Augmentation of Anticancer Drug Efficacy in Murine Hepatocellular Carcinoma Cells by a Peripherally Acting Competitive N-Methyl- d -aspartate (NMDA) Receptor Antagonist

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Augmentation of Anticancer Drug Eﬃcacy in Murine Hepatocellular

Carcinoma Cells by a Peripherally Acting Competitive

N‑Methyl‑^D‑aspartate (NMDA) Receptor Antagonist

Mikko Gynther,*^,⊥,‡Ilaria Proietti Silvestri,^†,⊥Jacob C. Hansen,^†Kasper B. Hansen,^§Tarja Malm,^∥

Yevheniia Ishchenko,^∥Younes Larsen,^†Liwei Han,^†Silke Kayser,^†Seppo Auriola,^‡Aleksanteri Petsalo,^‡

Birgitte Nielsen,^†Darryl S. Pickering,^†and Lennart Bunch*^,†

^†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen

2100, Denmark

^‡School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland

^§Department of Biomedical and Pharmaceutical Sciences and Center for Biomolecular Structure and Dynamics, University of

Montana, Missoula, Montana 59812, United States

^∥Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70211 Kuopio, Finland

S

* Supporting Information

ABSTRACT: The most common solid tumors show intrinsic multidrug resistance (MDR) or inevitably acquire such when

treated with anticancer drugs. In this work, we describe the discovery of a peripherally restricted, potent, competitive NMDA

receptor antagonist 1l by a structure−activity study of the broad-acting ionotropic glutamate receptor antagonist 1a.

Subsequently, we demonstrate that 1l augments the cytotoxic action of sorafenib in murine hepatocellular carcinoma cells. The

underlying biological mechanism was shown to be interference with the lipid signaling pathway, leading to reduced expression of

MDR transporters and thereby an increased accumulation of sorafenib in the cancer cells. Interference with lipid signaling

pathways by NMDA receptor inhibition is a novel and promising strategy for reversing transporter-mediated chemoresistance in

cancer cells.

the cancer cells.⁸⁻¹⁰The general strategy to reverse MDR has

INTRODUCTION

■

been to coadminister ABC transporter inhibitors with

Over time, solid tumors inevitably acquire resistance against

anticancer drugs.¹¹However, several complications limit the

anticancer therapy, a phenomenon known as multidrug

resistance (MDR).¹However, more devastating are cancers,

use of ABC transporter inhibitors, among them the fact that

ABC transporters are also present in normal cells, leading to

such as hepatocellular carcinoma (HCC), that exert intrinsic

undesired drug accumulation.¹¹Thus, a change of strategy is

needed to overcome ABC transporter-mediated chemoresist-

ance.

drug-resistance.²A key mechanism underlying MDR is

increased expression of ATP-binding cassette (ABC) trans-

porters, which expel a broad range of chemotherapeutic agents

from the cancer cells.³According to current knowledge, the

principal ABC transporters responsible for chemoresistance in

humans are ATP-binding cassette subfamily B member 1

(ABCB1, Pgp), ATP-binding cassette subfamily G member 2

(ABCG2, BCRP), and ATP-binding cassette subfamily C

(ABCC, MRP).⁴⁻⁷In regard to HCC, these transporters are

responsible for expelling the ﬁrst line HCC drug sorafenib from

One such strategy is to downregulate eﬄux transporter

expression by alternating the cell lipid signaling pathway by

blocking the N-methyl-^D-aspartate (NMDA) receptors.¹²

NMDA receptors are a subclass of ionotropic glutamate

Received: November 3, 2017

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receptors (iGluRs), which also comprises the α-amino-3-

hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors

and the kainic acid (KA) receptors.¹³NMDA receptor

activation increases calcium entry, which activates cytoplasmic

phospholipase A2 (cPLA2), leading to increased production of

arachidonic acid (Figure 1).^14,15This fatty acid is further

NMDA receptors are overexpressed on the cell membrane of

many types of cancer cells, including HCC.¹⁸The non-

competitive NMDA receptor antagonist MK-801 (Figure 2)

has previously been shown to augment the antiproliferative

eﬃcacy of antiestrogens in melanoma cells¹⁹and suppress the

growth of HCC cells.²⁰The antiproliferative eﬀect of MK-801

in HCC cells was shown to be mediated through the FOXO/

XTNIP pathway, which is not connected to pro-inﬂammatory

lipid signaling. Moreover, the impact of NMDA receptor

antagonists on transporter expression, anticancer drug accu-

mulation, and eﬃcacy in cancers has not been studied. Most

importantly, a competitive NMDA receptor antagonist for use

as an augmentative drug for the treatment of peripheral solid

tumors must not be capable of crossing the blood−brain barrier

(BBB), as this would otherwise lead to severe adverse eﬀects,

including psychosis.²¹

In the present study, we report the design and synthesis of

the novel peripherally acting potent NMDA receptor antagonist

1l. We describe the ability of 1l to modulate the cPLA2

activation-dependent lipid signaling pathway, downregulate

ABC transporter expression, and thereby augment the cytotoxic

eﬃcacy of the anticancer drug sorafenib in murine HCC cells.

Figure 1. Activation of the lipid signaling pathway by GluN1/GluN2A

receptor and the creation of transporter mediated drug resistance.

NMDA binding to the GluN1/GluN2A receptor allows the inﬂux of

Ca²⁺into the cell, which leads to cPLA2 activation and the release of

arachidonic acid (AA) from phospholipids. Arachidonic acid is

metabolized to PGE₂, which is transported by ABCC transporters

out of the cell allowing the PGE₂binding to EP-R. The EP-R

activation leads to nuclear translocation of NF-κB followed by ABCG2

protein transcription and MDR.^12,14−17

RESULTS AND DISCUSSION

■

The two commonly studied competitive NMDA receptor

antagonists that do not penetrate the BBB are D-AP5 and (R)-

CPP (Figure 2). Both are amino acid analogs and highly polar

with calculated partition coeﬃcient values in octanol/water

(cLogP(o/w)) well below zero (−1.7 and −2.2, respectively).

In comparison with recommended cLogP(o/w) values of −0.4

to 5.6²²for oral bioavailability, we believed that these NMDA

receptor antagonists were not attractive candidates for this

study.

converted to the proinﬂammatory lipid, prostaglandin-E2

(PGE₂), and actively transported into the extracellular space

by ABCC transporters, where it binds to the prostaglandin E

receptors (EP-Rs), leading to NF-κB activation and creating a

positive feedback loop creating an inﬂammation microenviron-

ment.^12,14It has been shown that NF-κB activation results in

increased cancer cell survival and proliferation as well as

chemoresistance due to elevated ABC transporter and CYP

enzyme expression.^12,16,17

We therefore turned to the previously reported nonselective

iGluR antagonist 1a (Figure 2 and Table 1).²³The facts that 1a

does not penetrate the BBB in mouse in situ brain perfusion

and that its cLogP(o/w) value is calculated at 1.3 make it an

attractive starting point for the purpose of developing an orally

available, peripherally restricted, competitive NMDA receptor

antagonist.²²

Figure 2. Chemical structures of Glu, selective uncompetitive NMDA receptor antagonist MK-801, selective competitive NMDA receptor

antagonists D-AP5, (R)-CPP, and competitive iGluR antagonist 1a, including published analogs 1b,c with relevance to the SAR study reported

herein.

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Table 1. Binding Aﬃnities of 1a−n at Native AMPA, KA, and NMDA Receptors (Rat Synaptosomes) and Cloned Homomeric

a

Receptors GluK1−3

a

−: not tested. Radio ligands: AMPA, [³H]AMPA; KA, [³H]KA; NMDA, [³H]CGP-39653; GluK1, [³H]SYM2081; GluK2 and GluK3, [³H]KA.

Data are mean values of three to six individual experiments performed in triplicate. For AMPA and KA: pIC₅₀values with SEM in brackets. For

NMDA: pK_ivalues with SEM in brackets.

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We have previously reported the ﬁrst structure−activity

relationship (SAR) study on 1a, which disclosed the 4-position

on the aryl ring as a hotspot for induction of KA receptor

subtype selectivity (compound 1b, Figure 2 and Table 1). Also,

it was seen that simple lipophilic substituents in the 5-position

did not lead to any signiﬁcant improvement in receptor

selectivity or higher aﬃnity (structures not shown).²⁴The 3-

carboxylic acid functionality was displaced with a phosphonic

acid group, compound 1c (Figure 2), which led to a signiﬁcant

reduction in binding aﬃnity at the iGluRs (Table 1).²⁴

Scheme 1. Synthesis of Key Alcohol Intermediates 6b,d−f,j

via Rhodium(I)-Catalyzed 1,4 Addition of Respective

a

Boronic Acids 3a,b,d−f,j to Enone 2

With this SAR information in hand, we set out to investigate

two strategies with the aim of improving iGluR class selectivity.

First, we wanted to substitute the 3-carboxylic acid functionality

with diﬀerent functional groups (analogs 1d−j). While it is

well-accepted that the γ-carboxylate functionality in Glu is

mandatory for agonist activity at the iGluRs,¹³it remains an

open question if nonionizable distal functional groups can

stabilize the NMDA receptor in its open antagonist state. Based

on synthetic tractability, the following seven analogs were thus

designed: 3-chloro (1d), 3-triﬂuoromethyl (1e), 3-amino (1f),

3-cyano (1g), 3-carbamido (1h), 3-boronic acid (1i), and 3,4-

dihydroxy (1j). The synthesis of 1d−j was carried out by a

stereoselective rhodium(I)-catalyzed addition of an arylboronic

acid to protected enone 2²⁴as the key step (Scheme 1).²⁵

Hereby, the 2,3-trans stereochemistry on the proline ring was

set, and subsequent functional group transformations in

accordance with earlier reported strategies (1d−f, Scheme 2;

1g,h, Scheme 3; 1i, Scheme 4; 1j, Scheme 5) gave the free

amino acids 1d−j ready for pharmacological evaluation.^23,24

The second series of analogs, compounds 1k−n, aimed to

further explore the impact of substituents in the 4-position of

the aryl ring. We have previously shown that introduction of a

4-hydroxy group (analog 1b) resulted in generally enhanced

aﬃnity for iGluRs with a 10-fold improvement for the GluK3

subunit (Table 1).²⁴Given the hydrogen bond donor and

acceptor abilities of the 4-hydroxyl group, three new analogs

were designed to address the inﬂuence of these features: 4-

ﬂuoro (1k), 4-chloro (1l), and 4-methyl (1m). For 1k and 1l,

the syntheses were based on the stereoselective conjugate

addition of an in situ formed aryl cuprate to enone 2,^24,26

whereas for 1m the afore-applied protocol of rhodium(I)-

catalyzed addition of a boronic ester to enone 2 was used

(Scheme 6). Subsequent functional group transformations to

obtain the free amino acids 1k−m followed the strategy for

comparable analogs previously reported by us.^23,24

a

Reagents and conditions: (a) [Rh(cod)Cl]₂, H₂O, Cs₂CO₃, enone 2,

THF or dioxane, rt (52−67%); (b) LiBEt₃H, THF, −78 °C; (c)

HSiEt₃, BF₃·Et₂O, DCM, −78 °C (31−78%).

Scheme 2. Synthesis of 1d−f from Alcohols 6d−f,

Respectively

a

Binding aﬃnities of the synthesized amino acids 1d−n were

determined at native AMPA, KA, and NMDA receptors (rat

synaptosomes) and cloned rat homomeric subtypes GluK1−3,

and results are summarized in Table 1. The 3-chloro, 3-

triﬂuoromethyl, 3-amino, and 3-cyano analogs, 1d−g.,

respectively, all showed insigniﬁcant binding aﬃnity for any

of the iGluRs (IC₅₀or K_i> 100 μM). Furthermore, 3-

carbamido analog 1h and 3-boronic acid analog 1i did not

display notable binding aﬃnities for native iGluRs nor for

homomeric GluK1−3 receptors (IC₅₀or K_i> 100 μM). Finally,

the 3,4-dihydroxy analog 1j was a weak binder at the NMDA

receptors (K_i= 35 μM). While these results were all together

disappointing, the aﬃnity proﬁles for the 4-substituted analogs

1k−m were in contrast exciting. In comparison with the 4-

hydroxy analog 1b, the 4-ﬂuoro analog 1k displayed the ﬁrst

a

Reagents and conditions: (a) RuCl₃·xH₂O, NaIO₄, EtOAc/MeCN/

H₂O (49−86%); (b) TFA, DCM, rt; (c) 1 M HCl (12−78%).

steps toward selectivity for the NMDA receptors by showing a

lower aﬃnity for native AMPA and KA receptors as well as for

homomeric GluK1−3 receptors. This trend was boosted

signiﬁcantly for 4-chloro analog 1l, which proved to be

selective for native NMDA receptors with submicromolar

binding aﬃnity (K_i= 0.63 μM). Also for 4-bromo analog 1n,²⁷

full selectivity for NMDA receptors was observed with binding

aﬃnity similar to 1l (K_i= 0.62 μM). In contrast to this

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a

Scheme 3. Synthesis of 3-Cyano Analog 1g from Alcohol 6b and 3-Carbamido Analog 1h from 7g

a

Reagents and conditions: (a) TBSCl, imidazole, DMF, rt (73%, three steps from 4b); (b) Zn(CN)₂, Pd₂(dba)₃, dppf, DMA, 120 °C; (c) TBAF,

THF, rt (71%, two steps); (d) RuCl₃·xH₂O, NaIO₄, EtOAc/MeCN/H₂O (76%); (e) TFA, DCM, rt; (f) 1 M HCl (81%, two steps); (g) 30% H₂O₂,

K₂CO₃, EtOH/H₂O, rt (66%); (h) TFA, DCM, rt; (i) 1 M HCl (59% after three steps).

a

Scheme 4. Synthesis of 3-Boronic Acid Analog 1i, Starting

from Bromine 8b

Scheme 5. Synthesis of 1j from Alcohol 6j

a

Reagents and conditions: (a) IBX, DMSO, rt; (b) NaClO₂,

NaH₂PO₄, 2-methyl-2-butene, tert-BuOH/H₂O, rt (66%, two steps);

(c) BBr₃, DCM (8% after recrystallization from MeOH).

a competitive antagonist with K_ivalues of 4.7, 10, 24, and 41

μM, respectively, and the selectivity proﬁle of 1l was similar to

D-AP5 (K_ivalues of 0.39, 2.8, 5.9, and 21, respectively²⁸).

Pharmacokinetics of Compound 1l in Mice. With the

attractive pharmacological proﬁle of 1l in hand, we turned to

determine its pharmacokinetics and brain permeation. The

pharmacokinetic analysis was performed by 10 mg/kg i.p.

injection at ﬁve time points between 10 and 240 min. The

apparent pharmacokinetic parameters, area under the concen-

tration−time curve from time zero to 240 min (AUC_{0−240 min}),

the maximum concentration after dosing (C_max), time to reach

a

Reagents and conditions: (a) KOAc, Pd₂(dba)₃, dppf, DMF (61%);

(b) TBAF, THF, rt (60%); (c) RuCl₃·xH₂O, NaIO₄, EtOAc/MeCN/

H₂O (65%); (d) TFA, DCM, rt; (e) 1 M HCl (40%, two steps).

important ﬁnding, the binding aﬃnity for native NMDA

receptors dropped 30-fold for the 4-methyl analog 1m.

The cLogP(o/w) values of 1l and 1n were calculated at 1.9

and 2.1, respectively, placing both analogs in the recommended

cLogP interval of −0.4 to 5.6 for good bioavailability.²²Given

that an aryl bromide is a less attractive functional group in

compounds for biological administration compared to an aryl

chloride, compound 1l was selected for further functional

studies at NMDA receptors.

Functional Characterization of 1l at NMDA Receptor

Subtypes. Compound 1l was next evaluated in a functional

assay at the four NMDA receptor subtypes, GluN1/GluN2A-D

(Figure 3 and Table 2). At all four subtypes, 1l was found to be

C

_max(t_max), and elimination half-life (t_1/2β) in plasma, liver, and

brain, calculated from the in vivo data are presented in Table 3.

The compound was absorbed from the injection site, and

concentrations above the K_ivalue were detected from both

plasma and liver. The K_{p liver/plasma}value was 0.19. Importantly,

compound 1l did not exhibit BBB permeation and was detected

only at 30 and 60 min time points with concentrations of 0.3

and 0.1 nmol/g, respectively. We conﬁrmed the poor BBB

permeation using an in situ mouse brain perfusion technique.

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Scheme 6. Synthesis of 1k−l via Copper(I)-Catalyzed Addition to Enone 2 and 1m via Rhodium(I)-Catalyzed Addition to

a

Enone 2

a

Reagents and conditions: (a) for 3k,l, n-BuLi, CuCN, then enone 2, Et₂O, −78 to −42 °C (46% and 73%); (b) for 3m, (Bpin)₂, KOAc,

(PPh₃)₂PdCl₂(quant); (c) [Rh(cod)Cl]₂, H₂O, Cs₂CO₃, enone 2, rt (41%); (d) for 4k,l, LiBEt₃H, THF, −78 °C, then HSiEt₃, BF₃·Et₂O, DCM,

−78 °C; (e) for 4m, BH₃·SMe₂, THF, reﬂux; (f) TBAF, THF, rt. (20% and 49%); (g) RuCl₃·xH₂O, NaIO₄, EtOAc/MeCN/H₂O (20−97%); (h)

TFA, DCM, rt; (i) 1 M HCl (56−60%).

Table 2. Inhibition of Recombinant NMDA Receptor

a

Subtypes by 1l

IC₅₀(μM)

n_H

estimated K_i(μM)

N

GluN1/GluN2A

GluN1/GluN2B

GluN1/GluN2C

GluN1/GluN2D

15

29

1

8

1.3

1.0

1.3

4.7 0.1

7

5

4

7

10

24

41

1

2

3

110

170 10

a

IC₅₀values for inhibition of current responses activated by

coapplication of 100 μM glycine and Glu to Xenopus oocytes

expressing recombinant rat GluN1/GluN2A−D NMDA receptors.

Responses were activated by Glu concentrations 2- to 3-fold higher

than the EC₅₀at the respective NMDA receptor subtypes; 10 μM Glu

was used for GluN1/GluN2A, 3 μM Glu for GluN1/GluN2B and

GluN1/GluN2C, and 1 μM Glu for GluN1/GluN2D receptors. K_i

values were estimated using the Cheng−Prusoﬀ relationship²⁹and

previously determined Glu EC₅₀values.³⁰IC₅₀and K_ivalues are mean

SEM, n_His the Hill slope, and N is the number of oocytes.

Figure 3. Concentration−inhibition data for 1l at recombinant

NMDA receptor subtypes GluN1/GluN2A-D. Responses were

measured using two-electrode voltage-clamp electrophysiology and

were activated by coapplication of 100 μM glycine and Glu to Xenopus

oocytes expressing recombinant NMDA receptors subtypes. Ten

micromolar Glu was used for GluN1/GluN2A, 3 μM Glu for GluN1/

GluN2B and GluN1/GluN2C, and 1 μM Glu for GluN1/GluN2D

receptors. Data are mean

SD (error bars are mostly contained

within the symbols). See Table 2 for IC₅₀and estimated K_ivalues.

Table 3. Pharmacokinetic Parameters of Compound 1l in

Plasma, Liver, and Brain Calculated from in Vivo Data after a

Single Dose of 10 mg/kg i.p. in Mice

The brain concentration of compound 1l following perfusion

with 100 μM compound was below the detection limit of our

analytical method (5 pmol/g). Moreover, there were no

observable changes in the behavior of the mice after 1l

injection. This supports our ﬁndings from the in vivo

pharmacokinetic experiments that 1l is a fully peripherally

restricted NMDA receptor antagonist suitable for aﬀecting

peripheral tumors. In addition to hepatocellular carcinoma,

NMDA receptor expression has been reported to be elevated in

colon, prostate, and breast cancers compared to healthy

tissues.¹⁸Moreover, the lack of pharmacological eﬀect in the

CNS provides the possibility to utilize the compound as a

research tool for investigating the role of peripheral NMDA

receptors in other diseases and/or conditions such as chronic

pain.

plasma

liver

brain

AUC_{(0−240 min)}(nmol/g·min)

C_max(nmol/g)

3020

43.4

15

575

8.7

30

13

0.3

30

t_max(min)

t_1/2β(min)

30

36

ND

Ability of Compound 1l To Inhibit NMDA Receptor

Expressed in Murine HCC Cells. With compound 1l in hand,

we proceeded to studies of its in vitro eﬃcacy in mice. First, we

conﬁrmed the expression of the GluN1 NMDA receptor

subunit in the murine HCC cell line by Western immunoblot-

ting (data not shown). In order to assess the function of

NMDA receptors in the murine HCC cell culture we used

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Figure 4. Ca²⁺transients via NMDA receptors in murine HCC cells. (A) Ca²⁺transients in HCC cells activated by NMDA applications (15 s) with

and without NMDA receptor antagonists (1l and D-AP5). (B) Histogram showing percentage of induced Ca²⁺transients in a response to NMDA

application and absence of the activation while applied together with 1l at 25 or 41 μM concentration and D-AP5 at 20 μM concentration.

