Design, synthesis and evaluation of derivatives based on pyrimidine scaffold as potent Pan-Raf inhibitors to overcome resistance

Article abstract of DOI:10.1016/j.ejmech.2017.02.041

Simutaneous targeting all Raf isoforms offers the prospect of enhanced efficacy as well as reduced potential for resistance. Described herein is the discovery and characterization of a series of pyrimidine scaffold with DFG-out conformation as potent Pan-Raf inhibitors. Among them, I-41 with excellent Pan-Raf potency demonstrates inhibitory activity against BRaf^WTphenotypic melanoma and BRaf^V600Ephenotypic colon cells. The western blot results for the Erk inhibition in human melanoma SK-Mel-2?cell lines showed I-41 inhibited the proliferation of SK-Mel-2?cell lines without paradoxical activation of Erk, which supported I-41 may become a good candidate compound to overcome the resistance of melanoma against the current BRaf^V600Einhibitor therapy. I-41 also have a favorable pharmacokinetic profile in rat. Synthesis, SAR, lead selection, and evaluation of the key compounds studies are described.

Full text of DOI:10.1016/j.ejmech.2017.02.041

Accepted Manuscript
Design, synthesis and evaluation of derivatives based on pyrimidine scaffold as
potent Pan-Raf inhibitors to overcome resistance
Lu Wang, Qing Zhang, Gaoyuan Zhu, Zhimin Zhang, Yanle Zhi, Li Zhang, Tianxiao
Mao, Xiang Zhou, Yadong Chen, Tao Lu, Weifang Tang
PII:
S0223-5234(17)30114-9
10.1016/j.ejmech.2017.02.041
EJMECH 9237
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 January 2017
Revised Date: 14 February 2017
Accepted Date: 16 February 2017
Please cite this article as: L. Wang, Q. Zhang, G. Zhu, Z. Zhang, Y. Zhi, L. Zhang, T. Mao, X. Zhou, Y.
Chen, T. Lu, W. Tang, Design, synthesis and evaluation of derivatives based on pyrimidine scaffold as
potent Pan-Raf inhibitors to overcome resistance, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.02.041.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of derivatives based on pyrimidine
scaffold as potent Pan-Raf inhibitors to overcome resistance
a
b
a
a
a
a
Lu Wang , Qing Zhang
, Gaoyuan Zhu , Zhimin Zhang , Yanle Zhi , Li Zhang , Tianxiao Mao
a
a
a
a,c,
a,
,
Xiang Zhou , Yadong Chen , Tao Lu * , Weifang Tang *
a
School of Basic Science, China Pharmaceutical University, 639 Longmian Avenue,
Nanjing, Jiangsu 211198, China.
b
School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 639
Longmian Avenue, Nanjing, Jiangsu, 211198, China.
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing, Jiangsu 210009, China.
c
Abstract
Simutaneous targeting all Raf isoforms offers the prospect of enhanced efficacy as well as
reduced potential for resistance. Described herein is the discovery and characterization of a series
of pyrimidine scaffold with DFG-out conformation as potent Pan-Raf inhibitors. Among them,
WT
I-41 with excellent Pan-Raf potency demonstrates inhibitory activity against BRaf phenotypic
V600E
melanoma and BRaf
phenotypic colon cells. The western blot results for the Erk inhibition in
human melanoma SK-Mel-2 cell lines showed I-41 inhibited the proliferation of SK-Mel-2 cell
lines without paradoxical activation of Erk, which supported I-41 may become a good candidate
V600E
compound to overcome the resistance of melanoma against the current BRaf
inhibitor therapy.
I-41 also have a favorable pharmacokinetic profile in rat. Synthesis, SAR, lead selection, and
evaluation of the key compounds studies are described.
1
.
Introduction
Targeted therapy constitutes the use of agents specific for regulating signal transduction
networks of cancer cells. The Ras/Raf/Mek/Erk (mitogen-activated protein kinase MAPK)
signaling pathway, transduces input from cell surface receptors to nuclear transcription factors
thereby regulating cellular proliferation, differentiation and survival [1]. As Raf phosphorylation is
an important step in the cellular MAPK signal transduction pathway, targeting abnormal Raf
activity has become an attractive targeted therapy for the treatment of cancer [2]. There are three
subtype Raf proteins in cells, ARaf, BRaf and CRaf, and studies have shown that the formation of
complexes by these different isoforms has an important role in their activation [3-5]. Mutations of
1

ACCEPTED MANUSCRIPT
BRaf are the most frequently detected in human cancers [6], including 50−80% of melanoma, 11%
of colorectal cancer [7], ∼100% of hairy cell leukemia [8], 45−50% of papillary thyroid
carcinoma [9] and others. Notably, over 90% of BRaf mutation is the substitution of valine to
glutamate at residue 600 (V600E). This substitution mimics phosphorylation of the activation loop,
elevating the in vitro kinase activity by up to 500~700-fold compared with its wild-type
WT
counterpart BRaf [10]. Vemurafenib (PLX4032) is classified as type I inhibitors with high
V600E
selectivity against BRaf
, which bind to the DFG (a conserved amino acid sequence of D549,
F595 and G596)-in “active” conformation of the ATP binding site [11]. In contrast, sorafenib
shown in Fig. 1 is the Raf kinase inhibitor that binds to the DFG-out “inactive” conformation of
WT
V600E
BRaf
and BRaf
. This compound is classified as type II inhibitor, which bind to the
DFG-out “inactive” conformation at the ATP binding site and inhibits all subtypes of Raf proteins
[12]. LY3009120 is also known as type II Pan-Raf inhibitor to slow cell proliferation [13].
V600E
Fig. 1. Binding mode of BRaf
(A) DFG-out conformation for PDB 1UWJ (crystalized with
sorafenib); (B) DFG-in conformation for PDB 3OG7 (crystalized with vemurafenib)
V600E
V600E
The approval of selective BRaf
inhibitors, for treating BRaf
-derived solid tumors
validate the approach of targeting the MAPK pathway as an effective way of treating cancer [14].
V600E
However, the rapid development of secondary malignancies in BRaf
tumors is reported as a
V600E
well-known side effect among these selective BRaf
inhibitors [15, 16]. The potency of
WT
vemurafenib in BRaf tumor models unexpectedly diverge [17]. A majority of colorectal cancer
patients display inherent resistance against vemurafenib, although they were detected to harbor
V600E
BRaf
mutation [18]. The exact mechanism for the resistance remains complicated [19], but
development of new compounds of DFG-out conformation with Pan-Raf potency may be a
valuable strategy to overcome or delay the occurrence of this challenging medical dilemma [20].
Herein, the optimal BRaf inhibitor should be DFG-out type that preserves potency against
2

ACCEPTED MANUSCRIPT
V600E
WT
oncogenic BRaf
without driving activation of BRaf or CRaf [21]. Hence we develop a
series of DFG-out conformation pyrimidine derivates I-01–I-43 and II-01–II-13 with Pan-Raf
potency targeting all subtypes of Raf proteins to avoid paradoxical stimulation of MAPK signaling
WT
in BRaf phenotypic melanoma.
2
.
Results and discussion
A total of 56 newly synthesized compounds I-01–I-43 and II-01–II-13 were designed and
synthesized. Compounds were evaluated through enzyme and cell-based assay. Furthermore, SAR
structure-activity relationship) had also been established. I-14 and I-41 with optimal potency at
(
enzymatic as well as cellular level was chosen to study the possible biological mechanisms and
pharmacokinetic profiles.
2
.1. Chemistry
As summarized in Scheme 1, 56 compounds were synthesized in total. The synthetic routes
were illustrated in Scheme 1–4 (see Supporting Information). The chemical structures of these
1
13
compounds were confirmed by H-NMR, C-NMR, HRMS spectra and the results were presented
in Experimental section.
The target pyrimidine derivatives I-01–I-43 and II-01–II-13 were prepared by the method as
shown in Scheme 1. The reaction of commercially available 4, 6-dichloropyrimidine with diethyl
malonate in the presence of sodium hydride gave diethyl 2-(6-chloropyrimidin-4-yl) malonate I-a.
The prepared I-a was allowed to react with sodium ethoxide to afford the desired I-b. Subsequent
alkylation of I-b with 1,2-dibromoethane or iodomethane in the presence of sodium hydroxide
afforded the corresponding alkylated derivatives ethyl 2-(6-chloropyrimidin-4-yl)acetate I-c or
ethyl 2-(6-chloropyrimidin-4-yl)-2-methylpropanoate II-c. The reaction of I-c or II-c with
commercially available amines through SNAr substitution reaction provided I-d-01–I-d-21 or
II-d-1–II-d-9 in 63.8–92.1% or 70.8–90.1%, respectively. Amide-forming reactions of I-d-01–
I-d-21 and II-d-1–II-d-9 with corresponding anilines A1–A23 were carried out in the presence of
trimethylaluminium under the condition of nitrogen atmosphere to give I-01–I-43 and II-01–II-13
in 70.8–88.6% or 69.7–88.1%, respectively.
3

