Novel type of prodrug activation through a long-range O,N-acyl transfer: A case of water-soluble CREB inhibitor

Article abstract of DOI:10.1021/ml500330n

CREB (cAMP response element binding protein) has been shown to play an important role in tumor initiation, progression, and metastasis. We discovered that naphthol AS-E, a cell-permeable CREB inhibitor, presented antiproliferative activity in a broad panel of cancer cell lines in vitro. However, it has limited aqueous solubility. In this report, we described a water-soluble inhibitor (compound 6) of CREB-mediated gene transcription with in vivo anticancer activity. Unexpectedly, compound 6 was found to be a prodrug of compound 12 necessitating an unprecedented long-range O,N-acyl transfer. The rate of this transfer was pH- and temperature-dependent. To the best of our knowledge, this is the first time to show that a long-range O,N-acyl transfer could be exploited as a prodrug activation strategy to improve aqueous solubility. This type of prodrug may be applicable to other structures with spatially arranged hydroxyl amide to improve their aqueous solubility.

Full text of DOI:10.1021/ml500330n

Letter
pubs.acs.org/acsmedchemlett
Novel Type of Prodrug Activation through a Long-Range O,N‑Acyl
Transfer: A Case of Water-Soluble CREB Inhibitor
Bingbing X. Li,†,‡,# Fuchun Xie,†,‡,# Qiuhua Fan,†,‡ Kerry M. Barnhart,∥ Curtis E. Moore,⊥
Arnold L. Rheingold,⊥ 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 97239, United States
∥Transmed Oncology, Cave Creek, Arizona 85327, United States
⊥Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States
S
* Supporting Information
ABSTRACT: CREB (cAMP response element binding
protein) has been shown to play an important role in tumor
initiation, progression, and metastasis. We discovered that
naphthol AS-E, a cell-permeable CREB inhibitor, presented
antiproliferative activity in a broad panel of cancer cell lines in
vitro. However, it has limited aqueous solubility. In this report,
we described a water-soluble inhibitor (compound 6) of
CREB-mediated gene transcription with in vivo anticancer
activity. Unexpectedly, compound 6 was found to be a prodrug of compound 12 necessitating an unprecedented long-range O,N-
acyl transfer. The rate of this transfer was pH- and temperature-dependent. To the best of our knowledge, this is the first time to
show that a long-range O,N-acyl transfer could be exploited as a prodrug activation strategy to improve aqueous solubility. This
type of prodrug may be applicable to other structures with spatially arranged hydroxyl amide to improve their aqueous solubility.
KEYWORDS: CREB, prodrug, water-soluble inhibitor, anticancer, O,N-acyl transfer
Figure 1) was recently identified as an inhibitor of KIX−KID
mong the various transcription factors that are involved in
Athe pathogenesis of cancer is cyclic-AMP-response
element binding protein (CREB).1−3 The signaling pathways
leading to phosphorylation and subsequent activation of CREB
start with extracellular signals through a variety of intracellular
oncogenic protein serine/threonine kinases.1,3 The critical
residue to be phosphorylated in CREB for its activation is
Ser133.4 Once phosphorylated, it binds to histone acetyl-
transferase and transcription coactivator, CREB-binding protein
(CBP), and its closely related paralog p300.5 Further
recruitment of additional components in the transcription
machinery to the gene promoter initiates CREB-dependent
gene transcription.3,5 However, three protein phosphatases with
known tumor suppressor functions can dephosphorylate CREB
to attenuate CREB-mediated gene transcription. These are
protein phosphatase 1 (PP1),6 protein phosphatase 2A
(PP2A),7 and phosphatase and tensin homologue (PTEN).8
The mechanisms leading to CREB activation and inactivation
suggest that CREB plays a critical role in tumorigenesis. Indeed,
CREB has been found to be overexpressed and overactivated in
a variety of cancers including breast,9,10 prostate,11 lung,12
brain,13 and acute leukemia.14 Therefore, CREB has been
proposed as a promising drug target for a variety of cancers.1,15
An essential step for activation of CREB-dependent gene
transcription is to recruit CBP.5 The CREB−CBP interaction is
mediated by kinase-inducible domain (KID) from CREB and
KIX (KID-interacting) domain from CBP.16 Naphthol AS-E (1,
interaction and a cell-permeable inhibitor of CREB-mediated
Figure 1. Chemical structure of naphthol AS-E (1).
