Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 4. Exploration of a novel binding pocket

Article abstract of DOI:10.1021/jm5001979

Structure-activity relationship investigations conducted at the 5-position of the N-pyridine ring of a series of N-arylsulfonyl-N′-2-pyridinyl- piperazines led to the identification of a novel bis-pyridinyl piperazine sulfonamide (51) that was a potent disruptor of the glucokinase-glucokinase regulatory protein (GK-GKRP) interaction. Analysis of the X-ray cocrystal of compound 51 bound to hGKRP revealed that the 3-pyridine ring moiety occupied a previously unexplored binding pocket within the protein. Key features of this new binding mode included forming favorable contacts with the top face of the Ala27-Val28-Pro29 ("shelf region") as well as an edge-to-face interaction with the Tyr24 side chain. Compound 51 was potent in both biochemical and cellular assays (IC₅₀ = 0.005 μM and EC ₅₀ = 0.205 μM, respectively) and exhibited acceptable pharmacokinetic properties for in vivo evaluation. When administered to db/db mice (100 mg/kg, po), compound 51 demonstrated a robust pharmacodynamic effect and significantly reduced blood glucose levels up to 6 h postdose.

Full text of DOI:10.1021/jm5001979

Article
pubs.acs.org/jmc
Small Molecule Disruptors of the Glucokinase−Glucokinase
Regulatory Protein Interaction: 4. Exploration of a Novel Binding
Pocket
Fang-Tsao Hong,*,† Mark H. Norman,*,† Kate S. Ashton,† Michael D. Bartberger,‡ Jie Chen,§
Samer Chmait,‡ Rod Cupples,∥ Christopher Fotsch,† Steven R. Jordan,‡ David J. Lloyd,∥ Glenn Sivits,∥
Seifu Tadesse,† Clarence Hale,∥ and David J. St. Jean, Jr.†
‡
§
†Departments of Therapeutic Discovery−Medicinal Chemistry, Therapeutic Discovery−Molecular Structure, Pharmacokinetics
∥
and Drug Metabolism, and Metabolic Disorders, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799,
United States
S
* Supporting Information
ABSTRACT: Structure−activity relationship investigations
conducted at the 5-position of the N-pyridine ring of a series
of N-arylsulfonyl-N′-2-pyridinyl-piperazines led to the identi-
fication of a novel bis-pyridinyl piperazine sulfonamide (51)
that was a potent disruptor of the glucokinase−glucokinase
regulatory protein (GK−GKRP) interaction. Analysis of the X-
ray cocrystal of compound 51 bound to hGKRP revealed that
the 3-pyridine ring moiety occupied a previously unexplored binding pocket within the protein. Key features of this new binding
mode included forming favorable contacts with the top face of the Ala27-Val28-Pro29 (“shelf region”) as well as an edge-to-face
interaction with the Tyr24 side chain. Compound 51 was potent in both biochemical and cellular assays (IC50 = 0.005 μM and
EC50 = 0.205 μM, respectively) and exhibited acceptable pharmacokinetic properties for in vivo evaluation. When administered
to db/db mice (100 mg/kg, po), compound 51 demonstrated a robust pharmacodynamic effect and significantly reduced blood
glucose levels up to 6 h postdose.
INTRODUCTION
■
Glucokinase (GK), which is mainly expressed in the liver and
pancreas, is the primary regulator of glucose homeostasis and is
responsible for the phosphorylation of glucose to form glucose 6-
phosphate (G6P).1 While pancreatic GK plays a role in
regulating insulin secretion; hepatic GK functions as an
important regulator for glucose uptake, glycogen synthesis, and
hepatic glucose output.2 The activity of GK in the liver is partially
controlled by glucokinase regulatory protein (GKRP).3 During
fasting, GKRP sequesters GK in the nucleus of hepatocytes and
Figure 1. Piperazine-propynyl lead compounds 1 and 2.
renders it inactive. When glucose levels increase by dietary
intake, the GK−GKRP complex is destabilized and GK
dissociates from GKRP, translocates into the cytoplasm, and
initiates the hepatic glucose metabolic process.4 Increasing GK
activity through the use of glucokinase activators (GKAs) has
emerged as a promising approach to treat diabetes, and several
small molecules are currently under investigation in clinical
trials.5 While this strategy is attractive, there is an increased risk of
hypoglycemia with the use of GKAs. One approach that has been
taken to mitigate this risk has been to design hepatoselective GK
activators that are taken up by the liver exclusively and thereby
eliminating pancreatic GK activation.6 We have investigated an
alternative approach where GK activity is modulated through the
disruption of the GK−GKRP interaction.7−11
optimization of the N-aryl carbinol moiety, which we refer to as
the C-ring.11 Compounds 1 and 2 were found to potently inhibit
the GK−GKRP interaction (IC50 = 0.013 and 0.010 μM,
respectively), and they effectively caused GK translocation in
mouse hepatocytes (EC50 = 0.160 and 0.421 μM, respectively).
In addition, compound 1 was shown to be effective in reducing
blood glucose levels in db/db mice (up to 70% at 100 mg/kg, po).
In our efforts to further improve the potency and increase
structural diversity of this class of GK−GKRP disruptors, we
examined the crystal structure of these piperazine-propynyl
derivatives in more detail with the aim of identifying areas where
In a previous report we identified compounds 1 and 2 (Figure
1) as potent and efficacious GK−GKRP disruptors through
Received: February 6, 2014
Published: July 8, 2014
© 2014 American Chemical Society
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Figure 2. Model of compound 2 based on the cocrystal structure of full-length human GKRP obtained with a structurally similar compound in this series
(i.e., phenyl C-ring).10 (a) Side view illustrating the main interactions of the ligand with the protein. Note: the labeled dihedral angle is between the
propynyl-bearing piperazine carbon and the pyridyl ring carbon (ϕ = −11°). (b) Front view showing the unoccupied shelf region next to Tyr24 and
above Ala27-Pro29 (highlighted by the dashed red oval). The solid red arrow indicates the proposed substitution on the 5-position of the pyridyl C-ring.
additional binding interactions might be gained.12,13 As shown in
Figure 2a, compound 2 binds to GKRP and forms key
interactions with the protein in three main areas: (1) the
amino group of the aminopyridine sulfonamide forms a bidentate
hydrogen bonding interaction with the carbonyl groups of
Gly181 and Met213; (2) the propynyl methyl group occupies a
hydrophobic pocket formed by Val10 and Leu520; and (3) the
bis-trifluoromethyl carbinol oxygen forms a hydrogen bonding
interaction with Arg525. In addition to these three main areas, a
fourth region was identified within the binding site that was not
occupied by compound 2. We refer to this area as the “shelf
region” (highlighted by the red oval in Figure 2b) and is
composed of the floor residues Ala27, Val28, and Pro29 and a
side wall created by the π-face of the Tyr24 side chain. We
postulated that an improvement in binding affinity might be
achieved by targeting this unexplored pocket and that this shelf
region could be accessed by substituting on the 5-position of the
pyridyl C-ring of compound 2 (as indicated by the red arrow in
Figure 2b). To test this hypothesis, we prepared the bis-propynyl
derivative, 3 (Figure 3), and evaluated it for its ability to disrupt
the hGK−hGKRP interaction.
and the pyridyl carbinol C-ring since we had found from our
previous structure−activity relationship (SAR) investigations
that compounds that could more easily adopt a planar
arrangement were preferred.10 Therefore, to better understand
the stark difference in activity between compound 2 and its bis-
propynyl derivative 3, a quantum mechanical study was
performed to analyze the conformational preferences with
respect to the dihedral angle between the central piperazine
and the pyridine C-rings of three propynyl substituted
compounds (Figure 4).14 For computational efficiency, the
carbinol moiety on the pyridyl C-ring was eliminated and a
Unfortunately, the hGK−hGKRP AlphaScreen data of
compound 3 was surprisingly low (IC50 = 8.6 μM). We
speculated that this decrease in activity may be due to an
unfavorable dihedral angle between the central piperazine ring
Figure 4. Dihedral angle plot (B3LYP/6-31G* with SCRF aqueous
solvation) for model substituted N-pyridylpiperazines. Scanned dihedral
depicted in bold and defined as 0° for the depicted conformer. Each
point on the plot corresponds to the lowest-energy structure arising
from a full stochastic conformational search, followed by full B3LYP/6-
31G* geometry optimization, each subject to the given dihedral
constraint. See Supporting Information for full details.
Figure 3. Bis-propynyl derivative 3.
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generic phenyl sulfonamide was used in place of the amino-
pyridine sulfonamide group.
resulting acyl chlorides with two equivalents of trifluoromethyl-
trimethylsilane in the presence of tetramethylammonium
fluoride.15 For the synthesis of compound 3, the chloride
group at the 6-position of compound 8 was displaced with (S)-1-
benzyl-3-(prop-1-yn-1-yl)piperazine (11)16 under palladium
(0)-catalyzed conditions17 to generate the N-pyridinyl-N′-
benzylpiperazine intermediate 12, and the benzyl protecting
group was removed to provide compound 13. Treatment of
intermediate 13 with tert-butyl (5-(chlorosulfonyl)pyridin-2-
yl)carbamate 1416 afforded sulfonamide 15 in good yield. The
second alkynyl group was introduced by the palladium-mediated
coupling18 of compound 15 with 1-propynyl trimethylsilane and
subsequent removal of the tert-butoxycarbonyl (Boc) group gave
the requisite bis-alkynyl derivative, 3.
For the syntheses of the unsubstituted piperazine derivatives,
tert-butyl piperazine 1-carboxylate was utilized as the nucleo-
philic coupling partner and was reacted with pyridinyl bis-
trifluoromethylcarbinols 8−10. The resulting tert-butyl carba-
mate protecting groups were removed to furnish intermediates
16−18, respectively. Sulfonamide intermediates 19−21 were
obtained by coupling 16−18 with sulfonyl chloride 14 in the
presence of triethylamine.
The final targets (23−67) were prepared from intermediates
19, 20, or 21 by following one of two general methods: by a
palladium-catalyzed coupling reaction19 to introduce the
substituent at the 5-position of the pyridine ring followed by
removal of the Boc-protecting group (via 22) or by reversing the
coupling/deprotection sequence (via 23). For Suzuki coupling
reactions, the chloro and bromo intermediates (19, 20, and 23)
were viable coupling partners; however, for Sonogashira-type
alkynylation reactions, bromo intermediate 20 was required.
Finally, the unsubstituted derivative (26) was prepared by
palladium-mediated reduction of chloro intermediate 23 with
formic acid in the presence of triethylamine.20
The dihedral angle plot for the compound with the alkyne
substitution only on the piperazine ring (compound (a) shown in
green; Figure 4) shows that the lowest energy conformers reside
between 0 and 20°; 180 and 220°; and 350 and 360° (Erel ≤ 0.5
kcal/mol; Figure 4). This is consistent with the crystallographic
data, where a near-coplanar orientation (−11°) was observed for
compounds similar to compound 2 in hGKRP.10 However, the
analogous coplanar conformation is highly inaccessible to
compound 3 as is shown by the dihedral angle plot for the bis-
propynyl substituted derivative (curve (b) in red; Figure 4).
When the piperazine and the pyridyl rings of this bis-substituted
compound are in a coplanar orientation, the compound resides
near a maximum on its potential energy surface, approximately
2.5 kcal/mol higher in energy than its global minimum (∼60°).
In addition to examining the compounds with substitution
patterns such as 2 and 3, we also generated the dihedral angle plot
of the compound containing the propynyl substitution only on
the pyridyl C-ring (compound (c) shown in blue; Figure 4).
Interestingly, we found that compound (c) was predicted to
exhibit a similar conformational preference to that of compound
(b), even in the absence of substitution on the piperazine ring,
with the global minima being ∼45−65°. We attribute the
significant conformational changes of (b) and (c) [versus
compound (a)] to the repulsive steric interactions occurring
between the propynyl group on the pyridyl C-ring and the
methine hydrogen (in compound (b)) or the methylene
hydrogens (in compound (c)) adjacent to the proximal
piperazine nitrogen. These computational results suggest that
the deviation from coplanarity predicted for (b) and (c) would
prohibit access to both the upper hydrophobic pocket and the
shelf regions simultaneously without engaging in a significant
steric clash with the protein. This observation is consistent with
the empirical result obtained for compound 3. Therefore, to
gather information solely from targeting the unexplored shelf
region, we prepared and evaluated a set of compounds where the
5-position of the pyridine ring was substituted with various
groups and the propynyl group on the piperazine was removed
entirely (i.e., general scaffold 4, Figure 5). We postulated that the
RESULTS AND DISCUSSION
■
The compounds described in this article were evaluated for their
ability to disrupt the human GK−GKRP binding interaction
using an in vitro AlphaScreen assay, and the data are reported in
Tables 1−4.21 The cellular activities were determined in primary
mouse hepatocytes by treating the cells with test compound and
measuring the difference between the nuclear and cytoplasmic
GK using an Operetta platform. Compounds that were effective
disruptors of the GK−GKRP interaction increased GK trans-
location, and therefore, decreased amounts of GK were observed
in the nucleus. In addition to the biochemical binding and cellular
translocation assays, the compounds were incubated in human
and rat liver microsomes (HLM and RLM) for an initial
assessment of oxidative metabolism. The microsomal data are
reported in Tables 1−4 as intrinsic clearance (CLint (μL/min/
mg)), and these data were utilized to help prioritize potent
compounds for further pharmacokinetic studies (vide infra).
