Systematic structure modifications of imidazo[1,2-a]pyrimidine to reduce metabolism mediated by aldehyde oxidase (AO)

Article abstract of DOI:10.1021/jm2010942

N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4- tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carboxamide (1) was recently identified as a full antagonist of the androgen receptor, demonstrating excellent in vivo tumor growth inhibition in castration-resistant prostate cancer (CRPC). However, the imidazo[1,2-a]pyrimidine moiety is rapidly metabolized by aldehyde oxidase (AO). The present paper describes a number of medicinal chemistry strategies taken to avoid the AO-mediated oxidation of this particular system. Guided by an AO protein structure-based model, our investigation revealed the most probable site of AO oxidation and the observation that altering the heterocycle or blocking the reactive site are two of the more effective strategies for reducing AO metabolism. These strategies may be useful for other drug discovery programs.

Full text of DOI:10.1021/jm2010942

ARTICLE
pubs.acs.org/jmc
Systematic Structure Modifications of Imidazo[1,2-a]pyrimidine to
Reduce Metabolism Mediated by Aldehyde Oxidase (AO)
Angelica Linton,*,† Ping Kang,‡ Martha Ornelas,† Susan Kephart,† Qiyue Hu,† Mason Pairish,† Ying Jiang,‡
and Chuangxing Guo*,†
†Oncology Medicinal Chemistry; ‡Pharmacokinetics and Drug Metabolism, Pfizer Worldwide Research & Development, San Diego,
California 92121, United States
S Supporting Information
b
ABSTRACT: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-
2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-
carboxamide (1) was recently identified as a full antagonist of
the androgen receptor, demonstrating excellent in vivo tumor
growth inhibitionincastration-resistant prostate cancer(CRPC).
However, the imidazo[1,2-a]pyrimidine moiety is rapidly meta-
bolized by aldehyde oxidase (AO). The present paper describes a number of medicinal chemistry strategies taken to avoid the AO-
mediated oxidation of this particular system. Guided by an AO protein structure-based model, our investigation revealed the most
probable site of AO oxidation and the observation that altering the heterocycle or blocking the reactive site are two of the more
effective strategies for reducing AO metabolism. These strategies may be useful for other drug discovery programs.
’ INTRODUCTION
above indicated that the site of oxidation was somewhere on the
imidazopyrimidine ring, rather than elsewhere in the molecule,
but the exact position could not be determined by this assay.
Clearance mediated by cytochrome P450 enzymes is currently
better understood and utilized today than in previous years. It has
become a common practice in drug discovery to use the data
from liver microsome assays to predict human clearance. How-
ever, in vitro HLM assays cannot accurately predict clearance for
compounds for which AO plays a significant role in metabolism.8
The present paper describes several medicinal chemistry strate-
gies applied to mitigate the AO liability of the imidazopyrimidine
ringin compound 1. Species differences ofAO activity exist among
human, rat, and dog, in that rats have lower AO activity and dogs
possess no AO activity. Hence human AO metabolism is generally
underestimated when based on animal studies. Furthermore, mul-
tiple AO isoforms exist in rat (Aox1, AOX3, Aox4, and Aox3l1)
while only one AO isoform (AOX1) is present in human, which
can result in the formation of different AO metabolites between
rat and human.9 Because of the species differences, it was deter-
mined that a more appropriate approach would be to study AO
metabolism of these compounds in human in vitro system
(human liver S9/cytosol).
N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-tetra-
methylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carboxamide 1 is
a novel, selective pure antagonist of androgen receptor (AR),
which has the potential for therapeutic value in castration-resis-
tant prostate cancer (Figure 1).1 Compound 1 is metabolically
stable in vitro in both human liver microsome and hepatocyte
systems (ER < 0.3). In a rat PK study, compound 1 displayed
acceptable oral absorption (bioavailability F = 31%), low clear-
ance (Clplasma = 11.6 mL/min/kg), and a reasonable half-life
(T1/2 = 3.6 h). The imidazo[1,2-a]pyrimidine-3-carboxamide
moiety in compound 1 is crucial for achieving potent AR full
antagonism. Heteroaryls, such as imidazopyrimidines, are versatile
synthetic building blocks commonly used in medicinal chemistry
because they are often capable of binding to diverse biological
targets with high affinity and providing useful pharmacological
activities.2 In addition, electron-deficient heteroaryls are often
resistant to cytochrome P450-mediated metabolism. However,
an electron-deficient nature may also make the ring carbons sus-
ceptible to nucleophilic attack by aldehyde oxidase (AO), parti-
cularly when they are adjacent to heterocyclic nitrogen(s).3
The presence of a polyaza heteroaryl group in 1 suggested it
could be a substrate for AO metabolism, so the compound was
tested for metabolic stability in human S9 (HS9) fraction. Though
only trace metabolism had been detected in the HLM assay,4 a
significant single mono-oxygenated metabolite M1, was observed
in HS9 without NADPH, suggesting AO oxidation (Figure 1).5
This finding is consistent with the fact that the liver microsome
assay does not contain cytosolic enzymes such as AO.6 Incuba-
tion of 1 in the presence of an AO inhibitor inhibited the forma-
tion of M1, consistent with the involvement of AO in the meta-
bolism of this compound.7 The incubation method described
A common strategy to reduce aromatic oxidation by cyto-
chrome P450 is to replace a carbon in the aromatic ring with a
nitrogen, but doing so can increase the susceptibility of the adja-
cent carbons to AO oxidation.10 The mechanism of AO oxidation
is believed to involve the molybdenum pyranopterin cofactor
moiety (MoCo) of the enzyme in a nucleophilic addition-like
mechanism.11 This theory is supported by the observation that
Received: August 15, 2011
Published: September 28, 2011
r
2011 American Chemical Society
7705
dx.doi.org/10.1021/jm2010942 J. Med. Chem. 2011, 54, 7705–7712
|

Journal of Medicinal Chemistry
ARTICLE
Table 1. Incorporation of a Remote Change in the Molecule
Figure 1. Androgen receptor antagonist 1 and potential metabolite M1.
electron-deficient heteroaromatic rings are common AO sub-
strates. In a heteroaryl system with more than one potential site
for AO metabolism, such as the imidazopyrimidine ring of 1, the
susceptibility of nucleophilic attack by AO will depend not only
on how electron-deficient the reactive site is but also on the shape
of the substrate molecule and the spatial accessibility of the
potentially reactive carbon toward the MoCo moiety. The im-
portance of the interaction between the enzyme and its sub-
strates has been previously addressed by Dastmalchi et al., who
have constructed a homology model of human AO that can be
used for computational studies.12
At the present, there are few publications describing methods
to predict and mitigate AO metabolism, nor are there reliable
computational tools to predict which molecules may be suscep-
tible. Such tools would be useful in drug discovery.13 It has been
hypothesized that the most likely site of AO metabolism is the
most positively charged aromatic carbon adjacent to an aromatic
nitrogen, given that the CꢀH is spatially accessible to AO
enzyme.10 Compound 1 contains three possible sites for AO
oxidation, C22, C27, and C29. However, only one oxidized meta-
bolite (M1, retention time 12.40 min, Figure 1)14 was observed
by LC/MS when compound 1 was incubated with HS9 NADPH,5
suggesting that only one of these carbons is sufficiently electron-
deficient and/or sufficiently accessible to the enzyme.
A number of analogues of 1 were designed to avoid AO
metabolism. Strategies explored to reduce AO oxidation included
introducing a remote structural change in the molecule (Table 1),
evaluating alternative heterocycles (ring connectivity, nitrogen
position, Table 2), and incorporating hindrance either close to or
directly at the suspected site of AO oxidation (Table 3). The
preparation of these analogues is outlined in Scheme 1. Synthesis
begins with nucleophilic displacement of various commercially
available aryl fluorides 2 with the anion of Boc-protected cyclo-
butyl amino alcohol 3, followed by protecting group removal
under acidic conditions to yield amines 5, which serve as versatile
templates for coupling with various carboxylic acids 6 to provide
amides 7 in good yields.1
The first strategy pursued was to introduce a structural change
remote to the potential AO oxidation sites in compound 1 that
would perturb binding to the AO catalytic site. With the assump-
tion that the AR binding site and AO enzyme catalytic site are
different in the region hosting the cyanopyridylether (left-hand)
side of 1, the modification could potentially result in minimal
reduction to AR protein binding affinity but decreased affinity to
the AO enzyme. A number of cyanoaryl ether groups have been
shown to be tolerated for AR binding,1 so analogues with different
arylethers were tested for AO metabolism (Table 1). When the
2-methylnicotinonitrile in 1 was replaced with 2-chlorobenzonitrile
(8), phthalonitrile (9), and 2-methoxynicotinonitrile (10, Table 1),
the AR antagonism potency was maintained, but AO-mediated
oxidation remained the main pathway for their metabolism.
a “yes” indicates an oxygenated metabolite was observed in HS9 without
NADHP suggesting AO oxidation. “no” means no metabolite was
observed under the aforementioned conditions. The spectra for all
compounds can be found in the Supporting Information.
