Discovery of M-808 as a Highly Potent, Covalent, Small-Molecule Inhibitor of the Menin-MLL Interaction with Strong in vivo Antitumor Activity

Abstract

Targeting the menin-MLL protein-protein interaction is a new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein the structure-based optimization of a class of covalent menin inhibitors, which led to the discovery of M-808 (16) as a highly potent and efficacious covalent menin inhibitor. M-808 effectively inhibits leukemia cell growth at low nanomolar concentrations and is capable of achieving partial tumor regression in an MV4;11 xenograft tumor model in mice at a well-tolerated dose schedule. Determination of the co-crystal structure of M-808 in complex with menin provides a structural basis for their high-affinity, covalent interactions. M-808 represents a promising, covalent menin inhibitor for further optimization and evaluation toward developing a new therapy for the treatment of MLL leukemia.

Graphical Abstract

Introduction

The chromosomal translocations of the mixed lineage leukemia (MLL) gene are observed in approximately 10% of adult acute myeloid leukemia (AML) and >70% of infant acute lymphoblastic leukemias (ALL).¹ The most common MLL translocation is its fusion with one of more than 70 translocation partner genes.^2-3 Patients with leukemia bearing a MLL translocation (MLL leukemia) have a particularly poor prognosis and fail to respond to currently available treatments^4-5, highlighting the urgent need for new treatment options for MLL leukemia.

In all the identified MLL fusion proteins, approximately 1400 amino acid residues from the wild-type MLL N-terminus are retained, which interact directly with menin protein.⁶ The menin-MLL protein-protein interaction has been shown to be critical for the expression of the HOXA and MEIS1 genes, which drive leukemogenesis in MLL leukemia.^7-8 Therefore, targeting the protein-protein interaction between menin and MLL represents a promising therapeutic strategy for the treatment of MLL leukemia.^{7, 9}

Analysis of a co-crystal structure of the menin-MLL complex suggests that targeting the menin-MLL protein-protein interaction with non-peptide small-molecule inhibitors (herein called menin inhibitors) is challenging but achievable^{6, 10}. In the last few years, several classes of reversible small-molecule menin inhibitors have been reported.¹¹ For example, inhibitors 1 (MI-503)^12-13, 2 (MI-3454)¹⁴ and 3 (VTP-50469)¹⁵ (Figure 1) containing the thienopyrimidine or pyrimide, bind to menin protein with low nanomolar affinities, and demonstrate in vivo activity in an MLL leukemia model in mice. The aminomethylpiperidine class of inhibitor 4 (MIV-6)¹⁶ shows moderate inhibitory activity toward the menin-MLL interaction and leukemia cell growth. Currently, two orally bioavailable menin inhibitors such as KO-539¹⁷ and SNDX-5613¹⁸, have progressed to clinical trials, although their chemical structure were not disclosed.

Figure 1.

Previously reported reversible and irreversible menin inhibitors.

Recently, using MIV-6 as the initial lead compound, our laboratory has reported the discovery of compounds 5 (M-89)¹⁹ and 6 (M-470)²⁰ as potent, reversible menin inhibitors. In addition to these reversible menin inhibitors, we also reported the discovery of M-525²⁰ as the first-in-class irreversible, covalent menin-MLL inhibitor (Figure 1). M-525 demonstrated high potency targeting the menin-MLL interaction and achieves potent activity in inhibition of leukemia cell growth in MLL leukemia cell lines.²⁰

In our continuous efforts toward identification of a highly potent and efficacious menin inhibitor for advanced preclinical development and future clinical trials, we have carried out further optimization of M-525. Our efforts have yielded a set of highly potent covalent menin inhibitors with M-808 identified as the most promising compound. M-808 achieves IC₅₀ values of 1 and 4 nM, respectively, in inhibition of cell growth in the MV4;11 and MOLM13 cell lines carrying MLL fusion and is capable of achieving partial tumor regression in the MV4;11 leukemia xenograft tumor model in mice. M-808 warrants extensive evaluation as a potential new therapy for the treatment of MLL leukemia.

Results and Discussion

Analysis of the co-crystal structure of M-525 complexed with the menin protein shows that although the nitrile group on the quaternary carbon atom in M-525 is directed towards a solvent exposed environment, it is adjacent to two negatively charged Asp180 and Glu359 residues.²⁰ Indeed, in our previous study, we have shown that a positively charged amino group at this site can significantly enhance the binding affinities and the cellular potencies of our designed reversible menin inhibitors¹⁹. Accordingly, we have employed a series of basic groups to replace the nitrile. For the ease of synthesis, these basic groups are connected to the quaternary carbon atom via a methylene group (Table 1).

Table 1.

Binding affinity and cell growth inhibition of menin inhibitors 7-12.

Compd.	R1	Binding Affinity (IC50, nM)a	Cell growth inhibition assay (7 days) (IC50, nM)b
			MV4;11 (MLL-AF4 fusion)	MOLM13 (MLL-AF9 fusion)	HL60 (No MLL fusion)
M-525c	-CN	3.3 ± 0.4	4 ± 3	19 ± 010	1500 ± 40
7		1.7 ± 0.3	2 ± 1	11 ± 5	> 10,000
8		2	2 ± 1	14 ± 5	4800 ± 500
9		5	1.1 ± 0.1	2 ± 1	700 ± 110
10		3	3 ± 2	8 ± 4	3000 ± 700
11		2	5 ± 2	15 ± 2	1400 ± 200
12		2	40 ± 14	157 ± 87	600 ± 30

^aIC₅₀ values were determined using a FP-based competitive binding assay. The binding affinity (IC₅₀) of all compounds is ≤ 5 nM, which exceeds the lower assay limit.

^bCell viability was determined using a WST-8 assay after 7 days of treatment for each compound, with average values and SD calculated from three independent experiments.

^cIC₅₀ values for M-525 were reported previously.²⁰

Replacement of the nitrile group with a basic heteroaromatic 2-ethyl imidazole group produced compound 7 (Table 1), which has IC₅₀ = 1.7 nM to menin in our fluorescence-polarization (FP) based, competitive binding assay²¹. However, since these compounds were designed as covalent menin inhibitors, the IC₅₀ values obtained in our FP-based binding assay result from both their binding to menin and their chemical reactivity with menin. We have therefore employed the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion to test their cellular potencies and the HL-60 leukemia cell line lacking the MLL fusion to evaluate their cellular specificity as the primary assays. Compound 7 was found to achieve IC₅₀ values of 2 nM and 11 nM in inhibition of cell growth in the MV4;11 and MOLM-13 cell lines and to have an IC₅₀ value of >10 μM in the HL-60 cell line. Hence, 7 demonstrates a potent cellular activity in two leukemia cell lines carrying MLL fusion proteins and a very high selectivity over a leukemia cell line lacking MLL fusion. We concluded that 7 is a potent and specific menin inhibitor.

Encouraged by the promising data for compound 7, we made additional compounds in which the nitrile group in M-525 was replaced by different basic groups. Changing the imidazole group in compound 7 with a triazole group led to 8, which has IC₅₀ values of 2 nM and 14 nM in the MV4;11 and MOLM-13 cell lines and displays an IC₅₀ value of 4.8 μM in the HL-60 cell line. Hence, compound 8 is also a potent and specific menin inhibitor. Replacing the triazole group in 8 with a much more basic dimethyl amine led to compound 9, which achieves IC₅₀ values of 1.1 nM and 2 nM in the MV4;11 and MOLM-13 cell lines, respectively. Compound 9 has an IC₅₀ value of 0.7 μM in the HL-60 cell line, thus demonstrating a cellular selectivity of >300-times for the MV4;11 and MOLM-13 cell lines over the HL-60 cell line. In direct comparison, 9 is 4-times more potent than M-525 in the MV4;11 cell line and 9.5-times more potent than M-525 in the MOLM-13 cell line, respectively. Hence, our data on compound 9 showed that a basic amino group can significantly improve the cellular activity in leukemia cells carrying MLL fusion, while retaining high cellular selectivity over leukemia cells lacking MLL fusion. Accordingly, we have made additional compounds containing a basic amine group.

Compound 10 containing an azetidine group has IC₅₀ values of 3 nM and 8 nM, respectively, in the MV4;11 and MOLM-13 cell lines, and is thus 2-3 times less potent than 9. Compound 10 has an IC₅₀ value of 3.0 μM in the HL-60 cell line, thus displaying a cellular selectivity of >300-times for the MV4;11 and MOLM-13 cell lines over the HL-60 cell line. Changing the 4-membered ring azetidine group in 10 to a 5-membered ring pyrrolidine yielded 11, which is 2-times less potent than 10 in both the MV4;11 and MOLM-13 cell lines. Interestingly, while 11 still retains a good cellular selectivity for MLL leukemia cells over non-MLL leukemia cells, it is less selective than 10. Changing the 4-membered ring azetidine group in compound 10 to a 6-membered ring piperidine resulted in 12, which is >10-times less potent than 10 in both the MV4;11 and MOLM-13 cell lines. However, 12 is more potent than 10 in the HL-60 cell line lacking MLL fusion and is therefore much less selective than 10.

