SAR Matrices Enable Discovery of Mixed Efficacy μ-Opioid Receptor Agonist Peptidomimetics with Simplified Structures through an Aromatic-Amine Pharmacophore

pubs.acs.org/chemneuro

Research Article

SAR Matrices Enable Discovery of Mixed Eﬃcacy μ‑Opioid Receptor

Agonist Peptidomimetics with Simpliﬁed Structures through an

Aromatic-Amine Pharmacophore

Sean Henry, Jessica P. Anand, Ashley C. Brinkel, Douglas M. McMillan, Jack. J. Twarozynski,

Christian E. Loo, John R. Traynor, and Henry I. Mosberg *

Cite This: https://dx.doi.org/10.1021/acschemneuro.0c00693

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: We previously described the development of potent

μ-opioid receptor (MOR)-agonist/δ-opioid receptor (DOR)-

antagonist peptidomimetic ligands as an approach toward eﬀective

analgesics with reduced side eﬀects. In this series, a tetrahy-

droquinoline (THQ) or substituted phenyl is employed to link two

key pharmacophore elements, a dimethyltyrosine amino acid and

typically an aromatic pendant. Using new and previously reported

analogues, we constructed a structure−activity relationship (SAR)

matrix that probes the utility of previously reported amine

pendants. This matrix reveals that the MOR-agonist/DOR-

antagonist properties of these ligands do not change when a tetrahydroisoquinoline (THIQ) pendant is used, despite removal of

substituents on the core phenyl ring. Based on this observation, we retained the THIQ pendant and replaced the phenyl core with

simpler aliphatic chain structures. These simpler analogues proved to be potent MOR-agonists with high variability in their eﬀects at

the DOR and the κ-opioid receptor (KOR). These data show that the amine of the THIQ pendant may be a novel pharmacophore

element that favors high MOR-eﬃcacy, whereas the aromatic ring of the THIQ pendant may produce high MOR-potency.

Combined, the two pharmacophores within the THIQ pendant may be a structurally eﬃcient means of converting opioid peptides

and peptidomimetics into potent and eﬃcacious MOR-agonists.

KEYWORDS: μ-Opioid receptor, δ-opioid receptor, κ-opioid receptor, peptidomimetics, structure−activity relationship, bifunctional ligands

has been shown to produce altered pharmacological proﬁles

compared to activation of a single opioid receptor type.¹⁰MOR-

agonists in conjunction with DOR-antagonists have been shown

to reduce the addictive side eﬀects of MOR-activation while

retaining their analgesic properties.¹¹⁻¹⁶MOR-agonist/DOR-

agonists have also shown these beneﬁcial eﬀects,¹⁷⁻¹⁹in

addition to reduced convulsions associated with DOR-

activation.²⁰

In the course of our development of a MOR-agonist/DOR-

antagonist peptidomimetic series with a reduced abuse

proﬁle,²¹⁻²⁹we found that the core tetrahydroquinoline

structure in this series was a major metabolic liability. As such,

we opted to simplify the core to a benzene ring.³⁰Short chain

ethers attached to this core aromatic ring provided the greatest

improvement in stability while producing a MOR-agonist/

INTRODUCTION

■

The opioid receptors are an important family of G protein-

coupled receptors (GPCRs) that aﬀect various behavioral

processes in the central nervous system. Activation of the μ-

opioid receptor (MOR) yields analgesia, euphoria, and

symptoms associated with drug addiction.^1,2Stimulation of

the δ-opioid receptor (DOR) is known to produce convulsions,

while activation of the κ-opioid receptor (KOR) is known to

produce dysphoria.^1,2These opioid receptors are acted on by

endogenous peptides, and structure−activity relationship

(SAR) studies of these peptides found that the ﬁrst four

residues are responsible for opioid receptor activation whereas

subsequent residues are responsible for receptor selectivity.³

This led to the development of selective opioid peptides such as

DAMGO,⁴DPDPE,⁵and DIPP-NH₂[Ψ],⁶which are used to

study the opioid receptors. Frequently, the exploration of opioid

peptide SAR has produced increasingly bulky and complicated

structures to induce their eﬀects.

Received: October 26, 2020

Accepted: December 3, 2020

Mixed eﬃcacy opioid ligands, i.e., ligands that aﬀord varying

degrees of activation across diﬀerent opioid receptors, have

recently become an increasingly attractive avenue of re-

search,⁷⁻⁹as targeting multiple opioid receptors simultaneously

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

A

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

Figure 1. Design rationale that led to the initial SAR study reported in this work. This work stems from two previously reported SAR campaigns aimed

at (A) converting a metabolically unstable tetrahydroquinoline core to a more stable aromatic core and (B) fully restoring lost MOR-eﬃcacy, MOR-

potency, and improving their stability through the use of amine pendants. The data from these two studies lead to (C) the current study that aims to

demonstrate how the incorporation of these amine pendants into the peptidomimetics allows us to alter the core structure. Initially, this was manifested

in (D) the construction of an SAR matrix aimed at understanding the interplay between the amine pendants and the aromatic R groups.

DOR-antagonist proﬁle (Figure 1A). However, the ability of the

compounds to operate as MOR-agonists was highly dependent

on the size of the ether, and MOR-potency and eﬃcacy were

limited compared to previous analogues. Since we were

interested in improving MOR-eﬃcacy, MOR-potency, and

metabolic stability further, we reexamined our original THQ

core series for modiﬁcations that might be useful. In that series,

there were cyclic amine pendant analogues that improved MOR-

eﬃcacy while reducing the cLogP of the ligands.²³Since

lowering cLogP is known to reduce metabolism by CYP

enzymes,³¹we took the ether with the greatest level of MOR-

agonism and replaced the benzyl pendant with a cyclic amine

(Figure 1B). This was found to improve MOR-eﬃcacy while

also improving the metabolic stability of these ligands.³²

Furthermore, the addition of an aromatic ring onto this cyclic

amine pendant improved MOR-potency at the cost of some of

the improved metabolic stability.

Given the considerable improvement in MOR-potency and

MOR-eﬃcacy that these amine pendants yielded in our series,

we opted to retain some of these amine pendants and again

varied the core structure using modiﬁcations from our initial

benzyl core study (Figure 1C). We were interested in the degree

to which the MOR-agonism of this series can be maintained with

these amine pendants without further structural additions to the

peptidomimetic. To narrow down our syntheses, two example

amine pendants were selected, namely, the piperidine and the

B

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

Scheme 1. (A) Synthesis of Aromatic Core Analogues to Produce SAR Matrices and (B) Analogue Codes for the Intermediate

a

Amides and Final Compounds for Each Aromatic Core Modiﬁcation and Amine Pendant Organized into Matrices

a

Previously reported compounds are encoded with Greek letters. Reaction conditions: (a) MeI or Alk-Br, K₂CO₃, DMF. (b) (1) (R)-(+)-2-methyl-

2-propanesulﬁnamide, Ti(OEt)₄, THF; (2) NaBH₄. (c) LiOH, THF, EtOH, H₂O. (d) (1) 1,2,3,4-tetrahydroisoquinoline, NMM, PyBOP, DMF;

(2) conc. HCl, dioxane. (e) BH₃*Me₂S, THF, 75 C°. (f) (1) DiBocDMT, DIEA, PyBOP, 6-Cl-HOBt, DMF; (2) TFA, DCM.

tetrahydroisoquinoline (THIQ) structures. The THIQ pendant

was chosen as it showed the highest MOR-aﬃnity and potency

of those previously reported, whereas the piperidine pendant

was selected due to its enhanced metabolic stability. These

pendants were then synthesized with three previously reported

core ethers (methyl, n-propyl, cyclopropylmethyl) and an

unmodiﬁed aromatic core, yielding a total of eight new

analogues. These analogues were compiled into an SAR matrix

aimed at producing a tighter SAR campaign that addresses the

following questions: To what degree can these new amine

pendants stimulate MOR? How dependent is this activity on

their respective aromatic core modiﬁcations?

aromatic structure. With this surprising discovery, we opted

then to remove the core aromatic structure and replace it with

simple alkyl, heteroalkyl, and peptide structures to link the

THIQ pharmacophore to the 2′6′-dimethyltyrosine pharmaco-

phore. This produced a series of potent and eﬃcacious MOR-

agonists that were either selective for MOR or were mixed

eﬃcacy MOR/DOR or MOR/KOR ligands. Herein, the two

SAR campaigns that describe the development of the THIQ

pharmacophore are discussed.

RESULTS AND DISCUSSION

■

The Eﬀect of Aromatic Core Substituents on Amine

Pendant Analogues. The syntheses of these new analogues

followed the same synthetic pathway described previously³²and

are shown in Scheme 1A. Here, diﬀerent ethers were introduced

by alkylating methyl 3-formyl-4-hydroxybenzoate (1) using

In answering these questions, we found that incorporation of

the THIQ pendant into these analogues produced potent and

eﬃcacious MOR-agonist/DOR antagonist ligands whose

properties were insensitive to modiﬁcations on the core

C

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

c

Table 1. Binding Aﬃnity Matrix of Aromatic Core Compounds at MOR, DOR, and KOR (in nM)

a

b

c

From ref 30. From ref 32. Binding aﬃnities (K_i) were obtained by competitive displacement of radiolabeled [³H]diprenorphine in membrane

preparations from CHO cells expressing single human opioid receptors. Included are previously reported benzyl pendant (a) and ethyl ether (b)

analogues for comparison. Selectivity was calculated by dividing the K_iof each receptor by the K_iat MOR for a given compound. All data are from

three separate experiments, performed in duplicate unless otherwise noted. These data are reported as the average standard error of the mean.

Cells colored in progressively darker shades of blue indicate progressively higher binding aﬃnity.

appropriate alkyl iodides or bromides. These ethers, in addition

to methyl 3-formylbenzoate (2), were then treated with Ellman’s

chiral auxiliary to introduce a protected amine in place of the

aldehyde. The subsequent ethyl ester was then cleaved using

LiOH and used as a common intermediate for incorporation of

our two selected amine pendants. These pendants were

incorporated into each aromatic core scaﬀold using PyBOP,

and the Ellman auxiliary was removed with conc. HCl and

puriﬁed by reversed-phase chromatography, yielding the

intermediate as a TFA salt. The subsequent tertiary amide was

then reduced with BH₃*Me₂S at 75 °C and the primary amine

was coupled to Boc-protected 2′,6′-dimethyltyrosine. The

synthesis was completed by deprotection of the Boc group

using TFA. The analogues synthesized in this manner are

organized into matrices (Scheme 1B) that include previously

reported analogues to probe this SAR space.

determine selectivity. Included also are previously reported

analogues (Greek letters) placed in positions appropriate for

their structure. Finally, the cells that correspond to each

analogue are color coded with darker shades of blue to illustrate

higher levels of binding aﬃnity to illustrate trends within these

data. Regarding MOR-binding aﬃnity, two trends readily

emerge. The ﬁrst is that there is a relatively ﬂat binding

landscape within all three pendants across diﬀerent R-groups, in

which there exists a maximum of a 5-fold spread in binding

aﬃnity. The second trend is that the piperidine pendant (A−D,

γ₂) possesses single-digit nanomolar binding aﬃnity, whereas

the THIQ pendant (E−H, γ₃) yields a log improvement in

aﬃnity into the subnanomolar range. The previously reported

benzyl pendant analogues (α, β, γ₁, δ, ε) possess binding aﬃnity

values between those of the piperidine and THIQ pendants.

DOR-binding aﬃnity in the piperidine pendants favored

progressively larger ethers and ranged from low triple-digit

nanomolar (A) to low double-digit nanomolar (B−D, γ₂)

We began our SAR analyses evaluating the binding aﬃnity of

these new analogues (Table 1) at MOR, DOR, and KOR to

D

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

c

Table 2. Potency Matrix of Aromatic Core Compounds at MOR, DOR, and KOR (in nM)

a

b

c

From ref 30. From ref 32. Potency data were obtained using agonist induced stimulation of [³⁵S] GTPγS binding in CHO cells expressing single

human opioid receptors. Potency is represented as EC₅₀(nM). Included are previously reported benzyl pendant (a) and ethyl ether (b) analogues

for comparison. All data are from three separate experiments, performed in duplicate unless otherwise noted. These data are reported as the average

standard error of the mean. Cells colored in progressively darker shades of blue indicate progressively higher potency. DNS = Does not stimulate.

aﬃnity values. However, there was little variance in DOR-

binding aﬃnity with the THIQ pendant, with all analogues

showing low nanomolar binding aﬃnities. At KOR, a reverse

trend is observed with the piperidine pendants in that improved

aﬃnity was observed with the shorter core analogues,

particularly the methyl ether (B). The THIQ pendants also

presented a ﬂat SAR at KOR in the double-digit nanomolar

range, though an exception exists with the lower aﬃnity n-propyl

ether analogue (G). Notably, both amine-containing pendants

possessed a signiﬁcantly higher binding aﬃnity for KOR over the

benzyl pendant. The binding aﬃnities between MOR and DOR

tended to become more similar as the size of the core

modiﬁcation increased across all pendants, with the notable

exception here being analogue H, likely due to its exceptional

MOR aﬃnity. Finally, the diﬀerences in binding aﬃnity between

MOR and KOR across all pendants reached a minimum with the

methyl ethers and a maximum with the n-propyl ethers.

Interestingly, the benzyl pendant and THIQ pendant possessed

more similar binding aﬃnity ratios to each other than to the

piperidine pendant. Within the piperidines, the smaller

analogues in fact had greater binding aﬃnity to KOR than to

DOR.

less potent than the benzyl pendants and like the benzyl

pendants, their potency was also dependent on the identity of

the aromatic R-group. Conversely, the THIQ pendants

possessed consistent single-digit nanomolar potency regardless

of the identity of the aromatic R-group. Curiously, both amine

pendants possessed very high MOR-eﬃcacy, save piperidine A,

whose measured maximal eﬀect was likely limited by its low

MOR-potency. This is in stark contrast to the benzyl pendant

analogues;, whose degree of maximal stimulation was highly

dependent on the identity of the aromatic R-group. In total,

these potency and relative eﬃcacy data illustrate that the amine

may be an important pharmacophore for acquiring high MOR-

eﬃcacy, whereas the reattachment of the aromatic ring as

exempliﬁed in the THIQ pendant may be an important

pharmacophore for acquiring high MOR-potency.

At DOR, the piperidine analogues generally functioned as

weak partial agonists, a contribution that appears to come from

the presence of an ether substituent. Where present, potency

consistently was around 100 nM and maximal stimulation values

were between 18 and 40% of the peptidic agonist DPDPE. The

THIQ pendant was found to produce moderate DOR eﬃcacy

with an unmodiﬁed core (analogue E), though the potency of

this analogue was particularly weak. These analogues generally

possessed no activity at KOR, though some very modest activity

was present with analogue F.

In addition to the binding aﬃnity data collected above, the

compounds were analyzed for their potency (Table 2) and

relative eﬃcacy (Table 3) at MOR, DOR, and KOR. For the

piperidine pendants, potency at MOR was drastically lower than

that of THIQ pendants. Furthermore, the piperidines were also

Elimination of the Aromatic Core. Given the surprisingly

potent and eﬃcacious MOR-agonist/DOR-antagonists the

E

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

c

Table 3. Eﬃcacy Matrix of Aromatic Core Compounds at MOR, DOR, and KOR (in %)

a

b

c

From ref 30. From ref 32. Relative eﬃcacy data were obtained using agonist-induced stimulation of [³⁵S] GTPγS binding in CHO cells

expressing human opioid receptors. Eﬃcacy is represented as percent maximal stimulation relative to standard agonist DAMGO (MOR), DPDPE

(DOR), or U69,593 (KOR) at 10 μM. Included are previously reported benzyl pendant (a) and ethyl ether (b) analogues for comparison. All data

were from three separate experiments, performed in duplicate unless otherwise noted. These data are reported as the average standard error of

the mean. DNS = Does not stimulate.

