Synthesis and Biological Evaluation of B-Cell Lymphoma 6 Inhibitors of N-Phenyl-4-pyrimidinamine Derivatives Bearing Potent Activities against Tumor Growth

Article abstract of DOI:10.1021/acs.jmedchem.9b01618

The transcriptional repressor B-cell lymphoma 6 (BCL6) is frequently misregulated in diffuse large B-cell lymphoma (DLBCL) and has emerged as an attractive drug target for the treatments of lymphoma. In this article, a series of N-phenyl-4-pyrimidinamine derivatives were designed and synthesized as potent BCL6 inhibitors by optimizing hit compound N⁴-(3-chloro-4-methoxyphenyl)-N²-isobutyl-5-fluoro-2,4-pyrimidinediamine on the basis of the structure-activity relationship. Among them, compound 14j displayed the most potent activities, which significantly blocked the interaction of BCL6 with its corepressors, reactivated BCL6 target genes in a dose-dependent manner, and had better effects compared with the two positive controls. Further studies indicated that a low dose of 14j could effectively inhibit germinal center formation. More importantly, 14j not only showed potent inhibition of DLBCL cell proliferation in vitro but also strongly suppressed the growth of DLBCL in vivo.

Full text of DOI:10.1021/acs.jmedchem.9b01618

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Article
Synthesis and Biological Evaluation of B-Cell Lymphoma
6
Inhibitors of N-phenyl-4-pyrimidinamine Derivatives
Bearing Potent Activities against Tumor Growth.
Weikai Guo, Yajing Xing, qiansen zhang, Jiuqing Xie, Dongxia Huang, haijun Gu, peng
he, Miaoran Zhou, Shifen Xu, Xiufeng Pang, Mingyao Liu, Zhengfang Yi, and Yihua Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01618 • Publication Date (Web): 02 Jan 2020
Downloaded from pubs.acs.org on January 2, 2020
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Synthesis and Biological Evaluation of B‑Cell
Lymphoma 6 Inhibitors of N-Phenyl-4-
pyrimidinamine Derivatives Bearing Potent
Activities against Tumor Growth
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Weikai Guo, †,# Yajing Xing, †,# Qiansen Zhang, Jiuqing Xie, Dongxia Huang, Haijun
†
†
†
†
†
‡
‡
†
†, *
Gu, Peng He, Miaoran Zhou, Shifen Xu, Xiufeng Pang, Mingyao Liu, Zhengfang
Yi,†, * Yihua Chen†,*
†East China Normal University and Shanghai Fengxian District Central Hospital Joint
Center for Translational Medicine, Shanghai Key Laboratory of Regulatory Biology,
Institute of Biomedical Sciences and School of Life Sciences, East China Normal
University, 200241 Shanghai, China.
‡Shanghai University of Traditional Chinese Medicine, Shanghai, 200030, China
ABSTRACT
The transcriptional repressor B-cell lymphoma 6 (BCL6) is frequently misregulated in
diffuse large B-cell lymphoma (DLBCL) and has emerged as an attractive drug target
for the treatments of lymphoma. In this article, a series of N-phenyl-4-pyrimidinamine
derivatives were designed and synthesized as potent BCL6 inhibitors by optimizing hit
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compound
N -(3-chloro-4-methoxyphenyl)-N -isobutyl-5-fluoro-2,4-
pyrimidinediamine on the basis of the structure-activity relationship. Among them,
compound 14j displayed the most potent activities, which significantly blocked the
interaction of BCL6 with its corepressors, reactivated BCL6 target genes in a dose-
dependent manner and had better effects compared with the two positive controls.
Further studies indicated that a low dose of 14j could effectively inhibit germinal center
formation. More importantly, 14j not only showed potent inhibition of DLBCL cell
proliferation in vitro but also strongly suppressed the growth of DLBCL in vivo.
INTRODUCTION
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B-cell lymphoma 6 (BCL6), as a transcriptional repressor and a member of the
BTB/POZ/zinc finger family, was first identified as an oncogene in diffuse large B-cell
lymphoma (DLBCL),1-3 which is necessary for germinal center (GC) formation and T-
cell dependent adaptive immune responses.4, 5 BCL6 mediates a series of key cellular
functions by repressing direct target genes mainly involved in the control of the cell
6
-8
cycle, gene transcription and DNA damage responses, among others. Several lines of
evidence suggest that DLBCL cells can be killed by delivering BCL6 shRNA, peptide
inhibitors or small molecular inhibitors.9-11 Most of the previous research results
indicated that BCL6 was critical for the survival of DLBCL cells and could be a
promising potential drug target for DLBCL therapy. In addition, BCL6 overexpression
is found in many other tumors, which can induce lethality by targeting BCL6, such as
breast cancer,12-14 glioblastoma,15, 16 ovarian cancer,14, 17 and non–small cell lung cancer
1
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(
NSCLC). More importantly, BCL6 is also associated with drug resistance of tyrosine
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kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cells and Ph-positive (Ph+)
acute lymphoblastic leukemia (ALL) cells.19-21 For example, BCL6 is upregulated after
+
treatments of TKIs in CML cells or Ph ALL cells, which is required for a basic level
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of imatinib-resistance and CML stem cell survival as well as self-renewal in CML and
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Ph ALL. Therefore, targeting BCL6 may also be a promising strategy to overcome
+
resistance for TKI-treated CML and Ph ALL. In addition, Dupont, T et al. have
indicated that combination of BCL2 with BCL6 inhibitors may overcome the intrinsic
resistance of BH3 mimetic inhibitors.22
The human proto-oncogene BCL6 encodes a 95-kDa protein,4, 23, 24 which contains
three regions: the N-terminal broad-complex/tramtrack/bric-a-brac (BTB) domain, a
central unstructured region, and a set of six C-terminal zinc finger domains. The
dimerization of the BCL6 BTB domain is necessary for its autonomous repressor
activities, which can interact with three known corepressors, namely thyroid hormone
receptors (SMRT),4, 25-27 nuclear receptor corepressor (N-CoR) and BCL6 corepressor
(
B-CoR) in a mutually exclusive manner.4, 25, 28 All three corepressors can bind to a
commonly exposed surface at the interface of the two chains of the dimer, the lateral
2
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groove (LG). Blocking the interaction of BCL6 with corepressors by mutation of the
BCL6 BTB domain or BCL6 peptide inhibitors can inhibit GC formation and reactivate
its target genes without any inflammation or other toxic effects.8, 11, 27, 29, 30 The results
indicate that the BCL6 BTB domain can be an excellent binding site for developing
novel BCL6 inhibitors.
To date, several small molecular BCL6 inhibitors have been reported (Figure 1). For
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example, compound 1 (79-6) was reported as the first small molecular BCL6 inhibitor
that could bind to a pocket in the LG of the BCL6BTB domain with K of 138 M.
