Study of E/Z isomerization in a series of novel non-ligand binding pocket androgen receptor antagonists

Article abstract of DOI:10.1021/ci300299n

We report the conformational analysis of a series of 3-hydroxy-N′- ((naphthalen-2-yl)methylene)naphthalene-2-carbohydrazides. This class of compounds has recently been reported as androgen receptor (AR)-coactivator disruptors for potential application in prostate cancer therapy. Definition of the E/Z isomerism around the imine linker group (hydrazide) is significant from a mechanistic point of view. A detailed study using theoretical calculations coupled with experimental techniques has allowed us determine an initial preference for the E isomer. The biological activity of newly synthesized compounds at the androgen receptor, along with a series of structural analogs, was determined and provides the basis for preliminary qualitative structure-activity relationship analysis.

Full text of DOI:10.1021/ci300299n

Article
pubs.acs.org/jcim
Study of E/Z Isomerization in a Series of Novel Non-ligand Binding
Pocket Androgen Receptor Antagonists
Fernando Blanco,*,† Billy Egan,‡ Laura Caboni,† Jose Elguero,§ John O’Brien,⊥ Thomas McCabe,⊥
́
Darren Fayne,† Mary J. Meegan,‡ and David G. Lloyd*,†
†Molecular Design Group, School of Biochemistry and Immunology, and ‡School of Pharmacy and Pharmaceutical Sciences, Trinity
⊥
Biomedical Science Institute, and School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
§Instituto de Química Med
́
ica, CSIC, Juan de la Cierva, 3, E-28006 Madrid, Spain
S
* Supporting Information
ABSTRACT: We report the conformational analysis of a series of 3-
hydroxy-N′-((naphthalen-2-yl)methylene)naphthalene-2-carbohydra-
zides. This class of compounds has recently been reported as
androgen receptor (AR)-coactivator disruptors for potential applica-
tion in prostate cancer therapy. Definition of the E/Z isomerism
around the imine linker group (hydrazide) is significant from a
mechanistic point of view. A detailed study using theoretical
calculations coupled with experimental techniques has allowed us
determine an initial preference for the E isomer. The biological
activity of newly synthesized compounds at the androgen receptor,
along with a series of structural analogs, was determined and provides
the basis for preliminary qualitative structure−activity relationship analysis.
The first systematic studies of the E/Z isomerism of
1. INTRODUCTION
hydrazones were carried out by Karabatsos.4−14 Other studies
are summarized in Scheme 1.
In a recent study, we demonstrated the activity of members of
the 3-hydroxy-N′-((naphthalen-2-yl)methylene)naphthalene-2-
carbohydrazide family as novel androgen receptor (AR)
antagonists and their potential application in prostate cancer
therapy.1 AR is a member of the highly homologous family of
steroidal nuclear receptors (NRs) that regulate transcription of
target genes in a ligand dependent fashion.2 NRs are composed
of a modular structure including an N-terminal domain (NTD),
a DNA binding domain (DBD), and a ligand binding domain
(LBD) which encloses a central ligand binding pocket (LBP)
where endogenous hormones bind.3 These include testosterone
(Tes) and dihydrotestosterone (DHT) in the specific case of
AR. Antiandrogens, in a pharmaceutical setting, typically act by
displacing the natural hormones from the LBP; however,
acquired drug resistance after prolonged use is a major concern
which impairs their application in advanced stages of prostate
cancer. The ligands we recently reported1 function via an
alternative non-LBP mediated mechanism which targets AR
association with coactivators. Coactivators are essential for AR
activity as transcription factors, and the direct disruption of this
interaction can potentially overcome therapeutic liabilities of
solely targeting the LBP. All of these compounds share the
presence of an N-acylhydrazone linker, which can exist in two
different E/Z isomeric forms. Characterization of the E/Z
isomerism around the linker is substantial for the elucidation of
the most probable bioactive conformation in the interaction
with the target.
Scheme 1. Previously Studied Hydrazone Family Members
The simpler hydrazones (I, N−NH2) were studied by
Minabe,15 Lemal,16 and Alkorta.17 N,N-Dimethyl derivatives
(II) were reported by Elguero18 and Lu.19 N-Aryl derivatives,
usually N-phenyl III, are the most common hydra-
zones.4−14,20−22 Less frequent, but closely related to the
compounds of the present study, are the N-acylhydrazones
IV.23−26
The most interesting result, in the context of the present
work, is a report on the crystal structure of V and the two
hydrogen bonds (HB), N−H···O and O−H···N, present in its
structure.27
Received: June 28, 2012
Published: August 2, 2012
© 2012 American Chemical Society
2387
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
Figure 1. Compounds studied in this work.
Figure 2. Optimized geometries and relative energies of the N-acylhydrazone tautomers/rotamers at the B3LYP/6-311++G** level.
The impact of substitution on the conformational isomerism
around the hydrazone and subsequent impact on conformer
ratios is of particular interest. In the absence of a protein
cocrystallized structure for these compounds, we carried out a
conformational study, both theoretical (DFT calculations) and
experimental (synthesis, NMR, and X-ray characterization), of
the most representative active ligands of the AR non-LBP
series, the 3-hydroxy-N′-(naphthalen-2-ylmethylene)-2-naph-
thohydrazide (1) and some related systems (2a−c). Biological
and qualitative structure−activity relationship (SAR) analysis of
both the studied compounds and some interesting analogs has
also been included in the discussion.
tional enantiomers due to the top/bottom arrangement of the
planes defined by the aromatic rings (Figure 3).
2. RESULTS AND DISCUSSION
2.1. Conformational Analysis. A theoretical study of the
E/Z isomerism in a series of 3-hydroxy-N′-((naphthalen-2-
yl)methylene)naphthalene-2-carbohydrazides (Figure 1) has
been performed using DFT calculations.
Figure 3. E/Z isomerism in 3-hydroxy-N′-(naphthalen-2-ylmethy-
lene)-2-naphthohydrazide derivatives.
As an initial approach, we evaluated the tautomeric/
conformational geometries of the simple linker (N-acylhy-
drazone) to establish the most favorable amide/imidic acid
tautomeric form (Figure 2). Relative energies at B3LYP/6-311+
+G** level show that the most stable conformation of
tautomer T1 (IIT1 amide, NHCHO) is clearly favored over
the most stable tautomer T2 (IVT2 imidic acid, NCHOH)
with an energy difference above 40.0 kJ mol−1; so that, to carry
out the subsequent conformational study of our compounds of
interest, we only focused on the possible geometries derived
from T1 series.
