Pyridinylpyrimidines selectively inhibit human methionine aminopeptidase-1

Abstract

Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure-activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of fifty-eight analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5′-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMet AP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a–30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.

1. Introduction

With few exceptions, cellular protein synthesis is initiated with either an N-formylmethionine in eubacteria, mitochondria and plastids or a methionine in archaea and eukaryotes¹. In eubacteria, the initiator methionine at the N-terminus of a significant portion of proteins is irreversibly removed,² following the cotranslational deformylation catalyzed by peptide deformylase.^3,4 In eukaryotes, the process of N-terminal methionine excision (NME) no longer requires peptide deformylase and became cotranslational.⁵ In all cells, NME is catalyzed by a family of ubiquitous enzymes named methionine aminopeptidases (MetAPs).⁶ As metalloproteases, MetAPs utilize one or two divalent metals to proteolytically cleave the initiator methionine from nascent peptides,⁷ with strict substrate specificity. Cleavage will only occur when the P1 residue is methionine and the side chain of P1′ residue is small and uncharged.^8,9 Residues at the positions of P2′ and beyond also contribute to the efficiency of cleavage.^8c,9

MetAPs are classified into two types. Type 1 enzymes originated from eubacteria, and type 2 originated from archaea. Only type 2 MetAP contains a characteristic insertion in its catalytic domain.⁶ Although archaea only have type 2 MetAPs and eubacteria only have type 1 MetAPs,^7b,10,11 at least two genes encoding one cytosolic type 1 MetAP (MetAP1) and one cytosolic type 2 MetAP (MetAP2), respectively, can be found in all eukaryotic genomes.^12,13 There is limited sequence identity between type 1 and type 2 MetAPs,^12,13 but crystallographic analysis demonstrated that the catalytic domains of all MetAPs adopt the same pita-bread fold, in which the N- and C-terminal halves display pseudosymmetry about the active site.⁶ Moreover, the five metal binding residues and the shape of methionine-binding pocket are also highly conserved.^6,14

The ubiquitous distribution of MetAP, their highly conserved structures and substrate specificity, suggest that NME may play an important role in cellular life. Mycoplasma genitalium has the smallest genome to support cellular growth in pure medium. One gene (map) encoding a MetAP has been found among its 382 essential genes.¹⁵ In eubacteria, the importance of MetAP is underscored by the lethality when the single map gene in Escherichia coli or Salmonella typhimurium was disrupted.^10,11 In Saccharomyces cerevisiae, deletion of either map1 or map2 gene caused a slow growth phenotype, and double deletion of both map genes was lethal.¹² In Arabidopsis thaliana, cytosolic MetAP1 and MetAP2s are functionally redundant. However, the complete inactivation of NME in the cytosol blocked the plant's development after germination.¹⁶ In animals, abrogation of cytosolic MetAP2 leads to organism-specific and tissue-specific developmental defects. Most of the defects caused either lethality or infertility.¹⁷ Small-interfering RNA (siRNA) mediated downregulation of either MetAP1 or MetAP2 significantly inhibited the proliferation of human umbilical vein endothelial cells, suggesting that both human MetAPs (HsMetAPs) are essential for cell proliferation.¹⁸ More recently, Hu et al. demonstrated that MetAP1 siRNA duplexes could delay the cell cycle progression of synchronized HeLa cells.¹⁹ Bengamides, a family of natural products isolated from marine sponges,^20,21 were identified as nonselective inhibitors of both MetAP1 and MetAP2.²² The antitumor activities of bengamides in vitro²¹ and in vivo²³ evince a pivotal role of MetAPs in human cells.

Over two decades ago, fumagillin, a natural metabolite of Aspergillus fumigatus, and its synthetic analogue TNP-470 (also known as AGM-1470) were discovered as potent inhibitors of angiogenesis.^24,25 Subsequently, we and others identified MetAP2 as the primary target of fumagillin and TNP-470,²⁶ and found that both inhibitors specifically inhibit MetAP2 without affecting MetAP1.^27,28 Because the ablation of MetAP2's enzymatic activity preferentially inhibited endothelial cell proliferation, hence angiogenesis,^29,30 TNP-470 displayed broad-spectrum activities against the growth of both primary and metastatic tumors in various animal models (for reviews, see^31,32). TNP-470 was also effective against nutritionally induced obesity³³, pulmonary hypertension³⁴ and arthritis³⁵. Inspired by fumagillin and its derivatives, there has been a renewed interest in developing MetAP2-selective inhibitors to treat cancer, rheumatoid arthritis and collagen-induced arthritis. Besides serving as a valuable prototype of angiogenesis inhibitors, fumagillin and its derivatives also greatly facilitated the investigation of MetAP2's functions in signal transduction, cell cycle progression and development of higher eukaryotes.

The shortage of potent and isoform-selective inhibitors of MetAP1 not only hampered the advancement of MetAP1 biology, but also restrained the evaluation of MetAP1 as a novel target for anti-antineoplastic therapy. Peptidyl hydroxamic acids were shown to inhibit human MetAP1 (HsMetAP1) in vitro. However, their inhibitory potency was low and lacked isoform selectivity with similar K_i values for the inhibition of both purified HsMetAP1 and human MetAP2 (HsMetAP2).³⁶ Later, several barbiturate-based compounds were identified in an in vitro assay as low micromolar inhibitors of HsMetAP1. But their isoform selectivity is still unclear.³⁷ We and others have previously reported that the derivatives of pyridine-2-carboxamide (such as compound 1, Figure 1) inhibited the purified type 1 MetAP from either prokaryotes (Escherichia coli) or eukaryotes (Saccharomyces cerevisiae and human) in vitro.^19,38,39 Intriguingly, this class of inhibitors not only have low micromolar IC₅₀ values against HsMetAP1 but also showed great selectivity for HsMetAP1 over HsMetAP2.¹⁹ However, we subsequently found that at low micromolar concentrations, 1 also had unexpected off-target celllular effects (a corrigendum will be published later). In addition to pyridine-2-carboxamides, a novel class of inhibitors containing 2-pyridinylpyrimidine (such as compound 2, Figure 1) was discovered in our group via a high-throughput screening of a commercial library.⁴⁰ These pyridinylpyrimidines potently inhibited both purified HsMetAP1 and purified HsMetAP2 with moderate selectivity for HsMetAP1. Subsequently, crystallographic analysis of the catalytic domain of HsMetAP1 in complex with inhibitors revealed that both 1 and 2 mainly depended on the same auxiliary Co(II) (M3) in the active site to inhibit HsMetAP1 (Figure 2A and Figure 2B).^19,40 However, Hu et al. showed that 1 and 2 could inhibit HsMetAPs inside HeLa cells.^19,40 In addition, Chai and Ye demonstrated that an auxiliary metal-mediated quinolinyl sulfonamide (compound 3, Figure 1) inhibited cellular MetAP in bacteria as well, albeit at a high concentration.⁴¹

Figure 1.

Structures of MetAP inhibitors 1-3.

Figure 2.

The X-ray crystal structures of the N-terminally truncated human MetAP1 (tHs MetAP1) in complex with 1 (2NQ6 [19]) (A) or 2 (2G6P [40]) (B) and (C). For all non-carbon atoms, nitrogen is blue, oxygen is red, sulfur is yellow and chlorine is green. In the active site of tHsMetAP1, Co(II) ions are shown as hot pink spheres, water molecules are shown as small red spheres and metal interactions are shown as dashed lines. In (A) and (B), 1 (carbon green), 2 (carbon purple) and the surrounding residues (carbon white) are shown as sticks. (C) In a surface-filling model of tHs MetAP1 (carbon white) in complex with Co(II) and 2, a HEPES molecule (carbon magenta) from the buffer is accommodated in a groove formed by Y196, G352 and W353. The HEPES molecule resides on the exterior of the phenyl group of 2, and generally parallels to the phenyl group.

Herein we describe a systematic medicinal chemistry approach to probing the structural requirements of pyridinylpyrimidine scaffold for selective inhibition of HsMetAP1. The X-ray crystal structure of compound 2 bound to the Co(II) form of the N-terminally truncated HsMetAP1⁴⁰ was used to guide our SAR studies. We set out to address: (1) the minimum elements of the pyridinylpyrimidine skeleton needed for inhibition of HsMetAP1; (2) the SAR of substitution at the C3′–C6′ positions of the pyridine ring; and (3) the optimal side chain at the C4 position of the pyrimidine ring. In addition to eight compounds purchased from commercial sources, we designed and synthesized fifty analogues based on the pyridinylpyrimidine scaffold and characterized their inhibitory activity with purified HsMetAP1 and HsMetAP2. We also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1 (PDB access code 4HXX).

2. Chemistry

Analogous to the procedure described by Medwid et al.,⁴² we started our synthesis of 2-pyridinylpyrimidine derivatives by transforming substituted picolinonitriles 4 into the corresponding picolinimidamides 5 (Scheme 1). Suitably substituted ethyl 3-oxobutanoates were condensed with 5 under basic condition using sodium methoxide and the resulting pyrimidinones 7 were refluxed with phosphoryl chloride to obtain 4-chloropyrimidines 8. The displacement of the 4-chloro group on pyrimidine 8 by phenethylamine was accomplished by refluxing the reaction mixture in methanol containing potassium carbonate. Thus, by using ethyl 2-fluoroacetoacetate, ethyl 2-methylacetoacetate, or ethyl acetoacetate in the condensation step to generate pyrimidinone 7, 2-pyridin-2ylpyrimidines 9a, 9b or 9c, respectively, were obtained as the final products. Pyrimidine derivatives carrying an ethylene diamine side chain 11, were synthesized by treating chloropyrimidine 8 with Boc-protected ethylyene diamine 10, and by treating the resulting product with trifluoroacetic acid. The pyrimidine derivatives 11 served as the intermediate in the synthesis of other side chain analogues of 2-pyridin-2-ylpyrimidine. Compound 13 was prepared by alkylating 5-chloro-6-methyl-2-(pyridin-2-yl)-3,4-dihydropyrimidine-4-thione 12 with phenethyl bromide as shown in Scheme 2.

Scheme 1.

Synthesis of 2-pyridinyl-6-methylpyrimidines. Reagents & Conditions: (i) MeONa/MeOH, rt; (ii) NH₄Cl/MeOH, 65-74% yields for two steps; (iii) H₃C-COCH(X)CO₂Et (6), MeONa/MeOH, 45-53% yields; (iv) POCl₃, reflux, 65-72% yields; (v) PhCH₂CH₂NH₂, K₂CO₃/MeOH, 65 °C, 85-92% yield; (vi) (R)-H₂NCH₂CH(Ph)NHBoc (10), Et₃N, THF, 82-95% yields; (vii) CF₃CO₂H/CH₂Cl₂, then aqueous NaHCO₃, 81-86% yields. Yield for 9a, 9b, and 9c were 56%, 62%, and 49% over three steps, respectively.

Scheme 2.

Synthesis of 13. Reagents & Conditions: (i) K₂CO₃, PhCH₂CH₂Br, toluene, 60 °C, 92% yield.

Acylation of amines 11 provided chlorides 14 (Scheme 3) which were reacted with piperazine derivative 15 to afford amino alcohols 16a and 16b after desilylation. Additionally, replacement of chloride in 14 with Boc-protected piperazine 16 and subsequent deprotection produced a series of biaryl compounds (18a–18o) with different substituents on the pyridine ring. In a parallel scheme (Scheme 4), amine 11a was reacted with two piperazine-containing alkyl bromides (19a and 19b) and the resulting adducts were dprotected with TFA and TBAF respectively to give compounds 20a and 20b. Reductive amination of 11a with two piperazine-containing aldehydes (21a and 21b) furnished compounds 22a and 22b, after removal of the protecting groups. The sulfonamides 24a and 24b were assembled from condensation of the amine 11a with ethenesulfonyl chloride (Scheme 5). The resultant sulfonamide 23 was reacted with piperazines 15 and 17 to afford 24a and 24b upon deprotection with TFA or TBAF.

Scheme 3.

Synthesis of 2-pyridinylpyrimidine derivatives 16a, 16b and 18a–18p. Reagents & Conditions: (i) Cl(CH₂)_nCH₂COCl, Et₃N, THF, ∼80% yield; (ii) NaI, Na₂CO₃, DMF then TBAF, 67-74% yields; (iii) NaI, Na₂CO₃, DMF, then CF₃CO₂H, 62-79% yields.

Scheme 4.

Synthesis of additional 2-pyridinylpyrimidine derivatives 20a, 20b, 22a and 22b. Reagents & Conditions: (i) NaI, DMF then TFA or TB AF, 84-88% yields; (ii) NaBH₃CN, HO Ac, MeOH, then TFA or TBAF, 55-60% yields.

Scheme 5.

Synthesis of sulfonamide derivatives 24a and 24b. Reagents & Conditions: (i) CH₂=CHSO₂Cl, Et₃N, CH₂Cl₂, 91% yield; (ii) 15 or 17, EtOH 50 °C, 80-90% yields; (iii) TFA or TBAF, 80-84% yields.

Amides 25a–25e were prepared by simple condensation of pyridinylpyrimidine 11b with carboxylic acids (Scheme 6). A series of secondary amines (26a–26f and 27a–27h) were obtained via either reductive amination or a simple displacement reaction (Scheme 6). To synthesize compounds 30a–30d, we carried out alkylation reaction of 2-phenylacetonitrile with alkyl bromides (Scheme 7). The resultant alkylation products 28 were reduced with LAH to afford primary amines 29, which were coupled with the aryl chloride 11b to yield 30a–30d in good yields.

Scheme 6.