31
D
Compound 2 (FX1), with a K of 7 M, was obtained via subsequent structural
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modification of compound 1 by the same research group. Both of these proteins could
disrupt BCL6 transcriptional complexes and reactivate BCL6 target genes. Moreover,
compound 2 could disrupt GC formation, inhibit the proliferation of DLBCL cells in
3
2
vitro and suppress the growth of DLBCL in vivo at a dose of 25 mg/kg/day. However,
compounds 1 and 2 with the same skeleton of rhodanine may be a detrimental, and they
potentially have limitations for further drug development.33, 34 Compound 3 could bind
to the LG of the BCL6BTB homodimer and disrupt the interaction between BCL6 and
the B-CoR peptide in a micromolar range. Compound 4 could also bind to BCL6BTB–
3
5
3
6
LG in a nanomolar range. Unfortunately, there are on reports further studies examing
whether they could reactivate BCL6 target genes or inhibit GC formation, so it is not
known whether these compounds could actually disrupt BCL6-mediated formation of
repression complexes and translate into the de-repression of target genes. Compound 5
could bind to BCL6BTB–LG and occupy the HDCH site with a K value of 44 M, but
D
it has not shown any antitumor growth effect, possibly due to its insufficient
33
BTB
permeability. Compound 6 (BI-3802) was reported to bind to BCL6
– LG and
induce degradation of BCL6 in a nanomolar range as well as reactivate BCL6 target
genes. Nevertheless, the functions of compound 6 have not been fully validated in vivo
3
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due to its poor bioavailability. Therefore, the design and search for novel BCL6
inhibitors with better bioactivities both in vitro and in vivo remain a tremendous
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challenge in this research field.
Based on our previous research work on diaminopyrimidine derivatives as anticancer
agents,38 we continuously modified this series of compounds and explored their
anticancer potencies by studying whether they can interact with BCL6 given that most
of the reported BCL6 inhibitors shared similar diaminopyrimidine scaffold. To discover
more novel BCL6 inhibitors with high affinities, a preliminary screening program was
initially carried out in our laboratory via the homogeneous time-resolved fluorescence
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(HTRF) assay. In brief, approximately 230 compounds containing diaminopyrimidine
scaffold and other skeletons in in-house libraries were selected for screening at a
concentration of 100 M. Subsequently, 32 compounds with obvious inhibitory
activities were screened at 50 M. Finally, the active compounds were screened again
4
2
at 25 M. As a result, compound 7 (N -(3-chloro-4-methoxyphenyl)-N -isobutyl-5-
fluoro-2,4-pyrimidinediamine) (Figure 2A) was identified, which could directly block
BCL6 and SMRT interactions with an IC value of 19.4 ± 3.4 M. In the present study,
5
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a series of novel N-phenyl-4-pyrimidinamine derivatives, with different amino-
substitutions on hit 7, were identified as potent BCL6 inhibitors through the elucidation
of the structure−activity relationship. Among the investigated compounds, compound
1
4j showed the best activities in vitro and in vivo, which was better than positive
controls 2 and 3. Compound 14j significantly inhibited BCL6 and its corepressor
interaction at low concentrations via direct binding to BCL6BTB, and biological research
subsequently showed that 14j could also effectively reactivate BCL6 target genes in a
dose-dependent manner. More importantly, compound 14j significantly inhibited GC
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formation and impaired immunoglobulin affinity maturation in mice at a low dose, in
addition, it strongly suppressed the growth of DLBCL in vivo.
CHEMISTRY
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Compounds 9a-n were prepared in accordance with the procedures described in
Scheme 1. 3-Chloro-4-methoxyaniline was coupled with 2,4-dichloro-5-fluoro-
pyrimidine to afford key intermediate 8. Then, 8 was substituted with various amines
to obtain target compounds 9a-n. Compounds 13a-f were obtained via the following
procedures listed in Scheme 2. The key intermediate 11a was synthesized through
similar procedures with compounds 9a-n, and the only exception was to replace 3-
chloro-4-methoxyaniline with 4-methoxy-3-nitroaniline. Compound 12a was gained by
the reduction of compound 11a. Then, compounds 13a-f were obtained by the coupling
reaction of intermediate 12a with various acids in the presence of EDC/HOBt or other
coupling reagents.
Scheme 1α
O
F
O
F
O
F
N
N
b
N
a
+
R2
Cl
N
H
9a-n
N
N
R1
Cl
N
H
N
Cl
Cl
NH2
Cl
N
Cl
8
α
1
2
Reagents and conditions: (a) DIEA, EtOH, 40 ℃; (b) R R NH, DIEA, n-BuOH, reflux.
Scheme 2α
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R2
R1
R2
R1
R2
R1
F
F
F
a or b
N
c
N
N
+
NH2 Cl
N
Cl
N
H
10a-b
N
Cl
N
H
N
N
NH
1
1a-b
0a R1 = -NO , R = -OCH
10b R = -OCH , R = -NO
2
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O
F
d
11a R =-NO , R = -OCH
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O
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1b R = -OCH , R = -NO2
3
R3
e or f or g
N
H
N
H
N
N
NH
NH
R2
R1
F
1
3a-f
N
N
H
N
N
R4
O
O
F
NH
N
12a-b
O
N
N
N
1
2
H
12a R =-NH2, R = -OCH3
e or f or g
1
2
12b R = -OCH , R = -NH
3 2
14a-e
α
Reagents and conditions: (a) DIEA, EtOH, 40 ℃; (b) Cs CO , DMF, r.t; (c) (2S,6R)-
2
3
.
2,6-dimethylpiperazine, DIEA, n-BuOH, reflux; (d) Pd/C, H , MeOH; (e) EDC HCl,
2
HOBt, DMF, 0 ℃ then to r.t; (f) HATU, DMF, 0 ℃ then to r.t; (g) CDI, DMF, 0 ℃ then
to r.t.
Compounds 14a-e were prepared via a similar procedure as compounds 13a-f (As
shown in scheme 2), except for changing 4-methoxy-3-nitroaniline to 3-methoxy-4-
nitroaniline. Compounds 14f-l were synthesized according to the procedures described
in Scheme 3 by Suzuki coupling reactions of methyl 5-bromothiophene-2-carboxylate
with different boronic acids in the presence of Pd(PPh ) under the corresponding
3
4
conditions, which were followed by acylation of the thiophene-2-carboxylic acid
derivatives with compound 12b to yield compounds 14f-l.
Scheme 3α
R5
S
HN
O
F
N
OH
OH
b or c
a
O
R5
+
B
O
Br
R5 or d
S
HOOC
N
H
N
N
S
O
NH
14f-l
α
.
Reagents and conditions:(a) Pd(PPh ) , Na CO , EtOH, DME, 95 ℃; (b) EDC HCl,
3
4
2
3
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HOBt, DMF, 0 ℃ then to r.t; (c) HATU, DMF, 0 ℃ then to r.t; (d) CDI, DMF, 0 ℃
then to r.t.
