Synthesis, in vitro covalent binding evaluation, and metabolism of 14C-labeled inhibitors of 11β-hsd1

Article abstract of DOI:10.1021/ml500331y

In this letter, we reported the design and synthesis of three potent, selective, and orally bioavailable 11β-HSD1 inhibitors labeled with ¹⁴C: AMG 456 (1), AM-6949 (2), and AM-7715 (3). We evaluated the covalent protein binding of the labeled i

Full text of DOI:10.1021/ml500331y

Letter
pubs.acs.org/acsmedchemlett
Synthesis, in Vitro Covalent Binding Evaluation, and Metabolism of
14C‑Labeled Inhibitors of 11β-HSD1
Daqing Sun,*,† Qiuping Ye,*,§ Xuelei Yan,† Yosup Rew,† Peter Fan,§ Xiao He,† Min Jiang,§
Dustin L. McMinn,† Mario Monshouwer,§ Hua Tu,‡ and Jay P. Powers†
‡
§
†Departments of Therapeutic Discovery, Metabolic Disorders, and Pharmacokinetics and Drug Metabolism, Amgen, Inc., 1120
Veterans Boulevard, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: In this letter, we reported the design and synthesis of three
potent, selective, and orally bioavailable 11β-HSD1 inhibitors labeled with
14C: AMG 456 (1), AM-6949 (2), and AM-7715 (3). We evaluated the
covalent protein binding of the labeled inhibitors in human liver microsomes
in vitro and assessed their potential bioactivation risk in humans. We then
studied the in vitro mechanism of 2 in human hepatocytes and the
formation of reactive intermediates. Our study results suggest that 1 and 3
have low potential for metabolic bioactivation in humans, while 2 has
relatively high risk.
KEYWORDS: 11β-HSD1, 11β-HSD2, diabetes, metabolic syndrome, hydroxysteroid dehydrogenase, arylsulfonylpiperazine,
diarylsulfone, 4,4-disubstituted cyclohexylbenzamides, radiolabeled inhibitor, covalent protein binding, bioactiviation, reactive metabolite
efficacy observed with 1 and 2,15,16 compound 3 achieved a
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a
nice dose-dependent inhibition of 11β-HSD1 compared to the
vehicle in a cynomolgus monkey ex vivo pharmacodynamic
model19 when administered orally (Figure 2).
key enzyme that acts as an NADPH-dependent reductase
converting inactive glucocorticoids such as cortisone into their
active form (e.g., cortisol) in specific tissues including liver,
adipose, and brain.1−4 However, 11β-hydroxysteroid dehydro-
genase type 2 (11β-HSD2), an oxidase structurally related to
11β-HSD1, catalyzes the conversion of cortisol to the inactive
cortisone using NAD+ as a cofactor. Excessive levels of
glucocorticoids greatly contributes to metabolic syndrome,
including insulin resistance, visceral obesity, and type 2
diabetes.5−9 Selective inhibition of 11β-HSD1 provides a
promising therapeutic strategy for the treatment of obesity
and the other elements of the metabolic syndrome. To date,
several small molecule inhibitors of 11β-HSD1 have been
tested in the clinic for the treatment of such conditions.10−14
Previously, we reported three series of potent, selective, and
orally bioavailable 11β-HSD1 inhibitors exemplified by
arylsulfonylpiperazine 1 (AMG 456),15 diarylsulfone 2 (AM-
6949),16 and cyclohexylbenzamide 3 (AM-7715) (Figure 1).17
All three compounds inhibited 11β-HSD1 significantly in the
biochemical enzyme assay18 with an IC50 of 0.5−0.9 nM but
had no inhibition against HSD2 at a concentration of 10 μM.
These molecules were also found to be extremely potent 11β-
HSD1 inhibitors in the cell-based enzyme assays with human
HEK 293 cells and fat cells.18 Furthermore, like 1 and 2,15,16
compound 3 has a low clearance (CL = 0.12−0.41 L/h/kg) and
excellent oral bioavailability (%F = 38−88) across three
preclinical species (Table 1). Consistent with the ex vivo
Nevertheless, small molecule drugs can undergo metabolic
bioactivation.20 Reactive drug metabolites may covalently bind
to biological proteins to form a drug−protein adduct. The
bioactivation is generally considered to be related to drug
toxicity, although mechanism involved in the toxicity induced
by the adduct is largely undefined.21 In vitro covalent protein
binding (CPB) study offers direct evidence of covalent binding
of drug to proteins and is commonly used to measure
bioactivation and assess potential toxicity risk in humans.22,23
Radiolabeling a drug provides a convenient way to measure the
metabolism of a drug and to quantify the extent of covalent
binding to proteins. In this article, we describe the design and
synthesis of the labeled 11β-HSD1 inhibitors 1−3, their CPB
evaluation, and the in vitro metabolism of 2 in human
hepatocytes.
