Beta-N-cyanoethyl acyl hydrazide derivatives: a new class of beta-glucuronidase inhibitors.

Article abstract of DOI:10.1248/cpb.50.1443

Eight new beta-N-substituted acyl hydrazides along with their corresponding acyl derivatives were synthesized and screened for in vitro beta-glucuronidase inhibition and found to be active against the enzyme. All of these compounds were found to be noncom

Full text of DOI:10.1248/cpb.50.1443

November 2002
Chem. Pharm. Bull. 50(11) 1443—1446 (2002)
1443
b
b
-N-Cyanoethyl Acyl Hydrazide Derivatives: A New Class of
-Glucuronidase Inhibitors
Khalid Mohammed KHAN,* Shahida SHUJAAT, Shagufta RAHAT, Safdar HAYAT, ATTA-UR-RAHMAN, and
Muhammad Iqbal CHOUDHARY
H.E.J. Research Institute of Chemistry, International Centre for Chemical Sciences, University of Karachi; Karachi-75270,
Pakistan. Received May 16, 2002; accepted July 23, 2002
Eight new b-N-substituted acyl hydrazides along with their corresponding acyl derivatives were synthesized
and screened for in vitro b-glucuronidase inhibition and found to be active against the enzyme. All of these com-
pounds were found to be noncompetitive inhibitors except for Nꢀ-(2-cyanoethyl)-4-hydroxy benzohydrazide (10),
which was found to be an uncompetitive inhibitor. Structure–activity relationship studies indicated that the ben-
zyloxy group present in compounds 12 and 13 is responsible for the b-glucuronidase inhibition activity.
Key words b-N-cyanoethylacyl hydrazide; b-glucuronidase; enzyme inhibition
b-Glucuronidase (EC 3.2.1.31) is an exoglycosidase en- and 9—16 for IC50 and kinetic studies. The IC50 and Ki val-
zyme that catalyzes the cleavage of glucuronosyl-O-bonds.1) ues and mode of inhibitions are given in Table 2. The Ki val-
The enzyme is present in many organs, body fluids, blood ues (inhibitory constants) were determined directly from the
cells, liver, spleen, kidney, gastric juice, lung, muscle, bile, Dixon plots.11) These values were also confirmed, first by
urine, and serum.2,3) It has been reported that in certain dis- plotting the 1/Vmaxapp against different concentrations of the
eases such as cancer, inflammatory joint disease, some he- respective inhibitor. 1/Vmaxapp was calculated at each intersec-
patic diseases, and AIDS the activity of b-glucuronidase in- tion point of lines of every inhibitory concentration on the Y-
creases.1) Human b-glucuronidase also has a role in the de- axis of the Lineweaver–Burk plot.12) Second, the slope of
conjugation of glycosaminoglycans. Endogenous biliary b- each line of inhibitory concentration on the Lineweaver–
glucuronidase deconjugates the glucuronides of bilirubin and Burk plot was plotted against inhibitor concentrations. For
causes the development of cholelithiasis in human bile.4) the uncompetitive type of inhibition, Kmapp was determined
Many b-glucuronidase inhibitors such as 8-hydroxytricetin- from the intersection of the lines for the inhibitory concentra-
7-glucuronide and isovitexin,5) trihydroxy pipecolic acid,6)
and scoparic acid A and C7) have already been isolated from
different plants and some are used clinically.
During random screening of synthetic compounds, we
found that hydrazide derivatives can inhibit b-glucuronidase
enzyme. Although these hydrazides are not stronger than
other inhibitors but have comparable inhibitory activity, to
the best of our knowledge, this is the first example of syn-
thetic inhibitors of this enzyme. The present report describes
the synthesis of a series of hydrazide derivatives and their b-
glucuronidase inhibitory activity with respect to structure–
activity relationships.7)
Results and Discussion
The synthesis of b-N-substituted acyl hydrazide deriva-
tives from their corresponding acyl hydrazides were carried
out as illustrated in Chart 1. The parent acyl hydrazides 1—8
were synthesized by treating their corresponding ethyl or
methyl esters with hydrazine hydrate in ethanol. The Michael
reaction between resulting acyl hydrazides and acrylonitrile
gave exclusively b-N-substituted cyanoethyl derivatives 9—
16 in high yields.8) The formation of double Michael prod-
ucts was not observed. This may be due to a decline in nucle-
ophilicity of the nitrogen atom after the first substitution.9)
The structures of synthesized compounds were identified and
determined through their various physical and spectroscopic
properties.