gene transcription.17 Subsequent studies also demonstrated that
compound 1 inhibited proliferation of cancer cells of different
organs with a GI50, which is the concentration required to
inhibit the cancer cell proliferation by 50%, in the low
micromolar range.18 Importantly, normal cells tolerated 1 very
well,18 consistent with the idea of cancer cells’ addiction to
CREB.1 One of the major limitations associated with
compound 1 is its poor aqueous solubility. In this report, we
describe our results in generating a water-soluble inhibitor of
CREB-mediated gene transcription (compound 6) with in vivo
anticancer activity. Unexpectedly, compound 6 was found to be
a prodrug to be activated by a long-range O,N-acyl transfer
reaction under physiological conditions.
Received: May 30, 2014
Accepted: August 22, 2014
Published: August 22, 2014
© 2014 American Chemical Society
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In order to improve the aqueous solubility of 1, compound 2
was designed (Scheme 1), which contains a free phenol group
living HEK 293T cells (IC50 = 1.95 μM, Table 1) as evaluated
by a CREB-Renilla luciferase reporter assay.17 Similarly, the
antiproliferative activity (GI50) of 5 in cancer cell lines (A549,
MCF-7, MDA-MB-231, and MDA-MB-468) was not improved
compared to the original compound 1 (Table 1). In order to
improve these two inherent limitations associated with 5, a bis-
aminoalkylated compound 6 was designed (Figure 2). The
Scheme 1. Synthesis of Compounds 2 and 5
Figure 2. Design of a water-soluble compound 6.
envisioned to be a facile point to attach polar groups to
enhance its aqueous solubility. Therefore, commercially
available acid 3 was coupled with aniline 4 in the presence of
BOP/DIPEA. In addition to the desired compound 2 (39%), a
bisacylated product 5 was also obtained from the reaction
mixture in 55% yield. The structure of 5 was initially deduced
from its 1H NMR, 13C NMR, MS data, and then confirmed by
single crystal X-ray analysis (Scheme 1). Both 2 and 5 were
evaluated for in vitro inhibition of KIX−KID interaction using a
split Renilla luciferase complementation assay we recently
described (Table 1).17 This assay directly monitored KIX−KID
interaction in vitro with a purified KIX fusion and KID fusion-
containing cell lystaes. Even though compound 2 was inactive
in this assay (IC50 >50 μM), the bisacylated product 5
displayed potent activity in inhibiting KIX−KID interaction
with an IC50 = 0.17 μM. This compound represents one of the
most potent inhibitors of KIX−KID interaction reported to
date18−21 and is 17-fold more potent than the initial lead
compound 1 (Table 1).
charged polar amino groups were added to improve its aqueous
solubility. In addition, the aminopropyl groups are sterically
more hindered than the methyl groups. Thus, it was predicted
that compound 6 would be hydrolytically more stable than 5
because esterase-mediated hydrolysis of esters is known to be
sensitive to sterics.22
The synthesis of 6 is presented in Scheme 2. Naphthol 7,
prepared from methylation of the corresponding acid,20 was
Scheme 2. Synthesis of Compound 6
The potent in vitro activity of compound 5, however, is
plagued by its poor aqueous solubility (<0.1 mg/mL in H2O).