RESULTS AND DISCUSSION
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Structure activity relationship (SAR) analysis. The activities of target compounds
to block the interaction of the co-repressor SMRT with the BTB domain of BCL6 were
evaluated using the well-established HTRF assay. Compounds 2 and 3 were selected as
the positive controls, and their IC values with BCL6 binding affinities were 37.7 ±
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0
35
3
.6 and 0.45 ± 0.1 M, respectively, which were consistent with reported data. In our
preliminary data, compound 7 showed to mild inhibition with an IC of 19.4 ± 3.4 M
5
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in the HTRF assay. Compound 7 shared a similar key scaffold to several reported BCL6
inhibitors, as shown in Figure 1, and it was selected as a hit and first modified with
different amino-substitutions at the pyrimidine 2-position to increase the binding
affinities. Compounds 9a−d were first synthesized and then evaluated their IC values
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by HTRF assay (Table 1). The BCL6 inhibitory activities of ethylamine analogue 9a
and isopropylamine analogue 9b were increased compared to that of 7 with IC values
5
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of 4.96 ± 0.8 and 4.83 ± 0.4 M, respectively. However, substitutions with butylamine
c or diisobutylamine 9d did not result in notable inhibitory effects (Table 1). To further
9
increase the binding affinities of the target compounds, different tails of amino-alcohols
and heterocyclic groups were introduced to the pyrimidinyl-2-position of compound 7,
and compounds 9e-n were designed and synthesized (Table 1). Interestingly, the
inhibitory activities of these compounds were greatly increased except for compounds
9
i and 9n. Amino alcohol residues were superior to the corresponding substitutions of
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aliphatic chain amines (9e-g vs 9a-d). Furthermore, the substitution with
propanolamine 9f seemed to be more beneficial for inhibitory activity than
ethanolamine 9e or butanolamine 9g. Substitutions with piperazine analogs (9h, 9j, 9k
and 9m) or morpholine analogue 9l demonstrated significantly increased BCL6
inhibitory effects compared to hit 7. Piperidine analogs (9i and 9n) did not significantly
inhibit the BCL6-SMRT interaction (Table 1). These results indicated that the existence
of heteroatoms (such as NH or O) at the end of the substituents was preferred and
critical for maintaining or increasing the inhibitory activities of the target compounds
(9h vs 9i, 9m vs 9n, 9e vs 9a and 9g vs 9c). Among them, compound 9h, with (2S,6R)-
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,6-dimethylpiperazinyl substitution, showed the best inhibitory activity, with an IC50
value of 0.77 ± 0.05 M, which displayed comparable activity to positive control 3 and
an activity level almost 45-fold higher than that of positive control 2 (Table 1).
Next, the substitutions in the left part of the phenyl ring of compound 9h were
investigated, and different substituents were introduced to the meta (m-) and para (p-)
positions of the phenyl ring. First, the m-chloro atom of the phenyl ring was replaced
with acyl residues, as shown in Figure 2B. Compounds 13a-f were designed and
synthesized (Table 2), and their IC50 values were tested via the HTRF assay.
Unfortunately, all of these compounds greatly reduced or almost completely abolished
BCL6 affinities compared with the effects of 9h. The BCL6 inhibitory activities of
bromophenyl analog 13c and pyridyl analogs 13d-f conferred weaker activities,
whereas bromothienyl analogue 13a and bromofuranyl analogue 13b led to great loss
in activities (IC > 20 M). These results suggested that the introduction of acyl
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residues to the m-position of the phenyl ring of 9h were not conducive to improving
BCL6 inhibitory activities.
Instead, our attention was next focused on the substitutions of the p-position of the
left part of the phenyl ring of compound 9h by changing its methoxy group from the p-
position to the m-position (Figure 2B). To account for the efficacies of the target
compounds containing acyl residues at this position interacting with BCL6BTB, several
docking simulations were conducted on the newly designed compounds to evaluate
their potential interactions with the BCL6BTB domain (Figure 3). The high-resolution
X-ray crystal structure of the BCL6BTB domain with the pyrazolo-pyrimidine
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macrocyclic ligand (PDB ID: 5N1Z) was used for docking simulations. As shown in
Figure 3A-C, the docking score of 14a, which only exchanged the position of methoxy
and acyl residues of compounds 13a or 13c between the p- and m-positions, was higher
than that of 13a and 13c. To validate the prediction, compounds 14a-e were synthesized
by replacing the m-chlorine atom of the phenyl ring with m-methoxy, and introducing
acyl residues at the corresponding position (Table 3). The BCL6 inhibitory activities of
most of these compounds dramatically increased compared to the corresponding 13-
series derivatives (such as 14a vs 13c and 14c vs 13f), except for compounds 14b and
1
4d (Table 3). The results demonstrated that the introduction of acyl residues to the p-
position of the phenyl ring was beneficial to improve the activities. Further
modifications of substitutions at this position were explored and compounds 14f-l were
synthesized (Table 3). After converting the bromo atom of 5-bromothienyl of
compound 14d to phenyl (14f-i), cyclopentenyl (14j), cyclohexenyl (14k) or furanyl
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(14l) via palladium-catalyzed coupling reactions, their potencies were increased to
almost 10 to 20-fold compared to that of 14d (Table 3). These results indicated that
several cycloalkenyl-heteroaromatic or aryl-heteroaromatic groups were more suitable
for enhancing inhibitory activities among the investigated compounds. Among these
compounds, compound 14j has the most potent activity with an IC of 0.47 ± 0.08 M,
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with comparable activity to positive control 3 and was almost 80-fold more potent than
the positive control 2 (Table 3).
To further elucidate how these potent compounds interacted with BCL6BTB
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compounds 14f, 14j and 14l, which showed the highest activities among the tested
compounds in the HTRF assay except for the above-mentioned compounds (13a, 13c
and 14a), were also selected to conduct docking simulations. Before the docking
simulations were carried out, quantum chemical calculations were performed on these
new compounds in an attempt to provide molecular and electronic- based explanations
for inhibitor binding (Figure S1). Quantum chemical calculations showed that the N
atom on the piperazine ring has a negative charge and the H atom on N atom has a
positive charge.
The docking results of 14a, 14f, 14j and 14l suggested that these compounds could
bind to the BCL6BTB domain, and an analysis of the docking configurations reveals
that these newly synthesized compounds assume similar binding models (Figure 3C-
F). In the docking simulations, the N-H atom with a positive charge over the piperazine
ring is bound to the non-protonated N atom in the residue His14 imidazole ring, which
partly explains that the existence of a heteroatom in the terminal of the tail is preferred
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in Table 1. The two nitrogen atoms from the pyrimidine ring interact with the side chain
of N21 and H116 (Figure 3C-F). R24 is a relevant amino acid for the binding of
compounds 14a, 14f, 14j and 14l, as its side chain interacts with the carbonyl oxygen
of the acyl residues at the p-position of the phenyl ring on the left part of compounds
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(Figure 3C-F). Moreover, the annular segment located at the end group of compounds
14a, 14f, 14j and 14l can be stabilized by hydrophobic pockets formed by L25, M51
and Y58, among others (Figure 3C-F). Compared with these compounds, compounds
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3a and 13c, containing acyl residues at the m-position of the phenyl ring, neither
interact with R24 nor exert hydrophobic effects at the end group with the BCL6 BTB
domain (Figure 3A-B). These docking results may partly explain why the activities of
acyl residues introduced at the p-position of the phenyl ring on the left part of
compounds are better than those of the corresponding analogs introduced at the m-
position.
Compound 14j induces de-repression of BCL6 target genes in BCL6 dependent
DLBCL cells. BCL6 mediates its biological effects by repressing its target genes,
which are enriched in the modulators of the cell cycle, gene transcription, DNA damage
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responses and terminal plasma cell differentiation. When knockdown of BCL6 is
accomplished by delivering shRNA in two DLBCL cell lines (SUDHL4 and Farage),
significant de-repression of BCL6 target genes (p53, ATR and CDKN1A) was observed
compared with the control (Figure S2A-B). According to our above results with highly
potent inhibition of BCL6, compounds 9h, 14a, 14j and 14l were selected to determine
their abilities reactivate BCL6 target genes by real-time quantitative PCR assay in
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SUDHL4 (a BCL6 dependent DLBCL cell line), and mRNA was collected after 24 h.
