Discovery and characterization of an inhibitor of glucosylceramide synthase

Article abstract of DOI:10.1021/jm300122u

Targeting glycosphingolipid synthesis has emerged as a novel approach for treating metabolic diseases. 32 (EXEL-0346) represents a new class of glucosylceramide synthase (GCS) inhibitors. This report details the elaboration of hit 8 with the goal of achieving and maintaining maximum GCS inhibition in vivo. 32 inhibited GCS with an IC₅₀ of 2 nM and achieved maximum hepatic GCS inhibition after four or five daily doses in rodents. Robust improvements in glucose tolerance in DIO mice and ZDF rats were observed after 2 weeks of q.d. dosing. Four weeks of dosing resulted in decreased plasma triglycerides and reduced hepatic fat deposition. Thus, 32 provides insight into the amount of metabolic regulation that can be restored following achievement of maximal target knockdown.

Full text of DOI:10.1021/jm300122u

Article
pubs.acs.org/jmc
Discovery and Characterization of an Inhibitor of Glucosylceramide
Synthase
Steven Richards,*,† Christopher J. Larson,† Elena S. Koltun, Art Hanel, Vicky Chan, Jason Nachtigall,
Amanda Harrison, Naing Aay, Hongwang Du, Arlyn Arcalas, Adam Galan, Jeff Zhang, Wentao Zhang,
Kwang-Ai Won, Danny Tam, Fawn Qian, Tao Wang, Patricia Finn, Kathy Ogilvie, Jon Rosen,
Ron Aoyama, Artur Plonowski, Belinda Cancilla, Frauke Bentzien, Michael Yakes, Raju Mohan,
Peter Lamb, John Nuss, and Patrick Kearney
Exelixis, Inc., 210 E. Grand Avenue, South San Francisco, California 94080, United States
ABSTRACT: Targeting glycosphingolipid synthesis has
emerged as a novel approach for treating metabolic diseases.
32 (EXEL-0346) represents a new class of glucosylceramide
synthase (GCS) inhibitors. This report details the elaboration
of hit 8 with the goal of achieving and maintaining maximum
GCS inhibition in vivo. 32 inhibited GCS with an IC50 of 2 nM
and achieved maximum hepatic GCS inhibition after four or
five daily doses in rodents. Robust improvements in glucose
tolerance in DIO mice and ZDF rats were observed after 2 weeks of q.d. dosing. Four weeks of dosing resulted in decreased
plasma triglycerides and reduced hepatic fat deposition. Thus, 32 provides insight into the amount of metabolic regulation that
can be restored following achievement of maximal target knockdown.
INTRODUCTION
■
Type 2 diabetes is a condition that affects millions in the
United States. The prevalence of the disease has increased as
the “Western” culture of calorie rich diet and sedentary lifestyle
has spread throughout the world. In 2007, diabetes was the
seventh leading cause of death in the U.S. and cost well over
$100 billion to treat.1 Current pharmaceutical interventions
work, alone or in combination, via a variety of mechanisms to
lower hepatic glucose output, inhibit glucose uptake, slow gastric
emptying, increase insulin sensitivity, and stimulate insulin secre-
tion.2 Despite these advances, there remains significant interest in
new molecular targets whose modulation could regulate glucose
and lipid metabolism.
One such target is glucosylceramide synthase (GCS). GCS
regulates the production of glycosphingolipid conjugates called
gangliosides (such as 3 (GM3)) via glucosyl transfer to 1
(ceramide, Figure 1). Improper regulation of the direct product
of this reaction, 2 (glucosylceramide), results in a rare
metabolic disorder known as Gaucher disease. This lysosomal
storage disease arises from a genetic lack of the enzyme
glucosylcerebrosidase, which degrades glucosylceramide. The clinical
manifestations of this disorder include thrombocytopenia, spleno-
megaly, increased liver size, and debilitating bone pain.3 In this
context, small molecule inhibition of GCS has proven effective.
Iminosugar 4 (migustat), shown in Figure 2, is used as substrate
reduction therapy (SRT) for Gaucher patients in lieu of enzyme
replacement therapy (ERT).4 Ceramide analogue 5 (Genz-112638)
is in clinical development to treat type 1 Gaucher disease.5
Figure 1. GCS mediated glycosphingolipid synthesis.
Received: January 27, 2012
Published: April 12, 2012
© 2012 American Chemical Society
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Glucose tolerance, as demonstrated by an IGTT, was improved
after 10 weeks of dosing at 125 mpk. These animals had lower
whole body fat mass, decreased liver triglycerides, and decreased
liver fat deposition.14
Inspired by these reports, we structured our research
program to provide further support for the clinical management
of type 2 diabetes via GCS inhibition. One aspect of our
approach was an interest in leveraging our screening platform
to identify novel scaffold classes. Early work in this area has
been previously reported.15 The other pillar of our approach
was an interest in achieving sustained GCS inhibition in vivo in
an attempt to determine how quickly antidiabetic efficacy
would result. This report details the achievement of the second
goal, the discovery and characterization of 32 (EXEL-0346), a
potent, efficacious GCS inhibitor.16
RESULTS AND DISCUSSION
■
Figure 2. Representative GCS inhibitors.8
Our HTS campaign surveyed our collection of over 4 million
compounds. One of the hits was the racemic tryptophan based
dipeptide (8) shown in Figure 3. When both enantiomers of
In the past decade, excess ganglioside GM3 has been shown
to disrupt insulin receptor function. Mice lacking GM3
exhibited increased glucose tolerance and improved insulin
receptor (IR) phosphorylation.6 The mechanistic hypothesis
for this dysregulation is that excess GM3 in the cell membrane
forms lipid rafts that disrupt the membrane environment
around the insulin receptor. Because of this perturbation, the
insulin receptor does not signal properly resulting in insulin
resistance.7 In this context, small molecule GCS inhibition has
also been beneficial.
Iminosugar 6 (AMP-DNM) both decreased cell surface GM3
and increased insulin stimulated IR phosphorylation in 3T3L1
adipoctes. In vivo effects include improved oral glucose
tolerance and decreased glycated hemoglobin (% HbA1c).9
The effects were observed after dosing for 10 weeks at 25 mpk
in Zucker diabetic fatty (ZDF) rats and correlated with
decreased hepatic glucosylceramide levels. Additional metabolic
improvements were measured after dosing 6 for 5 weeks at
100 mpk in another diabetic rodent model, the ob/ob mouse.
Increased insulin receptor activation, represented by increased
hepatic pAkt and pmTOR, and normalization of the expression
of several proteins related to hepatic lipid metabolism and
gluconeogenesis were observed. Additionally, hepatic fat depots
were decreased after treatment with 6.10 Iminosugar 6 is also a
potent inhibitor of intestinal glucosidases which contributes to
the observed antidiabetic efficacy.11
Ceramide analogue 7 (Genz-123346) has also demonstrated
metabolic improvements after chronic administration.12 In ZDF
rats, a striking decrease in HbA1c was observed after 6 weeks
of dosing at 75 mpk. Decreased hepatic glucosylceramide was
measured after 10 weeks of dosing 7 at 85 mpk. In diet induced
obese (DIO) mice, near normalization of glucose excursion
during an oral glucose challenge was achieved after dosing 7 for
10 weeks at 125 mpk. Again, this was accompanied by a marked
improvement in HbA1c. In both rodent models, efficacy was
driven by restoration of insulin receptor function. Treatment
with 7 (125 mpk for 12 or 20 weeks) also reversed hepatic
steatosis in DIO mice.13
Figure 3. HTS hit.
the HTS hit (8) were synthesized, the S enatiomer (8a, derived
from L-tryptophan) was found to be responsible for the
observed activity against GCS. Epimeric 8b was inactive. This
strong correlation between GCS activity and amino acid stereo-
chemistry was found to be general after several analogous
enantiomer pairs were synthesized (data not shown). Thus, we
proceeded with SAR development with analogues derived from
L-amino acids.
Further characterization of 8a revealed that it was also active in
our GM1 depletion cell assay. ADME profiling further revealed
instability to mouse microsomal oxidation and a modest
CYP3A4 liability. We were first interested in understanding the
impact of tryptophan replacement on potency and ADME
properties. A summary of these efforts is presented in Table 1.
The results of a broad survey of amino acids is not pre-
sented here, as most were not tolerated. However, benzylserine
analogue 11, while less potent than 8a, was tolerated.
Additionally, it showed that the ADME profile could be
influenced by the amino acid. A slight improvement in micro-
somal stability and increased affinity for CYP3A4 were the
result. Phenylalanine derivative 17 was also tolerated but
was 15-fold less potent than hit 8a with no ADME benefit.
