Use of small-molecule crystal structures to address solubility in a novel series of g protein coupled receptor 119 agonists: Optimization of a lead and in vivo evaluation

Article abstract of DOI:10.1021/jm300310c

G protein coupled receptor 119 (GPR119) is viewed as an attractive target for the treatment of type 2 diabetes and other elements of the metabolic syndrome. During a program toward discovering agonists of GPR119, we herein describe optimization of an initial lead compound, 2, into a development candidate, 42. A key challenge in this program of work was the insolubility of the lead compound. Small-molecule crystallography was utilized to understand the intermolecular interactions in the solid state and resulted in a switch from an aryl sulphone to a 3-cyanopyridyl motif. The compound was shown to be effective in wild-type but not knockout animals, confirming that the biological effects were due to GPR119 agonism.

Full text of DOI:10.1021/jm300310c

Article
pubs.acs.org/jmc
Use of Small-Molecule Crystal Structures To Address Solubility in a
Novel Series of G Protein Coupled Receptor 119 Agonists:
Optimization of a Lead and in Vivo Evaluation
James S. Scott,*,†† Alan M. Birch,† Katy J. Brocklehurst,† Anders Broo,‡ Hayley S. Brown,† Roger J. Butlin,†
‡
§
†
†
̈
David S. Clarke, Ojvind Davidsson, Anne Ertan, Kristin Goldberg, Sam D. Groombridge,
Julian A. Hudson,† David Laber,† Andrew G. Leach,† Philip A. MacFaul,† Darren McKerrecher,†
Adrian Pickup,† Paul Schofield,† Per H. Svensson,§ Pernilla Sorme,† and Joanne Teague†
̈
†Cardiovascular & Gastrointestinal Innovative Medicines Unit, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10
4TG, United Kingdom
‡Cardiovascular & Gastrointestinal Innovative Medicines Unit, AstraZeneca, Pepparedsleden 1, 431 83 Molndal, Sweden
̈
§Pharmaceutical Development, AstraZeneca R&D, S-151 85 Sodertalje, Sweden
̈
̈
S
* Supporting Information
ABSTRACT: G protein coupled receptor 119 (GPR119) is
viewed as an attractive target for the treatment of type 2
diabetes and other elements of the metabolic syndrome.
During a program toward discovering agonists of GPR119, we
herein describe optimization of an initial lead compound, 2,
into a development candidate, 42. A key challenge in this
program of work was the insolubility of the lead compound.
Small-molecule crystallography was utilized to understand the
intermolecular interactions in the solid state and resulted in a switch from an aryl sulphone to a 3-cyanopyridyl motif. The
compound was shown to be effective in wild-type but not knockout animals, confirming that the biological effects were due to
GPR119 agonism.
into clinical trials (Figure 1), including MBX-298216a
INTRODUCTION
■
(Metabolex/Sanofi-Aventis) and GSK-129226316b (GSK).
The compounds APD-597 (JNJ-38431055)9b and APD-6689b
have also been in clinical trials (Arena/Johnson & Johnson),
with initial clinical data recently being published on the first of
these compounds.17
G protein coupled receptor 119 (GPR119) is a class A type
receptor that is mainly expressed in pancreatic islets and
intestinal enteroendocrine cells.1 Recent work has led to the
putative identification of natural agonists of this receptor such
as oleoylethanolamide (OEA)2 and related compounds such as
N-oleoyldopamine (OLDA).3 Biological studies have shown
that agonism of the GPR119 receptor results in incretin
secretion from the gut (e.g., glucagon-like peptide-1 (GLP-1)
release from L-cells)4 in addition to the stimulation of insulin
release from β-cells in the pancreas.5 This dual mechanism of
action has considerable potential in the mediation of glucose
homeostasis, and as a result, GPR119 agonism has been viewed
with considerable interest as a novel approach to therapeutic
intervention in the treatment of diabetes.6,7 Interest in this
target was further increased by an initial disclosure by Arena
that a synthetic GPR119 agonist controlled glucose excursions
in preclinical animal models.8 A number of research groups,
including Arena,9 GSK,10 Pfizer,11 Merck,12 and Astellas,13 have
subsequently published their work in this area on a number of
different chemotypes. Other groups are also known to be active
on the basis of filed patent applications,14 and a pharmacophore
model has been proposed based on similarities in structural
features of published chemical series.15 A number of
compounds with disclosed structures have been progressed
We recently reported our own efforts to identify a series of
GPR119 agonists that were compounded by off-target effects
observed in vivo in GPR119 knockout mice.18 We herein report
the synthesis and structure−activity relationships of an
alternative, chemically distinct series from initial high-
throughput screening hit 1 to development candidate 42. In
contrast to our previous efforts, we report that in vivo effects
were observed in wild-type mice, but crucially not in GPR119
knockout mice, leading us to conclude that the biological effects
observed were mediated by agonism of the GPR119 receptor.
RESULTS AND DISCUSSION
■
A high-throughput screen of the AstraZeneca compound
collection identified a small cluster of hits exemplified by
compound 1 (Scheme 1). This compound showed reasonable
potency and intrinsic activity (IA)19 against both human (EC50
Received: March 5, 2012
Published: April 30, 2012
© 2012 American Chemical Society
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Figure 1. Structures of selected GPR119 agonists progressed into clinical development.
Scheme 1. Structures of the High-Throughput Screening Hit 1 and the Initial Lead 2
Figure 2. Three views of the X-ray structure of compound 2 showing views of the crystal packing around the molecule colored light blue. In (a) the
molecule is viewed from the side, showing its flat shape and the molecules stacking above and below. In (b) the molecule is viewed from the end,
showing the near vertical stacking with the molecules above and below. In (c) the molecule is shown from above, with the set of interactions shown
in green that hold together the plane that stacks in (a) and (b). Distances shown are in angstroms..
= 78 nM, IA = 81%) and murine (EC50 = 808 nM, IA = 78%)
forms of the receptor. The lipophilicity20 was high (log D7.4
3.3% free) and metabolic stability in human microsomes (Clint
= 47 (μL/min)/mg), but the solubility remained low (<1 μM).
Further experimentation revealed that compound 2 was
highly insoluble (0.03 μM) for its measured lipophilicity (log
=
4.2), leading to poor physicochemical properties21 (solubility
<1 μM, mouse plasma protein binding (PPB) 0.1% free) and
metabolic instability in human microsomes (Clint = 259 (μL/
min)/mg). Reordering of the structure to disrupt the three
contiguous rings, together with a switch from cyano to a
lipophilicity-lowering sulfone resulted in the initial lead 2. This
compound showed similar human potency (EC50 = 65 nM, IA
= 83%) but much improved mouse potency (EC50 = 41 nM, IA
= 99%) at a reduced lipophilicity (log D7.4 = 3.2). Improve-
ments were also observed in the levels of free drug (mouse PPB
D
7.4 = 3.2) and melting point (201−202 °C).22 To understand
any interactions that contribute to the stability of the solid
state23 and hence to the low solubility, crystals of 2 suitable for
X-ray structural determination were grown (Figure 2). Figure
2a shows a view from the side of one molecule of 2 (highlighted
with light blue carbon) amidst the crystal lattice and illustrates
how the molecule is rather flat and stacks very effectively with
molecules above and below. The tert-butyl groups that might
disrupt this packing pack together well and so do not provide
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a
Scheme 2
a
Reagents and conditions: (a) (i) bis(pinacolato)diborane, KOAc, Pd(OAc)2, DMF, 85 °C, 16 h; (ii) NaBO3·4H2O, THF/H2O, 25 °C, 16 h, 23−
56%; (b) BrCH2(p-SO2Me)Ph, Cs2CO3, DMF, 40 °C, 2 h, 28−94%; (c) (iPr)2NEt, toluene, reflux, 4 h, 26−58%; (d) HOCH2(p-SO2Me)Ph,
Cs2CO3, CH3CN, 150 °C, 30 h, 14−36%; (e) EtOH, 150 °C, microwave, 24 h, 63%; (f) Br2, H2O, 25 °C, 20 min, 74%; (g) HOCH2(p-SO2Me)Ph,
Cs2CO3, CuI, tetramethylphenanthroline, 1,4-dioxane, 100 °C, 20 h, 9%; (h) NaH, DMF, 25 °C, 16 h, 61−87%; (i) Boc-piperazine, Pd(dba)2,
BINAP, NaOtBu, toluene, 80 °C, 16 h, 43−55%.
a
Scheme 3
a
Reagents and conditions: (a) dichloro(1,1-bis(diphenylphosphino)ferrocene)palladium(II), K2CO3, DME/H2O, 85 °C, 18 h, 27%; (b) H2, 10%
Pd/C, EtOH, 20 °C, 2 h, 97%; (c) BrCH2(p-SO2Me)Ph, Cs2CO3, DMF, 20 °C, 18 h, 67%.
any significant disruption to the lattice. Figure 2b shows the
molecule viewed from the end and illustrates how the
molecules stack on top of one another. In Figure 2c an almost
orthogonal view shows the molecules surrounding the light
blue one along with potential interactions between them. For
instance, the pyrimidine nitrogens have potential weak
interactions with polarized C−H bonds in the adjacent
pyrimidine ring (N−H distance ∼3.06 Å) and in an adjacent
piperazine ring (N−H distance ∼3.26 Å). The tert-butyl groups
stack against tert-butyl groups in other molecules and therefore
benefit from hydrophobic interactions. Notably, the tightest set
of polar interactions are between sulfone oxygens and the C−H
bonds in the methyl groups in the sulfone moiety in adjacent
molecules. If the tert-butyl groups can be said to be interacting
tail-to-tail, then the sulfone groups not only interact head-to-
head to form a one-dimensional chain, but also interact in an
almost orthogonal direction to hold together the whole of the
two-dimensional plane that then stacks as shown in Figure 2a.
The network of interactions involving the sulfones prompted
a search of the Cambridge Crystallographic Database to see if
these were a common motif.24 Of the methyl sulfone structures
that were examined, 18% showed interactions like the head-to-
head one linking the light blue molecule to the molecule to its
right shown in brown. Also, 18% showed ladderlike interactions
similar to those between the light blue molecule and the
molecule shown in pink. A further 23% of methyl sulfones were
involved in both of these types of interaction. This left only
41% of methyl sulfones in the database that did not form either
of these types of lattice interaction. A clear majority of crystal
structures containing methyl sulfones involve them interacting
with other methyl sulfones and the weak hydrogen bond
acceptor of the sulfone interacting with at least one weak
hydrogen bond donor of the C−H bonds of an adjacent methyl
group.
Our medicinal chemistry strategy to improve the solubility of
this lead molecule 2 was therefore to investigate the
heterocyclic core and the piperazine (with a view to introducing
asymmetry and disrupting edge−face interactions) followed by
the aryl substituent with a view to removing the sulfone (and its
associated intermolecular interactions stabilizing the solid
state). Once we had identified an optimal structure, we
envisaged a final round of optimization to replace the acid-labile
Boc group. A compound that was potent against the mouse as
well as the human receptor was viewed as essential to enable in
vivo evaluation and to confirm that any biological effects were
being mediated through GPR119 agonism. The synthetic
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a
Scheme 4
a
Reagents and conditions: (a) K2CO3, CH3CN, 150 °C, microwave, 1 h, 16−96%; (b) (i) bis(pinnacol)diborane, KOAc, Pd(OAc)2, DMF, 85 °C,
16 h; (ii) NaBO3·4H2O, THF/H2O, 25 °C, 16 h, 10−78%; (c) BrCH2(p-SO2Me)Ph, Cs2CO3, DMF, 40 °C, 2 h, 25−94%.
a
Scheme 5
a
Reagents and conditions: (a) R2CH2Br, Cs2CO3, CH3CN, 100 °C, 1 h, 32−84%; (b) R2CH2Cl, Cs2CO3, DMF, 40 °C, 2 h, 31−51%; (c)
R2CH2OMs, Cs2CO3, DMF, 55 °C, 16 h, 85−91%; (d) R2CH2OH, DIAD, PPh3, THF, 20 °C, 16 h, 27−61%; (e) CH3SO2Na, CuI, L-proline,
NaOH, DMSO, 80 °C, 2 d, 26−58%; (f) Zn(CN)2, Pd(dba)2, xantphos, DMF, 130 °C, 1 h, microwave, 49−93%.
routes developed in the course of this program are described in
Schemes 2−8.
Piperidine variants 11−19 were made by nucleophilic
displacement of the 2-chloro substituent of 2-chloro-5-
bromopyrimidine in the presence of diisopropylethylamine as
a base with microwave heating. The bromo substituent was
converted to the phenol using a palladium-catalyzed borylation
followed by sodium perborate oxidation. This was then
alkylated using p-(methylsulfonyl)benzyl chloride using cesium
carbonate as the base (Scheme 4).
Synthesis. Synthesis of the various heterocycles (2−9) was
carried out as shown in Scheme 2. Compounds 2 and 6 were
synthesized from the bromides via a palladium-catalyzed
borylation followed by sodium perborate oxidation to the
phenol. The phenol was then alkylated using p-
(methylsulfonyl)benzyl chloride using cesium carbonate as
the base. Phenyl 8 was made via alkylation of the known
phenol25 (Scheme 2a). Pyridazine 4 and pyrazine 5 were made
from monodisplacement of the symmetrical dichlorides with
Boc-piperazine followed by displacement of the second chloride
with p-methylanesulfonylbenzyl alcohol using cesium carbonate
and microwave heating (Scheme 2b). The triazine 9 was made
via methanethiolate displacement with Boc-piperazine under
microwave irradiation followed by a regioselective bromination
and a copper-catalyzed bromide displacement with p-
(methylsulfonyl)benzyl alcohol (Scheme 2c). Pyrimidine 3
and pyridine 7 were made from the dibromides with an initial
regioselective nucleophilic aromatic substitution of the 2-bromo
substituent with p-(methylsulfonyl)benzyl alcohol followed by a
palladium-catalyzed amination with Boc-piperazine (Scheme
2d).