A related strategy was to introduce a substituent on C22 that
could both (1) change the dihedral angle between the amide and
the azapyrimidine ring, resulting in significant change in the most
stable conformation of these molecules and (2) block a potential
oxidation site. Blockage of C22 by a methyl group (11ꢀ12,
Table 1) had no effects on the AO metabolism, indicating that
the AO active site is flexible enough to accommodate significant
conformational changes in this region and that the site of oxi-
dation in these particular molecules may be located at C27 or
C29. Unlike P450-mediated metabolism, there is no apparent
correlation between AO metabolism and lipophilicity, consistent
with the observations reported by Pryde et al.3
Because remote structural changes or substitution to change
the dihedral angle of the amide bond did not reduce the propen-
sity of the molecules to be metabolized by AO, more significant
structural changes were investigated. The next strategy pursued
was to incorporate alternative heterocycles with different hetero-
atom placement, connection points, or ring systems. A similar
strategy has been reported to successfully eliminate AO metabo-
lism for a series of azetidinyl ketolide antibiotics.13 Several com-
pounds with significantly reduced AO liability were identified by
this strategy (Table 2). Compound 13, in which N30 of com-
pound 1 is replaced with a carbon, was designed to eliminate the
potential for AO oxidation at the adjacent C29. This compound
shows no oxidation by AO, but it becomes more susceptible to
7706
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712

Journal of Medicinal Chemistry
ARTICLE
Table 2. Evaluation of Alternative Heterocycles
Table 3. Direct Effect on C28 and C29
a “yes” indicates an oxygenated metabolite was observed in HS9 without
NADHP suggesting AO oxidation. “no” means no metabolite was
observed under the aforementioned conditions. The spectra for all
compounds can be found in the Supporting Information.
(AR), the alternative heterocycle strategy may be applied to
other target designs for mitigating AO risk.
Because drastic changes of the right-hand heterocycle had a
negative effect on potency against the primary target, a final
strategy was pursued in which the connection point and hetero-
atom arrangement of the imidazopyrimidine ring of 1 were
preserved but substitution was introduced to block the reactive
sites (Table 3). It has already been shown that substitution at
C22 does not prevent AO oxidation, leaving C27 or C29 as the
most likely oxidations sites. Thus, introduction of steric hin-
drance at C28 has the potential to interfere with AO metabolism.
Compounds 22 and 23 (Table 3) were prepared to test the
strategy. The steric bulk of a methyl (compound 22) or bromo
group (compound 23) at C28 did not have any significant effect
on the oxidation at C27 and C29, as the rate of metabolism
remained high. Electronic effects (withdrawing or donating) of
bromo, fluoro, and methyl substitutents at C28 (22ꢀ24) did
not change the rate of oxidation either. On the other hand,
substitution directly at C29 with a methoxy group (25) or a
morpholino group (26) completely eliminated AO oxidation.
From these observations, we can conclude that the C29 position
of 1 is the most probable site of AO oxidation. As such, the
structure of the single observed metabolite M1 may be com-
pound 1 with its C29 hydroxylated (Figure 1).
Compound 1 was submitted to a computational docking
protocol described by Pryde et al.3 in which a homology model
of human AO is used to predict the site of metabolism. The 3D
structure of the docked pose, and the potential sites of metabo-
lism of compound 1 are shown in Figure 2. The docking result
showed that the predicted site of metabolism C29 is at 2.1 Å away
from the MoCo moiety. Taking into consideration the rigidity of
compound 1, C22 or C27 are unlikely the sites of metabolism
because, according to the modeled complex, they are not acces-
sible to the MoCo moiety. Figure 2 also shows that there is
a “yes” indicates oxygenated metabolite(s) was qualitatively observed in
HS9 without NADHP suggesting AO oxidation. “no” means no
metabolite was observed under the aforementioned conditions. b Com-
pound 15 indicated a very small peak corresponding to a second
oxygenated species. The spectra for all compounds can be found in
the Supporting Information.
P450-mediated oxidation (HLM ER 0.5) and is poorly soluble in
water (kinetic solubility < 0.4uM), likely due to the increase in
lipophilicity (cLogP 5.51 vs 4.51 [1]). Interestingly, AO oxida-
tion remained when the heterocyclic nitrogen was moved from
N30 to N29 (compound 14, Table 2). In this case, even though
there are more aromatic CꢀH next to a nitrogen in the 5/6
heteroaryl ring, only one oxidized metabolite was observed in the
AO assay as well. Moving the amide connecting point from the
3-position (as in 1) to the 2 position of the imidazopyrimidine
(as in 15) did not reduce AO oxidation, but replacement of C22
with a nitrogen (compound 16) seemed to prevent the oxidation
at the remaining positions (C27 and C29). Saturation of the six-
membered aromatic ring (compound 18) also eliminated AO
metabolism. Compounds 17 and 19ꢀ21 all still contain poten-
tial AO metabolism sites as unsubstituted aromatic CꢀH are pre-
sent adjacent to aromatic nitrogens, but these compounds were
nevertheless all free of AO liability. Perhaps the significant
structural changes to the right-hand side region prevent recogni-
tion by the AO enzyme. Although the right-hand side replace-
ments resulted in loss of potency against our target of interest
7707
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Cyclobutyl Analoguesa
a Reagents and conditions: (i) NaH, DMF; (ii) HCl, EtOAC; iii) HBTU, DMF.
General Procedure for the Preparation of Cyclobutyl Ana-
logues. Preparation of 1: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)-
oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carbox-
amide. Step 1: 6-((1r,3r)-3-Amino-2,2,4,4-tetramethylcyclobutoxy)-
2-methylnicotinonitrile Hydrochloride. To a cooled stirred solution of
compound 31 (4.8 g, 19.7 mmol) in anhydrous tetrahydrofuran (80 mL)
was added sodium hydride (1.6 g, 39.4 mmol) in portions and at a tem-
perature below 10 °C. After the addition, the mixture was stirred at room
temperature for 30 min. Then the reaction mixture was cooled to 10 °C,
and 6-fluoro-2-methylnicotinonitrile (Ryan Scientific, PC5881) (3.2 g,
23.6 mmol) was added in portions. After the addition, the resulting mixture
was stirred at reflux for 1 h under N2. TLC (4:1 petroleum ether/ethyl
acetate) showed the reaction was complete. The reaction was cooled to
room temperature and quenched with cold water (10 mL). The resulting
mixture was evaporated, and the residue was diluted with ethyl acetate
(100 mL) and water (100 mL). The organic layer was separated, and the
aqueous layer was re-extracted with ethyl acetate (100 mL ꢁ 2). The
combined organic layers were washed with brine (50 mL), dried over
sodium sulfate, filtered, and evaporated to give the crude product, which
was purified by column chromatography (petroleum ether/ethyl acetate =
20:1 to 10:1) to give Boc-protected intermediate: tert-butyl(1r,3r)-3-(5-
cyano-6-methylpyridin-2-yloxy)-2,2,4,4 tetramethylcyclobutylcarbamate
Figure 2. Docking poses for compound 1.
1
potential room to accommodate small substitutions at C22, like
the methyl group of compounds 11 and 12, helping to explain
why C22 substitution in compound 11 and 12 did not affect the
(4.2 g, 59%) as white solid. H NMR (400 MHz, DMSO-d6) δ ppm
1.03 (s, 6 H) 1.11 (s, 6 H) 1.40 (s, 9 H) 2.55 (s, 3 H) 3.51 (d, J = 9.60 Hz,
1 H) 4.56 (s, 1 H) 6.66 (d, J = 9.35 Hz, 1 H) 6.82 (d, J = 8.59 Hz, 1 H) 8.04
(d, J = 8.59 Hz, 1 H).