ACCEPTED MANUSCRIPT
Scheme 1. The synthetic routes of compounds I-01–I-43 and II-01–II-13. Reagents and
o
conditions: (a) diethyl malonate, NaH, THF, 0 C, 0.5 h, then r.t., 2.5 h; (b) EtONa, EtOH, reflux,
2
9
.5 h; (c) 1,2-dibromoethane, NaOH, DMF, r.t., 5 h, 90.2%; iodomethane, NaOH, DMF, r.t., 5 h,
5
4.3%; (d) CH NH or C H NH , EtOH, r.t., 2 h; R NH , DIPEA, EtOH, reflux, 2 h; I-d-01–
3
2
2
5
2
2
I-d-21: 63.8–92.1%, II-d-1–II-d-9: 70.8–90.1%; (e) A1–A23, Al(CH ) , toluene, nitrogen
3
3
o
atmosphere, 80 C, 5 h; I-01–I-43: 67.7–89.6%, II-1–II-13: 69.7–88.1%, respectively. The
substituents are specified in Table 1.
A total of 23 important intermediates of amide analogs A1–A23 were synthesized according to
Scheme 2–4 via a two-five steps process (see Supporting Information).
4

ACCEPTED MANUSCRIPT
2
2
.2. Inhibitors design and molecular modeling
.2.1. Inhibitor design and optimization
Initiating a kinase-based drug discovery program, which has led to the identification of
compound P-1 developed in our laboratory. Importantly, P-1 showed Pan-Raf potency (ARaf IC50
WT
V600E
=
194 nM, BRaf IC = 287 nM, BRaf
IC = 77.2 nM, CRaf IC = 43.4 nM). Modification
50 50
5
0
of P-1 has led to the development of potent lead compound I-01 shown in Fig. 2. I-01 displayed
WT
V600E
Pan-Raf potency (ARaf IC = 17.8 nM; BRaf IC = 17.2 nM, BRaf
IC = 2.8 nM, CRaf
50
5
0
50
IC = 0.7 nM).
50
Fig. 2. The modification of P-1 to I-01 and the binding mode of I-01–I-43,II-01–II-13
We determined the important structural features essential for activity. The binding mode of I-01
V600E
V600E
to DFG-out conformation of BRaf
revealed that I-01 binds to the BRaf
ATP binding
pocket with the pyrimidine core forming two essential hydrogen bonds with the hinge region
Cys532 (Fig. 3). In addition to these hydrogen bonds, the amide group forms two hydrogen bonds
with Asp594 and Glu501 in the DFG-out region. The binding conformation of I-01 was consistent
with sorafenib which contains the same original essential hydrogen bonds shown in Fig. 3.
Before setting out to optimize I-01, we turned our attention toward the chlorine atom appended
to the terminal phenyl ring and the methylamino group appended to the pyrimidine core. More
importantly, they directed to the solvent accessible region of the enzyme and form hydrophilic
5

ACCEPTED MANUSCRIPT
interactions with the surrounding amino acid residues as shown in Fig. 2 and Fig. 3. In order to get
a better occupation of the solvent accessible region, we started optimization program to modify
I-01 with an aim to improve in vitro potency and ameliorate physicochemical properties.
The cyclopropyl carboxamide Q (Fig. 2) was replaced by 2, 2-dimethyl acetamide, which leads
to the discovery of series II compounds. Subsequent structure-based optimization of I-01 focused
on potency at enzymatic as well as cellular activity. We described the design and optimization of
pyrimidine derivatives I-01–I-43 and II-01–II-13 as new DFG-out Pan-Raf inhibitors. The SAR
1
2
3
4
on R and R (the hydrophobic moiety), R (the hydrophobic moiety), R (the solvent accessible
5
moiety), R (the other solvent accessible moiety) and Q (cyclopropyl formamide fragment) were
investigated.
Fig. 3. The predicted binding mode of compound I-01. Alignment of I-01 with (A) lead P-1 and
V600E
V600E
(B) sorafenib with BRaf
(PDB ID: 3IDP) and the binding pocket of BRaf
2
.2.2. Selectivity mechanism of lead P-1 and I-01
As shown in Fig.2 and Fig.3, the lead P-1 (BRaf
V600E
V600E
IC = 77.2 nM) and I-01 (BRaf
IC50
5
0
=
2.8 nM) only show slightly difference and the binding mode predicted from molecular docking
is also almost the same. However, their enzymatic activity is about 11, 17, 28 and 62 times
WT
V600E
difference against ARaf, BRaf , Raf
and CRaf, respectively. As the drug resistance mainly
occurred due to protein mutation, we focused on the selectivity of these two compounds against
V600E
BRaf
. Accordingly, 20ns molecular dynamics (MD) was conducted to simulate the binding
V600E
process and calculate the binding energy of these compounds with BRaf
(PDB: 3IDP). The
root-mean-square deviation (RMSD) for both the protein (Fig. 4A) and ligands (Fig. 4B) (here
referred to the two compounds) are shown in Fig. 4 where I-01 (red) demonstrated a more stable
RMSD than that of lead P-1 (black). Particularly, in the last 6ns, the lead P-1 showed a great
increase of RMSD from about 1Å to more than 2Å for the ligand RMSD. In addition, the number
6

ACCEPTED MANUSCRIPT
of hydrogen bonds formed during the simulation process were calculated where both the
compounds achieved one to five hydrogen bonds; however, I-01 mainly formed three to four
hydrogen bonds while lead P-1 only captured two to three, indicating a stronger binding for I-01
than that of lead P-1 (Fig. 4C). Moreover, the binding free energy calculated from MM/PBSA was
-41.8 kcal/mol for I-01 which was slightly lower than that of lead P-1 (-39.3 kcal/mol). From the
DeltaG distribution (Fig. 4D), both compounds showed a normal distribution and I-01 shifted left
slightly compared with leap P-1. All there MD results explained why I-01 possess a stronger
binding affinity against than that of leap P-1 even through their highly similar 2D structures and
binding mode form molecular docking.
Fig. 4. MD results for lead P-1(black) I-01 (red). RMSD values during the 20ns MD for the (A)
proteins and (B) ligands; (C) the number of hydrogen bonds. (D) the distribution of deltaG
V600E
calculated by MM/PBSA. Crystal structures of BRaf
used is PDB 3IDP.
2
.3. Investigation the enzymatic activity of compounds
1
2
Structure transformation of I-01 was focused on L (linkage moiety), R and R (hydrophobic
3
4
pocket moiety), R (hydrophobic back pocket moiety), R (solvent accessible region moiety) and
5
R (the other solvent accessible region moiety) for further biological evaluation and SAR
exploration. Fragment Q was investigated on rigid factor.
V600E
2
.3.1. Compounds screened for enzyme activity against BRaf
V600E
The compounds were evaluated as BRaf
inhibitors using vemurafenib and sorafenib as
7

ACCEPTED MANUSCRIPT
positive control compounds. Most of the compounds showed IC50 values at nanomolar ranges
shown in Table 1.
Q group of different rigidity was investigated. Compared to I-01, II-01 showed 8-fold decrease
in enzyme activity. II-01, still comparable with the positive control, was used as template for
futher modification.
Disruption of hydrogen bonds by replacement the L as amide of I-01 with “reverse” amide of
I-02 or urea of I-03 led to decreased potency. The decreased potency of I-02–I-03 to I-01 and
V600E
II-02–II-03 to II-01 confirmed the importance of the amide group for potent Raf
activity.
Table 1
1
2
3
4
5
V600E
SAR of Q, L, R , R , R R , and R in terms of BRaf
inhibitory assay
,
V600E
BRaf
1
2
3
4
5
Compd
Q
L
R
R
R
R
R
a
activity% IC50 (nM)
I-01
I-02
I-03
I-04
I-05
I-06
I-07
I-08
I-09
I-10
CONH
NHCO
CH
3
3
3
H
H
H
H
F
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
3
3
3
3
3
3
3
3
3
3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
-1.0
0.7
2.8
CH
4.3
NHCONH CH
0.9
18.5
38.6
152.0
39.1
30.8
237.0
25.1
178.0
CONH
CONH
CONH
CONH
CONH
CONH
CONH
F
H
2.4
5.1
Cl
H
H
H
F
0.4
5.1
F
14.5
0.04
7.6
CH
H
H
3
3
CH
8

