Rational design, synthesis, and biological evaluation of Pan-Raf inhibitors to overcome resistance

Article abstract of DOI:10.1039/c7ob00518k

Selective BRaf^V600E inhibitors with DFG-in conformation have been proven effective against a subset of melanoma. However, representative inhibitor vemurafenib rapidly acquires resistance in the BRaf^WT cells through a CRaf or BRaf^WT dependent manner. Simultaneous targeting of all subtypes of Raf proteins offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Herein, we describe the design and characterization of a series of compounds I-01-I-22, based on a pyrimidine scaffold with DFG-out conformation as Pan-Raf inhibitors. Among them, I-15 binds to all Raf protomers with IC₅₀ values of 12.6 nM (BRaf^V600E), 30.1 nM (ARaf), 19.7 nM (BRaf^WT) and 17.5 nM (CRaf) and demonstrates cellular activity against BRaf^WT phenotypic melanoma and BRaf^V600E phenotypic colorectal cancer cells. The western blot results for the P-Erk inhibition in human melanoma SK-Mel-2 cell line showed that I-15 inhibited the proliferation of the SK-Mel-2 cell line at concentrations as low as 400 nM, without paradoxical activation of Erk as vemurafenib, which supported that I-15 may become a good candidate compound to overcome the resistance of melanoma induced by vemurafenib. I-15 also has a favorable pharmacokinetic profile in rats. Rational design, synthesis, SAR, lead selection and evaluation of the key compounds studied are described.

Full text of DOI:10.1039/c7ob00518k

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DOI: 10.1039/C7OB00518K
Rational design, synthesis, and biological evaluation
of Pan-Raf inhibitors to overcome resistance
Lu Wang a, Gaoyuan Zhu a, Qing Zhang b, Chunqi Duan, Yanmin Zhang a, Zhimin
Zhang a, Yujun Zhou a, Tao Lu a,c* , Weifang Tang a,*
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.
c State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing, Jiangsu 210009, China.
Abstract: Selective BRafV600E inhibitors with DFGꢀin conformation have been
proven effective against a subset of melanoma. However, representative inhibitor
vemurafenib rapidly acquire resistance in the BRafWT cells through a CRaf or BRafWT
dependent manner. Simutaneous targeting all subtypes of Raf proteins offer the
prospect of enhanced efficacy as well as reduced potential for acquired resistance.
Described herein is the design and characterization of a series of compounds
I-01~I-22 which based on pyrimidine scaffold with DFGꢀout conformation as
PanꢀRaf inhibitors. Among them, I-15 binds to all Raf protomers with IC50 values of
12.6 nM (BRafV600E), 30.1 nM (ARaf), 19.7 nM (BRafWT) and 17.5 nM (CRaf),
respectively, demonstrates cellular activity against BRafWT phenotypic melanoma and
BRafV600E phenotypic colorectal cancer cells. The western blot results for the PꢀErk
inhibition in human melanoma SKꢀMelꢀ2 cell line showed I-15 inhibited the
proliferation of SKꢀMelꢀ2 cell line at concentration as low as 400 nM, without
paradoxical activation of Erk as vemurafenib, which supported I-15 may become a
good candidate compound to overcome the resistance of melanoma induced by
vemurafenib. I-15 also have a favorable pharmacokinetic profile in rat. Rational
design, synthesis, SAR, lead selection and evaluation of the key compounds studies
are described.
Keywords: Pan-Raf inhibitors; cyclopropyl formamide; resistance
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1. Introduction
The RasꢀRafꢀMekꢀErk (mitogenꢀactivated protein kinase MAPK) cascade is a
central signaling component that plays a role in the initiation and regulation of most
of the stimulated cellular processes such as proliferation, survival and differentiation.1
As a downstream effector of Ras in the MAPK signaling pathway, Raf proteins are
cytosolic protein kinases that regulate cell responses to extracellular signals.2 There
are three 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.3ꢀ5 BRaf, unlike ARaf or CRaf, can be converted into an active oncogenic
protein BRafV600E (Val600
mimics phosphorylation of the activation loop, elevating the in vitro enzymatic
activity by up to 500~700
fold compared with its wildꢀtype counterpart.6 According
– ꢀpoint mutation. This substitution
Glu600, V600E) by single
–
to the binding modes of DFG (a conserved amino acid sequence of D594, F595 and
G596), which derived from crystallographic analysis, Raf kinase inhibitors were
categorized into two types, DFGꢀout and DFGꢀin series.7 The DFGꢀout inhibitors,
such as sorafenib8 and LY3009120,9 engage the protein in DFGꢀout conformation and
inhibit all subtypes of Raf proteins (Figure 1). The DFGꢀin inhibitors, such as
vemurafenib, bind to the ATP binding site of the kinase with a DFGꢀin conformation
and inhibits BRafV600E with high selectivity.10
Figure 1. Binding mode of BRafV600E (A) DFGꢀout conformation for PDB 1UWJ
(crystalized with sorafenib) (B) DFGꢀin conformation for PDB 3OG7 (crystalized
with vemurafenib)
Vemurafenib have shown significant clinical efficacy in melanoma patients
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harboring BRafV600E 11ꢀ13
However, after 6 months, the development of acquired
.
