Imidazopyridazine hepatitis C virus polymerase inhibitors. Structure-activity relationship studies and the discovery of a novel, traceless prodrug mechanism

Article abstract of DOI:10.1021/jm401337x

By reducing the basicity of the core heterocycle in a series of HCV NS5B inhibitors, the hERG liability was reduced. The SAR was then systematically explored in order to increase solubility and enable dose escalation while retaining potency. During this e

Full text of DOI:10.1021/jm401337x

Article
pubs.acs.org/jmc
Imidazopyridazine Hepatitis C Virus Polymerase Inhibitors.
Structure−Activity Relationship Studies and the Discovery of a
Novel, Traceless Prodrug Mechanism
Martin Leivers,*,† John F. Miller,† Stephanie A. Chan,† Ryan Lauchli,† Sebastian Liehr,†,∥ Wenyan Mo,†,§
Tony Ton,†,⊥ Elizabeth M. Turner,† Michael Youngman,†,# J. Greg Falls,† Susan Long,†
Amanda Mathis,†,‡ and Jill Walker†
†GlaxoSmithKline Research & Development, 5 Moore Drive, Research Triangle Park, North Carolina 27709, United States
‡Nonclinical and Clinical Pharmacology, Salix Pharmaceuticals, Inc., 8510 Colonnade Center Drive, Raleigh, North Carolina 27615,
United States
§ChemPartner Co., 998 Halei Road, Shanghai 201203, China
∥Santen Inc., 2100 Powell Street, Suite 1600, Emeryville, California 94608, United States
⊥Principia Biopharma, Inc., 400 East Jamie Court, Suite 302, South San Francisco, California 94080, United States
#Lieber Institute For Brain Development, 855 North Wolfe Street, Suite 300, Baltimore, Maryland 21205, United States
S
* Supporting Information
ABSTRACT: By reducing the basicity of the core heterocycle in a series of HCV NS5B inhibitors, the hERG liability was
reduced. The SAR was then systematically explored in order to increase solubility and enable dose escalation while retaining
potency. During this exploration, a facile decarboxylation was noted and was exploited as a novel prodrug mechanism. The
synthesis and characterization of these prodrugs and their utilization in chronic toxicity studies are presented.
combined with the large number of virions produced per day,
results in a mutation frequency that is very high. It is estimated
that in an infected individual, every single point mutation is
present as a HCV quasispecies.11 Thus the virus can escape
selection pressure by a direct acting antiviral relatively easily,
resulting in viral rebound and treatment failure.12−15 This has
been seen in clinical trials where resistant mutations to DAAs
were found with monotherapy even after 1 day of treatment.16
As a result, a combination of agents with different antiviral
mechanisms and different resistant mutations is required for
effective treatment.17
Recently, a novel DAA from Gilead Sciences, compound 1
(GS-9190, Tegobuvir) has been disclosed with attractive
antiviral activity in clinical trials against HCV (Figure 1).18
Compounds in this class have been shown to bind to an
allosteric site in the NS5B polymerase. Interestingly, although
the compounds select for resistance in the NS5B polymerase,
they do not show activity in the isolated polymerase enzyme
assay. This class of compounds only shows activity when tested
INTRODUCTION
■
The hepatitis C virus (HCV) currently infects an estimated
270−300 million people worldwide.1−3 The virus is normally
transmitted by blood-to-blood contact and progresses to a
chronic infection in ∼85% of those acutely infected. The
disease is normally asymptomatic for years but eventually can
progress to liver fibrosis and cirrhosis and is the leading cause
of liver cancer.4 The advent of the subgenomic HCV replicon
model enabled compounds to be screened and developed that
function as direct acting antivirals (DAA).5 Boceprevir and
Telaprevir are the first approved drugs developed using this
approach, and these compounds are active site inhibitors of the
HCV protease NS3.6,7 The current standard of care consists of
pegylated interferon-α and ribavirin in combination with a
protease inhibitor and is only ∼70% effective in the prevailing
genotype in the U.S., which is genotype 1.8 Although the
addition of the protease inhibitors increased the effectiveness of
the treatment, there is still need for new therapies to improve
the cure rate and to address the needs of the many patients who
cannot tolerate the side effects of the standard 24−48 week
treatment.9,10
Special Issue: HCV Therapies
HCV replication results in quasispecies of viral RNAs due to
the low fidelity polymerase encoded by the virus. This fact,
Received: August 30, 2013
Published: November 13, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of Compounds 6 and 7
Figure 1. Structure of compound 1.
in the subgenomic replicon system with a hepatocyte as the
host cell. This apparent contradiction was explained by
researchers at Gilead who elegantly elucidated the mechanism
of action. It was found that these inhibitors were first activated
by epoxidation of the D-ring with CYP 1A, reacting with
glutathione and subsequently inhibiting the NS5B through
covalent modification. In the initial phase I trials, the
compound was shown to cause QT prolongation and this
limited the dosing levels.19 Inhibition of the hERG ion channel
is known to result in QT prolongation. We hypothesized that
the hERG inhibition could be reduced through structural
modifications that would allow higher doses be used in the
clinic, thereby realizing full potential of this mechanism. Herein
we describe SAR studies directed toward removing the hERG
liability associated with 1. In addition, the discovery of a novel,
traceless prodrug mechanism is discussed.
a
Reagents and conditions: (a) 2.3 equiv 50% aqueous hydroxylamine,
EtOH; (b) 2.0 equiv NCS or NaOCl, 1.0 equiv propargyl chloride,
DCM; (c) 1.0 equiv diaminomaleonitrile, H2SO4 (cat.), THF, RT, 90
min; (d) 1.5 equiv NCS, 1.5 equiv nicotinamide, DMF, RT, 1 h; (e)
6.5 equiv DIBAL-H, THF, −78 °C, then 3.0 equiv hydrazine hydrate;
(f) 3.0 equiv Cs2CO3, DMF, 120 °C, 10 min.
DISCOVERY AND PROFILING OF LEAD
■
COMPOUND 7
C-Ring SAR. A classic hERG pharmacophore consists of a
basic group flanked by hydrophobic groups.20 The C-ring of 1
is calculated to have a pKa of 6.6 and is flanked by two
hydrophobic arms (A- and D- rings). Several strategies have
proven successful in eliminating hERG inhibition. These
include steric blocking, addition of acidic functionality, and
reducing basicity.21 Our preliminary SAR work focused on
replacing the C-ring of 1 with less basic moieties in
combination with alternate A- and B-rings. These efforts led
to the isoxazole derivative 6 (Scheme 1), which showed an
EC50 of 0.17 μM in the genotype 1b replicon assay. A focused
scan of A- and D-ring substitutions resulted in compound 7,
which showed a nearly 10-fold improvement in 1b replicon
activity with respect to 6, comparable to 1 (0.005 μM in
genotype 1b). The synthesis of 6 and 7 (Scheme 1) is based on
the penultimate alkylation of the chloromethyl AB fragment 3
with the CD imidazopyridazine 5. The alkylation reaction was
found to be regioselective with <1% alkylation on the imidazole
nitrogen.
Biological Profiling of Lead Compound 7. Because of its
promising 1b replicon activity, compound 7 was examined in
several additional biological assays. In addition to its potent
activity against HCV genotype 1a and 1b replicons, it showed
no toxicity in three diverse cell lines (Table 1). Compound 7
was found to be highly protein bound (99.8%) and was inactive
in both HCV NS5B and NS3 enzyme assays. Importantly,
testing for inhibition of the hERG channel showed a marked
improvement over 1 or the close structural analogue 8 (Table
2). It can be seen that from the calculated pKa values that, as
initially hypothesized, attenuation of the C-ring basicity
correlates to a reduction in the hERG liability in this series.
Compound 7 was progressed to pharmacokinetic assessment
in both rat and dog (Table 3). After oral dosing in rat at 1 mg/
kg, the compound showed low clearance and high %F. In dog at
1 mg/kg oral dosing, 7 again displayed low clearance but
delivered a %F significantly lower than in rat. It was
hypothesized that the higher pH of the dog stomach resulted
in poorer solubility of the compound, leading to attenuated
absorption and lower systemic exposures.
In anticipation of toxicology studies, a dose escalation study
was performed in rats with an optimized solution formulation
with doses up to 300 mg/kg. Results were disappointing, with
maximal exposure seen at 100 mg/kg (Table 4). To understand
this result, solubility measurements were performed on
compound 7. The solubility was found to be quite low
(<0.01 μg/mL at pH 6.8). The poor aqueous solubility is likely
the primary factor in limiting the maximal exposures observed
in these studies. We then turned our attention toward further
structural diversification with a particular emphasis on
improving aqueous solubility.
