Pyridinylpyrimidines selectively inhibit human methionine aminopeptidase-1

Article abstract of DOI:10.1016/j.bmc.2013.02.023

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)

Full text of DOI:10.1016/j.bmc.2013.02.023

Bioorganic & Medicinal Chemistry 21 (2013) 2600–2617
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyridinylpyrimidines selectively inhibit human methionine
aminopeptidase-1 q
Pengtao Zhang a, , Xinye Yang b, , Feiran Zhang c, , Sandra B. Gabelli d,e,f, Renxiao Wang a, Yihua Zhang b,
Shridhar Bhat c, Xiaochun Chen c,à, Manuel Furlani e, L. Mario Amzel e, Jun O. Liu c,f, , Dawei Ma a,
⇑
⇑
a State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
c Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA
d Department of Medicine, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA
e Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA
f Department of Oncology, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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 pyridinyl-
pyrimidine 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 58 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 identi-
fied (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of
HsMetAP1; (2) 50-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 HsMetAP1-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 representa-
tive inhibitor (26d) in complex with N-terminally truncated HsMetAP1.
Received 20 December 2012
Revised 3 February 2013
Accepted 11 February 2013
Available online 21 February 2013
Keywords:
Methionine aminopeptidase
Pyridinylpyrimidine
Anti-cancer
Ó 2013 Elsevier Ltd. All rights reserved.
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
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 eukaryotes.1 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 re-
quires 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, Me-
tAPs 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 methi-
onine and the side chain of P10 residue is small and uncharged.8,9
Residues at the positions of P20 and beyond also contribute to the
efficiency of cleavage.8a,9
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); Ki, 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, eukary-
otic cytosolic type 2 methionine aminopeptidase; NME, N-terminal methionine
excision; SAR, structure–activity relationship; tHsMetAP1, N-terminally truncated
human methionine aminopeptidase.
q
Atomic coordinates for the N-terminally truncated human MetAP1 in complex
with 26d and Co(II) have been deposited in the Protein Data Bank (www.pdb.org)
under the access code 4HXX.
⇑
Corresponding authors. Tel.: +1 410 955 4619; fax: +1 410 955 4620 (J.O.L.);
tel.: +86 21 54925130; fax: +86 21 64166128 (D.M.).
E-mail addresses: joliu@jhu.edu (J.O. Liu), madw@mail.sioc.ac.cn (D. Ma).
These authors contributed equally to the manuscript.
Current address: Department of Pediatrics, University of Maryland School of
à
Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.023

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2601
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,7a,10,11 at least two genes encoding one cyto-
solic type 1 MetAP (MetAP1) and one cytosolic type 2 MetAP (Me-
tAP2), 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 essen-
tial genes.15 In eubacteria, the importance of MetAP is underscored
by the lethality when the single map gene in Escherichia coli or Sal-
monella typhimurium was disrupted.10,11 In Saccharomyces cerevisi-
ae, 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 func-
tionally 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-spe-
cific and tissue-specific developmental defects. Most of the defects
caused either lethality or infertility.17 Small-interfering RNA (siR-
NA) mediated downregulation of either MetAP1 or MetAP2 signif-
icantly 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. dem-
onstrated 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 anti-
tumor activities of bengamides in vitro21 and in vivo23 evince a piv-
otal role of MetAPs in human cells.
compounds were identified in an in vitro assay as low micromolar
inhibitors of HsMetAP1. But their isoform selectivity is still un-
clear.37 We and others have previously reported that the deriva-
tives of pyridine-2-carboxamide (such as compound 1, Fig. 1)
inhibited the purified type 1 MetAP from either prokaryotes (Esch-
erichia 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 sub-
sequently found that at low micromolar concentrations, 1 also had
unexpected off-target cellular effects (a corrigendum will be pub-
lished later). In addition to pyridine-2-carboxamides, a novel class
of inhibitors containing 2-pyridinylpyrimidine (such as compound
2, Fig. 1) was discovered by 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, crystallo-
graphic 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 (Fig. 2A and B).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 quin-
olinyl sulfonamide (compound 3, Fig. 1) inhibited cellular MetAP in
bacteria as well, albeit at a high concentration.41
Herein we describe a systematic medicinal chemistry approach
to probing the structural requirements of pyridinylpyrimidine scaf-
fold for selective inhibition of HsMetAP1. The X-ray crystal structure
of compound 2 bound to the Co(II) form of the N-terminally trun-
cated 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 substitu-
tion at the C30–C60 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 50 analogues based on the pyridinylpyrimidine
scaffold and characterized their inhibitory activity with purified
HsMetAP1 and HsMetAP2. We also performed crystallographic anal-
ysis of one representative inhibitor (26d) in complex with N-termi-
nally truncated HsMetAP1 (PDB access code 4HXX).
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 angio-
genesis,29,30 TNP-470 displayed broad-spectrum activities against
the growth of both primary and metastatic tumors in various
animal models (for reviews, see Refs. 31,32). TNP-470 was also
effective against nutritionally induced obesity,33 pulmonary hyper-
tension34 and arthritis.35 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 Me-
tAP1 not only hampered the advancement of MetAP1 biology,
but also restrained the evaluation of MetAP1 as a novel target for
anti-neoplastic 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 Ki values for the inhibition of both purified HsMetAP1 and
human MetAP2 (HsMetAP2).36 Later, several barbiturate-based
2. Chemistry
Analogous to the procedure described by Medwid et al.,42 we
started our synthesis of 2-pyridinylpyrimidine derivatives by trans-
forming substituted picolinonitriles 4 into the corresponding pico-
linimidamides
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 dis-
placement of the 4-chloro group on pyrimidine 8 by phenethyl-
amine was accomplished by refluxing the reaction mixture in
methanol containing potassium carbonate. Thus, by using ethyl 2-
fluoroacetoacetate, ethyl 2-methylacetoacetate, or ethyl acetoace-
tate in the condensation step to generate pyrimidinone 7, 2-pyri-
din-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 ana-
logues of 2-pyridin-2-ylpyrimidine. Compound 13 was prepared by
alkylating 5-chloro-6-methyl-2-(pyridin-2-yl)-3,4-dihydropyrimi-
dine-4-thione 12 with phenethyl bromide as shown in Scheme 2.
Acylation of amines 11 provided chlorides 14 (Scheme 3) which
were reacted with piperazine derivative 15 to afford amino

