The discovery of VX-745: A novel and selective p38α kinase inhibitor

Article abstract of DOI:10.1021/ml2001455

The synthesis of novel, selective, orally active 2,5-disubstituted 6H-pyrimido[1,6-b]pyridazin-6-one p38α inhibitors is described. Application of structural information from enzyme-ligand complexes guided the selection of screening compounds, leading to t

Full text of DOI:10.1021/ml2001455

LETTER
pubs.acs.org/acsmedchemlett
The Discovery of VX-745: A Novel and Selective p38r Kinase Inhibitor
John P. Duffy,*,† Edmund M. Harrington,‡ Francesco G. Salituro,§ John E. Cochran,† Jeremy Green,†
Huai Gao,† Guy W. Bemis,† Ghotas Evindar,|| Vincent P. Galullo,^ Pamella J. Ford,† Ursula A. Germann,†
Keith P. Wilson,# Steven F. Bellon,r Guanging Chen,‡ Paul Taslimi,‡ Peter Jones,† Cassey Huang,†
S. Pazhanisamy,† Yow-Ming Wang,O Mark A. Murcko,† and Michael S.S. Su§
†Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, Massachusetts 02139-4242, United States
S Supporting Information
b
ABSTRACT: The synthesis of novel, selective, orally active 2,5-disubstitut-
ed 6H-pyrimido[1,6-b]pyridazin-6-one p38R inhibitors is described. Appli-
cation of structural information from enzymeꢀligand complexes guided the
selection of screening compounds, leading to the identification of a novel
class of p38R inhibitors containing a previously unreported bicyclic hetero-
cycle core. Advancing the SAR of this series led to the eventual discovery
of 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]
pyridazin-6-one (VX-745). VX-745 displays excellent enzyme activity and
selectivity, has a favorable pharmacokinetic profile, and demonstrates good
in vivo activity in models of inflammation.
KEYWORDS: p38 inhibitor, VX-745, inflammatory diseases
ammalian mitogen-activated protein (MAP) kinases are
serine/threonine kinases that mediate pro-inflammatory
inhibit the closely related p38γ or p38δ kinases or other MAP
kinase family members such as the extracellular-signal regulated
kinases (ERK) and most isoforms of the c-Jun N-terminal kinases
(JNKs).16ꢀ18
Compound 2 has been shown to bind in the ATP binding site
of p38R and is a selective and potent inhibitor with a Ki of
60 nM.19 However, it has been previously reported that the
presence of the pyridyl moiety in 1 and 2 leads to significant
inhibition of hepatic cytochrome p450 isozymes in vitro,20
rendering these compounds unsuitable for development in the
treatment of chronic disease.
Analysis of our previously reported crystal structure of
unphosphorylated p38R MAP kinase bound to 221 facilitated
the early design and eventual discovery of novel p38 inhibitors.
We utilized virtual screening and a diverse selection of shape
similarity methods to search commercially available compound
databases for molecules with similar configuration, yet different
chemical connectivity when compared to 2. The selected com-
pounds were screened for p38 inhibition, and based on this
approach, we identified several inhibitors of p38R in the range of
IC50 = 1ꢀ30 μM. We report here the optimization of one
pyridazine-containing class of inhibitors, leading to the identifi-
cation of VX-745 (3), a first-generation p38 inhibitor clinical
candidate.
