Enantioselective inhibition of squalene synthase by aziridine analogues of presqualene diphosphate

Article abstract of DOI:10.1021/jo100718z

(Figure Presented) Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates (6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation-reduction sequence of (±)-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldimethylsilyl ethers. S_N2 displacements of the corresponding methanesulfonates with pyrophosphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and methanediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(α-methylene)bishomogeranyl substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute stereochemistry to PSPP proved to be the most potent inhibitor (IC₅₀ 1.17 ± 0.08 M in the presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The other aziridine analogues bearing the N-(α-methylene)bishomogeranyl and N-undecyl substituents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene synthase.

Full text of DOI:10.1021/jo100718z

pubs.acs.org/joc
Enantioselective Inhibition of Squalene Synthase by Aziridine Analogues
of Presqualene Diphosphate
Ali Koohang,†,§ Jessica L. Bailey,†, Robert M. Coates,*,† Hans K. Erickson,‡,^
David Owen,‡,# and C. Dale Poulter‡
†Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, and
‡Department of Chemistry, University of Utah, Salt Lake City, Utah 84112. §Current address: Abbott
Laboratories, Abbott Park, IL. Current address: GlaxoSmithKline, King of Prussia, PA. ^Current address:
ImmunoGen Inc., Waltham, MA. #Current address: Starpharma Pty, Melbourne, Australia.
rmcoates@uiuc.edu
Received April 13, 2010
Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to
squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this
novel reaction constitutes the first committed step in sterol biosynthesis, there has been consider-
able interest and research on the stereochemistry and mechanism of the process and in the design of
selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five
racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as
inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates
(6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation-reduction
sequence of (()-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldi-
methylsilyl ethers. SN2 displacements of the corresponding methanesulfonates with pyropho-
sphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and metha-
nediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(R-methylene)bishomogeranyl
substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S
diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute
stereochemistry to PSPP proved to be the most potent inhibitor (IC50 1.17 ( 0.08 μM in the
presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The
other aziridine analogues bearing the N-(R-methylene)bishomogeranyl and N-undecyl substitu-
ents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene
synthase.
Introduction
the biosynthesis of cholesterol and other essential sterols.
The conversion of FPP to squalene occurs in two steps by
way of the stable, enzyme-bound intermediate, presqualene
diphosphate (PSPP, 4 in eq 2).2 In the first and nonreductive
step, a formal stereospecific 1,1-elimination of HOPP from
one FPP is effected with two alkylations of the 2,3-double
bond of the second FPP molecule to form the cyclopropane
ring. Rearrangements and ring cleavage via carbocation inter-
mediates terminated by hydride capture from the NADPH
Squalene synthase (SS, EC 2.5.1.21) catalyzes the reduc-
tive head-to-head coupling of two molecules of (E,E)-farne-
syl diphosphate (FPP, 1-OPP) to squalene (2, eq 1).1 The
acyclic polyene product is a precursor to most polycyclic
triterpenes, and its formation is the first committed step in
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J. Org. Chem. 2010, 75, 4769–4777 4769
DOI: 10.1021/jo100718z
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Published on Web 06/15/2010
2010 American Chemical Society

Koohang et al.
JOCArticle
cofactor constitute a chemically reasonable mechanism for
the second step that now has considerable experimental
support.3
products led to the expectation that its inhibition would
selectively block triterpene and sterol biosynthesis.8 This
rationale has motivated a worldwide search for new inhibitors
of squalene synthase9 as potential anticholesterolemic,10a-c
antiparasitic,10d and antimicrobial agents.11 Some examples
of natural products discovered in screening programs are
the zaragozic acids,12a,b phomoidrides,12c,d viridiofungin,11b
schizostatin,12e and bisabaquals.12f Potent synthetic SS inhi-
bitors include FPP mimics (bisphosphonates, (phosphinyl-
methyl)phosphonates, and R-(phosphono)sulfonates)13 as
well as some heterocyclic compounds.14 Various ammo-
nium15,16 and sulfonium17 ion inhibitors designed to mimic
the substrate, carbocationic intermediates, or the product of
the conversion of FPP to squalene have been reported. Crystal
structures of human squalene synthase18a,b and S. aureous
dehydrosqualene synthase18c are now available to guide in-
hibitor design and to comprehend the role of the enzymes in
the catalytic mechanism.
PSPP and its analogues are intermediates in other related
biochemical processes. This vinyl cyclopropylcarbinyl diphos-
phate is considered to be the progenitor of the botryococcenes,
a family of acyclic triterpenes formed by an unusual 10-3
connection of farnesyl units.4 PSPP serves as an intercellular
signal for down-regulation of superoxide formation in neutra-
phils.5 The corresponding C40 compound prephytoene di-
phosphate is an intermediate in phytoene formation in the
biosynthesis of the carotenoid plant pigments.6 Chrysanthemyl
diphosphate, the C10 relative, is a key intermediate in biosynth-
esis of irregular monoterpenes such as artemesia ketone.7
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6-OPP was synthesized in both racemic and enantiopure forms to
assess the stereoselectivity of its interaction with the enzyme. The
related N-undecylaziridines (()-8-OPP and (()-8-OMDP were
also prepared as simpler models to evaluate the importance of the
isoprenoid polyene chains in the binding interaction.
