Docking-based structural splicing and reassembly strategy to develop novel deazapurine derivatives as potent B-Raf V600E inhibitors

Article abstract of DOI:10.1038/aps.2016.173

The mutation of B-Raf V600E is widespread in a variety of human cancers. Its inhibitors vemurafenib and dabrafenib have been launched as drugs for treating unresectable melanoma, demonstrating that B-Raf V600E is an ideal drug target. This study focused on developing novel B-Raf V600E inhibitors as drug leads against various cancers with B-Raf V600E mutation. Using molecular modeling approaches, 200 blockbuster drugs were spliced to generate 283 fragments followed by molecular docking to identify potent fragments. Molecular structures of potential inhibitors of B-Raf V600E were then obtained by fragment reassembly followed by docking to predict the bioactivity of the reassembled molecules. The structures with high predicted bioactivity were synthesized, followed by in vitro study to identify potent B-Raf V600E inhibitors. A highly potent fragment binding to the hinge area of B-Raf V600E was identified via a docking-based structural splicing approach. Using the fragment, 14 novel structures were designed by structural reassembly, two of which were predicted to be as strong as marketed B-Raf V600E inhibitors. Biological evaluation revealed that compound 1m is a potent B-Raf V600E inhibitor with an IC 50 value of 0.05 μmol/L, which was lower than that of vemurafenib (0.13 μmol/L). Moreover, the selectivity of 1m against B-Raf WT was enhanced compared with vemurafenib. In addition, 1m exhibits desirable solubility, bioavailability and metabolic stability in in vitro assays. Thus, a highly potent and selective B-Raf V600E inhibitor was designed via a docking-based structural splicing and reassembly strategy and was validated by medicinal synthesis and biological evaluation.

Full text of DOI:10.1038/aps.2016.173

Acta Pharmacologica Sinica 2017: 1–10
2017 CPS and SIMM All rights reserved 1671-4083/17
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©
Original Article
Docking-based structural splicing and reassembly
strategy to develop novel deazapurine derivatives as
V600E
potent B-Raf inhibitors
1
, 2, #
3, #
1, #
1, 2, #
1
1
1
1,
*
,
Gui-min WANG
, Xiang WANG , Jian-ming ZHU , Bin-bin GUO
, Zhuo YANG , Zhi-jian XU , Bo LI , He-yao WANG
3,
1,
3
*
*
Ling-hua MENG , Wei-liang ZHU , Jian DING
1
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
2
3
China; University of Chinese Academy of Sciences, Beijing 100049, China; National Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Abstract
The mutation of B-Raf
V600E
is widespread in a variety of human cancers. Its inhibitors vemurafenib and dabrafenib have been launched
V600E
as drugs for treating unresectable melanoma, demonstrating that B-Raf
novel B-Raf
is an ideal drug target. This study focused on developing
V600E
V600E
inhibitors as drug leads against various cancers with B-Raf
mutation. Using molecular modeling approaches, 200
blockbuster drugs were spliced to generate 283 fragments followed by molecular docking to identify potent fragments. Molecular
V600E
structures of potential inhibitors of B-Raf
were then obtained by fragment reassembly followed by docking to predict the bioactivity
of the reassembled molecules. The structures with high predicted bioactivity were synthesized, followed by in vitro study to identify
V600E
V600E
potent B-Raf
inhibitors. A highly potent fragment binding to the hinge area of B-Raf
was identified via a docking-based
structural splicing approach. Using the fragment, 14 novel structures were designed by structural reassembly, two of which were
V600E
V600E
predicted to be as strong as marketed B-Raf
inhibitors. Biological evaluation revealed that compound 1m is a potent B-Raf
inhibitor with an IC50 value of 0.05 μmol/L, which was lower than that of vemurafenib (0.13 μmol/L). Moreover, the selectivity of 1m
WT
against B-Raf was enhanced compared with vemurafenib. In addition, 1m exhibits desirable solubility, bioavailability and metabolic
V600E
stability in in vitro assays. Thus, a highly potent and selective B-Raf
inhibitor was designed via a docking-based structural splicing
and reassembly strategy and was validated by medicinal synthesis and biological evaluation.