In the initial phase of this investigation we examined a small set
of analogues that contained nonaromatic substituents at the 5-
position of the bistrifluoromethylcarbinol-pyridyl ring, which we
refer to as the C-ring (Table 1). The unsubstituted derivative
(26) and the halogen-substituted analogues (23 and 24) were
modestly active in the biochemical binding assay (IC50 values of
∼100−200 nM), yet were fairly weak in the GK translocation
assay (EC50 values of ≥5 μM). It is worth noting that compound
26 lost ∼18-fold activity in biochemical potency by not accessing
the upper hydrophobic pocket when compared with compound
2.22 Compounds with small alkoxy or alkyl substituents, such as
Figure 5. General scaffold 4 designed to project substituents on the
pyridyl C-ring toward the targeted shelf region.
N-pyridinylpiperazine moiety of 4 would provide a conforma-
tionally rigid scaffold and project the “R” substituent on the
pyridine ring toward the targeted shelf region while, at the same
time, retaining a low energy conformation devoid of steric clashes
in other regions of the ligand binding site.
CHEMISTRY
■
The syntheses of bis-trifluoromethylcarbinols 3 and 23−67 are
outlined in Scheme 1. Commercially available 2,3-disubstituted
nicotinic acids 5−7 were converted to their corresponding bis-
trifluoromethylcarbinols 8−10 via a two-step sequence; first by
treatment with oxalyl chloride followed by the reaction of the
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Scheme 1. Synthesis of 2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-substituted-pyridin-3-yl)-3-bis-
a
trifluoromethylcarbinols
a
Reagents and conditions: (a) oxalyl chloride, DCM, DMF, rt; (b) 2 equiv of TMSCF3, Me4NF, DME, −78 °C to rt; (c) from compound 8, Ruphos,
Ruphos first generation precatalyst, NaOtBu, 1,4-dioxane, 100 °C; (d) 1-chloroethyl chloroformate, K2CO3, DCM, rt, then MeOH, rt; (e) Et3N,
DCM, rt; (f) TMSCCCH3, Xphos precatalyst, Cs2CO3, MeCN, 95 °C; (g) 4 N aq HCl, 1,4-dioxane, rt; (h) tert-butyl piperazine-1-carboxylate,
iPr2EtN, DMF, microwave, 140 °C; (i) TFA, DCM, rt; (j) 14, Et3N, DCM, rt; (k) from compound 19 or 20, ArB(OH)2 or ArB(OR)2,
Pd(amphos)Cl2, K2CO3, aq 1,4-dioxane, Δ; (l) from compound 20, terminal alkynes, Pd(PPh3)4, CuI, Et3N, DME, 60 °C, or Xphos precatalyst,
Cs2CO3, MeCN, Δ; (m) from compound 23, ArB(OH)2 or ArB(OR)2 (or 2-cyclopropyl-6-methyl-1,3,6,2-dioxazaborocane-4,8-dione),
Pd(amphos)Cl2, K2CO3, aq 1,4-dioxane, Δ; (n) HCO2H, Et3N, Pd(amphos)Cl2, DMF, Δ, microwave.
methoxy and cyclopropyl derivatives 25 and 27, showed inferior
biochemical activities; however, potency was enhanced signifi-
cantly by extending further into the shelf region of the active site
by the addition of alkynyl substituents to the 5-position of the
pyridine ring (i.e., compounds 28−32). For example, an 18-fold
increase in potency was achieved when the cyclopropyl group of
27 was extended further into the pocket with an alkynyl spacer to
give cyclopropylalkyne 28 (IC50 = 0.033 μM). This result was
encouraging in that this was the first compound in this series with
sub-100 nM biochemical potency; however, a large cellular shift
was observed in the hepatocyte assay for this particular analogue
(∼100-fold shift). The activities were further enhanced by either
addition of a hydroxymethylene group (29) or a methoxy-
methylene moiety (30) at the ethynene terminal position.
However, gem-dimethyl substitution or homologation of the
hydroxymethylene alkyne group in compound 29 resulted in
decreased activities (compounds 31 and 32, respectively),
particularly in the cellular assay. Among these derivatives, the
methylene-methoxyalkynyl analogue (30) was the most potent
moderately active in the biochemical binding assay. However,
the 1-methyl-4-1H-pyrazole derivative (34) showed excellent
potencies both in the biochemical and cellular assays (IC50
=
0.001 μM; EC50 = 0.052 μM). This exciting result prompted us to
screen other N-alkyl-pyrazole derivatives. The SAR trend from
the data of alkyl pyrrazoles 34−37 clearly revealed that steric
hindrance plays an important role, as the potencies dropped with
increasing size of N-alkyl group. This rank order appeared to hold
as far as the metabolic stabilities were concerned as well (i.e.,
lower metabolic stabilities were observed from methyl (34) →
ethyl (35) → iso-propyl (36) → tert-butyl (37)). Alkyl-
substitutions at the position alpha to the pyrazole nitrogen
were not beneficial to activity (38 and 39). Replacement of 1-
methyl-1H-pyrazole with either 3-furan (40) or 4-methyl-2-
thiofuran (41) resulted in significantly decreased potencies in the
cell-based assay (EC50 = 1.73 and 1.19 μM, respectively).
In attempts to more fully occupy the shelf region of hGKRP,
we expanded our SAR studies by examining the effect of
substituting 6-membered aromatic groups on the 5-position of
the pyridine C-ring (Table 3). The unsubstituted phenyl
analogue (42) potently disrupted the hGK−hGKRP interaction
(IC50 = 0.007 μM) and it also showed submicromolar activity in
the cellular GK translocation assay (EC50 = 0.776 μM). In
addition, this derivative was reasonably stable in microsomes
(CLint = 14/49 μL/min/mg in HLM/RLM), encouraging us to
survey the effects of the substitutions on the aromatic ring. To
gain more potency/stability insights, several substituted phenyl
compound in both the biochemical and cellular assays (IC50
=
0.005 μM and EC50 = 0.254 μM, respectively), and it was over
200-fold more active than the derivative with methoxy group
directly attached to the pyridine ring (25). Unfortunately,
compound 30 showed rapid clearance in rat liver microsomes.
The shelf region SAR was expanded by preparing a small set of
5-membered aromatic heterocyclic derivatives. As shown in
Table 2, the first example, 3-1H-pyrazole 33, was only
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Table 1. SAR of Nonaromatic Substituted Analogues 23−32
Table 2. SAR of 5-Membered Heteroaromatic Analogues 33−
41
a
AlphaScreen data reported as an average (n ≥ 3). Standard deviations
b
a
are reported in the Supporting Information. Unless otherwise noted,
n = 1. In vitro microsomal stability measurements were conducted in
AlphaScreen data reported as an average (n ≥ 3). Standard deviations
c
b
are reported in the Supporting Information. Unless otherwise noted,
n = 1. In vitro microsomal stability measurements were conducted in
c
the presence of NADPH at 37 °C for 30 min at a final compound
d
e
concentration of 1 μM. Average of 2 experimental values. n = 2.
the presence of NADPH at 37 °C for 30 min at a final compound
concentration of 1 μM. Average of 2 experimental values. n = 2.
d
e
metabolic stability of the 3-pyridine derivative (51), we also
explored additional substitutions on the 3-pyridyl ring (i.e.,
fluoro-, methyl-, and methoxy-derivatives; 52−57). Unfortu-
nately, each of the additional substituents that we examined
resulted in a loss in potency (3−8-fold) in the AlphaScreen assay.
In addition, losses in cellular activity were also observed by
adding lipophilic fluoro- and methyl-substituents to the pyridine
ring, with only the 3-methyl derivative 54 showing submicro-
molar activity (EC50 = 0.794 μM). Pyridyl derivatives with polar
methoxy-substituents, such as pyridines 56 and 57, lost activity as
well, especially in the cellular translocation assay (EC50 = 1.40 vs
0.205 μM for the unsubstituted pyridyl compound, 51).
Encouraged by the activity of pyridine 51, we obtained a
cocrystal structure of this compound bound to hGKRP to better
understand the key binding interactions between the ligand and
the protein (Figure 6a). While the main anchoring hydrogen
bonding interactions from the aminopyridinylsulfonamide and
bis-trifluorometylcarbinol moieties remain the same in 51 as
those observed for compound 2, the 3′-pyridyl group on the 5-
position of the C-ring occupies the Ala27-Val28-Pro29 shelf
region as designed. The nitrogen atom of this pyridine ring
derivatives (43−49) were synthesized and examined. For
example, a series of ortho-, meta-, and para-fluorinated analogues
(43−45, respectively) gave similar potencies in the AlphaScreen
binding assay, but unfortunately cellular activity was diminished
and no improvements were obtained in regards to metabolic
stability. In addition to fluoro groups, we investigated polar
functionalities on the phenyl ring (compounds 46−49). With the
exception of the ortho-hydroxyl derivative (46), these polar and
relatively large functionalities (sulfonamide, amide, and sulfone
groups) had a negative impact on activities, and in general,
microsomal stabilities also decreased.
Next we evaluated the disruption of the hGK−hGKRP
interaction by compounds with 6-membered heteroaromatic
substituents to the 5-position of the pyridyl C-ring (Table 3, 50−
57). For the unsubstituted pyridines, the 3-pyridine derivative
(51) not only displayed favorable in vitro activities, both in the
biochemical and cellular assays, over its 4-aza isomer 50 but it
also provided improved cellular potency (∼4 fold) and rat
microsomal stability (∼2−3-fold) when compared with the
phenyl analogue (42). Because of the promising activity and
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points toward the more polar solvent-exposed environment,
while the pyridine hydrogens on the 3 and 4 positions are
engaged in an edge-to-face interaction with the Tyr24 side chain.
Figure 6b shows the superimposition of compounds 2 and 51
bound to GKRP, illustrating the conformational differences
between these two ligands as well as some small changes in the
surrounding residues of the GKRP protein. To make room for
the sterically demanding 3′-pyridyl group in 51, the central N,N′-
disubstituted piperazine tilts away from the 3′-pyridyl moiety
creating a ∼47° dihedral angle between the piperazine carbon
and the pyridyl ring carbon. This result is in good agreement with
the dihedral angle for the low energy conformation predicted
from quantum mechanical calculations with a compound
containing a 5-substituent on the pyridine C-ring (vide supra;
compound (c), Figure 4). The 47° tilt observed in 51 relieves the
torsional strain between the central two rings, and because there
is enough vertical room in the active site, the methylene
hydrogens of the piperazine ring do not clash with the ceiling
residues. Furthermore, it is clear that an additional propargyl
group on the piperazine ring would not be accommodated with
such a twist between the rings and that a significant steric clash
would result (see red arrow in Figure 6b).23 In addition to
differences observed in the conformation of the two ligands,
some small changes were also noted in the GKRP protein. While
most of the residues remained unchanged compared to the
protein bound to compound 2, small movements were observed
in the shelf region residues upon binding to compound 51. For
example, Tyr24 moved back and Pro29 moved forward slightly
to accommodate the 3′-pyridyl substituient of 51 (green vs gray,
Figure 6b).24
Table 3. SAR of 6-Membered Aromatic and Heteroaromatic
Analogues 42−57
Finally, we explored various [6.5]- and [5.6]-aromatic
heterocyclic derivatives (58−67) to probe whether or not the
substituents at the 5 position of the pyridyl ring could be
extended further into the shelf region, and the data is summarized
in Table 4. The first of the [6.5]-bicyclic aromatics that we
examined was the benzo[b]thiophene derivative (58), which
only showed modest biochemical activity when compared with
simple substituted phenyl analogues (42−45, Table 3).
Replacement of a carbon in the thiophene ring of 58 with a
nitrogen atom provided the benzo[d]thiazole analogue (59) in
which the cellular activity was dramatically improved (EC50
=
1.15 μM). Activity in both in vitro assays was improved further
with the corresponding benzo[d]oxazole derivatives 60 and 61
(0.020 and 0.015 μM in enzyme and 0.416 and 0.351 μM in cell,
respectively). Oxindole analogues 62 and 63 were the most
potent [6.5]-bicyclic systems that we surveyed, and they had
relatively low enzyme-to-cell shifts (∼10-fold in both cases);
however, these derivatives showed rapid clearance in rat liver
microsomes. Unfortunately, diminished activities were obtained
with other bicyclic heterocycles that we examined, such as
indazoles 64 and 65, indole 66, and benzthiazole 67. Of the
indazole regioisomers, the more “linear” [6.5]-aromatic hetero-
cycle (5-substituted 1H-indazole 64, IC50 = 0.060 μM) was better
suited for engagement in the shelf region than its corresponding
“angular” isomer (4-substituted 1H-indazole 65, IC50 = 1.95
μM). In addition, of the [5.6]-bicyclic derivatives, the smaller and
more polar 2-benzoindole analogue (66) was slightly more
favorable than the larger and more lipophilic, 2-benzothiophene
derivative (67) (∼2-fold in both the enzymatic and cellular
assays). The addition of the benzene ring of the benzothiophene
was detrimental to activity when compared to the corresponding
methyl thiophene derivative (i.e., 67 (IC50 = 0.048 μM and EC50
= 2.75 μM) vs 41 (Table 2; IC50 = 0.005 μM and EC50 = 1.19
a
AlphaScreen data reported as an average (n ≥ 3). Standard deviations
b
are reported in the Supporting Information. Unless otherwise noted,
n = 1. 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. Average of 2 experimental values. n = 2. n =
c
d
e
f
3.
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Figure 6. (a) X-ray cocrystal structure of compound 51 (green) bound to hGKRP determined at 2.20 Å resolution. The gray surface indicates the van der
Waals surface area. The proteins around the targeted “shelf region” are labeled and highlighted (dark green). (b) Superimposition of compound 51
(green) with compound 2 (gray). The green hGKRP protein corresponds to compound 51, and the gray hGKRP protein corresponds to compound 2.
The red arrow indicates the direction of the propargyl group if it were substituted on compound 51; a steric clash with the protein would result.22
μM). In general, all of the bicyclic moieties that we examined
showed lower metabolic stabilities, particularly in rat liver
microsomes.