resistance (the resistance caused by vemurafenib) is reported as a wellꢀknown side
effect. The mechanism for acquired resistance remains complicated. In cells, wild type
Raf kinases signal as dimers comprising various ARaf, BRaf, and CRaf homodimers
or heterodimers. The binding mode of inhibitors like vemurafenib (and others which
cause paradoxical activation) fail to occupy both protomers of these cellular dimers.
Furthermore, the binding of vemurafenib and analogous inhibitors to a cellular BRaf
protomer conformationally transactivates the unbound protomer (usually CRaf or
another BRaf) to increased enzymatic activity and downstream signaling through
ERK.14ꢀ16 PanꢀRaf inhibitors can bind to cellular Raf dimers and occupy both
protomers of these dimers (Figure 2).
Figure 2. The mechanism of acquired resistance
About 20% of patients had intrinsic resistance and didn’t respond to the drug
despite the presence of BRafV600E 17
Ras mutantꢀdriven cancers signal through
.
BRaf/CRaf heterodimers, and that a PanꢀRaf inhibitor capable of inhibiting these
cellular forms would find utility in the treatment of Ras mutant driven cancers,
comprising approximately 1/3 of human cancers.18ꢀ20 Clinically, paradoxical
activation promotes drug induced skin lesions including karatoacanthomas and
squamous cell carcinoma.12, 13
It is demonstrated that the feedback activation in BRafWT bearing cancers can be
significantly suppressed by DFGꢀout Raf inhibitors.21 The optimal Raf inhibitor
should be DFGꢀout type that preserves potency against oncogenic BRafV600E without
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driving activation of BRafWT or CRaf.22, 23 Hence we develop a series of DFGꢀout
conformation pyrimidine derivates I-01~I-22 having PanꢀRaf potency with low IC50
values, they without agonistically affect the MAPK pathway in BRafWT extraordinary
expression melanoma cell line (The data were obtained on compound I-14~I-15).
Furthermore, the compounds also displayed inhibition on the proliferation of a panel
of vemurafenibꢀ
resistant cancer cells harboring overexpressed BRafV600E (A375: WB
assay, acquired resistance; COLOꢀ205: IC50 = 1.92 ꢁM, SKꢀMelꢀ2: IC50 = 0.58 ꢁM,
intrinsic resistance), representing new leads for further development of PanꢀRaf
inhibitors to overcome the resistance caused by vemurafenib.
2. Results and discussion
These compounds were evaluated with enzyme and cellꢀbased assay. Furthermore,
SAR (structureꢀactivity relationship) had also been established. I-14 and I-15 with
optimal potency at enzymatic as well as cellular level was chosen to study the
possible biological mechanisms. I-15 achieving solubility of 0.228 mg/mL was also
studied some pharmacokinetic profiles compared to Y-1.
2.1. Inhibitors design and molecular modeling
Previously, lead compound Y-1 is found as a BRafV600E inhibitor with enzymatic
activity (IC50 = 3.2 nM) and antiproliferative activity against cell line A375 (IC50 = 8.8
ꢁM).24 However, its solubility and chemical novelty still need to be improved (Figure
4). Thus, on the basis of the fragmentꢀbased approach25 which decomposes
compounds from several databases, we tried to recombine those fragments by the
retrosynthetic combinatorial analysis procedure (RECAP) algorithm.26 Databases
consist of those novel compounds were again decomposed into fragments by the
fragmentꢀbased approach,25 which may greatly improve the novelty of the chemical
space for the databases. Fragments generated in this way were then docked to the
active binding site. A 1ꢀ(2ꢀ(methylamino)pyrimidinꢀ4ꢀyl)ꢀNꢀ phenylcyclopropaneꢀ1ꢀ
carboxamide (the red colored part of I-01 as shown in Figure 3) was found to be in
great alignment with the Nꢀphenylꢀ3ꢀ(9Hꢀpurinꢀ6ꢀyl) pyridinꢀ2ꢀamine part of the lead
Y-1 (Figure 3A) as well as the crystalized ligand of PDB 3IDP (BRafV600E IC50 = 1.6
nM) (Figure 3B). Furthermore, I-01 was designed by combining the novel scaffold
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with lead Y-1 through a scaffoldꢀmerging strategy. As shown in Figure3C and Figure
3D, I-01 adopts a similar binding conformation with lead Y-1 and the crystalized
ligand of PDB 3IDP, which maintains with the original essential hydrogen bonds in
both the hinge region and the DFG part. The cyclopropyl formamide part overlaps
well with pyridine moiety occupying this small hydrophobic binding pocket omitted
in the binding conformation of sorafenib.
