Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01651

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inbibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

Full text of DOI:10.1021/acs.jmedchem.5b01651

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Article
Discovery, Optimization, and in vivo Evaluation of Benzimidazole
Derivatives AM-8508 and AM-9635 as Potent and Selective PI3K# Inhibitors
Youngsook Shin, Julia Suchomel, Mario Cardozo, Jason Duquette, Xiao He, Kirk Henne, Yi-Ling
Hu, Ron C. Kelly, John D McCarter, Lawrence R McGee, Julio C. Medina, Daniela Metz, Tisha San
Miguel, Deanna Mohn, Thuy Tran, Christine Vissinga, Simon Wong, Sharon Wannberg, Douglas A.
Whittington, John Whoriskey, Gang Yu, Leeanne Zalameda, Xuxia Zhang, and Timothy D. Cushing
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01651 • Publication Date (Web): 10 Dec 2015
Downloaded from http://pubs.acs.org on December 14, 2015
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Discovery, Optimization, and in vivo Evaluation of
Benzimidazole Derivatives AM-8508 and AM-9635 as
Potent and Selective PI3Kδ Inhibitors
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Youngsook Shin,*,† Julia Suchomel,† Mario Cardozo,† Jason Duquette,† Xiao He,† Kirk Henne,§ Yiꢀ
Ling Hu,# Ron C. Kelly,⊥ John McCarter,‡ Lawrence R. McGee,† Julio C. Medina,† Daniela Metz,#
Tisha San Miguel,‡ Deanna Mohn,# Thuy Tran,§ Christine Vissinga,# Simon Wong,§ Sharon Wannberg,#
Douglas A. Whittington, ǁ John Whoriskey,# Gang Yu,# Leeanne Zalameda,‡ Xuxia Zhang,# and Timothy
D. Cushing†
†Department of Therapeutic Discovery, §Department of Pharmacokinetics and Drug Metabolism, and
⊥Drug Product Technologies, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California
94080, United States
‡Department of Therapeutic Discovery, and #Department of Inflammation Research, Amgen Inc., One
Amgen Center Drive, Thousand Oaks, California 91320, United States
ǁDepartment of Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts
02142, United States
ABSTRACT
Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable
PI3Kδ inhibitors, 1 (AMꢀ8508) and 2 (AMꢀ9635) with good pharmacokinetic properties. The comꢀ
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pounds inhibit B cell receptor (BCR)ꢀmediated AKT phosphorylation (pAKT) in PI3Kδꢀdependent in
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vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when adꢀ
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ministered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence
of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds
demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of
PI3Kδ activity by inbibitors 1 and 2 led to effective inhibition of antigenꢀspecific IgG and IgM forꢀ
mation after immunization with KLH.
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INTRODUCTION
Phosphoinositide 3ꢀkinases (PI3Ks) belong to a large family of lipid kinases that regulate numerous bioꢀ
logical functions by generating lipid second messengers.1 PI3Ks utilize ATP to phosphorylate the 3ꢀOH
of the inositol ring moiety, converting the phosphatidylinositol 4,5ꢀbisphosphate (PIP2) to phosphatidylꢀ
inositol 3,4,5ꢀtrisphosphate (PIP3).2 PIP3 induces AKT phosphorylation, which acts as a secondary
messenger in the control of a wide number of cellular functions including metabolism, cell growth and
motility.3 PI3Ks are divided into three classes (I, II, and III) based on functional and sequence homoloꢀ
gy.4,1 Class I PI3Ks are further divided into subclasses IA and IB based on their signaling pathways
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and the regulatory proteins to which they bind. The class IA isoforms PI3Kα, PI3Kβ, and PI3Kδ, are
primarily activated by protein tyrosine kinaseꢀcoupled receptors,5 whereas the class IB member PI3Kγ is
activated by Gꢀprotein coupled receptors (GPCRs), such as the chemokine receptors.6 PI3Kα and
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PI3Kβ are ubiquitously expressed and play a role in cell growth, division and survival.7 The expression
pattern of PI3Kδ and PI3Kγ is more restricted, with both isoforms found primarily in leukocytes.8
PI3Kδ and PI3Kγ have been identified as promising therapeutic targets for the treatment of immune
cellꢀmediated diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and heꢀ
matological malignancies.9 Given these findings, PI3Kδ or PI3Kγ selective inhibitors and dual inhibiꢀ
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tors of PI3Kβ/δ and PI3Kδ/γ have been utilized in research both to understand the biological roles of
PI3Ks, and as potential drugs for the treatment of inflammatory disorders.10 We recently disclosed the
discovery of dual PI3Kβ/δ inhibitors10c, 11 and PI3Kδ isoform selective inhibitors with a diverse range of
core moieties, including pyrazolopyrimidine,12 quinoline and quinoxaline,13 reversed quinoline,14 4ꢀ
substitutedꢀ3ꢀlinkedꢀquinoline and quinoxaline,15 naphthyridine,16 pyridopyrimidinone,17 benzimidazꢀ
ole,18 imidazopyridine,18 and thienylpyridine.19 Among these, AMG 31920 has advanced into clinical
development for the treatment of lymphoid malignancies and AMG 35721 has similarly advanced for the
treatment of inflammatory disease. Herein, we communicate the discovery and optimization of a unique
series18 of PI3Kδ selective inhibitors and their potential application in the treatment of inflammatory
disease.
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O
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N
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3
PI3Kαa IC50 = 4610 nM
PI3Kβa IC50 = 78 nM
PI3Kδa IC50 = 20 nM
PI3Kγa IC50 = 825 nM
B Cellb IC50 = 9 nM
pAKTc IC50 = 36.2 nM
Figure 1. Crystal structure of PI3Kγ in complex with 3 (PDB code 4FJZ). Amino acid labels correspond to the PI3Kγ isoform. Dashed
lines indicate hydrogen bonds. aBiochemical: Alphascreen assay. bCellular: In vitro antiꢀIgM/CD40Lꢀinduced B cell proliferation (as
measured by thymidine incorporation) assay. cPI3Kβ counterscreen assay: phosphorylation of AKT in MDAꢀMBꢀ468 cells.
RESULTS AND DISCUSSION
Earlier reports from our PI3Kδ program have demonstrated that the dual PI3Kβ/δ inhibitor 3 was well
tolerated and efficacious in animal models of inflammation.10c A crystal structure of 3 in complex with
PI3Kγ indicates that the compound adopts a propellerꢀlike conformation in which the quinoline and
hexahydrospiro[pyranꢀ4,3'ꢀpyrrolo[3,2ꢀb]pyridine] ring are at an angle of approximately 90° (Figure 1).
Previous reports have stated that similar propellerꢀshaped inhibitors induce a conformational change in
the ATPꢀbinding pocket where a methionine (Met804) is displaced to accommodate the quinoline ring
in a newlyꢀformed hydrophobic pocket.22 This ligandꢀinduced conformational change is critical for
achieving the selectivity of 3 for PI3K β/δ over other protein kinases.
In an attempt to optimize 3 as a PI3Kδ selective inhibitor, alternative hinge binders and bicyclic hetꢀ
eroaryl moieties were further explored and have been described in previous reports.16ꢀ19 These studies
identified a 4ꢀaminopyrimidineꢀ5ꢀcarbonitrile moiety as an alternative hinge binder that favorably interꢀ
acts with Val882 that shows potency and selectivity similar to the established morpholine and purine
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analogs.17,14c Analogs that contain this promising hinge binder along with quinoline and quinoxaline
core moieties were designed,13c and as predicted, the compounds showed good PI3Kδ potency and imꢀ
proved selectivity over other PI3K isoforms when compared to compound 3.
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As part of our medicinal chemistry efforts towards the identification of inhibitors of the CXCR3 recepꢀ
tor, we showed that fiveꢀsix fused heterocyclic systems, such as benzimidazole and imidazopyridine,
could be suitable replacements of the sixꢀsix fused heterocycles.23 It was therefore hypothesized that
these heterocyclic moieties could serve as isosteres of the quinoline and quinoxaline heterocycles within
the context of the PI3K program.
