Design, synthesis, and biological evaluation of conformationally constrained analogues of naphthol AS-E as inhibitors of CREB-mediated gene transcription

Article abstract of DOI:10.1021/jm300043c

Cyclic AMP response element binding protein (CREB) is often dysregulated in cancer cells and is an attractive cancer drug target. Previously, we described naphthol AS-E (1) as a small molecule inhibitor of CREB-mediated gene transcription. To understand its bioactive conformation, a series of conformationally constrained analogues of 1 were designed and synthesized. Biological evaluation of these analogues suggests that the global energy minimum of 1 is the likely bioactive conformation.

Full text of DOI:10.1021/jm300043c

Brief Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Conformationally
Constrained Analogues of Naphthol AS-E as Inhibitors of CREB-
Mediated Gene Transcription
Min Jiang,†,‡ Bingbing X. Li,†,‡ Fuchun Xie,†,‡ Frances Delaney,†,‡ and Xiangshu Xiao*,†,‡,§
‡
§
†Program in Chemical Biology, Department of Physiology and Pharmacology, and Knight Cancer Institute, Oregon Health &
Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon, United States
S
* Supporting Information
ABSTRACT: Cyclic AMP response element binding protein (CREB) is often dysregulated in cancer cells and is an attractive
cancer drug target. Previously, we described naphthol AS-E (1) as a small molecule inhibitor of CREB-mediated gene
transcription. To understand its bioactive conformation, a series of conformationally constrained analogues of 1 were designed
and synthesized. Biological evaluation of these analogues suggests that the global energy minimum of 1 is the likely bioactive
conformation.
INTRODUCTION
■
Cyclic-AMP response element (CRE) binding protein (CREB)
is a stimulus-activated nuclear transcription factor enabling cells
to respond to various extracellular stimuli.1 Its DNA-binding
domain, located at the C-terminus, binds the consensus CRE
sequence 5′-TGACGTCA-3′.2,3 Even though CREB is able to
bind CRE in the absence of any posttranslational modifications,
its transcription activity is not activated until its activation
domain called kinase-inducible domain (KID) is phosphory-
lated at Ser133.4 The thus phosphorylated CREB (p-CREB)
Figure 1. Potential conformations of 1. The yellow dashed lines
indicate hydrogen bonds. The dihedral angle of a−b−c−d in 1a is 0.1°,
allows for its subsequent binding with transcription coactivator
while that in 1b is 10.6°.
CBP (CREB-binding protein).5 The CREB−CBP interaction is
mediated by KID in CREB and KIX (KID-interacting) domain
in CBP.5 A variety of protein serine/threonine kinases
including protein kinase A (PKA),2 protein kinase B (PKB/
Akt),6 protein p90 ribosomal S6 kinase (pp90RSK),7 and
mitogen-activated protein kinase (MAPK)8,9 could phosphor-
ylate and activate CREB. Since these kinase signaling pathways
are often deregulated in cancer cells, CREB was consistently
found to be overactivated in cancer tissues from patients with
non-small-cell lung cancer (NSCLC),10 prostate cancer,11
breast cancer,12 acute myeloid leukemia, and acute lympho-
blastic leukemia.13,14 Thus, CREB has been proposed as an
intriguing target for the development of novel cancer
therapeutics15 and various strategies have been pursued to
identify potential small molecule modulators of CREB-
mediated gene transcription.15−17
RESULTS AND DISCUSSION
■
To investigate the conformational preference of 1, it was
subjected to a systematic conformational search by rotating all
the rotatable bonds in MacroModel. This search resulted in two
alternatively hydrogen-bonded conformations 1a and 1b
(Figure 1) as the energy minima. Conformer 1a, with a
dihedral angle of a−b−c−d of 0.1°, is fully conjugated and
adopts a coplanar conformation. On the other hand, the
dihedral angle of a−b−c−d in conformer 1b is 10.6°. As a
consequence, the naphthyl ring and the chlorophenyl ring are
not coplanar. Counterintuitively, conformer 1b is the global
minimum and is energetically slightly more favorable than
conformer 1a by 2.243 kJ/mol (Figure 1). To examine which
conformation might represent the biologically active one,
conformationally constrained analogues 2−7 (Chart 1) were
designed. Compounds 2 and 3 were designed to mimic
conformer 1a, while 4−7 were designed to mimic conformer
1b.
