Some hydrazones of 2-aroylamino-3-methylbutanohydrazide: Synthesis, molecular modeling studies, and identification as stereoselective inhibitors of HIV-1

Article abstract of DOI:10.1002/ardp.201200311

In accordance with our antiviral drug development attempt, acylhydrazone derivatives bearing amino acid side chains were synthesized for the evaluation of their antiviral activity against various types of viruses. Among these compounds, 8_S, 11_S, and 12_S showed anti-HIV-1 activity with a 50% inhibitory concentration (IC₅₀) = 123.8 μM (selectivity index, SI > 3), IC₅₀ = 12.1 μM (SI > 29), IC₅₀ = 17.4 μM (SI > 19), respectively. Enantiomers 8 _R, 11_R, and 12_R were inactive against the HIV-1 strain III_B. Hydrazones 8_S, 11_S, and 12 _S which were active against HIV-1 wild type showed no inhibition against a double mutant NNRTI-resistant strain (K103N;Y181C). Molecular docking calculations of R- and S-enantiomers of 8, 11, and 12 were performed using the hydrazone-bound novel site of HIV-1 RT. Copyright

Full text of DOI:10.1002/ardp.201200311

140
Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
Full Paper
Some Hydrazones of 2-Aroylamino-3-methylbutanohydrazide:
Synthesis, Molecular Modeling Studies, and Identification as
Stereoselective Inhibitors of HIV-1
1
Esra Tatar , Ilkay Ku¨c¸u¨kgu¨zel , Dirk Daelemans , Tanaji T. Talele , Neerja Kaushik-Basu ,
1
2
3
4
˙
Erik De Clercq2, and Christophe Pannecouque2
1
˙
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Marmara University, Haydarpas¸a, Istanbul,
Turkey
2 Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
3 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University,
Queens, NY, USA
4 Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA
In accordance with our antiviral drug development attempt, acylhydrazone derivatives bearing
amino acid side chains were synthesized for the evaluation of their antiviral activity against
various types of viruses. Among these compounds, 8S, 11S, and 12S showed anti-HIV-1 activity with
a 50% inhibitory concentration (IC50) ¼ 123.8 mM (selectivity index, SI > 3), IC50 ¼ 12.1 mM (SI > 29),
IC50 ¼ 17.4 mM (SI > 19), respectively. Enantiomers 8R, 11R, and 12R were inactive against the HIV-1
strain IIIB. Hydrazones 8S, 11S, and 12S which were active against HIV-1 wild type showed no inhibition
against a double mutant NNRTI-resistant strain (K103N;Y181C). Molecular docking calculations of R-
and S-enantiomers of 8, 11, and 12 were performed using the hydrazone-bound novel site of HIV-1 RT.
Keywords: Acylhydrazones / HIV-1 / Molecular modeling / Non-nucleoside reverse transcriptase inhibitors /
Stereoselective activity
Received: August 22, 2012; Revised: October 10, 2012; Accepted: October 17, 2012
DOI 10.1002/ardp.201200311
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
tors (NNRTIs), protease inhibitors (PIs), cell entry inhibitors
[fusion inhibitors (FIs) and co-receptor inhibitors (CRIs)], and
integrase inhibitors (INIs) [1]. Despite these advances, there
exists a pressing need to develop new treatment strategies
for AIDS due to the emergence of drug-resistant HIV variants.
To infect its hosts, HIV uses three essential enzymes: HIV
reverse transcriptase (HIV-RT), integrase (IN), and protease
(PR) [2]. Among these, HIV-1 RT has been a major target for
antiretroviral drug development and more than half of the
currently approved drugs for the treatment of HIV-1 infection
are RT inhibitors [3]. Conversion of the single stranded RNA
genome into a double stranded DNA is the primordial func-
tion of HIV-1 RT. HIV-1 RT consists of a p66 and a p51 subunit
and thus acts as a heterodimer. Despite the structural role of
the p51 subunit as elucidated by means of conformational
modifications, the NNRTI-binding pocket (NNRTI-BP), poly-
merase, and RNase H domains are located in the p66 subunit
[4]. The NNRTI-BP with hydrophobic and hydrophilic residues
Infection with human immunodeficiency virus (HIV), the etio-
logical agent of acquired immune deficiency syndrome (AIDS),
is a significant global concern. Drug development efforts aimed
at suppressing the viability, replication, and virulence of HIV
have resulted in a number of active anti-HIV compounds, 26 of
which have been FDA-approved to date. These clinically avail-
able compounds can be broadly classified into six groups
according to their targets and mode of action in the HIV
replicative cycle. These include the nucleoside reverse tran-
scriptase inhibitors (NRTIs), nucleotide reverse transcriptase
inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibi-
˙
Correspondence: Ilkay Ku¨c¸u¨kgu¨zel, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Marmara University, Haydarpas¸a, 34668
˙
Istanbul, Turkey.
E-mail: ikucukguzel@marmara.edu.tr
Fax: þ90 216 345 29 52
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Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
L-Valine Hydrazones as Stereoselective Inhibitors of HIV-1 RT
141
˚
is nearly 10 A away from the catalytic site and the amino acid
[30–35] and analysis of their structures has revealed the
presence of amino acid side chains (Fig. 2).
residues lysine at position 103 (K103N) and tyrosine at
position 181 (Y181C) are prone to mutation.
Keeping these structural parameters in view, we synthesized
a series of 2-aroylamino-3-methylbutanohydrazide-hydrazones
and evaluated them against diverse viruses. Compounds found
to be active against the HIV-1(IIIB) strain were further inves-
tigated for their mechanism of action. Finally, molecular
modeling was employed to gain insight into the structural
parameters for binding and stereoselectivity.
There are five NNRTIs approved for clinical use: nevirapine,
delavirdine, efavirenz, etravirine, and rilpivirine (Fig. 1).
Crystallographic analysis of the HIV-1 RT–NNRTI complex of
the first-generation NNRTIs such as nevirapine and delavirdine
has revealed a common ‘‘butterfly-like’’ conformation despite
the chemical diversity between these compounds [5, 6]. By
contrast, diarylpyrimidine (DAPY) analogues such as etravirine
and rilpivirine, belonging to the third generation NNRTIs, Results and discussion
adopt different conformational modes popularly called ‘‘giggle
and wiggle’’ through their torsional flexibility and ability to
reposition within the NNRTI binding pocket [1, 7, 8].
Chemistry
Compounds 1S and 1R were prepared by benzoylation of
L-valine or D-valine. Compounds 2S and 2R were obtained
by esterification of compounds 1S and 1R. Compounds 3S
and 3R were obtained by heating compounds 2S and 2R with
hydrazine hydrate. (2S)-2-(Benzoylamino)-3-methyl-N’-(arylme-
thylene)butanohydrazides (4–7, 8S, 9–10, 11S, 12S, 13–24) were
synthesized by refluxing compound 3S with various aromatic
aldehydes (4-chlorobenzaldehyde, 2-chlorobenzaldehyde, 2,6-
dichlorobenzaldehyde, 2,4–dichlorobenzaldehyde, 2-fluoro-
benzaldehyde, 3-fluorobenzaldehyde, 4-fluorobenzaldehyde,
The goal of this study was to identify and synthesize new
NNRTIs which potentially prevent the emergence of resistant
HIV-1 RT variants. Toward this end, we focused on the acyl-
hydrazone scaffold as our antiviral development strategy [9].
Acylhydrazones have served as starting compounds for
diverse heterocyclic ring structures and have been attributed
with various biological activities [10] such as antituberculosis
[11–18], antibacterial [19, 20], antiparasitic [21, 22], anti-
cancer [23–26], analgesic, and anti-inflammatory [27–29].
Further, the anti-HIV activity of acylhydrazone scaffold com-
pounds has been appraised in a limited number of studies
2,6-difluorobenzaldehyde,
2-chloro-6-fluorobenzaldehyde,
4-(trifluoromethyl)benzaldehyde, 4-methoxybenzaldehyde,
Figure 1. Structures of approved NNRTIs.
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Figure 2. Chemical structures and anti-HIV activity of acylhydrazones with amino acid side chains.
2,6-dimethoxybenzaldehyde, 4-methylbenzaldehyde, 2,6-
dimethylbenzaldehyde, 2,4,6-trimethylbenzaldehyde, 4-
hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde,
4-nitrobenzaldehyde, N,N-dimethyl-4-aminobenzaldehyde,
cinnamaldehyde, 5-nitrofurfural) in ethanolic medium
(Scheme 1). (2R)-2-(Benzoylamino)-3-methyl-N’-(arylmethyle-
ne)butanohydrazides (8R, 11R, 12R) were synthesized by
refluxing compound 3R with 2-fluorobenzaldehyde, 2,6-
difluorobenzaldehyde and 2-chloro-6-fluorobenzaldehyde in
ethanolic medium (Scheme 1).
compounds and they were in accordance with the literature
[19, 37].