The positively charged amino “head” groups in compounds 9, 10, 11 and 12 have calculated pKa values of 9.16-9.18²², indicating a very similar basicity. Based upon our design, these head groups were also expected to be exposed to a solvent environment. However, these four compounds have significant differences in their cellular potencies in inhibition of cell growth in MV4;11 and MOLM-13 cell lines with compound 9 being the most potent and compound 12 being the least potent. To shed light on the structural basis for their different cellular potencies, we modeled their binding models with menin, based upon the co-crystal structure of M-525 in complex with menin (Figure 2), followed by a 10 ns molecular dynamic (MD) simulation for each compound. Our modeling showed that while these 4 compounds bind to menin with similar binding models, the interactions between their respective, positively charged amino “head’ group and the negatively charged Asp180 carboxylic acid differ for these four compounds. The average distance between the positively charged amino group in 9 and the nearest negatively charged oxygen atom of the carboxylic acid group of Asp180 in menin is 3.56 ± 0.38 Å, indicating a very strong charge-charge interaction. In comparison, the corresponding distances in the binding models for 10, 11 and 12 are 4.06 ± 0.60, 5.83 ± 1.38 and 10.15 ± 0.97 Å, respectively. Hence, our modeling suggested that among these compounds, compound 9 has the strongest charge-charge interactions between its positively charged amino “head’ group and the negatively charged Asp180 carboxylic acid in menin, followed by compounds 10 and 11. In contrast, compound 12 has a minimal interaction between its positively charged amino “head’ group and the negatively charged Asp180 carboxylic acid in menin. Our modeling thus provides an explanation for their different cellular potencies in MV4;11 and MOLM-13 cell lines for compounds 9-12.

Figure 2.

Predicted binding models of compounds 9-12 with menin (PDB ID: 6B41). The distance between the respective amino group of each inhibitor and the nearest oxygen atom of Asp180 in menin is shown by a black dash line.

In our previous study, we have found the cellular potency of our covalent inhibitors can be significantly enhanced by introducing the “tail” dimethyl amine group in M-525, which is able to serve as intermolecular catalytic base in the addition reaction between Michael acceptor and cysteine^{20, 23}. Because 10 (M-734) has a very potent cellular activity in the MV4;11 and MOLM-13 cell lines and an excellent cellular selectivity over the HL-60 cell line, we have employed this compound as the template for tail modifications.

First, we examined the importance of the dimethylaminomethylene group in 10 by removal of this group, which led to 13. Compound 13 has IC₅₀ = 23 nM in the MV4;11 cell line and IC₅₀ = 374 nM in the MOLM-13 cell line (Table 2), which is 7- and 46-times less potent than 10, respectively. These data further confirmed the importance of the dimethylaminomethylene group in enhancing the cellular activity of our covalent menin inhibitors in leukemia cells carrying MLL fusion. We have therefore modified the dimethyl amine group with other groups containing an amine group.

Table 2.

Binding affinity and cell growth inhibition of menin inhibitors 13-20.

Compd.	R2	Binding Affinity (IC50, nM)	Cell growth inhibition assay (7 days) (IC50, μM)
			MV4;11 (MLL-AF4 fusion)	MOLM13 (MLL-AF9 fusion)	HL60 (No MLL fusion)
13		2	0.023 ± 0.009	0.374 ± 0.093	2.6 ± 0.7
14		2	0.002 ± 0.001	0.003 ± 0.001	2.1 ± 0.1
15		2	0.002 ± 0.0003	0.009 ± 0.001	2.1 ± 0.4
16 (M-808)		2.6 ± 0.5	0.001 ± 0.0002	0.004 ± 0.001	2.8 ± 0.6
17		2	0.001 ± 0.0004	0.003 ± 0.001	2.0 ± 0.3
18		1	0.004 ± 0.001	0.015 ± 0.004	2.8 ± 0.9
19		4	0.003 ± 0.0004	0.021 ± 0.002	3.6 ± 0.5
20		2	0.004 ± 0.001	0.020 ± 0.002	3.2 ± 0.5

We replaced the dimethylamino group in 10 with azetidine, which led to 14. Compound 14 achieves IC₅₀ values of 2 nM and 3 nM in the MV4;11 and MOLM-13 cell lines, respectively. Furthermore, 14 displays an IC₅₀ value of 2 μM in the HL-60 cell line, hence showing a cellular selectivity >700-fold for MLL leukemia cells over non-MLL leukemia cells. Encouraged with the excellent cellular potency in MLL leukemia cells and selectivity in non-MLL cells, we replaced the 4-membered ring azetidine with a 5-membered ring pyrrolidine, which yielded 15. Compound 15 is equally potent as 14 in inhibition of cell growth in the MV4;11 cell line (IC₅₀ = 2 nM) but is 3-times less potent than 14 in the MOLM-13 cell line. 15 demonstrates a >200-fold cellular selectivity over the HL-60 cell line. Replacement of the azetidine in 15 with a 6-membered ring piperidine resulted in 16, which is essentially equally potent as 14. Compound 16 and 14 are also equally selective over the HL-60 cell line.

We next installed one F atom or two F atoms in the 3-position of azetidine in 14 to reduce the basicity of the basic nitrogen to examine the effect on the cellular potency, which yielded 17 and 18. The calculated pK_a value of the basic nitrogen of the azetidine in 14 was reduced from 8.65 to 7.06 with installation of one F atom in 17. Interestingly, 17 is equally potent and selective as compared to 14. With installation of two F atoms in the 3-position of azetidine, the calculated pK_a value was reduced to 5.47 in 18. Compound 18 is 4-5 times less potent than 17 in inhibition of cell growth in both the MV4;11 and MOLM-13 cell lines.

Since 16 (M-808) is highly potent and selective, we also investigated the effect of reducing the basicity of the basic nitrogen of its tail piperidine by installation of one or two F atoms in the 4-position of the piperidine ring. Installation of one F atom yielded 19, which achieves IC₅₀ values of 3 and 21 nM in inhibition of cell growth in the MV4;11 and MOLM-13 cell lines, respectively. Hence, 19 is 3- and 5-times less potent than 16 in the MV4;11 and MOLM-13 cell lines, respectively. Installation of two F atoms at the 4-position of the piperidine ring resulted in 20, which has IC₅₀ values of 4 and 20 nM in inhibition of cell growth in the MV4;11 and MOLM-13 cell lines, respectively, and is similarly potent as 19. Hence, installation of one or two F atoms in the 4-position of the piperidine ring in 16 resulted in modest reduction (3-5 times) in cellular potencies in inhibition of cell growth in both the MV4;11 and MOLM-13 cell lines.

To understand the mode of action of our designed covalent inhibitors, we performed mass-spectrometry analyses of the reactivity for a number of representative covalent menin inhibitors with human menin protein in vitro (Table 3). Consistent with our previous data, M-525 readily forms a covalent complex with menin protein. Within 1 h of incubation, 95% of the protein forms a covalent complex with M-525, and 100% of protein reacts with M-525 with overnight incubation. Compounds 7 and 10 also readily form a covalent complex with menin. Compound 13, which has a much reduced cellular potency in both MV4;11 and MOLM-13 cell lines as compared to M-525, also has a slower reaction kinetics with menin than M-525.

Table 3.

Mass-spectrometry analyses of the reactivity of our designed covalent menin inhibitors with human menin protein in vitro.

Menin Inhibitor	% of menin protein forming a covalent complex with the inhibitor
	1 h	Overnight
M-525	95	100
7	100	100
10	75	100
13	50	75
14	75	100
15	100	100
16 (M-808)	75	100
17	20	75
18	0	0
19	20	100
20	0	0

Compounds 14, 15 and 16, all of which are very potent menin inhibitors, have a rapid reaction kinetics with menin. Compound 17, in which a F atom was installed on the 4-membered azetidine in 14, and 19, in which a F atom was onto the 6-membered piperidine in 16, have a much slower reaction kinetics than 14 and 16. For both 17 and 19, only 20% of menin protein forms a covalent complex with either compound with 1 h incubation time. Compound 18, in which two F atom were installed onto the 4-membered azetidine in 14, and 20, in which two F atom were installed onto the 6-membered piperidine in 16, fail to form a covalent complex with menin, even with overnight incubation time. These data strongly suggested that although 18 and 20 still potently inhibit cell growth in the MV4;11 and MOLM-13 cell lines and contain a Michael acceptor, they most likely work as reversible menin inhibitors in cells and not as covalent menin inhibitors.

To gain structural insights for their interactions with menin protein for these new covalent inhibitors, we determined a co-crystal structure of 16 (M-808) complexed with menin protein at 2.10 Å resolution (Figure 3, PDB code: 6MN9). Consistent with its design and our mass spectrometry data, M-808 forms a covalent bond between its acrylamide and the sulfur atom of Cys329 in menin, same as we have observed in the co-crystal complex for M-525 with menin, confirming its covalent binding nature with menin. While the azetidine “head” group is largely exposed to solvent, the basic nitrogen atom is 4.3 Å away from the negatively charged oxygen atom of the carboxylic acid group of Asp180 side chain, indicating a strong charge-charge interaction, consistent with our modeling results. The tail 6-membered piperidine is exposed to solvent and has no specific interactions with menin. The other interactions between M-808 and menin are essentially same as those observed in the co-crystal structure of M-525 in complex with menin. Specifically, the reverse carbamate group on the cyclopentyl ring inserts into a pocket formed by Asn282, Cys241, Met278, Tyr276 and Tyr323 in menin and its carbonyl group forms a hydrogen bond with the hydroxyl group of Tyr276, and the methyl group has hydrophobic contacts with the side chains of Met278 and Cys241. The fluorine substituted phenyl ring inserts into a large hydrophobic pocket in menin. The cyclopentyl group interacts with a hydrophobic pocket formed primarily by Phe159, Phe328 and Ala242. The 4-membered azetidine group in the middle of M-808 sandwiches between Tyr319 and Tyr323 and positions the sulfonylphenyl into a proper position and orientation for the sulfonyl group to form a strong hydrogen bond with the NH group of the indole of Trp341. This co-crystal structure provides a solid structural basis for the high-affinity, covalent binding of M-808 with menin.