The results showed that these compounds (5 M) could significantly induce de-
repression of BCL6 key target genes (ATR, CD69 and CXCR4) compared with the
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vehicle, especially for 14j (Figure S3). Since compound 14j was the most active
compound of reactivation of BCL6 target genes as well as possessed the best binding
affinity of BCL6 among the investigated genes, two BCL6 dependent DLBCL cell lines
(SUDHL4 and Farage) were further exposed to 14j (5 M), positive controls 2 (50 M,
32
as previously described ) and 3 (5 M). The results showed that compound 14j could
significantly induce de-repression of BCL6 target genes (p53, ATR, CXCR4, CD69
and CDKN1A) compared with the vehicle,39 which induced greater target gene
reactivation compared with positive control 3 at the same concentration (5 M) and
positive control 2 even at a 10-fold concentration (50 M) (Figure 4A). To further
explore the selectivity of 14j, two BCL6-dependent DLBCL cell lines (SUDHL4 and
Farage) and a BCL6-independent DLBCL cell line (TOLEDO) were chosen to expose
1
4j at concentrations of 1.25 M, 2.50 M and 5.00 M, and mRNA was collected
after 24 h. Compound 14j resulted in an increase in the mRNA level of BCL6 target
genes (p53, ATR, CDKN1A, CXCR4, CD69 and CD80) in a dose-dependent manner
in the two BCL6-dependent DLBCL cell lines (Figure 4B). In sharp contrast to these
phenomena, 14j had little effect on any of these genes in the BCL6-independent
DLBCL cell line (TOLEDO, Figure 4B). In general, these data indicated that 14j could
significantly and selectively reactivate BCL6 target genes in BCL6-dependent cancer
cell lines in a dose-dependent manner.
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Compound 14j binds the BCL6 BTB domain with potent affinity. Encouraged
by these results, biolayer interferometry (BLI)40, 41 was further chosen to study the
kinetics of the BCL6 BTB domain binding affinity of compound 14j on a ForteBIO
Octet (Menlo Park, CA). Compounds 2 and 3 were also selected as the positive controls.
As shown in Figure 5, compound 14j could directly bind to the BCL6 BTB domain
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with a dissociation constant K value of 366 nM (Figure 5A), with a more than 20-fold
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greater affinity than positive control 2 (K of 7.88 M), and a 3-fold greater affinity
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than positive control 3 (K of 1.19 M) (Figure 5B-C).
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Compound 14j significantly inhibits DLBCL growth in vitro. BCL6 favors the
survival and proliferation of DLBCL. The loss of BCL6 function, which is mediated by
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delivering shRNA or BCL6 inhibitors, can kill DLBCL cells. To verify the phenotype,
DLBCL cells (SUDHL4 and Farage) were transfected with lentivirus expressing BCL6
shRNA and a nontargeted control shRNA. The results showed that BCL6 shRNA led
to significantly reduced viabilities of DLBCL cells compared with the control (Figure
S2A and S2C). Therefore, compound 14j was further studied in a panel of four GCB-
DLBCL cell lines (SUDHL-4, Farage, DOHH2 and OCI-LY7) and an ABC-DLBCL
cell line (TOLEDO) to measure its antiproliferative effects via CCK8 assay for 72 h.
The results are summarized in Table 4, and compounds 2 and 3 were selected as the
positive controls. The results showed that compound 14j displayed significant
antiproliferative efficacies with average IC values of approximately 1 M on four
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GCB-DLBCL cell lines, which was about 15 to 55-fold more potent than positive
controls 2 and 3 (Table 4). TOLEDO was slightly less sensitive than the GCB-DLBCL
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cell lines for compound 14j, with an IC value of 3.25 M, and TOLEDO cells
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responded to similar doses of positive controls 2 and 3, which was consistent with
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previously reported results (Table 4), and the knockdown of BCL6 by delivering
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shRNA was just as detrimental to ABC-DLBCL cells as to GCB-DLBCL cells.32
To further illustrate the relevance between the biological effects of 14j and the
function of BCL6, more experiments through knockdown or overexpression of BCL6
in different DLBCL cells were carried out to validate whether the inhibitory effects of
1
4j on DLBCL cell growth were dependent or not by inhibiting the functions of BCL6.
Farage cells with lentivirus expressing BCL6 shRNA were treated with 14j. The results
indicated that 14j could effectively inhibit control vector cells in the investigated range
of 0.5-2.0 µM in dose-dependent manner, while it showed little effect on BCL6
knockdown cells in the same concentration range (Figure S4A). However, positive
controls 2 and 3 still showed similar antiproliferative effects against these DLBCL cells
with or without knockdown of BCL6 (Figure S4A). In addition, we also transduced
lentivirus overexpression of BCL6 in TOLEDO cells, showing an almost complete loss
of BCL6 expression. The results demonstrated that 14j had little effect against control
vector cells in the range of 0.25-2.0 µM but showed significant antiproliferation against
the DLBCL cell with overexpression of BCL6 in a dose-dependent manner at the same
concentrations (Figure S4B). Compared to the effects observed with 14j, positive
control 2 showed weaker inhibition against this DLBCL cell with overexpression of
BCL6, and 3 showed similar antiproliferative effects against these two DLBCL cells
with or without overexpression of BCL6 in a range of 0-20 µM (Figure S4B),
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supporting the notion that 14j inhibits DLBCL cell growth by targeting BCL6. To
subsequently evaluate the selectivity of 14j against tumor cells, the cytotoxicities of
compound 14j were tested on four human normal cell lines (LO2, HAF, NCM460 and
PNT1A), and the IC values of 14j for DLBCL cells were approximately 20-fold lower
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than that of normal cells (Table 4), which demonstrated that 14j results in specific levels
of lethality on DLBCL cells.
One of the most accurate methods to measure cell proliferation is by directly
measuring DNA synthesis. The EdU Flow Cytometry assay was conducted, which
detects cell growth especially the cell population in the S phase. Treatment with
compound 14j for 48 h caused a sharp reduction in living cells in SUDHL4 cells in a
dose-dependent manner, and fewer living cells and S phase cells were detected than in
the counterparts of compounds 2 and 3 (Figure 6A). Previous studies showed that
inhibition of BCL6 and its corepressor interaction by RI-BPI (a known BCL6 peptide
inhibitor) or compound 2 led to DLBCL apoptosis.11, 32 Compound 14j was also
selected to determine its effect on DLBCL apoptosis using flow cytometry. The results
indicated that 14j caused a dose-dependent induction of apoptosis at low doses of 0.625,
1
.25 and 2.50 M, while positive controls 2 and 3 did not induce apoptosis at the same
concentrations (Figure 6B). Collectively, these results demonstrated that 14j
significantly inhibited DLBCL growth and induced apoptosis in vitro.
Compound 14j inhibits germinal center formation in vivo. The germinal center is
a specialized structure that develops after T-cell-dependent antigen stimulation, where
activated B cells proliferate, undergo somatic hyper-mutation, affinity maturation and
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class-switch recombination, ultimately generating memory B cells and plasma cells that
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produce high-affinity antibodies. The corepressors are unable to be recruited when
there are mutations in the BCL6 BTB domain, which leads to the failure of GC
formation and immunoglobulin affinity maturation in mice.30 To identify whether
compound 14j could recapitulate this phenotype, we immunized C57/BL6 mice with 4-
hydroxy-3-nitrophenylacetyl (NP)-coupled chicken gamma globulin (CGG), a T cell-
dependent antigen. Two days later, compound 14j with a 10 mg/kg/3d dose or vehicle
was treated by tailꢀintravenousꢀinjection (i.v.). A previous article reported that GC B
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cells were significantly depleted by exposure to compound 2. To verify the feasibility
of the animal experiments, since compound 2 was not available with i.v. due to the
limitation of its poor solubility, which was then given with a 50 mg/kg dose via
intraperitonealꢀinjection (i.p.) daily. Mice were euthanized and spleens were collected
after 12 days of treatment with the compounds (when GCs are normally at their peak).
