Discovery of selective menaquinone biosynthesis inhibitors against Mycobacterium tuberculosis

Article

pubs.acs.org/jmc

Discovery of Selective Menaquinone Biosynthesis Inhibitors against

Mycobacterium tuberculosis^†

Joy Debnath,^‡Shajila Siricilla,^‡Bajoie Wan,^§Dean C. Crick,^∥Anne J. Lenaerts,^∥Scott G. Franzblau,^§

and Michio Kurosu*^,‡

^‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, 881 Madison, Memphis, Tennessee 38163,

United States

^§Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois

60612, United States

^∥Department of Microbiology, Immunology and Pathology, Colorado State University, 200 West Lake Street, Fort Collins, Colorado

80523, United States

S

* Supporting Information

ABSTRACT: Aurachin RE (1) is a strong antibiotic that was recently found to

possess 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) and bacterial

electron transport inhibitory activities. Aurachin RE is the only molecule in a

series of aurachin natural products that has the chiral center in the alkyl side

chain at C9′-position. To identify selective MenA inhibitors against

Mycobacterium tuberculosis, a series of chiral molecules were designed based on

the structures of previously identiﬁed MenA inhibitors and 1. The synthesized

molecules were evaluated in in vitro assays, including MenA enzyme and

bacterial growth inhibitory assays. We could identify novel MenA inhibitors that

showed signiﬁcant increase in potency of killing nonreplicating M. tuberculosis in

the low oxygen recovery assay (LORA) without inhibiting other Gram-positive bacterial growth even at high concentrations. The

MenA inhibitors reported here are useful new pharmacophores for the development of selective antimycobacterial agents with

strong activity against nonreplicating M. tuberculosis.

reactivated, causing the risk of fatal diseases.⁷⁻¹⁰Thus, in

addition to the necessity of drugs for the treatment of MDR-

Mtb, the development of drugs that kill Mtb in any state is very

important. However, no current TB drugs are eﬀective in killing

INTRODUCTION

■

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and

is responsible for nearly two million deaths annually.^1,2

Moreover, the emergence of multidrug-resistant (MDR) strains

of Mtb seriously threatens TB control and prevention eﬀorts.

the dormant form of Mtb in vivo. Therefore, ideal novel

antituberculosis compounds should show: (1) compatibility

One-third of the 42 million people living with HIV/AIDS

worldwide are coinfected with Mtb.³Recent studies have

and lack of cross-resistance with other anti-Mtb agents because

combination therapy remains mandatory to combat Mtb, (2)

antimicrobial spectrum focused against Mtb, (3) strong growth

inhibitory activity against nonreplicating Mtb, and (4) no

interactions with HAART.

shown that infection with Mtb enhances replication of HIV and

may accelerate the progression of HIV infection to AIDS.

There are signiﬁcant problems associated with treatment of

AIDS and Mtb coinfected patients.⁴Rifampicin and isoniazid

(key components of the local directly observed treatment

strategy) induce the cytochrome P450 3A4 enzyme, which

shows signiﬁcant interactions with anti-HIV drugs such as

protease inhibitors. In addition, rifampicin strongly interacts

with non-nucleoside reverse transcriptase and protease

inhibitors for HIV infections. Therefore, clinicians avoid

starting highly active antiretroviral therapy (HAART), which

consists of three or more highly potent reverse transcriptase

inhibitors and protease inhibitors, until the TB infection has

been cleared.^5,6M. tuberculosis is recognized to lie in a

nonreplicating state (dormancy), particularly in the caseous

pulmonary nodules where the lesions have little access to

oxygen, and can survive for many years in the host by entering a

dormant state. About 10% of patients with latent Mtb are

Function of ubiquinone (coenzyme Q₁₀) as a component of

the mitochondrial respiratory chain in human is well established

(“the chemiosmotic theory”, Mitchell, 1978).¹¹⁻¹³In prokar-

yotes, especially in Gram-positive bacteria, menaquinone

transfers two electrons in a process of either aerobic or

anaerobic respiration (Figure 1). On the other hand, a majority

of Gram-negative organisms utilize ubiquinone under aerobic

conditions and menaquinone under anaerobic conditions in

their electron transport systems.¹⁴⁻¹⁶Therefore, inhibitors of

menaquinone biosynthesis or speciﬁc inhibitors of enzymes

associated with electron transport systems have great potential

Received: November 29, 2011

Published: March 26, 2012

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Figure 1. The electron ﬂow system of M. tuberculosis. Menaquinone (MK) mediated energetic pathway (blue).

Figure 2. Biosynthesis of menaquinone from chorismate (classic pathway).

for the development of novel and selective drugs against MDR

Gram-positive bacteria.¹⁷The electron transport system

couples with ATP synthase to produce ATP through oxidative

phosphorylation. Bacterial ATP synthase, F₁F₀-ATPase, is a

viable target for treatment of MDR Mtb infections. A

diarylquinolone, a phase II clinical drug, is an inhibitor of

Mtb ATP synthase that exhibited a remarkable activity against

Mtb.¹⁸However, only few studies have investigated the

electron transport system for development of new antibacterial

drugs.¹⁷Weinstein and co-workers reported the inhibitors of

type II NADH:menaquinone oxidoreductase that eﬀectively

killed Mtb in vitro, and they concluded that type II NADH

dehydrogenase could be a unique and interesting antimicrobial

target.¹⁹We have reported that inhibition of 1,4-dihydroxy-2-

naphthoate prenyltransferase (MenA), which catalyzes a formal

decarboxylative prenylation of 1,4-dihydroxy-2-naphthoate

(DHNA) to form demethylmenaquinone (DMMK) in

menaquinone biosynthesis (Figure 2), showed signiﬁcant

growth inhibitory activities against drug resistant Gram-positive

bacteria including M. tuberculosis.^20,21In menaquinone biosyn-

thesis, 2-succinyl-5-enoylpyruvyl-6-hydroxy-3-cyclohexane-1-

carboxylic acid synthase (MenD), MenE (an acyl-CoA

synthase), and 1,4-dihydroxynaphthoyl-CoA synthase (MenB)

have recently been studied for the development of novel drug

lead for Gram-positive pathogens including M. tuberculosis.²²⁻²⁵

In an attempt at ﬁnding a new pharmacophore for MenA

inhibitors, we recently identiﬁed a new quinolone natural

product, aurachin RE (1, Figure 3),²⁶which exhibited MenA

enzyme inhibitory activity as well as a wide antimicrobial

spectrum against Gram-positive bacteria. As there is an overall

structural resemblance between aurachin RE and our ﬁrst

generation MenA inhibitors (e.g., 10 in Figure 4) or

menaquinone, aurachin RE’s antibacterial activity could be

attributed to a synergistic eﬀect of respiratory chain and

menaquinone biosynthesis inhibitory activities. The identiﬁca-

tion of aurachin RE’s MenA enzyme inhibitory activity

encouraged us to redesign and synthesize chiral MenA inhibitor

molecules, in which primary or secondary alcohol was

introduced in the side chain of the ﬁrst-generation MenA

inhibitor molecules.^20,21To date, we have synthesized over 400

molecules with >95% purity either in solution or on polymer-

support, and these molecules were evaluated in an enzymatic

assay in vitro (IC₅₀) against MenA and in bacterial growth

inhibitory assays (MIC). Figure 3 illustrates our assay scheme

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Figure 3. Assay to identify selective MenA inhibitors against M.

tuberculosis.

Figure 4. Rational of selective antimycobacterial MenA inhibitors.

to identify selective MenA inhibitors against M. tuberculosis. In

these molecules, 26 compounds were identiﬁed to exhibit the in

vitro biological activities which met the assay criteria

summarized in Figure 3. On the basis of obtained SAR from

a 400-membered library, it became evident that the topology of

the N atom in the inhibitor molecules plays an important role

in selectivity of the MenA enzymatic and bactericidal activities

(Mtb vs Staphylococcus aureus). As summarized in Figure 4,

selective mycobactericidal molecules (2−6) possess the

secondary or tertiary amine in the near center of the molecules

(highlighted moieties in 1−6 in Figure 4), whereas the

topology of the N atom of the molecules possessing

antibacterial activities against both Mtb and S. aureus (7−10)

locates the right half of the molecules (highlighted moieties in

7−10 in Figure 4). We have identiﬁed selective antimyco-

bacterial MenA inhibitors in their racemic forms. To obtain

insight into the eﬀect of chirality of new MenA inhibitors (2−

6), we commenced syntheses of the optically active forms of

the identiﬁed inhibitors. Herein we report the synthesis and in

vitro biological activity evaluation of optically active molecules

of 2−6 and their derivatives. The results disclosed in this article

identify novel chiral antimycobacterial MenA inhibitors with

signiﬁcant activity in killing nonreplicating Mtb.

