Synthesis and Antitubercular Activity of Novel Amino Acid Derivatives

Article abstract of DOI:10.1111/j.1747-0285.2011.01269.x

In this work, 17 new N-acylhydrazone derivatives of amino acids have been evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv. The compounds 8b, 8e, 8f, 9a-d, and 10c exhibited an important minimum inhibitory concentration activity between 12.5 and 50μg/mL, which can be compared with that of the tuberculostatic drug d-cycloserine (20μg/mL). In this work 17 new N-acylhydrazone derivatives of amino acids have been evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv. Eight compounds were non-cytotoxic and exhibited an important MIC activity between 12.5 and 50μg/mL, which can be compared with that of the tuberculostatic drug d-cycloserine (20μg/mL).

Full text of DOI:10.1111/j.1747-0285.2011.01269.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01269.x
Chem Biol Drug Des 2012; 79: 216–222
Research Letter
Synthesis and Antitubercular Activity of Novel
Amino Acid Derivatives
Cristiane F. Da Costa1,2, Alessandra C.
found in peptidoglycan precursors, interfering in the transpeptida-
tion step (2,4).
Pinheiro1, Mauro V. De Almeida2, Maria C.
S. Lourenc¸o3 and Marcus V. N. De Souza1,*
In our continuous search of new potent and safe antitubercular
agents, we decided to synthesize a new class of N-acylhydrazone
derivatives of amino acids as attractive D-cycloserine analogs.
N-acylhydrazones are also described with a wide range of pharma-
cological activities, such as antibacterial agents (5–11).
1Instituto de Tecnologia em Fꢀrmacos – Farmanguinhos, FioCruz –
Fundażo Oswaldo Cruz, R. Sizenando Nabuco, 100 Manguinhos,
21041-250 Rio de Janeiro, RJ, Brazil
2Departamento de Quꢁmica, Instituto de CiÞncias Exatas,
Universidade Federal de Juiz de Fora, Cidade Universitꢀria, 36036-
900 Juiz de Fora, MG, Brazil
3Instituto de Pesquisas Clꢁnicas Evandro Chagas, Departamento de
Bacteriologia, FioCruz – Fundażo Oswaldo Cruz, Av. Brasil, 4365
Manguinhos, Rio de Janeiro, Brazil
Materials and Methods
General procedures
NMR spectra were obtained using a Bruker Avance spectrometer
operating at 400 or 500 MHz (1H) and 100 or 125 MHz (13C), and a
Bruker Avance DRX300 spectrometer operating at 300 MHz (1H) and
75 MHz (13C), in deuterated methanol, chloroform, or dimethylsulfox-
ide. Chemical shifts are reported in ppm relative to tetramethylsilane.
Proton and carbon spectra were typically obtained at room tempera-
ture. Thin-layer chromatography was carried out on silica gel plates,
using chloroform ⁄ methanol mixtures as eluents. For column chro-
matographic purification, column grade silica gel (0.063–0.200 mm
mesh size) was employed. Mass spectra (ESI) were obtained on a ZQ
electrospray spectrometer simple quadrupole. Melting points were
determined using a Microquimica MQAPF-301 (Microquimica, Santa
Catarina, Brazil) digital melting point apparatus.
*Corresponding author: Marcus V. N. De Souza,
marcos_souza@far.fiocruz.br
In this work, 17 new N-acylhydrazone derivatives
of amino acids have been evaluated for their in
vitro antibacterial activity against Mycobacterium
tuberculosis H37Rv. The compounds 8b, 8e, 8f,
9a–d, and 10c exhibited an important minimum
inhibitory concentration activity between 12.5
and 50 lg ⁄ mL, which can be compared with that
of
the
tuberculostatic
drug
D-cycloserine
(20 lg ⁄ mL).
Key words: amino acids, cell wall, hydrazones, tuberculosis
Chemistry
Received 12 February 2011, revised 16 September 2011 and accepted
for publication 29 October 2011.
The synthetic route used for the preparation of the title compounds
is outlined in Scheme 1. The amino acids L-phenylalanine 1a, L-leu-
cine 1b, and L-alanine 1c were employed as starting materials,
and t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz) protecting
groups were included in the core structures of 8a–j and 9a–d
because of an intrinsic instability observed in non-protected deriva-
tives.
Tuberculosis (TB) is a serious airborne disease caused by Mycobac-
terium tuberculosis, being considered since 1993 a global health
emergency by the World Health Organization (WHO).a Control and
prevention of TB are major challenge as one-third of the world's
population is infected with Mycobacterium (1). Some antibiotics
employed on tuberculosis treatment affect the metabolism of bacte-
rial cell wall construction, which is a very important cellular compo-
nent surrounding the cell membrane, providing additional support
and protection (2). Basically, it is comprised of three covalently
linked substructures: mycolic acids, peptidoglycan, and arabinogalac-
tan, which represent over 60% of cell dry weight (3). The peptido-
glycan is formed by extensive chains of polysaccharides and amino
acid residues. The D-cycloserine, a second-line drug for tuberculosis
treatment, binds to the terminal portion of D-ala-D-ala of a peptide
Compounds 8a–j, 9a–d, and 10a–c were prepared from the
appropriate amino acids 1a–c, by esterification, leading to 2a–c,
followed by N-protection to furnish 3a–c or 4a–c, using (BOC)2O
or CbzCl, respectively. These compounds were converted into their
acyl hydrazine derivatives 5a–c, 6a–b, and 7a–b using 80%
aqueous hydrazine hydrate. After washing with cold ethanol the
pure products were obtained in 66–85% overall yield. Finally, after
condensation reactions of these compounds with substituted alde-
hydes, the desired derivatives 8a–j, 9a–d, and 10a–c were
obtained in 50–84% yield.
216

Synthesis and Antitubercular Activity of Amino Acid Derivatives
O
O
O
R
R
R
a
b,c
OH
OMe
OMe
NH2
NH2
2a-c
NHR1
3a-c: R1 = BOC
4a-c: R1 = Cbz
1a: R = C6H5CH2-;
1b: R = (CH3)2CHCH2-;
1c: R = CH3-;
d
O
O
N
Ar/Het
NH2
R
R
e
N
H
N
H
NHR1
NHR1
8a-j: R1 = BOC
9a-d: R1= Cbz
10a-c: R1= H
5a-c: R1 = BOC
6a-b: R1 = Cbz
7a-b: R1 = H
Scheme 1: Reagents and conditions: (a) SOCl2, MeOH, 60 ꢀC, 4 h; (b) (BOC)2O, Et3N, THF, rt; (c) CbzCl, THF, H2O, K2CO3, 0 ꢀC, 3 h; (d)
NH2-NH2, EtOH, rt, 24 h; (e) Ar ⁄ Het–CHO, EtOH, rt, 24 h.
Experimental
J = 7.2 Hz, CH3); 3.84 (3H, s, OCH3); 4.11 (1H, q, J = 7.2 Hz,
CHNH2) ppm NMR- 13C (125 MHz, CD3OD) d: 16.3; 49.9; 53.8;
171.6. IR (m, cm-1, NaCl): 2743, 1726. MS ⁄ ESI (m ⁄ z) calcd: 103.1
(M+, free base), found: 104.2 (M+H).
General procedures for the synthesis of methyl
ester derivatives of L-amino acids (2a–c)
To a stirred solution of thionyl chloride (200 mmol) in methanol
(100 mL) at 0 ꢀC was added the appropriated L-amino acid
(40 mmol). The reaction mixture was stirred for 24 h at room tem-
perature, and the solvent was removed to give 2a–c in quantitative
yield.
