Alutacenoic acids A and B, rare naturally occurring cyclopropenone derivatives isolated from fungi: Potent non-peptide factor XIIIa inhibitors [21]

Full text of DOI:10.1021/ja992355s

1842
J. Am. Chem. Soc. 2000, 122, 1842-1843
Alutacenoic Acids A and B, Rare Naturally
Occurring Cyclopropenone Derivatives Isolated from
Fungi: Potent Non-Peptide Factor XIIIa Inhibitors
Hiroshi Kogen,*,† Toshihiro Kiho,† Keiko Tago,†
Shuichi Miyamoto,† Tomoyuki Fujioka,‡ Noriko Otsuka,‡
Keiko Suzuki-Konagai,§ and Takeshi Ogita*,§
Figure 1.
potent specific inhibitors of factor XIIIa (see Supporting Informa-
tion). Antibacterial activities are absent in both the 1a and 1b.
Surprisingly, structure determination by spectroscopic methods
Exploratory Chemistry Research Laboratories
Pharmacological and Molecular Biological Research
Laboratories, and Biomedical Research Laboratories
Sankyo Co., Ltd., 2-58, Hiromachi 1-chome
Shinagawa-ku Tokyo, 140-8710 Japan
(IR, MS, H,13C NMR, DQFCOSY, HOHAHA, and HMBC)12
1
revealed that these two compounds are monosubstituted cyclo-
propenone derivatives with aliphatic tethers which attach to a
terminal carboxylic acid (Figure 1). Cyclopropenones have
attracted considerable attention from chemists during the past four
decades. In fact, there have been many efforts toward the synthesis
of cyclopropenone derivatives.13-15 However, only three cyclo-
propenone derivatives, penitricin16 and two sesquiterpenes,17 have
been reported to be isolated from natural sources. Among them,
penitricin is the only naturally occurring cyclopropenone which
shows biological activities as an antibiotic. It is particularly
interesting that these cyclopropenone derivatives 1a and 1b are
isolated from a common fungus, Eupenicillium. We applied the
synthesis of 1a, 1b, and their derivatives to confirm these unique
ring systems and study the structure-activity relationships for
chain length between the ring and carboxyl group.
ReceiVed July 7, 1999
Factor XIII is a plasma transglutaminase which acts on the
final step in the blood coagulation cascade.1 Factor XIII exists in
plasma as a zymogen and is activated to factor XIIIa by thrombin
in the presence of Ca2+ in a process that reveals a cysteine active
center.2 The enzyme covalently cross-links fibrin monomers and
converts soft fibrin clots to hard clots.3 These clots are less rapidly
lysed by agents such as plasmin or tissue plasminogen activator.
In addition, factor XIIIa is also known to cross-link fibrin to
extracellular matrixes, such as vitronectin, fibronectin, and
collagen, thereby forming additional clots to the vessel wall.
Specific inhibitors of factor XIIIa are therefore thought to offer
possibilities in the therapy for thrombosis,1a atherosclerosis, and
coronary heart disease,4 and a few such inhibitors have already
been reported. In addition to several peptides5 and the synthetic
compound L-722,151,6 a known antimicrobial agent, cerulenin,7
has also been reported by Tymiak to show inhibitory activity (10
µM)8 against factor XIIIa. To our knowledge, cerulenin is the
only naturally occurring non-peptide factor XIIIa inhibitor. Here
we report the isolation of novel specific non-peptide inhibitors
of factor XIIIa, alutacenoic acids A (1a) and B (1b), from fungi.
Both compounds have an extremely rare cyclopropenone ring
isolated from natural sources.
Total syntheses of cyclopropenone derivatives 1 (n ) 5-11)
were accomplished according to the method of Nakamura,15
establishing the construction of substituted cyclopropenone rings
as shown in Scheme 1. Readily available cyclopropenone acetal
(9) Alutacenoic acid A (1a): colorless oil; HRMS (FAB) calcd for C9H12O3
(M + H)+ 169.0865, found 169.0875; IR νmax (CHCl3) 3084, 2944, 2867,
1826, 1740, 1711, 1591, 1414 cm-1; H NMR (360 MHz, CD3OD) δ (ppm)
1
1.43-1.53 (m, 2H), 1.67 (m, 2H), 1.77 (m, 2H), 2.32 (t, J ) 7.5 Hz, 2H),
2.75 (t, J ) 7.3 Hz, 2H), 8.74 (s, 1H); 13C NMR (90 MHz, CD3OD) δ (ppm)
25.7, 26.5, 27.8, 29.6, 34.9, 150.0, 161.4, 170.9, 177.8. 1a gradually
decomposed within a few months on storage at -20 °C. We stored a solution
of 1a in a mixture of benzene and t-BuOH at -20 °C without decomposition
for at least 6 months. Alutacenoic acid B (1b): colorless needles; mp 66-69
°C, HRMS (FAB) calcd for C11H16O3 (M + H)+ 197.1178, found 197.1169;
IR νmax (KBr) 3059, 1821, 1732, 1609, 1567 cm-1; 1H NMR (360 MHz, CD3-
OD) δ (ppm) 1.33-1.51 (m, 6H), 1.61 (m, 2H), 1.75 (m, 2H), 2.28 (t, J )
7.4 Hz, 2H), 2.74 (t, J ) 7.2 Hz, 2H), 8.73 (s, 1H); 13C NMR (90 MHz,
CD3OD) δ (ppm) 26.5, 27.1, 28.1, 30.2, 30.4, 30.5, 35.5, 150.2, 161.8, 171.4,
178.2. 1b was stored in a freezer (-20 °C) without decomposition for at least
2 years.