Figure 3.

Co-crystal structure of compound 16 (M-808) (cyan carbons) complexed with menin at 2.1 Å resolution (PDB code: 6MN9). Hydrogen bonds depicted as black dashed lines.

In our previous study²⁰, we have shown that a single dose of M-525 is capable of achieving strong suppression of the expression of MEIS1 and HOXA9 genes in the MV4;11 xenograft tumor tissue at 50 mg/kg intravenous dosing. Based upon the potent cellular activity in both the MV4;11 and MOLM-13 cell lines, we have selected compounds M-525, 9, 10, 14-17 for their pharmacodynamics (PD) in mice bearing the MV4;11 xenograft tumors (Figure 4). Our PD data showed that 10, 15 and 16 are most effective in suppression of the expression of MEIS1 and HOXA9 genes in the MV4;11 tumors among these 7 menin inhibitors tested. Compounds 10, 15 and 16 at 10 mg/kg are effective in reducing the expression of MEIS1 gene by >2-fold at 24 hr time-point. Compounds 10, 15 and 16 have a significant but modest effect in suppressing the expression of HOXA9 gene.

Figure 4.

Pharmacodynamic effect of Menin inhibitors on expression of HOXA9 and MEIS1 genes in MV4;11 xenograft tumors. Mice bearing MV4;11 tumors were treated with a single intravenous of each menin inhibitor at 10 mg/kg and tumors were collected at 24 hr time point for qRT-PCR analysis. ** (p < 0.01).

Based upon the PD data, we selected 10, 15 and 16 (M-808) to evaluate their tolerability in mice. It was found that 10, 15 and 16 were well tolerated in SCID mice with intravenous administration of 10 mg/kg, every other day dosing (three times a week) for one week. However, 10 and 15 induced animal weight loss (>10%) at 25 mg/kg every other day dosing (three times a week) for one week. In comparison, 16 was found to be well tolerated at 25 mg/kg every other day dosing (three times a week) for one week. Based upon the tolerability data, we decided to evaluate 16 further for its pharmacodynamic effect and for its efficacy in vivo.

Mice bearing MV4;11 tumors were treated with a single intravenous dose of M-808 at 25 mg/kg. Mice were sacrificed at 6, 24 and 48 h time-points and tumors were collected for qRT-PCR analysis of HOXA9 and MEIS1 gene expression (Figure 5). The PD data showed that a single dose of M-808 at 25 mg/kg effectively suppresses the expression of MEIS1 and HOXA9 genes in the tumor tissue at all the three time-points. While M-808 is equally effective in suppressing HOXA9 gene expression at 6, 24 and 48 h, it becomes more effective in reducing the expression of MEIS1 gene at later time points. The long-lasting PD effect of M-808 on MV4;11 tumors provided a basis for us to evaluate the in vivo efficacy of M-808 at a dosing schedule less frequent than daily administration.

Figure 5.

Pharmacodynamic effect of 16 (M-808) on expression of HOXA9 and MEIS1 genes in MV4;11 xenograft tumors. Mice bearing MV4;11 tumors were treated with a single intravenous of M-808 at 25 mg/kg and tumors were collected at 6, 24 and 48 hr time points for qRT-PCR analysis. ** (p < 0.01).

M-808 was tested for its in vivo antitumor efficacy in the MV4;11 xenograft model in SCID mice. Mice were treated with M-808 at 10 or 25 mg/kg every other day, 3 times per week for a total of 11 doses. While M-808 at 10 mg/kg has no effect on tumor growth over the vehicle control, M-808 at 25 mg/kg is very efficacious. M-808 at 25 mg/kg achieves a maximum tumor growth inhibition (TGI) of 97% during treatment (day 35) (Figure 6) and reduces the average tumor volume from 92 mm³ at the beginning of the treatment to 59 mm³ at day 35. Mice treated with M-808 at both doses have no significant weight change and show no signs of drug-related toxicity during and after the treatment. Hence, our efficacy data show that M-808 is very efficacious in vivo at a well-tolerated dose-schedule.

Figure 6.

In vivo antitumor efficacy of compounds 16 (M-808) in the MV4;11 xenograft model in SCID mice. Compound was administered IV at the indicated dose schedules. (A) Tumor growth inhibition. (B) Body weight of mice.

Chemistry

The synthetic routes to these compounds are shown below. The intermediates 21, 27 and compound M-525 were prepared according to published methods²⁰. The syntheses of compounds 7-20 are shown in Scheme 1. Intermediate 21 was reduced successively by DIBAL-H and NaBH₄ to give the amine 22. The free amine of compound 22 was modified to yield 23a-f, which were further deprotected with trifluoroacetic acid to form 24a-f. Compounds 24a-f were reacted with dimethyl dicarbonate, then the benzyl protecting group was removed to afford the deprotected piperidine intermediates 26a-f, which were reacted with the separately prepared²⁰ tail 27, to form compounds 28a-f. Removal of the Boc protecting group of 28a-f produced the corresponding amine intermediates, which reacted with diverse α,β-unsaturated carboxylic acids or acryloyl chloride afforded the final products 7-20.

Scheme 1:

Syntheses of compounds 7-20.

Reagents and conditions: (a) DIBAL-H, toluene, 0 °C to rt; (b) NaBH₄, MeOH, 0 °C to rt; (c) propionaldehyde, glyoxal, ammonium acetate, MeOH, 60 °C (23a); p-toluenesulfonyl hydrazide, glyoxal, AcOH, MeOH, 60 °C (23b); HCHO, NaBH(AcO)₃, DCM, rt (23c); 1,3-dibromopropane, K₂CO₃, MeCN, 80 °C (23d); 1,4-dibromobutane, K₂CO₃, MeCN, 80 °C (23e); 1,5-dibromopentane, K₂CO₃, MeCN, 80 °C (23f); (d) TFA, DCM, 0 °C to rt; (e) dimethyl dicarbonate, DIPEA, DCM, 0 °C to rt; (f) 10% Pd/C, H₂, MeOH, rt; (g) K₂CO₃, KI, MeCN, 80 °C; (h) TFA, DCM, 0 °C to rt; (i) α,β-unsaturated carboxylic acids, HATU, DIPEA, DCM, 0 °C to rt (7-12, 14-20); acryloyl chloride, DIPEA, DCM, 0 °C to rt (13).

Conclusion

In the present study, we have described the optimization of a class of covalent menin inhibitors based upon our previously published covalent inhibitor M-525. Our efforts have yielded M-808 as a highly potent and efficacious covalent menin inhibitor. Mass spectrometric analysis and co-crystal structure of M-808 complexed with menin clearly show that M-808 readily forms a covalent bond with Cys329 in menin. M-808 achieves IC₅₀ values of 1-4 nM in inhibition of cell growth in MV4;11 and MOLM13 leukemia cell lines carrying MLL fusion proteins and displays an excellent selectivity over the HL-60 cell line lacking MLL fusion. M-808 effectively suppresses expression of MEIS1 and HOXA9 genes in the MV4;11 xenograft tumor tissues and is capable of achieving partial tumor regression in mice bearing the MV4;11 xenograft tumor at a well-tolerated dose-schedule. Taken together, our study shows that M-808 is a promising covalent menin inhibitor for further evaluations and optimization toward developing a new therapy for the treatment MLL leukemia.

EXPERIMENTAL SECTION

General Methods for Chemistry.

Unless otherwise noted, commercial solvents and reagents were used without further purification with the exception of THF which was freshly distilled from sodium wire. Proton nuclear magnetic resonance (¹H NMR) was recorded on a Bruker Advance 400 MHz spectrometer. ¹H NMR spectra were 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, m = multiplet. MS analyses were carried out with a Waters UPLC–mass spectrometer. The final compounds were all purified by C18 reverse phase preparative HPLC column with solvent A (0.1% TFA in H₂O) and solvent B (0.1% TFA in MeCN) as eluents. The purity of all the final compounds was confirmed to be > 95% by UPLC analysis (10% to 100% MeCN in H₂O containing 0.1% TFA in 10 min).

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-((E)-4-(dimethylamino)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-2-(2-ethyl-1H-imidazol-1-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (7).