Quantitative data were expressed as mean SEM (n = 5). The statistical signiﬁcance was assessed with the Student-paired t test or Mann−Whitney t

test for nonparametric data. Statistically signiﬁcant diﬀerences were set at *P < 0.05.

calcium imaging (Figure 4). To compare the abilities of 1l and

D-AP5 to inhibit NMDA receptor activation, we measured the

intracellular Ca²⁺transients induced by 150 μM NMDA alone

and in the presence of the compound 1l (25 and 40 μM) or D-

AP5 (20 μM). Both compounds were able to inhibit NMDA

receptor activation signiﬁcantly in the murine HCC cells

suggesting their ability to antagonize the lipid signaling pathway

in cancer cells.

Ability of Compound 1l To Decrease PGE₂Concen-

tration in Murine HCC Cells. The ability of compound 1l to

decrease the extracellular PGE₂concentration in murine HCC

cells was investigated by incubating the cells in 24-well plates

for 24 h with 100 μM 1l. In addition, the PGE₂concentration

was measured from HCC cells with lipopolysaccharide (LPS)

induced inﬂammation using the same compound 1l concen-

tration and incubation time (Figure 5). Compound 1l

signiﬁcantly decreased the PGE₂concentration in both the

presence and absence of LPS, conﬁrming that the compound is

able to interfere with the synthesis of PGE₂. This inhibition of

PGE₂production will reduce the proinﬂammatory lipid

signaling pathway in HCC cells, which has been suggested to

downregulate ABC transporters in cancer cells.^12,14−16

Figure 5. Eﬀect of compound 1l and D-AP5 on PGE₂levels in HCC

cells. Compounds 1l and D-AP5 at 10 μM and compound 1l at 100

μM reduced the concentration of PGE₂after 24 h incubation to 68

5%, 58 6%, and 48 6%, respectively. The addition of LPS (2.5 μg/

mL) increased the PGE₂concentration to 162 7%, and the addition

of compounds 1l and D-AP5 at 10 μM as well as compound 1l at 100

μM prevented the eﬀect of LPS, reducing the PGE₂concentrations to

Eﬀect of the Compound 1l on Transporter Expression

in Mouse HCC Cells. Expression levels of Abcb1, Abcg2,

Abcc2, and Abcc4 as well as Slc7a5 and Slc2a1 in HCC cells

were determined by selected/multiple reaction monitoring

(SRM/MRM) analysis in liquid chromatography−tandem mass

spectrometry (LC−MS/MS),³¹following administration of 10

μM 1l and compared to control. Compound 1l reduced the

expression levels of Abcb1 and Abcg2 transporters in the crude

membrane fraction by 39% and 34%, respectively (Table 4).

Abcc4 protein expression was reduced by 14%, but the

reduction was not statistically signiﬁcant, and Abcc2 protein

expression was below the lower limit of quantiﬁcation.

Interestingly, the expression of Slc2a1 and Slc7a5 transporter

proteins was decreased by 42% and 24%, respectively.

However, the diﬀerence in the case of Slc7a5 was not

statistically signiﬁcant. In all, these ﬁndings evidence that

NMDA receptor antagonism results in downregulation of ABC

transporter expression in HCC cells. In addition, the expression

of the two investigated nutrient transporters was reduced. In

127

4%, 110

7%, and 96

17%, respectively. The data is

presented as mean

SEM, n = 3. The statistical signiﬁcance of

diﬀerences in PGE₂concentrations between treatments was

determined using one-way ANOVA and Tukey’s test (*P < 0.05,

**P < 0.01).

order to further study the signiﬁcance of the transporter

downregulation, we investigated the cell accumulation of

transporter probes and sorafenib.

Ability of NMDA Receptor Antagonist 1l To Alter the

Cell Accumulation of Transporter Probes and Sorafenib.

To investigate the ability of compound 1l to reverse ABC

transporter mediated MDR and decrease the uptake of essential

nutrients in murine HCC cells, we determined the intracellular

accumulation of known ABC transporter substrates: Abcb1

probe [³H]-digoxin,³²Abcc1−5 probe ﬂuorescein,³³Abcb1 and

Abcg2 substrate sorafenib (Figure 6) as well as Slc7a5 substrate

[¹⁴C]-L-leucine and Slc2a1 substrate [¹⁴C]-D-glucose (Figure

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Table 4. ABC Transporter Expression Levels in Mouse HCC Cell Crude Membrane Fraction Measured by SMR/MRM Analysis

protein expression level (fmol/μg of total protein)

Abcb1 (Pgp)

0.64 0.08

Abcg2 (Bcrp)

Abcc2 (Mrp2)

Abcc4 (Mrp4)

Slc2a1 (Glut1)

Slc7a5 (Lat1)

control

1.88 0.04

a

0.31 0.04

0.27 0.04

22.7 0.84

7.55 0.28

5.72 1.61

a

10 μM 1l

0.39 0.06*

1.24 0.13**

13.2 0.53***

a

below the lower limit of quantiﬁcation. The statistical diﬀerence between compound 1l-treated cells and the control cells was determined using

unpaired two-tailed t test (*P < 0.05, **P < 0.01, ***P < 0.001). Data are presented as mean SEM (n = 4).

Figure 6. Eﬀect of compound 1l on cell accumulation of eﬄux probes [³H]-digoxin, ﬂuorescein, and sorafenib. The incubation of the HCC cells with

10 μM compound 1l increased the cell accumulation of (A) [³H]-digoxin, (B) ﬂuorescein, and (C) sorafenib to 149% 10%, 201% 9%, and 152%

11%, respectively. The analyte concentrations are normalized by the amount of protein in each sample. The statistical diﬀerence between

compound 1l-treated cell and the control cells was determined using unpaired two-tailed t test (**P < 0.01, ***P < 0.001). Data are presented as

mean SEM (n = 3).

7).⁸⁻¹⁰The cells were incubated with or without 10 μM of

compound 1l in the growth medium for 24 h. Accumulation of

the eﬄux probes in the cells was signiﬁcantly increased after

incubating the cells with compound 1l. In addition, the cell

uptake of [¹⁴C]-L-leucine was reduced signiﬁcantly. Therefore,

the data provides evidence that the reduced transporter

expression levels are signiﬁcant enough to lead to reduced

transporter activity. The risk of compound 1l to interfere with

transporter function was minimized by washing the cells before

uptake experiments. Thus, the increased cell accumulation of

transporter probes is not likely due to compound 1l acting as a

transporter inhibitor, but as a modulator of transporter

expression.

Eﬀect of Compound 1l on Sorafenib Cytotoxicity in

Mouse HCC Cells. The half maximal inhibitory concentration

(IC₅₀) value of sorafenib on HCC cell viability was investigated

at 72 h using concentration range from 0.1 to 200 μM (Figure

8A). The eﬀect of 1l on HCC cell proliferation and the possible

potentiating eﬀect on sorafenib cytotoxicity were evaluated by

incubating the HCC cells for 72 h at diﬀerent sorafenib

concentrations with and without a 100 μM concentration of 1l

(Figure 8B). Interestingly, 1l signiﬁcantly augmented the

eﬃcacy of sorafenib at 1, 2.5, and 5 μM concentrations. At

10 μM sorafenib concentration, NMDA receptor antagonist 1l

also augmented sorafenib eﬃcacy, although the eﬀect was less

pronounced. On its own the NMDA antagonist 1l was only

able to reduce HCC cell proliferation slightly. In addition, D-

AP5 had a potentiating eﬀect on sorafenib cytotoxicity at all

sorafenib concentrations investigated (Figure 8C). Previously it

was shown that at above 100 μM concentrations a non-

competitive NMDA receptor antagonist has antiproliferative

eﬀects on HCC cells aﬀecting the FOXO/TXNIP pathway.

Therefore, the antiproliferative eﬀect of 1l alone is likely

mediated via the same pathway. However, as sorafenib exerts its

eﬀect through the RAF/MEK/ERK pathway,³⁴the potentiating

eﬀect of 1l is more likely mediated through the lipid signaling

pathway and downregulation of eﬄux transporter expression.

Figure 7. Eﬀect of compound 1l on cell accumulation of Slc2a1 and

Slc7a5 transporter substrates [¹⁴C]-D-glucose and [¹⁴C]-L-leucine. The

incubation of the HCC cells with 10 μM compound 1l reduced the

cell accumulation of (A) [¹⁴C]-D-glucose and (B) [¹⁴C]-L-leucine to

84% 8% and 81% 6%, respectively. The analyte concentrations are

normalized by the amount of protein in each sample. The statistical

diﬀerence between compound 1l-treated cell and the control cells was

determined using unpaired two-tailed t test (*P < 0.05). Data are

presented as mean SEM (n = 4).

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Figure 8. (A) Concentration-dependent antiproliferative eﬃcacy of sorafenib in HCC cells after 72 h. IC₅₀value was 7.4 1.7 μM. Ability of

compound 1l to potentiate the cytotoxic eﬃcacy of sorafenib in mouse HCC cells. (B) Potentiating eﬀect of compound 1l on sorafenib

antiproliferative eﬃcacy in murine HCC cells. Compared to sorafenib eﬀect alone, the cell viability was further reduced in the presence of 100 μM

compound 1l from 88 3% to 33 3%, 82 3% to 30 2%, 72 1% to 35 2%, and 31 2% to 24 1%, at 1, 2.5, 5, and 10 μM sorafenib,

respectively. Compound 1l at 100 μM without sorafenib was able to reduce the cell viability to 84 2%. (C) Potentiating eﬀect of D-AP5 on

sorafenib antiproliferative eﬃcacy in murine HCC cells. The cell viability was reduced to 70 3%, 63 1%, 44 1%, and 16 1%, in combination

of 100 μM D-AP5 and 1, 2.5, 5, and 10 μM sorafenib, respectively. D-AP5 at 100 μM without sorafenib was able to reduce the cell viability to 79

2%. The remaining viability is presented as mean SEM (n = 3−6). The statistical diﬀerence between groups were determined by one-way ANOVA

followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).

Zorbax 300 SB-C18 (21.2 × 250 mm, 7 μm) column. LC−MS was

performed using an Agilent 1200 HPLC system coupled to an Agilent

6400 triple quadrupole mass spectrometer equipped with an

electrospray ionization source. A Zorbax Eclipse XDB-C18 (4.6 ×

50 mm) column and gradients of 10% aqueous acetonitrile + 0.05%

formic acid (buﬀer A) and 90% aqueous acetonitrile + 0.046% formic

acid (buﬀer B) were employed. Optical rotation was measured using a

PerkinElmer 241 spectrometer, with a Na lamp at 589 nm. Melting

points were measured using an automated melting point apparatus,

MPA100 OptiMelt (SRS), and are uncorrected. Compounds were

dried either under high vacuum or freeze-dried using a Holm and

Halby, Heto LyoPro 6000 freeze drier. Compounds for pharmaco-

logical characterization were all with a purity of >95%, determined by

HPLC (254 nM).

(2S,3R)-2-Carboxy-3-(3-chlorophenyl)pyrrolidin-1-ium Chloride

(1d). Acid 7d (120 mg, 0.37 mmol, 1.0 equiv) was dissolved in a

1:1 mixture of TFA/DCM (7.2 mL). The reaction mixture was

allowed to stir for 1 h at rt, then evaporated to dryness under reduced

pressure. The solid was dissolved in 1 M HCl (2 mL) and evaporated

to dryness (3×) to give the crude product, which was recrystallized

from MeOH/CHCl₃to aﬀord the title compound as white needles (17

mg, 18%). ¹H NMR (CDCl₃) δ (two rotamers) 9.64 (br s, 1H), 7.27−

7.22 (m, 3H), 7.15−7.13 (m, 1H), 4.40 (d, J = 5.5 Hz, 0.4H), 4.25 (d,

J = 6.5 Hz, 0.6H), 3.79−3.44 (m, 3H), 2.38−2.28 (m, 1H), 2.06−1.96

(m, 1H), 1.49 (s, 4H), 1.42 (s, 5H). ¹³C NMR (CDCl₃) δ (two

rotamers) 177.6, 175.7, 155.2, 153.7, 142.9, 142.4, 134.6, 130.1, 127.5,

127.4, 127.1, 125.2, 81.2, 80.1, 65.4, 64.9, 49.4, 47.5, 46.1, 45.9, 32.7,

32.2, 28.3, 28.2. MS (m/z) calcd. for C₁₁H₁₃ClNO₂[M + H]⁺226.1,

found 226.1. Mp: decomposition.

(2S,3R)-2-Carboxy-3-(3-(triﬂuoromethyl)phenyl)pyrrolidin-1-ium

Chloride (1e). Compound 7e (135 mg) was dissolved in a 1:1 mixture

TFA/DCM (7.0 mL). The reaction mixture was allowed to stir for 1 h

at rt, then the solvent was evaporated under reduced pressure. The

crude was dissolved in HCl 1 M (30 mL), and the solvent was

evaporated to aﬀord the corresponding HCl salt (63 mg) as a white

solid. The compound was recrystallized from MeOH/CHCl₃to aﬀord

the title compound as a white powder (51 mg, 46%). ¹H NMR

(CDCl₃) δ (two rotamers) 9.64 (br s, 1H), 7.27−7.22 (m, 3H), 7.15−

7.13 (m, 1H), 4.40 (d, J = 5.5 Hz, 0.4H), 4.25 (d, J = 6.5 Hz, 0.6H),

3.79−3.44 (m, 3H), 2.38−2.28 (m, 1H), 2.06−1.96 (m, 1H), 1.49 (s,

4H), 1.42 (s, 5H). ¹³C NMR (CDCl₃) δ (two rotamers) 177.6, 175.7,

155.2, 153.7, 142.9, 142.4, 134.6, 130.1, 127.5, 127.4, 127.1, 125.2,

CONCLUSION

■

Starting from the broad-acting iGluR antagonist 1a, we have

developed a potent and selective competitive NMDA receptor

antagonist, compound 1l, whose action is restricted to

peripheral tissues and which displays good drug-like properties.

Application of 1l to murine HCC cells reduced the intracellular

concentration of PGE₂and thereby interfered with the

proinﬂammatory lipid signaling pathway resulting in down-

regulation of MDR transporters. It was subsequently shown

that 1l augments cell accumulation of MDR transporter

substrates and that cell accumulation of the HCC anticancer

agent and ABC transporter substrate sorafenib was signiﬁcantly

increased leading to augmented cytotoxic eﬃcacy. Another

important ﬁnding was the signiﬁcant downregulation of

nutrient transporters, which are essential for the rapid growth

of cancer cells. In summary, this proof-of-concept study

demonstrates that competitive NMDA receptor antagonists

represent a novel and promising strategy to reverse MDR in

peripheral solid tumors, such as HCC.

EXPERIMENTAL SECTION

■

Chemistry. All reagents were obtained from commercial suppliers

and used without further puriﬁcation. Dry solvents were obtained

diﬀerently. THF was distilled over sodium/benzophenone. Et₂O was

dried over neatly cut sodium. All solvents were tested for water

content using a Carl Fisher apparatus. Water- or air sensitive reactions

were conducted in ﬂame-dried glassware under nitrogen with syringe-

septum cap technique. Puriﬁcation by dry column vacuum

chromatography (DCVC) was performed with silica gel size 25−40

μm (Merck, Silica gel 60). For TLC, Merck TLC Silica gel F254 plates

were used with appropriate spray reagents: KMnO₄or molybdenum

blue. ¹H NMR and ¹³C NMR spectra were obtained on a Varian

Mercury Plus (300 MHz) and a Varian Gemini 2000 instrument (75

MHz), respectively, unless otherwise noted. Dioxane was used as

internal reference for NMR spectra run in D₂O. Preparative HPLC

was performed using either a Spectraseries UV100 detector with a

JASCO 880-PU HPLC pump and an XTerraPrep MS C18 (10 μm, 10

× 300 mm) column or an Agilent Prep HPLC system, equipped with a

1100 series pump, a 1200 series multiple wavelength detector, and a

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81.2, 80.1, 65.4, 64.9, 49.4, 47.5, 46.1, 45.9, 32.7, 32.2, 28.3, 28.2. MS

128.7, 65.2, 48.2, 45.8, 33.1. LC−MS (m/z) calcd. for C₁₁H₁₅BNO₄

[M + H]⁺236.1, found 236.1. Mp: decomposition.

D

(m/z) calcd. for C₁₂H₁₃F₃NO₂[M + H]⁺295.06, found 295.1. [α]₂₅

(2S,3R)-3-(3,4-Dihydroxyphenyl)pyrrolidine-2-carboxylic Acid

(1j). In a ﬂame-dried ﬂask a solution of 7j (70 mg, 0.21 mmol, 1.0

equiv) in dry DCM (3 mL) was cooled to 0 °C. A 1 M solution of

BBr₃(in DCM) (0.53 mL, 0.522 mmol, 2.5 equiv) was added

dropwise, and the reaction mixture was allowed to stir 3 h at rt. The

reaction mixture was quenched with H₂O (1 mL), and the solvent

evaporated. The crude product was puriﬁed by preparative HPLC and

then recrystallized from MeOH to aﬀord the title compound as a white

+58.9 (c = 0.38, MeOH). Mp: 180.7−182.5 °C.

(2S,3R)-3-(3-Aminophenyl)pyrrolidine-2-carboxylic Acid Dihydro-

chloride (1f). Acid 7f (41 mg, 0.101 mmol, 1 equiv) was dissolved in a

1:1 mixture TFA/DCM (2.0 mL). The reaction mixture was allowed

to stir for 1.5 h at rt, then the solvent was evaporated under reduced

pressure. The oily residue was dissolved in 1 M HCl (10 mL), and the

solvent was evaporated (3×) to give the crude product, which was

recrystallized from MeOH/CHCl₃to aﬀord the title compound as a

1

solid (3.8 mg, 8% yield). H NMR (CDCl₃) δ 6.89−6.94 (m, 2H),

white solid (22 mg, 78%). H NMR (MeOD) δ 7.58−7.31 (m, 3H),

6.79−6.86 (m, 1H), 4.02 (d, J = 9.03 Hz, 1H), 3.54−3.62 (m, 1H),

3.36−3.51 (m, 2H), 2.43 (dtd, J = 3.64, 6.73, 13.52 Hz, 1H), 2.09−

2.23 (m, 1H). MS (m/z) calcd. for C₁₁H₁₃NO₄[M + H]⁺224.09,

found 224.1.

7.31 (d, J = 7.2 Hz, 1H), 4.45 (d, J = 9.1 Hz, 1H), 3.77−3.58 (m, 2H),

3.57−3.42 (m, 1H), 2.66−2.49 (m, 1H), 2.36−2.17 (m, 1H). ¹³C

NMR (CDCl₃) δ 168.5, 144.4, 140.6, 131.18, 131.17, 124.8, 118.6,

66.7, 49.6, 46.8, 34.4. MS (m/z) calcd. for C₁₁H₁₅N₂O₂[M + H]⁺

D

(2S,3R)-2-Carboxy-3-(3-carboxy-4-ﬂuorophenyl)pyrrolidin-1-ium

Chloride (1k). Acid 7k (85 mg, 0.24 mmol, 1 equiv) was dissolved in a

1:1 mixture TFA/DCM (4.66 mL). The reaction mixture was allowed

to stir for 2 h at rt, then the solvent was evaporated under reduced

pressure. The oily residue was dissolved in HCl 1 M (10 mL), and the

solvent was evaporated (3×) to aﬀord the title compound as a white

solid (42 mg, 60%). ¹H NMR (D₂O, 400 MHz) δ 7.97 (dd, J = 6.9, 2.6

Hz, 1H), 7.69 (ddd, J = 8.6, 4.6, 2.5 Hz, 1H), 7.31 (dd, J = 11.0, 8.6

Hz, 1H), 4.36 (d, J = 9.7 Hz, 1H), 3.77−3.66 (m, 2H), 3.57 (ddd, J =

11.8, 10.2, 6.9 Hz, 1H), 2.59 (dtd, J = 13.9, 7.0, 3.3 Hz, 1H), 2.29 (dtd,

J = 13.4, 10.4, 8.2 Hz, 1H). ¹³C NMR (D₂O) δ 172.0, 168.4, 162.5,

160.8, 135.34, 135.31, 135.2, 135.1, 131.5, 119.2, 119.1, 118.2, 118.1,

67.2, 65.9, 47.8, 46.3, 33.5. MS (m/z) calcd. for C₁₂H₁₃FNO₄[M +

H]⁺254.1, found 254.1. Mp: 196.8 → dec.