In addition, the ester group present in 5 was not expected to be
stable toward serum esterases. Indeed, we found that the half-
life (t1/2) of compound 5 in regular tissue culture media
(Dulbecco’s Modified Eagle Medium, DMEM, pH = 7.70)
containing 10% fetal bovine serum (FBS) was ∼20 h (data not
shown). These two factors may explain why 5 was still a low
micromolar inhibitor of CREB-mediated gene transcription in
coupled with alcohol 8 under standard Mitsunobu condition
(Ph3P, DEAD) to give 9 uneventfully. Saponification of methyl
ester 9 generated acid 10, which was then used for double
acylation with ortho-aminophenol 4 to yield 11. Removal of the
Table 1. Biological Activities of Newly Synthesized Compounds
a
b
IC50 (μM)
GI50 (μM)
c
d
compd
KIX−KID inhibition
CREB inhibition
A549
MCF-7
MDA-MB-231
MDA-MB-468
1
2.90 0.81
>50
2.29 0.31
>50
2.90 0.33
>100
2.81 0.35
>100
2.35 0.60
>100
1.46 0.30
>100
2
5
0.17 0.12
4.40 0.19
19.72 1.78
>50
1.95 1.14
2.27 0.28
2.22 0.38
26.25 13.64
8.78 2.55
0.59 0.02
0.29 0.014
2.73 0.20
3.38 0.49
0.42 0.15
0.14 0.03
1.85 1.23
23.51 2.27
1.30 0.66
0.37 0.13
2.19 0.34
8.11 5.47
0.33 0.004
0.22 0.07
1.20 0.20
6
12
14
a
The IC50 is presented as mean SD of at least two independent experiments or >50 in the cases where IC50 did not reach at the highest tested
b
concentration (50 μM). GI50 is the concentration required to inhibit the cancer cell growth by 50% as evaluated by the MTT assay. The
compounds were incubated with cells for 72 h. The GI50 is presented as mean SD of at least two independent experiments or >100 in the cases
c
where GI50 did not reach at the highest tested concentration (100 μM). KIX−KID interaction inhibition was evaluated by an in vitro Renilla
d
luciferase complementation assay. CREB inhibition refers to inhibition of CREB-mediated gene transcription in HEK 293T cells using a CREB
reporter assay.
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Boc protecting groups in 10 under acidic condition delivered
desired compound 6.
Consistent with the design rationale, compound 6 was found
to be highly water-soluble (>100 mg/mL in H2O), which is a
more than 1000-fold improvement over 5. However, this
compound was surprisingly found to be extremely unstable in
regular tissue culture media with t1/2 < 5 min (Figure 3). Unlike
that the proteins and enzymes in the tissue culture media were
not required for this conversion. Therefore, compound 6 was
treated with PBS, and the newly generated product was isolated
in 50% yield.24 Extensive NMR and MS analyses indicated that
the new product was O,N-acyl transferred product 12 (Scheme
3). To further confirm its structural assignment, compound 12
was synthesized through an independent route (Scheme 3).
Thus, acid 10 was coupled with ortho-aminophenol 4 at 1:1
ratio to give 13, whose Boc was removed under acidic condition
to give amine 14. Coupling of 14 with another copy of acid 10
followed by acidic hydrolysis provided 12. The two samples of
12 individually or a mixture thereof were identical in NMR and
HPLC, confirming the original structural assignment of 12.
To investigate if the conversion from 6 to 12 was an
intramolecular or intermolecular process, the kinetics of this
conversion was studied in detail in PBS at room temperature.
At physiological pH of 7.40, the decay of 6 followed a first-
order kinetics (Figure 4A), which was further confirmed by the
linearity of the semilog plot (r2 = 0.9892) (Figure S2,
Supporting Information). These results support that 12 was
formed from intramolecular aminolysis of 6, which would
require a 13-membered ring transition state. To understand
why this unusual transition state was favored, compound 6 was
subjected to molecular dynamics simulation in MacroModel.
During the 1000 ps production run, the distance between Ca
and Nb in 6 was monitored (Figure 5). It is evident from this
simulation that these two reaction centers are very close to each
other averaged at ∼4 Å during the majority of the simulation
time. This optimal distance may facilitate the intramolecular
aminolysis reaction to generate 12. Similar to other intra-
molecular aminolysis,25 conversion from 6 to 12 was pH-
dependent. Thus, the t1/2 of 6 in PBS at room temperature was
27.3, 37.7, 114.6, and 447.7 min at pH of 7.40, 6.98, 6.48, and
6.01, respectively (Figures 4 and S2, Supporting Information).
