Small molecule disruptors of the Glucokinase-Glucokinase regulatory protein interaction: 2. Leveraging structure-based drug design to identify analogues with improved pharmacokinetic profiles

Article abstract of DOI:10.1021/jm4016747

In the previous report, we described the discovery and optimization of novel small molecule disruptors of the GK-GKRP interaction culminating in the identification of 1 (AMG-1694). Although this analogue possessed excellent in vitro potency and was a useful tool compound in initial proof-of-concept experiments, high metabolic turnover limited its advancement. Guided by a combination of metabolite identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor (27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.

Full text of DOI:10.1021/jm4016747

Article
pubs.acs.org/jmc
Small Molecule Disruptors of the Glucokinase−Glucokinase
Regulatory Protein Interaction: 2. Leveraging Structure-Based Drug
Design to Identify Analogues with Improved Pharmacokinetic
Profiles
David J. St. Jean, Jr.,*,† Kate S. Ashton,† Michael D. Bartberger,‡ Jie Chen,∥ Samer Chmait,‡
Rod Cupples,§ Elizabeth Galbreath,⊥ Joan Helmering,§ Fang-Tsao Hong,† Steven R. Jordan,‡
Longbin Liu,† Roxanne K. Kunz,† Klaus Michelsen,‡ Nobuko Nishimura,† Lewis D. Pennington,†
Steve F. Poon,† Darren Reid,# Glenn Sivits,§ Markian M. Stec,† Seifu Tadesse,† Nuria Tamayo,†
Gwyneth Van,⊥ Kevin C. Yang,† Jiandong Zhang,‡,▽ Mark H. Norman,† Christopher Fotsch,†
David J. Lloyd,§ and Clarence Hale§
‡
†Department of Therapeutic DiscoveryMedicinal Chemistry, Department of Therapeutic DiscoveryMolecular Structure and
§
∥
⊥
Characterization, Department of Metabolic Disorders, Department of Pharmacokinetics and Drug Metabolism, Department of
#
Pathology, Department of Pharmaceutics Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California, 91320 and 360
Binney Street, Cambridge, Massachusetts, 02142, United States
S
* Supporting Information
ABSTRACT: In the previous report, we described the
discovery and optimization of novel small molecule disruptors
of the GK-GKRP interaction culminating in the identification
of 1 (AMG-1694). Although this analogue possessed excellent
in vitro potency and was a useful tool compound in initial
proof-of-concept experiments, high metabolic turnover limited
its advancement. Guided by a combination of metabolite
identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor
(27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK
translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.
metabolites complicated the interpretation of subsequent rat
toxicology studies involving analogue 1. Along with the poor rat
pharmacokinetic (PK) profile, this molecule also exhibited low
exposure in mice. At a 100 mg/kg dose in C57BL/6 mice, the
maximum unbound concentration of 1 was significantly less (≈
50-fold) than the cellular EC50 value.10 The combination of
poor potency and low exposure of 1 made it difficult to explore
small molecule-mediated GK-GKRP disruption in mouse
models of diabetes (e.g., db/db mice). In addition to the
poor rodent PK profiles, this compound was rapidly
metabolized in the presence of human liver microsomes
(CLint = 353 μL/min/mg). Given the liabilities of 1, our
focus turned toward the identification of analogues with
improved metabolic profiles across species while maintaining
or improving biochemical and cellular activity.
INTRODUCTION
■
Small molecule-mediated reduction of blood glucose by
allosteric activation of glucokinase (GK) is a well validated
pathway.1−4 Unfortunately, some glucokinase activators
(GKAs) have also been linked to a number of undesirable
side effects (e.g., hypoglycemia), which have limited their
therapeutic potential.1,5 In an effort to avoid the hypoglycemia
observed with GKAs, we focused on identifying compounds
that disrupt the glucokinase−glucokinase regulatory protein
(GK-GKRP) interaction. By targeting this pathway, the kinetic
parameters (i.e., S0.5 and Vmax) of GK would remain unchanged,
which could potentially mitigate the hypoglycemia associated
with overactivation.6 Additionally, because GKRP is predom-
inately expressed in liver hepatocytes, GK-GKRP modulation
would potentially avoid the insulin-mediated reduction in
glucose seen with certain GKAs.7
In the preceding report, we described the discovery of a
novel proof-of-concept tool molecule 1 (AMG-1694).8
Although this molecule was the first example of a potent GK-
GKRP disruptor to demonstrate in vivo activity in a rodent
model of diabetes,9 1 displayed a number of unfavorable
characteristics. First, methyl morpholine 1 was extensively
metabolized in rats. The formation of a large number of
RESULTS AND DISCUSSION
■
To address the poor metabolic profile of 1, metabolite
identification studies were performed using both rat and
Received: October 28, 2013
© XXXX American Chemical Society
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Scheme 1. In Vitro Metabolism of 1
a
Scheme 2. Synthesis of Analogues with Methyl Morpholine Replacements (See Table 1)
a
Reagents and conditions: (a) secondary amine, K2CO3, MeCN, 140 °C; (b) BCl3, CH2Cl2, 0 °C.
human liver microsomes (RLM/HLM). These experiments
revealed that cleavage of the morpholine ring was the primary
route of oxidative metabolism (Scheme 1). With a goal to
improve the in vitro/in vivo PK profile, we initially sought to
replace the metabolically liable methyl morpholine with groups
that maintained the overall steric footprint and hydrogen bond
acceptor features of 1. To that end, the syntheses of these
analogues were straightforward (Scheme 2). Starting from
known mesylate 2,11 SN2 displacement with a variety of
secondary amines provided the desired analogues. The
synthesis of the hydroxylated bridged morpholine derivatives
(9 and 10) required an additional manipulation (BCl3) to
remove a benzyl protecting group installed prior to the amine
coupling.
The GKRP activities were evaluated using both a biochemical
and cellular assay. The AlphaScreen was a binding assay that
measured the ability of a small molecule to disrupt the GK-
GKRP interaction.8 For our primary cellular assay, nuclear to
cytoplasmic GK translocation in mouse primary hepatocytes
was quantified using an Operetta platform.9,12 In addition to
the primary assays, the in vitro metabolic stabilities were
determined using both rat and human liver microsomes.13 The
in vitro profiles of the analogues which contained methyl
morpholine replacements are shown in Table 1. To allow for a
direct comparison between analogues, 1 was prepared as a
mixture of epimers at the tertiary carbinol (3). The 4,4-difluoro
piperidine analogue 4 showed significantly weaker potency (≈
60-fold loss) relative to 3 with no gain in metabolic stability.
The sulfone derivative (5) demonstrated an improved LM
profile, albeit with a substantial loss in activity. In our previous
SAR studies, we demonstrated that the incorporation of a
methyl group on the methylene adjacent to the nitrogen atom
of the morpholine ring could improve in vitro activity.8
Application of this strategy to 5 resulted in a modest increase in
biochemical activity (6, IC50 = 0.090 μM). Spirocyclic analogue
7 showed an improvement in rat metabolic stability relative to
methyl morpholine 3, but a significant drop in activity was once
again observed. Bicyclic morpholine 8 was as potent as
analogue 3 but suffered from high microsomal turnover. In
an attempt to improve the metabolic stability of 8, hydroxylated
analogues 9 and 10 were prepared.14 Compound 10 showed a
significant improvement in LM stability relative to 8, therefore
the four stereoisomers of 10 were isolated by chiral SFC (11−
14). Unfortunately, despite the promising in vitro activity and
improved in vitro stability relative to 3, these analogues
displayed poor rat in vivo PK profiles. All four compounds
displayed high in vivo clearances (CL), high volumes of
distributions (Vss), and short mean resonance times (MRT).15
Given the poor rat in vivo PK profiles of 11−14, a second
metabolite identification study was initiated with bridged
morpholine 11 (Scheme 3). Unlike analogue 1, no major
metabolites derived from oxidation of the bicyclic morpholine
ring were detected. Instead, it was found that metabolism had
shifted to the thiophene sulfonamide. Oxidation of the
thiophene ring (the molecular structures of these metabolites
were not unambiguously assigned) as well as cleavage of the
sulfonamide were identified as the major metabolic pathways.
Because both the thiophene and the methyl morpholine had
been identified as metabolic liabilities, a revision of our overall
strategy was necessary. Rather than attempting to block specific
sites of metabolism, we chose to revisit the SAR of our initial
HTS hit (15, Table 3).9 By optimizing the sulfonamide present
in 15, we hoped to identify a metabolically stable scaffold for
which potency could be optimized. Although this analogue
contained a metabolically liable thiophene sulfonamide, the in
vitro stability and potency were reasonable starting points given
its comparatively simple structure relative to 1. To that end,
numerous reports have indicated that lowering lipophilicity
(i.e., lower clogP) can have beneficial effects on a compound’s
metabolic profile.16−18 Additionally, based on the analysis of
the cocrystal structure of 1 bound to hGKRP (Figure 1),9 we
rationalized that a hydrogen bond donor could be incorporated
B
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hydrogen bond donor functionality, which would not only
engage in electrostatically favorable contacts but would serve to
lower lipophilicity as well. Hence, we shifted our focus to the
synthesis of compounds that possessed a simplified piperazine
core and hydrogen bond donors on the arylsulfonamide
functionality.
Table 1. Potency and Microsomal Turnover for Analogues
with Methyl Morpholine Replacements
The syntheses of the analogues which incorporated hydrogen
bond donors on the arylsulfomamides are outlined in Scheme
4. To avoid the separation of racemic mixtures, all of the
analogues were synthesized with the achiral bis-trifluoromethyl
(rather than the methyl-trifluoromethyl) carbinol because these
two functionalities were generally equipotent.9 Starting from
1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)phenyl)-2-propanol,9
the formation of thiophenesulfonamide 15 and phenyl-
sulfonamide 16 was straightforward since the sulfonylchlorides
were commercially available. The syntheses of the amino-
containing analogues required two steps. Aminothiophene
analogue 17 was synthesized via a Pd-catalyzed cross-coupling
of the 5-bromosulfonamide intermediate with benzophenone
imine followed by acidic hydrolysis of the intermediate imine.
For analogues 18−21, sulfonamide formations were accom-
plished utilizing a nitro-substituted sulfonylchloride followed by
reduction of the nitro group using 10% palladium on carbon.
The aminopyridine and aminopyrimidine analogues (22−24)
were synthesized using the corresponding chloro-substituted
sulfonyl chlorides followed by aminolysis of the heterocyclic
chloride intermediates with concentrated ammonium hydrox-
ide.