All the synthesized compounds 1—16 were screened for
their b-glucuronidase inhibition activity.10) On the basis of
the results obtained from the preliminary screening of this
series of compounds (Table 1), we selected compounds 4,
Chart 1
∗ To whom correspondence should be addressed. e-mail: hassaan2@super.net.pk
© 2002 Pharmaceutical Society of Japan

1444
Vol. 50, No. 11
Table 1. Results of Preliminary Screening of Compounds 1—16 against
b-Glucuronidase at 0.5 mM/ml Concentration
tions on the X-axis of the Lineweaver–Burk plot and plotted
against inhibitor concentrations. The majority of the com-
pounds exhibited a pure noncompetitive type of inhibition as
the Vmax values decreased without affecting the Km values.
Substitution of a benzyloxy group greatly enhanced the ac-
tivity against b-glucuronidase, as in compounds 12 and 13. It
was also clear that substitution at the C-3 position is more ef-
fective than that at C-4. Similar results were obtained when a
hydroxyl group (compounds 11, 10) was substituted at the C-
3 and C-4 positions. The effect of a benzenesulfonyl group
(compound 15) on enzyme activity was same as in com-
pound 10, but the mode of action of compound 10 was found
to be different from that of compound 15, i.e., uncompetitive.
The graphic analysis of steady-state inhibition data of com-
pound 13 for noncompetitive inhibition and of compound 10
for uncompetitive inhibition are shown in Figs. 1 and 2, re-
spectively, providing an example of each type of inhibition.
Sr. no.
Compound no.
% inhibition
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
48.9
19.0
20.0
81.8
44.2
55.3
42.0
33.3
94.1
86.9
93.0
88.2
98.4
82.0
70.8
63.9
Table 2. IC50 and Ki Values of Compounds 4 and 9—16 against b-Glucuronidase
IC50 (mM)
Kia) (mM)
Sr. no
Compound
Type of inhibition
MeanꢀS.E.M.
MeanꢀS.E.M.b)
1
2
3
4
5
6
7
8
9
4
9
420.0ꢀ0.250
55.4ꢀ0.002
12.0ꢀ0.033
7.8ꢀ0.100
2.2ꢀ0.05
580.0ꢀ0.15
64.0ꢀ0.1
8.0ꢀ0.001
8.7ꢀ0.02
Noncompetitive
Noncompetitive
Uncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
10
11
12
13
14
15
16
5.0ꢀ0.2
1.6ꢀ0.12
3.25ꢀ0.01
11.0ꢀ0.33
21.0ꢀ0.023
120.0ꢀ0.031
6.0ꢀ0.066
11.6ꢀ0.02
277.0ꢀ0.012
a) Ki is the mean of three values calculated using the Dixon plot and Lineweaver–Burk secondary plots. b) S.E.M. is the mean of five values in case of IC50, and three val-
ues in case of Ki. IC50 of D-saccharic acid 1,4-lactone (standard)ꢁ0.8 mMꢀ0.002.
Fig. 1. Steady-State Inhibition of b-Glucuronidase by Compound 13 (3-(Benzyloxy)-Nꢂ-(2-cyanoethyl)benzohydrazide)
(A) Lineweaver–Burk plot of reciprocal of the initial velocities versus various substrate (p-nitrophenyl-b-D-glucuronide) concentrations. (B) Respective secondary replots of the
Lineweaver–Burk plots, i.e., 1/Vmaxapp and slope versus various compound concentrations. (C) Dixon plot of reciprocal of the initial velocities versus various compound concentra-
tions. (D) Secondary replot of the Dixon plot: slope versus reciprocal of substrate concentrations. Each point in the graphs represents the mean of three experiments.

November 2002
1445
Fig. 2. Steady-State Inhibition of b-Glucuronidase by Compound 10 (Nꢂ-(2-cyanoethyl)-4-hydroxybenzohydrazide)
(A) Lineweaver–Burk plot of reciprocal of the initial velocities versus various substrate (p-nitrophenyl-b-D-glucuronide) concentrations. (B) Respective secondary replots of the
Lineweaver–Burk plots, i.e., 1/Kmaxapp and 1/Vmaxapp versus various compound concentrations. (C) Dixon plot of reciprocal of the initial velocities versus various compound concen-
trations. Each point in the graphs represents the mean of three experiments.
were then calculated using the EZ-Fit Enzyme Kinetic program (Perrella
The present study provides a basis for further research on
acyl hydrazides as b-glucuronidase inhibitors.
