Discovery of M-1121 as an Orally Active Covalent Inhibitor of Menin-MLL Interaction Capable of Achieving Complete and Long-Lasting Tumor Regression

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Discovery of M‑1121 as an Orally Active Covalent Inhibitor of

Menin-MLL Interaction Capable of Achieving Complete and Long-

Lasting Tumor Regression

Meng Zhang, Angelo Aguilar, Shilin Xu, Liyue Huang, Krishnapriya Chinnaswamy, Taryn Sleger,

Bo Wang, Stefan Gross, Brandon N. Nicolay, Sebastien Ronseaux, Kaitlin Harvey, Yu Wang,

Donna McEachern, Paul D. Kirchho ﬀ , Zhaomin Liu, Jeanne Stuckey, Adriana E. Tron, Tao Liu,

and Shaomeng Wang *

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ABSTRACT: Targeting the menin-MLL protein−protein interaction is

being pursued as a new therapeutic strategy for the treatment of acute

leukemia carrying MLL-rearrangements (MLLr leukemia). Herein, we

report M-1121, a covalent and orally active inhibitor of the menin-MLL

interaction capable of achieving complete and persistent tumor

regression. M-1121 establishes covalent interactions with Cysteine 329

located in the MLL binding pocket of menin and potently inhibits growth

of acute leukemia cell lines carrying MLL translocations with no activity

in cell lines with wild-type MLL. Consistent with the mechanism of

action, M-1121 drives dose-dependent down-regulation of HOXA9 and

MEIS1 gene expression in the MLL-rearranged MV4;11 leukemia cell

line. M-1121 is orally bioavailable and shows potent antitumor activity in

vivo with tumor regressions observed at tolerated doses in the MV4;11

subcutaneous and disseminated models of MLL-rearranged leukemia.

Together, our ﬁndings support development of an orally active covalent menin inhibitor as a new therapy for MLLr leukemia.

development, and encouraging early clinical activities have

been recently reported for both compounds.^31,32

INTRODUCTION

■

Chromosomal translocations of the mixed lineage leukemia 1

(MLL1, also known as MLL) are found in 5−10% of acute

leukemias in adults and in approximately 70% of acute

lymphoid leukemia (ALL) in infants.^1,2Acute myeloid

leukemias (AML) carrying MLL rearrangements (MLLr

leukemia) have poor clinical prognosis with a 5 year survival

rate of about 35%.^1,2MLLr leukemias are resistant to current

therapies, highlighting the need for developing new therapeutic

strategies for this disease.

Upon chromosomal translocations, the MLL gene is fused

with one of over 80 partner genes, resulting in chimeric genes

that encode oncogenic MLL fusion proteins.⁴The protein−

protein interaction between these MLL fusion proteins and the

oncogenic co-factor menin is critical for overexpression of

MEIS1 and HOXA genes that led to the development and

maintenance of MLLr leukemia.⁵⁻⁹Thus, targeting the menin-

MLL protein−protein interaction is being pursued as a new

therapeutic strategy for MLLr leukemia.^3,8,10−13To date,

potent small-molecule inhibitors of the menin-MLL protein−

protein interaction (hereafter called menin inhibitors, Figure

1) have been reported.¹⁴⁻²⁸Two of those small-molecule

inhibitors have been advanced into early-phase clinical

In 2018, we published the structure-based discovery of M-

525, the ﬁrst-in-class, potent, covalent small-molecule menin

inhibitor.²⁶We demonstrated that M-525 is more potent than

its noncovalent inhibitor counterparts in reducing the

expression of HOXA9 and MEIS1 genes and in inhibiting

growth of leukemia cells carrying MLL translocations.

Optimization of M-525 yielded M-808,²⁸a potent, covalent

menin inhibitor with antitumor activity in in vitro and in in vivo

models of MLLr leukemia (Figure 1). Despite its superior

cellular potency, M-808 was discontinued for further develop-

ment due to the low oral bioavailability demonstrated in mice.

In this study, we describe our eﬀorts to further improve M-808

oral bioavailability, which resulted in the discovery of M-1121

as the ﬁrst, potent, and orally active covalent menin inhibitor,

Received: April 30, 2021

Published: July 1, 2021

https://doi.org/10.1021/acs.jmedchem.1c00789

J. Med. Chem. 2021, 64, 10333−10349

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Figure 1. Representative menin inhibitors.

capable of achieving complete and long-lasting tumor

regression.

Table 1. Binding Aﬃnity and Cell Growth Inhibition Values

of Menin Inhibitors (7−13)

RESULTS AND DISCUSSION

■

Design and Synthesis of New Noncovalent Menin

Inhibitors. As can be seen from the chemical structures of M-

525 and M-808 in Figure 1, a covalent menin inhibitor consists

of a noncovalent menin-binding scaﬀold and an electrophile

for the formation of a covalent bond with a cysteine residue in

menin. We reasoned that superior oral bioavailability for a

covalent inhibitor could be achieved by improving the oral

bioavailability of the noncovalent portion of the molecule. We

modiﬁed the inhibitor portion of M-525 and M-808 lacking the

electrophile group to ﬁrst obtain a potent and orally

bioavailable noncovalent inhibitor that could later be added

an electrophile group for establishing covalent interactions.

Based upon M-525, compound 7 lacking an electrophile was

designed and synthesized as a noncovalent menin inhibitor.

Compound 7 binds to menin with an IC₅₀value of 6.9 nM as

determined by the ﬂuorescence polarization (FP)-based

competitive binding assay.²⁷This compound was then tested

for its ability to inhibit cell proliferation of the acute leukemia

cell lines MV4;11 and MOLM-13 carrying MLL-AF4 and

MLL-AF9 fusion, respectively. Compound 7 showed moderate

antiproliferative activities in MV4;11 and MOLM-13 cells with

IC₅₀values of 798 and 840 nM, respectively.

Based on the co-crystal structure of the M-808-menin

complex, the positively charged amino group in M-808

establishes charge−charge interaction with the negatively

charged carboxyl group of Asp180.¹²Accordingly, we have

synthesized and evaluated a series of new menin inhibitors

containing diﬀerent positively charged groups, with the results

summarized in Table 1.

a

IC₅₀values were determined using an FP-based competitive binding

b

assay from at least three independent experiments. Cell viability was

determined using a CellTiter-Glo Luminescent Cell Viability Assay

after 4 days of treatment for each compound, with average values and

SDs calculated from three independent experiments.

Compound 8 containing a primary amino group binds to

menin with an IC₅₀value of 4.0 nM and inhibits cell growth

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with IC₅₀values of 192 and 553 nM in the MV4;11 and

MOLM-13 cell lines, respectively. Dimethylation of the

primary amine group in 8 led to compound 9, which binds

to menin with an IC₅₀value of 1.7 nM and displays IC₅₀values

of 178 nM for MV4;11 cells and 703 nM for MOLM-13 cells.

Replacing the dimethylamino group with an azetidine group

yielded compound 10, which binds to menin with an IC₅₀

value of 3.5 nM and achieves IC₅₀values of 105 and 274 nM in

the MV4;11 and MOLM-13 cell lines, respectively. Expanding

the 4-membered ring in compound 10 to a 5-membered ring

generated compound 11, which resulted in an IC₅₀value of 2.8

nM for menin binding and antiproliferative activities for

MV4;11 and MOLM-13 cell lines with IC₅₀values of 516 and

599 nM, respectively. Next, we synthesized compounds 12 and

13, both of which contain a 6-membered ring. Compounds 12

and 13 have binding aﬃnity and cell growth inhibitory activity

similar to compound 11. Based upon the antiproliferative

activity in both MV4;11 and MOLM-13 cell lines, compound

10 is the most potent noncovalent inhibitor among the tested

compounds shown in Table 1.

compound 15, which binds to menin with an IC₅₀value of 2.9

nM. Compound 15 has antiproliferative activity in the MV4;11

and MOLM-13 cell lines with IC₅₀values of 272 and 544 nM,

respectively. Introduction of a bridge hydroxyl or methoxyl

group at the same bridge carbon in 10 resulted in compounds

16 and 17, which bind to menin with IC₅₀values of 3.6 and 2.5

nM, respectively. Compound 16 has IC₅₀values of 379 nM in

MV4;11 cells and 833 nM in the MOLM-13 cell line, while

compound 17 is about 2-fold more potent than 16. Addition of

a bridging methyl ester group to 10 led to compound 18,

which is more than 10-fold less potent than 10 in its binding

aﬃnity to menin. Consistent with its lower binding aﬃnity,

compound 18 has a weak cell growth inhibitory activity in both

the MV4;11 and MOML-13 cell lines. Hence, among these

analogues with a substituted bridging atom, compound 17 with

a bridging methoxyl group is the most potent compound based

upon its cell growth inhibitory activity in both the MV4;11 and

MOLM-13 cell lines.

We then evaluated the oral exposure of compound 17 in

mice. A single oral administration of compound 17 at 25 mg/

kg achieves average plasma compound concentrations of 8265,

5750, and 2195 ng/mL at 1, 3, and 6 h post-dosing,

respectively. Hence, while compound 17 is slightly less potent

than compound 10 in inhibiting cell growth in both the

MV4;11 and MOLM-13 cell lines, 17 displays much improved

oral plasma exposure when compared to 10 in mice.

We then evaluated the pharmacokinetic (PK) properties of

compound 10, when dosed orally at 25 mg/kg in mice (for

details, see the Supporting Information). We found that

compound 10 achieves an encouraging oral exposure with

average plasma concentrations of 624, 735, and 928 ng/mL at

1, 3, and 6 h post-dosing, respectively.

We next modiﬁed the “linker” region in compound 10 to

further improve oral exposure while retaining its antiprolifer-

ative activity in the MV4;11 and MOLM-13 cell lines. We

reasoned that diﬀerent R₂groups on the bridge atom of the

azetidine will aﬀect the pK_aof the nitrogen atom of the

piperidine. This in turn could have a signiﬁcant eﬀect on the

binding aﬃnity to menin and antiproliferative activity in MLL

cell lines, as well as on the PK of the resulting compounds.

Accordingly, we synthesized and evaluated several analogues of

compound 10 with diﬀerent groups on the bridge atom of the

azetidine. These results are summarized in Table 2.

Introduction of a bridge ﬂuorine atom in 10 yielded

compound 14, which shows an IC₅₀value of 3.2 nM for

binding to menin and inhibits cell growth in the MV4;11 and

MOLM-13 cell lines with IC₅₀values of 222 and 948 nM,

respectively. Adding a bridge methyl group in 10 generated

Design of Covalent Inhibitors Based upon Com-

pound 17. These promising cellular and oral exposure data

for compound 17 prompted us to design and synthesize a

series of covalent menin inhibitors based on this noncovalent

inhibitor with the objective of improving cellular potency in

MLLr cell lines. We tested these covalent inhibitors for their

binding aﬃnities to menin by the FP-based assay and their

antiproliferative activities in the MV4;11 and MOLM-13 cell

lines. These results are summarized in Table 3.

We replaced the cyclopropyl group in 17 with the Michael

acceptor used in M-525 to obtain compound 19. Compound

19 shows improved antiproliferative activity with IC₅₀values of

2.3 and 49 nM in MV4;11 and MOLM-13 cell lines,

respectively. Compound 19 thus is 72- and 9-fold more potent

than compound 17 in inhibiting proliferation of MV4;11 and

MOLM-13 cell lines, respectively, suggesting that compound

19 behaves as a covalent menin inhibitor in cells.

In both M-525 and M-808, a positively charged group was

attached to the Michael acceptor. Our data show that the

positively charged group in M-525 and M-808 is critical in

increasing the reactivity of the Michael acceptor for rapid

formation of a covalent bond with menin, and such enhanced

reactivity leads to improved antiproliferative activity in MLLr

leukemia cells.^26,28However, we hypothesized that the

positively charged group attached to the Michael acceptor in

M-525 and M-808 molecules diminishes the oral bioavailability

of these compounds. Therefore, we decided to synthesize and

test a series of potential covalent menin inhibitors lacking the

positively charged group attached to the Michael acceptor.

Removal of the positively charged group attached to the

Michael acceptor in compound 19 yielded compound 20. As

expected and consistent with our previous data, compound 20

is 49- and 8-fold less potent than compound 19 in inhibiting

growth of MV4;11 and MOLM-13 cell lines, respectively.

Therefore, while compounds 19 and 20 contain the same

acrylamide Michael acceptor, compound 20 has a much

weaker cell growth inhibitory potency in both the MV4;11 and

Table 2. Binding Aﬃnity and Cell Growth Inhibition Values

a

of Menin Inhibitors (14−18)

cell growth inhibition

(nM)

binding aﬃnity to menin

compd.