General procedures for the synthesis of tert-
butoxycarbonylamino derivatives 3a–c
To a reaction mixture containing the appropriate methyl ester deriv-
ative 2a–c (9 mmol) and triethylamine (10.8 mmol) in anhydrous
THF (20 mL) at room temperature was added (BOC)2O (13.5 mmol).
The reaction mixture was stirred for 24 h at room temperature,
quenched with water (40 mL), and extracted with ethyl acetate. The
combined organic phases were dried over anhydrous sodium sulfate
and concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (hexane ⁄ ethyl acetate 7:3)
affording 3a–c in 65–75% yield.
(2S)-1-metoxy-1-oxo-3-phenyl-2-propylamonium hydro-
chloride 2a: Yield: 98%, MP: 157–158 ꢀC. NMR-1H (500 MHz,
CD3OD) d: 2.03 (2H, s, NH); 3.19 (1H, dd, J1 = 6.2 Hz, J2 = 14.3 Hz,
CHPh); 3.26 (1H, dd, J1 = 6.2 Hz, J2 = 14.3 Hz, CHPh); 3.80 (3H, s,
OCH3); 4.32 (1H, t, J 6.8 Hz, CHNH2); 7.27 (2H, d, J = 7.1 Hz, Ph (H2
=
and H6); 7.31 (1H, dd, J1 = 4.9 Hz, J2 = 9.6 Hz, H4); 7.37 (2H, t,
J = 7.1 Hz, Ph (H3 and H5) ppm NMR- 13C (125 MHz, CD3OD) d: 37.5;
53.7; 55.4; 129.0; 130.2; 130.6; 133.4; 135.5; 170.5 ppm IR (m, cm-1,
KBr): 2983, 2841, 2702, 1745. MS ⁄ESI (m⁄z) calcd: 179.0 (M+, free
base), found: 179.9 (M+H).
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-phenyl-
propanoate 3a: Yield: 70%, oil. NMR-1H (500 MHz, CD3OD) d:
1.38 (9H, s, (CH3)3); 2.89 (1H, dd, J1 = 6.2 Hz, J2 = 13.5 Hz, CHPh);
3.09 (1H, dd, J1 = 6.2 Hz, J2 = 13.5 Hz, CHPh); 3.68 (3H, s, OCH3);
4.35 (1H, dd, J1 = 5.5 Hz, J2 = 9.0 Hz, CHNH); 5.07 (1H, d,
J = 7.2 Hz, NH); 7.20 (3H, t, J = 7.5 Hz, H2, H4 and H6); 7.27 (2H,
t, J = 7.5 Hz, H3 and H5) ppm NMR- 13C (125 MHz, CD3OD) d:
29.3; 38.8; 52.7; 56.7; 80.7; 127.3–131.0; 138.5; 157.9; 174.3 ppm
IR (m, cm-1, NaCl): 3380, 3028, 2978, 1712, 1496. MS ⁄ ESI (m ⁄ z)
calcd: 279.3 (M+), found: 301.9 (M+Na).
(2S)-4-methyl-1-metoxy-1-oxo-2-pentylamonium hydro-
chloride 2b: Yield: 98%, MP: 134–136 ꢀC. NMR-1H (500 MHz,
CD3OD) d: 1.07 (6H, q, J = 7.0 Hz, (CH3)2); 1.30 (1H, m, CH(CH3)2); 3.85
(3H, s, OCH3); 3.94 (1H, d, J = 4.7 Hz, CHNH2) ppm NMR- 13C
(125 MHz, CD3OD) d: 22.5; 22.7; 25.7; 40.8; 52.6; 53.8; 171.5 ppm IR
(m, cm-1, KBr): 2937, 2870, 1737. MS ⁄ESI (m⁄z) calcd: 144.9 (M+, free
base), found: 145.9 (M+H).
(2S)-1-metoxy-1-oxo-2-propylamonium hydrochloride
2c: Yield: 96%, oil. NMR-1H (500 MHz, CD3OD) d: 1.54 (3H, d,
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-4-methyl-
pentanoate 3b: Yield: 75%, oil. NMR-1H (400 MHz, CDCl3) d:
Chem Biol Drug Des 2012; 79: 216–222
217

Da Costa et al.
1.37 (6H, q, J1 = 4.5 Hz, J2 = 8.0 Hz, (CH3)2); 1.44 (9H, s, (CH3)3);
1.50 (1H, m, CH(CH3)2); 1.59 (2H, m, CH2); 3.79 (3H, s, OCH3); 4.32
(1H, m, CHNH); 4.89 (1H, d, J = 7.2 Hz, NH) ppm NMR- 13C
(125 MHz, CDCl3) d: 22.0; 22.1; 25.0; 28.5; 42.1; 52.2; 52.3; 80.0;
155.6; 174.2 ppm IR (m, cm-1, NaCl): 3334, 2956, 1728, 1527.
MS ⁄ ESI (m ⁄ z) calcd: 245.1 (M+), found: 268.1 (M+Na).
CD3OD) d: 1.36 (9H, s, (CH3)3); 2.83 (1H, dd, J1 = 9.2 Hz;
J2 = 13.5 Hz; CHPh); 3.04 (1H, dd, J1 = 9.2 Hz; J2 = 13.5 Hz,
CHPh); 4.25 (1H, t, J = 6.5 Hz; CHNH); 7.18–7.28 (5H, m, Ph) ppm
NMR- 13C (125 MHz, CD3OD) d: 29.3; 39.6; 56.3; 80.8; 127.2;
128.5; 128.9; 129.8; 130.2; 131.1; 138.6; 157.6; 173.5 ppm IR (m,
cm-1, KBr): 3327, 3058, 3030, 1689, 1537. MS ⁄ ESI (m ⁄ z) calcd:
279.2 (M+), found: 302.0 (M+Na).
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-propano-
ate 3c: Yield: 65%, oil. NMR-1H (500 MHz, CDCl3) d: 1.37 (3H, t,
J = 8.0 Hz, CH3); 1.43 (9H, s, (CH3)3); 3.79 (3H, s, OCH3); 4.29 (1H,
m, CHNH) ppm NMR- 13C (125 MHz, CDCl3) d: 19.2; 28.0; 49.3; 525;
80.1; 155.3; 174.1 ppm IR (m, cm-1, NaCl): 3325, 2943, 1731.
MS ⁄ ESI (m ⁄ z) calcd: 203.1 (M+), found: 204.2 (M+H).
Tert-butyl (2S)-2-hydrazino-1-isobutyl-2-oxoethylcar-
bamate 5b: Yield: 85%, MP: 73.4–75.0 ꢀC. NMR-1H (300 MHz,
DMSO-d6) d: 1.79 (6H, m, (CH3)2); 1.83 (9H, m, (CH3)3); 2.18 (2H, m,
CH2); 2.26 (2H, m, NH2); 2.81 (1H, m, CH(CH3)2); 4.31 (1H, m, CHNH);
8.25 (1H, s, OCONH); 9.70 (1H, s, NHN) ppm NMR- 13C (125 MHz,
DMSO-d6) d: 17.1; 24.9; 28.1; 30.7; 78.7; 153.2 ppm IR (m, cm-1,
KBr): 3354, 3273, 2958, 1681, 1631. MS ⁄ ESI (m ⁄ z) calcd: 245.1
(M+), found: 268.1 (M+Na).
General procedures for the synthesis of
benzyloxycarbonylamino derivatives 4a–b
To a reaction mixture containing the appropriate methyl ester deriv-
ative 2a–b (9 mmol), water (50 mL), diethyl ether (40 mL), and
sodium bicarbonate (50 mmol) at 0 ꢀC was added dropwise benzyl
chloroformate (14 mmol). After 2 h at 0 ꢀC and 1 h at room temper-
ature, the reaction mixture was quenched with pyridine (8 mL), and
water was added to solubilize all salts (40 mL). The organic layer
was washed with HCl (2.5 N), dried (MgSO4), filtered, and concen-
trated. The residue was chromatographed on silica gel (hex-
ane ⁄ ethyl acetate 8:2) affording 4a–b in 63–75% yield.