RESULTS AND DISCUSSION

■

Selective M. tuberculosis MenA Inhibitors: Assay

Strategy. Antimicrobial spectrum focused against Mtb

(selective antimycobacterial agent) is preferable for TB

chemotherapy.²⁷We realized that the peptide sequences of

the Mtb menA and S. aureus menA gene products are only 32%

identity and 50% similarity in the BLAST experiment.²⁸Indeed,

we have identiﬁed several molecules that exhibit selective MenA

enzyme and bacterial growth inhibitory activities against Mtb,

more than a 10-fold higher inhibitory activity against Mtb than

S. aureus. To develop MenA inhibitors which are selective

against Mtb, molecules generated in this program were ﬁrst

evaluated in MenA enzyme inhibitory assays. Only molecules

exhibited Mtb MenA activity over S. aureus MenA (IC₅₀< 20

μM, >60 μM against Mtb MenA and S. aureus MenA,

respectively) were evaluated in bacterial growth inhibitory

assays (MICs) using Mtb, S. aureus, and Escherichia coli (Figure

3). The molecules exhibited good activity only against Mtb

(MICs for Mtb, S. aureus, and E. coli are <12.5, >60, and >125

μg/mL, respectively) were evaluated in E. coli growth inhibitory

assays under anaerobic conditions followed by menaquinone

supplementation assays (E. coli utilize only menaquinone in

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a

Scheme 1. Syntheses of Optically Active MenA Inhibitors, Class A

a

Reagents and conditions: (a) Ac₂O/Py. (1:1), room temperature; (b) BnBr, NaH, DMF/THF (4:1), 0 °C; (c) TMSNCO, DMAP, CH₂Cl₂, room

temperature.

their electron transport system under anaerobic condi-

tions).^29,30This assay conﬁrmed that the molecules killed

bacteria by targeting MenA enzyme or electron transport

systems (vide infra). Oxygen consumption assay using Mtb

identiﬁes electron transport system inhibitors;^31,32in this assay,

selective MenA inhibitors should not show activity at

concentrations below the MIC against Mtb. Selective

antimycobacterial MenA inhibitors conﬁrmed via E. coli growth

inhibitory (under anaerobic conditions), and oxygen con-

sumption assays were evaluated for their activity against

nonreplicating Mtb via the low oxygen recovery assays

(LORA).³³

Chemistry. As mentioned above, we have identiﬁed several

antimycobacterial MenA inhibitors in their racemic forms. To

obtain insight into the eﬀect of chirality of 2−6 (Figure 4) and

functional groups around the chiral center on biological activity,

we synthesized both enantiomers of 2−6 and their analogues in

which the secondary alcohols were functionalized with acyl,

carbamate, and benzyl groups. Benzophenone functional group

shows a moderate electrophilicity and thus may require

chemical modiﬁcation to improve its physicochemical property

in early stage of drug development. In this study, we have

introduced benzophenone O-methyl oxime derivatives to

obtain preliminary SAR and their cytotoxicity data.

Syntheses of optically active molecules of 2−6 and their

analogues are summarized in Schemes 1−5. The Cl-substituted

benzophenone derivatives, 21 and 23, were synthesized

according to the previously reported procedures.²⁰O-Methyl

oxime derivatives 22 and 24 were prepared by the treatment of

21 or 23 with MeONH₂·HCl in pyridine at 105 °C. The

phenolic alcohol of 21 was subjected to the Mitsunobu reaction

with Boc-protected piperidin-4-ylmethanol 25 to provide the

piperidinyl ethers 26 in over 90% yield after deprotection of the

Boc group.^34,35Optically active (2R)- or (2S)-alkyloxiranes, 28

and 29, were synthesized via Jacobsen’s kinetic resolutions of

the racemic epoxides.³⁶Opening of the epoxides (S)-29 with

the piperidine derivatives 26 was achieved by using a

stoichiometric amount of AlMe₃at room temperature to

yield the amino-alcohols (S)-11. The generated optically active

alcohol (S)-11 was functionalized with Ac₂O, BnBr, and

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a

Scheme 2. Syntheses of Optically Active MenA Inhibitors, Class B

a

Reagents and conditions: (c) TMSNCO, DMAP, CH₂Cl₂, room temperature; (d) 1N NaOH, CH₃CN, 0 °C.

Scheme 3. Syntheses of Optically Active MenA Inhibitors, Class C

TMSNCO to aﬀord the corresponding acetate (S)-38, benzyl

ether (S)-40, and carbamate (S)-12 (class A molecules, Scheme

1). To synthesize optically active piperidinyl-benzyl alcohol

derivatives 3 (class B molecules), (S)- and (R)-benzaldehyde

derivatives 45 were synthesized in four steps from 44 via CBS

reductions (Scheme 2).³⁷The starting material 44 was readily

synthesized via a Grignard reaction of 42 with the Weinreb

amide 43.³⁸Reductive aminations of 26 with (S)-45 was

achieved with NaBH(OAc)₃in the absence of acid to provide

the desired tertiary amine (S)-46 without contamination of the

diphenylmethanol byproduct. Deacetylation of (S)-46 aﬀorded

the secondary alcohol (S)-3, whose alcohol was functionalized

with TMSNCO to aﬀord the corresponding carbamate (S)-14.

Optically active amino-alcohols (S)- and (R)-4 (class C

molecules) were synthesized via the resolution of rac-epoxide

49 followed by Zn(ClO₄)₂-catalyzed selective opening of

epoxide with the primary amine (Scheme 3).³⁹The other

optically active secondary alcohols (class D and E molecules)

were successfully synthesized via the same synthetic procedures

developed for class A and B molecules (Schemes 4 and 5).

Syntheses of the molecules having (R)-conﬁguration were also

achieved by using the antipodes of the building blocks utilized

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a

Scheme 4. Syntheses of Optically Active MenA Inhibitors, Class D

a

Reagents and conditions: (a) Ac₂O/Py (1:1), room temperature; (b) BnBr, NaH, DMF/THF (4:1), 0 °C; (c) TMSNCO, DMAP, CH₂Cl₂, room

temperature.

a

Scheme 5. Syntheses of Optically Active MenA Inhibitors, Class E

a

Reagents and conditions: (c) TMSNCO, DMAP, CH₂Cl₂, room temperature; (d) 1N NaOH, CH₃CN, 0 °C.

for the synthesis for the (S)-conﬁguration molecules. Similarly,

syntheses of optically active benzophenone O-methyl oxime

derivatives 13, 15, 18, 19, 59, 63, and 65 were successfully

achieved. Thus, we have synthesized both (S)- and (R)- forms

of 2−6 and their derivatives in short number of steps. Optical

purity and purity of each molecule were established via HPLC

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Table 1. Antibacterial Activities against M. tuberculosis and S. aureus, and MenA Enzyme Inhibitory Activities of the Selected

Molecules

a

b

c

d

e

The agar plate dilution method was used. MABA: microplate alamar blue assay. LORA: Low-oxygen recovery assay. RFP: rifampicin. INH:

f

isoniazid. EMB: ethanbutol.

analyses of Mosher esters of secondary alcohols or alcohols

using a chiral column.⁴⁰

MenA Enzyme Inhibitory Assays. MenA enzymatic

assays of generated molecules were originally performed with

[³H]rated farnesyl diphosphate and MenA containing mem-

brane fraction.⁴¹This assay requires a signiﬁcant eﬀort to

separate demethylmenaquinone (DMMK) from the reaction

mixtures and includes the signiﬁcant cost for [³H]farnesyl

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diphosphate. To simplify the procedure and to reduce the cost

of MenA enzyme inhibitory assays, we have recently developed

a MenA assay using HPLC. In new MenA assay, the MenA

product, demethylmenaquinone, can readily be quantitated via

UV absorbance (DMMK: λ₂325 nm).⁴²

the molecules in class A, the molecules possessing S-

conﬁguration (in class D) exhibited better antimycobacterial

activity than the corresponding R-conﬁguration molecules. The

eﬀect of carbamate group on enzyme and bacterial growth

inhibitory activities was not observed in the molecules in class

D; the MABA MIC value of the carbamate (S)-16 was equal to

the corresponding alcohol (S)-17. Nonetheless, the MABA

MIC for the rac-alcohol 5 could be improved 2-fold by the

formation of carbamate; the MABA MICs of rac-5 and rac-16

were 6.25 and 3.25 μg/mL, respectively. Antimycobacterial

activity was improved by increasing the hydrophobicity of the

side chain (C8 vs C6) in the molecules of class A, whereas

noticeable bactericidal eﬀect by increasing hydrophobicity of

the side chain was not observed in the molecules of class D. All

molecules summarized in Table 1 did not exhibit bactericidal

activity against S. aureus at 60 μg/mL concentrations; lack of

antistaphylococcal activity of these molecules was well-

correlated with their S. aureus MenA enzyme inhibitory activity

(IC₅₀>60 μM against S. aureus MenA).⁴⁵

E. coli Growth Inhibitory Assays under Anaerobic

Conditions. M. tuberculosis or S. aureus treated with the MenA

inhibitors could not be rescued completely even at higher

concentrations of exogenous menaquinone (VK₂). In contrast,

growth inhibition of E. coli by the MenA inhibitor could be

rescued by supplementation of VK₂(50 μM) under “anaerobic

conditions” (vide supra).⁴⁶E. coli growth inhibition rescued by

addition of VK₂may be attributed to the degree of permeability

of VK₂through the cell envelop. Although lack of activity of

MenA inhibitors against Gram-negative bacteria grown under

aerobic conditions have been demonstrated, all MenA

inhibitors identiﬁed in this program showed growth inhibition

of E. coli at 5−10 μg/mL concentrations under anaerobic

conditions, and the inhibitory eﬀect of MenA inhibitor was

rescued by supplementation of VK₂. Therefore, these

convenient cell-based assays using E. coli under anaerobic

conditions can be utilized to conﬁrm that the inhibitor

molecules kill Gram-positive bacteria including Mtb by

targeting MenA biosynthesis or bacterial electron transport

systems.