+
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As expected, GC B cells (GL7 FAS B220 ) were significantly decreased by exposure
to 14j treatment group, which is similar to the BCL6 BTB domain mutant phenotype
(Figure 7A). Compared with the control group, whose frequency of GC B cells was
1
.97%, the group treated with 14j exhibited better inhibitory activities against GC
formation with a frequency of 0.45% (Figure 7A), and the compound 2 group exhibited
a frequency of 0.73% (Figure S5A).
Splenic architecture was also examined by immunofluorescent staining. Staining
with IgD antibody showed normal B cell follicular structures, and staining with peanut
agglutinin (PNA) revealed GCs. Consistent with the results of the flow cytometry
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analysis, both the number and size of GCs were decreased dramatically in the spleens
of mice treated with compound 14j compared with control mice (Figure 7B).
Compound 2 also has an effect in impairing GC B cell development and GC formation
(Figure S5B).
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The development of Tfh cells in the germinal center was examined after treatment
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and maturation.44 The results showed that the proportion of Tfh cells
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CXCR5 PD1 CD4 ) was significantly depleted by exposure to the 14j group (Figure
7C). Consistent with the better inhibitory activity of 14j against the GCs, the proportion
of Tfh cells was much lower in the 14j group (0.99%) than in the control group (2.25%)
(Figure 7C).
Recruitment of corepressors to the BTB domain was indispensable for BCL6 to drive
30
the GC reaction to generate high-affinity immunoglobulins. We determined whether
compound 14j could impair immunoglobulin affinity maturation by enzyme-linked
immunosorbent assay (ELISA) (Figure 7D). As expected, compared to the control
counterparts, mice treated with 14j had much lower titers of high-affinity
immunoglobulin G1 that was able to bind NP5-BSA (NP conjugated to bovine serum
albumin (BSA) at a molecular ratio of 5:1), which recognizes high-affinity
immunoglobulin (Figure 7D). This effect was also observed in the production of total
NP-specific immunoglobulin G1 specific to NP23-BSA (NP conjugated to BSA at a
molecular ratio of 23:1) (Figure 7D). Collectively, these results demonstrated that 14j
inhibited germinal center formation and impaired immunoglobulin affinity maturation.
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Compound 14j potently suppresses BCL6 dependent DLBCLs in vivo. To further
evaluate the anticancer efficacy of compound 14j in vivo, we established an SCID
DLBCL xenograft model using SUDHL4 DLBCL cells. When tumors reached a
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volume of approximately 100 mm , mice were randomly assigned to four groups (n =
7
per group) according to the dosing regimen: the control group (20% of (2-
hydroxypropyl)-β-cyclodextrin by i.v.) and the 14j treated groups (5 or 10 mg/kg/3d by
i.v.). We also administered 25 mg/kg/d of compound 2 via i.p. as described to verify
32
the feasibility of the animal experiments (Figure S6). Animals were sacrificed when
the control group reached the maximal permitted tumor burden. To our delight,
compound 14j significantly suppressed the tumor weight and volume even at the lower
dosage (5 mg/kg/3d) compared with the control (Figure 8A-C). In addition, little
difference was observed in body weight between 14j-treated groups and control group
(Figure 8D). Moreover, major organs including hearts, livers, spleens, lungs and
kidneys were analyzed by H&E staining. No obvious damage to these organs was
shown by H&E staining (Figure S7), which suggested that 14j did not cause apparent
adverse events with the treated doses. Ki67 staining was also explored in the tumors
(Figure 8E). In comparison with the control, the expression of Ki67 was decreased
prominently in the mice treated with 14j (Figure 8E), which showed that 14j
significantly induced DLBCL cell growth arrest in vivo. To detect whether compound
1
4j could de-repress BCL6 target genes in vivo, mRNA extracted from tumor tissue
was evaluated for the abundance of BCL6 key target genes CD69, CXCR4 and ATR
by real-time quantitative PCR (Figure 8F). Remarkably, 14j significantly induced the
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up-regulation of BCL6 target genes in DLBCL xenografts (Figure 8F). Taken together,
these data underlined that 14j potently suppressed BCL6 dependent DLBCLs in vivo.
Preliminary in vitro ADME study. Encouraged by these excellent biological results,
compound 14j was selected for a preliminary in vitro ADME assessment. As shown in
Table 5, in vitro metabolism studies with human liver microsomes, revealed that
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compound 14j exhibited low microsomal CL (Clint(mic) < 9.6 μL/min/mg and Clint(liver)
int
<
38.0 mL/min/kg) and high metabolic stabilities (T1/2 > 60 min). The plasma protein
binding (PPB) of humans was also measured. Excitingly, compound 14j was also
indicated modest plasma protein binding (7.6% free fraction).
CONCLUSION
BCL6 is necessary for GC formation and lymphomagenesis and has emerged as a
critical therapeutic target in DLBCL. BCL6 mediates tumorigenesis via silencing its
target genes. With knockdown of BCL6 by delivering shRNA, the BCL6 target genes
were significantly reactivated and the DLBCL proliferation was obviously inhibited.
Previous research results elucidated that BCL6 peptidomimetic inhibitor (RI-BPI) and
small molecule inhibitors (79-6 and FX1) can inhibit BCL6 and its corepressor
interaction, resulting in de-repression of BCL6 target genes. More importantly, these
BCL6 inhibitors can kill DLBCL cell lines as well as primary human DLBCL cells. All
of these results demonstrated that BCL6 is important for the survival of DLBCL cells
and that the antiproliferation effects of BCL6 inhibitors are largely due to the release
from BCL6-mediated transcriptional repression. Therefore, one of the key factors
inhibiting DLBCL proliferation by BCL6 inhibitors is to disrupt BCL6-mediated
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formation of repression complexes and translate into de-repression of target genes.
In this study, a novel series of N-phenyl-4-pyrimidinamine derivatives were designed
4
and optimized as potent BCL6 inhibitors based on the hit compound N -(3-chloro-4-
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2
methoxyphenyl)-N -isobutyl-5-fluoro-2,4-pyrimidinediamine 7, which was identified
to have mild BCL6 inhibition in the initial screening of a small-molecule library.
Compound 14j, as one of the most potent BCL6 inhibitors among the investigated
compounds, could directly bind to the BCL6 BTB domain and significantly disrupt the
BCL6-SMRT interaction. A real-time quantitative PCR assay showed that 14j could
specifically induce the de-repression of BCL6 target genes only in BCL6-dependent
DLBCL cells with a dose-dependent manner. Further studies demonstrated that 14j
could significantly inhibit germinal center formation in vivo. Flow cytometry analysis
and ELISA assay indicated that 14j greatly reduced the proportion of Tfh cells and
impaired immunoglobulin affinity maturation. More importantly, 14j also strongly
suppressed DLBCL growth in vitro and in vivo. In addition, we performed docking
studies to simulate the binding mode of 14j and BCL6BTB. However, the exact binding
mode of compound 14j and BCL6BTB is unclear. Currently, we are trying to construct
the mutant of BCL6BTB to identify the specific binding sites of compound 14j to
BCL6BTB, these experiments are in progress.