7c), and good exposure levels were maintained during this time
(Figure 7d).26
In summary, we have designed, synthesized, and characterized
a new subseries of potent hGK−hGKRP disruptors capable of
accessing a previously unexplored binding pocket in the hGKRP
protein. On the basis of the N-pyridinylpiperazine lead
compound, 2, a systematic SAR exploration at the 5-position
of the pyridine C-ring led to the discovery of 51, which displayed
favorable enzymatic and cellular potencies as well as acceptable
pharmacokinetic properties. While we lost the interaction with
the upper hydophobic pocket (by removing the propargyl group
of compound 2), the additional interactions that we gained by
engaging the shelf region with the 3-pyrindyl group of compound
51 more than compensated for this loss. The effect on the
binding contributions of the two regions can be seen by
examining the activities of compound 2 (which engages the
upper hydrophobic pocket), compound 51 (which engages the
shelf region), and compound 26 (which does not engage either
the hydrophobic pocket or the shelf region). The propargyl
derivative, 2 (IC50 = 0.010 μM), is ∼18-fold more active than the
unsusbtituted derivatve 26 (IC50 = 0.176 μM), while the pyridyl
derivative, 51 (IC50 = 0.005 μM), is ∼35-fold more active than
26. In addition to increasing potency and structural diversity, we
gained an increased understanding of the conformational
requirements needed for this series and have illustrated the
new interactions in the Ala27-Val28-Pro29/Tyr24 shelf region
(as shown in the crystal structure of compound 51 bound to
hGKRP). Furthermore, compound 51 was found to be
efficacious when administered orally to diabetic db/db mice,
significantly lowering blood glucose levels at the 6 h time point.
In addition to the sulfonamide compounds describe herein, a
novel class of GK−GKRP disruptors that access the unique shelf-
region binding pocket has also been developed, and the results
will be reported in due course.
The promising in vitro potencies and stabilities of compounds
34 and 51 prompted us to evaluate their in vivo pharmacokinetic
(PK) profiles (Table 5). Although there was very little difference
between the pyridine 51 and methylpyrazole 34 in terms of CYP
inhibition liabilities, compound 51 was found to be less protein-
bound in rat plasma as compared to 34 (Fu 2.1% vs 1.1%).
Pyridine 51, which is more metabolically stable in rat liver
microsomes, also showed a better overall in vivo PK profile with a
lower clearance, a higher volume of distribution, and a longer
mean residence time compared to methylpyrazole 34. In
addition, compound 51 was superior to 34 in an oral PK study
as well, showing higher bioavailability (F = 43% vs 30%) and a
greater exposure (AUC 4.74 vs 2.80 μM·h).
Given its encouraging in vitro cellular potency and favorable
pharmacokinetic profile, pyridine 51 was evaluated further in
efficacy and pharmacodynamic studies in db/db mice (Figure 7).
In the efficacy study, 51 was administrated orally to db/db mice at
10, 30, or 100 mg/kg, and blood glucose levels were measured at
various time points (3, 6, and 24 h postdose) (Figure 7a). A
statistically significant reduction in glucose levels was achieved
(up to 25%) with the administration of 51 at 6 h postdose when
compared to the vehicle control group.25 In addition, the
exposure of compound 51 correlated well with the amount of
efficacy observed (Figure 7b). Finally, compound 51 was also
examined in a pharmacodymanic assay to evaluate its ability to
promote GK translocation out of the nucleus of hepatocytes at
the efficacious dose in db/db mice. In this pharmacodymanic
assay, mice were orally administered 100 mg/kg of compound
51, and GK translocation (as determined by immunohistochem-
istry (IHC)) was measured in liver slices isolated at 1 and 6 h
postdose. An IHC score of 0 indicated essentially complete
nuclear to cytoplasmic GK translocation. Pyridine 51 produced a
strong pharmacodymanic effect for up to 6 h postdose (Figure
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Table 4. SAR of Bicyclic Heteroaromatic Analogues 58−67
Table 5. Pharmacokinetic Profiles of Selected Compounds
a
in vitro properties
in vivo rat PK
rat
plasma
CYP
protein
b
c
inhibition binding
iv
po
3A4/2D6
IC50
(μM)
AUC
cmpd
no.
CL
Vdss
MRT
(h)
F
(%)
(μM·
Fu (%) (L/kg/h) (L/kg)
h)
d
d
34
51
27/18
27/27
1.1
2.1
2.0
1.5
2.57
3.36
1.34
2.18
30
2.80
43
4.74
a
Pharmacokinetic parameters following administration in male
Sprague−Dawley rat: unless otherwise indicated, 3 animals per
b
c
study. Dosed at 2 mg/kg as a solution in DMSO. Dosed at 10
mg/kg as a suspension in 2% hydroxypropyl methylcellulose (HPMC),
d
0.1% Tween 80 pH 7 1. Two animals were used in this study.
for 1 h. Three equal aliqouts from each concentration of whole blood
and plasma were transferred to fresh tubes (0.8−1 mL). Whole blood
and plasma samples were centrifuged at 2600−3000 rpm (approx-
imately 700−1000 × g) for 10 min at 22 °C and after that sample plasma
and reference plasma were collected. High concentration samples
required dilution in plasma to bring concentrations into the dynamic
range of the standard curve. Eight volumes of MeCN with internal
standard were added to reference and sample plasma to precipitate
protein. The reference and sample plasma samples with internal
standard were centrifuged at 14,000 rpm (approximately 22,000 × g) for
10 min at 4 °C. The supernatants were analyzed by liquid
chromatography−mass spectrometry (LC−MS).
Chemistry. General Procedure. Unless otherwise noted, all
materials were obtained from commercial suppliers and used without
further purification. Anhydrous solvents were obtained from Aldrich,
Acros, or EM Science and used directly. All reactions involving air- or
moisture-sensitive reagents were performed under a nitrogen or argon
atmosphere. Microwave-assisted reactions were conducted either with
an Initiator from Biotage, Uppsala, Sweden, or Explorer from CEM,
Matthews, North Carolina. Silica gel chromatography was performed
using either glass columns packed with silica gel (200−400 mesh,
Aldrich Chemical) or prepacked silica gel cartridges (Biotage or Redi-
Sep). Nuclear magnetic resonance (NMR) spectra were determined
with a Bruker 400 MHz spectrometer. Chemical shifts are reported in
parts per million (ppm, δ units). All final compounds were purified to
>95% purity as determined by LC−MS obtained on an Agilent 1100
spectrometer using the following methods: [A] Agilent SB-C18 column
(50 × 3.0 mm, 2.5 μm) at 40 °C with a 1.5 mL/min flow rate using a
gradient of 5−95% [0.1% TFA in MeCN] in [0.1% TFA in H2O] over
3.5 min; [B] Phenomenex Gemini NX C18 column (50 × 3.0 mm, 3 μm)
at 40 °C with a 1.5 mL/min flow rate using a gradient of 5−95% [0.1%
formic acid in MeCN] in [0.1% formic acid in H2O] over 3.5 min. Low-
resolution mass spectral (MS) data were obtained at the same time of
the purity determination on the LC−MS instrument using ES ionization
mode (positive).
a
AlphaScreen data reported as an average (n ≥ 3). Standard deviations
b
c
are reported in the Supporting Information. n = 1. 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
d
μM. Average of 2 experimental values.
(S)-2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)-2-(prop-1-yn-1-yl)-
piperazin-1-yl)-5-(prop-1-yn-1-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluor-
opropan-2-ol (3). A solution of (S)-tert-butyl (5-((4-(3-chloro-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-2-yl)-3-(prop-1-
yn-1-yl)piperazin-1-yl)sulfonyl)pyridin-2-yl)carbamate (15) (0.100 g,
0.152 mmol), 1-(trimethylsilyl)-1-propyne (0.085 g, 0.760 mmol),
Cs2CO3 (0.014 g, 0.456 mmol), and palladium(II) phenethylamine
chloride (Xphos) (0.006 g, 7.60 μmol) in MeCN (3.0 mL) was heated to
95 °C in a sealed tube overnight. The reaction was diluted with EtOAc
and H2O, and the organic layer was dried over Na2SO4, filtered, and
concentrated. The crude material was stirred in an excess amount of 4 N
HCl in 1,4-dioxane for 2 h before being concentrated onto silica gel and
purified by column chromatography on silica gel eluting with 0−20% (2
M NH3 in MeOH) in DCM. The compound was further purified via
reverse-phase preparative HPLC using a Phenomenex Gemini C18
column (30 × 150 mm, 10 μm) eluting with 0.1% TFA in MeCN/
EXPERIMENTAL SECTION
Biology. hGK−hGKRP AlphaScreen, GK Translocation in Mouse
Hepatocytes, and in Vivo Studies in db/db Mice. These assays were
performed as described previously.8−10
Measurement of B/P Ratio. Mice (CD-1) plasma and whole blood
(K2EDTA) were purchased from Bioreclamation. Partitioning between
blood and plasma was determined in vitro using the plasma depletion
assay27 in which compound 51 levels in plasma prepared by direct
addition to plasma (reference plasma) were compared with levels in
plasma prepared by the addition to whole blood (sample plasma). Stock
solutions of compound 51 in DMSO were first diluted by 20-fold into
plasma and then diluted by 50-fold into whole blood or plasma to
achieve final target concentrations of 0.1, 1.0, and 10 μM in 3 mL total
volume. Samples of whole blood and plasma were gently mixed at 37 °C
■
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Figure 7. (a) Blood glucose measurements (3, 6, and 24 h) postoral administration of 51 (10, 30, and 100 mg/kg) to db/db mice. The statistical
significance of the blood glucose measurements were based on comparison to the individual vehicle control groups (black, n ≥ 8; * = p < 0.05, ** = p <
0.01, *** = p < 0.001). (b) Unbound plasma concentrations (3, 6, and 24 h) postoral administration of 51 (10, 30, and 100 mg/kg) to db/db mice. The
plasma concentrations were determined based on the whole blood concentration measurements via DBS technique. (c) In vivo GK translocation in db/
db mice following oral administration of 51 (100 mg/kg, po), n = 4. (d) Unbound plasma concentration of 51 from the phamacodynamic assay shown in
panel c. Plasma samples were taken at terminal time points through a cardio puncture.
H2O (20−80% over 15 min). The collected fractions were neutralized
with aqueous saturated NaHCO3 and extracted with EtOAc. The
separated organic layer was dried over Na2SO4, filtered, and
concentrated to give the title compound (0.015 g, 18%) as a yellow
solid. MS (ESI pos. ion) m/z calcd for C23H21F6N5O3S, 561; found, 562
(M + H). 1H NMR (400 MHz, CD3OD) δ 8.57−8.53 (m, 1H), 8.24 (d,
J = 2.3 Hz, 1H), 8.12−8.08 (m, 1H), 7.67 (dd, J = 2.4, 8.9 Hz, 1H), 6.56
(d, J = 8.8 Hz, 1H), 4.77−4.71 (m, 1H), 4.37−4.30 (m, 1H), 3.93−3.87
(m, 1H), 3.69−3.63 (m, 1H), 3.39−3.35 (m, 2H), 2.39−2.29 (m, 1H),
2.09 (s, 3 H), 2.03 (s, 3H). Three exchangeable protons were not
observed.
2-(5,6-Dichloropyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(8). To a suspension of 5,6-dichloronicotinic acid (5) (3.00 g, 15.63
mmol) in DCM (20.0 mL) at room temperature was added oxalyl
chloride (15.63 mL, 31.3 mmol) followed by a drop of DMF (0.1 mL).
After gas evolution ceased, the solution was clear (2 h), and LC−MS
showed complete conversion, the reaction was concentrated to give the
crude 5,6-dichloronicotinoyl chloride (3.30 g, 100%) as an off-white
solid, which was used directly without further purification.
To a suspension of the crude acyl chloride isolated above (3.30 g,
15.68 mmol) and tetramethylammonium fluoride (3.21 g, 34.50 mmol)
in DME (100 mL) at −78 °C was added (trifluoromethyl)-
trimethylsilane (5.10 mL, 34.50 mmol). The reaction was allowed to
warm to room temperature for 1 h, then the reaction was diluted with
EtOAc and aq 1 N HCl. The organic layer was separated, dried over
Na2SO4, filtered, and concentrated. The crude material was purified by
column chromatography eluting with 0−50% hexanes in EtOAc to yield
the title compound (3.56 g, 72%) as an orange solid.
2-(5,6-Dibromopyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(9). To a stirred suspension of 5,6-dibromonicotinic acid (6) (2.80 g,
9.97 mmol) in DCM (25.0 mL) was added oxalyl chloride (1.33 mL,
14.95 mmol), and then DMF (0.04 mL, 0.50 mmol) was slowly added at
0 °C. The foaming mixture was stirred at room temperature for 5 h and
concentrated to give the crude 5,6-dibromonicotinoyl chloride (some 5-
bromo-6-chloronicotinoyl chloride was also observed) as a beige solid.
DME (8.00 mL) was added to this crude material and the resulting
mixture was treated with tetramethylammonium fluoride (2.01 g, 21.58
mmol) followed by the slow addition of (trifluoromethyl)-
trimethylsilane (3.19 mL, 21.58 mmol) at 0 °C. The suspension was
allowed to gradually warm up to room temperature and was stirred
overnight. The reaction mixture was diluted with 1 N HCl (25 mL) and
H2O (15 mL) and extracted with EtOAc (3 × 35 mL). The organic
extracts were washed with brine and dried over Na2SO4. The solution
was filtered and concentrated in vacuo to give the crude material as a
brown oil. This material was absorbed onto silica gel and purified by
chromatography through a Redi-Sep prepacked silica gel column (40 g),
eluting with a gradient of 0−30% EtOAc in hexanes, to provide a mixture
of the title compound and the corresponding 2-(5-bromo-6-
chloropyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (3.83 g total) as
a light-brown solid. MS (ESI pos. ion) m/z calcd for C8H3Br2F6NO,
403; found, 404; m/z calcd for C8H3BrClF6NO, 359; found, 360.