For aryl variation, the phenols (R1 = H, (R)-Me) were
alkylated with benzylic bromides, chlorides, or mesylates to
provide final compounds 22 and 27, 28 and 35, and 20 and 21,
respectively (Scheme 5). For substituted sulfones 24 and 25,
the intermediate aryl bromides were synthesized and then
subjected to a copper-catalyzed displacement with NaSO2Me.
Alternatively, Mitsunobu chemistry could be used to form
either final compounds (23, 29−31, 34) or a functionalized 3-
bromopyridine which could be further converted to either
sulfone 33 or cyanopyridyls 32 and 36. Mitsunobu chemistry
was also used to build a regioisomeric bromopyridine that was
treated with NaSO2Me as above to produce 26.
Treatment of final compound 36 with hydrochloric acid
removed the Boc group and gave the piperazine salt that could
be converted to carbamates by treatment with either isopropyl
chloroformate (to give 37) or various phenyl carbonates to give
alkyl carbamates 38−40. The amine could also be converted
into heterocycles, for example, the 5-fluoropyrimidine 41, using
nucleophilic aromatic substitution chemistry (Scheme 6).
Piperidine 10 was made by a palladium-catalyzed coupling
between the alkenyl boronate and 2-chloropyrimidin-5-ol
followed by a hydrogenation of the alkene and alkylation of
the phenol under standard conditions (Scheme 3).
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a
Scheme 6
a
Reagents and conditions: (a) HCl, 1,4-dioxane/CH2Cl2, 20 °C, 30 min, 74%; (b) iPrOCOCl, (iPr)2NEt, CH2Cl2, 25 °C, 16 h, 40%; (c) ROCO2Ph,
NEt3, CHCl3, 110 °C, 18 h, 10−39%; (d) 2-chloro-5-fluoropyrimidine, (iPr)2NEt, CH3CN, 150 °C, 3 h, 29%.
a
Scheme 7
a
Reagents and conditions: (a) HCl, 1,4-dioxane/CH2Cl2, 25 °C, 1 h 89%; (b) CNBr, NaHCO3, CH2Cl2/H2O, 25 °C, 2 h, 79%; (c) (i) NH2OH,
Na2CO3, DMF, 80 °C, 1 h; (ii) RCOOH, (iPr)2NEt, HOBt, EDAC, DMF, 20 °C, 18 h; (iii) toluene, 120 °C, 30 min, 54−66%; (d) Zn(CN)2,
Pd(dba)2, xantphos, DMF, 130 °C, 165 min, 32−88%.
a
Scheme 8
a
Reagents and conditions: (a) HCl, 1,4-dioxane/CH2Cl2, 20 °C, 3 d, 100%; (b) CNBr, NaHCO3, CH2Cl2/H2O, 0−25 °C, 1 h, 59%; (c) (i)
NH2OH·HCl, Na2CO3, DMF, 80 °C, 30 min; (ii) (R1CO)2O, pyridine, toluene, 45 °C, 40 min, 38−87%; (d) (4-(3-cyano)pyridyl)CH2Cl, Cs2CO3,
DMF, 20 °C, 70 h, 30−68%; (e) (i) R1C(NOH)NH2, ZnCl2, EtOAc/THF, 20 °C, 3 h; (ii) concentrated HCl, EtOH, 100 °C, 18 h, 47−68%.
Oxadiazoles are typically synthesized via the N-cyanamide,
but in this case, the presence of the cyanopyridyl group
presented chemoselectivity issues. To circumvent this, we
elected to carry the pyridyl with a bromo substituent which
could be converted into the cyanide in the final step.
Deprotection of the Boc group and conversion into the
cyanamide using cyanogen bromide proceeded in good yield to
give the key N-cyanamide intermediate. 3-Amino-1,2,4-
oxadiazoles were constructed by treatment with hydroxylamine
followed by coupling with the appropriate alkyl acid and
heating to form the heterocycle. Final products 42−44 were
produced through a palladium-catalyzed reaction of the pyridyl
bromide with zinc cyanide (Scheme 7).
When the above sequence was attempted in the presence of
(fluoroalkyl)oxadiazoles, the cyanation reactions were found to
be capricious. An alternative approach was therefore devised
that involved forming the heterocycle in the presence of the
phenol and then alkylation to produce final compounds
(Scheme 8). The Boc group was removed under acidic
conditions, and then the resultant amine was reacted with
cyanogen bromide to give the cyanamide. Oxadiazole formation
was carried out according to the methods described above, and
then the phenol group was alkylated with the requisite chloride
to produce compounds 45 and 46. This stategy was also used
to produce the regioisomeric 5-amino-1,2,4-oxadiazoles 47−49
through zinc chloride mediated coupling with the requisite
amidoximes followed by ring closure under acidic conditions.
Medicinal Chemistry. Our initial point of variation was the
heteroaryl core of pyrimidine 2 (Table 1). The isomeric
pyrimidine 3 was significantly less potent in both mouse and
human assays. Of the alternative diazines, the pyridazine 4
retained some potency but lost intrinsic activity in the human
assay, and in contrast, the pyrazine 5 showed no agonism at all.
The pyridine isomers 6 and 7 retained submicromolar potency
but were both less potent and less efficacious than the
pyrimidine lead compound 2 despite being of similar
lipophilicity. Removal of all heterocyclic nitrogens gave the
phenyl compound 8, which, despite being the most lipophilic
compound in the set, showed only weak activity at high
concentrations. The triazine isomer 9 was less active than the
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Table 1. Heterocyclic Core Variation
Table 2. Piperazine Variation
original hit (EC50 = 161 nM, IA = 61%) but was lower in
lipophilicity (log D7.4 = 2.9), leading to a similar value for
ligand-lipophilicity efficiency (LLE),26,27 pEC50 − log D (3.9 vs
4.0 for 2). On the basis of these results together with
consideration of synthetic tractability, the original pyrimidine
was considered optimal, and this group was fixed for further
investigation.
We next turned our attention to the piperazine heterocycle of
the initial lead 2 (Table 2). A switch to piperidine 10 caused a
small reduction in lipophilicity (log D7.4 = 2.8)28 and a large
drop in potency (EC50 = 893 nM), leading to an LLE reduction
(3.2 vs 4.0 for 2). A systematic variation of the methyl position
on the piperazine (11−14) revealed interesting subtleties in the
structure−activity relationship and also potency differences
between species. Against the human receptor, the 2(R)-isomer
12 was the most potent (EC50 = 19 nM) and showed
significantly increased intrinsic activity (150%) relative to the
start point 2. Against the mouse isoform, the 2-isomers both
showed similar EC50 values and full agonism, but by contrast,
the 3-isomers displayed only partial agonism in mouse.
Substitution at the 2-position was further explored with
geminal dimethyl 15, but this resulted in a reduction in
potency in human (EC50 = 466 nM) and a complete loss of
agonism in mouse. The 2,6-cis-dimethyl 16 showed a small
reduction in human potency (EC50 = 64 nM) but was a full
agonist in mouse and was the most potent compound identified
against the mouse receptor (EC50 = 35 nM, IA = 95%). The
2,4-trans-dimethyl 17 was potent in human (EC50 = 39 nM, IA
= 152%) but had no murine activity. Further exploration of
conformationally restricted piperazines 18 and 19 resulted in a
desired reduction in lipophilicity relative to that of their methyl
and dimethyl counterparts, but while 19 displayed reasonable
human activity (EC50 = 156 nM, IA = 85%), both showed no
activity against the mouse isoform. A common theme emerging
from these results was the sensitivity of the mouse receptor to
alterations in this region of the molecule and in particular an
intolerance for substitution proximal to the carbamate (13, 14,
17−19). Observations of similar effects have been reported by
others working on GPR119 with compounds containing
piperazine carbamates.12a These were attributed to different
conformational states, and this work further emphasizes the
nuances of structural modification of ligands on GPCR
pharmacology. On balance, the 2(R)-methyl substitution
present in 12 was selected for incorporation into further
compounds on the basis of the increased potency and intrinsic
activity in human and mouse with comparable LLEs (4.2 vs 4.0
for 2).
The solubility of crystalline 12 (melting point 173−174 °C)
was determined experimentally and found to be 1.7 μM.
Although this represents a distinct (>50-fold) improvement
from the initial lead 2 (0.03 μM), the solubility remained low.
To understand the effect of the methyl group on the solid state,
crystals suitable for X-ray crystallography were grown. The
structure of compound 12 was obtained and is shown in Figure
3. The most striking feature of this structure is that the methyl
group on the piperazine adopts an axial conformation.29 In
Figure 3a is shown the stacking view most closely related to
that in Figure 2a and in Figure 3b that most closely related to
Figure 2b. These views show that while most of the molecule
remains quite flat, the axial methyl group on the piperazine ring
protrudes orthogonal to the plane of the molecule. The
consequence of this is clear in Figure 3b, where the stacking of
the molecules of 12 is significantly changed from the vertical
arrangement of molecule 2 to one where the molecules are
offset to accommodate the axial methyl group (which is
highlighted with purple circles). In Figure 3c it is clear that
although the stacking has been disrupted, the set of orthogonal
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Figure 3. Three views of the X-ray structure of compound 12 showing views of the crystal packing around the molecule colored light blue. In (a) the
molecule is viewed from the side along with the molecules stacking above and below. In (b) the molecule is viewed from the end, showing the
significantly offset stacking with the molecules above and below; the methyl group on the piperazine is highlighted with purple circles. In (c) the
molecule is shown from above, with the set of interactions shown in green that hold together the plane that stacks in (a) and (b). Distances shown
are in angstroms.
interactions (hydrophobic between tert-butyl groups and polar
between methyl sulfones) has been maintained.
An aryl sulfone is a structural motif common to a number of
published GPR119 agonist chemotypes,9,10 and in an effort to
improve the solubility of our compounds, we sought to replace
it (Table 3). Piperidinesulfonamide 20 was a suitable
29 nM) and mouse (EC50 = 78 nM) and a similar LLE (4.0).
By contrast, the aryl sulfonamide 21 was less potent and
efficacious despite significantly increased lipophilicity. Cyano
22 and tetrazole 23 were also effective GPR119 agonists, with
the tetrazole being more potent than the sulfone in both human
(EC50 = 6 nM) and mouse (EC50 = 33 nM). Unfortunately, this
increase in potency was achieved at the expense of increasing
lipophilicity, and a log D7.4 > 4 was viewed as unacceptably
high. A return to the sulfone and investigation of aryl variation
revealed that the position meta to the sulfone tolerated
substitution and the fluoro sulfone 24 matched the tetrazole
for potency at lower log D7.4 (3.7), resulting in an improvement
in LLE (4.8). Efforts to lower lipophilicity with a switch to
cyano 25 and pyridyl 26 substitution did achieve the desired
reduction in log D7.4 but at the expense of lower potencies and
diminishing LLE (4.5 and 4.3, respectively). Notably, and in
contrast to the results obtained with the piperazines, variation
in this region did not lead to marked differences between
potency across species. A key issue at this stage was the low
aqueous solubility of the compounds (all measured as <5 μM
for 20−26). This presented us with a conundrum in that
although the sulfones were the most efficient groups in terms of
potency/lipophilicity, they suffered from poor solubility/
lipophilicity. Efforts to replace the sulfone with groups such
as cyano 22 and tetrazole 23 had delivered the required
potency but at the cost of higher lipophilicity, which in turn was
manifest in poor solubility.
replacement with similar potencies in both human (EC50
=
Table 3. Aryl Sulfone Variation
Faced with this dilemma, we elected to investigate
heteroaromatic alternatives to the sulfone that contained an
acceptor atom within the aryl ring, hypothesizing that this
should retain the acceptor without increasing lipophilicity.
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Table 4. Pyridine Variation
human GPR119 EC50 human GPR119 IA mouse GPR119 EC50 mouse GPR119 IA
log
D7.4
LLE (pEC50
−
solubility
(μM)
entry
R
X
(μM)
(%)
(μM)
(%)
log D)
27
28
29
30
31
32
33
34
35
36
H
H
0.621
>1.937
0.075
71
19
0.719
41
3.4
3.7
3.8
3.8
3.6
2.8
2.4
3.7
3.8
3.3
2.8
23
66
3.8
11
5.1
32
7
H
2-Me
H
3-Me
101
22
0.103
0.071
1.159
0.12
60
5
3.3
1.9
3.6
4.9
H
3-OMe
3-F
1.925
H
0.063
137
155
1
157
102
H
3-CN
3-SO2Me
3,5-F,F
3-Me
0.021
H
>30.000
0.154
H
94
0.342
0.236
0.036
70
60
97
3.1
2.9
5.0
11
45
24
Me
Me
0.184
68
3-CN
0.005
171
Figure 4. Three views of the X-ray structure of compound 27 showing views of the crystal packing around the molecule colored light blue. In (a) the
molecule is viewed from the side along with the molecules stacking above and below. In (b) the molecule is viewed from the end, showing the
slightly offset stacking with the molecules above and below. In (c) the molecule is shown from above, with the set of interactions shown in green that
hold together the plane that stacks in (a) and (b). Distances shown are in angstroms.
Initial work was carried out in the unsubstituted piperazine
series, and the pyridine 27 in which the hydrogen bond
acceptor present in the sulfone of 2 is replaced with a pyridyl
nitrogen was quickly identified. This was notable for having
significantly improved solubility (23 μM) at a similar log D7.4
(3.4) but lower melting point (147−148 °C) compared to the
sulfone 12 (Table 4). Crystallography was again used to
understand the solid-state interactions, and the structure that
was obtained is shown in Figure 4.
Figure 4a shows that 27 adopts a flat shape like that of 2
shown in Figure 2a. Figure 4b shows that the stacking of 27,
while not directly on top of one another as for 2 in Figure 2b, is
not offset to the same degree as that of the methylated 12 in
Figure 3b. The most striking difference between the structure
of compound 27 and those of the two sulfones in Figures 2 and
3 is the absence of the network of weak polar interactions that
the methyl sulfone is able to form. This presumably contributes
to the lower melting point and to the solubility improvements
for pyridyl compounds compared to the corresponding methyl
sulfones of similar lipophilicities.