Scientific Inc., Amherst, MA, U.S.A. Dissociation constants (Ki values) were
determined by the interpretation of Dixon plots.11) Lineweaver–Burk plots11)
and their secondary plots using initial velocities were drawn over a substrate
concentrations between 0.166 and 0.666 mM. Triplicate assays were con-
ducted at each concentration, and graphs were plotted using the above
Experimental
Melting points were determined in open capillaries on a Büchi 535 appa-
ratus and are uncorrected. Column chromatographic separations were car- method.
ried out on silica gel (70—230 mesh, E. Merck). Infrared spectra (KBr)
General Procedure for the Preparation of Acyl Hydrazides (1—8)
were recorded on a Shimadzu-IR-460 and IR (CHCl3) were recorded on The corresponding ethyl or methyl esters (0.06 mol) in small portions were
Jasco-A-302 spectrophotometer. The 1H-NMR spectra were recorded on added to hydrazine hydrate 99% (0.01 mol) in ethanol (3.7 ml) and then re-
Bruker AM 300 and 400 spectrometers using the UNIX data system at 300
fluxed for 28 h. The solvents were removed in vacuo and the residues were
and 400 MHz, respectively. Electron impact (EI)-MS were recorded on MAT crystallized overnight in a cold room.
311 A (Finnigan).
Benzohydrazide (1): Colorless compound (1.24 g, 80%). mp 111 °C. UV
lmax MeOH: 219 (log eꢁ4.21) nm. IR (KBr): 3306, 3011, 1662, 1235, 1078,
Material and Methods b-Glucuronidase (E.C. 3.2.1.31, from bovine
liver, G-0251) and p-nitrophenyl-b-D-glucuronide (N-1627) were purchased
1
748 cmꢃ1. H-NMR (400 MHz, DMSO-d6): d 9.63 (1H, s, CONH), 7.61—
from Sigma Chemical Co. (U.S.A.). Sodium carbonate anhydrous from 7.40 (5H, m, Ar-H), 4.50 (2H, s, NH2). FD-MS: m/z 136. Anal. Calcd for
Fluka and all other reagents were obtained from E. Merck and were of ana- C7H8N2O (136): C, 61.75; H, 5.92; N, 20.57. Found: C, 61.73; H, 5.90; N,
lytical grade. Anhydrous CHCl3 and EtOH were dried using the standard 20.56.
methods. All other solvents and reagents were of reagent grade and used di-
4-Hydroxybenzohydrazide (2): Colorless compound (1.32 g, 80%). mp
rectly without purification, except for benzoyl chloride, which was distilled 268 °C. UV lmax MeOH: 245 (log eꢁ3.84) nm. IR (KBr): 3351, 3013, 1622,
before use.
1185, 1034, 832 cmꢃ1 1H-NMR (300 MHz, DMSO-d6): d 9.5 (1H, s,
.
b-Glucuronidase Assay Protocol b-Glucuronidase activity was deter-
mined by measuring the absorbance at 405 nm of p-nitrophenol formed from
CONH), 7.68 (2H, d, Jꢁ8.8 Hz, H-2/H-6), 6.78 (2H, d, Jꢁ8.8 Hz, H-3/H-5),
4.35 (2H, s, NH2). FD-MS: m/z 152. Anal. Calcd for C7H8N2O2 (152): C,
the substrate by the spectrophotometric method developed by Collins et al.10) 55.26; H, 5.3; N, 18.41. Found: C, 55.22; H, 5.32; N, 18.37.
with the following modifications. The total reaction volume was reduced to
250 ml with 190 ml of 0.1 M acetate buffer (13.608 g/l of sodium acetate, pH
adjusted to 5.0 with 0.1 M acetic acid), and 5 ml of enzyme (30 U), 50 ml of
0.4 mM p-nitrophenyl-b-D-glucuronide were incubated at 37 °C for 30 min
and 50 ml of 0.2 M Na2CO3 was used to quench the reaction. The plates were
read on a multiplate reader (spectra MAX-340) at 405 nm. The inhibitory
activities were determined as described previously,7) except that 5 ml each of
test compound was dissolved in DMSO, mixed with enzyme, and incubated
at 37 °C for 15 min before the addition of substrate to start the reaction. The
3-Hydroxybenzohydrazide (3): Colorless compound (0.43 g, 75%). mp
153 °C. UV lmax MeOH: 251 (log eꢁ4.46) nm. IR (KBr): 3315, 3114, 1623,
1191, 1064, 751 cmꢃ1 1H-NMR (400 MHz, DMSO-d6): d 9.65 (1H, s,
.
