Synthesis and biological activity of Schiff base series of valproyl, N-valproyl glycinyl, and N-valproyl-4-aminobenzoyl hydrazide derivatives

Article abstract of DOI:10.1248/cpb.c14-00143

Series of Schiff bases of valproic acid hydrazide, N-valproylglycine hydrazide and N-valproyl-4-aminobenzoyl hydrazide derivatives were synthesized and characterized by IR, NMR (¹H- and ¹³C-NMR) and elemental analysis. The prepared compounds were evaluated in transgenic zebrafish embryos (Tg: flil-1: enhanced green fluorescent protein (EGFP)) for antiangiogenic activity and in HepG2 liver carcinoma cell line for anti cancer activity. The Schiff bases of N-valproylglycine hydrazide derivatives were most potent in term of suppressing angiogenic blood vessels formation and anticancer activity at very low concentration, followed by the Schiff bases of valproic acid hydrazide derivatives which exhibited moderate activity, while the Schiff bases of N-valproyl-4-aminobenzoyl hydrazide derivatives, ethyl 4-(2-propylpentanamido)benzoate (VABE) and N-(4-(hydrazinecarbonyl)phenyl)-2- propylpentanamide (VABH) in contrast exhibited pro-angiogenic activity and weaker anticancer activity which mean that these derivatives targeted a common signaling pathway in term of affecting the blood vessels formation.

Full text of DOI:10.1248/cpb.c14-00143

June 2014
Regular Article
Chem. Pharm. Bull. 62(6) 591–599 (2014)
591
Synthesis and Biological Activity of Schiff Base Series of Valproyl,
N-Valproyl Glycinyl, and N-Valproyl-4-aminobenzoyl Hydrazide
Derivatives
,a,b,c
,d
Ayman El-Faham,*
Muhammad Farooq,* Sherine Nabil Khattab,b Ahmed Mohamed Elkayal,b
Mahmoud Fawzy Ibrahim,b Nael Abutaha,d Mohammad Ahmad Wadaan,d and
Ezzat Awad Hamedb,e
d
a Department of Chemistry, College of Science, King Saud University; Bioproducts Research Chair (BPRC),
Department of Zoology, College of Science, King Saud University; P.O. Box 2455, Riyadh 11451, Kingdom of
b
Saudi Arabia: Department of Chemistry, Faculty of Science, Alexandria University; P.O. Box 426, Alexandria
c
21321, Egypt: Institute for Research in Biomedicine-Barcelona; Baldiri Reixac 10, Barcelona 08028, Spain: and
e Department of Chemistry, Faculty of Science, Beirut Arab University; Beirut 11–5020, Lebanon.
Received February 10, 2014; accepted March 11, 2014
Series of Schiff bases of valproic acid hydrazide, N-valproylglycine hydrazide and N-valproyl-4-ami-
nobenzoyl hydrazide derivatives were synthesized and characterized by IR, NMR (1H- and 13C-NMR) and
elemental analysis. The prepared compounds were evaluated in transgenic zebrafish embryos (Tg: flil-1: en-
hanced green fluorescent protein (EGFP)) for antiangiogenic activity and in HepG2 liver carcinoma cell line
for anti cancer activity. The Schiff bases of N-valproylglycine hydrazide derivatives were most potent in term
of suppressing angiogenic blood vessels formation and anticancer activity at very low concentration, followed
by the Schiff bases of valproic acid hydrazide derivatives which exhibited moderate activity, while the Schiff
bases of N-valproyl-4-aminobenzoyl hydrazide derivatives, ethyl 4-(2-propylpentanamido)benzoate (VABE)
and N-(4-(hydrazinecarbonyl)phenyl)-2-propylpentanamide (VABH) in contrast exhibited pro-angiogenic
activity and weaker anticancer activity which mean that these derivatives targeted a common signaling path-
way in term of affecting the blood vessels formation.
Key words valproic acid; valproic acid hydrazide; Schiff base; antiangiogenic activity; anticancer activity
Valproic acid (VPA, 1) is a widely used antiepileptic agent platelet properties.9–16) In addition, they were reported to elicit
that is undergoing clinical evaluation for anticancer therapy. anticancer17–24) and anti-human immunodeficiency virus (HIV)
Given the remarkable value of valproic acid as a potent anti- properties25) and hence they gained an important place in me-
epileptic molecule, there is tremendous interest in the search dicinal chemistry.26–33)
for derivatives with improved pharmacokinetic or safety
The aim of the study is designing series of hydrazide-Schiff
profiles for epilepsy condition, such as bipolar disorders and bases with a wide spectrum of pharmaceutical applications,
epilepsy (valnoctamide 2),1) diisopropyl acetamide 3 (PID), having the valproic acid unit at the N-terminal and differ-
valrocemide (valproyl glycinamide 4), N-2,2,3,3-pentamethyl- ent Schiff bases at the C-terminal; considering some factors
cyclopropane carboxamide 5, 2,2,3,3-tetramethylcyclopropyl- responsible for such activity which are i) the presence of
carbonyl urea 6a, isovaleramide 6b (NPS-1776), and arundic electron-rich aromatic moieties; ii) the presence of amide and
acid 7 (ONO-2506),2,3) Fig. 1. Beside an affective epileptic hydrazido functionality, iii) the valproic acid drug moiety. All
drug, valproic acid inhibits angiogenesis both in vitro and in the prepared compounds were tested for antiangiogenic activ-
vivo,4–8) but no attempt has been made to synthesize valproic ity as well as anticancer activity in HepG2 cell.
acid derivatives with improved antiangiogenic potential.
Valproic acid 1 was treated with methanol in the presence
Hydrazide-hydrazones derivatives have been reported of conc. H2SO4 to afford the ester derivative which was used
with different pharmaceutical applications, where they were directly in the next step for preparation of the valproic acid
considered as antibacterial, antifugal, antimicrobial and anti- hydrazide 8.34–36) Compound 8 was condensed with substituted
convulsant agents. Many of them showed analgesic and anti- benzaldehyde and acetophenone in the presence of 2–3 drops
Fig. 1. Valproic Acid and Derivatives with Improved Pharmacokinetic or Safety Profiles for Epilepsy Condition
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: aymanel_faham@hotmail.com;
fmuhammad@ksu.edu.sa

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Chart 1. Synthesis of Schiff Bases of Valproic Acid Hydrazide
Fig. 2. The Expected Geometrical Isomers, Anti and Syn Forms, of
Compound 10a
of glacial HOAc in methanol as solvent to afford products
10a–g (Chart 1). The structure of synthesized compounds 8
and 10a–g was confirmed by spectral data.