Figures 4 and 5 show the geometries of the structures studied
for compounds 1 and 2a−c. In general, we will call the
orientation shown in Figure 3 by the A1A2 rings horizontal (h)
and that shown by the B1B2 rings vertical (v). With this in
mind we consider it useful to make some conformational
clarifications before the energy discussion:
• Naphthalene ring A presents four possible orientations
for each E/Z isomer as a consequence of the rotation of
the r1 and r2 axes: Ah1 [Z1, Z5, E1, E5], Ah2 [Z3, Z7, E3,
E7], Av1 [Z2, Z6, E2, E6] and Av2 [Z4, Z8, E4, E8].
The combination of the E/Z imine isomerism (N-
acylhydrazone) and three rotational axes, r1 (C1A1CCO),
r2 (CCONNH), and r3 (CNCC1B1), afforded a total of 32
potential geometries corresponding to 16 pairs of conforma-
• Naphthalene ring B presents two possible orientations
for each E/Z isomer as a consequence of the rotation of
2388
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
Figure 4. (Z)-Conformers of compounds 1 [R1 = H, R2 = OH] and 2a−c [R1 = H, CH3 and Ph, respectively, R2 = H].
Figure 5. (E)-Conformers of compounds 1 [R1 = H, R2 = OH] and 2a−c [R1 = H, CH3 and Ph, respectively, R2 = H].
the r3 axis: Bh [Z1, Z2, Z3, Z4, E1, E2, E3, E4] and Bv [Z5,
Z6, Z7, Z8, E5, E6, E7, E8].
E5, with a difference of 37.4 kJ mol−1 compared to the most
favorable Z conformation (Z3). In series 2a [R1 = H, R2 = H],
this corresponds to the conformer E7 with a lower difference of
19.3 kJ mol−1 (Z3). Therefore, in both cases the E/Z energy gap
suggests an almost quantitative shift of the equilibrium to the E
isomer which should appear a priori as the most abundant
product in experimental conditions. The possibility of
establishing a highly stabilized intramolecular hydrogen bond
interaction (IMHB) between the OH substituent of the
naphthalene ring B1 and the imine nitrogen lone pair
(OHB1···NNC) explains the increase in the energy E/Z ratio
of compound 1 (E5) with regard to 2a (E7) (Figure 6).
2.2. DFT Energy Analysis. Sixteen minima were optimized
for compounds 1 and 2a−c at the B3LYP/6-311++G** level.
As expected, most of the conformers presented a twisted
geometry to avoid steric repulsions between the naphthalene
rings and/or the linker. Conformers E2 and E6 were an
exception, showing an almost planar arrangement of the rings
due to the more favorable anti orientation of the aromatic
groups and a stabilizing orientation of the hydroxyl groups.
The relative free energies of the minima are gathered in
Table 2. The gas phase values show that in series 1 [R1 = H, R2
= OH], the absolute minimum corresponds to the conformer
2389
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
Table 2. Relative Free Energies (kJ mol−1) of All Optimized Geometries at the B3LYP/6-311++G** Level in Gas Phase and
PCM (Water) Continuum Model
conformer
Grel (1) gas
Grel (1) water
Grel (2a) gas
Grel (2a) water
Grel (2b) gas
Grel (2b) water
Grel (2c) gas
Grel (2c) water
Z1
Z2
Z3
Z4
Z5
Z6
Z7
Z8
E1
E2
E3
E4
E5
E6
E7
E8
42.2
61.5
37.4
54.1
44.2
65.9
40.0
54.0
22.5
38.6
20.4
36.2
0.0
37.2
48.8
44.5
55.1
37.6
51.1
46.2
52.6
18.8
23.3
27.0
37.4
0.0
24.1
40.0
19.3
34.5
23.8
61.4
19.8
34.9
5.8
24.8
33.8
30.4
38.8
24.1
52.9
31.8
38.1
3.8
6.4
25.8
4.6
3.5
14.4
12.3
19.7
4.7
3.4
22.4
4.0
3.0
14.0
11.9
25.7
20.1
7.0
20.6
a
a
3.9
3.0
a
a
26.6
4.5
15.0
12.3
20.4
0.3
22.4
14.0
a
a
4.0
11.9
a
a
20.6
6.6
20.6
25.7
4.4
23.7
4.9
6.0
15.4
12.9
22.2
0.0
25.0
3.0
10.6
11.0
21.5
0.0
22.8
2.2
10.3
7.4
19.7
2.4
17.0
2.8
15.6
0.0
21.2
0.0
14.9
6.2
12.1
14.0
29.4
22.0
0.0
15.2
7.6
18.8
0.0
5.7
19.9
1.2
9.3
5.5
9.6
29.6
16.8
18.7
15.2
12.7
17.9
25.3
a
The geometries Z5, Z6, Z7, and Z8 evolve spontaneously to the same minima Z1, Z2, Z3, and Z4, respectively.
Figure 6. Most stable E and Z conformers for compounds 1 and 2a, respectively.
Focusing on series 2, the increase in the size of the R1
substituent produces a reduction in the E/Z energy gap from
19.3 kJ mol−1 in 2a [R1 = H, R2 = H] to 4.6 kJ mol−1 in 2b [R1
= CH3, R2 = H]. In series 2c [R1 = Ph, R2 = H], a near parity in
the energy differenceE5 is the absolute minimum, only 3.4 kJ
mol−1 below the best Z conformation (Z1)is observed. The
small energy difference for 2c suggests that the distribution of E
and Z isomers will be fairly similar, a result that could be
reflected experimentally as an isomeric mixture. It can be
inferred that in series 2 the steric hindrance determines the E/Z
Figure 7. IMHB interactions of naphthalene ring A.
distribution ratio.
A major consideration for us is to simulate the compounds in
condition of biological relevance. Accordingly, all the con-
formers have also been calculated in water solvent phase using
the pcm continuum model. As would be expected, slight
differences are presented in the relative energies with regard to
the gas phase results. However, the general trend confirms the
prevalence of the E isomer for compounds 1 and 2a, even in
aqueous conditions. Moreover, the potential E/Z isomeric
mixture due to the narrow energy range between isomers for
compound 2c is similarly observed in water phase calculations.