2-(6-Chloro-5-methoxypyridin-3-yl)-1,1,1,3,3,3-hexafluoropro-
pan-2-ol (10). To a stirred suspension of 6-chloro-5-methoxynicotinic
acid (7) (0.99 g, 5.30 mmol) in DCM (5.0 mL) was added oxalyl
chloride (0.71 mL, 7.95 mmol). DMF (0.021 mL, 0.27 mmol) was
added slowly, and the foaming mixture was stirred at room temperature
for 3 h and concentrated to give the crude acyl chloride as a beige solid.
DME (8.0 mL) and tetramethylammonium fluoride (1.09 g, 11.66
mmol) were added followed by the slow addition of (trifluoromethyl)-
trimethylsilane (1.73 mL, 11.66 mmol) at 0 °C. The resulting
suspension was allowed to gradually warm up to room temperature
and stirred overnight. The reaction mixture was diluted with aq 1 N HCl
(15 mL) and H2O (10 mL) and extracted with EtOAc (3 × 25 mL). The
organic extracts were washed with 1 N NaOH (1 × 10 mL) and brine
and dried over Na2SO4. The solution was filtered and concentrated in
vacuo to give the crude material as a brown oil. The material was
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Article
absorbed onto silica gel and purified by chromatography through a Redi-
Sep prepacked silica gel column (40 g), eluting with a gradient of 0−30%
EtOAc in hexanes, to provide the title compound (0.97 g, 59% yield) as
an off-white solid.
To a stirred mixture of tert-butyl 4-(3-bromo-5-(1,1,1,3,3,3-
hexafluoro-2-hydroxypropan-2-yl)pyridin-2-yl)piperazine-1-carboxy-
late (1.62 g, 3.19 mmol) in DCM (3.0 mL) was added TFA (3.0 mL),
and the mixture was stirred at room temperature for 1 h and
concentrated. The mixture was concentrated and then diluted with
H2O (5 mL) and then aq 1 N NaOH was added carefully until neutral.
The resulting beige precipitate was collected and dried. The aqueous
solution was extracted with EtOAc (2 × 50 mL) and washed with brine.
The organic phases were dried over Na2SO4 and concentrated. The
solids were combined to give 2-(5-bromo-6-(piperazin-1-yl)pyridin-3-
yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (17). To the crude solid (17) was
added DCM (30.0 mL) followed by EtiPr2N (1.11 mL, 0.286 mmol) and
tert-butyl (5-(chlorosulfonyl)pyridin-2-yl)carbamate (14)16 (1.12 g,
3.820 mmol) subsequently at room temperature. The mixture was
stirred at 1 h and quenched with saturated aqueous NH4Cl (30 mL) and
H2O (50 mL). The separated aqueous layer was extracted with DCM (3
× 50 mL). The organic extracts were washed with brine and dried over
Na2SO4. The solution was filtered and concentrated in vacuo to give the
crude material as a yellow solid. The residue was absorbed onto silica gel
and purified by chromatography through a Redi-Sep prepacked silica gel
column (80 g), eluting with a gradient of 0−30% 2 M NH3·MeOH in
DCM, to provide the title compound (1.97 g, 93% yield) as a pale-yellow
solid. MS (ESI pos. ion) m/z calcd for C22H24BrF6N5O5S, 663; found,
664 (M + H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-chloro-
pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (23). To a solution of
2-(5-chloro-6-(piperazin-1-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropro-
pan-2-ol (16) (3.90 g, 10.72 mmol) and Et3N (5.98 mL, 42.90 mmol) in
DCM (30.0 mL) at room temperature was added tert-butyl (5-
(chlorosulfonyl)pyridin-2-yl)carbamate (14)16 (3.45 g, 11.80 mmol).
After 10 min the reaction was diluted with H2O and DCM, and the
organic layer was separated, dried over Na2SO4, filtered, and
concentrated. The resulting residue was dissolved in DCM (30 mL)
and TFA (0.836 mL, 10.72 mmol) and stirred at room temperature for
20 min. The reaction was not complete, so 5 more equivalents of TFA
was added, and complete conversion was observed after an additional 20
min. The reaction was quenched with solid NaHCO3 and diluted with
DCM and H2O. The organic layer was separated, dried over Na2SO4,
filtered, and concentrated to give a pale-yellow foam. This material was
slurried with DCM and filtered, and 2-(6-(4-((6-aminopyridin-3-
yl)sulfonyl)piperazin-1-yl)-5-chloropyridin-3-yl)-1,1,1,3,3,3-hexafluor-
opropan-2-ol (23) (1.50 g, 27%) was obtained as an off-white solid. MS
(ESI pos. ion) m/z calcd for C17H16ClF6N5O3S, 519; found, 520 (M +
H). 1H NMR (400 MHz, CD3OD) δ 8.47−8.39 (m, 1H), 8.33−8.28 (m,
1H), 7.95−7.89 (m, 1H), 7.83 (dd, J = 2.3, 9.0 Hz, 1H), 6.75 (d, J = 9.0
Hz, 1H), 3.58−3.50 (m, 4H), 3.23−3.16 (m, 4H). Three exchangeable
protons were not observed.
(S)-2-(6-(4-Benzyl-2-(prop-1-yn-1-yl)piperazin-1-yl)-5-chloropyri-
din-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (12). A solution of (S)-1-
benzyl-3-(prop-1-yn-1-yl)piperazine (11)16 (1.00 g, 4.67 mmol), 2-(5,6-
dichloropyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (8) (1.47 g,
4.67 mmol), Ruphos precatalyst (0.19 g, 0.233 mmol), Ruphos (0.11
g, 0.233 mmol), and NaOtBu (1.35 g, 14.0 mmol) in 1,4-dioxane (10.0
mL) was heated in a sealed tube at 100 °C for 30 min. The reaction was
diluted with H2O and extracted with EtOAc. The organic layer was dried
over Na2SO4, filtered, and concentrated. The crude material was purified
by column chromatogrpahy eluting with 0−100% EtOAc in hexanes to
give the title compound (0.98 g, 43%).
(S)-2-(5-Chloro-6-(2-(prop-1-yn-1-yl)piperazin-1-yl)pyridin-3-yl)-
1,1,1,3,3,3-hexafluoropropan-2-ol (13). To a suspension of (S)-2-(6-
(4-benzyl-2-(prop-1-yn-1-yl)piperazin-1-yl)-5-chloropyridin-3-yl)-
1,1,1,3,3,3-hexafluoropropan-2-ol (12) (0.98 g, 1.992 mmol) and
K2CO3 (0.55 g, 3.98 mmol) in DCM (20.0 mL) at room temperature
was added 1-chloroethyl chloroformate (0.65 mL, 5.98 mmol). After 30
min the reaction was filtered and concentrated. MeOH (20.0 mL) was
added to the resulting oil, and the solution was stirred at room
temperature overnight. The reaction was then concentrated to yield the
title compound (0.80 g, 100%) as an oil that was used directly in the next
reaction.
(S)-tert-Butyl (5-((4-(3-chloro-5-(1,1,1,3,3,3-hexafluoro-2-hydrox-
ypropan-2-yl)pyridin-2-yl)-3-(prop-1-yn-1-yl)piperazin-1-yl)-
sulfonyl)pyridin-2-yl)carbamate (15). To a solution of (S)-2-(5-
chloro-6-(2-(prop-1-yn-1-yl)piperazin-1-yl)pyridin-3-yl)-1,1,1,3,3,3-
hexafluoropropan-2-ol (13) (0.80 g, 1.99 mmol) and Et3N (0.83 mL,
5.97 mmol) in DCM (10.0 mL) at room temperature was added tert-
butyl (5-(chlorosulfonyl)pyridin-2-yl)carbamate (14)16 (0.64 g, 2.19
mmol). After 10 min the reaction was quenched with the addition of
H2O and extracted with DCM. The organic layer was dried over
Na2SO4, filtered, and concentrated. The residue was purified by column
chromatography eluting with 0−40% EtOAc in hexanes to yield the title
compound (0.90 g, 69%) as a light-yellow solid.
2-(5-Chloro-6-(piperazin-1-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoro-
propan-2-ol (16). A 400 mL sealable vessel was charged with 2-(5,6-
dichloropyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (8) (43.5 g, 139
mmol), tert-butyl piperazine-1-carboxylate (31.0 g, 166 mmol), DMF
(100.0 mL), and iPr2EtN (36.3 mL, 208 mmol). The vessel was sealed
and heated at 100 °C overnight. After the mixture was allowed to cool to
room temperature, the reaction was poured into H2O (750 mL). The
resulting solid was collected and recrystallized from iPrOH to give tert-
butyl 4-(3-chloro-5-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-
pyridin-2-yl)piperazine-1-carboxylate (37.5 g, 58% yield) as a white
solid.
A solution of tert-butyl 4-(3-chloro-5-(1,1,1,3,3,3-hexafluoro-2-
hydroxypropan-2-yl)pyridin-2-yl)piperazine-1-carboxylate (5.0 g,
10.78 mmol) in DCM (20.0 mL) and TFA (20.0 mL, 269 mmol) was
stirred at room temperature for 20 min. The reaction was quenched with
excess solid NaHCO3 and diluted with EtOAc and H2O. The organic
layer was separated, dried over Na2SO4, filtered, and concentrated. The
title compound (16) (3.9 g, 100%) was used in the next reaction without
further purification.
tert-Butyl (5-((4-(3-bromo-5-(1,1,1,3,3,3-hexafluoro-2-hydroxy-
propan-2-yl)pyridin-2-yl)piperazin-1-yl)sulfonyl)pyridin-2-yl)-
carbamate (20). To a mixture of 2-(5,6-dibromopyridin-3-yl)-
1,1,1,3,3,3-hexafluoropropan-2-ol (9) (3.83 g, mixture with 2-(5-
bromo-6-chloropyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol) and
tert-butyl piperazine-1-carboxylate (1.59 g, 8.54 mmol) in DMF (1.0
mL) was added iPr2EtN (1.86 mL, 10.7 mmol), and the reaction mixture
was stirred and heated in a microwave reactor at 140 °C for 40 min. After
allowing to cool to room temperature, the mixture was added to ice−
water, and the resulting precipitate was filtrated and dried under vacuum
to provide crude tert-butyl 4-(3-bromo-5-(1,1,1,3,3,3-hexafluoro-2-
hydroxypropan-2-yl)pyridin-2-yl)piperazine-1-carboxylate (2.92 g,
58% from 6) as an off-white solid.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-bromo-
pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (24). To a stirred
mixture of tert-butyl (5-((4-(3-bromo-5-(1,1,1,3,3,3-hexafluoro-2-hy-
droxypropan-2-yl)pyridin-2-yl)piperazin-1-yl)sulfonyl)pyridin-2-yl)-
carbamate (20) (0.020 g, 0.03 mmol) in DCM (1.0 mL) was added TFA
(1.0 mL) at room temperature, and the yellow solution was stirred for 30
min and concentrated. The crude material was dissolved in 5% 2 M
NH3·MeOH in DCM, absorbed onto silica gel, and purified by
chromatography through a Redi-Sep prepacked silica gel column (4 g),
eluting with a gradient of 0−30% 2 M NH3·MeOH in DCM, to provide
the title compound (0.017 g, quantitative) as a white solid. MS (ESI pos.
1
ion) m/z calcd for C17H16BrF6N5O3S, 563; found, 564 (M + H). H
NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.47 (s, 1H), 8.25 (d, J =
2.35 Hz, 1H), 8.11−8.00 (m, 1H), 7.66 (dd, J = 2.45, 8.90 Hz, 1H), 7.03
(s, 2H), 6.55 (d, J = 8.80 Hz, 1H), 3.35−3.52 (m, 4H), 3.03 (br s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-methox-
ypyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (25). A glass micro-
wave reaction vessel was charged with 2-(6-chloro-5-methoxypyridin-3-
yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (10) (0.118 g, 0.381 mmol), tert-
i
butyl piperazine-1-carboxylate (0.078 g, 0.419 mmol), and Pr2EtN
(0.100 mL, 0.572 mmol) in DMF (0.2 mL). The reaction mixture was
stirred and heated in a microwave reactor at 140 °C for 100 min and then
160 °C for 30 min. After the mixture was allowed to cool to room
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temperature, H2O was added, and the resulting precipitate was collected,
washed with H2O, and dried under vacuum to give tert-butyl 4-(5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-3-methoxypyridin-2-yl)-
piperazine-1-carboxylate (98 mg, 56%) as an off-white solid. This
material was dissolved in DCM (2.0 mL), and then TFA (2.0 mL) was
added at room temperature. The yellow solution was stirred at room
temperature for 30 min and concentrated to give the crude 4-(5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-3-methoxypyridin-2-yl)-
piperazine (18). MS (ESI pos. ion) m/z calcd for C13H15F6N3O2, 359;
found, 360 (M + H).
yl)sulfonyl)pyridin-2-yl)carbamate (20) (0.040 g, 0.06 mmol) and
DMF (0.5 mL). The solution was purged with argon for 15 min, and
ethynylcyclopropane (1.5 equiv), Pd(PPh3)4 (0.003g, 0.003 mmol),
Et3N (0.041 mL, 0.300 mmol), and CuI (0.001 g, 0.006 mmol) were
added. After the additions were completed, the reaction mixture was
purged again with argon for 15 min, sealed, and stirred at ambient
temperature for 12 h. The reaction was quenched with an excess of ice/
H2O (10 mL), and the product was extracted with EtOAc (2 × 10 mL).