Synthesis of 2,3-Aziridinofarnesol (9-OH). The aziridine
ring in (()-9-OH was introduced by Atkinson’s hydroxyl-
directed imination21 of (E,E)-farnesol with 3-amino-4-(3H)-
quinazolone (Pb(OAc)4, CH2Cl2, -23 ꢀC, 2 h, 73%) (eq 3).
The quinazolone substituent was removed by reductive clea-
vage of the N-N bond (6 equiv of Li, NH3, THF, -33 ꢀC,
67%), conditions found to be general in a series of N-
quinazolonylaziridines.22
FIGURE 1. Aza analogues of hypothetical acyclic and bridged
carbocation/OPP- ion pairs 3A and 3B, respectively, proposed as
intermediates in different mechanisms for squalene synthase-
catalyzed formation of the three-membered ring of presqualene
PP from two molecules of farnesyl PP (eq 2). R=homogeranyl.
occur in biosynthetic processes catalyzed by terpene syn-
thases.15,19,20 These isoprenoid amines are potentially useful as
selective inhibitors and as mechanistic probes to elucidate the
structure, stereochemistry, charge delocalization, and active-site
interactions of the fleeting carbocation intermediates. In the case
of PSPP, it is reasonable to propose that the cyclopropane ring is
formed by electrophilic attack of C1 of one FPP molecule on the
2,3-double bond of the other, generating a carbocation/OPP-
ion pair within the enzyme active site (eq 2). In one mechanistic
scenario, an acyclic, tertiary carbocation (3A) with localized
positive charge would undergo 1,3 elimination of proton HB (C1
pro-S hydrogen). Alternatively, the intermediate might be better
represented as a bridged ion (protonated cyclopropane 3B) with
partial bonding to C2 and C3 and more charge delocalization.
The aza bifarnesyl and aziridinyl diphosphates (()-5-OPP,
(()-6-OPP, and (()-7-OPP (Figure 1) were synthesized pre-
viously in racemic form as mechanistic probes for the first step in
the squalene synthase reaction, and preliminary results on their
inhibitory properties were reported.15c,20 The protonated form of
the acyclic amino PP was intended to mimic tertiary ion 3A where-
as the aziridinium PPs would resemble more closely bridged ion
3B. The nordihydro and β-methylene isoprenoid substituents on
nitrogen in 6 and 7 were chosen as more stable surrogates for the
N-vinyl group in the exact aziridine aza analogue of PSPP.
In this paper we report syntheses of aziridines 6-OPP and 7-
OPP together with the corresponding hydrolytically more stable
methanediphosphonates 6-OMDP and 7-OMDP, and assays of
their inhibition of a truncated, soluble form of recombinant squa-
lene synthase from yeast. The more potent aziridine inhibitor
The enantiopure forms of 2,3-aziridino farnesol 9-OH
were prepared from their 2,3-epoxy precursors according to a
method developed for the synthesis of a series of racemic trisub-
stituted isoprenoid aziridines (eq 4).23 (E,E)-Farnesol was con-
verted to both enantiomeric 2,3-epoxides by the Sharpless asym-
metric epoxidation24 in excellent yields and high stereopurities
1
(96:4 ( 0.4 er) according to H NMR analyses of the (1S)-
camphanate derivatives (see Supporting Information). (þ)-2,3-
Epoxyfarnesol, (þ)-10, was converted to the corresponding
azidohydrin by regio- and stereospecific azide opening of the
epoxide with diethylaluminum azide25 (Et2AlCl, NaN3, toluene,
-78 to 0 ꢀC, 80%). Selective silylation at the primary hydroxyl
position (TBDMSCl, imidazole, DMAP, DMF, 0 ꢀC, 82%)
followed by conversion to the corresponding mesylate (MsCl,
NEt3, CH2Cl2, 0 ꢀC, 81%) and reductive cyclization (LiAlH4,
ether, 83%) afforded enantiopure aziridine (-)-9-OH [R]D
-19.2 (c 1.42, CHCl3). The enantiomeric NH-aziridino alcohol
(þ)-9-OH [R]D þ19.3 (c 1.21, CHCl3) was prepared from
epoxide (-)-10 in the same way.
(E)-6,10-Dimethyl-5,9-undecadienoic acid (14b), the pre-
cursor to the nordihydroisoprenoid chain in 6-OH, was
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synthesized by homologation of homogeraniol (13a) (eq 5).26
Conversion to the iodide (I2, Ph3P, imidazole, THF; 88%)27
followed by alkylation of tert-butyl lithioacetate (HMPA,
THF, -78 ꢀC, 77%)28 afforded tert-butyl ester 14a. Saponifica-
tion under strongly basic conditions, namely Claisen’s alkali29
(KOH, H2O, EtOH, 3 h), provided isoprenoid acid 14b. The
corresponding acid chloride 14c (oxalyl chloride, pyridine,
benzene, 0 ꢀC, 24 min) was used immediately in the N-acylation
of the racemic and enantiopure forms of silyl ether aziridine 9-
OTBDMS (eq 6).
converted to the N-acylaziridine 18 by reaction with acid
chloride 14c (Et3N, CH2Cl2). Although 18 was purified once
by rapid silica gel flash column chromatography, this material
was used in crude form immediately in most reactions owing to
its instability on silica gel or to prolonged storage even at -20 ꢀC.