ACCEPTED MANUSCRIPT
I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
I-23
I-24
I-25
I-26
I-27
I-28
I-29
I-30
I-31
I-32
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF
CF
CF
CF
CF
H
3
3
3
3
3
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
3
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
2.0
29.1
88.6
24.1
20.5
26.5
388.0
8.4
2.5
0.1
3.7
0.3
H
Cl
H
F
36.9
0.7
H
Cl
H
0.6
0.3
24.7
10.3
14.1
84.6
32.5
20.8
36.7
0.2
201.0
59.5
143.0
F
H
H
CN
H
b
ND
H
ND
CF
CF
CF
CF
CF
CF
CF
CF
CF
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OCH
CH
OCH
251.0
441.0
44.1
46.1
140.0
43.1
35.8
31.5
3
2
2
3
3
3
O(CH ) OCH
2 3 3
3
3
3
3
3
3
3
3
NHCH CH
2 3
NHCH
CH
CH
0.1
2
2
NHCH(CH
)
2.9
3
2
NHCH
CH
OH
0.2
2
2
3
NH(CH
)
OCH
0.3
2
3
NHCH
CH OH
2
0.4
2
b
NH(CH ) NHH
2 3 3
35.3
ND
9

ACCEPTED MANUSCRIPT
I-33
I-34
I-35
I-36
I-37
I-38
I-39
I-40
I-41
I-42
I-43
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
H
H
H
H
H
H
H
H
H
H
H
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
3
3
3
3
3
3
3
3
3
3
3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
NH(CH
NH(CH
NH(CH
2
2
2
)
)
)
2
2
3
CO
2
H
6.6
3.0
0.05
0.2
0.08
1.0
0.9
0.7
1.4
2.6
1.5
80.2
68.2
18.7
19.5
23.4
21.4
14.3
13.9
12.2
21.6
18.3
N(CH
)
)
3
3
2
2
N(CH
II-1
II-2
II-3
II-4
CONH
NHCO
CH
3
3
3
H
H
H
F
CF
CF
CF
CF
3
3
3
3
Cl
Cl
Cl
Cl
CH
CH
CH
CH
3
3
3
3
NH
NH
NH
NH
1.5
22.4
ND
ND
ND
CH
16.1
41.7
67.1
NHCONH CH
CONH
CONH
F
II-5
CH
3
H
CF
3
CH
3
NH
41.1
135.0
II-06
II-07
II-08
II-09
II-10
CONH
CONH
CONH
CONH
CONH
CH
CH
CH
CH
CH
3
3
3
3
3
H
H
H
H
H
CF
CF
CF
CF
CF
3
3
3
3
3
Cl
Cl
Cl
Cl
Cl
NHCH
NHCH CH
NH(CH OCH
2
CH
3
11.1
ND
ND
ND
ND
ND
CH
38.5
14.8
34.4
19.3
2
2
3
)
2
3
3
NH(CH
)
N(CH
)
2
2
2
3
3
3
2
2
NH(CH
)
N(CH
)
II-11
II-12
II-13
CONH
CONH
CONH
CH
CH
CH
3
3
3
H
H
H
CF
CF
CF
3
3
3
Cl
Cl
Cl
38.3
34.3
12.4
ND
ND
145.0
1
0

ACCEPTED MANUSCRIPT
sorafenib
38.1
31.6
vemurafenib
a
b
compounds with the concentration of 0.5 µM
not determination
1
2
R and R group on central phenyl ring occupied a small hydrophobic pocket typically closed
3
off the gatekeeper region. R substituted terminal phenyl ring was exposed to the hydrophobic
pocket, which was made by a rearrangement of the activation loop and subsequent movement of
4
phenylalanine side chain of DFG loop [22]. R substituent in the terminal phenyl ring was directed
toward the solvent accessible region. Based on these information, totally 23 compounds were
designed and synthesized.
1
2
The influence of R and R substituent at central phenyl ring was studied. Halogen atoms,
including fluorine and chloride, were employed to afford compounds I-04–I-06 and I-08.
Hydrogen was explored to afford I-07. All the modifications led to significantly decreased activity.
V600E
With I-05 and I-08 being completely inactive in the BRaf
assay, suggesting a strict steric and
electrical requirement to exist in small hydrophobic pocket closed off the gatekeeper region. I-01,
1
2
V600E
using methyl as R and hydrogen atom as R , displayed optimal BRaf
activity.
We hypothesized the poor antiproliferative activity of I-01 against A375 (IC = 10.6 µM) was
5
0
4
due to its meager aqueous solubility and cellular permeability. As described above, R toward the
4
solvent accessible region. The modification introducing solubilizing functional groups at R
position afforded I-09–I-11 to get a better occupation of solvent accessible region. Due to lower
lipophilicity compared to I-01, I-09–I-15 resulted in 8–60 fold decreased enzyme activity. I-09,
I-11 and I-13–I-15 were tolerated.
Having found the modification strategy of introducing hydrophilic groups at terminal phenyl
couldn’t enhace the enzyme activity, we turned our attention toward the strategy of removing
lipophilic groups. Then, I-16–I-21 were designed and synthesized. Due to I-16 loss of potency, it
was necessary to sustain substituents. I-17–I-20 introduced halogen atoms including fluorine and
V600E
chloride were more active in BRaf
assay compared to I-16.
4
3
Subsequently, the influence of R substituent (I-17 and I-18) and R substituent (I-18 and I-20)
3
on terminal phenyl ring were studied. It should be noted that the R substituent at C-3 position was
4
beneficial for the activity than R substituent at C-4 position. This result was consistent with the
1
1

ACCEPTED MANUSCRIPT
4
3
hypothesis that the R and R group occupy solvent accessible region domain and a small
V600E
hydrophobic pocket in BRaf
, respectively. The comparison of I-17 to I-19 and I-18 to I-20
3
indicated that the chloride in R position was beneficial for the potency than fluorine. I-18 using
3
V600E
chloride atom as R substituent, displayed optimal BRaf
activity.
3
With the aim to explore the electrical requirement in R , electron withdrawing group cyanogen
was introduced to achieve I-21. However, I-21 displayed significantly decreased potency against
V600E
BRaf
with IC50 as 143.0 nM to I-18 with IC50 as 0.3 nM. This result confirmed that the
electron donating group would be better suited to increase enzyme activity.
I-11–I-12 removed of lipophilic groups trifluoromethyl (-CF ) afforded I-22–I-23. The results
3
3
indicated that R substituent was necessary for potency, too.
5
Furthermore, we were interested to know wheather the introduction of R attached to the
pyrimidine ring could increase the enzymatic activity.
5
R of different steric sizes was explored. We envisioned introduction of solubilizing functional
5
groups R bearing methoxyl or amino functionality into pyrimdine ring. It has been reported that
the presence of methoxyl or basic amino functionality can properly occupy the solvent accessible
region [23-25], so we introduced these functional groups through two or three carbon atoms
linkage into the 4-position pyrimidine core. The carbon linkage would be tethered to the
pyrimidine ring through an ether or imidogen bridge. Compared to imidogen bridge (I-29 and
I-30), an ether bridge led to loss of activity (I-24 and I-25). This result suggested that the
imidogen bridge would be more suitable to enter the solvent exposed region of the enzyme.
Placed at pyrimidine ring, smaller alkyl groups I-26–I-28 were well tolerated. The strength
V600E
order against BRaf
is methyl > ethyl ≈ propyl >isopropyl. These results displayed that the
linear alkyl would be better suited to enter the solvent exposed region of the enzyme. One primary
amine I-32 was introduced through a three-carbon linker at the pyrimidine ring, which resulted in
V600E
loss of BRaf
activity. The decreased enzyme activity of I-32 confirmed the importance of the
V600E
location of the secondary amine for potent BRaf
activity. Compared to I-32, the potency at
enzymatic level of I-35 was significantly increased. Cyclic secondary amines I-36–I-43 could
V600E
sustain the BRaf
activity compared to I-01. Due to lower lipophilicity compared to I-01,
I-26–I-43 resulted in a decreased enzyme activity.
1
2

ACCEPTED MANUSCRIPT
V600E
Q group of different rigidity were investigated. II-1, II-4 and II-5 showed decreased BRaf
activity to I-01, I-08 and I-14. This result showed that cyclopropyl into the Q would be better
sustained kinase inhibition.
V600E
II-01 was introduced fragments screened from I-24–I-43 in terms of BRaf
activity to
5
4
-position pyrimidine ring as R substituent to achieve II-06–II-13.When cyclopropyl (I-26–I-27,
I-30, I-34–I-37, I-39) was replaced by dimethyl (II-06–II-13), the enzyme activity significantly
declined. These results indicateed that the Q fragment of cyclopropyl would be better suited to
enter the enzyme. The SAR tendency of series II compounds was in a range similar to series I
compounds.
5
These results showed that the steric accommodation was the core factor for R to affect
enzymatic activity. Bulky groups could get a better occupation of solvent accessible region leaning
the hinge moiety of pyrimidine.
2
.3.2. The Pan-Raf activity of chosen compounds
As mentioned earlier, Pan-Raf inhibitors with similar activity against all Raf isoforms have
potential to overcome the resistance of vemurafenib, so the Pan-Raf activity of chosen compounds
V600E
with potent BRaf
activity was evaluated. The result indicated that their potency were superior
V600E
WT
to positive control, especially against BRaf
Table 2
, BRaf and CRaf as shown in Table 2.
The Pan-Raf activity of chosen compounds
IC50 (nM)
Compd
V600E
WT
BRaf
ARaf
194.0
17.8
43.4
37.6
40.7
34.1
46.9
57.4
BRaf
287.0
17.2
14.2
10.5
21.2
33.8
52.1
60.0
CRaf
43.4
0.7
P-1
I-01
I-13
I-14
I-15
I-35
I-36
I-37
77.2
2.8
24.1
20.5
26.5
18.7
19.5
23.4
8.5
6.3
14.4
18.7
21.9
34.5
1
3