Synthesis of 2-(5-chloro-pyridin-2-yl)-pyrimidine derivatives 25a–25e, 26a–26f and 27a–27h. Reagents & Conditions: (i) Ph(CH₂)_nCO₂H, EDCI, HOBt, DIPEA, 70-85% yields; (ii) Ar(CH2)_nCHO, NaBH₃CN, HO Ac, MeOH, 25-35% yields; (iii) NaI, Et₃N, CH₃CN, 37% yields.

Scheme 7.

Synthesis of 2-(5-chloro-pyridin-2-yl)-pyrimidine derivatives 30a–30d. Reagents & Conditions: (i) Ph(CH₂)_nBr, NaH, DMF, 0 °C, 50-65% yields; (ii) LAH, ether, 80-90% yields; (iii) 8, Et₃N, THF, reflux, 70-80% yields.

3. Results and discussion

3.1 Improvement in potency and isoform selectivity of pyridinylpyrimidine derivatives against human MetAP1

The starting point for this work was compound 2, one of the best inhibitors of both Plasmodium falciparum MetAP1b and human cytosolic MetAPs (HsMetAP1 and HsMetAP2) identified through a high-throughput screening against approximately 175,000 compounds.^40,43 According to the crystal structure of N-terminally truncated HsMetAP1 in complex with Co(II) and 2, compound 2 mainly relied on an auxiliary Co(II)(M3) in the active site of HsMetAP1 to inhibit the enzyme (Figure 2B).⁴⁰ This auxiliary Co(II) adopted a distorted octahedral geometry, and was hexacoordinated by a pyrimidine nitrogen and a pyridine nitrogen from the inhibitor, an imidazole nitrogen from His212 of HsMetAP1 and three water molecules (W1, W2 and W3 in Figure 2B). In agreement with this vital role of pyridinylpyrimidine in coordinating the auxiliary Co(II), replacement of 2-pyridinyl group in 2 with phenyl, 3-pyridinyl or 4-pyridinyl group gave compounds exhibiting very poor potency against Plasmodium falciparum MetAP1b.⁴³ Similarly, compound 31, a 2-(3-pyridinyl)-pyrimidine analogue, had no effect on human MetAP at concentrations up to 100 μM (Table 1). Together, these lines of evidence suggested that the 2-(2-pyridinyl)-pyrimidine capable of chelating a metal ion is a key pharmacophore for the inhibition of MetAP enzymes.

Table 1. Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives.

Cmpd	R1	R2	IC50a (μM)		IC50 ratio (type 2/1)
			HsMetAP1	HsMetAP2
2	Cl	NHCH2CH2Ph	0.86 ± 0.04	9.0 ± 0.9	10
9a	F	NHCH2CH2Ph	4.6 ± 0.1	62 ± 9	13
9b	Me	NHCH2CH2Ph	>100	>100	NAb
9c	H	NHCH2CH2Ph	>100	>100	NAb
13	Cl	SCH2CH2Ph	10 ± 1	>100	>10
31	Cl	NHCH2Ph	>100	>100	NAb
32	Cl	SCH2Ph	11 ± 1	>100	>9.1
33	Cl	N(Me)CH2Ph	1.5 ± 0.1	13 ± 0.1	8.7
34	Cl	NHMe	8.9 ± 0.3	14 ± 1	1.6
35	Cl		1.0 ± 0.1	>100	>100
36	Cl		2.0 ± 0.1	>100	>50
37	Cl	4-(2-hydroxyl-ethyl)-piperazinyl	1.1 ± 0.2	>100	>91
38	Cl		0.36 ± 0.03	>100	>278

^aIC₅₀ values of compounds to inhibit human MetAPs were shown as mean ± SD of three determinations. The bottom values of all inhibitory dose-response curves were smaller than 10%.

^bNA: not available.

To determine the role of chlorine substitution at C5 of the pyrimidine ring (Table 1), three analogues were synthesized. Replacement of chlorine (2) with fluorine (9a) decreased the potency against HsMetAP1 and HsMetAP2 by 5.3-fold and 6.9-fold, respectively. Moreover, replacement of the chlorine with a methyl group (9b) or hydrogen (9c) led to inactive analogues, suggesting that chlorine is the optimal substituent at C5 of the pyrimidine ring.

Next, we assembled eight analogues with different substituents at C4 of the pyrimidine ring (Table 1). Swapping the secondary amine for thioether linkages (13 and 32) resulted in a significant decrease in potency against both HsMetAP1 and HsMetAP2 without any appreciable change in the isoform selectivity in favor of HsMetAP1. High similarity between the structures of the catalytic domains of HsMetAP1 and HsMetAP2 presents a formidable challenge in the design of type 1-selective inhibitors. In our previous report of Plasmodium falciparum MetAP1b inhibitors, a wide range of substituents at C4 were well tolerated by PfMetAP1 enzyme. However, when the X-ray crystal structure of HsMetAP1 in complex with 2 was superimposed on the structure of HsMetAP2, several potential steric clashes between the inhibitor and HsMetAP2 were revealed.⁴⁰ The most severe steric clash was between the C4 side chain of 2 and the Tyr444 residue of HsMetAP2. Presumably, an unfavorable interaction like this could be exploited to improve the inhibitor's selectivity for HsMetAP1 over HsMetAP2. Indeed, decreases in size of C4 substituents diminished HsMetAP1 selectivity (33 and 34). On the contrary, incorporating longer and more polar C4 side chains generally yielded substantial increases in the selectivity for HsMetAP1 over HsMetAP2, and at the same time the potency against HsMetAP1 was retained (35–38).

The crystal structure of compound 2 in complex with N-terminally truncated HsMetAP1 also revealed an unexpected 2-[4-(2-hydroxylethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) molecule from the buffer bound in a groove formed by Tyr196, Gly352 and Trp353. This HEPES molecule resided on the exterior of the phenyl group of 2 (Figure 2C). We postulated that the binding of pyridinylpyrimidine analogues to HsMetAP1 might benefit from extending C4 substituent of 2 all the way to the piperazine ring of HEPES. One such compound (24b in Table 2) was synthesized by fusing HEPES moiety to compound 2 via the C4 side chain. Although 24b did not gain appreciable potency against HsMetAP1, there was a considerable decrease in its potency against HsMetAP2. As a result, 24b achieved 158-fold selectivity for HsMetAP1 over HsMetAP2. In light of this increased selectivity for HsMetAP1, nine more pyridinylpyrimidine analogues were synthesized (Table 2). When the sulfonamide in the linker in 24b was replaced by either a secondary amine (20b) or an amide (16a), there was little change in potency and HsMetAP1-selectivity. Extending the amine linker and the amide linker by one carbon further improved the HsMetAP1-selectivity (22b and 16b). On the contrary, compared to 24b and 16a, shortening the side chain by removal of the 2-hydroxylethyl group led to 13-fold and 4-fold decreases in the HsMetAP1-selectivity of 24a and 18a, respectively. However, removing the same 2-hydroxylethyl group did not have any appreciable effect on two analogues with secondary amine linkers (20a vs. 20b, and 22a vs. 22b) and one analogue with a longer amide linker (18b vs. 16b). Thus, installation of a long side chain containing a piperazinyl group to the C4 position of the pyrimidine ring resulted in the largest improvement in HsMetAP1-selectivity.

Table 2. Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives.

Cmpd	R	IC50a (μM)		IC50 ratio (type 2/1)
		HsMetAP1	HsMetAP2
16a		0.8 ± 0.1	114 ± 12	143
16b		1.1 ± 0.1	>300	>272
18a		0.6 ± 0.1	22 ± 4	37
18b		1.1 ± 0.2	>300	>272
20a		1.4 ± 0.2	108 ± 11	77
20b		1.7 ± 0.5	101 ± 12	59
22a		1.3 ± 0.5	188 ± 17	145
22b		0.8 ± 0.2	197 ± 22	246
24a		0.8 ± 0.1	9.4 ± 1.0	12
24b		0.6 ± 0.1	95 ± 14	158

^aIC₅₀ values of compounds to inhibit human MetAPs were shown as mean ± SD of three determinations. The bottom values of all inhibitory dose-response curves were smaller than 10%.

After a comparative analysis of twenty analogues with various side chains, we selected 18b as a lead to explore the SAR of substituents on the pyridine ring of pyridinylpyrimidine (Table 3). Compared to 18b, installation of either a methoxyl group or a chloro group at C6′ or C3′ reduced the potency against HsMetAP1 to different degrees (18c, 18d, 18e and 18f). On the contrary, the potency was enhanced by 4.1-fold via the introduction of a methoxyl substituent at the adjacent C5′ position (18g). Similarly, introduction of fluoro (18h), chloro (18i), bromo (18j), nitro (18k) or dimethylamino (18l) group at C5′ led to 3.3-fold to 31-fold increases in the anti-HsMetAP1 activities relative to 18b. Unfortunately, the potency against HsMetAP2 was simultaneously restored for 18h, 18i, 18k and 18l, significantly compromising HsMetAP1-selectivity. A pyrrolidinyl substituent, which is bigger than a dimethylamino group, was not well tolerated at C5′, as indicated by the reduced inhibitory potency and the loss of HsMetAP1-selectivity of 18m. In contrast to the substituents at C6′ and C3′, analogues with methoxy (18n) and chloro (18o) substituents at C4′ maintained their potency against HsMetAP1. However, the HsMetAP1-selectivity of 18n was attenuated by the relatively bigger C4′ substituent.

Table 3. Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives.

Cmpd	Substituents (when not specified, R# = H)	EC50a (μM)		EC50 ratio (type 2/1)
		HsMetAP1	HsMetAP2
18c	R1 = OMe	20 ± 6	>30b	>1.5
18d	R1 = Cl	7.2 ± 2.4	>30b	>4.2
18e	R4 = OMe	2.5 ± 1.3 (Bc = 0.15)*	>300	>120
18f	R4 = Cl	7.6 ± 0.4	>300	>39
18g	R2 = OMe	0.27 ± 0.04	>300	>1111
18h	R2 = F	0.049 ± 0.07	1.2 ± 0.3	24
18i	R2 = Cl	0.036 ± 0.006	1.2 ± 0.3	33
18j	R2 = Br	0.33 ± 0.01	53 ± 5	161
18k	R2 = NO2	0.30 ± 0.06	14 ± 1	47
18l	R2 = N(CH3)2	0.14 ± 0.05	1.7 ± 0.7	12
18m	R2 = pyrrolidinyl	1.7 ± 0.3 (B = 0.36)	1.1 ± 0.1 (B = 0.22)	1.1
18n	R3 = OMe	0.53 ± 0.07	14 ± 9	26
18o	R3 = Cl	0.73 ± 0.03	>300	>411

^aEC₅₀ values of compounds to inhibit human MetAPs were shown as mean ± SD of two to three determinations.

^bHigher concentrations have not been tested for these compounds.

^cB: bottom value of the inhibitory dose-response curve. It has been noted in parentheses only when B>10%.

As 18i exhibited impressive potency against HsMetAP1 and moderate selectivity over HsMetAP2, we decided to further explore the C4 side chain to improve HsMetAP1-selectivity (Table 4). First, swapping of the piperazinyl group in 18i to a phenyl group greatly improved HsMetAP1-selectivity (25a–25e), best exemplified by 25b and 25c. Except 25d, other compounds in this series retained submicromolar IC₅₀ values for HsMetAP1. Secondly, changing the carbonyl group in side chain of 25a–25e to a methylene group afforded a new series of analogues (26a–26f) with submicromolar IC₅₀ values and comparable selectivity for HsMetAP1 in comparison to 25a–25e. Thirdly, derivatizing 26d to 27a–27d or replacing the phenyl group at the end of the side chain with various heterocyclic aromatic rings (27e–27g) only led to modest (1.6-fold to 6.0-fold) reductions in the potency against HsMetAP1. Except 27c that potently inhibited the coupling enzyme in BcProIP-coupled HsMetAP2 assay, the remaining eight analogues in this series were inactive against HsMetAP2 at the highest concentrations tested. And lastly, 30a–30d were generated by using 3–6 carbon aliphatic linkers to replace the secondary amino linker that tied the two phenyl groups together in the C4 side chain of 26d. All analogues in this series bore excellent HsMetAP1-selectivity. And when the aliphatic linker was shortened, the compound appeared to be more potent (compare 30a with 30d), with analogue 30a being among the most selective and potent inhibitor of HsMetAP1 reported to date.

Table 4. Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives.

Cmpd	R	EC50a (μM)		IC50 ratio (type 2/1)
		HsMetAP1	HsMetAP2
25a	NHCOCH2Ph	0.70 ± 0.52	28 ± 6	40
25b	NHCO(CH2)2Ph	0.093 ± 0.042	>30b	>323
25c	NHCO(CH2)3Ph	0.16 ± 0.12	>30b	>188
25d	NHCO(CH2)4Ph	1.3 ± 0.2 (Bc = 0.12)	>30b	>23
25e	NHCO(CH2)5Ph	0.67 ± 0.30	>30b	>45
26a	NHCH2Ph	0.22 ± 0.7	31 ± 5	141
26b	NH(CH2)2Ph	0.26 ± 0.05	>100	>384
26c	NH(CH2)3Ph	0.26 ± 0.06	>300	>1154
26d	NH(CH2)4Ph	0.20 ± 0.03	>100	>500
26e	NH(CH2)5Ph	0.78 ± 0.19	>100	>138
26f	NH(CH2)6Ph	0.71 ± 0.21	>30b	>42
27a	NH(CH2)4-(4-phenolyl)	0.42 ± 0.06	>300	>714
27b	NH(CH2)4-(4-chlorophenyl)	0.57 ± 0.10	>300	>526
27c	NH(CH2)4-(4-cyanophenyl)	0.52 ± 0.07	16 ± 7d	31
27d	NH(CH2)4-(4-propoxylphenyl)	1.2 ± 0.2	>300	>250
27e	NH(CH2)4-(2-furyl)	0.33 ± 0.08	>300	>909
27f	NH(CH2)4-(2-thienyl)	0.38 ± 0.18	>300	>789
27g	NH(CH2)4-(2-quinolyl)	0.69 ± 0.08	>300	>435
27h	NH(CH2)4-(1-pyrrol)	0.50 ± 0.09	>100	>200
30a	(CH2)2Ph	0.063 ± 0.024 (Bc = 0.14)	>200	>3175
30b	(CH2)3Ph	0.16 ± 0.01 (Bc = 0.13)	>200	>1250
30c	(CH2)4Ph	0.20 ± 0.11	>200	>1000
30d	(CH2)5Ph	2.3 ± 1.8	>200	>87

^aEC₅₀ values of compounds to inhibit human MetAPs were shown as mean ± SD of two to three determinations.