Unfortunately, the human potency of 27 was significantly
compromised (EC50 = 621 nM, IA = 71%), leading to an
erosion in the LLE (2.8). Substitution with a methyl group at
the 2-position (28) resulted in a loss of activity. However, at
the 3-position (29), methyl significantly improved potency
(EC50 = 75 nM, IA = 101%), leading to an increase in LLE
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(3.3). An exploration of groups at the 3-position (30−33)
identified that the cyano group 32 had the doubly desirable
effect of significantly increasing potency (EC50 = 21 nM, IA =
155%) while simultaneously lowering log D7.4 (2.8). This led to
an increase in LLE (4.9) and maintained the good solubility (32
μM). Substitution on both sides of the aromatic ring
exemplified by the 3,5-difluoropyridyl 34 did not significantly
increase either potency or solubility. Similar pyridyl substitution
in the 2(R)-methylpiperazine series (35 and 36) showed
additivity in the SAR, leading to compound 36 that had
potency and intrinsic activity (EC50 = 5 nM, IA = 171%)
comparable to those of either the tetrazole 23 or fluorosulfone
24 but with markedly improved solubility (24 μM vs <5 μM). It
is notable that although the structural difference between
pyridines 27 and 36 is merely the addition of a methyl and a
cyano group, and while the compounds have similar measured
solubility and log D7.4 values, pyridine 36 is over 100 times
more potent than the starting point 27 as reflected in the high
LLE (5.0).
Table 5. Carbamate and Heterocycle Variation
Our final task in the optimization of this series involved
replacement of the Boc group, and our initial focus was on the
alkyl substituent of the carbamate (Table 5). The isopropyl
carbamate 37 was significantly less potent but showed greater
stability under acidic conditions than the tert-butyl carbamate
36. The cyano analogue 38 showed a significant reduction in
potency (EC50 50 nM) but a concomitant drop in log D7.4 (2.3)
in line with a constant LLE (5.0). Disappointingly, the
reduction in lipophilicity did not result in increased solubility
(9 μM). The trifluoroethyl carbamate 39 showed only a small
decrease in potency (EC50 = 20 nM, IA = 132%) with solubility
(23 μM) and LLE (4.7) similar to those of 36. Fluorination at
the β-position relative to an oxygen atom can have profound
effects on the lipophilicity,30 and in this case difluoroethyl
carbamate 40 resulted in a significant reduction in log D7.4 (Δ
log D(CF −CHF ) = 0.7) and a corresponding drop in potency
3
2
(EC50 = 100 nM) in line with a constant LLE (4.7). In
common with 38, however, no benefits were observed in terms
of improved solubility. Certain heterocycles are well-known
isosteres of carbamates, and this tactic has been used by other
groups in their efforts to develop agonists of GPR119.9,10 We
found that six-membered heterocycles as exemplified by
pyrimidine 41 suffered a dramatic fall in solubility (<1 μM)
that we attributed to the symmetry and rigidity of the three
contiguous rings. A move to the less symmetrical five-
membered heterocycles, such as isopropyloxadiazole 42
resulted in a compound that was approximately equipotent
(EC50 = 8 nM, IA = 269%) and isolipophilic (3.3) with the Boc
carbamate 36 with only a small errosion in terms of solubility
(6 μM). Close analogues such as the cyclopropyl 43 retained
potency; however, trimming of the alkyl substituent to methyl
44 resulted in a worsening of LLE (4.0), although the reduced
lipophilicity did increase the measured solubility (64 μM). The
(trifluoromethyl)oxadiazole 45 resulted in the most potent and
efficaceous human GPR119 agonist identified in this series
(EC50 = 2 nM, IA = 243%) but at the expense of a higher than
desired log D7.4 (4.0). Efforts to lower lipophilicity by switching
to the (difluoromethyl)oxadiazole 46 resulted in a dramatic
reduction of log D7.4 (Δ(CF −CHF ) = 1.1) and constant LLE
fold higher solubility potentially due to solid-state interactions
of the CF2H group.31 The isomeric oxadiazoles 47−49 were
also profiled and found to be marginally less potent than their
matched pairs with the same rank order of substituents in terms
of potency (CF3 > Pr > Pr). The (trifluoromethyl)oxadiazole
49 represents the most efficient compound in terms of LLE
(5.2) and shows a remarkable reduction in log D7.4 (−0.9)
relative to its positional isomer 45 that has been attributed to
differences in hydrogen bond acceptor strength.31
Compound 42 was selected for further profiling and
evaluation in terms of its suitability for further development.
The compound displayed no inhibition toward five major
isoforms of cytochrome P450 enzymes (CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4) in a high-throughput
fluorescence assay, with IC50 values >30 μM. Plasma protein
i
c
3
2
(4.7), however no apparent benefit in terms of solubility (5
μM). The contrast between the (difluoromethyl)oxadiazole 46
and methyloxadiazole 44 is noteworthy in that they are of
similar lipophilicity and yet the methyl compound exhibits >10-
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binding was consistent across species (mouse, 1.9% free; rat,
1.8% free; dog, 2.0% free; human, 1.3% free). Compound 42
exhibited low solubility as measured on crystalline material (6
μM) but good cellular permeability as measured in an in vitro
CACO-2 assay (apical to basolateral Papp = 85 × 10−6 cm/s at a
compound concentration of 10 μM) with no evidence of efflux.
In contrast to Boc-containing compounds such as 12,
compound 42 showed no evidence of hydrolytic instability
across the pH range 1−10. Weak activity was observed against
the hERG channel (IC50 = 10 μM);32 however, the high margin
(>1000) relative to primary potency and lack of activity against
other ion channels screened (NaV1.5, IKs, Ito, CaV1.2) gave us
confidence to progress this compound further. Compound 42
was relatively clean against a panel of 167 other targets, with
only four hits (acetylcholinesterase, adenosine transporter,
5HT2B, and vesicular monoamine transporter 2) showing
between 50% and 75% activity at 10 μM.
The low bioavailability and solubility of this compound
caused concern that dose escalation would not lead to an
increase in exposure, potentially precluding further in vivo
evaluation in preclinical toxicity studies. As a consequence,
high-dose pharmacokinetic studies were carried out in all three
preclinical species on fully crystalline material (as judged by X-
ray powder diffraction). Pleasingly, the results shown in Table 7
show significant increases in exposure up to 100 mg/kg in
rodents and up to 50 mg/kg in dog (Table 7).
Compound 42 was investigated in vivo for its potential to
control the glucose excursion in a mouse (C57BL6/JAX) oral
glucose tolerance test (OGTT).33 As shown in Figure 5a, 42
significantly enhances glucose disposal at doses of 5 mg/kg and
above. In separate experiments, the amount of total GLP-1 in
systemic circulation was measured as a more proximal
pharmacodynamic marker of GPR119 receptor agonism. In
lean mice, 42 increases total GLP-1 levels 30 min after oral
administration of the compound, with statistically significant
increases observed at doses of 10 mg/kg and above (Figure 5b).
To verify that the pharmacodynamic effects were indeed
mediated through GPR119 agonism, compound 42 was tested
in both wild-type (WT) and GPR119 knockout (KO) mice. In
stark contrast to results obtained with a previous series,18 42
showed no effect on either glucose disposal (at a dose of 100
mg/kg) or GLP-1 secretion (at a dose of 50 mg/kg) in the
knockout mice, with wild-type controls displaying the expected
response (Figure 6).
Having demonstrated that compound 42 enhances glucose
disposal and GLP-1 secretion at a dose of 15 mg/kg, we studied
the pharmocology of compound 42 in conjunction with the
dipeptidyl peptidase-4 (DPP-IV) inhibitor sitagliptin at a dose
of 1 mg/kg. DPP-IV inhibitors prevent inactivation of GLP-1 to
an inactive form,34 and our hypothesis was that this would
augment the effects of our GPR119 agonist. As shown in Figure
7, in the mouse OGTT the glucose lowering achieved with the
combination demonstrates a clear, statistically significant
benefit of the combination over each monotherapy. On the
basis of the overall compound profile and the encouraging in
vivo results, 42 was selected as a candidate for further
development, the results of which will be reported in due
course.
Pharmacokinetic parameters (intravenous (iv) and per os
(po)) for compound 42 were determined in C57BL6 mice,
Han Wistar rats, and beagle dogs. The compound was
administered orally as a suspension in aqueous 0.1% (w/v)
pluronic F127 at relevant pharmacological doses (2−13 mg/
kg). As shown in Table 6, compound 42 displayed modest
a
Table 6. Pharmacokinetic Parameters for Compound 42
po
half-
life
iv
half-
life
Clp
Vss
bioavailability
(%)
species [(mL/min)/kg] (L/kg)
(h)
(h)
Fabs
mouse
rat
49
28
2.4
5.1
4.1
1
6
5
1
3
0.3
0.5
0.3
22
29
24
dog
3.6
11
a
Compounds were dosed at 2 mg/kg (iv) and 5 mg/kg (po, mouse
and rat) and 13 mg/kg (po, dog) in 5% DMSO/95% (hydroxypropyl)-
β-cyclodextrin and a 0.1% pluronic F127 suspension, respectively, at
volumes of 5 mL/kg (mouse and rat) and 2 mL/kg (dog).
fraction absorbed (Fabs) and bioavailability (F) across the three
preclinical species. The volume of distribution (Vss) was
relatively constant across the three species. Clearance (Clp) was
high in the rodent species and low in dog. This was consistent
with in vitro measurements that showed that compound 42 is
metabolically unstable in mouse and rat hepatocytes (Clint = 67
and 29 (μL/min)/106 cells, respectively) yet relatively stable in
dog hepatocytes (Clint = 5.6 (μL/min)/106 cells). In bile duct
cannulated rats, compound 42 was found to be eliminated
predominantly by metabolism, with no parent compound
detected in bile or urine. This was consistent with the good in
vitro−in vivo correlations of metabolic clearance observed in all
three preclinical species.
CONCLUSION
■
In conclusion, we have described the optimization of an initial
lead compound, 2, into a development candidate, 42. A key
challenge in this program of work was the insolubility of the
lead compound. Small-molecule crystallography was utilized to
understand the intermolecular interactions in the solid state
and resulted in a switch from an aryl sulfone to a 3-
cyanopyridyl motif. Compound 42 was shown to be effective
a
Table 7. Exposure from Rising Dose Pharmacokinetic Studies for Compound 42
species
dose (mg/kg)
Cmax (μM)
AUC0−t (μM·h)
species
dose (mg/kg)
Cmax (μM)
AUC0−t (μM·h)
mouse
mouse
mouse
rat
5
30
0.4
3.5
14
0.7
6.4
42
dog
dog
dog
dog
1
5
0.6
2.5
1.3
10
4.0
14
100
5
10
50
11
0.7
6.3
2.7
97
119
rat
100
a
AUC0−t is AUC0−24 in all rat and dog studies; however, due to LOQ issues in the mouse as a result of the microsampling technique, the 30 mg/kg
dose is AUC0−6 and the 5 and 100 mg/kg doses are AUC0−12
.
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Figure 5. Oral administration of compound 42 enhances glucose disposal and GLP-1 secretion in lean mice. (A) Percent reduction in OGTT blood
glucose AUC. Compound 42 was administered 30 min prior to a glucose load of 2 g/kg, and glucose levels were monitored for 90 min. (B) Fold
increase in GLP-1 secretion. Compound 42 was administered to fasted animals 30 min prior to terminal blood sampling for total GLP-1 levels.
Figure 6. Compound 42 enhances glucose disposal (OGTT) and GLP-1 secretion in WT but not GPR119 KO mice. (A) Percent reduction in
OGTT blood glucose AUC. Compound 42 was administered 30 min prior to a glucose load of 2 g/kg, and glucose levels were monitored for 90 min.
(B) Fold increase in GLP-1 secretion. Compound 42 was administered to fasted animals 30 min prior to terminal blood sampling for total GLP-1
levels.
EXPERIMENTAL SECTION
■
General Procedures. All solvents and chemicals used were
reagent grade. Flash column chromatography was carried out using
prepacked silica cartridges (from 4 to 330 g) from Redisep, Biotage, or
Crawford and eluted using an Isco Companion system. 1H NMR were
recorded on a Bruker Avance DPX400 (400 MHz) and were
determined in CDCl3 or DMSO-d6. 13C NMR spectra were recorded
at 101 or 175 MHz. Chemical shifts are reported in parts per million
relative to the peak for tetramethylsilane (TMS) (0.00 ppm) or solvent
peaks as the internal reference, and coupling constant (J) values are
reported in hertz. Splitting patterns are indicated as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. Merck precoated thin-layer
chromatography (TLC) plates (silica gel 60 F254, 0.25 mm, article
5715) were used for TLC analysis. The purity of compounds
submitted for screening was >95% as determined by UV analysis of
liquid chromatography−mass spectroscopy (LC−MS) chromatograms
at 254 nM and substantiated using the TAC (total absorption
chromatogram). Further support for the purity statement was
provided using the MS TIC (total ion current) trace in ESI +ve and
Figure 7. Combination of compound 42 and sitagliptin shows benefit
in glucose disposal over monotherapy. Percent reduction in OGTT
blood glucose AUC. Compound 42 (15 mg/kg) and/or sitagliptin (1
mg/kg) was administered 30 min prior to a glucose load of 2 g/kg, and
−ve ion modes, HRMS, and NMR analysis. Solutions were dried over
anhydrous magnesium sulfate, and solvent was removed by rotary
glucose levels were monitored for 90 min.
evaporation under reduced pressure. Optical rotations were measured
on a polarimeter at a wavelength of 589 nM corresponding to the
sodium D-line in solutions of methanol. Melting points were
in WT but not KO animals, confirming that the biological
effects were due to GPR119 agonism.
determined on a Mettler FP62 automatic melting point apparatus.
