Structure-Based Discovery of M-89 as a Highly Potent Inhibitor of the Menin-Mixed Lineage Leukemia (Menin-MLL) Protein-Protein Interaction

Article

pubs.acs.org/jmc

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

Structure-Based Discovery of M‑89 as a Highly Potent Inhibitor of

the Menin-Mixed Lineage Leukemia (Menin-MLL) Protein−Protein

Interaction

Angelo Aguilar,^†,‡,○Ke Zheng,^†,‡,○Tianfeng Xu,^†,‡,○Shilin Xu,^†,‡,○Liyue Huang,^†,‡,○

Ester Fernandez-Salas,^†,⊥Liu Liu,^†,‡Denzil Bernard,^†,‡Kaitlin P. Harvey,^†,‡Caroline Foster,^†,‡

Donna McEachern,^†,‡Jeanne Stuckey,^∇Krishnapriya Chinnaswamy,^∇James Delproposto,^∇

Jeﬀ W. Kampf,^#and Shaomeng Wang*^{,†,‡,§,∥}

^†Rogel Cancer Center, ^‡Departments of Internal Medicine, ^§Pharmacology, ^∥Medicinal Chemistry, ^⊥Pathology, and ^#Chemistry, and

^∇Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States

S

* Supporting Information

ABSTRACT: Inhibition of the menin-mixed lineage leukemia

(MLL) protein−protein interaction is a promising new

therapeutic strategy for the treatment of acute leukemia carrying

MLL fusion (MLL leukemia). We describe herein our structure-

based design, synthesis, and evaluation of a new class of small-

molecule inhibitors of the menin-MLL interaction (hereafter

called menin inhibitors). Our eﬀorts have resulted in the

discovery of highly potent menin inhibitors, as exempliﬁed by

compound 42 (M-89). M-89 binds to menin with a K_dvalue of 1.4 nM and eﬀectively engages cellular menin protein at low

nanomolar concentrations. M-89 inhibits cell growth in the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion

with IC₅₀values of 25 and 55 nM, respectively, and demonstrates >100-fold selectivity over the HL-60 leukemia cell line lacking

MLL fusion. The determination of a co-crystal structure of M-89 in a complex with menin provides the structural basis for their

high-aﬃnity interaction. Further optimization of M-89 may lead to a new class of therapy for the treatment of MLL leukemia.

molecules, hereafter also called menin inhibitors, could be quite

challenging. However, recent eﬀorts have led to the discovery of

potent, nonpeptide small-molecule menin inhibitors (Figure

1).¹⁸⁻²²The most promising noncovalent menin inhibitors

reported to date are 1 (MI-463), 2 (MI-503), 3 (MI-538), and 5

(MI-1481), which belong to the same chemical class of 5-

(piperidin-1-ylmethyl)-1H-indoles. These menin inhibitors

bind to menin with a K_dvalue of ∼10 nM and achieve sub-

micromolar cellular activity in MLL leukemia cell lines and good

antitumor activity in vivo against MLL leukemia tumors.²³

Importantly, small-molecule menin inhibitors can eﬀectively

block oncogenic transformation by MLL fusion proteins in a

manner independent of the MLL fusion partner,²⁴suggesting a

broad therapeutic potential of menin inhibitors for the treatment

of MLL leukemia. These studies have provided an important

preclinical proof-of-concept that small-molecule inhibitors

targeting the menin-MLL protein−protein interaction may

have a promising therapeutic potential for the treatment of

human MLL leukemia.

INTRODUCTION

■

Mixed lineage leukemia (MLL, also called MLL1 to distinguish

it from MLL2-4) protein is a histone methyltransferase and

speciﬁcally methylates histone H3 lysine 4 residue (H3K4).

MLL gene rearrangement is found in 5−10% of acute myeloid

leukemia in adults and almost 70% of acute lymphoblastic

leukemia in infants.¹⁻⁵Adult leukemia patients carrying a MLL

rearrangement, or MLL leukemia, have very poor prognosis with

current treatments.^6,7Accordingly, there is an urgent need to

develop new and eﬀective therapies for the treatment of MLL

leukemia.

The most common MLL rearrangements are balanced MLL

translocations, in which only one MLL allele is truncated and

fused with one of over 70 partners.^8,9Approximately 1400 amino

acids from the MLL N-terminus are retained in all of the MLL

fusion proteins and interact directly with the oncogenic co-

factor menin.^3,8,10−14The menin-MLL protein−protein inter-

action is essential for the expression of HOX and MEIS1 genes,

which drive leukemogenesis in MLL leukemia.^12,14,15Con-

sequently, targeting the menin-MLL protein−protein inter-

action has been proposed to be a promising, targeted therapeutic

strategy for the treatment of MLL leukemia.

Recently, we reported the discovery of M-525 as a ﬁrst-in-

class, highly potent, covalent menin inhibitor.²⁵M-525 binds to

menin covalently with an IC₅₀value of 3 nM and achieves low

nanomolar cellular potencies in inhibition of cell growth in a

Structural studies have shown that menin protein has an

approximately 5000 Å³cavity, which serves as the binding site

for MLL protein.¹⁶⁻¹⁸This very large binding site suggests that

targeting the menin-MLL interaction using nonpeptide small-

Received: January 4, 2019

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Figure 1. Previously reported reversible and irreversible small-molecule menin inhibitors.

Figure 2. (A) Superposition of the co-crystal structure of menin in complex with menin binding motif 1 (MBM1 in yellow lines, PDB ID 4GQ6) from

MLL onto the co-crystal structure of menin in complex with MIV-6 (magenta sticks) (PDB ID 4OG8). Menin protein is shown in the gray surface. (B)

Our proposed four modiﬁcation sites in MIV-6.

panel of leukemia cell lines carrying MLL fusion.²⁵M-525

represents a promising covalent menin inhibitor for further

optimization.

based on the following considerations: (1) MIV-6 has a good

binding aﬃnity to menin with a reported K_d= 85 nM; (2) MIV-6

is cell permeable and inhibits growth of the human leukemia

Despite these major progresses, the development of menin

inhibitors for the treatment of MLL leukemia is still in its early

stage. In the present study, we describe our structure-based

design and synthesis of menin inhibitors, which has yielded a

class of potent, noncovalent menin inhibitors. The most potent

compound, 42 (M-89), binds to menin with a K_dvalue of 1.4

nM, achieves low nanomolar potency in the inhibition of cell

growth in human MLL leukemia cell lines, and demonstrates

>100-fold selectivity over leukemia cells lacking the MLL fusion.

M-89 represents a class of promising noncovalent menin

inhibitors.

MV4;11 cell line with MLL-AF4 fusion with a reported IC₅₀

=

2.8 μM; (3) Its available co-crystal structure in a complex with

menin provides a solid foundation for eﬀective structure-based

optimization.

Comparison of previously published co-crystal structures of

MIV-6/menin and MLL/menin shows that MIV-6 captures the

interactions of the Phe9, Pro10, and Pro13 residues in MLL with

menin (Figure 2). Additionally, the nitrile of the benzonitrile

“tail” group in MIV-6 forms an additional hydrogen bond with

the NH of Trp341 of the menin protein.

Conformational constraint of a small-molecule inhibitor not

only can enhance the binding aﬃnity to its intended target

protein by reducing conformational entropic costs upon binding

but also can improve the binding selectivity by reducing

accessible, low-energy conformational space. Therefore, we

RESULTS AND DISCUSSION

■

We employed a previously reported menin inhibitor 4 (MIV-6,

Figure 1)²⁰as the starting point for our optimization eﬀorts

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Table 1. Optimization of MIV-6 through Cyclization of the Core, Rigidiﬁcation of the Linker, and Replacement of the Nitrile Tail

a

Group with a Substituted Sulfonyl Group

a

Values are average of at least two tests. K_i’s were determined from averaged IC₅₀values.

investigated the possibilities of locking the bound conformation

of MIV-6 to menin. Our analysis of the co-crystal structure of

MIV-6/menin suggested that the primary amino group in MIV-

6 can be cyclized with its adjacent phenyl group, which led to the

design of compound 6 (Table 1). This compound (6) was

synthesized and was found to bind to menin with a K_i= 22 nM,

which is 2−3 times more potent than MIV-6 in our binding assay

(Table 1), supporting our conformational constraint strategy.

Encouraged by the successful design of compound 6, we next

focused on the rigidiﬁcation of the ﬂexible linker (Table 1).

Since the ﬂexible linker in the MIV-6/menin complex does not

adopt an extended conformation, we investigated if other linker

lengths can adopt a favorable conformation for interaction with

the menin protein and give insight to the design of the rigid

linker. We tested compounds with a linker one atom shorter (7)

and a linker with one atom longer (8) than that in 6. Both

modiﬁcations signiﬁcantly reduced the binding aﬃnity to menin,

suggesting that a linker with four atoms has the optimal length

between the core-piperidine nitrogen and the para-position of

the benzonitrile group. To rigidify the linker, we explored several

cyclic amines, compounds 9−12, that maintain approximately

the four-atom linker length. Compounds 9 and 10, containing a

piperidine in the linker, are approximately 10- and 100-times less

potent than 6, whereas compound 11, containing a pyrrolidine

in the linker, is as potent as 6. Compound 12 containing an

azetidine is 2 times more potent than 6. Hence, through

cyclization of the core and rigidiﬁcation of the linker, we

obtained compound 12, which is 5 times more potent than MIV-

6 (Table 1).

The co-crystal structure of MIV-6/menin (Figure 2) shows

that the terminal nitrile group forms a hydrogen bond with the

indole NH group in Trp341. Additionally, there is a well-deﬁned

hydrophobic pocket formed by Trp341, Val371, Cys329,

Val367, and Ala325 adjacent to the nitrile group, which can be

utilized to further enhance the binding aﬃnity to menin. To

maintain the hydrogen bonding interaction and at the same time

to capture additional interactions with this well-deﬁned

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a

Table 2. Structure−Activity Relationship of the N-Substituents

a

Values are average of at least two tests. K_iwas calculated from an averaged IC₅₀value in the binding experiments for each compound.

hydrophobic pocket, we replaced the nitrile group in MIV-6 with

a substituted sulfonyl group (Table 1).

MV4;11 cell line (Table 1). For example, compounds 18−21

have IC₅₀values 0.9, 1.1, 0.5, and 1.3 μM, respectively, and are

3−7 times more potent than MIV-6. However, the most potent

compound 20 in this series only achieves a sub-micromolar

cellular potency, indicating the need for further optimization.

Although compound 20 is more potent than 21 in the

inhibition of cell growth in the MV4;11 cell line, 21 is more

soluble than 20. Accordingly, we used compound 21 as the

template for further optimization.

We moved the nitrogen atom in the tetrahydroisoquinoline

core one atom away from the quaternary carbon center in

compound 21 to render the functionalization of this nitrogen

atom more synthetically feasible, which yielded compound 22

(Table 2). Compound 22 binds to menin with a K_i= 1.7 nM and

is, therefore, 2−3 times more potent than 21. However, 22 is

only slightly more potent than 21 in the inhibition of cell growth

in the MV4;11 cell line. To further improve the cellular potency,

we introduced a variety of small hydrophobic groups as a

substituent on the nitrogen atom (Table 2). Substitution of the

nitrogen with a small, aliphatic group, such as methyl (23), ethyl

(24), isopropyl (25), and cyclobutyl (26), resulted in

compounds with high binding aﬃnities to menin, with K_ivalues

= 1−2 nM. Substitution of the nitrogen atom with an electron-

withdrawing group, such as benzylpyridine (29), however,

signiﬁcantly reduces the binding aﬃnity to menin. Despite the

high binding aﬃnities to menin, compounds 23−26 only

achieve IC₅₀values of ∼0.4−1 μM in the inhibition of cell

growth in the MV4;11 cell line, suggesting that a much greater

improvement in binding aﬃnity is needed to achieve a much

more potent cellular activity.

Replacing the terminal nitrile group with a simple

methylsulfonyl group resulted in 13, which has a K_ivalue of

45 nM to menin and is thus 4 times less potent than 12.

However, replacing the methyl group in 13 with an ethyl group,

which yielded 14, restored the binding aﬃnity (K_i= 10 nM).

Encouraged with the strong binding aﬃnity for 14, we next

systematically explored this site using a variety of alkyl- or aryl-

substituted sulfonyl groups and obtained the results shown in

Table 1. Replacing the methyl group in 13 with an isopropyl

group led to 15, which is 2 times more potent than 13 but 2

times less potent than 14 in binding to menin. Changing the

isopropyl group in 15 to a cyclopropyl group led to 16, which is

2−3 times more potent than 15. Encouraged by the high binding

aﬃnity of 16, we replaced the cyclopropyl group in 16 with a

cyclobutyl (17), cyclopentyl (18), or a cyclohexyl group (19).

Compounds 17−19 are all high-aﬃnity menin inhibitors with K_i

values of 11, 4.6, and 2.4 nM to menin, respectively. We next

replaced the cyclohexyl group in 19 with a phenyl or a pyridinyl

group, which yielded 20 and 21, respectively. Compounds 20

and 21 bind to menin with K_ivalues of 5.6 and 4.6 nM,

respectively.

We next evaluated these menin inhibitors for their cell growth

inhibitory activity in the MV4;11 cell line carrying MLL-AF4

fusion, which was shown to be sensitive to menin inhibitors.²³

The data are summarized in Table 1.

Consistent with their improved binding aﬃnity to menin over

MIV-6, a number of these new menin inhibitors are also more

potent than MIV-6 in the inhibition of cell growth in the

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Figure 3. Modiﬁcations of the cyclopentyl group of MIV-6 to access the P1 pocket, which was utilized by MBM1 (PDB ID 4GQ6) but not by MIV-6

(PDB ID 4OG8).

a

Table 3. Structure−Activity Relationship of Cyclopentyl-Substituted Compounds

a

IC₅₀values are average of at least three independent experiments. K_ivalues were calculated from averaged IC₅₀values.

In the co-crystal structures of MIV-6/menin and MLL/menin

(Figure 3), there is a well-deﬁned binding pocket (the P1

pocket) adjacent to the cyclopentyl group and formed by

Cys241, Tyr276, and Met278 residues of menin, and this pocket

is accessed by an alanine residue (Ala11) in MLL (Figure 3). We

reasoned that the installation of an appropriate group onto the

cyclopentyl ring to capture additional interactions with the

residues forming the P1 pocket should greatly enhance the

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a

Table 4. Design of Compound 42 (M-89) by Combination of the Best Structural Features in Compounds 23 and 39

binding aﬃnity to menin

cell growth inhibition (IC₅₀, μM)

MOLM-13 (μM)

compound

IC₅₀(μM)

K_i(μM)

MV4-11 (μM)

HL-60 (μM)

23

0.011 0.0002

0.005 0.001

0.002

<0.001

0.384 0.09

0.025 0.004

4.3 0.11

0.054 0.015

2.5 0.71

10.2 1.5

42 (M-89)

a

IC₅₀values are the average of at least three independent experiments. K_ivalues for binding aﬃnities were calculated from average IC₅₀values.

binding aﬃnity. Since it was synthetically diﬃcult to install

diverse groups onto the cyclopentyl ring in both MIV-6 and 23,

we synthesized 31, in which the quaternary amino group in the

core of MIV-6 is replaced by a nitrile group, but the rigid linker

in 23 is retained. Compound 31 binds to menin with a modest

aﬃnity (K_i= 0.96 μM) and is, thus, 16 times weaker than MIV-6,

suggesting that the basic nitrogen in 23 and its analogues greatly

enhance the binding aﬃnity to menin as compared to the neutral

nitrile group in 31. Despite the modest binding aﬃnity for 31, it

provided us with a synthetically more accessible template

molecule with which to explore the P1 pocket.

Since the P1 pocket is hydrophobic in nature, we decided to

install a small hydrophobic group onto the cyclopentyl ring, with

the results summarized in Table 3. An alkoxy group was placed

on the cyclopentyl ring, adjacent to the bond linking the

cyclopentyl ring to the tertiary carbon atom, and this produced

two diastereomers (32 and 33) with relative stereochemistry.

Compounds 32 and 33 bind to menin with similar aﬃnities (K_i

values = 4.9 and 6.1 μM), which are weaker than compound 31.

When the methoxy group in 32 is replaced by an ethoxy group,

the resulting compound 34 has an improved binding aﬃnity (K_i

= 1.7 μM) over 32 but is still a weaker binder than 31. When the

methoxy group in 32 was replaced by an acetoxyl group, the

resulting compound 35 has a K_i= 12 nM and is thus 80 times

more potent than 31. However, when the methoxy group in 33,

the diastereomer of 32, was replaced by an acetoxyl group, the

resulting compound (36) has a K_ivalue of 1.1 μM and is

equipotent with 31. The diﬀerence in binding aﬃnities of the

diastereomers 35 and 36 to menin clearly demonstrates the

stereospeciﬁc nature of the binding to menin.

