Discovery of novel, induced-pocket binding oxazolidinones as potent, selective, and orally bioavailable tankyrase inhibitors

Article abstract of DOI:10.1021/jm4000038

Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates β-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of β-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).

Full text of DOI:10.1021/jm4000038

Article
pubs.acs.org/jmc
Discovery of Novel, Induced-Pocket Binding Oxazolidinones as
Potent, Selective, and Orally Bioavailable Tankyrase Inhibitors
Howard Bregman,*,† Nagasree Chakka,† Angel Guzman-Perez,† Hakan Gunaydin,∥ Yan Gu,∇
Xin Huang,∥ Virginia Berry,‡ Jingzhou Liu,‡ Yohannes Teffera,‡ Liyue Huang,‡ Bryan Egge,⊥
Erin L. Mullady,⊥ Steve Schneider,⊥ Paul S. Andrews,⊥ Ankita Mishra,§ John Newcomb,§ Randy Serafino,§
Craig A. Strathdee,§ Susan M. Turci,§ Cindy Wilson,§ and Erin F. DiMauro†
§
†Department of Chemistry Research and Discovery; ‡Department of Pharmacokinetics and Drug Metabolism; Oncology Research;
⊥
∥Department of Molecular Structure; Bioassay and Profiling; and ∇Protein Technologies, Amgen Inc., 360 Binney Street,
Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein
levels and elevates β-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1
and 2) may reduce the levels of β-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously
described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the
utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with
improved pharmacokinetic properties (39, 40).
effectively facilitate the phosphorylation of β-catenin, resulting
in its nuclear translocation and the overexpression of β-catenin/
TCF-regulated genes. Mutations in axin similarly lead to a
nonfunctional β-catenin destruction complex while activating
mutations in β-catenin prevent its phosphorylation and
subsequent degradation.
INTRODUCTION
■
Colorectal cancer (CRC) is the third most commonly
diagnosed cancer and the third leading cause of cancer death
in both men and women in the United States and the fourth
leading cause of cancer death worldwide.1 The dysregulation of
the wnt/β-catenin signaling pathway is believed to be the
initiating event for the vast majority of CRCs where truncating
mutations of the Adenomatous polyposis coli (APC) gene are
the most common alteration, occurring in approximately 80%
of cases, while loss-of-function AXIN2 (axis inhibition protein
2) mutations and activating mutations in CTNNB1 (the gene
which encodes β-catenin) account for the aberrant Wnt
pathway activity in most of the remaining 20%.2−5 Many
wnt/β-catenin pathway alterations lead to sustained β-catenin
stabilization in the absence of exogenous wnt signals, thereby
blocking the normal differentiation of colonic epithelial stem
cells and promoting uncontrolled proliferation and tumor
initiation.6 The concentration of β-catenin is regulated by a
cytoplasmic multicomponent complex known as the β-catenin
destruction complex. The tumor suppressor proteins axin and
APC bind β-catenin and enable phosphorylation events
catalyzed by glycogen synthase kinase 3β (GSK3β) and casein
kinase 1α (CK1α). Phosphorylated β-catenin is subsequently
degraded by the proteosome. Truncated APC is unable to
TNKS1 and 2 (PARP-5a, PARP-5b) are members of the
poly-ADP-ribose polymerase (PARP) family of enzymes which
catalyze the transfer of NAD+ units to their substrate
proteins.7−10 The resulting poly-ADP-ribose tagged (PARsy-
lated) proteins are altered in size, charge, and function.
Tankyrase was first described as a positive regulator of telomere
length, acting by PARsylating telomere repeat binding factor 1
(TRF1), which subsequently dissociates from telomeric DNA
to allow access to telomerase.11 More recently, TNKS was
identified as a positive regulator of the wnt signaling pathway
via its interaction with axin protein. Tankyrase catalyzes the
PARsylation of axin, ultimately resulting in axin protein
degradation and thus elevated levels of β-catenin and increased
expression of β-catenin/TCF-regulated oncogenes. The inhib-
ition of TNKS produces elevated axin protein levels and
Received: January 3, 2013
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Lactam Molecules 3 and 5
a
Reagents and conditions: (a) MP-BH4, DCM/MeOH, 60 °C, 37%; (b) PhSeH, TFA, DCM, reflux, 7%; (c) quinolin-8-amine, HATU, DIEA, DMF,
24%.
a
Scheme 2. Synthesis of Modified Lactam Analogues 7−18
a
Reagents and conditions: (a) HATU, DIEA, DMF, 64%; (b) substituted lactam or oxazolidinone, CuI, DMEDA, K3PO4, 1,4-dioxane, microwave,
140 °C (conditions used for preparations of 7, 12, 13, 17, 18, yields in scheme); (c) substituted lactam or oxazolidinone, CuI, DMEDA, Cs2CO3,
1,4-dioxane, microwave, 140 °C (conditions used for preparations of 8−11, 15, 16, yields in scheme); (d) substituted lactam or oxazolidinone, CuI,
(1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine, Cs2CO3, toluene, 100 °C (conditions used for preparation of 14, yield in scheme); (e) chiral
separation.
reduced levels of cellular β-catenin, ultimately inhibiting the
growth of APC mutant cell colonies.12,13 Therefore, it is
hypothesized that the inhibition of TNKS may offer a novel
approach toward the treatment of CRC. Herein we describe the
identification of potent, selective, and orally bioavailable TNKS
inhibitors utilizing structure-based design principles.
subsequent reduction via trifluoroacetic acid (TFA) activation
and reaction with phenyl selenol afforded lactam 3. Lactam 5,
devoid of a bridged bicycle, was prepared under standard 2-(7-
Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HATU)-mediated amide formation condi-
tions with commercially available carboxylic acid 4 and
quinolin-8-amine.
The modified and substituted lactam analogues 7−18 were
synthesized by a general protocol (Scheme 2). The common
aryl iodide intermediate (6) was prepared by standard HATU
coupling conditions with 4-iodobenzoic acid and quinolin-8-
amine. Copper-mediated carbon−nitrogen bond formation
CHEMISTRY
■
The synthesis of lactam variants of a previously described
inhibitor IWR1 (1) are described in Scheme 1.14,15 The
synthesis of racemic lactam (3) was accomplished by a stepwise
reduction protocol wherein borohydride reduction with solid-
supported borohydride (MP-BH4) afforded alcohol 2 and
B
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a
Scheme 3. Synthesis of Naphthyridine Amide 21
a
Reagents and conditions: (a) methyl 4-iodobenzoate, CuI, DMEDA, Cs2CO3, 1,4-dioxane, microwave, 140 °C, 93%; (b) (1) LiOH, THF/MeOH/
H2O, 50 °C, 48%; (2) SOCl2, DCM; (c) 1,5-naphthyridin-4-amine, DIEA, DCM, 40 °C, 96%.
a
Scheme 4. Synthesis of trans-Cyclohexyl Amide 24
a
Reagents and conditions: (a) (1R,4R)-methyl 4-aminocyclohexanecarboxylate hydrochloride, NaBH(OAc)3, DCE, 30%; (b) (1) triphosgene,
DIEA, THF, 0 °C; (2) enantiomer separation; (3) LiOH, THF/MeOH/H2O, 50 °C, 100%; (c) quinolin-8-amine, HATU, DIEA, DMF, 38%.
conditions were broadly utilized to deliver 7−18 in varying
yields.
5. Reductive amination of (S)-methyl 2-amino-2-phenylacetate
hydrochloride with tert-butyl (4-oxocyclohexyl)carbamate
produced amino ester 25 in moderate yield as a mixture of
geometric isomers. The addition of methyl Grignard yielded
tertiary alcohol 26, which was cyclized to oxazolidinone 27 with
triphosgene. It was at this stage that the cis and trans cyclohexyl
isomers (∼1:1 cis/trans) were separated by silica gel
chromatography. Removal of the tert-butoxycarbonyl (Boc)
group under standard acidic conditions afforded penultimate
amine 28. Partial racemization of the oxazolidinone phenyl
group occurred during this sequence. (Studies with a related
analogue indicated that partial racemization occurred at the
reductive amination step. Reversal of the reductive amination
and Grignard reaction sequence prevented racemization
entirely.) Standard peptide coupling conditions utilizing either
HATU or propylphosphonic anhydride (T3P) furnished
amides 29−31.
The synthesis of naphthyridine amide 21 is described in
Scheme 3. Copper-mediated coupling of commercial (S)-5,5-
dimethyl-4-phenyloxazolidin-2-one with methyl 4-iodobenzoate
afforded ester 19 in good yield. Subsequent ester hydrolysis and
treatment with thionyl chloride provided penultimate acid
chloride 20, which was transformed smoothly to naphthyridine
amide 21 in high yield.
The synthesis of saturated amide 24 is described in Scheme
4. Reductive amination of commercial 2-hydroxy-2-methyl-1-
phenylpropan-1-one with (1R,4R)-methyl 4-aminocyclohexa-
necarboxylate hydrochloride provided amino alcohol 22.
Triphosgene-mediated cyclization, separation of the enan-
tiomers, and ester hydrolysis yielded oxazolidinone 23. Chiral
separations were accomplished using preparative SFC (super-
critical fluid) chromatography with the stationary phase and
percent cosolvent used for each separation selected through
method development screening. Standard peptide coupling
conditions furnished saturated amide analogue 24.
The benzimidazolone containing analogues 34−41 were
prepared by a robust, three-step protocol depicted in Scheme 6.
Nucleophilic aromatic substitution reaction (SNAr) of primary
amine 28 with the appropriately substituted ortho-halo
nitroarene substrate provided nitro amine 32. The nitro
Analogues in which the amide is “reversed” relative to 24
were prepared in a convergent manner, as described in Scheme
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a
Scheme 5. Synthesis of Cyclohexyl “Reverse” Amides 29 − 31
a
Reagents and conditions: (a) NaBH(OAc)3, DME, RT, 67%; (b) MeMgBr, THF, 0 °C to RT, 43%; (c) triphosgene, DIEA, THF, 0 °C to RT, 50%;
(d) (1) TFA, DCM, RT, 100%; (2) enantiomer separation; (e) (for 29) quinoline-8-carboxylic acid, HATU, DIEA, DCM, RT, 73%; (f) (for 30) 1,5-
naphthyridine-4-carboxylic acid, HATU, DIEA, DMF, RT, 17%; (g) (for 31) 7-fluoro-1,5-naphthyridine-4-carboxylic acid,16 T3P, Et3N, DMF, 35%.
a
Scheme 6. General Synthetic Route toward Benzimidazolones 34−41
a
Reagents and conditions: (a) Et3N, CH3CN, 80 °C; (b) Raney Ni, NH2NH2−H2O, EtOH, 40 °C (toward 34, 35, 38); (c) SnCl2, EtOH, 80 °C
(toward 36, 37, 39, 40); (d) triphosgene, DIEA, THF, 0 °C to RT or RT (3-step yields in scheme); (e) Zn(CN)2, Pd(PPh3)4, DMF, 120 °C, 94%.
group was reduced to aniline 33 in the presence of Raney nickel
and hydrazine hydrate or stannous chloride in ethanol.17
Cyclization to benzimidazoles 34−40 was effected with
carbonyldiimidazole (CDI) and diisopropylethyl amine
(DIEA) in tetrahydrofuran. Cyanation of bromo-substituted
benzimidazolone 38 with zinc cyanide under palladium
catalyzed conditions afforded 41 in high yield.
Analogues containing benzimidazolone moieties were
alternately prepared by synthesizing the amino-cyclohexyl
benzimidazolone portion prior to constructing the oxazolidi-
none, as outlined in Scheme 7. Amines 43 and 44 were
prepared beginning with an SNAr reaction of tert-butyl (1R,4R)-
4-aminocyclohexyl)carbamate with the appropriately substi-
tuted ortho-fluoro nitroarene under basic conditions. Sub-
D
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a
Scheme 7. Synthesis of aza-Benzimidazolone 46
a
Reagents and conditions: (a) (toward 43) (1) 2-chloro-3-nitropyridine, Et3N, CH3CN, 80 °C, 60%; (2) SnCl2, EtOH, 80 °C, 62%; (b) (toward 44)
(1) 4-chloro-3-nitrobenzonitrile, K2CO3, DMF, 60 °C, 90%; (2) Zn powder, NH4Cl, THF/MeOH/H2O, RT, 89%; (c) (toward 43) (1) CDI, DIEA,
THF, 0 °C, 67%; (2) TFA, DCM, RT, 82%; (d) triphosgene, DIEA, THF, RT, 22%; (e) HCl in 1,4-dioxane, 10% MeOH in CHCl3, 0 °C, 100%; (f)
NaBH(OAc)3, CH3COOH, DMF, 60 °C, 47%; (g) (1) triphosgene, DIEA, THF, RT, 15%; (2) enantiomer separation.
a
Scheme 8. Synthesis of aza-Benzimidazolones 48 and 51
a
Reagents and conditions: (a) (1) methyl 3-fluoroisonicotinate, Et3N, CH3CN, 80 °C, 69%; (2) LiOH, THF/MeOH/H2O, 50 °C, 75%; (b) DPPA,
Et3N, THF, 80 °C, 12%; (c) phthalic anhydride, Et3N, THF/toluene (1:1), 100 °C, 77%; (d) (1) 28, Et3N, CH3CN, 80 °C, 68%, (2) NH2NH2/
H2O, EtOH, 100 °C, 73%; (e) CDI, DIEA, THF, RT, 20%.
sequent nitro reduction afforded diamine 42, which was
cyclized to the benzimidazolone using CDI. Cleavage of the
Boc protecting group afforded amines 43 and 44 in good
overall yields. Reductive amination of amine 43 with 2-hydroxy-
2-methyl-1-phenylpropan-1-one provided racemate 45 in
modest yield. Cyclization with triphosgene and chiral
separation provided oxazolidinone 46.18
The syntheses of aza-benzimidazolone analogues 48 and 51
are described in Scheme 8. SNAr of amine 28 with methyl 3-
fluoroisonicotinate and subsequent lithium hydroxide-mediated
ester hydrolysis provided amino acid 47. Exposure of 47 to
diphenylphosphoryl azide (DPPA) in the presence of triethyl-
amine effected a Curtius rearrangement, with the resulting
isocyanate undergoing intramolecular cyclization with the
proximal amine to produce benzimidazolone 48. Protection
of 4-chloropyrimidin-5-amine as phthalimide 49 followed by
SNAr with amine 28 and phthalimide cleavage afforded diamine
50. Cyclization with CDI provided purinone 51.
RESULTS AND DISCUSSION
■
To validate the hypothesis that tankyrase inhibition is a suitable
approach for the treatment of CRC, potent and selective
pharmacological tools must be identified for proof of concept
studies in tumor-bearing mice. Toward this end, we focused on
driving cellular potency in an APC mutant CRC cell line while
optimizing rodent pharmacokineticsfirst in vitro with routine
monitoring of stability in liver microsomes and plasma, and
later in vivo toward achieving suitable unbound exposure levels
following oral dosing in mice.
Known TNKS inhibitor IWR1 (1) was the starting point for
our structure activity relationship (SAR) evaluations.14,15 In our
hands, benchmark 1 was found to inhibit TNKS1,2 at
submicromolar levels while demonstrating high levels of
selectivity over PARP 1,2 (>100-fold). In cellular assays,
treatment with 1 effected the accumulation of axin modestly
(EC50 = 2.5 μM) and more strongly inhibited β-catenin (IC50
0.25 μM) (Table 1). Inhibition of β-catenin-dependent gene
=
E
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a
Table 1. Profile of 1
Table 2. IWR to Phenyl Lactam SAR
assay
potency (μM)
TNKS1, TNKS2 IC50
PARP1, PARP2 IC50
0.15, 0.039
>85, >170
2.5
SW480 Axin EC50
SW480 β-catenin IC50
HLM, RLM CLint (μL/min/mg)
mouse plasma (t1/2, min)
0.25
41, 69
15
transcription was observed in non-APC mutant HEK-293-STF
cells, but only weakly in APC mutant SW480 cells.
The cocrystal structure of 1 with TNKS1 was obtained,
revealing a nontraditional PARP inhibitory binding motif.19,20
The critical hydrogen bonding contacts identified were one
hydrogen bond between TNKS residue Tyr1213 and one of the
imide carbonyl groups as well as one hydrogen bonding contact
between Asp1198 and the amide carbonyl. The bicyclic moiety
engages in significant hydrophobic contacts, as suggested by the
observed stereopreference (exo diastereomer TNKS1 IC50 = 1
μM vs 0.15 μM for endo). The quinoline moiety engages in
hydrophobic stacking interactions with His1201 (see Figure 1).
a
NA = not measured. *See the Supporting Information for individual
data variance and n values.
was replaced with a methylene (3, racemic mixture) which
reduced potency (Table 2). Further modification of the bicyclic
moiety to an aromatic group (5) abrogated activity.
Investigation of 5-membered succinimide replacements con-
taining one H-bond acceptor and a hydrophobic substituent
were explored using focused libraries. While replacing the fused
aromatic ring with a pendant phenyl group α (7) or β to the
carbonyl (8) did not provide measurable potency, attachment
at the γ position (racemate 9) afforded weak TNKS inhibition
(1.6 μM). This represented the first nonbicyclic analogue
prepared which displayed inhibitory activity. Resolution and
analysis of the separated enantiomers revealed a strong
stereopreference for a single enantiomer (10: IC50 = 0.54
μM; 11: IC50 > 20 μM). The importance of the phenyl group
was confirmed by reduced activity observed with small
substituents gem-dimethyl (12) and methyl substitution (13).
Phenyl lactam 10 suffered from poor microsomal stability,
particularly in rat liver microsomes (CLint > 399 μL/min/mg).
On the basis of the possibility that the phenyl ring may provide
sites for oxidative metabolism, a fluoro-walk was performed
Figure 1. Representation of the cocrystal structure of 1 with TNKS1.