The organic layers were separated, dried over anhydrous Na2SO4,
filtered, and evaporated under reduced pressure. The crude material was
purified by column chromatography (100−200 silica gel) eluting with
20−27% EtOAc in hexanes to give an off-white solid, which was
dissolved in DCM (1.0 mL). The resulting solution was cooled to 0 °C,
and TFA (0.5 mL) was added dropwise. The reaction was stirred at
ambient temperature for 2 h until the deprotection was complete as
indicated by TLC (50% EtOAc/hexanes). The reaction mixture was
diluted with DCM (10 mL), basified with saturated aqueous NaHCO3,
and extracted with EtOAc (10 mL). The organic layer was washed with
H2O (2 × 10 mL) and brine (10 mL) then separated, dried over Na2SO4,
and concentrated to give the title compound (0.030 g, 37%) as an off-
white solid. MS (ESI pos. ion) m/z calcd for C22H21F6N5O3S, 549;
found, 550 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.61(s, 1H),
8.29 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 2 Hz, 1H),
7.64 (dd, J = 2.4, 8.8 Hz, 1H), 7.03 (s, 1H), 6.53 (d, J = 8.8 Hz, 1H),
3.63−3.62 (m, 4H), 2.96−2.95 (m, 4H), 1.57−1.56 (m, 1H), 0.91−0.88
(m, 2H), 0.70−0.67 (m, 2H). One exchangeable proton was not
observed.
The crude material (18) isolated above was dissolved in DCM (1.0
mL), and iPr2EtN (0.041 g, 0.320 mmol) and tert-butyl (5-
(chlorosulfonyl)pyridin-2-yl)carbamate (14)16 (0.125 g, 0.427 mmol)
were added. The mixture was stirred at room temperature for 1 h and
concentrated to give the crude material tert-butyl (5-((4-(5-(1,1,1,3,3,3-
hexafluoro-2-hydroxypropan-2-yl)-3-methoxypyridin-2-yl)piperazin-1-
yl)sulfonyl)pyridin-2-yl)carbamate (21). DCM (2.0 mL) and TFA (2.0
mL) were added, and the resulting yellow solution was stirred at room
temperature for 30 min. The solution was concentrated and the resulting
residue was dissolved in 5% 2 M NH3·MeOH in DCM, absorbed onto
silica gel, and purified by chromatography through a Redi-Sep prepacked
silica gel column (12 g), eluting with a gradient of 0−7% 2 M NH3·
MeOH in DCM, to provide the title compound (0.004 g, 2% overall
yield from 10) as a white solid. MS (ESI pos. ion) m/z calcd for
1
C18H19F6N5O4S, 515; found, 516 (M + H). H NMR (400 MHz,
DMSO-d6) δ 8.80 (s, 1H), 8.22 (d, J = 2.15 Hz, 1H), 7.99 (s, 1H), 7.63
(dd, J = 2.54, 8.80 Hz, 1H), 7.30 (s, 1H), 7.03 (s, 2H), 6.53 (d, J = 8.41
Hz, 1H), 3.79 (s, 3H), 3.50−3.44 (m, 4H), 3.00−2.91 (m, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)pyridin-3-
yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (26). To a mixture of 2-(6-(4-
((6-aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-chloropyridin-3-yl)-
1,1,1,3,3,3-hexafluoropropan-2-ol (23) (0.057 g, 0.11 mmol) and
Pd(amphos)Cl2 (0.004 g, 5.48 μmol) was added Et3N (76 μL, 0.55
mmol), DMF (0.5 mL), and HCO2H (13 μL, 0.33 mmol). The reaction
mixture was stirred and heated in a microwave reactor at 120 °C for 90
min. After the solution was allowed cooled to room temperature, the
mixture was diluted with saturated aqueous NH4Cl (5 mL) and
extracted with EtOAc (3 × 30 mL). The combined organic extracts were
washed with brine (1 × 20 mL) and dried over Na2SO4. The solution
was filtered and concentrated in vacuo to give the crude material as a
light-yellow oil. The crude product was absorbed onto silica gel and
purified by chromatography through a Redi-Sep prepacked silica gel
column (12 g), eluting with a gradient of 0−70% EtOAc in hexanes, to
yield the title compound (0.010 g, 18%) as a white solid. MS (ESI pos.
ion) m/z calcd for C17H17F6N5O3S, 485; found, 486 (M + H). 1H NMR
(400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.31 (d, J = 2.35 Hz, 1H), 8.22 (d,
J = 2.35 Hz, 1H), 7.69 (d, J = 10.76 Hz, 1H), 7.62 (dd, J = 2.54, 9.00 Hz,
1H), 6.98 (s, 2H), 6.91 (d, J = 9.19 Hz, 1H), 6.51 (d, J = 8.41 Hz, 1H),
3.73−3.59 (m, 4H), 2.95 (t, J = 4.89 Hz, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-cyclopro-
pylpyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (27). This material
was prepared according to the general procedure described for the
synthesis of 26 from 23 using 2-cyclopropyl-6-methyl-1,3,6,2-
dioxazaborocane-4,8-dione (0.028 g, 0.144 mmol). The reaction was
heated to 120 °C for 60 h, and the crude product was purified by column
chromatography eluting with 0−7% MeOH in DCM. The material was
further purified via reverse-phase preparative HPLC using a
Phenomenex Gemini C18 column (30 × 150 mm, 10 μm) eluting
with 0.1% TFA in MeCN/H2O (10−80% over 15 min) followed by
column chromatography on silica gel eluting with 0−100% EtOAc in
hexanes to give the title compound (0.005 g, 7%) as a colorless film. MS
(ESI pos. ion) m/z calcd for C20H21F6N5O3S, 525; found, 526 (M + H).
1H NMR (400 MHz, CD3OD) δ 8.33−8.29 (m, 2H), 7.75 (dd, J = 2.4,
3-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)prop-2-
yn-1-ol (29). This material was prepared according to the general
procedure described for the synthesis of 28 from 20 (0.100 g, 0.150
mmol) using prop-2-yn-1-ol (0.013 g, 0.226 mmol) and isolated (0.030
g, 37%) as an off-white solid. MS (ESI pos. ion) m/z calcd for
1
C20H19F6N5O4S, 539; found, 540 (M + H). H NMR (400 MHz,
DMSO-d6) δ 8.39 (d, J = 2.4 Hz, 1H), 8.30 (d, J = 2 Hz, 1H), 7.88 (d, J =
2 Hz, 1H), 7.75 (dd, J = 2.8, 9.2 Hz, 1H), 6.65 (d, J = 8.8 Hz, 1H), 4.43
(s, 2H), 3.78−3.76 (m, 4H), 3.16−3.14 (m, 4H). Four exchangeable
protons were not observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(3-me-
thoxyprop-1-yn-1-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(30). To a mixture of tert-butyl (5-((4-(3-bromo-5-(1,1,1,3,3,3-
hexafluoro-2-hydroxypropan-2-yl)pyridin-2-yl)piperazin-1-yl)-
sulfonyl)pyridin-2-yl)carbamate (20) (0.059 g, 0.089 mmol),
palladium(II) phenethylamine chloride (Xphos) (0.066 g, 0.089
mmol), and Cs2CO3 (0.203 g, 0.622 mmol) was added methyl propargyl
ether (0.037 mL, 0.444 mmol) and MeCN (1.0 mL). The mixture was
stirred at 80 °C overnight, the reaction mixture was allowed to cool to
room temperature, and the mixture was passed through a plug of Celite.
The filter cake was washed with EtOAc and DCM (3 × 10 mL each).
The combined organic phases were concentrated to give a crude residue,
which was dissolved in DCM (1 mL). TFA (1 mL) was added, and the
yellow solution was stirred at room temperature for 30 min then
concentrated. The crude material was dissolved in 5% 2 M NH3·MeOH
in DCM and absorbed onto silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (12 g), eluting with a
gradient of 0−30% 2 M NH3·MeOH in DCM. The material was purified
further by a preparative TLC purification (EtOAc/DCM, 2:3) to
provide the title compound (0.002 g, 5%) as a white solid. MS (ESI pos.
ion) m/z calcd for C21H21F6N5O4S, 553; found, 554 (M + H). 1H NMR
(400 MHz, CD3OD) δ 8.42 (d, J = 1.96 Hz, 1H), 8.32 (d, J = 2.15 Hz,
1H), 7.91 (d, J = 2.15 Hz, 1H), 7.75 (dd, J = 2.45, 8.90 Hz, 1H), 6.66 (d, J
= 8.80 Hz, 1H), 4.37 (s, 2H), 3.81−3.73 (m, 4H), 3.41 (s, 3H), 3.20−
3.12 (m, 4H). Three exchangeable protons were not observed.
4-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)-2-meth-
ylbut-3-yn-2-ol (31). This material was prepared according to the
general procedure described for the synthesis of 28 from 20 (0.100 g,
0.150 mmol) using 2-methylbut-3-yn-2-ol (0.019 g, 0.223 mmol) and
isolated (0.009 g, 11%) as an off-white solid. MS (ESI pos. ion) m/z
8.9 Hz, 1H), 7.50−7.45 (m, 1H), 6.64 (d, J = 9.0 Hz, 1H), 3.49−3.42 (m,
4H), 3.24−3.16 (m, 4H), 2.01−1.91 (m, 1H), 1.10−1.03 (m, 2H),
0.72−0.64 (m, 2H). Three exchangeable protons were not observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(cyclopropylethynyl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(28). In a 10 mL sealable tube was added tert-butyl (5-((4-(3-bromo-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-2-yl)piperazin-1-
1
calcd for C22H23F6N5O4S, 567; found, 568 (M + H). H NMR (400
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MHz, DMSO-d6) δ 8.39 (d, J = 2.4 Hz, 1H), 8.30 (d, J = 2.4 Hz, 1H),
7.85 (d, J = 2.4 Hz, 1H), 7.75 (dd, J = 2.4, 8.8 Hz, 1H), 6.65 (d, J = 8.8
Hz, 1H), 3.76−3.74 (m, 4H), 3.16−3.14 (m, 4H), 1.54 (s, 6H). Four
exchangeable protons were not observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1-iso-
propyl-1H-pyrazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-
2-ol (36). This material was prepared according to the procedure
described for the synthesis of 33 using 1-isopropyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.071 g, 0.301
mmol). The TFA salt of the title compound was isolated (0.048 g,
45%) as a tan solid. MS (ESI pos. ion) m/z calcd for C23H25F6N7O3S,
593; found, 594 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.89−8.79
(m, 1H), 8.36−8.29 (m, 1H), 8.30−8.23 (d, J = 2.2 Hz, 1H), 8.17−8.10
(m, 1H), 7.83−7.75 (d, J = 2.0 Hz, 2H), 7.74−7.67 (m, 2H), 7.61−7.53
(dd, J = 2.5, 9.0 Hz, 1H), 6.70−6.62 (d, J = 9.0 Hz, 1H), 3.15 (d, J = 4.50
Hz, 4H), 3.01−2.96 (m, 4H), 2.85(m, 1H), 1.41−1.39 (m, 6H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1-(tert-
butyl)-1H-pyrazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-
2-ol (37). This material was prepared according to the procedure
described for the synthesis of 33 using 1-(tert-butyl)-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.075 g, 0.301
mmol). The TFA salt of the title compound was isolated (0.043 g,
39%) as a tan solid. MS (ESI pos. ion) m/z calcd for C24H27F6N7O3S,
607; found, 608 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.98−8.77
(m, 1H), 8.36−8.32 (m, 1H), 8.26−8.23 (d, J = 1.83 Hz, 1H), 8.11 (s,
1H), 7.84−7.81 (d, J = 2.0 Hz, 2H), 7.71−7.65 (dd, J = 2.5, 9.0 Hz, 2H),
7.60−7.57 (m, 1H), 6.73−6.54 (d, J = 9.0 Hz, 1H), 3.17 (br s, 4H), 2.99
(br s, 4H), 0.84 (dd, J = 2.74, 6.65 Hz, 9H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1,5-di-
methyl-1H-pyrazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-
2-ol (38). This material was prepared according to the procedure
described for the synthesis of 33 using 1,5-dimethyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.067 g, 0.301
mmol). The TFA salt of the title compound was isolated (0.010 g,
10%) as a tan solid. MS (ESI pos. ion) m/z calcd for C22H23F6N7O3S,
579; found, 580 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.73−8.93
(m, 1H), 8.36 (s, 1H), 8.23 (d, J = 2.15 Hz, 1H), 8.15 (s, 1H), 7.97 (s,
1H), 7.76−7.61 (m, 1H), 7.52 (br s, 2H), 6.69−6.52 (m, 1H), 3.76 (s,
3H), 3.17 (d, J = 4.69 Hz, 4H), 2.87 (br s, 4H), 2.12 (s, 3H).
