147°C; IR: 3318, 3234
1
(NH), 1726 (C=O, urethane), 1689 (C=O, amide), 1672 (C=O, amide) cm-1; H-NMR (DMSO-d6): δ
0.91 (d, 6H, 2 CH3), 1.38 (s, 9H, 3 CH3), 1.51 (m, 2H, CH2), 1.72 (m, 1H, CH), 4.12 (m, 1H, CH),
13
6.85 (d, 1H, 1 NH), 7.2-7.77 (m, 5H, aromatic), 10.10, 10.40 (2 br.s, 2H, 2 NH); C-NMR (DMSO-
d6): δ 21.88, 23.00, 23.29, 24.47, 28.54, 51.64, 79.20, 126.00, 127.50, 131.65, 153.65, 156.11, 171.15;
Anal. Calcd for C18H27N3O4: C, 61.87; H, 7.79; N, 12.03. Found: C, 62.05; H, 7.92; N, 12.26.
Boc-phenylalanyl(N`-benzoyl)-hydrazide (5d): White powder, 0.31 g (81 % yield); mp 168°C; IR:
1
3314, 3233 (NH), 1725 (C=O, urethane), 1692 (C=O, amide), 1673 (C=O, amide) cm-1; H-NMR
(DMSO-d6): δ 1.30 (s, 9H, 3 CH3), 2.82, 3.08 (2m, 2H, CH2), 4.30 (m, 1H, CH), 7.04 (d, 1H, 1 NH),
7.18-7.92 (m, 10H, aromatic), 10.18, 10.29 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 28.50, 37.68,
54.85, 126.58, 127.80, 128.40, 128.81, 129.59, 132.18, 153.62, 164.9, 173.10; Anal. Calcd for
C21H25N3O4: C, 65.78; H, 6.57; N, 10.96. Found: C, 65.98; H, 6.78; N, 10.71.
Boc-(o-benzyl)-tyrosyl(N`-benzoyl)-hydrazide (5e): White crystals, 0.47 g (96 % yield); mp 177°C; IR:
1
3322, 3245 (NH), 1726 (C=O, urethane), 1693 (C=O, amide), 1673 (C=O, amide) cm-1; H-NMR
(DMSO-d6): δ 1.31 (s, 9H, 3 CH3), 2.85, 3.05 (2m, 2H, CH2), 4.25 (m, 1H, CH), 5.05 (br.s, 2H, CH2),
6.92-7.93 (m, 15H, aromatic, 1NH), 10.15, 10.46 (2s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 26.52,

Molecules 2005, 10
1225
32.01, 53.48, 68.50, 77.52, 112.81, 125.90, 126.02, 126.81, 128.69, 130.21, 135.6, 153.62, 155.04,
163.81, 170.00; Anal. Calcd for C28H31N3O5: C, 68.69; H, 6.38; N, 8.58. Found: C, 68.51; H, 6.15; N,
8.73.
General Procedure for the Reaction of N-Boc-L-Amino Acids with Nicotinic Acid Hydrazide.
A mixture of N-Boc-L-amino acid (1 mmol), HATU (0.38 g, 1 mmol) and Et3N (0.15 mL, 1 mmol)
was stirred at 0°C for 3 minutes in DMF (2 mL). Then nicotinic acid hydrazide (4, 0.123 g, 1 mmol)
was added. The reaction mixture was stirred at 0°C for 1 hour and left overnight at room temperature.
The reaction mixture was diluted with methylene chloride (80 mL) and the mixture was washed with
5% aqueous citric acid solution (2×10 mL), saturated sodium bicarbonate solution (2×10 mL) and
saturated sodium chloride solution (2×10 mL). The organic layer was dried over anhydrous sodium
sulphate, filtered and the solvent was removed in vacuo. The crude product was crystallized from
methylene chloride-hexane.