CONH), 7.48 (1H, dt, Jꢁ8.3 Hz, Jꢁ1.7 Hz, H-6), 7.24 (1H, t, Jꢁ8.3 Hz, H-
5), 7.18 (1H, t, Jꢁ1.7 Hz, H-2), 6.90 (1H, dt, Jꢁ8.3 Hz, Jꢁ1.7 Hz, H-4),
4.44 (2H, s, NH2). FD-MS: m/z 152. Anal. Calcd for C7H8N2O2 (152): C,
55.26; H, 5.3; N, 18.4. Found: C, 55.22; H, 5.32; N, 18.37.
4-Benzyloxybenzohydrazide (4): Colorless compound (1.64 g, 73%). mp
140 °C. UV lmax MeOH: 272 (log eꢁ4.69) nm. IR (KBr): 3293; 1687; 1611,
percentage inhibitory activity was calculated as follows: (EꢃS)/Eꢄ100, 1213, 1053, 837 cmꢃ1
.
1H-NMR (300 MHz, DMSO-d6): d 9.58 (1H, s,
where E is the activity of enzyme without test material and S is the activity CONH), 7.78 (2H, d, Jꢁ8.8 Hz, H-2/H-6), 7.45—7.30 (5H, m, Ar-H), 7.04
of enzyme with test material. All assays were run in triplicate.
(2H, d, Jꢁ8.8 Hz, H-3/H-5), 5.14 (2H, s, PhCH2O), 4.47 (2H, s, NH2). FD-
Determination of IC50 Values and Kinetic Parameters The concentra- MS: m/z 242. Anal. Calcd for C14H14N2O2 (242): C, 69.40; H, 5.82; N,
tions of test compounds that inhibit the hydrolysis of p-nitrophenyl-b-D-glu-
curonide by 50% (IC50 value) were determined by monitoring the effect of
increasing concentrations of these compounds in the assay. The IC50 values
11.56. Found: C, 69.37; H, 5.84; N, 11.47.
3-Benzyloxybenzohydrazide (5): Colorless compound (1.81 g, 72%). mp
155 °C. UV lmax MeOH: 265 (log eꢁ4.73) nm. IR (KBr): 3312, 1663, 1613,

1446
Vol. 50, No. 11
(295): C, 69.14; H, 5.80; N, 14.23. Found: C, 69.10; H, 5.86; N, 14.17.
1375, 1043, 763 cmꢃ1
.
1H-NMR (300 MHz, DMSO-d6): d 9.63 (1H, s,
3-(Benzyloxy)-Nꢂ-(2-cyanoethyl)benzohydrazide (13): Colorless com-
pound (2.58 g, 72%). mp 68 °C. UV lmax MeOH: 263 (log eꢁ4.58) nm. IR
(KBr): 3307, 2241, 1682, 1625, 1134, 1058, 779 cmꢃ1. 1H-NMR (300 MHz,
DMSO-d6): d 9.79 (1H, s, N–H), 9.59 (1H, s, CONH), 7.73—7.48 (5H, m,
Ar-H), 7.33 (1H, dt, Jꢁ8.2 Hz, Jꢁ1.8 Hz, H-6), 7.22 (1H, t, Jꢁ8.2 Hz, H-5),
7.19 (1H, t, Jꢁ1.8 Hz, H-2), 6.85 (1H, dt, Jꢁ8.2 Hz, Jꢁ1.8 Hz, H-4), 5.18
(2H, s, PhCH2O), 3.05 (2H, dd, Jꢁ10.8 Hz, Jꢁ6.1 Hz, H2-10), 2.31 (2H, t,
Jꢁ6.1 Hz, H2-11). FD-MS: m/z 295. Anal. Calcd for C17H17N3O2 (295): C,
69.14; H, 5.80; N, 14.23. Found: C, 69.12; H, 5.75; N, 14.19.
