Pharmacokinetic optimization of class-selective histone deacetylase inhibitors and identification of associated candidate predictive biomarkers of hepatocellular carcinoma tumor response

Article abstract of DOI:10.1021/jm3011838

Herein, we describe the pharmacokinetic optimization of a series of class-selective histone deacetylase (HDAC) inhibitors and the subsequent identification of candidate predictive biomarkers of hepatocellular carcinoma (HCC) tumor response for our clinical lead using patient-derived HCC tumor xenograft models. Through a combination of conformational constraint and scaffold hopping, we lowered the in vivo clearance (CL) and significantly improved the bioavailability (F) and exposure (AUC) of our HDAC inhibitors while maintaining selectivity toward the class I HDAC family with particular potency against HDAC1, resulting in clinical lead 5 (HDAC1 IC₅₀ = 60 nM, mouse CL = 39 mL/min/kg, mouse F = 100%, mouse AUC after single oral dose at 10 mg/kg = 6316 h·ng/mL). We then evaluated 5 in a biomarker discovery pilot study using patient-derived tumor xenograft models, wherein two out of the three models responded to treatment. By comparing tumor response status to compound tumor exposure, induction of acetylated histone H3, candidate gene expression changes, and promoter DNA methylation status from all three models at various time points, we identified preliminary candidate response prediction biomarkers that warrant further validation in a larger cohort of patient-derived tumor models and through confirmatory functional studies.

Full text of DOI:10.1021/jm3011838

Article
pubs.acs.org/jmc
Pharmacokinetic Optimization of Class-Selective Histone
Deacetylase Inhibitors and Identification of Associated Candidate
Predictive Biomarkers of Hepatocellular Carcinoma Tumor Response
Jason C. Wong,*,†,# Guozhi Tang,† Xihan Wu,† Chungen Liang,† Zhenshan Zhang,† Lei Guo,†
Zhenghong Peng,† Weixing Zhang,† Xianfeng Lin,† Zhanguo Wang,† Jianghua Mei,† Junli Chen,†
Song Pan,† Nan Zhang,† Yongfu Liu,† Mingwei Zhou,† Lichun Feng,† Weili Zhao,† Shijie Li,†
Chao Zhang,† Meifang Zhang,† Yiping Rong,† Tai-Guang Jin,† Xiongwen Zhang,† Shuang Ren,† Ying Ji,†
Rong Zhao,† Jin She,† Yi Ren,† Chunping Xu,‡ Dawei Chen,‡ Jie Cai,‡ Song Shan,§ Desi Pan,§
Zhiqiang Ning,§ Xianping Lu,§ Taiping Chen,‡ Yun He,† and Li Chen†
†Roche R&D Center (China) Ltd., 720 Cai Lun Road, Building 5, Pudong, Shanghai 201203, P. R. China
‡Crown Bioscience Inc., Light Muller Building, Changping Sector of Zhongguancun Science Park, No.21 Huoju Street, Changping
District, Beijing 102200, P. R. China
§Chipscreen Biosciences Ltd., 2-601, BIO-Incubator, Gaoxin C, first Ave., Shenzhen High-Tech Industrial Park, Nanshan District,
Shenzhen, Guangdong 518057, P. R. China
S
* Supporting Information
ABSTRACT: Herein, we describe the pharmacokinetic
optimization of a series of class-selective histone deacetylase
(HDAC) inhibitors and the subsequent identification of
candidate predictive biomarkers of hepatocellular carcinoma
(HCC) tumor response for our clinical lead using patient-
derived HCC tumor xenograft models. Through a combination
of conformational constraint and scaffold hopping, we lowered
the in vivo clearance (CL) and significantly improved the bioavailability (F) and exposure (AUC) of our HDAC inhibitors while
maintaining selectivity toward the class I HDAC family with particular potency against HDAC1, resulting in clinical lead 5
(HDAC1 IC50 = 60 nM, mouse CL = 39 mL/min/kg, mouse F = 100%, mouse AUC after single oral dose at 10 mg/kg = 6316
h·ng/mL). We then evaluated 5 in a biomarker discovery pilot study using patient-derived tumor xenograft models, wherein two
out of the three models responded to treatment. By comparing tumor response status to compound tumor exposure, induction of
acetylated histone H3, candidate gene expression changes, and promoter DNA methylation status from all three models at
various time points, we identified preliminary candidate response prediction biomarkers that warrant further validation in a larger
cohort of patient-derived tumor models and through confirmatory functional studies.
in a wide variety of other post-translationally acetylated
proteins. The HDAC family of enzymes can be divided into
two major groups, the Rpd3/Hda1-like NAD-independent
lysine deacetylases and the NAD-dependent sirtuins.4 The
Rpd3/Hda1-like deacetylase group has been further organized
into class I enzymes (HDACs 1, 2, 3, and 8) and class II
enzymes (HDACs 4, 5, 6, 7, 9, 10) based on sequence similarity
to the yeast HDACs Rpd3 and Hda1, respectively.5
With respect to HDACs as potential targets for HCC
treatment, HDAC1 in particular has been found to be
significantly overexpressed in HCC patient tumor samples
and its expression in this context is negatively correlated to
overall survival.6 Moreover, hepatitis B virus (HBV), which has
been strongly implicated in HCC pathogenesis, has been
demonstrated to induce HDAC1 overexpression through the
INTRODUCTION
■
Hepatocellular carcinoma (HCC) is among the leading causes
of cancer death worldwide with an overall 5-year median
survival rate that remains below 12% in the United States.1
Most HCC patients are first diagnosed when the disease has
reached an advanced and/or unresectable stage where either
systemic or local chemotherapy are the standard treatment
options.2 Currently, systemic treatment with the multitargeted
kinase inhibitor sorafenib is considered the standard of care for
advanced HCC, providing a median overall survival of 10.7
months as compared to 7.9 months with placebo.3 In light of
the poor overall survival rates associated with HCC and the
limited efficacy of sorafenib, there is a great need to identify
new targets and corresponding drug candidates for HCC
treatment.
The histone deacetylases (HDACs) are a family of Zn2+-
dependent hydrolases that remove the acetyl group from the
acetylated ε-amino side chain of lysine residues in histones and
Received: August 11, 2012
Published: October 12, 2012
© 2012 American Chemical Society
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HBV HBx protein in hepatocellular carcinoma cells, leading to
an enhancement of pro-tumor hypoxia signaling.7 Functionally,
small molecule HDAC inhibition in HCC cells induces
antiproliferative and pro-apoptotic effects that correlate with
in-cell target inhibition as assessed by classical pharmacody-
namic markers such as p21 and acetylated histone H3.8,9
Moreover, in certain cellular contexts, HDAC inhibition can
reactivate the expression of tumor suppressor genes such as
TMS1/ASC10 and TFPI-211 that have been epigenetically
silenced through promoter CpG island DNA hypermethylation
which ultimately recruits multiprotein complexes including
HDAC1 to enforce a transcriptionally repressed heterochro-
matin state.12 Furthermore, in preclinical in vivo models,
HDAC inhibitors can significantly inhibit the growth of
established HCC tumors.13 As a result of the evidence linking
HDAC activity with HCC pathogenesis and progression,
several pan-HDAC inhibitors (Figure 1A) are currently in or
recently have been in early stage clinical trials in HCC either as
single agents or in combination with sorafenib or traditional
chemotherapy.14
first published example employing mRNA and DNA methyl-
ation profiling in patient-derived xenograft models to identify
candidate predictive biomarkers of HCC tumor response for a
class I-selective HDAC inhibitor.
We previously identified compound 1 (Figure 2) as an o-
aminoanilide-based selective class I HDAC inhibitor with
particular potency against HDAC1 and good cell-based activity
as assessed by p21 reporter gene assay, endogenous p21 and
acetylated histone H3 induction assays, and antiproliferative
assays in various cancer cell lines.19 While 1 and its closely
related analogues had reasonable enzyme- and cell-based
activities, these compounds were not suitable for further drug
candidate development due to high in vivo clearance and
limited bioavailability. We therefore undertook a two-stage
campaign to optimize compound 1. In the first stage, we
attempted to replace the conformationally flexible and
metabolically labile morpholine side chain with more compact
and rigid moieties to balance the overall properties of our lead
series. In the second stage, motivated by the modest, but still
suboptimal, property improvement attained by replacing the
morpholine side chain, we further rigidified our lead structure
through a scaffold-hopping approach.20 This two-staged effort
ultimately led to the discovery of clinical candidate compound
5 and its evaluation in primary patient tumor-derived HCC
xenograft models for the identification of candidate predictive
tumor response biomarkers, as detailed below.
CHEMISTRY
■
General Comments on Stereochemistry. Unless other-
wise noted, compounds were synthesized as racemic mixtures.
General Synthesis of Morpholine Side-Chain Replace-
ment Analogues of Compound 1 (Scheme 1). Synthesis of
morpholine side-chain replacement analogues of compound 1
began with esterification of commercially available 2-(4-
bromophenyl)-2-hydroxyacetate in methanol with sulfuric
acid as catalyst to obtain methyl ester I. Subsequent Heck
coupling of I with acrylamide III (obtained by acylation of Boc-
protected aniline II with the mixed anhydride of acrylic acid)
using tri-o-tolylphosphine as ligand provided cinnamide IV in
moderate yield. Mesylation of IV followed by nucleophic
substitution on the resulting mesylates with alkyl amines gave
α-amino esters which were hydrolyzed to the corresponding
acids and then coupled with various amines using carbodiimide
chemistry to obtain α-amino amides VI. Final Boc group
deprotection in methanolic HCl gave target compounds 2−4
and 6−10 (see Supporting Information Table S1).
Figure 1. Representative HDAC inhibitors. (A) HDAC inhibitors that
have been or are being evaluated in the clinic for HCC therapy; (B) o-
aminoanilide HDAC inhibitors under clinical development.
On the basis of the evidence implicating HDAC1 in HCC in
particular, and in contrast to the pan-HDAC inhibitors
currently under clinical evaluation in HCC, we set out to
develop a selective class I HDAC inhibitor based on the o-
aminoanilide pharmacophore15 (Figure 1B) with particular
potency toward HDAC1 for HCC therapy. Furthermore,
consistent with our general strategy of developing companion
predictive and/or pharmacodynamic biomarkers for our drug
discovery programs, we incorporated biomarker hypothesis
generation16 as part of our preclinical candidate research and
selection efforts, as detailed below, to ultimately inform and
improve our future clinical trial design. In particular, in light of
the relatively limited clinical efficacy of HDACi in solid tumor
treatment thus far, we envisioned that the identification of
predictive biomarkers of tumor response could help guide the
selection of HCC patients more likely to benefit from
treatment with our class I-selective HDACi in either a
monotherapy or combination therapy setting.17 While other
researchers have previously sought to identify predictive
biomarkers for HDACi in various cell models and/or clinical
isolates,18 to our knowledge the research disclosed herein is the
General Synthesis of R1 Analogues of the rac-(trans-
3,4)-Disubstituted Pyrrolidine Series (Scheme 2). Syn-
thesis of R1 analogues of the rac-(trans-3,4)-disubstituted
pyrrolidine series began with iminium ylide cycloaddition21 of
commercially available (E)-3-(4-bromophenyl)acrylic acid ethyl
ester with sarcosine and paraformaldehyde to obtain pyrrolidine
VII. Pyrrolidine VII was then reacted with acrylamide III using
Heck coupling conditions as described above to synthesize
cinnamide VIII. Hydrolysis of VIII using lithium hydroxide in
methanol furnished the corresponding carboxylic acid which
was then coupled with various amine/aniline building blocks
and subsequently subjected to methanolic HCl to provide final
compounds 11−42 as racemic mixtures of trans diastereomers.
General Synthesis of R2 Analogues of the rac-(trans-
3,4)-Disubstituted Pyrrolidine Series via Alkylation,
Epoxidation, Reductive Amination, Acylation, or Nucle-
ophilic Substitution (Scheme 3). Synthesis of R2 analogues
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Figure 2. Summary of compound 1 optimization history leading to identification of clinical lead 5 and the associated trans-3,4-disubstituted
pyrrolidine lead series. P21_RP3 is the relative fold increase in luciferase signal in the p21 reporter gene assay compared to clinical HDACi entinostat
(both test compound and comparator were evaluated at 3 μM in duplicate or triplicate) and normalized by internal GFP transfection control. CLh
(MLM) is the calculated hepatic clearance based on mouse liver microsome intrinsic clearance. CL (mouse) is the in vivo systemic clearance
measured by intravenous pharmacokinetic study in BALB/C nude mice. F is the bioavailability determined by single dose intravenous (2 mpk) and
oral (10 mpk) pharmacokinetic study in nude mice. See Experimental Section for further details.
a
Scheme 1. Synthesis Morpholine Side-Chain Replacement Analogues of Compound 1
a
(a) H2SO4, MeOH, 50 °C, 3 h, 77%; (b) Boc2O, THF, rt, 16 h, 77%; (c) acrylic acid, N-methylmorpholine, isobutyl chloroformate, DCM, 0 °C, 30
min and then add intermediate II at reflux over 30 min, stir 2 h, 34%; (d) tri-o-tolylphosphine, TEA, Pd2(dba)3, DMF, 100 °C, 6 h, 60%; (e) MsCl,
TEA, DCM, 0 °C, 1 h; (f) R2NH2 or R2R3NH, TEA, DCM, rt, 16 h; (g) LiOH, MeOH, rt, 5 h, 81% (three steps); (h) R1NH2, PyBrop, DIPEA,
DCM, rt, 12 h; (i) 1.25 M HCl in MeOH, rt, 2 h, and then NaHCO3, 20−25% (two steps).
of the rac-(trans-3,4)-disubstituted pyrrolidine series in which
the R2 group was installed via alkylation, epoxidation, reductive
amination, acylation, or nucleophilic substitution began with
orthoboric acid-catalyzed iminium ylide cycloaddition of
commercially available (E)-3-(4-bromophenyl)acrylic acid
ethyl ester with N-(methoxymethyl)-N-(trimethylsilylmethyl)-
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a
Scheme 2. Synthesis of R1 Analogues of the rac-(trans-3,4)-Disubstituted Pyrrolidine Series
a
(a) Sarcosine, paraformaldehyde, toluene, reflux, 4 h, 46%; (b) III, Pd2(dba)3, tri-o-tolylphosphine, TEA, DMF, 110 °C (sealed tube), 16 h, 87%;
(c), LiOH, MeOH, rt, 16 h; (d) R1 (substituted amine, aniline, or aminopyridine), HATU, TEA, DMF:DCM (1:10), rt, 3 h, 88% (two steps); (e) 1
N HCl in MeOH, rt, 4 h, 43%.
Scheme 3. Synthesis of R2 Analogues of the rac-(trans-3,4)-Disubstituted Pyrrolidine Series via Alkylation, Epoxidation,
a
Reductive Amination, Acylation, or Nucleophilic Aromatic Substitution
a
(a) N-(Methoxymethyl)-N-(trimethylsilylmethyl)benzylamine, orthoboric acid, DCM, rt, 2 d then 45 °C for 2 h, 71%; (b) 2,2,2-trichloroethyl
chloroformate, K2CO3, CH3CN, 60 °C, 3 h, 66%; (c) Zn, acetic acid, rt, 3 h; (d) for alkylation (R2X where X is halide, tosylate, or mesylate): K2CO3,
KI, R2X, DMF, 130 °C (microwave) 20 min; for epoxidation (2,2-dimethyloxirane): 2,2-dimethyloxirane, EtOH, 80 °C (sealed tube), 5 h, 96%; for
reductive amination (R2 = R3R4CH after reduction from R3R4CO): R3R4CO, NaBH(OAc)3, catalytic acetic acid, MeOH, rt, 16 h, 64%; for acylation:
triphosgene and R3R4NH (R2 = CONR3R4), DIPEA, THF, 0 °C to rt, 12 h; for nucleophilic aromatic substitution (2-chloropyrimidine): 2-
chloropyrimidine, DIPEA, i-PrOH, 130 °C (microwave), 10 min, 60%; (e) N-(2-(acryloylamino)phenyl)carbamic acid tert-butyl ester (III),
Pd2(dba)3, tri-o-tolylphosphine, TEA, DMF, 100 °C, 3 h; (f) R1 (substituted amine, aniline, or aminopyridine), LiOH, MeOH, rt, 16 h; (g) HATU,
TEA, DMF:DCM (1:10), rt, 3 h; (h) 1 N HCl in MeOH, rt, 4 h.
a
Scheme 4. Synthesis of the rac-(trans-3,4)-Disubstituted Pyrrolidine Series Analogue 53 (R2 = 2-hydroxy-1,1-dimethylethyl)
a
(a) NaOH, CH3CN, rt, 2 h then Na2CO3, Boc2O, rt, 16 h, 50%; (b) 4-chloroaniline, HOBt, EDCI, DCM, rt, 16 h; (c) 1.25 M HCl in MeOH, rt, 3
h then Na2CO3; (d) 2-bromo-2-methylpropionic acid methyl ester, K2CO3, DMF, 50 °C, 5 h; (e) LiBH4, THF, rt, 2 h (17%, four steps); (f) N-(2-
(acryloylamino)phenyl)carbamic acid tert-butyl ester (III), Pd2(dba)3, tri-o-tolylphosphine, TEA, DMF, 100 °C, 3 h; (g) 1.25 M HCl, MeOH, rt, 3 h
(24%, two steps).
benzylamine in DCM to afford N-benzylpyrrolidine IX. Except
for the synthesis of final compound 58, where N-benzyl
pyrrolidine IX was directly reacted with acrylamide III to
obtain cinnamide XII, the N-benzyl group was then removed in
a two-step process involving conversion of the N-benzyl group
to trichloroethyl carbamate with trichloroethyl chloroformate
followed by carbamate cleavage mediated by zinc metal in
acetic acid to provide pyrrolidine X. Pyrrolidine X was then
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a
Scheme 5. Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine Analogue 52 (R2 = tert-butyl)
a
(a) N-tert-butylglycine hydrochloride, paraformaldehyde, toluene, reflux, 4 h, 12%; (b) N-(2-(acryloylamino)phenyl)carbamic acid tert-butyl ester
(III), Pd2(dba)3, tri-o-tolylphosphine, TEA, DMF, 110 °C (sealed tube), 12 h, 53%; (c) LiOH, THF, rt, 16 h; (d) 4-chloroaniline, HATU, TEA,
DCM, rt, 16 h, 66% (two steps); (e) 1.25 M HCl in MeOH, rt, 4 h, 27%.