Oxygen Consumption Assays. The basic concept under-

lying bacterial oxygen consumption assay is that changes in the

rate of oxygen uptake result in a change in the oxygen

concentrations. The oxygen consumption by bacterial concen-

trations greater than 10⁸cfu/mL bacteria is proportional to the

concentration of bacteria. Eﬀect of the inhibitor molecule on

electron transport by the quantitation of decolorization of

methylene blue, which is a well-known redox dye, has been

unambiguously demonstrated.³⁰Oxygen-consumption assays of

(R)-12 and (S)-17 showed decolorization of methylene blue at

concentrations (12.5 μg/mL) above the MIC of each molecule

(MICs 2.31 and 1.50 μg/mL, respectively) against Mtb. Thus,

we have concluded that (R)-12 and (R)-17 are very weak (or

are not) electron transport system inhibitors, and thus

antimycobacterial activity of these molecules are attributed by

targeting menaquinone biosynthesis.

Cytotoxicity of MenA Inhibitors. To obtain insight into

potential toxicity of identiﬁed inhibitor molecules, all

antimycobacterial MenA inhibitors were evaluated in in vitro

cytotoxicity assays using Vero monkey kidney cells and HepG2

human hepatoblastoma cells.⁴⁷Most MenA inhibitors possess-

ing the benzophenone group showed IC₅₀of 1−6.5 μg/mL

against two mammalian cell lines; selectivity indexes (IC₅₀in

mammalian cells/MIC against Mtb) of a series of benzophe-

We performed a preliminary screening of the activity of

compounds synthesized in Schemes 1−5 at the single

concentrations of 100 μM. In these MenA enzyme inhibitory

assays, the optically active molecules were assayed against M.

tuberculosis and S. aureus MenA. IC₅₀values of the molecules

which exhibited activity against Mtb MenA and inactivity

against S. aureus MenA were calculated. Of 86 optically active

molecules synthesized in Schemes 1−5, 26 molecules exhibited

Mtb MenA inhibitory activity and were inactive against S.

aureus MenA at 100 μM concentrations. Dose−response plots

(DMMK formation vs concentrations of inhibitor) were

obtained for 26 molecules to determine IC₅₀values. Signiﬁcant

eﬀect of chirality in the MenA enzyme inhibitory activity was

observed in the molecules in classes A and D. On the contrary,

an obvious eﬀect of chirality in MenA enzyme inhibitory

activity of the molecules in classes B, C, and E was not

observed; the racemic forms and each enantiomer did not show

noticeable diﬀerence in MenA enzyme inhibitory activity. It is

noteworthy that the carbamate analogue (R)-12 group showed

6-fold better MenA enzyme inhibitory activity compared to the

alcohol from (R)-11. Similar improvement of enzymatic

inhibition by modiﬁcation of the alcohol with the carbamate

group is observed in the molecules in class D (Table 1).

Bacterial Growth Inhibitory Assays. All molecules that

showed activity against MenA in a preliminary assay at 100 μM

concentrations were evaluated for their mycobacterial growth

inhibitory activity via the microplate alamar blue assay (MABA)

and low oxygen recovery assay (LORA).^33,43,44Brieﬂy, the

MABA assay is a colorimetric assay that uses the color change

of rezasurin to evaluate M. tuberculosis (Mtb) growth inhibitory

activity under aerobic conditions. On the other hand, the

LORA assay evaluates potency against nonreplicating Mtb cells

under low oxygen conditions using a luminescent stain.

Signiﬁcantly, in all cases the MIC values obtained from the

LORA assays are lower than those from the MABA assays. To

the best of our knowledge, it is the ﬁrst observations that the

molecules killed nonreplicating Mtb at concentration below the

MIC against Mtb grown under aerobic conditions; in all cases

the values of MIC_LORA/MIC_MABAwere <1 (Table 1). In the

class A molecules, (R)-2 was 2-fold more potent than rac-2 and

(S)-2 in the MABA assays. Similar to an observed trend in

MenA enzyme inhibitory assays, the carbamate (R)-12 could

improve over 5-fold increase in mycobactericidal activity

compared to rac-2. (R)-12 exhibited a signiﬁcant activity in

the LORA assay; the MIC value of (R)-12 is 1.7-fold more

eﬀective in killing nonreplicating bacteria than rifampicin

(MIC_LORA1.47 μg/mL). It is worth mentioning that (R)-12

was the most active in killing nonreplicating Mtb in vitro

among antimycobacterial drugs (approved by FDA) and

preclinical drugs tested in our laboratory. Regardless of the

stereochemistry of the chiral centers, the molecules in class B,

C, and E did not show noticeable diﬀerence in antimycobacte-

rial activity in the both MABA and LORA assays. The

molecules in class D are regioisomers at the benzophenone

moiety of the class A molecules. Similar to the molecules in

class A, pronounced eﬀect of the stereochemistry of secondary

alcohol on antimycobacterial activity was observed in the

molecules in class D. Contrary to the chirality eﬀect observed in

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none MenA inhibitors identiﬁed in this program were less than

2.⁴⁸On the other hand, O-methyl oxime derivatives in class A

showed approximately 10-fold less cytotoxicity than the

corresponding benzophenone derivatives in in vitro cytotoxicity

assays (Figure 5); (R)-13 showed an encouraging in vitro

relatively low concentrations (3.0−6.5 μg/mL). Thus, we

concluded that new MenA inhibitors identiﬁed in this program

kills Mycobacterium species selectively and are especially

eﬀective in killing Mtb at low concentrations. MenA inhibitors

(R)-12 and (R)-13 eﬀectively inhibited growth of drug-resistant

Mycobacterium species, indicating that MenA is a valid drug

target to develop new drugs for MDR-Mycobacterium tuber-

culosis.

CONCLUSIONS

■

Through asymmetric synthesis of a series of optically active

molecules followed by screening these molecules by the assay

methods described here, we have identiﬁed new MenA (1,4-

dihydroxy-2-naphthoate prenyltransferase) biosynthesis inhib-

itors that showed very weak (or no) inhibitory activities of

bacterial electron transport systems. A series of MenA

inhibitors identiﬁed in this program exhibited antimicrobial

spectrum focused against Mtb. Selective activity against Mtb is

ideal in TB drug discovery due to the fact that TB

chemotherapy requires long regimen, so that broad-spectrum

anti-TB agents may cause resistant to other bacteria during TB

chemotherapy. The carbamates (R)-12 and (R)-13 in class A

(Scheme 1) showed signiﬁcant growth inhibitory activities

against nonreplicating Mtb (MIC_LORA, 0.85 μg/mL) with the

MIC_LORA/MIC_MABAvalue of 0.37 (MIC_LORA/MIC_MABA= 7.35

for rifampin). Eﬀectiveness in nonreplicating Mtb was also

conﬁrmed via assays using a modiﬁed Wayne model.⁴⁹⁻⁵¹

Among antimycobacterial agents tested in our laboratories, the

inhibitor (R)-12 and (R)-13 were the most active in killing

nonreplicating Mtb in vitro. MenA inhibitors identiﬁed in this

program strongly suggested that menaquinone biosynthesis is

important in maintaining mycobacterial viability under

conditions of restricted oxygen.⁵²MenA inhibitors seem to

be able to block the electron ﬂow, consequently inhibiting the

bacterial growth. It is conceptually very interesting that MenA

inhibitors can be developed as indirect ATP synthesis

inhibitors.¹⁷The assay data for the identiﬁed MenA inhibitors

indicate that menaquinone biosynthesis is a unique and new

antimycobacterial target. In addition, in tuberculosis, the key to

shortening the long regimen lies in targeting the nonreplicating

persistence subpopulation (vide supra). Thus, the discovery of

molecules that kill nonreplicating Mtb at lower concentrations

than MIC against Mtb under aerobic conditions is expected to

be of signiﬁcance in terms of discovering new lead molecules

that can be developed into new drugs to kill Mtb in any state.

Moreover, over the last several years, we are unable to isolate

resistant bacteria to the MenA inhibitors in the mutation

frequency studies. Therefore, we concluded that unlike the

other known bacterial molecular targets menA shows extremely

low mutation frequency.

Figure 5. In vitro assay data for (R)-12 and (R)-13.

activity/toxicity ratio (a SI value >10). It is believed that

electrophilicity of the benzopheneone moiety of (R)-12 can be

diminished by O-methyl oxime formation of the benzophe-

neone. Thus, the benzophenone O-methyl oxime may not be a

good electron acceptor that interferes with redox systems of

mammalian cells.