Figure 2. (A) Design of analogues with elimination of the aromatic core. (B) Design of more rigid analogues that use the THIQ pendant. These were

inspired by previously described piperazine core analogues.³⁶

THIQ pendants produced, we next examined the ability of the

THIQ pendants to produce this MOR/DOR proﬁle in simpler

structures. As such, we opted to remove the entire core aromatic

ring and replace it with simple alkyl or peptide chains (Figure

2A). The feasibility of this approach was reinforced by the fact

that many opioid ligands are peptides that frequently use glycine

F

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

a

Scheme 2. Synthesis of Analogues I−M

a

(A) 1. HO₂C-X-Boc, NMM, PyBOP, DMF. (B) Conc. HCl, dioxane, or TFA, DCM. (C) BH₃*Me₂S, THF, 65 C°. (D) (1) DiBocDMT, DIEA,

PyBOP, 6-Cl-HOBt, DMF; (2) TFA, DCM.

a

Scheme 3. Synthesis of Peptide Core Analogues N−P

a

(A) (1) HO₂C−CH₂−NXBoc, NMM, PyBOP, DMF. (B) Conc. HCl, dioxane, or TFA, DCM. (C) BH₃*Me₂S, THF, 65 C°. (D) (1)

DiBocDMT, DIEA, PyBOP, 6-Cl-HOBt, DMF; (2) TFA, DCM.

or D-alanine residues^4,33−35to link the tyrosine and phenyl-

alanine pharmacophores.³In conjunction with these analogues,

we previously reported a series of piperazine and piperidine core

analogues that terminate in a simple benzene ring (Figure 2B).³⁶

Notably, the MOR-eﬃcacy of a representative analogue in this

prior study peaked at 43% of the full agonist DAMGO. We then

hypothesized that the eﬃcacy of these structures may be

improved by replacing the terminal benzene ring with the amine-

containing THIQ pendant.

synthesized to conﬁrm that the compound eﬃcacy produced at

MOR is a product of the amine itself. The synthesis here began

with peptide coupling of tetrahydroisoquinoline to an

appropriate Boc-protected δ-amino acid. The Boc group was

removed with conc. HCl, and the tertiary amide was reduced

with BH₃*Me₂S at 65 °C if necessary. The primary amine was

then coupled to Boc-protected 2′,6′-dimethyltyrosine, at which

point the Boc-groups were removed with TFA, yielding the ﬁnal

peptidomimetics.

The synthesis of many of these new analogues required very

few steps and most are described in Scheme 2. These include a

simple alkyl chain, a chain possessing an ether, and the

piperidine and piperazine core structures aimed at reintroducing

conformational restrictions. An amide analogue K was also

The analogues that contained a peptide bond or amine in the

chain required a modiﬁed procedure that is described in Scheme

3. Analogue N, which possesses a tertiary amine, was synthesized

using peptide coupling of tetrahydroisoquinoline to N-Boc

sarcosine using PyBOP, at which point the Boc group was

G

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

a

Table 4. Binding Aﬃnity of Simple Core Compounds at MOR, DOR, and KOR

a

Binding aﬃnities (K_i) and selectivities were obtained as described in Table 1. Included is E for comparison. Selectivity was calculated by dividing

the K_iof each receptor by the K_iat MOR for a given compound. All data are from three separate experiments, performed in duplicate unless

otherwise noted. These data are reported as the average standard error of the mean.

removed with conc. HCl. This was then coupled to N-Boc

glycine and deprotected using the same method. Both peptide

bonds were then reduced with BH₃*Me₂S and the primary

amine was then coupled to Boc-protected 2′,6′-dimethyltyr-

osine, at which point the Boc-groups were removed with TFA,

yielding N.

Regarding MOR-binding, each analogue in this new subseries

possessed single-digit or subnanomolar binding-aﬃnity except

for the N-methyl amide analogue O. At DOR, elimination of the

core aromatic ring produced well over a 10-fold drop in aﬃnity.

The exceptions here are the rigid piperazine M and the two

amide analogues O and P. KOR-binding aﬃnity does not change

signiﬁcantly from the aromatic core analogue E, the only

exceptions being the N-methyl amide analogue O, which has low

KOR-aﬃnity, and the tertiary amine analogue N, which has

improved KOR-aﬃnity compared to E. Notably, this series

generated analogues that deviate from our original MOR/DOR

bifunctional proﬁle and instead appear to promote a MOR/

KOR proﬁle. This is illustrated with the alkyl (I), piperidine (L),

and tertiary amine (N) analogues. Finally, the ether analogue (J)

possessed a more monofunctional MOR selective proﬁle.

The potency and eﬃcacy relative to standard full agonists of

these analogues were also screened at each of these receptors

and are shown in Table 5. Signiﬁcantly, each of the amine

pendant compounds possessed moderate to high MOR-eﬃcacy.

The amide analogue K, while showing signiﬁcantly reduced

MOR-eﬃcacy compared to its amine counterpart J, retained

upward of 48% stimulation compared to the standard DAMGO.

Curiously, removal of the aromatic ring did not produce a

signiﬁcant drop in MOR-potency upon simpliﬁcation, even

when the amine is converted to an amide as with analogue K.

Losses in potency only occurred with the analogues that

possessed a reformed ring structure in the core (L and M) or

upon possession of an N-methyl substituent (N and O).

Two additional analogues containing peptide bonds in the

chain were also synthesized. These two analogues largely used

the same steps as those for the simpler analogues in Scheme 2

but used slightly diﬀerent synthetic strategies. As described in

Scheme 3, tetrahydroisoquinoline was coupled to N-Boc

protected glycine or sarcosine. These intermediates were

isolated before Boc deprotection, at which point the synthetic

schemes diverged. The sarcosine intermediate 27 was then

subject to reduction with borane before removal of the Boc

group, yielding intermediate 29. This was coupled to glycine and

deprotected. No extensive puriﬁcation of this intermediate was

performed, as this analogue had poor UV absorbance properties

and the impurities present were peptide coupling side products,

which were present in the subsequent coupling to DMT. As

such, DMT coupling was performed on this impure mixture and

subsequent deprotection yielded the N-methyl amide analogue

O. For the glycine intermediate 28, Boc deprotection was

performed before reduction, as the secondary carbamate was

unstable under these reduction conditions. This was then

coupled to an additional glycine residue, yielding intermediate

33 with better UV absorbance properties than its sarcosine

counterpart. After Boc deprotection, the intermediate was then

coupled to DMT, yielding P after ﬁnal Boc deprotection.

The binding aﬃnities of these analogues were determined and

are shown in Table 4. For comparison, analogue E is included.

DOR eﬃcacy was low in this series. Analogues that did

stimulate DOR were weak partial agonists with low potency and

eﬃcacy, and included the ether (J), tertiary amine (N), and the

H

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

a

Table 5. Potency and Eﬃcacy of Simple Core Compounds at MOR, DOR, and KOR

a

Potency and eﬃcacy data were obtained as described in Tables 2 and 3. Potency is represented as EC₅₀(nM) and eﬃcacy as percent maximal

stimulation relative to standard agonist DAMGO (MOR), DPDPE (DOR), or U69,593 (KOR) at 10 μM. Included is E for comparison. All data

are from three separate experiments, performed in duplicate unless otherwise noted. These data are reported as the average standard error of the

mean. DNS = Does Not Stimulate.

Figure 3. Structural comparison between the opioid peptide Met-enkephalin and analogue I. Relative positions of the amine and aromatic ring from

the tyrosine pharmacophore are numbered. (a) From ref 37.

most potent secondary amide analogue (P). At KOR, these

analogues either could not stimulate this receptor or were weak

partial agonists with very low potency (J and N).

The high MOR-potency in most of these simpliﬁed analogues

illustrate that the aromatic part of the THIQ pendant is an

important pharmacophore for MOR-potency, and tolerates a

variety of core structures, including those previously reported.

The exception here is the N-methyl amide analogue O, whose

reduced potency is likely due to the combination of both the N-

methyl group and the carbonyl, as each of these elements alone

had a modest or no eﬀect on MOR-potency (N and P

respectively).

We opted to compare MOR-potency and eﬃcacy of the

simplest analogue I to one of the simpler endogenous opioid

peptides endomorphin-2 (Figure 3).³⁷Here, each position

moving away from the tyrosine residue was numbered, and the

amine of analogue I was found to be in the same position as the

amido nitrogen of the fourth position phenylalanine residue.

Furthermore, the aromatic ring of the THIQ pendant lines up

nicely with the aromatic ring of the same phenylalanine. A

I

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

Biotage SNAP Ultra C18 column was utilized for some intermediate

amine salts. Puriﬁcation of ﬁnal compounds was performed using a

Waters semipreparative HPLC with a Vydac protein and peptide C18

reversed-phase column, using a linear gradient of 0% to 100% solvent B

in solvent A at a rate of 1%/min, monitoring UV absorbance at 230 nm.

The purity of ﬁnal compounds was assessed using a Waters Alliance

2690 analytical HPLC instrument with a Vydac protein and peptide

C18 reversed-phase column. A linear gradient (gradient A) of 0% to

70% solvent B in solvent A in 70 min, measuring UV absorbance at 230

nm was used to determine purity. All ﬁnal compounds used for testing

phenylalanine is typical of many opioid peptides and is a key

pharmacophore element necessary for opioid receptor activa-

tion.³As such, the enhancements in MOR-potency we observed

in the THIQ pendants are consistent with classical opioid

peptide SAR.

However, the amines represent a deviation from this classical

SAR, as this nitrogen is tied up in an amide structure that is

largely conserved throughout these opioid peptides. The

conversion to an amine in this position may be an eﬃcient

means of acquiring high MOR-eﬃcacy in these structurally

simpliﬁed peptidomimetics. This is illustrated by comparing

analogues J and K (Table 5), and the previously reported benzyl

pendant analogues and amine pendant analogues presented in

the matrices (Table 3). The conversion of the amide in K to an

amine in J results in considerably increased eﬃcacy at MOR

(from 49% to 90.7%), while the pendant amines presented in

Table 3 express higher MOR-eﬃcacy than their benzyl pendant

counterparts As such, the reduction of the amide to an amine

may prove eﬀective in the development of future high eﬃcacy

MOR-agonist peptides. Furthermore, the results illustrated in

Tables 4 and 5 point toward the development of selective MOR-

agonists or bifunctional MOR-agonists that may also be

produced with small deviations in structure.

were ≥95% pure, as determined by analytical HPLC. ¹H NMR and ¹³

C

NMR data were obtained on a 500 or 400 MHz Varian spectrometer

using CDCl₃, CD₃OD, DMSO-d6, or D₂O as solvents. The identities of

ﬁnal compounds were veriﬁed by mass spectrometry using an Agilent

6130 LC−MS mass spectrometer in positive ion mode, or an Agilent

6230 TOF HPLC-MS in the positive ion mode.

General Procedure for the Synthesis of 6-position Ethers

(Procedure A). To a ﬂame-dried ﬂask containing methyl 3-formyl-4-

hydroxybenzoate (1) was added 3 equiv of potassium carbonate. The

ﬂask was purged with argon, and 4 mL of DMF was added. Three

equivalents of an alkyl iodide or bromide was then added, and the

solution was stirred at room temperature overnight. The solution was

then concentrated in vacuo, partitioned between ethyl acetate and

saturated sodium carbonate, and extracted with ethyl acetate. The

organic layers were combined, dried with magnesium sulfate, ﬁltered,

and concentrated in vacuo, yielding the desired ether.

General Procedure for Ellman Reductions (Procedure B). A ﬂamed-

dried round-bottom ﬂask containing 1 equiv of aldehyde and 3 equiv of

(R)-(+)-2-methyl-2-propanesulﬁnamide was attached to a reﬂux

condenser and ﬂushed with argon. Four mL of THF was added and

cooled to 0 °C. Six equivalents of titanium(IV) ethoxide was added,

followed by an additional 4 mL of THF. The solution was stirred and

heated to 75 °C overnight with TLC monitoring until all ketone or

aldehyde was consumed. A separate ﬂame-dried ﬂask containing 6 equiv

of sodium borohydride was ﬂushed with argon. A volume of 4 mL of

THF was added, at which point the solution was cooled to −78 °C. The

solution containing Ellman adduct was cooled to room temperature and

slowly transferred to the sodium borohydride solution via syringe. This

ﬁnal solution was then allowed to warm to room temperature and

stirred for 2 h, at which point the reaction mixture was quenched with

methanol to consume the sodium borohydride, followed by DI water to

precipitate the titanium. The solution was vacuum ﬁltered, and the

precipitate was washed with ethyl acetate. The ﬁltrate was the

concentrated in vacuo and puriﬁed via column chromatography (0−

100% EtOAc in hexanes).

General Procedure for the Saponiﬁcation of Esters (Procedure C).

To a ﬂask containing 1 equiv of the desired ester was added 7 equiv of

LiOH, 2 mL of THF, 2 mL of EtOH, and 2 mL of H₂O. The reaction

was stirred overnight under ambient atmosphere and temperature.

Upon completion, the solvent was concentrated in vacuo, suspended in

acetone, and ﬁltered. The precipitate was washed with additional

acetone, and the ﬁltrate was concentrated in vacuo, yielding the

saponiﬁed product as a lithium carboxylate.

General Procedure for Amine Pendant Attachment and Cleavage

of Ellman Auxiliaries (Procedure D). To a ﬂask containing 1 equiv of

the lithium carboxylate or carboxylic acid was added 1 equiv of PyBOP

and 1 equiv of the desired amine. The ﬂask was ﬂushed with argon,

DMF was added as solvent, and 10 equiv of N-methylmorpholine was

added. The reaction was stirred overnight, at which point it was

concentrated in vacuo and puriﬁed via column chromatography (0−

10% methanol in DCM). To the protected amine was immediately

added 2 mL of dioxane and 0.2 mL concentrated HCl. The solution was

stirred at room temperature for 1 min and concentrated in vacuo. The

ensuing salt was triturated with diethyl ether, and then was puriﬁed

using a reversed-phase chromatography (0−100% B in A), yielding the

product as a TFA salt.

CONCLUSION

■

The two SAR campaigns illustrated here describe the develop-

ment of a potentially novel pharmacophore element that can be

used in the development of future opioid peptides and

peptidomimetics. The SAR matrices that describe the ﬁrst

campaign illustrate that an aromatic ring as represented in the

THIQ pendant produces high MOR-potency when compared

to the corresponding piperidine pendant. Furthermore, the high

MOR-eﬃcacy in both the piperidine and THIQ pendant

analogues compared to the previously reported benzyl pendant

illustrate that the amine in these pendants is responsible for this

eﬃcacy. In this ﬁrst series, the analogues acted as potent

bifunctional MOR-agonist/DOR antagonist ligands.

Given the promising proﬁle the THIQ pendants oﬀered, we

pursued a second SAR campaign aimed at simplifying the

peptidomimetics while simultaneously exploring the scope and

power that the THIQ pendants oﬀer to our peptidomimetic

series. These modiﬁcations consistently produced potent and

eﬃcacious MOR-agonists. Simultaneously, we found that slight

changes to the core structure can produce selective MOR-

agonists, bifunctional MOR-agonist/DOR-antagonists, and

bifunctional MOR/KOR analogues. Overall, the amine

component of the THIQ pendant may be a structurally eﬃcient

means of introducing MOR-eﬃcacy in opioid peptides and

peptidomimetics. Furthermore, the linking segment between the

THIQ pendant and the dimethyltyrosine pharmacophore can be

varied to ﬁne-tune selectivity and enable monofunctional or

bifunctional opioid eﬀects that can be used to develop future

opioid therapeutics.

