Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities

Article

pubs.acs.org/jmc

Further Optimization and Evaluation of Bioavailable, Mixed-Eﬃcacy

μ‑Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR)

Antagonists: Balancing MOR and DOR Aﬃnities

Aubrie A. Harland,^†Larisa Yeomans,^‡Nicholas W. Griggs,^§Jessica P. Anand,^§Irina D. Pogozheva,^‡

Emily M. Jutkiewicz,^§John R. Traynor,^§and Henry I. Mosberg*^,†,‡

‡

§

^†Interdepartmental Program in Medicinal Chemistry, Department of Medicinal Chemistry, College of Pharmacy, and Department

of Pharmacology, Medical School, University of Michigan, Ann Arbor, Michigan 48109, United States

S

* Supporting Information

ABSTRACT: In a previously described peptidomimetic

series, we reported the development of bifunctional μ-opioid

receptor (MOR) agonist and δ-opioid receptor (DOR)

antagonist ligands with a lead compound that produced

antinociception for 1 h after intraperitoneal administration in

mice. In this paper, we expand on our original series by

presenting two modiﬁcations, both of which were designed

with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing aﬃnities between MOR

and DOR while reducing aﬃnity and eﬃcacy at the κ-opioid receptor (KOR), and (3) improving in vivo eﬃcacy. Here, we

establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR aﬃnity and increase

selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist proﬁle. From initial in vivo studies, one

compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved

duration of action of longer than 3 h.

incorporated the bulky aromatic moieties that enhanced the

MOR agonist/DOR antagonist proﬁle we desired. We reported

a series of mixed-eﬃcacy opioid peptidomimetics employing a

THQ scaﬀold that displayed a MOR agonist/DOR antagonist

proﬁle and whose lead compound produced antinociception for

a duration of approximately 1 h in a mouse warm water tail

withdrawal (WWTW) assay after peripheral (intraperitoneal,

ip) administration.¹Although our original opioid peptidomi-

metic series displayed the desired proﬁle and validated our

THQ scaﬀold as being a suitable and bioavailable template,

there remained opportunity to improve the in vitro and in vivo

properties. For example, compounds in the original series

displayed a 10- to 130-fold binding aﬃnity preference for MOR

over DOR, and many also exhibited considerable aﬃnity and

eﬃcacy at the κ-opioid receptor (KOR). In the subsequent

generation of compounds, we explored two diﬀerent mod-

iﬁcations to our original series in an attempt to (1) probe and

improve bioavailability and metabolic stability, (2) balance the

aﬃnity at MOR and DOR while reducing KOR aﬃnity and

eﬃcacy, and (3) increase in vivo eﬃcacy and duration of action.

Here, we display our ﬁndings from two parallel series of

analogues, which include replacement of our THQ scaﬀold

with a tetrahydronaphthalene (THN) scaﬀold or N-acetylation

of the nitrogen in the THQ core of our original series (Figure

1).

INTRODUCTION

■

Several studies have reported the utility and application of

compounds with μ-opioid receptor (MOR) activation and

reduced δ-opioid receptor (DOR) signaling.¹⁻⁵Co-adminis-

tration of a MOR agonist with a DOR antagonist⁶⁻⁸produces

antinociception with reduced risk of tolerance and dependence,

implicating the importance of reduced DOR signaling in

mitigating these undesired properties. However, multidrug

regimens may produce pharmacokinetic complications and lead

to low patient compliance.⁹⁻¹¹Thus, we^1,2,12−14and

others^3−5,15,16have pursued the development of bifunctional,

mixed-eﬃcacy MOR agonist/DOR antagonist ligands. We ﬁrst

reported the synthesis of a high-aﬃnity (nanomolar binding)

opioid receptor peptidomimetic with selectivity for MOR in

1998. In this initial report, a tetrahydroquinoline (THQ)

scaﬀold was implemented to mimic the binding conformations

of the high-aﬃnity tetrapeptide JOM-13 (Tyr-c[^D-Cys-Phe-D-

Pen]OH) and related peptides while eliminating the disulﬁde-

containing moiety of the peptide.¹⁷Since then, we have

developed several additional mixed-eﬃcacy peptides that have

helped elucidate which functionalities yield high aﬃnity binding

across the receptor subtypes.^13,14,18,19For example, we have

found that, in our peptides, bulky aromatics (1-naphthyl, 2-

naphthyl, and 2-indanyl groups) are especially useful for

obtaining a more balanced MOR agonist/DOR antagonist

proﬁle.^13,14Given the promising results from the peptide series,

we sought to transfer the key binding elements from a peptide

scaﬀold to a more bioavailable peptidomimetic scaﬀold that

Received: August 12, 2015

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Figure 1. Modiﬁcations to the THQ scaﬀold.

Through both of these modiﬁcations, our goal was to reduce

the metabolic lability associated with the nitrogen heteroatom

in the THQ ring, as two common metabolic concerns

associated with this moiety include N-oxidation and oxidation

α to the heteroatom.²⁰⁻²⁴Furthermore, the amine in the THQ

core is also part of an aniline system, which is susceptible to

aromatization, not only in vivo,²⁵but potentially in ambient

atmosphere with trace acid present.²⁶As described below, we

observed that N-acetylation of our parent compound 14i to

form 14e improved DOR aﬃnity without altering MOR

aﬃnity, resulting in a better balance of MOR and DOR binding.

Consequently, we decided to explore parallel series with

variable substituent R (Figure 1) with and without N-

acetylation of the core THQ nitrogen to examine eﬀects on

bioavailability and relative MOR and DOR aﬃnities. We also

explored the eﬀect of eliminating the aniline nitrogen entirely

by replacing the THQ scaﬀold with THN. As described below,

the N-acetylation of the THQ scaﬀold improved DOR aﬃnity

across our parallel series without signiﬁcantly altering eﬃcacy

proﬁles or binding at MOR, thereby creating bifunctional

ligands with a more balanced MOR agonist/DOR antagonist

proﬁle. In addition, one of the new analogues, 14a, displays full

antinociception in the mouse WWTW assay for a duration of

longer than 3 h after peripheral administration, a signiﬁcant

improvement on our original in vivo results for parent 14i.

Scheme 1. Synthesis of THQ-Containing Analogues

Diversiﬁed via Suzuki Coupling

a

3-Bromopropionyl chloride (1.02 equiv), K₂CO₃(2.05 equiv), DCM,

b

c

RT. NaOtBu (1.05 equiv), DMF, RT. TfOH (3 equiv), DCE, RT.

d

e

Neat Ac₂O (excess), reﬂux. For synthesis of 6a and 6d: R₂-Bpin (2

equiv), Pd(dppf)Cl₂(0.1 equiv), K₂CO₃(3 equiv), 3:1 acetone:H₂O,

f

MW 100 °C, 300 W. For synthesis of 6c and 6d: R₂-B(OH)₂(2

equiv), Ag₂O (2.5 equiv), Pd(dppf)Cl₂(0.1 equiv), K₂CO₃(3 equiv),

g

THF, MW 100 °C, 300 W. Boc₂O (1.2−2 equiv), DMAP (0.1 equiv),

RESULTS

■

DIPEA (1.2−2 equiv), DCM, 60 °C.

Diversiﬁcation of THQ-Containing Analogues via

Suzuki Coupling. The core THQ intermediate 4 (Scheme

1) was synthesized using the methodology developed by

Schmidt et al.²⁷and described in our previous paper¹with the

single modiﬁcation of using p-bromoaniline as the starting

material. Brieﬂy, p-bromoaniline 1 was acylated using 3-

bromopropionyl chloride to yield 2, cyclized under basic

conditions to form lactam 3, and then treated with

triﬂuoromethanesulfonic acid to form intermediate 4. The

nitrogen of 4 was protected with a tert-butyloxycarbonyl (Boc)

group or an acetyl group (later referred to as “R₁”) to form

intermediates 5a and 5b, which were then subjected to Suzuki

cross-coupling^28,29to yield compounds 6a−d with diverse R₂

substituents.

sized as a racemic mixture using a slight modiﬁcation to the

methodology originally developed by Kolanos et al.³⁰that

begins with a condensation reaction between indene and 4-

acetamidobenzaldehyde, the product of which was hydro-

genated and deprotected to yield aniline intermediate 7g. The

aniline intermediates 7e−g were then acylated, cyclized to form

lactams 9e−g, and treated with triﬂuoromethanesulfonic acid to

form 10e−g, as shown previously^1,27(Scheme 2). Intermedi-

ates 10e−g were then protected with Boc or an acetyl group

(later referred to as “R₁”) to form intermediates 6e−h with

diverse substituents.

Completion of Synthesis of THQ-Containing Ana-

logues from a Common Intermediate. Once the R₂

substituent was implemented in the THQ scaﬀold, the

synthesis of ﬁnal compounds 14a−h converged beginning

with 6a−h (Scheme 3). Intermediates 6a−h were treated with

(R)-t-butanesulﬁnamide and Ti(OEt)₄to yield imines 11a−h,

which were reduced in situ with NaBH₄to form the desired R-

stereochemistry of intermediates 12a−h.³¹⁻³³The Ellman

auxiliary was cleaved using concentrated hydrochloric acid,

forming primary amines 13a−h,³¹⁻³³which were then coupled

to di-Boc-protected 2,6-dimethyl-^L-tyrosine (diBoc-DMT) and

Diversiﬁcation of THQ-Containing Analogues via

Condensation Chemistry. Although starting material for

7e, which bears a benzyl R₂substituent, was commercially

available, compounds containing a THQ core with a 1- or 2-

methylindanyl substituent were synthesized by incorporating

the R₂substituent during the ﬁrst synthetic step via a

condensation reaction. Speciﬁcally, the 2-methylindanyl ana-

logue was synthesized via an aldol condensation followed by a

hydrogenation as previously described¹to form aniline

intermediate 7f. The 1-methylindanyl analogues were synthe-

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Scheme 2. Synthesis of THQ-Containing Analogues via Condensation Reactions

a

b

c

d

3-Bromopropionyl chloride (1.02 equiv), K₂CO₃(2.05 equiv), DCM, RT. NaOtBu (1.05 equiv), DMF, RT. TfOH (3 equiv), DCE, RT. For

e

synthesis of 6e, 6g, and 6g: neat Ac₂O (excess), reﬂux. For synthesis of 6h: Boc₂O (1.2−2 equiv), DMAP (0.1 equiv), DIPEA (1.2−2 equiv), DCM,

60 °C.

Scheme 3. Completion of Synthesis of THQ-Containing

Analogues

Scheme 4. Synthesis of THN-Containing Analogues

a

15 (1 equiv), (R)-t-butanesulﬁnamide (2−3 equiv), THF, Ti(OEt)₄

b

(4−6 equiv) 0 °C, then reﬂux at 75 °C. NaBH₄(4−6 equiv), THF,

c

−50 °C to RT, then MeOH, RT. For synthesis of 18m, 18o, and 18p:

R₂-Bpin (1 equiv), Pd(dppf)Cl₂(0.1 equiv), K₂CO₃(3 equiv), 3:1

d

acetone:H₂O, MW 110 °C, 300 W. For synthesis of 18n: R₂-

B(OH)₂(2 equiv), Ag₂O (2.5 equiv), Pd(dppf)Cl₂(0.1 equiv), K₂CO₃

e

(3 equiv), THF, MW 80 °C, 300 W. HCI (6 equiv), dioxane, RT.

f

diBoc-DMT (1.05 equiv), PyBOP (1 equiv), 6Cl-HOBt (1 equiv),

g

a

DIPEA (10 equiv), DMF, RT. 1:1 TFA:DCM (excess).

Dihydroquinolinone intermediate, 6a−h (1 equiv), (R)-t-butanesulﬁ-

namide (2−3 equiv), THF, Ti(OEt)₄(4−6 equiv), 0 °C, then reﬂux at

b

75 °C. NaBH₄(6 equiv), THF, −50 °C to RT, then MeOH, RT.

c

d

treated with concentrated hydrochloric acid to cleave the

Ellman auxiliary, aﬀording primary amines 19m−p.³¹⁻³³In the

ﬁnal step, amines 19m−p were coupled to diBoc-DMT and

deprotected to yield compounds 14m−p.

HCl (6 equiv), dioxane, RT. diBoc-DMT (1.05 equiv), PyBOP (1

e

equiv), 6Cl-HOBt (1 equiv), DIPEA (10 equiv), DMF, RT. 1:1

TFA:DCM (excess). Compounds 14i−14l previously published, see

ref 1. * indicates starting material was recovered, but not included in

yield calculation.

Opioid Receptor Binding and Eﬃcacy. Binding aﬃnities

(K_i) for the ﬁnal compounds were determined from

competitive displacement of radiolabeled [³H]diprenorphine

in membrane preparations from C6 cells stably expressing

MOR (C6-MOR) or DOR (C6-DOR) or CHO cells stably

deprotected to yield compounds 14a−h. The syntheses for

analogues 14i−l, which also contain a THQ core, were

previously published.¹

Complete Synthesis of THN Peptidomimetic Ana-

logues. Synthesis of THN compounds 14m−p began by

converting the ketone of commercially available 7-bromo-

dihydronaphthalenone 15 to chiral imine 16 using (R)-t-

butanesulﬁnamide and Ti(OEt)₄, followed by in situ reduction

to form 17 using NaBH₄to aﬀord the desired R-stereo-

chemistry of the sulfonamide³¹⁻³³(Scheme 4). Next, various

R₂substituents were incorporated via Suzuki coupling to form

intermediates 18m−p.^28,29Intermediates 18m−p were then

expressing KOR (CHO-KOR), as previously described^34,35

-

(Table 1). Eﬃcacy of the compounds was assessed by agonist-

stimulated [³⁵S]GTPγS binding to G protein³⁶in cell

membrane preparations of C6-MOR, C6-DOR, and CHO-

KOR (Table 1).

Although a total of six compound sets were synthesized

(each set deﬁned by the R₂substituent), only four of these sets

incorporated all three of the diﬀerent scaﬀolds; these sets

include analogues in which the R₂substituent is a benzyl, a 1-

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Table 1. Opioid Receptor Binding Aﬃnities and Eﬃcacies of Peptidomimetics

a

b

Binding aﬃnities (K_i(nM)) were obtained by competitive displacement of radiolabeled [³H]diprenorphine in membrane preparations. Eﬃcacy

data were obtained using agonist-induced stimulation of [³⁵S]GTPγS binding; eﬃcacy is represented as percent maximal stimulation relative to

standard agonist DAMGO (MOR), DPDPE (DOR), or U69,593 (KOR) at 10 μM. All values are expressed as the mean with SEM in parentheses

for n = 3 independent assays in duplicate unless otherwise noted. In duplicate, n = 2 independent assays; dns: does not stimulate. Syntheses and

c

d

e

data for these compounds were previously published (see ref 1).

methylnaphthyl, a 2- methylnaphthyl, or a methylcyclohexyl.

The eﬀects of both scaﬀold modiﬁcations on the binding

aﬃnities are shown in Table 1. The most noticeable trend

across these four sets of compounds is seen with the N-

acetylated THQ series at DOR. This N-acetylated series

(regardless of R₂) shows a signiﬁcantly higher aﬃnity at DOR

than the unsubstituted THQ counterpart. Additionally, N-

acetylation maintains, and in some cases slightly improves,

aﬃnity at MOR and slightly decreases aﬃnity at KOR. The

eﬀect of the N-acetylated THQ scaﬀold on eﬃcacy (Table 1)

across all receptors is minimal with most compounds retaining

similar eﬃcacy as seen with the parent THQ series. When

considering the eﬀects of the N-acetylation on the THQ

scaﬀold as a whole, this modiﬁcation appears to balance the

MOR/DOR binding proﬁle while maintaining the desired

MOR agonist/DOR antagonist eﬃcacy proﬁle.

In contrast, the eﬀect of the THN scaﬀold on binding

aﬃnities at all three receptors (Table 1), when compared with

the parent THQ scaﬀold, showed no consistent or advanta-

geous trends. For example, when comparing 14m (THN

scaﬀold) with 14i (THQ scaﬀold), 14m shows increased

aﬃnity across all three receptors. Additionally, when comparing

14n (THN scaﬀold) with 14b (THQ scaﬀold), 14n has

decreased aﬃnity across all three receptors. Furthermore, a

decrease in eﬃcacy is seen at MOR regardless of the R₂

substituent when compared to the THQ scaﬀold. Because the

THN scaﬀold presented no apparent advantage, the 1- and 2-

indanyl THN analogues were not pursued.

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a

Table 2. In Vivo Activity of Select Peptidomimetics

a

Summary of antinociceptive eﬀects of select analogues (n = 3 for all analogues, except for 14a n = 6) in mouse WWTW assay following

b

c

intraperitoneal (ip) administration with a cutoﬀ time of 20 s. See ref 1. Full antinociception indicates that the cutoﬀ time was reached.