Compound 7 was synthesized using the method described for 10 from the intermediate 23a. ¹H NMR (400 MHz, MeOD) δ 7.70 (d, J = 8.8 Hz, 2H), 7.57-7.52 (m, 1H), 7.50-7.44 (m, 2H), 7.33 (s, 1H), 7.20 (t, J = 8.4 Hz, 1H), 6.97 (s, 1H), 6.77-6.70 (m, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 15.2 Hz, 1H), 4.84 (d, J = 16.0 Hz, 1H), 4.59 (d, J = 15.6 Hz, 1H), 4.51 (d, J = 6.0 Hz, 2H), 4.29-4.15 (m, 5H), 3.99-3.93 (m, 3H), 3.78-3.73 (m, 2H), 3.71-3.69 (m, 1H), 3.65 (s, 3H), 3.50-3.42 (m, 3H), 3.28-3.21 (m, 1H), 3.14-3.07 (m, 2H), 3.05-2.99 (m, 1H), 2.89 (s, 6H), 2.87-2.84 (m, 1H), 2.71-2.64 (m, 2H), 2.31 (d, J = 13.6 Hz, 1H), 2.02 (d, J = 15.6 Hz, 1H), 1.96-1.90 (m, 1H), 1.66-1.61 (m, 1H), 1.58-1.52 (m, 1H), 1.44-1.35 (m, 5H), 1.24-1.16 (m, 2H), 1.07-0.96 (m, 1H); ESI-MS calculated for C₄₄H₆₀FN₇O₅S [M + H]⁺ = 818.44, found: 818.61.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-((E)-4-(dimethylamino)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethyl)cyclopentyl)carbamate (8).

Compound 8 was synthesized using the method described for 10 from the intermediate 23b. ¹H NMR (400 MHz, MeOD) δ 8.01 (s, 1H), 7.79 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.47-7.37 (m, 3H), 7.09 (t, J = 8.0 Hz, 1H), 6.78-6.70 (m, 1H), 6.54 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 15.2 Hz, 1H), 5.28 (s, 2H), 4.52 (d, J = 10.4 Hz, 2H), 4.29-4.23 (m, 2H), 4.20-4.17 (m, 4H), 3.93 (d, J = 6.4 Hz, 2H), 3.76-3.74 (m, 2H), 3.55-3.47 (m, 3H), 3.42 (s, 3H), 3.26-3.20 (m, 1H), 3.07-2.94 (m, 2H), 2.90 (s, 6H), 2.85-2.78 (m, 1H), 2.52 (t, J = 11.2 Hz, 1H), 2.23 (d, J = 14.0 Hz, 1H), 1.95-1.92 (m, 1H), 1.86-1.79 (m, 1H), 1.60-1.51 (m, 2H), 1.49-1.42 (m, 1H), 1.38-1.15 (m, 4H), 0.78-0.67 (m, 1H); ESI-MS calculated for C₄₁H₅₅FN₈O₅S [M + H]⁺ = 791.40, found: 791.51.

Methyl ((1S,2R)-2-((S)-2-(dimethylamino)-1-(1-((1-(4-((1-((E)-4-(dimethylamino)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (9).

Compound 9 was synthesized using the method described for 10 from the intermediate 23c. ¹H NMR (400 MHz, MeOD) δ 7.71 (d, J = 8.8 Hz, 2H), 7.52-7.46 (m, 1H), 7.42-7.39 (m, 2H), 7.17-7.13 (m, 1H), 6.78-6.70 (m, 1H), 6.54 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 15.2 Hz, 1H), 4.52 (d, J = 6.4 Hz, 2H), 4.29-4.16 (m, 5H), 3.95-3.86 (m, 5H), 3.82-3.78 (m, 2H), 3.68-3.60 (m, 2H), 3.50-3.45 (m, 4H), 3.31 (s, 3H), 3.14-2.94 (m, 6H), 2.90 (m, 6H), 2.87-2.81 (m, 1 H), 2.71-2.63 (m, 1H), 2.50-2.40 (m, 1H), 2.22-2.12 (m, 2H), 1.98-1.96 (m, 1H), 1.76-1.64 (m, 3H), 1.59-1.50 (m, 3H), 1.44-1.37 (m, 1H); ESI-MS calculated for C₄₁H₅₉FN₆O₅S [M + H]⁺ = 767.43, found: 767.54.

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)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (10).

The trifluoroacetic acid salt of 29d (300 mg, 0.38 mmoL) was dissolved in dry DCM (10 mL) and MeCN (1 mL). Then, DIPEA (0.2 mL, 1.15 mmol), (E)-4-(dimethylamino)but-2-enoic acid hydrochloride (76 mg, 0.46 mmol) and HATU (175 mg, 0.46 mmol) were added at 0 °C. After stirring for 30 min at rt, the reaction mixture was concentrated under vacuum. The residue was purified by reverse phase preparative HPLC to give the title compound as a trifluoroacetate salt (200 mg, 67%). ¹H NMR (400 MHz, MeOD) δ 7.70 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.13 (m, 2H), 7.05 (d, J = 7.6 Hz, 1H), 6.77-6.70 (m, 1H), 6.53 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 15.2 Hz, 1H), 4.56-4.50 (m, 4H), 4.39-4.32 (m, 2H), 4.29-4.22 (m, 1H), 4.19-4.12 (m, 5H), 3.95-3.93 (m, 2H), 3.81-3.74 (m, 3H), 3.56-3.47 (m, 3H), 3.40 (d, J = 7.8 Hz, 2H), 3.25-3.19 (m, 1H), 3.04-2.95 (m, 2H), 2.90 (s, 6H), 2.81-2.74 (m, 1H), 2.57-2.50 (m, 1H), 2.48-2.41 (m, 1H), 2.07 (d, J = 14.8 Hz, 2H), 2.01-1.97 (m, 2H), 1.92-1.86 (m, 1H), 1.81-1.75 (m, 1H), 1.71-1.61 (m, 3H), 1.50-1.41 (m, 1H), 1.14-1.04 (m, 1H); ESI-MS calculated for C₄₂H₅₉FN₆O₅S [M + H]⁺ = 779.43, found: 779.51.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-((E)-4-(dimethylamino)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)-2-(pyrrolidin-1-yl)ethyl)cyclopentyl)carbamate (11).

Compound 11 was synthesized using the method described for 10 from the intermediate 23e. ¹H NMR (400 MHz, MeOD) δ 7.70 (d, J = 8.8 Hz, 2H), 7.50-7.44 (m, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.16-7.11 (m, 1H), 6.77-6.70 (m, 1H), 6.52 (d, J = 9.2 Hz, 2H), 6.47 (d, J = 15.2 Hz, 1H), 4.51 (d, J = 6.4 Hz, 2H), 4.29-4.22 (m, 1H), 4.21-4.15 (m, 4H), 4.12-4.06 (m, 1H), 3.95-3.93 (m, 2H), 3.85 (d, J = 15.6 Hz, 1H), 3.78-3.71 (m, 5H), 3.57 (d, J = 11.6 Hz, 2H), 3.48-3.33 (m, 5H), 3.31 (s, 3H), 3.28-3.22 (m, 1H), 3.07-3.01 (m, 1H), 2.98-2.92 (m, 1H), 2.89 (s, 6H), 2.85-2.79 (m, 1H); ESI-MS calculated for C₄₃H₆₁FN₆O₅S [M + H]⁺ = 793.44, found: 793.52.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-((E)-4-(dimethylamino)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)-2-(piperidin-1-yl)ethyl)cyclopentyl)carbamate (12).

Compound 12 was synthesized using the method described for 10 from the intermediate 23f. ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.47-7.41 (m, 1H), 7.36 (d, J = 8.8 Hz, 2H), 7.14-7.10 (m, 1H), 6.77-6.70 (m, 1H), 6.51 (d, J = 8.8 Hz, 2H), 6.46 (d, J = 15.6 Hz, 1H), 4.50 (d, J = 6.4 Hz, 2H), 4.28-4.14 (m, 5H), 4.04-4.00 (m, 1H), 3.94-3.92 (m, 2H), 3.79-3.74 (m, 3H), 3.69-3.51 (m, 5H), 3.40-3.32 (m, 5H), 3.29-3.22 (m, 4H), 3.02 (t, J = 12.0 Hz, 1H), 2.95-2.81 (m, 2H), 2.89 (s, 6H), 2.15-2.03 (m, 5H), 1.91-1.71 (m, 8H), 1.59-1.46 (m, 3H); ESI-MS calculated for C₄₄H₆₃FN₆O₅S [M + H]⁺ = 807.46, found: 807.50.

Methyl ((1S,2R)-2-((S)-1-(1-((1-(4-((1-acryloylazetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-2-(azetidin-1-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (13).

Compound 29d (41 mg, 0.061 mmol) was dissolved in dry DCM (5 mL) and DIPEA (16 mg, 0.122 mmol) and acryloyl chloride (7 mg, 0.074 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 reverse phase preparative HPLC to give the title product (30 mg, 59%) as the trifluoroacetate salt. ¹H NMR (400 MHz, MeOD) δ 7.70 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.12 (m, 2H), 7.06 (d, J = 7.6 Hz, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.33-6.20 (m, 2H), 5.77-5.74 (m, 1H), 4.51-4.44 (m, 4H), 4.39-4.32 (m, 2H), 4.26-4.11 (m, 6H), 3.80-3.72 (m, 3H), 3.56-3.48 (m, 3H), 3.41-3.39 (m, 2H), 3.25-3.20 (m, 1H), 3.04-2.92 (m, 2H), 2.81-2.74 (m, 1H), 2.57-2.41 (m, 2H), 2.08-1.97 (m, 4H), 1.89-1.77 (m, 2H), 1.71-1.63 (m, 3H), 1.51-1.42 (m, 1H), 1.15-1.08 (m, 1H); ESI-MS calculated for C₃₉H₅₂FN₅O₅S [M + H]⁺ = 722.37, found: 722.16.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-((1-(4-((1-((E)-4-(azetidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (14).