R₂

IC₅₀(nM)

MV4;11

MOLM13

10

14

15

16

17

18

−H

−F

−Me

−OH

−OMe

−COOMe

3.5 1.2

3.2 0.2

2.9 0.5

3.6 0.8

2.5 0.3

42.4 4.0

105 11 274 36

222 31 948 305

272 17 544 145

379 54 833 179

166

9

443 120

>10,000

>1000

a

IC₅₀values are averages of three independent experiments.

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Since compounds 21−23 are all much less potent than

compound 19, these compounds are still likely incapable of

eﬃciently forming a covalent bond with menin in cells.

We hypothesized that further conformational restriction of

the linker may lock the acrylamide Michael acceptor group in

an optimal position and orientation for eﬃcient formation of a

covalent bond with the thiol group of Cys329 in menin.

Computational modeling suggested that a highly conforma-

tionally constrained (1S,4S)-2,5-diazabicyclo[2.2.1]heptane

linker would lock the acrylamide in an optimal position and

orientation for eﬃcient formation of a covalent bond with the

thiol group of Cys 329 (Supporting Information). Interest-

ingly, the enantiomer (1R,4R)-2,5-diazabicyclo[2.2.1]heptane

linker was predicted to place the acrylamide much further away

(4.7 Å) from the thiol group of the Cys 329 residue, suggesting

that a formation of a covalent bond is unlikely.

Table 3. Binding Aﬃnity and Cell Growth Inhibition Values

of Menin Inhibitors 19-26^a

To test our predictions, we synthesized compounds 24 and

25 using these two enantiomeric 2,5-diazabicyclo[2.2.1]-

heptane linkers. Consistent with our predictions, compound

24 containing the (1S,4S)-2,5-diazabicyclo[2.2.1]heptane link-

er is a very potent inhibitor with IC₅₀values of 10.3 and 51.5

nM in the MV4;11 and MOLM-13 cell lines. In comparison,

the stereoisomer compound 25 containing the (1R,4R)-2,5-

diazabicyclo[2.2.1]heptane is a much weaker inhibitor and

displays IC₅₀values of 436 and 504 nM in the MV4;11 and

MOLM-13 cell lines, respectively. Hence, compound 24,

which was named M-1121, is 40- and 9-fold more potent than

its stereoisomer compound 25 in the MV4;11 and MOLM-13

cell lines, respectively. These data show that the stereo-

chemistry of the linker in M-1121 is critically important for its

potent activity in MLLr leukemia cells. M-1121 is also 8−10-

fold more potent than compound 20 in both MV4;11 and

MOLM-13 cell lines.

We synthesized compound 26 by converting the acrylamide

in compound 24 into propionamide to further test the

importance of covalent bond formation for cell growth

inhibition (Table 3). Compound 26 is >10 times less potent

than 24 in inhibition of cell growth in MV4;11 and MOLM-13

cell lines (Table 3), highlighting the importance of covalent

formation for achieving high cellular potency in MLLr

leukemia cell lines.

a

IC₅₀values are averages of three independent experiments.

To gain further insights into their mode of action and

cellular activity, we analyzed compounds 19−26 for covalent

complex formation with recombinant human menin protein by

mass spectrometry using four diﬀerent incubation times (10,

30, 60 min, and overnight) and obtained the data summarized

in Table 4.

MOLM-13 cell lines, indicating that compound 20 probably

does not form a covalent bond with menin very eﬃciently in

cells.

We reasoned that the Michael acceptor in compound 20

may not able to adopt an optimal position and/or orientation

for eﬃcient formation of a covalent bond with menin upon

binding. Therefore, we synthesized compounds 21−23 with

diﬀerent conformationally constrained linking groups between

the SO₂group and the acrylamide to determine if other linkers

would place the Michael acceptor in a more optimal position

and orientation for eﬃcient covalent bond formation with

menin protein.

Compound 21 employing a 6-membered ring piperidine

linker is slightly less potent than compound 20 in cell growth

inhibition in the MV4;11 and MOLM-13 cell lines. Changing

the acrylamide from the 4-position in compound 21 to the 3-

position in the piperidine linker with either an (S)- or (R)-

conﬁguration generated compounds 22 and 23, respectively.

Compounds 22 and 23 are both 2 times more potent than

compound 21 in the MV4;11 and MOLM-13 cell lines and

marginally more potent than compound 20 in both cell lines.

In general, the reaction kinetic data for compounds 19−26

with menin protein (Table 4) correlate nicely with their

potencies in inhibition of cell growth in both MV4;11 and

MOLM-13 cell lines (Table 3). Compound 19 is the most

potent inhibitor in cell growth inhibition and has the fastest

kinetics in formation of a covalent complex with menin protein

among compounds 19−26. With 10, 30, 60 min, and overnight

incubation times, 36.9, 83.4, 85.4, and 90.9% of menin protein

form a covalent complex with compound 19, respectively. M-

1121 has the second fastest kinetics in formation of a covalent

complex with menin protein and is also the second most

potent inhibitor. This is followed by compound 20 as the third

most potent compound with the third fastest kinetics.

Compounds 22 and 23 have a slower reaction kinetics than

compounds 19, 20, and M-1121, consistent with their weaker

cellular activities than compounds 19, 20, and M-1121.

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Gly326. The nitrogen atom of the azetidine head group is 4.1

Å from the negatively charged carboxylic acid group of the

Asp180 side chain in menin, indicating a charge−charge

interaction. The methoxy group on the azetidine linker in M-

1121 forms a hydrogen bond with the phenol group of Tyr323.

M-1121 was evaluated for its antiproliferative activity in 4

MLLr and 3 MLL wild-type cell lines to further deﬁne its

cellular activity and selectivity. We found that M-1121 potently

inhibits proliferation of MLL rearranged but not MLL wild-

type cell lines up to 10 μM, highlighting the selective activity of

M-1121 for cells carrying MLL translocations (Table 5).

Table 4. Analysis of Covalent Complex Formation of Menin

Inhibitors with Recombinant Human Menin Protein by

Mass Spectrometry

incubation time with menin protein

10 min

30 min

1 h

overnight

compound

19

20

21

22

% of menin covalent complex

36.9%

24.1%

0%

8.8%

9.8%

27%

0%

83.4%

40.9%

0%

85.4%

59.4%

0%

90.9%

81.5%

27.0%

93.1%

90.0%

100%

45.1%

0%

27.9%

22.6%

59%

0%

37.4%

40.5%

66%

0%

23

24 (M-1121)

25

26

Table 5. Cell Growth Inhibitory Activities of M-1121 in a

Panel of Leukemia Cell Lines after 4 Days of Treatment

Determined by the CellTiter-Glo

0%

disease

cell growth inhibition IC₅₀

(nM)

a

cell line

MV4;11

MLL status

indication

Compounds 21 and 25 have a very slow reaction kinetics, and

no covalent complex was detected within 1 h incubation time.

Consistent with its lack of a Michael acceptor, compound 26

does not form a covalent complex with menin even with

overnight incubation. Of note, the stoichiometry for each

inhibitor:menin covalent complex was 1:1.2.

MLL/AF4

MLL/AF9

MLL-ENL

MLL/AF4

MLL/ENL

MLL/AF4

MLLwt

ALL

10

52

3

51

MOLM-13

OCI-AML4

SEM

AML

ALL

AML

CML

KOPN8

RS4;11

HL-60

K562

MEG-1

110

1000

>10,000

A glutathione (GSH) reactivity assay was employed to test

the intrinsic reactivity of M-1121 toward glutathione (GSH).

Incubation of M-1121 under conditions mimicking intra-

cellular glutathione levels (4.5 mM GSH, 37 °C, pH 7.4)

revealed a GSH conjugation half-life of 89 min. These data

indicated that M-1121 has only mild reactivity with GSH.

To further understand the precise binding mode, we

determined the co-crystal structure for M-1121 in complex

with menin at 2.74 Å resolution (Figure 2.). Consistent with

our design and mass spectrometry data, M-1121 forms a

covalent bond between the acrylamide Michael acceptor and

the thiol group of Cys329 in menin. The 2,5-diazabicyclo

[2.2.1] ring orients and places the acrylamide group in a

position that is optimal for the formation of a covalent bond

with the sulfur atom in the Cys329 of menin. In addition, the

2,5-diazabicyclo [2.2.1] group establishes hydrophobic inter-

actions with menin, speciﬁcally with Val371, Ala325, and

MLLwt

a

AML: acute myeloid leukemia; ALL: acute lymphocytic leukemia;

CML: chronic myeloid leukemia.

In our previous studies, we showed that menin inhibitors

suppress the expression of HOXA9 and MEIS1 in MLLr

leukemia cells.^24,28We thus evaluated M-1121 for its eﬀect on

the expression of HOXA9 and MEIS1 in MV4;11 cells by qRT-

PCR. Our data showed that M-1121 suppresses HOXA9 and

MEIS1 gene transcription in a dose-dependent manner and

eﬀectively modulates the expression of HOXA9 and MEIS1

genes at concentrations as low as 10 and 30 nM, respectively

(Figure 3). Hence, M-1121 is potent in inhibiting the

expression of HOXA9 and MEIS1 gene transcription in the

Figure 2. Co-crystal structure of compound 24 (M-1121) complexed with menin at 2.74 Å resolution (PDB code: 7M4T). Side chains of menin

residues within 4 Å from the compound are shown as sticks. Hydrogen bonds are shown as dashed lines.

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M-1121 led to complete tumor regression in 10 out of 10 mice

with no tumor regrowth detected up to a month after last

treatment (day 45 after treatment start) (Figure 4c and Figure

S2 in the Supporting Information). Treatment with M-1121

was well tolerated with no signiﬁcant body weight loss (Figure

4d) or other signs of toxicity.

Since MLLr leukemias are bone marrow diseases, we next

investigated whether M-1121 has activity in this compartment

by evaluating the eﬀect of M-1121 in bone marrow CD45⁺

leukemic cells in NCG mice engrafted with the Luciferase-

tagged MV4;11 disseminated model. Mice were dosed with M-

1121 at 150 mg/kg once daily by oral gavage for 4 days to

achieve steady-state drug levels, and we evaluated the eﬀect at

a 48 h time-point post-last dose. We found that M-1121

suppresses the expression of the MEIS1 gene to less than 2%

compared to the vehicle group with concomitant induction of

the cell diﬀerentiation marker ITGAM (∼67-fold) and CD11b

protein in human CD45⁺cells isolated from bone marrow

(Figure 5a,b). It is interesting to note that even though mice

were treated for only 4 days, M-1121 was able to exert

antitumor activity with reduction in the number of human

CD45⁺CD33⁺leukemic cells in bone marrow as detected by

ﬂow cytometry (Figure 5c) and decrease the intensity of the

whole-body bioluminescence signal (Figure 5d). No signiﬁcant

changes in body weight were observed (data not shown).

Chemistry. The synthetic routes to these compounds are

shown below (Scheme 1). Compounds 8−13 were synthesized

in a convergent manner. The Boc group in the known

compound 27²⁸was deprotected with TFA, and the resulting

amine was converted into a methyl carbamate 28. A relay

reduction of 28 with DIBAL-H followed by NaBH₄was

employed to give primary amine 29, which was subjected to

Boc protection. Removal of the benzyl protecting group

followed by substitution and removal of the Boc group

aﬀorded compound 8, whose subsequent functionalization

gave 9−13. Intermediate 36 was synthesized in ﬁve steps

starting from 4-ﬂuorobenzenethiol.

Synthesis of compounds 14−18 (Scheme 2) started with

compound 29. A ring-closure/substitution reaction gave an

azetidine compound 37. Removal of the benzyl protecting

group followed by substitution with 41 aﬀorded compound 39.

Then the Boc group was cleaved, and the resulting product was

submitted to a nucleophilic aromatic substitution reaction with

34 to give 14−18 as the ﬁnal compounds.

Starting from 39d, compounds 19−26 were synthesized in a

similar manner (Scheme 3). Removal of Boc protection from

39d followed by aromatic substitution with 42 gave compound

40. Removal of the Boc protection from 40 produced the

corresponding amine intermediates, which reacted with diverse

carbonyl chlorides or anhydrides to aﬀord the ﬁnal products

19−26.

Figure 3. Gene expression changes induced by M-1121. Quantitative

real-time polymerase chain reaction (qRT-PCR) analysis of the eﬀect

of M-1121 on the mRNA levels of HOXA9 and MEIS1genes in

MV4;11 cells after 24 h of treatment.

M4;11 cell line, consistent with the expected mechanism of

action of a menin inhibitor.