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Early SAR
To test this hypothesis, the benzimidazole analog 5 was first synthesized and tested in an in vitro Alꢀ
phascreen biochemical assay (Table 1). Gratifyingly, it was found that this analog with the aminopyꢀ
rimidine as a hinge binder exhibited good inhibition of the PI3Kδ enzyme as well as promising selecꢀ
tivity over other PI3K isoforms, especially the PI3Kβ isoform and the replacement of quinoline bicycle
by the benzimidazole isostere substantially improved selectivity over the PI3Kγ isoform compared to
compound 3. Consistent with our previous results, the Rꢀenantiomers, 4 and 6, were much less potent
towards PI3Kδ than their corresponding Sꢀenantiomers, 5 and 7.20b This experimental result can be raꢀ
tionalized by invoking a steric clash between the methyl group of Rꢀenantiomers and the protein, which
is not present in the corresponding Sꢀenantiomers. This initial optimization work also revealed that the
6ꢀfluoro analogs, 4 and 5, were more potent than the corresponding 5ꢀfluoro analogs 6 and 7, a trend
that continued throughout the series.
Table 1. SAR of benzimidazole analogs.
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Biochemical potencya IC50 (ꢀM)
Selectivity
PI3Kα/
PI3Kδ
PI3Kβ/
PI3Kδ
PI3Kγ/
PI3Kδ
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cmpd
R
X
PI3Kα
PI3Kβ
PI3Kδ
PI3Kγ
3
4
5
6
7
4.61 ± 5.46
49.6
0.0786 ± 0.0917
0.0202 ± 0.0139
18.3
0.825 ± 0.698
29.5
230
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42
2
RꢀMe
SꢀMe
RꢀMe
SꢀMe
6ꢀF
6ꢀF
5ꢀF
5ꢀF
>125
3.58
>125
31.8
>7
52
>125
0.069
2.88
>1800
42
>125
>125
>125
33.2
0.1760 ± 0.0732
14.0 ± 9.16
189
181
79
aBiochemical: Alphascreen assay.
The exceptional selectivity of 5 over other PI3K isoforms can be explained by examining the Xꢀray
crystal structure of the compound in complex with PI3Kγ (Figure 2). The aminopyrimidine ring interꢀ
acts with the Val882 residue in the hinge region of PI3Kγ and the nitrile is projected into the affinity
pocket. The benzimidazole ring occupies the specificity pocket and adopts the desired propellerꢀlike
conformation in which the aminopyrimidine and benzimidazole rings are at an angle of approximately
90°, a conformation that has been shown previously to result in PI3Kδꢀselective inhibitors.22b The 3ꢀ
methylsulfonylꢀphenyl ring sits in the ribose pocket, orthogonal to the benzimidazole ring. Having
demonstrated the potential of the benzimidazole moiety as a promising isostere of the quinoline, subseꢀ
quent optimization efforts were pursued to further improve upon the lead compound 5.
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Affinity
Pocket
NH2
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Ribose
Pocket
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S
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5
αa
ꢀ
PI3K IC50 = >125
M
PI3Kβa IC50 = 3.58 ꢀM
PI3Kδa IC50 = 0.069 ꢀM
PI3Kγa IC50 = 2.88 ꢀM
Figure 2. Crystal structure of PI3Kγ in complex with 5 (PDB code 5EDS). Amino acid labels correspond to the PI3Kγ isoform; blue labels
indicate the corresponding residue in PI3Kδ. Dashed lines indicate hydrogen bonds. aBiochemical: Alphascreen assay.
Chemistry
A general synthetic route for the benzimidazole analogs of 5 is described in Scheme 1. Nucleophilic
aromatic substitution of 2,4ꢀdifluoroꢀ1ꢀnitrobenzene (8) with the relevant anilines was followed by reꢀ
duction of the nitro group in 9a-g using tin chloride dihydrate to provide 10a-g. Coupling of 10a-c,e-g
with BocꢀLꢀalanine afforded 11a-c,e-g which then underwent cyclodehydration in refluxing acetic acid.
During the cyclodehydration, the Bocꢀprotecting group was cleaved and subsequently acylated in situ to
provide benzimidazole intermediate 12a-c,e-g. Epimerization was later found to occur at the stereogenꢀ
ic carbon attached to the methyl group. Alternatively, 10d was coupled to FmocꢀLꢀalanine to give 11d,
which was cyclodehydrated in refluxing acetic acid to produce 12d.
2ꢀ(Methylthio)phenyl analog 12b was oxidized to the sulfone analog 12b’. Deacylation of the Nꢀ
acetates 12a-c, e-g with hydrochloric acid afforded 13a-c, e-g. The Fmoc group on 12d was removed by
piperidine to provide 13d. Treatment of 13a-g with 4ꢀaminoꢀ6ꢀchloropyrimidineꢀ5ꢀcarbonitrile afforded
the racemic coupling products 14a-g, which were then subjected to chiral separation to provide 5, 15-18,
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1, and 2. This route was designed as a regioꢀand stereoꢀselective synthetic route; however the stereoseꢀ
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lectivity was not retained due to the epimerization during cyclodehydration.
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Scheme 1a
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aReagents and conditions: (i) Aniline, 130 oC; (ii) SnCl2 ·2H2O, EtOAc, 90 °C; (iii) Nꢀmethylmorpholine, isobutyl chloroformate,
BocꢀLꢀAlaꢀOH, DCM (for 11d, FmocꢀLꢀAlaꢀOH), ꢀ10 °C; (iv) AcOH, 100 °C; (v) Oxone, THFꢀwater (3:1), rt; (vi) 2 N HCl, 100
°C; (vii) piperidine, DCM, rt; (viii) 4ꢀaminoꢀ6ꢀchloropyrimidineꢀ5ꢀcarbonitrile, DIPEA, microwave, nꢀbutanol, 120 °C; (ix) chiral
SFC chromatography.
A second synthetic route solved the stereoselectivity issue, but the regioselectivity was not addressed
(Scheme 2). Cyclodehydration of BocꢀLꢀAlaꢀOH and 19 afforded 20, which was then coupled with the
relevant boronic acids to give intermediates 21a-b as a mixture of regioisomers. The Bocꢀprotecting
group on 21a-b was cleaved with TFA to provide the amines 22a-b. Coupling of 20 with 2ꢀ
fluoropyridine at 150 °C afforded deprotected amine 22c. Treatment of 22a-c with 4ꢀaminoꢀ6ꢀ
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chloropyrimidineꢀ5ꢀcarbonitrile afforded a mixture of 5 and 6ꢀ fluorosubstituted benzimidazoles 23a-c,
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5
which were then separated by SFC purification to give 6ꢀfluorobenzimidazoles 24-26.
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Scheme 2a
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aReagents and conditions: (i) BocꢀLꢀAlaꢀOH, EDC·HCl, pyridine, rt; (ii) RB(OH)2, Cu (OAc)2, 2,2’ꢀbipyridyl, Cs2CO3, dichloroꢀ
ethane, 70 oC; (iii) TFA, DCM, rt; (iv) 2ꢀfluoropyridine, DMA, Cs2CO3, 150 oC; (v) 4ꢀaminoꢀ6ꢀchloropyrimidineꢀ5ꢀcarbonitrile,
DIPEA, microwave, nꢀbutanol, 120 °C; (vi) SFC separation.
Scheme 3a
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aReagents and conditions: (i) 2ꢀaminoꢀ5ꢀfluoropyridine, EtOH, reflux; (ii) NIS, CH3CN, rt; (iii) RB(OH)2, Pd(PPh3)4, Na2CO3,
CH3CNꢀwater (3:1), 100 °C or RSnBu3, Pd(PPh3)4, toluene, 110 °C; (iv) Oxone, THFꢀwater (4:1), rt; (v) DMS, TFA, rt; (vi) 4ꢀ
aminoꢀ6ꢀchloropyrimidineꢀ5ꢀcarbonitrile, DIPEA, nꢀbutanol, 110 °C.
The regioꢀ and stereoselective synthetic route employed for the synthesis of the imidazopyridine series
is shown in Scheme 3. Condensation of 27 with 2ꢀaminoꢀ5ꢀfluoropyridine afforded the protected imidꢀ
azopyridine 28, which was subsequently iodinated with NIS to give 29. Palladiumꢀcatalyzed coupling
of 29 with the relevant boronic acids or esters afforded 30a-h. 2ꢀ(Methylthio)phenyl analog 30g was
oxidized to the sulfone 30g’. The Cbzꢀprotecting group on 30a-h was cleaved with dimethyl sulfide and
TFA to provide 31a-h. Nucleophilic displacement of 4ꢀaminoꢀ6ꢀchloropyrimidineꢀ5ꢀcarbonitrile with
the amine 31a-h generated the desired imidazopyridine analogs 32-39.