(2S,3R)-3-(3-Carboxy-4-chlorophenyl)pyrrolidine-2-carboxylic

Acid Hydrochloride (1l). Diacid 7l (190 mg, 0.514 mmol, 1 equiv) was

dissolved in a 1:1 mixture TFA/DCM (10 mL). The reaction mixture

was allowed to stir for 2 h at rt, then the solvent was evaporated under

reduced pressure. The oily residue was dissolved in 1 M HCl (10 mL),

and the solvent was evaporated (3 times) to aﬀord the corresponding

HCl salt (135 mg). The crude product was recrystallized from H₂O/

acetone to aﬀord the title compound as an oﬀ-white solid (89 mg,

56%). ¹H NMR (D₂O) δ 7.85 (d, J = 1.8 Hz, 1H), 7.68−7.49 (m, 2H),

4.37 (d, J = 9.7 Hz, 1H), 3.71 (ddd, J = 11.8, 9.2, 5.1 Hz, 2H), 3.62−

3.52 (m, 1H), 2.60 (dtd, J = 13.9, 7.1, 3.4 Hz, 1H), 2.35−2.23 (m,

1H). ¹³C NMR (D₂O) δ 171.9, 170.7, 138.3, 132.6, 131.9, 131.8,

131.7, 130.3, 65.7, 47.8, 46.2, 33.4. MS (m/z) calcd. for C₁₂H₁₃ClNO₄

[M + H]⁺270.0, found 270.0. Mp 239.4−241.3 °C.

(2S,3R)-3-(3-Carboxy-4-methylphenyl)pyrrolidine-2-carboxylic

Acid Hydrochloride (1m). A solution of NaIO₄(638 mg, 2.98 mmol,

8.2 equiv) and RuCl₃·xH₂O (4.5 mg, 0.022 mmol, 0.06 equiv) in H₂O

(4.5 mL) was added to a solution of 6m (117 mg, 0.36 mmol) in

MeCN/EtOAc (1:1, 5.2 mL) cooled to 0 °C. The reaction mixture

was stirred for 30 min and then ﬁltered through Celite, and the ﬁlter

cake was washed with EtOAc. The aqueous layer was extracted with

EtOAc, and the combined organic layers were washed with brine, dried

over MgSO₄, and concentrated to give the corresponding diacid, which

was used without further puriﬁcation. The diacid 7m was dissolved in a

1:1 mixture of TFA/DCM (6.0 mL), the reaction mixture was allowed

to stir for 2.5 h at rt, and the solvent was evaporated under reduced

pressure. The oily residue was dissolved in 1 M HCl (10 mL) and

evaporated to dryness (3×). The crude product was puriﬁed by

preparative HPLC (0 to 40% B in A) and then evaporated with 1 M

HCl (3 × 1 mL) to aﬀord the title compound, as a white solid (21 mg,

20% over two steps). ¹H NMR (600 MHz, D₂O) δ 7.78 (d, J = 2.0 Hz,

1H), 7.45 (dd, J = 7.9, 2.1 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 4.39 (d, J

= 9.8 Hz, 1H), 3.69−3.61 (m, 2H), 3.51 (ddd, J = 11.7, 10.2, 6.9 Hz,

1H), 2.50 (dtd, J = 10.5, 7.1, 3.2 Hz, 1H), 2.44 (s, 3H), 2.25−2.17 (m,

1H). ¹³C NMR (151 MHz, D₂O) δ 171.54, 170.63, 139.15, 135.72,

132.31, 131.28, 130.16, 129.04, 64.56, 47.25, 45.81, 32.82, 20.04. MS

(m/z) calcd. for C₁₃H₁₆NO₄[M + H]⁺250.3, found 250.3. Mp:

decomposition.

207.1, found 207.1. [α]₂₅+62.7° (c = 0.22, MeOH). Mp:

decomposition.

(2S,3R)-3-(3-Cyanophenyl)pyrrolidine-2-carboxylic Acid Hydro-

chloride (1g). Acid 7g (65 mg, 0.205 mmol, 1 equiv) was dissolved

in a 1:1 mixture of TFA/DCM (2.0 mL). The reaction mixture was

allowed to stir for 3 h at rt, then the solvent was evaporated under

reduced pressure. The oily residue was dissolved in HCl 1 M (10 mL)

and evaporated to dryness (3×) to aﬀord the title compound as a

white solid (42 mg, 81%). ¹H NMR (D₂O) δ 7.82 (t, J = 1.6 Hz, 1H),

7.78−7.70 (m, 2H), 7.59 (t, J = 7.8 Hz, 1H), 4.44 (d, J = 9.9 Hz, 1H),

3.80−3.68 (m, 2H), 3.57 (ddd, J = 11.8, 10.3, 6.9 Hz, 1H), 2.61 (dtd, J

= 10.5, 7.1, 3.3 Hz, 1H), 2.35−2.22 (m, 1H). ¹³C NMR (D₂O +

dioxane) δ 171.4, 140.3, 133.4, 132.5, 132.1, 130.5, 120.0, 112.3, 65.41,

48.0, 46.4, 33.4. LC−MS (m/z) calcd. for C₁₂H₁₃N₂O₂[M + H]⁺

217.10, found 217.0. Mp: 210.8−212.9 °C.

(2S,3R)-3-(3-Carbamoylphenyl)-2-carboxypyrrolidin-1-ium Chlor-

ide (1h). H₂O₂(4.23 mmol, 0.43 mL of 30% solution in H₂O, 6 equiv)

was added dropwise to a solution of 7g (223 mg, 0.71 mmol, 1.0

equiv) and K₂CO₃(390 mg, 2.82 mmol, 4 equiv) in EtOH/H₂O (1:1,

4.86 mL). The reaction mixture was stirred at rt for 70 min, then

slowly acidiﬁed with 1 M HCl. The aqueous phase was extracted with

EtOAc (3 × 10 mL) and the combined organic layers washed with

brine (10 mL), dried over MgSO₄, and concentrated to give a pale

yellow solid. The crude was dissolved in DCM (5.8 mL), and TFA

(5.8 mL) was added. The reaction mixture was stirred for 2 h at rt.

The solvent was evaporated, and the oily residue was dissolved in 1 M

HCl and evaporated to dryness (3 × 10 mL). The crude pink solid was

recrystallized from H₂O/acetone to aﬀord the title compound as a

white solid (95 mg, 59% over two steps). ¹H NMR (D₂O) δ 7.85 (d, J

= 1.6 Hz, 1H), 7.83−7.77 (m, 1H), 7.70−7.65 (m, 1H), 7.59 (t, J = 7.7

Hz, 1H), 4.38 (d, J = 9.7 Hz, 1H), 3.77−3.67 (m, 2H), 3.64−3.52 (m,

1H), 2.61 (dtd, J = 13.9, 7.1, 3.4 Hz, 1H), 2.39−2.25 (m, 1H). ¹³C

NMR (D₂O) δ 173.4, 172.3, 139.9, 134.1, 132.2, 130.1, 127.4, 127.2,

66.1, 48.7, 46.3, 33.7. LC−MS (m/z) calcd. for C₁₂H₁₅N₂O₃[M + H]⁺

235.1, found 235.1. Mp: decomposition.

(2S,3R)-3-(3-Boronophenyl)pyrrolidine-2-carboxylic Acid Hydro-

chloride (1i). To a solution of 7i (50 mg, 0.120 mmol, 1.0 equiv) in

acetone (9.4 mL) was added 0.1 M NH₄OAc (27.7 mg, 0.36 mmol, 3.0

equiv, in H₂O) and NaIO₄(76.8 mg, 0.36 mmol, 3 equiv). The

reaction mixture was stirred for 48 h at rt. Acetone was removed under

reduced pressure, and the aqueous layer was diluted with 2 M NaOH

(4 mL) and washed with EtOAC. The aqueous phase was then

acidiﬁed with 1 M HCl until pH ≈ 3 and extracted with EtOAc (4 × 5

mL). The combined organic layers were washed with brine (10 mL),

dried over MgSO₄, and concentrated to dryness. The crude product

was dissolved in DCM (1.0 mL) and TFA (1.0 mL), and the reaction

mixture stirred for 2.5 h at rt. The solvents were evaporated, and the

oily residue was dissolved in 1 M HCl and evaporated to dryness (3 ×

10 mL) to give an oﬀ-white solid, which was recrystallized from H₂O/

acetone to give the title compound as an oﬀ-white solid (13 mg, 43%

over two steps). ¹H NMR (D₂O) δ 7.80−7.72 (m, 2H), 7.58−7.46 (m,

2H), 4.41 (d, J = 9.8 Hz, 1H), 3.75−3.65 (m, 2H), 3.56 (ddd, J = 11.7,

10.3, 6.9 Hz, 1H), 2.57 (dtd, J = 13.9, 7.1, 3.4 Hz, 1H), 2.38−2.20 (m,

1H). ¹³C NMR (D₂O + dioxane) δ 171.4, 137.9, 133.1, 132.6, 130.1,

(2S,3R)-3-(3-Carboxy-4-bromophenyl)pyrrolidine-2-carboxylic

1

Acid Hydrochloride (1n). H NMR (600 MHz, D₂O) δ 7.67 (d, J =

8.3 Hz, 1H), 7.63 (d, J = 2.3 Hz 1H), 7.36 (dd, J = 8.3, 2.4 Hz, 1H),

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4.18 (d, J = 9.4 Hz, 1H), 3.61−3.54 (m, 1H), 3.48−3.43 (m, 1H),

2.51−2.46 (m, 1H), 2.21−2.14 (m, 1H). ¹³C NMR (600 MHz, D₂O)

δ 171.9, 171.6, 138.6, 134.8, 134.2, 131.4, 128.8, 118.4, 65.6, 47.5, 45.6,

32.8. MS (m/z) calcd. for C₁₂H₁₂BrNO₄[M + H]⁺312.9 and 314.9,

found 313.9 and 315.9. Mp: 250.1−257.3 °C.

rt for 4 h. The reaction mixture was diluted with H₂O, and the aqueous

phase was extracted with EtOAc (3 × 15 mL). The combined organic

layers were washed with brine (1 × 20 mL), dried over MgSO₄, and

concentrated to give 1.56 g. The crude product was puriﬁed by ﬂash

chromatography (heptanes/EtOAc, 9:1) to aﬀord the title compound

1

5-Bromo-2-methylbenzyl)oxy)(tert-butyl)dimethylsilane (3m). To

a ﬂame-dried round-bottom ﬂask was charged with 5-bromo-2-

methylphenyl)methanol³⁵(4.34 g, 21.6 mmol), anhydrous DMF (50

mL), imidazole (4.41 g, 64.8 mmol), TBSCl (6.51 g, 43.2 mmol), and

DMAP (264 mg, 2.16 mmol). The reaction mixture was stirred for 16

h at rt and subsequently diluted with EtOAc. The resulting mixture

was washed twice with 1 M HCl, twice with brine, dried over MgSO₄,

ﬁltered, and concentrated in vacuo. The crude product was puriﬁed by

consecutive ﬂash chromatography (heptane/EtOAc, 2:1 and 5:1) to

as a white solid (803 mg, 54%). H NMR (CDCl₃, 400 MHz) δ 7.40

(ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.34 (t, J = 1.8 Hz, 1H), 7.21 (t, J = 7.8

Hz, 1H), 7.14−7.09 (m, 1H), 4.06 (dt, J = 3.9, 2.0 Hz, 1H), 3.99 (dd, J

= 10.5, 3.9 Hz, 1H), 3.80 (dd, J = 10.5, 2.2 Hz, 1H), 3.42 (dt, J = 9.6,

2.2 Hz, 1H), 3.14 (dd, J = 17.9, 9.6 Hz, 1H), 2.50 (dd, J = 17.9, 2.6 Hz,

1H), 1.53 (s, 9H), 0.91 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H). ¹³C NMR

(CDCl₃, 400 MHz) δ 173.6, 150.0, 146.6, 130.8, 130.5, 129.9, 125.0,

123.2, 83.4, 66.6, 63.8, 39.94, 38.7, 28.2, 26.0, 18.3, −5.3, −5.4. R_f0.21

(heptanes/EtOAc, 9:1).

1

provide title compound as a clear oil (4.63 g, 68%). H NMR (600

(2S,3R)-tert-Butyl-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-(3-

chlorophenyl)-5-oxopyrrolidine-1-carboxylate (4d). To a solution of

(S)-tert-butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxo-2,5-dihy-

dro-1H-pyrrole-1-carboxylate (2) (500 mg, 1.53 mmol, 1.0 equiv) and

(3-chlorophenyl)boronic acid (3d, 382 mg, 2.44 mmol, 1.6 equiv) in

THF/H₂O (9:1, 15 mL) under nitrogen at rt was added [Rh(cod)Cl]₂

(37.6 mg, 0.076 mmol, 0.05 equiv). Then, 1 M NaOH (2.5 mL, 1.6

equiv) was added dropwise, and the reaction mixture was stirred at rt

for 4.5 h. The reaction mixture was diluted with H₂O, and the aqueous

phase was extracted with EtOAc (3 × 15 mL). The combined organic

layers were washed with brine (1 × 20 mL), dried over MgSO₄, and

concentrated to give 690 mg. The crude product was puriﬁed by ﬂash

chromatography (heptanes/EtOAc, 4:1) to aﬀord the title compound

MHz, CDCl₃) δ 7.56 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 8.0, 2.2 Hz,

1H), 6.98 (d, J = 8.0 Hz, 1H), 4.65 (s, 2H), 2.19 (s, 3H), 0.95 (s, 9H),

0.11 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 141.60, 133.72, 131.50,

129.77, 129.30, 119.78, 62.76, 26.09, 18.56, 18.16, −5.17. R_f0.84

(heptane/EtOAc, 1:1).

tert-Butyldimethyl((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)benzyl)oxy)silane (3n). A dry, Ar-ﬁlled 20 mL vial was

charged with bromine 3m (419 mg, 1.33 mmol), (Bpin)₂(354 mg,

1.40 mmol), KOAc (394 mg, 4.06 mmol), and (PPh₃)₂PdCl₂(46.9

mg, 66.5 μmol). The vial was then evacuated and backﬁlled with Ar,

followed by addition of degassed dioxane (13.2 mL). The vial was

capped with a screw cap and stirred for 16 h at 100 °C. The reaction

mixture was then diluted with EtOAc and washed three times with

H₂O. The organic partition was washed with brine, dried over MgSO₄,

ﬁltered through Celite, and evaporated. The crude product was

puriﬁed by DCVC (heptane/EtOAc, 10:1) to yield the title compound

(407 mg, contains 5 mol % PPh₃), which was used without further

puriﬁcation. ¹H NMR (600 MHz, CDCl₃) δ 7.76 (s, 1H), 7.62 (dd, J =

7.4, 1.0 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 4.70 (s, 2H), 2.34 (s, 3H),

1.33 (s, 12H), 0.93 (s, 9H), 0.09 (s, 6H). ¹³C NMR (151 MHz,

CDCl₃) δ 139.94, 138.40, 134.16, 134.03, 129.75, 83.71, 64.14, 26.11,

25.01, 19.19, 18.57, −5.11. R_f0.54 (heptane/EtOAc, 10:1).

1

as a white solid (387 mg, 58%). H NMR (CDCl₃) δ 7.55−7.38 (m,

4H, Ar−H), 4.09 (m, 1H, CH−N), 4.01 (m, 1H), 3.83 (dd, J = 10.5,

2.3 Hz, 1H), 3.54 (dt, J = 9.5, 2.3 Hz, 1H), 3.20 (dd, J = 17.9, 9.7 Hz,

1H), 2.53 (dd, J = 17.8, 2.5 Hz, 1H), 1.54 (s, 9H), 0.92 (s, 9H), 0.09

(s, 3H), 0.08 (s, 3H). ¹³C NMR (CDCl₃) δ 173.3, 149.8, 145.1, 131.3

(q, J = 32 Hz, C−CF₃), 129.7, 129.4, 124.0 (q, J = 3.7 Hz, C−Ar),

123.9 (q, J = 270 Hz, CF₃), 123.6 (q, J = 3.7, C−Ar), 83.3, 66.3, 63.3,

39.8, 38.7, 28.0, 25.8, 18.1, −5.5, −5.6. LC−MS (m/z) calcd. for

D

C₂₂H₃₅ClNO₄Si [M + H]⁺440.2, found 340.1 [(M + H)-Boc]⁺. [α]₂₅

−26.5 (c = 0.27, MeOH). Mp: 71.4−73.9 °C. R_f0.29 (heptanes/

EtOAc, 9:1).

(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(3-

(ethoxycarbonyl)phenyl)-5-oxopyrrolidine-1-carboxylate (4a). [Rh-

(cod)Cl]₂(37.6 mg, 0.076 mmol, 0.05 equiv), (S)-tert-butyl 2-(((tert-

butyldimethylsilyl)oxy)methyl)-5-oxo-2,5-dihydro-1H-pyrrole-1-car-

boxylate (2) (500 mg, 1.53 mmol, 1.0 equiv) and (3-(ethoxycarbonyl)-

phenyl)boronic acid (3a, 474 mg, 2.44 mmol, 1.6 equiv) were placed

in a 50 mL ﬂask, which was evacuated and backﬁlled with N₂.

Degassed dioxane (13.0 mL) was added, and to the stirred clear

solution, 1 M NaOH (2.44 mL, 1.6 equiv, degassed water) was added

dropwise. The reaction mixture was stirred at rt for 4.5 h. The reaction

mixture was diluted with H₂O, and the aqueous phase was extracted

with EtOAc (3 × 15 mL). The combined organic layers were washed

with brine (1 × 20 mL), dried over MgSO₄, and concentrated to give

743 mg. The crude product was puriﬁed by ﬂash chromatography

(heptanes/EtOAc, 9:1) to aﬀord the title compound as an oﬀ-white

(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-oxo-

3-(3-(triﬂuoromethyl)phenyl)pyrrolidine-1-carboxylate (4e). To a

solution of (S)-tert-butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-5-

oxo-2,5-dihydro-1H-pyrrole-1-carboxylate (2) (1.0 g, 3.053 mmol, 1

equiv) and commercially available (3-(triﬂuormomethyl)phenyl)-

boronic acid (3e, 928 mg, 4.89 mmol, 1.6 equiv) in THF/H₂O (9:1,

30.0 mL) under nitrogen at rt was added [Rh(cod)Cl]₂(75.3 mg,

0.153 mmol, 0.05 equiv). Then, 1 M NaOH (5.0 mL, 1.6 equiv) was

added dropwise, and the reaction mixture was stirred at rt for 4 h. The

reaction mixture was diluted with H₂O, and the aqueous phase was

extracted with EtOAc (3 × 20 mL). The combined organic layers were

washed with brine (1 × 30 mL), dried over MgSO₄, and concentrated.

