Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase IV (DPP IV) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.08.057

In this paper the synthesis and structure-activity relationships of two classes of electrophile-based DPP IV inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The in vitro potency, stability and ex vivo experiments were performed on select compounds within these series to determine their inhibitory capacity in plasma. In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1′ binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the α-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6 h.

Full text of DOI:10.1016/j.bmcl.2004.08.057

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5579–5583
Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase
IV (DPP IV) inhibitors
Dana Ferraris,* Yao-Sen Ko, David Calvin, Tiffany Chiou, Susan Lautar, Bert Thomas,
Krystyna Wozniak, Camilo Rojas, Vincent Kalish and Sergei Belyakov
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD 21224, USA
Received 30 July 2004; revised 20 August 2004; accepted 26 August 2004
Available online 17 September 2004
Abstract—In this paper, the synthesis and structure–activity relationships (SAR) of two classes of electrophile-based dipeptidyl
peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SARof these series demonstrate that
the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S10 binding groups for potency against DPP IV. In addition, both
cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series.
Stereochemistry at the a-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the keto-
azetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining
their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for
up to 6h.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
(2) irreversible inhibitors such as phosphonic esters 2
(Ki = 15nM);7 (3) slow binding, reversible inhibitors
such as boronic acid 3 (Ki = 2nM),6 and nitrile 4
(IC50 = 8nM).8 In general, the incorporation of an
electrophilic, serine binding group into this proline-
derived pharmacophore can increase the potency by as
much as an order of magnitude over the unsubstituted
pyrrolidines 1a and 1b.7,9
Dipeptidyl peptidase IV (DPP IV) is a proline-specific
serine protease responsible for the in vitro and in vivo
cleavage and degradation of many endogenous peptides
and neuropeptides.1 The most prominent substrates for
this enzyme are GLP-1, neuropeptide Y, substance P,
and casmorphin.2 The inhibition of DPP IV and preven-
tion of the degradation of these substrates can ulti-
mately produce pharmacological effects in therapeutic
areas as wide ranging as diabetes,3 pain,4 and cognition
enhancement.5
Aryl and heteroaryl ketones have also been used effec-
tively as reversible inhibitors of serine proteases.10 In
the following text we will discuss two series of inhibitors,
the ketopyrrolidines (5a–n) and ketoazetidines (7a–i), as
potent DPP IV inhibitors. Structure–activity relation-
ships as well as stability studies and ex vivo DPP IV
inhibition experiments will be discussed below.
A common strategy for the inhibition of serine proteases
is to use substrate based analogs containing a chemically
reactive electrophile to covalently bind the catalytic ser-
ine at the active site. Examples of several electrophile-
and non-electrophile-based inhibitors of DPP IV are
outlined in Figure 1. These inhibitors can be divided into
three major categories based on their mode of inhibi-
tion: (1) competitive inhibitors such as pyrrolidine 1a
(IC50 = 125nM)6 and thiazolidine 1b (IC50 = 150nM);
The preparation of ketopyrrolidines 5 is outlined in
Scheme 1. Commercially available Weinreb amide 8
was alkylated with either an aryl lithium or an alkyl
Grignard reagent to afford the ketones 11a–h in 31–
72% yield. The deprotection and subsequent coupling
of the amino ketones 11a–h with various Boc-amino
acids 17–20 (Fig. 2) led to the Boc-protected ketopyrroli-
dines 14a–n in moderate yields (26–79%). The rapid
deprotection of compounds 14a–n followed by removal
of the solvent, triturating with Et2O, and filtration
Keywords: DPP IV; Diabetes; Serine protease.
*
Corresponding author. Tel.: +1 410 631 8126; fax: +1 410 631
6797; e-mail: ferrarisd@guilfordpharm.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.057

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D. Ferraris et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5579–5583
O
R
O
OH
B
OH
X
OPh
N
P
OPh
N
n
N
N
N
N
O
O
R1
O
O
O
NH
R2
NH2
NH2
NH2
NH2
4 (IC50 = 8nM)
1a (X = CH2, IC50 = 125nM)
1b (X = S, IC50 = 150nM)
2 (Ki = 15nM)
3 (Ki = 2nM)
5 (n = 1)
6 (n = 2)
7 (n = 0)
Figure 1. Examples of electrophile- and non-electrophile-based DPP IV inhibitors.