V600E
Keywords: B-Raf
inhibitor; anticancer; vemurafenib; deazapurine; fragment reassembly; molecular docking; structure–activity
relationship
Acta Pharmacologica Sinica advance online publication, 17 Apr 2017; doi: 10.1038/aps.2016.173
[
4]
Introduction
of malignant melanoma , ~58% of papillary thyroid cancer
[5]
[6, 7]
The RAS-RAF-MEK-ERK signaling pathway is the most (PTC) , 4.7% to 10% of colorectal cancer , 12.5% of Grade 1
extensively characterized cascade of three mitogen-activated serous ovarian carcinoma , ~94% of papillary craniopharyngi-
protein kinase (MAPK) pathways in the human body, which oma and a wide variety of other cancers . Compared with
transduces the signals from extracellular spaces to intracel- other isoforms of the RAF kinase family, A-Raf and C-Raf (also
lular locations and plays a prominent role in cell proliferation, known as Raf-1), the regulation of B-Raf activation requires
[8]
[9]
[10]
[
1–3]
differentiation and survival . Approximately one-third of less molecular events, resulting in increased kinase activity,
[
3]
human cancers possess mutations in this pathway , among and its activity is more frequently induced by a single point
V600E
[11]
V600E
which the mutation of B-Raf
has been identified in ~34% mutation . The B-Raf
mutation has ~500-fold greater
[
12]
activity compared with wild type in vitro . Persistent B-Raf
activation stimulates cancer cell proliferation and protects cells
from apoptosis, which makes it an attractive anticancer drug
inhibitors have been heavily investigated by
both academia and the pharmaceutical industry. Two small
#
These authors contributed equally to this work.
*
To whom correspondence should be addressed.
E-mail wlzhu@simm.ac.cn (Wei-liang ZHU);
hywang@simm.ac.cn (He-yao WANG);
V600E
target. B-Raf
lhmeng@simm.ac.cn (Ling-hua MENG)
Received 2016-11-01 Accepted 2016-12-24
molecule drugs, vemurafenib (PLX4032, brand name: Zel-

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2
Wang GM et al
[
22]
boraf) and dabrafenib (GSK2118436, brand name: Tafinlar), gets the B-cell lymphoma 2 (BCL-2) protein . In research,
have been approved by the FDA for the treatment of multiple docking-based fragment screening and fragment reassembly,
V600E
V600E
malignant cancers with the B-Raf
mutation; thus, B-Raf
ie, fragment growing, fragment linking and fragment merg-
[
13-16]
is a novel anticancer drug target
. There are some other ing, were also applied and exhibited high efficiency in lead
V600E
[23]
B-Raf
inhibitors in clinical trials or at various stages of discovery and optimization . In addition to virtual screening
drug development . However, new B-Raf
still urgently required due to unaffordable cost and emerging facilitate fragment reassembly. For instance, the AutoT&T2
[
3]
V600E
inhibitors are programs, computational approaches have been developed to
[16]
resistance to present inhibitors
.
software suite developed by Wang RX at the State Key Labo-
All protein kinases contain a segment connecting the amino- ratory of Bioorganic and Natural Products Chemistry achieves
and carboxy-terminal kinase catalytic domain, known as automatic tailoring and transplanting with the hits from
the hinge region (Supplementary Figure S1), which could screening, which establishes a complete pipeline of FBDD and
[
24]
form hydrogen bonding interactions with the adenine ring provides insight into de novo drug design . Accordingly, it
of ATP, playing a central role in ATP-binding and substrate is clear that appropriate application of FBDD could accelerate
[
17, 18]
phosphorylation
. Accordingly, hindering ATP from the drug discovery process.
binding to the hinge region should be a straightforward and
effective approach to inhibit kinase activity. This approach ment that can bind to the hinge region of B-Raf
In this context, we sought to identify a novel molecular frag-
V600E
with high
has been confirmed by its use in numerous successful kinase affinity and then performed further optimization using the
inhibitors, eg, PD166326 (ABL1 inhibitor), axitinib (ABL1 and FBDD strategy, as described in Figure 1.