Table 2. SAR: Potency and selectivity of analogs at 1-position of benzimidazole
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Mouse B
Human B
cell
HWB
HWB
HWB_unbound
(pAKT)f
Biochemical potencya IC50 (ꢀM)
cellb
(CD69)d
(pAKT)e
(pAKT)c
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cmpd
5
R
IC50 (nM)
PI3Kα
>125
55.9
8.6
PI3Kβ
3.58
8.96
0.51
0.45
0.54
0.03
1.08
3.53
1.78
2.33
PI3Kδ
0.069
PI3Kγ
2.9
IC50 (ꢀM)
0.0291
0.25
IC50 (ꢀM)
IC50 (ꢀM)
IC50 (ꢀM)
3ꢀSO2Meꢀphenyl
2ꢀSO2Meꢀphenyl
3ꢀFꢀphenyl
4ꢀFꢀphenyl
3,5ꢀdiꢀFꢀphenyl
cyclopropyl
phenyl
ꢀ
ꢀ
15
16
17
18
24
25
26
1
0.107
61.9
0.118
0.191
0.27
0.144
1.4
ꢀ
ꢀ
0.0008
0.0036
0.0004
0.0019
0.0022
0.012
0.0083
0.0573
0.0044
ꢀ
ꢀ
ꢀ
ꢀ
30.6
>125
2.5
ꢀ
0.0045
ꢀ
0.019
ꢀ
0.084
10.1
40.4
29.4
58.2
27.2
0.0019
0.016
0.0025
ꢀ
0.022
0.107
ꢀ
0.032
0.131
2.4
35.4
1.0
2ꢀpyridyl
12.6
5.8
3ꢀpyridyl
0.016
0.0064
0.0065
0.0046
0.0042
0.0027
0.0191
2
5ꢀFꢀ3ꢀpyridyl
0.019
5.9
0.0195
5.7
aBiochemical: Alphascreen assay. bCellular: In vitro antiꢀIgM/CD40Lꢀinduced human B cell proliferation (as measured by thymidine incorporation)
assay. cCellular: Ability of compound to inhibit antiꢀIgM induced AKT phosphorylation (pAKTSer473) in mouse B cells; phosphoꢀAKT (pAKT) exꢀ
pression on B220+gated B cells was determined by flow cytometry in mouse splenocytes. dCellular: Compound pretreated human whole blood
(HWB) was stimulated with antiꢀIgD to induce CDꢀ69 expression on B cells (6 hours) and was evaluated by flow cytometry. eCompound pretreated
human whole blood (HWB) was stimulated with antiꢀIgD to induce phosphorylation of AKT (pAKTSer473). fThe unbound human whole blood
(HWB_unbound) potency was derived by multiplying the human plasma protein binding (PPB) fraction unbound by the total HWB potency ( fu ×
HWB). fu values are found in Table 4. gSee Supporting Information for standard deviations.
SAR
Attempts to optimize the benzimidazole series were focused on modification of the 3ꢀ
methylsulfonylphenyl group in 5 (Table 2). Introduction of a 2ꢀsulfonylmethyl group (15) decreases
PI3Kδ activity (<2ꢀfold). Subsequent replacement of 3ꢀsulfonylmethyl group in 5 with a fluorine atom
in 16 led to substantial improvement of both biochemical potency and cellular potency, while maintainꢀ
ing good selectivity over other PI3K isoforms (>10,000ꢀfold over PI3Kα, 643ꢀfold over PI3Kβ, and
148ꢀfold over PI3Kγ). 4ꢀFluorophenyl 17 exhibited good biochemical potency and reasonably good seꢀ
lectivity (8500ꢀfold over PI3Kα, 126ꢀfold over PI3Kβ, and 53ꢀfold over PI3Kγ), but showed reduced
cellular potency. 3,5ꢀDifluorophenyl 18 was very potent in both biochemical and cellular assays and
showed great selectivity over other PI3K isoforms (>312,500ꢀfold over PI3Kα, 1343ꢀfold over PI3Kβ,
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and 675ꢀfold over PI3Kγ). Cyclopropyl analog 24 showed good biochemical potency, but the selectivity
2
3
4
5
6
7
8
9
over PI3Kβ dropped (1337ꢀfold over PI3Kα, 15ꢀfold over PI3Kβ, and 76ꢀfold over PI3Kγ). Unsubstiꢀ
tuted phenyl analog 25 was potent in both biochemical and cellular assays and showed good selectivity
(18,364ꢀfold over PI3Kα, 491ꢀfold over PI3Kβ, and 614ꢀfold over PI3Kγ). As we observed in an earlier
report,10c phenyl 25 and substituted phenyl analogs 5, 15-18 significantly improved selectivity over othꢀ
er PI3K isoforms when compared to the cyclopropyl analog 24. A 2ꢀpyridyl analog 26 was well toleratꢀ
ed, showing reasonable biochemical potency, cellular potency, and good selectivity (2450ꢀfold over
PI3Kα, 294ꢀfold over PI3Kβ, and 1050ꢀfold over PI3Kγ). 3ꢀPyridyl analog 1 and 5ꢀfluoroꢀ3ꢀpyridyl
analog 2 were also well tolerated, and demonstrated a high level of potency, and selectivity over other
PI3K isoforms (3638ꢀfold and 1432ꢀfold over PI3Kα, 111ꢀfold and 123ꢀfold over PI3Kβ, and 363ꢀfold
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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42
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45
46
47
48
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50
51
52
53
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55
56
57
58
59
60
and 312ꢀfold over PI3Kγ, respectively). Both 1 and 2 showed excellent kinase selectivity (KIꢀ
NOMEscan's selectivity score, SꢀScore(35) = 0) in a large panel of 442 protein kinases tested at 10 ꢁM
drug concentration.24 The analogs 18, 25, 26, 1, and 2 that showed good cellular potency in B cell proꢀ
liferation assay were further profiled in additional cellular assays, including antiꢀIgMꢀinduced phosꢀ
phorylation of AKT in mouse B cells (pAKTSer473), antiꢀIgDꢀinduced CDꢀ69 expression on B cells in
90% human whole blood [HWB (CD69)], and antiꢀIgDꢀinduced phosphorylation of AKT (pAKTSer473
)
in 90% human whole blood [HWB (pAKT)]. 18, 25, 1, and 2 showed good cellular potency (in vitro
pAKT IC50 = 4.5 nM, 2.5 nM, 4.6 nM, and 4.2 nM, respectively), which were similar to potency in the
B cell proliferation assay. 18, 25, 1, and 2 were potent in HWB (CD69 and pAKT) assays, but 26
showed weaker HWB cellular potency compared to other analogs. In particular, 1 exhibited excellent
HWB potency (HWB (pAKT) IC50 = 2.7 nM).
An alternative series of imidazopyridines was also explored (Table 3). While imidazopyridine analogs
showed promising PI3Kδ enzyme potencies and increased selectivities over other isoforms, they were
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consistently less potent, in both biochemical and cellular assays, compared to their benzimidazole counꢀ
2
3
4
5
terparts.
6
7
Table 3. SAR of Imidazopyridine substitution
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B Cellb
Biochemical potencya IC50 (ꢀM)
cmpd
32
R
PI3Kα
46.1
PI3Kβ
10.40 ± 4.46
24.7
PI3Kδ
0.103 ± 0.144
0.612
PI3Kγ
7.92 ± 3.08
82.5
IC50 (ꢀM)
0.103 ± 0.0872
ꢀ
2ꢀpyridyl
33
3ꢀpyridyl
>125
>125
>125
>125
>125
>125
>125
34
5ꢀFꢀ3ꢀpyridyl
phenyl
18.1 ± 13.4
18.7
0.57 ± 0.68
0.109
24.8 ± 18.6
27.5
0.0355
0.0576
0.0905
0.036
35
36
4ꢀFꢀphenyl
3,5ꢀdiꢀFꢀphenyl
2ꢀSO2Meꢀphenyl
3ꢀSO2Meꢀphenyl
5.93
0.017
1.42
37
14.10 ± 5.44
30.2
0.0228 ± 0.00956
0.08
3.550 ± 0.988
63.4
38
0.282
39
5.51
0.113
3.34
0.142
aBiochemical: Alphascreen assay. bCellular: In vitro antiꢀIgM/CD40Lꢀinduced B cell proliferation (as measured by thymidine incorporation)
assay.
In Vivo PK
The pharmacokinetic profiles of several promising analogs were evaluated and despite their desirable
potency and selectivity, the phenyl and substituted phenyl analogs (5, 16, 17, 18, and 25) were found
unsuitable for advancement due to a poor PK profile, namely undesirable clearance in the rat (Table 4).
Alternatively, it was observed that replacing the phenyl moiety with a pyridine ring, as with 26, 1, and
2, resulted in compounds with good total clearance and good oral bioavailability (F = 58%, 45%, and
41%, respectively). In fact, the unbound clearance of these pyridyl analogs was around 10ꢀfold lower
than the phenyl analogs. All analogs showed good solubility and did not inhibit CYP3A4.