As described for naphthalene B, in compound 1, the relative
orientation of the hydroxyl group of ring A plays a major role in
the stabilization of the conformers due to the possibility of
establishing up to three different types of IMHB interactions
(Figure 7): (1) OHA1···OCO [Z1, Z3, Z5, Z7, E1, E3, E5, E7]; (2)
NH···OHA1 [Z2, Z6, E2, E6], and (3) OHA1···NNC [Z4, Z8, E4,
E8]. Clustering those conformers that share the same
arrangement of naphthalene ring B, we observe systematically
that the relative free energies in gas phase follow the trend Z/E3
< Z/E1 < Z/E4 < Z/E2 and Z/E7 < Z/E5 < Z/E8 < Z/E6 for the
Bh and Bv conformers, respectively. An exception are the E5−E8
conformers of compound 1 where the presence of the hydroxyl
group in B1 distorts this general trend. This pattern is not
observed in water phase due to the decrease of the hydrogen
bonding stabilizing contribution to the energy due to solvation
effects. The main conclusion is that the degree of stabilization
induced by the HB interaction type OHA1···OCO is
significantly higher than that induced by OHA1···NNC or
NH···OHA1, which is in a narrow energy range playing a
significant role in the observed rearrangement determined by
the NMR structural study.
Although E1 and E5 conformers suffer an energy penalty
resulting from repulsion between the H1A1 and NH hydrogen
2390
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
Figure 8. OH···OCO (E1 and E5) vs NH···OOH (E2 and E6) stabilization.
Scheme 2. General Synthetic Pathway of 3-Hydroxy-N′-(naphthalen-2-ylmethylene)-2-aromatic Hydrazides Derivatives (R = H,
CH3, Ph)
atoms which breaks the planarity of the system, they show in all
cases a substantial increase in stability when compared to their
respective planar rotamers E2 and E6, emphasizing the
prevalence of the OHA1···OCO stabilization (Figure 8) both
in gas and water phase.
2.4. NMR Characterization. A combination of different
NMR spectroscopic methodologies has been employed for the
unequivocal characterization of the compounds studied in this
work. Figure 9 shows the 1H NMR spectroscopic signals within
the range of 7.0−12.4 ppm corresponding to the series 2a−c.
This region includes all the aromatic hydrogen atoms as well as
NH/OH hydrogen atoms present in these compounds. Two
important conclusions are evident, corroborating our theoreti-
cal predictions:
2.3. Synthesis of 3-Hydroxy-N′-((naphthalen-2-yl)-
methylene)naphthalene-2-carbohydrazide Derivatives.
A series of 3-hydroxy-N′-((naphthalen-2-yl)methylene)-
naphthalene-2-carbohydrazides (2a−c) analogs and the closely
related N′-benzylidene-3-hydroxy-2-naphthohydrazide (3) were
synthesized following a standard synthetic route (Scheme 2).
Starting from the commercially available 3-hydroxy-2-naphthoic
acid (I), an esterification was performed in the presence of
hydrochloric acid to afford the corresponding methyl 3-
hydroxynaphthalene-2-carboxylate intermediate (II). Treat-
ment of II with hydrazine hydrate afforded the hydrazide III.
Condensation of III with the appropriate aldehyde or ketone
(IV) afforded the products (V) in good yields. Systems 2a, 2b,
and 3 were isolated as a single isomer, and subsequently
1. The spectra of compounds 2a [R1 = H] and 2b [R1 =
CH3] correspond to an almost quantitative single isomer
(a minimal trace of the second isomer is observed);
conversely, compound 2c [R1 = Ph] presents duplicated
signals for all protonsindicating, as predicted, a
mixture of isomers.
2. OH hydrogen atoms are strongly displaced downfield
with chemical shift values above 10.0 ppm clearly
indicating their participation in an intramolecular
hydrogen bond with the lone pairs of the carbonyl
oxygen atom.
1
characterized by H NMR as the E isomer. Compound 2c,
Figure 10 illustrates the most significant intramolecular
interactions which were probed by NMR and employed for the
correct assignment of the experimentally obtained isomers for
however, resulted as an inseparable mixture of E/Z isomers (see
structure V) consistent with our theoretical predictions.
2391
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
1
Figure 9. H NMR spectra of compounds 2a−c.
Figure 10. Relevant spectroscopic data used for NMR structure
elucidation (2a).
compound 2a. Irradiation of the hydrogen corresponding to the
R2 group H (8.64 ppm) in NOE experiments resulted in the
perturbation of the NH signal at 12.09, demonstrating the E
character of the isolated isomer (a hypothetical Z isomer could
not show this NOE interaction due to the greater distance
between the involved groups). A similar result was obtained in
the NOE analysis of compound 2b when the R2 signal of CH3
(2.52 ppm) was irradiated.
Figure 11. Crystal structure of (E)-3-hydroxy-N′-((naphthalen-2-
yl)methylene)naphthalene-2-carbohydrazide (2a) obtained in this
work (Ortep representation).
With reference to the specific orientation of the naphthalene
ring A, both in the newly obtained structure (2a) and in that
previously reported in the literature, with the exception of the
structures RUJJEM and CICNUY, the preferential conforma-
tion observed corresponds to the E2/E6 conformers, observed
in almost all cases (See Figure 5 and Table 2 for comparison),
and not to the expected absolute minimum E7. This
discrepancy may be explained by the fact that crystal packing
forces tend to favor the most planar geometry, and on this
basis, E2/E6 would be the preferential conformation as
discussed above. However, we can not assume that this
constitutes a complete explanation because the observed
arrangement of the compounds analyzed does not follow a
standard stacking pattern (See packing geometry of referenced
structures). Another interesting observation is that the
substitution of the hydroxyl group (HUGPOP) by a methoxy
one (RUJKOX) induces the rotation of the phenyl ring in order
to favor the most stable geometry based on the hydrogen
Moreover, additional NOE experiments confirmed that R1 ≫
H1′ singlet [B1 ring] and NH ≫ H1 singlet [A1 ring]
interactions, in addition to the observed OHA1···OCO IMHB,
all of which confirm the most probable/favorable conformation
of the studied compounds under the experimental conditions.
2.5. X-ray Analysis. Confirmation of the NMR exper-
imental data and computational predictions was achieved with
the X-ray crystal analysis. Slow recrystallization of compound
2a from methanol provided the crystal structure of (E)-3-
hydroxy-N′-((naphthalen-2-yl)methylene)-naphthalene-2-car-
bohydrazide as an isolated E isomer (Figure 11).
A review of available data identified eight other crystal
structures of very similar compounds deposited within the
Cambridge Crystallographic Database (CSD).28 As shown
(Figure 12), all of them present an E geometry, consistent with
our results.
2392
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
Figure 12. Crystallographic structures of related compounds found in the CSD (compound labels correspond to the CSD codes).
Figure 13. Series of diarylhydrazides assayed on AR TR-FRET.
bonding network, so it is possible to achieve some conforma-
tional control of the rotational state, depending on the
properties of the substituent, which is very interesting from a
pharmacological point of view.
On the basis of our results, and the data found in the
literature, we can conclude the following for this class of
compounds:
structures are found as E isomers, which is consistent
with our theoretical studies.
