Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2-PCCA: Synthesis, Structure-Activity Relationships, and Molecular Modeling Studies

Article abstract of DOI:10.1021/acschemneuro.6b00182

GPR88, an orphan receptor richly expressed in the striatum, is implicated in a number of basal ganglia-associated disorders. In order to elucidate the functions of GPR88, an in vivo probe appropriate for CNS investigation is required. We previously reported that 2-PCCA was able to modulate GPR88-mediated cAMP production through a Gα_i-coupled pathway. Early structure-activity relationship (SAR) studies suggested that the aniline moiety of 2-PCCA is a suitable site for diverse modifications. Aimed at elucidating structural requirements in this region, we have designed and synthesized a series of analogues bearing a variety of substituents at the phenyl ring of the aniline moiety. Several compounds (e.g., 5j, 5o) showed improved or comparable potency, but have lower lipophilicity than 2-PCCA (clogP 6.19). These compounds provide the basis for further optimization to probe GPR88 in vivo functions. Computational studies confirmed the SAR trends and supported the notion that 4′-substituents on the biphenyl ring exit through a largely hydrophobic binding site to the extracellular loop.

Full text of DOI:10.1021/acschemneuro.6b00182

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Article
Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2-PCCA:
Synthesis, Structure-Activity Relationships and Molecular Modeling Studies
Chunyang Jin, Ann M. Decker, Danni L. Harris, and Bruce E. Blough
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00182 • Publication Date (Web): 06 Aug 2016
Downloaded from http://pubs.acs.org on August 7, 2016
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Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2-PCCA: Synthesis, Structure-
Activity Relationships and Molecular Modeling Studies
Chunyang Jin,* Ann M. Decker, Danni L. Harris, and Bruce E. Blough
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Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2-PCCA: Synthesis, Structure-
Activity Relationships and Molecular Modeling Studies
Chunyang Jin,* Ann M. Decker, Danni L. Harris, and Bruce E. Blough
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Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, North Carolina 27709
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Corresponding author:
Dr. Chunyang Jin
Research Triangle Institute
Post Office Box 12194
Research Triangle Park, NC 27709
Telephone: 919 541ꢀ6328
Fax: 919 541ꢀ8868
Eꢀmail: cjin@rti.org
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Abstract
GPR88, an orphan receptor richly expressed in the striatum, is implicated in a number of basal gangliaꢀ
associated disorders. In order to elucidate the functions of GPR88, an in vivo probe appropriate for CNS
investigation is required. We previously reported that 2ꢀPCCA was able to modulate GPR88−mediated cAMP
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production through a Gα −coupled pathway. Early structureꢀactivity relationship (SAR) studies suggested that
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the aniline moiety of 2ꢀPCCA is a suitable site for diverse modifications. Aimed at elucidating structural
requirements in this region, we have designed and synthesized a series of analogues bearing a variety of
substituents at the phenyl ring of the aniline moiety. Several compounds (e.g., 5j, 5o) showed improved or
comparable potency, but have lower lipophilicity than 2ꢀPCCA (clogP 6.19). These compounds provide the
basis for further optimization to probe GPR88 in vivo functions. Computational studies confirmed the SAR
trends and supported the notion that 4’ꢀsubstituents on the biphenyl ring exit through a largely hydrophobic
binding site to the extracellular loop.
Keywords: Orphan GPR88, 2ꢀPCCA, SAR, molecular modeling
INTRODUCTION
The G proteinꢀcoupled receptors (GPCRs) constitute the largest family of membrane proteins and
mediate most cellular responses to hormones and neurotransmitters. GPCRs also play important roles in
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disease pathogenesis and account for more than 30% of targets of marketed drugs. Analysis of the human
genome revealed more than 800 GPCR sequences, of which around 140 GPCRs, excluding olfactory
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receptors, are still classified as orphan receptors for which endogenous ligands are unknown. The complex
biology and potential for drug therapy within this class provide strong incentives for small molecule probe
development to enable modulation of individual receptors and facilitate elucidation of their biological
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functions.
GPR88 is an orphan receptor highly expressed in the CNS, with particularly robust expression in the
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striatum throughout the dorsal and ventral areas. Transcriptional profiling studies have revealed Gpr88 gene
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expression is altered by conditions related to Parkinson’s disease, schizophrenia, bipolar disorder,
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depression, and drug addiction. Interestingly, both receptor upꢀ and downꢀregulation have been observed
depending on the treatment and brain region. For example, in dopamineꢀdepleted striatum, Gpr88 expression
was differentially regulated in striatonigral and striatopallidal medium spinal neurons (MSNs), and LꢀDOPA
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treatment normalized Gpr88 expression levels. Gpr88 gene expression was upꢀregulated in the prefrontal
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b
cortex following methamphetamine exposure and downꢀregulated in the amygdala following treatment with
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the NMDA receptor antagonist MKꢀ801.
More direct evidence of GPR88 function was obtained from GPR88 knockout studies. GPR88 knockout
mice demonstrated disrupted prepulse inhibition of startle response, a phenotype of schizophrenia, and
exhibited D receptor hypersensitivity, which were normalized to control levels by antipsychotic drug
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administration. In another study, the GPR88 knockout animals exhibited increased locomotion, poor motor
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coordination and impaired cueꢀbased learning. Examination of MSNs electrophysiology in brain slices
revealed decreased tonic GABAergic inhibition and increased glutamatergic excitation, which promoted
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enhanced firing rates observed in vivo. In addition, GPR88 reꢀexpression normalized these impaired
behaviors and electrophysiological properties, indicating that GPR88 dysfunction may contribute to abnormal
behaviors observed in basal gangliaꢀassociated disorders such as Parkinson’s disease and schizophrenia.
Interestingly, a recent study showed that the GPR88ꢀdeficient mice performed better in spatial tasks and
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reduced levels of anxiety, indicating GPR88 may also play a role in cognitive and anxiety disorders.
Despite emerging pharmacological implications of GPR88, the signaling mechanisms and biological
functions of this orphan receptor are still largely unknown due to the lack of potent and selective native or
synthetic ligands. We previously reported that 2ꢀPCCA [(1R*,2R*)ꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropanecarboxylic
acid ((2S,3S)ꢀ2ꢀaminoꢀ3ꢀmethylpentyl)ꢀ(4′ꢀpropylbiphenylꢀ4ꢀyl)ꢀamide (1); Figure 1] was able to modulate
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GPR88ꢀmediated cAMP production through a Gα −coupled pathway. Unfortunately, 2ꢀPCCA has a high
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calculated lipophilicity (clogP 6.19) and was reported to be a Pꢀglycoprotein substrate, which might limit its
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effectiveness as a tool. In order to elucidate the functions of GPR88, an improved in vivo agonist probe
appropriate for CNS investigation is required. Early structureꢀactivity relationship (SAR) studies suggest that
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the aniline moiety of 2ꢀPCCA is a suitable site for diverse modifications.
Aimed at elucidating SAR
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requirements in this region, we have designed and synthesized a series of analogues (4a−g, Figure 1 and
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a−aa, Figure 2) bearing a variety of substituents at the phenyl ring of the aniline moiety. In this paper, we
report the SAR and computational studies of these compounds with the goal of improving potency and
lowering the lipophilicity.
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RESULTS AND DISCUSSION
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Chemistry. The overall approach to the synthesis followed methods detailed in our earlier work. Synthesis
of the designed compounds 4a−g is outlined in Scheme 1. Reductive amination of aldehyde 7, prepared by
DessꢀMartin oxidation of commercially available (−)ꢀ(2S,3S)ꢀNꢀBocꢀ2ꢀaminoꢀ3ꢀmethylꢀ1ꢀpentanol, with 4ꢀ
substituted aniline 6a−h afforded amine 8a−h in 46−77% yields. Amide formation with the acid chloride of
racemic (±)ꢀtransꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropanecarboxylic acid gave 9a−h in 32−72% yields. Removal of the
Boc protecting group of 9b−h with 4 M HCl in dioxane provided target compounds 4a-g in 83−97% yields.
Synthesis of the biphenyl derivatives 5a−aa is illustrated in Scheme 2. Suzuki coupling of the bromo
compound 9a with an appropriate arylboronic acid under microwave conditions gave intermediates 10a−aa in
the range of 51−81% yields. Deprotection of the Boc group furnished 5a−aa in 86−100% yields. Target
compounds 4a−g and 5a−aa were determined to be 1:1 mixture of (1R,2R)ꢀ and (1S,2S)ꢀenantiomers,
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differentiating at the configuration of the transꢀsubstituted cyclopropane ring, by H NMR and HPLC
analyses.
Biological Results and SAR Studies. We previously demonstrated that 2ꢀPCCA (1, Figure 1) inhibited
isoproterenolꢀstimulated cAMP accumulation with EC = 911 nM in HEK293 cells stably expressing the
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human GPR88 receptor and the GloSensor −22F cAMP construct, indicating that GPR88 is coupled to the
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Gα subunits. Recently, Bi et al. reported that the (1R,2R)ꢀenantiomer of 2ꢀPCCA exhibited an EC of 3.1
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nM in a HTRF (Cisbio Bioassays) cAMP assay. The discrepancy between the EC values of 2ꢀPCCA is
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possibly due to the sensitivity of the two different assay systems. In order to facilitate the SAR study and
discovery of potent GPR88 ligands, a reliable and sensitive assay is needed. Thus, we created a Chinese
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Hamster Ovary (CHO) cell line stably expressing the PPLSꢀHAꢀGPR88 construct and measured cAMP levels
using the Lance assay platform (PerkinElmer). A preꢀprolactin leader sequence (PPLS) and an influenza
hemagglutinin (HA) tag were used in the construct to facilitate membrane expression and immunostaining of
GPR88 in CHO cells. In the stable PPLSꢀHAꢀGPR88 CHO cells, 2ꢀPCCA and its pure enantiomer 2 had EC50
values of 116 nM and 56 nM, respectively, shown in Figure 1. The phenyl analogue 3 possessed an EC of
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33 nM.
Early SAR studies suggest that the aniline moiety of 2ꢀPCCA is a suitable site for optimization.
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It
appears that the phenyl ring of the aniline moiety can tolerate substitution at the 4ꢀposition, in some cases
leading to improved potency. In order to gain additional information about the structural requirements in this
region for obtaining high potency ligands with possibly improved drugꢀlike properties (e.g., clogP <5), we
have synthesized a series of analogues (4a−g, Figure 1 and 5a−aa, Figure 2) and tested their agonist activity
in the Lance cAMP assay. These compounds bear a variety of substituents at the phenyl ring of the aniline
moiety to systematically evaluate the effects of size, polarity, lipophilicity, and steric and electronic tolerance.