2602
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
Cl
O
O
H
N
5
4
NH
6
1 N
N 3
O
N
N
2
N
2'
O
HN
1'N
6'
3'
4'
HN
S
S
CH3
O
5'
1
2
3
Figure 1. Structures of MetAP inhibitors 1–3.
A
B
H212
H212
2
1
M3
W3
W2
W5
M3
W2
W3
H310
H310
W1
W1
M1
W5
W4
W4
D303
D303
M1
H303
H303
M2
M2
E336
E336
D240
D240
E367
E367
C
Figure 2. The X-ray crystal structures of the N-terminally truncated human MetAP1 (tHsMetAP1) in complex with 1 (2NQ619) (A) or 2 (2G6P40) (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 tHsMetAP1 (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.
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 depro-
tected 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 ethe-
nesulfonyl 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.
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.
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
Amides 25a–25e were prepared by simple condensation of
pyridinylpyrimidine 11b with carboxylic acids (Scheme 6). A series

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2603
R1
R3
R1
R1
R3
N
N
N
N
NH•HCl
i, ii
iii
iv
R2
X
R2
R2
CN
R2
NH
NH2
R4
O
R3
R1
R4
R4
4
5
7
R1
N
N
N
9a: R1-R4 = H, X = F
9b: R1-R4 = H, X = CH3
9c: R1-R4 = H, X = H
N
N
N
v
R2
X
X
R3
R1
R4
NH
R3
R4
Cl
9
8
vi, vii
N
N
R2
X
N
R3
R4
11
NH
NH2
Scheme 1. Synthesis of 2-pyridinyl-6-methylpyrimidines. Reagents and 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.
molecules (W1, W2 and W3 in Fig. 2B). In agreement with this vital
N
N
N
N
N
i
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
Cl
Cl
HN
S
S
13
12
MetAP at concentrations up to 100 lM (Table 1). Together, these
Scheme 2. Synthesis of 13. Reagents and conditions: (i) K2CO3, PhCH2CH2Br,
toluene, 60 °C, 92% yield.
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.
high-throughput screening against approximately 175,000 com-
pounds.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 HsMe-
tAP1 to inhibit the enzyme (Fig. 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
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 po-
tency 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.
Cl
Ph
O
Cl
Ph
O
Cl
Ph
H
N
H
N
H
N
OTBS
()n
()n
N
H
N
N
H
Cl
NH2
HN
N
ii
N
N
N
N
N
N
N
N
N
N
i
15
R4
R3
R4
R3
R4
R3
OH
R1
R1
R1
R2
R2
Cl
R2
14
11
16a: R1-R4 = H, n = 1
16b: R1-R4 = H, n = 2
HN
NBoc
iii
17
Ph
O
H
N
()n
N
N
H
N
N
NH
R4
R3
N
R1
R2
18a-18o
Scheme 3. Synthesis of 2-pyridinylpyrimidine derivatives 16a, 16b and 18a–18p. Reagents and 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.

2604
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
R1
N
Cl
Ph
Br
N
H
N
N
H
N
19a: R1 = Boc
19b: R1 = (CH2)2OTBS
N
N
N
N
R2
19
20a: R2 = H
20b: R2 = (CH2)2OH
i
Cl
Ph
H
N
NH2
N
N
N
R4
Cl
Ph
N
21
ii
H
N
N
N
H
11a
N
N
N
R3
N
OHC
N
22a: R4 = H
22b: R4 = (CH2)2OH
21a: R3 = Boc
21b: R3 = (CH2)2OTBS
Scheme 4. Synthesis of additional 2-pyridinylpyrimidine derivatives 20a, 20b, 22a and 22b. Reagents and conditions: (i) NaI, DMF then TFA or TBAF, 84–88% yields; (ii)
NaBH3CN, HOAc, MeOH, then TFA or TBAF, 55–60% yields.
Cl
Ph
Cl
Ph
O
Cl
Ph
O
O
H
N
H
N
H
N
Ph
S
()n
NH2
N
H
N
H
i
N
N
N
N
N
N
N
N
N
N
N
25a-25e
11a
23
i
Cl
Cl
Ph
Cl
N
O
O
H
N
Cl
Cl
Ph
Ph
S
H
N
H
N
N
N
Ar
H
()n
NH2
N
H
ii, iii
N
N
N
R
N
N
N
N
N
ii
24a: R = H
24b: R = (CH2)2OH
26a-26f, 27a-27g
11b
iii
Scheme 5. Synthesis of sulfonamide derivatives 24a and 24b. Reagents and
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.
Cl
Cl
Ph
H
N
N
N
MsO
()3
()3
N
H
N
N
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 with-
out 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 chal-
lenge 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 com-
plex with 2 was superimposed on the structure of HsMetAP2, sev-
eral 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. Presum-
ably, an unfavorable interaction like this could be exploited to im-
prove 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).
27h
Cl
Scheme 6. Synthesis of 2-(5-chloro-pyridin-2-yl)-pyrimidine derivatives 25a–25e,
26a–26f and 27a–27h. Reagents and conditions: (i) Ph(CH2)nCO2H, EDCI, HOBt,
DIPEA, 70–85% yields; (ii) Ar(CH2)nCHO, NaBH3CN, HOAc, MeOH, 25–35% yields;
(iii) NaI, Et3N, CH3CN, 37% yields.
The crystal structure of compound 2 in complex with N-termi-
nally truncated HsMetAP1 also revealed an unexpected 2-[4-(2-
hydroxylethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) mole-
cule 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 (Fig. 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,