Representative compounds (12, 42, 45) were examined by X-ray
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powder diffraction and found to have sharp peaks indicative of
crystalline compounds.
described for 2 using tert-butyl 4-(5-hydroxypyridin-2-yl)piperazine-1-
carboxylate as the starting material in 28% yield: 1H NMR (400 MHz,
CDCl3) δ 8.00−7.93 (m, 3H), 7.62 (d, J = 8.5 Hz, 2H), 7.20 (dd, J =
3.1, 9.1 Hz, 1H), 6.64 (d, J = 9.1 Hz, 1H), 5.13 (s, 2H), 3.59−3.59 (m,
4H), 3.45−3.36 (m, 4H), 3.05 (s, 3H), 1.48 (s, 9H); HRMS (ESI)
calcd for C22H30O5N3S (M + H)+ 448.1901, found 448.1898.
tert-Butyl 4-(5-{[4-(Methylsulfonyl)benzyl]oxy}pyrimidin-2-
yl)piperazine-1-carboxylate (2). Cesium carbonate (2.092 g, 6.42
mmol) was added to tert-butyl 4-(5-hydroxypyrimidin-2-yl)piperazine-
1-carboxylate (0.6 g, 2.14 mmol) and 1-(bromomethyl)-4-
(methylsulfonyl)benzene (0.587 g, 2.35 mmol) in DMF (10 mL).
The resulting mixture was stirred at 40 °C for 2 h. The reaction
mixture was quenched with water (150 mL) and extracted with Et2O
(2 × 200 mL), and the organic layer was dried over MgSO4, filtered,
and evaporated to afford a cream solid. Upon addition of water and
EtOAc/ether, a white solid was filtered off and dried. The cream solid
was triturated with CH2Cl2 to give a white solid. The two white solids
were combined to give tert-butyl 4-(5-{[4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (0.421 g, 43%): mp 201−
202 °C; 1H NMR (400 MHz, DMSO) δ 8.30 (s, 2H), 7.96 (d, J = 8.4
Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H), 5.25 (s, 2H), 3.62 (dd, J = 6.2, 4.2
Hz, 4H), 3.42−3.35 (m, 4H), 3.22 (s, 3H), 1.43 (s, 9H); HRMS (ESI)
calcd for C21H29O5N4S (M + H)+ 449.1853, found 449.1852.
tert-Butyl 4-(2-{[4-(Methylsulfonyl)benzyl]oxy}pyrimidin-5-
yl)piperazine-1-carboxylate (3). 5-Bromo-2-(4-(methylsulfonyl)-
benzyloxy)pyrimidine (748.8 mg, 2.18 mmol), tert-butyl piperazine-
1-carboxylate (447 mg, 2.40 mmol), tris(dibenzylideneacetone)-
dipalladium(0) (100 mg, 0.11 mmol), 2,2′-bis(diphenylphosphino)-
1,1′-binaphthyl (67.9 mg, 0.11 mmol), and sodium 2-methylpropan-2-
olate (315 mg, 3.27 mmol) were mixed with toluene (20 mL).
Nitrogen was bubbled through the solvent for 15 min, and the
resulting mixture was stirred at 80 °C for 16 h. The reaction mixture
was filtered through Celite, then evaporated to dryness, redissolved in
EtOAc (75 mL), and washed sequentially with water (2 × 75 mL) and
saturated brine (100 mL). The organic layer was dried over MgSO4,
filtered, and evaporated to afford crude product that was purified by
flash silica chromatography, elution gradient 0−100% EtOAc in
isohexane and then by preparative HPLC (Phenomenex Gemini C18
110A (axia) column, 5 μm silica, 21 mm diameter, 150 mm length)
using decreasingly polar mixtures of water (containing 0.5% NH3 and
MeCN) as eluants. Fractions containing the desired compound were
evaporated to dryness to afford tert-butyl 4-(2-{[4-(methylsulfonyl)-
benzyl]oxy}pyrimidin-5-yl)piperazine-1-carboxylate (0.538 g, 55%) as
a white solid: 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 2H), 7.93 (d, J =
8.4 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H), 5.48 (s, 2H), 3.65−3.50 (m,
4H), 3.05 (m, 7H), 1.48 (s, 9H); HRMS (ESI) calcd for C21H29O5N4S
(M + H)+ 449.1853, found 449.1850.
tert-Butyl 4-(6-{[4-(Methylsulfonyl)benzyl]oxy}pyridin-3-yl)-
piperazine-1-carboxylate (7). Compound 7 was synthesized as
described for 3 using 5-bromo-2-(4-(methylsulfonyl)benzyloxy)-
1
pyridine as the starting material in 43% yield: H NMR (400 MHz,
CDCl3) δ 7.96−7.89 (m, 2H), 7.76 (d, J = 2.8 Hz, 1H), 7.63 (d, J = 8.5
Hz, 2H), 7.32 (dd, J = 9.0, 3.0 Hz, 1H), 6.82−6.75 (m, 1H), 5.43 (s,
2H), 3.71−3.50 (m, 4H), 3.03 (d, J = 8.4 Hz, 7H), 1.48 (s, 9H);
HRMS (ESI) calcd for C22H30O5N3S (M + H)+ 448.1900, found
448.1897.
tert-Butyl 4-(4-{[4-(Methylsulfonyl)benzyl]oxy}phenyl)-
piperazine-1-carboxylate (8). Compound 8 was synthesized as
described for 2 using tert-butyl 4-(4-hydroxyphenyl)piperazine-1-
carboxylate as the starting material in 94% yield: mp 190−191 °C;
1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.4 Hz, 2H), 7.56 (d, J =
8.5 Hz, 2H), 6.82 (s, 4H), 5.04 (s, 2H), 3.56−3.46 (m, 4H), 2.98 (s,
3H), 2.97−2.92 (m, 4H), 1.41 (s, 9H); HRMS (ESI) calcd for
C13H31O5N2S (M + H)+ 447.1948, found 447.1944.
tert-Butyl 4-(6-{[4-(Methylsulfonyl)benzyl]oxy}-1,2,4-triazin-
3-yl)piperazine-1-carboxylate (9). To a stirred suspension of 4-
(methylsulfonyl)benzyl alcohol (465 mg, 2.50 mmol), tert-butyl 4-(6-
bromo-1,2,4-triazin-3-yl)piperazine-1-carboxylate (172 mg, 0.50
mmol), 3,4,7,8-tetramethyl-1,10-phenanthroline (23.62 mg, 0.10
mmol), copper(I) iodide (9.52 mg, 0.05 mmol), and cesium carbonate
(488 mg, 1.50 mmol) in a microwave vial was added 1,4-dioxane (5
mL). The mixture was heated at 100 °C for 20 h and cooled to
ambient temperature, and the dioxane was evaportated in vacuo to a
residue which was treated with saturated ammonium chloride solution
(15 mL) and extracted with ethyl acetate (3 × 15 mL). The combined
ethyl acetate extracts were washed with brine, dried (MgSO4), and
evaporated in vacuo to a residue which was chromatographed on silica
with 50% ethyl acetate in isohexane as the eluant and then by reversed-
phase chromatography to give tert-butyl 4-(6-{[4-(methylsulfonyl)-
benzyl]oxy}-1,2,4-triazin-3-yl)piperazine-1-carboxylate (21 mg, 9%):
1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H),
7.59 (d, J = 8.5 Hz, 2H), 5.47 (s, 2H), 3.73−3.42 (m, 8H), 2.98 (s,
3H), 1.42 (s, 9H); HRMS (ESI) calcd for C20H28O5N5S (M + H)+
450.1806, found 450.1803.
tert-Butyl 4-(6-{[4-(Methylsulfonyl)benzyl]oxy}pyridazin-3-
yl)piperazine-1-carboxylate (4). To a stirred solution of tert-butyl
4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate (300 mg, 1.00
mmol) and 4-(methylsulfonyl)benzyl alcohol (224 mg, 1.20 mmol)
in acetonitrile (10 mL) in a microwave vial was added cesium
carbonate (981 mg, 3.01 mmol). The stirred mixture was heated at
150° for a total of 30 h and then cooled to ambient temperature. The
solvent was evaporated to give a residue which was partitioned
between ethyl acetate (100 mL) and water (50 mL), and the ethyl
acetate layer was washed with brine, dried (MgSO4), and evaporated
to give a residue which was chromatographed on silica with 75% ethyl
acetate in isohexane as the eluant and then crystallized from methanol
to give tert-butyl 4-(6-{[4-(methylsulfonyl)benzyl]oxy}pyridazin-3-
yl)piperazine-1-carboxylate (63.0 mg, 14%): mp 208−209 °C; 1H
NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4
Hz, 2H), 6.99 (d, J = 9.6 Hz, 1H), 6.89 (d, J = 9.6 Hz, 1H), 5.50 (s,
2H), 3.56−3.36 (m, 8H), 2.97 (s, 3H), 1.42 (s, 9H); HRMS (ESI)
calcd for C21H29O5N4S (M + H)+ 449.1853, found 449.1849.
tert-Butyl 4-(5-{[4-(Methylsulfonyl)benzyl]oxy}pyrazin-2-yl)-
piperazine-1-carboxylate (5). Compound 5 was synthesized as
described for 2 using tert-butyl 4-(5-chloropyrazin-2-yl)piperazine-1-
carboxylate as the starting material in 36% yield: 1H NMR (400 MHz,
CDCl3) δ 7.98−7.91 (m, 2H), 7.69 (s, 1H), 7.65 (s, 1H), 7.64−7.59
(m, 2H), 5.41 (s, 2H), 3.52 (s, 8H), 3.05 (s, 3H), 1.49 (s, 9H); HRMS
(ESI) calcd for C21H29O5N4S (M + H)+ 449.1853, found 449.1850.
tert-Butyl 4-(5-{[4-(Methylsulfonyl)benzyl]oxy}pyridin-2-yl)-
piperazine-1-carboxylate (6). Compound 6 was synthesized as
tert-Butyl 4-(5-{[4-(Methylsulfonyl)benzyl]oxy}pyrimidin-2-
yl)piperidine-1-carboxylate (10). Cesium carbonate (303 mg,
0.93 mmol) was added to tert-butyl 4-(5-hydroxypyrimidin-2-
yl)piperidine-1-carboxylate (130 mg, 0.47 mmol) and 4-
(methylsulfonyl)benzyl chloride (105 mg, 0.51 mmol) in DMF (7.0
mL). The resulting suspension was stirred at 20 °C for 18 h. It was
diluted with ethyl acetate (100 mL), washed with water (20 mL) and
brine (20 mL), dried (MgSO4), concentrated in vacuo, and adsorbed
onto silica. The crude product was purified by flash silica
chromatography, elution gradient 0−100% EtOAc in isohexane. Pure
fractions were evaporated to dryness to afford tert-butyl 4-(5-{[4-
(methylsulfonyl)benzyl]oxy}pyrimidin-2-yl)piperidine-1-carboxylate
1
(140 mg, 67%) as a white solid: mp 144−145 °C; H NMR (400
MHz, DMSO) δ 8.56 (s, 2H), 7.96 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.2
Hz, 2H), 5.37 (s, 2H), 3.99 (d, J = 12.7 Hz, 2H), 3.21 (s, 3H), 3.08−
2.65 (m, 3H), 1.88 (d, J = 11.0 Hz, 2H), 1.70−1.45 (m, 2H), 1.40 (s,
9H); HRMS (ESI) calcd for C22H29N3O5S (M − tBu + H)+ 392.1275,
found 392.1273.
tert-Butyl (3S)-3-Methyl-4-(5-{[4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (11). Compound
11 was synthesized as described for 2 using (S)-tert-butyl 4-(5-
hydroxypyrimidin-2-yl)-3-methylpiperazine-1-carboxylate in 94%
1
yield: H NMR (400 MHz, CDCl3) δ 8.05 (s, 2H), 7.90 (d, J = 8.4
Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 5.04 (s, 2H), 4.77−4.61 (m, 1H),
4.24 (d, J = 13.3 Hz, 1H), 4.16−3.72 (m, 2H), 3.08 (ddd, J = 13.3,
12.1, 3.6 Hz, 1H), 2.99 (s, 3H), 2.94−2.78 (m, 2H), 1.42 (s, 9H), 1.08
5372
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(d, J = 6.7 Hz, 3H); HRMS (ESI) calcd for C22H31O5N4S (M + H)+
463.2010, found 463.2011.
tert-Butyl (1R,4R)-5-(5-{[4-(Methylsulfonyl)benzyl]oxy}-
pyrimidin-2-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate
(18). Compound 18 was synthesized as described for 2 using tert-butyl
(1R,4R)-5-(5-hydroxypyrimidin-2-yl)-2,5-diazabicyclo[2.2.1]heptane-
tert-Butyl (3R)-3-Methyl-4-(5-{[4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (12). To a stirred
solution of (R)-tert-butyl 4-(5-hydroxypyrimidin-2-yl)-3-methylpiper-
azine-1-carboxylate (5.0 g, 16.99 mmol), and 1-(chloromethyl)-4-
(methylsulfonyl)benzene (3.65 g, 17.84 mmol) in acetonitrile (170
mL) at ambient temperature was added potassium carbonate (7.04 g,
50.96 mmol). The mixture was heated under reflux at 80 °C for 2 h
and cooled to ambient temperature, and the acetonitrile was
evaporated in vacuo to give a residue which was partitioned between
ethyl acetate (160 mL) and water (80 mL). The ethyl acetate layer was
washed with brine, dried (MgSO4), and evaporated in vacuo to a
residue which was crystallized from ethyl acetate/isohexane to give
tert-butyl (3R)-3-methyl-4-(5-{[4-(methylsulfonyl)benzyl]oxy}-
pyrimidin-2-yl)piperazine-1-carboxylate (7.25 g, 92%): mp 173−174
°C; 1H NMR (400 MHz, DMSO, 100 °C) δ 8.26 (s, 2H), 7.94 (d, J =
8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 5.22 (s, 2H), 4.81−4.61 (m,
1H), 4.25 (dt, J = 13.4, 3.2 Hz, 1H), 3.99−3.88 (m, 1H), 3.79 (dt, J =
13.2, 2.0 Hz, 1H), 3.17 (s, 3H), 3.15−3.05 (m, 2H), 2.94−2.87 (m,
1H), 1.44 (s, 9H), 1.08 (d, J = 6.6 Hz, 3H); HRMS (ESI) calcd for
C22H31O5N4S (M + H)+ 463.2010, found 463.2009.