M
cytokine biosynthesis at the transcriptional and translational
level.1ꢀ3 Tumor necrosis factor R (TNFR) and interleukin-1β
(IL-1β) are cytokines involved in various cellular functions and,
when present in elevated levels, are associated with the patho-
genesis of a variety of inflammatory diseases, including rheuma-
toid arthritis (RA).4 It is known that small molecule inhibitors
of p38R MAP kinase (i.e., inhibitors of the p38R and p38β
isoforms) suppress the production of TNFR and IL-1β in vitro
and in animal models,5ꢀ8 suggesting that the stress-activated
signal transduction pathway leading to these cytokines is at least
partially regulated by p38. The clinical success in the treatment
of RA patients by targeting TNFR mediated inflammatory
pathways using, for example, the soluble TNFR receptor fusion
protein etanercept (Enbrel) and the monoclonal anti-TNFR
products infliximab (Remicade) and adalimumab (Humira)
provides the rationale of targeting these cytokines for therapeutic
effect.9ꢀ14 Thus, novel orally administered small molecules that
selectively inhibit p38 would be desirable to provide thera-
peutic intervention for inflammatory disorders and to more
clearly define the physiological role of this protein kinase-
mediated pathway.
First disclosed in 1994, pyridinylimidazole compounds, ex-
emplified by SB 203580 (1) and VRT-19911 (2) (see Figure 1),
block the production of TNFR and IL-1β from monocytes
stimulated by bacterial lipopolysaccharides (LPS) through the
inhibition of p38 MAP kinase.2 Compound 1 has been shown to
be effective in animal models of arthritis, bone resorption, and
endotoxin shock.15 Compound 1 is a selective inhibitor of p38R
and p38β (IC50 = 0.1 and 0.43 μM respectively, but does not
Structure 4 (see Figure 2), a commercially procured sample,
emerged as a p38R enzyme inhibitor screening hit22 selected for
further evaluation. In an attempt to resynthesize 4, we performed
Received: June 15, 2011
Accepted: July 28, 2011
Published: July 28, 2011
r
2011 American Chemical Society
758
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|

ACS Medicinal Chemistry Letters
LETTER
Figure 1. Inhibitors of p38 MAP kinase.
blood mononuclear cells (PBMCs). For other 2,6-disubstituted
systems examined (19, 20), potency was approximately 10-fold
weaker than that of 21.
With 21 representing optimal substitution of the 5-aryl ring,
we focused on the S-linked phenyl ring at the pyrimido-
[1,6-b]pyridazin-6-one 2-position (Table 2) using commercially
available thiophenols. Addition of a 4-fluoro group, exemplified
by 23, resulted in a slight improvement in enzyme and cellular
activity versus the corresponding unsubstituted thiophenyl ana-
logue 21 (Table 1). Small alkyl groups or halogens at the meta
position resulted in a reduction of affinity, as in 24, 25, 26, 30, 31,
and 32 without regard to the nature of the substituent at the para
position. Small polar functionalities at the ortho position were
tolerated, as in 29 and 33. However, the most significant
improvement was observed with a 2,4-dihalo substitution of
the thiophenol. The 2-chloro-4-fluoro compound 34 showed
excellent potency, while the 2,4-difluoro exhibited the best
potency, resulting in 3. As before, cellular data tracked enzyme
inhibition. Compound 3 exhibits PBMC IL-1β and TNFR IC50
values of 45 and 51 nM, respectively. Compound 3 is also
effective in whole blood, blocking IL-1β and TNFR release with
IC50 values of 150 and 180 nM, respectively. Conversely,
compound 3 does not affect proliferation of phytohemagluti-
nin-stimulated PBMCs up to 20 μM, indicating cellular selec-
tivity for p38 versus other kinases.
The X-ray cocomplex of 3 and p38R at 2.4 Å resolution
(Figure 3) shows favorable van der Waals interactions that
stabilize the interaction of the 2,6-dichloro phenyl ring with
p38.25 Each chlorine atom occupies a hydrophobic pocket with
contact to residues V30, L108, A157, and L167. The phenyl ring
itself makes favorable van der Waals interactions with backbone
residue atoms G110, A111, and D112. The para position of the
ring faces bulk solvent. The 2,4-difluorophenyl ring of 3 occupies
a hydrophobic pocket at the gatekeeper (T106) residue and
makes extensive van der Waals contacts. In addition, the enzyme
facilitates the binding of 3 by flipping the G110 backbone.26 This
flip is possible because glycine lacks an R-carbon substitution.