Highlighted are the hydrogen bonding contacts between an imide
carbonyl and the amide carbonyl of the ligand and protein as well as
the hydrophobic interactions of the bicyclic moiety with the protein.
Compound 1 functioned as a useful in vitro tool compound;
however, it suffered from poor pharmacokinetic properties
along with moderate cellular potency and was therefore
unsuitable for use as an in vivo tool. Consistent with previously
reported plasma stability results, studies indicated that both the
imide and amide moieties were labile sites (t1/2 = 15 min,
mouse plasma).15,21
With the information gleaned from the plasma stability study
and the cocrystal structure, we proceeded with the goals of
stabilizing the molecule and improving potency. Because only
one imide carbonyl group engaged the protein and the imide
functionality was deemed unstable, one imide carbonyl group
F
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Table 3. Phenyl Lactam SAR
Table 4. Phenyl Lactam to gem-Dimethyl Phenyl
Oxazolidinone
a
a
NA = not measured. *See the Supporting Information for individual
data variance and n values.
Table 5. Modification of the Biaryl Amide Motif
*
See the Supporting Information for individual data variance and n
values.
Figure 2. Representation of the cocrystal structure of 18 with TNKS1
highlighting hydrogen bonding contacts between the ligand and
protein.
a
MDR1-LLC-PK1/LLC-PK1 or porcine renal epithelial cells express-
(14−16) which presented the para position (16) as optimal for
potency. Whereas 16 was not stable in microsomes, markedly
improved mouse plasma stability was observed (t1/2 16 = 262
min vs 1 = 15 min). Subsequent efforts were directed toward
attenuating potential oxidation by systematically replacing or
substituting the methylene units of the lactam moiety (Table
3). Replacement of the α methylene with an oxygen (17) did
not yield improvements in intrinsic stability or potency;
however, further substituting the β methylene with geminal
dimethyl groups (18) improved intrinsic stability (HLM, RLM
CLint = 41, 83 μL/min/mg), resulting in modest clearance in
rat (1.3 L/hr/kg) (see Table 4).
b
ing human Pgp. CD-1 mice were dosed orally at 30 mpk in 10 mL/kg
of vehicle (10% pluronic F68, 30% HPBCD, 60% water, pH 2.5
adjusted with methanesulfonic acid for 21, 24, 30; pH adjusted to 10.0
*
with sodium hydroxide for 31). NA = not measured. See the
Supporting Information for individual data variance and n values.
and 6 (>10 μM).22 We obtained a cocrystal structure of 18 with
TNKS which indicated an aryl C−H−carbonyl interaction
between the C−H of the 6-position of the quinoline and the
carbonyl group of Gly1196 (Figure 2). Toward enhancing that
interaction by providing a more polarized C−H bond relative
to quinoline 18, naphthyridine 21 was prepared. Gratifyingly,
this point mutation afforded an order of magnitude boost in
enzyme and cellular potencies. Improvements in solubility in
Although 18 represented an improvement in PK relative to
lead 10, the cellular potency was weak (SW480 TBC = 2.1
μM). Additionally, 18 was very selective over PARPs 1, 2, 3,
G
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Figure 3. (A) Chemdraw representation of the hybridization of 18 with HTS hit 52 to produce 46. (B) Overlay of the TNKS1 cocrystal structure of
18 (pink) and 52 (yellow).
acidic media, and permeability were also realized along with
moderate plasma stability and clearance (Rat IV CL = 1.3 L/
hr/kg) (see Table 5).
oxazolidinone amide series, we sought isosteric amide replace-
ments in hopes of improving microsomal stability and oral
exposure. An interesting observation presented the possibility
of using a benzimidazolone in this regard based on the
identification of 52 from a TNKS1 auto-PARsylation high
throughput screen. We speculated that 52 may bind in an
induced pocket20 with the amide and benzimidazolone
carbonyl groups engaging the protein in a similar manner as
18, which was subsequently confirmed by the TNKS1/52
cocrystal structure. Overlay of the TNKS cocrystal structure of
HTS hit molecule 52 with the cocrystal structure of 18 revealed
that the carbonyl groups of the benzimidazolone and amide
moieties both engage ASP1198 in a hydrogen bonding contact.
Furthermore, the electron poor quinoline ring of 18 engaged in
a favorable stacking interaction with His1201 and overlaid well
with the benzimidazolone phenyl ring of 52. Hybridization of
the oxazolidinone portion of 18 with the cyclohexyl
benzimidazolone of 52 afforded 46 (Figure 3).
Lead benzimidazolone hybrid 46 possessed good enzyme
and β-catenin potency but weak axin activity (IC50 = 3.6 μM).
Toward improving stacking interactions with His1201, we
performed an aza-walk, preparing all four possible aza-
benzimidazolone analogues (Table 6).26 In general, good
enzyme potency (≤12 nM) was observed, with notable
improvements in cellular potency with the 3-aza analogue 34
(β-catenin, Axin IC50 = 0.14, 0.23 μM, respectively) as well as
improved oral exposure (∼3-fold greater Cmax vs 31). The
cellular potency was on par with our top amide lead (31, β-
catenin, Axin IC50 = 0.27, 0.19 μM, respectively), with
The cocrystal structure suggested that the central phenyl
moiety did not undergo any direct interaction with the protein.
We speculated that replacing the phenyl group with a trans
cyclohexyl might result in retention of potency and gains in
plasma stability and solubility.15,23,24 As hypothesized, saturated
derivative 24 was equipotent to 18 and offered improved
plasma stability (t1/2 > 500) and solubility (Table 5). Toward
eliminating potential aniline metabolites while reducing the
oxidative potential of the quinoline moiety, the reversed amide
analogue was prepared (29).25 Compound 29 had retained
potency with a significantly increased free fraction (2.7% vs
0.1% unbound for 18).
Toward further improving potency via the H-bond donor
properties of the hydrogen at the 2-position, as described
earlier, naphthyridine 30 was prepared, affording improved
enzyme and cellular potency (∼2-fold) but poor oral exposure
in mouse PO PK studies (Cmax @ 30 mg/kg = 0.22 μM, AUC =
0.258 μM h). Further blocking sites of potential oxidative
metabolism yielded fluorinated analogue 31, which featured
retained potency with improved solubility relative to 29.
Furthermore, improvements in oral exposure were observed in
PO PK studies (Cmax @ 30 mg/kg =2.24 μM, AUC = 1.3 μM
h).
Despite the vast improvements in cellular potency and PK
achieved, the amide containing molecules generally suffer from
moderate intrinsic clearance. In addition to optimizing the
H
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respectively). However, the mean blood concentration of 40
was only 2.2 and 0.1 μM at 1 and 6 h postdose, respectively,
after 3 days of BID administration at 50 mg/kg to female mice.
The low exposure after multiple dosing may potentially be due
to the induction of metabolic enzymes, as the compound was a
strong activator of rat pregnane X receptor (PXR).
Table 6. Benzimidazolone SAR
In conclusion, efforts to modify IWR-like TNKS inhibitors
toward improved cellular potency and pharmacokinetics in
rodents for potential in vivo proof of concept studies have led
to the identification of lead molecules within amide and
benzimidazoline series (Table 7) that have good selectivities
Table 7. Expanded Data Set and Comparison of Top Lead
Molecules
a
b
Assay run in the presence of 10% FBA. CD-1 mice were dosed orally
at 30 mpk in 10 mL/kg of vehicle (10% pluronic F68, 30% HPBCD,
60% water, pH 2.5 adjusted with methanesulfonic acid for 39; pH
c
adjusted to 10.0 with sodium hydroxide for 31, 40). fu in 10% FBS
*
>0.5. NA = not measured. See the Supporting Information for
individual data variance and n values.
over PARP1/2. Amide containing lead (31) had moderate
cellular potencies but poor oral exposure in mice. Disubstituted
benzimidazoles (39, 40) had significantly improved cellular
potencies relative to 1 (∼40-fold more potent Axin EC50
values) and produced unbound exposure levels exceeding
cellular IC50 values when dosed orally in mice (mouse PO
a
CD-1 mice were dosed orally at 30 mpk in 10 mL/kg of vehicle (10%
pluronic F68, 30% HPBCD, 60% water, pH 2.0 adjusted with
methanesulfonic acid for 34; pH 2.5 adjusted with methanesulfonic
acid for 39; pH adjusted to 10.0 with sodium hydroxide for 31, 36,
40). NA = not measured. See the Supporting Information for
individual data variance and n values.
C
max(unbound)/Axin EC50 = 23 for 40). Subsequent efforts
toward producing molecules in this series with extended target
coverage suitable for proof-of-concept pharmacodynamic
studies in mice will be reported in due course.
*
EXPERIMENTAL SECTION
■
improved microsomal stability. Toward realizing an additive
effect, pyrimidine 51 was prepared, but no improvements in
potency were observed relative to 34.
Axin2 Accumulation Assay. TNKS inhibition results in Axin2
accumulation into distinct cytoplasmic foci which were visualized and
quantified using a high content imaging system (Cellomics
ArrayScan). SW480 cells were grown under normal culture conditions
(RPMI 1640, 10% HI FBS, and 1× Sodium Pyruvate). On the day of
the assay, cells were plated at 2500 cells per well in 60 μL of assay
media in Perkin-Elmer Black 384 ViewPlates (Fisher, 509052489).
TNKS compounds were diluted to generate a 22-point dose titration
in media and incubated with the cells for 24 h at 37 °C, 5% CO2. The
next day the cells were fixed for 15 min in 4% paraformaldehyde and
0.1% Triton, washed in PBS, and blocked in PBS with 0.1% Tween-20
and 1% Normal Goat Serum. The cells were stained with an Axin2
primary antibody (Sigma, SAB1100677-200UL) at 1:10000 overnight
at 4 °C and an Alexa 488-labeled secondary antibody (Invitrogen,
A11008). The nuclei were visualized with Hoechst dye. The Axin2 foci
were quantified on the Cellomics ArrayScan (a variation of the
compartmental analysis protocol was optimized, and data was analyzed
The introduction of a cyano group at the 3-position of the
benzimidazole (36) provided improved enzyme and cellular
potencies (3-fold) and increased exposure in mouse PK studies
(Cmax = 6.0 μM with a 30 mg/kg PO dose). Cyano substitution
at the 4-position (37) reduced potency (∼4-fold) relative to 36.
Lead 36 was incubated with fresh rat hepatocytes, and
metabolite analysis suggested that the primary oxidative liability
was the benzimidazolone aryl moiety. While replacement of the
C−H at the 1 position with nitrogen (41) afforded reduced
potency (TBC potency: 172 nM vs 54 nM), substituting the
positions ortho to the cyano group with a fluorine (39, 40)
yielded retained potency with significantly improved oral
exposure for 40 (C = 16.3 and 5.9 μM at 1 and 6 h postdose,
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using MEAN_RingSpotAvgIntenCh2), and EC50 values were calcu-
lated. Assay standards: 18 runs with a standard which yielded a mean
of 0.311 μM with a standard deviation of 0.173. Eighteen runs with
another standard which yielded a mean of 0.114 μM with a standard
deviation of 0.065.
liquid chromatography (HPLC) systems with UV detection at
254 and 215 nm (System A: Agilent Zorbax SB-C18 3.0 mm ×
50 mm, 3.5 μm, 5−95% CH3CN in H2O with 0.1% TFA for 3.6
min at 1.5 mL/min or Halo Phenyl-Hexyl, 3 mm × 50 mm, 2.7
μm, 5−95% CH3CN in H2O with 0.1% TFA for 1.01 min at 2.0
mL/min; System B: Waters Xbridge C18, 3 mm × 50 mm, 3.5
μm, 5−95% CH3CN in H2O with 0.1% formic acid for 3.6 min
at 1.5 mL/min). Purities for final compounds were >95%. Exact
mass confirmation was performed on an Agilent 1200 series
high performance liquid chromatography (HPLC) system
(Santa Clara, CA, US) by flow injection analysis, eluting with
a binary solvent system A and B (A, water with 0.1% FA; B,
ACN with 0.1% FA) under isocratic conditions (50% A/50%
B) at 0.2 mL/min with MS detection by an Agilent 6510-Q
time-of-flight (TOF) mass spectrometer (Santa Clara, CA, US).
Silica gel chromatography was generally performed with
prepacked silica gel cartridges (Biotage, Teledyne-Isco, or
Interchim). Chiral method development was performed on an
analytical Thar SFC/MS. Preparative chiral separations were
performed on Thar SFC Prep 80 or SFC Prep 350 instruments.
Library purification methods: Preparative LC/MS: Waters
autopurification system; liquid transfer system, Tecan; drying
system, Genevac; preparative column, Xbridge (19 mm × 100
mm, C18, 10 μm); flow rate, 40 mL/min; general gradient, 5−
95% B, 0.1% additive in both A and B, 10 min gradient; mobile
phase A, water; mobile phase B, acetonitrile; additive, TFA or
NH4OH. 1H NMR spectra were recorded on a Bruker AV-400
(400 MHz) spectrometer or a Varian 400 MHz spectrometer at
ambient temperature, or the NMR spectra were collected with a
Bruker Avance III spectrometer operating at a proton frequency
of 500.13 MHz using a 10 mL Protasis CapNMR flow probe.
NMR samples were delivered to the flow probe using a Protasis
One-Minute NMR Automation system comprised of a
Discovery Tower Sample Manager and a Waters Liquid
Handler made by CTC, Switzerland (model 2777). All
observed protons are reported as parts per million (ppm)
downfield from tetramethylsilane (TMS) or other internal
reference in the appropriate solvent indicated. Data are
reported as follows: chemical shift, multiplicity (s = singlet, d
= doublet, t = triplet, q = quartet, br = broad, m = multiplet),
coupling constants, and number of protons. Low-resolution
mass spectral (MS) data were determined on an Agilent 1100
Series LCMS with UV detection at 254 and 215 nm and a low
resonance electrospray mode (ESI).
Synthetic Details. 4-((3aR,4S,7R,7aS)-1-Oxo-3a,4,7,7a-
tetrahydro-1H-4,7-methanoisoindol-2(3H)-yl)-N-(quino-
lin-8-yl)benzamide (3). To a vial charged with 1 (122 mg,
0.298 mmol) was added MeOH (1192 μL) and DCM (1192
μL) and MP-BH4 (macroporous borohydride) (114 mg, 0.358
mmol). The mixture was shaken overnight at 60 °C, providing
about 60% conversion to desired product. An additional aliquot
of DCM, MeOH, and MP-BH4 (same quantities as above) was
added, and the mixture was heated and shaken at 80 °C for 4 h,
providing complete conversion. The mixture was filtered
through cotton and washed with DCM and MeOH. The
filtrate was dried under reduced pressure and the crude material
purified by MPLC, 25g column ramping DCM/MeOH/
NH4OH (90:10:1) in DCM from 0% to 100%, leading to
coelution of product with impurities. The material was
repurified using a 15 μm spherical silica column (Interchim)
with the same gradient, leading to complete isolation of desired
product cleanly, obtained as a yellow oil ( )-4-((3aS,4R,7-
S,7aR)-1-hydroxy-3-oxo-3a,4,7,7a-tetrahydro-1H-4,7-methanoi-
Quantitative Total β-Catenin Assay. TNKS inhibition results in
degradation of the total pool of β-catenin in SW-480 colorectal cells.
SW480 cells do not express E-cadherin and thus do not have
membrane associated (“non-signaling”) β-catenin which interferes
with Wnt pathway activity analysis. Cells were seeded at 10,000/well
in CellBIND 96-well in 60 μL of normal growth medium (MEM alpha
supplemented with 10% heat inactivated FBS, GlutaMAX, pyruvate,
and 10 mM HEPES). A 10-point, 3-fold dilution series for each TNKS
inhibitor was constructed, and 20 μL of each diluted compound was
transferred to the plated SW-480 cells (resulting in a final vehicle
(DMSO) concentration of 0.1%). The plates were incubated at 37 °C
for 40−48 h, after which the media was removed and the cells were
lysed with 75 μL/well MSD lysis buffer. A goat anti-rabbit MSD plate
(catalog no. L41RA-1) was coated with 25 μL of 5 μg/mL of Cell
Signaling antitotal β-catenin polyclonal (catalog no. 9562, lyophilized,
carrier-free special order) and incubated overnight in a cold room with
gentle shaking. The plate was then blocked with 150 μL of Blocker “A”
per well and washed 4 times with 150 μL/well TBS-T wash buffer
(150 mM NaCl, 50 mM Tris, pH 7.5, 0.02% Tween-20). Cell lysates
(75 μL) were transferred to prepared MSD plates and incubated at 4
°C overnight with gentle shaking. The next day MSD plates were
washed with TBS-T wash buffer and the antitotal β-catenin mAb
(catalog no. 610153) detection antibody conjugated to SULFO-TAG
was added. The detection antibody was incubated for 1 h at room
temperature with vigorous shaking, after which plates were washed and
processed for analysis by the addition of 150 μL/well MSD Read 4X
Buffer T with surfactant. The plates were read, and the data was
captured on the SECTOR Imager 6000. Assay standards: 54 runs with
a standard which yielded a mean of 0.157 μM with a standard
deviation of 0.116; 38 runs with another standard which yielded a
mean of 0.063 μM with a standard deviation of 0.041.
Tankyrase 1 and 2 Assays. The tankyrase 1 biochemical activity
of the compounds was assayed in the following assay buffer (50 mM
MOPS pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20,
0.05% BSA, and 1 mM DTT) as follows: 0.25 nM of 6XHIS-
tankyrase1 (1091-1325) was incubated in the presence of compound
(DMSO 1.85% final) in a Perkin-Elmer 384 well Proxiplate PlusTM
(cat. no. 6008289) with 400 nM of NAD for 60 min at RT. The assay
was then stopped with the above assay buffer containing a 0.6 μM
inhibitor and the following detection components: 0.05 μg/mL
monoclonal anti-PAR antibody (Trevigen cat. no. 4335-MC-01K-AC)
prebound for 60 min with 0.63 μg/mL protein G AlphaLisa acceptor
bead (Perkin-Elmer cat. no. AL102M) and 5 μg/mL AlphaLisa nickel
chelate donor bead (Perkin-Elmer cat. no. AS101M). The assay was
incubated for 16 h at RT in the dark and read on a Perkin-Elmer
Envision multi label reader using the default program set with laser
excitation at 680 nM and emission at 615 nM. Assay standards: 66
runs with a standard which yielded a mean of 0.0086 μM with a
standard deviation of 0.00038; 42 runs with a different standard which
yielded a mean of 0.0013 μM with a standard deviation of 0.00064.