Tert-butyl (2S)-2-hydrazino-1-methyl-2-oxoethylcar-
bamate 5c: Yield: 70%, MP: 138.9–142.6 ꢀC. NMR-1H (500 MHz,
CDCl3) d: 1.43 (9H, s, (CH3)3); 2.10 (2H, s, NH2); 4.23 (1H, m, CHNH);
5.37 (1H, d, J = 7.0 Hz, CHNH); 9.49 (1H, s, NHN) ppm NMR- 13C
(125 MHz, CDCl3) d: 18.3; 28.0; 47.9; 79.6; 155.6; 175.3 ppm IR (m,
cm-1, KBr): 3329, 3253, 2985, 1676, 1639. MS ⁄ ESI (m ⁄ z) calcd:
203.1 (M+), found: 226.3 (M+Na).
Benzyl
(1S)-1-benzyl-2-hydrazino-2-oxoethylcarba-
mate 6a: Yield: 66%, MP: 170.5–171.8 ꢀC. NMR-1H (500 MHz,
CDCl3) d: 2.87 (1H, dd, J1 = 9.2 Hz, J2 = 13.5 Hz, CHPh); 3.07 (1H,
dd, J1 = 9.2 Hz, J2 = 13.5 Hz, CHPh); 4.32 (1H, dd, J1 = 6.0 Hz,
J2 = 8.5 Hz, CHNH); 4.99 (2H, s, CH2O); 5.02 (2H, m, CHNH); 7.19–
7.33 (10H, m, Ph) ppm NMR- 13C (100 MHz, CDCl3) d: 37.8, 54.9,
65.2, 126.2, 127.4, 127.6, 128.0, 128.2, 129.0, 129.2, 137.0, 138.0,
155.7, 170.7 ppm IR (m, cm-1, KBr): 3300, 3058, 3030, 1689, 1537.
MS ⁄ ESI (m ⁄ z) calcd: 313.1 (M+), found: 336.1 (M+Na).
Methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-phenyl-
propanoate 4a: Yield: 75%, oil. MR-1H (400 MHz, CDCl3) d:
2.92 (1H, dd, J1 = 9.2 Hz, J2 = 13.6 Hz CHPh); 3.13 (1H, dd,
J1 = 9.2 Hz, J2 = 13.6 Hz, CHPh); 3.68 (3H, s, OCH3); 4.44 (1H, dd,
J1 = 5.6 Hz, J2 = 8.8 Hz, CHNH); 5.06 (2H, s, CH2O); 7.18–7.33
(10H, m, Ph) ppm NMR- 13C (100 MHz, CDCl3) d: 38.7; 52.8; 57.1;
67.7; 128.0; 128.8; 129.1; 129.6; 130.4; 138.3; 138.4; 158.5; 174.1.
IR (m, cm-1, NaCl): 3354, 3030, 2951, 1728, 1518. MS ⁄ ESI (m ⁄ z)
calcd: 313.3 (M+), found: 336.0 (M+Na).
Benzyl (2S)-2-hydrazino-1-isobutyl-2-oxoethylcarba-
mate 6b: Yield: 71%, MP: 120.9–130.0 ꢀC. NMR-1H (500 MHz,
CD3OD) d: 0.93 (6H, q, J1 = 6.5 Hz, J2 = 11.5 Hz, (CH3)2); 1.50 (2H,
m, CH2); 1.66 (1H, m, CH(CH3)2); 4.13 (1H, dd, J1 = 6.0 Hz,
J2 = 9.5 Hz, CHNH); 5.08 (2H, m, CH2O); 7.29–7.34 (5H, m, Ph) ppm
NMR- 13C (125 MHz, CD3OD) d: 23.0; 26.4; 42.3; 54.4; 67.8; 128.4–
130.3; 138.3; 158.5; 174.6 ppm IR (m, cm-1, KBr): 3305, 3057, 2947,
1683, 1537. MS ⁄ ESI (m ⁄ z) calcd: 279.2 (M+), found: 302.1 (M+Na).
Methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-methyl-
pentanoate 4b: Yield: 63%, oil. NMR-1H (500 MHz, CDCl3) d:
0.94 (6H, m, (CH3)2); 1.53 (1H, m, CH(CH3)2); 1.67 (2H, m, CH2); 3.74
(3H, s, OCH3); 4.41 (1H, m, CHNH); 5.12 (2H, s, CH2O); 5.17 (1H, d,
J = 8.0 Hz, CHNH); 7.36 (5H, s, Ph) ppm NMR- 13C (125 MHz,
CDCl3) d: 22.5; 25.4; 42.0; 51.9; 53.2; 67.2; 127.7–129.3; 156.1;
173.8 ppm IR (m, cm-1, NaCl): 3361, 2958, 2872, 1712, 1516.
MS ⁄ ESI (m ⁄ z) calcd: 279.2 (M+), found: 302.2 (M+Na).
(2S)-2-amino-3-phenylpropanehydrazide 7a: Yield: 80%,
MP: 91.2–92.4 ꢀC. NMR-1H (400 MHz, CD3OD) d: 2.80 (1H, dd,
J1 = 7.2 Hz, J2 = 13.2 Hz, CHPh); 2.97 (1H, m, J1 = 13.2 Hz,
J2 = 11.5 Hz CHPh); 3.48 (1H, m, CHNH2); 7.24 (5H, m, Ph) ppm
NMR- 13C (100 MHz, CD3OD) d: 42.7; 56.8; 127.9; 129.7; 130.5;
138.9; 175.7 ppm IR (m, cm-1, KBr): 3348, 3178, 3024, 1670, 1566.
MS ⁄ ESI (m ⁄ z) calcd: 179.1 (M+), found: 180.2 (M+H).
General procedures for the synthesis of N-acyl
hydrazine derivatives 5a–c, 6a–b and 7a–b
To a stirred solution of 3a–c or 4a–c (10 mmol) in ethanol (20 mL)
at room temperature was added N2H4ÆH2O (80%, 40 mmol). The
reaction mixture was stirred for 24 h at room temperature and con-
centrated under reduced pressure. The residue was purified by
chromatography column (dichloromethane ⁄ methanol 9.5:0.5), leading
the pure derivative 5a–c, 6a–b, or 7a–b in 59–85% yield.
(2S)-2-amino-4-methylpentanehydrazide 7b: Yield: 60%,
oil. NMR-1H (500 MHz, CD3OD) d: 1.02 (6H, m, (CH3)2); 1.76 (2H, m,
CH2); 1.92 (1H, m, CH(CH3)2); 2.10–2.11 (2H, m, NHNH2); 4.12 (1H,
m, CHNH) ppm NMR- 13C (125 MHz, CD3OD) d: 23.8; 24.7; 25.7;
42.0; 51.6; 172.4 ppm IR (m, cm-1, NaCl): 3302, 2964, 1678. MS ⁄ ESI
(m ⁄ z) calcd: 131.1 (M+), found: 169.1 (M+Na).