VEGFR2 inhibitor), lapatinib (EGFR and ERBB2 inhibitor), and
[
18–20]
sorafenib (a dual RAF-KDR inhibitor)
. As a member of Materials and methods
the kinase family, apart from the hinge region, the B-Raf cata- Fragment preparation, molecular docking and assembly
lytic area also includes a glycine-rich loop (G-loop), αC helix, Molecular fragments were derived from the small molecular
[12]
and activation loop (A-loop) (Supplementary Figure S1) . To drugs listed in the top 200 pharmaceutical products by US
V600E
our knowledge, the vast majority of B-Raf
inhibitors bind retail sales in 2011. In consideration of the hinge-binding areas
to the hinge region in kinase catalytic areas and overlap with of vemurafenib and dabrafenib, we filtered the fragments gen-
the ATP-binding region to some extent. One or more criti- erated by Pipeline Pilot 7.5 with the component named Gener-
cal hydrogen bonds are found between the hinge region of ate Fragments using the following criteria: molecular weight
V600E
B-Raf
and the inhibitors, which make a significant contri- ranges from 50 to 300 and number of heavy atoms ranges from
[3, 15]
[25]
bution to the binding affinity
tal structures of B-Raf
. In accordance with the crys- 5 to 16 . Molecular fragments were prepared using LigPrep
V600E
and the drugs, both the 7-azaindole with all possible protonation states generated at pH 7.0±3.0
[
13]
[26–28]
moiety of vemurafenib (PDB code: 3OG7 , Supplementary by Epik
Figure S1B) and the 2-amino pyrimidine moiety of dabrafenib docking in its SP mode with the post-docking minimization
PDB code: 4XV2 , Supplementary Figure S1D) form two including 10000 poses per ligand, and the remaining param-
critical hydrogen bonds with the hinge region, which greatly eters were set to default. The X-ray structure of the B-Raf
supports the central role of the hinge-binding area. In our pre- binding by vemurafenib (PDB code: 3OG7) was retrieved from
vious work, combining pharmacophore-based virtual screen- the PDB as the docking structure in this study. To predict the
. Then, Glide was utilized to perform molecular
[
15]
(
V600E
ing and scaffold hopping, a series of compounds were identi- binding modes of the new compounds, molecular docking
V600E
fied as B-Raf
inhibitors. This study showed that forming was performed using Glide in its SP mode in a standard pro-
[29–31]
strong hydrogen bonding with the hinge region results in cedure
enhanced IC50 values for the series of inhibitors
. The docked conformations of the molecules with
[12]
.
the lowest energy were selected for further studies.
Fragment-based drug discovery (FBDD) has been widely
used in the pharmaceutical industry during recent years, typi- Chemistry
cally as a mainstream alternative to high-throughput screen- All starting materials and solvents were purchased from com-
ing. With a high hit rate and satisfactory ligand efficiency (LE) mercial suppliers and used without further purification unless
of the hits, numerous inhibitors have advanced to clinical trials otherwise noted. The chemical synthesis of all the designed
[
21]
or have been approved using the FBDD approach . Vemu- compounds is fully described in the Experimental Section of
1
13
rafenib is a remarkable representative of successful FBDD the Supplementary Information. The H and C spectra were
cases, which implies the applicability and high-efficiency of obtained on Bruker Avance III (Karlsruhe, Germany) with 300,
V600E
FBDD targeting B-Raf
. The 7-azaindole moiety of vemu- 400, 500 and 600 NMR spectrometers operating at 300 MHz,
1
rafenib was derived from high-concentration screening and 400 MHz or 600 MHz for H NMR and 100 MHz or 125 MHz
was shown to form hydrogen bonding with the hinge area for C NMR, respectively. The deuterated solvents, such as
by crystallographic analysis. Another successful FBDD drug CDCl and DMSO-d , were used with the internal standard
1
3
3
6
is the first approved PPI inhibitor Venetoclax (brand name: of tetramethylsilane (TMS). Chemical shifts are provided in
Venclexta), which was approved in April 2016 for the treat- δ values of ppm. The abbreviation s indicates singlet, d indi-
ment of patients with chronic lymphocytic leukemia (CLL). cates doublet, t indicates triplet, and m indicates multiplet.
Venetoclax was the first FDA-approved treatment that tar- Coupling constants (J) were measured in hertz (Hz). The
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Wang GM et al
3
V600E
Figure 1. Schematic representation of the B-Raf
inhibitor discovery process with FBDD.
LRMS and HRMS were recorded on a ThermoFinnigan LCQ IC50 values were from at least three independent tests.
Deca (San Jose, CA, USA) and a Micromass Q-TOF Ultima (in
ESI mode, Manchester, UK) spectrometer, respectively.