The crude product was puriﬁed by ﬂash chromatography (heptanes/

EtOAc, 9:1) to aﬀord the title compound as a white solid (727 mg,

1

solid (382 mg, 52%). H NMR (CDCl₃) δ 7.95 (dt, J = 7.4, 1.6 Hz,

56%). H NMR (CDCl₃) δ 7.55−7.38 (m, 4H), 4.09 (m, 1H), 4.01

1H), 7.87 (s, 1H), 7.39 (tt, J = 4.6, 3.3 Hz, 2H), 4.38 (q, J = 7.1 Hz,

2H), 4.09 (dt, J = 4.0, 2.1 Hz, 1H), 4.01 (dd, J = 10.6, 3.9 Hz, 1H),

3.82 (dd, J = 10.5, 2.2 Hz, 1H), 3.54 (dt, J = 9.6, 2.4 Hz, 1H), 3.17 (dd,

J = 17.9, 9.6 Hz, 1H), 2.53 (dd, J = 17.9, 2.8 Hz, 1H), 1.53 (s, 9H),

1.39 (t, J = 7.1 Hz, 3H), 0.91 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H). ¹³C

NMR (CDCl₃) δ 173.7, 166.4, 150.0, 144.6, 131.4, 130.6, 129.4, 128.5,

128.0, 83.3, 66.6, 63.7, 61.3, 40.1, 38.8, 28.2, 26.0, 18.3, 14.5, −5.3.

LC−MS (m/z) calcd. for C₂₅H₃₉NO₆Si [M + H]⁺478.2, found 378.2

[(M + H)-Boc]⁺. R_f0.25 (heptanes/EtOAc, 8:2).

(m, 1H), 3.83 (dd, J = 10.5, 2.3 Hz, 1H), 3.54 (dt, J = 9.5, 2.3 Hz, 1H),

3.20 (dd, J = 17.9, 9.7 Hz, 1H), 2.53 (dd, J = 17.8, 2.5 Hz, 1H), 1.54

(s, 9H), 0.92 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H). ¹³C NMR (CDCl₃) δ

173.3, 149.8, 145.1, 131.3 (q, J = 32 Hz), 129.7, 129.4, 124.0 (q, J = 3.7

Hz), 123.9 (q, J = 270 Hz), 123.6 (q, J = 3.7 Hz), 83.3, 66.3, 63.3, 39.8,

38.7, 28.0, 25.8, 18.1, −5.5, −5.6. LC−MS (m/z) calcd. for

C₂₃H₃₅F₃NO₄Si [M + H]⁺474.2, found 374.1 [(M + H)-Boc]⁺.

D

[α]₂₅−26.4 (c = 0.32, MeOH). Mp: 95.3−96.9 °C. R_f0.29

(heptanes/EtOAc, 9:1).

(2S,3R)-tert-Butyl 3-(3-Bromophenyl)-2-(((tert-butyldimethylsilyl)-

oxy)methyl)-5-oxopyrrolidine-1-carboxylate (4b). To a solution at rt

of (S)-tert-butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxo-2,5-

dihydro-1H-pyrrole-1-carboxylate (2) (1.0 g, 3.05 mmol, 1.0 equiv)

and (3-bromophenyl)boronic acid (3b, 981 mg, 4.89 mmol, 1.6 equiv)

in degassed THF (26.3 mL) under nitrogen was added [Rh(cod)Cl]₂

(75.3 mg, 0.153 mmol, 0.05 equiv). A 1 M NaOH (4.9 mL, 1.6 equiv)

solution was then added dropwise, and the reaction mixture stirred at

(2S,3R)-tert-Butyl 3-(3-((tert-Butoxycarbonyl)amino)phenyl)-2-

(((tert-butyldimethylsilyl)oxy)methyl)-5-oxopyrrolidine-1-carboxy-

late (4f). [Rh(cod)Cl]₂(37.6 mg, 0.076 mmol, 0.05 equiv), (S)-tert-

butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxo-2,5-dihydro-1H-

pyrrole-1-carboxylate (500 mg, 1.53 mmol, 1.0 equiv), and 3-(tert-

butoxycarbonylamino)boronic acid (3f, 579 mg, 2.44 mmol, 1.6 equiv)

were placed in a 50 mL ﬂask, which was evacuated and backﬁlled with

N₂. Degassed dioxane (13.0 mL) was added followed by dropwise

K

DOI: 10.1021/acs.jmedchem.7b01624

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

addition of 1 M NaOH (2.44 mL, 1.6 equiv, degassed water). The

reaction mixture was stirred at rt for 3.5 h. The reaction mixture was

diluted with H₂O, and the aqueous phase was extracted with EtOAc (3

× 15 mL). The combined organic layers were washed with brine (1 ×

20 mL), dried over MgSO₄, and concentrated to give 743 mg. The

crude product was puriﬁed by ﬂash chromatography (heptanes/

EtOAc, 9:1) to aﬀord the title compound as a pale yellow oil (471 mg,

126.64, 126.61, 126.25, 126.19, 115.6, 115.4, 83.2, 66. 9, 63.7, 59.1,

59.0, 40.3, 38.3, 28.2, 26.1, 26.0, 18.6, 18.3, −5.17, −5.20, −5.3. R_f0.32

(heptanes/EtOAc, 9:1).

(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(3-

(((tert-butyldimethylsilyl)oxy)methyl)-4-chlorophenyl)-5-oxopyrroli-

dine-1-carboxylate (4l). A ﬂamed-dry round-bottomed ﬂask was

charged with a solution of tert-BuLi in pentane (8.98 mL, 15.265

mmol, 5.0 equiv) and cooled to −78 °C. A solution of 3l (2.56 g, 7.63

mmol, 2.5 equiv) in dry Et₂O (25 mL) was added dropwise, and the

clear, yellow/orange solution was stirred at −78 °C for 10 min. A

suspension of CuCN in dry Et₂O (2.4 mL) was added portion-wise at

−78 °C. The resulting suspension was stirred at −78 °C for 5 min and

then at −42 °C for 10 min (clear solution), after which it was recooled

to −78 °C. Enone 2 was dissolved in dry Et₂O (3.0 mL) and added

dropwise to the cuprate mixture at −78 °C, which resulted in a color

change to bright dark red. The temperature was raised to −42 °C, and

the reaction mixture was stirred at this temperature for 1 h. The brown

solution was quenched by addition of a freshly prepared sat. NaHCO₃

(5 mL), allowed to warm up to rt, and then transferred to a separating

funnel with water (15 mL) and EtOAc (15 mL). The organic layer was

separated, and the aqueous layer was extracted with EtOAc (2 × 15

mL). The combined organic layers were washed with brine (1 × 15

mL), dried over anhydrous MgSO₄, ﬁltered, and evaporated in vacuo to

dryness. The crude product was puriﬁed by column chromatography

(heptanes/EtOAc, 9:1) to aﬀord the title compound as a low-melting

1

59% yield). H NMR (CDCl₃) δ 7.19−7.30 (m, 3H), 6.82−6.90 (m,

1H), 6.49 (s, 1H), 4.07−4.12 (m, 1H), 4.02 (dd, J = 3.51, 10.54 Hz,

1H), 3.80 (dd, J = 1.88, 10.67 Hz, 1H), 3.43 (td, J = 1.88, 9.54 Hz,

1H), 3.15 (dd, J = 9.54, 17.82 Hz, 1H), 2.50 (dd, J = 2.26, 17.82 Hz,

1H), 1.52 (s, 9H), 0.91 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H). ¹³C NMR

(CDCl₃) δ 174.1, 152.6, 149.9, 145.4, 138.9, 129.7, 120.7, 117.2, 116.6,

83.0, 80.7, 66.6, 63.8, 40.2, 38.9, 28.3, 28.1, 25.8, 18.2, −5.5. LC−MS

(m/z) calcd. for C₂₇_DH₄₅N₂O₆Si [M + H]⁺521.3, found 365.2 [(M +

H)-Boc-t-Bu]. [α]₂₅−19.8 (c = 0.55, MeOH). R_f0.28 (heptanes/

EtOAc, 8:2).

(2S,3R)-tert-Butyl 3-(Benzo[d][1,3]dioxol-5-yl)-2-(((tert-

butyldimethylsilyl)oxy)methyl)-5-oxopyrrolidine-1-carboxylate (4j).

[Rh(cod)Cl]₂(112.9 mg, 0.229 mmol, 0.05 equiv) was added to a

solution of (S)-tert-butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-5-

oxo-2,5-dihydro-1H-pyrrole-1-carboxylate (2) (1.50 g, 4.58 mmol, 1.0

equiv) and benzo[d][1,3]dioxol-5-ylboronic acid (1.22 g, 7.33 mmol,

1.6 equiv) in THF (39.0 mL) under nitrogen at rt. Aqueous 1 M

NaOH (7.5 mL, 1.6 equiv) was then added dropwise, and the reaction

mixture was stirred at rt for 4 h. The reaction mixture was diluted with

H₂O, and the aqueous phase was extracted with EtOAc (3 × 30 mL).

The combined organic layers were washed with brine (1 × 50 mL),

dried over MgSO₄, and concentrated. The crude product was puriﬁed

by ﬂash chromatography (heptanes/EtOAc, 9:1) to aﬀord the title

1

white solid (1.31 g, 73%). H NMR (CDCl₃) δ 7.39 (d, J = 2.3 Hz,

1H), 7.26 (d, J = 8.2 Hz, 1H), 7.01 (dd, J = 8.2, 2.3 Hz, 1H), 4.77 (s,

2H), 4.04 (dt, J = 3.9, 2.0 Hz, 1H), 4.00 (dd, J = 10.4, 3.9 Hz, 1H),

3.78 (dd, J = 10.4, 2.0 Hz, 1H), 3.45 (dt, J = 9.6, 2.3 Hz, 1H), 3.14 (dd,

J = 17.9, 9.6 Hz, 1H), 2.50 (dd, J = 17.9, 2.8 Hz, 1H), 1.52 (s, 9H),

0.96 (s, 9H), 0.90 (s, 9H), 0.13 (s, 3H), 0.13 (s, 3H), 0.07 (s, 3H),

0.06 (s, 3H). ¹³C NMR (CDCl₃) δ 173.7, 150.0, 143.1, 139.6, 130.2,

129.6, 125.9, 125.8, 83.2, 66.7, 63.7, 62.4, 40.1, 38. 6, 28.2, 26.1, 26.0,

18.6, 18.4, −5.1, −5.2, −5.34, −5.35. R_f0.47 (heptanes/EtOAc, 8:2).

tert-Butyl (2S,3R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(3-

(((tert-butyldimethylsilyl)oxy)methyl)-4-methylphenyl)-5-oxopyrro-

lidine-1-carboxylate (4m). [Rh(cod)Cl]₂(23.2 mg, 0.047 mmol, 0.05

equiv), (S)-tert-butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxo-

2,5-dihydro-1H-pyrrole-1-carboxylate (2) (300 mg, 0.92 mmol), tert-

butyldimethyl((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)benzyl)oxy)silane (3n) (532 mg, 1.47 mmol), and Cs₂CO₃(477

mg, 1.46 mmol) were placed in a 25 mL ﬂask, which was evacuated

and backﬁlled with Ar. Degassed, anhydrous THF (8.9 mL) was

added, followed by addition of degassed H₂O (21 μL, 1.1 mmol). The

reaction mixture was stirred at rt for 24 h. The reaction mixture was

diluted with H₂O, and the aqueous phase was extracted with EtOAc (3

× 15 mL). The combined organic layers were washed with brine, dried

over MgSO₄, and concentrated. The crude product was puriﬁed by

ﬂash chromatography (heptane:EtOAc, 10:1 to 5:2) to aﬀord the title

1

compound as a white solid (1.38 g, 67%). H NMR (CDCl₃) δ 6.75

(d, J = 8.0 Hz, 1H), 6.68 (d, J 1.8 Hz, 1H), 6.64 (dd, J = 8.0, 1.8 Hz,

1H), 5.95 (s, 2H), 4.03 (m, 1H), 3.98 (m, 1H), 3.78 (dd, J = 10.4, 2.1

Hz, 1H), 3.37 (dt, J = 9.5, 2.3 Hz, 1H), 3.12 (dd, J = 17.8, 9.5 Hz, 1H),

2.47 (dd, J = 17.8, 2.8 Hz, 1H), 1.53 (s, 9H), 0.91 (s, 9H), 0.08 (s,

3H), 0.07 (s, 3H). ¹³C NMR (CDCl₃) δ 173.9, 149.8, 148.2, 146.6,

138.1, 119.3, 108.5, 106.7, 101.1, 83.0, 66.9, 63.5, 40.2, 38.5, 28.1, 25.8,

18.2, −5.5. LC−MS (m/z) calcd. for C₂₃H₃₆NO₆Si [M + H]⁺450.2,

D

found 350.1 [(M + H)-Boc]⁺. [α]₂₅−25.6 (c = 0.42, MeOH). Mp:

106.6−107.6 °C. R_f0.36 (heptanes/EtOAc, 8:2).

(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(3-

(((tert-butyldimethylsilyl)oxy)methyl)-4-ﬂuorophenyl)-5-oxopyrroli-

dine-1-carboxylate (4k). A ﬂamed-dry round-bottomed ﬂask was

charged with a solution of tert-BuLi in pentane (8.98 mL, 15.265

mmol, 5.0 equiv) and cooled to −78 °C. A solution of bromine 3k

(2.44 g, 7.634 mmol, 2.5 equiv) in dry Et₂O (25 mL) was added

dropwise, and the clear, yellow solution was stirred at −78 °C for 10

min. A suspension of CuCN in dry Et₂O (2.5 mL) was added portion-

wise at −78 °C. The resulting suspension was stirred at −78 °C for 5

min and then at −42 °C for 10 min (clear solution), after which it was

recooled to −78 °C. Enone 2 was dissolved in dry Et₂O (3.0 mL) and

added dropwise to the cuprate mixture at −78 °C, which resulted in a

color change to bright dark red. The temperature was raised to −42

°C, and the reaction mixture was stirred at this temperature for 1 h.

The brown solution was quenched by addition of a freshly prepared

sat. NaHCO₃(5 mL), allowed to warm up to rt, and then transferred

to a separating funnel with water (15 mL) and EtOAc (15 mL). The

organic layer was separated, and the aqueous layer was extracted with

EtOAc (2 × 15 mL). The combined organic layers were washed with

brine (1 × 15 mL), dried over anhydrous MgSO₄, ﬁltered, and

evaporated in vacuo to dryness. The crude product was puriﬁed by

column chromatography (heptanes/EtOAc, 9:1) to aﬀord the title

1

compound as a pale yellow oil (211 mg, 41% yield). H NMR (400

MHz, CDCl₃) δ 7.24 (d, J = 1.4 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H),

7.00 (dd, J = 7.7, 2.0 Hz, 1H), 4.68 (s, 2H), 4.11−4.05 (m, 1H), 4.01

(dd, J = 10.5, 3.8 Hz, 1H), 3.79 (dd, J = 10.5, 2.0 Hz, 1H), 3.46−3.41

(m, 1H), 3.13 (dd, J = 17.9, 9.6 Hz, 1H), 2.53 (dd, J = 17.8, 2.8 Hz,

1H), 2.25 (s, 3H), 1.52 (s, 9H), 0.94 (s, 9H), 0.91 (s, 9H), 0.11 (s,

3H), 0.10 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 174.29, 150.02, 141.83, 139.94, 134.14, 130.65, 124.93,

124.83, 82.97, 67.01, 63.70, 63.41, 40.18, 38.57, 28.19, 26.08, 25.97,

18.52, 18.32, 18.26, −5.14, −5.15, −5.38, −5.40. R_f0.43 (heptanes/

EtOAc, 5:1).

(2S,3R)-tert-Butyl 3-(3-Chlorophenyl)-2-(hydroxymethyl)-

pyrrolidine-1-carboxylate (6d). In a ﬂame-dried ﬂask a solution of

4d (368 mg, 0.84 mmol, 1.0 equiv) in dry THF (2.2 mL) was cooled

to −78 °C. LiBEtH₃1 M (1.0 mL, 1.00 mmol, 1.2 equiv) was added

via syringe dropwise, and the reaction mixture was stirred for 1 h,

quenched with NaHCO₃sat. sol. (3 mL), and warmed to rt. The

aqueous phase was extracted with EtOAc (3 × 5 mL), and the

combined organic layers were washed with brine (10 mL), dried over

MgSO₄, and concentrated to give the corresponding hemiaminal 5d as

1

compound as a pale yellow oil (793 mg, 1.40 mmol, 46% yield). H

NMR (CDCl₃) δ 7.30 (dd, J = 6.8, 2.3 Hz, 1H), 7.04 (ddd, J = 7.5, 4.9,

2.5 Hz, 1H), 6.95 (dd, J = 9.6, 8.5 Hz, 1H), 4.77 (s, 2H), 4.04 (dd, J =

3.9, 2.0 Hz, 1H), 3.99 (dd, J = 10.4, 4.0 Hz, 1H), 3.79 (dd, J = 10.4, 2.1

Hz, 1H), 3.45 (dt, J = 9.6, 2.3 Hz, 1H), 3.13 (dd, J = 17.9, 9.6 Hz, 1H),

2.49 (dd, J = 17.9, 2.7 Hz, 1H), 1.52 (s, 9H), 0.94 (s, 9H), 0.91 (s,

9H), 0.11 (s, 3H), 0.11 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H). ¹³C NMR

(CDCl₃) δ 173.9, 159.8, 158.1, 150.0, 140.12, 140.10, 129.2, 129.1,

L

DOI: 10.1021/acs.jmedchem.7b01624

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a colorless oil, which was used in the next step without further

puriﬁcation.

chromatography (heptanes/EtOAc, 2:1) to aﬀord the title compound

as a colorless oil (110 mg, 31% over two steps). H NMR (CDCl₃) δ

1

In a ﬂame-dried ﬂask a solution of the crude hemiaminal 5d (0.84

mmol, 1.0 equiv) in dry DCM (2.6 mL) was cooled to −78 °C. HSiEt₃

(0.27 mL, 1.670 mmol, 2 equiv) and BF₃·Et₂O (0.23 mL, 1.837 mmol,

2.2 equiv) were added sequentially via syringe, and the reaction

mixture stirred for 6 h. The reaction mixture was quenched with sat.

NaHCO₃(4 mL) and warmed to rt. The mixture was diluted with

DCM, and the organic phase was separated, washed with sat. NH₄Cl

(5 mL), dried over MgSO₄, and concentrated. The crude product was

puriﬁed by ﬂash chromatography (heptanes/EtOAc, 2:1) to aﬀord the

title compounds as a colorless gummy oil (154 mg, 59% over two

7.39 (s, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.89 (d,

J = 7.6 Hz, 1H), 6.57 (s, 1H), 3.90 (d, J = 6.8 Hz, 1H), 3.79−3.68 (m,

2H), 3.60 (dd, J = 11.5, 6.7 Hz, 1H), 3.39−3.27 (m, 1H), 2.92 (s, 1H),

2.14 (dtd, J = 9.1, 6.5, 2.8 Hz, 1H), 1.96 (dtd, J = 12.4, 10.1, 8.1 Hz,

1H), 1.51 (s, 9H), 1.49 (s, 9H). ¹³C NMR (CDCl₃) δ 152.8, 141.1

(b), 139.0, 129.4, 122.3, 117.7, 117.4, 80.7, 80.6, 67.0, 48.0, 47.2, 32.9,

28.6, 28.5. MS (m/z) calcd. for C₂₁H₃₂N₂O₅[M + H]⁺392.23, found

D

293.1 [(M-Boc) + H]⁺. [α]₂₅−8.6 (c = 0.31, MeOH). R_f0.16

(heptanes/EtOAc, 2:1).

(2S,3R)-tert-Butyl 3-(3-Cyanophenyl)-2-(hydroxymethyl)-

pyrrolidine-1-carboxylate (6g). Under nitrogen atmosphere, TBAF

(1.74 mmol, 3 equiv, 1.74 mL of 1 M solution in THF) was added to a

solution of 8g (242 mg, 0.581 mmol, 1 equiv) in dry THF (5.6 mL).

The reaction mixture was stirred at rt for 2 h, then diluted with water

(10 mL) and sat. NaHCO₃(10 mL). The aqueous layer was extracted

with EtOAc (2 × 10 mL), and the combined organic layers were

washed with brine, dried over MgSO₄, and concentrated. The crude

product was puriﬁed by column chromatography (heptanes/EtOAc,

1:1) to give the title compound as a pale yellow oil (202 mg, 63% yield

1

steps). H NMR (CDCl₃) δ 7.31−7.26 (m, 3H, Ar−H), 7.16−7.14

(m, 1H, Ar−H), 3.94 (m, 1H, CH−N), 3.78 (m, 1H), 3.67 (m, 1H),

3.40 (m, 1H), 3.05 (broad s, 1H), 2.20 (m, 1H), 1.97 (m, 1H), 1.54 (s,

9H). ¹³C NMR (CDCl₃) δ 156.5, 143.0, 134.5, 130.0, 127.6, 127.2,

125.7, 80.5, 66.7, 65.5, 47.2, 46.8, 32.6, 28.4. MS (m/z) calcd. for

D

C₁₆H₂₃ClNO₃[M − H]⁺312.14, found 212.1 [(M + H)-Boc]⁺. [α]₂₅

−13.1 (c = 0.92, MeOH). R_f0.49 (heptanes/EtOAc, 1:1 + 1% AcOH).

