Optimization of Tetrahydronaphthalene Inhibitors of Raf with Selectivity over hERG

Article abstract of DOI:10.1016/j.bmcl.2016.01.049

Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.

Full text of DOI:10.1016/j.bmcl.2016.01.049

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1156–1160
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of tetrahydronaphthalene inhibitors of Raf
with selectivity over hERG
⇑
Shih-Chung Huang , Sharmila Adhikari, Roushan Afroze, Katherine Brewer, Emily F. Calderwood,
Jouhara Chouitar, Dylan B. England, Craig Fisher, Katherine M. Galvin, Jeffery Gaulin, Paul D. Greenspan,
Sean J. Harrison, Mi-Sook Kim, Steven P. Langston, Li-Ting Ma, Saurabh Menon, Hirotake Mizutani,
Mansoureh Rezaei, Michael D. Smith, Dong Mei Zhang, Alexandra E. Gould
Takeda Pharmaceuticals International Co. 40 Landsdowne Street, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo
activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe
our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide function-
ality. The resulting compounds have improved selectivity against the hERG channel, good pharmacoki-
netic properties and potently inhibit the Raf pathway in vivo.
Received 20 November 2015
Revised 15 January 2016
Accepted 18 January 2016
Available online 21 January 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
B-Raf inhibitor
hERG
Raf, a serine/threonine protein kinase, is an integral part of
the MAP kinase signaling pathway and is a component of the
Raf-MEK-ERK signaling cascade.1 Understanding the complex and
intricate role of Raf pathway signaling in cancer is the continuing
aim of intensive research.2 One component of this pathway, the
Raf isoform B-Raf, has a high rate of activating mutation in mela-
noma (50–70%) and other cancers, including papillary thyroid
(49%), colorectal (ꢀ15%), and ovarian (ꢀ30%).3,4 The V600E activat-
ing mutation is most common and significantly increases basal
level activity of the enzyme.5 Suppression of B-Raf (V600E) activity
in human melanoma cells leads to a down-regulation of the MAP
kinase signaling pathway and apoptosis,2,6 and recently, selective
B-Raf inhibitors have shown significant antitumor activity in
patients with B-Raf-mutant melanoma.7 Thus, inhibition of
mutated B-Raf could prove to be a useful therapeutic strategy
against melanoma as well as a number of other cancers.
to increase the risk of cardiac arrhythmia, including torsades de
points (TdP), possibly resulting in sudden death, further optimiza-
tion of this series was focused on reducing the hERG channel
activity while retaining the desirable Raf activity.
There have been several ligand-based quantitative structure–
activity relationships (QSAR) and pharmacophore models built
using structurally diverse compounds known to bind and block
the hERG channels.14 These models clearly suggest that lipophilic
amines are often hERG binders. As we focused on reducing the
hERG activity of our molecules, we concentrated our efforts on
modification of the benzylic amine moiety present in 1. Compound
1 has excellent pharmacokinetic (PK) properties and inhibits the
growth of B-Raf mutant tumor xenografts in mice.8 While we were
interested in modifying the hERG activity of this compound, we
We recently reported the discovery and evolution of a novel
series of tetrahydronaphthalenes as potent inhibitors for Raf, typi-
fied by compound 1 (Fig. 1).8 However, this compound inhibits
N
hERG ion channel activity (IC50 = 6.3 l
M)9 at concentrations near
H
O
N
CF3
those required for effects on the Raf pathway. The blockade of
hERG channels has been cited as a major factor in the drug-induced
alteration of cardiac ventricular repolarization that results in
prolongation of QT interval.10 Since QT prolongation is believed
N
O
HN
O
1
⇑
Corresponding author.
Figure 1. Structure of lead compound 1.
