Optimizing the physicochemical properties of Raf/MEK inhibitors by nitrogen scanning

Article abstract of DOI:10.1021/ml400379x

Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and 13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic stability. The impa

Full text of DOI:10.1021/ml400379x

Letter
pubs.acs.org/acsmedchemlett
Optimizing the Physicochemical Properties of Raf/MEK Inhibitors by
Nitrogen Scanning
Toshihiro Aoki,† Ikumi Hyohdoh,† Noriyuki Furuichi,† Sawako Ozawa,† Fumio Watanabe,†
Masayuki Matsushita,† Masahiro Sakaitani,† Kenji Morikami,‡ Kenji Takanashi,† Naoki Harada,†
Yasushi Tomii,† Koji Shiraki,‡ Kentaro Furumoto,‡ Mitsuyasu Tabo,‡ Kiyoshi Yoshinari,† Kazutomo Ori,†
Yuko Aoki,† Nobuo Shimma,† and Hitoshi Iikura*,†,‡
†Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
‡Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
S
* Supporting Information
ABSTRACT: Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and
13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic
stability. The impact after nitrogen substitution on interactions between a derivative and its on- and off-target proteins (Raf/
MEK, CYPs, and hERG channel) was also detected, most of them contributing to weaker interactions. After identifying the
positions that kept inhibitory activity on HCT116 cell growth and Raf/MEK, compound 1 (CH5126766/RO5126766) was
selected as a clinical compound. A phase I clinical trial is ongoing for solid cancers.
KEYWORDS: Nitrogen scan, Raf, MEK, kinase inhibitor, CH5126766, RO5126766
n the hit-to-lead process in medicinal chemistry, critical
aspects in a compound are compatibility between the
to the resulting PK profile.7 Decreasing hERG inhibitory
activity of lipophilic leads10−12 was found using nitrogen
substitution of a benzene moiety.13,14 Because CH−π
interaction between a drug and the hERG channel are reported
to be key regardless of the CLogP values,15 nitrogen
substitution of a phenyl ring could also be effective from this
point of view. Effects on CYP interactions by nitrogen
substitutions are still being elucidated: pyridine moiety has
the potential to inhibit CYP by the interaction of the nitrogen
to Fe,16,17 while a report showed a compound after nitrogen
substitution had reduced CYP inhibition compared to the
parent.6 Metabolic stability was improved in derivatives by
nitrogen substitution of aromatic moieties,8 which made them
less susceptible to oxidative metabolism and also less lipophilic.
We recently reported SAR studies of a Raf and MEK
inhibitor2,18 that inhibits one of the most important signal
transduction pathways in human cancer, the Ras/Raf/MEK/
ERK pathway.19 Introducing a sulfamide moiety to our
coumarin hit afforded the compatibility of Raf/MEK activity
and oral bioavailability.18 Fluorine scanning allowed us to
identify better leads;2 because all the derivatives retained the
physicochemical properties, we focused on identifying the
positions for enhancing Raf/MEK activity (Scheme 1). Here we
I
physicochemical properties, safety profiles, and bioactivity.
However, a derivatization favorable for one factor (bioactivity,
physicochemical properties, toxicity, etc.) often means that
another factor fails the criteria set for its advancement to the
clinic. To solve this paradox, one could attempt to improve one
factor by using a chemical modification that minimally changes
the conformation of a lead because the impact on other factors
might be minimized. A fluorine atom substitution of a hydrogen
atom attached to a carbon atom (fluorine substitution) is a
representative example of such derivatization: when subsets of
neighboring functional groups to the introduced fluorine are
absent, changes in 3D conformation would be minimal
compared to other modifications, and those of electronic
properties would also be limited.1−5
Another approach is nitrogen substitution of aromatic
moieties.6−8 In nitrogen substitution, just as in fluorine
substitution, 3D conformational changes of a lead compound
could be smaller than other possible chemical modifications, if
no critical functional groups that cause electronic repulsions or
interactions are located in proximity to the introduced nitrogen.