Boc-valyl(N`-nicotinoyl)-hydrazide (6a): White crystals, 0.28 g (83.3 % yield); mp 209°C; IR: 3344,
1
3242 (NH), 1726 (C=O, urethane), 1708 (C=O, amide), 1661 (C=O, amide) cm-1; H-NMR (DMSO-
d6): δ 0.86, 0.94 (2d, 6H, 2 CH3), 1.39 (s, 9H, 3 CH3), 1.96 (m, 1H, CH), 3.93 (m, 1H, CH), 6.81 (d,
1H, 1 NH), 7.77 (d, 2H, aromatic), 8.75 (d, 2H, aromatic), 10.12, 10.68 (2 br.s, 2H, 2 NH); 13C-NMR
(DMSO-d6): δ 18.7, 19.5, 28.6, 30.7, 58.7, 78.4, 121.7, 139.8, 150.7, 155.7, 164.1, 171.1; Anal. Calcd
for C16H24N4O4: C, 57.13; H, 7.19; N, 16.66. Found: C, 57.34; H, 7.35; N, 16.41.
Boc-(o-benzyl)-glutamyl(N`-nicotinoyl)-hydrazide (6b): White powder, 0.42 g (92 % yield); mp
120°C; IR: 3334, 3243 (NH), 1740 (C=O, ester), 1727 (C=O, urethane), 1681 (C=O, amide) cm-1; 1H-
NMR (DMSO-d6): δ 1.38 (s, 9H, 3 CH3), 1.84, 2.04 (2m, 2H, CH2), 2.32 (m, 2H, CH2-CO), 4.07 (m,
1H, CH), 5.14 m, 2H, CH2-O), 7.02 (m, 1H, 1 NH), 7.34 (br.s, 5H, aromatic), 7.77 (d, 2H, aromatic),
8.75 (d, 2H, aromatic), 10.13, 10.66 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 26.6, 28.3, 30.0,
53.6, 66.2, 78.7, 121.6, 128.1, 128.4, 128.5, 128.7, 136.3, 139.7, 150.8, 156.0, 164.3, 171.1, 172.6,
175.8; Anal. Calcd for C23H28N4O6: C, 60.52; H, 6.18; N, 12.27. Found: C, 60.37; H, 6.32; N, 12.45.
Boc-leucyl(N`- nicotinoyl)-hydrazide (6c): White crystals, 0.25 g (71.3 % yield); mp 210°C; IR: 3301,
1
3222 (NH), 1710 (C=O, urethane), 1662 (C=O, amide) cm-1; H-NMR (DMSO-d6): δ 0.91 (d, 6H, 2
CH3), 1.38 (s, 9H, 3 CH3), 1.53 (m, 2H, CH2), 1.72 (m, 1H, CH), 4.13 (m, 1H, CH), 6.85 (d, 1H, 1
NH), 7.77 (d, 2H, aromatic), 8.71 (d, 2H, aromatic), 10.10, 10.70 (2 br.s, 2H, 2 NH); 13C-NMR
(DMSO-d6): δ 21.88, 23.00, 23.29, 24.47, 28.54, 51.64, 78.75, 121.64, 139.77, 150.48, 155.55, 163.97,
172.38; Anal. Calcd for C17H26N4O4: C, 58.27; H, 7.48; N, 15.99. Found: C, 58.02; H, 7.21; N, 16.26.
Boc-phenylalanyl(N`-nicotinoyl)-hydrazide (6d): White powder, 0.29 g (75.4 %) yield, mp 145°C; IR:
1
3309, 3234 (NH), 1726 (C=O, urethane), 1695 (C=O, amide), 1677 (C=O, amide) cm-1; H-NMR
(DMSO-d6): δ 1.29 (s, 9H, 3 CH3), 2.82, 3.07 (2m, 2H, CH2), 4.31 (m, 1H, CH), 7.05-7.37 (m, 6H,
aromatic, 1 NH), 7.81 (d, 2H, aromatic), 8.76 (d, 2H, aromatic), 10.33, 10.82 (2 br.s, 2H, 2 NH)); 13C-
NMR (DMSO-d6): δ 28.12, 28.48, 37.82, 54.87, 78.4, 121.68, 126.62, 128.42, 129.57, 138.30, 139.70,

Molecules 2005, 10
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150.77, 155.67, 164.18, 171.70; Anal. Calcd for C20H24N4O4: C, 62.49; H, 6.29; N, 14.57. Found: C,
62.65; H, 6.50; N, 14.83.
