Synthesis of a new class of cathepsin B inhibitors exploiting a unique reaction cascade

Article abstract of DOI:10.1016/S0040-4039(00)00177-5

Chiral 5-substituted 3-pyrrolin-2-ones bearing L-Ile-L-Pro-OH or L-Phe- NHCH₂Ph were successfully synthesized by utilizing a characteristic reaction cascade based on alkaline hydrolysis of the corresponding diethyl α-hydroxy- α-(β-propiolamide)malonates. Among the synthesized chiral pyrrolinones, compound 5S-9 proved to be the most potent inhibitor against cathepsin B. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00177-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2419–2424
Synthesis of a new class of cathepsin B inhibitors exploiting a
unique reaction cascade
Yoshimitsu Nagao,a,∗ Shigeki Sano,a Kenji Morimoto,a Hisao Kakegawa,b
Tadanobu Takatani,a Motoo Shiro c and Nobuhiko Katunuma b
aFaculty of Pharmaceutical Sciences, The University of Tokushima, Sho-machi, Tokushima 770-8505, Japan
bInstitute for Health Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
cRigaku Corporation, 3-9-12 Matsubara-cho, Akishima-shi, Tokyo 196-8666, Japan
Received 27 December 1999; revised 17 January 2000; accepted 21 January 2000
Abstract
Chiral 5-substituted 3-pyrrolin-2-ones bearing L-Ile-L-Pro-OH or L-Phe-NHCH2Ph were successfully synthe-
sized by utilizing a characteristic reaction cascade based on alkaline hydrolysis of the corresponding diethyl α-
hydroxy-α-(β-propiolamide)malonates. Among the synthesized chiral pyrrolinones, compound 5S-9 proved to be
the most potent inhibitor against cathepsin B. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: alkynes; cyclization; enzyme inhibitor; lactams; X-ray crystal structures.
Cathepsins B, H, L, S, and K are mammalian lysosomal cysteine proteases that belong to the papain
superfamily. These cathepsins most probably play an important role in the regulation of the amount of
specific enzymes and hormones. Katunuma and colleagues have discussed the participation of cathepsin
B in antigen processing and the important role of cathepsin L in bone collagenolytic activity in bone
resorption.1 Our recent focus of research has been the development of new specific inhibitors against
cathepsin B.2 There have been several epoxysuccinic acid derivatives bearing the L-isoleucyl-L-proline
(L-Ile-L-Pro-OH) moiety as the irreversible inhibitors to cathepsin B;3 many kinds of peptidyl aldehydic
reversible and other irreversible inhibitors to various cathepsins.4 However, novel characteristic lead
compounds are still extensively required in the research field of specific enzyme inhibitors against several
selected cathepsins. We have focused on the functionalized chiral 3-pyrrolin-2-one derivatives as the new
active center moiety of various SH-enzyme inhibitors on the basis of earlier research on tenuazonic
acid and N-ethylmaleimide, and to develop an efficient synthetic methodology for them.5 We describe
here the synthesis of a new class of cathepsin B inhibitors, 3-pyrrolin-2-one L-Ile-L-Pro-OH derivatives,
which can be obtained by a unique reaction cascade exploiting the particular trigger moiety, diethyl α-
alkynylmalonate. This diethyl α-alkynylmalonate trigger system could be useful for the construction of
∗
Corresponding author. Fax: +81-88-633-9503; e-mail: ynagao@ph2.tokushima-u.ac.jp (Y. Nagao)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00177-5
tetl 16457

