Fluorine scanning by nonselective fluorination: Enhancing Raf/MEK inhibition while keeping physicochemical properties

Article abstract of DOI:10.1021/ml4002419

A facile methodology effective in obtaining a set of compounds monofluorinated at various positions (fluorine scan) by chemical synthesis is reported. Direct and nonselective fluorination reactions of our lead compound 1a and key intermediate 2a worked efficiently to afford a total of six monofluorinated derivatives. All of the derivatives kept their physicochemical properties compared with the lead 1a and one of them had enhanced Raf/MEK inhibitory activity. Keeping physicochemical properties could be considered a benefit of monofluorinated derivatives compared with chlorinated derivatives, iodinated derivatives, methylated derivatives, etc. This key finding led to the identification of compound 14d, which had potent tumor growth inhibition in a xenograft model, excellent PK profiles in three animal species, and no critical toxicity.

Full text of DOI:10.1021/ml4002419

Letter
pubs.acs.org/acsmedchemlett
Fluorine Scanning by Nonselective Fluorination: Enhancing Raf/MEK
Inhibition while Keeping Physicochemical Properties
Ikumi Hyohdoh,† Noriyuki Furuichi,† Toshihiro Aoki,† Yoshiko Itezono,† Haruyoshi Shirai,†
Sawako Ozawa,† Fumio Watanabe,† Masayuki Matsushita,† Masahiro Sakaitani,† Pil-Su Ho,§
Kenji Takanashi,† Naoki Harada,† Yasushi Tomii,† Kiyoshi Yoshinari,† Kazutomo Ori,† Mitsuyasu Tabo,‡
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
§C&C Research Laboratories, DRC Natural Sciences Campus, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon,
Gyeonggi-do 440-746, Republic of Korea
S
* Supporting Information
ABSTRACT: A facile methodology effective in obtaining a set of compounds monofluorinated at various positions (fluorine
scan) by chemical synthesis is reported. Direct and nonselective fluorination reactions of our lead compound 1a and key
intermediate 2a worked efficiently to afford a total of six monofluorinated derivatives. All of the derivatives kept their
physicochemical properties compared with the lead 1a and one of them had enhanced Raf/MEK inhibitory activity. Keeping
physicochemical properties could be considered a benefit of monofluorinated derivatives compared with chlorinated derivatives,
iodinated derivatives, methylated derivatives, etc. This key finding led to the identification of compound 14d, which had potent
tumor growth inhibition in a xenograft model, excellent PK profiles in three animal species, and no critical toxicity.
KEYWORDS: Fluorine scan, Raf, MEK, kinase inhibitor, anticancer
fluorine atom substitution of a hydrogen atom attached to
a carbon atom (fluorine substitution) has been intensively
chemical modifications, changes in 3D conformation would be
minimal and those of electronic properties would also be
limited.5,12,13
A
utilized to adjust molecular properties in many fields.1−3 In the
area of medicinal chemistry, fluorine substitutions of a lead
compound are one of the most utilized modifications during
lead optimizations,4−7 and about 20% of marketed drugs
contain fluorine atoms.6,8 By fluorine substitution at appro-
priate positions, adjustments to the acidity or basicity of
functional groups such as the hydroxyl and amino groups have
been reported to improve permeability.4,7,9 A fluorine block of
metabolically unstable hydrogen can improve the metabolic
stability.7 Enhanced binding affinity to the target protein was
also reported especially when X-ray crystallography was
available and the particular positions of fluorination could be
anticipated.10,11
In the latter situation, one of the possible advantages of
fluorine substitution is that the changes in physicochemical
parameters (water solubility, membrane permeability, metabolic
stability, etc.) could be minimal because of the limited changes
in 3D and electronic structures. In fact, fluorine substitution
could solve the paradox often encountered in medicinal
chemistry that a derivatization favorable for one factor
(bioactivity, physicochemical properties, toxicity, etc.) derives
unacceptable change for another factor in developing a clinical
candidate. Fluorine substitution has the potential to improve
bioactivity with the essence of the drug untouched.