In Vivo WWTW Assay. Several compounds were chosen

based on in vitro data to be assayed for antinociception in the

mouse WWTW assay following intraperitoneal (ip) injection.³⁷

Of the compounds assayed (Table 2, Figure 2), only three

Figure 3. Time course of antinociception of 14a and 14i (n = 6) in the

mouse WWTW assay following ip administration of 10 mg/kg. Plotted

as average SEM. Data for 14i from ref 1.

antinociception after ip administration, 14d displayed no

antinociception at the same dose after ip administration,

Figure 2. Cumulative antinociceptive dose response curves of select

suggesting that subtle diﬀerences in chemical structure create

analogues (n = 3 for all analogues, except for 14a where n = 6) in the

substantial diﬀerences in pharmacokinetics.

mouse WWTW assay following ip administration. Plotted as average

SEM ****, p < 0.0001 for 14a, 14f, 14i, and 14j for the 10 mg/kg

DISCUSSION AND CONCLUSIONS

dose when compared to baseline. Data for 14i from ref 1.

■

Our previously described results¹were promising in that we

were able to transfer pharmacophore elements from a MOR

agonist/DOR antagonist peptide to a peptidomimetic scaﬀold,

as seen in our parent peptidomimetic 14i, which was shown to

have antinociceptive activity after peripheral administration in

mice. Although our original series of compounds displayed the

desired MOR agonist/DOR antagonist proﬁle, there remained

opportunity to improve the balance of binding aﬃnities at

MOR and DOR and reduce any remaining KOR binding and

eﬃcacy. Although the optimal balance of “MOR agonism” with

“DOR antagonism” is yet to be determined, we reasoned that a

low nanomolar, balanced aﬃnity proﬁle (∼1:1 MOR K_i:DOR

K_i) was a logical place to start, because this would ensure that

both MOR agonist and DOR antagonist character would be

represented in the in vivo outcome and would provide useful

information for future studies. Our initial series displayed

relatively unbalanced MOR and DOR aﬃnities, where our

compounds were 10- to 130-fold more selective for MOR over

DOR (Table 1). In the present study, we set out to improve

compounds displayed dose-dependent antinociceptive activity.

Compounds 14f and 14j displayed partial antinociception at

the maximum dose tested, whereas 14a displayed full

antinociception with ED₅₀= 4.72

0.01 mg/kg. By

comparison, the ED₅₀of morphine, under the same conditions,

was 4.73 0.001 mg/kg. Because compound 14a displayed full

antinociception at 10 mg/kg in the initial WWTW assay, a time

course study was completed to determine the duration of action

(Figure 3). As can be seen in Figure 3, 14a produces maximal

antinociception with a rapid onset, which is maintained for

approximately 200 min. This 200 min duration of action is

greater than 3-times longer compared to the duration of action

of our original lead peptidomimetic 14i¹(and similar to that

observed for the same dose of morphine¹), suggesting better

bioavailability or metabolic stability. Interestingly, despite being

structural isomers with a similar in vitro proﬁle, 14a and 14d

displayed drastically diﬀerent in vivo results when administered

via ip injection (Figure 2). Although 14a displayed full

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Figure 4. (A) 14j (purple) in DORinactive (DORi) with key residues shown. (B) 14a (teal) in DORi with key residues shown. Dashed lines

represent calculated distances between ligand and receptor residues, indicating a possible interaction between N-acetyl moiety and Tyr¹²⁹that may

increase binding aﬃnity at DORi. (C) Overlay of 14j (purple) and DIPP-NH₂(orange) in DORi. (D) Overlay of 14a (teal) and DIPP-NH₂

(orange) in DORi.

upon our initial compounds in two ways. First, we wished to

explore modiﬁcation of the THQ scaﬀold to avoid metabolic

susceptibility due to the THQ ring nitrogen while also

examining the eﬀect of these modiﬁcations on binding aﬃnity

and eﬃcacy. Second, we wished to explore the additional eﬀect

of modifying the R₂substituent in the search for a MOR

agonist/DOR antagonist with a better MOR/DOR aﬃnity

balance.

To explain the results of the N-acetylated THQ series, we

looked at computational docking of one of the THQ/N-

acetylated ligand pairs docked in the binding pocket of the

inactive state of DOR (DORi), as previously described.^13,38

Modeling 14j and 14a in the DORi binding pocket (Figure 4),

as a representative example, it can be seen that the N-acetyl of

14a extends further into the DORi binding pocket to create a

polar contact with Tyr¹²⁹in transmembrane helix 3 (TM3) of

the receptor (Figure 4, panel B), an interaction that appears to

be unavailable with the unsubstituted THQ core (Figure 4,

panel A). It seems that the N-acetyl can increase DORi binding

through three modes: (1) the carbonyl of the N-acetyl forms a

hydrogen bond with the hydroxyl in Tyr¹²⁹, and once the

hydrogen bond is formed, this could orient the ligand in the

receptor to form a tighter hydrogen bonding network between

(2) the primary amine of the 2,6-dimethyl tyrosine (Dmt)

moiety of the ligand and Asp¹²⁸of the receptor, and (3) the

Dmt hydroxyl moiety of the ligand and His²⁷⁸. It has been

previously reported³⁹⁻⁴⁴that ligand interaction with His²⁷⁸and

Asp¹²⁸is important for opioid ligand binding in all three

classical receptors. Although the DOR Tyr¹²⁹residue is

conserved in MOR (Tyr¹⁴⁸), the N-acetylation does not appear

to have as profound of an eﬀect in the MOR binding pocket

with the measured distance between 14j and 14a to the Tyr

residue 4.7 to 4.3 Å, respectively (not pictured). When both 14j

and 14a are overlaid with the crystal structure of the recently

reported⁴⁴MOR agonist/DOR antagonist, DIPP-NH₂(Figure

4, panels C and D, respectively) in DORi, it can be seen that

14a aligns better with the Dmt and free amine moiety of DIPP-

NH₂than 14j. This suggests that the N-acetyl moiety has a

subtle, yet impactful eﬀect on the orientation of the ligand

resulting in increased binding aﬃnity at DORi. We hypothesize

that the interaction between Tyr¹²⁹in the binding pocket of

DORi and N-acetylated ligand 14a (and other N-acetylated

analogues) may bring 14a into closer proximity to His²⁷⁸and

Asp¹²⁸, facilitating a better binding network within the DORi

The initial in vitro results for the parallel series of scaﬀold

modiﬁcations yield interesting insight into both the electronic

and steric requirements for optimal binding. The data in Table

1 indicates that the series incorporating the THN scaﬀold,

when compared to the unsubstituted THQ scaﬀold, leads to

greater disparity in the binding aﬃnities at MOR and DOR.

These disparities are caused by either increasing aﬃnity at

MOR more so than at DOR or by decreasing aﬃnity at DOR

more than at MOR relative to the parallel analogue with a

THQ core. Both of these eﬀects result in a less desirable aﬃnity

proﬁle and sometimes result in a proﬁle with reduced aﬃnity at

both MOR and DOR. Furthermore, the THN scaﬀold does not

improve selectivity over KOR across all sets of compounds. In

addition to the eﬀects that the THN scaﬀold has on the binding

aﬃnities at MOR, DOR, and KOR, the THN core also results

in reduced eﬃcacy at MOR when compared to our parent

THQ series. In contrast, the N-acetylated THQ series appears

to be superior to both the THN series and the original THQ

series. When compared to the original THQ scaﬀold, the

corresponding N-acetylated analogues not only maintain MOR

agonist activity and DOR antagonism but also show increased

DOR binding aﬃnity (regardless of R₂substituent), whereas

MOR aﬃnity remains relatively unchanged, thereby creating a

series of ligands with more balanced MOR and DOR aﬃnities

(Table 1). The only exception to this trend is in the case of

compound 14d, where the MOR and DOR aﬃnity both

increase, with the MOR aﬃnity increasing to a greater extent,

resulting in a higher preference for MOR over DOR.

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Figure 5. Summary of in vivo activity following ip administration, cLogP, and molecular weight (MW, g/mol) for select compounds. Compounds

14e, 14o, 14p, 14l, and 14m do not display antinociception following ip administration, and structures are not shown. ¹See ref 1 for in vivo activity.

binding pocket. Although the increase in DOR aﬃnity across

the N-acetylated analogues is the most apparent trend when

considering the in vitro data in their entirety, it is clear that

some substituents display superior proﬁles over others. For

example, the methylcyclohexyl (14b, 14c, 14n) and 1- and 2-

methylindanyl (14h, 14g, and 14l, 14f, respectively) analogues

pick up considerable undesired KOR aﬃnity and agonism,

whereas the 1- and 2-methylnaphthyl analogues (14k, 14d,

14p, and 14j, 14a, 14o, respectively) display agonist activity at

MOR, little or no agonist activity at DOR, and signiﬁcantly

reduced binding and eﬃcacy at KOR, suggesting that these

compounds behave as functional antagonists at DOR. To

conﬁrm antagonist activity at DOR, we tested 14a and 14d

against the DOR agonist DPDPE, and as expected from the

high binding aﬃnity and lack of stimulation of [³⁵S]GTPγS

binding at DOR, 14a and 14d both antagonized the DOR

agonist with antagonist aﬃnity constants (K_e) of 1.98 nM and

27.5 nM, respectively (data not shown).

On the basis of the results from our initial in vitro screening,

we chose 12 candidates for in vivo screening evaluation in the

mouse WWTW assay. As seen in Figure 5, the in vitro results

and cLogP calculations were both poor predictors of in vivo

activity. Surprisingly, 14d displayed no in vivo activity at 10

mg/kg ip whereas 14a, a structural isomer of 14d, displayed full

antinociception at 10 mg/kg ip. To explore this unexpected

result, we submitted both 14a and 14d for plasma stability

testing performed by Quintara Discovery (San Francisco, CA,

USA). These results revealed that both analogues tested were

fully stable in plasma after 30 min, suggesting that compound

degradation in the plasma does not account for the diﬀering

activities in vivo. Planned pharmacokinetic studies on 14a and

14d will be helpful for understanding the basis of the disparate

in vivo results.

In summary, we have expanded upon our original THQ

scaﬀold by incorporating a THN and an N-acetylated THQ

scaﬀold with the intention of (1) probing and improving

bioavailability and metabolic stability, (2) balancing further the

aﬃnity at MOR and DOR while reducing KOR aﬃnity and

eﬃcacy, and (3) increasing in vivo activity and duration of

action. Through N-acetylation of the THQ nitrogen, not only

were we able to better balance the aﬃnity at MOR and DOR

while maintaining the desired MOR agonist/DOR antagonist

proﬁle, but we were also able to decrease binding aﬃnity and

eﬃcacy at KOR, thereby creating a more desirable compound

proﬁle. Furthermore, three of the compounds presented in this

paper show in vivo activity when administered peripherally, one

of which (14a) displays full antinociception in the mouse

WWTW assay for >3 h, a promising improvement upon our

original lead compound.¹

EXPERIMENTAL SECTION

■

Chemistry. All reagents and solvents were obtained from

commercial sources and used without additional puriﬁcation. Suzuki

couplings were performed on a Discover S-class (CEM) microwave in

a closed vessel with maximum power input of 300 W and temperature

set at 110 °C for 10−60 min under the standard method from their

Synergy software. Hydrogenations were performed on a Parr

hydrogenator apparatus from Parr Instrument Company, model

3916EA, at the pressures speciﬁed using 10% of Pd/C as the catalyst.

Flash column chromatography was carried out using P60 silica gel

(230−400 mesh). Puriﬁcation of ﬁnal compounds was performed

using a Waters semipreparative HPLC with a Vydac protein and

peptide C18 reverse phase column, using a linear gradient of 15% of

solvent B (0.1% of TFA in acetonitrile) in solvent A (0.1% of TFA in

water) to 50% of solvent B in solvent A at a rate of either 0.5% or 1%

G

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per minute and monitoring UV absorbance at 230 nm. Purity of

synthesized compounds was determined on a Waters Alliance 2690

analytical HPLC instrument and a Vydac protein and peptide C18

reverse phase column, using a linear gradient of 0% of solvent B in

solvent A to 45, 70, or 90% of solvent B in solvent A in 45, 70, or 90

min, respectively, and UV absorbance at 230 nm (gradient A). Purities

of the ﬁnal compounds used for testing were ≥95% as determined by

HPLC. ¹H NMR and ¹³C NMR data were obtained on either a 400 or

500 MHz Varian spectrometer using CDCl₃or CD₃OD solvents. The

identity of each compound was veriﬁed by mass spectrometry using an

Agilent 6130 LC−MS mass spectrometer in positive mode.

General Procedure A for the Synthesis of the 3-Bromo-N-

propanamides.^1,27To a ﬂame-dried round-bottom ﬂask under Ar

were added the aniline compound (1.0 equiv) and K₂CO₃(2.05

equiv). The reaction vessel was placed back under vacuum, and anhyd

DCE was added via syringe. The reaction solution was stirred under

vacuum for 5 min. After 5 min, the reaction vessel was then ﬂooded

with Ar, and 3-bromopropionyl chloride (1.02 equiv) was added via

syringe. The reaction was stirred under Ar at RT for 1 h and was

monitored by TLC using a ninhydrin stain for disappearance of the

aniline compound. Once the reaction was complete, it was quenched

with dI H₂O, and the layers were separated. The organic layer was

washed with dI H₂O (1 × 50 mL) followed by brine (1 × 30 mL) and

then dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure to yield the pure product.

General Procedure B for the Synthesis of Phenylazetidin-2-

ones.^1,27To a round-bottom ﬂask already containing the dried,

desiccated 3-bromo-N-propanamide (1.0 equiv) was added NaOtBu

(1.05 equiv). The reaction vessel was placed under vacuum, and anhyd

DMF was added via syringe. The solution was stirred under vacuum

for 5 min and was then ﬂooded with Ar. The reaction was stirred

under Ar at RT for up to 3 h and was monitored by TLC. Once

complete, the solvent was removed under reduced pressure; the

resulting crude residue was resuspended in DCM and dI H₂O, and the

layers were separated. The organic layer was washed once with dI H₂O

(1 × 30 mL) and then brine (1 × 30 mL) and then dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure to yield the crude

product, which was then puriﬁed using silica gel chromatography to

yield the pure product.

General Procedure C for the Synthesis of 2,3-Dihydroqui-

nolin-4(1H)-ones.^1,27To a round-bottom ﬂask already containing

the dried, desiccated phenylazetidin-2-ones (1.0 equiv) was added

anhyd DCE under vacuum. The reaction vessel was stirred under

vacuum for 5 min and then ﬂooded with Ar. Next, triﬂic acid (TfOH)

(3.0 equiv) was added via syringe. The reaction was stirred under Ar at

RT for up to 3 h and was monitored by TLC. Once complete, the

reaction was quenched with dI H₂O (20 mL) and solid K₂CO₃(one

spatula full), and the layers were separated. The organic layer was

washed once with dI H₂O (1 × 30 mL), sat. NaHCO₃(1 × 30 mL),

and brine (1 × 30 mL) and then dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure to yield the crude product, which

was then puriﬁed using silica gel chromatography to yield the pure

product.

min under vacuum. The round-bottom ﬂask was ﬂooded with Ar, and

DIPEA (1.2−2.0 equiv) was added via syringe. The reaction vessel was

equipped with a condenser and placed in an oil bath at 60 °C. The

reaction was stirred at reﬂux for 12−16 h under Ar and was monitored

by TLC. Once signiﬁcant conversion to product was seen, the reaction

was quenched using dI H₂O (20 mL), and the layers were separated.

The organic layer was washed with sat. NaHCO₃solution (1 × 20

mL) and brine (1 × 20 mL) and then dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure to yield a crude yellow oil that

was puriﬁed using silica gel chromatography to obtain the pure

product.

General Procedure F for Suzuki Coupling Using an Aromatic

Boronic Ester. A solution of 3:1 acetone:dI H₂O was degassed for 1

h, and then Ar was bubbled through the solution for 1 h to ensure

removal and displacement of ambient oxygen. When all the reagents

are solid: aromatic bromide (1.0 equiv), boronic ester (2.0 equiv),

K₂CO₃(3.0 equiv), and Pd(dppf)Cl₂(0.1 equiv) were added to a

microwave tube, and the tube was placed under vacuum for 15 min

and then ﬂooded with Ar. Roughly 1−2 mL of the 3:1 acetone:dI H₂O

solution was added to the tube via syringe; then, the tube was placed in

a microwave for 30−60 min with a maximum power of 300 W and a

maximum temperature of 100 °C with the “PowerMax” option

enabled. When the boronic ester is liquid: aromatic bromide (1.0

equiv), K₂CO₃(3.0 equiv), and Pd(dppf)Cl₂(0.1 equiv) were added

to a microwave tube, and the tube was placed under vacuum for 15

min and then ﬂooded with Ar. Roughly 1−2 mL of the 3:1 acetone:dI

H₂O solution was added to the tube via syringe, followed by addition

of the boronic ester (2.0 equiv) via syringe. The tube was placed in a

microwave for 30−60 min with a maximum power of 300 W and a

maximum temperature of 100 °C with the “PowerMax” option

enabled. Once the microwave reaction was complete, the reaction

mixture was ﬁltered through a pad of Celite to remove palladium, and

solvents were removed under reduced pressure to yield a crude brown

residue that was puriﬁed using silica gel chromatography to obtain the

pure product. This procedure was adapted from ref 28.