Fig. 3. Geometry Optimizations Using the DFT Level (B3LYP/6-
31G**) of Theory for Schiff’s Base 10a, Anti and Syn Forms
1
The H- and 13C-NMR spectra of products 10a–g indicate
the presence of two isomeric forms. As a prototype, the as base at retention time (r.t.) in dichloromethane as solvent
1H-NMR spectrum of 10a showed two singlet peaks equiva- (Chart 3). Hydrazinolysis of 14 was carried out in methanol
lent to one proton at δ 8.20 and 7.97ppm, in ratio 54.8%: under reflux for 4h; to afford the corresponding hydrazide 15
45.2%, corresponding to the sp2 CH proton. In addition, two in 87% yield (Chart 3). The structure of compound 14 and 15
singlet peaks, which are D2O exchangeable, were observed were confirmed by NMR spectra and was in agreement with
at 11.31 and 11.21ppm corresponding to the NH proton. The the reported data in the literature.38) The reaction of 15 with
13C-NMR spectrum showed two carbonyl signals at δ 172.24 substituted aromatic aldehydes 16a–e as well as with ketones
and 177.86ppm, which is a further confirmation of the pres- 18a–e in the presence of drops of glacial acetic acid afforded
ence of two isomers. It is expected that compound 10a could the corresponding Schiff bases 17a–e and 19a–e in good to
adopt two different geometrical isomers as shown in Fig. 2. excellent yields as shown in Chart 3. The structures of the
Therefore, it is considered worthwhile to carry out quantum synthesized products 17a–e were confirmed by spectral data.
chemical calculations with the GAUSSIAN 98 suite of pro-
The synthesized Schiff bases of valproylglycine hydrazide
grams. Geometry optimizations were carried out using the in this study (12a–e, Chart 2) showed significant level of
density functional theory (DFT) level (B3LYP/6-31G**) of antiangiogenic activity in transgenic zebrafish embryos (Tg:
theory to assess the relative stability of the anti–syn isomeric fli1: enhanced green fluorescent protein (EGFP)) by blocking
species. Calculated relative energies of 10a anti–syn isomers 70–80% of intersegmental (ISV) and 100% of subintestinal
are −768.8173146au and −768.7634624au, respectively. Com- vein (SIV) blood vessels formation. The level of antiangio-
puted energies indicate the stability of the anti isomer over the genic activity varied with each compound. As shown in Table
syn one by 0.0538522au (33.79279kcal/mol), Fig. 3.
The glycinate derivatives were synthesized by the reaction angiogenic blood vessels at minimum EC50 value.
of the freshly prepared valproylchloride37) with glycine methyl
Compopund 12a suppressed 72% of intrasegmental and
1, compound 12a was the most potent in term of inhibition of
ester in the presence of triethylamine as a base. The valproyl- 100% of subintestinal blood vessels formation process at EC50
glycine ester obtained was treated with hydrazine hydrate in value of only 5µM, followed by compound 12b by blocking
methanol to afford the hydrazide derivative 11 as white crys- 72% of ISV and 100% of SIV with EC50 value of 7µM. Com-
talline solid. The hydrazide 11 was condensed with substituted pound 12c inhibited more than 83% of ISV and 100% of SIV
benzaldehyde and acetophenone to afford the products 12a–e but with EC50 value of 10µM. The ISV inhibition was not ob-
in good yield (Chart 2). The structures of the synthesized served with compound 12d; however, it blocked 100% of SIV
products 12a–e were confirmed by spectral data.
at 3dpf embryos. Compound 12e failed to show any antiangio-
The benzoate derivatives were synthesized by the reaction genic activity in treated zebrafish embryos even using 10 fold
of the freshly prepared valproylchloride with ethyl 4-amino- more concentration (50µM) as compared to 12a.
benzoate hydrochloride 13 in the presence of triethyl amine
Compounds 12a–e were also screened for anticancer activ-

June 2014
593
Chart 2. Synthesis of Schiff Bases of Valproylglycine Hydrazide
Chart 3. Synthesis of Schiff Bases of N-Valproyl-4-aminobenzoyl Hydrazide Derivatives
compound 12a suppressed maximum level of cell growth in-
hibition of HepG2 cells by suppressing more than 60% of cell
growth at 40µM after 24h of treatment followed by compound
12c which suppressed 40% of cancer cell proliferation. Com-
pound 12b suppressed only 30% the cell growth. Compounds
12d and e exhibited moderate level of anticancer activity at
40µM by suppressing 20% and 15% of cell survival of HepG2
cancer cells at 40µM. Valproic acid compound 1 could only
block 13% of HepG2 cell proliferation at 40µM, which means
that the newly synthesized derivatives turnout to be more po-
tent in term of anticancer activity and antiangiogenic activity
as well. We have chosen the liver cancer cell line to evaluate
the anticancer profile of valproic acid derivatives as valproic
acid is finally metabolized in liver39–41) and also the heptotox-
icity is one of the major issue with valproic acid.39,42–47)
Table 1. Comparative Antiangiogenic and Anticancer Profile of Schiff
Bases of Valporyglycine Hydrazide Derivatives and Valproic Acid
Anti-angiogenic activity
Anticancer activity
EC50
concentration
% Survival in
HepG2 cells treated
with 40µM of comp.
Compd.
% ISVa)
inhibition
% SIVb)
inhibition
(µM)
12a
12b
5
72 1.667c) 100 0c)
36.88 0.008c)
71.34 0.009
62.17 0.017
78.62 0.009
84.76 0.010
87.26 0.004
83.47 0.010
100.00 0.021
7
72 0.666
83 1.527
100
0
12c
10
32
50
50
80
100 0.333
12d
0
0
0
0
100
0
0
0
0
0
0
12e
VPA
VALPH
17 0.577
100
0
0
0
0
0
Control 1% DMSO v/v
0
a) ISV: intersegmental vessels. b) SIV; subintestinal vein. c) Values are standard
error of three different replicates.
Compared to Schiff bases of valproylglycine hydrazide
12a–e, the Schiff bases of valproic acid hydrazide (10a–g)
showed moderate level of antiangiogenic activity. As shown
ity in HepG2 liver cancer cell line. The cells were treated with in Table 2, most of these compounds failed to affect the ISV
40µM of compounds 12a–e for 24h and the effect on cell pro- blood vessels formation process in treated embryos, however
liferation was checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-di- they affected the SIV formation with EC50 value ranging from
phenyltetrazolium bromide (MTT) assay. As shown in Table 1, 40–50µM. Compounds 10a–d blocked 100% of subintestinal

594
Vol. 62, No. 6
Table 2. Comparative Antiangiogenic and Anticancer Profile of Schiff
Bases of Valproic Acid Hydrazide Derivatives
Schiff bases 19a–e could not modulate the angiogenic process
in zebrafish embryos even at very high concentration (500µM).
However, these compounds did not show any teratogenic or
toxic affect at this very high concentration. One of the rational
to design and synthesis new derivatives of 1, is to overcome
the severe toxicity attributed to it and to get effective epileptic
compounds with minimum or no toxicity.
Anti-angiogenic activity in
Anticancer activity
in HepG2 cells
zebrafish embryos
Compd.
EC50
concentration
(µM)
% Survival in
HepG2 cells treated
with 40µM of comp.
% ISVa)
inhibition
% SIVb)
inhibition
The Schiff bases of N-valproyl-4-aminobenzoyl hydrazide
derivatives (14, 15, 17a–e, 19a–d) showed a weaker level
of anticancer activity i.e., only 15–30% of suppressing the
HepG2 cell survival (Table 3).
10a
10b
10c
10d
10e
10f
40
45
50
50
40
80
80
0
0
0
0
0
0
0
0c)
0
100
100
100
100
0
0
0
0
57.58 0.025c)
68.30 0.044
75.25 0.004
72.96 0.009
52.46 0.001
75.85 0.007
78.08 0.014
0
0
0
50 1.66
Conclusion
0
0
0
0
0
In conclusion, the synthesized compounds in this study
modulated the angiogenic blood vessels formation in devel-
oping zebrafish embryos significantly. The Schiff bases of
valproylglycine hydrazide exhibited strong antiangiogenic and
anticancer activities, while on contrary Schiff bases of N-
valproyl-4-aminobenzoyl hydrazide derivatives promoted the
angiogenic process. Beside possessing a strong antiangiogenic
activity these compounds did not show strong toxicity or
teratogenecity in zebrafish embryos and specifically disrupted
the angiogenic process, which mean that these compounds are
targeted to those molecular and proteins involved in blood
vessels formation process. The compounds have the potential
to be promoted to pharmaceutical formulation as anticancer
therapeutics or in condition which require the promotion of
angiogenic blood vessels such as tissue regeneration process
during wound healing.