Antibacterial Activities of (R)-12 and (R)-13 against

Drug-Resistant M. tuberculosis and other Mycobacte-

rium Species. Two MenA inhibitors molecules (R)-12 and

(R)-13 showed MIC values of 2.31 μg/mL against Mtb

(H37Rv, a common laboratory strain). We resynthesized (R)-

12 and (R)-13 and determined MICs against several other

Mycobacterium species and drug-resistant Mtb. As summarized

in Table 2, (R)-12 and (R)-13 showed mycobactericidal activity

against rifampicin- and isoniazid-resistant strains at slightly

lower concentrations (entries 2 and 3). (R)-12 and (R)-13 also

killed other Mycobacterium species such as Mycobacterium. bovis,

Mycobacterium intracellulare, and Mycobacterium smegmatis at

Table 2. MICs of (R)-12, (R)-13, and Representative

Antimycobacterial Agents (Clinically Used) for

Mycobacterium Species Including Drug-Resistant Strains

In conclusion, robustness of our in vitro assay approaches to

identify novel and selective MenA inhibitors against M.

tuberculosis (summarized in Figure 3) has been demonstrated

a

MIC (μg/mL)

d

e

entry

species and strain

(R)-12 (R)-13 RFP

INH

by the identiﬁcation of a lead molecule (R)-13 (MIC_LORA

/

1

2

3

4

6

7

M. tuberculosis H37Rv

2.31

2.91

3.00

6.50

2.31

1.52

1.51

2.95

6.50

0.07

0.03

>8

MIC_MABAvalue of 0.37, SI >10). Further studies are underway

to thoroughly characterize the activity of (R)-13 against

multidrug resistant strains of Mtb and to investigate activities

of (R)-13 and its related molecules against the other

menaquinone biosynthesis enzymes.⁵³The MenA inhibitors

described here can be synthesized in short steps with high yield,

and structural modiﬁcations to improve in vitro eﬃcacy will be

achieved by modifying the hydrophobic side chain and

benzophenone O-methyl oxime moieties of (R)-13. Thorough

b

M. tuberculosis H37Rv INHr

0.03

>4

c

M. tuberculosis H37Rv RFPr

M. bovis BCG

0.05

M. intracellulare ATCC15984

M. smegmatis

a

b

The agar plate dilution method was used. INH-resistant M.

tuberculosis. RFP-resistant M. tuberculosis. RFP: rifampicin. INH:

isoniazid.

c

d

e

3747

dx.doi.org/10.1021/jm201608g | J. Med. Chem. 2012, 55, 3739−3755

Journal of Medicinal Chemistry

Article

TPP (3.42 g, 13.05 mmol) in THF (40 mL) was added DIAD (4.79 g,

13.05 mmol). After 3 h, all volatiles were evaporated in vacuo.

Puriﬁcation by silica gel chromatography (4:1, hexanes:EtOAc)

provided tert-butyl 4-((3-(4-chlorobenzoyl)phenoxy)methyl)-

in vitro biological evaluation of (R)-13 and its analogues and in

vivo evaluation of promising menaquinone biosynthesis

inhibitors will be reported elsewhere.⁵³

1

piperidine-1-carboxylate (3.61 g, 97%) as a colorless oil. H NMR

EXPERIMENTAL SECTION

(500 MHz, CDCl₃): δ 1.64−1.25 (m, 2H), 1.39 (s, 9H), 1.75 (d, J =

13.5 Hz, 2H), 1.87−1.90 (m, 1H), 2.67 (bs, 2H), 3.77 (d, J = 6.5 Hz,

2H), 4.08 (bs, 2H), 7.04 (dd, J = 2, 8 Hz, 1H), 7.20−7.22 (m, 2H),

7.29 (t, J = 8 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.66−7.68 (m, 2H).

¹³C NMR (125 MHz, CDCl₃): δ 28.6, 29.0, 36.4, 72.7, 79.6, 115.0,

■

Chemistry. General Information. All glassware were oven-

dried, assembled hot, and cooled under a stream of nitrogen

before use. Reactions with air sensitive materials were carried

out by standard syringe techniques. Commercially available

reagents were used as received without further puriﬁcation.

Thin layer chromatography was performed using 0.25 mm silica

gel 60 (F254, Merck) plates visualizing at 254 nm or developed

with ceric ammonium molybdate or anisaldehyde solutions by

heating with a hot-air gun. Speciﬁed products were puriﬁed by

ﬂash column chromatography using silica gel 60 (230−400

mesh, Merck). IR absorptions on NaCl plates were run on a

Perkin-Elmer FT-IR 1600. ¹H NMR spectral data were

obtained using Varian 300, 400, and 500 MHz instruments.

The residual solvent signal was utilized as an internal reference.

¹³C NMR spectral data were obtained using a Varian 100, 125

MHz spectrometer. Chemical shifts were reported in parts per

million (ppm) downﬁeld from TMS, using the middle

resonance of CDCl₃(77.0 ppm) as an internal standard. For

all NMR spectra, δ values are given in ppm and J values in Hz.

Mass spectra were obtained at University of Tennessee Central

Instrument Facility. HPLC analyses were performed with a

Shimadzu LC-20AD HPLC system. All compounds were

puriﬁed by PTLC or reverse HPLC to be ≥95% purity

whose purities were established by HPLC.

119.6, 122.8, 128.8, 129.5, 131.6, 136.0, 138.7, 139.1, 155.0, 159.2,

195.4. LRMS (ESI) m/z: 430.1 (M + H)⁺.

(4-Chlorophenyl)(3-(piperidin-4-ylmethoxy)phenyl)methanone

(26). tert-Butyl 4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidine-1-

carboxylate (3.61 g, 8.50 mmol) was dissolved in 50% triﬂuoroacetic

acid (TFA) in CH₂Cl₂(35 mL) and stirred for 2 h at rt. All volatiles

were evaporated in vacuo. The reaction mixture was dissolved in

CH₂Cl₂and washed with 1N NaOH (twice). The combined organic

phase was dried over Na₂SO₄and evaporated in vacuo. Puriﬁcation by

silica gel chromatography (4:1, CHCl₃:MeOH) aﬀorded 26 (2.66 g,

95%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): δ 1.24−1.32 (m,

2H), 1.79 (d, J = 13 Hz, 2H), 1.86−2.10 (m, 1H), 2.79−2.64 (m, 2H),

3.11 (d, J = 12.5 Hz, 2H), 3.78 (d, J = 6.5 Hz, 2H), 7.05 (dd, J = 2, 8

Hz, 1H), 7.20−7.23 (m, 2H), 7.30 (t, J = 8 Hz, 1H), 7.39 (d, J = 9 Hz,

2H), 7.69 (d, J = 8.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃): δ 29.2,

29.9, 31.1, 36.3, 46.2, 73.2, 115.1, 119.7, 122.8, 128.8, 129.5, 131.6,

136.1, 138.7, 139.1, 159.4, 195.5. LRMS (ESI) m/z: 330.1 (M + H)⁺.

(S)-(4-Chlorophenyl)(3-((1-(2-hydroxyoctyl)piperidin-4-yl)-

methoxy)phenyl)methanone ((S)-11). To a stirred solution of 26

(100.0 mg, 0.31 mmol) in CH₂Cl₂was added Al(CH₃)₃(0.2 M in

CH₂Cl₂, 0.43 mmol) at 0 °C. After 15 min, (S)-1,2-epoxyoctane (29,

60.0 mg, 0.43 mmol) was added. After 4 h at rt, the reaction mixture

was quenched with aq NaHCO₃and extracted with CH₂Cl₂. The

combined organic phase was dried over Na₂SO₄and evaporated in

vacuo. Puriﬁcation by silica gel chromatography (3:2, CHCl₃:MeOH)

aﬀorded (S)-2 (103.0 mg, 73%) as a colorless oil. [α]²⁰_D= +13.9 (c 1.0

(4-Chlorophenyl)(3-methoxyphenyl)methanone. 4-Chlorobenzo-

yl chloride (5.00 g, 28.58 mmol) was dissolved in CH₂Cl₂(240 mL)

and cooled to 0 °C. N,O-Dimethylhydroxyl amine (3.07 g, 31.43

mmol) and triethylamine (6.36 g, 62.85 mmol) were added into the

reaction mixture. After 4 h at rt, the reaction mixture was quenched

with water and the organic phase was washed with 1N HCl. The

combined organic phase was dried over Na₂SO₄and evaporated in

vacuo to aﬀord crude 4-chloro-N-methoxy-N-methylbenzamide (6.41

g). This was used for further puriﬁcation. To a stirred solution of 1-

iodo-3-methoxybenzene (21.48 g, 91.80 mmol) in THF (10 mL) was

added isopropyl magnesium chloride (2M, 1.50 mmol) at −20 °C.

After 1 h at the same temperature, the reaction mixture was cooled to

−78 °C and 4-chloro-N-methoxy-N-methylbenzamide (100 mg, 0.50

mmol) in THF (1 mL). The reaction mixture was warmed to 0 °C

over 30 min. After 3 h at 0 °C, the reaction mixture was quenched with

aq satd NH₄Cl. The water phase was extracted with EtOAc, and the

combined organic phase was dried over Na₂SO₄and evaporated in

vacuo. Puriﬁcation by silica gel chromatography (4:1, hexanes:EtOAc)

aﬀorded (4-chlorophenyl)(3-methoxyphenyl)methanone (97.0 mg,

1

in CHCl₃). H NMR (300 MHz, CDCl₃): δ 0.90 (s, 3H), 1.31−1.49

(m, 11H), 1.84−1.99 (m, 4H), 2.28−2.39 (m, 3H), 2.87 (d, J = 10.2

Hz, 1H), 3.10 (d, J = 10.2 Hz, 1H), 3.69 (s, 1H), 3087 (d, J = 3.9 Hz,

2H), 7.14 (d, J = 7.2 Hz, 1H), 7.29−7.49 (m, 5H), 7.77 (dd, J = 2.1,

8.4 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 13.5, 22.0, 25.0, 28.5,

28.8, 28.9, 31.2, 34.5, 35.3, 51.3, 54.7, 64.0, 65.8, 72.3, 114.4, 118.8,

122.0, 128.1, 128.8, 130.8, 135.4, 138.1, 138.3, 158.7, 194.6. HRMS

(ESI⁺): m/z calcd for C₂₇H₃₆ClNO₃(M + H)⁺, 458.2405; found,

458.2403. The purity of (S)-11 was determined to be >99% by reverse

HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60

mm; CH₃CN/0.05%TFA in H₂O = 25/1). The optical purity of (S)-

11 was determined to be >99% by HPLC analyses (Daicel Chiralcel

OD-H (0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0

mL/min and a UV detector at 245 nm; (S)-enantiomer, t_R= 21.0 min;

(R)-enantiomer, t_R= 26.7 min).