Tert-butyl (1S)-1-benzyl-2-hydrazino-2-oxoethylcarba-
mate 5a: Yield: 83%, MP: 126.7–127.7 ꢀC. NMR-1H (500 MHz,
218
Chem Biol Drug Des 2012; 79: 216–222

Synthesis and Antitubercular Activity of Amino Acid Derivatives
General procedures for the synthesis of N-acyl
J = 4.4 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 7.14 (d, J = 6.8 Hz) and 7.54 (d,
J = 4.4 Hz) (1H, H3¢ (E ⁄ Z)-isomer); 8.11 (1H, m, CHNH); 8.17 and
8.51 (1H, s, CH=N (E ⁄ Z)-isomer); 11.66 and 11.89 (H, s, NHN (E ⁄ Z)-
isomer) ppm NMR- 13C (125 MHz, DMSO-d6) d: 25.6; 28.1; 41.9;
50.2; 80.3; 127.6; 127.9; 137.4; 145.9; 145.4; 152.3; 156.0; 156.5;
170.3 ppm IR (m, cm-1, KBr): 3350, 3134, 2958, 2872, 1687, 1516,
1334. MS ⁄ ESI (m ⁄ z) calcd: 368.2.1 (M+), found: 367.2 (M-H).
hydrazone derivatives 8a–j, 9a–d, and 10a–c
To a stirred solution of 5a–c, 6a–b, or 7a–b (0.5 mmol) in etha-
nol (10 mL) at room temperature was added the appropriate benzal-
dehyde (0.52 mmol). The reaction mixture was stirred for 24 h at
room temperature and concentrated under reduced pressure. The
residue was purified by chromatography column (dichlorome-
thane ⁄ methanol 9:1), affording the derivatives 9a–c or 10a–c in
25–84% yield.
Tert-butyl (1S)-1-isobutyl-2-{(2E/Z)-2-[(2-furyl)methy-
lene]hydrazine]-2-oxoethylcarbamate 8e: Yield: 60%.
MP: 140.1–141.5 ꢀC. NMR-1H (500 MHz, CDCl3) d: 0.94 and 1.07
(6H, m, (CH3)2 (E ⁄ Z)-isomer); 1.44 (9H, s, (CH3)3); 1.60 and 1.71 (2H,
m, CH2); 1.80 (1H, m, CH(CH3)2; 4.25 and 5.43 (1H, m, CHNH (E ⁄ Z)-
isomer); 5.22 (1H, m, H3¢); 6.43 (d, J = 3.2 Hz) and 6.72 (d,
J = 3.6 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 6.47 (m) and 6.76 (d, J = 4.0 Hz)
(1H, H2¢ (E ⁄ Z)-isomer); 7.46 and 7.48 (1H, s, CHNH (E ⁄ Z)-isomer);
7.78 and 8.13 (1H, s, CH=N (E ⁄ Z)-isomer); 9.95 and 10.53 (1H, s,
NHN (E ⁄ Z)-isomer) ppm NMR- 13C (125 MHz, CD3OD) d: 21.6; 23.4;
24.7; 28.4; 41.0; 42.1; 50.0; 52.2; 79.5; 138.7; 144.4; 144.6; 149.3;
149.5; 155.6; 156.3; 169.6; 175.8 ppm IR (m, cm-1, KBr): 3331, 3064,
2966, 2899, 1666. MS ⁄ ESI (m ⁄ z) calcd: 323.2 (M+), found: 322.1
(M-H).
Tert-butyl (1S)-1-benzyl-2-{(2E/Z)-2-[(5-nitro-2-furyl)-
methylene]hydrazine]-2-oxoethylcarbamate 8a: Yield:
70%. MP: 174.7–175.6 ꢀC. NMR-1H (400 MHz, DMSO-d6) d: 1.32
(9H, s, (CH3)3); 2.79 (1H, m, CHPh); 2.92 (1H, m, CHPh); 4.21 and
4.97 (1H, m, CHNH, (E ⁄ Z)-isomer); 7.30 (5H, m, Ph); 7.20 and 7.53
(1H, m, H4¢ (E ⁄ Z)-isomer); 7.22 and 7.56 (1H, m, H3¢ (E ⁄ Z)-isomer);
8.13 (1H, s, CHNH); 7.96 and 8.16 (1H, s, CH=N (E ⁄ Z)-isomer); 11.77
and 11.94 (1H, s, NHN (E ⁄ Z)-isomer) ppm NMR- 13C (75 MHz,
DMSO-d6) d: 28.2, 36.2, 37.1, 54.1, 55.3, 78.1, 78.4, 114.7, 115.0,
115.5, 126.4, 128.2, 129.3, 129.5, 131.2, 134.9, 137.7, 138.7, 151.6,
151.7, 151.9, 155.5, 155.7, 169.2, 173.9 ppm IR (m, cm-1, KBr): 3327,
3026, 2983, 1670, 1523, 1489, 1352. MS ⁄ ESI (m ⁄ z) calcd: 402.1
(M+), found: 425.1 (M+Na).
Tert-butyl
(1S)-1-isobutyl-2-{(2E/Z)-2-[(2-thienyl)-
Tert-butyl
(1S)-1-benzyl-2-{(2E/Z)-2-[(5-nitro-2-thie-
8b:
methylene]hydrazine]-2-oxoethylcarbamate 8f: Yield:
69%. MP: 169.5–173.1 ꢀC. NMR-1H (400 MHz, DMSO-d6) d: 0.88
and 0.98 (6H, m, (CH3)2 (E ⁄ Z)-isomer); 1.38 (9H, s, (CH3)3); 1.46 (2H,
m, CH2); 1.62 and 1.72 (2H, m, CH(CH3)2 (E ⁄ Z)-isomer); 4.00 and
4.83 (1H, m, CHNH (E ⁄ Z)-isomer); 7.11 (1H, m, H4¢); 7.40 (d,
J = 3.0 Hz) and 7.62 (m) (1H, H3¢ (E ⁄ Z)-isomer); 7.44 (d, J = 3.2 Hz)
and 7.64 (m) (1H, H2¢ (E ⁄ Z)-isomer); 8.15 (1H, s, CHNH); 8.19 and
8.44 (1H, s, CH=N (E ⁄ Z)-isomer); 11.22 and 11.46 (1H, s, NHN (E ⁄ Z)-
isomer) ppm NMR- 13C (100 MHz, DMSO-d6) d: 23.7; 24.5; 28.2;
49.4; 51.9; 77.8; 129.0; 129.4; 130.8; 138.0; 138.2; 139.0; 139.5;
142.0; 155.4; 1556.6; 169.2; 174.1 ppm IR (m, cm-1, KBr): 3367,
2958, 2870, 1689. MS ⁄ ESI (m ⁄ z) calcd: 339.2 (M+), found: 362.1
(M+Na).
nyl)methylene]hydrazine]-2-oxoethylcarbamate
Yield: 84%. MP: 180.9–182.8 ꢀC. NMR-1H (300 MHz, DMSO-d6) d:
1.32 (9H, s, (CH3)3); 2.79 (1H, m, CHPh); 2.92 (1H, m, CHPh); 4.21
and 4.97 (1H, m, CHNH (E ⁄ Z)-isomer); 7.20 and 7.53 (1H, m, H4¢
(E ⁄ Z)-isomer); 7.30 (5H, m, Ph); 7.22 and 7.56 (1H, m, H3¢ (E ⁄ Z)-iso-
mer); 8.12 and 8.13 (1H, s, CHNH); 8.14 and 8.46 (1H, s, CH=N,
(E ⁄ Z)-isomer); 11.77 and 11.94 (1H, s, NHN (E ⁄ Z)-isomer) ppm
NMR- 13C (75 MHz, DMSO-d6) d: 28.1; 36.4; 52.7; 55.2; 78.1; 126.4;
128.1; 129.2; 137.7; 138.1; 140.5; 146.6; 150.5; 155.4; 169.0;
173.5 ppm IR (m, cm-1, KBr): 3336, 2983, 2889, 1676, 1527, 1330.
MS ⁄ ESI (m ⁄ z) calcd: 418.1 (M+), found: 457.1 (M+K).