AO metabolism.
In summary, the imidazo[1,2-a]pyrimidine ring system has
been identified as a representative example of heteroaryl systems
which are substrates of AO-mediated metabolism. Multiple medi-
cinal chemistry approaches were employed to reduce the AO lia-
bility, and our investigation suggests that alternative heterocycles
or direct blockade of reactive site are two of the more effective
strategies. The potential reactive site(s) may be predicted by the
AO protein structure-based model. The medicinal chemistry ap-
proaches described here may be applicable to other drug discovery
programs in which similar AO metabolism problems are encoun-
tered.
To a stirred solution of above intermediate tert-butyl(1r,3r)-3-
(5-cyano-6-methylpyridin-2-yloxy)-2,2,4,4 tetramethylcyclobutylcarba-
mate (4.2 g, 11.6 mmol) in anhydrous dioxane (60 mL) was added 4 N
HCl/dioxane (80 mL) dropwise at 5 °C. After the addition, the resulting
mixture was stirred at room temperature for 3 h. The suspension was
filtered, and the filter cake was washed with petroleum ether/ethyl
acetate = 10:1 (50 mL), dried in vacuo to give the hydrochloride salt of
the title compound (3.47 g, 91%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.08 (s, 6 H) 1.30 (s, 6 H) 2.56 (s, 3 H) 3.05 (q, J =
5.56 Hz, 1 H) 4.66 (s, 1 H) 6.86 (d, J = 8.59 Hz, 1 H) 8.07 (d, J = 8.59 Hz,
1 H) 8.33 (br s, 3 H). MS (APCI): 260.2 (M + H)+.
Step 2: Coupling Reaction: N-((1r,3r)-3-(5-Cyano-6-methylpyri-
din-2-yloxy)-2,2,4,4-tetramethylcyclobutyl)imidazo[1,2-a]pyrimidine-
3-carboxamide (1). 6-((1r,3r)-3-Amino-2,2,4,4-tetramethylcyclobut-
oxy)-2-methylnicotinonitrile hydrochloride from step 1 (70.0 mg,
0.237 mmol, 1.00 equiv), imidazo[1,2-a]pyrimidine-3-carboxylic acid
(Ryan Scientific, CA10088) (44.9 mg, 0.275 mmol, 1.16 equiv), and
HBTU (O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-
phosphate CAS no. 94790-37-1, 113.9 mg, 0.29 mmol, 1.2 equiv) were
dissolved in 1.0 mL of dimethyl formamide (0.237 M). Triethyl amine
(0.10 mL 0.72 mmol, 3.0 equiv) was added, and the reaction was stirred
at room temperature for 2.5 h. LCMS after 2 h at room temperature
showed clean conversion to amide coupling product (1.759 min, (M +
H)+ 405, Rf = 0.16 in 100% EtOAc, UV+). The reaction was partitioned
between 10 mL of deionized water and 20 mL of ethyl acetate. The
organic layer was washed with 5 mL of brine, dried over magnesium
sulfate, filtered, and concentrated to 131.9 mg of a yellow wax, which was
’ EXPERIMENTAL SECTION
General. All starting materials and chemicals reagents were pur-
chased from commercial suppliers and used without further purification
unless otherwise indicated. 1H NMR spectra were recorded on a Bruker
instrument operating at 400 or 600 MHz. NMR spectra are obtained as
CDCl3, DMSO-d6 solutions (reported in ppm), using CDCl3 as the ref-
erence standard (7.25 ppm), or DMSO-d6 (2.50 ppm). Other NMR
solvents were used as needed. The mass spectra were obtained using
LC/MS or HRMS. The purity of all final compounds was determined
to be at least 95% pure by a combination of HPLC, LCMS, NMR
(no extra peaks in the proton NMR spectrum), and combustion analysis
(all final compounds have satisfying CHN results consistent with high
purity).
7708
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712

Journal of Medicinal Chemistry
ARTICLE
dissolved in ethyl acetate, adsorbed on silica, and purified on 12 g silica
column, eluting with 100% ethyl acetate. The corresponding fractions
were combined and concentrated in vacuo to afford the product as a
white solid 62.8 mg (66%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.15
(s, 6 H), 1.25 (s, 6 H), 2.58 (s, 3 H), 4.12 (d, J = 9.60 Hz, 1 H), 4.79 (s,
1 H), 6.88 (d, J = 8.59 Hz, 1 H), 7.26 (dd, J = 6.95, 4.17 Hz, 1 H), 7.95 (d,
J = 9.60 Hz, 1 H), 8.07 (d, J = 8.59 Hz, 1 H), 8.67ꢀ8.73 (m, 2 H), 9.70
(dd, J = 6.95, 1.89 Hz, 1 H). HRMS: [M + H]+ calcd 405.203351, found
in step 2. 1H NMR (500 MHz, DMSO-d6) δ ppm 8.61 (br s, 1 H), 7.89
(d, J = 8.78 Hz, 1 H), 7.46 (s, 1 H), 7.22 (s, 1 H), 7.17 (br s, 1 H), 7.02
(d, J = 8.78 Hz, 1 H), 4.33 (s, 1 H), 3.98 (s, 1 H), 2.66 (s, 3 H), 1.26 (s, 6
H), 1.18 (s, 6 H). MS (APCI): 438.0 (M + H)+. Anal. Calcd For
C23H24ClN5O2 2.0H2O: C, 58.29; H, 5.95; N, 14.78. Found: C, 57.94;
3
H, 5.93; N, 14.47.
Preparation of 13: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)-
oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyridine-3-carbox-
amide. Prepared by the method of compound 1, using imidazo[1,2-a]-
pyridine-3-carboxylic acid (Ryan scientific, CC25401) instead of
405.202098, error ꢀ3.09 ppm. Anal. Calcd For C22H24N6O2 0.5 H2O:
3
C, 63.91; H, 6.09; N, 20.32. Found: C, 63.85; H, 5.92; N, 20.28.
Characterization of Final Analogues. Preparation of 8: N-
((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-
imidazo[1,2-a]pyrimidine-3-carboxamide. Prepared by the method of
compound 1, using 2-chloro-4-fluoro-benzonitrile (Aldrich, 344265)
1
imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. H NMR (400
MHz, DMSO-d6) δ ppm 1.07 (s, 6 H), 1.16 (s, 6 H), 2.49 (s, 3 H), 4.03
(d, J = 9.35 Hz, 1 H), 4.70 (d, J = 0.51 Hz, 1 H), 6.79 (d, J = 8.84 Hz,
1 H), 7.03 (td, J = 6.88, 1.14 Hz, 1 H), 7.38 (ddd, J = 8.97, 6.82, 1.39 Hz,
1 H), 7.58ꢀ7.67 (m, 1 H), 7.73 (d, J = 9.60 Hz, 1 H), 7.98 (d, J = 8.84
Hz, 1 H), 8.46 (s, 1 H), 9.25ꢀ9.39 (m, 1 H). MS (APCI): 404.0 (M +
1
instead of 6-fluoro-2-methylnicotinonitrile in step 1. H NMR (400
MHz, CDCl3) δ ppm 1.25 (s, 6 H), 1.32 (s, 6 H), 4.10 (s, 1 H), 4.18 (d,
J = 8.34 Hz, 1 H), 6.09 (d, J = 8.08 Hz, 1 H), 6.83 (dd, J = 8.59, 2.53 Hz,
1 H), 6.99 (d, J = 2.53 Hz, 1 H), 7.09 (dd, J = 6.95, 4.17 Hz, 1 H), 7.59 (d,
J = 8.59 Hz, 1 H), 8.22 (s, 1 H), 8.73 (dd, J = 4.29, 2.02 Hz, 1 H), 9.76 (d,
J = 5.05 Hz, 1 H). MS (APCI): 424 (M + H)+. Anal. Calcd For
H)+. Anal. Calcd For C23H25N5O2 1.0H2O: C, 65.54, H, 6.46; N, 16.62.