(2S,3R)-tert-Butyl 2-(Hydroxymethyl)-5-oxo-3-(3-

(triﬂuoromethyl)phenyl)pyrrolidine-1-carboxylate (6e). In a ﬂame-

dried ﬂask a solution of 4e (500 mg, 1.06 mmol, 1.0 equiv) in dry THF

(2.8 mL) was cooled to −78 °C. LiBEtH₃1 M (solution in THF)

(1.27 mL, 1.27 mmol, 1.2 equiv) was added via syringe dropwise, and

the reaction mixture was stirred for 40 min, quenched with NaHCO₃

sat. sol. (3 mL), and warmed to rt. The aqueous phase was extracted

with EtOAc (3 × 10 mL), and the combined organic layers were

washed with brine (10 mL), dried over MgSO₄, and concentrated to

give the corresponding hemiaminal 5e as a colorless oil, which was

used in the next step without further puriﬁcation.

1

over two steps). H NMR (CDCl₃) δ 7.51−7.34 (m, 4H), 4.78 (br s,

1H), 3.86 (br s, 1H), 3.73−3.54 (m, 3H), 3.33 (ddd, J = 11.1, 8.8, 6.8

Hz, 1H), 3.01 (bs, 1H), 2.17 (m, 1H), 1.95−1.81 (m, 1H), 1.43 (s,

9H). ¹³C NMR (CDCl₃) δ 156.2, 154.2, 144.6, 142.9, 132.0, 131.1,

130.7, 129.6, 118.6, 112.7, 80.6, 80.3, 66.5, 65.5, 64.9, 62.4, 46.9, 46.8,

46.3, 32.5, 31.7, 28.4. R_f0.21 (heptanes/EtOAc, 1:1).

(2S,3R)-tert-Butyl 2-(Hydroxymethyl)-3-(3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxylate (6i).

Under nitrogen atmosphere, TBAF (1.311 mmol, 3 equiv, 1.31 mL

of 1 M solution in THF), was added to a solution of 9 (226 mg, 0.437

mmol, 1 equiv) in dry THF (5.3 mL). The reaction mixture was stirred

at rt for 2.5 h, then was diluted with sat. NaHCO₃(10 mL). The

aqueous layer was extracted with EtOAc (3 × 10 mL), and the

combined organic layers were washed with brine, dried over MgSO₄,

and concentrated. The crude product was puriﬁed by column

chromatography (heptanes/EtOAc, 2:1) to give the title compound

In a ﬂame-dried ﬂask, a solution of the crude hemiaminal 5e (1.06

mmol, 1 equiv) in dry DCM (3.3 mL) was cooled to −78 °C. HSiEt₃

(0.34 mL, 2.11 mmol, 2 equiv) and BF₃·Et₂O (0.29 mL, 2.315 mmol,

2.2 equiv) were sequentially added via syringe, and the reaction

mixture was stirred for 6 h. After the complete consumption of the

substrate, the reaction was quenched with sat. NaHCO₃(4 mL) and

warmed to rt. The mixture was diluted with DCM, and the organic

phase was separated, washed with sat. NH₄Cl (5 mL), dried over

MgSO₄, and concentrated to give 371 mg (crude). The crude was

puriﬁed by ﬂash chromatography (heptanes/EtOAc, 2:1) to aﬀord the

title compound as a colorless gummy oil (286 mg, 78% yield over two

1

as a colorless oil (106 mg, 63%). H NMR (CDCl₃) δ 7.72−7.63 (m,

2H), 7.32−7.29 (m, 2H), 3.96 (bs, 1H), 3.72 (m, 2H), 3.59 (dd, J =

11.5, 6.7 Hz, 1H), 3.33 (td, J = 10.5, 6.5 Hz, 1H), 2.89 (bs, 1H), 2.10

(bs, 1H), 2.04−1.89 (m, 1H), 1.48 (s, 9H), 1.33 (s, 12H). ¹³C NMR

(CDCl₃) δ 156.9, 139.9, 133.9, 133.7, 130.7, 129.6, 128.2, 83.9, 80.5,

67.1, 66.0, 48.0, 47.2, 33.1, 28.5, 24.9. LC−MS (m/z) calcd. for

C₁₇H₂₃N₂O₃[M + H]⁺302.17, found 203.1 [(M + H)-Boc]⁺. R_f0.17

(heptanes/EtOAc, 2:1).

1

steps). H NMR (CDCl₃) 7.49−7.39 (m, 4H), 4.94 (br s, 1H, OH),

3.92 (br s, 1H), 3.75−3.58 (m, 3H), 3.35 (ddd, J = 11.2, 8.9, 6.8, 1H),

3.04 (m, 1H), 2.18 (br s, 1H), 1.99−1.89 (m, 1H), 1.46 (s, 9H). ¹³C

NMR (CDCl₃) δ (two rotamers) 156.3, (154.3), (143.9), 142.1, 130.9

(q, J = 31.5 Hz, C−CF₃), 130.8, 129.1, 124.1, 123.9 (q, J = 272.2 Hz,

CF₃), 123.8, 80.4, (80.1), 66.6, (65.6), 65.0, (62.5), 47.1, 46.7, (46.2),

(32.6), 31.7. MS (m/z) calcd. for C₁₇H₂₃F₃NO₃[M + H]⁺346.2,

found 246.1 [(M + H)-Boc]⁺. [α]₂₅^D−10.3 (c = 0.43, MeOH). R_f0.23

(heptanes/EtOAc, 2:1).

(2S,3R)-tert-Butyl 3-(Benzo[d][1,3]dioxol-5-yl)-2-(hydroxymethyl)-

5-oxopyrrolidine-1-carboxylate (6j). In a ﬂame-dried ﬂask a solution

of 4j (687 mg, 1.53 mmol, 1.0 equiv) in dry THF (4 mL) was cooled

to −78 °C. LiBEtH₃1 M (THF solution) (1.83 mL, 1.834 mmol, 1.2

equiv) was added via syringe dropwise, and the reaction mixture was

stirred for 45 min, quenched with NaHCO₃sat. sol. (4 mL), and

warmed to rt. The aqueous phase was extracted with EtOAc (3 × 10

mL), and the combined organic layers were washed with brine (10

mL), dried over MgSO₄, and concentrated to give the corresponding

hemiaminal 5j as a colorless oil, which was used in the next step

without further puriﬁcation.

(2S,3R)-tert-Butyl 3-(3-((tert-Butoxycarbonyl)amino)phenyl)-2-

(hydroxymethyl)pyrrolidine-1-carboxylate (6f). In a ﬂame-dried

ﬂask a solution of 4f (371 mg, 0.71 mmol, 1.0 equiv) in dry THF

(1.86 mL) was cooled to −78 °C. LiBEtH₃1 M (solution in THF)

(1.7 mL, 1.72 mmol, 2.4 equiv) was added via syringe dropwise, and

the reaction mixture was stirred for 1 h, quenched with sat. NaHCO₃

(3 mL), and warmed to rt. The aqueous phase was extracted with

EtOAc (3 × 5 mL), and the combined organic layers were washed

with brine (10 mL), dried over MgSO₄, and concentrated to give the

corresponding hemiaminal 4f as a colorless oil, which was used in the

next step without further puriﬁcation.

In a ﬂame-dried ﬂask a solution of the crude hemiaminal 4f (0.714

mmol, 1 equiv) in dry DCM (2.2 mL) was cooled to −78 °C. HSiEt₃

(0.23 mL, 1.43 mmol, 2.0 equiv) and BF₃·Et₂O (0.27 mL, 2.14 mmol,

3.0 equiv) were sequentially added via syringe, and the reaction

mixture was stirred for 5 h. After the complete consumption of the

substrate, the reaction was quenched with NaHCO₃sat. sol. (4 mL)

and warmed to rt. The mixture was diluted with DCM, and the organic

phase was separated, washed with sat. NH₄Cl (5 mL), dried over

MgSO₄, and concentrated. The crude product was puriﬁed by ﬂash

In a ﬂame-dried ﬂask a solution of the crude hemiaminal (1.53

mmol, 1 equiv) in dry DCM (4.8 mL) was cooled to −78 °C. HSiEt₃

(0.49 mL, 3.06 mmol, 2 equiv) and BF₃·Et₂O (0.42 mL, 3.37 mmol,

2.2 equiv) were added sequentially via syringe, and the reaction

mixture was stirred for 6 h. The reaction was quenched with sat.

NaHCO₃(4 mL) and warmed to rt. The mixture was diluted with

DCM, and the organic phase was separated, washed with sat. NH₄Cl

(5 mL), dried over MgSO₄, and concentrated. The crude product was

puriﬁed by ﬂash chromatography (heptanes/EtOAc, 2:1) to aﬀord the

title compound as a pale yellow oil (334 mg, 69% yield over two

1

steps). H NMR (CDCl₃) δ 6.69−6.64 (m, J = 4.7, 3.2 Hz, 2H), 6.61

(dd, J = 8.0, 1.6 Hz, 1H), 5.85 (s, 2H), 3.82−3.58 (m, 3H), 3.53 (dd, J

= 11.4, 6.0 Hz, 1H), 3.26 (ddd, J = 11.0, 9.4, 6.7 Hz, 1H), 2.88−2.73

(m, 1H), 2.05 (br s, 1H), 1.89−1.72 (m, 1H), 1.42 (s, 9H). ¹³C NMR

M

DOI: 10.1021/acs.jmedchem.7b01624

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(CDCl₃) δ (two rotamers) 156.4, 154.4, 147.9, 146.4, 136.7, 134.7,

120.7, 120.4, 108.2, 107.5, 100. 9, 80.3, 80.0, 66.9, 66.0, 65.2, 62.4,

47.3, 46.8, 46.4, 32.8, 32.0, 28.3. MS (m/z) calcd. for C₁₇H₂₄NO₅[M

aﬀord the title compounds as a colorless oil (370 mg, 49% over three

steps). ¹H NMR (CDCl₃) δ 7.37 (d, J = 1.9 Hz, 1H), 7.25 (d, J = 8.2

Hz, 1H), 7.04 (dd, J = 8.2, 2.2 Hz, 1H), 4.69 (s, 2H), 4.00−3.48 (m,

6H), 3.41−3.24 (m, 1H), 2.92 (s, 1H), 2.12 (dtd, J = 9.4, 6.4, 2.9 Hz,

1H), 1.89 (dq, J = 12.3, 9.7 Hz, 1H), 1.47 (s, 9H). ¹³C NMR (CDCl₃)

δ 156.7, 139.9, 139.0, 130.9, 129.5, 127.5, 127.5, 80.7, 66.8, 65.5, 62.3,

47.2, 47.0, 32.8, 28.5. MS (m/z) calcd. for C₁₇H₂₅ClNO₄[M + H]⁺

342.1, found 242.1 [(M-Boc) + H]⁺. R_f0.25 (heptanes/EtOAc, 6:4).

tert-Butyl (2S,3R)-2-(Hydroxymethyl)-3-(3-(hydroxymethyl)-4-

methylphenyl)pyrrolidine-1-carboxylate (6m). Compound 4m (211

mg, 0.037 mmol) was dissolved in dry THF (3.1 mL), and

dimethylsulﬁde borane complex (0.11 mL, 10.5 M in THF) was

added dropwise. The reaction mixture was reﬂuxed for 2.5 h, after

which it was allowed to cool down to rt and diluted with Et₂O,

quenched with sat. NH₄Cl, and the organic portion separated. The

organic partition was washed with brine, dried over MgSO₄, ﬁltered

through Celite, and concentrated in vacuo. The crude product was

dissolved in THF (4.7 mL) and treated with TBAF (1 M, 2.24 mL) at

rt for 23 h. The mixture was diluted with EtOAc and washed

consecutively with NH₄Cl (1 M), H₂O, and brine, and dried over

MgSO₄. The solution was concentrated under reduced pressure and

puriﬁed by ﬂash column chromatography (heptanes/EtOAc, 10:1 →

EtOAc) to provide the title compound as a colorless syrup (117 mg,

D

+ H]⁺322.16, found 222.1 [(M + H)-Boc]⁺. [α]₂₅−18.3 (c = 1.2,

MeOH). R_f0.38 (heptanes/EtOAc, 2:1).

(2S,3R)-tert-Butyl 3-(4-Fluoro-3-(hydroxymethyl)phenyl)-2-

(hydroxymethyl)pyrrolidine-1-carboxylate (6k). In a ﬂame-dried

ﬂask a solution of 4k (793 mg, 1.40 mmol, 1.0 equiv) in dry THF

(4.3 mL) was cooled to −78 °C. LiBEtH₃1 M (1.68 mL, 1.675 mmol,

1.2 equiv) was added via syringe dropwise, and the reaction mixture

was stirred for 1 h, then quenched with sat. NaHCO₃(5 mL). The

aqueous phase was extracted with EtOAc (3 × 10 mL), and the

combined organic layers were washed with brine (10 mL), dried over

MgSO₄, and concentrated to give the corresponding hemiaminal 5k as

a colorless oil, which was used in the next step without further

puriﬁcation.

In a ﬂame-dried ﬂask a solution of the crude hemiaminal (1.40

mmol, 1.0 equiv) in dry DCM (4.43 mL) was cooled to −78 °C.

HSiEt₃(0.44 mL, 2.79 mmol, 2 equiv) and BF₃·Et₂O (0.38 mL, 3.07

mmol, 2.2 equiv) were added sequentially via syringe, and the reaction

mixture stirred for 5 h at −78 °C. The reaction mixture was quenched

with sat. NaHCO₃(5 mL), warmed to rt, and diluted with DCM. The

organic phase was separated, washed with sat. NH₄Cl (5 mL), dried

over MgSO₄, and concentrated. The crude was dissolved in dry THF

under N₂atmosphere at rt, and 1 M TBAF (THF solution) (1.02 mL,

1.02 mmol, 3 equiv) was added dropwise. The reaction mixture was

allowed to stir overnight, quenched with sat. NaHCO₃(5 mL), and

portioned between EtOAc (10 mL) and H₂O (10 mL). The layers

were separated, and the aqueous layer was extracted with EtOAc (2 ×

10 mL). The combined organic layers were washed with brine (1 × 10

mL), dried over MgSO₄, and concentrated. The crude product was

puriﬁed by ﬂash chromatography (heptanes/EtOAc, 2:3) to aﬀord the

1

97%). H NMR (400 MHz, CDCl₃) δ (two rotamers) 7.25 (s, 1H),

7.11−7.07 (m, 1H), 7.06−7.01 (m, 1H), 4.68−4.50 (m, 2H), 3.99−

3.84 (m, 1H), 3.72 (m, 2H), 3.65−3.55 (m, 1H), 3.38−3.26 (m, 1H),

2.87 (m, 1H), 2.30−2.25 (m, 3H), 2.15−2.05 (m, 1H), 2.00−1.86 (m,

1H), 1.49 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 156.93, 139.42,

130.66, 128.87, 126.69, 126.45, 114.21, 80.66, 67.02, 65.78, 63.11,

47.56, 47.22, 33.00, 28.57, 18.27. R_f0.05 (heptanes/EtOAc, 10:1).

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-chlorophenyl)pyrrolidine-2-

carboxylic Acid (7d). A solution of NaIO₄(434 mg, 2.03 mmol, 4.1

equiv) and RuCl₃·xH₂O (3.1 mg, 0.015 mmol, 0.03 equiv) in H₂O (6.2

mL) was added to a solution of 6d (154 mg, 0.494 mmol, 1 equiv) in

MeCN/EtOAc (1:1, 7.0 mL) cooled to 0 °C. The reaction mixture

was stirred for 30 min, then ﬁltered through Celite, and the ﬁlter cake

was washed with EtOAc. The aqueous layer was extracted with EtOAc,

and the combined organic layers were washed with brine, dried over

MgSO₄, and concentrated. The crude product was puriﬁed by ﬂash

chromatography (heptanes/EtOAc, 1:1 + 1% AcOH) to aﬀord the

title compound as an oﬀ-white solid (138 mg, 86%). ¹H NMR

(CDCl₃) δ (two rotamers) 9.64 (br s, 1H), 7.27−7.22 (m, 3H), 7.15−

7.13 (m, 1H), 4.40 (d, J = 5.5 Hz, 0.4H), 4.25 (d, J = 6.5 Hz, 0.6H),

3.79−3.44 (m, 3H), 2.38−2.28 (m, 1H), 2.06−1.96 (m, 1H), 1.49 (s,

4H), 1.42 (s, 5H). ¹³C NMR (CDCl₃) δ (two rotamers) 177.6, 175.7,

155.2, 153.7, 142.9, 142.4, 134.6, 130.1, 127.5, 127.4, 127.1, 125.2,

81.2, 80.1, 65.4, 64.9, 49.4, 47.5, 46.1, 45.9, 32.7, 32.2, 28.3, 28.2. LC−

MS (m/z) calcd. for C₁₆H₂₁ClNO₄[M + H]⁺326.12, found 226.0 [(M

1

title compound as a colorless oil (92 mg, 20% over three steps). H

NMR (CDCl₃, 400 MHz) δ 7.35−7.26 (m, 1H), 7.08 (ddd, J = 7.7,

5.0, 2.4 Hz, 1H), 6.95 (dd, J = 9.7, 8.4 Hz, 1H), 5.13 (br s, 1H), 4.68

(s, 2H), 3.95−3.53 (m, 4H), 3.31 (td, J = 10.4, 6.4 Hz, 1H), 2.90 (br s,

1H), 2.22−2.05 (m, 1H), 1.89 (ddd, J = 12.6, 10.5, 8.2 Hz, 1H), 1.47

(s, 9H). ¹³C NMR (CDCl₃, 600 MHz) δ 160.3, 158.6, 156.8, 154.7,

138.3, 136.6, 128.6, 128.5, 128.13, 128.10, 115.5, 115.4, 80.7, 67.1,

66.1, 65.6, 62.8, 58.94, 58.91, 47.2, 47.1, 46.7, 46.5, 33.0, 32.3, 28.5.

MS (m/z) calcd. for C₁₇H₂₅FNO₄[M + H]⁺326.2, found 226.1 [(M-

Boc) + H]⁺. R_f0.18 (heptanes/EtOAc, 1:1).

(2S,3R)-tert-Butyl 3-(4-Chloro-3-(hydroxymethyl)phenyl)-2-

(hydroxymethyl)pyrrolidine-1-carboxylate (6l). In a ﬂame-dried

ﬂask a solution of 4l (1.31 g, 2.24 mmol, 1 equiv) in dry THF

(6.85 mL) was cooled to −78 °C. LiBEtH₃1 M (2.69 mL, 2.69 mmol,

1.2 equiv) was added via syringe dropwise, and the reaction mixture

was stirred for 1 h, quenched with sat. NaHCO₃(6 mL), and warmed

to rt. The aqueous phase was extracted with EtOAc (3 × 10 mL), and

the combined organic layers were washed with brine (10 mL), dried

over MgSO₄, and concentrated to give the corresponding hemiaminal

5l as a colorless oil, which was used in the next step without further

puriﬁcation.

In a ﬂame-dried ﬂask a solution of the crude hemiaminal 5l (2.24

mmol, 1 equiv) in dry DCM (7.1 mL) was cooled to −78 °C. HSiEt₃

(0.71 mL, 4.476 mmol, 2 equiv) and BF₃·Et₂O (0.62 mL, 4.94 mmol,

2.2 equiv) were sequentially added via syringe, and the reaction

mixture was stirred for 5 h at −78 °C. After the complete consumption

of the substrate, the reaction was quenched with sat. NaHCO₃(4 mL)

and warmed to rt. The mixture was diluted with DCM, and the organic

phase was separated, washed with sat. NH₄Cl (5 mL), dried over

MgSO₄, and concentrated to give 1.12 g. The crude product was

dissolved in dry THF under N₂atmosphere at rt, and 1 M TBAF−

THF solution (6.7 mL, 6.71 mmol, 3 equiv) was added dropwise. After

2 h, the reaction mixture was quenched with sat. NaHCO₃(5 mL),

EtOAc (10 mL), and H₂O (10 mL). The layers were separated, and

the aqueous layer was extracted with EtOAc (2 × 10 mL). The

combined organic layers were washed with brine (1 × 10 mL), dried

over MgSO₄, and concentrated to dryness. The crude product was

puriﬁed by ﬂash chromatography (heptanes/EtOAc, 2:1 → 3:2) to

D

+ H)-Boc]⁺. [α]₂₅+40.5 (c = 0.55, MeOH). R_f0.27 (heptanes/

EtOAc, 1:1 + 1% AcOH). Mp: decomposition.