In vitro B-Raf kinase assay
Given that B-Raf catalyzes the phosphorylation of MEK1/2, an
ELISA-based MEK1/2 phosphorylation assay was developed
and widely used in the evaluation of the kinase activity of
Biological evaluation
Cell viability assessment
The human melanoma cell line A375 (B-Raf
V600E
) and human B-Raf or the inhibitory activity of B-Raf inhibitors. To deter-
colorectal cancer cell line HCT116 (B-Raf ) were used in the mine the IC50 values of compounds in this study at the molecu-
WT
preliminary evaluation of the anti-tumor activity of the com- lar level, we performed the ELISA-based MEK1/2 phosphory-
[
32]
[33]
pounds using the MTT assay . The cells were dissociated lation assay as previously described . A sigmoidal dose
into single suspension with H-DMEM culture medium con- response curve was generated with duplicate or triplicate
V600E
taining 10% fetal bovine serum and then seeded into 96-well measurements at each inhibitor concentration using B-Raf
plates with 2500 (A375) or 3500 (HCT116) cells per well. After or B-Raf protein. Accordingly, IC50 values were generated.
WT
2
4 h of cultivation, compounds were treated at the initial con- In addition, Western blot assays were also performed to esti-
centration of 10 μmol/L, and an equal amount of DMSO was mate the phosphorylation level of MEK1/2 using a standard
used as the vehicle control. Cells were further incubated for procedure.
7
2 h, and then MTT solution was added to the well to a final
concentration of 0.5 mg/mL. The plates were incubated at In vitro ADME profile and solubility
7°C for 4 h. The medium was discarded, followed by addi- Human liver microsome assays were performed to evaluate
3
tion of 100 μL DMSO. The plates were shaken for 10 min and in vitro metabolic stability. The concentrations of the parent
absorbance was detected at 492 nm with a multi-well spectro- compound in reaction systems were determined by LC-MS/
photometer.
MS to estimate the stability (the detailed experimental proce-
The sulforhodamine B (SRB) staining assay was performed dures and data analyses are included in the Supplementary
to detect the activity of compounds on the proliferation of Information). Solubility was measured in different buffer
HT-29, COLO205, LOVO, HCT-15, HCT-8, HCT116, SW1116, solutions using the classical shake test method. Permeability
SW620 and SW480 human colorectal cancer cells. In brief, determination was performed using bidirectional permeability
cells were seeded in 96-well plates at 3000 to 4000 cells/well, assays. In addition, metabolic evaluation with cytochrome
followed by treatment of serially diluted compounds for 72 h. P450 was also performed to assess the metabolic stability of
The medium was replaced by 10% trichloroacetic acid, and the compound.
then cells were stained with SRB. After cells being washed
with 1% acetic acid, remaining SRB was dissolved in 100 μL Results and discussion
of buffer containing 10 mmol/L Tris-base, and the OD value Fragment generation and evaluation
was measured at 560 nm with a multi-well spectrophotometer. Based on the structures of the top 200 drugs, 283 fragments
The inhibitory rate of cell proliferation was calculated using were generated. Taking into account the different protonation
the formula (ODcontrol–ODtreatment)/ODcontrol×100%. The average states, 429 fragment structures were prepared for docking. All
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Wang GM et al
V600E
of the fragment structures were then docked against B-Raf
1a (Figure 3C). The docking study revealed that 1a is a potent
V600E
with one pose output for each structure (Supplementary Table inhibitor of B-Raf
given its similar docking score and bind-
S1). The top 10 fragments with the highest score (Figure 2) all ing mode as vemurafenib (Table 1 and Figure 3D). Indeed,
formed hydrogen bonding with the hinge region, except for primary biological evaluation indicated that the IC50 of 1a was
fragments f3, f6 and f7. In particular, the fragment of peme- 0.80 μmol/L against the A375 cell line, whereas that of vemu-
trexed (7-deazaguanine) f1 with the highest docking score of rafenib is 0.56 μmol/L. Given the availability of the reagents
-
7.920 caught our attention given its 5 hydrogen bond accep- in our laboratory, compounds 1b-1n were designed and syn-
V600E
tor/donors. We re-docked the fragment to B-Raf
and thesized for exploring the structure-activity relationship (Table
output 3 poses to search for more binding modes. All 3 bind- 1). Among the new compounds, 1m was predicted to have
V600E
ing poses were located around the hinge region. Of note, in similar B-Raf
addition to the first pose with a docking score value of -7.920,
inhibitory potential as 1a.