General Procedure G for Suzuki Coupling Using an Aliphatic

Boronic Acid. The aromatic bromide (1.0 equiv), boronic acid (1.1−

2.0 equiv), K₂CO₃(3.0 equiv), Ag₂O (2.5 equiv), and Pd(dppf)Cl₂

(0.1 equiv) were added to a microwave tube, and the tube was placed

under vacuum for 15 min and then ﬂooded with Ar. Roughly 1−2 mL

of anhyd THF was added to the tube via syringe, and then the tube

was placed in a microwave for 1 h with a maximum power of 300 W

and a maximum temperature of 80 °C with the “PowerMax” option

enabled. Once the microwave reaction was complete, the reaction

mixture was ﬁltered through a pad of Celite to remove palladium, and

the solvents were removed under reduced pressure to yield a crude

brown residue that was puriﬁed using silica gel chromatography to

obtain the pure product. This procedure was adapted from ref 29.

General Procedure H for the Synthesis of (R, R) THQ and

THN Sulﬁnamides.³¹⁻³³To a round-bottom ﬂask already containing

dried, desiccated 6-substituted dihydroquinolinone intermediate (1.0

equiv) was added (R)-2-methylpropane-2-sulﬁnamide (2.0−3.0

equiv); then, the round-bottom ﬂask was placed under vacuum for

10 min. Meanwhile, a reﬂux condenser was ﬂame-dried under vacuum

and then ﬂooded with Ar. Next, anhyd THF (∼20 mL) was added to

the reaction vessel containing starting reagents via syringe. The

reaction solution was allowed to stir under vacuum for ∼5 min and

was then ﬂooded with Ar. The round-bottom ﬂask was placed in an ice

bath and allowed to equilibrate. Next, Ti(OEt)₄(4.0−6.0 equiv) was

added slowly via syringe. Once the addition was complete, the reaction

vessel was taken out of the ice bath and placed in an oil bath at 70−75

°C, equipped with condenser, and stirred for 16−40 h under Ar. The

reaction was monitored by TLC for loss of ketone. Once suﬃcient

conversion to the tert-butanesulﬁnyl imine was observed, the reaction

vessel was taken out of the oil bath and cooled to ambient

temperature. Meanwhile, an additional round-bottom ﬂask containing

a stir bar was ﬂame-dried under vacuum and then ﬂooded with Ar;

then, NaBH₄was added quickly, and then the reaction vessel was

placed back under vacuum for 5 min. Minimal anhyd THF was added

(∼5 mL), and the vessel was allowed to stir under vacuum for ∼5 min

General Procedure D for the Synthesis of 1-Acetyl-2,3-

dihydroquinolin-4(1H)-ones. To a ﬂame-dried round-bottom ﬂask

under Ar was added 2,3-dihydroquinolin-4(1H)-one. The reaction

vessel was placed under vacuum for 5 min; then, excess Ac₂O was

added via syringe, and the solution was stirred for 5 min. The round-

bottom ﬂask was ﬂooded with Ar, equipped with a condenser, and

placed in an oil bath at 100 °C. The reaction was stirred at reﬂux for

12−20 h under Ar and was monitored by TLC. Once the reaction was

complete, the solvent was removed under reduced pressure, yielding a

crude yellow oil that was puriﬁed using silica gel chromatography to

obtain the pure product.

General Procedure E for the Synthesis of tert-Butyl 4-Oxo-

3,4-dihydroquinoline-1(2H)-carboxylates. To a ﬂame-dried

round-bottom ﬂask under Ar were added 2,3-dihydroquinolin-

4(1H)-one (1.0 equiv), Boc₂O (1.2−2.0 equiv), and DMAP (0.1

equiv). The reaction vessel was placed under vacuum for 5 min; then,

anhyd DCM was added via syringe, and the solution was stirred for 5

H

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and then ﬂooded with Ar. The round-bottom ﬂask was placed in a dry

ice/xylene bath and allowed to equilibrate. Contents from the round-

bottom ﬂask containing the imine intermediate were transferred to a

round-bottom ﬂask containing NaBH₄via cannula. Once the contents

were completely added, the reaction was taken out of the dry ice/

xylene bath and allowed to warm to room temperature. The reaction

was stirred at ambient temperature for 2−3 h. Once the reaction was

complete, MeOH was added for quenching. The solvent was removed

under reduced pressure, yielding a solid residue. The residue was

resuspended in DCM and the remaining solid was removed by

ﬁltration through a cotton plug, and the mother liquor was

concentrated and puriﬁed using silica gel chromatography to yield

pure sulﬁnamide.

General Procedure I for the Synthesis of (R)-1,2,3,4-

Tetrahydroquinolin-4-amines and (R)-1,2,3,4-Tetrahydronaph-

thalen-1-amines. To a round-bottom ﬂask already containing the

sulﬁnamide intermediate was added 15−20 mL of dioxane followed by

concd HCl (6.0 equiv). The reaction was stirred at RT for up to 3 h.

Solvent was removed under reduced pressure to yield a slightly yellow,

clear residue. The residue was resuspended in Et₂O. If a white solid

precipitated (the HCl salt of the amine): the solid was removed via

ﬁltration as product without any further puriﬁcation necessary. If a

white solid did not precipitate, but a residue remained as a ﬁlm on the

ﬂask: the residue was washed with fresh Et₂O (3 × 5 mL) and dried

without any further puriﬁcation necessary.

General Procedure J for diBoc-DMT Coupling to Form Final

Product. The (R)-amine intermediate, diBoc-DMT (1.05 equiv), and

the coupling reagents PyBOP (1.0 equiv) and HOBt-Cl (1.0 equiv)

were dissolved in DMF (10−15 mL) followed by the addition of

DIPEA (10.0 equiv). The reaction mixture was stirred for 18 h at room

temperature. After being concentrated under reduced pressure, the

crude residue was dissolved in a 1:1 mixture of DCM and TFA (10

mL) and stirred for 1 h. The mixture was concentrated and puriﬁed by

semipreparative HPLC to yield the ﬁnal compound. Note that

although other coupling reagents could be used, the coupling reagents

used here were chosen to minimize racemization. Additionally, diBoc-

DMT was used instead of monoBoc-DMT to prevent any possible

ester formation at the phenol of tyrosine that might occur under these

coupling conditions, especially at longer reaction times.

solvent, the resulting crude yellow oil was puriﬁed using silica gel

chromatography (equilibrated in 100%, run in 1:3 EA:hex) to yield the

title compound as a white waxy solid (704 mg, 80.4%). ¹H NMR (500

MHz, CDCl₃): δ 8.10 (d, J = 2.4 Hz, 1H), 7.64 (dd, J = 8.7, 2.5 Hz,

1H), 7.47 (s, 1H), 4.21 (t, J = 6.3 Hz, 2H), 2.81 (t, J = 6.3 Hz, 2H),

2.35 (s, 3H). ¹³C NMR (126 MHz, CDCl₃): δ 192.4, 169.0, 142.6,

136.6, 130.3, 127.0, 125.8, 118.7, 44.2, 39.1, 23.1.

tert-Butyl 6-Bromo-4-oxo-3,4-dihydroquinoline-1(2H)-carboxy-

late (5b). This compound was synthesized following general

procedure E using 4 (300 mg, 1.33 mmol, 1.0 equiv), Boc₂O (348

mg, 1.59 mmol, 1.2 equiv), DMAP (16.2 mg, 0.13 mmol, 0.1 equiv),

and DIPEA (0.277 mL, 1.59 mmol, 1.2 equiv). Following the

quenching and workup, the crude product was chromatographed on

silica gel (equilibrated in 100% hex, run in 2:3 EA:hex) to yield pure

product as a white solid (380 mg, 87.8%). ¹H NMR (500 MHz,

CDCl₃): δ 8.10 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.57 (dd,

J = 9.0, 2.4 Hz, 1H), 4.15 (t, J = 6.3 Hz, 2H), 2.77 (t, J = 6.3 Hz, 2H),

1.55 (s, 9H). ¹³C NMR (126 MHz, CDCl₃): δ 192.8, 152.4, 143.1,

136.6, 129.9, 126.1, 125.5, 117.1, 82.6, 44.2, 38.7, 28.3.

1-Acetyl-6-(naphthalen-2-ylmethyl)-2,3-dihydroquinolin-4(1H)-

one (6a). This compound was synthesized following general

procedure F using 5a (50 mg, 0.19 mmol, 1.0 equiv), 4,4,5,5-

tetramethyl-2-(naphthalen-2-ylmethyl)-1,3,2-dioxaborolane (100 mg,

0.37 mmol, 2.0 equiv), K₂CO₃(78 mg, 0.56 mmol, 3.0 equiv), and

Pd(dppf)Cl₂(14 mg, 0.019 mmol, 0.1 equiv). The contents were

placed in a microwave tube and reacted in a microwave with a max

temp of 110 °C and max power of 250 W for 30 min with the

“Powermax” option enabled. Once the crude mixture was ﬁltered

through Celite, the solvent was removed, and the residue was puriﬁed

via silica gel chromatography (equilibrated in 100% hex, run in 1:1

EA:hex) to yield the title compound (51 mg, 83.6%) as a clear,

colorless oil. ¹H NMR (500 MHz, CDCl₃): δ 7.91 (s, 1H), 7.80−7.74

(m, 3H), 7.63 (s, 1H), 7.48−7.36 (m, 3H), 7.31−7.26 (m, 1H), 4.24−

4.15 (m, 2H), 4.13 (s, 2H), 2.80−2.69 (m, 2H), 2.29 (s, 3H). ¹³C

NMR (126 MHz, CDCl₃): δ 194.0, 169.2, 142.1, 138.6, 137.4, 134.6,

133.5, 132.1, 128.3, 127.60, 127.57, 127.5, 127.3, 127.2, 127.1, 126.1,

125.9, 125.5, 124.2, 43.9, 41.3, 39.4, 23.0.

tert-Butyl 6-(Cyclohexylmethyl)-4-oxo-3,4-dihydroquinoline-

1(2H)-carboxylate (6b). This compound was synthesized following

general procedure G using 5b (100 mg, 0.31 mmol, 1.0 equiv),

(cyclohexylmethyl)boronic acid (87 mg, 0.61 mmol, 2.0 equiv),

K₂CO₃(127 mg, 0.92 mmol, 3.0 equiv), Ag₂O (178 mg, 0.77 mmol,

2.5 equiv), and Pd(dppf)Cl₂(22 mg, 0.031 mmol, 0.1 equiv). The

contents were placed in a microwave tube and reacted in a microwave

with a max temp of 80 °C and max power of 300 W for 60 min with

the “Powermax” option disabled. Once ﬁltered through Celite, the

solvent was removed, and the crude residue puriﬁed via silica gel

chromatography (equilibrated in 100% petroleum ether, run in 5:1

petroleum ether:Et₂O) to yield the title compound as a clear, colorless

3-Bromo-N-(4-bromophenyl)propanamide (2). This compound

was synthesized following general procedure A starting from

commercially available 1 (2.0 g, 11.6 mmol, 1.0 equiv), K₂CO₃(3.29

g, 23.8 mmol, 2.05 equiv), and 3-bromopropionyl chloride (1.20 mL,

11.9 mmol, 1.02 equiv) to yield the title compound as an oﬀ-white

solid (3.53 g, 98.9%) with no additional puriﬁcation necessary after

1

quenching and workup. H NMR (500 MHz, CDCl₃): δ 7.47−7.39

(m, 4H), 3.70 (t, J = 6.5 Hz, 2H), 2.94 (t, J = 6.5 Hz, 2H). ¹³C NMR

(126 MHz, CDCl₃): δ 167.9, 136.4, 132.0, 128.3, 121.6, 40.7, 26.8.

1-(4-Bromophenyl)azetidin-2-one (3). This compound was

synthesized following general procedure B starting with 2 (3.53 g,

11.5 mmol, 1.0 equiv) and NaOtBu (1.16 g, 12.1 mmol, 1.05 equiv).

Following solvent removal and workup, the crude product was

chromatographed on silica gel (equilibrated in 100% hex, run in 1:4

EA:hex) to yield the title compound as a pure white ﬂaky solid (1.36 g,

1

oil (81 mg, 77.1%). H NMR (500 MHz, CDCl₃): δ 7.75 (d, J = 2.2

Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 2.3 Hz, 1H), 4.19−4.08

(m, 2H), 2.82−2.71 (m, 2H), 2.47 (d, J = 7.1 Hz, 2H), 1.77−1.59 (m,

5H), 1.55 (s, 9H), 1.51 (s, 1H), 1.18−1.12 (m, 2H), 0.97−0.90 (m,

3H). ¹³C NMR (126 MHz, CDCl₃): δ 194.5, 152.8, 137.1, 135.0,

133.9, 125.5, 124.6, 123.3, 82.0, 44.3, 43.2, 39.6, 39.1, 36.0, 33.0, 28.3,

26.2.

1-Acetyl-6-(cyclohexylmethyl)-2,3-dihydroquinolin-4(1H)-one

(6c). This compound was synthesized following general procedure G

using 5a (100 mg, 0.37 mmol, 1.0 equiv), (cyclohexylmethyl)boronic

acid (58 mg, 0.41 mmol, 1.1 equiv), K₂CO₃(155 mg, 1.12 mmol, 3.0

equiv), Ag₂O (216 mg, 0.93 mmol, 2.5 equiv), and Pd(dppf)Cl₂(27

mg, 0.37 mmol, 0.1 equiv). The contents were placed in a microwave

tube and reacted in a microwave with a max temp of 80 °C and max

power of 300 W for 60 min with the “Powermax” option disabled.

Once ﬁltered through Celite, the crude residue was puriﬁed via silica

gel chromatography (equilibrated in 100% hex, run in 1:3 EA:hex) to

yield the title compound as a clear, colorless oil (30 mg, 28.3%).

Additionally, 51 mg of 5a was recovered; this was not considered when

calculating the percent yield. ¹H NMR (500 MHz, CDCl₃): δ 7.78 (s,

1H), 7.33 (d, J = 6.8 Hz, 1H), 4.29−4.19 (m, 2H), 2.79 (t, J = 6.2 Hz,

1

52.1%). H NMR (500 MHz, CDCl₃): δ 7.46−7.42 (m, 2H), 7.27−

7.22 (m, 2H), 3.62 (td, J = 4.6, 2.0 Hz, 2H), 3.13 (td, J = 4.6, 1.9 Hz,

2H). No ¹³C spectrum acquired.

6-Bromo-2,3-dihydroquinolin-4(1H)-one (4). This compound was

synthesized following general procedure C starting with 3 (1.36 g, 6

mmol, 1.0 equiv) and TfOH (1.60 mL, 18.0 mmol, 3.0 equiv) to yield

a crude yellow oil. Following workup, the crude material was

chromatographed on silica gel (equilibrated in 100% hex, run in 2:3

EA:hex) to yield the title compound as a pure yellow powder (739 mg,

54.5%). ¹H NMR (500 MHz, CDCl₃): δ 7.95 (d, J = 2.4 Hz, 1H), 7.35

(dd, J = 8.7, 2.4 Hz, 1H), 6.58 (d, J = 8.7 Hz, 1H), 4.43 (s, 1H), 3.58

(t, 2H), 2.69 (t, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 192.3, 150.6,

137.6, 130.0, 120.5, 117.7, 110.2, 42.0, 37.6.

1-Acetyl-6-bromo-2,3-dihydroquinolin-4(1H)-one (5a). This com-

pound was synthesized following general procedure D starting with 4

(739 mg, 3.27 mmol, 1.0 equiv) for 16 h. Following removal of

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2H), 2.50 (d, J = 7.1 Hz, 2H), 2.33 (s, 3H), 1.73−1.59 (m, 5H), 1.52

(m, 1H), 1.19−1.14 (m, 3H), 0.99−0.90 (m, 2H). ¹³C NMR (126

MHz, CDCl₃): δ 194.4, 169.3, 141.7, 139.1, 135.0, 127.8, 125.7, 123.8,

43.2, 39.6, 35.3, 34.1, 33.0, 26.4, 26.2, 23.1.