EXPERIMENTAL SECTION

■

Chemistry. General Methods. All reagents and solvents were

obtained commercially and were used without further puriﬁcation.

Intermediates were puriﬁed by ﬂash chromatography using a Biotage

Isolera One instrument. Most puriﬁcation methods utilized a hexanes/

ethyl acetate solvent system in a Biotage SNAP KP-Sil column, with a

linear gradient between 0 and 100% ethyl acetate. Reversed-phase

column chromatography using a linear gradient of 0% to 100% solvent

B (0.1% TFA in acetonitrile) in solvent A (0.1% TFA in water) using a

General Procedure for the Reduction of Amides (Procedure E). To

a dried ﬂask containing 1 equiv of the desired amide under argon was

added THF and 7 equiv of 2 M BH₃*Me₂S complex in THF. The

reaction was heated at 75 °C for 3 h, at which point the reaction was

J

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

quenched with MeOH and heated for an additional 15 min. The

reaction was then cooled, concentrated in vacuo, and was used in

Procedure F without further puriﬁcation.

6.9 Hz, 2H), 3.80 (s, 3H), 1.31−1.20 (m, 1H), 0.65−0.56 (m, 2H), 0.32

(dt, J = 6.2, 4.8 Hz, 2H). ¹³C NMR (126 MHz, chloroform-d) δ 188.81,

165.84, 164.29, 136.86, 130.06, 124.34, 122.48, 112.43, 73.58, 51.97,

9.83, 3.18.

General Procedure for the Coupling of 2′,6′-Dimethyltyrosine to

Functionalized Amine Salts (Procedure F). To a dried ﬂask containing

the amine under argon was added 3 mL of DMF and 10 equiv of

Hunig’s base. One equivalent of PyBOP and 1 equiv of 6-Cl-HOBt were

added, followed by a 1 equiv of doubly Boc protected 2′,6′-

dimethyltyrosine in 1.5 mL of DMF. The solution was stirred overnight

at room temperature, concentrated in vacuo, and puriﬁed via

semipreparative reversed-phase HPLC (0.1% TFA in water: 0.1%

TFA in acetonitrile). Then 2 mL of TFA and 2 mL of DCM were added,

and the solution was stirred for an additional hour. The reaction

mixture was concentrated in vacuo and puriﬁed via an additional

semipreparative reversed-phase HPLC (0.1% TFA in water: 0.1% TFA

in acetonitrile). The product was concentrated in vacuo and lyophilized

overnight to yield the ﬁnal peptidomimetic.

General Procedure for Coupling of Tetrahydroisoquinoline

Analogues to N-Boc Protected Carboxylic Acids (Procedure G). To

a ﬂask containing 1 equiv of N-Boc protected carboxylic acid was added

1 equiv of PyBOP and 1 equiv of tetrahydroisoquinoline or the

tetrahydroquinoline analogue. The ﬂask was ﬂushed with argon, DMF

was added as the solvent, and 10 equiv of N-methylmorpholine was

added. The reaction was stirred overnight, at which point it was

concentrated in vacuo. The residue was then partitioned between

EtOAc and sat. Na₂CO₃and extracted with EtOAc, the organic layers

were combined, dried with MgSO₄, ﬁltered, and concentrated in vacuo.

Puriﬁcation by column chromatography (0−5% MeOH in DCM, or 0−

66% EtOAc in hexanes) yielded the coupled product.

General Procedure for the Deprotection of Boc-Groups Using

Concentrated HCl (Procedure Ha). To the Boc protected compound

was added 2−6 mL of dioxane and 0.2−0.6 mL concentrated HCl. The

solution was stirred at room temperature for 1−5 min and concentrated

in vacuo. The ensuing salt was suspended in solvent A and was either

concentrated in vacuo and triturated with hexanes, or puriﬁed by

reversed-phase chromatography (0−100% B in A), yielding the product

as a TFA salt.

General Procedure for the Deprotection of Boc-Groups Using TFA

(Procedure Hb). To the Boc protected compound was added 2 mL of

TFA and 2 mL of DCM. The solution was stirred at room temperature

for 1−5 min and concentrated in vacuo. The ensuing salt was either

continued without further puriﬁcation or suspended in solvent A and

was puriﬁed via reversed-phase chromatography (0−100% B in A),

yielding the product as a TFA salt.

Ethyl (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)benzoate (6). See

Procedure B: Step 1: 220 mg (1.34 mmol) of methyl 3-formylbenzoate

(2), 490 mg (4.04 mmol, 3.02 equiv) of (R)-(+)-2-methyl-2-

propanesulﬁnamide, 1.7 mL (1.8 g, 8.1 mmol, 6.1 equiv) of Ti(OEt)₄,

5 + 5 mL THF. Step 2: 331 mg (8.75 mmol, 6.08 equiv) of sodium

borohydride in 5 mL of THF. Compound 6 (278 mg, yield = 73%) was

isolated as a colorless oil. ¹H NMR (500 MHz, chloroform-d) δ 7.93 (t,

J = 1.8 Hz, 1H), 7.86 (dt, J = 7.8, 1.5 Hz, 1H), 7.46 (dt, J = 7.7, 1.4 Hz,

1H), 7.32 (td, J = 7.7, 2.5 Hz, 1H), 4.33−4.18 (m, 4H), 3.80 (dd, J = 6.9,

4.6 Hz, 1H), 1.30 (t, J = 7.1 Hz, 3H), 1.17 (d, J = 1.4 Hz, 9H). ¹³C NMR

(126 MHz, chloroform-d) δ 166.24, 139.00, 132.41, 130.68, 128.97,

128.66, 128.57, 60.95, 55.94, 48.79, 22.63, 14.24.

Ethyl (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)-4-methoxyben-

zoate (7). See Procedure B: Step 1: 234 mg (1.21 mmol) of 3, 441

mg (3.64 mmol, 3.02 equiv) of (R)-(+)-2-methyl-2-propanesulﬁna-

mide, 1.55 mL (1.69 g, 7.39 mmol, 6.14 equiv) of Ti(OEt)₄, 4 + 4 mL of

THF. Step 2: 274 mg (7.24 mmol, 6.01 equiv) of sodium borohydride

in 4 mL THF. Compound 7 (374 mg, yield = 99%) was isolated as a

colorless oil. ¹H NMR (500 MHz, chloroform-d) δ 7.96−7.90 (m, 2H),

6.84 (d, J = 9.3 Hz, 1H), 4.36 (dd, J = 14.3, 5.6 Hz, 1H), 4.28 (q, J = 7.1

Hz, 2H), 4.16 (dd, J = 14.3, 7.6 Hz, 1H), 3.85 (s, 3H), 3.73 (t, J = 6.6

Hz, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.17 (s, 9H). ¹³C NMR (126 MHz,

chloroform-d) δ 166.19, 160.92, 131.07, 130.63, 127.08, 122.66,

109.83, 60.65, 55.88, 55.58, 44.92, 22.56, 21.94, 14.31.

Ethyl (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)-4-propoxyben-

zoate (8). See Procedure B: Step 1: 181 mg (0.81 mmol) of 4, 299

mg (2.5 mmol, 3.0 equiv) of (R)-(+)-2-methyl-2-propanesulﬁnamide,

1025 μL (1115 mg, 4.9 mmol, 6.0 equiv) of Ti(OEt)₄, 4 + 4 mL of THF.

Step 2: 187 mg (4.9 mmol, 6.1 equiv) of sodium borohydride in 4 mL

THF. Compound 8 (272 mg, yield = 98%) was isolated as a yellow oil.

¹H NMR (500 MHz, chloroform-d) δ 7.89 (d, J = 2.2 Hz, 1H), 7.86 (dd,

J = 8.5, 2.3 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 4.32 (dd, J = 14.4, 5.5 Hz,

1H), 4.23 (q, J = 7.0 Hz, 2H), 4.13 (dd, J = 14.3, 7.7 Hz, 1H), 3.91 (t, J =

6.4 Hz, 2H), 3.76 (t, J = 6.6 Hz, 1H), 1.75 (h, J = 7.4 Hz, 2H), 1.27 (t, J

= 7.1 Hz, 3H), 1.13 (s, 9H), 0.97 (t, J = 7.4 Hz, 3H). ¹³C NMR (126

MHz, chloroform-d) δ 166.11, 160.35, 130.90, 130.46, 127.11, 122.34,

110.40, 69.73, 60.20, 55.74, 44.88, 22.52, 22.40, 14.25, 10.52.

Ethyl (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)-4-

(cyclopropylmethoxy)benzoate (9). See Procedure B: Step 1: 230

mg (0.98 mmol) of 5, 359 mg (2.96 mmol, 3.02 equiv) of (R)-(+)-2-

methyl-2-propanesulﬁnamide, 1.25 mL (1.36 g, 5.96 mmol, 6.07 equiv)

of Ti(OEt)₄, 4 + 4 mL of THF. Step 2: 225 mg (4.26 mmol, 5.95 equiv)

of sodium borohydride in 4 mL of THF. Compound 9 (333 mg, yield =

96%) was isolated as a colorless oil. ¹H NMR (500 MHz, chloroform-d)

δ 7.97−7.87 (m, 2H), 6.79 (d, J = 8.5 Hz, 1H), 4.42 (dd, J = 14.3, 5.7

Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.20 (dd, J = 14.3, 7.8 Hz, 1H), 3.91−

3.81 (m, 3H), 1.34 (t, J = 7.1 Hz, 4H), 1.29−1.20 (m, 2H), 1.19 (s,

10H), 0.66−0.58 (m, 2H), 0.36−0.29 (m, 2H). ¹³C NMR (126 MHz,

chloroform-d) δ 166.23, 160.41, 131.01, 130.59, 127.24, 122.48,

110.58, 72.99, 60.65, 55.83, 45.46, 22.59, 14.33, 10.10, 3.24, 3.20.

Lithium (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)benzoate (10).

See Procedure C: 285 mg (1.01 mmol) of 6, 143 mg (5.97 mmol, 5.94

equiv) of LiOH, 2 mL of THF, 2 mL of EtOH, and 2 mL of H₂O. The

compound was suspended and ﬁltered in EtOH instead of acetone.

Compound 10 (240 mg, yield = 91%) was isolated as a white solid. ¹H

NMR (400 MHz, methanol-d₄) δ 7.92 (d, J = 1.8 Hz, 1H), 7.85 (dt, J =

7.7, 1.5 Hz, 1H), 7.45−7.39 (m, 1H), 7.33 (t, J = 7.6 Hz, 1H), 4.34 (d, J

= 14.4 Hz, 1H), 4.26 (d, J = 14.4 Hz, 1H), 1.24 (s, 9H). ¹³C NMR (101

MHz, methanol-d₄) δ 173.83, 138.51, 137.91, 129.60, 128.53, 127.93,

127.48, 55.72, 48.76, 21.79.

Methyl 4-Methoxy-3-formylbenzoate (3). See Procedure A: 160

mg (0.88 mmol) of methyl 3-formyl-4-hydroxybenzoate (1), 368 mg

(2.66 mmol, 3.00 equiv) of K₂CO₃, 170 μL (388 mg, 2.73 mmol, 3.08

equiv) of MeI, 4 mL of DMF. Compound 3 (162 mg, yield = 94%) was

isolated as a yellow solid. ¹H NMR (500 MHz, chloroform-d) δ 10.44

(s, 1H), 8.49 (d, J = 2.3 Hz, 1H), 8.23 (dd, J = 8.8, 2.3 Hz, 1H), 7.04 (d,

J = 8.8 Hz, 1H), 4.00 (s, 3H), 3.90 (s, 3H). ¹³C NMR (126 MHz,

chloroform-d) δ 188.84, 165.96, 164.71, 137.10, 130.67, 124.44,

122.92, 111.54, 56.06, 52.12.

Methyl 4-Propoxy-3-formylbenzoate (4). See Procedure A: 168 mg

(0.93 mmol) of methyl 3-formyl-4-hydroxybenzoate (1), 388 mg (2.81

mmol, 3.01 equiv) of K₂CO₃, 260 μL (352 mg, 2.86 mmol, 3.07 equiv)

of nPrBr, 4 mL of DMF. Compound 4 (188 mg, yield = 91%) was

1

isolated as a colorless oil that turns to a white solid on standing. H

NMR (500 MHz, chloroform-d) δ 10.44 (s, 1H), 8.43 (d, J = 2.3 Hz,

1H), 8.15 (dd, J = 8.8, 2.3 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 4.07 (t, J =

6.4 Hz, 2H), 3.86 (s, 3H), 1.87 (h, J = 7.2 Hz, 2H), 1.05 (t, J = 7.4 Hz,

3H). ¹³C NMR (126 MHz, chloroform-d) δ 188.78, 165.94, 164.41,

136.98, 130.24, 124.36, 122.56, 112.27, 70.44, 52.03, 22.30, 10.41.

Methyl 4-(Cyclopropylmethoxy)-3-formylbenzoate (5). See Pro-

cedure A: 181 mg (1.00 mmol) of methyl 3-formyl-4-hydroxybenzoate

(1), 417 g (3.02 mmol, 3.00 equiv) of K₂CO₃, 290 μL (404 mg, 2.99

mmol, 2.98 equiv) of cyclopropylmethyl bromide, 4 mL of DMF.

Lithium (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)-4-methoxyben-

zoate (11). See Procedure C: 181 mg (0.58 mmol) of 7, 86 mg (3.59

mmol, 6.21 equiv) of LiOH, 2 mL of THF, 2 mL of EtOH, and 2 mL of

H₂O. Compound 11 (128 mg, yield = 76%) was isolated as a white

solid. ¹H NMR (500 MHz, methanol-d₄) δ 7.95−7.88 (m, 2H), 6.93 (d,

J = 8.4 Hz, 1H), 4.32 (d, J = 14.4 Hz, 1H), 4.22 (d, J = 14.4 Hz, 1H),

1

Compound 5 (233 mg, yield = 99%) was isolated as a yellow oil. H

NMR (500 MHz, chloroform-d) δ 10.41 (s, 1H), 8.35 (d, J = 2.3 Hz,

1H), 8.07 (dd, J = 8.8, 2.3 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 3.91 (d, J =

K

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

3.87 (s, 3H), 1.22 (s, 9H). ¹³C NMR (126 MHz, Methanol-d₄) δ

173.83, 159.18, 130.41, 130.20, 129.69, 125.95, 109.03, 55.64, 54.63,

44.23, 21.74.

158.30, 129.68, 129.29, 127.96, 121.38, 111.51, 73.30, 38.45, 24.01,

9.50, 2.24.

(3-(1,2,3,4-Tetrahydroisoquinoline-2-carbonyl)phenyl)-

methanaminium Triﬂuoroacetate (18). See Procedure D: Step 1: 16

mg (0.061 mmol) of 10, 36 mg (0.069 mmol, 1.13 equiv) of PyBOP, 20

μL (21 mg, 0.16 mmol, 2.6 equiv) of 1,2,3,4-tetrahydroisoquinoline, 80

μL (74 mg, 0.73 mmol, 11.9 equiv) of NMM, and 5 mL of DMF. Step 2:

2 mL of dioxane and 0.2 mL of conc. HCl. Compound 18 (17 mg, yield

Lithium (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)-4-propoxyben-

zoate (12). See Procedure C: 272 mg (0.80 mmol) of 8, 114 mg (4.76

mmol, 6.0 equiv) of LiOH, 2 mL of THF, 2 mL of EtOH, and 2 mL of

H₂O. Compound 12 (226 mg, yield = 89%) was isolated as a colorless

oil. ¹H NMR (400 MHz, methanol-d₄) δ 7.92 (d, J = 2.2 Hz, 1H), 7.87

(dd, J = 8.5, 2.2 Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 4.35 (d, J = 14.3 Hz,

1H), 4.23 (d, J = 14.3 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H), 1.85 (h, J = 7.4

Hz, 2H), 1.22 (s, 9H), 1.08 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz,

methanol-d₄) δ 173.91, 158.57, 130.34, 130.11, 129.48, 125.96, 109.75,

69.35, 55.63, 44.22, 22.32, 21.73, 9.67.