2.6. Biological Study. We present the biological evaluation
of ten different diarylhydrazide compounds (Figure 13),
including those conformationally studied in this work (1−2),
for their ability to displace AR:coactivator interaction using TR-
FRET techniques.
1. The E/Z isomeric equilibrium is strongly governed by
the ratio of the size (steric hindrance) of the substituents
on the imine group.
2. Free rotation around the r1 and r3 axes in solution favors
a higher representation of the thermodynamically most
stable conformer as determined by the balance between
the stabilizing (IMHB)/repulsive effect of the substi-
tuents.
The TR-FRET technique measures the recruitment of a
fluorescent (Fl) labeled coactivator (of the AR specific FxxLF
type: acceptor) to a Terbium (Tb) labeled GST tagged AR-
LBD (donor). The technique is dependent on the distance
between the donor/acceptor pair.29 In the case of an agonist
bound LBD, the Fl labeled coactivator is associated with LBD,
so when Tb is excited at 340 nm, energy is transferred to Fl
resulting in an increase in TR-FRET signal. In the presence of
an antagonist, the Fl coactivator is not bound to the LBD;
therefore, energy cannot be successfully transferred from Tb to
Fl resulting in a decrease of the TR-FRET signal. Data is
presented as a percentage maximum of activity as described
3. The available crystallographic data suggests that the
preferential conformation in the solid state is strongly
determined by packing forces, but all the reported
2393
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
Figure 14. (a) Dose response curve for (1) and its closest analog (2a). Compounds were tested using a maximum concentration of 100 μM in the
presence of a concentration of DHT (endogenous agonist) equal to its EC80 concentration (see the Experimental Section). Data points are
presented as standard error of mean (SEM) of at least two independent experiments in triplicate. Data was fitted using the dose−response curve
(variable slope). (b) Diarylhydrazides activity toward AR-LBD. Values are presented as IC50 SEM of at least two independent experiments where
each well was included as triplicate.
previously,30 and the binding data of the tested compounds is
presented in Figure 14.
Qualitative SAR Analysis. Derived from our biological
evaluation of targeted modifications of the diarylhydrazide
scaffold, we can determine some initial qualitative SAR
conclusions:
surface of AR LBD. With this unambiguous determination of
molecular conformational preference, and ahead of resolution
of any X-ray cocrystal structure of the protein:ligand complex
for these compounds, this study now affords a significant
starting point for structure-based expansion of this molecular
series.
1. The OH group on naphthalene ring B seems important
for activity when comparing 1/2a, 7/3, and 5/4 pairs of
analogs. If not present, it abolishes (2a and 3) or
diminishes (4) the activity.
2. Naphthalene ring A2 seems to be required when
comparing 7/8 pair since the substitution of naphthalene
by a benzene ring abolishes the activity (8).
3. Compounds 1, 5, and 6 are equipotent, demonstrating
that the presence of naphthalene ring B2 (1) or a
hydrophobic group like chlorine (5) or bromine (6)
increase the activity on AR.
4. EXPERIMENTAL SECTION
4.1. General Experimental Data.31 All commercially
available reagents were purchased and used without further
purification unless otherwise indicated. Dichloromethane was
dried by distillation from calcium hydride prior to use. IR
spectra were recorded as thin films on NaCl plates or as KBr
discs on a 100 FT-IR spectrometer. H and 13C NMR spectra
1
1
were obtained at 20 °C, 400.13 MHz for H spectra, 100.61
MHz for 13C spectra, in (CD3)2SO (internal standard
tetramethylsilane). High resolution accurate mass determina-
tions for all final target compounds were obtained and recorded
by electrospray ionization mass spectrometry (ESI-MS), which
was performed in the positive ion mode on a liquid
chromatography time-of-flight mass spectrometer. Thin layer
chromatography was performed using Silica gel 60 TLC
aluminum sheets with fluorescent indicator visualizing with UV
light at 254 nm. Flash chromatography was carried out using
standard silica gel 60 (230−400 mesh).
3. CONCLUSIONS
The preferred molecular conformations for four 3-hydroxy-N′-
((naphthalen-2-yl)methylene)naphthalene-2-carbohydrazides
have been modeled using DFT theoretical calculations and
experimentally determined through solution NMR and
crystallographic diffraction analysis. Their isomerism and
conformational space, including that of some simpler models,
have been fully determined. The energy ranges between
isomers depend on a number of factors, the most important
of which are the formation of intramolecular hydrogen bonds
and steric effects. Under experimental conditions, we have
demonstrated that the E-isomer is formed under the described
synthetic conditions for this family of compounds. The Z-
isomer is formed when the steric hindrance of the second imine
substituent is sufficiently high, in this case as an equal mixture
with the corresponding E-isomer. This is in full agreement with
the theoretical predictions.
Additionally, the biological activity of ten diarylhydrazides
analogs as non-ligand binding pocket antagonists of the
androgen receptor, including those synthesized in this work,
has been determined using TR-FRET. This preliminary SAR
evaluation highlights the importance of naphthalene ring
placement for compound binding and should be considered
when advancing future series of non-LBP AR binding analogs.
