Synthesis and evaluation of novel 8,6-fused bicyclic peptidomimetic compounds as interleukin-1b converting enzyme inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.08.109

Two novel 8,6-fused bicyclic peptidomimetic ring systems were synthesized utilizing olefin metathesis as the key reaction for the formation of the eight-membered ring. Both peptidomimetic scaffolds were further elaborated into potent ICE inhibitors, with

Full text of DOI:10.1016/j.bmcl.2005.08.109

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5434–5438
Synthesis and evaluation of novel 8,6-fused bicyclic
peptidomimetic compounds as interleukin-1b
converting enzyme inhibitors
Steven V. OꢀNeil,* Yili Wang, Michael C. Laufersweiler, Kofi A. Oppong,
David L. Soper, John A. Wos, Christopher D. Ellis, Mark W. Baize,
Gregory K. Bosch, Amy N. Fancher, Wei Lu, Maureen K. Suchanek,
Richard L. Wang, Biswanath De and Thomas P. Demuth, Jr.
Procter & Gamble Pharmaceuticals, Inc., 8700 Mason-Montgomery Road, Mason, OH 45040, USA
Received 3 August 2005; accepted 30 August 2005
Available online 10 October 2005
Abstract—Two novel 8,6-fused bicyclic peptidomimetic ring systems were synthesized utilizing olefin metathesis as the key reaction
for the formation of the eight-membered ring. Both peptidomimetic scaffolds were further elaborated into potent ICE inhibitors,
with numerous compounds exhibiting caspase-1 IC50s less than 10 nM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Cytokines are important signaling molecules that are
essential to immune and inflammatory responses in
mammals. Much attention in recent years has been given
to interleukin-1b (IL-1b), a cytokine known to play an
important role in the pathophysiology of articular joint
destruction.1,2 Modulating the levels of IL-1b is there-
fore believed to be a promising strategy for the treat-
ment of inflammatory diseases such as rheumatoid
arthritis (RA) and osteoarthritis (OA). IL-1b converting
enzyme (ICE), also known as caspase-1, is known to be
involved in the processing of biologically inactive proIL-
1b to the mature cytokine.3,4 Therefore, direct inhibition
of ICE should provide a means of controlling IL-1b
levels therapeutically.
reported.8 Pralnacasanꢁ (2), a selective reversible ICE
inhibitor which progressed into late stage clinical trials
for RA before being withdrawn for toxicological issues,
is perhaps the most studied and successful inhibitor de-
signed to date.9 Pralnacasanꢁ possesses a bicyclic core
which effectively constrains the P2–P3 region of the
inhibitor in a preferred conformation and utilizes a pro-
drug to mask the acidic character of the required aspar-
tic acid recognition element, making the compound
orally bioavailable. The lactone acetal prodrug of 2 is
reported to undergo enzymatic hydrolysis under physio-
logical conditions to provide the bioactive form of the
drug (3) (see Fig. 1).
We elected to follow a related approach for the synthesis
of ICE inhibitors, constraining the P2–P3 region in the
form of an 8,6-fused bicyclic ring system. We believed
that bicyclic peptidomimetic compounds such as 4
would possess all the necessary features to function as
ICE inhibitors, providing the desired conformational
constraint for properly orienting the P1 aspartate resi-
due and the P4 residue believed to impart selectivity be-
tween caspase isoforms.9,6 Inhibitors such as 4 should be
readily accessible from versatile bicyclic cores such as 5
(Scheme 1). We envisioned 5 coming from an eight-
membered ring closure on 6 via olefin metathesis.10
Compounds 5a and b (Scheme 2) are ideally functional-
ized for elaboration into potential inhibitors. Both
ICE is one member of a family of at least 12 human
enzymes referred to as ꢁcaspases.ꢀ The term caspase
was proposed to describe cysteine proteases sharing se-
quence homology and a preference for an aspartate res-
idue at the P1 position of their substrates.5,6 Since the
report of the potent reversible tetrapeptide ICE inhibitor
Ac-YVAD-CHO (1),7 numerous other reversible and
irreversible peptidomimetic inhibitors have been
Keywords: Interleukin-1 converting enzyme inhibitors; Caspase;
Peptidomimetic; Olefin metathesis.
*
Corresponding author. E-mail: oneil.sv@pg.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.109

S. V. OꢀNeil et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5434–5438
5435
OH
O
O
O
O
H
N
N
N
N
P3
P2
N
H
OH
H
O
P1
P4
NH
O
N
O
N
O
N
H
O
OR
H
O
H
O
O
2, R = Et
3, R = H
1
Figure 1. Known ICE inhibitors.