The SAR and clogPs of analogues containing functionalized
sulfonamides are shown in Table 2. Both the 5- and 4-
aminothiophene derivatives (17 and 18) exhibited modest
increases in biochemical activity albeit with no improvement in
metabolic stability relative to 15. Replacing the aminothiophene
present in these analogues with an aniline resulted in
equipotent compounds (19−21). Although the potencies
were similar, the 3- and 4-amino substituted analogues (20
and 21) showed encouraging improvements in rat metabolic
stability relative to the unsubstituted parent compound 16. To
further reduce the clogP, additional nitrogen atoms were
incorporated into both 20 and 21. Although aminopyridine 22
displayed a significant loss of activity relative to 20, the
regioisomeric analogue 23 showed a roughly 6-fold increase in
biochemical potency relative to 21 with no detectable oxidative
metabolism. Aminopyrimidine 24 also exhibited excellent in
vitro stability although the potency was slightly lower relative to
analogue 23. Analysis of the X-ray cocrystal structure of 23
bound to hGKRP (Figure 2a) confirmed that this analogue
formed two new hydrogen bonding interactions with the
backbone carbonyls of Gly181 and Met213. Given the
biochemical potency and excellent metabolic stability, this
analogue was progressed into a rat in vivo PK study. Relative to
15,8 aminopyridine 23 represented a significant improvement
in essentially all measurable PK parameters (CL = 0.12 L/h/kg,
Vss = 1.41 L/kg, MRT = 8.4 h, %F = 71). Despite the excellent
rat PK profile, 23 displayed no measurable activity in our
cellular assay (EC50 = >12.5 μM). Nevertheless, we believed
that this analogue provided a metabolically stable scaffold from
which potency could be optimized.
a
All compounds were synthesized as mixtures of stereoisomers at the
b
tertiary carbinol unless otherwise noted. AS = hGK-hGKRP
AlphaScreen, data reported as an average (n ≥ 3). Standard deviations
are reported in the Supporting Information. Compounds with
activities >12.5 μM, n = 1. For all others, standard deviations (n ≥
3) are reported in the Supporting Information. In vitro microsomal
stability measurements were conducted in the presence of NADPH at
37 °C for 30 min at a final compound concentration of 1 μM. (R)-
Stereoisomer at the CF3-tertiary carbinol. The stereochemistry of the
secondary alcohol was tentatively assigned based on the analysis of the
cocrystal structure with hGKRP. (S)-Stereoisomer at the tertiary CF3-
carbinol. The stereochemistry of the secondary alcohol was tentatively
assigned based on the analysis of the cocrystal structure with hGKRP.
c
d
e
f
We have previously reported the existence of a small,
hydrophobic subpocket located near Ile11 of the GKRP ligand
binding site.8 Furthermore, we have also shown that occupying
this pocket with small alkyl groups can lead to increases in
activity. From the analysis of the X-ray cocrystal structures of
on the thiophene ring. This portion of the GKRP binding site
possesses a pair of backbone carbonyl acceptor groups (Gly181
and Met213), conveniently oriented upward toward the
thiophene ring. We postulated that the presence of these
acceptors in the protein target would permit the introduction of
C
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Scheme 3. In Vitro Metabolism of 11
Table 2. SAR and cLogPs of Analogues Containing
Hydrogen Bond Donating Sulfonamides
Figure 1. Compound 1 (yellow) bound to hGKRP. Key residues are
highlighted in white, and hydrogen bonding interactions are
represented by green dashed lines.
a
Scheme 4. Synthesis of Sulfonamide Derivatives
a
Reagents and conditions: (a) 2-thiophenesulfonyl chloride or
benzenesulfonyl chloride, NEt3, CH2Cl2, rt; (b) 5-bromo-2-thiophe-
nesulfonyl chloride, NEt3, CH2Cl2, rt; (c) benzophenone imine, t-
BuONa, Pd2dba3, BINAP, toluene, 80 °C then 5 M HCl/THF, rt; (d)
4-nitro-2-thiophenesulfonyl chloride or nitrobenzenesulfonyl chloride,
NEt3, CH2Cl2, rt; (e) 10% Pd/C, H2 (1 atm) EtOH, rt; (f) 6-chloro-3-
pyridinesulfonyl chloride, 2-chloro-4-pyridinesulfonyl chloride, or 2-
chloro-5-pyrimidinesulfonyl chloride, NEt3, CH2Cl2, rt; (g) NH4OH,
EtOH, rt or 120 °C.
a
AS = hGK-hGKRP AlphaScreen, data reported as an average (n ≥ 3).
Standard deviations are reported in the Supporting Information. All
compounds demonstrated cellular EC50 values of >12.5 μM. In vitro
microsomal stability measurements were conducted in the presence of
NADPH at 37 °C for 30 min at a final compound concentration of 1
μM. cLogP values were calculated using the PCModels software,
version 4.9.2, from Daylight, which is developed and supported by
BioByte, Inc., Claremont, CA.
b
c
23 and 1 bound to hGKRP (Figure 2b), we rationalized that a
substituted alkyne could replace the metabolically liable methyl
morpholine present in 1. It was proposed that this small
binding pocket, occupied by the methyl group of 1, could
potentially be accessed by a propynyl substituent (i.e.,
).
reduce the molecular weight but also lower the number of
aliphatic C−H bonds, which could minimize the metabolic
liability. Furthermore, the minimal steric bulk of the narrow
propynyl substituent would allow a solvating water molecule
The linear trajectory of the alkyne function would serve to
present the terminal methyl group in nearly perfect overlap
with the methyl group of 1. This would not only significantly
D
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Figure 2. (a) Analogue 23 (green) bound to hGKRP. Key residues are highlighted in white, and the hydrogen bonding interactions are represented
as green dashed lines. (b) Overlay of compounds 23 (green) and 1 (yellow) bound to hGKRP (rotated relative to (a)). The hydrophobic subpocket
is highlighted by a white oval.
a
Scheme 5. Synthesis of Alkynyl-Substituted Piperazines (See Table 3)
a
Reagents and conditions: (a) benzyl chloroformate, Na2CO3, dioxane/water, rt; (b) (Boc)2O, NEt3, DMAP, CH2Cl2, rt; (c) alkynyl Grignard
reagents, THF, 0 °C; (d) TFA, CH2Cl2 then NaBH(OAc)3, rt; (e) 2-(4-bromophenyl)-1,1,1,3,3,3-hexafluoro-2-propanol or 2-(4-bromophenyl)-
1,1,1-trifluoropropan-2-ol, RuPhos first generation precatalyst, RuPhos, NaOt-Bu, toluene, 100 °C; (f) TfOH, TFA, rt; (g) 6-chloropyridine-3-
sulfonyl chloride, NEt3, CH2Cl2, rt; (h) NH4OH/EtOH, 110−120 °C; (i) chiral SFC.
(rather than the morpholine oxygen atom in 1) to satisfy the
backbone donor NH of Ile11.19 Access to this methyl-
accommodating subpocket directly from the piperazine core
would require an axial-like disposition of the linear substituent.
Gratifyingly, this orientation was predicted from quantum
mechanical calculations to be the lowest-energy conformation
in the unbound state.20
The syntheses of these alkynyl-substituted derivatives are
shown in Scheme 5. Starting from the commercially available 2-
piperazinone, two sequential protections (Cbz followed by
Boc) proceeded in high yield. A variety of alkynyl Grignard
reagents were added into the imide carbonyl to provide the
corresponding ketone intermediates. Trifluoroacetic acid-
mediated removal of the Boc group, followed by in situ
reduction of the resultant imine, provided the racemic
piperazines. N-Arylation,21 removal of the Cbz protecting
group, and sulfonamide formation using 6-chloropyridine-3-
sulfonyl chloride22 delivered the penultimate intermediates.
Formation of the aminopyridine functionality was accom-
plished by treating the chloropyridines with ammonium
hydroxide in ethanol. For the most promising analogues, the
individual stereoisomers were isolated using chiral SFC.
The GKRP activities and the metabolic stabilities of the
alkynyl-substituted piperazine analogues are shown in Table 3.
Incorporation of the methyl substituted alkyne (25) had a
dramatic effect on the biochemical potency when compared to
the unsubstituted piperazine (23). More importantly, the
cellular potency of 25 was significantly improved without a
deleterious drop in metabolic stability. The more active
enantiomer, 27 (AMG-3969), exhibited both potent cellular
activity (EC50 = 0.202 μM) and excellent metabolic stability
when exposed to rat/human liver microsomes (42 and <14 μL/
min/mg, respectively). Analysis of the cocrystal structure of 27
bound to hGKRP (Figure 3) confirmed that the terminal
E
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Table 3. Potency and Microsomal Turnover of Alkynyl-Substituted Piperazines
a
AS = hGK-hGKRP AlphaScreen, data reported as an average (n ≥ 3). Standard deviations are reported in the Supporting Information.
b
c
Compounds with activities >12.5 μM, n = 1. For all others, standard deviations (n ≥ 3) are reported in the Supporting Information. In vitro
microsomal stability measurements were conducted in the presence of NADPH at 37 °C for 30 min at a final compound concentration of 1 μM.
29 also showed promising in vitro activities and excellent rat
and human in vitro metabolic stabilities. Unfortunately,
attempts to further improve activity by incorporating larger
hydrophobic groups at the terminal end of the alkyne (i.e., 30−
32) did not lead to further improvements in activity.
Given the promising activity and metabolic profiles of 27−
29, each of these molecules were progressed into rat in vivo PK
studies (Table 4). Compounds 27 and 29 displayed excellent
PK profiles when compared to methylmorpholine 1. Both
compounds had in vivo clearances significantly less than liver
blood flow (10−20%) with excellent bioavailabilities (>75%).
Table 4. In Vivo PK and PPB Data for Compounds 27, 28,
29, and 1
CL
(L/h/kg)
Vss
(L/kg)
MRT
b
f
u c
a
a
b
species compd
(h)
%F
(%)
rat
27
28
29
1
0.75
1.8
3.6
3.7
1.6
8.3
1.1
1.0
6.2
2.9
4.6
4.4
9.2
5.3
75
57
82
7
1.5
3.6
2.7
3.7
1.2
2.3
rat
rat
0.39
4.1
Figure 3. Overlay of analogues 27 (blue, shown with an active site
water molecule (pink)) and 1 (yellow) bound to hGKRP. Key protein
residues are highlighted in white, while hydrogen bonding interactions
are represented as green dashed lines.
rat
mouse
mouse
27
29
0.11
0.18
40
39
a
b
2 mg/kg intravenous dose (100% DMSO). 10 mg/kg oral dose (1%
c
methyl group occupied the same hydrophobic pocket as the
methyl group on the morpholine ring of 1. Analogues 28 and
Tween 80, 2% HPMC, 97% water, pH = 7.0). Fraction unbound was
determined via rapid equilibrium dialysis.
F
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Figure 4. (a) Blood glucose measurements 6 h postoral administration of 27 (green bars) and 29 (red bars) (10, 30, 100, mg/kg) to db/db mice.
The statistical significance of the blood glucose measurements were based on comparison to the individual vehicle (Veh) control groups (blue bars, n
≥ 6, * = p < 0.05, *** = p < 0.001). (b) In vivo GK translocation (green bars, left y-axis) and unbound plasma concentrations (black triangles, right y-
axis) in db/db mice following oral administration of 27 (10, 30, 100 mg/kg, 6 h postdose). The dotted line represents the GK translocation EC50
value for 27 (0.202 μM) in mouse hepatocytes. (c) Blood glucose measurements of db/db mice following administration of either 27 (green bars,
100 mg/kg, po) or GKA 33 (black bars, 50 mg/kg, po) at 4 and 8 h postdose. The statistical significance of the blood glucose measurements were
determined based on comparison to the individual vehicle (Veh) control groups (blue bars, n ≥ 7, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns =
not significant). (d) Molecular structures of 27, 29, and 33.
G
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2. GK Translocation in Mouse Hepatocytes. Mouse hepatocytes29
in maintenance media (Williams E, 10% FBS, 1 × PSG, 1 μg/mL
insulin, 100 nM dexamethasone) were plated into a 96-well plate
(50000 cells/well) and incubated overnight. Cells were washed twice
(200 μL of DMEM no glucose/0.2% BSA), then 100 μL of DMEM
(no glucose)/0.2% BSA was added and cells were incubated for 3−4 h
at 37 °C. Cells were again washed with DMEM (no glucose) only, and
test compounds diluted in DMEM (no glucose) were added,
incubated for 20 min at 37 °C, followed by addition of 10 μL of 25
mM glucose and further incubated for an additional 40 min at 37 °C.
