Discovery and Optimization of a Porcupine Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.5b00507

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleate

Full text of DOI:10.1021/acs.jmedchem.5b00507

Article
pubs.acs.org/jmc
Discovery and Optimization of a Porcupine Inhibitor
Athisayamani Jeyaraj Duraiswamy,†,§ May Ann Lee,† Babita Madan,‡ Shi Hua Ang,†
Eldwin Sum Wai Tan,† Wei Wen Vivien Cheong,† Zhiyuan Ke,† Vishal Pendharkar,† Li Jun Ding,†
Yun Shan Chew,† Vithya Manoharan,† Kanda Sangthongpitag,† Jenefer Alam,† Anders Poulsen,†
Soo Yei Ho,*,† David M. Virshup,‡ and Thomas H. Keller†
†Experimental Therapeutics Centre, 31 Biopolis Way, No. 03-01 Nanos, 138669, Singapore
‡Duke-NUS Graduate Medical School Singapore, 8 College Road, 169857, Singapore
S
* Supporting Information
ABSTRACT: Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life.
Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase
that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a
therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt
secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN
inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model.
Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that
such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.
of AXIN, GSK3, APC, and CK1α and translocation of β-
catenin into the nucleus where it forms a complex with LEF/
TCF to drive downstream gene expression.9,10
INTRODUCTION
■
Wnts are secreted glycoproteins that act as growth factors and
regulate various cellular functions1 including proliferation,2
differentiation,3 death,4 migration and polarity,5 through the
activation of multiple intracellular signaling cascades by binding
to various receptors including Frizzleds. There are 19 human
Wnt proteins and 10 Frizzled (Fzd) receptors, all exhibiting
unique expression patterns and serving distinct roles in normal
development and throughout life.6 This complexity has
hampered progress toward viable therapeutics, even though
aberrant Wnt signaling is associated with a number of diseases.
The canonical Wnt-induced activation of the β-catenin-TCF
transcriptional complexes holds promise for drug discovery,
since dysregulation of this pathway has been implicated in
neurological diseases, inflammatory and fibrotic diseases,
metabolic diseases, and a variety of cancers.6
During biosynthesis, Wnt ligands undergo palmitoleation by
Porcupine (PORCN), a membrane-bound O-acyltransferase
enzyme that is specifically required for the acylation of newly
synthesized Wnt ligands in the endoplasmic reticulum (ER).7
Palmitoleated Wnt ligands then undergo glycosylation in the
ER-Golgi system before secretion.8 Once outside the cell, Wnt
glycoproteins bind to receptors formed by Frizzleds and LRP5/
6 co-receptors,9 resulting in phosphorylation of the Dishevelled
protein. In the canonical Wnt pathway this eventually leads to
the disruption of the β-catenin degradation complex consisting
The development of small molecules targeting Wnt-secretion
or Wnt-signaling may be beneficial for the treatment of
cancer11 or fibrotic diseases.6 While a number of tool
compounds are available, clinical candidates have only recently
emerged but none of them have reached the later stages of
clinical development.6 Our approach to disrupt Wnt signaling
was to block the secretion of all Wnt proteins through
inhibition of a key biosynthetic enzyme, Porcupine (PORCN).
Inhibitors of this enzyme have also been pursued by other
research groups, and a few tool compounds that inhibit
PORCN activity with nanomolar activity (e.g., 1 (IWP-L6),12,13
Figure 1) have been reported. Novartis disclosed the PORCN
inhibitors 2 (C59)11 and 3 (LGK974)14 (Figure 1), the latter
having reached the stage of a proof or concept clinical trial
(ClinicalTrials.gov NCT01351103).
Herein, we describe the identification of a Wnt secretion
inhibitor from cellular high throughput screening. The hit was
characterized and optimized to develop a potent PORCN
inhibitor with excellent bioavailability and good in vivo efficacy
Received: March 30, 2015
© XXXX American Chemical Society
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Hydrolysis of the ester group followed by amidation yielded the
chiral alanine derivatives (S)-20 and (R)-20.
Compounds 25−27 were synthesized in racemic form, and
then the enantiomers were separated by chiral HPLC. The
synthesis (Scheme 5) of these three compounds started with
the Suzuki reaction of 2-bromo-5-nitropyridine with 1-methyl-
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
providing the intermediate 21. Then the nitro group was
reduced and the resulting aniline reacted with ethyl 2-
bromopropanoate yielding racemic ester 23. Hydrolysis of the
ester group provided compound 24. Generally amide couplings
between acid 24 and anilines do not proceed well, and it is
often advisible to use the corresponding N-oxides for this
reaction, as demonstrated for the synthesis of 26 and 27
(Scheme 5).
Figure 1. Structures of published PORCN inhibitors and hit 4.
RESULTS AND DISCUSSION
■
in a Wnt-dependent murine mammary cancer model, MMTV-
Wnt1.
Lead Finding and Hit Evaluation. PORCN is a
membrane bound O-acyltransferase that so far has not yielded
a biochemical assay suitable for high throughput screening,
leaving cellular pathway screening as the only alternative. A
HEK293-STF cell line15 that expresses Wnt3A and contains a
luciferase reporter for β-catenin mediated transcriptional
activation was employed for high throughput screening
(HTS) of 226 000 compounds. In order to eliminate hits that
act downstream of surface receptor activation by Wnt (e.g.,
tankyrase inhibitors), a second assay was performed. In this
case a HEK293-STF cell line without Wnt3A expression was
used and screening was done in the presence of Wnt-
conditioned medium16 (HEK293-STF/Wnt) (for a schematic
that explains the screening strategy12 see Supporting
Information Figure S1). Hits that were active in the second
assay were eliminated from consideration and the remaining
compounds examined for general cytotoxicity. Compounds that
passed these hurdles and exhibited IC50’s of <100 nM were
further evaluated for advancement to the hit-to-lead stage (see
Figure S2 for the HTS flowchart). One of the most potent hits
4 displayed an interesting profile with a cellular IC50 of 19 nM
and a molecular weight of 369 g/mol. Physicochemical
parameters were suboptimal (Table 1); however, it was thought
that the low solubility and the cytochrome P450 (Cyp)
inhibition could be improved during the optimization and the
molecular weight suggested that the permeability and
bioavailability issues were likely due to the limited solubility
of the chemical starting point.
CHEMISTRY
■
The achiral Wnt pathway modulators were synthesized as
shown in Scheme 1. [1,1′-Biphenyl]-4-amine was coupled with
chloro- or bromoacetyl chloride in the presence of triethyl-
amine, providing intermediate 5, which was further reacted with
substituted phenols in the presence of K2CO3 to give the final
compounds 4 and 6−10. Substituted aminoacetamide 11 was
synthesized using the same reaction sequence described in
Scheme 1.
The two enantiomers of 14 were synthesized starting with
ethyl lactate (12) (Scheme 2). Mitsunobu reaction with p-
imidazophenol provided the intermediate 13, which after
hydrolysis of the ethyl ester was coupled with [1,1′-
biphenyl]-4-amine. The conformationally restricted proline
derivatives were prepared as described in Scheme 3. (S)-
Proline or (R)-proline were N-arylated with dibromobenzene
in the presence of CuI to yield intermediates 15, which were
coupled with [1,1′-biphenyl]-4-amine giving the amides 16.
Finally copper-catalyzed reaction of the bromo substituent with
imidazole in DMSO provided the chiral proline derivatives (S)-
17 and (R)-17. The chiral integrity of the compounds was
maintained during the last step, despite the relatively strong
basic conditions (see Experimental Section and Supporting
Information Figure S9).
Compound 20 was synthesized in racemic form, and then the
enantiomers were separated by chiral HPLC (Scheme 4). The
reaction of 4-bromoaniline and ethyl 2-bromopropanoate
provided the intermediate 18, which undergoes copper-
catalyzed reaction with imidazole to yield intermediate 19.
A comparison of the low energy conformations of HTS hit 4
and the known PORCN inhibitors 3 and 1 (Figure 2) suggests
that the three inhibitors fit into a common pharmacophore.17
All three compounds are substituted acetamides, and the
a
Scheme 1. Synthesis of Compounds 4 and 6−10
a
Reagents and conditions: (a) NaHCO3, dioxane, chloro- or bromoacetyl chloride, rt, 16 h; (b) substituted phenol, DMF, K2CO3, NaI, rt, 16 h.