Scheme 6. Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine Analogues 47 and 48 (R2 = 1,1-difluoroethyl and 1,1,1-
a
trifluoroethyl)
a
(a) For Y = CF3: triflic anhydride, TEA, DCM, 0 °C, 30 min and then to rt, 1 h, 68%; for Y = CF2H: difluoroacetyl chloride, DCM, rt, 1 h, 97%; (b)
for Y = CF3: BH3, THF, 0 °C, 2 h, 30%; for Y = CF2H: BH3, THF, 60 °C, 5 h, 63% (c) NaOH H2O/MeOH, rt, 4 h; (d) 4-chloroaniline, PyBrop,
DIPEA, DCM, rt, 16 h, 90%; (e) N-(2-(acryloylamino)phenyl)carbamic acid tert-butyl ester (III), Pd2(dba)3, tri-o-tolylphosphine, TEA, DMF, 110
°C (sealed tube), 4 h, 86%; (f) TFA, DCM, rt, 2 h.
converted to R2-substituted pyrrolidine XI via one of five
general methods depending on the nature of the R2 substituent.
To obtain the R2-substituted pyrrolidines XIa corresponding to
final compounds 45, 46, 49, 50, and 54, pyrrolidine X was
alkylated under microwave conditions with potassium iodide as
catalyst with various R2X building blocks, where X was a
suitable leaving group such as bromide or tosylate. Over-
alkylation was generally not an issue in these cases presumably
due to the deactivating effect that these R2 substituents had on
the nucleophilicity of the resulting tertiary pyrrolidine amine.
To obtain the corresponding pyrrolidine intermediate (XIb) for
compound 51, pyrrolidine X underwent SN2 reaction with 2,2-
dimethyloxirane in ethanol. For pyrrolidine intermediates XIc
corresponding to final compounds 43, 44, and 59, pyrrolidine
X underwent reductive amination with acetaldehyde, acetone,
and tetrahydropyran-4-one, respectively. For the pyrrolidine
intermediate XId corresponding to final compounds 55 and 56,
pyrrolidine X was acylated with triphosgene and the
appropriate alkyl amine. For the pyrrolidine intermediate XIe
corresponding to final compound 57, pyrrolidine X underwent
nucleophilic aromatic substitution with 2-chloropyrimidine.
Pyrrolidine intermediates XIa−e from all five general methods
were then coupled with acrylamide III via the Heck conditions
described above to obtain cinnamide esters XII. Esters XII were
then hydrolyzed to the corresponding cinnamide acids with
lithium hydroxide in methanol followed by HATU-mediated
condensation with 4-chloroaniline and final deprotection in
methanolic HCl to afford final compounds 43−46, 49−51, and
54−59 as racemic mixtures of trans diastereomers.
the pyrrolidine acid XIII. Carbodiimide-mediated coupling of
4-chloroaniline with acid XIII furnished pyrrolidine amide XIV.
Amide XIV was deprotected in methanolic HCl to yield
pyrrolidine amine HCl salt, which was then reacted with 2-
bromo-2-methylpropionic acid methyl ester in DMF and
subsequently reduced with lithium borohydride in THF to
the corresponding alcohol XV. Heck coupling of acrylamide III
with XV provided the corresponding cinnamide which was then
deprotected in methanolic HCl to give compound 53.
Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine
Analogue 52 (R2 = tert-butyl) (Scheme 5). Synthesis of
compound 52 began with iminium ylide cycloaddition of
commercially available (E)-3-(4-bromophenyl)acrylic acid ethyl
ester with N-tert-butylglycine hydrochloride salt and parafor-
maldehyde in refluxing toluene to obtain N-tert-butylpyrroli-
dine XVI. Heck coupling of XVI with acrylamide III then
furnished cinnamide ester XVII. Ester XVII was hydrolyzed
with lithium hydroxide in THF, then the corresponding acid
underwent HATU-mediated coupling with 4-chloroaniline
followed by Boc deprotection with methanolic HCl to obtain
final compound 52.
Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine
Analogues 47 and 48 (R2 = 1,1-difluoroethyl and 1,1,1-
trifluoroethyl) (Scheme 6). Synthesis of compounds 47 and
48 began with acylation of pyrrolidine X with difluoroacetyl
chloride and triflic anhydride, respectively, followed by selective
amide reduction with borane in THF to yield the
corresponding N-alkyl pyrrolidines XVIII. N-Alkyl pyrrolidines
XVIII were then treated with lithium hydroxide followed by
PyBrop-mediated coupling of 4-chloroaniline to the resulting
acids to obtain pyrrolidine amides XIX. These pyrrolidine
intermediates XIX then underwent Heck coupling with
Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine
Analogue 53 (R2 = 2-hydroxy-1,1-dimethylethyl)
(Scheme 4). Synthesis of compound 53 began with one-pot
hydrolysis and Boc protection of pyrrolidine ester X to obtain
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a
Scheme 7. Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine 5-Vinylpyridin-2-yl Linker Analogue 64
a
(a) i-PrMgCl, THF, −20 °C, 2 h and then add DMF, 0 °C to rt, 1 h, 90%; (b) triethyl phosphonoacetate, TEA, LiBr, CH3CN, rt, 16 h, 67%; (c)
sarcosine, paraformaldehyde, toluene, reflux, 4 h, 77%; (d) N-(2-(acryloylamino)phenyl)carbamic acid tert-butyl ester (III), Pd2(dba)3, tri-o-
tolylphosphine, TEA, DMF, 100 °C, 3 h, 75%; (e) LiOH, THF/H2O (3:1), rt, 5 h, 86%; (f) 4-chloroaniline, HATU, TEA, DCM, rt, 16 h, 54%; (g) 2
N HCl, MeOH, rt, 4 h.
a
Scheme 8. Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine 6-Vinylpyridin-3-yl Linker Analogue 61
a
(a) Ethane-1,2-diol, TsOH, toluene, reflux, 6 h; (b) ethyl acrylate, Pd2(dba)3, tris-o-tolylphosphine, TEA, 110 °C, 3 h; (c) N-methylglycine,
paraformaldehyde, toluene, reflux, 6 h; (d) LiCl, DMSO/H2O (3:1), 150 °C (microwave), 10 min; (e) diethyl tert-butyl phosphonoacetate, n-
butyllithium, THF, −78 to 0 °C, 2 h; (f) LiOH, THF/H2O (3:1), rt, 3 h; (g) 4-chloroaniline, HATU, TEA, DCM, rt, 24 h; (h) TFA, DCM, 0 °C to
rt, 2 h; (i) 1,2-diaminobenzene, HATU, TEA, DCM, rt, 12 h.
acrylamide III followed by Boc deprotection in methanolic HCl
to afford final compounds 47 and 48.
sarcosine and paraformaldehyde in refluxing toluene yielded N-
methylpyrrolidine XXIII. Removal of the acetal protecting
group using lithium chloride in a DMSO−H2O solvent system
under microwave conditions furnished the corresponding
aldehyde which was then converted to (E)-3-pyridin-2-ylacrylic
acid tert-butyl ester XXIV via HWE reaction with diethyl tert-
butyl phosphonoacetate. Selective lithium hydroxide-mediated
hydrolysis of the ethyl ester followed by HATU-mediated
coupling to 4-chloroaniline gave pyrrolidine amide XXV.
Cleavage of the tert-butyl ester with TFA followed by coupling
to 1,2-diaminobenzene using HATU furnished target com-
pound 61.
Synthesis of Compounds 5, 60, 62, and 63. Schemes
and experimental methods for the synthesis of rac-(trans-3,4)-
disubstituted pyrrolidine 2-fluoro-4-vinylphenyl linker analogue
60, rac-(trans-3,4)-disubstituted pyrrolidine 5-vinylpyrazin-2-yl
linker analogue 62, and rac-(trans-3,4)-disubstituted pyrrolidine
5-vinylpyradizin-2-yl linker analogue 63 can be found in the
Supporting Information. A scheme and experimental methods
for the enantioselective synthesis of compounds 5 (3R,4S) and
Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine
5-Vinylpyridin-2-yl Linker Analogue 64 (Scheme 7).
Synthesis of compound 64 began with isopropyl magnesium
chloride-mediated formylation of 5-bromo-2-iodopyridine with
DMF followed by HWE reaction to furnish substituted ethyl
acrylate XX. Iminium ylide cycloaddition of ethyl acrylate XX
with sarcosine and paraformaldehyde in refluxing toluene
produced the N-methyl pyrrolidine ester XXI. Intermediate
XXI was then converted to final compound 64 in a series of
transformations analogous to those for the conversion of
intermediate XI to rac-(trans-3,4)-disubstitued pyrrolidine R2
analogues (Scheme 3).
Synthesis of rac-(trans-3,4)-Disubstituted Pyrrolidine
6-Vinylpyridin-3-yl Linker Analogue 61 (Scheme 8).
Synthesis of compound 61 began with protection of 4-
bromobenzaldehyde as its [1,3]dioxolane acetal followed by
Heck reaction with ethyl acrylate to furnish substituted ethyl
acrylate XXII. Iminium ylide cycloaddition of XXII with
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65 (3S,4R), the corresponding enantiomers of rac-(trans-3,4)-
disubstituted pyrrolidine 15, are available in the Supporting
Information.
RESULTS AND DISCUSSION
■
Lead Optimization Process and Parameters. We
selected the p21 reporter gene assay and liver microsome
stability assay as a primary screening system to guide our
medicinal chemistry efforts because the goal of our
optimization campaign was to improve compound metabolic
stability while maintaining in-cell activity against the class I
HDACs comparable or superior to entinostat (Figure 1B), a
clinical stage o-aminoanilide HDAC inhibitor. Representative
compounds from structurally distinct subseries were also tested
in a solubility assay to focus optimization efforts on subseries
with comparable or improved solubility relative to 1 so as to
broaden future formulation and dosing administration options.
Compounds with good in-cell HDAC inhibitory activity,
comparable or improved solubility relative to 1, and improved
in vitro metabolic stability were then assessed in single-dose
intravenous and oral pharmacokinetic studies in rodents
(mostly BALB/C nude mice). Compounds with good
pharmacokinetic profiles were then further prioritized through
Figure 3. Docked conformational alignment by Glide software (see
Experimental Section) of a prototypical trans-3,4-disubstituted
pyrrolidine scaffold core (blue sticks) with that of the pyrrolidine-
based side-chain analogues of 4 (yellow sticks). For clarity, HDAC1
homology model used for docking is not shown.
Figure 4. Generalized structure of the trans-3,4-disubstituted
pyrrolidine series.
̀
cytochrome P450 (CYP) and human ether-a-go-go-related
gene (hERG) counter-screening. The optimized leads arising
from this process were then spot-checked in a cell-free
biochemical HDAC inhibition panel before proceeding to in
vivo efficacy and biomarker studies.
and a pharmacokinetic profile suitable for in vivo efficacy and
biomarker studies.
R1 Position SAR. Our optimization effort began with the
exploration of various R1 substituents while fixing R2 and the
linker region as a simple methyl group and the standard
cinnamide linker, respectively. Compounds with aromatic R1
substituents containing electron-withdrawing groups in the para
position of the aromatic ring generally had reasonable in vitro
microsomal stability (compounds 12, 13, 16, 19, 20 in Table 1)
whereas the p-fluoro and p-chloro substituted compounds 12
and 13 were preferred for obtaining both moderate microsomal
stability and in-cell target activity. In contrast, meta and ortho
monosubstituted aromatic R1 substituents were well-tolerated
in terms of target activity but did not result in good microsomal
stability (compounds 21−25). Combining meta or ortho
substituents with p-fluoro or p-chloro substituents (compounds
26−33) did not lead to any obvious improvement in activity or
microsomal stability as compared to the monosubstituted p-
fluoro or p-chloro derivatives 12 and 13. Simple pyridinyl R1
analogues 35 and 37 also displayed reasonable activity and
stability, whereas pyridinyl R1 analogue 34 had decreased in-cell
target activity. Finally, we explored various aliphatic R1
substituents but found that none of these compounds (39−
42) had both robust in-cell target activity and good microsomal
stability. In summary, these results indicated that the optimal
choice of substituent for the R1 position is either a p-fluoro- or
p-chloro-substituted phenyl ring. Accordingly, in our subse-
quent studies of the R2 and linker region SAR, we generally
fixed the R1 position as one of these two substituents.
Modification of the Morpholine Side Chain. Metabolite
identification studies indicated that the 2-morpholin-4-
ylethylamino side chain is a major site of oxidative metabolism
for compound 1 (see Supporting Information for details). We
initially explored two approaches to reduce oxidative
metabolism at this site: (1) replacement of the morpholine
moiety; (2) conformational restriction of the alkyl amino side
chain. Whereas replacement of the morpholine moiety with a
simple primary hydroxyl group to generate 2 led to improved
microsomal stability (Figure 2), not surprisingly the removal of
the basic amine also lowered compound solubility. Alter-
natively, side-chain cyclization (compound 3) combined with
addition of a solubilizing dialkylamino group to give 4 led to
both improved in vitro metabolic stability and vastly improved
solubility (Figure 2). Notably, five-membered ring cyclization
was found to be superior to four- or six-membered ring
cyclization in terms of p21 induction activity (see compounds
6−10 in Supporting Information Table S1). Despite the
improved in vitro metabolic stability and solubility of 4, this
compound still had high in vivo clearance, albeit lower than
compound 1 (Figure 2). Therefore, our subsequent efforts
focused on a distinct trans-3,4-disubstituted pyrrolidine-based
scaffold inspired by our previous experience with the trans-3,4-
disubstituted pyrrolidine core in another lead optimization
program and supported by docked conformational alignment
modeling with 4 (Figure 3).
Systematic trans-3,4-Disubstituted Pyrrolidine Opti-
mization. As briefly summarized above, due to the difficulties
we encountered in optimizing either compound 1 or its side-
chain-cyclized analogues, we focused the majority of our efforts
on systematically exploring and optimizing the trans-3,4-
disubstituted pyrrolidine scaffold. In this respect, we sequen-
tially varied the R1, R2, and linker (L) positions (Figure 4) to
identify an optimal combination of R1, R2, and linker
substructures to achieve both good in-cell target inhibition
R2 Position SAR. While fixing R1 and the linker region as a
p-chloro-substituted phenyl ring and cinnamide linker,
respectively, we explored a range of saturated and unsaturated
R2 substituents (Table 2), with the goal of probing both the
steric requirements of this region as well as the effect of
modulating the basicity of the pyrrolidine nitrogen on the
overall properties of the resulting analogues. In terms of steric
requirements, we found that a relatively wide range of
substituents were well-tolerated in this region, from simple
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Table 1. Structure−Activity and Structure−Property
Relationships for trans-3,4-Disubstituted Pyrrolidines in the
R1 Region
Table 2. Structure−Activity and Structure−Property
Relationships for trans-3,4-Disubstituted Pyrrolidines in the
R2 Region
a
P21 RP3 is the relative fold increase in luciferase signal in the p21
reporter gene assay compared to clinical HDACi entinostat (both test
compound and comparator were evaluated at 3 μM in duplicate or
b
triplicate) and normalized by internal GFP transfection control. HLM
and MLM are the human and mouse hepatic clearance in units of mL/
min/kg as calculated from the intrinsic microsome clearance (see
Experimental Section for details).
modifications on the basis of in vitro assays alone, so we
selected representative R2 analogues with good p21 induction
activity and microsomal stability for single-dose pharmacoki-
netic evaluation (see below).
