Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of prolyl oligopeptidase (POP)

Article abstract of DOI:10.1016/j.bmcl.2008.06.067

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Full text of DOI:10.1016/j.bmcl.2008.06.067

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4360–4363
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of
prolyl oligopeptidase (POP)
a
b
b
b
Curt D. Haffner a, , Caroline J. Diaz , Aaron B. Miller , Robert A. Reid , Kevin P. Madauss ,
*
Annie Hassell b, Mary H. Hanlon c, David J. T. Porter c, , J. David Becherer c, Luke H. Carter d
a Department of Medicinal Chemistry, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA
b Department of Computational and Structural Chemistry, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA
c Department of Biochemistry and Analytical Pharmacology, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA
d Department of Biological Reagents and Assay Development, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as
potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized
within the catalytic site of a human chimeric POP protein which provided a more detailed understanding
of how these inhibitors interacted with the key residues within the catalytic pocket.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 27 May 2008
Revised 18 June 2008
Accepted 19 June 2008
Available online 24 June 2008
Keywords:
Prolyl oligopeptidase
Serine protease
Pyridone
pyrazinone
Pyrrolidine
Prolyl endopeptidase
Prolyl oligopeptidase (POP: E.C. 3.4.21.26) is a serine protease
first identified in 1971 in human uterus as an oxytocin-cleaving
enzyme.1 It has since been found to be widely expressed in various
tissues with the highest concentrations found in the brain.2 Three
different types of POP have been described in mammals: cytoplas-
mic,2c,e,3 serum,4 and membrane.5 Although POP has been thor-
oughly characterized enzymatically and structurally its exact
physiological role still remains obscure. However, due to its struc-
ture, which includes a b-propeller domain, POP catalysis is con-
trolled by a gating filter mechanism which only allows smaller
proteins to gain access to the catalytic site (30 amino acids or
less).6 POP has been shown to cleave internal proline containing
peptides at the C-terminus.7 A number of bioactive peptides which
contain an internal proline have been identified as potential sub-
strates for POP, although many of these have not been validated
in vivo. Interestingly, many of these are neuropeptides, such as
substance P, thyrotropin releasing hormone, arginine vasopressin
and oxytocin.8 It is not surprising then that studies have linked
POP to various neurodegenerative disorders like bipolar disorder,
amnesia, and Alzheimer’s and Parkinson’s disease.9 It has also been
shown to be involved in inositol-1,4,5-triphosphate signaling.8 Not
surprisingly this has led, since the late 1970s, to the search for spe-
cific potent inhibitors of POP. Indeed, many small molecule POP
inhibitors have now been reported,10 for example, Z-pro-prolinal,11
S-1709212 and JTP-481913 (Fig. 1).
We report herein our efforts to identify novel POP inhibitors.
The goal was to not only identify potent novel inhibitors, but at-
tempt to remove as much of the peptidic nature as possible from
these inhibitors. Many of the previously reported POP inhibitors
H
N
N
O
N
N
O
O
N
O
O
O
OHC
Z-Pro-prolinal
OH
JT-4819
Ph
O
H
H
O
N
N
S
* Corresponding author. Tel.: +1 919 483 6247; fax: +1 919 315 0430.
E-mail address: curt.d.haffner@gsk.com (C.D. Haffner).
David Porter has since retired from GlaxoSmithKline.
S-17092
Figure 1. Examples of known POP inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.067

C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4360–4363
4361
O
O
O
O
O
PhSO2
d
a or b, c
HN
MeO2C
c
N
a, b
N
MeO2C
R1
1
2
N
CO2Me
HO
O
R1
O
O
CO2Me
O
27, R1 = H
28, R1 = F
O
e, f
g, h, i
PhSO2
4
N
N
O
O
N2
MeO2C
HO
CO2Me
3
4, R1 = C(O)Me
5, R1 = SO2Ph
d, e
N
N
O
R1
O
N
CO2Me
R1
O
R1
31, R1 = H
32, R1 = F
j
29, R1 = H
30, R1 = F
N
(for R1 = SO2Ph)
R3
R2O
N
O
R2O
R3
O
O
O
Scheme 2. Reagents and conditions: (a) Tf2NPh, NEt3, CH2Cl2, 0 °C to rt; (b) 2-
arylvinylboronic acid, dppfꢀPdCl2ꢀCH2Cl2, 2 M aq Na2CO3, toluene, EtOH, 80 °C; (c)
H2 (1 atm), dioxane/EtOH; (d) LiOHꢀH2O, dioxane/water; (e) pyrrolidine, HATU,
DIEA, DMF.
