Discovery of potent and orally active MTP inhibitors as potential anti-obesity agents

Article abstract of DOI:10.1016/j.bmcl.2006.02.058

We have successfully identified a number of novel MTP inhibitors with single digit nanomolar potency. Analogues 10aq and 10dq demonstrated in vivo efficacy in a murine gut retention assay.

Full text of DOI:10.1016/j.bmcl.2006.02.058

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3039–3042
Discovery of potent and orally active MTP inhibitors as
potential anti-obesity agents
Jin Li,* Peter Bertinato, Hengmiao Cheng, Bridget M. Cole, Brian S. Bronk,
Burton H. Jaynes, Anne Hickman, Michelle L. Haven, Nicole L. Kolosko,
Chris J. Barry and Tara B. Manion
Pfizer Inc., Groton, CT 06340, USA
Received 20 January 2006; revised 17 February 2006; accepted 20 February 2006
Available online 10 March 2006
Abstract—We have successfully identified a number of novel MTP inhibitors with single digit nanomolar potency. Analogues 10aq
and 10dq demonstrated in vivo efficacy in a murine gut retention assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
As in human health, obesity is a growing health prob-
lem in companion animals, with 25–40% of the pet
population estimated to be overweight and 5–10%
considered severely obese.1 Obesity predisposes dogs
and cats to a number of harmful conditions including
diabetes, hepatic lipidosis, cancer, osteoarthritis, der-
matitis and musculoskeletal problems such as cruciate
and inter-vertebral disk rupture. Obesity also nega-
tively impacts veterinary patients with cardiovascular
and respiratory disease and limits the efficacy of
pharmaceutical therapy in these conditions.2–11 Cur-
rent therapy for obesity is based on food restriction
and/or exercise and affords limited success in most
patients. The failure of weight loss programs is large-
ly the result of poor owner compliance due to hunger
and begging of the pet. Because there are no veteri-
nary drugs currently available for the treatment of
obesity, there is a major opportunity for a safe, effi-
cacious agent.
lipid assembly and transport.12 Inhibition of MTP
has been shown to be an effective method for
reducing serum cholesterol.15 Recently we disclosed
the use of MTP inhibitors for the treatment of
obesity by inhibition of fat absorption.16
Several potent MTP inhibitors have been disclosed,
including CP-346086 (1),17 implitapide (2),18,19 JNJ-
4506463 (3),20 diaminohydroindan derivative21 (4)
and BMS-212122 (5).22 Starting from 1 as a lead,
we successfully identified a new class of potent MTP
inhibitors, represented by the indole amide
6
(Fig. 1).23 In order to further explore the chemical
space and ADME properties in this series many ana-
logs have been prepared by either replacing the indole
moiety with other fragments or varying the terminal
amines. In this paper, we would like to disclose the
syntheses and SAR of phenyl/substituted phenyl moi-
eties.24 This research effort resulted in the discovery
of a number of highly potent MTP inhibitors for
the potential treatment of obesity, highlighted by ana-
log 10dq (entry 35, Table 1).
Microsomal triglyceride transfer protein (MTP)12 is
involved in the assembly of triglyceride-rich chylo-
microns in enterocytes and very low-density lipopro-
teins (VLDL) in hepatocytes.12–14 MTP is located in
intestinal and liver tissues where it plays a role in
Two factors were considered in replacing the indole
fragment in 6: (1) the rigidity and (2) the size of the
new fragments. A parallel synthesis approach was em-
ployed in order to quickly explore the SAR of the
new templates. As depicted in Figure 2, the acid deriv-
atives 8a–8i were chosen to replace the indole moiety
in 6 based on the considerations mentioned above.
Keywords: MTP; Microsomal triglyceride transfer protein; Obesity.
*
Corresponding author. Tel.: +1 860 715 3552; fax: +1 860 715
9259; e-mail: jin.li@pfizer.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.058