1
2-carboxylate in 59% yield: mp 202−203 °C; H NMR (400 MHz,
DMSO) δ 8.23 (s, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.3 Hz,
2H), 5.20 (s, 2H), 4.81 (s, 1H), 4.45 (s, 1H), 3.49 (dd, J = 10.2, 1.8
Hz, 1H), 3.44−3.32 (m, 2H), 3.16 (s, 4H), 1.88 (s, 2H), 1.39 (s, 9H);
HRMS (ESI) calcd for C22H28O5N4S (M + H)+ 461.1853, found
461.1854.
tert-Butyl (1R,4S)-5-(5-{[4-(Methylsulfonyl)benzyl]oxy}-
pyrimidin-2-yl)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate
(19). Compound 19 was synthesized as described for 2 using tert-butyl
(1S,4S)-5-(5-hydroxypyrimidin-2-yl)-2,5-diazabicyclo[2.2.2]octane-2-
carboxylate in 48% yield: mp 154−155 °C; 1H NMR (400 MHz,
CDCl3) δ 8.04 (s, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.2 Hz,
2H), 5.03 (s, 2H), 4.86−4.73 (m, 1H), 4.40−4.14 (m, 1H), 3.77−3.63
(m, 1H), 3.61−3.35 (m, 3H), 2.99 (s, 3H), 2.08−1.85 (m, 2H), 1.85−
1.63 (m, 2H), 1.30 (s, 9H); HRMS (ESI) calcd for C23H31O5N4S (M
+ H)+ 475.2010, found 475.2006.
tert-Butyl (3R)-3-Methyl-4-(5-{[1-(methylsulfonyl)piperidin-
4-yl]methoxy}pyrimidin-2-yl)piperazine-1-carboxylate (20).
To a stirred solution of (R)-tert-butyl 4-(5-hydroxypyrimidin-2-yl)-3-
methylpiperazine-1-carboxylate (8.83 g, 30.0 mmol) and (1-
(methylsulfonyl)piperidin-4-yl)methyl methanesulfonate (8.95 g,
33.0 mmol) in DMF (150 mL) was added cesium carbonate (19.55
g, 60.00 mmol), and the mixture was heated at 55 °C for 16 h. The
mixture was cooled to ambient temperature, the dimethylformamide
evaporated in vacuo to a residue which was partitioned between water
(270 mL) and ethyl acetate (450 mL), and the ethyl acetate layer
washed with brine, dried (MgSO4), and evaporated in vacuo to a
residue which was crystallized from ethyl acetate/isohexane to give
tert-butyl (3R)-3-methyl-4-(5-{[1-(methylsulfonyl)piperidin-4-yl]-
methoxy}pyrimidin-2-yl)piperazine-1-carboxylate (9.88 g, 70%). The
mother liquors from the crystallization were evaporated in vacuo to a
residue which was chromatographed on silica with 50% ethyl acetate in
isohexane as the eluant to give a solid which was crystallized from ethyl
acetate/isohexane to give a further 2.1 g of product, giving a total yield
tert-Butyl (2R)-2-Methyl-4-(5-{[4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (13). Compound
13 was synthesized as described for 2 using (R)-tert-butyl 4-(5-
hydroxypyrimidin-2-yl)-2-methylpiperazine-1-carboxylate as the start-
1
ing material in 40% yield: mp 145−146 °C; H NMR (400 MHz,
CDCl3) δ 8.11 (s, 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.6 Hz,
2H), 5.11 (s, 2H), 4.48−4.39 (m, 1H), 4.40−4.27 (m, 2H), 3.95−3.86
(m, 1H), 3.22−3.09 (m, 2H), 3.06 (s, 3H), 2.97 (td, J = 12.1, 3.7 Hz,
1H), 1.48 (s, 9H), 1.15 (d, J = 6.7 Hz, 3H); HRMS (ESI) calcd for
C22H31O5N4S (M + H)+ 463.2009, found 463.2008.
tert-Butyl (2S)-2-Methyl-4-(5-{[4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (14). Compound
14 was synthesized as described for 2 using (S)-tert-butyl 4-(5-
hydroxypyrimidin-2-yl)-2-methylpiperazine-1-carboxylate as the start-
ing material in 29% yield: 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 2H),
8.00−7.95 (m, 2H), 7.64−7.59 (m, 2H), 5.11 (s, 2H), 4.48−4.41 (m,
1H), 4.39−4.26 (m, 2H), 3.94−3.86 (m, 1H), 3.20−3.11 (m, 2H),
3.06 (s, 3H), 3.01−2.93 (m, 1H), 1.48 (s, 9H), 1.15 (d, J = 6.7 Hz,
3H); HRMS (ESI) calcd for C22H31O5N4S (M + H)+ 463.2010, found
463.2007.
tert-Butyl 3,3-Dimethyl-4-(5-{[4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (15). Compound
15 was synthesized as described for 2 using tert-butyl 4-(5-
hydroxypyrimidin-2-yl)-3,3-dimethylpiperazine-1-carboxylate as the
starting material in 28% yield: mp 140−141 °C; 1H NMR (400
MHz, CDCl3) δ 8.11 (s, 2H), 8.01−7.94 (m, 2H), 7.62 (d, J = 8.5 Hz,
2H), 5.11 (s, 2H), 4.04−3.94 (m, 2H), 3.62−3.41 (m, 4H), 3.06 (s,
3H), 1.51 (s, 6H), 1.48 (s, 9H); HRMS (ESI) calcd for C23H33O5N4S
(M + H)+ 477.2166, found 477.2161.
1
of 85%: mp 162−164 °C; H NMR (400 MHz, DMSO, 100 °C) δ
8.19 (s, 2H), 4.77−4.63 (m, 1H), 4.24 (dt, J = 13.3, 3.2 Hz, 1H), 3.89
(d, J = 6.0 Hz, 2H), 3.79 (dt, J = 13.2, 1.9 Hz, 1H), 3.62 (d, J = 12.1
Hz, 2H), 3.20−3.02 (m, 2H), 2.95 (s, 3H), 2.95−2.87 (m, 2H), 2.77
(d, J = 2.5 Hz, 1H), 1.87 (m, 4H, 1.49−1.44 (m, 2H), 1.44 (s, 9H),
1.08 (d, J = 6.6 Hz, 3H); HRMS (ESI) calcd for C21H36O5N5S (M +
H)+ 470.2432, found 470.2429.
tert-Butyl (3R)-4-(5-{[4-(Dimethylsulfamoyl)benzyl]oxy}-
pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (21). Com-
pound 21 was synthesized as described for 20 using (R)-tert-butyl 4-
(5-((4-bromo-2-cyanobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-
1-carboxylate and 4-(N,N-dimethylsulfamoyl)benzyl methanesulfonate
1
as starting materials in 91% yield: H NMR (400 MHz, DMSO, 100
°C) δ 8.26 (s, 2H), 7.78−7.75 (m, 2H), 7.71−7.66 (m, 2H), 5.20 (s,
2H), 4.76−4.66 (m, 1H), 4.32−4.20 (m, 1H), 3.98−3.88 (m, 1H),
3.84−3.74 (m, 1H), 3.20−3.06 (m, 2H), 2.98−2.86 (m, 1H), 2.67 (s,
6H), 1.44 (s, 9H), 1.08 (d, J = 6.6 Hz, 3H); HRMS (ESI) calcd for
C23H34O5N5S (M + H)+ 492.2275, found 492.2274.
tert-Butyl (3R,5S)-3,5-Dimethyl-4-(5-{[4-(methylsulfonyl)-
benzyl]oxy}pyrimidin-2-yl)piperazine-1-carboxylate (16). Com-
pound 16 was synthesized as described for 2 using tert-butyl (3R,5S)-
4-(5-hydroxypyrimidin-2-yl)-3,5-dimethylpiperazine-1-carboxylate as
1
the starting material in 25% yield: H NMR (400 MHz, CDCl3) δ
tert-Butyl (3R)-4-{5-[(4-Cyanobenzyl)oxy]pyrimidin-2-yl}-3-
methylpiperazine-1-carboxylate (22). To a mixture of (R)-tert-
butyl 4-(5-hydroxypyrimidin-2-yl)-3-methylpiperazine-1-carboxylate
(295 mg, 1.00 mmol), 4-(bromomethyl)benzonitrile (216 mg, 1.10
mmol), and cesium carbonate (980 mg, 3.01 mmol) under an
atmosphere of nitrogen in a microwave vial was added acetonitrile
(13.5 mL). The mixture was heated at 100 °C for 1 h, and the
acetonitrile was evaporated in vacuo to a residue which was taken up in
ethyl acetate (50 mL), washed with water and brine, dried (MgSO4),
and evaporated in vacuo to a residue which was chromatographed on
silica with 30% ethyl acetate in isohexane as the eluant to give a solid
which was crystallized from ethyl acetate/isohexane to give tert-butyl
(3R)-4-{5-[(4-cyanobenzyl)oxy]pyrimidin-2-yl}-3-methylpiperazine-1-
8.15 (s, 2H), 8.01−7.94 (m, 2H), 7.62 (d, J = 8.5 Hz, 2H), 5.11 (s,
2H), 4.66 (s, 3H), 4.22−3.82 (m, 3H), 3.06 (s, 3H), 1.50 (s, 9H), 1.23
(s, 3H), 1.21 (s, 3H); HRMS (ESI) calcd for C23H33O5N4S (M + H)+
477.2166, found 477.2164.
tert-Butyl (2R,5S)-2,5-Dimethyl-4-(5-{[4-(methylsulfonyl)-
benzyl]oxy}pyrimidin-2-yl)piperazine-1-carboxylate (17). Com-
pound 17 was synthesized as described for 2 using tert-butyl (2R,5S)-
4-(5-hydroxypyrimidin-2-yl)-2,5-dimethylpiperazine-1-carboxylate as
1
the starting material in 30% yield: H NMR (400 MHz, CDCl3) δ
8.11 (s, 2H), 8.00−7.95 (m, 2H), 7.62 (d, J = 8.5 Hz, 2H), 5.11 (s,
2H), 4.84−4.70 (m, 1H), 4.48 (s, 1H), 4.32−4.15 (m, 1H), 3.86−3.67
(m, 1H), 3.36−3.23 (m, 2H), 3.06 (s, 3H), 1.48 (s, 9H), 1.15 (d, J =
5.6 Hz, 6H); HRMS (ESI) calcd for C23H33O5N4S (M + H)+
477.2166, found 477.2163.
1
carboxylate (343 mg, 84%): mp 144−145 °C; H NMR (400 MHz,
5373
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DMSO, 100 °C) δ 8.24 (s, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.62 (d, J =
8.0 Hz, 2H), 5.19 (s, 2H), 4.76−4.62 (m, 1H), 4.24 (d, J = 13.4 Hz,
1H), 3.97−3.87 (m, 1H), 3.83−3.73 (m, 1H), 3.19−3.04 (m, 3H),
2.98−2.89 (m, 1H), 1.43 (s, 9H), 1.08 (d, J = 6.6 Hz, 3H); HRMS
(ESI) calcd for C22H28O3N5 (M + H)+ 410.2187, found 410.2187.
tert-Butyl (3R)-3-Methyl-4-(5-{[4-(1H-tetrazol-1-yl)benzyl]-
oxy}pyrimidin-2-yl)piperazine-1-carboxylate (23). Diisopropyl
azodicarboxylate (0.176 mL, 0.89 mmol) was added to a stirred
solution of (R)-tert-butyl 4-(5-hydroxypyrimidin-2-yl)-3-methylpiper-
azine-1-carboxylate (0.21 g, 0.71 mmol), and triphenylphosphine
(0.281 g, 1.07 mmol) in THF (15 mL) under nitrogen. The resulting
solution was stirred at 20 °C for 30 min, and then (4-(1H-tetrazol-1-
yl)phenyl)methanol (0.157 g, 0.89 mmol) was added. The resulting
solution was stirred at room temperature overnight under nitrogen.