It is known that the hydrazones may exist as E/Z geometri-
–
cal isomers due to the rotational restriction about the N C
–
double bond and cis/trans amide conformers [34, 38]. On the
basis of literature, hydrazones comprising aldehyde and sub-
stituted hydrazide moieties prefer existing as E isomers in
dimethyl-d6 sulfoxide solution [34, 38, 39]. The chemical shift
of the azomethine proton in the synthesized compounds was
detected in the range of 8.01–8.69 ppm and two singlet peaks
at 11.20–12.35 ppm were attributed to –NH– protons of the
acylhydrazone moiety for compounds 4–24. According to
Easmon et al. [40] the N–H proton belonging to E-isomeric
forms of acylhydrazones appears in the range 9–12 ppm;
however, the same proton belonging to Z-isomeric forms of
acylhydrazones appears in the range of 14–15 ppm in the
1H NMR spectra. In the light of the foregoing, we propose that
the compounds 4–24 exist as E-form. Two sets of signals
belonging to cis/trans conformers of NH– protons of the acyl-
hydrazone moiety were detected between 11.20–11.88 ppm
and 11.36–12.35 ppm; the up-field signal of the mentioned
proton was assigned to the cis conformer while the down-field
While elemental analysis and HPLC data confirmed
the purity of compounds 4–24 and 1–24, respectively,
their structures were identified by IR and 1H NMR
spectroscopy.
IR spectral data of compounds 4–24 were also confirmed on
the basis of IR spectral data in the literature [30, 36]. The
bands between 1676 and 1666 cmꢀ1 were attributed to
–
the C O stretching bands and the bands between 1612
–
–
and 1577 were assigned to C N stretching bands of acyl-
–
hydrazones 4–24.
1
The chemical shifts obtained from the H NMR spectra of
compounds 4–24 all supported the proposed structures of the
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L-Valine Hydrazones as Stereoselective Inhibitors of HIV-1 RT
143
Scheme 1. Synthetic route to compounds 1S, 2S, 3S, 4–24, 8S, 11S, 12S. Reagents and conditions: (a) C6H5COCl/NaOH; (b) MeOH/
H2SO4; (c) NH2NH2.H2O; (d) Ar–CHO/EtOH, reflux.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
signal was assigned to the trans isomer (possible tautomeric
forms of acylhydrazones 4–24 according to 1H NMR data are
depicted in Scheme 2). 13C NMR spectrum of compound 11
was also recorded for further support. Detecting CHCH(CH3)2,
(EC50 ¼ 26.8 mM) and feline herpes virus (EC50 ¼ 60.6 mM,
Supporting Information Table S4).
The anti-HCV activity of the compounds was investigated
employing the in vitro HCV NS5BRdRp inhibition assay as
described in the Experimental section. The compounds 4–
5, 7, 8S, 9, 12S, 14–18, and 21–22 exhibited <20% inhibition of
HCV NS5B RdRp activity at a compound concentration of
100 mM (Supporting Information Table S5), thus suggesting
that these compounds are specific to HIV.
–
CHCH(CH3)2 acylhydrazone C O and some of the aromatic
–
C-atoms as two peaks instead of one, thus, provided confir-
matory evidence for the presence of both isomers. Specific
rotation ([a]D) of all compounds was measured to judge opti-
cal purity. The specific optical rotation values of L-valine
derived acylhydrazones 4–7, 8S, 9–10, 11S, 12S, 13–24
were determined and ranged between 243.311 and 122.387
while D-valine derived acylhydrazones 8R, 11R, and 12R exhib-
ited specific optical rotation values of ꢀ185.584, ꢀ154.594,
and ꢀ151.258, respectively.
Anti-HIV and cytotoxicity data were obtained with the
synthesized compounds using the strains HIV-1 (IIIB) and
HIV-2 (ROD) in an MT-4/MTT based assay [41] (see
Supporting Information Table S6). The results of the anti-
HIV study indicated that compounds 8S, 11S, and 12S showed
appreciable anti HIV-1 (IIIB) activity with 50% inhibitory con-
centration (IC50) of 123.8 mM and a selectivity index (SI) > 3,
IC50 ¼ 12.1 mM and SI > 29, IC50 ¼ 17.4 mM and SI > 19,
respectively (Table 1). Compounds 8R, 11R, and 12R as enan-
tiomers of three active hydrazones, were synthesized in order
to evaluate their activity against the HIV-1 (IIIB) strain in
parallel with their S-enantiomers but none of them exhibited
any significant activity. None of the evaluated compounds
were found to be active against HIV-2 (strain ROD). Moreover,
the hydrazones 8s, 11s, and 12s, which were active against
HIV-1 wild type, showed no activity against a double mutant
NNRTI resistant strain (K103N; Y181C; Table 1). It is note-
worthy that the antiretroviral activity of compounds 8, 11, 12
is stereoselective and mainly imparted by their S-enantiom-
ers. Although compound 8S with 2-fluorophenyl moiety was
found moderately active against HIV-1 (IIIB), IC50 values of
its analogous compounds 9 and 10 with 3-fluorophenyl
Antiviral evaluation
In view of the antiviral activity ascertained for similar acyl-
hydrazones, the synthesized compounds were subjected to a
preliminary screening for their antiviral effects against var-
ious types of viruses in HEL, HeLa, Vero, and CRFK (Crandell-
Rees feline kidney) cell cultures. Compounds 4–24 were not
found to be active against HSV–1(KOS), HSV-1(TKꢀKOSACVr),
HSV-2(G), vaccinia virus, vesicular stomatitis virus, varicella
zoster virus TKþVZV, varicella zoster virus TKꢀVZV, cytome-
galovirus, vesicular stomatitis virus, respiratory syncytial
virus, Coxsackie B4 virus, parainfluenza-3 virus, reovirus-1,
Sindbis virus, and Punta Toro virus (in vitro antiviral
evaluation and cytotoxicity data for compounds 4–24 in
HEL, HeLa, and Vero cell cultures are available as
Supporting Information Tables S1–S3). Compound 14 was
found to have marginal activity against feline corona virus
Scheme 2. Possible tautomeric forms of acylhydrazones 4–24 according to 1H NMR data.
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L-Valine Hydrazones as Stereoselective Inhibitors of HIV-1 RT
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Table 1. Anti-HIV evaluation of selected compounds against wild-
type HIV-1 (IIIB), an HIV-1 NNRTI-resistant strain (NNRTIR), and
HIV-2 (ROD) in MT4 cells.
a polyanionic entry inhibitor, and clinically used RT inhibi-
tors, AZT and nevirapine, were included as references. Both
compounds, 11S and 12S, completely block the replication of
the virus in the same time frame as the established RT
inhibitors, confirming their supposed NNRTI mode of action
(Fig. 3).
Compound
8S
Strain
IC50 (mM)
CC50 (mM)
SI
IIIB
NNRTIR
ROD
IIIB
123.8 ꢁ 116.5
>366
>366
>366
>366
>366
>366
>366
>348
>348
>348
>348
>348
>332
>332
>332
>332
>332
>3
–
–
Molecular docking studies
8R
>366
>366
–
–
>29
–
–
–
–
>19
–
–
–
ROD
IIIB
To evaluate the probable binding conformation of the rep-
resentative chiral hydrazone derivatives, we performed dock-
ing study using Glide docking software v5.0 (Schro¨dinger,
Inc., New York, NY, 2009). Since the hydrazone derivatives
have been previously shown to inhibit HIV-1 RT through
binding to a novel site located between the NNRTI-BP and
the active site, the docking calculations of the chiral hydra-
zone derivatives were performed using the DHBNH (dihy-
droxy benzoyl naphthyl hydrazone)-HIV-1 RT co-crystal
structure. It has been reported that, although DHBNH prim-
11S
12.1 ꢁ 6.3
NNRTIR
ROD
IIIB
>348
>348
11R
12S
>348
>348
ROD
IIIB
NNRTIR
ROD
IIIB
17.4 ꢁ 11.2
>332
>332
12R
>332
>332
ROD
–
˚
arily inhibits the RNase H activity, it binds >50 A away from
the RNase H subdomain, at a site that partially overlaps the
NNRTI-binding pocket [42]. As a preliminary step, we validated
the accuracy of our docking approach by determining how
closely the lowest energy binding conformation predicted by
the Glidescore (Gscore) function resembles an experimental
binding conformation as determined by X-ray crystallo-
graphy. This validation resulted in a very good agreement
between the localization of the inhibitor from docking and
and 4-fluorophenyl moieties were determined as >117.5 and
>116.5 mg/mL, respectively. Among 2,6-halogenosubstituted
acylhydrazones (compounds 6, 11, 12); compounds 11S and
12S with 2,6-difluorophenyl and 2-chloro-6-fluorophenyl moi-
eties showed satisfactory anti-HIV-1 (IIIB) activity; however,
compound 6 carrying the 2,6-chlorophenyl moiety was
thoroughly inactive with an IC50 value of >113 mg/mL.
The compounds that displayed anti-HIV-1 activity in cell
culture and their respective inactive enantiomer were eval-
uated for their inhibitory action on the RT polymerase
activity in an enzymatic assay. In this assay the compounds
11S and 12S proved to be inhibitory with IC50 values of
162.2 ꢁ 6.3 mM and 79.5 ꢁ 9.7 mM, respectively, whereas
the other compounds were completely devoid of activity even
at concentrations of 3200 mM (Table 2).