^bHigher concentrations have not been tested for these compounds.

^cB: bottom value of the inhibitory dose-response curve. It has been noted in parentheses only when B>10%.

^dIn the counter screening, compound 27c was identified as a potent inhibitor of the coupling enzyme, BcProIP. 10 μM and 100 μM of 27c inhibited 27% and 97% of the activity of BcProIP in MetAP assay buffer, in the presence of 600 μM Pro-pNA and 10 μM CoCl₂.

3.2 The X-ray crystal structure of N-terminally truncated human MetAP1 in complex with compound 26d

The crystal structure of N-terminally truncated (Δ1–80) human MetAP1 (tHsMetAP1) in complex with Co(II) and 26d was determined to 2.09 Å resolution (Table 5). The locations of cobalt ions were identified by a difference electron density map computed with the anomalous diffraction signal (Figure 3A). Two Co(II) are presented at the bottom of the active site (Figure 2C), the ubiquitous positions for metal ions (M1 and M2) in the MetAP family. In addition, a third Co(II) (Co3) is located at a place (M3) close to the active site, which has previously been identified in the complexes of tHsMetAP1 with two specific classes of inhibitors.^19,40 Among them, compound 2 has a pyridinylpyrimidine core similar to that of compound 26d. In the crystal structures of tHsMetAP1 in complex with 2 (PDB access code 2G6P, reported in [40]) and 26d, both pyridine nitrogen (N1′) and pyrimidine nitrogen (N1) are bound to Co3 (Figure 3B), displaying a near-perfect octahedral coordination which is completed by the nitrogen atom from the side chain of His212 and three water molecules.

Table 5. X-ray data collection and refinement statistics of the N-terminal truncated (Δl–80) human MetAP1 (tHsMetAP1) in complex with 26d.

Crystal	tHsMetAP1 w. 26d
Space group	P21
Cell dimensions
a (Å)	47.5
b (Å)	77.4
c (Å)	48.0
β (deg)	90.9
X-ray data collection statistics
X-ray Source	FR-E+/Raxis IV
Wavelength (Å)	1.54178
Resolution range (Å) (HighRes shell)	50.00-2.09 (2.16-2.09)
Collected Reflections	71,126
Unique Reflections	20,226
I/σ	29.6 (2.8)
Completeness (%)	97.9 (86.6)
Rmerge (%)	11.7 (49.0)
Refinement statistics
Rcryst (%)	0.18 (0.27)
Rfree (%)	0.25 (0.33)
R.m.s deviations
Bond length (Å)	0.013
Angle (deg)	1.1533
Monomer in ASU	1
Total Atoms	2,586
Protein atoms	2,400
Water molecules	152
Ligand	34
B-factor (ΔHsMetAP1)(Å2)	41.8
B-factor (26d)(Å2)	62.0
B-factor (H2O)(Å2)	55.0

Figure 3.

The X-ray crystal structure (4HXX) of the N-terminally truncated human MetAP1 (tHs MetAP1) in complex with 26d (cyan) (A) and (C), and a superimposition of 4HXX with the crystal structure (2G6P [40]) of tHsMetAP1 with 2 (purple) (B) and (D). A HEPES molecule (carbon purple) in 2G6P was from the buffer. In (A) and (B), the stereo views of the active site of tHsMetAP1 show that both 26d and 2 (sticks) mainly depend on an auxiliary Co(II) (Co3, green sphere) to bind the H212 residue (sticks) of HsMetAP1. Water molecules are shown as red spheres and metal interactions are shown as dashed lines. In (C) and (D), the surface-filling views of tHsMetAP1 (surface colored gray) show the cavity in the active site which accommodates 26d and 2. The C4 side chains of 26d and 2 interact with different parts of the enzyme. For all non-carbon atoms, nitrogen is blue, oxygen is red, sulfur is yellow and chlorine is green.

26d buries about 468 Ų upon binding tHsMetAP1 from a total accessible area of 753 Ų. Similar to 2, the pyridine ring of 26d is fitted in a hydrophobic pocket which is normally occupied by the side chain of the initiator methionine (Figure 3C). But when compared with 2, an extra chloro group is present at the C5′ position of the pyridine ring in 26d. This chloro group points towards the phenyl group of Phe198 which constitutes the bottom of the hydrophobic pocket for methionine side chain. In comparison to the pyridinylpyrimidine core of 2 in the structure 2G6P, 26d is displaced up to 1 Å out of the hydrophobic pocket for methionine side chain to accommodate the C5′ chloro substituent (Figure 3B). Therefore for 26d, the distances from C5′ of the pyridine ring of the inhibitor to C1 and C4 of the phenyl group in Phe198 increase from 3.87 Å and 4.64 Å in 2G6P to 4.77 Å and 5.70 Å, respectively. However, the pyridinylpyrimidine core of 26d still maintains the octahedral interaction with Co3 and Co3 is in a slightly changed position. Just like 2 in the structure 2G6P,⁴⁰ the pyridine ring and the pyrimidine ring of 26d are still coplanar. In the complex of tHsMetAP1 with 26d, the distances from Co3 to the imidazole nitrogen of His212 and from Co3 to the pyridine nitrogen (N1′) of 26d remain the same as the two distances in 2G6P (2.20 Å, 2.13 Å). However, the distance from Co3 to the pyrimidine nitrogen (N1) of the inhibitor decreases from 2.32 Å in 2G6P to 2.27 Å. The inhibitor binding site does not change significantly, as the main chain remains within 0.25 rmsd of the previously described structure of tHsMetAP1 with 2. The fact that the pyridinylpyrimidine core of 26d is forced out of the hydrophobic pocket for methionine side chain up to 1 Å does not necessarily mean that the chloro substitute at C5′ sabotages the inhibitory potency of 26d. To the contrary, since 18i was 22 times more potent against purified HsMetAP1, the interaction between this chloro group and the hydrophobic pocket may even contribute to the binding affinity of 26d to HsMetAP1. But to satisfy the geometry of the metal coordination, there would be a limit for the displacement of the pyridinylpyrimidine core. This limit may help to explain why the inhibitors with larger substituents at C5′ (18g, 18j, 18k, 18l and 18m) were less potent against purified HsMetAP1 than the inhibitors with smaller substituents such as a fluoro group (18h) or a chloro group (18i).

In the crystal structure of tHsMetAP1 with 2 (PDB access code 2G6P), an unexpected HEPES molecule from the buffer is accommodated in a hydrophobic groove formed by Tyr196, Gly352 and Trp353 on the exterior of the phenyl group of 2.⁴⁰ Because extra interactions with the enzyme may lead to higher potency, we extended the C4 side chain of the inhibitors to exploit potential hydrophobic interactions with this groove. In the structure 2G6P, the phenyl group in the C4 side chain of 2 curls back and stacks over the pyrimidine ring of the same compound. Although the C4 side chain in compound 26d is exactly an extended version of the equivalent in 2, it does not curls back and interact with Tyr196, Gly352 and Trp353. Instead, the C4 side chain of 26d adopts a wild-open conformation in which the additional aliphatic segment of the side chain and the second phenyl group at its end extend out towards the external surface of the enzyme and binds to a small hydrophobic pocket created by the side chain of Lys132 (Figure 3C). To accommodate the end of the C4 side chain of 26d, the side chain of Lys132 moves up to 4.74 Å towards the external surface of the enzyme. This interaction most likely keeps the C4 side chain of 26d in the open conformation and prevents it from curling onto the pyridinylpyrimidine core of the inhibitor.

A superimposition of the crystal structure of tHsMetAP1 in complex with compound 26d and the crystal structure of HsMetAP2 in complex with L-methionine (PDB access code 1KQ9, reported in [44]) revealed a possible structural basis for the selectivity of 26d for HsMetAP1 over HsMetAP2 (Figure 4). Compared with HsMetAP1, the 63-amino acid insertion (from residue 382 to 444) in the catalytic domain of HsMetAP2 results in a smaller entrance to its active site. In addition, the smaller distance between His231 and His339 of HsMetAP2 (5.38 Å vs. 5.89 Å between the equivalent His212 and His310 in HsMetAP1) also narrows the entrance. In the present conformation, the long C4 side chain of 26d is likely to clash with Tyr444 of HsMetAP2, and the C5′ chloro substituent on the pyridine ring of 26d might clash with Pro220 which defines the bottom of the binding pocket for methionine side chain in HsMetAP2.

Figure 4.

A superimposition of the crystal structure of N-terminally truncated HsMetAP1 (surface colored grey) in complex with 26d (sticks, carbon cyan) and the crystal structure of HsMetAP2 (wires and ribbons, colored teal) in complex with L-methionine (1KQ9 [44]). The residues of HsMetAP2 are shown as teal sticks. The numbering throughout is based on HsMetAP2. Compared with the catalytic domain of HsMetAP1, the insertion (382–444) in the catalytic domain of HsMetAP2 results in a smaller entrance to the active site. In particular, Y444 clashed with 26d. D442 and H339 on the other side of the inhibitor also narrow the binding site for 26d. The L-methionine and the surface filling model of HsMetAP2 are not shown for the sake of clarity. For all non-carbon atoms, nitrogen is blue and chlorine is green.

4. Conclusion

Starting from 5-chloro-6-methyl-N-(2-phenylethyl)-2-pyridin-2-ylpyrimidine-4-amine (2) as a hit in a high-throughput screening and based on the X-ray crystal structure of N-terminally truncated HsMetAP1 (tHsMetAP1) in complex with Co(II) and 2, we set out to identified the key pharmacophore for the inhibition of MetAP enzymes. The deleterious effects on inhibitor potency by the replacement of the 2-pyridin-2-yl group with a 2-pyridin-3-yl group, as well as the crystal structure of 26d bound in the active site,of tHsMetAP1, confirm the key interactions between the auxiliary cobalt ion and the N1 and N1′ nitrogens. We also established chlorine as the optimal substituent at C5 of the pyrimidine ring. Therefore, 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine has been revealed as the key pharmacophore for the potent inhibition of HsMetAP1. A potential steric clash between the C4 substituent of 2 and the Tyr444 residue of HsMetAP2 has been exploited to improve the inhibitor's selectivity for HsMetAP1 over HsMetAP2. Assays with a variety of commercially available pyridinylpyrimidine analogues demonstrated that decreases in size of C4 substituents diminished the selectivity for HsMetAP1, while the installation of longer side chains at C4 increased the selectivity in general. In the effort to extend the C4 side chain to exploit hydrophobic interactions with the enzyme, we discovered that a long C4 side chain containing a piperazinyl group greatly enhanced selectivity for HsMetAP1 (18b). Based on 18b, a variety of substituents on the pyridine ring have been explored and we found that small substituents at C4′ could be tolerated, substituents at C6′ or C3′ reduced the potency against HsMetAP1, and small substituents at C5′ greatly improved the potency but compromised the HsMetAP1 selectivity (18h and 18i). In the further exploration of the C4 side chain on the basis of 18i, we successfully restored the HsMetAP1 selectivity of inhibitors by swapping the piperazinyl group to various aromatic groups. In addition, we uncovered that the replacement of the carbonyl group with a methylene group and the exchange of the secondary amino linker for aliphatic linkers in the C4 side chain were both well tolerated. In the end, we obtained a group of compounds containing both high nanomolar potency against 0.15 μM of purified HsMetAP1 and hundreds/thousands fold selectivity over HsMetAP2. Thus, these compounds may serve as leads for the development of therapeutical agents against cancer.

5. Experimental

5.1. Chemistry

5.1.1. General methods

Unless stated otherwise, all non-aqueous reactions were carried out under ambient atmosphere in oven-dried glassware. Indicated reaction temperatures refer to those of the reaction bath while room temperature (rt) is noted as 25 °C. All solvents were of reagent grade purchased from Fisher Scientific or VWR and used as received. Commercially available starting materials and reagents were purchased from Acros, Aldrich, or TCI America and were used as received. Eight commercially available 2-pyridinylpyrimidines (31–38) were purchased from Maybridge (Cambridge, United Kingdom), and their masses were confirmed by API-MS.

Analytical thin layer chromatography (TLC) was performed using Analtech Uniplates (silica gel HLF, W/UV254, 250 μm). Visualization was achieved using 254 nm UV light, or additionally by staining with iodine, or ceric ammonium molybdate stain. Crude products were purified by air-flashed column chromatography over silica gel (0.06–0.2 mm, 60 Å, from Acros) using indicated eluents. Melting points were recorded on a Mel-Temp II apparatus and are uncorrected. NMR data were collected on a Varian Unity-400 (400 MHz 1H, 100.6 MHz 13C) or Bruker-Spectrospin 400 MHz spectrometers. Chemical shifts are reported in ppm (δ) with the solvent resonance or 0.1% tetramethylsilane contained in the deuterated solvent as the internal reference. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet, br = broad, app = apparent, exch = exchangeable), coupling constants (J, reported in Hertz, Hz), and number of protons. Low resolution mass spectra were acquired on a Thermo-Finnigan MAT, LCQ Classic ESI-mass spectrometer or Voyager DE-STR, MALDI-TOF (Applied Biosystems) instruments. The MALDI-samples were prepared by mixing the droplets of the sample solutions in chloroform or methanol and 2,5-dihydroxybenzoic acid solution in acetone, where the latter served as the matrix. Data are reported in the form m/z (molecular ion).