3040
J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3039–3042
Table 1. In vitro canine MTP inhibition data
F
F
CF3
H
F
N
HO
O
O
R2
N
O
N
N
O
N
N
N
R1
N
N
X
N
N
H
H
O
2 implitapide
1
CP-346086
Entry Compound NH2–x–COOH NR1R2 MTP inhibition
IC50 (nM)
Cl
O
N
N
O
O
1
2
3
4
5
6
7
8
9
10aj
12j
16.93
2.53
S
10ak
10al
12k
12l
N
N
O
22.46
29.51
69.39
35.40
12.97
17.38
13.29
F
N
O
O
10am
10an
10ao
10ap
10aq
10ar
12m
12n
12o
12p
12q
12r
F
F
OH
4 diaminoindanes
N
H2N
N
N
O
8a
3 JNJ-4506463
N
N
N
10
11
12
13
14
15
16
17
18
10bj
12j
20.51
CF3
CF3
H
10bk
10bl
12k
12l
ND
CF3
N
R1
N
OMe O
13.27
6.23
O
O
O
O
OH
10bm
10bn
10bo
10bp
10bq
10br
12m
12n
12o
12p
12q
12r
HN
R2
H N
2
N
14.78
1.01
N
N
X
H
H
O
N
8b
N
R3
23.13
6.47
5.28
6
5 BMS-212122
Figure 1. Selected MTP inhibitors.
19
20
21
22
23
24
25
26
27
10cj
12j
10ck
10cl
12k
12l
2.0
A diverse set of amines was selected in order to quick-
ly explore SAR (Fig. 3).
O
3.85
6.38
10cm
10cn
10co
10cp
10cq
10cr
12m
12n
12o
12p
12q
12r
OH
ND
H N
2
OMe
The preparation of the analogues is outlined in
Scheme 1. The 40-(trifluoromethyl)-2-biphenylcarboxy-
lic acid (7) was reacted with 8a–8i to provide the es-
ter intermediates, which were then hydrolyzed under
basic conditions to furnish the acids (9a–9i). The
phenylglycine derivatives 13j–13r were prepared by
coupling Boc-protected phenylglycine 11 with amines
12j–12r. Several standard amide coupling reaction
conditions were screened in order to avoid epimer-
ization of the chiral center of the phenylglycine.
The coupling condition, PyBroP/DIPEA/DCM,
proved to be the most robust for the coupling pro-
cess without epimerization as monitored by chiral
HPLC. Subsequently coupling the acid 9a–9i with
the phenylglycine derivatives 13j–13r provided the
final analogues represented by 10 for biology screen-
ing Table 2.
1.68
7.14
1.75
2.34
8c
28
29
30
31
32
33
34
35
36
10dj
12j
ND
10dk
10dl
12k
12l
1.64
3.50
O
10dm
10dn
10do
10dp
10dq
10dr
12m
12n
12o
12p
12q
12r
ND
ND
1.78
OH
H2N
8d
4.62
3.37
37
38
39
40
41
42
43
44
45
10ej
12j
85.5
5.53
26.8
10ek
10el
12k
12l
O
10em
10en
10eo
10ep
10eq
10er
12m
12n
12o
12p
12q
12r
29
72.7
OH
HN
All analogues were tested in a canine MTP in vitro
binding assay.25 The results are summarized in Table
1. In general, analogues prepared from the mono-aryl
templates (8a–8d, entries 1–36) demonstrated good
in vitro potency despite their decreased size compared
to the indole analog 6. The analogues derived from
3-methoxy 8c and 3-methyl 8d templates showed the
most potent inhibition toward MTP, suggesting a
lipophilic binding pocket for these substituents. An
electron-donating group on the 2-position of the
phenyl ring was tolerated (entries 19–27). Electron
deficient pyridyl acid template 8a showed good but
decreased potency compared to analogues 10bj–10dr
ND
8e
26.6
14.9
18.3
46
47
48
49
50
51
52
53
54
10fj
12j
15.6
61.15
2.97
5.67
11.78
16.94
4.11
8.15
5.05
10fk
10fl
12k
12l
O
10fm
10fn
10fo
10fp
10fq
10fr
12m
12n
12o
12p
12q
12r
OH
H N
2
8f

