1,3-Disubstituted 4-aminopiperidines as useful tools in the optimization of the 2-aminobenzo[a]quinolizine dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.03.072

In a search for novel DPP-IV inhibitors, 2-aminobenzo[a]quinolizines were identified as submicromolar HTS hits. Due to the difficult synthetic access to this compound class, 1,3-disubstituted 4-aminopiperidines were used as model compounds for optimizatio

Full text of DOI:10.1016/j.bmcl.2007.03.072

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2966–2970
1,3-Disubstituted 4-aminopiperidines as useful tools in
the optimization of the 2-aminobenzo[a]quinolizine
dipeptidyl peptidase IV inhibitors
Thomas Lubbers,* Markus Bohringer, Luca Gobbi, Michael Hennig, Daniel Hunziker,
¨
¨
Bernd Kuhn, Bernd Lo¨ffler, Patrizio Mattei, Robert Narquizian, Jens-Uwe Peters,
Yves Ruff, Hans Peter Wessel and Pierre Wyss
Discovery Research, Pharmaceutical Division, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
Received 5 February 2007; revised 20 March 2007; accepted 22 March 2007
Available online 25 March 2007
Abstract—In a search for novel DPP-IV inhibitors, 2-aminobenzo[a]quinolizines were identified as submicromolar HTS hits. Due to
the difficult synthetic access to this compound class, 1,3-disubstituted 4-aminopiperidines were used as model compounds for opti-
mization. The developed synthetic methodology and the SAR could be transferred to the 2-aminobenzo[a]quinolizine series, leading
to highly active DPP-IV inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
The incretin hormone glucagon-like peptide-1 (GLP-1)
is released from the gastrointestinal tract in response
to nutrient ingestion, and stimulates insulin secretion
from the pancreas in a glucose-dependent manner.
GLP-1 is degraded by dipeptidyl peptidase IV (DPP-
IV), a multifunctional type II transmembrane serine
protease which cleaves a dipeptide from the N-terminus
of peptide substrates with either proline or alanine at the
penultimate position. Once in circulation, GLP-1 has a
half-life of only few minutes. Thus, inhibitors of DPP-
IV increase levels of GLP-1 and subsequently insulin,
and are therefore promising therapeutic agents for the
treatment of type 2 diabetes.1
H
H
NH2
N
HO
N
HO
N
H
O
CN
CN
O
vildagliptin 1
saxagliptin 2
F
O
N
O
F
O
NH2
N
N
N
N
N
CN
N
F
F3C
NH2
There are both covalent and non-covalent inhibitors of
DPP-IV in late clinical development (Fig. 1). The cyano-
pyrrolidines vildagliptin 1 (Novartis, FDA approval
filed)2 and saxagliptin 2 (Bristol-Myers Squibb, phase
3)3 bind covalently to the active serine in the S1 pocket
of DPP-IV, whereas the non-covalent inhibitors sitaglip-
tin 3 (Merck, launched 2006)4 and alogliptin 4 (Takeda,
phase 3)5 rely entirely on non-covalent protein–ligand
sitagliptin 3
alogliptin 4
Figure 1. Selected clinically studied DPP-IV inhibitors.
interactions and have substituted phenyl groups as
ligands for the S1 pocket of DPP-IV.
Clinical trials have proven that DPP-IV inhibitors
reduce blood glucose and HbA1c levels in diabetic
patients and an excellent safety profile with minimal side
effects can be achieved.6
Keywords: Dipeptidyl peptidase IV; DPP-IV inhibitor; 2-Aminobenzo-
[a]quinolizine.
*
In a high throughput screening the 2-aminobenzo[a]-
quinolizine 5 and the aminopyrimidine derivative 6 were
Corresponding author. Tel.: +41 616883095; fax: +41 616886459;
e-mail: thomas.luebbers@roche.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.072

