Discovery of potent Pan-Raf inhibitors with increased solubility to overcome drug resistance

Article abstract of DOI:10.1016/j.ejmech.2018.11.033

Despite various applications of kinase inhibitors in oncology and inflammatory diseases, the emergence of resistance still remains the major barrier to achieve long-term remission in cancer treatment. With the aim of overcoming the resistance induced by type IIB BRaf ^V600E selective inhibitor vemurafenib, and further ameliorating the antiproliferative activity, a novel type IIA Pan-Raf inhibitors Ia–Io based on pyrrolo[2,3-d] pyrimidine scaffold were designed and evaluated in this work. Herein, we tried to improve the cellular potency of the target compounds by increasing their solubility. Among them, Il, with the solubility of 0.107 mg/mL, demonstrated favorable cellular activity against vemurafenib-resistant carcinoma cells including BRaf^WT phenotypic melanoma SK-MEL-2 and BRaf^V600E phenotypic colorectal cancer HT-29 cell lines. Based on the well solubility, Il exhibited good metabolic stability compared to sorafenib and showed favorable pharmacokinetic profiles in rats. As for the biological mechanism research, Il had the similar P-ERK kinase inhibitory activity in A375 and SK-Mel-2 cells as our previously lead P-2. Il may become a good candidate compound to overcome the resistance associated with vemurafenib.

Full text of DOI:10.1016/j.ejmech.2018.11.033

Accepted Manuscript
Discovery of potent Pan-Raf inhibitors with increased solubility to overcome drug
resistance
Lu Wang, Yanmin Zhang, Qing Zhang, Gaoyuan Zhu, Zhimin Zhang, Chunqi Duan,
Tao Lu, Weifang Tang
PII:
S0223-5234(18)30992-9
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.033
EJMECH 10890
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 October 2018
Revised Date: 10 November 2018
Accepted Date: 14 November 2018
Please cite this article as: L. Wang, Y. Zhang, Q. Zhang, G. Zhu, Z. Zhang, C. Duan, T. Lu, W. Tang,
Discovery of potent Pan-Raf inhibitors with increased solubility to overcome drug resistance, European
Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.033.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract (for Table of contents use only)
Discovery of Potent Pan-Raf Inhibitors with Increased
Solubility to Overcome Drug Resistance
Lu Wang a,1,Yanmin Zhang a,1,Qing Zhang b, Gaoyuan Zhu a, Zhimin Zhang a,Chunqi
Duan a, Tao Lu a,c,*, and Weifang Tang a,*
1

ACCEPTED MANUSCRIPT
Discovery of Potent Pan-Raf Inhibitors with Increased
Solubility to Overcome Drug Resistance
Lu Wang a,1,Yanmin Zhang a,1,Qing Zhang b, Gaoyuan Zhu a, Zhimin Zhang a,Chunqi
Duan a, Tao Lu a,c,*, and Weifang Tang a,*
a School of 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.
1 The authors contributed equally to this work and should be considered as co-first
authors
Abstract
Despite various applications of kinase inhibitors in oncology and inflammatory
diseases, the emergence of resistance still remains the major barrier to achieve
long-term remission in cancer treatment. With the aim of overcoming the resistance
V600E
induced by type IIB BRaf
selective inhibitor vemurafenib, and further
ameliorating the antiproliferative activity, a novel type IIA Pan-Raf inhibitors Ia–Io
based on pyrrolo[2,3-d] pyrimidine scaffold were designed and evaluated in this work.
Herein, we tried to improve the cellular potency of the target compounds by
increasing their solubility. Among them, Il, with the solubility of 0.107 mg/mL,
demonstrated favorable cellular activity against vemurafenib-resistant carcinoma cells
including BRafWT phenotypic melanoma SK-MEL-2 and BRafV600E phenotypic
colorectal cancer HT-29 cell lines. Based on the well solubility, Il exhibited good
metabolic stability compared to sorafenib and showed favorable pharmacokinetic
profiles in rats. As for the biological mechanism research, Il had the similar P-ERK
kinase inhibitory activity in A375 and SK-Mel-2 cells as our previously lead P-2. Il
may become a good candidate compound to overcome the resistance associated with
vemurafenib.
Keywords: Pan-Raf inhibitors; pyrrolo[2,3-d] pyrimidine; solubility;
pharmacokinetics; drug resistance
1

ACCEPTED MANUSCRIPT
1. Introduction
The Ras-Raf-MEK-ERK cascade (mitogen-activated protein kinase, MAPK) is
an essential signaling pathway, with activation typically mediated through cell surface
receptors thereby regulating cellular proliferation, differentiation and survival1.
Mutations that lead to continuous activation of the MAPK pathway are among the
most common aspect in human cancers2, 3. BRafWT (wild-type BRaf) can be converted
into an active oncogenic protein BRafV600E by single-point mutation4. According to
their binding modes derived from crystallographic analysis, BRaf kinase inhibitors are
classified as type I (DFG-in/αC-helix-in), type IIA (DFG-out/αC-helix-in) and type
IIB (DFG-in/αC-helix-out)5. Type IIA inhibitors, such as sorafenib6 and LY30091207,
perform weakly in selectivity as these inhibitors harbor some degree of tyrosine
kinase activities. The representative type IIB inhibitor vemurafenib, shows excellent
BRafV600E selectivity, and displays significant clinical efficacy in melanoma patients
harboring BRafV600E 8. While vemurafenib have been developed to treat BRafV600E
mutant tumors, these inhibitors are ineffective against tumors with wild-type BRaf
and in fact can accelerate the growth of Ras mutant tumors through paradoxical
activation of Raf dimers9, 10
.
There are two possible mechanisms (Figure 1). Firstly, it is hypothesized that
vemurafenib triggers BRafV600E/CRaf heterodimer, acquired resistance then appears
when vemurafenib leaves unoccupied CRaf protomer activated for signaling in cells11;
secondly, vemurafenib induces conformational changes of BRafWT kinase domain
leading to heterodimerization with CRaf. In melanoma SK-MEL-2, vemurafenib
activates the MAPK pathway through the activation of BRafWT/CRaf, which enhances
tumor growth in xenograft model12. Ras mutant-driven cancers signal through
BRafV600E/CRaf or BRafWT/CRaf heterodimer, comprise approximately 1/3 of human
cancers13. About 20% of patients have intrinsic resistance and do not respond to
vemurafenib despite the presence of BRafV600E, for instance, colon carcinoma HT-29
patients14. The acquired resistance against BRafWT phenotypic melanoma SK-MEL-2
and intrinsic resistance against BRafV600E phenotypic colon carcinoma HT-29 of
vemurafenib are challenging medical dilemma. Type IIA Pan-Raf inhibitors capable
2

ACCEPTED MANUSCRIPT
of inhibiting all Raf dimers cellular forms would be useful in the treatment of Ras
mutant driven cancers. They preserve potency against oncogenic BRafV600E without
activating the MAPK pathway through BRafWT or CRaf (Figure 1)12, 15. According
to Novartis’ statistical data, there was also a consistent trend toward type IIA
inhibitors exhibiting less paradoxical activation compared to type I16.
Figure 1. The mechanism for acquired resistance
Previously, pan-Raf inhibitor P-1 was designed based on purine scaffold through
a fragment-based method17. However, the solubility of P-1 is far from satisfactory.
P-1 cannot dissolve in CH3OH and dissolves only slightly in DMSO. In this article,
we further described the design, synthesis, and structure-activity relationships (SAR)
of a novel series of IIA type pan-RAF inhibitor based on pyrrolo[2,3-d] pyrimidine
scaffold. First, we removed lipophilic group trifluoromethyl(-CF3) to enhance
solubility. Ih exhibited favorable BRafV600E activity (IC50 = 0.2 nM) was submitted to
the National Cancer Institute (NCI)-60 platform. However, the poor solubility of Ih
(0.029 mg/mL) lead to the poor cellular activity (the mean cellular activity is 79%
under 10 µM). Then, hydrophilic groups were introduced to ameliorate solubility. Il
with the solubility of 0.107 mg/mL had the excellent antiproliferative activity.
Meanwhile, the cellular spectrum of Il was broad. Finally, pharmacokinetics of Il and
possible biological mechanisms were investigated.
2. Results and discussion
2.1. Design and molecular modeling
Previously, we obtained multitarget kinase inhibitor P-1 based on purine
scaffold17. Since P-1 can avoid the repair and compensatory mechanism leading to
3