The ultimate proof confirming the NNRTI mode of action
on HIV-1 strain IIIB replication in MT-4 cell culture was
generated by performing a time-of-addition experiment with
the most active compounds, 11S and 12S. Dextran sulfate,
Table 2. Selected compounds assayed against wild-type HIV-1 RT
polymerase activity.
Compound
IC50 (mM)a)
11R
12R
11S
12S
Nevirapine
Efavirenz
>3200
>3200
162.2 ꢁ 6.3
79.5 ꢁ 9.7
12.3 ꢁ 3.8
0.17 ꢁ 0.06
Figure 3. Time-of-addition experiment. MT-4 cells were infected with
HIV-1, and the test compounds were added at different time points
after infection. Virus production was determined by p24 Ag production
in the supernatant at 31 h post infection. Crosses, control; diamonds,
dextran sulfate 8000 (12.5 mM); stars, AZT (1.9 mM); triangles, nevir-
apine (7.5 mM); inverted triangles, 11S (95 mM); discs, 12S (125 mM).
(This figure can be viewed in color mode in the online version.)
a)
Compound concentration required to inhibit HIV-1 RT wild-
type activity by 50%.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
from the crystal structure as exemplified by a RMSD value of Conclusion
˚
0.5 A between the predicted conformation and the observed
X-ray crystallographic conformation of DHBNH. Analysis of
the binding energy data for the docked conformations of
representative chiral hydrazone derivatives (compounds 11R
and 11S) at the novel site showed that the (S)-isomer
(Gscore ¼ ꢀ7.73 kcal/mol) binds more favorably than its
(R)-counterpart (Gscore ¼ ꢀ7.18 kcal/mol). Based on Glide
docking scores, the E-isomer of compound 11S was found
to bind more favorably than the Z-isomer.
Among 21 newly designed and synthesized acylhydrazone
compounds, 8S, 11S, and 12S were shown to possess anti-
HIV-1 (IIIB) activity with IC50 ¼ 123.8 mM and SI > 3,
IC50 ¼ 12.1 mM and SI > 29, IC50 ¼ 17.4 mM and SI > 19,
respectively. The 8R, 11R, and 12R enantiomers of the three
active hydrazones were synthesized and evaluated for their
activity against the HIV-1 (IIIB) strain, but none of them
exhibited any significant antiviral activity. None of the com-
pounds tested (4–24) were found to be active against the HIV-2
strain ROD. Moreover, the hydrazones 8s, 11s, and 12s, which
were active against HIV-1 wild type, showed no activity against
a double mutant NNRTI resistant strain (K103N;Y181C). The
NNRTI activity of compounds 8, 11, and 12 is stereoselective,
and is exclusively due to the activity of the S-enantiomer. It was
clearly established, as could be expected, that the anti-HIV-1
activity results from an NNRTI mode of action.
The binding mode of compound 11S within the novel site
of HIV-1 RT showed four key interactions (Fig. 4A). The phenyl
ring of the benzoyl moiety is stabilized through the hydro-
phobic contacts with the methylene groups of Lys223 and the
isopropyl group of Val108. The isopropyl group forms hydro-
phobic contacts with the side chains of Val108 and Leu234.
The benzamide –NH and hydrazide –NH, respectively, forms
hydrogen bonding interaction with the carboxylate group of
the highly conserved Asp186. The highly electron-deficient
2,6-difluorophenyl ring forms a face-to-face pi–pi interaction
with the electron-rich indole ring of the highly conserved
primer grip residue Trp229, in addition to forming hydro-
phobic interactions with the side chains of Pro95, Leu100,
and Tyr181.
Based on the results from anti-HIV screening and enzyme
inhibition studies as well as molecular modeling findings,
novel hydrazone derivatives targeting HIV-1 RT at lower con-
centrations with higher selectivity have been developed.
Experimental
The binding mode of compound 11R within the novel site
of HIV-1 RT is shown in Fig. 4B. The binding conformation of
compound 11R maintained all of the interactions of 11S
except that the phenyl ring of the benzoyl moiety is fully
solvent-exposed and does not participate in hydrophobic
interaction with the methylene groups of the side chain of
Lys223 and the isopropyl group of Val108.
Synthesis
All solvents and reagents were obtained from commercial sour-
ces and used without purification. All melting points (8C, uncor-
rected) were determined using a Kleinfeld SMP-II basic model
melting point apparatus. Specific rotation was measured on a
Autopol V Plus digital polarimeter (serial number 85123, man-
ufactured by Rudolph Research Analytical) with a 2 dm optical
Figure 4. Glide-XP predicted binding model of compound (S)-11 (A) and (R)-11 (B) within the hydrazone binding site of HIV-1 RT.
Amino acid residues are shown as stick model (please view this figure in color mode in the online version with the atoms colored as
carbon – green, hydrogen – white, nitrogen – blue, and oxygen – red) whereas compounds are shown as ball and stick model with
the same color scheme as above except that carbon atoms are represented in orange and the fluoro atoms are shown in green color.
Dotted black line indicates hydrogen bonding interaction whereas dotted red line indicates electrostatic interactions.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
L-Valine Hydrazones as Stereoselective Inhibitors of HIV-1 RT
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length cell at 25.38C. Elemental analyses were obtained using
Leco CHNS-932 and are consistent with the assigned structures.
Infrared spectra were recorded on a Shimadzu FTIR 8400S and
data are expressed in wavenumber y (cmꢀ1). NMR spectra were
recorded on a Bruker AVANCE-DPX 400 at 400 MHz for 1H NMR
and 100 MHz for 13C NMR (DEPT and decoupled), the chemical
shifts were expressed in d (ppm) downfield from tetramethyl-
silane (TMS) using DMSO-d6 as solvent. The liquid chromato-
graphic system consists of an Agilent Technologies 1100 series
instrument equipped with a quaternary solvent delivery system
and a model Agilent series G1315 A photodiode array detector.
A Rheodyne syringe loading sample injector with a 50 mL
sample loop was used for the injection of the analytes.
Chromatographic data were collected and processed using
Agilent Chemstation Plus software. The separation was per-
formed at ambient temperature by using a reversed phase
Nova-Pak C18 (3.9 mm ꢂ 150 mm, 5 mm particle size) column.
All experiments were performed in isocratic mode. The mobile
phase was prepared by mixing acetonitrile and bi-distilled water
(40:60 v/v) and filtered through a 0.45 mm pore filter and sub-
sequently degassed by ultrasonication, prior to use. Solvent
delivery was employed at a flow rate of 1 mL/min. Detection
of the analytes was carried out at 280 nm.
(2R)-2-(Benzoylamino)-3-methylbutyric acid methyl ester
(2R)
Compound 1R (2.21 g, 0.01 mol) was dissolved in 20 mL meth-
anol and 1 mL conc. H2SO4 solution was added. The reaction
mixture was heated under reflux for 3 h. The crude product was
precipitated by using NaHCO3 solution (5% w/v), filtered, dried,
and crystallized from petroleum ether. Yield 50%. m.p. 1118C
[46]. [a]D ꢀ15.954 (c: 1.0, DMF). HPLC tR (min.): 3.54. IR, y (cmꢀ1):
3074 (aromatic CH str), 2966 (alkyl CH str), 2874 (alkyl CH str),
–
–
1735 (ester C O str), 1639 (amide C O str), 1240 (C–O str).
–
–
(2S)-2-(Benzoylamino)-3-methylbutyric acid hydrazide
(3S)
Compound 2S (2.35 g, 0.01 mol) and hydrazine hydrate were
heated under reflux for 1 h and 25 mL methanol was added to
the reaction medium, subsequently the mixture was further
heated under reflux for 1 h. The crude product was filtered and
washed with boiling petroleum ether. Yield 77%. m.p. 210–2118C
[47]. [a]D 40.094 (c: 1.0, DMF). HPLC tR (min.): 1.88. IR, y (cmꢀ1):
–
3269 and 3184 (NH str), 1660 (hydrazide C O str), 1624 (amide
–
–
C O str).
–
(2R)-2-(Benzoylamino)-3-methylbutyric acid hydrazide
(3R)
(2S)-2-(Benzoylamino)-3-methylbutyric acid (1S)
(2S)-2-Amino-3-methylbutyric acid (L-valine, 1.17 g, 0.01 mol) was
dissolved in sodium hydroxide solution (100 mL, 0.02 mol) and
benzoyl chloride (1.40 g, 0.01 mol) was added to the reaction
medium with stirring in an ice bath. The crude product was
precipitated by addition of conc. HCl solution, filtered, dried,
and washed with boiling petroleum ether. Yield 38%. m.p. 136–
1378C [43]. [a]D 31.859 (c: 1.0, DMF (dimethylformamide)).
HPLC tR (min.): 1.5. IR, y (cmꢀ1): 3298 (amide NH str), 3228
(H-bonded OH str), 3066 (aromatic CH str), 2960 (alkyl CH str),
Compound 2R (2.35 g, 0.01 mol) and hydrazine hydrate was
heated under reflux for 1 h and 25 mL methanol was added
to the reaction medium, before further heating under reflux
for 1 h. The crude product was filtered and washed with boiling
petroleum ether. Yield 60%. m.p. 210–2118C [46]. [a]D ꢀ38.269 (c:
1.0, DMF). HPLC tR (min.): 1.88. IR, y (cmꢀ1): 3269 and 3184 (NH
–
–
str), 1660 (hydrazide C O str), 1624 (amide C O str).