It is important to note that these diarylhydrazides have been
characterized as non-LBP AR antagoniststhey do not bind in
an enclosed pocket. The proposed mechanism of action is a
functional disruption of AR interaction with coactivators on the
4.2. General Procedure for the Synthesis of 3-
Hydroxy-N′-(naphthalen-2-ylmethylene)-2-naphthohy-
drazides (Scheme 2). 4.2.1. Methyl 3-Hydroxynaphthalene-
2-carboxylate (II). 3-Hydroxy-2-naphthoic acid (3.76 g, 20
mmol) was added to a solution of MeOH (20 mL) and HCl
37% (20 mL). The reaction mixture was heated at 90 °C and
the esterification was monitored on TLC until complete
consumption of the starting material was observed. Solvent was
then removed in vacuo, and the reaction mixture was diluted
with CH2Cl2 (30 mL) and washed with H2O, CH2Cl2 (2 × 30
mL), and saturated aqueous NaHCO3. The aqueous phase was
extracted and the combined organic layers were dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to give
the crude product. The crude mixture was purified by flash
column chromatography over silica gel (eluent 9:1, CH2Cl2/
MeOH) to afford II (Scheme 2) as a colorless solid (2.17 g,
65%, lit. MP: 73−75 °C).32
4.2.2. 3-Hydroxynaphthalene-2-carbohydrazide (III). To a
solution of II (2.02 g, 10 mmol) in EtOH (10 mL), hydrazine
hydrate was added (1.245 mL, 40 mmol) and the reaction
mixture was heated to 120 °C until complete precipitation of
2394
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
hydrazide occurred (15 min). Completion of the reaction was
confirmed by TLC. The reaction mixture was allowed to cool
to 20 °C, filtered and washed with hexane to give III as a beige
powder (2.01 g, 99%, lit. MP: 205−208 °C).33
brown resin identified as a mixture of E/Z isomers 2c (50 mg,
5%, MP: 295−298 °C). IR (KBr) υmax: 3482.27 (OH stretch),
3149.78 (NH stretch), 1620.74 (CO stretch) cm−1. 1H
NMR (600 MHz, DMSO-d6): δ 11.56 (s, NH), 11.51 (s, NH),
11.06 (s, OH), 10.81 (s, OH), 8.68 (s, 1H, H2), 8.66 (s, 1H,
H1), 7.71 (t, 1H, H21), 7.44 (m, 1H, H32), 7.73 (t, 1H, H20),
8.20 (d, 1H, H5), 7.97 (d, 1H, H18), 7.96 (d, 1H, H19), 7.86 (d,
1H, H22), 8.07 (d, 1H, H26), 7.43 (m, 1H, H27), 7.49 (m, 1H,
H30), 7.46 (t, 1H, H31), 7.49 (m, 1H, H29), 7.69 (m, 1H, H28),
8.01 (d, 1H, H25), 8.10 (d, 1H, H24), 8.05 (s, 1H, H23), 7.96 (d,
1H, H17), 7.59 (d, 1H, H15), 7.69 (d, 1H, H16), 7.57 (t, 1H,
H14), 7.64 (t, 1H, H13), 7.52 (s, 1H, H12), 7.69 (m, 1H, H10),
7.63 (t, 1H, H9), 7.51 (m, 1H, H11), 7.35 (d, 1H, H8), δ 7.33 (t,
1H, H7), δ 8.14 (d, 1H, H6), δ 7.21 (s, 1H, H4) δ 7.10 (s, 1H,
H3) ppm. 13C NMR (600 MHz, DMSO-d6): δ 128.53 (CH Ar,
C52), 129.80 (CH Ar, C51), 120.20 (CH Ar, C50), 110.50 (CH
Ar, C49), 110.54 (CH Ar, C48), 120.19 (CH Ar, C47), 123.86
(CH Ar, C46), 123.93 (CH Ar, C45), 123.95 (CH Ar, C44),
125.32 (CH Ar, C43), 125.63 (CH Ar, C42), 125.67 (CH Ar,
C41), 126.75 (CH Ar, C40), 126.96 (CH Ar, C39), 127.17 (CH
Ar, C38), 127.20 (CH Ar, C37), 127.23 (CH Ar, C36), 127.37
(CH Ar, C35), 127.58 (CH Ar, C34), 127.63 (CH Ar, C33),
128.07 (CH Ar, C32), 128.09 (CH Ar, C31), 128.14 (CH Ar,
C30), 128.21 (CH Ar, C29), 128.29 (CH Ar, C28), 128.39 (CH
Ar, C27), 128.44 (CH Ar, C26), 128.52 (CH Ar, C25), 128.58
(CH Ar, C24), 129.02 (CH Ar, C23), 129.06 (CH Ar, C22),
129.51 (CH Ar, C21), 129.82 (CH Ar, C20), 130.04 (CH Ar,
C19), 130.18 (CH Ar, C18), 132.51 (CH Ar, C17), 132.61 (CH
Ar, C16), 132.97 (CH Ar, C15), 133.03 (CH Ar, C14), 133.06
(CH Ar, C13), 133.22 (CH Ar, C12), 133.50 (CH Ar, C11),
134.83 (CH Ar, C10), 135.78 (CH Ar, C9), 135.85 (CH Ar, C8),
137.38 (CH Ar, C7), 152.19 (CH Ar, C6), 152.25 (CH Ar, C5),
153.73 (CH Ar, C4), 153.81 (CH Ar, C3), 160.97 (CH Ar, C2),
161.12 (CH Ar, C1). HRMS: C28H20N2NaO2 [M + Na]+
requires (m/e) 439.14225, found 439.141.
4.2.3. (E)-3-Hydroxy-N′-((naphthalen-2-yl)methylene)-
naphthalene-2-carbohydrazide (2a). To a solution of III
(1.534 g, 7.5 mmol) in EtOH (20 mL), 2-naphthaldehyde was
added (1.1713 g, 7.5 mmol), the reaction was heated to 120 °C
and monitored by TLC until complete consumption of the
starting material was observed (6 h). The reaction mixture was
allowed to cool to 20 °C, filtered, and washed with hexane to
give a pale yellow powder identified as 2a (2.2 g, 86%, MP:
276−280 °C). IR (KBr) υmax: 3439.93 (OH stretch), 3054.38
1
(NH stretch), 1625.69 (CO stretch) cm−1. H NMR (600
MHz, DMSO-d6): δ 12.09 (s, NH), 11.33 (s, OH), 8.64 (s, 1H,
HCN), 8.50 (s, 1H, H1), 8.21 (s, 1H, H1′), 8.04 (d, 1H, H3′),
8.03 (d, 1H, H8′), 8.02 (d, 1H, H4′), 7.98 (d, 1H, H5′), 7.95 (d, J
= 8.3 Hz, 1H, H8), 7.79 (d, J = 8.2 Hz, 1H, H5), 7.59 (dd, 1H,
H6′), 7.58 (dd, 1H, H7′), 7.53 (dd, J1 = 8.2 Hz, J2 = 7.5 Hz, 1H,
H6), 7.39 (dd, J1 = 8.3 Hz, J2 = 7.5 Hz, 1H, H7), 7.36 (s, 1H,
H4) ppm. 13C NMR (600 MHz, DMSO-d6): δ 163.8 (q, CCO),
154.1 (q, C3), 148.4 (q, CCN), 135.8 (q Ar, C10), 133.8 (q Ar,
C10′), 131.9 (q Ar, C2′), 130.2 (CH Ar, C1), 129.0 (CH Ar, C1′),
128.6 (CH Ar, C8), 128.5 (CH Ar, C4′), 128.4 (CH Ar, C8′),
128.2 (CH Ar, C6), 127.8 (CH Ar, C5′), 127.2 (CH Ar, C6′),
126.8 (q Ar, C9), 126.8 (q Ar, C9′), 126.8 (CH Ar, C7′), 125.8
(CH Ar, C5), 123.8 (CH Ar, C7), 122.7 (CH Ar, C3′), 120.4 (q