Fortunately, modification of the phenylalanine restored the
biochemical and cell potency to this subseries, as evidenced by
compounds 18−20. Unfortunately, compounds 18−20 were no
more stable in mouse microsomes than 8a and were all about
10-fold more potent inhibitors of CYP3A4. Thus, our efforts to try
to modify the amino acid moiety were successful only in part.
In recent studies supported by Genzyme, treatment with
ceramide analogue 5 modulated hepatic ceramide, glucosylcer-
amide, and GM3 in lean mice. These effects were observed after
dosing these animals for 22 weeks at 120 mpk. In DIO mice,
5 lowered HbA1c after 12 weeks of dosing at either 75 or 125 mpk.
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To that end, compound 24 was prepared. This “ring opened”
piperazine analogue was biochemically about 5-fold more
potent than 18. Cellular potency was about 30 fold lower (GCS
cell IC50 = 120 nM). The CYP profile and susceptibility to
microsomal oxidation were not improved. The bromopheny-
lalanine analogue 25 (GCS IC50 = 4 nM) was equipotent to 24
biochemically. Fortunately, the modification of the phenyl-
alanine ring provided a 7-fold improvement in cellular potency
relative to 24. Compound 25 also showed about a 5-fold
decrease in CYP3A4 potency relative to 18. However,
microsomal stability was not improved. From this set of
compounds, we learned that the piperazine could indeed be
replaced and that modifications to the amino acid core could
have a beneficial impact on cell potency and CYP profile.
We also confirmed that a basic amine was necessary for
potency in this series. A representative example of this is
compound 26. With a hydroxyl group, 26 was tolerated (GCS
IC50 = 260 nM) but was 100-fold less potent than 4-amin-
opiperidide 27 (GCS IC50 = 3 nM). Additionally, compound
27 had a better CYP profile. Metabolic stability of the two
compounds was similar.
Table 1. Amino Acid Derivatives
a
b
Average value of duplicate experiments reported. Average value of
c
Compound 27 also taught us that a 1,5-diamino substitution
pattern was well tolerated. This observation was confirmed by
the synthesis of 3-aminopiperidide 28. While the metabolic
stability of this compound was quite good, it was a very potent
CYP inhibitor (CYP3A4 IC50 = 420 nM). Further constraint of
the 1,5-diamino motif into an aminotropane resulted in 29.
Compound 29 (GCS IC50 = 4 nM) was 4-fold more potent
than 28 and especially potent in cells (GCS cell IC50 = 2 nM).
However, like 28, 29 was a potent CYP3A4 inhibitor. An
attempt to modify the amino acid portion to improve the
CYP3A4 profile, as we had with 25, was not successful.
Compound 30 had a biochemical and ADME profile identical
to that of 29 while losing 20-fold cellular potency. Fortunately,
modification of the cyclopropylaryl moiety provided a way
forward. In general, modification of this region of the scaffold
was very poorly tolerated. One of the few reasonable
substituents was the 4-trifluoromethoxy group. When incorpo-
rated, this group helped compound 31 (GCS IC50 = 3 nM)
maintain the biochemical potency observed in compounds 29
and 30. Cell activity of 31 (GCS cell IC50 = 8 nM), while 4-fold
worse than 29, was still within a desirable range. The most
significant profile improvement was the 5-fold decrease in
CYP3A4 potency exhibited by 31 (CYP3A4 IC50 = 2.4 μM).
Combining the bromophenylalanine substituent with the
4-trifluoromethoxyphenylcyclopropamide led to compound 32.
Again, biochemical and cell potency were comparable to 31 as was
the ADME profile. Since it possessed a balanced in vitro profile, 32
was advanced into PK to compare with compound 18. The aim was
to help us understand to what extent in vitro ADME improvements
could help us achieve the desired in vivo exposure profile.
Our goal was to discover a GCS inhibitor that could achieve
sustained target knockdown between doses. Theoretically, the
resulting cell surface ganglioside depletion would restore insulin
receptor function and lead to improved glucose control.
Table 4 is a summary of rodent PK data obtained for 32. In
the mouse screening PK experiment, a 10 mpk dose of 32
resulted in 24 h exposure in liver, muscle, and fat well in excess
of the cellular IC50 of the compound. In order to achieve
comparable 5.9 μM liver exposure of 18 at 24 h, a 30−75 mpk
dose was required. Additionally, 32 plasma exposure was
relatively low. This was thought to be an advantage for the
compound, as the benefit of GCS inhibition would be in tissues
triplicate experiments reported. n.d. = value not determined.
With compounds in hand that represented two subseries, an
effort was made to differentiate them in vivo. As shown in
Table 2, 100 mpk doses of hit 8a and 18 were given orally to
Table 2. Mouse Plasma Exposure of Lead Compounds 8a
and 18
a
a
compd
1 h [plasma] (μM)
4 h [plasma] (μM)
8a
18
0.9 0.5
4.2 1.6
0.7 0.3
5.8 1.7
a
Female nude mouse (n = 3), 100 mpk, po. Values reported are the
mean SD.
mice, and compound levels were measured 1 and 4 h after
dosing. In these experiments, 18 had higher plasma levels at
both measured time points than 8a. Our shift in research focus
to phenylalanine analogues was driven largely by the results of
this screening PK experiment. Our goal became to address the
ADME liabilities of 18 with an analogue that had comparable or
improved potency and exposure.
With the amino acid core in place, we turned our attention to
the other poles of the scaffold. A summary of these efforts is
presented in Table 3. A metabolite identification experiment
performed on 18 determined that the N-methylpiperazine
moiety was the primary site of oxidative metabolism (data not
shown). Piperazine 21, one of the putative metabolites, was
10-fold less potent than 18. What was of more concern was
the potent CYP3A4 inhibition exhibited by this compound
(CYP3A4 IC50 < 200 nM). When the methyl group was
transposed from the N4 to the C3 position on the piperazine
ring, the resulting diastereomers 22 and 23 were also tolerated.
Compound 22 (GCS IC50 = 20 nM) was equipotent to 18, and
23 (GCS IC50 = 190 nM) was equipotent to 21. Unfortunately,
both compounds had the same severe CYP3A4 liability.
Given the potent CYP3A4 inhibition for this set of compounds,
it was difficult to interpret the increased metabolic stability they
exhibited relative to 18. Taken together, these data suggested
that the piperazine moiety had to be replaced rather than
modified.
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Table 3. Identification of 32
a
b
c
Average value of duplicate experiments reported. Average value of triplicate experiments reported. n.d. = value not determined.
Table 4. Rodent PK Paramaters of 32
Rat PK is also shown in Table 4. The plasma Cmax after a
5 mpk dose was relatively low. The low clearance and high
volume of distribution were in line with the desire to have
systemic effects. The excellent oral bioavailability coupled with
the long half-life further supported the advancement of 32,
dosed once daily, into our PD models.
For our initial PD experiment, we measured the modulation of
liver glycosphingolipid levels in mice after dosing with GCS
inhibitors 32, 6, and 7. The ceramide species measured contained
the C16 (palmitoyl) acyl chain. As is shown in Figure 4A,
a 10 mpk dose of 32 was effective at significantly lowering
glucosylceramide [p < 0.05 vs vehicle], lactosylceramide [p < 0.05
vs vehicle], and GM3 [p < 0.05 vs vehicle] levels. This effect was
comparable to a 75 mpk dose of 7. Ceramide levels were
unchanged from vehicle. Additionally, 32 was more potent than 6
in this experiment.17
a
b
mouse
rat
[plasma] (μM)
[liver] (μM)
[muscle] (μM)
[fat] (μM)
0.5 0.1
5.9 1.1
1.8 0.2
0.8 0.2
Cmax (μM)
Tmax (h)
0.3
8.0
AUC(0−t) (μM·h)
CL (mL h−1 kg−1
Vss (L/kg)
5.8
)
574.8
19.3
25.3
88.1
T1/2 (h)
F (%)
a
Male C57BL6 mouse (n = 3), 10 mpk, po; data collected 24 h after
b
dose. Values reported are the mean SD. Female CD rat (n = 3),
5 mpk dose. The HCl salt of 32 dissolved in saline was used for both
oral and iv formulations.
primarily responsible for glucose metabolism (liver, muscle, and
fat) rather than in circulation.