10g
0
a) ISV: intersegmental vessels. b) SIV; subintestinal vein. c) Values are standard
error of three different replicates.
Table 3. Comparative Antiangiogenic and Anticancer Profile of Schiff
Bases of N-Valproyl-4-aminobenzoyl Hydrazide Derivatives
Pro-angiogenic activity in
Anticancer activity in
HepG2 cells
zebrafish embryos
Compd.
EC50
concentration
# of blood vessels
arcades in SIVa)
basket
% Survival in
HepG2 cells treated
with 40µM of comp.
(µM)
17a
17b
17c
17d
17e
19a
19b
19c
19d
1
100
100
100
100
100
500
500
500
500
50
13 0.33b)
12 0.58
86.72 0.27b)
119.37 0.37
79.45 0.25
119.25 0.37
112.48 0.35
89.63 0.28
119.30 0.37
97.21 0.30
115.46 0.36
122.78 0.38
95.78 0.30
92.80 0.29
100.00 0.31
12
0
12 0.33
12 0.33
Chemistry
8
8
8
8
8
0
General The solvents used were of HPLC reagent grade.
Melting points were determined with a Mel-Temp apparatus
and are uncorrected. Infrared (IR) spectra were recorded on
a Perkin-Elmer 1600 series Fourier transform instrument as
KBr pellets. Nuclear magnetic resonance spectra (1H- and
13C-NMR spectra) were recorded on 400MHz JEOL spec-
trometer at room temperature. Chemical shifts are reported in
0.33
0
0.33
0.33
15
40
18 0.33
15 0.33
14
40
Control 1% DMSO v/v
8 0.57
a) ISV: intersegmental vessels. b) Values are standard error of three different parts per million (ppm) and are referenced relative to residual
replicates.
solvent (e.g. CHCl3 at δH 7.26ppm for CDCl3, dimethylsulfox-
ide (DMSO) at δH 2.50ppm for DMSO-d6). Spin multiplicities
vein, however compounds 10e–g did not show significant level are represented by the following signals: singlet (s), broad
of antiangiogenic activity and only compound 10e could in- singlet (brs), doublet (d), broad doublet (brd), doublet of dou-
hibit 50% of SIV formation at 40µM.
blets (dd), triplet (t), doublet of triplets (dt), quartet (q), sextet
The anticancer activity of Schiff bases of valproic acid (sex) and multiplet (m). Elemental analyses were performed on
hydrazide are shown in Table 2. An inhibition of 25 to 40% Perkin-Elmer 2400 elemental analyzer, and the values found
of cell proliferation in HepG2 was observed with compounds were within 0.3% of the theoretical values. Follow-up of
10a–g at 40µM.
the reactions and checks of the purity of the compounds was
The comparative angiogenic profile of valproyl-4-ami- done by TLC on silica gel-protected aluminum sheets (Type
nobenzoyl hydrazide derivatives 17a–e and 19a–e with 1, 60 GF254, Merck) and the spots were detected by exposure to
revealed that in contrast to 1, compounds 14, 15 and 17a–e UV-lamp at λ 254nm for a few seconds. The compounds were
promoted the angiogenic process in zebrafish embryos. These named using Chem. Draw Ultra version 11, Cambridge Soft
compounds modulated the subintestinal vein formation pro- Corporation.
cess by enhancing the number of blood vessels in SIV.
Synthesis of Valproic Hydrazide
8
Valproic acid
In Tg (fli-1: EGFP) transgenic zebrafish embryos SIV (0.01mol) was dissolved in 50mL of methanol, and 3–4 drops
can easily be visualized at three days post fertilization as a of conc. sulphuric acid was added. The reaction mixture was
smooth basket-like structure with 5–6 arcades. In zebrafish refluxed for 12–14h on water bath. The progress of the reac-
embryos treated with compounds 14, 15 and 17a–e and 19a– tion was checked by TLC using hexane–ethyl acetate (4:6) as
d, a mesh of blood vessels with 15–18 arcades formed (Table a mobile phase. After the reaction was completed, excess of
3). This pro-angiogenic activity was more prominent in em- methanol was removed under reduced pressure and the crude
bryos treated with 40µM of either compound 14 or 15 at 3dpf. product was dissolved in ethyl acetate (30mL), washed with

June 2014
595
5% sodium bicarbonate solution (20mL×2), water (20mL×2), 9.82 (s, 1H, OH), 10.99 (s, 1H, NH). 13C-NMR (DMSO-d6) δ
dried over anhydrous sodium sulphate, filtered, and then the (ppm): 14.65, 20.80, 20.89, 34.94, 35.43, 44.72. 116.30, 126.00,
solvent was removed under reduced pressure to afford the 126.13, 129.32, 143.24, 147.06, 159.88, 171.83, 177.47. Anal.
product as a viscous liquid in 90% yield.34)
Calcd for C15H22N2O2: C, 68.67; H, 8.45; N, 10.68. Found: C,
The crude product was used for the next step. Hydrazine 68.81; H, 8.55; N, 10.51.
hydrate (15mL) was added to a solution of methyl valproate
N′-(4-Methoxybenzylidene)-2-propylpentanehydrazide
(0.01mol) in methanol (20mL) and the reaction mixture was (10d): The product was obtained as white crystals, in yield
refluxed for 12–14h. The reaction mixture was cooled to r.t. to 0.29g (83%), mp 110–112°C. IR (KBr): 3216 (NH), 1656
1
give the hydrazide 8 as a white colored shining fluffy product, (CO, amide) cm−1. H-NMR (DMSO-d6): Isomer A (53.9%)
in yield 85%, mp 123–124°C.35) IR (KBr): 3284 (NH), 1631 δ (ppm): 0.82–0.86 (m, 6H, 2CH3), 1.18–1.33 (m, 6H, 3CH2),
1
(CO, amide) cm−1. H-NMR (CDCl3) δ (ppm): 0.86 (t, 6H, 1.47–1.54 (m, 2H, CH2), 2.19–2.22 (m, 1H, CH), 3.78 (s,
2CH3), 1.14–1.39 (m, 6H, 3CH2), 1.53–1.604 (m, 2H, CH2), 3H,OCH3), 6.96–6.98 (m, 2H, Ar-H), 7.54–7.60 (m, 2H, Ar-H),
1.95–2.01 (m, 1H, CH), 3.95 (brs, 2H, NH2), 7.05 (s, 1H, NH). 8.13 (m, 1H, CH), 11.17 (s, 1H, NH). Isomer B (46.1%) δ
13C-NMR (CDCl3) δ (ppm): 14.11, 20.86, 35.05, 45.61, 177.06.
(ppm): 0.82–0.86 (m, 6H, 2CH3), 1.18–1.33 (m, 6H, 3CH2),
General Method for Preparation of Schiff’s Base of 1.47–1.54 (m, 2H, CH2), 3.39–3.43 (m, 1H, CH), 3.78 (s, 3H,
Valproic Hydrazide Derivatives 10a–g36) A solution of val- OCH3), 6.96–6.98 (m, 2H, Ar-H), 7.54–7.60 (m, 2H, Ar-H),
proic hydrazide 8 (0.2g, 1.27mmol) in methanol (25mL) was 7.90 (m, 1H, CH), 11.07 (s, 1H, NH). 13C-NMR (DMSO-d6) δ
added to a slotution of substituted benzaldehyde and acetophe- (ppm): 14.65, 20.80, 20.89, 34.93, 35.41, 44.73, 55.95, 114.94,
none (1.27mmol) in methanol (10mL), and glacial acetic acid 129.18, 142.82, 142.84, 146.66, 171.96, 177.60. Anal. Calcd for
(2 drops), the reaction mixture was refluxed for 8h. The prod- C16H24N2O2: C, 69.53; H, 8.75; N, 10.14. Found: C, 69.71; H,
uct was separated out on cooling, filtered, recrystallized from 8.54; N, 10.41.
ethanol to give 2-propylpentanehydrazide derivatives 10a–g.