This unique enzyme conformation allows the carbonyl oxygen of
3 to accept an additional H-bond from the p38 backbone. This
combination of interactions is a key determinant of enzyme
specificity.19,21
Figure 2. Structure of screening hit, as reported by vendor.
the synthesis outlined in Scheme 1.23 2,4-Dichlorophenylaceto-
nitrile (5) was treated with sodium tert-butoxide followed by
3,6-dichloropyridizine to give the corresponding pyridazinyl-
phenylacetonitrile (6). Displacement of the second chloro group
with phenylthiosodium gave the 3,6-disubstituted pyridazine (7).
Compound 7 was subsequently hydrated to amide 8 using
concentrated sulfuric acid. The attempted synthesis of 4 from
8 using DMFꢀDMA in toluene at 100 ꢀC did not result in the
desired product but afforded a product in which the N,N-
1
dimethyl group was absent from the H NMR spectrum. This
was confirmed by mass spectrometry, which showed m/z = 400
(M + H), not 445 as might be expected, indicating the loss
of dimethylamine. We hypothesized the in situ formation of
compound 4, and tautomerization of the R-pyridazine ring
system to compound 9 allowed for nucleophilic collapse of the
pyridazine ring nitrogen on the ethanamide carbon to afford
compound 10.
Small molecule X-ray crystallography24 of a closely related
analogue (23), also prepared via Scheme 1, confirmed the
structure we pursued was not 4, as assigned by the vendor, but 5-(2,4-
dichlorophenyl)-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-
one (10). The core bicyclic system, 6H-pyrimido[1,6-b]pyridazin-6-
one, was, at this time, unreported in the literature.
Having developed a practical synthetic route, we initiated
exploration of the SAR of the two pendant aryl rings. Initial
studies focused on the substitutions of the directly attached
phenyl ring (5-position substituent of pyrimido[1,6-b]pyridazin-
6-one: Table 1), prepared from commercially available phenyla-
cetonitriles, by the same methods shown in Scheme 1. Evaluation
of the products 10ꢀ21 (Table 1) indicated that the unsubsti-
tuted phenyl (11) and the 4-substituted systems 16, 17, and 18
showed poor enzyme inhibitory activity. Introduction of a
2-substituent (12ꢀ15) showed modest potency, similar to the
screening hit 10, suggesting that, of the two substituents, the
2-chloro is the key to enzyme activity. The 2,6-dichloro substitu-
tion emerged as optimal, as indicated by compound 21, which
also demonstrated corresponding inhibition of IL-1β and TNFR
release from lipopolysaccharide (LPS) treated human peripheral
The lead compound, 3, was tested against the different p38
isozymes and closely related MAP kinases (ERK, JNK) as well as
a panel of 50 other kinases to examine its selectivity profile.
Compound 3 showed a promising selectivity profile, with 20-fold
selectivity for p38R over p38β (Ki = 220 nM),27 and no
significant inhibition of other MAP kinases (with the excep-
tion of MKK6, itself an activator of p38)28 or any of the other
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ACS Medicinal Chemistry Letters
LETTER
Scheme 1a
a Reagents and conditions: (a) 3,6-dichloropyridazine, NaOtBu, THF, 15ꢀ54%; (b) thiophenol, K2CO3, THF, reflux, 40ꢀ92%; (c) cH2SO4, 100 ꢀC,
45ꢀ100%; (d) DMFꢀDMA, 100 ꢀC, 41%.
Table 1. Inhibition of p38r and PBMC Cytokine Release by 5-Aryl-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-ones
compd
R1
Cl
R2
R3
R4
Cl
p38R IC50 (μM)a
PBMC IL-1β IC50b (μM)
PBMC TNFR IC50b (μM)
10
11
12
13
14
15
16
17
18
19
20
21
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
Cl
H
H
H
5
20
20
H
H
>20
4.4
12
NDc
3.5
2.8
2.1
5.0
15
ND
9.7
Me
OMe
CF3
Cl
H
H
>20
>20
19.9
>20
>20
>20
3.1
H
11
H
4.1
>20
>20
>20
1.7
3.5
0.25
H
F
H
OMe
Cl
H
20
H
>20
1.0
3.7
0.40
CF3
F
F
H
15.3
0.46
Cl
Cl
H
a Assayed according to ref 22. b See Supporting Information for assay details. c ND = not determined.