We recently described naphthol AS-E (1, Figure 1) as a small
molecule inhibitor of KIX−KID interaction and CREB-
mediated gene transcription with potencies in the low
micromolar range.18 However, its bioactive conformation,
which would be very useful in facilitating the design of more
potent analogues, is currently unknown. In this report, a series
of conformationally constrained analogues of 1 were designed
and synthesized to interrogate the bioactive conformation of 1
as inhibitors of KIX−KID interaction and CREB-mediated gene
transcription.
Compound 2 was synthesized in one step by treating 1 with
oxalyl chloride.19 The synthesis of benzo[g]quinazolinedione 3
would require precursor 11 (Scheme 1). Therefore, the amino
Received: January 12, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
yldiimidazole23 and Mitsunobu reagent (Ph3P/DEAD)24 were
also tried, but they both failed to give 14.
The apparent difficulty in synthesizing 14 prompted us to
investigate an alternative route to make 4 as presented in
Scheme 3. Thus, 3-amino-2-naphthoic acid (8) was converted
Chart 1. Structures of Designed Conformationally
Constrained Compounds 2−7
Scheme 3. Synthesis of 4−6
Scheme 1. Synthesis of 3
group in 3-amino-2-naphthoic acid (8) was first protected by a
Boc group to give acid 9, which was then coupled with 4-
chloroaniline to provide amide 10. Removal of Boc under acidic
condition (TFA) yielded amine 11, which was then treated
with triphosgene to afford analogue 3 uneventfully.
The synthesis of analogue 4 was less straightforward. Initially,
it was envisioned that an SNAr reaction between 4-chloroaniline
and chloride 15 would deliver 4.20 Thus, a synthetic route
depicted in Scheme 2 was designed. Hydroxamide 13 was
into fluoride 19 through a three-step sequence involving methyl
esterification, diazonium salt formation, and thermal decom-
position of the diazonium tetrafluoroborate. The ester group in
19 was then reduced with DIBAL-H to provide aldehyde 20,
which was then transformed into the corresponding oxime. It
was found that the oxime could be oxidized by NCS to give N-
hydroxyimidoyl chloride 21,25 which was then coupled with 4-
chloroaniline to yield N-hydroxyimidamide 22. Intramolecular
SNAr reaction of 22 under basic condition (K2CO3/DMF)
delivered analogue 4.
Scheme 2. Attempted Synthesis of 14
The intermediate ester 19 was also utilized to synthesize 5
and 6 (Scheme 3). Saponification of ester in 19 gave acid 23,
which was then coupled with 4-chloroaniline with BOP reagent
to afford amide 24. The thioamide 25 was formed by treating
24 with Lawesson’s reagent. Ring closure between 25 and
hydrazine26 delivered analogue 5. Methylation of 5 with NaH/
MeI afforded 6. The regiochemistry of the methyl group in 6
was confirmed by the positive NOE between the methyl
protons and one of the naphthyl protons, while no NOE was
observed between the protons in the chlorophenyl ring and the
methyl group.
Finally, 7 was prepared by a sequence of reactions presented
in Scheme 4. Intermediate 26 was prepared by condensing acid
8 with formamide under solvent-free conditions.27 7 was made
by an SNAr reaction between 4-chloroaniline and chloride 27,
which was derived from 26 after being treated with POCl3.
