Discovery of EBI-907: A highly potent and orally active B-RafV600Einhibitor for the treatment of melanoma and associated cancers

Article abstract of DOI:10.1016/j.bmcl.2015.12.086

A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-Raf^V600Einhibitors through scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the discovery of potent B-Raf^V600

Full text of DOI:10.1016/j.bmcl.2015.12.086

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of EBI-907: A highly potent and orally active B-RafV600E
inhibitor for the treatment of melanoma and associated cancers
a
b
a
a
b
b
a
Biao Lu a, , Hu Cao , Jingsong Cao , Song Huang , Qiyue Hu , Dong Liu , Ru Shen , Xiaodong Shen ,
Weikang Tao a, Hong Wan a, Dan Wang a, Yinfa Yan b, Liuqing Yang b, Jiayin Zhang b, Lei Zhang a,
Lianshan Zhang c, Minsheng Zhang b
⇑
a Shanghai Hengrui Pharmaceutical Co., Ltd, 279 Wenjing Rd, Minhang Hi-tech Zone, Shanghai 200245, China
b Eternity Bioscience Inc., 2005 EastPark Blvd, Cranbury, NJ 08512, USA
c National Engineering and Research Center for Targeted Drugs, Jiangsu Hengrui Medicine Co., Ltd, 7 Kunlun Shan Road, Lianyungang Economic and Technology Development
Zone, Jiangsu Province, China
a r t i c l e i n f o
a b s t r a c t
A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-RafV600E inhibitors through
scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the
discovery of potent B-RafV600E inhibitors with good oral bioavailability in rats and dogs. One of the com-
pounds EBI-907 (13g) demonstrated excellent in vivo efficacy in B-RafV600E dependent Colo-205 tumor
xenograft models in mouse and is under preclinical studies for the treatment of melanoma and B-
RafV600E associated cancers.
Article history:
Received 19 October 2015
Revised 21 December 2015
Accepted 24 December 2015
Available online xxxx
Keywords:
Ó 2015 Published by Elsevier Ltd.
B-RafV600E
Kinase
Pyrazolo[3,4-c]isoquinoline
Scaffold hopping
Cancer
The Ras/Raf/MEK/ERK pathway plays a key role in regulating
cell proliferation, differentiation, and growth. The Raf family of ser-
ine/threonine kinases comprises A-Raf, B-Raf, and C-Raf. There is a
well established biological mechanism that Raf proteins can signal
through phosphorylation and activation of a downstream kinase,
mitogen-activated protein MAPK/ERK kinase (MEK), which subse-
quently phosphorylates and activates extracellular signal-regu-
lated kinase (ERK)1 that regulates cell proliferation and survival.
Mutation in B-Raf drives constitutive activation of the Raf–MEK–
ERK pathway which often leads to alteration of gene expression
and results in many cancers.2 The most common B-Raf mutation
is a glutamate instead of a valine substitution at position 600
(V600E). So far the oncogenic mutation of B-RafV600E has been
found in 7% of all human cancers, including more than 60% of mel-
anoma, 45% of papillary thyroid cancer, 10% of colorectal cancers,
and a small subset of ovarian, breast, lung cancers, and leukemia.2
Based on its association with human malignancies, B-Raf has been
a target of high interest to treat cancer in recent decades.3 Herein,
we report our discovery of a highly potent and orally efficacious B-
RafV600E inhibitor EBI-907 (13g) with a novel biaryl scaffold fea-
tured pyrazolo[3,4-c]isoquinoline.
There are many Braf-specific inhibitors with different scaffolds
reported in the literature. Vemurafenib (PLX4032)4 with an aza-
indole core was developed by Plexxikon and Roche and became
the first marketed drug for treatments of patients with unresectable
and metastatic melanoma. Dabrafenib (GSK2118436)5 with a
pyrimidine substituted thiazole core was later approved as well
by FDA. It is noteworthy that the analogue PLX4720 showed similar
activity in vitro and in vivo as Vemurafenib. The latter was selected
over PLX4720 because it has more scalable pharmacokinetic prop-
erties.6 Besides, there are other chemotypes of B-RafV600E inhibitors
at different clinical stages including BGB283 (BeiGene),7 LGX818
(Novartis),8 LY3009120 (Lilly),9 MLN248010 (Takeda) and others in
preclinical development11 (Fig. 1).