T. Lu¨bbers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2966–2970
2967
identified as inhibitors of DPP-IV (Fig. 2). It turned out
that the HTS hits 5 and 6 share a similar structural rec-
ognition motif for DPP-IV with sitagliptin 3, whose
structure was not known to us when we performed the
research reported in this paper.
The diastereomer 7 of the screening hit 5 had an interest-
ing drug-like profile and was significantly active in an
oral glucose tolerance test (OGTT) in fa/fa rats
(Table 1).
Particularly, interesting was the 3-butyl substituent in
compound 7, which according to its X-ray complex
structure with DPP-IV pointed into the S1 specificity
pocket (Fig. 3).7 The butyl group did not fill the hydro-
phobic S1 pocket optimally. Synthesis of derivatives
with different substituents in 3-position was often
cumbersome. Therefore, we looked for a simplified
open-chain analogue. The fact that in the 2-amin-
obenzo[a]quinolizine 8 annulation of a cyclohexyl ring
is tolerated suggested 1-substituted 4-amino-3-butylpi-
peridines 9 as potential DPP-IV inhibitors (Fig. 4).
Figure 3. X-ray structure of the binding pocket of the complex of
2-aminobenzo[a]quinolizine 7 with human DPP-IV.
NH2
NH2
Me
H
Me
MeO
MeO
N
H
N
R
H
The 4-aminopiperidines 9 were easily accessible in six
steps starting from the commercially available b-ketoest-
er 10 using literature known procedures (Scheme 1).8
The residue R was introduced through double Hofmann
elimination and Michael addition of the amine to the
ammonium salt 11. Oxime-formation and reduction
8, IC50 = 140 nM
Figure 4. Simplification of the 2-aminobenzo[a]quinolizine structure.
9
O
O
N
O
yielded the 4-aminopiperidines
9
as mixtures of
O
a,b,c
N+
Cl
NH2
NH2 NH2
N
H
I
Me
2
3
H
MeO
MeO
O
N
10
11
N
O
d
5, IC50 = 0.64 μM
6, IC50 = 10 μM
NH2
O
Figure 2. DPP-IV inhibitory activity of selected HTS screening hits.
e,f
N
R
N
R
Table 1. Profile of the 2-aminobenzo[a]quinolizine 7
NH2
9
12
Me
2
3
H
Scheme 1. Reagents and conditions: (a) 1.9 equiv BuI, 4.5 equiv
K2CO3, 1 equiv i-Pr2NEt, acetone, reflux, 12 h; (b) 20% aq HCl, reflux,
24 h, 67% over two steps; (c) 1.2 equiv MeI, acetone, rt, 3 h, 78%; (d)
0.8 equiv RNH2, 0.13 equiv K2CO3, EtOH, reflux, 3 h, 57–100%; (e)
1.1 equiv NH2OH*HCl, 1.1 equiv NaOAc, EtOH, rt, 2 h; (f) Ra–Ni
alloy, EtOH, H2O, NaOH, rt, 2 h, 23–71% over two steps.
MeO
MeO
N
DPP-IV IC50 (nM)
Solubility (mg/L)
520
>414
0.8
2.5
logD (pH 7.4)
Permeation coefficient Pe (10À6 cm sÀ1) (PAMPA)
CLmicr (mL/min/mg protein) rat/human
IC50 CYPs (lM) (2C9, 2D6, 3A4)
cis/trans-diastereomers, which could be often separated
by silica gel chromatography.9
4.7/0.0
>50
À16
OGTT (Dglucose, 40 min) (%)
A phenyl substituent (9c) was more active than a cyclo-
hexyl (9a) or a benzylic substituent (9b) (Table 2).10
Generally, the cis-diastereomer was more potent than
the trans-diastereomer. Various substitutions with elec-
tron-rich (9j) as well as electron-poor (9i) residues on
the aryl moiety were allowed. The most active derivative
PAMPA, parallel artificial membrane permeation assay; CLmicr
,
intrinsic clearance in liver microsome preparations; CYPs, cytochrom
P450 enzyme; OGTT, oral glucose tolerance test in fa/fa rats (reduc-
tion of glucose levels 40 min after glucose challenge (2 g/kg) compared
to non-treated animals; 0.3 mg/kg of 7 given 2 h before glucose
challenge).