the second pose, with a docking score of -7.603 and ligand Chemistry
efficiency (the docking score divided by the number of heavy The synthetic route of deazapurine derivatives studied in this
non-hydrogen) atoms) of -0.691, was predicted to superim- study is outlined in Figure 4. 2-Bromo-1,1-dimethoxyethane
pose well with the hinge-binding fragment of vemurafenib was hydrolyzed under concentrated hydrochloride to obtain
Figure 3A). In addition, we also docked the hinge-binding bromoacetaldehyde, which was then heated to condense
(
(
fragment of vemurafenib, and the second conformation could 2,6-diaminopyrimidin-4(3H)-one (2) to give compound 3
reproduce the crystal structure of vemurafenib (Supplemen- under sodium acetate. Compound 3 was acetylized under
tary Figure S2). Impressively, the corresponding docking acetic anhydride to obtain 5a, which was then hydrolyzed to
score (-7.742) is slightly lower than f1 (-7.603), but the ligand give 6a under ammonium hydroxide without further purifica-
efficiency (-0.484) is significantly increased compared with the tion. Compound 3 was methylated to obtain 4 under dimethyl
latter (-0.691). Therefore, f1 should be an ideal fragment to sulfate, which was acetylized to achieve 6b via 5b (Figure 4A).
replace the 7-azaindole moiety of vemurafenib.
2,6-Difluorobenzoic acid (7) was nitrified to obtain 8 under
the mixture of nitric acid and sulfuric acid, and 8 reacted
with methanol under a catalytic amount of concentrated sul-
Hit compounds derived from fragment reassembly
Using f1 as an ideal hinge-binding fragment, we sought to furic acid to form 9, which was reduced by iron powder in
replace the hinge-binding area of vemurafenib with f1. To the solvent of acetic acid to obtain 10. Then, 10 reacted with
ensure the validity of the aforementioned fragment reas- two equivalents of sulfonyl chloride to afford 11, which was
sembly strategy, we performed molecular docking using the hydrolyzed under the aqueous solution of NaOH to achieve
remaining vemurafenib moieties with the same parameters 12. The derivatives of benzoic acid (8, 12a and 12b) were
as the aforementioned fragment docking. As expected, the chloridized to afford the corresponding acyl chlorides (13a-c)
binding mode similar to that of vemurafenib was ranked third (Figure 4B). Compounds 1a-d, 1m and 1n were synthesized
in the docking results with a docking score of -7.368 (Figure from the fragments obtained from procedures A and B (6 and
3
B). In this context, we found that no fragment apart from 13a-c) via the Friedel-Crafts reaction in the presence of AlCl
3
V600E
f3 could bind to B-Raf
at the same position with a higher (Figure 4C). Compound 14 was hydrolyzed by NaOH aque-
docking score than -7.368. For synthesis, we replaced the ous solution to achieve 15, which was then protected by Boc to
-azaindole moiety of vemurafenib and obtained compound give 16. Compounds 17a-c were obtained by the reaction of 16
7
Figure 2. Structure of the top 10 molecular fragments ranked by docking score.
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Wang GM et al
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V600E
Figure 3. Molecular docking and fragment reassembly identified 1a as a potential B-Raf
fragment of vemurafenib (pink) and (D) 1a (green) on vemurafenib (gray) with B-Raf
inhibitor. Overlay of (A) f1 (cyan), (B) hinge-binding
V600E
(PDB entry 3OG7). The protein is shown in cartoon form and
the ligands are in stick form. (C) Molecular tailoring and fragment reassembly.
with CH
deprotected to give the important intermediates 18a-c (Figure 1d with a chlorine atom, resulting in 1e, 1f, 1g and 1h, respec-
D). Compounds 1e-l were synthesized from the fragments tively. As expected, the activities of chlorine-substituted com-
15 or 18a-c and 13a-c) obtained from the procedures B and D pounds became weaker compared with the amino-substituted
3
I, 4-nitrobenzyl bromide or allyl bromide, which was ing affinity, we replaced the amino groups of 1a, 1b, 1c and
4
(
via the Friedel-Crafts reaction in the presence of AlCl
E).
3
(Figure compounds except for 1g, whose inhibitory activity was
4
extremely inconsistent with the docking score (Table 1). In
view of the structure of 1g, we hypothesized that the activity
V600E
Structure-activity relationship study
of 1g in cells was not achieved by targeting B-Raf
given
Vemurafenib forms 3 hydrogen bonds with residues D594, that its selectivity between A375 and HCT116 cell lines was
F595 and G596 by the sulfonamide moiety (Figure 5A). If 1a lost.