On the basis of the co-crystal structures of Vemurafenib and its
analog PLX4720 published (pdb codes 3C4C, Fig. 2),12 we were able
to identify the key binding interactions of PLX4720 to an ‘active’
conformation unique to the RAF proteins, in which the DFG motif
is in the ‘DFG-in’ conformation but the aC helix is moved outward
from the active site by the lipophilic alkyl group. The sulfonamide
moiety is engaged in very important interactions with key residues
such as Asp-594 and Phe-595, Lys-483 via hydrogen bonds.12 The
N and NH of the aza-indole core are making interactions with
the hinge residues Thr-529, Gln-530, and Trp-531. By breaking
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.12.086
0960-894X/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Lu, B.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.086

2
B. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Scheme 1. Synthesis of compound 1. Reagents and conditions: (a) BH3ꢀTHF, THF,
reflux; (b) methyl 2,2-diethoxyacetimidate, 70 °C; (c) concd H2SO4, 40 °C; (d) bis
(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane 120 °C; (e) 7, Pd(dppf)Cl2, Na2CO3,
wet DME, 120 °C, microwave.
from a crystallographic water molecule (HOH44), as shown in
Figure 2 and increase potency based on Plexxikon’s pivotal work.12
We thought that hydrogen bonding acceptor capability of the
carbonyl group may be mimicked by an oxygen-bearing group
such as a methoxyl at the 6-position on the isoquinoline ring. On
the other hand, the amine group of compound 1 may be substi-
tuted by employing LGX818’s side chain to enhance potency as
well.15 Consequently, we started from different 4-substituted
3-bromo-benzonitrile to synthesize corresponding analogs 8a
and 8b by similar route. Meanwhile, compound 1 and compounds
8a/8b were reacted with methyl N-(1-oxopropan-2-yl) carbamate
via reductive amination to obtain N-substituted analogs of com-
pounds 9–12 (Scheme 2).
A summary of SAR for the isoquinoline series is shown in
Table 1. Compound 10 bearing LGX-818’s side chain was 10 times
more potent than compound 1 in biochemical assay although its
cellular activity was not potent enough. However, compound 9
with the wrong configuration tail showed no activity at all. Fortu-
nately, 6-methoxyl substituted isoquinoline analog 8a exhibited 10
Figure 1. Structures of representative B-RafV600E inhibitors.
the indole 5-membered ring and building a new fused ring from
the carbonyl position, we envisioned novel scaffold of
a
3-aminoisoquinoline which will make the same hinge interaction
with Trp-531 and pick up new interactions with Cys-532 (Fig. 2)
from which
a
new series of B-RafV600E inhibitors could be
developed.13
To test this idea, we rapidly synthesized compound 1 as shown
in Scheme 1. 3-Bromo-benzonitrile 2 was reduced by using
BH3ꢀTHF to its benzylic amine 3, which was condensed with ace-
timidate to afford acetimidamide intermediate 4. The subsequent
ring closure reaction was carried out in concd sulfuric acid to give
the isoquinoline core 5,14 which was converted to the boronic ester
6 via Miyaura reaction. The boronic ester was coupled with aryl
bromide piece via Suzuki reaction to give the target product 1.
Compound 1 showed 8.7 lM potency in biochemical assays
times more potent enzymatic activity and 1.2 lM cellular potency.
performed using LanthaScreenTM. It was 39 times weaker than
PLX4720 (220 nM). The micromolar enzymatic activity revealed
that additional interactions would be necessary to achieve enough
potency. The carbonyl oxygen of PLX4720 could accept an H-bond
Next, we examined compound 11 with both 6-substituted meth-
oxyl and LGX-818’s side chain. Its enzymatic activity and cellular
activity were dramatically improved to 100 nM and 20 nM, respec-
tively. To understand the role of methoxyl group, we synthesized
ethyl substituted analog 12, which was 6 times less potent than
Scheme 2. Synthesis of analogs of compound 1. Reagents and conditions:
(a) BH3ꢀTHF, THF, reflux; (b) methyl 2,2-diethoxyacetimidate, 70 °C, (c) concd
H2SO4, 40 °C; (d) bis-(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 120 °C; (e) 7,
Pd(dppf)Cl2, K2CO3, wet DMF, 120 °C; (f) R2-aldehyde, NaBH3CN, HOAc, MeOH.