3
Found: C, 65.63; H, 6.40; N, 16.55.
Preparation of 14: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-
2,2,4,4-tetramethylcyclobutyl}-2-methylimidazo[1,2-a]pyrazine-3-car-
boxamide. Prepared by the method of compound 1, using 2-methyl-
imidazo[1,2-a]pyrazine-3-carboxylic acid (ChemICHIBA, COL-00748)
instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.09 (s, 6 H), 1.18 (s, 6 H), 2.48
(s, 3 H), 2.60 (s, 3 H), 3.88 (d, J = 8.08 Hz, 1 H), 4.64 (s, 1 H), 6.79 (d,
J = 8.34 Hz, 1 H), 7.68 (d, J = 8.34 Hz, 1 H), 7.94 (d, J = 4.55 Hz, 1 H),
7.99 (d, J = 8.59 Hz, 1 H), 8.75 (dd, J = 4.55, 1.26 Hz, 1 H), 9.01 (d, J =
1.26 Hz, 1 H). MS (APCI): 419.0 (M + H)+. Anal. Calcd For C23H26-
N6O2: C, 66.01; H, 6.26; N, 20.08. Found: C, 65.87; H, 6.29; N, 19.85.
Preparation of 15: N-[trans-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl]imidazo[1,2-a]pyrimidine-2-carboxamide. Pre-
pared bythe method of compound 1, using 2-chloro-4-fluoro-benzonitrile
(Aldrich, 344265) instead of 6-fluoro-2-methylnicotinonitrile in step 1
and imidazo[1,2-a]pyrimidine-2-carboxylic acid (Oakwood Products,
043463) instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step
2. 1H NMR (400 MHz, CDCl3) δ ppm 1.24 (s, 6 H) 1.33 (s, 6 H) 4.10 (s,
1 H) 4.21 (d, J = 8.84 Hz, 1 H) 6.83 (dd, J = 8.72, 2.40 Hz, 1 H) 6.94ꢀ7.04
(m, 1 H) 7.58 (d, J = 8.84 Hz, 1 H) 7.74 (d, J = 9.09 Hz, 1 H) 8.11 (s, 1 H)
8.51 (dd, J = 6.82, 2.02 Hz, 1 H) 8.67 (dd, J = 4.04, 2.02 Hz, 1 H). MS
C22H22ClN5O2 1.15H2O: C, 59.43; H, 5.51; N, 15.75. Found: C, 59.19;
3
H, 5.18; N, 15.60.
Preparation of 9: N-[trans-3-(3,4-Dicyanophenoxy)-2,2,4,4-tetra-
methylcyclobutyl]imidazo[1,2-a]pyrimidine-3-carboxamide. Prepared
by the method of compound 1, using 4-fluorophthalonitrile (Aldrich,
1
47410) instead of 6-fluoro-2-methylnicotinonitrile in step 1. H NMR
(400 MHz, CDCl3) δ ppm 9.68 (dd, J = 6.82, 2.02 Hz, 1 H), 8.65 (dd, J =
4.04, 2.02 Hz, 1 H), 8.17 (s, 1 H), 7.66 (d, J = 8.84 Hz, 1 H), 7.17 (d, J =
2.53 Hz, 1 H), 7.08 (dd, J = 8.84, 2.53 Hz, 1 H), 7.02 (dd, J = 6.95, 4.17 Hz,
1 H), 6.06 (d, J = 8.08 Hz, 1 H), 4.13 (d, J = 8.08 Hz, 1 H), 4.07 (s, 1 H),
1.26 (s, 6 H), 1.18 (s, 6 H). MS (APCI): 415.0 (M + H)+. Anal. Calcd For
C23H22N6O2 1.0H2O: C, 63.88; H, 5.59; N, 19.43. Found: C, 63.73; H,
3
5.55; N, 19.34
Preparation of 10: N-{trans-3-[(5-Cyano-6-methoxypyridin-2-yl)-
oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carbo-
xamide. Prepared by the method of compound 1, using 6-chloro-2-
methoxynicotinonitrile (ArrayChem, P21111) instead of 6-fluoro-2-
1
methylnicotinonitrile in step 1. H NMR (400 MHz, DMSO-d6) δ
ppm 1.16 (s, 6 H), 1.26 (s, 6 H), 4.01 (s, 3 H), 4.15 (d, J = 9.60 Hz, 1 H),
4.81 (s, 1 H), 6.60 (d, J = 8.34 Hz, 1 H), 7.26 (dd, J = 6.82, 4.04 Hz, 1 H),
7.93 (d, J = 9.35 Hz, 1 H), 8.08 (d, J = 8.34 Hz, 1 H), 8.69 (s, 1 H), 8.71
(dd, J = 4.04, 2.02 Hz, 1 H), 9.70 (dd, J = 6.82, 2.02 Hz, 1 H). Anal.
(APCI): 424.0 (M + H)+. Anal. Calcd For C22H22ClN5O2 0.1H2O: C,
3
62.07; H, 5.26; N, 16.45. Found: C, 61.88; H, 5.30; N, 16.21.
Preparation of 16: N-[trans-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl][1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide.
Prepared by the method of compound 1, using 2-chloro-4-fluoro-ben-
zonitrile (Aldrich, 344265) instead of 6-fluoro-2-methylnicotinonitrile
in step 1 and [1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylic acid (Ryan
Scientific, B001275) instead of imidazo[1,2-a]pyrimidine-3-carboxylic
acid in step 2. 1H NMR (400 MHz, CDCl3) δ ppm 1.25 (s, 6 H), 1.34
(s, 6 H), 4.11 (s, 1 H), 4.27 (d, J = 8.84 Hz, 1 H), 6.83 (dd, J = 8.72,
2.40 Hz, 1 H), 6.99 (d, J =2.53 Hz, 1 H), 7.29 (s, 1 H), 7.59 (d, J =8.84Hz,
1 H), 7.74 (d, J = 9.35 Hz, 1 H), 8.91ꢀ9.04 (m, 2 H). MS (APCI): 425.0
Calcd. For C22H24N6O3 0.1H2O: C, 62.58; H, 5.78; N, 19.90. Found:
3
C, 62.89; H, 6.09; N, 19.51.
Preparation of 11: N-[trans-3-(3,4-Dicyanophenoxy)-2,2,4,4-tetra-
methylcyclobutyl]-2-methylimidazo[1,2-a]pyrimidine-3-carboxamide.
Prepared by the method of compound 1, using 4-fluorophthalonitrile
(Aldrich, 47410) instead of 6-fluoro-2-methylnicotinonitrile in step 1
and 2-methyl-imidazo[1,2-a]pyrimidine-3-carboxylic acid (Ryan Scien-
tific, CA10041) instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid
in step 2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.17 (s, 6 H), 1.28 (s, 6
H), 2.66 (s, 3 H), 3.98 (d, J = 7.83 Hz, 1 H), 4.39 (s, 1 H), 7.18 (dd, J =
6.95, 4.17 Hz, 1 H), 7.36 (dd, J = 8.84, 2.53 Hz, 1 H), 7.54 (d, J = 7.83 Hz,
1 H), 7.67 (d, J = 2.53 Hz, 1 H), 8.06 (d, J = 8.59 Hz, 1 H), 8.63 (dd, J =
4.29, 2.02 Hz, 1 H), 9.29 (dd, J = 6.82, 2.02 Hz, 1 H). HRMS: calcd For
C24H24N6O2 (M + H)+: 429.203351, found 429.201887, error ꢀ3.41
(M + H)+. Anal. Calcd For C21H21ClN6O2 0.1H2O: C, 59.11; H, 5.01;
3
N, 19.70. Found: C, 59.05; H, 4.98; N, 19.66.