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-(triﬂuoromethyl)phenyl)-

pyrrolidine-2-carboxylic Acid (7e). A solution of NaIO₄(380.6 mg,

1.78 mmol, 4.1 equiv) and RuCl₃·xH₂O (2.7 mg, 0.013 mmol, 0.03

equiv) in H₂O (5.45 mL) was added to a solution of 6e in MeCN/

EtOAc (1:1, 6.2 mL) cooled to 0 °C. The reaction mixture was stirred

for 30 min, then was ﬁltered through Celite, and the ﬁlter cake was

washed with EtOAc. The aqueous layer was extracted with EtOAc, and

the combined organic layers were washed with brine, dried over

MgSO₄, and concentrated to give the title compound as a colorless

sticky oil (95 mg, 61%). ¹H NMR (CDCl₃) δ (two rotamers) 9.26 (br

s, 1H), 7.55−7.44 (m, 4H), 4.43 (d, J = 5.8 Hz, 0.4H), 4.27 (d, J = 6.5

Hz, 0.6H), 3.81−3.52 (m, 3H), 2.42−2.31 (m, 1H), 2.10−1.99 (m,

1H), 1.50 (s, 4H), 1.42 (s, 5H). ¹³C NMR (CDCl₃) δ (two rotamers)

177.4, 175.4, 155.3 153.7, 141.8, 141.3, 131.1 (q, J = 33.0 Hz, C-CF₃),

130.4, 130.3, 129.4, 129.3, 124.2, 124.1, 123.9 (q, J = 272.2 Hz, CF₃),

123.8, 123.7, 81.3, 81.1, 65.4, 65.0, 49.5, 47.6, 46.1, 45.9, 32.8, 32.3,

28.3, 28.1. [α]₂₅^D+ 27.9 (c = 0.64, MeOH). R_f0.26 (heptanes/EtOAc,

1:1 + 1% AcOH).

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-((tert-butoxycarbonyl)-

amino)phenyl)pyrrolidine-2-carboxylic Acid (7f). A solution of

N

DOI: 10.1021/acs.jmedchem.7b01624

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Journal of Medicinal Chemistry

Article

NaIO₄(179 mg, 0.836 mmol, 4.1 equiv) and RuCl₃·xH₂O (1.27 mg,

0.006 mmol, 0.03 equiv) in H₂O (2.55 mL) was added to a solution of

6f (80 mg, 0.204 mmol, 1 equiv) in MeCN/EtOAc (1:1, 2.9 mL)

cooled to 0 °C. The reaction mixture was stirred for 30 min, then

ﬁltered through ﬁlter paper, and the ﬁlter cake was washed with

EtOAc. The aqueous layer was extracted with EtOAc, and the

combined organic layers were washed with brine, dried over MgSO₄,

and concentrated to give 128 mg. The crude product was puriﬁed by

ﬂash chromatography (heptanes/EtOAc, 1:1 + 1% AcOH) to aﬀord

(CDCl₃) δ (two rotamers) 178.1, 175.5, 155.7, 153.8, 140.2, 139.76,

134.0, 133.8, 133.3, 133.3, 130.2, 129.6, 128.3, 84.0, 81.3, 80.8, 65.8,

65.3, 50.1, 47.8, 46.4, 46.2, 33.3, 32.8, 28.5, 28.4, 25.0. LC−MS (m/z)

calcd. for C₂₂H₃₃BNO₆[M + H]⁺418.2, found 318.1 [(M + H)-Boc]⁺.

Mp: decomposition. R_f0.40 (heptanes/EtOAc, 1:2 + 1% AcOH).

(2S,3R)-3-(Benzo[d][1,3]dioxol-5-yl)-1-(tert-butoxycarbonyl)-5-

oxopyrrolidine-2-carboxylic Acid (7j). IBX (340 mg, 1.21 mmol, 2

equiv) was added to a solution of 6j (195.0 mg, 0.607 mmol, 1 equiv)

in DMSO (2.4 mL) at rt. The reaction mixture was allowed to stir

until the total consumption of the starting material (3.5 h), then was

quenched with sat. NaHCO₃(3 mL). The aqueous phase was

extracted with EtOAc (3 × 5 mL), and the combined organic layers

were washed with brine (10 mL), dried over MgSO₄, and

concentrated. The crude aldehyde (colorless oil) was used in the

next step without further puriﬁcation.

1

the title compound as a colorless oil (41 mg, 49% yield). H NMR

(CDCl₃) δ (two rotamers) 10.51 (s, 1H), 7.30 (bs, 1H), 7.25−7.16

(m, 2H), 6.94−6.89 (m, 1H), 6.73 (bs, 1H), 4.41 (d, J = 5.4 Hz,

0.4H), 4.25 (d, J = 6.5 Hz, 0.6H), 3.85−3.35 (m, 3H), 2.42−2.18 (m,

1H), 2.07−1.96 (m, 1H), 1.51 (s, 9H), 1.49 (s, 4H), 1.41 (s, 5H). ¹³C

NMR (CDCl₃) δ (two rotamers) 178.1, 177.1, 175.8, 155.6, 153.9,

142.0, 141.5, 139.0, 138.9, 129.5, 121.7, 117.7, 117.4, 81.3, 80.9, 65.7,

The crude aldehyde (0.607 mmol, 1 equiv), NaH₂PO₄·2H₂O (284

mg, 1.82 mmol, 3 equiv), and 2-methyl-2-butene (0.32 mL, 3.04

mmol, 5 equiv) were dissolved in tert-BuOH/H₂O (3:1, 3 mL).

NaClO₂was then added, and the reaction mixture was stirred for 1.5 h

at rt. After the complete consumption of the starting material, the

reaction was quenched with pH 7 phosphate buﬀer (3 mL), and the

aqueous phase was extracted with EtOAc (3 × 10 mL). The combined

organic layers were washed with brine (10 mL), dried over MgSO₄,

and concentrated. The crude product was puriﬁed by ﬂash

chromatography (heptanes/EtOAc, 2:1 + 1% AcOH) to aﬀord the

title compound as an oﬀ-white solid (135 mg, 66% over two steps). ¹H

NMR (CDCl₃) δ (two rotamers) 7.93 (br s, 1H), 6.72 (m, 3H), 5.94

(s, 2H), 4.33 (d, J = 5.3 Hz, 0.4H), 4.18 (d, J = 6.6 Hz, 0.6H), 3.80−

3.36 (m, 3H), 2.33−2.22 (m, 1H), 2.07−1.88 (m, 1H), 1.50 (s, 4H),

1.42 (s, 5H). ¹³C NMR (CDCl₃) δ (two rotamers) 178.2, 175.1, 155.9,

153. 8, 148.2, 146.9, 146.80, 134.8, 134.3, 120.4, 120.3, 108.5, 107.4,

101.2, 81.6, 80.9, 66.0, 65.6, 49.9, 47.4, 46.4, 46.1, 33.1, 32.7, 28.5,

28.4. LC−MS (m/z) calcd. for C₁₇H₂₂NO₆[M + H]⁺336.1, found

D

65.3, 60.6, 50.0, 47.8, 46.4, 46.1, 32.9, 32.5, 28.5, 28.5, 28.4. [α]₂₅

+48.4 (c = 0.28, MeOH). R_f0.21 (heptanes/EtOAc, 1:1 + 1% AcOH).

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-cyanophenyl)pyrrolidine-2-

carboxylic Acid (7g). A solution of NaIO₄(435 mg, 2.03 mmol, 4.1

equiv) and RuCl₃·xH₂O (3.09 mg, 0.015 mmol, 0.03 equiv) in H₂O

(6.19 mL) was added to a solution of 6g (150 mg, 0.496 mmol, 1

equiv) in MeCN/EtOAc (1:1, 7.04 mL) cooled to 0 °C. The reaction

mixture was stirred for 1 h, then ﬁltered through ﬁlter paper, and the

ﬁlter cake was washed with EtOAc. The aqueous layer was extracted

with EtOAc (3 × 5 mL), and the combined organic layers were

washed with brine, dried over MgSO₄, and concentrated. The crude

product was puriﬁed by ﬂash chromatography (heptanes/EtOAc, 1:2 +

1% AcOH) to aﬀord the title compound as a white solid (119 mg,

76%). ¹H NMR (CDCl₃) δ (two rotamers) 10.56 (bs, 1H), 7.66−7.37

(m, 4H), 4.37 (d, J = 5.6 Hz, 0.5H), 4.23 (d, J = 6.6 Hz, 0.5H), 3.83−

3.40 (m, 3H), 2.36 (dt, J = 11.9, 6.5 Hz, 1H), 2.01 (dq, J = 12.7, 7.9

Hz, 1H), 1.48 (s, 4H), 1.41 (s, 5H). ¹³C NMR (CDCl₃) δ (two

rotamers) 177.3, 175.3, 155.4, 153.7, 142.5, 142.1, 131.8, 131. 7, 131.2,

131.1, 130.8, 130.7, 129.9, 118.6, 113.1, 81.7, 81.3, 65.4, 65.1, 49.3,

47.4, 46.2, 46.0, 32.7, 32.3, 28.5, 28.3. MS (m/z) calcd. for

C₁₇H₂₁N₂O₄[M + H]⁺317.15, found 217.1 [(M + H)-Boc]⁺. Mp:

141.4−143.1 °C. R_f0.26 (heptanes/EtOAc, 1:2 + 1% AcOH).

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carbamoylphenyl)-

pyrrolidine-2-carboxylic Acid (7h). H₂O₂30% (w/w) in H₂O (0.089

mL, 0.870 mmol, 6 equiv) was added dropwise to a solution of 7g (46

mg, 0.145 mmol, 1 equiv) and K₂CO₃(80 mg, 0.58 mmol, 4 equiv) in

EtOH/H₂O (1:1, 1 mL). The reaction mixture was stirred for 3 h at rt,

then cautiously acidiﬁed with HCl 1 M (until pH ≈ 3) and extracted

with EtOAc (3 × 5 mL). The combined organic layers were washed

with brine (1 × 15 mL), dried over MgSO₄, and concentrated to give

the title compound (32 mg, 66%). ¹H NMR (CDCl₃) δ (two

rotamers) 8.61 (br s, 1H), 7.87−7.61 (m, 2H), 7.48−7.30 (m, 2H),

7.12−6.72 (m, 2H), 4.50 (d, J = 6.6 Hz, 0.5H), 4.21 (d, J = 7.3 Hz,

0.5H), 3.83−3.40 (m, 3H), 2.44−2.17 (m, 1H), 2.12−1.94 (m, 1H),

1.47 (s, 5H), 1.40 (s, 4H). ¹³C NMR (CDCl₃) δ (two rotamers) 176.4,

175.0, 171.0, 170.9, 155.4, 154.0, 141.2, 141.1, 133.6, 133.3, 131.2,

130.7, 129.3, 129.2, 127.3, 126.8, 126.6, 126.5, 81.3, 81.0, 66.1, 65.3,

50.1, 48.5, 46.7, 46.2, 33.4, 32.4, 28.6, 28.4. LC−MS (m/z) calcd. for

C₁₇H₂₃N₂O₅[M + H]⁺335.16, found 235.1 [(M + H)-Boc]⁺. Mp:

136.9−139.4 °C. R_f0.13 (heptanes/EtOAc, 1:3 + 1% AcOH).

D

236.1 [(M + H)-Boc]⁺. [α]₂₅+43.8 (c = 1.0, MeOH). Mp: 129.5−

131.7. R_f0.32 (heptanes/EtOAc, 1:1 + 1% AcOH).

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-4-ﬂuorophenyl)-

pyrrolidine-2-carboxylic Acid (7k). A solution of NaIO₄(496 mg, 2.32

mmol, 8.2 equiv) and RuCl₃·xH₂O (3.5 mg, 0.017 mmol, 0.06 equiv)

in H₂O (3.51 mL) was added to a solution of 6k (92 mg, 0.283 mmol,

1 equiv) in MeCN/EtOAc (1:1, 3.98 mL) cooled to 0 °C. The

reaction mixture was stirred for 2 h, then was ﬁltered through ﬁlter

paper, and the ﬁlter cake was washed with EtOAc. The aqueous layer

was extracted with EtOAc (3 × 10 mL), and the combined organic

layers were washed with brine (10 mL), dried over MgSO₄, and

concentrated to give 93 mg. The crude was puriﬁed by column

chromatography (eluent: hept/EtOAc 1/3 + 1% AcOH) to aﬀord the

title compound as white needles (89 mg, 89% yield). ¹H NMR

(CDCl₃, 400 MHz) δ (two rotamers) 7.91 (dd, J = 6.8, 2.5 Hz, 1H),

7.49 (ddd, J = 8.6, 4.4, 2.5 Hz, 1H), 7.15 (dt, J = 14.4, 7.2 Hz, 1H),

4.39 (d, J = 6.4 Hz, 0.5H), 4.22 (d, J = 7.3 Hz, 0.5H), 3.88−3.45 (m,

3H), 2.33 (dt, J = 12.4, 6.1 Hz, 1H), 2.10−1.99 (m, 1H), 1.51 (s, 5H),

1.43 (s, 4H). ¹³C NMR (MeOD) δ (two rotamers) 177.9, 176.4,

174.4, 168.4, 168.3, 162.7, 162.6, 161.0, 160.9, 156.2, 153.7, 136.7,

136.2, 134.53, 134.47, 134.0, 133.9, 131.8, 131.4, 118.0, 117.90,

117.86, 117.7, 82.2, 81.3, 65.8, 65. 6, 49.3, 46.8, 46.6, 46.2, 33.0, 32.7,

28.5, 28.4, 20.7. LC−MS (m/z) calcd. for C₁₇H₂₁FNO₆[M + H]⁺

354.1, found 254.1 [(M + H)-Boc]⁺. Mp: 168.5−170.6 °C. R_f0.16

(heptanes/EtOAc, 3:1 + 1% AcOH).

(2S,3R)-1-(tert-butoxycarbonyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)phenyl)pyrrolidine-2-carboxylic Acid (7i). A sol-

ution of NaIO₄(231 mg, 1.08 mmol, 4.1 equiv) and RuCl₃·xH₂O

(1.63 mg, 0.008 mmol, 0.03 equiv) in H₂O (3.27 mL) was added to a

solution of 6i (106 mg, 0.263 mmol, 1.0 equiv) in MeCN/EtOAc (1:1,

3.7 mL) cooled to 0 °C. The reaction mixture was stirred for 30 min,

then ﬁltered through ﬁlter paper, and the ﬁlter cake was washed with

EtOAc. The aqueous layer was extracted with EtOAc (3 × 5 mL), and

the combined organic layers were washed with brine, dried over

MgSO₄, and concentrated to aﬀord the title compound as an oﬀ-white

(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-4-chlorophenyl)-

pyrrolidine-2-carboxylic Acid (7l). A solution of NaIO₄(164 mg, 7.68

mmol, 8.2 equiv) and RuCl₃·xH₂O (11.6 mg, 0.056 mmol, 0.06 equiv)

in H₂O (11.6 mL) was added to a solution of 6l (320 mg, 0.936 mmol,

1 equiv) in MeCN/EtOAc (1:1, 13.2 mL) cooled to 0 °C. The

reaction mixture was stirred for 2 h, then was ﬁltered through ﬁlter

paper, and the ﬁlter cake was washed with EtOAc. The aqueous layer

was extracted with EtOAc (3 × 10 mL), and the combined organic

layers were washed with brine (10 mL), dried over MgSO₄, and

concentrated. The crude product was puriﬁed by column chromatog-

raphy (heptanes/EtOAc, 1:3 + 1% AcOH) to aﬀord the title

1

solid (72 mg, 66%). H NMR (CDCl₃) δ (two rotamers) 7.77−7.65

(m, 2H), 7.35−7.32 (m, 2H), 4.46 (d, J = 5.6 Hz, 0.4H), 4.30 (d, J =

6.7 Hz, 0.6H), 3.85−3.42 (m, 3H), 2.43−2.23 (m, 1H), 2.07 (dt, J =

16.9, 8.1 Hz, 1H), 1.50 (s, 4H), 1.42 (s, 5H), 1.34 (s, 12H). ¹³C NMR

1

compound as a white solid (205 mg, 51% yield). H NMR (CDCl₃)

O

DOI: 10.1021/acs.jmedchem.7b01624

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Journal of Medicinal Chemistry

Article

δ (two rotamers) 7.88 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 8.1, 4.4 Hz,

1H), 7.39 (d, J = 8.3 Hz, 1H), 6.91 (br s, 2H), 4.40 (d, J = 6.2 Hz,

0.5H), 4.24 (d, J = 7.2 Hz, 0.5H), 3.85−3.48 (m, 3H), 2.41−2.30 (m,

1H), 2.03 (dt, J = 9.1, 8.6 Hz, 1H), 1.51 (s, 5H), 1.44 (s, 4H). ¹³C

NMR (MeOD) δ (two rotamers) 175.7, 175.3, 169.0, 168.9, 156.0,

155.6, 141.7, 141.2, 132.93, 132.87, 132.80, 132.76, 132.7, 132.31,

132.27, 132.2, 131.1, 130.9, 82.0, 81.6, 67.2, 66.6, 50.5, 47.3, 47.1, 33.8,

33.2, 28.7, 28.5. LC−MS (m/z) calcd. for C₁₇H₂₁ClNO₆[M + H]⁺

370.1, found 270.0 [(M + H)-Boc]⁺. Mp: 184.2−185.6 °C. R_f0.26

(heptanes/EtOAc, 3:1 + 1% AcOH).

(2S,3R)-tert-Butyl 3-(3-Bromophenyl)-2-(((tert-butyldimethylsilyl)-

oxy)methyl)pyrrolidine-1-carboxylate (8b). In a ﬂame-dried ﬂask a

solution of 4b (803 mg, 1.66 mmol, 1 equiv) in dry THF (5.1 mL) was

cooled to −78 °C. LiBEtH₃1 M (THF solution) (1.99 mL, 1.99

mmol, 1.2 equiv) was added via syringe dropwise, and the reaction

mixture was stirred for 1 h, quenched with sat. NaHCO₃(3 mL), and

warmed to rt. The aqueous phase was extracted with EtOAc (3 × 5

mL), and the combined organic layers were washed with brine (10

mL), dried over MgSO₄, and concentrated to give the corresponding

hemiaminal 5b, which was used in the next step without further

puriﬁcation.

was evacuated and backﬁlled with nitrogen. A solution of aryl bromide

8 (640 mg, 1.36 mmol, 1.0 equiv) in dry DMF (8.2 mL) was added via

syringe, and the reaction mixture was stirred at 80 °C for 5 h. The

reaction mixture was cooled to rt and diluted with H₂O (10 mL). The

aqueous layer was extracted with Et₂O (3 × 10 mL), and the

combined organic layers were washed with HCl 1 M (1 × 10 mL),

brine (1 × 10 mL), dried over MgSO₄, and concentrated. The crude

product was puriﬁed by column chromatography (heptanes/EtOAc,

95:5) to give the title compound as a colorless oil (431 mg, 61%). ¹H

NMR (CDCl₃) δ 7.71−7.64 (m, 2H), 7.36−7.27 (m, 2H), 4.10−3.49

(m, 5H), 3.43−3.23 (m, 1H), 2.31−2.19 (m, 1H), 1.99−1.88 (m, 1H),

1.49 (s, 9H), 1.34 (s, 12H), 0.89 (s, 9H), 0.04 (s, 6H). ¹³C NMR

(CDCl₃) δ (two rotamers) 154.4, 154.3, 143.2, 142.7, 133.9, 133.7,

133.0, 130.5, 130.3, 129.4 (bs), 128.2, 83.9, 79.5, 79.1, 65.5, 65.4, 63.0,

61.5, 47.2, 46.7, 46.5, 45.7, 33.0, 31.8, 28.7, 26.0, 25.0, 25.0, 18.3, −5.3.