In contrast to fluorine substitution, nitrogen substitution
derives inherently dramatic changes in the electronic properties.
Reflecting its ability to make such changes in electronic
properties, nitrogen substitution has been utilized to modify
various physicochemical properties and safety profiles.
Improved water solubility has been reported,9 with benefits
Received: September 24, 2013
Accepted: January 22, 2014
© XXXX American Chemical Society
A
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Scheme 2. Coumarin with nitrogen substituted at X2−X5 or Y
was synthesized effectively in a manner similar to that reported
previously.21 Compounds 4c−f, which were obtained by the
alkylation of ethyl acetoacetate with bromomethylpyridines
3c−f, were converted to coumarins 6c−f via a typical
Pechmann reaction with resorcinol 5a or 5f. After introducing
the R1 moiety, reaction with N-methylsulfamoyl chloride or N-
methyl-2-oxooxazolidine-3-sulfonamide afforded the targets
11b−e or 12f. The fluorinated compound 4g (X2 = CF),
which was obtained by the alkylation of ethyl acetoacetate with
compound 3g, was converted to target 1 or 13h−i, as above. A
coumarin substituted to nitrogen at the Y position was also
obtained via Pechmann reaction of resorcinol (5a) and
compound 4j, which was prepared from the reaction of 3-
nitroaniline (3j) with compound 922 and subsequent
hydrolysis. Converting to the target 11j was smoothly achieved
by reducing the nitro group and by subsequent sulfamoylation.
In the case of the phenyl group at R1, coumarin was formed by
the reaction of compound 5g instead of resorcinol (5a). Using
Zn(OTf)2 in MeOH for the Pechmann reaction instead of
H2SO4 was crucial for obtaining coumarin 6b, which has
nitrogen at X1.23 Synthesis of 5- (X6 = N) or 6-azacoumarin (X7
= N) derivative was unsuccessful. An attempted Pechmann
reaction using a typical condition (H2SO4) or modified
conditions24 (NH2SO3H, ZrCl4, ZnCl2, Sm(NO3)3, and
AlCl3) did not give the desired coumarin.
Scheme 1. Fluorine Scanning and Nitrogen Scanning Led to
Clinical Compound 1
report another strategy, a nitrogen scan, in which conforma-
tional change of a lead would be smaller than other possible
chemical modifications (C−H → C−NH2, CH → C−OH,
etc.). Nitrogen-substituted compounds of our leads 11a and
13g, which had room for improved solubility and metabolic
stability, afforded positive effects on them, regardless of the
nitrogen substitution positions (Scheme 1). This allowed us to
focus on finding the positions that kept bioactivity, and we
identified the clinical compound 1 (CH5126766/RO5126766),
which showed superior antitumor effects compared to a pure
MEK inhibitor in a mouse xenograft.20
In the five compounds in Table 1 with nitrogen substituted at
different positions, only one position (X3) maintained
inhibitory activity on HCT116 cell growth and Raf/MEK
A set of coumarin derivatives possessing nitrogens
(compounds 1, 11−13, and 15) was synthesized as shown in
a
Scheme 2. Synthesis of Coumarins 1, 11−13, and 15
a
Reagents and conditions: (a) (Boc)2O, 60 °C, then (Boc)2O, THF, DMAP, rt; (b) NBS, benzoylperoxide or AIBN, CCl4, reflux; (c) ethyl
acetoacetate, NaH, THF, 0 °C; (d) 5, conc. H2SO4; (e) LDA, THF, DMF, −78 to 0 °C, then NaBH4, 0 °C, 58%; (f) TBSCl, imidazole, THF, rt,
93%; (g) benzophenone imine, NaOtBu, Pd2(dba)3, rac-BINAP, toluene, 60 °C; (h) TBAF, THF, 76% (2 steps); (i) MsCl, LiOtBu, THF, 0 °C, then
ethyl acetoacetate, LiOtBu, NaI, THF, 50 °C, 3 h, 95%; (j) 5a, MsOH, CF3CH2OH; (k) 9, THF, 70 °C, 12 h, 78%; (l) TiCl3, 20−30% HCl aq.,
acetone, rt, 30 min, 71%; (m) SnCl2·2H2O, EtOAc, 75 °C; (n) R1X, Cs2CO3 or K2CO3 or NaH, DMF; (o) R1X, CuI, N,N’-
dimethylethylenediamine, Cs2CO3, DMF, 100 °C; (p) N-methylsulfamoyl chloride, pyridine, DMF; (q) N-methyl-2-oxooxazolidine-3-sulfonamide,
Et3N, MeCN, 80 °C; (r) 5b, Zn(OTf)2, MeOH, reflux; (s) NaOH or KOH, MeOH.