B
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a
Scheme 2. Synthesis of the R- and S-Enantiomers of 14
a
Reagents and conditions: (a) p-imidazophenol, diisopropyl azodicarboxylate, Ph3P, THF, rt, 16 h; (b) LiOH, MeOH, THF, water, rt, 4 h; (c) [1,1′-
biphenyl]-4-amine, HATU, (iPr)2NEt, rt, 16 h.
a
Scheme 3. Synthesis of the Proline Derivatives (R)-17 and (S)-17
a
Reagents and conditions: (a) p-dibromobenzene, CuI, K2CO3, 140 °C, 16 h; (b) [1,1′-biphenyl]-4-amine, HATU, (iPr)2NEt, rt, 16 h; (c) CuI,
imidazole, Cs2CO3, DMSO, 90 °C, 16 h.
a
Scheme 4. Synthesis of the Alanine Derivatives (R)-20 and (S)-20
a
Reagents and conditions: (a) K2CO3, 70 °C, 16 h; (b) imidazole, CuI, L-proline, Cs2CO3, 90 °C, 16 h; (c) LiOH, 1:1:1 THF−H2O−MeOH, rt, 2 h;
(d) 5-phenylpyridin-2-amine, HATU, N-methylmorpholine, DMF, rt, 16 h.
pharmacophore elements of the biaryl and heterobiaryl
substituents on the amide overlap very well (see Supporting
Information Figure S6). The similarity of the substituents on
the -CH2- group is less obvious, but an analysis of the
pharmacophore model17 in Figure 2 suggests that the terminal
pyridine nitrogen of 3 and the imidazole nitrogen of 4 serve as
hydrogen bond acceptors in PORCN, while the heterocyclic
carbonyl atom of 1 likely uses a water molecule (denoted by the
plum pharmacophore element in Figure 2) to make the same
interaction with the PORCN protein.
To confirm the hypothesis that 4 is a PORCN inhibitor, a
cellular assay was developed to determine whether 4 can inhibit
the palmitoleation of Wnt proteins. In this assay exogenous V5-
tagged Wnt3a18 is overexpressed in HeLa cells and cultured in
medium containing hexadec-15-ynoic acid (ω-alkynyl palmi-
tate). When the cells are cultured, the PORCN acylates the V5-
tagged Wnt3a with ω-alkynyl palmitate, and this modified
protein can then be reacted (in the cell lysate) with biotin azide
through copper-catalyzed azide−alkyne cycloaddition.19 The
total Wnt3a or palmitoleated Wnt3a can then be detected using
an anti-V5 antibody or a streptavidin conjugated dye,
respectively. Figure 3 depicts the Western blot demonstrating
that 4 blocks the palmitoleation of Wnt3A, consistent with its
inhibition of PORCN activity.
Lead Optimization. Having established that 4 is a PORCN
inhibitor, the initial stages of the optimization were focused on
the imidazole substituent that likely is responsible for the
potent Cyp inhibition (Table 2). Nitrogen heterocycles often
interact with the heme active site of the Cyp,20 leading to
potent inhibition. The emerging structure−activity relationship
(SAR) clearly indicated that the terminal nitrogen is essential
for activity in our assay (e.g., 6). This is also supported by our
pharmacophore model17 (Figure 2). Nevertheless it was
possible to remove the undesired activity on the Cyp enzymes
by modulating the steric and electronic environment around
the terminal nitrogen. Both the oxazole 7 and the N-
C
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a
Scheme 5. Synthesis of Compounds 25, 26, and 27
Figure 2. Putative bioactive conformation of 4 (gray carbon), 3 (green
carbon), and 1 (plum carbon) superimposed. A site point (plum ball)
was constructed 2.8 Å from the oxygen atom (red) in the direction of
the carbonyl lone pair on 1. The site point overlays well with the
pyridine acceptor of 3 and imidazole acceptor of 4.17
a
Reagents and conditions: (a) 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole, Pd(PPh3)4, K3PO4, 100 °C, 16 h; (b)
H2, 10% Pd/C, EtOAc, rt, 12 h; (c) ethyl bromopropionate, NaOAc,
ACN, reflux, 48 h; (d) LiOH, 1:1:1 THF−H2O−MeOH, rt, 2 h; (e)
2,3′-bipyridin-5-amine, HATU, DIPEA, rt, 16 h; (f) 2-amino-5-
phenylpyridine 1-oxide, HATU, DIPEA, rt, 16 h; (g) Fe, AcOH, reflux,
2 h; (h) 2-amino-5-bromopyridine 1-oxide, HATU, DIPEA, DMF, rt,
16 h; (i) pyridine-3-boronic acid, Pd(dppf)Cl2.DCM, K3PO4, 4:1
dioxane−H2O, 100 °C, 16 h.
however we observed a clear effect on the biological activity.
With the lactate linker (14, Table 3) the S-enantiomer is about
20-fold more active than the R-enantiomer, an effect that could
also be observed in the palmitoleation assay (Figure 3). The
chiral discrimination is more pronounced with the more rigid
proline derivatives (17) leading to a >500-fold difference in
biological activity again in favor of the S-enantiomer (Table 3).
Since the introduction of a chiral center did not have the
desired effect, the decision was taken to reduce the clogP,
which is 4.5 for (S)-14. As a first step we synthesized (S)-20
which exhibited a clogP of 3.1. Gratifyingly potency was
retained (Table 3) and both microsomal stability and solubility
(Supporting Information Table S1) were improved. At this
point of the optimization we introduced the N-methylpyrazole
(see Table 2) into the scaffold and further reduced the clogP
with the judicious introduction of further aromatic nitrogens
(Table 4). Overall the introduction of the nitrogen atoms led to
a sizable reduction in clogP, providing compounds with good
solubility and good overall physical properties (Table 4).
Compound (S)-27, with a molecular weight of 400 g/mol,
exhibits both good solubility and permeability, suggesting that
the low PAMPA value and the limited bioavailability of hit 4
might have been caused by the low solubility. The optimized
inhibitor (S)-27 displays good in vivo pharmacokinetics as
shown in Table 5 (see also Supporting Information Figure S7).
As discussed previously, the hit evaluation suggested that the
chemical starting point 4 is a PORCN inhibitor, and Figure 3
shows that the optimization did not affect the mechanism of the
compound. (S)-27 inhibits the palmitoleation of Wnt proteins
(Figure 3) with similar potency as the literature compound 2.11
Further evidence that (S)-27 is a potent PORCN inhibitor was
provided by a cell line overexpressing this Wnt acyltransferase.
Table 1. Profile of HTS Hit 4
assay/property
value
a
b
HEK293-STF (μM)
0.019
a
b
HEK293-STF/Wnt (μM)
>30
>50
a
b
CC50 (μM)
molecular weight (g/mol)
solubility (μg/mL)
PAMPA, pH 7.4 (10−6 cm/s)
369.4
<1
c
<1
d
CYP 3A4, 2D6 inhibition (μM)
0.02/0.1
c
mouse oral bioavailability (%)
<1
a
b
c
See Experimental Section. Average of three determinations. May
d
have been impacted by poor solubility. Cytochrome P450 enzyme.
methylpyrazole 10 were devoid of activity on the Cyp enzymes,
but the latter was clearly more attractive with single digit
nanomolar activity in the Wnt secretion assay. While the
discussed modifications (Table 2) improved the side effect
profile of the scaffold, solubility was not impacted by these
changes (see Supporting Information Table S1).
Two strategies can be considered to improve solubility in
drug candidates: (a) reduce the lipophilicity of the compounds;
(b) disrupt solid state interactions that are the cause of low
intrinsic solubility.21 During the optimization, we attempted to
disrupt crystal packing interactions via the addition of
substitutuents around the linker (Table 3).
In the present case, the chiral center had no influence on the
solubility at pH 7.4 (see Supporting Information Table S1);
D
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Figure 3. Inhibition of Wnt palmitoleation by Wnt secretion inhibitors in HeLa cells. (a) Total V5-Wnt3A visualized using an anti-V5 antibody
followed by anti-mouse Dylight 680. (b) Palmitoleated V5-Wnt3A is reacted in the cell lysate with biotin azide and then detected with streptavidine-
Dylight 800.
Table 2. Imidazole SAR
Table 3. Optimization of the Glycolate Linker
a
b
Data represent mean value of triplicate experiments. n.d. = not
determined.
For this experiment wild type HT1080 cells were treated with
either a control plasmid or a murine PORCN expression
plasmid in combination with a Wnt/β-catenin reporter plasmid
and Wnt3a. Treatment of the wild-type cells with (S)-27 led to
a dose dependent inhibition of the luciferase activity, while cells
overexpressing PORCN blunted the effect of the inhibitor
(Figure 4).
a
b
Data represent mean value of triplicate experiments. Mouse liver
c
microsomes. Not determined.