ACCEPTED MANUSCRIPT
resistance, we developed P-2 through structural optimization17. However, the
antiproliferative activity of P-2 is still not satisfactory, which may due to its relatively
higher polar surface area (PSA). According to Wenglowsky S18, reduction of hetero
atoms of the purine ring can decrease the PSA of a molecule. Together with our
former SAR19, 20, we retained the nitrogen atom of the purine ring that forms a key
hydrogen bond with the hinge region. Then, by adopting a scaffold hopping method,
we obtained (Ia, a pyrrolo[2,3-d] pyrimidine-based) scaffold with unnecessary N
atoms. The tPSA of Ia, decreasing from 107.31 Å2 to 94.95 Å2 (Figure 2), is more
close to that of two known FDA-approved BRaf inhibitors sorafenib and vemurafenib,
for which their tPSA values are 91.82 Å2 and 87.63 Å2, respectively. Ia had improved
antiproliferative effect and measurable solubility relative to P-1 (Figure 2). SAR of
the bottom phenyl portion of Ia was investigated next, with an emphasis on improving
the cellular potency while maintaining Raf potency. Incorporation hydrophilic group
4-(methoxymethyl)-1-methylpiperidine (Il) in the solvent accessible area enabled an
increased ClogP value, solubility and a concomitant boost in cellular activity.
4

ACCEPTED MANUSCRIPT
Figure 2. (A) The structural modification of P-1 to Ia; (B) Predicted binding mode of
Ia (color orange) with BRafV600E (PDB ID: 3IDP) and alignment with sorafenib (color
green); (C) Binding mode of Ia–Io.
Ia was docked to the BRafV600E binding pockets and results showed that the
conformation of this novel scaffold was consistent with sorafenib. Ia contained the
essential hydrogen bonds and a better occupation of the cavity above the phenyl part
shown in Figure 2B. Before optimizing Ia, we explored the important structural
features essential for enzymatic activity and identified possible modification sites.
The pyrrolo[2,3-d]pyrimidine core formed two essential hydrogen bonds with Cys532
(Figure 2C). In addition to these hydrogen bonds, the amide group formed two
hydrogen bonds with Asp594 and Glu501. R1 and R2 substituents of terminal phenyl
ring directed toward the solvent accessible region and the hydrophobic back pocket
formed by the rearrangement of the activation loop and subsequent movement of the
phenylalanine side chain of DFG loop21, respectively. The cyclopropyl formamide
fragment occupied another hydrophobic pocket as shown in Figure 2C. Using
medicinal chemistry approaches, we had designed and synthesized a series of type IIA
derivatives Ia–Io based on pyrrolo[2,3-d]pyrimidine scaffold. The optimization of Ia
focused on L (linker domain), R1, R2 and Q with an aim to improve solubility and
cellular potency.
2.2.Chemistry
As summarized in Scheme 3, Ia
–Io were synthesized. The synthetic routes were
illustrated in Scheme 1 3. The chemical structures of these compounds were
–
13
elucidated by 1H-NMR, C-NMR and HRMS spectra and the results were presented
in Experimental section.
Aa
–
Ah were prepared by the method as shown in Scheme 1.
chloride, prepared in the presence of
4-methyl-3-nitrobenzoyl
4-methyl-3-nitrobenzoic acid and oxalyl chloride, was allowed to react with
appropriate anilines to give appropriate amides. Reduction of amides with Fe/NH4Cl
gave the corresponding anilines Aa
–
Af in 68.6–80.4% yields. Ag was prepared from
5

ACCEPTED MANUSCRIPT
4-chloro-3-(trifluoromethyl) benzoic acid and 4-methyl-3-nitroaniline, in a similar
manner to Aa. Urea Ah was generated from condensation reaction using CDI.
Scheme 1. Synthesis of intermediates Aa–Ah
Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, r.t., 2 h; (b) appropriate anilines, Et3N,
CH2Cl2, r.t., 5 h; (c) Fe, NH4Cl, 75% EtOH, reflux, 3 h; (d) CDI, CH2Cl2, r.t., 34 h.
The synthetic route of intermediates Ai
reaction of 1-fluoro-4-nitro-2-(trifluoromethyl) benzene with morpholine or
N-methylpiperazine provided 1a 1b in 94.2% or 91.4% yield, respectively. Reduction
of the nitro group in 1a 1b using Fe/NH4Cl gave the corresponding aniline.
Subsequent treatment of the resulting aniline with intermediate
–Am was outlined in Scheme 2. The
–
–
4-methyl-3-nitrobenzoyl chloride provided substituted amide, which was reduced to
afford the desired anilines Ai-Aj in 85.4% or 76.2% yield, respectively. The reaction
of commercially available 1-fluoro-4-nitro-2-(trifluoromethyl) benzene with
1-methylpi-peridin-4-ol,
nopropan-1-ol in the presence of sodium hydride gave 1c
were same as previous reactions (c, d and e) for making Ai
anilines Ak Am were afforded in 89.4, 86.4 and 85.2% yields, respectively.
(1-methylpi-peridin-4-yl)methanol
1e. The last three steps
Aj. Then, the desired
or
3-morpholi-
–
–
–
Scheme 2. Synthesis of intermediates Ai–Am
6

ACCEPTED MANUSCRIPT
Reagents and conditions: (a) morpholine or N-methyl piperazine, DMSO, 100oC, 5 h, 94.2% or
91.4% yield, respectively; (b) 1-methylpi-peridin-4-ol, (1-methylpi-peridin-4-yl) methanol or
3-morpholi-nopropan-1-ol, NaH, DMF, ice bath, 0.5 h, then 100oC, 5 h; (c) Fe, NH4Cl, 75% EtOH,
reflux, 2 h; (d) 4-methyl-3-nitrobenzoyl chloride, Et3N, CH2Cl2, r.t., 5 h; (e) Fe, NH4Cl, 75%
EtOH, reflux, 2 h.
Tosylation of the 7-nitrogen atom on the 4-chloro-7H-pyrrolo [2,3-d]pyrimidine
ring afforded 2a. The reaction of 2a with diethyl malonate in the presence of sodium
hydride gave diethyl 2-(7-tosyl-7H-pyrrolo [2,3-d] pyrimidin-4-yl) malonate 3a in
86.2% yield. The prepared 3a was allowed to react with sodium ethoxide to afford 4a
in 92.4% yield. Subsequent alkylation of 4a with 1, 2-dibromoethane or iodomethane
in the presence of sodium hydroxide afforded the corresponding alkylated derivatives
5a or 5b in 90.2% or 93.4% yield, respectively. Amide-forming reactions of 5a or 5b
with the corresponding anilines Aa–Am were carried out in the presence of Al(CH3)3
(2 M solution of toluene) under the condition of nitrogen atmosphere. Ts, the
protecting group used in 7H-pyrrolo [2, 3-d] pyrimidine derivatives, was removed
under the alkali condition to afford Ia
–Io in 70.6–84.2% yields.
Scheme 3. The synthetic route of Ia Io
–
7

ACCEPTED MANUSCRIPT
Reagents and conditions: (a) TsCl, Et3N, CH2Cl2, r.t., 5 h; (b) diethyl malonate, NaH, THF, 0oC,
0.5 h, then reflux, 2.5 h, 86.2% yield; (c) EtONa, EtOH, reflux, 2.5 h, 92.4% yield; (d)
1,2-dibromoethane, NaOH, DMF, r.t., 5 h, 90.2% yield; iodomethane, NaOH, DMF, r.t., 5 h, 94.3%
yield; (e) Aa–Am, Al(CH3)3, toluene, nitrogen atmosphere, 80oC, 5 h, 72.8–87.4% yields; (f)
NaOH, EtOH, r.t., 1.5h, 70.6–84.2% yields.
2.3. Enzymatic and cellular potency
Our previous work reported P-1 inhibits the MAPK pathway of upstream and
downstream multiple targets with similar potency(DDR1 IC50 = 3.0 nM, DDR2 IC50 =
2.6 nM, EPHA2 IC50 = 13.5 nM, FGFR2 IC50 = 39.2 nM, FMS IC50 = 3.9 nM, LCK
IC50 = 40.9 nM, PDGFRb IC50 = 25.3 nM, Ret IC50 = 5.2 nM, ARaf IC50 = 1.0 nM,
BRafWT IC50 = 1.0 nM, BrafV600E IC50 = 1.9 nM, CRaf IC50 = 0.5 nM)17. The broad
kinase spectrum of P-1 ensured Ia-Il could avoid the repair and compensatory
mechanism induced by vemurafenib.
2.3.1. Solubility and enzymatic activity against BRafV600E
Using Ia as a starting point, derivatives Ib−Il with different groups attached at
8