4-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)but-3-yn-
1-ol (32). This material was prepared according to the general procedure
described for the synthesis of 28 from 20 (0.100 g, 0.150 mmol) using
but-3-yn-1-ol (0.016 mg, 0.226 mmol) and isolated (0.007 g, 8%) as an
off-white solid. MS (ESI pos. ion) m/z calcd for C21H21F6N5O4S, 553;
found, 554 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.36 (d, J = 2.8
Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.86 (d, J = 2 Hz, 1H), 7.77 (dd, J =
2.4, 6.4 Hz, 1H), 6.67 (d, J = 9.2 Hz, 1H), 3.79−3.77 (m, 6H), 3.16 (t, J =
5.2 Hz, 4H), 2.38 (t, J = 6.4 Hz, 2H). Four exchangeable protons were
not observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1H-pyr-
azol-3-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (33). A sol-
ution of tert-butyl (5-((4-(3-bromo-5-(1,1,1,3,3,3-hexafluoro-2-hydrox-
ypropan-2-yl)pyridin-2-yl)piperazin-1-yl)sulfonyl)pyridin-2-yl)-
carbamate (20) (0.100 g, 0.151 mmol), (1H-pyrazol-3-yl)boronic acid
(0.034 g, 0.301 mmol), Pd(amphos)Cl2 (0.005 g, 0.008 mmol), and
K2CO3 (0.062 g, 0.452 mmol) in 1,4-dioxane (5.0 mL) and H2O (0.2
mL) was heated in a sealed tube at 120 °C overnight. The reaction
mixture was allowed to cool to room temperature and then passed
through a short path of Celite. The filter cake was washed with EtOAc
and DCM (3 × 25 mL each). The filtrate was concentrated in vacuo, the
residue was treated with DCM (10 mL) and TFA (10 mL), and the
mixture was stirred for 1 h. The solution was concentrated, and the crude
residue was purified using Xbridge 19 × 100 mm, 10 μm Column, 40
mL/min flow rate, 0.1% TFA in H2O/MeCN. The TFA salt of the title
compound was isolated (0.056 g, 56%) as a tan solid. MS (ESI pos. ion)
m/z calcd for C20H19F6N7O3S, 551; found, 552 (M + H). 1H NMR (400
MHz, DMSO-d6) δ 9.03−8.69 (m, 1H), 8.41 (d, J = 2.35 Hz, 1H), 8.28
(br s, 1H), 8.06 (s, 1H), 7.77 (d, J = 2.15 Hz, 2H), 7.57 (s, 2H), 6.78 (d, J
= 7.43 Hz, 1H), 6.75−6.68 (m, 1H), 6.61 (s, 1H), 3.19 (d, J = 4.69 Hz,
4H), 3.01 (br s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1-meth-
yl-1H-pyrazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(34). A solution of tert-butyl (5-((4-(3-chloro-5-(1,1,1,3,3,3-hexafluoro-
2-hydroxypropan-2-yl)pyridin-2-yl)piperazin-1-yl)sulfonyl)pyridin-2-
yl)carbamate (19) (0.500 g, 0.806 mmol), Pd(amphos)Cl2 (0.057 g,
0.081 mmol), K2CO3 (0.446 g, 3.23 mmol), and 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.336 g, 1.613
mmol) in 1,4-dioxane (8.0 mL)/H2O (0.8 mL) was heated in a sealed
tube at 140 °C for 1 h in the microwave. The solution was then filtered
through a pad of Celite with MeOH, and the filtrate was concentrated.
The residue was redissolved in DCM (20 mL) and TFA (10 mL) and
stirred at room temperature for 30 min. The reaction was quenched with
solid NaHCO3 and diluted with H2O and EtOAc. The organic layer was
separated, dried over Na2SO4, filtered, and concentrated. The crude
material was purified by column chromatography eluting with 0−7% (2
M NH3 in MeOH) in DCM to give the title compound (0.170 g, 37%) as
an off-white solid. MS (ESI pos. ion) m/z calcd for C21H21F6N7O3S,
562; found, 563 (M + H). 1H NMR (400 MHz, CD3OD) δ 8.40−8.36
(m, 1H), 8.28 (d, J = 2.2 Hz, 1H), 7.97−7.92 (m, 1H), 7.83 (d, J = 2.0
Hz, 1H), 7.79−7.76 (m, 1H), 7.72 (dd, J = 2.5, 9.0 Hz, 1H), 6.64 (d, J =
9.0 Hz, 1H), 3.90 (s, 3H), 3.28−3.23 (m, 4H), 3.10−3.04 (m, 4H).
Three exchangeable protons were not observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1-ethyl-
1H-pyrazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
2,2,2-trifluoroacetate (35). This material was prepared according to the
procedure described for the synthesis of 33 using 1-ethyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.067 g, 0.301
mmol). The TFA salt of the title compound was isolated (0.048 g,
46%) as a tan solid. MS (ESI pos. ion) m/z calcd for C22H23F6N7O3S,
579; found, 580 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.90−8.79
(m, 1H), 8.33 (s, 1H), 8.27 (d, J = 2.2 Hz, 1H), 8.14 (s, 1H), 7.78 (d, J =
2.0 Hz, 2H), 7.76−7.70 (m, 2H), 7.61−7.53 (dd, J = 2.5, 9.0 Hz, 1H),
6.74−6.63 (d, J = 9.0 Hz, 1H), 3.16 (d, J = 4.69 Hz, 4H), 3.01 (br s, 4H),
3.06−2.99 (m, 2H), 1.38−1.33 (m, 3H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1-meth-
yl-3-(trifluoromethyl)-1H-pyrazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexa-
fluoropropan-2-ol (39). This material was prepared according to the
procedure described for the synthesis of 33 using 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)-1H-pyrazol
(0.083 g, 0.301 mmol). The TFA salt of the title compound was isolated
(0.059 g, 52%) as a tan solid. MS (ESI pos. ion) m/z calcd for
1
C22H20F9N7O3S, 633; found, 634 (M + H). H NMR (400 MHz,
DMSO-d6) δ 9.00−8.78 (m, 1H), 8.44 (d, J = 2.35 Hz, 1H), 8.24 (d, J =
2.15 Hz, 1H), 8.15 (s, 1H), 7.97 (s, 1H), 7.70 (dd, J = 2.35, 9.00 Hz, 1H),
7.60 (d, J = 1.76 Hz, 1H), 6.65 (d, J = 9.00 Hz, 1H), 3.96 (s, 1H), 3.93 (s,
3H), 3.24−3.10 (m, 4H), 2.88 (br s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(furan-3-
yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (40). This material
was prepared according to the procedure described for the synthesis of
33 using furan-3-ylboronic acid (0.051 g, 0.452 mmol). The TFA salt of
the title compound was isolated (0.024 g, 19%) as a tan solid. MS (ESI
pos. ion) m/z calcd for C21H19F6N5O4S, 551; found, 552 (M + H). 1H
NMR (400 MHz, DMSO-d6) δ 8.98−8.81 (m, 1H), 8.39 (d, J = 1.96 Hz,
1H), 8.23 (d, J = 2.35 Hz, 1H), 8.10 (s, 1H), 7.77 (s, 2H), 7.705−7.55
(m, 1H), 7.01 (s, 2H), 6.82 (d, J = 0.78 Hz, 1H), 6.55 (d, J = 9.00 Hz,
1H), 3.21 (d, J = 4.69 Hz, 4H), 3.05−2.91 (m, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(4-meth-
ylthiophen-2-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(41). This material was prepared according to the procedure described
for the synthesis of 34 from 19 (0.200 g, 0.323 mmol) using (4-
methylthiophen-2-yl)boronic acid (0.092 g, 0.645 mmol). The title
compound 41 was isolated (0.025 g, 13%) as an off-white solid. MS (ESI
pos. ion) m/z calcd for C22H21F6N5O3S2, 581; found, 582 (M + H). 1H
NMR (400 MHz, CD3OD) δ 8.43−8.39 (m, 1H), 8.28 (d, J = 2.2 Hz,
1H), 7.90−7.85 (m, 1H), 7.72 (dd, J = 2.4, 8.9 Hz, 1H), 7.18−7.12 (m,
1H), 7.07−7.01 (m, 1H), 6.65 (d, J = 8.8 Hz, 1H), 3.29−3.23 (m, 4H),
3.10−3.03 (m, 4H), 2.24 (s, 3H). Three exchangeable protons were not
observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-phenyl-
pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (42). This material
5960
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was prepared according to the procedure described for the synthesis of
34 from 19 (0.060 g, 0.097 mmol) using phenyl boronic acid (0.018 g,
0.145 mmol). The title compound 42 was isolated (0.047 g, 86% yield)
as a white solid. MS (ESI pos. ion) m/z calcd for C23H21F6N5O3S, 561;
found, 562 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H),
8.42 (s, 1H), 8.21 (d, J = 2.35 Hz, 1H), 7.71−7.59 (m, 2H), 7.55−7.47
(m, 2H), 7.47−7.36 (m, 3H), 7.16 (br s, 2H), 6.60 (d, J = 8.80 Hz, 1H),
3.17 (d, J = 4.69 Hz, 4H), 2.85 (br s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(2-
fluorophenyl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (43).
Following a similar procedure as described previously in the synthesis
of 33, tert-butyl (5-((4-(3-bromo-5-(1,1,1,3,3,3-hexafluoro-2-hydrox-
ypropan-2-yl)pyridin-2-yl)piperazin-1-yl)sulfonyl)pyridin-2-yl)-
carbamate 20 (0.100 g, 0.151 mmol), and (2-fluorophenyl)boronic acid
(0.063 g, 0.452 mmol) were used. The crude material was purified
(Xbridge 19 × 100 mm, 10 μm Column, 40 mL/min flow rate, 0.1%
NH4OH in H2O/MeCN) to give the title compound (0.038 g, 29%) as a
light-yellow solid. MS (ESI pos. ion) m/z calcd for C23H20F7N5O3S,
579; found, 580 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.87 (s,
1H), 8.46 (d, J = 2.15 Hz, 1H), 8.18 (d, J = 2.35 Hz, 1H), 7.67 (s, 1H),
7.59 (dd, J = 2.54, 8.80 Hz, 1H), 7.49 (d, J = 7.82 Hz, 2H), 7.28 (d, J =
8.02 Hz, 2H), 7.03 (s, 2H), 6.55 (d, J = 8.80 Hz, 1H), 3.19 (d, J = 5.09
Hz, 4H), 2.85−2.72 (m, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(3-
fluorophenyl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (44).
This material was prepared according to the procedure described for
the synthesis of 43 from 20 using (3-fluorophenyl)boronic acid (0.063 g,
0.452 mmol). The title compound 44 was isolated (0.027 g, 26%) as a
light-yellow solid. MS (ESI pos. ion) m/z calcd for C23H20F7N5O3S,
579; found, 580 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.99−8.72
(m, 1H), 8.44 (d, J = 2.15 Hz, 1H), 8.20 (d, J = 2.35 Hz, 1H), 7.75−7.65
(m, 1H), 7.59 (d, J = 2.54 Hz, 2H), 7.55−7.41 (d, J = 7.82 Hz, 1H), 7.36
(s, 2H), 7.28−7.18 (m, 1H), 7.02 (s, 1H), 6.55 (d, J = 9.00 Hz, 1H), 3.19
(br s, 4H), 2.85 (br s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(4-
fluorophenyl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (45).
This material was prepared according to the procedure described for
the synthesis of 43 from 20 using (4-fluorophenyl)boronic acid (0.063 g,
0.452 mmol). The title compound 45 was isolated (0.035 g, 26%) as a
light-yellow solid. MS (ESI pos. ion) m/z calcd for C23H20F7N5O3S,
579; found, 580 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.94−8.79
(m, 1H), 8.42 (d, J = 1.96 Hz, 1H), 8.20 (d, J = 2.54 Hz, 1H), 7.56 (d, J =
3.13 Hz, 4H), 7.25 (s, 2H), 7.02 (s, 2H), 6.54 (d, J = 8.80 Hz, 1H), 3.16
(br s, 4H), 2.85 (d, J = 4.11 Hz, 4H).
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)phenol
(46). This material was prepared according to the procedure described
for the synthesis of 43 from 20 using 2-hydroxyphenylboronic acid
pinacol ester (0.066 g, 0.301 mmol). The crude product was adsorbed
onto silica gel and chromatographed through a Redi-Sep prepacked
silica gel column (40 g), eluting with a gradient of 0−20% 2 M NH3·
MeOH in DCM, to provide the title compound (0.056 g, 64%) as a tan
solid. MS (ESI pos. ion) m/z calcd for C23H21F6N5O4S, 577; found, 578
(M + H). 1H NMR (400 MHz, DMSO-d6) δ 9.61−9.54 (m, 1H), 9.22−
9.13 (m, 1H), 8.79−8.72 (m, 1H), 8.41−8.33 (d, J = 4.50 Hz, 1H),
8.22−8.12 (ddd, J = 1.66, 7.58, 9.73 Hz, 1H), 7.71−7.62 (m, 1H), 7.62−
7.53 (dd, J = 2.45, 8.90 Hz, 1H), 7.23−7.08 (m, 1H), 7.08−7.00 (m,
1H), 6.95−6.86 (m, 1H), 6.86−6.74 (m, 1H), 6.56−6.48 (d, J = 8.80 Hz,
1H), 5.80−5.69 (m, 1H), 3.20 (br s, 4H), 2.77 (br s, 4H).
N-(3-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)phenyl)-
methanesulfonamide (47). To a mixture of 23 (0.050 g, 0.096 mmol),
3-(methylsulfonylamino)phenylboronic acid (0.031 g, 0.144 mmol),
1,1-bis[(di-tert-butyl-p-methylaminophenyl]palladium(II) chloride
(6.81 mg, 0.0096 mmol), and potassium carbonate (53.2 mg, 0.385
mmol) was added 1,4-dioxane (3 mL)/water (0.30 mL). The vial was
sealed and heated at 140 °C for 45 min. The mixture was allowed to cool
to room temperature, concentrated on silica gel, and purifed by
chromatography through a Redi-Sep prepacked silica gel column (40 g),
eluting with a gradient of 0−7% 2 M NH3·MeOH in DCM, to provide
the title compound isolated (0.005 g, 8%) as a white solid. MS (ESI pos.
ion) m/z calcd for C24H24F6N6O5S2, 654; found, 655 (M + H). 1H NMR
(400 MHz, CD3OD) δ 8.49−8.43 (m, 1H), 8.23 (d, J = 2.2 Hz, 1H),
7.76−7.72 (m, 1H), 7.66 (dd, J = 2.5, 9.0 Hz, 1H), 7.45−7.39 (m, 2H),
7.25−7.20 (m, 2H), 6.64 (d, J = 9.0 Hz, 1H), 3.26−3.19 (m, 4H), 3.01−
2.95 (m, 4H), 2.93 (s, 3H). Four exchangeable protons were not
observed.
