1. Introduction
In orienting experiments, we used the parent structure of the
HTS hit compound 1a to modify the substituents of the pyrazole
moiety (Scheme 1). Saponification of the ethyl ester delivered
carboxylic acid 2, which did not show any substantial mATGL
inhibitory activity anymore. The importance of the ester moiety
was further corroborated by the decent inhibitory activities of the
methyl and butyl esters 3a-b. Alkylation of the parent HTS-hit
compound 1a with methyliodide produced methoxypyrazole 4,
which showed a significantly improved IC50 value. In addition to
IC50 values, activity of new compounds was further determined as
% of inhibition at a single concentration of 200 µM (I200).
Variation of the substitution pattern (1b-p) of the bottom ring of
the parent hit structure 1a led to activity loss (see SI). Further we
tested if a substituent is necessary at all at the 4-position. The
Adipose tissue (AT) constitutes the body’s largest energy store.
The prevailing cell type of AT is the adipocyte which major
function is to store and to liberate energy substrates in form of free
fatty acids (FA) on demand. To fulfill their functions, adipocytes
comprise a set of lipogenic and lipolytic enzymes and regulatory
proteins, which are tightly regulated by feeding/fasting cycle of the
organism. In the postprandial state a surplus of energy is stored as
triacylglycerol within cytoplasmic lipid droplets. Upon fasting,
adipose triglyceride lipase (ATGL) initiates the breakdown of
stored TG to diacylglycerol (DG) and FA.1 Subsequently the
activities
of
hormone-sensitive
lipase
(HSL)
and
monoacylglycerol lipase (MGL) generate glycerol and two FA.2
Adipocytes release the lipolytic products into the circulation to
provide energy substrates for oxidative tissues. A constantly
positive energy balance causes an increase in AT mass due to an
increase in adipocyte number and adipocyte size, and obesity.
However, the expandability of AT is not infinite. If the supply of
energy substrates exceeds the storage capacity of AT, FA
concentrations in the circulation increase and lipids are deposited
in ectopic tissues, like muscle and liver. These ectopically stored
lipids are responsible for the development of obesity associated
insulin resistance and non-alcoholic fatty liver disease (NAFLD),
finally leading to tissue dysfunction and early death.3,4 It is
suggested that an increased release of FA from hypertrophic
adipocytes contributes to the elevation of plasma and ectopic lipid
concentrations.5 As ATGL is the initiating enzyme in the liberation
of FA from TG stores of adipocytes, reducing FA in the circulation
by inhibiting ATGL represents an attractive strategy to counteract
the metabolic complications of obesity. Indeed, as mouse models
of global as well as adipose tissue specific genetic ATGL
deficiency showed, reduced plasma FA concentrations
accompanied by a resistance to diet induced obesity and insulin
resistance.6,7
HTS-Hit
O
O
N
O
N
O
O
N
X
B(OH)2
MeO
W
V
O
HO
HO
HO
OR
OH
O
O
O
U
X
N
W
O
a
N
N
O
N
O
N
b
a
c
8a-g
V
U
O
Br
U = O, S, CH
V,W,X = N, CH
O
O
O
10a-g
9
3ab
1a
IC50 = 120 µM
2
4
O
O
3a
R = Me
IC50 = 130 µM
200 = 64 %
IC50 = > 200 µM
200 = 15 %
IC50 = 90 µM
200 = 63 %
O
O
I
I
200 = 67 %
I
N
NH
I
b
11
8b
3b
R = nBu
IC50 = 100 µM
200 = 74 %
I
O
O
O
O
NH2
N
O
N
c-d
12
N
O
13
Br
O
N
O
a
N
O
These findings prompted us to develop and characterize small
molecule inhibitors of ATGL. The versatile synthetic strategies we
applied resulted in the development of Atglistatin. Atglistatin
selectively, competitively, and transiently inhibits murine ATGL
(mATGL) activity in vitro, in cultured cells, and in vivo.8 In
contrast to complete ATGL deficiency in mice and humans, long
term Atglistatin treatment does not cause cardiac steatosis, cardiac
failure or a premature death. Importantly, Atglistatin treatment
reduced circulating plasma lipids and protected from diet induced
obesity, insulin resistance and NAFLD in mice.9 At present no 3D-
structure of ATGL is available and any effort to identify inhibitors
of ATGL has to rely on the traditional approach of synthesizing
and testing compounds.10 In this manuscript we describe the
structure-activity relationship (SAR) studies which allowed us in
O
N
N
O
H
O
Sch6eme 3. Introducing variations into the top ring fragments. Reagents and
7
conditions: (a) 0.05 equiv PdCl2(dppf)*DCM,52.1 equiv CsF, DME, 80 °C; (b)
IC50 = 40 µM
I200 = 89 %
1.6 equiv PtO2, 1 bar H2, EtOH/DCM, RT, 27 h, 59%; (c) 1.5 equiv NaNO2,
H2O, conc. HCl, 0 °C, 1 h, then 2.0 equiv NaN3, H2O, 0 °C to RT, 1 h, 84%;
(d) 1.0 equiv ethyl propiolate, 0.2 equiv sodium ascorbate, 0.07 equiv
CuSO4*5H2O, H2O/ACN, RT, 15 h
desired
compound 5 was prepared via CuI-catalyzed N-arylation of ethyl
3-pyrazolecarboxylate (6) and p-bromophenetole (7) (Scheme
2).11 5 exhibited the best inhibitory activity so far. Unfortunately,
we also observed a considerable inhibitory activity of 5 against
MGL (see SI, Figure S1).
Scheme 1. HTS-hit and explorative modifications. Reagents and conditions:
(a) 5 M NaOH, EtOH, 60 °C, 30 min, 41%; (b) R-OH, cat. conc. H2SO4, reflux;
(c) 1.5 equiv K2CO3, DMF, overnight, 93%.
2.2. Exchange of the top ring system
In order to improve lipase selectivity, the pyrazole ring was
replaced by several different ring systems, while keeping the 1,3-
arrangement of the 4-ethoxyphenyl and ethyl ester substituents
constant (8a-g). 3-Bromoaryl esters 10a-g were coupled with
4-ethoxyphenylboronic acid (9) in a Suzuki coupling reaction with
PdCl2(dppf)*DCM as catalyst and CsF as base in DME (Scheme
3).12,13 In order to test if a non-planar compound could also fit into
the binding pocket, piperidine derivative 11 was produced from
pyridine ester 8b via catalytic hydrogenation.14 Triazole ester 12
was produced by converting aniline 13 via diazonium chemistry
into the corresponding azide,15 which was reacted with ethyl
iterative rounds of optimization to develop biaryl hit compound 1
(IC50 (half maximal inhibitory concentration) = 120 µM, resulting
from a high-throughput screening (HTS) campaign originally
aimed to identify HSL inhibitors) to Atglistatin, which has proven
to serve as a valuable tool compound to validate ATGL as a drug
target. The presented SAR-data give valuable insight about the
nature of the ATGL binding pocket and might provide
opportunities for applying in silico methods to develop ATGL
inhibitors.
2. Results and discussion
propiolate in
a
Cu(I)-catalyzed azide-alkyne 1,3-dipolar
2.1. Preliminary experiments