R2
O
X
X
X
N
N
N
O
R1
NH
O
BocHN
O
BocHN
O
N
OMe
OMe
OH
O
O
O
H
O
5
6
4
Scheme 1. Retrosynthesis.
MeO
Boc
X
f
X
X
X
X
e
a,b
c,d
BocHN
O
N
O
HN
O
N
N
BocHN
O
BocHN
O
OMe
OMe
OMe
OMe
OMe
O
O
7a, X = O
7b, X = NCbz
8a, X = O
8b, X = NCbz
9a, X = O
9b, X = NCbz
6a, X = O
6b, X = NCbz
5a, X = O
5b, X = NCbz
Scheme 2. Synthesis of 5a,b. Reagents and conditions: (a) O3, 10:1 CH2Cl2/MeOH, À78 ꢂC; then DMS; (b) TsOH, MeOH; (c) allyl trimethylsilane,
BF3ÆOEt2, CH2Cl2, À78 ꢂC; (d) SOCl2, MeOH or TFA, CH2Cl2; (e) N-Boc-allylglycine, IIDQ, THF; (f) first generation Grubbs catalyst, CH2Cl2.
dipeptide scaffolds possess an ester terminus that can be
easily hydrolyzed, providing a handle for attachment of
the required aspartic acid aldehyde recognition element.
Additionally, the nitrogen terminus can be deprotected
and derivatized to explore the P4 region of our inhibi-
tors. Furthermore, 5b, where X = NCbz, possesses one
additional site of diversification to explore further bind-
ing in the S2 pocket of the enzyme.
or SOCl2/MeOH yielded a single stereoisomer (9a,b) in
each case in good yield (60–70%).13 The coupling of
morpholine 9a and piperazine 9b with N-Boc-allylgly-
cine was originally problematic under standard
carbodiimide coupling conditions. We were eventually
able to obtain acceptable yields of 6a,b (40–60%) in this
coupling by employing isobutyl 1,2-dihydro-2-isobut-
oxy-1-quinoline carboxylate (IIDQ) with excess N-Boc-
allyglycine (3-5 equiv.) in very concentrated THF solu-
tions.14 Completion of the bicyclic scaffolds (5a,b) was
accomplished via olefin metathesis. Typical metathesis
conditions involved refluxing 6a,b in CH2Cl2 (0.5–0.75
M) in the presence of first generation Grubbs catalyst
(15–30 mol%) and delivered the desired bicyclic core
(5a,b)15,16 in 50–60% yield after purification.
The synthesis of the bicyclic cores 5a and b commenced
from readily available allylated amino acids 7a11 and
7b,12 respectively (Scheme 2). Ozonolysis of 7a,b in
10:1 CH2Cl2/MeOH followed by reduction of the ozon-
ide with dimethylsulfide yielded a crude mixture of the
hemiaminal and 8a,b. The aldehyde was never observed,
due to rapid cyclization of the Boc protected nitrogen
onto the newly formed aldehyde. To drive the product
mixture to the desired methyl aminal 8a,b, the crude
reaction solution was concentrated and treated with
TsOH in MeOH, yielding crude 8a,b in 50–78% yield.
Although methyl aminals 8a,b were isolable, they were
rather labile and were typically used immediately with-
After removal of the Boc protecting group upon treat-
ment of 5a,b with TFA in CH2Cl2, the crude amines
were converted to a variety of aryl amides through either
carbodiimide mediated couplings with the required car-
boxylic acid or by reaction of the crude amine with com-
mercially available acid chlorides (Scheme 3). The
isolated yields for this two step sequence were generally
between 80% and 95%. Once the P4 aryl amide was
introduced, the piperazine nitrogen of 11b was deprotec-
.
out purification. Allylation of 8a,b with BF3 OEt2 and
allyltrimethylsilane in CH2Cl2 at À78 ꢂC, and subse-
quent treatment of the crude product with TFA/CH2Cl2

5436
S. V. OꢀNeil et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5434–5438
b or c
Ar
a
d
NH
X
X
X
N
N
N
N
Ar
NH
O
BocHN
O
H2N
O
NH
O
OMe
OMe
OMe
OMe
O
O
O
O
O
O
5a, X = O
10a, X = O
11a, X = O
12
5b, X = NCbz
10b, X = NCbz
11b, X = NCbz
b, e, f, or g
h
C10
C9
R
X
R
N
R
O
i, j
h
X
N
N
O
N
O
Ar
Ar
Ar
NH
O
NH
NH
O
OH
OMe
N
OH
O
O
O
H
O
O
O
15, X = O
16, X = N
14a, X = O
14b, X = N
13
O
O
O
O
N
H
OEt
17
Scheme 3. ICE inhibitor synthesis. Reagents: (a) TFA, CH2Cl2; (b) acid chloride, Et3N, CH2Cl2; (c) ArCO2H, HOBt, EDCI, CH2Cl2; (d) H2, Pd/C,
MeOH; (e) sulfonyl chloride, Et3N, THF; (f) alkyl halide, Et3N, THF; (g) organo-isocyanate, THF; (h) LiOH, THF/H2O; (i) 17, 1,3-
dimethylbarbituric acid, Pd(Ph3P)4, then HOBt and EDCI; (j) TFA, CH3CN/H2O.