Cells were then fixed (100 μL of 8% formaldehyde/PBS), and the
plates were incubated at rt for 15 min, washed twice with 200 μL of
PBS, then permeabilized (75 μL of 0.3% Triton X-100/PBS) for 10
min. Following two washes with 200 μL of PBS, 50 μL of blocking
solution (Li-Cor) was added and incubated for 1 h at rt. Cells were
washed (100 μL of 1% goat serum/0.1% Tween in PBS), incubated
with 50 μL of anti-GK antibody diluted 1:125 in 1:1 Li-Cor
blocking:wash buffer, incubated at 4 °C overnight, and, after washing,
50 μL of goat antirabbit AlexaFluor 555 diluted 1:100 and 1 μg/mL of
Hoechst 33342 in wash buffer was added to each well and incubated
for 1 h at rt. Following washing, 200 μL of PBS was added to each well
and imaging was performed using an Operetta system (PerkinElmer).
3. In Vivo Studies in db/db Mice. Diabetic db/db mice were
purchased from Jackson Laboratories at 8−9 weeks of age and cared
for in accordance to the Guide for the Care and Use of Laboratory
Animals, 8th ed. Animals were housed four per cage at an AAALAC,
Intl-accredited facility in a ventilated microisolator on corn cob
bedding. All research protocols were approved by the Amgen. Inc.,
Institutional Animal Care and Use Committee. Animals had ad libitum
access to pelleted chow and water and were maintained on a 12:12 h
light:dark cycle with nestlet enrichment opportunities and acclimatized
for 1 week. All animals were determined specific pathogen free. At
8:00 AM, mice were bled via retro-orbital sinus puncture and blood
glucose values were determined (AlphaTRAK glucose meter) and used
to randomize the animals in which their averages were similar, and
only mice with blood glucose ranges between 300 and 500 mg/dL
were included. Vehicle (2% hydroxypropyl methycellulose, 1% Tween
80, pH 2.2 adjusted with MSA), 27, 29, or 33 formulations were
gavaged at 9:00 AM. Blood glucose was measured at 4, 6, or 8 h post-
treatment. At each time point, a 15 μL sample of whole blood was
analyzed for drug exposure. SAS version 9.2 on a Linux system was
used to perform all statistical analyses by ANCOVA.
For compound 28, the modestly higher microsomal turnover
(71 μL/min/mg) translated to an increase in in vivo clearance
relative to both 27 and 29. Both 27 and 29 also demonstrated
excellent in vivo PK profiles in mice (Table 4). The unbound
drug concentrations (10 mg/kg, po) in the mouse PK study
approached or exceeded the cellular EC50 values for both 27
and 29 (concentrations at Cmax were 0.068 and 0.292 μM,
respectively). Because the mouse PK profiles allowed for in vivo
coverage of the in vitro EC50 values, both of these analogues
were administered to db/db mice to determine the effect of
small molecule-mediated GK-GKRP disruption on fed blood
glucose levels.
Genetically obese, insulin resistant diabetic db/db mice are
commonly used to monitor the efficacy of glucose lowering
agents.23 At 6 h postoral administration with either 27 or 29, a
statistically significant dose-dependent reduction in fed blood
glucose levels was observed (Figure 4a). Because analogue 27
demonstrated a more robust in vivo response when compared
to 29,24 the livers of the animals treated with compound 27
were examined to determine the extent of the GK translocation
via immunohistochemistry (IHC). An IHC score of four
represented no pharmacodynamic (PD) response, while a score
of zero indicated essentially complete nuclear translocation of
GK. As shown in Figure 4b, an IHC score of approximately
zero was observed at both the 30 and 100 mg/kg doses of 27,
which corresponded to 47% and 60% reductions in blood
glucose, respectively. The exposure from this efficacy experi-
ment also demonstrated a reasonable correlation to the in vitro
potency. Statistically significant reductions in blood glucose
were observed at systemic free drug concentrations near the
mouse hepatocytes cellular EC50 values for both 27 and 29.25 In
a subsequent experiment, we investigated two additional time
points following an oral dose of compound 27 with the purpose
of comparing to a known GKA (33) published by Roche
(Figure 4c).26 As shown in Figure 4c, analogue 27 (100 mg/kg)
demonstrated significant reductions in blood glucose with
robust efficacy (56% reduction) observed at the 8 h time point.
Although GKA 33 (50 mg/kg)26 was efficacious at the early (4
h) time point, no statistically significant reduction in fed blood
glucose was observed at 8 h postdose.27
For IHC studies, mice were sacrificed following anesthesia, and liver
samples were immediately excised (2−3 mm thick and 7−8 mm long,
from the right medial and the left lateral lobe), fixed in cold
paraformaldehyde for 3 h, and transferred to 70% EtOH prior to
processing and embedding in paraffin. Then 5 μm thick sections of
paraffin-embedded liver were deparaffinized and hydrated in DI H2O.
Sections were pretreated with Diva Decloaker (Biocare Medical) in a
pressure cooker, blocked with CAS-Block (Invitrogen), and incubated
with rabbit anti-GK antibody (Santa Cruz Biotechnologies) at 1:100
for 1 h at rt. Slides were quenched with 3% H2O2, followed with
EnVision + HRP labeled polymer antirabbit (DAKO). Reaction sites
were visualized with DAB (DAKO), and slides were counterstained
with hematoxylin. Slides were independently evaluated for nuclear GK
intensity by two observers without knowledge of treatment group.
B. Chemistry. Unless otherwise noted, all reagents were
commercially available and used as received. All final compounds
possessed purity ≥95% as determined by high performance liquid
chromatography (HPLC). The HPLC methods used the following
conditions: Zorbax SB-C18 column (50 mm × 3.0 mm, 3.5 μm) at 40
°C with a 1.5 mL/min flow rate; solvent A of 0.1% TFA in water,
solvent B of 0.1% TFA in MeCN; 0.0−3.0 min, 5−95% B in A; 3.0−
3.5 min, 95% B in A; 3.5−3.51 min, 5% B in A. Flow from the UV
detector was split (50:50) to the MS detector which was configured
CONCLUSION
■
By relying heavily on metabolite identification and structure-
based drug design, we systemically eliminated a number of
metabolic liabilities present in 1. By replacing both the
thiophene and the methyl morpholine functionalities, we
successfully indentified a potent and metabolically stable GK-
GKRP disruptor (27). This compound represents the first
compound from this class to demonstrate in vivo efficacy in a
mouse model of diabetes. Furthermore, analogue 27 did not
bind to GK itself, hence the robust in vivo efficacy observed
with this compound was not a result of GK activation.28 Also,
consistent with our initial report, no signs of hypoglycemia
were observed in animals (either normal or hypergylcemic)
when treated with compound 27.9 Hence, the lack of
hypoglycemia combined with the significant glucose lowering
observed in db/db mice provide compelling evidence to
support GK-GKRP disruption as a potential treatment for
diabetes.
1
with APIES as ionizable source. H NMR spectra were recorded on a
300 or 400 MHz Bruker NMR spectrometer at ambient temperature.
Data are reported as follows: chemical shift (ppm, δ units) from an
internal standard, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, and br = broad), coupling constant (Hz), and
EXPERIMENTAL SECTION
■
A. Biology. 1. hGK-hGKRP AlphaScreen. This assay was
conducted as previously reported.9,10
H
dx.doi.org/10.1021/jm4016747 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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integration. All microwave-assisted reactions were performed in sealed
reaction vials using a Personal Chemistry Emrys Optimizer microwave
synthesizer. Analytical thin-layer chromatography (TLC) was
performed using JT Baker silica gel plates precoated with a fluorescent
indicator. Silica gel chromatography was performed using either an
ISCO Companion or Biotage medium pressure liquid chromatography
system.
3.11 (m, 1 H), 2.97−2.83 (m, 1 H), 2.66 (dd, J = 2.8, 11.4 Hz, 1 H),
2.57 (dt, J = 3.8, 11.4 Hz, 1 H), 2.36 (br d, J = 12.1 Hz, 1 H), 1.69 (s, 3
H), 1.24−1.19 (m, 1 H), 1.06−0.99 (m, 1 H). m/z (ESI, +ve ion)
532.1 (M + H)+.
1,1,1-Trifluoro-2-(4-((2S)-2-(3-oxa-8-azabicyclo[3.2.1]oct-8-yl-
methyl)-4-(2-thiophenylsulfonyl)-1-piperazinyl)phenyl)-2-propanol
(8). Following the procedure outlined above for 3, the reaction of 3-
oxa-8-azabicyclo[3.2.1]octane and ((2R)-4-(2-thiophenylsulfonyl)-1-
(4-(2,2,2-trifluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)-
methyl methanesulfonate produced the title compound (10% yield).
1H NMR (400 MHz, CDCl3) δ 7.64 (dd, J = 1.2, 5.1 Hz, 1 H), 7.61−
7.55 (m, 1 H), 7.42 (d, J = 8.8 Hz, 2 H), 7.17 (dd, J = 3.7, 4.9 Hz, 1
H), 6.80 (d, J = 8.8 Hz, 2 H), 4.21 (d, J = 11.2 Hz, 1 H), 3.95−3.75
(m, 2 H), 3.66−3.53 (m, 2 H), 3.52−3.37 (m, 3 H), 3.20 (dt, J = 3.4,
12.0 Hz, 1 H), 3.09−2.99 (m, 1 H), 2.97−2.86 (m, 1 H), 2.71−2.47
(m, 3 H), 2.34 (br s, 1 H), 2.19 (dd, J = 3.3, 12.5 Hz, 1 H), 1.89−1.49
(m, 7 H). m/z (ESI, +ve ion) 546.0 (M + H)+.
1,1,1-Trifluoro-2-(4-((2S)-2-(((3S)-3-methyl-4-morpholinyl)-
methyl)-4-(2-thiophenylsulfonyl)-1-piperazinyl)phenyl)-2-propanol
(3). A 20 mL microwave vial was charged with ((2R)-4-(2-
thiophenylsulfonyl)-1-(4-(2,2,2-trifluoro-1-hydroxy-1-methylethyl)-
phenyl)-2-piperazinyl)methyl methanesulfonate11 (0.50 g, 0.95
mmol), (S)-3-methylmorpholine (0.191 g, 1.89 mmol), and 7 mL of
MeCN. The vial was sealed and heated in a microwave synthesizer at
125 °C for 1 h. The mixture was allowed to cool to rt and then diluted
with 10 mL of EtOAc and filtered. The filtrate was concentrated and
purified via silica gel chromatography (0−70% EtOAc in hexanes) to
produce the title compound (0.050 g, 0.094 mmol, 10% yield) as a
white solid. 1H NMR (400 MHz, CD3OD) δ 7.90 (dd, J = 1.2, 5.1 Hz,
1 H), 7.66 (dd, J = 1.4, 3.7 Hz, 1 H), 7.46 (d, J = 8.8 Hz, 2 H), 7.28
(dd, J = 3.8, 5.0 Hz, 1 H), 6.94 (d, J = 8.8 Hz, 2 H), 4.12−3.92 (m, 2
H), 3.85−3.40 (m, 6 H), 3.29−3.07 (m, 2 H), 2.87 (d, J = 11.7 Hz, 1
H), 2.70−2.45 (m, 2 H), 2.34 (t, J = 6.0 Hz, 1 H), 2.19−2.00 (m, 1
H), 1.88 (d, J = 12.3 Hz, 1 H), 1.69 (s, 3 H), 1.02 (d, J = 6.2 Hz, 3 H).
m/z (ESI, +ve ion) 534.0 (M + H)+.