(39), an acetamide (40), or a reverse carbamate (41).

Compounds 39−41 bind to menin with K_ivalues of 2.7, 3.2,

and 8.5 nM, respectively (Table 3), and are, therefore, high-

aﬃnity menin inhibitors.

These menin inhibitors in Table 3 were evaluated for their

inhibition of cell growth in the MV4;11 cell line, giving the

results in Table 3. The majority of these menin inhibitors has

IC₅₀values only in the micromolar range, but compound 39 with

the highest binding aﬃnity achieves the best IC₅₀value of 0.3

μM in the inhibition of cell growth in the MV4;11 cell line

among this series of compounds.

Taken together, the data show that the substitution of a

methyl carbamate in the cyclopentyl group of the modestly

potent inhibitor 31, which generates 39, enhances the binding to

menin by a factor of >100 and the cellular activity in the MV4;11

cell line by a factor of >50. These data indicate that the

additional interactions with residues in the P1 pocket greatly

enhance the binding aﬃnity to menin and cellular activity.

We next combined the best structural modiﬁcations in 23 and

39 and designed and synthesized compound 42 (M-89) (Table

4).

In our ﬂuorescence polarization (FP)-based binding assay, M-

89 achieves an IC₅₀value of 5 nM with an estimated K_ivalue <1

nM. Because the binding aﬃnity of M-89 exceeds the lower limit

in our FP-based assay, we employed a biolayer interferometry

(BLI) assay to further evaluate its binding aﬃnity. The BLI assay

determined that M-89 has a K_dvalue of 1.4 nM, a k_offvalue of 2.9

× 10⁻⁴s⁻¹, and a k_onvalue of 2.3 × 10⁵M⁻¹s⁻¹. In comparison,

MIV-6 has a K_dvalue of 110 nM, a k_offvalue of 460 × 10⁻⁴s⁻¹,

and a k_onvalue of 4.1 × 10⁵M⁻¹s⁻¹in our experiments. Hence,

M-89 has a high aﬃnity to menin and an oﬀ-rate, which is 158

times slower than that of MIV-6 (Table 5).

Encouraged by its excellent binding aﬃnity, we performed

further modiﬁcations of 35 to optimize the interactions with the

P1 pocket (Table 3). Changing the acetoxyl group in 35 to a

propionoxyl generated 37, which has a K_ivalue of 9.7 nM,

slightly more potent than 35. However, converting the

propionoxyl group in 37 to a butyroxyl group, producing 38,

results in a 30-fold loss in binding aﬃnity to menin, consistent

with the limited space in the P1 pocket.

M-89 was evaluated for its ability to inhibit cell growth in the

MV4;11 leukemia cell line carrying MLL-AF4 fusion and in the

HL-60 leukemia cell line lacking the MLL fusion. Our data

showed that M-89 potently inhibits cell growth in the MV4;11

cell line, achieving an IC₅₀value of 25 nM. In comparison, M-89

has an IC₅₀value of 10.2 μM in the HL-60 cell line. We further

evaluated M-89 for its cell growth inhibitory activity in the

MOLM-13 leukemia cell line carrying MLL-AF9 fusion and

Because ester groups are typically not metabolically stable, we

converted the acetoxyl group in 37 into a methyl carbamate

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Table 5. Determination of K_d, k_on, and k_offValues for Two

Menin Inhibitors with a Label Free, Biolayer Interferometry

Assay Using an Octet RED System

IC₅₀(nM) to

compound menin by FP

K_d(nM)

k

_on(10⁵M⁻¹s⁻¹

)

k

_off(10⁻⁴s⁻¹

)

MIV-6

M-89

229 29

5.0 1.0

110 29

1.4 0.6

4.1 1.1

2.3 1.1

460 102

2.9 0.2

found that it achieves an IC₅₀value of 54 nM. Thus, M-89

displays a potent cell growth inhibitory activity in the MV4;11

and MOLM-13 leukemia cell lines carrying MLL fusion and

>100-fold speciﬁcity over the HL-60 cell line lacking MLL

fusion.

To gain structural insights into the high binding aﬃnity of M-

89 with menin, we determined their co-crystal structure (PDB

ID: 6E1A). This co-crystal structure (Figure 4) shows that M-89

not only maintains the key interactions observed for MIV-6 but

captures additional interactions. Consistent with our design, the

carbamate group on the cyclopentyl ﬁts precisely into the

available P1 pocket in menin, and the carbonyl group forms a

strong hydrogen bond with the hydroxyl group of Tyr276 of

menin. The sulfonyl group forms a strong hydrogen bond with

the NH of Trp341, and the pyridyl group has additional

hydrophobic contacts with menin. The inﬂexible azetidine linker

constrains the molecule into a conformation ideal for eﬀective

interactions with menin. This co-crystal structure provides

structural insights into the high-aﬃnity binding of M-89 with

menin and a solid structural basis for further structure-based

optimization.

The cellular thermal shift assay (CETSA) is a powerful assay

with which to examine cellular protein thermal stability and to

determine if a small-molecule inhibitor targets a speciﬁc protein

in cells.^26,27We employed the CETSA assay to assess if M-89

stabilizes menin protein in cells. Our CETSA data (Figure 5A)

show that M-89 stabilizes cellular menin protein in both

MV4;11 and MOLM-13 cells in a dose-dependent manner. It

signiﬁcantly enhances the thermal stability of cellular menin

protein at concentrations as low as 3.7 nM and reaches a

maximal eﬀect at 33−100 nM. Since the cells were treated with

M-89 for only 1 h, the thermal stabilization of cellular menin by

the compound is expected to be a direct eﬀect. Our CETSA data,

thus, provide clear evidence that M-89 targets cellular menin

protein at low nanomolar concentrations, consistent with its

Figure 5. (A) Stabilization of cellular menin protein by M-89 as

assessed using the cellular thermal shift assay (CETSA) in MV4;11

cells. (B) Quantitative real-time polymerase chain reaction (qRT-PCR)

analysis of the eﬀect of M-89 on the mRNA levels of Hoxa9 and MEIS1

genes in MV4;11 cells. (C) Flow cytometry analysis of the eﬀect of M-

89 on apoptosis and cell diﬀerentiation in MV4;11 cells.

excellent cellular potency in the inhibition of cell growth in

leukemia cells carrying MLL fusion.

The menin-MLL protein−protein interaction has been shown

to play a key role in the regulation of Hoxa9 and MEIS1 gene

expression in leukemia cells carrying an MLL fusion.²³

Accordingly, we evaluated M-89 for its ability to suppress the

expression of Hoxa9 and MEIS1 in the MV4;11 cells carrying

MLL-AF4 fusion and in MOLM-13 cells carrying MLL-AF9

fusion by qRT-PCR. Our data (Figure 5B) showed that M-89

potently and eﬀectively inhibits Hoxa9 and MEIS1 gene

transcription in both cell lines, consistent with its potencies in

cell growth inhibition assay.

Using ﬂow cytometry, we evaluated the ability of M-89 to

induce apoptosis and cell diﬀerentiation in the MV4;11 cell line.

Figure 4. Co-crystal structure of M-89 bound to menin (PDB ID 6E1A) (left panel) and superposition of MIV-6 and M-89 based upon their co-crystal

structures (right panel).

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Treatment of the MV4;11 cells with M-89 for 24 or 48 h resulted

in time- and dose-dependent induction of apoptosis (Figure 5C,

left panel). Robust apoptosis induction was observed at

concentrations as low as 100−300 nM with a 48 h treatment.

Although M-89 only has a modest eﬀect on cell diﬀerentiation

with 24 h treatment, it eﬀectively induces cell diﬀerentiation

with 48 h treatment in a dose-dependent manner in the MV4;11

cells (Figure 5C, right panel).

We evaluated the pharmacodynamic (PD) eﬀect of M-89 in

mice bearing MV4;11 xenograft tumors. Previous study showed

that for reversible menin inhibitors, repeated administration was

needed to observe their PD eﬀect in vivo.²³Therefore, mice

bearing MV4;11 xenograft tumors were dosed with M-89 at 50

mg/kg daily for 3 days with intraperitoneal (IP) administration.

Mice were sacriﬁced at diﬀerent time points to harvest tumor

tissues for RT-PCR analysis of the expression levels of Hoxa9

and MEIS1 genes, with the data provided in Figure 6. Our data

showed that M-89 signiﬁcantly decreases the expression of

Hoxa9 and MEIS1 genes in the MV4;11 xenograft tumor tissue

at 6, 24, and 48 h time points.

To explore the replacement of the tail nitrile with sulfones, we

used p-ﬂuorophenyl sulfones (67−75) in the ﬁnal S_NAr

reactions to produce compounds 13−21. The p-ﬂuorophenyl

sulfones that are not commercially available were synthesized, as

shown in Scheme 2.

p-Fluorothiophenol (56) was used to substitute alkyl

bromides or in an S_NAr reaction with 4-bromopyridine to

produce the sulﬁdes (62−66). Subsequent oxidation to the

sulfones (67−71) was accomplished with mCPBA for thioalkyls

(62−65) and potassium peroxymonosulfate (Oxone) for the

thiopyridine (66).

For further exploration, we took advantage of the pyridine

sulfone and nitrile tail groups. Their reactive intermediates (78

and 79) were synthesized (Scheme 3) and used in a convergent

method to install on our modiﬁed cores. To obtain these

intermediates, ﬁrst an S_NAr reaction of azetidin-3-ylmethanol to

either 71 or 56 produced alcohols (76 and 77) that were

converted to their corresponding mesylates 78 and 79.

Compounds in Table 2 were synthesized according to the

route shown in Scheme 4. Condensation of 80 and 81 followed

by the reduction of the double bond in compound 82 yielded the

intermediate (83). Deprotonation of the hydrogen next to the

nitrile group generated a nucleophilic carbon that was reacted

with cyclopentyl bromide to yield 84. The nitrile in 84 was

reduced in two steps. The treatment with diisobutylaluminium

hydride (DIBALH) in toluene yielded the imine that was

suﬃciently stable to be isolated. This imine was treated with

NaBH₄to obtain the amine (85) that was then converted to a

methyl carbamate (86). An intramolecular Friedel Crafts

reaction produced the dihydroisoquinolin-1(2H)-one (87),

which was eﬃciently reduced with the soluble aluminum reagent

Red-Al to produce the tetrahydroisoquinoline (88). Boc

protection of 88 produced 89, which was debenzylated by

catalytic hydrogenation, and the resulting piperidine was reacted

with 78 in an second-order nucleophilic substitution (S_N2)

reaction to produce an intermediate (90). Acidic removal of the

Boc yielded 22, whose free nitrogen was then substituted by

either an S_N2 reaction or by reductive amination to produce the

ﬁnal compounds (23−30).

Functionalization of the cyclopentyl group extending from a

core structure that has a diversely substituted quaternary center

was necessary and would result in the formation of three

stereogenic centers. To simplify the situation, we decided to

explore this modiﬁcation with intermediate 83, which has an

easily generated nucleophilic carbon atom and is an early

intermediate in the synthesis of tetrahydroisoquinolines.

Reacting the carbanion of compound 83 with either a

cyclopentene-epoxide (93) or cyclopentene-N-Boc-aziridine

(94) produced a (1:1) diastereomeric mixture of intermediates

95a and 95b or 96a and 96b, respectively. Acid removal of Boc

from 96a followed by the reaction with acetic anhydride or

methyl chloroformate produced 97a and 98a, respectively. The

removal of the benzyl protecting group on the piperidine

followed by S_N2 reaction with the tail intermediate 79 produced

target molecules 31 (from 84b), 40, and 41 and the

intermediates 102a and 102b with a hydroxyl that could be

substituted, as a handle. Compounds 32−39 were produced by

the substitution of the hydroxyl group. Consistently, the

diastereomer with the same relative stereochemistry as the

intermediate (95a) was the more potent, and its stereochemistry

was conﬁrmed by the single-crystal structure of the compound

(103), which was obtained from the diastereomeric compound

(95a) (Scheme 5).

Figure 6. Pharmacodynamic eﬀect of M-89 on the expression of Hoxa9

and MEIS1 genes in the MV4;11 xenograft tissue in mice. Mice bearing

MV4;11 xenograft tumors were dosed with M-89 for 3 days and tumors

were harvested at 6, 24, and 48 h after the last administration for qRT-

PCR analysis of Hoxa9 and MEIS1 gene expression.

Chemistry. The compounds in Table 1 were synthesized, as

shown in Schemes 1 and 2. Amide coupling of 43 with 44

yielded 45 that was cyclized to produce an imine (46). A

Grignard reaction of 46 with cyclopentyl magnesium bromide

produced an intermediate (47) that was deprotected by catalytic

hydrogenation to produce a core intermediate (48) with a

reactive piperidine that could be used as a synthetic handle for

exploration of tail groups. The tail groups were installed using

either a convergent or a linear synthetic method. In the

convergent method, the tail portion (49, 50, or 51) with a

chloride or bromide leaving group was reacted with the

intermediate (48) to produce compounds 6−8. In the linear

method, the piperidine compound (48) was reacted with keto-

or aldehyde-N-Boc-protected cyclic amine linker groups in a

reductive amination which was followed by acid deprotection of

their corresponding Boc-amino groups to produce intermedi-

ates 52−55. The ﬁnal compounds 9−12 were obtained through

a nucleophilic aromatic substitution (S_NAr) reaction between

the free amino groups in compounds 52−55 and p-

ﬂuorobenzonitrile (56).

H

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a

Scheme 1. Synthesis of Compounds 6−12

a

Reagents and conditions: (a) i. 43, dichloromethane (DCM), dimethylformamide (DMF) (cat.), oxalyl chloride; ii. DCM, Et₃N, 44, 0 °C to room

temperature (rt) overnight; (b) toluene, POCl₃, P₂O₅, reﬂux; (c) tetrahydrofuran (THF), BF₃·Et₂O, cyclopentyl magnesium bromide; (d) MeOH,

Pd−C (cat.), H₂(1 atm); (e) CH₃CN, K₂CO₃, KI·H₂O (cat.), 49 or 50 or 51, reﬂux; (f) DCM/AcOH (1:1), N-Boc-cyclic-amine-ketone or

-aldehyde, NaBH(OAc)₃; (g) DCM/triﬂuoracetic acid (TFA) (1:1) 15 min; (h) dimethyl sulfoxide (DMSO), K₂CO₃, 56, 90 °C overnight.

a

Scheme 2. Synthesis of Compounds 13−21

a

Reagents and conditions: (a) For R = alkyl, t-BuONa, DMSO, R-Br, 90 °C overnight; for R = 4-bromopyridine, K₂CO₃, DMSO, 90 °C overnight;

(b) for 62−65, DCM, meta-chloroperbenzoic acid (mCPBA), overnight; for 66 Me₂CO/H₂O (5:1), oxone, overnight; (c) DMSO, K₂CO₃, 4-F-Ph-

SO₂-R, 90 °C overnight.

a

Scheme 3. Synthesis of Intermediates 78 and 79

a

Reagents and conditions: (a) DMSO, K₂CO₃, azetidin-3-ylmethanol·HCl, 80 °C, overnight; (b) DCM, Et₃N, MsCl, 0 °C to rt, 30 min.

After determining the appropriate substituents and stereo-

chemistry of the cyclopentyl group, this modiﬁcation was

applied to the more active tetrahydroisoquinoline core. First, the

hydroxyl group of the active diastereomer (95a) was benzylated,

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a

Scheme 4. Synthesis of Compounds 22−30

a

Reagents and conditions: (a) MeOH, NaOMe, reﬂux; (b) MeOH, NaBH₄; (c) i. THF, lithium bis(trimethylsilyl)amide (LiHMDS), −78 °C 30

min; ii. cyclopentyl bromide, −78 °C to rt; (d) toluene, DIBALH, 30 min, rtwork-up with aqueous (aq) NaOH; (e) MeOH, NaBH₄; (f) DCM,

Et₃N, ClCO₂Me, 0 °C, 1 h; (g) polyphosphoric acid (PPA), 150 °C, 1 h; (h) toluene, Red-Al, 30 min, rt; (i) DCM, Et₃N, Boc₂O, 0 °C to rt; (j)

MeOH, Pd−C (cat.), H₂(1 atm); (k) CH₃CN, 78, K₂CO₃, KI·H₂O, reﬂux; (l) DCM/CF₃CO₂H, 15 min; (m) CH₃CN, alkyl-Br, KI·H₂O, reﬂux or

DCM/AcOH (1:1), alkyl aldehyde or ketone, NaBH(OAc)₃.

and this was followed by the reduction of the nitrile group using

DIBAL to produce an amine (105), which was converted to a

methyl carbamate to facilitate the Pictet−Spengler cyclization

forming the tetrahydroisoquinoline (107) whose methyl

carbamate was reduced with the soluble aluminum hydride

reagent Red-Al to produce the intermediate core compound

(108). Upon hydrogenation, both benzyl protecting groups

were removed, and the piperidine ring in the product (109) was

regioselectively reacted with 78 in an S_N2 reaction to produce

compound 110. The reaction of 110 with methyl isocyanate

produced the ﬁnal compound 42 (M-89) (Scheme 6).

the MV4;11 cell line. Our pharmacodynamic experiment in mice

bearing MV4;11 xenograft tumors showed that M-89 eﬀectively

downregulates the expression of MEIS1 and Hoxa9 genes in the

MV4;11 xenograft tumor tissue. The determination of the co-

crystal structure of M-89 in a complex with menin provides a

solid structural basis for its high-aﬃnity binding and for further

structure-based optimization.