Table 4. Physicochemical Properties of the Benzimidazole leads
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2
3
4
5
6
7
8
9
Microsomal Stabilityc
(%Turnover)
Protein Binding
(fu)
humand
Rat PKf
IVg
Solubilitya
CYP3A4b
POl
%F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CL
(L/h/Kg)
CLuk
(L/h/Kg)
cmpd
R
(% inhibition)
human
rat
rate
(ꢀM)
5
3ꢀSO2Meꢀphenyl
3ꢀFꢀphenyl
4ꢀFꢀphenyl
3,5ꢀdiꢀFꢀphenyl
Phenyl
500
401
353
305
374
500
474
500
20
15
<10
15
<10
28
0.40
0.12
0.11
0.12
0.07
0.27
0.36
0.30
0.35
0.10
0.10
0.13
0.09
0.22
0.36
0.32
4.3h
2.76i
1.78
2.64
2.41
0.97
0.93j
0.99
12.3
ꢀ
ꢀ
16
17
18
25
26
1
27.6
18.0
20.3
26.8
4.4
<10
16
<10
11
22
ꢀ
30
ꢀ
<10
<10
<10
<10
<10
<10
<10
<10
26
ꢀ
2ꢀpyridyl
<10
<10
<10
58
45
41
3ꢀpyridyl
2.6
2
5ꢀFꢀ3ꢀpyridyl
3.1
aSingle experimental values. Aqueous equilibrium solubility assay: compound was equilibrated at room temperature for 72 hours in PBS buffer (pH
7.4), supernatant was centrifuged and analyzed by HPLC for reporting solubility as ꢀM. bCYP: cytochrome P450 assay competitive (midazolam as
probe substrate, 5 ꢀM) reported as % inhibition at 3 ꢀM (LC/MS). cSingle experimental values. %Turnover was measured by LC/MS after incubation
of parent compound (1 ꢀM) in liver microsome (0.25 mg/mL) in potassium phosphate (66.7 mM) buffered with NADPH (1 mM) at 37 oC for 30 min.
d5 ꢀM concentration in human plasma, protein binding measured by ultracentrifugation and LC/MS. e5 ꢀM concentration in rat plasma, protein bindꢀ
ing measured by ultracentrifugation and LC/MS. fPharmacokinetic parameters following administration in male SpragueꢀDawley rats: two animals per
study for IV and three animals per study for PO. gDosed at 0.5 mg/kg as a solution in 100% DMSO. hDose IV, 0.8 mg/kg. iDose IV, 0.72 mg/kg. jDose
IV, 0.71 mg/kg. kUnbound clearance equals the total clearance divided by the rat fraction unbound (CLu = CL/fu). lDosed at 2.0 mg/kg as a suspension
in 1% Tween 80, 0.5% methylcellulose, 98.5% water.
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Figure 3. Inhibition of activation induced AKT phosphorylation in B cells in vivo. Vehicle, 1 (AM-8508), 2 (AM-9635), and benchmark
compound (Dꢀ073)25 in 2%HPMC+1%Tween80, pH 2.0, was administered to IgMm mice orally 15 minutes before intravenous (IV) injecꢀ
tion of FITCꢀlabeled antiꢀIgM to stimulate B cells. Mice were sacrificed 30 minutes after stimulation to measure pAKT in the spleen and
whole blood of FITCꢀIgM positive B cells by flow cytometry. (a) pAKT of 1 (AM-8508) in whole blood. (b) pAKT of 1 (AM-8508) in
splenocytes. (c) pAKT of 2 (AM-9635) in whole blood. (d) pAKT of 2 (AM-9635) in splenocytes. tꢀTest was used to evaluate differences
between groups. The asterisk (*) denotes statistical significance (p < 0.05) when compared to the control group.
In Vivo Pharmacology
Given the potency, selectivity, and pharmacokinetic profile of 1 and 2, these compounds were advanced
for evaluation in our in vivo animal models of inflammation. To establish a correlation between the in
vitro and in vivo activity of 1 and 2, a singleꢀdose in vivo assay was established that simulated the in
vitro antiꢀIgM stimulated pAKT assay. In this assay, the murine B cellꢀtransgenic line 3751, which has
only surfaceꢀbound IgM and lacks secreted/circulating IgM was employed (IgMmembrane only; IgMm).26
These transgenic mice were used to avoid IgM/antiꢀIgM immune complex formation that could promote
serum sickness in normal mice. Compound 1 or 2 was administered to IgMm mice orally 15 minutes
before intravenous (IV) injection of FITCꢀlabeled antiꢀIgM to stimulate B cells. Mice were sacrificed
30 minutes after stimulation to measure pAKT in the spleen and whole blood of FITCꢀIgM positive B
cells by flow cytometry (Figure 3). 1 and 2 showed doseꢀdependent inhibition of AKT phosphorylation
in antiꢀIgM stimulated B cells isolated from the whole blood and spleen compartments of IgMm mice.
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The calculated ED50 values of 1 in whole blood and spleen were <0.03 mg/kg (Figure 3a and Figure 3b).
The calculated ED50 values of 2 in mouse whole blood and spleen were 0.16 mg/kg and 0.027 mg/kg,
respectively (Figure 3c and Figure 3d). Data from the in vivo pAKT assay was subsequently utilized to
select doses for the multiꢀdose rat proteinꢀantigen (Keyhole Limpet Hemocyanin, KLH) studies. A mulꢀ
tiple dose rat study was performed to determine whether 1 and 2 impacted B cell function in vivo. For
this purpose, the effect of 1 on a humoral immune response was studied at oral doses of 0.003, 0.01,
0.03, 0.1, and 0.3 mg/kg per day and the effect of 2 on a humoral immune response was studied at oral
doses of 0.01, 0.03, 0.1, 0.3, and 1 mg/kg per day to determine whether the compounds inhibit antigenꢀ
specific Ig secretion by B cells. 1 inhibited KLHꢀspecific IgG and IgM in a doseꢀdependent manner
(Figure 4a and Figure 4b).a The calculated ED50 values were 0.08 mg/kg and 0.12 mg/kg, respectively.
Exposure at these doses would be expected to significantly inhibit PI3Kδ given the free drug concentraꢀ
2
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tions achieved relative to the HWB_unbound pAKT IC50 (1.0 nM) and PI3Kδ in vitro mouse
pAKT_unbound IC50 (3.1 nM) over the 24 hours dosing interval (Figure 4c). 2 inhibited KLHꢀspecific
IgG and IgM in a doseꢀdependent manner (Figure 5a and Figure 5b).b The calculated ED50 values were
0.11 mg/kg and 0.04 mg/kg, respectively. Exposure at these doses would be expected to significantly
inhibit PI3Kδ given the free drug concentrations achieved relative to the HWB_unbound pAKT IC50
(5.7 nM) and PI3Kδ in vitro mouse pAKT_unbound IC50 (3.2 nM) over the 24 hours dosing interval
(Figure 5c). Exposure at all doses of 1 and 2 were below PI3Kβ in vitro human pAKT_unbound IC50
(650 nM and 1928 nM, respectively) over a 24 hours dosing interval (Figure 4c and Figure 5c). Both
compounds 1 and 2 were well tolerated at all doses and exhibited significantly reduced IgG and IgM
a The effect on IgM was not statistically significant.
b The effect on IgM was only statistically significant at the 1 mg/kg dose.
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specific antibodies (Figure 4 and Figure 5). The KLHꢀmediated humoral immune response allowed us
2
3
4
5
to select 1 and 2 as candidates for further animal disease studies.
6
7
8
CONCLUSION
9
10
11
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18
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23
24
25
26
27
28
29
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A novel series of potent and selective PI3Kδ inhibitors were designed by introducing an optimized 4ꢀ
aminopyrimidineꢀ5ꢀcarbonitrile moiety as a hinge binder on a benzimidazole ring in the specificity
pocket. The alternative hinge binder 4ꢀaminopyrimidineꢀ5ꢀcarbonitrile moiety substantially improved
selectivity over the PI3Kβ isoform. The replacement of the quinoline ring to benzimidazole ring signifꢀ
icantly improved selectivity over the PI3Kγ isoform. The 90° angle conformation of the aminopyrimiꢀ
dine and benzimidazole rings resulted in excellent kinase selectivity. SAR efforts led to the optimized
compounds 1 (AMꢀ8508) and 2 (AMꢀ9635). Notably, analogs 1 and 2 had excellent PI3Kδ potency and
selectivity over other PI3K isoforms and protein kinases, as well as acceptable PK properties, which
made them suitable for evaluation in in vivo efficacy studies. These experiments demonstrated that both
1 and 2 could inhibit KLHꢀspecific antibodies in animal models, signifying their potential for the treatꢀ
ment of human inflammatory diseases.