Linker Region SAR. While fixing R1 and R2 as a p-chloro-
substituted phenyl ring and simple methyl group, respectively,
we explored various fine-tuning modifications of the cinnamide
phenyl ring in the linker region (L), including a nitrogen walk
(compounds 61−64) and fluorine substitution (compound 60)
(Table 3). Because these relatively conservative changes were
all well-tolerated in terms of both in-cell target activity and
microsomal stability, we selected representative compounds for
single-dose pharmacokinetic evaluation (see below).
a
P21 RP3 is the relative fold increase in luciferase signal in the p21
reporter gene assay compared to clinical HDACi entinostat (both test
compound and comparator were evaluated at 3 μM in duplicate or
triplicate) and normalized by internal GFP transfection control. HLM
and MLM are the human and mouse hepatic clearance in units of mL/
min/kg as calculated from the intrinsic microsome clearance (see
Experimental Section for details).
b
Pharmacokinetic Evaluation of Representative trans-
3,4-Disubstituted Pyrrolidines. We selected 14 trans-3,4-
disubstituted pyrrolidines, with various R1, R2, and linker region
moieties for intravenous and oral single-dose pharmacokinetic
studies (Table 4) based on the aforementioned in vitro SAR
results. Compounds 12, 44, 63, and 64 had particularly high in
vivo clearance generally associated with low to moderate
exposure. We presumed that the high in vivo clearance of these
compounds was due to clearance pathways acting on these
compounds that were not present in the in vitro microsomal
assay system. Compounds 53 and 59 had moderate in vivo
methyl substitution (compound 13), to tert-butyl (compound
52), pyrimidinyl (compound 57), and tetrahydropyranyl
(compound 59) substitutions. Moreover, electron-withdrawing
alkoxy substituents (for example, compounds 49, 51, 53) were
also generally well-tolerated in terms of both in-cell target
activity and microsomal stability. In contrast, acyl substituents
such as those found in compounds 55 and 56 were less well-
tolerated, either leading to suboptimal p21 induction activity or
poorer microsomal stability, respectively. Overall, we were
unable to fully distinguish beneficial from harmful R2
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Table 3. Structure−Activity and Structure−Property Relationships for Linker Variation (L) within the trans-3,4-Disubstituted
Pyrrolidine Scaffold
a
P21 RP3 is the relative fold increase in luciferase signal in the p21 reporter gene assay compared to clinical HDACi entinostat (both test compound
b
and comparator were evaluated at 3 μM in duplicate or triplicate) and normalized by internal GFP transfection control. HLM and MLM are the
human and mouse hepatic clearance in units of mL/min/kg as calculated from the intrinsic microsome clearance (see Experimental Section for
c
details). Estimated to be at least 100% based on 4-F-phenyl analogue and SAR correlation between 4-F-phenyl and 4-Cl-phenyl subseries (data not
shown).
Table 4. Pharmacokinetic Parameters for Selected trans-3,4-Disubstituted Pyrrolidines with Acceptable p21 Activity and Mouse
Liver Microsome Stability
a
compound
Tmax (h)
Cmax (ng/mL)
T1/2 (h)
AUC (h·ng/mL)
CL (mL/min/kg)
Vss (L)
F (%)
12
2
170
324
893
450
262
331
957
1100
113
337
315
828
33
5.0
14
624
2946
6316
2544
911
127
39
39
58
53
86
20
33
60
27
39
23
120
79
16
8.6
6.9
9.2
7.2
32
4.1
3.7
31
6.6
5.6
3
95
75
13
1
5 (3R,4S)
0.25
1
3.4
6.2
4.0
5.4
15
100
89
65 (3S,4R)
26
44
49
51
52
53
59
60
63
64
1
29
2
2077
7096
5653
813
100
58
0.25
1
6.5
6.8
1.1
4.9
3.4
ND
4.6
100
59
2
2
743
12
0.17
2
1002
3566
116
23
49
4
19
4
9
0.17
60
204
19
a
Compounds were dosed in nude mice at 10 mpk po and 2 mpk iv except for 12 and 64 which were dosed at 5 mpk po and 1 mpk iv and 63 which
was dosed in rat rather than nude mouse. More details for PK studies can be found in the Experimental Section.
clearance but relatively low bioavailability resulting in moderate
exposure. Compounds 26 and 52 had moderate bioavailability
but also moderately high in vivo clearance. In the case of
compound 52, we presumed that the relatively high lip-
ophilicity of this compound (cLogP = 5.4) relative to the other
analogues under investigation contributed to its higher-than-
expected in vivo clearance. The remaining compounds, 13, 5
(3R,4S) and its corresponding enantiomer 65 (3S,4R), 49, 51,
and 60, demonstrated reasonable overall pharmacokinetic
properties, resulting in good exposure and prompting us to
conduct more detailed in vitro selectivity assays to select
potential preclinical leads for in vivo efficacy and biomarker
studies.
In Vitro Selectivity Panel Assessment for Selected
trans-3,4-Disubstituted Pyrrolidines. To select compounds
not only suitable for in vivo efficacy and biomarker studies but
also having good potential to ultimately progress to the clinic,
we evaluated the CYP and hERG inhibition profiles for the
aforementioned compounds 5, 65, 49, 51, and 60 that had
reasonable pharmacokinetic properties (Table 5). Compounds
49 and 51 demonstrated significant inhibition (less than 10
μM) of several CYP isoforms and were therefore deprioritized.
Compounds 5, 65, and 60 were found to have acceptable CYP
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Table 5. In Vitro Selectivity of Representative trans-3,4-Disubstituted Pyrrolidines toward HDACs, CYPs, and hERG
a
b
HDAC isoform IC50
CYP isoform IC50
hERG
c
compound
1
2
3
4
5
6
7
8
9
10
3.4
11
14
3A4 2D6 2C9 2C19 1A2
IC50
5
0.06
0.14
0.1
0.25
0.27
0.22
0.23
0.20
0.23
13
36
3.5
>30
>30
>30
ND
ND
>30
>30
>30
ND
ND
>30
>30
>30
ND
ND
16
13
14
21
4
5
32
9
29
23
13
3
43
43
21
10
7
42
50
28
26
7
10
5
65
60
49
51
1.1
5.3
1.6
5
20
>30
ND
ND
46
43
7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2
2
15
ND
3
0.7
4
a
HDAC enzyme inhibition assays were performed by Reaction Biology Corporation according to their standard assay protocols (www.
b
reactionbiology.com, see Experimental Section for additional information). ND = not determined. CYP isoform inhibition assays were performed
using the LCMS method.31 hERG inhibition assay was performed using PatchXpress methodology.32 All IC50 measurements are reported in units of
c
μM. ND = not determined.
inhibition profiles and were thus assessed in an HDAC isoform
panel selectivity assay. Consistent with our expectation of
compounds with the o-aminoanilide zinc-binding group, these
molecules were confirmed to be selective inhibitors of HDAC
isoforms 1, 2, and 3. Moreover, compound 5 was particularly
active toward HDAC1. Therefore, based on the overall in vitro
and in vivo properties of 5, 65, and 60, we selected compound
5 as our lead for in vitro and in vivo efficacy and biomarker
studies as well as further preclinical development.
In Vitro Efficacy and Mechanistic Studies of Com-
pound 5. While the intended primary indication for our class I
HDAC-selective inhibitors was hepatocellular carcinoma, we
decided to use both hepatocellular carcinoma (HCC) and
gastric cancer (GC) cell lines to increase our confidence in the
basic in vitro efficacy and cell-based mechanistic and functional
studies for 5. Therefore, the antiproliferative activity of 5 was
evaluated in a panel of HCC and GC cell lines, using the
clinical HDAC inhibitor entinostat and the FDA-approved
HDAC inhibitor vorinostat as comparators. As assessed by the
concentration required to inhibit 90% of cell growth at 72 h, 5
demonstrated better antiproliferative activity than either
entinostat or vorinostat in all cell lines tested (Table 6).
Mechanistically, the antiproliferative effect of 5 correlated with
the dose-dependent induction of endogenous acetylated
histone H3 and p21 after 24 h treatment within a concentration
range consistent with the GI90 observed in the same cell line
(Figure 5A). Functionally, induction of endogenous acetylated
histone H3 and p21 corresponded with a dose-dependent
decrease in S phase cells at 24 h (consistent with G1/G2 cell
cycle arrest typically observed for HDAC inhibitors) and
subsequent induction of apoptosis at 48 h (representative cell
lines, Figure 5B and 5C). Taken together, these results
demonstrate that the potent antiproliferative efficacy of 5 is
due to HDAC-dependent in-cell induction of histone
acetylation leading to concomitant increase in the expression
of endogenous cyclin-dependent kinase inhibitor p21, ulti-
mately resulting in cell cycle arrest and apoptosis.
In Vivo Efficacy and PK/PD Studies of 5. Having
demonstrated that 5 can potently inhibit HCC and GC cancer
cell growth in vitro, we next set out to assess its tumor growth
inhibitory effects in subcutaneous cell line and primary tumor-
derived xenograft models.
We first evaluated 5 in the HCC cell line-derived xenograft
model BEL-7404. At an oral dose of 50 mg/kg once daily or
100 mg/kg every other day, 5 significantly inhibited the growth
of established BEL-7404 tumors (tumor growth inhibition rate
>60%) with acceptable body weight loss (less than 10% relative
to the mean starting weight of the mice). Terminal
pharmacokinetic analysis indicated that both dosing schedules
resulted in total trough concentrations of 5 in the tumor that
were 4-fold above the in vitro GI90 for the corresponding BEL-
7404 cell line. This corresponded to consistent pharmacody-
namic induction of acetylated histone H3 in the tumor from
zero to 24 h after administration of the last dose of 5, as
assessed by Western blot (Figure 6). Taken together, these
results demonstrate that 5 has significant in vivo antitumor
activity and, while not ruling out unforeseen off-target effects,
suggest that this efficacy is linked to target engagement in the
tumor as assessed by pharmacokinetic and pharmacodynamic
measurements.
Table 6. Antiproliferative Activity of Compound 5 versus
Clinical Comparators Entinostat and Vorinostat in Asian
Hepatocellular Carcinoma and Gastric Cancer Cell Lines
With preliminary in vivo efficacy results for 5 in hand, we
proceeded to conduct a pilot study with 5 to assess the
feasibility of using primary tumor-derived xenograft models to
identify candidate predictive biomarkers that could indicate
which tumors (or patients) are more likely to respond to
treatment with 5, with the future goal of developing a
companion diagnostic for patient selection. We chose to
conduct this pilot study with primary tumor-derived subcuta-
neous xenograft models because, compared to traditional cell
line-derived subcutaneous xenograft models, primary tumor-
derived models better reflect the histology of the original
patient tumor and are presumably therefore more likely to
resemble the genetic and epigenetic status of the original
tumor.24
a
a
a
cell line
5 (GI90)
entinostat (GI90)
vorinostat (GI90)
SMMC-7721
BEL-7402
BEL-7404
SKHEP1
1.9
1.2
3.8
2.5
1.3
1.4
2
11
8
20
17
>30
13
18
17
QGY-7701
SGC-7901
MGC8−03
BGC-823
HGC-27
>30
2.5
8.9
7.1
>30
>30
9.4
10.8
7.5
>30
2.2
1
a
GI90 is the concentration (reported in μM) required to inhibit cell
growth by 90% after 72 h compound treatment using the water-soluble
tetrazolium (WST) dye assay with eight-point dose−response (half-
log serial dilutions in triplicate). See Experimental Section for further
details.
We selected three patient-derived HCC tumor models with
different associated clinical and molecular characteristics to
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Figure 5. Mechanistic and functional effects of compound 5 in representative hepatocellular carcinoma and gastric cancer cell lines. (A) Dose-
dependent induction of endogenous p21 and acetylated histone H3 in response to 24 h treatment with clinical HDACi comparator mocetinostat or
compound 5 in SGC-7901 cells. (B) Dose-dependent induction of cell cycle arrest by compound 5 in SGC-7901 cells at 24 h. (C) Induction of
apoptosis at 48 h by compound 5 at 5 μM in SMMC-7721 cells. See Experimental Section for details.
Figure 6. In vivo antitumor efficacy and PK/PD correlation for compound 5. (A) Effect of compound 5 on growth of an established BEL-7404
tumor in nude mice (n = 9−10) over four weeks. (B) Concentration of compound 5 in tumor tissue over 24 h period subsequent to the last dose.
(C) Acetylated histone H3 induction in tumor tissue sampled at various time points after sacrificing mice subsequent to the last dose of compound 5.
assess response toward 5 and to identify candidate predictive
biomarkers (see Supporting Information Table S5). Each
model was treated with 5 at 100 mpk every other day (the
efficacious dose in cell line-derived liver cancer xenografts) for
approximately four weeks. In regards to biomarker hypothesis
generation, while we recognized that global expression and
methyl-ome profiling are ideal for unbiased biomarker
discovery, practical considerations led us to choose instead
the candidate gene approach for our pilot study. Thus, the
promoter CpG methylation status and mRNA expression level
for 22 candidate genes (Table 7) were assessed on treatment
days 0, 7, 15 and the last day of the study (see Supporting
Information for more details on the study design) using
Methyl-Profiler PCR arrays allowing for parallel analysis of up
to 24 genes at a time (our rationale for the selection of these 22
candidate genes is detailed below). Histone-H3 acetylation and
pharmacokinetic parameters were assessed by immunohisto-
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Table 7. Twenty-Two Candidate Genes Evaluated as Potential Biomarkers for Tumor Response Prediction
number of
methylation frequency in
patients
detected in
microarray?
up-regulated by 5 in
a
b
c
c
candidate gene category description
gene ID
function
HCC patients, %
evaluated
microarray?
TSGs methylated in patients; up-
regulated by 5
CACNA1G
proliferation
20−40
85
Y
Y
CCND2
CDKN1C
CDKN2B
CTGF
cell cycle
cell cycle
cell cycle
adhesion
adhesion
apoptosis
adhesion
invasion
cell cycle
60−80
20−40
40−60
0−20
40−60
60−80
0−20
26
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
20
301
24
FN1
24
SOCS1
THBS1
TIMP3
RASSF1
250
79
20−40
60−80
238
648
TSGs methylated in patients; not up-
regulated by 5
TFPI2
invasion
40−60
40−60
20−40
80−100
34
Y
Y
Y
N
N
N
N
N
CDKN2A
CDH1
cell cycle
1380
574
26
adhesion
TSGs methylated in patients; not
detected in microarray
SPINT2
cell movement
HIC1
cell cycle
apoptosis
apoptosis
80−100
20−40
not known
67
N
N
Y
N
N
Y
PYCARD
IGFBP3
36
up-regulated by 5; no patient
methylation information available
NA
CEBPA
CDKN1A
SDC2
differentiation
cell cycle
not known
not known
not known
not known
not known
NA
NA
NA
NA
NA
Y
Y
Y
Y
Y
Y
Y
proliferation
growth
Y
oncogenes down-regulated by 5
WNT5A
CFLAR
down-regulated
down-regulated
apoptosis
a
The candidate genes were grouped into five categories according to their promoter CpG island methylation status in HCC patient tissues, their
response to treatment with compound 5 in in vitro microarray studies, their detect-ability in our microarray studies, and their functional classification
b
c
as tumor suppressor genes (TSGs) or oncogenes (OGs). Microarray data were analyzed by Ingenuity Pathway Analysis.25 Information from
PubMeth public database of promoter CpG methylation profiling in cancer tissues.26
chemistry and compound 5 bioanalysis, respectively, on the last
day of the study.
of 5 and the induction of acetylated histone H3 resulting from
target engagement (as measured by immunohistochemistry
over multiple time points) were somewhat lower in the
nonresponding model (Figure 7B and 7C). This result suggests
that the apparent lack of response toward 5 for model A may
have been in part or in whole due to differences in compound
exposure and target engagement in this model compared to the
other two models.
Analysis of the CpG methylation status of the 22 candidate
genes in the three primary tumor models revealed a significant
difference in the methylation pattern between the responding
models and the nonresponding model. In particular, in the
responding models B and C, only one and two genes had CpG
promoter methylation, respectively. In contrast, in the non-
responding model A, seven out of the twenty-two candidate
genes were methylated (Table 8). These results suggest that the
severity of baseline epigenetic silencing of these candidate
(mostly tumor suppressor) genes may be predictive of tumor
response to 5, or at least may contribute to the refractory
nature of nonresponding model A. Hypothetically, if the
nonresponsiveness of model A were functionally confirmed to
be due to heavy promoter DNA methylation, then one
potential approach to overcome this therapeutic resistance
would be to treat patients with such heavy baseline methylation
with a combination of HDAC and DNA methyltransferase
inhibitors.27
The 22 candidate genes were selected based upon the results
of microarray gene expression profiling of 5 in liver cancer cell
lines, qRT-PCR confirmation of treatment-responsive genes,
and a public database of liver cancer tumor suppressor genes
found to be methylated frequently in patients26 (see Supporting
Information for further details). Broadly, the candidate genes
were grouped into five categories: (1) tumor suppressor genes
(TSGs) with frequent CpG island promoter methylation in
patient tissue and up-regulated by 5 in microarray profiling
studies; (2) TSGs with frequent CpG island promoter
methylation in patient tissue and not up-regulated by 5 in
microarray profiling studies; (3) TSGs with frequent CpG
island promoter methylation in patient tissue but not detected
in microarray profiling studies due to low hybridization signal;
(4) TSGs not known to be methylated in patient tissue but up-
regulated by 5 in microarray profiling studies; (5) candidate
oncogenes (OGs) down-regulated by 5 in microarray profiling
studies (Table 7). We hypothesized that responsive tumors
would have a qualitatively distinct epigenetic signature from
nonresponsive tumors and that such a signature, if confirmed
through functional studies and a larger tumor cohort, could
potentially be used to select patients more likely to respond to
treatment with 5.