ACCEPTED MANUSCRIPT
different portion were prepared. Ia–Io were evaluated against BRafV600E potency
using vemurafenib, sorafenib and LY3009120 as controls. Vemurafenib and sorafenib
are available on the market and LY3009120 is a representative Pan-Raf inhibitor. All
of the compounds showed IC50 values at nanomolar range shown in Table 1.
Table 1. SAR of linkage L, R1, R2 and Q
Solubility BRafV600E
Compd
Ia
Q
L
R1
Cl
Cl
Cl
H
R2
CF3
CF3
CF3
H
mg/mL
IC50 (nM)
CONH
NHCO
NHCONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
0.012
3.5
0.009
0.002
0.025
0.034
0.038
0.025
0.029
0.081
0.092
0.105
0.107
0.096
0.009
0.089
2.3
2.1
Ib
Ic
34.1
17.0
12.3
1.2
Id
Ie
F
H
H
F
If
Cl
H
H
Ig
Cl
0.2
Ih
Ii
CF3
CF3
CF3
CF3
CF3
CF3
CF3
13.4
34.5
15.8
13.4
7.4
Ij
Ik
Il
CON
H
Im
In
Io
CONH
CONH
Cl
6.2
43.7
9

ACCEPTED MANUSCRIPT
sorafenib
vemurafenib
LY3009120
38.1
31.6
5.5
Replacement of the amide function of Ia with either a “reverse” amide or urea
linkage Ib Ic (Table 1), which led to slightly weaker in the solubility assay. Amide
–
was used as linkage group L. We hypothesized the poor antiproliferative activity of Ia
against A375 (IC50 = 8.6 µM, shown in Figure 2) was due to its meager solubility. At
first, the strategy was to remove lipophilic group trifluoromethyl (-CF3) to enhance
the solubility and reduce the molecular weight. So, Id
synthesized. As Id showed a 10-fold decrease in enzymatic activity compared to Ia, it
was necessary to sustain substituents on terminal phenyl. Ie Ih, introduced halogen
–Ih were designed and
–
atom including fluorine and chloride, were more active than Id against BRafV600E. The
influence of R1 substituent (Ie and Ig) and R2 substituent (If and Ih) on terminal
phenyl ring were explored. The results displayed that compounds with R2 substituent
at C-3 position were potent than those with R1 substituent at C-4 position. This result
was consistent with the hypothesis that R1 and R2 group occupied solvent accessible
region domain and a small hydrophobic pocket in BRafV600E, respectively. The
comparison of Ie to Ig and If to Ih indicated that the chloride atom in R2 position was
potent than fluorine. Ih, adopting cyclopropyl formamide fragment as Q group, amide
group as a linker, and chloride atom as R2 substituent on terminal phenyl ring,
displayed the optimal BRafV600E enzymatic activity. To test the idea that removal of
lipophilic groups could enhance the potency of compounds against different cell lines
or not, Ih was submitted to NCI (National Cancer Institute) for screening a panel of
57 cell lines as shown in Table 2. The detailed experimental method and process were
presented in Experimental section (see Supporting Information).
Table 2. The growth percent of Ih on 57 cell lines
Cell line
Growth Percent
Cell line
Growth Percent
CCRF-CEM
87.9
64.7
6.4
SF-268
SF-295
SF-539
95.2
89.3
73.0
HL-60 (TB)
K-562
Leukemia
CNS Cancer
MOLT-4
RPMI-8226
SR
72.7
183.2
87.9
SNB-19
SNB-75
U251
120.0
93.6
93.6
10

ACCEPTED MANUSCRIPT
A549 (ATCC) 82.4
IGROV1
90.3
EKVX
HOP-62
91.8
67.4
67.4
68.2
115.7
96.2
61.0
59.1
60.5
44.5
88.8
52.5
82.7
67.0
42.1
35.0
60.0
OVCAR-3
OVCAR-4
102.9
101.5
83.6
Non-Small Cell NCI-H226
Ovarian Cancer OVCAR-5
OVCAR-8
Lung Cancer
Colon Cancer
Melanoma
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO205
HCC-2998
HCT-116
HCT-15
89.7
NCI/ADR-RES 92.0
SK-OV-3
MCF7
91.1
87.1
MDA-MB-231 54.8
Breast Cancer
HS 578T
BT-549
T-47D
74.1
93.4
74.1
HT-29
MDA-MB-468 85.8
KM12
786-0
109.7
104.6
98.8
SW-620
A498
LOX IMVI
MALME-3M
M14
ACHN
RXF393
SN12C
TK-10
UO-31
Renal Cancer
91.5
91.2
MDA-MB-435 74.0
131.6
51.6
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
14.5
83.3
74.3
54.5
Prostate Cancer PC-3
DU-145
MEAN = 79
92.2
105.6
Although the enzymatic inhibitory activity of Ih against BRafV600E (IC50 = 0.2
nM) was more potent than vemurafenib (IC50 = 31.6 nM), sorafenib (IC50 = 38.1 nM)
and LY3009120 (IC50 = 5.5 nM), its cellular activity was completely different. The
mean growth percent of Ih was 79% (the concentration of Ih was 0.5 µM) against 57
cell lines tested. We hypothesized that the poor antiproliferative activity of Ih against
various cells was probably due to its poor solubility(0.029 mg/mL). Moreover,
removal of lipophilic groups could not enhance its cellular potency, we then
introduced hydrophilic groups. Introduction of solubility enhancing functionality
groups R1 on terminal phenyl ring toward the solvent accessible region of BRafV600E
gave Ii–Im, whose solubility was improved compared to Ia–Ih (Solubility
determination was presented in Supporting Information)22. The solubility of Il was
0.107 mg/mL (Table 1). Q group with different rigidity was explored. In showed
comparable BRafV600E activity with Ia. However, introduction of
4-methoxyl-1-methylpi-peridine (the fragment was screened from Id–Im in terms of
11

ACCEPTED MANUSCRIPT
cellular activity) as R1 substituent to terminal phenyl ring in Io resulted a 3-fold loss
of BRafV600E activity. This was attributed to Thorpe-Ingold effect23. Balancing
solubility, Raf biochemical assay and cellular proliferation assay, we provided Ii
to achieved the IC50 values of the chosen cell lines.
–Im
2.3.2. Antiproliferative activity against different cell lines in vitro
As shown in Table 2, Ih demonstrated potential antiproliferative activities
against Melanoma, Colon Cancer and Leukemia cell lines at 10 µM. Based on this
result, compounds with potent enzymatic activity were selected to test against A37524,
HT-2925, SK-MEL-226 and MV4-1127 cell lines with extraordinary expression of
BRafV600E or BRafWT. Since these compounds were type IIA Raf inhibitors similar to
sorafenib, HepG2 and SW57928 cells were also selected. The IC50 values of the
chosen compounds were summarized in Table 3.
Table 3. The cell activity IC50 values of the selected compounds
IC50a (µM)
Compd
A375
8.6±0.9
SK-Mel-2
3.3±0.2
1.3±0.1
0.8±0.0
0.7±0.0
0.4±0.0
1.4±0.2
11.4±2.2
5.6±0.2
0.2±0.0
HT-29
MV4-11
3.2±0.1
1.6±0.0
1.1±0.1
0.4±0.1
0.2±0.0
0.8±0.3
0.3±0.1
NAb
HepG2
4.1±1.0
4.3±0.3
1.6±0.2
0.4±0.0
0.8±0.1
2.3±0.2
2.8±0.3
5.5±0.2
2.6±0.0
SW579
3.0±0.3
3.3±0.5
2.5±0.1
0.9±0.1
0.5±0.1
3.4±0.4
1.7±0.2
NA
Ia
9.2±1.1
5.6±0.2
5.2±0.2
3.5±0.1
2.2±0.1
6.3±0.3
6.6±1.7
4.9±0.3
0.9±0.1
Ii
5.4±0.4
4.3±0.3
3.6±0.3
2.0±0.1
6.4±0.8
13.6±2.4
0.7±0.1
0.3±0.0
Ij
Ik
Il
Im
Sorafenib
Vemurafenib
LY3009120
0.1±0.0
0.9±0.1
a n=3; b NA :not determination
12

ACCEPTED MANUSCRIPT
Figure 3 Comparison of cell activity IC50 values for selected compounds
As shown in Figure 3, although Ii
–
Im showed in 2
–
10 fold decrease against
Ih
BRafV600E compared to Ia, their cellular potency was increased compared to Ia
–
whose solubility was poor, probably due to improved solubility. Compared to Ii and
Im, the antiproliferative activity of Ij was slightly increased against all cell lines
tested, indicating that cyclic tertiary amines would be beneficial to cellular potency.
Next we explored the effect of linker with different length to cellular potency. When
the length of linker increased from zero atom (e.g., Ij) to one atom (e.g., Ik) or two
atoms (e.g., Il), the cellular potencies were increased. Il was as potent as vemurafenib
and sorafenib in A375 and MV4-11 cell lines. What’s more, Il was even more potent
than vemurafenib and sorafenib in SK-Mel-2, HT-29, HepG2 and SW579 cell lines.
Overall, all the compounds with solubilizing functional group demonstrated improved
cellular potency compared to Ia. Cellular potencies correlated well with inhibitory
activity.
2.3.3. The Pan-Raf potency of selected compounds
As mentioned earlier, Pan-Raf inhibitors with similar activity against all Raf
protomer might overcome the resistance induced by vemurafenib. The IC50 values
(Table 4 and Figure 4) of Ia, Ik and Il to different Raf protomer were comparable to
vemurafenib and sorafenib, especially against BRafV600E and CRaf. But less potent
than LY3009120.
13

