Carbazates as potent inhibitors of hormone-sensitive lipase

Article abstract of DOI:10.1016/j.bmcl.2004.01.038

The central role of adipose tissue hormone-sensitive lipase in regulating fatty acid metabolism makes it a potential pharmacological target for the prevention of peripheral insulin resistance in obese, prediabetic and diabetic individuals. The synthesis of a new series of carbazates is presented. Modification of the phenolic 4-position in a series of 1,2,3,4- tetrahydroisoquinoline and morpholine derived carbazates, yielded inhibitors of the catalytic activity of this enzyme with nanomolar potency.

Full text of DOI:10.1016/j.bmcl.2004.01.038

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1741–1744
Carbazates as potent inhibitors of hormone-sensitive lipase
Johannes C. de Jong,a Lotte G. Sørensen,b Hans Tornqvistc and Poul Jacobsena,*
aNovo Nordisk A/S, Medicinal Chemistry, Novo Nordisk Park, 2760 Maaloev, Denmark
bNovo Nordisk A/S Assay and Cell Technology, Novo Alle, 2880 Bagsvaerd, Denmark
cNovo Nordisk A/S, Diabetes Biology, Novo Nordisk Park, 2760 Maaloev, Denmark
Received 13October 2003; revised 16 January 2004; accepted 16 January 2004
Abstract—The central role of adipose tissue hormone-sensitive lipase in regulating fatty acid metabolism makes it a potential
pharmacological target for the prevention of peripheral insulin resistance in obese, prediabetic and diabetic individuals. The synthesis
of a new series of carbazates is presented. Modification of the phenolic 4-position in a series of 1,2,3,4-tetrahydroisoquinoline and
morpholine derived carbazates, yielded inhibitors of the catalytic activity of this enzyme with nanomolar potency.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
against water-soluble esters such as p-nitrophenyl ace-
tate or butyrate. The activity of HSL is acutely regu-
lated by hormones which influence the levels of cAMP
and hence the activity of protein kinase A, which acti-
vates HSL by phosphorylation of specific serine resi-
dues. The prime players are catecholamines and insulin
where cetecholamines via induction of cAMP activates
HSL and lipolysis while insulin potently reduces cAMP
levels and hence lipolysis.7 Thus, the central role of HSL
regulating the release of fatty acids stored as triglycer-
ides in adipose tissue makes it a potential pharmacolo-
gical target for the prevention of peripheral insulin
resistance in obese, prediabetic and diabetic individuals.
In type 2 diabetes, hyperglycemia is accompanied by
abnormalities in lipid metabolism. The elevation of cir-
culating free fatty acids (FFA), in particular, has
received much attention and is widely considered a
possible pathogenetic factor in the disease.1À3 Increased
influx of FFA leads to peripheral insulin resistance seen
as decreased glucose uptake in muscle and increased
hepatic glucose production. The mechanism for this
effect has been attributed to glucolipotoxia.4 Further-
more, increased FFA flux to the liver results in enhanced
triglyceride synthesis and assembly into secreted VLDL,
contributing to the abnormal lipoprotein profile seen in
diabetics and insulin resistant individuals.5
Common structural features of lipases and several
esterases are that they adopt the so-called a/b hydrolase
fold and perform catalysis using a catalytic triad con-
sisting of a serine, an aspartic or glutamic acid and a
histidine residue. Most members of this esterase-lipase
family utilize a serine catalytic mechanism, reminiscent
of the serine protease mechanism. The mechanism for
inhibition of choline esterases by aryl carbamate esters
has been studied in detail, involving covalent carba-
moylation of the serine-OH and subsequent hydrolysis
of the enzyme-inhibitor complex.8 In this paper the
synthesis of some novel carbazates along with in vitro
inhibitory effects on HSL activity are presented.
The driving force behind the increased flux of plasma FFA
is an overflow, in adipose tissue, of fatty acids derived
from increased intracellular lipolysis and from hydrolysis
of chylomicron triglycerides of direct dietary origin.
The key rate-limiting enzyme in regulating lipolysis in
adipose tissue is hormone-sensitive lipase (HSL) which
catalyses the first and second step in the breakdown of
triglycerides, releasing two molecules of fatty acid and
one molecule of monoacylglycerol, which is then hydro-
lysed by a separate, specific monoacylglycerol lipase.6
Cholesterol fatty acid esters can also be readily hydro-
lysed by HSL. As all lipases HSL has a low activity
2. Enzyme assay
Keywords: Hormone-sensitive lipase; Inhibition; Carbazates.
* Corresponding author. Tel.: +45-4443-4882; fax: +45-4443-4547;
e-mail: pjac@novonordisk.com
Hormone-sensitive lipase activity was measured as
the release over time of 3H-oleic acid from
a
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.038