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Figure 4. Effects of GCS inhibition on the liver levels of ceramide (Cer), glucosylceramide (GluCer), lactosylceramide (LacCer), and GM3 in lean
mice (C57/B6, n = 4) after 4 days of dosing q.d., po (A). Data were collected 4 h after last dose. Data are presented as the mean SD: (∗) p < 0.05
vs vehicle, one-way ANOVA followed by Dunnett’s test. Also shown are the effects of GCS inhibition in the livers of lean rats (Sprague−Dawley,
n = 3) after 5 days of dosing (q.d., po) (B). Data were collected 24 h after last dose. Maximum GCS inhibition was observed at the 30 mpk dose (B).
Data are depicted as the mean SD: (∗) p < 0.05 vs vehicle, one-way ANOVA followed by Dunnett’s test.
While the mouse experiment was a promising snapshot of the
ability of 32 to inhibit GCS in vivo, it was also necessary to find
the dose at which maximum GCS inhibition could be maintained
for 24 h after dose. The requisite dose response experiment was
performed in rats (Figure 4B). Liver glycosphinoglipid levels were
measured 24 h after a 5-day regimen of 3, 10, 30, or 100 mpk
doses. A clear dose response was observed for all measured signals
downstream of GCS. Statistically significant decreases in
glucosylceramide [p < 0.05 vs vehicle] were observed at all
doses. This led to significant reductions in lactosylceramide [p <
0.05 vs vehicle] and GM3 [p < 0.05 vs vehicle]. The maximum
effect was observed at the 30 mpk dose with no further benefit
gained at 100 mpk. These results were used to determine dosing
for the subsequent rodent efficacy experiments.
After observing efficacy in the DIO mouse model, we looked
for confirmation in a genetic diabetes model. For this
experiment, we chose the Zucker diabetic fatty (ZDF) rat. 32
was administered orally, once daily, at either 10 or 30 mpk
doses for 29 days (Figure 8). The first OGTT was performed
after 15 days of dosing (Figure 8A,B). In this experiment, the
10 mpk dose did not have a significant effect. However, the
30 mpk dose improved glucose tolerance comparable to 33
[p < 0.05 vs ZDF vehicle]. A second OGTT, performed at
27 days, showed an improvement in efficacy at the 10 mpk dose
of 32 (Figure 8C,D). This effect did not reach statistical signifi-
cance. By 27 days, the 30 mpk dose of 32 nearly normalized
glucose excursion and disposal in this model. However, the
body weight data for this study were of concern (Figure 9A).
Even though all the animals gained weight during the study,
the compound 32 (30 mpk) dose group gained weight more
slowly than the ZDF, vehicle treated controls. The decreased
body weight gain was statistically significant [p < 0.05 vs ZDF
vehicle] from day 7 onward. This decreased body weight gain
was linked to decreased food intake.
When 32 was dosed in diet induced obese (DIO) mice,
robust glucose lowering and insulin sensitization were observed.
Thiazolidinedione 33 (rosiglitazone), shown in Figure 5, was
Because this decreased body weight gain could have
contributed to the efficacy signal measured in the OGTT, we
opted to perform a pair-fed efficacy study. In this 29-day
experiment, three ZDF control groups were used. One control
group was fed ad libitum and dosed with vehicle. A second was
pair-fed to the first control group and also dosed with vehicle.
The third control group was pair-fed to the 32 (30 mpk)
treated group and dosed with vehicle. After 14 days of dosing,
HbA1c was measured (Figure 9B). Fortunately, the compound
32 group showed a statistically significant decrease relative
to the pair-fed control group [p < 0.06 vs pair-fed vehicle].20
This indicated to us that 32 was indeed improving glucose
processing in this rat study. In addition, 32 had beneficial
effects on lipid trafficking (Figure 9C). Decreased plasma
triglycerides were observed relative to pair-fed control [p < 0.05
vs pair-fed vehicle]. In addition, abdominal fat pad mass was
decreased relative to pair-fed controls [p < 0.05 vs pair-fed
control] (Figure 9D).
Figure 5. Positive control used for rodent efficacy experiments.
run as a positive control.18 A 10 mpk dose of 32, once daily,
increased oral glucose tolerance in 14 days (Figure 6A,B). After
28 days, 32 still significantly [p < 0.05] improved glucose
tolerance (Figure 6C,D). These effects were observed without a
confounding decrease in body weight (Figure 7A). Food intake
was not measured. Fasting glucose was found to be significantly
lower [p < 0.05], relative to vehicle treated control, after both
14 and 28 days of dosing (Figure 7B). Further evidence
of increased insulin sensitivity was provided by an end of
study measurement of insulin stimulated Akt phosphorylation
(Figure 7C). Animals chronically treated with 32 and then
given insulin had markedly increased pAkt [p < 0.05] relative to
treatment naive animals. Increased pAkt implies increased
insulin receptor function.19 These effects correlated well with
decreased liver glycosphingolipid and GM3 levels measured at
the end of the study (Figure 7D). The robust efficacy shown in
this model inspired us to further characterize 32.
Further evidence of decreased lipid accumulation mediated
by 32 was found upon examination of liver samples. Livers were
isolated from animals in both the ZDF rat and DIO mouse
efficacy studies (Figure 10). When untreated, both of these
animal models develop liver steatosis.21 The fat droplets in the
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Figure 6. 32 was dosed once daily, by oral gavage, in diet induced obese (DIO) mice for 28 days (n = 8 per group). A control group was treated with
33. Day 14 OGTT (2 h after dose) data are presented. 32 dose group reached statistical significance [p < 0.05 vs DIO vehicle, two-way ANOVA
followed by Bonferroni’s test] at 15, 30, and 60 min time points. Lean vehicle and 33 groups were statistically different from DIO vehicle group at all
measured time points [p < 0.05 vs DIO vehicle] (A). Also shown are day 14 AUC data with a glucose baseline of 135 mg/dL: (∗) p < 0.05 vs DIO
vehicle, one-way ANOVA followed by Dunnett’s test (B). Day 28 OGTT (2 h after dose) data are presented. 32 dose group reached statistical
significance [p < 0.05 vs DIO vehicle, two-way ANOVA followed by Bonferroni’s test] at 15, 30, 60, and 120 min time points. Lean vehicle and 33
groups were statistically different from DIO vehicle group at all measured time points [p < 0.05 vs DIO vehicle, two-way ANOVA followed by
Bonferroni’s test] (C). Day 28 AUC data with a glucose baseline of 97 mg/dL are presented: (∗) p < 0.05 vs DIO vehicle, one-way ANOVA
followed by Dunnett’s test (D).
little as 2 weeks of dosing. Furthermore, decreased circulating
hepatocytes were visualized using hematoxylin−eosin (H&E)
and Oil Red O staining.22 The negative, or white, space in the
triglycerides and fat pad mass and decreased liver fat accumula-
tion were observed upon continued dosing. Thus, 32 represents
further proof of the concept that GCS inhibition could be a
viable treatment for type 2 diabetes.
H&E stained images generally represents fat. In the Oil Red O
stained images, fat is red. The lean animal livers showed very
little fat accumulation. In rat, the white spaces in the H&E
stained images were not fat but sinusoidal tracts between
hepatocytes. In contrast, the insulin resistant animals had
marked fat accumulation. Four-week treatment with 32 showed
decreased fat accumulation in both species. A quantitative
measurement of this effect was not made.
We met our goal of finding a GCS inhibitor that had
sufficient potency and exposure to achieve sustained target
inhibition. Once daily dosing with compound 32 in rodent
models resulted in decreased glucosylceramide and ganglioside
levels. Robust efficacy, in the form of improved glucose
tolerance in both DIO mice and ZDF rats, was observed after as
CHEMISTRY
■
Preparation of hit 8a proceeded as described in Scheme 1.
Activated BOC tryptophan (9) was subjected to EDC/HOBt
mediated coupling with N-methylpiperazine. Following acidic
BOC deprotection, the resulting amine was coupled with acid
14b, again under EDC/HOBt conditions. Benzylserine
analogue 11 was prepared in a similar manner.
The phenylalanine analogues were prepared by one of
two methods, both of which are described in Scheme 2. In the
first approach, BOC phenylalanines 12a−d were coupled with
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Figure 7. 32 was dosed once daily, by oral gavage, in diet induced obese (DIO) mice for 28 days (n = 8 per group). A control group was treated with
33. Shown is the body weight change over the course of the study. Data are depicted as the mean SD (A). Fasted glucose measured at days 14 and
28 is shown: (∗) p < 0.05 vs DIO vehicle, one-way ANOVA followed by Dunnett’s test (B). Increased pAkt was observed 28 days after dosing in
liver, fat, and muscle. Data are depicted as the mean SD. For insulin naive 32 group, (∗) p < 0.05 vs DIO saline control, one-way ANOVA
followed by Bonferroni’s test. For 32 + insulin group, (∗) p < 0.05 vs DIO insulin group, one-way ANOVA followed by Bonferroni’s test (C). End of
study liver ceramide and glycosphingolipid levels were measured 24 h after final dose. Data are depicted as the mean SD: (∗) p < 0.05 vs DIO
vehicle group, one- way ANOVA followed by Dunnett’s test (D).