The unsubstituted analogue 4a was significantly less potent than 2ꢀPCCA (2760 nM vs. 116 nM, Figure 1).
Addition of a hexyl group at the 4ꢀposition of the phenyl ring led to 4b, which regained most of the potency
(EC = 421 nM), indicating a hydrophobic pocket may be present in the binding site to interact with this
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region. Replacing the hexyl group with cyclohexyl (4c) or phenyl (4d) further improved the EC to 283 nM.
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However, moving the distal phenyl group in 4d away from the aniline phenyl ring by replacement with
phenoxy (4e), benzyl (4f) or phenethyl (4g) resulted in deteriorated activity. These findings suggested that an
aromatic stacking interaction between the biphenyl moiety and the receptor might contribute to the agonist
activity of 2ꢀPCCA.
Substituent effect of the distal phenyl ring of 4d was next examined and the results are summarized in
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Table 1. Consistent with the previous findings, the 4ꢀposition of the distal phenyl ring tolerated small to
medium sized alkyl substitutions with the ethyl analogue 5c (EC = 85 nM) being the most potent compound
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and the bulk tꢀbutyl 5f (EC = 515 nM) being the least potent compound in the series. Interestingly, the large
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alkyl substitutions, such as hexyl (5g), cyclohexyl (5h) and phenyl (5i), were also well tolerated giving good
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to moderate (EC = 136−289 nM) activity. This provided further evidence to support the previous
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hypothesis that the 4’ꢀsubstitution at the biphenyl group likely extends into a hydrophobic pocket.
Improvement of the potency of 4d was also observed by adding small alkoxy groups at the 4ꢀposition
(5j−m). Attachment of an additional methoxy group at the 3ꢀposition of 5j led to the 3,4ꢀdimethoxy analogue
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n, resulting in approximately 10ꢀfold loss of activity (96 nM vs. 917 nM). The loss in potency of 5n was
probably due to the limited steric tolerance proximal to the distal phenyl ring, as evidenced in 5f. Somewhat
surprisingly, the 3,4ꢀmethylenedioxy analogue 5o possessed good activity with an EC of 204 nM. The
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electronꢀwithdrawing groups (5b, 5p-u) were well tolerated at the 4ꢀpostion, with EC values ranging from
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187 nM to 351 nM. However, substituents containing a hydrogenꢀbond donor (5v and 5w) led to a significant
decrease in activity. The dimethylamino group (5x) had an improved potency compared to the amino group in
5w (294 nM vs. 1120 nM), suggesting that both basicity and lipophilicity of the substituents at the 4ꢀpostion
may have a marked influence on the activity. Finally, substitution at the 4ꢀposition with an amide group (5y,
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z and 5aa) gave the least activity in the series.
Calculated physiochemical properties such as lipophilicity (clogP), topological polar surface area
(TPSA), and derived values such as logBB are useful indicators of a compound’s potential to penetrate the
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brain. In general, CNS drugs have clogP in the range of 2−4, TPSA less than 76 Å , and logBB greater
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than −1. These molecular descriptors were calculated for 2ꢀPCCA, as well as selected compounds 5a, 5c, 5j,
l, 5m, and 5o (Table 2). 2ꢀPCCA has the highest clogP value of 6.19. All other calculated compounds have
clogP greater than 4, among which 5o has the lowest clogP = 4.41. 2ꢀPCCA and all of the analogues, except
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o, have TPSA less than 76 Å . Even 5o has a TPSA = 77.68 Å , just above the recommended threshold (a
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TPSA cutoff of 90 Å has also been suggested for CNS drugs ). All compounds have logBB values greater
than −1, predicting potential brain penetration.
QSAR Analysis. In a complementary effort to assess the SAR, we computed a series of properties of the
substituents and analyzed their importance in explaining the observed variations in lnEC values. We
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computed optimized geometries of the hydrogen capped substituted biphenyl fragments using MOPAC7 and
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then computed shape/steric (Sterimol parameters/globularity), polar surface area (PSA), volume, solvent
accessible area, total hydrophobicity (TotalHyd) and HOMO/LUMO frontier orbital energetics using these
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semiempirical results in addition to GRAPHA assessments of the varied substituted biphenyl fragments.
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LogP and Molar refractivity and number of rotatable bonds were computed using OpenBabel 2.3.2.
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Isotropic polarizabilities of the substituents were computed using GAMESSꢀUK at 6ꢀ31G** B3LYP DFT
optimized geometries.
The covariance matrix of all the descriptors was computed to facilitate exploring QSAR employing
descriptors with reasonable ‘orthogonality’ in variance. In this way we maximized the linear independence of
the descriptors and sought to explain the lnEC variations via the QSAR description with the smallest
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number of possible terms while probing multiple facets of each of the substitutions. While many models were
explored in an attempt to find explanations for the lnEC ’s in terms of all the computed metrics, we focused
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on a buildup of three models and examined at each stage the impact of an additional descriptor.
Table 3 reports the best (model C) of three successive models, while the Supporting Information
contains the description and statistics for the other two models A and B upon which model C was based. In
the model C, lnEC ~ −1.0 L + 1.7 TotalHyd + 0.04 V_S + 0.1 PSA – 0.9 LogP + 1.1 B4 + 8.0. The
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significance of both the ‘metric’ within the given QSAR model as well as the ANOVA computations allowed
one to decide on the significance of any improvement in the multivariateꢀlinear least squares with model
expansion. The model table provides the coefficients of the metrics in the model QSAR equation, the standard
error (an error level of the model computed from the sumsꢀofꢀsquares of deviations from the theoretical fit
and the number of cases), the tꢀvalue (the tꢀstatistic providing an indication of whether the coefficient is
different than zero), and Pr (the pꢀvalue for the hypothesis test for which the tꢀvalue is the test statistic). As
shown in Table 3, the pꢀvalue was < 0.05 and indicated significance. At a 95% confidence limit, the model
had significance in explaining the variations of lnEC in terms of the physiochemical descriptors probed. The
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R value illustrated reasonable correlations in the fit described by the theoretical QSAR equation to the
observed lnEC value, and this coefficient of determination showed that the variables employed provided a
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8% explanation for the variations in lnEC . Figure 3 shows a plot of the predicted versus the experimental
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lnEC values.
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The model C contains six physiochemical descriptors. PSA, logP, and total hydrophobicity are common
features used in the assessment of drug candidates, but the Lꢀ and B4ꢀSterimol (L and B4, respectively) and
the vibrational thermochemical (V_S) descriptors are much less well known. An example of the Lꢀ and B4ꢀ
Sterimol metrics for two ligands 5j and 5o is shown in Figure 4. The LꢀSterimol axis is the longest dimension
spanning the molecular surface of the substituent. The B4ꢀSterimol descriptor is orthogonal to this Lꢀ
dimension and provides a metric of a width of the substituent to the Lꢀaxis. Such orthogonal descriptor
captures the width of the binding pocket orthogonal to the long axis of the substituent.
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The vibrational parameter (V_S) is a descriptor that captures a dynamical aspect of the ligand and
possibly indicates a propensity for cooperative binding. While differential binding to the orthosteric site of a
GPCR is not likely to result in significant receptor structural changes near the orthosteric site, small changes
in a ligand’s architecture may result in differential stabilization of activated conformations of the GPCR. The
effect of the local fluctuations in a portion of a ligand substituent may bias larger cooperative structural
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changes at the intracellular side to conformations constituting an ‘active state’.
Small changes in a
ligand’s dynamics may in this way alter ionic locks/loop and helical structure at the intracellular region that
modifies interactions with GPCR partners (e.g., G proteins, βꢀarrestin). While such a rationale for V_S
remains speculative, there was a plausible connection of the vibrational parameter to the lnEC in our QSAR
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model.
Docking Analysis of (1R,2R)-2-PCCA. While the QSAR model compared reasonably with the qualitative
facets of the SAR deductions at the aniline moiety, we sought to discover if a homology model, based on
existing crystallographic templates, would give useful information about the receptor binding site
characteristics for this region. The focus of this limited structural exploration was to ascertain whether the
QSAR is consistent with structural principles/orthosteric ligand binding site based on the docking and
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MMGBSA (Molecular Mechanics Generalized Born Surface Area) scoring analysis of (1R,2R)ꢀ2ꢀPCCA.
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Sequence alignments of human GPR88 with selected GPCR templates of known structure were
performed using a BLOSUM62 matrix. While no GPCR crystal structures with high or moderate sequence
identity (>40% by our own experience) to GPR88 have yet been solved, the β1ꢀadrenergic receptor (PDB:
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5A8E) has a 26% sequence identity to GPR88 deduced from a pairwise alignment. This template also has one
of the highest sequence identities in the alignment of 9 GPCRs with GPR88 (~20%, see Supporting
Information). The β1ꢀadrenergic receptor template was used to construct an initial backbone model of GPR88
26
employing Sali’s MODELLER v9.11, refining the backbone model ‘energetically’ using simulated
2
7
annealing with topological constraints. We next employed SCWRL rotamer exploration to find nonꢀclashing
sidechain orientations for nonꢀconserved residues with conserved residues between the target and template
employing conservation assessments shown in the alignment. Conserved residue sidechain from the β1ꢀ
adrenergic receptor template were initially retained in the SCWRL rotamer exploration. This initial level
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model was then energy minimized using AMBER12 to provide a homology model for docking and freeꢀ
energy scoring.
2
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We docked (1R,2R)ꢀ2ꢀPCCA using both Autodock VINA with AMBER12 MMGBSA rescoring as
30
well as GLIDEꢀSP (Schrödinger
) followed by PrimeꢀMMGBSA rescoring. As shown in Figure 5, the 4’ꢀ
propylbiphenyl group in (1R,2R)ꢀ2ꢀPCCA pointed out of the orthosteric site into the extracellular loop region.
The residue character along the binding site region housing the biphenyl is quite hydrophobic. The initial
GPR88 homology model appears to be consistent with the results of QSAR analysis in that the longꢀspatial
axis, parameterized at the LꢀSterimol, is an important facet as is the overall hydrophobicity. The model is not,
at this stage, predictive of lnEC . Refinement of the model using pure (R,R)ꢀdiastereomers is currently under
50
investigation.
CONCLUSIONS
In summary, we have designed and synthesized a series of 2ꢀPCCA analogues bearing a variety of
substituents at the phenyl ring of the aniline moiety to determine the SAR in this region. The target
compounds were evaluated in a Lance cAMP assay using stable PPLSꢀHAꢀGPR88 CHO cells. SAR studies
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suggested that there is a hydrophobic pocket in the GPR88 binding site interacting with the aniline region of
ꢀPCCA. In addition, the 4’ꢀposition of the biphenyl group was well tolerated with alkyl, alkoxy and electronꢀ
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withdrawing groups, but less tolerated with the substituents having a hydrogenꢀbond donor. Several
compounds had a slightly improved or comparable potency, but lower calculated lipophilicity than 2ꢀPCCA.