1742
J. C. de Jong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1741–1744
followed by reduction of the nitroso compound 4 with
lithium aluminium hydride. The N-methyl analogue 6
was prepared by reaction of 5 (R1, R2=H) with formic
acid18 followed by reduction of the formylhydrazine
intermediate with lithium aluminium hydride.19
Scheme 1. Reagents and conditions: (i) H2NNHCO2R, Et3N, DMF,
60 ꢀC, 2–15%.
A third method of synthesizing carbazates is by reaction
of a carbamoyl chloride with an alcohol or phenol. This
method was successfully applied on solid phase sup-
ported synthesis (Scheme 3). N-Amino-1,2,3,4-tetra-
hydroisoquinoline was attached to the solid support20
by reductive amination with sodium cyanoborohydride.
Reaction with triphosgene afforded the corresponding
hydrazinecarbonyl chloride 11, which was reacted with
a variety of phenols to give the resin bound carbazates
12. Reaction with 25% trifluoroacetic acid in dichloro-
methane gave the free carbazates in high purity.
3H-trioleoylglycerol emulsion stabilized with phospho-
lipids in the presence of bovine serum albumin as fatty
acid acceptor9 using purified recombinant human hor-
mone-sensitive lipase with a specific acꢀtivity of 2.6 U/mg
protein and triolein as substrate at 37 C.10,11
3. Chemistry
The synthesis of the carbazates starts either from 1-(2-
chloroethyl)-2-chloromethylbenzene (1)12,13 and a hydra-
zinecarboxylic acid ester14,15 (Scheme 1) or more practi-
cally via reaction of a chloroformate with a (substituted)
2-amino-3,4-dihydro-1H-isoquinoline 5 (Scheme 2). This
compound is readily prepared from the corresponding
1,2,3,4-tetrahydroisoquinoline 3 by reaction with isoamyl
nitrite16 or with sodium nitrite and sulphuric acid,17
4. Results
From our initial results with compounds 14–17 (Table
1), which were prepared according to Scheme 1, it
became clear that simple aliphatic carbazates showed
very low potency in inhibiting HSL. Only with the aro-
Scheme 2. Reagents and conditions: (i) isoamyl nitrite or NaNO2, H2SO4; (ii) LiAlH4, Et2O, reflux, 2 h, 54–56% over 2 steps; (iii) for R1, R2=H (a)
HCO2H, reflux, 1 h; (b) LiAlH4, Et2O, 19%; (iv) phenyl chloroformate, iPr2NEt or Et3N, dichloromethane, 2–63%.
Scheme 3. Reagents and conditions: (i) triphosgene, iPr2NEt, dichloromethane; (ii) DABCO, phenol, DMF; (iii) 25% TFA in dichloromethane, 24–
31% overall yield.