Figure 2. Our initial design. Crystal structure conformation of PLX4720 (yellow)
and modeled binding conformation of
1 (cyan) within the crystal structure
conformation of B-Raf protein from pdb:3C4C.
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B. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
Selected SAR of isoquinoline series
O
S
HN
O
F
R1
F
R2
N
N
H
Compd R1
R2
H
BrafV600E Lanthaa
A375 cell growtha
IC50
(l
M)
IC50
(lM)
1
9
H
H
8.7
n.db
O
O
>30
n.d
MeO
N
H
Crystal structure conformation of Wenglowsky’s compound 17
from pdb:3TV6 (Orange) and modeled binding conformation of
11 (Green) within the crystal structure conformation of B-Raf
protein from pdb:3OG7.
10
8a
H
0.8
0.8
2.7
1.2
MeO
N
H
MeO
H
O
O
11
12
MeO
Et
0.1
0.6
0.02
0.3
MeO
MeO
N
H
N
H
a
Data represent mean value of at least two experiments.
n.d. = not determined.
b
compound 11. These results further supported our hypothesis that
the methoxyl group at the 6-position of isoquinoline ring may
indeed increase the binding affinity via polar or H-bond
interactions.
Figure 3. Our modified design.
Although compound 11 was potent enough for in vivo study, we
were concerned about its carbamate fragment for potential meta-
bolic instability. At that time Wenglowsky et al. published a set
of B-Raf inhibitors.11c One of them, compound 17, was co-
crystalized with B-Raf (pdb:3TV6). Based on the 3D alignment of
their compound and the modeled pose of our compound 11 in
the crystal structure (Fig. 3), the pyrazole ring is in a good
position to fuse with our isoquinoline core. It seems that it will
not only lock in the NH hydrogen bonding interaction with Cys-
532, but also form a
p–p stacking interaction with Trp-531. We
ventured to replace the carbamate side chain with pyrazolo[3,4-
c]isoquinoline core by fusing another 5 membered ring onto the
isoquinoline analog 11 (Fig. 3).
The synthetic approach for the preparation of pyrazolo[3,4-c]
isoquinoline analogs is outlined in Scheme 3. 4-Bromo-3-methoxyl
benzoic acid 14 was reduced to its benzylic alcohol, followed by
bromination and cyanation to afford benzylic nitrile intermediate
17. After treatment with sodium hydride (or LiHMDS), it was con-
densed with methyl formic ester (or acyl chloride) smoothly to
give functionalized nitrile 18a–b which was condensed with ben-
zyl hydrazine to form benzylic pyrozole intermediate 19a–b.16
The following assembly of its isoquinoline ring underwent through
Pictet–Spengler type reaction.17 The obtained bromo piece was
converted to its boronic ester which was further coupled with
another bromo fragment 22 to give biaryl intermediate 23a–h
via Suzuki reaction. Subsequent debenzylation reaction turned
out to be difficult. Initial attempts using hydrogenation (Pd/C, H2)
or KOtBu/DMSO with O2 was not successful.17 Finally, upon heating
23a–h in formic acid via palladium mediated transfer hydrogena-
tion gave analogs 13a–h in modest yields.18
Scheme 3. Synthesis of analogs 13. Reactions and conditions: (a) BH3, THF; (b) BBr3,
Et2O; (c) KCN/18-crown-6, DMEO; (d) melhyl formate or cydopropanecarbonyl
chloride, NaH or LiHMDSJHF; (e) benzyl hydrazine, HOAc, IPrOH, 70 °C; (f) (HCHO)n,
TFA, reilux; (g) bis (pinacolato)diboron, Pd(dppf)2Cl2, KOAc, dioxane, 110 °C; (h) 22,
Pd(dppf)2Cl2, K3PO4, dioxane, 110 °C; (i) 2W 20% Pd(OH)2, ammonium formic, fomic
acid, 100 °C.