Hydride reduction (10 equiv of LiAlH4, ether, reflux, 48 h) was
accompanied by concomitant removal of the silyl group. Puri-
fication by silica gel chromatography afforded N-alkylaziridine
6-OH (32%), recovered NH-aziridino alcohol 9-OH (17%), and
trishomogeraniol 13a (27%) as shown in eq 6. The acylation-
reduction approach was used to prepare aziridine 6-OH in
both enantiomerically pure forms (-)-6-OH [R]D -3.7 (c 0.85,
CHCl3) and (þ)-6-OH [R]D þ4.9 (c 1.09, CHCl3) from aziridine
(-)-9-OH and its enantiomer (þ)-9-OH, respectively.
The formation of substantial amounts of acyl cleavage
products 9-OH and trishomogeraniol (13a) in the LiAlH4
reduction of N-acyl-O-silyl aziridine 18 prompted some
experiments to determine whether this side reaction could
be suppressed and to try to understand the competing
processes. Reductions of 18 with other hydride reducing
agents and/or conditions had no significant effect on the
product ratios and yields. The model substrate N-pentanoyl-
O-silyl aziridino alcohol 19 was prepared in the same manner
by sequential silylation and acylation with pentanoyl chlo-
ride (80%). An attempt to remove the TBDMS protecting
group with Bu4NF afforded the O-pentanoyl ester 20 (68%)
instead of the N-pentanoyl aziridino alcohol isomer (eq 7).
Evidently, rapid N-to-O acyl migration followed attack of
fluoride on the silyl group. Prolonged reduction of 19 (10
equiv LiAlH4, ether, reflux, 60 h) furnished the N-pentyla-
ziridino alcohol 21 (46%) together with cleavage byproduct
9-OH (28%) in accord with the previous results with 18.
The (E)-6,10-dimethyl-2-methylene-5,9-undecadienol pre-
cursor 16-OH for the N-substituent in aziridino alcohol 7-OH
was prepared by means of Marshall’s R-methylene alcohol
synthesis.30 Alkylation of diethyl sodiomalonate with iodide
13b provided homogeranyl malonate 15 (NaOEt, EtOH, reflux,
1 h, 62%) accompanied by a substantial amount of the cyclo-
propylcarbinyl ethyl ether (31%, structure not shown) presum-
ably arising from 13b by competing homoallylic participation
and nucleophilic attack of ethanol on the cyclopropylcarbinyl
ion. Deprotonation of malonate 15 with 1.2 equiv of NaH
(DME, reflux) followed by hydride reduction (2.6 equiv of
LiAlH4, reflux, 3 h, 79%) provided a 3:1 mixture allylic alcohol
16-OH and its dihydro derivative 17 based on 1H NMR inte-
grations. The resulting mixture of alcohols was converted to the
corresponding mesylate derivatives (MsCl, NEt3, ether, -78
to -15 ꢀC). Immediate N-alkylation of the TBDMS ether
(9-OTBDMS)31 of (()-aziridine 9-OH followed by fluoride-
induced desilation afforded (()-7-OH in 34% yield (eq 6).
LiAlH4 reductions of the unstable N,O-dipentanoyl deri-
vative 22, prepared by acylation of aziridino alcohol 9-OH
with pentanoyl chloride (2.1 equiv of Et3N, ether, 0 ꢀC,
80%), proved to be time dependent (eq 8). After 1 h, the sole
product obtained was the starting NH-aziridino alcohol 9-
OH (79%). No effort was made to isolate the C5 alcohol
coproduct. However, after 24 h, the N-pentyl aziridino
alcohol 21 was isolated in 32% yield, along with 51% of
recovered 9-OH. Continuation of the slow reduction for 48 h
Installation of the N-alkyl substituent in 6-OH was effected
by a three-step sequence: O-silylation-N-acylation-hydride
reduction (eq 6). Racemic aziridine 9-OH was again O-silylated
to 9-OTBDMS (TBDMSCl, imidazole, DMF, 75%)20,31 and
(28) (a) Heathcock, C. H.; Piettre, S.; Ruggeri, R. B.; Ragan, J. A.; Kath,
J. C. J. Org. Chem. 1992, 57, 2554–2556. (b) Heathcock, C. H.; Hansen, M. M.;
Ruggeri, R. B.; Kath, J. C. J. Org. Chem. 1992, 57, 2544–2453. (c) Heathcock,
C. H.; Piettre, S.; Kath, J. C. Pure Appl. Chem. 1990, 62, 1911–1920.
(29) Claisen, L.;Eisleb, O.;Kremers, F.Liebigs Ann. Chem 1919, 418, 96–120.