LC−MS (m/z) calcd. for C₂₈H₄₉BNO₅Si [M + H]⁺518.3, found 336.2

[(M + H)-Boc-Pin]⁺. R_f0.16 (heptanes/EtOAc, 95:5).

Pharmacological Studies. All reagents and solvents were

commercial and of high purity analytical grade or ultragradient

HPLC-grade purchased from Sigma, (St. Louis, MO, USA), J.T. Baker

(Denventer, The Netherlands), Merck (Darmstadt, Germany), or

In a ﬂame-dried ﬂask a solution of the crude hemiaminal 5b (1.66

mmol, 1 equiv) in dry DCM (5.3 mL) was cooled to −78 °C. HSiEt₃

(0.53 mL, 3.31 mmol, 2 equiv) and BF₃·Et₂O (0.46 mL, 3.65 mmol,

2.2 equiv) were added sequentially via syringe, and the reaction

mixture stirred for 4.5 h. The reaction was quenched with sat.

NaHCO₃(4 mL) and warmed to rt. The mixture was diluted with

DCM, and the organic phase was separated, washed with sat. NH₄Cl

(5 mL), dried over MgSO₄, and concentrated to dryness to give crude

alcohol 6b.

TBSCl (300 mg, 1.99 mmol, 1.2 equiv) was added to a solution of

the crude alcohol 6b (1.66 mmol, 1 equiv) and imidazole (282 mg,

4.14 mmol, 2.5 equiv) in dry DMF (11.7 mL) under nitrogen at rt.

The reaction mixture was stirred overnight, then poured into H₂O.

The aqueous layer was extracted with Et₂O (3 × 10 mL), and the

collective organic layers were washed with 1 M HCl (10 mL) and

brine (10 mL), dried over MgSO₄and concentrated to give 839 mg

(crude). The crude product was puriﬁed by ﬂash chromatography

(heptanes/EtOAc, 9:1) to aﬀord the title compound as a colorless oil

(572 mg, 73% over three steps). ¹H NMR (CDCl₃, 400 MHz) δ 7.39−

7.32 (m, 2H), 7.16 (dd, J = 16.8, 9.0 Hz, 2H), 4.03−3.27 (m, 6H),

2.35−2.19 (m, 1H), 1.96−1.80 (m, 1H), 1.48 (s, 9H), 0.89 (s, 9H),

0.04 (s, 3H), 0.04 (s, 3H). ¹³C NMR (CDCl₃) δ 154.3, 146.6, 146.2,

130.7, 130.6, 130.6, 130.3, 129.7, 126.1, 126.0, 122.8, 79.8, 79.4, 65.5,

65.4, 63.2, 61.8, 47.0, 46.6, 46.4, 45.5, 32.8, 31.7, 28.7, 26.0, 18.3, −5.3.

R_f0.27 (heptanes/EtOAc, 9:1).

Riedel-de Haen (Seelze, Germany). Water was puriﬁed using a Milli-Q

̈

Gradient system (Millipore, Milford, MA, USA). LPS from Escherichia

coli 055:B5 and ﬂuorescein were purchased from Sigma (St. Louis,

MO, USA). [³H]-digoxin, 250 μCi (9.25 MBq), and Emulsiﬁer safe

liquid scintillation cocktail were purchased from PerkinElmer (Boston,

MA, USA). Prostaglandin E2 was purchased from Biotechne Ltd.

(Abingdon, UK) and sorafenib from Cayman Chemical (Ann Arbor,

MI, USA).

Liquid Chromatographic and Mass Spectrometric (LC−MS/

MS) Analyses. An Agilent 1200 Series Rapid Resolution LC System

was used together with a Poroshell 120 EC-C-18 column (50 mm ×

2.1 mm, 2.7 μm) for liquid chromatography prior to MS analysis of

compound 1l, sorafenib, and PGE₂with Agilent 6410 triple

quadrupole mass spectrometer equipped with an electrospray

ionization source (Agilent Technologies Inc., Wilmington, DE). The

high-performance liquid chromatography eluents were water (A)

containing 0.1% (v/v) formic acid and acetonitrile (B). For the

compound 1l, a gradient elution with 5−60% B was applied over 1−3

min, followed by 1 min of isocratic elution with 60% B and 3 min

column equilibration, giving a total time of 7 min/injection. For

sorafenib, a gradient elution with 20−90% B was applied over 1−4

min, followed by 1 min of isocratic elution with 90% B and 3 min

column equilibration, giving a total time of 8 min/injection. For PGE₂

an isocratic method with 10% B was used. For all compounds the

eluent ﬂow rate was 0.2 mL/min, the column temperature was 40 °C,

and injection volume was 5 μL. The following mass spectrometry

conditions were used for the compound 1l, sorafenib, and PGE₂.

Electrospray ionization: positive ion mode for compound 1l, sorafenib

and negative ion mode for PGE₂; drying gas (nitrogen) temperature,

300 °C; drying gas ﬂow rate, 8 L/min; nebulizer pressure, 20 psi; and

capillary voltage, 3500 V. Analyte detection was performed using

multiple reaction monitoring, the transitions being 270.1 → 114.5 and

236.1 → 190 for compound 1l and 1a (internal standard), respectively.

The transitions for sorafenib and the used internal standard,

diclofenac, were 465.1 → 252.2 and 296.1 → 250, respectively. The

transition for PGE₂was 315.4 → 314.5. Fragmentor voltages used for

compound 1l, 1a, sorafenib, diclofenac, and PGE₂were 60, 140, 140,

100, and 180 V, and the collision energies were 40, 16, 30, 10, and 6 V,

respectively. Agilent MassHunter Workstation Acquisition software

(Data Acquisition for Triple Quadrupole Mass Spectometer, version

B.03.01) was used for data acquisition, and Quantitative Analysis

(B.04.00) software was used for the data processing and analysis. The

compound 1l lower limit of quantiﬁcation for the brain, liver, and

plasma samples were 0.02 nmol/g, 0.02 nmol/g, and 0.5 μM

respectively. The lower limit of quantiﬁcation for sorafenib and

PGE₂in cell samples was 1 nM. Linearity of the calibration curves was

evaluated by a quadratic regression analysis. The method was also

selective, accurate, and precise over the calibration range. Within-run

accuracy and precision were calculated from the results of the quality

(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(3-

cyanophenyl)pyrrolidine-1-carboxylate (8g). Aryl bromide 8b (500

mg, 1.06 mmol, 1 equiv), Zn(CN)₂(75 mg, 0.64 mmol, 0.6 equiv), Zn

powder (8.3 mg, 0.127 mmol, 0.12 equiv), Pd₂(dba)₃(48.6 mg, 0.053

mmol, 0.05 equiv), and dppf (58.9 mg, 0.106 mmol, 0.1 equiv) were

placed in a 10 mL oven-dried ﬂask, which was evacuated and backﬁlled

with N₂. Dry dimethylacetamide (2.1 mL) was added, and the reaction

mixture was stirred at 120 °C for 75 min, then cooled to rt and diluted

with EtOAc (5 mL). The organic phase was washed with sat.

NaHCO₃, brine, dried over MgSO₄, and concentrated. The crude

product was puriﬁed by column chromatography (heptanes/EtOAc,

1

95:5) to give the title compound a colorless oil. H NMR (CDCl₃) δ

7.53−7.35 (m, 4H), 3.99−3.58 (m, 4H), 3.57−3.49 (m, 1H), 3.42−

3.29 (m, 1H), 2.30 (dtd, J = 12.8, 7.3, 5.6 Hz, 1H), 1.87 (dq, J = 14.3,

7.2 Hz, 1H), 1.47 (s, 9H), 0.86 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H). ¹³C

NMR (CDCl₃) δ 154.1, 145.5, 145.3, 131.9, 131.7, 131.0, 130.2, 129.5,

118.8, 112.7, 79.9, 79.5, 65.2, 63.2, 61.8, 46.7, 46.6, 46.1, 45.4, 32.5,

31.6, 28.6, 25.8, 18.2, −5.4. LC−MS (m/z) calcd. for C₁₈H₂₉N₂OSi

[(M + H)-Boc]⁺317.2, found 317.2. R_f0.59 (heptanes/EtOAc, 2:1)

(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(3-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-

carboxylate (8i). (BPin)₂(380 mg, 1.50 mmol, 1.1 equiv),

PdCl₂(dppf) (49.7 mg, 0.068 mmol, 0.05 equiv), and KOAc (400

mg, 4.08 mmol, 3.0 equiv) were placed in an oven-dried ﬂask, which

P

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Journal of Medicinal Chemistry

Article

Table 5. Probe Peptides and MRM Transitions for the LC−MS/MS Analysis of Investigated Transporters

MRM transitions (m/z)

transporter

Abcb1

St/Is

probe peptide sequence

NTTGALTTR

Q1

Q3.1

Q3.2

Q3.3

St

Is

St

Is

St

Is

St

Is

St

Is

St

Is

467.8

472.6

522.8

527.8

899.0

904.0

482.8

487.8

460.8

464.8

571.8

576.8

618.4

628.4

757.5

767.5

1356.7

1366.7

796.4

806.4

693.4

701.4

894.4

904.4

719.4

729.4

644.3

654.4

678.9

683.9

697.4

707.4

578.3

586.3

537.3

547.3

516.3

571.3

529.3

539.4

a

NTTGALTTR

Abcg2

Abcc2

Abcc4

Slc7a5

Slc2a1

SSLLDVLAAR

a

SSLLDVLAAR

LTIIPQDPILFSGNLR

a

LTIIPQDPILFSGNLR

APVLFFDR

584.3

594.3

a

APVLFFDR

VQDAFAAAK

a

VQDAFAAAK

TFDEIASGFR

a

TFDEIASGFR

a

Denotes ¹³C-labeled arginine and lysine.

control samples at the three concentrations. The accuracies and

precisions for quality control concentrations of 20% were considered

to be acceptable.

and negative ionization: high-pressure ion funnel RF voltage 200 V,

and low-pressure ion funnel RF voltage 100 V. An injection volume of

15 μL was used, and analytes were separated by AdvanceBio Peptide

Map 2.1 × 250 mm, 2.7 μm. Mobile phases A and B, respectively,

consisted of 0.1% formic acid in milli-Q water and acetonitrile. The

gradient sequence was as follows: ﬂow rate of 0.30 mL/min, 50 min of

total run time, 97:3 to 60:40 (A:B) during 40 min after injection, 5:95

at 44 min, 97:3 at 45 min, and constant 97:3 for 5 min. The eluted

peptides were selectively and simultaneously analyzed by SRM/MRM

mode with LC−MS/MS. For each target protein, one unique peptide

was chosen according to previously published work.³⁶These peptides

were monitored with two or three diﬀerent SRM/MRM transition sets

(Table 5) derived from one set of stable isotope-labeled peptides

purchased from JPT Peptide Technologies GmbH (Berlin, Germany)

and unlabeled peptides.

Ability of Compound 1l To Alter the Cell Accumulation of

Transporter Probes and Sorafenib. The ability of compound 1l to

increase the cell accumulation of Abcb1 probe [³H]-digoxin, Abcc1−5

probe ﬂuorescein, and Abcb1 and Abcg2 substrate sorafenib as well as

to decrease cell accumulation of Slc7a5 probe [¹⁴C]-L-leucine and

Slc2a1 probe [¹⁴C]-D-glucose was determined in mouse Nras driven

p53^−/−HCC cells. The cells were cultured in DMEM supplemented

with ^L-glutamine (2 mM), heat-inactivated fetal bovine serum (10%),

penicillin (50 U/mL), and streptomycin (50 μg/mL). HCC cells were

seeded at the density of 1 × 10⁵cells/well onto 24-well plates. After

seeding, the cells were incubated for 24 h with or without 10 μM of

compound 1l. In order to ensure that compound 1l does not inhibit

the function of the eﬄux transporters, the incubation medium was

removed and the cells were carefully washed three times with

prewarmed Hank’s balance salt solution (HBSS) containing 125 mM

choline chloride, 4.8 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 1.3

mM CaCl₂, 5.6 mM glucose, 100 μM leucine, and 25 mM HEPES (pH

7.4), after which a transporter probe, [³H]-digoxin, [¹⁴C]-L-leucine,

[¹⁴C]-D-glucose, 20 μM ﬂuorescein, or 10 μM sorafenib, was added in

prewarmed HBSS buﬀer (250 μL) on the top of the cell layer and

incubating at 37 °C for 30 min for [³H]-digoxin, ﬂuorescein and

sorafenib or 5 min for [¹⁴C]-L-leucine and [¹⁴C]-D-glucose.

Subsequently, the cells were washed three times with ice-cold HBSS

and lysed with 500 μL of 0.1 M NaOH. The protein concentrations in

each well were determined by Bio-Rad Protein Assay (EnVision,

PerkinElmer, Inc., Waltham, MA, USA). The lysate from the [³H]-

digoxin, [¹⁴C]-L-leucine, and [¹⁴C]-D-glucose samples was mixed with

3.5 mL of emulsiﬁer safe liquid scintillation cocktail. The radioactivity

was measured by liquid scintillation counting (Wallac 1450 MicroBeta;

Wallac Oy, Finland). The ﬂuorescein samples were measured using

ﬂuorescein detector (EnVision, PerkinElmer, Inc., Waltham, MA,

USA), and the sorafenib concentrations were determined by the LC−

MS/MS.

Preparation of Crude Membrane Fractions of Mouse HCC

Cells. Mouse Nras driven p53^−/−HCC cells were cultured in

Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with ^L-

glutamine (2 mM), heat-inactivated fetal bovine serum (10%),

penicillin (50 U/mL), and streptomycin (50 μg/mL). The cells

were incubated for 48 h with or without 10 μM of compound 1l,

washed three times with HBSS, followed by centrifugation of the cells

into a pellet and rapid freezing in liquid nitrogen. ProteoExtract

Subcellular Proteome Extraction Kit (Merck KGaA, Darmstadt,

Germany) was used for the isolation of crude membrane fraction

from the cell pellets following the manufacturer’s instructions. The

protein concentrations in the crude membrane fractions were

determined as mean of three samples by Bio-Rad Protein Assay

(EnVision, PerkinElmer, Inc., Waltham, MA, USA), and aliquots

containing 50 μg of total protein were taken for further sample

preparation.

Protein Quantiﬁcation by Multiplexed Selected/Multiple

Reaction Monitoring by LC−MS/MS. The protein expressions of

the investigated transporters were simultaneously determined by

means of multiplexed multiple reaction monitoring analysis. The

aliquots containing 50 μg of protein from the crude membrane

fractions were solubilized in 500 mM Tris−HCl (pH 8.5), 7 M

guanidine hydrochloride, and 10 mM EDTA, and the proteins were S-

carbamoylmethylated with iodoacetamide following dithiothreitol

treatment. The alkylated proteins were precipitated using methanol

and chloroform. The precipitates were dissolved in 6 M urea in 100

mM Tris−HCl (pH 8.5), diluted ﬁve-fold with 100 mM Tris−HCl

(pH 8.5), spiked with internal standard peptides, and treated with

Protease-Max surfactant (Promega, Madison, WI, USA). The dilutions

were treated with lysyl endopeptidase (Lys-C: Wako Pure Chemical

Industries, Osaka, Japan) at rt for 3 h, after which tosylphenylalanyl

chloromethyl ketone-treated trypsin (Promega, Madison, WI, USA) at

an enzyme/substrate ratio of 1:100 was added into the samples and

incubated at 37 °C for 16 h. The tryptic digests were mixed formic

acid and then centrifuged at 4 °C and 14 000g for 5 min. The

supernatant was mixed with water prior to LC−MS/MS analysis.

LC−MS/MS analysis was performed by coupling an Agilent 1290

Inﬁnity LC (Agilent Technologies, Waldbronn, Germany) instrumen-

tation to an Agilent 6495 Triple Quadrupole Mass Spectrometer with

an electrospray ionization (ESI) source (Agilent Technologies, Palo

Alto, CA, USA) using multiple reaction monitoring (MRM). The

following conditions were applied: positive ionization mode, the

drying gas (nitrogen) was maintained at 210 °C, drying gas ﬂow rate

was 16 L/min, nebulizer gas pressure was 45 psi, sheath gas

temperature 300 °C, sheath gas ﬂow 11 L/min, fragmentor voltage

was 380 V, and the mass-spectrometer (MS) capillary voltage was 3.0

kV. The following ion funnel parameters were used for both positive

Live Imaging of Murine HCC Cell Culture. The cells were

loaded with the Ca²⁺sensitive ﬂuorescent dye Fluo-4-AM (5 μM) for

Q

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Journal of Medicinal Chemistry

Article

45 min followed by a 20 min washout in the BSS containing the

following (in millimolar): 2.5 KCl, 152 NaCl, 10 glucose, 2 CaCl₂, and

10 HEPES at pH 7.4. Fluorescence was visualized using the imaging

setup (TILL Photonics GmbH) consisting of a monochromatic light

source and a CCD camera (SensiCam). Cells loaded with Fluo-4 were

imaged with an excitation light of 495 nm (exposure time 100 ms,

binning 2). Chemicals were applied using a fast perfusion system

(Rapid Solution Changer RSC-200, BioLogic Science). Data were

analyzed oﬄine using the TILL Photonics and Origin 8 software.

Ability of 1l To Decrease PGE₂Concentration in the Cells.

The ability of compound 1l to decrease PGE₂synthesis was studied in

HCC cells with and without LPS-induced inﬂammation. The LPS

concentration in the growth medium was 2.5 μg/mL. The cells were

incubated 24 h with or without 100 μM compound 1l. Subsequently,

the cells were washed three times with ice-cold HBSS, and then lysed

with 500 μL of acetonitrile on ice. The PGE₂concentrations from the

cell lysates were determined by the LC−MS/MS.

Antiproliferative Activity in Vitro. The mouse HCC cells were

seeded at the density of 2 × 10⁴cells/well onto collagen-coated 96-

well plates, and the cells were used for the experiments 1 day after

seeding. A concentration of 1, 2.5, 5, and 10 μM sorafenib, 100 μM

compound 1l, and the combination of all the mentioned sorafenib

concentrations with 100 μM compound 1l were added into the growth

medium and incubated for 3 days. Each day the medium was changed,

and after the 72 h incubation, the cell viability was determined by

resazurin cell proliferation kit (Sigma, St. Louis, MO, USA), which is

directly proportional to aerobic respiration and cellular metabolism of

cells. The samples were measured ﬂuorometrically by monitoring the

increase in ﬂuorescence at a wavelength of 590 nm using an excitation

wavelength of 560 nm (EnVision, PerkinElmer, Inc., Waltham, MA,

USA). The cell death was conﬁrmed in the decrease of cell amount by

visualizing the wells with microscopy.

of supernatant was mixed with 100 μL of ultrapure water prior to LC−

MS/MS analysis.

Two-Electrode Voltage-Clamp Electrophysiology. Rat cDNAs

encoding GluN1−1a (Genbank accession number U11418 and

U08261; hereafter GluN1), GluN2A (D13211), GluN2B (U11419),

GluN2C (M91563), and GluN2D (L31611) were generously provided

by Dr. S. Heinemann (Salk Institute, La Jolla, CA), Dr. S. Nakanishi

(Osaka Bioscience Institute, Osaka, Japan), and Dr. P. Seeburg

(University of Heidelberg). The cDNA encoding rat GluN2B was

modiﬁed without changing the amino acid sequence to remove a T7

RNA polymerase termination site located in the C-terminal domain.³⁸

For expression in Xenopus laevis oocytes, cDNAs were linearized using

restriction enzymes and used as templates to synthesize cRNA using

the mMessage mMachine kit (Ambion, Life Technologies, Paisley,

UK). Xenopus oocytes were obtained from Rob Weymouth (Xenopus

1, Dexter, MI) and prepared as previously described.³⁹The oocytes

were injected with cRNAs encoding GluN1 and GluN2 in a 1:2 ratio

and maintained at 18 °C in Barth’s solution containing 88 mM NaCl, 1

mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂,

0.91 mM CaCl₂, 10 mM HEPES (pH 7.5 with NaOH) supplemented

with 100 IU/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL

gentamycin (Invitrogen, Life Technologies, Paisley, UK). Two-

electrode voltage-clamp recordings were performed on Xenopus

oocytes at rt 2−6 days postinjection with the extracellular recording

solution comprising 90 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.5

mM BaCl₂, and 0.01 mM EDTA (pH 7.4 with NaOH) at a holding

potential of −40 mV essentially as previously described.³⁹NMDA

receptor ligands were dissolved in extracellular recording solution and

applied to the oocyte by gravity-driven perfusion.