1

79%) as a white powder. H NMR (500 MHz, CDCl₃): δ 3.88 (s,

3H), 7.16 (dd, J = 1.5, 8 Hz, 1H), 7.31−7.35 (m, 2H), 7.40 (t, J = 8

Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H). ¹³C NMR

(125 MHz, CDCl₃): δ 55.6, 114.4, 119.2, 122.8, 128.8, 129.5, 131.6,

136.1, 138.7, 139.1, 159.8, 195.4. LRMS (ESI) m/z: 247.0 (M + H)⁺.

(4-Chlorophenyl)(3-hydroxyphenyl)methanone (21). To a stirred

solution of (4-chlorophenyl)(3-methoxyphenyl)methanone (5.30 g,

21.5 mmol) in AcOH (15 mL) was added HBr (48% in water, 200

mL). The reaction mixture was gently reﬂuxed at 125 °C for 36 h. The

reaction mixture was cooled to rt, and all volatiles were distilled oﬀ.

Puriﬁcation by silica gel chromatography (4:1, hexanes:EtOAc)

aﬀorded 21 (3.84 g, 78%) as a white solid, and the unreacted starting

(R)-(4-Chlorophenyl)(3-((1-(2-hydroxyoctyl)piperidin-4-yl)-

methoxy)phenyl)methanone ((R)-11). Colorless oil. [α]²⁰= −12.8

D

1

(c 0.5 in CHCl₃). H NMR (500 MHz, CDCl₃): δ 0.81 (t, J = 7 Hz,

3H), 1.18−1.42 (m, 12H), 1.76−1.78 (m, 3H), 1.90 (t, J = 11 Hz,

1H), 2.17−2.28 (m, 3H), 2.77 (d, J = 11 Hz, 1H), 3.00 (d, J = 11.5 Hz,

1H), 3.58−3.62 (m, 1H), 3.78 (d, J = 6 Hz, 2H), 7.05 (dd, J = 2, 8 Hz,

1H), 7.19−7.23 (m, 2H), 7.30 (t, J = 8 Hz, 1H), 7.39 (d, J = 8.5 Hz,

2H), 7.68 (d, J = 8.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃): δ14.3,

14.3, 22.8, 22.9, 25.9, 29.3, 29.6, 29.7, 32.1, 32.1, 34.5, 35.3, 36.1, 52.0,

55.5, 64.7, 66.5, 73.0, 110.2, 115.1, 119.7, 122.8, 128.9, 129.6, 131.7,

136.2, 138.8, 139.1, 159.4, 195.5. HRMS (ESI⁺): m/z calcd for

C₂₇H₃₆ClNO₃(M + H)⁺, 458.2404; found, 458.2403. The purity of

(R)-11 was determined to be >99% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm; CH₃CN/

0.05%TFA in H₂O = 25/1). The optical purity of (R)-11 was

determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 21.0 min; (R)-

enantiomer, t_R= 26.7 min).

1

material (15%) was recovered. H NMR (500 MHz, CDCl₃): δ 5.84

(s, 1H), 7.02−7.04 (m, 1H), 7.19−7.21 (m, 1H), 7.25−7.29 (m, 2H),

7.37−7.39 (m, 2H), 7.67−7.69 (m, 2H). ¹³C NMR (125 MHz,

CDCl₃): δ 116.6, 120.4, 122.9, 128.9, 129.9, 131.8, 135.8, 138.7, 139.4,

156.2, 195.9; LRMS (ESI) m/z: 233.0 (M + H)⁺.

tert-Butyl 4-((3-(4-Chlorobenzoyl)phenoxy)methyl)piperidine-1-

carboxylate. To a stirred solution of (4-chlorophenyl)(3-

hydroxyphenyl)methanone (21, 2.00 g, 8.71 mmol), tert-butyl 4-

(hydroxymethyl)piperidine-1-carboxylate (25, 3.75 g, 17.4 mmol), and

3748

dx.doi.org/10.1021/jm201608g | J. Med. Chem. 2012, 55, 3739−3755

Journal of Medicinal Chemistry

Article

(S)-1-(4-((3-(4-Chlorobenzoyl)phenoxy)methyl)piperidin-1-yl)-

octan-2-yl carbamate ((S)-12). To a stirred solution of (S)-11 (10

mg, 0.02 mmol) and DMAP (5.0 mg, 0.04 mmol) in CH₂Cl₂was

added trimethylsilylisocyanate (TMSNCO) (7.0 mg, 0.06 mmol).

After 4 h at rt, the reaction mixture was quenched with aq NaHCO₃,

and extracted with CH₂Cl₂. The combined organic phase was dried

over Na₂SO₄and evaporated in vacuo. Puriﬁcation by silica gel

NMR (125 MHz, CDCl₃): δ −5.2, 14.1, 18.4, 22.6, 25.8, 26.0, 29.2,

29.5, 31.8, 39.1, 65.0, 74.8, 123.6, 124.5, 125.3, 128.3, 141.7, 144.9.

LRMS (ESI) m/z: 338.2 (M + H)⁺.

(S)-1-(3-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)octan-1-ol.

1

Colorless oil. [α]²⁰= −34.8 (c 1 in CHCl₃). H NMR (500 MHz,

D

CDCl₃): δ 0.10 (s, 6H), 0.87 (t, J = 6.5 Hz, 3H), 0.94 (s, 9H), 1.25−

1.31 (m, 9H), 1.37−1.42 (m, 1H), 1.67−1.83 (m, 3H), 4.66 (t, J = 6.5

Hz, 1H), 4.74 (s, 3H), 7.21−7.26 (m, 2H), 7.29−7.32 (m, 2H). ¹³C

NMR (125 MHz, CDCl₃): δ −5.2, 14.1, 18.4, 22.6, 25.8, 26.0, 29.2,

29.5, 31.8, 39.1, 65.0, 74.8, 123.6, 124.5, 125.3, 128.3, 141.7, 144.9.

LRMS (ESI) m/z: 338.2 (M + H)⁺.

chromatography (9:1, CHCl₃:MeOH) aﬀorded (S)-12 (7.0 mg, 63%)

1

as a colorless oil. [α]²⁰= +9.4° (c 0.9 in CHCl₃). H NMR (300

D

MHz, CDCl₃): δ 0.89 (d, J = 7.2 Hz, 3H), 1.31−1.58 (m, 12H), 1.90

(d, J = 10.8 Hz, 3H), 2.09 (m, 1H), 2.37−2.44 (m, 3H), 2.96 (m, 1H),

3.15 (m, 1H)3.76−3.89 (m, 3H), 7.13−7.16 (m, 1H), 7.28−7.50 (m,

5H), 7.78(dd, J = 1.8, 8.4 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃): δ

13.5, 22.0, 25.0, 28.1, 28.4, 28.9, 29.1, 31.2, 34.5, 35.1, 51.5, 54.7, 64.1,

65.7, 72.1, 114.5, 118.8, 122.1, 128.1, 128.8, 130.8, 135.4, 138.1, 138.4,

158.6, 194.6. MS (ESI⁺): m/z calcd for C₂₈H₃₇ClN₂O₄(M + H)⁺,

501.2400; found, 501.2309. The purity of (S)-12 was determined to be

>99% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18

100A, 100 mm × 4.60 mm; CH₃CN/0.05%TFA in H₂O = 25/1). The

optical purity of (S)-12 was determined to be >99% by HPLC analyses

(Daicel Chiralcel OD-H (0.46 cm × 25 cm; hexanes/tBuOH = 20/1

with ﬂow rate = 1.0 mL/min and a UV detector at 245 nm; (S)-

enantiomer, t_R= 18.0 min; (R)-enantiomer, t_R= 20.0 min).

(R)-1-(3-(Hydroxymethyl)phenyl)octyl Acetate. (R)-1-(3-(((tert-

Butyldimethylsilyl)oxy)methyl)phenyl)octan-1-ol (70 mg, 0.2 mmol)

was dissolved in pyridine (1 mL) and acetic anhydride (1 mL). After 5

h at rt, all volatiles were evaporated in vacuo. Puriﬁcation by silica gel

chromatography (9.5:0.5, hexanes:EtOAc) aﬀorded (R)-1-(3-(((tert-

butyldimethylsilyl)oxy)methyl)phenyl)octyl acetate (78.4 mg, 100%)

as a colorless oil. To a stirred solution of (R)-1-(3-(((tert-

butyldimethylsilyl)oxy)methyl)phenyl)octyl acetate (25 mg, 0.06

mmol) in THF (0.5 mL) was added TBAF (1 M in THF, 0.12

mmol). After 4 h at rt, the reaction was quenched with water. The

water phase was extracted with EtOAc, and the combined extract was

washed with brine, dried over Na₂SO₄, and concentrated in vacuo.

