Synthesis and structure-activity relationship for a novel class of potent and selective carbamate-based inhibitors of hormone selective lipase with acute in vivo antilipolytic effects

Article abstract of DOI:10.1021/jm0607653

Hormone-sensitive lipase (HSL) is an intracellular enzyme that has a central role in the regulation of fatty acid metabolism. The enzyme, therefore, is a potentially interesting pharmacological target for the treatment of insulin resistance and dyslipidem

Full text of DOI:10.1021/jm0607653

J. Med. Chem. 2007, 50, 5449-5456
5449
Synthesis and Structure-Activity Relationship for a Novel Class of Potent and Selective
Carbamate-Based Inhibitors of Hormone Selective Lipase with Acute In Vivo Antilipolytic
Effects
Søren Ebdrup,* Hanne Hoffmann Frølund Refsgaard, Christian Fledelius, and Poul Jacobsen
NoVo Nordisk A/S, NoVo Nordisk Park, 2760 MåløV, Denmark
ReceiVed June 28, 2006
Hormone-sensitive lipase (HSL) is an intracellular enzyme that has a central role in the regulation of fatty
acid metabolism. The enzyme, therefore, is a potentially interesting pharmacological target for the treatment
of insulin resistance and dyslipidemic disorders. Based on a high throughput screening, a carbamate based
HSL inhibitor was identified and optimized into the selective HSL inhibitors 4-hydroxymethyl-piperidine-
1-carboxylic acid 4-(5-trifluoromethylpyridin-2-yloxy)-phenyl ester (13f) and 4-hydroxy-piperidine-1-
carboxylic acid 4-(5-trifluoromethylpyridin-2-yloxy)-phenyl ester (13g), with IC50 values of 110 and 500
nM, respectively. Both inhibitors were active in acute antilipolytic experiments in vivo and none of the
inhibitors inhibited the cytochrome P450 (CYP) isoforms 2D6, 3A4, and 1A2.
Introduction
out mice20,21 and the accumulation of diglycerides, not triglyc-
erides, in their adipose tissue22 suggest that there may be another
lipase involved in the regulation of lipid storage and mobiliza-
tion. Thus, it has been suggested that although HSL catalyzes
the rate-limiting step in triglyceride hydrolysis and is able to
hydrolyze both tri- and diglycerides, the major physiological
substrate for this enzyme is diglyceride. The recently identified
adipose triglyceride lipase (ATGL), is suggested to hydrolyze
triglycerides to generate diglycerides that are then further
hydrolyzed by HSL.23 Selective HSL inhibitors may provide
useful tools to further clarify the role of HSL and ATGL in
lipolysis.
The causes of type 2 diabetes, including the metabolic
syndrome, are complex and involve both genetic and acquired
factors.1 Among the acquired defects, lipotoxicity,2 glucotox-
icity,3 and obesity4 all have been shown to exacerbate insulin
resistance and contribute to the decline in â-cell function.
Increased levels of plasma and tissue fatty acids and triglyc-
erides are often present in patients with type 2 diabetes.5 The
elevated level of plasma fatty acids (FA) are thought to play a
major role in the pathogenesis of insulin resistance and type 2
diabetes6-8 by inhibiting glucose uptake and utilization by
muscle9,10 and causing increased glucose output by the liver.11-13
These events combine to give the hyperglycemia that is
characteristic of type 2 diabetes.14 Moreover, elevated triglyc-
erides, insulin resistance, and hyperinsulinemia, cardinal char-
acteristics of type 2 diabetes, may be involved in the develop-
ment of cardiovascular disease in type 2 diabetic patients.15
Triglycerides are a major source of stored energy. Energy is
released from these lipids by hydrolysis followed by oxidation,
primarily â-oxidation, of the liberated FA during periods of low-
energy intake. Regulation of the synthesis and mobilization of
triglycerides is, therefore, essential for energy homeostasis. Most
of the increase in plasma FA levels seen in type 2 diabetic
patients is the result of increased nocturnal and postprandial
adipose tissue lipolysis.16 Hormone-sensitive lipase (HSL) is
thought to be the rate-limiting enzyme in adipose tissue lipolysis
and net FA mobilization.17 The activity of HSL is acutely
activated via cAMP and protein kinase A mediated phospho-
rylation18 and its regulation in adipocytes is the primary means
by which lipolytic agents, such as adrenalin/noradrenalin, control
the circulating levels of FA. During periods of high-energy
intake, insulin stimulation of adipocytes prevents HSL activa-
tion, leading to a decrease in the release of FA and glycerol.19
The pivotal role of elevated plasma FA in the development of
insulin resistance and type 2 diabetes has led to the proposal
that HSL may be a potential therapeutic target for this disease,
lowering plasma FA levels and, thereby, reducing insulin
resistance. However, the nonobese phenotype of HSL knock-
During the last couple of years, a range of different classes
of HSL inhibitors have been described by different companies.
