Synthesis and evaluation of tricyclic pyrrolopyrimidinones as dipeptide mimetics: Inhibition of interleukin-1β-converting enzyme

Article abstract of DOI:10.1016/j.bmcl.2005.06.046

The application of a tricyclic pyrrolopyrimidinone scaffold for the synthesis of peptidomimetic inhibitors of interleukin-1β-converting enzyme (ICE) is reported. The synthesis of the tricyclic scaffold and conversion of it to a variety of target ICE inhib

Full text of DOI:10.1016/j.bmcl.2005.06.046

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4322–4326
Synthesis and evaluation of tricyclic pyrrolopyrimidinones
as dipeptide mimetics: Inhibition of
interleukin-1b-converting enzyme
Michael C. Laufersweiler,* Yili Wang, David L. Soper, Maureen K. Suchanek,
Amy N. Fancher, Wei Lu, Richard L. Wang, Kofi A. Oppong, Christopher D. Ellis,
Mark W. Baize, Steven V. OÕNeil, John A. Wos and Thomas P. Demuth, Jr.
Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason Montgomery Road, Mason, OH 45040, USA
Received 19 April 2005; revised 14 June 2005; accepted 15 June 2005
Available online 19 July 2005
Abstract—The application of a tricyclic pyrrolopyrimidinone scaffold for the synthesis of peptidomimetic inhibitors of interleukin-
1b-converting enzyme (ICE) is reported. The synthesis of the tricyclic scaffold and conversion of it to a variety of target ICE inhib-
itors were accomplished in 4–5 steps. In vitro biological evaluation of the tricyclic pyrrolopyrimidinones revealed fair to good ICE
inhibitors, with the most active compound exhibiting an IC50 of 14 nM in a caspase-1 enzyme binding assay.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Interleukin-1b (IL-1b) has been implicated in a number
of disease states, including rheumatoid arthritis, osteo-
arthritis (OA), and sepsis.1 In recent years, significant
attention has been given to IL-1b, as it is widely believed
to contribute significantly to the cartilage degradation,
joint inflammation and pain associated with OA.2,3
Interleukin-1b-converting enzyme (ICE) is known to
be responsible for processing pro-IL-1b to its active
form (IL-1b) and then releasing it extracellularly.4,5
Therefore, inhibition of ICE should provide a means
of controlling IL-1b levels therapeutically, providing
disease modification and symptomatic relief for OA
patients.
is essential for selective inhibition of caspase-1 over
other caspases.6
Since MerckÕs first report of a potent reversible tetrapep-
tide ICE inhibitor, 17 (Fig. 1), there have been several po-
tent inhibitors described.8 While most of the focus for
inhibitor design has been on constrained dipeptide mim-
ics such as 29, there have been reports of non-peptidic
pyrimidinone and pyridone-based inhibitors of ICE, such
as 3–5.10–13 These heteroaryl-based inhibitors are report-
ed to exhibit modest potency for ICE inhibition, demon-
strating that constraining the P3 region of the inhibitor in
the form of a planar amino pyrimidinone/pyridone ring
maintains activity. Tricyclic pyrimidinone 5 effectively
constrains the P2 region as well as the P3, but sacrifices
a key hydrogen bond donor in the P3 residue.
ICE, or caspase-1, is a member of the Caspase family
of enzymes, a class of cysteine proteases sharing se-
quence homology and a preference for an aspartate
residue at the P1 position of their substrates.6 As a re-
sult of this preference, most ICE inhibitors reported to
date possess an aspartic acid derived cysteine trap that
is essential for inhibition. Additionally, it is well
known that a hydrophobic P4 residue in the substrate
The key binding elements for ICE inhibition are well
known based on the reported crystal structure of tetra-
peptide 1 bound to the active site of ICE.14 A successful
scaffold for inhibition of ICE must properly orient the
aspartate-derived trap and the P4 hydrophobic group,
while maintaining certain hydrogen bonds with the pep-
tide backbone in the S1 and S3 residues (Fig. 2). The P3
carbonyl and NH both participate in critical hydrogen
bonds that help to orient the inhibitor in the active site.
It was our belief that an appropriately substituted
heteroaryl-fused pyrimidinone could effectively mimic
Keywords: Caspase-1; Interleukin-1b-converting enzyme; Heterocycles;
Pyrrolopyrimidinones; Peptidomimitics.
*
Corresponding author. Tel.: +1 513 622 3062; fax: +1 513 622
2675; e-mail: Laufersweiler.mc@pg.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.046