Next, we evaluated the oral exposure of M-1121 in mice. A

single oral administration of M-1121 at 25 mg/kg achieves

average plasma concentrations of 3797, 4640, and 2055 ng/mL

at 1, 3, and 6 h post-dosing, respectively, indicating excellent

oral exposure. Subsequently, a PK study with both intravenous

and oral administrations was performed with M-1121. The PK

data showed that M-1121 has a low clearance and a moderate

volume of distribution (Table 6). M-1121 dosed orally at 5

mg/kg achieves a C_maxvalue of 4153 ng/mL and AUC_0−∞of

43,567 h·ng/mL. Together, M-1121 has an acceptable PK

proﬁle in mice with 49.4% oral bioavailability.

We determined the plasma protein binding data for M-1121

and found that M-1121 has 90.7, 87.5, and 99.3% binding in

human, rat, and mouse plasma, respectively. The PPB data

showed that while M-1121 has an excellent PPB in human and

rat plasma, it has a very high PPB in mouse plasma, suggesting

that high doses may be needed to achieve strong antitumor

activity in mice.

We next evaluated M-1121 for its in vivo antitumor activity

in SCID mice harboring MV4;11 subcutaneous tumors. In the

ﬁrst experiment, when xenograft tumors reached an average

volume of 100 mm³, mice were treated with M-1121 at 100

mg/kg daily for 26 days via oral gavage (Figure 4a,b). M-1121

reduced the average tumor volume from 157 mm³at the

beginning of the treatment to 106 mm³on day 26 of the

treatment, a reduction of tumor volume of 32%. Signiﬁcantly,

M-1121 caused no animal weight loss or other signs of toxicity

during and after the treatment.

Because of the high PPB (99.3%) of M-1121 in mouse

plasma and importantly its lack of any signs of toxicity in mice

at 100 mg/kg, we further tested its antitumor activity in the

MV4;11 subcutaneous tumor model at a higher dose to

determine if M-1121 can achieve an even stronger antitumor

activity. In the second experiment, when tumors reached an

average volume of 200 mm³, mice were treated with M-1121 at

300 mg/kg once daily for 15 days via oral gavage (Figure 4c,d).

a

Table 6. Pharmacokinetics of M-1121 in Mice

1 mg/kg (IV)

5 mg/kg (PO)

compd.

M-1121

CL (L/h/kg)

0.0567

Vss (L/kg)

AUC_0−∞(h·ng/mL)

t_max(h)

C_max(ng/mL)

AUC_0−∞(h·ng/mL)

F%

0.330

17,644

2.00

4153

43,567

49.4

a

Female C57BL/6 mice were dosed either intravenously with a solution in 10% NMP, 10% Solutol HS15, and 80% saline or orally at 5 mg/kg in

0.5% methyl cellulose + 0.2% Tween 80 (w/w).

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Figure 4. M-1121 exhibits potent antitumor eﬃcacy in the MV4;11 (MLL rearranged) subcutaneous tumor xenograft model. The compound was

administered orally at the indicated dose schedules.

Methyl ((1S,2R)-2-((S)-2-Amino-1-(1-((1-(4-(cyclopropylsulfonyl)-

EXPERIMENTAL SECTION

■

phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-ﬂuorophenyl)-

General Methods for Chemistry. Unless otherwise noted,

commercial solvents and reagents were used without further

puriﬁcation. Proton nuclear magnetic resonance (¹H NMR) spectra

were recorded on a Bruker Advance 400 MHz spectrometer and are

reported in parts per million (ppm) downﬁeld from tetramethylsilane

(TMS). In the spectral data reported, the format (δ) chemical shift

(multiplicity, J values in Hz, integration) was used with the following

abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, and m =

multiplet. Mass spectrometric (MS) analysis was carried out with a

Waters ultraperformance liquid chromatography (UPLC)−mass

spectrometer. The ﬁnal compounds were all puriﬁed by a C18

reversed-phase preparative high-performance liquid chromatography

(HPLC) column with solvent A (0.1% TFA in H₂O) and solvent B

(0.1% TFA in MeCN). The purity of all the ﬁnal compounds was

conﬁrmed to be >95% by UPLC analysis (10−100% MeCN in H₂O

containing 0.1% TFA in 10 min).

ethyl)cyclopentyl)carbamate (8). Compound 31 (680 mg, 0.954

mmol) was dissolved in DCM (3 mL), and TFA (1.5 mL) was added

slowly. After stirring for 3 h at room temperature (rt), the reaction

mixture was concentrated under vacuum. The residue was puriﬁed

with preparative HPLC to give the title compound (495 mg, 85%).

¹H NMR (400 MHz, MeOH-d4) δ 7.64−7.59 (m, 2H), 7.44 (q, J =

7.6 Hz, 1H), 7.24−7.16 (m, 2H), 7.13 (t, J = 8.8 Hz, 1H), 6.77−6.71

(m, 2H), 3.81−3.66 (m, 4H), 3.66−3.53 (m, 2H), 3.53−3.45 (m,

1H), 3.31−3.20 (m, 4H), 3.20−3.09 (m, 3H), 3.02 (t, J = 12.3 Hz,

1H), 2.93 (t, J = 12.5 Hz, 1H), 2.89−2.70 (m, 2H), 2.57 (m, 1H),

2.41 (s, 1H), 2.18−2.08 (m, 1H), 2.04−1.94 (m, 3H), 1.94−1.82 (m,

1H), 1.82−1.67 (m, 2H), 1.67−1.54 (m, 3H), 1.21−1.12 (m, 2H),

1.06−0.97 (m, 2H); ESI-MS calcd for C₃₃H₄₆FN₄O₄S [M + H]⁺=

613.32, found: 613.17.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(Cyclopropylsulfonyl)phenyl)-

azetidin-3-yl)methyl)piperidin-4-yl)-2-(dimethylamino)-1-(3-

ﬂuorophenyl)ethyl)cyclopentyl)carbamate (9). Triethylamine (23

μL, 0.163 mmol), acetic acid (15 μL, 0.261 mmol), and formaldehyde

(3 μL, 0.196 mmol) were added to a solution of compound 8 (40 mg,

0.0653 mmol) in MeCN/THF (0.5/0.5 mL). After 3 h, sodium

triacetoxyborohydride (42 mg, 0.196 mmol) was added. The mixture

was stirred overnight, quenched with H₂O, and concentrated under

vacuum. The residue was puriﬁed by reversed-phase preparative

Methyl ((1S,2R)-2-((S)-Cyano(1-((1-(4-(cyclopropylsulfonyl)-

phenyl)azetidin-3-yl)methyl)piperidin-4-yl)(3-ﬂuorophenyl)-

methyl)cyclopentyl)carbamate (7). Compound 7 was prepared from

compounds 28 and 36 with the procedure that was used to produce

1

compound 31. H NMR (400 MHz, MeOH-d4) δ 7.49−7.44 (m,

2H), 7.25 (td, J = 8.1, 6.1 Hz, 1H), 7.15 (d, J = 7.9 Hz, 1H), 7.07 (d, J

= 10.2 Hz, 1H), 6.98−6.92 (m, 1H), 6.36−6.26 (m, 2H), 3.95 (td, J =

8.0, 2.0 Hz, 2H), 3.68 (q, J = 6.8 Hz, 1H), 3.53 (dd, J = 8.1, 5.6 Hz,

2H), 3.38 (t, J = 11.6 Hz, 1H), 3.25 (s, 1H), 3.23 (s, 3H), 3.13 (p, J =

1.6 Hz, 1H), 3.08−2.94 (m, 1H), 2.92−2.79 (m, 2H), 2.67 (q, J = 7.9

Hz, 1H), 2.37 (tt, J = 7.9, 4.8 Hz, 1H), 2.26 (t, J = 12.1 Hz, 1H),

2.10−1.91 (m, 2H), 1.83−1.75 (m, 1H), 1.70−1.59 (m, 1H), 1.58−

1.31 (m, 5H), 1.29−1.13 (m, 1H), 1.00−0.93 (m, 2H), 0.84−0.77

(m, 2H); ESI-MS calcd for C₃₃H₄₂FN₄O₄S [M + H]⁺= 609.29,

found: 609.14.

1

HPLC to give the title compound (22 mg, 53%). H NMR (400

MHz, MeOH-d4) δ 7.71−7.66 (m, 2H), 7.56−7.48 (m, 1H), 7.46−

7.37 (m, 2H), 7.21−7.14 (m, 1H), 6.58−6.51 (m, 2H), 4.21 (t, J =

8.0 Hz, 2H), 4.01−3.83 (m, 3H), 3.79 (ddd, J = 8.3, 5.6, 2.9 Hz, 2H),

3.72−3.59 (m, 2H), 3.57−3.43 (m, 4H), 3.31−3.19 (m, 2H), 3.16−

3.01 (m, 3H), 3.01−2.82 (m, 4H), 2.69 (d, J = 7.4 Hz, 1H), 2.58 (tt, J

= 8.0, 4.8 Hz, 1H), 2.44 (s, 1H), 2.28−2.11 (m, 2H), 2.06−1.93 (m,

1H), 1.70 (m, 3H), 1.60 (m, 3H), 1.42 (m, 1H), 1.33 (t, J = 7.3 Hz,

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Figure 5. M-1121 shows activity in the disseminated Luciferase-tagged MV4;11 (MLL rearranged) xenograft model. M-1121 was administered via

oral gavage at 150 mg/kg once daily for 4 days, and data are presented as mean SEM, and ﬁve mice were evaluated per group. Readouts in a, b,

and c were measured 48 h after the last dose. MEIS1 and ITGAM gene expression in human CD45⁺cells isolated from bone marrow evaluated by

RT-qPCR (a), CD11b mean ﬂuorescence intensity (MFI) measured in human CD45⁺CD33⁺cells from bone marrow by ﬂow cytometry (b), the

number of human CD45⁺CD33⁺cells measured by ﬂow cytometry (c) and whole-body bioluminescence intensity determined in vehicle or M-

1121-treated mice (d). The dotted line indicates the last day of dosing.

1H), 1.21−1.14 (m, 2H), 1.05−0.98 (m, 2H); ESI-MS calcd for

C₃₅H₅₀FN₄O₄S [M + H]⁺= 641.35, found: 641.20.

prepared from compound 8 and 1,4-dibromobutane according to

the procedure used to produce compound 10 (26 mg, 60%). ¹H

NMR (400 MHz, MeOH-d4) δ 7.71−7.65 (m, 2H), 7.49 (td, J = 8.0,

6.3 Hz, 1H), 7.39−7.30 (m, 2H), 7.20−7.12 (m, 1H), 6.56−6.50 (m,

2H), 4.17 (t, J = 8.0 Hz, 2H), 3.91−3.67 (m, 6H), 3.64−3.50 (m,

3H), 3.49−3.40 (m, 3H), 3.38−3.35 (m, 2H), 3.29−3.18 (m, 2H),

3.05 (t, J = 12.4 Hz, 1H), 2.96 (t, J = 12.5 Hz, 1H), 2.90−2.77 (m,

1H), 2.58 (tt, J = 7.9, 4.8 Hz, 1H), 2.32−1.98 (m, 8H), 1.98−1.86

(m, 1H), 1.86−1.49 (m, 6H), 1.44−1.25 (m, 1H), 1.20−1.13 (m,

2H), 1.06−0.97 (m, 2H); ESI-MS calcd for C₃₇H₅₂FN₄O₄S [M + H]⁺

= 667.37, found: 667.25.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(Cyclopropylsulfonyl)phenyl)-

azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-ﬂuorophenyl)-2-

morpholinoethyl)cyclopentyl)carbamate (12). Compound 12 was

prepared from compound 8 and bis(2-bromoethyl) ether according to

the procedure used to prepare compound 10 (20 mg, 45%). ¹H NMR

(400 MHz, MeOH-d4) δ 7.70−7.64 (m, 2H), 7.45 (q, J = 7.6 Hz,

1H), 7.39 (d, J = 9.6 Hz, 2H), 7.17−7.06 (m, 1H), 6.56−6.49 (m,

2H), 4.18 (t, J = 8.0 Hz, 2H), 3.92 (s, 4H), 3.80−3.68 (m, 4H),

3.64−3.52 (m, 4H), 3.49−3.36 (m, 6H), 3.29−3.10 (m, 2H), 3.04 (t,

J = 12.5 Hz, 1H), 2.92 (t, J = 12.4 Hz, 1H), 2.81 (s, 1H), 2.58 (tt, J =

7.9, 4.8 Hz, 1H), 2.24−2.07 (m, 3H), 2.07−1.93 (m, 2H), 1.70 (s,

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-((1-(4-

(cyclopropylsulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-

(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate (10). 1,3-Dibromo-

propane (8 μL, 0.0784 mmol), K₂CO₃(27 mg, 0.196 mmol), and

KI (1 mg, 0.00653 mmol) were added to a solution of compound 8

(40 mg, 0.0653 mmol) in MeCN (1.5 mL). The mixture was stirred at

80 °C overnight, quenched with H₂O, and concentrated under

vacuum. The residue was puriﬁed by reversed-phase preparative

1

HPLC to give the title compound (26 mg, 61%). H NMR (400

MHz, MeOH-d4) δ 7.69−7.63 (m, 2H), 7.47 (td, J = 8.1, 6.2 Hz,

1H), 7.19−7.11 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.55−6.49 (m,

2H), 4.60−4.46 (m, 2H), 4.42−4.28 (m, 2H), 4.21−4.10 (m, 3H),

3.79 (d, J = 15.6 Hz, 1H), 3.72 (ddd, J = 8.0, 5.6, 2.4 Hz, 2H), 3.61−

3.44 (m, 3H), 3.41 (d, J = 7.1 Hz, 2H), 3.26−3.16 (m, 1H), 2.99 (dt,

J = 24.9, 12.4 Hz, 2H), 2.79 (d, J = 9.4 Hz, 1H), 2.57 (tt, J = 7.9, 4.8

Hz, 2H), 2.50−2.39 (m, 1H), 2.13−1.96 (m, 5H), 1.93−1.83 (m,

1H), 1.83−1.74 (m, 1H), 1.73−1.57 (m, 3H), 1.47 (q, J = 12.9 Hz,

1H), 1.19−1.12 (m, 2H), 1.04−0.96 (m, 2H); ESI-MS calcd for

C₃₆H₅₀FN₄O₄S [M + H]⁺= 653.35, found: 653.10.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(Cyclopropylsulfonyl)phenyl)-

azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-ﬂuorophenyl)-2-(pyrroli-

din-1-yl)ethyl)cyclopentyl)carbamate (11). Compound 11 was

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a

Scheme 1. Synthesis of Compounds 8−13

a

Reagents and conditions: (a) TFA, DCM, rt; (b) dimethyl dicarbonate, Et₃N, DCM, rt; (c) DIBAL-H, toluene, 0 °C; (d) NaBH₄, MeOH, 0 °C to

rt; (e) Boc₂O, DCM, rt; (f) H₂, Pd/C, MeOH, rt; (g) 36, K₂CO₃, KI, MeCN, 80 °C; (h) TFA, DCM, rt; (i) formaldehyde, NaBH(OAc)₃, MeCN/

THF, rt (9); 1,4-dibromobutane, K₂CO₃, MeCN, 80 °C (10); 1,5-dibromopentane, K₂CO₃, MeCN, 80 °C (11); 1-bromo-2-(2-

bromoethoxy)ethane, K₂CO₃, MeCN, 80 °C (12); methallyltrimethylsilane, formaldehyde, H₂O, 45 °C (13); (j) bromocyclopropane, t-

BuONa, DMSO, 80 °C; (k) m-CPBA, DCM, rt; (l) methyl azetidine-3-carboxylate hydrochloride, K₂CO₃, DMSO, 80 °C; (m) LiAlH₄, THF, 0 °C;

(n) MsCl, Et₃N, DCM, rt.

a

Scheme 2. Synthesis of Compounds 14−18

a

Reagents and conditions: (a) 1,3-dibromopropane, K₂CO₃, KI, MeCN, 80 °C; (b) H₂, Pd/C, MeOH, rt; (c) 41, K₂CO₃, KI, MeCN, 80 °C; (d)

TFA, DCM, rt; (e) 34, K₂CO₃, DMSO, 80 °C.

7H), 1.20−1.13 (m, 2H), 1.05−0.97 (m, 2H); ESI-MS calcd for

C₃₇H₅₂FN₄O₅S [M + H]⁺= 683.36, found: 683.23.

stirred at 45 °C overnight and then was puriﬁed by reversed-phase

preparative HPLC to give the title compound (60 mg, 69%). ¹H

NMR (400 MHz, MeOH-d4) δ 7.71−7.64 (m, 2H), 7.52−7.44 (m,

1H), 7.44−7.33 (m, 2H), 7.18−7.10 (m, 1H), 6.55−6.47 (m, 2H),

4.15 (t, J = 8.0 Hz, 2H), 4.03 (s, 1H), 3.87−3.71 (m, 3H), 3.71−3.61

(m, 3H), 3.61−3.48 (m, 3H), 3.48−3.38 (m, 3H), 3.38−3.35 (m,

1H), 3.31−3.14 (m, 3H), 3.04 (t, J = 12.4 Hz, 1H), 2.98−2.79 (m,

2H), 2.58 (tt, J = 7.9, 4.8 Hz, 1H), 2.33−1.98 (m, 5H), 1.98−1.86

(m, 2H), 1.85−1.45 (m, 8H), 1.25 (s, 3H), 1.20−1.12 (m, 2H),

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(Cyclopropylsulfonyl)phenyl)-

azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-ﬂuorophenyl)-2-(4-hy-

droxy-4-methylpiperidin-1-yl)ethyl)cyclopentyl)carbamate (13).

Compound 13 was prepared according to a reported procedure.²⁹

Methallyltrimethylsilane (32 μL, 0.184 mmol) and formaldehyde (41

μL, 0.488 mmol) were added to a solution of compound 8 (75 mg,

0.1223 mmol) in MeCN (1 mL) and H₂O (1 mL). The mixture was

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Scheme 3. Synthesis of Compounds 19−26

a

Reagents and conditions: (a) TFA, DCM, rt; (b) 42, K₂CO₃, DMSO, 80 °C; (c) TFA, DCM, rt; (d) (2E)-4-(dimethylamino)-2-butenoyl chloride

hydrochloride, DIPEA, DCM, 0 °C to rt (19); acrylic anhydride, DIPEA, DCM, rt (20−25); propionic anhydride, DIPEA, DCM, rt (26).

1.05−0.96 (m, 2H); ESI-MS calcd for C₃₉H₅₆FN₄O₅S [M + H]⁺=

711.40, found: 711.20.

Hz, 1H), 1.22−1.12 (m, 2H), 1.06−0.97 (m, 2H); ESI-MS calcd for

C₃₆H₅₀FN₄O₅S [M + H]⁺= 669.35, found: 669.11.

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-((1-(4-

(cyclopropylsulfonyl)phenyl)-3-ﬂuoroazetidin-3-yl)methyl)-

piperidin-4-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(14). Compound 14 was prepared from compounds 37, 41a, and 34

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-((1-(4-

(cyclopropylsulfonyl)phenyl)-3-methoxy-azetidin-3-yl)methyl)-

piperidin-4-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(17). 10% Pd/C (344 mg, 10 wt %) was added to a solution of

compound 37 (1.6 g, 3.24 mmol) in MeOH (50 mL) under a N₂

atmosphere. The solution was brieﬂy vacuumed to remove the N₂

atmosphere and then put under a H₂atmosphere; this was repeated

three times. The mixture was then stirred for 2 h at rt under a H₂

atmosphere. The Pd/C catalyst was then ﬁltered, and the solvent was

removed by rotary evaporation to give compound 38, which was not

1

according to the procedure used to prepare compound 17. H NMR

(400 MHz, MeOH-d4) δ 7.74−7.68 (m, 2H), 7.52−7.42 (m, 1H),

7.20−7.11 (m, 2H), 7.11−7.02 (m, 1H), 6.63−6.58 (m, 2H), 4.61−

4.45 (m, 2H), 4.42−4.30 (m, 2H), 4.30−4.10 (m, 6H), 3.85−3.73

(m, 4H), 3.60 (t, J = 12.1 Hz, 2H), 3.55−3.42 (m, 1H), 3.16 (dt, J =

24.0, 12.4 Hz, 2H), 2.87−2.73 (m, 1H), 2.63−2.51 (m, 2H), 2.51−

2.39 (m, 1H), 2.14−1.95 (m, 5H), 1.95−1.85 (m, 1H), 1.85−1.74

(m, 1H), 1.73−1.48 (m, 5H), 1.20−1.13 (m, 2H), 1.05−0.98 (m,

2H); ESI-MS calcd for C₃₆H₄₉F₂N₄O₄S [M + H]⁺= 671.34, found:

671.26.

1

puriﬁed further. H NMR (400 MHz, MeOH-d₄) δ 7.48−7.43 (m,

1H), 7.16−7.06 (m, 3H), 4.51−4.45 (m, 2H), 4.38−4.27 (m, 2H),

4.10 (d, J = 15.6 Hz, 1H), 3.77 (d, J = 15.2 Hz, 1H), 3.55−3.52 (m,

1H), 3.40−3.33 (m, 2H), 3.31 (s, 3H), 3.01−2.89 (m, 2H), 2.78−

2.72 (m, 1H), 2.58−2.48 (m, 1H), 2.46−2.39 (m, 1H), 2.05−1.93

(m, 5H), 1.78−1.70 (m, 1H), 1.68−1.54 (m, 3H), 1.39−1.30 (m,

1H), 1.08−1.02 (m, 1H); ESI-MS calculated for C₂₃H₃₄FN₃O₂[M +

H]⁺= 404.26, found: 404.42.

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-((1-(4-

(cyclopropylsulfonyl)phenyl)-3-methylazetidin-3-yl)methyl)-

piperidin-4-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(15). Compound 15 was prepared from compounds 37, 41b, and 34

1

according to the method used to produce compound 17. H NMR

(400 MHz, MeOH-d4) δ 7.69−7.62 (m, 2H), 7.46 (td, J = 8.3, 6.3

Hz, 1H), 7.19−7.10 (m, 2H), 7.10−7.00 (m, 1H), 6.56−6.49 (m,

2H), 4.58−4.42 (m, 2H), 4.41−4.21 (m, 2H), 4.12 (d, J = 15.6 Hz,

1H), 3.92−3.71 (m, 6H), 3.59−3.42 (m, 3H), 3.38 (s, 3H), 3.18−

2.98 (m, 2H), 2.87−2.72 (m, 1H), 2.56 (tt, J = 7.9, 4.8 Hz, 2H),

2.49−2.35 (m, 1H), 2.10−1.93 (m, 5H), 1.93−1.81 (m, 1H), 1.81−

1.72 (m, 1H), 1.72−1.52 (m, 5H), 1.49 (s, 3H), 1.18−1.12 (m, 2H),

1.03−0.97 (m, 2H); ESI-MS calcd for C₃₇H₅₂FN₄O₄S [M + H]⁺=

667.37, found: 667.27.

Compound 38 (200 mg, 0.496 mmol), K₂CO₃(206 mg, 1.49

mmol), and KI (8 mg, 0.0496 mmol) were added to a solution of

compound 41d (176 mg, 0.595 mmol) in MeCN (4 mL). The

mixture was reﬂuxed overnight. Then, the mixture was ﬁltered

through celite, concentrated, and puriﬁed with preparative HPLC to

give 39d (155 mg, 52%) as a white solid.

Compound 39d (155 mg, 0.257 mmol) was dissolved in DCM (4

mL), and then TFA (380 μL, 5.14 mmol) was added. After stirring for

2 h at rt, the reaction mixture was then evaporated. The crude product

was dissolved in DMSO (3 mL). Compound 34 (62 mg, 0.308 mmol)

and K₂CO₃(142 mg, 1.03 mmol) were added, and the mixture was

stirred at 80 °C overnight. The mixture was quenched with H₂O and

puriﬁed by reversed-phase preparative HPLC to give compound 17

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-((1-(4-

(cyclopropylsulfonyl)phenyl)-3-hydroxyazetidin-3-yl)methyl)-

piperidin-4-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(16). Compound 16 was prepared from compounds 37, 41c, and 34

1

according to the procedure used to prepare compound 17. H NMR

1

(112 mg, 64%). H NMR (400 MHz, MeOH-d4) δ 7.69−7.62 (m,

(400 MHz, MeOH-d4) δ 7.72−7.66 (m, 2H), 7.49 (td, J = 8.2, 6.2

Hz, 1H), 7.20−7.13 (m, 2H), 7.11−7.02 (m, 1H), 6.61−6.55 (m,

2H), 4.61−4.44 (m, 2H), 4.44−4.25 (m, 2H), 4.24−4.05 (m, 3H),

3.94 (d, J = 8.8 Hz, 2H), 3.80 (d, J = 15.6 Hz, 1H), 3.62 (t, J = 12.8

Hz, 2H), 3.49 (s, 3H), 3.22−3.02 (m, 2H), 2.90−2.75 (m, 1H), 2.58

(tt, J = 8.0, 4.8 Hz, 2H), 2.53−2.39 (m, 1H), 2.15−1.94 (m, 5H),

1.85 (d, J = 24.0 Hz, 3H), 1.69 (d, J = 12.5 Hz, 4H), 1.55 (d, J = 12.7

2H), 7.48−7.39 (m, 1H), 7.16−7.08 (m, 2H), 7.04 (d, J = 8.0 Hz,

1H), 6.59−6.53 (m, 2H), 4.57−4.39 (m, 2H), 4.38−4.20 (m, 4H),

4.11 (d, J = 15.6 Hz, 1H), 3.96−3.87 (m, 2H), 3.75 (d, J = 15.6 Hz,

1H), 3.61−3.50 (m, 4H), 3.50−3.39 (m, 5H), 3.15−2.97 (m, 2H),

2.84−2.70 (m, 1H), 2.59−2.46 (m, 2H), 2.46−2.33 (m, 1H), 2.10−

1.90 (m, 5H), 1.90−1.71 (m, 3H), 1.71−1.57 (m, 4H), 1.56−1.44

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(m, 1H), 1.18−1.08 (m, 2H), 1.03−0.93 (m, 2H); ESI-MS calculated

for C₃₇H₅₂FN₄O₅S [M + H]⁺= 683.36, found: 683.16.