ACCEPTED MANUSCRIPT
I-39
I-41
14.3
12.2
31.6
38.1
42.2
25.3
2.1
39.8
29.7
6.9
21.4
13.3
135.0
6.2
vemurafenib
sorafenib
7.1
22.6
Against all the subtypes of Raf isoforms, I-01 exhibited superior inhibitory activity to P-1. This
result displayed the importance of nitrogen atom position on pyrimidine ring.
2
.4. Antiproliferative activity of chosen compounds against various cell lines
As described above, Rafs are important targets in developing small-molecular inhibitors for
cancer therapies, especially melanoma, colon cancer, leukemia, hepatoma and thyroidcarcinoma
[6]. A375 [26], SK-Mel-2 [27] and COLO-205 [28] cell lines have extraordinary expression of
V600E
BRaf
. MV4-11 [8], HepG2 and SW579 [29] cell lines have extraordinary expression of
WT
BRaf
.
5
2
.4.1. SAR exploration of R group in terms of antiproliferative activity
Antiproliferative activities of compounds with good enzyme activity against different cell lines
were tested, and the results were summarized in Fig. 5.
Fig. 5. The cellular activity of compounds against cell lines under the concentration of 10 µM
Initially, morpholine substituent was introduced at the terminal phenyl to achieve I-09, which
resulted in improved potency against all the cell lines compared to I-01. Having successfully
improved the cellular potency, the effect of increasing the length of the linker chain bearing the
solubilizing functionality was explored. When the linker length was increased from zero atom
(I-09) to one atom (I-11), four atoms (I-15) or one atom (I-13) to two atoms (I-14), the potency
increased at the cellular level. The comparison between morpholine and N-methylpiperidine
1
4

ACCEPTED MANUSCRIPT
substituent displayed that the bulky space and electron donating N-methylpiperidine would be
better suited to increase cellular activity.
V600E
Although I-18 displaying optimal BRaf
activity (IC = 0.3 nM) of this series compounds
50
was more potent than vemurafenib (IC = 31.6 nM) and sorafenib (IC = 38.1 nM), its cellular
5
0
50
activity was completely different. To a certain extent, I-17–I-18 displayed better cellular potency
compared to I-01. Howerver, the improved degree was far less than I-13–I-15.
Interestingly, within this series of compounds, good correlation wasn’t observed between
enzyme inhibition and cell inhibition. The strategy of introducing hydrophilic groups at terminal
phenyl was better than the strategy of removing lipophilic groups in cell activity.
The antiproliferative activities of I-13–I-15 against A375, COLO-205, MV4-11 and SW579 cell
lines were comparable to positive control. When I-13–I-15 were investigated on SK-Mel-2 and
HepG2 cell lines, their potency proved to be superior to positive control. It should be noted that
I-14 was superior to others.
Placed at pyrimidine ring, smaller alkyl groups I-26–I-27 were beneficial to cellular inhibition,
indicating that the long-chained alkyl would be better suited to increase cell activity. The change
in celluar inhibition profile was in a range close to that for I-29–I-30 compared to I-01.
To explore the electrical requirement in solvent accessible region, electron donating group
hydroxyl and electron withdrawing group carboxyl was introduced to the terminal side chain of
I-01 to achieve I-31 and I-33, respectively. Comparison of these two compounds showed that
introduction of a hydroxyl group resulted in improved potency against all the cell lines. However,
introduction of carboxyl group resulted in loss of antiproliferative activity, demonstrating that the
electron donating group would be better suited to increase potency at the cellular level.
Then, various secondary amines of different steric sizes were explored. Initially, N, N-dimethyl
amino substitution I-34 was introduced through a two-carbon linker at the pyrimidine ring, which
resulted in improved potency against all the cell lines compared to I-01. Having successfully
improved the cellular potency, we investigated the introduction of cyclic secondary amines such as
pyrollidine I-36, piperidine I-37, N-methylpiperazine I-38–I-41, and morpholine I-42–I-43 as the
amino functionality. I-36–I-43 (cyclic secondary amines) exhibited significantly improved
antiproliferative activity compared to I-34. This result displayed that the cyclic secondary amines
would be better suited to increase cell activity.
1
5

ACCEPTED MANUSCRIPT
The effect of increasing the length of the linker carbon chain bearing the solubilizing amino
functionality was explored. When the linker carbon length was increased from zero carbon (I-38)
to one caron (I-39) or two carbons (I-29, I-34, I-40 and I-42) to three carbons (I-30, I-35, I-41
and I-43), the potency increased at enzyme as well as cell level.
2
.4.2. The IC values of chosen compounds with well antiproliferative activity
50
Then, the compounds with well antiproliferative activity were tested on different cell lines to
obtain the IC values. The datas showed in Table 3.
5
0
Table 3
The IC values of chosen compounds against different cell lines
50
IC50 (µM)
Compd
A375
2.68
0.68
4.73
3.74
3.60
2.83
2.27
1.81
1.02
0.70
13.64
SK-Mel-2 COLO-205 MV4-11
HepG2 SW579
I-13
I-14
1.49
0.71
2.97
1.58
1.61
1.45
1.54
0.83
0.36
5.64
11.35
1.62
1.36
2.70
2.35
2.71
1.96
0.93
0.73
0.75
5.16
7.04
1.13
0.24
0.82
1.44
1.28
0.75
0.50
0.54
0.29
NA
1.67
0.47
1.81
3.28
3.41
2.76
1.45
1.81
1.20
5.48
2.84
1.42
0.53
0.94
2.34
2.48
1.91
1.49
0.71
0.39
NA
I-15
I-36
I-37
I-38
I-39
I-40
I-41
vemurafenib
sorafenib
0.30
1.73
The antiproliferative activities of compounds I-13–I-15 and I-36–I-41 against A375,
COLO-205 and MV4-11 cell lines were comparable to positive control. When compounds I-13–
I-15 and I-36–I-41 were studied on SK-Mel-2, HepG2 and SW579 cell lines, their potency proved
to be superior to positive control.
Overall, all the compounds bearing the solubilizing functional group showed improved
antiproliferative activity compared to the initial lead I-01. Interestingly, within this series of
compounds, good correlation was observed between enzyme inhibition and cellular inhibition.
1
6

ACCEPTED MANUSCRIPT
The data in Fig. 4 and Table 3 indicated the importance of solubilizing functional groups in the
solvent exposed pocket for cellular inhibition. Although introducing of solubilizing functional
groups resulted in a decrease in enzyme inhibition, their potency were still comparable to positive
control. Meanwhile, probably due to enhanced aqueous solubility improved cellular permeability,
the modification successfully improved the cellular potency.
2
.4.3. The anti-tumor spectrum of representative compound I-39
In order to expand spectrum of tumor cells, representative compound I-39 was submitted to
NCI (National Cancer Institute) to test on a panel of 57 cell lines as shown in Table 4. I-39 was
found to display potent antiproliferative activity against Leukemia, Colon Cancer, CNS Cancer
and Melanoma. The experimental process was presented in Experimental section (see Supporting
Information).
Table 4
The growth percent of I-39 on 57 cell lines by NCI
Cell line
GI50 (µM)
2.02
0.32
2.03
1.61
2.29
2.46
2.00
1.21
1.36
1.94
2.00
2.13
2.01
1.85
2.39
0.61
1.60
1.73
2.58
2.54
Cell line
GI50 (µM)
2.08
CCRF-CEM
K-562
SF-268
Leukemia
SF-539
1.61
MOLT-4
RPMI-8226
A549
CNS Cancer
SNB-19
SNB-75
U251
2.18
0.96
1.63
EKVX
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
2.17
HOP-62
2.57
Non-Small Cell
Lung Cancer
HOP-92
13.6
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO205
HCC-2998
HCT-116
HCT-15
Ovarian Cancer
3.28
3.72
NCI/ADR-RES 5.46
SK-OV-3
MCF7
2.17
1.97
MDA-MB-231 1.42
Breast Cancer
HS 578T
BT-549
T-47D
1.99
3.01
4.14
Colon Cancer
HT-29
MDA-MB-468 1.83
KM12
786-0
A498
1.72
2.52
SW-620
1
7