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Figure 4. Inhibition of KLHꢀspecific antibodies and deltaꢀspecific coverage. Vehicle or 1 (AM-8508) in 2% HPMC, 1% Pluronic F68,
10% Captisol, pH 2.0, was administered (0.003, 0.01, 0.03, 0.1, and 0.3 mg/kg) q.d. po for 10 days in female Lewis rats (N = 8/dose
group). 2 hours after the first dosing, 200 ꢁL of PBS containing 60 ꢁg of KLH was administered to each rat intravenously. Ten days after
the KLH priming, blood was collected for the measurement of KLH specific IgG (a) and IgM (b) by ELISA. The yꢀaxis is represented as a
mean serum dilution factor. Error bars represent the standard error of the mean (SEM) of eight rats. (c) After administration of 1 (AM-
8508), plasma was also harvested at day 10 to assess exposures in each dose group. Unbound drug concentrations were measured by
LC−MS/MS and plotted relative to HWB_unbound pAKT IC50 = 1.0 nM (in Table 2) represented as a blueꢀdotted line, PI3Kδ in vitro
mouse pAKT_unbound IC50 3.1 nM [calculated from 4.6 nM (PI3Kδ in vitro mouse pAKT IC50) × 0.674 (fu in pAKT assay media)] repreꢀ
sented as a blue line, and PI3Kβ in vitro human pAKT_unbound IC50 650 nM [calculated from 964 nM (PI3Kβ in vitro human pAKT IC50)
× 0.674 (fu in pAKT assay media)] represented as a red line.
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Figure 5. Inhibition of KLHꢀspecific antibodies and deltaꢀspecific coverage. Vehicle or 2 (AM-9635) in 2% HPMC, 1% Pluronic F68,
10% Captisol, pH 2.0, was administered (0.01, 0.03, 0.1, 0.3, and 1 mg/kg) q.d. po for 10 days in female Lewis rats (N = 8/dose group). 2
hours after the first dosing, 200 ꢁL of PBS containing 60 ꢁg of KLH was administered to each rat intravenously. Ten days after the KLH
priming, blood was collected for the measurement of KLH specific IgG (a) and IgM (b) by ELISA. The yꢀaxis is represented as a mean
serum dilution factor. Error bars represent the standard error of the mean (SEM) of eight rats. (c) After administration of 2 (AM-9635),
plasma was also harvested at day 10 to assess exposures in each dose group. Unbound drug concentrations were measured by LC−MS/MS
and plotted relative to HWB_unbound pAKT IC50 = 5.7 nM (in Table 2) represented as a blueꢀdotted line, PI3Kδ in vitro mouse
pAKT_unbound IC50 3.2 nM [calculated from 4.2 nM (PI3Kδ in vitro mouse pAKT IC50) × 0.771 (fu in pAKT assay media)] represented
as a blue line, and PI3Kβ in vitro human pAKT_unbound IC50 1928 nM [calculated from 2500 nM (PI3Kβ in vitro human pAKT IC50) ×
0.771 (fu in pAKT assay media)] represented as a red line.
EXPERIMENTAL
General Chemistry
All solvents and chemicals used were reagent grade. Anhydrous solvents were purchased from Sigmaꢀ
Aldrich and used as received. Analytical thin layer chromatography (TLC) and silica gel column chroꢀ
matography were performed on Merck silica gel 60 (230ꢀ400 mesh). Removal of solvents was conductꢀ
ed by using a rotary evaporator and residual solvents were removed from nonvolatile compounds using
a vacuum manifold maintained at approximately 1 Torr. All yields reported are isolated yields. Preparꢀ
ative reverse–phase high pressure liquid chromatography (RP–HPLC) was performed using an Agilent
1100 series HPLC and Phenomenex Gemini C18 column (5 ꢀm, 100 mm × 30 mm i.d.), eluting with a
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binary solvent system, A and B, using a gradient elution [A, H2O with 0.1% TFA, B, CH3CN with 0.1%
TFA] with UV detection at 220 nm. All final compounds were purified to ≥95% purity as determined
by an Agilent 1100 series HPLC with UV detection at 220 nm using the following method: Zorbax SBꢀ
C8 column (3.5 ꢀm, 150 mm × 4.6 mm i.d.), eluting with a binary solvent system, A and B, using a 5ꢀ
95% B (0ꢀ15 min) gradient elution [A, H2O with 0.1% TFA, B, CH3CN with 0.1% TFA]; flow rate 1.5
mL/min. Mass spectral data was recorded on an Agilent 1100 series LCMS with UV detection at 254
nm. All accurate mass data (Highꢀresolution mass spectra: HRMS) were acquired on a Synapt G2 ToF
instrument operating in positive electrospray ionization mode, over the m/z range 50ꢀ1200. Lock mass
correction was performed on the leucineꢀenkephalin ion m/z 556.2771. The instrument resolution was
28,000 at FWHM. The compounds were introduced into the mass spectrometer using an Agilent 1200
operated with a C4 Bridgedꢀethylꢀhybrid (BEH) analytical column (2.1 x 50 mm) at 0.25 mL/min.
NMR spectra were recorded on a Bruker Avance 400 MHz and 500 MHz NMR spectrometer. Chemiꢀ
cal shifts (δ) are reported in parts per million (ppm) relative to residual undeuterated solvent as internal
reference and coupling constants (J) are reported in hertz (Hz). Splitting patterns are indicated as folꢀ
lows: s = singlet; d = doublet; t = triplet; q = quartet; qn = quintet; dd = doublet of doublets; dt = doublet
2
3
4
5
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of triplets; tt = triplet of triplets; m = multiplet; br = broad peak.
Benzimidazole Analogs
General Procedure for the Synthesis of 9a-g. A mixture of 2,4ꢀdifluoronitrobenzene 8 (1 equiv) and
substituted aniline 9aꢀg (1 equiv) was stirred at 130 °C for 24 hours. The mixture was cooled to room
temperature and precipitates were noted. The mixture was dissolved in DCM and washed with saturated
aqueous NaHCO3 solution. The organic layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude material was purified by chromatography on a silica gel column using 0ꢀ
50% gradient of EtOAc in hexane as eluent to give 9a-g.
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1
5-Fluoro-N-(3-(methylsulfonyl)phenyl)-2-nitroaniline (9a). 50.5 % yield as a bright yellow solid. H
NMR (400 MHz, DMSO-d6) δ ppm 9.65 (1 H, s), 8.25 (1 H, dd, J=9.6, 6.1 Hz), 7.85 ꢀ 7.89 (1 H, m),
7.64 ꢀ 7.75 (3 H, m), 6.98 (1 H, dd, J=11.4, 2.6 Hz), 6.79 ꢀ 6.87 (1 H, m), 3.24 (3 H, s). Mass spectrum
(ESI) m/z 311.0 [M+H]+.
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5-Fluoro-N-(2-(methylthio)phenyl)-2-nitroaniline (9b). 43.3 % yield as an orange solid. H NMR
(400 MHz, DMSO-d6) δ ppm 9.52 (1 H, s), 8.26 (1 H, dd, J=9.4, 6.1 Hz), 7.34 ꢀ 7.45 (3 H, m), 7.26 ꢀ
7.32 (1 H, m), 6.67 ꢀ 6.75 (1 H, m), 6.35 (1 H, dd, J=11.6, 2.6 Hz), 2.43 (3 H, s). Mass spectrum (ESI)
m/z 279.0 [M+H]+.
1
5-Fluoro-N-(3-fluorophenyl)-2-nitroaniline (9c). 20.9% yield as an orange solid. H NMR (400
MHz, DMSO-d6) δ ppm 9.54 (1 H, s), 8.24 (1 H, dd, J=9.4, 6.1 Hz), 7.40 ꢀ 7.50 (1 H, m), 7.18 ꢀ 7.27 (2
H, m), 7.05 (1 H, td, J=8.5, 2.5 Hz), 6.94 (1 H, dd, J=11.6, 2.6 Hz), 6.79 (1 H, ddd, J=9.6, 7.3, 2.6 Hz).
Mass spectrum (ESI) m/z 251.1 [M+H]+.