7.21−7.13 (m, 2H), 7.08 (d, J = 8.0 Hz, 1H), 6.73 (dd, J = 16.8, 10.7

Hz, 1H), 6.66−6.59 (m, 2H), 6.16 (d, J = 16.8 Hz, 1H), 5.79−5.69

(m, 1H), 4.64 (d, J = 12.8 Hz, 1H), 4.53 (s, 2H), 4.44−4.22 (m, 5H),

4.16 (d, J = 15.6 Hz, 1H), 3.98 (dd, J = 9.6, 6.7 Hz, 3H), 3.80 (d, J =

15.6 Hz, 1H), 3.65−3.54 (m, 4H), 3.51 (s, 5H), 3.19−3.02 (m, 3H),

2.96 (t, J = 11.7 Hz, 1H), 2.91−2.76 (m, 1H), 2.64−2.50 (m, 1H),

2.50−2.38 (m, 1H), 2.22−2.10 (m, 1H), 2.10−1.96 (m, 5H), 1.96−

1.85 (m, 2H), 1.85−1.75 (m, 2H), 1.75−1.59 (m, 4H), 1.59−1.42

(m, 2H), 1.17 (s, 1H); ESI-MS calcd for C₄₂H₅₉FN₅O₆S [M + H]⁺=

780.42, found: 780.23.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(((R)-1-Acryloylpiperidin-3-yl)-

sulfonyl)phenyl)-3-methoxyazetidin-3-yl)methyl)piperidin-4-yl)-2-

(azetidin-1-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(23). Compound 23 was prepared from compounds 39d and 42d

according to the procedure used to produce compound 24. ¹H NMR

(400 MHz, MeOH-d4) δ 7.74−7.66 (m, 2H), 7.49 (td, J = 8.3, 6.4

Hz, 1H), 7.21−7.13 (m, 2H), 7.08 (d, J = 8.0 Hz, 1H), 6.73 (dd, J =

16.9, 10.8 Hz, 1H), 6.67−6.59 (m, 2H), 6.16 (d, J = 16.7 Hz, 1H),

5.79−5.70 (m, 1H), 4.64 (d, J = 12.8 Hz, 1H), 4.53 (s, 2H), 4.44−

4.21 (m, 5H), 4.16 (d, J = 15.6 Hz, 1H), 3.98 (dd, J = 9.7, 6.6 Hz,

3H), 3.80 (d, J = 15.6 Hz, 1H), 3.57 (s, 4H), 3.52 (s, 5H), 3.19−3.02

(m, 3H), 2.96 (t, J = 11.6 Hz, 1H), 2.89−2.75 (m, 1H), 2.65−2.51

(m, 1H), 2.51−2.40 (m, 1H), 2.24−2.11 (m, 1H), 2.11−1.98 (m,

5H), 1.97−1.75 (m, 4H), 1.75−1.59 (m, 4H), 1.59−1.43 (m, 2H),

1.17 (s, 1H); ESI-MS calcd for C₄₂H₅₉FN₅O₆S [M + H]⁺= 780.42,

found: 780.19.

Methyl 3-((4-((S)-2-(Azetidin-1-yl)-1-(3-ﬂuorophenyl)-1-((1R,2S)-

2-((methoxycarbonyl)amino)cyclopentyl)ethyl)piperidin-1-yl)-

methyl)-1-(4-(cyclopropylsulfonyl)phenyl)azetidine-3-carboxylate

(18). Compound 18 was prepared from compounds 37, 41e, and 34

1

according to the procedure used to prepare compound 17. H NMR

(400 MHz, MeOH-d₄) δ 7.73−7.67 (m, 2H), 7.52−7.41 (m, 1H),

7.20−7.10 (m, 2H), 7.06 (d, J = 7.9 Hz, 1H), 6.62−6.53 (m, 2H),

4.60−4.43 (m, 2H), 4.41−4.32 (m, 2H), 4.28 (dd, J = 8.3, 2.3 Hz,

2H), 4.13 (d, J = 15.6 Hz, 1H), 4.01 (dd, J = 8.4, 6.5 Hz, 2H), 3.88−

3.79 (m, 4H), 3.79−3.72 (m, 3H), 3.69−3.60 (m, 2H), 3.55−3.40

(m, 1H), 3.25−3.08 (m, 2H), 2.83−2.72 (m, 1H), 2.58 (tt, J = 8.0,

4.8 Hz, 2H), 2.49−2.38 (m, 1H), 2.12−1.94 (m, 5H), 1.89 (s, 1H),

1.84−1.72 (m, 1H), 1.72−1.59 (m, 4H), 1.59−1.42 (m, 1H), 1.21−

1.11 (m, 2H), 1.06−0.97 (m, 2H). ESI-MS calcd for C₃₈H₅₂FN₄O₆S

[M + H]⁺= 711.36, found: 711.15.

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-((1-(4-((1-((E)-4-

(dimethylamino)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)-3-me-

thoxyazetidin-3-yl)methyl)piperidin-4-yl)-1-(3-ﬂuorophenyl)ethyl)-

cyclopentyl)carbamate (19). Compound 19 was prepared from

compounds 39d, 42a, and (2E)-4-(dimethylamino)-2-butenoyl

chloride hydrochloride instead of acrylic anhydride according to the

method used to produce compound 24. ¹H NMR (400 MHz, MeOH-

d₄) δ 7.70−7.64 (m, 2H), 7.44−7.36 (m, 1H), 7.12−7.04 (m, 2H),

7.00 (d, J = 8.0 Hz, 1H), 6.68 (dt, J = 15.3, 7.1 Hz, 1H), 6.58−6.52

(m, 2H), 6.41 (dt, J = 15.2, 1.3 Hz, 1H), 4.53−4.37 (m, 4H), 4.35−

4.20 (m, 6H), 4.21−4.02 (m, 4H), 3.96−3.85 (m, 4H), 3.76−3.67

(m, 1H), 3.49 (s, 4H), 3.46−3.38 (m, 5H), 3.11−2.95 (m, 2H), 2.84

(s, 6H), 2.73 (d, J = 9.2 Hz, 1H), 2.48 (dt, J = 19.0, 9.5 Hz, 1H),

2.42−2.30 (m, 1H), 2.06−1.87 (m, 5H), 1.87−1.76 (m, 1H), 1.76−

1.67 (m, 1H), 1.67−1.54 (m, 3H), 1.54−1.40 (m, 1H), 1.20−1.00

(m, 1H); ESI-MS calcd for C₄₃H₆₂FN₆O₆S [M + H]⁺= 809.44,

found: 809.28.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-Acryloylazetidin-3-yl)-

sulfonyl)phenyl)-3-methoxyazetidin-3-yl)methyl)piperidin-4-yl)-2-

(azetidin-1-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(20). Compound 20 was prepared from compounds 39d and 42a

according to the procedure used to produce compound 24. ¹H NMR

(400 MHz, MeOH-d4) δ 7.79−7.73 (m, 2H), 7.54−7.43 (m, 1H),

7.21−7.13 (m, 2H), 7.08 (d, J = 8.0 Hz, 1H), 6.32 (dd, J = 17.0, 9.5

Hz, 1H), 6.25 (dd, J = 17.0, 2.7 Hz, 1H), 5.77 (dd, J = 9.5, 2.7 Hz,

1H), 4.61−4.44 (m, 4H), 4.42−4.34 (m, 1H), 4.34−4.23 (m, 4H),

4.23−4.10 (m, 3H), 3.98 (dd, J = 9.7, 5.8 Hz, 2H), 3.79 (d, J = 15.6

Hz, 1H), 3.57 (s, 4H), 3.54−3.42 (m, 5H), 3.19−3.01 (m, 2H),

2.87−2.75 (m, 1H), 2.62−2.51 (m, 1H), 2.51−2.39 (m, 1H), 2.13−

1.95 (m, 4H), 1.93−1.75 (m, 2H), 1.75−1.61 (m, 4H), 1.61−1.46

(m, 1H), 1.27−1.04 (m, 1H); ESI-MS calcd for C₄₀H₅₅FN₅O₆S [M +

H]⁺= 752.39, found: 752.21.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-Acryloylpiperidin-4-yl)-

sulfonyl)phenyl)-3-methoxyazetidin-3-yl)methyl)piperidin-4-yl)-2-

(azetidin-1-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(21). Compound 21 was prepared from compounds 39d and 42b

according to the procedure used to produce compound 24. ¹H NMR

(400 MHz, MeOH-d₄) δ 7.65−7.59 (m, 2H), 7.43 (td, J = 8.3, 6.2

Hz, 1H), 7.17−7.07 (m, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.69 (dd, J =

16.8, 10.7 Hz, 1H), 6.60−6.53 (m, 2H), 6.13 (dd, J = 16.8, 1.9 Hz,

1H), 5.69 (dd, J = 10.7, 1.9 Hz, 1H), 4.58 (d, J = 13.5 Hz, 1H), 4.47

(s, 2H), 4.38−4.22 (m, 5H), 4.22−4.04 (m, 2H), 3.92 (dd, J = 9.7,

5.9 Hz, 2H), 3.74 (d, J = 15.5 Hz, 1H), 3.60−3.49 (m, 4H), 3.45 (s,

5H), 3.37−3.31 (m, 1H), 3.16−2.95 (m, 3H), 2.83−2.72 (m, 1H),

2.72−2.61 (m, 1H), 2.57−2.46 (m, 1H), 2.45−2.29 (m, 1H), 2.11−

1.89 (m, 6H), 1.88−1.79 (m, 1H), 1.78−1.70 (m, 1H), 1.70−1.54

(m, 4H), 1.54−1.35 (m, 3H), 1.13 (s, 1H); ESI-MS calcd for

C₄₂H₅₉FN₅O₆S [M + H]⁺= 780.42, found:780.33.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(((1S,4S)-5-Acryloyl-2,5-

diazabicyclo[2.2.1]heptan-2-yl)sulfonyl)phenyl)-3-methoxyazeti-

din-3-yl)methyl)piperidin-4-yl)-2-(azetidin-1-yl)-1-(3-ﬂuorophenyl)-

ethyl)cyclopentyl)carbamate (24). Compound 39d (2.20 g, 3.48

mmol) was dissolved in DCM (50 mL), and then triﬂuoroacetic acid

(5.0 mL, 73.1 mmol) was added. After stirring for 2 h at rt, the

reaction mixture was evaporated. The crude product was dissolved in

DMSO (30 mL), and compound 42e (1.49 g, 4.18 mmol) and K₂CO₃

(1.92 g, 13.9 mmol) were added. The mixture was stirred at 80 °C

overnight. The mixture was quenched with H₂O and puriﬁed by

reversed-phase preparative HPLC to give 40e (1.66 g, 59%).