http://dx.doi.org/10.1016/j.bmcl.2016.01.049
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1156–1160
1157
Table 1
9
900
800
Biological activity of tetrahydronaphthalene analogs
R
700
600
500
400
300
200
100
0
2
H
O
N
CF3
N
O
5
10
8
HN
14
12
15
O
7
13
6
4
1
3
11
16
b,c
b,d
b,e
Ra
V600E B-Raf IC50
(nM)
pERK IC50
hERG IC50
(lM)
0%
20%
40%
60%
80%
100%
(nM)
hERG % Inhibiꢀon
N
Figure 2. hERG percent inhibition at 11.1 l
M and Raf activity.9
1
2
3
2.7 0.4
3.1
48 26
487
6.3 1.1
>11.1
H
for cellular potency and the unsubstituted compound 2 (Table 1) is
significantly less active in our cell-based pathway assay. To
measure the cellular activity of our compounds, we monitored
the inhibition of the downstream marker, pERK, rather than the
direct marker of Raf activity, pMEK. We found that inhibition of
pERK and pMEK correlated well for a small handful of compounds
(data not shown) and that the pERK assay was more robust for
screening large numbers of compounds. We also noted that while
our compounds in general potently inhibited the enzymatic Raf
activity, inhibition of the cellular activity was more variable.
Although we have not rigorously confirmed this, our observations
indicate poor compound solubility and permeability15 as possible
explanations for the lack of cellular activity.
H
N
2.2 0.5
45 12
10 7.1
H
N
4
1.9 0.2
56 25
5.6 3.0
H2N
H2N
5
6
7
8
2.9
274
6.6 3.1
7.4 2.0
3.1 1.2
13.8 3.6
2.1 0.5
1.8 0.2
1.7
56 30
67 19
207 59
H2N
H2N
OH
Efforts to alter hERG activity in bioactive molecules have been
well documented and we took a cue from the literature in our
initial efforts. Attempts to modify the pKa of our compounds by
modifying the amine’s alkyl substituents had no effect on either
hERG activity or B-Raf enzyme potency (compounds 3–5).
Attempts to alter the electron density of the benzyl ring itself, by
adding electron-withdrawing substituents to the phenyl ring did
not alter hERG activity but did decrease Raf enzyme potency (data
not shown). Increasing the steric bulk around the basic nitrogen
had no effect on hERG activity (compounds 6 and 7). The addition
of additional polar functionality near the basic site has been shown
in some cases to reduce hERG activity,16 however in our case the
introduction of an alcohol moiety offered little benefit with respect
to hERG and led to a decrease in cellular potency (compounds 8
and 9), most likely due to decreased cellular permeability.
NH
HO
O
9
2.2
4.0
854
254
4.9 1.1
>100
OH
10
N
N
HN
11
12
13
2.0 0.5
9.5
38 12
121 39
55 64
>33
HN
N
>33
HN
N
2.9 0.6
>11.1
These relatively extensive explorations of the benzyl amine
moiety led us to conclude that the structure–activity relationships
for hERG activity and the Raf activity were closely linked as we
were only able to reduce hERG activity in this series when we
added functionality that also reduced Raf inhibition in cells.
We were fortunate however in that many types of substitution
are tolerated at this position on the phenyl ring and therefore we
expanded our search for benzyl amine replacements to include
N
14
15
16
2.1 0.2
1.5
123 53
74 38
34 17
>100
>11.1
>100
N
N
N
N
N
2.1 0.8
a
All compounds were isolated as either the HCl or bis-HCl salt except compound
10, which was isolated as free base.11
Table 2
Pharmacokinetic properties of compounds 1 and 16a
b
Standard deviations are reported for N P 3. Otherwise IC50 values are mean
values of two determinations.
Compound 1
Compound 16
c
Raf enzyme activity was determined using a Flash Plate Assay.12
d
Clp (L/h/kg)
Vss (L/kg)
0.23 0.01
0.75 0.10
98.6 37.5
100
0.08 0.00
0.41 0.05
125.7 3.5
54
Cellular Raf pathway activity was determined with a whole cell ELISA assay
utilizing a human melanoma cell line (A375) possessing the mutation BRaf V600E.13
hERG activity was determined with the PredictorTM hERG assay (Invitrogen).9
e
po AUC (lM h)
po% F
a
Pharmacokinetic experiments were performed by dosing either intravenously
were also keen to maintain its excellent physicochemical and PK
properties and its in vivo activity. Earlier work established that
substitution at the 5 position of the benzoic amide was important
(1 mg/kg) or orally (10 mg/kg) in male SD rats.17 Plasma clearance (Clp); Steady-
state volume of distribution (Vss); Area under the curve for oral administration
(po AUC); Oral bioavailability (po% F).