Preparation of 17: N-[trans-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl]imidazo[1,5-a]pyridine-8-carboxamide. Pre-
pared by the method of compound 1, using 2-chloro-4-fluoro-benzoni-
trile (Aldrich, 344265) instead of 6-fluoro-2-methylnicotinonitrile in
step 1 and imidazo[1,5-a]pyridine-8-carboxylic acid (Best PharmaTec,
BP26399) instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step
2. 1H NMR (500 MHz, DMSO-d6) δ ppm 8.48 (d, J = 6.83 Hz, 1 H),
8.45 (s, 1 H), 7.84ꢀ7.92 (m, 2 H), 7.55 (s, 1 H), 7.27 (d, J = 6.34 Hz,
1 H), 7.21 (d, J = 2.44 Hz, 1 H), 7.01 (dd, J = 8.78, 1.95 Hz, 1 H), 6.76
(t, J = 6.83 Hz, 1 H), 4.33 (s, 1 H), 4.07 (d, J = 8.78 Hz, 1 H), 1.24
(s, 6 H), 1.16 (s, 6 H). MS (APCI): 423.2 (M + H)+. Anal. Calcd For
ppm. C24H24N6O2 0.25H2O: C, 66.57; H, 5.70; N, 19.41. Found: C,
3
66.40; H, 5.47; N, 19.35.
Preparation of 12: N-[trans-3-(2-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl]-2-methylimidazo[1,2-a]pyrimidine-3-carbox-
amide. Prepared by the method of compound 1, using 3-chloro-4-fluoro-
benzonitrile (Aldrich, 376582) instead of 6-fluoro-2-methylnicotinonitrile in
step 1 and 2-methyl-imidazo[1,2-a]pyrimidine-3-carboxylic acid (Ryan
Scientific, CA10041) instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid
7709
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712

Journal of Medicinal Chemistry
ARTICLE
C23H23ClN4O2 0.75H2O: C, 63.30; H, 5.66; N, 12.84. Found: C, 63.22;
H, 5.50; N, 12.65.
and then poured over saturated aqueous sodium bicarbonate (100 mL)
and extracted with ethyl acetate (2 ꢁ 100 mL). The organic layer was
dried over anhydrous sodium sulfate, and the filtrate was concentrated in
vacuo. The resultant residue was diluted in dichloromethate and loaded
into a silica column. The column was eluted with heptane/ethyl acetate
0ꢀ50%. The fractions containing the product were combined and
evaporated to give ethyl 6-methylimidazo[1,2-a]pyrimidine-3-carboxy-
late as a yellow solid (2.28 g, 80%). 1H NMR (acetone, 400 MHz): δ
(ppm) 9.23ꢀ9.49 (m, 1H), 8.65 (d, J = 2.5 Hz, 1H), 8.31 (s, 1H), 4.41
(q, J = 7.2 Hz, 2H), 2.49 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H).
To a solution of ethyl 6-methylimidazo[1,2-a]pyrimidine-3-carboxy
late (from above) (2.28 g, 11.11 mmol) in tetrahydrofuran (20 mL) a 1N
lithium hydroxide solution (16.7 mL, 16.7 mmol) was added. The
mixture was stirred at room temp overnight. The reaction was taken to
pH = 5 with 6 N HCl, and the volatiles were removed. The solid ob-
tained was filtered and the cake washed with water. The solid was dried
in a vacuum oven to give the titled product as a yellow solid (302 mg,
66%).
Step 2: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-
tetramethylcyclobutyl}-6-methylimidazo[1,2-a]pyrimidine-3-carbox-
amide 22. Prepared by the method of compound 1, using 6-
methylimidazo[1,2-a]pyrimidine-3-carboxylic acid (from step 1 above)
instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. 1H NMR
(DMSO-d6, 400 MHz): δ ppm 9.53 (s, 1H), 8.62 (s, 1H), 8.60 (d, J =
2.5 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 9.3 Hz, 1H), 6.88 (d, J =
8.6 Hz, 1H), 4.78 (s, 1H), 4.11 (d, J = 9.1 Hz, 1H), 2.58 (s, 3H), 2.38 (s,
3H), 1.25 (s, 6H), 1.15 (s, 6H). MS (APCI): 419.50 (M + H)+. Anal.
3
Preparation of 18: N-((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyridine-
3-carboxamide. Prepared by the method of compound 1, using
2-chloro-4-fluoro-benzonitrile (Aldrich, 344265) instead of 6-fluoro-2-
methylnicotinonitrile in step 1 and 5,6,7,8-tetrahydro-[1,2,4]triazolo-
[4,3-a]pyridine-3-carboxylic acid (Anichem H10612) instead of imi-
dazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.04 (d, J = 9.35 Hz, 1 H), 7.91 (d, J = 8.84 Hz, 1 H),
7.24 (d, J = 2.27 Hz, 1 H), 7.03 (dd, J = 8.72, 2.40 Hz, 1 H), 4.45 (s, 1 H),
4.25 (t, J = 5.94 Hz, 2 H), 3.99 (d, J = 9.09 Hz, 1 H), 2.91 (t, J = 6.32 Hz,
2 H), 2.68 (dt, J = 3.73, 1.80 Hz, 1 H), 2.28ꢀ2.38 (m, 1 H), 1.75ꢀ1.97
(m, 2 H), 1.24 (s, 6 H), 1.15 (s, 6 H). MS (APCI): 428.2 (M + H)+.
HRMS: calcd For C22H26ClN5O2 (M + H)+: 427.177506, found
427.1769798. Anal. Calcd For C22H26ClN5O2 0.5H2O: C, 60.48; H,
3
6.23; N, 16.03. Found: C, 60.30; H, 6.15; N, 15.93.
Preparation of 19: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-
2,2,4,4-tetramethylcyclobutyl}-6-methoxyimidazo[1,2-b]pyridazine-3-
carboxamide. Prepared by the method of compound 1, 6-meth-
oxyimidazo[1,2-b]pyridazine-3-carboxylic acid (preparation reported
in Tetrahedron 1967, 23, 2739ꢀ2746) instead of imidazo[1,2-a]-
1
pyrimidine-3-carboxylic acid in step 2. H NMR (acetonitrile-d3, 400
MHz): δ (ppm) 8.31 (d, J = 8.6 Hz, 1H), 8.14 (s, 1H), 7.97 (d,
J = 9.6 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 6.96 (d, J = 9.6 Hz, 1H), 6.77 (d,
J = 8.6 Hz, 1H), 4.76 (s, 1H), 4.27 (d, J = 8.1 Hz, 1H), 4.12 (s, 3H), 2.59
(s, 3H), 1.31 (s, 6H), 1.16 (s, 6H). MS (APCI): 435.2 (M + H)+. Anal.
Calcd For C23H26N6O3: C, 63.58; H, 6.03; N, 19.34. Found: C, 63.32;
H, 5.99; N, 19.14.
Calcd For C23H26N6O2 0.1H2O: C, 65.73; H, 6.28; N, 20.00. Found: C,
3
65.41; H, 6.29; N, 19.94.
Preparation of 23: 6-Bromo-N-{trans-3-[(5-cyano-6-methylpyri-
din-2-yl)oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-
3-carboxamide. Prepared by the method of compound 1, using 6-
bromoimidazo[1,2-a]pyrimidine-3-carboxylic acid (Anichem, H13576)
instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. 1H
NMR (acetonitrile-d3, 400 MHz): δ ppm 9.84 (d, J = 2.5 Hz, 1H),
8.66 (d, J = 2.5 Hz, 1H), 8.40 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 6.76 (d, J =
8.6 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 4.75 (s, 1H), 4.11 (d, J = 9.1 Hz,
1H), 2.60 (s, 3H), 1.28 (s, 6H), 1.18 (s, 6H). MS (APCI): 484.3 (M +
Preparation of 20: N-[trans-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide. Pre-