Taken together, our study has led to the discovery of M-89 as

a highly potent and speciﬁc menin inhibitor. M-89 represents a

promising lead compound for further optimization toward the

development of a menin inhibitor for the treatment of MLL

leukemia.

CONCLUSIONS

■

EXPERIMENTAL SECTION

Starting from a previously reported menin inhibitor (MIV-6),

we have performed extensive structure-based optimization to

dramatically improve its binding aﬃnity, cellular potency, and

selectivity. Through the systematic optimization of four diﬀerent

sites in the molecule, we have successfully obtained M-89 as a

high-aﬃnity menin inhibitor. M-89 has a K_dvalue of 1.4 nM to

menin and is >50 times more potent than MIV-6. M-89 achieves

IC₅₀values of 25 and 54 nM, respectively, in the MV4;11 and

MOLM-13 cell lines carrying MLL fusion and is >100 times

more potent than MIV-6. Signiﬁcantly, M-89 demonstrates

>100-fold cellular selectivity in its inhibition of cell growth in the

MV4;11 and MOLM-13 leukemia cell lines over the HL-60

leukemia cell line lacking MLL fusion. M-89 stabilizes cellular

menin protein and eﬀectively suppresses the expression of

Hoxa9 and MEIS1 genes at low nanomolar concentrations in

both MV4;11 and MOLM-13 leukemia cell lines. M-89 is also

eﬀective in the induction of apoptosis and cell diﬀerentiation in

■

General Information. Unless otherwise stated, all commercial

reagents were used as supplied without further puriﬁcation, and all

reactions were performed under a nitrogen atmosphere in a dry solvent

under anhydrous conditions. NMR spectra were obtained on a Bruker

1

400 Ascend spectrometer at a H frequency of 400 MHz and a ¹³C

frequency of 100 MHz. Chemical shifts (δ) are reported in parts per

million (ppm) relative to an internal standard. The ﬁnal products were

puriﬁed on a preparative high-performance liquid chromatography

(HPLC) column (Waters 2545, Quaternary Gradient Module) with a

SunFire Prep C18 OBD 5 μm 50 × 100 mm²reverse phase column. The

mobile phase was a gradient of solvent A (H₂O with 0.1% TFA) and

solvent B (MeCN with 0.1% TFA) at a ﬂow rate of 60 mL/min and 1%/

min increase of solvent B. All ﬁnal compounds have purity ≥95% as

determined by Waters ACQUITY ultra-performance liquid chromato-

graph (UPLC) using reverse phase column (SunFire, C18, 5 μm, 4.6 ×

150 mm²) and a solvent gradient of A (H₂O with 0.1% of TFA) and

solvent B (CH₃CN with 0.1% of TFA). Electrospray ionization (ESI)

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a

Scheme 5. Synthesis of Compounds 31−41

a

Reagents and conditions: (a) MeOH, Et₃N, Boc₂O; (b) THF, PPh₃, diisopropylazodicarboxylate, −78 °C to rt overnight; (c) 83, THF, LiHMDS,

−78 °C for 30 min then 93, −78 °C to rt overnight; (d) 83, 18-crown-6, toluene, potassium bis(trimethylsilyl)amide, −78 °C for 30 min then 94,

−78 °C to rt overnight; (e) DCM/TFA, 30 min; (f) DCM, Et₃N, Ac₂O or ClCO₂Me; (g) MeOH, Pd−C (cat.), H₂(1 atm); (h) CH₃CN, K₂CO₃,

KI·H₂O, 79, reﬂux; (i) NaH, DMF, alkyl iodide; (j) DCM, Et₃N, alkyl anhydride or MeNCO; (k) DCM, 1-[bis(dimethylamino)methylene]-

1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexaﬂuorophosphate (HATU), Et₃N, 4-Br-PhCO₂H, 0 °C to rt.

a

Scheme 6. Synthesis of M-89

a

Reagents and conditions: (a) THF/toluene (1:1), NaH, BnBr, Bu₄NI; (b) toluene, DIBALH; (c) DCM, Et₃N, ClCO₂Me, 0 °C to rt; (d) AcOH,

CF₃CO₂H, paraformaldehyde; (e) toluene, Red-Al; (f) MeOH, Pd−C (cat.), H₂(1 atm); (g) CH₃CN, K₂CO₃, KI·H₂O, 78, reﬂux; (h) DCM,

NaHCO₃, Et₃N, MeNCO, rt.

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mass spectral (MS) analysis was performed on a Thermo Scientiﬁc

LCQ Fleet mass spectrometer.

lyophilized to give 12 (14 mg, 42%) as a white powder. ¹H NMR (400

MHz, MeOH-d₄) δ 7.48 (d, J = 7.3 Hz, 2H), 7.45 (d, J = 9.7 Hz, 1H),

7.41−7.28 (m, 3H), 6.47 (d, J = 7.5 Hz, 2H), 4.17 (t, J = 8.0 Hz, 2H),

3.80−3.71 (m, 2H), 3.63 (d, J = 11.7 Hz, 1H), 3.60−3.40 (m, 7H),

3.12−2.95 (m, 3H), 2.87−2.71 (m, 1H), 2.65−2.50 (m, 1H), 2.23 (d, J

= 14.9 Hz, 1H), 2.01−1.87 (m, 2H), 1.86−1.40 (m, 8H), 1.22 (s, 1H).

ESI-MS m/z 455.83 (M + H)⁺.

1-Cyclopentyl-1-(1-((1-(4-(methylsulfonyl)phenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (13). Starting

with intermediates 55 and 72, the target molecule was prepared

according to the procedure described for compound 12. ¹H NMR (400

MHz, MeOH-d₄) δ 7.71 (d, J = 8.8 Hz, 2H), 7.48−7.41 (m, 1H), 7.41−

7.28 (m, 3H), 6.52 (d, J = 8.8 Hz, 2H), 4.19 (t, J = 8.0 Hz, 2H), 3.80−

3.74 (m, 2H), 3.64 (d, J = 11.7 Hz, 1H), 3.61−3.42 (m, 6H), 3.15−3.04

(m, 4H), 3.02 (s, 3H), 2.86−2.71 (m, 1H), 2.62 (t, J = 12.2 Hz, 1H),

2.25 (d, J = 14.2 Hz, 1H), 2.03−1.89 (m, 2H), 1.86−1.39 (m, 8H),

1.28−1.12 (m, 1H). ESI-MS m/z 508.83 (M + H)⁺.

1-Cyclopentyl-1-(1-((1-(4-(ethylsulfonyl)phenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (14). Starting

with intermediates 55 and 73, the target molecule was prepared

according to the procedure described for compound 12. ¹H NMR (400

MHz, MeOH-d₄) δ 7.66 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 6.8 Hz, 1H),

7.41−7.29 (m, 3H), 6.52 (d, J = 8.4 Hz, 2H), 4.19 (t, J = 8.0 Hz, 2H),

3.78 (t, J = 6.5 Hz, 2H), 3.65 (d, J = 12.0 Hz, 1H), 3.61−3.42 (m, 5H),

3.19−2.97 (m, 6H), 2.78 (dt, J = 18.1, 9.6 Hz, 1H), 2.64 (t, J = 12.1 Hz,

1H), 2.25 (d, J = 14.0 Hz, 1H), 2.02−1.89 (m, 2H), 1.86−1.72 (m,

3H), 1.73−1.42 (m, 5H), 1.36−1.08 (m, 5H). ESI-MS m/z 522.50 (M

+ H)⁺.

1-Cyclopentyl-1-(1-((1-(4-(isopropylsulfonyl)phenyl)azetidin-3-

yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (15). Start-

ing with intermediates 55 and 74, the target molecule was prepared

according to the procedure described for compound 12. ¹H NMR (400

MHz, MeOH-d₄) δ 7.63 (d, J = 8.8 Hz, 2H), 7.48−7.42 (m, 1H), 7.41−

7.28 (m, 3H), 6.53 (d, J = 8.8 Hz, 2H), 4.23−4.16 (m, 2H), 3.78 (ddd, J

= 8.0, 5.6, 2.2 Hz, 2H), 3.65 (d, J = 12.3 Hz, 1H), 3.61−3.40 (m, 5H),

3.29−3.24 (m, 1H), 3.24−3.14 (m, 1H), 3.13−2.98 (m, 4H), 2.79 (p, J

= 9.6 Hz, 1H), 2.64 (d, J = 12.2 Hz, 1H), 2.25 (d, J = 14.0 Hz, 1H),

2.02−1.89 (m, 2H), 1.87−1.40 (m, 8H), 1.27−1.14 (m, 7H). ESI-MS

m/z 536.67 (M + H)⁺.

1-Cyclopentyl-1-(1-((1-(4-(cyclopropylsulfonyl)phenyl)azetidin-

3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (16).

Starting with intermediates 55 and 67, the target molecule was

prepared according to the procedure described for compound 12. ¹H

NMR (400 MHz, MeOH-d₄) δ 7.66 (d, J = 7.5 Hz, 2H), 7.45 (d, J = 7.4

Hz, 1H), 7.41−7.27 (m, 3H), 6.52 (d, J = 7.8 Hz, 2H), 4.19 (t, J = 7.9

Hz, 2H), 3.82−3.73 (m, 2H), 3.64 (d, J = 12.0 Hz, 1H), 3.60−3.41 (m,

5H), 3.18−2.94 (m, 4H), 2.86−2.71 (m, 1H), 2.69−2.50 (m, 2H), 2.25

(d, J = 14.7 Hz, 1H), 2.04−1.88 (m, 2H), 1.87−1.40 (m, 8H), 1.32−

1.09 (m, 4H), 1.06−0.90 (m, 2H); ESI-MS m/z 534.50 (M + H)⁺.

1-(1-((1-(4-(Cyclobutylsulfonyl)phenyl)azetidin-3-yl)methyl)-

piperidin-4-yl)-1-cyclopentyl-1,2,3,4-tetrahydroisoquinoline (17).

Starting with intermediates 55 and 68, the target molecule was

prepared according to the procedure described for compound 12. ¹H

NMR (400 MHz, MeOH-d₄) δ 7.63 (d, J = 8.8 Hz, 2H), 7.48−7.42 (m,

1H), 7.41−7.29 (m, 3H), 6.52 (d, J = 8.8 Hz, 2H), 4.18 (t, J = 7.9 Hz,

2H), 3.88 (pd, J = 8.2, 0.8 Hz, 1H), 3.77 (ddd, J = 8.0, 5.6, 2.2 Hz, 2H),

3.65 (d, J = 12.1 Hz, 1H), 3.61−3.40 (m, 5H), 3.29−3.20 (m, 1H),

3.15−2.97 (m, 4H), 2.79 (p, J = 9.8 Hz, 1H), 2.63 (t, J = 12.8 Hz, 1H),

2.48−2.33 (m, 2H), 2.25 (d, J = 13.9 Hz, 1H), 2.22−2.08 (m, 2H),

2.04−1.85 (m, 4H), 1.85−1.40 (m, 8H), 1.28−1.13 (m, 1H); ESI-MS

m/z 548.3 [M + H]⁺.

4-(3-(4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-

piperidin-1-yl)propoxy)-benzonitrile (6). Compound 49 (75 mg,

0.384 mmol), K₂CO₃(88 mg, 0.640 mmol), and KI (catalytic) were

added to a solution of the intermediate 48 (91 mg, 0.320 mmol) in

MeCN (2 mL). The mixture was stirred at 80 °C overnight and then

extracted with EtOAc, washed with brine, dried over Na₂SO₄, and the

solvent was evaporated. The residue was puriﬁed with preparative

HPLC to give the title compound (80 mg, 56%). ¹H NMR (400 MHz,

CDCl₃) δ 7.57 (d, J = 8.4 Hz, 2H), 7.36−7.29 (m, 2H), 7.23−7.15 (m,

2H), 6.90 (d, J = 8.5 Hz, 2H), 4.10 (s, 2H), 3.84−3.71 (m, 1H), 3.67−

3.52 (m, 2H), 3.49−3.36 (m, 1H), 3.27−2.93 (m, 4H), 2.90−2.72 (m,

3H), 2.58−2.43 (m, 1H), 2.36−2.17 (m, 4H), 2.06−1.84 (m, 2H),

1.77−1.45 (m, 4H), 1.37−1.12 (m, 2H); ESI-MS m/z 444.3 [M + H]⁺.

4-(2-(4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-

piperidin-1-yl)ethoxy)benzonitrile (7). Compound 7 was prepared

from compounds 48 and 50 according to the procedure used to

produce compound 6. ¹H NMR (400 MHz, MeOH-d₄) δ 7.69 (d, J =

8.9 Hz, 2H), 7.47−7.42 (m, 1H), 7.41−7.28 (m, 3H), 7.13 (d, J = 9.0

Hz, 2H), 4.44 (t, J = 4.8 Hz, 2H), 3.82−3.65 (m, 2H), 3.63−3.51 (m,

3H), 3.51−3.41 (m, 1H), 3.23−3.05 (m, 4H), 2.81 (p, J = 9.7 Hz, 1H),

2.67−2.53 (m, 1H), 2.24 (d, J = 14.1 Hz, 1H), 2.06−1.90 (m, 2H),

1.90−1.51 (m, 7H), 1.52−1.37 (m, 1H), 1.32−1.17 (m, 1H); ESI-MS

m/z 430.26 [M + H]⁺.

4-(4-(4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-

piperidin-1-yl)butoxy)-benzonitrile (8). Compound 8 was prepared

from compounds 48 and 51 according to the procedure used to make

compound 6. ¹H NMR (400 MHz, MeOH-d₄) δ 7.65 (d, J = 8.9 Hz,

2H), 7.47−7.41 (m, 1H), 7.40−7.29 (m, 3H), 7.05 (d, J = 8.9 Hz, 2H),

4.11 (t, J = 5.5 Hz, 2H), 3.73−3.59 (m, 2H), 3.59−3.40 (m, 2H), 3.21−

3.06 (m, 4H), 3.06−2.94 (m, 2H), 2.87−2.73 (m, 1H), 2.58 (t, J = 12.0

Hz, 1H), 2.23 (d, J = 14.1 Hz, 1H), 2.01−1.79 (m, 7H), 1.79−1.51 (m,

6H), 1.51−1.37 (m, 1H), 1.32−1.15 (m, 1H); ESI-MS m/z 458.30 [M

+ H]⁺.

4-(4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-[1,4′-bi-

piperidin]-1′-yl)benzonitrile (9). Compound 9 was prepared according

1

to the procedure used to make compound 12. H NMR (400 MHz,

MeOH-d₄) δ 7.53 (d, J = 9.0 Hz, 2H), 7.47−7.41 (m, 1H), 7.40−7.29

(m, 3H), 7.04 (d, J = 9.0 Hz, 2H), 4.11 (d, J = 13.2 Hz, 2H), 3.70−3.51

(m, 3H), 3.51−3.39 (m, 2H), 3.17−3.04 (m, 4H), 2.93 (t, J = 12.7 Hz,

2H), 2.86−2.74 (m, 1H), 2.60 (t, J = 12.0 Hz, 1H), 2.27 (d, J = 14.0 Hz,

1H), 2.16 (d, J = 12.1 Hz, 2H), 2.02−1.51 (m, 11H), 1.52−1.39 (m,

1H), 1.28−1.16 (m, 1H); ESI-MS m/z 469.31 [M + H]⁺.

4-(3-((4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-

piperidin-1-yl)methyl)piperidin-1-yl)benzonitrile (10). Compound

10 was prepared according to the procedure used to make compound

12. ¹H NMR (400 MHz, MeOH-d₄) δ 7.54−7.41 (m, 3H), 7.42−7.27

(m, 3H), 7.04 (dd, J = 9.2, 2.3 Hz, 2H), 3.96 (d, J = 12.9 Hz, 1H), 3.84−

3.58 (m, 3H), 3.57−3.43 (m, 2H), 3.20−2.86 (m, 7H), 2.75 (dd, J =

12.6, 9.8 Hz, 2H), 2.65 (t, J = 12.6 Hz, 1H), 2.30−2.01 (m, 3H), 2.01−

1.87 (m, 3H), 1.87−1.42 (m, 9H), 1.42−1.27 (m, 1H), 1.24−1.07 (m,

1H); ESI-MS m/z 483.3 [M + H]⁺.