PARP 1 and 2 Assays. PARP1 biochemical assay was purchased as
a kit from Trevigen (cat. no. 4676-096-K) and used per the
manufacturer’s recommendations. PARP2 biochemical assay was
purchased as a kit from BPS (cat. no. 80552) and used per the
manufacturer’s recommendations.
CHEMISTRY
■
General. Unless otherwise noted, all materials were
obtained from commercial suppliers and used without further
purification. Anhydrous solvents were obtained from Aldrich
and used directly. All reactions involving air- or moisture-
sensitive reagents were performed under a nitrogen or argon
atmosphere. Purity (3 min methods only) and reaction analyses
were measured using Agilent 1100 Series high performance
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soindol-2(3H)-yl)-N-(quinolin-8-yl)benzamide (45 mg, 0.109
mmol, 36.7% yield). m/z (ESI) 412.2 (M + H)+.
(ESI) m/z: 409.2. HRMS calcd for C25H20N4O2 (M + H)+
409.1664, found 409.1657.
4-((4R)-2-Oxo-4-phenyl-1-pyrrolidinyl)-N-8-quinolinyl-
benzamide (8). Compound 8 was prepared as described for
compound 9 using (4R)-phenylpyrrolidin-2-one (1 equiv) to
afford 4-((4R)-2-oxo-4-phenyl-1-pyrrolidinyl)-N-8-quinolinyl-
To a flask charged with 4-((3aS,4R,7S,7aR)-1-hydroxy-3-oxo-
3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindol-2(3H)-yl)-N-
(quinolin-8-yl)benzamide (36 mg, 0.087 mmol) was added
DCM (700 μL), TFA (10.11 μL, 0.131 mmol) and
benzeneselenol (37.2 μL, 0.350 mmol). The orange solution
was refluxed overnight. The resulting orange solution was dried
under reduced pressure and the crude residue purified by a 25g
column ramping DCM/MeOH/NH4OH (90:10:1) in DCM
from 0% to 100%, leading to coelution of product with a
selenium containing impurity. The mixture was repurified using
the Gilson RP-HPLC, ramping ACN in H2O (10−90%, 0.1%
TFA), providing product as a white film upon drying. The
material was converted to its free-base using a 2g SCX-2
(strong cation exchange) column, washing first with MeOH
and then with 2 M NH3 in MeOH, providing a white film upon
drying. The film obtained was lyophilized from MeOH/H2O,
providing product as a fluffy light yellow solid 4-((3aR,4S,7-
R,7aS)-1-oxo-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindol-
2(3H)-yl)-N-(quinolin-8-yl)benzamide (2.5 mg, 6.32 μmol,
7.23% yield). 1H NMR (500 MHz, DMSO-d6) δ 10.61 (s, 1H),
8.95−9.02 (m, 1H), 8.72 (d, J = 7.5 Hz, 1H), 8.47 (d, J = 8.2
Hz, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H),
7.72−7.77 (m, 1H), 7.62−7.72 (m, 2H), 6.22−6.28 (m, 1H),
6.17 (dd, J = 2.8, 5.7 Hz, 1H), 3.94 (t, J = 10.1 Hz, 1H), peak
under water, 2.90−3.00 (m, 1H), 1.42−1.53 (m, 2H). m/z
(ESI) 396.2 (M + H)+, HRMS calcd for C25H21N3O2 (M + H)+
396.1712, found 396.1708.
1
benzamide (0.19 g, 0.466 mmol, 58% yield). H NMR (400
MHz, DMSO-d6) δ 10.65 (s, 1H), 8.99 (dd, J = 4.2, 1.7 Hz, 1
H), 8.73 (dd, J = 7.6, 1.3 Hz, 1H), 8.47 (dd, J = 8.4, 1.6 Hz,
1H), 8.04−8.10 (m, 2H), 7.92−7.99 (m, 2H), 7.72−7.77 (m,
1H), 7.63−7.72 (m, 2H), 7.41−7.47 (m, 2H), 7.35−7.41 (m,
2H), 7.25−7.32 (m, 1H). LC-MS (ESI) m/z: 408.2 (M + 1).
HRMS calcd for C26H21N3O2 (M + H)+ 408.1712, found
408.1706.
4-(2-Oxo-5-phenylpyrrolidin-1-yl)-N-(quinolin-8-yl)-
benzamide (9). A mixture of 4-iodobenzoic acid (9.26 g, 37.3
mmol), DIEA (13.05 mL, 74.7 mmol), HATU (7.10 g, 18.67
mmol), and quinolin-8-amine (7 g, 48.6 mmol) in DMF (124
mL) was stirred under nitrogen overnight at room temperature.
The reaction mixture was diluted with saturated aqueous
sodium bicarbonate (100 mL) and water (300 mL), yielding a
yellow/brown precipitate which was filtered, washed with water
(100 mL), and dried under high vacuum to obtain 4-iodo-N-
(quinolin-8-yl)benzamide (9 g, 24.05 mmol, 64.4% yield) as a
1
yellow brown solid. H NMR (400 MHz, DMSO-d6) δ 10.65
(s, 1 H), 8.98 (dd, J = 1.6, 4.3 Hz, 1 H), 8.70 (dd, J = 1.4, 7.6
Hz, 1 H), 8.47 (dd, J = 1.7, 8.3 Hz, 1 H), 8.03−7.98 (m, 2 H),
7.83−7.79 (m, 2 H), 7.76 (dd, J = 1.3, 8.2 Hz, 1 H), 7.71−7.62
(m, 2 H).
To a flask charged with 4-iodo-N-(quinolin-8-yl)benzamide
(0.30 g, 0.802 mmol), 5-phenylpyrrolidin-2-one (0.13 g, 0.802
mmol), and cesium carbonate (0.784 g, 2.405 mmol) was
added dioxane (9 mL), and the mixture was purged with
nitrogen prior to the addition of copper(I) iodide (0.153 g,
0.802 mmol) and N1,N2-dimethylethane-1,2-diamine (0.141 g,
1.604 mmol) and repurging with nitrogen and then heating in
the microwave at 140 °C for 2 h. The resulting reaction mixture
was filtered through a frit and purified by MPLC by eluting
with EtOAc/hexanes and DCM (5%) as an additive to obtain
compound 9 (0.25 g, 0.614 mmol, 77% yield) as a white solid.
Compound 9 was separated using ChiralPak AD-H (2 × 15
cm), 35% isopropanol (0.1%, DEA), 100 bar, 60 mL/min, 220
nm, inj vol: 1−3 mL, 15 mg/mL methanol, to obtain
4-(1-Oxoisoindolin-2-yl)-N-(quinolin-8-yl)benzamide
(5). A resealable tube was charged with phthalaldehyde (0.474
g, 3.54 mmol), 4-aminobenzoic acid (0.485 g, 3.54 mmol), 4-
aminobenzoic acid (0.485 g, 3.54 mmol), and EtOH (10 mL).
The mixture was heated at 90 °C for 6 h. The pale yellow
precipitate was isolated via vacuum filtration and then washed
with EtOH and diethyl ether and dried under reduced pressure
to afford crude 4-(1-oxoisoindolin-2-yl)benzoic acid (0.222 g,
0.877 mmol, 25%), which was used without further purification.
To a flask charged with crude 4-(1-oxoisoindolin-2-yl)-
benzoic acid (0.217 g, 0.857 mmol) was added DMF (5.0 mL)
followed by DIPEA (0.449 mL, 2.57 mmol), 8-aminoquinoline
(0.130 g, 0.900 mmol), and HATU (0.326 g, 0.857 mmol). The
mixture was stirred overnight at RT. The resulting yellow
suspension was then filtered through a membrane, washing
with MeOH to afford a solid. Purification by SFC afforded 4-
(1-oxoisoindolin-2-yl)-N-(quinolin-8-yl)benzamide (0.079 g,
0.208 mmol, 24%) as a pale yellow solid in 95% HPLC purity.
1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1 H), 9.02 (d, J =
2.7 Hz, 1 H), 8.76 (d, J = 7.6 Hz, 1 H), 8.49 (d, J = 8.2 Hz, 1
H), 8.21−8.14 (m, 4 H), 7.85 (dd, J = 7.6 Hz, 1 H), 7.77−7.66
(m, 5 H), 7.60 (m, 1 H), 5.15 (s, 2 H). m/z (ESI) 380.2 (M +
H)+, HRMS calcd for C24H17N3O2 (M + H)+ 380.1399, found
380.1392.
1
compounds 10 and 11 as white solids. H NMR (400 MHz,
DMSO-d6) δ ppm 10.54 (s, 1H), 8.95 (dd, J = 4.3, 1.7 Hz, 1H),
8.67 (dd, J = 7.6, 1.4 Hz, 1H), 8.44 (dd, J = 8.4, 1.6 Hz, 1H),
7.90−7.95 (m, 2H), 7.60−7.75 (m, 5H), 7.29−7.37 (m, 4H),
7.20−7.27 (m, 1H), 5.58 (dd, J = 7.8, 4.8 Hz, 1H), 2.56−2.74
(m, 3H), 1.85−1.93 (m, 1H). LC-MS (ESI) m/z: 408.2 (M +
1). HRMS calcd for C26H21N3O2 (M + H)+ 408.1712, found
408.1711.
4-(2,2-Dimethyl-5-oxo-1-pyrrolidinyl)-N-8-quinolinyl-
benzamide Trifluoroacetic Acid (12). Compound 12 was
prepared as described for compound 9 using 5,5-dimethylpyr-
rolidin-2-one (1 equiv) to afford 4-(2,2-dimethyl-5-oxo-1-
pyrrolidinyl)-N-8-quinolinylbenzamide trifluoroacetic acid (1.2
mg, 0.003 mmol, 0.5% yield) as a tan solid following
4-(2-Oxo-3-phenyl-1-imidazolidinyl)-N-8-quinolinyl-
benzamide (7). Compound 7 was prepared as described for
compound 9 using 1-phenylimidazolidin-2-one (1 equiv) to
afford 4-(2-oxo-3-phenyl-1-imidazolidinyl)-N-8-quinolinylben-
1
purification with TFA modified RP-HPLC. H NMR (400
1
zamide (0.04 g, 98 mmol, 12% yield). H NMR (400 MHz,
MHz, DMSO-d6) δ 10.68 (s, 1 H), 9.02−8.95 (m, 1 H), 8.73
(dd, J = 1.3, 7.6 Hz, 1 H), 8.49 (dd, J = 1.7, 8.3 Hz, 1 H), 8.13−
8.04 (m, 2 H), 7.77 (dd, J = 1.3, 8.3 Hz, 1 H), 7.73−7.62 (m, 2
H), 7.45−7.37 (m, 2 H), 2.58−2.51 (m, 2 H), 2.04 (m, 2 H),
1.27 (s, 6 H). m/z (ESI) 360.0 (M + H)+.
DMSO-d6) δ 10.64 (s, 1H), 9.00 (dd, J = 4.2, 1.7 Hz, 1H), 8.74
(dd, J = 7.6, 1.3 Hz, 1H), 8.47 (dd, J = 8.3, 1.6 Hz, 1H), 8.05−
8.10 (m, 2H), 7.87−7.92 (m, 2H), 7.63−7.77 (m, 5H), 7.37−
7.44 (m, 2H), 7.07−7.14 (m, 1H), 4.02−4.10 (m, 4H). LC-MS
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4-(2-Methyl-5-oxo-1-pyrrolidinyl)-N-8-quinolinylben-
zamide (13). Compound 13 was prepared as described for
compound 9 using 5-methylpyrrolidin-2-one (1 equiv) to afford
4-(2-methyl-5-oxo-1-pyrrolidinyl)-N-8-quinolinylbenzamide
(0.008 g, 24 mmol, 4% yield). 1H NMR (500 MHz, DMSO-d6)
δ ppm 10.64 (s, 1H), 8.96−9.00 (m, 1H), 8.73 (d, J = 7.6 Hz,
1H), 8.46 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 8.7 Hz, 2H), 7.72−
7.80 (m, 3H), 7.63−7.71 (m, 2H), 4.50−4.58 (m, 1H), 2.65
(ddd, J = 16.9, 9.4, 7.4 Hz, 1H), 2.42−2.49 (m, 1H), 2.28−2.38
(m, 1H), 1.73 (td, J = 8.3, 4.8 Hz, 1H), 1.21 (d, J = 6.2 Hz,
3H). LC-MS (ESI) m/z: 346.2 (M + 1). HRMS calcd for
C21H19N3O2 (M + H)+ 346.1555, found 346.1561.
4-(2-(4-Fluorophenyl)-5-oxopyrrolidin-1-yl)-N-(quino-
lin-8-yl)benzamide (14). Compound 14 was prepared as
described for compound 9 using 5-(4-fluorophenyl)pyrrolidin-
2-one (1 equiv), affording 4-(2-(4-fluorophenyl)-5-oxopyrroli-
din-1-yl)-N-(quinolin-8-yl)benzamide (29 mg, 0.068 mmol,
42.1% yield). 1H NMR (500 MHz, DMSO-d6) δ 10.54 (s, 1 H),
8.95 (dd, J = 1.7, 4.3 Hz, 1 H), 8.67 (d, J = 6.5 Hz, 1 H), 8.44
(dd, J = 1.5, 8.3 Hz, 1 H), 7.94 (s, 2 H), 7.74−7.61 (m, 5 H),
7.37 (dd, J = 5.4, 8.7 Hz, 2 H), 7.15 (t, J = 8.8 Hz, 2 H), 5.61−
5.57 (m, 1 H), 2.74−2.55 (m, 3 H), 1.92−1.86 (m, 1 H). m/z
(ESI) 426.2 (M + H)+. HRMS calcd for C26H20FN3O2 (M +
H)+ 426.1618, found 426.1617.
4-(2-(3-Fluorophenyl)-5-oxopyrrolidin-1-yl)-N-(quino-
lin-8-yl)benzamide (15). Compound 15 was prepared as
described for compound 9 using 5-(3-fluorophenyl)pyrrolidin-
2-one (1 equiv), affording 4-(2-(3-fluorophenyl)-5-oxopyrroli-
din-1-yl)-N-(quinolin-8-yl)benzamide (460 mg, 1.081 mmol,
60.5% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1 H),
8.95 (dd, J = 1.7, 4.2 Hz, 1 H), 8.67 (dd, J = 1.2, 7.6 Hz, 1 H),
8.45 (dd, J = 1.6, 8.3 Hz, 1 H), 7.95 (d, J = 8.8 Hz, 2 H), 7.75−
7.60 (m, 5 H), 7.41−7.33 (m, 1 H), 7.21−7.14 (m, 2 H), 7.06
(dt, J = 2.2, 8.4 Hz, 1 H), 5.61 (dd, J = 5.0, 7.5 Hz, 1 H), 2.76−
2.56 (m, 3 H), 1.95−1.87 (m, 1 H). m/z (ESI) 426.2 (M +
H)+. HRMS calcd for C26H20FN3O2 (M + H)+ 426.1618, found
426.1619.
12 h at 100 °C. Reaction progress was monitored by the TLC
system: 40% ethyl acetate in hexane. Upon completion, the
reaction was concentrated under reduced pressure, and then
the crude material was purified by column chromatography
using 20% ethyl acetate in hexane as the eluent to afford 4-(2-
(2-fluorophenyl)-5-oxopyrrolidin-1-yl)-N-(quinolin-8-yl)-
1
benzamide (30 mg, 52.8% yield) as a white solid. H NMR
(400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.95 (d, J = 2.9 Hz,
1H), 8.67 (d, J = 7.4 Hz, 1H), 8.45 (d, J = 8.1 Hz, 1H), 7.96 (d,
J = 8.7 Hz, 2H), 7.81−7.55 (m, 5H), 7.52−7.09 (m, 3H),
7.09−7.03 (m, 1H), 5.80 (d, J = 7.5 Hz, 1H), 3.05−2.60 (m,
4H), 2.01−1.63 (m, 1H). m/z (ESI) 426.0 (M + H)+, HRMS
calcd for C26H20FN3O2 (M + H)+ 426.1618, found 426.1611.
4-((4S)-2-Oxo-4-phenyl-1,3-oxazolidin-3-yl)-N-8-qui-
nolinylbenzamide Trifluoroacetic Acid (17). Compound
17 was prepared as described for compound 9 using (R)-5-
phenylpyrrolidin-2-one (1 equiv) to afford 4-((4S)-2-oxo-4-
phenyl-1,3-oxazolidin-3-yl)-N-8-quinolinylbenzamide trifluoro-
acetic acid as a white solid following purification with TFA
modified RP-HPLC (57 mg, 0.109 mmol, 20% yield). 1H NMR
(500 MHz, DMSO-d6) δ 10.55 (s, 1 H), 8.95 (dd, J = 1.5, 4.1
Hz, 1 H), 8.67 (d, J = 6.8 Hz, 1 H), 8.44 (dd, J = 1.5, 8.4 Hz, 1
H), 7.97 (d, J = 8.8 Hz, 2 H), 7.76−7.58 (m, 5 H), 7.45−7.35
(m, 4 H), 7.32 (d, J = 6.9 Hz, 1 H), 5.83 (dd, J = 5.4, 8.6 Hz, 1
H), 4.90 (t, J = 8.6 Hz, 1 H), 4.20 (dd, J = 5.3, 8.5 Hz, 1 H). m/
z (ESI) 410.4 (M + H)+. HRMS calcd for C25H19N3O3 (M +
H)+ 410.1504, found 410.15.