Patient tumor models B and C demonstrated a significant
response to 5 (∼50% tumor growth inhibition) while patient
tumor model A did not respond at all to compound treatment
(Figure 7A). PK/PD analysis revealed that the tumor exposure
With regard to the ability of 5 to reactivate epigenetically
silenced tumor suppressor genes, one hypermethylated TSG in
one responder, SPINT2, was significantly up-regulated upon
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Figure 7. In vivo efficacy with PK/PD correlation for compound 5 in three patient tumor-derived xenograft models (top row = patient A; middle
row = patient B; bottom row = patient C): (A) tumor growth curves for patients A−C treated with vehicle (15% Kleptose, pH 4.5 buffered with
tartaric acid) or compound 5 Q2D PO 100 mpk. (B) Tumor concentration of compound 5 vs time for patients A−C (log 10 scale). Each square
represents the concentration of 5 measured from a single mouse tumor. (C) Acetylated histone H3 IHC score (0−3) vs time for patients A−C
(representative IHC sections scored as 0, 1, 2, or 3 available in Supporting Information Figure S6). Each square represents the IHC score from a
single mouse tumor. See Experimental Section for further detail.
Table 8. Promoter CpG Island Methylation Status for Genes
Methylated in at Least One out of Three Patients on Day 0,
Start of Weeks 2 and 3, and Last Day of Study
functional consequences of SPINT2 reactivation. Validation of
the SPINT2 biomarker would suggest a rational means of
selecting patients for combination therapy with 5 and MET
kinase inhibitors.
a
b
b
b
gene ID
patient A
patient B
patient C
In summary, the pilot epigenetic biomarker study of 5 in
three primary liver cancer xenograft models indicated a
potential correlation between baseline promoter methylation
status of key tumor suppressor genes and tumor response, with
heavy promoter methylation potentially predictive of poor
response and suggesting combination therapy with DNA
methyltransferase inhibitors. Furthermore, in one responder, a
specific tumor suppressor gene with CpG island hyper-
methylation, SPINT2, was reactivated in response to
compound treatment. The expression and promoter DNA
methylation status of this gene deserves further assessment as a
potential biomarker for response prediction and for combina-
tion therapy of 5 with MET kinase inhibitors. These
preliminary findings should be interpreted in the context of
the PK/PD data, which indicated somewhat lower compound 5
tumor exposure and target modulation for the nonresponding
model.
CACNA1G
CCND2
CDKN1C
SOCS1
X
X
X
X
X
X
X
−
−
−
−
X
−
−
−
−
−
−
X
−
X
RASSF1
TFPI2
SPINT2
a
Only 7 out of 22 candidate genes are listed (the remaining 15 genes,
CDKN2B, CTGF, FN1, THBS1, TIMP3, CDKNA2, CDH1, HIC1,
PYCARD, IGFBP3, CEBPA, CDKN1A, SDC2, WNT5A, CFLAR,
were not methylated in any of the three patients). “X” = methylated
as determined by methylation-specific PCR; “−” = not methylated as
determined by methylation-specific PCR. Methylation status did not
change over the course of the study. See Experimental Section for
details.
b
treatment with 5. While a few other genes were up-regulated in
both responding models at one or more time points (e.g.,
TIMP3, p21/CDKN1A), only the SPINT2 gene was both
hypermethylated and responsive to treatment with 5 (Figure
8). Intriguingly, SPINT2 is an inhibitor of the HGF/MET
pathway and has been identified as a TSG in certain cancers.28
Future follow-up studies to these pilot experiments should
evaluate the utility of SPINT2 as a potential biomarker in
additional primary HCC tumor models as well as probe the
CONCLUSION
■
Through a combination of scaffold hopping and structure−
property relationship studies, we have developed an orally
bioavailable HDAC inhibitor with selectivity toward the class I
HDAC family and particular potency toward HDAC1. We
demonstrated that this compound is more potent at inhibiting
liver cancer cell growth in vitro as compared to clinical HDAC
inhibitors entinostat and vorinostat and that its antiproliferative
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Figure 8. mRNA expression levels of candidate genes with absolute fold change greater than two at any of three time points (day 7, day 15, and last
day of study) in the three patient models. CCND2, CDKN2B, FN1, RASSF1, CDKN2A, HIC1, CEBPA, SDC2, and CFLAR are not shown because
these genes did not change significantly throughout the course of the experiment. Expression levels were determined by qRT-PCR, normalized to
GAPDH, and reported as fold-change relative to day 0 (expression level before treatment). See Experimental Section for further details.
phosphate buffer (pH = 7.4), 10 mM NADPH, and 1 μM of test
compound in a total volume of 400 μL. After prewarming at 37 °C for
10 min, the NADPH was added to initiate the reaction. The reaction
was terminated after 0, 3, 6, 9, 15, and 30 min by adding 150 μL of 100
ng/mL of tolbutamide (internal standard) in ice-cold methanol into
300 μL of incubation mixtures. Samples were then centrifuged at 4000
rpm for 10 min at 4 °C. The supernatant was then analyzed by LC-
MS/MS.
effects correlate to markers of in-cell target engagement such as
acetylated histone H3 and p21/CDKN1A. Moreover, these in
vitro effects translated into significant efficacy in both cell-based
and patient tumor-derived xenograft models that appeared to
correlate well to compound exposure levels and to changes in
the same pharmacodynamic markers used to assess in-cell
target engagement. One out of the three patient tumor models
did not respond to treatment with our optimized HDAC
inhibitor and, compared to the two responding models, had
much higher levels of DNA methylation in the promoter
regions of a panel of tumor suppressor genes known to be
frequently epigenetically silenced in liver cancer patients.
Furthermore, in one of the responding models, we identified
SPINT2, an endogenous inhibitor of the progrowth HGF/
MET pathway, as an epigenetically silenced gene whose
expression could be reactivated by treatment with our HDAC
inhibitor. Future research should seek to confirm the potential
clinical utility of our candidate DNA methylation and tumor
suppressor gene biomarkers in a larger cohort of patient tumor-
derived models, through functional studies of these biomarkers,
and in the context of combination therapy with either MET
kinase inhibitors, DNA methyltransferase inhibitors, or the
current standard of care in HCC, sorafenib.
Solubility Assay. DMSO stock solution (10 mM) of compound
was prepared and divided into two portions. For one portion, the
DMSO was evaporated to dryness and the remaining was redissolved
in 50 mM phosphate buffer (pH 6.5). The mixture was stirred and
shaken for 1 and 2 h, respectively. After the mixture was allowed to
stand overnight, the solution was filtered before HPLC analysis. The
other portion was used for the calibration curve preparation by diluting
the DMSO stock solution using the same PBS buffer mentioned above
to a series of solutions with concentration range of 50−500 μM.
Cytochrome P450 Inhibition Assay. A cocktail method that
enables simultaneous incubation and measurement of compound
inhibitory activity against CYP isoforms CYP3A4, CYP2D6, CYP2C9,
CYP2C19, and CYP1A2 was developed with modification from
previously reported methods.31 Each incubated mixture contained
0.125 mg/mL human liver microsome (protein content), 5 mM
MgCl2, 100 mM potassium phosphate buffer (pH= 7.4), substrate
cocktail, various concentrations of test compound, and 2 mM NADPH
in a total volume of 200 μL. The final concentrations of each CYP
substrate were at the reported literature Km values31 (see Supporting
Information Table S2 for further details). Before addition of NADPH
to initiate the reaction, mixtures were prewarmed at 37 °C for 10 min,
and the reaction was terminated after 15 min of incubation by adding
100 μL of ice cold methanol containing 3 μM dextrorphan as an
internal standard (IS). Samples were then centrifuged at 4000 rpm for
10 min at 4 °C. The supernatant was then analyzed by LC-MS/MS.
hERG Inhibition Assay. The inhibitory effect of compounds
against the hERG channel was measured on an automated patch clamp
system.32 CHO cells expressing hERG were loaded into the system.
Cell solutions and buffer solutions were transferred to specially
designed chips, where cells were attached to each well. Pressure
changes were applied in each well until a gigaseal status and a whole
cell configuration were achieved. After a brief stabilization process, a
predefined pulse procedure was applied in each well, opening hERG
channels and triggering tail currents. The peak of a tail current was
measured. Compounds at various concentrations were then
coincubated with the cell, and the same pulse procedure was repeated
EXPERIMENTAL SECTION
■
Molecular Docking. OPLS_2005 force field of MacroModel29
was used to optimize the initial geometries of the compounds with a
final root-mean-square gradient of 0.01 kcal/mol/Å. The default values
of the optimization parameters and thresholds were retained. Glide XP
was used for compound docking because of its robust energetic terms
on metal−ligand interaction.30 All torsion angles of compound were
released to freely rotate, and 10000 docking runs were adopted during
the XP docking with NE2 atom of His178 and zinc ion as docking
constraints after trying different constraint combinations. Docking
poses were clustered based on heavy atom RMSD values (1 Å), and
top-ranked poses of each compound were selected and viewed
graphically within the Maestro of Schrodinger program (Schrodinger,
̈
̈
L.L.C., New York, NY), and finally displayed in Pymol (Schrodinger,
L.L.C., New York, NY).
̈
Microsomal Stability Assay. Each incubated mixture contained
0.5 mg/mL liver microsome (human or mouse), 100 mM potassium
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to generate tail current data. Amoxicillin and amitriptyline were
employed as negative and positive controls, respectively. Generally, a
compound was tested at a minimum of two concentrations in
duplicate, and thus IC20 or IC50 can be estimated to evaluate
compound inhibitory potential on hERG channels.
PK Analyses. All compounds were evaluated in BALB/C nude
mice (15−20 g, N = 16 per arm) except for compound 63 which was
tested in male Sprague−Dawley rats (250 20 g; N = 3). Compounds
were administered by iv bolus at 2 mg/kg in 5% DMSO, 40% PEG400,
and 55% saline and/or PO (gavage) at 10 mg/kg in 0.445%
microcrystalline cellulose microsuspension. In each species, blood
samples (150 μL) were collected at eight time points (po) or nine
time points (iv) into sodium heparin centrifuge tubes, and plasma
samples were then isolated by centrifugation and stored at −20 °C
prior to analysis. Plasma concentrations were determined by LCMS/
MS and data were analyzed by noncompartmental methods using
WinNonlin Pro (Pharsight Corp., Mountain View, CA).
Biology General Comments. Brief descriptions of the biological
protocols used to generate data in this manuscript can be found below.
For further details regarding experimental protocols, cell line origins,
culture conditions, and reagent manufacturers, see Supporting
Information.
HDAC Enzyme Inhibition Assay. HDAC enzyme inhibition
assays were conducted by Reaction Biology Corporation as described
(www.reactionbiology.com) using a 10-point dose response curve with
half-log serial dilutions, fluorogenic peptide from p53 residues 379−
382 (RHKKAc) at 50 μM as substrate, and trichostatin A as a positive
control.
p21 Reporter Gene Assay. Compounds were tested for their
ability to induce p21 gene expression using a reporter gene assay
involving HeLa cells transfected with a p21 promoter-luciferase
construct. The p21 promoter contained the Sp1/Sp3 binding site for
HDAC but not the upstream p53 binding site. Briefly, the day before
transfection, HeLa cells were seeded at 11 000 cells/well in a 96-well
culture plate and incubated at 37 °C in 5% CO2 overnight. For
transfection, the medium was removed and replaced with 100 μL/well
transfection media previously prepared as follows: 5 μL of serum-free
DMEM, 0.15 μL of Fugene 6 reagent, 40 ng of p21-luc, 10 ng of GFP
were mixed gently and incubated at room temperature for 30 min, and
then 98 μL of DMEM (with 10% FBS, 1% penicillin, and
streptomycin) was added to the DNA:Fugene 6 reagent complex
and mixed gently. After the cells were incubated for 24 h at 37 °C in
5% CO2, fresh media and test compounds were added to the wells and
the cells further incubated for 15 h at 37 °C in 5% CO2. Cells were
lysed by adding 80 μL/well of a cell culture lysis reagent (Promega). A
50 μL amount of each lysate was taken for GFP detection using an
excitation wavelength of 486 nm and detection at 527 nm. A 100 μL
amount of Luciferase assay reagent (Promega) was then added to
every 20 μL of cell lysate for luminometer detection.
Cell Proliferation (water-soluble tetrazolium dye) Assay.
Cells were seeded in 96-well culture plates (200 μL/well at different
seeding concentrations depending on cell type) and incubated
overnight at 37 °C in 5% CO2. After adding compound dilutions
(typically eight-point half-log serial dilutions in triplicate) to the cells
(DMSO concentration kept below 0.5%), the cells were incubated at
37 °C in 5% CO2 for 72 h. The effects on proliferation were
determined by addition of CCK-8 reagent (Dojindo) according to the
manufacturer’s instruction, followed by incubation for 2 h at 37 °C in
5% CO2, and finally recording the absorbance at 450 nm using an
ELISA plate reader.
Apoptosis Assay. An amount of 1 × 105 SMMC-7721 cells was
treated with 5 μM of mocetinostat or 5 for 48 h. Cells were
trypsinized, washed twice with cold PBS, and then resuspended in 1X
Annexin V binding buffer. A 100 μL amount of the cell solution was
incubated with 5 μL of FITC/Annexin V for 15 min at room
temperature. Cell samples were resuspended in 400 μL of 1X Annexin
V binding buffer, and FITC/Annexin V positive cells were quantified
using flow cytometer and CellQuest software (BD Biosciences).
Western Blotting for p21 and Acetylated Histone H3 from
Cells. An amount of 1 × 106 SGC-7901 cells was seeded overnight
and then incubated with indicated concentrations of mocetinostat and
5 for 24 h. Cell extracts were prepared by lysing cultured cells into a
mammalian protein extraction reagent supplemented with EDTA-free
protease inhibitor on ice for 15 min. Supernatants were collected
following centrifugation of lysed cells (15 000 g) for 10 min at 4 °C.
For analysis of total cell lysates, 30 μg of total protein per sample was
resolved by SDSPAGE and transferred onto nitrocellulose membrane.
Membranes with immobilized proteins were probed with indicated
antibodies. The reactive protein bands were visualized using enhanced
chemiluminescence (ECL) detection system.
BEL-7404 Xenograft Efficacy Study. The use and care of
experimental animals was approved by the Institutional Animal Care
and Use Committee, Roche R&D Center (China). Female BALB/c
nu/nu nude mice aged 6−7 weeks were obtained from Beijing
Academy of Military Medical Science, Green violet blue Lab Animal
Technology Co. Ltd. (Beijing, China), and housed in specific
pathogen-free (SPF) conditions. Animals were held for a minimum
of three days for acclimation prior to beginning the study. BEL-7404
suspended in serum-free RPMI-1640 (Invitrogen, Carlsbad, CA) was
injected into the right flank of the nude mouse at 2.0 × 106 cells/
mouse. After the tumor was established and reached 100−150 mm3,
the mice were randomized into 10 mice per group and started to
receive dosing of compound 5 (either QD PO 50 mpk or Q2D PO
100 mpk prepared in (15%) Kleptose-tartaric buffer (pH = 4.5)). The
tumor growth was recorded three times a week from the measurement
of length (L) and width (W) with caliper and calculated as tumor
volume (TV, mm3) = 0.5 × L × W × W (mm). The tumor growth
inhibition (TGI) was calculated as TGI% = (1 − (mean tumor volume
of the treatment group on the first day − mean tumor volume of the
treatment group on the end day)/(mean tumor volume of the control
group on the first day − mean tumor volume of the control group on
the end day)) × 100%.
For PD biomarker evaluation, 50 mg of frozen tumor sample was
dissected on dry ice and lysed in 500 μL of tissue protein extraction
reagent (Thermo Scientific) supplemented with EDTA-free protease
inhibitors in MagNA lyser green beads and homogenized at 5000 rpm
for 4 × 25 s (4 °C) using MagNA lyser (Roche). Tissue proteins were
obtained from the supernatant of tissue homogenate by centrifugation
at 15 000 rpm for 15 min (4 °C) and analyzed by Western blot (50 μg
of total protein per sample).
Patient Tumor-Derived Xenograft Model and PK/PD
Analysis. The use and care of experimental animals was approved
by the Institutional Animal Care and Use Committee, Roche R&D
Center (China). Female BALB/c nu/nu nude mice aged 6−7 weeks
were obtained from Shanghai SLAC Laboratory Animal Company
(Shanghai, China) and housed in specific pathogen-free (SPF)
conditions. The tumor fragments (3 × 3 × 3 mm3) derived from
three HCC cancer patients were harvested for tumor inoculation.