4-(3-((4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-

piperidin-1-yl)methyl)-pyrrolidin-1-yl)benzonitrile (11). Compound

11 was prepared according to the procedure used to make compound

12. ¹H NMR (400 MHz, MeOH-d₄) δ 7.50−7.43 (m, 3H), 7.36 (tt, J =

12.0, 7.0 Hz, 3H), 6.62 (d, J = 8.7 Hz, 2H), 3.85−3.70 (m, 2H), 3.66 (t,

J = 8.8 Hz, 1H), 3.58−3.44 (m, 3H), 3.42−3.34 (m, 1H), 3.26 (d, J =

6.8 Hz, 2H), 3.19−2.97 (m, 5H), 2.89−2.72 (m, 2H), 2.69−2.55 (m,

1H), 2.37−2.20 (m, 2H), 2.12−1.73 (m, 6H), 1.73−1.40 (m, 5H),

1.27−1.14 (m, 1H); ESI-MS m/z 469.3 [M + H]⁺.

1-Cyclopentyl-1-(1-((1-(4-(cyclopentylsulfonyl)phenyl)azetidin-

3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (18).

Starting with intermediates 55 and 69, the target molecule was

prepared according to the procedure described for compound 12. ¹H

NMR (400 MHz, MeOH-d₄) δ 7.65 (d, J = 8.7 Hz, 2H), 7.47−7.41 (m,

1H), 7.40−7.27 (m, 3H), 6.52 (d, J = 8.8 Hz, 2H), 4.18 (t, J = 8.0 Hz,

2H), 3.82−3.73 (m, 2H), 3.64 (d, J = 11.8 Hz, 1H), 3.60−3.40 (m,

6H), 3.17−2.96 (m, 4H), 2.79 (dt, J = 17.7, 8.0 Hz, 1H), 2.70−2.54 (m,

4-(3-((4-(1-Cyclopentyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-

piperidin-1-yl)methyl)azetidin-1-yl)benzonitrile (12). p-Fluoroben-

zonitrile (56) (18 mg, 0.148 mmol) was added to a solution of

compound 55 (26 mg, 0.074 mmol) and K₂CO₃(41 mg, 0.295 mmol)

in DMSO (2 mL), then stirred and heated to 80 °C. After being stirred

overnight, the reaction was quenched with TFA (0.5 mL), diluted with

3:1 MeOH/H₂O, and puriﬁed by preparative HPLC. The pure

fractions were combined, concentrated, rediluted with H₂O, frozen, and

L

DOI: 10.1021/acs.jmedchem.9b00021

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 2.25 (d, J = 14.2 Hz, 1H), 2.03−1.40 (m, 19H), 1.31−1.10 (m,

1H). ESI-MS m/z 562.92 (M + H)⁺.

d₄) δ 8.80−8.72 (m, 2H), 7.82 (d, J = 6.2 Hz, 2H), 7.76 (d, J = 8.9 Hz,

2H), 7.61−7.55 (m, 1H), 7.51−7.43 (m, 1H), 7.35 (ddd, J = 16.2, 7.9,

6.6 Hz, 2H), 6.51 (d, J = 8.9 Hz, 2H), 4.51−4.11 (m, 4H), 3.90−3.60

(m, 4H), 3.58−3.37 (m, 6H), 3.26−3.15 (m, 1H), 3.10−2.91 (m, 2H),

2.87−2.66 (m, 1H), 2.59−2.26 (m, 2H), 2.16 (d, J = 14.0 Hz, 1H),

2.10−1.92 (m, 1H), 1.92−1.80 (m, 1H), 1.80−1.52 (m, 6H), 1.48 (t, J

= 7.3 Hz, 3H), 1.36−1.16 (m, 1H), 1.16−0.99 (m, 1H); ESI-MS m/z

599.67 (M + H)⁺.

1-(1-((1-(4-(Cyclohexylsulfonyl)phenyl)azetidin-3-yl)methyl)-

piperidin-4-yl)-1-cyclopentyl-1,2,3,4-tetrahydroisoquinoline (19).

Starting with intermediates 55 and 70, the target molecule was

prepared according to the procedure described for compound 12. ¹H

NMR (400 MHz, MeOH-d₄) δ 7.61 (d, J = 7.4 Hz, 2H), 7.45 (d, J = 6.5

Hz, 1H), 7.41−7.29 (m, 3H), 6.52 (d, J = 7.6 Hz, 2H), 4.19 (t, J = 8.0

Hz, 2H), 3.78 (t, J = 6.7 Hz, 2H), 3.65 (d, J = 11.9 Hz, 1H), 3.60−3.44

(m, 5H), 3.29−3.22 (m, 1H), 3.16−3.01 (m, 4H), 2.98−2.87 (m, 1H),

2.84−2.73 (m, 1H), 2.70−2.58 (m, 1H), 2.25 (d, J = 14.8 Hz, 1H),

2.06−1.91 (m, 4H), 1.89−1.73 (m, 5H), 1.72−1.60 (m, 4H), 1.58−

1.43 (m, 2H), 1.38−1.19 (m, 5H), 1.19−1.05 (m, 1H); ESI-MS m/z

576.4 [M + H]⁺.

1-Cyclopentyl-1-(1-((1-(4-(phenylsulfonyl)phenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (20). Starting

with intermediates 55 and 75, the target molecule was prepared

according to the procedure described for compound 12. ¹H NMR (400

MHz, MeOH-d₄) δ 7.90−7.83 (m, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.61−

7.49 (m, 3H), 7.47−7.40 (m, 1H), 7.40−7.27 (m, 3H), 6.47 (d, J = 8.9

Hz, 2H), 4.14 (t, J = 8.0 Hz, 2H), 3.73 (ddd, J = 8.0, 5.5, 2.3 Hz, 2H),

3.62 (d, J = 12.2 Hz, 1H), 3.59−3.36 (m, 5H), 3.24 (p, J = 6.6 Hz, 1H),

3.17−2.95 (m, 4H), 2.77 (p, J = 9.6 Hz, 1H), 2.68−2.54 (m, 1H), 2.24

(d, J = 14.0 Hz, 1H), 2.02−1.88 (m, 2H), 1.86−1.39 (m, 8H), 1.27−

1.09 (m, 1H). ESI-MS m/z 570.50 (M + H)⁺.

1-Cyclopentyl-1-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)azetidin-

3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (21).

Starting with intermediates 55 and 71, the target molecule was

prepared according to the procedure described for compound 12. ¹H

NMR (400 MHz, MeOH-d₄) δ 8.75 (d, J = 5.2 Hz, 2H), 7.88−7.79 (m,

2H), 7.76 (d, J = 8.9 Hz, 2H), 7.48−7.40 (m, 1H), 7.42−7.27 (m, 3H),

6.51 (d, J = 8.9 Hz, 1H), 4.19 (t, J = 7.9 Hz, 2H), 3.77 (t, J = 6.7 Hz,

2H), 3.67−3.41 (m, 7H), 3.17−2.97 (m, 4H), 2.89−2.72 (m, 1H),

2.68−2.50 (m, 1H), 2.24 (d, J = 14.5 Hz, 1H), 2.04−1.86 (m, 2H),

1.88−1.37 (m, 8H), 1.35−1.16 (m, 1H); ESI-MS m/z 571.67 (M +

H)⁺.

4-Cyclopentyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)azetidin-

3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (22).

Compound 90 (20 mg, 0.0298 mmol) was dissolved in TFA (1 mL).

After 30 min, the TFA was removed in vacuo, and the crude was puriﬁed

by preparative HPLC to yield 22−TFA salt (12 mg) as a yellow solid.

¹H NMR (400 MHz, MeOH-d₄) δ 8.76 (s, 2H), 7.83 (d, J = 5.1 Hz,

2H), 7.76 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.47−7.37 (m,

1H), 7.38−7.22 (m, 2H), 6.50 (d, J = 8.6 Hz, 2H), 4.33−4.22 (m, 2H),

4.23−4.11 (m, 2H), 3.76 (t, J = 6.8 Hz, 2H), 3.66−3.37 (m, 7H), 3.09−

2.90 (m, 2H), 2.64−2.47 (m, 1H), 2.34−2.19 (m, 1H), 2.14 (d, J = 14.0

Hz, 1H), 1.89−1.70 (m, 3H), 1.72−1.44 (m, 6H), 1.36−1.21 (m, 1H),

1.18−1.00 (m, 1H); ESI-MS m/z 571.58 (M + H)⁺.

4-Cyclopentyl-2-methyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)-

azetidin-3-yl)methyl)-piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline

(23). Crude 22 (0.0298 mmol) was dissolved in MeCN (1 mL),

followed by the addition of K₂CO₃(16 mg, 0.119 mmol) and MeI (8.4

mg, 0.0596 mmol) at rt. After 1 h, the reaction was ﬁltered through

celite, concentrated, and puriﬁed by preparative HPLC to produce 23−

TFA salt. ¹H NMR (400 MHz, MeOH-d₄) δ 8.75 (d, J = 6.3 Hz, 2H),

7.82 (d, J = 6.2 Hz, 2H), 7.76 (d, J = 8.9 Hz, 2H), 7.60−7.55 (m, 1H),

7.46 (t, J = 7.5 Hz, 1H), 7.36 (td, J = 7.4, 1.1 Hz, 1H), 7.31−7.25 (m,

1H), 6.50 (d, J = 8.8 Hz, 2H), 4.46−4.23 (m, 2H), 4.18 (t, J = 8.1 Hz,

2H), 3.76 (s, 2H), 3.68−3.46 (m, 3H), 3.46−3.37 (m, 2H), 3.29−3.18

(m, 1H), 3.15 (s, 3H), 3.07−2.90 (m, 2H), 2.52−2.29 (m, 1H), 2.22−

2.01 (m, 2H), 1.95−1.41 (m, 10H), 1.40−1.20 (m, 2H); ESI-MS m/z

585.26 [M + H]⁺.

4-Cyclopentyl-2-ethyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)-

azetidin-3-yl)methyl)-piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline

(24). Acetaldehyde (3.9 mg, 0.0894 mmol) was added to a solution of

crude 22 (0.0298 mmol) in DCM/AcOH (1:1, 2 mL). After 10 min,

NaBH(OAc)₃(38 mg, 0.179 mmol) was slowly added to the reaction.

After standing overnight, the reaction was slowly quenched with

saturated NaHCO₃, extracted with EtOAc, dried over Na₂SO₄, ﬁltered,

and concentrated to produce a crude product that was puriﬁed by

preparative HPLC to yield 24−TFA salt. ¹H NMR (400 MHz, MeOH-

4-Cyclopentyl-2-isopropyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)-

phenyl)azetidin-3-yl)methyl)-piperidin-4-yl)-1,2,3,4-tetrahydroiso-

quinoline (25). Compound 25 was prepared from crude 22 and

1

Me₂CO according to the procedure described for compound 24. H

NMR (400 MHz, MeOH-d₄) δ 8.75 (s, 2H), 7.90−7.79 (m, 2H), 7.76

(d, J = 8.9 Hz, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.46 (td, J = 8.0, 7.5, 1.8 Hz,

1H), 7.40−7.25 (m, 2H), 6.49 (d, J = 8.7 Hz, 2H), 4.45−4.25 (m, 2H),

4.25−4.05 (m, 2H), 3.87−3.60 (m, 5H), 3.56−3.38 (m, 4H), 3.09−

2.89 (m, 2H), 2.84−2.64 (m, 1H), 2.64−2.45 (m, 1H), 2.45−2.24 (m,

1H), 2.24−2.03 (m, 2H), 1.93−1.78 (m, 2H), 1.78−1.59 (m, 6H), 1.51

(d, J = 6.6 Hz, 6H), 1.38−1.08 (m, 2H); ESI-MS m/z 613.58 (M + H)⁺.

2-Cyclobutyl-4-cyclopentyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)-

phenyl)azetidin-3-yl)methyl)-piperidin-4-yl)-1,2,3,4-tetrahydroiso-

quinoline (26). Compound 26 was prepared from crude 22 and

cyclobutanone according to the procedure described for compound 24.

¹H NMR (400 MHz, MeOH-d₄) δ 8.75 (d, J = 5.9 Hz, 2H), 7.82 (d, J =

6.1 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.47 (t, J

= 7.5 Hz, 1H), 7.36 (q, J = 7.8 Hz, 2H), 6.50 (d, J = 8.8 Hz, 2H), 4.45−

4.26 (m, 1H), 4.17 (t, J = 7.8 Hz, 2H), 4.04−3.89 (m, 1H), 3.83−3.68

(m, 2H), 3.68−3.36 (m, 5H), 3.24−3.15 (m, 1H), 3.09−2.92 (m, 2H),

2.88−2.73 (m, 1H), 2.65−2.19 (m, 6H), 2.15 (d, J = 15.8 Hz, 1H),

2.03−1.80 (m, 4H), 1.80−1.37 (m, 8H), 1.34−1.17 (m, 2H). ESI-MS

m/z 625.58 (M + H)⁺.

4-Cyclopentyl-2-(cyclopropylmethyl)-4-(1-((1-(4-(pyridin-4-

ylsulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetra-

hydroisoquinoline (27). Compound 27 was prepared from crude 22

and (bromomethyl)cyclopropane according to the procedure described

for compound 23. ¹H NMR (400 MHz, MeOH-d₄) δ 8.75 (d, J = 5.6

Hz, 2H), 7.82 (d, J = 6.1 Hz, 2H), 7.76 (d, J = 8.9 Hz, 2H), 7.58 (d, J =

7.8 Hz, 1H), 7.47 (t, J = 7.4 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 7.32 (d, J

= 6.7 Hz, 1H), 6.50 (d, J = 8.9 Hz, 2H), 4.62−4.24 (m, 2H), 4.17 (t, J =

8.3 Hz, 2H), 4.07−3.85 (m, 1H), 3.83−3.69 (m, 2H), 3.58−3.37 (m,

5H), 3.10−2.89 (m, 2H), 2.88−2.66 (m, 1H), 2.60−2.25 (m, 2H), 2.17

(d, J = 14.0 Hz, 1H), 1.94−1.36 (m, 10H), 1.36−1.17 (m, 3H), 1.16−

0.97 (m, 1H), 0.94−0.70 (m, 2H), 0.59−0.43 (m, 2H). ESI-MS m/z

625.75 (M + H)⁺.

4-Cyclopentyl-2-(oxetan-3-ylmethyl)-4-(1-((1-(4-(pyridin-4-

ylsulfonyl)phenyl)azetidin-3-yl)-methyl)piperidin-4-yl)-1,2,3,4-tet-

rahydroisoquinoline (28). Compound 28 was prepared from crude 22

and oxetane-3-carbaldehyde according to the procedure described for

compound 24. ¹H NMR (400 MHz, MeOH-d₄) δ 8.76 (d, J = 5.8 Hz,

2H), 7.84 (d, J = 6.1 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.60−7.49 (m,

1H), 7.45 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.4 Hz, 1H), 7.30 (d, J = 7.4

Hz, 1H), 6.50 (d, J = 8.8 Hz, 2H), 4.18 (t, J = 8.1 Hz, 2H), 3.88−3.70

(m, 5H), 3.70−3.54 (m, 5H), 3.54−3.37 (m, 4H), 3.09−2.88 (m, 2H),

2.68−2.42 (m, 2H), 2.41−2.23 (m, 1H), 2.17 (d, J = 14.3 Hz, 1H),

1.93−1.75 (m, 3H), 1.75−1.21 (m, 10H), 1.12−0.93 (m, 1H). ESI-MS

m/z 641.93 (M + H)⁺.

4-Cyclopentyl-2-(pyridin-4-ylmethyl)-4-(1-((1-(4-(pyridin-4-

ylsulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetra-

hydroisoquinoline (29). Compound 29 was prepared from crude 22

and isonicotinaldehyde according to the procedure described for

compound 24. ¹H NMR (400 MHz, MeOH-d₄) δ 8.78 (dd, J = 12.1, 3.8

Hz, 4H), 8.07 (d, J = 5.7 Hz, 2H), 7.84 (d, J = 4.7 Hz, 2H), 7.76 (d, J =

8.7 Hz, 2H), 7.41 (d, J = 7.6 Hz, 1H), 7.24 (t, J = 7.1 Hz, 1H), 7.14 (t, J

= 7.4 Hz, 1H), 6.99 (d, J = 7.2 Hz, 1H), 6.50 (d, J = 8.8 Hz, 2H), 4.26−

4.06 (m, 3H), 3.93 (d, J = 15.6 Hz, 1H), 3.84−3.70 (m, 2H), 3.68−3.37

(m, 7H), 3.10−2.95 (m, 2H), 2.90 (t, J = 12.7 Hz, 1H), 2.78 (d, J = 12.0

Hz, 1H), 2.58−2.44 (m, 1H), 2.40 (d, J = 13.8 Hz, 1H), 2.25−2.10 (m,

1H), 2.02−1.80 (m, 2H), 1.73 (d, J = 14.3 Hz, 1H), 1.66−1.38 (m,

6H), 1.38−1.27 (m, 1H), 1.27−1.12 (m, 1H). ESI-MS m/z 662.58 (M

+ H)⁺.