50 kinases tested at 2 μM concentration (see Supporting
Information). Similar selectivity results have been reported
elsewhere.29
The pharmacokinetic parameters obtained for 3 in three
species are summarized in Table 3. Systemic clearance was
slightly higher than hepatic blood flow for rat and dog, and 3
appears to have significant extravascular distribution in all
three species studied, as indicated by the volume of distribu-
tion at steady state. The oral pharmacokinetic profile of 3 in
TPGS/PEG-400/water (2:7:1) was characterized in male BALB/c
mice, SpragueꢀDawley rats, and beagle dogs. The results of
these single dose studies showed that 3 has excellent bioavail-
ability in all three species (87%, 56%, and 69%, respectively).
Compound 3 demonstrated a longer half-life following oral
administration as compared to IV administration, suggesting an
absorption-rate limited elimination process in the three species.
Compound 3 is neither a significant inhibitor nor an inducer
of human hepatic cytochrome p450 isozymes. The IC50
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LETTER
Table 2. Inhibition of p38r and PBMC Cytokine Release by 5-(2,6-Dichlorophenyl)-2-(arylthio)-6H-pyrimido[1,6-b]pyridazin-
6-ones
compd
R5
R6
R7
p38R IC50 (μM)
PBMC IL-1β IC50 (μM)
PBMC TNFR IC50 (μM)
21
22
23
24
25
26
27
28
29
30
31
32
33
34
3
H
H
H
Me
F
0.25
1.4
0.40
0.37
0.099
0.44
0.35
0.21
0.98
0.33
0.14
0.97
2.4
0.45
0.87
0.293
0.72
0.90
0.28
2.8
H
H
H
H
0.123
0.45
0.28
1.4
H
F
H
H
H
H
H
H
Cl
Me
F
H
Cl
Me
H
H
Et
0.75
0.20
0.36
0.8
Me
OH
H
H
3.7
H
0.49
1.2
Cl
Me
Cl
H
H
1.9
3.5
H
0.5
0.18
0.28
0.053
0.045
0.18
0.69
0.48
1.2
NH2
Cl
F
F
0.22
0.036
0.009
0.18
0.26
H
F
0.275
0.051
0.16
0.32
H
F
35
36
Me
Me
H
Cl
Me
H
Table 3. Pharmacokinetic Properties of 3
parameter
rat
mouse
dog
IV dose (mg/kg)
n
4.8
3
2
13.8
4
3
T1/2 (h)
1.9
3.9
1.26
63
2.6
2.2
1.54
21
1.5
2.3
6.12
39
Vss (L/kg)
AUC (μg h/mL)
3
Cl (mL/min/kg)
PO dose (mg/kg)
n
43
40
33
3
3
4
T1/2 (h)
4.5
0.89
6.3
56
4.5
7.85
26.96
87
4.5
1.37
4.34
69
Cmax (μg/mL)
Figure 3. Structure of 3 bound to p38.
AUC (μg h/mL)
3
F (%)
values for the inhibition of CYPs 3A4, 2D6, 2C19, 2C9, and 1A2
were determined to be >40 μM. The fraction of 3 bound to
plasma protein was 98% and 92% in the rat and the dog,
respectively.
To evaluate the in vivo therapeutic potential of compound 3,
we employed the murine collagen-induced arthritis model of
human rheumatoid arthritis, in which DBA/1J mice were im-
munized twice with chick type II collagen. Treatment was
initiated once an inflammation score of 2 in each paw had been
reached, corresponding to focal swelling of the wrist joint.