Under sterilized conditions, fragments were subcutaneously implanted
into the right flank of nude mice for tumor development. Mice were
randomized (n = 10 per dosing arm and 15 for biomarker satellite
arm) and treatment initiated when tumors reached approximately 200
(150−250) mm3 as indicated (see Supporting Information for more
study design details). Measurements were taken in the same way as in
the BEL-7404 model study. Single-dose administration was also given
as indicated on the last day of treatment, and the tumors were isolated
at assigned time points (0 to 24 h postdosing) and cut into two pieces.
The first piece from each tumor was subjected to pharmacokinetic
analysis (see PK Analysis). The second piece from each tumor was
subjected to immunohistochemistry. The 8 μm paraffin embedded
Cell Cycle Analysis. An amount of 1 × 105 SGC-7901 cells was
cultured overnight prior to treatment at about 50% confluency. Cells
were treated with indicated concentrations of mocetinostat or 5 for 24
h. Cells were trypsinized and fixed with chilled 70% ethanol for 1 h at
4 °C. The cells were then resuspended in 500 μL of propidium iodide
(PI) solution (50 μg/mL PI, 0.1 mg/mL RNase A and 0.05% Triton
X-100) and incubated for 1 h at room temperature. Cells were washed
three times with PBS and suspended in 500 μL of PBS. PI positive
cells were quantified using flow cytometer and CellQuest software
(BD Biosciences).
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tumor tissue slides were heated in a dry oven at 60 °C for 1 h and then
deparaffinized and hydrolyzed by a Leica autostainer XL ST5010 at
room temperature. Antigen retrieval was carried out at 95−99 °C in
citrate buffer (0.01 M, pH 6.0) heated by a medical microwave at 92−
98 °C for 5 min and then cooled to room temperature. Then
peroxidase-blocking reagent (DAKO, DK-2600, Glostrup Denmark)
was applied to quench the endogenous peroxidase. The slides were
incubated in blocking serum for 20 min and then covered by 100 of μL
rabbit antihuman Ac-H3 (Lys9) antibody (Cell Signaling #9671, 1:200
in TBS/T) at 4 °C overnight. The slides were covered by 100 μL of
DAKO Envision+/HRP, rabbit/mouse after being washed thoroughly
with TBS. One hundred microliters of substrate−chromogen solution
(DAB) was applied to the slides and incubated at room temperature
for 10 min. The slides were rinsed gently with tap water for 15 min.
Counterstain and dehydration were performed with an XL ST5010 at
room temperature. Finally, the slides were mounted with Permount
mounting medium (Fisher Scientific, Miami, FL). The scoring was
carried out by two pathologists independently (see Supporting
Information for representative images and scores).
mRNA and Methylation Analysis by Real Time PCR. Human
xenograft total RNA was extracted with TRIzol (Invitrogen), treated
with DNAaseI (Progmega), and further purified by RNA clean kit
(Qiagen) before PCR. The 22-gene customized qRT-PCR array was
designed by SABiosciences. Genomic DNA control, positive PCR
control, and reverse transcription control were included in the array to
monitor the PCR quality. GAPDH and β-glucuronidase were
housekeeping genes used as internal controls. The reaction system
and conditions were suggested by SABiosciences protocols. Bio-Rad
Chromo4 was used for the qPCR reaction and detection.
Amplification plots and dissociation curves were checked for reaction
validity. ΔCt values were calculated against the average of two
housekeeping genes, and then the fold changes were calculated using
lower expressed samples as control.
DNA methylation status was detected by using the customized
“Methyl-Profiler” DNA methylation PCR array system (SABio-
science). The experiments and analysis were performed by following
the manufacturer’s manual. Briefly, the real time PCR primers for the
22 genes were designed, covering CpG islands in the DNA promoter
regions. Four separate restriction enzyme digestion reactions were
carried out to differentiate the methylation levels. Genomic DNA was
extracted with a DNeasy kit (Qiagen) before the restriction digestions
with the Methyl-Profiler enzyme kit (SABiosciences). The real-time
PCR was performed under the suggested conditions from
SABiosciences on a Bio-Rad Chromo4. The relative amount of each
methylated DNA fraction is calculated using the standard ΔCT
method, normalizing the amount of DNA in each digestion to the total
amount of input DNA in the mock digestion. The quality control for
the digestions was checked by the refractory DNA percentage during
the analysis. Methylation superarray data were analyzed following Web
site instructions (http://www.sabiosciences.com/dna_methylation_
data_analysis.php).
was collected. The mother liquor was concentrated, and the crude
product was recrystallized with ethyl acetate/petroleum ether = 1/4
(v/v). The combined solids were dried in vacuo at 40 °C for 4 h. An
off-white solid (80 g, 77%) was obtained. MS: calcd 209 (MH+), exp
209 (MH+). 1H NMR (CDCl3, 400 MHz) δ ppm 7.25 (m, 1H), 7.00
(m, 1H), 6.77 (m, 2H), 6.29 (broad m, 1H), 3.60 (broad m, 2H), 1.51
(s, 9H).
N-(2-(Acryloylamino)phenyl)carbamic Acid tert-Butyl Ester
(III). To a solution of acrylic acid (2.50 g, 34.7 mmol) in
dichloromethane (80 mL) at 0 °C was added N-methylmorpholine
(4.73 g, 46.8 mmol), followed by isobutyl chloroformate (6.37 g, 46.8
mmol). After 30 min, a solution of (2-aminophenyl)carbamic acid tert-
butyl ester (II) (5.80 g, 27.8 mmol) in dichloromethane (50 mL) was
added dropwise to the refluxing reaction mixture over 30 min. After
the reaction was completed (2 h later), the reaction mixture was
allowed to cool down to room temperature, poured into ice−water,
and extracted with dichloromethane (30 mL × 3). The organic layer
was washed with water, dilute sodium bicarbonate solution, 0.1 M
HCl, water, and brine in turn. The organic layer was dried over sodium
sulfate, filtered, and concentrated in vacuo. The crude solid was
recrystallized from ethyl acetate/petroleum ether = 1/4 (v/v) to
obtain the desired product (2.5 g, 34%). MS: calcd 263 (MH+), exp
1
263 (MH+). H NMR (d6-DMSO, 400 MHz) δ ppm 9.66 (s, 1 H),
8.45 (s, 1 H), 7.47 - 7.63 (m, 2 H), 7.03 - 7.23 (m, 2 H), 6.42 - 6.57
(m, 1 H), 6.27 (dd, J = 17, 2 Hz, 1 H), 5.79 (dd, J = 10, 2 Hz, 1 H),
1.46 (s, 9 H).
(E)-{4-[2-(2-tert-Butoxycarbonylaminophenylcarbamoyl)-
vinyl]phenyl}hydroxyacetic Acid Methyl Ester (IV). To a solution
of (4-bromophenyl)hydroxyacetic acid (31.4 g, 136 mmol) in
methanol (200 mL) was slowly added concentrated sulfuric acid (10
mL) dropwise, and then the reaction was heated to 50 °C for 3 h. The
reaction was neutralized with sodium carbonate, concentrated by
rotary evaporation, then extracted with ethyl acetate. Desired product I
was recrystallized from hexanes/ethyl acetate (25.6 g, 77%) and used
directly in the following step.
A mixture of N-(2-(acryloylamino)phenyl)carbamic acid tert-butyl
ester (III) (23 g, 87.7 mmol), methyl-2-(4-bromophenyl)-2-
hydroxyacetate (25.6 g, 104.5 mmol), tri-o-tolylphosphine (2.8 g, 9.2
mmol), Et3N (35.8 g, 353.8 mmol), and Pd2(dba)3 (4.3 g, 4.7 mmol)
in DMF (400 mL) was heated at 100 °C for 6 h under N2 atmosphere
and monitored by TLC. After being cooled to room temperature, the
mixture was poured into a saturated aqueous solution of NH4Cl and
extracted with ethyl acetate. The organic layer was washed with brine,
dried over Na2SO4, filtered, concentrated in vacuo, and purified by
flash column chromatography (ethyl acetate/petroleum ether 1:2) to
obtain pale yellow solid (IV) (22.4 g, 60%). MS: calcd 427 (MH+),
exp 427 (MH+). 1H NMR (DMSO-d6, 400 MHz) δ ppm 9.71 (s, 1H),
8.48 (s, 1H), 7.64 (d, 2H, J = 8.0 Hz), 7.59 (d, 1H, J = 15.6 Hz), 7.57
(m, 1H), 7.48 (d, 2H, J = 8.0 Hz), 7.14 (m, 2H), 6.92 (d, 1H, J = 15.6,
6.17 (d, 1H, J = 5.2 Hz), 5.21 (d, 1H, J = 4.8 Hz), 3.64 (s, 3H), 1.47
(s, 9H).
Synthetic Chemistry General Comments. All starting materials
were obtained commercially and were used without further
purification. All reported yields are of isolated products and are not
optimized. All final compounds were purified by preparative HPLC on
a reverse phase column using Waters XBridge OBD Phenyl (30 × 100
mm, 5 μm) column or OBD RP18 (30 × 100 mm, 5 μm) column.
LC/MS spectra were obtained using a MicroMass Platform LC
(Waters alliance 2795-ZQ2000). For further details regarding
preparative HPLC and LCMS protocols, see Supporting Information.
NMR spectra were obtained using a Bruker AVANCE 400 MHz NMR
spectrometer. All final compounds have purities greater than 95%
based upon LC/MS and 1H NMR. Enantiomeric excess was
determined by chiral HPLC (see Supporting Information for details).
(2-Aminophenyl)carbamic Acid tert-Butyl Ester (II). To a
solution of o-phenylenediamine (54.0 g, 0.500 mol) in THF (500 mL)
was added (Boc)2O (109 g, 0.500 mol) in THF (150 mL) dropwise,
and the mixture was stirred at room temperature overnight. After
concentration under vacuum, the residue was diluted with ethyl
acetate/petroleum ether = 1/4 (v/v) (150 mL), and the precipitate
(E)-{4-[2-(2-tert-Butoxycarbonylaminophenylcarbamoyl)-
vinyl]phenyl}-((S)-3-hydroxypyrrolidin-1-yl)acetic Acid Methyl
E s t e r ( V ) . T o
a
s o l u t i o n o f { 4 - [ 2 - ( 2 - t e r t -
butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}hydroxyacetic
acid methyl ester (IV) (9.00 g, 21.1 mmol) and triethylamine (3.20 g,
31.6 mmol) in CH2Cl2 (135 mL) cooled to −5 °C was added
dropwise methanesulfonyl chloride (3.14 g, 27.4 mmol) under N2
atmosphere. The reaction was stirred at 0 °C until the starting material
had been consumed according to TLC (about 1 h). The mixture was
washed with water (90 mL) and brine (90 mL), dried with MgSO4,
filtered, and evaporated in vacuo to obtain 11.2 g (quantitative yield)
of {4-[2-(2-tert-butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-
methanesulfonyloxyacetic acid methyl ester as light yellow crystals
which were used in the following step without further purification. MS:
calcd 505 (MH+), exp 505 (MH+).
To a solution of {4-[2-(2-tert-butoxycarbonylaminophenyl-
carbamoyl)vinyl]phenyl}methanesulfonyloxyacetic acid methyl ester
(1.26 g, 2.5 mmol) and Et3N (0.76 g, 7.5 mmol) in CH2Cl2 (15 mL)
was added (S)-3-hydroxypyrrolidine (0.27 g, 3 mmol). This mixture
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was stirred overnight at room temperature, diluted with CH2Cl2 (50
mL), washed with saturated aqueous NaHCO3 solution, water and
brine, dried with Na2SO4, filtered, and evaporated to obtain a yellow
solid (V) which was directly used in the next reaction without further
purification. MS: calcd 496 (MH+), exp 496 (MH+).
starting material was consumed. The mixture was adjusted to pH = 6−
8 with 6 N HCl. The solvent was removed to give crude product rac-
(trans-3,4)-4-{4-[2-(2-aminophenylcarbamoyl)vinyl]phenyl}-1-meth-
ylpyrrolidine-3-carboxylic acid which was used in the next step without
1
further purification. H NMR (CD3OD, 400 MHz) δ ppm 7.81 (d,
(E)-N-(2-Aminophenyl)-3-{4-[(4-bromophenylcarbamoyl)-
((S)-3-hydroxypyrrolidin-1-yl)methyl]phenyl}acrylamide (3).
To a solution of {4-[2-(2-tert-butoxycarbonylaminophenylcarbamoyl)-
vinyl]phenyl}-(3-hydroxypyrrolidin-1-yl)acetic acid methyl ester (V)
(1.23 g, 2.5 mmol) in MeOH (30 mL) was added aqueous LiOH (1
N, 10 mL). This mixture was stirred for about 5 h at room temperature
and then evaporated to remove most of the MeOH. The aqueous layer
was acidified with concentrated HCl to pH 5−6. The acidified aqueous
layer was extracted with ethyl acetate (3 × 30 mL). The combined
organic layer was washed with brine, dried with Na2SO4, filtered, and
1H, J = 15.6 Hz), 7.76 (d, 2H, J = 7.6 Hz), 7.70 (d, 1H, J = 7.6 Hz),
7.62 (d, 1H, J = 7.6 Hz), 7.58 (d, 2H, J = 8.0 Hz), 7.36 (t, 1H, J = 7.2
Hz), 7.29 (t, 1H, J = 7.2 Hz), 6.97 (d, 1H, J = 15.6 Hz), 4.02−3.84 (m,
2H), 3.82−3.64 (m, 2H), 3.33−3.26 (m, 2H), 3.12 (s, 3H), 1.63 (s,
9H).
HATU (13.3 g, 35 mmol) was added to a solution of compound
rac-(trans-3,4)-4-{4-[2-(2-aminophenylcarbamoyl)vinyl]phenyl}-1-
methylpyrrolidine-3-carboxylic acid (crude material, 28.8 mmol) and
4-fluorophenylamine (3.55 g, 32 mmol) in TEA (10 mL, 72 mmol),
DMF (30 mL), and dichloromethane (300 mL) at rt. Then the
mixture was stirred at rt for 3 h. LC-MS indicated that the reaction was
completed. The mixture was partitioned between water and dichloro-
methane. The organic phase was dried and concentrated. The residue
was purified by chromatography on silica gel to give 14.1 g of rac-[2-
(3-{4-[(trans-3,4)-4-(4-fluorophenylcarbamoyl)-1-methylpyrrolidin-3-
yl]phenyl}acryloylamino)phenyl]carbamic acid tert-butyl ester (88%
over two steps). MS: calcd (MH+) 559, exp (MH+) 559.
evaporated to obtain 0.85
g (81%) of {4-[2-(2-tert-
butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-(3-hydroxypyr-
rolidin-1-yl)acetic acid as a yellow solid product used directly in the
following step. MS: calcd 482 (MH+), exp 482 (MH+).
To a solution of {4-[2-(2-tert-butoxycarbonylaminophenyl-
carbamoyl)vinyl]phenyl}-(3-hydroxypyrrolidin-1-yl)acetic acid (2.3 g,
4.78 mmol), PyBrop (3 g, 6.4 mmol) and DIPEA (1 mL, 5.68 mmol)
in CH2Cl2 (50 mL) was added 4-bromoaniline (1 g, 5.8 mmol). This
mixture was stirred overnight at room temperature and then diluted
with CH2Cl2, washed with saturated aqueous NaHCO3 solution,
water, and brine, dried with Na2SO4, and evaporated to obtain a yellow
residue (VI). Hydrochloric acid in methanol (1.25 M, 30 mL) was
added to the residue, the mixture was stirred for about 3 h, and then
NaHCO3 was added to the reaction system. After filtration of solids,
the crude mixture was purified by preparative HPLC to obtain 520 mg
of compound 3 as a pale-yellow solid (20%). MS: calcd 535 (MH+),
exp 535 (MH+). 1H NMR (MeOD-d4, 400 MHz) δ ppm 7.83 (d, 2H,
J = 8.0 Hz), 7.81 (d, 1H, J = 15.6 Hz), 7.75 (d, 2H, J = 8.0 Hz), 7.55
(d, 2H, J = 8.8 Hz), 7.49 (d, 2H, J = 8.8 Hz), 7.40−7.30 (m, 4H), 6.98
(d, 1H, J = 15.6 Hz), 5.24 (m, 1H), 4.60 (broad s, 1H), 4.00−3.80
(broad m, 2H), 3.60−3.20 (broad m, 2H), 2.35−2.15 (broad m, 2H).
rac-(trans-3,4)-4-(4-Bromophenyl)-1-methylpyrrolidine-3-
carboxylic Acid Ethyl Ester (VII). A mixture of sarcosine (44.5 g, 0.5
mol, 2.5 equiv), paraformaldehyde (60 g, 2 mol, 10 equiv), and (E)-3-
(4-bromophenyl)acrylic acid ethyl ester (50 g, 196 mmol, 1 equiv) was
heated under reflux in toluene (500 mL). The H2O formed was
removed with the aid of a Dean−Stark trap. After 4 h, the cooled
mixture was filtered. The filtrate was concentrated. The residue was
purified by chromatography on silica gel eluted with hexane−EtOAc to
give 28 g of product (yield was 46%) and 26 g of starting material ethyl
rac-[2-(3-{4-[(trans-3,4)-4-(4-Fluorophenylcarbamoyl)-1-methyl-
pyrrolidin-3-yl]phenyl}acryloylamino)phenyl]carbamic acid tert-butyl
ester (14.5 g, 25.3 mmol) was dissolved in 150 mL of MeOH (with 1
N HCl) and stirred for 4 h. LC-MS indicated that the reaction was
completed. The solvent was removed, and the residue was basified
with TEA and purified by prep-HPLC to give 5 g of product 12 (43%).