SAR DIVERSIFICATION IN THE
IMIDAZOPYRIDAZINE SERIES
■
A-Ring SAR. With compound 7 as a starting point, we first
explored modifications of the A-ring (Table 5). Synthesis of
these analogues was analogous to that of 7 (Scheme 1). The
requisite A-ring aldehyde was converted to an oxime (2),
oxidized to a nitrile oxide by treatment with NCS or
hypochlorite, and then cyclized with propargyl chloride to
give the chloromethyl AB fragment (3). This was then coupled
to the CD imidazopyridazine (5) to generate the final products.
Because high hydrophobicity and low aqueous solubility were
problems with this series, we replaced the phenyl A-ring with
isosteric nitrogen heterocycles (9−11); however, this resulted
in generally poor activity. Substitution at the 2- and 4-positions
of the A-ring generated analogues with the best activities, with
alkyl and alkoxy groups being preferred (12−16). As no groups
bestowed both acceptable solubility and good potency, the 2,4-
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Table 1. In Vitro Profiling of Compound 7
a
assay results (μM)
b
b
EC50 (1a) (Huh 7) EC50 (1b) (Huh 7) CC50 (Huh 7) CC50 (MT4) CC50 (HEPG2) human PPB (%) NS5B enzyme IC50 NS3 enzyme IC50
0.122
0.016
>50
>50
>50
99.8
>50
>50
a
b
Cell-based HCV activity (genotypes 1a and 1b) measured in the subgenomic replicon system. Enzyme assays were conducted on genotype 1b
sequences.
Table 2. Comparison of Calculated Basicity and Inhibition of hERG Current for Different C-Ring Derivatives
a
b
For description of the assays, see Supporting Information. pKa values calculated from quantum mechanical protonation energies for model
c
compounds that include the B, C, and D rings, excluding the A ring. See Supporting Information for details. From ref 30.
a
b
Table 3. In Vitro and in Vivo Pharmacokinetic Profiling of Compound 7 in Rat and Dog
microsomal clearance
CLint
species
(mL/min/kg)
relative to standards IV in vivo CL (mL/min/kg) IV volume of distribution (L/kg) IV t1/2 (h) PO AUC (h·μg/mL) PO %F
rat
<10
<10
low
low
3
0.57
0.83
3.1
6.9
6.3
2.0
109
18.8
dog
2.7
a
b
Rat administered 0.5 mg/kg IV and 1 mg/kg PO. Dog administered 1 mg/kg IV and PO.
a
B-Ring SAR. To further improve potency and/or aqueous
solubility, a number of B-ring modifications were explored in
combination with several different A-rings that were known to
be active. Compounds 25−27, 29, 34, and 35 (Table 7) were
prepared by alkylation of imidazopyridazine intermediate 5b
with various AB mesylates (Scheme 3, see Supporting
Information for mesylate syntheses). Compounds 28, 31, and
33 were prepared via routes shown in Scheme 3. The reversed
isoxazole derivative 25 retained activity but was unlikely to
solve the solubility problem associated with 7. Replacing the
isoxazole with the more polar imidazole ring (26) resulted in a
loss of potency. Replacing the aryl B ring with an aliphatic
tether (27) also resulted in a loss of activity. Moving to 6-
membered ring, aryl and heteroaryl replacements retained
activity as exemplified by compounds 28, 29, 31, and 33−35.
The pyridine derivative 31 was chosen for further profiling.
The potency was similar to that of the initial lead 7, but the
intrinsic solubility was improved. Even in neutral aqueous
media (pH 6.8), solubility was measured at 9 μg/mL.
Progression to low dose rat studies demonstrated a favorable
PK profile with low clearance and high bioavailability (Table 8).
Dose escalation in the rat was then performed (Table 9). The
compound was administered as a suspension in carboxyme-
thylcellulose. At increasing dosages, we saw increasing
exposures, with an AUC of 280 μg·h/mL at the top dose
tested (300 mg/kg). However, testing in the hERG patch-
clamp assay showed that 31 inhibited the hERG channel at a
pEC50 of 5.1−6 . Introduction of the basic pyridine23 moiety
Table 4. Dose Escalation of Compound 7 in Rat
dose (mg/kg)
Cmax (μg/mL)
AUC (h·μg/mL)
10
30
1.9
7.2
7.4
2.9
25
98
100
300
102
31
a
Formulated as the free base in 2% NMP, 2.5% Tween 80, 25% PEG
400, 70.5% 0.1N HCl in H2O, pH 2−3.
bis(trifluoromethyl)phenyl A-ring in 7 was retained and other
parts of the molecule were explored.
D-Ring SAR. The D-ring was varied using either the
chloromethyl AB alkylation protocol (Scheme 1) for
compound 18 or a Suzuki-based approach (Scheme 2) for
compounds 22−24. The SAR at this position was found to be
steep, with minor changes in structure resulting in large losses
in potency (Table 6). The 2-fluoro derivative (18) retained
some replicon activity characteristic of lead compound 7, while
the closely related 3-fluoro and 2,6-difluoro analogues (22,23)
showed a complete loss of activity. At the time of this work, the
MOA associated with 1 was not known but has recently been
shown to be due to irreversible, covalent inhibition via
metabolic activation of the D-ring.18 In retrospect, the steep
SAR seen in the D-ring can be explained by the requirement of
metabolic activation at this site. Interestingly, analogues bearing
groups known for their metabolic instability, such as the
thiophene derivative 24, did show a modest level of activity.22
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Table 5. A-Ring Analogues
Table 6. D-Ring Analogues
Table 7. B-Ring Analogues
a
Scheme 2. Synthesis of D-Ring Analogues 22−24
a
Reagents and conditions: (a) 4.9 equiv DIBAL-H THF, −78 °C, then
3.0 equiv hydrazine hydrate, 17%; (b) 3.0 equiv K2CO3, DMF 40 °C, 1
h, 68%; (c) 1.3 equiv ArB(OH)2, Pd(Ph3P)4, 3.0 equiv K2CO3, iPrOH,
microwave, 120 °C.
into the B-ring position of our scaffold solved our solubility and
dose escalation problems but reintroduced an unacceptable
hERG liability by reintroducing the hERG pharmacophore
(albeit at a different site).
Linker SAR. The only remaining part of the molecule left
unexplored was the linker carbon between the B and C-rings.
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a
Scheme 3. Synthesis of B-Ring Analogues
Table 9. Dose Escalation of 31 in Rat
dose (mg/kg)
Cmax (μg/mL)
AUC (h·μg/mL)
5
10
1.1
2.7
8
24
30
2.8
24
100
300
7.6
69
27.3
280
a
Scheme 4. Synthesis of Linker Analogues 37 and 39
a
Reagents and conditions: (a) 3.0 equiv K2CO3, DMF, 80 °C; (b) 1.0
equiv 4-bromobenzyl bromide, 2.0 equiv K2CO3, DMF, 80 °C; (c) 1.5
equiv 2,4-bis(trifluoromethyl)phenylboronic acid, Pd(Ph3P)4, 2.0
equiv Na2CO3, H2O, PhMe, 100 °C; (d) Pd(Ph3P)4, THF, microwave,
130 °C, 16%; (e) 1.5 equiv NBS, 0.17 equiv benzoyl peroxide CCl4, 80
°C; (f) 5b, 1.5 equiv K2CO3, DMF, 45 °C, 14%; (g) 0.40 equiv
trichloroisocyanuric acid, CCl4, 60 °C, 95%; (h) 5b, 1.7 equiv K2CO3,
DMF, 80 °C, 25%; (i) 1.0 equiv 4-propoxy-2-(trifluoromethyl)-
phenyl)boronic acid pinacol ester, Pd(Ph3P)4, 3.0 equiv Na2CO3,
dioxane, H2O, 120 °C.
a
Reagents and conditions: (a) NaOCl, DCM, reflux; (b) 3.9 equiv
NaBH4, EtOH, 66%; (c) 1.4 equiv MsCl, 1.9 equiv DIPEA, DCM; (d)
5b, 2.0 equiv K2CO3, DMF, 80 °C; (e) 5b, 5.0 equiv K2CO3, DMF,
110 °C.
The racemic methyl analogue 37 and gem-dimethyl derivative
39 were prepared as described in Scheme 4. Both syntheses
were based on dipolar cycloadditions with the appropriately
functionalized alkynes. The penultimate alkylation reaction of
the more sterically hindered gem-dimethyl chloride leading to
39 required both higher temperature and a longer reaction
time.