B
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Table 1. Enzymatic and Cellular Activity and Pharmaceutical Properties of Coumarin Derivatives
IC50 (nM)
solubility
(μg/mL)
CLint human
(μL/min/mg)
PAMPA
a
compd
R2
X1
X2
X3
X4
X5
HCT116
HT-29
C-Raf
MEK1
(10−6 cm/s) AUC
(μM·h)
po
b
11a
11b
11c
11d
11e
12a
12f
H
CH
N
CH
CH
N
CH
CH
CH
N
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
24
9
300
110
32
20
3
6
78
H
1100
270
600
73
2500
70
440
43
3
ND
314
134
ND
18
H
CH
CH
CH
CH
CH
110
320
55
7
6
H
CH
CH
CH
CH
32
25
32
99
6
7
H
CH
CH
CH
>10000
22
>10000
8
>50000
78
>50000
29
240
11
ND
17
ND
ND
4
Me
Me
CH
CH
>10000
5700
27000
37000
425
ND
ND
a
Compounds were evaluated in 24 h exposure studies in mice at 100 mg/kg and formulated as solutions of 5% DMSO, 5% Cremophor EL, 15%
b
PEG400, 15% HPCD, and 60% water. Sodium salt was used.
Table 2. Enzymatic and Cellular Activity of Coumarin Derivatives
IC50 (nM)
C-Raf
compd
X3
X8
X9
X10
Y
R3
HCT116
MEK1
13g
13h
13i
1
N
CH
N
CH
CH
CH
N
CH
CH
N
CH2
CH2
CH2
CH2
CH2
NH
F
F
F
F
F
H
110
45
21
76
N
81
190
650
160
97
N
N
900
40
56
N
N
CH
CH
CH
56
14
CH
CH
N
N
17
23
15j
N
N
>10000
>50000
>50000
(compound 11d), and none of the positions enhanced it.
Introducing nitrogen at X2 position resulted in decreased cell
growth inhibitory activity by a factor of 10 (compound 11c)
compared with the parent 11a. Compounds with a nitrogen
substitution at X1, X4, and X5 showed larger than 50-fold
decreases in cell growth inhibitory activity.
acceptable cell inhibitory activity (IC50 = 110 nM in HCT116
cell line) after random modification of the R1 part by fixing R3
to fluorine2 and X3 to nitrogen. Because the solubility as
evaluated by the LYSA method was lower (18 μg/mL),
nitrogen scanning at the R1 position was performed. Compared
to the parent compound 13g, compound 13h with nitrogen at
X8 position maintained the inhibitory activity on HCT116 cell
growth and Raf/MEK. Although introducing an additional
nitrogen at X10 position caused weaker inhibitory activity on
cell growth (compound 13i), introducing one at X9 position
(compound 120) afforded similar bioactivity to the parent 13g.