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
Ph
2605
and the amide linker by one carbon further improved the HsMe-
tAP1-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 appre-
ciable effect on two analogues with secondary amine linkers (20a
vs 20b, and 22a vs 22b) and one analogue with a longer amide lin-
ker (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.
Ph
()n
()n
2-phenylacetonitrile
CN
NH2
ii
i
CN
29
28
Cl
Ph
H
N
Ph
()n
N
N
N
iii
30a: n = 2
30b: n = 3
30c: n = 4
30d: n = 5
After a comparative analysis of twenty analogues with various
side chains, we selected 18b as a lead to explore the SAR of substit-
uents on the pyridine ring of pyridinylpyrimidine (Table 3). Com-
pared to 18b, installation of either a methoxyl group or a chloro
group at C60 or C30 reduced the potency against HsMetAP1 to differ-
ent degrees (18c, 18d, 18e and 18f). On the contrary, the potency
was enhanced by 4.1-fold via the introduction of a methoxyl substi-
tuent at the adjacent C50 position (18g). Similarly, introduction of
fluoro (18h), chloro (18i), bromo (18j), nitro (18k) or dimethylamino
(18l) group at C50 led to 3.3-fold to 31-fold increases in the anti-
HsMetAP1activities relative to 18b. Unfortunately, the potency
against HsMetAP2 was simultaneously restored for 18h, 18i, 18k
and 18l, significantly compromising HsMetAP1-selectivity. A pyrro-
lidinyl substituent, which is bigger than a dimethylamino group,
was not well tolerated at C50, as indicated by the reduced inhibitory
Cl
Scheme 7. Synthesis of 2-(5-chloro-pyridin-2-yl)-pyrimidine derivatives 30a–30d.
Reagents and 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.
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
Table 1
Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives
R1
R1
5
R2
4
R2
6
N
N
N
1 N
N3
2
N
For other compounds
For 31
a
Compd
R1
R2
IC50
(lM)
IC50 ratio (type 2/1)
HsMetAP1
HsMetAP2
2
Cl
F
Me
H
Cl
Cl
Cl
Cl
Cl
NHCH2CH2Ph
NHCH2CH2Ph
NHCH2CH2Ph
NHCH2CH2Ph
SCH2CH2Ph
NHCH2Ph
SCH2Ph
N(Me)CH2Ph
NHMe
0.86 0.04
4.6 0.1
>100
9.0 0.9
10
13
9a
9b
9c
13
31
32
33
34
62
9
>100
>100
>100
>100
>100
13 0.1
NAb
NAb
>10
NAb
>9.1
8.7
>100
10
1
>100
11
1
1.5 0.1
8.9 0.3
14
1
1.6
NH
HN
35
Cl
1.0 0.1
>100
>100
N
N
CF3
CF3
NH
HN
36
37
Cl
Cl
2.0 0.1
1.1 0.2
>100
>100
>50
>91
4-(2-Hydroxyl-ethyl)-piperazinyl
N
HN
N
CF3
38
Cl
0.36 0.03
>100
>278
Cl
IC50 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
a
smaller than 10%.
b
NA: not available.

2606
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
Table 2
Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives
Cl
5
H
N
R
4
N
H
6
1 N
N 3
2
N
a
Compd
R
IC50
(lM)
IC50 ratio (type 2/1)
HsMetAP1
HsMetAP2
16a
16b
18a
18b
20a
20b
22a
22b
24a
24b
CO(CH2)2
CO(CH2)3
CO(CH2)2
CO(CH2)3
N
N
N
N
0.8 0.1
114 12
143
>272
37
N
(CH2)2OH
(CH2)2OH
N
1.1 0.1
0.6 0.1
1.1 0.2
1.4 0.2
1.7 0.5
1.3 0.5
0.8 0.2
0.8 0.1
0.6 0.1
>300
NH
NH
22
4
>300
>272
77
(CH2)3
(CH2)3
(CH2)4
(CH2)4
N
NH
108 11
101 12
188 17
197 22
9.4 1.0
95 14
N
N
N
N
(CH2)2OH
59
145
246
12
NH
N
(CH2)2OH
NH
SO2(CH2)2
SO2(CH2)2
N
N
N
(CH2)2OH
158
a
IC50 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%.
potency and the loss of HsMetAP1-selectivity of 18m. In contrast to
the substituents at C60 and C30, analogues with methoxy (18n) and
chloro (18o) substituents at C40 maintained their potency against
HsMetAP1. However, the HsMetAP1-selectivity of 18n was attenu-
ated by the relatively bigger C40 substituent.
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.
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 compari-
son to 25a–25e. Thirdly, derivatizing 26d to 27a–27d or replacing
the phenyl group at the end of the side chain with various hetero-
cyclic 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
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 (D1–80) human
MetAP1 (tHsMetAP1) in complex with Co(II) and 26d was deter-
mined 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 (Fig. 3A). Two Co(II) are pre-
sented at the bottom of the active site (Fig. 2C), the ubiquitous
positions for metal ions (M1 and M2) in the MetAP family. In addi-
tion, a third Co(II) (Co3) is located at a place (M3) close to the ac-
tive site, which has previously been identified in the complexes of
tHsMetAP1with two specific classes of inhibitors.19,40 Among them,
compound 2 has a pyridinylpyrimidine core similar to that of com-
pound 26d. In the crystal structures of tHsMetAP1 in complex with
2 (PDB access code 2G6P, reported in Ref. 40) and 26d, both pyri-
dine nitrogen (N10) and pyrimidine nitrogen (N1) are bound to

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2607
Table 3
Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives
O
N
Cl
H
N
N
N
H
N
N
2'
R4
R3
1'
N
3'
6'
4'
R1
5'
R2
a
Compd
Substituents (when not specified, R# = H)
EC50
(lM)
EC50 ratio (type 2/1)
HsMetAP1
20
HsMetAP2
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
R1 = OMe
R1 = Cl
R4 = OMe
R4 = Cl
R2 = OMe
R2 = F
R2 = Cl
6
>30b
>1.5
>4.2
>120
>39
>1111
24
33
161
47
12
1.1
26
>411
7.2 2.4
>30b
2.5 1.3 (Bc = 0.15)
7.6 0.4
>300
>300
>300
1.2 0.3
1.2 0.3
0.27 0.04
0.049 0.07
0.036 0.006
0.33 0.01
0.30 0.06
0.14 0.05
1.7 0.3 (B = 0.36)
0.53 0.07
0.73 0.03
R2 = Br
53
14
5
1
R2 = NO2
R2 = N(CH3)2
R2 = pyrrolidinyl
R3 = OMe
R3 = Cl
1.7 0.7
1.1 0.1 (B = 0.22)
18m
18n
18o
14
9
>300
a
b
c
EC50 values of compounds to inhibit human MetAPs were shown as mean SD of two to three determinations.
Higher concentrations have not been tested for these compounds.
B: bottom value of the inhibitory dose–response curve. It has been noted in parentheses only when B > 10%.
Co3 (Fig. 3B), displaying a near-perfect octahedral coordination
which is completed by the nitrogen atom from the side chain of
His212 and three water molecules.
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 coor-
dination, there would be a limit for the displacement of the pyrid-
inylpyrimidine core. This limit may help to explain why the
inhibitors with larger substituents at C50 (18g, 18j, 18k, 18l and
18m) were less potent against purified HsMetAP1 than the inhibi-
tors with smaller substituents such as a fluoro group (18h) or a
chloro group (18i).
Compound 26d buries about 468 Å2 upon binding tHsMetAP1
from a total accessible area of 753 Å2. 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 (Fig. 3C). But when
compared with 2, an extra chloro group is present at the C50 posi-
tion 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 dis-
placed up to 1 Å out of the hydrophobic pocket for methionine side
chain to accommodate the C50 chloro substituent (Fig. 3B). There-
fore for 26d, the distances from C50 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. How-
ever, the pyridinylpyrimidine core of 26d still maintains the octa-
hedral 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 (N10)
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 struc-
ture 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 sub-
stitute at C50 sabotages the inhibitory potency of 26d. To the con-
trary, since 18i was 22 times more potent against purified
In the crystal structure of tHsMetAP1 with 2 (PDB access code
2G6P), an unexpected HEPES molecule from the buffer is accom-
modated 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 ex-
tended 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 (Fig. 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 com-
plex with compound 26d and the crystal structure of HsMetAP2 in
complex with L-methionine (PDB access code 1KQ9, reported in