N′-(1-Phenylethylidene)-2-propylpentanehydrazide
(10e):
N-Benzylidene-2-propylpentanehydrazide (10a): The prod- The product was obtained as white crystals, in yield 0.30g
uct was obtained as white crystals, in yield 0.27g (87%), mp (91%), mp 118–119°C. IR (KBr): 3201 (NH), 1666 (CO,
105–106°C. IR (KBr): 3203 (NH), 1659 (CO, amide) cm−1. amide) cm−1. 1H-NMR (DMSO-d6): Isomer A (55.2%) δ
1H-NMR (DMSO-d6): Isomer A (54.8%) δ (ppm): 0.82–0.86 (ppm): 0.81–0.87 (m, 6H, 2CH3), 1.20–1.35 (m, 6H, 3CH2),
(m, 6H, 2CH3), 1.19–1.36 (m, 6H, 3CH2), 1.48–1.56 (m, 2H, 1.48–1.60 (m, 2H, CH2), 2.21 (s, 3H, CH3), 2.50–2.51 (m,
CH2), 2.20–2.27 (m, 1H, CH), 7.38–7.41 (m, 3H, Ar-H), 1H, CH), 7.37–7.39 (m, 3H, Ar-H), 7.72–7.77 (m, 2H, Ar-H),
7.60–7.66 (m, 2H, Ar-H), 8.20 (s, 1H, CH), 11.31 (s, 1H, NH). 10.36 (s, 1H, NH). Isomer B (44.8%) δ (ppm): 0.81–0.87 (m,
Isomer B (45.2%) δ (ppm): 0.82–0.86 (m, 6H, 2CH3), 1.19–1.36 6H, 2CH3), 1.20–1.35 (m, 6H, 3CH2), 1.48–1.60 (m, 2H, CH2),
(m, 6H, 3CH2), 1.48–1.56 (m, 2H, CH2), 3.38–3.46 (m, 1H, 2.24 (s, 3H, CH3), 3.48–3.52 (m, 1H, CH), 7.37–7.39 (m, 3H,
CH), 7.38–7.41 (m, 3H, Ar-H), 7.60–7.66 (m, 2H, Ar-H), 7.97 Ar-H), 7.72–7.77 (m, 2H, Ar-H), 10.24 (s, 1H, NH). 13C-NMR
(m, 1H, CH), 11.21 (s, 1H, NH). 13C-NMR (DMSO-d6) δ (DMSO-d6) δ (ppm): 14.69, 14.74, 20.83, 20.87, 34.92, 35.49,
(ppm): 14.64, 20.80, 20.89, 34.94, 35.38, 44.77, 127.17, 127.61, 43.99, 126.46, 126.95, 128.92, 129.08, 147.22, 152.38, 172.72,
129.43, 129.53, 142.96, 146.78, 172.24, 177.86. Anal. Calcd for 178.58. Anal. Calcd for C16H24N2O: C, 73.81; H, 9.29; N, 10.76.
C15H22N2O: C, 73.13; H, 9.00; N, 11.37. Found: C, 72.91; H, Found: C, 73.61; H, 9.53; N, 10.48.
8.79; N, 11.58.
N′-(1-(4-Hydroxyphenyl)ethylidene)-2-propylpentane-
N′-(4-Chlorobenzylidene)-2-propylpentanehydrazide (10b): hydrazide (10f): The product was obtained as white crystals,
The product was obtained as white crystals, in yield 0.31g in yield 0.30g (86%), mp 183–184°C. IR (KBr): 3450–3200
1
(87%), mp 155°C. IR (KBr): 3208 (NH), 1661 (CO, amide) (OH), 3281 (NH), 1660 (CO, amide) cm−1. H-NMR (DMSO-
cm−1. 1H-NMR (DMSO-d6): Isomer A (53.7%) δ (ppm): d6): Isomer A (48.9%) δ (ppm): 0.80–0.86 (m, 6H, 2CH3),
0.82–0.84 (m, 6H, 2CH3), 1.20–1.55 (m, 8H, 4CH2), 2.21–2.26 1.17–1.37 (m, 6H, 3CH2), 1.47–1.58 (m, 2H, CH2), 2.14 (s,
(m, 1H, CH), 7.48–7.91 (m, 4H, Ar-H), 7.93 (s, 1H, CH), 11.38 3H, CH3), 2.45–2.50 (m, 1H, CH), 6.74–6.83 (m, 2H, Ar-H),
(s, 1H, NH). Isomer B (46.3%) δ (ppm): 0.82–0.84 (m, 6H, 7.56–7.62 (m, 2H, Ar-H), 9.68 (s, 1H, OH), 10.17 (s, 1H, NH).
2CH3), 1.20–1.55 (m, 8H, 4CH2), 2.21–2.26 (m, 1H, CH), Isomer B (51.1%) δ (ppm): 0.80–0.86 (m, 6H, 2CH3), 1.17–1.37
7.48–7.91 (m, 4H, Ar-H), 8.19 (m, 1H, CH), 11.28 (s, 1H, NH). (m, 6H, 3CH2), 1.47–1.58 (m, 2H, CH2), 2.16 (s, 3H, CH3),
13C-NMR (DMSO-d6) δ (ppm): 14.63, 14.65, 20.79, 20.88, 3.43–3.52 (m, 1H, CH), 6.74–6.83 (m, 2H, Ar-H), 7.56–7.62
34.95, 35.36, 44.77, 129.39, 131.80, 134.05, 138.41, 141.74, (m, 2H, Ar-H), 9.68 (s, 1H, OH), 10.10 (s, 1H, NH). 13C-NMR
145.49, 167.13, 172.34. Anal. Calcd for C15H21ClN2O: C, 64.16; (DMSO-d6): δ (ppm): 14.71, 14.73, 20.83, 20.88, 34.94, 35.53,
H, 7.54; N, 9.98. Found: C, 63.94; H, 7.79; N, 9.78.
44.03, 115.65, 115.79, 115.81, 127.95, 128.51, 159.27, 172.34,
N′-(4-Hydroxybenzylidene)-2-propylpentanehydrazide (10c): 178.28. Anal. Calcd for C16H24N2O2: C, 69.53; H, 8.75; N,
The product was obtained as white crystals, in yield 0.30g 10.14. Found: C, 69.69; H, 8.63; N, 10.38.