2-(4-((2S)-2-((4,4-Difluoro-1-piperidinyl)methyl)-4-(2-thiophenyl-
sulfonyl)-1-piperazinyl)phenyl)-1,1,1-trifluoro-2-propano1 (4). Fol-
lowing the procedure outlined above for 3, the reaction of 4,4-
difluoropiperidine and ((2R)-4-(2-thiophenylsulfonyl)-1-(4-(2,2,2-tri-
fluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl metha-
nesulfonate produced the title compound (51% yield). 1H NMR
(400 MHz, CD3OD) δ 7.89 (dd, J = 1.2, 5.1 Hz, 1 H), 7.66 (dd, J =
1.2, 3.7 Hz, 1 H), 7.45 (d, J = 8.8 Hz, 2H), 7.27 (dd, J = 3.8, 5.0 Hz, 1
H), 6.93 (d, J = 8.8 Hz, 2 H), 4.19−4.01 (m, 1 H), 3.96 (d, J = 11.2
Hz, 1 H), 3.79 (d, J = 9.4 Hz, 1 H), 3.45−3.24 (m, 2 H), 3.24 (dt, J =
3.5, 12.0 Hz, 1 H), 2.78 (dd, J = 9.2, 12.9 Hz, 1 H), 2.71−2.55 (m, 3
H), 2.55−2.44 (m, 2 H), 2.38 (dd, J = 4.8, 12.8 Hz, 1 H), 1.91−1.74
(m, 4 H), 1.69 (s, 3 H). m/z (ESI, +ve ion) 554.2 (M + H)+.
2-(4-((2S)-2-((1,1-Dioxido-4-thiomorpholinyl)methyl)-4-(2-thio-
phenylsulfonyl)-1-piperazinyl)phenyl)-1,1,1-trifluoro-2-propanol
(5). Following the procedure outlined above for 3, the reaction of
thiomorpholine 1,1-dioxide and ((2R)-4-(2-thiophenylsulfonyl)-1-(4-
(2,2,2-trifluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl
methanesulfonate produced the title compound (12% yield) as an off-
white solid. 1H NMR (400 MHz, CD3OD) δ 7.91 (dd, J = 1.3, 5.0 Hz,
1 H), 7.68 (dd, J = 1.4, 3.7 Hz, 1 H), 7.48 (d, J = 8.8 Hz, 2 H), 7.29
(dd, J = 3.7, 5.1 Hz, 1 H), 6.98−6.92 (m, 2 H), 4.15 (br. s, 1 H), 3.98
(br d, J = 11.2 Hz, 1 H), 3.83 (br d, J = 11.3 Hz, 1 H), 3.47 (d, J = 12.5
Hz, 1 H), 3.32−3.22 (m, 1 H), 3.09−2.75 (m, 9 H), 2.74−2.50 (m, 3
H), 1.70 (s, 3 H). m/z (ESI, +ve ion) 567.8 (M + H)+.
8-(((2S)-4-(2-Thiophenylsulfonyl)-1-(4-(2,2,2-trifluoro-1-hydroxy-
1-methylethyl)phenyl)-2-piperazinyl)methyl)-3-oxa-8-exo-
azabicyclo[3.2.1]octan-6-ol (9). Following the two-step procedure
reported for 10, 3-oxa-8-exo-azabicyclo[3.2.1]octan-6-ol hydrochlor-
ide14 delivered the title compound as a white solid (mixture of four
1
isomers, 10% yield over two steps). H NMR (400 MHz, CD3OD) δ
7.89 (d, J = 5.1 Hz, 1 H), 7.67 (br s, 1 H), 7.44 (dd, J = 8.8, 12.5 Hz, 2
H), 7.28 (t, J = 4.0 Hz, 1 H), 6.92 (dd, J = 6.8, 8.8 Hz, 2 H), 4.64−4.37
(m, 1 H), 4.28−3.72 (m, 3 H), 3.62−2.83 (m, 10 H), 2.70−2.50 (m, 3
H), 2.24−2.03 (m, 1 H), 1.76−1.64 (m, 3 H). m/z (ESI, +ve ion)
562.1 (M + H)+.
8-(((2S)-4-(2-Thiophenylsulfonyl)-1-(4-(2,2,2-trifluoro-1-hydroxy-
1-methylethyl)phenyl)-2-piperazinyl)methyl)-3-oxa-8-endo-
azabicyclo[3.2.1]octan-6-ol (10). A 10 mL vial was charged with
((2R)-4-(2-thiophenylsulfonyl)-1-(4-(2,2,2-trifluoro-1-hydroxy-1-
methylethyl)phenyl)-2-piperazinyl)methyl methanesulfonate11 (0.207
g, 0.391 mmol), 6-(benzyloxy)-3-oxa-8-endo-azabicyclo[3.2.1]octane
hydrochloride14 (0.100 g, 0.391 mmol), potassium carbonate (0.162 g,
1.17 mmol), and 3 mL of MeCN. The tube was sealed and heated in a
microwave reactor at 150 °C for 1 h. The mixture was allowed to cool
to rt and then diluted with 5 mL of EtOAc and filtered. The filtrate
was concentrated and purified via silica gel chromatography (0−50%
EtOAc in hexanes) to give 2-(4-((2S)-2-((6-(benzyloxy)-3-oxa-8-endo-
azabicyclo[3.2.1]oct-8-yl)methyl)-4-(2-thiophenylsulfonyl)-1-
piperazinyl)phenyl)-1,1,1-trifluoro-2-propanol as a white solid (mix-
ture of four isomers, 0.130 g, 37% yield). 1H NMR (400 MHz,
CD3OD) δ 7.89 (br d, J = 5.1 Hz, 1 H), 7.69−7.63 (m, 1 H), 7.46 (d, J
= 8.6 Hz, 2 H), 7.40−7.24 (m, 6 H), 6.91 (dd, J = 2.0, 9.0 Hz, 2 H),
4.61−4.41 (m, 2 H), 4.26−3.69 (m, 6 H), 3.67−3.38 (m, 3 H), 3.20
(dq, J = 3.3, 11.6 Hz, 1 H), 3.07−2.95 (m, 2 H), 2.87−2.68 (m, 1 H),
2.67−2.44 (m, 3 H), 2.38−2.10 (m, 1 H), 1.79−1.58 (m, 4 H).
A 100 mL round-bottomed flask was charged with 2-(4-((2S)-2-((6-
(benzyloxy)-3-oxa-8-endo-azabicyclo[3.2.1]oct-8-yl)methyl)-4-(2-thio-
phenylsulfonyl)-1-piperazinyl)phenyl)-1,1,1-trifluoro-2-propanol (0.35
g, 0.54 mmol) and 5 mL of CH2Cl2. After cooling to 0 °C, BCl3 (1 M
in CH2Cl2, 2.15 mL, 2.15 mmol) was added dropwise. This mixture
was allowed to warm to rt and then stirred for an additional 15 min.
Then 10 mL of MeOH was added, and the mixture was concentrated
onto silica gel and then purified via silica gel chromatography (0−7%
MeOH in CH2Cl2) to give the title compound (0.21 g, 70% yield) as a
1,1,1-Trifluoro-2-(4-((2S)-2-((3-methyl-1,1-dioxido-4-
thiomorpholinyl)methyl)-4-(2-thiophenylsulfonyl)-1-piperazinyl)-
phenyl)-2-propanol (6). Following the procedure outlined above for
3, the reaction of 3-methylthiomorpholine 1,1-dioxide30 and ((2R)-4-
(2-thiophenylsulfonyl)-1-(4-(2,2,2-trifluoro-1-hydroxy-1-methylethyl)-
phenyl)-2-piperazinyl)methyl methanesulfonate produced the title
1
compound (35% yield) as an off-white solid. H NMR (400 MHz,
CD3OD) δ 7.90 (dd, J = 1.2, 5.1 Hz, 1 H), 7.73−7.59 (m, 1 H), 7.55−
7.37 (m, 2 H), 7.27 (dd, J = 3.7, 4.9 Hz, 1 H), 6.94 (dd, J = 2.9, 9.0 Hz,
2 H), 4.14−3.97 (m, 2 H), 3.79 (d, J = 11.0 Hz, 1 H), 3.47−3.17 (m, 3
H), 3.11−2.73 (m, 7 H), 2.65−2.37 (m, 3 H), 1.68 (s, 3 H), 1.28−1.21
(m, 3 H). m/z (ESI, +ve ion) 581.9 (M + H)+.
1,1,1-Trifluoro-2-(4-((2S)-2-(2-oxa-6-azaspiro[3.3]hept-6-ylmeth-
yl)-4-(2-thiophenylsulfonyl)-1-piperazinyl)phenyl)-2-propanol (7).
Following the procedure outlined above for 3, the reaction of 2-oxa-
6-azaspiro[3.3]heptane and ((2R)-4-(2-thiophenylsulfonyl)-1-(4-
(2,2,2-trifluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl
methanesulfonate produced the title compound (32% yield). 1H NMR
(400 MHz, CD3OD) δ 7.89 (d, J = 5.1 Hz, 1 H), 7.65 (d, J = 3.7 Hz, 1
H), 7.47 (d, J = 8.8 Hz, 2 H), 7.33−7.21 (m, 1 H), 6.97−6.85 (m, 2
H), 4.69 (br. s, 4 H), 3.90−3.66 (m, 3 H), 3.52−3.38 (m, 3 H), 3.27−
1
white solid (mixture of four isomers). H NMR (400 MHz, CD3OD)
δ 7.92 (d, J = 4.9 Hz, 1 H), 7.67 (d, J = 3.5 Hz, 1 H), 7.54 (t, J = 7.2
Hz, 2 H), 7.28 (t, J = 5.3 Hz, 1 H), 7.06 (t, J = 9.1 Hz, 2H), 4.43−4.62
(m, 2H), 3.39−4.24 (m, 13H), 2.66−2.98 (m, 2H), 2.55 (d, J = 3.91
Hz, 1H), 1.69 (br s, 3H). m/z (ESI, +ve ion) 562.1 (M + H)+.
The individual isomers (11−14) were isolated using two sequential
chiral SFC purifications. The first separation used a Chiralpak column
(21 mm × 250 mm, 5 μm) with 30% MeOH (w/20 mM NH3) in
supercritical CO2 at a flow rate of 70 mL/min. The second SFC
purification used a Chiralcel OJH column (21 mm × 250 mm, 5 μm)
with 25% MeOH (w/20 mM NH3) in supercritical CO2 and a flow
rate of 70 mL/min. This sequence produced the four isomers with
both diastereomeric and enantiomeric excesses >95%. The absolute
I
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stereochemistries were tentatively assigned based on the X-ray
cocrystal structures with hGKRP.
(diphenylphosphino)-1,1′-binaphthyl (BINAP) (0.25 g, 0.40 mmol).