Tert-butyl (1S)-1-isobutyl-2-{(2E/Z)-2-[(5-nitro-2-furyl)-
methylene]hydrazine]-2-oxoethylcarbamate 8c: Yield:
54%. MP: 112.4–113.5 ꢀC. NMR-1H (500 MHz, CDCl3) d: 0.95 and
1.08 (6H, m, (CH3)2 (E ⁄ Z)-isomer); 1.46 (9H, s, (CH3)3); 1.54 (2H, m,
CH2); 1.72 and 1.84 (1H, m, CH(CH3)2 (E ⁄ Z)-isomer); 4.24 and 5.16
(1H, m, CHNH (E ⁄ Z)-isomer); 6.89 (d, J = 3.8 Hz) and 7.33 (d,
J = 3.5 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 7.09 (d, J = 3.5 Hz) and 7.36 (d,
J = 3.5 Hz) (1H, H3¢ (E ⁄ Z)-isomer); 7.83 (1H, m, CHNH); 8.31 and
8.39 (1H, s, CH=N (E ⁄ Z)-isomer); 10.24 and 10.70 (1H, s, NHN (E ⁄ Z)-
isomer) ppm NMR- 13C (125 MHz, CDCl3) d: 21.0; 22.8; 24.2; 25.6;
28.1; 49.2; 52.0; 77.8; 129.2; 129.4; 130.4; 130.5; 136.5; 140.3;
146.4; 146.6; 150.3; 150.7; 155.4; 155.5; 169.8; 171.6; 174.5 ppm IR
(m, cm-1, KBr): 3215, 2958, 2872, 1693, 1516, 1352. MS ⁄ ESI (m ⁄ z)
calcd: 368.2.1 (M+), found: 367.2 (M-H).
Tert-butyl (1S)-1-methyl-2-{(2E/Z)-2-[(5-nitro-2-furyl)-
methylene]hydrazine]-2-oxoethylcarbamate 8g: Yield:
54%. MP: 157.5–159.0 ꢀC. NMR-1H (500 MHz, CDCl3) d: 1.44
(9H, s, (CH3)3); 1.46 (3H, s, CH3); 4.33 and 5.10 (1H, m, CHNH
(E ⁄ Z)-isomer); 6.93 (d, J = 3.5 Hz) and 7.33 (m) (1H, H4¢ (E ⁄ Z)-
isomer); 7.08 (m) and 7.36 (d, J = 3.5 Hz) (1H, H3¢ (E ⁄ Z)-isomer);
7.89 and 8.28 (1H, s, CH=N (E ⁄ Z)-isomer); 10.56 and 10.80 (1H,
s, NHN, (E ⁄ Z)-isomer) ppm NMR- 13C (125 MHz, CDCl3) d: 16.9;
17.5, 28.2; 46.6; 48.1; 80.3; 113.3; 113.4; 114.2; 132.1; 133.0;
155.8; 156.4; 170.4; 176.2 ppm IR (m, cm-1, KBr): 3327, 3064,
2981, 1670, 1525, 1352. MS ⁄ ESI (m ⁄ z) calcd: 326.1 (M+), found:
325.1 (M-H).
Tert-butyl (1S)-1-methyl-2-{(2E/Z)-2-[(5-nitro-2-thienyl)
methylene]hydrazine]-2-oxoethylcarbamate 8h: Yield:
62%. MP: 143.1–145.9 ꢀC. NMR-1H (500 MHz, CD3OD) d: 1.23 (3H,
m, CH3); 1.38 (9H, s, (CH3)3); 4.01 and 4.74 (1H, m, CHNH (E⁄ Z)-iso-
mer); 7.11 (d, J = 7.6 Hz) and 7.52 (d, J = 4.0 Hz) (1H, H4¢ (E⁄ Z)-iso-
mer); 7.20 (d, J = 7.6 Hz) and 7.54 (d, J = 4.0 Hz) (1H, d, H3¢ (E⁄ Z)-
isomer); 8.12 and 8.13 (1H, s, CHNH (E⁄ Z)-isomer); 8.17 and 8.49 (1H,
Tert-butyl (1S)-1-isobutyl-2-{(2E/Z)-2-[(5-nitro-2-thie-
nyl)methylene]hydrazine]-2-oxoethylcarbamate
8d:
Yield: 71%. MP: 103.0–105.4 ꢀC. NMR-1H (500 MHz, DMSO-d6) d:
0.88 and 0.99 (6H, m, (CH3)2 (E ⁄ Z)-isomer); 1.38 (9H, s, (CH3)3); 1.51
(2H, m, CH2); 1.63 and 1.73 (1H, m, CH(CH3)2 (E ⁄ Z)-isomer); 4.01
and 4.84 (1H, m, CHNH (E ⁄ Z)-isomer); 7.01 (m) and 7.51 (d,
Chem Biol Drug Des 2012; 79: 216–222
219

Da Costa et al.
s, CH=N (E ⁄Z)-isomer); 11.74 and 11.88 (1H, s, NHN, (E⁄ Z)-isomer)
ppm NMR- 13C (125 MHz, CD3OD) d: 16.7; 17.6; 28.2; 46.7; 49.1;
79.2; 129.0; 130.7; 136.5; 140.3; 146.7;v150.4; 150.8; 155.2; 170.1;
174.6 ppm IR (m, cm-1, KBr): 3338, 3103, 2983, 2933, 1678, 1525,
1332. MS⁄ ESI (m⁄ z) calcd: 342.1 (M+), found: 343.1 (M+H).
Benzyl
(1S)-1-isobutyl-2-{(2E/Z)-2-[(5-nitro-2-furyl)-
methylene]hydrazine]-2-oxoethylcarbamate 9c: Yield:
70%. MP: 89.3–92.6 ꢀC. NMR-1H (500 MHz, DMSO-d6) d: 0.89 and
0.99 (6H, m, (CH3)2 (E ⁄ Z)-isomer); 1.42 (2H, m, CH(CH3)2); 1.56 (1H, m,
CH2); 4.11 and 4.93 (1H, m, H2, (E ⁄ Z)-isomer); 5.03 (2H, m, CH2O);
7.14 (d, J = 4.0 Hz) and 7.54 (d, J = 8.0 Hz) (1H, H4¢ (E ⁄ Z)-isomer);
7.35 (5H, m, Ph); 7.22 (d, J = 4.0 Hz) and 7.68 (d, J = 7.8 Hz) (1H,
H3¢ (E ⁄ Z)-isomer); 7.77 (1H, m, CHNH); 7.93 and 8.21 (1H, s, CH=N
(E ⁄ Z)-isomer); 11.72 and 11.96 (1H, s, NHN (E ⁄ Z)-isomer) ppm NMR-
13C (500 MHz, DMSO-d6) d: 20.8, 21.5, 22.8, 23.2, 24.2, 24.6, 50.0,
52.4, 114.4, 115.2, 127.7, 128.3, 131.1, 135.0, 137.6, 151.7, 156.2,
169.7, 174.6 ppm IR (m, cm-1, KBr): 3209, 3032, 2956, 1691, 1680,
1517, 1352. MS ⁄ ESI (m ⁄ z) calcd: 402.1 (M+), found: 425.1 (M+Na).