Results showed that mice treated with 2.5, 5, and 10 mg/kg of
compound 3 twice daily for 20 days had 27%, 31%, and 44%
improvement in the inflammatory scores, respectively, when
compared to vehicle-treated mice at the end of treatment
(Table 4; see Supporting Information for details). In addition,
histological scores showed a 32ꢀ39% protection of bone and
cartilage erosion.
Compound 3 (VX-745) and other analogues described in
this study represent the first known examples of 5-phenyl-
2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-ones as p38
inhibitors. These compounds show potent inhibition of the key
inflammatory mediators, IL-1β and TNFR, production in vitro in
isolated PBMCs and whole blood. Compound 3 has also been
shown to demonstrate anti-inflammatory efficacy in an animal
model of rheumatoid arthritis and was designated for further
development.30
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LETTER
Table 4. Therapeutic Effect of 3 in Type II Murine Collagen-Induced Arthritis (CIA) Model
clinical score on paw inflammationa
histology scoreb
SEM
dose
meana
SEM
% inh
meanb
% inh
vehicle (100% polyethylene glycol)
compound 3, 2.5 mg/kg
compound 3, 5 mg/kg
3.81
2.77
2.65
2.13
0.13
0.17
0.28
0.13
NA
27
2.04
1.33
1.38
1.25
0.09
0.09
0.11
0.09
NA
35
31
32
compound 3, 10 mg/kg
44
39
a Clinical scores measured on final day 20. Clinical scores are defined in the Supporting Information. b Histology scores are defined in the Supporting
Information.
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Supporting Information. Full experimental details for
b
representative compounds synthesized, HRMS results, descrip-
tion of assays, animal efficacy studies, and X-ray crystallographic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: 617 444 6813. Fax: 617 444 7827. E-mail address:
john_duffy@vrtx.com.
Present Addresses
‡Novartis, 250 Massachusetts Avenue, Cambridge, MA 02139.
§Agios Pharmaceuticals, 38 Sidney Street, Cambridge, MA 02139.
GlaxoSmithKline, 830 Winter Street, Waltham, MA 02451.
^AstraZeneca, 35 Gatehouse Drive, Waltham, MA 02451.
#Takeda, 10410 Science Center Drive, San Diego, CA 92121.
rConstellation Pharmaceuticals, 215 First Street, Suite 200,
Cambridge, MA 02142.
OAmgen, 1 Amgen Center Drive, Thousand Oaks, CA 91320.
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’ ACKNOWLEDGMENT
We thank Dr. Mariusz Krawiec for X-ray crystallographic
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support, Dr. Patricia McCaffrey and Dr. Karyn Cepek for cellular
assays support, and Dr. George Ku for in vivo pharmacology
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(27) Although p38β is homologous to p38R in both primary
sequence and tertiary structure, conformational differences in the ATP
binding site lead to different selectivities of small molecule inhibitors.
Patel, S. B.; Cameron, P. M.; O’Keefe, S. J.; Frantz-Wattley, B.;
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(28) Remy, G.; Risco, A. M.; I~nesta-Vaquera, F. A.; Gonzꢀalez-Terꢀan,
B.; Sabio, G.; Davis, R. J.; Cuenda, A. Differential activation of p38
MAPK isoforms by MKK6 and MKK3. Cell. Signalling 2010, 22, 660–667.
(29) Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.;
Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen,
P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.;
Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lꢀelias, J.-M.; Mehta,
S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.;
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(30) Compound 3 (VX-745) attained clinical proof-of-principle
correlating inhibition of p38 with anti-inflammatory effect, but clinical
development was suspended following adverse effects findings in non-
clinical studies. http://www.prnewswire.com/news-releases/vertex-moves-
to-re-allocate-resources-from-vx-745-in-p38-map-kinase-program-to-
accelerate-development-of-second-generation-drug-candidates-vx-702-
and-vx-850-72104487.html.
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