Combined with calculated TPSA and logBB values, compound 5j may provide the basis for further
optimization to develop an in vivo probe for GPR88 functional studies.
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Exploration of both a computational QSAR and an initial homology model and docking of (1R,2R)ꢀ2ꢀ
PCCA supported the SAR conclusions. The findings of statistically sound models including both Lꢀ and B4ꢀ
Sterimol parameters in the QSAR were consistent with the positioning of the 4’ꢀsubstituted biphenyl encased
in a largely hydrophobic GPR88 homology model binding site.
EXPERIMENTAL SECTION
Chemistry. General Methods. Melting points were determined using a MELꢀTEMP II capillary melting point
1
13
apparatus and are uncorrected. Nuclear magnetic resonance ( H NMR and C NMR) spectra were obtained
on a Bruker Avance DPXꢀ300 MHz NMR spectrometer. Chemical shifts are reported in parts per million
13
(
ppm) with reference to internal solvent. C NMR data of diastereomeric mixtures were not reported due to
the complicity of the spectra. Mass spectra (MS) were run on a PerkinꢀElmer Sciex API 150 EX mass
spectrometer. HRMS spectra were run on a Waters Synapt G2 HDMS QꢀTOF mass spectrometer, using
electrospray ionization in positive ion mode. Analytical thinꢀlayer chromatography (TLC) was carried out
using EMD silica gel 60 F254 TLC plates. TLC visualization was achieved with a UV lamp or in an iodine
chamber. Flash column chromatography was done on a CombiFlash Companion system using Isco prepacked
silica gel columns. Unless otherwise stated, reagentꢀgrade chemicals were obtained from commercial sources
and were used without further purification. All moistureꢀ and airꢀsensitive reactions and reagent transfers
were carried out under dry nitrogen. Synthesis and characterization of compounds 1−3, 5a−c, 5e, 5h, 5p, 5q
12
and 5t have been previously reported. All synthesized compounds were ≥95% pure as determined by HPLC
analyses (see Supporting Information).
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tert-Butyl {(2S,3S)-1-[(4-Bromophenyl)amino]-3-methylpentan-2-yl}carbamate (8a). To a solution of
−)ꢀ(2S,3S)ꢀNꢀBocꢀ2ꢀaminoꢀ3ꢀmethylꢀ1ꢀpentanol (2.17 g, 10.0 mmol) in waterꢀsaturated CH Cl (10 mL) at
(
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2
room temperature was added DessꢀMartin reagent (8.90 g, 21.0 mmol) and the reaction was stirred for 1 h.
Additional waterꢀsaturated CH Cl (5 mL) was added every 15 min during the reaction time. The mixture was
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diluted with Et O (100 mL) and poured into a solution of Na S O (17 g) in 80% saturated NaHCO (100
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2
3
3
mL). After stirring for 10 min, the layers were separated and the aqueous layer was extracted with Et O (100
2
mL). The combined organic layers were washed with iceꢀcold saturated NaHCO (30 mL) and water (30
3
mL). The solution was dried (Na SO ) and concentrated under reduced pressure to give the crude aldehyde 7.
2
4
To a solution of 4ꢀbromoaniline (6a) (1.72 g, 10.0 mmol) in dichloroethane (60 mL) was added the above
crude aldehyde, followed by NaBH(OAc) (4.24 g, 20.0 mmol). The mixture was stirred at room temperature
3
overnight. Saturated NaHCO (20 mL) was added and the layers were separated. The aqueous layer was
3
extracted with CH Cl (2 x 30 mL). The combined organic layers were washed with brine (3 x 30 mL), dried
2
2
(
Na SO ) and concentrated under reduced pressure. Flash column chromatography of the crude product on
2 4
1
silica gel using 0−30% EtOAc in hexanes afforded 8a (2.78 g, 75%) as a white solid: mp 103−105 ºC; H
NMR (300 MHz; CDCl ) δ 7.24 (d, J = 9.0 Hz, 2H), 6.45 (d, J = 9.0 Hz, 2H), 4.52 (d, J = 9.0 Hz, 1H), 4.20
3
(
br s, 1H), 3.82−3.65 (m, 1H), 3.30−3.14 (m, 1H), 3.05−2.89 (m, 1H), 1.60−1.47 (m, 1H), 1.44 (s, 9H),
1
3
1
.23−1.10 (m, 1H), 0.95 (d, J = 6.0 Hz, 3H), 0.95 (t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl ) δ 154.9,
3
+
79
145.7, 130.1, 112.4, 106.9, 77.9, 52.9, 45.0, 35.7, 26.6, 23.6, 13.8, 9.9;MS (ESI) m/z 371.3 [M + H] ( Br),
+
81
3
73.3 [M + H] ( Br).
tert-Butyl [(2S,3S)-1-Phenylamino-3-methylpentan-2-yl]carbamate (8b). The procedure for 8a was
1
followed using 140 mg (1.5 mmol) of 6b to give 336 mg (77%) of 8b as a white solid: mp 77−79 ºC; H NMR
(300 MHz; CDCl ) δ 7.16 (t, J = 7.5 Hz, 2H), 6.69 (t, J = 7.5 Hz, 1H), 6.59 (d, J = 9.0 Hz, 2H), 4.57 (d, J =
3
9
1
.0 Hz, 1H), 4.13 (br s, 1H), 3.82−3.68 (m, 1H), 3.26 (dd, J = 12.0, 3.0 Hz, 1H), 3.02 (t, J = 10.5 Hz, 1H),
1
3
.68−1.46 (m, 1H), 1.45 (s, 9H), 1.28−1.10 (m, 1H), 0.98ꢀ0.89 (m, 6H); C NMR (75 MHz; CDCl ) δ 156.6,
3
+
148.3, 129.2, 117.4, 112.8, 79.5, 54.7, 46.6, 37.5, 28.4, 25.4, 15.5, 11.7; MS (ESI) m/z 293.3 [M + H] .
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tert-Butyl {(2S,3S)-1-[(4-Hexylphenyl)amino]-3-methylpentan-2-yl}carbamate (8c). The procedure for
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8
6
1
a was followed using 355 mg (2.0 mmol) of 6c to give 526 mg (70%) of 8c as an oil: H NMR (300 MHz;
7
CDCl ) δ 6.98 (d, J = 9.0 Hz, 2H), 6.53 (d, J = 9.0 Hz, 2H), 4.52 (d, J = 9.0 Hz, 1H), 3.86 (br s, 1H),
8
9
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3.80−3.66 (m, 1H), 3.30−3.18 (m, 1H), 3.06−2.90 (m, 1H), 2.48 (t, J = 6.0 Hz, 2H), 1.70−1.53 (m, 2H), 1.44
13
(
s, 9H), 1.38−1.10 (m, 8H), 1.00−0.80 (m, 9H); C NMR (75 MHz; CDCl ) δ 156.5, 146.5, 131.8, 129.1,
3
1
12.8, 79.4, 54.8, 46.7, 37.5, 35.1, 31.9, 31.8, 29.0, 28.4, 25.4, 22.7, 15.5, 14.1, 11.7; MS (ESI) m/z 377.3 [M
+
+
H] .
tert-Butyl {(2S,3S)-1-[(4-Cyclohexylphenyl)amino]-3-methylpentan-2-yl}carbamate (8d). The procedure
for 8a was followed using 263 mg (1.5 mmol) of 6d to give 345 mg (61%) of 8d as a white solid: mp
1
1
08−110 ºC; H NMR (300 MHz; CDCl ) δ 7.02 (d, J = 9.0 Hz, 2H), 6.55 (d, J = 9.0 Hz, 2H), 4.53 (d, J = 9.0
3
Hz, 1H), 4.00 (br s, 1H), 3.80−3.66 (m, 1H), 3.32−3.20 (m, 1H), 3.10−2.90 (m, 1H), 2.46−2.31 (m, 1H), 1.90ꢀ
1
3
1.70 (m, 5H), 1.70−1.10 (m, 16H), 0.98−0.90 (m, 6H); C NMR (75 MHz; CDCl ) δ 156.5, 146.5, 137.3,
3
1
27.5, 122.8, 79.4, 54.8, 46.7, 43.7, 37.4, 34.8, 28.4, 27.1, 26.3, 25.4, 15.5, 11.7; MS (ESI) m/z 375.2 [M +
+
H] .
tert-Butyl [(2S,3S)-1-Biphenylamino-3-methylpentan-2-yl]carbamate (8e). The procedure for 8a was
1
followed using 545 mg (2.5 mmol) of 6e to give 560 mg (76%) of 8e as a white solid: mp 98−100 ºC; H
NMR (300 MHz; CDCl ) δ 7.56−7.50 (m, 2H), 7.46−7.34 (m, 4H), 7.28−7.21 (m, 1H), 6.66 (d, J = 9.0 Hz,
3
2
1
H), 4.47 (d, J = 9.0 Hz, 1H), 4.18 (br s, 1H), 3.81−3.71 (m, 1H), 3.32−3.22 (m, 1H), 3.10−2.96 (m, 1H),
13
.68−1.46 (m, 1H), 1.45 (s, 9H), 1.30−1.10 (m, 1H), 0.98 (d, J = 9.0 Hz, 3H), 0.95 (t, J = 7.5 Hz, 3H); C
NMR (75 MHz; CDCl ) δ 156.7, 148.1, 141.4, 130.2, 128.7, 127.6, 126.6, 126.1, 133.0, 79.6, 54.8, 46.6, 37.5,
3
+
2
8.5, 25.5, 15.6, 11.7; MS (ESI) m/z 369.4 [M + H] .
tert-Butyl {(2S,3S)-1-[(4-Phenoxyphenyl)amino]-3-methylpentan-2-yl}carbamate (8f). The procedure for
1
8
a was followed using 370 mg (2.0 mmol) of 6f to give 350 mg (46%) of 8f as an oil: H NMR (300 MHz;
CDCl ) δ 7.32−7.25 (m, 2H), 7.05−6.98 (m, 1H), 6.96−6.86 (m, 4H), 6.59 (d, J = 9.0 Hz, 2H), 4.56 (d, J = 9.0
3
Hz, 1H), 4.02 (br s, 1H), 3.81−3.72 (m, 1H), 3.31−3.20 (m, 1H), 3.06−2.95 (m, 1H), 1.66−1.50 (m, 1H), 1.45
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(
s, 9H), 1.26−1.10 (m, 1H), 1.02−0.90 (m, 6H); C NMR (75 MHz; CDCl ) δ 159.3, 156.7, 147.5, 145.3,
3
+
1
29.9, 121.9, 121.3, 117.1, 113.7, 79.4, 54.8, 46.9, 37.5, 28.5, 25.4, 15.6, 11.7; MS (ESI) m/z 385.4 [M + H] .