J. C. de Jong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1741–1744
1743
Table 1. Inhibition of HSL by initially synthesised (3,4-dihydro-1H-
isoquinolin-2-yl)-carbamic acid esters
compound 25 versus 26. After attack of serine on the
carbazate the substituted phenol acts as a leaving group.
Clearly a carbonyl substituent in the 4-position stabi-
lizes the negative charge of the phenolate, and thereby
makes it a better leaving group. The same is true for
compound 27 with an ester functionality in 4-position.
Also in this case the negative charge is stabilized by the
substituent. Based on the stabilizing effect of the sub-
stituents, compound 25 should be the best inhibitor but
actually the opposite is found, indicating that the
polarity of the compounds plays a major role in the
inhibition of HSL. Although the result obtained with
acetamide 28 was very disappointing and seemed to
indicate that an amine functionality in the para-position
of the phenolic part of the carbazate was not advanta-
geous, a more lipophilic group like the Boc-protected
amine in 29 proved to give a compound with very high
potency against HSL. However, with these lipophilic
substituents the solubility of our compounds might
become a severe problem. Therefore, several analogues
of compound 21 were prepared, in which the tetra-
hydroisoquinoline group was replaced by a smaller and
less lipophilic heterocycle (Table 3). Although some of
Compda
R
% Inhibition
IC50 (nM)
14
15
16
17
tBu
Et
CH2Ph
Ph
8
9
4
NT
NTb
NT
NT
205
a All new compounds gave satisfactory analytic and spectral data.
b Not tested.
matic carbazate 17 a strong inhibition was observed.
This prompted us to synthesize a number of derivatives
of this compound and to developing a SAR around this
class of compounds.
From the results obtained with the compounds 18–29
(Table 2) it became clear that steric as well as electronic
effects play an important role in the inhibition of HSL.
Ortho-substituents have a dramatic influence on the
activity of the carbazates as is clearly demonstrated by
the much lower activity of compound 18 compared to
compound 19. The two ortho-substituents in compound
20 are protecting the carbazate functionality completely
from nucleophilic attack by the serine in the active site,
making it no longer a pseudo-substrate for the enzyme.
Also a small substituent on the nitrogen atom of the
carbazate changes the molecule from a highly active
inhibitor (21) into a completely inactive compound (24).
On the other hand, small substituents in the tetra-
hydroisoquinoline part of the molecule (compounds 22
and 23) hardly have any influence on the activity.
the activity was lost changing from
a
tetra-
hydroisoquinoline to a morpholine (19 versus 184 nM)
as in compound 31, we were convinced that the activity
could be improved by changing the lipophilic sub-
stituent in the aromatic part of the molecule.
Based on the promising results obtained with the com-
pounds presented in Tables 2 and 3 we decided to con-
centrate our efforts on the synthesis of carbazates
Table 3. Inhibition of HSL by carbamic acid 4-chlorophenyl esters
The importance of polar effects on the activity of the
carbazates as inhibitors is clearly demonstrated with
Compda
R1, R2
% Inhibition
IC50 (nM)
30
31
32
33
(CH2)5
(CH2)2O(CH2)2
(CH2)2NMe(CH2)2
(CH2)4
99
99
98
100
50
184
515
24
Table 2. Inhibition of HSL activity by a series of (3,4-dihydro-1H-
isoquinolin-2-yl)-carbamic acid phenyl esters
a All new compounds gave satisfactory analytic and spectral data.
Table 4. Inhibition of HSL by morpholin-4-yl-carbamic acid phenyl
esters
Compda
R
R1 R2 RN % Inhibition IC50 (nM)
18
19
20
21
22
23
24
25
26
27
28
29
2-MeO
4-MeO
2,6-di-Cl
4-Cl
4-Cl
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
76
97
3NT
6400
360
b
Compda
R
% Inhibition
IC50 (nM)
98
99
91
2
21
97
57
7
19
35
32
NTb
NTb
97
9952
NTb
3
34
35
36
37
38
39
40
41
CH2CO2Me
CH2CO2Et
CH2CO2CH2Ph
NHCOMe
NHCO-nPr
95
99
100
20
84
99
99
100
716
138
5
NTb
3200
47
4-Cl
4-Cl
4-COPh
4-CH2Ph
4-CO2Me
4-NHCOMe
4-NHCO2-tBu
NHCOCH2-tBu
NHCOcyclohexyl
NHCO(4-tBu-cyclohexyl)
10
1
98
a All new compounds gave satisfactory analytic and spectral data.
b Not tested.
a All new compounds gave satisfactory analytic and spectral data.
b Not tested.