J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3039–3042
3041
Table 1 (continued)
CF3
Entry Compound NH2–x–COOH
NR1R2 MTP inhibition
IC50 (nM)
X
a
+
O
H2N
CO2Me
55
56
57
58
59
60
61
62
63
10gj
12j
17.27
35.92
OH
10gk
10gl
12k
12l
>100
55.84
>100
O
8a-i
7
10gm
10gn
10go
10gp
10gq
10gr
S
12m
12n
12o
12p
12q
12r
OH
CF3
CF3
H
N
2
69.84
7.33
8.27
8g
13
O
O
R2
N
O
b, c
N
X
N
R1
ND
X
H
H
N
CO2H
O
H
64
65
66
67
68
69
70
71
72
10hj
12j
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
9a-i
10hk
10hl
12k
12l
O
10hm
10hn
10ho
10hp
10hq
10hr
12m
12n
12o
12p
12q
12r
OH
R2
N
N
H
H
N
O
d
H2N
+
R1
Boc
OH
R1
R2
H2N
8h
N
O
H
O
13j-r
11
12j-r
Scheme 1. Reagents and conditions: (a) i—PyBroP, DIPEA, DCM,
0 ꢁC to rt, ii—LiOH, THF/H2, reflux, >95%; (b) EDC, HOBT,
DIPEA, DCM, rt, >85%; (c) LiOH, THF/H2O, reflux, >98%;
(d) i—PyBroP, DIPEA, DCM, 0 ꢁC to rt, ii—4 N HCl/dioxane, 100%.
73
74
75
76
77
78
79
80
81
10ij
12j
23.56
10ik
10il
12k
12l
96.71
69.52
95.89
O
10im
10in
10io
10ip
10iq
10ir
12m
12n
12o
12p
12q
12r
OH
HN
>100
>100
76.52
>100
9.43
8i
Table 2. In vivo data for compounds 10aq and 10dq
Entry
Compound
ED25 (mg/kg, rat)
8
35
10aq
10dq
6.59
3
ND, not determined.
(entries 10–36). When a conformationally restricted
template 8e was used, all analogues prepared showed
a significant drop in potency toward MTP. Templates
in which the aniline functionality was replaced with
more flexible benzylic amines (8f, 8g, 8h and 8i) were
in general less potent toward MTP.
O
O
OMe O
OH
OH
OH
H2N
H2N
N
H2N
HN
OMe
8c
8a
8b
O
O
O
OH
OH
Several potent analogs were progressed into in vivo
studies. The murine gut retention assay23 was used to as-
sess a compound’s ability to inhibit intestinal MTP. In
this assay, compounds 10aq and 10dq were potent inhib-
itors of intestinal MTP, with ED25s of 6.93 and 3 mg/kg,
respectively.
OH
H2N
H2N
8d
8g
8e
8f
8i
O
O
O
OH
S
N
H
OH
OH
H2N
O
HN
In summary, we have successfully identified a number of
novel and potent MTP inhibitors. Analogues 10aq and
10dq also demonstrated in vivo activity when tested in
a murine gut retention assay.
H2N
8h
Figure 2. Acid derivatives.
H2N
H2N
References and notes
HN
H2N
12k
12j
12l
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O
NH
12n
HN
NH
12o
12r
12m
HN
NH
12q
12p
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Figure 3. Amine derivatives.