Samples were analyzed for purity on an Agilent LC-MS system consisting of 1200 Infinity Series LC equipped with an autoinjector, 1260 Diode-array detector (DAD), and 6120 Quadrupole MS detector. A Pursuit XRs C18 column (3 μm, 4.6 × 150 mm) was used while eluting with 60:40 acetonitrile/water (with 0.1 % TFA) as an isocratic mobile phase set at a flow rate of 0.8 mL/min. DAD output was processed for 254, 274, 294 nm while simultaneously screening for the desired mass in API-ES mode of the MS detector (mass range: 100 to 1000, positive mode, fragmenter set at 200). Purity of final compounds was determined to be ≥94%, using a 20 μL injection (approximately 1 mM in acetonitrile) with quantitation by area under the curve (AUC) at 254 and 274 nm. The retention time (tR) is reported in minutes with the AUC given in parentheses.

5.1.2. Synthesis of tetrasubstituted pyrimidines (9a–9c)

Tetrasubstituted pyrimidines 9a–9c were synthesized following a three-step procedure described by Medwid et al.⁴⁵ Briefly, amidine hydrochloride 5 was prepared from 2-cyanopyridine by treating with 1.25 M HCl in methanol for 18 h and the resulting white solid was used without further purification to condense with different acetoacetates (ethyl 2-fluoroacetoacetate, ethyl 2-methylacetoacetate and ethyl acetoacetate in the cases of 9a, 9b and 9c, respectively) under basic conditions. The pyrimidinones 7 were reacted with phosphorus oxychloride at 65 °C for 24 h and after a basic work-up they were purified by flash column chromatography over silica gel. Subsequent displacement of the 4-chloro group on pyrimidines 8 with phenethylamine under refluxing conditions with potassium carbonate in methanol yielded the final products 9a-9c which were purified by column chromatography over silica gel (eluent: 5–10% EtOH—DCM). Pyridinyl pyrimidines 9a– 9c were analyzed for purity using an Agilent LC-MS system described in the general methods section. The t_R values refer to the retention time when the samples were analyzed using Pursuit XRs C18 column (3 μm, 4.6 × 150 mm) with 60:40 acetonitrile/water (0.1% TFA) as an isocratic mobile phase.

5.1.2.1. 5-Fluoro-6-methyl-N-phenethyl-2-(pyridin-2-yl)pyrimidin-4-amine (9a)

Yield: 56% (over three steps). t_R: 4.029 min (99.1%). ¹H NMR (400 MHz, acetone-d6): δ 2.38 (d, J = 1.8 Hz, 3H), 2.96 (t, J = 6.5 Hz, 2H), 3.03 (br s, 1H), 3.55 (t, J = 6.5 Hz, 2H ), 7.21 (m, 5H), 7.61 (ddd, J = 8.2, 2.5, & 1.9 Hz, 1H), 8.05 (ddd, J = 8.2, 2.5, & 1.9 Hz, 1H), 8.42 (d, J = 8.2 Hz, 1H), 8.72 (d, J = 5.6 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃): δ 170.25, 161.72, 155.19, 149.83, 147.46, 139.03, 137.90, 136.94, 129.00, 128.88, 126.77, 124.35, 123.64, 40.31, 35.79, 17.19. MALDI-TOF: m/z 309 (M+H) ⁺, 331 (M+Na) ⁺.

5.1.2.2 5,6-Dimethyl-N-phenethyl-2-(pyridin-2-yl)pyrimidin-4-amine (9b)

Yield: 62% (over three steps). t_R: 7.163 min (94.6%). ¹H NMR (400 MHz, acetone-d6): δ 2.25 (s, 3H), 2.71 (s, 3H), 2.93 (t, J = 6.8 Hz, 2H), 3.42 (t, J = 6.8 Hz, 2H ), 7.13 (m, 5H), 7.41 (ddd, J = 8.1, 5.5, & 2.1 Hz, 1H), 7.85 (ddd, J = 8.1, 5.5, & 2.1 Hz, 1H), 8.1 (d, J = 8.1 Hz, 1H), 8.72 (d, J = 5.5 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃): δ 168.13, 164.17, 157.05, 154.93, 149.46, 139.76, 136.58, 128.91, 128.37, 126.33, 124.91, 123.69, 117.94, 41.29, 34.67, 25.11, 14.73. MALDI: m/z 308 (M+H) ⁺, 330 (M+Na) ⁺, 346 (M+K) ⁺.

5.1.2.3. 6-Methyl-N-phenethyl-2-(pyridin-2-yl)pyrimidin-4-amine (9c).⁴⁶

Yield: 49% (over three steps). t_R: 6.328 min (95.8%). ¹H NMR (400 MHz, acetone-d₆): δ 2.32 (s, 3H), 2.93 (t, J = 7.6 Hz, 2H), 3.65 (m, 2H ), 6.92 (t, J = 7.8 Hz, 2H), 7.24 (t, J = 8.1 Hz, 3H), 7.58 (d, J = 7.8 Hz, 3H), 7.66 (ddd, J = 8.2, 5.6, & 1.9 Hz, 1H), 7.86 (dd, J = 8.2 & 1.9 Hz, 1H), 8.08 (dd, J = 5.6 & 1.9 Hz, 1H), 8.13 (br s, 1H), 8.76 (d, J = 5.6 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃): δ 168.02, 165.51, 162.41, 153.22, 149.13, 147.95, 137.98, 129.05, 126.85, 122.50, 113.48, 38.98, 30.62, 24.19. MALDI-TOF: m/z 291 (M+H) ⁺.

5.1.3. Synthesis of 5-chloro-4-methyl-6-(phenethylthio)-2-(pyridin-2-yl)pyrimidine (13)

Anhydrous potassium carbonate (415 mg, 3 mmol) was added to a solution of 5-Chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-thione (238 mg, 1 mmol) in toluene (15 mL) and the suspension was stirred at 60 °C for 20 min. Phenethyl bromide (222 mg, 1.2 mmol) was added to the reaction mixture and the stirring was continued for an additional 8 h. The reaction mixture was cooled to rt, quenched with with water (25 mL) and the mixture was extracted with EtOAc (2×20 mL). The combined organic layer was concentrated and the crude product was purified by flash column chromatography over silica gel (eluent: EtOH/CHCl₃=5:95) to afford pyrimidine as an off-white solid. Yield: 314 mg (92%). t_R: 6.103 min (96.1%). ¹H NMR (400 MHz, acetone-d₆): δ 2.71 (s, 3H), 2.91 (t, J = 7.6 Hz, 2H), 3.45 (t, J = 7.6 Hz, 2H ), 7.15 (m, 5H), 7.37 (app. dd, J = 8.2 & 5.6 Hz, 1H), 7.84 (app t, J = 8.2 Hz, 1H), 8.1 (d, J = 8.2 Hz, 1H), 8.75 (d, J = 5.6 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃): δ 171.50, 158.82, 155.79, 154.93, 140.36, 136.49, 129.07, 128.89, 126.95, 125.03, 111.45, 35.81, 32.22, 22.83. MALDI-TOF: m/z 343 (M+H) ⁺, 365 (M+Na) ⁺.

5.1.4. Typical procedure for synthesizing compounds 16 and 18

To the solution of 4,5-dichloro-6-methyl-2-(pyridin-2-yl)pyrimidine (8a, 612 mg, 2.5 mmol) and (R)-tert-butyl-2-amino-1-phenylethylcarbamate (783 mg, 3.3 mmol) in THF (10 mL) was added Et₃N (516 mg, 5.1 mmol). The mixture was heated at 50 °C for 12 h, at which point TLC analysis indicated complete transformation. The reaction mixture was cooled to room temperature and diluted with ethyl acetate. After washed with water and brine, the organic phase was dried (Na₂SO₄), filtered and concentrated to give the corresponding condensation product as a light yellow solid, which was treated at room temperature with TFA (3 mL) and CH₂Cl₂ (10 mL) for 1 h before being concentrated in vacuo. The resulting TFA salt was dissolved in EA, and then washed with aq. NaHCO₃ and brine. The organic phase was dried over Na₂SO₄. Evaporation and column chromatography on silica gel (eluent: 5–10% EtOH—DCM or similar conditions) afforded 11a (R1–R4 = H, X = Cl) as the foamy solid (722 mg, 85%): [α]_D²⁰ = -43.6 (c 0.1, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.73 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.27-7.36 (m, 6H), 6.02 (s, 1H), 4.28 (s, 1H), 3.88-3.92 (m, 1H), 3.70-3.72 (m, 1H), 3.34 (br s, 2H), 2.52 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 161.5, 159.5, 157.8, 154.9, 149.8, 142.8, 136.7, 128.8, 127.8, 126.4, 124.4, 123.5, 112.7, 55.1, 48.3, 22.2; ESIMS m/z 340.1 [M + H]⁺; HR-ESIMS (m/z) Calcd. for C₁₈H₁₉N₅Cl [M + H]⁺ 340.1324, found 340.1323.

4-chlorobutanoyl chloride (345 mg, 2.5 mmol) was added drop wisely to the solution of 11a (426 mg, 1.3 mmol) and TEA (254 mg, 2.5 mmol) in anhydrous THF (6 mL) at 0 °C. After being stirred for 0.5 h, reaction mixture was diluted with ethyl acetate, washed with brine. The organic phase was dried over Na₂SO₄, filtered and concentrated to give the crude product, which was dissolved in DME and then added to a solution of NaI (195 mg, 1.3 mmol), anhydrous Na₂CO₃ (207 mg, 1.95 mmol) and 1-(2-(tert -butyldimethylsilyloxy)ethyl)piperazine (1.4 mmol) or tert-butyl piperazine-1-carboxylate (254 mg, 1.37 mmol) in DME. After being stirred at 90 °C for 18 h, the mixture was cooled to rt, and diluted with ethyl acetate, washed with water and brine. Treatment of crude product with a solution of TB AF in THF or a mixture of TFA and CH₂Cl₂ followed by ethyl acetate work up afforded 16a or 18a in about 30% overall yield.

N-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-3-(4-(2-hydroxyethyl)piperazin-1-yl)propanamide (16a) [α]_D²⁰ = -41.6 (c 0.16, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 9.36 (d, J = 8.2 Hz, 1H), 8.79 (d, J = 4.5 Hz, 1H), 8.40-8.46 (m, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.30-7.44 (m, 5H), 6.29 (t, J = 4.8 Hz, 1H), 5.29-5.37 (m, 1H), 3.91-4.13 (m, 2H), 3.56 (t, J = 5.4 Hz, 2H), 2.60 (s, 3H), 2.48-2.55 (m, 2H), 2.14-2.46 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 172.9, 161.6, 159.5, 158.2,154.9, 149.8, 139.4, 136.8, 129.0, 128.1, 126.8, 124.5, 123.6, 112.7, 59.2, 57.8, 53.7, 53.5, 52.6,52.3, 47.4, 31.8, 22.3; ESI-MS m/z 524.3 (M + H) ⁺; HRMS (ESI) calcd for C₂₇H₃₅N₇O₂Cl (M + H) ⁺ 524.2535, found 524.2531.

N-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-4-(4-(2-hydroxyethyl)piperazin-1-yl)butanamide (16b) [α]_D²⁰ = -34.4 (c 0.25, CHCl₃); ¹H NMR (300 MHz, CDCl3) δ 8.83-8.79 (m, 1H), 8.44 (d, J = 7.8 Hz, 1H), 7.84 (dt, J = 1.5 Hz, 7.5 Hz, 1H), 7.76 (d, J = 6.6 Hz, 1H), 7.42-7.28 (m, 5H), 6.06 (t, J = 5.4 Hz, 1H), 5.32-5.23 (m, 1H), 4.15-4.03 (m, 1H), 3.95-3.86 (m, 1H), 3.57 (t, J = 5.4 Hz, 2H), 2.61 (s, 3H), 2.51-2.11 (m, 12H), 1.76-1.63 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 161.8, 1595, 158.4, 154.8, 149.9, 139.8, 136.8, 128.9, 127.9, 126.7, 124.6, 123.5, 59.1, 57.7, 57.1, 54.9, 52.9, 52.7, 46.9, 34.2, 29.7, 22.5, 22.3. ESI-MS m/z 538.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₈H₃₇ClN₇O₂ (M + H) ⁺ 538.2692, found 538.2700.

N-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-3-(piperazin-1-yl)propanamide (18a) [α]_D²⁰ = -44.6 (c 0.3, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 9.37 (d, J = 7.8 Hz, 1H), 8.77-8.81 (m, 1H), 8.43 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.39-7.42 (m, 4H), 7.31-7.38 (m, 2H), 6.24-6.32 (m, 1H), 5.28-5.38 (m, 2H), 4.03-4.14 (m, 1H), 3.89-4.01 (m, 1H), 2.64-2.79 (m, 4H), 2.64 (s, 3H), 2.45-2.52 (m, 2H), 2.23-2.41 (m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 172.6, 161.6, 159.5, 158.2, 154.8, 154.5, 149.8, 139.4, 136.8, 129.0, 128.1, 126.6, 124.5, 123.6, 112.6, 79.9, 53.9, 52.2, 47.2, 32.0, 28.4, 22.3; ESI-MS m/z 480.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₅H₃₁N₇OCl (M + H) ⁺ 480.2278, found 480.2278.