(30) Marshall, J. A.; Andersen, N. H.; Hochstetler, A. R. J. Org. Chem.
1967, 32, 113–118.
(26) Leopold, E. J. Org. Synth. 1986, 64, 164–174. Organic Synthesis;
Wiley: New York, 1990; Collect. Vol. VII, p 258-263.
(27) Marshall, J. A.; Dehoff, B. S. Tetrahedron 1987, 43, 4849–4860.
(31) Corey, E. J.; Venkateswarlu, A. J. Am. Chem. Soc. 1972, 92, 6190–6191.
4772 J. Org. Chem. Vol. 75, No. 14, 2010

Koohang et al.
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resulted in further conversion to 21 (67%), and the amount
NH,OH aziridino alcohol 9-OH decreased to 13%.
31P NMR analysis of the crude products revealed a 1:1 mixture
of aziridinyl diphosphates to inorganic diphosphate. Similar
reactions with methanediphosphonate anion (2 equiv ((Bu4N)3-
OMDP, CH3CN, molecular sieves, rt, 60 h) were considerably
slower, and in most cases, 1:3 mixtures of the aziridine methane-
diphosphates to methanediphosphonate were obtained. The
N-undecylaziridine analogues 8-OH, 8-OPP, and 8-OMDP
were prepared from (()-9-OH in the same way.
The relatively slow conversions of the N-acylaziridines to the
N-alkylaziridines in the preceding reductions is presumably
attributable to angle strain associated with formation of the
required aziridinium ion intermediates. This allows time for the
initially formed aziridino alkoxides to undergo fragmentation
to the corresponding aldehydes and aziridino alcohol 9-OH,
and the former would be rapidly reduced to the primary alcohol
byproduct. In fact, the partial hydride reduction of N-acyl
derivatives of the parent ethyleneimine is known to give
aldehydes.32 The facile N-to-O acyl transfer observed in the
desilylation of 19 suggests the possibility of similar silyl transfers
in the hydride reductions of the N-acyl-O-silyl aziridino alcohols
(eq 9). The simultaneous loss of the TBDMS groups in these
reductions and the known stability of this protecting group to
The crude aziridinyl diphosphates obtained from these reac-
tions were 1:1 mixtures of aziridine diphosphates and inorganic
pyrophosphate according to 31P NMR spectra. Purification and
characterization of organic diphosphates prepared by the dis-
placement method are typically carried out after ion exchange to
the more tractable ammonium salts, at which stage remaining
inorganic pyrophosphate can be separated by precipitation from
methanol.34 Unfortunately, initial attempts to effect ion ex-
change caused partial conversion to a new compound tenta-
tively assigned as the 5- or 6-membered cyclic monophosphates
24 or 25 (δP -1.75, s) arising from aziridinium ion opening of 8-
OPP, cyclization onto the proximal phosphate, and hydrolysis
of the terminal phosphate. Accordingly we opted to forego ion
exchange and to conduct the enzyme inhibition assays with the
unpurified aziridinyl PPs as their tetrabutylammonium salts,
with the consequences that 1H NMR characterization would be
limited since the spectra are dominated by the intense resonances
for the butyl groups and that approximately 1 equiv of inorganic
OPP- would necessarily be present in the incubation medium.
Nevertheless, it proved possible to discern the peaks for the
33
LiAlH4 provide support for this assumption. It therefore
seems reasonable to propose the sequence of reactions shown
in eq 9 (23a f 23b f 23c), i.e., hydride addition to the
aziridinamide carbonyl, silyl transfer, C-O bond heterolysis
to the aziridine iminium ion, and finally, a second hydride attack
to form the N-alkylaziridino alkoxide.
CH2OPP protons in the 1H NMR spectra of 6-OPP (Bu4N)3
3
Formation of Diphosphates and Methanediphosphates.
Phosphorylations of N-alkylaziridino alcohols were carried out
by activation of the primary alcohols followed by SN2 dis-
placement with pyrophosphate or methanediphosphonate an-
ions (eq 10).34 Reaction of enantiopure and racemic aziridines
(()-6-OH, (-)-6-OH, and (þ)-6-OH and racemic 7-OH with
MsCl provided the corresponding mesylates 6-OMs and 7-OMs
(Et3N, CH2Cl2, -15 ꢀC, 0.5 h, 85-97%). Displacement by
inorganic pyrophosphate (2 equiv of (Bu4N)3OPP, CH3CN,
molecular sieves, rt, 48 h) afforded the aziridinyl diphosphates.
salt, as well as resonances ascribable to the ring C-CH3, twoCH-
N protons, and three vinyl protons.