Taken together, our results indicated that compound 14j inhibited the proliferation
of DLBCL in vitro and in vivo by directly binding to the BCL6BTB to block the
interaction between BCL6 and SMRT and up-regulate BCL6 target genes. These new
generations of N-phenyl-4-pyrimidinamine BCL6 inhibitors could be identified and
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developed as novel potential anticancer agents against DLBCL growth in the future.
EXPERIMENTAL SECTION
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General Methods for Chemistry. All commercially available reagents were
purchased from bidepharm.com., Sigma-Aldrich Inc., J&K Inc., or Aladdin-Reagents
Inc. and used without further purification. All reactions except those in H atmosphere
2
media were carried out with the use of standard techniques under an inert atmosphere
(
N ). NMR spectra were generated on a Bruker 500 MHz instrument. High-resolution
2
mass spectra were gathered on Bruker Micro-TOF-Q II LCMS instrument operating in
electrospray ionization (ESI). High-pressure liquid chromatography (HPLC, Agilent
Technologies 1200 Series) was conducted on an Eclipse XDB C18 column (5 μm, 4.6
mm × 150 mm) for purity determination, eluting with water (with 0.1% trifluoroacetic
acid) and methanol (with 0.1% trifluoroacetic acid) at 40 °C for column temperature;
gradient of 40-70% methanol (0-5 min), 70-90% methanol (5-15 min), 90-40%
methanol (15-20 min) or 10-50% methanol (0-8 min), 50-90% methanol (8-15 min),
90-10% methanol (15-20 min) at the flow rate of 1.5 mL/min. All compounds purities
were ≥ 95%, as verified by HPLC.
2
-Chloro-N-(3-chloro-4-methoxyphenyl)-5-fluoro-4-pyrimidinamine (8). To a
solution of 3-chloro-4-methoxyaniline (1.58 g, 10 mmol) and DIEA (5.2 mL, 30 mmol)
in anhydrous EtOH (10 mL), then 2,4-dichloro-5-fluoropyrimidine (1.84 g, 11 mmol)
was added, the mixture was stirred at 40 ℃ for overnight, the reaction mixture was
concentrated under reduced pressure. The residue was partitioned between CH Cl and
2
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H O. The separated CH Cl layer was washed with brine, dried over anhydrous Na SO
4
2
2
2
2
and evaporated to dryness, and then the residue was purified by column
chromatography (eluting with PE/EA by 8:1-4:1) to give compound 8 (2.71 g, 94%
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yield). H NMR (500 MHz, DMSO-d ) δ 9.97 (s, 1H), 8.31 (d, J = 3.5 Hz, 1H), 7.78
6
(d, J = 2.6 Hz, 1H), 7.61 (dd, J = 9.0, 2.6 Hz, 1H), 7.19 (d, J = 9.0 Hz, 1H), 3.86 (s,
+
3H). HR MS (ESI): calcd for C H Cl FN O [M + H] 288.0101, found 288.0102.
1
1
9
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4
2
N -(3-Chloro-4-methoxyphenyl)-N -ethyl-5-fluoro-2,4-pyrimidinediamine (9a).
To a solution of intermediate 8 (144.1 mg, 0.5 mmol) and DIEA (0.26 mL, 1.5 mmol)
in n-BuOH (5 mL), then ethylamine (0.32 mL, 5 mmol) was added, the mixture was
stirred at 120 ℃ for overnight, the reaction mixture was concentrated under reduced
pressure. The residue was partitioned between CH Cl and H O. The separated CH Cl
2
2
2
2
2
layer was washed with brine, dried over anhydrous Na SO and evaporated to dryness,
2
4
and then the residue was purified by column chromatography (eluting with PE/EA by
4:1 followed by 100:1-20:1 CH Cl /MeOH) to give compound 9a (115 mg, 77% yield).
2
2
1
H NMR (500 MHz, DMSO-d ) δ 9.10 (s, 1H), 8.06 (s, 1H), 7.91 (d, J = 3.9 Hz, 1H),
6
7.69 – 7.68 (m, 1H), 7.09 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 3.83 (s, 3H), 3.26 – 3.19 (m,
2
H), 1.11 (t, J = 7.1 Hz, 3H). HPLC purity: 99.8%, R = 4.15 mim. HR MS (ESI): calcd
t
+
for C H ClFN O [M + H] 297.0913, found 297.0918.
1
3
15
4
4
2
N -(3-Chloro-4-methoxyphenyl)-N -isopropyl-5-fluoro-2,4-pyrimidinediamine
(9b). Compound 9b (33% yield) was obtained through the similar synthetic route of
1
compound 9a, except replacing ethylamine with isopropylamine. H NMR (500 MHz,
DMSO-d ) δ 9.09 (s, 1H), 8.05 (s, 1H), 7.91 (d, J = 3.8 Hz, 1H), 7.69 (d, J = 7.9 Hz,
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1H), 7.09 (d, J = 9.0 Hz, 1H), 6.57 (d, J = 7.1 Hz, 1H), 3.90 – 3.89 (m,1H), 3.83 (s,
3
H), 1.14 (d, J = 6.5 Hz, 6H). HPLC purity: 96.7%, R = 4.98 mim. HR MS (ESI): calcd
t
+
for C H ClFN O [M + H] 311.1069, found 311.1073.
1
4
17
4
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N -(3-Chloro-4-methoxyphenyl)-N -butyl-5-fluoro-2,4-pyrimidinediamine (9c).
Compound 9c (94% yield) was obtained through the similar synthetic route of
1
compound 9a, except replacing ethylamine with butylamine. H NMR (500 MHz,
DMSO-d ) δ 9.10 (s, 1H), 8.07 (s, 1H), 7.90 (d, J = 3.8 Hz, 1H), 7.67 (d, J = 6.1 Hz,
6
1
H), 7.08 (d, J = 9.0 Hz, 1H), 6.77 (s, 1H), 3.82 (s, 3H), 3.21 – 3.17 (m, 2H), 1.54 –
1.45 (m, 2H), 1.38 – 1.28 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). HR MS (ESI): calcd for
+
C H ClFN O [M + H] 325.1226, found 325.1228.
1
5
19
4
4
2
2
N -(3-Chloro-4-methoxyphenyl)-N ,N -diisobutyl-5-fluoro-2,4-pyrimidine-
diamine (9d). Compound 9d (52% yield) was obtained through the similar synthetic
1
route of compound 9a, except replacing ethylamine with diisobutylamine. H NMR
(500 MHz, DMSO-d ) δ 9.13 (s, 1H), 8.03 (d, J = 2.6 Hz, 1H), 7.97 (d, J = 3.8 Hz, 1H),
6
7
.59 – 7.57 (m, 1H), 7.10 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H), 3.32 – 3.31 (m, 4H), 2.13 –
2.03 (m, 2H), 0.84 (d, J = 6.7 Hz, 12H). HR MS (ESI): calcd for C H ClFN O [M +
1
9
27
4
+
H] 381.1852, found 381.1857.