N-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18b) [α]_D²⁰ = -52.3 (c 1.8, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.82 (d, J = 4.5 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 7.89-7.76 (m, 2H), 7.43-7.28 (m, 6H), 6.1 (t, J = 6.0 Hz, 1H), 5.33-5.23 (m, 1H), 4.15-4.02 (m, 1H), 3.96-3.86 (m, 1H), 2.77 (t, J = 4.8 Hz, 4H), 2.60 (s, 3H), 2.33-2.11 (m, 10H), 1.76-1.64 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 161.6, 159.4, 158.3, 154.7, 149.7, 139.8, 136.8, 128.8, 127.8, 126.6, 124.6, 123.5, 112.6, 57.6, 54.9, 52.8, 51.2, 46.8, 45.6, 34.0, 22.2; ESI-MS m/z 494.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₃₃ClN₇O (M + H) ⁺ 494.2430, found 494.2440.

N-((R)-2-(5-chloro-2-(6-methoxypyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18c) [α]_D²⁰ = -65.9 (c 0.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.23 (d, J = 6 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.37-7.30 (m, 5H), 6.88 (d, J = 8.1 Hz, 1H), 6.05 (t, J = 6.3 Hz, 1H), 5.19 (m, 1H), 4.24 (m, 1H), 4.12 (s, 3H), 3.72 (m, 1H), 2.75 (m, 4H), 2.57 (s, 3H), 2.35 (m, 1H), 2.20 (m, 4H), 1.99 (m, 4H), 1.58 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 169.0, 159.8, 157.5,155.1, 154.4, 147.9, 135.9, 135.1, 124.6, 123.6, 122.3, 112.8, 108.4, 108.0, 53.6, 52.1, 49.6, 49.2, 42.3, 41.5, 30.1, 18.1, 18.0. ESI-MS m/z 524.3 (M + H) ⁺. HRMS (ESI) calcd for C₂₇H₃₅ClN₇O₂ (M + H) ⁺ 524.2535, found 524.2521.

N-((R)-2-(5-chloro-2-(6-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18d) [α]_D²⁰ = -68.1 (c 0.4, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.36 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 6.6 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.48-7.25 (m, 5H), 6.28-6.18 (m, 1H), 5.29-5.18 (m, 1H), 4.50-4.21 (m, 2H), 4.21-4.07 (m, 1H), 3.77-3.65 (m, 1H), 2.87-2.75 (m, 4H), 2.57 (s, 3H), 2.38-2.20 (m, 4H), 2.20-2.03 (m, 4H), 1.71-1.57 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 161.4, 158.4, 157.9, 155.2, 151.4, 140.1, 139.4, 128.6, 127.6, 126.5, 125.3, 121.8, 112.7, 57.7, 55.3, 53.8, 46.7, 45.6, 34.0, 222, 22.0; ESI-MS m/z 528.1 (M + H) ⁺. HRMS (ESI) calcd for C₂₆H₃₂Cl₂N₇O (M + H) ⁺ 528.2040, found 528.2036.

N-((R)-2-(5-chloro-2-(3-methoxypyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18e) ¹H NMR (300 MHz, CDCl₃) δ 8.34 (m, 1H), 7.78 (d, J = 6.9, 1H), 7.38 (m, 2H), 7.36-7.23 (m, 5H), 6.05 (m, 1H), 5.24 (m, 1H), 4.10 (m, 1H), 3.88 (s, 3H), 3.68 (m, 1H), 2.79 (m, 4H), 2.35-2.21 (m, 4H), 2.22-2.01 (m, 4H), 1.66 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 161.3, 160.2, 158.2, 154.0, 146.0, 141.1, 139.9, 128.6, 127.6, 126.5, 124.8, 119.4, 111.9, 57.7, 55.7, 55.1, 53.5, 46.2, 45.5, 33.8, 22.2, 22.0. ESI-MS m/z 524.3 (M + H) ⁺. HRMS (ESI) calcd for C₂₇H₃₅ClN₇O₂ (M + H) ⁺ 524.2535, found 524.2526.

N-((R)-2-(5-chloro-2-(3-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18f) [α]_D²⁰ = -49.0 (c 3.5, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.62 (d, J = 4.8 Hz, 1H), 7.88-7.82 (m, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.39-7.24 (m, 5H), 6.24 (t, J = 5.4 Hz, 1H), 5.29-5.19 (m, 1H), 4.10-3.97 (m, 1H), 3.85-3.73 (m, 1H), 2.84-2.72 (m, 4H), 2.57 (s, 3H), 2.37-2.09 (m, 8H), 1.78-1.64 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.8, 161.6, 160.1, 158.4, 154.0, 147.6, 139.8, 138.7, 130.7, 129.0, 128.1, 126.9, 124.9, 112.7, 57.8, 54.8, 53.9, 46.9, 45.7, 34.3, 22.4, 22.3. ESI-MS m/z 528.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₃2Cl₂N₇O (M + H) ⁺ 528.2040, found 528.2036.

N-((R)-2-(5-chloro-2-(5-methoxypyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18g) [α]_D²⁰ = -21.2 (c 0.4, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.48 (d, J = 2.1 Hz, 1H), 8.42 (d, J = 9 Hz, 1H), 7.83 (d, J = 6.6 Hz, 1H), 7.38-7.30 (m, 6H), 6.02 (t, J = 5.4 Hz, 1H), 5.30-5.23 (m, 1H), 4.10-4.02 (m, 1H), 3.93-3,86 (m, 4H), 2.78-2.75 (m, 4H), 2.58 (s, 3H), 2.36-2.13 (m, 8H), 1.73-1.65 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 161.7, 159.3, 158.3, 156.7, 147.4, 139.9, 137.8, 128.9, 127.9, 126.7, 124.4, 120.5, 112.0, 57.7, 55.8, 55.0, 54.0, 46.8, 45.8, 34.2, 22.3; ESI-MS m/z 524.3(M + H)⁺. HRMS (ESI) calcd for C₂₇H₃₅ClN₇O₂ (M + H)⁺ 524.2535, found 524.2546.

N-((R)-2-(5-chloro-2-(5-fluoropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18h) [α]_D²⁰ = -39.1 (c 0.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.64 (d, J = 2.4 Hz, 1H), 8.49 (dd, J = 4.8, 8.7 Hz, 1H), 7.70 (d, J = 6.9 Hz, 1H), 7.54 (dt, J = 2.7 Hz, 8.1 Hz, 1H), 7.41-7.28 (m, 5H), 6.18 (t, J = 5.4 Hz, 1H), 5.28 (m, 1H), 4.05 (m, 1H), 3.88 (m, 1H), 2.76 (t, J = 4.5 Hz, 4H), 2.58 (s, 3H), 2.27-2.12 (m, 8H), 1.72 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 161.6, 160.2 (d, J = 260 Hz), 158.5, 158.3, 151.0, 139.6, 138.0 (d, J = 24 Hz), 128.9, 127.9, 126.6, 125.0 (d, J = 5.1 Hz), 123.4 (d, J = 18 Hz), 112.6, 57.6, 54.6, 54.1, 46.9, 45.8, 34.1, 22.2. ESI-MS m/z 512.3 (M + H) ⁺. HRMS(ESI) calcd for C₂₆H₃₂ClFN₇O (M + H) ⁺ 512.2335, found 512.2344.

N-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18i) [α]_D²⁰ = -45.2 (c 1.9, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.73 (m, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.85-7.72 (m, 2H), 7.41-7.29 (m, 5H), 6.20 (m, 1H), 5.28 (m, 1H), 4.06 (m, 1H), 3.86 (m, 1H), 2.76 (m, 4H), 2.58 (s, 3H), 2.31-2.09 (m, 8H), 1.71 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 161.5, 158.5, 158.1, 152.8, 148.5, 139.5, 136.4, 133.2, 128.8, 127.9, 126.5, 124.3, 112.7, 57.5, 54.5, 53.9, 46.8, 45.7, 34.0, 22.1. ESI-MS m/z 528.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₃₂Cl₂N₇O (M + H) ⁺ 528.2040, found 528.2039.

N-((R)-2-(2-(5-bromopyridin-2-yl)-5-chloro-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18j) [α]_D²⁰ = -47.8 (c 2.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.83 (m, d, J = 2.1 Hz, 1H), 8.38-8.31 (m, 1H), 8.00-7.92 (m, 1H), 7.83 (d, J = 6.9 Hz, 1H), 7.41-7.25 (m, 5H), 6.30-6.20 (m, 1H), 5.31-5.21 (m, 1H), 4.12-3.96 (m, 1H), 3.92-3.79 (m, 1H), 2.80 (m, 4H), 2.57 (s, 3H), 2.43-2.09 (m, 8H), 1.81-1.62 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 161.6, 158.7, 158.3, 153.3, 150.7, 139.6, 139.4, 128.9, 127.9, 126.6, 124.8, 122.3, 112.8, 57.6, 54.7, 54.0, 46.9, 45.7, 34.1, 22.21, 22.16. ESI-MS m/z 572.0 (M + H) ⁺. HRMS (ESI) calcd for C₂₆H₃₂BrClN₇O (M + H) ⁺ 572.1535, found 572.1541.

N-((R)-2-(5-chloro-6-methyl-2-(5-nitropyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18k) [α]_D²⁰ = -25.8 (c 1.3, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 9.58 (m, 1H), 8.68 (m, 1H), 8.60 (dd, J = 2.7 Hz, 8.7 Hz, 1H), 7.43-7.30 (m, 6H), 6.42 (t, J = 4.2 Hz, 1H), 5.31 (m, 1H), 4.08 (m, 1H), 3.90 (m, 1H), 2.75 (m, 4H), 2.62 (s, 3H), 2.35-2.17 (m, 8H), 1.77 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.7, 161.8, 159.7, 158.3, 157.6, 145.0, 144.4, 139.3, 131.9, 129.1, 128.2, 126.7, 123.9, 113.8, 57.4, 54.2, 54.0, 47.2, 45.8, 34.1, 22.2, 22.1. ESI-MS m/z 539.3 (M + H) ⁺. HRMS (ESI) calcd for C₂₆H₃2ClN₈O₂ (M + H) ⁺ 539.2280, found 539.2271.

N-((R)-2-(5-chloro-2-(5-(dimethylamino)pyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18l) [α]_D²⁰ = -52.0 (c 0.3, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.35-8.26 (m, 2H), 8.07 (m, 1H), 7.41-7.33 (m, 4H), 7.30 (m, 1H), 7.04 (dd, J = 2.7 Hz, 9.0 Hz, 1H), 5.88 (t, J = 5.7 Hz, 1H), 5.23 (m, 1H), 4.10 (m, 1H), 3.86 (m, 1H), 3.09 (s, 6H), 2.79 (t, J = 4.8 Hz, 4H), 2.27 (m, 4H), 2.13 (m, 4H), 1.66 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 161.8, 160.0, 158.4, 146.8, 142.6, 140.3, 134.6,129.0, 127.9, 126.8, 124.2, 118.2, 111.3, 57.9, 55.7, 53.5, 46.8, 45.6, 40.2, 34.4, 22.5. ESI-MS m/z537.3 (M + H) ⁺.

N-((R)-2-(5-chloro-6-methyl-2-(5-(pyrrolidin-1-yl)pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18m) [α]_D²⁰ = -66.3 (c 1.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, J = 8.7 Hz, 1H), 8.16-8.05 (m, 2H), 7.40-7.29 (m, 5H), 6.89 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 5.86 (m, 1H), 5.22 (m, 1H), 4.09 (m, 1H), 3.86 (m, 1H), 3.40 (m, 4H), 2.76 (m, 4H), 2.56 (s, 3H), 2.23 (m, 4H), 2.16-2.00 (m, 8H), 1.65 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 161.4, 159.8, 158.2, 144.2, 141.5, 140.1, 134.0, 128.7,127.6, 126.6, 124.2, 117.6, 110.8, 57.8, 55.4, 54.0, 47.4, 46.5, 45.7, 34.2, 25.4, 22.3, 22.2. ESI-MS m/z 563.3 (M + H) ⁺. HRMS (ESI) calcd for C₃₀H₄₀ClN_8O (M + H) ⁺ 563.3008, found 563.3033.

N-((R)-2-(5-chloro-2-(4-methoxypyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18n) [α]_D²⁰ = -48.5 (c 0.9, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.62 (d, J = 6.0 Hz, 1H), 8.00 (d, J = 2.1 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.41-7.28 (m, 5H), 6.91 (dd, J = 2.4 Hz, 6.0 Hz, 1H), 6.03 (t, J = 4.8 Hz, 1H), 5.31-5.22 (m, 1H), 4.18-4.06 (m, 1H), 3.99-3.84 (m, 4H), 2.82-2.74 (m, 4H), 2.59 (s, 3H), 2.32-2.12 (m, 8H), 1.74-1.63 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 166.4, 161.4, 159.2, 158.2, 156.3, 150.8, 139.7, 128.7, 127.6, 126.5, 112.5, 110.7, 109.4, 57.4, 55.2, 54.8, 52.8, 51.1, 46.5, 45.1, 33.9, 22.1. ESI-MS m/z 524.3 (M + H) ⁺. HRMS (ESI) calcd for C₂₇H₃₅ClN₇O₂ (M + H) ⁺ 524.2535, found 524.2552.