Ion-exchange chromatography (AG 50W-X8, 100-200
mash, NH4þ) of 8-OPP Bu4N using 25 mM NH4CO3 as buffer
3
effected complete conversion to the NH4þ salt. The crude mate-
rial was washed with CH3OH, and inorganic pyrophosphate
was removed by filtration. 31P NMR analysis of the methanol-
soluble material showed a 1:1 mixture of the aziridinyl diphos-
phate (δP -5.85, d, 1P, J=20.7 Hz and δP -7.19, d, 1P, J=
20.7 Hz) and an upfield resonance (δP -1.75, 1P, singlet) attri-
buted to a ring-opened monophosphate product. A 2:1 mixture
(31P NMR) favoring the aziridinyl diphosphate was obtained
(32) Brown, H. C.; Tsukamoto, A. J. Am. Chem. Soc. 1961, 83, 4549–
4552.
(33) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synth-
esis, 3rd ed.; Wiley: New York, 1999; p 710.
(34) (a) Davisson, V. J.; Woodside, A. B.; Poulter, C. D. Methods
Enzymol. 1984, 110, 130–144. (b) Woodside, A. B.; Huang, Z.; Poulter,
C. D. Org. Synth. 1988, 66, 211–219. Organic Synthesis; Wiley: New York,
1993; Collect. Vol. VIII, pp 616-620. (c) Davisson., V. J.; Woodside, A. B.;
Neal, T. R.; Stremler, K. E.; Muehlbacher, M.; Poulter, C. D. J. Org. Chem.
1986, 51, 4768–4779. (d) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter,
C. D. J. Org. Chem. 1987, 52, 1794–1801.
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TABLE 1. Inhibition of Recombinant Yeast Squalene Synthase by Azir-
idino Diphosphates and Methanediphosphonates (Figure 1) in the Absence
and Presence of Added Inorganic Pyrophosphate (PPi) at pH 7.2a
values for each compound (Table 1) were measured in the
absence and presence of inorganic pyrophosphate (1.5 mM) on
the expectation that the aziridinium/pyrophosphate ion pairs
might exhibit stronger interaction with the catalytic site on the
enzyme. However ca. 1 equivalent of OPP- anion was already
present in the unpurified NBu4þ salts of 6-OPP, 7-OPP, and 8-
OPP, and 2-3 equiv of OMDP- anion was present in the
corresponding methanediphosphonates. In contrast, the inhibi-
tion of squalene synthase by the acyclic aza analogue 5-OPP was
determined on the purified ammonium salt established to be free
of OPP- anion.15c,20
In the absence of added PPi, (2R,3S)-6-OPP obtained from
(-)-6-OH resembling the proper enantiomer of PSPP was a
much stronger inhibitor of squalene synthase (about 16-fold)
than the (2S,3R)-enantiomer obtained from (þ)-6-OH.
Although the synergistic effect of the PPi additive on the
inhibitory properties of the enantiopure aziridino diphosphates
was more pronounced for the “wrong” enantiomer (2S,3R)-6-
OPP (about 4-fold vs almost no change), the 2R,3S enantiomer
remained 4 times more potent under these conditions. Addi-
tional kinetic experiments established a Ki of 0.21 μM (com-
petitive against FPP, Km for FPP = 20 μΜ) for racemic 6-OPP
in the presence of PPi. The selectivity of the interactions of 6-
OPP and 7-OPP with the recombinant enzyme was revealed by
the much lower affinity exhibited by 8-OPP bearing the N-
C11H23 substituent (55% inhibition of squalene synthase at
219 μM in the absence of PPi) instead of the unsaturated
isoprenoid groups in 6-OPP and 7-OPP. Among the methane-
diphosphonate derivatives, aziridine 6-OMDP and 7-OMDP
inhibited squalene synthase in the presence of PPi additive with
IC50 values of 13.8 and 17.4 μM, respectively.
IC50b (μM)
inhibitor
-PPi
þPPic
11.4 ( 2.2
1.9 ( 0.2
Kid (μM)
(()-5-OPP
(()-6-OPP
9.7 ( 2.7
7.2 ( 0.8
0.21
(2R,3S)-6-OPP
(2S,3R)-6-OPP
(()-6-OMDP
(()-7-OPP
(()-7-OMDP
(()-8-OPP
1.19 ( 0.05
18.7 ( 4.9
3.2 ( 0.2
1.17 ( 0.08
4.8 ( 0.9
13.8 ( 3.8
4.0 ( 0.4
17.4 ( 5.3
80% inhibition
at 219 μM
no inhibition
6.9 ( 1.6
99.8 ( 18.5
55% inhibition
at 219 μM
no inhibition
(()-8-OMDP
aAssay conditions: 50 mM MOPS, pH 7.2, 20 mM MgCl2, 2% (v/v)
Tween 80, l mM DTT, 1 img/mL bovine serum albumin. 1.0 mM NADPH,
0 or 1.5 mM PPi, 100 uM [3H]FPP (7.5 μCi/μmol). 0-300 μM inhibitor, and
0.1 μg of protein in a total volume of 200 μM. Samples were incubated at
30 ꢀC for 5 min before addition of enzyme and stopped after an additional
10 min by addition of 40% aqueous KOH in methanol. Product
[3H]squalene was isolated by extraction with hexanes, purified by filtration
over alumina, and analyzed by liquid scintillation spectrometry. bIC50 values
were interpolated from plots of specific activity at [I] = 0.001, 0.50, 1.00,
3.00, 10.0, 30.0, and 300 μM using Graf it (Erithicus Software, Staines, UK).