Compounds 2−7 were then evaluated for inhibition of KIX−
KID interaction in vitro by a Renilla luciferase complementa-
tion assay that we recently developed (Table 1).18 In this assay,
1 inhibits KIX−KID interaction with IC50 = 2.90 μM.18 2 and 3
were designed to mimic conformation 1a. Compound 3 also
retains a hydrogen-bond donor as seen in 1. However, neither
prepared by condensing hydroxylamine with the methyl ester of
acid 12. However, when 13 was treated with thionyl chloride,21
isoxazole 14 was not obtained as expected. Instead, oxazolone
18 was isolated in 37% yield, whose structure is consistent with
the observed positive NOE between Ha and Hb. The formation
of 18 is perhaps through a process analogous to the Lossen
rearrangement22 of chloride 16 generated from chlorination of
13 to provide isocyanate 17, cyclization of which produced 18.
The electron-rich nature of the naphthyl ring may facilitate the
Lossen type rearrangement pathway instead of direct ring
closure to form 14. Other reagents including carbon-
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Brief Article
when optimized at a lower level of theory (e.g., HF/3-21G).
Similar nitrogen pyramidization was also observed in other
systems experimentally28 and in theoretical calculations.29 With
these optimized structures, we then generated molecular
electrostatic potential (MEP) surface for each molecule from
their respective electron densities (Figure 2). The major
Scheme 4. Synthesis of 7
a
Table 1. Biological Activities of 1−7
KIX−KID
CREB
inhibition (μM)
b
c
compd
inhibition (μM)
1
2
3
4
5
6
7
2.90 0.81
>50
2.29 0.31
30.84 8.40
>50
>50
>50
24.45 16.80
4.75 3.88
>50
8.74 1.68
5.19 2.20
9.46 4.15
18.96 6.09
a
All the biological activities are presented as IC50 with mean SD of
at least two independent experiments or >50 if IC50 was not reached at
b
the highest tested concentration, which is 50 μM. KIX−KID
inhibition was evaluated by an in vitro Renilla luciferase
c
complementation assay. CREB inhibition refers to inhibition of
CREB-mediated gene transcription in HEK 293T cells using a CREB
reporter assay.
of these compounds showed any activity in inhibiting KIX−
KID interaction in vitro, suggesting that conformation 1a may
not be biologically relevant. This lack of activity also translates
into low or no inhibitory activity against CREB-mediated gene
transcription in HEK 293T cells using a CREB reporter assay.18
Compounds 4−7 were designed to interrogate if conformation
1b is a potential bioactive one. Although 4 was inactive in
inhibiting KIX−KID interaction, 5 with a nitrogen linkage
showed only slightly reduced activity (IC50 = 8.74 μM)
compared to 1. The loss of activity with 4 does not seem to be
due to elimination of a hydrogen-bond donor present in 1
because the methylated 6 (IC50 = 5.19 μM) and benzo[g]-
quinazoline 7 (IC50 = 9.46 μM) were in the same range of
activity as 1. In the cell-based CREB reporter assay, the oxygen-
linked 4 had very weak activity, as expected from its lack of
activity in the KIX−KID interaction assay. Compound 5, on the
other hand, retained activity in inhibiting CREB-mediated gene
transcription with IC50 = 4.75 μM. The intermediate potency of
inhibiting KIX−KID interaction by 7 was also reflected in its
Figure 2. Molecular electrostatic potential (MEP) surfaces of 1, 4, and
5 mapped to their electron densities calculated at the HF/6-31G**
level of theory in Jaguar. The blue indicates electrostatically positively
charged regions, while the red is negatively charged. All the surfaces
are normalized from −60 to +60 kcal/mol.
difference observed among 1, 4, and 5 is located in the linkage
section as highlighted in green circles in Figure 2. In both of the
active compounds 1 and 5, this area is represented by positively
and negatively charges surfaces. However, only electrostatically
negatively charged (−40 to −50 kcal/mol) surfaces are present
in the corresponding area in the inactive 4 (Figure 2). It could
be that the binding site in KIX for the circled area is
electrostatically negatively charged. As a consequence, 4 does
not fit well in that binding site, contributing to its loss of
inhibition of KIX−KID interaction. On the other hand, the
mixed surfaces from 1 and 5 are well accommodated.