N-((R)-2-(5-chloro-2-(4-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide (18o) [α]_D²⁰ = -36.0 (c 2.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.70 (d, J = 5.4 Hz, 1H), 8.47-8.43 (m, 1H), 7.65 (d, J = 6.9 Hz, 1H), 7.42-7.28 (m, 5H), 6.24-6.15 (m, 1H), 5.33-5.23 (m, 1H), 4.14-4.01 (m, 1H), 3.96-3.85 (m, 1H), 2.82-2.72 (m, 4H), 2.59 (s, 3H), 2.38-2.13 (m, 8H), 1.79-1.66 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 161.6, 158.3, 156.3, 150.5, 145.0, 139.7, 128.9, 127.9, 126.6, 124.8 123.9, 113.0, 57.5, 54.6, 53.5, 46.9, 45.4, 34.0, 22.2. ESI-MS m/z 528.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₃₂Cl₂N₇O (M + H) ⁺ 528.2040, found 528.2027.

5.1.5. Typical procedure for synthesizing compounds 20

5-chloro-6-methyl-N-((R)-2-phenyl-2-(3-(piperazin-1-yl)propylamino)ethyl)-2-(pyridin-2-yl)pyrimidin-4-amine (20a)

To a solution of 11a (115 mg, 0.34 mmol) and bromide 19a (156 mg, 0.51 mmol) in dry DMF (1 mL) were added NaI (152 mg, 1.02 mmol) and anhydrous K₂CO₃ (140 mg, 1.02 mmol). After being stirred overnight at 100 °C for 24 h, the reaction mixture was cooled to rt, and diluted with ethyl acetate, and washed with water and brine. The organic phase was dried (Na₂SO₄), filtered and concentrated. The crude product was treated at with TFA (0.3 mL) and CH₂Cl₂ (1 mL) for 1 h at rt. After diluting with ethyl acetate, the solution was washed with aq. NaHCO₃ and brine, and dried over Na₂SO₄. Evaporation and column chromatography on silica gel (eluent: 5–10% EtOH—DCM or similar conditions) afforded 20 as the foamy solid (60 mg, 38% yield). [α]_D²⁰ = -23.1 (c 0.15, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.79 (d, J = 4.8 Hz, 1H), (8.42-8.36, m, 1H), 7.79 (dt, J = 1.8, 7.5 Hz, 1H), 7.42-7.29 (m, 6H), 6.18-6.10 (m, 1H), 3.97-3.86 (m, 2H), 3.72-3.60 (m, 1H), 2.91-2.82 (m, 4H), 2.68-2.52 (m, 9H), 2.48-2.31 (m, 4H), 1.74-1.60 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 161.4, 159.6, 157.8, 155.0, 149.8, 141.6, 136.6, 128.7, 127.7, 127.0, 124.3, 123.5, 112.7, 62.1, 57.5, 54.5, 47.0, 46.1, 46.0, 26.7, 22.3; ESI-MS m/z 466.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₅H₃₃ClN₇ (M + H) ⁺ 466.2481, found 466.2496.

2-(4-(3-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethylamino)propyl)piperazin-1-yl)ethanol (20b)

Following a similar procedure from 19a to 20a, 20b was obtained from 19b. [α]_D²⁰ = -13.9 (c 0.65, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.80 (d, J = 4.8 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 7.83-7.75 (m, 1H), 7.43-7.28 (m, 5H), 6.15-6.08 (m, 1H), 3.98-3.87 (m, 2H), 3.70-3.56 (m, 3H), 2.68-2.57 (m, 6H), 2.56-2.35 (m, 9H), 1.76-1.61 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 161.4, 159.7, 157.8, 155.1, 149.9, 141.7, 136.6, 128.7, 127.7, 127.0, 124.3, 123.5, 112.7, 62.1, 59.2, 57.7, 56.9, 53.4, 52.9, 47.0, 46.2, 27.1, 22.3. ESI-MS m/z 510.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₇H₃₇ClN₇O (M + H) ⁺ 510.2743, found 510.2751.

5.1.6. Typical procedure for synthesizing compounds 22

5-chloro-6-methyl-N-((R)-2-phenyl-2-(4-(piperazin-1-yl)butylamino)ethyl)-2-(pyridin-2-yl)pyrimidin-4-amine (22a)

To a solution of 11a (85 mg, 0.25 mmol) and aldehyde 21a (79 mg, 0.25 mmol) in MeOH (0.7 mL) was added acetic acid (72 μL, 1.25 mmol) under argon. The reaction mixture was stirred for 10 min at rt, and then NaBH₃CN (47 mg, 0.75 mmol) was added at 0 °C. After being stirred for 1 h, the reaction mixture was diluted with CH₂Cl₂, and washed with water. The organic phase was dried over Na₂SO₄ and concentrated. The crude product was treated at with TFA (0.3 mL) and CH₂Cl₂ (1 mL) for 1 h at rt. After diluting with ethyl acetate, the solution was washed with aq. NaHCO₃ and brine, and dried over Na₂SO₄. Evaporation and column chromatography on silica gel (eluent: 5–10% EtOH—DCM or similar conditions) afforded 22a (58 mg, 49% yield). [α]_D²⁰ = -11.6 (c 1.95, CHCl₃); 1H NMR (300 MHz, CDCl₃) δ 8.80 (d, J = 4.2 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H), 7.79 (dt, J = 1.8, 7.5 Hz, 1H), 7.42-7.27 (m, 6H), 6.14-6.06 (m, 1H), 3.97-3.85 (m, 2H), 3.72-3.61 (m, 1H), 2.88 (t, J = 4.8 Hz, 4H), 2.61 (s, 3H), 2.60-2.50 (m, 2H), 2.46-2.24 (m, 6H), 1.60-1.41 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 161.2, 159.5, 157.7, 154.9, 149.7, 141.5, 136.5, 128.6, 127.6, 126.9, 124.3, 123.4, 112.6, 62.0, 58.8, 54.0, 47.1, 46.9, 45.6, 28.0, 24.2, 22.1. ESI-MS m/z 480.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₃₄ClN₇ (M + H) ⁺ 480.2637, found 480.2630.

2-(4-(4-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethylamino)butyl)piperazin-1-yl)ethanol (22b)

Following a similar procedure from 21a to 22a, 22b was obtained from 21b. [α]_D²⁰ = -28.1 (c 0.15, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.80 (d, J = 4.8 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 7.83-7.75 (m, 1H), 7.42-7.28 (m, 6H), 6.10-6.02 (m, 1H), 3.96-3.84 (m, 2H), 3.73-3.55 (m, 4H), 2.61 (s, 3H), 2.59-2.37 (m, 10H), 2.36-2.22 (m, 4H), 1.58-1.40 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 161.4, 159.6, 157.8, 149.8, 141.6, 136.6, 128.7, 127.7, 127.0, 124.3, 123.5, 112.7 62.0, 59.1, 58.4, 57.6, 53.2, 52.8, 47.2, 46.9, 28.2, 24.6. ESI-MS m/z 524.2 (M + H) ⁺; HRMS (ESI) calcd for C₂₈H₃9ClN₇O (M + H) ⁺ 524.2899, found 524.2908.

5.1.7. Typical procedure for synthesizing compounds 24

N-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-2-(piperazin-1-yl)ethanesulfonamide (24a)

To a cooled solution (0 °C) of 11a (594 mg, 1.75 mmol) and Et₃N (0.53 mL, 3.85 mmol) in CH₂Cl₂ (3 mL) was added 2-chloroethanesulfonyl chloride (627 mg, 3.85 mmol) with stirring. After addition, the mixture was stirred at rt for 5 h, and then reaction was quenched with ice water. The reaction mixture was extracted with CH₂Cl₂, and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was dissolved in EtOH before tert-butyl piperazine-1-carboxylate (171 mg, 0.4 mmol) was added. The resultant solution was heated at 50 °C for 16 h, and then concentrated in vacuo. The residue was treated at with TFA (0.3 mL) and CH₂Cl₂ (1 mL) for 1 h at rt. After diluting with ethyl acetate, the solution was washed with aq. NaHCO₃ and brine, and dried over Na₂SO₄. Evaporation and column chromatography on silica gel (eluent: 5–10% EtOH—DCM or similar conditions) afforded 24a. [α]_D²⁰ = -38.9 (c 4.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.86-8.80 (m, 1H), 8.47-8.39 (m, 1H), 7.83 (t, J = 4.2 Hz, 1H), 7.42-7.27 (m, 5H), 5.79 (t, J = 5.7 Hz), 4.98-4.58 (m, 3H), 4.12-3.87 (m, 2H), 3.02-2.85 (m, 2H), 2.84-2.71 (m, 2H), 2.71-2.61 (m, 2H), 2.57 (s, 3H), 2.32-2.18 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 161.7, 159.4, 158.0, 154.4, 149.8, 139.4, 136.9, 128.9, 128.2, 126.9, 124.8, 123.5, 112.6, 58.0, 52.6, 52.4, 50.4, 47.6, 45.0, 22.2; ESI-MS m/z 516.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₄H₃₁N₇O₂SCl₂ (M + H) ⁺ 516.1943, found 516.1955.

N-((R)-2-(5-chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-2-(4-(2-hydroxyethyl)piperazin-1-yl)ethane sulfonamide (24b)

Following a similar procedure from 11a to 24a, 24b was obtained from 11a. [α]_D²⁰ = -46.5 (c 0.8, CHCl₃);¹H NMR (300 MHz, CDCl₃) δ 8.85 (d, J = 4.8 Hz, 1H), 8.45 (d, J = 7.8 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H) 7.42-7.28 (m, 6H), 5.79 (t, J = 5.7 Hz, 1H), 4.97-4.89 (m, 1H), 4.13-4.01 (m, 1H), 4.00-3.90 (m, 1H), 3.55 (t, J = 5.4 Hz, 2H), 3.03-2.91 (m, 2H), 2.74-2.65 (m, 2H), 2.58 (s, 3H), 2.47-2.15 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 161.8, 159.4, 158.1, 154.4, 149.9, 139.3, 136.9, 128.9, 128.2, 126.9, 124.8, 123.5, 112.6, 59.1, 58.0, 57.7, 52.7, 52.4, 52.1, 50.4, 47.7, 22.2. ESI-MS m/z 560.3 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₃₅N₇O₃SCl (M + H) ⁺ 560.2205, found 560.2199.

5.1.8. General procedure for preparing compounds 25

To a solution of acid (0.232 mmol), 11b (0.193 mmol), HOBt (0.232 mmol) and EDCI (0.232 mmol) in CH₂Cl₂ (1 mL) was added DIPEA (0.483 mmol). The resultant solution was stirred for 20 h at rt before it was concentrated. The residue was dissolved in ethyl acetate, and washed with saturated NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, concentrated and then purified by flash chromatography (eluent: 5–10% EtOH—DCM or similar conditions) to give the corresponding amide.

N-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-2-phenylacetamide (25a) [α]_D²⁰ = -4.2 (c 0.4, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J = 2.4 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.79 (dd, J = 2.1, 8.4 Hz, 1H), 7.40-7.29 (m, 3H), 7.29-7.22 (m, 2H), 7.18-7.10 (m, 3H), 7.08-6.97 (m, 3H), 5.89 (m, 1H), 5.27 (m, 1H), 4.02 (m, 1H), 3.85 (m, 1H), 3.50 (s, 2H), 2.61 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.3, 161.8, 158.6, 158.2, 152.9, 148.7, 139.4, 136.5, 134.7, 133.3, 128.91, 128.86, 128.7, 127.9, 127.0, 126.4, 124.3, 112.7, 54.9, 46.4, 43.6, 22.3. ESI-MS m/z 492.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₂₄Cl₂N₅O (M + H) ⁺ 492.1352, found 492.1344.

N-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-3-phenylpropanamide (25b) white solid (70% yield): [α]_D²⁰ = -3.1 (c 1.5, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.64 (m, 1H), 8.40 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.62 (brs, 1H), 7.30 (m, 3H), 7.18 (m, 5H), 6.98 (m, 2H), 6.00 (brs, 1H), 5.18 (m, 1H), 3.91 (m, 1H), 3.79 (m, 1H), 2.82 (t, J = 7.8 Hz, 2H), 2.59 (s, 3H), 2.42 (t, J = 7.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 161.7, 158.5, 158.3, 152.8, 148.6, 140.7, 139.6, 136.5, 133.4, 128.8, 128.2, 127.7, 126.5, 126.0, 124.3, 112.8, 55.3, 46.8, 38.0, 31.6, 22.2. ESIMS m/z 506.0 (M + H)⁺; HR-ESIMS (m/z): Calcd. For C₂₇H₂₆Cl₂N₅O (M + H)⁺ 506.1509, found 506.1504.

N-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-phenylbutanamide (25c) white solid (76% yield): [α]_D²⁰ = -57.1 (c 0.9, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 8.69 (d, J = 2.7 Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.80 (m, 1H), 7.45-7.28 (m, 6H), 7.25-7.11 (m, 3H), 7.03 (m, 2H), 6.06 (brs, 1H), 5.26 (m, 1H), 4.04 (m, 1H), 3.84 (m, 1H), 2.57 (s, 3H), 2.46 (m, 2H), 2.12 (m, 2H), 1.83 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 173.3, 161.7, 158.4, 152.8, 148.6, 141.4, 139.7, 136.5, 133.4, 128.9, 127.9, 126.6, 125.9, 124.3, 112.8, 55.1, 46.8, 35.7, 35.1, 27.1, 22.2.

N-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-5-phenylpentanamide (25d) white solid (80% yield): ¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J = 2.4 Hz, 1H), 8.42 (d, J = 7.5 Hz, 1H), 7.80 (m, 1H), 7.44-7.28 (m, 6H), 7.24 (m, 2H), 7.16 (m, 1H), 7.08 (m, 2H), 6.05 (brs, 1H), 5.25 (m, 1H), 4.02 (m, 1H), 3.84 (m, 1H), 2.58 (s, 3H), 2.48 (m, 2H), 2.12 (m, 2H), 1.62-1.39 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 173.4, 161.6, 158.3, 152.7, 148.5, 142.0, 139.6, 136.5, 133.3, 128.8, 128.18, 128.16, 127.8, 126.4, 125.9, 124.3, 112.7, 55.0, 46.7, 36.2, 35.5, 30.9, 25.3, 22.1. ESIMS m/z 556.1 (M + H)⁺; HR-ESIMS (m/z): Calcd. For C₂₉H₃0Cl₂N₅O (M + H)⁺ 534.1822, found 534.1819.