tert-Butyl {(2S,3S)-1-[(4-Benzylphenyl)amino]-3-methylpentan-2-yl}carbamate (8g). The procedure for
0
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8a was followed using 275 mg (1.5 mmol) of 6g to give 390 mg (68%) of 8g as an offꢀ white solid: mp 65−66
1
ºC; H NMR (300 MHz; CDCl ) δ 7.30−7.15 (m, 5H), 6.99 (d, J = 9.0 Hz, 2H), 6.54 (d, J = 9.0 Hz, 2H), 4.54
3
(
d, J = 9.0 Hz, 1H), 3.95 (br s, 1H), 3.87 (s, 2H), 3.83−3.70 (m, 1H), 3.30−3.18 (m, 1H), 3.06−2.95 (m, 1H),
1
3
1.65−1.50 (m, 1H), 1.44 (s, 9H), 1.28−1.10 (m, 1H), 1.01−0.90 (m, 6H); C NMR (75 MHz; CDCl ) δ 156.5,
3
1
46.6, 142.1, 130.0, 129.7, 128.8, 128.3, 125.8, 113.0, 79.5, 54.7, 46.8, 41.1, 37.5, 28.4, 25.4, 15.5, 11.7; MS
+
(
ESI) m/z 383.5 [M + H] .
tert-Butyl {(2S,3S)-1-[(4- Phenethylphenyl)amino]-3-methylpentan-2-yl}carbamate (8h). The procedure
for 8a was followed using 290 mg (1.5 mmol) of 6h to give 325 mg (55%) of 8h as a white solid: mp 55−57
1
ºC; H NMR (300 MHz; CDCl ) δ 7.30−7.10 (m, 5H), 6.99 (d, J = 9.0 Hz, 2H), 6.59 (d, J = 9.0 Hz, 2H), 4.62
3
(
d, J = 9.0 Hz, 1H), 3.95 (br s, 1H), 3.80−3.68 (m, 1H), 3.25 (dd, J = 12.0, 3.0 Hz, 1H), 3.02 (t, J = 10.5 Hz,
1
3
1
H), 2.90−2.76 (, 4H), 1.65−1.50 (m, 1H), 1.44 (s, 9H), 1.23−1.10 (m, 1H), 0.98−0.88 (m, 6H); C NMR (75
MHz; CDCl ) δ 156.6, 145.9, 142.1, 131.4, 129.2, 128.5, 128.3, 125.8, 113.5, 79.5, 54.6, 47.2, 38.3, 37.5,
3
+
3
7.1, 28.4, 25.4, 15.5, 11.7; MS (ESI) m/z 397.5 [M + H] .
tert-Butyl [(2S,3S)-1-{4-Bromophenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9a). To a solution of (±)ꢀtransꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropanecarboxylic acid
0.40 g, 2.0 mmol) in CH Cl (20 mL) at room temperature was added oxalyl chloride (0.35 mL, 4.0 mmol)
(
2
2
and DMF (50 µL). The mixture was stirred at 40 °C for 2 h, then cooled to room temperature and
concentrated under reduced pressure. The residue was dissolved in CH Cl (20 mL) and treated with 8a (0.74
2
2
g, 2.0 mmol) and Et N (1.1 mL, 8.0 mmol). The resulting solution was stirred at room temperature overnight.
3
Saturated NaHCO (10 mL) was added and the layers were separated. The aqueous layer was extracted with
3
CH Cl (3 x 10 mL). The combined organic layers were washed with brine (3 x 20 mL), dried (Na SO ) and
2
2
2
4
concentrated under reduced pressure. Flash column chromatography of the crude product on silica gel using
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−25% EtOAc in hexanes afforded 9a (0.74 g, 72%, 1:1 diastereomeric mixture) as a light yellow foam: H
5
NMR (300 MHz; CDCl ) δ 8.30 (d, J = 6.0 Hz, 1H), 7.58−7.34 (m, 3H), 7.22−6.98 (m, 4H), 4.96 (d, J = 9.0
6
7
8
9
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Hz, 1H), 4.45−4.25 (m, 1H), 3.80−3.78 (m, 1H), 3.21−3.06 (m, 1H), 2.71−2.62 (m, 0.5H), 2.58−2.48 (m,
0
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8
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.5H), 1.98−1.86 (m, 1H), 1.75−1.62 (m, 1H), 1.60−1.35 (m, 3H), 1.45 and 1.40 (2s, 9H), 1.15−1.02 (m, 1H),
+
79
+ 81
0
.92−0.80 (m, 6H); MS (ESI) m/z 516.7 [M + H] ( Br), 518.6 [M + H] ( Br).
tert-Butyl [(2S,3S)-1-{Phenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9b). The procedure for 9a was followed using 102 mg (0.35 mmol) of 8b to
1
give 85 mg (56%) of 9b (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.26 (d, J = 6.0
3
Hz, 1H), 7.54−7.46 (m, 1H), 7.38−7.10 (m, 6H), 7.03−6.96 (m, 1H), 4.96 (t, J = 7.5 Hz, 1H), 4.48−4.32 (m,
1H), 3.81−3.62 (m, 1H), 3.22−3.08 (m, 1H), 2.72−2.62 (m, 0.5H), 2.58−2.48 (m, 0.5H), 2.00−1.88 (m, 1H),
1
.72−1.48 (m, 4H), 1.46 and 1.42 (2s, 9H), 1.15−1.00 (m, 1H), 0.91−0.78 (m, 6H); MS (ESI) m/z 438.5 [M +
+
H] .
tert-Butyl [(2S,3S)-1-{4-Hexylphenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9c). The procedure for 9a was followed using 188 mg (0.5 mmol) of 8c to give
1
140 mg (54%) of 9c (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.28−8.22 (m, 1H),
3
7
.53−7.45 (m, 1H), 7.20−6.92 (m, 6H), 5.10 (t, J = 9.0 Hz, 1H), 4.45−4.31 (m, 1H), 3.80−3.60 (m, 1H),
3
.18−3.06 (m, 1H), 2.70−2.61 (m, 0.5H), 2.60−2.47 (m, 2.5H), 2.00−1.89 (m, 1H), 1.71−1.30 (m, 24H),
+
1.20−1.02 (m, 1H), 0.96−0.80 (m, 6H); MS (ESI) m/z 522.6 [M + H] .
tert-Butyl [(2S,3S)-1-{4-Cyclohexylphenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9d). The procedure for 9a was followed using 131 mg (0.35 mmol) of 8d to
1
give 104 mg (57%) of 9d (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.30−8.20 (m,
3
1H), 7.52−7.45 (m, 1H), 7.20−6.92 (m, 6H), 5.12 (dd, J = 12.0, 9.0 Hz, 1H), 4.46−4.30 (m, 1H), 3.80−3.60
(
m, 1H), 3.20−3.06 (m, 1H), 2.70−2.60 (m, 0.5H), 2.58−2.47 (m, 1.5H), 2.00−1.65 (m, 7H), 1.55−1.20 (m,
+
1
7H), 1.18−1.00 (m, 1H), 0.92−0.78 (m, 6H); MS (ESI) m/z 520.6 [M + H] .
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tert-Butyl [(2S,3S)-1-{Biphenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9e). The procedure for 9a was followed using 184 mg (0.5 mmol) of 8e to give
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1
30 mg (51%) of 9e (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.29−8.23 (m, 1H),
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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0
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7.60−7.15 (m, 11H), 7.01−6.95 (m, 1H), 5.11 (t, J = 9.0 Hz, 1H), 4.50−4.37 (m, 1H), 3.86−3.68 (m, 1H),
3
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.26−3.17 (m, 1H), 2.75−2.68 (m, 0.5H), 2.60−2.51 (m, 0.5H), 2.10−1.98 (m, 1H), 1.75−1.67 (m, 0.5H),
.66−1.58 (m, 0.5H), 1.57−1.40 (m, 3H), 1.47 and 1.43 (2s, 9H), 1.20−1.03 (m, 1H), 0.93−0.80 (m, 6H); MS
+
(ESI) m/z 514.7 [M + H] .
tert-Butyl [(2S,3S)-1-{4-Phenoxyphenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9f). The procedure for 9a was followed using 130 mg (0.34 mmol) of 8f to
1
give 57 mg (32%) of 9f (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.33−8.27 (m,
3
1
4
2
H), 7.52−7.43 (m, 1H), 7.40−7.30 (m, 2H), 7.22−7.10 (m, 4H), 7.02−6.78 (m, 5H), 5.08 (t, J = 9.0 Hz, 1H),
.55−4.40 (m, 1H), 3.82−3.66 (m, 1H), 3.20−3.02 (m, 1H), 2.68−2.60 (m, 0.5H), 2.52−2.43 (m, 0.5H),
.02−1.90 (m, 1H), 1.75−1.45 (m, 4H), 1.44 and 1.41 (2s, 9H), 1.18−1.00 (m, 1H), 0.92−0.80 (m, 6H); MS
+
(
ESI) m/z 530.7 [M + H] .
tert-Butyl [(2S,3S)-1-{4-Benzylphenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9g). The procedure for 9a was followed using 100 mg (0.26 mmol) of 8g to
1
give 65 mg (47%) of 9g (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.30−8.26 (m,
3
1
H), 7.60−7.52 (m, 1H), 7.38−6.95 (m, 11H), 5.06 (t, J = 9.0 Hz, 1H), 4.48−4.30 (m, 1H), 3.92 and 3.88 (2s,
2H), 3.80−3.66 (m, 1H), 3.20−3.05 (m, 1H), 2.70−2.62 (m, 0.5H), 2.50−2.42 (m, 0.5H), 1.98−1.90 (m, 1H),
1
.76−1.46 (m, 4H), 1.44 and 1.42 (2s, 9H), 1.20−1.02 (m, 1H), 0.93−0.82 (m, 6H); MS (ESI) m/z 528.8 [M +
+
H] .
tert-Butyl [(2S,3S)-1-{4-Phenethylphenyl-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methylpentan-2-yl]carbamate (9h). The procedure for 9a was followed using 139 mg (0.35 mmol) of 8h to
1
give 96 mg (51%) of 9h (1:1 diastereomeric mixture) as an oil: H NMR (300 MHz; CDCl ) δ 8.30−8.25 (m,
3
1
H), 7.55−7.46 (m, 1H), 7.30−6.96 (m, 11H), 5.10 (dd, J = 9.0, 3.0 Hz, 1H), 4.46−4.32 (m, 1H), 3.80−3.62
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m, 1H), 3.20−3.06 (m, 1H), 2.90−2.80 (m, 4H), 2.71−2.632 (m, 0.5H), 2.56−2.47 (m, 0.5H), 1.98−1.90 (m,
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H), 1.72−1.47 (m, 4H), 1.46 and 1.42 (2s, 9H), 1.16−1.02 (m, 1H), 0.90−0.78 (m, 6H); MS (ESI) m/z 528.8
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+
[M + H] .