1

= 73%) was isolated as a colorless oil. H NMR (400 MHz, 50 °C,

methanol-d₄) δ 7.62−7.41 (m, 5H), 7.17 (s, 3H), 4.77 (br s, 2H), 4.18

(s, 2H), 3.68 (br s, 2H), 2.92 (br s, 2H). ¹³C NMR (101 MHz, 50 °C,

methanol-d₄) δ 170.64,136.84, 133.78, 132.44, 130.11, 129.20, 128.32,

127.16, 127.00, 126.57, 126.18, 42.63.

(2-Methoxy-5-(1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-

phenyl)methanaminium Triﬂuoroacetate (19). See Procedure D:

Step 1: 19 mg (0.065 mmol) of 11, 34 mg (0.065 mmol, 1.0 equiv) of

PyBOP, 40 μL (42 mg, 0.32 mmol, 4.8 equiv) of 1,2,3,4-

tetrahydroisoquinoline, 75 μL (69 mg, 0.68 mmol, 10.0 equiv) of

NMM, and 4 mL of DMF. Step 2: 2 mL of dioxane and 0.2 mL of conc.

HCl. Compound 19 (23 mg, yield = 86%) was isolated as a colorless oil.

¹H NMR (400 MHz, 50 °C, methanol-d₄) δ 7.56 (dd, J = 8.5, 2.2 Hz,

1H), 7.47 (d, J = 2.1 Hz, 1H), 7.20−7.13 (m, 4H), 7.07 (br s, 1H), 4.74

(br s, 2H), 4.15 (s, 2H), 3.98 (s, 3H), 3.80 (br s, 2H), 2.92 (t, J = 6.1 Hz,

2H). ¹³C NMR (101 MHz, 50 °C, methanol-d₄) δ 159.24, 134.20,

132.60, 130.00, 129.60, 128.31, 128.17, 126.52, 126.14, 121.34, 110.66,

55.13, 38.75.

(2-Propoxy-5-(1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-

phenyl)methanaminium Triﬂuoroacetate (20). See Procedure D:

Step 1: 30 mg (0.094 mmol) of 12, 49 mg (0.094 mmol, 1.0 equiv) of

PyBOP, 20 μL (21 mg, 0.16 mmol, 1.7 equiv) of 1,2,3,4-

tetrahydroisoquinoline, 110 μL (101 mg, 1.0 mmol, 10.7 equiv) of

NMM, and 4 mL of DMF. Step 2: 2 mL of dioxane and 0.2 mL of conc.

HCl. Compound 20 (33 mg, yield = 80%) was isolated as a colorless oil.

¹H NMR (400 MHz, 50 °C, methanol-d₄) δ 7.53 (dd, J = 8.5, 2.2 Hz,

1H), 7.48 (d, J = 2.1 Hz, 1H), 7.21−7.12 (m, 4H), 7.07 (br s, 1H), 4.73

(br s, 2H), 4.17 (s, 2H), 4.14 (t, J = 6.6 Hz, 2H), 3.80 (br s, 2H), 2.92

(br t, J = 6.1 Hz, 2H), 1.90 (h, J = 7.2 Hz, 2H), 1.08 (t, J = 7.4 Hz, 3H).

¹³C NMR (101 MHz, 50 °C, Methanol-d₄) δ 158.55, 134.20, 132.61,

Lithium (R)-3-(((tert-Butylsulﬁnyl)amino)methyl)-4-

(cyclopropylmethoxy)benzoate (13). See Procedure C: 333 mg

(0.94 mmol) of 9, 135 mg (5.64 mmol, 5.98 equiv) of LiOH, 2 mL

of THF, 2 mL of EtOH, and 2 mL of H₂O. Compound 13 (271 mg,

yield = 87%) was isolated as a white amorphous solid. ¹H NMR (500

MHz, Methanol-d₄) δ 7.93 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.5, 2.2 Hz,

1H), 6.89 (d, J = 8.6 Hz, 1H), 4.37 (d, J = 14.3 Hz, 1H), 4.25 (d, J = 14.4

Hz, 1H), 3.90 (d, J = 7.2 Hz, 2H), 1.30 (dddd, J = 11.6, 8.0, 4.7, 1.2 Hz,

2H), 1.23 (s, 12H), 0.67−0.59 (m, 2H), 0.41−0.35 (m, 2H). ¹³C NMR

(126 MHz, methanol-d₄) δ 173.84, 158.58, 130.38, 130.15, 129.60,

126.06, 110.05, 72.54, 55.65, 44.55, 21.77, 9.87, 2.28, 2.27.

(3-(Piperidine-1-carbonyl)phenyl)methanaminium Triﬂuoroace-

tate (14). See Procedure D: Step 1:20 mg (0.077 mmol) of 10, 41 mg

(0.079 mmol, 1.03 equiv) of PyBOP, 20 μL (17 mg, 0.20 mmol, 2.6

equiv) of piperidine, 90 μL (83 mg, 0.82 mmol, 10.7 equiv) of NMM,

and 4 mL of DMF. Step 2: 2 mL of dioxane and 0.2 mL conc. HCl.

Compound 14 (15 mg, yield = 59%) was isolated as a colorless oil. ¹H

NMR (500 MHz, methanol-d₄) δ 7.60−7.51 (m, 2H), 7.47 (d, J = 1.8

Hz, 1H), 7.44 (dt, J = 7.1, 1.6 Hz, 1H), 3.72 (d, J = 5.9 Hz, 2H), 3.37 (t,

J = 5.4 Hz, 2H), 1.76−1.63 (m, 4H), 1.54 (s, 2H). ¹³C NMR (126 MHz,

methanol-d₄) δ 170.04, 136.87, 133.77, 129.94, 129.13, 126.96, 126.91,

48.62, 42.94, 42.53, 26.13, 25.28, 23.97.

(2-Methoxy-5-(piperidine-1-carbonyl)phenyl)methanaminium

Triﬂuoroacetate (15). See Procedure D: Step 1: 23 mg (0.079 mmol)

of 11, 42 mg (0.081 mmol, 1.02 equiv) of PyBOP, 20 μL (17 mg, 0.20

mmol, 2.5 equiv) of piperidine, 90 μL (83 mg, 0.82 mmol, 10.4 equiv)

of NMM, and 4 mL of DMF. Step 2: 2 mL of dioxane and 0.2 mL conc.

HCl. Compound 15 (23 mg, yield = 80%) was isolated as a colorless oil.

¹H NMR (500 MHz, methanol-d₄) δ 7.49 (dd, J = 8.5, 2.2 Hz, 1H), 7.41

(d, J = 2.1 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 4.13 (s, 2H), 3.97 (s, 3H),

3.69 (br s, 2H), 3.44 (br s, 2H), 1.74−1.51 (m, 6H). ¹³C NMR (126

MHz, methanol-d₄) δ 170.18, 158.97, 129.82, 129.49, 128.10, 121.24,

110.47, 55.08, 48.43, 38.67, 24.01, 20.05.

(5-(Piperidine-1-carbonyl)-2-propoxyphenyl)methanaminium

Triﬂuoroacetate (16). See Procedure D: Step 1: 25 mg (0.078 mmol)

of 12, 41 mg (0.079 mmol, 1.0 equiv) of PyBOP, 20 μL (17 mg, 0.20

mmol, 2.6 equiv) of piperidine, 90 μL (83 mg, 0.82 mmol, 10.5 equiv)

of NMM, and 4 mL of DMF. Step 2: 2 mL of dioxane and 0.2 mL conc.

HCl. Compound 16 (24 mg, yield = 79%) was isolated as a colorless oil.

¹H NMR (500 MHz, methanol-d₄) δ 7.46 (dd, J = 8.5, 2.2 Hz, 1H), 7.41

(d, J = 2.1 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 4.14 (s, 2H), 4.11 (t, J = 6.6

Hz, 2H), 3.68 (br s, 2H), 3.44 (br s, 2H), 1.89 (h, J = 7.4 Hz, 2H),

1.75−1.61 (m, 4H), 1.55 (br s, 2H), 1.08 (t, J = 7.4 Hz, 3H). ¹³C NMR

(126 MHz, methanol-d₄) δ 170.32, 158.30, 129.73, 129.29, 127.79,

121.27, 111.30, 70.03, 38.34, 23.98, 21.89, 20.07, 9.34.

(2-(Cyclopropylmethoxy)-5-(piperidine-1-carbonyl)phenyl)-

methanaminium Triﬂuoroacetate (17). See Procedure D: Step 1:31

mg (0.094 mmol) of 13, 49 mg (0.094 mmol, 1.01 equiv) of PyBOP, 20

μL (17 mg, 0.20 mmol, 2.2 equiv) of piperidine, 100 μL (92 mg, 0.91

mmol, 9.7 equiv) of NMM, and 4.5 mL of DMF. Step 2:2 mL of dioxane

and 0.2 mL conc. HCl. Compound 17 (25 mg, yield = 69%) was

isolated as a colorless oil. ¹H NMR (500 MHz, methanol-d₄) δ 7.45 (d, J

= 8.4 Hz, 1H), 7.42 (s, 1H), 7.12 (d, J = 8.4 Hz, 1H), 4.17 (s, 2H), 4.00

(d, J = 6.9 Hz, 2H), 3.68 (br s, 2H), 3.44 (br s, 2H), 1.72 (br s, 2H), 1.64

(br s, 2H), 1.56 (br s, 2H), 1.42−1.30 (m, 1H), 0.71−0.61 (m, 2H),

0.48−0.35 (m, 2H). ¹³C NMR (126 MHz, methanol-d₄) δ 170.20,

129.88, 129.46, 128.31, 127.98, 126.53, 126.14, 121.42, 111.50, 70.23,

38.43, 21.90, 9.22.

(2-(Cyclopropylmethoxy)-5-(1,2,3,4-tetrahydroisoquinoline-2-

carbonyl)phenyl)methanaminium Triﬂuoroacetate (21). See Proce-

dure D: Step 1: 28 mg (0.085 mmol) of 13, 46 mg (0.089 mmol, 1.0

equiv) of PyBOP, 20 μL (21 mg, 0.16 mmol, 1.9 equiv) of 1,2,3,4-

tetrahydroisoquinoline, 100 μL (92 mg, 0.91 mmol, 10.8 equiv) of

NMM, and 4 mL of DMF. Step 2: 2 mL of dioxane and 0.2 mL of conc.

HCl. Compound 21 (30 mg, yield = 79%) was isolated as a colorless oil.

¹H NMR (400 MHz, 50 °C, methanol-d₄) δ 7.52 (dd, J = 8.5, 2.1 Hz,

1H), 7.48 (d, J = 1.9 Hz, 1H), 7.20−6.97 (m, 5H), 4.73 (s, 2H), 4.19 (s,

2H), 4.04 (d, J = 7.0 Hz, 2H), 3.80 (s, 2H), 2.92 (t, J = 6.1 Hz, 2H),

1.41−1.29 (m, 1H), 0.77−0.60 (m, 2H), 0.42 (qd, J = 4.6, 2.3 Hz, 2H).

¹³C NMR (101 MHz, 50 °C, methanol-d₄) δ 158.58, 134.19, 132.61,

129.86, 129.42, 128.31, 128.04, 126.52, 126.14, 121.52, 111.81, 73.45,

38.59, 9.53, 2.22.

(S)-3-(4-Hydroxy-2,6-dimethylphenyl)-1-oxo-1-((3-(piperidin-1-

ylmethyl)benzyl)amino)propan-2-aminium Triﬂuoroacetate (A).

See Procedures E and F: 16 mg (0.048 mmol) of 14, 170 μL (0.34

mmol, 7.08 equiv) of 2 M BH₃*Me₂S in THF, and 4 mL of THF. Step 1

of F: 90 μL (67 mg, 0.52 mmol, 10.7 equiv) of N,N-diisopropylethyl-

amine, 29 mg (0.056 mmol, 1.16 equiv) of PyBOP, 9 mg (0.053 mmol,

1.10 equiv) of 6-Cl-HOBt, 21 mg (0.051 mmol, 1.07 equiv) of Boc-O-

Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF. Step 2 of F: 2 mL of

TFA and 2 mLof DCM. Compound A (11.5 mg, yield = 47%) was

isolated as a white solid. (MS)EI: 396.3 (M + H). Retention time: 15.67

min. ¹H NMR (500 MHz, deuterium oxide) δ 7.95 (t, J = 6.0 Hz, 1H),

7.33−7.18 (m, 2H), 6.96 (s, 1H), 6.90 (d, J = 7.2 Hz, 1H), 6.28 (s, 2H),

4.38−4.28 (m, 1H), 4.13 (d, J = 13.2 Hz, 1H), 4.06 (d, J = 13.2 Hz, 1H),

3.95−3.82 (m, 2H), 3.29 (t, J = 12.4 Hz, 2H), 3.03 (dd, J = 13.9, 11.8

Hz, 1H), 2.94 (dd, J = 14.0, 5.0 Hz, 1H), 2.78 (qd, J = 13.1, 3.0 Hz, 2H),

L

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

1.93 (s, 6H), 1.78 (d, J = 12.7 Hz, 2H), 1.67 (dt, J = 13.4, 3.3 Hz, 1H),

1.60−1.46 (m, 2H), 1.32 (qt, J = 12.6, 3.5 Hz, 1H).

11.6, 4.7 Hz, 1H), 3.76 (s, 1H), 3.42 (s, 1H), 3.25−3.14 (m, 3H), 3.01

(dd, J = 13.8, 4.8 Hz, 1H), 2.19 (s, 6H).

(S)-3-(4-Hydroxy-2,6-dimethylphenyl)-1-((2-methoxy-5-(piperi-

din-1-ylmethyl)benzyl)amino)-1-oxopropan-2-aminium Triﬂuoroa-

cetate (B). See Procedures E and F: 23 mg (0.063 mmol) of 15, 220 μL

(0.44 mmol, 6.93 equiv) of 2 M BH₃*Me₂S in THF, and 4 mL of THF.

Step 1 of F: 110 μL (82 mg, 0.63 mmol, 9.95 equiv) of N,N-

diisopropylethylamine, 33 mg (0.063 mmol, 1.00 equiv) of PyBOP, 13

mg (0.077 mmol, 1.21 equiv) of 6-Cl-HOBt, 28 mg (0.068 mmol, 1.08

equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF.

Step 2 of F: 2 mL of TFA and 2 mL of DCM. Compound B (9.1 mg,

yield = 27%) was isolated as a white solid. 426.3 (M + H). Retention

time: 16.13 min. ¹H NMR (500 MHz, methanol-d₄) δ 7.94 (t, J = 6.0

Hz, 1H), 7.41 (dd, J = 8.4, 2.3 Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.00 (d,

J = 8.4 Hz, 1H), 6.33 (s, 2H), 4.35 (dd, J = 14.5, 6.8 Hz, 1H), 4.22−4.12

(m, 3H), 3.89 (dd, J = 11.7, 4.4 Hz, 1H), 3.74 (s, 3H), 3.42 (t, J = 12.3

Hz, 2H), 3.12 (dd, J = 13.7, 11.8 Hz, 1H), 2.96−2.84 (m, 3H), 2.04 (s,

6H), 1.93 (d, J = 14.9 Hz, 2H), 1.84 (d, J = 13.7 Hz, 1H), 1.78−1.66 (m,

2H), 1.50 (qt, J = 12.5, 3.7 Hz, 1H).