CONH), 7.70—7.30 (5H, m, Ar-H), 7.51 (1H, dt, Jꢁ8.3 Hz, Jꢁ1.5 Hz, H-
6), 7.24 (1H, t, Jꢁ8.3 Hz, H-5), 7.15 (1H, t, Jꢁ1.5 Hz, H-2), 6.95 (1H, dt,
Jꢁ8.3 Hz, Jꢁ1.5 Hz, H-4), 5.21 (2H, s, PhCH2O), 4.49 (2H, s, NH2). FD-
MS: m/z 242. Anal. Calcd for C14H14N2O2 (242): C, 54.90; H, 4.60; N, 9.14.
Found: C, 54.87; H, 4.52; N, 9.17.
4-Benzoyloxybenzohydrazide (6): Colorless compound (1.74 g, 69%). mp
255 °C. UV lmax MeOH: 283 (log eꢁ5.28) nm. IR (KBr): 3350, 1700, 1690,
1640 cmꢃ1. 1H-NMR (300 MHz, DMSO-d6): d 9.53 (1H, s, CONH), 7.91—
7.42 (5H, m, Ar-H), 7.71 (2H, d, Jꢁ8.1 Hz, H-2/H-6), 6.72 (1H, d, Jꢁ8.1
Hz, H-3/H-5), 4.42 (2H, s, NH2). FD-MS: m/z 256. Anal. Calcd for
C14H12N2O3 (256): C, 65.61; H, 4.72; N, 10.93. Found: C, 65.60; H, 4.68; N,
10.90.
4-(Benzoyloxy)-Nꢂ-(2-cyanoethyl)benzohydrazide (14): Colorless com-
pound (1.85 g, 50%). mp 128 °C. UV lmax MeOH: 274 (log eꢁ4.87) nm. IR
(KBr): 3315, 2234, 1725, 1672, 1625, 1142, 1063, 747 cmꢃ1 1H-NMR
.
(300 MHz, DMSO-d6): d 9.78 (1H, s, N–H), 9.58 (1H, s, CONH), 7.90—
7.43 (5H, m, Ar-H), 7.68 (2H, d, Jꢁ7.9 Hz, H-2/H-6), 6.79 (1H, d,
Jꢁ7.9 Hz, H-3/H-5), 3.02 (2H, dd, Jꢁ11.2 Hz, Jꢁ6.4 Hz, H2-10), 2.62 (2H,
t, Jꢁ6.4 Hz, H2-11). FD-MS: m/z 309. Anal. Calcd for C17H15N3O3 (309): C,
66.01; H, 4.89; N, 13.59. Found: C, 66.03; H, 4.81; N, 13.61.
4-(4ꢂ-Methylbenzenesulfonyl)benzohydrazide (7): Colorless compound
(2.83 g, 63%). mp 105 °C. UV lmax MeOH: 280 (log eꢁ5.11) nm. IR (KBr):
3354, 1693, 1645, 1382, 1053, 773 cmꢃ1. 1H-NMR (300 MHz, DMSO-d6): d
9.51 (1H, s, CONH), 7.95 (2H, d, Jꢁ8.7 Hz, H-2/H-6), 7.76 (2H, d, Jꢁ8.9
Hz, H-3ꢂ/H-5ꢂ), 7.48 (2H, d, Jꢁ8.9 Hz, H-2ꢂ/H-6ꢂ), 6.77 (2H, d, Jꢁ8.7 Hz,
H-3/H-5), 4.49 (2H, s, NH2), 2.40 (3H, s, CH3). FD-MS: m/z 306. Anal.
Calcd for C14H14N2O4S (306): C, 54.90; H, 4.60; N, 9.14. Found: C, 54.87;
H, 4.51; N, 9.11.
4-(4ꢂ-Methylbenzenesulfonyl)-Nꢂ-(2-cyanoethyl)benzohydrazide (15): Col-
orless compound (1.25 g, 61%). mp 103 °C. UV lmax MeOH: 278 (log eꢁ
4.94) nm. IR (KBr): 3309, 2243, 1694, 1625, 1136, 1063, 749 cmꢃ1 1H-
.