N-methylpiperazine using EDC/HOBt. BOC deprotection and
coupling of the resulting amine with 14b furnished analogues
17−20. Alternatively, BOC phenylalanines 12b and 12d were
esterified and deprotected to furnish amino esters 13a,b.
This was accomplished in two steps by EDC coupling with
EtOH followed by HCl mediated cleavage of the BOC group.
Or the esterification and deprotection were accomplished
simultaneously by refluxing acids 12 in SOCl2. The amino
esters 15a,b were coupled, using either EDC/HOBt or
HATU, to acids 14a,b. Acid 14a was prepared in two steps
from 13a. The arylacetonitrile 13a was treated with NaH in
THF, and the resulting anion was trapped with dibromo-
ethane. Nitrile hydrolysis under forcing conditions provided
the desired acid (14a). Ester hydrolysis and EDC coupling
with R1 amines provided analogues 24−26. Final BOC
deprotection with 4 M HCl in dioxane provided analogues
21−23 and 27−32.
EXPERIMENTAL METHODS
■
Chemistry. All reagents and solvents were used as obtained from
vendors unless specified in the text. Starting materials 12a−d and 14b
were commercially available. Proton NMR spectra were collected
using a Varian Mercury Plus 400 MHz spectrometer. Reported MS
data were collected using a Waters Micromass ZQ or Agilent 1100
series MSD or similar instrument. All final compounds were at least
95% pure as determined by analytical HPLC (Waters Alliance or
Shimadzu Prominence) using a Phenomenex Gemini C18 column
(50 mm × 4.60 mm, 3 μm) or similar column.
1-(2,4-Dichlorophenyl)-N-[(2S)-3-(1H-indol-3-yl)-1-(4-meth-
ylpiperazin-1-yl)-1-oxopropan-2-yl]cyclopropanecarboxamide
(8a). (S)-2,5-Dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylamino)-3-
(1H-indol-3-yl)propanoate (9) (Advanced Chemtech, 803 mg,
2 mmol) was dissolved in dry acetonitrile (10 mL). N-Methylpiper-
azine (233 μL, 2.1 mmol) was added and the solution stirred for 2 h.
A complete reaction was indicated by LC/MS analysis. A 4 M solution
of HCl in dioxane (2 mL) was added and the solution stirred
overnight. A precipitate was formed that was filtered off and washed
with acetonitrile. LC/MS analysis of the solid was consistent with the
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Figure 8. 32 was dosed once daily, by oral gavage, in Zucker diabetic fatty (ZDF) rats for 29 days (n = 8 per group). A control group was treated
with 33. Shown are day 15 OGTT curves, 2 h after dose. 32 (10 mpk) dose group reached statistical significance [p < 0.05 vs ZDF vehicle, two-way
ANOVA followed by Bonferroni’s test] at 30 and 60 min time points. 32 (30 mpk) dose group reached statistical significance [p < 0.05 vs ZDF
vehicle, two-way ANOVA followed by Bonferroni’s test] at 30, 60, 120, and 180 min time points. 33 dose group reached statistical significance [p <
0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test] at 30, 60, 120, and 180 min time points. Lean vehicle group was statistically
different from ZDF vehicle group at all measured time points [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test] (A). Day 15
AUC data are shown: (∗) p < 0.05 vs ZDF vehicle, one-way ANOVA followed by Dunnett’s test (B). Day 27 OGTT curves are shown, 2 h after
dose. 32 10 mpk dose group reached statistical significance [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test] at the 30 min
time point. 32 30 mpk dose group reached statistical significance [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test] at 15,
30, 60, 120, and 180 min time points. 33 dose group reached statistical significance [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by
Bonferroni’s test] at 15, 30, 60, 120, and 180 min time points. Lean vehicle group was statistically different from ZDF vehicle group at 15, 30, 60,
120, and 180 min time points [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test] (C). Day 27 OGTT AUC data with a
glucose baseline of 145 mg/dL are shown: (∗) p < 0.05 vs ZDF vehicle, one-way ANOVA followed by Dunnett’s test (D).
mass of (S)-2-amino-3-(1H-indol-3-yl)-1-(4-methylpiperazin-1-yl)-
propan-1-one. After the sample was dried in vacuo, 690 mg of
the amine was obtained (1.92 mmol of the monohydrochloride salt).
2.95 (m, 2H), 2.17 (m, 1H), 2.09 (m, 1H), 2.05 (s, 3H), 1.94 (m, 1H),
1.70 (m, 1H), 1.45 (m, 2H), 0.97 (m, 2H). MS (EI) for
C26H28Cl2N4O2, found 499.0 (MH+).
1-(2,4-Dichlorophenyl)-N-[(1S)-2-(4-methylpiperazin-1-yl)-2-
o x o - 1 - { [ ( p h e n y l m e t h y l ) o x y ] m e t h y l } e t h y l ] -
cyclopropanecarboxamide (11). Compound 11 was prepared in an
analogous manner to compound 8a. 1H NMR (400 MHz, DMSO-d6):
δ 7.66 (d, 1H), 7.47 (m, 2H), 7.34 (m, 2H), 7.29 (m, 1H), 7.23 (m,
2H), 6.85 (d, 1H), 4.92 (m, 1H), 4.41 (m, 2H), 3.49 (m, 6H), 2.43
(m, 4H), 2.29 (s, 3H), 1.49 (m, 2H), 1.04 (m, 2H). MS (EI) for
C25H29Cl2N3O3, found 491 (MH+).
1-(4-(Trifluoromethoxy)phenyl)cyclopropanecarboxylic
Acid (14a). NaH (60% in mineral oil, 14.9 g, 372 mmol) was added to
a dry flask. The flask was cooled in an ice bath, and THF (100 mL)
was added. The slurry was stirred for ∼5 min. To that slurry was
added a solution of 2-(4-(trifluoromethoxy)phenyl)acetonitrile (13a)
This material was dissolved in dry acetonitrile (10 mL). Hunig’s base
̈
(1.64 mL, 9.6 mmol), 1-(2,4-dichlorophenyl)cyclopropanecarboxylic
acid (Acros, 443 mg, 1.92 mmol), and HATU (Applied Biosystems,
802 mg, 2.11 mmol) were added. The resulting reaction solution was
stirred overnight. The product was isolated by reverse-phase
preparative HPLC (aqueous ammonium acetate buffer solution,
pH ≈ 4−5, acetonitrile; 20−70% eluant gradient). Pure product
containing fractions were collected, combined, concentrated, and
1
lyophilized to obtain 466 mg (41%) of the title compound. H NMR
(400 MHz, DMSO-d6): δ10.84 (s, 1H), 7.61 (m, 1H), 7.41(m, 1H),
7.35 (m, 2H), 7.31 (m, 1H), 7.04 (m, 1H), 7.01 (m, 1H), 6.85
(m, 1H), 6.68 (m, 1H), 5.93 (m, 1H), 3.25 (s, 3H), 3.08 (m, 1H),
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Figure 9. 32 was dosed once daily, by oral gavage, in Zucker diabetic fatty (ZDF) rats for 29 days (n = 8 per group). Body weight change over the
course of the study is shown. Data are depicted as the mean SEM. 32 (30 mpk) dose group was statistically different from ZDF vehicle group from
day 7 onward [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test]. 33 dose group was statistically different from ZDF vehicle
group from day 7 onward [p < 0.05 vs ZDF vehicle, two-way ANOVA followed by Bonferroni’s test] (A). Shown are results from day 14 HbA1c
analysis, pair-fed ZDF efficacy experiment with 30 mpk 32: (∗) p < 0.06 vs ZDF pair-fed vehicle, one-way ANOVA followed by Bonfferoni’s test (B).
Plasma triglyceride levels following 28 days of dosing with 32 at 30 mpk (ZDF rats, pair-fed) are shown: (∗) p < 0.05 vs pair-fed vehicle, one-way
ANOVA followed by Bonfferoni’s test (C). Retroperitoneal WAT weight is shown following 28 days of dosing with 32 at 30 mpk (ZDF rats, pair-
fed): (∗) p < 0.05 vs pair-fed vehicle, one-way ANOVA followed by Bonfferoni’s test (D).