1
MS: calcd (MH+) 459, exp (MH+) 459. H NMR (CD3OD, 400
MHz) δ ppm 7.63−7.58 (m, 3H), 7.53−7.50 (m, 2H), 7.40 (d, 2H, J =
8.0 Hz), 7.21 (d, 1H, J = 7.6 Hz), 7.08−7.00 (m, 3H), 6.90−6.74 (m,
3H), 3.85 (m, 1H), 3.21−3.11 (m, 3H), 2.97−2.93 (m, 2H), 2.50 (s,
3H).
rac-(trans-3,4)-1-Benzyl-4-(4-bromophenyl)pyrrolidine-3-
carboxylic Acid Ethyl Ester (IX). To a mixture of ethyl trans-4-
bromocinnamate (0.95 g, 3.73 mmol) and N-(methoxymethyl)-N-
(trimethylsilylmethyl)benzylamine (1.06 g, 4.48 mmol) in dichloro-
methane (10 mL) was added orthoboric acid (23 mg, 0.37 mmol), and
the mixture was stirred at room temperature for 2 days and heated at
45 °C for 2 h. The resulting mixture was concentrated in vacuo, and
the crude product was purified by flash chromatography (PET:EtOAc
= 10:1) to get product as colorless oil (1.08 g, 71%). MS: calcd 388
(MH+), exp 388 (MH+).
rac-(trans-3,4)-4-(4-Bromophenyl)pyrrolidine-3-carboxylic
Acid Ethyl Ester (X). To a solution of rac-(trans-3,4)-1-benzyl-4-(4-
bromophenyl)pyrrolidine-3-carboxylic acid ethyl ester (IX) (3.97 g,
10.2 mmol) in acetonitrile (40 mL) was added K2CO3 (2.25 g, 16.3
mmol) followed by 2,2,2-trichloroethyl chloroformate (3.03 g, 14.3
mmol), and the mixture was heated at 60 °C for 3 h. The mixture was
diluted with ethyl acetate and filtered. The filtrate was concentrated in
vacuo, and the crude product was purified by flash chromatography
(Pet:EtOAc = 10:1) to get rac-(trans-3,4)-4-(4-bromophenyl)-
pyrrolidine-1,3-dicarboxylic acid 3-ethyl ester 1-(2,2,2-trichloroethyl)
ester as a colorless oil (3.2 g, 66.2%). MS: calcd 472 (MH+), exp 472
(MH+).
1
cinnamate (52%). MS: calcd (MH+) 312, exp (MH+) 312. H NMR
(CDCl3, 400 MHz) δ ppm 7.48 (d, 1H, J = 8.4 Hz), 7.29 (d, 2H, J =
8.4 Hz), 4.15 (q, 2H, 6.8 Hz), 3.51 (m, 1H), 3.30 (m, 2H), 3.20 (m,
2H), 2.72 (s, 3H), 1.21 (t, 3H, 6.8 Hz).
rac-(trans-3,4)-4-{4-[2-(2-tert-Butoxycarbonylaminophenyl-
carbamoyl)vinyl]phenyl}-1-methylpyrrolidine-3-carboxylic
Acid Ethyl Ester (VIII). The mixture of compound rac-(trans-3,4)-4-
(4-bromophenyl)-1-methylpyrrolidine-3-carboxylic acid ethyl ester
(VII) (10.3 g, 33 mmol), N-(2-(acryloylamino)phenyl)carbamic acid
tert-butyl ester (III) (9.51 g, 36.3 mmol), Pd2(dba)3 (300 mg, 0.33
mmol), and P(o-tolyl)3 (1.0 g, 3.3 mmol) in DMF (50 mL) and TEA
(9 mL, 66 mmol) was stirred at 110 °C under N2 in a sealed tube
overnight. LC-MS indicated that the reaction was completed. The
cooled mixture was partitioned between water and ethyl acetate. The
organic phase was dried and concentrated. The residue was purified by
chromatography on silica gel eluted by dichloromethane to give 14.22
g of desired product (87%). MS: calcd (MH+) 494, exp (MH+) 494.
rac-(trans-3,4)-4-{4-[2-(2-Aminophenylcarbamoyl)vinyl]-
phenyl}-1-methylpyrrolidine-3-carboxylic Acid (4-
fluorophenyl)amide (12). Lithium hydroxide monohydrate (2.42
g, 57.6 mmol) in water (20 mL) was added to a solution of rac-(trans-
3,4)-4-{4-[2-(2-tert-butoxycarbonylaminophenylcarbamoyl)vinyl]-
phenyl}-1-methylpyrrolidine-3-carboxylic acid ethyl ester (VIII)
(14.22 g, 28.8 mmol) in methanol (80 mL). Then the mixture was
stirred at room temperature overnight. LC-MS indicated that the
To a solution of rac-(trans-3,4)-4-(4-bromophenyl)pyrrolidine-1,3-
dicarboxylic acid 3-ethyl ester 1-(2,2,2-trichloroethyl) ester (3.2 g, 6.76
mmol) in acetic acid (15 mL) was added Zn (1.28 g, 19.6 mmol)
under Ar, and the mixture was stirred for 3 h at room temperature.
The resulting mixture was diluted with dichloromethane and filtered.
The filtrate was concentrated in vacuo. The crude product X (2.5 g)
was obtained and used directly without further purification. MS: calcd
298 (MH+), exp (MH+) 298. 1H NMR (CD3OD, 400 MHz), 7.45 (d,
2H, J = 8.4 Hz), 7.21 (d, 2H, J = 8.4 Hz), 4.09 (t, 2H, J = 7.2 Hz),
3.47−3.33 (m, 3H), 3.26−3.21 (m, 1H), 3.09−3.05 (m, 1H), 2.89−
2.84 (m, 1H), 1.16 (t, 3H, J = 7.2 Hz).
Representative Alkylation Procedure for R2 Analogues of
the 3,4-Disubstituted Pyrrolidine Series: rac-(trans-3,4)-4-(4-
Bromophenyl)-1-(2-fluoroethyl)pyrrolidine-3-carboxylic Acid
Ethyl Ester (XIa). A 20 mL microwave process vial was charged
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with rac-(trans-3,4)-4-(4-bromophenyl)pyrrolidine-3-carboxylic acid
ethyl ester (X) (1.49 g, 5 mmol), K2CO3 (1.38 g, 10 mmol), KI
(41.5 mg, 0.25 mmol), and 1-bromo-2-fluoroethane (0.76 g, 6 mmol)
in DMF (15 mL). The vial was sealed and heated at 130 °C for 20 min
under microwave irradiation. After cooling, the solution was poured
into water (30 mL), and the aqueous layer was extracted with ethyl
acetate (3 × 50 mL). The combined organic layer was washed with
brine, dried with Na2SO4, filtered, and evaporated. The crude product
was purified by column chromatography (PE:EtOAc = 1:1) to give
product as a yellow solid. MS: calcd 344 (MH+), exp 344 (MH+).
Representative Epoxidation Procedure for R2 Analogues of
the 3,4-Disubstituted Pyrrolidine Series: rac-(trans-3,4)-4-(4-
Bromophenyl)-1-(2-hydroxy-2-methylpropyl)pyrrolidine-3-
carboxylic Acid Ethyl Ester (XIb). To a solution of rac-(trans-3,4)-
4-(4-bromophenyl)pyrrolidine-3-carboxylic acid ethyl ester (X) (5.0 g,
16.78 mmol) in ethanol (60 mL) was added 2,2-dimethyloxirane (8
mL). This mixture was heated for about 5 h at 80 °C in a sealed tube
until the starting material had been consumed and then evaporated in
vacuo to give 6.0 g (96%) of crude product as a yellow oil. MS: calcd
370 (MH+), exp 370 (MH+).
was purified by column chromatography (PE:EtOAc = 1:1) to give
yellow solid product. MS: calcd 526 (MH+), exp 526 (MH+).
rac-(trans-3,4)-4-{4-[2-(2-Aminophenylcarbamoyl)vinyl]-
phenyl}-1-(2-fluoroethyl)pyrrolidine-3-carboxylic Acid (4-
chlorophenyl)amide (46). To a solution of rac-(trans-3,4)-4-{4-[2-
(2-tert-butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-1-(2-
fluoroethyl)pyrrolidine-3-carboxylic acid ethyl ester (XII) (1.05 g, 2.0
mmol) in MeOH/H2O (20 mL/6 mL) was added LiOH·H2O (252
mg, 6.0 mmol). This mixture was stirred at room temperature
overnight and then evaporated to remove most of the MeOH. The
mixture was extracted with ethyl acetate (3 × 50 mL). The combined
organic layer was washed with brine, dried with Na2SO4, filtered, and
evaporated to get yellow solid product rac-(trans-3,4)-4-{4-[2-(2-tert-
butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-1-(2-
fluoroethyl)pyrrolidine-3-carboxylic acid. MS: calcd 498 (MH+), exp
498 (MH+).
To
a solution of rac-(trans-3,4)-4-{4-[2-(2-tert-
butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-1-(2-
fluoroethyl)pyrrolidine-3-carboxylic acid (300 mg, 0.6 mmol), HATU
(342 mg, 0.9 mmol), and Et3N (181.8 mg, 1.8 mmol) in CH2Cl2 (15
mL) was added 4-chloroaniline (114.3 mg, 0.9 mmol). The reaction
was stirred at room temperature overnight. The mixture was diluted
with CH2Cl2 (15 mL), washed with water (15 mL) and brine (15 mL),
dried with Na2SO4, filtered, and evaporated in vacuo to obtain light
yellow residue which was used without further purification. MS: calcd
607 (MH+), exp 607 (MH+).
Representative Reductive Amination Procedure for R2
Analogues of the 3,4-Disubstituted Pyrrolidine Series: rac-
(trans-3,4)-4-(4-Bromophenyl)-1-isopropylpyrrolidine-3-car-
boxylic Acid Ethyl Ester (XIc). To a solution of rac-(trans-3,4)-4-(4-
bromophenyl)pyrrolidine-3-carboxylic acid ethyl ester (X) (2.5 g, 8.39
mmol) in methanol were added acetone (9.73 g, 16.78 mmol),
NaBH(OAc)3 (3.56 g, 16.78 mmol), and catalytic acetic acid. This
mixture was stirred at rt overnight until the starting material had been
consumed. Water was added, and the organic phase was washed by
brine, dried over Na2SO4, and concentrated to give 1.8 g of product
(yield 64%). MS: calcd 340 (MH+), exp 340 (MH+).
Hydrochloric acid in methanol (1.25 M, 5 mL) was added to the
rac-[2-(3-{4-[(trans-3,4)-4-(4-chlorophenylcarbamoyl)-1-(2-
fluoroethyl)pyrrolidin-3-yl]phenyl}acryloylamino)phenyl]carbamic
acid tert-butyl ester residue, the mixture was stirred for about 3 h, and
then NaHCO3 was added to the reaction system. After filtration of
solids, the crude mixture was purified by preparative HPLC to obtain
Representative Acylation Procedure for R2 Analogues of the
3,4-Disubstituted Pyrrolidine Series: rac-(trans-3,4)-4-(4-Bro-
mophenyl)-1-ethylcarbamoylpyrrolidine-3-carboxylic Acid
Ethyl Ester (XId). To a solution of DIPEA (0.87 mL, 5 mmol) in
dry THF was added triphosgene (530 mg, 1.8 mmol) at 0 °C, the
resulting solution was stirred for 5 min, and then a solution of rac-
(trans-3,4)-4-(4-bromophenyl)pyrrolidine-3-carboxylic acid ethyl ester
(X) (1.485 g, 5 mmol) in THF and DIPEA (0.87 mL, 5 mmol) was
added dropwise. After the mixture was stirring for an additional 20
min, ethylamine (450 mg, 10 mmol) was added dropwise and stirred
overnight at room temperature before it was poured into H2O (25
mL) and extracted with EtOAc (2 × 15 mL). The organic layer was
washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo to give a crude product which was purified by
flash column chromatography to yield the title compound (1.14 g,
62%) as a white solid. MS: calcd 369 (MH+), exp 369 (MH+).
Representative Nucleophilic Aromatic Substitution Proce-
dure for R2 Analogues of the 3,4-Disubstituted Pyrrolidine
Series: rac-(trans-3,4)-4-{4-[(E)-2-(2-Aminophenylcarbamoyl)-
vinyl]phenyl}-1-pyrimidin-2-ylpyrrolidine-3-carboxylic Acid
(4-Chlorophenyl)amide (XIe). To a solution of rac-(trans-3,4)-4-
(4-bromophenyl)pyrrolidine-3-carboxylic acid ethyl ester (X) (466
mg, 1.56 mmol) in 2-propanol (6 mL) were added DIPEA (412 μL,
2.34 mmol) and 2-chloropyrimidine (228 mg, 2 mmol). The solution
was heated to 130 °C for 10 min by microwave and then concentrated
in vacuo and purified by flash chromatography to obtain 357 mg of
product as an oil (60%). MS: calcd 376 (MH+), exp 376 (MH+).
rac-(trans-3,4)-4-{4-[2-(2-tert-Butoxycarbonylaminophenyl-
carbamoyl)vinyl]phenyl}-1-(2-fluoroethyl)pyrrolidine-3-car-
boxylic Acid Ethyl Ester (XII). To a solution of rac-(trans-3,4)-4-(4-
bromophenyl)-1-(2-fluoroethyl)pyrrolidine-3-carboxylic acid ethyl
ester (XIa) (2.0 g, 5.81 mmol), Pd2(dba)3 (265.8 mg, 0.29 mmol),
tri-(o-tolyl)phosphine (88 mg, 0.29 mmol), and Et3N (17.5 g, 17.4
mmol) in DMF (20 mL) was added N-(2-(acryloylamino)phenyl)-
carbamic acid tert-butyl ester (III) (1.82 g, 6.97 mmol). This mixture
was stirred at 100 °C for about 3 h until the starting material had been
consumed, and then the reaction mixture was cooled and filtered. The
solution was poured into water (40 mL) and extracted with ethyl
acetate (3 × 60 mL). The combined organic layer was washed with
brine, dried with Na2SO4, filtered, and evaporated. The crude product
1
light yellow solid. MS: calcd 507 (MH+), exp 507 (MH+). H NMR
(CD3OD, 400 MHz), 7.66 (d, 1H, J = 15.6 Hz), 7.60 (d, 2H, J = 8.0
Hz), 7.53 (d, 2H, J = 8.8 Hz), 7.42 (d, 2H, J = 8.0 Hz), 7.21 (d, 1H, J
= 8.0 Hz), 7.08−7.04 (m, 3H), 6.90−6.82 (m, 2H), 6.78−6.74 (m,
1H), 4.69 (t, 1H, J = 4.8 Hz), 4.57 (t, 1H, J = 4.8 Hz), 3.84−3.78 (m,
1H), 3.30−3.16 (m, 3H), 3.03−2.87 (m, 4H).
rac-(trans-3,4)-4-(4-Bromophenyl)pyrrolidine-1,3-dicarbox-
ylic Acid 1-tert-Butyl Ester (XIII). To a solution of rac-(trans-3,4)-4-
(4-bromophenyl)pyrrolidine-3-carboxylic acid ethyl ester (X) (5.0 g,
16.8 mmol) in 20 mL of acetonitrile was added 20 mL of 2.5 M
NaOH, and the resulted mixture was stirred at room temperature for 2
h. Then Na2CO3 (3.56 g 33.6 mmol) and Boc2O (11 g, 50.4 mmol)
were added, and the mixture was stirred at rt overnight. After removal
of acetonitrile by vacuum, the residue was adjusted to pH 3. The solid
was collected and triturated in hexane twice and dried by vacuum
(50% yield). MS: calcd 370 (MH+), exp 370 (MH+).
r a c - ( t r a n s - 3 , 4 ) - 3 - ( 4 - B r o m o p h e n y l ) - 4 - ( 4 -
chlorophenylcarbamoyl)pyrrolidine-1-carboxylic Acid tert-
Butyl Ester (XIV). A suspension of rac-(trans-3,4)-4-(4-
bromophenyl)pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester
(XIII) (3.1 g, 8.4 mmol), 4-chloroaniline (1.6 g, 12.6 mmol), HOBt
(1.7 g, 12.6 mmol), and EDCI (2.4g, 12.6 mmol) in 50 mL of
dichloromethane was stirred at rt overnight, and the reaction mixture
was washed with 1 M NaOH, water, and brine. The organic layer was
dried over Na2SO4 and concentrated to give yellow oil, which was used
directly in the next step without purification. MS: calcd 479 (MH+),
exp 479 (MH+).
rac-(trans-3,4)-4-(4-Bromophenyl)-1-(2-hydroxy-1,1-
dimethylethyl)pyrrolidine-3-carboxylic Acid (4-Chlorophenyl)-
amide (XV). rac-(trans-3,4)-3-(4-Bromophenyl)-4-(4-
chlorophenylcarbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester
(XIV) was dissolved in 15 mL of HCl/MeOH (1.25 M) and stirred at
rt for 3 h. The reaction mixture was treated with saturated Na2CO3
solution and was extracted with dichloromethane. The organic layer
was concentrated to give yellow oil, which was used without further
purification. MS: calcd 379 (MH+), exp 379 (MH+).