The solvent was evaporated and the residue diluted with EtOAc and
brine. A white precipitate was filtered off and dried under vacuum. The
aqueous layer was extracted with EtOAc (50 mL), and the combined
organics were concentrated in vacuo to afford crude product. The
crude product was purified by flash silica chromatography, elution
gradient 1−4% MeOH in CH2Cl2. The crude product was repurified
by flash silica chromatography, elution gradient 40−100% EtOAc in
isohexane. Pure fractions were evaporated to dryness to afford tert-
butyl (3R)-3-methyl-4-(5-{[4-(1H-tetrazol-1-yl)benzyl]oxy}pyrimidin-
2-yl)piperazine-1-carboxylate (87 mg, 27%) as a white solid: mp 202−
8.27 (dd, J = 2.3, 8.2 Hz, 1H), 8.17 (s, 2H), 7.76 (d, J = 8.2 Hz, 1H),
5.24 (s, 2H), 4.84−4.71 (m, 1H), 4.32 (d, J = 10.8 Hz, 1H), 4.23−3.82
(m, 2H), 3.22−3.14 (m, 2H), 3.12 (s, 3H), 3.01−2.77 (m, 1H), 1.49
(s, 9H), 1.15 (d, J = 6.7 Hz, 3H); HRMS (ESI) calcd for C21H30O5N5
S (M + H)+ 464.1962, found 464.1962.
tert-Butyl 4-[5-(Pyridin-4-ylmethoxy)pyrimidin-2-yl]-
piperazine-1-carboxylate (27). Compound 27 was synthesized as
described for 22 using tert-butyl 4-(5-hydroxypyrimidin-2-yl)-
piperazine-1-carboxylate and 4-(bromomethyl)nicotinonitrile as start-
1
ing materials in 32% yield: mp 147−148 °C; H NMR (400 MHz,
CDCl3) δ 8.68−8.58 (m, 2H), 8.12 (s, 2H), 7.37−7.29 (m, 2H), 5.04
(s, 2H), 3.71 (dd, J = 6.2, 4.3 Hz, 4H), 3.52−3.46 (m, 4H), 1.48 (s,
9H); HRMS (ESI) calcd for C19H26O3N5 (M + H)+ 372.2030, found
372.2029.
tert-Butyl 4-{5-[(2-Methylpyridin-4-yl)methoxy]pyrimidin-2-
yl}piperazine-1-carboxylate (28). Cesium carbonate (2.111 g, 6.48
mmol) was added to a mixture of 4-(chloromethyl)-2-methylpyridine
hydrochloride (0.288 g, 1.62 mmol) and tert-butyl 4-(5-hydroxypyr-
imidin-2-yl)piperazine-1-carboxylate (0.454 g, 1.62 mmol) in DMF (5
mL). The resulting mixture was stirred at 40 °C for 2 h. The reaction
mixture was diluted with EtOAc (75 mL) and washed sequentially
with water (20 mL) and saturated brine (20 mL). The organic layer
was dried over Na2SO4, filtered, and evaporated to afford crude
product. The crude product was purified by flash alumina
chromatography, elution gradient 0−70% EtOAc in isohexane. Pure
fractions were evaporated to dryness to afford tert-butyl 4-{5-[(2-
methylpyridin-4-yl)methoxy]pyrimidin-2-yl}piperazine-1-carboxylate
(194 mg, 31%) as a yellow solid: mp 97−98 °C; 1H NMR (400 MHz,
DMSO) δ 8.49 (d, J = 5.06 Hz, 1H), 8.34 (s, 2H), 7.36−7.33 (m, 1H),
7.29−7.25 (m, 1H), 5.19 (s, 2H), 3.71−3.63 (m, 4H), 3.47−3.40 (m,
4H), 1.48 (s, 9H); HRMS (ESI) calcd for C20H28O3N5 (M + H)+
386.2187, found 386.2185.
1
203 °C; H NMR (400 MHz, CDCl3) δ 8.99 (s, 1H), 8.14 (s, 2H),
7.80−7.22 (m, 2H), 7.68−7.60 (m, 2H), 5.11 (s, 2H), 4.84−4.69 (m,
1H), 4.32 (d, J = 13.1 Hz, 1H), 4.13 (s, 2H), 3.20−3.03 (m, 2H),
3.01−2.76 (m, 1H), 1.49 (s, 9H), 1.15 (d, J = 6.7 Hz, 3H); HRMS
(ESI) calcd for C22H29O3N8 (M + H)+ 453.2357, found 453.2358.
tert-Butyl (3R)-4-(5-{[2-Fluoro-4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (24).
Copper(I) iodide (2.58 μL, 0.07 mmol) was added to (R)-tert-butyl
4-(5-((4-bromo-2-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpipera-
zine-1-carboxylate (330 mg, 0.69 mmol), methanesulfinic acid sodium
salt (84 mg, 0.82 mmol), L-proline (15.79 mg, 0.14 mmol), and
sodium hydroxide (4.06 μL, 0.14 mmol) in DMSO (3.0 mL) under
nitrogen. The resulting solution was degassed with nitrogen for 20 min
and stirred at 80 °C for 2 d. It was cooled to room temperature and
partitioned between 50% brine (50 mL) and ethyl acetate (150 mL).
The aqueous portion was extracted with ethyl acetate (2 × 100 mL),
and all of the combined organics were washed with brine (50 mL),
dried (sodium sulfate), concentrated in vacuo, and adsorbed onto
silica. The crude product was purified by flash silica chromatography,
elution gradient 0−75% EtOAc in isohexane. Pure fractions were
evaporated to dryness to afford tert-butyl (3R)-4-(5-{[2-fluoro-4-
(methylsulfonyl)benzyl]oxy}pyrimidin-2-yl)-3-methylpiperazine-1-car-
tert-Butyl 4-{5-[(3-Methylpyridin-4-yl)methoxy]pyrimidin-2-
yl}piperazine-1-carboxylate (29). Compound 29 was synthesized
as described for 23 using tert-butyl 4-(5-hydroxypyrimidin-2-yl)-
piperazine-1-carboxylate and (3-methylpyridin-4-yl)methanol as start-
1
ing materials in 61% yield: mp 154−155 °C; H NMR (400 MHz,
DMSO) δ 8.40 (d, J = 5.1 Hz, 2H), 8.31 (s, 2H), 7.39 (d, J = 4.9 Hz,
1H), 5.15 (s, 2H), 3.74−3.52 (m, 4H), 3.45−3.32 (m, 4H), 2.28 (s,
3H), 1.41 (s, 9H); HRMS (ESI) calcd for C20H27O3N5 (M + H)+
386.2187, found 386.2185.
tert-Butyl 4-{5-[(3-Methoxypyridin-4-yl)methoxy]pyrimidin-
2-yl}piperazine-1-carboxylate (30). Compound 30 was synthe-
sized as described for 23 using tert-butyl 4-(5-hydroxypyrimidin-2-
yl)piperazine-1-carboxylate and (3-methoxypyridin-4-yl)methanol as
starting materials in 37% yield: mp 126−127 °C; 1H NMR (400 MHz,
DMSO) δ 8.38 (s, 1H), 8.27 (s, 2H), 8.23 (d, J = 4.7 Hz, 1H), 7.41 (d,
J = 4.7 Hz, 1H), 5.11 (s, 2H), 3.93 (s, 3H), 3.61 (dd, J = 6.2, 4.3 Hz,
4H), 3.40−3.35 (m, 4H), 1.41 (s, 9H); HRMS (ESI) calcd for
C20H27O4N5 (M + H)+ 402.2136, found 402.2135.
1
boxylate (190 mg, 58%) as a white solid: mp 124−125 °C; H NMR
(400 MHz, DMSO) δ 8.31 (s, 2H), 7.93−7.74 (m, 3H), 5.25 (s, 2H),
4.77−4.57 (m, 1H), 4.23 (d, J = 13.0 Hz, 1H), 3.94 (s, 1H), 3.80 (d, J
= 13.1 Hz, 1H), 3.27 (s, 3H), 3.14−2.96 (m, 2H), 2.86 (s, 1H), 1.41
(s, 9H), 1.03 (d, J = 6.6 Hz, 3H); HRMS (ESI) calcd for
C22H29O5N4SF (M + H)+ 481.1916, found 481.1912.
tert-Butyl 4-{5-[(3-Fluoropyridin-4-yl)methoxy]pyrimidin-2-
yl}piperazine-1-carboxylate (31). Compound 31 was synthesized
as described for 23 using tert-butyl 4-(5-hydroxypyrimidin-2-yl)-
piperazine-1-carboxylate and (3-fluoropyridin-4-yl)methanol as start-
tert-Butyl (3R)-4-(5-{[2-Cyano-4-(methylsulfonyl)benzyl]-
oxy}pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (25).
Compound 25 was synthesized as described for 24 using (R)-tert-
butyl 4-(5-((4-bromo-2-cyanobenzyl)oxy)pyrimidin-2-yl)-3-methylpi-
perazine-1-carboxylate as the starting material in 50% yield: mp 159−
160 °C; 1H NMR (400 MHz, DMSO, 100 °C) δ 8.39 (d, J = 1.8 Hz,
1H), 8.30 (s, 2H), 8.25 (dd, J = 8.2, 1.9 Hz, 1H), 7.98 (d, J = 8.2 Hz,
1H), 5.35 (s, 2H), 4.78−4.67 (m, 1H), 4.27 (dt, J = 13.4, 3.3 Hz, 1H),
3.93 (d, J = 12.9 Hz, 1H), 3.80 (dt, J = 13.2, 1.8 Hz, 1H), 3.13 (ddd, J
= 13.4, 12.3, 3.7 Hz, 2H), 2.95 (s, 3H), 3.01−2.90 (m, 1H), 1.44 (s,
9H), 1.10 (d, J = 6.6 Hz, 3H); HRMS (ESI) calcd for C23H30O5N5S
(M + H)+ 488.1962, found 488.1960.
tert-Butyl (3R)-3-Methyl-4-(5-{[5-(methylsulfonyl)pyridin-2-
yl]methoxy}pyrimidin-2-yl)piperazine-1-carboxylate (26).
Compound 26 was synthesized as described for 24 using (R)-tert-
butyl 4-(5-((5-bromopyridin-2-yl)methoxy)pyrimidin-2-yl)-3-methyl-
piperazine-1-carboxylate as the starting material in 26% yield: mp
219−220 °C; 1H NMR (400 MHz, CDCl3) δ 9.12 (d, J = 2.3 Hz, 1H),
1
ing materials in 46% yield: mp 148−148 °C; H NMR (400 MHz,
CDCl3) δ 8.46 (s, 1H), 8.41−8.37 (m, 1H), 8.09 (s, 2H), 7.43 (m,
1H), 5.06 (s, 2H), 3.68 (dd, J = 6.2, 4.3 Hz, 4H), 3.43 (dd, J = 6.2, 4.3
Hz, 4H), 1.41 (s, 9H); HRMS (ESI) calcd for C19H25O3N5F (M +
H)+ 390.1935, found 390.1936.
tert-Butyl 4-{5-[(3-Cyanopyridin-4-yl)methoxy]pyrimidin-2-
yl}piperazine-1-carboxylate (32). tert-Butyl 4-(5-((3-bromopyri-
din-4-yl)methoxy)pyrimidin-2-yl)piperazine-1-carboxylate (0.95 g,
2.11 mmol), zinc cyanide (0.198 g, 1.69 mmol), tris-
(dibenzylideneacetone)dipalladium(0) (0.077 g, 0.08 mmol), and
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (xantphos; 0.098 g,
0.17 mmol) were suspended in DMF (20 mL) and sealed into a
microwave tube (evacuated and purged with nitrogen). The reaction
was heated to 130 °C for 60 min in the microwave reactor and cooled
to room temperature. The reaction mixture was filtered through Celite.
The reaction mixture was diluted with EtOAc (100 mL) and washed
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sequentially with water (100 mL) and saturated brine (100 mL). The
organic layer was dried over Na2SO4, filtered, and evaporated to afford
crude product that was purified by flash silica chromatography, elution
gradient 1−4% MeOH in CH2Cl2 and by flash silica chromatography,
elution gradient 20−80% EtOAc in isohexane. Pure fractions were
evaporated to dryness to afford tert-butyl 4-{5-[(3-cyanopyridin-4-
yl)methoxy]pyrimidin-2-yl}piperazine-1-carboxylate (410 mg, 49%) as
1H), 4.22−3.80 (m, 2H), 3.18−2.99 (m, 2H), 2.97−2.79 (m, 1H),
1.20 (d, J = 6.2 Hz, 6H), 1.09 (d, J = 6.7 Hz, 3H); HRMS (ESI) calcd
for C20H25O3N6 (M + H)+ 397.1983, found 397.1981.
1-Cyano-1-methylethyl (3R)-4-{5-[(3-Cyanopyridin-4-yl)-
methoxy]pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate
(38). Triethylamine (0.251 mL, 1.80 mmol) was added to (R)-4-((2-
(2-methylpiperazin-1-yl)pyrimidin-5-yloxy)methyl)nicotinonitrile
(0.28 g, 0.90 mmol) and 2-cyanopropan-2-yl phenyl carbonate (0.278
g, 1.35 mmol) in chloroform (2 mL) under nitrogen. The resulting
solution was stirred at 110 °C for 18 h, during which time all the
solvent was evaporated to leave a thick gum. The reaction mixture was
diluted with CH2Cl2 (50 mL) and washed with 2 M K2CO3 aq. (20
mL). The organic layer was dried over Na2SO4, filtered, and
evaporated to afford crude product that was purified by flash silica
chromatography, elution gradient 0−5% MeOH in CH2Cl2. The crude
solid was triturated with Et2O and then crystallized from hot EtOH to
give 1-cyano-1-methylethyl (3R)-4-{5-[(3-cyanopyridin-4-yl)-
methoxy]pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate (62 mg,
1
a white solid: mp 107−108 °C; H NMR (400 MHz, CDCl3) δ 8.90
(s, 1H), 8.84 (d, J = 5.2 Hz, 1H), 8.17 (s, 2H), 7.65 (d, J = 5.2 Hz,
1H), 5.21 (s, 2H), 3.80−3.70 (m, 4H), 3.54−3.45 (m, 4H), 1.49 (s,
9H); HRMS (ESI) calcd for C20H25O3N6 (M + H)+ 397.1983, found
397.1981.
tert-Butyl 4-(5-{[3-(Methylsulfonyl)pyridin-4-yl]methoxy}-
pyrimidin-2-yl)piperazine-1-carboxylate (33). Compound 33
was synthesized as described for 24 using tert-butyl 4-(5-((3-
bromopyridin-4-yl)methoxy)pyrimidin-2-yl)piperazine-1-carboxylate
as the starting material in 33% yield: mp 148−149 °C; 1H NMR (400
MHz, CDCl3) δ 9.14 (s, 1H), 8.84 (d, J = 5.1 Hz, 1H), 8.12 (s, 2H),
7.76−7.58 (m, 2H), 5.06 (s, 2H), 5.37 (s, 2H), 3.67 (dd, J = 6.2, 4.3
Hz, 4H), 3.43 (dd, J = 6.2, 4.3 Hz, 4H), 3.11 (s, 3H), 1.42 (s, 9H);
HRMS (ESI) calcd for C20H28O5N5S (M + H)+ 450.1806, found
450.1803.