Table 10. Linker Analogues
Replicon data for the linker substituted analogues is shown in
Table 10. Monosubstitution on the linker was well tolerated,
affording 37 which showed a small drop in potency (5−7×)
relative to the unsubstituted variant 7. The loss of activity
observed for 39 clearly shows that disubstitution is deleterious.
Given that monosubstitution was tolerated, we decided to
prepare methyl ester 42 (Scheme 5) as a potential intermediate
for the attachment of solubility enhancing groups to the linker
methylene. The potency of 42 was remarkably similar to that of
the unsubstituted inhibitor 16. Intriguingly, all attempts to
saponify the ester to generate the corresponding free acid
resulted in decarboxylation to 16. We then considered the
possibility that the ester group could be hydrolyzing and
decarboxylating under the assay conditions thus liberating the
unsubstituted parent compound, accounting for the similarity
in potencies between 42 and 16 (Scheme 6).
DISCOVERY OF TRACELESS ESTER PRODRUGS
We hypothesized that the methyl ester’s activity in the HCV
replicon system was due to conversion to the corresponding
■
a
Table 8. In Vitro and in Vivo Pharmacokinetic Profiling of Compound 31 in Rat
microsomal clearance
CLint (mL/min/kg) relative to standards IV in vivo CL (mL/min/kg) IV volume of distribution (L/kg) IV t1/2 (h) PO AUC (h·μg/mL) PO %F
22
medium
9
2.04
3.14
1.5
78
a
Rat administered 0.5 mg/kg IV and 1 mg/kg PO.
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a
exposure of the corresponding parent 16 was high. Remarkably,
the exposure of the parent was higher when dosed as the
methyl ester than when dosed as the parent itself at equivalent
dose levels (Figure 2).
Scheme 5. Synthesis of Methyl Ester 42
We sought to exploit these findings by incorporating ester R
groups that would confer improved aqueous solubilities and
hopefully even higher systemic exposures of parent compound.
If successful, this approach would constitute a traceless prodrug
mechanism at a carbon−hydrogen bond, and would be, to our
knowledge, the first example of such an approach.24−27
For this prodrug mechanism to be operative, decarboxylation
of the intermediate free acid must be facile under physiological
conditions. It is unlikely that this strategy could be exploited to
many systems due to the relative stability of most carboxylic
acids. Simply substituting off of a diaryl substituted methylene
is not sufficient. The prototypical compound diphenylacetic
acid is stable at its melting point of 144−145 °C.28 Additional
electron withdrawing groups will likely be required in order for
this mechanism to be operative.
To avoid linear syntheses for each ester prodrug analogue, a
divergent synthesis based on a late stage intermediate was
desirable. It was found that under basic conditions, 42 could be
readily transesterified in the presence of an excess of the desired
alcohol (Scheme 7). In this manner, a number of prodrugs were
prepared. They were ranked by exposure of the corresponding
parent in low dose rat and dog studies as well as a high dose of
300 mg/kg as a suspension in rat studies. For ease of
downstream formulation and manufacturing, only crystalline
derivatives were progressed into animal studies. The two
alcohol derivatives 43 and 44 (Scheme 7 and Table 12)
emerged as frontrunners from this triage, with the diethylene
glycol ester 44 being chosen for further progression.
a
Reagents and conditions: (a) 2.0 equiv 50% aqueous NH2OH, EtOH,
84%; (b) 1.05 equiv NCS, DMF, 0 °C; (c) 1.5 equiv 3-butyn-1-ol,
TEA, 1,2-DCE, reflux, 95% for 2 steps; (d) 2.2 equiv periodic acid, cat.
PCC, MeCN, 0 °C to RT, 95%; (e) SOCl2, MeOH, RT, 2 h, 99%; (f)
2.2 equiv NBS, 0.10 equiv AIBN, CCl4, reflux, 5 h, 57%; (g) 5b, 3.3
equiv K2CO3, 0 °C to RT, 54%.
Scheme 6. Proposed Mechanism for Conversion of 42 to 16
No hERG inhibition was observed for the parent 16,
presumably due to the absence of basic groups in the scaffold.
Systemic exposure of the intact prodrugs were <1% that of the
corresponding parent. Installation of the prodrug moiety
conferred ∼10× greater solubility, leading to high exposures
in both rat and dog. Because the prodrug 44 delivered the
highest AUC of the parent in high dose rat profiling, it was
progressed into chronic toxicity studies. The results for the 7
day rat toxicology study showed no adverse findings, with
exposures 100× that predicted for clinical efficacy in humans.
Unfortunately, in 7 day dog studies, liver toxicity was seen with
insufficient margins, thereby precluding further development of
this series.
CONCLUSION
■
parent 16. This was corroborated by the detection of 16 in the
supernatant of the replicon assay after treatment with 42. This
conversion of ester to parent was found to take place in
biological media, but the ester was stable in pH 2 or 7 water
(Table 11).
Intrigued by this serendipitous finding, we then examined the
in vivo performance of this compound. In both rat and dog,
after low oral doses, the systemic exposure of 42 was very low
and the compound was rapidly converted. However, the
A series of HCV NS5B inhibitors was discovered that
demonstrated reduced inhibition of the hERG channel through
variation of the core heterocycle. Substitutions around the core
were then explored in order to increase solubility and exposures
required for toxicity studies. During SAR studies of the linker
portion of the scaffold, a facile decarboxylation was noted. This
was exploited as a novel, traceless prodrug mechanism through
a series of ester prodrugs. The utility of these prodrugs was
Table 11. Conversion of Ester Prodrug 42 to Parent 16 in Vitro
compd
pH 2
pH 7
blood
stable
microsomes (rat)
hepatocytes (rat)
stable
16 (parent)
42 (ester)
stable
stable
stable
stable
stable, low clearance
a
a
a
unstable
unstable, high clearance
unstable
a
t1/2 < 15 min.
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Figure 2. Comparison of in vivo exposure of parent (16) when rats (left) or dogs (right) are dosed with parent (pink) or methyl ester prodrug (42,
blue) at 5 mg/kg parent or parent equivalent.
EXPERIMENTAL SECTION
Scheme 7. Synthesis of Prodrugs 43 and 44
■
All commercially obtained solvents and reagents were used as received.
1H NMR spectra were taken on a Varian (Agilent) Inova 400 NMR
spectrometer. Chemical shifts are reported in parts per million (ppm,
δ) using the residual solvent line as a reference. Splitting patterns are
designated using the following abbreviations: s, singlet; d, doublet; t,
triplet; dd, doublet of doublet; m, multiplet; br, broad. Coupling
constants (J) are reported in hertz (Hz). Mass spectrometric analyses
and compound purity determinations were conducted on a Waters
Acquity UPLC system (Phenomenex Kinetex column at 40 °C, mobile
phase of water with 0.2% v/v formic acid, and acetonitrile with 0.15%
v/v formic acid) and Waters Acquity SQD with alternating positive/
negative electrospray ionization scanning from 125 to 1000 amu, with
a scan time of 105 ms and an interscan delay of 20 ms. All final target
compounds were found to have purities of ≥95%. High resolution
mass spectrometric analysis was performed on a Waters qTOF
Premiere mass spectrometer using flow injection operating in W
mode. Reverse phase HPLC purifications were performed on an
Agilent 1100 preparative system.
General Procedure for the Synthesis of 3-Aryl-5-
(chloromethyl)isoxazole Intermediates (3). A. Arylaldehyde
Oxime (2). A solution of the aldehyde starting material in EtOH
(1.3 M) was treated with 50% aqueous hydroxylamine (2.3 equiv).
After stirring at RT for 2 h, the solution was concentrated to dryness at
demonstrated through increased exposure of the parent
compound when the prodrugs were dosed in vivo. This
facilitated progression of the prodrugs into chronic toxicity
studies.
Table 12. PK Profiles of Parent Compound 16 and Ester Prodrugs 42, 43, and 44
a
b
c
CLND solubility: see Supporting Information for details. Rat administered 1 mg/kg IV and 5 mg/kg PO. Dog administered 1 mg/kg IV and 5
mg/kg PO.
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reduced pressure to give the arylaldehyde oxime (2), which was used
in the next step without purification.
HRMS m/z calcd for C23H11F8N5O (M + 1), 526.0909; found,
526.0915.
B. 3-Aryl-5-(chloromethyl)isoxazole (3). A solution of the
arylaldehyde oxime in DCM (1.0 M) was cooled to 0 °C and treated
with propargyl chloride (1.0 equiv). The solution was then treated
with 6.5% aqueous NaOCl (2.0 equiv) by dropwise addition. After
stirring at 0 °C for 15 min, the solution was heated to reflux for 3 h.