The in vivo efficacy of (S)-27 was tested in a well-established
Wnt-driven murine tumor model. For this purpose MMTV-
Wnt1 tumor fragments were implanted in the mammary fat
pads of Balb/c nude mice. After 14 days, mice bearing
established MMTV-Wnt1 tumors were treated with vehicle or
three different doses of (S)-27 by oral gavage. For comparison
one group of animals received 3 mg/kg of 2, a compound that
previously had been shown to prevent tumor growth in this
animal model.11 Figure 5 shows that (S)-27 inhibited tumor
growth in a dose-dependent manner and with similar potency
as 2 (for a comparison of the in vitro pharmacokinetic profile of
2 and (S)-27 see Supporting Information Table S3). The
percentage of tumor growth inhibition was statistically
significant at all three doses and ranged from 50% to 91%
(Figure 5).
CONCLUSION
■
Modulation of aberrant Wnt signaling is becoming a very
attractive drug discovery strategy for cancer, vascular disease,
and tissue fibrosis. Unfortunately, the progress toward viable
therapeutics has been rather slow, mainly because of the
complexity of Wnt signaling and the dearth of tool compounds
to dissect the pathway.
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Table 4. Optimization of Physical Properties
a
b
Determined using Stardrop. Data represent mean value of triplicate
c
d
experiments. Thermodynamic solubility at pH 7.4. Determined at
pH 7.4. Mouse liver microsomes.
e
Figure 5. (S)-27 inhibits the growth of MMTV-Wnt1 tumors. Tumor
growth inhibition was 50% at 1 mg/kg, 71% at 3 mg/kg, and 91% at 10
mg/kg. Tumor growth inhibition compared with vehicle was
statistically significant for all doses with a p value of <0.0001.
a
Table 5. In Vivo Pharmacokinetics for (S)-27
F (%)
Cmax (ng/mL)
T1/2 (h)
AUC0−t(last) (ng·h/mL)
(S)-27
106
4268
1.9
12266
useful for mode of action studies on the PORCN O-
acyltransferase.
a
Mouse. Dose of 1 mg/kg for iv studies and 5 mg/kg for po studies
(see Experimental Section and Supporting Information Table S2).
EXPERIMENTAL SECTION
■
General. All reagents were purchased from commercial sources and
used as received. Reaction progress was monitored by TLC using
Merck silica gel 60 F254 on glass plates with detection by UV at 254
nm. LC−MS analysis was carried out with Shimadzu LC-20AD and
LCMS-2020 instruments. The column used was a Phenomenex
Kinetex (2.6 μm, 50 mm × 2.10 mm). Proton nuclear magnetic
resonance (1H NMR) spectra were obtained using a Bruker
Ultrashield 400 PLUS/R system, operating at 400 MHz. All resonance
bands were referenced to tetramethylsilane (internal standard).
Splitting patterns are indicated as follows: s, singlet; d, doublet; t,
triplet; m, multiplet; br, broad peak. The compounds’ purities were
≥95% determined by a VARIAN ProStar HPLC instrument.
Figure 4. Effect of (S)-27 on HT1080 cells treated with control
plasmid in combination with Wnt/β-catenin reporter plasmid and
Wnt3a (black bars) and effect of (S)-27 on HT1080 cells treated with
murine PORCN expression plasmid in combination with Wnt/β-
catenin reporter plasmid and Wnt3a (gray bars).
Thermodynamic Solubility Studies. A 6 μL volume of 50 mM
DMSO stock from the stock plate is added to the reaction deep well
plate containing 600 μL of pH 7.4 or pH 4.0 pION buffer, mixed, and
incubated for 18 h. The plate is sealed well during the incubation
process. The test compound concentration is 500 μM. The DMSO
content in the sample was 1.0%. At the end of the incubation period,
100 μL of sample from the storage plate is vacuum filtered using a
filter plate. This step wets the filters, and the filtrate is discarded.
Another 200 μL of the sample from the deep well plate is vacuum
filtered using the same filter block but a clean filter plate. A 75 μL
volume of the filtrate from the filter plate is transferred to a UV sample
plate. A 75 μL volume of 1-propanol is added to this UV plate. The
solution is mixed, and the spectrum is read using the UV
spectrophotometer (Spectramax-Molecular Devices). The analysis is
carried out by using pION μSOL EXPLORER software, version 3.3.
N-([1,1′-Biphenyl]-4-yl)-2-chloroacetamide (5). To biphenyl-4-
amine (500 mg, 2.95 mmol) in anhydrous DCM (15 mL) at 0 °C were
added triethylamine (495 μL, 3.54 mmol) and 2-chloroacetyl chloride
(282 μL, 1.418 mmol) dropwise. The reaction mixture was allowed to
warm to room temperature and stirred for 2 h, and then it was diluted
with H2O (30 mL) and extracted with DCM (3 × 15 mL). The
combined organic layer was dried over Na2SO4 and was concentrated
under reduced pressure. The crude product 5 (600 mg, brown solid)
was used without further purification. 1H NMR (400 MHz, DMSO-d6)
Our approach to disrupt Wnt signaling involves blocking the
secretion of Wnt proteins through inhibition of a key
biosynthetic enzyme. High throughput screening of a β-catenin
reporter assay followed by the removal of compounds that work
downstream of the Wnt binding to Frizzled receptor and its co-
receptors provided a hit that was shown to be a PORCN
inhibitor. Classical SAR based cellular optimization led to a
compound that inhibits Wnt secretion with nanomolar potency.
Undesirable properties of the original hit, namely, limited
solubility and Cyp inhibition, were removed through targeted
modification of substituents and modulation of the overall
lipophilicity, providing us with a PORCN inhibitor with
excellent bioavailability and potent activity in a Wnt-driven
murine tumor model. Finally we also discovered that
enantiomeric PORCN inhibitors show very different activity
in our reporter assay, suggesting that such compounds may be
F
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δ 10.38 (s, 1H), 7.70−7.63 (m, 6H), 7.46−7.42 (m, 2H), 7.35−7.31
(m, 1H), 4.27 (s, 2H); MS (ESI) m/z 246 [C14H12ClNO + H]+.
General Synthesis Route for Compounds 6−10. To a solution
of N-([1,1′-biphenyl]-4-yl)-2-chloroacetamide (0.316 mmol) and the
respective phenols (0.316 mmol) in DMF (2 mL) was added K2CO3
(153 mg, 1.105 mmol). The solution was heated at 70 °C for 16 h, and
then it was diluted with H2O (25 mL) and extracted with EtOAc (3 ×
25 mL). The combined organic layer was dried over Na2SO4 and was
concentrated under reduced pressure to afford the crude product. The
crude residue was purified by preparative HPLC (C18, eluent ACN,
water, formic acid 0.1%) to afford the purified product.
2-(4-(1H-Imidazol-1-yl)phenoxy)-N-([1,1′-biphenyl]-4-yl)-
acetamide (4). 4 was prepared according to the general procedure
using commercially available 4-(1H-imidazol-1-yl)phenol and N-
([1,1′-biphenyl]-4-yl)-2-chloroacetamide to give a white solid (yield,
31%). 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.13 (s, 1H),
7.77−7.72 (m, 2H), 7.68−7.61 (m, 5H), 7.61−7.55 (m, 2H), 7.48−
7.41 (m, 2H), 7.36−7.30 (m, 1H), 7.18−7.12 (m, 2H), 7.08 (s, 1H),
4.79 (s, 2H); MS (ESI) m/z 370 [C23H19N3O2 + H]+. Melting point:
206−208 °C.
126.23, 123.22, 120.00, 114.93, 105.36, 67.17, 37.33; MS (ESI) m/z
384 [C24H21N3O2 + H]+. Melting point: 166−168 °C.
2-((4-(1H-Imidazol-1-yl)phenyl)amino)-N-([1,1′-biphenyl]-4-
yl)acetamide (11). 11 was synthesized according to the general
procedure using 4-(1H-imidazol-1-yl)aniline and N-([1,1′-biphenyl]-4-
1
yl)-2-chloroacetamide to give a white solid (yield, 45%). H NMR
(400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.01 (s, 1H), 7.74−7.68 (m,
2H), 7.67−7.59 (m, 4H), 7.52 (s, 1H), 7.44 (t, J = 7.2 Hz, 2H), 7.37−
7.29 (m, 3H), 7.04 (s, 1H), 6.72 (d, J = 8.8 Hz, 2H), 6.28 (t, J = 6.4
Hz, 1H), 3.94 (d, J = 6.0 Hz, 2H); MS (ESI) m/z 368.9 [C23H20N4O
+ H]+. Melting point: 230−232 °C.