Deviations shown are standard errors. cIncubations in the presence of
1.5 mM PPi. d5-10 μM [3H]FPP (7.5 μCi/μmol).
when 1 M (NH4)2CO3 was used as ion exchange buffer, and
the receiving tubes were basified immediately with NH4OH.
Further purification of the mixture by cellulose chromato-
graphy (8:1:1 of 2-propanol/concd. NH4OH/H2O as eluent)
provided a white powder, the structure of which was assigned
as an organic monophosphate by MS [(þ)FAB, m/z 472.4
(Mþ1)]. Cyclic amino monophosphates 24 or 25 are tenta-
tively proposed as possible structures for this byproduct.
Enzyme Inhibition Assays. The apparent instability of the
aziridine PPs during ion exchange in the presence of aqueous
NH4HCO3 buffer, and the known tendency of aziridines to
undergo solvolytic ring-opening under acidic conditions,35,36
prompted control experiments to evaluate the stability of the
compounds in the standard squalene synthase assay condi-
tions (pH 7.2, Mg2þ). 31P NMR spectra of solutions (D2O,
NH4OD, pH 9) of 8-OMDP were unchanged after 3 days,
whereas the spectra of diphosphate 8-OPP showed a new
singlet (δP -1.8, s) similar to those seen in the spectra of the
aziridinyl PPs after ion exchange. Fortunately, the rate was
slow enough (t1/2 ∼4 days at pH 9 and 18 h at pH 7) that this
process was unlikely to occur to a significant extent during the
typical 1-h incubations.
Discussion
Aziridine 6-OPP proved to be one of the most potent
mechanism-based squalene synthase inhibitors known. The
IC50 value of 1.2 μM for the 2R,3S enantiomer and the
submicromolar Ki determined previously for the racemate20
suggest quite strong interactions with the enzyme, despite the
absence of the proximal double bond and the methyl group
in the side chain on the aziridine nitrogen. The heightened
inhibition exerted by the 2R,3S enantiomer matching the
configuration of PSPP over that of the “wrong” 2S,3R stereo-
isomer in both the absence and presence of the PPi additive
(16-fold and 4-fold, respectively) signify substantial stereo-
selectivity in their association with the protein. The enhanced
affinities of these aza analogue inhibitors for the enzyme
compared to those of FPP substrate (S0.5FPP =19 ( 6 μM)
and the PSPP intermediate (KiPSPP= 75 ( 20 μM)38 encou-
rage the view that the compounds may be valid mimics of
transient carbocation intermediates. Furthermore, the grea-
ter potency of the aziridine inhibitors 6-OPP and 7-OPP,
compared to that of the acyclic aza bifarnesyl compound 5-
OPP, is consistent with a protonated cyclopropane inter-
mediate 3B analogous to that proposed in the biosynthesis of
related C10 isoprenoids.7b,c Nevertheless, the kinetics and
mechanism of the squalene synthase reaction are quite
complex,38 and the precise orientation of the compounds in
the catalytic site is uncertain. The reduced affinity of the
three methanediphosphonates (()-6-OMDP, (()-7-OMDP,
Kinetic assays of recombinant yeast squalene synthase
activity37 with [3H]FPP (100 μM, 7.5 μCi/μmol) as substrate at
pH 7.2 were carried out at various concentrations of the
aziridines as Bu4Nþ salts. Product [3H]squalene was isolated
by extraction with hexanes, purified by filtration over silica gel,
and analyzed by liquid scintillation spectrometry. The IC50
(35) (a) Lacourciere, G. M.; Vakharia, V. N.; Tan, C. P.; Morris, D. I.;
Edwards, G. H.; Moos, M.; Armstrong, R. N. Biochemistry 1993, 32, 2610–
2616. (b) Tomasz, M.; Lipman, R. J. Am. Chem. Soc. 1979, 101, 6063–6067.
(c) Beijnen, J. H.; Van der Houwen, O. A. G. J.; Rosing, H.; Underberg,
W. J. M. Chem. Pharm. Bull. 1986, 34, 2900–2913.
(36) (a) Earley, J. E.; O’Rourke, C. E.; Clapp, L. B.; Edwards, J. O.;
Lawes, B. J. Am. Chem. Soc. 1958, 80, 3458–3462. (b) Schatz, V. B.; Clapp,
L. B. J. Am. Chem. Soc. 1955, 77, 5113–5116.
(37) Zhang, D.; Jennings, S. M.; Robinson, G. W.; Poulter, C. D. Arch.
Biochem. Biophys. 1993, 304, 133–143.
(38) Radisky, E. S.; Poulter, C. D. Biochemistry 2000, 39, 1748–1760.
4774 J. Org. Chem. Vol. 75, No. 14, 2010

Koohang et al.