(S)-1-((5-((3,4-Dihydroisoquinolin-2(1H)-yl)methyl)-2-

methoxybenzyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxo-

propan-2-aminium Triﬂuoroacetate (F). See Procedures E and F: 22

mg (0.054 mmol) of 19, 190 μL (0.38 mmol, 7.09 equiv) of 2 M

BH₃*Me₂S in THF, and 4 mL of THF. Step 1 of F: 100 μL (74 mg, 0.57

mmol, 10.7 equiv) of N,N-diisopropylethylamine, 28 mg (0.054 mmol,

1.00 equiv) of PyBOP, 10 mg (0.059 mmol, 1.10 equiv) of 6-Cl-HOBt,

23 mg (0.056 mmol, 1.05 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyr-

osine, 3 + 1.5 mL of DMF. Step 2 of F: 2 mL of TFA and 2 mL of DCM.

Compound F (3.4 mg, yield = 11%) was isolated as a white solid.

1

(MS)EI: 474.3 (M + H). Retention time: 20.56 min. H NMR (500

MHz, deuterium oxide) δ 7.39 (dd, J = 8.5, 2.3 Hz, 1H), 7.25−7.19 (m,

1H), 7.19−7.14 (m, 3H), 7.03 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 8.5 Hz,

1H), 6.11 (s, 2H), 4.32 (d, J = 13.5 Hz, 3H), 4.24 (s, 2H), 3.88 (d, J =

14.1 Hz, 1H), 3.84 (dd, J = 11.6, 5.1 Hz, 1H), 3.52 (s, 5H), 3.07 (t, J =

6.4 Hz, 2H), 2.96−2.81 (m, 2H), 1.76 (s, 6H).

(S)-1-((5-((3,4-Dihydroisoquinolin-2(1H)-yl)methyl)-2-

propoxybenzyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxo-

propan-2-aminium Triﬂuoroacetate (G). See Procedures E and F: 33

mg (0.075 mmol) of 20, 260 μL (0.52 mmol, 6.93 equiv) of 2 M

BH₃*Me₂S in THF, and 4 mL of THF. Step 1 of F: 130 μL (96 mg, 0.75

mmol, 9.95 equiv) of N,N-diisopropylethylamine, 39 mg (0.075 mmol,

1.00 equiv) of PyBOP, 13 mg (0.077 mmol, 1.02 equiv) of 6-Cl-HOBt,

31 mg (0.076 mmol, 1.01 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyr-

osine, 3 + 1.5 mL of DMF. Step 2 of F: 2 mL of TFA and 2 mL of DCM.

Compound G (7 mg, yield = 15%) was isolated as a white solid.

(S)-3-(4-Hydroxy-2,6-dimethylphenyl)-1-oxo-1-((5-(piperidin-1-

ylmethyl)-2-propoxybenzyl)amino)propan-2-aminium Triﬂuoroa-

cetate (C). See Procedures E and F: 24 mg (0.062 mmol) of 16, 220

μL (0.44 mmol, 7.15 equiv) of 2 M BH₃*Me₂S in THF, and 4 mL of

THF. Step 1 of F: 110 μL (82 mg, 0.63 mmol, 10.3 equiv) of N,N-

diisopropylethylamine, 28 mg (0.061 mmol, 1.00 equiv) of PyBOP, 11

mg (0.065 mmol, 1.05 equiv) of 6-Cl-HOBt, 26 mg (0.063 mmol, 1.03

equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF.

Step 2 of F: 2 mL of TFA and 2 mL of DCM. Compound C (4 mg, yield

= 12%) was isolated as a white solid. (MS)EI: 454.3 (M + H). Retention

time: 21.64 min. ¹H NMR (500 MHz, methanol-d₄) δ 7.66 (t, J = 5.8

Hz, 1H), 7.39 (dd, J = 8.4, 2.3 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 6.99 (d,

J = 8.4 Hz, 1H), 6.35 (s, 2H), 4.39 (dd, J = 14.7, 6.8 Hz, 1H), 4.24−4.11

(m, 3H), 3.98−3.82 (m, 3H), 3.41 (d, J = 13.6 Hz, 2H), 3.12 (dd, J =

13.7, 11.9 Hz, 1H), 2.98−2.83 (m, 3H), 2.04 (s, 6H), 1.94 (d, J = 10.4

Hz, 2H), 1.84 (d, J = 13.5 Hz, 1H), 1.79−1.65 (m, 4H), 1.50 (qt, J =

12.6, 3.4 Hz, 1H), 1.01 (t, J = 7.4 Hz, 3H).

(S)-1-((2-(Cyclopropylmethoxy)-5-(piperidin-1-ylmethyl)benzyl)-

amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-ami-

nium Triﬂuoroacetate (D). See Procedures E and F: 25 mg (0.062

mmol) of 17, 220 μL (0.44 mmol, 7.10 equiv) of 2 M BH₃*Me₂S in

THF, and 4 mL of THF. Step 1 of F: 110 μL (82 mg, 0.63 mmol, 10.2

equiv) of N,N-diisopropylethylamine, 32 mg (0.061 mmol, 0.99 equiv)

of PyBOP, 12 mg (0.071 mmol, 1.14 equiv) of 6-Cl-HOBt, 25 mg

(0.061 mmol, 0.98 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 +

1.5 mL of DMF. Step 2 of F: 2 mL TFA and 2 mL DCM. Compound D

(1.8 mg, yield = 5%) was isolated as a white solid. (MS)EI: 466.3 (M +

H). Retention time: 22.01 min. ¹H NMR (500 MHz, methanol-d₄) δ

7.63 (t, J = 5.9 Hz, 1H), 7.37 (dd, J = 8.4, 2.3 Hz, 1H), 7.12 (d, J = 2.3

Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.34 (s, 2H), 4.42 (dd, J = 14.7, 6.8

Hz, 1H), 4.24−4.13 (m, 3H), 3.90 (dd, J = 11.7, 4.4 Hz, 1H), 3.78 (d, J

= 6.9 Hz, 2H), 3.47−3.37 (m, 3H), 3.13 (dd, J = 13.6, 11.9 Hz, 2H),

2.95 (dd, J = 14.1, 4.3 Hz, 2H), 2.93−2.82 (m, 2H), 2.05 (s, 6H), 1.97−

1.91 (m, 2H), 1.84 (d, J = 13.6 Hz, 1H), 1.72 (p, J = 12.7 Hz, 2H), 1.51

(dddd, J = 16.9, 13.0, 8.2, 3.7 Hz, 1H), 1.16 (dddd, J = 15.5, 12.3, 7.6,

4.6 Hz, 1H), 0.59 (dd, J = 8.1, 1.8 Hz, 2H), 0.30 (pd, J = 4.2, 2.8 Hz,

2H).

1

(MS)EI: 502.3 (M + H). Retention time: 26.01 min. H NMR (500

MHz, deuterium oxide) δ 7.51 (q, J = 7.5 Hz, 1H), 7.39 (dt, J = 8.6, 3.1

Hz, 1H), 7.27−7.15 (m, 4H), 7.05 (t, J = 7.9 Hz, 1H), 6.92 (dd, J = 8.5,

3.0 Hz, 1H), 6.17 (s, 2H), 4.39−4.19 (m, 6H), 3.99−3.84 (m, 3H),

3.75−3.62 (m, 3H), 3.36−3.25 (m, 1H), 3.13−3.02 (m, 2H), 2.96−

2.78 (m, 2H), 1.72 (s, 6H), 1.47 (dtdd, J = 27.3, 14.1, 6.8, 2.1 Hz, 2H),

0.81 (t, J = 7.4 Hz, 3H).

(S)-1-((2-(Cyclopropylmethoxy)-5-((3,4-dihydroisoquinolin-

2(1H)-yl)methyl)benzyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-

1-oxopropan-2-aminium Triﬂuoroacetate (H). See Procedures E and

F: 30 mg (0.067 mmol) of 21, 230 μL (0.46 mmol, 6.91 equiv) of 2 M

BH₃*Me₂S in THF, and 4 mL of THF. Step 1 of F: 120 μL (89 mg, 0.69

mmol, 10.3 equiv) of N,N-diisopropylethylamine, 35 mg (0.067 mmol,

1.01 equiv) of PyBOP, 11 mg (0.065 mmol, 0.97 equiv) of 6-Cl-HOBt,

27 mg (0.066 mmol, 0.99 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyr-

osine, 3 + 1.5 mL of DMF. Step 2 of F: 2 mL of TFA and 2 mL of DCM.

Compound H (7.0 mg, yield = 17%) was isolated as a white solid.

1

(MS)EI: 514.3 (M + H). Retention time: 26.36 min. H NMR (500

MHz, deuterium oxide) δ 7.63 (q, J = 7.3 Hz, 1H), 7.38 (dq, J = 8.4, 3.1,

2.7 Hz, 1H), 7.28−7.13 (m, 4H), 7.05 (t, J = 8.4 Hz, 1H), 6.89 (dd, J =

8.5, 2.7 Hz, 1H), 6.16 (s, 2H), 4.43−4.17 (m, 5H), 4.00 (dt, J = 13.9, 4.1

Hz, 1H), 3.87 (dt, J = 10.6, 5.2 Hz, 1H), 3.71 (dd, J = 12.3, 5.3 Hz, 1H),

3.56 (d, J = 7.1 Hz, 2H), 3.38−3.25 (m, 1H), 3.18−3.03 (m, 2H),

3.02−2.76 (m, 2H), 1.72 (s, 6H), 0.99−0.85 (m, 1H), 0.52−0.36 (m,

2H), 0.27−0.02 (m, 2H).

5-(3,4-Dihydroisoquinolin-2(1H)-yl)-5-oxopentan-1-aminium

Triﬂuoroacetate (22). See Procedures G and Ha: 85 mg (0.39 mmol)

of 5-((tert-butoxycarbonyl)amino)pentanoic acid, 204 mg (0.39 mmol,

1.0 equiv) of PyBOP, 50 μL (53 mg, 0.39 mmol, 1.0 equiv) of 1,2,3,4-

tetrahydroisoquinoline, 430 μL (396 mg, 3.9 mmol, 10 equiv) of NMM,

and 4 mL of DMF. The intermediate was puriﬁed by column

chromatography (0−50% EtOAc in hexanes) and used without further

puriﬁcation. Procedure Ha: 5 mL of dioxane and 0.5 mL of conc. HCl

were added, and the solution was concentrated in vacuo after 5 min.

Compound 22 (99 mg, yield = 73%) was isolated as a colorless oil. ¹H

NMR (500 MHz, methanol-d₄) δ 7.23−7.09 (m, 4H), 4.69 (s, 1H),

4.68 (s, 1H), 3.78 (t, J = 6.0 Hz, 1H), 3.74 (t, J = 6.0 Hz, 1H), 2.93 (p, J

= 6.4 Hz, 3H), 2.84 (t, J = 6.0 Hz, 1H), 2.55 (q, J = 6.6 Hz, 2H), 1.76−

1.67 (m, 4H). ¹³C NMR (126 MHz, methanol-d₄) δ 172.33, 172.29,

160.32, 160.02, 134.68, 134.26, 132.96, 132.60, 128.24, 127.98, 126.60,

126.38, 126.18, 126.08, 126.02, 125.81, 117.21, 114.90, 43.94, 43.15,

39.96, 38.96, 32.12, 31.87, 28.82, 27.93, 26.74, 26.70, 21.30.

(S)-1-((3-((3,4-Dihydroisoquinolin-2(1H)-yl)methyl)benzyl)-

amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-ami-

nium Triﬂuoroacetate (E). See Procedures E and F: 11 mg (0.029

mmol) of 18, 100 μL (0.20 mmol, 6.92 equiv) of 2 M BH₃*Me₂S in

THF, and 4 mL of THF. Step 1 of F: 50 μL (37 mg, 0.29 mmol, 9.93

equiv) of N,N-diisopropylethylamine, 17 mg (0.033 mmol, 1.13 equiv)

of PyBOP, 5 mg (0.029 mmol, 1.02 equiv) of 6-Cl-HOBt, 12 mg (0.029

mmol, 1.01 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL

of DMF. Step 2 of F: 2 mL of TFA and 2 mL of DCM. Compound E

(5.0 mg, yield = 31%) was isolated as a white solid. (MS)EI: 444.3 (M +

H). Retention time: 20.66 min. ¹H NMR (500 MHz, methanol-d₄) δ

8.31 (t, J = 5.7 Hz, 1H), 7.44 (d, J = 6.3 Hz, 2H), 7.37−7.25 (m, 3H),

7.20 (s, 1H), 7.16 (d, J = 7.4 Hz, 1H), 7.06 (d, J = 6.6 Hz, 1H), 6.45 (s,

2H), 4.50−4.34 (m, 5H), 4.17 (dd, J = 15.1, 4.2 Hz, 1H), 3.89 (dd, J =

M

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

2-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethoxy)ethan-1-

aminium Triﬂuoroacetate (23). See Procedures G and Ha: 94 mg

(0.43 mmol) of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid,

223 mg (0.43 mmol, 1.0 equiv) of PyBOP, 60 μL (63 mg, 0.47 mmol,

1.1 equiv) of 1,2,3,4-tetrahydroisoquinoline, 470 μL (432 mg, 4.3

mmol, 10 equiv) of NMM, and 4 mL of DMF. The intermediate was

puriﬁed by column chromatography (0−5% MeOH in DCM) and used

without further puriﬁcation. Procedure Ha: 5 mL of dioxane and 0.5 of

mL conc. HCl were added, and the solution was concentrated in vacuo

after 5 min. Compound 23 (121 mg, yield = 81%) was isolated as a

colorless oil. ¹H NMR (400 MHz, methanol-d₄) δ 7.21−7.12 (m, 4H),

4.69 (s, 1H), 4.55 (s, 1H), 4.43 (s, 1H), 4.42 (s, 1H), 3.82−3.74 (m,

3H), 3.59 (t, J = 6.0 Hz, 1H), 3.15 (q, J = 4.5 Hz, 2H), 2.91 (t, J = 6.0

Hz, 1H), 2.85 (t, J = 6.0 Hz, 1H). ¹³C NMR (101 MHz, methanol-d₄) δ

169.39, 169.35, 161.16, 160.80, 160.44, 160.08, 134.54, 134.14, 132.52,

131.97, 128.26, 127.98, 126.70, 126.50, 126.26, 126.15, 126.03, 125.87,

120.64, 117.75, 114.86, 68.20, 45.19, 43.79, 41.68, 39.89, 39.22, 28.46,

27.74.

4-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)piperidin-1-

ium Hydrochloride (24). See Procedures G and Ha: 75 mg (0.31

mmol) of 2-(1-(tert-butoxycarbonyl)piperidin-4-yl)acetic acid, 161 mg

(0.31 mmol, 1.0 equiv) of PyBOP, 40 μL (42 mg, 0.32 mmol, 1.0 equiv)

of 1,2,3,4-tetrahydroisoquinoline, 340 μL (313 mg, 3.1 mmol, 10 equiv)

of NMM, and 4 mL of DMF. The intermediate was puriﬁed by column

chromatography (0−5% MeOH in DCM) and used without further

puriﬁcation. Procedure Ha: 5 mL of dioxane and 0.5 mL of conc. HCl

were, added and the solution was concentrated in vacuo after 1 min.