Figure 3. The binding conformation of the novel scaffold and I-01
Alignment of lead scaffold and I-01 with Y-1 and the crystalized ligand of PDB 3IDP
(3A) Alignment of lead scaffold of I-01 (orange colored carbons) with Y-I (green
colored carbons). (3B) Alignment of lead scaffold of I-01 with crystallized ligand
(green) from PDB 3IDP. (3C/D) Fully elaborated structures from lead scaffold of I-01,
showing further overlap in the back hydrophobic pocket.
The structure shown for Y-1 is a docked structure, and not an actual coꢀcrystal
structure. The structures of I-01~I-22 were shown in the same manner to Y-1.
However, I-01 didn’t display comparable enzymatic activities with Y-1 or the
ligand of PDB 3IDP (Figure 4). Hence the structure optimization was performed to
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identify potent compounds.
Figure 4. The structure modification of Y-1 to I-01
Before optimizing the lead I-01, we explored the binding mode of I-01. The
modeled binding mode of I-01 to the DFGꢀout conformation of BRafV600E revealed
that I-01 binds to the BRafV600E ATP binding pocket with the pyrimidine core forming
two essential hydrogen bonds with the hinge region Cys532 (Figure 5). In addition to
these hydrogen bonds, the amide group forms two hydrogen bonds with Asp594 and
Glu501. Then, we turned our attention toward the methylamino group proximal to the
pyrimidine core. The binding mode showed it to form hydrophilic interactions with
the surrounding amino acid residues. More importantly, the methylamino group in the
pyrimidine ring was directed toward the solvent accessible region of BRafV600E
(Figure 5). The chlorine atom in the terminal phenyl ring was directed toward another
solvent accessible region, too. In order to get a better occupation of these two solvent
accessible regions, we started optimization program to modify I-01 with an aim to
improve solubility and cellular potency.
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H
B
N
Glu501
O
O
O
Solvent
accessible region
HS
N
R3
R2
H
N
N
HN
L
Hydrophobic
pocket
O
Cys532
Q
O
HN
R1
accessible region
H
O
Solvent
N
Hydrophobic
pocket
O
Asp594
O
I-01~I-22
Figure 5. (A) The binding pockets of BRafV600E. (B) The binding mode of I-01~I-22
and sorafenib.
The further modification depended on the important structural features essential for
enzymatic activity. L as linkage using different groups was attempted. Fragment Q
was investigated on rigid and steric factors. R1 substituent was directed toward the
solvent accessible region of BRafV600E proximal to the hinge region. R2 substituent
was exposed to the hydrophobic back pocket, which was formed by a rearrangement
of the activation loop and subsequent movement of a phenylalanine side chain of
DFG loop.27 R3 substituent in the terminal phenyl ring was directed toword another
solvent accessible region of BRafV600E. Based on these information, we described the
design and synthesis of pyrimidine derivatives I-01~I-22 as new DFGꢀout BRafV600E
inhibitors.
2.2. Chemistry
As summarized in Scheme 1, totally 22 compounds were synthesized. The
synthetic routes were illustrated in Scheme 1–3. The chemical structures of these
1
compounds were confirmed by HꢀNMR, 13CꢀNMR, HRMS spectra and results were
presented in experimental section.
The target pyrimidine derivatives I-01~I-22 were prepared by the method shown in
Scheme 1. The reaction of commercially available 2, 4ꢀdichloropyrimidine with
diethyl malonate in the presence of sodium hydride gave diethyl
2ꢀ(2ꢀchloropyrimidinꢀ4ꢀyl) 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,
2ꢀdibromoethane, iodomethane or 1, 4ꢀdibromobutane in the presence of sodium
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hydroxide afforded the corresponding alkylated derivatives I-c-1~I-c-3 in 90.2%, 93.4%
or 90.2%, respectively.
Amide
were carried out in the presence of trimethylaluminium (2 M solution of toluene)
under the condition of nitrogen atmosphere to give I-d-1~I-d-12 in 75.6 91.4%.
Finally, the reaction of I-d-1~I-d-12 with commercially available amines through
ꢀforming reactions of I-c-1~I-c-3 with the corresponding anilines A1–A10
–
SNAr substitution reaction provided I-01~I-22 in 80.3
–93.1%.