Additionally, 122 mg of starting material 10g was recovered; this was

not considered when calculating the percent yield. H NMR (500

1

MHz, CDCl₃): δ 7.70 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 5.9 Hz, 2H),

7.15 (d, J = 8.3 Hz, 4H), 4.16 (t, J = 6.3 Hz, 2H), 3.43 (p, J = 6.9 Hz,

1H), 3.13 (dd, J = 13.7, 5.4 Hz, 1H), 2.88−2.83 (m, 1H), 2.83−2.78

(m, 1H), 2.78−2.74 (t, J = 6.3 Hz, 2H), 2.67 (dd, J = 13.6, 9.5 Hz,

1H), 2.13 (dq, J = 13.1, 7.8 Hz, 1H), 1.74 (dq, J = 13.9, 7.3 Hz, 1H),

1.56 (s, 9H). ¹³C NMR (126 MHz, CDCl₃): δ 194.3, 152.1, 146.4,

144.0, 142.2, 136.5, 134.8, 127.2, 126.5, 126.0, 124.6, 124.5, 123.6,

123.5, 82.0, 77.3, 77.0, 76.8, 46.2, 44.3, 40.5, 39.0, 31.6, 31.0, 28.3.

4-((2,3-Dihydro-1H-inden-2-yl)methyl)aniline (7f). This com-

pound was synthesized according to a published procedure¹to yield

the title compound. NMR data matched previously reported literature

values.¹

4-((2,3-Dihydro-1H-inden-1-yl)methyl)aniline (7g). This com-

pound was synthesized according to a published procedure²⁶to

yield the title compound. NMR data matched previously reported

literature values.²⁶

N-(4-Benzylphenyl)-3-bromopropanamide (8e). This compound

was synthesized according to general procedure A using commercially

available 7e (2.15 g, 11.7 mmol, 1.0 equiv) to yield the title compound

as a tan solid (2.63 g, 98.0%) with no additional puriﬁcation necessary.

¹H NMR (500 MHz, CDCl₃): δ 7.43 (d, J = 8.3 Hz, 2H), 7.29 (d, J =

7.4 Hz, 2H), 7.22−7.12 (5H, m), 3.95 (s, 2H), 3.71 (t, J = 6.6 Hz,

2H), 2.92 (t, J = 6.6 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 167.7,

141.9, 137.7, 135.4, 129.5, 128.9, 128.5, 126.1, 120.3, 41.3, 40.7, 27.1.

3-Bromo-N-(4-((2,3-dihydro-1H-inden-2-yl)methyl)phenyl)-

propanamide (8f). This compound was synthesized following general

procedure A using 7f (2.1 g, 9.5 mmol, 1.0 equiv) to yield the title

compound as a brown solid (2.87 g, 84.4%) with no additional

puriﬁcation necessary to yield the title compound. NMR data matched

previously reported values.¹

1-Acetyl-6-(naphthalen-1-ylmethyl)-2,3-dihydroquinolin-4(1H)-

one (6d). This compound was synthesized following general

procedure F using 5a (50 mg, 0.19 mmol, 1.0 equiv), 4,4,5,5-

tetramethyl-2-(naphthalen-1-ylmethyl)-1,3,2-dioxaborolane (100 mg,

0.37 mmol, 2.0 equiv), K₂CO₃(78 mg, 0.56 mmol, 3.0 equiv), and

Pd(dppf)Cl₂(14 mg, 0.019 mmol, 0.1 equiv). Contents were placed in

a microwave tube and reacted in a microwave with a max temp of 110

°C and max power of 250 W for 30 min with “Powermax” enabled.

Once the crude mixture was ﬁltered through Celite, the residue was

puriﬁed via silica gel chromatography (equilibrated in 100% hex, run in

9:1 EA:hex) to yield the title compound as a clear, colorless oil (31

1

mg, 50.8%). H NMR (500 MHz, CDCl₃): δ 7.96−7.90 (m, 2H),

7.89−7.83 (m, 1H), 7.80−7.75 (m, 1H), 7.49−7.40 (m, 3H), 7.32 (d, J

= 7.1 Hz, 2H), 4.44 (s, 2H), 4.23−4.13 (m, 2H), 2.75 (t, J = 6.2 Hz,

2H), 2.28 (s, 3H). ¹³C NMR (126 MHz, CDCl₃): δ 194.0, 169.2,

142.1, 138.3, 135.4, 134.2, 134.0, 133.6, 131.8, 128.8, 127.6, 127.5,

127.4, 126.1, 125.7, 125.5, 124.2, 124.0, 43.9, 39.5, 38.3, 23.0.

1-Acetyl-6-benzyl-2,3-dihydroquinolin-4(1H)-one (6e). This com-

pound was synthesized following general procedure D using 10e (170

mg, 0.72 mmol, 1.0 equiv) and excess Ac₂O. The reaction was stirred

at 100 °C for 16 h. Once complete, the solvent was removed under

reduced pressure, and the crude product was chromatographed on

silica gel (equilibrated in 100% hexane (hex), run in 3:2 EA:hex) to

1

yield the title compound as a clear, colorless oil (168 mg, 84.0%) H

NMR (400 MHzCDCl₃): δ 7.87−7.84 (bs, 1H), 7.37−7.33 (m, 2H),

7.31−7.24 (m, 2H), 7.23−7.15 (m, 3H), 4.20 (t, J = 6.2 Hz, 2H), 3.98

(s, 2H), 2.76 (t, J = 6.2 Hz, 2H), 2.30 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃): δ 194.0, 169.3, 142.1, 140.0, 138.9, 134.6, 128.8, 128.7, 127.6,

126.4, 126.0, 124.2, 43.9, 41.2, 39.5, 23.1.

3-Bromo-N-(4-((2,3-dihydro-1H-inden-1-yl)methyl)phenyl)-

propanamide (8g). This compound was synthesized following general

procedure A using 7g (473 mg, 2.1 mmol, 1.0 equiv), K₂CO₃(600 mg,

4.3 mmol, 2.05 equiv), and 3-bromopropionyl chloride (220 mL, 2.2

mmol, 1.02 equiv) to yield the title compound as an oﬀ-white solid

1-Acetyl-6-((2,3-dihydro-1H-inden-2-yl)methyl)-2,3-dihydroqui-

nolin-4(1H)-one (6f). This compound was synthesized following

general procedure D using 10f (55 mg, 0.24 mmol, 1.0 equiv). The

reaction was stirred at reﬂux for 20 h. Once the reaction was complete,

the solvent was removed, and the crude residue was puriﬁed using

silica gel chromatography (equilibrated in 100% hex, run in 2:3

1

(759 mg, quant.) with no additional puriﬁcation necessary. H NMR

(500 MHz, CDCl₃): δ 7.44 (d, J = 7.0 Hz, 2H), 7.34 (s, 1H), 7.26 (d, J

= 1.5 Hz, 0H), 7.22 (d, J = 6.8 Hz, 1H), 7.14 (dd, J = 19.7, 9.8 Hz,

6H), 5.29 (d, J = 1.5 Hz, 2H), 3.71 (t, J = 6.5 Hz, 2H), 3.41 (p, J = 7.1

Hz, 1H), 3.09 (dd, J = 13.7, 5.6 Hz, 1H), 2.94 (d, J = 6.6 Hz, 1H),

2.90−2.84 (m, 1H), 2.79 (dt, J = 15.7, 7.7 Hz, 1H), 2.66 (dd, J = 13.6,

9.2 Hz, 1H), 2.17−2.08 (m, 1H), 1.74 (dq, J = 15.1, 8.0, 7.6 Hz, 1H).

¹³C NMR (126 MHz, CDCl₃): δ 167.8, 146.7, 144.1, 137.4, 135.3,

1

EA:hex) to yield the title compound as a clear oil (39 mg, 50.4%). H

NMR (400 MHz, CDCl₃): δ 7.86 (s, 1H), 7.41 (d, J = 6.7 Hz, 2H),

7.20−7.07 (m, 4H), 4.24 (t, J = 6.3 Hz, 2H), 3.00 (dd, J = 15.3, 6.3 Hz,

2H), 2.87−2.76 (m, 5H), 2.67 (dd, J = 15.4, 6.2 Hz, 2H), 2.35 (s, 3H).

¹³C NMR (101 MHz, CDCl₃): δ 194.2, 169.4, 142.7, 141.9, 139.0,

134.7, 127.5, 126.2, 125.9, 124.4, 124.0, 43.9, 41.1, 40.7, 39.4, 38.7,

23.1.

129.6, 126.5, 126.0, 124.5, 123.7, 120.0, 53.4, 46.4, 40.8, 31.8, 31.1,

27.1.

1-Acetyl-6-((2,3-dihydro-1H-inden-1-yl)methyl)-2,3-dihydroqui-

nolin-4(1H)-one (6g). This compound was synthesized following

general procedure D using 10g (245 mg, 0.88 mmol, 1.0 equiv). The

reaction was stirred at reﬂux for 16 h. Once the reaction was complete,

the solvent was removed, and the crude residue was puriﬁed using

silica gel chromatography (equilibrated in 100% hex, run in 2:3

EA:hex) to yield the title compound as a clear, colorless oil (190 mg,

67.4%). ¹H NMR (500 MHz, CDCl₃): δ 7.88 (s, 1H), 7.36 (d, J = 6.2

Hz, 1H), 7.21 (d, J = 5.7 Hz, 1H), 7.18−7.09 (m, 4H), 4.23 (s, 2H),

3.44 (p, J = 7.0 Hz, 1H), 3.15 (dd, J = 13.7, 5.5 Hz, 1H), 2.88 (ddd, J =

14.1, 8.2, 5.5 Hz, 2H), 2.84−2.75 (m, 4H), 2.69 (dd, J = 13.7, 9.5 Hz,

1H), 2.34 (s, 3H), 2.13 (ddd, J = 13.0, 10.4, 6.7 Hz, 1H), 1.81−1.67

(m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 194.0, 169.1, 146.1, 143.8,

141.9, 138.3, 134.7, 127.5, 126.5, 125.9, 125.7, 124.5, 123.9, 123.5,

46.0, 43.8, 40.4, 39.4, 31.5, 30.9, 23.0.

tert-Butyl 6-((2,3-Dihydro-1H-inden-1-yl)methyl)-4-oxo-3,4-dihy-

droquinoline-1(2H)-carboxylate (6h). This compound was synthe-

sized following general procedure E using 10g (214 mg, 0.77 mmol,

1.0 equiv), Boc₂O (337 mg, 1.54 mmol, 2.0 equiv), DMAP (9 mg,

0.077 mmol, 0.1 equiv), and DIPEA (0.268 mL, 1.54 mmol, 2.0 equiv).

The reaction was stirred at reﬂux for 16 h. Once enough starting

material was converted to product, the crude yellow oil was puriﬁed

using silica gel chromatography (equilibrated in 100% hex, run in 2:3

EA:hex) to yield the title compound as a yellow oil (83 mg, 28.5%).

1-(4-Benzylphenyl)azetidin-2-one (9e). This compound was

synthesized following general procedure B using 8e (3.63 g, 1.1

mmol, 1.0 equiv) and NaOtBu (1.15 g, 1.2 mmol, 1.05 equiv) in anhyd

DMF. Following aqueous washes, the title compound was isolated as a

tan solid (2.70 g, quant.) and was taken ahead to the next step

(formation of 10e) without puriﬁcation, isolation, or characterization.

1-(4-((2,3-Dihydro-1H-inden-2-yl)methyl)phenyl)azetidin-2-one

(9f). This compound was synthesized following general procedure B

using 8f (2.87 g, 8.0 mmol, 1.0 equiv) and NaOtBu (808 mg, 8.41

mmol, 1.05 equiv) to yield the title compound as a brown solid (2.22

g, quant.) with no additional puriﬁcation necessary. NMR data

matched previously reported values.¹

1-(4-((2,3-Dihydro-1H-inden-1-yl)methyl)phenyl)azetidin-2-one

(9g). This compound was synthesized following general procedure B

using 8g (759 mg, 2.12 mmol, 1.0 equiv) and NaOtBu (214 mg, 2.22

mmol, 1.05 equiv) to yield the title compound as a light tan solid (512

1

mg, 87.4%) with no additional puriﬁcation necessary. H NMR (500

MHz, CDCl₃): δ 7.29 (d, J = 8.3 Hz, 2H), 7.25−7.05 (m, 8H), 3.60 (t,

J = 4.4 Hz, 2H), 3.40 (p, J = 7.1 Hz, 1H), 3.09 (t, J = 4.5 Hz, 3H), 3.06

(d, J = 6.0 Hz, 1H), 2.90−2.73 (m, 3H), 2.66 (dd, J = 13.6, 9.0 Hz,

1H), 2.11 (ddd, J = 15.9, 7.9, 5.4 Hz, 1H), 1.73 (dq, J = 15.1, 7.2 Hz,

1H). ¹³C NMR (126 MHz, CDCl₃): δ 164.2, 146.6, 144.0, 136.6,

J

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136.4, 129.6, 126.4, 125.9, 124.5, 123.7, 116.0, 46.4, 40.8, 38.0, 36.0,

31.7, 31.1.

reaction mixture was transferred to a round-bottom ﬂask containing

NaBH₄(30 mg, 0.785 mmol, 4.0 equiv) and stirred at room

temperature for 3 h before being quenched with MeOH. Once the

resultant solid was removed, the crude residue was puriﬁed using silica

gel chromatography (equilibrated in 100% hex, run in 4:1 EA:hex) to

yield the title compound as a clear, yellow oil (20 mg, 26.1% from 6c)

that was taken ahead to the next step (formation of 13c) without

further characterization.

6-Benzyl-2,3-dihydroquinolin-4(1H)-one (10e). This compound

was synthesized following general procedure C using 9e (2.70 g, 1.1

mmol, 1.0 equiv) and TfOH (3.0 mL, 3.4 mmol, 3.0 equiv). Following

column puriﬁcation (equilibrated in 100% hex, run in 2:3 EA:hex), the

title compound was isolated as a yellow solid (1.91 g, 70.7%). NMR

data matched previously reported values.¹

6-((2,3-Dihydro-1H-inden-2-yl)methyl)-2,3-dihydroquinolin-

4(1H)-one (10f). This compound was synthesized following general

procedure C using 9f (2.22 g, 8.00 mmol, 1.0 equiv) and TfOH (2.12

mL, 24.0 mmol, 3.0 equiv) to yield a crude dark red oil. Crude residue

was puriﬁed using silica gel chromatography (equilibrated in 100%, run

in 1:3 EA:hex) to yield the title compound as a yellow powder (1.55 g,

69.7%). NMR data matched previously reported values.¹

(R)-N-((R)-1-Acetyl-6-(naphthalen-1-ylmethyl)-1,2,3,4-tetrahy-

droquinolin-4-yl)-2-methylpropane-2-sulﬁnamide (12d). This com-

pound was synthesized following general procedure H using 6d (31

mg, 0.0941 mmol, 1.0 equiv), (R)-2-methylpropane-2-sulﬁnamide (34

mg, 0.282 mmol, 3.0 equiv), and Ti(OEt)₄(0.118 mL, 0.565 mmol,

6.0 equiv) to form the (R)-tert-butanesulﬁnyl imine intermediate

(11d) in situ. Once suﬃcient ketone was converted to imine

intermediate (after 40 h), the reaction mixture was transferred to a

round-bottom ﬂask containing NaBH₄(21 mg, 0.565 mmol, 6.0 equiv)

and stirred at room temperature for 3 h before being quenched with

MeOH. The resultant solid was removed to yield a clear, colorless

residue (41 mg, quant.) that was taken ahead to the next step

(formation of 13d) without further puriﬁcation, isolation, or

characterization.

6-((2,3-Dihydro-1H-inden-1-yl)methyl)-2,3-dihydroquinolin-

4(1H)-one (10g). This compound was synthesized following general

procedure C using 9g (512 mg, 1.85 mmol, 1.0 equiv) and TfOH

(0.490 mL, 5.54 mmol, 3.0 equiv) to yield the title compound as a

slightly impure yellow solid (459 mg, 89.6%) with no additional

1

puriﬁcation necessary. H NMR (500 MHz, CDCl₃): δ 7.70 (s, 1H),

7.19 (s, 1H), 7.11 (m, 4H), 6.60 (d, J = 8.3 Hz, 1H), 3.52 (t, J = 6.5

Hz, 2H), 3.37−3.32 (m, 1H), 3.01 (dd, J = 13.7, 5.0 Hz, 1H), 2.85 (m,

1H), 2.77 (m, 1H), 2.67 (m, 2H), 2.56 (m, 1H), 2.11 (dt, J = 12.3, 6.5

Hz, 1H), 1.72 (dq, J = 13.8, 7.4, 6.9 Hz, 1H). ¹³C NMR (126 MHz,

CDCl₃): δ 193.9, 150.5, 146.6, 144.0, 136.2, 130.2, 127.1, 126.3, 125.9,

124.4, 123.6, 119.0, 115.8, 46.3, 42.3, 40.2, 38.1, 31.5, 31.0.