(25 g, 124 mmol) in 120 mL of THF via dropping funnel. The
resulting mixture was warmed to room temperature and stirred for
40 min before cooling again in an ice bath. Dibromoethane (70 g,
372 mmol) was added dropwise via syringe. The reaction mixture was
warmed to room temperature and stirred overnight. The reaction was
quenched by the addition of 1 M HCl and extracted with EtOAc. The
organics were washed with water and brine and dried with MgSO4.
The solvent was removed, and the residue was used crude in the next
step. To a solution of NaOH (35 g, 795 mmol) in 400 mL of water
was added 1-(4-(trifluoromethoxy)phenyl)cyclopropanecarbonitrile
(14.9 g, 65.0 mmol). The reaction mixture was heated to 120 °C
for 48 h. The resulting solution was cooled to 0 °C and acidified to pH
3 via addition of concentrated HCl. The resulting precipitate was
filtered, washed with water, and dried. The dry residue was redissolved
in diethyl ether (500 mL), filtered from salts, and concentrated under
reduced pressure. The resulting off-white solid was used in the next
step without further purification (15.6 g, 97.5%). 1H NMR (400 MHz,
DMSO-d6): δ 12.47 (m, 1H), 7.68−7.34 (m, 2H), 7.25 (m, 2H),
1.45 (m, 2H), 1.29−0.91 (m, 2H). MS (EI) for C11H9F3O3, found
247.2 (MH+).
dichlorophenyl)propanoic acid (10b) (5 g, 15 mmol, Fluka) in
dichloromethane (100 mL) were added 1-hydroxybenzotriazole (3 g,
22.5 mmol), N-methylmorpholine (16.5 mL, 150 mmol), 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (4.3 g,
22.8 mmol), and ethyl alcohol (13.8 mL, 300 mmol). The mixture
was stirred at room temperature for 18 h. The resulting solution was
extracted with water (25 mL), saturated sodium bicarbonate solution
(25 mL), and 1 M HCl solution (25 mL). The layers were separated,
and the organic layer was dried over MgSO4, filtered, and concentrated
in vacuo. The resulting oil was dissolved in methanol (25 mL)
followed by the addition of 4 M HCl in dioxane (25 mL, Aldrich).
The mixture was stirred at room temperature for 18 h. The resulting
solution was concentrated in vacuo to give 3.0 g (60%) of the title
compound which was used in the next step without further
1
purification. H NMR (400 MHz, DMSO-d6): δ 8.86 (s, 3H), 7.65
(m, 1H), 7.46 (m, 2H), 4.14 (m, 1H), 4.13−3.92 (m, 2H), 3.53−3.02
(m, 2H), 1.05 (m, 3H). MS (EI) for C11H13Cl2NO2, found 263.2
(MH+).
(S)-Ethyl 2-Amino-3-(4-bromophenyl)propanoate (15b). To
a solution of BOC-L-4-bromophenylalanine (10d) (30.0 g, 87.16 mmol,
Chem-Impex) in ethanol (450 mL) was added thionyl chloride
(8.0 mL, 109.67 mmol). The mixture was warmed at 90 °C for 18 h.
(S)-Ethyl 2-Amino-3-(2,4-dichlorophenyl)propanoate (15a).
To a solution of (S)-2-(tert-butoxycarbonylamino)-3-(2,4-
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Figure 10. Liver preparation and staining from DIO mouse and ZDF rat efficacy experiments. With hematoxylin−eosin (H&E) stain, nuclei appear
blue and fat droplets white. With Oil Red O stain, nuclei appear blue and fat droplets stain red. After 4 weeks of treatment, 32 showed a qualitative
decrease in fat accumulation.
a
NMR (400 MHz, DMSO-d6): δ 7.44 (m, 2H), 7.36−7.21 (m, 4H),
7.08 (m, 3H), 4.44 (m, 1H), 4.04 (m, 2H), 3.11−2.79 (m, 2H), 1.31−
1.21 (m, 2H), 1.17−1.07 (m, 3H), 1.06−0.88 (m, 2H). MS (EI) for
C22H21BrF3NO4, found 501.3 (MH+). To a solution of the ester
(17.4 g, 34.8 mmol) in 300 mL of MeOH was added 100 mL of 2 N
NaOH solution, and the reaction mixture was stirred at room
temperature for 18 h. The resulting solution was concentrated under
reduced pressure, acidified with concentrated HCl to pH 3, and
extracted with 300 mL of EtOAc. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo to give 14.5 g (88%) of the
title compound which was used in the next step without further
Scheme 1
a
1
Reagents and conditions: (a) (i) N-methylpiperazine, MeCN, rt, 2 h;
purification. H NMR (400 MHz, DMSO-d6): δ 12.57 (s, 1H), 7.44
(ii) 4 M HCl in dioxane, rt, 18 h; (iii) 14b, HATU, MeCN, Hunig’s
base, rt, 18 h.
̈
(m, 2H), 7.34−7.16 (m, 4H), 7.04 (m, 2H), 6.87 (m, 1H), 4.42 (m,
1H), 3.09−2.68 (m, 2H), 1.25 (m, 2H), 1.10−0.80 (m, 2H). MS (EI)
for C20H17BrF3NO4, found 473.3 (MH+).
Compounds 16a−c were made in an analogous manner to 16d.
(S)-3-(2,4-Dichlorophenyl)-2-(1-(2,4-dichlorophenyl)-
cyclopropanecarboxamido)propanoic Acid (16a). 1H NMR
(400 MHz, DMSO-d6): δ 12.73 (s, 1H), 7.62−7.46 (m, 1H), 7.37
(m, 3H), 7.17 (m, 1H), 6.99 (d, 1H), 4.50 (m, 1H), 3.17 (m, 1H),
2.92 (m, 1H), 1.49−1.11 (m, 2H), 1.06−0.79 (m, 2H). MS (EI) for
C19H15Cl4NO3, found 448.5 (MH+).
(S)-3-(2,4-Dichlorophenyl)-2-(1-(4-(trifluoromethoxy)-
phenyl)cyclopropanecarboxamido)propanoic Acid (16b). 1H
NMR (400 MHz, DMSO-d6): δ 12.65 (s, 1H), 7.52 (m, 1H), 7.45−
7.28 (m, 5H), 7.16 (m, 2H), 4.70−4.35 (m, 1H), 3.25−2.83 (m, 2H),
1.33−1.08 (m, 2H), 1.08−0.70 (m, 2H). MS (EI) for
C20H16Cl2F3NO4, found 461.9 (MH+).
The resulting solution was concentrated in vacuo to give 27.0 g (90%)
of the title compound as the HCl salt which was used in the next step
without further purification. H NMR (400 MHz, DMSO-d6): δ 8.76
1
(s, 3H), 7.53 (m, 2H), 7.24 (m, 2H), 4.23 (m, 1H), 4.16−4.00 (m,
2H), 3.15 (m, 2H), 1.12 (m, 3H). MS (EI) for C11H14BrNO2, found
273.1 (MH+).
(S)-3-(4-Bromophenyl)-2-(1-(4-(trifluoromethoxy)phenyl)-
cyclopropanecarboxamido)propanoic Acid (16d). To a solution
of (S)-ethyl 2-amino-3-(4-bromophenyl)propanoate (15b) (11.5 g,
42.26 mmol) in acetonitrile (150.0 mL) were added 1-(4-
(trifluoromethoxy)phenyl)cyclopropanecarboxylic acid (14a) (10.4 g,
42.26 mmol), Hunig’s base (13.9 mL, 84.52 mmol), and HATU
̈
(19.3 g, 50.71 mmol). The mixture was stirred at room temperature
for 1 h. The resulting solution was concentrated in vacuo, dissolved
in dichloromethane (350 mL), and extracted with water (350 mL)
2 times. The layers were separated, and the organic layer was dried
over MgSO4. The resulting solution was concentrated in vacuo and
chromatographed on silica gel, eluting with 15% EtOAc in hexane
to give 17.4 g (82%) of (S)-ethyl 3-(4-bromophenyl)-2-(1-(4-tri-
fluoromethoxy)phenyl)cyclopropanecarboxamido)propanoate. 1H
(S)-3-(4-Bromophenyl)-2-(1-(2,4-dichlorophenyl)-
cyclopropanecarboxamido)propanoic Acid (16c). 1H NMR
(400 MHz, DMSO-d6): δ 7.60−7.52 (m, 1H), 7.44−7.31 (m, 4H),
7.03 (m, 2H), 6.73 (d, 1H), 4.38 (m, 1H), 3.05−2.75 (m, 2H), 1.42−
1.29 (m, 2H), 1.07−0.83 (m, 2H).