3-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)-
benzamide (48). This material was prepared according to the procedure
described for the synthesis of 47 from 23 (0.050 g, 0.096 mmol) using
(3-carbamoylphenyl)boronic acid (0.024 g, 0.144 mmol), and the
mixture was heated in a sealed tube at 120 °C overnight. The product
was purified by column chromatography twice eluting with 0−10%
MeOH in DCM. This was followed by recryallization from EtOAc and
hexanes. A final purification by column chromatography eluting with 0−
100% EtOAc in hexanes followed by 0−7% MeOH in CH2Cl2 over 30
min gave the title compound (0.002 g, 4%) as an amorphous white solid.
MS (ESI pos. ion) m/z calcd for C24H22F6N6O4S, 604; found, 605 (M +
H). 1H NMR (400 MHz, CD3OD) δ 8.49−8.45 (m, 1H), 8.22 (d, J = 2.2
Hz, 1H), 8.04−8.00 (m, 1H), 7.89−7.86 (m, 1H), 7.80−7.78 (m, 1H),
7.69−7.62 (m, 2H), 7.55−7.49 (m, 1H), 6.63 (d, J = 9.0 Hz, 1H), 3.24−
3.19 (m, 4H), 2.94−2.88 (m, 4H). Five exchangeable protons were not
observed.
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(4-
(methylsulfonyl)phenyl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-
2-ol (49). This material was prepared according to the procedure
described for the synthesis of 48 from 23 (0.050 g, 0.096 mmol) using 4-
(methanesulfonyl)benzeneboronic acid (0.029 g, 0.144 mmol). The
crude material was purified by column chromatography, eluting with 0−
7% MeOH in DCM, followed by recrystallization from Et2O and
hexanes. The title compound was isolated (10 mg, 16%) as a white solid.
MS (ESI pos. ion) m/z calcd for C24H23F6N5O5S2, 639; found, 640 (M +
H). 1H NMR (400 MHz, CD3OD) δ 8.51−8.48 (m, 1H), 8.25−8.22 (m,
1H), 8.01−7.97 (m, 2H), 7.80−7.75 (m, 3H), 7.66 (dd, J = 2.4, 8.9 Hz,
1H), 6.64 (d, J = 8.8 Hz, 1H), 3.26−3.22 (m, 4H), 3.17 (s, 3H), 2.97−
2.91 (m, 4H). Three exchangeable protons were not observed.
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-[3,4′-bipyr-
idin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (50). This material was
prepared according to the procedure described for the synthesis of 34
from 19 (0.158 g, 0.255 mmol) using pyridin-4-ylboronic acid (0.063 g,
0.510 mmol). The crude material was purified by preparative TLC
eluting with 10% 2 M MH3/MeOH in DCM, to give the desired product
(0.026 g, 20%) as a tan solid. MS (ESI pos. ion) m/z calcd for
1
C23H20F6N6O3S, 562; found, 563 (M + H). H NMR (400 MHz,
DMSO-d6) δ 8.94 (br s, 1H), 8.62 (d, J = 2.15 Hz, 2H), 8.46 (d, J = 1.96
Hz, 1H), 8.19 (d, J = 2.15 Hz, 1H), 7.71 (br s, 1H), 7.66−7.42 (m, 3H),
7.05 (br s, 2H), 6.54 (d, J = 8.41 Hz, 1H), 3.19 (br s, 4H), 2.85 (br s,
4H).
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-[3,3′-bipyr-
idin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (51). This material was
prepared according to the procedure described for the synthesis of 34
from 19 (0.892 g, 1.439 mmol) using 3-pyridylboronic acid (0.708 g,
5.760 mmol). The title compound was isolated (0.577 g, 71%) as a pale-
yellow solid. MS (ESI pos. ion) m/z calcd for C22H20F6N6O3S, 562;
found, 563 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H),
8.72 (d, J = 1.56 Hz, 1H), 8.58 (d, J = 3.33 Hz, 1H), 8.45 (s, 1H), 8.17 (d,
J = 2.35 Hz, 1H), 7.94 (d, J = 7.63 Hz, 1H), 7.68 (s, 1H), 7.58 (dd, J =
2.35, 8.80 Hz, 1H), 7.44 (dd, J = 4.69, 7.82 Hz, 1H), 7.04 (s, 2H), 6.52
(d, J = 8.80 Hz, 1H), 3.15 (br s, 4H), 2.81 (br s, 4H).
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-4′-fluoro-
[3,3′-bipyridin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (52). This
material was prepared according to the procedure described for the
synthesis of 43 from 20 (0.100 g, 0.151 mmol) using (4-fluoropyridin-3-
yl)boronic acid (0.064 g, 0.452 mmol). The title compound was isolated
(0.07 g, 6%) as a tan solid. MS (ESI pos. ion) m/z calcd for
1
C22H19F7N6O3S, 580; found, 581 (M + H). H NMR (400 MHz,
DMSO-d6) δ 9.00−8.85 (m, 1H), 8.75−8.69 (m, 1H), 8.68−8.61 (d, J =
2.15 Hz,1H), 8.54−8.48 (d, J = 2.15 Hz, 1H), 8.20−8.14 (d, J = 2.35 Hz,
1H), 7.79−7.72 (m, 1H), 7.61−7.54 (dd, J = 2.45, 8.90 Hz, 1H), 7.48−
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7.37 (m, 1H), 7.06−6.99 (m, 2H), 6.60−6.40 (d, J = 8.80 Hz, 1H),
3.28−3.10 (m, 4H), 2.86−2.73 (m, 4H).
7.73 (br s, 1H), 7.66−7.46 (m, 2H), 7.39 (br s, 1H), 7.02 (s, 2H), 6.53
(d, J = 8.82 Hz, 1H), 5.74 (s, 1H), 3.17 (s, 4H), 2.80 (br s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(benzo-
[d]thiazol-5-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (59).
This material was prepared according to the procedure described for the
synthesis of 34 from 19 (0.100 g, 0.192 mmol) using 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazole (0.100 g, 0.385
mmol). The title compound was isolated (0.009 g, 8%) as a light-yellow
solid. MS (ESI pos. ion) m/z calcd for C24H20F6N6O3S2, 618; found, 619
(M + H). 1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.44 (s, 1H),
8.31−8.18 (m, 3H), 7.76 (br s, 1H), 7.64 (d, J = 8.08 Hz, 1H), 7.55 (dd, J
= 8.85 Hz, 1H), 7.00 (s, 2H), 6.52 (d, J = 8.88 Hz, 1H), 5.75 (s, 1H), 3.18
(br s, 4H), 2.82 (br s, 4H).
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5′-fluoro-
[3,3′-bipyridin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (53). This
material was prepared according to the procedure described for the
synthesis of 43 from 20 (0.100 g, 0.151 mmol) using (5-fluoropyridin-3-
yl)boronic acid (0.064 g, 0.452 mmol). The title compound was isolated
(0.034 g, 26%) as a tan solid. MS (ESI pos. ion) m/z calcd for
1
C22H19F7N6O3S, 580; found, 581 (M + H). H NMR (400 MHz,
DMSO-d6) δ 9.00−8.82 (m, 1H), 8.69−8.54 (m, 2H), 8.49 (d, J = 2.15
Hz, 1H), 8.20 (d, J = 2.35 Hz, 1H), 8.00−7.90 (m, 1H), 7.83−7.68 (m,
1H), 7.60 (dd, J = 2.54, 8.80 Hz, 1H), 7.02 (s, 2H), 6.55 (d, J = 8.80 Hz,
1H), 3.18 (br s, 4H), 2.85 (d, J = 4.30 Hz, 4H).
2-(6-(4-((6-aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(benzo-
[d]oxazol-5-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (60).
This material was prepared according to the procedure described for
the synthesis of 34 from 19 (0.100 g, 0.192 mmol) using 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole (0.094 g, 0.385
mmol). The title compound was isolated (0.008 g, 6%) as a tan solid. MS
(ESI pos. ion) m/z calcd for C24H20F6N6O4S, 602; found, 603 (M + H).
1H NMR (400 MHz, DMSO-d6) δ 8.92−8.70 (m, 1H), 8.27−8.10 (d, J
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5′-methyl-
[3,3′-bipyridin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (54). This
material was prepared according to the procedure described for the
synthesis of 33 from 20 (0.100 g, 0.151 mmol) using (5-methylpyridin-
3-yl)boronic acid (0.041 g, 0.301 mmol). The TFA salt of the title
compound was isolated (0.019 g, 19%) as a tan solid. MS (ESI pos. ion)
m/z calcd for C23H22F6N6O3S, 576; found, 577 (M + H). 1H NMR (400
MHz, DMSO-d6) δ 8.98−8.78 (m, 1H), 8.58 (d, J = 2.15 Hz, 1H), 8.45
(d, J = 1.96 Hz, 1H), 8.20 (d, J = 2.35 Hz, 1H), 7.89−7.82 (m, 1H),
7.64−7.70 (m, 1H), 7.63−7.56 (m, 1H), 7.37−7.22 (m, 1H), 7.02 (s,
2H), 6.55 (d, J = 8.61 Hz, 1H), 3.19 (d, J = 4.69 Hz, 4H), 2.86 (br s, 4H),
2.31 (br s, 3H).
= 2.35 Hz, 1H), 7.83−7.63 (dd, J = 2.35, 8.80 Hz, 2H), 7.65−7.51 (m,
2H), 7.09−6.92 (d, J = 5.10 Hz, 1H), 6.84−6.71 (m, 1H), 6.59−6.45 (d,
J = 8.61 Hz, 2H), 3.74−3.55 (m, 2H), 3.26−3.06 (m, 4H), 2.96 (s, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(2-
methylbenzo[d]oxazol-5-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropro-
pan-2-ol (61). This material was prepared according to the procedure
described for the synthesis of 34 from 19 (0.100 g, 0.192 mmol) using 2-
methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]-
oxazole (0.069 g, 0.385 mmol). The title compound was isolated (0.012
g, 12%) as a light-yellow solid. MS (ESI pos. ion) m/z calcd for
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-6′-methyl-
[3,3′-bipyridin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (55). This
material was prepared according to the procedure described for the
synthesis of 43 from 20 (0.100 g, 0.151 mmol) using (6-methylpyridin-
3-yl)boronic acid (0.062 g, 0.452 mmol). The title compound was
isolated (0.017 g, 13%) as a tan solid. MS (ESI pos. ion) m/z calcd for
1
C25H22F6N6O4S, 616; found, 617 (M + H). H NMR (400 MHz,
1
C23H22F6N6O3S, 576; found, 577 (M + H). H NMR (400 MHz,
DMSO-d6) δ 8.96−8.70 (m, 1H), 8.47−8.34 (m, 1H), 8.22−8.09 (m,
1H), 7.78−7.71 (m, 1H), 7.71−7.63 (m, 2H), 7.59−7.52 (m, 2H),
7.52−7.37 (m, 1H), 7.00 (s, 1H), 6.85−6.68 (m, 1H), 6.59−6.46 (m,
1H), 3.22−3.05 (m, 4H), 2.96 (s, 3H), 2.80 (br s, 3H).
DMSO-d6) δ 8.98−8.78 (m, 1H), 8.58 (d, J = 2.15 Hz, 1H), 8.45 (d, J =
1.96 Hz, 1H), 8.20 (d, J = 2.35 Hz, 1H), 7.89−7.82 (m, 1H), 7.70−7.64
(m, 1H), 7.63−7.56 (m, 1H), 7.37−7.22 (m, 1H), 7.02 (s, 2H), 6.55 (d,
J = 8.61 Hz, 1H), 3.19 (d, J = 4.69 Hz, 4H), 2.86 (br s, 4H), 2.46 (br s,
3H).
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-6′-me-
thoxy-[3,3′-bipyridin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (56).
This material was prepared according to the procedure described for
the synthesis of 43 from 20 (0.100 g, 0.151 mmol) using (6-
methoxypyridin-3-yl)boronic acid (0.069 g, 0.52 mmol). The title
compound was isolated (0.008 g, 6%) as a tan solid. MS (ESI pos. ion)
m/z calcd for C23H22F6N6O4S, 592; found, 593 (M + H). 1H NMR (400
MHz, DMSO-d6) δ 8.91−8.82 (m, 1H), 8.49−8.36 (d, J = 2.15 Hz, 1H),
8.32−8.27 (d, J = 1.96 Hz, 1H), 8.22−8.17 (d, J = 2.35 Hz, 1H), 7.91−
7.84 (m, 1H), 7.68−7.63 (m, 1H), 7.63−7.57 (m, 1H), 7.05−6.99 (m,
2H), 6.88−6.82 (m, 1H), 6.60−6.46 (d, J = 8.61 Hz, 1H), 3.89 (s, 3H),
3.18 (d, J = 5.28 Hz, 4H), 2.90−2.76 (m, 4H).
2-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5′-me-
thoxy-[3,3′-bipyridin]-5-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (57).