Nitrogen gas was bubbled through the solution for 2 min, and then the
vial was sealed and heated at 80 °C for 12 h. The black mixture was
then diluted with EtOAc (10 mL) and filtered, and the filtrate was
concentrated to give a black tar. Purification via column chromatog-
raphy on silica gel (0−60% EtOAc in hexanes) provided the
intermediate imine. To this was added 5 M HCl (15 mL) and THF
(15 mL). After 15 min, 5 M NaOH (15 mL) was added and the
mixture was extracted with EtOAc (50 mL). The organics were dried
(MgSO4), filtered, and concentrated to give an orange foam.
Purification via silica gel chromatography (0−70% EtOAc in hexanes)
gave an orange oil. Diethylether (25 mL) and hexanes (50 mL) were
added, and the resulting white solid was collected by filtration to
deliver 2-(4-(4-((5-amino-2-thiophenyl)sulfonyl)-1-piperazinyl)-
phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (0.32 g, 40% yield). 1H
NMR (400 MHz, CD3OD) δ 7.57 (d, J = 8.8 Hz, 2 H), 7.22 (d, J = 4.1
Hz, 1 H), 7.03 (d, J = 9.2 Hz, 2 H), 6.07 (d, J = 4.1 Hz, 1 H), 3.39−
3.26 (m, 4 H), 3.21−3.10 (m, 4 H). m/z (ESI, +ve ion) 490.0 (M +
H)+.
2-(4-(4-((4-Amino-2-thiophenyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (18). 1,1,1,3,3,3-Hexafluoro-2-(4-
(1-piperazinyl)phenyl)-2-propanol dihydrochloride8 (0.31 g, 0.84
mmol) was dissolved in CH2Cl2 (20 mL) and cooled to 0 °C. To
this solution was added triethylamine (0.21 g, 2.10 mmol) followed by
4-nitrothiophene-2-sulfonyl chloride (0.21 g, 0.92 mmol). The
reaction was allowed to warm to rt and stirred for 2 h. The mixture
was filtered, and the filtrate was concentrated and then purified via
reverse-phase preparative HPLC (Phenomenex Gemini C18 column
(150 mm × 30 mm, 10 μm) eluting with 0.1% TFA in CH3CN/H2O
(5−100% over 15 min)) to give 2-(4-(4-((4-nitro-2-thiophenyl)-
sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol
(0.086 g, 20%). m/z (ESI, +ve ion) 520.0 (M + H)+.
A 50 mL round-bottomed flask was charged with 2-(4-(4-((4-nitro-
2-thiophenyl)sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-
propanol (0.080 g, 0.154)mmol), ammonium formate (0.049 g, 0.770
mmol), and 10% Pd/C (0.02 g) in EtOH (10 mL). This mixture was
stirred under an atmosphere of H2 (1 atm) for 18 h at rt. The reaction
mixture was filtered through a plug of Celite (diatomaceous earth),
and the filtrate was concentrated, dissolved in CH2Cl2, and then
filtered for a second time through a small plug of silica gel. The filtrate
was concentrated to give 2-(4-(4-((4-amino-2-thiophenyl)sulfonyl)-1-
piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol as an off-white
solid (0.023 g, 31% yield). 1H NMR (400 MHz, CD3OD) δ 7.58 (d, J
= 8.8 Hz, 2 H), 7.19 (d, J = 2.0 Hz, 1 H), 7.03 (d, J = 9.0 Hz, 2 H),
6.60 (d, J = 1.8 Hz, 1 H), 3.40−3.35 (m, 4 H), 3.23−3.17 (m, 4 H).
m/z (ESI, +ve ion) 490.1 (M + H)+.
2-(4-(4-((2-Aminophenyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (19). To a 25 mL round-bottomed
flask was added 1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)phenyl)-2-
propanol dihydrochloride8 (0.25 g, 0.62 mmol), triethylamine (0.19 g,
0.26 mL, 1.9 mmol), and CH2Cl2 (6 mL). While stirring at rt, 2-
nitrobenzenesulfonyl chloride (0.14 g, 0.62 mmol) was added. The
mixture was stirred for 20 min and then concentrated onto silica gel.
Purification by silica gel chromatography (0−6% MeOH in CH2Cl2)
afforded 1,1,1,3,3,3-hexafluoro-2-(4-(4-(2-nitrophenylsulfonyl)-
piperazin-1-yl)phenyl)-2-propanol (0.13 g, 41% yield) as a yellow
solid. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1 H), 7.97−8.11 (m,
2 H), 7.83−7.97 (m, 2 H), 7.47 (d, J = 8.6 Hz, 2 H), 7.03 (d, J = 9.2
Hz, 2 H), 3.31 (br s, 8 H).
(1R,5R,6R)-8-(((2S)-4-(2-Thiophenylsulfonyl)-1-(4-((1R)-2,2,2-tri-
fluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl)-3-
oxa-8-azabicyclo[3.2.1]octan-6-ol (11). 1H NMR (400 MHz,
CD3OD) δ 7.78 (d, J = 4.9 Hz, 1 H), 7.60−7.45 (m, 1 H), 7.34 (d,
J = 8.8 Hz, 2 H), 7.16 (t, J = 4.4 Hz, 1 H), 6.80 (s, 2 H), 4.31−4.07
(m, 3 H), 3.92 (d, J = 11.2 Hz, 2 H), 3.81−3.61 (m, 2 H), 3.52 (s, 2
H), 3.37 (br s, 1 H), 3.10 (d, J = 3.5 Hz, 1 H), 2.89 (br s, 1 H), 2.79−
2.63 (m, 2 H), 2.60−2.15 (m, 4 H), 1.58 (s, 3 H). m/z (ESI, +ve ion)
562.1 (M + H)+.
(1R,5R,6R)-8-(((2S)-4-(2-Thiophenylsulfonyl)-1-(4-((1S)-2,2,2-tri-
fluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl)-3-
oxa-8-azabicyclo[3.2.1]octan-6-ol (12). 1H NMR (400 MHz,
CD3OD) δ 7.90 (dd, J = 1.2, 5.1 Hz, 1 H), 7.66 (d, J = 2.7 Hz, 1
H), 7.46 (d, J = 8.8 Hz, 2 H), 7.34−7.21 (m, 1 H), 6.91 (d, J = 8.8 Hz,
2 H), 4.42−4.27 (m, 1 H), 4.09−3.93 (m, 2 H), 3.88 (d, J = 10.2 Hz, 1
H), 3.79 (d, J = 11.3 Hz, 1 H), 3.69−3.56 (m, 2 H), 3.47 (d, J = 12.7
Hz, 1 H), 3.39−3.35 (m, 1 H), 3.22 (dt, J = 3.5, 12.1 Hz, 1 H), 3.01 (s,
1 H), 2.89−2.74 (m, 2 H), 2.70−2.48 (m, 4 H), 2.47−2.30 (m, 1 H),
1.69 (s, 3 H). m/z (ESI, +ve ion) 562.1.
(1S,5S,6S)-8-(((2S)-4-(2-Thiophenylsulfonyl)-1-(4-((1R)-2,2,2-tri-
fluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl)-3-
oxa-8-azabicyclo[3.2.1]octan-6-ol (13). 1H NMR (400 MHz,
CD3OD) δ 7.81−7.73 (m, 1 H), 7.58−7.50 (m, 1 H), 7.34 (d, J =
8.6 Hz, 2 H), 7.19−7.12 (m, 1 H), 6.80 (d, J = 8.8 Hz, 2 H), 4.35−
4.20 (m, 1 H), 3.99 (d, J = 11.2 Hz, 1 H), 3.84−3.58 (m, 4 H), 3.45
(d, J = 10.6 Hz, 1 H), 3.42−3.28 (m, 2 H), 3.07 (dt, J = 3.1, 12.0 Hz, 1
H), 2.83 (d, J = 6.8 Hz, 1 H), 2.73 (d, J = 6.1 Hz, 1 H), 2.62 (dd, J =
9.9, 12.6 Hz, 1 H), 2.57−2.31 (m, 4 H), 2.12 (m, 1 H), 1.57 (s, 3 H).
m/z (ESI, +ve ion) 562.1 (M + H)+.
(1S,5S,6S)-8-(((2S)-4-(2-Thiophenylsulfonyl)-1-(4-((1S)-2,2,2-tri-
fluoro-1-hydroxy-1-methylethyl)phenyl)-2-piperazinyl)methyl)-3-
oxa-8-azabicyclo[3.2.1]octan-6-ol (14). 1H NMR (400 MHz,
CD3OD) δ 7.78 (d, J = 4.9 Hz, 1 H), 7.55 (d, J = 2.7 Hz, 1 H),
7.36 (d, J = 8.6 Hz, 2 H), 7.21−7.08 (m, 1 H), 6.82 (d, J = 8.6 Hz, 2
H), 4.33 (br s, 1 H), 4.01−3.58 (m, 5 H), 3.56−3.43 (m, 1 H), 3.42−
3.30 (m, 2 H), 3.16−2.38 (m, 7 H), 2.25−2.03 (m, 1 H), 1.65−1.44
(m, 4 H). m/z (ESI, +ve ion) 562.1 (M + H)+.
1,1,1,3,3,3-Hexafluoro-2-(4-(4-(phenylsulfonyl)-1-piperazinyl)-
phenyl)-2-propanol (16). A 100 mL round-bottomed flask was
charged with 1,1,1,3,3,3-hexafluoro-2-(4-(piperazin-1-yl)phenyl)-
propan-2-ol8 (2.19 g, 6.67 mmol), 20 mL of CH2Cl2, and triethylamine
(0.91 g, 1.25 mL, 9.01 mmol). To this was added 2-benzenesulfonyl
chloride (1.30 g, 6.67 mmol). After stirring at rt for 2 h, the mixture
was diluted with water and extracted with EtOAc. The combined
organics extracts were dried (MgSO4), filtered, and concentrated to
give an oil. This oil was purified by silica gel chromatography (0−50%
EtOAc in hexanes) to give 1,1,1,3,3,3-hexafluoro-2-(4-(4-(phenyl-
sulfonyl)-1-piperazinyl)phenyl)-2-propanol (1.85 g, 59% yield) as a
1
white solid. H NMR (400 MHz, CDCl3) δ = 7.85−7.77 (m, 2 H),
7.67−7.54 (m, 5 H), 7.01−6.92 (m, 2 H), 3.39−3.28 (m, 4 H), 3.27−
3.21 (m, 4 H). m/z (ESI, +ve ion) 469.0 (M + H)+.
2-(4-(4-((5-Amino-2-thiophenyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (17). A 100 mL round-bottomed
was charged with 1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)phenyl)-2-
propanol8 (0.54 g, 1.7 mmol), 10 mL of CH2Cl2, and 5-bromo-2-
thiophenesulfonyl chloride (0.43 g, 1.7 mmol). To this was added
triethylamine (0.20 g, 0.28 mL, 2.1 mmol). After 15 min at rt, the
mixture was diluted with 10 mL of CH2Cl2 and filtered. The filtrate
was concentrated and purified by silica gel chromatography (0−50%
EtOAc in hexanes) to give 2-(4-(4-((5-bromo-2-thiophenyl)sulfonyl)-
1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (0.83 g, 91%
To
a solution of 1,1,1,3,3,3-hexafluoro-2-(4-(4-(2-
nitrophenylsulfonyl)piperazin-1-yl)phenyl)-2-propanol (0.13 g, 0.25
mmol) in EtOH (5 mL) was added 10% Pd/C (0.026 g, 0.24 mmol).