medium potency in CREB transcription reporter assay (IC50
=
18.96 μM). However, the methylated 6 was inactive in cell-
based CREB reporter assay even though it was a reasonable
inhibitor of KIX−KID interaction. This discrepancy could
result from unfavorable cellular permeation and/or subcellular
distribution of 6, which has a higher cLogP (cLogP = 5.298)
than 5 (cLogP = 4.433). Taken together, these results suggest
that the likely bioactive conformation of 1 resembles conformer
1b.
To further understand the potential reasons for the observed
activity among 1, 4, and 5, these structures were geometrically
optimized at the HF/6-31G** level of theory in Jaguar. We
found that the N1 atom in 5 (Chart 1) prefers a pyramidal
configuration instead of a planar one even though it is planar
CONCLUSION
■
A series of compounds (2−7) were designed and synthesized
to mimic two alternative conformations of 1 to derive its
potential bioactive conformation in inhibiting KIX−KID
interaction. These compounds were then biologically evaluated
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7.34 (d, J = 8.8 Hz, 2 H), 7.32 (t, J = 8.4 Hz, 1 H); 13C NMR (100
MHz, DMSO-d6) δ 145.2, 142.1, 139.9, 133.3, 129.5, 129.0, 127.9,
127.5, 126.3, 123.0, 122.9, 119.1, 117.9, 104.3; ESI-MS m/z 292 (M −
H)−; HRESI-MS for C17H12ClN3 − H, calcd 292.0636, found
292.0629.
for their capability to inhibit KIX−KID interaction in vitro and
CREB-mediated gene transcription in HEK 293T cells. These
results suggest that the energetically favored conformer 1b may
represent the bioactive conformation. Molecular modeling
studies suggest that the electrostatic potential surface of the
newly created heterocycle needs to be at least partially
positively charged. Further investigations are underway to
utilize these concepts to design more potent derivatives in
inhibiting KIX−KID interaction and CREB-mediated gene
transcription.
N-(4-Chlorophenyl)-1-methyl-1H-benzo[f ]indazol-3-amine
(6). NaH (60% in mineral oil, 6 mg, 0.15 mmol) was added to a stirred
solution of 5 (40 mg, 0.136 mmol) in DMF (0.5 mL) at 0 °C under
Ar. The mixture was stirred at 0 °C for 30 min, when CH3I (8.6 μL,
0.136 mmol) was added. The mixture was stirred at room temperature
for another 1 h and poured into cooled water (10 mL). The precipitate
was collected by filtration. The yellow solid was purified by silica gel
flash column chromatography, eluting with hexanes/ethyl acetate, 4:1,
to yield a yellow solid (19 mg, 45%): mp 121−122 °C. 1H NMR (400
MHz, DMSO-d6) δ 9.47 (s, 1 H), 8.64 (s, 1 H), 8.00 (d, J = 8.8 Hz, 1
H), 7.96 (d, J = 8.4 Hz, 1 H), 7.91 (s, 1 H), 7.83 (d, J = 8.8 Hz, 2 H),
7.54 (t, J = 8.4 Hz, 1 H), 7.37 (d, J = 8.8 Hz, 2 H), 7.32 (t, J = 8.0 Hz,
1 H), 3.98 (s, 3 H); 13C NMR (100 MHz, DMSO-d6) δ 143.8, 141.3,
139.3, 132.9, 129.1, 128.6, 127.3, 126.9, 126.1, 122.7, 122.6, 119.1,
117.7, 117.5, 103.1, 35.3; ESI-MS m/z 305.9 (M − H)−; HRESI-MS
for C18H14ClN3 − H, calcd 306.0793, found 306.0785.
EXPERIMENTAL SECTION
General. See Supporting Information for details. All final
compounds were confirmed to be of >95% purity based on HPLC
analysis.