Tert-butyl (1S)-1-methyl-2-{(2E/Z)-2-[(2-furyl)methyle-
ne]hydrazine]-2-oxoethylcarbamate 8i: Yield: 65%. MP:
158.0–159.6 ꢀC. NMR-1H (500 MHz, CD3OD) d: 1.24 (3H, d,
J = 7.0 Hz, CH3); 1.44 (9H, s, (CH3)3); 4.14 and 4.96 (1H, m, CHNH,
(E ⁄ Z)-isomer); 7.08 (1H, m, H3¢); 7.31 (d, J = 3.5 Hz) and 7.49 (d,
J = 5.0 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 7.38 (d, J = 3.2 Hz) and 7.52 (d,
J = 5.0 Hz) (1H, H2¢ (E ⁄ Z)-isomer); 8.12 and 8.35 (1H, s, CH=N
(E ⁄ Z)-isomer); 10.88 and 11.30 (1H, s, NHN, (E ⁄ Z)-isomer) ppm
NMR- 13C (125 MHz, CD3OD) d: 18.0; 28.8; 50.8; 80.8; 128.7; 129.5;
130.4; 132.4; 139.6; 140.6; 141.0; 145.4; 157.8; 172.5; 176.6 ppm IR
(m, cm-1, KBr): 3332, 3043, 2981, 2889, 1675. MS ⁄ ESI (m ⁄ z) calcd:
281.1 (M+), found: 280.2 (M-H).
Benzyl (1S)-1-isobutyl-2-{(2E ⁄ Z)-2-[(5-nitro-2-thienyl)-
methylene]hydrazine]-2-oxoethylcarbamate 9d: Yield:
75%. MP: 90.8–94.0 ꢀC. NMR-1H (400 MHz, Acetone-d6) d: 0.89
and 0.99 (6H, m, (CH3)2 (E ⁄ Z)-isomer); 1.42 (2H, m, CH2); 1.42 and
1.54 (1H, m, CH(CH3)2 (E ⁄ Z)-isomer); 4.12 and 4.92 (1H, m, CHNH
(E ⁄ Z)-isomer); 5.04 (2H, m, CH2O); 7.36 (5H, m, Ph); 7.52 (d,
J = 4.4 Hz) and 7.58 (d, J = 8.0 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 7.55 (d,
J = 4.4 Hz) and 7.70 (d, J = 7.8 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 8.12 (1H,
m, CHNH); 8.18 and 8.51 (1H, s, CH=N (E ⁄ Z)-isomer); 11.76 and
11.98 (1H, s, NHN (E ⁄ Z)-isomer) ppm NMR- 13C (100 MHz, Acetone-
d6) d: 21.1; 23.8; 24.5; 29.1; 49.8; 52.4; 65.4; 127.8; 128.4; 129.5;
130.7; 136.9; 140.7; 146.4; 150.5; 150.9; 156.2; 170.0; 174.4 ppm IR
(m, cm-1, KBr): 3331, 3130, 2958, 2870, 1691, 1647, 1516, 1334.
MS ⁄ ESI (m ⁄ z) calcd: 418.1 (M+), found: 441.1 (M+Na).
Tert-butyl (1S)-1-methyl-2-{(2E/Z)-2-[(2-thienyl)methylene]
hydrazine]-2-oxoethylcarbamate 8j: Yield: 80%. MP:
160.1–161.9 ꢀC. NMR-1H (400 MHz, CD3OD) d: 1.36 (3H, d,
J = 7.2 Hz, CH3); 1.44 (9H, s, (CH3)3); 4.15 and 5.00 (1H, m, CHNH
(E ⁄ Z)-isomer); 6.55 (1H, m, H3¢); 6.78 (d, J = 3.0 Hz) and 7.62 (m)
(1H, H4¢ (E ⁄ Z)-isomer); 6.92 (d, J = 3.0 Hz) and 7.64 (m) (1H, H2¢
(E ⁄ Z)-isomer); 7.82 and 8.06 (1H, s, CH=N (E ⁄ Z)-isomer) ppm NMR-
13C (100 MHz, CD3OD) d: 18.1; 18.5; 28.5; 48.0; 80.8; 113.3; 136.1;
139.9; 146.0; 146.5; 151.0; 151.3; 157.8; 172.6; 176.9 ppm IR (m,
cm-1, KBr): 3331, 3057, 2983, 2875, 1678. MS ⁄ ESI (m ⁄ z) calcd:
297.1 (M+), found: 320.2 (M+Na).
N’-[(2E/Z )-4-nitrobenzylidene]-(2S)-amino-3-phenyl-
propanehydrazide 10a: Yield: 41%. NMR-1H (400 MHz,
CF3COOD) d: 2.89 (2H, s, NH2); 2.89 (1H, m, CHPh); 3.07 (1H, m,
CHPh); 4.58 and 5.16 (1H, m, CHNH (E ⁄ Z)-isomer); 7.82–7.92 and
8.11–8.55 (9H, m, Ph); 9.43 and 9.53 (1H, s, CH=N (E ⁄ Z)-isomer);
10.77 (1H, s, NHN) ppm NMR- 13C (100 MHz, CF3COOD) d: 40.2;
56.7; 112.7; 115.5; 121.2; 126.7; 127.0; 133.9; 135.0; 138.2; 142.1;
168.7; 170.6; 183.5 ppm IR (m, cm-1, KBr): 3530, 1597, 1525, 1346.
MS ⁄ ESI (m ⁄ z) calcd: 312.1 (M+), found: 335.1 (M+Na).
Benzyl
(1S)-1-benzyl-2-{(2E/Z)-2-[(5-nitro-2-furyl)-
methylene]hydrazine]-2-oxoethylcarbamate 9a: Yield:
25%. MP: 179.5–181.4 ꢀC. NMR-1H (400 MHz, DMSO-d6) d: 2.72
(1H, m, CHPh); 3.04 (1H, m, CHPh); 4.33 and 4.90 (1H, m, CHNH
(E ⁄ Z)-isomer); 4.90 (2H, s, CH2O); 7.23–7.31 (10H, m, 2 Ph); 7.51
(1H, m, H4¢); 7.87 (1H, m, H3¢); 7.88 (1H, m, CHNH (E ⁄ Z)-isomer);
7.98 and 8.18 (1H, s, CH=N (E ⁄ Z)-isomer); 11.89 and 12.03 (1H, s,
NHN (E ⁄ Z)-isomer) ppm NMR- 13C (100 MHz, DMSO-d6) d: 36.2,
37.4, 54.5, 55.6, 6.2, 114.5, 115.5, 126.4, 127.5, 128.1, 128.2, 129.2,
131.3, 135.0, 137.0, 138.5, 151.3, 151.6, 156.0, 168.7, 173.4 ppm IR
(m, cm-1, KBr): 3306, 3089, 3032, 1693, 1676, 1535, 1352. MS ⁄ ESI
(m ⁄ z) calcd: 436.1 (M+), found: 459.1 (M+Na).
N’-[(2E/Z )-4-nitrobenzylidene]-(2S )-amino-4-methyl-
pentanehydrazide 10b: Yield: 39%. MP: 133.5–135.2 ꢀC.
NMR-1H (500 MHz, DMSO-d6) d: 0.98 (6H, m, (CH3)2); 1.63 (1H, m,
CH(CH3)2); 1.81 (2H, m, CH2); 2.80 (2H, s, NH2); 3.51 and 4.21 (1H, m,
CHNH2 (E⁄Z)-isomer), 7.17 (1H, d, J = 8.5 Hz, H2 and H6); 7.98 (2H, d,
J = 8.5 Hz, H3 and H5); 7.54 and 7.82 (1H, s, CH=N (E⁄Z)-isomer);
11.75 and 11.82 (1H, s, NHN (E⁄Z)-isomer) ppm NMR- 13C (125 MHz,
DMSO-d6) d: 17.0; 19.5; 31.9; 59.1; 77.7; 123.4; 124.7; 127.2; 128.5;
141.1; 148.7; 174.0 ppm IR (m, cm-1, KBr): 3622, 3342, 2954, 2870,
1695, 1517, 1336. MS ⁄ESI (m⁄z) calcd: 278.1 (M+), found: 277.3 (M-H).