After cooling to RT, the mixture was partitioned between DCM and
water and the phases separated. The aqueous phase was extracted with
DCM (3×). The combined DCM extracts were washed with saturated
brine, dried over MgSO4, and concentrated to dryness at reduced
pressure to afford the crude 3-aryl-5-(chloromethyl)isoxazole (3),
which was used without purification.
2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]pyridazine (5b).
A. 2-Amino-3-((E)-(2,3-difluorobenzylidene)amino)maleonitrile. To
a solution of diaminomaleonitrile (15.0 g, 139 mmol) was added 2,3-
difluorobenzaldehyde (19.8 g, 139 mmol) followed by catalytic sulfuric
acid (4 drops). After stirring at RT for 1.5 h, the solvent was
evaporated at reduced pressure. The solid was triturated with 1:1
ether/hexanes, collected by filtration, and dried in vacuo to afford the
title compound (31.3 g, 96%).
B. 2-(2,3-Difluorophenyl)-1H-imidazole-4,5-dicarbonitrile (4b). A
stirred solution of 2-amino-3-((E)-(2,3-difluorobenzylidene)amino)-
maleonitrile (30.8 g, 133 mmol) in DMF (400 mL) was treated with
NCS (26.5 g, 199 mmol) followed by nicotimamide (24.3 g, 199
mmol). After 1 h, the precipitated nicotimamide hydrochloride was
removed by filtration and the filtrate concentrated at reduced pressure.
The residue was partitioned between EtOAc and water and the phases
separated. The EtOAc solution was washed with saturated brine (1×),
dried over MgSO4, and concentrated to afford a black oil. This material
was dissolved in DCM and filtered through a silica gel plug washing
with 4:1 DCM/MeOH. The resulting filtrate was concentrated to
dryness at reduced pressure to give 4b (14.1 g, 46%). ES-LCMS m/z:
231 (M + 1).
5-((2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]pyridazin-5-yl)-
methyl)-3-(6-(trifluoromethyl)pyridin-3-yl)isoxazole Hydro-
1
chloride (9). H NMR (300 MHz, DMSO-d6): δ 10.24 (s, 1 H),
9.58 (s, 1 H), 9.24 (s, 1 H), 8.50−8.57 (m, 1 H), 8.11−8.19 (m, 1 H),
8.02−8.09 (m, 1 H), 7.56−7.68 (m, 1 H), 7.34−7.44 (m, 2 H), 6.29
(s, 2 H). HRMS m/z calcd for C21H11F5N6O (M + 1), 459.0987;
found, 459.0990.
5-((2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]pyridazin-5-yl)-
methyl)-3-(4-(trifluoromethyl)phenyl)isoxazole Hydrochloride
(12). 1H NMR (300 MHz, DMSO-d6): δ 10.46 (s, 1 H), 9.73 (s, 1 H),
8.04−8.20 (m, 3 H), 7.84−7.91 (m, 2 H), 7.64−7.77 (m, 1 H), 7.34−
7.50 (m, 2 H), 6.35 (s, 2 H). HRMS m/z calcd for C22H12F5N5O (M +
1), 458.1035; found, 458.1038.
3-(4-Butylphenyl)-5-((2-(2,3-difluorophenyl)-5H-imidazo-
1
[4,5-d]pyridazin-5-yl)methyl)isoxazole Hydrochloride (13). H
NMR (300 MHz, DMSO-d6): δ 10.26 (s, 1 H), 9.60 (s, 1 H), 8.11−
8.20 (m, 1 H), 7.74 (dd, J = 7.5 Hz, 1.4 Hz, 2 H), 7.55−7.69 (m, 1 H),
7.27−7.45 (m, 3 H), 7.18 (m, 1 H), 6.23 (s, 2 H), 2.62 (t, J = 7.1 Hz, 2
H), 1.48−1.63 (m, 2 H), 1.21−1.36 (m, 2 H), 0.90 (t, J = 7.9 Hz, 3
H). HRMS m/z calcd for C25H21F2N5O (M + 1), 446.1787; found,
446.1790.
5-((2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]pyridazin-5-yl)-
methyl)-3-(4-propoxy-2-(trifluoromethyl)phenyl)isoxazole Hy-
drochloride (16). 1H NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1 H),
9.50 (s, 1 H), 8.08−8.24 (m, 1 H), 7.48−7.65 (m, 2 H), 7.27−7.43
(m, 3 H), 6.91 (s, 1 H), 6.20 (s, 2 H), 4.07 (t, J = 7.1 Hz, 2 H), 1.64−
1.85 (m, 2 H), 0.98 (t, J = 7.9 Hz, 3 H). ES-LCMS m/z: 516 (M + 1).
3-(2,4-Bis(trifluoromethyl)phenyl)-5-((2-(2-fluorophenyl)-
5H-imidazo[4,5-d]pyridazin-5-yl)methyl)isoxazole Hydrochlor-
ide (18). 1H NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1 H), 9.60 (s, 1
H), 8.30 (m, 1 H), 8.20 (m, 2 H), 7.90 (d, J = 7.4 Hz, 1 H), 7.60 (m, 1
H), 7.40 (m, 2 H), 7.00 (s, 1 H), 6.30 (s, 2 H). ES-LCMS m/z: 508
(M + 1).
3-(2,4-Bis(trifluoromethyl)phenyl)-5-((2-(thiophen-2-yl)-5H-
imidazo[4,5-d]pyridazin-5-yl)methyl)isoxazole Hydrochloride
(24). A. 2-Bromo-1H-imidazo[4,5-d]pyridazine (20). A solution of
2-bromo-1H-imidazole-4,5-dicarbonitrile (19) (2.00 g, 10.2 mmol, see
ref 29) in THF (100 mL) was cooled to −78 °C and treated with 1 M
DIBAL-H/THF (50 mL, 50.0 mmol) over 10 min. The mixture was
stirred at −78 °C for 15 min and then quenched by addition of 10%
aqueous potassium sodium tartrate (80 mL). The solution was then
treated with hydrazine hydrate (1.53 g, 30.6 mmol). The mixture was
warmed to RT and stirred for 1 h. The reaction mixture was then
cooled to 0 °C overnight and filtered to remove solids. The filter cake
was washed with MeOH (2 × 100 mL) and the filtrate concentrated to
dryness at reduced pressure. The crude product was subjected to flash
chromatography (silica gel, 0−60% MeOH (containing 10%
NH4OH)/DCM) to give 20 (0.350 g, 17%). ES-LCMS m/z: 199 +
201 (M + 1).
B. 3-(2,4-Bis(trifluoromethyl)phenyl)-5-((2-bromo-5H-imidazo-
[4,5-d]pyridazin-5-yl)methyl)isoxazole (21). A mixture of 20 (0.350
g, 1.76 mmol), 3a (0.600 g, 1.82 mmol), and K2CO3 (0.500 g, 3.62
mmol) in DMF (5 mL) was heated to 40 °C with stirring. After 1 h,
the mixture was cooled to RT and poured into cold water (30 mL).
The resulting solid was collected by filtration and dried in vacuo to
afford 21 (0.590 g, 68%) as a yellow solid.
C. 3-(2,4-Bis(trifluoromethyl)phenyl)-5-((2-(thiophen-2-yl)-5H-
imidazo[4,5-d]pyridazin-5-yl)methyl)isoxazole Hydrochloride (24).
A mixture of 21 (0.100 g, 0.203 mmol), thiophen-2-ylboronic acid
(33.8 mg, 0.264 mmol), 2 M aqueous K2CO3 (0.31 mL, 0.61 mmol),
and Pd(Ph3P)4 (11.7 mg, 10.2 μmol) in 2-propanol (2 mL) was
subjected to microwave heating at 120 °C for 20 min. The reaction
mixture was filtered to remove solids and the filtrate concentrated to
dryness at reduced pressure. The crude residue was subjected to RP-
HPLC purification (C18, MeCN/water with 0.1% TFA) followed by
treatment with 1N aqueous HCl to afford 24 as the hydrochloride salt.
1H NMR (300 MHz, DMSO-d6): δ 10.34 (s, 1 H), 9.65 (s, 1 H),
C. 2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]pyridazine (5b). A
solution of 2-(2,3-difluorophenyl)-1H-imidazole-4,5-dicarbonitrile
(14.1 g, 61.3 mmol) in THF (80 mL) was cooled to −78 °C and
treated with 1 M DIBAL-H/THF (400 mL, 400 mmol) by rapid
dropwise addition. The solution was then quenched by careful
addition of water until gas evolution ceased. Hydrazine hydrate (5.77
mL, 184 mmol) was added, and the solution was allowed to warm to
RT. The solution was treated with MeOH (61 mL) and the solid
aluminum salts removed by filtration. The filter cake was washed with
an additional portion (50 mL) of MeOH. The filtrate was
concentrated to dryness at reduced pressure and the residue subjected
to flash chromatography (silica gel, 10−30% MeOH (containing 10%
1
NH4OH)/DCM) to give 5b as a solid. H NMR (300 MHz, DMSO-
d6): δ 9.58 (s, 2 H), 8.11 (m, 1 H), 7.58 (m, 1 H), 7.37 (m, 1 H). ES-
LCMS m/z: 233 (M + 1).