(R)-N-((R)-1-Acetyl-6-(naphthalen-2-ylmethyl)-1,2,3,4-tetrahy-

droquinolin-4-yl)-2-methylpropane-2-sulﬁnamide (12a). This com-

pound was synthesized following general procedure H from 6a (51

mg, 0.155 mmol, 1.0 equiv), (R)-2-methylpropane-2-sulﬁnamide (56

mg, 0.464 mmol, 3.0 equiv), and Ti(OEt)₄(0.195 mL, 0.929 mmol,

6.0 equiv) to form the (R)-tert-butanesulﬁnyl imine intermediate

(11a) in situ. Once suﬃcient ketone was converted to imine

intermediate (after 40 h), the reaction mixture was transferred to a

round-bottom ﬂask containing NaBH₄(35 mg, 0.929 mmol, 6.0 equiv)

and stirred at room temperature for 3 h before being quenched with

MeOH. Once the resultant solid was removed, the crude residue was

puriﬁed using silica gel chromatography (equilibrated in 100% hex, run

in 100% EA) to yield the title compound as a clear, colorless oil (67

mg, quant. from 6a), which was taken ahead to the next step

(formation of 13a) without further characterization.

tert-Butyl(R)-4-(((R)-tert-butylsulﬁnyl)amino)-6-(cyclohexylmeth-

yl)-3,4-dihydroquinoline-1(2H)-carboxylate (12b). This compound

was synthesized following general procedure H using 6b (78 mg, 0.227

mmol, 1.0 equiv), (R)-2-methylpropane-2-sulﬁnamide (55 mg, 0.454

mmol, 2.0 equiv), and Ti(OEt)₄(0.191 mL, 0.909 mmol, 4.0 equiv) to

form the (R)-tert-butanesulﬁnyl imine intermediate (11b) in situ.

Once suﬃcient ketone was converted to imine intermediate (after 40

h), the reaction mixture was transferred to a round-bottom ﬂask

containing NaBH₄(34 mg, 0.909 mmol, 4.0 equiv) and stirred at room

temperature for 3 h before being quenched with MeOH. Once the

resultant solid was removed, the crude residue was puriﬁed using silica

gel chromatography (equilibrated in 100% hex, run in 1:3 EA:hex) to

yield the title compound as a dark, yellow oil (102 mg, 23.5% from

6b). ¹H NMR (500 MHz, CDCl₃): δ 7.69 (d, J = 8.6 Hz, 1H), 7.10 (s,

1H), 7.02 (d, J = 8.5 Hz, 1H), 4.55 (s, 1H), 3.99 (d, J = 12.8 Hz, 1H),

3.55 (t, J = 12.1 Hz, 1H), 3.27 (s, 1H), 2.46−2.37 (m, 2H), 2.17 (d, J

= 13.3 Hz, 1H), 1.97 (t, J = 12.8 Hz, 1H), 1.71−1.59 (m, 5H), 1.52 (s,

9H), 1.46 (d, J = 7.5 Hz, 1H), 1.22 (s, 9H), 1.17−1.12 (m, 2H), 0.99−

0.87 (m, 3H). ¹³C NMR (126 MHz, CDCl₃): δ 153.6, 136.9, 136.1,

129.3, 128.9, 128.0, 123.5, 81.0, 55.6, 50.3, 43.2, 40.0, 39.6, 33.1, 33.0,

29.6, 28.3, 26.5, 26.2, 22.6.

(R)-N-((R)-1-Acetyl-6-benzyl-1,2,3,4-tetrahydroquinolin-4-yl)-2-

methylpropane-2-sulﬁnamide (12e). This compound was synthe-

sized following general procedure H using 6e (168 mg, 0.601 mmol,

1.0 equiv), (R)-2-methylpropane-2-sulﬁnamide (219 mg, 1.80 mmol,

3.0 equiv), and Ti(OEt)₄(0.757 mL, 3.61 mmol, 6.0 equiv) to form

the (R)-tert-butanesulﬁnyl imine intermediate (11e) in situ. Once

suﬃcient ketone was converted to imine intermediate (after 40 h), the

reaction mixture was transferred to a round-bottom ﬂask containing

NaBH₄(137 mg, 3.61 mmol, 6.0 equiv) and stirred at room

temperature for 3 h before being quenched with MeOH. Once the

resultant solid was removed, the crude residue was puriﬁed using silica

gel chromatography (4:1 EA:hex) to yield the title compound as a

clear, colorless oil (105 mg, 45.5% from 6e), which was taken ahead to

the next step (formation of 13e) without further characterization.

(R)-N-((R)-1-Acetyl-6-((2,3-dihydro-1H-inden-2-yl)methyl)-1,2,3,4-

tetrahydroquinolin-4-yl)-2-methylpropane-2-sulﬁnamide (12f).

This compound was synthesized following general procedure H

using 6f (39 mg, 0.122 mmol, 1.0 equiv), (R)-2-methylpropane-2-

sulﬁnamide (30 mg, 0.244 mmol, 2.0 equiv), and Ti(OEt)₄(0.102 mL,

0.488 mmol, 4.0 equiv) to form the (R)-tert-butanesulﬁnyl imine

intermediate (11f) in situ. Once suﬃcient ketone was converted to

imine intermediate (after 24 h), the reaction mixture was transferred

to a round-bottom ﬂask containing NaBH₄(19 mg, 0.488 mmol, 4.0

equiv) and stirred at room temperature for 3 h before being quenched

with MeOH. Once the resultant solid was removed, the crude residue

was puriﬁed using silica gel chromatography (equilibrated in 100% hex,

run in 9:1 EA:hex) to yield the title compound as a clear, colorless oil

(46 mg, 88.9% from 6f). ¹H NMR (500 MHz, CDCl³): δ 7.40 (s, 1H),

7.29 (s, 1H), 7.21−7.15 (m, 2H), 7.15−7.09 (m, 3H), 4.77−4.66 (m,

1H), 4.57 (bs, 1H), 3.01 (dd, J = 15.3, 6.0 Hz, 2H), 2.83−2.72 (m,

3H), 2.67 (dd, J = 15.2, 5.7 Hz, 2H), 2.43−2.34 (m, 1H), 2.28−2.20

(m, 4H), 2.13−2.02 (m, 1H), 1.22 (s, 9H). No ¹³C spectrum acquired.

(R)-N-((4R)-1-Acetyl-6-((2,3-dihydro-1H-inden-1-yl)methyl)-

1,2,3,4-tetrahydroquinolin-4-yl)-2-methylpropane-2-sulﬁnamide

(12g). This compound was synthesized as a mixture of diastereomers

following general procedure H using 6g (176 mg, 0.551 mmol, 1.0

equiv), (R)-2-methylpropane-2-sulﬁnamide (200 mg, 1.65 mmol, 3.0

equiv), and Ti(OEt)₄(0.693 mL, 3.31 mmol, 6.0 equiv) to form the

(R)-tert-butanesulﬁnyl imine intermediate (11g) in situ. Once

suﬃcient ketone was converted to imine intermediate (after 16 h),

the reaction mixture was transferred to a round-bottom ﬂask

containing NaBH₄(125 mg, 3.31 mmol, 6.0 equiv) and stirred at

room temperature for 3 h before being quenched with MeOH. Once

the resultant solid was removed, the crude residue was puriﬁed using

silica gel chromatography (equilibrated and run in 100% EA) to yield

the title compound as a clear, colorless oil of a mixture of

diastereomers (219 mg, 93.6% from 6g). ¹H NMR (500 MHz,

CDCl₃): δ 7.29−7.25 (m, 1H), 7.24−7.19 (m, 1H), 7.17−7.09 (m,

(R)-N-((R)-1-Acetyl-6-(cyclohexylmethyl)-1,2,3,4-tetrahydroquino-

lin-4-yl)-2-methylpropane-2-sulﬁnamide (12c). This compound was

synthesized following general procedure H using 6c (56 mg, 0.196

mmol, 1.0 equiv), (R)-2-methylpropane-2-sulﬁnamide (48 mg, 0.392

mmol, 2.0 equiv), and Ti(OEt)₄(0.165 mL, 0.785 mmol, 4.0 equiv) to

form the (R)-tert-butanesulﬁnyl imine intermediate (11c) in situ. Once

suﬃcient ketone was converted to imine intermediate (after 40 h), the

K

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4H), 7.09−7.05 (m, 1H), 4.54 (bs, 1H), 3.99−3.87 (m, 2H), 3.84−

3.72 (m, 1H), 3.44 (bs, 1H), 3.19−3.04 (m, 1H), 2.96−2.75 (m, 2H),

2.68 (q, J = 12.4 Hz, 1H), 2.25 (s, 3H), 2.23−2.19 (m, 1H), 2.19−2.12

(m, 1H) 2.12−2.05 (m, 1H), 1.84−1.71 (m, 1H), 1.24−1.14 (s, 9H).

¹³C NMR (126 MHz, CDCl₃): δ 169.7, 146.3, 143.8, 138.1, 136.4,

128.7, 128.6, 126.4, 125.9, 125.8, 124.4, 124.3, 123.6, 123.5, 55.6, 55.0,

50.9, 46.1, 46.0, 40.6, 40.5, 31.7, 31.6, 30.9, 30.9, 30.5, 23.2, 22.4, 22.0,

20.9, 14.0.

5H), 1.61−1.53 (m, 1H), 1.26−1.19 (m, 3H), 1.02−0.94 (m, 2H). No

¹³C spectrum acquired.

(R)-1-Acetyl-6-(naphthalen-1-ylmethyl)-1,2,3,4-tetrahydroquino-

lin-4-aminium Chloride (13d). This compound was synthesized

following general procedure I using 12d (41 mg (from theoretical yield

of 12d), 0.0943 mmol, 1.0 equiv) and concd HCl (14 μL, 0.566 mmol,

6.0 equiv). After removing the solvent, the residue was resuspended in

Et₂O, and the solid crashed out. The solid was ﬁltered oﬀ, washed 3×

with fresh Et₂O, and dried to yield the product as a white solid (22 mg,

tert-Butyl(4R)-4-(((R)-tert-butylsulﬁnyl)amino)-6-((2,3-dihydro-

1H-inden-1-yl)methyl)-3,4-dihydroquinoline-1(2H)-carboxylate

(12h). This compound was synthesized as a mixture of diastereomers

following general procedure H using 6h (83 mg, 0.220 mmol, 1.0

equiv), (R)-2-methylpropane-2-sulﬁnamide (80 mg, 0.660 mmol, 3.0

equiv), and Ti(OEt)₄(0.277 mL, 0.132 mmol, 6.0 equiv) to form the

(R)-tert-butanesulﬁnyl imine intermediate (11h) in situ. Once

suﬃcient ketone was converted to imine intermediate (after 16 h),

the reaction mixture was transferred to a round-bottom ﬂask

containing NaBH₄(50 mg, 0.132 mmol, 6.0 equiv) and stirred at

room temperature for 3 h before being quenched with MeOH. Once

the resultant solid was removed, the crude residue was puriﬁed using

silica gel chromatography (equilibrated in 100%, run in 2:3 EA:hex) to

yield the title compound as a clear, colorless oil of a mixture of

diastereomers (27 mg, 25.7% from 6h). ¹H NMR (500 MHz, CDCl₃):

δ 7.73 (d, J = 8.0 Hz, 1H), 7.31−7.03 (m, 7H), 4.55 (s, 1H), 4.00 (d, J

= 12.8 Hz, 1H), 3.58 (t, J = 12.0 Hz, 1H), 3.44−3.35 (m, 1H), 3.28 (s,

1H), 3.07 (ddd, J = 29.4, 13.6, 5.2 Hz, 1H), 2.94−2.84 (m, 1H), 2.83−

2.74 (m, 1H), 2.64 (ddd, J = 19.0, 13.7, 9.6 Hz, 1H), 2.23−2.10 (m,

2H), 1.99 (dd, J = 23.8, 10.6 Hz, 1H), 1.76 (tt, J = 14.4, 7.6 Hz, 2H),

1.53 (s, 9H), 1.44 (bs, 3H), 1.22 (s, 9H). ¹³C NMR (126 MHz,

CDCl₃): δ 153.6, 146.7, 144.1, 136.41, 136.37, 136.3, 129.22, 129.16,

128.9, 128.8, 128.31, 128.28, 126.5, 126.0, 125.9, 124.49, 124.46,

123.8, 123.72, 123.69, 81.1, 55.6, 50.3, 46.4, 46.3, 40.6, 40.5, 40.03,

39.99, 31.9, 31.7, 31.06, 31.05, 29.5, 28.4, 24.2, 22.6.

(R)-1-Acetyl-6-(naphthalen-2-ylmethyl)-1,2,3,4-tetrahydroquino-

lin-4-aminium Chloride (13a). This compound was synthesized

following general procedure I using 12a (56 mg, 0.135 mmol, 1.0

equiv). After removing the solvent, the residue was resuspended in

Et₂O, and the solid did not crash out. The residue was washed 3× with

fresh Et₂₁O and dried to yield the product as a clear, yellow oil (56 mg,

quant.). H NMR (500 MHz, CD₃OD): δ 7.83−7.67 (m, 4H), 7.54−

7.24 (m, 6H), 4.56 (dd, J = 7.7, 4.0 Hz, 1H), 4.16 (s, 2H), 3.96−3.81

(m, 2H), 2.46−2.35 (m, 1H), 2.26 (s, 3H), 2.13−2.09 (m, 1H). ¹³C

NMR (126 MHz, CD₃OD): δ 172.5, 140.8, 139.6, 138.1, 135.1, 133.6,

130.8, 129.2, 128.6, 128.5, 128.4, 128.1, 127.1, 126.5, 48.2, 42.3, 29.8,

23.3, 21.5.

1

62.9%). H NMR (500 MHz, CD₃OD): δ 8.02 (d, J = 7.1 Hz, 1H),

7.87 (d, J = 8.1, 1H), 7.78 (d, J = 7.1 Hz, 1H), 7.50−7.43 (m, 4H),

7.43−7.39 (m, 2H), 7.30−7.23 (m, 1H), 4.53 (t, J = 6.7 Hz, 1H), 4.49

(s, 2H), 3.87 (dtd, J = 8.8, 6.7, 6.2, 3.4 Hz, 2H), 2.39 (dq, J = 12.5, 5.7

Hz, 1H), 2.25 (s, 3H), 2.04 (dd, J = 14.2, 7.1 Hz, 1H). ¹³C NMR (126

MHz, CD₃OD): δ 172.5, 137.3, 135.6, 134.6, 133.3, 130.5, 129.8,

128.8, 128.7, 128.5, 127.08, 127.07, 126.71, 126.70, 126.6, 126.5,

125.3, 48.2, 39.4, 29.9, 23.3.

(R)-1-Acetyl-6-benzyl-1,2,3,4-tetrahydroquinolin-4-aminium

Chloride (13e). This compound was synthesized following general

procedure I using 12e (105 mg 0.273 mmol, 1.0 equiv) and concd

HCl (40 μL, 1.64 mmol, 6.0 equiv). After removing the solvent, the

residue was resuspended in Et₂O, and the solid crashed out. The solid

was ﬁltered oﬀ, washed 3× with fresh Et₂O, and dried to yield the

product as a white solid (68 mg, 78.2%). ¹H NMR (500 MHz,

CD₃OD): δ 7.43−7.37 (m, 1H), 7.32−7.21 (m, 4H), 7.20−7.15 (m,

1H), 4.62−4.53 (m, 1H), 4.01 (s, 2H), 3.95−3.86 (m, 2H), 2.49−2.38

(m, 1H), 2.28 (s, 3H), 2.07 (s, 1H). ¹³C NMR (126 MHz, CD₃OD): δ

172.4, 142.1, 140.9, 138.1, 130.7, 129.9, 129.6, 129.0, 127.3, 126.5,

48.3, 42.2, 29.9, 23.3.

(R)-1-Acetyl-6-((2,3-dihydro-1H-inden-2-yl)methyl)-1,2,3,4-tetra-

hydroquinolin-4-aminium Chloride (13f). This compound was

synthesized following general procedure I using 12f (46 mg 0.108

mmol, 1.0 equiv) and concd HCl (16 μL, 0.650 mmol, 6.0 equiv).

After removing the solvent, the residue was resuspended in Et₂O, and

the residue crashed out. The residue was gummy and sticky so it was

washed 3× with fresh Et₂O and dried to yield the product as a tan

solid (39 mg, quant.). The solid was taken ahead to the next reaction

(formation of 14f) without further isolation, puriﬁcation, or character-

ization.