This material was prepared according to the procedure described for
the synthesis of 43 from 20 (0.100 g, 0.151 mmol) using (5-
methoxypyridin-3-yl)boronic acid (0.069 g, 0.452 mmol). The title
compound was isolated (0.029 g, 22%) as a tan solid. MS (ESI pos. ion)
m/z calcd for C23H22F6N6O4S, 592; found, 593 (M + H). 1H NMR (400
MHz, DMSO-d6) δ 8.95−8.83 (m, 1H), 8.52−8.40 (d, J = 2.15 Hz, 1H),
8.20 (d, J = 1.96 Hz, 2H), 7.79−7.68 (d, J = 2.35 Hz, 1H), 7.64−7.51 (m,
1H), 7.15−7.10 (m, 1H), 7.03 (s, 2H), 6.94 (d, J = 8.61 Hz, 1H), 6.62−
6.50 (m, 1H), 3.88 (s, 3H), 3.21 (br s, 4H), 2.86 (d, J = 4.11 Hz, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(benzo-
[b]thiophen-5-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(58). This material was prepared according to the procedure described
for the synthesis of 34 from 19 (0.100 g, 0.192 mmol) using 2-
(benzo[b]thiophen-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(0.100 g, 0.385 mmol). The title compound was isolated (0.009 g, 8%)
as a tan solid. MS (ESI pos. ion) m/z calcd for C25H21F6N5O3S2, 617;
found, 618 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 1H),
8.15 (d, J = 2.23 Hz, 1H), 8.08−7.97 (m, 2H), 7.83 (d, J = 5.21 Hz, 1H),
5-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)-1-meth-
ylindolin-2-one (62). This material was prepared according to the
procedure described for the synthesis of 48 from 23 (0.100 g, 0.192
mmol) using 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
indolin-2-one (0.106 g, 0.385 mmol). The title compound was isolated
(0.009 g, 7%) as a tan solid. MS (ESI pos. ion) m/z calcd for
1
C26H24F6N6O4S, 630; found, 631 (M + H). H NMR (400 MHz,
DMSO-d6) δ 8.91−8.661 (m, 1H), 8.36 (s, 1H), 8.28−8.19 (d, J = 2.35
Hz, 1H), 7.71−7.57 (dd, J = 2.35, 8.80 Hz, 2H), 7.57−7.47 (br, s, 1H),
7.47−7.34 (m, 1H), 7.12−6.92 (d, J = 5.10 Hz, 1H), 6.93−6.80 (m, 1H),
6.59−6.49 (d, J = 8.76 Hz, 1H), 3.74−3.55 (m, 1H), 3.43 (s, 1H), 3.17
(m, 7H), 3.02−2.94 (m, 1H), 2.84 (d, J = 3.33 Hz, 4H).
5-(2-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-
(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)pyridin-3-yl)indolin-
2-one (63). This material was prepared according to the procedure
described for the synthesis of 34 from 19 (0.100 g, 0.192 mmol) using 5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indolin-2-one (0.100 g,
0.385 mmol). The title compound was isolated (0.015 g, 13%) as a
white solid. MS (ESI pos. ion) m/z calcd for C25H22F6N6O4S, 616;
found, 617 (M + H). 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H),
8.91−8.66 (m, 1H), 8.36 (s, 1H), 8.20 (d, J = 2.35 Hz, 1H), 7.62 (dd, J =
2.35, 8.80 Hz, 2H), 7.35 (s, 1H), 7.06−6.92 (m, 2H), 6.93−6.80 (m,
1H), 6.53 (d, J = 8.61 Hz, 1H), 3.74−3.55 (m, 1H), 3.43 (s, 1H), 3.17
(br s, 4H), 3.02−2.94 (m, 1H), 2.84 (d, J = 3.33 Hz, 4H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1H-in-
dazol-5-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (64). This
material was prepared according to the procedure described for the
synthesis of 33 from 20 (0.100 g, 0.151 mmol) using tetramethyl-1,3,2-
dioxaborolane-2-yl)-1H-indazole (0.073 g, 0.301 mmol). The TFA salt
of the title compound was isolated (0.013 g, 14%) as a tan solid. MS (ESI
pos. ion) m/z calcd for C24H21F6N7O3S, 601; found, 602 (M + H). 1H
NMR (400 MHz, DMSO-d6) δ 8.98−8.71 (m, 1H), 8.41 (s, 2H), 8.20
(d, J = 2.15 Hz, 1H), 8.08 (s, 1H), 7.87 (s, 1H), 7.70 (s, 1H), 7.68−7.60
5962
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Journal of Medicinal Chemistry
Article
(m, 1H), 7.59−7.54 (m, 1H), 7.51 (d, J = 1.17 Hz, 1H), 6.62 (s, 1H),
3.16 (d, J = 4.69 Hz, 4H), 2.84 (br s, 4H), 1.15 (d, J = 13.30 Hz, 2H).
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1H-in-
dazol-4-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (65). This
material was prepared according to the procedure described for the
synthesis of 48 from 23 (0.050 g, 0.096 mmol) using (1H-indazol-4-
yl)boronic acid (0.023 g, 0.144 mmol). The crude product was purified
by column chromatography eluting with 0−10% MeOH in DCM. This
was followed by another purification by column chromatography eluting
with 0−100% EtOAc in hexanes followed by another purification by
column chromatography eluting with 0−7% MeOH in DCM to yield
the title compound (0.002 g, 3%) as a white solid. MS (ESI pos. ion) m/
z calcd for C24H21F6N7O3S, 601; found, 602 (M + H). 1H NMR (400
MHz, CD3OD) δ 8.55−8.49 (m, 1H), 8.13 (d, J = 2.2 Hz, 1H), 7.91−
7.87 (m, 1H), 7.79−7.75 (m, 1H), 7.60−7.54 (m, 2H), 7.49−7.42 (m, J
= 7.0 Hz, 1H), 7.21 (d, J = 7.0 Hz, 1H), 6.60 (d, J = 9.0 Hz, 1H), 3.23−
3.16 (m, 4H), 2.73−2.65 (m, 4H). Four exchangeable protons were not
observed.
phamacodynamic assay. We would also like to thank Scott
Simonet, Murielle Veniant, Philip Tagari, and Randall Hungate
́
for their support of this research.
ABBREVIATIONS USED
■
GK, glucokinase; GKA, glucokinase activator; GKRP, glucoki-
nase regulatory protein; RLM, rat liver microsomes; HLM,
human liver microsomes; alpha, amplified luminescent proximity
homogeneous assay; SFC, supercritical fluid chromatography;
PK, pharmacokinetics; CL, clearance; MRT, mean residence
time; AUC, area under curve; Vdss, volume of distribution; F,
bioavailability; B/P, blood to plasma ratio; Fu, fraction unbound;
IHC, immunohistochemistry; CYP, cytochrome P450; EC50, half
maximal effective concentration; IC50, half maximal inhibitory
concentration
2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(1H-
indol-2-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (66). This
material was prepared according to the procedure described for the
synthesis of 34 from 19 (0.100 g, 0.192 mmol) using 2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (0.094 g, 0.385 mmol).
The title compound was isolated (0.016 g, 14%) as a tan solid. MS (ESI
pos. ion) m/z calcd for C25H22F6N6O3S, 600; found, 601 (M + H). 1H
NMR (400 MHz, DMSO-d6) δ 11.30 (br s, 1H), 8.40 (s,1H) 8.19 (d, J =
2.23 Hz, 1H), 7.94 (br s, 1H), 7.58 (dd, J = 8.91 Hz, 1H), 7.51 (d, J =
7.45 Hz, 1H), 7.39−7.26 (m, 1H), 7.13 (t, J = 7.45 Hz, 1H), 7.08−6.95
(m, 2H), 6.76 (d, J = 7.33 Hz, 1H), 6.69 (s, 1H), 6.57 (d, J = 8.99 Hz,
1H), 5.74 (s, 1H), 3.18 (br s, 4H), 3.06 (m, 4H).
REFERENCES
■
(1) Agius, L. Glucokinase and Molecular Aspects of Liver Glycogen
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2-(6-(4-((6-Aminopyridin-3-yl)sulfonyl)piperazin-1-yl)-5-(benzo-
[b]thiophen-2-yl)pyridin-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(67). This material was prepared according to the procedure described
for the synthesis of 34 from 19 (0.100 g, 0.192 mmol) using
benzo[b]thiophen-2-ylboronic acid (0.068 g, 0.385 mmol). The title
compound was isolated (0.007 g, 7%) as a tan solid. MS (ESI pos. ion)
1
m/z calcd for C25H21F6N5O3S2, 617; found, 618 (M + H). H NMR
(400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.20 (d, J = 2.29 Hz, 1H), 7.97 (br
s, 1H), 7.92 (d, J = 7.68 Hz, 1H), 7.82 (d, J = 7.33 Hz, 1H), 7.76 (s, 1H),
7.61 (d, J = 8.88 Hz, 1H), 7.48−7.28 (m, 2H), 7.04 (s, 1H), 6.55 (d, J =
8.88 Hz, 1H), 5.74 (s, 1H), 3.27 (s, 4H), 3.17 (br s, 1H), 2.98 (br s, 4H).
ASSOCIATED CONTENT
* Supporting Information
■
S
Standard deviations for Tables 1−4; blood glucose measure-
ments of postoral administration of compounds 51 and 1 (10, 30,
100, mg/kg) to db/db mice; unbound plasma concentrations of
51 from the efficacy study (Figure 7b) and the unbound plasma
concentrations from the pharmacodynamic study (Figure 7d)
plotted on the same graph; and calculation details for dihedral
angle plot in Figure 4. This material is available free of charge via
the Internet at http://pubs.acs.org. The cocrystal structure of
hGKRP + compound 51 has been deposited in the Protein Data
Bank with PDB code: 4OLH (see ref 7 for experimental details of
crystallography).
AUTHOR INFORMATION
Corresponding Authors
*(F.-T.H.) Tel: 805-447-4992. E-mail: fhong@amgen.com.
*(M.H.N.) Tel: 805-447-1552. E-mail: markn@amgen.com.
■
(8) 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,
Notes
The authors declare no competing financial interest.
́
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.
ACKNOWLEDGMENTS
■
The authors acknowledge Kyung Gahm, Wesley Barnhart, and
Samuel Thomas for conducting the chiral SFC separations and
Elizabeth Galbreath and Gywneth Van for their assistance in the
(9) 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.;
5963
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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, 57, 309−324.
(10) St. Jean, D. J., Jr.; Ashton, K. S.; Bartberger, M. D.; Chen, J.;
Chmait, S.; Cupples, R.; Galbreath, E.; Helmering, J.; Hong, F.-T.;
Jordan, S. R.; Liu, L.; Kunz, R. K.; Michelsen, K.; Nishimura, N.;
Pennington, L. D.; Poon, S. F.; Reid, D.; Sivits, G.; Stec, M. M.; Tadesse,
S.; Tamayo, N.; Van, G.; Yang, K.; Zhang, J.; Norman, M. H.; Fotsch, C.;
Lloyd, D. J.; Hale, C. Small Molecule Disruptors of the Glucokinase−
Glucokinase Regulatory Protein Interaction: 2. Leveraging Structure-
Based Drug Design to Identify Analogs with Improved Pharmacokinetic
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(24) Similar changes in Tyr24 and Pro29 residues were also observed
in X-ray cocrystal structure with the 1-methyl-4-1H-pyrazole substituted
analogue, 34.
(25) For comparison, the efficacy of compound 1 is also reported (see
Supporting Information).
(26) Note: Different methods were used to measure the exposure
levels of compound 51 in the efficacy and pharmacodynamic
experiments (Figure 7b vs Figure 7d). For Figure 7b, the whole blood
was taken by a dry blood spot (DBS) technique at nonterminal time
points, while for Figure 7d plasma samples were taken at terminal time
points through a cardio puncture. Although lower exposure levels of
compound 51 were observed in the pharmacodynamic study than were
obtained in the efficacy study at 100 mg/kg, the unbound exposure
levels were sufficient in both studies to cover the mouse EC50 value
obtained from the GK translocation assay. For illustration, the unbound
plasma concentrations of compound 51 from both the pharmocody-
namic and efficacy studies are plotted on the same graph (see
Supporting Information).
(11) Nishimura, N.; Norman, M. H.; Liu, L.; Yang, K.; Ashton, K. S.;
Bartberger, M. D.; Chen, J.; Chmait, S.; Cupples, R.; Fotsch, C.;
Helmering, J.; Jordan, S. R.; Kunz, R. K.; Poon, S. F.; Siegmund, A.;
Sivits, G.; Lloyd, D. J.; Hale, C.; St. Jean, D. J., Jr. Small Molecule
Disruptors of the Glucokinase−Glucokinase Regulatory Protein
Interaction: 3. Structure−Activity Relationships within the Aryl
Carbinol Region of the N-Arylsulfonamido-N′-aryl-piperazine Series.
J. Med. Chem. 2014, 57, 3094−3116.
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analysis, see ref 7.
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Nar, H.; Reinert, D.; Lenter, M.; Heckel, A.; Schnapp, G.; Kauschke, S.
G. Crystal Structure of Glucokinase Regulatory Protein. Biochemistry
2013, 52, 3523−3531.
(14) See Supporting Information for details of calculations and data
analysis of this quantum mechanical study.
(15) This two-step sequence has been utilized to prepare structurally
similar intermediates, see ref 6.
(16) Ashton, K.; Bourbeau, M. P.; Hong, F.-T.; Liu, L.; Nishimura, N.;
Norman, M. H.; Poon, S. F.; Stec, M. M.; St. Jean, D. J., Jr.; Tamayo, N.
A.; Yang, K. C. Preparation of Cyclic Sulfonyl Compounds That Interact
with Glucokinase Regulatory Protein for Use in Treating Type 2
Diabetes and Other Diseases. PCT Int. Appl. WO 2013123444, 2013.
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Activated Palladium Precatalysts for Facile C−N Cross-Coupling
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(21) See Experimental Section for details.
(22) This result is consistent with the data previously reported for an
analogous benzene C-ring series (IC50 = 0.004 μM with the propargyl
group and IC50 = 0.087 μM without the propargyl group, i.e., a ∼22-fold
difference) (see ref 10).
(23) The direct (S)-propargyl-piperazine analogue of 51 was not
prepared; however, the des-nitrogen version of 51 (i.e., phenyl C-ring)
and its corresponding (S)-methyl-piperazine analogue were prepared
and evaluated (see below). As illustrated below, the difference of
activities between these two compounds in the AlphaScreen binding
assay is striking (0.028 vs 5.5 μM). This data supports the assertion that
the upper hydrophobic pocket and the shelf regions cannot be
simultaneously occupied without engaging in a significant steric clash
with the protein.
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