M. C. Laufersweiler et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4322–4326
4323
Figure 1. Selected inhibitors of ICE.
Figure 2. ICE inhibitor binding elements.
the preferred conformation for the P3–P4 region of the
tetrapeptide while retaining the key hydrogen-bonding
residues, thereby giving a potentially potent, non-pep-
tide ICE inhibitor such as 6. The R substituent of 6
may additionally provide the hydrophobic interactions
essential for selectivity of caspase-1 over other caspases.
thesis of the related tricyclic core of 5 involved the diaz-
otization of anthranilic acid, followed by acid chloride
formation to yield 7. Coupling of 7 with the lithium an-
ion of t-butyl pyroglutamate gave compound 9. The cen-
tral ring was closed with an intramolecular aza-Wittig
reaction to yield the tricyclic core 10 in four total steps.13
(Scheme 1).
Though the proposed inhibitor 6 shares some structural
similarity with compound 5, we felt that incorporation
of a pyrrole ring into our inhibitors would necessitate
designing a new synthetic pathway. The reported syn-
We believed that the tricyclic core of 6 could be prepared
in a single step from the readily available amino esters
11 and chloroimine 12. To test the validity of this
Scheme 1. Reagents and conditions: (a) 1: NaNO2/HCl/NaN3/NaOAc, 2: SOCl2; (b) LDA; (c) PPh3, xylene, reflux.

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M. C. Laufersweiler et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4322–4326
approach, compound 16, possessing the same tricyclic
core as 10, was synthesized from methyl anthranilate
and ethyl pyroglutamate, 13. A mixture of methyl
anthranilate and ethyl pyroglutamate in dichloroethane
was treated with POCl3 and heated at reflux for 3 h. It is
believed that the pyroglutamate reacts with POCl3 to
form intermediate 14, which rapidly reacts with the ani-
line nitrogen of methyl anthranilate to form amidine 15.
Intermediate 15 then undergoes spontaneous cyclization
to form the desired tricyclic core, 16, in a single step in
91% yield (Scheme 2).
solvent exchange. After initial reaction of pyrroles
20a–e with ethyl pyroglutamate and POCl3 in dichloro-
ethane, the crude reaction mixture containing the inter-
mediate amidine was concentrated in vacuo and re-
dissolved in DMF. Heating the DMF solution at
110 ꢁC affected the cyclization to provide tricyclic core,
21a–e,17 in 40–50% yield. Compounds 21a–e were then
carried on to the final inhibitors in a fashion analogous
to that described for 18.18
These novel ICE inhibitors were screened in a THP-1
whole cell assay19 measuring IL-1b production to evalu-
ate potency. Out of the five pyrrole scaffolds synthesized,
four had whole cell IC50 < 1000 nM. These compounds
were then evaluated in isolated enzyme assays20 to
evaluate selectivity for caspase-1 over caspase-3 and -8
(Table 1). The tricyclic pyrrolopyrimidinone-based
inhibitors exhibited selectivity for caspase-1 of >100-fold
over caspase-3 and >50-fold over caspase-8.
The ethyl ester, 16, was subsequently hydrolyzed with
NaOH to give carboxylic acid 17. After in situ deprotec-
tion of the Alloc protecting group, aspartate-derived
cysteine trap 1915 was coupled with 17. Acetal hydrolysis
with TFA gave the final inhibitor 18, a reversible ICE
inhibitor analogous to 5. These synthetic investigations
provided us with good confidence that the new method-
ology could be applied to the readily available amino
esters, 20a–e to give our desired tricyclic core.
In summary, it has been demonstrated that hetero-aro-
matic rings can function as peptidomimetic backbones
for ICE inhibition. The pyrrolopyrimidinone inhibitors
described here were synthesized in 3–4 steps from readily
available starting materials. The most potent inhibitor,
6b, exhibited good activity (14 nM at caspase-1 and
252 nM in THP-1) while maintaining excellent selectivity
over caspase-3 and -8.
The synthesis of pyrroles 6a–e (Scheme 3) from aryl
substituted pyrrole amino esters 20a–e16 under the same
conditions initially failed to provide the desired tricyclic
core in appreciable amounts, presumably due to prob-
lems with the solubility of intermediates after the addi-
tion of POCl3. This problem was accounted for with a
Scheme 2. Reagents and conditions: (a) POCl3, DCE, reflux, 3 h; (b) 1 N NaOH, MeOH; (c) 1: 19, N,N-dimethylbarbituric acid, Pd(PPh3)4, HOBt,
EDCI; 2: TFA, H2O, AcCN.
Scheme 3. Reagents and conditions: (a) 1: POCl3, DCE, RT, 10 min; 2: DMF, 110 ꢁC, 3 h; (b) 1: 1 N NaOH, MeOH; 2: 19, N,N-dimethylbarbituric
acid, Pd(PPh3)4, HOBt, EDCI; 3: TFA, H2O, AcCN.