pared by the method of compound 1, using 2-chloro-4-fluoro-benzonitrile
(Aldrich, 344265) instead of 6-fluoro-2-methylnicotinonitrile in step 1
and 1H-pyrrolo[2,3-b]pyridine-5-carboxylic acid (Anichem T12723) ins-
1
tead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. H NMR
(400 MHz, DMSO-d6) δ ppm 1.16 (s, 6 H), 1.26 (s, 6 H), 4.13 (d, J = 9.09
Hz, 1 H), 4.33 (s, 1 H), 6.58 (d, J = 3.54 Hz, 1 H), 7.02 (dd, J = 8.84, 2.53
Hz, 1 H), 7.22 (d, J = 2.27 Hz, 1 H), 7.57 (d, J = 3.28 Hz, 1 H), 7.84ꢀ7.94
(m, 2 H), 8.45 (d, J = 2.27 Hz, 1 H), 8.71 (d, J = 2.02 Hz, 1 H), 11.91 (s,
1 H). HRMS: (M + H)+ calcd 423.15823, found 423.158189. Anal. Calcd
H)+. Anal. Calcd For C22H23BrN6O2 0.2EtOAc: C, 54.66; H, 4.95; N,
3
16.77. Found: C, 54.84; H, 5.22; N, 16.43.
Preparation of 24: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-
2,2,4,4-tetramethylcyclobutyl}-6-fluoroimidazo[1,2-a]pyrimidine-3-
carboxamide. Step 1: 6-Fluoroimidazo[1,2-a]pyrimidine-3-carbo-
xylic Acid. To a solution of (E)-ethyl 3-ethoxyacrylate (Aldrich,
250120) (638 mg, 4.42 mmol) in a mixture of water and 1,4-dioxane
0.5:1 (10 mL) at room temperature, N-Bromosuccinamide was added
(750 mg, 4.21 mmol). The mixture was stirred at room temperature for
10 min, and 5-fluoropyrimidin-2-amine (Cheminstock Ltd., C4795) was
added (500 mg, 4.42 mmol). The reaction was heated in a microwave to
100 °C for 10 min. The reaction was poured over saturated aqueous
sodium bicarbonate, extracted with ethyl acetate, and dried over anhy-
drous sodium sulfate. The volatiles were removed, and the residue was
diluted in dichloromethane and loaded into a silica column. The column
was eluted with heptane/ethyl acetate 0ꢀ50%. The fractions containing
the product were combined and evaporated to give ethyl 6-fluoro-
imidazo[1,2-a]pyrimidine-3-carboxylateas a yellow solid (529 mg, 57%).
1H NMR (acetonitrile-d3, 400 MHz): δ ppm 9.50 (dd, J = 4.3, 3.0 Hz,
1H), 8.75 (d, J = 3.0 Hz, 1H), 8.40 (s, 1H), 4.42 (q, J = 7.1 Hz, 2H), 1.39
(t, J = 7.1 Hz, 3H). MS (APCI): 210 (M + H)+.
For C23H23ClN4O2 0.5H2O: C, 66.82; H, 5.73; N, 10.16. Found:. C,
66.57; H, 5.64; N, 10.13.
3
Preparation of 21: N-[trans-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl][1,2,4]triazolo[4,3-a]pyridine-6-carboxamide.
Prepared by the method of compound 1, using 2-chloro-4-fluoro-
benzonitrile (Aldrich, 344265) instead of 6-fluoro-2-methylnicotinoni-
trile in step 1 and [1,2,4]triazolo[4,3-a]pyridine-6-carboxylic acid
(ACC, CHM0079893) instead of imidazo[1,2-a]pyrimidine-3-car-
boxylic acid in step 2. 1H NMR (400 MHz, CDCl3) δ ppm 1.18 (s, 6
H), 1.24 (s, 6 H), 4.01 (s, 1 H), 4.11 (d, J = 8.08 Hz, 1 H), 6.12 (d, J =
7.83 Hz, 1 H), 6.74 (dd, J = 8.72, 2.40 Hz, 1 H), 6.90 (d, J = 2.27 Hz,
1 H), 7.42 (dd, J = 9.60, 1.26 Hz, 1 H), 7.51 (d, J = 8.84 Hz, 1 H), 7.80 (d,
J = 9.35 Hz, 1 H), 8.71 (s, 1 H), 8.87 (s, 1 H). MS (APCI): 424.2 (M +
H)+. Anal. Calcd For C22H22ClN5O2 0.5H2O: C, 61.04; H, 5.36; N,
3
16.18. Found: C, 60.89; H, 5.32; N, 15.80.
Preparation of 22: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)-
oxy]-2,2,4,4-tetramethylcyclobutyl}-6-methylimidazo[1,2-a]pyrimidine-
3-carboxamide. Step 1: 6-Methylimidazo[1,2-a]pyrimidine-3-car-
boxylic Acid. (E)-Ethyl 3-ethoxyacrylate (Aldrich, 250120) (991 mg,
6.87 mmol) was dissolved in a mixture of water and dioxane 0.5:1
(10 mL) at rt, N-bromosuccinamide (1160 mg, 6.54 mmol) was added
and the mixture was stirred at room temperature for 10 min, and 5-
methylpyrimidin-2-amine (Anichem, H12650) (750 mg, 6.87 mmol)
was added. The reaction was heated in a microwave to 100 °C for 10 min
To a solution of ethyl 6-fluoroimidazo[1,2-a]pyrimidine-3-carboxy-
late (described above) (529 mg, 2.53 mmol) in tetrahydrofuran (10 mL)
a 1N lithium hydroxyde solution (3.79 mL, 3.79 mmol) was added and
10 mL of water. The mixture was stirred at room temp overnight. The
reaction was taken to pH = 5 with 6 N HCl (0.650 mL, 4 mmol), and
7710
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712

Journal of Medicinal Chemistry
ARTICLE
the volatiles were removed. The solid obtained was filtered and the cake
washed with water. The solid was dried in a vacuum oven to give the prod-
uct as a yellow solid (302 mg, 66%). 1H NMR (DMSO-d6, 400 MHz): δ
ppm 9.48 (dd, J = 4.3, 3.0 Hz, 1H), 8.89 (d, J = 3.0 Hz, 1H), 8.35 (s, 1H).
MS (APCI): 182 (M + H)+.
Step 2: N-((1r,3r)-3-(5-Cyano-6-methylpyridin-2-yloxy)-2,2,4,4-
tetramethylcyclobutyl)-6-fluoroimidazo[1,2-a]pyrimidine-3-carbox-
amide 24. Prepared by the method of compound 1, using 6-fluoro-
imidazo[1,2-a]pyrimidine-3-carboxylic acid (from step 1 above) instead
of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2. 1H NMR
(acetonitrile-d3, 400 MHz): δ ppm 9.71 (dd, J = 4.5, 3.0 Hz, 1H),
8.70 (d, J = 3.0 Hz, 1H), 8.46 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 6.76 (d, J =
8.6 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 4.75 (s, 1H), 4.12 (d, J = 9.1 Hz,
1H), 2.60 (s, 3H), 1.28 (s, 6H), 1.13ꢀ1.19 (m, 6H). MS (APCI): 423.4
(M + H)+. Anal. Calcd For C22H23FN6O2: C, 62.55; H, 5.49; N, 19.89.
Found: C, 62.32; H, 5.56; N, 19.66.
(APCI): 435.5 (M + H)+. Anal. Calcd For C23H26N6O3 0.1EtOAc: C,
63.40; H, 6.09; N, 18.96. Found: C, 63.33; H, 6.08; N, 18.70.
3
Preparation of 26: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)-
oxy]-2,2,4,4-tetramethylcyclobutyl}-7-(morpholin-4-yl)imidazo[1,2-a]-
pyrimidine-3-carboxamide. Step 1: 4-Morpholinopyrimidin-2-amine.
To a solution of 4-chloropyrimidin-2-amine (Aldrich, 661325) (100 mg,
0.772 mmol) in DMSO (2 mL), morpholine (0.202 mL, 1.36 mmol) and
potassium carbonate (320 mg, 2.32 mmol) were added. The reaction was
heatedin a microwave to 100 °C for 20 min. The reaction was diluted with
water, saturated with anhydrous sodium sulfate, and extracted with ethyl
acetate (3 ꢁ 10 mL). The organics were dried over anhydrous sodium
sulfate and evaporated to give the product as a crystalline solid (139 mg,
93%). 1H NMR (DMSO-d6, 400 MHz): δ ppm 8.38 (d, J = 6.1 Hz, 1H),
6.53 (d, J = 6.1 Hz, 1H), 5.68 (br s, 2H), 4.16ꢀ4.27 (m, 4H), 3.97ꢀ4.09
(m, 4H). MS (APCI): 181.2 (M + H)+.
Step 2: 7-Morpholinoimidazo[1,2-a]pyrimidine-3-carboxylic Acid.