Boc-(o-benzyl)-tyrosyl(N`-nicotinoyl)-hydrazide (6e): White crystals, 0.38 g (77.5 %) yield, mp 141°C;
1
IR: 3311, 3237 (NH), 1727 (C=O, urethane), 1695 (C=O, amide), 1677 (C=O, amide) cm-1; H-NMR
(DMSO-d6): δ 1.33 (s, 9H, 3 CH3), 2.83, 3.05 (2m, 2H, CH2), 4.26 (m, 1H, CH), 5.10 (br.s, 2H, CH2),
6.96-7.47 (m, 10H, aromatic, 1NH), 7.83 (d, 2H, aromatic), 8.78 (d, 2H, aromatic),10.36, 10.63 (2m,
2H, 2 NH); 13C-NMR (DMSO-d6): δ 28.20, 28.54, 37.03, 55.19, 69.50, 78.38, 114.77, 121.70, 127.99,
128.77, 130.63, 137.60, 140.06, 150.72, 155.68, 157.32, 164.08, 171.64; Anal. Calcd for C27H30N4O5:
C, 66.11; H, 6.16; N, 11.42. Found: C, 65.82; H, 5.95; N, 11.59.
General Procedure for the Preparation of the Hydrochloride Salts 7 and 8.
5 or 6 (1 mmol) were dissolved in a mixture of methylene chloride (4 mL) and ether (4 mL). HCl
gas was passed through the solution for 2 hours. A white precipitate that formed was filtered and
washed with anhydrous ether. The crude product was recrystallized from methyl alcohol-anhydrous
ether to give white crystals. The amino acid-(N`- nicotinoyl) hydrazide hydrochloride salts 8a-8e were
obtained as hygroscopic salts.
General Procedure for the Preparation of the Cu Complexes.
A solution of Cu(NO3)2 (0.48 gm, 2 mmol) in methyl alcohol (2 mL) was added to a solution of 5,
6, 7 or 8 (1 mmol) in methyl alcohol (4 mL). A change was observed in the color of the solution. The
Cu complexes were obtained as green crystals. The Cu complexes were later decomposed and their Cu
content was analyzed by atomic absorption to determine the ratio of complex formation of Cu to
ligand. The atomic absorption analysis showed the formation of complexes Cu:L in the ratio (1:1).
General Procedure for the Preparation of the Cd Complexes
A solution of Cd(CH3COO)2 (0.46 gm, 2 mmol) in methyl alcohol (2 mL) was added to a solution
of 5, 6, 7 or 8 (1 mmol) in methyl alcohol (4 mL). The Cd complexes were obtained as white crystals.
The Cd complexes were later decomposed and their Cd content was analyzed by atomic absorption to
determine the ratio of complex formation of Cd to ligand. The atomic absorption analysis showed the
formation of complexes Cd:L in the ratio (1:1).
In vitro antimicrobial activity
The microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and Sabouraud Liquid
Medium (Oxoid) were used for the determination of antibacterial and antifungal activity [17, 18]. The
utilized test organisms were: Escherichia coli (E. coli) ATCC 25922 as an example of Gram-negative
bacteria, Staphylococcus aureus (S. aureus) ATCC 19433 as an example of Gram-positive bacteria and
Candida albicans (C. albicans) as yeast-like fungi. Ampicillin trihydrate and clotrimazole were used
as standard antibacterial and antifungal agents, respectively. Solutions of the test compounds,

Molecules 2005, 10
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ampicillin trihydrate and clotrimazole were prepared in DMSO to a concentration of 1600 µg/mL.
Twofold dilutions of the compounds were prepared (800, 400, … 6.25 µg/mL). Microorganism
suspensions at 106 CFU/mL (Colony Forming Unit/mL) concentrations were inoculated to the
corresponding wells. Plates were incubated at 36°C for 24 h to 48 h. The incubation chamber was kept
sufficiently humid. At the end of the incubation period, the minimal inhibitory concentrations (MIC)
were determined.
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Sample availability: Available from the author.
© 2005 by MDPI (http:www.mdpi.org). Reproduction is permitted for noncommercial purposes.