N-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-ylamino)-1-phenylethyl)-6-phenylhexanamide (25e) white solid (75% yield): ¹H NMR (300 MHz, CDCl₃) δ 8.72 (m, 1H), 8.41 (d, J = 7.8 Hz, 1H), 7.79 (m, 1H), 7.43-7.30 (m, 5H), 7.30-7.21 (m, 3H), 7.20-7.08 (m, 3H), 6.06 (brs, 1H), 5.26 (m, 1H), 4.04 (m, 1H), 3.84 (m, 1H), 2.59 (s, 3H), 2.50 (t, J = 7.5 Hz, 2H), 2.08 (m, 2H), 1.49 (m, 4H), 1.17 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 173.7, 161.7, 158.4, 152.8, 148.6, 142.4, 139.7, 136.6, 133.4, 128.9, 128.3, 128.2, 127.9, 126.5, 125.6, 124.4, 112.8, 55.1, 46.8, 36.5, 35.7, 31.1, 28.8, 25.6. 22.2.

5.1.9. General procedure for preparing compounds 26 and 27

To a solution of 1 1f (0.401 mmol) and aldehyde (0.401 mmol) in MeOH (3 mL) was added acetic acid (0.441 mmol) under argon. The reaction mixture was stirred for 10 min at rt, and then NaBH₃CN (26 mg, 0.401 mmol) was added at 0 °C. After being stirred for 1 h, the reaction mixture was diluted with CH₂Cl₂, and washed with saturated NaHCO₃. The organic phase was dried over Na₂SO₄ and concentrated. Purification by flash chromatography (eluent: 5–10% EtOH—DCM or similar conditions) gave the corresponding amine.

N-((R)-2-(benzylamino)-2-phenylethyl)-5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-amine (26a) [α]_D²⁰ = -15.0 (c 2.5, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 8.70 (d, J = 2.4 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 7.73 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.23-7.44 (m, 11H), 6.13 (t, J = 4.8 Hz, 1H), 3.86-3.97 (m, 2H), 3.80 (d, J = 13.2 Hz, 1H), 3.60-3.69 (m, 2H), 2.61 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 161.4, 157.7, 153.1, 148.6, 141.2, 140.0, 136.3, 133.0, 128.9, 128.5, 128.2, 127.9, 127.1, 127.1, 124.3, 61.0, 51.1, 47.0, 22.3. ESI-MS m/z 464.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₅H₂₄N₅Cl₂ (M + H) ⁺ 464.1403, found 464.1393.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-(phenethylamino)-2-phenylethyl)pyrimidin-4-amine (26b) foamy solid, [α]_D²⁰ = -16.5 (c 1.7, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.4 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.70 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.13-7.39 (m, 11H), 6.00 (t, J = 5.1 Hz, 1H), 3.81-3.96 (m, 2H), 3.58-3.65 (m, 1H), 2.76-3.87 (m, 4H), 2.60 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 161.4, 158.7, 157.7, 153.1, 148.6, 141.3, 139.8, 136.4, 133.0, 128.8, 128.6, 128.4, 127.8, 127.0, 126.2, 124.4, 112.8, 61.9, 48.3, 47.0, 36.3, 22.3. ESI-MS m/z 478.0 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₂₆N₅Cl₂ (M + H) ⁺ 478.1560, found 478.1544.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-phenyl-2-(3-phenylpropylamino)ethyl)pyrimidin-4-amine (26c) foamy solid, [α]_D²⁰ = -13.8 (c 2.4, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.4 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.08-7.41 (m, 11H), 6.16 (t, J = 5.1 Hz, 1H), 3.85-3.94 (m, 2H), 3.58-3.68 (m, 1H), 2.49-2.70 (m, 7H), 1.75-1.85 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.4, 158.7, 157.7, 153.1, 148.6, 142.0, 141.5, 136.3, 133.0, 128.7, 128.3, 128.2, 127.8, 126.9, 125.8, 124.4, 112.8, 61.9, 46.9, 46.6, 33.4, 31.8, 22.3. ESI-MS m/z 492.0 (M + H) ⁺; HRMS (ESI) calcd for C₂₇H₂₈N₅Cl₂ (M + H) ⁺ 492.1716, found 492.1708.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-phenyl-2-(4-phenylbutylamino)ethyl)pyrimidin-4-amine (26d) foamy solid, [α]_D²⁰ = -20.8 (c 0.3, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.1 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.1 Hz, 8.4 Hz, 1H), 7.12-7.41 (m, 11H), 6.15 (d, J = 5.1 Hz, 1H), 3.85-3.96 (m, 2H), 3.67-3.72 (m, 1H), 2.50-2.66 (m, 7H), 1.60-1.70 (m, 2H), 1.48-1.58 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.4, 158.7, 157.7, 153.1, 148.6, 142.3, 136.4, 133.1, 128.8, 128.4, 128.3, 127.8, 127.0, 125.7, 124.4, 112.9, 62.1, 47.1, 46.9, 35.7, 29.6, 29.0, 22.3. ESI-MS m/z 506.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₈H₃₀N₅Cl₂ (M + H) ⁺ 506.1873, found 506.1879.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-phenyl-2-(5-phenylpentylamino)ethyl)pyrimidin-4-amine (26e) foamy solid, [α]_D²⁰ = -9.9 (c 1.2, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 8.73 (d, J = 1.5 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 1.5 Hz, 8.4 Hz, 1H), 7.13-7.41 (m, 10H), 6.16 (s, 1H), 3.85-3.95 (m, 2H), 3.62-3.70 (m, 1H), 2.49-2.60 (m, 7H), 1.47-1.64 (m, 4H), 1.33-1.40 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 158.8, 157.8, 153.3, 148.8, 142.6, 136.5, 133.2, 128.9, 128.5, 128.4, 127.9, 127.1, 125.8, 124.5, 113.0, 62.1, 47.3, 47.0, 36.0, 31.4, 30.0, 27.0, 22.4. ESI-MS m/z 520.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₉H₃₂N₅Cl₂ (M + H) ⁺ 520.2029, found 520.2034.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-phenyl-2-(6-phenylhexylamino)ethyl)pyrimidin-4-amine (26f) foamy solid, [α]_D²⁰ = -12.3 (c 1.4, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 8.73 (d, J = 2.4 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.14-7.43 (m, 10H), 6.15 (t, J = 4.8 Hz, 1H), 3.84-3.94 (m, 2H), 3.61-3.70 (m, 1H), 2.46-2.60 (m, 7H), 1.53-1.63 (m, 2H), 1.42-1.47 (m, 2H), 1.25-1.33 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 158.8, 157.8, 153.3, 148.8, 142.8, 136.5, 133.2, 128.9, 128.5, 128.4, 127.9, 127.1, 125.7, 124.5, 62.1, 47.4, 47.0, 36.0, 31.6, 30.1, 29.3, 27.2, 22.4. ESI-MS m/z 556.1 (M + H) ⁺; HRMS (ESI) calcd for C₃₀H₃₄N₅Cl₂ (M + H) ⁺ 534.2186, found 534.2175.

4-(4-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethylamino)butyl)phenol (27a) ¹H NMR (300 MHz, CDCl₃): δ 8.68 (d, J = 1.2 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.75 (m, 1H), 7.26-7.40 (m, 5H), 6.94 (d, J = 7.6 Hz, 2H), 6.71 (d, J = 8.1 Hz, 2H), 6.02 (t, J = 5.1 Hz, 1H), 3.85-3.92 (m, 1H), 3.71-3.83 (m, 2H), 2.93 (s, 3H), 2.46-2.55 (m, 7H), 1.56-1.60 (m, 2H), 1.48-1.53 (m, 2H). ESI-MS m/z 522.3 (M + H) ⁺; HRMS (ESI) calcd for C₂₈H₃₀N₅Cl₂O (M + H) ⁺ 522.1835, found 522.18219.

5-chloro-N-((R)-2-(4-(4-chlorophenyl)butylamino)-2-phenylethyl)-2-(5-chloropyridin-2-yl)pyrimidin-4-amine (27b) foamy solid (53% yield): ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.73 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.28-7.40 (m, 5H), 7.22 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 6.09 (t, J = 5.4 Hz, 1H), 3.83-3.92 (m, 2H), 3.62-3.70 (m, 1H), 2.59 (s, 3H), 2.47-2.56 (m, 4H), 1.57-1.67 (m, 2H), 1.43-1.53 (m, 2H). ¹³ (100 MHz, CDCl₃): δ 161.5, 158.8, 157.9, 153.2, 148.7, 141.6, 140.8, 136.5, 133.2, 131.5, 129.8, 129.7, 128.9, 128.4, 127.9, 127.1, 124.5, 112.9, 62.2, 47.2, 47.0, 35.1, 29.7, 29.0, 22.3. ESI-MS m/z 540.3 (M + H) ⁺; HRMS (ESI) calcd for C₂₈H₂₉N₅Cl₃ (M + H) ⁺ 540.1500, found 540.1483.

4-(4-((R)-2-(5-chloro-2-(5-chloropyridin-2-yl)pyrimidin-4-ylamino)-1-phenylethylamino)butyl)benzonitrile (27c) ¹H NMR (300 MHz, CDCl₃): δ 8.71 (d, J = 1.2 Hz, 1H), 8.36 (d, J = 8.7 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.29-7.42 (m, 5H), 7.21 (d, J = 7.8 Hz, 2H), 6.21 (s, 1H), 3.87-3.98 (m, 2H), 3.69-3.76 (m, 1H), 2.51-2.65 (m, 7H), 1.61-1.71 (m, 2H), 1.51-1.57 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.1, 157.8, 157.7, 152.3, 148.3, 147.7, 139.3, 133.6, 132.1, 129.1,129.0, 128.3, 127.1, 124.5, 118.9, 109.6, 62.1, 35.6, 28.7, 28.3, 22.1. ESI-MS m/z 531.4 (M + H) ⁺;HRMS (ESI) calcd for C₂₉H₂₉N₆Cl₂ (M + H) ⁺ 531.1844, found 531.18253.

5-chloro-2-(5-chloropyridin-2-yl)-N-((R)-2-phenyl-2-(4-(4-propoxyphenyl)butylamino)ethyl)pyrimidin-4-amine (27d) ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.26-7.40 (m, 5H), 7.03 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 6.10 (t, J = 5.4 Hz, 1H), 3.82-3.93 (m, 4H), 3.62-3.70 (m, 1H), 2.59 (s, 3H), 2.49-2.56 (m, 4H), 1.75-1.85 (m, 2H), 1.57-1.66 (m, 2H), 1.47-1.54 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 158.8, 157.8, 157.3, 153.2, 148.7, 141.7, 136.4, 134.3, 133.1, 129.3, 128.8, 127.8, 127.1, 124.5, 114.4, 112.9, 69.6, 62.1, 47.3, 47.0, 34.9, 29.8, 29.4, 22.7, 22.3, 10.6. ESI-MS m/z 564.5 (M + H) ⁺; HRMS (ESI) calcd for C₃₁H₃₆N_5OCl₂ (M + H) ⁺ 564.2307, found 564.2291.

5-chloro-2-(5-chloropyridin-2-yl)-N-((R)-2-(4-(furan-2-yl)butylamino)-2-phenylethyl)-6-methylpyrimidin-4-amine (27e) [α]_D²⁰ = -9.0 (c 1.50, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.1 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.1 Hz, 8.4 Hz, 1H), 7.27-7.41 (m, 5H), 6.26 (t, J = 2.1 Hz, 1H), 6.12 (m, 1H), 6.73 (d, J = 5.1 Hz, 1H), 5.94 (d, J = 3.3 Hz, 1H), 3.85-3.96 (m, 2H), 3.66-3.75 (m, 1H), 2.50-2.64 (m, 7H), 2.13 (s, 1H), 1.63-1.73 (m, 2H), 1.48-1.58 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 158.5, 157.9, 156.1, 153.2, 148.7, 141.2, 140.9, 136.5, 133.2, 128.9, 128.0, 127.2, 124.5, 113.0, 110.2,104.9, 62.2, 47.0, 46.9, 29.5, 27.8, 25.8, 22.4. ESI-MS m/z 496.4 (M + H) ⁺; HRMS (ESI) calcd forC₂₆H₂₈N_5OCl₂ (M + H) ⁺ 496.1666, found 496.1665.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-phenyl-2-(4-(thiophen-2-yl)butylamino)ethyl)pyrimidin-4-amine (27f) [α]_D²⁰ = -10.9 (c 1.35, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.1 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.1 Hz, 8.4 Hz, 1H), 7.26-7.42 (m, 5H), 7.09 (dd, J = 0.9 Hz, 5.1 Hz, 1H), 6.87-6.90 (m, 1H), 6.73 (d, J = 3 Hz), 6.15 (t, J = 4.8 Hz, 1H), 3.86-3.98 (m, 2H), 3.69-3.77 (m, 1H), 2.79 (t, J = 7.5 Hz, 2H), 2.53-2.67 (m, 5H), 2.30 (s, 1H), 1.66-1.76 (m, 2H), 1.52-1.62 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.6, 158.8, 157.9, 153.2, 148.7, 145.2, 136.5, 133.2, 129.0, 128.9, 128.1, 127.2, 126.8, 124.5, 124.2, 123.0, 113.1, 62.2, 47.0, 46.9, 29.8, 29.5, 29.3, 22.4. ESI-MS m/z 512.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₂₈N₅SCl₂ (M + H) ⁺ 512.1447, found 512.1437.