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and (()-8-OMDP should presumably be attributed to the
higher pKa values for methanediphosphonic acids and their
weaker interaction with the PP binding pocket.39
tion of protein structure by affinity-labeling or X-ray
crystallography.43
Squalene synthase has two nonequivalent PP binding sites
that accommodate both FPP substrates. A strong interac-
tion of the PP group of the inhibitors with either one would
effectively prevent squalene synthesis. The rate-determining
step of the normal enzymatic reaction is proposed to be
either PSPP formation or a conformational change of the
enzyme during the catalytic cycle.38 The protonation state,
hybridization, and charge at the amino nitrogen of the aza
analogues no doubt affect the resemblance of the inhibitors
to the carbocations they are intended to mimic, i.e., 3A and
3B. Aza-bifarnesyl PP 5-OPP very likely exists predomi-
nantly in the tetrahedral ammonium form at pH 7.2 (pKa
ca. 10-11). However, the pKa values for highly substituted
aziridines are rather low (pKa 4-6),35,40 and the aziridine
inhibitors 6-OPP and 7-OPP probably associate with the
protein in their unprotonated, but still tetrahedral, forms.
Clearly subtle differences such as these will influence the
interactions of the inhibitors with the catalytic apparatus of
the protein.
Some of the ambiguities are highlighted by the crystal
structure of bornyl diphosphate synthase complexed with
amine analogues of substrate, a carbocation intermediate,
and the bornyl PP product.19g 3-Aza-2,3-dihydrogeranyl PP
is bound in the ammonium form with the dimethylallyl tail in
an orientation differing from that predicted for the geranyl
PP substrate. (R)-7-Aza-7,8-dihydrolimonene, an amine
corresponding in structure and stereochemistry to the (R)-
R-terpinyl carbocation intermediate, is observed in the active
site in an inverted position. Hydrogen bonds with an incor-
porated water molecule may be one important factor in
the distortions observed in the crystal structures of these
inhibitor-enzyme complexes.
The stability of the enzyme-bound aziridine PPs is also
uncertain. The facility with which aziridinium ions undergo
ring-opening to potential electrophiles,35,36 and the observed
conversion of 8-OPP to proposed cyclic monophosphates 24
or 25 suggest that similar transformations might occur under
the influence of the enzyme. Although the kinetic behavior
was inconsistent with appreciable inactivation during the
measurements, the structural integrity of the aziridines under
the incubation conditions remains unknown.
Despite the forgoing imponderables, aziridine analogues
of bridged, nonclassical carbocations proposed as inter-
mediates in various terpene synthase-catalyzed reactions
are novel, mechanism-based inhibitors for investigation
of these enzymes.41,42 This concept for inhibitor design
may lead to useful active site probes for characteriza-
Experimental Section
The purity of purified products was established by analysis of
1H NMR spectra to be g95% pure unless stated otherwise.
Complete experimental procedures and spectral data are out-
lined below for synthesis of enantiopure (-)-(2R,3S)-6-OH and
(2R,3S)-6-OPP. Yields or abbreviated procedures, and in some
cases optical rotations, are presented for intermediates and the
final product in synthesis of ent-6-OH and ent-6-OPP in the
Supporting Information. NMR spectra, physical data, and
purity evaluations agree well with those reported previously
for the racemic series.20,23 Physical and spectral data for the
enantiomers were identical.
(þ)-(2R,3R)-trans-2,3-Epoxy-3,7,11-trimethyl-(E)-6,10-dode-
cadien-1-ol (trans-2,3-Epoxyfarnesol) ((þ)-10). The asymmetric
epoxidation was carried out according to a procedure reported
for (þ)-2,3-epoxygeraniol.24 A suspension of powdered mole-
˚
cular sieves (1.83 g, 3A, activated) in CH2Cl2 (50 mL) and D-(-)-
diethyl tartrate (6.16 g, 28.9 mmol) was stirred and cooled at
-20 ꢀC under N2 as titanium (IV) isopropoxide (7.07 g, 24.9
mmol) in CH2Cl2 (7 mL) and 5-6 M tert-butyl hydroperoxide
in isooctane (12 mL, 70 mmol) were added sequentially. After
30 min at -20 ꢀC, trans, trans-farnesol (10.06 g, 45.2 mmol,
˚
dried over 3 A molecular sieves for 40 min) in CH2Cl2 (10 mL)
was added dropwise, and the mixture was stirred at -20 ꢀC for
2 h and 1 h at rt to generate a pale yellow oil after workup.
Purification by flash column chromatography (50% EtOAc/
hexanes) afforded 10.78 g (97%) of epoxy alcohol (þ)-10 as a
1
colorless oil. The H and 13C NMR spectral data and assign-
ments correspond to those reported in the literature.44 1H NMR
analysis of the (S)-camphanate ester derivative indicated that
the enantiomeric ratio was 94:6 (see Supporting Information):
[R]D = þ6.35, (c 1.73, CHCl3). The magnitude of the optical
rotation corresponds to that of enantioenriched (þ)-2,3-epoxy-
farnesol (10) with minor deviations due to concentration differ-
ences [lit.44 [R]D = þ6.53 (c 4.21, CHCl3)].