2
-((4-((3-Chloro-4-methoxyphenyl)amino)-5-fluoro-2-pyrimidinyl)amino)-1-
ethanol (9e). Compound 9e (79% yield) was obtained through the similar synthetic
1
route of compound 9a, except replacing ethylamine with 2-aminoethanol. H NMR
(500 MHz, DMSO-d ) δ 9.11 (s, 1H), 7.98 (s, 1H), 7.91 (d, J = 3.9 Hz, 1H), 7.72 – 7.71
6
(m, 1H), 7.09 (d, J = 9.0 Hz, 1H), 6.61 (t, J = 5.7 Hz, 1H), 4.63 (t, J = 5.4 Hz, 1H), 3.83
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(s, 3H), 3.53 – 3.49 (m, 2H), 3.29 – 3.26 (m, 2H). HPLC purity: 97.7%, R = 7.83 mim.
t
+
HR MS (ESI): calcd for C H ClFN O [M + H] 313.0862, found 313.0865.
1
3
15
4
2
3
-((4-((3-Chloro-4-methoxyphenyl)amino)-5-fluoro-2-pyrimidinyl)amino)-1-
1
1
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propanol (9f). Compound 9f (89% yield) was obtained through the similar synthetic
1
route of compound 9a, except replacing ethylamine with 3-aminopropanol. H NMR
(500 MHz, DMSO-d ) δ 9.10 (s, 1H), 8.03 (s, 1H), 7.90 (d, J = 3.9 Hz, 1H), 7.72 – 7.70
6
(m, 1H), 7.09 (d, J = 9.0 Hz, 1H), 6.73 (s, 1H), 4.43 (t, J = 5.1 Hz, 1H), 3.83 (s, 3H),
3
.47 (m, 2H), 3.25 – 3.24 (m, 2H), 1.71 – 1.63 (m, 2H). HPLC purity: 97.2%, R = 8.43
t
+
mim. HR MS (ESI): calcd for C H ClFN O [M + H] 327.1019, found 327.1019.
1
4
17
4
2
4
-((4-((3-Chloro-4-methoxyphenyl)amino)-5-fluoro-2-pyrimidinyl)amino)-1-
butanol (9g). Compound 9g (85% yield) was obtained through the similar synthetic
1
route of compound 9a, except replacing ethylamine with 4-aminobutanol. H NMR
(
500 MHz, DMSO-d ) δ 9.10 (s, 1H), 8.06 (s, 1H), 7.90 (d, J = 3.9 Hz, 1H), 7.69 (d, J
6
= 8.0 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 6.78 (s, 1H), 4.36 (t, J = 5.1 Hz, 1H), 3.83 (s,
H), 3.40 (dd, J = 11.6, 6.3 Hz, 2H), 3.19 (dd, J = 13.1, 6.7 Hz, 2H), 1.57 – 1.50 (m,
3
2H), 1.49 – 1.43 (m, 2H). HPLC purity: 96.9%, R = 8.95 mim. HR MS (ESI): calcd for
t
+
C H ClFN O [M + H] 341.1175, found 341.1178.
1
5
19
4
2
N-(3-Chloro-4-methoxyphenyl)-2-((3S,5R)-3,5-dimethyl-1-piperazinyl)-5-
fluoro-4-pyrimidinamine (9h). Compound 9h (79% yield) was obtained through the
similar synthetic route of compound 9a, except replacing ethylamine with (2S,6R)-2,6-
1
dimethylpiperazine. H NMR (500 MHz, DMSO-d ) δ 9.28 (s, 1H), 8.11 (d, J = 2.6 Hz,
6
1
H), 7.99 (d, J = 3.7 Hz, 1H), 7.51 – 7.49 (m, 1H), 7.13 (d, J = 9.0 Hz, 1H), 4.34 (d, J
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= 11.8 Hz, 2H), 3.83 (s, 3H), 2.71 – 2.70 (m, 2H), 2.35 – 2.30 (m, 2H), 1.02 (d, J = 6.2
Hz, 6H). HPLC purity: 97.9%, R = 8.60 mim. HR MS (ESI): calcd for C H ClFN O
t
17 22
5
+
[
M + H] 366.1491, found 366.1495.
0
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N-(3-Chloro-4-methoxyphenyl)-2-((3R,5S)-3,5-dimethyl-1-piperidinyl)-5-
fluoro-4-pyrimidinamine (9i). Compound 9i (87% yield) was obtained through the
similar synthetic route of compound 9a, except replacing ethylamine with (3R,5S)-3,5-
1
dimethylpiperidine. H NMR (500 MHz, DMSO-d ) δ 9.24 (s, 1H), 8.11 (d, J = 2.4 Hz,
6
1
H), 7.98 (d, J = 3.7 Hz, 1H), 7.50 (dd, J = 8.9, 2.5 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H),
4.48 (d, J = 11.8 Hz, 2H), 3.83 (s, 3H), 2.29 – 2.26 (m, 2H), 1.76 (d, J = 12.7 Hz, 1H),
1
.58 – 1.47 (m, 2H), 0.89 (d, J = 6.5 Hz, 6H), 0.77 (d, J = 12.0 Hz, 1H). HR MS (ESI):
+
calcd for C H ClFN O [M + H] 365.1539, found 365.1540.
1
8
23
4
N-(3-Chloro-4-methoxyphenyl)-5-fluoro-2-piperazinyl-4-pyrimidinamine (9j).
Compound 9j (98% yield) was obtained through the similar synthetic route of
1
compound 9a, except replacing ethylamine with piperazine. H NMR (500 MHz,
DMSO-d ) δ 9.24 (s, 1H), 8.00 (d, J = 3.7 Hz, 1H), 7.91 (d, J = 2.6 Hz, 1H), 7.64 – 7.61
6
(m, 1H), 7.13 (d, J = 9.0 Hz, 1H), 5.76 (s, 1H), 3.83 (s, 3H), 3.58 – 3.49 (m, 4H), 2.77
–
2.67 (m, 4H). HPLC purity: 97.3%, R = 6.43 mim. HR MS (ESI): calcd for
t
+
C H ClFN O [M + H] 338.1178, found 338.1179.
1
5
18
5
N-(3-Chloro-4-methoxyphenyl)-5-fluoro-2-(4-methyl-1-piperazinyl)-4-
pyrimidinamine (9k). Compound 9k (97% yield) was obtained through the similar
synthetic route of compound 9a, except replacing ethylamine with 1-methylpiperazine.
1
H NMR (500 MHz, DMSO-d ) δ 9.27 (s, 1H), 8.00 (d, J = 3.6 Hz, 1H), 7.91 (d, J =
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2.5 Hz, 1H), 7.62 – 7.61 (m, 1H), 7.13 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H), 3.65 – 3.57 (m,
4
H), 2.36 – 2.31 (m, 4H), 2.20 (s, 3H). HPLC purity: 98.9%, R = 6.54 mim. HR MS
t
+
(
ESI): calcd for C H ClFN O [M + H] 352.1335, found 352.1336.
1
6
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9
0
N-(3-Chloro-4-methoxyphenyl)-5-fluoro-2-morpholino-4-pyrimidinamine (9l).
Compound 9l (91% yield) was obtained through the similar synthetic route compound
1
9a, except replacing ethylamine with morpholine. H NMR (500 MHz, DMSO-d ) δ
6
9
7
.30 (s, 1H), 8.03 (d, J = 3.7 Hz, 1H), 7.87 (d, J = 2.6 Hz, 1H), 7.67 – 7.62 (m, 1H),
.13 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H), 3.67 – 3.61 (m, 4H), 3.60 – 3.54 (m, 4H). HPLC
+
purity: 98.0%, R = 9.11 mim. HR MS (ESI): calcd for C H ClFN O [M + H]
t
15 17
4
2
3
39.1019, found 339.1021.