Compound 24 (86 mg, yield = 95%) was isolated as an oﬀ white

amorphous solid. ¹H NMR (500 MHz, methanol-d₄) δ 7.22−7.11 (m,

4H), 4.70 (s, 1H), 4.68 (s, 1H), 3.78 (t, J = 6.0 Hz, 1H), 3.75 (t, J = 5.9

Hz, 1H), 3.37 (ddd, J = 12.8, 6.3, 3.1 Hz, 2H), 3.05−2.94 (m, 2H), 2.92

(t, J = 5.9 Hz, 1H), 2.84 (t, J = 6.0 Hz, 1H), 2.49 (t, J = 7.3 Hz, 2H), 2.14

(ddtt, J = 14.4, 10.7, 6.9, 3.8 Hz, 1H), 2.05−1.95 (m, 2H), 1.55−1.41

(m, 2H). ¹³C NMR (126 MHz, methanol-d₄) δ 170.84, 170.78, 160.78,

160.49, 134.67, 134.24, 132.94, 132.62, 128.26, 127.99, 126.62, 126.38,

126.19, 126.08, 126.03, 125.80, 117.46, 115.14, 43.92, 43.73, 43.71,

43.26, 39.96, 38.72, 38.52, 30.66, 30.63, 28.87, 28.46, 28.42, 27.92.

1-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)piperazine-

1,4-diium Triﬂuoroacetate (25). See Procedures G and Ha: 78 mg

(0.32 mmol) of 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)acetic acid,

172 mg (0.33 mmol, 1.0 equiv) of PyBOP, 50 μL (53 mg, 0.39 mmol,

1.2 equiv) of 1,2,3,4-tetrahydroisoquinoline, 350 μL (322 mg, 3.2

mmol, 10 equiv) of NMM, and 4 mL of DMF. The intermediate was

puriﬁed by column chromatography (0−5% MeOH in DCM) and used

without further puriﬁcation. Procedure Ha: 5 mL of dioxane and 0.5 mL

conc. HCl were added and the solution was concentrated in vacuo after

5 min. Compound 25 (156 mg, quantitative yield) was isolated as a

white waxy solid. ¹H NMR (500 MHz, methanol-d₄) δ 7.25−7.09 (m,

4H), 4.70 (s, 1H), 4.63 (s, 1H), 4.40 (s, 2H), 3.79 (t, J = 6.1 Hz, 1H),

3.69−3.57 (m, 9H), 2.96 (t, J = 6.0 Hz, 1H), 2.87 (t, J = 6.1 Hz, 1H).

¹³C NMR (126 MHz, methanol-d₄) δ 163.34, 163.26, 161.82, 161.54,

119.71, 117.40, 115.09, 112.77, 49.10, 49.00, 45.56, 43.93, 42.14, 40.08,

32.25, 28.34, 27.67.

tert-Butyl (2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)-

(methyl)carbamate (27). See Procedure G: 99 mg (0.52 mmol) of

N-(tert-butoxycarbonyl)-N-methylglycine, 271 mg (0.52 mmol, 1.0

equiv) of PyBOP, 70 μL (74 mg, 0.55 mmol, 1.1 equiv) of 1,2,3,4-

tetrahydroisoquinoline, 580 μL (534 mg, 5.3 mmol, 10 equiv) of NMM,

and 4 mL of DMF. The product was puriﬁed by column

chromatography (0−50% EtOAc in hexanes), yielding compound 27

(152 mg, yield = 95%) as a colorless oil. ¹H NMR (500 MHz,

chloroform-d) δ 7.22−7.02 (m, 4H), 4.71 (d, J = 9.6 Hz, 1H), 4.57 (s,

1H), 4.13 (d, J = 6.2 Hz, 1H), 4.05 (s, 1H), 3.84−3.77 (m, 1H), 3.67−

3.59 (m, 1H), 2.92 (d, J = 5.4 Hz, 3H), 2.85 (dt, J = 15.7, 5.5 Hz, 2H),

1.46 (s, 6H), 1.39 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d) δ

167.55, 167.46, 156.22, 134.87, 133.97, 133.23, 132.16, 129.01, 128.85,

128.28, 126.94, 126.62, 126.57, 126.36, 126.07, 125.96, 79.95, 51.02,

50.56, 50.44, 46.29, 44.36, 42.43, 39.93, 35.46, 29.30, 28.36, 28.29.

tert-Butyl (2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)-

carbamate (28). See Procedure G: 91 mg (0.52 mmol) of (tert-

butoxycarbonyl)glycine, 270 mg (0.52 mmol, 1.0 equiv) of PyBOP, 70

μL (74 mg, 0.55 mmol, 1.1 equiv) of 1,2,3,4-tetrahydroisoquinoline,

570 μL (524 mg, 5.2 mmol, 10 equiv) of NMM, and 4 mL of DMF. The

product was puriﬁed by column chromatography (0−66% EtOAc in

hexanes), yielding compound 28 (136 mg, yield = 90%) as a yellow oil.

¹H NMR (500 MHz, chloroform-d) δ 7.22−7.03 (m, 4H), 5.59 (s, 1H),

4.71 (s, 1H), 4.52 (s, 1H), 4.02 (dd, J = 8.5, 4.4 Hz, 2H), 3.81 (t, J = 6.0

Hz, 1H), 3.58 (t, J = 5.9 Hz, 1H), 2.86 (dt, J = 22.9, 6.0 Hz, 2H), 1.43 (s,

9H). ¹³C NMR (126 MHz, chloroform-d) δ 167.29, 155.83, 134.70,

133.80, 132.83, 131.68, 128.84, 128.30, 127.12, 126.75, 126.70, 126.55,

126.53, 126.11, 79.61, 45.93, 44.38, 42.64, 42.48, 42.03, 40.06, 29.09,

28.34, 28.29.

tert-Butyl (2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)(methyl)-

carbamate (29). See Procedure E: 48 mg (0.16 mmol) of 27, 550

μL (1.1 mmol, 7.0 equiv) of 2 M BH₃*Me₂S in THF, and 4 mL THF.

The concentrated product was puriﬁed by column chromatography

(0−33% EtOAc in hexanes), yielding compound 29 (30 mg, yield =

66%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d) δ 7.26−7.13

(m, 3H), 7.02 (d, J = 7.1 Hz, 1H), 4.23 (d, J = 15.6 Hz, 1H), 3.96−3.58

(m, 3H), 3.35−2.78 (m, 9H), 1.40 (s, 4H), 1.34 (s, 5H). ¹³C NMR

(126 MHz, chloroform-d) δ 129.75, 128.59, 127.60, 127.36, 127.00,

126.78, 80.00, 79.82, 62.10, 61.03, 54.96, 54.79, 54.10, 53.87, 44.29,

43.94, 35.01, 34.70, 28.32, 24.04.

2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethan-1-aminium Tri-

ﬂuoroacetate (30). Procedure Ha: 54 mg (0.19 mmol) of 28, 5 mL

of dioxane and 0.5 mL of conc. HCl were added, and the solution was

concentrated in vacuo after 5 min. Compound 30 (32 mg, yield = 57%)

was isolated as a pinkish white solid. ¹H NMR (500 MHz, methanol-d₄)

δ 7.24−7.10 (m, 4H), 4.69 (s, 1H), 4.60 (s, 1H), 4.04 (s, 1H), 4.03 (s,

1H), 3.78 (t, J = 6.1 Hz, 1H), 3.64 (t, J = 6.0 Hz, 1H), 2.94 (t, J = 6.0 Hz,

1H), 2.86 (t, J = 6.1 Hz, 1H). ¹³C NMR (126 MHz, methanol-d₄) δ

164.95, 164.90, 161.74, 161.46, 161.18, 160.89, 134.48, 134.04, 132.13,

131.57, 128.28, 128.07, 126.91, 126.69, 126.40, 126.32, 126.08, 125.98,

119.96, 117.64, 115.32, 113.00, 45.64, 44.15, 42.23, 40.39, 28.33, 27.66.

2-((2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)(methyl)-

amino)-2-oxoethan-1-aminium Triﬂuoroacetate (31). See Proce-

dures G and Ha: 47 mg (0.15 mmol) of 26, 80 mg (0.15 mmol, 1.0

equiv) of PyBOP, 29 mg (0.17 mmol, 1.1 equiv) of N-(tert-

butoxycarbonyl)glycine, 170 μL (156 mg, 1.5 mmol, 10 equiv) of

NMM, and 4 mL of DMF. The intermediate was puriﬁed by column

chromatography (0−5% MeOH in DCM). Procedure Ha: 5 mL of

dioxane and 0.5 mL conc. of HCl were added, and the solution was

concentrated in vacuo after 5 min. Compound 31 (24 mg, yield = 43%)

161.25, 134.44, 134.05, 132.07, 131.59, 128.26, 128.05, 126.90, 126.70,

126.39, 126.31, 126.07, 125.99, 117.61, 115.29, 57.02, 56.93, 49.41,

49.38, 45.84, 44.05, 42.45, 40.69, 40.31, 28.35, 27.64.

2-(3,4-Dihydroisoquinolin-2(1H)-yl)-N-methyl-2-oxoethan-1-

aminium Triﬂuoroacetate (26). See Procedures G and Ha: 104 mg

(0.55 mmol) of N-(tert-butoxycarbonyl)-N-methylglycine, 288 mg

(0.55 mmol, 1.0 equiv) of PyBOP, 70 μL (74 mg, 0.55 mmol, 1.0 equiv)

of 1,2,3,4-tetrahydroisoquinoline, 600 μL (552 mg, 5.5 mmol, 10 equiv)

of NMM, and 4 mL of DMF. The intermediate was puriﬁed by column

chromatography (0−5% MeOH in DCM) and used without further

puriﬁcation. Procedure Ha: 6 mL of dioxane and 0.6 mL of conc. HCl

were added, and the solution was concentrated in vacuo after 5 min.

Compound 26 (132 mg, yield = 75%) was isolated as a colorless oil that

1

was isolated as a colorless oil. H NMR (500 MHz, methanol-d₄) δ

1

7.23−7.12 (m, 4H), 4.71−4.66 (m, 2H), 4.45−4.40 (m, 2H), 4.00 (s,

2H), 3.79 (q, J = 6.0, 5.6 Hz, 1H), 3.71 (td, J = 6.0, 1.8 Hz, 1H), 3.11−

3.02 (m, 3H), 2.98−2.96 (m, 1H), 2.90−2.81 (m, 1H). ¹³C NMR (126

MHz, Methanol-d₄) δ 167.14, 166.71, 160.08, 159.78, 134.59, 134.22,

133.52, 132.63, 128.81, 128.23, 127.97, 127.93, 127.26, 126.68, 126.58,

126.50, 126.30, 126.24, 126.15, 126.00, 125.89, 117.07, 114.76, 55.11,

turned purple overtime. H NMR (500 MHz, methanol-d₄) δ 7.23−

7.10 (m, 4H), 4.69 (s, 1H), 4.60 (s, 1H), 4.16 (s, 1H), 4.15 (s, 1H), 3.79

(t, J = 6.1 Hz, 1H), 3.63 (t, J = 6.0 Hz, 1H), 2.94 (t, J = 6.0 Hz, 1H), 2.85

(t, J = 6.0 Hz, 1H), 2.78 (s, 3H). ¹³C NMR (126 MHz, methanol-d₄) δ

164.05, 164.01, 161.03, 160.74, 160.46, 160.16, 134.42, 134.00, 132.18,

131.60, 128.27, 128.05, 126.81, 126.61, 126.33, 126.24, 126.02, 125.92,

N

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

49.47, 49.32, 45.76, 44.24, 44.08, 42.29, 42.23, 41.87, 40.16, 39.64,

39.47, 34.68, 34.63, 34.27, 28.49, 27.82, 27.25.

isolated as a white solid. ¹H NMR (500 MHz, methanol-d₄) δ 8.14 (dt, J

= 22.7, 5.2 Hz, 1H), 7.23−7.11 (m, 4H), 6.46 (s, 1H), 6.43 (s, 1H), 4.67

(s, 1H), 4.55 (d, J = 2.7 Hz, 1H), 4.16 (qd, J = 15.0, 6.9 Hz, 2H), 3.85

(dd, J = 11.5, 4.9 Hz, 1H), 3.83−3.70 (m, 1H), 3.63−3.54 (m, 1H),

3.54−3.48 (m, 1H), 3.37−3.31 (m, 1H), 3.28−3.15 (m, 3H), 3.04−

2.83 (m, 3H), 2.25 (s, 2H), 2.24 (s, 4H). (MS)EI: 427.3 (M + H).

Retention time: 26.81 min.

(S)-1-(4-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)piperidin-1-

yl)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-aminium Tri-

ﬂuoroacetate (L). See Procedures E and F: 25 mg (0.067 mmol) of 24,

250 μL (0.50 mmol, 7.4 equiv) of 2 M BH₃*Me₂S in THF, and 4 mL of

THF. Step 1 of Procedure F: 120 μL (89 mg, 0.69 mmol, 10 equiv) of

N,N-diisopropylethylamine, 35 mg (0.067 mmol, 1.0 equiv) of PyBOP,

13 mg (0.077 mmol, 1.1 equiv) of 6-Cl-HOBt, 29 mg (0.071 mmol, 1.1

equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF.

Step 2 of Procedure F: 2 mL of TFA and 2 mL of DCM. Compound L

(2.8 mg, yield = 8%) was isolated as a white solid. ¹H NMR (500 MHz,

methanol-d₄) δ 7.37−7.15 (m, 4H), 6.56 (s, 1H), 6.49 (s, 1H), 4.66−

4.44 (m, 3H), 4.38−4.20 (m, 1H), 3.79 (s, 1H), 3.28−3.10 (m, 5H),

3.10−3.00 (m, 1H), 2.87 (td, J = 12.9, 2.6 Hz, 1H), 2.50 (dtd, J = 44.8,

13.0, 2.4 Hz, 1H), 2.29 (s, 4H), 2.23 (s, 2H), 1.73 (dd, J = 33.7, 13.1 Hz,

2H), 1.67−1.38 (m, 3H), 1.21 (d, J = 13.2 Hz, 1H), 1.17−1.00 (m,

1H), 0.84 (qd, J = 13.0, 4.7 Hz, 1H), −0.30 − −0.63 (m, 1H). (MS)EI:

436.3 (M + H). Retention time: 20.44 min.

2-(3,4-Dihydroisoquinolin-2(1H)-yl)-N-methylethan-1-aminium

Triﬂuoroacetate (32). See Procedure Ha: 30 mg (0.16 mmol) of 29, 5

mL of dioxane, and 0.5 mL of conc. HCl were added, and the solution

was concentrated in vacuo after 5 min. The residue was then partitioned

between EtOAc and sat. Na₂CO₃and then extracted with EtOAc. The

organic layers were then combined, dried with MgSO₄, ﬁltered, and

concentrated in vacuo. The residue was suspended in solvent A,

concentrated in vacuo, and triturated with hexanes. Compound 32 (28

mg, yield = 89%) was isolated as a colorless oil. ¹H NMR (500 MHz,

methanol-d₄) δ 7.33−7.25 (m, 3H), 7.22−7.18 (m, 1H), 4.53 (s, 2H),

3.71−3.63 (m, 4H), 3.63−3.53 (m, 2H), 3.24 (t, J = 6.4 Hz, 2H), 2.82

(s, 3H). ¹³C NMR (126 MHz, methanol-d₄) δ 130.47, 128.46, 128.18,

128.15, 127.09, 126.93, 126.88, 126.43, 126.38, 53.31, 53.24, 52.14,

50.94, 50.47, 50.22, 49.57, 42.85, 42.24, 32.58, 27.17, 24.88.

2-((2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)amino)-2-oxoe-

than-1-aminium Triﬂuoroacetate (33). See Procedures E, G, and Hb:

24 mg (0.079 mmol) of 30, 280 μL (0.56 mmol, 7.1 equiv) of 2 M

BH₃*Me₂S in THF, and 4 mL THF. Procedure G: 43 mg (0.083 mmol,

1.0 equiv) of PyBOP, 14 mg (0.080 mmol, 1.0 equiv) of N-(tert-

butoxycarbonyl)glycine, 90 μL (83 mg, 0.82 mmol, 10 equiv) of NMM,

and 4 mL of DMF. Procedure Hb: 2 mL of TFA and 2 mL of DCM were

added, and the solution was concentrated in vacuo after 1 min. The

residue was then puriﬁed by reversed-phase chromatography.