1
16%) as a white solid: H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H),
8.85 (d, J = 5.2 Hz, 1H), 8.19 (s, 2H), 7.71−7.62 (m, 1H), 5.21 (s,
2H), 4.89−4.75 (m, 1H), 4.46−4.34 (d, J = 13.5 Hz, 1H), 4.05−3.89
(m, 1H), 3.80 (d, J = 13.4 Hz, 1H), 3.20 (td, J = 13.3, 3.5, Hz, 2H),
3.12−2.92 (m, 1H), 1.80 (s, 6H), 1.18 (d, J = 6.1 Hz, 3H); HRMS
(ESI) calcd for C21H24O3N7 (M + H)+ 422.1935, found 422.1932.
2,2,2-Trifluoroethyl (3R)-4-{5-[(3-Cyanopyridin-4-yl)-
methoxy]pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate
(39). Compound 39 was synthesized as described for 38 using (R)-4-
((2-(2-methylpiperazin-1-yl)pyrimidin-5-yloxy)methyl)nicotinonitrile
and 2,2,2-trifluoroethyl phenyl carbonate as starting materials in 39%
tert-Butyl 4-{5-[(3,5-Difluoropyridin-4-yl)methoxy]-
pyrimidin-2-yl}piperazine-1-carboxylate (34). Compound 34
was synthesized as described for 23 using tert-butyl 4-(5-hydroxypyr-
imidin-2-yl)piperazine-1-carboxylate and (3,5-difluoropyridin-4-yl)-
1
methanol as starting materials in 50% yield: H NMR (400 MHz,
CDCl3) δ 8.41 (s, 1H), 8.14 (s, 1H), 7.26 (s, 1H), 5.13 (s, 2H), 3.78−
3.68 (m, 4H), 3.53−3.44 (m, 4H), 1.48 (s, 9H); HRMS (ESI) calcd
for C19H24O3N5F2 (M + H)+ 408.1842, found 408.1840.
1
yield: H NMR (400 MHz, DMSO) δ 8.99 (s, 1H), 8.85 (d, J = 4.9
Hz, 1H), 8.31 (s, 2H), 7.72 (d, J = 4.9 Hz, 1H), 5.31 (s, 2H), 4.85−
4.60 (m, 3H), 4.40−4.27 (m, 1H), 4.03−3.91 (m, 1H), 3.89−3.77 (m,
1H), 3.33−3.03 (m, 3H), 1.10 (d, J = 6.5 Hz, 3H); HRMS (ESI) calcd
for C19H20O3N6F3 (M + H)+ 437.1543, found 437.1542.
2,2-Difluoroethyl (3R)-4-{5-[(3-Cyanopyridin-4-yl)methoxy]-
pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate (40). Com-
pound 40 was synthesized as described for 38 using (R)-4-((2-(2-
methylpiperazin-1-yl)pyrimidin-5-yloxy)methyl)nicotinonitrile and
2,2-difluoroethyl phenyl carbonate as starting materials in 10% yield:
1H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H), 8.85 (d, J = 5.2 Hz, 1H),
8.18 (s, 2H), 7.71−7.61 (m, 1H), 5.97 (t, J = 55.4 Hz, 1H), 5.21 (s,
2H), 4.90−4.78 (m, 1H), 4.48−4.23 (m, 3H), 4.23−3.86 (m, 2H),
3.20 (td, J = 13.1, 3.5 Hz, 2H), 3.13−2.95 (m, 1H), 1.17 (d, J = 6.6 Hz,
3H); LRMS (ES+) m/z (M + H)+ 419.
tert-Butyl (3R)-3-Methyl-4-[5-(pyridin-4-ylmethoxy)-
pyrimidin-2-yl]piperazine-1-carboxylate (35). Compound 35
was synthesized as described for 28 using (R)-tert-butyl 4-(5-
hydroxypyrimidin-2-yl)-3-methylpiperazine-1-carboxylate and 3-meth-
yl-4-(chloromethyl)pyridine hydrochloride as starting materials in 51%
yield: mp 126−128 °C; 1H NMR (400 MHz, DMSO, 100 °C) δ 8.57
(d, J = 6.0 Hz, 2H), 8.25 (s, 2H), 7.40 (d, J = 5.9 Hz, 2H), 5.14 (s,
2H), 4.70 (ddd, J = 6.5, 3.9, 2.5 Hz, 1H), 4.25 (dt, J = 13.4, 3.2 Hz,
1H), 3.92 (dt, J = 12.9, 3.9 Hz, 1H), 3.79 (dt, J = 13.2, 1.0 Hz, 1H),
3.16−3.05 (m, 2H), 2.99−2.85 (m, 1H), 1.44 (s, 9H), 1.08 (d, J = 6.6
Hz, 3H); HRMS (ESI) calcd for C20H28O3N5 (M + H)+ 386.2187,
found 386.2187.
tert-Butyl (3R)-4-{5-[(3-Cyanopyridin-4-yl)methoxy]-
pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate (36). Com-
pound 36 was synthesized as described for 32 using (R)-tert-butyl 4-
(5-((3-bromopyridin-4-yl)methoxy)pyrimidin-2-yl)-3-methylpipera-
zine-1-carboxylate as the starting material in 93% yield: mp 91−93 °C;
1H NMR (400 MHz, CDCl3) δ 8.93−8.88 (m, 1H), 8.84 (d, J = 5.2
4-[({2-[(2R)-4-(5-Fluoropyrimidin-2-yl)-2-methylpiperazin-1-
yl]pyrimidin-5-yl}oxy)methyl]pyridine-3-carbonitrile (41). To a
stirred solution of (R)-4-((2-(2-methylpiperazin-1-yl)pyrimidin-5-
yloxy)methyl)nicotinonitrile dihydrochloride (1.31 g, 3.42 mmol)
and 2-chloro-5-fluoropyrimidine (0.498 g, 3.76 mmol) in acetonitrile
(54 mL) in a microwave vial was added N,N-diisopropylethylamine
(1.905 mL, 10.94 mmol). The stirred mixture was heated at 150 °C for
3 h and cooled to ambient temperature, and the reaction was diluted
with EtOAc (300 mL), washed with water (150 mL) and then brine
(100 mL), and evaporated in vacuo to a residue which was
chromatographed on silica with 0−70% EtOAc in isohexane as the
eluant to give a solid which was triturated with CH2Cl2/ether to give
4-[({2-[(2R)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl]-
pyrimidin-5-yl}oxy)methyl]pyridine-3-carbonitrile (400 mg, 29%): mp
Hz, 1H), 8.18 (s, 2H), 7.66 (dd, J = 5.2, 0.8 Hz, 1H), 5.25−5.18 (m,
2H), 4.85−4.68 (m, 1H), 4.34 (d, J = 13.3 Hz, 1H), 4.22−3.84 (m,
2H), 3.21−3.05 (m, 2H), 3.01−2.74 (m, 1H), 1.49 (s, 9H), 1.16 (d, J
= 6.7 Hz, 3H); HRMS (ESI) calcd for C21H27O3N6 (M + H)+
411.2139, found 411.2138.
1-Methylethyl (3R)-4-{5-[(3-Cyanopyridin-4-yl)methoxy]-
pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate (37). To a
stirred solution of (R)-4-((2-(2-methylpiperazin-1-yl)pyrimidin-5-
yloxy)methyl)nicotinonitrile (320 mg, 1.03 mmol) and N,N-
diisopropylethylamine (0.360 mL, 2.06 mmol) in dichloromethane
(37 mL) under nitrogen at 0 °C was added isopropyl chloroformate
(1.24 mL, 1.24 mmol). The mixture was stirred at 0 °C for 1 h and
then at ambient temperature for 16 h. The dichloromethane was
evaporated in vacuo to a residue which was taken up in ethyl acetate
(50 mL), washed with 2 M sodium carbonate solution and brine, dried
(MgSO4), and evaporated in vacuo to a residue which was
chromatographed on silica with 50−100% ethyl acetate in isohexane
as the eluant to give 1-methylethyl (3R)-4-{5-[(3-cyanopyridin-4-
yl)methoxy]pyrimidin-2-yl}-3-methylpiperazine-1-carboxylate (165
1
140−141 °C; H NMR (400 MHz, DMSO) δ 1.05 (d, J = 6.6 Hz,
3H), 3.04 (td, J = 12.0, 3.7 Hz, 1H), 3.27−3.14 (m, 2H), 4.35 (dt, J =
13.2, 3.1 Hz, 1H), 4.55−4.40 (m, 2H), 4.79 (ddd, J = 6.5, 3.9, 2.4 Hz,
1H), 5.34 (s, 2H), 7.74 (dd, J = 5.1, 0.6 Hz, 1H), 8.34 (s, 2H), 8.45 (d,
J = 0.8 Hz, 2H), 8.87 (d, J = 5.1 Hz, 1H), 9.05 (d, J = 0.6 Hz, 1H);
HRMS (ESI) calcd for C20H20ON8F (M + H)+ 407.1739, found
407.1737.
4-{[(2-{(2R)-2-Methyl-4-[5-(1-methylethyl)-1,2,4-oxadiazol-3-
yl]piperazin-1-yl}pyrimidin-5-yl)oxy]methyl}pyridine-3-car-
bonitrile (42). (R)-3-(4-(5-((3-Bromopyridin-4-yl)methoxy)-
pyrimidin-2-yl)-3-methylpiperazin-1-yl)-5-isopropyl-1,2,4-oxadiazole
(1.09 g, 2.30 mmol), zinc cyanide (0.270 g, 2.30 mmol),
tris(dibenzylideneacetone)dipalladium(0) (7.80 mg, 8.52 μmol), and
1
mg, 40%): H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 8.78 (d, J
= 5.2 Hz, 1H), 8.11 (s, 2H), 7.59 (d, J = 5.2 Hz, 1H), 5.14 (s, 2H),
4.90 (hept, J = 6.2 Hz, 1H), 4.79−4.65 (m, 1H), 4.29 (d, J = 12.9 Hz,
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xantphos (0.106 g, 0.18 mmol) were placed in a dry flask and stirred
under a stream of nitrogen . After 20 min degassed DMF (1 mL) was
added and the resultant black mixture degassed by vacuum/nitrogen
six times. The reaction was heated to 130 °C for 165 min in an oil
bath, cooled to room temperature, then diluted with CH2Cl2 (100
mL), and washed with 2 M K2CO3(aq) (50 mL). The organic layer
was dried over MgSO4, filtered, and evaporated to afford crude
product as a brown oil which solidified on standing. The crude product
was purified by flash silica chromatography, elution gradient 0−100%
EtOAc in isohexane. Pure fractions were evaporated to dryness to
afford 4-{[(2-{(2R)-2-methyl-4-[5-(1-methylethyl)-1,2,4-oxadiazol-3-
yl]piperazin-1-yl}pyrimidin-5-yl)oxy]methyl}pyridine-3-carbonitrile
(925 mg, 88%) as a very pale yellow solid: mp 117−118 °C; [α]D18
1615, 1583, 1274, 1163, 1088, 1061, 1035, 991, 909, 837, 791 cm−1;
HRMS (ESI) calcd for C19H18O2N8F3(M + H)+ 447.1499, found
447.1497.
4-{[(2-{(2R)-4-[5-(Difluoromethyl)-1,2,4-oxadiazol-3-yl]-2-
methylpiperazin-1-yl}pyrimidin-5-yl)oxy]methyl}pyridine-3-
carbonitrile (46). Compound 46 was synthesized as described for 45
using (R)-2-(4-(5-(difluoromethyl)-1,2,4-oxadiazol-3-yl)-2-methylpi-
perazin-1-yl)pyrimidin-5-ol as the starting material in 34% yield: mp
1
126−127 °C; [α]1D8 −60.9; H NMR (400 MHz, CDCl3) δ 8.91 (s,
1H), 8.85 (d, J = 5.2 Hz, 1H), 8.20 (s, 2H), 7.66 (dd, J = 5.2, 0.8 Hz,
1H), 6.65 (t, J = 52.4 Hz, 1H), 5.22 (s, 2H), 4.87−4.85 (m, 1H),
4.56−4.47 (m, 1H), 4.07−3.98 (m, 1H), 3.88 (dt, J = 12.8, 1.7 Hz,
1H), 3.38−3.28 (m, 2H), 3.13 (td, J = 12.3, 3.7 Hz, 1H), 1.25 (d, J =
6.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ = 170.6, 167.4 (t, J =
29.5 Hz), 157.9, 153.5, 153.0, 148.8, 146.5 (2C), 144.8, 121.8, 114.8,
108.1, 105.6 (t, J = 244.4 Hz), 68.8, 50.3, 46.6, 45.8, 37.9, 14.3; IR
(Nujol) νmax 2231, 1609, 1583, 1289, 1270, 1228, 1179, 1115, 1081,
1063, 1041, 910, 833, 822 cm−1; HRMS (ESI) calcd for C19H18O2N8F2
(M + H)+ 429.1594, found 429.1590.
1
−67.6; H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H), 8.85 (d, J = 5.2
Hz, 1H), 8.20 (s, 2H), 7.66 (dd, J = 5.2, 0.6 Hz, 1H), 5.22 (s, 2H),
4.96−4.86 (m, 1H), 4.47 (ddd, J = 13.4, 3.3, 2.2 Hz, 1H), 4.01 (ddt, J
= 12.4, 3.5, 1.8 Hz, 1H), 3.85 (dt, J = 12.6, 1.7 Hz, 1H), 3.31 (ddd, J =
13.4, 12.3, 3.7 Hz, 1H), 3.22 (dd, J = 12.7, 4.0 Hz, 1H), 3.14−2.99 (m,
2H), 1.36 (d, J = 7.0 Hz, 6H), 1.25 (d, J = 6.7 Hz, 3H); 13C NMR
(101 MHz, CDCl3) 183.0, 170.8, 158.0, 153.4, 153.0, 148.9, 146.6,
144.7, 121.8, 114.8, 108.1, 68.8, 50.3, 46.6, 45.9, 38.2, 27.7, 20.1 (2C),
14.3; IR (Nujol) νmax 2234, 1586, 1548, 1461, 1376, 1287, 1269, 1223,
1176, 1036, 927, 834 cm−1; HRMS (ESI) calcd for C21H25O2N8 (M +
H)+ 421.2095, found 421.2092.