Scheme 1. The synthetic route of I-01~I-22
Reagents and conditions: (a) diethyl malonate, NaH, THF, 0oC, 0.5 h, then reflux, 2.5 h; (b)
EtONa, EtOH, reflux, 2.5 h; (c) 1,2ꢀdibromoethane, NaOH, DMF, r.t., 5 h, 90.2%; iodomethane,
NaOH, DMF, r.t., 5 h, 94.3%; 1,4ꢀdibromobutane, NaOH, DMF, r.t., 5 h, 90.1%; (d) A1–A10,
Al(CH3)3, toluene, nitrogen atmosphere, 80oC, 5 h, 70.8–88.6%; (e) R1NH2, DIPEA, EtOH, reflux,
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2 h, 80.3–92.7%.
Totally 10 important intermediates A1
2 and Scheme 3 via a two five step process.
Intermediates of anilines A1 A2 were prepared by the method shown in Scheme 2.
–A10 were synthesized according to Scheme
ꢀ
–
Substituted benzoyl chloride, which prepared in the presence of benzoic acid with
various substituent and oxalyl chloride, were allowed to react with
4ꢀchloroꢀ3ꢀ(trifluoromethyl) aniline or 4ꢀmethylꢀ3ꢀnitroꢀsubstituted amides to afford
the corresponding nitroaniline. Subsequent treatment of the resulting substituted
amide with Fe/NH4Cl gave the corresponding aniline A1–A2 in 72.6% or 80.4%,
respectively. A3 was generated from condensation reaction using CDI (N,
Nꢀcarbonyldiimidazole). Then, achieved crude product was converted to A3 through
reduction reaction in 92.6%.
Scheme 2. Synthesis of intermediates A1–A3
Reagents and conditions: (a) (COCl)2, CH2Cl2, r.t., 2 h; (b) 4ꢀchloroꢀ3ꢀ(trifluoromethyl) aniline or
4ꢀmethylꢀ3ꢀnitroaniline, Et3N, CH2Cl2, r.t., 5 h; (c) CDI, CH2Cl2, r.t., 34 h; (d) Fe, NH4Cl, 75%
EtOH, reflux, 3 h.
The reaction of 1ꢀfluoroꢀ4ꢀnitroꢀ2ꢀ(trifluoromethyl) benzene with morpholine or
Nꢀmethyl piperazine in solvent DMSO (dimethyl sulfoxide) through SNAr substitution
reaction provided 1a
–
1b in 94.2% or 91.4%, respectively (Scheme 3). Reduction of
the nitro group in 1a
–
1b using Fe/NH4Cl gave the corresponding aniline. Subsequent
treatment of the resulting aniline with intermediate 4ꢀmethylꢀ3ꢀnitrobenzoyl chloride
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provided substituted amide, was reduced with Fe/NH4Cl to afford the desired anilines
A4 A5 in 85.4% or 76.2%, respectively.
The desired aniline A6 A7 utilized 4ꢀnitrotoluene or 4ꢀmethylꢀ3ꢀtrifluoromethyl
–
–
nitrobenzene as starting material. With NBS (Nꢀbromosuccinimide) in solvent DCE
(1,2ꢀdichloroethane), they were converted to 2a or 2b in 86.4% or 89.2%, respectively.
2a
as solvent. The last three steps of A6
adopted for A4 A5.
–
2b were converted to 3a
–
3b in the presence of NEt3 as base, THF (tetrahydrofuran)
–
A7 were same as previous reaction (e, f and g)
–
The reaction of commercially available 1ꢀfluoroꢀ4ꢀnitroꢀ2ꢀ(trifluoromethyl)
benzene with 1ꢀmethylpiperidinꢀ4ꢀol, (1ꢀmethylpiperidinꢀ4ꢀyl) methanol or
3ꢀmorpholinopropanꢀ1ꢀol in the presence of sodium hydride in solvent DMF (N,
Nꢀdimethylformamide) gave 4a
reaction (e, f and g) adopted for A4
were afforded in 89.4%, 86.4% or 85.2%, respectively.
Scheme 3. Synthesis of intermediates A4 A10
–
4c. The last three steps were same as previous
–
A5. Then, the desired intermediates A8 A10
–
–
Reagents and conditions: (a) morpholine or Nꢀmethyl piperazine, DMSO, 100oC, 5 h, 94.2% or
91.4%; (b) NBS, DCE, reflux, 5 h, 86.4%; (c) morpholine, NEt3, THF, reflux, 2 h; (d)
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1ꢀmethylpiperidinꢀ4ꢀol, (1ꢀmethylpiperidinꢀ4ꢀyl) methanol or 3ꢀmorpholinopropanꢀ1ꢀol, NaH,
DMF, ice bath, 0.5 h, then 100oC, 5 h; (e) Fe, NH4Cl, 75% EtOH, reflux, 2 h; (f)
4ꢀmethylꢀ3ꢀnitrobenzoyl chloride, Et3N, CH2Cl2, r.t, 5 h; (g) Fe, NH4Cl, 75% EtOH, reflux, 2 h.