(S)-2-(4-(1H-Imidazol-1-yl)phenoxy)-N-(biphenyl-4-yl)-
propanamide ((S)-14). To a solution of (+)-R-ethyl lactate (3.12
mmol) in THF (250 mL) were added DIAD (3.44 mmol) and TPP
(3.12 mmol) at room temperature, and the reaction mixture was
stirred for 16 h. The reaction mixture was poured into ice−water (100
mL) and extracted with EtOAc (2 × 100 mL). The combined organics
were washed with water, brine, dried over Na2SO4, filtered, and
concentrated to give crude product. The crude product was washed
with diethyl ether several times to remove TPPO. The combined
organics were concentrated to give (S)-ethyl 2-(4-(1H-imidazol-1-
yl)phenoxy)propanoate as a crude product. MS (ESI) m/z 261.12
[C14H16N2O3 + H]+.
2-(4-(1H-Pyrrol-1-yl)phenoxy)-N-(biphenyl-4-yl)acetamide
(6). 6 was prepared according to the general procedure using
commercially available 4-(1H-pyrrol-1-yl)phenol and N-([1,1′-biphen-
yl]-4-yl)-2-chloroacetamide to give a beige solid (yield, 65%). 1H
NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 7.76−7.74 (m, 2H),
7.66−7.64 (m, 4H), 7.52−7.42 (m, 4H), 7.35−7.31 (m, 1H), 7.25 (t, J
= 2.0 Hz, 2H), 7.11−7.08 (m, 2H), 6.22 (t, J = 2.0 Hz, 2H), 4.75 (s,
2H); 13C NMR (400 MHz, DMSO-d6) δ 166.53, 155.45, 139.59,
137.80, 135.36, 134.14, 128.87, 127.06, 126.90, 126.25, 120.84, 120.04,
119.06, 115.62, 109.93, 67.48; MS (ESI) m/z 369 [C24H20N2O2 +
H]+. Melting point: 248−250 °C.
To a solution of (S)-ethyl 2-(4-(1H-imidazol-1-yl)phenoxy)-
propanoate crude product (35.02 mmol) in 30 mL of THF−
MeOH−H2O was added LiOH (4.62 mmol) at rt. The reaction
mixture was stirred at rt for 4 h. The reaction mixture was
concentrated and diluted with water. The aqueous layer was acidified
with sat. KHSO4 solution and concentrated to give crude material
which was extracted with 10% MeOH in CHCl3 (3 × 100 mL). The
combined organics were dried over Na2SO4, filtered, and concentrated
to give (S)-2-(4-(1H-imidazol-1-yl)phenoxy)propanoic acid (S)-13
N-(Biphenyl-4-yl)-2-(4-(oxazol-5-yl)phenoxy)acetamide (7).
7 was prepared according to the general procedure using commercially
available 4-(oxazol-5-yl)phenol and N-([1,1′-biphenyl]-4-yl)-2-chlor-
1
(yield, 31%). H NMR (400 MHz, DMSO-d6) δ 8.5 (s, 1H), 8.06 (s,
1H), 7.58 (s, 1H), 7.42 (d, J = 9.2 Hz, 2H), 7.04 (s, 1H), 6.90 (d, J =
8.7 Hz, 2H), 4.30 (m, 1H) 2.50 (d, J = 1.8 Hz, 3H); MS (ESI) m/z
234.2 [C12H12N2O3 + H]+.
1
oacetamide to give an off-white solid (yield, 66%). H NMR (400
MHz, DMSO-d6) δ 10.20 (s, 1H), 8.38 (s, 1H), 7.77−7.72 (m, 2H),
7.72−7.67 (m, 2H), 7.67−7.62 (m, 4H), 7.55 (s, 1H), 7.48−7.41 (m,
2H), 7.36−7.30 (m, 1H), 7.16−7.10 (m, 2H), 4.79 (s, 2H); MS (ESI)
m/z 371 [C23H18N2O3 + H]+. Melting point: 244−246 °C.
(S)-13 (0.65 mmol) was taken up in DMF (20 mL). DIPEA (1.96
mmol), HATU (1.30 mmol), and biphenyl-4-amine (0.65 mmol) were
added to the reaction mixture. The reaction mixture was stirred for 16
h at room temperature. Upon completion, the reaction mixture was
poured into ice−water (100 mL) and stirred for 30 min. The
precipitated solid was filtered, washed with water, and dried to afford
(S)-2-(4-(1H-imidazol-1-yl)phenoxy)-N-(biphenyl-4-yl)propanamide
(S)-14 as an off white solid (yield, 24%). 1H NMR (400 MHz,
DMSO-d6) δ 10.27 (s, 1H), 8.13 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H),
7.64 (m, 5H), 7.57 (d, J = 8.4 Hz, 2H), 7.44 (t, J = 7.25 Hz, 2H),
7.34−7.31 (m, 1H) 7.11 (d, J = 8.3 Hz, 3H), 4.97 (d, J = 6.6 Hz, 1H),
1.6 (d, J = 6.6 Hz, 3H); MS (ESI) m/z 384.14 [C24H21N3O2 + H]+.
Melting point: 238−240 °C.
2-(4-(1H-Pyrazol-3-yl)phenoxy)-N-(biphenyl-4-yl)acetamide
(8). 8 was prepared according to the general procedure using
commercially available 4-(1H-pyrazol-3-yl)phenol and N-([1,1′-
biphenyl]-4-yl)-2-chloroacetamide to give an off-white solid (yield,
24%). 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.44 (s, 1H),
7.76 (d, J = 2.4 Hz, 1H), 7.71−7.58 (m, 8H), 7.44 (t, J = 7.6 Hz, 2H),
7.32 (t, J = 7.6 Hz, 1H), 6.78−6.76 (m, 2H), 6.59 (d, J = 2.4 Hz, 1H),
5.04 (s, 2H); 13C NMR (400 MHz, DMSO-d6) δ 165.54, 156.87,
150.67, 139.50, 137.99, 135.17, 133.07, 128.79, 126.97, 126.94, 126.33,
126.16, 124.39, 119.48, 115.25, 101.75, 54.55; MS (ESI) m/z 370
[C23H19N3O2 + H]+. Melting point: 213−215 °C.
(R)-2-(4-(1H-Imidazol-1-yl)phenoxy)-N-(biphenyl-4-yl)-
propanamide ((R)-14). (R)-14 was synthesized similarly to (S)-14
using (−)-S-ethyl lactate as the starting material to afford an off white
N-(Biphenyl-4-yl)-2-(4-(2-methyl-1H-imidazol-1-yl)-
phenoxy)acetamide (9). 9 was prepared according to the general
procedure using commercially available 4-(2-methyl-1H-imidazol-1-
yl)phenol and N-([1,1′-biphenyl]-4-yl)-2-bromoacetamide to give a
1
solid (yield, 28%). H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H),
8.13 (s, 1H), 7.80−7.56 (m, 9H), 7.44 (t, J = 7.25 Hz, 2H), 7.34−7.32
(m, 1H) 7.11 (d, J = 8.3 Hz, 3H), 4.97 (d, J = 6.5 Hz, 1H), 1.6 (d, J =
5.7 Hz, 3H); MS (ESI) m/z 384.4 [C24H21N3O2 + H]+. Melting point:
169−171 °C.
1
white solid (yield, 14%). H NMR (400 MHz, DMSO-d6) δ 10.23 (s,
1H), 7.76−7.74 (m, 2H), 7.66−7.64 (m, 4H), 7.46−7.31 (m, 5H),
7.20 (d, J = 1.6 Hz, 1H), 7.15−7.13 (m, 2H), 6.87 (d, J = 1.6 Hz, 1H),
4.80 (s, 2H), 2.24 (s, 3H); 13C NMR (400 MHz, DMSO-d6) δ 166.28,
157.19, 143.64, 139.52, 137.73, 135.31, 131.05, 128.80, 127.00, 126.87,
126.84, 126.51, 126.18, 120.93, 119.95, 115.30, 67.27, 13.31; MS (ESI)
m/z 384 [C24H21N3O2 + H]+. Melting point: 184−186 °C.
N-(Biphenyl-4-yl)-2-(4-(1-methyl-1H-pyrazol-5-yl)phenoxy)-
acetamide (10). 10 was prepared according to the general procedure
using commercially available 4-(1-methyl-1H-pyrazol-5-yl)phenol and
N-([1,1′-biphenyl]-4-yl)-2-chloroacetamide to give a white solid
(yield, 91%). 1H NMR (400 MHz, DMSO-d6) δ 10.24 (bs, 1H),
7.78−7.73 (m, 2H), 7.68−7.62 (m, 4H), 7.52−7.41 (m, 5H), 7.37−
7.30 (m, 1H), 7.15−7.10 (m, 2H), 6.33 (d, J = 1.6 Hz, 1H), 4.80 (s,
2H), 3.83 (s, 3H); 13C NMR (400 MHz, DMSO-d6): 166.39, 157.83,
142.32, 139.58, 137.80, 137.71, 135.34, 129.75, 128.84, 127.04, 126.88,
(R)-1-(4-Bromophenyl)pyrrolidine-2-carboxylic Acid ((R)-15).