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(
1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(phenyl-4-
yl)amide (4a). A solution of 9b (85 mg, 0.19 mmol) and 4 M HCl in dioxane (2 mL) in CH Cl (5 mL) was
2
2
stirred at room temperature for 6 h. The solvent was removed under reduced pressure. The resulting residue
was triturated with hexanes to give 4a dihydrochloride (76 mg, 96%, 1:1 diastereomeric mixture) as an offꢀ
1
white solid: H NMR (300 MHz; CD OD) δ 8.66−8.56 (m, 1H), 8.40−8.28 (m, 1H), 7.85−7.74 (m, 1H),
3
7
.61−7.33 (m, 6H), 4.40 (dd, J = 15.0, 9.0 Hz, 0.5H), 4.29 (dd, J = 15.0, 9.0 Hz, 0.5H), 3.78−3.56 (m, 1.5H),
3
.39−3.30 (m, 0.5H), 3.08−2.99 (m, 0.5H), 2.98−2.89 (m, 0.5H), 2.11−1.84 (m, 2H), 1.82−1.58 (m, 2H),
+
1
.40−1.10 (m, 2H), 1.00−0.76 (m, 6H); HRMS (ESI) calcd. for C H N O [M + H] : 338.2227. Found:
21 27
3
338.2241.
(
1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
hexylphenyl-4-yl)amide (4b). The procedure for 4a was followed using 135 mg (0.26 mmol) of 9c to give 121
1
mg (95%) of 4b dihydrochloride as a 1:1 diastereomeric mixture: H NMR (300 MHz; CD OD) δ 8.26−8.16
3
(m, 1H), 7.70−7.56 (m, 1H), 7.28−7.08 (m, 6H), 4.32−4.16 (m, 1H), 3.70−3.58 (m, 1H), 3.36−3.20 (m, 1H),
2
.66−2.50 (m, 3H), 1.92−1.80 (m, 1H), 1.78−1.48 (m, 4H), 1.48−1.10 (m, 9H), 1.00−0.76 (m, 9H); HRMS
+
(
ESI) calcd. for C H N O [M + H] : 422.3166. Found: 422.3170.
27
39
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(
1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
cyclohexylphenyl-4-yl)amide (4c). The procedure for 4a was followed using 104 mg (0.2 mmol) of 9d to give
1
93 mg (95%) of 4c dihydrochloride as a 1:1 diastereomeric mixture: H NMR (300 MHz; CD OD) δ
3
8
.63−8.52 (m, 1H), 8.38−8.24 (m, 1H), 7.85−7.72 (m, 1H), 7.60−7.48 (m, 1H), 7.48−7.20 (m, 4H), 4.36 (dd, J
15.0, 9.0 Hz, 0.5H), 4.25 (dd, J = 15.0, 9.0 Hz, 0.5H), 3.78−3.56 (m, 1.5H), 3.40−3.30 (m, 0.5H), 3.04−2.95
=
(m, 0.5H), 2.94−2.83 (m, 0.5H), 2.60−2.42 (m, 1H), 2.10−2.00 (m, 1H), 1.98−1.52 (m, 7H), 1.52−1.05 (m,
+
8
H), 1.00−0.76 (m, 6H); HRMS (ESI) calcd. for C H N O [M + H] : 420.3009. Found: 420.3022.
2
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(
1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(biphenyl-4-
yl)amide (4d). The procedure for 4a was followed using 125 mg (0.24 mmol) of 9e to give 115 mg (97%) of
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d dihydrochloride as a 1:1 diastereomeric mixture: H NMR (300 MHz; CD OD) δ 8.28−8.21 (m, 1H),
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
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0
7.72−7.52 (m, 5H), 7.48−7.32 (m, 5H), 7.30−7.23 (m, 1H), 7.19−7.10 (m, 1H), 4.40−4.22 (m, 1H), 3.80−3.66
(
m, 1H), 3.40−3.22 (m, 1H), 2.70−2.53 (m, 1H), 2.05−1.90 (m, 1H), 1.84−1.62 (m, 2H), 1.50−1.15 (m, 3H),
+
0
.99 and 0.97 (2d, J = 6.0 Hz, 3H), 0.90−0.80 (m, 3H); HRMS (ESI) calcd. for C H N O [M + H] :
2
7
31
3
414.2540. Found: 414.2541.
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
phenoxyphenyl-4-yl)amide (4e). The procedure for 4a was followed using 50 mg (0.09 mmol) of 9f to give 46
1
mg (97%) of 4e dihydrochloride as a 1:1 diastereomeric mixture: H NMR (300 MHz; CD OD) δ 8.60−8.46
3
(
m, 1H), 8.32−8.20 (m, 1H), 7.78−7.64 (m, 1H), 7.60−7.20 (m, 5H), 7.12−7.02 (m, 1H), 7.00−6.76 (m, 4H),
4
.32 (dd, J = 15.0, 9.0 Hz, 0.5H), 4.13 (dd, J = 15.0, 9.0 Hz, 0.5H), 3.68−3.40 (m, 1.5H), 3.35−3.25 (m,
0
.5H), 3.02−2.90 (m, 0.5H), 2.88−2.78 (m, 0.5H), 2.08−1.54 (m, 4H), 1.42−1.08 (m, 2H), 0.98−0.68 (m, 6H);
+
HRMS (ESI) calcd. for C H N O [M + H] : 430.2489. Found: 430.2505.
2
7
31
3
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
benzylphenyl-4-yl)amide (4f). The procedure for 4a was followed using 65 mg (0.12 mmol) of 9g to give 51
1
mg (83%) of 4f dihydrochloride as a 1:1 diastereomeric mixture: H NMR (300 MHz; CD OD) δ 8.52−8.46
3
(
m, 1H), 8.22−8.10 (m, 1H), 7.70−7.60 (m, 1H), 7.50−7.32 (m, 3H), 7.30−7.06 (m, 7H), 4.32 (dd, J = 15.0,
9.0 Hz, 0.5H), 4.21 (dd, J = 15.0, 9.0 Hz, 0.5H), 3.92 and 3.90 (2s, 2H), 3.75−3.65 (m, 1.5H), 3.38−3.26 (m,
0
.5H), 3.00−2.90 (m, 0.5H), 2.88−2.80 (m, 0.5H), 2.08−1.58 (m, 4H), 1.42−1.12 (m, 2H), 0.98−0.72 (m, 6H);
+
HRMS (ESI) calcd. for C H N O [M + H] : 428.2696. Found: 428.2711.
2
8
33
3
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
phenethylphenyl-4-yl)amide (4g). The procedure for 4a was followed using 96 mg (0.18 mmol) of 9h to give
1
87 mg (95%) of 4g dihydrochloride as a 1:1 diastereomeric mixture: H NMR (300 MHz; CD OD) δ
3
8
.66−8.56 (m, 1H), 8.38−8.25 (m, 1H), 7.82−7.70 (m, 1H), 7.58−7.42 (m, 1H), 7.40−7.30 (m, 2H), 7.30−7.05
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(
m, 7H), 4.42 (dd, J = 15.0, 9.0 Hz, 0.5H), 4.27 (dd, J = 15.0, 9.0 Hz, 0.5H), 3.75−3.56 (m, 1.5H), 3.38−3.28
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(
m, 0.5H), 3.06−2.96 (m, 0.5H), 2.96−2.78 (m, 4.5H), 2.10−1.82 (m, 2H), 1.80−1.58 (m, 2H), 1.40−1.10 (m,
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+
2H), 1.00−0.70 (m, 6H); HRMS (ESI) calcd. for C H N O [M + H] : 442.2853. Found: 442.2859.