Ar, C2), 110.5 (CH Ar, C4) ppm. HRMS: C22H17N2O2 [M +
H]+ requires (m/e) 341.1290, found 341.1296
4.2.4. (E)-3-Hydroxy-N′-(1-(naphthalen-2-yl)ethylidene)-
naphthalene-2-carbohydrazide (2b). To a solution of III
(1.01 g, 5 mmol) in EtOH (20 mL), 2-acetonaphthone was
added (850 mg, 5 mmol), the reaction was heated to 120 °C
and monitored by TLC until complete consumption of the
starting material was observed (3 h). The reaction mixture was
allowed to cool to 20 °C, filtered, and washed with hexane to
give a pale yellow powder identified as 2b (1.48 g, 84%, MP:
275−280 °C). IR (KBr) υmax: 3369.86 (OH stretch), 3052.55
4.2.6. (E)-N′-Benzylidene-3-hydroxynaphthalene-2-carbo-
hydrazide (3). To a solution of III (1.01 g, 5 mmol) in EtOH
(20 mL), benzaldehyde was added (0.507 mL, 5 mmol); the
reaction was heated to 120 °C and monitored by TLC until
complete consumption of the starting material was observed (2
h). The reaction mixture was allowed to cool to 20 °C, filtered,
and washed with hexane to give a pale brown powder identified
as 3 (1.24 g, 85%, MP: 227−230 °C, lit. MP: 224−225 °C).34
IR (KBr) υmax: 3242.11 (OH stretch), 3047.11 (NH stretch),
1
(NH stretch), 1627.45 (CO stretch) cm−1. H NMR (600
MHz, DMSO-d6): δ 11.69 (s, OH), 11.67 (s, NH), 8.67 (s, 1H,
H1), 8.37 (s, 1H, H1′), 8.20 (d, J = 8.52 Hz, 1H, H3′), 8.05 (d,
1H, H8′), 8.02 (d, J = 8.0 Hz, 1H, H8), 7.98 (d, 1H, H4′), 7.97
(d, 1H, H5′), 7.80 (d, J = 8.0 Hz, 1H, H5), 7.58 (dd, 1H, H6′),
7.57 (dd, 1H, H7′), 7.54 (dd, J1 = 8.0 Hz, J2 = 7.3 Hz, 1H, H6),
7.40 (s, 1H, H4), 7.39 (dd, J1 = 8.0 Hz, J2 = 7.3 Hz, 1H, H7),
2.52 (s, 3H, HMe) ppm. 13C NMR (600 MHz, DMSO-d6): δ
161.6 (q, CCO), 152.8 (q, C3), 152.0 (q, CCN), 135.8 (q Ar, C10),
135.3 (q Ar, C2′), 133.4 (q Ar, C10′), 132.8 (q Ar, C9′), 132.3
(CH Ar, C1), 128.9 (CH Ar, C8), 128.6 (CH Ar, C8′), 128.3
(CH Ar, C6), 127.8 (CH Ar, C4′), 127.5 (CH Ar, C5′), 127.2(q
Ar, C9), 126.9 (CH Ar, C6′), 126.6 (CH Ar, C1′), 126.5 (CH Ar,
C7′), 125.7 (CH Ar, C5), 123.9 (CH Ar, C7), 123.7 (CH Ar,
C3′), 120.8 (q Ar, C2), 110.7 (CH Ar, C4), 13.7 (CH3) ppm.
HRMS: C23H19N2O2 [M + H]+ requires (m/e) 355.1447, found
355.1459.
1
1625.80 (CO stretch) cm−1. H NMR (600 MHz, DMSO-
d6): δ 11.99 (s, OH), 11.30 (s, NH), 8.48 (s, 1H, HCN), 8.47 (s,
1H, H1), 7.93 (d, J = 8.2 Hz, 1H, H8), 7.79 (d, J = 7.2 Hz, 1H,
H2′), 7.78 (d, J = 7.2 Hz, 1H, H5), 7.53 (dd, J1 = 7.3 Hz, J2 = 7.3
Hz, 1H, H6), 7.50 (dd, J = 7.52 Hz, 1H, H3′), 7.49 (ddd, H4′),
7.38 (dd, J1 = 8.2 Hz, J2 = 7.3 Hz, 1H, H7), 7.34 (s, 1H, H4)
ppm. 13C NMR (600 MHz, DMSO-d6): δ 163.8 (q, CCO), 154.1
(q, C3), 148.5 (q, CCN), 135.8 (q Ar, C10), 134.1 (q Ar, C1′),
130.3 (CH Ar, C4′), 130.2 (CH Ar, C1), 128.9 (CH Ar, C3′),
128.7 (CH Ar, C8), 128.3 (CH Ar, C6), 126.8 (CH Ar, C2′),
126.8 (q Ar, C9), 125.7 (CH Ar, C5), 123.9 (CH Ar, C7), 120.4
(q Ar, C2), 110.6 (CH Ar, C4) ppm. HRMS: C18H15N2O2 [M +
H]+ requires (m/e) 291.1134, found 291.1141.
4.2.5. (E/Z)-3-Hydroxy-N′-((naphthalen-2-yl)(phenyl)-
methylene)naphthalene-2-carbohydrazide (2c). To a solu-
tion of III (404 mg, 2 mmol) in EtOH (10 mL), 2-
benzoylnaphthalene was added (464 mg, 2 mmol), the reaction
was heated to 120 °C and monitored by TLC until complete
consumption of the starting material was observed (5 h). The
reaction mixture was purified by flash column chromatography
over silica gel (eluent 9:1, CH2Cl2/MeOH) to give a pale
4.3. Biological Assays. TR-FRET AR Coactivator Assay
kit was used to screen for AR binders following the
manufacturer’s protocol.35 In brief, serial dilutions of each
compound were prepared in 100× DMSO and then diluted in
coregulator buffer A to achieve 1% DMSO concentration in the
plate. TR-FRET signal was measured with apppropiate
2395
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
equipment using a optic module excitation 335 nm, emission
520 nm-channel A and 495 nm-channel B.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +353-1-896 2304. Fax: +353-1-896 2303. E-mail:
lloyddg@tcd.ie (D.G.L.); blancof@tcd.ie (F.B.).
TR-FRET values were calculated at 10 flashes per well, using
a delay time of 100 μs and integration time 200 μs as
recommended by the Invitrogen assay guidelines. The ratio
520/495 nm was then calculated and plotted against the
concentration. A 10 μL porting of compound solution was
added to a 384 black well plate, following addition of 5 μL 4x
AR-LBD and 5 μL of D11-FxxLF/Tb Anti-GST antibody in
agonist mode and 5 μL of D11-FxxLF/Tb anti-GST antibody/
DHT (included at a concentration equal to EC80 as determined
by running the assay in agonist mode first).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work has been funded by the Health Research Board
(HRB/2007/2) and the European Commission (Marie-Curie
grant, People FP7, Project Reference: 274988). The Trinity
Biomedical Sciences Institute is supported a capital infra-
structure investment from Cycle 5 of the Irish Higher
Education Authority’s Programme for Research in Third
Level Institutions (PRTLI). Calculations were performed on
the Stokes cluster maintained by the Irish Centre for High-End
Computing (ICHEC). We would like to thank Dr Martin
Peters for his assistance.