2.3. Investigation the enzymatic activity of compounds
2.3.1. Compounds screened for solubility and enzymatic activity against BRafV600E
Newly synthesized compounds were evaluated as BRafV600E inhibitors using
vemurafenib, sorafenib and LY3009120 as reference compounds (vemurafenib and
sorafenib have launched, LY3009120 is a PanꢀRaf inhibitor). Most of compounds
showed IC50 values in the submicromolar range (Table 1).
Table 1. SAR exploration of compounds against BRafV600E activity
BRafV600E
Solubility
Compd
L
Q
R1
R2
R3
activity%a IC50(nM) c
mg/mL
0.016
I-01
I-02
I-03
I-04
I-05
I-06
I-07
I-08
I-09
I-10
CONH
NHCO
NHCONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
7.1
77.2±2.4
0.015
0.009
0.005
0.005
0.102
0.241
0.187
0.065
0.204
7.3 166.0±4.6
8.4 154.0±3.1
CH3
CH3
92.1
97.1
13.6
3.6
ND b
CH3
ND
CH2CH2OH
CH2CH2OCH3
(CH2)3OCH3
44.8±11.2
95.7±18.7
13.3 133.0±17.5
CH2CH2NHCH3 CF3
63.2
9.0
ND
CH2CH2N(CH3)2 CF3
75.3±7.3
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I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
Cl
Cl
Cl
Cl
Cl
0.282
0.243
0.261
0.197
0.228
0.214
0.241
0.198
0.211
0.176
0.150
0.203
7.7
46.1±21.6
73.8±27.7
59.6±13.1
48.8±7.9
12.6±15.8
ND
14.1
11.4
10.5
3.0
CH3
CH3
CH3
CH3
CH3
CH3
CH3
30.4
35.6
37.7
94.4
ND
195.0±9.8
ND
CF3
CF3
CF3
21.7 154.0±16.3
40.0
ND
26.2 144.0±22.2
vemurafenib
sorafenib
31.6±2.4
38.1±3.0
5.5±3.9
LY3009120
acontrol activity: compounds with concentration of 0.5 ꢁM; b not determination; c n=3
Replacement of the amide function of I-01 with either a “reverse” amide or urea
linkage I-02~I-03 (Table 1), led to at least twoꢀfold reduction in potency. In order to
facilitate the comparison with Y-1, amide remained as linkage group L.
Q fragment was investigated on rigid and steric factors. I-04 exhibited significant
loss of kinase inhibitory potency. This result was attributed to the effect of πꢀπ
hyperconjugation between the cyclopropyl CꢀC bonds and the amide C=O exerting a
conformational bias based on the increased π character of cyclopropyl CꢀC bonds
which can optimally interact with the amide C=O moiety at 0o and 180o (the
ThorpeꢀIngold effect).28 Moreover, the 116 bond angle enforced by the cyclopropyl
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ring is closer to the 120 vector inherent to the pyridine core. Expanding the
cyclopropyl of I-01 to cyclopentyl of I-05 resulted in significant loss of kinase
inhibitory potency. The cyclopentyl ring also didn’t provide for the aforementioned
electronic stabilization (hyperconjugation) that the cyclopropyl anologs provide. I-01
using amide group and cyclopropyl as L and Q displayed optimal enzymatic activity.
We hypothesized that the poor antiproliferative activity of I-01 against A375 cell
lines (IC50 = 18.4 ꢁM) was due to its meager solubility and cellular permeability. In
order to get a better occupation of the solvent accessible region proximal to the
pyrimidine ring, we introduced the solubilizing functional group R1. I-06~I-22
increased in solubilities, compared to those analogs I-01~I-05 lacking solubility
enhancing functional group at R1 (Solubility determination was presented in
29
supporting information). The goal to ameliate solubility was achieved with analog
I-15, achieving solubility of 0.228 mg/mL (Table 1).
Different steric size of R1 was explored. We envisioned introduction of hydroxyl,
methoxyl or amino functional group through carbon linker into pyrimdine ring. It has
been reported that the presence of these groups can properly occupy the solvent
accessible region.30ꢀ32 I-06~I-08 weren’t achieve apparent change of enzymatic
activity. Secondary amine I-09 was introduced through a twoꢀcarbon linker at the
pyrimidine ring, which resulted in loss of BRafV600E inhibition. Compared to I-09, the
enzymatic activity of I-10 was significantly increased. This result confirmed the
importance of the tertiary amine for Raf kinase activity. Due to steric compatibility
with BRafV600E, expanding the tertiary amine of I-10 to cyclic amine of I-11 resulted
in 2ꢀfold increase in enzymatic activity.