1744
J. C. de Jong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1741–1744
derived from N-aminomorpholine and phenols having a
lipophilic substituent in 4-position of the aromatic part
of the molecule (Table 4).
3. Mulder, H.; Holst, L. S.; Svensson, H.; Sundler, F.;
Ahren, B.; Rorsman, P.; Holm, C. Diabetes 1999, 48, 228.
4. Prentki, M.; Corkey, B. E. Diabetes 1996, 45, 273.
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1996, 90, 243.
6. Fredrikson, G.; Tornqvist, H.; Belfrage, P. Biochim. Bio-
phys. Acta 1986, 876, 288.
7. Holm, C.; Osterlund, T.; Laurell, H.; Contreras, J. A.
Annu. Rev. Nutr. 2000, 20, 365.
Changing the size of the ester from a methyl into an
ethyl and benzyl (34, 35 and 36) increased the binding
affinity from 716 nM to 5 nM. The same trend was
found with the amides 37–41. Changing the substituent
from an acetamide into a butyramide (37 vs 38) or even
8. Feaster, S. R.; Lee, K.; Baker, N.; Hui, D. Y.; Quinn,
D. M. Biochemistry 1996, 35, 16723.
a
more bulky 3,3-dimethylbutyramide as in 35,
increased the inhibition of HSL from only 20% to
almost complete inhibition. Increasing the size of the
substituent even further resulted in complete inhibition
of HSL with an IC50=1 nM for compound 41.
9. Tornqvist, H.; Krabisch, L.; Belfrage, P. J. Lipid Res.
1974, 15, 291.
10. For a detailed description of the assays see: Beltrandel-
Rio, H.; Jacobsen, P.; de Jong, J. C. WO Patent 01/87843,
2001; Chem. Abstr. 2001, 135, 371653.
11. Contreras, J. A.; Danielsson, B.; Johansson, C.; Oster-
lund, T.; Langin, D.; Holm, C. Protein Expr. Purif. 1998,
12, 93 .
5. Summary
12. Gade, T.; Streek, M.; Voss, J. Chem. Ber. 1992, 125, 127.
13. Laurenco, C.; Villien, L.; Kaufmann, G. Tetrahedron
1984, 40, 2731.
In conclusion we have identified carbazates derived
from N-amino-1,2,3,4-tetrahydroisoquinoline and N-
aminomorpholine as a new class of highly potent inhi-
bitors of human HSL. Polar as well as steric effects play
a major role in the activity of these compounds. The
binding site of the enzyme tolerates a large number of
different substituents in the 4-position of the phenyl
ester. Most importantly, highly lipophilic substituents in
this position lead to a very strong inhibition of the
lipase.
14. Hoft, E.; Rieche, A. Angew. Chem. 1961, 73, 807.
¨
15. Carpino, L. A. J. Org. Chem. 1965, 30, 321.
16. Sakane, K.; Terayama, K. I.; Haruki, E.; Otsuji, Y.;
Imoto, E. Bull. Chem. Soc. Jpn. 1974, 47, 1297.
17. Biel, J. H.; Drukker, A. E.; Mitchell, T. F.; Sprengeler,
E. P.; Nuhfer, P. A.; Conway, A. C.; Horita, A. J. Am.
Chem. Soc. 1959, 81, 2805.
18. Hajos, Gy.; Messmer, A. J. Heterocycl. Chem. 1978, 15, 463 .
19. Biel, J. H.; Drukker, A. E.; Mitchell, T. F., Jr. J. Am.
Chem. Soc. 1960, 82, 2204.
20. 3-(3,5-dimethoxy-4-formylphenoxy)propanoyl
amino-
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