M

DOI: 10.1021/acs.jmedchem.9b00021

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Journal of Medicinal Chemistry

Article

4-(2-(4-Cyclopentyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)-

azetidin-3-yl)methyl)piperidin-4-yl)-3,4-dihydroisoquinolin-2(1H)-

yl)ethyl)morpholine (30). Compound 30 was prepared from crude 22

and 4-(2-bromoethyl)morpholine according to the procedure

described for compound 23. ¹H NMR (400 MHz, MeOH-d₄) δ 8.75

(d, J = 5.4 Hz, 2H), 7.82 (d, J = 6.1 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H),

7.41 (d, J = 7.7 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.7 Hz,

1H), 7.12 (d, J = 6.7 Hz, 1H), 6.50 (d, J = 8.9 Hz, 2H), 4.18 (t, J = 7.9

Hz, 2H), 3.98−3.85 (m, 4H), 3.83 (s, 1H), 3.75 (dd, J = 9.6, 4.0 Hz,

2H), 3.62−3.38 (m, 9H), 3.27−3.17 (m, 1H), 3.16−2.81 (m, 6H), 2.70

(d, J = 12.3 Hz, 1H), 2.48 (dt, J = 17.6, 8.7 Hz, 1H), 2.32 (d, J = 14.4 Hz,

1H), 2.23−2.10 (m, 1H), 2.01−1.85 (m, 1H), 1.85−1.73 (m, 1H), 1.67

(d, J = 13.9 Hz, 1H), 1.63−1.37 (m, 7H), 1.37−1.24 (m, 1H), 1.24−

1.09 (m, 1H). ESI-MS m/z 684.50 (M + H)⁺.

rac-4-(3-((4-(Cyano(cyclopentyl)(phenyl)methyl)piperidin-1-yl)-

methyl)azetidin-1-yl)benzonitrile (31). Compound 31 was synthe-

sized using the method described for compound 102a from the

intermediates 84 and 79. ¹H NMR (400 MHz, MeOH-d₄) δ 7.50−7.42

(m, 6H), 7.40−7.36 (m, 1H), 6.46 (d, J = 8.8 Hz, 2H), 4.16−4.12 (m,

2H), 3.72−3.69 (m, 2H), 3.54 (t, J = 14.1 Hz, 2H), 3.41 (d, J = 7.2 Hz,

2H), 3.24−3.16 (m, 1H), 3.12−2.98 (m, 2H), 2.97−2.89 (m, 1H),

2.42−2.36 (m, 1H), 2.28 (d, J = 14.3 Hz, 1H), 2.08−2.00 (m, 2H),

1.76−1.67 (m 2H), 1.65−1.55 (m, 4H), 1.53−1.38 (m, 2H), 1.29−

1.16 (m, 1H); ESI-MS m/z 439.42 (M + H)⁺.

(d, J = 7.1 Hz, 2H), 3.24−3.13 (m, 2H), 3.10−3.01 (m, 2H), 2.41 (t, J =

12.3 Hz, 1H), 2.28−2.25 (m, 2H), 2.06 (d, J = 14.3 Hz, 1H), 1.87−1.77

(m, 3H), 1.76 (s, 3H), 1.73−1.62 (m, 2H), 1.59−1.48 (m, 1H), 1.45−

1.34 (m, 1H); ESI-MS m/z 497.42 [M + H]⁺.

rac-(1S,2R)-2-((R)-Cyano(1-((1-(4-cyanophenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-(phenyl)methyl)cyclopentyl Acetate (36).

Compound 36 was synthesized from the intermediate 95b using the

method described for compound 35. ¹H NMR (400 MHz, MeOH-d₄) δ

7.50−7.44 (m, 6H), 7.42−7.38 (m, 1H), 6.46 (d, J = 8.8 Hz, 2H),

5.21−5.18 (m, 1H), 4.16−4.11 (m, 2H), 3.73−3.69 (m, 2H), 3.56 (t, J

= 14.0 Hz, 2H), 3.42 (d, J = 7.1 Hz, 2H), 3.23−3.18 (m, 1H), 3.16−

3.09 (m, 2H), 2.96 (t, J = 12.4 Hz, 1H), 2.54 (t, J = 12.1 Hz, 1H), 2.31−

2.27 (m, 1H), 2.11 (s, 3H), 2.04 (d, J = 14.3 Hz, 1H), 1.99−1.91 (m,

1H), 1.90−1.82 (m, 1H), 1.76−1.68 (m, 2H), 1.67−1.58 (m, 1H),

1.56−1.41 (m, 2H), 1.33−1.22 (m, 1H). ESI-MS m/z 497.38 [M +

H]⁺.

rac-(1S,2R)-2-((S)-Cyano(1-((1-(4-cyanophenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-(phenyl)methyl)cyclopentyl Propionate (37).

Compound 37 was obtained according to the procedure described for

compound 35. ¹H NMR (400 MHz, MeOH-d₄) δ 7.51−7.36 (m, 7H),

7.46 (d, J = 8.8 Hz, 2H), 4.93−4.88 (m, 1H), 4.16−4.12 (m, 2H), 3.71

(t, J = 6.7 Hz, 2H), 3.54 (t, J = 9.8 Hz, 2H), 3.41 (d, J = 7.1 Hz, 2H),

3.22−3.13 (m, 2H), 3.09−3.01 (m, 2H), 2.40 (t, J = 12.3 Hz, 1H),

2.27−2.23 (m, 2H), 2.15−1.96 (m, 3H), 1.88−1.74 (m, 3H), 1.73−

1.61 (m, 2H), 1.58−1.47 (m, 1H), 1.43−1.29 (m, 1H), 0.93 (t, J = 7.5

Hz, 3H); ESI-MS m/z 511.38 [M + H]⁺.

rac-(1S,2R)-2-((S)-Cyano(1-((1-(4-cyanophenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-(phenyl)methyl)cyclopentyl Butyrate (38).

Compound 38 was obtained according to the procedure described

for compound 35. ¹H NMR (400 MHz, MeOH-d₄) δ 7.51−7.42 (m,

6H), 7.40−7.36 (m, 1H), 6,46 (d, J = 8.8 Hz, 2H), 4.93−4.88 (m, 1H),

4.16−4.12 (m, 2H), 3.71 (t, J = 5.9 Hz, 2H), 3.54 (t, J = 9.9 Hz, 2H),

3.41 (d, J = 7.1 Hz, 2H), 3.23−3.12 (m, 2H), 3.09−3.00 (m, 2H), 2.39

(t, J = 12.4 Hz, 1H), 2.27−2.24 (m, 2H), 2.11−1.94 (m, 3H), 1.88−

1.61 (m, 5H), 1.57−1.50 (m, 1H), 1.49−1.40 (m, 2H), 1.39−1.29 (m,

1H), 0.84 (t, J = 7.4 Hz, 3H); ESI-MS m/z 525.37 [M + H]⁺.

rac-(1S,2R)-2-((S)-Cyano(1-((1-(4-cyanophenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-(phenyl)methyl)cyclopentyl Methyl Carba-

mate (39). Methyl isocyanate (8 mg, 0.145 mmol) was added at 0

°C to a solution of the intermediates 102a (22 mg, 0.048 mmol) and

Et₃N (15 mg, 0.145 mmol) in DCM (2 mL). The reaction mixture was

stirred overnight at rt. Then, the reaction was concentrated under

vacuum. The residue was puriﬁed directly by reverse phase preparative

HPLC to give the title compound as a salt of TFA (13 mg, 34%). ¹H

NMR (400 MHz, MeOH-d₄) δ 7.51−7.37 (m, 7H), 6.46 (d, J = 8.8 Hz,

2H), 4.88−4.86 (m, 1H), 4.17−4.12 (m, 2H), 3.73−3.70 (m, 2H), 3.58

(d, J = 12.2 Hz, 1H), 3.52 (d, J = 12.3 Hz, 1H), 3.43 (d, J = 7.1 Hz, 2H),

3.23−3.17 (m, 1H), 3.08−3.01 (m, 3H), 2.56 (s, 3H), 2.45 (t, J = 12.0

Hz, 1H), 2.32 (d, J = 14.3 Hz, 1H), 2.22−2.16 (m, 1H), 1.97 (d, J = 14.1

Hz, 1H), 1.74−1.63 (m, 4H), 1.60−1.47 (m, 3H); ESI-MS m/z 512.40

[M + H]⁺.

rac-4-(3-((4-((S)-Cyano((1R,2S)-2-methoxycyclopentyl)(phenyl)-

methyl)piperidin-1-yl)-methyl)azetidin-1-yl)benzonitrile (32). NaH

(60% dispersion in mineral oil, 5.3 mg, 0.132 mmol) at 0 °C was added

to a solution of the intermediate 102a (30 mg, 0.066 mmol) in DMF (1

mL). After stirring for 10 min, MeI (9.4 mg, 0.066 mmol) was added.

The reaction mixture was stirred for additional 3 h, then was quenched

with H₂O. The mixture was puriﬁed directly by reverse phase

preparative HPLC to give the title compound as a TFA salt (14 mg,

1

38%). H NMR (400 MHz, MeOH-d₄) δ 7.53 (d, J = 7.2 Hz, 2H),

7.49−7.44 (m, 4H), 7.43−7.39 (m, 1H), 6.46 (d, J = 8.8 Hz, 2H),

4.17−4.12 (m, 2H), 3.74−3.70 (m, 2H), 3.58 (d, J = 12.2 Hz, 1H), 3.51

(d, 12.5 Hz, 1H), 3.42 (d, J = 7.1 Hz, 2H), 3.39−3.35 (m, 1H), 3.25−

3.15 (m, 1H), 3.11−3.00 (m, 2H), 2.98 (s, 3H), 2.89−2.83 (m, 1H),

2.50−2.44 (m, 1H), 2.39−2.36 (m, 1H), 2.14−2.08 (m, 1H), 1.96 (d, J

= 14.4 Hz, 1H), 1.78−1.72 (m, 1H), 1.71−1.65 (m, 2H), 1.62−1.45

(m, 4H); ESI-MS m/z 469.41 [M + H]⁺.

rac-4-(3-((4-((R)-Cyano((1R,2S)-2-methoxycyclopentyl)(phenyl)-

methyl)piperidin-1-yl)-methyl)azetidin-1-yl)benzonitrile (33). Com-

pound 33 was synthesized from the intermediate 95b using the method

described for compound 32. ¹H NMR (400 MHz, MeOH-d₄) δ 7.54−

7.42 (m, 6H), 7.41−7.36 (m, 1H), 6.46 (d, J = 8.8 Hz, 2H), 4.16−4.11

(m, 2H), 3.93−3.88 (m, 1H), 3.72−3.69 (m, 2H), 3.58−3.51 (m, 2H),

3.41 (d, J = 7.2 Hz, 2H), 3.39 (s, 3H), 3.23−3.09 (m, 2H), 2.99−2.90

(m, 2H), 2.49−2.43 (m, 1H), 2.30 (d, J = 14.4 Hz, 1H), 2.21 (d, J = 14.1

Hz, 1H), 1.89−1.76 (m, 2H), 1.75−1.55 (m, 3H), 1.49−1.34 (m, 2H),

1.29−1.16 (m, 1H); ESI-MS m/z 469.41 [M + H]⁺.

rac-4-(3-((4-((S)-Cyano((1R,2S)-2-ethoxycyclopentyl)(phenyl)-

methyl)piperidin-1-yl)-methyl)azetidin-1-yl)benzonitrile (34). Com-

pound 34 was obtained according to the procedure described for

compound 32 using EtI. ¹H NMR (400 MHz, MeOH-d₄) δ 7.54 (d, J =

7.3 Hz, 2H), 7.49−7.38 (m, 5H), 6.46 (d, J = 8.8 Hz, 2H), 4.17−4.12

(m, 2H), 3.73−3.70 (m, 2H), 3.60−3.47 (m, 3H), 3.42 (d, J = 7.1 Hz,

2H), 3.36−3.32 (m, 1H), 3.23−3.16 (m, 1H), 3.09−3.00 (m, 2H),

2.90−2.81 (m, 2H), 2.48−2.35 (m, 2H), 2.15−2.09 (m, 1H), 1.98 (d, J

= 14.4 Hz, 1H), 1.74−1.67 (m, 3H), 1.65−1.56 (m, 1H), 1.54−1.42

(m, 3H), 0.95 (t, J = 7.0 Hz, 3H); ESI-MS m/z 483.42 [M + H]⁺.

rac-(1S,2R)-2-((S)-Cyano(1-((1-(4-cyanophenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)(phenyl)methyl)cyclopentyl Acetate (35). Ace-

tic anhydride (9 mg, 0.088 mmol) was added at 0 °C to a solution of the

intermediate 102a (20 mg, 0.044 mmol) and Et₃N (13 mg, 0.131

mmol) in DCM (2 mL). The reaction mixture was stirred for 1 h at rt.

Then, the reaction was concentrated under vacuum. The residue was

puriﬁed directly by reverse phase preparative HPLC to give the title

compound as a TFA salt (13 mg, 50%). ¹H NMR (400 MHz, MeOH-

d₄) δ 7.51−7.36 (m, 7H), 6.46 (d, J = 8.8 Hz, 2H), 4.91−4.88 (m, 1H),

4.16−4.12 (m, 2H), 3.72−3.69 (m, 2H), 3.55 (t, J = 11.6 Hz, 2H), 3.41

rac-N-((1S,2R)-2-((S)-Cyano(1-((1-(4-cyanophenyl)azetidin-3-yl)-

methyl)piperidin-4-yl)-(phenyl)methyl)cyclopentyl)acetamide (40).

Compound 40 was synthesized using the method described for

1

compound 102a from the intermediates 97a and 79. H NMR (400

MHz, MeOH-d₄) δ 7.54 (d, J = 7.7 Hz, 2H), 7.51−7.36 (m, 5H), 6.48

(d, J = 8.8 Hz, 2H), 4.26−4.12 (m, 3H), 3.77−3.68 (m, 2H), 3.59 (d, J

= 12.4 Hz, 1H), 3.53 (d, J = 12.2 Hz, 1H), 3.43 (d, J = 7.1 Hz, 2H),

3.26−3.14 (m, 1H), 3.04 (t, J = 13.1 Hz, 2H), 2.84 (q, J = 7.7 Hz, 1H),

2.54 (t, J = 12.2 Hz, 1H), 2.25 (d, J = 14.7 Hz, 1H), 2.20−2.10 (m, 1H),

1.99−1.90 (m, 1H), 1.79−1.67 (m, 3H), 1.66 (s, 3H), 1.65−1.41 (m,

4H); ESI-MS m/z 496.47 [M + H]⁺.

rac-Methyl ((1S,2R)-2-((S)-Cyano(1-((1-(4-cyanophenyl)azetidin-

3-yl)methyl)piperidin-4-yl)(phenyl)methyl)cyclopentyl)carbamate

(41).²⁵Compound 41 was synthesized using the method described for

compound 102a starting from the intermediate 101a and 79. ¹H NMR

(400 MHz, MeOH-d₄) δ 7.52 (d, J = 7.2 Hz, 2H), 7.47 (d, J = 8.8 Hz,

2H), 7.45−7.36 (m, 3H), 6.47 (d, J = 8.8 Hz, 2H), 4.17−4.12 (m, 2H),

3.93−3.88 (m, 1H), 3.73−3.70 (m, 2H), 3.60−3.51 (m, 2H), 3.44 (s,

3H), 3.42 (s, 2H), 3.23−3.17 (m, 1H), 3.07−2.99 (m, 2H), 2.89−2.83

(m, 1H), 2.50 (t, J = 10.2 Hz, 1H), 2.28 (d, J = 14.6 Hz, 1H), 2.16−2.10

N

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(m, 1H), 1.94 (d, J = 14.3 Hz, 1H), 1.79−1.44 (m, 7H); ESI-MS m/z

512.42 [M + H]⁺.

3H), 3.00 (t, J = 5.9 Hz, 2H), 2.96−2.89 (m, 1H), 2.86−2.74 (m, 2H),

2.41−2.30 (m, 1H), 1.98 (d, J = 13.1 Hz, 1H), 1.89−1.79 (m, 1H),

1.70−1.39 (m, 7H), 1.35−1.18 (m, 3H); ESI-MS m/z 285.2 [M + H]⁺.