Puriﬁcation by silica gel chromatography (7:3, hexane:EtOAc)

(R)-1-(4-((3-(4-Chlorobenzoyl)phenoxy)methyl)piperidin-1-yl)-

octan-2-yl carbamate ((R)-12). A colorless oil. [α]²⁰= +-8.6 (c 0.8

D

aﬀorded (R)-1-(3-(hydroxymethyl)phenyl)octyl acetate (14.0 mg,

in CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 0.88−0.97 (m, 3H),

1.28−1.49 (m, 13H), 1.93 (d, J = 10.8 Hz, 3H), 2.21 (s, 2H), 2.49 (s,

2H), 3.10 (s, 1H), 3.27 (s, 1H), 3.90 (d, J = 5.4 Hz, 2H), 7.13−7.15

(m, 1H), 7.25−7.50 (m, 5H), 7.76−7.79 (m, 2H). ¹³C NMR (75

MHz, CDCl₃): δ 13.5, 22.0, 23.3, 24.9, 27.7, 28.8, 29.1, 31.2, 34.5,

34.9, 51.7, 54.7, 64.1, 65.6, 71.9, 114.5, 118.8, 122.1, 128.1, 128.8,

130.8, 138.2, 138.4, 158.5, 194.7. HRMS (ESI⁺): m/z calcd for

C₂₈H₃₇ClN₂O₄(M + H)⁺, 501.2400; found, 501.2309. The purity of

(R)-12 was determined to be >99% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm; CH₃CN/

0.05%TFA in H₂O = 25/1). The optical purity of (R)-12 was

determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 18.0 min; (R)-

enantiomer, t_R= 20.0 min).

1

79%) as a colorless oil. [α]²⁰= +38.0 (c 0.7 in CHCl₃). H NMR

D

(500 MHz, CDCl₃): δ 0.87 (t, J = 7 Hz, 3H), 1.20−1.34 (m, 10H),

1.73−1.79 (m, 1H), 1.86−1.93 (m, 1H), 2.06 (s, 3H), 4.70 (s, 2H),

5.71 (t, J = 7 Hz, 1H), 7.24−7.29 (m, 2H), 7.32−7.35 (m, 2H). ¹³C

NMR (125 MHz, CDCl₃): δ 14.1, 21.3, 22.6, 25.6, 29.1, 29.3, 31.8,

36.3, 65.3, 76.2, 125.1, 125.8, 126.4, 128.7, 141.1, 141.3, 170.5. LRMS

(ESI) m/z: 279.2 (M + H)⁺.

(S)-1-(3-(Hydroxymethyl)phenyl)octyl Acetate. Colorless oil.

1

[α]²⁰= −35.0 (c 0.8 in CHCl₃). H NMR (500 MHz, CDCl₃): δ

D

0.87 (t, J = 7 Hz, 3H), 1.20−1.34 (m, 10H), 1.73−1.79 (m, 1H),

1.86−1.93 (m, 1H), 2.06 (s, 3H), 4.70 (s, 2H), 5.71 (t, J = 7 Hz, 1H),

7.24−7.29 (m, 2H), 7.32−7.35 (m, 2H). ¹³C NMR (125 MHz,

CDCl₃): δ 14.1, 21.3, 22.6, 25.6, 29.1, 29.3, 31.8, 36.3, 65.3, 76.2,

125.1, 125.8, 126.4, 128.7, 141.1, 141.3, 170.5. LRMS (ESI) m/z:

279.2 (M + H)⁺.

1-(3-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)octan-1-one

(44). To a stirred solution of tert-butyl-(3-iodo-benzyloxy)-dimethylsi-

lane (42, 0.70 g, 2.0 mmol) in THF (3 mL) was added

isopropylmagnesium chloride (2 M in THF, 3.0 mmol) at 0 °C.

After 2.5 h, the reaction mixture was cooled to −78 °C and N-

methoxy-N-methyloctanamide (43, 0.12 g, 0.66 mmol) in THF (1

mL) was added. After 3 h at 0 °C, the reaction mixture was quenched

with aq NH₄Cl. The water phase was extracted with CH₂Cl₂. The

combined organic phase was dried over Na₂SO₄and evaporated in

vacuo. Puriﬁcation by silica gel chromatography (9.8:0.2, hexanes:E-

(R)-1-(3-((4-((3-(4-Chlorobenzoyl)phenoxy)methyl)piperidin-1-yl)-

methyl)phenyl)octyl Acetate ((R)-46). To a stirred solution of DMSO

(113.0 mg, 1.44 mmol) in CH₂Cl₂(2 mL) at −78 °C was added oxalyl

chloride (91.0 mg, 0.72 mmol). After 30 min, (R)-1-(3-

(hydroxymethyl)phenyl)octyl acetate (100.0 mg, 0.36 mmol) in

CH₂Cl₂(0.5 mL) was added. After 30 min at −78 °C, Et₃N (220.0

mg, 2.16 mmol) was added and the reaction mixture was warmed to rt

over 1 h. The reaction mixture was quenched with water, and the

water phase was extracted with CH₂Cl₂. The combined extract was

washed with brine, dried over Na₂S₂O₄, and evaporated in vacuo.

Puriﬁcation by silica gel chromatography aﬀorded the corresponding

aldehyde (R)-1-(3-formylphenyl)octyl acetate ((R)-45, 95.0 mg) as a

colorless oil. To a stirred solution of (R)-45 (95.0 mg, 0.34 mmol) and

(4-chlorophenyl)(3-(piperidin-4-ylmethoxy)phenyl)methanone (26,

98.7 mg, 0.30 mmol) in CH₂Cl₂was added NaBH(OAc)₃(150.0

mg, 0.72 mmol). After 8 h at rt, the reaction was quenched with aq

satd NaHCO₃, and the water phase was extracted with CH₂Cl₂. The

combined extract was washed with brine, dried over Na₂S₂O₄, and

evaporated in vacuo. Puriﬁcation by silica gel chromatography (3:7,

hexanes:EtOAc) aﬀorded (R)-46; (170.0 mg, 96%) as a colorless oil.

[α]²⁰_D= −17.9 (c 0.8 in CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 0.86

(t, J = 7 Hz, 3H), 1.23−1.29 (m, 10H), 1.43−1.45 (m, 2H), 1.73−1.91

(m, 5H), 1.99−2.04 (m, 2H), 2.07 (s, 3H), 2.93 (d, J = 10 Hz, 2H),

3.53 (d, J = 1.5 Hz, 2H), 3.85 (d, J = 6 Hz, 2H), 5.72 (t, J = 7 Hz, 1H),

7.12 (dd, J = 1.5, 7.5 Hz, 1H), 7.21−7.30 (m, 6H), 7.36 (t, J = 8 Hz,

1H), 7.45 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H). ¹³C NMR (125

MHz, CDCl₃): δ 14.1, 21.4, 22.6, 25.6, 29.0, 31.8, 35.9, 36.4, 53.3,

53.3, 63.2, 72.9, 76.2, 114.9, 119.5, 122.5, 125.1, 127.3, 128.3, 128.6,

128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 140.8, 159.2, 170.4, 195.3.

1

tOAc) aﬀorded 44 (0.21 g, 91%) as a colorless oil. H NMR (500

MHz, CDCl₃): δ 0.11 (s, 6H), 0.88 (t, J = 7.5 Hz, 3H), 0.96 (s, 9H),

1.29−1.39 (m, 8H), 1.73 (d, J = 7 Hz, 2H), 2.95 (t, J = 7 Hz, 2H), 4.79

(s, 2H), 7.42 (t, J = 7 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8

Hz, 1H), 7.91 (s, 1H). ¹³C NMR (125 MHz, CDCl₃): δ −5.3, 14.1,

18.4, 22.6, 24.5, 25.9, 29.1, 29.4, 31.7, 38.7, 64.5, 125.6, 126.7, 128.5,

130.4, 137.1, 142.0, 200.7. LRMS (ESI) m/z: 336.2 (M + H)⁺.

(R)-1-(3-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)octan-1-ol.

To a stirred solution of 44 (0.2 g, 0.57 mmol) in THF (2 mL) at −78

°C was added (S)-2-methyl-CBS (0.12 g, 0.43 mmol) and BH₃-Me₂S

(43.0 mg, 0.57 mmol). The reaction mixture was kept at −15 °C for an

additional 1.5 h and quenched with MeOH followed by water. The

water phase was extracted with EtOAc. The combined organic phase

was dried over Na₂SO₄and evaporated in vacuo. Puriﬁcation by silica

gel chromatography (9:1, hexanes:EtOAc) aﬀorded (R)-1-(3-(((tert-

butyldimethylsilyl)oxy)methyl)phenyl)octan-1-ol (180.0 mg, 89%) as

1

a colorless oil. [α]²⁰= +35.1 (c 1 in CHCl₃). H NMR (500 MHz,

D

CDCl₃): δ 0.10 (s, 6H), 0.87 (t, J = 6.5 Hz, 3H), 0.94 (s, 9H), 1.25−

1.31 (m, 9H), 1.37−1.42 (m, 1H), 1.67−1.83 (m, 3H), 4.66 (t, J = 6.5

Hz, 1H), 4.74 (s, 3H), 7.21−7.26 (m, 2H), 7.29−7.32 (m, 2H). ¹³C

3749

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1

HRMS (ESI⁺): m/z calcd for C₃₆H₄₄ClNO₄(M + H)⁺, 590.3010;

found, 590.3015.