2608
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
Table 4
Inhibition of purified recombinant human MetAPs by pyridinylpyrimidine derivatives
Cl
Cl
5
H
5
H
N
N
4
4
R
R
6
6
1 N
N3
1 N
N3
2
2
N
N
Cl
Cl
For 30
For 25, 26 and 27
a
Compd
R
EC50
(
lM)
IC50 ratio (type 2/1)
HsMetAP1
HsMetAP2
25a
25b
25c
25d
25e
26a
26b
26c
26d
26e
26f
27a
27b
27c
27d
27e
27f
NHCOCH2Ph
0.70 0.52
0.093 0.042
0.16 0.12
28
6
40
NHCO(CH2)2Ph
NHCO(CH2)3Ph
NHCO(CH2)4Ph
NHCO(CH2)5Ph
NHCH2Ph
NH(CH2)2Ph
NH(CH2)3Ph
NH(CH2)4Ph
NH(CH2)5Ph
>30b
>30b
>30b
>30b
31
>323
>188
>23
>45
141
1.3 0.2 (Bc = 0.12)
0.67 0.30
0.22 0.7
5
0.26 0.05
0.26 0.06
0.20 0.03
0.78 0.19
0.71 0.21
0.42 0.06
0.57 0.10
0.52 0.07
1.2 0.2
0.33 0.08
0.38 0.18
0.69 0.08
0.50 0.09
>100
>300
>100
>100
>30b
>300
>300
16 7d
>300
>300
>300
>300
>100
>200
>200
>200
>200
>384
>1154
>500
>138
>42
>714
>526
31
>250
>909
>789
>435
>200
>3175
>1250
>1000
>87
NH(CH2)6Ph
NH(CH2)4-(4-phenolyl)
NH(CH2)4-(4-chlorophenyl)
NH(CH2)4-(4-cyanophenyl)
NH(CH2)4-(4-propoxylphenyl)
NH(CH2)4-(2-furyl)
NH(CH2)4-(2-thienyl)
NH(CH2)4-(2-quinolyl)
NH(CH2)4-(1-pyrrol)
(CH2)2Ph
27g
27h
30a
30b
30c
30d
0.063 0.024 (Bc = 0.14)
0.16 0.01 (Bc = 0.13)
0.20 0.11
(CH2)3Ph
(CH2)4Ph
(CH2)5Ph
2.3 1.8
a
b
c
EC50 values of compounds to inhibit human MetAPs were shown as mean SD of two to three determinations.
Higher concentrations have not been tested for these compounds.
B: bottom value of the inhibitory dose–response curve. It has been noted in parentheses only when B > 10%.
d
In the counter screening, compound 27c was identified as a potent inhibitor of the coupling enzyme, BcProIP. 10
M Pro-pNA and 10 M CoCl2.
lM and 100
l
M of 27c inhibited 27% and 97% of the
activity of BcProIP in MetAP assay buffer, in the presence of 600
l
l
Ref. 44) revealed a possible structural basis for the selectivity of
26d for HsMetAP1 over HsMetAP2 (Fig. 4). Compared with HsMe-
tAP1, 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 en-
trance. In the present conformation, the long C4 side chain of
26d is likely to clash with Tyr444 of HsMetAP2, and the C50 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.
well as the crystal structure of 26d bound in the active site of
tHsMetAP1, confirm the key interactions between the auxiliary co-
balt ion and the N1 and N10 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 ex-
tend the C4 side chain to exploit hydrophobic interactions with the
enzyme, we discovered that a long C4 side chain containing a pip-
erazinyl 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 C40 could
be tolerated, substituents at C60 or C30 reduced the potency against
HsMetAP1, and small substituents at C50 greatly improved the po-
tency but compromised the HsMetAP1selectivity (18h and 18i). In
the further exploration of the C4 side chain on the basis of 18i, we
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 en-
zymes. The deleterious effects on inhibitor potency by the replace-
ment of the 2-pyridin-2-yl group with a 2-pyridin-3-yl group, as

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2609
Table 5
linker for aliphatic linkers in the C4 side chain were both well tol-
erated. In the end, we obtained a group of compounds containing
both high nanomolar potency against 0.15 lM of purified HsMe-
tAP1 and hundreds/thousands fold selectivity over HsMetAP2.
Thus, these compounds may serve as leads for the development
of therapeutical agents against cancer.
X-ray data collection and refinement statistics of the N-terminal truncated (
human MetAP1 (tHsMetAP1) in complex with 26d
D1–80)
Crystal
tHsMetAP1 w. 26d
Space group
Cell dimensions
a (Å)
b (Å)
c (Å)
P21
47.5
77.4
48.0
90.9
5. Experimental
5.1. Chemistry
b (deg)
X-ray data collection statistics
X-ray source
Wavelength (Å)
Resolution range (Å) (HighRes shell)
Collected reflections
Unique reflections
FR-E+/Raxis IV
1.54178
5.1.1. General methods
50.00–2.09 (2.16–2.09)
71,126
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 re-
agent 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 King-
dom), and their masses were confirmed by API-MS.
20,226
29.6 (2.8)
97.9 (86.6)
11.7 (49.0)
I/
r
Completeness (%)
Rmerge (%)
Refinement statistics
Rcryst (%)
0.18 (0.27)
0.25 (0.33)
Rfree (%)
R.m.s deviations
Bond length (Å)
Angle (deg)
Monomer in ASU
Total atoms
Protein atoms
Water molecules
Ligand
0.013
1.1533
1
2586
2400
152
Analytical thin layer chromatography (TLC) was performed using
Analtech Uniplates (silica gel HLF, W/UV254, 250 lm). Visualization
was achieved using 254 nm UV light, or additionally by stainingwith
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 uncor-
rected. 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 (d) with the solvent resonance
or 0.1% tetramethylsilane contained in the deuterated solvent as
the internal reference. Data are reported as follows: chemical shift,
34
B-factor (
D
HsMetAP1) (Å2)
41.8
62.0
55.0
B-Factor (26d) (Å2)
B-Factor (H2O) (Å2)
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
A
B
HEPES
2
26d
26d
H212
H212
Co3
Co3
C
D
26d
26d
2
HEPES
Figure 3. The X-ray crystal structure (4HXX) of the N-terminally truncated human MetAP1 (tHsMetAP1) in complex with 26d (cyan) (A) and (C), and a superimposition of
4HXX with the crystal structure (2G6P40) 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.