N O
O
R
O
R
R
ii
iii
i
N
Boc
N
Boc
N
O
N
O
n
n
n
n
Boc a.a.
a.a.
11a-h (n = 1)
12 (n = 2)
13a-b (n = 0)
8 (n = 1)
9 (n = 2)
10 (n = 0)
14a-n (n = 1)
15a-b (n = 2)
16a-i (n = 0)
5a-n (n = 1)
6a-b (n = 2)
7a-i (n = 0)
Scheme 1. Reagents and conditions: (i) RLi or RMgBr, THF, ꢀ78 to ꢀ40°C, 31–72%; (ii) (1) TFA (2) HATU, 17–20, DMA, DIEA, 26–79%;
(iii) 4.0M HCl in dioxane, 53–91%. See Supplementary data for experimental details.
binding mode, three variables were taken into account
O
O
to optimize the enzymatic potency0: (1) the nature of
the ketone Rgroup bound to the S1 site; (2) the hydro-
phobic S2 binding group; (3) the size and stereochemis-
try in the central ring, binding to the S1 site. Synthetic
efforts systematically addressed each of these factors
in turn and the results are outlined in Tables 1–3.
O
O
N
H
R
NH OH
OH
H2N
OH H2N
OH
19a (R = cyclohexyl, N-CHG)
19b (R = t-Butyl, N-tBuG)
17 (Ile)
20 (OIC)
18 (CHG)
Figure 2. Representative hydrophobic amino acids.
Potency data for several alkyl and aryl pyrrolidinyl ke-
tones are outlined in Table 1. The S2 binding group and
the ring size and stereochemistry (S1 binding group)
were kept constant while the ketone substituent was al-
tered. These results indicated that the heteroaryl ketones
(i.e., 2-thiazole 5a, 2-benzothiazole 5b, and 2-pyridyl 5c)
were necessary to gain substantial potency. The simple
methyl ketone 5d was inactive as was the phenyl deriva-
tive 5e. Interestingly, the 3-pyridyl derivative 5f was over
an order of magnitude less potent than the 2-pyridyl
afforded the ketopyrrolidines 5a–n.11 The ketopiperi-
dines 6a–b and ketoazetidines 7a–i were synthesized in
a similar manner to the ketopyrrolidines except that
the Weinreb amides 9 and 10 were synthesized from
either (R)- or (S)-Boc-azetidine-2-carboxylic acid or
(R)- or (S)-Boc pipecolic acid according to the
literature.12
2. Biological evaluation and SAR discussion
Table 1. Optimization of S10 binding groups for DPP IV inhibition
The binding mode for these proline-derived inhibitors is
outlined in Figure 3 based on previous studies with
other proline mimetics.7,13 The electrophile portion (E)
of the molecule resides in the S10 pocket while the cen-
tral ring lies in a very tight proline binding pocket
(S1). The hydrophobic amino acid side chain occupies
the spacious S2 pocket of the enzyme. Based on this
O
N
R
O
NH
HCl
Compounds
RGroup
2-Thiazole
IC (nM)a
50
5a
5b
5c
5d
5e
5f
44 ( 8)b
30 ( 5)b
2000
S1
S1'
2-Benzothiazole
2-Pyridiyl
Methyl
Ser630
HO
H
E
>100,000
>100,000
>100,000
50,000
N
n
H
N
Phenyl
3-Pyridyl
O
R1
NH
N
5g
5h
2-Thiophenyl
N-Methyl imidazole
Arg125
R2
H
>100,000
S2
a See Supplementary data for details of the DPP IV inhibitory assay.
b Average of three values.