ACCEPTED MANUSCRIPT
Table 4. The Pan-Raf potency of chosen compounds
IC50 (nM)
Compd
BRafV600E
ARaf
BRafWT
CRaf
Ia
Ik
3.5
15.8
13.4
31.6
38.1
5.5
8.6
52.4
35.7
2.1
9.4
43.1
39.6
6.9
2.7
31.7
20.0
135.0
6.2
Il
Vemurafenib
Sorafenib
LY3009120
7.1
22.6
1.5
1.2
3.8
Figure 4 Comparison of Pan-Raf potency of chosen compounds
Whole cell KiNativ assays were developed by ActiveX Biosiences Inc, which
used whole cell lysates of A375 cells as described (Table 5)29. Il inhibited ARaf, BRaf
and CRaf native dimers with IC50 values of 276, 274−318 and 182 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. These results showed Il was superior to vemurafenib in terms of “KiNativ
Assays”. LY3009120 bound ARaf, BRaf and CRaf potently with IC50 values of 44,
31−47 and 42 nM, respectively.
Table 5. Raf KiNativ data in A375 cells
IC50 (nM)
Compd
ARaf
276
BRaf
CRaf
182
Il
274
−318
vemurafenib 1130
LY3009120 44
280-320
>10000
42a
31−47
a Cite the reference 7
14

ACCEPTED MANUSCRIPT
2.4. Possible Mechanisms of Action
As Il exerting the best solubility and cellular activity, we tested Il for its ability
to inhibit p-ERK in A375 and SK-MEL-2 cell lines. Il, as potent as vemurafenib,
effectively and dose-dependently inhibited p-ERK in A375 cell line with BRafV600E
phenotype at concentration as low as 400 nM, indicating that Il inhibited the
proliferation of A375 through MAPK pathway. In the SK-Mel-2 cell line with BRafWT
phenotype, vemurafenib activated p-ERK with the increase of its concentration.
However, Il dramatically inhibited p-ERK at the concentration of 400 nM. It is
noteworthy that, unlike vemurafenib, Il inhibited the proliferation of melanoma A375
cells through ERK pathway, without paradoxical activation MAPK pathway in
SK-Mel-2 cells (Figure 5). Il is a promising compound to overcome the intrinsic or
acquired resistance induced by vemurafenib.
Figure 5. (A) ERK kinase inhibition in A375 cells (B) ERK kinase inhibition in
SK-MEL-2 cells
2.5. Human microsomal stability
Having optimized the potency of pyrrolo[2,3-d] pyrimidine series inhibitors,
examination of T1/2 with a representative inhibitor Il in rat human liver microsomal
was undertaken (Figure 6). The marketed Sorafenib was selected as the standard. The
half-life (T1/2) data was used to evaluate the metabolic stabilities. The results indicated
that the half-life of Il 6.5 h was longer than Sorafenib 5.6 h, which indicated that it
was metabolized slowly than Sorafenib. The favorable in vitro profiles prompted us to
evaluate Il in vivo.
15

ACCEPTED MANUSCRIPT
Figure 6. Human microsomal stability of Il and sorafenib
Based on the results of metabolic stability profiles, Il was further investigated by
profiling intravenous (IV) and oral (PO) administrations pharmacokinetics in male
Sprague-Dawley (SD) rats. The pharmacokinetics parameters were listed in Table 6. Il
was orally bioavailable with AUC0−∞ of 522 µg/L·h and half-life T1/2 = 4.6 h at a 10
mg/kg oral dose. After oral administration at 10 mg/kg, Cmax reached 72.1 µg/L at 4.0
h post dosing and oral bioavailability F was 23%. Il exhibited low clearance (CL) and
high volume of distribution (Vss).
Table 6. Pharmacokinetic profiles of Il in rat
PK parameters (n=3) IV (2.5 mg/Kg) PO (10 mg/Kg)
AUC(0-t) (ug.h/L)
AUC(0-∞) (ug.h/L)
t1/2 (h)
549.1±35.1
606.9±48.7
7.7±1.7
503.4±29.5
521.9±41.5
4.6±2.1
4.0±0.0
aNA
Tmax (h)
0.083±0.0
4.1±0.6
CL (L/h/Kg)
V (L/Kg)
45.7±9.4
272.5±69.2
NA
Cmax (ug/L)
F
72.1±3.3
22.9±0.02
a NA: not applicable
The Plasma Concentration-time curve of Il was shown in Figure 7, the
experimental process was presented in Experimental section. These data suggested a
favorable pharmacokinetic profile of Il in preparation for efficacy studies in rat.
16

ACCEPTED MANUSCRIPT
Figure 7. The Plasma Concentration-time curve of compound Il
3. Conclusion
Our previous studies focused on targeted anticancer agents had included the
discovery of P-1. Since P-1 avoided the resistance caused by compensatory and repair
mechanism, the study was carried out to develop Ia as the bioisostere of P-1.
Although the enzymatic activity of Ia was favorable, the antiproliferative activity of
Ia is not comparable to vemurafenib. We hypothesized that the poor antiproliferative
activity was due to its meager solubility. According to analysis of the binding mode of
Ia, a series of novel pyrrolo[2,3-d] pyrimidine derivatives Ia-Il were designed and
evaluated as type IIA Pan-Raf inhibitors in this work. The strategy of removing
lipophilic groups or introducing hydrophilic groups obtained the representative
compounds Ih and Il. Although Ih was more potent than positive control, its
antiproliferative activity was poor against the cell spectrum of NCI.
Physicochemical properties such as CLogP, total polar surface area(tPSA) and
solubility for Il was in acceptable ranges. Its solubility correlated well with cellular
potency. Il, with the solubility of 0.107 mg/mL, not only demonstrated favorable
cellular activity against vemurafenib-resistant carcinoma cells including SK-MEL-2
and HT-29 cell lines, but also exhibited the comparable IC50 values to positive
control against A375, MV4-11, HepG2 and SW579 cells. Meanwhile, the
antiproliferative activity of Il was improved than P-2. As for the biological
mechanism research, Il had the similar P-ERK kinase inhibitory activity in A375 and
SK-Mel-2 cells as P-2. The favorable in vitro profiles prompted us to evaluate Il in
vivo. Il exhibited good metabolic stability compared to sorafenib and favorable
17

ACCEPTED MANUSCRIPT
pharmacokinetic profiles in rats. Based on these results, Il may become a good
candidate compound to overcome the resistance associated with vemurafenib.
Experimental section
4.1. Molecular docking and fragment-based strategy
Crystal structures of BRaf (PDB code 3IDP) were downloaded from Protein Data
Bank (PDB) and prepared by the Protein Preparation Wizard in the Schrödinger suite.
The small molecules were initially minimized by the LigPrep module in Schrödinger.
The Glide module (extra precision [XP] mode) was selected for molecular docking
due to its excellent performance through a self-docking analysis30, 31 and the 10 best
poses of each ligand were minimized by a post docking program with the best pose
saved for further analysis.
4.2. Chemistry synthesis
All starting materials were obtained from commercial suppliers and used without
further purification. NMR spectrum recorded, on a BrukerDPX-300 spectrometer, in
DMSO-d6 using 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, 0.25 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 Waters Quattro
Micromass instrument using electrospray ionization (ESI) technique.
4.2.1. 4-chloro-7-tosyl-7H-pyrrolo [2, 3-d] pyrimidine (2a)
The mixture of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (3.0 g, 20 mM), NEt3 (6.1 g,
60 mM) and p-toluene sulfonyl chloride (2.6 g, 24 mM) were stirred at room
temperature in dichloromethane (100 mL). After 5 h, TLC analysis showed the
complete consumption of 4-chloro-7H-pyrrolo [2, 3-d] pyrimidine. The reaction
mixture was quenched by the addition of water (70 mL). After quenching the reaction,
the reaction mixture was poured into separator funnel and the aqueous layer was
extracted with dichloromethane (100 mL×3). The combined organic layers were
evaporated in vacuo and purified by silica gel column chromatography to get the
1
product 2a (4.9 g, 79.2%) as a white solid. ESI-MS m/z: 308.0 [M+H]+. H-NMR
(300 MHz, DMSO-d6): δ 2.37 (3H, s, CH3), 6.96 (1H, d, pyrrolyl-H, J = 4.0 Hz), 7.48
(2H, d, ArH, J = 8.1 Hz), 8.05 (2H, d, ArH, J = 8.1 Hz), 8.12 (1H, d, pyrrolyl-H, J =
4.0 Hz), 8.82 (1H, s, pyrimidiny-H).
18