Benzyl
(1S)-1-benzyl-2-{(2E/Z)-2-[(5-nitro-2-thienyl)-
methylene]hydrazine]-2-oxoethylcarbamate 9b: Yield:
77%. MP: 147.6–149.9 ꢀC. NMR-1H (400 MHz, DMSO-d6) d: 2.83
(1H, m, CHPh); 2.99 (1H, m, CHPh); 4.30 and 5.05 (1H, m, CHNH
(E ⁄ Z)-isomer); 4.96 (2H, s, CH2O); 7.21–7.35 (10H, m, 2 Ph); 7.53 (d,
J = 3.2 Hz) and 7.76 (d, J = 6.8 Hz) (1H, H4¢ (E ⁄ Z)-isomer); 7.56 (d,
J = 3.2 Hz) and 7.85 (d, J = 6.4 Hz) (1H, H3¢ (E ⁄ Z)-isomer); 8.13 (1H,
s, CHNH); 8.16 and 8.47 (1H, s, CH=N (E ⁄ Z)-isomer); 11.86 and
12.01 (1H, s, NHN (E ⁄ Z)-isomer) ppm NMR- 13C (100 MHz, DMSO-
d6) d: 36.5, 37.1, 53.2, 55.6, 65.3, 65.4, 126.5, 127.6, 127.8, 128.1,
128.3, 129.2, 129.4, 129.8, 130.5, 136.9, 137.0, 137.6, 138.0, 140.8,
146.3, 150.6, 150.9, 156.0, 168.8, 173.2. ppm IR (m, cm-1, KBr):
3305, 3062, 2956, 1647, 1531, 1334. MS ⁄ ESI (m ⁄ z) calcd: 452.1
(M+), found: 475.1 (M+Na).
N¢-{(2E ⁄ Z )-[(5-nitro-2-furyl)metylene]}-(2S )-amino-3-
methylbutanehidrazide 10c: Yield: 44%. MP: 125.2–127.4 ꢀC.
NMR-1H (500 MHz, DMSO-d6) d: 0.96 (6H, t, J = 7.0 Hz; (CH3)2); 1.65
(1H, m, CH(CH3)2); 2.09 (2H, m, CH2); 2.55 (2H, s, NH2); 3.01 and 3.86
(1H, m, CHNH2 (E ⁄ Z)-isomer); 6.20 and 6.70 (7.17 (1H, m, H4¢ (E ⁄ Z)-
isomer); 6.31 and 6.97 (1H, m, H3¢ (E⁄ Z)-isomer); 7.50 and 7.58 (1H,
s, CH=N (E⁄ Z)-isomer); 9.02 and 9.22 (1H, s, NHN (E⁄ Z)-isomer) ppm
NMR- 13C (125 MHz, DMSO-d6) d: 26.3, 41.9, 70.2, 113.9, 114.1,
220
Chem Biol Drug Des 2012; 79: 216–222

Synthesis and Antitubercular Activity of Amino Acid Derivatives
115.4, 116.8, 138.0, 139.4, 140.7, 152.7, 153.1, 156.1, 156.8,
Table 2: The in vitro activity of N-acyl hydrazones L-phenylala-
nine derivatives 8a–8b, 9a–b and 10a against Mycobacterium
tuberculosis H37Rv strain (ATCC 27294, susceptible to cycloserine)
O
173.6 ppm IR (m, cm-1, KBr): 3134, 2958, 2872, 1712, 1529, 1352.
MS⁄ ESI (m⁄ z) calcd: 268, 1.1 (M+), found: 267.2 (M-H).
N
Ar/Het
Biologic evaluation
N
H
The antimycobacterial activities of the compounds 5a–c, 6a–b,
7a–b, 8a–j, 9a–d, and 10a–c have been assessed against
Mycobacterium tuberculosis ATTC 27294 using the microplate Ala-
mar Blue assay (12). This methodology is nontoxic, uses a thermally
stable reagent, and shows good correlation with proportional and
BACTEC radiometric methods (13,14). The method is described as
follows: 200 mL of sterile deionized water was added to all outer-
perimeter wells of 96-sterile-well plates (falcon, 3072; Becton Dick-
inson, Lincoln Park, NJ, USA) to minimize evaporation of the med-
ium in the test wells during incubation. The 96 plates received
100 mL of the Middlebrook 7H9 broth (Difco laboratories, Detroit,
MI, USA), and successive dilution of the compounds 5a–c, 6a–b,
7a–b, 8a–j, 9a–d and 10a–c was made directly on the plate.
The final drug concentrations tested were 0.01–20.0 lg ⁄ mL. Plates
were covered and sealed with parafilm and incubated at 37 ꢀC for
5 days. 25 mL of a freshly prepared 1:1 mixture of Alamar Blue
(Accumed International, Westlake, OH, USA) reagent and 10%
Tween-80 was then added to the plate and incubated for 24 h. A
blue color in the well was interpreted as no bacterial growth, and
a pink color was scored as growth. The minimal inhibition concen-
tration (MIC) was defined as the lowest drug concentration, which
prevented a color change from blue to pink.
NHR1
Compound
R1
Ar ⁄ Het
logPa
MIC (lg ⁄ mL)
8a
8b
BOC
BOC
Cbz
Cbz
H
5-nitro-2-furyl
5.137
5.779
5.546
6.188
2.013
–
100
50
12.5
50
Resistant
20
5-nitro-2-thienyl
5-nitro-2-thienyl
5-nitro-2-thienyl
p-nitrobenzyl
–
9a
9b
10a
Cycloserine
–
aCalculated by http://www.molinspiration.com/cgi-bin/properties.
Table 3: The in vitro activity of N-acyl hydrazones L-leucine
derivatives compounds 8c–f, 9c–d and 10b–c against Mycobacte-
rium tuberculosis H37Rv strain (ATCC 27294, susceptible to cycloser-
ine)
O
N
Ar/Het
N
H
NHR1
Ar ⁄ Het
R1
logPa
MIC (lg ⁄ mL)
Results and Discussion
Compound
8c
8d
BOC
BOC
BOC
BOC
Cbz
Cbz
H
2-furyl
2-thienyl
4.903
5.545
4.986
5.627
5.395
6.037
1.862
1.242
Resistant
Resistant
The in vitro antitubercular activity of the synthesized compounds is
shown in Tables 1–4. The N-acyl hydrazine synthesized compound
7a, an L-phenylalanine derivative, was the only intermediate prod-
uct active against M. tuberculosis, with a MIC of 100 lg ⁄ mL
(Table 1). The compounds 5a and 6a were used as starting materi-
als for the synthesis of 8a–b and 9a–b, respectively. No reaction
8e
5-nitro-2-furyl
5-nitro-2-thienyl
5-nitro-2-furyl
5-nitro-2-thienyl
p-nitrobenzyl
5-nitro-2-furyl
–
50
50
25
25
8f
9c
9d
10b
Resistant
25
10c
Cycloserine
H
–
Table 1: The in vitro activity of N-acyl hydrazines amino acid
derivatives 5a–c, 6a–b, and 7a–b against Mycobacterium tuber-
20
aCalculated by http://www.molinspiration.com/cgi-bin/properties.
culosis H37Rv strain (ATCC 27294, susceptible to D-cycloserine)
O
was observed between the unprotected hydrazine 7a and heteroa-
romatic aldehydes. However, the coupling reaction between 7a and
p-nitrobenzaldehyde was successfully achieved, being the desired
compound 10a inactive against M. tuberculosis. Among the 8a
and 8b N-BOC-protected compounds, the derivative 8b, synthesized
from the 5-nitrothiophenecarboxaldehyde, showed a moderate activ-
ity, with a MIC of 50 lg ⁄ mL. The 5-nitrofuran derivative 9a, with a
MIC of 12.5 lg ⁄ mL, exhibited the highest activity among all the
compounds synthesized in this study, being more potent when com-
pared with that of the tuberculostatic drug D-cycloserine
(20 lg ⁄ mL). These data highlight the importance of 5-nitrofuranyl
groups for the tuberculostatic activity observed (Table 2).