Various synthetic methodologies to introduce fluorine were
reported including regioselective and enantioselective reac-
tions.14−16 These methods are powerful when the target
The effects of fluorine substitution could vary depending on
the presence or absence of subsets of neighboring functional
groups. When such functional groups are located in proximity
to the introduced fluorine, changes in 3D conformation
resulting from electronic repulsions and/or changes in
properties such as basicity can be derived. Conversely, if
there are no such functional groups in the vicinity, of all
Received: June 25, 2013
Accepted: September 22, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
ab c
, ,
position for fluorination has been identified. However,
exhaustive reaction steps are required when the target position
is unknown, and random scanning of fluorinated positions is
used to identify the best. An effective method for a fluorine scan
was reported in 2009 using mutant CYP metabolism,17 but to
the best of our knowledge, there are no reports of facile
chemical methodologies effective for fluorine scanning.18 We
previously reported that compound CH5126766/RO5126766
showed both Raf and MEK inhibitory activity of the Ras/Raf/
MEK/ERK pathway, and it showed superior antitumor effect
compared to that of a pure MEK inhibitor in a mouse
xenograft.19−21 Here we report the facile strategy of a fluorine
scan utilized during our hit-to-lead chemistry, and the excellent
effects of monofluorination on the bioactivity of our lead while
retaining its physicochemical properties.
Scheme 1. Direct and Nonselective Monofluorination
To achieve a fluorine scan of a lead, we chose the direct and
nonselective fluorination reaction of a lead compound (or a key
intermediate) (Figure 1). Usually selective chemical reactions
Figure 1. Fluorine scanning by direct and nonselective monofluori-
nation or stepwise synthesis.
to afford only one derivative are preferable, but this method of
stepwise chemical synthesis requires multisteps to obtain only
one derivative, and exhaustive chemical steps need to be
conducted to achieve fluorine scanning. In contrast, the
advantage of the nonselective method is that a single reaction
could afford several compounds. Direct fluorination of a lead is
a powerful method for two reasons: it can be applied to
compounds with a set of functional groups, and many
fluorination reagents are commercially available,14 so the
desired nonselectivity could be achieved by fine-tuning the
reactivity for each reactant.22,23
a
Reagents and conditions: (a) LiHMDS (1.0 equiv), THF, −78 °C, 30
min, then, 5, 2 h, 53%. (b) N,N-dimethylcarbamoyl chloride (1.2
equiv), NaH (1.1 equiv), THF, rt, 1 h; (c) SnCl2 (5.0 equiv), EtOH/
EtOAc 1:1, 80 °C, 3 h; (d) chlorosulfonyl isocyanate (2.7 equiv),
Fluorination of lead compound 1a21 and key intermediate
2a21 afforded a total of six monofluorinated derivatives
(Scheme 1; 1b, 1c, 1d, 2e, 2f, and 2h). Because fluorination
of lead 1a by reagent 5 did not proceed (entry 1), we chose the
more reactive reagent 6. This afforded compound 1b in a
selective manner (entry 3). However, reagent 7, which has the
highest reactivity among the three reagents, afforded the three
monofluorinated compounds in a nonselective manner (ratio of
1b/(1c + 1d) = 32/26 (entry 5)), and isolated yields after
chromatography were 15% (1b), 4.3% (1c), and 3.4% (1d).
Fluorinated positions of these three compounds were identified
b
HCOOH (1.3 equiv), pyridine (2.0 equiv), CH2Cl2, rt, 16 h. The
ratio of 2e−h was determined by HPLC analysis (UV area% at 200−
400 nm) using 2,4-difluoro-nitrobenzene as an internal standard. The
ratio of 1b−d was determined by LC/MS analysis (MS area%) taking
1a intensity before the reaction started as 100%. The peaks of 1c and
1d overlapped. Isolated yields are described in parentheses.
c
CF3CH2OH was used as a reaction solvent. NaOTf was used as an
additive.
their coupling constants in 1H NMR spectrum (see Supporting
Information). We demonstrated that direct and nonselective
monofluorination of both compounds 2a and 1a worked by
tuning the reactivity of the fluorinating reagents. The more
reactive substrate 2a required milder fluorinating reagent 6, and
less reactive substrate 1a required more reactive 7.
Compound 1g was synthesized from 3a because fluorinated
intermediate 2g was not observed and compound 2h was
isolated by the fluorination of compound 2a (Scheme 1).
Fluorination of 3a21 by LiHMDS and reagent 5 afforded
compound 4g, which was converted to 1g in the following 2
steps.