4-{[(2-{(2R)-2-Methyl-4-[3-(1-methylethyl)-1,2,4-oxadiazol-5-
yl]piperazin-1-yl}pyrimidin-5-yl)oxy]methyl}pyridine-3-car-
bonitrile (47). Compound 47 was synthesized as described for 45
using (R)-2-(4-(3-isopropyl-1,2,4-oxadiazol-5-yl)-2-methylpiperazin-1-
yl)pyrimidin-5-ol as the starting material in 68% yield: mp 115−116
°C; [α]1D8 −79.2; 1H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H), 8.85 (d,
J = 5.2 Hz, 1H), 8.20 (s, 2H), 7.68−7.63 (m, 1H), 5.22 (s, 2H), 4.99−
4.89 (m, 1H), 4.56−4.44 (m, 1H), 4.20−4.08 (m, 1H), 3.96 (dd, J =
14.7, 1.6 Hz, 1H), 3.41 (dd, J = 13.0, 4.0 Hz, 1H), 3.35−3.20 (m, 2H),
2.91 (hept, J = 7.0 Hz, 1H), 1.30 (d, J = 7.0 Hz, 6H), 1.23 (d, J = 6.7
Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 175.8, 171.4, 157.7, 153.4,
152.9, 148.7, 146.5, 144.9, 121.8, 114.7, 108.1, 68.7, 50.1, 46.5, 45.7,
37.9, 27.0, 20.4 (2C), 14.1; IR (Nujol) νmax 2230, 1621, 1592, 1552,
1460, 1408, 1347, 1281, 1265, 1222, 1180, 1034, 1017, 839 cm−1;
HRMS (ESI) calcd for C21H25O2N8 (M + H)+ 421.2095, found
421.2091.
4-[({2-[(2R)-4-(5-Cyclopropyl-1,2,4-oxadiazol-3-yl)-2-methyl-
piperazin-1-yl]pyrimidin-5-yl}oxy)methyl]pyridine-3-carboni-
trile (43). Compound 43 was synthesized as described for 42 using
(R)-3-(4-(5-((3-bromopyridin-4-yl)methoxy)pyrimidin-2-yl)-3-meth-
ylpiperazin-1-yl)-5-cyclopropyl-1,2,4-oxadiazole as the starting material
1
in 54% yield: mp 132−133 °C; H NMR (400 MHz, DMSO) δ 9.11
(s, 1H), 8.94 (d, J = 5.1 Hz, 1H), 8.40 (s, 2H), 7.81 (d, J = 5.1 Hz,
1H), 5.40 (s, 2H), 4.98−4.74 (m, 1H), 4.52−4.34 (m, 1H), 3.88 (d, J
= 12.5 Hz, 1H), 3.75 (d, J = 12.7 Hz, 1H), 3.30−3.11 (m, 2H), 3.00
(td, J = 12.3 3.7, Hz, 1H), 2.29−2.14 (m, 1H), 1.23 (dt, J = 7.9, 3.3 Hz,
2H), 1.17 (d, J = 6.6 Hz, 3H), 1.13−1.07 (m, 2H).
4-[({2-[(2R)-4-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)-2-methyl-
piperazin-1-yl]pyrimidin-5-yl}oxy)methyl]pyridine-3-carboni-
trile (48). Compound 48 was synthesized as described for 45 using
(R)-2-(2-methyl-4-(3-(cyclopropyl)-1,2,4-oxadiazol-5-yl)piperazin-1-
4-[({2-[(2R)-2-Methyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)-
piperazin-1-yl]pyrimidin-5-yl}oxy)methyl]pyridine-3-carboni-
trile (44). Compound 44 was synthesized as described for 42 using
(R)-3-(4-(5-((3-bromopyridin-4-yl)methoxy)pyrimidin-2-yl)-3-meth-
ylpiperazin-1-yl)-5-methyl-1,2,4-oxadiazole as the starting material in
32% yield: mp 109−110 °C; 1H NMR (400 MHz, DMSO, 100 °C) δ
8.99 (s, 1H), 8.86 (d, J = 5.1 Hz, 1H), 8.31 (s, 2H), 7.72 (d, J = 5.1
Hz, 1H), 5.31 (s, 2H), 4.91−4.76 (m, 1H), 4.39 (dt, J = 13.5, 3.0 Hz,
1H), 3.85 (dd, J = 12.5, 1.6 Hz, 1H), 3.73 (dt, J = 12.8, 1.8 Hz, 1H),
3.34−3.14 (m, 2H), 3.08−2.97 (m, 1H), 2.44 (s, 3H), 1.16 (d, J = 6.6
Hz, 3H); HRMS (ESI) calcd for C19H21O2N8 (M + H)+ 393.1782,
found 393.1780.
4-{[(2-{(2R)-2-Methyl-4-[5-(trifluoromethyl)-1,2,4-oxadiazol-
3-yl]piperazin-1-yl}pyrimidin-5-yl)oxy]methyl}pyridine-3-car-
bonitrile (45). Cesium carbonate (5.49 g, 16.86 mmol) was added to
4-(chloromethyl)nicotinonitrile (2.14 g, 14.05 mmol) and (R)-2-(2-
methyl-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)piperazin-1-yl)-
pyrimidin-5-ol (4.64 g, 14.05 mmol) in DMF (60 mL). The resulting
mixture was stirred at 20 °C for 70 h. The reaction mixture was
quenched with water (15 mL) and extracted with EtOAc (2 × 20 mL),
and the organic layer was dried over MgSO4, filtered, and evaporated
to afford a beige solid that was purified by flash silica chromatography,
elution gradient 10−30% EtOAc in CH2Cl2. The oil was triturated
with isohexane/Et2O to give a solid which was collected by filtration
and dried under vacuum to give 4-{[(2-{(2R)-2-methyl-4-[5-
(trifluoromethyl)-1,2,4-oxadiazol-3-yl]piperazin-1-yl}pyrimidin-5-yl)-
oxy]methyl}pyridine-3-carbonitrile (3.43 g, 55%) as a white solid: mp
83−84 °C; [α]1D8 −46.8; 1H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H),
8.85 (d, J = 5.2 Hz, 1H), 8.20 (s, 2H), 7.66 (dd, J = 5.1, 0.7 Hz, 1H),
5.22 (s, 2H), 5.02−4.91 (m, 1H), 4.52 (ddd, J = 13.4, 3.2, 2.3 Hz, 1H),
4.10−3.98 (m, 1H), 3.88 (dt, J = 12.8, 1.7 Hz, 1H), 3.39−3.26 (m,
2H), 3.15 (td, J = 12.3, 3.7 Hz, 1H), 1.25 (d, J = 6.7 Hz, 3H); 13C
NMR (101 MHz, CDCl3) δ 170.4, 164.5 (q, J = 43.8 Hz), 157.8,
153.4, 152.9, 148.8, 146.5 (2C), 144.8, 121.8, 115.4 (q, J = 274.8 Hz),
114.7, 108.1, 68.8, 50.2, 46.5, 45.7, 37.8, 14.2; IR (Nujol) νmax 2234,
1
yl)pyrimidin-5-ol as the starting material in 48% yield: H NMR (400
MHz, CDCl3) δ 8.91 (s, 1H), 8.85 (d, J = 5.2 Hz, 1H), 8.19 (s, 2H),
7.71−7.61 (m, 1H), 5.22 (s, 2H), 4.93 (ddt, J = 6.4, 4.1, 2.4 Hz, 1H),
4.58−4.43 (m, 1H), 4.16−4.02 (m, 1H), 3.92 (dt, J = 13.0, 1.7 Hz,
1H), 3.38 (dd, J = 13.0, 4.0 Hz, 1H), 3.35−3.14 (m, 2H), 1.88 (tt, J =
8.0, 5.4 Hz 1H), 1.21 (d, J = 6.7 Hz, 3H), 1.01−0.91 (m, 4H); HRMS
(ESI) calcd for C21H23O2N8 (M + H)+ 419.1938, found 419.1935.
4-{[(2-{(2R)-2-Methyl-4-[3-(trifluoromethyl)-1,2,4-oxadiazol-
5-yl]piperazin-1-yl}pyrimidin-5-yl)oxy]methyl}pyridine-3-car-
bonitrile (49). Compound 49 was synthesized as described for 45
using (R)-2-(2-methyl-4-(3-(trifluoromethyl)-1,2,4-oxadiazol-5-yl)-
piperazin-1-yl)pyrimidin-5-ol as the starting material in 30% yield:
1
mp 150−151 °C; H NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 8.86
(d, J = 5.2 Hz, 1H), 8.22 (s, 2H), 7.66 (d, J = 5.1 Hz, 1H), 5.23 (s,
2H), 5.07−4.96 (m, 1H), 4.65−4.51 (m, 1H), 4.26−4.12 (m, 1H),
4.01 (d, J = 13.1 Hz, 1H), 3.51 (dd, J = 13.1, 4.0 Hz, 1H), 3.41−3.25
(m, 2H), 1.24 (d, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3)
172.3, 161.9 (q, J = 39.0 Hz), 157.5, 153.4, 153.0, 148.6, 146.4, 145.1,
121.8, 118.1 (q, J = 272.1 Hz), 114.7, 108.1, 68.7, 50.3, 46.3, 45.9, 37.7,
14.0; IR (Nujol) νmax 2227, 1648, 1590, 1546, 1465, 1401, 1345, 1293,
1234, 1155, 1089, 1058, 908, 837 cm−1; HRMS (ESI) calcd for
C19H18O2N8F3(M + H)+ 447.1499, found 447.1499.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the syntheses of the intermediates,
crystallographic information, biological protocols, and proce-
dures for the determination of physicochemical properties. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Article
(8) Semple, G.; Fioravanti, B.; Pereira, G.; Calderon, I.; Uy, J.; Choi,
K.; Xiong, Y.; Ren, A.; Morgan, M.; Dave, V.; Thomsen, W.; Unett, D.
J.; Xing, C.; Bossie, S.; Carroll, C.; Chu, Z.; Grottick, A. J.; Hauser, E.
K.; Leonard, J.; Jones, R. M. Discovery of the first potent and orally
efficacious agonist of the orphan G-protein coupled receptor 119. J.
Med. Chem. 2008, 51, 5172−5175.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +44 (0)1625 232567. Fax: +44 (0)1625 516667. E-
mail: jamie.scott@astrazeneca.com.
Notes
(9) Semple, G.; Ren, A.; Fioravanti, B.; Pereira, G.; Calderon, I.;
Choi, K.; Xiong, Y.; Shin, Y.; Gharbaoui, T.; Sage, C. R.; Morgan, M.;
Xing, C.; Chu, Z.; Leonard, J. N.; Grottick, A. J.; Al-Shamma, H.;
Liang, Y.; Demarest, K. T.; Jones, R. M. Discovery of fused bicyclic
agonists of the orphan G-protein coupled receptor GPR119 with in
vivo activity in rodent models of glucose control. Bioorg. Med. Chem.
Lett. 2011, 21, 3134−3141. (b) Semple, G.; Lehmann, J.; Wong, A.;
Ren, A.; Bruce, M.; Shin, Y.; Sage, C. R.; Morgan, M.; Chen, W.;
Sebring, K.; Chu, Z.; Leonard, J. N.; Al-Shamma, H.; Grottick, A. J.;
Du, F.; Liang, Y.; Demarest, K.; Jones, R. M. Discovery of a second
generation agonist of the orphan G-protein coupled receptor GPR119
with an improved profile. Bioorg. Med. Chem. Lett. 2012, 22, 1750−
1755.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Wendy Snelson, Helen Pointon, and Ruth Poultney are
thanked for their synthetic contribution. Members of the
AstraZeneca Animal Science & Welfare group are thanked both
for their work on breeding the mice and for their technical
support in running the experiments. Johnathan Bright is
thanked for statistical analysis of the cAMP assay. Michael
Waring is thanked for providing helpful comments on the
manuscript, and the reviewers are thanked for constructive
comments on agonist pharmacology.
(10) Wu, Y.; Kuntz, J. D.; Carpenter, A. J.; Fang, J.; Sauls, H. R.;
Gomez, D. J.; Ammala, C.; Xu, Y.; Hart, S.; Tadepalli, S. 2,5-
Disubstituted pyridines as potent GPR119 agonists. Bioorg. Med.
Chem. Lett. 2010, 20, 2577−2581.
ABBREVIATIONS USED
■
GPR119, G protein coupled receptor 119; WT, wild type; KO,
knockout; OEA, oleoylethanolamide; OLDA, N-oleoyldop-
amine; GLP-1, glucagon-like peptide-1; PPB, plasma protein
binding; LLE, ligand-lipophilicity efficiency; SAR, structure−
activity relationship; hERG, human ether-a-go-go-related gene;
OGTT, oral glucose tolerance test; DPP-IV, dipeptidyl
peptidase-4
(11) (a) McClure, K. F.; Darout, E.; Guimaraes, C. R. W.; DeNinno,
̃
M. P.; Mascitti, V.; Munchhof, M. J.; Robinson, R. P.; Kohrt, J.; Harris,
A. R.; Moore, D. E.; Li, B.; Samp, L.; Lefker, B. A.; Futatsugi, K.; Kung,
D.; Bonin, P. D.; Cornelius, P.; Wang, R.; Salter, E.; Hornby, S.;
Kalgutkar, A. S.; Chen, Y. Activation of the G-protein-coupled receptor
119: A conformation-based hypothesis for understanding agonist
response. J. Med. Chem. 2011, 54, 1948−1952. (b) Mascitti, V.;
Stevens, B. D.; Choi, C.; McClure, K. F.; Guimaraes, C. R. W.; Farley,
̃
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