Bayer has published work on 2H-isoxazol-5-ones (1),24 Aventis
has published work on oxadiazolones (2),25,26 Ontogene has
published work on pyrrolopyrazinediones (3),27 and Novo
Nordisk has published work on carbazates (4),28 carbamoyl-
triazoles (5),29 and aryl boronic acids (6; Figure 1).30 In this
paper, a lead optimization program for a range of carbamate-
* To whom correspondence should be addressed. Phone: + 45 46752549.
Fax: + 45 39179714. E-mail: sebdrup@yahoo.dk.
Figure 1. Chemical structures of related HSL inhibitors.
10.1021/jm0607653 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/05/2007

5450 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Ebdrup et al.
based HSL inhibitors found in high-throughput screening is
presented together with acute rat in vivo data.
Chemistry
The compounds synthesized were independently prepared by
three different synthetic methods as described below and in the
Experimental Section. Different phenols (7) were converted to
different chloroformates (8) with phosgene or triphosgene. The
respective chloroformates were converted to different carbamates
(9-13). The respective phenols are for some cases also
converted to carbamates (9-13) via a reaction with a 1-methyl-
3H-imidazol-1-ium iodide or a carbamoyl chloride as shown
in Figure 2. The yields, preparation, and purification methods
are given in Tables 1-5. The 1H and 13C NMR data show that
a range of compounds exist as isomers due to hindered rotation
around the carbamate group. The isomers cannot be resolved
by liquid chromatography.
Figure 2. Synthetic route for carbamates. (i) Diphosgene, DIPEA, CH2-
Cl2; (ii) amine:
THF, or DCM; (iii) DABCO, carbamoyl chloride, THF; (iv) 1-methyl-
3H-imidazol-1-ium iodide, acetonitrile.
Table 1. Enzyme Activity and Selectivity for 2-Alkoxy-3,5-dichloropyridine-Based HSL Inhibitors
a N,N-Dimethylcarbamoyl chloride. b 4-Morpholincarbamoyl chloride. c N-Methyl-N-phenylcarbamoyl chloride. d IC50 ( SEM (n ) 2). e 1-Pyrrolidin-
carbamoyl chloride.
Table 2. Enzyme Activity and Selectivity for Methyl-phenyl Carbamates-Based HSL Inhibitors
a N-Methyl-N-phenylcarbamoyl chloride. b IC50 ( SEM (n ) 2). c IC50 ( SEM (n ) 5).

Carbamate-Based Inhibitors of SelectiVe Lipase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5451
Table 3. Enzyme Activity and Selectivity for 2-alkoxy-5-trifluoromethyl Based HSL Inhibitors
a Reflux 18 h. b 3-(Isopropyl-methyl-carbamoyl)-1-methyl-3H-imidazol-1-ium; iodide. c 3-(tert-Butyl-methyl-carbamoyl)-1-methyl-3H-imidazol-1-ium; iodide.
d 3-(Cyclohexyl-methyl-carbamoyl)-1-methyl-3H-imidazol-1-ium; iodide. e 3-(Benzyl-methyl-carbamoyl)-1-methyl-3H-imidazol-1-ium; iodide. f IC50 ( SEM
(n ) 2). g 3-(Ethyl-phenyl-carbamoyl)-1-methyl-3H-imidazol-1-ium; iodide. h Room temperature for 4 days. i 3-(1,3-Dihydro-isoindole-2-carbonyl)-1-methyl-
3H-imidazol-1-ium; iodide. j 3-(2,3-Dihydro-indole-1-carbonyl)-1-methyl-3H-imidazol-1-ium; iodide. k 4-Morpholinecarbonyl chloride. l 3-(2,6-Dimethyl-
morpholine-4-carbonyl)-1-methyl-3H-imidazol-1-ium; iodide. m IC50 ( SEM (n ) 5). n 1-Methyl-3-(piperidine-1-carbonyl)-3H-imidazol-1-ium; iodide. o Room
temperature for 2 days.