M. C. Laufersweiler et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4322–4326
4325
Table 1. Caspase inhibition data
Compound
Ar
R
THP-1 IC50 (nM)
Casp-1 IC50 (nM)
Casp-3 IC50 (nM)
Casp-8 IC50 (nM)
18
6a
6b
6c
6d
6e
Ph
H
Ph
10,000
1064
252
4583
264
14
10,000
10,000
1841
10,000
3046
336
Pyrrole
Pyrrole
Pyrrole
Pyrrole
Pyrrole
2-ClPh
3-ClPh
4-ClPh
tBu
529
830
55
97
4394
10,000
2293
1823
4811
7540
975
421
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E. WO 97/22619, 1997.
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8.08 mmol) in 100 mL 1,2-dichloroethane, POCl3
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1
21b as a yellow solid. H (CD3OD) d 7.66 (m, 1H), 7.60
(m, 1H), 7.45 (m, 2H), 6.77 (s, 1H), 5.27 (dd, J = 9.9,
3.0 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 3.42–3.28 (br m,
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18. 2-(2-Chlorophenyl)-8-oxo-5,6,7,8-tetrahydro-1H-1,4,7a-
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tetrahydro-furan-3-yl)-amide (6b):
a solution of 21b
(500 mg, 1.40 mmol) in methanol (75 mL) was treated
with excess 1 N NaOH (14.0 mL) and stirred for 15 h at rt.
The solution was acidified with 1 N HCl and extracted
with EtOAc. The crude acid of 21b was isolated as a
yellow solid, 446 mg (96%), MS 330 (M + H)+. Catalytic
Pd(PPh3)4 was added to a solution of 19 (670 mg,
2.92 mmol) and N,N-dimethylbarbituric acid (2.0 g,
12.81 mmol) in 75 mL CH2Cl2 at rt. The solution was
stirred at rt for 15 min and then the crude carboxylic acid
of 21b prepared above was added as a solution in 15 mL
DMF followed by HOBt (1.1 g, 8.14 mmol), and EDCI
(1.4 g, 7.3 mmol). The solution was stirred for 3 h, diluted
with CH2Cl2, washed with saturated NaHCO3 and brine,
dried (MgSO4) and concentrated. After purification by
preparative reverse phase HPLC the product was hydro-
lyzed with TFA in CH3CN/H2O. Further purification by
preparative reverse phase HPLC yielded 6b as a white
1
solid, 346 mg (60%). H NMR (CD3OD) d 7.66 (m, 1H),
7.60 (m, 1H), 7.46 (m, 2H), 6.76 (s, 1H), 5.30 (m, 1H), 4.71
(m, 1H), 4.35 (m, 1H), 3.35 (m, 2H), 2.70 (m, 4H); MS 429
(M + H)+.
19. A suspension of human monocytic cells (THP-1, ATCC
strain TIB202, 2 · 106/ml in RPMI 1640 medium from
Gibco BRL) were 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 ꢁC for future use.
15. Batchelor, M. J.; Bebbington, D.; Bemis, G. W.; Fridman,
W. H.; Gillespie, R. J.; Golec, J. M. C.; Gu, Y.; Lauffer,

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20. The isolated Caspase enzyme (caspase-1, 3, and 8)
assays were performed in 96 well format using
measuring fluorescence using a BMG Fluostar plate
reader (excitation filter 390 nm, emission filter 460 nm).
IC50 values were calculated based on the equation
IC50 = [I]/(Vo/Vi)À1, 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.
a
fluorogenic substrates, enzymes and control peptide
inhibitors purchased from BioMol Research Laborato-
ries (Plymouth Meeting, PA). The assay was conducted
according to the manufacturerÕs instructions. Enzyme
inhibition was monitored over 30 min at 37 ꢁC by