Communications
J . Org. Chem., Vol. 64, No. 1, 1999
7
Ta ble 1. In h ibition of Recom bin a n t Yea st Squ a len e
Syn th a se by Aza Bifa r n esyl Dip h osp h a te (4) a n d
Azir id in o Dip h osp h a tes 12a ,b in th e Absen ce a n d
P r esen ce of In or ga n ic Dip h osp h a te a t p H 7.2a
Sch em e 4
b
IC50 (µM)
c
inhibitor
-PPi
+PPi
Kid (µM)
4
12a
12b
9.7 ( 2.7
7.2 ( 0.8
6.9 ( 1.6
11.4 ( 2.2
1.9 ( 0.2
4.0 ( 0.4
0.21c
a
Assay conditions: 50 mM MOPS, pH 7.2, 20 mM MgCl2, 2%
(v/v) Tween 80, 1 mM DTT, 1 mg/mL of bovine serum albumin,
1.0 mM NADPH, 0 or 1.5 mM PPi, 100 µM [3H]FPP (7.5 µCi/µmol),
0-300 µM inhibitor, and 0.1 µg protein in a total volume of 200
µL. Samples were incubated at 30 °C for 5 min before enzyme was
added and stopped after an additional 10 min by adding 40%
aqueous KOH in methanol. Product [3H]squalene was isolated by
extraction with hexanes, purified by filtration over alumina, and
cyclic substituent on nitrogen was detached by reductive
cleavage (6 equiv of Li, NH3-THF, -33 °C, 67%), conditions
found to be general for a series of N-quinazolonylaziridines.19
b
analyzed by liquid scintillation spectrometry.7 IC50 values were
Installation of the N-(E)-6,10-dimethyl-5,9-undecadienyl
substituent in 12a and its 2-methylene analogue in 12b
resembling the triene isoprenoid chains of PSPP was effected
in three steps by O-silylation (t-BuMe2SiCl, imidazole, DMF,
75%),20 N-acylation,21 and hydride reduction (10 equiv of
LiAlH4, ether, reflux, 48h, 68%) or by O-silylation, N-
alkylation21 (ether, rt), and desilylation (Bu4NF‚3H2O, THF,
rt; 77%), respectively. SN2 displacements of the correspond-
ing aziridino mesylates with pyrophosphate anion [2 equiv
of (n-Bu4N)3HP2O7, CH3CN, rt, 48 h]22 afforded aziridino
diphosphates 12a (66%) and 12b (64%) as n-Bu4N+ salts that
interpolated from plots of specific activity at [I] ) 0.001, 0.50, 1.00,
3.00, 10.0, 30.0, 100, and 300 µM using Grafit (Erithicus Software,
Staines, UK). Deviations shown are standard errors. c Incubations
d
in the presence of 1.5 mM PPi. 5-100 µM [3H]FPP (7.5 µCi/
µmol).
The selectivity of the interactions of these three aza
analogue inhibitors with SS is validated by the much lower
affinity exhibited by a similar aziridino diphosphate having
a straight-chain N-C11H23 substituent (55% inhibition of SS
at 219 µM) instead of the unsaturated isoprenoid groups in
12a ,b. It is noteworthy that the two aziridino diphosphates
are substantially stronger inhibitors than their acyclic
analogue 4, despite the lack of the proximal double bond,
the additional absence of a methyl group in 12a , and the
lower basicity of the heterocyclic nitrogen. The enhanced
potency of the aziridine inhibitors presumably manifests
better spacial congruency with the SS active site than that
of the acyclic aza analogue and may indicate a nonclassical,
protonated cyclopropane intermediate in the formation of
2. It is not known whether the aziridine inhibitors bind to
SS in the aziridinium ion forms (5a and 5b)24 or the aziridine
forms (12a and 12b). If the latter is true, the aziridine
diphosphates may be better regarded as PSPP analogues
rather than mimics for a charged transition state. In any
case, aziridines structurally analogous to bridged nonclas-
sical ions proposed as intermediates in cyclizations catalyzed
by terpene synthases are interesting candidates for novel,
mechanism-based inhibitors of these enzymes.25
1
were characterized by H and 31P NMR spectroscopy.23
Although the aziridino diphosphates underwent slow
solvolytic ring opening in aqueous solution at pH 7 (D2O,
rt, t1/2 ca. 24 h), the compounds were sufficiently stable for
evaluation of their inhibitory properties toward squalene
synthase. Kinetic assays of recombinant yeast SS activity
with [3H]FPP as substrate at pH 7.2 (see Table 1, footnote
a for complete conditions)8 were carried out at various
concentrations of 4,10 12a , and 12b as Bu4N+ salts. The IC50
values for each compound were measured in the absence and
presence of inorganic pyrophosphate (1.5 mM PPi) on the
expectation that the ammonium/diphosphate ion pairs might
interact more strongly with the catalytic site on the enzyme.9
In fact, synergistic effects of the PPi additive on the inhibi-
tory potencies of the aziridino diphosphates 12a (ca. 4-fold)
and 12b (ca. 2-fold) were observed, although the IC50 of the
aza bifarnesyl inhibitor was unaffected by PPi addition
within experimental error. A Ki value of 0.21 µM (competi-
FPP
tive against FPP, Km
) 20 µM) was determined for the
most potent aziridino diphosphate inhibitor 12a in the
presence of PPi.