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25. Canine in vitro MTP assays. (A) Canine hepatic microsome
isolation: canine microsomes are first isolated from canine
liver by thawing frozen liver on ice and rinsing several times
with 0.25 M sucrose. A 50% liver homogenate (w/v) is made
in 0.25 M sucrose. The homogenate is diluted 1:1 with
0.25 M sucrose, and centrifuged at 10,000g at 4 ꢁC for
20 min. The supernatant is saved. The pellet is re-suspended
in a minimal volume of 0.25 M sucrose and re-centrifuged at
10,000g for 20 min at 4 ꢁC. The supernatants are combined
and centrifuged at 105,000g for 75 min at 4 ꢁC. The
supernatant is discarded and the resulting microsomal
pellet is saved. The microsomal pellet is re-suspended in a
minimum volume of 0.25 M sucrose and diluted to 3 ml/g
liver weight in 0.15 M Tris–HCl, pH 8.0. The resulting
suspension is divided into 12 tubes and centrifuged at
105,000g for 75 min. The resulting microsomal pellets are
stored at ꢀ80 ꢁC until needed. MTP is isolated by thawing
the microsomal pellet tube and suspending in 12 ml/tube of
cold 50 mM Tris–HCl, 50 mM KCl, 2 mM MgCl, pH 7.4,
and slowly adding 1.2 ml of a 0.54% deoxycholate, pH 7.4
solution. After 30 min incubation on ice with gentle mixing,
the solution is centrifuged at 105,000g for 75 min at 4 ꢁC.
The supernatant, containing soluble MTP, is dialyzed for 2–
3 days with 5 changes of assay buffer (15.0 mM Tris–HCl,
40 mM NaCl, 1 mM EDTA, 0.02% NaN3, pH 7.4). (B)
MTP activity assay reagents: donor liposomes are created
by adding 447 mM egg phosphatidylcholine (68/20 ml),
83 mM bovine heart cardiolipin (169/20 ml) and 0.91 mM
[14C]triolein (110 Ci/mol) (20/20 ml). The lipids are avail-
able in chloroform and are first dried under nitrogen and
then hydrated in assay buffer to the volume needed. To
create liposomes, lipids are sonicated for ꢁ7 min. Lipids are
centrifuged at 105,000g for 2 h and liposomes are harvested
by removing the top ꢁ80% of supernatant into separate
tube. Acceptor liposomes are created by adding 1.33 mM
egg phosphatidylcholine (404/40 ml), 2.6 mM triolein (100/
40 ml) and 0.5 nM [3H]egg phosphatidylcholine (50 Ci/mol)
(10/40 ml). The lipids are available in chloroform and are
first dried under nitrogen and then hydrated in assay buffer
to the volume needed. To create liposomes, lipids are
sonicated for ꢁ20 min. Lipids are centrifuged at 105,000g
for 2 h and are harvested by removing the top ꢁ80% of
supernatant into separate tube. (C) MTP in vitro lipid
transfer inhibition assay. Appropriately diluted drug or
control samples in 100 ml assay buffer containing 5% BSA
are added to reaction tubes containing assay buffer, 50 ml
donor liposomes, 100 ml acceptor liposomes, and partially
purified liver MTP. The tubes are vortexed and incubated
on a tube shaker for 1 h at 37 ꢁC to allow lipid transfer
reaction to occur. Donor liposomes are precipitated by
adding 300 ml of a 50% (w/v) DEAE cellulose suspension in
assay buffer to each tube, followed by gentle/repeated
inversion for5 min at room temperature. Tubes are then
centrifuged at ꢁ1000 rpm to pellet resin. Four hundred
milliliters of supernatant is transferred into a scintillation
vial with scintillation fluid and DPM counts for both [3H]
and [14C] are determined. Triolein transfer is calculated by
comparing the amount of [14C] and [3H] remaining in the
supernatant to [14C] and [3H] in the original donor and
acceptor liposomes, respectively. % Triolein transfer =
([14C]supernatant/[14C]donor) · ([3H]acceptor/[3H]superna-
tant) · 100 IC50 values are obtained using standard methods
and first order kinetic calculations.