Compound 40e (1.28 g, 1.50 mmol) was dissolved in DCM (25

mL), and then TFA (2.2 mL, 30.0 mmol) was added slowly. After

stirring for 2 h at rt, the reaction mixture was evaporated to give the

crude product without further puriﬁcation. Acrylic anhydride (206 μL,

1.80 mmol) and Et₃N (623 μL, 4.50 mmol) were added to a solution

of this crude product in DCM (20 mL). After stirring for 3 h at rt, the

reaction mixture was evaporated and the residue was puriﬁed by

reversed-phase preparative HPLC to give 24 (927 mg, 78%). ¹H

NMR (400 MHz, MeOH-d4) δ 7.69−7.62 (m, 2H), 7.45 (q, J = 7.6

Hz, 1H), 7.18−7.08 (m, 2H), 7.07−6.98 (m, 1H), 6.66−6.59 (m,

0.5H), 6.59−6.52 (m, 2H), 6.32 (dd, J = 16.8, 10.0 Hz, 0.5H), 6.23

(ddd, J = 16.8, 2.1, 1.2 Hz, 1H), 5.72 (ddd, J = 10.0, 3.4, 2.1 Hz, 1H),

4.73 (s, 1H), 4.58−4.41 (m, 2H), 4.40−4.29 (m, 2H), 4.29−4.21 (m,

2H), 4.13 (d, J = 15.6 Hz, 1H), 3.91 (t, J = 8.7 Hz, 2H), 3.76 (d, J =

15.6 Hz, 1H), 3.63−3.54 (m, 2H), 3.54−3.50 (m, 3H), 3.50−3.45

(m, 4H), 3.44−3.38 (m, 1H), 3.34 (dd, J = 11.6, 2.2 Hz, 0.5H), 3.27

(s, 3H), 3.18 (dd, J = 9.6, 2.1 Hz, 0.5H), 3.14−2.97 (m, 2H), 2.85−

2.71 (m, 1H), 2.61−2.47 (m, 1H), 2.47−2.34 (m, 1H), 2.13−1.90

(m, 4H), 1.89−1.72 (m, 4H), 1.72−1.57 (m, 4H), 1.49 (d, J = 12.8

Hz, 1H), 1.41−1.23 (m, 1H), 1.18−0.98 (m, 1H); ¹³C NMR (101

MHz, MeOH-d4) δ 165.14, 162.70, 159.82, 154.38 (d, J = 4.4 Hz),

131.20 (d, J = 8.4 Hz), 130.26 (d, J = 6.2 Hz), 129.25, 129.10, 129.05,

128.76, 126.37 (d, J = 3.9 Hz), 125.56, 117.05 (d, J = 23.1 Hz),

115.61 (d, J = 21.2 Hz), 112.28, 74.16, 61.38, 60.53, 59.72, 58.61 (d, J

= 6.7 Hz), 57.53, 56.72, 55.74, 55.06, 54.84, 52.96, 51.73, 50.94,

41.32, 37.54, 35.90, 33.72, 26.90, 26.20, 25.77, 21.21, 17.08; ESI-MS

calcd for C₄₂H₅₈FN₆O₆S [M + H]⁺= 793.41, found: 793.16.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(((1R,4R)-5-Acryloyl-2,5-

diazabicyclo[2.2.1]heptan-2-yl)sulfonyl)phenyl)-3-methoxyazeti-

din-3-yl)methyl)piperidin-4-yl)-2-(azetidin-1-yl)-1-(3-ﬂuorophenyl)-

ethyl)cyclopentyl)carbamate (25). Compound 25 was prepared from

compounds 39d and 42f according to the procedure used to produce

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-(((S)-1-Acryloylpiperidin-3-yl)-

sulfonyl)phenyl)-3-methoxyazetidin-3-yl)methyl)piperidin-4-yl)-2-

(azetidin-1-yl)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate

(22). Compound 22 was prepared from compounds 39d and 42c

according to the procedure used to produce compound 24. ¹H NMR

(400 MHz, MeOH-d4) δ 7.73−7.67 (m, 2H), 7.53−7.43 (m, 1H),

1

compound 24. H NMR (400 MHz, MeOH-d4) δ 7.72−7.67 (m,

2H), 7.49 (q, J = 7.7 Hz, 1H), 7.21−7.12 (m, 2H), 7.07 (d, J = 8.0

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Hz, 1H), 6.69−6.62 (m, 0.5H), 6.62−6.57 (m, 2H), 6.36 (dd, J =

16.8, 10.0 Hz, 0.5H), 6.30−6.23 (m, 1H), 5.75 (ddd, J = 10.0, 3.8, 2.1

Hz, 1H), 4.77 (s, 1H), 4.53 (s, 2H), 4.36 (s, 2H), 4.32−4.24 (m, 2H),

4.16 (d, J = 15.7 Hz, 1H), 3.95 (dd, J = 9.5, 4.8 Hz, 2H), 3.84−3.75

(m, 1H), 3.67−3.57 (m, 2H), 3.57−3.54 (m, 3H), 3.54−3.49 (m,

4H), 3.48−3.42 (m, 1H), 3.38 (dd, J = 11.7, 2.1 Hz, 0.5H), 3.31−3.27

(m, 3H), 3.22 (dd, J = 9.5, 2.1 Hz, 0.5H), 3.18−3.00 (m, 2H), 2.81

(d, J = 9.5 Hz, 1H), 2.62−2.38 (m, 2H), 2.13−1.95 (m, 4H), 1.93−

1.76 (m, 3H), 1.76−1.61 (m, 4H), 1.54 (d, J = 12.8 Hz, 1H), 1.46−

1.35 (m, 1H), 1.35−1.27 (m, 1H), 1.15 (s, 1H); ESI-MS calcd for

C₄₂H₅₈FN₆O₆S [M + H]⁺= 793.41, found: 793.12.

Methyl ((1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(3-ﬂuorophenyl)-1-(1-

((3-methoxy-1-(4-(((1S,4S)-5-propionyl-2,5-diazabicyclo[2.2.1]-

heptan-2-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-

ethyl)cyclopentyl)carbamate (26). Compound 26 was prepared from

compounds 39d, 42e, and propionic anhydride instead of acrylic

anhydride according to the procedure used to produce compound 24.

¹H NMR (400 MHz, MeOH-d4) δ 7.72−7.66 (m, 2H), 7.53−7.44

2.72 (m, 1H), 2.60 (q, J = 9.4 Hz, 1H), 2.00−1.92 (m, 1H), 1.90 (s,

2H), 1.89−1.68 (m, 4H), 1.64−1.52 (m, 2H), 1.52−1.41 (m, 2H),

1.40−1.30 (m, 1H); ESI-MS calcd for C₂₇H₃₇FN₃O₂[M + H]⁺

=

454.29, found: 454.24.

Methyl ((1S,2R)-2-((S)-1-(1-Benzylpiperidin-4-yl)-2-((tert-

butoxycarbonyl)amino)-1-(3-ﬂuorophenyl)ethyl)cyclopentyl)-

carbamate (30). Compound 29 (2.68 g, 5.91 mmol) was dissolved in

dry dichloromethane (50 mL). Then, Et₃N (1.2 mL, 8.87 mmol) and

di-tert-butyl dicarbonate (2.71 mL, 11.8 mmol) were added. After

stirring for 2 h at rt, the reaction mixture was concentrated under

vacuum. The residue was puriﬁed by column chromatography (silica

1

gel, DCM/MeOH 20:1 to 8:1) to give 30 (2.30 g, 70%). H NMR

(400 MHz, MeOH-d4) δ 7.42−7.18 (m, 8H), 6.99 (td, J = 8.1, 2.3

Hz, 1H), 4.10 (q, J = 6.8 Hz, 1H), 3.84−3.73 (m, 1H), 3.70−3.60 (m,

1H), 3.54 (s, 3H), 3.49 (s, 2H), 3.00−2.85 (m, 2H), 2.45 (q, J = 8.4

Hz, 1H), 2.05−1.92 (m, 3H), 1.92−1.78 (m, 2H), 1.67−1.51 (m,

4H), 1.47 (s, 9H), 1.42−1.30 (m, 3H), 1.25−1.15 (m, 1H); ESI-MS

calcd for C₃₂H₄₅FN₃O₄[M + H]⁺= 554.34, found: 554.09.

(m, 1H), 7.20−7.12 (m, 2H), 7.08 (d, J = 8.0 Hz, 1H), 6.64−6.55 (m,

2H), 4.74−4.43 (m, 4H), 4.43−4.31 (m, 2H), 4.31−4.24 (m, 2H),

4.21−4.10 (m, 1H), 3.96 (t, J = 8.5 Hz, 2H), 3.84−3.75 (m, 1H),

3.56 (s, 3H), 3.54−3.44 (m, 6H), 3.42−3.35 (m, 0.5H), 3.30−3.25

(m, 2H), 3.19 (dd, J = 9.5, 2.0 Hz, 0.5H), 3.17−3.02 (m, 2H), 2.81

(d, J = 9.4 Hz, 1H), 2.61−2.49 (m, 1H), 2.50−2.40 (m, 1H), 2.40−

2.29 (m, 1H), 2.22−2.08 (m, 2H), 2.08−1.95 (m, 5H), 1.94−1.84

(m, 1H), 1.84−1.73 (m, 2H), 1.73−1.59 (m, 4H), 1.59−1.48 (m,

1H), 1.37−1.26 (m, 1H), 1.11 (t, J = 7.5 Hz, 2H), 1.05 (t, J = 7.5 Hz,

2H); ESI-MS calcd for C₄₂H₆₀FN₆O₆S [M + H]⁺= 795.43, found:

827.32.

Methyl ((1S,2R)-2-((S)-2-((tert-Butoxycarbonyl)amino)-1-(1-((1-

(4-(cyclopropylsulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-

1-(3-ﬂuorophenyl)ethyl)cyclopentyl)carbamate (31). Palladium on

carbon (150 mg, 10 wt %) was added to a solution of compound 30

(500 mg, 0.903 mmol) in MeOH (10 mL) under a N₂atmosphere.

The solution was brieﬂy vacuumed to remove the N₂atmosphere and

then put under a H₂atmosphere; this was repeated three times. The

mixture was then stirred for 6 h at rt under a H₂atmosphere. The Pd/

C catalyst was then ﬁltered, and the solvent was removed by rotary

evaporation. The residue was then redissolved in acetonitrile (5 mL).

K₂CO₃(374 mg, 2.71 mmol), KI (15 mg, 0.0903 mmol), and 36 (374

mg, 1.08 mmol) were added to the solution. The mixture was stirred

at 80 °C overnight. Then, the mixture was concentrated in vacuum

and puriﬁed by reversed-phase preparative HPLC to give 31 (399 mg,

62%). ESI-MS calcd for C₃₈H₅₄FN₄O₆S [M + H]⁺= 713.37, found:

713.15.

Cyclopropyl(4-ﬂuorophenyl)sulfane (33). To a stirred solution of

32 (2.0 mL, 18.7 mmol) in DMSO (50 mL) under a N₂atmosphere

were added cyclopropyl bromide (1.6 mL, 20.6 mmol) and t-BuONa

(4.49 g, 46.8 mmol). Then the reaction mixture was heated at 80 ° C

for 24 h. After cooling to rt, the mixture was poured into H₂O (250

mL) and extracted with Et₂O three times. The combined organic

phases were washed with brine, dried over Na₂SO₄, and concentrated

in vacuum. The residue was puriﬁed by column chromatography

(silica gel, hexane/EtOAc 100:1 to 15:1) to give 33 (1.32 g, 42%).

Spectral data was identical to the literature compound.³⁰

Methyl ((1S,2R)-2-((S)-(1-Benzylpiperidin-4-yl)(cyano)(3-

ﬂuorophenyl)methyl)cyclopentyl)-carbamate (28). Compound 27

(2.28 g, 4.64 mmol) was dissolved in dichloromethane (14 mL), and

TFA (6.9 mL, 92.9 mmol) was added slowly. After stirring for 2 h at

rt, the reaction mixture was concentrated under vacuum. The crude

product was dissolved in dry dichloromethane (50 mL). Then, Et₃N

(5.2 mL, 37.1 mmol) and dimethyl dicarbonate (1.24 mg, 9.28 mmol)

were added at 0 °C. After stirring for 2 h at rt, the reaction mixture

was concentrated under vacuum. The residue was puriﬁed by column

chromatography (silica gel, DCM/MeOH 20:1 to 10:1) to give 28

1

(1.86 g, 89%). H NMR (400 MHz, MeOH-d4) δ 7.43 (td, J = 8.0,

6.0 Hz, 1H), 7.38−7.23 (m, 7H), 7.11 (tdd, J = 8.3, 2.5, 1.0 Hz, 1H),

3.88 (q, J = 7.0 Hz, 1H), 3.49 (s, 2H), 3.46 (s, 3H), 2.99−2.89 (m,

2H), 2.84 (td, J = 8.3, 6.9 Hz, 1H), 2.15−2.04 (m, 2H), 2.04−1.94

(m, 3H), 1.88−1.76 (m, 1H), 1.72−1.60 (m, 3H), 1.60−1.48 (m,

2H), 1.43−1.34 (m, 1H), 1.26 (td, J = 12.7, 4.0 Hz, 1H); ESI-MS

calcd for C₂₇H₃₃FN₃O₂[M + H]⁺= 450.26, found: 450.08.