General Procedure for the Alkylation of 2-Substituted 5H-
Imidazo[4,5-d]pyridazines (5) with 3-Aryl-5-(chloromethyl)-
isoxazoles (3) to Give Compounds 6, 7, 9, 12, 13, 16, and
18. A mixture of 3-aryl-5-(chloromethyl)isoxazole (3), 2-substituted
5H-imidazo[4,5-d]pyridazine (5) (1.0 equiv), and Cs2CO3 (3.0 equiv)
in DMF (0.04 M) was subjected to microwave heating at 120 °C for
10 min. The reaction mixture was filtered to remove solids and the
filtrate subjected to RP-HPLC purification (C18, MeCN/water with
0.1% TFA) followed by treatment with 1N aqueous HCl to afford the
desired compound as the hydrochloride salt.
3-(4-Chlorophenyl)-5-((2-(2-fluorophenyl)-5H-imidazo[4,5-
1
d]pyridazin-5-yl)methyl)isoxazole Hydrochloride (6). H NMR
(300 MHz, DMSO-d6): δ 10.40 (s, 1 H), 9.71 (s, 1 H), 8.33 (m, 1 H),
7.84 (dd, J = 7.5 Hz, 1.4 Hz, 2 H), 7.73 (m, 1 H), 7.44−7.63 (m, 2 H),
7.42 (dd, J = 7.5 Hz, 1.3 Hz, 2 H), 7.26 (s, 1 H), 6.30 (s, 2 H). HRMS
m/z calcd for C23H13ClFN5O (M + 1), 406.0865; found, 406.0870.
3-(2,4-Bis(trifluoromethyl)phenyl)-5-((2-(2,3-difluorophen-
yl)-5H-imidazo[4,5-d]pyridazin-5-yl)methyl)isoxazole Hydro-
1
chloride (7). H NMR (300 MHz, DMSO-d6): δ 10.17 (s, 1 H),
9.55 (s, 1 H), 8.12−8.26 (m, 3 H), 7.93 (d, J = 7.5 Hz, 1 H), 7.54−
7.65 (m, 1 H), 7.33−7.41 (m, 1 H), 7.06 (s, 1 H), 6.26 (s, 2 H).
8.17−8.25 (m, 2 H), 7.97 (dd, J = 4.8 Hz, 1.0 Hz, 1 H), 7.90 (d, J = 8.4
Hz, 1 H), 7.65−7.48 (m, 2H), 7.33 (dd, J = 5.1 Hz, 3.8 Hz, 1 H), 7.07
1971
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Journal of Medicinal Chemistry
Article
(s, 1 H), 6.35 (s, 2 H). HRMS m/z calcd for C21H11F6N5OS (M + 1),
496.0661; found, 496.0665.
D. 2-(2,3-Difluorophenyl)-5-((5-(4-propoxy-2-(trifluoromethyl)-
phenyl)pyrazin-2-yl)methyl)-5H-imidazo[4,5-d]pyridazine (34).
Compound 34 was prepared from (5-(4-propoxy-2-(trifluoromethyl)-
phenyl)pyrazin-2-yl)methyl methanesulfonate and 5b as described
herein for the synthesis of 29, step C. The crude product was purified
by flash chromatography (silica gel, 0−15% MeOH/DCM) to afford
34 (31 mg, 20% for 2 steps). 1H NMR (300 MHz, DMSO-d6): δ 10.54
(s, 1 H), 9.73 (s, 1 H), 8.90 (d, J = 1.2 Hz, 1 H), 8.63 (d, J = 1.0 Hz, 1
H), 8.10−8.14 (m, 1 H), 7.65−7.76 (m, 1 H), 7.40−7.51 (m, 2 H),
7.27−7.31 (m, 2 H), 6.30 (s, 2 H), 4.00 (t, J = 6.6 Hz, 2 H), 1.64−1.75
(m, 2 H), 0.93 (t, J = 7.4 Hz, 3 H). HRMS m/z calcd for
C26H19F5N6O (M + 1), 527.1613; found, 527.1615.
3-(2,4-Bis(trifluoromethyl)phenyl)-5-(1-(2-(2,3-difluorophen-
yl)-5H-imidazo[4,5-d]pyridazin-5-yl)ethyl)isoxazole Hydro-
chloride (37). A. 1-(3-(2,4-Bis(trifluoromethyl)phenyl)isoxazol-5-
yl)ethanone (36). To a stirred solution of 2a (1.03 g, 4.00 mmol) and
but-3-yn-2-one (0.272 g, 4.00 mmol) in DCM at 0 °C was added 10%
aqueous NaOCl (6.30 mL, 8.40 mmol). The solution was stirred at 0
°C for 10 min and then heated to reflux. After 1 h, the mixture was
cooled to RT and partitioned between water and DCM and the phases
separated. The DCM solution was washed with saturated aqueous
brine (1×), dried over Na2SO4, and concentrated to dryness at
reduced pressure to give 36 (0.74 g, 57%) which was used in the next
step without purification.
B. 1-(3-(2,4-Bis(trifluoromethyl)phenyl)isoxazol-5-yl)ethanol. A
solution of 36 (0.660 g, 2.04 mmol) in EtOH (40 mL) was treated
with NaBH4 (0.300 g, 7.93 mmol) and the resulting solution stirred at
RT. After 20 min, the solution was diluted with water and extracted
with EtOAc (3×). The combined EtOAc extracts were concentrated to
dryness and the residue purified by flash chromatography (silica gel,
0−40% EtOAc/hexanes) to afford the title compound (0.44 g, 66%).
C. 1-(3-(2,4-Bis(trifluoromethyl)phenyl)isoxazol-5-yl)ethyl meth-
anesulfonate. A solution of 1-(3-(2,4-bis(trifluoromethyl)phenyl)-
isoxazol-5-yl)ethanol (0.200 g, 0.615 mmol) in DCM (5 mL) was
treated with DIPEA (200 μL, 1.15 mmol) followed by MsCl (65 μL,
0.834 mmol). After stirring at RT for 5 min, the solution was
partitioned between water and EtOAc and the phases separated. The
EtOAc solution was washed with saturated brine (1×), dried over
sodium sulfate, and concentrated to dryness at reduced pressure to
give the title compound, which was used in the next step without
purification.
D. 3-(2,4-Bis(trifluoromethyl)phenyl)-5-(1-(2-(2,3-difluorophenyl)-
5H-imidazo[4,5-d]pyridazin-5-yl)ethyl)isoxazole (37). A stirred
mixture of 1-(3-(2,4-bis(trifluoromethyl)phenyl)isoxazol-5-yl)ethyl
methanesulfonate (0.100 g, 0.248 mmol), 5b (48.0 mg, 0.207
mmol), and K2CO3 (69.0 mg, 0.496 mmol) in DMF was subjected
to microwave heating at 80 °C for 10 min. The reaction mixture was
filtered to remove solids and the filtrate subjected to RP-HPLC
purification (C18, MeCN/water with 0.1% TFA) followed by
treatment with 1N aqueous HCl to afford 37 as the hydrochloride
salt. 1H NMR (300 MHz, DMSO-d6): δ 10.33 (s, 1 H), 9.65 (s, 1 H),
8.11−8.26 (m, 3 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.59−7.21 (m, 1 H),
7.37−7.45 (m, 1 H), 7.01 (s, 1 H), 6.64 (q, J = 6.9 Hz, 1 H), 2.10 (d, J
= 6.9 Hz, 3 H). HRMS m/z calcd for C24H13F8N5O (M + 1),
540.1065; found, 540.1068.
5-((5-(2,4-Bis(trifluoromethyl)phenyl)pyridin-2-yl)methyl)-2-
(2,3-difluorophenyl)-5H-imidazo[4,5-d]pyridazine Hydrochlor-
ide (29). A. (5-(2,4-Bis(trifluoromethyl)phenyl)pyridin-2-yl)-
methanol. A mixture of (2,4-bis(trifluoromethyl)phenyl)boronic
acid (0.387 g, 1.50 mmol), (5-bromopyridin-2-yl)methanol (0.188 g,
1.00 mmol), Pd(Ph3P)4 (57.8 mg, 0.0500 mmol), and 2 M aqueous
Na2CO3 (1.00 mL, 2.00 mmol) in toluene (4 mL) was sparged with
nitrogen for several minutes and heated to 100 °C with stirring. After 1
h, the mixture was cooled to RT, partitioned between water and
EtOAc, and the phases separated. The EtOAc solution was washed
with saturated brine, dried over Na2SO4, and concentrated to dryness
at reduced pressure. The crude material was subjected to flash
chromatography (silica gel, 20−100% EtOAc/hexanes) to afford the
title compound (0.225 g, 70%).