(c 0.5 in CHCl₃). H NMR (500 MHz, CDCl₃): δ 0.85 (t, J = 7 Hz,

3H), 1.26 (m, 9H), 1.35−1.46 (m, 3H), 1.59−1.74 (m, 2H), 1.82 (d, J

= 10.5 Hz, 3H), 2.02 (m, 2H), 2.94 (d, J = 8 Hz, 2H), 3.54 (s, 2H),

3.85 (d, J = 6 Hz, 2H), 4.60 (q, J = 5 Hz, 1H), 7.11−7.12 (m, 1H),

7.18−7.19 (m, 2H), 7.24−7.29 (m, 4H), 7.36 (t, J = 8.5 Hz, 1H), 7.46

(d, J = 8 Hz, 2H), 7.75 (d, J = 9 Hz, 2H). ¹³C NMR (125 MHz,

CDCl₃): δ 14.1, 22.7, 26.0, 29.0, 29.3, 29.5, 31.9, 35.9, 40.7, 53.2, 72.9,

75.1, 114.9, 119.4, 122.5, 126.8, 128.0, 128.6, 129.3, 131.4, 135.9,

138.5, 138.9, 159.2, 195.3. HRMS (ESI⁺): m/z calcd for

C₃₅H₄₃ClN₂O₄(M + H)⁺, 590.2901; found, 590.2899. The purity of

(S)-14 was determined to be >95% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm; CH₃CN/

0.05%TFA in H₂O = 25/1). The optical purity of (S)-14 was

determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 14.0 min; (R)-

enantiomer, t_R= 15.0 min).

(4-Chlorophenyl)(3-((3-vinylbenzyl)oxy)phenyl)methanone. To a

stirred solution of (4-chlorophenyl)(3-hydroxyphenyl)methanone (21,

26.0 mg, 1.13 mmol), (3-vinylphenyl)methanol (48, 230.0 mg, 1.7

mmol), and TPP (0.45 g, 1.7 mmol) in THF (10 mL) was added

DIAD (0.62 g, 1.7 mmol). After 4 h at rt, all volatiles were evaporated

in vacuo to aﬀord the crude product. Puriﬁcation by silica gel

chromatography (4:1, hexanes:EtOAc) provided (4-chlorophenyl)(3-

((3-vinylbenzyl)oxy)phenyl)methanone (334.3 mg, 85%) as a yellow

oil. ¹H NMR (500 MHz, CDCl₃): δ 5.12 (s, 3H), 5.28 (d, J = 10.5 Hz,

1H), 5.77 (d, J = 17.5 Hz, 1H), 6.73 (q, J = 11 Hz, 1H), 7.22 (dd, J =

2.5, 8.5 Hz, 1H), 7.31−7.46 (m, 8H), 7.71−7.73 (m, 2H). ¹³C NMR

(125 MHz, CDCl₃): δ 69.9, 70.1, 70.4, 114.3, 114.4, 114.5, 114.5,

114.5, 114.6, 115.3, 115.6, 119.8, 120.0, 122.7, 123.0, 125.2, 125.5,

125.8, 126.1, 126.7, 127.0, 128.5, 128.8, 129.0, 129.4, 129.6, 131.3,

131.5, 135.8, 136.5, 136.5, 136.8, 138.1, 138.5, 138.9, 158.7, 195.1.

LRMS (ESI) m/z: 349.1 (M + H)⁺.

(4-Chlorophenyl)(3-((3-(oxiran-2-yl)benzyl)oxy)phenyl)-

methanone (rac-49). To a stirred solution of (4-chlorophenyl)(3-((3-

vinylbenzyl)oxy)phenyl)methanone (250.0 mg, 0.70 mmol) in

acetonitrile (60 mL) was added Na₂EDTA (0.09%, 8 mL), oxone

(86.0 mg, 0.28 mmol), NaHCO₃(0.056 g, 0.67 mmol), and

triﬂuoroacetone (250.0 mg, 2.24 mmol) at 0 °C. After 1 h, an

additional oxone (86.0 mg, 0.28 mmol) and NaHCO₃(56.0 mg, 0.67

mmol) were added every 1 h; this process was repeated 8 times, during

which the reaction temperature was kept at 0 °C. After completion of

the reaction all volatiles were evaporated in vacuo. The residue was

dissolved in water, and the water phase was extracted with CH₂Cl₂.

The combined organic phase was dried over Na₂SO₄and evaporated

in vacuo. Puriﬁcation by silica gel chromatography (9:1, hexane:E-

tOAc) aﬀord rac-49 (148.0 mg, 74%) as a colorless oil. rac-49 was

resolute via Jocobsen’s kinetic resolution with (S,S)-salene Co(III) or

(R)-(4-Chlorophenyl)(3-((1-(3-(1-hydroxyoctyl)benzyl)piperidin-4-

yl)methoxy)phenyl) Methanone ((R)-3). To a stirred solution of (R)-

46 (20.0 mg, 0.03 mmol) in acetonitrile (1 mL) was added 1N NaOH

(1 mL). After 2 h at rt, the reaction mixture was quenched with water

and extracted with CHCl₃. The combined organic phase was washed

with brine and Na₂S₂O₄and evaporated in vacuo. Puriﬁcation by silica

gel chromatography (1:3, hexanes:EtOAc) aﬀorded (R)-3 (10.0 mg,

1

54%) as a colorless oil. [α]²⁰= +25.8 (c 0.8 in CHCl₃). H NMR

D

(500 MHz, CDCl₃): δ 0.86 (t, J = 7 Hz, 3H), 1.21−1.28 (m, 10H),

1.40−1.47 (m, 3H), 1.68−1.82 (m, 5H), 2.02 (t, J = 12 Hz, 2H), 2.94

(d, J = 11 Hz, 2H), 3.53 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.66 (t, J = 7

Hz, 1H), 7.12 (dd, J = 2.5, 8 Hz, 1H), 7.22−7.31 (m, 6H), 7.36 (t, J =

8 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H). ¹³C

NMR (125 MHz, CDCl₃): δ 14.1, 22.7, 25.9, 29.0, 29.2, 29.5, 31.8,

35.9, 39.2, 45.6, 53.3, 53.3, 63.3, 72.9, 74.7, 114.9, 119.5, 122.6, 124.6,

126.8, 128.3, 128.4, 128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 145.0,

159.2, 195.3. HRMS (ESI⁺): m/z calcd for C₃₄H₄₂ClNO₃(M + H)⁺,

548.2906; found, 548.2909. The purity of (R)-3 was determined to be

>95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18

100A, 100 mm × 4.60 mm; CH₃CN/0.05%TFA in H₂O = 25/1). The

optical purity of (R)-3 was determined to be 95% by HPLC analyses

(Daicel Chiralcel OD-H (0.46 cm × 25 cm; hexanes/tBuOH = 20/1

with ﬂow rate = 1.0 mL/min and a UV detector at 245 nm; (S)-

enantiomer, t_R= 15.0 min; (R)-enantiomer, t_R= 16.0 min).

(S)-(4-Chlorophenyl)(3-((1-(3-(1-hydroxyoctyl)benzyl)piperidin-4-

yl)methoxy)phenyl) Methanone ((S)-3). Colorless oil, [α]²⁰_D= −25.6

1

(c 0.5 in CHCl₃). H NMR (500 MHz, CDCl₃): δ 0.86 (t, J = 7 Hz,

3H), 1.21−1.28 (m, 10H), 1.40−1.47 (m, 3H), 1.68−1.82 (m, 5H),

2.02 (t, J = 12 Hz, 2H), 2.94 (d, J = 11 Hz, 2H), 3.53 (s, 2H), 3.85 (d,

J = 6 Hz, 2H), 4.66 (t, J = 7 Hz, 1H), 7.12 (dd, J = 2.5, 8 Hz, 1H),

7.22−7.31 (m, 6H), 7.36 (t, J = 8 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H),

7.75 (d, J = 8.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃): δ 14.1, 22.7,

25.9, 29.0, 29.2, 29.5, 31.8, 35.9, 39.2, 45.6, 53.3, 53.3, 63.3, 72.9, 74.7,

114.9, 119.5, 122.6, 124.6, 126.8, 128.3, 128.4, 128.6, 129.3, 131.4,

135.9, 138.5, 138.9, 145.0, 159.2, 195.3. HRMS (ESI⁺): m/z calcd for

C₃₄H₄₂ClNO₃(M + H)⁺, 548.2907; found, 548.3001. The purity of

(S)-3 was determined to be >95% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm;

CH₃CN/0.05%TFA in H₂O = 25/1). The optical purity of (S)-3 was

determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 15.0 min; (R)-

enantiomer, t_R= 16.0 min).