The reaction was stirred under a hydrogen atmosphere (1 atm) for 4 h
at rt. The solution was filtered through Celite (diatomaceous earth),
and the filtrate was concentrated onto silica gel. Purification by silica
gel chromatography (0−6% MeOH in CH2Cl2) afforded 2-(4-(4-(2-
aminophenylsulfonyl)piperazin-1-yl)phenyl)-1,1,1,3,3,3-hexafluoro-2-
1
yield) as a white solid. H NMR (400 MHz, CD3OD) δ 7.57 (d, J =
8.8 Hz, 2 H), 7.44 (d, J = 4.1 Hz, 1 H), 7.31 (d, J = 3.9 Hz, 1 H),
7.09−6.94 (m, 2 H), 3.39−3.34 (m, 4 H), 3.24−3.18 (m, 4 H).
A 20 mL vial was charged with 2-(4-(4-((5-bromo-2-thiophenyl)-
sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol
(0.89 g, 1.61 mmol), 1,1-diphenylmethanimine (0.35 g, 1.93 mmol),
sodium tert-butoxide (0.37 g, 3.87 mmol), and 6 mL of toluene. To
this was added Pd2(dba)3 (0.15 g, 0.16 mmol) and 2,2′-bis-
1
propanol (0.079 g, 64% yield) as a white solid. H NMR (400 MHz,
DMSO-d6) δ 8.40 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.41 (dd, J = 1.2,
8.0 Hz, 1H), 7.28−7.35 (m, 1H), 7.00 (d, J = 9.0 Hz, 2H), 6.87 (d, J =
J
dx.doi.org/10.1021/jm4016747 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
8.0 Hz, 1H), 6.66 (t, J = 7.2 Hz, 1H), 6.09 (s, 2H), 3.22−3.29 (m,
4H), 3.06−3.14 (m, 4H). m/z (ESI, +ve ion) 483.8 (M + H)+.
2-(4-(4-((3-Aminophenyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (20). Following the procedure
outlined above for 19, the reaction of 3-nitrobenzenesulfonyl chloride
and 1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)phenyl)-2-propanol di-
hydrochloride8 followed by reduction with 10% Pd/C produced the
filtered, and concentrated to give an oil. Purification via silica gel
chromatography (0−70% EtOAc in hexanes) delivered 2-(4-(4-((6-
amino-3-pyridinyl)sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexa-
1
fluoro-2-propanol (1.65 g, 55% yield) as an off-white solid. H NMR
(400 MHz, CD3OD) δ 8.30 (d, J = 2.3 Hz, 1 H), 7.74 (dd, J = 2.4, 8.9
Hz, 1 H), 7.56 (d, J = 8.8 Hz, 2 H), 7.00 (d, J = 9.0 Hz, 2 H), 6.64 (d, J
= 8.8 Hz, 1 H), 3.35−3.30 (m, 4 H), 3.17−3.11 (m, 4 H). m/z (ESI,
+ve ion) 484.9 (M + H)+.
1
title compound as a white solid (48% yield over two steps). H NMR
(400 MHz, DMSO-d6) δ = 8.39 (s, 1 H), 7.46 (d, J = 8.8 Hz, 2 H),
7.25 (t, J = 7.9 Hz, 1 H), 7.09−6.89 (m, 3 H), 6.87−6.70 (m, 2 H),
5.65 (s, 2 H), 3.35−3.26 (m, 4H), 3.05−2.91 (m, 4 H). m/z (ESI, +ve
ion) 484.1 (M + H)+.
2-(4-(4-((2-Amino-5-pyrimidinyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (24). A 100 mL round-bottomed
flask was charged with 1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)-
phenyl)-2-propanol dihydrochloride8 (0.45 g, 1.13 mmol), 10 mL of
CH2Cl2, triethylamine (0.46 g, 0.63 mL, 4.51 mmol), and 2-chloro-5-
pyrimidinesulfonyl chloride (0.24 g, 1.13 mmol). After stirring for 15
min at rt, the mixture was concentrated and diluted with 10 mL of
EtOH and 10 mL of NH4OH. The mixture was stirred at rt for 1 h and
then diluted with 50 mL of EtOAc. The organics were separated, dried
(MgSO4), filtered, and concentrated. The residue was purified by
column chromatography on silica gel (0−65% EtOAc in hexanes) to
give 2-(4-(4-((2-amino-5-pyrimidinyl)sulfonyl)-1-piperazinyl)phenyl)-
2-(4-(4-((4-Aminophenyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (21). Following the procedure
outlined above for 19, the reaction of 4-nitrobenzenesulfonyl chloride
and 1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)phenyl)-2-propanol di-
hydrochloride8 followed by reduction with 10% Pd/C produced the
1
title compound as a white solid (30% yield over two steps). H NMR
(400 MHz, CD3OD) δ 7.54 (d, J = 8.8 Hz, 2 H), 7.48 (d, J = 8.6 Hz, 2
H), 6.98 (d, J = 9.0 Hz, 2 H), 6.73 (d, J = 8.6 Hz, 2 H), 3.31−3.26 (m,
4 H), 3.11−3.03 (m, 4 H). m/z (ESI, +ve ion) 484.1 (M + H)+.
2-(4-(4-((2-Amino-4-pyridinyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (22). A 250 mL round-bottomed
flask was charged with 4-bromo-2-chloropyridine (1.00 g, 5.20 mmol)
and 20 mL of dry diethylether under a nitrogen atmosphere. The
solution was then cooled to −78 °C, where n-BuLi (2.5 M in hexanes,
2.08 mL, 5.20 mmol) was added. After stirring for 5 min at −78 °C,
sulfur dioxide gas was bubbled through the solution for approximately
1 min. The resulting suspension was allowed to warm to rt and
concentrated. To the oily solid was added 20 mL of CH2Cl2 and NCS
(0.69 g, 5.20 mmol). After stirring for additional 30 min at rt,
1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)phenyl)-2-propanol dihy-
drochloride8 (2.09 g, 5.20 mmol) and triethylamine (2.10 g, 2.90
mL, 20.8 mmol) were added. After an additional 30 min at rt, 50 mL
of water was added and the layers were separated. The organics were
dried (MgSO4), filtered, concentrated, and purified via silica gel
chromatography (0−50% EtOAc in hexanes) to give 2-(4-(4-((2-
chloro-4-pyridinyl)sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexa-
1
1,1,1,3,3,3-hexafluoro-2-propanol (0.22 g, 40% yield). H NMR (400
MHz, CD3OD) δ 8.58 (s, 2 H), 7.57 (d, J = 8.8 Hz, 2 H), 7.03 (d, J =
9.2 Hz, 2 H), 3.38−3.34 (m, 4 H), 3.22−3.17 (m, 4 H). m/z (ESI, +ve
ion) 485.9 (M + H)+.
2-(4-(4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-
piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (25).9 A 2 L
Erlenmeyer flask was charged with 2-piperazinone (36.5 g, 364 mmol),
sodium carbonate (116 g, 1.10 mol), 600 mL of dioxane, and 150 mL
of water at room temperature. To this was slowly added benzyl
chloroformate (62.1 g, 364 mmol) over 20 min. After the addition was
complete, the mixture was stirred for 2 h and then diluted with water
and extracted with 2 L of EtOAc. The combined organic extracts were
dried (MgSO4), filtered, and concentrated to give a white solid. To this
solid was added 500 mL of CH2Cl2, triethylamine (92.6 g, 128 mL,
911 mmol), DMAP (4.45 g, 36.4 mmol), and di-tert-butyl dicarbonate
(119 g, 546 mmol). After 1 h at room temperature, the mixture was
diluted with water and the organics were separated, dried (MgSO4),
filtered, and concentrated to give a brown oil. To this oil was added
100 mL of CH2Cl2 followed by 1 L of hexane. The resulting white
solid was collected by filtration to give 4-benzyl 1-tert-butyl 2-oxo-1,4-
piperazinedicarboxylate (101 g, 81% yield). 1H NMR (400 MHz,
CDCl3) δ 7.45−7.29 (m, 5 H), 5.15 (s, 2 H), 4.24 (s, 2 H), 3.88−3.74
(m, 2 H), 3.74−3.59 (m, 2 H), 1.54 (s, 9 H).
1
fluoro-2-propanol (1.60 g, 61% yield) as an off-white solid. H NMR
(400 MHz, CD3OD) δ 8.68 (d, J = 5.1 Hz, 1 H), 7.82 (s, 1 H), 7.72
(dd, J = 1.4, 5.1 Hz, 1 H), 7.56 (d, J = 8.8 Hz, 2 H), 7.00 (d, J = 9.0
Hz, 2 H), 3.36−3.33 (m, 4 H), 3.29−3.23 (m, 4 H).
A 350 mL pressure vessel was charged with 2-(4-(4-((2-chloro-4-
pyridinyl)sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-
propanol (1.60 g, 3.18 mmol), 15 mL of EtOH, and concentrated
ammonium hydroxide (15 mL, 385 mmol). The vessel was sealed, and
the mixture was heated at 125 °C for 18 h. After the mixture was
allowed to cool to rt, water (50 mL) was added and the organics were
extracted with CH2Cl2 (3 × 100 mL). The combined organic extracts
were dried (MgSO4), filtered, and concentrated. The residue was
purified by silica gel chromatography (0−70% EtOAc in hexanes). The
resulting solid was washed with CH2Cl2 to give 2-(4-(4-((2-amino-4-
pyridinyl)sulfonyl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-
A 150 mL round-bottomed flask charged with a solution of 4-benzyl
1-tert-butyl 2-oxo-1,4-piperazinedicarboxylate (1.41 g, 4.22 mmol) in 5
mL of THF was cooled to 0 °C. 1-Propynylmagnesium bromide (0.5
M in THF, 20.0 mL, 10.0 mmol) was added dropwise over 15 min.
The mixture was stirred at 0 °C for 2 h, and then saturated aqueous
NH4Cl (40 mL) was added. The aqueous phase was extracted with
EtOAc (200 mL, then 2 × 100 mL). The organic extracts were dried
(Na2SO4), filtered, and concentrated. The crude product was purified
by silica gel chromatography (0−50% EtOAc in hexanes) to afford
benzyl (2-((tert-butoxycarbonyl)amino)ethyl)(2-oxo-3-pentyn-1-yl)-
1
1
carbamate (1.55 g, 99% yield) as a clear oil. H NMR (400 MHz,
propanol (0.10 g, 7% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.39−7.27 (m, 5 H), 5.13 (d, J = 19.4 Hz, 2 H), 4.97 (d, J =
3.9 Hz, 1 H), 4.18 (s, 1 H), 4.16−4.08 (m, 1 H), 3.44 (td, J = 5.8, 11.7
Hz, 2 H), 3.32−3.16 (m, 2 H), 2.00 (d, J = 13.3 Hz, 3 H), 1.42 (d, J =
4.7 Hz, 9 H).
CD3OD) δ = 7.97 (d, J = 5.5 Hz, 1 H), 7.39 (d, J = 8.6 Hz, 2 H), 6.84
(d, J = 9.2 Hz, 2 H), 6.75−6.65 (m, 2 H), 3.19−3.12 (m, 4 H), 3.08−
3.02 (m, 4 H). m/z (ESI, +ve ion) 484.8 (M + H)+.