2610
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
over silica gel. Subsequent displacement of the 4-chloro group on
D442
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
Section 5.1.1. The tR values refer to the retention time when the
Y444
26d
samples were analyzed using Pursuit XRs C18 column (3 lm,
H339
4.6 ꢁ 150 mm) with 60:40 acetonitrile/water (0.1% TFA) as an iso-
cratic mobile phase.
H339
5.1.2.1. 5-Fluoro-6-methyl-N-phenethyl-2-(pyridin-2-yl)pyrimi-
din-4-amine (9a). Yield: 56% (over three steps). tR: 4.029 min
(99.1%). 1H NMR (400 MHz, acetone-d6): d 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): d 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)+.
Y444
26d
D442
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 (1KQ944). 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
5.1.2.2. 5,6-Dimethyl-N-phenethyl-2-(pyridin-2-yl)pyrimidin-4-
amine (9b). Yield: 62% (over three steps). tR: 7.163 min (94.6%).
1H NMR (400 MHz, acetone-d6): d 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): d 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)+.
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.
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-dihydroxyben-
zoic acid solution in acetone, where the latter served as the matrix.
Data are reported in the form m/z (molecular ion).
5.1.2.3.
6-Methyl-N-phenethyl-2-(pyridin-2-yl)pyrimidin-4-
Yield: 49% (over three steps). tR: 6.328 min
amine (9c)45
.
(95.8%). 1H NMR (400 MHz, acetone-d6): d 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
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
(125 MHz, CDCl3):
d 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,
Quadrupole MS detector. A Pursuit XRs C18 column (3 lm,
24.19. MALDI-TOF: m/z 291 (M+H)+.
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: 100–1000, positive
mode, fragmenter set at 200). Purity of final compounds was
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 water (25 mL) and the mixture was ex-
tracted 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 af-
ford pyrimidine as an off-white solid. Yield: 314 mg (92%). tR:
6.103 min (96.1%). 1H NMR (400 MHz, acetone-d6): d 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):
d 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)+.
determined to be P94%, using a 20 lL 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.42 Briefly, ami-
dine hydrochloride 5 was prepared from 2-cyanopyridine by treat-
ing with 1.25 M HCl in methanol for 18 h and the resulting white
solid was used without further purification to condense with dif-
ferent acetoacetates (ethyl 2-fluoroacetoacetate, ethyl 2-methy-
lacetoacetate 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

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2611
5.1.4. Typical procedure for synthesizing compounds 16 and 18
To the solution of 4,5-dichloro-6-methyl-2-(pyridin-2-yl)pyrim-
idine (8a, 612 mg, 2.5 mmol) and (R)-tert-butyl-2-amino-1-phenyl-
ethylcarbamate (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 corre-
sponding 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
5.1.4.3.
N-((R)-2-(5-Chloro-6-methyl-2-(pyridin-2-yl)pyrimi-
din-4-ylamino)-1-phenylethyl)-3-(piperazin-1-yl)propan-
amide (18a).
½
a 2D0
ꢂ
ꢃ44.6 (c 0.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d 9.37 (d, J = 7.8 Hz 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) d 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.
5.1.4.4. N-((R)-2-(5-Chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-
4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butanamide
(18b).
½
a 2D0
ꢂ
ꢃ52.3 (c 1.8, CHCl3); 1H NMR (300 MHz, CDCl3) d 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)
d 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.
(722 mg, 85%): ½a D20
ꢂ
ꢃ43.6 (c 0.1, CHCl3); 1H NMR (300 MHz, CDCl3)
d 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)
d 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, the 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-butyldi-
methylsilyloxy)ethyl)piperazine (1.4 mmol) or tert-butyl pipera-
zine-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 TBAF in THF or a mixture of TFA
and CH2Cl2 followed by ethyl acetate work up afforded 16a or
18a in about 30% overall yield.
5.1.4.5. N-((R)-2-(5-Chloro-2-(6-methoxypyridin-2-yl)-6-meth-
ylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)buta-
namide (18c).
½
a 2D0
ꢂ
ꢃ65.9 (c 0.1, CHCl3). 1H NMR (300 MHz,
CDCl3) d 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) d 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.
5.1.4.6. N-((R)-2-(5-Chloro-2-(6-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-
yl)butanamide (18d).
½
a 2D0
ꢂ
ꢃ68.1 (c 0.4, CHCl3). 1H NMR
(300 MHz, CDCl3) d 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)
d 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,
22.2, 22.0; ESI-MS m/z 528.1 (M+H)+. HRMS (ESI) calcd for
5.1.4.1. 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).
½
a 2D0
ꢂ
ꢃ41.6 (c 0.16, CHCl3); 1H NMR
(300 MHz, CDCl3): d 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) d 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.
C
26H32Cl2N7O (M+H)+ 528.2040, found 528.2036.
5.1.4.7. N-((R)-2-(5-Chloro-2-(3-methoxypyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butana-
mide (18e). 1H NMR (300 MHz, CDCl3) d 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) d 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.
5.1.4.2. 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).
½
a 2D0
ꢂ
ꢃ34.4 (c 0.25, CHCl3); 1H NMR (300 MHz,
CDCl3) d 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) d 173.3, 161.8,
159.5, 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.
5.1.4.8. N-((R)-2-(5-Chloro-2-(3-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)buta-
namide (18f).
CDCl3)
½
a 2D0
ꢂ
ꢃ49.0 (c 3.5, CHCl3). 1H NMR (300 MHz,
d
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),