Results and Discussion. Chemistry. We recently reported
the design and synthesis of nonradiolabeled 1−3;15−17
however, those synthetic routes were not suitable for the
synthesis of radiolabeled compounds. Since carbon and
hydrogen atoms are present in all drug molecules, 14C and
Received: August 12, 2014
Accepted: September 23, 2014
Published: September 23, 2014
© 2014 American Chemical Society
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Figure 2. Compound 3 achieved a nice dose-dependent inhibition of
11β-HSD1 compared to the vehicle. *p < 0.07. Ex vivo 11β-HSD1
enzyme activity in intact mesenteric fat collected from cynomolgus
monkeys dosed orally with compound 3. Plasma and mesenteric fat
samples were collected 4 h after 3 was administered orally in dosing
vehicle (15% HPβCD in 50% Ora-plus). 11β-HSD1 enzyme activity
was measured as the [3H]-cortisol formed after 1 h incubation of fat
samples in reaction buffer containing [3H]-cortisone at 37 °C.
Compound concentration in plasma was determined by LC−MS/MS.
Average concentrations of 3 in plasma were measured to be 64 ng/mL
(1 mg/kg), 524 ng/mL (5 mg/kg), and 4320 ng/kg (25 mg/kg),
respectively.
Figure 1. Chemical structures and potencies of 1, 2, and 3. (a) IC50 in
the human 11β-HSD1 biochemical scintillation proximity assay (SPA).
(b) IC50 in the cell-based enzyme assays with human HEK 293 cells
(serum free). (c) IC50 in the cell-based enzyme assays with human
HEK 293 cells in the presence of 3% human serum albumin (HSA).
(d) IC50 in the cell-based enzyme assays with human fat cells (serum
free). (e) IC50 in the human 11β-HSD2 biochemical scintillation
proximity assay. Values of IC50 are means of at least two
determinations. Standard deviation was 30%.
a
Scheme 1. Synthesis of [14C]-1
Table 1. Preclinical Pharmacokinetics of 3
iv
Vdss
(L/kg)
T1/2
(h)
po
F (%)
Cmax
(ng/mL)
CL (L/kg/h)
ab
,
rat
0.041
0.029
0.012
2.3
1.0
1.2
43
27
78
72
38
88
1130
211
ac
,
cyno
ab
,
dog
1840
a
iv dose (0.5 mg/kg) in 10% N,N-dimethylacetamide, 10% ethanol,
b
40% propylene glycol, and 40% water. po dose (2.0 mg/kg) as a
solution in 15% HPβCD in Ora-Plus. po dose (2.0 mg/kg) as a
c
suspension in 1% Tween 80/99% and 1% methylcellulose (4000 cps)
in deionized water. Values are an average for three animals. Standard
deviation was 30%.
tritium (3H) are widely used radioisotopes for labeling drug
molecule. Because of the potential for label loss by
metabolism,24 we chose 14C over tritium as a label isotope
for the synthesis of radiolabeled compounds 1−3. The
strategies used to synthesize the radiolabeled 1−3 were (a)
selecting appropriate label positions away from the metabolic
soft spots; (b) design and synthesis of labeling precursors that
allow 14C incorporation; and (c) incorporating 14C isotope at
the end of the synthetic routes to minimize a number of
radioactive intermediates in the synthesis. On the basis of the
aforementioned strategies as well as the availabilities of 14C
labeling reagents, the label positions were designated and
shown in the structures of [14C]-1, [14C]-2, and [14C]-3,
respectively. The synthesis of labeling precursors 9, 16, and 19
is described below in Schemes 1−3.
a
Reagents and conditions: (a) 4-acetylbenzene-1-sulfonyl chloride,
triethylamine, 48%; (b) TMS−CF3, TBAF, THF, 67%; (c) MsCl,
triethylamine, CH2Cl2, 62%; (d) NaN3, DMSO, 98%; (e)
triphenylphosphine, THF, water, 79%; (f) 1-(iodomethyl)-
cyclopropanecarbonitrile, N-ethyldiisopropyl amine, acetonitrile,
87%; (g) chiral separation, then ceric ammonium nitrate (CAN),
acetonitrile, water, 15%; (h) [14C]1,2-dibromoethane, K2CO3,
acetonitrile; (i) KOH, t-BuOH, 90 °C, 23% from 9.
The synthesis of [14C]-1 and its labeling precursor 9 is
outlined in Scheme 1. Sulfonylation of 4 with 4-acetylbenze-
nesulfonyl chloride provided the sulfonamide 5. Treatment of 5
with trifluoromethyltrimethylsilane afforded the carbinol 6,
which was directly converted to the methanesulfonate 7.