Data Analysis. All statistical analyses were performed using

GraphPad Prism v. 5.03 software (GraphPad Software, San Diego, CA,

USA). Statistical diﬀerences between groups were tested using two-

tailed t test (Figures 1 and 2). Concentration−inhibition data were

measured using two-electrode voltage-clamp electrophysiology ﬁtted

to the Hill equation to obtain IC₅₀values for individual oocytes as

previously described.³⁹The sorafenib IC₅₀value for cytotoxicity in

murine HCC cells was calculated by nonlinear regression analysis and

Animals. Adult male mice weighing 25 5 g were supplied by the

National Laboratory Animal Centre (Kuopio, Finland). Mice were

housed in stainless steel cages on a 12 h light (07:00−19:00) and 12 h

dark (19:00−07:00) cycle at an ambient temperature of 22 1 °C

with a relative humidity of 50−60%. All experiments were carried out

during the light phase. Tap water and food pellets (Lactamin R36;

presented as the mean

SEM. The pharmacokinetic parameters,

AUC_{0−240 min}, C_max, t_max, and t_1/2βin plasma, brain, and liver, were

obtained from the pharmacokinetic data.

Lactamin AB, Sodertalje, Sweden) were available ad libitum. All

̈

procedures with the animals were performed according to European

Community Guidelines and Guide for the Care and Use of Laboratory

Animals (National Institutes of Health publication no. 85−23, revised

in 1985). The procedures were reviewed and approved by the Finnish

National Animal Experiment Board.

Radioligand Binding. Ligand aﬃnities at native AMPA, KA, and

NMDA receptors (rat brain synaptosomes) were determined using

[³H]AMPA, [³H]KA, and [³H]CGP-39653, respectively, as previously

described.⁴⁰Ligand aﬃnities at recombinant homomeric rat GluK1−3

were determined using [³H]KA as the radioligand as previously

detailed (Sagot et al. and Alcaide et al.).^41,42Data were analyzed using

GraphPad Prism 7 (GraphPad Software, San Diego, CA) to determine

ligand IC₅₀and K_ivalues.

In Situ Mouse Brain Perfusion. Mice were anesthetized with

intraperitoneal (i.p.) injections of ketamine (120 mg/kg) and xylazine

(8 mg/mL), and their right carotid artery system was exposed. The

right external carotid artery was ligated, and the right common carotid

artery was cannulated with a catheter ﬁlled with 100 IU/mL heparin.

The perfusions were performed at 37 °C with a ﬂow rate of 2.5 mL/

min for 60 s followed by the washing of the capillaries for 2 s with 4 °C

drug-free perfusion buﬀer. The method is described in more detail by

Gynther et al.³⁷

In Vivo Pharmacokinetics of Compound 1l. A 5.0 mM

concentration of compound 1l was dissolved in a vehicle containing

0.9% (w/v) NaCl in water. A dose of 10 mg/kg of compound 1l was

administered as a bolus injection (i.p.) to mice. The mice were

sacriﬁced by decapitation at selected time points between (10−240

min), and plasma, brain, and liver were collected for analysis.

Plasma and Tissue Sample Preparation. Plasma samples were

prepared by precipitating 100 μL of plasma with 200 μL of acetonitrile

containing the internal standard (1a). Samples were vortexed and

centrifuged for 10 min at 14 000g at 4 °C. Then 100 μL of supernatant

was mixed with 100 μL of ultrapure water prior to LC−MS/MS

analysis. Tissue samples were weighed and homogenized with

ultrapure water (1:3). An aliquot of 100 μL was taken, and the

analyte was isolated from the samples by protein precipitation with

300 μL of acetonitrile containing the internal standard. Samples were

vortexed and centrifuged for 10 min at 14 000g at 4 °C. Then 200 μL

In Silico Studies. Calculation of LogP(o/w) and total polar surface

area (TPSA) was done in MOE version 2016.08.02 released by

Chemcomp corporation.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b01624.

SMILES (CSV)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: lebu@sund.ku.dk. Phone: +45 35 33 62 44.

*E-mail: mikko.gynther@uef.ﬁ.

■

ORCID

Kasper B. Hansen: 0000-0002-3303-4819

Darryl S. Pickering: 0000-0001-8687-6094

Lennart Bunch: 0000-0002-0180-4639

R

DOI: 10.1021/acs.jmedchem.7b01624

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(8) Rigalli, J. P.; Ciriaci, N.; Arias, A.; Ceballos, M. P.; Villanueva, S.

S. M.; Luquita, M. G.; Mottino, A. D.; Ghanem, C. I.; Catania, V. A.;

Ruiz, M. L. Regulation of Multidrug Resistance Proteins by Genistein

in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity.

PLoS One 2015, 10, e0119502.

Author Contributions

^⊥These authors contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

(9) Huang, W. C.; Hsieh, Y. L.; Hung, C. M.; Chien, P. H.; Chien, Y.

F.; Chen, L. C.; Tu, C. Y.; Chen, C. H.; Hsu, S. C.; Lin, Y. M.; Chen,

Y. J. BCRP/ABCG2 Inhibition Sensitizes Hepatocellular Carcinoma

Cells to Sorafenib. PLoS One 2013, 8, e83627.

(10) Gnoth, M. J.; Sandmann, S.; Engel, K.; Radtke, M. In Vitro to in

Vivo Comparison of the Substrate Characteristics of Sorafenib

Tosylate toward P-Glycoprotein. Drug Metab. Dispos. 2010, 38,

1341−1346.

(11) Callaghan, R.; Luk, F.; Bebawy, M. Inhibition of the Multidrug

Resistance P-Glycoprotein: Time for a Change of Strategy? Drug

Metab. Dispos. 2014, 42, 623−631.

(12) Fletcher, J. I.; Haber, M.; Henderson, M. J.; Norris, M. D. ABC

Transporters in Cancer: More than Just Drug Efflux Pumps. Nat. Rev.

Cancer 2010, 10, 147−156.

ACKNOWLEDGMENTS

■

We would like to thank the Lundbeck Foundation and the

Danish Medical Research Council for ﬁnancial support of the

medicinal chemistry work reported herein. Emil Aaltonen

Foundation and The Finnish Cultural Foundation are acknowl-

edged for the funding regarding the pharmacokinetic analysis,

transporter expression modulation, and cytotoxicity studies.

Finally, the National Institutes of Health (P20GM103546 and

R01NS097536) is acknowledged for ﬁnancial support in

regards to the functional characterization of 1l at NMDA

receptors. Mrs. Sari Ukkonen is acknowledged for technical

assistance. The murine hcc cells were a kind donation by Prof.

(13) Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.;

Vance, K. M.; Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S. J.;

Dingledine, R. Glutamate Receptor Ion Channels: Structure,

Regulation, and Function. Pharmacol. Rev. 2010, 62, 405−496.

(14) Qosa, H.; Miller, D. S.; Pasinelli, P.; Trotti, D. Regulation of

ABC Efflux Transporters at Blood-Brain Barrier in Health and

Neurological Disorders. Brain Res. 2015, 1628, 298−316.

(15) Leslie, C. C. Properties and Regulation of Cytosolic

Phospholipase A2. J. Biol. Chem. 1997, 272, 16709−16712.

(16) Maeda, S.; Omata, M. Inflammation and Cancer: Role of

Nuclear Factor-kappaB Activation. Cancer Sci. 2008, 99, 836−842.

(17) Shi, Y.; Wang, S.-Y.; Yao, M.; Sai, W.-L.; Wu, W.; Yang, J.-L.;

Cai, Y.; Zheng, W.-J.; Yao, D.-F. Chemosensitization of HepG2 Cells

by Suppression of NF-kappaB/p65 Gene Transcription with Specific-

siRNA. World J. Gastroenterol. 2015, 21, 12814−12821.

Lars Zender, Tubingen University.

̈

ABBREVIATIONS USED

■

ABC, ATP-binding cassette; Abcb1, ATP-binding cassette

subfamily B member 1; Abcc, ATP-binding cassette subfamily

C; Abcg2, ATP-binding cassette subfamily G member 2; AUC,

area under the concentration curve; BBB, blood−brain barrier;

C

_max, maximum concentration; cPLA2, cytoplasmic phospholi-

pase A2; DCM, dichloromethane; DCVC, dry collum vacuum

chromatography; DMAP, N,N-dimethyl-4-aminopyridine;

DMEM, Dulbecco’s modiﬁed Eagle medium; DMF, N,N-

dimethylformamide; DMSO, N,N-dimethylsulfonamide; HBSS,

Hank’s balance salt solution; HCC, hepatocellular carcinoma;

IBX, 2-iodoxybenzoic acid; LC−MS/MS, liquid chromatog-

raphy−mass spectrometry; LPS, lipopolysaccharide; MDR,

multidrug resistance; MRM, multiple reaction monitoring;

PGE₂, prostaglandin E2; SRM, selected reaction monitoring;

TBAF, tetrabutylammonium ﬂuoride; TBSCl, tert-butyldime-

thylsilyl chloride; TFA, triﬂuoroacetic acid; T_max, time to reach

the maximum concentration; t_1/2β, elimination half-life

(18) Deutsch, S. I.; Tang, A. H.; Burket, J. A.; Benson, A. D. NMDA

Receptors on the Surface of Cancer Cells: Target for Chemotherapy?

Biomed. Pharmacother. 2014, 68, 493−496.

́

(19) Ribeiro, M. P. C.; Nunes-Correia, I.; Santos, A. E.; Custodio, J.

B. A. The Combination of Glutamate Receptor Antagonist MK-801

with Tamoxifen and Its Active Metabolites Potentiates Their

Antiproliferative Activity in Mouse Melanoma K1735-M2 Cells. Exp.

Cell Res. 2014, 321, 288−296.

(20) Yamaguchi, F.; Hirata, Y.; Akram, H.; Kamitori, K.; Dong, Y.;

Sui, L.; Tokuda, M. FOXO/TXNIP Pathway Is Involved in the

Suppression of Hepatocellular Carcinoma Growth by Glutamate

Antagonist MK-801. BMC Cancer 2013, 13, 468.

(21) Bergeron, R.; Coyle, J. T. NAAG, NMDA Receptor and

Psychosis. Curr. Med. Chem. 2012, 19, 1360−1364.

REFERENCES

■

(1) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P.

G. Cancer Drug Resistance: An Evolving Paradigm. Nat. Rev. Cancer

2013, 13, 714−726.

(2) Rudalska, R.; Dauch, D.; Longerich, T.; McJunkin, K.;

Wuestefeld, T.; Kang, T.-W. T.-W. T.-W.; Hohmeyer, A.; Pesic, M.;

Leibold, J.; von Thun, A.; Schirmacher, P.; Zuber, J.; Weiss, K.-H. K.-

H.; Powers, S.; Malek, N. P. N. P.; Eilers, M.; Sipos, B.; Lowe, S. W. S.

W. S. W.; Geffers, R.; Laufer, S.; Zender, L. In Vivo RNAi Screening

Identifies a Mechanism of Sorafenib Resistance in Liver Cancer. Nat.

Med. 2014, 20, 1138−1146.

(22) Ghose, A. K.; Viswanadhan, V. N.; Wendoloski, J. J. A

Knowledge-Based Approach in Designing Combinatorial or Medicinal

Chemistry Libraries for Drug Discovery. 1. A Qualitative and

Quantitative Characterization of Known Drug Databases. J. Comb.

Chem. 1999, 1, 55−68.

́

(23) Larsen, A. M.; Venskutonyte, R.; Valades, E. A.; Nielsen, B.;

̇

Pickering, D. S.; Bunch, L. Discovery of a New Class of Ionotropic

Glutamate Receptor Antagonists by the Rational Design of (2S,3R)-3-

(3-Carboxyphenyl)-Pyrrolidine-2-Carboxylic Acid. ACS Chem. Neuro-

sci. 2011, 2, 107−114.

́

(3) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.;

Gottesman, M. M. Targeting Multidrug Resistance in Cancer. Nat. Rev.

Drug Discovery 2006, 5, 219−234.

(4) Zhang, Y.-K.; Wang, Y.-J.; Gupta, P.; Chen, Z.-S. Multidrug

Resistance Proteins (MRPs) and Cancer Therapy. AAPS J. 2015, 17,

802−812.

(24) Krogsgaard-Larsen, N.; Storgaard, M.; Møller, C.; Demmer, C.

S.; Hansen, J.; Han, L.; Monrad, R. N.; Nielsen, B.; Tapken, D.;

Pickering, D. S.; Kastrup, J. S.; Frydenvang, K.; Bunch, L. Structure-

Activity Relationship Study of Ionotropic Glutamate Receptor

Antagonist (2S,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-Carboxylic

Acid. J. Med. Chem. 2015, 58, 6131−6150.

(25) Ogura, A.; Yamada, K.; Yokoshima, S.; Fukuyama, T. Total

Synthesis of (−)-Anisatin. Org. Lett. 2012, 14, 1632−1635.

(26) Bunch, L.; Krogsgaard-Larsen, P.; Madsen, U. Mixed Cyano-

Gilman Cuprates-Advances in Conjugate Addition to Alpha, Beta-

Unsaturated Pyroglutaminol. Synthesis 2002, 2002, 31−33.

(5) Stacy, A. E.; Jansson, P. J.; Richardson, D. R. Molecular

Pharmacology of ABCG2 and Its Role in Chemoresistance. Mol.

Pharmacol. 2013, 84, 655−669.

(6) Gillet, J.-P.; Efferth, T.; Remacle, J. Chemotherapy-Induced

Resistance by ATP-Binding Cassette Transporter Genes. Biochim.

Biophys. Acta, Rev. Cancer 2007, 1775, 237−262.

(7) Wu, C. P.; Hsieh, C. H.; Wu, Y. S. The Emergence of Drug

Transporter-Mediated Multidrug Resistance to Cancer Chemotherapy.

Mol. Pharmaceutics 2011, 8, 1996−2011.

S

DOI: 10.1021/acs.jmedchem.7b01624

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(27) Synthesis will be reported elsewhere.

(28) Hansen, K. B.; Brauner-Osborne, H.; Egebjerg, J. Pharmaco-

̈

logical Characterization of Ligands at Recombinant NMDA Receptor

Subtypes by Electrophysiological Recordings and Intracellular Calcium

Measurements. Comb. Chem. High Throughput Screening 2008, 11,

304−315.

(29) Yung-Chi, C.; Prusoff, W. H. Relationship between the

Inhibition Constant (KI) and the Concentration of Inhibitor Which

Causes 50 per Cent Inhibition (I50) of an Enzymatic Reaction.

Biochem. Pharmacol. 1973, 22, 3099−3108.

(30) Hedegaard, M.; Hansen, K. B.; Andersen, K. T.; Brauner-

̈

Osborne, H.; Traynelis, S. F. Molecular Pharmacology of Human

NMDA Receptors. Neurochem. Int. 2012, 61, 601−609.

(31) Uchida, Y.; Tachikawa, M.; Obuchi, W.; Hoshi, Y.; Tomioka, Y.;

Ohtsuki, S.; Terasaki, T. A Study Protocol for Quantitative Targeted

Absolute Proteomics (QTAP) by LC−MS/MS: Application for Inter-

Strain Differences in Protein Expression Levels of Transporters,

Receptors, Claudin-5, and Marker Proteins at the Blood−Brain Barrier

in ddY, FVB, an. Fluids Barriers CNS 2013, 10, 21.

(32) Rautio, J.; Humphreys, J. E.; Webster, L. O.; Balakrishnan, A.;

Keogh, J. P.; Kunta, J. R.; Serabjit-Singh, C. J.; Polli, J. W. In Vitro P-

Glycoprotein Inhibition Assays for Assessment of Clinical Drug

Interaction Potential of New Drug Candidates: A Recommendation

for Probe Substrates. Drug Metab. Dispos. 2006, 34, 786−792.

(33) Loscher, W.; Potschka, H. Blood-Brain Barrier Active Efflux

̈

Transporters: ATP-Binding Cassette Gene Family. NeuroRx 2005, 2,

86−98.

(34) Liu, L.; Cao, Y.; Chen, C.; Zhang, X.; McNabola, A.; Wilkie, D.;

Wilhelm, S.; Lynch, M.; Carter, C. Sorafenib Blocks the RAF/MEK/

ERK Pathway, Inhibits Tumor Angiogenesis, and Induces Tumor Cell

Apoptosis in Hepatocellular Carcinoma Model PLC/PRF/5. Cancer

Res. 2006, 66, 11851−11858.

(35) Si, C.; Myers, A. G. A Versatile Synthesis of Substituted

Isoquinolines. Angew. Chem., Int. Ed. 2011, 50, 10409−10413.

(36) Akazawa, T.; Uchida, Y.; Tachikawa, M.; Ohtsuki, S.; Terasaki,

T. Quantitative Targeted Absolute Proteomics of Transporters and

Pharmacoproteomics-Based Reconstruction of P-Glycoprotein Func-

tion in Mouse Small Intestine. Mol. Pharmaceutics 2016, 13, 2443−

2456.

(37) Gynther, M.; Pickering, D. S.; Spicer, J. A.; Denny, W. A.;

Huttunen, K. M. Systemic and Brain Pharmacokinetics of Perforin

Inhibitor Prodrugs. Mol. Pharmaceutics 2016, 13, 2484−2491.

(38) Hansen, K. B.; Ogden, K. K.; Yuan, H.; Traynelis, S. F. Distinct

Functional and Pharmacological Properties of Triheteromeric GluN1/

GluN2A/GluN2B NMDA Receptors. Neuron 2014, 81, 1084−1096.

(39) Hansen, K. B.; Tajima, N.; Risgaard, R.; Perszyk, R. E.;

Jørgensen, L.; Vance, K. M.; Ogden, K. K.; Clausen, R. P.; Furukawa,

H.; Traynelis, S. F. Structural Determinants of Agonist Efficacy at the

Glutamate Binding Site of N-Methyl-D-Aspartate Receptors. Mol.

Pharmacol. 2013, 84, 114−127.

(40) Assaf, Z.; Larsen, A. P.; Venskutonyte, R.; Han, L.; Abrahamsen,

̇

B.; Nielsen, B.; Gajhede, M.; Kastrup, J. S.; Jensen, A. a.; Pickering, D.

S.; Frydenvang, K.; Gefflaut, T.; Bunch, L. Chemoenzymatic Synthesis

of New 2,4-Syn-Functionalized (S)-Glutamate Analogues and

Structure-Activity Relationship Studies at Ionotropic Glutamate

Receptors and Excitatory Amino Acid Transporters. J. Med. Chem.

2013, 56, 1614−1628.

(41) Sagot, E.; Pickering, D. S.; Pu, X.; Umberti, M.; Stensbøl, T. B.;

Nielsen, B.; Chapelet, M.; Bolte, J.; Gefflaut, T.; Bunch, L. Chemo-

Enzymatic Synthesis of a Series of 2,4-Syn-Functionalized (S)-

Glutamate Analogues: New Insight into the Structure-Activity Relation

of Ionotropic Glutamate Receptor Subtypes 5, 6, and 7. J. Med. Chem.

2008, 51, 4093−4103.

(42) Alcaide, A.; Marconi, L.; Marek, A.; Haym, I.; Nielsen, B.;

Møllerud, S.; Jensen, M.; Conti, P.; Pickering, D. S.; Bunch, L.

Synthesis and Pharmacological Characterization of the Selective

GluK1 Radioligand (S)-2-Amino-3-(6-[ 3 H]-2,4-Dioxo-3,4-

dihydrothieno[3,2-D]pyrimidin-1(2H)-Yl)propanoic Acid ([3H]-

NF608). MedChemComm 2016, 7, 2136−2144.

T

DOI: 10.1021/acs.jmedchem.7b01624

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Augmentation of Anticancer Drug Efficacy in Murine Hepatocellular Carcinoma Cells by a Peripherally Acting Competitive N-Methyl- d -aspartate (NMDA) Receptor Antagonist

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