50)+((1/hill slope)×log(80/(100−80))]
EC80 = 10[(log EC
D11-FxxLF and Tb antibody were premixed in light
protected vials just prior to use. A final concentration of
DTT 5 mM was used in the assay buffer in order to prevent
protein degradation. All plates (agonist and antagonist mode)
were incubated between 2 and 4 h at room temperature
protected from light prior to TR-FRET measurement. IC50
values were determined by testing each ligand at concentrations
ranging from 100 μM to 45 nM using 2-fold and 3-fold
dilutions to generate a 12 point dose response curve.
REFERENCES
■
(1) Caboni, L.; Kinsella, G. K.; Blanco, F.; Fayne, D.; Jagoe, W. N.;
Carr, M.; Williams, D. C.; Meegan, M. J.; Lloyd, D. True”
AntiandrogensSelective Non-Ligand-Binding Pocket Disruptors of
Androgen Receptor−Coactivator Interactions: Novel Tools for
Prostate Cancer. J. Med. Chem. 2012, 55 (4), 1635−1644.
(2) Evans, R. M. The steroid and thyroid hormone receptor
superfamily. Science 1988, 240, 889−895.
(3) Bain, D. L.; Heneghan, A. F.; Connaghan-Jones, K. D.; Miura, M.
T. Nuclear receptor structure: implications for function. Annu. Rev.
Physiol. 2007, 69, 201−220.
(4) Karabatsos, G. J.; Graham, J. D.; Vane, F. M. Syn-anti Isomer
Determination of 2,4-Dinitrophenylhydrazones and Semicarbazones
by N.M.R. J. Am. Chem. Soc. 1962, 84, 753−755.
(5) Karabatsos, G. J.; Shapiro, B. L.; Fleming, J. S.; Ratka, J. S.
Structural Studies by Nuclear Magnetic Resonance. II. Aldehyde 2,4-
Dinitrophenylhydrazones. J. Am. Chem. Soc. 1963, 85, 2784−2788.
(6) Karabatsos, G. J.; Taller, R. A. Structural Studies by Nuclear
Magnetic Resonance. V. Phenylhydrazones. J. Am. Chem. Soc. 1963,
85, 3624−3629.
(7) Karabatsos, G. J.; Vane, F. M.; Taller, R. A.; Hsi, N. Structural
studies by nuclear magnetic resonance. VIII. Ring-Substituted
Phenylhydrazones, Semicarbazones, and Thiosemicarbazones. J. Am.
Chem. Soc. 1964, 86, 3351−3357.
(8) Karabatsos, G. J.; Hsi, N. Structural studies by nuclear magnetic
resonance-XI: Conformations and configurations of oxime o-methyl
ethers. Tetrahedron 1967, 23, 1079−1095.
Data was fitted using the sigmoidal dose response (variable
slope) available from Graphpad Prism 5.36
50−X)hill slope)]
Y = bottom + (top − bottom)/(1 + 10[(logIC
In line with the assay protocol, a known agonist,
dihydrotestosterone (DHT, cat no. A8380, Sigma) and a
known antagonist, cyproterone acetate (cat no. C3412, Sigma),
were used as controls. A control with no AR-LBD present was
included to account for diffusion enhanced FRET or ligand-
independent coactivator recruitment. A negative control with
2× DMSO was present to account for any solvent vehicle
effects.
4.4. Crystallographic Data of Compound 2a. Cam-
bridge Database Deposition number: CCDC 864271,
C44H32N4O4, M = 680.74, monoclinic, a = 15.352(3), b =
6.4600(13), c = 8.4600(17) Å, α = 90°, β = 91.48(3)°, γ = 90°,
U = 838.7(3)Å3, T = 123 K, space group Pc, Z = 1, μ (Mo Kα)
= 0.09 mm−1, ρ = 1.348 g cm−3, 12 088 reflections collected,
a
2680 unique, (Rint = 0.0654), R1 = 0.1855, wR2 [I > 2σ(I)] =
a
0.5210, Gof = 2.164, R1 = ∑∥Fo| − |Fc∥/∑|Fo|, wR2
=
[∑w(Fo − Fc2)2/∑w(Fo )2]1/2
.
2
2
(9) Karabatsos, G. J.; Lande, S. S. Structural studies by nuclear
magnetic resonance-XVIII: Conformational and configurational
isomerism of N-alkyl imines. Tetrahedron 1968, 24, 3907−3922.
(10) Karabatsos, G. J.; Taller, R. A. Structural studies by nuclear
magnetic resonance-XV: Conformations and configurations of oximes.
Tetrahedron 1968, 24, 3347−3360.
5. COMPUTATIONAL DETAILS
The systems have been optimized by the Gaussian-09
package37 using the B3LYP38,39 functional with the 6-311+
+G(d,p)40 basis set. Frequency calculations have been
performed to confirm that the resulting optimized structures
are energetic minima. Effects of water solvation have been
included by means of the SCRF-PCM41 approaches imple-
mented in Gaussian-09.
(11) Karabatsos, G. J.; Taller, R. A. Structural studies by nuclear
magnetic resonance-XVII: Conformations and configurations of N-
methylhydrazones. Tetrahedron 1968, 24, 3557−3568.
(12) Karabatsos, G. J.; Osborne, C. E. Structural studies by nuclear
magnetic resonance-XVI: Conformations and configurations of
hydrazones. Tetrahedron 1968, 24, 3361−3368.
(13) Stassinopoulou, C. I.; Zioudrou, C.; Karabatsos, G. J.
Temperature dependence of the C-methyl chemical shifts in N-
substituted hydrazones of acetone. Tetrahedron Lett. 1972, 13, 3671−
3674.
(14) Stassinopoulou, C. I.; Zioudrou, C.; Karabatsos, G. J. Solvent
effects on the syn-anti isomerization rates of hydrazones of acetone.
Tetrahedron 1976, 32, 1147−1151.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supporting Information associated with this article includes 1H
NMR, 13C NMR, and NOE spectra, crystallographic data,
calculated absolute energies, and Cartesian coordinates of
optimized structures. This material is available free of charge via
Internet at http://pubs.acs.org.