Azetidine (4.3 mg, 0.074 mmol) was added at room temperature to a solution of (E)-4-bromobut-2-enoic acid (12 mg, 0.074 mmol) and DIPEA (19 mg, 0.150 mmol) in DMF (1 mL). After stirring for 1 h at 60 °C, compound 29d (25 mg, 0.037 mmol) and HATU (28 mg, 0.074 mmol) were added at 0 °C. After stirring for 30 min at rt, the reaction mixture was concentrated under vacuum. The residue was purified by reverse phase preparative HPLC to give the title compound as a trifluoroacetate salt (15 mg, 44%). ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.12 (m, 2H), 7.05 (d, J = 7.2 Hz, 1H), 6.65-6.58 (m, 1H), 6.53 (d, J = 8.8 Hz, 2H), 6.39 (d, J = 15.6 Hz, 1H), 4.59-4.49 (m, 4H), 4.37-7.31 (m, 2H), 4.28-4.21 (m, 3H), 4.18-4.15 (m, 4H), 4.13-4.09 (m, 2H), 4.01-3.99 (m, 2H), 3.81-3.74 (m, 3H), 3.57-3.44 (m, 3H), 3.40 (d, J = 6.8 Hz, 2H), 3.31 (s, 3H), 3.26-3.19 (m, 1H), 3.04-2.90 (m, 3H), 2.80-2.74 (m, 1H), 2.60-2.42 (m, 4H), 2.08-1.97 (m, 4H), 1.92-1.85 (m, 1H), 1.82-1.74 (m, 1H), 1.71-1.58 (m, 3H), 1.51-1.41 (m, 1H), 1.18-1.05 (m, 1H); ESI-MS calculated for C₄₃H₅₉FN₆O₅S [M + H]⁺ = 791.43, found: 791.44.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(3-fluorophenyl)-1-(1-((1-(4-((1-((E)-4-(pyrrolidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)ethyl)cyclopentyl)carbamate (15).

Compound 15 was synthesized using the method described for 10 from the intermediate 29d and (E)-4-(pyrrolidin-1-yl)but-2-enoic acid hydrochloride. ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.48-7.43 (m, 1H), 7.15-7.12 (m, 2H), 7.06 (d, J = 7.2 Hz, 1H), 6.78-6.71 (m, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.45 (d, J = 15.2 Hz, 1H), 4.52-4.50 (m, 4H), 4.37-4.28 (m, 2H), 4.27-4.22 (m, 1H), 4.20-4.11 (m, 5H), 4.00 (d, J = 6.0 Hz, 2H), 3.80-3.73 (m, 3H), 3.66-3.60 (m, 2H), 3.56-3.48 (m, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.31 (s, 3H), 3.26-3.20 (m, 1H), 3.14-3.09 (m, 2H), 3.04-2.92 (m, 2H), 2.81-2.74 (m, 1H), 2.56-2.49 (m, 1H), 2.46-2.40 (m, 1H), 2.19-2.13 (m, 2H), 2.08-1.91 (m, 7H), 1.80-1.73 (m, 1H), 1.70-1.59 (m, 3H), 1.52-1.43 (m, 1H), 1.20-1.09 (m, 1H); ESI-MS calculated for C₄₄H₆₁FN₆O₅S [M + H]⁺ = 805.44, found: 805.57.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(3-fluorophenyl)-1-(1-((1-(4-((1-((E)-4-(piperidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)ethyl)cyclopentyl)carbamate (16).

Compound 16 was synthesized using the method described for 10 from the intermediate 29d and (E)-4-(piperidin-1-yl)but-2-enoic acid hydrochloride. ¹H NMR (400 MHz, MeOD) δ 7.70 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.13 (m, 2H), 7.05 (d, J = 6.8 Hz, 1H), 6.78-6.71 (m, 1H), 6.53 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 15.2 Hz, 1H), 4.53-4.49 (m, 4H), 4.39-4.32 (m, 2H), 4.28-4.12 (m, 6H), 3.91 (d, J = 6.4 Hz, 2H), 3.80-3.73 (m, 3H), 3.56-3.46 (m, 5H), 3.40 (d, J = 6.8 Hz, 2H), 3.31 (s, 3H), 3.26-3.20 (m, 1H), 3.04-2.92 (m, 4H), 2.81-2.74 (m, 1H), 2.57-2.45 (m, 2H), 2.09-1.95 (m, 6H), 1.87-1.84 (m, 2H), 1.79-1.73 (m, 3H), 1.69-1.56 (m, 3H), 1.53-1.41 (m, 2H), 1.15-1.05 (m, 1H); ¹³C NMR (100 MHz, MeOD) δ 165.8, 165.2, 162.7, 162.3, 162.0, 161.6, 159.8, 156.0, 139.8, 132.9, 131.3, 128.3, 125.6, 123.9, 122.2, 119.3, 117.2, 116.9, 116.4, 115.8, 115.5, 111.6, 62.0, 60.8, 60.3, 58.0, 56.1, 54.8, 54.3, 54.0, 53.0, 52.2, 51.9, 51.0, 50.1, 41.3, 33.7, 26.9, 26.7, 26.4, 26.0, 24.3, 22.5, 21.2, 17.0; ESI-MS calculated for C₄₅H₆₃FN₆O₅S [M + H]⁺ = 819.46, found: 819.06.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-((1-(4-((1-((E)-4-(3-fluoroazetidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (17).

Compound 17 was synthesized using the method described for 14 from the intermediate 29d and 3-fluoroazetidine hydrochloride. ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.12 (m, 2H), 7.05 (d, J = 7.6 Hz, 1H), 6.67-6.60 (m, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.42 (d, J = 15.6 Hz, 1H), 5.51-5.32 (m, 1H), 4.62-4.55 (m, 2H), 4.53-4.47 (m, 4H), 4.42-4.28 (m, 4H), 4.26-4.22 (m, 1H), 4.19-4.15 (m, 4H), 4.09 (d, J = 6.4 Hz, 2H), 3.80-3.73 (m, 3H), 3.56-3.48 (m, 3H), 3.40 (d, J = 6.8 Hz, 2H), 3.31 (s, 3H), 3.30-3.19 (m, 2H), 3.04-2.90 (m, 2H), 2.80-2.74 (m, 1H), 2.57-2.41 (m, 2H), 2.08-1.97 (m, 4H), 1.92-1.86 (m, 1H), 1.81-1.75 (m, 1H), 1.70-1.59 (m, 3H), 1.51-1.41 (m, 1H), 1.17-1.06 (m, 1H); ESI-MS calculated for C₄₃H₅₈F₂N₆O₅S [M + H]⁺ = 809.42, found: 809.50.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-((1-(4-((1-((E)-4-(3,3-difluoroazetidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (18).

Compound 18 was synthesized using the method described for 14 from the intermediate 29d and 3,3-difluoroazetidine hydrochloride. ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 9.2 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.12 (m, 2H), 7.05 (d, J = 7.6 Hz, 1H), 6.70-6.63 (m,, 1H), 6.53 (d, J = 8.8 Hz, 2H), 6.36 (d, J = 15.6 Hz, 1H), 4.55-4.49 (m, 8H), 4.38-4.32 (m, 2H), 4.28-4.21 (m, 1H), 4.18-4.11 (m, 5H), 3.98 (d, J = 6.4 Hz, 2H), 3.80-3.73 (m, 3H ), 3.56-3.47 (m, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.31 (s, 3H), 3.26-3.19 (m, 1H), 3.04-2.92 (m, 2H), 2.80-2.74 (m, 1H), 2.54-2.42 (m, 2H), 2.08-1.97 (m, 4H), 1.92-1.86 (m, 1H), 1.80-1.75 (m, 1H), 1.71-1.58 (m, 3H), 1.51-1.45 (m, 1H), 1.15-1.05 (m, 1H); ESI-MS calculated for C₄₃H₅₈F₃N₆O₅S [M + H]⁺ = 827.41, found: 827.49.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(3-fluorophenyl)-1-(1-((1-(4-((1-((E)-4-(4-fluoropiperidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)ethyl)cyclopentyl)carbamate (19).

Compound 19 was synthesized using the method described for 14 from the intermediate 29d and 4-fluoropiperidine hydrochloride. ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.12 (m, 2H), 7.06 (d, J = 7.6 Hz, 1H), 6.79-6.72 (m, 1H), 6.53 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 15.2 Hz, 1H), 5.07-5.00 (m, 1H), 4.53-4.47 (m, 4H), 4.37-4.32 (m, 2H), 4.27-4.11 (m, 6H), 3.97 (d, J = 6.8 Hz, 2H), 3.80-3.74 (m, 3H), 3.56-3.39 (m, 7H), 3.31 (s, 3H), 3.27-3.22 (m, 2H), 3.04-2.92 (m, 2H), 2.83-2.74 (m, 1H), 2.55-2.44 (m, 2H), 2.24-1.89 (m, 9H), 1.82-1.63 (m, 5H), 1.51-1.41 (m, 1H), 1.16-1.11 (m, 1H); ESI-MS calculated for C₄₅H₆₂F₂N₆O₅S [M + H]⁺ = 837.45, found: 837.60.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-((1-(4-((1-((E)-4-(4,4-difluoropiperidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (20).