To a solution of 1,4-dibromobenzene (8.47 mmol) in 50 mL of DMF
were added CuI (1.69 mmol), K2CO3 (25.43 mmol), and (R)-proline
(9.32 mmol) at room temperature under argon. The reaction mixture
was heated at 140 °C for 16 h. Upon completion, the reaction mixture
was cooled to room temperature, poured into ice cold water (500
mL), and extracted with EtOAc (2 × 100 mL). The aqueous layer was
acidified with sat. KHSO4 solution and then extracted with EtOAc (2
× 100 mL). The combined organic layer was washed with water
followed by brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure to afford (R)-1-(4-bromophenyl)pyrrolidine-2-
1
carboxylic acid (R)-15 (yield, 54%). H NMR (400 MHz, DMSO-
d6) δ 12.90 (s, 1H), 7.95 (br s, 2H), 7.30 (br s, 2H), 4.01 (m, 1H),
G
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Journal of Medicinal Chemistry
Article
3.32 (m, 2H), 2.05 (m, 4H); MS (ESI) m/z 271.8 [C11H12BrNO2 +
H]+. MS (ESI) m/z 271.8 [C11H12BrNO2 + H]+.
7.20 (d, J = 6.8 Hz, 2H), 6.50 (d, J = 6.8 Hz, 2H), 6.19 (d, J = 8.4 Hz,
1H), 4.11−3.99 (m, 3H), 1.36 (d, J = 6.8 Hz, 3H), 1.15 (t, J = 6.8 Hz,
3H); MS (ESI) m/z 204.9 [C11H14BrNO2 + H]+.
(S)-1-(4-Bromophenyl)pyrrolidine-2-carboxylic Acid ((S)-15).
(S)-15 was synthesized similarly using 1,4-dibromobenzene and (S)-
proline (yield, 54%). 1H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H),
7.95 (br s, 2H), 7.30 (br s, 2H), 4.01 (m, 1H), 3.32 (m, 2H), 2.05 (m,
4H); MS (ESI) m/z 271.8 [C11H12BrNO2 + H]+.
Ethyl 2-(4-(1H-Imidazol-1-yl)phenylamino)propanoate (19).
Imidazole (2.9 mmol), ethyl 2-(4-bromophenylamino)propanoate 18
(1.5 mmol), CuI (0.15 mmol), L-proline (2.9 mmol), and Cs2CO3 (2.9
mmol) were dissolved in DMSO (3.5 mL). The reaction mixture was
heated at 90 °C for 16 h, and then it was diluted with water and
extracted with EtOAc. The combined organic extracts were
concentrated and the residue was purified by column chromatography
to afford the desired product as yellow oil (yield, 67%). 1H NMR (400
MHz, DMSO-d6) δ 7.98 (s, 1H), 7.50 (s, 1H), 7.30 (d, J = 6.8 Hz,
2H), 7.02 (s, 1H), 6.64 (d, J = 6.8 Hz, 2H), 6.20 (d, J = 8.4 Hz, 1H),
4.13−4.07 (m, 3H), 1.39 (d, J = 6.8 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H);
MS (ESI) m/z 260 [C14H17N3O2 + H]+.
(R)-N-(Biphenyl-4-yl)-1-(4-bromophenyl)pyrrolidine-2-car-
boxamide ((R)-16). To a solution of (R)-1-(4-bromophenyl)-
pyrrolidine-2-carboxylic acid (R)-15 (4.46 mmol) in DMF (25 mL)
were added DIPEA (13.38 mmol), HATU (8.92 mmol), and biphenyl-
4-amine (4.46 mmol), and the reaction mixture was stirred at room
temperature for 16 h. After completion, the reaction mixture was
poured into ice−water (100 mL) and stirred for 30 min. The
precipitated solid was filtered and washed with water and dried to
afford (R)-N-(biphenyl-4-yl)-1-(4-bromophenyl)pyrrolidine-2-carbox-
2-(4-(1H-Imidazol-1-yl)phenylamino)-N-(5-phenylpyridin-2-
yl)propanamide (20). To ethyl 2-(4-(1H-imidazol-1-yl)-
phenylamino)propanoate 19 0.96 mmol) in H2O (1 mL) and THF
(1 mL) was added lithium hydroxide (1.9 mmol). The reaction
mixture was stirred at room temperature for 1 h and was then acidified
using 1 M HCl solution. Solvent was then evaporated in vacuo, and
the crude residue was used without further purification. 1H NMR (400
MHz, DMSO-d6) δ 9.54 (s, 1H), 8.11 (s, 1H), 7.85 (s, 1H), 7.47 (d, J
= 9.2 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 4.06 (q, J = 7.2 Hz, 1H), 1.40
(d, J = 6.8 Hz, 3H); MS (ESI) m/z 232 [C12H13N3O2 + H]+.
To the crude material (0.48 mmol) in DMF (2.5 mL) were added
HATU (0.72 mmol) and N-methylmorpholine (1.92 mmol). The
reaction mixture was stirred at room temperature under inert
atmosphere for 1 h, followed by the addition of 5-phenylpyridin-2-
amine (0.72 mmol). The reaction mixture was left to stir for 16 h, and
then it was diluted with H2O and extracted with EtOAc. The
combined organic layer was dried over Na2SO4 and was concentrated
under reduced pressure. The crude residue was purified by column
chromatography to afford racemate compound 20 as an off-white solid
(yield, 10%). Isomers were separated by chiral HPLC (CHIRALPAK
ASH column; hexane/EtOH/isopropyamine 20:80:0.1) to afford the
required compound (S)-20 (yield, 35%) and (R)-20 (yield, 32%).
1
amide (R)-16 (yield, 41%). H NMR (400 MHz, DMSO-d6) δ 10.08
(s, 1H), 7.70−7.68 (m, 2H), 7.64−7.60 (m, 4H), 7.44 (t, J = 7.7 Hz,
2H), 7.34−7.30 (m, 3H), 6.50−6.48 (d, J = 8.8 Hz, 2H), 4.23−4.20
(d, J = 8.4 Hz, 1 H), 3.60 (m, 1H), 3.32 (m, 1H), 1.40−1.25 (m, 1H),
2.08−2.00 (m, 3H); MS (ESI) m/z 421.09 [C23H21BrN2O + H]+.
(S)-N-(Biphenyl-4-yl)-1-(4-bromophenyl)pyrrolidine-2-car-
boxamide ((S)-16). (S)-16 was synthesized similarly via amide
coupling between (S)-1-(4-bromophenyl)pyrrolidine-2-carboxylic acid
(S)-15 and biphenyl-4-amine (yield, 54%). 1H NMR (400 MHz,
DMSO-d6) δ 10.08 (s, 1H), 7.70−7.68 (m, 2H), 7.64−7.60 (m, 4H),
7.44 (t, J = 7.7 Hz, 2H), 7.34−7.30 (m, 3H), 6.50−6.48 (d, J = 8.8 Hz,
2H), 4.23−4.20 (d, J = 8.4 Hz, 1 H), 3.60 (m, 1H), 3.32 (m, 1H),
1.40−1.25 (m, 1H), 2.08−2.00 (m, 3H); MS (ESI) m/z 421.09
[C23H21BrN2O + H]+.
(R)-1-(4-(1H-Imidazol-1-yl)phenyl)-N-(biphenyl-4-yl)-
pyrrolidine-2-carboxamide ((R)-17). Imidazole (1.227 mmol), (R)-
N-(biphenyl-4-yl)-1-(4-bromophenyl)pyrrolidine-2-carboxamide (R)-
16 (0.356 mmol), CuI (0.036 mmol), L-proline (0.712 mmol), and
Cs2CO3 (0.712 mmol) were dissolved in DMSO (2 mL). The reaction
mixture was heated at 90 °C for 16 h. After completion, it was diluted
with water (25 mL) and extracted with EtOAc (25 mL × 3). The
combined organic extracts were concentrated and the residue was
purified by preparative HPLC (C18, eluent ACN, water, formic acid
0.1%) to afford (R)-1-(4-(1H-imidazol-1-yl)phenyl)-N-(biphenyl-4-
(S)-20. [α]20 −61.6 (c 0.26, MeOH). 1H NMR (400 MHz,
D
DMSO-d6) δ 10.59 (s, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.17−8.15 (m,
1H), 8.10−8.07 (m, 1H), 7.97 (s, 1H), 7.70−7.68 (m, 2H), 7.50−7.45
(m, 3H), 7.40−7.31 (m, 3H), 7.00 (s, 1H), 6.73 (d, J = 8.8 Hz, 2H),
6.25 (d, J = 8.4 Hz, 1H), 4.31−4.23 (m, 1H), 1.44 (d, J = 6.8 Hz, 3H);
MS (ESI) m/z 384 [C23H21N5O + H]+. Melting point: 88−90 °C.
yl)pyrrolidine-2-carboxamide (R)-17 as brown solid (yield, 29%).