ACCEPTED MANUSCRIPT
LOX IMVI
MALME-3M
M14
1.67
ACHN
RXF393
SN12C
TK-10
UO-31
PC-3
2.16
1.49
2.56
3.89
1.82
3.30
1.58
1.20
1.23
1.84
0.90
1.73
1.61
2.00
1.96
Renal Cancer
MDA-MB-435
SK-Mel-2
SK-Mel-28
SK-Mel-5
UACC-257
UACC-62
Melanoma
Prostate Cancer
DU-145
LC50 > 100 µM
2
.5. Research on biological mechanisms
I-14 and I-41 with well enzyme and cell activity was selected to study the possible biological
mechanisms, which displayed dose dependent inhibition against the phosphorylation of ERK in
A375 and SK-Mel-2 cancer cells by Western blot analysis. A key finding from this class of
compounds was that, unlike vemurafenib, they dramatically inhibited the proliferation of
melanoma A375 cells through ERK pathway, without paradoxical activation of the MAPK in
SK-Mel-2 cells. These results were shown as Fig. 6.
Our results supported the hypothesis that the Pan-Raf inhibition might be a tractable strategy to
V600E
overcome the intrinsic and acquired resistance of melanoma cancer against the current BRaf
inhibitor therapy.
Fig. 6. (A) ERK kinase inhibition in A375 cell (B) ERK kinase inhibition in SK-Mel-2 cell
2
.6. Research on pharmacokinetics behavior
The metabolic stability results of 0.5 µM I-01, I-14, I-41 and reference compound sorafenib in
.8 mg/mL human liver microsomal were shown in Fig. 7. I-01, I-14, I-41 and sorafenib had T1/2
0
1
8

ACCEPTED MANUSCRIPT
as 1.1 h, 3.1 h, 5.0 h and 5.6 h, respectively. The experimental process was presented in
Experimental section (see Supporting Information).
Fig. 7. The metabolic stability results of I-01, I-14, I-41 and sorafenib
The favorable in vitro profiles prompted us to evaluate I-14 and I-41 in vivo. The
pharmacokinetic (PK) parameters of I-14 and I-41 were assessed in male SD rat following
intravenous (IV) and oral (PO) administrations as described in Table 5 and Table 6.
I-14 possesses favorable pharmacokinetic profiles with high oral exposures (AUC0−∞) of over
3
9
103.6 µg/L·h at a 10 mg/kg oral dose and acceptable half-life (T1/2 = 9.1 h).
After IV administration I-41 at a dose of 2.5 mg/kg, maximum concentration (Cmax) reached
7.3
µg/L, and area under the curve (AUC0−∞) was 428.4
µg/L·h. After oral administration at 10
mg/kg, Cmax reached 61.0
µg/L at 6.7 h post dosing, and AUC0−∞ was 734.5 µg/L·h. These results
demonstrate good pharmacokinetic properties of I-41 in rat with high oral bioavailability F = 44%
and a sustained plasma concentration exceeding in vitro EC during at least 13 h after oral dosing.
5
0
I-14 and I-41 also exhibited low clearance (CL) and high volume of distribution (Vss).
Table 5
Pharmacokinetic profile of I-14 in rat
PK parameters (n=3)
AUC(0-t) (ug.h/L)
AUC(0-∞) (ug.h/L)
IV (2.5 mg/Kg)
235.2
SD
77.3
102.6
PO (10 mg/Kg)
1247.6
SD
442.9
317.8
305.5
3103.6
1
9

ACCEPTED MANUSCRIPT
t1/2 (h)
Tmax (h)
2.2
0.22
8.9
1.1
0.24
3.2
9.1
6.7
1.4
2.3
CL (L/h/Kg)
V (L/Kg)
Cmax (ug/L)
3.2
1.1
25.5
131.9
11.1
70.5
42.5
177.9
14.2
73.0
Table 6
Pharmacokinetic profile of I-41 in rat
PK parameters (n=3)
AUC(0-t) (ug.h/L)
AUC(0-∞) (ug.h/L)
t1/2 (h)
IV (2.5 mg/Kg)
399.7
428.4
6.3
SD
36.0
61.2
1.7
PO (10 mg/Kg)
SD
698.2
734.5
4.9
38.7
47.4
1.2
Tmax (h)
0.083
5.9
0
6.7
1.3
CL (L/h/Kg)
V (L/Kg)
0.8
13.9
96.6
61.0
44
2.5
52.9
6.5
21.8
5.9
Cmax (ug/L)
F (%)
97.3
13.0
a
N/A
6.6
a
N/A : not applicable
The Plasma Concentration-time curve of I-14 and I-41 was shown in Fig. 8, the experimental
process was presented in Experimental section (see Supporting Information).
Fig. 8. The Plasma Concentration-time curve of I-14 and I-41
From the Plasma Concentration-time curve, we know I-14 was eliminated faster in serum
than I-41 and absorbed slower by oral. The SD value of I-14 was higher than I-41,too. These data
indicate a favorable pharmacokinetic profile of I-41 in preparation for efficacy studies in rat.
3
.
Conclusion
2
0

ACCEPTED MANUSCRIPT
Replacement of 2,4-substituted pyrimidine group of P-1 by 4,6 -substituted pyrimidine group in
hinge moiety afforded I-01 as a novel Pan-Raf inhibitory template, inhibits all subtypes of Rafs
V600E
WT
with IC values of 17.2 nM (BRaf
), 17.8 nM (ARaf), 2.8 nM (BRaf ) and 0.7 nM (CRaf),
5
0
respectively. On the basis of insights obtained from the binding mode of I-01, various solubilizing
4
functional groups were introduced to the terminal phenyl ring as R group or the 4-position
5
pyrimidine ring as R group to get a better occupation of solvent accessible region. This
optimization campaign led to the identification of I-14 and I-41 as potent Pan-Raf inhibitors with
well enzymatic and cellular activity. As the representative compounds, I-14 inhibited all subtypes
V600E
WT
of Raf proteins with IC values of 20.5 nM (BRaf
), 37.6 nM (ARaf), 10.5 nM (BRaf ) and
5
0
6
.3 nM (CRaf); I-41 inhibited all subtypes of Raf proteins with IC50 values of 12.2 nM
V600E
WT
(BRaf
), 25.3 nM (ARaf), 29.7 nM (BRaf ) and 13.3 nM (CRaf), more potent than positive
control. In particular, I-14 exhibited the better IC values of 0.71 µM, 1.36 µM, 0.53 µM and 0.47
5
0
µM against SK-Mel-2, COLO205, SW579 and HepG2 cell lines, which also displayed potency
against A375 and MV4-11 cell lines comparable to positive control. These selected cell lines were
V600E
WT
extraordinarily expressed BRAF
or BRaf . The anti-proliferation activities of I-41 against
different cell lines was similar to I-14. Representative compound I-39 also has a wide anti-tumor
spectrum.
Futher mechanism investigation in terms of the Erk inhibition in human melanoma A375 and
SK-Mel-2 cell lines by Western blot is investigated. The results revealed that I-14 and I-41 inhibit
the proliferation of melanoma A375 cells through Erk pathway, without paradoxical activation of
Erk in melanoma SK-Mel-2 cells. This result supports the hypothesis that the Pan-Raf inhibition
might be a tractable strategy to overcome the intrinsic and acquired resistance of melanoma
V600E
against the current BRaf
inhibitor therapy. The metabolic stability results of I-41 in human
liver microsomal and better pharmacokinetics profile in rat proved I-41 was suitable in
preparation for efficacy studies in rat.
Thus, I-41 may be a promising compound to be developed as an alternative or aid for current
sorafenib and vemurafenib therapeutics [30-34].
4
.
Experimental section
4
.1. Computer Aided Drug Design
4
.1.1. Molecular docking and fragment-based strategy
2
1