B. (5-(2,4-Bis(trifluoromethyl)phenyl)pyridin-2-yl)methyl Metha-
nesulfonate. A solution of (5-(2,4-bis(trifluoromethyl)phenyl)-
pyridin-2-yl)methanol (0.180 g, 0.560 mmol) in DCM (5 mL) was
treated with DIPEA (200 μL, 1.15 mmol) followed by MsCl (65 μL,
0.834 mmol). After stirring at RT for 1 h, the solution was partitioned
between water and EtOAc and the phases separated. The EtOAc
solution was washed with saturated brine (1×), dried over sodium
sulfate, and concentrated to dryness at reduced pressure to give the
title compound which was used in the next step without purification.
C. 5-((5-(2,4-Bis(trifluoromethyl)phenyl)pyridin-2-yl)methyl)-2-
(2,3-difluorophenyl)-5H-imidazo[4,5-d]pyridazine Hydrochloride
(29). A stirred mixture of (5-(2,4-bis(trifluoromethyl)phenyl)pyridin-
2-yl)methyl methanesulfonate (60.0 mg, 0.150 mmol), 5b (45.4 mg,
0.195 mmol), and K2CO3 (60.2 mg, 0.436 mmol) in DMF (4 mL) was
heated to 80 °C. After 1 h, the reaction mixture was cooled to RT,
filtered to remove solids, and the filtrate subjected to RP-HPLC
purification (C18, MeCN/water with 0.1% TFA) followed by
treatment with 1N aqueous HCl to afford 29 as the hydrochloride
salt. 1H NMR (300 MHz, DMSO-d6): δ 10.40 (s, 1 H), 9.68 (s, 1 H),
8.47 (d, J = 2.1 Hz, 1 H), 8.10−8.18 (m, 3 H), 7.92 (dd, J = 8.1 Hz, 2.0
Hz, 1 H), 7.64−7.75 (m, 3 H), 7.39−7.50 (m, 1 H), 6.23 (s, 2 H).
HRMS m/z calcd for C25H13F8N5 (M + 1), 536.1116; found,
536.1118.
2-(2,3-Difluorophenyl)-5-((5-(4-propoxy-2-(trifluoromethyl)-
phenyl)pyrazin-2-yl)methyl)-5H-imidazo[4,5-d]pyridazine (34).
A. Methyl 5-(4-Propoxy-2-(trifluoromethyl)phenyl)pyrazine-2-car-
boxylate. A mixture of methyl 5-chloropyrazine-2-carboxylate (0.173
g, 1.00 mmol), (4-propoxy-2-(trifluoromethyl)phenyl)boronic acid
(0.298 g, 1.20 mmol), Pd(Ph3P)4 (58.0 mg, 0.0500 mmol), and 2 M
aqueous K2CO3 (0.902 mL, 1.80 mmol) in toluene (1.8 mL) was
sparged with argon for several minutes and then stirred at 95 °C. After
1 h, the mixture was cooled to RT, partitioned between EtOAc and
water, and the phases separated. The EtOAc solution was washed with
brine (1×), dried over Na2SO4, and concentrated to dryness at
reduced pressure. The crude material was subjected to flash
chromatography (silica gel, 20−70% EtOAc/hexanes) to afford the
title compound (0.328 g, 96%) as a waxy white solid.
B. (5-(4-Propoxy-2-(trifluoromethyl)phenyl)pyrazin-2-yl)-
methanol. To a stirred solution of methyl 5-(4-propoxy-2-
(trifluoromethyl)phenyl)pyrazine-2-carboxylate (0.150 g, 0.441
mmol) in 7:2 THF/H2O (9 mL) was added NaBH4 portionwise
(1.50 g, 39.6 mmol). The reaction was briefly warmed and then stirred
without heating for 30 min. The solution was partitioned between
water and DCM and the phases separated. The DCM solution was
dried over Na2SO4 and evaporated to dryness in the presence of
Celite. The crude material was subjected to flash chromatography
(silica gel, 30−50% EtOAc/hexanes) to give the title compound (90.0
mg, 65%).
3-(2,4-Bis(trifluoromethyl)phenyl)-5-(2-(2-(2,3-difluorophen-
yl)-5H-imidazo[4,5-d]pyridazin-5-yl)propan-2-yl)isoxazole Hy-
drochloride (39). A. 3-(2,4-Bis(trifluoromethyl)phenyl)-5-(2-chlor-
opropan-2-yl)isoxazole (38). A suspension of 2a (1.00 g, 3.89 mmol)
and 3-chloro-3-methylbut-1-yne (0.798 g, 7.78 mmol) in DCM (5
mL) at 0 °C was treated with 10% aqueous NaOCl (4.00 mL, 5.38
mmol) by dropwise addition. After stirring at 0 °C for 15 min, the
solution was heated to reflux. After 3 h, the reaction mixture was
cooled to RT, partitioned between DCM and water, and the phases
separated. The DCM solution was washed with saturated brine, dried
over sodium sulfate, and concentrated to dryness at reduced pressure
to give crude 38, which was used in the next step without purification.
B. 3-(2,4-Bis(trifluoromethyl)phenyl)-5-(2-(2-(2,3-difluorophenyl)-
5H-imidazo[4,5-d]pyridazin-5-yl)propan-2-yl)isoxazole (39). A
stirred mixture of 38 (0.179 g, 0.500 mmol), 5b (0.116 g, 0.500
mmol), and K2CO3 (0.338 g, 2.45 mmol) in DMF (5 mL) was heated
C. (5-(4-Propoxy-2-(trifluoromethyl)phenyl)pyrazin-2-yl)methyl
Methanesulfonate. The title compound was prepared from (5-(4-
propoxy-2-(trifluoromethyl)phenyl)pyrazin-2-yl)methanol (90 mg,
0.288 mmol) as described herein for the synthesis of compound 29,
step B. The crude product was used in the next step without
purification.
1972
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Journal of Medicinal Chemistry
Article
to 80 °C for 18 h, at which point LCMS indicated very little
conversion of the starting materials to the desired product. The
mixture was then heated to 100 °C for 3 h and then briefly at 110 °C.
The reaction mixture was cooled to RT, filtered to remove solids, and
the filtrate subjected to RP-HPLC purification (C18, MeCN/water
with 0.1% TFA) followed by treatment with 1N aqueous HCl to afford
39 as the hydrochloride salt. 1H NMR (300 MHz, DMSO-d6): δ 10.44
(s, 1 H), 9.69 (s, 1 H), 8.21−8.27 (m, 2 H), 8.09−8.18 (m, 1 H),
7.91−7.99 (m, 1 H), 7.65−7.26 (m, 1 H), 7.40−7.49 (m, 1 H), 7.12
(s, 1 H), 2.30 (s, 6 H). HRMS m/z calcd for C25H15F8N5O (M + 1),
554.1222; found, 554.1226.
= 6.6 Hz, 2 H), 3.98 (s, 2 H), 1.71−1.82 (m, 2 H), 1.00 (t, J = 7.4 Hz,
3 H). ES-LCMS m/z: 330 (M + 1).
D. Methyl 2-(3-(4-Propoxy-2-(trifluoromethyl)phenyl)isoxazol-5-
yl)acetate (41). Thionyl chloride (4 mL) was slowly added to a stirred
20 mL portion of MeOH. After 10 min, a solution of 2-(3-(4-propoxy-
2-(trifluoromethyl)phenyl)isoxazol-5-yl)acetic acid (0.530 g, 1.61
mmol) in MeOH (6 mL) was added and the resulting solution was
stirred at RT. After 2 h, the solution was concentrated to dryness at
reduced pressure and the residue dissolved in EtOAc. The solution
was washed with saturated aqueous sodium bicarbonate (2×) and
brine (1×), dried over Na2SO4, and concentrated to dryness at
1
reduced pressure to afford 41 (0.55 g, 99%) as a light-yellow oil. H
Methyl 2-(2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]-
pyridazin-5-yl)-2-(3-(4-propoxy-2-(trifluoromethyl)phenyl)-
isoxazol-5-yl)acetate (42). A. 4-Propoxy-2-(trifluoromethyl)-
benzaldehyde Oxime (2b). A solution of 4-(propyloxy)-2-
(trifluoromethyl)benzaldehyde (16.3 g, 70.2 mmol) in EtOH (70
mL) was treated with 50% aqueous hydroxylamine (10.0 mL, 140
mmol), and the resulting solution was stirred at RT. After 2 h, TLC
(silica gel, 8:2 hexane/EtOAc) indicated complete conversion of the
aldehyde starting material to a new, lower Rf spot. The solution was
concentrated by rotary evaporation to a volume of approximately 25
mL, at which point a white solid had precipitated. The suspension was
diluted with 60 mL of water and stirred for 45 min. The solid was
collected by vacuum filtration on a medium frit. The filter cake was
washed twice with water, suction dried for 20 min, and then dried in
vacuo overnight to afford 16.9 g of a very pale-yellow crystalline solid.