NMR (300 MHz, DMSO-d6): d 9.79 (1H, s, N–H), 9.54 (1H, s, CONH),
7.89 (2H, d, Jꢁ8.5 Hz, H-2/H-6), 7.65 (2H, d, Jꢁ8.8 Hz, H-3ꢂ/H-5ꢂ), 7.45
(2H, d, Jꢁ8.8 Hz, H-2ꢂ/H-6ꢂ), 6.79 (2H, d, Jꢁ8.5 Hz, H-3/H-5), 3.07 (2H,
dd, Jꢁ10.5 Hz, Jꢁ6.2 Hz, H2-10), 2.63 (2H, t, Jꢁ6.2 Hz, H2-11), 2.38 (3H,
s, CH3). FD-MS: m/z 359. Anal. Calcd for C17H17N3O4S (359): C, 56.83; H,
4.77; N, 11.70. Found: C, 56.84; H, 4.69; N, 11.71.
Nꢂ-(2-Cyanoethyl)-2-(1H-indol-3-yl)acetohydrazide (16): Colorless com-
pound (1.64 g, 75%). mp 123 °C. UV lmax MeOH: 256 (log eꢁ3.98) nm. IR
(KBr): 3308, 2253, 1662, 1621, 1137, 1061, 823 cmꢃ1. 1H-NMR (300 MHz,
DMSO-d6): d 9.83 (1H, s, N–H), 9.57 (1H, s, CONH), 7.61 (1H, dd,
Jꢁ8.3 Hz, Jꢁ1.9 Hz, H-7), 7.34 (1H, dt, Jꢁ8.3 Hz, Jꢁ1.9 Hz, H-6), 7.13
(1H, dt, Jꢁ8.3 Hz, Jꢁ1.9 Hz, H-5), 7.01 (1H, d, Jꢁ8.3 Hz, H-4), 5.12 (2H,
s, H2-8), 3.45 (H, s, H-2), 2.98 (2H, dd, Jꢁ11.4 Hz, Jꢁ6.3 Hz, H2-12), 2.52
(2H, t, Jꢁ6.3 Hz, H2-13). FD-MS: m/z 242. Anal. Calcd for C13H14N4O
(242): C, 64.45; H, 5.82; N, 23.13. Found: C, 64.41; H, 5.75; N, 23.09.
2-(1H-Indol-3-yl)acetohydrazide (8): Colorless compound (1.66 g, 68%).
mp 137 °C. UV lmax MeOH: 261 (log eꢁ4.32) nm. IR (KBr): 3317, 1649,
1623, 1235, 1063, 812 cmꢃ1. 1H-NMR (300 MHz, DMSO-d6): d 9.65 (1H, s,
CONH), 7.59 (1H, dd, Jꢁ7.8 Hz, Jꢁ1.6 Hz, H-7), 7.30 (1H, dt, Jꢁ7.8 Hz,
Jꢁ1.6 Hz, H-6), 7.09 (1H, dt, Jꢁ7.8 Hz, Jꢁ1.6 Hz, H-5), 6.98 (1H, d,
Jꢁ7.8 Hz, H-4), 4.45 (2H, s, NH2), 4.22 (2H, s, H2-8), 3.40 (H, s, H-2). FD-
MS: m/z 189. Anal. Calcd for C10H11N3O (189): C, 63.48; H, 5.86; N, 22.21.
Found: C, 63.41; H, 5.80; N, 22.17.
General Procedure for the Preparation of Substituted Benzoyl[2-(2-
cyanoethyl)hydrazines] (9—16) Acrylonitrile (1.5 mol) was added to a
mixture of the corresponding acyl hydrazides 1—8 (0.5 mol) in ethanol
(172 ml) and the mixture was heated under reflux conditions for 48 h. The
solvent was removed in vacuo and the resulting residue was chro-
matographed over silica gel eluted with hexane–ethyl acetate (7 : 3) to obtain
the crystalline substituted hydrazines 9—16.
Nꢂ-(2-Cyanoethyl)benzohydrazide (9): Colorless compound (1.08 g,
80%). mp 105 °C. UV lmax MeOH: 213 (log eꢁ4.11) nm. IR (KBr): 3314,
3011, 2315, 1663, 1624, 1135, 1062, 748 cmꢃ1. 1H-NMR (400 MHz, DMSO-
d6): d 10.01 (1H, s, N–H), 9.75 (1H, s, CONH), 7.81—7.41 (5H, m, Ar-H),
3.04 (2H, dd, Jꢁ11.3 Hz, Jꢁ6.4 Hz, H2-10), 2.61 (2H, t, Jꢁ6.4 Hz, H2-11).