1
(see Supporting Information) by their coupling pattern in H
NMR (discrimination of 1d,24 from 1b and 1c), and correlation
in CH-HMBC spectrum (discrimination of 1b and 1c). For key
intermediate 2a, nonselective monofluorination was achieved
by reagent 6, while the more reactive reagent 7 gave only
multifluorinated compounds (entry 6). The fluorinated
intermediates 2e and 2f were further modified to targets 1e
and 1f by 3 steps as shown in Scheme 1 in a total isolated yield
of 2.5% and 2.8%, respectively, from intermediate 2a.
Fluorinated positions of 1e24 and 1f were discriminated by
B
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Table 1. Enzymatic and Celluar Activities and Pharmaceutical Properties of Monofluorinated Compounds
IC50 (nM)
solubility
(μg/mL)
CL human
(μL/min/mg)
PAMPA
a
compd X1
X2
X3
X4
X5
X6
R2
HCT116 MEK1 C-Raf
(10−6 cm/s)
AUC
(μM·h)
po
c
1a
1b
1c
1d
1e
1f
H
F
H
H
F
H
H
H
F
H
H
H
H
F
H
H
H
H
H
F
H
H
H
H
H
H
F
H
95
950
610
18
230
940
44
530
830
1100
13
32
9
6
6
6
5
104
176
ND
40
H
H
H
H
H
H
H
F
H
37
6
6
bc
,
H
H
H
H
H
H
H
H
H
H
53
273
114
ND
14
6
6
b
H
H
H
H
H
F
H
87
110
240
60
180
120
220
300
2400
8
6
5
61
H
H
H
H
H
F
H
140
200
24
ND
7
ND
4
ND
84
1g
8a
8b
8d
8e
8g
H
H
H
H
H
H
H
c
c
H
H
H
H
F
Me
Me
Me
Me
Me
110
2900
38
32
20
22
13
13
22
6
78
550
9
51
5
75
H
H
H
81
5
70
H
H
22
64
ND
120
55
5
99
H
30
66
72
4
86
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
c
PEG400, 15% HPCD, and 60% water. At 50 mg/kg. Sodium salt was used.
a
Among the six fluorinated positions, one position (X3)
contributed to stronger inhibitory activity on HCT116 cell
growth and MEK/Raf in both the compounds (R2 = H, Me)
used to evaluate the fluorination effects (Table 1). For
compounds 1d and 8d (X3 = F), 3- to 5-fold enhancement
of cell growth and MEK inhibitory activity and 40-fold
enhancement of Raf inhibitory activity were observed. Three
positions (X4, X5, and X6) kept that level of activity after
fluorination, and two positions (X1 and X2) resulted in
decreased bioactivity.
Scheme 2. Synthesis Suitable for Large Quantities
Evaluation of water solubility, metabolic stability, and
membrane permeability for the fluorinated compounds showed
a
Reagents and conditions: (a) CsF (1.5 equiv), DMSO, 140 °C, 10 h,
82%; (b) NBS (1.2 equiv), benzoyl peroxide (0.1 equiv), CCl4, reflux,
5 h; (c) NaH (1.0 equiv), ethyl acetoacetate (1.0 equiv), THF, 0 °C,
12 h, 42% (2 steps); (d) resorcinol (1.0 equiv), conc. H2SO4, 0 °C, 12
h, 64%; (e) SnCl2 (5.0 equiv), EtOAc, reflux, 1.5 h, 71%; (f) 2-
bromopyrimidine or 2-bromothiazole (4.0 equiv), Cs2CO3, DMF, 100
°C; (g) N-methyl-2-oxooxazolidine-3-sulfonamide (3.0 equiv), Et3N
(5.0 equiv), MeCN, 75 °C, 3.5 h.
that all of these parameters were not significantly changed from
our lead compound 1a by monofluorination. Though the
solubility of two compounds (1b and 1g) was a third less than
that of lead 1a, the other seven derivatives kept or increased the
solubility (four compounds had more than double the value).
Because no specified fluorinated positions led to decreased
water solubility (compounds 8b and 8e had comparable
solubility to the parent 8a), we concluded that monofluorinated
compounds of our lead 1a tend to keep their solubility at all
positions. All of nine compounds kept the metabolic stability, as
evaluated from human liver microsome. In spite of identifying
oxidized metabolites at aromatic positions attached to
sulfamide,21 no clear improvement in microsomal stability
was observed by fluorination at X1, X2, or X3 (see below).