ACCEPTED MANUSCRIPT
4.2.2. Diethyl 2-(7-tosyl-7H-pyrrolo [2, 3-d] pyrimidin-4-yl) malonate (3a)
Sodium hydride (0.5 g, 13.2 mM, 60%) was successively added to a solution of
diethyl malonate (1.1 g, 6.6 mM) in THF (tetrahydrofuran, 50 mL), while keeping
internal temperature between 0–5oC. The mixture was stirred at 0oC for 0.5 h,
followed by addition of the required 2a (1.0 g, 3.3 mM) and heating to reflux for
another 2.5 h. After cooling the reaction, the mixture was diluted in 50 mL saturated
ammonium chloride solution, the aqueous layer was extracted with ethyl acetate (50
mL×3). The combined organic layers were washed with brine (30 mL×3), and then
dried over anhydrous MgSO4. The mixture was evaporated in vacuo and purified by
silica gel column chromatography to obtain 3a (1.4 g, 98.6%) as colorless oil.
ESI-MS m/z: 432.1 [M+H]+.
4.2.3. Ethyl 2-(7-tosyl-7H-pyrrolo [2, 3-d] pyrimidin-4-yl) acetate (4a)
3a (4.3 g, 10 mM) and sodium ethoxide (0.07 mg, 1 mM) in ethanol (50 mL) were
heated to reflux for 2.5 h. After cooling the reaction, the pH was adjusted to 7 by
progressively adding HCl (1 M). The mixture was evaporated in vacuo and purified
by column chromatography on silica gel eluted with ethyl acetate and petroleum to
1
give 4a (2.8 g, 77%) as a white solid. ESI-MS m/z: 360.1 [M+H]+. H-NMR (300
MHz, DMSO-d6): δ 1.19 (3H, t, CH2CH3, J = 7.1 Hz), 2.36 (3H, s, ArCH3), 4.08 (2H,
q, CH2CH3, J = 7.1 Hz), 4.34 (2H, s, CH2CO), 7.03 (1H, d, pyrrolyl-H, J = 4.0 Hz),
7.46 (2H, d, ArH, J = 8.2 Hz), 7.99 (1H, d, pyrrolyl-H , J = 4.0 Hz), 8.04 (2H, d, ArH,
J = 8.2 Hz), 8.87 (1H, s, pyrimidiny-H).
4.2.4. General procedure for the preparation of ester (5a–5b)
4.2.4.1. Ethyl 1-(7-tosyl-7H-pyrrolo [2,3-d]pyrimidin-4-yl)cyclopropane-1-
carboxylate (5a)
The mixture of 4a (1.0 g, 2.7 mM), sodium hydroxide (0.3 g, 8.3 mM) and 1,
2-dibromoethane (2.8 g, 14 mM) were stirred at room temperature in N,
N-dimethylformamide (DMF, 150 mL). After 5 h, TLC analysis showed the complete
consumption of 4a. 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 5a (1.0
1
g, 89.1%) as a white solid. ESI-MS m/z: 386.1 [M+H]+. H-NMR (300 MHz,
DMSO-d6): δ 1.04 (3H, t, CH2CH3, J = 7.0 Hz), 1.51-1.54 (2H, m, CH2-cyclopropyl),
1.57-1.62 (2H, m, CH2-cyclopropyl), 2.36 (3H, s, ArCH3), 3.35 (3H, t, CH2CH3, J =
7.0 Hz), 4.06 (2H, t, CH2CH3, J = 7.0 Hz), 6.84 (1H, d, pyrrolyl-H, J = 4.0 Hz), 7.47
19

ACCEPTED MANUSCRIPT
(2H, d, ArH, J = 8.1 Hz), 7.98 (1H, d, pyrrolyl-H, J = 4.0 Hz), 8.06 (2H, d, ArH, J =
8.1 Hz), 8.86 (1H, s, pyrimidiny-H).
4.2.4.2. Ethyl 2-methyl-2-(7-tosyl-7H-pyrrolo [2,3-d]pyrimidin-4-yl) propanoate(5b)
5b (3.8 g, 94.3%) was prepared from 4a (3.7 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 5a. ESI-MS m/z:
388.1 [M+H]+.
4.2.5. General procedure for the preparation of Ia–Io
4.2.5.1. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-
chloro-3-(trifluoromet-hyl)phenyl)-4-meth-ylbenzamide (Ia)
The mixture of 5a (0.4 g, 1 mM) and the intermediate Aa (0.3 g, 1 mM) in
anhydrous toluene (5 mL) were degassed for 10 min and with nitrogen gas refilled. A
solution of Al (CH3)3 (2 M solution in toluene, 1.5 mL) was added dropwise to the
stirred reaction mixture via syringe over 5 min at 0oC. The resulting suspension was
stirred for 0.5 h, then heated to 80oC and stirred until all solid was dissolved. Reaction
mixture was further stirred for 4–6 h at 80oC until TLC analysis showed the complete
consumption of Aa. The mixture, quenched with C2H5OH (10 mL) , was evaporated
in vacuo and purified by silica gel column chromatography to get 6a (0.6 g, 83.6%) as
a yellow solid. Afterward, the mixture of 6a and sodium hydroxide (1 M solution in
ethanol, 3.1 mL) were stirred at room temperature. After 3 h, TLC analysis showed
the complete consumption of 6a. The mixture was evaporated in vacuo and purified
by silica gel column chromatography to get the target compound Ia (0.3 g, 74.4%) as
1
yellow solid. mp: 223–224°C. H NMR (300 MHz, DMSO-d6): δ 1.61-1.62 (2H, m,
CH2-cyclopropyl), 1.68-1.71 (2H, m, CH2-cyclopropyl), 2.28 (3H, s, ArCH3), 6.58
(1H, dd, pyrrolyl-H, J = 1.8 Hz, J = 3.6 Hz), 7.37 (1H, d, ArH, J = 8.0 Hz), 7.57 (1H,
m, ArH), 7.71 (2H, m, ArH), 8.08 (1H, d, pyrrolyl-H, J = 1.8 Hz), 8.12 (1H, dd, ArH,
J = 2.5 Hz, J = 8.7 Hz), 8.35 (1H, d, ArH, J = 2.5 Hz), 8.75 (1H, s, pyrimidiny-H),
13
10.10 (1H, s, CONH), 10.57 (1H, s, CONH), 12.19 (1H, s, pyrrolyl-NH). C NMR
(75 MHz, DMSO) δ 169.46, 165.58, 158.14, 151.63, 150.11, 138.67, 136.78,
136.27,131.96, 131.92, 130.22, 126.75, 124.89, 124.56, 124.25, 124.15, 118.96,
118.89, 117.26, 116.15, 99.38, 30.92, 17.88, 16.65. HRMS (ESI) calcd for
C25H20ClF3N5O2 [M+H]+, 514.1179; found, 514.1264.
20

ACCEPTED MANUSCRIPT
4.2.5.2. N-(3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methylphenyl)-4-chloro-3-(trifluoromethyl) benzamide (Ib)
6b (0.58 g, 87.4%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ag
(0.33 g, 1 mM), in a manner same as 6a. Ib (0.35 g, 77.3%) was prepared from 6b, in
a manner same as Ia. mp: >250°C. 1H NMR (300 MHz, DMSO-d6): δ 1.60-1.63 (2H,
m,CH2-cyclopropyl), 1.67-1.70 (2H, m, CH2-cyclopropyl), 2.11 (3H, s, ArCH3), 6.58
(1H, dd, pyrrolyl-H, J = 1.6 Hz, J = 3.6 Hz), 7.18 (1H, d, ArH, J = 8.4 Hz), 7.90-7.95
(2H, m, ArH, pyrrolyl-H), 8.27 (1H, dd, ArH, J = 2.2 Hz, J = 8.4 Hz), 8.40 (1H, d,
ArH, J = 2.2 Hz), 8.67 (1H, dd, ArH, J = 1.4 Hz, J = 8.4 Hz), 8.75 (1H, s,
pyrimidiny-H), 8.78 (1H, dd, ArH, J = 1.4 Hz, J = 4.4 Hz), 10.05 (1H, s, CONH),
13
10.50 (1H, s, CONH), 12.19 (1H, s, pyrrolyl-NH). C NMR (75 MHz, DMSO) δ
162.93, 158.16, 152.02, 151.72, 150.08, 136.56, 136.54, 134.65, 134.47, 134.08,
133.35, 131.85, 130.07, 129.40, 126.97, 126.69, 121.48, 117.30, 116.67, 105.32,
99.35, 31.02, 17.25, 16.68. HRMS (ESI) calcd for C25H20ClF3N5O2 [M+H]+,
514.1179; found, 514.1254.
4.2.5.3. N-(5-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-2-methylphenyl)-1-(7H
-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamide (Ic)
6c (0.56 g, 82.1%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ah
(0.34 g, 1 mM), in a manner same as 6a. Ic (0.31 g, 70.6%) was prepared from 6c, in
1
a manner same as Ia. mp: 184–185oC. H NMR (300 MHz, DMSO-d6): δ 1.64-1.65
(2H, m, CH2-cyclopropyl), 1.71- 1.72 (2H, m, CH2-cyclopropyl), 2.11 (3H, s, ArCH3),
6.61 (1H, dd, pyrrolyl-H, J = 1.8 Hz, J = 3.6 Hz), 7.11 (1H, d, ArH, J = 7.8 Hz), 7.25
(1H, d, ArH, J = 7.4 Hz), 7.58 (d, ArH, J = 7.8 Hz), 7.61-7.63 (2H, m, ArH), 7.78 (1H,
d, ArH, J = 7.4 Hz), 8.14 (1H, d, pyrrolyl-H, J = 1.8 Hz), 8.45 (1H, s, H-NHCONH),
8.78 (1H, s, pyrimidiny-H), 9.05 (1H, s, H-NHCONH), 10.15 (1H, s, NHCO), 12.23
13
(1H, s, pyrrolyl-NH). C NMR (75 MHz, DMSO) δ 169.16, 158.80, 152.28, 151.74,
149.99, 139.35, 137.10, 136.69, 131.85, 130.20, 126.63, 124.62, 122.90, 122.15,
120.98, 116.67, 115.89, 115.23, 114.37, 99.46, 30.95, 17.08, 16.83. HRMS (ESI)
calcd for C25H20ClF3N5O2 [M]+, 529.1288; found, 529.1357.
4.2.5.4. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methyl-N-phenylbenzam-de (Id)
6d (0.49 g, 86.1%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ab
21