The crude material was recrystallized from hexane/EtOAc to afford 2b
(14.5 g, 84%) as a white crystalline solid. 1H NMR (400 MHz,
DMSO-d6) δ 11.58 (s, 1 H), 8.19−8.23 (m, 1 H), 7.93 (d, J = 8.6 Hz,
1 H), 7.21−7.30 (m, 2 H), 4.03 (t, J = 6.6 Hz, 2H), 1.68−1.80 (m,
2H), 0.98 (t, J = 7.4 Hz, 3H). ES-LCMS m/z: 248 (M + 1).
B. 2-(3-(4-Propoxy-2-(trifluoromethyl)phenyl)isoxazol-5-yl)-
ethanol (40). A stirred solution of 2b (3.00 g, 12.1 mmol) in DMF
(25 mL) was cooled to 0 °C and treated with NCS (1.70 g, 12.7
mmol) by slow addition over 1 min. After 1 h, the solution was diluted
with EtOAc, washed with water (2×) and brine (1×), dried over
Na2SO4, and concentrated to dryness at reduced pressure. The residue
was dissolved in 1,2-DCE (35 mL) and the solution treated with 3-
butyn-1-ol (1.30 g, 18.2 mmol) followed by TEA (2.60 mL, 18.2
mmol). A solid immediately precipitated (TEA-HCl salt). The
suspension was heated to reflux with stirring, at which point the
solid had gone into solution. After 30 min, the solution was cooled to
RT and stirred overnight. The solution was diluted 2-fold with DCM,
washed with water (3×) and brine (1×), dried over Na2SO4, and
concentrated to dryness at reduced pressure to afford 40 (4.02 g, 95%)
as a viscous, yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.6
Hz, 1 H), 7.27 (d, J = 2.3 Hz, 1 H), 7.07 (dd, J = 8.5 Hz, 2.5 Hz, 1 H),
6.27 (s, 1 H), 3.94−4.02 (m, 4 H), 3.06 (t, J = 6.2 Hz, 2 H), 1.78−1.88
(m, 2 H), 1.76 (br s, 1H) 1.04 (t, J = 7.4 Hz, 3 H). ES-LCMS m/z:
316 (M + 1).
NMR (400 MHz, DMSO-d6) δ 7.53 (d, J = 8.4 Hz, 1 H), 7.27−7.34
(m, 2 H), 6.55 (s, 1 H), 4.07 (s, 2 H), 4.03 (t, J = 6.44 Hz, 2 H), 3.64
(s, 3 H), 1.67−1.78 (m, 2 H), 1.00 (t, J = 7.4 Hz, 3 H). ES-LCMS m/
z: 344 (M + 1).
E. Methyl 2-Bromo-2-(3-(4-propoxy-2-(trifluoromethyl)phenyl)-
isoxazol-5-yl)acetate. A mixture of methyl 2-(3-(4-propoxy-2-
(trifluoromethyl)phenyl)isoxazol-5-yl)acetate (0.383 g, 1.12 mmol),
NBS (0.238 g, 1.34 mmol), and AIBN (10 mg, 0.061 mmol) in CCl4
(20 mL) was heated to reflux with stirring under a nitrogen
atmosphere. After 2 h, TLC (silica gel, 1:1 hexane/EtOAc) indicated
approximately 50% conversion of the starting material to a slightly
higher Rf component. The mixture was treated with an additional
portion of NBS (0.200 g, 1.12 mmol) followed by AIBN (10 mg, 0.061
mmol), and stirring at reflux continued. After 3 more hours of
refluxing, the reaction mixture was cooled to RT and stirred overnight.
The mixture was diluted with DCM and the solids removed by
filtration through a medium fritted funnel. The filtrate was
concentrated to dryness at reduced pressure and the residue subjected
to flash chromatography (silica gel, 0−60% EtOAc/hexanes) to afford
1
the title compound (0.268 g, 57%) as an oil. H NMR (400 MHz,
DMSO-d6) δ 7.63 (d, J = 8.4 Hz, 1 H), 7.33−7.42 (m, 2 H), 6.90 (s, 1
H), 6.44 (s, 1 H), 4.09 (t, J = 6.45 Hz, 2 H), 3.81 (s, 3 H), 1.73−1.82
(m, 2 H), 1.00 (t, J = 7.4 Hz, 3 H).
F. Methyl 2-(2-(2,3-Difluorophenyl)-5H-imidazo[4,5-d]pyridazin-
5-yl)-2-(3-(4-propoxy-2-(trifluoromethyl)phenyl)isoxazol-5-yl)-
acetate (42). A mixture of 5b (0.313 g, 1.35 mmol) in DMF (10 mL)
was briefly warmed to dissolve the starting material. The resulting
solution was treated with K2CO3 (0.622 g, 4.50 mmol) and cooled in
an ice water bath. A solution of methyl 2-bromo-2-(3-(4-propoxy-2-
(trifluoromethyl)phenyl)isoxazol-5-yl)acetate (0.380 g, 0.900 mmol)
in DMF (4 mL) was added dropwise over 3 min. The mixture was
allowed to warm to RT. After 1.5 h, the mixture was partitioned
between 10% aqueous NaCl and EtOAc and the phases separated. The
aqueous phase was back extracted with EtOAc (3×). The combined
EtOAc solutions were washed with 10% aqueous NaCl (2×) and
saturated aqueous brine (1×), dried over Na2SO4, and concentrated to
dryness at reduced pressure. The crude product was subjected to flash
chromatography (silica gel, 0−10% MeOH/DCM) followed by
reverse phase HPLC purification (C18, 10−100% MeCN/water with
0.1% TFA). Fractions containing pure product were combined and
concentrated to dryness at reduced pressure. The residue was
dissolved in EtOAc. The solution was washed with saturated aqueous
sodium bicarbonate (1×) and brine (1×), dried over sodium sulfate,
and concentrated to dryness at reduced pressure to afford 42 (0.280 g,
C. 2-(3-(4-Propoxy-2-(trifluoromethyl)phenyl)isoxazol-5-yl)acetic
Acid. Periodic acid (4.58 g, 20.1 mmol) was added to anhydrous
MeCN (70 mL) and the mixture stirred at RT for 15 min. The solid
reagent slowly dissolved to afford a clear solution. A solution of 40
(2.88 g, 9.13 mmol) in MeCN (15 mL) was added, and the resulting
solution was cooled in an ice water bath. The solution was then treated
with PCC (40 mg, 0.18 mmol). A light-yellow precipitate was rapidly
produced. The ice bath was removed, and the reaction mixture was
allowed to warm to RT with stirring. After 3 h, LCMS indicated
complete conversion to the desired product. The mixture was
subjected to rotary evaporation to a volume of approximately 15 mL
and was then diluted with 100 mL of 9:1 chloroform/iPrOH. The
rapidly stirred suspension was treated with 10% aqueous sodium
bisulfate (100 mL) and the mixture stirred vigorously for a short time.
The mixture was transferred to a separatory funnel and the phases
separated. The organic solution was washed with water (3×) and brine
(1×), dried over sodium sulfate, and concentrated to dryness at
reduced pressure to afford the title compound (2.85 g, 95%) as a light-
1
54%) as a white foam. H NMR (400 MHz, CDCl3) δ 9.40 (s, 1 H),
9.29 (s, 1 H), 8.15−8.23 (m, 1 H), 7.58 (d, J = 8.6 Hz, 1 H), 7.17−
7.34 (m, 3 H), 7.12 (dd, J = 8.6 Hz, 2.4 Hz, 1 H), 6.88 (s, 1 H), 6.85
(s, 1 H), 4.00 (t, J = 6.5 Hz, 2 H), 3.94 (s, 3 H), 1.78−1.91 (m, 2 H),
1.05 (t, J = 7.4 Hz, 3 H). HRMS m/z calcd for C27H20F5N5O4 (M +
1), 574.1508; found, 574.1504.