4
2
N -(3-Chloro-4-methoxyphenyl)-5-fluoro-N -(1-methyl-4-piperidinyl)-2,4-
pyrimidinediamine (9m). Compound 9m (43% yield) was obtained through the
similar synthetic route of compound 9a, except replacing ethylamine with 1-
1
methylpiperidin-4-amine. H NMR (500 MHz, DMSO-d ) δ 9.23 (s, 1H), 7.98 (d, J =
6
3
.6 Hz, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.58 – 7.57 (m, 1H), 7.12 (d, J = 9.0 Hz, 1H),
4.33 (d, J = 13.2 Hz, 2H), 4.12 – 4.05 (m, 1H), 3.83 (s, 3H), 3.32 (s, 3H), 2.96 – 2.91
(m, 2H), 2.83 – 2.78 (m, 1H), 1.73 – 1.70 (m, 2H), 1.19 – 1.12 (m, 2H). HPLC purity:
+
9
6.1%, R = 6.41 mim. HR MS (ESI): calcd for C H ClFN O [M + H] 366.1491,
t
17 22
5
found 366.1493.
4
2
N -(3-Chloro-4-methoxyphenyl)-N -cyclohexyl-5-fluoro-2,4-
pyrimidinediamine (9n). Compound 9n (98% yield) was obtained through the similar
synthetic route of compound 9a, except replacing ethylamine with cyclohexanamine.
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H NMR (500 MHz, DMSO-d ) δ 9.09 (s, 1H), 8.08 (s, 1H), 7.90 (d, J = 3.8 Hz, 1H),
6
7
.65 (s, 1H), 7.08 (d, J = 9.0 Hz, 1H), 6.60 (d, J = 4.7 Hz, 1H), 3.83 (s, 3H), 2.09 (s,
H), 1.89 (d, J = 10.7 Hz, 2H), 1.71 (d, J = 12.9 Hz, 2H), 1.60 (d, J = 12.7 Hz, 1H),
1
0
1
2
3
4
5
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8
9
0
1
2
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7
8
9
0
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9
0
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8
9
0
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2
3
4
5
6
7
8
9
0
1.31 – 1.30 (m, 2H), 1.25 – 1.18 (m, 2H), 1.15 – 1.06 (m, 1H). HPLC purity: 98.7%, Rt
+
=
6.73 mim. HR MS (ESI): calcd for C H ClFN O [M + H] 351.1382, found
1
7
21
4
351.1384.
2
-Chloro-5-fluoro-N-(4-methoxy-3-nitrophenyl)-4-pyrimidinamine (10a). Com-
pound 10a was obtained through the similar synthetic route of compound 8, except
1
replacing 3-chloro-4-methoxyaniline with 4-methoxy-3-nitroaniline (91% yield). H
NMR (500 MHz, DMSO-d ) δ 10.18 (s, 1H), 8.36 (d, J = 3.4 Hz, 1H), 8.27 (d, J = 2.7
6
Hz, 1H), 7.96 (dd, J = 9.1, 2.7 Hz, 1H), 7.44 (d, J = 9.2 Hz, 1H), 3.94 (s, 3H). HR MS
+
(ESI): calcd for C H ClFN NaO [M + H] 321.0161, found 321.0165.
1
1
8
4
3
2
-Chloro-5-fluoro-N-(3-methoxy-4-nitrophenyl)-4-pyrimidinamine (10b). To a
solution of 3-methoxy-4-nitroaniline (840.8 mg, 5.0 mmol) and Cs CO (4.882 g, 15.0
2
3
mmol) in anhydrous DMF (5 mL), then 2,4-dichloro-5-fluoropyrimidine (1.67 g, 10.0
mmol) was added, the mixture was stirred at r.t for overnight. The residue was
partitioned between CH Cl and H O. The separated CH Cl layer was washed with
2
2
2
2
2
brine, dried over anhydrous Na SO and evaporated to dryness, and then the residue
2
4
was purified by column chromatography (eluting with PE/EA by 8:1-4:1) to give
1
compound 10b (725.0 mg, 49% yield). H NMR (500 MHz, DMSO-d ) δ 8.95 (s, 1H),
6
8.94 (s, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.24 (dd, J = 8.7, 1.9
+
Hz, 1H), 3.85 (s, 3H). HR MS (ESI): calcd for C H ClFN NaO [M + H] 321.0161,
1
1
8
4
3
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found 321.0157.
-((3S,5R)-3,5-Dimethyl-1-piperazinyl)-5-fluoro-N-(4-methoxy-3-nitrophenyl)-
-pyrimidinamine (11a). Compound 11a (83% yield) was obtained through the similar
2
4
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0
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0
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0
synthetic route of compound 9a, except replacing compound 8 with compound 10a
1
instead of and replacing ethylamine with (2S,6R)-2,6-dimethylpiperazine. H NMR
(500 MHz, DMSO-d ) δ 9.51 (s, 1H), 8.72 (d, J = 2.7 Hz, 1H), 8.03 (d, J = 3.6 Hz, 1H),
6
7
.78 (dd, J = 9.1, 2.7 Hz, 1H), 7.37 (d, J = 9.2 Hz, 1H), 4.37 (d, J = 11.0 Hz, 2H), 3.91
(s, 3H), 2.71 – 2.69 (m, 2H), 2.32 – 2.30 (m, 2H), 1.02 (d, J = 6.2 Hz, 6H). HR MS
+
(ESI): calcd for C H FN O [M + H] 377.1732, found 377.1737.
1
7
22
6
3
2
-((3S,5R)-3,5-Dimethyl-1-piperazinyl)-5-fluoro-N-(3-methoxy-4-nitrophenyl)
-4-pyrimidinamine (11b). Compound 11b (86% yield) was obtained through the
similar synthetic route of compound 11a, except replacing compound 10a with
1
compound 10b. H NMR (500 MHz, DMSO-d ) δ 9.77 (s, 1H), 8.16 (d, J = 3.4 Hz,
6
1H), 7.98 (dd, J = 5.5, 3.5 Hz, 2H), 7.43 (dd, J = 9.1, 2.1 Hz, 1H), 4.37 (d, J = 10.5 Hz,
2
H), 3.94 (s, 3H), 2.71 – 2.67 (m, 2H), 2.36 – 2.31 (m, 2H), 1.00 (d, J = 6.2 Hz, 6H).
+
HR MS (ESI): calcd for C H FN O [M + H] 377.1732, found 377.1730.
1
7
22
6
3
1
N -(2-((3S,5R)-3,5-Dimethyl-1-piperazinyl)-5-fluoro-4-pyrimidinyl)-4-methoxy
-benzene-1,3-diamine (12a). To a solution of compound 11a (1.694 g, 4.5 mmol) in
anhydrous MeOH (50 mL) was added Pd-C under N . The suspension was degassed
2
under vacuum and purged with H several times. The mixture was stirred under H at
2
2
r.t for overnight. The suspension was filtered and the filtrate was concentrated by rotary
1
evaporation to give compound 12a (95%). H NMR (500 MHz, DMSO-d ) δ 8.87 (s,
6
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