V600E
interacts with B-Raf
in a similar manner, replacing the
On account of the similar binding mode of 1a and vemu-
sulfonamide moiety with amino (1b) should lead to loss of rafenib, increased substitution at the 3-position was proposed
the inhibitory activity. The nitro substituted derivative 1c as rational (Figure 5D). From the organic synthetic perspec-
exhibited weak inhibitory activity, but the selectivity against tive, methyl, allyl and 4-nitrobenzyl were added to 1e, lead-
WT
B-Raf disappeared, which is consistent with the notion that ing to 1i, 1j and 1k, respectively. The activities of the three
the deprotonated sulfonamide favors an interaction with compounds were all improved compared with 1e. Moreover,
V600E
[34]
the B-Raf
mutation other than the wild type . Using 1i had the best performance with respect to both activity and
2
,6-difluorobenzenesulfonamido, the fragment of dabrafenib selectivity. The methyl substituted derivative of 1h, 1l, also
substituted at the 1’-position, 1d, also exhibited weak inhibi- had a higher inhibition rate, implying that methyl substitution
tory activity on A375 cells (Table 1). is applicable. Then, we synthesized two additional methyl-
Compared with vemurafenib, 1a was predicted by a dock- substituted derivatives, 1m (Figure 5C) and 1n. Compound
ing study to form one more hydrogen bond with C532 on the 1n exhibited increased activity against the A375 cell line com-
hinge region via the amino of 7-deazaguanine (Figure 5B). To pared with 1d, whereas 1m had the same IC50 value as 1a (0.80
explore how the hydrogen bonding contributes to the bind- μmol/L). At the molecular level, the IC50 values of 1a and 1m
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Wang GM et al
Figure 4. Synthesis of deazapurine derivatives. Reagents and conditions: (a) Bromoacetaldehyde dimethyl acetal, HCl, sodium acetate trihydrate,
8
(
0°C, over night; (b) Acetic anhydride, acetic acid, 130°C, 2 h; (c) 0.1 mol/L NaOH, dimethyl sulfate; (d) Acetic anhydride, acetic acid, 130°C, 2 h;
e) Ammonium hydroxide, methanol; (f) 65%–68% HNO , H SO ; (g) Conc sulfuric acid, methanol; (h) Acetic acid, ethanol, Fe powder, 110°C, 0.5 h; (i)
Triethylamine, 3-chloropropylsulfonyl chloride, 3.5 h; (j) NaOH (aq) THF, reflux, 2 h; (k1) SOCl , toluene, reflux, 3 h; (k2) Oxalyl chloride, cat. DMF, DCM;
l) AlCl , MeNO , 60°C, 80–90°C or 100–105°C, over night; (m) Acetic acid, ethanol, Fe powder, 110°C, 0.5 h; (n) NaOH (aq) 80°C, over night; (o)
Triethylamine, DMAP, Boc O, THF, RT, over night; (p1) KF, MeI, acetonitrile; (p2) Anhydrous DME and DMF, NaH, LiBr, 4-Nitrobenzyl bromide or allyl
bromide; (q) Conc HCl, RT, over night; (r) AlCl , MeNO or nitrobenzene, 60°C, 80–90°C or 100–105°C, over night; (s) AlCl , MeNO , 60°C, over night; (t)
Acetic acid, ethanol, Fe powder, 110°C, 0.5 h.
3
2
4
2
(
3
2
2
3
2
3
2
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Wang GM et al
7
V600E
Figure 5. The binding mode of B-Raf
binding to B-Raf
inhibitors (docked with Glide in SP mode, PDB entry 3OG7). (A) Crystal structure of vemurafenib (orange)
V600E
V600E
V600E
. (B) Predicted binding mode of 1a (green) to B-Raf
.
(C) Predicted binding mode of 1m (magenta) to B-Raf
. (D)
Superimposition view of all the three compounds. The protein is show in cartoon form with critical residues and the inhibitors in stick form.
V600E
V600E
on B-Raf
were 0.50 μmol/L and 0.05 μmol/L, respectively, interactions with B-Raf
than vemurafenib, which might be
whereas that of vemurafenib was 0.13 μmol/L. In addition, another reason why 1m has lower activity against wild type
V600E
the selectivity of 1m (9 times) for B-Raf
was increased com- B-Raf.