2-(4-(4-((6-Amino-3-pyridinyl)sulfonyl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (23). A 250 mL round-bottomed
flask was charged with 1,1,1,3,3,3-hexafluoro-2-(4-(1-piperazinyl)-
phenyl)-2-propanol dihydrochloride8 (2.50 g, 6.23 mmol), 20 mL of
CH2Cl2, triethylamine (2.52 g, 3.47 mL, 24.9 mmol), and 6-
chloropyridine-3-sulfonyl chloride22 (1.32 g, 6.23 mmol). After stirring
at rt for 30 min, 150 mL of water was added and the layers were
separated. The organics were dried (MgSO4), filtered, and
concentrated to give an oily solid. To this material was added 10
mL of EtOH and concentrated ammonium hydroxide (9.95 mL, 255
mmol). The vessel was sealed and heated at 120 °C for 12 h. After the
mixture was allowed to cool to rt, 100 mL of EtOAc and 50 mL of
water were added. The organics were separated, dried (MgSO4),
A 3 L round-bottomed flask was charged with 2-((tert-
butoxycarbonyl)amino)ethyl)(2-oxo-3-pentyn-1-yl)carbamate (82.2 g,
219 mmol) and 300 mL of CH2Cl2. After cooling to −10 °C, TFA
(169 mL, 2.20 mol) was added, and the resulting dark solution was
stirred at room temperature for 15 min. Sodium triacetoxyborohydride
(186 g, 878 mmol) was added portionwise over 10 min. After 2 h, the
mixture was concentrated, diluted with EtOAc (1 L), and neutralized
with 5 N NaOH. The layers were separated, and the organic extracts
were washed with brine, dried (MgSO4), filtered, and concentrated.
The resulting orange oil was purified via silica gel chromatography (0−
4.5% MeOH in CH2Cl2) to give benzyl 3-(1-propyn-1-yl)-1-
piperazinecarboxylate (43.7 g, 77% yield) as a brown foam. 1H
K
dx.doi.org/10.1021/jm4016747 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
NMR (400 MHz, CDCl3) δ 7.43−7.30 (m, 5 H), 5.28−5.04 (m, 2 H),
4.05−3.61 (m, 5 H), 3.37 (br s, 1 H), 3.03 (br s, 1 H), 1.79 (br s, 3 H)
(one exchangeable proton was not observed).
eluting peak) were assigned based on the cocrystal structure with
hGKRP.
2-(4-((2R)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-
piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (26). 1H
NMR (400 MHz, CDCl3) δ 8.49 (d, J = 1.8 Hz, 1 H), 7.78 (dd, J
= 2.3, 8.8 Hz, 1 H), 7.59 (d, J = 8.6 Hz, 2 H), 6.97 (d, J = 9.0 Hz, 2 H),
6.54 (d, J = 8.8 Hz, 1 H), 4.97 (s, 2 H), 4.46 (br s, 1 H), 3.77 (t, J =
11.7 Hz, 2 H), 3.67 (br s, 1 H), 3.51−3.33 (m, 2 H), 2.82 (dd, J = 3.3,
11.0 Hz, 1 H), 2.68 (dt, J = 3.9, 11.1 Hz, 1 H), 1.79 (d, J = 2.0 Hz, 3
H). m/z (ESI, +ve ion) 523.2 (M + H)+.
2-(4-((2S)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-
piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (27). 1H
NMR (400 MHz, CDCl3) δ 8.48 (d, J = 2.3 Hz, 1 H), 7.77 (dd, J
= 2.5, 8.8 Hz, 1 H), 7.57 (d, J = 8.8 Hz, 2 H), 6.95 (d, J = 9.2 Hz, 2 H),
6.52 (d, J = 8.8 Hz, 1 H), 4.94 (s, 2 H), 4.44 (br s, 1 H), 3.82−3.71
(m, 2 H), 3.58−3.33 (m, 3 H), 2.81 (dd, J = 3.2, 11.1 Hz, 1 H), 2.67
(dt, J = 3.9, 11.0 Hz, 1 H), 1.78 (d, J = 2.2 Hz, 3 H). m/z (ESI, +ve
ion) 523.2 (M + H)+.
(2S)-2-(4-((2S)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-
yl)-1-piperazinyl)phenyl)-1,1,1-trifluoro-2-propanol (28). Com-
pound 28 was synthesized according to the procedure described for
the synthesis of 25 using (S)-2-(4-bromophenyl)-1,1,1-trifluoro-2-
propanol8 in place of 2-(4-bromophenyl)-1,1,1,3,3,3-hexafluoropro-
pan-2-ol. The individual isomers were isolated using preparative SFC
(Chiralpak AD-H column (4.6 mm × 150 mm, 5 μm) eluting with
60% supercritical CO2 in 40% MeOH with 40 mM ammonia at a flow
rate of 60 mL/min) to give the title compound (first eluting peak) in
>99% diastereomeric excess. 1H NMR (300 MHz, CDCl3) δ 8.50 (s, 1
H), 7.83−7.74 (m, 1 H), 7.47 (d, J = 8.6 Hz, 2 H), 6.94 (d, J = 8.8 Hz,
2 H), 6.54 (d, J = 8.6 Hz, 1 H), 5.01 (s, 2 H), 4.42 (br s, 1 H), 3.74 (t,
J = 10.2 Hz, 2 H), 3.44−3.31 (m, 2 H), 2.86 (dd, J = 3.1, 11.2 Hz, 1
H), 2.78−2.60 (m, 1 H), 2.38 (br s, 1 H), 1.79 (d, J = 1.9 Hz, 3 H),
1.76 (s, 3 H). m/z (ESI, +ve ion) 469.2 (M + H)+.
A 150 mL reaction vessel was charged with benzyl 3-(prop-1-yn-1-
yl)piperazine-1-carboxylate (2.88 g, 11.2 mmol), 2-(4-bromophenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol31 (4.36 g, 13.5 mmol), dicyclohexyl-
(2′,6′-diisopropoxy-[1,1′-biphenyl]-2-yl)phosphine (RuPhos) (0.530
g, 1.14 mmol), chloro(2-dicyclohexylphosphino-2′,6′-di-i-propoxy-
1,1′-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), methyl tert-bu-
tylether adduct (RuPhos first generation precatalyst) (0.417 g, 0.572
mmol), sodium tert-butoxide (2.73 g, 28.4 mmol), and toluene (35
mL). Argon was bubbled through the solution for 10 min. The vessel
was sealed and heated at 100 °C for 1.5 h. After the reaction was
allowed to cool to room temperature, water (100 mL) was added. The
aqueous phase was extracted with EtOAc (3 × 100 mL), and the
combined organic extracts were washed with brine, dried (Na2SO4),
filtered, and concentrated. The crude product was purified by silica gel
chromatography (0−50% EtOAc in hexanes) to afford benzyl 3-(1-
propyn-1-yl)-4-(4-(2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl)-
phenyl)-1-piperazinecarboxylate (3.84 g, 69% yield) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.6 Hz, 2 H), 7.44−7.29
(m, 5 H), 6.99 (d, J = 9.0 Hz, 2 H), 5.32−5.08 (m, 2 H), 4.48−4.17
(m, 3 H), 3.50−3.02 (m, 5 H), 1.69 (br s, 3 H).
A 500 mL round-bottomed flask was charged with benzyl 3-(1-
propyn-1-yl)-4-(4-(2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl)-
phenyl)-1-piperazinecarboxylate (3.13 g, 6.25 mmol) and TFA (40
mL). Trifluoromethanesulfonic acid (TfOH) (1.25 mL, 14.1 mmol)
was added dropwise at room temperature. After 5 min, additional
TfOH (0.45 mL, 5.1 mmol) was added. After an additional 10 min,
solid NaHCO3 was carefully added in portions until effervescence
ceased. Saturated aqueous NaHCO3 (250 mL) was then added slowly
to bring the pH to approximately 7. The aqueous phase was then
extracted with EtOAc (100 mL). The organic layers were combined
and washed with water (200 mL) and brine (200 mL) then dried
(Na2SO4), filtered, and concentrated to afford a tan solid. To this solid
was added CH2Cl2 (30 mL) and triethylamine (3.63 g, 5.00 mL, 35.9
mmol). 6-Chloropyridine-3-sulfonyl chloride22 (1.58 g, 7.43 mmol)
was then added in potions at 0 °C. The brown mixture was stirred at 0
°C for 10 min, and then the reaction mixture was concentrated and
purified by silica gel chromatography (twice, 0−50% EtOAc in
hexanes) to afford 2-(4-(4-((6-chloro-3-pyridinyl)sulfonyl)-2-(1-pro-
pyn-1-yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol
(3.46 g, 100% yield) as an off-white solid. 1H NMR (400 MHz,
CDCl3) δ 8.84 (d, J = 2.2 Hz, 1 H), 8.06 (dd, J = 2.3, 8.4 Hz, 1 H),
7.60 (d, J = 8.6 Hz, 2 H), 7.52 (d, J = 8.4 Hz, 1 H), 7.01−6.93 (m, 2
H), 4.46 (br s, 1 H), 3.85 (t, J = 11.2 Hz, 2 H), 3.52−3.33 (m, 3 H),
2.95 (dd, J = 3.1, 11.3 Hz, 1 H), 2.81 (dt, J = 3.7, 11.3 Hz, 1 H), 1.78
(d, J = 1.6 Hz, 3 H).
(2R)-2-(4-((2S)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-
yl)-1-piperazinyl)phenyl)-1,1,1-trifluoro-2-propanol (29). Com-
pound 29 was synthesized according to the procedure described for
25 using (R)-2-(4-bromophenyl)-1,1,1-trifluoro-2-propanol8 in place
of 2-(4-bromophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol. The individ-
ual isomers were isolated using preparative SFC (Chiralpak AD-H
column (21 mm × 150 mm, 5 μm) eluting with 50% liquid CO2 in
50% MeOH with 20 mM ammonia at a flow rate of 70 mL/min) to
give the title compound (first eluting peak) with >99% diastereomeric
excess. 1H NMR (300 MHz, CDCl3) δ 8.49 (s, 1 H), 7.78 (dd, J = 2.3,
8.8 Hz, 1 H), 7.46 (d, J = 8.6 Hz, 2 H), 7.00−6.87 (m, 2 H), 6.60−
6.47 (m, 1 H), 4.99 (s, 2 H), 4.41 (d, J = 2.3 Hz, 1 H), 3.82−3.65 (m,
2 H), 3.37 (dd, J = 2.8, 7.3 Hz, 2 H), 2.85 (dd, J = 3.4, 11.1 Hz, 1 H),
2.70 (td, J = 7.3, 11.4 Hz, 1 H), 2.39 (s, 1 H), 1.77 (d, J = 2.0 Hz, 3
H), 1.75 (d, J = 0.7 Hz, 3 H). m/z (ESI, +ve ion) 469.0 (M + H)+.
2-(4-((2R)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-butyn-1-yl)-1-
piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (30). Follow-
ing the procedure described above for 25, chloro(1-butyn-1-yl)
magnesium (generated from 1-butyne and i-PrMgCl) produced the
A 20 mL sealed tube was charged with 2-(4-(4-((6-chloro-3-
pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-piperazinyl)phenyl)-
1,1,1,3,3,3-hexafluoro-2-propanol (0.340 g, 0.627 mmol), concentrated
ammonium hydroxide (5.00 mL, 38.5 mmol), and EtOH (5 mL). The
reaction mixture was heated in a microwave reactor at 120 °C for 1 h.