4-((4S)-5,5-Dimethyl-2-oxo-4-phenyl-1,3-oxazolidin-
3-yl)-N-8-quinolinylbenzamide (18). Compound 18 was
prepared as described for compound 9 using (S)-5,5-dimethyl-
4-phenyloxazolidin-2-one from Aldrich. Purification was done
by preparative LC-MS with 0.1% NH4OH in ACN and water as
mobile phase. The pure fractions were dried under reduced
pressure to afford ((4S)-5,5-dimethyl-2-oxo-4-phenyl-1,3-ox-
azolidin-3-yl)-N-8-quinolinylbenzamide as a brown solid (82
1
mg, 0.187 mmol, 35% yield). H NMR (400 MHz, DMSO-d6)
δ 10.55 (s, 1 H), 8.95 (dd, J = 1.6, 4.2 Hz, 1 H), 8.67 (dd, J =
1.2, 7.6 Hz, 1 H), 8.45 (dd, J = 1.6, 8.3 Hz, 1 H), 7.96 (d, J =
8.8 Hz, 2 H), 7.81−7.56 (m, 5 H), 7.48−7.17 (m, 5 H), 5.57 (s,
1 H), 3.17 (d, J = 5.1 Hz, 1 H), 1.66 (s, 3 H), 0.93 (s, 3 H). m/
z (ESI) 438.3 (M + H)+. HRMS calcd for C27H23N3O2 (M +
H)+ 438.1817, found 438.1813.
4-(2-(2-Fluorophenyl)-5-oxopyrrolidin-1-yl)-N-(quino-
lin-8-yl)benzamide (16). To a solution of ethyl 4-(2-
fluorophenyl)-4-oxobutanoate (500 mg, 2.2 mmol) in methanol
(5 mL) were added ammonium acetate (4.62 g, 60 mmol) and
sodium borohydride (250 mg, 4.4 mmol) at ambient
temperature, and the reaction mixture was stirred for 2 h.
The resulting reaction mixture was stirred at 80 °C for 12 h.
Reaction progress was monitored by the TLC system: 50%
ethyl acetate in hexane. After reaction completion the reaction
mixture was concentrated under reduced pressure. The
resulting reaction mixture was adjusted to pH ∼12 by treatment
with 6 N NaOH solution and then ethyl acetate. The ethyl
acetate layer was concentrated and the crude material purified
by column chromatography with 30% ethyl acetate in hexane as
the eluent to afford 5-(2-fluorophenyl)pyrrolidin-2-one (300
(S)-4-(5,5-Dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-N-
(1,5-naphthyridin-4-yl)benzamide (21). To a round-
bottom flask was added (S)-5,5-dimethyl-4-phenyloxazolidin-
2-one (1.717 g, 8.98 mmol, Sigma Aldrich), methyl 4-
iodobenzoate (2.353 g, 8.98 mmol), and dioxane (90 mL)
followed by potassium phosphate, tribasic (9.53 g, 44.9 mmol),
and copper(I) iodide (1.710 g, 8.98 mmol). The vessel was
purged with nitrogen, followed by the addition of N,N′-
dimethylethylenediamine (1.933 mL, 17.96 mmol). The
suspension was heated to reflux overnight, yielding a tan
suspension. The mixture was filtered through Celite, and the
filtrate was dried under reduced pressure, providing a light
green oil (S)-methyl 4-(5,5-dimethyl-2-oxo-4-phenyloxazolidin-
3-yl)benzoate (19) (2.72 g, 8.36 mmol, 93% yield), which will
be used as is in the next step. 1H NMR (400 MHz, chloroform-
d) δ 7.94−7.90 (m, 2 H), 7.55−7.51 (m, 2 H), 7.39−7.32 (m, 3
H), 7.21−7.16 (m, 2 H), 5.04 (s, 1 H), 3.86 (s, 3 H), 1.70 (s, 3
H), 1.02 (s, 3 H).
1
mg, 75% yield) as a colorless liquid. H NMR (300 MHz,
DMSO-d6) δ 8.08 (s, 1H), 7.47−7.10 (m, 4H), 5.00−4.83 (m,
1H), 2.24 (t, J = 8.0 Hz, 2H), 2.02−1.67 (m, 1H). m/z (ESI)
179 (M + H)+.
To a mixture of 4-iodo-N-(quinolin-8-yl)benzamide (see
protocol for compound 9) (50 mg, 0.13 mmol), 5-(2-
fluorophenyl)pyrrolidin-2-one (23 mg, 0.13 mmol), and
(1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (36 mg, 0.26
mmol) in toluene (2 mL) was added CuI (24 mg, 0.13 mmol)
and cesium carbonate (210 mg, 0.65 mmol) at ambient
temperature, and the resulting reaction mixture was stirred for
To a flask charged with (S)-methyl 4-(5,5-dimethyl-2-oxo-4-
phenyloxazolidin-3-yl)benzoate (2.70 g, 8.30 mmol) was added
THF (48.5 mL), MeOH (48.5 mL), and water (48.5 mL),
L
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respectively. To the resulting turbid solution was added LiOH
(0.994 g, 41.5 mmol), and the resulting mixture was heated at
50 °C overnight. The resulting mixture was cooled in an ice
water bath and brought to pH ∼2 by the addition of 2 N HCl
(∼15 mL), leading to a light yellow solution to which water was
added (∼100 mL), leading to the formation of a white
precipitate which was collected via vacuum filtration and
washed with excess water. After drying under high vacuum
overnight, product was obtained as a white solid (S)-4-(5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)benzoic acid (1.25 g,
4.02 mmol, 48.4% yield). NMR indicates that the product
present as well, possibly imine. The material was used without
further purification. m/z (ESI) 306.3 (M + H)+.
To a flask charged with (1R,4R)-methyl 4-((2-hydroxy-2-
methyl-1-phenylpropyl)amino)cyclohexanecarboxylate (2.785
g, 9.12 mmol) was added tetrahydrofuran (36.5 mL) and
DIEA (4.78 mL, 27.4 mmol). The mixture was cooled in an ice
water bath prior to the addition of triphosgene (2.71 g, 9.12
mmol), yielding a white suspension which was allowed to stir
and slowly warm to room temperature (2 h, ice melt). LC-MS
indicated about 75% conversion, which was the same as after 1
h. The mixture was cooled back to 0 °C, and an additional 0.5
equiv of triphosgene was added. After 1 h additional conversion
was not observed. To the mixture was added additional DIEA
(1.5 equiv). After 30 min LC-MS indicated complete
conversion to desired product. The suspension was added
carefully to a stirred flask containing aqueous NH4Cl and ice.
The mixture was transferred to a separatory funnel and
extracted with EtOAc (2×). The combined organics were dried
with Na2SO4, filtered, and dried under reduced pressure,
providing a light yellow crude solid (quantitative yield).
Enantiomer separation: system, Prep SFC-2, Chiralpak AD-H,
2 cm × 25 cm, 20% methanol w/0.2% DEA; 80 mL/min flow
rate (pressure drop = 55 bar); 220 nm detection; 100 bar BPR;
sample dissolved in 50 mL of methanol. Test Injections: 500
μL, 1 mL, 1.5 mL, sample processed with 1.5 mL injection
volume and 3.5 min cycle time. Absolute stereochemical
determination: Compound 24 had previously been prepared in
a more stepwise asymmetrical manner (not described). One of
the separated enantiomers from this separation was converted
to 24, and the retention time compared with that of 24
prepared from the asymmetric route to determine absolute
stereochemistry. m/z (ESI) 332.3 (M + H)+.
To a vial charged with (1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-
phenyloxazolidin-3-yl)cyclohexanecarboxylic acid (23, 0.028 g,
0.088 mmol) was added HATU (0.034 g, 0.088 mmol),
quinolin-8-amine (0.013 g, 0.088 mmol), DMF (0.353 mL),
and DIEA (0.015 mL, 0.088 mmol), respectively. The vessel
was sealed and shaken at room temperature overnight. The
resulting mixture was dried under reduced pressure and purified
with a 25 HP spherical silica column, ramping DCM/MeOH
(90:10) in DCM (0−40%, isocratic at 40%, detection at 215
nm), providing product as a white film, (1R,4R)-4-((R)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-N-(quinolin-8-yl)-
cyclohexanecarboxamide (0.015 g, 0.034 mmol, 38.3% yield).
1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1 H), 8.90 (dd, J =
1.7, 4.3 Hz, 1 H), 8.57 (dd, J = 1.3, 7.7 Hz, 1 H), 8.39 (dd, J =
1.7, 8.4 Hz, 1 H), 7.66−7.59 (m, 2 H), 7.57−7.50 (m, 1 H),
7.46−7.40 (m, 2 H), 7.40−7.15 (m, 3 H), 4.62 (s, 1 H), 3.51−
3.41 (m, 1 H), 2.58−2.52 (m, 1 H), 2.03−1.95 (m, 1 H), 1.92−
1.75 (m, 3 H), 1.64 (d, J = 10.9 Hz, 1 H), 1.59−1.50 (m, 1 H),
1.50−1.47 (m, 3 H), 1.47−1.36 (m, 1 H), 1.27−1.13 (m, 1 H),
0.81 (s, 3 H). m/z (ESI) 444.3 (M + H)+, HRMS calcd for
C27H29N3O3 (M + H)+ 444.2287, found 444.2288.
1
formed as a THF adduct with a 3:2 product to THF ratio. H
NMR (400 MHz, DMSO-d6) δ 12.76 (br s, 1 H), 7.87−7.81
(m, 2 H), 7.63−7.57 (m, 2 H), 7.41−7.34 (m, 2 H), 7.34−7.30
(m, 1 H), 7.30−7.19 (m, 2 H), 5.51 (s, 1 H), 1.63 (s, 3 H), 0.91
(s, 3 H).
To a vial charged with (S)-4-(5,5-dimethyl-2-oxo-4-phenyl-
oxazolidin-3-yl)benzoic acid (0.200 g, 0.642 mmol) was added
DCM (2.57 mL) followed by thionyl chloride (0.469 mL, 6.42
mmol). The resulting solution was sealed and shaken at 40 °C
for 2 h. LC-MS indicated near complete conversion to desired
product (primarily methyl ester peak identified). The mixture
was dried under reduced pressure, providing product (20) as a
light yellow solid. m/z (ESI) 326.2 (M + H)+ (methyl ester
ionization observed).
To a vessel charged with (S)-4-(5,5-dimethyl-2-oxo-4-
phenyloxazolidin-3-yl)benzoyl chloride (20) (50 mg, 0.152
mmol) was added DCM (606 μL), 1,5-naphthyridin-4-amine
(Astatech) (24 mg, 0.167 mmol), and DIEA (55.6 μL, 0.318
mmol), respectively. The vessel was sealed and shaken
overnight at 40 °C. The mixture was dried under reduced
pressure and purified using a 25 g, 15 μm spherical silica
column (Interchim) ramping 0−100% EtOAc in heptane to
afford product, which was lyophilized from MeOH/H2O to
yield (S)-4-(5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-N-
(1,5-naphthyridin-4-yl)benzamide (64 mg, 96%) as an off-
1
white powder. H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1
H), 9.01 (dd, J = 1.6, 4.2 Hz, 1 H), 8.93 (d, J = 5.0 Hz, 1 H),
8.54 (d, J = 5.1 Hz, 1 H), 8.46 (dd, J = 1.6, 8.5 Hz, 1 H), 8.01−
7.96 (m, 2 H), 7.89 (dd, J = 4.3, 8.6 Hz, 1 H), 7.76−7.70 (m, 2
H), 7.43−7.37 (m, 2 H), 7.36−7.22 (m, 3 H), 5.57 (s, 1 H),
1.67 (s, 3 H), 0.94 (s, 3 H). m/z (ESI) 439.2 (M + H)+. HRMS
calcd for C26H22N4O3 (M + H)+ 439.1770, found 438.1775.
(1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazolidin-
3-yl)-N-(quinolin-8-yl)cyclohexanecarboxamide (24). To
a flask charged with 2-hydroxy-2-methyl-1-phenylpropan-1-one
(1.570 mL, 10.33 mmol) was added DCE (20.65 mL) and
methyl trans-4-aminocyclohexanecarboxylate hydrochloride
(1.00 g, 5.16 mmol), and the reaction mixture was cooled in
an ice water bath prior to the addition of sodium
triacetoxyhydroborate (1.094 g, 5.16 mmol). The mixture was
stirred for 1 h. The mixture was diluted with water, transferred
to a separatory funnel, and extracted with DCM (2×). The
combined organics were dried with Na2SO4, filtered, and dried
under reduced pressure, yielding a colorless oil. The oil was
purified with a 10g SCX-2 column, washing with MeOH and
then with 2 M NH3 in MeOH. LC-MC of the basic wash shows
m/z of 304/306, suggestive of an imine product, which is
strange, since the initial crude LC-MS indicated the amine mass
cleanly. NMR suggests amine product, obtained as a sticky
white solid ( )-(1R,4R)-methyl 4-((2-hydroxy-2-methyl-1-
phenylpropyl)amino)cyclohexanecarboxylate (22; 0.570 g,
1.866 mmol, 36.1% yield). There were about 20% impurity
(S)-3-((1R,4S)-4-Aminocyclohexyl)-5,5-dimethyl-4-
phenyloxazolidin-2-one (28). To a flask charged with 4-N-
boc-aminocyclohexanone (Combi-Blocks) (11.00 g, 51.6
mmol) was added DCE (206 mL) followed by (S)-(+)-2-
phenylglycine methyl ester hydrochloride (Aldrich) (10.40 g,
51.6 mmol). The resulting mixture was stirred at room
temperature for 15 min prior to the addition of sodium
triacetoxyborohydride (21.86 g, 103 mmol). The resulting
suspension was stirred overnight at room temperature, leading
to conversion to desired product (∼1:1 cis/trans). To the
M
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mixture was added water, and the resulting mixture was
transferred to a separatory funnel. The mixture was extracted
with EtOAc (2×). The combined organics were dried with
Na2SO4, filtered, and dried under reduced pressure. The crude
material was purified with a 100g SNAP column, ramping
DCM/MeOH (90:10) in DCM (0−25%), then isocratic at
25% (monitoring at 215 nm) to yield product as a mixture of
isomers as a yellow solid ( )-(S)-methyl 2-(((1R,4S)-4-((tert-
butoxycarbonyl)amino)cyclohexyl)amino)-2-phenylacetate
(12.43 g, 34.3 mmol, 66.5% yield).
1.25 mL injections. Absolute stereochemistry establishment:
only partial racemization had occurred in this sequence. We
began with enantiopure (S) material, and therefore, the major
1
enantiomer was the (S) enantiomer. H NMR (400 MHz,
DMSO-d6) δ 7.79−7.64 (m, 2 H), 7.45−7.38 (m, 2 H), 7.38−
7.32 (m, 1 H), 7.32−7.10 (m, 2 H), 4.59 (s, 1 H), 3.41−3.32
(m, 1 H), 2.84 (tt, J = 3.8, 11.6 Hz, 1 H), 1.94 (td, J = 3.2, 12.5
Hz, 1 H), 1.86−1.74 (m, 3 H), 1.59−1.52 (m, 1 H), 1.45 (s, 3
H), 1.37−1.07 (m, 3 H), 0.78 (s, 3 H). m/z (ESI) 289.2 (M +
H)+.
A flask charged with ( )-(S)-methyl 2-(((1R,4S)-4-((tert-
butoxycarbonyl)amino)cyclohexyl)amino)-2-phenylacetate
(12.4 g, 34.2 mmol) was dried under high vacuum and placed
under nitrogen. Then, THF (98 mL) was added and the
resulting solution cooled in an ice water bath prior to the
addition of methylmagnesium bromide (1 M in THF) (16.32 g,
137 mmol) over 15 min. The mixture was allowed to slowly
warm to room temperature overnight, providing a yellow
suspension. The mixture was carefully added to a mixture of ice,
saturated aqueous NH4Cl, and EtOAc with stirring. The
mixture was transferred to a separatory funnel with the aid of
MeOH and extracted with EtOAc (2×). The combined
organics were dried with MgSO4, filtered, and dried under
reduced pressure. The residue was purified with a 100g SNAP
column, ramping DCM/MeOH (90:10) in DCM from 0 to
30%, then isocratic at 30% (monitoring at 215 nm), providing
product as a yellow solid as a 3:1 mixture of isomers ( )-tert-
butyl ((1S,4R)-4-(((S)-2-hydroxy-2-methyl-1-phenylpropyl)-
amino)cyclohexyl)carbamate (5.28 g, 14.57 mmol, 42.6%
yield) (note: partial racemization of the phenyl bonded center
occurs during this two-step protocol).
To a flask charged with ( )-tert-butyl ((1S,4R)-4-(((S)-2-
hydroxy-2-methyl-1-phenylpropyl)amino)cyclohexyl)carbamate
(5.2 g, 14.34 mmol) was added tetrahydrofuran (57.4 mL) and
DIEA (12.53 mL, 71.7 mmol). The mixture was cooled in an
ice water bath prior to the addition of triphosgene (4.26 g,
14.34 mmol). The resulting yellow suspension was allowed to
stir and warm slowly to room temperature overnight. LC-MS of
the yellow suspension indicated product as the main peak
(observed as carbamic acid) with consumption of starting
material.
To the mixture was added water, and the resulting orange
solution was transferred to a separatory funnel, diluted with
brine, and extracted with EtOAc (2×). The combined organics
were dried under reduced pressure and purified with a 200g (15
μm spherical silica, Interchim) column, flow rate 65 mL/min,
ramping DCM/MeOH (90:10) in DCM, from 0 to 30%, then
isocratic at 30% (monitoring at 215 nm), providing some
separation of regioisomers with peak slicing yielding the trans/
cis product in 50% yield (∼4:1).