2
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tert-Butyl [(2S,3S)-1-{(4’-Methylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10a). A mixture of 9a (30 mg, 0.058
mmol), 4ꢀmethylphenylboronic acid (12 mg, 0.087 mmol), Pd(dppf)Cl •CH Cl (4.35 mg, 0.0058 mmol) and
2
2
2
K PO (38 mg, 2.7 mmol) in dimethoxyethane (1 mL) and water (0.3 mL) was heated in a sealed vessel by
3
4
microwave irradiation at 160 °C for 6 min. The resulting mixture was poured into 1 N NaOH solution (5 mL)
and extracted with CH Cl (3 x 10 mL). The combined organic layers were dried (Na SO ) and concentrated
2
2
2
4
under reduced pressure. Flash column chromatography of the crude product on silica gel using 0 → 20%
1
EtOAc in hexanes afforded 10a (20 mg, 65%) as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ
3
8
.29−8.22 (m, 1H), 7.58−7.38 (m, 5H), 7.37−7.16 (m, 5H), 7.02−6.92 (m, 1H), 5.13−5.06 (m, 1H), 4.48−4.37
(
m, 1H), 3.83−3.68 (m, 1H), 3.24−3.12 (m, 1H), 2.73−2.65 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.39 (s, 3H),
2
.08−1.96 (m, 1H), 1.76−1.55 (m, 1H), 1.54−1.37 (m, 3H), 1.47 and 1.43 (2s, 9H), 1.17−1.00 (m, 1H),
+
0.92−0.78 (m, 6H); MS (ESI) m/z 528.7 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Trifluoromethylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10b). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 17 mg (0.087 mmol) of 4ꢀtrifluoromethylphenylboronic acid to give 25
1
mg (74%) of 10b as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H),
3
7
.72−7.40 (m, 7H), 7.38−7.16 (m, 3H), 7.02−6.93 (m, 1H), 5.13−5.02 (m, 1H), 4.50−4.38 (m, 1H), 3.84−3.65
(
m, 1H), 3.28−3.15 (m, 1H), 2.76−2.65 (m, 0.5H), 2.61−2.48 (m, 0.5H), 2.08−1.96 (m, 1H), 1.76−1.55 (m,
1
H), 1.54−1.37 (m, 3H), 1.47 and 1.43 (2s, 9H), 1.18−1.00 (m, 1H), 0.94−0.78 (m, 6H); MS (ESI) m/z 582.7
+
[M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Ethylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10c). The procedure for 10a was followed
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using 30 mg (0.058 mmol) of 9a and 13 mg (0.087 mmol) of 4ꢀethylphenylboronic acid to give 25 mg (80%)
1
of 10c as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.23 (m, 1H), 7.58−7.38 (m, 5H),
3
7.37−7.15 (m, 5H), 7.04−6.95 (m, 1H), 5.15−5.05 (m, 1H), 4.50−4.37 (m, 1H), 3.85−3.68 (m, 1H), 3.25−3.14
0
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0
1
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5
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8
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0
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2
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4
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8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
m, 1H), 2.76−2.66 (m, 2.5H), 2.60−2.48 (m, 0.5H), 2.10−1.96 (m, 1H), 1.76−1.54 (m, 1H), 1.53−1.37 (m,
3
H), 1.47 and 1.43 (2s, 9H), 1.28 (t, J = 7.5 Hz, 3H), 1.17−1.00 (m, 1H), 0.93−0.78 (m, 6H); MS (ESI) m/z
+
5
42.6 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Isopropylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10d). The procedure for 10a was followed
using 68 mg (0.13 mmol) of 9a and 34 mg (0.2 mmol) of 4ꢀisopropylphenylboronic acid to give 50 mg (69%)
1
of 10d as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.26−8.20 (m, 1H), 7.54−7.38 (m, 5H),
3
7
.35−7.12 (m, 5H), 7.03−6.92 (m, 1H), 5.14−5.05 (m, 1H), 4.48−4.35 (m, 1H), 3.88−3.68 (m, 1H), 3.26−3.17
(
m, 1H), 3.02−2.88 (m, 1H), 2.72−2.66 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.10−1.95 (m, 1H), 1.76−1.40 (m,
4
5
H), 1.47 and 1.43 (2s, 9H), 1.29 (d, J = 6.0 Hz, 6H), 1.18−1.02 (m, 1H), 0.95−0.82 (m, 6H); MS (ESI) m/z
+
56.9 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Isobutylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10e). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 16 mg (0.087 mmol) of 4ꢀisobutylphenylboronic acid to give 23 mg
1
(
(
77%) of 10e as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.23 (m, 1H), 7.60−7.38
3
m, 5H), 7.34−7.15 (m, 5H), 7.04−6.95 (m, 1H), 5.14−5.05 (m, 1H), 4.50−4.36 (m, 1H), 3.86−3.68 (m, 1H),
3.24−3.14 (m, 1H), 2.74−2.65 (m, 0.5H), 2.60−2.46 (m, 2.5H), 2.10−1.82 (m, 2H), 1.76−1.54 (m, 1H),
1
.53−1.36 (m, 3H), 1.47 and 1.43 (2s, 9H), 1.16−1.00 (m, 1H), 0.98−0.78 (m, 12H); MS (ESI) m/z 570.6 [M +
+
H] .
tert-Butyl [(2S,3S)-1-{(4’-tert-butylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10f). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 16 mg (0.087 mmol) of 4ꢀtertꢀbutylphenylboronic acid to give 20 mg
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61%) of 10f as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.20 (m, 1H), 7.58−7.45
3
5
(
m, 7H), 7.30−7.12 (m, 3H), 7.00−6.92 (m, 1H), 5.12−5.04 (m, 1H), 4.45−4.33 (m, 1H), 3.82−3.66 (m, 1H),
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3.25−3.15 (m, 1H), 2.72−2.65 (m, 0.5H), 2.60−2.48 (m, 0.5H), 2.08−1.95 (m, 1H), 1.75−1.40 (m, 4H), 1.47
0
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and 1.43 (2s, 9H), 1.36 (s, 9H), 1.16−1.02 (m, 1H), 0.96−0.80 (m, 6H); MS (ESI) m/z 570.8 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-hexylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10g). The procedure for 10a was followed
using 68 mg (0.13 mmol) of 9a and 42 mg (0.2 mmol) of 4ꢀhexylphenylboronic acid to give 40 mg (51%) of
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1
0g as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.21 (m, 1H), 7.58−7.40 (m, 5H),
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7
.36−7.15 (m, 5H), 7.05−6.94 (m, 1H), 5.16−5.08 (m, 1H), 4.50−4.38 (m, 1H), 3.88−3.68 (m, 1H), 3.27−3.15
(
m, 1H), 2.76−2.50 (m, 3H), 2.10−1.95 (m, 1H), 1.72−1.22 (m, 12H), 1.47 and 1.43 (2s, 9H), 1.18−1.02 (m,
+
1
H), 0.98−0.80 (m, 9H); MS (ESI) m/z 599.0 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Cyclohexylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10h). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 18 mg (0.087 mmol) of 4ꢀcyclohexylphenylboronic acid to give 28 mg
1
(81%) of 10h as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.21 (m, 1H), 7.58−7.40
3
(
m, 5H), 7.32−7.12 (m, 5H), 7.02−6.92 (m, 1H), 5.15−5.05 (m, 1H), 4.50−4.36 (m, 1H), 3.88−3.68 (m, 1H),
3
.26−3.13 (m, 1H), 2.75−2.64 (m, 0.5H), 2.60−2.48 (m, 1.5H), 2.05−1.71 (m, 7H), 1.69−1.25 (m, 8H), 1.47
+
and 1.43 (2s, 9H), 1.17−1.00 (m, 1H), 0.96−0.80 (m, 6H); MS (ESI) m/z 596.9 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Phenylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10i). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 18 mg (0.087 mmol) of 4ꢀbiphenylboronic acid to give 22 mg (65%) of
1
1
0i as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H), 7.70−7.42 (m, 10H),
3
7.42−7.16 (m, 5H), 7.03−6.95 (m, 1H), 5.15−5.05 (m, 1H), 4.50−4.38 (m, 1H), 3.88−3.68 (m, 1H), 3.30−3.18
(
(
m, 1H), 2.78−2.65 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.10−1.98 (m, 1H), 1.80−1.40 (m, 4H), 1.48 and 1.44
+
2s, 9H), 1.20−1.02 (m, 1H), 0.95−0.80 (m, 6H); MS (ESI) m/z 590.4 [M + H] .
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tert-Butyl [(2S,3S)-1-{(4’-Methoxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10j). The procedure for 10a was followed
using 68 mg (0.13 mmol) of 9a and 30 mg (0.2 mmol) of 4ꢀmethoxyphenylboronic acid to give 52 mg (74%)
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0
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of 10j as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.25 (m, 1H), 7.56−7.36 (m, 5H),
3
7
1
.32−7.16 (m, 3H), 7.04−6.93 (m, 3H), 5.15−5.06 (m, 1H), 4.50−4.37 (m, 1H), 3.85 (s, 3H), 3.84−3.68 (m,
H), 3.28−3.12 (m, 1H), 2.76−2.68 (m, 0.5H), 2.60−2.52 (m, 0.5H), 2.10−1.98 (m, 1H), 1.76−1.40 (m, 4H),
+
1.47 and 1.43 (2s, 9H), 1.20−1.02 (m, 1H), 0.95−0.80 (m, 6H); MS (ESI) m/z 544.7 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Trifluoromethoxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10k). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 18 mg (0.087 mmol) of 4ꢀtrifluoromethoxyphenylboronic acid to give
1
1
8 mg (52%) of 10k as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H),
3
7.58−7.40 (m, 5H), 7.38−7.16 (m, 5H), 7.04−6.96 (m, 1H), 5.10−5.03 (m, 1H), 4.50−4.38 (m, 1H), 3.82−3.68
(
m, 1H), 3.28−3.16 (m, 1H), 2.76−2.68 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.08−1.94 (m, 1H), 1.75−1.40 (m,
+
4
H), 1.47 and 1.43 (2s, 9H), 1.20−1.05 (m, 1H), 0.96−0.82 (m, 6H); MS (ESI) m/z 598.9 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Ethoxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10l). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 14 mg (0.087 mmol) of 4ꢀethoxyphenylboronic acid to give 22 mg
1
(
(
68%) of 10l as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.20 (m, 1H), 7.55−7.40
3
m, 5H), 7.30−7.12 (m, 3H), 7.02−6.92 (m, 3H), 5.12−5.05 (m, 1H), 4.48−4.38 (m, 1H), 4.15−4.04 (m, 2H),
3.86−3.68 (m, 1H), 3.26−3.15 (m, 1H), 2.76−2.68 (m, 0.5H), 2.60−2.52 (m, 0.5H), 2.08−1.96 (m, 1H),
1
.78−1.40 (m, 4H), 1.47 and 1.42 (2s, 9H), 1.20−1.04 (m, 1H), 0.94−0.82 (m, 9H); MS (ESI) m/z 558.8 [M +
+
H] .
tert-Butyl [(2S,3S)-1-{(4’-Isopropoxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10m). The procedure for 10a was
followed using 30 mg (0.058 mmol) of 9a and 16 mg (0.087 mmol) of 4ꢀisopropoxyphenylboronic acid to
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give 20 mg (60%) of 10m as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.22 (m, 1H),
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8
9
1
1
1
1
1
1
1
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.54−7.38 (m, 5H), 7.32−7.15 (m, 3H), 7.05−6.92 (m, 3H), 5.15−5.06 (m, 1H), 4.68−4.55 (m, 1H), 4.50−4.38
(m, 1H), 3.86−3.68 (m, 1H), 3.25−3.15 (m, 1H), 2.76−2.68 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.10−1.96 (m,
0
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H), 1.77−1.40 (m, 4H), 1.47 and 1.43 (2s, 9H), 1.37 (d, J = 6.0 Hz, 6H), 1.20−1.05 (m, 1H), 0.95−0.80 (m,
+
H); MS (ESI) m/z 572.9 [M + H] .
tert-Butyl [(2S,3S)-1-{(3’,4’-Dimethoxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10n). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 16 mg (0.087 mmol) of 3,4ꢀdimethoxyphenylboronic acid to give 19 mg
1
(
(
57%) of 10n as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.20 (m, 1H), 7.55−7.40
3
m, 2H), 7.32−6.90 (m, 8H), 5.15−5.08 (m, 1H), 4.50−4.38 (m, 1H), 3.96 (s, 3H), 3.33 (s, 3H), 3.85−3.68 (m,
1H), 3.26−3.15 (m, 1H), 2.78−2.68 (m, 0.5H), 2.62−2.53 (m, 0.5H), 2.10−1.98 (m, 1H), 1.76−1.40 (m, 4H),
+
1
.47 and 1.43 (2s, 9H), 1.20−1.03 (m, 1H), 0.96−0.80 (m, 6H); MS (ESI) m/z 574.9 [M + H] .