FD-MS: m/z 189. Anal. Calcd for C10H11N3O (189): C, 63.48; H, 5.9; N,
22.0. Found: C, 63.46; H, 5.92; N, 22.06.
Acknowledgments This work was financially supported by the Pakistan
Science Foundation (Grant no. SKU/CHEM/372).
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7) Hayashi T., Kawasaki M., Okamura K., Tamada Y., Morita N., Tezuka
Y., Kikuchi T., Miwa Y., Taga T., J. Nat. Prod., 55, 1748—1755
(1992).
8) Mazouz F., Gueddari S., Burstein C., Mansy D., Milcent R., J. Med.
Chem., 36, 1157—1167 (1993).
DMSO-d6): d 9.95 (1H, s, N–H), 9.72 (1H, s, CONH), 7.69 (2H, d,
Jꢁ8.5 Hz, H-2/H-6), 6.79 (2H, d, Jꢁ8.5 Hz, H-3/H-5), 3.01 (2H, dd, Jꢁ
11.1 Hz, Jꢁ6.4 Hz, H2-10), 2.51 (2H, t, Jꢁ6.4 Hz, H2-11). FD-MS: m/z 205.
Anal. Calcd for C10H11N3O2 (205): C, 58.53; H, 5.40; N, 20.48. Found: C,
58.46; H, 5.28; N, 20.56.
Nꢂ-(2-Cyanoethyl)-3-hydroxybenzohydrazide (11): Colorless compound
(1.46 g, 70%). mp 115 °C. UV lmax MeOH: 246 (log eꢁ4.15) nm. IR (KBr):
3511, 3017, 2306, 1625, 1143, 1034, 762 cmꢃ1 1H-NMR (400 MHz,
.
DMSO-d6): d 9.82 (1H, s, N–H), 9.54 (1H, s, CONH), 7.54 (1H, dt, Jꢁ8.4
Hz, Jꢁ1.9 Hz, H-6), 7.28 (1H, t, Jꢁ8.4 Hz, H-5), 7.14 (1H, t, Jꢁ1.9 Hz, H-
2), 6.97 (1H, dt, Jꢁ8.4 Hz, Jꢁ1.9 Hz, H-4), 3.01 (2H, dd, Jꢁ11.2 Hz,
Jꢁ6.1 Hz, H2-10), 2.28 (2H, t, Jꢁ6.1 Hz, H2-11). FD-MS: m/z 205. Anal.
Calcd for C10H11N3O2 (205): C, 58.53; H, 5.40; N, 20.48. Found: C, 58.46;
H, 5.28; N, 20.56.
9) Khan K. M., Rahat S., Choudhary M. I., Atta-ur-Rahman, Ghani U.,
Perveen S., Khatoon S., Dar A., Malik A., Helv. Chem. Acta, 85,
559—569 (2002).
10) Collins R. A., Ng T. B., Fong W. P., Wan C. C., Yeung H. W., Biochem.
Mol. Biol. Int., 42, 1163—1169 (1997).
11) Dixon M., Biochem. J., 55, 170—171 (1953).
12) Segel I. H. (ed.), “Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady State Enzyme System,” John Wiley and Sons,
New York, 1993, pp. 125—143.
4-(Benzyloxy)-Nꢂ-(2-cyanoethyl)benzohydrazide (12): Colorless com-
pound (1.77 g, 66%). mp 139 °C. UV lmax MeOH: 263 (log eꢁ4.58) nm. IR
(KBr): 3312, 1682, 1633, 1135, 1053, 821 cmꢃ1 1H-NMR (300 MHz,
.
DMSO-d6): d 9.90 (1H, s, N–H), 9.61 (1H, s, CONH), 7.80 (2H, d, Jꢁ8.7
Hz, H-2/H-6), 7.42—7.30 (5H, m, Ar-H), 7.05 (2H, d, Jꢁ8.7 Hz, H-3/H-5),
5.25 (2H, s, PhCH2O), 3.04 (2H, dd, Jꢁ10.8 Hz, Jꢁ6.3 Hz, H2-10), 2.62
(2H, t, Jꢁ6.3 Hz, H2-11). FD-MS: m/z 295. Anal. Calcd for C17H17N3O2