To a solution of rac-(trans-3,4)-4-(4-bromophenyl)pyrrolidine-3-
carboxylic acid (4-chlorophenyl)amide (1.12 g, 2.98 mmol) in 5 mL of
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DMF were added K2CO3 (1.24 g, 8.9 mmol) and 2-bromo-2-
methylpropionic acid methyl ester (0.81 g, 4.47 mmol), and the
mixture was heated at 50 °C for 5 h. The reaction mixture was diluted
with water and extracted with EtOAc, and the organic layer was
washed with water and brine. After removal of solvent, the residue was
dissolved in 30 mL of dry THF under N2 atmosphere, and then LiBH4
(2 M in THF, 2.9 mL, 5.8 mmol) was added dropwise at room
temperature over 5 min. The resulting mixture was kept at this
temperature for 2 h and then quenched by adding water carefully. The
mixture was extracted with EtOAc, and the crude product was purified
by column chromatography. A 0.23 g amount of product XV was
obtained with a yield of 17%. MS: calcd 451 (MH+), exp 451 (MH+).
rac-(trans-3,4)-4-{4-[2-(2-Aminophenylcarbamoyl)vinyl]-
phenyl}-1-(2-hydroxy-1,1-dimethylethyl)pyrrolidine-3-carbox-
ylic Acid (4-Chlorophenyl)amide (53). The mixture of rac-(trans-
3,4)-4-(4-bromophenyl)-1-(2-hydroxy-1,1-dimethylethyl)pyrrolidine-
3-carboxylic acid (4-chlorophenyl)amide (0.23 g, 0.5 mmol) (XV), N-
(2-(acryloylamino)phenyl)carbamic acid tert-butyl ester tert-butyl ester
(III) (0.14 g, 0.53 mmol), Pd2(dba)3 (37 mg, 0.04 mmol), and tri-(o-
tolyl)phosphine (27 mg, 0.08 mmol) in DMF (3 mL) and TEA (0.35
mL, 2.5 mmol) was stirred at 100 °C under N2 in a sealed tube for 3 h.
The cooled mixture was partitioned between water and ethyl acetate.
The organic phase was washed with water and brine, dried over
Na2SO4, and concentrated to give crude yellow oil. The crude oil was
dissolved in 3 mL of HCl/MeOH (1.25 M) and stirred at room
temperature for 3 h. The resultant mixture was neutralized with NH3/
EtOH solution and purified by prep-HPLC to obtain 66 mg yellow
solid as product. Yield was 24% over two steps. MS: calcd 533 (MH+),
exp 533 (MH+). 1H NMR (DMSO-d6, 400 MHz), 10.09 (s, 1H), 9.36
(s, 1H), 7.50−7.61 (m, 5H), 7.33−7.39 (m, 5H), 6.84−6.94 (m, 2H),
6.75 (d, 1H, J = 8.0 Hz), 6.58 (t, 1H, J = 7.2 Hz), 4.93 (s, 2H), 4.46 (s,
1H), 3.62 (q, 1H, J = 7.6 Hz), 3.30 (m, 2H), 3.21−3.29 (m, 2H), 3.04
(q, 1H, J = 7.6 Hz), 2.78−2.88 (m, 2H), 1.03 (s, 3H), 1.01 (s, 3H)
rac-(trans-3,4)-4-(4-Bromophenyl)-1-tert-butylpyrrolidine-3-
carboxylic Acid Ethyl Ester (XVI). A mixture of N-tert-butylglycine
hydrochloride salt (0.9 g, 6.8 mmol), paraformaldehyde (1.5 g, 50
mmol), and ethyl cinnamate (0.9 g, 35 mmol) was heated under reflux
in toluene (30 mL). The H2O formed was removed with the aid of a
Dean−Stark trap. After 4 h, the cooled mixture was filtered. The
filtrate was concentrated. The residue was purified by chromatography
on silica gel eluted with hexane−EtOAc to give 300 mg of product
(yield was 12%). MS: calcd 354 (MH+), exp 354 (MH+).
phenyl}-1-tert-butylpyrrolidine-3-carboxylic acid (crude material, 0.2
mmol) and 4-chloroaniline (0.1 g, 0.78 mmol) in TEA (0.1 mL), and
dichloromethane (7 mL) at rt, and the mixture was stirred at rt
overnight. LC-MS indicated that the reaction was completed. The
mixture was partitioned between water and dichloromethane. The
organic phase was dried and concentrated. The residue was purified by
chromatography on silica gel to give 80 mg of rac-[2-(3-{4-[(trans-
3,4)-1-tert-butyl-4-(4-chlorophenylcarbamoyl)pyrrolidin-3-yl]phenyl}-
acryloylamino)phenyl]carbamic acid tert-butyl ester (yield was 66%).
MS: calcd 617 (MH+), exp 617 (MH+).
rac-[2-(3-{4-[(trans-3,4)-1-tert-Butyl-4-(4-chlorophenylcarbamoyl)-
pyrrolidin-3-yl]phenyl}acryloylamino)phenyl]carbamic acid tert-butyl
ester (80 mg, 0.13 mmol) was dissolved in MeOH (1.25 M HCl, 10
mL) and stirred for 4 h. LC-MS indicated that the reaction was
completed. The solvent was removed, and the residue was basified
with TEA and purified by prep-HPLC to give 18 mg of product. MS:
1
calcd (MH+) 517, exp (MH+) 517. H NMR (CD3OD, 400 MHz),
7.64 (d, 1H, J = 15.4 Hz), 7.55 (d, 2H, J = 8.0 Hz), 7.48 (d, 2H, J = 8.8
Hz), 7.39 (d, 2H, J = 8.0 Hz), 7.25 (d, 2H, J = 8.8 Hz), 7.19 (d, 1H, J
= 8.0 Hz), 7.04 (t, 1H, J = 7.2 Hz), 6.87 (d, 1H, J = 8.0 Hz), 6.82 (d,
1H, J = 15.6 Hz), 6.74 (t, 1H, J = 7.6 Hz), 3.72 (m, 1H), 3.18−3.07
(m, 3H), 2.91 (m, 2H), 1.16 (s, 9H).
rac-(trans-3,4)-4-(4-Bromophenyl)-1-(2,2,2-trifluoroethyl)-
pyrrolidine-3-carboxylic Acid Ethyl Ester (XVIII). To a suspension
of rac-(trans-3,4)-(4-(4-bromophenyl)pyrrolidine-3-carboxylic acid
ethyl ester hydrochloride (X) (1.10 g, 3.28 mmol) in DCM (10
mL) was added Et3N (1.4 mL, 9.86 mmol). This mixture was stirred
for about 5 min at room temperature and then cooled to 0 °C. To the
resulting mixture was added trifluoroacetate anhydride (0.9 g, 4.28
mmol). The mixture was stirred for 30 min at 0 °C, warmed to room
temperature, and stirred for 1 h. The resulting mixture was washed
with water and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography
(Pet:EtOAc = 10:1) to get rac-(trans-3,4)-4-(4-bromophenyl)-1-
(2,2,2-trifluoroacetyl)pyrrolidine-3-carboxylic acid ethyl ester as a
colorless oil product (0.88 g, 68.1%).
To a solution of rac-(trans-3,4)-4-(4-bromophenyl)-1-(2,2,2-
trifluoroacetyl)pyrrolidine-3-carboxylic acid ethyl ester (0.82 g, 2.08
mmol) was added a solution of borane in THF (3.3 mL, 3.3 mmol) at
0 °C. Then the mixture was refluxed for 2 h. The resulting mixture was
cooled and quenched by aqueous HCl (6 N). The mixture was
concentrated in vacuo to remove THF. The residue was basified by
solid Na2CO3 and then extracted with EtOAc. The organic layer was
washed with brine, dried with Na2SO4, and concentrated in vacuo. The
crude product was purified by flash chromatography (Pet:EtOAc =
20:1) to get colorless oil product XVIII (230 mg, 30%); 188 mg of
starting material was recovered. MS: calcd 380 (MH+), exp 380 (MH
rac-(trans-3,4)-4-{4-[2-(2-tert-Butoxycarbonylaminophenyl-
carbamoyl)vinyl]phenyl}-1-tert-butylpyrrolidine-3-carboxylic
Acid Ethyl Ester (XVII). A mixture of XVI (0.5 g, 1.4 mmol), N-(2-
(acryloylamino)phenyl)carbamic acid tert-butyl ester (III) (0.5 g, 1.9
mmol), Pd2(dba)3 (100 mg, 0.01 mmol), and P(o-tolyl)3 (100 mg,
0.33 mmol) in DMF (5 mL) and TEA (1 mL) was stirred at 110 °C
under N2 in a sealed tube overnight. LC-MS indicated that the reaction
was completed. The cooled mixture was partitioned between water
and ethyl acetate. The organic phase was dried and concentrated. The
residue was purified by chromatography on silica gel eluted by
dichloromethane to give 0.4 g of product (yield was 53%). MS: calcd
536 (MH+), exp 536 (MH+).
1
+). H NMR (CDCl3, 400 MHz), 7.45 (d, 2H, J = 8.4 Hz), 7.23 (d,
2H, J = 8.4 Hz), 4.13−4.19 (m, 2H), 3.61−3.66 (m, 1H), 3.34−3.37
(m, 1H), 3.04−3.24 (m, 6H), 1.24 (t, 3H, J = 6.8 Hz).
rac-(trans-3,4)-4-(4-Bromophenyl)-1-(2,2,2-trifluoroethyl)-
pyrrolidine-3-carboxylic Acid (4-Chlorophenyl)amide (XIX). To
a
solution of rac-(trans-3,4)-4-(4-bromophenyl)-1-(2,2,2-
trifluoroethyl)pyrrolidine-3-carboxylic acid ethyl ester (440 mg, 1.16
mmol) in methanol (3 mL) was added aqueous NaOH (2.9 mL, 2 M).
The mixture was stirred at rt for 4 h. Then the mixture was
concentrated in vacuo to remove methanol, and the residue was
acidified by aqueous HCl and lyophilized to offer a colorless solid. MS:
calcd 352 (MH+), exp 352 (MH+), calcd 350 (M − H), exp 350 (M
− H).
To a suspension of the crude acid in DCM (10 mL) were added
PyBrop (811 mg, 1.74 mmol), 4-chlorophenylamine (177 mg, 1.39
mmol), and DIPEA (0.5 mL, 2.84 mmol). The reaction was stirred at
room temperature overnight and diluted with CH2Cl2 (10 mL). The
mixture was washed with water (10 mL) and brine (10 mL), dried
with Na2SO4, filtered, and concentrated in vacuo. The crude product
was purified by flash chromatography on silica gel (Pet:EtOAc = 5:1−
2:1) to get the product (483 mg, 90%). MS: calcd 461 (MH+), exp
461 (MH+).
rac-(trans-3,4)-4-{4-[2-(2-Aminophenylcarbamoyl)vinyl]-
phenyl}-1-tert-butylpyrrolidine-3-carboxylic Acid (4-
Chlorophenyl)amide (52). A solution of lithium hydroxide
monohydrate (2.1 g, 50 mmol) in water (50 mL) was added to a
s o l u t i o n o f r a c - ( t r a n s - 3 , 4 ) - 4 - { 4 - [ 2 - ( 2 - t e r t -
butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-1-tert-butylpyr-
rolidine-3-carboxylic acid ethyl ester (XVII) (0.4 g, 0.74 mmol) in
THF (50 mL), and the mixture was stirred at room temperature
overnight. LC-MS indicated that the starting material was consumed.
The mixture was adjusted to pH = 6−8 with 6 N HCl. The solvent was
removed to give crude rac-(trans-3,4)-4-{4-[2-(2-tert-
butoxycarbonylaminophenylcarbamoyl)vinyl]phenyl}-1-tert-butylpyr-
rolidine-3-carboxylic acid, which was used in the next step without
further purification. MS: calcd 508 (MH+), exp 508 (MH+).
HATU (0.2 g, 0.52 mmol) was added to a solution of rac-(trans-
3,4)-4-{4-[2-(2-tert-butoxycarbonylaminophenylcarbamoyl)vinyl]-
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rac-(trans-3,4)-4-{4-[2-(2-Aminophenylcarbamoyl)vinyl]-
phenyl}-1-(2,2,2-trifluoroethyl)pyrrolidine-3-carboxylic Acid
(4-Chlorophenyl)amide (48). The mixture of rac-(trans-3,4)-4-(4-
bromophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidine-3-carboxylic acid (4-
chlorophenyl)amide (XIX) (483 mg, 1.05 mmol), N-(2-
(acryloylamino)phenyl)carbamic acid tert-butyl ester (302 mg, 1.15
mmol), Pd2(dba)3 (28.8 mg, 0.03 mmol), and P(o-tolyl)3 (38.4 mg,
0.12 mmol) in DMF (50 mL) and TEA (0.42 mL, 3 mmol) was
heated at 110 °C under Ar in a sealed tube for 4 h. The cooled mixture
was concentrated in vacuo to remove DMF. The residue was purified
by chromatography on silica gel (DCM:methanol = 40:1−20:1) to
give rac-[2-(3-{4-[(trans-3,4)-4-(4-chlorophenylcarbamoyl)-1-(2,2,2-
trifluoroethyl)pyrrolidin-3-yl]phenyl}acryloylamino)phenyl]carbamic
acid tert-butyl ester (580 mg, 86%). MS: calcd 643(MH+), exp 643
(MH+).
(acryloylamino)phenyl)carbamic acid tert-butyl ester (III) (2.1 g, 8.05
mmol), tris-o-tolylphosphine (0.41 g, 1.34 mmol), Pd2(dba)3 (0.62 g,
0.67 mmol), and Et3N (2.7 g, 26.8 mmol). The resulting mixture was
stirred for 3 h at 100 °C under argon. After cooling, the mixture was
filtered, and the solvent was removed, diluted with EtOAc (100 mL),
washed with water and brine, dried with Na2SO4, filtered, and
evaporated to give an oil. The crude product was purified by column
chromatography (PE: EtOAc = 1:1) to give 2.5 g (75%) of rac-(trans-
3,4)-4-{5-[2-(2-tert-butoxycarbonylaminophenylcarbamoyl)vinyl]-
pyridin-2-yl}-1-methylpyrrolidine-3-carboxylic acid ethyl ester as a
white solid product. MS: calcd 495 (MH+), exp 495 (MH+).
To
a solution of rac-(trans-3,4)-4-{5-[2-(2-tert-
butoxycarbonylaminophenylcarbamoyl)vinyl]pyridin-2-yl}-1-methyl-
pyrrolidine-3-carboxylic acid ethyl ester (2.5 g, 5 mmol) in THF/H2O
(45 mL/15 mL) was added LiOH monohydrate (1.05 g, 25 mmol).
This mixture was stirred for about 5 h at room temperature and then
evaporated to remove most of the THF. The aqueous solution was
acidified with 2 N HCl to pH 6−7 and then extracted with ethyl
acetate (3 × 70 mL). The combined organic layer was washed with
brine, dried with Na2SO4, filtered, and evaporated to obtain 2.0 g
( 8 5 . 6 % ) o f r a c - ( t r a n s - 3 , 4 ) - 4 - { 5 - [ 2 - ( 2 - t e r t -
butoxycarbonylaminophenylcarbamoyl)vinyl]pyridin-2-yl}-1-methyl-
pyrrolidine-3-carboxylic acid as a yellow solid product. MS: calcd 467
(MH+), exp 467 (MH+).
To a solution of rac-[2-(3-{4-[(trans-3,4)-4-(4-chlorophenylcarba-
moyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl]phenyl}acryloylamino)-
phenyl]carbamic acid tert-butyl ester (580 mg, 0.9 mmol) in DCM (6
mL) was added trifluoroacetic acid (2 mL), and the mixture was stirred
for 2 h. The solvent was removed, and the residue was basified with
TEA and purified by prep-HPLC. MS: calcd 543 (MH+), exp 543
1
(MH+). H NMR (CD3OD, 400 MHz), 7.66 (d, 1H, J = 15.6 Hz),
7.60 (d, 2H, J = 8.0 Hz), 7.53 (d, 2H, J = 8.4 Hz), 7.42 (d, 2H, J = 8.0
Hz), 7.29 (d, 2H, J = 8.4 Hz), 7.21 (d, 1H, J = 7.6 Hz), 7.08−7.04 (m,
1H), 6.90−6.82 (m, 2H), 6.78−6.74 (m, 1H), 3.92−3.60 (m, 1H),
3.44−3.38 (m, 2H), 3.29−3.24 (m, 4H), 3.10−3.06 (m, 1H).