(4R)-1-Acetyl-6-((2,3-dihydro-1H-inden-1-yl)methyl)-1,2,3,4-tetra-

hydroquinolin-4-aminium Chloride (13g). This compound was

synthesized as a mixture of diastereomers following general procedure

I using 12g (219 mg 0.516 mmol, 1.0 equiv) and concd HCl (76 μL,

3.10 mmol, 6.0 equiv). After removing the solvent, the residue was

resuspended in Et₂O, and the solid crashed out. The solid was ﬁltered

oﬀ, washed 3× with fresh Et₂O, and dried to yield the title compound

(R)-1-(tert-Butoxycarbonyl)-6-(cyclohexylmethyl)-1,2,3,4-tetrahy-

droquinolin-4-aminium Chloride (13b). This compound was

synthesized following general procedure I using 12b (22 mg, 0.0490

mmol, 1.0 equiv). After removing the solvent, the residue was

resuspended in Et₂O, and the solid crashed out. After washing the

solid 3× with fresh Et₂O, the remaining Et₂O was decanted, yielding

1

as a white solid (135 mg, 73.4%). H NMR (500 MHz, CD₃OD): δ

7.46−7.38 (1H), 7.27 (bs, 1 H), 7.22−7.17 (m, 1H), 7.16−7.08 (m,

3H), 4.60 (s, 1H), 3.93 (qt, J = 8.3, 4.4 Hz, 2H), 3.53−3.44 (m, 1H),

3.17 (dt, J = 13.4, 6.6 Hz, 1H), 2.94−2.84 (m, 1H), 2.84−2.74 (m,

1H), 2.74−2.67 (m, 1H), 2.46 (dq, J = 11.5, 4.7 Hz, 1H), 2.30 (s, 3H),

2.18−2.05 (m, 2H), 1.78 (dp, J = 13.2, 6.6 Hz, 1H). ¹³C NMR (126

MHz, CD₃OD): δ 172.43, 172.41, 147.7, 147.6, 145.11, 145.08, 138.1,

131.0, 129.44, 129.37, 127.7, 127.10, 127.06, 126.3, 125.48, 125.47,

124.73, 124.70, 48.3, 47.6, 41.8, 41.8, 32.9, 32.8, 31.9, 30.0, 23.4, 23.3.

(4R)-1-(tert-Butoxycarbonyl)-6-((2,3-dihydro-1H-inden-1-yl)-

methyl)-1,2,3,4-tetrahydroquinolin-4-aminium Chloride (13h). This

compound was synthesized as a mixture of diastereomers following

general procedure I using 12h (27 mg 0.0559 mmol, 1.0 equiv) and

concd HCl (8 μL, 0.336 mmol, 6.0 equiv). After removing the solvent,

the residue was resuspended in Et₂O, and the solid crashed out. The

solid was ﬁltered oﬀ, washed 3× with fresh Et₂O, and dried to yield the

title compound as a white solid (10 mg, 43.5%). ¹H NMR (500 MHz,

CD₃OD): δ 7.71 (d, J = 8.5 Hz, 1H), 7.28 (s, 1H), 7.24−7.17 (m,

2H), 7.10 (m, 3H), 4.56 (d, J = 4.7 Hz, 1H), 3.95−3.78 (m, 2H),

3.51−3.40 (m, 1H), 3.13 (dt, J = 12.8, 6.4 Hz, 1H), 2.95−2.74 (m,

2H), 2.68 (dd, J = 13.5, 9.2 Hz, 1H), 2.39−2.30 (d, J = 4.1 Hz, 1H),

2.18−2.03 (m, 2H), 1.78 (dp, J = 13.2, 6.5 Hz, 1H), 1.54 (d, J = 1.4

Hz, 9H). ¹³C NMR (126 MHz, CD₃OD): δ 154.8, 147.7, 145.1,

1

the title compound as an oﬀ-white solid (10 mg, 53.5%). H NMR

(500 MHz, CDCl₃): δ 8.79 (s, 3H), 7.70 (d, J = 8.4 Hz, 1H), 7.32 (s,

1H), 7.06 (d, J = 8.4 Hz, 1H), 4.39 (s, 1H), 4.08 (d, J = 13.5 Hz, 1H),

3.64−3.54 (m, 1H), 2.40 (d, J = 7.0 Hz, 2H), 2.19 (s, 2H), 1.70−1.56

(m, 5H), 1.51 (s, 10H), 1.19−1.08 (m, 3H), 0.98−0.85 (m, 2H). ¹³C

NMR (126 MHz, CDCl₃): δ 153.1, 137.1, 136.3, 130.0, 129.4, 123.9,

122.1, 81.4, 47.6, 43.2, 39.9, 39.5, 33.1, 33.0, 28.3, 27.8, 26.5, 26.3,

26.3.

(R)-1-Acetyl-6-(cyclohexylmethyl)-1,2,3,4-tetrahydroquinolin-4-

aminium Chloride (13c). This compound was synthesized following

general procedure I using 12c (20 mg, 0.0512 mmol, 1.0 equiv) and

concd HCl (7.5 μL, 0.31 mmol, 6.0 equiv). After removing the solvent,

the residue was resuspended in Et₂O, and the solid crashed out. After

washing the solid 3× with fresh Et₂O, the remaining Et₂O was

1

decanted, yielding an oﬀ-white solid (12 mg, 72.7%). H NMR (500

MHz, CD₃OD): δ 7.27 (s, 1H), 7.21 (d, J = 8.2 Hz, 1H), 4.58 (t, J =

6.7 Hz, 1H), 3.91 (h, J = 8.3, 7.9 Hz, 2H), 2.53 (d, J = 7.1 Hz, 2H),

2.48−2.39 (m, 1H), 2.28 (s, 3H), 2.10−1.99 (m, 1H), 1.75−1.65 (m,

L

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Journal of Medicinal Chemistry

Article

138.41, 138.37, 138.0, 131.0, 130.9, 129.5, 129.4, 127.7, 127.1, 127.0,

125.7, 125.5, 124.7, 83.0, 47.6, 41.9, 41.7, 32.8, 31.9, 29.2, 28.5.

(S)-N-((R)-1-Acetyl-6-(naphthalen-2-ylmethyl)-1,2,3,4-tetrahydro-

quinolin-4-yl)-2-amino-3-(4-hydroxy-2,6-dimethylphenyl)-

propanamide (14a). This compound was synthesized following

general procedure J starting from the (R) amine intermediate 13a (56

mg, 0.153 mmol, 1.0 equiv) to yield crude product, which was puriﬁed

by semipreparative HPLC and then lyophilized to yield the title

compound as a TFA salt (17 mg, 30.4%). Starting material 13a was

recovered but not considered when calculating percent yield. ¹H NMR

(500 MHz, CD₃OD): δ 7.80−7.71 (m, 4H), 7.60 (s, 1H), 7.47−7.36

(m, 2H), 7.28 (dd, J = 8.6, 1.8 Hz, 1H), 7.19 (s, 1H), 7.12 (dd, J = 8.6,

2.1 Hz, 1H), 6.51 (s, 2H), 4.94 (q, J = 6.8 Hz, 1H), 4.07 (s, 2H), 3.87

(dd, J = 11.5, 5.0 Hz, 1H), 3.24 (dd, J = 13.7, 11.5 Hz, 1H), 3.04 (dd, J

= 13.7, 5.0 Hz, 1H), 2.26 (s, 6H), 2.17 (s, 3H), 1.89−1.79 (m, 1H),

1.51−1.41 (m, 1H). ¹³C NMR (126 MHz, CD₃OD): δ 172.5, 169.2,

157.4, 151.9, 140.1, 139.8, 135.1, 133.6, 129.5, 129.1, 128.6, 128.5,

128.4, 127.9, 127.1, 126.5, 125.8, 123.3, 116.5, 53.5, 47.2, 47.1, 42.3,

31.9, 31.2, 23.4, 20.4. HPLC (gradient A): retention time = 38.8. ESI-

MS: 522.3 [M + H]⁺.

(S)-2-Amino-N-((R)-6-(cyclohexylmethyl)-1,2,3,4-tetrahydroqui-

nolin-4-yl)-3-(4-hydroxy-2,6-dimethylphenyl)propanamide (14b).

This compound was synthesized following general procedure J starting

from the (R) amine intermediate 13b (10 mg, 0.0262 mmol, 1.0

equiv) to yield crude product, which was puriﬁed by semipreparative

HPLC and then lyophilized to yield the title compound as a TFA salt

(9 mg, 63.2%). ¹H NMR (500 MHz, CD₃OD): δ 8.09 (d, J = 8.0 Hz,

1H), 6.85 (d, J = 2.0 Hz, 1H), 6.83 (s, 1H), 6.61 (d, J = 8.1 Hz, 1H),

6.39 (s, 2H), 4.89 (q, J = 5.0 Hz, 1H), 3.78 (dd, J = 11.6, 5.1 Hz, 1H),

3.16 (dd, J = 13.6, 11.6 Hz, 1H), 2.99 (dt, J = 12.5, 4.3 Hz, 1H), 2.93

(dd, J = 13.7, 5.1 Hz, 1H), 2.52 (td, J = 11.7, 2.6 Hz, 1H), 2.31−2.22

(m, 2H), 2.18 (s, 6H), 1.77−1.67 (m, 1H), 1.61−1.48 (m, 5H), 1.47−

1.40 (m, 1H), 1.37−1.27 (m, 1H), 1.12−1.02 (m, 3H), 0.87−0.74 (m,

2H). ¹³C NMR (126 MHz, CD₃OD): δ 168.6, 157.4, 140.0, 136.3,

131.8, 131.0, 123.3, 118.9, 116.4, 53.4, 45.8, 44.3, 41.2, 39.0, 34.3, 34.1,

31.9, 28.7, 27.6, 27.38, 27.36, 20.5. HPLC (gradient A): retention time

= 31.0. ESI-MS: 458.2 [M + Na]⁺.

(S)-N-((R)-1-Acetyl-6-(cyclohexylmethyl)-1,2,3,4-tetrahydroquino-

lin-4-yl)-2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanamide

(14c). This compound was synthesized following general procedure J

starting from the (R) amine intermediate 13c (12 mg, 0.0372 mmol,

1.0 equiv) to yield crude product, which was puriﬁed by semi-

preparative HPLC and then lyophilized to yield the title compound as

a TFA salt (12 mg, 55.5%). ¹H NMR (500 MHz, CD₃OD): δ 8.15 (d,

J = 8.1 Hz, 1H), 7.06−7.00 (m, 2H), 6.51 (s, 2H), 4.99−4.93 (m, 1H),

3.88 (dd, J = 11.5, 5.0 Hz, 1H), 3.79 (s, 1H), 3.26 (dd, J = 13.6, 11.5

Hz, 1H), 3.22−3.10 (m, 1H), 3.05 (dd, J = 13.7, 5.0 Hz, 1H), 2.48−

2.38 (m, 2H), 2.28 (s, 6H), 2.19 (s, 3H), 1.90−1.82 (m, 1H), 1.74−

1.59 (m, 5H), 1.53−1.37 (m, 2H), 1.26−1.12 (m, 3H), 0.99−0.87 (m,

2H). ¹³C NMR (126 MHz, CD₃OD): δ 172.5, 169.2, 157.5, 140.1,

129.5, 125.4, 123.3, 116.5, 53.5, 47.0, 44.4, 41.0, 34.3, 34.2, 32.0, 31.3,

27.6, 27.36, 27.35, 23.33, 20.44. HPLC (gradient A): retention time =

39.5. ESI-MS: 478.3 [M + Na]⁺.

compound was synthesized following general procedure J starting from

the (R) amine intermediate 13e (68 mg, 0.215 mmol, 1.0 equiv) to

yield crude product, which was puriﬁed by semipreparative HPLC and

then lyophilized to yield the title compound as a TFA salt (16 mg,

12.7%). Starting material 13e was recovered but not considered when

calculating the percent yield. ¹H NMR (500 MHz, CD₃OD): δ 7.26−

7.20 (m, 2H), 7.18−7.12 (m, 4H), 7.06 (d, J = 8.3 Hz, 1H), 6.51 (s,

2H), 4.94 (t, J = 6.0 Hz, 1H), 3.94−3.83 (m, 3H), 3.27−3.22 (m, 2H),

3.16 (m, 1H), 3.05 (dd, J = 13.8, 5.0 Hz, 1H), 2.27 (s, 6H), 2.18 (s,

3H). No ¹³C NMR spectrum acquired. HPLC (gradient A): retention

time = 32.7. ESI-MS: 494.3 [M + Na]⁺.

(S)-N-((R)-1-Acetyl-6-((2,3-dihydro-1H-inden-2-yl)methyl)-1,2,3,4-

tetrahydroquinolin-4-yl)-2-amino-3-(4-hydroxy-2,6-

dimethylphenyl)propanamide (14f). This compound was synthesized

following general procedure J starting from the (R) amine

intermediate 13f (39 mg, 0.108 mmol, 1.0 equiv) to yield crude

product, which was puriﬁed by semipreparative HPLC and then

lyophilized to yield the title compound as a TFA salt (14 mg, 20.6%).

¹H NMR (500 MHz, CD₃OD): δ 7.12 (s, 4H), 7.09−7.04 (m, 2H),

6.51 (s, 2H), 4.97 (s, 1H), 3.85 (dd, J = 11.2, 4.3 Hz, 1H), 3.29−3.19

(m, 2H), 3.04 (dd, J = 13.7, 4.1 Hz, 2H), 2.98−2.87 (m, 2H), 2.73 (d,

J = 10.3 Hz, 3H), 2.62 (d, J = 15.6 Hz, 2H), 2.28 (s, 6H), 2.21 (s, 3H).

No ¹³C NMR spectrum acquired. HPLC (gradient A): retention time

= 38.4. ESI-MS: 534.3 [M + Na]⁺.

(S)-N-((R)-1-Acetyl-6-(((R/S)-2,3-dihydro-1H-inden-1-yl)methyl)-

1,2,3,4-tetrahydroquinolin-4-yl)-2-amino-3-(4-hydroxy-2,6-

dimethylphenyl)propanamide (14g). This compound was synthe-

sized following general procedure J starting from the (R) amine

intermediate 13g (20 mg, 0.056 mmol, 1.0 equiv) to yield crude

product, which was puriﬁed by semipreparative HPLC and then

lyophilized to yield the title compound as the TFA salt of a mixture of

diastereomers (8 mg, 22.9%). Starting material 13g was recovered but

1

not considered when calculating the percent yield. H NMR (500

MHz, CD₃OD): δ 7.21−6.98 (m, 7H), 6.52 (s, 2H), 5.03−4.93 (1,

2H), 3.87 (dt, J = 10.5, 4.9 Hz, 1H), 3.81 (s, 1H), 3.40 (q, J = 7.3 Hz,

1H), 3.30−3.21 (m, 1H), 3.08−3.00 (m, 2H), 2.90−2.81 (m, 1H),

2.80−2.70 (m, 1H), 2.67−2.55 (m, 1H), 2.28 (s, 6H), 2.21 (s, 3H),

2.15−2.03 (m, 1H), 1.94−1.82 (m, 1H), 1.71 (dq, J = 14.3, 7.4 Hz,

1H), 1.45 (s, 1H). No ¹³C NMR spectrum acquired. HPLC (gradient

A): retention time = 38.4. ESI-MS: 534.3 [M + Na]⁺.

(S)-2-Amino-N-((R)-6-(((R/S)-2,3-dihydro-1H-inden-1-yl)methyl)-

1,2,3,4-tetrahydroquinolin-4-yl)-3-(4-hydroxy-2,6-dimethylphenyl)-

propanamide (14h). This compound was synthesized following

general procedure J starting from the (R) amine intermediate 13h (10

mg, 0.0241 mmol, 1.0 equiv) as a mixture of diastereomers to yield

crude product, which was puriﬁed by semipreparative HPLC and then

lyophilized to yield the title compound as the TFA salt of a mixture of

diastereomers (5 mg, 35.7%). ¹H NMR (500 MHz, CD₃OD): δ 7.18−

7.14 (m, 1H), 7.12−7.06 (m, 2H), 7.05−7.00 (m, 1H), 6.97−6.91 (m,

2H), 6.62 (dd, J = 8.4, 3.4 Hz, 1H), 6.49 (s, 2H), 4.97 (t, J = 4.7 Hz,

1H), 3.86 (dt, J = 11.7, 4.8 Hz, 1H), 3.38−3.31 (m, 1H) 3.29−3.22

(m, 1H), 3.09−2.97 (m, 3H), 2.88−2.79 (m, 1H), 2.78−2.69 (m, 1H),

2.64−2.43 (m, 3H), 2.28 (s, 6H), 2.13−1.98 (m, 1H), 1.79 (t, J = 12.6

Hz, 1H), 1.69 (ddd, J = 15.1, 13.0, 7.0 Hz, 1H), 1.58−1.49 (m, 1H).