Figure 3. Binding mode for proline-based DPP IV inhibitors.

D. Ferraris et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5579–5583
Table 2. Optimization of S2 binding groups for DPP IV inhibition
5581
O
α
N
N
n
S
a.a.
Compounds
n
Isomera
Amino acid group
IC50 (nM)b
5i
1
1
1
1
0
0
0
0
S
S
S
S
S
S
S
S
Ile
OIC
100
5b
5j
44 ( 8)c
50
300
CHG
N-CHG
Ile
5k
7a
7b
7c
7d
200
OIC
CHG
N-CHG
80 ( 14)c
42 ( 4)c
190 ( 10)c
a Denotes the stereochemistry at the a-position.
Figure 4. A model depicting the predicted binding mode of compound
5a.
b See Supplementary data for details of the DPP IV inhibitory assay.
c Average of three values.
Based on previous SARstudies of nitrile based DPP IV
inhibitors,7,8 a panel of Boc-protected hydrophobic ami-
no acids were coupled to the central ring. Compounds
17–20 (Fig. 2) are representative hydrophobic amino
acids since they have the substituents on the a-carbon,
for example, 17 (Ile) and 18 (CHG), on the amino acid
nitrogen, for example, 19a and 19b (N-CHG and N-t-
BuG) and on both the a-carbon and the nitrogen, for
example, 20 (OIC).
Table 3. Optimization of S1 binding group for DPP IV inhibition
O
α
n
N
Ar
a.a.
Compounds
n
Isomera Aryl group
Amino IC50 (nM)b
acid
group
The inhibitory data outlined in Table 2 indicates that the
two optimal amino acid groups for potency are OIC and
CHG. The most potent ketopyrrolidine derivatives are
5b and 5j while the most potent ketoazetidine derivatives
are 7b and 7c.
5l
1
1
2
2
0
0
0
0
S
R
S
R
S
R
S
R
Thiazole
Thiazole
Thiazole
Thiazole
Thiazole
Thiazole
CHG
CHG
CHG
CHG
CHG
CHG
42
1000
5m
6a
6b
7e
7f
>100,000
>100,000
30
40
80 ( 14)c
115
7b
7g
Benzothiazole OIC
Benzothiazole OIC
Table 3 outlines the differences in potency upon chang-
ing the size of the central ring (S1 binding group) and
the stereochemistry at the a-position. The six-membered
piperidines 6a and 6b were notably less potent than the
ketopyrrolidines 5l–m. The ketoazetidine 7e, however,
displayed a similar potency to the five-membered pyrrol-
idine 5l. The stereochemistry at the a-position is neces-
sary for potency within the ketopyrrolidine series as
the 2-(R) derivative 5m was less potent than its diaste-
reomer 5l. Consequently, both the size and stereochem-
istry results were mirrored in a similar series of DPP IV
inhibitors, the cyanopyrrolidines, and cyanopiper-
idines.7 Interestingly, the diastereomer of 7e, the 2-(R)
azetidine 7f, were equipotent, indicating that there was
no difference in potency between the 2-(R) and 2-(S)
diastereomers within the ketoazetidine series. Testing
the diastereomers 7b and 7g, both of which had simi-
lar potencies against DPP IV, reconfirmed this
observation.
a Denotes the stereochemistry at the a-position of the ring.
b See Supplementary data for details of the DPP IV inhibitory assay.
c Average of three values.
derivative 5c. In addition, the 2-thiophenyl derivative 5g
was three orders of magnitude less potent than the thiaz-
ole derivative 5a. Taken together, these results indicate
the necessity of a nitrogen atom in the 2-position of
the aryl group in order to maintain optimal potency.