1158
S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1156–1160
140
120
100
80
decreased significantly. We next explored the effect of alkylation
on these heterocycles and found the optimal combination of cellu-
lar Raf inhibition with minimal hERG activity in compound 16. The
limited solubility of these compounds affected our ability to deter-
mine an exact IC50 in the hERG assay however we were able to
compare the hERG percent inhibition at the lower concentration
as shown in Figure 2.
Our attempts to modify the hERG potency of our Raf inhibitors
led us a few conclusions. First, the basic amine substituent of the
benzamide moiety in compound 1 is responsible for the hERG
activity of this compound and attempts to modify this substituent
while maintaining its basic and solubilizing nature did not lead to
improved hERG selectivity. The SAR for the Raf and hERG activities
track closely for the basic amine substituted analogs. Substitution
of benzyl amine with a heterocycle led to more selective com-
pounds and further substitution of these heterocycles provided
an optimal combination of Raf activity with hERG selectivity indi-
cating that both reduction of basicity and addition of steric bulk
were required to achieve the desired selective compound.
Compound 16 has good cellular potency without any hERG
60
40
20
0
Veh
30m
1h
2h
4h
8h
24h
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
activity at 11.1 lM. We were pleased also to find that despite its
poor solubility, it maintains good PK properties in rodents as
shown in Table 2.
Compound 16 was found to have pharmacodynamic (PD) and
antitumor activity in vivo. As shown in Figure 3, strong inhibition
of the Raf pathway is observed following a 33 mg/kg oral dose. Fur-
thermore, twice daily dosing for 21 days at 33 mg/kg induces par-
tial regressions of an A375M tumor xenograft with significant
tumor growth reduction at lower daily doses. These doses are well
tolerated by the mice, and no indication of toxicity or weight loss
as compared to vehicle treated animals was observed (Fig. 4).
Our synthetic exploration of this series required the preparation
of a number of benzoic acid derivatives which were then coupled
to amino tetralin 178 as illustrated in Scheme 1.11
The syntheses of the required benzoic acid derivatives are sum-
marized in Schemes 2 and 3. Scheme 2 details the preparation of
benzoic acids derived from aldehyde 20, which can be prepared
in two steps from benzoic acid 18. One-step reductive amination
followed by a deprotection/protection sequence allowed access
to a number of benzylamines including ethylamine 22, and the
substituted morpholino-benzoate used to prepare compound 10.
The primary benzylamine 24 was accessible through RaneyÒ-Ni-
mediated reduction of the aldoxime derived from 20. Aryl
0.0
30m
1h
2h
4h
8h
24h
Figure 3. PK/PD study of compound 16 following a 33 mg/kg oral dose.18
heterocycles. We initially hoped that we would be able to identify
a heterocycle that would also maintain the good physicochemical
properties of this series. Saturated heterocycles, such as pyrro-
lidine and piperidine at the 5 position provided some of our most
potent Raf inhibitors, but also were very potent against hERG (data
not shown), while the substituted morpholine 10 showed better
selectivity versus hERG with poor cellular activity. We were
pleased to find that many different 5- and 6-member aromatic
heterocycles are well tolerated at this position. Imidazole and
pyrazole in particular showed promise with respect to Raf potency
(compounds 11, 12, and 13); hERG activity for these compounds
Figure 4. Inhibition of subcutaneous A375M xenografts in immunocompromised mice by 16.19

S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1156–1160
1159
R
H
O
NH2
O
N
CF3
a
N
O
N
HN
HN
1-16
O
O
17
Scheme 1. Synthesis of amide-based Raf inhibitors. Reagents and conditions: (a) benzoic acid, EDCI, pyridine, rt then HCl, DCM, rt.
Boc
H
N
N
c
d, e
HO2C
HO2C
CF3
t-BuO2C
t-BuO2C
CF3
21
23
22
24
NHBoc
CF3
O
H
NH2
Br
Br
f, g
b
d, e
a
t-BuO2C
CF3
CF3
HO2C
CF3
t-BuO2C
CF3
18
19
20
NH
NH
N
N
d
h
HO2C
CF3
t-BuO2C
CF3
26
25
Scheme 2. Synthesis of benzoic acids. Reagents and conditions: (a) oxalyl chloride, DMF/DCM, rt, 18 h then KOtBu, THF, 0 °C, 1 h, 91%. (b) iPrMgClÁLiCl, THF, À20 °C, 1 h then
DMF, À20 °C, 30 min, 69%. (c) Ethylamine, NaB(OAc)3H, DCM, rt, overnight, 90%. (d) TFA, DCM, rt, 2 h then HCl, DCM, 90–100%. (e) (BOC)2O, NaOH, dioxane/H2O, rt, 2 h, 70–95%.