ACCEPTED MANUSCRIPT
Crystal structures of BRaf (PDB code 3IDP and 1UWJ) were downloaded from Protein Data
Bank (PDB) and prepared using the Protein Preparation Wizard in the Schrödinger suite [35]. All
compounds were initially minimized by the LigPrep module in Schrödinger. Because of its
excellent performance through a self-docking analysis, the Glide module with extra precision [XP]
mode was selected for molecular docking [36], and the 10 best poses of each ligand were
minimized by a post docking program with the best pose saved for further analysis. Fragmentation
of compounds from various databases were accordining to the method proposed before [37] and
the recombination of compounds were conducted using the Recap algorim in Pipeline Pilot 7.5
[38].
4
.1.2. Molecular dynamics and MM/PBSA
The molecular docking-based protein complexes were used for MD simulation which was
carried out using GROMACS [39] with AMBER03 force field [40] and TIP3P water model [41].
All simulations were conducted in a water filled dodecahedron box with the distance at least 12 Å
+
-
between the complex and the box edges. By adding counter ions, either Na or Cl by the genion
tool, the charges of the system were neutralized. Then, an energy minization process with
conjugate gradient algorithm followed by a steepest descent algorithm was used to relax the
system. Afterwards, an NVT simulation was performed by increasing the temperature going from
1
00 to 300 K and then the pressure was equilibrated by an NPT simulation. Finally, a 20 ns
production MD was conducted at the temperature of 300 K. Bond lengths were constrained using
LINCS algorithm [42]. The last 5 ns of each production simulation were extracted at every 10 ps
interval and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) was used to
calculated the binding energy with AMBER12 package [43, 44]. The RMSD, and number of
hydrogen bonds were analyzed in GROMACS package [45].
4
.2. Chemistry synthesis
All starting materials were obtained from commercial suppliers and used without further
purification. NMR spectrum recorded, on aBrukerDPX-300 spectrometer, in DMSO-d using
6
TMS as the internal standard. Chemical shifts (d) were reported in parts per million downfield
from the internal standard. The signals were quoted as s (singlet), d (doublet), t (triplet), m
(
multiplet). Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F254,
.25 mm per-coated TLC plates. TLC plates were visualized using UV254. Column
chromatography was conducted on silica gel (200–300 mesh). Mass spectra were obtained on a
0
2
2

ACCEPTED MANUSCRIPT
Waters Quattro Micromass instrument using electrospray ionization (ESI) technique.
4
.2.1. Diethyl 2-(6-chloropyrimidin-4-yl) malonate (I-a)
To a solution of diethyl malonate (16.5 g, 99 mM) in THF (300 mL) were added sodium
o
hydride (60%, 8.0 g, 198 mM) successively. The mixture was stirred at 0 C for 0.5 h, followed by
addition of the required 4, 6-dichloropyrimidine (9.8 g, 66 mM) and stirring at room temperature
for another 2.5 h. The reaction mixture was diluted in 300 mL saturated ammonium chloride
solution, the aqueous layer was extracted with EtOAc (500 mL×3). The combined organic layers
were washed with brine (300 mL×3), and then dried over anhydrous MgSO . The mixture was
4
evaporated in vacuo and purified by silica gel column chromatography to obtain I-a as oil.
+
ESI-MS m/z: 149.0 [M+H] .
4
.2.2. Ethyl 2-(6-chloropyrimidin-4-yl) acetate (I-b)
I-a (8.2 g, 30 mM) and sodium ethoxide (0.7 g, 3 mM) in ethanol (100 mL) were heated to
reflux for 2.5 h. After cooling the reaction, HCl (1 M) was added to adjust pH to 7. The mixture
was evaporated in vacuo and purified by silica gel column chromatography to get I-b as oil.
+
ESI-MS m/z: 201.0 [M+H] .
4
4
.2.3. General procedure for the preparation of ester (I-c–II-c)
.2.3.1. Ethyl 1-(6-chloropyrimidin-4-yl) cyclopropane-1-carboxylate (I-c)
The mixture of I-b (5.0 g, 25 mM), sodium hydroxide (2.0 g, 50 mM) and 1, 2-dibromoethane
(15.9 g, 100 mM) were stirred at room temperature in DMF (N, N-dimethylformamide, 150 mL)
for 5 h. The mixture was diluted in 150 mL water, the aqueous layer was extracted with ethyl
acetate (250 mL×3). The combined organic layers were evaporated in vacuo and purified by silica
+
gel column chromatography to get I-c as oil. ESI-MS m/z: 227.1 [M+H] .
4
.2.3.2. Ethyl 2-(6-chloropyrimidin-4-yl)-2-methylpropanoate (II-c)
II-c (2.1 g, 94.3%) was prepared from I-b (2.1 g, 10.3 mM), sodium hydroxide (0.8 g, 20.6 mM)
+
and iodomethane (5.9 g, 41.3 mM), in a manner similar to I-c. ESI-MS m/z: 229.1 [M+H] .
4
.2.4. General procedure for the preparation of methylamino analogs (I-d-01–II-d-01)
.2.4.1. Ethyl 1-(6-(methylamino) pyrimidin-4-yl) cyclopropane-1-carboxylate (I-d-01)
The mixture of I-c (6.3 g, 28 mM) and methylamine (33%, 24 mL, 170 mM) were stirred at
4
room temperature for 2 h. The mixture was evaporated in vacuo and purified by silica gel column
+
1
chromatography to get I-d (3.3 g, 87.9%) as a white solid. ESI-MS m/z: 222.1 [M+H] . H-NMR
300 MHz, DMSO-d ): δ 1.18 (3H, t, OCH CH , J = 7.1 Hz), 1.42-1.48 (4H, m, CH ), 2.81 (3H, d,
(
6
2
3
2
NHCH J = 4.2 Hz), 4.01-4.16 (2H, m, OCH CH ), 6.72 (1H, s, NH), 7.35 (1H, s, pyrimidine-H),
3,
2
3
8
.32 (1H, s, pyrimidine-H).
4
.2.4.2. Ethyl 2-methyl-2-(6-(methylamino)pyrimidin-4-yl) propanoate (II-d-01)
II-d (2.1 g, 94.3%) was prepared from II-c (2.1 g, 9.2 mM) and methylamine (33%, 6.5 mL, 46
+
mM), in a manner similar to I-d-01. ESI-MS m/z: 224.1 [M+H] .
4
.2.5. General procedure for the preparation of target compounds (I-01–I-43 and II-01–II-13)
4
.2.5.1. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)
2
3

ACCEPTED MANUSCRIPT
cyclopropane-1-carboxamido)benzamide (I-01)
The mixture of I-d-01 (0.33 g, 1.5 mM) and the intermediate A1 (0.49 g, 1.5 mM) in anhydrous
toluene (5 mL) was degassed for 10 min and with nitrogen gas refilled. To the stirred reaction
mixture was added a solution of Al (CH ) (2 M solution in toluene, 1.5 mL) dropwise via syringe
3
3
o
o
over 5 min at 0 C. The resulting suspension was stirred for 0.5 h, then heated to 80 C and stirred
o
until all solid was dissolved. Reaction mixture was further stirred for 4–6 h at 80 C until TLC
analysis showed the complete consumption of A1.The mixture was quenched with C H OH (10
2
5
mL). The mixture was evaporated in vacuo and purified by silica gel column chromatography to
o
1
get I-01 (0.66 g, 87.2%) as white solid. mp: 229–230 C. H-NMR (300 MHz, DMSO-d ): δ
6
1
3
.32-1.34 (2H, m, CH ), 1.58-1.65 (2H, m, CH ), 2.36 (3H, s, ArCH ), 2.81 (3H, d, NHCH J =
2 2 3 3,
.3 Hz), 6.32-6.43 (1H, m, NH), 7.40-7.42 (2H, m, ArH), 7.68-7.72 (2H, m, ArH), 8.13 (1H, dd,
ArH, J = 2.1 Hz, J = 8.7 Hz), 8.35-8.36 (2H, m, ArH, ArH), 8.47 (1H, s, ArH), 10.58 (1H, s,
+
NHCO), 11.21 (1H, s, NHCO). HRMS (ESI) calcd for C H ClF N O [M] , 504.1336; found,
2
4
22
3
5
2
5
04.1408.
.2.5.2. 4-chloro-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)cyclopropane-1-carboxamido
4
)phenyl)-3-(trifluoromethyl)benzamide (I-02)
I-02 (0.63 g, 83.4%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A12
o
1
(
0.49 g, 1.5 mM) as white solid, in a manner same as I-01. mp: 220–221 C. H-NMR (300 MHz,
DMSO-d ): δ 1.30-1.32 (2H, m, CH ), 1.55-1.57 (2H, m, CH ), 2.26 (3H, s, ArCH ), 2.80-2.82
6
2
2
3
(
3H, m, NHCH ), 6.35-6.38 (1H, m, NH), 7.21 (1H, d, ArH, J = 8.4 Hz), 7.43 (1H, q, ArH, J = 4.7
3
Hz), 7.55 (1H, dd, ArH, J = 2.0 Hz, J = 8.2 Hz), 7.92 (1H, d, ArH, J = 8.4 Hz), 8.19 (1H, s, ArH),
8
1
1
1
.27 (1H, dd, ArH, J = 1.7 Hz, J = 8.4 Hz) , 8.40 (1H, d, ArH, J = 1.5 Hz), 8.46 (1H, s, ArH),
1
3
0.50 (1H, s, NHCO), 11.50 (1H, s, NHCO). C-NMR (300 MHz, DMSO) δ 168.63, 163.26,
62.78, 157.04, 136.90, 136.66, 134.04, 133.31, 131.78, 130.05, 127.02, 126.95, 125.01, 124.44,
20.82, 116.56, 115.02, 101.66, 35.46, 29.55, 27.03, 17.57. HRMS (ESI) calcd for
+
C H ClF N O [M] , 504.1336; found, 504.1413.
2
4
22
3
5
2
4
.2.5.3. N-(5-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-2-methylphenyl)-1-(6-(methylamin)
pyrimidin-4-yl)cyclopropane-1-carboxamide (I-03)
I-03 (0.61 g, 78.7%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A13
o
1
(
0.52 g, 1.5 mM) as white solid, in a manner same as I-01. mp: 140–141 C. H-NMR (300 MHz,
2
4