1
[α]20 158 (c 0.1, MeOH). H NMR (400 MHz, CDCl3) δ 8.28 (s,
D
1H), 7.76 (br s, 1H), 7.59−7.53 (m, 6H), 7.41 (t, J = 7.2 Hz, 2H),
7.34−7.25 (m, 3H), 7.25−7.20 (m, 2H), 6.79 (d, J = 8.8 Hz, 2H),
4.15−4.12 (m, 1H), 3.84−3.80 (m, 1H), 3.37−3.30 (m, 1H), 2.44−
2.39 (m, 2H), 2.16−2.08 (m, 2H); 13C NMR (400 MHz, CDCl3) δ
171.59, 146.95, 140.39, 137.69, 136.40, 128.79, 127.64, 127.22, 126.85,
123.44, 120.26, 114.14, 65.39, 50.34, 31.68, 24.35; MS (ESI) m/z 409
[C26H24N4O+ H]+. Melting point: 182−184 °C.
(S)-1-(4-(1H-Imidazol-1-yl)phenyl)-N-(biphenyl-4-yl)-
pyrrolidine-2-carboxamide ((S)-17). (S)-17 was synthesized
similarly via coupling between (S)-N-(biphenyl-4-yl)-1-(4-
(R)-20. [α]20 78.2 (c 0.26, MeOH).1H NMR (400 MHz, DMSO-
D
d6) δ 10.59 (s, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.17−8.15 (m, 1H),
8.10−8.07 (m, 1H), 7.97 (s, 1H), 7.70−7.68 (m, 2H), 7.50−7.45 (m,
3H), 7.40−7.31 (m, 3H), 7.00 (s, 1H), 6.73 (d, J = 8.8 Hz, 2H), 6.25
(d, J = 8.4 Hz, 1H), 4.31−4.23 (m, 1H), 1.44 (d, J = 6.8 Hz, 3H); MS
(ESI) m/z 384 [C23H21N5O + H]+. Melting point: 98−100 °C.
2-(1-Methyl-1H-pyrazol-5-yl)-5-nitropyridine (21). To a well
stirred solution of 2-bromo-5-nitropyridine (4.9 mmol) in dioxane−
water (4:1, 50 mL) were added K3PO4 (9.8 mmol), 1-methyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (4.9 mmol),
and Pd(PPh3)4 (0.25 mmol) under Ar, and the reaction mixture was
heated at 100 °C for 16 h. The reaction mixture was evaporated under
reduced pressure. The residue was poured into ice−water, and the
compound was extracted with EtOAc (3 × 50 mL). The combined
organic layer was washed with ice−water, brine, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The crude
material was purified by column chromatography (silica gel, eluent
EtOAc/hexane 25:75) to afford 2-(1-methyl-1H-pyrazol-5-yl)-5-nitro-
pyridine 21 (yield, 85%). 1H NMR (400 MHz, DMSO-d6) δ 9.46 (d, J
= 2.8 Hz, 1H), 8.67 (dd, J = 2.4, 8.8 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H),
7.59 (d, J = 2 Hz, 1H), 9.09 (d, J = 2 Hz, 1H), 4.21 (s, 3H); MS (ESI)
m/z 204.9 [C9H8N4O2 + H]+.
bromophenyl)pyrrolidine-2-carboxamide (S)-16 and imidazole (yield,
1
41%; ee, 99.6%). [α]20 −181 (c 0.1, MeOH). H NMR (400 MHz,
D
CDCl3) δ 8.28 (s, 1H), 7.76 (br s, 1H), 7.59−7.53 (m, 6H), 7.41 (t, J
= 7.2 Hz, 2H), 7.34−7.25 (m, 3H), 7.25−7.20 (m, 2H), 6.79 (d, J =
8.8 Hz, 2H), 4.15−4.12 (m, 1H), 3.84−3.80 (m, 1H), 3.37−3.30 (m,
1H), 2.44−2.39 (m, 2H), 2.16−2.08 (m, 2H); 13C NMR (400 MHz,
CDCl3) δ 171.59, 146.95, 140.39, 137.69, 136.40, 128.79, 127.64,
127.22, 126.85, 123.44, 120.26, 114.14, 65.39, 50.34, 31.68, 24.35; MS
(ESI) m/z 409 [C26H24N4O+ H]+. Melting point: 227−229 °C.
Ethyl 2-(4-Bromophenylamino)propanoate (18). To a sol-
ution of 4-bromoaniline (5.8 mmol) and ethyl 2-bromopropanoate (7
mmol) in acetonitrile (15 mL) was added K2CO3 (8.7 mmol). The
solution was heated at 70 °C for 16 h. Solvent was then removed
under reduced pressure, and then it was diluted with H2O and
extracted with EtOAc. The combined organic layer was dried over
Na2SO4 and was concentrated under reduced pressure. The crude
residue was purified by column chromatography to afford the desired
product as yellow oil (yield, 57%). 1H NMR (400 MHz, DMSO-d6) δ
6-(1-Methyl-1H-pyrazol-5-yl)pyridin-3-amine (22). To a
stirred solution of 2-(1-methyl-1H-pyrazol-5-yl)-5-nitropyridine 21
(2.1 mmol) in EtOAc (20 mL) was added 10% Pd/C (20% by wt),
and the mixture was hydrogenated under H2 (balloon) at room
temperature for 12 h. After completion, the reaction mixture was
H
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Journal of Medicinal Chemistry
Article
filtered through Celite pad and washed with EtOAc. Filtrate was
concentrated under reduced pressure to afford 6-(1-methyl-1H-
pyrazol-5-yl)pyridin-3-amine 22 as a yellow solid (yield, 96%). 1H
NMR (400 MHz, DMSO-d6) δ 8.01 (d, J = 1.6 Hz, 1H), 7.39 (m, 2H),
6.99 (m, 1H), 6.47 (s, 1H), 5.58 (s, 2H), 4.03 (s, 3H); MS (ESI) m/z
175.1 [C9H10N4 + H]+.
To a stirred solution of 2-(2-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-
3-ylamino)propanamido)-5-phenylpyridine 1-oxide (1.44 mmol) was
added iron (7.2 mmol) in AcOH (1 mL), and the reaction mixture was
stirred at reflux for 2 h. The reaction mixture was concentrated under
reduced pressure and basified with sat. NaHCO3 and extracted with
EtOAc (3 × 10 mL) and purified by column chromatography (silica
gel, eluent MeOH/DCM 5:95) to afford 2-(6-(1-methyl-1H-pyrazol-5-
yl)pyridin-3-ylamino)-N-(5-phenylpyridin-2-yl)propanamide 26 race-
mate as an off-white solid. Chiral separation (CHIRALPAK IA
column; hexane/EtOH/TFA 20:80:0.1) gave the desired compound
(S)-26 (yield, 2%, chiral HPLC 99%). [α]20D −72.6 (c 0.5, CHCl3). 1H
NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.47 (s, 1H), 8.36 (d, J =
8.4 Hz, 1H), 8.19 (s, 1H), 7.95 (d, J = 10.8 Hz, 1H), 7.54 (d, J = 7.6
Hz, 2H), 7.48−7.36 (m, 5H), 7.0 (d, J = 11.2 Hz, 1H), 6.43 (s, 1H),
4.24 (s, 1H), 4.14 (s, 1H), 4.01 (s, 3H), 1.70 (d, J = 7.2 Hz, 3H,); MS
(ESI) m/z 399.25 [C23H22N6O + H]+. Melting point: 82−84 °C.
N-(3,3′-Bipyridin-6-yl)-2-(6-(1-methyl-1H-pyrazol-5-yl)-
pyridin-3-ylamino)propanamide ((S)-27). To a stirred solution of
2-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanoic acid 24
(4.1 mmol) in DMF (5 mL) were added HATU (8.1 mmol), 2-amino-
5-bromopyridine 1-oxide (4.1 mmol), and DIPEA (12.2 mmol). The
reaction mixture was stirred at room temperature for 16 h. The
reaction mixture was poured into ice−water and extracted with EtOAc
(3 × 10 mL). The combined organic layers were washed with water
and brine solution, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to afford 5-bromo-2-(2-(6-(1-
methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanamido)pyridine 1-
oxide as a liquid (yield, 60%).