20-26
6-19
Scheme 1. Reagents and conditions: (a) (phenylsulfonyl)acetyl chloride, benzene,
reflux; (b) (phenylthio)acetyl chloride, benzene, reflux; (c) oxone, MeOH/H2O; (d)
4-acetamidobenzenesulfonyl azide, NEt3, CH3CN, 0 °C to rt; (e) Rh2(OAc)4, methyl
vinyl ketone or phenyl vinyl sulfone, benzene, reflux; (f) p-TsOHꢀH2O, benzene,
reflux; (g) R2Br, K2CO3, DMF, 105 °C; (h) LiOHꢀH2O, dioxane/water; (i) HATU, DIEA,
DMF, pyrrolidine or pyrazole; (j) Raney Ni 2800, ethanol, reflux.
tives 29 and 30. These were then converted to the pyrrolidinyl car-
boxamides 31 and 32, employing the same conditions as described
previously (Scheme 2).
Based on the modeling of the pyridone in the catalytic site, it
appeared that insertion of nitrogen at the 4 position of the pyri-
done ring would not be deleterious in regard to POP inhibitory
activity. In order to test this hypothesis the pyrazinone core was
synthesized utilizing the known procedure.16 The synthesis started
utilize an acyl-prolyl-pyrrolidine structure, although there are
reports where P2 prolyl mimetics have been used successfully to
identify potent inhibitors.10e,g Our initial synthetic efforts utilized
a structure based design (SBD) approach using the published
liganded porcine structure (1QFS) reported by Polgar et al.6a This
entailed inspection of this X-ray crystal structure, utilization of
reported SAR, and new chemotype generation followed by a dock-
ing calculation using the program MVP.14 Results of the docking
calculation were visually inspected, and ideas were docked
iteratively until a satisfactory solution was generated. One of the
chemotypes that was designed via this approach was the pyridone
template shown in Scheme 1. It was felt that this chemotype would
accomplish two things: (1) remove some of the peptidic nature of
the molecule and (2) lock the P2-P3 portion of the molecule there-
by rigidifying it and making it more drug-like.
with the ethyl ester of L-pyrroglutamic acid which was protected
with di-tert-butyldicarbonate under basic conditions followed by
reduction with LiBEt3H in THF and methanolysis in the presence
of a catalytic amount of sulfuric acid to give BOC protected pyrrol-
idine 34. Pyrrolidine 34 was then treated with trimethylsilyl cya-
nide and BF3ꢀEt2O followed by exposure to TFA removing the
BOC protecting group yielding cyanopyrrolidine 35. The crude
material was then condensed with oxalyl chloride in toluene at
85 °C providing 3,5-dichloropyrazinone 36. The resulting pyrazi-
none was then hydrolyzed (LiOHꢀH2O, dioxane, H2O) followed by
amide formation as described previously to generate pyrrolidinyl
carboxamide 37. Nucleophilic addition via alcohols or amines oc-
curred exclusively at the C-3 position of the pyrazinone ring pro-
ducing the desired 5-chloropyrazinones 38–54 (Scheme 3).
The synthesis into these compounds utilized the prior work re-
ported by Padwa et al.15 Starting with the methyl ester of
L-pyrro-
glutamic acid 1, acylation with (phenylthio)acetyl chloride
followed by sulfide oxidation to the sulfone via oxone provided
compound 2. It was later found that the commercially available
(phenylsulfonyl)acetic acid when converted to the acid chloride
(benzene, oxalyl chloride) followed by acylation as before provided
2 in very good yield (85% vs 45%), thus eliminating the oxone oxi-
dation step which proved to be somewhat capricious in our hands.
The diazoimide was then synthesized via a diazo transfer reaction
with 4-acetamidobenzenesulfonyl azide under basic conditions
yielding diazosulfone 3. The dipolar cycloaddition was carried
out as previously described15 using Rh2(OAc)4 in refluxing benzene
whereby the dipolarophile chosen was either methyl vinyl ketone
or phenyl vinyl sulfone. Interestingly, it was found that after sev-
eral hours of reflux, removal of the catalyst followed by the addi-
tion of a catalytic amount of pTsOHꢀH2O and additional reflux
provided more consistent yields of the desired pyridones 4 and 5.
The 3-hydroxypyridones were then alkylated (K2CO3, DMF,
105 °C) followed by ester hydrolysis and amide formation (HATU,
DMF, iPr2NEt) providing carboxamides 6–19. The phenylsulfone
moiety could then be removed under Raney nickel conditions gen-
erating the C-5 hydridopyridones 20–26. In order to gain access to
the C-3 carbon linked analogues, the 3-hydroxypyridone was tri-
flated and then reacted under Suzuki conditions to provide the
styrenyl products 27 and 28. The alkene was then hydrogenated
under an atmosphere of hydrogen yielding the phenethyl deriva-
OMe
O
d,e
BOC
a, b, c
N
HN
EtO2C
EtO2C
34
33
Cl
N
CN
g, h
f
HN
N
Cl
CO2Et
Cl
EtO2C
O
36
35
Cl
i or j
N
N
R
N
N
X
Cl
O
O
38-54
N
O
N
O
37
Scheme 3. Reagents and conditions: (a) Boc2O, DMAP, CH3CN, 0 °C to rt; (b)
LiEt3BH, THF, ꢁ78 °C; (c) H2SO4, MeOH; (d) Me3SiCN, BF3ꢀEt2O, CH2Cl2, ꢁ78 °C; (e)
TFA, CH2Cl2; (f) (COCl)2, toluene, 85 °C; (g) LiOHꢀH2O, dioxane/water; (h) pyrrol-
idine, HATU, DIEA, DMF; (i) ROH, K2CO3, DMF (100 °C) or CH3CN (reflux); (j) RNH2,
EtOAc, reflux.

4362
C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4360–4363
Table 1
Table 2
Prolyl oligopeptidase inhibiton data for pyridones 6–26, 31 and 32
Prolyl oligopeptidase inhibition data for pyrazinones 37–54
R1
Cl
N
N
N
R2
R
R3
O
O
N
O
O
SEa (nM)
a
Compound R1
R2
R3
IC50 (nM) SEb (nM)
Compound
R
IC50 (nM)
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Cl
OPh
OCH2-4-F-Ph
OCH2-3,4-Cl-Ph
OCH2-3,4-F-Ph
OCH2-3-Cl-4-F-Ph
OCH2-2-naphthalene
O(CH2)2-3,4-Cl-Ph
OCH2-4-pyridyl
O(CH2)2-4-F-Ph
OCH2-Cyclohexyl
OCH2-4-CF3-Ph
OCH2-2,3,5-F-Ph
OCH2-3-Cl-Ph
NH(CH2)2Ph
NHCH2Ph
NHCH2-4-F-Ph
NHCH2-3,4-F-Ph
3100
1000
40
34
55
600
300
0.6
5
4
3
1000
300
100
200
20
10
10
5
S-17092
6
7
8
9
—
—
—
2
584
53
28
140
48
783
22
7
0.3
100
10
4.1
10
50
200
4
3
3
6
0.5
2
40
60
6
5.8
2
7
5
10
6
0.5
1
C(O)Me
C(O)Me
C(O)Me
C(O)Me
C(O)Me
C(O)Me
C(O)Me
C(O)Me
C(O)Me
C(O)Me
SO2Ph
SO2Ph
SO2Ph
C(O)Me
H
OCH2CF3
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrazole
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
OCH2-4-F-Ph
OCH2-4-CF3-Ph
OCH2-4-OMe-Ph
OCH2-4-Cl-Ph
OCH2-4-CN-Ph
OCH2-3,5-F-Ph
OCH2-3,4-F-Ph
OCH2-3-Cl-4-F-Ph
OCH2-3,4-Cl-Ph
OCH2-3,5-F-Ph
OCH2-4-CF3-Ph
OCH2-4-tBu-Ph
OCH2-4-F-Ph
OCH2-4-F-Ph
OCH2-3,5-F-Ph
OCH2-3,4-F-Ph
OCH2-2,5-F-Ph
OCH2-4-CF3-Ph
OCH2-4-tBu-Ph
OCH2-2,4-F-Ph
(CH2)2Ph
43
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
31
32
4000
2200
800
900
70
250
40
30
80
12
17
15
8
28
4
11
270
343
22
22
18
64
34
60
31
2
10
1
2
H
H
H
H
H
H
3
a
Standard error.
hydrophobic S1 pocket presumably occupying the same space as
proline containing endogenous substrates. There also appear to
be hydrogen bond interactions between the carboxamide carbonyl
and Arg-643, and the pyrazinone carbonyl and Trp-595. The struc-
ture also shows the S3 portion of the protein to be quite hydropho-
bic, with the 4-fluorophenyl substituent appearing to fit nicely into
this hydrophobic pocket. The C-5 chloro substituent occupies the
large cavity extending away from the catalytic site. This presum-
ably is the same region occupied by both the methyl ketone and
phenyl sulfone from the pyridone series, perhaps explaining why
they were equally tolerated versus their hydrogen counterparts.
This also could be an area which lends itself to further modifica-
tions in the inhibitor to improve its overall drug properties as
needed.
C(O)Me
C(O)Me
(CH2)2-4-F-Ph
3
a
A brief description of the conditions used to determine the IC50’s can be found
in Ref. 17.
b
Standard error.
The data shown in Table 1 compare the in vitro activity of the
pyridone analogues. Many of the compounds showed low nanomo-
lar potency against POP. The substituent at R1 tolerated bulk as can
be seen by comparing compounds 12 and 16 versus 21. In fact,
compound 16 which has the greatest bulk was 5-fold more active
than compound 21. We also found that replacing C for O at R2 in-
creased potency by 18-fold (compound 7 vs compound 32). Fur-
thermore, replacing the pyrrolidine ring in compound 7 with a
pyrrazole in compound 19 lowered activity by 6.5-fold. It does ap-
pear however that there are steric and electronic limitations within
the aryl groups substituted off the R2 position. Compounds 9 and
11 which contain either a 4-methoxy or 4-cyano moiety, respec-
tively, are less active than those analogues containing less polar
substituents such as the fluoro or trifluoromethyl groups. It was
also noted that compound 18 was 25-fold less active than com-
pound 17 suggesting possible steric limitations in this part of the
protein.
The data in Table 2 compare the in vitro activity of the pyrazi-
none analogues. As the R substituent increased in length the po-
tency increased as well as can be seen by comparing compounds
37, 38 and 39. However, as was seen in the pyridone series, there
were steric limitations as can be seen when comparing compounds
40 versus 44 and 39 versus 46 (65- and 23-fold difference, respec-
tively, in potency). The nitrogen linked analogues were also syn-
thesized and tested (compounds 51–54). All were quite potent,
but similar to that observed for the oxygen analogues, a drop in po-
tency was seen in the phenethyl derivative 51. There did appear to
be a small increase in potency when going from the O-linked to N-
linked compounds (54 vs 41 and 53 vs 39).
Figure 2. X-ray co-crystal structure of pyrazinone 39 (carbons highlighted in
darker green) bound at the catalytic site of the human POP protein. POP carbons are
colored in lighter green with the catalytic triad residues highlighted in magenta.
The semi-transparent white surface represents the molecular surface while
hydrogen bonds are depicted as yellow dashed lines. The coordinates have been
deposited in the Brookhaven Protein Data Bank, accession number 3DDU. This
figure was generated using PyMOL version 1.02 (Delano Scientific, www.
pymol.org).
We were also able to solve a single X-ray crystal structure of
compound 39 bound to a human chimeric protein (Fig. 2). As can
be seen from the structure, the pyrrolidine ring fits into the small

C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4360–4363
4363
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In conclusion, we have utilized a SBD approach to identify novel
pyrrolidinyl pyridone and pyrazinone analogues, many of which
were highly potent inhibitors, of POP. We were also able to solve
a liganded crystal structure with compound 39 bound into the cat-
alytic site. This structure has assisted in the understanding of how
these compounds interact with the protein. Undoubtedly, this
structure will provide valuable insight into further inhibitor de-
sign. We also found that the in vivo PK properties of some of these
inhibitors provided higher plasma and brain levels than S-17092
(data not reported) which may provide better tool molecules for
further validation work.
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was assayed in 15 mM potassium phosphate, pH 7.1, with 25
Pro-pNA (substrate). Cleavage of the peptide by POP was observed by the
increase in absorbance at 385 nm. Compound (1 in DMSO) was
assay buffer plus enzyme prior to
L substrate in assay buffer. The equation
lM Z-Gly-
l
L
incubated for 15 min in 50
initiation of the assay with 50
lL
l
Y ¼ Vmax ꢂ ð1 ꢁ ðxn=ðIC5n0ÞÞ þ Y2 was fit to the rate data from the
concentration response curves where Y2 is the background (no enzyme)
rate, Vmax is the uninhibited rate—Y2, and IC50 is the concentration of
compound at which the initial rate is Y2 + (Vmax/2).