Introducing nitrogen at the Y position derived almost no
bioactivity (compound 15j). Nitrogen substitution improved
solubility, and a high-throughput solubility assay of compounds
13g and 1 showed 18 and 159 μg/mL, respectively. The AUC
of compound 1 after oral administration to mice increased 36-
fold (2831 μM·h) compared to the original 11a. As just
described, we succeeded in replacing a carbamate with a
pyrimidyl moiety that was more metabolically stable. The effect
of nitrogen substitution was also observed when the properties
of compound 1 were compared to compound 14,2 which has
C−H group at X3 and nitrogen at X8 and X9. Metabolic stability
(1, 0.5 μL/min/mg; 14, 6.7 μL/min/mg), solubility (1, 159
μg/mL; 14, 13 μg/mL), and AUC (1, 2831 μM·h; 14, 425 μM·
h) of compound 1 were all superior to those of compound 14
by around a factor of 10. As a result, by nitrogen scans at four
additional positions (X8, X9, X10, and Y), we identified the
promising compound 1, which has an IC50 value of 40 nM on
On the other hand, all the evaluated compounds had
improved water solubility and metabolic stability compared
with the parent 11a or 12a (Table 1). Solubility was evaluated
by the LYSA method (high-throughput solubility assay),25 and
the values of the compounds 11b and 11d were slightly more
than that of 11a, and those of 11c, 11e, and 12f increased up to
40-fold. Metabolic stability was evaluated using human liver
microsome, and all evaluated compounds were more stable (by
more than 3-fold) than the parent 11a. Because one of the
metabolic positions of compound 11a was previously identified
as the phenyl ring bearing a sulfamide moiety,18 decreasing the
electron density of carbon atoms by replacing the aryl ring with
a pyridyl ring (11c and 11d) would be a reason for oxidative
metabolism to persist. Membrane permeability evaluated by the
PAMPA method26 had high enough and comparable values to
parent 11a. After oral administration of compound 11c or 11d
to mice, AUCs increased up to 4-fold, which could reflect the
improved metabolic stability and/or solubility.
We also modified the X8−10 (R1 part of Scheme 2) and Y part
because modifying the carbamate part (R1 part), which is a
major metabolic site, seemed fruitful (Table 2).18 Although the
carbamate moiety was key for the strong bioactivity in our
preliminary SAR,18 we obtained compound 13g (R1 = Ph) with
C
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HCT116 cell growth inhibitory activity and excellent solubility
and AUC in mouse.
CYP or hERG inhibitory activity in the nitrogen-introduced
derivatives was superior to that of the corresponding parents
(Table 3). Nitrogen-introduced derivative 11d showed almost
Table 3. CYP and hERG Inhibition of Coumarin
a
CYP inhibition IC50 (μM)
b
compd
2C9 (−/+)
3A4 (−/+)
hERG inhibiton (%)
11a
11d
14
29/96
13/5.6
ND
>100/>100
19/19
>100/>100
26/11
ND
25
1
12/60
>100/>100
no inhibition
a
IC50 (−) and IC50 (+) values were determined after a 30 min
b
preincubation without and with NADPH, respectively. At 10 μM.
no inhibition on CYP 2C9 and 3A4, while the parent 11a
showed CYP 3A4 inhibition at an IC50 of 13 μM. This tendency
is similar to a previous report,6 while pyridine moiety itself has
the potential to inhibit CYP. One possible explanation is that
reducing lipophilicity of the molecule and/or decreasing
electron density of carbon atoms on an aromatic ring by
nitrogen substitution might be key to reducing the interaction
to CYPs, which inherently have the function of modifying
lipophilic compounds to hydrophilic compounds.27−29 The
same tendency was observed when comparing compounds 1
and 14.
Our nitrogen scan at nine different positions resulted in us
identifying three positions that kept bioactivity (Raf/MEK), six
positions that decreased bioactivity at least 8-fold (Tables 1 and
2), and none that enhanced bioactivity. Because of the change
in electronic structure to a more hydrophilic compound,
derivatives after nitrogen substitution improved metabolic
stability as well as solubility. The key to obtaining candidates
with improved physicochemical properties is to identify the
positions for nitrogen substitution at which the bioactivity will
be acceptable. Decreased bioactivity could be explained by two
possible reasons: reason 1 is that change of the whole 3D
conformation after nitrogen substitution is critical and the
compound could not achieve an active conformation; reason 2
is that change of the whole 3D conformation is minimal but
electrostatic repulsion between the introduced nitrogen atom
and target proteins is critical.