–
–
General procedure for the synthesis of (2S)-2-
(benzoylamino)-3-methyl-N’-(arylmethylene)-
butanohydrazides (4–24) and (2R)-2-(benzoylamino)-3-
methyl-N’-(2-fluoro/2,6-difluoro/2-chloro-6-
–
2874 (alkyl CH str), 1722 and 1695 (carboxylic acid C O str), 1639
–
–
(amide C O str).
–
(2R)-2-(Benzoylamino)-3-methylbutyric acid (1R)
(2R)-2-Amino-3-methylbutyric acid (D-valine, 1.17 g, 0.01 mol) was
dissolved in sodium hydroxide solution (100 mL, 0.02 mol)
and benzoyl chloride (1.40 g, 0.01 mol) was added to the
reaction medium with stirring in ice bath. The crude product
was precipitated by addition of conc. HCl solution, filtered,
dried, and washed with boiling petroleum ether. Yield
35%. m.p. 136–1378C [44]. [a]D ꢀ27.785 (c: 1.0, DMF). HPLC tR
(min.): 1.5. IR, y (cmꢀ1): 3298 (amide NH str), 3228 (H-bonded
OH str), 3066 (aromatic CH str), 2960 (alkyl CH str), 2874 (alkyl
fluorobenzylidene)butanohydrazide (8R, 11R, and 12R)
Compound 3S was heated with various aromatic aldehydes under
reflux for 2 h in ethanol. The crude products (4–7, 8S, 9–10,
11S, 12S, 13–24) were filtered and crystallized from appropriate
solvents.
(2R)-2-(Benzoylamino)-3-methylbutyric acid hydrazide (3R) was
heated with 2–fluorobenzaldehyde, 2,6-difluorobenzaldehyde-2-
chloro-6-fluorobenzaldehyde under reflux for 2 h in ethanol. The
crude products (8R, 11R, 12R) were filtered and crystallized from
the appropriate solvents.
–
CH str), 1722 and 1695 (carboxylic acid C O str), 1639 (amide
–
–
C O str).
–
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-
chlorobenzylidene)butanohydrazide (4)
(2S)-2-(Benzoylamino)-3-methylbutyric acid methyl ester
(2S)
Yield 88%. m.p. 246–2478C (EtOH). [a]D 200.721 (c: 1.2, DMF).
HPLC tR (min.): 7.90. IR, y (cmꢀ1): 3281 and 3221 (NH str),
Compound 1S (2.21 g, 0.01 mol) was dissolved in 20 mL meth-
anol and 1 mL conc. H2SO4 solution was added. The reaction
mixture was heated under reflux for 3 h. The crude product was
precipitated using NaHCO3 solution (5% w/v), filtered, dried, and
crystallized from petroleum ether. Yield 82%. m.p. 111–1148C
[45]. [a]D 14.176 (c: 1.0, DMF). HPLC tR (min.): 3.54. IR, y (cmꢀ1):
3074 (aromatic CH str), 2966 (alkyl CH str), 2874 (alkyl CH str),
–
–
–
1674 (hydrazone C O str), 1631 (amide C O str), 1599 and
–
1579 (C N, C C str), 1091 (Ar–Cl str). 1H NMR: d (ppm) 0.94–
–
–
–
–
1.02 (m, 6H, CHCH(CH3)2), 2.17–2.18 (m, 1H, CHCH(CH3)2), 4.30
and 5.34–5.38 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2),
7.45–7.56 (m, 5H, ArH), 7.72 (d, J ¼ 8.6 Hz, 2H, ArH), 7.88–7.92
(m, 2H, ArH), 8.01 and 8.25 (s and s, 1H, amide NH), 8.31 and 8.55
–
–
–
1735 (ester C O str), 1639 (amide C O str), 1240 (C–O str).
(d, J ¼ 8.5 Hz and d, J ¼ 8.2 Hz, 1H, –CH N–), 11.55 and 11.75
–
–
–
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

148
E. Tatar et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
(s and s, 1H, hydrazone NH). Anal. calcd. for C19H20ClN3O2 (357.83):
C, 63.77; H, 5.63; N, 11.74%. Found C, 63.55; H, 4.26; N, 11.77%.
(2R)-2-(Benzoylamino)-3-methyl-N’-(2-
fluorobenzylidene)butanohydrazide (8R)
Yield 36%. m.p. 2328C (MeOH). [a]D ꢀ185.584 (c: 1.0, DMF). HPLC tR
(min.): 5.40. IR, y (cmꢀ1): 3246 and 3207 (NH str), 1672 (hydrazone
(2S)-2-(Benzoylamino)-3-methyl-N’-(2-
chlorobenzylidene)butanohydrazide (5)
C O str), 1631 (amide C O str), 1616 (C N). 1H NMR: d (ppm)
–
–
–
–
–
–
0.95–1.03 (m, 6H, CHCH(CH3)2), 2.18–2.32 (m, 1H, CHCH(CH3)2),
4.28 and 5.35–5.40 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H,
CHCH(CH3)2), 7.26–7.35 (m, 2H, ArH), 7.44–7.57 (m, 4H, ArH),
7.86–7.96 (m, 3H, ArH), 8.23 and 8.50 (s and s, 1H, amide NH),
Yield 42%. m.p. 2428C (EtOH). [a]D 159.331 (c: 0.96, DMF). HPLC tR
(min.): 8.03. IR, y (cmꢀ1): 3276 and 3214 (NH str), 1668 (hydrazone
–
–
–
–
–
–
C O str), 1631 (amide C O str), 1599 and 1579 (C N, C C str),
–
–
1091 (Ar–Cl str). 1H NMR: d (ppm) 0.96–1.03 (m, 6H, CHCH(CH3)2),
2.21–2.29 (m, 1H, CHCH(CH3)2), 4.28 and 5.36–5.40 (t, J ¼ 8.5 Hz,
J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.41–7.57 (m, 6H, ArH), 7.88–
8.02 (m, 3H, ArH), 8.34 and 8.58 (d, J ¼ 8.5 Hz and d, J ¼ 8.2 Hz,
–
8.32 and 8.57 (d, J ¼ 8.4 Hz and d, J ¼ 8.4 Hz, 1H, –CH N–),
–
11.61 and 11.84 (s and s, 1H, hydrazone NH). Anal. calcd.
for C19H20FN3O2 (341.38): C, 66.85; H, 5.91; N, 12.31%. Found
C, 66.00; H, 5.75; N, 12.33%.
–
1H, –CH N–), 8.41 and 8.67 (s and s, 1H, amide NH), 11.37 and
–
12.2 (s and s, 1H, hydrazone NH). Anal. calcd. for C19H20ClN3O2
(357.83): C, 63.77; H, 5.63; N, 11.74%. Found C, 63.49; H, 5.15;
N, 11.70%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(3-fluorobenzylidene)-
butanohydrazide (9)
Yield 69%. m.p. 242–2438C (EtOH/DMF (90:10 v/v)). [a]D 197.284
(c: 1.2, DMF). HPLC tR (min.): 5.54.IR, y (cmꢀ1): 3265 and 3213
(2S)-2-(Benzoylamino)-3-methyl-N’-(2,6-
dichlorobenzylidene)butanohydrazide (6)
–
–
–
(NH str), 1670 (hydrazone C O str), 1627 (amide C O str), 1581
–
1
–
–
–
(C N; C C str), 1219 and 1141 (Ar–F str). H NMR: d (ppm) 0.95–
–
Yield 90%. m.p. 260–2648C (EtOH/DMF (90:10 v/v)). [a]D 122.387
1.02 (m, 6H, CHCH(CH3)2), 2.00–2.44 (m, 1H, CHCH(CH3)2), 4.30
and 5.36–5.40 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2),
7.44–7.56 (m, 7H, ArH), 7.88–7.92 (m, 2H, ArH), 8.01 and 8.26
(s and s, 1H, amide NH), 8.35 and 8.58 (d, J ¼ 8.2 Hz and d,
(c: 0.70, DMF). HPLC tR (min.): 11.12 IR, y (cmꢀ1): 3246 and 3198
–
–
–
(NH str), 1674 (hydrazone C O str), 1626 (amide C O str), 1602
–
and 1579 (C N, C C str). 1H NMR: d (ppm) 0.96–1.01 (m, 6H,
–
–
–
–
CHCH(CH3)2), 2.28–2.33 (m, 1H, CHCH(CH3)2), 4.32 and 5.31–5.35
(t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.42–7.60
(m, 6H, ArH), 7.86 and 7.92 (d, J ¼ 7.1 Hz and d, J ¼ 7.1 Hz, 2H,
Ar–H), 8.32 and 8.47 (s and s, 1H, amide NH), 8.21 and 8.60
–
J ¼ 8.5 Hz, –CH N– 11.60 and 11.79 (s and s, 1H, hydrazone
–
NH). Anal. calcd. for C19H20FN3O2 (341.38): C, 66.85; H, 5.91;
N, 12.31%. Found C, 66.76; H, 4.88; N, 12.36%.