2-(2-Hydroxyethoxy)ethyl 2-(2-(2,3-Difluorophenyl)-5H-
imidazo[4,5-d]pyridazin-5-yl)-2-(3-(4-propoxy-2-
(trifluoromethyl)phenyl)isoxazol-5-yl)acetate (44). To a stirred
solution of compound 42 (2.00 g, 3.49 mmol) in 1:1 diethylene
glycol/DME (25 mL) was added K2CO3 (1.46 g, 10.5 mmol) and the
resulting mixture stirred at RT. After 18 h, the mixture was treated
with glacial acetic acid (3 mL) and diluted with EtOAc. The resulting
mixture was washed with water (4×) and brine (1×), dried over
1
tan solid. H NMR (400 MHz, DMSO-d6) δ ppm 12.90 (br s, 1 H),
7.58 (d, J = 8.4 Hz, 1 H), 7.31−7.39 (m, 2 H), 6.57 (s, 1 H), 4.08 (t, J
1973
dx.doi.org/10.1021/jm401337x | J. Med. Chem. 2014, 57, 1964−1975

Journal of Medicinal Chemistry
Article
Na2SO4, and concentrated to dryness at reduced pressure. The crude
material was subjected to flash chromatography (silica gel, 50−100%
EtOAc/hexanes) followed by recrystallization from EtOAc/hexanes to
(5) Lohmann, V.; Korner, F.; Koch, J.; Herian, U.; Theilmann, L.;
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Modern Drug Discovery: A Case Study of Boceprevir, an HCV
Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.
Acc. Chem. Res. 2008, 41, 50−59.
1
afford compound 44 (1.71 g, 76%) as a white solid. H NMR (400
MHz, DMSO-d6) δ 10.10 (s, 1 H), 9.55 (s, 1 H), 8.15−8.22 (m, 1 H),
7.72 (s, 1 H), 7.54−7.68 (m, 2 H), 7.33−7.42 (m, 3 H), 7.13 (s, 1 H),
4.55 (t, J = 5.2 Hz, 1 H), 4.33−4.49 (m, 2 H), 4.09 (t, J = 6.6 Hz, 2 H),
3.62 (t, J = 4.5 Hz, 2 H), 3.28−3.43 (m, 4 H), 1.71−1.84 (m, 2 H),
1.00 (t, J = 7.4 Hz, 3 H). HRMS m/z calcd for C30H27F5N5O6 (M +
1), 648.1882; found, 648.1888.
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development of telaprevir: an NS3-4A protease inhibitor for treating
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triple therapy for chronic HCV genotype 1 infection. Antiviral Ther.
2011, 16, 1187−1201.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental details for compound syntheses and
biological assays presented in this paper. This material is
available free of charge via the Internet at http://pubs.acs.org.
(9) Fried, M. Side effects of therapy of hepatitis C and their
management. Hepatology 2002, 36, S237−S244.
(10) Manns, M.; Wedemeyer, H.; Cornberg, M. Treating viral
hepatitis C: efficacy, side effects, and complications. Gut 2006, 55,
1350−1359.
AUTHOR INFORMATION
■
(11) Powdrill, M.; Tchesnokov, E.; Kozak, R.; Russell, R.; Martin, R.;
Svarovskaia, E.; Mo, H.; Kouyos, R.; Gotte, M. Contribution of a
mutational bias in hepatitis C virus replication to the genetic barrier in
the development of drug resistance. Proc. Natl. Acad. Sci. U. S. A. 2011,
108, 20509−20513.
Corresponding Author
*Phone: 919-483-3123. E-mail: martin.r.leivers@gsk.com.
Notes
The authors declare no competing financial interest.
(12) Kuntzen, T.; Timm, J.; Berical, A.; Lennon, N.; Berlin, A.;
Young, S.; Lee, B.; Heckerman, D.; Carlson, J.; Reyor, L.; Kleyman,
M.; McMahon, C.; Birch, C.; Schulze Zur Wiesch, J.; Ledlie, T.;
Koehrsen, M.; Kodira, C.; Roberts, A.; Lauer, G.; Rosen, H.; Bihl, F.;
Cerny, A.; Spengler, U.; Liu, Z.; Kim, A.; Xing, Y.; Schneidewind, A.;
Madey, M.; Fleckenstein, J.; Park, V.; Galagan, J.; Nusbaum, C.;
Walker, B.; Lake-Bakaar, G.; Daar, E.; Jacobson, I.; Gomperts, E.;
Edlin, B.; Donfield, S.; Chung, R.; Talal, A.; Marion, T.; Birren, B.;
Henn, M.; Allen, T. Naturally occurring dominant resistance
mutations to hepatitis C virus protease and polymerase inhibitors in
ACKNOWLEDGMENTS
■
We acknowledge the contributions of the following GSK
scientists to the manuscript: Iris Scherer, John Eaddy, Roy
Bowman, Shiping Xie, Mark Hatcher, Matt Toczko, Andrew
Brown, Susan Long, Christopher Nixon, Padmaja Vulimiri, Ping
Xiong, Mill Lambert, and Brian Donovan.
ABBREVIATIONS USED
̈
treatment-naive patients. Hepatology 2008, 48, 1769−1778.
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(13) Puyang, X.; Poulin, D.; Mathy, J.; Anderson, L.; Ma, S.; Fang, Z.;
Zhu, S.; Lin, K.; Fujimoto, R.; Compton, T.; Wiedmann, B.
Mechanism of Resistance of Hepatitis C Virus Replicons to
Structurally Distinct Cyclophilin Inhibitors. Antimicrob. Agents Chemo-
ther. 2010, 54, 1981−1987.
AIBN, azobisisobutyronitrile; CL, clearance; CYP, cytochrome
P-450; DAA, direct acting antiviral; DCE, dichloroethane;
DCM, dichloromethane; DIBAL, diisobutylaluminum hydride;
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylfor-
mamide; EtOAc, ethyl acetate; EtOH, ethanol; HCV, hepatitis
(14) Suzuki, F.; Sezaki, H.; Akuta, N.; Suzuki, Y.; Seko, Y.;
Kawamura, Y.; Hosaka, T.; Kobayashi, M.; Sait, S.; Arase, Y.; Ikeda,
K.; Kobayashi, M.; Mineta, R.; Watahiki, S.; Miyakawa, Y.; Kumada, H.
Prevalence of hepatitis C virus variants resistant to NS3 protease
inhibitors or the NS5A inhibitor (BMS-790052) in hepatitis patients
with genotype 1b. J. Clin. Virol. 2012, 54, 352−354.
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Patients With Hepatitis C Virus Infection. Gastroenterology 2010, 138,
447−462.
(16) McPhee, F.; Friborg, J.; Levine, S.; Chen, C.; Falk, P.; Yu, F.;
Hernandez, D.; Lee, M.; Chaniewski, S.; Sheaffer, A.; Pasquinelli, C.
Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor
Asunaprevir. Antimicrob. Agents Chemother. 2012, 56, 3670−3681.
(17) Gelman, M.; Glenn, J. Mixing the right hepatitis C inhibitor
cocktail. Trends Mol. Med. 2011, 17, 34−46.
(18) Hebner, C.; Han, B.; Brendza, K.; Nash, M.; Sulfab, M.; Tian,
Y.; Hung, M.; Fung, W.; Vivian, R.; Trenkle, J.; Taylor, J.; Bjornson,
K.; Bondy, S.; Liu, X.; Link, J.; Neyts, J.; Sakowicz, R.; Zhong, W.;
Tang, H.; Schmitz, U. The HCV Non-Nucleoside Inhibitor Tegobuvir
Utilizes a Novel Mechanism of Action to Inhibit NS5B Polymerase
Function. PLoS One 2012, 7, e39163.
̀
C virus; hERG, human ether-a-go-go-related gene; HPLC,
high-performance liquid chromatography; iPrOH, 2-propanol;
LCMS, liquid chromatography−mass spectrometry; MeCN,
acetonitrile; MeOH, methanol; MOA, mechanism of action;
MsCl, methanesulfonyl chloride; NBS, N-bromosuccinimide;
NCS, N-chlorosuccinimide; NMP, N-methylpyrrolidone; NS3,
nonstructural protein 3; NS5B, nonstructural protein 5B; PCC,
pyridinium chlorochromate; PEG, polyethylene glycol; RNA,
ribonucleic acid; RP-HPLC, reverse phase high performance
liquid chromatography; RT, room temperature; SAR, struc-
ture−activity relationship; TEA, triethylamine; TFA, trifluoro-
acetic acid; THF, tetrahydrofuran
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