To a stirred solution of 5-bromo-2-(2-(6-(1-methyl-1H-pyrazol-5-
yl)pyridin-3-ylamino)propanamido)pyridine 1-oxide (2.4 mmol) was
added iron (12 mmol) in AcOH (5 mL), and the reaction mixture was
stirred at reflux for 2 h. The reaction mixture was concentrated under
reduced pressure, basified with sat. NaHCO3, extracted with EtOAc (3
× 10 mL), and purified by column chromatography (silica gel, eluent
CH2Cl2/CH3OH 95:5) to afford N-(5-bromopyridin-2-yl)-2-(6-(1-
methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanamide as an off-
white solid (yield, 64%).
To a well stirred solution of N-(5-bromopyridin-2-yl)-2-(6-(1-
methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanamide (1.4 mmol)
in dioxane−water (4:1, 10 mL) were added K3PO4 (2.74 mmol),
pyridine-3-boronic acid (1.4 mmol), and Pd(dppf)Cl2·DCM (5 mol
%) under Ar, and the reaction mixture was heated at 100 °C for 16 h.
The reaction mixture was evaporated under reduced pressure. The
residue was poured into ice−water, and the compound was extracted
with EtOAc (3 × 50 mL). The combined organic layer was washed
with ice−water, brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The crude material was purified
prep-HPLC to afford N-(3,3′-bipyridin-6-yl)-2-(6-(1-methyl-1H-pyr-
azol-5-yl)pyridin-3-ylamino)propanamide 27 racemate. Chiral separa-
tion (CHIRALCEL OJ-H column; hexane/EtOH/IPA 50:50:0.1) gave
(S)-27 as an off white solid (yield, 11%). [α]20D −72.5 (c 0.5, CHCl3).
1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.93 (s, 1H), 8.74 (s,
Ethyl 2-(6-(1-Methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)-
propanoate (23). To a stirred solution of 6-(1-methyl-1H-pyrazol-
5-yl)pyridin-3-amine 22 (2 mmol) in acetonitrile (20 mL) was added
NaOAc (6 mmol) and ethyl bromopropionate (2 mmol), and the
reaction mixture was heated to reflux for 48 h. The reaction mixture
was concentrated under reduced pressure and the crude material was
purified by column chromatography (silica gel, eluent EtOAc/hexane
20:80) to afford ethyl 2-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-3-
1
ylamino)propanoate 23 as a colorless liquid (yield, 45%). H NMR
(400 MHz, DMSO-d6) δ 8.04 (d, J = 2.8 Hz, 1H), 7.48 (d, J = 8.8 Hz,
1H), 7.38 (d, J = 2.4 Hz, 1H), 6.98 (dd, J = 2.4, 8.8 Hz, 1H), 6.54 (d, J
= 8.4 Hz, 1H), 6.51 (s, 1H), 4.21 (q, J = 6.8 Hz, 1H), 4.14 (q, J = 7.6
Hz, 2H), 4.04 (s, 3H), 1.41 (d, J = 6.8 Hz, 3H), 1.18 (t, J = 7.6 Hz,
3H); MS (ESI) m/z 275.2 [C14H18N4O2 + H]+.
2-(6-(1-Methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanoic
Acid (24). To a stirred solution of ethyl 2-(6-(1-methyl-1H-pyrazol-5-
yl)pyridin-3-ylamino)propanoate 23 (1 mmol) in THF−H2O−MeOH
(1:1:1) (15 mL) was added LiOH (2 mmol), and the reaction mixture
was stirred at room temperature for 2 h. After completion, the volatiles
were removed under reduced pressure and the residue was acidified
with aq KHSO4 and extracted into EtOAc. The combined organic
layer was washed with brine, dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure to afford 2-(6-(1-methyl-1H-
pyrazol-5-yl)pyridin-3-ylamino)propanoic acid 24 (yield, 89%). 1H
NMR (400 MHz, DMSO-d6) δ 8.05 (d, J = 2.8 Hz, 1H), 7.61 (d, J =
8.8 Hz, 1H), 7.45 (s, 1H), 7.21 (br s, 1H), 6.56 (br s, 1H), 4.44 (br s,
1H), 4.29 (m, 1H), 4.17 (m, 1H), 3.99 (s, 3H), 1.40 (d, J = 7.2 Hz,
3H); MS (ESI) m/z 247.2 [C12H14N4O2 + H]+.
N-(2,3′-Bipyridin-5-yl)-2-(6-(1-methyl-1H-pyrazol-5-yl)-
pyridin-3-ylamino)propanamide ((S)-25). To a stirred solution of
2-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanoic acid 24
(0.8 mmol) in DMF (5 mL) were added HATU (1.2 mmol), 2,3′-
bipyridin-5-amine (0.8 mmol), and DIPEA (1.6 mmol). The reaction
mixture was stirred at room temperature for 16 h. After completion,
the reaction mixture was poured into ice−water and the compound
was extracted with EtOAc (3 × 50 mL). The combined organic layer
was washed with ice−water, brine, dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The crude material
was purified by column chromatography (silica gel, eluent MeOH/
chloroform 5:95) to afford N-(2,3′-bipyridin-5-yl)-2-(6-(1-methyl-1H-
pyrazol-5-yl)pyridin-3-ylamino)propanamide 25 racemate. Isomers
were separated by chiral HPLC to afford the required compound
1
(S)-25 (yield, 10%). [α]20 −51.4 (c 1.19, CHCl3). H NMR (400
D
MHz, CDCl3) δ: 10.52 (s, 1H), 9.21 (d, J = 1.8 Hz, 1H), 8.90 (d, J =
2.2 Hz, 1H), 8.59 (dd, J = 1.3 Hz, 4.8 Hz, 1H), 8.37 (dt, J = 1.7, 8.0
Hz, 1H), 8.22 (dd, J = 2.6, 8.6 Hz, 1H), 8.11 (d, J = 2.6 Hz, 1H), 8.03
(d, J = 8.8 Hz, 1H), 7.55−7.45 (m, 2H), 7.37 (d, J = 1.7 Hz, 1H), 7.04
(dd. J = 2.6, 8.30 Hz, 1H), 6.60 (d, J = 7.5 Hz, 1H), 6.50 (d, J = 1.8
Hz, 1H), 4.22 (t, J = 7.0 Hz, 1H), 4.03 (s, 3H), 1.49 (d, J = 6.6 Hz,
3H); MS (ESI) m/z 400.25 [C22H21N7O + H]+. Melting point: 98−
100 °C.
1H), 8.58 (d, J = 4.8 Hz, 1H), 8.20 (s, 2H), 8.12 (m, 2H), 7.50 (m,
2H), 7.36 (s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H),
6.49 (s, 1H), 4.28 (t, J = 6.8 Hz, 1H), 4.03 (s, 3H), 1.48 (d, J = 6.8 Hz,
3H); 13C NMR (400 MHz, CDCl3) δ 172.36, 150.78, 148.86, 147.64,
146.17, 141.48, 141.42, 141.26, 138.18, 137.19, 135.84, 134.58, 133.30,
130.16, 124.09, 123.74, 120.63, 114.20, 105.61, 55.87, 39.17, 19.74;
MS (ESI) m/z 400.47 [C22H21N7O + H]+. Melting point: 98−100 °C.
STF3A Reporter Assay. HEK293-STF3A cells and the assay used
to determine the IC50 of the compounds were described by Coombs
and co-workers.15 2 × 104 STF3A cells were seeded in each well of a
96-well plate (Greiner) and incubated overnight at 37 °C. An amount
of 25 μL of serially diluted compound was added to the cells to give
final concentrations of 50 μM to 1.5 nM. After 1 day of treatment, 100
μL of Steady-Glo luciferase assay reagent (Promega) was added to
each well and incubated for 10 min at room temperature.
Luminescence was measured using the Tecan Safire2 microplate
reader.
2-(6-(1-Methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)-N-(5-phe-
nylpyridin-2-yl)propanamide ((S)-26). To a stirred solution of 2-
(6-(1-methyl-1H-pyrazol-5-yl)pyridin-3-ylamino)propanoic acid 24 (1
mmol) in DMF (5 mL) were added HATU (2 mmol), 2-amino-5-
phenylpyridine 1-oxide (1 mmol), and DIPEA (3 mmol). The reaction
mixture was stirred at room temperature for 16 h. The reaction
mixture was poured into ice−water and extracted with EtOAc (3 × 10
mL). The combined organic layers were washed with water and brine
solution, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure to afford 2-(2-(6-(1-methyl-1H-pyrazol-5-
yl)pyridin-3-ylamino)propanamido)-5-phenylpyridine 1-oxide as an
off-white solid.