1-(1-(Azetidin-3-ylmethyl)piperidin-4-yl)-1-cyclopentyl-1,2,3,4-

tetrahydroisoquinoline (55). 1-Boc-azetidine-3-carboxaldehyde (920

mg, 4.96 mmol) was added to a solution of crude 48 (1.24 mmol) in

DCM/AcOH (1:1, 6 mL) and stirred. After 10 min, NaBH(OAc)₃

(2.10 g, 9.92 mmol) was slowly added to the reaction. After standing

overnight, the reaction was slowly quenched with saturated NaHCO₃,

extracted with EtOAc, dried over Na₂SO₄, ﬁltered, and concentrated to

produce crude Boc-protected-55. To remove the Boc protecting group,

the crude product was dissolved in TFA and stirred. After 10 min, the

TFA was removed in vacuo, the crude product was puriﬁed by reverse

phase preparative HPLC, and the pure product was lyophilized to give

55-TFA salt (569 mg) as white solid. ¹H NMR (400 MHz, MeOH-d₄) δ

7.47−7.39 (m, 1H), 7.39−7.27 (m, 3H), 4.27−4.13 (m, 2H), 4.08−

3.94 (m, 2H), 3.62 (d, J = 12.4 Hz, 1H), 3.57−3.37 (m, 6H), 3.14−2.93

(m, 4H), 2.80−2.58 (m, 2H), 2.24 (d, J = 14.3 Hz, 1H), 2.05−1.40 (m,

10H), 1.22−1.07 (m, 1H).

1-(Cyclopropylsulfonyl)-p-ﬂuorobenzene (67). Bromocyclopro-

pane (2.06 mL, 24.75 mmol) was added to a solution of p-

ﬂuorobenzenethiol (3.0 g, 23.41 mmol) and sodium tert-butoxide

(3.15 g, 37.77 mmol) in 60 mL of DMSO, and the reaction was heated

to 80 °C. After reacting overnight, the system was cooled, quenched

with saturated NH₄Cl, and extracted with Et₂O. The combined organic

layers were washed twice with saturated NaHCO₃, once with brine,

dried over Na₂SO₄, ﬁltered, and concentrated to produce crude 62

(2.20 g) that was used without further puriﬁcation. m-Chloroperben-

zoic acid (7.34 g, 32.74 mmol, of 77 wt %) was added to a solution, at 0

°C, of crude 62 in DCM (50 mL). After standing overnight at rt, the

reaction was quenched with saturated NaHCO₃, extracted with EtOAc,

and puriﬁed by column chromatography to give 67 (2.11 g, 45%) as a

white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.96−7.88 (m, 2H), 7.28−

7.19 (m, 2H), 2.45 (tt, J = 7.9, 4.8 Hz, 1H), 1.39−1.31 (m, 2H), 1.08−

1.01 (m, 2H).

1-(Cyclobutylsulfonyl)-p-ﬂuorobenzene (68). Compound 68 was

prepared according to the procedure used to make compound 67. ¹H

NMR (400 MHz, CDCl₃) δ 7.91−7.87 (m, 2H), 7.26−7.21(m, 2H),

3.83−3.75 (m, 1H), 2.61−2.51 (m, 2H), 2.25−2.15 (m, 2H), 2.05−

1.94 (m, 2H).

1-(Cyclopentylsulfonyl)-p-ﬂuorobenzene (69). Compound 69 was

prepared according to the procedure used to make compound 67. ¹H

NMR (400 MHz, CDCl₃) δ 7.98−7.87 (m, 2H), 7.29−7.19 (m, 2H),

3.47 (tt, J = 8.8, 7.2 Hz, 1H), 2.12−1.98 (m, 2H), 1.95−1.71 (m, 4H),

1.67−1.54 (m, 2H).

1-(Cyclohexylsulfonyl)-p-ﬂuorobenzene (70). Compound 70 was

prepared according to the procedure used to make compound 67. ¹H

NMR (400 MHz, CDCl₃) δ 7.90−7.86 (m, 2H), 7.26−7.22 (m, 2H),

2.93−2.85 (m, 1H), 2.08−2.05 (m, 2H), 1.89−1.85 (m, 2H), 1.69−

1.66 (m, 1H), 1.44−1.34 (m, 2H), 1.28−1.11 (m, 3H).

rac-(1S,2R)-2-((S)-2-Methyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)-

phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroiso-

quinolin-4-yl)cyclopentyl Methyl Carbamate 42 (M-89). Methyl

isocyanate (0.3 mmol) was added to a solution of compound 110 (30

mg, 0.05 mmol) and NEt₃(28 μL, 0.2 mmol) in DCM (1 mL), then

stirred at rt for 4 h. The reaction was diluted with 3:1 MeOH/H₂O

(10% TFA) and puriﬁed by preparative HPLC. The pure fractions were

combined, concentrated, diluted with H₂O, frozen, and lyophilized to

give 42 (M-89) as a yellow powder. ¹H NMR (400 MHz, MeOH-d₄) δ

8.75 (d, J = 5.6 Hz, 2H), 7.83 (d, J = 5.5 Hz, 2H), 7.75 (d, J = 8.6 Hz,

2H), 7.54 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.34 (t, J = 7.4

Hz, 1H), 7.24 (d, J = 7.4 Hz, 1H), 6.48 (d, J = 8.7 Hz, 2H), 5.17−5.04

(m, 1H), 4.44−4.25 (m, 3H), 4.15 (t, J = 8.2 Hz, 2H), 3.77−3.67 (m,

2H), 3.55 (d, J = 12.2 Hz, 1H), 3.38 (d, J = 7.3 Hz, 2H), 3.24−3.14 (m,

4H), 3.05−2.84 (m, 4H), 2.28 (s, 3H), 2.14−1.99 (m, 4H), 1.92−1.55

(m, 8H); ESI-MS m/z 658.4 [M + H]⁺.

1-Benzyl-N-phenethylpiperidine-4-carboxamide (45). DMF (one

drop) was added to a suspension of 1-benzylpiperidine-4-carboxylic

acid (15 g, 68.4 mmol) in DCM (100 mL), and this was followed by the

dropwise addition of oxalyl chloride (7 mL, 82 mmol). The mixture was

stirred for 4 h, then concentrated under vacuum, aﬀording an acid

chloride, which was rediluted with DCM (100 mL). Triethylamine

(23.8 mL, 171 mmol) was added to the mixture, followed by 2-

phenylethan-1-amine (8.29 g, 68.4 mmol) at 0 °C. The reaction mixture

was stirred at rt overnight. The organic phase was washed with brine,

dried over Na₂SO₄, and evaporated. The crude product was puriﬁed by

recrystallization in DCM to obtain the title compound (14.9 g, 68%).

¹H NMR (400 MHz, CDCl₃) δ 7.34−7.20 (m, 8H), 7.19−7.14 (m,

2H), 5.45 (s, 1H), 3.57−3.40 (m, 4H), 2.90 (dt, J = 11.2, 2.5 Hz, 2H),

2.80 (t, J = 6.8 Hz, 2H), 2.08−1.88 (m, 3H), 1.83−1.61 (m, 4H); ESI-

MS m/z 323.2 [M + H]⁺.

1-(1-Benzylpiperidin-4-yl)-3,4-dihydroisoquinoline (46). Phos-

phoryl chloride (3.3 mL, 35.4 mmol) and phosphorus pentoxide

(3.35 g, 23.6 mmol) were added to a solution of the intermediate 45

(3.82 g, 11.9 mmol) in toluene (15 mL). The reaction mixture was

stirred under reﬂux overnight, then the mixture was quenched and

basiﬁed with saturated NaHCO₃, extracted with DCM, and dried over

Na₂SO₄. The solvent was evaporated to give the title compound (3.5 g,

97%), which was used in the next step without further puriﬁcation; ESI-

MS m/z 305.3 [M + H]⁺.

1-(1-Benzylpiperidin-4-yl)-1-cyclopentyl-1,2,3,4-tetrahydroiso-

quinoline (47). Boron triﬂuoride diethyl etherate (0.6 mL) was added

to a solution of the intermediate 46 (0.5 g, 1.64 mmol) at 0 °C under a

N₂atmosphere. After the mixture was stirred for 5 min, a 2 M solution

of cyclopentyl magnesium bromide in Et₂O (3.3 mL, 6.6 mmol) was

added into the mixture dropwise at 0 °C. The reaction mixture was

stirred overnight, then warmed slowly to rt. The reaction was quenched

with saturated aqueous NH₄Cl, extracted with DCM, dried over

Na₂SO₄, and the solvent was evaporated. The crude product was

puriﬁed by preparative HPLC to give the title compound as its TFA salt

(740 mg, 60%). ¹H NMR (400 MHz, CDCl₃) δ 7.48−7.35 (m, 5H),

7.32−7.28 (m, 2H), 7.21−7.13 (m, 2H), 4.11 (dd, J = 25.6, 12.9 Hz,

2H), 3.65−3.37 (m, 4H), 3.16−2.91 (m, 2H), 2.86−2.75 (m, 2H), 2.69

(t, J = 11.5 Hz, 1H), 2.40 (d, J = 11.7 Hz, 1H), 2.27−2.12 (m, 2H),

1.96−1.80 (m, 2H), 1.75−1.57 (m, 4H), 1.46 (dd, J = 33.9, 3.1 Hz,

2H), 1.37−1.25 (m, 1H), 1.22−1.09 (m, 1H); ESI-MS m/z 375.2 [M +

H]⁺.

1-Cyclopentyl-1-(piperidin-4-yl)-1,2,3,4-tetrahydroisoquinoline

(48). Compound 47 (588 mg, 1.24 mmol) was dissolved in MeOH (5

mL), and the solution was vacuumed brieﬂy and then put under a N₂

atmosphere. This was repeated three times, then Pd/C (10% w/w)

(150 mg) was quickly added to the solution, which was again vacuumed

and put under a N₂atmosphere. The solution was brieﬂy vacuumed to

remove the N₂atmosphere and then put under H₂atmosphere. This

was repeated three times. After 30 min, the reaction was ﬁltered through

celite and concentrated to give a crude product (48) that was used

without further puriﬁcation. ¹H NMR (400 MHz, DMSO) δ 7.43−7.38

(m, 1H), 7.34−7.26 (m, 3H), 3.49−3.41 (m, 1H), 3.30 (t, J = 11.6 Hz,

4-((p-Fluorophenyl)sulfonyl)pyridine (71). 4-Bromopyridine·HCl

(4.02 g, 20.68 mmol) was added to a solution of p-ﬂuorobenzenethiol

(2.41 g, 18.80 mmol) and K₂CO₃(7.78 g, 56.4 mmol) in DMSO (20

mL), and the reaction was maintained at 110 °C overnight. Then, the

reaction was cooled, quenched with saturated NH₄Cl, and extracted

with EtOAc. The combined organic layers were washed twice with

saturated NaHCO₃, once with brine, dried over Na₂SO₄, ﬁltered, and

concentrated to produce crude 66 (4.01 g, quantitative yield) that was

used without further puriﬁcation. Oxone monopersulfate (15.03 g,

48.90 mmol) was added to a solution of crude compound 66 in

Me₂CO/H₂O (5:1, 84 mL). After standing overnight, the reaction was

quenched with saturated NaHCO₃, extracted with EtOAc, and puriﬁed

by column chromatography to give 71 (quantitative yield) as a white

solid. ¹H NMR (400 MHz, CDCl₃) δ 8.88−8.81 (m, 2H), 8.03−7.95

(m, 2H), 7.78−7.73 (m, 2H), 7.28−7.21 (m, 2H).

(1-(4-(Pyridin-4-ylsulfonyl)phenyl)azetidin-3-yl)methyl Methane-

sulfonate (78). Compound 78 was prepared according to the

procedure described for compound 79. ¹H NMR (400 MHz, CDCl₃)

δ 8.77 (d, J = 6.0 Hz, 2H), 7.75 (d, J = 8.9 Hz, 2H), 7.70 (d, J = 6.1 Hz,

2H), 6.40 (d, J = 8.9 Hz, 2H), 4.43 (d, J = 6.6 Hz, 2H), 4.10 (t, J = 8.2

O

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Hz, 2H), 3.81 (dd, J = 8.2, 5.0 Hz, 2H), 3.19 (dddd, J = 11.5, 7.9, 6.6, 2.7

Hz, 1H), 3.04 (s, 3H).

Na₂SO₄, ﬁltered and concentrated to give 1.77 g of the crude methyl

carbamate (86) that was used without further puriﬁcation. Poly-

phosphoric acid (PPA) (20 mL) was added to crude 86, and the

reaction heated to 150 °C. After 1.5 h, UPLC indicated that the reaction

was complete, and it was cooled to a moderate temperature, diluted

with H₂O, slowly quenched with saturated NaHCO₃, extracted with

EtOAc, dried over Na₂SO₄, ﬁltered, and concentrated to give crude 87

(1.82 g) that was used without further puriﬁcation. Red-Al (3.2 M in

toluene, 7.3 mL) was added dropwise to a solution, at rt, of crude 87

(1.82 g, 4.69 mmol) in toluene (30 mL) and stirred. After 30 min, the

reaction was quenched by the dropwise addition of 2 M NaOH, and the

aqueous solution was extracted with EtOAc and concentrated. The

crude 88 was puriﬁed by reverse phase preparative HPLC, and the pure

compound was lyophilized to produce 88-TFA salt as a white powder.

¹H NMR (400 MHz, MeOH-d₄) δ 7.60−7.20 (m, 9H), 4.33−4.16 (m,

(1-(4-Cyanophenyl)azetidin-3-yl)methyl Methanesulfonate

(79).²⁵K₂CO₃(8.8 g, 63.7 mmol) was added to a solution of p-

ﬂuorobenzonitrile (2.6 g, 21.2 mmol) and azetidin-3-ylmethanol

hydrochloride (3.4 g, 27.6 mmol) in DMSO. The mixture was stirred

at 80 °C overnight. Then, the reaction mixture was cooled to rt, poured

into ice, and extracted twice with EtOAc. The combined organic

solution was dried over Na₂SO₄, ﬁltered, and the solvent was

evaporated in vacuo. The residue was dissolved in DCM (100 mL),

and Et₃N (8.9 mL, 63.6 mmol) and methanesulfonyl chloride (2.5 mL,

31.8 mmol) were added dropwise at 0 °C. The reaction mixture was

allowed to warm to rt and stirred for 5 h. Then, the reaction mixture was

quenched with saturated NaHCO₃, washed with brine, dried over

Na₂SO₄, and the solvent was evaporated under vacuum. The residue

was puriﬁed by ﬂash chromatography to obtain the title compound as a

white solid (2.5 g, 44%). ¹H NMR (400 MHz, CDCl₃) δ 7.39 (d, J = 8.8

Hz, 2H), 6.31 (d, J = 8.8 Hz, 2H), 4.38 (d, J = 6.7 Hz, 2H), 4.02 (t, J =

8.2 Hz, 2H), 3.74−3.70 (m, 2H), 3.16−3.08 (m, 1H), 2.99 (s, 3H);

ESI-MS m/z 267.11 [M + H]⁺.

2-(1-Benzylpiperidin-4-ylidene)-2-phenylacetonitrile (82). So-

dium methoxide (25 wt % in MeOH) (46.8 mL, 205 mmol) was

added to a solution of 1-benzylpiperidin-4-one (32.3 g, 171 mmol) and

2-phenylacetonitrile (20 g, 171 mmol) in anhydrous MeOH (200 mL)

under N₂, and the mixture was stirred under reﬂux overnight. Then, the

reaction mixture was cooled to rt and poured into ice (200 g). The

resulting mixture was extracted with EtOAc. The separated organic

layer was dried with Na₂SO₄, ﬁltered, and the solvent was evaporated in

vacuum to yield the title compound (48 g, 95%); ESI-MS m/z 289.1 [M

+ H]⁺.