tert-Butyl [(2S,3S)-1-{(3’,4’-Methylenedioxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10o). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 14 mg (0.087 mmol) of 3,4ꢀmethylenedioxyphenylboronic acid to give
1
20 mg (62%) of 10o as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H),
3
7
.56−7.33 (m, 3H), 7.32−7.15 (m, 3H), 7.08−6.92 (m, 3H), 6.90−6.85 (m, 1H), 6.01 (s, 2H), 5.10−5.04 (m,
H), 4.50−4.38 (m, 1H), 3.85−3.68 (m, 1H), 3.26−3.16 (m, 1H), 2.76−2.66 (m, 0.5H), 2.60−2.50 (m, 0.5H),
1
2.10−1.98 (m, 1H), 1.76−1.40 (m, 4H), 1.47 and 1.42 (2s, 9H), 1.20−1.02 (m, 1H), 0.96−0.80 (m, 6H); MS
+
(
ESI) m/z 558.7 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Fluorobiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10p). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 12 mg (0.087 mmol) of 4ꢀfluorophenylboronic acid to give 20 mg (74%)
1
of 10p as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H), 7.56−7.48 (m, 4H),
3
7
.30−7.18 (m, 6H), 7.02−6.92 (m, 1H), 5.12−5.05 (m, 1H), 4.52−4.38 (m, 1H), 3.82−3.65 (m, 1H), 3.25−3.12
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(
m, 1H), 2.76−2.65 (m, 0.5H), 2.61−2.48 (m, 0.5H), 2.08−1.95 (m, 1H), 1.78−1.55 (m, 1H), 1.54−1.37 (m,
+
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H), 1.47 and 1.43 (2s, 9H), 1.18−1.00 (m, 1H), 0.95−0.80 (m, 6H); MS (ESI) m/z 532.5 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Chlorobiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
0
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0
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10q). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 14 mg (0.087 mmol) of 4ꢀchlorophenylboronic acid to give 22 mg
1
(
74%) of 10q as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.21 (m, 1H), 7.55−7.36
3
(m, 6H), 7.30−7.15 (m, 4H), 7.02−6.95 (m, 1H), 5.10−5.00 (m, 1H), 4.50−4.35 (m, 1H), 3.80−3.62 (m, 1H),
3
1
.26−3.15 (m, 1H), 2.76−2.65 (m, 0.5H), 2.61−2.48 (m, 0.5H), 2.08−1.95 (m, 1H), 1.75−1.55 (m, 1H),
.54−1.37 (m, 3H), 1.47 and 1.42 (2s, 9H), 1.16−1.00 (m, 1H), 0.95−0.80 (m, 6H); MS (ESI) m/z 548.5 [M +
+
H] .
tert-Butyl [(2S,3S)-1-{(4’-Cyanobiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10r). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 13 mg (0.087 mmol) of 4ꢀcyanophenylboronic acid to give 20 mg (64%)
1
of 10r as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.20 (m, 1H), 7.76−7.42 (m, 7H),
3
7
.40−7.16 (m, 3H), 7.02−6.92 (m, 1H), 5.10−5.00 (m, 1H), 4.50−4.38 (m, 1H), 3.80−3.64 (m, 1H), 3.26−3.14
(m, 1H), 2.76−2.66 (m, 0.5H), 2.61−2.52 (m, 0.5H), 2.08−1.95 (m, 1H), 1.75−1.40 (m, 4H), 1.47 and 1.42
+
(
2s, 9H), 1.18−1.00 (m, 1H), 0.96−0.80 (m, 6H); MS (ESI) m/z 539.3 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Nitrobiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10s). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 15 mg (0.087 mmol) of 4ꢀnitrophenylboronic acid to give 21 mg (65%)
1
of 10s as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.35−8.21 (m, 3H), 7.75−7.44 (m, 5H),
3
7
.40−7.16 (m, 3H), 7.02−6.95 (m, 1H), 5.08−4.98 (m, 1H), 4.50−4.38 (m, 1H), 3.80−3.62 (m, 1H), 3.28−3.18
(
(
m, 1H), 2.76−2.66 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.08−1.94 (m, 1H), 1.76−1.40 (m, 4H), 1.47 and 1.42
+
2s, 9H), 1.17−1.00 (m, 1H), 0.95−0.80 (m, 6H); MS (ESI) m/z 559.4 [M + H] .
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tert-Butyl [(2S,3S)-1-{(4’-Acetylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10t). The procedure for 10a was followed
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using 30 mg (0.058 mmol) of 9a and 14 mg (0.087 mmol) of 4ꢀacetylphenylboronic acid to give 25 mg (76%)
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of 10t as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.21 (m, 1H), 8.06−7.98 (m, 2H),
3
7
.68−7.48 (m, 5H), 7.38−7.15 (m, 3H), 7.02−6.93 (m, 1H), 5.10−5.00 (m, 1H), 4.50−4.36 (m, 1H), 3.80−3.62
(
m, 1H), 3.28−3.15 (m, 1H), 2.75−2.65 (m, 0.5H), 2.64 (s, 3H), 2.61−2.48 (m, 0.5H), 2.08−1.95 (m, 1H),
1.76−1.55 (m, 1H), 1.54−1.37 (m, 3H), 1.47 and 1.43 (2s, 9H), 1.16−0.98 (m, 1H), 0.92−0.78 (m, 6H); MS
+
(ESI) m/z 557.2 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Ethoxycarbonylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10u). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 17 mg (0.087 mmol) of 4ꢀethoxycarbonylphenylboronic acid to give 22
1
mg (65%) of 10u as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H),
3
8
.16−8.08 (m, 2H), 7.66−7.48 (m, 5H), 7.38−7.16 (m, 3H), 7.02−6.95 (m, 1H), 5.10−5.02 (m, 1H), 4.50−4.36
(
m, 3H), 3.85−3.66 (m, 1H), 3.28−3.16 (m, 1H), 2.75−2.65 (m, 0.5H), 2.62−2.50 (m, 0.5H), 2.10−1.98 (m,
1
H), 1.76−1.37 (m, 4H), 1.47 and 1.43 (2s, 9H), 1.20−1.02 (m, 1H), 0.95−0.81 (m, 9H); MS (ESI) m/z 586.5
+
[M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Hydroxybiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10v). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 25 mg (0.087 mmol) of 4ꢀ(tertꢀbutoxycarboxy)phenylboronic acid to
1
give 22 mg (72%) of 10v as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.52 (br s, 1H),
3
8.28−8.18 (m, 1H), 7.60−7.50 (m, 1H), 7.38−7.10 (m, 7H), 7.08−7.00 (m, 1H), 7.76−6.68 (m, 2H), 5.26−5.18
(
m, 1H), 4.52−4.40 (m, 1H), 3.82−3.68 (m, 1H), 3.20−3.08 (m, 1H), 2.78−2.68 (m, 0.5H), 2.62−2.52 (m,
0
.5H), 2.15−2.08 (m, 1H), 1.76−1.40 (m, 4H), 1.51 and 1.46 (2s, 9H), 1.20−1.02 (m, 1H), 0.96−0.80 (m, 6H);
+
MS (ESI) m/z 530.9 [M + H] .
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tert-Butyl [(2S,3S)-1-{[4’-(tert-Butoxycarbonylamino)biphenyl-4-yl]-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10w). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 21 mg (0.087 mmol) of 4ꢀ(tertꢀbutoxycarbonylamino)phenylboronic
1
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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8
9
0
1
2
3
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7
8
9
0
acid to give 22 mg (60%) of 10w as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.20
3
(
m, 1H), 7.52−7.40 (m, 7H), 7.30−7.12 (m, 3H), 7.02−6.94 (m, 1H), 6.68 (s, 1H), 5.12−5.06 (m, 1H),
.48−4.38 (m, 1H), 3.82−3.67 (m, 1H), 3.25−3.15 (m, 1H), 2.76−2.68 (m, 0.5H), 2.58−2.48 (m, 0.5H),
4
2.08−1.98 (m, 1H), 1.77−1.40 (m, 4H), 1.53 (s, 9H), 1.47 and 1.43 (2s, 9H), 1.20−1.02 (m, 1H), 0.95−0.80
+
(m, 6H); MS (ESI) m/z 629.8 [M + H] .
tert-Butyl [(2S,3S)-1-{[4’-(Dimethylamino)biphenyl-4-yl]-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10x). The procedure for 10a was followed
using 68 mg (0.13 mmol) of 9a and 35 mg (0.2 mmol) of 4ꢀ(dimethylamino)phenylboronic acid to give 52 mg
1
(
(
72%) of 10x as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.22 (m, 1H), 7.55−7.36
3
m, 5H), 7.30−7.12 (m, 3H), 7.02−6.96 (m, 1H), 6.84−6.76 (m, 2H), 5.18−5.10 (m, 1H), 4.50−4.38 (m, 1H),
3
.88−3.70 (m, 1H), 3.26−3.16 (m, 1H), 2.99 (s, 6H), 2.76−2.68 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.10−1.98
(
m, 1H), 1.72−1.40 (m, 4H), 1.47 and 1.43 (2s, 9H), 1.20−1.02 (m, 1H), 0.98−0.82 (m, 6H); MS (ESI) m/z
+
557.8 [M + H] .
tert-Butyl [(2S,3S)-1-{(4’-Acetamidobiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10y). The procedure for 10a was followed
using 30 mg (0.058 mmol) of 9a and 15.6 mg (0.087 mmol) of 4ꢀacetamidophenylboronic acid to give 18 mg
1
(
54%) of 10y as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.20 (m, 1H), 7.96−7.80
3
(m, 1H), 7.62−7.34 (m, 7H), 7.30−7.15 (m, 3H), 7.02−6.96 (m, 1H), 5.18−5.08 (m, 1H), 4.50−4.38 (m, 1H),
.82−3.66 (m, 1H), 3.28−3.15 (m, 1H), 2.76−2.67 (m, 0.5H), 2.60−2.50 (m, 0.5H), 2.18 (s, 3H), 2.08−1.95
3
(
m, 1H), 1.76−1.40 (m, 4H), 1.47 and 1.43 (2s, 9H), 1.20−1.02 (m, 1H), 0.96−0.80 (m, 6H); MS (ESI) m/z
+
571.6 [M + H] .