1-(Cyclopropylsulfonyl)-4-ﬂuorobenzene (34). m-CPBA (4.28 g,

17.4 mmol, 70%) was added to a stirred solution of 33 (1.46 g, 8.68

mmol) in DCM (80 mL) at 0 °C. After 2 h, the reaction mixture was

quenched with 1 M NaOH (aq.) and extracted with DCM three

times. The combined organic phases were washed with brine, dried

over Na₂SO₄, and concentrated in vacuum. The residue was puriﬁed

by column chromatography (silica gel, hexane/EtOAc 10:1 to 1:1) to

give 34 (1.58 g, 91%). ¹H NMR (400 MHz, CDCl₃) δ 7.97−7.91 (m,

2H), 7.28−7.22 (m, 2H), 2.47 (tt, J = 8.0, 4.8 Hz, 1H), 1.40−1.34

(m, 2H), 1.11−1.03 (m, 2H).

Methyl ((1S,2R)-2-((S)-2-Amino-1-(1-benzylpiperidin-4-yl)-1-(3-

ﬂuorophenyl)ethyl)cyclopentyl)carbamate (29). Compound 28

(7.62 g, 16.9 mmol) was added to a dry RB-ﬂask, which was then

covered with a Kimwipe and put in a desiccator that was put under

vacuum for 1−2 days. After the vacuuming step, the ﬂask was

removed from the desiccator and quickly capped with a septum and

the system was vacuumed under a N₂atmosphere. Anhydrous toluene

(150 mL) was added to the ﬂask and then was cooled to 0 °C in an

ice-bath. Diisobutylaluminum hydride (25% in toluene, 45.2 mL, 67.8

mmol) was injected into the reaction mixture slowly at 0 °C with a

syringe under stirring. After 4 h, the reaction was quenched with

potassium sodium tartrate (aq). The mixture was stirred vigorously

overnight and was extracted with EtOAc three times. The organic

solvent was washed with brine, dried with Na₂SO₄, ﬁltered, and

concentrated under rotatory vacuum. The residue was redissolved in

MeOH (200 mL), NaBH₄(2.56 g, 67.8 mmol) was added slowly at 0

°C, and the reaction mixture was stirred at rt overnight. The reaction

mixture was concentrated and diluted with water and extracted with

DCM three times. The organic solvent was washed with brine, dried

with Na₂SO₄, ﬁltered, and concentrated under rotatory vacuum to

give crude product 29 (6.52 g, 85%), which was used without further

Methyl 1-(4-(Cyclopropylsulfonyl)phenyl)azetidine-3-carboxy-

late (35). Compound 34 (819 mg, 4.09 mmol) was dissolved in

DMSO (20 mL), methyl azetidine-3-carboxylate hydrochloride (620

mg, 4.09 mmol), and K₂CO₃(2.26 g, 16.4 mmol) were added. The

mixture was quenched with water and extracted with EA three times.

The combined organic phases were washed with brine, dried over

Na₂SO₄, and concentrated in vacuum. The residue was puriﬁed by

column chromatography (silica gel, hexane/EtOAc 2:1 to 1:2) to give

1

35 (729 mg, 60%). H NMR (400 MHz, CDCl₃) δ 7.71−7.66 (m,

2H), 6.46−6.41 (m, 2H), 4.21−4.10 (m, 4H), 3.76 (s, 3H), 3.60 (tt, J

= 8.6, 6.0 Hz, 1H), 2.40 (tt, J = 8.0, 4.8 Hz, 1H), 1.32−1.22 (m, 2H),

1.01−0.90 (m, 2H); ESI-MS calcd for C₁₄H₁₈NO₄S [M + H]⁺

296.10, found: 259.82.

=

1

puriﬁcation. H NMR (400 MHz, MeOH-d₄) δ 7.37−7.24 (m, 6H),

(1-(4-(Cyclopropylsulfonyl)phenyl)azetidin-3-yl)methyl Metha-

nesulfonate (36). Compound 35 (729 mg, 2.47 mmol) was dissolved

in THF (30 mL), and LiAlH₄(4.9 mL, 1 M in THF) was added at 0

7.08−7.00 (m, 2H), 6.99−6.92 (m, 1H), 3.64−3.51 (m, 2H), 3.39−

3.25 (m, 2H), 3.23 (s, 3H), 3.08 (s, 2H), 2.93−2.81 (m, 1H), 2.81−

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°C. After 4 h, the reaction was quenched with potassium sodium

tartrate (aq). The mixture was stirred vigorously overnight and was

extracted with EtOAc three times. The organic solvent was washed

with brine, dried with Na₂SO₄, ﬁltered, and concentrated under

rotatory vacuum. The residue was redissolved in DCM (25 mL), and

Et₃N (1.0 mL, 7.41 mmol) and methanesulfonyl chloride (230 μL,

2.96 mmol) were added. After stirring under rt for 1 h, the reaction

was quenched with water and extracted with DCM three times. The

combined organic phases were washed with brine, dried over Na₂SO₄,

and concentrated in vacuum. The residue was puriﬁed by column

chromatography (silica gel, DCM/MeOH 20:1 to 15:1) to give 36

(699 mg, 82%). ¹H NMR (400 MHz, CDCl₃) δ 7.73−7.67 (m, 2H),

6.46−6.40 (m, 2H), 4.46 (d, J = 6.7 Hz, 2H), 4.10 (t, J = 8.1 Hz, 2H),

3.81 (dd, J = 8.0, 5.0 Hz, 2H), 3.25−3.13 (m, 1H), 3.06 (s, 3H), 2.41

(tt, J = 8.0, 4.8 Hz, 1H), 1.32−1.25 (m, 2H), 1.00−0.93 (m, 2H);

ESI-MS calcd for C₁₄H₂₀NO₅S₂[M + H]⁺= 346.08, found: 345.92.

tert-Butyl (1S,4S)-5-((4-Fluorophenyl)sulfonyl)-2,5-diazabicyclo-

[2.2.1]heptane-2-carboxylate (42e). (1S,4S)-2-Boc-2,5-diazabicyclo-

[2.2.1]heptane (1.0 g, 5.05 mmol) was added to a solution of 4-

ﬂuorobenzenesulfonyl chloride (1.08 g, 5.55 mmol) and Et₃N (2.1

mL, 15.2 mmol) in 40 mL of DCM. After 5 h, H₂O was added and

the reaction was extracted three times with DCM, concentrated, and

puriﬁed by column chromatography (silica gel, hexane/EtOAc 3:1 to

which were built in using MOE utilities. All three of the breaks were

distant from the binding site. Both the N- and C-termini were capped

due to unresolved residues. Missing sidechains were built in using

MOE utilities. Both termini and the missing sidechains were distant

from the binding site except for the sidechain of Arg332, which was

approximately 5 Å from the tail of the inhibitor. The system was

parameterized with AMBER 10. The Cys329 sulfur atom was

displayed as being covalently bound to the inhibitor. That bond

was removed, and the cysteine residue was allowed to move away

from the inhibitor using energy minimization keeping all other atoms

ﬁxed. Bond orders for the inhibitor were checked, the inhibitor was

protonated appropriately, and then partial charges for it were obtained

using the MMFF94 (modiﬁed) force ﬁeld since the inhibitor exceeds

the AM1-BCC size limits in MOE. All heavy atoms were ﬁxed, and

the positions of the hydrogen atoms were allowed to relax using

energy minimization. This prepared structure was used as the starting

point for the modeling of the inhibitors. Compounds 24 and 25 were

each separately built into the Menin binding site by modifying the M-

808 inhibitor. Once the new inhibitor had been built into the binding

site, the whole system was assigned AMBER 10 parameters. Following

that, the inhibitor was assigned partial charges using AM1-BCC.

(Because of the smaller sizes of compounds 24 and 25, it was possible

to use AM1-BCC.) A series of minimizations were then conducted to

generate the ﬁnal models for the 24 and 25 inhibitors in complex with

Menin. The positions of the hydrogen atoms were ﬁrst relaxed with

energy minimization while all heavy atoms were held ﬁxed. Next,

portions of the inhibitor that had been modiﬁed to create inhibitors

24 and 25 were relaxed using energy minimization. Sidechains of any

residue having at least one atom within 6 Å of the inhibitor were then

also allowed to relax. Finally, all of the atoms of the inhibitor and any

residue with at least one atom within 6 Å of the inhibitor were allowed

to relax with energy minimization.

Crystallization and Structure Determination. Menin (residues

2−610 containing a deletion from 460 to 519) was puriﬁed as

previously described.²⁶For crystallization, Menin (25 mg/mL in 25

mM Tris 8.0, 150 mM NaCl, and 5 mM DTT) was mixed with M-

1121 in a protein to compound ratio of 1:1.2 and then immediately

set up for crystallization at 4 °C. All crystals grew in drops containing

1 μL of the complex and 1 μL of well solution (1.96 M NaCl, 89 mM

Bis-Tris pH 6.8, 0.178 M MgCl₂, and 10.7 mM Pr Acetate). Crystals

were cryoprotected by progressively soaking crystals in well solution

with increasingly higher amounts of sodium formate (1−5 M in 1 M

steps). Diﬀraction data were collected on an Eiger 9 M detector at the

Advanced Photon Source LS-CAT 21-ID-D beamline at the Argonne

National Laboratory. All data were processed with HKL2000.³³The

structure of menin-1121 was solved by molecular replacement

(Molrep³⁴) using the protein structure from PDB ID 6WNH as the

search model. The structure went through iterative rounds of electron

density ﬁtting and structural reﬁnement using Coot³⁵and Buster,³⁶

respectively. The coordinates and restraint ﬁles for the ligands were

created from smiles in Grade³⁷with the mogul+qm option. The initial

Fo-Fc electron density map showed the presence of one compound

covalently bound to C329 (Figure S1). 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 S1.

1

1:2) to give 42e as a white solid. H NMR (400 MHz, CDCl₃) δ

7.93−7.80 (m, 2H), 7.21 (td, J = 8.7, 2.3 Hz, 2H), 4.50−4.30 (m,

2H), 3.40 (ddd, J = 23.1, 14.1, 9.2 Hz, 2H), 3.29−3.13 (m, 2H), 1.71

(dd, J = 15.1, 10.1 Hz, 1H), 1.41 (d, J = 12.2 Hz, 9H), 1.33 (t, J = 8.0

Hz, 1H). ESI-MS calcd for C₁₆H₂₂FN₂O₄S [M + H]⁺= 357.13,

found: 357.01.

Fluorescence Polarization (FP)-Based Binding Assay. Com-

pound binding was measured using an FP assay as described

previously.²⁷Brieﬂy, 5 μL of compounds at various concentrations in

dimethyl sulfoxide (DMSO) solution was added to 195 μL of a

mixture of menin and the ﬂuorescein-labeled tracer (compound 37 in

our previous publication²⁷) in the assay buﬀer (phosphate-buﬀered

saline, 100 μg/mL bovine γ-globulin, with 0.01% Triton X-100), and

the mixture was incubated for 1 h at rt. Final concentrations of the

menin protein and the ﬂuorescein-labeled tracer were 4 and 2 nM,

respectively. FP values were measured using an Inﬁnite M-1000 plate

reader (Tecan, Morrisville, NC). The IC₅₀values were determined by

nonlinear regression ﬁtting of the sigmoidal dose-dependent FP

decreases as a function of total compound concentrations using the

GraphPad Prism 5.0 software.

Cell Growth Evaluation. MLLr and MLL wild-type leukemia cell

lines were plated in 96-well tissue culture plates in triplicate.

Compounds were added in a 9-point dose response curve starting

from 10 μM top concentration with 1:3 serial dilutions. Compounds

were diluted in DMSO to a ﬁnal concentration of 0.1% DMSO in

media. One column on each plate was designated for the 0.1% DMSO

vehicle control. Total cellular ATP levels were measured using the

CellTiter-Glo (Promega) reagent on the day of cell plating (day 0

readout). The cells were then incubated at 37 °C in RPMI 1640

media (Gibco) and 5% CO₂for 4 days, and total cellular ATP levels

were measured again using the CellTiter-Glo reagent. ATP standard

curves were generated on both day 0 and day 4. IC₅₀was calculated

using the GraphPad Prism data analysis software. The catalog

numbers for each cell lines are MV4;11 (CRL-9591, ATCC), OCI-

AML4 (ACC-729, DSMZ), MOLM-13 (C0003003, AddexBio), SEM

(ACC-546, DSMZ), KOPN8 (ACC-552, DSMZ), RS4;11(CRL-

1873, ATCC), HL-60 (CCL-240, ATCC), K562 (CCL-243, ATCC),

and MEG-1 (CRL-2021, ATCC).

Mass Spectroscopic Analysis of the Human Menin Protein

Incubated with Menin Inhibitors. Samples of menin (25 mg/mL

in 25 mM Tris 8.0, 150 mM NaCl, and 5 mM DTT) were incubated

with compounds in a protein-to-compound molar ratio of 1:1.2 for 1

h or overnight at 4 °C. Following incubation, the sample was diluted

to 1 mg/mL with H₂O. Each sample (0.1 mL) was subjected to a

reversed-phase HPLC column (Phenomenex Aeris widepore C4

column 3.6 μM, 50 mm × 2.10 mm) at a ﬂow rate of 0.5 mL/min in

H₂O with 0.2% (v/v) HCOOH. The protein was eluted using a

gradient of 5−100% MeCN with 0.2% (v/v) HCOOH over 4 min.