3H), 3.58−3.37 (m, 4H), 3.11−2.88 (m, 2H), 2.62−2.45 (m, 1H),

2.29−2.17 (m, 1H), 2.13 (d, J = 14.1 Hz, 1H), 1.91−1.41 (m, 9H),

1.36−1.17 (m, 2H), 1.10 (p, J = 9.0 Hz, 1H).

tert-Butyl 4-(1-Benzylpiperidin-4-yl)-4-cyclopentyl-3,4-dihydroi-

soquinoline-2(1H)-carboxylate (89). At 0 °C, Et₃N (446 μL, 3.21

mmol) followed by Boc₂O (350 mg, 1.60 mmol) were added to a

stirring solution of 88 (300 mg, 0.802 mmol) in DCM (5 mL), then

allowed to warm to rt. After 1 h, the solvent was removed in vacuo then

puriﬁed by ﬂash column chromatography to produce 288 mg of 89. ¹H

NMR (400 MHz, MeOH-d₄) δ 7.41−7.25 (m, 6H), 7.23−7.08 (m,

3H), 4.65−4.33 (m, 2H), 3.74−3.52 (m, 4H), 3.16−2.88 (m, 2H),

2.52−2.35 (m, 1H), 2.34−1.82 (m, 4H), 1.73−1.56 (m, 2H), 1.50 (s,

9H), 1.48−1.35 (m, 6H), 1.35−1.20 (m, 2H), 1.06−0.92 (m, 1H).

tert-Butyl 4-Cyclopentyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)-

azetidin-3-yl)methyl)-piperidin-4-yl)-3,4-dihydroisoquinoline-

2(1H)-carboxylate (90). Compound 89 (288 mg, 0.608 mmol) was

dissolved in MeOH (5 mL), and the solution was vacuumed brieﬂy then

put under a N₂atmosphere. This was repeated three times, then Pd/C

(10% w/w, 200 mg) was quickly added to the solution that was

vacuumed and put under N₂atmosphere. The solution was brieﬂy

vacuumed to remove the N₂atmosphere then put under a H₂

atmosphere, this was repeated three times. After 30 min, the reaction

was ﬁltered through celite and concentrated to give crude debenzylated-

89 that was redissolved in MeCN (10 mL). K₂CO₃(420 mg, 3.04

mmol), 78 (301 mg, 0.790 mmol), KI (cat.) were added to the crude

solution which was then reﬂuxed. After overnight reﬂux, the reaction

was cooled, ﬁltered through celite, concentrated, and puriﬁed by ﬂash

column chromatography to produce 90 (176 mg, 43%). ¹H NMR (400

MHz, MeOH-d₄) δ 8.74 (dd, J = 4.6, 1.6 Hz, 2H), 7.80 (dd, J = 4.5, 1.6

Hz, 2H), 7.72 (d, J = 8.9 Hz, 2H), 7.40−7.29 (m, 1H), 7.25−7.07 (m,

3H), 6.46 (d, J = 8.9 Hz, 2H), 4.63−4.35 (m, 2H), 4.10 (t, J = 8.1 Hz,

2H), 3.70−3.55 (m, 4H), 3.17−2.93 (m, 3H), 2.87−2.69 (m, 2H),

2.55−2.38 (m, 1H), 2.36−2.06 (m, 2H), 2.06−1.84 (m, 2H), 1.76−

1.57 (m, 2H), 1.50 (s, 9H), 1.48−1.36 (m, 6H), 1.36−1.17 (m, 2H),

1.08−0.93 (m, 1H).

2-(1-Benzylpiperidin-4-yl)-2-phenylacetonitrile (83).²⁵Sodium

borohydride (12.6 g, 333 mmol) was added to a solution of

intermediate 82 (48 g, 166 mmol) in MeOH (100 ml). The mixture

was stirred at rt overnight. Then, a mixture of H₂O and ice (200 ml) was

added; a light yellow precipitate was formed and collected by ﬁltration.

The residue was washed with H₂O and dried in vacuum to yield a light

yellow product (38 g, 79%). ¹H NMR (400 MHz, CDCl₃) δ 7.31−7.25

(m, 3H), 7.24−7.20 (m, 5H), 7.20−7.17 (m, 2H), 3.52 (d, J = 7.6 Hz,

1H), 3.43 (s, 2H), 2.90−2.81 (m, 2H), 1.92−1.79 (m, 3H), 1.74−1.64

(m, 1H), 1.52−1.34 (m, 3H); ESI-MS m/z 291.19 [M + H]⁺.

2-(1-Benzylpiperidin-4-yl)-2-cyclopentyl-2-phenylacetonitrile

(84).²⁵LiHMDS (1 M in THF, 20.66 mL, 20.66 mmol) was added

dropwise at −78 °C to a solution of 83 (3 g, 10.33 mmol) in dry THF

(100 mL) and stirred. After 30 min at −78 °C, cyclopentyl bromide

(3.32 mL, 30.99 mmol) was added dropwise, and the reaction was

allowed to slowly warm to rt. After overnight at rt, the reaction was

quenched with saturated NH₄Cl, extracted with EtOAc, concentrated,

and puriﬁed by column chromatography to produce 3.64 g of

1

compound 84 as an oil. H NMR (400 MHz, CDCl₃) δ 7.31−7.24

(m, 4H), 7.23−7.15 (m, 6H), 3.39 (s, 2H), 2.88−2.83 (m, 2H), 2.71−

2.62 (m, 1H), 1.96−1.78 (m, 5H), 1.70−1.64 (m, 1H), 1.58−1.49 (m,

4H), 1.46−1.41 (m, 2H), 1.26−1.11 (m, 3H); ESI-MS m/z 359.32 [M

+ H]⁺.

tert-Butyl ((1S,2S)-2-Hydroxycyclopentyl)carbamate (92). The

title compound was prepared, as reported previously.²⁵¹H NMR

(400 MHz, CDCl₃) δ 4.66 (s, 1H), 4.00−3.96 (m, 2H), 3.66−3.59 (m,

1H), 2.12−2.06 (m, 1H), 2.05−1.98 (m, 1H), 1.81−1.74 (m, 1H),

1.70−1.63 (m, 2H), 1.45 (s, 9H), 1.38−1.31 (m, 1H).

tert-Butyl 6-Azabicyclo[3.1.0]hexane-6-carboxylate (94). The title

compound was prepared, as reported previously.²⁵¹H NMR (400

MHz, CDCl₃) δ 2.9 (s, 2H), 2.11−2.05 (m, 2H), 1.66−1.56 (m, 3H),

1.46 (s, 9H), 1.30−1.13 (m, 1H).

rac-(S)-2-(1-Benzylpiperidin-4-yl)-2-((1R,2S)-2-hydroxycyclopen-

tyl)-2-phenylacetonitrile (95a) and rac-(R)-2-(1-Benzylpiperidin-4-

yl)-2-((1R,2S)-2-hydroxycyclopentyl)-2-phenylacetonitrile (95b). To

a solution of 2-(1-benzylpiperidin-4-yl)-2-phenylacetonitrile (83) (1.4

g, 4.8 mmol) in dry THF (20 mL) was added dropwise LiHMDS (1 M

in THF, 14.3 mL, 14.3 mmol) at 0 °C under N₂. After stirring for 30

min, cyclopentene oxide (1.7 mL, 19.0 mmol) was added dropwise, and

the reaction mixture was allowed to warm slowly to rt. After stirring

overnight, the reaction was quenched with saturated NH₄Cl and

concentrated to remove the THF. The resulting mixture was extracted

with DCM twice, dried over Na₂SO₄, ﬁltered, and concentrated under

2-(1-Benzylpiperidin-4-yl)-2-cyclopentyl-2-phenylethan-1-amine

(85). DIBALH (25 wt % in toluene, 29 mL, 51.72 mmol) was added

dropwise to a solution of 84 (3.0 g, 8.37 mmol) in toluene (60 mL) and

stirred at rt. After 1 h, the reaction was quenched by the dropwise

addition of 2 M NaOH, and the aqueous was extracted with EtOAc and

concentrated. The crude imine was redissolved in MeOH, NaBH₄(786

mg, 20.68 mmol) was slowly added, and the reaction was stirred. After

overnight, the reaction was quenched with H₂O, extracted with EtOAc,

dried over Na₂SO₄, ﬁltered through celite, and concentrated to produce

crude compound 85 (3.15 g) that was used in the next step without

further puriﬁcation.

4-(1-Benzylpiperidin-4-yl)-4-cyclopentyl-1,2,3,4-tetrahydroiso-

quinoline (88). Methyl chloroformate (0.600 mL, 7.76 mmol) was

added to a solution, at 0 °C, of crude 85 (crude, 5.17 mmol) and Et₃N

(1.4 mL, 10.34 mmol) in DCM (50 mL) and stirred. After 30 min at 0

°C, the reaction was stirred at rt. After 30 min at rt, the reaction was

quenched with H₂O and brine, extracted with EtOAc, dried over

P

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vacuum. The residue was puriﬁed by ﬂash chromatography to obtain a

diasteromeric mixture (95a/95b). The mixture was further separated

by reverse phase preparative HPLC and basiﬁed with saturated

NaHCO₃to give the title compounds 95a (0.56 g, 32%) and 95b (0.5 g,

28%) as colorless oils. Data for 95a: ¹H NMR (400 MHz, MeOH-d₄) δ

7.50−7.47 (m, 2H), 7.42−7.38 (m, 2H), 7.36−7.32 (m, 1H), 7.30−

7.21 (m, 5H), 3.84−3.80 (m, 1H), 3.45 (s, 2H), 2.95−2.86 (m, 2H),

2.83−2.77 (m, 1H), 2.14−2.02 (m, 4H), 2.00−1.93 (m, 1H), 1.78−

1.57 (m, 5H), 1.54−1.44 (m, 1H), 1.31−1.19 (m, 3H); ESI-MS m/z

mmol) and HATU (320 mg, 0.844 mmol) were added at 0 °C to a

solution of intermediate 99a (200 mg, 0.703 mmol) and p-

bromobenzoic acid (169 mg, 0.844 mmol) in DCM. The reaction

mixture was stirred at rt for 3 h. Then, the mixture was extracted with

DCM, washed with brine, dried over Na₂SO₄, and the solvent was

evaporated under vacuum. The residue was puriﬁed by ﬂash

chromatography to obtain the title compound as a white solid (210

mg, 64%). ¹H NMR (400 MHz, MeOH-d₄) δ 7.59 (d, J = 8.3 Hz, 2H),

7.50 (d, J = 7.5 Hz, 2H), 7.43 (t, J = 7.1 Hz, 2H), 7.38−7.35 (m, 1H),

7.22 (d, J = 8.5 Hz, 2H), 4.69−4.60 (m, 1H), 3.87−3.84 (m, 1H),

3.75−3.64 (m, 1H), 3.17−3.09 (m, 1H), 2.86−2.76 (m, 2H), 2.54−

2.47 (m, 1H), 2.27−2.08 (m, 2H), 1.80−1.43 (m, 6H), 1.18−1.13 (m,

2H); ESI-MS m/z 467.27, 469.28 [M + H]⁺.

rac-(S)-2-((1R,2S)-2-(Benzyloxy)cyclopentyl)-2-(1-benzylpiperi-

din-4-yl)-2-phenylacetonitrile (104). NaH (65 wt % in oil, 263 mg,

6.59 mmol) was added to a solution of 95a (821 mg, 2.20 mmol), and

tetrabutylammonium iodide (8 mg, 0.220 mmol) dissolved in dry

THF/PhCH₃(1:1, 10 mL) at 0 °C and stirred. After 30 min at 0 °C,

benzyl bromide (0.286 mL, 2.63 mmol) was added dropwise, and the

reaction was allowed to warm to rt. After overnight at rt, the reaction

was quenched with saturated NH₄Cl, extracted with EtOAc,

concentrated, and puriﬁed by column chromatography to produce

104 (643 mg). ¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J = 7.8 Hz, 2H),

7.37−7.17 (m, 11H), 7.08 (dd, J = 7.4, 2.1 Hz, 2H), 4.20 (d, J = 11.0 Hz,

1H), 3.79 (d, J = 10.9 Hz, 1H), 3.58 (ddd, J = 5.8, 3.7, 2.1 Hz, 1H),

3.48−3.35 (m, 2H), 2.97−2.81 (m, 3H), 2.09−2.00 (m, 1H), 2.00−

1.91 (m, 2H), 1.89−1.80 (m, 3H), 1.79−1.57 (m, 5H), 1.31−1.07 (m,

2H).

rac-(S)-2-((1R,2S)-2-(Benzyloxy)cyclopentyl)-2-(1-benzylpiperi-

din-4-yl)-2-phenylethan-1-amine (105). DIBALH (3.92 mL, 6.89

mmol) was added dropwise to a solution of 104 (640 mg, 1.38 mmol)

in toluene (6 mL) and stirred at rt. After 1 h, the reaction was quenched

by the dropwise addition of 2 M NaOH, and the aqueous solution was

extracted with EtOAc, dried over Na₂SO₄, ﬁltered through celite, and

concentrated to produce the crude compound 105 (691 mg) that was

used in the next step without further puriﬁcation.

rac-(S)-4-((1R,2S)-2-(Benzyloxy)cyclopentyl)-4-(1-benzylpiperi-

din-4-yl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (108). Methyl

chloroformate (0.166 mL, 2.14 mmol) was added at 0 °C to a solution

of crude 105 (501 mg, 1.07 mmol) and Et₃N (0.595 mL, 4.28 mmol) in

DCM (10 mL) and stirred. After 30 min at 0 °C, the reaction was stirred

at rt. After 30 min at rt, the reaction was quenched with H₂O and brine,

extracted with EtOAc, dried over Na₂SO₄, ﬁltered, and concentrated to

give crude 106 (561 mg) that was used without further puriﬁcation.

Crude 106 (561 mg) was dissolved in AcOH (5 mL), followed by the

addition of paraformaldehyde (3 equiv) and concentrated TFA (2 mL)

at rt. After overnight at rt, the reaction was slowly quenched with

saturated NaHCO₃, extracted with EtOAc, dried over Na₂SO₄, ﬁltered,

and concentrated to give crude 107 (572 mg) that was used without

further puriﬁcation.

1

375.33 [M + H]⁺; Data for 95b: H NMR (400 MHz, MeOH-d₄) δ

7.43−7.37 (m, 4H), 7.34−7.23 (m, 6H), 4.27−4.23 (m, 1H), 3.50 (s,

2H), 2.98−2.94 (m, 1H), 2.91−2.87 (m, 1H), 2.83−2.77 (m, 1H),

2.30−2.22 (m, 1H), 2.18−2.11 (m, 1H), 2.09−2.01 (m, 2H), 1.90−

1.79 (m, 2H), 1.75−1.61 (m, 3H), 1.60−1.49 (m, 1H), 1.30−1.11 (m,

4H).; ESI-MS m/z 375.33 [M + H]⁺.

Mixture of rac-tert-Butyl ((1S,2R)-2-((S)-(1-Benzylpiperidin-4-yl)-

(cyano)(phenyl)methyl)cyclopentyl)carbamate (96a) and rac-tert-

Butyl ((1S,2R)-2-((R)-(1-Benzylpiperidin-4-yl)(cyano)(phenyl)-

methyl)cyclopentyl)carbamate (96b). A mixture of 96a and 96b

was obtained, as described in our previous publication.²⁵Preparative

HPLC separated the diastereomers, and their data are as follows. 96a:

¹H NMR (400 MHz, MeOH-d₄) δ 7.55−7.33 (m, 10H), 4.25 (s, 2H),

3.89−3.80 (m, 1H), 3.52 (d, J = 12.6 Hz, 1H), 3.45 (d, J = 12.8 Hz, 1H),

3.06−2.94 (m, 2H), 2.85−2.75 (m, 1H), 2.50 (t, J = 10.8 Hz, 1H), 2.28

(d, J = 14.3 Hz, 1H), 2.15−2.03 (m, 1H), 1.89 (d, J = 14.4 Hz, 1H),

1.72−1.40 (m, 7H), 1.32 (s, 9H). 96b: ¹H NMR (400 MHz, MeOH-

d₄) δ 7.41 (dd, J = 19.2, 4.0 Hz, 10H), 4.19−3.98 (m, 3H), 3.45−3.34

(m, 2H), 2.95−2.80 (m, 2H), 2.58−2.45 (m, 1H), 2.21 (d, J = 13.8 Hz,

1H), 2.00−1.84 (m, 2H), 1.82−1.71 (m, 1H), 1.69−1.52 (m, 3H), 1.46

(s, 9H), 1.42−1.15 (m, 4H). ESI-MS m/z 474.50 [M + H]⁺.

rac-N-((1S,2R)-2-((S)-(1-Benzylpiperidin-4-yl)(cyano)(phenyl)-

methyl) cyclopentyl)-acetamide (97a). Compound 96a (20 mg, 0.042

mmol) was dissolved and stirred in TFA. After 15 min, the TFA was

removed in vacuo and the crude compound was redissolved in DCM (3

mL), cooled to 0 °C, then Et₃N and acetic anhydride were added, and

the mixture was stirred. After 30 min at rt, the solvent was removed, and

the crude product was puriﬁed by preparative HPLC to produce 97a as

the TFA salt (8 mg, 47%). ¹H NMR (400 MHz, MeOH-d₄) δ 7.56−

7.31 (m, 10H), 4.24 (s, 2H), 4.17 (q, J = 6.7 Hz, 1H), 3.51 (d, J = 13.7

Hz, 1H), 3.44 (d, J = 12.7 Hz, 1H), 3.03 (td, J = 12.9, 3.0 Hz, 2H), 2.81

(q, J = 7.9 Hz, 1H), 2.51 (tt, J = 12.2, 9.0, 3.4 Hz, 1H), 2.27−2.18 (m,

1H), 2.18−2.08 (m, 1H), 1.96−1.86 (m, 1H), 1.81−1.65 (m, 3H), 1.64

(s, 3H), 1.61−1.36 (m, 4H).

rac-Methyl ((1S,2R)-2-((S)-(1-Benzylpiperidin-4-yl)(cyano)-

(phenyl)methyl)-cyclopentyl)-carbamate (98a). Starting with dia-

stereomer 96a, the title compound was obtained, as previously

reported.²⁵¹H NMR (400 MHz, MeOH-d₄) δ 7.51−7.34 (m, 10H),

4.24 (s, 2H), 3.91−3.86 (m, 1H), 3.53−3.46 (m, 2H), 3.43 (s, 3H),

3.06−2.96 (m, 2H), 2.86−2.80 (m, 1H), 2.47 (t, J = 12.1 Hz, 1H), 2.26

(d, J = 14.4 Hz, 1H), 2.13−2.07 (m, 1H), 1.90 (d, J = 14.4 Hz, 1H),

1.79−1.72 (m, 1H), 1.70−1.60 (m, 2H), 1.58−1.40 (m, 4H); ESI-MS

m/z 432.53 [M + H]⁺.

rac-(S)-2-((1R,2S)-2-Hydroxycyclopentyl)-2-phenyl-2-(piperidin-

4-yl)acetonitrile (99a). Compound 99a was obtained according to the

same procedure used for intermediate 48. ¹H NMR (400 MHz, MeOH-

d₄) δ 7.51−7.48 (m, 2H), 7.43−7.39 (m, 2H), 7.36−7.32 (m, 1H),

3.86−3.82 (m, 1H), 3.06−2.98 (m, 2H), 2.85−2.80 (m, 1H), 2.65−

2.58 (m, 1H), 2.57−2.50 (m, 1H), 2.28−2.20 (m, 1H), 2.13−2.03 (m,

2H), 1.80−1.71 (m, 1H), 1.69−1.56 (m, 4H), 1.54−1.47 (m, 1H),

1.20−1.08 (m, 2H). ESI-MS m/z 285.23 [M + H]⁺.

rac-4-(3-((4-((S)-Cyano((1R,2S)-2-hydroxycyclopentyl)(phenyl)-

methyl)piperidin-1-yl)methyl)azetidin-1-yl)benzonitrile (102a).