(90%), mp 190–192°C. IR (KBr): 3320–3200 (OH), 3218 (NH),
N′-(1-(4-Methoxyphenyl)ethylidene)-2-propylpentane-
1632 (CO, amide) cm−1. 1H-NMR (DMSO-d6): Isomer A hydrazide (10g): The product was obtained as white crystals,
(60.5%) δ (ppm): 0.82–0.85 (m, 6H, 2CH3), 1.18–1.32 (m, 6H, in yield 0.31g (85%), mp 117–118°C. IR (KBr): 3232 (NH),
3CH2), 1.46–1.54 (m, 2H, CH2), 2.17–2.22 (m, 1H, CH), 6.78 1649 (CO, amide) cm−1. 1H-NMR (DMSO-d6): Isomer A
(d, 2H,J=8.4Hz, Ar-H), 7.42–7.48 (m, 2H, Ar-H), 8.07 (s, 1H, (47.4%) δ (ppm): 0.81–0.87 (m, 6H, 2CH3), 1.20–1.36 (m, 6H,
CH), 9.82 (s, 1H, OH), 11.09 (s, 1H, NH). Isomer B (39.5%) δ 3CH2), 1.47–1.59 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.5–2.55
(ppm): 0.82–0.85 (m, 6H, 2 CH3), 1.18–1.32 (m, 6H, 3 CH2), (m, 1H, CH), 3.75–3.77 (m, 3H, OCH3), 6.93–6.95 (d, 2H,
1.46–1.54 (m, 2H, CH2), 3.37–3.42 (m, 1H, CH), 6.78 (d, 2H, Ar-H), 7.67–7.72 (m, 2H, Ar-H), 10.24 (s, 1H, NH). Isomer B
J=8.4Hz, Ar-H), 7.42–7.48 (m, 2H, Ar-H), 7.86 (m, 1H, CH), (52.6%) δ (ppm): 0.81–0.87 (m, 6H, 2CH3), 1.20–1.36 (m, 6H,

596
Vol. 62, No. 6
3CH2), 1.47–1.59 (m, 2H, CH2), 2.20 (s, 3H, CH3), 3.45–3.52 128.51, 128.58, 135.60, 136.03, 137.25, 168.91, 168.92. Anal.
(m, 1H, CH), 3.75–3.77 (m, 3H, OCH3), 6.93–6.95 (d, 2H, Calcd for C17H24ClN3O2: C, 60.44; H, 7.16; N, 12.44. Found:
Ar-H), 7.67–7.72 (m, 2H, Ar-H), 10.14 (s, 1H, NH). 13C-NMR C, 60.17; H, 6.95; N, 12.68.
(DMSO-d6) δ (ppm): 14.71, 14.74, 20.83, 20.88, 34.93, 35.52,
N-(2-(2-(4-Hydroxybenzylidene)hydrazinyl)-2-oxoethyl)-2-
55.88, 114.28, 114.46, 127.88, 127.43, 172.45, 178.36. Anal. propylpentanamide (12c): The product was obtained as white
Calcd for C17H26N2O2: C, 70.31; H, 9.02; N, 9.65. Found: C, crystals, in yield 0.27g (85%), mp 253–254°C. 1H-NMR
70.60; H, 8.83; N, 9.48.
(DMSO-d6) δ (ppm): 0.82 (t, 6H, 2CH3), 1.19–1.34 (m, 6H,
Synthesis of N-(2-Hydrazinyl-2-oxoethyl)-2-propylpen- 3CH2), 1.40–1.47 (m, 2H, CH2), 2.11–2.23 (m, 1H, CH), 3.59
tanamide 11 Ethyl glycinate hydrochloride (3.48g, 25mmol) (s, 2H, CH2), 7.58–7.68 (m, 3H, Ar-H+NH), 7.93–7.95 (d,
was mixed with 2eq triethylamine (7mL, 50mmol) in 200mL 2H, Ar-H), 8.70 (s, 1H, CH), 11.33 (s, 1H, NH). 13C-NMR
dichloromethane, while stirring continuously at 0°C. Freshly (DMSO-d6) δ (ppm): 14.57, 14.65, 14.68, 20.71, 20.81, 34.83,
distilled valproyl chloride (4.1g, 25mmol) dissolved in 50mL 35.56, 45.67, 62.20, 129.04, 129.58, 132.02, 134.48, 162.13.
of dichloromethane and added dropwise, after complete ad- Anal. Calcd for C17H25N3O3: C, 63.93; H, 7.89; N, 13.16.
dition, the reaction mixture was stirred overnight at room Found: C, 64.21; H, 8.05; N, 13.44.
temperature. The reaction mixture was diluted with CH2Cl2
N-(2-Oxo-2-(2-(1-phenylethylidene)hydrazinyl)ethyl)-2-
(50mL) and washed with water (2×20mL), dried over MgSO4 propylpentanamide (12d): The product was obtained as white
anhydrous, filtered and the solvent was removed under re- crystals, in yield 0.26g (82%), mp 105–106°C. 1H-NMR
duced pressure, the residue was recrystallized from acetone (DMSO-d6) δ (ppm): 0.82–0.85 (m, 6H, 2CH3), 1.23–1.26 (m,
to afford ethyl 2-(2-propylpentanamido)acetate in yield 5.5g 6H, 3CH2), 1.40–1.47 (m, 2H, CH2), 2.15–2.23 (m, 1H, CH),
(95%), mp 82°C.
2.26 (s, 3H, CH3), 4.25 (s, 2H, CH2), 7.38–7.40 (m, 3H, Ar-H+
To a solution of ethyl 2-(2-propylpentanamido)acetate (4.6g, NH), 7.75–7.78 (d, 2H, Ar-H), 7.98 (brs, 1H, NH), 11.17 (s,
20mmol) in 10mL methanol, 10mL hydrazine hydrate (80%) 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 14.60, 14.75, 15.40,
was added. The reaction mixture was refluxed for about 4h, 20.79, 20.83, 22.17, 34.92, 35.63, 45.33, 45.78, 127.15, 129.10,
and the excess of hydrazine was evaporated under reduced 130.43, 138.57, 157.98. Anal. Calcd for C18H27N3O2: C, 68.11;
pressure. The product that precipitated was filtrated off and H, 8.57; N, 13.24. Found: C, 67.97; H, 8.35; N, 13.48.
washed several time with ethanol. The product 11 was ob-
N-(2-(2-(1-(4-Chlorophenyl)ethylidene)hydrazinyl)-2-
tained as white crystals in yield 3.9g (90.6%), mp 245–246°C. oxoethyl)-2-propylpentanamide (12e): The product was ob-
1H-NMR (DMSO-d6) δ (ppm): 0.77 (m, 6H, 2CH3), 1.16 (m, tained as white crystals, in yield 0.31g (88%), mp 145–146°C.
6H, 3CH2), 1.37 (m, 2H, CH2), 2.16 (m, 1H, CH), 3.65 (s, 2H, 1H-NMR (DMSO-d6) δ (ppm): 0.81–0.84 (m, 6H, 2CH3),
CH2), 4.25 (brs, 2H, NH2), 8.03 (m, 1H, NH), 9.84 (s, 1H, 1.21–1.25 (m, 6H, 3CH2), 1.43–1.46 (m, 2H, CH2), 2.19–2.23
NH). 13C-NMR (DMSO-d6) δ (ppm): 8.99, 14.58, 20.62, 35.37, (m, 1H, CH), 2.25 (s, 3H, CH3), 4.23 (s, 2H, CH2), 7.44 (d,
35.42, 40.91, 45.53, 45.96, 169.09, 175.89.