The effect on dihedral angles (ϕ1−ϕ6) was estimated by
collecting crystal structures in the CSD data or by evaluation of
potential energy surfaces at the B3LYP/6-31G(d) level (Figure
1A). Crystal data processing a diaryl ether part in CSD showed
that stable conformations after nitrogen substitution at X8
(blue), X9 (green), and X10 (red) overlapped well with those
of the parent (black), and they had strong preferences for
twisted structures (ϕ1 on 0°, 180°, and 360° and ϕ2 on 90°
and 270° (Figure 1C)). Because lone pairs of N and O atoms
were repulsive, conformations of the parent except those
mentioned above could not be achieved (Figure S1 in
Supporting Information).30 Similarly, potential energy surfaces
of N-substituted compounds at X2 or X5 suggested its stable
conformation overlapped with that of the parent (around ϕ3
on 90°, and ϕ4 on 240°), but they could not take another
conformation because of N to O (carbonyl) repulsion (around
ϕ3 on 90°, and ϕ4 on 60°; Figure 1B and Supporting
Information Figure S5). The same tendency was observed at X1
of ϕ1−ϕ2 (see Supporting Information for calculated data of
Figure 1. (A) Rotatable bonds of our compound. (B) Potential energy
surfaces calculated at the B3LYP/6-31(d) level. The ΔE from 0 to 5
kJ/mol is colored in blue, 5−10 kJ/mol in green, 10−15 kJ/mol in
brown, and >15 kJ/mol in red. (C) Torsional distributions in small
molecule crystal structures from the CSD data (* = C−R or N).
this and other values below). Structural difference for ϕ3−ϕ4 at
X3 or X4 could be minimal. Fixation of sulfamide by
intramolecular hydrogen bonding was suggested in ϕ5−ϕ6 at
X2 or X3 by potential energy surfaces (Supporting Information
Figure S6), while there were smaller effects at X4 or X5. Thus,
two positions (X10 and X4) could afford smaller effects on the
whole 3D structure (most preferred conformations of the
parent would be acceptable), and the reason for their reduced
bioactivity would be attributed to reason 2. Compounds after
nitrogen substitution of six positions (X1, X2, X3, X5, X8, and
X9) could inherit some stable conformations of the parent, but
not others, because of intramolecular electrostatic repulsion
and/or formation of hydrogen bonds. Three of them (X3, X8,
and X9) retained bioactivity, and the other three reduced it
(though the reason for reduced bioactivity could not be
identified). We considered that our nitrogen scanning worked
because at least some stable conformations could overlap with
the parent in most derivatives after nitrogen substitution.
After further development of compounds 120 and 142 by
pharmacology and PK profiles, we selected compound 1 for
clinical trial. Compound 1 showed excellent PK data for mouse,
rat, and monkey with bioavailability values comparable to
compound 14 (compound 1, 93%, 66%, and 82%, and 14, 75%,
84%, and 59%, respectively) and better clearance values
(compound 1, 1.1, 0.7, and 0.1 mL/min/kg, and 14, 6.8, 6.6,
and 0.9 mL/min/kg, respectively). A potent antitumor effect of
both compounds was observed in the C32 xenograft model
(IC50s on C32 (B-Raf V600E) cell growth of compound 1, 47
nM, and 14, 57 nM): comparable maximum efficacy (TGI of
compound 1, 118%; 14, 96%) and 16-fold smaller doses in
compound 1 (ED50 of compound 1, 0.09 mg/kg, and 14, 1.44
mg/kg), which reflected the improvement in metabolic stability
after nitrogen substitution (Figure 2 and Supporting
Information). Neither compound showed serious effects on
body weight or any adverse clinical signs.