5-chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-phenyl-2-(4-(quinolin-2-yl)butylamino)ethyl)pyrimidin-4-amine (27g) foamy solid (32% yield): [α]_D²⁰ = -14.4 (c 1.50, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.1 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.04 (t, J = 8.7 Hz, 2H), 7.65-7.78 (m, 3H), 7.48 (t, J = 7.8 Hz, 1H), 7.23-7.37 (m, 6H), 6.13 (t, J = 5.1 Hz, 1H), 3.83-3.95 (m, 2H), 3.64-3.72 (m, 1H), 2.96 (t, J = 7.5 Hz, 2H), 2.60-2.67 (m, 2H), 2.57 (s, 3H), 2.14 (s, 1H), 1.82-1.92 (m, 2H), 1.56-1.66 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 162.6, 161.5, 158.8, 157.9, 153.3, 148.7, 148.0, 141.4, 136.5, 136.4, 133.2, 129.5, 128.9, 128.8, 127.9, 127.6, 127.1, 126.8, 125.9, 124.5, 121.4, 113.0, 62.1, 47.2, 47.0, 39.0, 29.9, 27.6, 22.3. ESI-MS m/z 557.3 (M + H) ⁺; HRMS (ESI) calcd for C₃₁H₃₁N₆Cl₂ (M + H) ⁺ 557.1992, found 557.1982.

N-((R)-2-(2-(1H-pyrrol-1-yl)ethylamino)-2-phenylethyl)-5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-amine (27h) [α]_D²⁰ = -13.7 (c 1.45, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 8.72 (d, J = 2.4 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.31-7.41 (m, 5H), 6.60 (m, 2H), 6.12 (m, 2H), 6.08 (t, J = 5.4 Hz, 1H), 3.82-3.93 (m, 4H), 3.65-3.73 (m, 1H), 2.59 (s, 3H), 2.45-2.57 (m, 2H), 2.10 (s, 1H), 1.74-1.84 (m, 2H), 1.41-1.51 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 160.6, 157.8, 156.9, 152.2, 147.7, 140.2, 135.5, 132.2, 127.9, 127.0, 126.1, 123.5, 119.5, 112.0, 107.0, 61.2, 48.5, 46.0, 45.8, 28.3, 26.3, 21.3. ESI-MS m/z 495.3 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₂₉N₆Cl₂ (M + H) ⁺ 495.1826, found 495.1825.

5.1.10. General procedure for preparing compounds 30

Phenylacetonitrile (11 mmol) was added dropwise to a suspension solution of NaH (11 mmol) in anhydrous DMF (20 mL) at 0 °C under nitrogen. After the reaction mixture was stirred for 30 min at rt, bromide (10 mmol) was added in a dropwise manner. The resultant solution was stirred overnight at rt before water was added to quench the reaction. After the mixture was extracted with ethyl acetate, the organic phase was washed with brine, and dried over Na₂SO₄. Evaporation and column chromatography on silica gel afforded the crude alkylation product, which was dissolved in anhydrous Et₂O. The resultant solution was added dropwise to a suspension of LiAlH₄ (4.4 mmol) in anhydrous Et₂O at rt under nitrogen. After being stirred for 1 h, the reaction was quenched by adding water. Ether extract work up followed by chromatography provided crude amine, which was dissolved in THF. To this solution were added 11b and TEA before it was refluxed for 24 h. Ethyl acetate work up followed by purification by flash chromatography (eluent: 5–10% EtOH—DCM or similar conditions) gave the corresponding condensation product.

5-chloro-2-(5-chloropyridin-2-yl)-N-(2,4-diphenylbutyl)-6-methylpyrimidin-4-amine (30a) ¹H NMR (300 MHz, CDCl₃): δ 8.74 (d, J = 2.1 Hz, 1H), 8.30 (d, J = 8.7 Hz, 1H), 7.76 (dd, J = 2.1 Hz, 8.7 Hz, 1H), 7.10-7.40 (m, 10H), 5.32 (t, J = 6.3 Hz, 1H), 3.96-4.05 (m, 1H), 3.59-3.68 (m, 1H), 2.94-3.00 (m, 1H), 2.50-2.61 (m, 5H), 2.04-2.14 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.4, 158.8, 157.7, 153.3, 148.7, 142.0, 141.8, 136.4, 133.1, 129.0, 128.4, 128.3, 127.9, 127.1, 125.9, 124.4, 112.7, 47.0, 45.0, 35.0, 33.4, 22.3. ESI-MS m/z 463.1 (M + H) ⁺; HRMS (ESI) calcd for C₂₆H₂₅N₄Cl₂ (M + H) ⁺ 463.1457, found 463.1451.

5-chloro-2-(5-chloropyridin-2-yl)-N-(2,5-diphenylpentyl)-6-methylpyrimidin-4-amine (30b) foamy solid : ¹H NMR (300 MHz, CDCl₃): δ 8.73 (d, J = 1.8 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.78 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 7.08-7.35 (m, 10H), 5.32 (m, 1H), 3.97-4.06 (m, 1H), 3.54-3.63 (m, 1H), 2.92-2.97 (m, 1H), 2.55-2.62 (m, 5H), 1.76-1.85 (m, 2H), 1.57-1.75 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 158.9, 157.7, 153.8, 142.5, 142.1, 136.5, 133.2, 133.1, 128.9, 128.5, 128.4, 127.9, 127.1, 125.9, 124.5, 112.9, 45.9, 45.7, 35.9, 33.0, 29.3, 22.4. ESI-MS m/z 477.4 (M + H) ⁺; HRMS (ESI) calcd for C₂₇H₂₇N₄Cl₂ (M + H) ⁺ 477.1618, found 477.1607.

5-chloro-2-(5-chloropyridin-2-yl)-N-(2,6-diphenylhexyl)-6-methylpyrimidin-4-amine (30c) foamy solid : ¹H NMR (300 MHz, CDCl₃): δ 8.74 (d, J = 1.8 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 7.07-7.37 (m, 10H), 5.33 (t, J = 5.4 Hz, 1H), 3.97-4.05 (m, 1H), 3.55-3.64 (m, 1H), 2.90-3.00 (m, 1H), 2.51-2.60 (m, 5H), 1.72-1.83 (m, 2H), 1.57-1.69 (m, 2H), 1.26-1.37 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 158.9, 157.7, 153.4, 148.8, 142.6, 142.5, 136.5, 133.2, 128.9, 128.4, 128.3, 127.9, 127.0, 126.8, 125.8, 124.4, 112.8, 47.0, 45.6, 35.8, 33.5, 31.5, 27.2, 22.3. ESI-MS m/z 491.4 (M + H)⁺; HRMS (ESI) calcd for C₂₈H₂₉N₄Cl₂ (M + H)⁺ 491.1767, found 491.1764.

5-chloro-2-(5-chloropyridin-2-yl)-N-(2,7-diphenylheptyl)-6-methylpyrimidin-4-amine (30d) foamy solid : ¹H NMR (300 MHz, CDCl₃): δ 8.75 (d, J = 2.7 Hz, 1H), 8.36 (d, J = 8.7 Hz, 1H), 7.75 (dd, J = 2.7 Hz, 8.7 Hz, 1H), 7.10-7.37 (m, 10H), 5.32 (t, J = 5.1 Hz, 1H), 3.96-4.05 (m, 1H), 3.56-3.63 (m, 1H), 2.88-2.93 (m, 1H), 2.51-2.57 (m, 5H), 1.70-1.76 (m, 2H), 1.54-1.59 (m, 2H), 1.29-1.31 (m, 4H). ¹³C NMR (100 MHz, CDCl3): δ 161.3, 158.6, 157.7, 153.3, 148.7, 142.6, 142.5, 136.4, 133.2, 128.8, 128.4, 128.3, 127.8, 127.0, 125.6, 124.4, 112.8, 46.9, 45.6, 35.8, 33.4, 31.2, 29.2, 27.3, 22.2. ESI-MS m/z 505.5 (M + H)⁺; HRMS (ESI) calcd for C₂₉₈H₃₁N₄Cl₂ (M + H)⁺ 505.19267, found 505.19203.

5.2 Biological assays

5.2.1. Materials

30% acrylamide/bis solution (37.5:1) (161-0158), ammonium persulfate (161-0700) and N,N,N′,N′-tetramethylethlenediamine (TEMED) (161-0800) for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was purchased from Bio-Rad Laboratories (Hercules, CA). CoCl₂·6H₂O (C-3169) was purchased from Sigma-Aldrich (St. Louis, MO). Other commercially available chemicals were purchased from Sigma-Aldrich and Fisher Scientific (Hampton, NH). Restriction enzymes were purchased from New England BioLabs (Ipswich, MA).

5.2.2. Cell lines and cell culture

High Five (BTI-TN-5B1-4) cells (Life Technologies B855-02, Carlsbad, CA) used for the expression of recombinant HsMetAP2 were grown in Grace's insect cell medium (Life Technologies 11605-094) supplemented with 10% FBS and were maintained in a nonhumidified incubator at 27°C as per manufacturer's instructions.

5.2.3. ProIP-coupled MetAP activity assay

MetAP is known to have substrate specificity completely orthogonal to that of ProIP. The BcProIP-coupled MetAP activity assay was adapted from [47] with modifications. In this assay, MetAP activity was measured by continuously monitoring the production of pNA with BcProIP. Aliquots of purified recombinant human MetAP1 and MetAP2 proteins stored at -80 °C were thawed on ice. Repeated freeze-thaw cycles were avoided. The coupling enzyme BcProIP was taken from the stock at -20 °C. The substrate methionylprolyl-p-nitroanilide (Met-Pro-pNA) and the test compounds were dissolved in 100% DMSO. The total reaction volume was 100 μL and the reaction was carried out in 96-well plates at room temperature. 0.15 μM of HsMetAP1 or 0.1 μM of HsMetAP2 was incubated with either vehicle (DMSO) or test compounds in MetAP assay buffer containing 15 U/mL of BcProIP and 10 μM of CoCl₂ at room temperature for 20 minutes. The enzymatic reaction was initiated by the addition of 600 μM of Met-Pro-pNA. Absorbance at 405 nm (ε = 1.06×10⁴ M^-1cm^-1) was recorded every 30 seconds within the first 30 minutes of reaction by the FLUOstar OPTIMA microplate reader. After subtracting the background hydrolysis determined in the absence of MetAP, the net increases in the absorbance were plotted versus the reaction time and the slope of the early linear portion (usually between 2 minutes and 15 minutes after the reaction started) of the curve was determined by the function “linear regression analysis” in GraphPad Prism 5. The initial rate was then calculated from the slope. In addition, to rule out the possibility that any inhibitor found by the BcProIP-coupled MetAP assay might work simply because it inhibited the coupling enzyme, compounds showing inhibitory effects were counter screened against 0.1 U/mL of BcProIP, 600 μM of Pro-pNA and 10 μM of CoCl₂ in the MetAP assay buffer. Although the initial rates of 0.1 U/mL of BcProIP were greatly decreased by 100 μM of 18m and 18n, the remaining activity of 15 U/mL of BcProIP was still enough to saturate the coupling reaction in MetAP activity assays (data no shown).

5.2.4. Determination of the IC₅₀ or EC₅₀ values of the inhibitors

For in vitro MetAP activity assays, the initial rates of the reactions determined by the function “linear regression analysis” in GraphPad Prism 5 were first normalized to the initial rates of the vehicle control, and then plotted versus the concentrations of the compound (in a logarithmic scale). For cell proliferation assays, the [³H]-thymidine counts determined by the MicroBeta plate reader were first normalized to the counts of the vehicle control, and then plotted versus the concentrations of the compound (in a logarithmic scale). The inhibitory dose-response curve was generated by the nonlinear regression function “log(inhibitor) vs. response-Variable slope” in GraphPad Prism 5, using the model “Y=Bottom+(Top-Bottom)/{1+10×[(LogEC₅₀-X)×HillSlope]}” and the fitting method “least squares”. Top and Bottom are plateaus in the units of the Y axis. The Top values of the dose-response curve were always constrained to 1 (100% of the vehicle control). The Bottom values of the dose-response curve were constrained to zero (a complete inhibition) when the calculated Bottom values were smaller than zero. Otherwise, the Bottom values were not constrained and will be reported when they were larger than 0.1 (10% of the vehicle control). EC₅₀ was used to define the concentration of an inhibitor that gave a response half way between Bottom and Top. When Top=1 and Bottom=zero, IC₅₀ was used to replace EC₅₀.

Abbreviations

BcProIP

Bacillus coagulans proline iminopeptidase

DMSO

dimethyl sulfoxide

EC₅₀

half maximal effective concentration

HEPES

2-[4-(2-hydroxylethyl)-1-piperazinyl]ethanesulfonic acid

HRP

horseradish peroxidase

HsMetAP1

human cytosolic type 1 methionine aminopeptidase

HsMetAP2

human cytosolic type 2 methionine aminopeptidase

IC₅₀

half maximal inhibitory concentration (IC₅₀ equals to EC₅₀ when the bottom value of an inhibitory dose-response curve is zero)

K_i

dissociation constant for inhibitor binding

map

gene encoding prokaryotic methionine aminopeptidase

map1

gene encoding eukaryotic cytosolic type 1 methionine aminopeptidase

map2

gene encoding eukaryotic cytosolic type 2 methionine aminopeptidase

MetAP

methionine aminopeptidase

MetAP1

eukaryotic cytosolic type 1 methionine aminopeptidase

MetAP2

eukaryotic cytosolic type 2 methionine aminopeptidase

NME

N-terminal methionine excision

SAR

structure-activity relationship

tHsMetAP1

N-terminally truncated human methionine aminopeptidase