ACCEPTED MANUSCRIPT
DMSO-d ): δ 1.29-1.30 (2H, m, CH ), 1.56-1.57 (2H, m, CH ), 2.23 (3H, s, ArCH ), 2.80-2.82
6
2
2
3
(
3H, m, NHCH ), 6.34-6.36 (1H, m, NH), 7.10-7.13 (1H, m, ArH), 7.20 (1H, dd, ArH, J = 2.0 Hz,
3
J = 8.2 Hz), 7.43-7.44 (1H, m, ArH), 7.58-7.64 (2H, m, ArH), 7.95 (1H, s, ArH), 8.12 (1H, s, ArH),
1
3
8
.46 (1H, s, ArH), 8.84 (1H, s, CONH), 9.03 (1H, s, NHCO),11.12 (1H, s, NHCO). C-NMR (300
MHz, DMSO) δ 168.59, 163.33, 157.05, 152.27, 139.37, 137.15, 137.02, 131.90, 130.24, 126.44,
1
24.60, 122.92, 122.11, 120.98, 116.64, 116.56, 114.99, 114.52, 112.94, 29.64, 27.06, 17.40.
+
HRMS (ESI) calcd for C H ClF N O [M] , 519.1445; found, 519.1423.
2
4
23
3
6
2
4
.2.5.4. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-fluoro-3-(1-(6-(methylamino)pyrimidin-4-yl)
cyclopropane-1-carboxamido)benzamide (I-04)
I-04 (0.57 g, 75.3%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A2 (0.50
o
1
g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 234–235 C. H-NMR (300 MHz,
DMSO-d ): δ 1.32-1.34 (2H, m, CH ), 1.60-1.61 (2H, m, CH ), 2.78-2.81 (3H, m, CH ), 6.31-6.34
6
2
2
3
(1H, m, NH), 7.44-7.52 (2H, m, ArH), 7.71-7.81 (2H, m, ArH), 8.10 (1H, dd, ArH , J = 2.1 Hz, J =
8
.8 Hz), 8.33 (1H, d, ArH, J = 2.1 Hz), 8.46 (1H, s, ArH), 8.61 (1H, d, ArH, J = 6.0 Hz), 10.69
1
3
(
1H, s, NHCO),11.90 (1H, s, NHCO). C NMR (300 MHz, DMSO) δ 169.47, 164.57, 162.95,
1
1
5
4
56.22, 153.26, 138.56, 131.95, 130.71, 126.67, 126.47, 124.90, 124.63, 124.54, 124.29, 123.10,
+
15.42, 115.26, 115.14, 101.28, 29.32, 26.46, 18.40. HRMS (ESI) calcd for C H ClF N O [M] ,
2
3
19
4
5
2
08.1085; found, 508.1164.
.2.5.5. N-(4-chloro-3-(trifluoromethyl)phenyl)-2-fluoro-3-(1-(6-(methylamino)pyrimidin-4-yl)
cyclopropane-1-carboxamido)benzamide (I-05)
I-05 (0.59 g, 78.1%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A3 (0.50
o
1
g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 201–203 C. H-NMR (300 MHz,
DMSO-d ): δ 1.33-1.34 (2H, m, CH ), 1.61-1.62 (2H, m, CH ), 2.80-2.83 (3H, m, CH ), 6.27-6.32
6
2
2
3
(1H, m, NH), 7.30 (1H, t, ArH, J = 7.9 Hz), 7.38-7.44 (2H, m, ArH), 7.73 (1H, d, ArH, J = 8.8 Hz),
8
.01 (1H, dd, ArH , J = 2.0 Hz, J = 8.8 Hz), 8.23 (1H, t, ArH, J = 7.1 Hz), 8.31 (1H, d, ArH, J =
1
3
2
.0 Hz), 8.45 (1H, s, ArH), 10.91 (1H, s, NHCO),12.03 (1H, s, NHCO). C NMR (300 MHz,
DMSO) δ 169.37, 162.89, 156.78, 151.53, 148.27, 138.26, 132.12, 127.21, 127.06, 124.97, 124.57,
1
24.42, 124.34, 124.25, 123.88, 120.80, 118.38, 118.31, 100.68, 29.16, 26.99, 18.48. HRMS (ESI)
+
calcd for C H ClF N O [M] , 508.1085; found, 508.1151.
2
3
19
4
5
2
4
.2.5.6. N-(4-chloro-3-(trifluoromethyl)phenyl)- 4-chloro-3-(1-(6-(methylamino)pyrimidin-4-yl)
2
5

ACCEPTED MANUSCRIPT
cyclopropane-1-carboxamido)benzamide (I-06)
I-06 (0.66 g, 84.3%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A4 (0.52
o
1
g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 209–210 C. H-NMR (300 MHz,
DMSO-d ): δ 1.31-1.35 (2H, m, CH ), 1.68-1.71 (2H, m, CH ), 2.81-2.84 (3H, m, CH ), 6.28-6.32
6
2
2
3
(1H, m, NH), 7.47-7.49 (1H, m, ArH), 7.72-7.74 (3H, m, ArH), 8.11 (1H, dd, ArH , J = 2.3 Hz, J =
8
1
1
1
5
4
.8 Hz), 8.33 (1H, d, ArH, J = 2.3 Hz), 8.53 (1H, s, ArH), 8.81 (1H, s, ArH), 10.73 (1H, s,NHCO),
1
3
2.53 (1H, s, NHCO). C NMR (300 MHz, DMSO) δ 170.71, 169.38, 165.01, 163.35, 138.45,
35.81, 133.57, 131.99, 129.39, 127.17, 124.97, 124.41, 123.76, 121.94, 120.90, 119.01, 118.95,
+
00.67, 29.01, 27.01, 18.84. HRMS (ESI) calcd for C H Cl F N O [M] , 524.0790; found,
2
3
19
2
3
5
2
24.0866.
.2.5.7. N-(4-chloro-3-(trifluoromethyl)phenyl)-3-(1-(6-(methylamino)pyrimidin-4-yl)
cyclopropane-1-carboxamido)benzamide (I-07)
I-07 (0.64 g, 87.1%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A5 (0.47
o
1
g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 218–220 C. H-NMR (300 MHz,
DMSO-d ): δ 1.35-1.38 (2H, m, CH ), 1.42-1.45 (2H, m, CH ), 2.78 (3H, d, CH , J = 3.9 Hz),
6
2
2
3
6
.37-6.39 (1H, m, NH), 7.34 (1H, s, ArH), 7.50 (1H, t, ArH, J = 8.0 Hz), 7.65-7.74 (2H, m, ArH),
.89 (1H, d, ArH, J = 8.0 Hz), 8.11 (1H, dd, ArH , J = 2.3 Hz, J = 8.8 Hz), 8.21 (1H, s, ArH), 8.35
7
1
3
(
1H, d, ArH, J = 2.3 Hz), 8.40 (1H, s, ArH), 10.66 (2H, s, NHCO). C NMR (300 MHz, DMSO)
δ 169.21, 165.89, 163.43, 162.86, 157.61, 139.32, 138.64, 134.84, 131.85, 128.66, 126.54, 124.75,
1
24.20, 123.12, 122.41, 119.25, 118.89, 118.75, 102.29, 31.57, 27.07, 16.41. HRMS (ESI) calcd
+
for C H ClF N O [M] , 490.1179; found, 490.1258.
23
20
3
5
2
4
.2.5.8. N-(4-chloro-3-(trifluoromethyl)phenyl)-2,4-difluoro-3-(1-(6-(methylamino)pyrimidin-4-
yl)cyclopropane-1-carboxamido)benzamide (I-08)
I-08 (0.54 g, 68.3%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A14
o
1
(
0.53 g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 156–157 C. H-NMR (300 MHz,
DMSO-d ): δ 1.38-1.43 (4H, m, CH CH ), 2.78 (3H, d, CH , J = 3.9 Hz), 6.44-6.46 (1H, m, NH),
6
2
2
3
7
.22 (1H, q, ArH, J = 9.0 Hz), 7.33-7.36 (1H, m, ArH), 7.52 (1H, q, ArH, J = 5.9 Hz), 7.94 (1H, d,
ArH, J = 8.3 Hz), 8.26 (1H, d, ArH, J = 8.3 Hz), 8.38-8.42 (2H, m, ArH), 10.24 (1H, s, NHCO),
1
3
1
0.53 (1H, s, NHCO). C NMR (300 MHz, DMSO) δ 169.81, 163.36, 162.93, 157.59, 153.88,
1
34.20, 133.44, 132.88, 132.07, 127.12, 127.06, 125.32, 125.19, 121.77, 115.49, 115.21, 110.96,
2
6