Our recent screening efforts have concentrated on the search
for specific inhibitors of factor XIIIa. As a result of our efforts,
we have isolated alutacenoic acids A (1a) and B (1b)9 (1a, IC50
) 1.9 µM; 1b, IC50 ) 0.61 µM),10 a pair of fungal metabolites
from Eupenicillium alutaceum Scott,11 and found them to be
† Exploratory Chemistry Research Laboratories.
(10) For the factor XIIIa assay utilized for this study, see: Usui, T.; Takagi,
J.; Saito, Y. J. Biol. Chem. 1993, 268, 12311-12316.
‡ Pharmacological and Molecular Biological Research Laboratories.
§ Biomedical Research Laboratories.
(11) Alutacenoic acid B (1b) is also isolated from Aspergillus fumigatus
Fresenius.
(1) (a) Leidy, E. M.; Stern, A. M.; Friedman, P. A.; Bush, L. R. Thromb.
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FASEB J. 1991, 5, 3071-3077. (c) Aeschliman, D.; Paulsson, M. Thromb.
Haemostasis 1994, 71, 402-415. (d) Yee, V. C.; Pedersen, L. C.; Trong, I.
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1994, 91, 7296-7300.
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(12) For a detailed study on structure determination by NMR, see the
Supporting Information.
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10.1021/ja992355s CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/10/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 8, 2000 1843
Scheme 1a
a (a) n-BuLi, -78 °C, HMPA, THF, then 3c, -23 °C; (b) TBAF,
AcOH, THF, 0 °C to rt; (c) Amberlyst15, THF-H2O, rt; (d) (COCl)2,
Me2SO, Et3N, -78 to 0 °C, CH2Cl2; (e) NaClO2, NaH2PO4, 2-methyl-
2-butene, t-BuOH-H2O, rt.
Figure 2. Complex structure model of 11b (green) and the factor XIII
binding site (black). The Connolly surface of the enzyme is shown in
gray. The blue dotted line indicates the hydrogen bond between the
carbonyl oxygen and the indole NH of Trp279. The docking study was
performed using the QUANTA/CHARMm system24 with the enzyme
structure (1F1322b in the Protein Data Bank25) fixed.
Scheme 2a
is a time-dependent (i.e., irreversible) inhibitor of factor XIIIa
with a second-order inhibition rate constant (kinact/Ki) of 305 000
min-1 M-1
.
Three-dimensional structures of factor XIII, solved recently by
X-ray crystallography, suggest the movement of the â-barrel-1
domain on activation, allowing the ligands access to the active
site.1d,22 In an attempt to obtain structural insight into the inhibition
mechanism for these cyclopropenone derivatives, we carried out
the docking study of the inhibitors into the crystal structure of
factor XIII22b with the amino-terminal activation peptide and the
â-barrel-1 domain removed. Figure 2 shows the stable complex
structure model of 11b and the enzyme binding site thus obtained,
indicating how the cyclopropenone ring fits highly complemen-
tarily into the active site located at the base of the binding cavity.
The carbonyl oxygen of the cyclopropenone ring forms a
hydrogen bond to the indole NH of Trp279 (the O-Nꢀ1 distance
is 2.9 Å), and the terminal carbon atom is in close proximity to
the sulfur atom of Cys314 (C-Sγ ) 2.9 Å).23
a (a) TEMPO, KBr, Aliquat336, NaClO, NaHCO3, CH2Cl2-H2O, 0
°C; (b) CDI, phenethylamine, CH2Cl2, 0 °C; (c) Amberlyst15, THF-
H2O.