Results and Discussion
an IC50 value of 630 nM, and fortunately, did not inhibit BChE
and acetylcholine esterase (AChE).
Optimization of HSL Inhibitors. A high-throughput screen-
ing was performed to identify HSL inhibitors. A range of
inhibitors were identified and subjected to different counter
screens, and compound 9a was selected as one of the lead
structures that should be investigated further. Compound 9a is
a potent HSL inhibitor with an IC50 value of 350 nM. It is also
a very weak inhibitor of butyrylcholine esterase (BChE), with
an IC50 value of 19 µM. (The enzyme source is recombinant
human enzyme.) We decided to prepare a few N,N-disubstituted
carbamate analogues of compound 9a as shown in Table 1. Only
the N-methyl-N-phenyl carbamate analogue of compound 9d
had a reasonable HSL inhibition profile as it inhibited HSL with
We synthesize a small range of N-methyl-N-phenyl carbamate
analogues of 9a as shown in Table 2. As can be seen, 10c, the
trisubstituted pyridine analogue, was a potent and selective
inhibitor of HSL, with an IC50 value of 350 nM. The disubsti-
tuted pyridine analogues 10d and 10e were even more interesting
as they were both able to inhibit HSL with an IC50 value of 46
nM. None of the compounds inhibited BChE and AChE to any
measurable extent. As 10e is at least 10 times more potent than
9d, we also evaluated the rat PK profile of 10e and, as can be
seen from Table 6, the compound had a quite high clearance
(CL) of 61 ng/mL/kg, a low peroral bioavailability (Fpo) of 16%,

5452 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Ebdrup et al.
Table 4. Enzyme Activity and Selectivity for Substituted Methyl-phenyl Carbamate-Based HSL Inhibitors
a IC50 ( SEM (n ) 5). b (2-Methyl-phenyl)-methyl-amine. c (3-Methyl-phenyl)-methyl-amine. d (4-Methyl-phenyl)-methyl-amine. e IC50 ( SEM (n )
2). f (2-Chloro-phenyl)-methyl-amine. g (3-Chloro-phenyl)-methyl-amine. h 3[(4-Chlorophenyl)-methyl-carbamoyl]-1-methyl-3H-imidazol-1-ium; iodide, reflux
18 h. i (2-Methoxy-phenyl)-methyl-amine. j (3-Methoxy-phenyl)-methyl-amine.
and a low maximum plasma concentration (Cmax) of 43 ng/mL
after a p.o. dose of 2.2 mg/kg and an i.v. dose of 11 mg/kg. It
was decided to use the phenol part of 10e in the search for other
preferred amines other than the N-methyl-N-phenyl amine part
of 9d. First we investigated different N-methyl-N-alkyl amines
(compound 11a-c; Table 3). The isobutyl, tert-butyl, and cyclo-
hexyl analogues were all weak inhibitors of HSL (IC50 > 5
µM) despite having a bulky substituent at the methyl amine
part of the carbamate. The N-methyl-N-benzyl 11d (IC50 ) 1.4
µM) and N-ethyl-N-phenyl carbamate 11e (IC50 > 5 µM) were
also not capable of inhibiting HSL to any degree of importance.
As these five quite close structural analogues of 10e are at least
50 times weaker inhibitors of HSL, it seems like the N-methyl-
N-phenyl amine part of the carbamate is a preferred structural
element.
We synthesize a range a cyclic amine based carbamates (entry
11f-11m) and, as can be seen the pyrrolidine (11f), morpholine
(11i), and piperidine (11k) carbamates, all are capable of
inhibiting HSL with IC50 values of 23, 250, and 65 nM,
respectively. The annelated analogues 11g and 11h of 11f were
both not capable of inhibiting HSL to any degree of importance
(IC50 > 3 µM). The two annelated analogues 11l (IC50 ) 1.1
µM) and 11m (IC50 > 5 µM) of 11k were also quite weak
inhibitors. These results demonstrate that the active site pocket
corresponding to this position seems to be quite narrow.
As the N-methyl-N-phenyl-amine part of 10e seems to be an
important element, it was decided to synthesize a range of
substituted phenyl analogues, as shown in Table 4. Compound
10e is quite a lipophilic compound with a PSA (polar surface
area) value of 48.4 Å2 and a cLogP value of 4.97 (Table 6).