Salt screening was executed to identify the active
pharmaceutical ingredient (API), and crystalline K salts from
compound 1 and 14 were found as the candidates.31 The
supersaturated solubility of the K salt of compound 1 in fasted
D
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AUTHOR INFORMATION
■
Corresponding Author
*(H.I.) Phone: +81-550-87-8597. Fax: +81-550-87-5326. E-
mail: iikurahts@chugai-pharm.co.jp.
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Y. Tachibana-Kondoh, K. Sakata, and T. Fujii for
biological assays, Y. Ishiguro and H. Suda for mass
spectrometry measurement, and Chugai Editing Services for
proofreading the manuscript.
Figure 2. In vivo efficacy of 1 (K salt) in the C32 human malignant
melanoma xenograft model. C32 cells were inoculated subcutaneously
into the right flank of BALB-nu/nu mice. Tumors were allowed to
establish growth after implantation before start of treatment.
Coumarin 1 was administered orally once daily for 11 days, from
day 0 to day 10. Tumor size was measured twice per week. Values are
mean SD, n = 4.
ABBREVIATIONS
■
AIBN, 2,2′-azodiisobutyronitrile; AUC, area under the curve;
BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; CL,
clearance; CSD, Cambridge Structural Database; CYP,
cytochrome P450; dba, dibenzylideneacetone; DMAP, 4-
dimethylaminopyridine; ERK, extracellular signal-regulated
kinase; hERG, human ether-a-go-go related gene; HPCD, 2-
hydroxypropyl-β-cyclodextrin; LDA, lithium diisopropylamide;
LYSA, lyophilized solubility assay; MEK, mitogen-activated
protein kinase kinase; NADPH, nicotinamide adenine
dinucleotide phosphate (reduced); NBS, N-bromosuccinimide;
ND, no data; PAMPA, parallel artificial membrane permeability
assay; PEG, polyethylene glycol; PK, pharmacokinetics; TBAF,
tetra-n-butylammonium fluoride; TGI, tumor growth inhibition
state simulated intestinal fluid (FaSSIF) after 4 h in a nonsink
condition using a mini-scale dissolution test afforded 5-fold
greater value (57 μg/mL) than that of compound 14 (12 μg/
mL).32 However, nitrogen substituted compound 1 has
comparable saturated solubility to the corresponding 14
regarding crystalline acids (free form). The saturated solubility
of compounds 1 and 14 was determined after 24 h equilibration
in FaSSIF, which gave 2.7 and 5.5 μg/mL, respectively.33 It is
interesting that nitrogen substitution does not always
contribute to increasing the saturated solubility in the free
crystalline form; however, it still has an advantage for drug
absorption in human because it contributes to increasing the
ability to generate and keep the supersaturated state. Judging
from these experiments, we selected the salt form of compound
1 (CH5126766/RO5126766)20 for clinical trial.
In summary, lead optimization of our leads 11a and 13g by
nitrogen scanning at nine different positions worked effectively
to improve the physicochemical properties such as metabolic
stability and solubility, as evaluated by high-throughput assay.
Changes by nitrogen substitution on the interactions between a
derivative and its on- and off-target proteins (Raf/MEK, CYPs,
and hERG channel) have an impact, and we focused on
identifying the positions for maintaining Raf/MEK activity.
Changes in electronic structure created synthetic difficulties
because of the difference in reactivity of each nitrogen-
containing building block. A candidate with nitrogen
introduced could have an advantage in drug absorption,
especially if supersaturated formulations, including a salt
formation, were developed. We have demonstrated that, in
late stage lead optimization, not only the fluorine scan but also
the nitrogen scan worked efficiently to select the best
compound for clinical use.
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NOTE ADDED AFTER ASAP PUBLICATION
■
Due to a production error, this paper published ASAP on
January 24, 2014 without its required corrections. The revised
version was reposted on January 27, 2014.
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