To a solution of (E)-ethyl 3-ethoxyacrylate (111 mg, 0.772 mmol) in a
mixture of water and dioxane 1:1 (1 mL) at room temperature, was added
N-bromosuccinamide (131 mg, 0.735 mmol). The mixture was stirred at
room temperature for 10 min, and 4-morpholinopyrimidin-2-amine
(from step 1 above) was added (139 mg, 0.772 mmol). The reaction
was heated in a microwave to 100 °C for 20 min. The reaction was
neutralized with aqueous sodium bicarbonate and extracted with ethyl
acetate (3 ꢁ 10 mL). The organics were dried over sodium sulfate and
evaporated. The residue was diluted in dichloromethane, loaded into a
12 g silica column, and eluted with ethyl acetate. The fractions containing the
product were combined to give 7-morpholinoimidazo[1,2-a]pyrimidine-3-
carboxylate as a white solid (60 mg, 28%).
Preparation of 25: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)-
oxy]-2,2,4,4-tetramethylcyclobutyl}-7-methoxyimidazo[1,2-a]pyrimi-
dine-3-carboxamide. Step 1: 4-Methoxypyrimidin-2-amine. To a
solution of 4-chloropyrimidin-2-amine (Aldrich, 661325) (1.5 g, 11.58
mmol) in methanol (100 mL), a 25% w/w solution of sodium meth-
oxide/methanol (6 mL, 30 mmol) was added portionwise. The reaction
was stirred at room temp for 24 h. To the reaction 1.93 mL of 6 N HCl
were added and the volatiles were removed. The residue was dissolved in
water, and the pH was adjusted to 7 using more HCl and sodium
bicarbonate. The aqueous mixture was extracted with ethyl acetate (4 ꢁ
20 mL), dried over anhydrous sodium sulfate, and evaporated to dryness
1
to give the crude product as a yellow solid (1.34 g, 92%). H NMR
To a solution of ethyl 7-morpholinoimidazo[1,2-a]pyrimidine-3-
carboxylate (described above) (60 mg, 0.22 mmol) in tetrahydrofuran
(1 mL) was added 1N lithium hydroxide solution (0.240 mL, 0.239 mmol).
The mixture was stirred at room temperature for 8 h. After the hydrolysis was
complete, 6 N HCl (0.040 mL) were added. The volatiles were removed,
and the residue was triturated in water. The supernatant was decanted and
the residue freeze-dried to give the product as a white solid (60 mg,
(acetone, 400 MHz): δ ppm 7.95 (d, J = 5.6 Hz, 1H), 6.00 (d, J = 5.6 Hz,
1H), 5.91 (br s, 2H), 3.81 (s, 3H). MS (APCI): 126 (M + H)+.
Step 2: 7-Methoxyimidazo[1,2-a]pyrimidine-3-carboxylic Acid.
To a solution of (E)-ethyl 3-ethoxyacrylate (Aldrich, 250120) (691 mg,
4.8 mmol) in a mixture of water and 1,4-dioxane 0.5:1 (10 mL) at room
temperature, N-bromosuccinamide (813 mg, 4.57 mmol) was added. The
mixture was stirred at room temperature until the yellow color disap-
peared (10 min). To this solution, 4-methoxypyrimidin-2-amine (from
step 1 above) (600 mg, 4.8 mmol) was added and the reaction was heated
in a microwave to 100 °C for 15 min. The reaction was poured into an
aqueous saturated sodium bicarbonate solution, and the aqueous solution
was extracted with ethyl acetate (3 ꢁ 50 mL). The organics were
combined and dried over anhydrous sodium sulfate. The volatiles were
removed, and the residue was loaded into a silica column and eluted with
heptane/ethyl acetate 0ꢀ50%. The fractions containing the ethyl 7-meth-
oxyimidazo[1,2-a]pyrimidine-3-carboxylate as a yellow solid (627 mg,
29.5%).
To a solution of ethyl 7-methoxyimidazo[1,2-a]pyrimidine-3-carboxy-
late (described above) (627 mg, 2.83 mmol) in methanol (20 mL), a 1N
lithium hydroxide solution (4.25 mL, 4.25 mmol) was added. The
mixture was stirred at room temp overnight. The solution was adjusted
to pH = 5 with 6 N HCl, the volatiles removed, and the solid obtained
filtered and washed with water. The solid was dried in a vacuum oven to
give 7-methoxyimidazo[1,2-a]pyrimidine-3-carboxylic acid as a white
solid (373 mg, 68%). 1H NMR (DMSO-d6, 400 MHz): δ ppm 9.31 (d,
J = 7.3 Hz, 1H), 8.31 (s, 1H), 6.95 (d, J = 7.3 Hz, 1H), 4.02 (s, 3H). MS
(APCI): 194 (M + H)+.
1
quantitative; contained 10% LiCl). H NMR (DMSO-d6, 400 MHz): δ
ppm 9.59 (dd, J = 4.4, 3.2 Hz, 1H), 8.82 (d, J = 3.0 Hz, 1H), 8.24 (s, 1H),
2.26ꢀ2.36 (m, 4H), 2.16ꢀ2.23 (m, 4H). MS (APCI): 249 (M + H)+.
Step 3: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-tet-
ramethylcyclobutyl}-7-(morpholin-4-yl)imidazo[1,2-a]pyrimidine-3-
carboxamide 26. Prepared by the method of compound 1, using
7-morpholinoimidazo[1,2-a]pyrimidine-3-carboxylic acid (prepared in
step 2 above) instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in
step 2. 1H NMR (acetonitrile-d3, 400 MHz): shift (ppm) 9.24 (d, J = 8.1
Hz, 1H), 8.01 (s, 1H), 7.85 (d, J = 8.6 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H),
6.65 (d, J = 7.8 Hz, 1H), 6.45 (d, J = 8.6 Hz, 1H), 4.72 (s, 1H), 4.07 (d,
J = 8.3 Hz, 1H), 3.71ꢀ3.78 (m, 4H), 3.61ꢀ3.70 (m, 4H), 2.59 (s, 3H),
1.26 (s, 6H), 1.15 (s, 6H). MS (APCI): 490.5 (M + H)+. Anal. Calcd
For C26H31N7O3 0.1H2O: C, 63.55; H, 6.40; N, 19.95. Found: C,
3
63.28; H, 6.36; N, 19.82.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
AO metabolic stability in human S9 (HS9) fraction spectra of
final analogues. This material is available free of charge via the
Internet at http://pubs.acs.org.
Step 3: N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-
tetramethylcyclobutyl}-6-methoxyimidazo[1,2-b]pyridazine-3-car-
boxamide 25. Prepared by the method of compound 1, using 7-
methoxyimidazo[1,2-a]pyrimidine-3-carboxylic acid (prepared in step
2 above) instead of imidazo[1,2-a]pyrimidine-3-carboxylic acid in step 2
of compound 1. 1H NMR (acetone-d, 400 MHz): δ ppm 9.53 (d, J = 7.6
Hz, 1H), 8.27 (s, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.20 (d, J = 9.3 Hz, 1H),
6.88 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 7.6 Hz, 1H), 4.82 (s, 1H), 4.19 (d, J =
8.8 Hz, 1H), 4.02 (s, 3H), 2.62 (s, 3H), 1.33 (s, 6H), 1.20 (s, 6H). MS
’ AUTHOR INFORMATION
Corresponding Author
*For A.L.: phone, (858) 622 7913; E-mail, angelica.linton@
pfizer.com. For C.G.: phone, (858) 622 5927; E-mail, alex.guo@
pfizer.com.
7711
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712

Journal of Medicinal Chemistry
ARTICLE
’ ACKNOWLEDGMENT
16.50 min, hold at 2% B from 16 to 20 min. The major operating
parameters for the LTQ ESI-MS methods are shown below: spray
voltage 3.5 kV; capillary temperature 350 °C; sheath gas flow rate 25
(arbitrary); auxiliary gas flow rate 10 (arbitrary); m/z scan range 120ꢀ
1500 amu. Ion-trap LC-MSn (n = 2ꢀ4) experiments were performed to
generate multistage mass spectra for selected molecular ions represent-
ing possible metabolites. At a constant pressure of 40 psi, helium was
used as the damping and collision gas for all MSn experiments. Precursor
isolation window, activation amplitude, activation Q and activation time
were set at 1.6 amu, 40%, 0.25 and 30 ms, respectively. The Xcalibur
software (Thermo Fisher Scientific, Waltham, MA) was used to control
both HPLC and MS systems to acquire and process all spectral data. The
UV chromatograms for each compound and its metabolite(s) can be
found in the Supporting Information.