The analogues shown in Table 4 are even more lipophilic and
probably unsuited as drug candidates, but the structure-activity
relationship is very valuable as a starting point for the introduc-
tion of more hydrophilic structural elements in the phenyl part
of the N-methyl-N-phenyl amine. A methyl group is well
tolerated in both the ortho (12a), meta (12b), and para (12c)
position, but the ortho methyl analogue 12a is the most potent
inhibitor with a IC50 value of 39 nM (12b; IC50 ) 250 nm and
12c; IC50 ) 320 nM). A more bulky and lipophilic chloro atom
is well tolerated in the ortho (12d; IC50 ) 26 nM) and meta
position (12e; IC50 ) 71 nM) but not in the para position (12f;
IC50 ) 1.5 µM). Contrary to this, a methoxy group is well
tolerated in both the ortho (12g; IC50 ) 51 nM) and para (12h;
IC50 ) 23 nM) position. In conclusion, substituents are tolerated
in all positions of the phenyl core.
As pyrrolidine- and piperidine-based cyclic amines are well
tolerated, we prepare a range of substituted piperidine carbam-
ates, as a large range of substituted piperidines are commercially
available. To investigate if it was at all possible to find a position
in the piperidine ring where a substitution is allowed, it was
decided to synthesize and test the 2-, 3-, and 4-methyl-
substituted analogues (compounds 13a-c; Table 5). As can be
seen, a methyl group is tolerated in the 4-position, as 13c is a
potent inhibitor of HSL, with an IC50 value of 120 nM.
Unfortunately, 13c is also a weak inhibitor of BChE, with an
IC50 value of 16 µM. Furthermore, 4-substituted piperidin-

Carbamate-Based Inhibitors of SelectiVe Lipase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5453
Table 5. Enzyme Activity and Selectivity for Substituted Piperidine Carbamate-Based HSL Inhibitors
a Room temperature for 4 days. b 3-(3,4-Dihydro-1H-isoquinoline-2-carbonyl)-1-methyl-3H-imidazol-1-ium; iodide. c Reflux, 20 h. d 1-Methyl-3-(7-
trifluoromethyl-3,4-dihydro-2H-quinoline-1-carbonyl)-3H-imidazol-1-ium; iodide. e 1-Methyl-3-(2-methyl-piperidine-1-carbonyl)-3H-imidazol-1-ium; iodide.
f 1-Methyl-3-(3-methyl-piperidine-1-carbonyl)-3H-imidazol-1-ium; iodide. g 1-Methyl-3-(4-methyl-piperidine-1-carbonyl)-3H-imidazol-1-ium; iodide. h Phenyl-
piperidin-4-yl-methanone. i 3-(4-Benzyl-piperidine-1-carbonyl)-1-methyl-3H-imidazol-1-ium; iodide. j Piperidin-4-yl-methanol. k 4-Piperidinol.
Table 6. Comparation of Calculated Properties and PK Properties for
Selected HSL Inhibitors
also of great importance. The introduction of polar groups will
in general increase PSA and decrease clogP.
% reduction
Fpo of glycerol
Compounds 11i, 13f, and 13g are selective and potent
PSA
Cmax
CL
exp
(ng/mL)
inhibitors of HSL and they all contain solubilizing hydroxyl
groups or ether groups that have a positive influence on the
hydrophilicity (increased solubility). The PSA level is increased
to about 61 Å2 for 11i and to 75 Å2 for 13f and 13g and the
clogP value is reduced to 2-3 compared to the PSA of 48 Å2
and clogP of 4.97 for 10e. The PK profile of 13f and 13g, which
have clogP and PSA properties close to the properties of RPM
127963, were evaluated further together with 11i (Figure 3).
No ClogP (Å2) (ng/mL) (mL/min/kg) (%)
(p.o.)
10e 4.97 48.4
11i 2.98 61.3
13f 2.78 75.4
43a
21b
60a
61a
203b
46a
9c
16a
12b
10a
56c
59d
42d
379d
13g 2.16 75.6 1220c
3202d
a Tested at 1.1 mg/kg (i.v.) and 2.2 mg/kg (p.o.). b Tested at 0.5 mg/kg
(i.v.) and 1.0 mg/kg (p.o.). c Tested at 1.2 mg/kg (i.v.) and 2.4 mg/kg (p.o.).
d 10 mg/kg p.o., n ) 4.