1
5-Fluoro-N-(4-fluorophenyl)-2-nitroaniline (9d). 80 % yield as an orange solid. H NMR (400 MHz,
DMSO-d6) δ ppm 9.56 (1 H, s), 8.24 (1 H, dd, J=9.5, 6.2 Hz), 7.36 ꢀ 7.44 (2 H, m), 7.25 ꢀ 7.33 (2 H, m),
6.67 ꢀ 6.74 (1 H, m), 6.64 (1 H, dd, J=11.8, 2.6 Hz). Mass spectrum (ESI) m/z 251.1 [M+H]+.
1
N-(3,5-Difluorophenyl)-5-fluoro-2-nitroaniline (9e). 17.6% yield as an orange solid. H NMR (400
MHz, DMSO-d6) δ ppm 9.48 (1 H, s), 8.23 (1 H, dd, J=9.4, 6.1 Hz), 7.05 ꢀ 7.17 (3 H, m), 6.99 (1 H, tt,
J=9.4, 2.3 Hz), 6.84 ꢀ 6.91 (1 H, m). Mass spectrum (ESI) m/z 267.0 [MꢀH]ꢀ.
N-(5-Fluoro-2-nitrophenyl)pyridin-3-amine (9f). 38.3 % yield as an orange solid. Mass spectrum
(ESI) m/z 234.1 [M+H]+.
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5-Fluoro-N-(5-fluoro-2-nitrophenyl)pyridin-3-amine (9g). 52.5 % yield as an orange solid. Mass
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5
spectrum m/z 252.1 [M+H]+.
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General Procedure for the Synthesis of 10a-g. A mixture of 9a-g (1 equiv) and Tin(II) chloride dihyꢀ
drate (5 equiv) in EtOAc (0.15 M) was stirred at reflux. After 5 hours, the mixture was cooled to room
temperature, and poured into 10 M aqueous NaOH solution. The mixture was extracted with EtOAc.
The combined organic layers were washed with water, brine, dried over MgSO4, filtered, and concenꢀ
trated under reduced pressure. The crude material was purified by column chromatography on a silica
gel column using a 0ꢀ20% gradient of EtOAc in hexane as eluent to give 10a-g.
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5-Fluoro-N1-(3-(methylsulfonyl)phenyl)benzene-1,2-diamine (10a). 100% yield as a colorless syrup.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.82 (1 H, s), 7.36 ꢀ 7.43 (1 H, m), 7.18 ꢀ 7.25 (2 H, m), 7.01 (1
H, ddd, J=8.2, 2.2, 1.1 Hz), 6.83 ꢀ 6.89 (1 H, m), 6.71 ꢀ 6.79 (2 H, m), 4.71 (2 H, s), 3.14 (3 H, s). Mass
spectrum (ESI) m/z 281.0 [M+H]+.
5-Fluoro-N1-(2-(methylthio)phenyl)benzene-1,2-diamine (10b). 92% yield as a pale yellow syrup.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.35 (1 H, dd, J=7.7, 1.5 Hz), 7.11 (1 H, td, J=7.7, 1.5 Hz), 6.91
(1 H, td, J=7.5, 1.2 Hz), 6.78 (1 H, dd, J=8.0, 1.4 Hz), 6.71 (1 H, dd, J=8.6, 5.9 Hz), 6.65 (1 H, s), 6.52 ꢀ
6.62 (2 H, m), 4.61 (2 H, s), 2.40 (3 H, s). Mass spectrum (ESI) m/z 249.0 [M+H]+.
1
5-Fluoro-N1-(3-fluorophenyl)benzene-1,2-diamine (10c). 69.4% yield as an orange oil. H NMR
(400 MHz, DMSO-d6) δ ppm 7.54 (1 H, s), 7.12 ꢀ 7.22 (1 H, m), 6.83 (1 H, dd, J=10.4, 2.7 Hz), 6.65 ꢀ
6.77 (2 H, m), 6.57 ꢀ 6.63 (1 H, m), 6.43 ꢀ 6.53 (2 H, m), 4.66 (2 H, s). Mass spectrum (ESI) m/z 221.1
[M+H]+.
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5-Fluoro-N1-(4-fluorophenyl)benzene-1,2-diamine (10d). 94% yield as a brown syrup. H NMR
(400 MHz, DMSO-d6) δ ppm 7.18 (1 H, s), 6.98 ꢀ 7.07 (2 H, m), 6.81 ꢀ 6.90 (2 H, m), 6.65 ꢀ 6.76 (2 H,
m), 6.54 ꢀ 6.62 (1 H, m), 4.61 (2 H, s). Mass spectrum (ESI) m/z 221.1 [M+H]+.
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N1-(3,5-Difluorophenyl)-5-fluorobenzene-1,2-diamine (10e). 96 % yield as a light yellow syrup. Hꢀ
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NMR (400 MHz, DMSO-d6) δ ppm 7.85 (1 H, s), 6.85 (1 H, dd, J=10.0, 2.3 Hz), 6.72 ꢀ 6.81 (2 H, m),
6.41 (1 H, tt, J=9.4, 2.3 Hz), 6.24 ꢀ 6.33 (2 H, m), 4.71 (2 H, s). Mass spectrum (ESI) m/z 239.1
[M+H]+.
5-Fluoro-N1-(pyridin-3-yl)benzene-1,2-diamine (10f). 77 % yield as a black solid. Mass spectrum
(ESI) m/z 204.1 [M+H]+.
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5-Fluoro-N1-(5-fluoropyridin-3-yl)benzene-1,2-diamine (10g). 95% yield as a dark solid. H NMR
(400 MHz, DMSO-d6) δ ppm 7.98 (1 H, t, J=2.0 Hz), 7.83 ꢀ 7.91 (2 H, m), 6.84 ꢀ 6.91 (1 H, m), 6.72 ꢀ
6.79 (3 H, m), 4.75 (2 H, s). Mass spectrum (ESI) m/z 222.1 [M+H]+.
General Procedure for the Synthesis of 11a-c, 11e-g. To a ꢀ10 °C solution (NaClꢀice bath) of BocꢀLꢀ
AlaꢀOH (2 equiv) and Nꢀmethylmorpholine (2.1 equiv) in DCM (0.2 M) was added isobutyl chloroforꢀ
mate (2 equiv). The resulting cloudy colorless mixture was stirred at ꢀ10 °C. After 1 hour, a solution of
10a-c, 10e-g (1 equiv) in DCM (0.2 M). was added to the mixture. The resulting mixture was stirred at
ꢀ10 °C for 40 minutes, then allowed to warm to room temperature. After 24 hours, saturated aqueous
NH4Cl solution was added to the mixture. and the organic layer separated. The aqueous mixture was
extracted with DCM. The combined organic layers were dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude material was purified by column chromatography on a silica gel colꢀ
umn using 0ꢀ50% gradient of EtOAc in hexane as eluent to give 11a-c, 11e-g.
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(S)-tert-Butyl 1-(4-fluoro-2-(3-(methylsulfonyl)phenylamino)phenylamino)-1-oxopropan-2-
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4
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ylcarbamate (11a). 84 % yield as a solid. H NMR (500 MHz, DMSO-d6) δ ppm 9.40 (1 H, s), 7.86 (1
H, s), 7.48 (2 H, q, J=8.0 Hz), 7.40 (1 H, br. s.), 7.35 (1 H, d, J=7.3 Hz), 7.23 (1 H, d, J=7.8 Hz), 7.13 (1
H, d, J=6.4 Hz), 7.09 (1 H, dd, J=10.5, 2.7 Hz), 6.87 (1 H, td, J=8.2, 1.5 Hz), 4.05 ꢀ 4.11 (1 H, m), 3.16
(3 H, s), 1.35 (9 H, s), 1.16 ꢀ 1.21 (3 H, m). Mass spectrum (ESI) m/z 452.1 [M+H]+ and m/e 450.1 [Mꢀ
H]ꢀ.
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(S)-tert-Butyl 1-(4-fluoro-2-(2-(methylthio)phenylamino)phenylamino)-1-oxopropan-2-
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ylcarbamate (11b). 94% yield as a white solid. HꢀNMR (400 MHz, DMSO-d6) δ ppm 9.56 (1 H, br.
s.), 7.36 (1 H, dd, J=7.8, 1.4 Hz), 7.26 (1 H, dd, J=8.6, 6.3 Hz), 7.12 ꢀ 7.19 (1 H, m), 7.10 (1 H, d, J=6.8
Hz), 6.97 ꢀ 7.07 (3 H, m), 6.70 (1 H, td, J=8.2, 2.3 Hz), 6.61 (1 H, d, J=11.0 Hz), 4.14 (1 H, quin, J=6.8
Hz), 2.37 (3 H, s), 1.34 (9 H, s), 1.26 (3 H, d, J=7.2 Hz). Mass spectrum (ESI) m/z 420.1 [M+H]+.