To a flask charged with ( )-tert-butyl ((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)carbamate
(2.81 g, 7.23 mmol) was added DCM (28.9 mL) followed by
TFA (5.57 mL, 72.3 mmol). The resulting mixture was stirred
for 3 h at room temperature.
N-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)quinoline-8-carboxamide (29). To a
vial charged with 28 (0.750 g, 2.60 mmol) was added DCM
(10.40 mL), DIEA (1.363 mL, 7.80 mmol), 8-quinoline
carboxylic acid (Aldrich) (0.450 g, 2.60 mmol), and HATU
(0.989 g, 2.60 mmol), respectively. The resulting suspension
was shaken overnight at room temperature, leading to
conversion to desired product according to LC-MS with
more minor and polar impurities visible.
The resulting brown solution was dried under reduced
pressure and purified with a 40g HP spherical silica column (15
μm spherical, Interchim), ramping DCM/MeOH (90:10) in
DCM (0−30%, then isocratic at 30%, monitoring at 215 nm),
providing product along with impurity coelution. The material
was repurified (same column type) with a ramp of EtOAc in
heptane (10% DCM throughout) (0−100%), leading to
separation of some minor impurities, but NMR revealed
∼10% starting quinoline carboxylic acid. The white solid was
dissolved in DCM and extracted with 1 M Na2CO3 (3×). The
organic phase was dried with Na2SO4, filtered, and dried under
reduced pressure, providing a N-((1S,4R)-4-((S)-5,5-dimethyl-
2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)quinoline-8-carboxa-
1
mide (0.846 g, 1.907 mmol, 73.3% yield) as a white solid. H
NMR (400 MHz, DMSO-d6) δ 10.80 (d, J = 7.6 Hz, 1 H), 9.00
(dd, J = 1.9, 4.3 Hz, 1 H), 8.53 (ddd, J = 1.7, 7.7, 8.9 Hz, 2 H),
8.17 (dd, J = 1.6, 8.2 Hz, 1 H), 7.72 (t, J = 7.7 Hz, 1 H), 7.66
(dd, J = 4.3, 8.3 Hz, 1 H), 7.48−7.40 (m, 2 H), 7.40−7.12 (m,
3 H), 4.67 (s, 1 H), 3.71 (s, 1 H), 3.58−3.46 (m, 1 H), 2.08
(td, J = 3.0, 12.4 Hz, 1 H), 1.96−1.82 (m, 3 H), 1.62−1.53 (m,
1 H), 1.51−1.37 (m, 4 H), 1.37−1.27 (m, 1 H), 1.26−1.14 (m,
1 H), 0.81 (s, 3 H). m/z (ESI) 444.2 (M + H)+, HRMS calcd
for C27H29N3O3 (M + H)+ 444.2287, found 444.2297.
N-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-1,5-naphthyridine-4-carboxamide
(30). To a vial charged with 28 (0.052 g, 0.180 mmol) was
added DCM (0.721 mL), DIEA (0.094 mL, 0.541 mmol), 1,5-
naphthyridine-4-carboxylic acid (Life Chemicals Building
Blocks) (0.031 g, 0.180 mmol), and HATU (0.069 g, 0.180
mmol), respectively. The resulting suspension was shaken
overnight at room temperature. The resulting mixture was dried
under reduced pressure and purified using a 25g HP-spherical
silica column (15 um), ramping DCM in MeOH (90:10) in
DCM from 0 to 30%, then isocratic at 30%, affording 32 mg of
product with minor impurities present. The material was
repurified using a 2g SCX-2 column, washing with MeOH and
then 2 M NH3 in MeOH. The basic wash was dried under
reduced pressure and lyophilized from MeOH/H2O to yield
product as a white powder N-((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)-1,5-naphthyridine-4-
The resulting mixture was dried under reduced pressure and
purified with a 40g HP spherical silica column (Interchim),
ramping DCM/MeOH (90:10) in DCM (0−100%) (monitor-
ing at 215 nm) to provide product as a white foam (quantitative
conversion). Chiral purification: column, Chiralpak AD-H, 5
cm × 30 cm; mobile phase, 20% methanol w/0.2% diethyl-
amine/80% CO2; flow rate, 350 mL/min; sample dissolution,
72 mg/mL in 1:1 dichloromethane/methanol, processed with
1
carboxamide (0.009 g, 0.020 mmol, 11.23% yield). H NMR
(500 MHz, DMSO-d6) δ 10.25 (d, J = 7.7 Hz, 1 H), 9.13 (d, J =
4.4 Hz, 1 H), 9.07 (dd, J = 1.7, 4.2 Hz, 1 H), 8.57 (dd, J = 1.7,
8.5 Hz, 1 H), 8.20 (d, J = 4.4 Hz, 1 H), 7.89 (dd, J = 4.2, 8.5
N
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Hz, 1 H), 7.46−7.40 (m, 2 H), 7.39−7.16 (m, 3 H), 4.66 (s, 1
H), 3.77−3.67 (m, 1 H), 3.56−3.45 (m, 1 H), 2.10−2.03 (m, 1
H), 1.94−1.83 (m, 3 H), 1.62−1.55 (m, 1 H), 1.48 (s, 3 H),
1.47−1.42 (m, 1 H), 1.34 (dq, J = 3.2, 12.5 Hz, 1 H), 1.26−
1.16 (m, 1 H), 0.81 (s, 3 H). m/z (ESI) 445.3 (M + H)+. m/z
(ESI) 444.3 (M + H)+. HRMS calcd for C26H28N4O3 (M + H)+
445.2239, found 445.2235.
reaction mixture was stirred for 16 h at 90 °C. After completion
of the reaction, monitored by TLC (TLC eluent: 50% EtOAc
in petroleum ether, UV active), the reaction mixture was cooled
to ambient temperature and concentrated. The crude material
was taken in 20% NaOH solution and extracted with ethyl
acetate. Then the aqueous layer was acidified with 1 N HCl at
0−5 °C and then extracted with ethyl acetate. The extract was
washed with water and saturated NaCl solution and dried over
sodium sulfate. The organic solvent was evaporated to afford 7-
N-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-7-fluoro-1,5-naphthyridine-4-car-
boxamide (31). To a solution of 5-fluoropyridin-3-amine (2 g,
0.01 mol) in ethanol (20 mL), 2, 2′-dimethyl-1, 3-dioxane-4,6-
dione (Meldrum’s acid) (2.44 g, 0.02 mol) and triethyl
orthoformate (2.64 g, 0.01 mol) were added, and the reaction
mixture was heated to reflux for 3 h with constant stirring. The
reaction was monitored by TLC (TLC eluent: 40% EtOAc in
petroleum ether, UV active). After the completion of the
reaction, the mixture was allowed to cool to ambient
temperature, yielding a white precipitate. The precipitate was
filtered, washed with ethanol (20 mL), and dried under vacuum
to afford 5-(((5-fluoropyridin-3-yl)amino)methylene)-2,2-di-
methyl-1,3-dioxane-4,6-dione as an off white solid (1.2 g,
1
fluoro-1,5-naphthyridine-4-carboxylic acid (950 mg, 66%). H
NMR (400 MHz, DMSO-d6): δ 14.2 (br s, 1H), 9.20 (d, J = 2.8
Hz, 1H), 9.16 (d, J = 4.4 Hz, 1H), 8.49 (dd, J = 2.8 Hz, 9.6 Hz,
1H), 7.98 (d, J = 4 Hz, 1H). m/z (ESI) 193.2 (M + H)+.
To a vial charged with (S)-3-((1R,4S)-4-aminocyclohexyl)-
5,5-dimethyl-4-phenyloxazolidin-2-one 28 (0.073 g, 0.253
mmol) was added DMF (1.013 mL), Et3N (0.039 mL, 0.278
mmol), 7-fluoro-1,5-naphthyridine-4-carboxylic acid (0.049 g,
0.253 mmol), and 1-propanephosphonic acid cyclic anhydride
(0.161 mL, 0.253 mmol), respectively. The resulting light
brown solution was shaken at room temperature. After 4 h
additional 1-propanephosphonic acid cyclic anhydride (0.161
mL, 0.253 mmol) was added, and the mixture was shaken
overnight at room temperature. The mixture was dried under
reduced pressure and purified using a 25g HP spherical silica
column (15 μm, Interchim), ramping DCM/MeOH (90:10) in
DCM from 0 to 100% with product elution occurring toward
the 100% polar eluent along with a minor impurity (10−20%),
which was more polar and visible by TLC and LC-MS. The
material was repurified by dissolving in MeOH and using RP-
HPLC (Gilson), ramping ACN in H2O (10−90%, 0.1% TFA),
affording separation of impurities. The product containing
eluents were transferred to a separatory funnel, diluted with
EtOAc, and extracted with saturated aqueous NaHCO3. The
organic layer was dried with Na2SO4, filtered, and dried under
reduced pressure. The film obtained was lyophilized from
MeOH/H2O, providing product as an off-white solid N-
((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-
cyclohexyl)-7-fluoro-1,5-naphthyridine-4-carboxamide (0.041 g,
1
25%). H NMR (400 MHz, DMSO-d6): δ 11.33 (d, J = 14.4
Hz, 1H), 8.71 (s, 1H), 8.65 (d, J = 14.4 Hz, 1H), 8.47 (d, J =
2.4 Hz, 1H), 8.17−8.13 (m, 1H), 1.68 (s, 6H).
To a stirred solution of dowtherm A at 250 °C was added 5-
(((5-fluoropyridin-3-yl)amino)methylene)-2,2-dimethyl-1,3-di-
oxane-4,6-dione (1 g, 0.003 mol) portionwise over a period of
10 min. The reaction was monitored by TLC (TLC eluent:
50% EtOAc in petroleum ether, UV active). After completion
of the reaction, the mixture was cooled to ambient temperature
and treated with diethyl ether to obtain a gray precipitate. The
precipitate was stirred for 5−10 min and filtered, washed with
diethyl ether, and dried under vacuum to obtain 7-fluoro-1,5-
naphthyridin-4-ol as a tan colored solid (0.4 g, 65%). 1H NMR
(400 MHz, DMSO-d6): δ 12.68 (br s, 1H), 9.43 (s, 1H), 8.75
(t, J = 6.4 Hz, 1H), 8.59 (d, J = 8, 1H), 6.99 (d, J = 17.2 Hz,
1H). m/z (ESI) 165.1 (M + H)+.
To a suspension of 7-fluoro-1,5-naphthyridin-4-ol (400 mg,
2.43 mmol) in DMF (8 mL) was added PBr3 (660 mg, 2.43
mmol) dropwise at 50 °C for 10 min. After complete addition
of PBr3, the suspension became homogeneous, and a brown
colored precipitate was obtained over a period of 30 min. The
reaction was monitored by TLC (TLC eluent: 50% EtOAc in
petroleum ether). After completion of reaction, the mixture was
cooled to ambient temperature to generate more precipitate.
The precipitate was filtered and washed with Et2O (15 mL) and
dried under vacuum to afford a brown colored hydrobromide
salt of 8-bromo-2-methoxy[1, 5]naphthyridine which was
converted to free base by treatment with saturated aqueous
bicarbonate solution (10 mL) and product extraction into
EtOAc (2 × 25 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated under reduced
pressure to afford 8-bromo-3-fluoro-1,5-naphthyridine as a
1
0.089 mmol, 35.0% yield). H NMR (400 MHz, DMSO-d6) δ
9.57 (d, J = 7.7 Hz, 1 H), 9.15 (d, J = 2.8 Hz, 1 H), 9.13 (d, J =
4.4 Hz, 1 H), 8.44 (dd, J = 2.8, 9.5 Hz, 1 H), 8.05 (d, J = 4.4
Hz, 1 H), 7.46−7.15 (m, 5 H), 4.66 (s, 1 H), 3.70 (tdt, J = 3.9,
7.6, 11.5 Hz, 1 H), 3.53−3.41 (m, 1 H), 2.08−1.99 (m, 1 H),
1.92−1.81 (m, 2 H), 1.61−1.53 (m, 1 H), 1.47 (s, 3 H), 1.45−
1.13 (m, 4 H), 0.80 (s, 3 H). m/z (ESI) 463.3 (M + H)+.
HRMS calcd for C26H27FN4O3 (M + H)+ 463.2145, found
463.215.
(S)-5,5-Dimethyl-3-((1R,4S)-4-(2-oxo-2,3-dihydro-1H-
imidazo[4,5-c]pyridin-1-yl)cyclohexyl)-4-phenyloxazoli-
din-2-one (34). To a flask charged with 28 (3.00 g, 7.46
mmol) was added acetonitrile (24.85 mL), triethylamine (2.078
mL, 14.91 mmol), and 4-chloro-3-nitropyridine (Aldrich)
(1.206 g, 7.60 mmol), respectively. The resulting orange
solution was shaken at 80 °C overnight, leading to 80−90%
conversion according to LC-MS. Additional 4-chloro-3-nitro-
pyridine was added (0.4 equiv, 480 mg), and stirring and
heating were continued. After 30 min no additional conversion
had occurred. Additional triethylamine (2.078 mL, 14.91
mmol) was added, and heating and stirring were continued at
80 °C. After 1 h LC-MS of the red/orange solution indicated
consumption of starting material. The mixture was cooled in an
ice water bath and stirred at 0 °C, and saturated aqueous
NH4Cl was added. The mixture was transferred to a separatory
1
brown solid (200 mg, 36%). H NMR (300 MHz, DMSO-d6):
δ 9.19 (d, J = 2.4 Hz, 1H), 8.84 (d, J = 4.8 Hz, 1H), 8.44 (dd, J
= 2.8, 9.2 Hz, 1H), 8.23 (d, J = 4.8 Hz, 1H). m/z (ESI) 227.1
(M + H)+.
In a sealed tube, to a solution of 8-bromo-3-fluoro-1,5-
naphthyridine (1.7 g, 0.0074 mol) in DMF (17 mL) were
added dppf (207 mg, 0.0003 mol) and palladium acetate (84
mg, 0.0003 mol). The mixture was purged with argon for 5−10
min. Then, DIPEA (3.4 mL, 2 times) and acetic formic
anhydride (3.4 mL, 2 times) were added slowly at RT. The
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funnel and extracted with EtOAc (2×). The combined organics
were dried with Na2SO4, filtered, and dried under reduced
pressure. The crude residue obtained was purified with an 80g
HP spherical silica column (15 μm spherical), ramping EtOAc
in heptane (0−100%, 5% DCM throughout), leading to the
isolation of product as a yellow solid 5,5-dimethyl-3-((1R,4R)-
4-((3-nitropyridin-4-yl)amino)cyclohexyl)-4-phenyloxazolidin-
mixture was dried under reduced pressure and purified using a
25g SNAP column (Biotage), ramping EtOAc in heptane (0−
100%, with 10% DCM throughout), providing product as a
yellow foam (S)-5,5-dimethyl-3-((1R,4S)-4-((2-nitropyridin-3-
yl)amino)cyclohexyl)-4-phenyloxazolidin-2-one (0.714 g, 1.739
mmol, 90% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.78 (dd,
J = 1.3, 3.9 Hz, 1 H), 7.66 (dd, J = 1.2, 8.9 Hz, 1 H), 7.53 (dd, J
= 4.0, 8.7 Hz, 1 H), 7.46−7.39 (m, 2 H), 7.38−7.15 (m, 3 H),
4.60 (s, 1 H), 3.53−3.41 (m, 2 H), 2.02 (td, J = 3.1, 12.8 Hz, 1
H), 1.98−1.79 (m, 3 H), 1.58 (d, J = 10.5 Hz, 1 H), 1.47 (s, 3
H), 1.46−1.37 (m, 1 H), 1.37−1.25 (m, 2 H), 0.78 (s, 3 H). m/
z (ESI) 411.2 (M + H)+.
To a flask charged with (S)-5,5-dimethyl-3-((1R,4S)-4-((2-
nitropyridin-3-yl)amino)cyclohexyl)-4-phenyloxazolidin-2-one
(0.702 g, 1.710 mmol) was added EtOH (34.2 mL), Raney
nickel, slurry, in water (1.884 mL, 286 mmol) (added using
pipet). The flask was sealed and placed under nitrogen, and
heated to 40 °C. To the resulting yellow solution was added
hydrazine hydrate solution (0.799 mL, 25.7 mmol) dropwise.
The resulting mixture was stirred overnight at 40 °C.
The resulting mixture was filtered through Celite which was
washed with MeOH. The filtrate was dried under reduced
pressure and the material purified with SCX-2 (5g), washing
with MeOH, then 2 M NH3 in MeOH, but much material came
through the column with the initial MeOH wash. The washes
were combined and dried under reduced pressure. The crude
residue was purified with a 50g SNAP column (Biotage),
ramping MeOH in DCM from 0 to 25%, leading to isolation of
product as a purple film (S)-3-((1R,4S)-4-((2-aminopyridin-3-
yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one
(0.580 g, 1.524 mmol, 89% yield). 1H NMR (400 MHz,
DMSO-d6) δ 7.45−7.38 (m, 2 H), 7.38−7.23 (m, 3 H), 7.21
(dd, J = 1.5, 4.9 Hz, 1 H), 6.52 (d, J = 6.8 Hz, 1 H), 6.38 (dd, J
= 4.9, 7.6 Hz, 1 H), 5.36 (s, 2 H), 4.61 (s, 1 H), 4.38 (d, J = 7.4
Hz, 1 H), 3.53−3.42 (m, 1 H), 3.06−2.95 (m, 1 H), 2.03 (td, J
= 3.2, 12.9 Hz, 1 H), 1.92−1.76 (m, 3 H), 1.58−1.49 (m, 1 H),
1.46 (s, 3 H), 1.26−1.03 (m, 3 H), 0.78 (s, 3 H). m/z (ESI)
381.4 (M + H)+.