ted with accompanying olefin reduction upon treatment
with H2 and 10% Pd/C in MeOH. The crude amine (12)
was isolated upon filtration and concentration of the
reaction mixture, but was used without purification. A
small sampling of substitutions for the piperazine nitro-
gen was selected and crude amine 12 was elaborated by
standard alkylation, acylation, or sulfonylation proce-
dures leading to 13 in generally good yield. The methyl
esters of intermediates 11a and 13 were hydrolyzed with
LiOH in 3:1 THF/H2O to yield carboxylic acids 14a,b in
near quantitative yield.
exhibited caspase-1 IC50s < 10 nM.21 These better per-
forming compounds were also screened in a THP-1
whole cell assay measuring IL-1b production.22 All five
compounds performed well in the whole cell assay, with
two exhibiting THP-1 IC50s < 100 nM. To explore the
effect of potential conformational changes invoked by
the unsaturation in the 8-membered ring on activity,
we hydrogenated compounds 15h–j to obtain 15k–m.
Direct comparison between these analogs and their
unsaturated counterparts showed only a slight improve-
ment in potency, perhaps indicating minimal conforma-
tional changes were induced by saturation of the
8-membered ring. This result was contrary to that
observed for a related 8,5-fused bicyclic scaffold.20
The aspartic acid aldehyde residue was introduced utiliz-
ing intermediate 17. The free amine of compound 1717
was prepared in situ upon treatment of 17 with dimeth-
ylbarbituric acid and Pd(Ph3P)4 in CH2Cl2 at room tem-
perature. Once deprotection of the amine was complete
(typically <15 min), acids 14a,b were added as solutions
in either DMF or CH2Cl2, followed by the addition of
HOBt and EDCI. The coupling was generally complete
within 2 h and the isolated crude product was treated
with TFA in CH3CN/H2O to effect hydrolysis of the eth-
yl acetal to obtain final compounds 1518 and 16.19
Next we decided to evaluate the effect of having a piper-
azine as the 6-membered ring of the bicyclic core. Com-
pounds 16a–h were synthesized and screened as
described previously. For our initial investigations on
the piperazine containing ring system (16), we elected
to hold the P4 substituent constant as a simple benzam-
ide. All eight of these compounds were found to be quite
potent, particularly in the caspase-1 enzyme assay
(IC50s < 10 nM), but significant fluctuations in activity
were observed in the THP-1 assay. Amides 16c and d,
urea 16g, and tertiary amine 16b all exhibited a 5–7
times loss in potency when compared to unsubstituted
16a. This result is probably not due to the inability of
the enzyme to accommodate substitution at this posi-
tion, because several substituents actually resulted in
slight increases in activity. Two different sulfonamides
(16e and f) and a benzyl carbamate (16h) were well tol-
erated. These fluctuations in the whole cell activity are
more likely related to changes in the cell permeability
of the inhibitors. We attempted to further optimize scaf-
fold 16 by incorporating a more potent P4 substituent in
It is well known in the area of ICE inhibitor synthesis
that caspase-1 has a strong affinity for large hydropho-
bic substituents in the P4 pocket, as well as the require-
ment for an aspartic acid residue at P1 previously
described. To evaluate the ability of our bicyclic dipep-
tide scaffolds to function as ICE inhibitor scaffolds, we
synthesized a small set of aromatic amides at P4. We
had previously established a preference for meta-substi-
tuted benzamides and fused bicyclic aromatic amides at
P420 and these trends were again observed for the mor-
pholine-containing ring system (15). Among the ten
compounds synthesized with P4 variations (15a–j), five

S. V. OꢀNeil et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5434–5438