Ack n ow led gm en t. We thank the National Institutes
of Health for financial support of this work through Grants
GM13956 (R.M.C.) and GM25521 (C.D.P.) and the National
Science Foundation for providing support for the VOICE
NMR Facility at the University of Illinois.
(19) Coates, R. M.; Stanchina, C. L.; Koohang, A. Unpublished results.
(20) Corey, E. J .; Venkateswarlu, A. J . Am. Chem. Soc. 1972, 92, 6190.
(21) Acid chloride 10 and mesylate 12 were synthesized by chain
extensions of (E)-1-iodo-4,8-dimethyl-3,7-nonadiene (homogeranyl iodide).21a
Alkylation of tert-butyl lithioacetate (THF, -78 °C, 77%),21b saponification
(KOH, H2O, EtOH, reflux, 60%), and acid chloride formation ((COCl)2,
pyridine, rt)21c afforded 10. Alkylation of diethyl sodiomalonate (ethanol,
62%) followed by LiAlH4 reduction of the sodium salt (10 equiv of LiAlH4,
DME, reflux)21d gave (E)-6,10-dimethyl-2-methylene-5,9-undecadienol con-
taining ca. 25% of the 2-methyl analogue, which was converted to mesylate
13 (MsCl, Et3N, ether, 0 °C): (a) Marshall, J . A.; Dehoff, B. S. Tetrahedron
1987, 43, 4849. (b) Heathcock, C. H.; Piettre, S.; Ruggeri, R. B.; Ragan, J .
A.; Kath, J . C. J . Org. Chem. 1992, 57, 2554. (c) Mori, K.; Matsui, M.
Tetrahedron 1970, 26, 2801. (d) Marshall, J . A.; Andersen, N. H.; Hoch-
stetler, A. R. J . Org. Chem. 1967, 32, 113.
Su p p or tin g In for m a tion Ava ila ble: The detailed experi-
mental procedures and characterization data for compounds 7-9,
11, 14, and 12a ,b, and unnumbered intermediates; 1H NMR
spectra for 7-9, 11, 14, and 12a ,b, and unnumbered intermedi-
ates; and 31P NMR spectra for 12a ,b (24 pages).
J O981833Z
(22) (a) Davisson, V. J .; Woodside, A. B.; Poulter, C. D. Methods Enzymol.
1984, 110, 130. (b) Woodside, A. B.; Huang, Z.; Poulter, C. D. Org. Synth.
1988, 66, 211. (c) Davisson. V. J .; Woodside, A. B.; Neal, T. R.; Stremler, K.
E.; Muehlbacher, M.; Poulter, C. D. J . Org. Chem. 1986, 51, 4768.
(23) The n-Bu4N+ salts 12a ,b were contaminated by an equal amount of
unreacted Bu4NHP2O7 . Selected data for 12a : 31P NMR (162 MHz, CD3CN)
δ -2.29 and -2.86 (2 d, J ) 19.4 Hz, 2P); 1H NMR (400 MHz, CD3CN) δ
3.73 and 3.65 (two dt, J ) 10.8, 6 Hz, 1H each, CH2OPP); Selected data for
12b: 31P NMR (162 MHz, CD3CN) δ -2.23 and -2.83 (2 d, J ) 19.4 Hz,
2P); 1H NMR (400 MHz, CD3CN) δ 3.78 and 3.69 (two dt, J ) 11, 6 Hz, 1H
each, CH2OPP), 4.98 and 4.78 (two singlets, 1H each, dCH2).
(24) pKa values of 8.3-8.6 have been reported for alkyl-substituted
aziridines: (a) O’Rourke, C. E.; Clapp, L. B.; Edwards, J . O. J . Am. Chem.
Soc. 1956, 78, 2159. (b) Searles, S.; Tamers, M.; Block, F.; Quarterman, L.
J . Am. Chem. Soc. 1956, 78, 4917.
(25) Steroids bearing aziridines in the side chain (24,25-epimino and
24,28-epimino) are considered to be aza analogues of protonated cyclopro-
pane intermediates in the side chain alkylations associated with phytosterol
biosynthesis.25a-c (a) Fujimoto, Y.; Morisaki, M.; Ikekawa, N. Steroids 1974,
24, 367. (b) Giner, J .-L.; Djerassi, C. J . Am. Chem. Soc. 1991, 113, 1386. (c)
Nes, W. D.; Guo, D.-a; Zhou, W. Arch. Biochem. Biophys. 1997, 342, 68.