(f) NH4OHÁHCl, pyridine, EtOH, rt, 5 h, quant. (g) RaneyÒ-Nickel, NH3, MeOH, H2, rt, 18 h, 66%. (h) TosMIC, NaCN, EtOH, rt, 30 min then NH3, MeOH, 110 °C, 20 min, 17%.
HO
BocHN
H2N
OTBDMS
d, e
OH
OTBDMS
CF3
f, g
b, c
a
t-BuO2C
CF3
t-BuO2C
CF3
HO2C
CF3
t-BuO2C
27
NH
N
28
29
30
NH
N
Br
h
f
t-BuO2C
CF3
t-BuO2C
CF3
HO2C
CF3
19
32
31
33
HO
X
X
HO
i
f
t-BuO2C
CF3
HO2C
CF3
34
Scheme 3. Synthesis of benzoic acids. Reagents and conditions: (a) potassiumvinyltrifluoroborate, PdCl2, PPh3, Cs2CO3, THF/H2O, 85 °C, overnight, 65%. (b) AD-mix-alpha,
tBuOH/H2O, rt, overnight, 78%. (c) TBDMS-Cl, imidazole, DMF, rt, 4 h, 86%. (d) PPh3, DPPA, DIAD, THF, 0 °C, 2 h, 83%. (e) PPh3, THF, H2O, 60 °C, 3 h, 72%. (f) TFA, DCM, rt then
HCl, DCM, 95–100%. (g) (BOC)2O, NaOH, dioxane/H2O, rt, 3 h, 46%. (h) 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(Ph3P)4, Na2CO3, dioxane, 100 °C,
10 min, 69%. (i) X = NCHPh2 iPrMgClÁLiCl, THF, À20 °C, 90 min then 1-benzhydrylazetidin-3-one, À20 °C to rt, 30 min, 31%.
imidazoles can be accessed through aldehyde 20 by a Van Leusen
reaction.20
Scheme 3 illustrates the elaboration of bromide 19 into addi-
tional benzoates. A sequence involving a modified Suzuki reac-
tion,21 Sharpless dihydroxylation and Mitsunobu reaction
allowed access to amino-alcohol 30. Suzuki coupling provided
pyrazole 31 and similar conditions provided access to additional
heteroaryl benzoates required for the synthesis of compounds 13,
15 and 16 (see Table 1). Grignard reaction of 19 and benzhydry-
lazetidin-3-one led to azetidine 33.
In summary, our focus in this study was the improvement of
hERG selectivity in the series characterized by lead compound 1.

1160
S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1156–1160
12. Enzyme assay details: 15 lL of a solution containing 50 mM 4-(2-
Modification of the benzyl amine-containing portion of the mole-
cule provided analogs with reduced hERG channel activity, leading
to optimized derivative 16. Furthermore, compound 16 was found
to exhibit good pharmacokinetic properties to display strong inhi-
bition of the Raf pathway in vivo and to potently inhibit tumor
growth in mice.
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 7.5, 0.025% Brij
35, 10 mM 1,4-dithiothreitol (DTT), and 10 nM BRafV600E were added to the
wells of an assay plate containing compound and the mixture was incubated
for 20 min. A substrate solution (15
0.025% Brij 35, 5 mM b-glycerol phosphate, 10 mM MnCl2,
(Biotin-DRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRGRARATSWYSPY-NH2,
New England Peptide), 1 M ATP, 0.1 mg/mL BSA, and 33P ATP 0.5 Ci/reaction
was then added. The reaction mixture was incubated for 3 h and then stopped
by the addition of 50 L of 100 mM ethylenediaminetetraacetic acid (EDTA).
The stopped reaction mixture (65 L) was transferred to Flash Plate
l
L) containing 50 mM HEPES, pH 7.5,
2 lM
peptide
l
l
l
References and notes
l
a
(Perkin-Elmer) and incubated for 2 h. The wells were washed three times
with 0.02% Tween-20. Plates were read on a TopCount analyzer.
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Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.