■
3-(4-Chlorophenyl)-2H-naphtho[2,3-e][1,3]oxazine-2,4-(3H)-
dione (2). Compound 1 (100 mg, 0.34 mmol) was dissolved in p-
xylene (4.0 mL), and the solution was stirred for 5 min at room
temperature. Oxalyl chloride (150 μL, 1.7 mmol) in p-xylene (1.0 mL)
was then added, and the mixture was heated at 120 °C overnight. The
reaction mixture was cooled to room temperature. The precipitate was
collected by filtration and washed with p-xylene to give a pink-white
N-(4-Chlorophenyl)benzo[g]quinazolin-4-amine (7). A mix-
ture of 4-chlorobenzo[g]quinazoline (27) (50 mg, 0.23 mmol), 4-
chloroaniline (36 mg, 0.28 mmol), and DMAP (29 mg, 0.23 mmol) in
p-xylene (1 mL) was stirred at 140 °C for 10 min. The mixture was
cooled to room temperature. Evaporation of p-xylene resulted in a
residue which was subjected to silica gel flash column chromatography,
eluting with hexanes/ethyl acetate (2:1) to yield a yellow solid (31 mg,
1
solid 17 (57 mg, 52%): mp 306−308 °C (dec). H NMR (400 MHz,
CDCl3) δ 8.77 (s, 1 H), 8.06 (d, J = 8.4 Hz, 1 H), 7.96 (d, J = 8.4 Hz,
1 H), 7.78 (s, 1 H), 7.72 (t, J = 9.6 Hz, 1 H), 7.61 (t, J = 7.3 Hz, 1 H),
7.55 (d, J = 8.8 Hz, 2 H), 7.33 (d, J = 8.4 Hz, 2 H); 13C NMR (100
MHz, DMSO-d6) δ 161.3, 148.5, 148.1, 137.0, 134.8, 133.9, 131.1,
130.3, 130.1, 130.1, 129.6, 127.9, 126.9, 114.9, 112.6.
1
44%): mp 251−252 °C. H NMR (400 MHz, CDCl3) δ 10.26 (s, 1
H), 9.32 (s, 1 H), 8.67 (s, 1 H), 8.47 (s, 1 H), 8.21 (d, J = 8.8 Hz, 1
H), 8.19 (d, J = 8.4 Hz, 1 H), 8.08 (d, J = 8.8 Hz, 2 H), 7.42 (t, J = 7.2
Hz, 1 H), 7.67 (t, J = 7.2 Hz, 1 H), 7.54 (d, J = 8.8 Hz, 2 H); 13C
NMR (100 MHz, CDCl3) δ 158.6, 154.4, 145.5, 138.6, 135.9, 131.4,
129.3, 128.9, 128.5, 128.3, 128.0, 126.8, 125.5, 124.3, 123.7, 115.5;
ESI-MS m/z 303.9 (M − H)−; HRESI-MS for C18H12ClN3 + H, calcd
306.0793, found 306.0805.
3-(4-Chlorophenyl)benzo[g]quinazoline-2,4-(1H,3H)-dione
(3). Triphosgene (7.5 mg, 0.025 mmol) was added to a stirred solution
of 11 (15 mg, 0.05 mmol) in THF (1.5 mL) at room temperature. The
resulting mixture was heated under reflux for 2 h. The mixture was
then cooled to room temperature and diluted with THF (5 mL). The
solid was collected by filtration to give a white solid (11 mg, 69%): mp
>400 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1 H), 8.70 (s, 1
H), 8.14 (d, J = 7.5 Hz, 1 H), 7.95 (d, J = 7.7 Hz, 1 H), 7.63 (dt, J =
7.5, 1.3 Hz, 1 H), 7.59 (s, 1 H), 7.57 (d, J = 9.0 Hz, 2 H), 7.47 (dt, J =
7.6, 1.3 Hz, 1 H), 7.43 (d, J = 8.8 Hz, 2 H); 13C NMR (100 MHz,
DMSO-d6) δ 162.2, 150.1, 136.39, 135.41, 134.7, 132.7, 131.2, 129.6,
129.6, 129.4, 128.8, 128.5, 126.7, 125.0, 115.1, 110.1; ESI-MS m/z
320.7 (M − H)−; HRESI-MS for C18H11ClN2O2 − H, calcd 321.0425,
found 321.0416.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for synthesis of and characterization
data for 9−11, 19−20, and 22−27; details of the biological
assays and molecular modeling. This material is available free of
charge via the Internet at http://pubs.acs.org.