The effect of increasing the length of the linker carbon chain bearing the
solubilizing amino functional group was explored. When the linker carbon length was
increased from two carbons to three carons (I-07 to I-08, I-11 to I-12), the enzymatic
activity decreased. The linker of two carbons was better suited for hydrophilic
interactions. Based on this information, cyclic tertiary amines pyrollidine, piperidine
and Nꢀmethylpiperazine were introduced to pyrimidine through two carbons chain to
achieve I-13~I-15. Compared to I-01, they increased enzymatic activity. Especially,
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I-15 displayed 6ꢀfold increase of enzymatic activity compared to I-01, with IC50 value
as 12.6 nM. This result showed that the steric accommodation was the core factor for
R1 to affect enzymatic activity. Bulky groups could get a better occupation of solvent
accessible region proximal to the hinge moiety of pyrimidine.
I-18 was removed of lipophilic groups trifluoromethyl (ꢀCF3) to afford I-19. The
loss of enzymatic activity indicated that R2 substituent was necessary for enzymatic
potency. As described above, R3 toward another solvent accessible region. The
modification introducing solubilizing functional groups at R3 position afforded
I-16~I-22 to get a better occupation of solvent accessible region. Perhaps due to not
occupy the solvent accessible region properly, I-16~I-22 resulted in significantly
decreased enzymatic activity. I-18, I-20 and I-22 were tolerated.
2.3.2. The Pan-Raf activity of chosen compounds
As mentioned earlier, PanꢀRaf inhibitors with similar activity against all subtypes
of Raf proteins might have potential to overcome the resistance caused by
vemurafenib. The Raf protomer IC50 values of chosen compounds were comparable to
vemurafenib and sorafenib, especially against BRafV600E and CRaf. Although their
poency weren’t comparable to LY3009120 as shown in Table 2, their potency in terms
of CRaf were all superior to vemurafenib.
Table 2. The Raf protomer activity of chosen compounds
IC50 (nM)a
Compd
BRafV600E
77.2±2.4
75.3±7.3
59.6±13.1
48.8±7.9
12.6±15.8
31.6±2.4
38.1±3.0
5.5±3.9
ARaf
BRafWT
CRaf
194.6±3.8
287.2±9.1
43.4±1.1
41.7±3.7
47.2±7.6
38.8±2.5
17.5±6.8
135.7±0.9
6.2±1.3
I-01
I-10
108.2±5.5 121.4±13.3
92.4±9.7
71.4±4.7
30.1±7.0
2.1±0.6
7.1±1.1
1.2±2.2
102.3±20.3
68.1±14.0
19.7±19.4
6.9±8.5
I-13
I-14
I-15
vemurafenib
sorafenib
LY3009120
22.6±10.4
1.5±12.1
3.8±0.5
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a n=3
Kinase Activity Measurement Using KiNativ Assays of ActivX Biosciences Inc.
Whole cell KiNativ assays were developed by ActivX Biosciences Inc, using whole
cell lysates of A375 cells as described (Table 3).33 I-15 bound ARaf, BRaf, and CRaf
native proteins with IC50 values of 284, 168−225, and 163 nM, respectively.
Vemurafenib was able to bind to BRaf and ARaf with IC50 values of 280−320 and
1130 nM, respectively; however, its binding affinity to CRaf was greater than 10,000
nM. LY3009120 bound ARaf, BRaf, and CRaf potently with IC50 values of 44, 31−47,
and 42 nM respectively.
Table 3. Raf KiNativ data in A375 cells
IC50 (nM)
Compd
ARaf
BRaf
168ꢀ225
280ꢀ320
31ꢀ47
CRaf
163
284
I-15
vemurafenib 1130
LY3009120 44
>10000
42a
a Cite the reference 15
Although the potency of I-15 wasn’t comparable to LY3009120, its potency was
superior to vemurafenib.
2.4. Antiproliferative activity against different cell lines in vitro
As described above, all isoforms of Raf are important targets in developing
smallꢀmolecular inhibitors for cancer therapies, especially melanoma, colon cancer
and hepatoma.34 A375,35 and COLOꢀ20536 cell lines have extraordinary expression of
BRafV600E. SKꢀMelꢀ223 and HepG2 cell lines have extraordinary expression of
BRafWT 37
.
Antiproliferative activities of compounds with good enzymatic activity against
A375, SKꢀMelꢀ2, COLOꢀ205 and HepG2 cell lines were evaluated. The results were
summarized in Figure 6.
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Figure 6. The cellular potency of compounds against cell lines with concentration
of 10 ꢁM
Introducing hydroxyl group through carbon linker at the pyrimidine ring to achieve
I-06 resulted in improved potency against all the cell lines compared to I-01 (The
solubility was improved from 0.016 mg/mL of I-01 to 0.102 mg/mL of I-06). Then,
various tertiary amines of different steric sizes were explored. Initially, I-10 with N,
Nꢀdimethyl amino substitution displayed significantly increased cellular potency
compared to I-06. Subsequently, I-11 and I-13~I-15 with cyclic tertiary amines were
investigated. I-13~I-15 were superior to I-10, while I-11 wasn’t comparablet to I-10.