2H, Ar-H), 7.78 (d, 2H, 2 Ar-H), 7.98 (s, 1H, NH), 11.22 (s,
General Procedure for Preparation of Schiff’s Base of 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 14.75, 15.36, 20.78,
N-(2-Hydrazinyl-2-oxoethyl)-2-propylpentanamide Deriva- 21.12, 35.62, 49.83, 129.00, 129.16, 135.28, 137.25, 157.61,
tives 12a–e A solution of N-(2-hydrazinyl-2-oxoethyl)-2-pro- 168.61. Anal. Calcd for C18H26ClN3O2: C, 61.44; H, 7.45; N,
pylpentanamide 11 (0.22g, 1mmol) in methanol (25mL) was 11.94. Found: C, 61.67; H, 7.21; N, 12.17.
added to substituted aldehydes or ketones (1mmol) in metha-
Synthesis of Ethyl 4-(2-Propylpentanamido)benzoate
nol (10mL), and glacial acetic acid (2 drops) and the reaction (VABE, 14)37) Ethyl 4-aminobenzoate 13 (25mmol) was
mixture was then refluxed for 8h. The product was separated mixed with triethylamine (25mmol) in 200mL dichlorometh-
out on cooling, filtered off, recrystallized from ethanol and ane. While a continuous stirring at 0°C; freshly distilled val-
dried to give the N-(2-hydrazinyl-2-oxoethyl)-2-propylpentan- proyl chloride (25mmol) mixed with 50mL dichloromethane
amide derivatives 12a–e.
was added dropwise and the reaction mixture was stirred
N-(2-(2-Benzylidenehydrazinyl)-2-oxoethyl)-2-propylpen- overnight at room temperature. The reaction mixture was
tanamide (12a): The product was obtained as white crystals, diluted with CH2Cl2 (50mL), the organic phase washed with
1
in yield 0.25g (83%), mp 80–81°C. H-NMR (DMSO-d6) δ water, and dried over MgSO4 anhydrous. Filtered and the
(ppm): 0.82 (t, 6H, 2CH3), 1.19–1.34 (m, 6H, 3CH2), 1.40–1.47 solvent was removed with a rotary evaporator to afford 14
1
(m, 2H, CH2), 2.11–2.23 (m, 1H, CH), 3.59 (s, 2H, CH2), in yield 6g (82%) as an oily product. H-NMR (DMSO-d6,
7.58–7.68 (m, 4H, Ar-H+NH), 7.93–7.95 (d, 2H, Ar-H), 8.70 400MHz) δ: 0.85 (t, 6H, J=7.3Hz, 2 CH3), 1.19–1.32 (m, 9 H,
(s, 1H, CH), 11.22 (s, 1H, NH). 13C-NMR (DMSO-d6) δ 3 CH2, CH3), 1.53–1.55 (m, 2H, CH2), 2.44–2.50 (m, 1H, CH),
(ppm): 14.57, 14.65, 14.68, 20.71, 20.81, 34.83, 35.56, 45.67, 4.26 (q, 2H, J=7.3Hz, CH2), 7.77 (d, 2H, J=8.8Hz, Ar-H),
62.20, 129.04, 129.58, 132.02, 134.48, 162.13. Anal. Calcd for 7.90 (d, 2H, J=8.8Hz, Ar-H), 10.25 (s, 1H, NH).
C17H25N3O2: C, 67.30; H, 8.31; N, 13.85. Found: C, 67.57; H,
8.25; N, 13.78.
Synthesis of N-(4-(Hydrazinecarbonyl)phenyl)-2-pro-
pylpentanamide (VABH, 15) To solution of ethyl
a
N-(2-(2-(4-Chlorobenzylidene)hydrazinyl)-2-oxoethyl)-2- 4-(2-propylpentanamido)benzoate 14 (5.82g, 20mmol) in
propylpentanamide (12b): The product was obtained as white 10mL methanol, 10mL hydrazine hydrate (80%) was added.
crystals, in yield 0.28g (83%), mp 130–131°C. 1H-NMR The reaction mixture was refluxed for about 4h then cooled
(DMSO-d6) δ (ppm): 0.79–0.83 (m, 6H, 2CH3), 1.15–1.30 to room temperature. The excess of hydrazine was evaporated
(m, 6H, 3CH2), 1.39–1.44 (m, 2H, CH2), 2.19–2.31 (m, 1H, under reduced pressure. The product that precipitated was
CH), 4.19 (s, 2H, CH2), 7.33 (d, 2H, Ar-H), 7.65–7.69 (m, 3H, filtrated off and washed several time by ethanol. The prod-
Ar-H+NH), 8.70 (s, 1H, CH), 11.26 (s, 1H, NH). 13C-NMR uct was obtained as white crystal in yield 4.8g (87%), mp
1
(DMSO-d6) δ (ppm): 14.74, 20.77, 21.98, 35.60, 45.76, 127.29, 235–236°C. H-NMR (DMSO-d6, 500MHz) δ: 0.83 (t, 6H,

June 2014
597
1
J=6.9Hz, 2CH3), 1.17–1.35 (m, 6H, 3CH2), 1.48–1.55 (m, 2H, (NO2) cm−1. H-NMR (DMSO-d6, 400MHz) δ: 0.86 (t, 6H,
CH2), 2.36–2.39 (m, 1H, CH), 4.42 (brs, 2H, NH2), 7.63 (d, J=7.2Hz, 2CH3), 1.23–1.38 (m, 6H, 3CH2), 1.52–1.55 (m, 2H,
2H, J=9.2Hz, Ar-H), 7.72 (d, 2H, J=9.2Hz, Ar-H), 9.60 (s, CH2), 2.41–2.43 (m, 1H, CH), 7.75 (d, 2H, J=8.8Hz, Ar-H),
1H, NH), 10.03 (s, 1H, NH). 13C-NMR (DMSO-d6, 125MHz) 7.89 (d, 2H, J=8.8Hz, Ar-H), 7.95–7.97 (m, 2H, Ar-H), 8.28
δ: 14.54, 20.77, 35.34, 46.78, 118.95, 128.17, 128.24, 142.19, (d, 2H, J=8.8Hz, Ar-H), 8.53 (s, 1H, CH), 10.14 (s, 1H,
166.06, 175.18.
NH), 12.02 (s, 1H, NH). 13C-NMR (DMSO-d6, 100MHz)
General Procedure for Preparation of Schiff’s Base δ: 14.68, 20.92, 35.46, 46.97, 119.17, 119.25, 124.77, 128.59,
of N-(4-(Hydrazinecarbonyl)phenyl)-2-propylpentanamide 129.38, 129.40, 141.45, 145.47, 148.46, 175.49. Anal. Calcd for
Derivatives 17a–e and 19a–d
A
solution of N-(4- C22H26N4O4: C, 64.37; H, 6.38; N, 13.65. Found: C, 64.14; H,
(hydrazinecarbonyl)phenyl)-2-propylpentanamide 15 (0.19g, 6.30; N, 13.38.
0.7mmol) in methanol (25mL) was added to a solution of sub-
N-(4-(2-4-Methoxybenzylidenehydrazinecarbonyl)-
stituted benzaldehyde or acetophenone (0.7mmol) in methanol phenyl)-2-propylpentanamide (17e): The product was obtained
(10mL), and glacial acetic acid (2 drops). The reaction mixture as white crystals, in yield 0.27g (95%), mp 259–260°C. IR
1
was refluxed for 8h. The product was separated out on cool- (KBr): 3306, 3218 (NH), 1647 (CO, amide) cm−1. H-NMR
ing, filtered off, recrystallized from ethanol and dried to give (DMSO-d6, 400MHz) δ: 0.85 (t, 6H, J=7.2Hz, 2CH3),
the N-(4-(hydrazinecarbonyl)phenyl)-2-propylpentanamide de- 1.19–1.39 (m, 6H, 3CH2), 1.50–1.57 (m, 2H, CH2), 2.40–2.42
rivatives 17a–e and 19a–d.