–
(d, J ¼ 8.5 Hz and d, J ¼ 8.3 Hz, 1H, –CH N–), 11.72 and 11.96
–
2-(Benzoylamino)-3-methyl-N’-(4-fluorobenzylidene)-
butanohydrazide (10)
(s and s, 1H, hydrazone NH). Anal. calcd. for C19H19Cl2N3O2
(392.28): C, 58.17; H, 4.88; N, 10.71%. Found C, 58.03; H, 3.80;
N, 10.81%.
Yield 86%. m.p. 233–2358C (EtOH/DMF (90:10 v/v)). [a]D 197.520
(c: 1.0, DMF). HPLC tR (min.): 5.13. IR, y (cmꢀ1): 3255 and 3213
–
–
–
(NH str), 1670 (hydrazone C O str), 1631 (amide C O str), 1602
–
–
–
(2S)-2-(Benzoylamino)-3-methyl-N’-(2,4-
dichlorobenzylidene)butanohydrazide (7)
1
(C N str), 1238, 1207, and 1153(Ar–F str). H NMR: d (ppm) 0.95–
1.02 (m, 6H, CHCH(CH3)2), 2.19–2.27 (m, 1H, CHCH(CH3)2), 4.29
and 5.35–5.39 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2),
7.26–7.33 (m, 2H, ArH), 7.44–7.56 (m, 3H, ArH), 7.74–7.78 (m, 2H,
ArH), 7.88–7.92 (m, 2H, ArH), 8.28 and 8.56 (t, J ¼ 8.6 Hz,
J ¼ 8.3 Hz and d, J ¼ 8.3 Hz, amide NH), 8.01 and 8.69 (s and s,
Yield 68%. m.p. 215–2208C (acetone). [a]D 178.141 (c: 1.0, DMF).
HPLC tR (min.): 5.60. IR, y (cmꢀ1): 3225 (NH str), 1672 (hydrazone
1
C O str), 1631 (amide C O str). H NMR: d (ppm) 0.95–1.02 (m, 6H,
–
–
–
–
CHCH(CH3)2), 2.19–2.29 (m, 1H, CHCH(CH3)2), 4.27 and 5.33–5.37
(t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.44–7.57
(m, 5H, ArH), 7.72 (d, J ¼ 2.1 Hz, 1H, ArH), 7.88–7.93 (m, 2H,
ArH), 7.94, 7.96, 7.99, 8.01 (4s, 1H, amide NH), 8.36 and 8.60
–
1H, –CH N–), 11.49 and 11.69 (s and s, 1H, hydrazone NH). Anal.
–
calcd. for C19H20FN3O2 (341.38): C, 66.85; H, 5.91; N, 12.31%.
Found C, 66.99; H, 5.13; N, 12.31%.
–
(d, J ¼ 8.2 Hz and d, J ¼ 7.7 Hz, 1H, –CH N–), 11.42 and 12.35
–
(s and s, 1H, hydrazone NH). Anal. calcd. for C19H19Cl2N3O2
(392.28): C, 58.17; H, 4.88; N, 10.71%. Found C, 57.53; H, 3.89;
N, 10.01%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(2,6-
difluorobenzylidene)butanohydrazide (11S)
Yield 98%. m.p. 246–2478C (EtOH/DMF (90:10 v/v)). [a]D 178.826 (c:
1.0, DMF). HPLC tR (min.): 6.11. IR, y (cmꢀ1): 3250 and 3211 (NH
(2S)-2-(Benzoylamino)-3-methyl-N’-(2-
fluorobenzylidene)butanohydrazide (8S)
–
–
–
–
str), 1670 (hydrazone C O str), 1631 (amide C O str), 1602 (C N
–
–
1
str), 1236, 1207, and 1153 (Ar–F str). H NMR: d (ppm) 0.95–1.01
(m, 6H, CHCH(CH3)2), 2.28–2.41 (m, 1H, CHCH(CH3)2), 4.27 and
5.29–5.33 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2),
7.17–7.24 (q, 2H, ArH), 7.45–7.56 (m, 4H, ArH), 7.87–7.92
(m, 2H, ArH), 8.16 and 8.43 (s and s, 1H, amide NH), 8.18 and
Yield 82%. m.p. 232–2338C (EtOH). [a]D 198.465 (c: 1.2, DMF).
HPLC tR (min.): 5.46.IR, y (cmꢀ1): 3244 and 3207 (NH str), 1670
–
–
–
–
(hydrazone C O str), 1629 (amide C O str), 1602 and 1579 (C N,
–
–
–
–
1
C C str). H NMR: d (ppm) 0.95–1.02 (m, 6H, CHCH(CH ) ), 2.19–
3 2
–
2.31 (m, 1H, CHCH(CH3)2), 4.28 and 5.35–5.39 (t, J ¼ 8.5 Hz,
J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.27–7.34 (m, 2H, ArH),
7.45–7.57 (m, 5H, ArH), 7.87–7.95 (m, 2H, ArH), 8.23 and 8.50
(s and s, 1H, amide NH), 8.33 and 8.59 (d, J ¼ 8.5 Hz and d,
8.59 (d and d, J ¼ 8.5 Hz, J ¼ 8.2 Hz 1H, –CH N–), 11.62 and
–
11.83 (s and s, 1H, hydrazone NH). 13C NMR-DEPT: d (ppm) 19.71
and 19.77 (CH3), 30.8 and 30.21 (CH), 55.46 and 59.22 (CH), 111.88
(Ar–Cq), 112.65, 112.75, 112.89, 112.99 (Ar–CH), 128.02 and
128.11 (Ar–CH), 128.65 (Ar–CH), 131.70 and 131.82 (Ar–CH),
–
J ¼ 8.2 Hz, 1H, –CH N–), 11.59 and 11.82 (s and s, 1H, hydrazone
–
–
134.46 and 134.70 (Ar–C ), 137.88 (CONHN CH–), 159.58 and
–
q
161.95 (Ar–C ), 167.23 and 168.52 (CONHN ), 173.83 (Ar–CONH).
NH). Anal. calcd. for C19H20FN3O2 (341.38): C, 66.85; H, 5.91;
N, 12.31%. Found C, 66.88; H, 4.67; N, 12.41%.
–
–
q
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
L-Valine Hydrazones as Stereoselective Inhibitors of HIV-1 RT
149
Anal. calcd. for C19H20F2N3O2 (359.37): C, 63.50; H, 5.33;
N, 11.69%. Found C, 63.40; H, 5.15; N, 11.73%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-
methoxybenzylidene)butanohydrazide (14)
Yield 93%. m.p. 255–2568C (EtOH/DMF (90:10 v/v)). [a]D 145.682
(c:1.0, DMF). HPLC tR (min.): 4.53. IR, y (cmꢀ1): 3275 and 3232 (NH
(2R)-2-(Benzoylamino)-3-methyl-N’-(2,6-
difluorobenzylidene)butanohydrazide (11R)
–
–
–
–
str), 1668 (hydrazone C O str), 1631 (amide C O str), 1608 (C N
–
–
str). 1H NMR: d (ppm) 0.94–1.01 (m, 6H, CHCH(CH3)2), 2.06–2.38
(m, 1H, CHCH(CH3)2), 3.80 (d, J ¼ 9.5 Hz, 3H, –OCH3), 4.28 and
5.35–5.37 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2),
6.99–7.04 (m, 2H, ArH), 7.44–7.49 (m, 2H, ArH), 7.52–7.55
(m, 1H, ArH), 7.65 (d, 2H, J ¼ 8.8 Hz, 2H, ArH), 7.88–7.92
(m, 2H, ArH), 7.95 and 8.19 (s and s, 1H, amide NH), 8.25 and
Yield 32%. m.p. 2488C (MeOH). [a]D ꢀ154.594 (c: 0.9, DMF). HPLC tR
(min.): 6.08. IR, y (cmꢀ1): 3257 and 3211 (NH str), 1672 (hydrazone
1
C O str), 1627 (amide C O str), 1232, 1209 (Ar–F str). H NMR: d
–
–
–
–
(ppm) 0.95–1.01 (m, 6H, CHCH(CH3)2), 2.18–2.33 (m, 1H,
CHCH(CH3)2), 4.27 and 5.28–5.34 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz
and m, 1H, CHCH(CH3)2), 7.16–7.25 (q, 2H, ArH), 7.44–7.57
(m, 4H, ArH), 7.87–7.92 (m, 2H, ArH), 8.16 and 8.42 (s and s,
1H, amide NH), 8.19 and 8.58 (d and d, J ¼ 8.4 Hz, J ¼ 8.1 Hz 1H,
–
8.54 (d, J ¼ 8.6 Hz and d, J ¼ 8.3 Hz 1H, –CH N–), 11.50 and
–
11.58 (s and s, 1H, hydrazone NH). Anal. calcd. for C20H23N3O3
(353.42): C, 67.97; H, 6.56; N, 11.89%. Found C, 67.75; H, 6.01; N,
11.92%.
–
–CH N– 11.64 and 11.83 (s and s, 1H, hydrazone NH). Anal. calcd.