Membrane permeability of all nine compounds, evaluated by
PAMPA method, showed high enough and comparable values
to our lead 1a. AUCs after oral administration to mice were
also similar to lead 1a, the BA of which was 62%. By fluorine
scanning of our lead 1a, compounds for oral drugs with higher
bioactivity, which retained their physicochemical properties,
were identified.
For further derivatizations with fixed fluorine at X3, a
synthetic method available for large scale synthesis was
established by modifying the reported procedures (Scheme
2).21 The fluorine was introduced by substituting the chlorine
with CsF to give compound 10d. Bromination of benzyl
position and the reaction with ethyl acetoacetate then afforded
derivative 11d. Converting to coumarin 12d via Pechmann
reaction with resorcinol, followed by reduction of a nitro group
by SnCl2, afforded aniline derivative 13d. After introducing the
pyrimidyl or thiazole group, sulfamide 14d and 15d were
obtained by the reaction with N-methyl-2-oxooxazolidine-3-
sulfonamide.
As shown in Table 2, over 10-fold increased inhibition of cell
growth and Raf/MEK by fluorination at X3 position was
observed in the case of pyrimidyl and thiazole moieties at R1.
Again, solubility change by fluorination tended to be small: by
half in the pyrimidine derivative and 1.5-fold in the thiazole
derivative. Metabolic stability was improved in both com-
pounds. Fluorination had the effect of blocking the aromatic
ring hydrogen, potentially because carbamate, which was the
major metabolic position, was substituted to more stable
pyrimidine and thiazole moieties (see above). AUC values after
C
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ACS Medicinal Chemistry Letters
Letter
tended to be decreased by fluorination (see Supporting
Information). Inhibition of hERG at 10 μM was 25%, which
was weak enough. The compound showed satisfactory PK data
for mouse, rat, and monkey (Table 4) with good BA values
Table 2. Enzymatic and Celluar Activities and
Pharmaceutical Properties of Monofluorinated Compounds
14 and 15
Table 4. PK Profile of Sodium Salt of 14d in Mouse, Rat, and
a
Monkey
parameter
mouse
rat
monkey
iv/po dose (mg/kg)
AUCinf (μM·h), po
t1/2 (h), iv
10/20
75
5.0/10
43
2.5/5.0
108
4.8
1.3
2.9
Cl (mL min−1 kg−1
)
6.8
6.6
0.9
bioavailability (%)
75
84
59
a
Vehicle: 5% DMSO/5% Cremophor EL/15% PEG400/15% HPCD/
60% water.
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% PEG400, 15% HPCD, and 60% water. Sodium salt was used.
b
(75%, 84%, and 59%, respectively) and good clearance values
(6.8, 6.6, and 0.9 mL min−1 kg−1, respectively). Potent
antitumor effect was observed in the HCT116 xenograft
model: ED50 of 1.4 mg/kg and TGI of 119% at 30 mg/kg
(Figure 2). It showed no serious effect on body weight or any
adverse clinical signs.
oral administration to mice increased 2-fold, reflecting an
improved metabolic stability.
The positions X3 and X4, which were identified as keeping
Raf/MEK inhibitory activity after fluorination, were further
modified in the larger functional groups because they could be
potential positions for enhancing bioactivity (Table 3). By
Table 3. Enzymatic and Celluar Activity and Pharmaceutical
Properties of Coumarins Modified at X3 or X4
Figure 2. In vivo efficacy of sodium salt of 14d in the HCT116 human
colon cancer xenograft model. HCT116 cells were inoculated
subcutaneously into the right flank of BALB-nu/nu mice. Tumors
were allowed to establish growth after implantation before treatment
was initiated. Pyrimidine derivative 14d 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.
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% PEG400, 15% HPCD, and 60% water. Sodium salt was used.
b
introducing a methyl group to X3, the bioactivity was decreased
(compound 16). However, various modifications were accept-
able at X4 position (compounds 17−21), and the strongest
activity was obtained at a chlorine21 or iodine. Fluorine
scanning can also be utilized for identifying intensive
modification positions because keeping bioactivity by fluorina-
tion may be a sign of vacant space existing between a drug and
its target protein.5 We noticed that all of the modifications in
Table 3 resulted in decreased values for at least one of the
physicochemical properties (solubility, metabolic stability, or
permeability). Larger groups than fluorine resulted in negative
factors for the physicochemical properties of our lead 1a,
proving the special nature of fluorine.