As expected, pyrazolo[3,4-c]isoquinoline analogs also showed
potent inhibition in both LanthaScreenTM and A375 cell prolifera-
tion assays. Their SAR is summarized in Table 2. Compound 13a
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4
B. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
Selected SAR of pyrazolo[3,4-c]isoquinoline series
R3
O
S
HN
O
R2
O
R1
N
R2'
N
N
H
0
Compd R1
R2/R2
R3
BrafV600E Lanthaa
IC50 (nM)
A375 cell growtha
IC50 (nM)
13a
13b
13c
13d
H
H
H
H
F/F
H
H
H
F
70
11
20
7
138
47
340
220
Cl/F
Cl/Cl
Cl/F
13e
13f
F/F
H
220
80
7
135
Figure 5. Colo-205 xenograft mice model.
Cl/F
Cl/F
Cl/F
H
313
13
13g
13h
F
dichloro substituted analog 13c was less potent than 13b. By put-
ting one fluorine atom into the side chain of 13b, enzymatic activ-
ity of compound 13d reached single digit nanomolar (7 nM), but its
cellular activity was lowered. To fix this issue and eliminate poten-
tial metabolic soft spot, we assembled a cyclopropyl onto 3-posi-
tion of the pyrazole.19 Compounds 13e, 13f and 13h were less
potent as well, but compound 13g demonstrated excellent A375
cellular activity with IC50 13 nM. In the other cell line Colo-205,
it also showed good potency with IC50 of 14 nM. In contrast, 13g
did not show meaningful anti-proliferative effects on the growth
of Calu-6 cells carrying a wild-type B-Raf, exhibiting at least 40-
fold (455 nM) selectivity towards B-RafV600E-dependent cells lines.
The modeled binding pose of 13g is shown in Figure 4. In addition
to the same key hinge interactions and backpocket sulfonamide
interactions as 1, 13g also forms the same key hydrogen bond
interaction with HOH44 as PLX4720. The terminal Fluoro group
is placed in close Van der Waals contacts with Phe-516, Leu-505
and tightly bound crystal water HOH14 as well.
CH3
17
435
a
Data represent mean value of at least two experiments.
Furthermore, compound 13g exhibited excellent liver microso-
mal stability in different species (T1/2 Human/Rat/Mouse:
350/148/81 min). Based on the above data, 13g became our candi-
date for in vivo study.20 It displayed desired in vivo pharmacoki-
netics as shown in Table 3. After a single 5 mg/kg oral dose of a
solution of 13g to rats, its AUC0–24 reached 14186 ng/ml * h. 13g
also showed AUC0–24 of 8906 ng/ml * h in dogs with a single
2 mg/kg oral dose. Its oral bioavailability for rats and dogs was
20% and 40%, respectively.
Figure 4. The binding pose of 13g to B-Raf protein.
The efficacy of 13g in vivo was evaluated in a B-RafV600E-depen-
dent human Colo-205 tumor xenograft mouse model as shown in
Figure 5. Compound 13g was administered orally at 20 mg/kg
and 60 mg/kg daily in a 14 days (QD) with the reference compound
PLX4720 60 mg/kg as a positive control. PLX4720 showed 85%
tumor growth inhibition. Compound 13g caused a partial (76%,
gave comparable enzyme activity to aminoisoquinoline analog 11.
When changing one of fluorine to chlorine on the right benzene
ring of 13a, enzymatic activity of 13b was dramatically improved
by 5 times although cellular activity was dropped a little. However,
Table 3
The PK parameters of 13g (EBI-907) in rats and dogs
Species/route/dose
Cmax (ng/ml)
AUC0.24 (ng/ml * h)
T1,2 (h)
Cl (ml/min/kg)
1.2
Vss (L/kg)
F (%)
Rat/iva/0.5 mg/kg
Rat/pob/5 mg/kg
Dog/iv/0.2 mg/kg
Dog/po/2 mg/kg
7224
14,186
2201
8906
3.4
0.34
1498
972
20
40
7.8
1.4
0.91
a
iv: intravenous injection.
po: oral administration.
b
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B. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
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Melanoma Res. 2015, 25, 91.
20 mg/kg) or complete tumor regression (95%, 60 mg/kg) in a dose-
dependent manner, with superior efficacy over PLX4720
(P = 0.012). It was noteworthy that no significant changes in body
weights of all treatment groups were observed.