N-(4-Chlorophenyl)naphtho[2,3-d]isoxazol-3-amine (4).
K2CO3 (7.9 mg, 0.057 mmol) was added to a stirred solution of 22
(6.0 mg, 0.019 mmol) in DMF (1 mL) at room temperature. The
resulting mixture was then heated at 110 °C for 4 h. The mixture was
cooled to room temperature, and DMF was removed in vacuo. The
residue was dissolved in EtOAc (50 mL), which was then washed with
H2O (2 × 10 mL) and brine (2 × 10 mL). The organic solution was
dried over anhydrous Na2SO4, filtered, and concentrated. The residue
was then subjected to column chromatography, eluting with hexanes/
EtOAc (10:1 to 4:1) to give a white solid (2.7 mg, 48%): mp 218−220
°C. 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 1 H), 7.98 (d, J = 8.8 Hz,
1 H), 7.95 (d, J = 8.4 Hz, 1 H), 7.86 (s, 1 H), 7.60 (d, J = 8.8 Hz, 2 H),
7.56 (td, J = 8.4, 1.2 Hz, 1 H), 7.47 (td, J = 8.0, 1.2 Hz, 1 H), 7.35 (d, J
= 8.8 Hz, 2 H), 6.60 (brs, 1 H); ESI-MS m/z 295 (M + H)+; HRESI-
MS for C17H11ClN2O − H, calcd 293.0476, found 293.0470.
AUTHOR INFORMATION
Corresponding Author
*Phone: 503-494-4748. Fax: 503-494-4352. E-mail: xiaoxi@
ohsu.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was made possible by financial support from NIH
(Grant R01GM087305) and Susan G. Komen for the Cure
(Grant KG100458). We thank Jenny Luo, Dr. Andrea
DeBarber, and Dr. Dennis Koop for running the mass
spectroscopic analyses.
N-(4-Chlorophenyl)-1H-benzo[f ]indazol-3-amine (5). A mix-
ture of thioamide 24 (60 mg, 0.19 mmol) in anhydrous hydrazine/
DMSO (0.5/0.5 mL) was stirred at 150 °C for 1 h. The mixture was
cooled to room temperature and poured into water (10 mL). The
organic compound was extracted with dichloromethane (3 × 10 mL).
The combined organic layers were washed with brine (2 × 10 mL),
dried with Na2SO4, filtered, and concentrated. The residue was
purified by silica gel flash column chromatography, eluting with
hexanes/ethyl acetate (4:1) to yield a yellow solid (10 mg, 18%): mp
ABBREVIATIONS USED
■
CRE, cyclic AMP response element; CREB, cyclic AMP
response element binding protein; CBP, CREB-binding
protein; KID, kinase-inducible domain; KIX, kinase-inducible
domain interacting; p-CREB, phosphorylated cyclic AMP
response element binding protein; PKA, protein kinase A;
PKB, protein kinase B; MAPK, mitogen-activated protein
kinase; pp90RSK, protein p90 ribosomal S6 kinase; NSCLC,
1
220−221 °C. H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1 H), 9.37
(s, 1 H), 8.62 (s, 1 H), 7.98 (d, J = 8.0 Hz, 1 H), 7.96 (d, J = 8.0 Hz, 1
H), 7.82 (s, 1 H), 7.81 (d, J = 8.8 Hz, 2 H), 7.44 (t, J = 8.0 Hz, 1 H),
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