(m, 1H, CH), 3.79 (s, 3H, OCH3), 6.99 (d, 2H, J=8.8Hz,
N-(4-(2-Benzylidenehydrazinecarbonyl)phenyl)-2- Ar-H), 7.64 (d, 2H, J=8.4Hz, Ar-H), 7.72 (d, 2H, J=8.8Hz,
propylpentanamide (17a): The product was obtained as white Ar-H), 7.85 (d, 2H, J=8.4Hz, Ar-H), 8.37 (s, 1H, CH),
crystals, in yield 0.23g (89%), mp 251–252°C. IR (KBr): 10.11 (s, 1H, NH), 11.58 (s, 1H, NH). 13C-NMR (DMSO-d6,
1
3450, 3290 (NH), 1646 (CO, amide) cm−1. H-NMR (DMSO- 100 MHz) δ: 14.68, 20.92, 35.47, 46.96, 55.98, 115.01, 119.15,
d6, 400MHz) δ: 0.86 (t, 6H, J=7.2Hz, 2CH3), 1.20–1.39 (m, 127.66, 129.29, 142.88, 142.89, 147.92, 161.45, 175.42. Anal.
6H, 3CH2), 1.50–1.59 (m, 2H, CH2), 2.38–2.47 (m, 1H, CH), Calcd for C23H29N3O3: C, 69.85; H, 7.39; N, 10.62. Found: C,
7.41–7.46 (m, 3H, Ar-H), 7.70–7.75 (m, 4H, Ar-H), 7.87 (d, 1H, 69.74; H, 7.60; N, 10.48.
J=8.4Hz, Ar-H), 8.44 (s, 1H, CH), 10.12 (s, 1H, NH), 11.72
N-(4-(2-(1-Phenylethylidene)hydrazinecarbonyl)phenyl)-2-
(s, 1H, NH). 13C-NMR (DMSO-d6, 100MHz) δ: 14.68, 20.92, propylpentanamide (19a): The product was obtained as white
35.47, 46.96, 119.16, 127.69, 127.73, 129.18, 129.51, 129.90, crystals, in yield 0.25g (92%), mp 220–221°C. IR (KBr): 3438,
130.64, 130.65, 130.69, 135.11, 143.01, 148.02, 148.05, 163.14, 3296 (NH), 1650 (CO, amide) cm−1. 1H-NMR (DMSO-d6,
175.44. Anal. Calcd for C22H27N3O2: C, 72.30; H, 7.45; N, 400MHz) δ: 0.86 (t, 6H, J=7.2Hz, 2CH3), 1.20–1.38 (m, 6H,
11.50. Found: C, 72.02; H, 7.25; N, 11.78.
3CH2), 1.50–1.59 (m, 2H, CH2), 2.34 (s, 3H, CH3), 2.38–2.45
N-(4-(2-4-Chlorobenzylidenehydrazinecarbonyl)phenyl)-2- (m, 1H, CH), 7.40–7.41 (m, 3H, Ar-H), 7.72 (d, 2H, J=8.8Hz,
propylpentanamide (17b): The product was obtained as white Ar-H), 7.80–7.85 (m, 4H, Ar-H), 10.10 (s, 1H, NH), 10.59 (s,
crystals, in yield 0.26g (90%), mp 265–266°C. IR (KBr): 1H, NH). 13C-NMR (DMSO-d6, 100MHz) δ: 14.68, 20.92,
1
3466, 3294 (NH), 1681 (CO, amide) cm−1. H-NMR (DMSO- 35.48, 46.97, 118.97, 118.98, 119.00, 119.02, 119.05, 127.03,
d6, 400MHz) δ: 0.82–0.86 (m, 6H, 2CH3), 1.22–1.35 (m, 6H, 129.02, 138.86, 175.41. Anal. Calcd for C23H29N3O2: C, 72.79;
2CH2), 1.50–1.55 (m, 2H, CH2), 2.35–2.42 (m, 1H, CH), 7.54 H, 7.70; N, 11.07. Found: C, 73.00; H, 7.44; N, 11.32.
(d, 2H, J=8.8Hz, Ar-H), 7.64 (d, 2H, J=8.8Hz, Ar-H), 7.74
N-(4-(2-(1-(4-Chlorophenyl)ethylidene)hydrazinecarbonyl)-
(d, 2H, J=8.8Hz, Ar-H), 7.91 (d, 2H, J=8.8Hz, Ar-H), 8.43 phenyl)-2-propylpentanamide (19b): The product was obtained
(s, 1H, CH), 9.60 (s, 1H, NH), 10.03 (s, 1H, NH). 13C-NMR as white crystals, in yield 0.24g (81%), mp 265–266°C. IR
1
(DMSO-d6, 100MHz) δ: 14.67, 20.90, 35.47, 46.93, 119.10, (KBr): 3463, 3250 (NH), 1663 (CO, amide) cm−1. H-NMR
119.15, 119.18, 128.31, 128.37, 129.39, 129.61, 131.80, 138.41, (DMSO-d6, 400MHz) δ: 0.85 (t, 6H, J=7.2Hz, 2CH3),
142.33, 166.21, 167.15, 175.31. Anal. Calcd for C21H26ClN3O: 1.18–1.39 (m, 6H, 3CH2), 1.50–1.59 (m, 2H, CH2), 2.33 (s,
C, 67.82; H, 7.05; N, 11.30. Found: C, 68.04; H, 7.28; N, 11.58. 3H, CH3), 2.38–2.45 (m, 1H, CH), 7.47 (d, 2H, J=8.4Hz,
N-(4-(2-4-Hydroxybenzylidenehydrazinecarbonyl)phenyl)-2- Ar-H), 7.71 (d, 2H, J=8.8Hz, Ar-H), 7.82–7.84 (m, 4H, Ar-H),
propylpentanamide (17c): The product was obtained as white 10.10 (s, 1H, NH), 10.63 (s, 1H, NH). 13C-NMR (DMSO-d6,
crystals, in yield 0.22g (82%), mp 220–221°C. IR (KBr): 100 MHz) δ: 14.68, 20.92, 35.48, 46.97, 118.97, 118.99, 119.01,
3300–3200 (OH), 3300, 3235 (NH), 1662 (CO, amide) cm−1. 119.03, 128.75, 128.78, 128.82, 129.07, 129.65, 137.69, 175. 41.
1H-NMR (DMSO-d6, 400MHz) δ: 0.84 (t, 6H, J=7.2Hz, Anal. Calcd for C23H28ClN3O2: C, 66.74; H, 6.82; N, 10.15.
2CH3), 1.16–1.38 (m, 6H, 3CH2), 1.47–1.59 (m, 2H, CH2), Found: C, 66.57; H, 6.58; N, 10.32.
2.37–2.44 (m, 1H, CH), 6.81 (d, 2H, J=8.4Hz, Ar-H), 7.53
N-(4-(2-(1-(4-Hydroxyphenyl)ethylidene)hydrazine-
(d, 2H, J=8.4Hz, Ar-H), 7.72 (d, 2H, J=8.8Hz, Ar-H), 7.85 carbonyl)phenyl)-2-propylpentanamide (19c): The product
(d, 2H, J=8.8Hz, Ar-H), 8.32 (s, 1H, CH), 9.98 (s, 1H, OH), was obtained as white crystals, in yield 0.24g (84%), mp
10.11 (s, 1H, NH), 11.51 (s, 1H, NH). 13C-NMR (DMSO-d6, 264–265°C. IR (KBr): 3300–3200 (OH), 3278 (NH), 1657
1
100 MHz) δ: 14.67, 20.92, 35.47, 46.96, 116.37, 116.49, 119.15, (CO, amide) cm−1. H-NMR (DMSO-d6, 400MHz) δ: 0.85 (t,
119.28, 119.30, 126.07, 129.06, 129.46, 142.83, 148.39, 160.01, 6H, J=7.2Hz, 2CH3), 1.20–1.39 (m, 6H, 3CH2), 1.50–1.59 (m,
162.91, 175.43. Anal. Calcd for C22H27N3O3: C, 69.27; H, 7.13; 2H, CH2), 2.27 (s, 3H, CH3), 2.38–2.45 (m, 1H, CH), 6.78 (d,
N, 11.02. Found: C, 69.04; H, 7.38; N, 11.28.