No ¹³C NMR spectrum acquired. HPLC (gradient A): retention time

= 30.7. ESI-MS: 492.2 [M + Na]⁺.

(S)-2-Amino-N-((R)-6-benzyl-1,2,3,4-tetrahydroquinolin-4-yl)-3-

(4-hydroxy-2,6-dimethylphenyl)propanamide (14i). This compound

was previously synthesized; see ref 1 for characterization details.

(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)-N-((R)-6-(naph-

thalen-2-ylmethyl)-1,2,3,4-tetrahydroquinolin-4-yl)propanamide

(14j). This compound was previously synthesized; see ref 1 for

characterization details.

(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)-N-((R)-6-(naph-

thalen-1-ylmethyl)-1,2,3,4-tetrahydroquinolin-4-yl)propanamide

(14k). This compound was previously synthesized; see ref 1 for

characterization details.

(S)-N-((R)-1-Acetyl-6-(naphthalen-1-ylmethyl)-1,2,3,4-tetrahydro-

quinolin-4-yl)-2-amino-3-(4-hydroxy-2,6-dimethylphenyl)-

propanamide (14d). This compound was synthesized following

general procedure J starting from the (R) amine intermediate 13d (22

mg, 0.060 mmol, 1.0 equiv) to yield crude product, which was puriﬁed

by semipreparative HPLC and then lyophilized to yield the title

compound as a TFA salt (4 mg, 12.9%). Starting material 13d was

1

recovered but not considered when calculating the percent yield. H

NMR (500 MHz, CD₃OD): δ 7.99−7.95 (m, 1H), 7.85 (d, J = 7.8 Hz,

1H), 7.78−7.74 (m, 1H), 7.48−7.37 (m, 4H), 7.27 (d, J = 7.0 Hz,

1H), 7.22−7.17 (m, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.51 (s, 2H), 4.93

(bs, 1H) 4.40 (s, 2H), 3.84 (dd, J = 11.4, 5.0 Hz, 1H), 3.24 (t, J = 12.6

Hz, 1H), 3.05−2.98 (m, 1H), 2.27 (d, J = 1.4 Hz, 6H), 2.17 (s, 3H),

1.86 (dt, J = 11.6, 5.7 Hz, 1H), 1.49 (bs, 1H). No ¹³C NMR spectrum

acquired. HPLC (gradient A): retention time = 38.3. ESI-MS: 544.3

[M + Na]⁺.

(S)-2-Amino-N-((R)-6-((2,3-dihydro-1H-inden-2-yl)methyl)-

1,2,3,4-tetrahydroquinolin-4-yl)-3-(4-hydroxy-2,6-dimethylphenyl)-

propanamide (14l). This compound was previously synthesized; see

ref 1 for characterization details.

(S)-N-((R)-1-Acetyl-6-benzyl-1,2,3,4-tetrahydroquinolin-4-yl)-2-

amino-3-(4-hydroxy-2,6-dimethylphenyl)propanamide (14e). This

M

DOI: 10.1021/acs.jmedchem.5b01270

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Journal of Medicinal Chemistry

Article

(S)-2-Amino-N-((R)-7-benzyl-1,2,3,4-tetrahydronaphthalen-1-yl)-

3-(4-hydroxy-2,6-dimethylphenyl) propanamide (14m). This com-

pound was synthesized following general procedure J starting from the

(R) amine intermediate 19m (44 mg, 0.161 mmol) to yield crude

product, which was puriﬁed by semipreparative HPLC and lyophilized

8.88 mmol, 2.0 equiv), and Ti(OEt)₄(3.73 mL, 17.771 mmol, 4 equiv)

to form the (R)-tert-butanesulﬁnyl imine intermediate (16) in situ.

Once suﬃcient ketone was converted to imine intermediate (after 48

h), the reaction mixture was transferred via cannula to a round-bottom

ﬂask containing NaBH₄(671.7 mg, 17.76 mmol, 4 equiv) and 13 mL

of THF in a xylene dry ice bath; after this addition, the solution was

stirred at room temperature for 3 h before being quenched with

MeOH. Once the resultant solid was removed, the crude residue was

puriﬁed using silica gel chromatography (1:9 EA:hex) to yield the title

1

to yield the title compound as a TFA salt (58 mg, 84%). H NMR

(400 MHz, CD₃OD): δ 8.09 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.8, 6.8

Hz, 2H), 7.16−7.07 (m, 3H), 7.04 (s, 1H), 6.91 (d, J = 1.2 Hz, 2H),

6.47 (s, 2H), 4.92 (s, 1H), 3.87 (d, J = 4.9 Hz, 1H), 3.83 (s, 2H), 3.24

(dd, J = 13.6, 11.5 Hz, 1H), 3.01 (dd, J = 13.7, 5.0 Hz, 1H), 2.62−2.51

(m, 2H), 2.26 (s, 6H), 1.69−1.45 (m, 2H), 1.45−1.34 (m, 1H), 1.32−

1.19 (m, 1H). ¹³C NMR (101 MHz, CD₃OD): δ 167.4, 155.9, 141.3,

138.8, 138.5, 135.14, 135.10, 135.0, 129.2, 128.8, 128.3, 127.9, 127.8,

125.5, 121.8, 115.0, 52.1, 48.2, 48.0, 47.8, 47.6, 47.4, 47.3, 47.2, 47.0,

40.9, 30.6, 29.2, 28.1, 19.1, 18.9. HPLC (gradient A): retention time =

40.6 ESI-MS: 451.2 [M + Na]⁺.

(S)-2-Amino-N-((R)-7-(cyclohexylmethyl)-1,2,3,4-tetrahydronaph-

thalen-1-yl)-3-(4-hydroxy-2,6-dimethylphenyl)propanamide (14n).

This compound was synthesized following general procedure J starting

from the (R) amine intermediate 19n (20 mg, 0.0715 mmol) to yield

crude product, which was puriﬁed by semipreparative HPLC and

lyophilized to yield the title compound as a TFA salt (35 mg, 89.7%).

¹H NMR (500 MHz, CD₃OD): δ 8.08−8.02 (m, 1H), 6.95−6.89 (m,

3H), 6.49 (s, 2H), 4.98−4.93 (m, 1H), 3.92−3.81 (m, 1H), 3.25 (d, J

= 12.2 Hz, 1H), 3.01 (d, J = 14.1 Hz, 1H), 2.60−2.54 (bs, 2H), 2.40−

4.33 (bs, 2H), 2.28 (s, 6H), 1.73−1.59 (m, 6H), 1.58−1.52 (m, 1H),

1.47−1.37 (m, 2H), 1.33−1.24 (m, 1H), 1.22−1.12 (m, 3H), 0.92 (m,

2H). ¹³C NMR (126 MHz, CD₃OD): δ 168.7, 157.3, 140.1, 139.9,

136.2, 136.0, 130.7, 129.8, 129.5, 123.2, 116.5, 53.53, 53.49, 48.8, 44.7,

41.1, 34.4, 34.2, 32.0, 30.7, 29.6, 27.7, 27.39, 27.38, 20.5, 20.3. HPLC

(gradient A): retention time = 47.3. ESI-MS: 457.3 [M + H]⁺.

(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)-N-((R)-7-(naph-

thalen-2-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)-

propanamide (14o). This compound was synthesized following

general procedure J starting from the (R) amine intermediate 19o (11

mg, 0.034 mmol) to yield crude product, which was puriﬁed by

semipreparative HPLC and lyophilized to yield the title compound as

a TFA salt (14 mg, 69.7%). ¹H NMR (500 MHz, CD₃OD): δ 8.09 (d,

J = 8.2 Hz, 1H), 7.74 (dd, J = 25.4, 7.7 Hz, 4H), 7.56 (s, 1H), 7.40 (p,

J = 7.0 Hz, 3H), 7.26 (d, J = 8.3 Hz, 1H), 7.11 (s, 1H), 7.03−6.94 (m,

3H), 6.49 (s, 2H), 5.01−4.93 (m, 1H), 4.03 (s, 2H), 3.85 (dd, J = 11.2,

4.7 Hz, 1H), 3.24 (t, J = 12.5 Hz, 1H), 3.00 (dd, J = 13.5, 4.7 Hz, 1H),

2.65−2.55 (m, 2H), 2.27 (s, 6H), 1.69−1.60 (m, 1H), 1.59−1.52 (m,

1H), 1.47−1.37 (m, 1H), 1.33−1.24 (bs, 1H). ¹³C NMR (126 MHz,

CD₃OD): δ 168.8, 157.3, 140.3, 140.0, 139.9, 136.7, 136.6, 135.1,

133.5, 130.7, 130.4, 129.5, 128.9, 128.6, 128.4, 127.7, 127.0, 126.4,

123.2, 116.5, 111.4, 53.5, 48.7, 42.5, 32.0, 30.6, 29.6, 20.5, 20.3. HPLC

(gradient A): retention time = 45.6. ESI-MS: 479.3 [M + H]⁺.

(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)-N-((R)-7-(naph-

thalen-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)-

propanamide (14p). This compound was synthesized following

general procedure J starting from the (R) amine intermediate 19p (7

mg, 0.0216 mmol) to yield crude product, which was puriﬁed by

semipreparative HPLC and lyophilized to yield the title compound as

a TFA salt (11 mg, 85.9%). ¹H NMR (500 MHz, CD₃OD): δ 7.97 (d,

J = 8.1 Hz, 1H), 7.87−7.82 (m, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.49−

7.34 (m, 3H), 7.22 (d, J = 7.0 Hz, 1H), 7.13 (s, 1H), 6.95−6.84 (m,

2H), 6.49 (d, J = 1.8 Hz, 2H), 4.99−4.91 (m, 1H), 4.35 (s, 2H), 3.84

(ddd, J = 11.5, 5.1, 1.8 Hz, 1H), 3.25 (ddd, J = 13.5, 11.4, 1.9 Hz, 1H),

3.00 (ddd, J = 13.9, 5.1, 1.7 Hz, 1H), 2.64−2.56 (m, 2H), 2.27 (s, 6H),

1.68−1.59 (m, 1H), 1.59−1.51 (m, 1H), 1.46−1.38 (m, 1H), 1.33−

1.24 (m, 1H). ¹³C NMR (126 MHz, CD₃OD): δ 167.2, 155.9, 138.5,

138.3, 136.6, 135.2, 135.1, 134.1, 131.9, 129.0, 128.8, 128.2, 127.6,

126.71, 126.69, 125.4, 125.1, 125.0, 123.8, 121.8, 115.0, 52.1, 47.4,

38.1, 30.6, 29.2, 28.1, 19.1, 18.9. No HPLC retention time data was

obtained. ESI-MS: 501.1 [M + Na]⁺.

1

compound as a clear, colorless oil (1126 mg, 77%) (from 15). H

NMR (500 MHz, CDCl₃): δ 7.56 (d, J = 2.1 Hz, 1H), 7.24 (dd, J =

8.2, 2.1 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 4.46 (q, J = 4.4 Hz, 1H),

3.31 (d, J = 4.0 Hz, 1H), 2.72 (dt, J = 17.0, 5.2 Hz, 1H), 2.61 (ddd, J =

17.0, 8.9, 5.7 Hz, 1H), 2.00−1.92 (m, 1H), 1.92−1.79 (m, 2H), 1.76−

1.67 (m, 1H), 1.18 (s, 9H). ¹³C NMR (126 MHz, CDCl₃): δ 138.9,

138.0, 132.07, 132.06, 130.8, 130.5, 119.6, 77.3, 77.0, 76.7, 55.4, 52.5,

30.2, 28.4, 22.5, 18.0.

(R)-N-((R)-7-Benzyl-1,2,3,4-tetrahydronaphthalen-1-yl)-2-methyl-

propane-2-sulﬁnamide (18m). This compound was synthesized

following general procedure F using 17 (110 mg, 0.33 mmol, 1.0

equiv), 2-benzyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (148.2 uL,

0.66 mmol, 2.0 equiv), K₂CO₃(138 mg, 0.99 mmol, 3.0 equiv), and

Pd(dppf)Cl₂(24.4 mg, 0.033 mmol, 0.1 equiv). The reagents were

placed in a microwave tube followed by 1−2 mL of the previously

degassed 3:1 acetone:water solvent system and reacted in a microwave

with a max temp of 110 °C and max power of 300 W for 60 min with

“Powermax” enabled. Once the crude mixture was ﬁltered through

Celite, the residue was puriﬁed via silica gel chromatography (1:1

EA:hex) to yield the title compound (64 mg, 56%) as a clear, colorless

1

oil. H NMR (500 MHz, CDCl₃): δ 7.35−7.31 (m, 1H), 7.31−7.24

(m, 2H), 7.23−7.15 (m, 3H), 7.02 (t, J = 1.4 Hz, 2H), 4.56 (d, J = 3.9

Hz, 1H), 3.95 (s, 2H), 3.23 (d, J = 3.2 Hz, 1H), 2.78 (dt, J = 16.9, 4.9

Hz, 1H), 2.69 (ddd, J = 16.5, 9.5, 5.6 Hz, 1H), 2.08−1.99 (m, 1H),

1.99−1.82 (m, 2H), 1.80−1.70 (m, 1H), 1.22 (s, 9H). ¹³C NMR (126

MHz, CDCl₃): δ 141.2, 139.5, 136.8, 135.5, 129.9, 129.5, 128.83,

128.82, 128.5, 128.2, 126.0, 77.3, 77.1, 76.8, 55.4, 52.5, 41.6, 30.3, 28.7,

22.6, 18.1.

(R)-N-((R)-7-(Cyclohexylmethyl)-1,2,3,4-tetrahydronaphthalen-1-

yl)-2-methylpropane-2-sulﬁnamide (18n). This compound was

synthesized following general procedure G using 17 (100 mg, 0.30

mmol, 1.0 equiv), (cyclohexylmethyl)boronic acid (86 mg, 0.61 mmol,

2.0 equiv), K₂CO₃(126 mg, 0.91 mmol, 3.0 equiv), Ag₂O (175 mg,

0.76 mmol, 2.5 equiv), and Pd(dppf)Cl₂(22 mg, 0.031 mmol, 0.1

equiv). The reagents were placed in a microwave tube followed by 1.5

mL of anhyd THF and reacted in a microwave with a max temp of 80

°C and max power of 300 W for 60 min with the “Powermax” option

disabled. Once the crude mixture was ﬁltered through Celite, the

residue puriﬁed via silica gel chromatography (equilibrated in 100%

hex, run in 2:3 EA:hex) to yield the title compound as a clear, colorless

oil (105 mg, quant), which was taken to the next step (formation of

19n) without further characterization.

(R)-2-Methyl-N-((R)-7-(naphthalen-2-ylmethyl)-1,2,3,4-tetrahy-

dronaphthalen-1-yl)propane-2-sulﬁnamide (18o). This compound

was synthesized following general procedure F using 17 (49 mg, 0.15

mmol, 1.0 equiv), 4,4,5,5-tetramethyl-2-(naphthalen-2-ylmethyl)-1,3,2-

dioxaborolane (80 mg, 3.0 mmol, 2.0 equiv), K₂CO₃(58 mg, 0.42

mmol, 3.0 equiv), and Pd(dppf)Cl₂(11 mg, 0.015 mmol, 0.1 equiv).

The reagents were placed in a microwave tube followed by 1−2 mL of

the previously degassed 3:1 acetone:water solvent system and reacted

in a microwave with a max temp of 110 °C and max power of 300 W

for 60 min with “Powermax” enabled. Once the crude mixture was

ﬁltered through Celite, the residue was puriﬁed via silica gel

chromatography (equilibrated in 100% hex, run in 1:1 EA:hex) to

yield the title compound (32 mg, 55.2%) as a clear, colorless oil, which

was taken ahead.

(R)-2-Methyl-N-((R)-7-(naphthalen-1-ylmethyl)-1,2,3,4-tetrahy-

dronaphthalen-1-yl)propane-2-sulﬁnamide (18p). This compound

was synthesized following general procedure F using 17 (46 mg, 0.14

mmol, 1 equiv), 4,4,5,5-tetramethyl-2-(naphthalen-1-ylmethyl)-1,3,2-

dioxaborolane (75 mg, 0.28 mmol, 2 equiv), K₂CO₃(58 mg, 0.42

(R)-N-((R)-7-Bromo-1,2,3,4-tetrahydronaphthalen-1-yl)-2-methyl-

propane-2-sulﬁnamide (17). This compound was synthesized

following general procedure H using commercially available 15 (1.0

g, 4.44 mmol, 1 equiv), (R)-2-methylpropane-2-sulﬁnamide (1076 mg,

N

DOI: 10.1021/acs.jmedchem.5b01270

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Journal of Medicinal Chemistry

Article

mmol, 3 equiv), and Pd(dppf)Cl₂(10 mg, 0.014 mmol, 0.1 equiv).