This result displayed that the cyclic tertiary amines would be better suited to increase
cellular potency. Eventually, the chosen compounds with good antiproliferative
activity were tested on the cell lines to acquire the IC50 values. The data showed in
Table 4.
Table 4. The IC50 values of chosen compounds against various cell lines
IC50 (ꢁM) a
Compd
A375
COLOꢀ205
3.36±0.62
3.95±1.48
3.62±0.66
1.92±0.34
5.16±1.39
7.04±0.87
SKꢀMelꢀ2
2.31±0.10
1.92±0.56
1.54±0.72
0.58±0.04
5.64±1.07
11.35±2.10
HepG2
4.52±0.13
I-10
I-13
3.26±0.41
2.37±0.17
1.78±0.38
1.07±0.10
0.70±0.09
13.64±2.44
3.67±0.94
2.13±0.70
2.46±0.11
5.48±1.01
2.84±0.41
I-14
I-15
vemurafenib
sorafenib
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LY3009120
0.31±0.09
0.96±0.13
0.22±0.03
2.60±0.91
a n=5
The antiproliferative activity of I-10 and I-13~I-15 against A375 and HepG2 cell
lines were comparable to vemurafenib and sorafenib. When tested on SKꢀMelꢀ2 and
COLOꢀ205, their potency proved to be superior to vemurafenib and sorafenib. Overall,
all the compounds bearing solubilizing functional group showed improved cellular
potency compared to I-01. Interestingly, within this series of compounds, cellular
potencies followed roughly the same rank order of biochemical potencies. The
improved cellular activity is probably due to enhanced solubility.
It should be noted that I-14 and I-15 had a similar PanꢀRaf activity and various
cellular potency as that of the compounds vemurafenib and sorafenib.
Although I-15 wasn’t comparable to LY3009120 against A375 cell line, its
potencies against COLOꢀ205, SKꢀMelꢀ2 and HepG2 cell lines were comparable.
2.5. Research on biological mechanisms
We tested I-14 and I-15 for its ability to inhibit PꢀERK in cancer cells by Western
blot analysis (Figure 7). I-14 and I-15 effectively and doseꢀdependently inhibits
PꢀERK in the A375 cell line with BRafV600E at concentration as low as 400 nM, which
is as potent as vemurafenib. In the SKꢀMelꢀ2 cell line with BRafWT, I-14 and I-15
effectively and doseꢀdependently inhibits PꢀERK at concentration as low as 400 nM,
too. However, vemurafenib activates PꢀERK with the increase of its concentration.
Figure 7. (A) ERK kinase inhibition in A375 (B) ERK kinase inhibition in SKꢀMelꢀ2
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Our results supported that PanꢀRaf inhibition might be a tractable strategy to
overcome the intrinsic or acquired resistance of melanoma cancer caused by
vemurafenib.
2.6. Research on pharmacokinetics behavior
The favorable in vitro profiles prompted us to evaluate I-15 in vivo, compared to
Y-1. The pharmacokinetic (PK) parameters of Y-1 and I-15 were assessed in male SD
rat following intravenous (IV) and oral (PO) administrations as described in Table 5
and Table 6.
I-15 possesses favorable pharmacokinetic profiles with high oral exposures
(AUC0−∞) of over 868.0 ꢁg/L·h at a 10 mg/kg oral dose and acceptable halfꢀlife (T1/2
= 5.9 h). After IV administration I-15 at a dose of 2.5 mg/kg, maximum concentration
(Cmax) reached 127.5 ꢁg/L, and area under the curve (AUC0−∞) was 695.7 ꢁg/L·h.
After oral administration at 10 mg/kg, Cmax reached 68.5 ꢁg/L at 6.0 h post dosing.
These results demonstrate good pharmacokinetic properties of I-15 in rat with high
oral bioavailability F = 33.4% and a sustained plasma concentration exceeding in
vitro EC50 during at least 11.8 h after oral dosing. I-15 also exhibited low clearance
(CL) and high volume of distribution (Vss).