WT
pared with vemurafenib (5 times) against B-Raf (Table 1).
Accordingly, we obtained a potential B-Raf
comparable activity and better selectivity than vemurafenib.
The phosphorylation level of MEK1/2, which are the down-
inhibitor with stream kinases of B-Raf, was determined in HT-29 cells to
V600E
V600E
validate B-Raf
inhibition by 1m at the cellular level. After
incubation with serially diluted 1m or vemurafenib for 2 h, the
Assessment of 1m against multiple colorectal cancer cell lines
phosphorylation level of MEK1/2 was measured by Western
For the high proportion (4.7%–10%) of colorectal cancer blot. Phosphorylated MEK1/2 significantly decreased with
V600E
patients bearing the B-Raf
mutation, we used multiple increasing concentrations of 1m (Figure 6), confirming its
V600E
colorectal cancer cell lines to evaluate the inhibitory activity target of B-Raf
in HT29 cells. Similar observations were
and selectivity of 1m (Table 2). The IC50 values of 1m were at obtained with vemurafenib.
V600E
the micromolar level in B-Raf
mutant HT-29 and COLO205
cells, and these values were comparable to vemurafenib. In vitro ADME properties
However, the IC50 values of 1m were over 10 μmol/L in Finally, we determined the solubility, permeability and
LOVO, HCT-15, HCT-8, SW1116, HCT116, SW620 and SW480 metabolic stability of 1m. The human liver microsome assay
cells, which harbor wild type B-Raf. The IC50 values of vemu- indicated that the remaining rate (proportion of compounds
rafenib in LOVO, HCT-15 and HCT-8 cells were 8.88 μmol/L, that are not decomposed) of 1m decreased slightly from 30
7
1
.75 μmol/L and 5.84 μmol/L, respectively, indicating that min to 2 h, implying that 1m was stable in human liver micro-
m exhibits superior selectivity against the V600E mutated somes (Supplementary Table S2 and Figure 7). Moreover, the
cells compared with vemurafenib. A molecular docking study metabolic stability evaluation concerning cytochrome P450
demonstrated that 1m has a similar binding mode as vemu- indicated that the metabolic bioavailability of 1m was greater
rafenib. Furthermore, the sulfonamide moiety of vemurafenib than 90% and varied slightly with different species. Further-
is retained in 1m, suggesting that the moiety preferred bind- more, 1m also exhibited good solubility, whereas the minimal
[
34]
ing to the V600E mutant , accounting for the selectivity of soluble concentration of 1m in buffers A and B was 50 μmol/L
m. In addition, the 7-deazaguanine moiety of 1m forms more and that in buffer C was 20 μmol/L. Regarding permeability,
1
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Wang GM et al
Table 1. Structures, docking scores and biological activities of compounds.
No
R
1
R
2
R
3
Docking score
-11.89
Inhibition rate (%) at 10 μmol/L or IC50 (μmol/L)
V600E
WT
A375
HCT116
B-Raf
B-Raf
a
1a
NH
2
H
0.80
21%
0.50
1.50
1
1
b
c
NH
NH
2
2
H
H
NH
NO
2
-9.75
-10.29
0.98%
9.13
-11.0%
6.71
NT
NT
NT
NT
2
1
d
e
NH
2
H
H
-10.85
-9.46
19.08%
-7.89%
7.67%
NT
NT
NT
NT
1
Cl
-0.04%
1
f
Cl
Cl
H
H
NH
NO
2
-7.43
-7.47
-0.11%
4.69
-3.09%
4.68
NT
NT
NT
NT
1g
2
1
1
1
h
Cl
Cl
Cl
H
-8.81
-9.67
-9.18
-8.94%
37.04%
21.50%
-3.61%
5.32%
1.49%
NT
NT
NT
NT
NT
NT
i
Me
j
1k
Cl
-10.31
31.16%
21.34%
NT
NT
1
l
Cl
Me
Me
-8.90
16.93%
0.80
18.37%
4.38%
NT
NT
1m
NH
2
-11.89
0.05
0.44
1n
NH
2
Me
-11.48
48.42%
19.26%
NT
NT
Vemurafenib
Sorafenib
-12.31
-7.25
0.56
10.01
36.42
8.70
0.13
NT
0.60
NT
a
Tested at 100 μmol/L.