The PK profile for the three compounds were measured
(Figure 3 and Table 6) and it was found that 11i had a very
high CL of 203 mg/min/kg, a low Fpo of 12%, and a low Cmax
of 21 ng/mL after an intravenous (i.v.) dose of 1.1 mg/kg and
a per oral (p.o.) dose of 2.2 mg/kg, which disqualified the
compound for further investigations. Compound 13f had a high
CL of 46 mg/min/kg, a low Cmax of 60 ng/mL and a low Fpo of
10 mg/kg after an i.v. dose of 0.5 mg/kg and a p.o. dose of 1.0
mg/kg. In contrast to these two compounds, 13g has a much
more promising PK profile with a CL of 9 mg/min/kg, a Cmax
of 1220 ng/mL, and a Fpo of 56% (i.v. dose of 1.2 mg/kg and
p.o. dose of 2.4 mg/kg). The promising PK profile seen for 13g
further support the hypothesis that introduction of polar groups
in saturated rings can greatly improve the PK profiles of
compounds. The challenge is to find positions where polar
groups also are tolerated potency vice. Both compounds 13f
and 13g were evaluated in an acute in vivo rat PK/PD model at
10 mg/kg and plasma glycerol was measured as lipolytic
endpoint. As can be seen from Table 6 and Figure 4, both
compounds were capable of significantly reducing the plasma
glycerol levels acutely. This correlates well with the in vivo
carbamates were synthesized (compound 13d-g; Table 5). The
4-carboxy-phenyl analogue 13d was only a very weak inhibitor
of HSL, but the 4-benzyl (13e; IC50 ) 210 nM), 4-hydroxy-
methyl (13f; IC50 ) 110 nM), and 4-hydroxy (13g; IC50 ) 450
nM) were all potent inhibitors of HSL. Unfortunately, the
4-benzyl analogue was also a potent inhibitor of BChE.
Nevertheless, the results demonstrate that large substituents are
well tolerated in the 4-position of the piperidine part of
compound 11k. Fortunately, the introduction of a hydroxyl
group as in 13f and 13g is not tolerated for inhibition of BChE
and AChE, making the two compounds selective.
As demonstrated by Wei31 and Yeh,32 the introduction of a
polar group in a lipophilic saturated ring system can in some
cases greatly improve the PK profile compared to the more
lipophilic nonsubstituted analogue (properties for the PK
preferred structure RPR 127963: ClogP, 2.47 and PSA, 88 Å2).
For lipophilic parts of molecules, it is expected that the
introduction of polar groups in some cases make saturated ring
systems less susceptible for CYP 3A4 derived degradation. The
overall properties for the respective compound is, of course,

5454 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Ebdrup et al.
Figure 3. PK profiles for selected carbamate-based HSL inhibitors. A, 10e and 11i; and B, 13f and 13g.
Figure 4. PK/PD profile (10 mg/kg) for 13f and 13g.
data published by Claus33 for 1. In line with the PK profile for
the two compounds, 13g has a longer duration of action. The
maximum exposure levels after 1 h were 379 ng/mL and 3202
ng/mL for 13f and 13g, respectively. Compounds 13f and 13g
did not inhibit the 3 CYP isoforms 1A2, 2C9, and 2D6 and
were also not capable of inhibiting PL, LPL, and HL.
values. Human recombinant HSL was prepared as described by
Holm et al.34 Human hepatic lipase and bovine lipoprotein lipase35
were kindly provided by Professor Gunilla Olivecrona, Umeå
University, Sweden. Apo CII was prepared as described by Holm
et al.36
Butyl-carbamic acid (4-(3,5-dichloropyridin-2-yloxy)-phenyl es-
ter was purchased from Specs. The respective 1-methyl-3H-
imidazol-1-ium iodides were prepared as described by Batey et al.37
Chloroformates were synthesized from the appropriate phenols and
phosgene or a phosgene substitute like, for example, trichloromethyl
chloroformate, as described in Konakahara et al.,38 except that the
crude product was separated from the diisopropylethylamine
hydrochloride by extraction with diethyl ether rather than with THF.
2-(4-Hydroxyphenoxy)-5-trifluoromethylpyridine was prepared as
described by Watson and Serban.39
HPLC-MS was measured on a Hewlett-Packard series 1100.