Compound 20 was synthesized using the method described for 14 from the intermediate 29d and 4,4-difluoropiperidine hydrochloride. ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.16-7.12 (m, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.79-6.72 (m, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 15.6 Hz, 1H), 4.52-4.51 (m, 4H), 4.40-4.32 (m, 2H), 4.28-4.22 (m, 1H), 4.20-4.10 (m, 5H), 4.02-4.00 (m, 2H), 3.80-3.73 (m, 3H), 3.56-3.39 (m, 8H), 3.31 (s, 3H), 3.25-3.19 (m, 2H), 3.04-2.92 (m, 2H), 2.81-2.74 (m, 1H), 2.56-2.45 (m, 2H), 2.41-2.32 (m, 4H), 2.08-1.97 (m, 4H), 1.91-1.75 (m, 2H), 1.71-1.58 (m, 3H), 1.51-1.41 (m, 1H), 1.13-1.02 (m, 1H); ESI-MS calculated for C₄₅H₆₁F₃N₆O₅S [M + H]⁺ = 855.44, found: 855.55.

tert-Butyl ((1S,2R)-2-((S)-2-amino-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)ethyl)-cyclopentyl)carbamate (22).

Intermediate 21²⁰ (3g, 6.1 mmol) was added to a dry round bottomed flask then covered with a kimwipe and placed in a desiccator that was maintained under vacuum for 1-2 days. Then the flask was removed from the desiccator and quickly capped with a septum and the system was vacuumed under an N₂ atmosphere. The anhydrous toluene (30 ml) was added to the flask, which was then cooled to 0 °C in an ice-bath. Diisobutylaluminiumhydride (25% in toluene, 16.4 mL, 24.4 mmol) was injected slowly into the reaction mixture with a syringe at 0 °C with stirring. Then the ice-bath was removed and the reaction was monitored using UPLC-Mass. After the intermediate 21 was consumed, in about 4 h, NaOH solution (1M, 20 mL) was added slowly into the reaction mixture at 0 °C to quench the reaction. After stirring for 5 min, the ice-bath was removed and additional saturated brine (20 mL) was added. Then EtOAc (~50 mL) was added, the solid was filtered with celite, and was washed with EtOAc. The solution was extracted with EtOAc and DCM twice respectively. The combined organic solvent was dried with Na₂SO₄, filtered, and concentrated in a rotatory evaporator. Then the residue was redissolved in MeOH (100 mL), NaBH₄ (461 mg, 12.2 mmol) was added slowly at 0 °C and the reaction mixture was stirred at rt for 2 days. Then the reaction mixture was concentrated, and diluted with H₂O. The solution was extracted with EtOAc and DCM twice respectively. The combined organic solvent was dried with Na₂SO₄, filter, and concentrated in a rotatory evaporator to give crude title product 22 (2.8 g, 93%) without further purification. ¹H NMR (400 MHz, MeOD) δ 7.41-7.35 (m, 1H), 7.33-7.23 (m, 6H), 7.18 (d, J = 11.6 Hz, 1H), 6.99-6.95 (m, 1H), 4.07-4.02 (m, 1H), 3.52-3.44 (m, 2H), 3.24 (d, J = 14.4 Hz, 1H), 3.09 (d, J = 14.4 Hz, 1H), 2.98 (d, J = 11.2 Hz, 1H), 2.91 (d, J = 10.8 Hz, 1H), 2.35-2.29 (m, 1H), 2.12-2.04 (m, 2H), 2.01-1.94 (m, 2H), 1.77-1.69 (m, 1H), 1.61-1.58 (m, 1H), 1.54-1.47 (m, 2H), 1.44 (s, 9H), 1.41-1.29 (m, 3H), 1.22-1.14 (m, 2H); ESI-MS calculated for C₃₀H₄₂FN₃O₂ [M + H]⁺ = 496.33, found: 496.48.

tert-Butyl ((1S,2R)-2-((S)-1-(1-benzylpiperidin-4-yl)-2-(2-ethyl-1H-imidazol-1-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (23a).

Propionaldehyde (0.75 mL, 10.09 mmol), glyoxal (0.35 mL, 3.03 mmol, 40%) and ammonium acetate (227 mg, 3.03 mmol) were added to a solution of 22 (0.5 g, 1.01 mmol) in MeOH (6 mL). The reaction mixture was stirred at 60 °C overnight under an N₂ atmosphere. Then the solution was concentrated and purified by reverse phase HPLC to afford the title product as the trifluoroacetate salt (323 mg, 46%). ¹H NMR (400 MHz, MeOD) δ 7.56-7.50 (m, 1H), 7.47-7.42 (m, 7H), 7.31 (s, 1H), 7.19 (t, J = 8.4 Hz, 1H), 6.96 (s, 1H), 4.80 (d, J = 15.6 Hz, 1H), 4.58 (d, J = 15.6 Hz, 1H), 4.22 (s, 2H), 3.92-3.87 (m, 1H), 3.57 (d, J = 12.0 Hz, 1H), 3.42 (d, J = 11.6 Hz, 1H), 3.10-3.02 (m, 3H), 2.96 (t, J = 12.0 Hz, 1H), 2.70-2.61 (m, 2H), 2.27 (d, J = 14.8 Hz, 1H), 2.00 (d, J = 14.8 Hz, 1H), 1.92-1.90 (m, 1H), 1.58-1.52 (m, 2H), 1.46 (s, 9H), 1.40 (t, J = 7.6 Hz, 3H), 1.35-1.26 (m, 3H), 1.04-0.90 (m, 2H); ESI-MS calculated for C₃₅H₄₇FN₄O₂ [M + H]⁺ = 575.37, found: 575.49.

tert-Butyl ((1S,2R)-2-((S)-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethyl)cyclopentyl)carbamate (23b).

p-Toluenesulfonylhydrazide (207 mg, 1.11 mmol), glyoxal (0.29 mL, 2.52 mmol, 40% wt in H₂O) and AcOH (12 mg, 0.2 mmol) were added to a solution of intermediate 22 (0.5 g, 1.01 mmol) in MeOH (10 mL). The reaction mixture was stirred overnight at 60 °C under an N₂ atmosphere. Then, the solution was concentrated and purified by reverse phase HPLC to afford the title product as the trifluoroacetate salt (520 mg, 78%). ¹H NMR (400 MHz, MeOD) δ 8.04 (s, 1H), 7.77 (s, 1H), 7.48-7.36 (m, 8H), 7.07 (t, J = 7.6 Hz, 1H), 5.27-5.11 (m, 2H), 4.21 (s, 2H), 4.20-4.10 (m, 1H), 3.44-3.38 (m, 2H), 3.04-2.94 (m, 2H), 2.80-2.74 (m, 1H), 2.55 (t, J = 11.6 Hz, 1H), 2.21 (d, J = 13.6 Hz, 1H), 1.90-1.76 (m, 2H), 1.55-1.45 (m, 2H), 1.40-1.35 (m, 1H), 1.31 (s, 9H), 1.28-1.05 (m, 3H), 0.72-0.61 (m, 1H); ESI-MS calculated for C₃₂H₄₂FN₅O₂ [M + H]⁺ = 548.33, found: 548.47.

tert-Butyl ((1S,2R)-2-((S)-1-(1-benzylpiperidin-4-yl)-2-(dimethylamino)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (23c).

Formaldehyde (0.12 mL, 1.61 mmol, 37% wt. in H₂O) and sodium triacetoxyborohydride (342 mg, 1.61 mmol) were added to a solution of intermediate 22 (0.16 g, 0.32 mmol) in DCM (10 mL). The reaction mixture was stirred at rt overnight. Then the solution was concentrated and purified by reverse phase HPLC to afford the title product as the trifluoroacetate salt (150mg, 73%). ¹H NMR (400 MHz, MeOD) δ 7.49-7.43 (m, 6H), 7.37 (d, J = 8.8 Hz, 2H), 7.14-1.10 (m, 1H), 4.24 (s, 2H), 3.97-3.93 (m, 1H), 3.83-3.77 (m, 2H), 3.52-3.47 (m, 2H), 3.07-2.90 (m, 8H), 2.75-2.65 (m, 1H), 2.34-2.20 (m, 1H), 2.14-2.03 (m, 2H), 2.00-1.93 (m, 1H), 1.68-1.46 (m, 7H), 1.29 (s, 9H); ESI-MS calculated for C₃₂H₄₆FN₃O₂ [M + H]⁺ = 524.36, found: 524.51.

tert-Butyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (23d).

1,3-Dibromopropane (0.74 ml, 7.26 mmol), K₂CO₃ (2.51 g, 18 mmol) and KI (100 mg, 0.6 mmol) were added to a solution of the intermediate 22 (3 g, 6.05 mmol) in MeCN (150 mL). The mixture was stirred at 80 °C for 1~2 days then it was filtered with celite to remove solid K₂CO₃. The filtrate was concentrated and dissolved in H₂O, extracted with EtOAc and DCM twice respectively, and dried over Na₂SO₄, and the solvent was evaporated under vacuum. The residue was purified by the chromatography column to afford the title product (3g, 93%). ¹H NMR (400 MHz, MeOD) δ 7.47-7.40 (m, 6H), 7.16-7.03 (m, 3H), 4.52-4.46 (m, 2H), 4.38-4.31 (m, 1H), 4.19-4.10 (m, 2H), 4.19 (s, 2H), 3.70-3.66 (m, 1H), 3.44-3.40 (m, 3H), 3.01-2.90 (m, 2H), 2.79-2.73 (m, 1H), 2.56-2.46 (m, 1H), 2.42-2.36 (m, 1H), 2.05-1.93 (m, 4H), 1.82-1.73 (m, 2H), 1.68-1.57 (m, 3H), 1.37-1.29 (m, 1H), 1.22 (s, 9H), 1.06-0.98 (m, 1H). ¹H NMR (400 MHz, MeOD) δ ; ESI-MS calculated for C₃₃H₄₆FN₃O₂ [M + H]⁺ = 536.36, found: 536.44.

tert-Butyl ((1S,2R)-2-((S)-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)-2-(pyrrolidin-1-yl)ethyl)cyclopentyl)carbamate (23e).