To a flask charged with (S)-3-((1R,4S)-4-((2-aminopyridin-
3-yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one
(0.560 g, 1.472 mmol) was added THF (5.89 mL), DIEA
(0.566 mL, 3.24 mmol), and CDI (0.525 g, 3.24 mmol),
respectively. The dark mixture was stirred at 45 °C overnight,
providing ∼70% conversion according to LC-MS, with clean
conversion and starting material remaining. An additional 1
equiv of DIEA and CDI were added, and heating continued at
45 °C for 2 h. The dark purple solution was dried under
reduced pressure and purified with a 50g SNAP column
(Biotage), ramping DCM/MeOH (90:10) in DCM from 0 to
30%, then isocratic at 30%, providing elution of product. Using
the following conditions the enantiomers were successfully
separated (AD-H (2 × 20 cm) column, 25% MeOH (0.1%
DEA)/CO2; 100 bar; 70 mL/min; 220 nm detection; injection
volume, 1 mL; 18 mg/mL, 4:1 MeOH/DCM). The NMR
showed the presence of substantial diethyl amine. Each of the
enantiomers were dissolved in DCM/trace MeOH and
extracted with H2O (2×). The organics were dried with
Na2SO4, filtered, and dried under reduced pressure, then
lyophilized from MeOH/H2O, affording the title compound 35
as a light pink solid (S enantiomer, peak 2, major enantiomer
−235 mg, 39%) 1H NMR (400 MHz, DMSO-d6) δ 11.47 (br s,
1 H), 7.86 (d, J = 3.9 Hz, 1 H), 7.67 (d, J = 7.9 Hz, 1 H), 7.42
(d, J = 6.5 Hz, 2 H), 7.39−7.13 (m, 3 H), 6.97−6.88 (m, 1 H),
1
2-one (1.83 g, 4.46 mmol, 59.8% yield). H NMR (400 MHz,
CDCl3) δ 9.19 (s, 1 H), 8.25 (d, J = 6.3 Hz, 1 H), 8.00 (d, J =
7.2 Hz, 1 H), 7.49−7.29 (m, 4 H), 7.23−7.06 (m, 1 H), 6.64
(d, J = 6.3 Hz, 1 H), 4.39 (s, 1 H), 3.50−3.34 (m, 2 H), 2.24−
2.05 (m, 3 H), 2.04−1.95 (m, 1 H), 1.91−1.80 (m, 1 H), 1.63−
1.56 (m, 1 H), 1.46−1.26 (m, 2 H), 0.94 (s, 3 H). m/z (ESI)
411.2 (M + H)+.
To a flask charged with (S)-5,5-dimethyl-3-((1R,4S)-4-((3-
nitropyridin-4-yl)amino)cyclohexyl)-4-phenyloxazolidin-2-one
(1.82 g, 4.43 mmol) was added EtOH (89 mL), Raney nickel,
slurry, in water (4.88 mL, 740 mmol) (added roughly using
pipet). The flask was sealed and placed under nitrogen, and the
reaction mixture was heated to 40 °C. To the resulting yellow
solution was added hydrazine hydrate solution (2.071 mL, 66.5
mmol) dropwise. The resulting mixture was stirred for 3 h at 40
°C. The mixture was filtered through Celite, which was washed
with MeOH. The filtrate was dried under reduced pressure,
providing an off-white foam after drying under high vacuum
overnight, which was used directly in the next step (quantitative
yield). m/z (ESI) 381.4 (M + H)+.
To a flask charged with 3-((1R,4R)-4-((3-aminopyridin-4-
yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one
(1.60 g, 4.21 mmol) was added THF (16.82 mL) and DIEA
(2.57 mL, 14.72 mmol). The mixture was cooled in an ice water
bath prior to the addition of CDI (2.387 g, 14.72 mmol). The
resulting mixture was allowed to stir and warm slowly to room
temperature (ice melt) over 2 h. To the yellow solution was
added saturated aqueous NH4Cl, and the mixture was
transferred to a separatory funnel and extracted with EtOAc
(2×). The combined organics were washed with brine, dried
with Na2SO4, filtered, and dried under reduced pressure. The
crude residue obtained was purified with a 40g HP spherical
silica column (Interchim), ramping DCM/MeOH/NH4OH
(90:10:1) in DCM from 0 to 45%, then isocratic at 45%,
affording the title compound as a white solid (1.3 g). 1H NMR
(400 MHz, DMSO-d6) δ 8.15 (d, J = 0.6 Hz, 1 H), 8.08 (d, J =
5.4 Hz, 1 H), 7.46−7.39 (m, 3 H), 7.39−7.35 (m, 1 H), 7.28
(br s, 2 H), 4.66 (s, 1 H), 4.04 (tt, J = 3.8, 12.4 Hz, 1 H), 3.68
(tt, J = 3.9, 11.6 Hz, 1 H), 2.25−2.03 (m, 2 H), 2.02−1.83 (m,
2 H), 1.77−1.68 (m, 1 H), 1.61 (s, 3 H), 1.48 (s, 3 H), 1.29
(dq, J = 3.1, 12.7 Hz, 1 H), 0.80 (s, 3 H). m/z (ESI) 407.2 (M
+ H)+. HRMS calcd for C23H26N4O3 (M + H)+ 407.2083,
found 407.2089.
(S)-5,5-Dimethyl-3-((1R,4S)-4-(2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-1-yl)cyclohexyl)-4-phenyloxazoli-
din-2-one (35). To a vial charged with (S)-3-((1R,4S)-4-
aminocyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one (28;
0.560 g, 1.942 mmol) (this intermediate 28 lot existed as a
∼3:1 S/R mixture of phenyl chirality) was added acetonitrile
(6.47 mL), triethylamine (0.541 mL, 3.88 mmol), and 3-fluoro-
2-nitropyridine (Matrix Scientific) (276 mg, 1.94 mmol),
respectively. The resulting orange solution was shaken at 80
°C for 4 h, leading to conversion to the desired product as the
major species according to LC-MS with about 50% conversion.
The mixture was shaken for 48 h at 80 °C, providing complete
conversion to desired product according to LC-MS. The
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4.66 (s, 1 H), 4.11−3.98 (m, 1 H), 3.74−3.60 (m, 1 H), 2.24−
1.86 (m, 4 H), 1.74 (d, J = 11.2 Hz, 1 H), 1.60 (t, J = 12.6 Hz, 2
H), 1.48 (s, 3 H), 1.28 (q, J = 11.3 Hz, 1 H), 0.80 (s, 3 H). m/z
(ESI) 407.1 (M + H)+. HRMS calcd for C23H26N4O3 (M + H)+
407.2083, found 407.2084.
solid was ground with a spatula and triturated again with ether,
providing a white solid 1-((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-
4-phenyloxazolidin-3-yl)cyclohexyl)-2-oxo-2,3-dihydro-1H-
benzo[d]imidazole-5-carbonitrile (790 mg, 85%). 1H NMR
(500 MHz, DMSO-d6) δ 7.53 (d, J = 8.3 Hz, 1 H), 7.49−7.34
(m, 4 H), 7.29 (d, J = 8.3 Hz, 3 H), 4.65 (s, 1 H), 4.12−4.04
(m, 1 H), 3.70−3.61 (m, 1 H), 2.27−2.07 (m, 2 H), 2.02−1.85
(m, 2 H), 1.73 (d, J = 11.6 Hz, 1 H), 1.68−1.54 (m, 2 H), 1.48
(s, 3 H), 1.36−1.25 (m, 1 H), 0.82−0.77 (m, 3 H). m/z (ESI)
431.1 (M + H)+. HRMS calcd for C25H26N4O3 (M + H)+
431.2083, found 431.2083.
1-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazole-4-carbonitrile (37). To a vial charged with 3-
((1R,4R)-4-aminocyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-
2-one 2,2,2-trifluoroacetate (28; 0.300 g, 0.746 mmol) (this lot
existed as a ∼3:1 S/R mixture of phenyl chirality) was added
acetonitrile (2.485 mL), DIEA (0.260 mL, 1.491 mmol), and 3-
chloro-2-nitrobenzonitrile (Biogene Organics) (0.136 g, 0.746
mmol), respectively. The vial was sealed and heated to 80 °C
overnight, providing an orange/brown solution and about 50%
conversion according to LC-MS. Additional DIEA (0.260 mL,
1.491 mmol) was added, and heating and shaking continued.
After 1 additional hourr at 80 °C, no additional conversion was
observed by LC-MS. The mixture was heated to 110 °C
overnight, leading to conversion to product along with two
major impurities which have similar retention times to product.
The mixture was dried under reduced pressure and purified
with a 25g column, ramping DCM/MeOH/NH4OH (90:10:1)
in DCM from 0 to 15%, then 15% isocratic, which yielded some
separation of impurities but not complete separation. The
material (∼50% purity) was used without further purification,
isolated as an orange/brown solid 3-(((1R,4R)-4-(5,5-dimethyl-
2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-nitroben-
zonitrile (0.180 g, 0.414 mmol, 55.6% yield). m/z (ESI) 435.0
(M + H)+.
To a flask charged with 3-(((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-nitrobenzo-
nitrile (0.180 g, 0.414 mmol) was added ethanol (2.76 mL)
followed by tin(II) chloride (0.236 g, 1.243 mmol). The
resulting yellow suspension was heated at 80 °C for 2 h,
providing an orange brown solution. LC-MS indicated product
as a major species along with impurities (present in starting
material) which are close to product in retention time. The
solution was cooled to room temperature and filtered through a
Si-thiol column. The filtrate was dried under reduced pressure
and purified with a 25g SNAP column, ramping DCM/MeOH
(90:10) in DCM from 0 to 50%, then isocratic at 50% to yield
product along with the impurities which coeluted (most
impurities present from crude). The material was further
purified with a 2g SCX-2 column, washing with MeOH and
then 2 M NH3 in MeOH to yield a brown oil 2-amino-3-
(((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-
cyclohexyl)amino)benzonitrile (0.124 g, 0.307 mmol, 74.0%
yield) which had been enriched in purity relative to after MPLC
(∼75% pure). The material was used as is without further
purification. m/z (ESI) 405.0 (M + H)+.
1-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazole-5-carbonitrile (36). To a vial charged with (S)-3-
((1R,4S)-4-aminocyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-
2-one (28; 0.150 g, 0.520 mmol) (this lot existed as a ∼3:1 S/R
mixture of phenyl chirality) was added acetonitrile (1.734 mL),
triethylamine (0.145 mL, 1.040 mmol), and 4-fluoro-3-
nitrobenzonitrile (Alfa Aesar) (0.086 g, 0.520 mmol),
respectively. The resulting orange solution was shaken at 80
°C overnight. The resulting mixture was dried under reduced
pressure and the crude material purified with a 25g SNAP
column, ramping EtOAc in heptane from 0 to 100% with 10%
DCM throughout, providing product as a yellow solid 4-
(((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-
cyclohexyl)amino)-3-nitrobenzonitrile (0.172 g, 0.396 mmol,
76% yield). 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 2.0 Hz,
1 H), 8.25 (d, J = 7.4 Hz, 1 H), 7.58−7.51 (m, 1 H), 7.47−7.28
(m, 4 H), 7.14 (br s, 1 H), 6.85 (d, J = 9.2 Hz, 1 H), 4.39 (s, 1
H), 3.50−3.33 (m, 2 H), 2.24−2.06 (m, 3 H), 2.02−1.93 (m, 1
H), 1.90−1.79 (m, 1 H), 1.62−1.49 (m, 4 H), 1.47−1.26 (m, 2
H), 0.92 (s, 3 H). m/z (ESI) 435.2 (M + H)+.
To a flask charged with 4-(((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-3-nitrobenzo-
nitrile (0.172 g, 0.396 mmol) was added ethanol (2.64 mL)
followed by tin(II) chloride (0.225 g, 1.188 mmol). The
resulting yellow suspension was heated overnight at 80 °C. LC-
MS of the resulting yellow solution indicates product as a major
species along with minor impurities which are close to product
in retention time. The solution was cooled to room
temperature and filtered through a Si-thiol column. The filtrate
was dried under reduced pressure and purified with a 25g
SNAP column, ramping DCM/MeOH (90:10) in DCM from 0
to 30%, then isocratic at 30% to yield product along with minor
impurities which had coeluted. The material obtained was used
without further purification. Product was obtained as a yellow
oil 3-amino-4-(((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenylox-
azolidin-3-yl)cyclohexyl)amino)benzonitrile (0.117 g, 0.289
mmol, 73.1% yield). m/z (ESI) 405.2 (M + H)+.
To a flask charged with 3-amino-4-(((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-
benzonitrile (0.878 g, 2.171 mmol) was added THF (8.68 mL)
and DIEA (1.327 mL, 7.60 mmol). The resulting solution was
cooled in an ice water bath prior to the addition of CDI (1.23 g,
7.60 mmol). The resulting mixture was stirred for 3 h and
allowed to slowly warm to ambient temperature (ice melt),
providing a greenish suspension. LC-MS indicated complete
conversion to product. The mixture was dried under reduced
pressure and purified with a 40g HP spherical silica column (15
μm spherical, Interchim), ramping DCM/MeOH (90:10) in
DCM from 0 to 30%, then isocratic at 30%, yielding partial
separation of clean product obtained as a white film as well as a
darker eluting mixture primarily containing product. The dark
mixture was purified with an SCX-2 column (5g), washing with
MeOH, leading to elution of a yellow solution containing
product. After drying, the pale yellow solid was triturated with
diethyl ether, and the off-white solid obtained was combined
with the clean material from the initial column. NMR revealed
still some very minor impurities in the aliphatic region. The
To a vial charged with 2-amino-3-(((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-
benzonitrile (0.124 g, 0.307 mmol) was added THF (1.226
mL), DIEA (0.187 mL, 1.073 mmol), and CDI (0.174 g, 1.073
mmol), respectively. The dark mixture was stirred for 1 h at
room temperature, yielding no conversion according to LC-MS.
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The mixture was heated to 60 °C. After 2 h LC-MS indicated
about 30% conversion. Heating and shaking at 60 °C was
continued overnight. LC-MS indicated product as the main
species.
2.137 mmol). The mixture was stirred at room temperature for
16 h. LC-MS indicated complete reaction. The solution was
dried under reduced pressure and purified with a 25g SNAP
column (Biotage), ramping DCM/MeOH (90:10) in DCM
from 0 to 40%, then isocratic at 40% to provide product 1-
((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)-
cyclohexyl)-6-fluoro-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-
5-carbonitrile as a light yellow solid (0.151 g, 0.337 mmol, 55%
yield).1H NMR (400 MHz, DMSO-d6) δ 11.34 (br s, 1 H),
7.73 (d, J = 10.5 Hz, 1H), 7.45−7.2 (m, 6 H), 4.65 (s, 1 H),
4.13−4.03 (m, 1 H), 3.77−3.68 (m, 1 H), 2.28−2.07 (m, 2 H),
2.02−1.83 (m, 2 H), 1.75−1.55 (m, 2 H), 1.48 (s, 3 H), 1.38−
1.25 (m, 1 H), 0.80 (s, 3 H). m/z (ESI) 449.1 (M + H)+.
HRMS calcd for C25H25FN4O3 (M + H)+ 449.1989, found
449.1985.
1-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-4-fluoro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazole-5-carbonitrile (40). To a vial charged
with (S)-3-((1R,4S)-4-aminocyclohexyl)-5,5-dimethyl-4-phe-
nyloxazolidin-2-one (28; 0.114 g, 0.395 mmol) was added
acetonitrile (1.318 mL) and triethylamine (0.110 mL, 0.791
mmol). The resulting solution was cooled in an ice water bath.
In a separate vial, 2,4-difluoro-3-nitrobenzonitrile (0.076 g,
0.415 mmol) was dissolved in ACN (200 μL) and added slowly
to the cooled solution of amine. The mixture was allowed to
slowly warm to room temperature overnight (ice melt). The
resulting orange solution was dried under reduced pressure and
purified with a 25g SNAP column, ramping DCM/MeOH
(90:10) in DCM from 0 to 30%, then isocratic at 30%
(detection at 215 nm) to provide product, obtained as a yellow
solid 4-(((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)amino)-2-fluoro-3-nitrobenzonitrile (0.161
g, 0.356 mmol, 90% yield) with about 20% impurity present.
The material was used as is for a subsequent step. m/z (ESI)
453.4 (M + H)+.
To a vial charged with 4-(((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-fluoro-3-ni-
trobenzonitrile (0.161 g, 0.356 mmol) was added EtOH (1.423
mL) and tin(II) chloride (0.202 g, 1.067 mmol), respectively.
The suspension was heated at 80 °C for 3 h, yielding an orange
solution. The resulting mixture was dried under reduced
pressure, and the residue obtained was purified with a 25g
SNAP column, ramping DCM/MeOH (90:10) in DCM (0−
30%, then isocratic at 30%, detection at 215 nm), leading to
isolation of product, obtained as an orange oil (145 mg, 96%).
m/z (ESI) 423.4 (M + H)+.
To a flask charged with 3-amino-4-(((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-
fluorobenzonitrile (0.145 g, 0.343 mmol) was added THF
(1.373 mL) and DIEA (0.210 mL, 1.201 mmol) and CDI
(0.195 g, 1.201 mmol), respectively. The mixture was shaken at
room temperature for 1.5 h. The mixture was dried under
reduced pressure and the residue purified with a 25g HP
spherical silica column (15 μm spherical silica, Interchim),
ramping DCM/MeOH (90:10) in DCM from 0 to 25%, then
isocratic at 25%, with detection at 215 nm, providing product as
a white solid (118 mg, 77%). 1H NMR (400 MHz, DMSO-d6)
δ 11.94 (br s, 1 H), 7.52−7.13 (m, 7 H), 4.67−4.62 (m, 1 H),
4.09 (tt, J = 3.4, 12.4 Hz, 1 H), 3.66 (tt, J = 3.8, 11.7 Hz, 1 H),
2.40−2.05 (m, 2 H), 2.03−1.84 (m, 2 H), 1.81−1.55 (m, 3 H),
1.48 (s, 3 H), 1.36−1.21 (m, 1 H), 0.80 (s, 3 H). m/z (ESI)
449.1 (M + H)+, HRMS calcd for C25H25FN4O3 (M + H)+
449.1989, found 449.1987. See Supporting Information Figures
The dark solution was dried under reduced pressure and
purified with RP-HPLC, ramping ACN in H2O (10−90%, 0.1%
TFA), affording incomplete purification. The product contain-
ing eluents were dried under reduced pressure and repurified.