(S)-tert-Butyl (1-((4-fluoro-2-((3-fluorophenyl)amino)phenyl)amino)-1-oxopropan-2-yl)carbamate
(11c). 93% yield as a white solid. Mass spectrum (ESI) m/z 390.1 [MꢀH]ꢀ.
(S)-(9H-Fluoren-9-yl)methyl 1-(4-fluoro-2-(4-fluorophenylamino)phenylamino)-1-oxopropan-2-
ylcarbamate (11d). To a ꢀ10 °C solution (NaClꢀice bath) of FmocꢀLꢀAlaꢀOH (2.1 equiv) and Nꢀ
methylmorpholine (2.1 equiv) in DCM (0.4 M) was added isobutyl chloroformate (2 equiv). The resultꢀ
ing cloudy colorless mixture was stirred at ꢀ10 °C for 30 minutes. To the mixture was then added a soluꢀ
tion of 5ꢀfluoroꢀN1ꢀ(4ꢀfluorophenyl)benzeneꢀ1,2ꢀdiamine (1 equiv) in DCM (0.5 M). The resulting
mixture was stirred at ꢀ10 °C. After 1 hour, to the cold mixture was added saturated NH4Cl. The resultꢀ
ing precipitate was collected by filtration and washed the solid with water to give 11d (quantitave yield)
1
as an offꢀwhite solid. H NMR (400 MHz, DMSO-d6) δ ppm 9.42 (1 H, s), 7.89 (2 H, d, J=7.6 Hz), 7.63
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ꢀ 7.78 (3 H, m), 7.22 ꢀ 7.46 (6 H, m), 6.96 ꢀ 7.14 (4 H, m), 6.83 (1 H, dd, J=11.2, 2.7 Hz), 6.67 (1 H, td,
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J=8.5, 2.8 Hz), 4.11 ꢀ 4.33 (4 H, m), 1.28 (3 H, d, J=7.0 Hz). Mass spectrum (ESI) m/z 514.2 [M+H]+.
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(S)-tert-butyl 1-(2-(3,5-difluorophenylamino)-4-fluorophenylamino)-1-oxopropan-2-ylcarbamate
9
1
(11e). 98 % yield as a white solid. H NMR (400 MHz, DMSO-d6) δ ppm 9.41 (1 H, br. s.), 7.87 (1 H,
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br. s.), 7.50 (1 H, dd, J=8.6, 6.5 Hz), 7.07 ꢀ 7.20 (2 H, m), 6.87 ꢀ 6.96 (1 H, m), 6.43 ꢀ 6.62 (3 H, m),
3.95 ꢀ 4.13 (1 H, m), 1.37 (9 H, s), 1.17 (3 H, d, J=7.0 Hz). Mass spectrum (ESI) m/z 410.2 [M+H]+.
(S)-Methyl 5-(2-(2-(tert-butoxycarbonylamino)propanamido)-5-fluorophenylamino)nicotinate
1
(11f). 99% yield as a dark brown solid. H NMR (400 MHz, DMSO-d6) δ ppm 9.44 (1 H, br s), 8.28 (1
H, br s), 8.08 (1 H, br d, J=4.1 Hz), 7.32 ꢀ 7.68 (1 H, m), 7.31 ꢀ 7.81 (1 H, m), 7.24 (1 H, dd, J=8.1, 4.6
Hz), 7.16 (1 H, br s), 6.99 (1 H, dd, J=10.8, 2.9 Hz), 6.72 ꢀ 6.85 (1 H, m), 4.07 (1 H, br s), 1.35 (9 H, s),
1.20 (3 H, br d, J=6.8 Hz). Mass spectrum (ESI) m/z 375.1 [M+H]+.
(S)-tert-Butyl 1-(4-fluoro-2-(5-fluoropyridin-3-ylamino)phenylamino)-1-oxopropan-2-ylcarbamate
1
(11g). 79 % yield as a light yellow solid. H NMR (400 MHz, DMSO-d6) δ ppm 9.45 (1 H, s), 8.11 (1
H, br s), 7.99 (1 H, d, J=2.0 Hz), 7.92 (1 H, s), 7.44 ꢀ 7.54 (1 H, m), 7.02 ꢀ 7.19 (2 H, m), 6.91 (1 H, br t,
J=7.1 Hz), 2.60 ꢀ 2.86 (1 H, m), 1.30 ꢀ 1.42 (9 H, m), 1.14 ꢀ 1.19 (3 H, m). Mass spectrum (ESI) m/z
393.1 [M+H]+.
General Procedure for the Synthesis of 12a-g. A solution of 11a-g in AcOH (0.3 M) was stirred and
heated at 100 °C. After 24 hours, the mixture was cooled to room temperature and poured into DCM
and saturated aqueous NaHCO3 solution. The aqueous layer was separated. The organic layer was
washed with saturated aqueous NaHCO3 solution, washed with water, brine, dried over MgSO4, filtered,
and concentrated under reduced pressure to give 12a-g.
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[NOTE]: Epimerization occurred during cyclization, and Bocꢀprotecting group was cleaved and the free
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amine was aceylated under reaction conditions.
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(S)-N-(1-(6-Fluoro-1-(3-(methylsulfonyl)phenyl)-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12a).
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83% yield as a pink solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.40 (1 H, d, J=8.0 Hz), 8.07 ꢀ 8.14 (1
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H, m), 8.04 (1 H, br. s.), 7.89 (2 H, d, J=4.5 Hz), 7.75 (1 H, dd, J=8.8, 4.9 Hz), 7.15 (1 H, ddd, J=9.9,
8.9, 2.5 Hz), 7.01 (1 H, dd, J=9.0, 2.3 Hz), 4.98 ꢀ 5.13 (1 H, m), 3.32 (3 H, s), 1.59 (3 H, s), 1.45 (3 H,
d, J=6.8 Hz). Mass spectrum (ESI) m/z 376.0 [M+H]+.
N-(1-(6-fluoro-1-(2-(methylthio)phenyl)-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12b). 29.3 %
1
yield as a pink solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.16 ꢀ 8.45 (1 H, m), 7.72 (1 H, dd, J=8.8,
4.9 Hz), 7.56 ꢀ 7.64 (1 H, m), 7.49 ꢀ 7.55 (1 H, m), 7.33 ꢀ 7.48 (2 H, m), 7.10 (1 H, ddd, J=9.8, 8.8, 2.5
Hz), 6.61 ꢀ 6.70 (1 H, m), 4.73 ꢀ 5.04 (1 H, m), 2.35 ꢀ 2.43 (3 H, m), 1.63 ꢀ 1.72 (3 H, m), 1.34 ꢀ 1.44 (3
H, m). Mass spectrum (ESI) m/z 344.0 [M+H]+.
N-(1-(6-fluoro-1-(2-(methylsulfonyl)phenyl)-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12b’). To
a mixture of 12b (1 equiv) in THFꢀwater (3:1, 1 M) was added Oxone (2.5 equiv) and the mixture was
stirred at room temperature. After 48 hours, to the mixture was diluted with water and extracted with
DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced
1
pressure to give 12b’ (98 % yield) as an orange solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.13 ꢀ
8.30 (2 H, m), 7.84 ꢀ 8.02 (2 H, m), 7.48 ꢀ 7.80 (2 H, m), 6.82 ꢀ 7.18 (2 H, m), 4.85 ꢀ 5.03 (1 H, m), 2.81
ꢀ 3.02 (3 H, m), 1.53 ꢀ 1.73 (3 H, m), 1.32 ꢀ 1.50 (3 H, m). Mass spectrum (ESI) m/z 376.0 [M+H]+.
N-(1-(6-Fluoro-1-(3-fluorophenyl)-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12c). Quantitative
yield as pink solid. Mass spectrum (ESI) m/z 316.1 [M+H]+.
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(S)-(9H-fluoren-9-yl)methyl 1-(6-fluoro-1-(4-fluorophenyl)-1H-benzo[d]imidazol-2-
yl)ethylcarbamate (12d). Quantitative yield as a dark brown syrup. Mass spectrum (ESI) m/z 496.1
[M+H]+.