The reagents were placed in a microwave tube followed by the

previously degassed 3:1 acetone:water solvent system and reacted in a

microwave with a max temp of 110 °C and max power of 250 W for 30

min with “Powermax” enabled. Once the crude mixture was ﬁltered

through Celite, the residue was puriﬁed via silica gel chromatography

(equilibrated in 100% hex, run in 1:4 EA:hex) to yield the title

In Vitro Pharmacology: Cell Lines and Membrane Prepara-

tions. All tissue culture reagents were purchased from Gibco Life

Sciences (Grand Island, NY, U.S.). C6-rat glioma cells stably

transfected with a rat μ (C6-MOR) or rat δ (C6-DOR) opioid

receptor³⁰and Chinese hamster ovary (CHO) cells stably expressing a

human κ (CHO-KOR) opioid receptor³¹were used for all in vitro

assays. Cells were grown to conﬂuence at 37 °C in 5% CO₂in

Dulbecco’s modiﬁed Eagle medium (DMEM) containing 10% fetal

bovine serum and 5% penicillin/streptomycin. Membranes were

prepared by washing conﬂuent cells 3× with ice cold phosphate

buﬀered saline (0.9% NaCl, 0.61 mM Na₂HPO₄, 0.38 mM KH₂PO₄,

pH 7.4). Cells were detached from the plates by incubation in warm

harvesting buﬀer (20 mM HEPES, 150 mM NaCl, 0.68 mM EDTA,

pH 7.4) and pelleted by centrifugation at 1600 rpm for 3 min. The cell

pellet was suspended in ice-cold 50 mM Tris-HCl buﬀer, pH 7.4, and

homogenized with a Tissue Tearor (Biospec Products, Incorporated;

Bartlesville, OK, USA) for 20 s. The homogenate was centrifuged at

15,000 rpm for 20 min at 4 °C. The pellet was rehomogenized in 50

mM Tris-HCl with a Tissue Tearor for 10 s followed by

recentrifugation. The ﬁnal pellet was resuspended in 50 mM Tris-

HCl and frozen in aliquots at 80 °C. Protein concentration was

determined via a BCA protein assay (Thermo Scientiﬁc Pierce;

Waltham, MA, USA) using bovine serum albumin as the standard.

Radioligand Binding Assays. Radiolabeled compounds were

purchased from PerkinElmer (Waltham, MA, USA). Opioid ligand

binding assays were performed by competitive displacement of 0.2 nM

[³H]diprenorphine (250 μCi, 1.85 TBq/mmol) by the peptidomi-

metic from membrane preparations containing opioid receptors as

described above. The assay mixture, containing membranes (20 μg

protein/tube) in 50 mM Tris-HCl buﬀer (pH 7.4), [³H]-

diprenorphine, and various concentrations of test peptidomimetic,

was incubated at room temperature for 1 h to allow binding to reach

equilibrium. The samples were rapidly ﬁltered through Whatman GF/

C ﬁlters using a Brandel harvester (Brandel; Gaithersburg, MD, USA)

and washed 5× with 50 mM Tris-HCl buﬀer. Bound radioactivity on

dried ﬁlters was determined by liquid scintillation counting after

saturation with EcoLume liquid scintillation cocktail in a Wallac 1450

MicroBeta (PerkinElmer; Waltham, MA, USA). Nonspeciﬁc binding

was determined using 10 μM naloxone. The results presented are the

1

compound (11 mg, 20.2%) as a clear, colorless oil. H NMR (500

MHz, CDCl₃): δ 8.03−7.97 (m, 1H), 7.87−7.81 (m, 1H), 7.74 (d, J =

8.2 Hz, 1H), 7.48−7.38 (m, 3H), 7.37 (s, 1H), 7.31 (d, J = 6.9 Hz,

1H), 6.96 (s, 2H), 4.53 (q, J = 3.4 Hz, 1H), 4.40 (s, 2H), 3.20 (s, 1H),

2.79−2.71 (m, 1H), 2.70−2.60 (m, 1H), 2.05−1.97 (m, 1H), 1.94−

1.79 (m, 2H), 1.77−1.65 (m, 1H), 1.17 (s, 9H). ¹³C NMR (126 MHz,

CDCl₃): δ 138.9, 136.8, 136.6, 135.5, 133.9, 132.0, 129.7, 129.4, 128.6,

127.9, 127.2, 127.1, 125.9, 125.54, 125.47, 124.2, 55.4, 52.4, 38.6, 30.3,

28.7, 22.6, 18.1.

(R)-7-Benzyl-1,2,3,4-tetrahydronaphthalen-1-aminium Chloride

(19m). This compound was synthesized following general procedure

I using 18m (112 mg, 0.33 mmol, 1.0 equiv) and 5 mL of 2 M HCl in

dioxane. The hydrochloride salt crashed out after the addition of the

acid. The Et₂O was ﬁltered oﬀ, leaving a white, ﬂaky precipitate that

was washed with cold Et₂O and dried under vacuum without any

1

further puriﬁcation to yield the title compound (78 mg, 87%). H

NMR (400 MHz, CDCl₃): δ 8.74 (s, 3H), 7.56 (s, 1H), 7.30−7.05 (m,

5H), 7.01 (s, 2H), 4.39 (dd, J = 6.7 Hz, 1H), 3.86 (s, 2H), 2.87−2.74

(m, 1H), 2.73−2.57 (m, 1H), 2.19−1.89 (m, 3H), 1.72 (dd, J = 14.7,

6.2 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 141.0, 139.5, 135.3,

131.1, 129.8, 129.5, 129.3, 128.9, 128.4, 126.0, 77.3, 77.0, 76.7, 49.6,

41.5, 28.3, 27.9, 18.7.

(R)-7-(Cyclohexylmethyl)-1,2,3,4-tetrahydronaphthalen-1-ami-

nium Chloride (19n). This compound was synthesized following

general procedure I using 18n (100 mg, 0.29 mmol, 1.0 equiv) and

concd HCl (42 μL, 1.7 mmol, 6.0 equiv). After removal of the solvent,

the residue was resuspended in Et₂O, and a white solid crashed out.

The Et₂O was ﬁltered oﬀ, leaving a white, ﬂaky precipitate that was

washed with cold Et₂O and dried under vacuum without any further

1

puriﬁcation to yield the title compound (45 mg, 55.9%). H NMR

(500 MHz, CDCl₃): δ 8.72 (s, 3H), 7.38 (s, 1H), 7.00 (s, 2H), 4.40 (q,

J = 5.4 Hz, 1H), 2.81 (dt, J = 16.7, 5.4 Hz, 1H), 2.71−2.61 (m, 1H),

2.40 (d, J = 7.1 Hz, 2H), 2.18−2.07 (m, 2H), 2.05−1.94(m, 1H),

1.79−1.69 (m, 1H), 1.68−1.55 (m, 5H), 1.54−1.46 m, 1H), 1.22−

1.08 (m, 3H), 1.01−0.84 (m, 2H). ¹³C NMR (126 MHz, CDCl₃): δ

139.7, 134.7, 130.6, 129.7, 129.6, 129.3, 49.7, 43.5, 39.5, 33.11, 33.09,

28.3, 27.93, 27.91, 26.6, 26.3, 26.3.

mean

standard error (S.E.M.) from at least three separate assays

performed in duplicate. K_i(nM) values were calculated using

nonlinear regression analysis to ﬁt a logistic equation to the

competition data using GraphPad Prism, version 6.0c, for Mac OS X

(GraphPad Software Incorporated; La Jolla, CA, USA).

Stimulation of [³⁵S]GTPγS Binding. Agonist stimulation of

[³⁵S]guanosine 5′-O-[γ-thio]triphosphate ([³⁵5S]GTPγS, 1250 Ci,

46.2 TBq/mmol) binding to G-protein was measured as described

previously.³²Brieﬂy, membranes (10−20 μg of protein/tube) were

incubated for 1 h at 25 °C in GTPγS buﬀer (50 mM Tris-HCl, 100

mM NaCl, 5 mM MgCl₂, pH 7.4) containing 0.1 nM [³⁵S]GTPγS, 30

μM guanosine diphosphate (GDP), and varying concentrations of test

peptidomimetic. G-protein activation following receptor stimulation of

[³⁵S]GTPγS (% stimulation) with peptidomimetic was compared with

10 μM of the standard compounds [^D-Ala2,N-MePhe4,Gly-ol]-

enkephalin (DAMGO) at MOR, ^D-Pen2,5-enkephalin (DPDPE) at

DOR, or U69,593 at KOR. The reaction was terminated by vacuum

ﬁltration of GF/C ﬁlters that were washed 10× with GTPγS buﬀer.

Bound radioactivity was measured as described above. The results are

presented as the mean standard error (S.E.M.) from at least three

separate assays performed in duplicate; potency (EC₅₀(nM)) and %

stimulation were determined using nonlinear regression analysis with

GraphPad Prism, as above.

In Vivo Pharmacology. Animals. Adult male C57BL/6 mice

weighing between 20 and 30 g at 8−16 weeks old were purchased

from Harlan (Indianapolis, IN, USA). Mice were group-housed and

had free access to food and water at all times. Experiments were

conducted in the housing room, which was maintained on a 12 h light/

dark cycle (with lights on at 0700). Each mouse was used only once,

and experiments were conducted between 10 a.m. and 5 p.m. Studies

were performed in accordance with the University of Michigan

(R)-7-(Naphthalen-2-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-

aminium Chloride (19o). This compound was synthesized following

general procedure I using 18o (30 mg, 0.077 mmol, 1.0 equiv) and

concd HCl (11 μL, 0.46 mmol, 6.0 equiv). After removal of the

solvent, the residue was resuspended in Et₂O, and the solid did not

crash out. The residue was washed 3× with fresh Et₂O and then dried

to yield the title compound as an oﬀ-white, sticky solid (11 mg,

44.3%). ¹H NMR (500 MHz, CD₃OD): δ 7.83−7.74 (m, 3H), 7.66 (s,

1H), 7.43 (p, J = 7.0 Hz, 2H), 7.35−7.29 (m, 2H), 7.23 (d, J = 8.0 Hz,

1H), 7.16 (d, J = 7.9 Hz, 1H), 4.45 (t, J = 5.6 Hz, 1H), 4.14 (s, 2H),

2.92−2.74 (m, 2H), 2.22−2.11 (m, 1H), 2.04−1.82 (m, 3H). ¹³C

NMR (126 MHz, CD₃OD): δ 141.2, 140.0, 136.9, 135.1, 133.6, 133.0,

131.2, 130.9, 129.8, 129.1, 128.6, 128.5, 128.4, 128.0, 127.1, 126.5,

50.3, 42.5, 29.4, 29.1, 19.6.

(R)-7-(Naphthalen-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-

aminium Chloride (19p). This compound was synthesized following

general procedure I using 18p (11 mg, 0.028 mmol, 1.0 equiv) and

concd HCl (4 μL, 0.17 mmol, 6.0 equiv). After removing the solvent,

the residue was resuspended in Et₂O, and the solid crashed out. After

washing the solid 3× with fresh Et₂O, the remaining Et₂O was

decanted, yielding the title compound as a white solid (7 mg, 77.8%).

¹H NMR (400 MHz, CD₃OD): δ 8.05−7.96 (m, 1H), 7.86 (dd, J =

7.0, 2.3 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.47−7.39 (m, 3H), 7.36 (d,

J = 7.0 Hz, 1H), 7.30 (s, 1H), 7.17−7.07 (m, 2H), 4.47−3.38 (m, 3H),

2.90−2.69 (m, 2H), 2.14 (ddt, J = 14.5, 9.6, 4.6 Hz, 1H), 1.92 (ddt, J =

43.3, 20.4, 8.1 Hz, 3H). No ¹³C NMR spectrum acquired.

O

DOI: 10.1021/acs.jmedchem.5b01270

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Committee on the Use and Care of Animals and the Guide for the

Care and Use of Laboratory Animals (National Research Council,

2011 publication).

Notes

The authors declare no competing ﬁnancial interest.

Antinociception. The antinociceptive eﬀects of select peptidomi-

metics were evaluated in the WWTW assay using a cumulative dosing

procedure.³³For determination of the tail withdrawal latencies, each

mouse was placed brieﬂy into a plastic, cylindrical restrainer, and 2−3

cm of the tail tip was placed into a water bath maintained at 50 °C.

The latency to withdraw of the tail was recorded with a maximum

cutoﬀ time of 20 s. If the mouse did not remove its tail by the cutoﬀ

time, the experimenter removed its tail from the water to prevent

tissue damage. Each animal received an injection of saline (intra-

peritoneal, ip), and then, 30 min later, the baseline withdrawal

latencies were recorded and ranged between 3 and 6 s. Following

baseline determinations, three increasing doses (1, 2.2, and 6.8 mg/kg)

of a ﬁnal compound 14 analogue was given at 30 min intervals to

provide ﬁnal doses of 1, 3.2, and 10 mg/kg. Thirty minutes after each

injection, the tail withdrawal latency was measured as described above.

To determine the duration of antinociceptive action, the tail-

withdrawal test was performed at varying times following admin-

istration of a ﬁnal compound 14 analogue (10 mg/kg, ip). To conﬁrm

that 14a produces antinociception via the opioid receptors, we

repeated the cumulative dose response over the doses 3.2, 10, and 32

mg/kg following a 30 min pretreatment with 1 mg/kg naltrexone or

saline (ip). The ED₅₀for mice receiving saline pretreatment is 4.73

0.08 mg/kg 14a, and the ED₅₀for the mice receiving 1 mg/kg

naltrexone is 15.07 1.03 mg/kg 14a, suggesting that the

antinociception 14a produces is MOR-mediated.

ACKNOWLEDGMENTS

■

This study was supported by NIH Grant DA003910 (H.I.M,

E.M.J., and J.R.T). J.P.A. was supported by the Substance Abuse

Interdisciplinary Training Program administered by NIDA 5

T32 DA007267, and N.W.G. was supported by the

Pharmacological Sciences Training Program T32 GM007767.

Tyler Trask, Evan Schramm, Aaron Chadderdon, and Chao

Gao are thanked for additional in vitro studies.

ABBREVIATIONS USED

■

MOR, μ-opioid receptor; DOR, δ-opioid receptor; KOR, κ-

opioid receptor; THQ, tetrahydroquinoline; THN, tetrahy-

dronaphthalene; CHO, Chinese hamster ovary; GTPγS,

guanosine 5′-O-[γ-thio]triphosphate; WWTW, warm water

tail withdrawal; TM, transmembrane; Boc₂O, di-tert-butyldicar-

bonate; Boc, tert-butyldicarbonate; DIPEA, N,N- diisopropyle-

thylamine; EA, ethyl acetate; hex, hexanes; TfOH, triﬂic acid;

PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexa-

ﬂuorophosphate; HOBt-Cl, 6-chlorohydroxybenzotriazole;

diBoc-Dmt, tert-butyloxycarbonyl-protected 2,6-dimethyltyro-

sine

Computational Modeling. Three-dimensional models of opioid

receptors in inactive conformation were produced as previously

described³⁹using X-ray structures of the mouse MOR (PDB ID:

4DKL),⁴¹the human DOR (PDB ID: 4N6H),⁴⁵and the human KOR

(PDB ID: 4DJH)⁴³as structural templates. The recently obtained

crystal structure of mouse MOR in the active conformation (PDB ID:

5C1M)⁴⁶was used as a template for homology modeling of active

conformations of DOR and KOR. Structures of receptor loops in the

active state were kept similar to those in the crystal structures of

corresponding receptors in the inactive state. N-termini of DOR

(residues 33−45) and KOR (residues 45−57) were modeled using the

structure of MOR N-terminus in the active conformation with a few

adjustment to satisfy the formation of Zn-binding centers involving

D216 (H54−D216) and H319 (C57−H319), which were previously

suggested for MOR.⁴⁷Structures of peptidomimetic ligands were

generated using 3D-Builder Application of QUANTA (Accelrys, Inc.)

followed by the Conformational Search included in the program

package. Ligand conformations that demonstrated the best super-

position of aromatic substituents of the THQ core with the

pharmacophore elements (Tyr¹and Phe³) of receptor-bound

conformations of cyclic tetrapeptides^40,48were selected and minimized

with CHARMm implemented in QUANTA (adopted-basis Newton−

Raphson method, 100 steps, ε = 10). Low energy conformations

(within 2 kcal/mol) were manually positioned inside the receptor

binding cavity to reproduce the binding modes of cyclic tetrapeptides.

The docking pose of each ligand was subsequently reﬁned using the

solid docking module of QUANTA. Models of opioid ligand−receptor

complexes are available upon request.

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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.5b01270.

Ki, EC₅₀, and percent stimulation for MOR, DOR, and

KOR for 14a−p (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: him@umich.edu; phone: (734)764-8117.

■

P

DOI: 10.1021/acs.jmedchem.5b01270

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Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities

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