–
for C19H20F2N3O2 (359.37): C, 63.50; H, 5.33; N, 11.69%. Found C,
62.62; H, 5.21; N, 11.67%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(2,6-
dimethoxybenzylidene)butanohydrazide (15)
(2S)-2-(Benzoylamino)-3-methyl-N’-(2-chloro-6-
fluorobenzylidene)butanohydrazide (12S)
Yield 60%. m.p. 248–2498C (EtOH/DMF (90:10 v/v)). [a]D 180.901
(c:1.2, DMF). HPLC tR (min.): 5.75. IR, y (cmꢀ1): 3275 and 3232 (NH
Yield 84%. m.p. 248–2498C (EtOH/DMF (90:10 v/v)). [a]D 163.498 (c:
–
–
–
–
str), 1668 (hydrazone C O str), 1631 (amide C O str), 1608 (C N
–
–
0.98, DMF). HPLC tR (min.): 8.33. IR, y (cmꢀ1): 3248 and 3201 (NH
str). 1H NMR: d (ppm) 0.95–1.01 (m, 6H, CHCH(CH3)2), 2.06–2.27
(m, 1H, CHCH(CH3)2), 3.79 (d, J ¼ 6.5 Hz, 6H, –OCH3), 4.28 and
5.21–5.25 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2),
6.70–7.75 (m, 2H, ArH), 7.32–7.37 (m, 1H, ArH), 7.43–7.55 (m,
3H, ArH), 7.84 and 7.90 (d, J ¼ 7.1 Hz and d, J ¼ 7.1 Hz 1H, ArH),
–
–
–
str), 1676 (hydrazone C O str), 1626 (amide C O str), 1602 and
–
1577 (C N; C C str). 1H NMR: d (ppm) 0.96–1.01 (m, 6H,
–
–
–
–
CHCH(CH3)2), 2.11–2.41 (m, 1H, CHCH(CH3)2), 4.29 and 5.29–
5.34 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.33–
7.55 (m, 6H, ArH), 7.87–7.93 (m, 2H, ArH), 8.22 and 8.60 (d,
–
–
8.15 and 8.44 (d, J ¼ 9.1 Hz and d, J ¼ 9.1 Hz 1H, –CH N–), 8.25
–
J ¼ 8.4 Hz, and d, J ¼ 8.2 Hz, 1H, CH N–), 8.28 and 8.51 (s and s,
–
and 8.41 (s and s, 1H, amide NH), 11.27 and 11.44 (s and s, 1H,
hydrazone NH). Anal. calcd. for C21H25N3O4 (383.44): C, 65.78; H,
6.57; N, 10.96%. Found C, 65.32; H, 5.97; N, 10.90%.
1H, amide NH), 11.68 and 11.92 (s and s, 1H, hydrazone NH). Anal.
calcd. for C19H19ClFN3O2 (375.82): C, 60.72; H, 5.10; N, 11.18%.
Found C, 60.99; H, 4.94; N, 11.29%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-
methylbenzylidene)butanohydrazide (16)
(2R)-2-(Benzoylamino)-3-methyl-N’-(2-chloro-6-
fluorobenzylidene)butanohydrazide (12R)
Yield 92%. m.p. 251–2538C (EtOH/DMF (90:10 v/v)). [a]D 185.166 (c:
1.0, DMF). HPLC tR (min.): 6.59.IR, y (cmꢀ1): 3276 and 3230 (NH
Yield 56%. m.p. 252–2538C (MeOH). [a]D ꢀ151.258 (c: 0.92, DMF).
HPLC tR (min.): 8.30. IR, y (cmꢀ1): 3248 and 3200 (NH str), 1676
–
–
–
–
–
str), 1670 (hydrazone C O str), 1635 (amide C O str), 1612 (C N
–
str). 1H NMR: d (ppm) 0.95–1.02 (m, 6H, CHCH(CH3)2), 2.19–2.21
(m, 1H, CHCH(CH3)2), 2.34 (s, 3H, Ar–CH3), 4.29 and 5.35–5.40
(t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2), 7.25–7.28
(m, 2H, ArH), 7.44–7.60 (m, 5H, ArH), 7.88–7.92 (m, 2H, ArH),
7.98 and 8.22 (s and s, 1H, amide NH), 8.25 and 8.54 (d, J ¼ 8.5 Hz
–
–
–
–
–
(hydrazone C O str), 1626 (amide C O str), 1600 (C N; C C str).
–
–
–
1H NMR: d (ppm) 0.96–1.01 (m, 6H, CHCH(CH3)2), 2.18–2.33
(m, 1H, CHCH(CH3)2), 4.29 and 5.29–5.34 (t, J ¼ 8.5 Hz,
J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.33–7.57 (m, 6H, ArH),
7.87–7.93 (m, 2H, ArH), 8.21 and 8.60 (d, J ¼ 8.4 Hz and d,
–
–
J ¼ 8.1 Hz, 1H, –CH N– 8.28 and 8.51 (s and s, 1H, amide NH),
–
and d, J ¼ 8.3 Hz 1H, –CH N–), 11.42 and 11.61 (s and s, 1H,
–
11.70 and 11.93 (s and s, 1H, hydrazone NH). Anal. calcd.
for C19H19ClFN3O2 (375.82): C, 60.72; H, 5.10; N, 11.18%. Found
C, 60.04; H, 4.96; N, 11.17%.
hydrazone NH). Anal. calcd. for C20H23N3O2 (337.42): C, 71.19;
H, 6.87; N, 12.45%. Found C, 71.11; H, 7.27; N, 12.49%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(2,6-
dimethylbenzylidene)butanohydrazide (17)
2-(Benzoylamino)-3-methyl-N’-(4-
trifluoromethylbenzylidene)butanohydrazide (13)
Yield 41%. m.p. 2428C (MeOH). [a]D 206.099 (c: 1.1, DMF). HPLC tR
(min.): 11.98. IR, y (cmꢀ1): 3273 and 3229 (NH str), 1672 (hydra-
Yield 57%. m.p. 243–2448C (EtOH/DMF (90:10 v/v)). [a]D 177.633
(c:1.0, DMF). HPLC tR (min.): 11.87. IR, y (cmꢀ1): 3244 and 3213
1
zone C O str), 1631 (amide C O str), 1609 (C N str). H NMR: d
–
–
–
–
–
–
–
–
–
(NH str), 1672 (hydrazone C O str), 1633 (amide C O str), 1602
–
–
(ppm) 0.96–1.01 (m, 6H, CHCH(CH3)2), 2.19–2.21 (m, 1H,
CHCH(CH3)2), 2.41 and 2.47 (s and s, 6H, Ar–CH3), 4.31 and
5.36–5.44 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2),
7.08–7.19 (m, 3H, ArH), 7.45–7.55 (m, 3H, ArH), 7.88 and 7.92
(dd, J ¼ 7.2 Hz, J ¼ 7.2 Hz, 2H, ArH), 8.20 and 8.48 (d, J ¼ 8.5 Hz
–
1
(C N str). H NMR: d (ppm) 0.95–1.03 (m, 6H, CHCH(CH ) ), 2.19–
3 2
2.29 (m, 1H, CHCH(CH3)2), 4.32 and 5.35–5.38 (t, J ¼ 8.5 Hz,
J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.45–7.57 (m, 4H, ArH),
7.81–7.87 (m, 2H, ArH), 7.88–7.93 (m, 4H, ArH), 8.35 (s, 1H, amide
–
–
NH), 8.09 and 8.61 (s and d, J ¼ 8.2 Hz 1H, –CH N–), 11.68 and
and d, J ¼ 8.3 Hz, 1H, –CH N–), 8.43 and 8.58 (s and s, 1H, amide
–
–
11.89 (s and s, 1H, hydrazone NH). Anal. calcd. for C20H20F3N3O2
(391.38): C, 61.38; H, 5.15; N, 10.74%. Found C, 61.77; H, 5.69; N,
10.74%.
NH), 11.35 and 11.61 (s and s, 1H, hydrazone NH). Anal. calcd.
for C21H25N3O2 (351.44): C, 71.77; H, 7.17; N, 11.96%. Found C,
71.09; H, 7.09; N, 11.88%.