Nucleophilic displacement of 7 with sodium azide, subsequent
reduction of the azide, and alkylation of the resultant amine
with 1-(iodomethyl)cyclopropane carbonitrile in acetonitrile
provided a nitrile 8 as a mixture of diastereomers at the tertiary
alcohol. The desired isomer was separated by chromatography
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a
Scheme 3. Synthesis of [14C]-3
on a chiral column, followed by removal of PMB group with
ceric ammonium nitrate (CAN), to yield the diamine (labeling
precursor 9). Finally, [14C]-1 was obtained by alkylation of the
precursor 9 with [14C]1,2-dibromoethane, followed by basic
hydrolysis of the cyano group.
The synthesis of [14C]-2 and its precursor 16 is detailed in
Scheme 2. Reaction of phenol 10 with dimethylcarbamothioic
a
Scheme 2. Synthesis of [14C]-2
a
Reagents and conditions: (a) cyclopropylamine, NaBH(OAc)3, acetic
acid, acetonitrile, 52%; (b) (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-
yl) benzoic acid, EDC, HOAt, NaHCO3, DMF, 44%; (c) [14C]
sodium cyanate, TFA, 40%.
coupled with (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-yl) ben-
zoic acid17 to afford the primary alcohol (labeling precursor
19), which was treated with [14C]sodium cyanate in the
presence of trifluoracetic acid to provide [14C]-3.
In Vitro Covalent Protein Binding (CPB) Evaluation of
[14C]-1, [14C]-2, and [14C]-3 in Human Liver Microsomes. To
measure drug bioactivation, an in vitro CPB study of 1−3 in
human liver microsomes was carried out by incubating 10 μM
of the labeled test article in the (−) or (+) NADPH-
regenerating system and in the presence of both 1 mM
NADPH and 1 mM glutathione for 1 h.25 In the study,
imipramine or naphthalene was used as a positive control and
tested along with compounds [14C]-1, [14C]-2, and [14C]-3.
CPB of the labeled inhibitors or positive controls in the (−)
NADPH was measured to be low in all cases (Figure 3),
showing that their CPB contained trace level of NADPH-
independent processes. In the presence of NADPH, the CPB of
1, 2, and 3 to human microsomal protein was determined to be
70, 370, and 15 pmol drug equiv/mg protein, respectively.
Imipramine had moderate CPB of 124.5 pmol drug equiv/mg
protein, while naphthalene showed relatively high CPB of 420
pmol drug equiv/mg protein, which are in the line with the data
reported in the literature.26 Both compounds 1 and 3 have a
considerably low level of CPB compared to imipramine and
naphthalene, respectively (70 pmol drug equiv/mg protein of 1
vs 125 pmol drug equiv/mg protein of imipramine in Figure 3a;
15 pmol drug equiv/mg protein of 3 vs 420 pmol drug equiv/
mg protein of naphthalene in Figure 3c). Moreover, the CPB of
1 and 3 in human microsomes is also lower than or comparable
to a proposed conservative threshold value of 50 pmol drug
equiv/mg protein, an upper limit considered to be low risk by
Evans et al.22 These findings suggest that both 1 and 3 have low
potential bioactivation risk in humans. In contrast, compound 2
exhibited a high level of CPB relative to imipramine (350 and
a
Reagents and conditions: (a) dimethylcarbamothioic chloride, 1,4-
diazabicyclo[2.2.2]octane (DABCO), DMF, 65 °C; 83%; (b) neat, 218
°C, 99%; (c) TMS−CF3, TBAF, 94%; (d) (i) 4-nitrochloroformate,
DMAP, CH3CN; (ii) (S)-1-(naphthalen-1-yl)ethanamine, crystalliza-
tion, 31%; (f) LiOH, 1,4-dioxane, H2O, 100%; (g) 2-chloro-1,4-
difluorobenzene, Cs2CO3, DMF, 110 °C, 74%; (h) m-CPBA, 82%; (i)
[14C]-KCN, 18-crown-6, DMF, 40%.
chloride in the presence of 1,4-diazabicyclo[2.2.2]octane
(DABCO) produced the carbamothioate 11, which was
transformed into the intermediate 12 by heating. Reaction of
12 with trifluoromethyltrimethylsilane in THF generated the
carbinol 13, which was successively treated with 4-nitro-
chloroformate and (S)-1-(naphthalen-1-yl)ethanamine to deliv-
er a carbamate as a mixture of the two diastereoisomers at the
tertiary alcohol. The desired isomer 14 was separated by
fractional crystallization and hydrolyzed with lithium hydroxide
to give the thiophenol 15. The coupling of thiophenol 15 to 2-
chloro-1,4-difluorobenzene in the presence of cesium carbonate
yielded diphenylsulfide (labeling precursor 16), which was
subjected to m-CPBA oxidation to afford a sulfone
intermediate. The preparation of [14C]-2 was achieved by
replacement of the fluorine group in the sulfonyl benzene ring
with [14C]potassium cyanide.