Fluorine scanning by direct and nonselective monofluorina-
tion can be a powerful method of identifying compounds that
have stronger efficacy than the parent compound while keeping
the physicochemical properties. This ability to keep the
physicochemical properties could be considered a particular
advantage of a fluorinated compound, if changes of electronic
structure by fluorination are limited, when compared with
compounds introducing other moieties such as chlorine, iodine,
methyl, etc. We demonstrated that the direct and nonselective
monofluorination method could be superior to the normal hit-
to-lead chemistry by stepwise synthesis because a single
reaction afforded several derivatives, making it time- and cost-
effective and also because utilizing both lead compound and
key intermediates widened the possible choice of fluorination
points.
We identified compound 14d as the best compound for
further drug development. No strong CYP inhibition activity
was observed in five different species, and the IC50 values
D
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ACS Medicinal Chemistry Letters
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Predicting and tuning physicochemical properties in lead optimization:
amine basicities. ChemMedChem 2007, 2, 1100−1115.
ASSOCIATED CONTENT
* Supporting Information
Experimental preparation of compounds, characterization,
biological, in vitro physicochemical properties, and in vivo
experimental data. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
(10) Olsen, J. A.; Banner, D. W.; Seiler, P.; Wagner, B.; Tschopp, T.;
Obst-Sander, U.; Kansy, M.; Muller, K.; Diederich, F. Fluorine
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interactions at the thrombin active site: protein backbone fragments
H−Ca−CO comprise a favorable C−F environment and inter-
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Fah, C.; Seiler, P.; Obst-Sander, U.; Wagner, B.; Kansy, M.; Diederich,
̈
AUTHOR INFORMATION
Corresponding Author
*(H.I.) Phone: +81-550-87-8597. Fax: +81-550-87-5326. E-
mail: iikurahts@chugai-pharm.co.jp.
■
F. A fluorine scan at the catalytic center of thrombin: C−F, C−OH,
and C−OMe bioisosterism and fluorine effects on pKa and logD
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2004, 5, 644−649.
Author Contributions
The manuscript was written through contributions of all
authors.
(13) For examples, CLogP values of benzene (2.14) and F−Ph
(2.29) are rather similar to each other than compared to other
substitutions such as Cl−Ph (2.86), Br−Ph (3.01), I−Ph (3.27) and
Me−Ph (2.64).
Notes
The authors declare no competing financial interest.
(14) Kirk, K. L. Fluorination in medicinal chemistry: methods,
strategies, and recent developments. Org. Process Res. Dev. 2008, 12,
305−321.
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.
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(18) After completion of our investigations (see ref 19), direct and
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(19) Iikura, H.; Aoki, T.; Hyoudoh, I.; Furuichi, N.; Watanabe, F.;
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Tomii, Y.; Aoki, Y.; Takanashi, K. Discovery of a novel specific MEK
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(20) Ishii, N.; Harada, N.; Joseph, E. W.; Ohara, K.; Miura, T.;
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ABBREVIATIONS
■
AUC, area under the curve; BA, bioavailability; CL, clearance;
CYP, cytochrome P450; DMF, N,N-dimethylformamide;
DMSO, dimethylsulfoxide; ED50, median effective dose; ERK,
extracellular signal-regulated kinase; hERG, human ether-a-go-
go related gene; HMBC, heteronuclear multiple bond
correlation spectroscopy; HPCD, 2-hydroxypropyl-β-cyclo-
dextrin; HPLC, high performance liquid chlomatography;
IC50, half maximal inhibitory concentration; LiHMDS, lithium
bis(trimethylsilyl)amide; MEK, mitogen-activated protein
kinase kinase; NBS, N-bromosuccinimide; ND, no data;
PAMPA, parallel artificial membrane permeability assay; PEG,
polyethylene glycol; PK, pharmacokinetics; SD, standard
deviation; t1/2, half-life period; Tf, trifluoromethanesulfonyl;
TGI, tumor growth inhibition; THF, tetrahydrofuran; UV,
ultraviolet
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