2612
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
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) d 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.
ESI-MS m/z 539.3 (M+H)+. HRMS (ESI) calcd for C26H32ClN8O2
(M+H)+ 539.2280, found 539.2271.
5.1.4.14. N-((R)-2-(5-Chloro-2-(5-(dimethylamino)pyridin-2-yl)-
6-methylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)
butanamide (18l).
½
a 2D0
ꢂ
ꢃ52.0 (c 0.3, CHCl3). 1H NMR (300 MHz,
CDCl3) d 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) d 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/z 537.3 (M+H)+.
5.1.4.9. N-((R)-2-(5-Chloro-2-(5-methoxypyridin-2-yl)-6-meth-
ylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)buta-
namide (18g).
½
a 2D0
ꢂ
ꢃ21.2 (c 0.4, CHCl3); 1H NMR (300 MHz,
CDCl3) d 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) d 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.
5.1.4.15. N-((R)-2-(5-Chloro-6-methyl-2-(5-(pyrrolidin-1-yl)pyri-
din-2-yl)pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)
butanamide (18m).
½
a 2D0
ꢂ
ꢃ66.3 (c 1.1, CHCl3). 1H NMR (300 MHz,
CDCl3) d 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) d 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)+.
5.1.4.10. N-((R)-2-(5-Chloro-2-(5-fluoropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)buta-
namide (18h).
½
a 2D0
ꢂ
ꢃ39.1 (c 0.1, CHCl3). 1H NMR (300 MHz,
CDCl3) d 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) d 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.
HRMS (ESI) calcd for
563.3033.
C
30H40ClN8O (M+H)+ 563.3008, found
5.1.4.16. N-((R)-2-(5-Chloro-2-(4-methoxypyridin-2-yl)-6-meth-
ylpyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)buta-
namide (18n).
½
a 2D0
ꢂ
ꢃ48.5 (c 0.9, CHCl3). 1H NMR (300 MHz,
CDCl3) d 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) d 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.
5.1.4.11. N-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butana-
mide (18i).
½
a 2D0
ꢂ
ꢃ45.2 (c 1.9, CHCl3). 1H NMR (300 MHz, CDCl3) d
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) d 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.
5.1.4.17. N-((R)-2-(5-Chloro-2-(4-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butana-
mide (18o).
½
a 2D0
ꢂ
ꢃ36.0 (c 2.1, CHCl3). 1H NMR (300 MHz, CDCl3) d
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) d
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.
5.1.4.12. N-((R)-2-(2-(5-Bromopyridin-2-yl)-5-chloro-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)butana-
mide (18j).
½
a 2D0
ꢂ
ꢃ47.8 (c 2.1, CHCl3). 1H NMR (300 MHz, CDCl3) d
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) d 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.
5.1.5. Typical procedure for synthesizing compounds 20
5.1.5.1. 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 over-
night at 100 °C for 24 h, the reaction mixture was cooled to rt, and di-
luted 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 NaH-
CO3 and brine, and dried over Na2SO4. Evaporation and column chroma-
tography on silica gel (eluent: 5–10% EtOH–DCM or similar conditions)
5.1.4.13.
N-((R)-2-(5-Chloro-6-methyl-2-(5-nitropyridin-2-yl)
pyrimidin-4-ylamino)-1-phenylethyl)-4-(piperazin-1-yl)buta-
namide (18k).
½
a 2D0
ꢂ
ꢃ25.8 (c 1.3, CHCl3). 1H NMR (300 MHz,
CDCl3) d 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) d 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.
afforded 20 as the foamy solid (60 mg, 38% yield). ½a D20
ꢃ23.1 (c 0.15,
ꢂ

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2613
CHCl3); 1H NMR (300 MHz, CDCl3) d 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) d 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
3.85 mmol) with stirring. After addition, the mixture was stir-
red at rt for 5 h, and then reaction was quenched with ice water.
The reaction mixture was extracted with CH2Cl2, and the com-
bined 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:
C
25H33ClN7 (M+H)+ 466.2481, found 466.2496.
5.1.5.2. 2-(4-(3-((R)-2-(5-Chloro-6-methyl-2-(pyridin-2-yl)pyr-
imidin-4-ylamino)-1-phenylethylamino)propyl)piperazin-1-yl)
ethanol (20b). Following a similar procedure from 19a to 20a,
20b was obtained from 19b. ½a D20
ꢂ
ꢃ13.9 (c 0.65, CHCl3); 1H NMR
5–10% EtOH–DCM or similar conditions) afforded 24a. ½a D20
ꢂ
(300 MHz, CDCl3) d 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) d
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
ꢃ38.9 (c 4.0, CHCl3); 1H NMR (300 MHz, CDCl3) d 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) d
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
C
27H37ClN7O (M+H)+ 510.2743, found 510.2751.
C
24H31N7O2SCl2 (M+H)+ 516.1943, found 516.1955.
5.1.6. Typical procedure for synthesizing compounds 22
5.1.6.1. 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
5.1.7.2. N-((R)-2-(5-Chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-
4-ylamino)-1-phenylethyl)-2-(4-(2-hydroxyethyl)piperazin-1-yl)
ethanesulfonamide (24b). Following a similar procedure from
11a to 24a, 24b was obtained from 11a. ½a D20
ꢂ
ꢃ46.5 (c 0.8, CHCl3);1H
(72
l
L, 1.25 mmol) under argon. The reaction mixture was stirred
NMR (300 MHz, CDCl3) d 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) d 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.
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 prod-
uct 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).
½
a 2D0
ꢂ
ꢃ11.6 (c 1.95, CHCl3); 1H NMR (300 MHz, CDCl3) d 8.80 (d,
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 condi-
tions) to give the corresponding amide.
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)
d 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.
5.1.6.2. 2-(4-(4-((R)-2-(5-Chloro-6-methyl-2-(pyridin-2-yl)pyr-
imidin-4-ylamino)-1-phenylethylamino)butyl)piperazin-1-yl)
ethanol (22b). Following a similar procedure from 21a to 22a,
5.1.8.1. N-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-2-phenylacetamide
(25a).
½
a 2D0
ꢂ
ꢃ4.2 (c 0.4, CHCl3). 1H NMR (300 MHz, CDCl3) d 8.73
22b was obtained from 21b. ½a D20
ꢂ
ꢃ28.1 (c 0.15, CHCl3); 1H NMR
(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) d
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
(300 MHz, CDCl3) d 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) d 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
C
26H24Cl2N5O (M+H)+ 492.1352, found 492.1344.
C
28H39ClN7O (M+H)+ 524.2899, found 524.2908.
5.1.8.2. N-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-
5.1.7. Typical procedure for synthesizing compounds 24
5.1.7.1. N-((R)-2-(5-Chloro-6-methyl-2-(pyridin-2-yl)pyrimidin-
4-ylamino)-1-phenylethyl)-2-(piperazin-1-yl)ethanesulfona-
pyrimidin-4-ylamino)-1-phenylethyl)-3-phenylpropanamide
(25b). White solid (70% yield): ½a D20
ꢂ
ꢃ3.1 (c 1.5, CHCl3). 1H
NMR (300 MHz, CDCl3) d 8.64 (m, 1H), 8.40 (d, J = 8.1 Hz, 1H),
7.79 (d, J = 8.7 Hz, 1H), 7.62 (br s, 1H), 7.30 (m, 3H), 7.18 (m,
5H), 6.98 (m, 2H), 6.00 (br s, 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,
mide (24a). To
1.75 mmol) and Et3N (0.53 mL, 3.85 mmol) in CH2Cl2 (3 mL)
was added 2-chloroethanesulfonyl chloride (627 mg,
a cooled solution (0 °C) of 11a (594 mg,