The synthesis of [14C]-3 and its precursor 19 is described in
Scheme 3. Reductive amination of 1717 with cyclopropylamine
resulted in a 1:1.1 mixture of cis- and trans-cyclopropylamine
intermediates, and the desired trans isomer 18 was separated by
silica-gel chromatography. The trans-amine 18 was then
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parent and its metabolites using tandem LC−MS/MS radio-
metric detection. M2 was identified as the major metabolite
(5.3% of administered dose) along with M3 (1.3% of
administered dose) and an O-glucuronide M1 (3.2% of
administered dose) (Figure 4). In this study, a 9.8% total
Figure 4. Major metabolites of [14C]-2 following incubation with
human hepatocytes. Structures shown in bracket represent proposed
reactive intermediates, which were not isolated.
Figure 3. In vitro covalent binding evaluation of radioactivity to
human liver microsomes upon incubation with [14C]-labeled inhibitors
at 10 μM concentration for 1 h. (a) [14C]-1 was incubated with human
liver microsomes in the (−) or (+) of NADPH, and in the presence of
both 1 mM NADPH and 1 mM glutathione, [14C] imipramine was
used as a positive control. (b) [14C]-2 was incubated with human liver
microsomes in the (−) or (+) of NADPH, and in the presence of both
1 mM NADPH and 1 mM glutathione, [14C] imipramine was used as
a positive control. (c) [14C]-3 was incubated with human liver
microsomes in the (−) or (+) of an NADPH, and in the presence of
both 1 mM NADPH and 1 mM glutathione, [14C] naphthalene was
used as a positive control. CPB values are means of at least two
determinations.
turnover was observed upon incubation of 2 with human
hepatocytes. Both metabolites M2 and M3 were isolated after
incubation of 2 with human microsomes for 2 h. The chemical
structures of M2 and M3 were resolved by comparing their
radiochromatographic retention times, mass spectra, NMR
proton chemical shift, and splitting patterns with those of 2
(see the Supporting Information). The reaction phenotyping
study indicated that CYP3A4-mediated oxidation of 2
generated the highly electrophilic epoxides shown in brackets,
which proceed by a 1,2-hydride shift followed by isomerization,
leading to the hydroxyl metabolites M2 and M3. These data
clearly indicated that 2 might cause metabolic activation in
humans by the formation of reactive epoxides, which covalently
modify proteins.
Conclusions. In summary, we successfully developed the
synthetic routes to three potent [14C]-labeled 11β-HSD1
inhibitors and used them for in vitro human microsomal
protein covalent binding assessment. The covalent binding data
suggest that both 1 and 3 have low potential for metabolic
bioactivation in humans, while 2 has a relatively high risk for
the associated liabilities of drug bioactivation. Furthermore, the
metabolism of 2 in human hepatocytes in vitro was
125 pmol drug equiv/mg protein for 2 and imipramine,
respectively), indicating a high potential bioactivation risk of 2
in humans (Figure 3b). Furthermore, formation of chemically
reactive intermediates was confirmed by a glutathione (GSH)
trapping study. The high CPB of 2 in human microsomes was
significantly reduced to 20 pmol drug equiv/mg protein by
adding a natural trapping agent GSH (Figure 3b).
In Vitro Metabolism of [14C]-2 in Human Hepatocyte
Incubation. To identify the possible reactive intermediate, we
decided to investigate the metabolic mechanism of 2 in human
hepatocytes. After incubation of [14C]-2 with human
hepatocytes, radiolabeled components were analyzed for the
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ASSOCIATED CONTENT
* Supporting Information
■
S
(i) In vitro biological assays. (ii) Cynomolgus monkey tissue ex
vivo protocol. (iii) Covalent binding assays. (iv) Metabolite ID.
(v) Synthesis and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(D.S.) Phone: 650-244-2195. Fax: 650-837-9369. E-mail:
daqings@amgen.com.
■
*(Q.Y.) Phone: (650) 244-2327. E-mail: qye@amgen.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Tim Carlson for helpful discussions.
■
ABBREVIATIONS
■
AcCN, acetonitrile; CAN, ceric ammonium nitrate; CL,
clearance; m-CPBA, m-chloroperoxybenzoic acid; CYP3A4,
cytochrome P450 3A4; DABCO, 1,4-diazabicyclo[2.2.2]octane;
DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylforma-
mide; DMSO, dimethyl sulfoxide;; EDC, N-ethyl-N′-(3-
(dimethylamino)propyl)carbodiimide; EtOAc, ethyl acetate;
HPβCD, hydroxypropyl-beta-cyclodextrin; MsCl, methanesul-
fonyl chloride; MTBE, methyl t-butyl ether; TBAF, tetrabuty-
lammonium fluoride; TEA, triethylamine; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TMS-CF3, trimethyl-
(trifloromethyl)silane
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