R
NH2
N
H
NHR1
Compound
R
R1
logPa
MIC (lg ⁄ mL)
5a
5b
C6H5CH2-
CH3(CH2)2CHCH2-
CH3-
C6H5CH2-
CH3(CH2)2CHCH2-
C6H5CH2-
BOC
BOC
BOC
Cbz
Cbz
H
2.380
2.229
0.919
2.790
2.638
)1.363
)1.514
–
Resistant
Resistant
Resistant
Resistant
Resistant
100
5c
6a
6b
7a
7b
Cycloserine
CH3(CH2)2CHCH2-
–
H
–
Resistant
20
The use of the L-leucine as starting material confirms the impor-
tance of the heteroaromatic nuclei containing 5-NO2 substituents
for the biologic activity of the synthesized compounds. Concerning
aCalculated by http://www.molinspiration.com/cgi-bin/properties.
Chem Biol Drug Des 2012; 79: 216–222
221

Da Costa et al.
Table 4: The in vitro activity of the N-acyl hydrazones L-alanine
derivatives 8g–8j against Mycobacterium tuberculosis H37Rv strain
(ATCC 27294, susceptible to cycloserine)
A., Coutinho E.C. (2010) Screening for in vitro antimycobacterial
activity and three-dimensional quantitative structure-activity rela-
tionship (3D-QSAR) study of 4-(arylamino)coumarin derivatives.
Chem Biol Drug Des;76:412–424.
O
2. Silveira G.P., Nome F., Gesser J.C., Sꢀ M.M. (2006) Estratꢁgias
N
Ar/Het
utilizadas no combate
Nova;29:844–855.
a
resistÞncia bacteriana. Quim
N
H
3. Souza M.V.N., Ferreira M.L., Pinheiro A.C., Saraiva M.F., Almeida
M.V., Valle M.S. (2008) Synthesis and biological aspects of my-
colic acids: an important target against Mycobacterium tubercu-
losis. TSWJ;8:720–751.
NHR1
Compound
R1
Ar ⁄ Het
logPa
MIC (lg ⁄ mL)
4. Souza M.V.N., Vasconcelos T.R.A. (2005) Fꢀrmacos no combate
ꢂ tuberculose: passado, presente e futuro. Quim Nova;28:678–
682.
5. Rollas S., KꢃÅꢃkgꢃzel S.G. (2007) Biological activities of hydraz-
one derivatives. Molecules;1:1910–1939.
8g
8h
BOC
BOC
BOC
BOC
–
2-furyl
2-thienyl
5-nitro-2-furyl
5-nitro-2-thienyl
–
3.593
4.235
3.676
4.318
–
100
100
Resistant
Resistant
20
8i
8j
Cycloserine
aCalculated by http://www.molinspiration.com/cgi-bin/properties.
6. Dimmock J.R., Vashishtha S.C., Stables J.P. (2000) Anticonvulsant
properties of various acetyhydrazones, oxamoylhydrazones and
semicarbazones derived from aromatic and unsatured carbonyl
compounds. Eur J Med Chem;35:241–248.
compounds containing BOC in their structures, 8e and 8f showed
activity at concentrations of 50 lg ⁄ mL. However, without nitro
groups, the compounds 8c and 8d proved to be inactive. Then, the
nitrated aldehydes were used to obtain the N-Cbz-protected analogs
9c and 9d, and the unprotected hydrazone 10c. The three com-
pounds displayed activities of 25 lg ⁄ mL. The analogous compound
containing the 5-nitro-2-thienyl subunit in their structures was not
successfully synthesized, and the p-nitrobenzyl derivative 10b was
not active against M. tuberculosis, confirming the importance of the
heteroaromatic nuclei to the activities observed.
˘
7. ꢄakır B., Dag ꢅ., Yıldırım E., Erol K., S¸ ahin M.F. (2001) Synthesis
and anticonvulsant activity of some hydrazones of 2-[(3H)-oxo-
benzoxazolin-3-yl-aceto]hydrazide. J Fac Pharm Gazi;18:99–106.
8. ErgenÅ N., Gꢃnay N.S. (1998) Synthesis and antidepressant eval-
uation of new 3-phenyl-5-sulfonamidoindole derivatives. Eur J
Med Chem;33:143–148.
9. Todeschini A.R., Miranda A.L., Silva C.M., Parrini S.C., Barreiro
E.J. (1998) Synthesis and evaluation of analgesic, antiinflamma-
tory and antiplatelet properties of new 2-pyridylarylhydrazone
derivatives. Eur J Med Chem;33:189–199.
10. Lima P.C., Lima L.M., Silva K.C., Leda P.H., Miranda A.L.P., Fraga
C.A.M., Barreiro E.J. (2000) Synthesis and analgesic activity of
novel N-acylhydrazones and isosters, derived from natural saf-
role. Eur J Med Chem;35:187–203.
Other amino acids were also used as starting materials, such as L-
alanine. However, no relevant activities were detected using the N-
BOC-protected hydrazones 8g, 8h, 8i, and 8j, and because of
these results, the protected analogs with Cbz and unprotected ana-
logs were not synthesized (Table 4).
11. Walcourt A., Loyevsky M., Lovejoy D.B., Gordeuk V.R., Richardson
D.R. (2004) Novel arylhydrazone and thiosemicarbazone iron che-
lators with anti-malarial activity against chloroquine-resistant
and -sensitive parasites. Int J Biochem Cell Biol;36:401–407.
12. Franzblau S.G., Witzig R.S., McLaughlin J.C., Torres P., Madico
G., Hernandez A., Degnan M.T., Cook M.B., Quenzer V.K., Fergu-
son R.M., Gilman R.H. (1998) Rapid, low-technology MIC deter-
mination with clinical Mycobacterium tuberculosis isolates by
Conclusion
In summary, this work describes the synthesis and biologic evalua-
tion of 17 N-acylhydrazone derivatives of amino acids from different
amino acids, such as L-phenylalanine, L-leucine, and L-alanine. The
protection of these amino acids with Cbz and BOC was necessary
for the stability of the derivatives synthesized. The compounds were
evaluated against M. tuberculosis furnishing promising results, dis-
playing a MIC between 12.5 and 50 lg ⁄ mL. Among then, the most
promising compound was 9a, being more potent when compared
with that of the tuberculostatic drug D-cycloserine (20 lg ⁄ mL).
using the Microplate Alamar blue assay.
biol;36:362–366.
J Clin Micro-
13. Vanitha J.D., Paramasivan C.N. (2004) Evaluation of microplate Al-
amar blue assay for drug susceptibility testing of Mycobacterium
avium complex isolates. Diagn Microbiol Infect Dis;49:179–182.
14. Reis R.S., Neves I. Jr, LourenÅo S.L.S., Fonseca L.S., LourenÅo
M.C.S. (2004) Comparison of flow cytometric and Alamar blue
tests with the proportional method for testing susceptibility of
Mycobacterium tuberculosis to rifampin and isoniazid. J Clin
Microbiol;42:2247–2248.
Acknowledgments
The authors gratefully acknowledge Farmanguinhos, IPEC, UFJF,
CAPES, and CNPq for fellowships.
Note
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Chem Biol Drug Des 2012; 79: 216–222