NT: Not test.
given that 1m might be a substrate of P-gp (efflux ratio was 35%) for the saturation of the efflux transporter (Table 3).
7.4), the in vivo absorption and bioavailability of 1m should Accordingly, 1m should have good ADME properties, includ-
be higher than the in vitro predictive value (mean Fabs was ing metabolism and bioavailability.
6
Acta Pharmacologica Sinica

www.chinaphar.com
Wang GM et al
9
V600E
WT
Table 2. The selectivity of 1m for B-Raf
colorectal cancer cell lines.
over B-Raf in multiple
Conclusion
In view of the structure and interaction analyses of approved
drugs and inhibitors in development, we utilized molecu-
lar tailoring, molecular docking and fragment reassembly
strategies to design and synthesize a series of deazapurine
IC50 (μmol/L)
B-Raf
Cell line
1m
Vemurafenib
derivatives, among which compound 1m was identified as
V600E
HT-29
COLO205
LOVO
HCT-15
HCT-8
SW1116
HCT116
SW620
SW480
0.55±0.08
1.64±0.08
>10
>10
>10
>10
>10
>10
>10
0.69±0.07
0.58±0.30
8.88±2.50
7.75±0.10
5.84±1.53
>10
V600E
a potential B-Raf
inhibitor with comparable activity and
superior selectivity compared with vemurafenib. Moreover,
m exhibits significant activity against the proliferation of
Wild type
1
V600E
HT-29 colorectal cancer cells harboring B-Raf
with an IC50
value of 0.55 μmol/L, which is lower than that of vemurafenib
WT
(
0.69 μmol/L), while sparing B-Raf cells (IC50 >10 μmol/L).
>10
>10
>10
An obvious decrease in the phosphorylation of MEK1/2 was
observed upon treatment with 1m, confirming that 1m inhib-
V600E
ited B-Raf
in cells. In addition, 1m also exhibits good solu-
bility, bioavailability and metabolic stability. These results of
m lay a solid foundation for further development. In addi-
tion, this study provides a novel strategy for designing inhibi-
1
V600E
tors targeting B-Raf
.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (81273435, 81302699 and 81321092),
the National Science and Technology Major Project
(2013ZX09103001001), the Ministry of Science and Technology
Figure 6. The phosphorylation level of MEK1/2 in HT-29 cells was (2012AA01A305), the Natural Science Foundation of Shanghai,
detected with Western blot.
China (14ZR1447800), the State Key Laboratory of Natural and
Biomimetic Drugs (K20150205) and the Special Program for
Applied Research on Super Computation of the NSFC-Guang-
dong Joint Fund (the second phase).
Author contribution
Wei-liang ZHU, Bo LI, He-yao WANG, Ling-hua MENG, and
Jian DING conceived and designed the research; Gui-min
WANG, Xiang WANG, Jian-ming ZHU, Bin-bin GUO, and
Zhuo YANG performed the research; Gui-min WANG, Xiang
WANG, Jian-ming ZHU, Bin-bin GUO, and Zhi-jian XU ana-
lyzed the data; Gui-min WANG, Wei-liang ZHU, Jian-ming
ZHU, Ling-hua MENG, and He-yao WANG wrote the paper.
Supplementary information
Supplementary information is available at the Acta Pharmaco-
logica Sinica’s website.
Figure 7. Metabolic stability of 1m in human liver microsomes.
Midazolam was a positive control and negative control was ultrapure-
water rather than NADPH.
Table 3. Solubility, permeability and metabolic stability of 1m.
Solubility
Permeability
Mean fabs (%)
Metabolic stability
Species
Buffer
Minimal soluble concentration (µmol/L)
Efflux ratio
MF (%)
A
B
C
50.0
50.0
20.0
35.0
67.4
Human
Mouse
Rat
100
91
90
Buffer A: Hanks balanced salt solution (HBSS) buffer containing N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) 10 mmol/L, pH 6.8 with
bovine serum albumin (BSA) up to 0.1%.
Buffer B: HBSS buffer containing HEPES 10 mmol/L, pH 7.4 with BSA up to 0.1%.
Buffer C: TRIS buffer with BSA up to 0.1%.
Acta Pharmacologica Sinica

www.nature.com/aps
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Wang GM et al
Discov Today 2009; 14: 291–7.
Li HF, Chen Y, Rao SS, Chen XM, Liu HC, Qin JH, et al. Recent advanc- 18 Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule ki-
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