I
DOI: 10.1021/acs.jmedchem.5b00507
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
STF/Wnt3A Conditioned Medium Assay. For this assay
HEK293-STF cells that do not express Wnt3A were used. To make
Wnt3A conditioned medium, mouse L-Wnt3A cells were cultured in
three T-175 flasks at 3 × 104 cells/mL in 30 mL culture medium per
flask. After 4 days of incubation, the Wnt3A-conditioned medium was
harvested. An amount of 2 ×104 HEK293-STF cells in 25 μL of culture
medium was added to each well of a 96 well plate (Greiner). 25 μL of
serially diluted compound was added. After 4 h of incubation, 100 μL
of Wnt-3A-conditioned medium was added to the cells. The final
concentration of compound ranged from 33 μM to 1 nM. After
incubation for 1 day at 37 °C, 100 μL of Steady-Glo luciferase assay
reagent (Promega) was added to each well and incubated for 10 min at
room temperature. Luminescence was measured using the Tecan
Safire2 microplate reader.
was monitored using calipers. Fourteen days after tumor implantation
(designated as day 1 of the study), mice bearing established orthotopic
MMTV-Wnt1 tumors were selected and randomly distributed into five
groups. Each group contained 10 mice. The group mean tumor
volume ranged from 103 to 107 mm3. After randomization, mice
bearing established orthotopic MMTV-Wnt1 tumors received vehicle
(50% polyethylene glycol (PEG) 400 in deionized water), 2, or (S)-27
once daily for 14 days via oral gavage. The volume of oral
administration was 10 mL/kg. The dosing level for 2 was 3 mg/kg,
and the dosing levels for (S)-27 were 1, 3, and 10 mg/kg. The end
point used to measure the response of the MMTV-Wnt1 tumors after
compound treatment was tumor growth inhibition at day 14 and was
expressed as % TGI and T/C ratio. The percentage of tumor growth
inhibition (%TGI) was calculated as follows:
Cell Viability Assay. 5000 cells in 75 μL of culture medium were
seeded in each well of black 96-well plates (Greiner no. 655090) and
incubated overnight at 37 °C. 25 μL of serially diluted compound was
added to the cells giving a final concentration of 50 μM to 1.5 nM.
After 1 day of treatment, 100 μL of CellTiter-Glo luminescent cell
viability assay reagent (no. G7571, Promega) was added to each well
and incubated for 10 min at room temperature. Luminescence was
measured using Tecan Safire2 microplate reader.
Cdaya − T
Cdaya − Cday1
daya
% TGI =
× 100
where Cday a is the mean tumor volume of the vehicle control group at
the indicated day a, Tday a is the mean tumor volume of the group
treated with the test compound at the indicated day a, and Cday 1 is the
mean tumor volume of the vehicle control group at day 1. The T/C
ratio was calculated as follows:
Wnt3A Palmitoleation Assay. The assay used to determine the
inhibition of the palmitoleation of Wnts by the compounds was
described by Yap et al.19 3 × 106 HeLa cells were seeded in a 10 cm
culture dish and incubated at 37 °C overnight. The cells were
transfected with 5 μg of pcDNA3.2/V5-Wnt3a vector18 to overexpress
V5-tagged Wnt3a. After 6 h, the cells were washed with PBS and
treated with 100 μM ω-alkynyl palmitate in medium with 5% fatty acid
free BSA. 100 nM compound or DMSO was added, and the cells were
incubated overnight at 37 °C. The cells were lysed, and 600 μg of cell
lysate was collected and incubated with anti-V5 antibody (Invitrogen)
followed by the pull down of V5-Wnt3a with the addition of protein
A/G agarose beads (Thermo Scientific). The pulled down lysates
containing V5-Wnt3a was click-reacted with biotin azide (Invitrogen).
The biotin-labeled protein lysate was then separated by SDS−PAGE
and transferred to a nitrocellulose membrane. The membrane was
incubated with primary anti-V5 antibody, followed by secondary anti-
mouse Dylight 680 (Thermo scientific) to detect V5-Wnt3a. The
membrane was then incubated with streptavidin-Dylight 800 (Thermo
Scientific) to detect biotin labeled Wnt3a. The signals were captured
on the Odyssey CLx infrared imaging system (LI-COR Bioscience).
PORCN Overexpression Assay. The human fibrosarcoma cell
line HT1080 (ATCC cat. no. CCL121) was maintained in DMEM
(Nacali Tesque, Japan) containing 4.5 g/L glucose, penicillin/
streptomycin, 10% FBS, and 1 mM sodium pyruvate in a humidified
37 °C atmosphere. The HT1080 cells were seeded at 150 000 per well
in 24-well culture dishes 1 day prior to transfection. The cells were
then transfected with either control plasmid or murine PORCN
expression plasmids (100 ng) in combination with Wnt/β-catenin
reporter plasmid (550 ng) and Wnt3a (50 ng) using Lipofectamine
2000 (Invitrogen/LifeTechnologies). The cells were treated with
serially diluted (S)-27 in concentrations from 625 to 0.2 nM at a final
DMSO concentration of 0.1%. The activation of the β-catenin reporter
gene activity was measured in 50 μL of cell lysate 24 h post-
transfection, using the luciferase assay kit (Promega) according to the
manufacturer’s recommendations, on a Tecan Infinite M200 plate
reader. Luminescence was read with no attenuation and an integration
time of 1000 ms.
T
T
C
daya
=
Cdaya
One-way ANOVA followed by Dunnett’s multiple comparison test
was used to determine statistically significant differences between the
tumor volumes of the vehicle control group and the tumor volumes of
the group treated with different dosing regimens of 2 and (S)-27.
ASSOCIATED CONTENT
■
S
* Supporting Information
Schematic representation of HTS strategy; H and 13C NMR
1
data for (S)-27; solubility for compounds 4, 6−11, (S)-14, and
(S)-17, and (S)-20; graphs for in vivo pharmacokinetics;
tabulated results for the MMTV-Wnt1 in vivo model for
compound (S)-27; mouse body weight for efficacy results; csv
file containing molecular formula strings. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.jmedchem.5b00507.
AUTHOR INFORMATION
Corresponding Author
*E-mail: syho@etc.a-star.edu.sg. Telephone: +65 6407 0670.
■
Present Address
§A.J.D.: GVK Biosciences Private Limited, Plot No. 28 A, IDA
Nacharam, Hyderabad, 500076, India.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare the following competing financial
interest(s): All authors are current or former employees of
the Experimental Therapeutics Centre or Duke-NUS Graduate
Medical School Singapore. Both institutes have a commercial
interest in the development of Wnt secretion inhibitors.
In Vivo Efficacy Studies. In vivo antitumor efficacy study was
conducted at Charles River Discovery Research Services North
Carolina, USA. The experimental protocols were in compliance with
the recommendations of the Guide for Care and Use of Laboratory
Animals.
The primary mammary tumors (MMTV-Wnt1) provided by
Experimental Therapeutics Centre, Singapore, were passaged in
female NCr nude mice prior to implantation. The tumor fragment
utilized in this experiment originated from working tumor bank no.
947 R75. Each test mouse received a 3 mm3 MMTV-Wnt1 fragment
implanted in the mammary fat pad number 4. The growth of tumors
ACKNOWLEDGMENTS
■
We acknowledge the technical assistance provided by members
of Experimental Therapeutics Centre including Jeff Hill,
Shermaine Q. Y. Lim, and Sifang Wang, and we acknowledge
Justina Fulwood and Horst Flotow for performing the high
throughput screening of our compound library. This work was
J
DOI: 10.1021/acs.jmedchem.5b00507
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(16) Willert, K.; Brown, J. D.; Danenberg, E.; Duncan, A. W.;
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H.; Tan, E. S.; Lin, G. R.; Cheong, V. W.; Ke, Z.; Lee, M. A.; Keller, T.
H. A pharmacophore model for Wnt/porcupine inhibitors and its use
for drug design. J. Chem. Inf. Model. 2015, DOI: 10.1021/
acs.jcim.5b00159.
(18) Najdi, R.; Proffitt, K.; Sprowl, S.; Kaur, S.; Yu, J.; Covey, T. M.;
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library reveals a shared secretory pathway and unique signaling
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(19) Yap, M. C.; Kostiuk, M. A.; Martin, D. D.; Perinpanayagam, M.
A.; Hak, P. G.; Siddam, A.; Majjigapu, J. R.; Rajaiah, G.; Keller, B. O.;
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supported in part by the Singapore Translational Research
Investigator Award (D.M.V.), funded by the National Research
Foundation and the National Medical Research Council of
Singapore.
ABBREVIATIONS USED
■
Cyp, cytochrome P450 enzyme; ER, endoplasmic reticulum;
HTS, high throughput screening; MMTV, mouse mammary
tumor virus; PORCN, porcupine Wnt O-acyltransferase; SAR,
structure−activity relationship; STF, super top flash
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