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T. Lu¨bbers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2966–2970
Table 2. DPP-IV inhibitory activity of 4-aminopiperidine derivatives 9
NH2
Me
N
R
Compound
Ra
IC50 (lM)
cis
trans
cis/trans
9a
9b
9c
9d
9e
9f
c-Hex
Ph2CH
45.2
18.6
Figure 6. Modeled overlay of 2-aminobenzo[a]quinolizine 13 with the
co-crystal structure of DPP-IV with inhibitor 14 illustrating the
asymmetric shape of the S1 pocket.
Ph
4-PhOPh
4-Tol
3.5
6.3
5.2
35.7
15.0
94.7
3,5-(CF3)2–Ph
3-MeO-5-CF3–Ph
3-Ph-4-MeO–Ph
4-Cl-3-CF3–Ph
3,4-(MeO)2–Ph
3,4,5-(MeO)3–Ph
3,4-Cl2–Ph
13.4
8.4
only low yields (5%). The Pd-catalyzed arylation13 of
15 was successful, however furnished 16 in low yield
(Scheme 2) because of the formation of the dehydro
by-products 17 and 18.
9g
9h
9i
2.1
5.8
2.8
4.9
2.3
3.3
1.5
142
2.6
9j
26
18.4
9k
9l
The SAR revealed that a small lipophilic group such as
methyl or possibly a halogen substituent in the meta-po-
sition of the aromatic residue was favored (Table 3). The
toluene derivative 19c had a IC50 value of 600 nM com-
bined with a low molecular weight. It was still a diaste-
reomeric mixture.
9m
9n
2-Naphthyl
5,6,7,8-Tetrahydro-
naphthalen-1-yl
1-Naphthyl
9.4
13.6
5.3
9o
a The numbering refers to the position of the substituent on the cor-
responding aromatic ring.
We were able to solve the X-ray complex structure of
14
˚
19c with DPP-IV to a resolution of 2.8 A (Fig. 7).
The binding mode revealed clear electron density for
the (3R,4S)-enantiomer of the cis-isomer. The proton-
ated primary amine is engaged in the three characteristic
hydrogen bonds with Glu 205, Glu 206, and Tyr 662, as
seen with other primary amines, such as sitagliptin 3.
The favorable 3-methyl phenyl substituent points into
the S1 pocket and perfectly occupies the small hydro-
phobic niche in the back. Some residual electron density
was the 2-naphthyl-compound cis-9m. Because of the
relatively flat SAR we decided to keep the more polar
3,4-dimethoxyphenyl group constant and vary the sub-
stituent in 3-position.
The phenyl substituted 2-aminobenzo[a]quinolizine 13
exhibited a nice inhibitory activity of 200 nM (Fig. 5).
The modeled overlay with the co-crystal structure of
DPP-IV with inhibitor 14,11 an optimized compound
from a previous lead series identified from the HTS hit
6, clearly showed that a substantial improvement in
affinity could be expected by the introduction of the cor-
rect substitution pattern on the 3-aryl substituent of the
4-aminopiperidines, respectively, the 2-aminobenzo-
[a]quinolizines (Fig. 6).
O
N
O
N
O
N
O
N
Ar
O
Ar
O
a
The employed synthetic route to 13 was not generally
applicable for the variation of the 3-aryl substituent.
Therefore, we decided to study the direct introduction
of an aryl substituent into the ketone-precursor. As a
model system we choose the 4-aminopiperidines.
Several methods such as the direct arylation of the
copper-enolate, generated from 15 with LDA and
CuCN, with a mixed bis aryl iodonium salt,12 gave
O
O
O
O
O
O
15
16
17
18
NH2
N
Ar
O
b,c
NH2 NH2
NH2
2
N
3
H
O
O
O
N
N
19
Cl
14, IC50 = 10 nM
Cl
Scheme 2. Reagents and conditions: (a) 1 equiv ArBr, 3 equiv NaOt-
Bu, 10 mol% Pd(OAc)2, 10 mol% Pdt-Bu3, THF, rt, overnight, 10–27%
(b) 1.1 equiv NH2OH*HCl, 1.1 equiv NaOAc, EtOH, rt, 2 h;
(c) Ra–Ni alloy, EtOH, H2O, NaOH, rt, 2 h, 28–96% over two steps.
13, IC50 = 200 nM
Figure 5. DPP-IV inhibitors.