The liquid chromatography−mass spectrometry (LC−MS) experi-

ment (Agilent Q-TOF 6545) was carried out under the following

conditions: fragmentor voltage, 300 V; skimmer voltage, 75 V; nozzle

voltage, 100 V; sheath gas temperature, 350 °C; and drying gas

Computational Modeling. This modeling is based on the crystal

structure of Menin bound to inhibitor M-808 (PDB id: 6WNH),

which was obtained from the RCSB. All of the modeling was

conducted using the software package MOE.³⁸The 6WNH structure

was imported into MOE and prepared for modeling in a standard

fashion. Brieﬂy, the crystallization additives were removed, and

crystallographic water molecules were retained. There were three

chain breaks due to unresolved residues. Ends for two of the breaks

were capped. The third break only involved three missing residues,

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temperature, 325 °C. The MassHunter Qualitative Analysis software

(Agilent) was used to analyze the data. Intact protein masses were

obtained using the maximum entropy deconvolution algorithm.

Real-Time PCR. Total RNA was isolated from either cell grown in

vitro or bone marrow samples enriched for human CD45⁺cells by an

EasySep Human CD45 Depletion Kit (STEMCELL Technologies)

using an RNeasy kit (QIAGEN) according to the manufacturer’s

protocol. The cDNA was generated using a High-Capacity cDNA

Reverse Transcription Kit (Applied Biosystems). Real-time PCR

ampliﬁcations of HOXA9, MEIS1, ITGAM, GAPDH, and

HPRT1genes were carried out with primers speciﬁc for each gene,

using TaqMan gene expression assays (Applied Biosystems). Relative

quantiﬁcation of each gene transcript was calculated by a comparative

cycle threshold (Ct) method. The results were presented as relative

expression to vehicle treatment after normalizing to an internal

loading control GAPDH or HPRT1. The catalog numbers for primers

of each genes are HOXA9 (Hs00365956_m1), MEIS1

(Hs00180020_m1), ITGAM (Hs00167304_m1), GAPDH

(Hs99999905_m1), and HPRT1 (Hs99999909_m1).

Plasma Protein Binding. To determine plasma protein binding

using a dialysis method, dialysis buﬀer was loaded into the receiver

side of a dialysis chamber and plasma or dialysis buﬀer spiked with M-

1121 (1 μM), or plasma spiked with warfarin (1 μM) or quinidine (1

μM) was loaded into the donor side of the dialysis chambers and the

dialysis block was shaken at 37 °C for 5 h. After incubation, samples

from both the donor and receiver sides of the dialysis apparatus were

added to a 96-well plate and mixed with the same volume of opposite

matrices (blank buﬀer to plasma and blank plasma to buﬀer). To

prepare 0 h samples, plasma or dialysis buﬀer spiked with M-1121 (1

μM) or 100 μL of plasma spiked with warfarin (1 μM) or quinidine

(1 μM) was added to a 96-well plate and mixed with the same volume

of blank buﬀer. Zero-hour samples were stored at −20 °C until

analysis. At the time of analysis, all samples were quenched with

acetonitrile containing the internal standard imipramine. After

quenching, assay plates were shaken followed by centrifugation.

Supernatants were removed and added to a new 96-well plate and

then diluted with Mill-Q water before being analyzed by liquid

chromatography with tandem mass spectrometry. The peak area ratio

between the sample and the internal standard was used to calculate

percent bound, percent unbound, and recovery.

mean standard error (SEM). Graphing and statistical analysis were

performed using GraphPad Prism 8.00 (GraphPad Software).

In Vivo Studies in a Luciferase-Tagged MV4;11 AML

Xenograft Model. Five−six-week-old female NCG mice obtained

from Charles River Laboratories were implanted systemically via tail

vein injection with Luciferase-tagged-MV;411 cells at an inoculum

level of 5 × 10⁶cells/mouse in serum free media. The severity of the

disease was monitored by an IVIS SpectrumCT Imaging System twice

weekly as recommended by the manufacturer (PerkinElmer) until

disease burden reached the log phase (27 days post implant.) Mice

were randomized into two groups, vehicle and M-1121, that were

formulated and administered as mentioned above. Mice were treated

for 4 days, and bone marrow samples were collected 48 h after the last

dose and subjected to either staining for ﬂow cytometry evaluation or

human CD45⁺cell enrichment for downstream gene expression

evaluation by RT-qPCR.

Bioluminescence Imaging. Mice implanted with Luciferase-

tagged MV4;11 cells were peritoneally injected with luciferin

potassium salt (PerkinElmer, cat#122799-5) at a dose of 150 mg/

kg before undergoing anesthesia with 3% isoﬂurane (Patterson

Veterinary Supply, cat#07-806-3204) in air in an anesthesia induction

chamber. Bioluminescence imaging was performed using an IVIS

imaging system, and mice were imaged approximately 5 min after

substrate injection. Acquisition settings (binning and duration) were

set up depending upon tumor activity at the time of acquisition.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00789.

■

sı

Crystallography data collection and reﬁnement statistics.

In vivo pharmacokinetics and eﬃcacy data of compounds

10, 17, 24 (M-1121) in mice. Purity and spectral data of

M-1121. Method for computational modeling for

compounds 24 and 25 (PDF)

A molecular string ﬁle for all the ﬁnal target compounds

(CSV)

Modeled structures of compounds 24 and 25 in complex

with the menin protein (PBD) (PBD)

Flow Cytometry. Bone marrow cells were collected, and cell

suspensions were ﬁltered through a 70 μM cell strainer and washed

with PBS. Red blood cells were removed by using 1× RBC lysis buﬀer

(Sigma, cat#R7757.) Samples were washed twice with pre-cold PBS

and stained with a ﬁxable viability stain (BD, cat# 565388) followed

by human FcR blocker treatment (BD, cat#564219). Next, cells were

stained with an Anti-human CD45 (BD, cat#563204), Anti-human

CD33 (BD, cat#555626), and Anti-human CD11b (BD, cat#562721)

antibody mixture at 4 °C for 40 min in the dark. Samples were

suspended with cell staining buﬀer and analyzed on a Thermo Attune

NxT ﬂow cytometer.

Accession Codes

The coordinates for M-1121 complexed with menin have been

deposited into the Protein Data Bank with the PDB code

7M4T. Authors will release the atomic coordinates upon

publication of this article.

AUTHOR INFORMATION

Corresponding Author

■

Shaomeng Wang − Rogel Cancer Center, Department of

Internal Medicine, Department of Pharmacology, and

Department of Medicinal Chemistry, Medical School,

University of Michigan, Ann Arbor, Michigan 48109, United

States; orcid.org/0000-0002-8782-6950;

Email: shaomeng@umich.edu

Animal Experiments. Animal experiments were performed under

the guidelines of the University of Michigan Committee for Use and

Care of Animals and using an approved animal protocol (PI,

Shaomeng Wang) or in accordance with ChemPartner approved

Institutional Animal Care and Use Committee (IACUC) protocols.

In Vivo Eﬃcacy Studies in an MV4;11 Human AML

Xenograft Model in Mice. Five−six-week-old female C.B.-17

SCID mice were purchased from Charles River and implanted

subcutaneously in the right ﬂank with MV4;11 cells at an inoculum of

5 × 10⁶cells/mouse (cell suspension and Matrigel at a 1:1 ratio.)

Once tumors reached 200 mm³, mice were randomized into vehicle

(0.5% MC (400 cP) + 0.2% tween 80 (w/w)) or M-1121-treated

groups. Treatment was administered daily by oral gavage (QD) at 10

mL/kg.

Authors

Meng Zhang − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Angelo Aguilar − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Shilin Xu − Rogel Cancer Center and Department of Internal

Medicine, University of Michigan, Ann Arbor, Michigan

48109, United States; orcid.org/0000-0003-0845-0260

Body weights were measured daily, and tumor volume was

measured three times a week using Vernier calipers. Tumor volume

was calculated using the formula 0.5 × W × W × L, with W being the

tumor width and L being the tumor length. Results were graphed as

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Journal of Medicinal Chemistry

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Article

Liyue Huang − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Krishnapriya Chinnaswamy − Life Sciences Institute,

University of Michigan, Ann Arbor, Michigan 48109, United

States

Taryn Sleger − Agios Pharmaceuticals, Inc., Cambridge,

Massachusetts 02139, United States

Bo Wang − Agios Pharmaceuticals, Inc., Cambridge,

Massachusetts 02139, United States

of patent applications on this class of menin inhibitors, which

have been licensed to Medsyn Biopharma, LLC. S. Wang, M.

Zhang, A. Aguilar, S. Xu, L. Huang, T. Xu, K. Zheng, D.

McEachern, and J. Stuckey are co-inventors on one or more of

these patents and receive royalties on these patents from the

University of Michigan. S. Wang is a co-founder of Medsyn

Biopharma LLC. The University of Michigan and S. Wang own

stock in Medsyn. T.S, B.W, S.G., B.N.N., S.R., A.E.T and T.L

are current or former employees and shareholders of Agios

Pharmaceuticals, Inc.

Stefan Gross − Agios Pharmaceuticals, Inc., Cambridge,

ACKNOWLEDGMENTS

We thank G.W. A. Milne for critical reading and editing of the

manuscript.

Massachusetts 02139, United States

■

Brandon N. Nicolay − Agios Pharmaceuticals, Inc.,

Cambridge, Massachusetts 02139, United States

Sebastien Ronseaux − Agios Pharmaceuticals, Inc.,

Cambridge, Massachusetts 02139, United States

Kaitlin Harvey − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Yu Wang − Rogel Cancer Center and Department of Internal

Medicine, University of Michigan, Ann Arbor, Michigan

48109, United States

Donna McEachern − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Paul D. Kirchhoﬀ − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Zhaomin Liu − Rogel Cancer Center and Department of

Internal Medicine, University of Michigan, Ann Arbor,

Michigan 48109, United States

Jeanne Stuckey − Life Sciences Institute, University of

Michigan, Ann Arbor, Michigan 48109, United States

Adriana E. Tron − Agios Pharmaceuticals, Inc., Cambridge,

Massachusetts 02139, United States

ABBREVIATIONS

■

ALL, acute lymphoblastic leukemia; AML, acute myeloid

leukemia; DCM, dichloromethane; DIBAL-H, diisobutylalu-

minum hydride; DIPEA, N,N-diisopropylethylamine; ESI-MS,

electrospray ionization mass spectrometry; EtOAc, ethyl

acetate; FP, ﬂuorescence polarization; MEN1, multiple

endocrine neoplasia 1; MLL, mixed lineage leukemia protein;

GSH, glutathione; PBS, phosphate buﬀered saline; PD,

pharmacodynamics; rt, room temperature; SCID, severe

combined immunodeﬁcient; TFA, triﬂuoroacetic acid; UPLC,

ultraperformance liquid chromatography

REFERENCES

■

(1) Dimartino, J. F.; Cleary, M. L. Mll rearrangements in

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proteins. Br. J. Haematol. 2011, 152, 141−154.

(3) Slany, R. K. The molecular biology of mixed lineage leukemia.

Haematologica 2009, 94, 984−993.

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00789

Author Contributions

M.Z., A.A., S.X., and L.H. contributed equally. T.S, B.W.,

S.G., B.N.N., S.R., and A.E.T. designed and/or executed in

vitro and in vivo studies. A.E.T. and T.L. contributed to writing

and review of the manuscript.

Funding

This work is supported in part by the National Institutes of

Health/National Cancer Institute (R01 CA208267 to S.W.),

the Prostate Cancer Foundation (to S.W.), the Rogel Cancer

Center Core Grant from the National Cancer Institutes, NIH

(P30 CA046592), and a research contract from Agios

Pharmaceuticals (to S.W.). Use of the Advanced Photon

Source, an Oﬃce of Science User Facility operated for the U.S.

Department of Energy (DOE) Oﬃce of Science by the

Argonne National Laboratory, was supported by the U.S. DOE

under Contract No. DE-AC02-06CH11357. Use of the LS-

CAT Sector 21 was supported by the Michigan Economic

Development Corporation and the Michigan Technology Tri-

Corridor (Grant 085P1000817).

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The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled a number

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Article Doi

Discovery of M-1121 as an Orally Active Covalent Inhibitor of Menin-MLL Interaction Capable of Achieving Complete and Long-Lasting Tumor Regression

DOI: 10.1021/acs.jmedchem.1c00789

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