1-(2,4-Dichlorophenyl)-N-[(1S)-2-(4-methylpiperazin-1-yl)-2-
oxo-1-(phenylmethyl)ethyl]cyclopropanecarboxamide (17).
To a solution of 10a (2.7 g, 10 mmol) dissolved in dichloromethane
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a
Scheme 2
a
Reagents and conditions: (a) (i) N-methylpiperazine, EDC, HOBt, N-methylmorpholine, CH2Cl2, rt, 18 h; (ii) 4 M HCl in dioxane;
(b) N-methylpiperazine, EDC, HOBt, N-methylmorpholine, CH2Cl2, rt, 18 h; (c) NaH, 1,2-dibromoethane, THF, 0 °C to rt, 18 h; (d) NaOH,
water, 120 °C, 48 h; (e) (i) EtOH, EDC, HOBt, N-methylmorpholine, rt, 18 h; (ii) 4 M HCl in dioxane or SOCl2, EtOH, 90 °C, 18 h; (f) EDC,
HOBt, N-methylmorpholine, CH Cl , rt, 18 h or HATU, Hunig’s base, MeCN, rt, 1 h; (g) 2 M NaOH, MeOH, rt, 18 h; (h) (i) amine, EDC, HOBt,
̈
2
2
N-methylmorpholine, CH2Cl2, rt, 18 h; (ii) 4 M HCl in dioxane.
(50 mL) were added 1-hydroxybenzotriazole (1.8 g, 13.7 mmol),
369 mg (8%) of the title compound. 1H NMR (400 MHz, DMSO-d6):
δ 7.51 (s, 1H), 7.44−7.34 (m, 2H), 7.30−7.20 (m, 3H), 7.13−7.08 (m,
1H), 6.57 (m, 1H), 5.09−5.00 (m, 1H), 3.64−3.48 (m, 3H), 2.88 (m,
2H), 2.65−2.47 (m, 3H), 2.23−2.15 (m, 1H), 1.63−1.52 (m, 2H),
1.18−1.04 (m, 2H). MS (EI) for C24H27Cl2N3O2, found 461.9 (MH+).
Compounds 18−20 were made in an analogous manner to
compound 17.
1-(2,4-Dichlorophenyl)-N-[(1S)-1-[(2,4-dichlorophenyl)-
methyl]-2-(4-methylpiperazin-1-yl)-2-oxoethyl]-
cyclopropanecarboxamide (18). 1H NMR (400 MHz, CD3OD): δ
7.51 (m, 1H), 7.48−7.35 (m, 3H), 7.35 (m, 1H), 7.15 (m, 1H), 5.18
(m, 1H), 3.78−3.63 (m, 2H), 3.58−3.44 (m, 2H), 3.05 (m, 1H), 2.96
(m, 1H), 2.78−2.55 (m, 4H), 1.58−1.43 (m, 2H), 1.13−1.10 (m, 2H).
MS (EI) for C24H25Cl4N3O2, found 530 (MH+).
Hunig’s base (2 mL, 12.2 mmol), and 1-[3-(dimethylamino)propyl]-3-
̈
ethylcarbodiimide hydrochloride (2.3 g, 12.2 mmol). After the mixture
was stirred for 10 min, N-methylpiperazine (1.2 mL, 11 mmol) was
added. The resulting solution was stirred for 18 h and was then diluted
with water and dichloromethane. The organic layer was separated and
dried over MgSO4. The solvent was removed, and the residue was
dissolved in EtOAc (50 mL). To this solution was added 4 M HCl
in dioxane (50 mL). The resulting solution was stirred at room
temperature for an additional 18 h. The solvents were removed, and
the residue was used without purification. Then 100 mg (0.3 mmol) of
this residue was dissolved in DMF (2 mL). To this solution were
added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlor-
ide (86 mg, 0.5 mmol), 1-hydroxybenzotriazole (69 g, 0.5 mmol),
Hunig’s base (0.5 mL, 3.1 mmol), and 14a (70 mg, 0.3 mmol). The
̈
N-[(1S)-1-[(4-Chlorophenyl)methyl]-2-(4-methylpiperazin-1-
y l ) - 2 - o x o e t h y l ] - 1 - ( 2 , 4 - d i c h l o r o p h e n y l ) -
cyclopropanecarboxamide (19). 1H NMR (400 MHz, DMSO-d6):
δ 0.7.63 (s, 1H), 7.40 (s, 2H), 7.26 (m, 2H), 7.09 (m, 2H), 6.83
(m, 1H), 4.87 (m, 1H), 3.46−3.25 (m, 4H), 2.86−2.73 (m, 2H),
reaction mixture was stirred at room temperature for 18 h. The
resulting solution was extracted with water (25 mL), saturated sodium
bicarbonate (25 mL), and 1 M HCl (25 mL). The organic layer
was separated and dried over MgSO4. The resulting solution was
concentrated in vacuo and purified by preparative HPLC to provide
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2.19−2.02 (m, 7H), 1.42−1.29 (m, 2H), 0.98−0.89 (m, 2H). MS (EI)
for C24H26Cl3N3O2, found 495.8 (MH+).
Compounds 21−23 and 27−31 were made in an analogous manner
to compound 32.
N-[(1S)-1-[(4-Bromophenyl)methyl]-2-(4-methylpiperazin-1-
y l ) - 2 - o x o e t h y l ] - 1 - ( 2 , 4 - d i c h l o r o p h e n y l ) -
cyclopropanecarboxamide (20). 1H NMR (400 MHz, DMSO-d6):
δ 7.63 (s, 1H), 7.43 (m, 4H), 7.05 (m, 2H), 6.86 (m, 1H), 4.90
(m, 1H), 3.46−3.33 (m, 4H), 2.86−2.73 (m, 2H), 2.24−2.10 (m, 7H),
1.44−1.34 (m, 2H), 1.01−0.96 (m, 2H). MS (EI) for
C24H26BrCl2N3O2, found 540.3 (MH+).
1-(2,4-Dichlorophenyl)-N-{(1S)-1-[(2,4-dichlorophenyl)-
m e t h y l ] - 2 - o x o - 2 - p i p e r a z i n - 1 - y l e t h y l } -
cyclopropanecarboxamide (21). 1H NMR (400 MHz, DMSO-d6):
δ 8.24 (s, 1H), 7.62 (m, 1H), 7.53 (m, 1H), 7.45−7.40 (m, 1H), 7.37−
7.35 (m, 1H), 7.23 (m, 1H), 6.94 (m, 1H), 5.04−4.98 (m, 1H), 3.43−
3.29 (m, 5H), 2.97−2.84 (m, 2H), 2.67−2.51 (m, 4H), 1.41−1.36 (m,
1H), 1.21−1.18 (m, 1H), 0.97−0.90 (m, 2H). MS (EI) for
C23H23Cl4N3O2, found 516.1 (MH+).
1-(2,4-Dichlorophenyl)-N-{(1S)-1-[(2,4-dichlorophenyl)-
methyl]-2-[(3R)-3-methylpiperazin-1-yl]-2-oxoethyl}-
cyclopropanecarboxamide (22). 1H NMR (400 MHz, CD3OD): δ
8.43 (s, 1H), 7.50−7.38 (m, 4H), 7.25−7.15 (m, 2H), 7.20−7.15 (m,
1H), 4.35 (m, 1H), 4.00 (m, 1H), 3.25−2.80 (m, 7H), 1.57−1.43 (m,
2H), 1.20−1.05 (m, 2H). MS (EI) for C24H25Cl4N3O2, found 529.9
(MH+).
1-(2,4-Dichlorophenyl)-N-{(1S)-1-[(2,4-dichlorophenyl)-
methyl]-2-[(3S)-3-methylpiperazin-1-yl]-2-oxoethyl}-
cyclopropanecarboxamide (23). 1H NMR (400 MHz, CD3OD): δ
8.42 (s, 1H), 7.53−7.39 (m, 4H), 7.33−7.13 (m, 2H), 5.20−5.15 (m,
1H), 4.40 (m, 1H), 4.02 (m, 1H), 3.25−2.84 (m, 6H), 2.80−2.58 (m,
1H), 1.55−1.40 (m, 2H), 1.23−1.00 (m, 5H). MS (EI) for
C24H25Cl4N3O2, found 530.1 (MH+).