23e was synthesized using the method described for 23d from the intermediate 22 and 1,4-dibromobutane. ¹H NMR (400 MHz, MeOD) δ 7.49-7.41 (m, 6H), 7.33-7.30 (m, 2H), 7.14-7.09 (m, 1H), 4.19 (s, 2H), 4.14-4.10 (m, 1H), 3.81-3.64 (m, 4H), 3.49-3.33 (m, 4H), 3.02-2.90 (m, 2H), 2.87-2.80 (m, 1H), 2.21-2.14 (m, 2H), 2.05-1.93 (m, 7H), 1.80-1.68 (m, 4H), 1.38-1.27 (m 2H), 1.19 (s, 9H). ESI-MS calculated for C₃₄H₄₈FN₃O₂ [M + H]⁺ = 550.37, found: 550.53.

tert-Butyl ((1S,2R)-2-((S)-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)-2-(piperidin-1-yl)ethyl)cyclopentyl)carbamate (23f).

23f was synthesized using the method described for 23d from the intermediate 22 and 1,5-dibromopentane. ¹H NMR (400 MHz, MeOD) δ 7.48-7.34 (m, 8H), 7.11-7.07 (m, 1H), 4.18 (s, 2H), 4.06-4.03 (m, 2H), 3.74-3.72 (m, 1H), 3.61-3.55 (m, 3H), 3.43-3.41 (m, 2H), 3.29-3.22 (m, 2H), 3.01-2.89 (m, 3H), 2.07-1.86 (m, 9H), 1.80-1.70 (m, 5H), 1.59-1.50 (m, 1H), 1.34-1.23 (m, 2H), 1.18 (s, 9H). ESI-MS calculated for C₃₅H₅₀FN₃O₂ [M + H]⁺ = 564.39, found: 564.56.

(1S,2R)-2-((S)-2-(Azetidin-1-yl)-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentan-1-amine (24d).

Compound 23d (2.55 g, 4.76 mmol) was dissolved in DCM (5 mL), then trifluoroacetic acid (10 mL) was added slowly at 0 °C. After stirring for 2 h at rt, the reaction mixture was concentrated under vacuum, and redissolved in DCM (100 mL). Amberlyst® a21 (3g) was added and stirred for 30 min to neutralize the remaining trifluoroacetic acid. Then the resin was filtered, and the organic solvent was evaporated to give the crude title product (1.8 g, 87%) without further purification. ESI-MS calculated for C₂₈H₃₈FN₃ [M + H]⁺ = 436.30, found: 436.32.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-benzylpiperidin-4-yl)-1-(3-fluorophenyl)-ethyl)cyclopentyl)carbamate (25d).

Compound 24d (2.07 g, 4.75 mmol) was dissolved in dry DCM (50 mL). Then, DIPEA (3.31 mL, 19 mmol) and dimethyl dicarbonate (764 mg, 5.7 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 reverse phase HPLC to give the title product (2.5 g, 87%) as a trifluoroacetate salt. ¹H NMR (400 MHz, MeOD) δ 7.48-7.40 (m, 6H), 7.14-7.10 (m, 2H), 7.02 (d, J = 7.6 Hz, 1H), 4.52-4.47 (m, 2H), 4.38-4.31 (m, 2H), 4.21 (s, 2H), 4.11 (d, J = 15. 6 Hz, 1H), 3.76 (d, J = 15.6 Hz, 1H), 3.46-3.41 (m, 3H), 3.29 (s, 3H), 3.02-2.90 (m, 2H), 2.77-2.71 (m, 1H), 2.55-2.48 (m, 1H), 2.46-2.40 (m, 1H), 2.05-2.02 (m, 2H), 1.99-1.95 (m, 2H), 1.88-1.82 (m, 1H), 1.77-1.73 (m, 1H), 1.69-1.61 (m, 3H), 1.43-1.34 (m, 1H), 1.07-0.97 (m, 1H); ESI-MS calculated for C₃₀H₄₀FN₃O₂ [M + H]⁺ = 494.31, found: 494.45.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(3-fluorophenyl)-1-(piperidin-4-yl)ethyl)cyclopentyl)carbamate (26d).

10% Pd/C (280 mg, 10% wt.) was added to a solution of the trifluoroacetate salt 25d (1.6 g, 2.63 mmol) in MeOH (50 mL) under an N₂ atmosphere. Then, the flask was degassed three times with stirring. Then the mixture was stirred for 2 h at room temperature under a normal pressure H₂ atmosphere. After the Pd/C catalyst was filtered off, the solvent was removed by rotary evaporation to give the title product (0.95 g, 89%). ¹H NMR (400 MHz, MeOD) 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.

tert-Butyl 3-((4-(3-((4-((S)-2-(azetidin-1-yl)-1-(3-fluorophenyl)-1-((1R,2S)-2-((methoxy-carbonyl)amino)cyclopentyl)ethyl)piperidin-1-yl)methyl)azetidin-1-yl)phenyl)sulfonyl)-azetidine-1-carboxylate (28d).

Compound 27 (548 mg, 1.19 mmol), K₂CO₃ (274 mg, 1.98 mmol) and KI (16 mg, 0.099 mmol) were added to a solution of the intermediate 26d (400 mg, 0.991 mmol) in MeCN (5 mL). The mixture was stirred at 80 °C overnight. 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 reverse phase preparative HPLC to give the trifluoroacetate salt of 28d (650 mg, 74%). ¹H NMR (400 MHz, MeOD) δ 7.68 (d, J = 8.8 Hz, 2H), 7.49-7.43 (m, 1H), 7.17-7.07 (m, 3H), 6.52 (d, J = 8.8 Hz, 2H), 4.51-4.46 (m, 2H), 4.39-4.28 (m, 2H), 4.18-4.07 (m, 8H), 3.81-3.74 (m, 3H), 3.55-3.51 (m, 3H), 3.41 (d, J = 6.8 Hz, 2H), 3.33(s, 3H), 3.26-3.20 (m 1H), 3.07-2.94 (m, 2H), 2.81-2.75 (m, 1H), 2.57-2.49 (m, 1H), 2.47-2.39 (m, 1H), 2.10-1.95 (m, 5H), 1.78-1.74 (m, 1H), 1.70-1.57 (m, 3H), 1.52-1.48 (m, 1H), 1.42 (s, 9H), 1.23-1.18 (m, 1H); ESI-MS calculated for C₄₁H₅₈FN₅O₆S [M + H]⁺ = 768.41, found: 768.50.

Methyl ((1S,2R)-2-((S)-2-(azetidin-1-yl)-1-(1-((1-(4-(azetidin-3-ylsulfonyl)phenyl)-azetidin-3-yl)methyl)piperidin-4-yl)-1-(3-fluorophenyl)ethyl)cyclopentyl)carbamate (29d).

The trifluoroacetic acid salt of 28d (650 mg, 0.737 mmol) was dissolved in DCM (5 mL) and trifluoroacetic acid (5 mL) was added at 0 °C. After stirring for 1 h at rt, the reaction mixture was concentrated under vacuum to give the trifluoroacetate salt of 29d (500 mg, 87%). ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.8 Hz, 2H), 7.48-7.43 (m, 1H), 7.15-7.11 (m, 2H), 7.07 (d, J = 7.2 Hz, 1H), 6.52 (d, J = 9.2 Hz, 2H), 4.52-4.47 (m, 2H), 4.41-4.26 (m, 7H), 4.19-4.11 (m, 3H), 3.80-3.74 (m, 3H), 3.56-3.51 (m, 3H), 3.41 (d, J = 7.2 Hz, 2H), 3.32 (s, 3H), 3.27-3.20 (m, 1H), 3.05-2.93 (m, 2H), 2.81-2.74 (m, 1H), 2.56-2.49 (m, 1H), 2.47-2.39 (m, 1H), 2.08-2.05 (m, 2H), 2.01-1.95 (m, 3H), 1.80-1.73 (m, 1H), 1.70-1.59 (m, 3H), 1.53-1.44 (m, 1H), 1.21-1.11 (m, 1H); ESI-MS calculated for C₃₆H₅₀FN₅O₄S [M + H]⁺ = 668.36, found: 668.53.

Fluorescence Polarization (FP)-Based Binding Assay.