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a conjugated allenyl ester system under mild basic conditions in an aqueous solution, as it has been
efficiently utilized to inhibit SH-enzymes,2a oxazole ring formation via 5-endo-mode cyclization,2a and
Myers-type cycloaromatization of some diethyl α-enediynyl-α-methoxymalonates.6
Based on the background described above and on a series of studies of the cyclization reactions of
the conjugated allenyl esters and ketones,2,7 we anticipated a novel reaction cascade for the conversion
of diethyl α-hydroxy-α-(β-propiolamide)malonates A into 3-pyrrolin-2-one derivatives E, as shown in
Fig. 1. Namely, alkaline hydrolysis of A, followed by decarboxylation, will initiate the desirable reaction
cascade to generate cumulenolates B and then hydroxy allenyl esters C. Cyclization in the resultant Z-
olefinic ketones D, obtained by stereospecific protonation at the sterically less hindered side of the enol
moiety of C, will afford 5-substituted 3-pyrrolin-2-ones E.
Fig. 1. Possible reaction cascade from diethyl α-alkynylmalonates A into 3-pyrrolin-2-ones E
Thus, the diethyl α-hydroxy-α-(β-propiolamide)malonates 3 or 4 were readily synthesized by a
reaction of diethyl ketomalonate 1 with 3-lithiopropiolic acid lithium salt followed by dehydrative
condensation [isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM)] of the resultant 2 with
L-Ile-L-Pro-OH·HCl 5 or L-Ile-L-Pro-OBut 6 obtained by conventional dipeptide synthetic methodology
(Scheme 1). Compound 4 was submitted to alkaline hydrolysis with 1N KOH in EtOH at 0°C for 30
min to give 3-pyrrolin-2-one 7 as an inseparable diastereomeric mixture in 90% yield. After treatment
of 7 with methoxymethyl chloride (MOMCl) in the presence of the Hünig base, the corresponding
MOM-diastereomers, 5R-8 and 5S-8, could be separated by silica gel column chromatography with
25
hexane:AcOEt (1:1) in 43% yield [5R-8: mp 107–108°C, [α]D −91.4 (c 1.03, CHCl3)] and 46% yield
24
[5S-8: mp 110.5–111.5°C, [α]D −106.7 (c 0.83, CHCl3)] as colorless needles from CH2Cl2–hexane,
respectively (Scheme 2). The absolute configuration of C5 of 5S-8 was determined by its X-ray
crystallographic analysis (Fig. 2),8 and, therefore, another diastereomer is 5R-8. Removal of MOM and
the t-Bu groups of 5R-8 and 5S-8 employing CF3CO2H proceeded smoothly to give 5R-9 [colorless
23
23
amorphous powder, [α]D −144.3 (c 1.44, CHCl3)] and 5S-9 [colorless amorphous powder, [α]D
−97.6 (c 0.54, CHCl3)] in a quantitative yield, respectively.
Similar alkaline hydrolysis of 3 furnished 3-pyrrolin-2-one 10 as a diastereomeric mixture in 86%
yield. Methoxymethylation of the mixture 10, followed by chromatographic separation of the resultant
C5–MOM derivatives on a silica gel column with hexane:AcOEt (1:3), gave the corresponding bis MOM
22
derivatives, 5R-11 [colorless oil, [α]D –144.2 (c 2.84, CHCl3)] in 43% yield and 5S-11 [colorless oil,
23
[α]D −88.0 (c 1.68, CHCl3)] in 27% yield. On the other hand, treatment of 10 with triethylsilyl (TES)
chloride in the presence of dimethylaminopyridine (DMAP) exclusively yielded the TES derivative 5R-
24
12 [colorless needles, mp 43–44°C, [α]D –173.5 (c 0.49, CHCl3)] in 55% yield. Its 5S diastereomer
might decompose through the silylation reaction. The stereochemistry of newly formed chiral C5 in the
compounds 5R-11, 5S-11 and 5R-12 was determined by chemical conversion of 5R-11 and 5R-12 to the
known compound 5R-9, as shown in Scheme 3.
Subsequently, we have tentatively prepared 3-pyrrolin-2-one derivatives bearing the L-Phe-NHCH2Ph
25
group. A precursor 13 [colorless oil, [α]D +6.4 (c 0.52, CHCl3)], obtained from the conventional