3-(5-Bromopyridin-2-yl)acrylic Acid Ethyl Ester (XX). To a
solution of 5-bromo-2-iodopyridine (14.2 g, 50 mmol) in dry THF
(160 mL) was added dropwise isopropyl magnesium chloride (30 mL,
60 mmol) at −20 °C under N2. After the addition, the reaction
mixture was stirred for about 2 h at the same temperature, and then
DMF (5.11 g, 70 mmol) was added while keeping the temperature
under 0 °C. The reaction temperature was allowed to rise to rt, the
mixture was stirred for 1 h and quenched with saturated ammonium
chloride (10 mL), and then the solution pH was adjusted to 7−8 with
2.0 M HCl. The solvent was removed and extracted with ethyl acetate
(2 × 200 mL). The combined organic layer was washed with brine,
dried with Na2SO4, filtered, and evaporated to obtain 8.5 g (90%) of 5-
bromopyridine-2-carbaldehyde as a yellow solid product which was
used directly in the next step without further purification. MS: calcd
186 (MH+), exp 186 (MH+).
To a solution of triethyl phosphonoacetate (10.2 g, 45.7 mmol),
Et3N (7 mL, 50.3 mmol), and lithium bromide (4.3 g, 50.3 mol) in
CH3CN (120 mL) was added 5-bromopyridine-2-carbaldehyde (8.5 g,
45.7 mmol). This mixture was stirred overnight at room temperature.
When LC-MS indicated the reaction was complete, it was quenched
with water (10 mL). Excess CH3CN was removed in vacuo, and then
the remaining mixture was extracted with EtOAc (2 × 100 mL). The
combined organic layer was washed with brine, dried with Na2SO4,
filtered, and evaporated. The crude product was purified by column
chromatography (PE: EtOAc = 5:1) to give 7.8 g (67%) of a yellow
solid product. MS: calcd 256 (MH+), exp 256 (MH+).
rac-(trans-3,4)-4-(5-Bromopyridin-2-yl)-1-methylpyrrolidine-
3-carboxylic Acid Ethyl Ester (XXI). To a solution of 3-(5-
bromopyridin-2-yl)acrylic acid ethyl ester (XX) (3 g, 11.7 mmol) and
paraformaldehyde (4.22 g, 140.4 mol) in toluene (200 mL) was added
sarcosine (2.1 g, 23.4 mmol). This mixture was heated to reflux for 4 h,
using Dean−Stark trap to remove the water formed in the reaction.
LC-MS indicated that the reaction was complete. After cooling, the
solution was filtered, and the solid was washed with EtOAc (2 × 50
mL). The combined organic layer was washed with water and brine,
dried with Na2SO4, filtered, and evaporated. The crude product was
purified by column chromatography (PE: EtOAc = 3:1) to give 2.8 g
(77%) of a white solid product. MS: calcd 313 (MH+), exp 313 (MH
+).
To
a solution of rac-(trans-3,4)-4-{5-[2-(2-tert-
butoxycarbonylaminophenylcarbamoyl)vinyl]pyridin-2-yl}-1-methyl-
pyrrolidine-3-carboxylic acid (350 mg, 0.75 mmol), 4-chloroaniline
(115 mg, 0.9 mmol), and Et3N (227 mg, 2.25 mol) in CH2Cl2 (50
mL) was added HATU (430 mg, 1.125 mmol). This mixture was
stirred overnight at room temperature and then diluted with CH2Cl2
(50 mL), washed with saturated aqueous NaHCO3 solution, water,
and brine, dried with Na2SO4, and evaporated to obtain 0.23 g (53%)
of rac-2-(3-{6-[(trans-3,4)-4-(4-chlorophenylcarbamoyl)-1-methylpyr-
rolidin-3-yl]pyridin-3-yl}acryloylamino)phenyl]carbamic acid tert-
butyl ester as a yellow solid. MS: calcd 576 (MH+), exp 576 (MH+).
The solution of rac-2-(3-{6-[(trans-3,4)-4-(4-chlorophenylcarbamo-
yl)-1-methylpyrrolidin-3-yl]pyridin-3-yl}acryloylamino)phenyl]-
carbamic acid tert-butyl ester (168 mg, 0.3 mmol) in 2 N HCl in
MeOH (10 mL) was stirred for 4 h at room temperature, and then the
pH of the solution was adjusted to 8−9 with NaHCO3 solution. Then
the mixture was diluted with water (50 mL) and extracted with EtOAc
(2 × 50 mL). The combined organic layer was washed with brine,
dried with Na2SO4, filtered, and evaporated. The crude product was
purified by preparative HPLC to give a yellow solid product. MS: calcd
476 (MH+), exp 476 (MH+). 1H NMR (DMSO-d6, 400 MHz), 10.16
(s, 1H), 9.44 (s, 1H), 8.76 (s, 1H), 7.97 (d, 1H J = 8.0 Hz,), 7.63−7.56
broad m, 3H), 7.41 (d, 1H, J = 8.4 Hz), 7.36−7.30 (broad m, 3H),
6.98−6.91 (broad m, 2H), 6.76 (d, 1H, J = 7.2 Hz), 6.58 (t, 1H, J = 7.2
Hz), 4.95 (broad s, 2H), 3.95 (m, 1H), 3.48 (m, 1H), 3.03 (broad m,
2H), 2.79 (m, 2H), 2.33 (s, 3H)
Ethyl 3-(2-[1,3]-Dioxolanpyridinyl)acrylate (XXII). To a
solution of 5-bromopyridine-2-aldehyde (3.72 g, 20 mmol) in toluene
(60 mL) were added TsOH monohydrate (190 mg, 1 mmol) and
ethane-1,2-diol (2.48 g, 40 mmol). The mixture was heated to reflux
for 6 h. The solvent was removed, and the residue was dissolved in
ethyl acetate and washed with sodium bicarbonate and brine. The
organic layer was dried with sodium sulfate and concentrated to give
the crude product 5-bromo-2-[1,3]dioxolan-2-ylpyridine which was
used in the next step without further purification. MS: calcd 230 (MH
+), exp 230 (MH+).
To a solution of 5-bromo-2-[1,3]dioxolan-2-ylpyridine (460 mg, 2.0
mmol) in DMF (10 mL) were added ethyl acrylate (240 mg, 2.4
mmol), Pd2(dba)3 (183 mg, 0.2 mmol), tris-o-tolylphosphine (122 mg,
0.4 mmol), and Et3N (606 mg, 6 mmol). The mixture was heated to
110 °C for 3 h under protection of nitrogen. The mixture was diluted
with ethyl acetate and washed with brine. The organic layer was dried
with sodium sulfate and concentrated. The residue was purified by
chromatography to give the pure product. MS: calcd 250 (MH+), exp
250 (MH+).
rac-(trans-3,4)-4-{5-[2-(2-Aminophenylcarbamoyl)vinyl]-
pyridin-2-yl}-1-methylpyrrolidine-3-carboxylic Acid (4-
Chlorophenyl)amide (64). To a solution of rac-(trans-3,4)-4-(5-
bromopyridin-2-yl)-1-methylpyrrolidine-3-carboxylic acid ethyl ester
(XXI) (2.1 g, 6.7 mmol) in dried DMF (50 mL) were added N-(2-
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rac-(trans-3,4)-4-(6-[1,3]Dioxolan-2-ylpyridin-3-yl)-1-methyl-
pyrrolidine-3-carboxylic Acid Ethyl Ester (XXIII). To a solution of
ethyl 3-{2-[1,3]-dioxolanpyridinyl}acrylate (6 g, 24 mmol) in toluene
(600 mL) was added N-methylglycine (10.7 g, 120 mmol) and
paraformaldehyde (14.4 g, 480 mmol). The mixture was heated to
reflux for 6 h. After cooling, the solvent was removed and the residue
was purified by chromatography to give the pure product. MS: calcd
307 (MH+), exp 307 (MH+).
rac-(trans-3,4)-4-[6-(2-tert-Butoxycarbonylvinyl)pyridin-3-
yl]-1-methylpyrrolidine-3-carboxylic Acid Ethyl Ester (XXIV).
To a solution of rac-(trans-3,4)-4-(6-[1,3]dioxolan-2-ylpyridin-3-yl)-1-
methylpyrrolidine-3-carboxylic acid ethyl ester (612 mg, 2 mmol) in
DMSO/H2O (12 mL/4 mL) was added LiCl (509 mg, 12 mmol). The
mixture was sealed in a microwave tube and heated to 150 °C for 10
min under microwave conditions. The mixture was diluted with ethyl
acetate and washed with brine. The organic layer was dried with
sodium sulfate and concentrated. The residue was purified by
chromatography to give the pure product rac-(trans-3,4)-4-(6-
formylpyridin-3-yl)-1-methylpyrrolidine-3-carboxylic acid ethyl ester.
MS: calcd 263 (MH+), exp 263 (MH+).
To the solution of rac-(trans-3,4)-4-(6-formylpyridin-3-yl)-1-meth-
ylpyrrolidine-3-carboxylic acid ethyl ester (1.31 g, 5 mmol) in THF
(35 mL) was added diethyl tert-butyl phosphonoacetate (1.78 g, 7
mmol) at rt. n-Butyllithium (4 mL, 8 mmol) was added dropwise to
the above solution at −78 °C. The solution was allowed to rise to 0 °C
slowly and stirred at 0 °C for 2 h. The reaction was quenched with
saturated ammonium chloride. The solvent was removed, and the
residue was dissolved in ethyl acetate. The organic layer was washed
with brine and dried with sodium sulfate. The solvent was
concentrated, and the residue was purified by chromatography to
give the pure product. MS: calcd 361 (MH+), exp 361 (MH+).
rac-3-{5-[(trans-3,4)-4-(4-Chlorophenylcarbamoyl)-1-methyl-
pyrrolidin-3-yl]pyridin-2-yl}acrylic acid tert-Butyl Ester (XXV).
To a solution of rac-(trans-3,4)-4-[6-(2-tert-butoxycarbonylvinyl)-
pyridin-3-yl]-1-methylpyrrolidine-3-carboxylic acid ethyl ester (500
mg, 1.39 mmol) in THF (9 mL) was added lithium hydroxide
monohydrate (234 mg, 5.56 mmol) and water (3 mL). The mixture
was stirred at rt for 3 h. The solution was neutralized to pH 6−7. The
solvent was removed, and the residue (rac-(trans-3,4)-4-[6-(2-tert-
butoxycarbonylvinyl)pyridin-3-yl]-1-methylpyrrolidine-3-carboxylic
acid) was used directly in the next step. MS: calcd 333 (MH+), exp
333 (MH+).
To a solution of rac-(trans-3,4)-4-[6-(2-tert-butoxycarbonylvinyl)-
pyridin-3-yl]-1-methylpyrrolidine-3-carboxylic acid (430 mg, 1.3
mmol) in dichloromethane (10 mL) were added HATU (990 mg,
2.6 mmol), Et3N (525 mg, 5.2 mmol), and 4-chloroaniline (249 mg,
1.95 mmol). The mixture was stirred at rt for 24 h. The solvent was
removed, and the residue was dissolved in ethyl acetate. The organic
layer was washed with brine and dried with sodium sulfate. The
solvent was removed, and the residue was purified by chromatography
to give the pure product. MS: calcd 442 (MH+), exp 442 (MH+).
rac-(trans-3,4)-4-{6-[2-(2-Aminophenylcarbamoyl)vinyl]-
pyridin-3-yl}-1-methylpyrrolidine-3-carboxylic Acid (4-
Chlorophenyl)amide (61). To a solution of rac-3-{5-[(trans-3,4)-
4-(4-chlorophenylcarbamoyl)-1-methylpyrrolidin-3-yl]pyridin-2-yl}-
acrylic acid tert-butyl ester (172 mg, 0.5 mmol) in dichloromethane (5
mL) was added trifluoroacetic acid (570 mg, 5 mmol) at 0 °C. The
solution was allowed to rise to rt and stirred for 2 h. The solvent was
removed under reduced pressure to give the crude product (rac-3-{5-
[(trans-3,4)-4-(4-chlorophenylcarbamoyl)-1-methylpyrrolidin-3-yl]-
pyridin-2-yl}acrylic acid) which was used directly in next step. MS:
calcd 386 (MH+), exp 386 (MH+).
HPLC to give pure product. MS: calcd 476 (MH+), exp 476 (MH+).
1H NMR (CD3OD, 400 MHz), 8.57 (s, 1H), 7.88 (d, 1H, J = 8.4 Hz),
7.68−7.62 (m, 2H), 7.53 (d, 2H, J = 8.8 Hz), 7.28 (d, 2H, J = 8.8 Hz),
7.22−7.18 (m, 2H), 7.05 (t, 1H, J = 7.6 Hz), 6.87 (d, 1H, J = 8.0 Hz),
6.76 (t, 1H, J = 7.6 Hz), 3.90 (q, 1H, J = 8.0 Hz), 3.32−3.18 (broad m,
3H), 3.01 (t, 2H, J = 8.4 Hz), 2.54 (s, 3H).
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional information regarding compound characterization
(LCMS and 1H NMR), supplemental SAR data, and
experimental details for the CYP inhibition assay, metabolite
identification, cell lines and biological reagents, patient-derived
xenograft model study design, microarray profiling study of
compound 5, and immunohistochemistry scoring images. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-510-710-4129. E-mail: jwong@alum.mit.edu.
■
Present Address
#14 Moon Beam Drive, Mountain View, CA 94043.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank members of the following departments of the Roche
R&D Center (China) Ltd. for research support: (1) the
department of analytical chemistry (Wenzhi Cheng, Lucy Lu,
Sterling Yuan, Harkin Gao, Dr. Penny Ding, Dr. Fred Li) for
support on purification and characterization of compounds; (2)
the DMPK department (Hongxia Qiu, Yuxia Zhang, Jian Xin)
for conducting microsome, solubility, and CYP inhibition
studies; (3) the compound management and inventory group
(Lana Liu, Bill Wang, Qinghong Lv, Dr. Lynn Lin) for support
on obtaining and tracking synthetic reagents and formatting
final compounds for biological assays.
ABBREVIATIONS USED
■
AFP, α-fetoprotein; AUC, area under curve; Boc, COOC-
(CH3)3; CL, systemic clearance rate; CYP, cytochrome P450;
dba, dibenzylideneacetone; DCM, dichloromethane; DIPEA,
diisopropylethylamine; DMAP, 4-dimethylaminopyridine;
DMF, dimethylformamide; DMPK, distribution, metabolism,
pharmacokinetics; DMSO, dimethyl sulfoxide; EDCI, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride; EtOH,
ethanol; F, bioavailability; GFP, green fluorescent protein;
HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium hexafluorophosphate 3-oxide; HBV, hepatitis
B virus; HCC, hepatocellular carcinoma; HDAC, histone
deacetylase; HDACi, histone deacetylase inhibitor; hERG,
̀
human ether-a-go-go related gene; HLM, human liver micro-
somes; HOBt, hydroxybenzotriazole; HWE, Horner−Wads-
worth−Emmons; IHC, immunohistochemistry; i-PrOH, 2-
propanol; MeOH, methanol; MLM, mouse liver microsomes;
mpk, milligrams per kilogram; Ms, mesyl; NAD, nicotinamide
adenine dinucleotide; PD, pharmacodynamics; PE, petroleum
ether; PK, pharmacokinetics; PO, oral administration; Q2D,
once every two days; QD, every day; qRT-PCR, quantitative
real time polymerase chain reaction; SAR, structure−activity
relationship; SPR, structure−property relationship; TEA,
To a solution of rac-3-{5-[(trans-3,4)-4-(4-chlorophenylcarbamoyl)-
1-methylpyrrolidin-3-yl]pyridin-2-yl}acrylic acid (54 mg, 0.14 mmol)
in dichloromethane (3 mL) were added 1,2-diaminobenzene (46 mg,
0.42 mmol), HATU (80 mg, 0.21 mmol), and Et3N (85 mg, 0.84
mmol). The mixture was stirred at rt for 12 h. The solvent was
removed, and the residue was dissolved in ethyl acetate. The solution
was washed with brine and dried with sodium sulfate. The solvent was
concentrated to give the residue, which was purified by preparative
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Grauslund, M.; Christensen, I. J.; Tjørnelund, J.; Buhl Jensen, P.;
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M. Constitutive activation of signal transducers and activators of
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(19) Wong, J. C.; Guo, L.; Peng, Z. H.; Zhang, W. X.; Zhang, N.; Lai,
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(20) Zhao, H. Scaffold selection and scaffold hopping in lead
generation: a medicinal chemistry perspective. Drug Discovery Today
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(21) Joucla, M.; Mortier, J. 2,5-Unsubstituted pyrrolidines from
formaldehyde and amino acids through in situ azomethine-ylide 1,3-
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triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TSG, tumor suppressor gene; Vss, volume of distribution at
steady state; WST, water-soluble tetrazolium dye
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