The rapid intramolecular aminolysis of 6 at physiological pH
suggested that 12 was the active species in all the cellular
experiments with 6, which is thus considered as a prodrug.
Other prodrugs involving intramolecular aminolysis has been
known for O-acyl-1,2-hydroxylamines such as the ester
derivatives of propranolol,26,27 paclitaxel,25 and peptidomimet-
ics.28 However, to the best of our knowledge, this type of
prodrug activation necessitating a long-range O,N-acyl transfer
has not been known before in the literature.
Figure 3. Compound 6 was converted into 12 in tissue culture media.
Compound 6 was incubated in DMEM containing 10% FBS at 37 °C.
At the indicated time point, an aliquot (10 μL) was withdrawn and
mixed with acetonitrile (90 μL) to precipitate the proteins. Then 10
μL of the supernatant was subjected to RP-HPLC analysis monitored
by UV absorbance at 254 nm. IS: internal standard. The other peaks
eluted between 12.0 and 12.5 min are unidentified components from
the tissue culture media.23
compound 5, the instability of 6 was not due to its ester
hydrolysis to produce phenol 14 (Scheme 3) because this
hydrolysis product only constituted a minor species in the
incubation mixture (Figure 3). Instead, the major species
generated was the peak eluted at ∼12.2 min in the HPLC
chromatogram (Figure 3). This new species became apparently
important because compound 6 was found to be a potent
inhibitor of all four cancer cell lines tested even though its
potencies of CREB inhibition in HEK 293T cells and in vitro
KIX−KID interaction inhibition remained modest (Table 1).
To facilitate identification of the new species, we investigated if
this conversion was an enzyme-mediated process by switching
the tissue culture media to phosphate buffered saline (PBS, pH
= 7.40). Similar instability of compound 6 in PBS was also
observed (Figure S1, Supporting Information), demonstrating
To further test the prodrug hypothesis, the newly synthesized
compound 12 was biologically evaluated, and the results are
presented in Table 1. Compound 12 was about as potent as 6
Scheme 3. Two Syntheses of Compound 12
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ase chain reaction (qRT-PCR) (Figure 6). As an endogenous
CREB target gene, forskolin (10 μM), robustly stimulated
Figure 6. Compounds 6 and 12 suppressed expression of endogenous
CREB target NR4A2. HEK 293T cells were treated with different
drugs for 1 h, when forskolin was added for another 45 min. The
relative mRNA level was quantified by qRT-PCR. The student t test
was used to evaluate significance.
NR4A2 expression level to ∼44-fold. Both compounds 6 (2.5
μM) and 12 (2.5 μM) suppressed the expression of NR4A2 to
∼10-fold (Figure 6).
Figure 4. Kinetics of conversion from 6 to 12 in PBS at room
temperature at different pH. (A) Conversion from 6 to 12 was a first-
order, unimolecular process and pH-dependent in PBS solution.
Compound 6 was incubated in PBS at different pH values at room
temperature. At the indicated time points, an aliquot was taken and
analyzed by RP-HPLC. The percentage of 6 remaining was quantified
from each chromatogram. The lines represent the best fit curves based
on first-order kinetics. (B) The t1/2 of 6 in PBS at different pH values
at room temperature. The data are presented as mean SD of two
independent experiments.
In suppressing cancer cell growth, 12 was also similar to or
only marginally more potent than 6 in A549, MCF-7, MDA-
MB-231, and MDA-MB-468 cells. Under these cellular
experimental conditions, the small amount of hydrolysis
product 14 generated did not likely contribute to the observed
activity for 6 because 14 was less potent in all the evaluated
assays (Table 1). It is clear that the antiproliferative activity
observed for 14 is not due to CREB inhibition or KIX−KID
interaction inhibition.