215c was treated with n-BuLi in the presence of HMPA in THF
at -78 °C, and a subsequent reaction with tert-butyldimethylsi-
lyloxy alkyl bromide 3 (n ) 5-11) gave conveniently coupling
product 4 (n ) 5-11) in 60-79% yield. Deprotection of a silyl
group of 4 with tetra-n-butylammonium fluoride (TBAF)-acetic
acid in THF at 0 °C provided alcohol 5 (n ) 5-11) in 91-97%
yield. Hydrolysis of an acetal group of the alcohol 5 with an acidic
ion-exchange resin, Amberlyst15, produced cyclopropenone 6 (n
) 5-11) in 57-92% yield. Oxidation of 6 using Swern conditions
provided aldehyde 7 (n ) 5-11) in 75-95% yield.
Careful oxidation of an aldehyde group under NaClO2 condi-
tions at room temperature afforded cyclopropenone derivatives
1 (n ) 5-11) in 63-86% yield. Synthetic 1a and 1b were
spectroscopically identical in all respects to the natural alutacenoic
acids A and B, respectively. Among all these synthetic com-
pounds, 1c (n ) 10, IC50 ) 0.31 µM) showed the most potent
inhibitory activity against factor XIIIa. Interestingly, the inhibitory
activity of disubstituted cyclopropenone derivative 1d18 (n ) 7)
was greatly reduced.
In summary, we isolated novel specific non-peptide inhibitors
of factor XIIIa, alutacenoic acids A and B, from fungi. Both
compounds are rare cyclopropenone derivatives isolated from
natural sources. Additionally, synthetic analogue 11b shows the
most potent inhibitory activity against factor XIIIa among these
cyclopropenone derivatives.
Supporting Information Available: HMBC connectivities of 1a and
1b; experimental details of the synthesis of 1 (n ) 5-11), 1d, and 11 (n
) 7-10); inhibitory activities of 1 (n ) 6, 8, 9, 11) and 11 (n ) 8-10)
and of 1b against several thiol enzymes; and NMR spectra (1H and 13C)
of synthetic and natural 1a and 1b (PDF). This material is available free
charge via the Internet at http://pubs.acs.org.
JA992355S
(18) Compound 1d was derived from 4d, which was prepared by meth-
ylation of 4b. Further details, see the Supporting Information.
(19) We also prepared various esters, amides, ethers, and acylamines.
Further detailed study will be reported elsewhere.
(20) Anelli, P. L.; Biffi, C.; Montanari, F.; Quici, S. J. Org. Chem. 1987,
52, 2559-2562.
(21) (a) Roush, W. R.; Gwaltney II, S. L.; Cheng, J.; Scheidt, K. A.;
McKerrow, J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994-10995.
Bieth, J. G. Methods Enzymol. 1995, 248, 59-84. Thornberry, N. A.; Peterson,
E. P.; Zhao, J. J.; Howard, A. D.; Griffin, P. R.; Chapman, K. T. Biochemistry
1994, 33, 3934-3940. Progress curves were obtained with three inhibitor
concentrations (0.34, 0.67, and 1.34 µM). We calculated the kobs value, the
rate constant for loss of enzyme activity, by using EnzFitter (Biosoft). A
second-order rate constant (kinact/Ki) was determined by using the program
because the obtained kobs varied hyperbolically with inhibitor concentration.
A detailed study will be reported elsewhere.
To further explore the inhibitory activity of this series of
compounds, various carboxyl derivatives19 were synthesized. A
desirable and improved inhibitory potency of 26 nM (11b) was
achieved by converting the terminal functional group from
carboxyl to phenethyl amide. The amide 11b was synthesized as
shown in Scheme 2. The acetal derivative 5b was used as the
starting material to modify the terminal carboxyl group because
of the instability of the cyclopropenone ring under even weakly
basic conditions. Oxidation of 5b with NaClO-2,2,6,6-tetrameth-
ylpiperidinyl-1-oxyl (TEMPO)20 afforded carboxylic acid 9b, and
a subsequent coupling reaction with phenethylamine using the
1,1-carbonyldiimidazole (CDI) method provided amide 10b in
58% yield from 5b. Hydrolysis of an acetal group of the amide
10b with an acidic ion-exchange resin, Amberlyst15, produced
phenethyl amide 11b in 75% yield.
(22) (a) Yee, V. C.; Pedersen, L. C.; Bishop, P. D.; Stenkamp, R. E.; Teller,
D. C. Thromb. Res. 1995, 78, 389-397. (b) Weiss, M. S.; Metzner, H. J.;
Hilgenfeld, R. FEBS Lett. 1998, 423, 291-296.
(23) Compound 1d does not fit the active site well because of the steric
repulsion of the methyl group.
(24) Molecular Simulations Inc., USA.
(25) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. F., Jr.;
Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J.
Mol. Biol. 1977, 112, 535.
A full kinetic analysis21 was performed on the most potent
inhibitor 11b, which confirmed that the phenethyl amide derivative