13. A375 cells seeded overnight were incubated with Raf inhibitors for 3 h. At the
end of the incubation, the cells were fixed, permeabilized, blocking buffer
added and the plates were incubated overnight. After the blocking buffer was
discarded, the plates were incubated with anti-phospho-ERK antibody for 1 h
followed by treatment with anti-horse radish peroxidase. Optical density was
read at 650 nm for tetramethylbenzidine as substrate.
14. Aronov, A. M. Drug Discov. Today 2005, 10, 149.
15. Permeability was determined with the in vitro PAMPA permeability assay
where test compounds are partitioned between two chambers divided by a
lipid membrane. Compounds are run in duplicate at one of three pHs: 5.0, 6.2,
4. Lee, J.-H.; Lee, E.-S.; Kim, Y.-S. Cancer 2007, 110, 38.
and 7.4. The donor plate (bottom) contains 50
specified pHs. A filter plate containing a lipid, a negatively charged mixture of
20% phospholipids and dodecane, which has been painted on porous
lM of the test compound at the
5. Gray-Schopfer, V.; Wellbrock, C.; Marais, R. Nature 2007, 445, 851.
6. Hingorani, S. R.; Jacobetz, M. A.; Robertson, G. P.; Herlyn, M.; Tuveson, D. A.
Cancer Res. 2003, 63, 5198.
a
membrane is then placed on top of the donor plate. The acceptor plate (top)
is filled with an acceptor buffer at pH 7.4 containing a chemical scavenger that
mimics in vivo carrier proteins. Test compounds are allowed to distribute
between the two chambers over the course of four hours. The relative
absorbances of the UV traces, 250–490 nm, collected from the blank, reference,
donor, and acceptor plates are used to calculate permeability in nm/sec.
16. Berglund, S.; Egner, B. J.; Gradén, H.; Gradén, J.; Morgan, D. G. A.; Inghardt, T.;
Giordanetto, F. Bioorg. Med. Chem. Lett. 2009, 19, 4274.
17. Compound 1 was dosed in 10% 2-hydroxypropyl-beta-cyclodextrin (HPBCD)
for both routes of administration. Compound 16 was dosed in 10% HPBCD and
in ethanol/PEG400/N,N-dimethylacetamide/water = 1:4:1:4 (by volume) for PO
and IV, respectively.
7. (a) Hauschild, A.; Grob, J.-J.; Demidov, L. V.; Jouary, T.; Gutzmer, R.; Millward,
M.; Rutkowski, P.; Blank, C. U.; Miller, W. H., Jr.; Kaempgen, E.; Martín-Algarra,
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Huang, S.-C.; Iartchouk, N.; Janowick, D.; Kim, M.-S.; Kulkarni, B.; Langston, S.
P.; Liu, J. X.; Ma, L.-T.; Menon, S.; Mizutani, H.; Paske, E.; Renou, C. C.; Rezaei,
M.; Rowland, R. S.; Sintchak, M. D.; Smith, M. D.; Stroud, S. G.; Tregay, M.; Tian,
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9. hERG activity was determined by the inhibition of Tracer Red fluorescent probe
binding to hERG channels expressed in PredictorTM hERG membranes
(Invitrogen).
18. The PK/PD study was performed in nude mice bearing A375M xenografts.
Compound 16 was dosed as solutions in 10% HPBCD. Xenografts were
harvested for analysis at the time listed after
a single 33 mg/kg dose.
Average of pERK levels determined by Li-Cor quantitative Western blots (3
or 4 animals per group) as compared to vehicle control set at 100%. Average of
compound levels in the plasma of mice was determined by LC/MS/MS
quantitation (3 or 4 animals per group).
19. 33 mg/kg BID is the maximum daily dose tolerated without body weight loss.
Compound 16 was administered PO/BID for 21 days using the formulation
described in the PK/PD studies.
20. Horne, D. A.; Yakusijin, K.; Büchi, G. Heterocycles 1994, 39, 139. N-
methylimidazole analog which ultimately leads to compound 14 is prepared
by using methylamine instead of ammonia.
10. In cardiology, the QT interval is a measure of the time between the start of the
Q wave and the end of the T wave in the heart’s electrical cycle. The QT interval
represents electrical depolarization and repolarization of the ventricles. (a) De
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11. For synthetic details see U.S. Pat. Appl. Publ. (2010), US 2010/0197924.
Compounds 1, 3 and 6 were prepared as described previously.8