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150
E. Tatar et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
(2S)-2-(Benzoylamino)-3-methyl-N0-(2,4,6-
trimethylbenzylidene)butanohydrazide (18)
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-
dimethylaminobenzylidene)butanohydrazide (22)
Yield 56%. m.p. 248–2538C (EtOH/DMF (90:10 v/v)). [a]D 149.004 (c:
Yield 46%. m.p. 289–2928C (EtOH/acetone (90:10 v/v)). [a]D
0.82, DMF). HPLC tR (min.): 20.15. IR, y (cmꢀ1): 3254 and 3205 (NH
155.281 (c: 1.0, DMF). HPLC tR (min.): 7.14. IR, y (cmꢀ1): 3253
1
str), 1668 (hydrazone C O str), 1629 (amide C O str). H NMR: d
–
–
–
–
–
and 3211 (NH str), 1666 (hydrazone C O str), 1633 (amide C O
–
–
–
(ppm) 0.96–1.00 (m, 6H, CHCH(CH3)2), 2.24–2.31 (q, 4H,
CHCH(CH3)2 and Ar–CH3), 2.38 and 2.44 (s and s, 6H, Ar–CH3),
4.29 and 5.35–5.37 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H,
CHCH(CH3)2), 6.91 and 6.94 (s and s, 2H, ArH), 7.45–7.56 (m,
3H, ArH), 7.87–7.93 (m, 2H, ArH), 8.20 and 8.40 (d, J ¼ 8.6 Hz
str). 1H NMR: d (ppm) 0.94–1.00 (m, 6H, CHCH(CH3)2), 2.16–2.29
(m, 1H, CHCH(CH3)2), 2.94 and 2.97 (s and s, 6H, –N(CH3)2), 4.37
and 5.35–5.39 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2),
6.72–6.80 (m, 2H, ArH), 7.45–7.56 (m, 5H, ArH), 7.88–7.89 (m, 2H,
ArH), 7.92 and 8.09 (s and s, 1H, amide NH), 8.16 and 8.49 (d,
–
–
and s, 1H, amide NH), 8.54 (s, 1H, –CH N– 11.28 and 11.53 (s
–
J ¼ 8.7 Hz and d, J ¼ 8.4 Hz 1H, –CH N–), 11.20 and 11.36 (s
–
and s, 1H, hydrazone NH). Anal. calcd. for C21H26N4O2 (366.45): C,
68.83; H, 7.15; N, 15.29%. Found C, 68.30; H, 7.99; N, 14.89%.
and s, 1H, hydrazone NH). Anal. calcd. for C22H27N3O2 (365.46): C,
72.30; H, 7.45; N, 11.50%. Found C, 72.20; H, 7.11; N, 11.38%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-
hydroxybenzylidene)butanohydrazide (19)
(2S)-2-(Benzoylamino)-3-methyl-N’-[3-[4-
(dimethylamino)phenyl]prop-2-en-1-ylidene]butano-
Yield 83%. m.p. 216–217 8C (EtOH). [a]D 153.713 (c: 0.97, DMF).
hydrazide (23)
Yield 67%. m.p. 2898C (EtOH/acetone (90:10 v/v)). [a]D 159.811
HPLC tR (min.): 2.39. IR, y (cmꢀ1): 3410 (OH str), 3254 and 3205
(c: 1.0, DMF). HPLC tR (min.): 13.44. IR, y (cmꢀ1): 3250 and
–
–
–
(NH str), 1670 (hydrazone C O str), 1630 (amide C O str).
–
1H NMR: d (ppm) 0.94–1.01 (m, 6H, CHCH(CH3)2), 2.16–2.28
(m, 1H, CHCH(CH3)2), 4.28 and 5.35–5.38 (t, J ¼ 8.6 Hz,
J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2), 6.83 (t, J ¼ 8.9 Hz,
J ¼ 8.9 Hz, 2H, ArH), 7.44–7.56 (m, 5H, ArH), 7.88–7.92 (m, 2H,
ArH), 8.14 (s, 1H, amide NH), 8.20 and 8.51 (d, J ¼ 8.7 Hz and
–
–
–
3207 (NH str), 1665 (hydrazone C O str), 1633 (amide C O
–
str). 1H NMR: d (ppm) 0.93–0.98 (m, 6H, CHCH(CH3)2), 2.16–2.38
(m, 1H, CHCH(CH3)2), 2.95 (s, 6H, –N(CH3)2), 2.97–2.99 (m, 1H,
–
–
–
–
–
Ar–CH CH–CH N–), 3.31 (s, 1H, Ar–CH CH–CH N–), 4.28 and
–
–
–
5.33–5.37 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2),
6.68–6.77 (m, 3H, ArH), 6.84, 6.88, 6.92 (s, s, s, 1H, ArH), 7.41–
7.49 (m, 3H, ArH), 7.52–7.56 (m, 1H, ArH), 7.77 and 7.94
(d, J ¼ 9.2 Hz and d, J ¼ 9.3 Hz, 1H, ArH), 7.88–7.91 (m, 1H,
amide NH), 8.12 and 8.48 (d, J ¼ 8.8 Hz and d, J ¼ 8.5 Hz 1H,
–
–
d, J ¼ 8.4 Hz 1H, –CH N–), 9.91 (s, 1H, Ar–OH), 11.28 and
11.46 (s and s, 1H, hydrazone NH). Anal. calcd. for C19H21N3O3
(339.38): C, 67.24; H, 6.24; N, 12.38%. Found C, 66.61; H, 5.89;
N, 12.03%.
–
–CH N–), 11.20 and 11.39 (s and s, 1H, hydrazone NH).
–
Anal. calcd. for C23H28N4O2 (392.49): C, 70.38; H, 7.19; N,
14.27%. Found C, 69.87; H, 7.11; N, 14.16%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-hydroxy-3-
methoxybenzylidene)butanohydrazide (20)
Yield 46%. m.p. 204–2068C (EtOH/acetone (90:10 v/v)). [a]D
196.947 (c: 1.0, DMF). HPLC tR (min.): 2.56. IR, y (cmꢀ1): 3413
(2S)-2-(Benzoylamino)-3-methyl-N’-(5-
nitrofurfurylidene)butanohydrazide (24)
–
(OH str), 3250 and 3210 (NH str), 1672 (hydrazone C O str), 1629
1
–
(amide C O str). H NMR: d (ppm) 0.94–1,01 (m, 6H, CHCH(CH ) ),
–
–
3 2
Yield 94%. m.p. 1888C (EtOH/DMF (90:10 v/v)). [a]D 188.737 (c: 1.0,
DMF). HPLC tR (min.): 3.86. IR, y (cmꢀ1): 3254 and 3205 (NH str),
2.09–2.31 (m, 1H, CHCH(CH3)2), 3.82 (s, 3H, –OCH3), 4.27 and
5.34–5.37 (t, J ¼ 8.6 Hz, J ¼ 8.6 Hz and m, 1H, CHCH(CH3)2), 6.82
and 6.84 (dd, J ¼ 2.5 Hz, J ¼ 2.5 Hz, 1H, ArH), 7.05–7.09 (m, 1H,
ArH), 7.26 and 7.30 (dd, J ¼ 1.8 Hz, J ¼ 1.8 Hz, 1H, ArH), 7.44–
7.56 (m, 3H, ArH), 7.87–7.92 (m, 2H, ArH), 8.13 (s, 1H, amide NH),
–
–
–
1672 (hydrazone C O str), 1635 (amide C O str), 1540 (NO str),
–
2
1365 (NO2 str). 1H NMR: d (ppm) 0.94–1.02 (m, 6H, CHCH(CH3)),
2.18–2.27 (m, 1H, CHCH(CH3)2), 4.29 and 5.25–5.29 (t, J ¼ 8.4 Hz,
J ¼ 8.4 Hz and m, 1H, CHCH(CH3)2), 7.24 (t, J ¼ 3.9 Hz, J ¼ 3.9 Hz,
1H, ArH), 7.46–7.57 (m, 3H, ArH), 7.79 (t, J ¼ 4.1 Hz, J ¼ 4.1 Hz, 1H,
ArH), 7.96 and 8.23 (s and s, 1H, amide NH), 8.32 and 8.64
–
8.22 and 8.52 (d, J ¼ 8.7 Hz and d, J ¼ 8.3 Hz 1H, –CH N–), 9.52
–
(s, 1H, Ar–OH), 11.32 and 11.48 (s and s, 1H, hydrazone NH). Anal.
calcd. for C20H23N3O4 (369.41): C, 65.03; H, 6.28; N, 11.37%. Found
C, 64.48; H, 6.09; N, 11.20%.
–
(d, J ¼ 8.3 Hz and d, J ¼ 8.1 Hz, 1H, –CH N–), 11.88 and 12.11
–
(s and s, 1H, hydrazone NH). Anal. calcd. for C17H18N4O5 (358.35):
C, 56.98; H, 5.06; N, 15.63%. Found C, 56.74; H, 4.88; N, 15.43%.
(2S)-2-(Benzoylamino)-3-methyl-N’-(4-nitrobenzylidene)-
butanohydrazide (21)
In vitro antiviral assays
Inhibition of HIV-induced cytopathicity in MT-4 cells
Yield 45%. m.p. 207–2088C (EtOH). [a]D 243.311 (c: 1.0, DMF).
HPLC tR (min.): 5.62. IR, y (cmꢀ1): 3254 and 3205 (NH str),
Evaluation of the antiviral activity of the compounds against
HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells was performed
using the MTT assay as previously described [41, 48]. Stock
solutions (10ꢂ final concentration) of test compounds were
added in 25 mL volumes to two series of triplicate wells so as
to allow simultaneous evaluation of their effects on mock- and
HIV-infected cells at the beginning of each experiment. Serial
fivefold dilutions of test compounds were made directly in flat-
bottomed 96-well microtiter trays using a Biomek 3000 robot
(Beckman Instruments, Fullerton, CA). Untreated control HIV-
and mock-infected cell samples were included for each sample.