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tert-Butyl [(2S,3S)-1-{(4’-Carbamoylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10z). The procedure for 10a was followed
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1
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using 30 mg (0.058 mmol) of 9a and 14 mg (0.087 mmol) of 4ꢀcarbamoylphenylboronic acid to give 18 mg
1
0
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4
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0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(56%) of 10z as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.30−8.21 (m, 1H), 7.92−7.86
3
(
m, 2H), 7.66−7.46 (m, 5H), 7.40−7.18 (m, 3H), 7.05−6.98 (m, 1H), 6.16 (br s, 1H), 5.80 (br s, 1H),
.10−5.05 (m, 1H), 4.50−4.38 (m, 1H), 3.82−3.64 (m, 1H), 3.28−3.16 (m, 1H), 2.76−2.68 (m, 0.5H),
5
2.60−2.50 (m, 0.5H), 2.08−1.96 (m, 1H), 1.76−1.40 (m, 4H), 1.47 and 1.42 (2s, 9H), 1.20−1.02 (m, 1H),
+
0
.95−0.81 (m, 6H); MS (ESI) m/z 558.1 [M + H] .
tert-Butyl [(2S,3S)-1-{[4’-(Dimethylcarbamoyl)biphenyl-4-yl]-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-3-methylpentan-2-yl]carbamate (10aa). The procedure for 10a was
followed using 30 mg (0.058 mmol) of 9a and 17 mg (0.087 mmol) of 4ꢀ(dimethylcarbamoyl)phenylboronic
1
acid to give 20 mg (56%) of 10aa as a 1:1 diastereomeric mixture: H NMR (300 MHz; CDCl ) δ 8.28−8.22
3
(
m, 1H), 7.60−7.38 (m, 7H), 7.32−7.12 (m, 3H), 7.02−6.95 (m, 1H), 5.10−5.00 (m, 1H), 4.50−4.38 (m, 1H),
.82−3.65 (m, 1H), 3.28−3.16 (m, 1H), 3.14 (s, 3H), 3.04 (s, 3H), 2.75−2.66 (m, 0.5H), 2.60−2.50 (m, 0.5H),
3
2.08−1.96 (m, 1H), 1.76−1.40 (m, 4H), 1.47 and 1.43 (2s, 9H), 1.20−1.02 (m, 1H), 0.96−0.80 (m, 6H); MS
+
(
ESI) m/z 585.8 [M + H] .
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
methylbiphenyl-4-yl)amide (5a). The procedure for 4a was followed using 20 mg (0.038 mmol) of 10a and 1
1
mL of 4 M HCl in dioxane to give 18 mg (95%) of 5a dihydrochloride as a 1:1 diastereomeric mixture: H
NMR (300 MHz; CD OD) δ 8.68−8.55 (m, 1H), 8.42−8.30 (m, 1H), 7.88−7.40 (m, 8H), 7.30−7.20 (m, 2H),
3
4
3
2
.50−4.40 (m, 0.5H), 4.40−4.28 (m, 0.5H), 3.88−3.56 (m, 1H), 3.48−3.38 (m, 1H), 3.12−3.05 (m, 0.5H),
.05−2.96 (m, 0.5H), 2.37 (s, 3H), 2.22−2.10 (m, 1H), 2.10−1.88 (m, 1H), 1.87−1.60 (m, 2H), 1.48−1.15 (m,
+
H), 1.06−0.80 (m, 6H); HRMS (ESI) calcd. for C H N O [M + H] : 428.2696. Found: 428.2701.
28 33
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(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
trifluoromethylbiphenyl-4-yl)amide (5b). The procedure for 4a was followed using 20 mg (0.034 mmol) of
2
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1
0b and 1 mL of 4 M HCl in dioxane to give 18 mg (95%) of 5b dihydrochloride as a 1:1 diastereomeric
1
mixture: H NMR (300 MHz; CD OD) δ 8.70−8.58 (m, 1H), 8.42−8.30 (m, 1H), 7.90−7.50 (m, 10H),
3
4.55−4.40 (m, 0.5H), 4.40−4.26 (m, 0.5H), 3.90−3.60 (m, 1H), 3.50−3.38 (m, 1H), 3.15−3.05 (m, 0.5H),
0
1
2
3
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0
1
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9
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
.05−2.92 (m, 0.5H), 2.25−2.10 (m, 1H), 2.10−1.92 (m, 1H), 1.92−1.62 (m, 2H), 1.50−1.15 (m, 2H),
+
1
.08−0.80 (m, 6H); HRMS (ESI) calcd. for C H F N O [M + H] : 482.2414. Found: 482.2416.
2
8
30
3
3
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
ethylbiphenyl-4-yl)amide (5c). The procedure for 4a was followed using 20 mg (0.037 mmol) of 10c and 1
1
mL of 4 M HCl in dioxane to give 18 mg (95%) of 5c dihydrochloride as a 1:1 diastereomeric mixture: H
NMR (300 MHz; CD OD) δ 8.70−8.58 (m, 1H), 8.45−8.30 (m, 1H), 7.88−7.45 (m, 8H), 7.32−7.20 (m, 2H),
3
4
.52−4.40 (m, 0.5H), 4.38−4.28 (m, 0.5H), 3.88−3.56 (m, 1H), 3.48−3.35 (m, 1H), 3.15−3.05 (m, 0.5H),
3.05−2.95 (m, 0.5H), 2.80−2.60 (m, 2H), 2.25−2.10 (m, 1H), 2.10−1.90 (m, 1H), 1.87−1.60 (m, 2H),
+
1
.50−1.10 (m, 5H), 1.08−0.80 (m, 6H); HRMS (ESI) calcd. for C H N O [M + H] : 442.2853. Found:
2
9
35
3
4
42.2867.
1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
(
isopropylbiphenyl-4-yl)amide (5d). The procedure for 4a was followed using 45 mg (0.08 mmol) of 10d and 2
1
mL of 4 M HCl in dioxane to give 40 mg (95%) of 5d dihydrochloride as a 1:1 diastereomeric mixture: H
NMR (300 MHz; CD OD) δ 8.60−8.50 (m, 1H), 8.30−8.18 (m, 1H), 7.70−7.48 (m, 8H), 7.36−7.28 (m, 2H),
3
4
.48−4.22 (m, 1H), 3.83−3.62 (m, 1H), 3.46−3.38 (m, 1H), 3.06−2.88 (m, 2H), 2.20−2.10 (m, 1H), 2.00−1.84
(m, 1H), 1.84−1.72 (m, 1H), 1.72−1.60 (m, 1H), 1.50−1.20 (m, 2H), 1.28 (d, J = 9.0 Hz, 6H), 1.05−0.80 (m,
+
6
H); HRMS (ESI) calcd. for C H N O [M + H] : 456.3009. Found: 456.3014.
30
37
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(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
isobutylbiphenyl-4-yl)amide (5e). The procedure for 4a was followed using 20 mg (0.035 mmol) of 10e and 1
1
mL of 4 M HCl in dioxane to give 17 mg (90%) of 5e dihydrochloride as a 1:1 diastereomeric mixture: H
NMR (300 MHz; CD OD) δ 8.68−8.58 (m, 1H), 8.40−8.28 (m, 1H), 7.92−7.43 (m, 8H), 7.30−7.20 (m, 2H),
3
4
.50−4.40 (m, 0.5H), 4.38−4.25 (m, 0.5H), 3.88−3.60 (m, 1H), 3.50−3.38 (m, 1H), 3.13−3.05 (m, 0.5H),
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.05−2.90 (m, 0.5H), 2.51 (d, J = 6.0 Hz, 2H), 2.25−2.10 (m, 1H), 2.05−1.60 (m, 4H), 1.50−1.20 (m, 2H),
5
+
1
.10−0.76 (m, 12H); HRMS (ESI) calcd. for C H N O [M + H] : 470.3166. Found: 470.3178.
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9
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1
1
1
1
1
1
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
butylbiphenyl-4-yl)amide (5f). The procedure for 4a was followed using 20 mg (0.035 mmol) of 10f and 1 mL
1
of 4 M HCl in dioxane to give 18 mg (95%) of 5f dihydrochloride as a 1:1 diastereomeric mixture: H NMR
(
300 MHz; CD OD) δ 8.68−8.58 (m, 1H), 8.42−8.30 (m, 1H), 7.88−7.46 (m, 10H), 4.50−4.38 (m, 0.5H),
3
4.38−4.22 (m, 0.5H), 3.88−3.60 (m, 1H), 3.46−3.38 (m, 1H), 3.14−3.05 (m, 0.5H), 3.05−2.94 (m, 0.5H),
2
6
.22−2.08 (m, 1H), 2.08−1.90 (m, 1H), 1.88−1.65 (m, 2H), 1.50−1.20 (m, 2H), 1.35 (s, 9H), 1.05−0.80 (m,
+
H); HRMS (ESI) calcd. for C H N O [M + H] : 470.3166. Found: 470.3178.
31
39
3
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
hexylbiphenyl-4-yl)amide (5g). The procedure for 4a was followed using 35 mg (0.058 mmol) of 10g and 1
1
mL of 4 M HCl in dioxane to give 30 mg (90%) of 5g dihydrochloride as a 1:1 diastereomeric mixture: H
NMR (300 MHz; CD OD) δ 8.52−8.42 (m, 1H), 8.22−8.10 (m, 1H), 7.65−7.32 (m, 8H), 7.25−7.10 (m, 2H),
3
4
.40−4.28 (m, 0.5H), 4.28−4.16 (m, 0.5H), 3.75−3.55 (m, 1H), 3.36−3.25 (m, 1H), 2.98−2.86 (m, 0.5H),
2
.86−2.68 (m, 0.5H), 2.60−2.45 (m, 2H), 2.10−1.95 (m, 1H), 1.90−1.60 (m, 2H), 1.60−1.45 (m, 3H),
+
1.40−1.05 (m, 8H), 0.95−0.68 (m, 9H); HRMS (ESI) calcd. for C H N O [M + H] : 498.3479. Found:
3
3
43
3
4
98.3496.
1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3S)-2-Amino-3-methylpentyl]-(4’-
(
cyclohexylbiphenyl-4-yl)amide (5h). The procedure for 4a was followed using 20 mg (0.034 mmol) of 10h
and 1 mL of 4 M HCl in dioxane to give 19 mg (98%) of 5h dihydrochloride as a 1:1 diastereomeric mixture:
1
H NMR (300 MHz; CD OD) δ 8.55−8.40 (m, 1H), 8.15−8.00 (m, 1H), 7.75−7.40 (m, 8H), 7.35−7.20 (m,
3
2
2
H), 4.48−4.20 (m, 1H), 3.88−3.68 (m, 1H), 3.42−3.35 (m, 1H), 2.98−2.88 (m, 1H), 2.68−2.48 (m, 1H),
.20−2.00 (m, 1H), 1.98−1.70 (m, 7H), 1.66−1.10 (m, 8H), 1.05−0.78 (m, 6H); HRMS (ESI) calcd. for
+
C H N O [M + H] : 496.3322. Found: 496.3323.
33
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3
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