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Scheme 1. (a) Li–^–CO2Li/THF/−78°C→rt; (b) IBCF/NMM/THF/−16–−18°C; (c) 5/NMM/THF–DMF/−18°C→rt; (d)
6/THF/−16°C→rt
Scheme 2. (a) 1N KOH/EtOH/0°C; (b) MOMCl/DIEA/CH2Cl2/rt; (c) silica gel column/hexane:AcOEt (1:1); (d) CF3CO2H/0°C;
(e) acetate buffer (pH 5.5)/37°C
dehydrative condensation reaction of 2 with L-Phe-NHCH2Ph 14, was allowed to react with 1N KOH in
EtOH at 0°C for 40 min. The desirable cascade reaction proceeded in a highly diastereoselective manner
23
to afford 5R-15 [colorless amorphous powder, [α]D +5.1 (c 0.93, CHCl3)] in 72% yield as the major
product with the minor 5S diastereomer in a 9.1:0.9 ratio (1H NMR analysis). The absolute configuration
of chiral C5 in 5R-15 was established by X-ray analysis (Fig. 2)8 of its crystalline MOM derivative 5R-
16 [mp 115–117°C (CH2Cl2–hexane), [α]D23 +19.5 (c 1.31, CHCl3)], which was converted to 5R-15 by
treatment with 4N HCl in dioxane, as shown in Scheme 4.
Finally, we examined the inhibition of several 3-pyrrolin-2-one derivatives, namely 5R-8, 5S-8, 5R-
9, 5S-9, 5R-11, 5S-11, 5R-12 and 5R-15, against enzymatic hydrolysis of Z-L-Phe-L-Arg-MCA (MCA:
methylcoumarylamino group) with human liver cathepsin B and rat liver cathepsin L in sodium acetate
buffer (pH 5.5) under the Barrett assay procedure.9 Compounds 5R-8 and 5S-8 did not inhibit enzymatic

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Fig. 2. Computer-generated drawing of 5S-8 and 5R-16 derived from the X-ray coordinates
Scheme 3. (a) 1N KOH/EtOH/0°C; (b) MOMCl/DIEA/CH2Cl2/0°C→rt; (c) silica gel column/hexane:AcOEt (1:3); (d)
TESCl/DMAP/CH2Cl2/rt; (e) CF3CO2H/0°C
Scheme 4. (a) IBCF/NMM/THF/−18°C; (b) 14/THF/−18°C→rt; (c) 1N KOH/EtOH/0°C; (d) MOMCl/DIEA/CH2Cl2/0°C→rt;
(e) 4N HCl/dioxane/0°C

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hydrolysis with both cathepsins B and L, even at their high concentration orders, 10−5 M and 10−6 M.
Compounds 5R-11, 5S-11, and 5R-12 exhibited weak inhibitory activities against cathepsin B (5R-11:
50.3% at 10−5 M, 5S-11: 40.7% at 10−5 M, 5R-12: 62.1% at 10−6 M) and cathepsin L (5R-11: 25.6%
at 10−5 M, 5S-11: 50.5% at 10−5 M, 5R-12: 37.5% at 10−5 M), respectively. Other selected enzyme-
inhibitory assay data are listed in Table 1. Among the 3-pyrrolin-2-one derivatives, compounds 5R-9 and
5S-9 bearing the 5-hydroxy and 5-ethoxycarbonyl groups exhibited selective inhibitory activities against
cathepsin B in comparison with those against cathepsin L. Specifically, 5S-9 proved to be the most potent
inhibitor against cathepsin B. Because a maleimide derivative 17 did not exhibit any inhibitory activity
against cathepsins B and L, the chiral 3-pyrrolin-2-one moiety, having the functional group(s) (e.g., OH,
CO2Et, etc.) at the C5 position, must be essential for the molecular recognition to the active center10
in the cathepsin B. Interestingly, a 3-pyrrolin-2-one derivative 5R-15 with L-Phe-NHCH2Ph exhibited
higher inhibition values against cathepsin L than those against cathepsin B, as shown in Table 1.
Table 1
Inhibition with 3-pyrrolin-2-ones against cathepsins B and L
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research (B) (2) (No. 10470470) from the
Ministry of Education, Science, Sports and Culture, of the Japanese Government.
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