102a was obtained using 99a and 79 according to the procedure

used to obtain compound 5. ¹H NMR (400 MHz, MeOH-d₄) δ 7.51 (d,

J = 7.2 Hz, 2H), 7.45−7.40 (m, 4H), 7.38−7.33 (m, 1H), 6.41 (d, J =

8.9 Hz, 2H), 4.07−4.03 (m, 2H), 3.85−3.82 (m, 1H), 3.60−3.56 (m,

2H), 3.00−2.93 (m, 3H), 2.85−2.79 (m, 1H), 2.65 (d, J = 7.2 Hz, 2H),

2.18−2.01 (m, 5H), 1.81−1.58 (m, 5H), 1.56−1.46 (m, 1H), 1.33−

1.22 (m, 2H); ESI-MS m/z 455.39 [M + H]⁺.

Red-Al (3.2 M in toluene, 3 equiv) was added dropwise to a solution,

at rt, of crude 107 in toluene (15 mL) and stirred. After 30 min, the

reaction was quenched by the dropwise addition of 2 M NaOH, and the

aqueous solution was extracted with EtOAc and concentrated. The

crude compound 108 was puriﬁed by reverse phase preparative HPLC,

and the pure compound was lyophilized to produce 108−TFA salt as a

white powder. ¹H NMR (400 MHz, MeOH-d₄) δ 7.64−7.10 (m, 14H),

4.71−4.48 (m, 1H), 4.48−4.26 (m, 3H), 4.28−4.05 (m, 3H), 3.90−

3.58 (m, 1H), 3.40 (d, J = 12.3 Hz, 2H), 3.22−2.67 (m, 5H), 2.66−2.49

(m, 1H), 2.48−2.38 (m, 1H), 2.37−2.08 (m, 1H), 2.05−1.06 (m,

10H).

rac-(1S,2R)-2-((S)-2-Methyl-4-(piperidin-4-yl)-1,2,3,4-tetrahydroi-

soquinolin-4-yl)-cyclopentan-1-ol (109). Compound 108 (110 mg,

0.223 mmol) was dissolved in MeOH (10 mL), and the solution was

vacuumed brieﬂy then put under a N₂atmosphere, this was repeated

three times. Pd/C (10% w/w, 100 mg) was quickly added to the

solution, which was then vacuumed and put under a N₂atmosphere.

The solution was brieﬂy vacuumed to remove the N₂atmosphere, then

put under a H₂atmosphere, this was repeated three times. After 4 h, the

rac-(S)-2-(1-(4-Bromobenzoyl)piperidin-4-yl)-2-((1R,2S)-2-hy-

droxycyclopentyl)-2-phenyl-acetonitrile (103). Et₃N (0.25 mL, 1.41

Q

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Journal of Medicinal Chemistry

Article

reaction was ﬁltered through celite and concentrated to give crude 109

(76 mg) that was used without further puriﬁcation.

In the cell growth experiments, cells were seeded in 96-well cell

culture plates at a density of 10 000−20 000 cells/well in 200 μL of

culture medium containing serial dilution of testing compounds. After 4

days of treatments, cell growth was measured by a lactate dehydrogen-

ase-based WST-8 assay (Dojindo Molecular Technologies) using a

Tecan Inﬁnite M-1000 multimode microplate reader (Tecan US,

Morrisville, NC). The WST-8 reagent was added to each well, and cells

were incubated for an additional 1−2 h and read at 450 nm. The

readings were normalized to the vehicle-treated cells, and the IC₅₀was

calculated by nonlinear regression analysis using the GraphPad Prism 6

software.

rac-(1S,2R)-2-((S)-2-Methyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)-

phenyl)azetidin-3-yl)-methyl)piperidin-4-yl)-1,2,3,4-tetrahydroiso-

quinolin-4-yl)cyclopentan-1-ol (110). Compound 78 (44 mg, 0.115

mmol), K₂CO₃(31 mg, 0.228 mmol), and KI (cat.) were added to a

solution of the intermediate 109 (18 mg, 0.057 mmol) in MeCN (3

mL). The mixture was reﬂuxed overnight. Then, the mixture was

ﬁltered through celite, concentrated, and puriﬁed with preparative

HPLC to give 110−TFA (29 mg) salt as a white solid.

Fluorescence Polarization (FP)-Based Binding Assay. Binding

aﬃnities of menin inhibitors to menin protein were determined using

our established ﬂuorescence polarization (FP) binding assay described

previously.^25,28The FP competitive binding assay was developed and

optimized using a novel FAM-labeled ﬂuorescent probe. Equilibrium

dissociation constant (K_d) value of FAM-probe to menin protein was

determined from protein saturation experiments by monitoring the

total ﬂuorescence polarization of mixtures composed with the

ﬂuorescent probe at a ﬁxed concentration and the protein with

increasing concentrations up to full saturation. FP values in

millipolarization units (mP) were measured using the Inﬁnite M-

1000 plate reader (Tecan US, Research Triangle Park, NC) in

Microﬂuor 1 96-well, black, v-bottom plates (Thermo Scientiﬁc,

Waltham, MA) at an excitation wavelength of 485 nm and an emission

wavelength of 530 nm. K_dvalue of FAM-probe, which was calculated by

ﬁtting the sigmoidal dose-dependent FP increases as a function of

protein concentrations using the GraphPad Prism 6.0 software

(GraphPad Software, San Diego, CA), is determined as 1.4 nM.

The IC₅₀and K_ivalues of compounds were determined in a

competitive binding experiment, with a ﬁnal concentration of the menin

protein at 4 nM and a ﬁnal probe concentration at 4 nM. Negative

controls containing protein/probe complex only (equivalent to 0%

inhibition) and positive controls containing only free probes

(equivalent to 100% inhibition) were included in each assay plate. FP

values were measured following the procedure described above. IC₅₀

values were determined by nonlinear regression ﬁtting of the

competition curves.²⁹

Cellular Thermal Shift Assay. MV4;11 or MOLM-13 cells were

treated with M-89 ranging from 3.7 to 300 nM for 1 h in six-well plates

at a density of 5 × 10⁶cells per well. Centrifugation followed by washing

with PBS produced cell pellets that were resuspended in 100 μL of PBS

containing Halt protease inhibitors, then transferred to PCR tubes, and

heated to 45 °C for 3 min. Two freeze−thaw cycles in liquid nitrogen

achieved cell lysis, and the lysates were clariﬁed by centrifugation at 15

000 rpm. In clean tubes, the transferred supernatants were mixed with

loading buﬀer, heated, and proteins were separated by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis. Membranes were blotted

with an antimenin antibody (CST, cat. No. 6891).

Real-Time PCR. MV4;11 or MOLM-13 cells were treated for 48 h

with vehicle or M-89 at 30, 100, and 300 nM concentrations for

MV4;11 cells or 100 nM, 300 nM, and 1 μM for MOLM-13 cells.

According to the manufacturer’s protocol, total RNA was isolated from

cells using the RNeasy kit (QIAGEN). A High Capacity cDNA Reverse

Transcription Kit (Applied Biosystems) was used to generate the

cDNA. Using TacMan gene expression assays (Applied Biosystems)

and primers speciﬁc for each gene real-time PCR ampliﬁcations of

Hoxa9 (Applied Biosystems, Hs00365956_m1), MEIS1 (Applied

Biosystems, Hs00180020_m1) and GAPDH (Applied Biosystems,

Hs02786624_g1) genes were carried out. Using the comparative cycle

threshold (Ct) method, the relative quantiﬁcation of each gene

transcript was calculated and the results were presented as relative

expression to vehicle treatment after normalization to the internal

control of GAPDH.

Biolayer Interferometry (BLI) Binding Assay. Biotinylation of

puriﬁed recombinant menin protein was accomplished by using the

Thermo EZ-Link long-chain biotinylation reagent. Menin protein and

biotinylation reagent were mixed with 1:1 molar ratio in phosphate-

buﬀered saline (PBS) at 4 °C. This reaction was incubated at 4 °C for

2h. The reaction mixture was then dialyzed using Fishersci 10K

MWCO dialysis cassettes to remove the unreacted biotinylation

reagent.

BLI binding assays were performed in 96-well microplates at room

temperature with continuous 1000 rpm shaking using the Octet Red 96

system (ForteBio, Menlo Park, CA). PBS with 0.1% bovine serum

albumin, 0.01% Tween-20, and 2% DMSO were used as the assay

buﬀer. Biotinylated menin protein was tethered on super streptavidin

(SSA) biosensors (ForteBio) by dipping sensors into 200 μL per well

10 μg/mL protein solutions. Average saturation response level of 8−10

nm was achieved in 20 min. The measurement processes were all under

computer control. Program procedures were established as follows: For

the initial step, biosensors were washed in assay buﬀer for 60 sec to form

a baseline; the biosensors labeled with biotin-menin were exposed to

100 nM compounds for association and were monitored for 1200 s, and

then, the biosensors were moved back into assay buﬀer to disassociate

for another 1800 s. Data were ﬁt globally and generated automatically

by the Octet User software (version 9.0; ForteBio).²⁵

Cell Growth Inhibition Assay. The human acute leukemia

MV4;11 cell line and HL-60 were purchased from the American

Type Culture Collection, and the human acute leukemia MOLM-13

cell line was purchased from the DSMZ German cell bank. In all

experiments, cultured human leukemia cells were used within 2 months

of thawing fresh vials. Cells were cultured in Roswell Park Memorial

Institute 1640 media (MOLM-13) or Iscove’s modiﬁed Dulbecco’s

media (MV4;11 and HL-60) supplemented with 10% fetal bovine

serum and 1% penicillin, streptomycin at 37 °C in a humidiﬁed

atmosphere containing 5% CO₂in air.

Cell Apoptosis and Diﬀerentiation Analysis by Flow

Cytometry. Flow cytometry was used to analyze eﬀects of M-89 on

apoptosis (annexin V and propidium iodide staining) or cell

diﬀerentiation using CD11b expression as an indicator. Cells were

treated with M-89 at the indicated concentrations for 24 or 48 h,

collected, stained with an apoptosis assay kit (Roche cat. No. 11 988

549 001) or a phycoerythrin-anti-mouse/human CD11b antibody

(BioLegend cat. No 101208), and analyzed by ﬂow cytometry.

In Vivo Pharmacodynamic Assay. All animal experiments were

performed under the guidelines of the University of Michigan

Committee for Use and Care of Animals using an approved animal

protocol. Female immunodeﬁcient (severe combined immunodeﬁ-

cient) mice obtained from Charles River were injected subcutaneously

in the right ﬂank with 5 million MV4;11 cells in a 5 mg/mL solution of

Matrigel. When tumors reached 100 mm³, mice were dosed by

intraperitoneal injection with either vehicle or M-89 at 50 mg/kg daily

for 3 days. Tumors were harvested at 6, 24, or 48 h after the third dose of

M-89 with three animals per time point. Tumors were immediately

frozen in liquid nitrogen, ground into a ﬁne powder, placed on dry ice,

and stored at −80°C for analysis. Expression levels of MEIS1 and

Hoxa9 were analyzed by RT-PCR, as described above.

Statistical analysis and graph creation were performed in the

GraphPad Prism version 7 for Windows (GraphPad Software, La Jolla

California), using t-test (nonparametric test) to calculate signiﬁcant

levels between treated groups and control groups. ** (p < 0.01).

Determination of the Co-crystal Structure of M-89 with

Recombinant Human Menin Protein. Menin (residues 2-610

containing a deletion from 460-519) was puriﬁed, as previously

described.²⁵For crystallization, menin (25 mg/mL in 20 mM Tris 8.0,

150 mM NaCl and 5 mM dithiothreitol) was incubated for 1 h at 4 °C in

a 1:1 molar ratio with M-89. Crystals grew at 4 °Ç in sitting drop vapor

diﬀusion experiments over wells containing 2.0 M NaCl, 90.9 M bis(2-

hydroxyethyl)amino−tris(hydroxymethyl)methane (Bis−Tris) pH

R

DOI: 10.1021/acs.jmedchem.9b00021

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

6.5, 0.182 M MgCl₂, and 9 mM Pr acetate. Crystals were annealed and

cryoprotected through serial soaking in solutions containing 1, 2, 3, 4,

and 5 M sodium formate in 0.1 M Bis−Tris pH 6.5, 2.2 M NaCl, 0.2 M

MgCl₂, and12 mM Pr acetate. Diﬀraction data were collected on at the

Advanced Photon Source LS-CAT 21-ID-G beamline at Argonne

National Laboratory with a wavelength of 0.9786 Å and processed with

HKL2000.³⁰The structure of menin bound to M-89 was solved by

molecular replacement (Molrep)³¹using the apo menin structure

(PDB ID 3U84) as the search model. The menin/M-89 co-crystal

structure was produced via iterative rounds of electron density ﬁtting

and structural reﬁnement using Coot³²and Buster,³³respectively. The

coordinates and restraint ﬁles for the ligand were created from SMILES

in Grade.³³The initial Fo−Fc electron density map showed the

presence of M-89 bound in the active site (Figure S2). The following

regions were disordered in the structure: 71-73, 386-401, 528-547, and

582-610. Data collection and structural reﬁnement statistics are shown

in Table S2.

Notes

The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled a patent

application on these menin inhibitors reported in this study,

which has been licensed to Medsyn Biopharma, LLC. S. Wang,

A. Aguilar, K. Zheng, T. Xu, S. Xu, L. Huang, E. Fernandez-Salas,

D. Bernard, and J. Stuckey are co-inventors on the patent

application, and receive royalties on the patent from the

University of Michigan. S. Wang is a co-founder of Medsyn

Biopharma. The University of Michigan and S. Wang own stock

in Medsyn Biopharma.

ABBREVIATIONS

■

Bis−Tris, bis(2-hydroxyethyl)amino−tris(hydroxymethyl)-

methane; BLI, biolayer interferometry; CETSA, cellular thermal

shift assay; ESI-MS, electrospray ionization mass spectrometry;

FP, ﬂuorescence polarization; HATU, 1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-

ﬂuorophosphate; LiHMDS, lithium bis(trimethylsilyl)amide;

MeOH, methanol; MLL, mixed lineage leukemia; Oxone,

potassium peroxymonosulfate; PPA, polyphosphoric acid;

qRT-PCR, quantitative real-time polymerase chain reaction;

rt, room temperature; UPLC, ultra-performance liquid

chromatography

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.9b00021.

Single-crystal structure of compound 103 and a table of

crystal data and structure reﬁnement of 103; electron

density maps of the co-crystal structure of M-89

complexed with menin and a table of crystallographic

data collection and reﬁnement statistics for the menin/M-

89 co-crystal (PDF)

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Corresponding Author

*E-mail: shaomeng@umich.edu.

ORCID

Shaomeng Wang: 0000-0002-8782-6950

Present Address

College of Chemistry, Sichuan University, Chengdu, Sichuan

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Article Doi

Structure-Based Discovery of M-89 as a Highly Potent Inhibitor of the Menin-Mixed Lineage Leukemia (Menin-MLL) Protein-Protein Interaction

DOI: 10.1021/acs.jmedchem.9b00021

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