Table 5
．Pharmacokinetic profile of Y-1 in rat
PK parameters (n=3)
AUC(0ꢀt) (ug.h/L)
AUC(0ꢀ∞) (ug.h/L)
t1/2 (h)
IV (2.5 mg/Kg)
378.4
405.8
8.3
SD
58.3
90.1
2.1
PO (10 mg/Kg)
509.1
590.6
7.3
SD
87.3
75.6
1.7
Tmax (h)
0.083
6.2
0.1
4.0
1.9
CL (L/h/Kg)
V (L/Kg)
1.2
N/A
1.8
73.4
7.6
N/A
32.5
23.4
6.0
Cmax (ug/L)
F
225.0
28.8
68.9
33.6
Table 6
．Pharmacokinetic profile of I-15 in rat
PK parameters (n=3)
AUC(0ꢀt) (ug.h/L)
AUC(0ꢀ∞) (ug.h/L)
IV (2.5 mg/Kg)
600.4
SD
PO (10 mg/Kg)
801.2
SD
37.4
57.1
45.2
38.7
695.7
868.0
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t1/2 (h)
Tmax (h)
7.3
0.083
3.6
1.5
0
5.9
6.0
0.9
0.9
CL (L/h/Kg)
V (L/Kg)
1.1
8.2
16.8
N/A
N/A
68.5
33.4
2.9
37.7
127.5
25.5
6.2
Cmax (ug/L)
F (%)
5.2
N/A a: not applicable
The Plasma Concentrationꢀtime curve of Y-1 and I-15 was shown in Figure 8, the
experimental process was presented in Experimental section (see Supporting
Information).
Figure 8. The Plasma Concentrationꢀtime curve of Y-1 and I-15
From the Plasma Concentrationꢀtime curve, we know the AUC0−∞ value of Y-1 was
lower than I-15. The SD value of Y-1 was higher than I-15,too. These data indicate a
favorable pharmacokinetic profile of I-15 in preparation for efficacy studies in rat.
3. Conclusion
Our previous studies toward targeted anticancer agents have included the discovery
of Y-1. Replacing the purine group in hinge moiety of Y-1 by pyrimidine group results
in I-01 as a novel PanꢀRaf inhibitory template, which inhibits all subtypes of Raf
protomer proteins with IC50 values as 77.2 nM (BRafV600E), 194 nM (ARaf), 287 nM
(BRafWT) and 43.4 nM (CRaf), respectively. On the basis of information obtained
from the binding mode of I-01, various solubilizing functional groups as R1 and R3
group were introduced to 4ꢀposition pyrimidine ring in hinge moiety or terminal
phenyl to achieve a better occupation of solvent accessible region, and this
optimization campaign lead to the identification of I-15 as a PanꢀRaf inhibitor with
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improved solubility. As the representative compound, I-15 inhibited all subtypes of
Raf protomer with IC50 values of 12.6 nM (BRafV600E), 30.1 nM (ARaf), 19.7 nM
(BRafWT) and 17.5 nM (CRaf), respectively. Although I-15 wasn’t comparable with
LY3009120 in terms of cellular potency, its potency was superior to or comparable
with vemurafenib and sorafenib (The selected cell lines were extraordinarily
expressed BRAFV600E or BRafWT). The western blot results for the PꢀERK inhibition
in human melanoma A375 and SKꢀMelꢀ2 cell lines displayed our compounds
inhibited the proliferation of A375 cell lines through ERK pathway, without
paradoxical activation of ERK in SKꢀMelꢀ2 cell lines. Our results supported the
hypothesis that the PanꢀRaf inhibiton might be a tractable strategy to overcome the
acquired resistance caused by vemurafenib. The better pharmacokinetics profile in rat
proved I-15 was suitable in preparation for efficacy studies in rat. Thus I-15 was a
promising lead compound to be developed as a potent PanꢀRaf inhibitor. 38ꢀ42
4. Experimental section
4.1. Molecular docking and fragment-based strategy
Crystal structures of BRafV600E (PDB 3IDP) were downloaded from Protein Data
Bank (PDB) and prepared by the Protein Preparation Wizard in the Schrödinger
suite.43 All compounds were initially minimized by the LigPrep module. Due to its
excellent performance through a selfꢀdocking analysis,44 the Glide module (extra
precision [XP] mode) was selected for molecular docking and the top 10 poses of
each ligand were minimized by a post docking program with the best pose saved for
further analysis. In the fragmentꢀbased strategy, detailed decomposition of the
compound databases was found in a method proposed before25 and the RECAP
algorithm were implemented in Pipeline Pilot 7.5 with the Generate Recap Fragments
protocol.26 Duplications were removed no matter for decomposing or resynthesing
compounds.
Acknowledgments
This work was financially supported by National Natural Science Foundation of
China (No. 21572273/ B020601), A Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions and College Student
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Innovation Project for the R&D of Novel Drugs (No. J1030830). We also express our
gratitude to Dr. Xiazhong Ren, Dr. Huajun Yang, Dr. Chunlan Dong
(Crown Bioscience Corporation) and Dr. Jamie Planck (RBC, Pennsylvania, USA) for
their helpful support in biological evaluation.
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Describe the design and characterization of a series of pyrimidine scaffold as
Pan-Raf inhibitors, which may overcome the resistance associated with current
BRafV600E inhibitors.