T. Lu¨bbers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2966–2970
2969
Table 3. DPP-IV inhibitory activity of 4-aminopiperidine derivatives
19
O
O
N
Ar
a
H
NH2
O
O
O
O
N
Ar
N
20
21
b,c
O
O
NH2
N
Compound
Ara
4-Me–Ph
IC50 (lM) cis/trans
Ar
H
19a
19b
19c
19d
19e
14.8
31.3
0.6
O
O
3,4-Me2–Ph
3-Me–Ph
3-MeO–Ph
2-Pyridyl
25.6
8.9
22
a The numbering refers to the position of the substituent on the cor-
responding aromatic ring.
Scheme 3. Reagents and conditions: (a) 1 equiv ArBr/Cl, 3 equiv
NaOtBu, 10 mol% Pd(OAc)2, 10 mol% Pdt-Bu3, THF, rt, overnight,
11–41%; (b) 1.1 equiv NH2OH*HCl, 1.1 equiv NaOAc, EtOH, rt, 2 h;
(c) Ra–Ni alloy, EtOH, H2O, NaOH, rt, 2 h, 52–73% over two steps.
Table 4. DPP-IV inhibitory activity of 2-aminobenzo[a]quinolizines 22
NH2
Ar
H
O
N
O
Compound
Ara
IC50 (nM)
cis
270
trans
cis/trans
22a
22b
22c
22d
22e
22f
22g
Ph
200
1520
370
3730
650
4
220
2-Pyridyl
4-Me–Ph
3,4-Me2–Ph
3-MeO–Ph
3-Me–Ph
3-Cl–Ph
990
10590
9380
5130
32
13
50
4
a The numbering refers to the position of the substituent on the cor-
responding aromatic ring.
Figure 7. X-ray complex crystal structure of human DPP-IV with
compound 19c (cis, (3R,4S)-enantiomer). Protein residues engaged in
hydrogen bonds (dashed red lines) with the ligand are shown.
Table 5. Profile of the 2-aminobenzo[a]quinolizine trans-22f
is, however, seen for a second ligand conformation in
which the 3-methyl phenyl substituent is rotated by
180° around the bond connecting the P1 substituent
with the piperidine ring.
NH2
H
MeO
N
The developed methodology could be nicely transferred
to the 2-aminobenzo[a]quinolizines (Scheme 3). Phenyl
iodides in general and electron-poor aromatic bromides
were not successful in the arylation of the ketone 20.
Aryl chlorides sometimes produced the products 22 in
low yields, when the corresponding aryl bromides and
iodides failed.
MeO
DPP-IV IC50 (nM)
Solubility (mg/L)
4.6
>469
1.3
3.4
logD (pH 7.4)
Permeation constant Pe (10À6 cm sÀ1) (PAMPA)
Clmicr (mL/min/mg protein) (rat/human)
IC50 CYPs (lM) (2C9, 2D6, 3A4)
OGTT (Dglucose, 40 min) (%)
1.0/3.0
15, 11, >50
À41
Small lipophilic groups in meta-position, such as methyl
or chlorine, were the best substituents, providing com-
pounds with an IC50 value of 4 nM (trans-22f and
trans-22g, Table 4). Trans-22f reduced glucose levels in
the oral glucose tolerance test (OGTT) in fa/fa rats by
41% (Table 5) 40 min after the glucose challenge at a
PAMPA, parallel artificial membrane permeation assay; CLmicr
,
intrinsic clearance in liver microsome preparations; CYPs, cytochrom
P450 enzyme; OGTT, oral glucose tolerance test in fa/fa rats (reduc-
tion of glucose levels 40 min after glucose challenge (2 g/kg) compared
to non-treated animals; 0.3 mg/kg of 22f given 2 h before glucose
challenge).

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T. Lu¨bbers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2966–2970
National Meeting of the American Chemical Society,
Atlanta, GA, March 26–30, 2006.
6. von Geldern, T. W.; Trevillyan, J. M. Drug Dev. Res.
2006, 67, 627.
7. Hunziker, D.; Hennig, M.; Peters, J.-U. Curr. Top. Med.
Chem. 2005, 5, 1623.
dose of 0.3 mg/kg (applied 120 min before glucose
challenge).
In summary, we describe a novel series of 4-aminopi-
peridine DPP-IV inhibitors. A methodology for the
introduction of 3-aryl substituents could be developed.
An aromatic residue with a small lipophilic group in
meta-position yielded the most active inhibitors. The
developed SAR and the synthetic procedures could be
applied to the 2-aminobenzo[a]quinazoline series, lead-
ing to highly active DPP-IV inhibitors, with trans-22f
and g as the best compounds (Table 4).
8. (a) Tortolani, D. R.; Poss, M. A. Org. Lett. 1999, 1, 1261;
(b) Bonjoch, J.; Linares, A.; Guardi, M. Heterocycles
1987, 26, 2165.
1
9. All new compounds were characterized by H NMR and
MS prior to submission for biological evaluation. For
detailed synthetic procedures, see (a) Boehringer, M.;
Hunziker, D.; Kuhn, B.; Loeffler, B. M.; Luebbers, T.;
Ricklin, F. WO2006066747; (b) Boehringer, M.; Kuhn, B.;
Luebbers, T.; Mattei, P.; Narquizian, R.; Wessel, H. P.
WO2005000846.
References and notes
10. DPP-IV inhibitors were measured for their ability to
inhibit DPP-IV mediated cleavage of Ala-Pro-7-amido-
4-trifluoromethylcoumarin in a fluorogenic assay. All
compounds were measured in triplicate at 5–7 con-
centrations in the range of 100 lM to 100 pM. IC50
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values were calculated with
a nonlinear best fit
regression model. All assays were calibrated with
NVP-DPP728 as internal standard inhibitor. NVP-
DPP728 under the conditions of the assay showed an
IC50 of 15 4 nM (M SD, n = 12) at 50 lM sub-
strate concentration and
a Ki of 11 3 nM deter-
mined at substrate concentration range of 10–600 lM.
IC50 values of unknown compounds were accepted
when the IC50 (x) measured for NVP-DPP728 in the
assay was 11 < x < 19 nM.
11. Peters, J.-U.; Weber, S.; Kritter, S.; Weiss, P.; Wallier, A.;
Boehringer, M.; Hennig, M.; Kuhn, B.; Loeffler, B.-M.
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14. The coordinates of the X-ray complex structure of human
DPP-IV with compound 19c were deposited to the Protein
Data Bank (PDBid: 2ijd).