2H, J=8.4Hz, Ar-H), 7.67–7.72 (m, 4H, Ar-H), 7.82 (d, 2H,
N-(4-(2-4-Nitrobenzylidenehydrazinecarbonyl)phenyl)-2- J=8.4Hz, Ar-H), 9.75 (s, 1H, OH), 10.09 (s, 1H, NH), 10.47
propylpentanamide (17d): The product was obtained as pale (s, 1H, NH). 13C-NMR (DMSO-d6, 100MHz) δ: 14.68, 20.93,
yellow crystals, in yield 0.24g (81%), mp 278–280°C. IR 35.48, 46.96, 115.75, 119.03, 128.67, 128.68, 129.63, 137.89,
(KBr): 3296, 3219 (NH), 1649 (CO, amide), 1548 and 1348 138.01, 142.58, 142.46, 142.67, 142.69, 142.75, 142.93, 159.51,

598
Vol. 62, No. 6
159.52, 175.37. Anal. Calcd for C23H29N3O3: C, 69.85; H, 7.39;
N, 10.62. Found: C, 69.59; H, 7.48; N, 10.39.
N-(4-(2-(1-(4-Nitrophenyl)ethylidene)hydrazinecarbonyl)-
phenyl)-2-propylpentanamide (19d): The product was obtained
as pale yellow crystals, in yield 0.25g (82%), mp 283–284°C.
% antiangiogenic activity
number of missing blood vessels in the trunk
=
×100
total number of blood vessels
IR (KBr): 3298, 3220 (NH), 1649 (CO, amide), 1548 and 1346
At least three biological replications were conducted with
(NO2) cm−1. H-NMR (DMSO-d6, 400MHz) δ: 0.86 (t, 6H, different clutches of embryos from different parents. A con-
J=7.2Hz, 2CH3), 1.23–1.38 (m, 6H, 3CH2), 1.52–1.55 (m, 2H, centration which was not inducing gross tearatological effects
CH2), 2.41–2.43 (m, 1H, CH), 2.50 (s, 3H, CH3), 7.75 (d, 2H, and only affecting the blood vessels outgrowth in zebrafish
J=8.8Hz, Ar-H), 7.89 (d, 2H, J=8.8Hz, Ar-H), 7.95–7.97 embryos was taken into consideration. A Compound was
(m, 2H, Ar-H), 8.28 (d, 2H, J=8.8Hz, Ar-H), 10.14 (s, 1H, scored as antiangiogenic when it blocked the angiognesis
NH), 12.02 (s, 1H, NH). 13C-NMR (DMSO-d6, 100MHz) blood vessels at least 70% of the treated embryos in all three
δ: 14.68, 20.92, 35.46, 46.97, 119.17, 124.77, 124.83, 128.59, biological replicates.
1
129.34, 129.38, 129.40, 141.45, 148.46, 175.49. Anal. Calcd for
Calculation of EC50 EC50 values were calculated by treat-
C23H28N4O4: C, 65.08; H, 6.65; N, 13.20. Found: C, 65.36; H, ing the embryos with serial dilution of the compound and a
6.48; N, 13.49.
mean value which inhibited the blood vessels formation in
50% of treated embryos in at least three replicates was taken
as EC50.
Biology
Animals Wild type (AB/Tuebingen TAB-14) and Tg (fli1:
Cell Culture and Proliferation Assay HepG2 derived
EGFP)48) zebrafish were obtained from zebrafish international from human liver cancer were cultured in high glucose Dul-
resource center (ZIRC University of Oregon, Oregon, U.S.A.) becco’s modified Eagle’s medium (DMEM: Life Technologies
and maintained in our facility under recommended conditions. Cat #11995073) supplemented with 10% fetal bovine serum
The embryos were obtained by natural pair wise mating and (FBS: Life Technologies Cat #16000044) in a humidified
treated following local and international guide lines for the incubator with 5% CO2 at 37°C. Around 2×103 cells were
use of laboratory animals.
seeded in each well of a 96-well cell culture plate and were
Treatment of Zebrafish Embryos with VPA and Its allowed to adhere and spread for 24h. The compounds were
Derivatives Stock Solutions: VPA and newly synthesized added to a final concentration of 40µM in triplicate, and the
derivatives were dissolved in water and molecular biology cells were cultured for another 24h at 37°C with the com-
grade dimethyl sulfoxide (DMSO) Sigma-Aldrich Cat # D8418 pounds. The proliferation was determined in each experiment
respectively to make a stock concentration of 20mg/mL. The using MTT 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan
calculated volume of the compounds was added directly to colorimetric assay. Briefly, the treated or untreated cells were
Embryo Medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2 trypsenized, centrifuged and the resulting pellet was resus-
& 0.33mM MgSO4) to obtain required working dilutions. The pended in 100µL of DMEM serum free medium in each well
mock (0.5% DMSO, v/v) treated embryos served as control.
of 96-well plates and incubated at 37°C for two hours. After
Animal Treatment: Synchronized AB wild type embryos incubation 20µL of MTT solution (5mg/mL in phosphate
were raised to shield stage: (ca. 6h post fertilization). The buffered saline (PBS): Sigma-Aldrich Cat #M2003) was added
embryos were staged according to Kimmel et al.49) Any un- to each well and further incubated for 2h. The plate was
fertilized or embryo that appeared developmentally delayed centrifuged at 40000rpm for 10min then the medium was re-
or otherwise abnormal were also excluded. Around fifty (50) moved from each well and isopropanol containing 0.04M HCl
embryos were placed in 35mm Petri dishes; in 10mL embryo was added to dissolve the formazan produced in the cells. The
medium containing desired quantity of compound. The em- optical density of the formazan product in solution was mea-
bryos were incubated in refrigerated air incubator at 28.5°C sured with a microplate reader at 540nm. The experiment was
overnight. On the following day any dead embryos either in conducted in triplicate. Data were calculated as percent of cell
control or treated groups were recorded or removed and em- viability by the following formula:
bryos were raised in compounds free embryo medium subse-
quently up to five days post fertilization (5dpf) with replace-
ment of embryo medium every day.
mean absorbance in test wells
mean absorbance in control wells
% cell viability=
×100
Antiangiogenic Assay in Zebrafish Embryos We have
scored the antiangiogenic activity of the compounds in the
Acknowledgment The authors thank the Deanship of
live transgenic zebrafish embryos by observing the reduction Scientific Research at King Saud University for funding this
in the outgrowth of blood vessels at two time points in the work through research group project “RGP-234.”
same embryo. i) ISV at 48h post fertilization and then ii) SIV
out growth at 72h post fertilization. Just before counting the
blood vessels, the embryos were anesthetized using 0.003%
tricane (ethyl 3-aminobenzoate methanesulfonate, Sigma-
Aldrich cat #E10521) in embryo medium. A scoring method
was used to assess the level of antiangiogenic activity of com-
pounds by counting total number of blood vessels in the trunk
area of treated embryos and any missing or un-developed
blood vessels. The percentage was calculated by using follow-
ing equation.
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