2396
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397

Journal of Chemical Information and Modeling
Article
(15) Minabe, M.; Takabayashi, Y.; Setta, Y.; Nakamura, H.; Kimura,
T.; Tsubota, M. Characterization of 9-(p-Substituted
Benzylidenehydrazono)fluorenes. Bull. Chem. Soc. Jpn. 1996, 69,
3633−3638.
(35) TR-FRET AR Coactivator Assay kit Manual. http://tools.
invitrogen.com/content/sfs/manuals/lanthascreen_AR.pdf (accessed
Aug 2012).
(36) GraphPad Prism, version 5 for Windows; GraphPad Software, La
Jolla, CA; www.graphpad.com (accessed Aug 10, 2012).
(16) Ramanathan, S.; Lemal, D. M. Conformational and configura-
tional dynamics of a highly fluorinated hydrazone. J. Org. Chem. 2007,
72, 1566−1569.
(37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr., J. A.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
(17) Blanco, F.; Alkorta, I.; Elguero, J. Barriers about Double Carbon-
Nitrogen Bond in Imine Derivatives Aldimines, Oximes, Hydrazones,
Azines). Croat. Chim. Acta 2009, 82, 173−183.
(18) Chapelle, J. P.; Elguero, J.; Jacquier, R.; Tarrago, G. Studies on
enehydrazines 2. Reaction of trimethylhydrazine with ketones −
Formation of enehydrazines and N,N-dimethylhydrazones. Bull. Soc.
Chim. Fr. 1970, 3145−3146.
(19) Lu, S. I. Gibbs energy of activation for thermal isomerization of
(1Z)-acetaldehyde hydrazone and (1Z)-acetaldehyde N,N-dimethyl-
hydrazone by Gaussian-4 theory and CCSD(T)/CBS computations. J.
Comput. Chem. 2009, 30, 2176−2180.
(20) Trabelsi, M.; Salem, M.; Champagne, B. Investigation of the
configuration of alkyl phenyl ketone phenylhydrazones from ab initio
1H NMR chemical shifts. Org. Biomol. Chem. 2003, 1, 3839−3844.
(21) Langa, F.; de la Cruz, P.; Delgado, J. L.; Haley, M. M.; Shirtcliff,
L.; Alkorta, I.; Elguero, J. The structure of p-nitrophenylhydrazones of
aldehydes: the case of the p-nitrophenylhydrazone of 2-diethylamino-
5-methoxy-2H-indazole-3-carboxaldehyde. J. Mol. Struct. 2004, 699,
17−21.
̈
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
Revision A.1, Gaussian, Inc.: Wallingford CT, 2009.
(38) Becke, A. D. Density-functional thermochemistry. III. The role
of exact exchange. J. Chem. Phys. 1993, 98, 5648−5652.
(39) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti
correlation-energy formula into a functional of the electron density.
Phys. Rev. B 1988, 37, 785−789.
(40) Frisch, M. J.; Pople, J. A.; Krishnam, R.; Binkley, J. S. Self-
Consistent Molecular Orbital Methods. 25. Supplementary Functions
for Gaussian Basis Sets. J. Chem. Phys. 1984, 80, 3265−3269.
(41) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum mechanical
continuum solvation models. Chem. Rev. 2005, 105, 2999−3093.
(22) Savini, L.; Chiasserini, L.; Gaeta, A.; Pellerano, C. Synthesis and
anti-tubercular evaluation of 4-quinolylhydrazones. Bioorg. Med. Chem.
2002, 10, 2193−2198.
(23) Wyrzykiewicz, E.; Prukala, D. New Isomeric N-substituted
Hydrazones of 2-, 3- and 4-Pyridinecarboxaldehydes. J. Heterocycl.
Chem. 1998, 35, 381−387.
(24) Brokaite, K.; Mickevicius, V.; Mikulskiene, G. Synthesis and
structural investigation of some 1,4-disubstituted-2-pyrrolidinones.
Arkivoc 2006, ii, 61−67.
(25) Kuodis, Z.; Rutavicius, A.; Matijoska, A.; Eicher-Lorka, O.
Synthesis and isomerism of hydrazones of 2-(5-thioxo-4,5-dihydro-
1,3,4-thiadiazol-2-ylthio)acetohydrazide. Centr. Eur. J. Chem. 2007, 5,
996−1006.
(26) Ershov, A. Y.; Lagoda, I. V.; Yakimovich, S. I.; Pakal’nis, V. V.;
Zerova, I. V.; Dobrodumov, A. V.; Shamanin, V. V. Tautomerism and
conformational isomerism of mercaptoacetylhydrazones of aliphatic
and aromatic aldehydes. Russ. J. Org. Chem. 2009, 45, 660−666.
(27) Monfared, H. H.; Bikas, R.; Mayer, P. (E)-3-Hydroxy-N′-(2-
hydroxybenzylidene)-2-naphthohydrazide. Acta Crystallogr. (E) 2010,
66, o236−o237.
(28) Cambridge Crystallographic Data Centre (CCDC), 12 Union
Road, Cambridge CB2 1EZ, England.
(29) Pope, A. J.; Haupts, U. M.; Moore, K. J. Homogeneous
fluorescence readouts for miniaturized high-throughput screening:
theory and practice. Drug Discov. Today. 1999, 4, 350−362.
(30) Gunther, J. R.; Du, Y.; Rhoden, E.; Lewis, I.; Revennaugh, B.;
Moore, T. W.; Kim, S. H.; Dingledine, R.; Fu, H.; Katzenellenbogen, J.
A. A set of time-resolved fluorescence resonance energy transfer assays
for the discovery of inhibitors of estrogen receptor-coactivator binding.
J. Biomol. Screen 2009, 14, 181−193.
(31) Compounds 2a−c and 3 were synthesized according to the
procedure described, all the other compounds evaluated in the
biological studies were purchased from the company Specs (http://
www.specs.net).
(32) Razniewska-Lazecka, G. 13C NMR spectra of some o-carbonyl
derivatives of naphthols. Magn. Reson. Chem. 1986, 24 (4), 365−367.
(33) Karpagam, J. Molecular structure, vibrational spectroscopic,
first-order hyperpolarizability and HOMO, LUMO studies of 3-
hydroxy-2-naphthoic acid hydrazide. J. Raman Spectrosc. 2010, 41 (1),
53−62.
(34) Pant, U. C.; Ramchandran, R.; Joshi, B. C. Studies on 1-
hydroxy-2-naphtoic and 3-hydroxy-2-naphtoic acid hydrazides. Rev.
Roum. de Chim 1979, 24 (3), 471−482.
2397
dx.doi.org/10.1021/ci300299n | J. Chem. Inf. Model. 2012, 52, 2387−2397