The reaction mixture was then heated in a heating block at 110 °C for
5 h and then was concentrated and purified by silica gel
chromatography (25 g of silica, 30−80% EtOAc in hexanes) to afford
2-(4-(4-((6-amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-
piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (0.289 g, 88%
yield) as a off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.49 (br s, 1
H), 7.80 (dd, J = 2.3, 8.8 Hz, 1 H), 7.59 (d, J = 8.8 Hz, 2 H), 6.97 (d, J
= 9.0 Hz, 2 H), 6.55 (d, J = 8.8 Hz, 1 H), 5.05 (s, 2 H), 4.46 (br s, 1
H), 3.85−3.72 (m, 2 H), 3.54 (br s, 1 H), 3.50−3.34 (m, 2 H), 2.83
(dd, J = 3.3, 11.0 Hz, 1 H), 2.69 (dt, J = 3.4, 11.0 Hz, 1 H), 1.80 (s, 3
H). m/z (ESI, +ve ion) 523.1 (M + H)+.
1
title compound as a mixture of enantiomers. H NMR (400 MHz,
CD3OD) δ 8.31 (d, J = 2.3 Hz, 1 H), 7.74 (dd, J = 2.4, 8.9 Hz, 1 H),
7.57 (d, J = 8.8 Hz, 2 H), 7.04 (d, J = 9.2 Hz, 2 H), 6.63 (d, J = 8.4 Hz,
1 H), 4.76−4.57 (m, 1 H), 3.82−3.68 (m, 2 H), 3.57−3.44 (m, 1 H),
3.29−3.20 (m, 1 H), 2.85−2.73 (m, 1 H), 2.68−2.45 (m, 1 H), 2.14
(dd, J = 1.9, 7.5 Hz, 2 H), 1.04 (t, J = 7.5 Hz, 3 H). m/z (ESI, +ve ion)
537.2 (M + H)+.
2-(4-((2S)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(cyclopropylethyn-
yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (31).
Following the procedure described above for 25, chloro-
(cyclopropylethynyl) magnesium (generated from ethynylcyclopro-
pane and i-PrMgCl) produced the title compound as a mixture of
1
enantiomers. H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1 H), 8.23
The individual enantiomers (26 and 27) were isolated using chiral
SFC (Chiralpak ADH column (21 mm × 250 mm, 5 μm) eluting with
35% MeOH in supercritical CO2 (total flow was 70 mL/min)). This
produced the two isomers with enantiomeric excesses of >98%. The
absolute stereochemistries of 26 (second eluting peak) and 27 (first
(br s, 1 H), 7.63 (d, J = 9.0 Hz, 1 H), 7.26 (d, J = 8.6 Hz, 1 H), 7.07−
6.94 (m, 3 H), 6.53 (d, J = 8.8 Hz, 1 H), 5.46 (br s, 1 H), 4.42 (br s, 1
H), 4.29 (br s, 1 H), 3.56 (br s, 1 H), 3.46−3.34 (m, 1 H), 3.24 (br s,
2 H), 2.94 (d, J = 10.8 Hz, 2 H), 2.84−2.69 (m, 1 H), 2.71−2.52 (m, 3
H), 2.06 (br s, 1 H). m/z (ESI, +ve ion) 549.2 (M + H)+.
L
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Journal of Medicinal Chemistry
Article
2-(4-(4-((6-Amino-3-pyridinyl)sulfonyl)-2-(3,3-dimethyl-1-butyn-
1-yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (32).
Following the procedure described above for 25, chloro(3,3-
dimethyl-1-butyn-1-yl)magnesium (generated from 3,3-dimethyl-1-
butyne and i-PrMgCl) produced the title compound as a mixture of
(5) Rees, M. G.; Gloyn, A. L. Small Molecular Glucokinase
Activators: Has Another New Anti-Diabetic Therapeutic Lost Favour?
Br. J. Pharmacol. 2013, 168 (2), 335−338.
(6) Pfefferkorn, J. A. Strategies For the Design of Hepatoselective
Glucokinase Activators to Treat Type 2 Diabetes. Expert Opin. Drug
Discovery 2013, 8, 319−330.
1
enantiomers. H NMR (400 MHz, CD3OD) δ 8.32 (d, J = 2.0 Hz, 1
H), 7.74 (dd, J = 2.4, 8.9 Hz, 1 H), 7.58 (d, J = 8.8 Hz, 2 H), 7.05 (d, J
= 9.0 Hz, 2 H), 6.63 (d, J = 9.0 Hz, 1 H), 4.68−4.58 (m, 1 H), 3.70 (d,
J = 11.2 Hz, 2 H), 3.46 (d, J = 12.1 Hz, 1 H), 3.28−3.22 (m, 1 H),
2.80 (dd, J = 3.3, 11.3 Hz, 1 H), 2.70−2.50 (m, 1 H), 1.11 (s, 9 H). m/
z (ESI, +ve ion) 565.1 (M + H)+.
(7) Matschinsky, F. M.; Van der Kooi, J. M.; Zelent, B.; Naji, A.;
Doliba, N.; Sarabu, R.; Li, C.; Grimsby, J. Glucokinase Activators for
Diabetes Therapy. Diabetes Care 2011, 34 (Suppl 2), S236−S243.
(8) Ashton, K. S.; Andrews, K. L.; Bryan, M. C.; Chen, J.; Chen, K.;
Chen, M.; Chmait, S.; Croghan, M.; Cupples, R.; Fotsch, C.;
Helmering, J.; Jordan, S. R.; Kurzeja, R. J. M.; Michelsen, K.;
Pennington, L. D.; Poon, S. F.; Sivits, G.; Van, G.; Vonderfecht, S. L.,
Wahl, R. C.; Zhang, J.; Lloyd, D. J.; Hale, C.; St. Jean, D. J., Jr. Small
Molecule Disruptors of the Glucokinase−Glucokinase Regulatory
Protein Interaction: 1. Discovery of a Novel Tool Compound for In
Vivo Proof-of-Concept. J. Med. Chem. 2014, DOI jm4016735.
(9) Lloyd, D. J.; St. Jean, D. J., Jr.; Kurzeja, R. J. M.; Wahl, R. C.;
Michelsen, K.; Cupples, R.; Chen, M.; Wu, J.; Sivits, G.; Helmering, J.;
Ashton, K. S.; Pennington, L. D.; Fotsch, C. H; Vazir, M.; Chen, K.;
Chmait, S.; Zhang, J.; Liu, L.; Norman, M. H.; Andrews, K. A.;
Bartberger, M. D.; Van, G.; Galbreath, E. J.; Vonderfecht, S. L.; Wang,
ASSOCIATED CONTENT
■
S
* Supporting Information
Standard deviations for the in vitro activities listed in Tables
1−3, rat in vivo PK data for analogues 11−14, and hGK SPR
data for 27, 29, and 33. This material is available free of charge
via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID codes for 1, 23, and 27 bound to hGKRP are 4LY9,
4MRO, and 4MQU, respectively.
́
M.; Jordan, S. R.; Veniant, M. M.; Hale, C. Anti-Diabetic Effects of a
Glucokinase Regulatory Protein Small Molecule Disruptor. Nature
2013, 504, 437−440.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 805-313-5153. E-mail: david01@amgen.com.
(10) The average unbound drug concentration at Cmax was 0.032 μM.
The EC50 value for 1 in mouse hepatocytes was 1.79 μM.
(11) Ashton, K.; Bartberger, M. D.; Bo, Y.; Bryan, M. C.; Croghan,
M.; Fotsch, C. H.; Hale, C. H; Kunz, R. K.; Liu, L.; Nishimura, N.;
Norman, M. H.; Pennington, L. D.; Poon, S. F.; Stec, M. M.; St. Jean,
D. J., Jr.; Tamayo, N. A.; Tegley, C. M.; Yang, K. Preparation of
sulfonylpiperazine derivatives that interact with glucokinase regulatory
protein for the treatment of diabetes and other diseases. PCT Int.
Appl. WO 2012027261, 2012.
(12) Wolff, M.; Kauschke, S. G.; Schmidt, S.; Heilker, R. Activation
and Translocation of Glucokinase in Rat Primary Hepatocytes
Monitored by High Content Image Analysis. J. Biomol. Screening
2008, 13, 837−846.
(13) Because of the greater accessibility of rat liver microsomes, RLM
data was used as a surrogate for in vitro mouse metabolism. We
observed a strong correlation between the two species (data not
shown).
(14) Chen, Z.; Venkatesan, A. M.; Dos Santos, O.; Delos Santos, E.;
Dehnhardt, C. M.; Ayral-Kaloustian, S.; Ashcroft, J.; McDonald, L. A.;
Mansour, T. S. Stereoselective Synthesis of an Active Metabolite of the
Potent PI3 Kinase Inhibitor PKI-179. J. Org. Chem. 2010, 75, 1643−
1651.
Present Address
▽For J.Z.: JD Consulting, 1969 Roadrunner Avenue,
Thousand Oaks, California 91360, United States.
Notes
The authors declare the following competing financial
interest(s): The authors declare competing financial interests
as employees of Amgen Inc.
ACKNOWLEDGMENTS
■
We acknowledge Kyung Gahm, Wesley Barnhart, and Samuel
Thomas for conducting the SFC separations, Robert Kurzeja
for providing the purified proteins, and Robert Wahl and Kathy
Chen for assay development. Additionally, we thank Scott
́
Simonet, Murielle Veniant, Dean Hickman, Philip Tagari, and
Randall Hungate for their support of this research.
ABBREVIATIONS USED
■
(15) See Supporting Information.
GK, glucokinase; GKA, glucokinase activator; GKRP, glucoki-
nase regulatory protein; S0.5, concentration at which the enzyme
is 50% active; Vmax, maximum velocity; RLM, rat liver
microsomes; HLM, human liver microsomes; CL, clearance;
MRT, mean resonance time; PPB, plasma protein binding; fu,
fraction unbound; IHC, immunohistochemistry; DMEM,
Dulbecco’s Modified Eagle Medium; AAALAC, Association
for Assessment and Accreditation of Laboratory Animal Care
(16) Gleeson, M. P. Generation of a Set of Simple, Interpretable
ADMET Rules of Thumb. J. Med. Chem. 2008, 51, 817−834.
(17) Gleeson, M. P.; Bravi, G.; Modi, S.; Lowe, D. ADMET Rules of
Thumb II: A Comparison of the Effects of Common Substituents on a
Range of ADMET Parameters. Bioorg. Med. Chem. 2009, 17, 5906−
5919.
(18) St. Jean, D. J., Jr.; Fotsch, C. Mitigating Heterocycle Metabolism
in Drug Discovery. J. Med. Chem. 2012, 55, 6002−6020.
(19) Indeed, an NH-engaging water molecule is observed in other in-
house cocrystal structures not possessing an occluding substituent in
this region.
(20) B3LYP/6-31G* optimizations of methyl- and propynyl
substituted, S-phenyl-N-phenylpiperazine sulfonamides give rise to
an axial−equatorial energy difference of ca. 0.9 and 1.3 kcal/mol,
respectively, suggesting a > 75% population of the axial conformer.
Additional CPCM single-point energy calculations in an aqueous
solvent model (UAKS radii; B3LYP/6-31G*; Gaussian 03, revision
D.01) further support the preference for the axial substituent
orientation (ca. 1.2 and 1.1 kcal/mol, respectively) regardless of the
polarity of the medium.
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