–
–
–
1670 (hydrazone C O str), 1635 (amide C O str), 1520 (NO
–
2
str), 1350 (NO2 str). 1H NMR: d (ppm) 0.96–1.23 (m, 6H,
CHCH(CH3)2), 2.08–2.29 (m, 1H, CHCH(CH3)2), 4.32 and 5.36–
5.40 (t, J ¼ 8.5 Hz, J ¼ 8.5 Hz and m, 1H, CHCH(CH3)2), 7.45–
7.57 (m, 3H, ArH), 7.88–7.98 (m, 4H, ArH), 8.12 and 8.61 (s and
d, J ¼ 8.1 Hz, 1H, ArH), 8.28 and 8.31 (dd, J ¼ 5.7 Hz, J ¼ 5.7 Hz,
–
–
1H, amide NH), 8.37 (d, J ¼ 8.7 Hz, 1H, –CH N–), 11.78 and
11.98 (s and s, 1H, hydrazone NH). Anal. calcd. for C19H20N4O4
(368.38): C, 61.95; H, 5.47; N, 15.21%. Found C, 62.02; H, 4.89;
N, 15.16%.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
L-Valine Hydrazones as Stereoselective Inhibitors of HIV-1 RT
151
HIV-1(IIIB) [49] or HIV-2 (ROD) [50] stock (50 mL) at 100–300
CCID50 (cell culture infectious dose) or culture medium was
added to either the infected or mock-infected wells of the micro-
titer tray. Mock-infected cells were used to evaluate the effect of
test compound on uninfected cells in order to assess the cyto-
toxicity of the test compound. Exponentially growing MT-4 cells
[51] were centrifuged for 5 min at 220 g and the supernatant was
discarded. The MT-4 cells were resuspended at 6 ꢂ 105 cells/mL,
and 50-mL volumes were transferred to the microtiter tray wells.
Five days after infection, the viability of mock- and HIV-infected
cells was examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
genase of metabolically active cells to a blue-purple formazan
that can be measured spectrophotometrically. The absorbances
were read in an eight-channel computer-controlled photometer
(Safire2, Tecan, Mechelen, Belgium), at two wavelenghths (540
and 690 nm). All data were calculated using the median OD
(optical density) value of three wells. The 50% cytotoxic concen-
tration (CC50) was defined as the concentration of the test com-
pound that reduced the absorbance (OD540) of the mock-infected
control sample by 50%. The concentration achieving 50% pro-
tection from the cytopathic effect of the virus in infected cells
was defined as the 50% effective concentration (EC50).
Signals are read using an excitation wavelength of 490 nm
and emission detection at 523 nm using a spectrofluorometer
(Safire2, Tecan). To test the activity of compounds against RT,
1 mL of compound in DMSO is added to each well before the
addition of RT enzyme solution. Control wells without com-
pound contain the same amount of DMSO. Results are expressed
as relative fluorescence, i.e., the fluorescence signal of the reac-
tion mix with compound divided by the signal of the same
reaction mix without compound.
NS5B inhibition assay
The biological activity of the compounds against NS5B polymer-
ase were evaluated in a reaction buffer containing 20 mM Tris–
HCl (pH 7.0), 100 mM NaCl, 100 mM sodium glutamate, 0.1 mM
DTT, 0.01% BSA, 0.01% Tween-20, 5% glycerol, 20 U/mL of RNase
Out, 0.25 mM of polyrA/U12, 25 mM UTP, 2 mCi [alpha-32P]UTP,
300 ng of NS5BCD21and 1.0 mM MnCl2 with or without inhibi-
tors (100 mM) in a total volume of 25 mL for 1 h at 308C as
previously described [56, 57].
Reactions were terminated by the addition of ice-cold 5% v/v
trichloroacetic acid (TCA) containing 0.5 mM pyrophosphate.
Reaction products were precipitated on GF-B filters and quanti-
fied on a liquid scintillation counter. NS5B activity in the pres-
ence of DMSO control was set at 100% and that in the presence of
the compounds was determined relative to this control.
Time-of-addition experiments
Molecular modeling
Ligand structure preparation
Time-of-addition experiments were adapted from previously
reported methods [52, 53]. Briefly, MT-4 cells were infected with
HIV-1(IIIB) at an m.o.i. of 0.5. Following a 1 h adsorption period
cells were distributed in a 96-well tray at 45 000 cells/well
and incubated at 378C. Test compounds were added at different
times (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 24, and 25 h) after infection. HIV-1
production was determined at 31 h post infection via a p24
enzyme-linked immunosorbentassay (Perkin Elmer, Brussels,
Belgium). Dextran sulfate was used at 12.5 mM, AZT at 1.9 mM,
nevirapine at 7.5 mM, compound 11S at 95 mM, and compound
12S at 125 mM.
Representative chiral hydrazone derivatives were built using the
fragment dictionary of Maestro 9.0 and energy minimized by
Macromodel program v9.7 (Schro¨dinger, Inc., New York, NY,
2009) using the OPLSAA force field with the steepest descent
followed by truncated Newton conjugate gradient protocol.
The low-energy 3D structures of the hydrazone derivatives were
generated with the following parameters present in LigPrep v2.3:
different protonation states at physiological pH, all possible
tautomers, ring conformations and stereoisomers. The output
obtained from the LigPrep run was further used for generating
100 ligand conformations using default parameters of the con-
formational search panel of the Macromodel submenu which
uses a combination of mixed torsional/low-mode sampling
function. Minimized structures were filtered with a maximum
relative energy difference of 5 kcal/mol to exclude redundant
conformers. The output conformational search file (CSearch file)
containing at most 100 unique conformers of the hydrazone
derivatives was used as input for docking simulations.
Reverse transcriptase assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and
purified as described previously [54]. The RT assay is performed
with the EnzCheck Reverse Transcriptase Assay kit (Molecular
Probes, Invitrogen), as described by the manufacturer. The assay
is based on the dsDNA quantitation reagent PicoGreen. This
reagent shows a pronounced increase in fluorescence signal
upon binding to dsDNA or RNA–DNA heteroduplexes. Single-
stranded nucleic acids generate only minor fluorescence signal
enhancement when a sufficiently high dye/base pair ratio is
applied [55]. This condition is met in the assay.
A poly(rA) template of approximately 350 bases long, and an
oligo(dT) 16 primer, are annealed in a molar ratio of 1:1.2
(60 min at room temperature). Fifty-two nanograms of the
RNA/DNA is brought into each well of a 96-well plate in a volume
of 20 mL polymerization buffer (60 mM Tris–HCl, 60 mM KCl,
8 mM MgCl2, 13 mM DTT, 100 mM dTTP, pH 8.1). Five microliters
of RT enzyme solution, diluted to a suitable concentration in
enzyme dilution buffer (50 mM Tris–HCl, 20% glycerol, 2 mM
DTT, pH 7.6), is added. The reactions are incubated at 258C for
40 min and then stopped by the addition of EDTA (15 mM fc).
Heteroduplexes are then detected by addition of PicoGreen.
Protein structure preparation
The X-ray co-crystal structure of HIV-1 RT-dihydroxy benzoyl
naphthyl hydrazone (DHBNH) (PDB ID: 2I5J) [42] obtained from
the RCSB Protein Data Bank was used for docking into the novel
non-nucleoside inhibitor binding site located between the
NNRTIBP and the active site of HIV-1 RT. The initial structure
thus obtained was refined by means of default parameters men-
tioned in the protein preparation tool implemented in Maestro
v9.0 and Impact program v5.5 (Schro¨dinger, Inc., New York, NY,
2009), in which the protonation states of residues were adjusted
to the dominant ionic forms at pH 7.4. Refined HIV-1 RT model
was further used to generate the grid by selecting the bound
inhibitor (DHBNH).
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www.archpharm.com

152
E. Tatar et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 140–153
[17] D. Sriram, P. Yogeeswari, R. V. Devakaram, Bioorg. Med. Chem.
2006, 14, 3113–3118.
Docking protocol
The conformational library of the chiral hydrazone derivatives
was docked at the hydrazone binding novel site of HIV-1 RT
using the ‘‘Extra Precision’’ (XP) Glide docking program v5.0
(Schro¨dinger, Inc., New York, NY, 2009) and the default
parameters. The top scoring pose of each hydrazone derivative
within the novel site was then subjected to energy minimization
using Macromodel program v9.7 and used for graphical analysis.
All computations were carried out on a Dell Precision 470n dual
processor with the Linux OS (Red Hat Enterprise WS 4.0).
[18] D. Sriram, P. Yogeeswari, K. Madhu, Bioorg. Med. Chem. Lett.
2005, 15, 4502–4505.
¨
[19] S¸. G. Ku¨c¸u¨kgu¨zel, A. Mazi, F. Sahin, S. Oztu¨rk, J. Stables, Eur.
J. Med. Chem. 2003, 38, 1005–1013.
[20] S. Rollas, N. Gu¨lerman, H. Erdeniz, Farmaco 2002, 57, 171–
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[21] J. M. dos Santos Filho, A. C. Leite, B. G. de Oliveira, D. R.
Moreira, M. S. Lima, M. B. Soares, L. F. Leite, Bioorg. Med. Chem.
2009, 17, 6682–6691.
This work is supported by the Research Fund of Marmara University,
project number: SAG-DKR-200407-0079 and the KULeuven (GOA/10/014).
HCV NS5B studies were supported by funds from the UMDNJ foundation to
N.K.-B. The authors thank K. Uyttersprot, K. Erven and C. Heens for fine
technical assistance.
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H. Cerecetto, I. Aldana, S. Perez-Silanes, A. Monge, E. J.
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2009, 17, 6158–6165.
The authors have declared no conflict of interest.
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