Compound 33 (16 mg, yield = 58%) was isolated as a colorless oil.

¹H NMR (500 MHz, methanol-d₄) δ 7.35−7.23 (m, 3H), 7.20 (d, J =

(S)-1-(4-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)piperazin-1-

yl)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-aminium Tri-

ﬂuoroacetate (M). See Procedures E and F: 70 mg (0.14 mmol) of 25,

720 μL (1.4 mmol, 10 equiv) of 2 M BH₃*Me₂S in THF, and 5 mL of

THF. Step 1 of Procedure F: 250 μL (186 mg, 1.4 mmol, 10 equiv) of

N,N-diisopropylethylamine, 76 mg (0.15 mmol, 1.0 equiv) of PyBOP,

25 mg (0.15 mmol, 1.0 equiv) of 6-Cl-HOBt, 59 mg (0.14 mmol, 1.0

equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 2.5 mL of DMF.

Step 2 of Procedure F: 2 mL of TFA and 2 mL of DCM. Compound M

7.2 Hz, 1H), 4.55 (br s, 2H), 3.99−3.68 (m, 5H), 3.65−3.40 (m, 3H),

3.22 (s, 2H). ¹³C NMR (126 MHz, methanol-d₄) δ 167.41, 160.53,

160.24, 159.94, 130.59, 128.43, 128.09, 127.21, 126.85, 126.41, 117.24,

114.93, 55.08, 53.22, 49.88, 40.13, 33.96, 24.78.

(S)-1-((5-(3,4-Dihydroisoquinolin-2(1H)-yl)pentyl)amino)-3-(4-

hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-aminium Triﬂuoroa-

cetate (I). See Procedures E and F: 30 mg (0.087 mmol) of 22, 300 μL

(0.60 mmol, 6.9 equiv) of 2 M BH₃*Me₂S in THF, and 4 mL of THF.

Step 1 of Procedure F: 150 μL (111 mg, 0.86 mmol, 10 equiv) of N,N-

diisopropylethylamine, 45 mg (0.086 mmol, 1.0 equiv) of PyBOP, 16

mg (0.094 mmol, 1.1 equiv) of 6-Cl-HOBt, 38 mg (0.093 mmol, 1.1

equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF.

Step 2 of Procedure F: 2 mL of TFA and 2 mL of DCM. Compound I

1

(2.9 mg, yield = 3.7%) was isolated as a white solid. H NMR (500

MHz, methanol-d₄) δ 7.35−7.23 (m, 3H), 7.22−7.12 (m, 1H), 6.52 (s,

2H), 4.53 (dd, J = 12.1, 4.4 Hz, 1H), 4.46 (s, 2H), 3.66 (d, J = 13.4 Hz,

1H), 3.60 (t, J = 6.4 Hz, 2H), 3.54 (t, J = 10.5 Hz, 1H), 3.39 (t, J = 6.1

Hz, 2H), 3.24−3.17 (m, 3H), 3.16−3.10 (m, 1H), 3.07 (dd, J = 13.8,

4.4 Hz, 1H), 2.80 (q, J = 6.4 Hz, 2H), 2.69−2.57 (m, 2H), 2.43−2.33

(m, 2H), 2.26 (s, 6H), 1.64−1.52 (m, 1H). (MS)EI: 437.3 (M+H).

Retention time: 17.60 min.

1

(13.1 mg, Yield = 29%) was isolated as a white solid. H NMR (500

(S)-1-((2-((2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)(methyl)-

amino)ethyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopro-

pan-2-aminium Triﬂuoroacetate (N). See Procedures E and F: 24 mg

(0.064 mmol) of 31, 300 μL (0.60 mmol, 9.4 equiv) of 2 M BH₃*Me₂S

in THF, and 4 mL of THF. Step 1 of Procedure F: 110 μL (82 mg, 0.63

mmol, 9.9 equiv) of N,N-diisopropylethylamine, 35 mg (0.67 mmol, 1.1

equiv) of PyBOP, 13 mg (0.077 mmol, 1.2 equiv) of 6-Cl-HOBt, 28 mg

(0.068 mmol, 1.1 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 +

1.5 mL of DMF. Step 2 of Procedure F: 2 mL of TFA and 2 mL of DCM.

Compound N (4.9 mg, yield = 14%) was isolated as a white solid. ¹H

NMR (500 MHz, methanol-d₄) δ 7.34−7.25 (m, 3H), 7.20 (d, J = 7.4

Hz, 1H), 6.51 (s, 2H), 4.49 (s, 2H), 3.84 (dd, J = 11.3, 5.1 Hz, 1H),

3.73−3.58 (m, 6H), 3.57−3.47 (m, 1H), 3.29−3.18 (m, 4H), 3.14 (dt, J

= 11.9, 5.7 Hz, 1H), 3.01 (ddd, J = 20.8, 13.6, 6.0 Hz, 2H), 2.83 (s, 3H),

2.25 (s, 6H). (MS)EI: 425.3 (M+H). Retention time: 16.72 min.

(S)-1-((2-((2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)(methyl)-

amino)-2-oxoethyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-

oxopropan-2-aminium Triﬂuoroacetate (O). See Procedures G, Hb,

and F: 28 mg (0.092 mmol) of 32, 47 mg (0.0.90 mmol, 0.98 equiv) of

PyBOP, 18 mg (0.10 mmol, 1.1 equiv) of N-(tert-butoxycarbonyl)-

glycine, 100 μL (92 mg, 0.91 mmol, 9.9 equiv) of NMM, and 4 mL of

DMF. The intermediate was puriﬁed by column chromatography (0−

5% MeOH in DCM), and 25 mg of intermediate was isolated as a crude

mixture with tri(pyrrolidin-1-yl)phosphine oxide (approximately 2.5:1

phosphine oxide:intermediate by NMR). No repuriﬁcation was

performed due to poor UV absorbance. Procedure Hb: 25 mg

(approximately 0.029 mmol) of crude, 2 mL of TFA, and 2 mL of DCM

were added, and the solution was concentrated in vacuo after 5 min. No

puriﬁcation by reversed-phase chromatography was performed. Step 1

of Procedure F: 50 μL (37 mg, 0.29 mmol, 1.0 equiv) of N,N-

MHz, methanol-d₄) δ 7.80 (t, J = 5.7 Hz, 1H), 7.36−7.24 (m, 3H), 7.22

(d, J = 7.4 Hz, 1H), 6.51 (s, 2H), 4.60 (dd, J = 14.5, 7.7 Hz, 1H), 4.32 (t,

J = 14.4 Hz, 1H), 3.90−3.75 (m, 2H), 3.40 (dd, J = 25.6, 15.6 Hz, 1H),

3.31−3.10 (m, 9H), 3.00 (dd, J = 13.8, 4.8 Hz, 1H), 2.97−2.87 (m,

1H), 2.27 (s, 6H), 1.81−1.65 (m, 2H), 1.35 (dp, J = 9.1, 6.5 Hz, 2H),

1.05 (dtt, J = 21.2, 13.2, 6.7 Hz, 2H). (MS)EI: 410.3 (M + H).

Retention time: 19.34 min.

(S)-1-((2-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethoxy)ethyl)-

amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-ami-

nium Triﬂuoroacetate (J). See Procedures E and F: 41 mg (0.12 mmol)

of 23, 420 μL (0.84 mmol, 7.1 equiv) of 2 M BH₃*Me₂S in THF, and 4

mL of THF. Step 1 of Procedure F: 210 μL (156 mg, 1.2 mmol, 10

equiv) of N,N-diisopropylethylamine, 63 mg (0.12 mmol, 1.0 equiv) of

PyBOP, 21 mg (0.12 mmol, 1.0 equiv) of 6-Cl-HOBt, 50 mg (0.12

mmol, 1.0 equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of

DMF. Step 2 of Procedure F: 2 mL of TFA and 2 mL of DCM.

Compound J (11.1 mg, yield = 18%) was isolated as a white solid. ¹H

NMR (500 MHz, methanol-d₄) δ 7.34−7.25 (m, 3H), 7.21 (d, J = 7.5

Hz, 1H), 6.50 (s, 2H), 4.56 (s, 1H), 4.37 (s, 1H), 3.89 (dd, J = 11.4, 4.9

Hz, 1H), 3.83−3.72 (m, 2H), 3.69 (ddd, J = 11.7, 7.7, 4.2 Hz, 1H),

3.50−3.34 (m, 5H), 3.29−3.16 (m, 4H), 3.01 (dd, J = 13.8, 4.9 Hz,

1H), 2.26 (s, 6H). (MS)EI: 413.3 (M + H). Retention time: 17.78 min.

(S)-1-((2-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-oxoethoxy)-

ethyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-2-

aminium Triﬂuoroacetate (K). See Procedure F: Step 1: 36 mg (0.10

mmol) of 23, 180 μL (134 mg, 1.0 mmol, 10 equiv) of N,N-

diisopropylethylamine, 56 mg (0.11 mmol, 1.0 equiv) of PyBOP, 19 mg

(0.11 mmol, 1.1 equiv) of 6-Cl-HOBt, 44 mg (0.11 mmol, 1.0 equiv) of

Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF. Step 2: 2 mL

of TFA and 2 mL of DCM. Compound K (15.1 mg, yield = 27%) was

O

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

diisopropylethylamine, 16 mg (0.031 mmol, 1.1 equiv) of PyBOP, 7 mg

(0.041 mmol, 1.4 equiv) of 6-Cl-HOBt, 13 mg (0.32 mmol, 1.1 equiv)

of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF. Step 2 of

Procedure F: 2 mL of TFA and 2 mL of DCM. Compound O (10.1 mg,

yield = 20% over four steps) was isolated as a white solid. ¹H NMR (500

MHz, methanol-d₄) δ 7.35−7.22 (m, 3H), 7.19 (d, J = 7.4 Hz, 1H), 6.48

(s, 2H), 4.67 (s, 1H), 4.42 (s, 1H), 4.16−3.74 (m, 6H), 3.60−3.41 (m,

3H), 3.25−3.14 (m, 3H), 3.05 (s, 3H), 3.01 (dd, J = 14.1, 5.3 Hz, 1H),

2.24 (s, 6H). (MS)EI: 439.3 (M + H). Retention time: 16.60 min.

(S)-1-((2-((2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl)amino)-2-

oxoethyl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-1-oxopropan-

2-aminium Triﬂuoroacetate (P). See Step 1 of Procedure F: 15 mg

(0.043 mmol) of 33, 80 μL (59 mg, 0.46 mmol, 11 equiv) of N,N-

diisopropylethylamine, 23 mg (0.044 mmol, 1.0 equiv) of PyBOP, 7 mg

(0.041 mmol, 0.96 equiv) of 6-Cl-HOBt, 19 mg (0.046 mmol, 1.1

equiv) of Boc-O-Boc-L-2′,6′-dimethyltyrosine, 3 + 1.5 mL of DMF.

Step 2 of Procedure F: 2 mL TFA and 2 mL DCM. Compound P (7.3

membranes (10 μg of protein/well) were incubated for 1 h at 25 °C in

GTPγS buﬀer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl₂, 1 mM

EDTA, pH 7.4) containing 0.1 nM [³⁵S]-GTPγS, 30 μM guanosine

diphosphate (GDP), and varying concentrations of test peptidomi-

metic. G protein activation following receptor activation by

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 through GF/C ﬁlters that

were washed ﬁve times with GTPγS buﬀer. Bound radioactivity was

measured as described above. The results are presented as the mean

standard error (SEM) from at least three separate assays performed in

duplicate; potency (EC₅₀(nM)) and percent stimulation were

determined using nonlinear regression analysis with GraphPad Prism,

as above.

ASSOCIATED CONTENT

1

■

mg, yield = 31%) was isolated as a white solid. H NMR (400 MHz,

sı

methanol-d₄) δ 7.38−7.24 (m, 3H), 7.21 (d, J = 6.6 Hz, 1H), 6.49 (s,

2H), 4.62 (s, 1H), 4.41 (s, 1H), 4.03−3.83 (m, 3H), 3.69 (s, 2H), 3.56

(d, J = 16.8 Hz, 1H), 3.41 (t, J = 5.8 Hz, 3H), 3.27−3.14 (m, 3H), 3.02

(dd, J = 14.0, 5.2 Hz, 1H), 2.24 (s, 6H). (MS)EI: 425.3 (M + H).

Retention time: 16.30 min.

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschemneuro.0c00693.

Molecular formula strings (XLSX)

In Vitro Pharmacology. Cell Lines and Membrane Preparations.

All tissue culture reagents were purchased from Gibco Life Sciences

(Grand Island, NY) unless otherwise noted. Chinese hamster ovary

(CHO) cells stably expressing human MOR (CHO-MOR), DOR

(CHO-DOR), or KOR (CHO-KOR) (a generous gift from Larry Toll)

were used for all in vitro assays. Cells were grown to conﬂuence at 37 °C

in 5% CO₂in Dulbecco’s modiﬁed Eagle’s medium/nutrient mixture F-

12 (DMEM-F12) containing 10% fetal bovine serum and 5%

penicillin/streptomycin. Membranes were prepared by washing

conﬂuent cells three times 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, Inc., Bartlesville, OK) 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) using bovine serum albumin as the

standard.

AUTHOR INFORMATION

■

Corresponding Author

Henry I. Mosberg − Department of Medicinal Chemistry,

College of Pharmacy and Edward F Domino Research Center,

University of Michigan, Ann Arbor, Michigan 48109, United

States; orcid.org/0000-0002-4107-8870; Phone: 734-

764-8117; Email: him@umich.edu

Authors

Sean Henry − Department of Medicinal Chemistry, College of

Pharmacy, University of Michigan, Ann Arbor, Michigan

48109, United States

Jessica P. Anand − Department of Pharmacology, Medical

School and Edward F Domino Research Center, University of

Michigan, Ann Arbor, Michigan 48109, United States

Ashley C. Brinkel − Department of Pharmacology, Medical

School, University of Michigan, Ann Arbor, Michigan 48109,

United States

Douglas M. McMillan − Department of Pharmacology, Medical

School, University of Michigan, Ann Arbor, Michigan 48109,

United States

Jack. J. Twarozynski − Department of Pharmacology, Medical

School, University of Michigan, Ann Arbor, Michigan 48109,

United States

Christian E. Loo − Department of Medicinal Chemistry, College

of Pharmacy, University of Michigan, Ann Arbor, Michigan

48109, United States

John R. Traynor − Department of Medicinal Chemistry, College

of Pharmacy, Department of Pharmacology, Medical School,

and Edward F Domino Research Center, University of

Michigan, Ann Arbor, Michigan 48109, United States

Radioligand Competition Binding Assays. Radiolabeled com-

pounds were purchased from PerkinElmer (Waltham, MA). Opioid

ligand binding assays were performed by competitive displacement of

0.2 nM [³H]-diprenorphine (250 μCi, 1.85 TBq/mmol) by the

peptidomimetic 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), 0.2 nM [³H]-diprenorphine and various concentrations of test

peptidomimetic, was incubated at room temperature on a shaker 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) and washed three times with 50 mM Tris-HCl

buﬀer, pH 7.4. Bound radioactivity on dried ﬁlters was determined by

liquid scintillation counting, after saturation with EcoLume liquid

scintillation cocktail, in a MicroBeta 2450 (PerkinElmer, Waltham,

MA). Nonspeciﬁc binding was determined using 10 μM naloxone. The

results presented are the mean standard error (SEM) 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, ver. 8.4 (GraphPad

Software Inc., La Jolla, CA).