(þ)-(2S,3S)-3-Azido-3,7,11-trimethyl-(E)-6,10-dodecadiene-
1,2-diol (11). The following procedure is based on those reported
by Davis23 and Benedetti.25 A suspension of NaN3 (2.41 g, 37.0
mmol) in toluene (42 mL) was stirred and cooled at 0 ꢀC as a
solution of Et2AlCl (18.8 mL, 33.6 mmol, 1.8 M in toluene) was
added dropwise over 10 min via syringe. After 6 h at rt, the
resulting suspension was cooled to -78 ꢀC under N2, and a
solution of (þ)-2,3-epoxyfarnesol ((þ)-10, 4.00 g, 16.8 mmol)
was added dropwise over 10 min. The mixture was stirred for 1 h
at -78 ꢀC and for 16 h at rt, cooled to 0 ꢀC, and diluted with
EtOAc (70 mL). NaF (31.8 g) and H2O (3.9 mL) were added
sequentially, and the heterogeneous mixture was allowed to stir
for 4 h at rt. Filtration through a pad of anhyd NaSO4 (ca. 100 g)
to remove salts and water followed by solvent evaporation gave
a pale yellow oil. Purification by flash column chromatography
(50% EtOAc/hexanes) afforded 3.80 g (80%) of azido diol (þ)-
11 as a colorless oil: 1H NMR (500 MHz, CDCl3) δ 1.36 (s, 3H),
1.47 (ddd, J = 14.0, 10.7, 6.1 Hz, 1H), 1.59 (s, 3H), 1.62 (s, 3H),
1.65 (m, 1H), 1.68 (s, 3H), 1.97 (3 peak m, 2H), 2.08 (8 peak m,
4H), 2.25 (br s, 1H), 2.75 (br s, 1H) 3.58 (app ddd, J = 7.9, 3.2,
1.9 Hz, 1H), 3.6 g (app dd, J = 10.7, 2.8 Hz, 1H), 3.76 (app ddd,
J = 10.9, 7.1, 3.2 Hz, 1H), 5.09 (m, 2H); 13C NMR (126 MHz,
CDCl3) δ 16.2, 17.9, 19.6. 22.4, 25.9, 26.8, 36.7, 39.8, 62.7, 65.8,
76.4, 123.3, 124.4, 131.7, 136.4; IR (neat) νmax 2104 (N3), 3392
(OH) cm-1; [R]D þ34.7, (c 1.62, CHCl3).
(39) (a) Blackburn, G. B.; England, D. A.; Kolkmann, F. Chem. Com-
mun. 1981, 930–931. (b) Stremler, K. E.; Poulter, C. D J. Am. Chem. Soc.
1987, 109, 5542–5544.
(40) (a) O’Rourke, C. E.; Clapp, L. B.; Edwards, J. O. J. Am. Chem. Soc.
1956, 78, 2159–2160. (b) Searles, S.; Tamers, M.; Block, F.; Quarterman,
L. A. J. Am. Chem. Soc. 1956, 78, 4917–4920.
(41) (a) Giner, J. L.; Djerassi, C. J. Am. Chem. Soc. 1991, 113, 1386–1393.
(b) Fujimoto, Y.; Morisaki, M.; Ikekawa, N.; Horie, Y.; Nakasone, S.
Steroids 1974, 24, 367–375. (c) Nes, W. D.; Guo, D.-a.; Zhou, W. Arch.
Biochem. Biophys. 1997, 342, 68–81. (d) Janssen, G. G.; Nes, W. D. J. Biol.
Chem. 1992, 267, 25856–25863. (e) Pinto, W. J.; Nes, W. R. J. Biol. Chem.
1983, 258, 44724476.
(42) (a) Roy, A.; Roberts, F. G.; Wilderman, P. R.; Zhou, K.; Peters,
R. J.; Coates, R. M. J. Am. Chem. Soc. 2007, 129, 12453–12460. (b) Hu, H.;
Faraldos, J. A.; Coates, R. M. J. Am. Chem. Soc. 2009, 131, 11998–12006.
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hedron 1986, 42, 3789–3792.
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(2S,3S)-3-Azido-1-(tert-butyldimethylsilyloxy)-3,7,11-trim-
ethyl-(E)-6,10-dodecadien-2-ol (11-OTBDMS). The following
procedure is based on ones reported by Davis23 and Fujii and
Ibuka.45 A solution of the azido diol (11, 3.42 g, 12.17 mmol),
imidazole (4.98 g, 73.2 mmol), and 4-DMAP (147 mg, 1.20
mmol) in DMF (20 mL) was stirred and cooled at 0 ꢀC under N2
as TBDMSCl (5.52 g, 36.7 mmol) in DMF (18 mL) was added
dropwise over 5 min. The solution was warmed to rt, and after
30 min, ethanolamine (2.33 g, 37.7 mmol) was added to scavenge
excess TBDMSCl. After 2 h, H2O was added, and the product
was extracted with benzene. The organic extracts were combined,
dried (MgSO4), and concentrated under reduced pressure to give a
colorless oil. Purification by flash column chromatography (5%
EtOAc/hexanes) afforded 3.22 g (66%) of the primary silyl ether
and 0.675 g (14%) of the secondary silyl ether isomer. