Discovery of novel conformationally restricted diazocan peptidomimetics as inhibitors of interleukin-1β synthesis

Article abstract of DOI:10.1016/j.bmcl.2005.06.050

A novel diazocan containing dipeptide mimetic was synthesized via reductive N-N bond cleavage of a pyrazolidino-pyrazolidine using Raney-Ni and evaluated as an ICE inhibitor. This versatile 8-membered ring containing scaffold possesses an N-5 ring nitroge

Full text of DOI:10.1016/j.bmcl.2005.06.050

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4291–4294
Discovery of novel conformationally restricted diazocan
peptidomimetics as inhibitors of interleukin-1b synthesis
Kofi A. Oppong,* Christopher D. Ellis, Michael C. Laufersweiler, Steven V. OÕNeil,
Yili Wang, David L. Soper, Mark W. Baize, John A. Wos, Biswanath De,
Gregory K. Bosch, Amy N. Fancher, Wei Lu, Maureen K. Suchanek,
Richard L. Wang and Thomas P. Demuth, Jr.
Procter & Gamble Pharmaceuticals, Inc., 8700 Mason-Montgomery Road, Mason, OH 45040, USA
Received 3 May 2005; revised 16 June 2005; accepted 20 June 2005
Available online 19 July 2005
Abstract—A novel diazocan containing dipeptide mimetic was synthesized via reductive N–N bond cleavage of a pyrazolidino-pyr-
azolidine using Raney-Ni and evaluated as an ICE inhibitor. This versatile 8-membered ring containing scaffold possesses an N-5
ring nitrogen that was used to explore structure-activity relationships in a cell-based assay measuring inhibition of interleukin-1b.
ꢀ 2005 Elsevier Ltd. All rights reserved.
In recent years, there has been a substantial effort to-
ward the development of therapeutic agents in the area
of arthritis.1 The cytokine (interleukin-1b), a critical
mediator in the inflammatory pathway, has been impli-
cated in the progression of this disease, and one method
for modulating the levels of the IL-1b is through inhibi-
tion of IL-1b converting enzyme (ICE).2,3 ICE, also
known as caspase-1, is a member of the caspase (cysteine
aspartate proteases) family of enzymes which require an
aspartic acid residue for recognition and is responsible
for the processing of IL-1b from an inactive pro-form
into the mature cytokine.3 It has been recently reported
that a selective peptide ICE inhibitor blocks IL-1b pro-
duction in a human cartilage explant culture.4 Further-
more, ICE inhibitors show both anti-inflammatory
and analgesic effects in animal models.5 The validity of
IL-1 modulation as a therapeutically relevant approach
gained additional credibility with the FDA approval of
Kineretꢁ, an injectable IL-1 receptor antagonist for
rheumatoid arthritis (RA) therapy.6
4 lM.7 A widely utilized approach for the development
of ICE inhibitors has been to constrain the Val-Ala por-
tion (Fig. 1, structure 2) of the tetrapeptide, thereby
maintaining the important hydrogen bond interactions
of the peptide backbone and properly orienting the
important P1 and P4 recognition regions of the molecule
in the enzyme pocket.8 One notable example of the
application of this strategy is Pralnacasanꢁ 3, a revers-
ible ICE inhibitor, which constricts the P2–P3 region of
5
the molecule as a pyridazinodiazepine. Pralnacasanꢁ
progressed into clinical trials,9 but the phase II program
was discontinued due to liver abnormalities in dogs after
a nine-month exposure to a high dose of Pralnacasanꢁ.
In this article, we describe the development of a novel
diazocan peptidomimetic 4, and the evaluation of it as
an ICE inhibitor. Our approach to this design was guid-
ed by four main criteria: (a) preservation of key binding
interactions with ICE, (b) structural novelty of scaffold,
(c) ease of synthesis, and (d) the ability to explore new
binding interactions in the S3 region of the enzyme
pocket. In addition, we believed that the secondary
nitrogen at N-5 may also serve as a handle for optimiza-
tion of the physical/chemical properties of the
molecule.10
At present, most small molecule inhibitors of ICE
have been based on the tetrapeptide (Ac-YVAD-CHO)
1 (Fig. 1), which is reported to block the release of IL-
1b from human whole blood cells with an IC50 of
Monocyclic 8-membered lactams have generally been
elusive targets due to the inability to overcome entropic
barriers associated with traditional lactam formation.11
Our approach hinged on a reductive N–N bond scission
Keywords: ICE; Caspase-1; Diazocan; Peptidomimetic; Interleukin-1b.
Corresponding author: Tel.:+1 513 622 0481; fax:+1 513 622 5338;
e-mail: Oppong.ka@pg.com
*
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.050

4292
K. A. Oppong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4291–4294
Figure 1.
of the intermediate pyrazolidino-pyrazolidine (8) with
Raney-Ni.12
cursor 15.15 Final acid hydrolysis of the acetal of 16 af-
fords the final compound 4.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC) coupling of pyrazolidine 6 with Boc-
(L)-serine led to 7 in 75% yield (Scheme 1). Standard
Mitsunobu13 reaction conditions were employed to con-
vert alcohol 7 into the intermediate pyrazolidino-pyraz-
olidine (8), which is carried forward without isolation
into the next sequence of reactions. Reductive cleavage
of the N–N bond in 814 with Raney-Ni in isopropanol
at 50 ꢂC for 2 h led to isolation of target diazocan inter-
mediate 9 in 75–84% yield over two steps. Dipeptide 11
was obtained by a sequence that involved initial protec-
tion of the secondary amine as the benzyl carbamate,
followed by alkylation of the amide nitrogen with ethyl
bromoacetate.
Using SAR developed for another series of ICE inhibi-
tors,16 we synthesized compounds bearing a small num-
ber of P4 substituent groups (18–23, Table 1), keeping
the N-5 substituent as a benzamide. For screening pur-
poses, we selected a THP-117 whole-cell assay measuring
IL-1b production as our primary tool to evaluate poten-
cy. Since ICE is an intracellular target, we believed that
a whole cell assay, taking into account properties such
as cell permeability, would be more predictive of the fu-
ture success in animal models. Among the six analogs
screened, compound 21 distinguished itself as the most
active with an IC50 of 205 nM.18 With these results in
hand, we retained the naphthyl amide as the preferred
P4 substituent and made variations at N-5 (24–36, Table
1). We observed a slight preference at this position for
small amide substituents, as exemplified by the formam-
ide (33) and the acetamide (34). The morpholino-urea
substituent (36) also proved to be a potent inhibitor in
the THP whole-cell assay at 224 nM. Overall, none of
the substituents screened at N-5 showed a remarkable
improvement compared to the parent benzamide (21).
A selection of diazocan ICE inhibitors was screened in
caspase-1, -3, and -819 enzyme inhibitor assays to
confirm their ability to inhibit ICE and to assess the
Conversion of dipeptide 11 into an ICE inhibitor (Scheme
2) involved an initial deprotection of the Boc-carbamate
with TFA in CH2Cl2, followed by acylation of the amine
with a variety of aryl acids or aryl acid chlorides. Standard
deprotection of the benzyl carbamate with H2/Pd-C was
followed by conversion of the resultant secondary amine
into a variety of amides and ureas. The final sequence in-
volved initial base hydrolysis of the ester of 13 using
LiOH, followed by EDC coupling to the cysteine trap pre-
Scheme 1.

K. A. Oppong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4291–4294
4293
Scheme 2.
Table 1. Diazocan SAR
Compound
Ar
R1
THP-1 IC50 (nM)
Casp-1 IC50 (nM)
Casp-3 IC50 (nM)
Casp-8 IC50 (nM)
18
19
20
21
22
23
24
25
27
28
29
30
31
32
33
34
35
36
3
Ph
3-CF3Ph
Ph
Ph
1015
1019
298
5-Cl-2-MePh
2-Naphthyl
1-Isoquinoline
2-Benzothiophene
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
2-Napthyl
Pralnacasanꢁ
Ph
Ph
205
280
19
30
>10,000
>10,000
>10,000
>10,000
Ph
Ph
560
246
Ph(2-OMe)
Ph(3-OMe)
Ph(3-F)
Ph(2-CF3)
Ph(3-CF3)
Ph(5-Cl-2-Me)
Piperonyl
CH2Ph
H
38
15
22
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
229
397
1525
1736
>2500
285
24
>10,000
>10,000
>10,000
>10,000
808
219
Me
Cyclohexyl
N-morpholine
216
577
224
120
44
2
selectivity for caspase-1 over other caspases. Overall, the
diazocan inhibitors showed an excellent selectivity pro-
file against caspase-3 and -8. The most active inhibitor
21 was less potent than Pralnacasanꢁ in the caspase-1
enzyme assay, but showed comparable activity in the
THP-1 whole-cell assay.
In summary, we have developed an efficient synthesis of
a potent novel diazocan ICE inhibitor starting from
Boc-serine and pyrazolidine. Though the diazocan scaf-
fold possessed a versatile secondary nitrogen at N-5,
variations at this position had little effect on the
observed activity. In the THP whole cell assay, we

4294
K. A. Oppong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4291–4294
14. The intermediate 8 is stable to column chromatography
but decomposes over a period of one week when kept neat
at room temperature. However it can be stored for
extended periods as a cold solution in isopropanol.
15. Batchelor, M. J.; Bebbington, D.; Bemis, G. W.; Fridman,
W. H.; Gillespie, R. J.; Golec, J. M. C.; Gu, Y.; Lauffer,
D. J.; Livingston, D. J.; Matharu, S. S.; Mullican, M. D.;
Murcko, M. A.; Murdoch, R.; Nyce, P. L.; Robidoux, A.
L. C.; Su, M.; Wannamaker, M.; Wilson, K. P.; Zelle, R.
E. PCT Intl. Appl. WO9722619, 1997.
16. Soper, D. L.; OÕNeil, S. V.; Wang, Y.; Oppong, K. A.;
Ellis, C. D.; Laufersweiler, M. C.; Demuth, T. P.; Fancher,
A. N.; Lu, W.; Wang, R. L.; Schwecke, W. P.; Cruze, C.
A.; Buchalova, M.; Belkin, M.; Wos, J. A. Abstracts of
Papers, 228th National Meeting of the American Chem-
ical Society, Philadelphia, PA; American Chemical Soci-
ety: Washington DC 2004; Abstract U956.
identified the naphthalene-2-carboxylic acid diazocan
amide (21)20 as the most potent inhibitor of ICE in this
series, comparable to Pralnacasanꢁ. We plan to discuss
data from ongoing in-vivo studies in the near future.
References and notes
1. For a review on IL-1 biology, see: (a) Dinarello, C. A.;
Wolff, S. M. N. Engl. J. Med. 1993, 328, 106; (b) Pelletier,
J. P.; DiBattistal, J. A.; Roughley, P.; McCollum, R.;
Martel-Pelletier, J. Rheum. Dis. Clin. North Am. 1993, 19,
545; (c) Fernandez-Madrid, F.; Karvonen, R. L.; Teitge,
R. A.; Miller, P. R.; Negendank, W. G. J. Magn. Reson.
Imaging 1994, 12, 703.
2. (a) Dinarello, C. A.; Wolff, S. M. N. Engl. J. Med. 1993,
328, 106; (b) Ator, M. A.; Dolle, R. E. Curr. Pharm. Des.
1995, 1, 191.
3. (a) Wilson, K. P.; Black, J.; Thompson, J. A.; Kim, E. E.;
Griffith, J. P.; Navia, M. A.; Murcko, M. A.; Chambers, S.
P.; Aldape, J. P.; Raybuck, S. A.; Livingston, D. J. Nature
1994, 370, 270; (b) Sleath, P. R.; Hendrickson, R. C.;
Kronheim, S. R.; March, C. J.; Black, R. A. J. Biol. Chem.
1990, 265, 14526.
4. Saha, N.; Moldovan, F.; Tardif, G.; Pelletier, J. P.;
Cloutier, J. M.; Martel-Pelletier, J. Arthritis Rheum. 1999,
42, 1577.
5. Rudolphi, K.; Gerwin, N.; Verzijl, N.; Van der Kraan, P.;
Van den Berg, W. Osteoarthritis and Cartilage 2003, 11, 738.
6. Bresnihan, B.; Alvaro-Gracia, J. M.; Cobby, M.; Doherty,
M.; Domljan, Z.; Emery, P.; Nuki, G.; Pavelka, K.; Rau,
R.; Rozman, B.; Watt, I.; Williams, B.; Aitchison, R.;
McCabe, D.; Musikic, P. Arthritis Rheum. 1999, 42, 1781.
7. Thornberry, N. A.; Peterson, E. P.; Zhao, J. J.; Howard,
A. D.; Griffin, P. R.; Chapman, K. T. Biochemistry 1992,
33, 3934.
8. Dolle, R. E.; Prasad, C. V. C.; Prouty, C. P.; Salvino, J.
M.; Awad, M. M. A.; Schmidt, S. J.; Hoyer, D.; Ross, T.
M.; Graybill, T. L.; Speier, G. J.; Uhl, J.; Miller, B. E.;
Helasek, C. T.; Ator, M. A. J. Med. Chem. 1997, 40, 1941.
9. Randle, J. C. Abstracts of Papers, 228th National Meeting
of the American Chemical Society, Philadelphia, PA;
American Chemical Society: Washington, DC, 2004;
Abstract U944.
17. Inhibition of IL-1b production in a cell-based assay: A
suspension of human monocytic cells (THP-1, ATCC
strain TIB202, 2 · 106/ml in RPMI 1640 medium from
Gibco BRL) was plated in 96-well plates, incubated with
or without compounds (administered as solutions in
DMSO, such that test concentrations ranged from 1 nM
to 10 lM) for 15 min, and then stimulated with LPS (1 lg/
ml) for a total of 4 h. Cells were centrifuged and the
conditioned media was collected to quantify the release of
IL-1b by an ELISA measurement according to the
manufacturerÕs instructions (R&D Systems, catalog num-
ber DLB50) or stored at À20 ꢂC for future use.
18. The values obtained for the free drug form of Pralnaca-
sanꢁ in our assays were comparable to those reported in
similar bioassays see Rudolphi, K.; Gerwin, N.; Verzijl,
N.; van der Kraan, P.; van den Berg, W. OsteoArthritis
Cartilage, 2003, 11, 738 reference 5.
19. Inhibition of caspase-1, -3, and -8 enzymes: The isolated
caspase enzyme (caspase-1, -3, and -8) assays were
performed in a 96-well format using fluorogenic sub-
strates, enzymes and control peptide inhibitors purchased
from BioMol Research Laboratories (Plymouth Meeting,
PA, USA). The assay was conducted according to the
manufacturerÕs instructions. Enzyme inhibition was mon-
itored over 30 min at 37 ꢂC by measuring fluorescence
using a BMG Fluostar plate reader (excitation filter
390 nm, emission filter 460 nm). IC50 values were calcu-
lated based on the equation IC50 = [I] / (Vo/Vi) À 1, where
Vi was the initial velocity of substrate cleavage in the
presence of inhibitor at concentration [I], and Vo was the
initial velocity in the absence of inhibitor.
10. Hanessian, S.; McNaughtonSmith, G.; Lombart, H. G.;
Lubell, W. D. Tetrahedron 1997, 53, 12789.
`
11. (a) Hocquemiller, R.; Cave, A.; Husson, H. P. Tetrahedron
1977, 33, 653; (b) Evans, P. A.; Holmes, B. Tetrahedron
1991, 47, 9131; (c) Nubbemeyer, U. Top. Curr. Chem.
2001, 216, 125; (d) Maier, M. E. Angew. Chem. Int. Ed.
2000, 39, 2073.
20. 1H NMR (CD3OD): d 8.38 (s, 1H), 7.97–7.85 (m, 4H),
7.62–7.48 (m, 8H), 5.60 (dd, J = 11.1, 3.9 Hz, 1H), 4.66
(dd, J = 6.0, 3.9 Hz, 1H), 4.48–4.25 (m, 2H), 3.99–3.68 (m,
4H), 3.50 (m, 1H), 2.73 (ddd, J = 16.2, 6.3, 5.4 Hz, 1H),
2.56 (ddd, J = 16.2, 8.1, 2.7 Hz, 1H), 2.38 (bs, 1H), 2.01
(bs, 1H); FAB HRMS 559.217560 (M + H)+.
12. Martinez-Perez, J. A.; Pickel, M. A.; Caroff, E.; Woggon,
W. Syn. Lett 1999, 12, 1875.
13. Mitsunobu, O. Synthesis 1981, 1, 1.