Compounds binding activity was measured using a FP assay described previously.²¹ Briefly, 5 μL of compounds with varying concentrations dissolved in DMSO was added to 195 μL of mixture of menin and the fluorescein-labeled tracer (compound 37 in our previous paper²¹) in the assay buffer (phosphate buffered saline, 100 μg/mL bovine γ-globulin, with 0.01% triton-x 100), and were incubated for 1 h at room temperture. Final concentrations of menin protein and the fluorescein-labeled tracer were 4 nM and 2 nM, respectively. FP values were measured using the Infinite M-1000 plate reader (Tecan, Morrisville, NC). The IC₅₀ values were determined by nonlinear regression fitting of the sigmoidal dose-dependent FP decreases as a function of total compound concentrations using Graphpad Prism 5.0 software.

Cell Growth Inhibition.

MV4;11 (catalog no. CRL-9591) and HL-60 (catalog no. CRL-9591) cell lines were purchased from the American Type Culture Collection (ATCC), and MOLM-13 (catalog no. ACC 554) cell line was purchased from the DSMZ German cell bank. Cells were cultured in either Iscove's Modified Dulbecco's Medium or RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum and 100 U/L penicillin-streptomycin and incubated at 37 °C under 5% CO₂, according to the cell supplier’s instructions.

For cell growth experiments, compounds were diluted in the corresponding medium and then serially diluted 3-fold in a 96-well tissue culture plate to a final volume of 100 μL/well. Two to three thousand cells/well in 100 μL were added to each well containing compound to a final volume of 200 μL/well. Cells were incubated for 7 days at 37 °C under 5% CO₂. Cell growth was evaluated utilizing a lactate dehydrogenase-based WST-8 assay (Dojindo Molecular Technologies, MD). An adequate volume of WST-8 reagent was added to each well, incubated for at least 1 h in the cell culture incubator, and read at 450 nm in a Tecan Infinite M-1000 multimode microplate reader (Tecan, Morrisville, NC). The readings were normalized to the untreated cells and fitted using a nonlinear regression analysis with the Graphpad Prism 5.0 software to obtain the IC₅₀ value for each compound.

Computational modeling.

The ligands of interest covalently bind to Menin protein, thus the binding modes were investigated using covalent docking mode available in GOLD (version 5.2)^24-25. Co-crystal structure of M-525/Menin (PDB code: 6B41) was used and “protonate 3D” module in MOE²⁶ was applied to assign the physiological protonation states considering pH = 7.0. Ligand 3D structures were drawn and the carbon-carbon double bonds in the Michael acceptor of the ligands were replaced by sulfur-carbon single bonds to mimic the status after covalently binding. This sulfur was also used to assign the covalently binding atom in GOLD for docking simulation. 15 runs were performed for each ligand and the top-ranked poses by ChemPLP scoring function was selected.

Considering the high flexibility of our ligands, a 10 ns MD simulation was performed for each of the predicted binding modes using AMBER18.²⁷ The covalent bonds of ligands-Cys329 require additional setup. RESP charge fitting²⁸ needs to be firstly given to the special ligand-cysteine fragment. Both backbone amine and carbonyl of the cysteine are capped by methyl group.²⁹ The charge profile are computed in HF/6-31G* using Gaussian09³⁰ and the atomic partial charge are assigned in antechamber package available in AMBER18. The partial charges on the backbone atoms (e.g. Ca, N, H, C, O) in Cys329 were reserved, in order to be consistent with natural amino acids, but charges on the side chain were assigned based on the different ligand binding. Required force field parameters such as bond, angles, torsions associated with the C-S covalent bond were added using the available parameters in small molecular force field GAFF2. The complex system was then solvated using TIP3P solvation model³¹ and neutralized by adding counter ions, followed by a 12000 steps minimization (first 3000 steps in steepest descent and followed by conjugate gradient). A 0.2 ns NVT simulation with a 5 kcal/mol/Ų force constraint was given to the protein-ligand system (not solvent) to gradually raised the temperature to physiological condition (310K); another constrained 0.2 ns NPT simulation with P = 1 atm was then performed. The whole system was then relaxed by gradually reducing the force constant to 0 in a 2 ns NPT simulation. A 10 ns unconstraint simulation was performed on the fully relaxed system. Over the simulation, the bond vibration associated with hydrogens are fixed using the SHAKE algorithm³², and the long-range non-bonding interactions were treated by applying PME method³³ with a cutoff of 10Å. Figures were prepared using the PyMOL program (www.pymol.org).

X-ray Crystallography.

Menin (residues 2-610 containing a deletion from 460-519) was purified as described previously.²⁰ Prior to crystallization, menin (25 mg/mL in 25 mM Tris 8.0, 150 mM NaCl and 5mM DTT) were incubated with M-808 in a protein to compound ratio of 1:1.2. Menin:M-808 was immediately allowed to crystallize at 4 °C.

All crystals were grown in drops containing 1 μL of 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). The crystals were cryoprotected by progressively soaking crystals in well solution containing increasingly higher amounts of sodium formate (1 M - 5 M in steps of 1M). Diffraction data were collected on a Mar300 detector at the Advanced Photon Source LS-CAT 21-ID-F beamlines at Argonne National Laboratory. All data were processed with HKL2000³⁴. The structures of the menin inhibitors were solved by molecular replacement (Molrep³⁵) using the apo menin structure (PDB ID 3U84) as the search model. Each structure went through iterative rounds of electron density fitting and structural refinement using Coot³⁶ and Buster³⁷, respectively. The coordinates and restraint files for the ligands were created from smiles in Grade³⁸ with the mogul+qm option. The initial Fo-Fc electron density map showed the presence of each compound covalently bound to C329 (Figure S1). The following regions were disordered in the structures: 582-610 in menin:M-808. Data collection and structural refinement statistics are provided in Table S1 in SI.

Mass-spectroscopic analysis of human menin protein incubated with menin inhibitors.

Samples of menin (25 mg/mL in 25 mM T ris 8.0, 150 mM NaCl and 5mM 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. 0.1 mL of each sample was applied to a reverse phase HPLC column (Phenomenex Aeris widepore C4 column 3.6 μM, 50 × 2.10 mm) at a flow rate of 0.5 mL/min in H₂O with 0.2% (v/v) HCOOH. Protein was eluted using a gradient of 5-100% MeCN with 0.2% (v/v) HCOOH over 4 min. The LC-MS experiment (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; drying gas temperature, 325 °C. MassHunter Qualitative Analysis Software (Agilent) was used to analyze the data. Intact protein masses were obtained using the maximum entropy deconvolution algorithm.

Animal Experiments.

All 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).

Tolerability studies in mice.

The compounds were tested at indicated doses, with three times a week (days 1, 3 and 5) for one week. Three female SCID mice were used per group. Mice were weighed and checked for clinical signs of toxicity. Greater than 10% weight loss was considered toxic.

In Vivo pharmacodynamics and efficacy studies in an MV4;11 human AML xenograft model in mice.

MV4;11 cells were grown in suspension and collected in the log phase. A cell sample was mixed 1:1 with Trypan Blue (GIBCO™, Invitrogen Corp.) and counted on a hemocytometer to determine the number of live/dead cells. Cells were washed twice with 1 × PBS (GIBCO™, Invitrogen Corp.) and resuspended in an ice cold mixture of 1:1 PBS and Matrigel (BD Biosciences, Invitrogen Corp.) for a final Matrigel protein concentration of 5 mg/mL. MV4;11 cells were inoculated into female C.B-17 SCID mice at 5 × 10⁶ cells in 0.1 mL with Matrigel. Cells were injected by subcutaneous route into the flank region of each mouse. The size of tumors growing in the mice was measured in two dimensions using calipers. Tumor volume (mm³) = (A×B²)/2 where A and B are the tumor length and width (mm), respectively.

For pharmacodynamics studies, mice bearing MV4;11 tumors were injected with a single dose of either vehicle or compound at 10 mg/kg or 25 mg/kg administered IV. Tumors were harvested at 6 h, 24 h or 48 h after the injection, with 3 mice/tumors for each time point. Tumor tissue was immediately frozen in liquid N₂, ground into fine powder, placed on dry ice and stored at −80 °C for RT-PCR analysis.

For efficacy studies, before treatment, tumors were allowed to grow to 60-140 mm³ in volume. Mice with tumors within the acceptable size range were randomized into treatment groups of 7 mice per group. Experimental compound M-808 was given IV, in a solution of 10% PEG 400:3% Cremophor EL:87% PBS. The control group was given vehicle only. During treatment, tumor volume and body weight was measured two or three times per week. After the treatment was stopped, tumor volume and body weight was measured at least once per week.

Real-Time PCR.

Total RNA was isolated from tumor samples from pharmacodynamic studies, using the 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, and GAPDH genes 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. The catalog numbers for primers of each genes are: Hs00365956_m1 (HOXA9), Hs00180020_m1 (MEIS1), and Hs02786624_g1 (GAPDH)

ABBREVIATIONS

ALL

acute lymphoblastic leukemia

AML

acute myeloid leukemia

DCM

dichloromethane

DIBAL-H

Diisobutylaluminium hydride

DIPEA

N,N-Diisopropylethylamine

ESI-MS

electrospray ionization mass spectrometry

FP

Fluorescence polarization

MD

molecular dynamic

MEN1

multiple endocrine neoplasia 1

MLL

mixed lineage leukemia protein

MTD

maximum tolerated dose

PBS

phosphate buffered saline

PD

pharmacodynamics

RT

room temperature

SCID

severe combined immunodeficient

TFA

trifluoroacetic acid

TGI

tumor growth inhibition

THF

tetrahydrofuran

UPLC

Ultra performance liquid chromatography