ACCEPTED MANUSCRIPT
(0.23 g, 1 mM), in a manner same as 6a. Id (0.38 g, 91.3%) was prepared from 6d, in
1
a manner same as Ia. mp: 231–232oC. H NMR (300 MHz, DMSO-d6): δ 1.59-1.61
(2H, m, CH2-cyclopropyl), 1.68-1.71 (2H, m, CH2-cyclopropyl), 2.20 (3H, s, ArCH3),
6.59 (1H, dd, pyrrolyl-H, J = 1.8 Hz, J = 3.6 Hz), 7.09 (1H, m, ArH), 7.32-7.37 (3H,
m, ArH), 7.58 (1H, m, ArH), 7.71 (1H, m, ArH), 7.76 (2H, m, ArH), 8.04 (1H, d,
pyrrolyl-H, J = 1.8 Hz), 8.75 (1H, s, pyrimidiny-H), 10.07 (1H, s, NHCO), 10.21 (1H,
s, NHCO), 12.21 (1H, s, pyrrolyl-NH). 13C NMR (75 MHz, DMSO) δ 169.51, 165.03,
158.50, 151.74, 150.15, 139.13, 136.63, 136.41, 135.74, 132.80, 130.07, 128.53,
126.72, 124.22, 123.55, 120.38, 116.23, 99.40, 30.97, 17.83, 16.53. HRMS (ESI)
calcd for C24H22N5O2 [M]+, 412.1695; found, 412.1767.
4.2.5.5. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-
chlorophenyl)-4-meth-ylbenzamide (Ie)
6e (0.55 g, 91.3%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ac
(0.26 g, 1 mM), in a manner same as 6a. Ie (0.39 g, 87.4%) was prepared from 6e, in
1
a manner same as Ia. mp:233–234oC. H NMR (300 MHz, DMSO-d6): δ 2.07-2.13
(4H, m, CH2-cyclopropyl), 2.62 (3H, s, ArCH3), 7.00 (1H, m, pyrrolyl-H), 7.63-7.82
(3H, m, ArH), 7.95-8.23 (4H, m, ArH, pyrrolyl-H), 8.48-8.52 (1H, m, ArH), 9.15-9.20
(1H, m, ArH), 10.58 (1H, s, NHCO), 10.73 (1H, s, NHCO), 12.61 (1H, s,
pyrrolyl-NH). 13C NMR (75 MHz, DMSO) δ 169.53, 165.16, 158.53, 151.77, 150.10,
138.12, 136.69, 135.80, 132.54, 130.12, 128.43, 127.18, 126.72, 124.17, 124.09,
121.87, 116.15, 99.41, 30.93, 17.85, 16.65. HRMS (ESI) calcd for C24H21ClN5O2
[M]+, 446.1306; found, 446.1387.
4.2.5.6. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(3-
chlorophenyl)-4-meth-ylbenzamide (If)
6f (0.53 g, 88.7%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ad
(0.26 g, 1 mM), in a manner same as 6a. If (0.38 g, 86.1%) was prepared from 6f, in a
manner same as Ia. mp:237–238oC. 1H NMR (300 MHz, DMSO-d6): δ 1.59-1.62 (2H,
m, CH2-cyclopropyl), 1.69-1.71 (2H, m, CH2-cyclopropyl), 2.21 (3H, s, ArCH3), 6.59
(1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.6 Hz), 7.16 (1H, dd, ArH, J = 2.0 Hz, J = 8.0
Hz), 7.35-7.41 (2H, m, ArH), 7.58 (1H, t, ArH, J = 2.9 Hz), 7.71 (2H, dt, ArH, J = 2.5
Hz, J = 8.0 Hz), 7.96 (1H, t, ArH, J = 2.0 Hz), 8.05 (1H, d, pyrrolyl-H, J = 1.7 Hz),
8.75 (1H, s, pyrimidiny-H), 10.10 (1H, s, NHCO), 10.36 (1H, s, NHCO), 12.20 (1H, s,
pyrrolyl-NH). 13C NMR (75 MHz, DMSO) δ 169.54, 165.32, 158.55, 151.79, 150.09,
140.66, 136.75, 135.88, 132.91, 132.42, 130.18, 130.15, 126.72, 124.19, 124.07,
22

ACCEPTED MANUSCRIPT
123.20, 119.74, 118.62, 116.13, 99.42, 30.92, 17.87, 16.72. HRMS (ESI) calcd for
C24H21ClN5O2 [M]+, 446.1306; found, 446.1385.
4.2.5.7. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(4-
fluorophenyl)-4-meth-ylbenzamide (Ig)
6g (0.54 g, 93.2%) was prepared from 5a (0.38 g, 1mM) and the intermediate Ae
(0.24 g, 1 mM), in a manner same as 6a. Ig (0.38 g, 89.2%) was prepared from 6g, in
1
a manner same as Ia. mp: 235–236oC. H NMR (300 MHz, DMSO-d6): δ 1.62-1.63
(2H, m, CH2-cyclopropyl), 1.68-1.71 (2H, m, CH2-cyclopropyl), 2.20 (3H, s, ArCH3),
6.59 (1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.6 Hz), 6.93 (1H, td, ArH, J = 2.6 Hz, J =
8.5 Hz), 7.35-7.39 (2H, m, ArH), 7.55-7.59 (2H, m, ArH), 7.69-7.78 (2H, m, ArH),
8.04 (1H, d, pyrrolyl-H, J = 1.7 Hz), 8.75 (1H, s, pyrimidiny-H), 10.08 (1H, s,
NHCO), 10.26 (1H, s, NHCO), 12.20 (1H, s, pyrrolyl-NH). 13C NMR (75 MHz,
DMSO) δ 169.52, 164.96, 158.51, 156.63, 151.74, 150.14, 136.66, 135.77, 135.49,
132.61, 130.10, 126.73, 124.15, 122.25, 122.15, 115.25, 114.95, 99.40, 30.96, 17.83,
16.56. HRMS (ESI) calcd for C24H21FN5O2 [M]+, 430.1601; found, 430.1673.
4.2.5.8. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-N-(3-
fluorophenyl)-4-meth-ylbenzamide (Ih)
6h (0.51 g, 87.5%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Af
(0.24 g, 1 mM), in a manner same as 6a. Ih (0.36 g, 84.8%) was prepared from 6h, in
1
a manner same as Ia. mp:240–241oC. H NMR (300 MHz, DMSO-d6): δ 1.62-1.63
(2H, m,CH2-cyclopropyl), 1.69-1.72 (2H, m, CH2-cyclopropyl), 2.21 (3H, s, ArCH3),
6.59 (1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.6 Hz), 7.19 (2H, m, ArH), 7.35 (1H, d,
ArH, J = 8.0 Hz), 7.57-7.59 (1H, m, ArH), 7.70 (1H, dd, ArH, J = 2.0 Hz, J = 8.0 Hz),
7.76-781(1H, m, ArH), 8.04 (1H, d, pyrrolyl-H, J = 1.7 Hz), 8.76 (1H, s,
pyrimidiny-H), 10.11 (1H, s, NHCO), 10.39 (1H, s, NHCO), 12.20 (1H, s,
pyrrolyl-NH). 13C NMR (75 MHz, DMSO) δ 169.53, 165.31, 163.60, 160.41, 158.50,
151.75, 150.13, 140.86, 136.70, 135.99, 132.43, 130.15, 126.74, 124.17, 115.98,
110.11, 109.83, 107.13, 106.78, 99.39, 30.95, 17.85, 16.59. HRMS (ESI) calcd for
C24H21FN5O2 [M]+, 430.1601; found, 430.1679.
4.2.5.9. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methyl-N-(4-morpholino-3-(trifluoromethyl)phenyl)benzamide (Ii)
6i (0.59 g, 82.7%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ai
(0.38 g, 1 mM), in a manner same as 6a. Ii (0.39 g, 83.5%) was prepared from 6i, in a
23