Column: Waters X-terra MS C-18 × 3 mm id. Gradient: 10-
100% acetonitrile (0.01% TFA) linear during 7.5 min at 1.0 mL/
min. Detection: 210 nm (analogue output from DAD). MS:
ionization mode API-ES, scan 100-1000 amu step 0.1 amu.
Preparative HPLC. The system consists of two Gilson 322
pumps and a Gilson manometric module 805. A Gilson 215
combined auto-injector and fraction collector performs injection
and fraction collection. Detection is performed with a Gilson Diode
array detector 170. A sample containing 25-100 mg of material
dissolved in 0.5-2.0 mL of solvent (minimum water concentra-
tion: 10%).
Separation is performed on Waters Xterra, RP18 7 µm, columns
19 mm × 150 mm, flow rate 15 mL/min (sample added with a
flow rate of 5 mL/min for about 1 min). The most widely used
gradient starts at 5% acetonitrile in water and ends after 14 min on
95% acetonitrile. This concentration is maintained for 6 min. The
system is buffered with 0.05% TFA. In special cases, the gradient
is altered to fit the separation need. The pooled fractions are
evaporated to dryness in vacuo.
Conclusion
We have identified a novel type of carbamate-based HSL
inhibitors. It is expected that the compounds are reversible
pseudo-substrate inhibitors like a range of other known HSL
inhibitors. Compounds within different classes of carbamate-
based HSL inhibitors are potent and selective and all compounds
have full efficacy. The N-methyl-N-phenyl carbamates are in
general the most potent inhibitors of HSL, with IC50 values for
a range of compounds below 50 nM.
The two piperidine carbamate based HSL inhibitors 13f and
13g were potent and selective HSL inhibitors, with IC50 values
of 110 and 450 nM, respectively. As both compounds have a
promising PK profile and increased solubility due to the
influence of the hydroxy groups, they were tested in an acute
PK/PD study in rats. Both compounds showed acute antilipolytic
activity at a dose of 10 mg/kg p.o., did not inhibit the 3 CYP
isoforms 1A2, 2C9 and 2D6 and were not capable of inhibiting
any other lipase or esterase tested (HL, PL, LPL, BChL and
AChL). Due to the promising profile seen for 13f and 13g
further studies will be conducted to evaluate the biological
importance of inhibiting HSL.
Experimental Section
Materials and Methods. NMR data were recorded on a 300
MHz and on a 400 MHz spectrometer. Assignments: quint, quintet;
sext, sextet. Elemental analyses were within 0.4% of the calculated

Carbamate-Based Inhibitors of SelectiVe Lipase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5455
The inhibition of the different lipases and esterases (HSL, HL,
LPL, PL, AChE, and BChE) were determined as described in
Ebdrup et al.29 The lipase assays were based on a new fluoro-
chrome-labeled triacylglyceride octadec-9-enoic acid 2-[12-(7-nitro-
benzo1,2,5oxadiazol-4-ylamino)-dodecanoyloxy]-1-octadec-9-enoy-
loxymethyl-ethyl ester, and the AChE and BChE assays were based
on acetylthiocholine or butyrylthiocholine, respectively. Maximum
concentration of inhibitors for determination of IC50 values was
100 µM, with 5-fold dilution steps in six concentrations. Each data
point was determined as the average of two measurements. IC50
values were calculated using Prism, version 4.0, by fitting the data
to a sigmoidal dose-response (variable slope) nonlinear regression
analyses.
General Procedure 1. The appropriate phenol was dissolved in
dry THF (30 mL) and 1,4-diazadicyclo[2.2.2]octane (0.89 g, 8.0
mmol) and a carbamoyl chloride (6.0 mol) was added, and the
reaction mixture was stirred overnight at room temperature. A 3%
aqueous citric acid solution (30 mL) was added and the phases
were separated. The aqueous phase was extracted with dichlo-
romethane (30 mL × 3) and the pooled organic phases were dried
and evaporated to dryness to give the crude product.