4-Bromo-N-α-{[1-(2,4-dichlorophenyl)cyclopropyl]-
carbonyl}-N-[2-(dimethylamino)ethyl]-N-methyl-L-phenylalani-
namide (25). To a solution of (S)-3-(4-bromophenyl)-2-(1-(2,4-
dichlorophenyl)cyclopropanecarboxamido)propanoic acid (16c) (87
mg, 0.19 mmol) in dichloromethane (5 mL) were added 1-hydro-
xybenzotriazole (25 mg, 0.19 mmol), N-methylmorpholine (0.172 mL,
1.57 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (36 mg, 0.19 mmol), and N1,N1-dimethylethane-1,2-diamine
(16 mg, 0.19 mmol, TCI America). The mixture was stirred at room
temperature for 18 h. The resulting solution was washed with water
(25 mL) and saturated sodium bicarbonate (25 mL). The layers were
separated, and the organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The product was then purified by preparative
HPLC (reverse-phase, acetonitrile/aqueous 10 mM formic acid buffer)
1
to give 41 mg (40%) of the title compound. H NMR (400 MHz,
DMSO-d6): δ 8.16 (s, 1H), 7.60 (m, 1H), 7.46−7.34 (m, 4H), 7.08−
6.98 (m, 2H), 4.84 (m, 1H), 3.45 (m, 1H), 3.25−3.08 (m, 1H), 2.93
(s, 2H), 2.85−2.70 (m, 3H), 2.37−2.20 (m, 2H), 2.21−2.08 (m, 6H),
1.43−1.29 (m, 2H), 1.03−0.88 (m, 2H). MS (EI) for
C24H28BrCl2N3O2, found 542.3 (MH+).
N-{(1S)-2-(4-Aminopiperidin-1-yl)-1-[(4-bromophenyl)-
m e t h y l ] - 2 - o x o e t h y l } - 1 - ( 2 , 4 - d i c h l o r o p h e n y l ) -
cyclopropanecarboxamide (27). 1H NMR (400 MHz, CD3OD): δ
8.37 (s, 1H), 7.58 (d, 1H), 7.40−7.35 (m, 3H), 7.00−6.90 (m, 2H),
4.85−4.80 (m, 1H), 4.18−4.05 (m, 2H), 3.86−3.78 (m, 2H), 3.05−
2.85 (m, 2H), 2.80−2.68 (m, 3H), 1.80−1.73 (m, 2H), 1.37−1.00 (m,
3H), 0.95−0.86 (m, 2H). MS (EI) for C24H26BrCl2N3O2, found 540.1
(MH+).
Compounds 24 and 26 were made in an analogous manner to
compound 25.
2,4-Dichloro-N-α-{[1-(2,4-dichlorophenyl)cyclopropyl]-
N-(3-Aminocyclohexyl)-2,4-dichloro-N-α-{[1-(2,4-
carbonyl}-N-[2-(dimethylamino)ethyl]-N-methyl-L-phenylalani-
1
dichlorophenyl)cyclopropyl]carbonyl}-L-phenylalaninamide
namide (24). H NMR (400 MHz, DMSO-d6): δ 8.18 (s, 1H), 7.58
1
(28). H NMR (400 MHz, CD3OD): δ 8.52 (s, 1H), 7.50−7.38 (m,
(m, 1H), 7.51 (m, 1H), 7.42 (m, 2H), 7.33 (m, 1H), 7.23 (m, 1H),
4.99 (m, 1H), 3.40 (m, 1H), 3.23 (m, 1H), 2.95 (s, 3H), 2.86 (m, 2H),
2.27 (m, 2H), 2.13 (s, 6H), 1.37 (m, 1H), 1.14 (m, 1H), 0.96 (m, 1H),
0.89 (m, 1H). MS (EI) for C24H27Cl4N3O2, found 532.4 (MH+).
N-[(1S)-1-[(4-Bromophenyl)methyl]-2-(4-hydroxypiperidin-
1 - y l ) - 2 - o x o e t h y l ] - 1 - ( 2 , 4 - d i c h l o r o p h e n y l ) -
cyclopropanecarboxamide (26). 1H NMR (400 MHz, DMSO-d6):
δ 7.61 (m, 1H), 7.40 (m, 4H), 7.01 (m, 2H), 6.75 (m, 1H), 4.88 (m,
1H), 4.74 (m, 1H), 3.86 (m, 1H), 3.84−3.56 (m, 4H), 3.19−2.63 (m,
2H), 1.60 (m, 2H), 1.40−1.11 (m, 7H), 0.96 (m, 2H). MS (EI) for
C24H25BrCl2N2O3, found 541.0 (MH+).
N-[(1S)-2-(3-Amino-8-azabicyclo[3.2.1]oct-8-yl)-1-[(4-
bromophenyl)methyl]-2-oxoethyl]-1-[4-[(trifluoromethyl)oxy]-
phenyl]cyclopropanecarboxamide (32). To a solution of (S)-3-
(4-bromophenyl)-2-(1-(4-(trifluoromethoxy)phenyl)-
cyclopropanecarboxamido)propanoic acid (16d) (100 mg, 0.2 mmol)
in acetonitrile (5.0 mL) were added tert-butyl 8-azabicyclo[3.2.1]-
4H), 7.23−7.15 (m, 2H), 4.82−4.75 (m, 1H), 3.67−3.58 (m, 1H),
2.20−2.13 (m, 1H), 2.02−1.83 (m, 3H), 1.77−1.40 (m, 5H), 1.25−
1.00 (m, 3H). MS (EI) for C24H27Cl4N3O2, found 544.2 (MH+).
N-{(1S)-2-(3-Amino-8-azabicyclo[3.2.1]oct-8-yl)-1-[(2,4-
dichlorophenyl)methyl]-2-oxoethyl}-1-(2,4-dichlorophenyl)-
cyclopropanecarboxamide (29). 1H NMR (400 MHz, CD3OD): δ
7.50−7.39 (m, 4H), 7.28−7.12 (m, 2H), 4.80−4.60 (m, 1H), 4.44−
4.27 (m, 1H), 3.25−3.08 (m, 1H), 3.02−2.95 (m, 2H), 2.63−2.58 (m,
1H), 2.23−2.00 (m, 1H), 1.83−1.72 (m, 3H), 1.62−1.40 (m, 4H),
1.20−1.00 (m, 2H). MS (EI) for C26H27Cl4N3O2, found 555.9 (M+).
N-{(1S)-2-(3-Amino-8-azabicyclo[3.2.1]oct-8-yl)-1-[(2,4-
dichlorophenyl)methyl]-2-oxoethyl}-1-{4-[(trifluoromethyl)-
oxy]phenyl}cyclopropanecarboxamide (30). 1H NMR (400
MHz, DMSO-d6): δ 7.54 (m, 1H), 7.35 (m, 5H), 7.23 (m, 1H),
7.04 (m, 1H), 4.79 (m, 1H), 4.28 (m, 2H), 3.12−2.94 (m, 1H), 2.87
(m, 2H), 2.12−1.87 (m, 3H), 1.88−1.70 (m, 1H), 1.60 (m, 2H), 1.47
(m, 2H), 1.30−1.09 (m, 2H), 0.94 (m, 2H). MS (EI) for
C27H28Cl2F3N3O3, found 571.4 (MH+).
octan-3-ylcarbamate (Oakwood, 45.2 mg, 0.2 mmol), Hunig’s base
̈
(1.1 mL, 6.1 mmol), and HATU (76 mg, 0.2 mmol). The mixture was
stirred at room temperature for 1 h. The resulting solution was
concentrated in vacuo. The residue was dissolved in dichloromethane
(300 mL). This organic solution was extracted with water (10 mL),
saturated sodium bicarbonate (10 mL), and saturated sodium chloride
(10 mL). The layers were separated, and the organic layer was dried
over MgSO4, filtered, and concentrated in vacuo. The resulting oil was
dissolved in methanol (10 mL), and 4 M HCl in dioxane (5 mL,
Aldrich) was added. The mixture was heated to 45 °C for 2 h. The
resulting solution was concentrated in vacuo. The product was purified
by preparative HPLC (reverse-phase, acetonitrile/water with 0.1%
formic acid) to give 20 mg (17%) of the title compound. Analytical
HPLC: YMC Pack Pro 150 mm × 4.6 mm, 5 μm C18 column; 10−
90% MeCN (0.1% TFA) in H2O (0.1% TFA); flow rate 1.5 mL/min
N-{(1S)-2-(3-Amino-8-azabicyclo[3.2.1]oct-8-yl)-1-[(4-
bromophenyl)methyl]-2-oxoethyl}-1-(2,4-dichlorophenyl)-
cyclopropanecarboxamide (31). 1H NMR (400 MHz, CD3OD): δ
8.57 (s, 1H), 7.50−7.38 (m, 3H), 7.15−7.00 (m, 3H), 4.84−4.80 (m,
1H), 3.63−3.43 (m, 2H), 4.18 (m, 1H), 3.23−3.18 (m, 1H), 3.19−2.99
(m, 2H), 2.83−2.78 (m, 3H), 2.58−2.50 (m, 1H), 2.39−2.30 (m, 1H),
2.16−2.03 (m, 1H), 1.98−1.80 (m, 1H), 1.60−1.40 (m, 5H), 1.20−
1.05 (m, 2H). MS (EI) for C26H28BrCl2N3O2, found 565.0 (MH+).