Table 1. Biological activity for compounds 15 and 16
5437
Compound
Ar
R1
C9–C10 olefin
THP-1 (nM)
Casp-1 (nM)
Casp-3 (nM)
Casp-8 (nM)
15a
15b
15c
15d
15e
15f
15g
15h
15i
Ph
2-ClPh
—
—
—
—
—
—
—
—
—
—
—
—
—
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
—
—
130
33
7
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
7900
448
3-ClPh
4-ClPh
118
—
1550
3770
670
74
50
9
2-CF3Ph
3-CF3Ph
4-CF3Ph
—
132
—
4190
6450
1600
2590
1585
429
135
3
2-Benzothiophenyl
2-Naphthyl
1-Isoquinolinyl
2-Benzothiophenyl
2-Naphthyl
1-Isoquinolinyl
57
62
3
15j
182
178
60
3
15k
15l
1
No
No
1
911
264
15m
101
1
16a
16b
16c
16d
16e
16f
16g
16h
16i
Ph
Ph
H
Me
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
No
No
Yes
245
1820
1260
1420
174
159
1690
125
75
10
5
6
7
6
2
5
1
1
1
1
1
1
1
1
1
>104
>104
>104
8070
>104
>104
>104
3260
>104
>104
>104
4850
>104
>104
>104
>104
384
247
481
38
Ph
Ph
Ac
COPh
SO2Me
SO2Ph
CONHPh
Cbz
Ph
Ph
384
106
601
13
Ph
Ph
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
1-Isoquinolinyl
1-Isoquinolinyl
1-Isoquinolinyl
1-Isoquinolinyl
H
506
283
50
16j
SO2Me
SO2Ph
Cbz
46
16k
16l
153
106
156
168
87
5
16m
16n
16o
16p
H
595
917
160
4
SO2Me
SO2Ph
Cbz
154
conjunction with the better performing piperazine P2
substituents (16i–p). For this investigation, we selected
the 2-naphthyl and 1-isoquinolinyl groups as our pre-
ferred P4 substituents, and hydrogen, methylsulfona-
mide, phenylsulfonamide, and Cbz as our preferred
piperazine substitutions. These efforts resulted in very
potent enzyme inhibitors, with all 8 analogs possessing
IC50s less than or equal to 1 nM. Unfortunately, only
modest improvements were observed in the THP-1
whole cell assay.
construction of the 8-membered ring. Multiple com-
pounds from both structural classes were identified with
good enzyme (<10 nM) and whole cell potency
(<150 nM). Further pharmacokinetic and in vivo evalu-
ation of these compounds will be reported elsewhere.
References and notes
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6. Wei, Y.; Fox, T.; Chambers, S. P.; Sintchak, J.; Coll, J. T.;
Golec, J. M. C.; Swenson, L.; Wilson, K. P.; Charifson, P.
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7. Thornberry, N. A.; Bull, H. G.; Calaycay, J. R.; Chap-
man, K. T.; Howard, A. D.; Kostura, M. J.; Miller, D. K.;
Molieaux, S. M.; Weidner, J. R.; Aunins, J.; Elliston, K.
O.; Ayala, J. M.; Casano, F. J.; Chin, J.; Ding, G. J.-F.;
To establish that our compounds were selectively inhib-
iting caspase-1 over other caspases, we screened our
compounds against caspase-3 and caspase-8. Though
many of our compounds were low nanomolars inhibi-
tors of caspase-1, nearly all had little or no activity at
caspase-3 and most of them were fairly selective against
caspase-8 (>100·). It is worth noting though that the
selectivity over caspase-8 did fluctuate significantly with
certain structural changes. The morpholine-containing
inhibitors 15 were more selective for caspase-1 over cas-
pase-8 than the piperazine-containing inhibitors 16. Fur-
thermore, several R substituents (R = COPh and Cbz) in
16 were found to erode selectivity significantly, yielding
potent (<40 nM) caspase-8 inhibitors (16d,h,l,p) (see
Table 1).
In summary, we have synthesized two novel 8,6-fused
bicyclic dipeptide scaffolds (5) that possessed utility as
ICE inhibitors. The core bicyclic scaffold was easily
prepared utilizing an olefin metathesis reaction for

5438
S. V. OꢀNeil et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5434–5438
Egger, L. A.; Gaffney, E. P.; Limjuco, G.; Palyha, O. C.;
Murcko, M. A.; Murdoch, R.; Nyce, P. L.; Robidoux, A.
L. C.; Su, M.; Wannamaker, M.; Wilson, K. P.; Zelle, R.
E. WO 97/22619, 1997.
Raju, S. M.; Rolando, A. M.; Salley, J. P.; Yamin, T.-T.;
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Schmidt, J. A.; Tocci, M. J. Nature 1992, 356, 786.