(R)-1-(3-((4-((3-(4-Chlorobenzoyl)phenoxy)methyl)piperidin-1-yl)-

methyl)phenyl)octyl Carbamate ((R)-14). To a stirred solution of

(R)-3 (20.0 mg, 0.04 mmol) and DMAP (100.0 mg, 0.08 mmol) in

CH₂Cl₂(0.5 mL) was added TMSNCO (100.0 mg, 0.09 mmol). After

4 h at rt, all volatiles were evaporated in vacuo. Puriﬁcation by silica gel

(R,R)-salene Co(III) to aﬀord (R)-49 and (S)-49, respectively. (R)-49:

1

[α]²⁰= +11.3° (c 0.5 in CHCl₃). H NMR (500 MHz, CDCl₃): δ

D

chromatography (9:1, CHCl₃:MeOH) aﬀorded (R)-14 (17.3 mg,

2.79 (dd, J = 2.5, 5.5 Hz, 1H), 3.15 (t, J = 4 Hz, 1H), 3.88 (t, J = 3 Hz,

1H), 5.10 (s, 2H), 7.21 (dd, J = 4.5, 8 Hz, 1H), 7.26−7.27 (m, 1H),

7.33−7.41 (m, 6H), 7.44 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H).

¹³C NMR (125 MHz, CDCl₃): δ 51.5, 52.4, 70.2, 115.6, 120.0, 123.1,

124.7, 125.5, 127.5, 128.8, 129.1, 129.7, 131.6, 136.0, 137.1, 138.5,

138.8, 139.1, 158.8, 195.3. LRMS (ESI) m/z: 365.1 (M + H)⁺. (S)-49:

1

80%) as a colorless oil. [α]²⁰= +21.1 (c 0.5 in CHCl₃). H NMR

D

(500 MHz, CDCl₃): δ 0.85 (t, J = 7 Hz, 3H), 1.26 (m, 9H), 1.35−1.46

(m, 3H), 1.59−1.74 (m, 2H), 1.82 (d, J = 10.5 Hz, 3H), 2.02 (m, 2H),

2.94 (d, J = 8 Hz, 2H), 3.54 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.60 (q, J

= 5 Hz, 1H), 7.11−7.12 (m, 1H), 7.18−7.19 (m, 2H), 7.24−7.29 (m,

4H), 7.36 (t, J = 8.5 Hz, 1H), 7.46 (d, J = 8 Hz, 2H), 7.75 (d, J = 9 Hz,

2H). ¹³C NMR (125 MHz, CDCl₃): δ 14.1, 22.7, 26.0, 29.0, 29.3, 29.5,

31.9, 35.9, 40.7, 53.2, 72.9, 75.1, 114.9, 119.4, 122.5, 126.8, 128.0,

128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 159.2, 195.3. HRMS (ESI⁺):

m/z calcd for C₃₅H₄₃ClN₂O₄(M + H)⁺, 590.2901; found, 590.2898.

The purity of (R)-14 was determined to be >95% by reverse HPLC

analysis (Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm;

CH₃CN/0.05%TFA in H₂O = 25/1). The optical purity of (R)-14 was

determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 14.0 min; (R)-

enantiomer, t_R= 15.0 min).

colorless oil; [α]²⁰= −9.3° (c 0.5 in CHCl₃).

D

(R)-(4-Chlorophenyl)(3-((3-(2-hydroxy-1-((4-methoxyphenethyl)-

amino)ethyl)benzyl)oxy)phenyl)methanone ((R)-4). A mixture of

(R)-49 (700.0 mg, 0.2 mmol), p-methoxyphenethyl amine (30.0 mg,

0.20 mmol) and Zn(ClO₄)₂·6H₂O (10.0 mg) is heated at 90 °C for 1

h. Puriﬁcation by silica gel chromatography (7:3, hexanes:EtOAc)

aﬀorded (R)-4 (27.0 mg, 27%) as a colorless oil and the corresponding

regiodisomer (15%). (R)-4: [α]²⁰= +20.3 (c 0.5 in CHCl₃). ¹H

D

NMR (500 MHz, CDCl₃): δ 2.68−2.76 (m, 3H), 2.84−2.95 (m, 3H),

3.78 (s, 3H), 4.72 (dd, J = 3.5, 9.5 Hz, 1H), 5.09 (s, 2H), 6.83 (d, J = 8

Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.20 (dd, J = 1.5, 8 Hz, 1H), 7.32−

7.44 (m, 9H), 7.72 (d, J = 8.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃):

δ 30.9, 35.2, 50.7, 55.3, 56.8, 70.2, 71.2, 114.0, 115.5, 119.8, 122.9,

124.9, 125.7, 126.7, 128.6, 128.7, 129.5, 129.6, 131.4, 135.8, 136.6,

(S)-1-(3-((4-((3-(4-Chlorobenzoyl)phenoxy)methyl)piperidin-1-yl)-

methyl)phenyl)octyl Carbamate (S)-14. Colorless oil. [α]²⁰_D= −21.9

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138.5, 138.9, 143.0, 158.2, 158.7, 195.2. HRMS (ESI⁺): m/z calcd for

C₃₁H₃₀ClNO₄(M + H)⁺, 516.1905; found, 516.2101. The purity of

(R)-4 was determined to be >95% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm;

CH₃CN/0.05%TFA in H₂O = 25/1). The optical purity of (R)-4 was

determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 13.5 min; (R)-

enantiomer, t_R= 15.0 min).

purity of (R)-17 was determined to be >95% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm; CH₃CN/

0.05%TFA in H₂O = 25/1). The optical purity of (R)-17 was

determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 18.0 min; (R)-

enantiomer, t_R= 19.5 min).

(S)-(2-Chloro-4-((1-(3-(1-hydroxyoctyl)benzyl)piperidin-4-yl)-

methoxy)phenyl)(4-chlorophenyl) Methanone ((S)-6). Colorless oil.

[α]²⁰_D= −31.7 (c 0.2 in CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 0.87

(t, J = 6.5 Hz, 3H), 1.25−1.50 (m, 12H), 1.68−1.86 (m, 5H), 2.05 (t, J

= 11.5 Hz, 3H), 2.97 (d, J = 11.5 Hz, 2H), 3.56 (s, 2H), 3.85 (d, J = 6

Hz, 2H), 4.67 (t, J = 7 Hz, 1H), 6.86 (dd, J = 2, 8.5 Hz, 1H), 6.96 (d, J

= 2.5 Hz, 1H), 7.24−7.26 (m, 2H), 7.29−7.35 (m, 3H), 7.42 (d, J =

8.5 Hz, 2H), 7.73 (d, J = 9 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃): δ

14.1, 22.7, 25.9, 28.8, 29.3, 29.5, 31.8, 35.7, 39.2, 53.2, 53.2, 63.3, 73.1,

76.6, 113.1, 116.1, 124.7, 126.8, 128.4, 128.4, 128.8, 030.0, 131.3,

131.4, 133.2, 135.8, 139.7, 145.0, 161.4, 193.7. HRMS (ESI⁺): m/z

calcd for C₃₄H₄₁Cl₂NO₃(M + H)⁺, 582.2504; found, 582.2302. The

purity of (S)-6 was determined to be >95% by reverse HPLC analysis

(Phenominex kinetex 2.6 μ C18 100A, 100 mm × 4.60 mm; CH₃CN/

0.05%TFA in H₂O = 25/1). The optical purity of (S)-6 was

determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H

(0.46 cm × 25 cm; hexanes/tBuOH = 20/1 with ﬂow rate = 1.0 mL/

min and a UV detector at 245 nm; (S)-enantiomer, t_R= 14.5 min; (R)-

enantiomer, t_R= 15.5 min).

(S)-(4-Chlorophenyl)(3-((3-(2-hydroxy-1-((4-methoxyphenethyl)-

amino)ethyl)benzyl)oxy)phenyl)methanone ((S)-4). A colorless oil.

1

[α]²⁰= −19.5 (c 0.5 in CHCl₃). H NMR (500 MHz, CDCl₃): δ

D

2.68−2.76 (m, 3H), 2.84−2.95 (m, 3H), 3.78 (s, 3H), 4.72 (dd, J =

3.5, 9.5 Hz, 1H), 5.09 (s, 2H), 6.83 (d, J = 8 Hz, 2H), 7.10 (d, J = 8.5

Hz, 2H), 7.20 (dd, J = 1.5, 8 Hz, 1H), 7.32−7.44 (m, 9H), 7.72 (d, J =

8.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃): δ 30.9, 35.2, 50.7, 55.3,

56.8, 70.2, 71.2, 114.0, 115.5, 119.8, 122.9, 124.9, 125.7, 126.7, 128.6,

128.7, 129.5, 129.6, 131.4, 135.8, 136.6, 138.5, 138.9, 143.0, 158.2,

158.7, 195.2. HRMS (ESI⁺): m/z calcd for C₃₁H₃₀ClNO₄(M + H)⁺,

516.1895; found, 516.19051. The purity of (S)-4 was determined to be

>95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18

100A, 100 mm × 4.60 mm; CH₃CN/0.05%TFA in H₂O = 25/1). The

optical purity of (S)-4 was determined to be >99% by HPLC analyses

(Daicel Chiralcel OD-H (0.46 cm × 25 cm; hexanes/tBuOH = 20/1

with ﬂow rate = 1.0 mL/min and a UV detector at 245 nm; (S)-

enantiomer, t_R= 13.5 min; (R)-enantiomer, t_R= 15.0 min).

(R)-(2-Chloro-4-((1-(3-(1-hydroxyoctyl)benzyl)piperidin-4-yl)-

methoxy)phenyl)(4-chlorophenyl) Methanone ((R)-6). Colorless oil.

[α]²⁰_D= +32.5 (c 0.2 in CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 0.87