2614
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
J = 7.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) d 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
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): d 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.
C
27H26Cl2N5O (M+H)+ 506.1509, found 506.1504.
5.1.8.3. N-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-4-phenylbutanamide
(25c). White solid (76% yield): ½a D20
ꢂ
ꢃ57.1 (c 0.9, CHCl3). 1H
5.1.9.3.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-
NMR (300 MHz, CDCl3) d 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 (br s, 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) d 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.
2-phenyl-2-(3-phenylpropylamino)ethyl)pyrimidin-4-amine
(26c). Foamy solid,
½
a 2D0
ꢂ
ꢃ13.8 (c 2.4, CHCl3). 1H NMR
(300 MHz, CDCl3): d 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): d
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.
5.1.8.4. N-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-5-phenylpentanamide
(25d). White solid (80% yield): 1H NMR (300 MHz, CDCl3) d
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 (br s, 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) d 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.
5.1.9.4.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-
2-phenyl-2-(4-phenylbutylamino)ethyl)pyrimidin-4-amine
(26d). Foamy solid, ½a D20
ꢂ
ꢃ20.8 (c 0.3, CHCl3). 1H NMR (300 MHz,
CDCl3): d 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): d 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.
5.1.8.5. N-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-
pyrimidin-4-ylamino)-1-phenylethyl)-6-phenylhexanamide
(25e). White solid (75% yield): 1H NMR1H NMR (300 MHz,
CDCl3) d 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
(br s, 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) d 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.
5.1.9.5.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-
2-phenyl-2-(5-phenylpentylamino)ethyl)pyrimidin-4-amine
(26e). Foamy solid,
½
a 2D0
ꢂ
ꢃ9.9 (c 1.2, CHCl3). 1H NMR
(300 MHz, CDCl3): d 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): d 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.
5.1.9. General procedure for preparing compounds 26 and 27
To a solution of 11f (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 sat-
urated NaHCO3. The organic phase was dried over Na2SO4 and con-
centrated. Purification by flash chromatography (eluent: 5–10%
EtOH–DCM or similar conditions) gave the corresponding amine.
5.1.9.6.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-
2-phenyl-2-(6-phenylhexylamino)ethyl)pyrimidin-4-amine
(26f). Foamy solid, ½a D20
ꢂ
ꢃ12.3 (c 1.4, CHCl3). 1H NMR (300 MHz,
CDCl3): d 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): d 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.
5.1.9.1. N-((R)-2-(Benzylamino)-2-phenylethyl)-5-chloro-2-(5-
chloropyridin-2-yl)-6-methylpyrimidin-4-amine (26a).
½ ꢂ
a 2D0
ꢃ15.0 (c 2.5, CHCl3). 1H NMR (300 MHz, CDCl3):
d 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): d 161.4, 158.7, 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, 112.8,
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.
5.1.9.7.
4-(4-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)pyrimi-
(27a). 1H
din-4-ylamino)-1-phenylethylamino)butyl)phenol
NMR (300 MHz, CDCl3): d 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
5.1.9.2.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-2-
(phenethylamino)-2-phenylethyl)pyrimidin-4-amine (26b). Foamy
solid, ½a 2D0
ꢂ
ꢃ16.5 (c 1.7, CHCl3). 1H NMR (300 MHz, CDCl3): d 8.72 (d,
C
28H30N5Cl2O (M+H)+ 522.1835, found 522.18219.

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2615
5.1.9.8.
5-Chloro-N-((R)-2-(4-(4-chlorophenyl)butylamino)-
5.1.9.13.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-
2-phenylethyl)-2-(5-chloropyridin-2-yl)pyrimidin-4-amine
(27b). Foamy solid (53% yield): 1H NMR (300 MHz, CDCl3): d
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): d
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.
2-phenyl-2-(4-(quinolin-2-yl)butylamino)ethyl)pyrimidin-4-
amine (27g). Foamy solid (32% yield) ½a D20
ꢃ14.4 (c 1.50,
ꢂ
CHCl3); 1H NMR (300 MHz, CDCl3): d 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): d 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.
5.1.9.9. 4-(4-((R)-2-(5-Chloro-2-(5-chloropyridin-2-yl)pyrimidin-
4-ylamino)-1-phenylethylamino)butyl)benzonitrile
(27c). 1H
5.1.9.14.
N-((R)-2-(2-(1H-Pyrrol-1-yl)ethylamino)-2-phenyl-
NMR (300 MHz, CDCl3): d 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): d 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.
ethyl)-5-chloro-2-(5-chloropyridin-2-yl)-6-methylpyrimidin-4-
amine (27h).
½
a 2D0
ꢂ
ꢃ13.7 (c 1.45, CHCl3); 1H NMR (300 MHz,
CDCl3): d 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): d 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
5.1.9.10.
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): d 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): d 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 C31H36N5OCl2 (M+H)+
564.2307, found 564.2291.
C
26H29N6Cl2 (M+H)+ 495.1826, found 495.1825.
5.1.10. General procedure for preparing compounds 30
Phenylacetonitrile (11 mmol) was added dropwise to a suspen-
sion 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 resul-
tant 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 chromatogra-
phy 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 chromatog-
raphy (eluent: 5–10% EtOH–DCM or similar conditions) gave the
corresponding condensation product.
5.1.9.11. 5-Chloro-2-(5-chloropyridin-2-yl)-N-((R)-2-(4-(furan-
2-yl)butylamino)-2-phenylethyl)-6-methylpyrimidin-4-amine
(27e).
½
a 2D0
ꢂ
ꢃ9.0 (c 1.50, CHCl3); 1H NMR (300 MHz, CDCl3): d
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): d 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 for C26H28N5OCl2 (M+H)+
496.1666, found 496.1665.
5.1.10.1. 5-Chloro-2-(5-chloropyridin-2-yl)-N-(2,4-diphenylbu-
tyl)-6-methylpyrimidin-4-amine (30a). 1H NMR (300 MHz,
CDCl3): d 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): d
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
5.1.9.12.
5-Chloro-2-(5-chloropyridin-2-yl)-6-methyl-N-((R)-
2-phenyl-2-(4-(thiophen-2-yl)butylamino)ethyl)pyrimidin-4-
amine (27f).
½
a 2D0
ꢂ
ꢃ10.9 (c 1.35, CHCl3); 1H NMR (300 MHz,
CDCl3): d 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): d 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.
C
26H25N4Cl2 (M+H)+ 463.1457, found 463.1451.
5.1.10.2. 5-Chloro-2-(5-chloropyridin-2-yl)-N-(2,5-diphenylpen-
tyl)-6-methylpyrimidin-4-amine (30b). Foamy solid : 1H NMR
(300 MHz, CDCl3): d 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): d 161.5, 158.9, 157.7, 153.8, 142.5, 142.1,