ACCEPTED MANUSCRIPT
manner same as Ia. mp:171–173oC. 1H NMR (300 MHz, DMSO-d6): δ 1.61-1.63 (2H,
m, CH2-cyclopropyl), 1.68-1.69 (2H, m, CH2-cyclopropyl), 2.20 (3H, s, ArCH3),
2.82-2.85 (4H, m, CH2NCH2), 3.69-3.72 (4H, m, CH2OCH2), 6.59 (1H, dd,
pyrrolyl-H, J = 1.7 Hz, J = 3.6 Hz), 7.36 (1H, d, ArH, J = 8.0 Hz), 7.57-7.61 (2H, m,
ArH), 7.72 (1H, d, ArH, J = 8.0 Hz), 8.07 (1H, d, pyrrolyl-H, J = 1.7 Hz), 8.08-8.16
(2H, m, ArH), 8.75 (1H, s, pyrimidiny-H), 10.08 (1H, s, CONH), 10.42 (1H, s,
13
CONH), 12.20 (1H, s, pyrrolyl-NH). C NMR (75 MHz, DMSO) δ 169.54, 165.22,
158.44, 151.75, 150.05, 147.15, 136.72, 136.46, 136.05, 132.19, 130.06, 129.50,
126.73, 125.20, 124.82, 124.10, 118.31, 116.26, 115.02, 112.98, 99.38, 66.61, 53.45,
31.03, 17.85, 16.61. HRMS (ESI) calcd for C29H28F3N6O3 [M]+, 565.2097; found,
565.2184.
4.2.5.10. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methyl-N-(4-(4-methylpi-perazin-1-yl)-3-(trifluoromethyl)phenyl)benzamide (Ij)
6j (0.57 g, 78.5%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Aj
(0.39 g, 1 mM), in a manner same as 6a. Ij (0.38 g, 83.9%) was prepared from 6j, in a
manner same as Ia. mp:180–182oC. 1H NMR (300 MHz, DMSO-d6): δ 1.59-1.63 (2H,
m, CH2- cyclopropyl), 1.68-1.71 (2H, m, CH2-cyclopropyl), 2.20 (3H, s, ArCH3), 2.24
(3H, s, NCH3), 2.46-2.51 (4H, m, CH2NCH2), 2.83-2.85 (4H, m, CH2NCH2), 6.59
(1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.6 Hz), 7.36 (1H, d, ArH, J = 8.0 Hz), 7.55-7.58
(2H, m, ArH), 7.71 (1H, dd, ArH, J = 2.0 Hz, J = 8.0 Hz), 8.03-8.06 (2H, m, ArH,
pyrrolyl-H), 8.14 (1H, d, ArH, J = 2.5 Hz), 8.75 (1H, s, pyrimidiny-H), 10.08 (1H, s,
CONH), 10.40 (1H, s, CONH), 12.20 (1H, s, pyrrolyl-NH). HRMS (ESI) calcd for
C30H31F3N7O2 [M]+, 578.2413; found, 578.2497.
4.2.5.11. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methyl-N-(4-((1-methylpi-peridin-4-yl)oxy)-3-(trifluoromethyl)phenyl)benzamide
(Ik)
6k (0.57 g, 76.2%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Ak
(0.41 g, 1 mM), in a manner same as 6a. Ik (0.38 g, 84.2%) was prepared from 6k, in
1
a manner same as Ia. mp:150–152oC. H NMR (300 MHz, DMSO-d6): δ 1.59-1.63
(2H, m, CH2-cyclopropyl), 1.65-1.71 (4H, m, CH2-cyclopropyl, CHCH2), 1.89-1.95
(2H, m, CHCH2), 2.17 (3H, s, NCH3), 2.20 (3H, s, ArCH3), 2.25-2.27 (2H, m, NCH2),
24

ACCEPTED MANUSCRIPT
2.51-2.52 (2H, m, NCH2), 4.58 (1H, s, OCH), 6.59 (1H, dd, pyrrolyl-H, J = 1.5 Hz, J
= 3.5 Hz), 7.30-7.37 (2H, m, ArH), 7.58 (1H, m, ArH), 7.71 (1H, dd,ArH, J = 1.9 Hz,
J = 7.9 Hz), 7.95-7.99 (1H, m, ArH), 8.06-8.09 (2H, m, ArH, pyrrolyl-H), 8.76 (1H, s,
pyrimidiny-H), 10.08 (1H, s, NHCO), 10.29 (1H, s, NHCO), 12.20 (1H, s,
pyrrolyl-NH). 13C NMR (75 MHz, DMSO) δ 169.53, 164.90, 158.50, 151.75, 150.82,
150.12, 136.69, 135.85, 132.39, 131.81, 130.13, 126.73, 125.65, 124.12, 118.97,
116.19, 115.17, 99.74, 72.23, 51.64, 45.81, 30.93, 30.10, 17.84, 16.60. HRMS (ESI)
calcd for C31H32F3N6O3 [M]+, 593.2410; found, 593.2488.
4.2.5.12. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methyl-N-(4-((1-methylpi-peridin-4-yl)methoxy)-3-(trifluoromethyl)phenyl)
benzamide (Il)
6l (0.55 g, 72.8%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Al
(0.42 g, 1 mM), in a manner same as 6a. Il (0.37 g, 83.8%) was prepared from 6l, in a
manner same as Ia. mp: 237–238oC. 1H NMR (300 MHz, DMSO-d6): δ 1.38-1.45 (4H,
m, CH2CHCH2), 1.59-1.61 (2H, m, CH2-cyclopropyl), 1.67-1.69 (2H, m,
CH2-cyclopropyl), 2.20 (3H, s, ArCH3), 2.28 (3H, s, NCH3), 2.67-2.73 (2H, m, NCH2),
2.92-2.95 (2H, m, NCH2), 3.96-3.97 (2H, m, OCH2) , 4.49-4.54 (1H, m, CH),6.58 (1H,
dd, pyrrolyl-H, J = 1.7 Hz, J = 3.5 Hz), 7.28 (1H, d, ArH, J = 9.0 Hz), 7.47 (1H, d,
ArH, J = 8.0 Hz), 7.56-7.58 (1H, m , ArH), 7.86-7.95 (2H, m, ArH), 8.00 (1H, dd,
ArH, J = 2.7 Hz, J = 9.0 Hz), 8.05-8.08 (2H, m, ArH, pyrrolyl-H ), 8.75 (1H, s,
pyrimidiny-H), 10.37 (1H, s,NHCO), 12.16 (1H, s, pyrrolyl-NH). 13C NMR (75 MHz,
DMSO) δ 172.02, 164.40, 160.04, 159.39, 159.30,152.46, 151.29, 150.86, 139.66,
138.92, 137.91, 133.03, 131.76, 130.79, 128.14, 126.64, 126.10, 125.83, 121.83,
113.78, 99.20, 72.54, 54.38, 48.82, 47.40, 45.29, 34.27, 27.54, 17.70. HRMS (ESI)
calcd for C32H34F3N6O3 [M]+, 607.2566; found, 607.2651.
4.2.5.13. 3-(1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclopropane-1-carboxamido)-4-
methyl-N-(4-(3-morpholi-nopropoxy)-3-(trifluoromethyl)phenyl)benzamide (Im)
6m (0.65 g, 84.3%) was prepared from 5a (0.38 g, 1 mM) and the intermediate Am
(0.44 g, 1 mM), in a manner same as 6a. Im (0.44 g, 83.6%) was prepared from 6m,
in a manner same as Ia. mp:169-171oC. 1H NMR (300 MHz, DMSO-d6): δ 1.59-1.63
25