In the in vitro KIX−KID interaction inhibition assay,
compound 6 was substantially more potent than 12 (4.40 vs
19.72 μM, Table 1). We attributed this difference to the fact
that this assay was conducted at 4 °C and conversion from 6 to
12 was temperature-dependent. We found that the t1/2 of 6 in
PBS (pH = 7.40) at 4 °C was about 24 h (data not shown).
Therefore, the IC50 of KIX−KID interaction presented in Table
1 for 6 actually represented a mixture of both 6 and 12. The
IC50 discordance between CREB transcription (IC50 = 2.22
μM) and KIX−KID interaction (IC50 = 19.72 μM) for 12
suggests that 12 inhibited CREB’s transcription activity through
a mechanism independent of inhibition of KIX−KID
interaction.
Although 12 was the active species of prodrug 6 under
cellular experimental conditions, it showed considerably
decreased aqueous solubility (∼0.12 mg/mL in H2O). On
the other hand, the prodrug 6 demonstrated excellent aqueous
solubility, and therefore, it was selected for in vivo antitumor
activity study. Preliminary studies showed that compound 6
was well-tolerated in mice receiving daily intravenous injection
of a saline solution at 10 mg/kg, and this dose was chosen for
antitumor efficacy study. To this end, a variant of breast cancer
MCF-7 cells designated as MCF-7ER−, which does not express
estrogen receptor (ER),30 was subcutaneously implanted at the
right flank of BALB/c nude mice. When the tumor sizes
reached ∼100 mm3 on day 16, the tumor-bearing mice were
Figure 5. Distance profile of Ca and Nb in 6 during 1000 ps molecular
dynamics simulation.
in inhibiting CREB’s activity in HEK 293T cells in the CREB
reporter assay. To further confirm their CREB inhibition, the
expression level of endogenous CREB target gene NR4A229
was evaluated using quantitative reverse transcription polymer-
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randomized and treated with either vehicle or a saline solution
of 6 at 10 mg/kg once a day for 28 days. The tumor volumes
from the drug-treated mice were significantly smaller than those
just receiving vehicle starting from 23 days post-treatment
(Figure 7A). At the end of the treatment, the average tumor
the crystal structure of 5. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(X.X.) E-mail: xiaoxi@ohsu.edu. Phone: 1-503-494-4748.
Author Contributions
#These authors (B.X.L. and F.X.) contributed equally to this
work.
Funding
This work was made possible by financial supports from Susan
G. Komen for the Cure (KG100458) and NIH
(R01GM087305).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank CrownBio for the help regarding the in vivo
xenograft studies. We thank Dr. Andrea DeBarber for running
the mass spectroscopic analyses.
ABBREVIATIONS
■
BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; CREB, cyclic-AMP-response element-
binding protein; CBP, CREB-binding protein; DEAD, diethyl
azodicarboxylate; DIPEA, diisoproylethylamine; DMEM, Dul-
becco’s Modified Eagle Medium; ER, estrogen receptor; FBS,
fetal bovine serum; KID, kinase-inducible domain; KIX, KID-
interacting; PBS, phosphate buffered saline; MS, mass spec-
troscopy; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide; NMR, nuclear magnetic resonance; PP1,
protein phosphatase 1; PP2A, protein phosphatase 2A; PTEN,
phosphatase and tensin homologue; qRT-PCR, quantitative
reverse transcription polymerase chain reaction; SD, standard
deviation; t1/2, half-life
Figure 7. In vivo antitumor activity of compound 6 in BALB/c nude
mice with MCF-7ER− xenograft. The female BALB/c nude mice were
xenografted with MCF-7ER− at the right flanks. When the average
tumor volume reached ∼100 mm3 (on day 16), the mice were
randomized to receive either 6 in saline or just saline (n = 8) once a
day for 28 days (qd × 28). The tumor volume (A) and body weight
(B) were measured twice a week during the treatment period. The
student t test was used to evaluate significance. *P < 0.05.
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ASSOCIATED CONTENT
* Supporting Information
Additional synthetic and biological evaluation procedures.
■
S
Detailed compound characterization data and a CIF file for
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