General Procedure 2. The phenol (2.0 mmol) was dissolved in
acetonitrile (30 mL) in a glass screw cap vessel. Triethylamine (2.0
mmol) was added together with the respective 1-methyl-3H-
imidazol-1-ium iodide (2.0 mmol) dissolved in acetonitrile (30 mL)
at room temperature. The reaction mixture was shaken for 16-48
h at reflux and evaporated. Dichloromethane (30 mL) and 0.1 N
aqueous hydrochloric acid solution (20 mL) was added, the organic
phase was separated, and the aqueous phase was extracted with
dichloromethane (2 × 30 mL). The combined organic phases were
washed with water (10 mL), dried, and evaporated to give the crude
product.
General Procedure 3. The appropriate aryl chloroformate (4.0
mmol) was dissolved in dry THF or dichloromethane (30 mL). An
appropriate amine (6.0 mmol) and diisopropylethylamine (01.05
g, 6.0 mmol) were added, and the reaction mixture was stirred
overnight at room temperature. The crude reaction mixture was
evaporated to dryness to give the crude product.
Dawley rats (Charles River Sulzfeld, Germany). Blood samples
were collected by heart puncture under carbon dioxide anaesthesia
in ethylene diamine tetraacetic acid EDTA-NaF tubes at prede-
termined time points. A rat was applied for each time point.
PK/PD Studies in Rats. Animals: Male Sprague-Dawley rats,
twelve weeks of age, were purchased from Charles River (Sulzfeld,
Germany). The rats were housed under constant humidity in a
temperature- (20 ( 2 °C) and light-controlled environment (12 h
light/dark cycle; lights on from 6 a.m.), with free access to food
(Altromin 1324) and water. All experimental procedures were
approved by the Danish Animal Experiment Inspectorate.
In Vivo Inhibition of Lipolysis. The acute effects of HSL
inhibition were tested in vivo in the overnight fasted rats using
plasma glycerol as antilipolytic biomarker. Briefly, overnight fasted
rats received vehicle (n ) 8 or HSL inhibitor (10 mg/kg body
weight p.o.; n ) 4 per group). Blood samples were collected at
baseline (0.5 h before dosing of compound) and again 1, 3, and 5
h after dosing of compound. Blood was drawn from orbital plexus
(anaesthetic: isofluran, Abbott Scandinivia AB, Sweden) into
EDTA-coated tubes containing 0.1% NaF (W/V) and was plasma
collected. Plasma samples were stored at -80 °C before analysis.
Plasma glycerol levels were determined using standard laboratory
procedures (Hitachi 912 automatic analyzer, Boehringer Man-
nheim).
Plasma Analysis. Plasma samples were analyzed by turbulent
flow chromatography combined with tandem mass spectrometry
(TFC-MS/MS). Plasma sample preparation included dilution (10%
methanol, 1:1), centrifugation (14 300 g for 20 min), and aliquo-
tation of a minimum of 100 µL to 96-well plates. TFC was
performed using a 2300 HTLC system (Cohesive Technologies)
consisting of an isocratic pump for sample cleanup and flush of
liquid lines, a binary pump for elution of retained analytes, and a
valve switching module with two Rheodyne six-port valves. The
autosampler was a CTC HTS PAL (CTC Analytics, Zingen,
Switzerland). Injection volumes were 50 µL. A single column
method with a Waters Oasis HLB, 1.0 × 50 mm, column was used,
except for 11i, where a Cohesive Turbo column, C8 50 µm, was
applied. The mobile phases were A, methanol-0.1% formic acid
(5:95, v/v), and B, methanol-0.1% formic acid (95:5, v/v), except
for 10e, where mobile phase A, 0.05% trifluoroacetic acid, and
mobile phase B, 0.05% trifluoroacetic acid in acetonitrile, were
used. Samples containing the compounds were extracted from the
plasma and eluted into the interface on the mass spectrometer, using
the following three-step method: step 1, the sample was loaded
onto the column with an aqueous mobile phase (100% A); step 2,
elution of analytes to detector with mobile phase B; and step 3,
system re-equilibration.
Samples were analyzed by MS-MS using a MDS Sciex API
3000 triple quadrupole tandem mass spectrometer (Toronto, Canada)
equipped with a Turbo Ion Spray interface. The mass spectrometer
was operated in positive-ion multiple reaction monitoring (MRM)
mode with key parameter settings: temperature, 350; ionization
current, 4500-5000; orifice potential, 25-70; and ring potential,
100-360. The molecular ions and production ions for the individual
compounds were 13f, 397.1 f 142.0; 10e, 389.1 f 134.0; 11i,
369.2 f 114.3; and 13g, 383.1 f 127.9. Calibration curves were
prepared for each compound dissolved in plasma in the concentra-
tion range 10-3000 ng/mL.