ACCEPTED MANUSCRIPT
(2H, m, CH2-cyclopropyl), 1.68-1.71 (2H, m, CH2-cyclopropyl), 1.84-1.92 (2H, m,
CH2- CH2CH2CH2), 2.20 (3H, s, ArCH3), 2.34-2.36 (4H, m, CH2NCH2), 2.43 (2H, t,
CH2N, J = 7.0 Hz), 3.57 (4H, t, CH2OCH2, J = 4.5 Hz), 4.13 (2H, t, OCH2CH2, J =
6.0 Hz), 6.59 (1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.6 Hz), 7.27 (1H, d, ArH, J = 9.0
Hz), 7.35 (1H, d, ArH, J = 8.0 Hz), 7.58 (1H, dd, ArH, J = 2.3 Hz, J = 3.6 Hz), 7.71
(1H, dd, ArH, J = 2.0 Hz, J = 7.9 Hz), 8.00 (1H, dd, ArH, J = 2.3 Hz, J = 9.0 Hz),
8.06-8.08 (2H, m, ArH, pyrrolyl-H), 8.75 (1H, s, pyrimidiny-H), 10.07 (1H, s,
CONH), 10.29 (1H, s, CONH), 12.19 (1H, s, pyrrolyl-NH). 13C NMR (75 MHz,
DMSO) δ 169.45, 164.96, 158.53, 154.42, 152.32, 151.76, 150.10, 136.86, 135.66,
132.19, 131.95, 129.96, 126.72, 125.74, 125.43, 124.09, 121.87, 118.80, 115.96,
113.93, 99.40, 66.64, 66.05, 54.51, 53.24, 30.81, 25.66, 17.69, 16.67. HRMS (ESI)
calcd for C32H34F3N6O4 [M]+, 623.2515; found, 623.2586.
4.2.5.14.N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-(2-methyl-2-(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)propanamido)benzamide (In)
6n (0.55 g, 82.8%) was prepared from 5b (0.39 g, 1 mM) and the intermediate Aa
(0.33 g, 1 mM), in a manner same as 6a. In (0.42 g, 81.3%) was prepared from 6n, in
a manner same as Ia. mp: 168–169oC. 1H NMR (300MHz, DMSO-d6): δ 1.73 (6H, s,
CH3), 2.02 (3H, s, ArCH3), 6.54 (1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.5 Hz), 7.34
(1H, d, ArH, J = 8.6 Hz), 7.53 (1H, d, ArH, J = 1.7 Hz), 7.70-7.75 (3H, m, ArH), 8.12
(1H, d, pyrrolyl-H, J = 1.7 Hz), 8.35 (1H, d, ArH, J = 2.2 Hz), 8.78 (1H, s,
pyrimidiny-H), 9.07 (1H, s, NHCO), 10.60 (1H, s, NHCO), 12.14 (1H, s,
pyrrolyl-NH). 13C NMR (75 MHz, DMSO) δ 173.40, 165.30, 152.12, 150.69, 146.09,
138.66, 138.33, 136.36, 134.09, 131.95, 131.86, 130.21, 126.67, 126.37, 125.04,
124.87, 124.25, 118.98, 117.64, 104.25, 25.25, 21.01, 17.43. HRMS (ESI) calcd for
C25H22ClF3N5O2 [M]+, 516.1336; found, 516.1411.
4.2.5.15. 4-methyl-3-(2-methyl-2-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)propanamido)-N
-(4-((1-methylpiperidi-n-4-yl)methoxy)-3-(trifluoromethyl)phenyl)benzamide (Io)
6o (0.57 g, 74.6%) was prepared from 5b (0.39 g, 1 mM) and the intermediate Al
(0.42 g, 1 mM), in a manner same as 6a. Io (0.52 g, 84.7%) was prepared from 6o, in
1
a manner same as Ia. mp:177–179oC. H-NMR (300 MHz, DMSO-d6): δ 1.33-1.37
(2H, m, CH2),1.70-1.73 (8H, m, CH3 and CH2), 1.84-1.92 (3H, m, CH2 and CH), 2.01
26

ACCEPTED MANUSCRIPT
(3H, s, ArCH3), 2.16 (3H, s, N-CH3), 2.78-2.82 (2H, m, CH2), 3.94-3.95 (2H, m, CH2),
6.55 (1H, dd, pyrrolyl-H, J = 1.7 Hz, J = 3.5 Hz), 7.23 (1H, d, ArH, J = 8.9 Hz), 7.31
(1H, d, ArH, J = 8.3 Hz), 7.53-7.54 (1H, m, ArH), 7.72-7.74 (1H, m, ArH), 7.98 (1H,
d, ArH, J = 8.3 Hz), 8.07 (1H, d, pyrrolyl-H, J = 1.7 Hz), 8.78 (1H, s, pyrimidiny-H),
9.06 (1H, s, NHCO), 10.30 (1H, s, NHCO), 12.15 (1H, s, pyrrolyl-NH). 13C NMR (75
MHz, DMSO) δ 174.02, 164.82, 163.12, 152.50, 151.74, 150.20, 137.58, 136.63,
132.21, 131.80, 130.03, 126.20, 126.04, 125.76, 125.45, 124.55, 118.92, 118.85,
115.00, 113.64, 99.67, 72.79, 54.83, 49.92, 46.03, 34.79, 28.14, 25.49, 17.45. HRMS
(ESI) calcd for C32H36F3N6O3 [M]+, 609.2723; found, 609.2798.
Supporting Information
Part I. The experimental section for biological activity assay (The Experimental
Section 4.). Part II. The chemical structures of P-1 and Ia-Io were elucidated by
1H-NMR and 13C-NMR
Corresponding Authors
For Weifang Tang: Tel.: +86-25-86185201. Fax: +86-25-86185182. E-mail: twf@cpu.edu.cn
For Tao Lu: Tel.: +86-25-86185180. Fax: +86-25-86185179. E-mail: lutao@cpu.edu.cn
Author Contributions
The manuscript was written through contributions of all authors. Lu Wang and
Yanmin Zhang contributed equally to this work and should be considered as co-first
authors. All authors have given approval to the final version of the manuscript.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
This work was financially supported by National Natural Science Foundation of
China (No. 21572273/B020601 and No. 81803370), National Natural Science
27

ACCEPTED MANUSCRIPT
Foundation of Jiangsu Province (No. BK20180559), Key Laboratory Innovation
Research and Cultivation Fund (No. SKLNMZZCX201812), a project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions
and College Student Innovation Project for the R&D of Novel Drugs (No. J1030830)
and the “Double World-classes” Construction Program of China Pharmaceutical
Unibersity (No. CPU2018GF02). We also express our gratitude to Dr. Xiazhong Ren,
Dr. Huajun Yang, Dr. Chunlan Dong (Crown Bioscience Corporation), Dr. Jamie
Planck (RBC, Pennsylvania, USA) and NCI for their helpful support in biological
evaluation.
Abbreviations
MAPK
NCI
PSA
mitogen-activated protein kinase
National Cancer Institute
polar surface area
SAR
NMR
Structure activity relationships
nuclear magnetic resonance
Reference
[1] L. Santarpia, S. M. Lippman, A. K. El-Naggar, Targeting the MAPK–RAS–RAF signaling
pathway in cancer therapy. Expert Opin. Ther. Targets 16 (1) (2012) 103-119.
[2] D. Vigil, J. Cherfils, K. L. Rossman, C. J. Der, Ras superfamily GEFs and GAPs: validated and
tractable targets for cancer therapy? Nat. Rev. Cancer 10 (2010) 842.
[3] Y. Pylayeva-Gupta, E. Grabocka, D. Bar-Sagi, RAS oncogenes: weaving a tumorigenic web. Nat.
Rev. Cancer 11 (2011) 761.
[4] P. T. C. Wan, M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz, V. M. Good, C. G. Project, C.
M. Jones, C. J. Marshall, C. J. Springer, D. Barford, R. Marais, Mechanism of Activation of the
RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF. Cell 116 (6) (2004) 855-867.
[5] X. Wang, J. Kim, Conformation-Specific Effects of Raf Kinase Inhibitors. J. Med. Chem. 55 (17)
(2012) 7332-7341.
[6] A. C. Backes, B. Zech, B. Felber, B. Klebl, G. Müller, Small-molecule inhibitors binding to
protein kinase. Part II: the novel pharmacophore approach of type II and type III inhibition. Expert.
Opin. Drug Discov. 3 (12) (2008) 1427-1449.
[7] J. R. Henry, M. D. Kaufman, S.-B. Peng, Y. M. Ahn, T. M. Caldwell, L. Vogeti, H. Telikepalli,
W.-P. Lu, M. M. Hood, T. J. Rutkoski, B. D. Smith, S. Vogeti, D. Miller, S. C. Wise, L. Chun, X.
Zhang, Y. Zhang, L. Kays, P. A. Hipskind, A. D. Wrobleski, K. L. Lobb, J. M. Clay, J. D. Cohen, J.
L. Walgren, D. McCann, P. Patel, D. K. Clawson, S. Guo, D. Manglicmot, C. Groshong, C. Logan,
J.
J.
Starling,
D.
L.
Flynn,
Discovery
of
1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-
6-yl)phenyl)urea (LY3009120) as a Pan-RAF Inhibitor with Minimal Paradoxical Activation and
Activity against BRAF or RAS Mutant Tumor Cells. J. Med. Chem. 58 (10) (2015) 4165-4179.
28