(6) Brandon, E. F.; Raap, C. D.; Meijerman, I.; Beijnen, J. H.;
Schellens, J. H. An update on in vitro test methods in human hepatic
drug biotransformation research: pros and cons. Toxicol. Appl. Pharma-
col. 2003, 189, 233–246.
(7) To confirm the role of AO, raloxifene (10 μM final), an AO
inhibitor, was coincubated with substrate following the same procedure
described in ref 5.
(8) Zientek, M.; Jiang, Y.; Youdim, K.; Obach, R, S. In VitroꢀIn Vivo
Correlation for Intrinsic Clearance for Drugs Metabolized by Human
Aldehyde Oxidase. Drug Metab. Dispos. 2010, 38, 1322–1327.
(9) Garattini, E.; Terao, M. Increasing recognition of the importance
of aldehyde oxidase in drug development and discovery. Drug Metab.
Rev. 2011, 43, 374–386.
(10) Torres, R. A.; Korzekwa, K. R.; McMasters, D. R.; Fandozzi,
C. M.; Jones, J. P. Use of Density Functional Calculations To Predict the
Regioselectivity of Drugs and Molecules Metabolized by Aldehyde
Oxidase. J. Med. Chem. 2007, 50, 4642–4647.
(11) Garattini, E.; Mendel, R.; Romao, M. J.; Wright, R.; Terao, M.
Mammalian molybdo-flavoenzymes, an expanding family of proteins:
structure, genetics, regulation, function and pathophysiology. Biochem. J.
2003, 372, 15–32.
(12) Dastmalchi, S.; Hamzeh-Mivehrod, M. Molecular modelling of
human aldehyde oxidase and identification of the key interactions in the
enzymeꢀsubstrate complex. Daru, J. Fac. Pharm., Tehran Univ. Med. Sci.
2005, 13, 82–93.
We thank Dr. Martin Edwards for the helpful discussion
during manuscript preparation and Dr. Chris Bi for coordinating
CRO synthesis of intermediates.
’ ABBREVIATIONS USED
AO, aldehyde oxidase; CRPC, castration-resistant prostate
cancer; AR, androgen receptor; HS9, human S9 fraction; MoCo,
molybdenum pyranopterin cofactor moiety; HLM, human liver
microsome
’ REFERENCES
(1) Guo, C.; Linton, M. A.; Kephart, S.; Ornelas, M.; Mason, P.;
Gonzalez, J.; Greasley, S.; Nagata, A.; Burke, B. J.; Edwards, M.; Hosea,
N.; Kang, P.; Hu, W.; Engebretsen, J.; Briere, D.; Shi, M.; Gukasyan, H.;
Richardson, P.; Dack, K.; Underwood, T.; Johnson, P.; Morell, A.;
Felstead, R.; Kuruma, H.; Matsimoto, H.; Zoubeidi, A.; Gleave, M.; Los,
G.; Fanjul, A. N. Discovery of Aryloxy Tetramethylcyclobutanes as
Novel Androgen Receptor Antagonists. J. Med. Chem. 2011, 54, DOI:
10.1021/jm201059s.
(2) (a) LaRocca, J. P.; Gibson, C. A.; Thompson, B. B. Synthesis and
characterization of some 3-nitroso-2-arylimidazo(1,2-a)pyrimidines.
J. Pharm. Sci. 1971, 60, 74–76. (b) Anaflous, A.; Benchat, N.; Mimouni,
M.; Abouricha, S.; Ben-Hadda, T.; El-Bali, B.; Hakkou, A.; Hacht, B.
Armed imidazo [1,2-a]pyrimidines (pyridines): Evaluation of antibac-
terial activity. Lett. Drug Des. Discovery 2004, 1, 224–229. (c) Steenackers,
H. P. L.; Ermolat’ev, D. S.; Savaliya, B.; De Weerdt, A.; De Costert, D.;
Shah, A.; Van der Eycken, E. V.; De Vos, D. E.; Vanderleyden, J.;
De Keersmaecker, S. C. J. Structureꢀactivity relationship of 4(5)-
aryl-2-amino-1H-imidazoles, N1-substituted 2-aminoimidazoles and
imidazo[1,2-a]pyrimidinium salts as inhibitors of biofilm formation by
Salmonella Typhimurium and Pseudomonas aeruginosa. J. Med. Chem.
2011, 54, 472–484.
(3) Pryde, D. C.; Dalvie, D.; Hu, Q.; Jones, P.; Obach, R. S.; Tran,
T.-D. Aldehyde Oxidase: An Enzyme of Emerging Importance in Drug
Discovery. J. Med. Chem. 2010, 53, 8441–8460.
(4) HLM corresponds to the intrinsic metabolic clearance (Clint) in
microsomes, in μL/min/mg of microsomal protein. Data interpretation
as following: Clint < 15 μL/min/mg (low clearance); Clint 15ꢀ40
μL/min/mg (moderate clearance); Clint > 40 (high clearance).
(5) Determination of metabolites formed in HS9 fraction without
NADPH was done by incubating the substrates (10 μM final concen-
tration) and human liver S9 (1 mg/mL final concentration) in 100 mM
potassium phosphate at pH 7.4. The reaction was initiated by addition of
human liver S9 without preincubation. The mixture (1 mL) was then
incubated at 37 °C for 1 h. The reaction was terminated by the addition
of acetonitrile (5 mL) followed by vortexing for 1 min and then cen-
trifuged. The supernatants were transferred into conical glass tubes for
evaporation to dryness under nitrogen at 37 °C. The residues were
reconstituted in 200 μL of 30:70 (v/v) acetonitrile:water (0.1% formic
acid) and aliquots (20 μL) were injected for HPLC-MS analysis. The
formation of AO metabolites was monitored by LCMS. Metabolite
profiling and structure elucidation were performed using HPLC coupled
in-line with UV and MS detection with ESI source in positive ion mode.
The instrumental components for the methods are as follows: surveyor
HPLC system and LTQ (Thermo Fisher Scientific, Waltham, MA),
Phenomenex C8 column (100 mm ꢁ 2 mm, Phenomenex Torrance,
CA). The mobile phase consisted of 0.1% formic acid in water (solvent
A) and 0.1% formic acid in acetonitrile (solvent B). The flow rate was
maintained at 0.4 mL/min. Parent drug and its metabolites were eluted
using a linear gradient in mobile phase composition summarized as
follows: equilibration for 0.75 min at 2% B, linear gradient to 25% B from
0.75 to 7.25 min, linear gradient to 55% B from 7.25 to 9 min, hold at
55% B from 9 to 10.25 min, linear gradient to 90% B from 10.25 to
15 min, hold at 90% B from 15 to 16 min, linear gradient to 2% B from 16 to
(13) Magee, T. V.; Ripp, S. L.; Li, B.; Buzon, R. A.; Chupak, L.;
Dougherty, T. J.; Finegan, S. M.; Girard, D.; Hagen, A. E.; Falcone, M. J.;
Farley, K. A.; Granskog, K.; Hardink, J. R.; Huband, M. D.; Kamicker,
B. J.; Kaneko, T.; Knickerbocker, M. J.; Liras, J. L.; Marra, A.; Medina, I.;
Nguyen, T.-T.; Noe, M. C.; Obach, R. S.; O’Donnell, J. P.; Penzien, J. B.;
Reilly, U. D.; Schafer, J. R.; Shen, Y.; Stone, G. G.; Strelevitz, T. J.; Sun, J.;
Tait-Kamradt, A.; Vaz, A. D. N.; Whipple, D. A.; Widlicka, D. W.;
Wishka, D. G.; Wolkowski, J. P.; Flanagan, M. E. Discovery of Azetidinyl
Ketolides for the Treatment of Susceptible and Multidrug Resistant
CommunityꢀAcquired Respiratory Tract Infections. J. Med. Chem.
2009, 52, 7446–7457.
(14) The UV chromatogram for compound 1 and its metabolite(s)
can be found in the Supporting Information.
7712
dx.doi.org/10.1021/jm2010942 |J. Med. Chem. 2011, 54, 7705–7712