CYP Inhibition Assay. The in vitro CYP-subtype activity in
the absence and presence of a test compound (the potential CYP-
subtype inhibitor) was determined using a CYP-subtype selective
substrate (dextromethorphan for CYP2D6, erythromycin for CYP3A4,
and phenacetin for CYP1A2, respectively). Incubations were utilized
(200 µL, 37 °C, 10 min) containing human liver microsomes (HLM,
0.1 mg), [O-methyl-14C]-dextromethorphan in the CYP2D6 assay
(total concn: 3 µM ) Km), [N-methyl-14C]erythromycin and
erythromycin in the CYP3A4 assay (total concn: 20µM ) Km), or
[O-ethyl-14C]phenacetin and phenacetin in the CYP1A2 assay (total
concn: 40 µM ) Km), the cofactor NADPH (1 mM), and the test
compound (20 µM). All incubations were performed in triplicate.
The HLM preparations used were a pool of g15 donors to obtain
The preparative and purification methods used and the yields
obtained are described in Tables 1-5 for each individual compound.
The Spectroscopic data, LC-MS and elemental analyses can be
found in the Supporting Information.
4-Hydroxymethyl-piperidine-1-carboxylic Acid 4-(5-Trifluo-
romethylpyridin-2-yloxy)-phenyl Ester, 13f. 1H NMR (400 MHz,
CDCl3) δ 1.20-1.35 (m, 2H), 1.68-1.78 (m, 1H), 1.71 (br s, 1H),
1.78-1.86 (m, 2H), 2.86 (t, J ) 12.1, 1H), 2.99 (t, J ) 12.1, 1H),
3.54 (d, J ) 6.0, 2H), 4.32 (t, J ) 14, 2H), 7.00 (d, J ) 8.6, 1H),
7.11-7.19 (m, 4H), 7.89 (dd, J ) 8.5 and 2.5, 1H), 8.43 (m, 1H).
13C NMR (100 MHz, CDCl3) δ 28.34 and 28.77 (ratio 1:1), 38.56,
44.19 and 44.49 (ratio 1:1), 67.31, 111.13, 121.56 (q, J ) 33.7
Hz), 122.23, 122.94, 123.65 (q, J ) 271.5 Hz), 136.67 (q, J ) 2.9
Hz), 145.44 (q, J ) 4.4), 148.66, 150.03, 153.53, 165.79. LC-MS
Rt 3.87 min, m/z: 397 (M + H)+, >99%. Anal. (C19H19F3N2O4)
C, H, N.
4-Hydroxy-piperidine-1-carboxylic Acid 4-(5-Trifluorometh-
1
ylpyridin-2-yloxy)-phenyl Ester, 13g. H NMR (CDCl3) 1.54-
1.66 (m, 2H), 1.73 (br s, 1H, minor), 1.79 (br s, 1H, major), 1.90-
1.99 (m, 2H), 3.26 (t, J ) 9, 1H), 3.37 (t, J ) 9, 1H), 3.90-4.07
(m, 3H), 7.00 (d, J ) 8.6, 1H), 7.11-7.21 (m, 4H), 7.89 (dd, J )
8.5 and 2.5, 1H), 8.42-8.46 (m, 1H). 13C NMR (100 MHz, CDCl3)
δ 33.90 (d, J ) 26.3), 41.65 (d, J ) 21.2), 66.97, 111.15, 121.59
(q, J ) 33), 122.16, 122.94, 123.62 (q, J ) 271.5), 136.73 (q, J )
3), 145.38 (q, J ) 4), 148.57, 150.05, 153.53, 165.74. LC-MS Rt
3.63 min, m/z: 383 (M + H)+, >98%. Anal. (C18H17F3N2O4) C,
H, N.
Pharmacokinetic Studies in Rat. The p.o. dose was around 2
mg/kg and the i.v. dose was around 1 mg/kg, and the dosing solution
contained 10% hydroxy propyl cyclodextrin (HPCD) in phosphate
buffer pH 7.0. However, 11i was dosed with 20% tween 80 and
5% glucose in phosphate buffer, pH 7.0. The compounds were
dosed p.o. by gavage and i.v. in the tail vein to male Sprague-

5456 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
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