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N-(1-(1-(3,5-Difluorophenyl)-6-fluoro-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12e). 86% yield
as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.38 (1 H, d, J=7.4 Hz), 7.72 (1 H, dd, J=8.8,
4.9 Hz), 7.51 (1 H, tt, J=9.5, 2.3 Hz), 7.41 (2 H, dd, J=7.9, 1.7 Hz), 7.13 (1 H, ddd, J=9.8, 8.8, 2.5 Hz),
7.03 ꢀ 7.09 (1 H, m), 5.07 (1 H, qd, J=7.1, 6.8 Hz), 1.64 (3 H, s), 1.44 (3 H, d, J=6.8 Hz). Mass specꢀ
trum (ESI) m/z 334.1 [M+H]+.
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N-(1-(6-Fluoro-1-(pyridin-3-yl)-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12f). 99 % yield as a
brown foam. Mass spectrum (ESI) m/z 299.1 [M+H]+.
N-(1-(6-Fluoro-1-(5-fluoropyridin-3-yl)-1H-benzo[d]imidazol-2-yl)ethyl)acetamide (12g). 69.7%
1
yield as a dark brown solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.20 ꢀ 8.39 (1 H, m), 8.13 (1 H, s),
7.98 (1 H, d, J=2.3 Hz), 7.47 (1 H, dd, J=8.8, 6.3 Hz), 7.02 ꢀ 7.18 (2 H, m), 6.88 (1 H, td, J=8.5, 2.7
Hz), 4.28 (1 H, br d, J=7.2 Hz), 1.83 (3 H, s), 1.18 (3 H, d, J=7.0 Hz). Mass spectrum (ESI) m/z 317.1
[M+H]+.
General Procedure for the Synthesis of 13a-c, 13e-g. A solution of 12a-c, 12e-g (1 equiv) and 2 N
HCl (16 equiv) was stirred and heated at 100 °C. After 24 hours, the mixture was cooled to room temꢀ
perature. The acidic aqueous mixture was washed with DCM to remove organic impurities and then
basified to pH 10 with aqueous 10 N NaOH and extracted with DCM. The combined organic layers
were washed with water, brine, dried over MgSO4, filtered, and concentrated under reduced pressure to
give 13a-c, 13e-g.
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1-(6-fluoro-1-(3-(methylsulfonyl)phenyl)-1H-benzo[d]imidazol-2-yl)ethanamine (13a). 80% yield
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3
4
5
1
as white solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.16 ꢀ 8.20 (1 H, m), 8.09 ꢀ 8.15 (1 H, m), 7.97 ꢀ
6
7
8
9
8.03 (1 H, m), 7.89 ꢀ 7.95 (1 H, m), 7.71 (1 H, dd, J=8.8, 4.9 Hz), 7.12 (1 H, ddd, J=10.0, 8.8, 2.5 Hz),
7.01 (1 H, dd, J=9.0, 2.3 Hz), 3.96 (1 H, q, J=6.7 Hz), 3.35 (3 H, s), 1.95 (2 H, br. s.), 1.36 (3 H, d,
J=6.7 Hz). Mass spectrum (ESI) m/z 334.0 [M+H]+.
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(S)-1-(6-Fluoro-1-(3-(methylsulfonyl)phenyl)-1H-benzo[d]imidazol-2-yl)ethanamine (13a). 80 %
1
yield as a white solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.16 ꢀ 8.20 (1 H, m), 8.09 ꢀ 8.15 (1 H, m),
7.97 ꢀ 8.03 (1 H, m), 7.89 ꢀ 7.95 (1 H, m), 7.71 (1 H, dd, J=8.8, 4.9 Hz), 7.12 (1 H, ddd, J=10.0, 8.8, 2.5
Hz), 7.01 (1 H, dd, J=9.0, 2.3 Hz), 3.96 (1 H, q, J=6.7 Hz), 3.35 (3 H, s), 1.95 (2 H, br. s.), 1.36 (3 H, d,
J=6.7 Hz). Mass spectrum (ESI) m/z 334.0 [M+H]+.
1-(6-Fluoro-1-(2-(methylsulfonyl)phenyl)-1H-benzo[d]imidazol-2-yl)ethanamine (13b). 80 % yield
1
as a tan solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.20 ꢀ 8.29 (1 H, m), 7.89 ꢀ 8.03 (2 H, m), 7.64 ꢀ
7.81 (2 H, m), 7.04 ꢀ 7.14 (1 H, m), 6.81 ꢀ 6.91 (1 H, m), 3.62 ꢀ 3.75 (1 H, m), 2.80 ꢀ 2.98 (3 H, m), 1.57
ꢀ 1.93 (2 H, m), 1.19 ꢀ 1.48 (3 H, m). Mass spectrum (ESI) m/z 334.0 [M+H]+.
1-(6-Fluoro-1-(3-fluorophenyl)-1H-benzo[d]imidazol-2-yl)ethanamine (13c). 81% yield an offꢀ
white solid. Mass spectrum (ESI) m/z 274.1 [M+H]+.
1-(6-Fluoro-1-(4-fluorophenyl)-1H-benzo[d]imidazol-2-yl)ethanamine (13d). To a solution of 12d
(1 equiv) in DCM (0.2 M) was added piperidine (3.6 equiv). The mixture was stirred at room temperaꢀ
ture for 5 hours, then concentrated under reduced pressure to give a brown solid. The brown solid was
purified by silica gel column chromatography using 0ꢀ100% gradient of DCM:MeOH:NH4OH (89:9:1)
1
in DCM as eluent to give 13d (46.5 % yield) over three steps as a pink semiꢀsolid. H NMR (400 MHz,
DMSO-d6) δ ppm 7.67 (3 H, td, J=8.8, 5.0 Hz), 7.48 (2 H, t, J=8.8 Hz), 7.09 (1 H, ddd, J=9.8, 8.8, 2.5
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Page 29 of 49
Journal of Medicinal Chemistry
11/25/2015
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Hz), 6.88 (1 H, dd, J=9.0, 2.3 Hz), 3.93 (1 H, q, J=6.8 Hz), 1.92 (2 H, br. s.), 1.33 (3 H, d, J=6.7 Hz).
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3
4
5
Mass spectrum (ESI) m/z 274.0 [M+H]+.
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1-(1-(3,5-Difluorophenyl)-6-fluoro-1H-benzo[d]imidazol-2-yl)ethanamine (13e). 79 % yield as a
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pink solid. H NMR (400 MHz, DMSO-d6) δ ppm 7.69 (1 H, dd, J=8.8, 4.9 Hz), 7.49 ꢀ 7.58 (3 H, m),
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7.04 ꢀ 7.15 (2 H, m), 4.01 (1 H, q, J=6.6 Hz), 1.95 (2 H, br. s.), 1.36 (3 H, d, J=6.5 Hz). Mass spectrum
(ESI) m/z 292.0 [M+H]+.
1-(6-fluoro-1-(pyridin-3-yl)-1H-benzo[d]imidazol-2-yl)ethanamine (13f). 61.2% yield as as a brown
foam. Mass spectrum (ESI) m/z 257.1 [M+H]+.
1-(6-Fluoro-1-(5-fluoropyridin-3-yl)-1H-benzo[d]imidazol-2-yl)ethanamine (13g). 95% yield as a
black tar. Mass spectrum (ESI) m/z 275.1 [M+H]+.
General Procedure for the Synthesis of 14a-g. A mixture of 4ꢀaminoꢀ6ꢀchloropyrimidineꢀ5ꢀ
carbonitrile (1 equiv), 13a-g (1 equiv), and DIPEA (3 equiv) in nꢀbutanol (0.1 M) was stirred at 120 °C.
After 24 hours, the mixture was cooled to room temperature and concentrated under reduced pressure to
give a brown solid. The brown solid was suspended in water, filtered, and washed with water to give a
tan solid. The tan solid was suspended in EtOAcꢀhexane (1:4), filtered, washed with EtOAcꢀhexane
(1:4), and dried to give 14a-g as a racemic mixture.
4-Amino-6-(1-(6-fluoro-1-(3-(methylsulfonyl)phenyl)-1H-benzo[d]imidazol-2-
1
yl)ethylamino)pyrimidine-5-carbonitrile (14a). 62.8 % yield as a tan solid. H NMR (400 MHz,
DMSO-d6) δ ppm 8.14 (1 H, t, J=1.9 Hz), 8.03 (1 H, dt, J=7.8, 1.4 Hz), 7.87 ꢀ 7.96 (1 H, m), 7.84 (1 H,
s), 7.72 ꢀ 7.83 (3 H, m), 7.09 ꢀ 7.25 (3 H, m), 6.97 (1 H, dd, J=8.9, 2.4 Hz), 5.50 (1 H, quin, J=7.0 Hz),
3.26 (3 H, s), 1.55 (3 H, d, J=6.8 Hz). Mass spectrum (ESI) m/z 452.0 [M+H]+.
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