This critical finding can be rationalized by examining a
model of the complex of 5a bound in the active site of
DPP IV (Fig. 4).13 The side chain of Arg125 forms a
hydrogen bond to the 2-thiazole nitrogen, as well as a
hydrogen bond to the prolyl amide oxygen. The forma-
tion of this extra hydrogen bond with the thiazolidine
could explain the difference in potency between the 2-
thiazole and 2-thiophenyl derivatives 5a and 5g, as well
as the 2- and 3-pyridyl derivatives 5c and 5f. Two other
prominent features seen in this protein–ligand model are
as follows: (1) the proximity of the ketone with the cata-
lytic serine (Ser630) and (2) the interaction the ketone
oxygen with residues in the oxyanion hole of DPP IV
(i.e., Tyr547 side chain and Tyr631 backbone).
3. Stability data and ex vivo experiments
Despite the potency of these amino ketones, major
issues involving their stability had to be addressed. A
simple intramolecular cyclization can occur between
the amine and ketone forming an imine bond. This

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D. Ferraris et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5579–5583
N
N
N
S
N
O
N
N
S
N
S
-H2O
[O]
N
NH2
O
O
O
21
5l
Scheme 2. Cyclization/oxidation of ketopyrrolidine 5l.
Table 4. DPP IV inhibition of sterically hindered ketones 7g and 7h
Compounds
n
Aryl groupa
Amino acid group
Time at rt (h)
Estimated % ketoneb
IC50 (nM)c
5l
1
1
Tzl
Tzl
CHG
CHG
0.25
24
95
<5
0
30
>20,000
>100,000
80
5l
21
7g
7g
7g
7h
7h
7h
0
0
0
0
0
0
Bntzl
Bntzl
Bntzl
Bntzl
Bntzl
Bntzl
OIC
OIC
0.25
24
99
75
55
99
75
70
250
500
OIC
168
N-t-BuG
N-t-BuG
N-t-BuG
0.25
168
504
100
200
900
a Tzl = thiazole, Bntzl = benzothiazole.
b Measured by 1H NMRintegration.
c See Supplementary data for details of the DPP IV inhibitory assay.
intermediate can either revert back to the parent ketone
in a reversible manner or oxidize into the dihydroketo-
pyrazine as outlined in Scheme 2. The major product
(>90%) of the decomposition of 5l is indeed the dihydro-
Compound 7g
100
80
60
40
20
1
ketopyrazine 21 as evidenced by H NMR, MS, and
Plasma
Brain
CHN (IC50 > 100lM). This conversion appears to be al-
most stoichiometric with minimal byproducts visible by
1H NMRand HPLC. Consequently, the azetidinyl
derivative 7g displayed a minimal increase in stability
with an estimated cyclization at 24h of 55% (vs 95%
for compound 5l, Table 4). This improvement was fur-
ther amplified by the N-t-BuG derivative 7h. The bulky
secondary tert-butyl amine reacts very sluggishly with
the internal aryl ketone and DPP IV inhibitory potency
is maintained over several days at room temperature
(Table 4).
0
0
2
4
6
8
Time after dose p.o. 50 mg/kg
(hours)
Figure 5. Ex vivo experiment with compound 7g.
tency in the ketopyrrolidine series, but not in the ketoaze-
tidine series. Finally, certain stabilized ketoazetidines
such as 7g can maintain their in vitro potency and inhi-
bit DPP IV in the plasma for up to 6h.
Ex vivo experiments were done in order to determine the
relative potency of these derivatives in the plasma and
brain of Sprague-Dawley rats. Animals were dosed
p.o. with compound 7g (50mg/kg). One animal was
sacrificed at each time point followed by collection of
both plasma and brain samples. As Figure 5 illustrates,
compound 7g displays sufficient inhibitory potency
against DPP IV over a 6h period in the plasma.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2004.08.057.
In conclusion, structure–activity relationships of the
ketopyrrolidines and ketoazetidines have determined
that the 2-thiazole, 2-benzothiazole, and 2-pyridylke-
tones are optimal S10 binding groups for potency against
DPP IV. This finding can be rationalized from the dock-
ing studies and crystal structure model. In addition,
both cyclohexyl glycine and octahydroindole carboxy-
late serve as potent S2 binding groups. Stereochemistry
at the a-position of the central ring is relevant to po-
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