1
18. Data for 15i: H NMR (CD3OD) d 8.50 (s, 1H), 7.98 (m,
8. For reviews of Caspase-1 inhibitors see: (a) Talanian, R.
V.; Brady, K. D.; Cryns, V. L. J. Med. Chem. 2000, 43,
3351; (b) Ashwell, S. Expert Opin. Ther. Patents 2001, 11,
1593.
9. 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.;
Helaszek, C. T.; Ator, M. A. J. Med. Chem. 1997, 40,
1941.
10. For the synthesis of a related bicyclic dipeptide mimetic,
see (a) Grossmith, C. E.; Senia, F.; Wagner, J. Synletters
1999, 10, 1660; (b) Harris, P. W. R.; Brimble, M. A.;
Gluckman, P. D. Org. Lett. 2003, 11, 1847.
11. Zumpe, F. L.; Kazmaier, U. Synthesis 1999, 10, 1785.
12. Warshawski, A. M.; Patel, M. V.; Chen, T.-M. J. Org.
Chem. 1997, 62, 6439.
4H), 7.62 (m, 2H), 5.80 (m, 1H), 5.69 (m, 1H), 4.75 (d,
J = 2.1 Hz, 1H), 4.68 (m, 1H), 4.55 (m, 1H), 4.49 (d,
J = 6.2 Hz, 1H), 4.37 (m, 1H), 3.65 (dd, J = 12, 4.5 Hz,
1H), 3.36–3.21 (m, 2H), 2.88 (m, 1H), 2.64 (m, 1H), 2.54
(m, 4H); MS 494 (M+H)+.
1
19. Data for 16j: H NMR (CD3OD) d 8.48 (s, 1H), 8.02 (m,
1H), 7.98 (m, 3H), 7.60 (m, 2H), 5.18 (m, 2H), 4.71 (m,
1H), 4.55 (d, J = 12.8 Hz, 1H), 4.33 (m, 1H), 4.23 (d,
J = 13.2 Hz, 1H), 3.69 (d, J = 12.4 Hz, 1H), 3.33 (m, 1H),
3.08 (m, 1H), 3.02 (s, 3H), 2.61 (m, 2H), 2.20 (m, 2H), 1.90
(m, 4H), 1.60 (m, 2H); MS 573 (M+H)+.
20. Manuscript in preparation.
21. The isolated caspase enzyme (caspase-1, -3, and -8) assays
were performed in a 96-well format using fluorogenic
substrates, enzymes, and control peptide inhibitors pur-
chased from BioMol Research Laboratories (Plymouth
Meeting, PA). The assay was conducted according to the
manufacturerꢀs 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 is the initial velocity of substrate cleavage in the
presence of inhibitor at concentration [I], and Vo is the
initial velocity in the absence of inhibitor.
22. 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 were collected to quantify the release of
IL-1b by an ELISA measurement according to the
manufacturerꢀs instructions (R&D Systems, catalog No.
DLB50) or stored at À20 ꢂC for future use.
13. Similar syn selectivity has been observed for allylation of a
related methyl aminal. See Shono, T.; Matsumura, Y.;
Tsubata, K.; Uchida, K. J. Org. Chem. 1986, 51, 2590.
14. Beal, L. M.; Liu, B.; Chu, W.; Moeller, K. D. Tetrahedron
2000, 56, 10113.
1
15. Data for 5a: H NMR (CDCl3) d 5.92 (br d, J = 6.9 Hz,
1H), 5.66 (br s, 2H), 4.90 (m, 2H), 4.55 (d, J = 11.7 Hz,
1H), 4.30 (br d, J = 11.8 Hz, 1H), 3.86–3.64 (m, 3H), 3.77
(s, 3H), 2.92 (m, 2H), 2.48 (m, 1H), 2.31 (br d, J = 16.5 Hz,
1H), 1.48 (s, 9H); 13C NMR (CDCl3) 172.9, 170.5, 155.5,
128.9, 124.3, 80.0, 70.7, 67.9, 52.6, 51.6, 51.2, 51.0, 36.3,
33.4, 28.6 (3C); MS 355 (M+H)+.
16. Data for 5b: 1H NMR (CDCl3) d 7.37 (m, 5H), 5.86 (br d,
J = 6.3 Hz, 1H), 5.63 (m, 2H), 5.17 (br s, 2H), 5.06 (d,
J = 4.2 Hz, 1H), 4.92 (m, 1H), 4.64 (d, J = 13.8, 1H), 4.40
(m, 1H), 3.98 (m, 1H), 3.65 (m, 1H), 3.25 (m, 2H), 2.92 (m,
1H), 2.60–2.30 (m, 3H), 1.45 (s, 9H); MS 488 (M+H)+.
17. 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.;