9. Henry, J. R.; Kaufman, M. D.; Peng, S.; Ahn, Y. M.; Caldwell, T. M.; Vogeti, L.;
Telikepalli, H.; Lu, W.; Hood, M. M.; Rutkoski, T. J.; Smith, B. D.; Vogeti, S.;
Miller, D.; Wise, S. C.; Chun, L.; Zhang, X.; Zhang, Y.; Kays, L.; Hipskind, P. A.;
Wrobleski, A. D.; Lobb, K. L.; Clay, J. M.; Cohen, J. D.; Walgren, J. L.; McCann, D.;
Patel, P.; Clawson, D. K.; Guo, S.; Manglicmot, D.; Groshong, C.; Logan, C.;
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Khabele, D.; Shamji, A. F.; Schreiber, S. L. Cancer Res. 2015, 75, 2897.
11. (a) Pulici, M.; Traquandi, G.; Marchionni, C.; Modugno, M.; Lupi, R.; Amboldi,
N.; Casale, E.; Colombo, N.; Corti, L.; Fasolini, M.; Gasparri, F.; Pastori, W.;
Scolaro, A.; Donati, D.; Felder, E.; Galvani, A.; Isacchi, A.; Pesenti, E.; Ciomei, M.
ChemMedChem 2015, 10, 276; (b) Cheng, H.; Chang, Y.; Zhang, L.; Luo, J.; Tu, Z.;
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Wenglowsky, S.; Ren, L.; Ahrendt, K. A.; Laird, E. R.; Aliagas, I.; Alicke, B.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Feng, B.; Gloor, S. L.; Gould, S. E.; Gross,
S.; Gunzner-Toste, J.; Hansen, J. D.; Hatzivassiliou, G.; Liu, B.; Malesky, K.;
Mathieu, S.; Newhouse, B.; Raddatz, N. J.; Ran, Y.; Rana, S.; Randolph, N.; Ri-
som, T.; Rudolph, J.; Savage, S.; Selby, L. T.; Shrag, M.; Song, K.; Sturgis, H. L.;
Voegtli, W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.; Young, W. B.;
Grina, J. ACS Med. Chem. Lett. 2011, 2, 342.
In summary, we developed a novel class of B-RafV600E inhibitor
with biaryl structure featured pyrazolo[3,4-c]isoquinoline core via
two consecutive scaffold hopping from the literature lead PLX4720.
One of the optimized analogs EBI-907 (13g) demonstrated both
excellent potency in vitro and superior efficacy in vivo. Further
development studies of this molecule are in progress and more
detailed results will be disclosed in due course.
Acknowledgements
We thank members of the analytical group of Shanghai Hengrui
Pharmaceutical Ltd for their analytical and spectral determina-
tions, and the Informatics group as well as Drs. Hejun Lv and Jun
Feng for their useful discussions and enormous support.
12. Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.;
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Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu,
C.; Settachat-gul, C.; Shellooe, R.; Cantwell, J.; Kim, S. H.; Schlessinger, J.; Zhang,
K. Y.; West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth, P.; Artis,
D. R.; Herlyn, M.; Bollag, G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3041.
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(400 MHz, DMSO-d6) d: 9.73 (s, 1H), 9.03 (s, 1H), 8.14 (s, 1H), 8.03 (s, 1H), 7.58
(dd, J = 8.8, 6.0 Hz, 1H), 7.40 (dd, J = 8.8, 8.4 Hz, 1H), 4.57 (dt, J = 47.2, 6.0 Hz,
2H), 3.99 (s, 1H), 3.26–3.30 (m, 2H), 2.59 (m, 1H), 2.11–2.23 (m, 2H), 1.10–1.15
(m, 2H), 0.96–1.0 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d 159.2, 158.0 (d,
J = 244.3 Hz), 152.3, 150.3, 146.0, 133.1, 132.6, 131.4 (d, J = 4.4 Hz), 130.7 (d,
J = 3.6 Hz), 129.1 (d, J = 8.8 Hz), 125.5 (d, J = 21 Hz), 120.4, 119.2, 114.6 (d,
J = 23.8 Hz), 106.3, 101.9, 81.9 (d, J = 161.9 Hz), 56.0, 49.2 (d, J = 5.1 Hz), 24.7 (d,
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Please cite this article in press as: Lu, B.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.086