ACCEPTED MANUSCRIPT
+
1
5
4
10.64, 30.55, 27.09, 16.50. HRMS (ESI) calcd for C H ClF N O [M] , 526.0991; found,
23 18 5 5 2
26.1065.
.2.5.9. 4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-
morpholino-3-(trifluoromethyl)phenyl)benzamide (I-09)
I-09 (0.73 g, 88.3%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A19
o
1
(
0.57 g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 142–143 C. H-NMR (300 MHz,
DMSO-d ): δ 1.31-1.33 (2H, m, CH ),1.57-1.59 (2H, m, CH2), 2.36 (3H, s, ArCH ), 2.82-2.83
6
2
3
(
8H, m, CH ), 3.70-3.71 (3H, m, NHCH ), 6.38-6.39 (1H, m, NH), 7.38-7.43 (2H, m, NH), 7.60
2
3
(1H, d, ArH, J = 8.8 Hz),7.67-7.68 (1H, m, ArH), 8.05-8.07 (1H, m, ArH), 8.16 (1H, d, ArH, J =
1
3
2
.2 Hz), 8.35 (1H, s, ArH), 8.47 (1H, s, ArH), 10.43 (1H, s, NHCO), 11.16 (1H, s, NHCO). C
NMR (300 MHz, DMSO) δ 168.99, 165.35, 163.64, 163.13, 157.01, 147.08, 136.98, 136.48,
1
5
5
4
34.11, 132.46, 130.22, 129.34, 126.19, 125.13, 124.77, 123.49, 122.51, 118.35, 118.28, 66.61,
+
3.44, 29.68, 26.99, 18.10, 17.67. HRMS (ESI) calcd for C H F N O [M] , 554.2253; found,
2
8
30
3
6
3
55.2333.
.2.5.10. 4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-(4-
methylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)benzamide (I-10)
I-10 (0.63 g, 74.1%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A20
o
1
(
0.59 g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 177–178 C. H-NMR (300 MHz,
DMSO-d ): δ 1.37-1.39 (2H, m, CH ),1.76-1.78 (2H, m, CH2), 2.27 (3H, s, ArCH ), 2.28-2.29
6
2
3
(
3H, m, NHCH ), 2.41 (3H, s, NCH ), 6.37-6.38 (1H, m, NH), 7.36-7.43 (2H, m, ArH), 7.56 (1H,
3 3
d, ArH, J = 8.8 Hz), 7.66-7.70 (1H, m, ArH), 8.06 (1H, dd, ArH, J = 2.2 Hz, J = 8.8 Hz), 8.14 (1H,
d, ArH, J = 2.2 Hz), 8.32 (1H, s, ArH), 8.46 (1H, s, ArH), 10.40 (1H,s,NHCO),11.16 (1H, s,
1
3
NHCO). C NMR (300 MHz, DMSO) δ 169.00, 165.39, 163.06, 157.30, 157.02, 146.21, 139.81,
1
5
5
4
36.98, 136.81, 132.44, 130.18, 125.06, 124.71, 123.49, 122.60, 121.92, 118.42, 118.33, 53.90,
+
1.22, 43.59, 29.71, 18.11. HRMS (ESI) calcd for C H F N O [M] , 568.2570; found,
2
9
33
3
7
2
68.2650.
.2.5.11. 4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-(
morpholinomethyl)-3-(trifluoromethyl)phenyl)benzamide (I-11)
I-11 (0.70 g, 82.1%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A17
o
1
(
0.59 g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 129–132 C. H-NMR (300 MHz,
2
7

ACCEPTED MANUSCRIPT
DMSO-d ): δ 1.32-1.33 (2H, m, CH ), 1.57-1.58 (2H, m, CH ), 2.36-2.38 (7H,m, N(CH ) and
6
2
2
2 2
NCH ), 2.80-2.81 (3H, m,NH), 3.58-3.59 (6H,m, NCH and O(CH ) ), 6.38-6.39 (1H, m, NH),
3
2
2 2
7
.41 (2H, m, ArH), 7.71 (2H, m, ArH), 8.06 (1H, d, ArH, J = 8.4 Hz), 8.21 (1H, s, ArH), 8.34 (1H,
1
3
s, ArH), 8.47 (1H, s, ArH), 10.47 (1H, s, NHCO), 11.16 (1H, s, NHCO). C NMR (300 MHz,
DMSO) δ 168.98, 165.51, 163.37, 160.37, 156.93, 142.16, 138.37, 136.97, 135.49, 134.19, 132.48,
1
1
4
31.27, 130.17, 123.47, 123.40, 122.50, 117.30, 117.16, 101.29, 66.19, 57.82, 53.24, 29.66, 26.98,
+
8.07, 17.48. HRMS (ESI) calcd for C H F N O [M] , 569.2410; found, 569.2485.
29
32
3
6
3
.2.5.12. 4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-((4-
methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide (I-12)
I-12 (0.59 g, 67.7%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A18
o
1
(
0.61 g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 165–166 C. H-NMR (300 MHz,
DMSO-d ): δ 1.31-1.33 (2H, m, CH ), 1.57-1.59 (2H, m, CH ), 2.10 (3H, s, ArCH ), 2.28-2.36
6
2
2
3
(
10H, m, N(CH ) ), 2.36 (3H, s, ArCH ), 2.73-2.82 (3H, m, NHCH ), 3.55-3.56 (3H, m, NCH ),
2 2 3 3 3
6
.38-6.39 (1H, m, NH), 7.41 (2H, t, ArH, J = 8.0 Hz), 7.69 (2H, d, ArH, J = 8.4 Hz), 8.05 (1H, d,
ArH, J = 8.4 Hz), 8.20 (1H, s, ArH), 8.33 (1H, s, ArH), 8.46 (1H, s, ArH), 10.46 (1H, s, NHCO),
1
3
1
1.16 (1H, s, NHCO). C NMR (300 MHz, DMSO) δ 168.97, 165.50, 163.68, 163.18, 156.99,
1
5
5
4
38.27, 137.01, 133.94, 132.51, 131.80, 131.06, 130.12, 123.40, 122.49, 122.37, 117.20, 117.12,
+
7.40, 54.64, 52.59, 45.57, 29.56, 27.06, 18.11, 17.62. HRMS (ESI) calcd for C H F N O [M] ,
3
0
35
3
7
2
82.2726; found, 582.2795.
.2.5.13. 4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-((1-
methylpiperidin-4-yl)oxy)-3-(trifluoromethyl)phenyl)benzamide (I-13)
I-13 (0.60 g, 68.2%) was prepared from I-d-01 (0.33 g, 1.5 mM) and the intermediate A21
o
1
(
0.61 g, 1.5 mM) as a white solid, in a manner same as I-01. mp: 91–94 C. H-NMR (300 MHz,
DMSO-d ): δ 1.31-1.32 (2H, m, CH ), 1.57-1.58 (2H, m, CH ), 1.90 (3H, s, ArCH ), 2.18-2.19
6
2
2
3
(
4H, m, CH CHCH ), 2.24-2.26 (2H, m, NCH ), 2.39 (3H, s, NCH ), 2.54-2.55 (2H, m, NCH ),
2 2 2 3 2
2
.88 (3H, d, NHCH , J = 3.3 Hz), 4.58 (1H, m, OCH) , 6.41-6.42 (1H, m, NH), 7.24-7.30 (1H, m,
3
ArH), 7.33-7.37 (1H, m, ArH), 7.39-7.45 (1H, m, ArH), 7.66 (1H, dd, ArH, J = 1.5 Hz, J = 7.9 Hz),
.97-8.00 (1H, m, ArH), 8.09 (1H, d, ArH, J = 2.5 Hz), 8.32 (1H, s, pyrimidine-H), 8.46 (1H, s,
7
1
3
pyrimidine-H), 10.30 (1H, s, NHCO), 11.13 (1H, s, NHCO). C NMR (300 MHz, DMSO) δ
1
68.97, 168.46, 165.15, 163.20, 156.97, 137.01, 133.61, 132.70, 131.90, 130.06, 125.56, 125.44,
2
8