Separation #1: Chiralpak OJ-H, 2 cm × 25 cm, 80% CO2/20%
methanol w/0.2% DEA, 80 mL/min, 254 nm; dissolution, 20
mL of 3:1 MeOH/DCM; sample processed with 0.25 mL
injections and 2.75 min cycle time. Separation #2: Chiralpak IA,
2 cm × 25 cm, 75% CO2/25% methanol w/0.2% DEA, 80 mL/
min, 215 nm; dissolution, 10 mL of 1:1 MeOH/DCM; sample
processed with 0.15 mL injections and 5.25 min cycle time.
Desired major enantiomer eluted first. Product contained a
minor amount of DEA. The material was passed through a 2g
SCX-2 column and the initial wash dried under reduced
pressure and lyophilized from MeOH/H2O, providing product
as a white solid 1-((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyl-
oxazolidin-3-yl)cyclohexyl)-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazole-4-carbonitrile (17 mg, 0.039 mmol, 12.88% yield).
1H NMR (500 MHz, DMSO-d6) δ 11.90 (s, 1 H), 7.66 (d, J =
7.8 Hz, 1 H), 7.46−7.39 (m, 2 H), 7.39−7.15 (m, 4 H), 7.07 (t,
J = 8.0 Hz, 1 H), 4.65 (s, 1 H), 4.11−4.02 (m, 1 H), 3.66 (d, J =
11.8 Hz, 1 H), 2.26−2.06 (m, 2 H), 2.03−1.84 (m, 2 H), 1.80−
1.69 (m, 1 H), 1.67−1.55 (m, 2 H), 1.48 (s, 3 H), 1.30 (dq, J =
3.7, 12.8 Hz, 1 H), 0.80 (s, 3 H). m/z (ESI) 431.1 (M + H)+.
HRMS calcd for C25H26N4O3 (M + H)+ 431.2083, found
431.2082.
1-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-6-fluoro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazole-5-carbonitrile (39). A vial charged with
(S)-3-((1R,4S)-4-aminocyclohexyl)-5,5-dimethyl-4-phenyloxa-
zolidin-2-one (28; 0.184 g, 0.638 mmol), acetonitrile (1.734
mL), and DIEA (0.181 mL, 1.04 mmol) was cooled to 0 °C,
and 2,4-difluoro-5-nitrobenzonitrile (Aldrich) (0.078 g, 0.342
mmol) was added. The vial was sealed, and the temperature
was slowly raised to room temperature over 16 h, providing a
brown solution with product as the main species according to
LC-MS. The solution was dried under reduced pressure and
purified with a 25g SNAP column (Biotage), ramping DCM/
MeOH (90:10) in DCM (0−25%, then isocratic at 30%, 215
nm detection), providing 4-(((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-fluoro-5-ni-
trobenzonitrile (0.282 g, 0.623 mmol, 98% yield) as a yellow
solid. m/z (ESI) 453.3 (M + H)+.
To a flask charged with 4-(((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-fluoro-5-ni-
trobenzonitrile (0.282 g, 0.623 mmol) was added ethanol (6.23
mL) and tin(II) chloride (0.019 mL, 0.405 mmol), and the
resulting yellow suspension was shaken for 3 h at 80 °C. LC-
MS indicated complete conversion. The solution was dried
under reduced pressure and purified with a 25g SNAP column
(Biotage), ramping DCM/MeOH (90:10) in DCM from 0 to
30%, then isocratic at 30% to provide 5-amino-4-(((1S,4R)-4-
((S)-5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)-
amino)-2-fluorobenzonitrile (0.258 g, 0.611 mmol, 98% yield)
as yellow brown solid. m/z (ESI) 423.3 (M + H)+.
To a flask charged with 5-amino-4-(((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-2-
fluorobenzonitrile (0.258 g, 0.611 mmol) was added THF
(2.043 mL), DIEA (0.372 mL, 2.137 mmol), and CDI (0.347 g,
R
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S1 and S2 for IR spectrum and NMR proton, carbon peak
assignments, respectively.
for 30 min at 160 °C. LC-MS indicated that no conversion had
occurred. To the mixture was added Pd(PPh3)4 (0.048 g, 0.041
mmol) and 2 equiv of Zn(CN)2. The vessel was purged with
argon and heated to 120 °C for 90 min, yielding complete
conversion according to LC-MS.
3-((1S,4R)-4-((S)-5,5-Dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)-2-oxo-2,3-dihydro-1H-imidazo[4,5-
b]pyridine-6-carbonitrile (41). To a vial charged with (S)-3-
((1R,4S)-4-aminocyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-
2-one (28; 0.194 g, 0.673 mmol) was added acetonitrile (2.242
mL), DIEA (0.235 mL, 1.345 mmol), and 5-bromo-2-chloro-3-
nitropyridine (Matrix Scientific) (0.168 g, 0.706 mmol,)
respectively. The vial was sealed and heated to 90 °C for 2 h,
providing a brown solution with product as the main species
according to LC-MS. The solution was dried under reduced
pressure and purified with a 25g SNAP column, ramping
DCM/MeOH (90:10) in DCM (0−30%, then isocratic at 30%,
detection at 215 nm), providing (S)-3-((1R,4S)-4-((5-bromo-
3-nitropyridin-2-yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyl-
oxazolidin-2-one (0.315 g, 0.644 mmol, 96% yield) as a yellow/
orange solid. m/z (ESI) 490.1 (M + H)+.
To a flask charged with (S)-3-((1R,4S)-4-((5-bromo-3-
nitropyridin-2-yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxa-
zolidin-2-one (0.310 g, 0.633 mmol) was added EtOH (12.67
mL), Raney 3202 nickel, slurry, in water (0.698 mL, 106 mmol)
(added roughly using pipet). The flask was sealed and placed
under nitrogen, and heated to 40 °C. To the resulting yellow
solution was added hydrazine hydrate solution (0.296 mL, 9.50
mmol) dropwise. The resulting mixture was stirred at 40 °C
overnight. LC-MS of the cloudy white suspension indicated
complete conversion to desired product. The mixture was
cooled to room temperature and filtered through Celite, which
was washed with MeOH. The filtrate was dried under reduced
pressure and then purified using a 5g SCX-2 column, washing
with MeOH and then 2 M NH3 in MeOH. The basic was dried
under reduced pressure, providing (S)-3-((1R,4S)-4-((3-amino-
5-bromopyridin-2-yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyl-
oxazolidin-2-one (0.270 g, 0.588 mmol, 93% yield) as a purple
film. m/z (ESI) 459.1 (M + H)+.
The suspension was cooled to room temperature and filtered
through a 5g SCX-2 column, washing with MeOH, then 2 M
NH3 in MeOH. The product eluted during the initial MeOH
wash. This wash was dried under reduced pressure and the
crude brown oil purified with a 25g HP spherical silica column
(15 μm, Interchim), ramping DCM/MeOH (90:10) in DCM
(0−30%, then isocratic at 30%, detection at 215 nm), yielding
an off-white solid with a minor yellow discoloration (168 mg).
The material was triturated with MeOH and washed with a
small volume of MeOH to yield 3-((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)-2-oxo-2,3-
dihydro-1H-imidazo[4,5-b]pyridine-6-carbonitrile as a white
solid (56 mg, 31%). 1H NMR (400 MHz, DMSO-d6) δ
11.56 (br s, 1 H), 8.38 (d, J = 1.9 Hz, 1 H), 7.67 (d, J = 1.9 Hz,
1 H), 7.47−7.17 (m, 5 H), 4.67 (s, 1 H), 4.13 (tt, J = 3.8, 12.3
Hz, 1 H), 3.53−3.41 (m, 1 H), 2.41−2.17 (m, 2 H), 2.01−1.84
(m, 2 H), 1.83−1.73 (m, 1 H), 1.71−1.58 (m, 2 H), 1.47 (s, 3
H), 1.35−1.20 (m, 1 H), 0.81 (s, 3 H). m/z (ESI) 432.0 (M +
H)+, HRMS calcd for C23H25BrN4O3 (M + H)+ 485.1188,
found 485.1185.
(S)-5,5-Dimethyl-3-((1R,4S)-4-(2-oxo-1H-imidazo[4,5-
b]pyridin-3(2H)-yl)cyclohexyl)-4-phenyloxazolidin-2-
one (46). To a flask charged with toluene (37.3 mL) was
added trans-N-boc-1,4-cyclohexanediamine (2.00 g, 9.33
mmol), potassium carbonate (1.290 g, 9.33 mmol), and 2-
fluoro-3-nitropyridine (Matrix Scientific) (1.326 mL, 9.33
mmol). The resulting bright yellow solution was heated
overnight at 120 °C, affording a bright yellow suspension.
The mixture was diluted with water, transferred to a separatory
funnel, and extracted with EtOAc (2×). The combined organics
were dried with Na2SO4, filtered, and dried under reduced
pressure, providing a bright yellow solid which was triturated
with DCM, providing product as a bright yellow solid tert-butyl
((1R,4R)-4-((3-nitropyridin-2-yl)amino)cyclohexyl)carbamate
(1.51 g, 4.49 mmol, 48.1% yield). 1H NMR (400 MHz, DMSO-
d6) δ 8.49 (dd, J = 1.8, 4.5 Hz, 1 H), 8.41 (dd, J = 1.8, 8.4 Hz, 1
H), 8.06 (d, J = 7.7 Hz, 1 H), 6.79−6.69 (m, 2 H), 4.13−4.00
(m, 1 H), 3.29−3.20 (m, 1 H), 2.01−1.92 (m, 2 H), 1.82 (d, J
= 10.6 Hz, 2 H), 1.55−1.41 (m, 2 H), 1.38 (s, 9 H), 1.35−1.21
(m, 2 H). m/z (ESI) 281.2 (M + H)+.
To a flask charged with (S)-3-(4-((3-amino-5-bromopyridin-
2-yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one
(0.270 g, 0.588 mmol) was added THF (2.351 mL), DIEA
(0.359 mL, 2.057 mmol), and CDI (0.334 g, 2.057 mmol),
respectively. The mixture was shaken at room temperature
overnight. LC-MS of the dark solution indicated complete
reaction. The mixture was dried under reduced pressure and
purified with a 25g HP 15 μm spherical silica column
(Interchim), ramping DCM/MeOH (90:10) in DCM (0−
30%, then isocratic at 30%, detection at 215 nm), yielding
product as an off-white solid, (S)-3-((1S,4R)-4-(6-bromo-2-
oxo-1H-imidazo[4,5-b]pyridin-3(2H)-yl)cyclohexyl)-5,5-di-
methyl-4-phenyloxazolidin-2-one (0.230 g, 0.474 mmol, 81%
To a flask charged with tert-butyl ((1R,4R)-4-((3-nitro-
pyridin-2-yl)amino)cyclohexyl)carbamate (1.500 g, 4.46 mmol)
was added EtOH (12.74 mL) and tin(II) chloride (2.54 g,
13.38 mmol). The resulting yellow suspension was heated to 80
°C under nitrogen overnight, providing a light brown solution
containing primarily product according to LC-MS. The mixture
was dried under reduced pressure and purified with a 50g
SNAP column (Biotage), ramping DCM/MeOH (90:10) in
DCM from 0 to 100% (215 nm detection), yielding product as
an orange foam. The material was purified with a 10g SCX-2
column washing with MeOH, then with NH3 in MeOH,
providing product as a brown flaky solid tert-butyl ((1R,4R)-4-
((3-aminopyridin-2-yl)amino)cyclohexyl)carbamate (0.840 g,
1
yield) upon drying. H NMR (400 MHz, DMSO-d6) δ 11.27
(br s, 1 H), 8.00 (d, J = 2.0 Hz, 1 H), 7.52−7.14 (m, 6 H), 4.67
(s, 1 H), 4.12−4.01 (m, 1 H), 3.52−3.40 (m, 1 H), 2.42−2.17
(m, 2 H), 2.01−1.83 (m, 2 H), 1.75 (d, J = 12.0 Hz, 1 H), 1.61
(t, J = 13.2 Hz, 2 H), 1.47 (s, 3 H), 1.33−1.16 (m, 1 H), 0.81
(s, 3 H). m/z (ESI) 485.1 (M + H)+.
To a 0.5−2.0 mL microwave vial charged with (S)-3-
((1S,4R)-4-(6-bromo-2-oxo-1H-imidazo[4,5-b]pyridin-3(2H)-
yl)cyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one (0.200 g,
0.412 mmol) was added DMF (1.648 mL) followed by zinc
cyanide (0.026 mL, 0.412 mmol) and [1,1-bis-
(diphenylphosphino)ferrocene]palladium(II) chloride, complex
with dichloromethane (1:1) (0.034 g, 0.041 mmol). The
resulting mixture was purged with argon, ,sealed and irradiated
1
2.74 mmol, 61.5% yield). H NMR (400 MHz, DMSO-d6) δ
7.31 (dd, J = 1.6, 5.2 Hz, 1 H), 6.71 (dd, J = 7.8, 12.8 Hz, 2 H),
6.36 (t, J = 6.2 Hz, 1 H), 4.87 (br s, 2 H), 3.73 (s, 1 H), 3.23
(br s, 1 H), 1.97 (d, J = 8.2 Hz, 2 H), 1.80 (d, J = 7.7 Hz, 2 H),
1.38 (s, 9 H), 1.35−1.17 (m, 4 H). m/z (ESI) 307.2 (M + H)+.
S
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Journal of Medicinal Chemistry
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To a flask charged with tert-butyl ((1R,4R)-4-((3-amino-
pyridin-2-yl)amino)cyclohexyl)carbamate (0.830 g, 2.71 mmol)
was added THF (10.84 mL) and triethylamine (0.793 mL, 5.69
mmol). The resulting suspension was cooled in an ice water
bath prior to the addition of triphosgene (0.804 g, 2.71 mmol),
leading to the immediate formation of a precipitate, product
according to LC-MS. To the glob of solid was added additional
THF (∼15 mL) and a small amount of water. The resulting
solid was collected via vacuum filtration and washed with water,
followed by diethyl ether, and dried under high vacuum,
providing product as a white solid (420 mg). The filtrate was
analyzed by LC-MS, which indicated the presence of additional
product. The mixture was transferred to a separatory funnel and
extracted with EtOAc (2×). The combined organics were dried
with Na2SO4, filtered, and dried under reduced pressure,
providing additional product (180 mg) as a light tan solid with
slightly lower purity than that of the precipitated material (total
−600 mg, 67%). 1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1
H), 7.93 (dd, J = 1.4, 5.2 Hz, 1 H), 7.26 (dd, J = 1.3, 7.7 Hz, 1
H), 6.97 (dd, J = 5.2, 7.7 Hz, 1 H), 6.77 (d, J = 7.3 Hz, 1 H),
4.18 (tt, J = 4.0, 12.4 Hz, 1 H), peak under H2O in DMSO
(1H), 2.47−2.27 (m, 2 H), 1.90 (d, J = 11.6 Hz, 2 H), 1.68 (d, J
= 10.8 Hz, 2 H), 1.39 (s, 9 H), 1.36−1.25 (m, 2 H). m/z (ESI)
355.2 (M + Na)+.
( )-3-((1R,4R)-4-((2-hydroxy-2-methyl-1-phenylpropyl)-
amino)cyclohexyl)-1H-imidazo[4,5-b]pyridin-2(3H)-one
(0.255 g, 0.670 mmol, 46.5% yield). m/z (ESI) 381.2 (M +
H)+.
A vial charged with ( )-3-((1R,4R)-4-((2-hydroxy-2-methyl-
1-phenylpropyl)amino)cyclohexyl)-1H-imidazo[4,5-b]pyridin-
2(3H)-one (0.245 g, 0.644 mmol) and a stir bar was dried
under reduced pressure and placed under nitrogen prior to the
addition of THF (2.58 mL) and DIEA (0.236 mL, 1.352
mmol). The resulting suspension was cooled in an ice water
bath prior to the addition of triphosgene (0.065 g, 0.219
mmol). The resulting suspension was stirred at 0 °C and
allowed to slowly warm to room temperature. The mixture was
cooled back in an ice water bath, and additional triphosgene
was added (130 mg) and stirring continued. After 15 min, LC-
MS indicated cyclization had occurred along with overacylation,
as indicated by the masses of the overacylated acid and ester.
After 20 min of stirring at room temperature, no additional
conversion was observed. Therefore, an additional 0.5 equiv of
triphosgene was added (98 mg), and stirring at room
temperature continued. After another hour, only minor
additional conversion was observed. To the reaction was
added MeOH, and the mixture was dried under reduced
pressure. The crude material was purified with a 25g HP
spherical silica column (15 μm), ramping DCM/MeOH
(90:10) in DCM from 0 to 30%, then isocratic at 30%, then
to 100%, providing product (38 mg, 15%) along with
overacylated material. Enantiomer resolution: column, Chir-
alpak AS, 5 μm, 2 cm i.d. × 40 cm length; mobile phase, 35%
methanol w/0.2% diethylamine/65% CO2; flow rate, 65 mL/
min; detection, 293 nm; injection size, 3 mg in 400 μL of 1:1
DCM/methanol. Absolute configurations not experimentally
elucidated but assigned on the basis of potency discrepancies (S
more potent than R) and elution order comparisons to related
analogues. 1H NMR (500 MHz, DMSO-d6) δ 11.02 (b. s, 1 H),
7.91−7.87 (m, 1 H), 7.46−7.40 (m, 2 H), 7.40−7.34 (m, 1 H),
7.34−7.19 (m, 3 H), 6.95 (dd, J = 5.2, 7.7 Hz, 1 H), 4.68 (s, 1
H), 4.14−4.06 (m, 1 H), 3.48 (t, J = 11.6 Hz, 1 H), 2.46−2.36
(m, 1 H), 2.35−2.24 (m, 1 H), 1.99−1.85 (m, 2 H), 1.74 (d, J
= 12.6 Hz, 1 H), 1.68−1.54 (m, 2 H), 1.48 (s, 3 H), 1.31−1.21
(m, 1 H), 0.82 (s, 3 H). m/z (ESI) 407.1 (M + H)+, HRMS
calcd for C23H26N4O3 (M + H)+ 407.2083, found 407.2087.