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschemneuro.0c00693

Author Contributions

Synthesis was carried out by S.P.H. and C.E.L. The research

project was designed by S.P.H. and H.I.M. In vitro assays were

carried out by J.P.A., J.J.T., D.M.M., and A.C.B., and supervised

by J.R.T. This manuscript was written through contributions by

all authors.

[³⁵S]-GTPγS Binding Assays. Agonist stimulation of [³⁵S]guanosine

5′-O-[γ-thio]triphosphate ([³⁵S]-GTPγS, 1250 Ci, 46.2 TBq/mmol)

binding to G protein was measured as described previously.³⁸Brieﬂy,

P

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

(10) Anand, J. P., and Montgomery, D. (2019) Multifunctional

Opioid Ligands. In Delta Opioid Receptor Pharmacology and Therapeutic

Applications, pp 21−51, Springer International Publishing, Cham,

Switzerland.

(11) Fundytus, M. E., Schiller, P. W., Shapiro, M., Weltrowska, G., and

Coderre, T. J. (1995) Attenuation of Morphine Tolerance and

Dependence with the Highly Selective δ -Opioid Receptor Antagonist

TIPP[Ψ ]. Eur. J. Pharmacol. 286, 105−108.

(12) Abdelhamid, E. E., Sultana, M., Portoghese, P. S., and Takemori,

A. E. (1991) Selective Blockage of Delta Opioid Receptors Prevents the

Development of Morphine Tolerance and Dependence in Mice. J.

Pharmacol. Exp. Ther. 258 (1), 299−303.

(13) Hepburn, M. J., Little, P. J., Gingras, J., and Kuhn, C. M. (1997)

Differential Effects of Naltrindole on Morphine-Induced Tolerance and

Physical Dependence in Rats. J. Pharmacol. Exp. Ther. 281 (3), 1350−

1356.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This study was supported by NIH Grant DA003910 (H.I.M, and

J.R.T), DA048129 (J.P.A.) and by the Edward F. Domino

Center. S.P.H. was supported by the Substance Abuse

Interdisciplinary Training Program administered by NIDA

(T32 DA007281). J.P.A. was supported by grants G020292

(Domino Center). C.E.L. was sponsored by the NSF

Interdisciplinary REU Program − University of Michigan −

College of Pharmacy, with support from the California State

University − Long Beach NIH MARC USTAR Program Grant

T34GM008074.

(14) Zhu, Y., King, M. A., Schuller, A. G. P., Nitsche, J. F., Reidl, M.,

Elde, R. P., Unterwald, E., Pasternak, G. W., and Pintar, J. E. (1999)

Retention of Supraspinal Delta-like Analgesia and Loss of Morphine

Tolerance in δ Opioid Receptor Knockout Mice. Neuron 24 (1), 243−

252.

(15) Vekariya, R. H., Lei, W., Ray, A., Saini, S. K., Zhang, S., Molnar,

G., Barlow, D., Karlage, K. L., Bilsky, E. J., Houseknecht, K. L., Largent-

Milnes, T. M., Streicher, J. M., and Ananthan, S. (2020) Synthesis and

Structure −Activity Relationships of 5 ′ -Aryl-14- Alkoxypyridomor-

phinans: Identification of a μ Opioid Receptor Agonist/δ Opioid

Receptor Antagonist Ligand with Systemic Antinociceptive Activity

and Diminished Opioid Side Effects. J. Med. Chem. 63, 7663−7694.

(16) Kest, B., Lee, C. E., McLemore, G. L., and Inturrisi, C. E. (1996)

An Antisense Oligodeoxynucleotide to the Delta Opioid Receptor

(DOR-1) Inhibits Morphine Tolerance and Acute Dependence in

Mice. Brain Res. Bull. 39 (3), 185−188.

(17) Lee, P. H. K., McNutt, R. W., and Chang, K.-J. (1993) A

Nonpeptidic Delta Opiold Receptor Agonist, BW373U86, Attenuates

the Development and Expression of Morphine Abstinence Precipitated

by Naloxone in Rat. J. Pharmacol. Exp. Ther. 267 (2), 883−887.

(18) Su, Y., McNutt, R. W., and Chang, K. (1998) Delta-Opioid

Ligands Reverse Alfentanil-Induced Respiratory Depression but Not

Antinociception. J. Pharmacol. Exp. Ther. 287 (3), 815−823.

(19) Yamazaki, M., Suzuki, T., Narita, M., and Lipkowski, A. W.

(2001) The Opioid Peptide Analogue Biphalin Induces Less Physical

Dependence than Morphine. Life Sci. 69, 1023−1028.

(20) O’Neill, S. J., Collins, M. A., Pettit, H. O., McNutt, R. W., and

Chang, K. (1997) Antagonistic Modulation Between the Delta Opioid

Agonist BW373U86 and the Mu Opioid Agonist Fentanyl in Mice. J.

Pharmacol. Exp. Ther. 282 (1), 271−277.

(21) Anand, J. P., Kochan, K. E., Nastase, A. F., Montgomery, D.,

Griggs, N. W., Traynor, J. R., Mosberg, H. I., and Jutkiewicz, E. M.

(2018) In Vivo Effects of μ Opioid Receptor Agonist/δ Opioid

Receptor Antagonist Peptidomimetics Following Acute and Repeated

Administration. Br. J. Pharmacol. 175, 2013−2027.

(22) Bender, A. M., Griggs, N. W., Gao, C., Trask, T. J., Traynor, J. R.,

and Mosberg, H. I. (2015) Rapid Synthesis of Boc-2 ′,6 ′-Dimethyl-L-

Tyrosine and Derivatives and Incorporation into Opioid Peptidomi-

metics. ACS Med. Chem. Lett. 6, 1199−1203.

(23) Bender, A. M., Griggs, N. W., Anand, J. P., Traynor, J. R.,

Jutkiewicz, E. M., and Mosberg, H. I. (2015) Asymmetric Synthesis and

in Vitro and in Vivo Activity of Tetrahydroquinolines Featuring a

Diverse Set of Polar Substitutions at the 6 Position as Mixed-E Fficacy μ

Opioid Receptor/ δ Opioid Receptor Ligands. ACS Chem. Neurosci. 6,

1428−1435.

ABBREVIATIONS USED

■

BCA, bicinchoninic acid; BH₃*Me₂S, borane-dimethyl sulﬁde

complex; CHO, Chinese hamster ovary; 6-Cl-HOBt, 6-chloro-

1-hydroxybenzotriazole; DAMGO, [^D-Ala²-N-Me-Phe⁴-Gly-

ol]-enkephalin; DI, deionized; DiBocDMT, N-Boc-O-Boc-

2′,6′-dimethyl-^L-tyrosine; DIEA, N,N-diisopropylethylamine;

DMEM, Dulbecco’s modiﬁed Eagle’s medium; DMT, 2′,6′-

dimethyl-^L-tyrosine; DNS, does not stimulate; DOR, δ-opioid

receptor; DPDPE, [D-Pen²-D-Pen⁵]enkephalin; EtBr, ethyl

bromide; GTPγS, guanosine 5′-O-[γ-thio]triphosphate;

HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;

KOR, κ-opioid receptor; MLM, mouse liver microsome;

MOR, μ-opioid receptor; NLX, naloxone; NMM, N-methyl

morpholine; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophos-

phonium hexaﬂuorophosphate; SEM, standard error of the

mean; THIQ, 1,2,3,4-tetrahydroisoquinoline; THQ, 1,2,3,4-

tetrahydroquinoline

REFERENCES

■

(1) Lutz, P.-E., and Kieffer, B. L. (2013) Opioid Receptors: Distinct

Roles in Mood Disorders. Trends Neurosci. 36 (3), 195−206.

(2) Fine, P. G. (2004) The Endogenous Opioid System. In A Clinical

Guide to Opioid Analgesia, pp 9−15, Chapter 2, McGraw-Hill, New

York.

(3) Janecka, A., Fichna, J., and Janecki, T. (2004) Opioid Receptors

and Their Ligands. Curr. Top. Med. Chem. 4, 1−17.

(4) Handa, B. K., Lane, A. C., Lord, J. A.H., Morgan, B. A., Rance, M.

J., and Smith, C. F.C. (1981) Analogues of β -LPH61 −64 Posessing

Selective Agonist Activity at μ -Opiate Receptors. Eur. J. Pharmacol. 70,

531−540.

(5) Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Yamamura, H. I.,

Galligan, J. J., and Burks, T. F. (1983) Bis-Penicillamine Enkephalins

Possess Highly Improved Specificity toward δ Opioid Receptors. Proc.

Natl. Acad. Sci. U. S. A. 80, 5871−5874.

(6) Schiller, P. W., Fundytus, M. E., Merovitz, L., Weltrowska, G.,

Nguyen, T. M., Lemieux, C., Chung, N. N., and Coderre, T. J. (1999)

The Opioid Mu Agonist/Delta Antagonist DIPP-NH(2)[Psi]

Produces a Potent Analgesic Effect, No Physical Dependence, and

Less Tolerance than Morphine in Rats. J. Med. Chem. 42 (18), 3520−

3526.

(7) Dietis, N., Guerrini, R., Calo, G., Salvadori, S., Rowbotham, D. J.,

and Lambert, D. G. (2009) Simultaneous Targeting of Multiple Opioid

Receptors: A Strategy to Improve Side-Effect Profile. Br. J. Anaesth. 103

(1), 38−49.

(8) Wtorek, K., Piekielna-Ciesielska, J., Janecki, T., and Janecka, A.

(2020) The Search for Opioid Analgesics with Limited Tolerance

Liability. Peptides 130, 170331.

(24) Harland, A. A., Yeomans, L., Griggs, N. W., Anand, J. P.,

Pogozheva, I. D., Jutkiewicz, E. M., Traynor, J. R., and Mosberg, H. I.

(2015) Further Optimization and Evaluation of Bioavailable, Mixed-

Efficacy μ -Opioid Receptor (MOR) Agonists/δ -Opioid Receptor

(DOR) Antagonists: Balancing MOR and DOR Affinities. J. Med.

Chem. 58, 8952−8969.

(9) Cunningham, C. W., Elballa, W. M., and Vold, S. U. (2019)

Bifunctional Opioid Receptor Ligands as Novel Analgesics. Neuro-

pharmacology 151, 195−207.

(25) Harland, A. A., Pogozheva, I. D., Griggs, N. W., Trask, T. J.,

Traynor, J. R., and Mosberg, H. I. (2017) Placement of Hydroxy Moiety

Q

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

on Pendant of Peptidomimetic Scaffold Modulates Mu and Kappa

Opioid Receptor Efficacy. ACS Chem. Neurosci. 8, 2549−2557.

(26) Harland, A. A., Bender, A. M., Griggs, N. W., Gao, C., Anand, J.

P., Pogozheva, I. D., Traynor, J. R., Jutkiewicz, E. M., and Mosberg, H. I.

(2016) Effects of N-Substitutions on the Tetrahydroquinoline (THQ)

Core of Mixed-Efficacy μ -Opioid Receptor (MOR)/δ -Opioid

Receptor (DOR) Ligands. J. Med. Chem. 59, 4985−4998.

(27) Mosberg, H. I., Yeomans, L., Harland, A. A., Bender, A. M.,

Sobczyk-Kojiro, K., Anand, J. P., Clark, M. J., Jutkiewicz, E. M., and

Traynor, J. R. (2013) Opioid Peptidomimetics: Leads for the Design of

Bioavailable Mixed Efficacy μ Opioid Receptor (MOR) Agonist/δ

Opioid Receptor (DOR) Antagonist Ligands. J. Med. Chem. 56, 2139−

2149.

(28) Nastase, A. F., Griggs, N. W., Anand, J. P., Fernandez, T. J.,

Harland, A. A., Trask, T. J., Jutkiewicz, E. M., Traynor, J. R., and

Mosberg, H. I. (2018) Synthesis and Pharmacological Evaluation of

Novel C-8 Substituted Tetrahydroquinolines as Balanced-Affinity Mu/

Delta Opioid Ligands for the Treatment of Pain. ACS Chem. Neurosci. 9,

1840−1848.

(29) Nastase, A. F., Anand, J. P., Bender, A. M., Montgomery, D.,

Griggs, N. W., Fernandez, T. J., Jutkiewicz, E. M., Traynor, J. R., and

Mosberg, H. I. (2019) Dual Pharmacophores Explored via Structure −

Activity Relationship (SAR) Matrix: Insights into Potent, Bifunctional

Opioid Ligand Design. J. Med. Chem. 62, 4193−4203.

(30) Henry, S. P., Fernandez, T. J., Anand, J. P., Griggs, N. W.,

Traynor, J. R., and Mosberg, H. I. (2019) Structural Simplification of a

Tetrahydroquinoline-Core Peptidomimetic μ -Opioid Receptor

(MOR) Agonist/δ -Opioid Receptor (DOR) Antagonist Produces

Improved Metabolic Stability. J. Med. Chem. 62, 4142−4157.

(31) Stepan, A. F., Mascitti, V., Beaumont, K., and Kalgutkar, A. S.

(2013) Metabolism-Guided Drug Design. MedChemComm 4, 631−

652.

(32) Henry, S., Anand, J. P., Twarozynski, J. J., Brinkel, A. C.,

Pogozheva, I. D., Sears, B. F., Jutkiewicz, E. M., Traynor, J. R., and

Mosberg, H. I. (2020) Aromatic −Amine Pendants Produce Highly

Potent and Efficacious Mixed Effiacy M-Opioid Receptor (MOR)/δ -

Opioid Receptor (DOR) Peptidomimetics with Enhanced Metabolic

Stability. J. Med. Chem. 63, 1671−1683.

(33) Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A.,

Morgan, B. A., and Morris, H. R. (1975) Identification of Two Related

Pentapeptides from the Brain with Potent Opiate Agonist Activity.

Nature 258, 577−579.

(34) Goldstein, A., and Lowery, P. J. (1975) Effect of the Opiate

Antagonist Naloxone on Body Temperature in Rats. Life Sci. 17, 927−

931.

(35) Naqvi, T., Haq, W., and Mathur, K. B. (1998) Structure −Activity

Relationship Studies of Dynorphin A and Related Peptides. Peptides 19

(7), 1277−1292.

(36) Bender, A. M., Clark, M. J., Agius, M. P., Traynor, J. R., and

Mosberg, H. I. (2014) Synthesis and Evaluation of 4-Substituted

Piperidines and Piperazines as Balanced Affinity μ Opioid Receptor

(MOR) Agonist/δ Opioid Receptor (DOR) Antagonist Ligands.

Bioorg. Med. Chem. Lett. 24, 548−551.

(37) McPherson, J., Rivero, G., Baptist, M., Llorente, J., Al-Sabah, S.,

Krasel, C., Dewey, W. L., Bailey, C. P., Rosethorne, E. M., Charlton, S. J.,

Henderson, G., and Kelly, E. (2010) μ -Opioid Receptors: Correlation

of Agonist Efficacy for Signalling with Ability to Activate Internal-

ization. Mol. Pharmacol. 78 (4), 756−766.

(38) Traynor, J. R., and Nahorski, S. R. (1995) Modulation by μ-

Opioid Agonists of Guanosine-5′-O-(3-[35S]thio)triphosphate Bind-

ing to Membranes from Human Neuroblastoma SH-SY5Y Cells. Mol.

Pharmacol. 47, 848−854.

R

https://dx.doi.org/10.1021/acschemneuro.0c00693

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Article Doi

SAR Matrices Enable Discovery of Mixed Efficacy μ-Opioid Receptor Agonist Peptidomimetics with Simplified Structures through an Aromatic-Amine Pharmacophore

DOI: 10.1021/acschemneuro.0c00693

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acschemneuro.0c00693

Full text of DOI:10.1021/acschemneuro.0c00693

Products guided by the article

R&D Labs maybe for 1428847-86-2

Relevant to this article

Hot Product