2616
P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
136.5, 133.2, 133.1, 128.9, 128.5, 128.4, 127.9, 127.1, 125.9, 124.5,
reaction was initiated by the addition of 600 lM Met-Pro-pNA.
112.9, 45.9, 45.7, 35.9, 33.0, 29.3, 22.4. ESI-MS m/z 477.4 (M+H)+;
Absorbance at 405 nm (
e
= 1.06 ꢁ 104 Mꢃ1 cmꢃ1) was recorded
HRMS (ESI) calcd for
477.1607.
C
27H27N4Cl2 (M+H)+ 477.1618, found
every 30 s within the first 30 min of reaction by the FLUOstar OP-
TIMA microplate reader. After subtracting the background hydroly-
sis 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 min and 15 min 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 pos-
sibility that any inhibitor found by the BcProIP-coupled MetAP as-
say might work simply because it inhibited the coupling enzyme,
compounds showing inhibitory effects were counter screened
5.1.10.3.
5-Chloro-2-(5-chloropyridin-2-yl)-N-(2,6-diph-
enylhexyl)-6-methylpyrimidin-4-amine (30c). Foamy solid :
1H NMR (300 MHz, CDCl3): d 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):
d 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.
against 0.1 U/mL BcProIP, 600
the MetAP assay buffer. Although the initial rates of 0.1 U/mL
BcProIP were greatly decreased by 100 M 18m and 18n, the
lM Pro-pNA and 10 lM CoCl2 in
l
remaining activity of 15 U/mL BcProIP was still enough to saturate
5.1.10.4. 5-Chloro-2-(5-chloropyridin-2-yl)-N-(2,7-diphenylhep-
tyl)-6-methylpyrimidin-4-amine (30d). Foamy solid : 1H NMR
(300 MHz, CDCl3): d 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): d 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.
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 reac-
tions 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 gen-
erated by the nonlinear regression function ‘log(inhibitor) versus
response-variable slope’ in GraphPad Prism 5, using the model
‘Y = Bottom + (Top ꢃ Bottom)/{1 + 10 ꢁ [(LogEC50 ꢃ X) ꢁ Hill-
Slope]}’ and the fitting method ‘least squares’. Top and Bottom are
plateaus in the units of the Y axis. The Top values of the dose–re-
sponse curve were always constrained to 1 (100% of the vehicle
control). The Bottom values of the dose–response curve were con-
strained to zero (a complete inhibition) when the calculated Bot-
tom 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 be-
tween Bottom and Top. When Top = 1 and Bottom = zero, IC50
5.2. Biological assays
5.2.1. Materials
30% acrylamide/bis solution (37.5:1) (161-0158), ammonium
persulfate (161-0700) and N,N,N0,N0-tetramethylethlenediamine
(TEMED) (161-0800) for sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) was purchased from Bio-Rad Lab-
oratories (Hercules, CA). CoCl2ꢄ6H2O (C-3169) was purchased from
Sigma–Aldrich (St. Louis, MO). Other commercially available chem-
icals 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
was used to replace EC50.
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
Author contributions
All authors were involved in designing experiments and inter-
preting data. P.Z. and X.Y. synthesized most of the new compounds
for this study and F.Z. carried out most of the biological experi-
ments. They contributed equally to this work. P.Z., X.Y., R.W., Y.Z.
and S.B. synthesized the new compounds. F.Z. and X.C. character-
ized the inhibitory activities of the compounds with human Me-
tAPs. S.B.G., M.F. and F.Z. performed the crystallographic analysis.
F.Z., S.B.G., J.O.L. and D.M. wrote the manuscript.
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 as-
say was adapted from Ref. 46 with modifications. In this assay, Me-
tAP activity was measured by continuously monitoring the
production of pNA with BcProIP. Aliquots of purified recombinant
human MetAP1 and MetAP2 proteins stored at ꢃ80 °C were
thawed on ice. Repeated freeze–thaw cycles were avoided. The
coupling enzyme BcProIP was taken from the stock at ꢃ20 °C.
The substrate methionylprolyl-p-nitroanilide (Met-Pro-pNA) and
the test compounds were dissolved in 100% DMSO. The total reac-
Notes
The authors report no conflicts of interest.
Acknowledgements
We thank Drs. Daisuke Tsuru and Ana Kitzono for providing us
with pPI-20 plasmid containing BcProIP gene. This work was sup-
ported in part by a grant under National Institutes of Health
(NIH) R01 CA078743. Molecular graphics and analyses were per-
formed in part with the UCSF Chimera package. Chimera is devel-
tion volume was 100
well plates at room temperature. 0.15 microM HsMetAP1 or
0.1 M HsMetAP2 was incubated with either vehicle (DMSO) or
test compounds in MetAP assay buffer containing 15 U/mL BcProIP
and 10 M CoCl2 at room temperature for 20 min. The enzymatic
lL and the reaction was carried out in 96-
l
l

P. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 2600–2617
2617
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