(S)-5,5-Dimethyl-3-((1R,4S)-4-(2-oxo-1H-imidazo[4,5-
c]pyridin-3(2H)-yl)cyclohexyl)-4-phenyloxazolidin-2-one
(48). To a vial charged with (S)-3-((1R,4S)-4-aminocyclohex-
yl)-5,5-dimethyl-4-phenyloxazolidin-2-one (28; 0.181 g, 0.628
mmol) was added acetonitrile (2.51 mL), triethylamine (0.262
mL, 1.883 mmol), and methyl 3-fluoroisonicotinate (Biogene
Organics) (0.107 g, 0.690 mmol). The vessel was sealed and
shaken overnight at 80 °C over 72 h. The mixture was dried
under reduced pressure and purified with a 25g SNAP column,
ramping DCM/MeOH (90:10) in DCM (0−25%, then
isocratic at 25%, detection at 215 nm), providing product
with minor impurities as a light brown oil methyl 3-(((1S,4R)-
4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)-
amino)isonicotinate (0.184 g, 0.434 mmol, 69.2% yield). m/z
(ESI) 424.4 (M + H)+
To a vial charged with tert-butyl ((1R,4R)-4-(2-oxo-1H-
imidazo[4,5-b]pyridin-3(2H)-yl)cyclohexyl)carbamate (0.600
g, 1.805 mmol) was added DCM (7.22 mL) and TFA (1.391
mL, 18.05 mmol), providing a light peach solution, which was
stirred at room temperature. After 1 h, LC-MS indicated
product as the primary species. The solution was dried under
reduced pressure, and the residue obtained was purified with a
5g SCX-2 column, washing with MeOH followed by 2 M NH3
in MeOH. The basic wash was dried under reduced pressure to
provide product 3-((1R,4R)-4-aminocyclohexyl)-1H-imidazo-
[4,5-b]pyridin-2(3H)-one (0.343 g, 1.477 mmol, 82% yield) as
1
a white solid. H NMR (400 MHz, DMSO-d6) δ 7.92 (dd, J =
1.5, 5.2 Hz, 1 H), 7.26 (dd, J = 1.5, 7.6 Hz, 1 H), 6.96 (dd, J =
5.2, 7.7 Hz, 1 H), 4.19 (tt, J = 3.9, 12.3 Hz, 1 H), 2.70−2.60
(m, 1 H), 2.41 (dq, J = 3.3, 12.8 Hz, 2 H), 1.88 (d, J = 11.5 Hz,
2 H), 1.64 (d, J = 11.2 Hz, 2 H), 1.25−1.11 (m, 2 H). m/z
(ESI) 233.2 (M + H)+.
To a flask charged with 3-((1R,4R)-4-aminocyclohexyl)-1H-
imidazo[4,5-b]pyridin-2(3H)-one (0.335 g, 1.442 mmol) was
added DMF (5.77 mL), 1-phenyl-2-hydroxy-2-methyl-1-prop-
anone (Aldrich) (0.219 mL, 1.442 mmol), and acetic acid
(0.091 mL, 1.586 mmol), respectively, and the resulting
suspension was stirred for 15 min prior to the addition of
sodium triacetoxyborohydride (0.611 g, 2.88 mmol). The
resulting suspension was stirred overnight at 60 °C, leading to
the formation of product along with significant reduced alcohol
and starting amine still remaining. To the mixture was added
additional ketone (2 equiv) and sodium triacetoxyborohydride
(0.611 g, 2.88 mmol), and stirring at 60 °C continued. After 2 h
of stirring, LC-MS indicated starting material consumption and
additional product present. To the mixture was added water,
and the material was transferred to a separatory funnel and
extracted with EtOAc (2×). The combined organics were dried
with Na2SO4, filtered, and dried under reduced pressure,
providing the crude residue as an oil. The crude residue was
purified with a 25g SNAP column (Biotage), ramping DCM/
MeOH (90:10) in DCM from 0 to 30%, then isocratic at 30%,
providing impurity elution, then ramping to 100% polar eluent
to provide product, obtained as a sticky white goo upon drying,
To a vial charged with methyl 3-(((1S,4R)-4-((S)-5,5-
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-
isonicotinate (0.182 g, 0.430 mmol) was added THF (2.51
mL), MeOH (2.51 mL), water (2.51 mL), and LiOH (0.051 g,
2.149 mmol), respectively. The mixture was heated to 50 °C for
2 h. The light brown solution was diluted with water, acidified
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with 2 N HCl, and extracted with EtOAc (2×) and DCM (2×).
The combined organics were dried with Na2SO4, filtered, and
dried under reduced pressure, providing product as an off-white
solid 3-(((1S,4R)-4-((S)-5,5-dimethyl-2-oxo-4-phenyloxazoli-
din-3-yl)cyclohexyl)amino)isonicotinic acid (0.132 g, 0.322
mmol, 75% yield). m/z (ESI) 410.3 (M + H)+.
dimethyl-2-oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)-
pyrimidin-5-yl)isoindoline-1,3-dione (0.435 g, 0.850 mmol,
67.5% yield). m/z (ESI) 512.3 (M + H)+.
To a vial charged with 2-(4-(((1R,4R)-4-(5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)pyrimidin-5-yl)-
isoindoline-1,3-dione (0.435 g, 0.850 mmol) was added ethanol
(3.40 mL) and hydrazine hydrate (0.132 mL, 4.25 mmol). The
vial was sealed and heated to 100 °C for 2 h, yielding a white
suspension. LC-MS indicated complete conversion to product
cleanly. The solid was collected via vacuum filtration and
washed with DCM/MeOH. The precipitate was primarily 2,3-
dihydrophthalazine-1,4-dione. The filtrate contained product
along with phthalazinedione. The filtrate was purified with a 5g
SCX-2 column, washing with MeOH, then with 2 M NH3 in
MeOH. The basic wash was dried under reduced pressure,
providing product as a white foam 3-((1R,4R)-4-((5-amino-
pyrimidin-4-yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxazo-
lidin-2-one (0.236, 73%). m/z (ESI) 382.3 (M + H)+.
To a vial charged with 3-((1R,4R)-4-((5-aminopyrimidin-4-
yl)amino)cyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one
(0.330 g, 0.865 mmol) was added THF (3.46 mL), DIEA
(0.529 mL, 3.03 mmol), and CDI (0.491 g, 3.03 mmol),
respectively. The mixture was shaken at room temperature for 2
h, providing a yellow solution which contained primarily
product according to LC-MS. The material was loaded directly
onto a load column and purified with a 25g HP spherical silica
gel column (Interchim) ramping DCM/MeOH/NH4OH
(90:10:1) in DCM from 0 to 35%, then isocratic at 35%,
yielding product as a white foam upon drying. NMR is
consistent with a product as a mixture of isomers. The material
was repurified twice more as follows: column, Chiralpak OD-H
(2 cm × 15 cm); gradient, 20% MeOH (0.2% DEA)/CO2; 100
bar; flow, 65 mL/min; detection, 220 nm; injection volume,
0.75 mL, 12 mg/mL; dissolution, 1:1 DCM/MeOH. The
desired enantiomer (major product) was the first eluting.
Second purification: column, Chiralpak AD-H, 2 cm × 15 cm;
mobile phase, 75% CO2/25% MeOH w/0.2% DEA; flow rate,
80 mL/min; detection, 279 nm; injection volume, 0.5 mL;
dissolution, 5 mL of 1:1 DCM/MeOH. The product was
lyophilized from MeOH/H2O yielding a white powder (S)-5,5-
dimethyl-3-((1R,4S)-4-(8-oxo-7H-purin-9(8H)-yl)cyclohexyl)-
4-phenyloxazolidin-2-one (0.069 g, 0.169 mmol, 19.58% yield).
1H NMR (400 MHz, DMSO-d6) δ 11.20 (br s, 1 H), 8.56−8.48
(m, 1 H), 8.21 (s, 1 H), 7.51−7.41 (m, 2 H), 7.40−7.10 (m, 3
H), 4.68 (s, 1 H), 4.07 (tt, J = 3.6, 12.3 Hz, 1 H), 3.54−3.43
(m, 1 H), 2.41−2.17 (m, 2 H), 2.02−1.86 (m, 2 H), 1.79 (d, J
= 12.4 Hz, 1 H), 1.64 (d, J = 10.6 Hz, 2 H), 1.48 (s, 3 H),
1.35−1.17 (m, 1 H), 0.81 (s, 3 H). m/z (ESI) 408.0 (M + H)+,
HRMS calcd for C22H25N5O3 (M+H)+ 408.2035, found
408.2044.
To a flask charged with 3-(((1S,4R)-4-((S)-5,5-dimethyl-2-
oxo-4-phenyloxazolidin-3-yl)cyclohexyl)amino)isonicotinic acid
(0.128 g, 0.313 mmol) was added THF (1.563 mL),
triethylamine (0.061 mL, 0.438 mmol), and diphenyl
phosphoryl azide (0.094 mL, 0.438 mmol), respectively. The
mixture was heated and shaken at 80 °C overnight. The
resulting mixture was dried under reduced pressure and purified
with a 25g SNAP column, ramping DCM/MeOH (90:10) in
DCM (0−100%), providing product eluting at 100% polar
eluent which had coeluted with phosphitic acid side product
from DPPA (∼20%). Repurification with a HP spherical silica
column (25g, 15 μm spherical, Interchim) was only able to
partially separate the impurity (∼70 mg with ∼5% impurity by
LC-MS). The material was dissolved in DMSO/MeOH and
purified with Gilson RP-HPLC, ramping ACN in H2O (25−
80%, 0.1% TFA throughout).
Product eluents were directly free-based with a 5g SCX-2
column washing with MeOH and then 2 M NH3 in MeOH.
The basic wash was dried under reduced pressure, then
lyophilized from MeOH/H2O, yielding product as a white solid
(S)-5,5-dimethyl-3-((1R,4S)-4-(2-oxo-1H-imidazo[4,5-c]-
pyridin-3(2H)-yl)cyclohexyl)-4-phenyloxazolidin-2-one (0.015
g, 0.037 mmol, 11.81% yield). 1H NMR (500 MHz, DMSO-d6)
δ 11.43−11.22 (m, 1 H), 8.56 (s, 1 H), 8.13 (d, J = 5.3 Hz, 1
H), 7.51−7.06 (m, 5 H), 7.01 (d, J = 5.1 Hz, 1 H), 4.66 (s, 1
H), 4.06 (t, J = 12.1 Hz, 1 H), 3.76−3.65 (m, 1 H), 2.26−2.04
(m, 2 H), 2.03−1.85 (m, 2 H), 1.76 (d, J = 11.9 Hz, 1 H),
1.71−1.53 (m, 3 H), 1.48 (s, 2 H), 1.39−1.20 (m, 1 H), 0.80 (s,
3 H). m/z (ESI) 407.4 (M + H)+, HRMS calcd for
C23H26N4O3 (M + H)+ 407.2083, found 407.2089.
(S)-5,5-Dimethyl-3-((1R,4S)-4-(8-oxo-7H-purin-9(8H)-
yl)cyclohexyl)-4-phenyloxazolidin-2-one (51). To a vial
charged with 4-chloropyrimidin-5-amine (Frontier Scientific)
(0.500 g, 3.86 mmol) was added THF (6.43 mL), toluene (6.43
mL), triethylamine (1.345 mL, 9.65 mmol), and phthalic
anhydride (1.310 mL, 13.51 mmol). The mixture was heated to
100 °C for 72 h. The crude mixture was dried under reduced
pressure and purified with a 50g SNAP column (Biotage),
ramping EtOAc in heptane from 0 to 45%, then isocratic at
45% to elute product, obtained as a yellow solid 2-(4-
chloropyrimidin-5-yl)isoindoline-1,3-dione (0.773 g, 2.98
1
mmol, 77% yield). H NMR (400 MHz, DMSO-d6) δ 9.23
(d, J = 0.4 Hz, 1 H), 9.09 (d, J = 0.4 Hz, 1 H), 8.12−8.05 (m, 2
H), 8.04−7.96 (m, 2 H). m/z (ESI) 260.1 (M + H)+.
To a vial charged with 2-(4-chloropyrimidin-5-yl)isoindoline-
1,3-dione (0.327 g, 1.259 mmol) was added acetonitrile (4.20
mL), triethylamine (0.369 mL, 2.64 mmol), and 3-((1R,4R)-4-
aminocyclohexyl)-5,5-dimethyl-4-phenyloxazolidin-2-one 2,2,2-
trifluoroacetate (0.507 g, 1.259 mmol) (28 as a mixture of S:R
enantiomers ∼3:2), respectively. The vessel was sealed and
shaken overnight at 80 °C, providing a dark solution containing
product as the primary species according to LC-MS. The
solution was dried under reduced pressure and purified with a
40g HP spherical silica column (Interchim), ramping EtOAc in
heptane (0−100%, 5% DCM throughout), leading to isolation
of product as a mixture of trans/cis isomers with minor
impurities as a light yellow foam 2-(4-(((1R,4R)-4-(5,5-
ASSOCIATED CONTENT
■
S
* Supporting Information
Table S1 provides enzyme and cellular data including individual
standard deviation and n values. Figure S1 provides the IR
spectrum of 40, and Figure S2 and Table S2 provide the
proton/carbon NMR assigments for 40. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
4K4F (18) and 4K4E (52)
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Journal of Medicinal Chemistry
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(10) Chang, P.; Coughlin, M.; Mitchison, T. J. Interaction between
Poly(ADP-ribose) and NuMA contributes to mitotic spindle pole
assembly. Mol. Biol. Cell 2009, 20, 4575−4585.
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poly(ADP-ribose) polymerase at human telomeres. Science 1998, 282,
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(12) Huang, S. M.; Mishina, Y. M.; Liu, S.; Cheung, A.; Stegmeier, F.;
Michaud, G. A.; Charlat, O.; Wiellette, E.; Zhang, Y.; Wiessner, S.;
Hild, M.; Shi, X.; Wilson, C. J.; Mickanin, C.; Myer, V.; Fazal, A.;
Tomlinson, R.; Serluca, F.; Shao, W.; Cheng, H.; Shultz, M.; Rau, C.;
Schirle, M.; Schlegl, J.; Ghidelli, S.; Fawell, S.; Lu, C.; Curtis, D.;
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 617-444-5351. E-mail: hbregman@amgen.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Dhan Bagal, Matthew Potter,
Paul H. Krolikowski, and Shawn Ayube for analytical support,
and Dr. Margaret Y. Chu-Moyer for proofreading the
manuscript.
(13) Waaler, J.; Machon, O.; Tumova, L.; Dinh, H.; Korinek, V.;
Wilson, S. R.; Paulsen, J. E.; Pedersen, N. M.; Eide, T. J.; Machonova,
O.; Gradl, D.; Voronkov, A.; von Kries, J. P.; Krauss, S. A novel
tankyrase inhibitor decreases canonical Wnt signaling in colon
carcinoma cells and reduces tumor growth in conditional APC mutant
mice. Cancer Res. 2012, 72, 2822−2832.
(14) Chen, B.; Dodge, M. E.; Tang, W.; Lu, J.; Ma, Z.; Fan, C. W.;
Wei, S.; Hao, W.; Kilgore, J.; Williams, N. S.; Roth, M. G.; Amatruda, J.
F.; Chen, C.; Lum, L. Small molecule-mediated disruption of Wnt-
dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol.
2009, 5, 100−107.
(15) Lu, J.; Ma, Z.; Hsieh, J. C.; Fan, C. W.; Chen, B.; Longgood, J.
C.; Williams, N. S.; Amatruda, J. F.; Lum, L.; Chen, C. Structure-
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(16) See the Experimental Section for the preparation of 31 for the
synthetic details of 7-fluoro-1,5-naphthyridine-4-carboxylic acid.
(17) For cyano-substituted substrates, Raney nickel was avoided in
favor of tin chloride to mitigate over-reduction.
ABBREVIATIONS USED
■
TNKS, tankyrase; PARP, poly(ADP-ribose) polymerase; CRC,
colorectal cancer; APC, adenomatous polyposis coli; AXIN2,
axis inhibition protein 2; CTNNB1, gene which encodes β-
catenin; GSK3β, glycogen synthase kinase 3β; CK1α, casein
kinase 1α; TCF, T-cell specific transcription factor; TRF1,
telomere repeat binding factor 1; HATU, O-(7-azabenzotriazol-
1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
DIEA, N-ethyl-N-isopropylpropan-2-amine; DMEDA, N1,N2-
dimethylethane-1,2-diamine; DEA, diethylamine; MP-BH4,
macroporous borohydride; ACN, acetonitrile; SFC, super-
critical fluid; SCX-2, strong cation exchange prepacked resin
(propylsulfonic acid functionalized); DPPA, diphenyl phos-
phoryl azide; T3P, 1-propanephosphonic anhydride; SNAR,
nucleophilic aromatic substitution; CDI, 1,1′-carbonyldiimida-
zole
(18) The absolute stereochemistry was established on the basis of the
knowledge that only partial racemization occurred in the synthetic
sequence toward common intermediate 28. We began with the (S),
and therefore, the major enantiomer must be the (S) enantiomer.
Some analogues were prepared using enantiopure 28, and some were
separated at the end of the synthetic sequence.
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