Biochemical Assay. The primary biochemical assay was a coupled
experiment in which GCS activity was measured as the amount
of UDP-glucose consumed during the synthase-catalyzed reaction.
Upon quenching, remaining UDP-glucose was processed by UDP-
glucose dehydrogenase, generating NADH. The NADH in turn
participated in the diaphorase mediated reduction of resazurin to
fluorescent resorufin. The more UDP-glucose remaining at the end
of the reaction (i.e., the better the inhibitor), the more fluorescence
was measured. Reactions were conducted in 384-well black, medium
binding microtiter plates (Greiner). Synthase reaction mixtures
1
× 25 min; retention time of 14.37 min at 254 nm. H NMR (400
MHz, DMSO-d6): δ 7.42 (m, 2H), 7.31 (m, 4H), 7.03(m, 3H), 4.67
(m, 1H), 4.27 (m, 2H), 3.10 (m, 1H), 2.78 (m, 2H), 1.81 (m, 8H),
1.22 (m, 2H), 0.98 (m, 2H). MS (EI) for C27H29BrF3N3O3, found
581.4 (MH+).
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Journal of Medicinal Chemistry
Article
(25 mM HEPES, pH 7.4, 50 mM KCl and 5 mM MgCl2) were
prepared by first adding 0.0005 mL of compound in 100% DMSO to
the plate. A volume V = 0.010 mL of 30 mol % C6-ceramide (Avanti
Polar Lipids, Inc.) and 70 mol % dioleoylphosphatidylcholine (DOPC,
Avanti Polar Lipids) with total concentration of 0.3 mM in 25 mM
HEPES, pH 7.4, was added to the wells.23 Full-activity controls (100%
activity) contained DMSO only. No-activity control reactions (0%
control) contained 0.010 mL of 100 mol % DOPC (0.3 mM) and
DMSO only. The reactions were initiated by the addition of V =
0.020 mL of 0.020 mM UDP-glucose, 1.5 mg/mL CHAPS, 0.17 mg/
mL GCS in 25 mM HEPES, pH 7.4, 75 mM KCl, and 7.5 mM MgCl2
to all wells of the plate. The plate was kept at 26 °C for 3 h. The UDP-
glucose that remains was measured by the addition of 0.015 mL of
0.30 mM NAD+, 0.30 mM resazurin, 0.11 mg/mL UDP-glucose
dehydrogenase and 0.030 mg/mL diaphorase in 25 mM HEPES, pH
7.4, 50 mM KCl, and 5 mM MgCl2. The plate was kept at 26 °C for
30 min or longer, and the increase in fluorescence with excitation at
530 nm and emission at 560 nm was measured with an Envision 1
plate reader (Perkin-Elmer). Consumption of UDP-glucose was
limited to 40−60% of the total UDP-glucose in the assay.
IC50 values were calculated by nonlinear regression analysis using
the four-parameter equation [Y = max − (max − min)/(1 + (X/
IC50)N)] where Y is the observed fluorescence, X is the concentration
of inhibitor, max is the fluorescence in the absence of C6-ceramide
(0% control), min is the fluorescence in the absence of inhibitor
(100% control), IC50 is the concentration of inhibitor that gave 50%
increase in fluorescence, and N is the empirical Hill slope. N should
approximate unity. Curve fitting was performed using commercial
software (IDBS ActivityBase XE).
Cellular Assay. The principal cellular readout used was the quantita-
tion of ganglioside GM1 on the surface of A549 cells. Decreased
glucosylceramide synthesis would lead to decreased GM1 synthesis.
A549 cells were seeded in a 96-well plate at a density of 1000 cells/well
in complete medium for 24 h at 37 °C. The cells were then treated with
compounds for 72 h in the presence of 10% serum. On day 3, the cells
were stained for 30 min at 4 °C with a fluorescent cholera toxin subunit
B (FL-CTB) that binds to the ganglioside GM1 on the plasma
membrane. The cells were then fixed with 3.7% formaldehyde prior to
the addition of Hoechst stain. The plate was then read on an automated
fluorescent microscope (ArrayScan from Cellomics) using a detection
software algorithm (Spot Detector) to quantify the amount of
gangliosides on the plasma membrane. The total intensity from the
binding of FL-CTB to the membrane was normalized to the amount of
cells in each well.
Microsomal Stability Assay. Microsomal oxidation of com-
pounds was performed in the presence of mouse liver microsomes.
In 96-well microtiter plates were added test compound (15 μM),
microsomal protein (0.5 mg/mL), 1 mM NADPH in 100 mM
potassium phosphate, pH 7.4 buffer, and 0.15% DMSO to a total
volume of 100 μL/well. The mixtures were incubated at 37 °C for
30 min before being terminated by the addition of 50 μL of
acetonitrile containing 20% acetic acid. Test compound concentrations
were measured by LC/MS/MS and compared to no-NADPH control
reactions to measure the extent of metabolism.
being separated into dose groups by body weight (n = 4 animasl/
group). Six-week-old ZDF rats (Charles River) were fed a high fat diet
(Purina 5008) and separated into groups of 8. The animals had ad
libitum access to food and water except for the ZDF animals in the
pair-fed study. In that study, two control groups were pair-fed to
match ad libitum fed 32 or vehicle treated groups. On dose days 14
and 28, the animals were fasted for 5 h prior to bolus oral
administration of glucose (2 g/kg), which was followed by a glucose
tolerance test (0−120 min after glucose). At the end of the studies,
animals were again fasted for 5 h prior to euthanasia. In the DIO
studies, the mice were dosed with insulin before sacrifice in order to
measure IR function. Plasma, liver, muscle, and white adipose tissue
(WAT) were prepared and collected for PK, glycosphingolipid, insulin,
glucose, and triglyceride analysis. Liver samples were also prepared for
histopathology.
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-650-837-7018. Fax: +1-650-837-8177. E-mail: sjr.
researcher@gmail.com.
■
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to Sean Wu, Jing Wang, Eric Brooks,
Lory Tan, and John Bui for collecting the reported ADME data.
We also acknowledge our colleagues in the compound
repository, Eliana Bustamante, Dorothy Trent, Melanie
Dimapasoc, Yelena Zherebina, Shaun Nguyen, and Josh Rulloda,
for their support of this project. Thanks are extended to Chip
Blazey, Jason Harris, and Henry Johnson for their review of this
manuscript. Finally, we thank Tom von Geldern and Keith
Woerpel for their guidance during many helpful discussions.
ABBREVIATIONS USED
■
GCS, glucosylceramide synthase; Cer, ceramide; GluCer,
glucosylceramide; LacCer, lactosylceramide; GM3, ganglioside
containing glucose, lactose, and sialic acid moieties; DIO, diet
induced obese; ZDF, Zucker diabetic fatty; mpk, milligram of
compound per kilogram of animal body weight; IR, insulin
receptor; HbA1c, a measure of glycated hemoglobin; CYP3A4,
cytochrome P450 isoform 3A4; WAT, white adipose tissue;
TG, triglyceride; OGTT, oral glucose tolerance test; IGTT,
intraperitoneal glucose tolerance test; SRT, substrate reduction
therapy; ERT, enzyme replacement therapy; HATU, O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate
CYP Inhibition Assay. This assay was performed using pooled
human liver microsomes and a cocktail of CYP substrates.24
Compounds were incubated for 45 min with human liver microsomes
(0.25 mg/mL) in the presence of NADPH (1 mmol) and substrates
(20 μM). Changes in testosterone metabolism were used to measure
compound inhibition of CYP3A4.
PD Experiment. C57Bl/6 mice were purchased from Taconic.
Sprague−Dawley rats were purchased from Charles River Laborato-
ries. The animals were fed normal chow (Purina 5001 or Purina 5010).
Animals were dosed q.d. by oral gavage with study compound or
vehicle. At the end of study, the animals were sacrificed by
exsanguination. Liver and blood were collected for sphingolipid and
glycosphongolipid analysis. The analysis was performed in a manner
similar to what has been reported.25
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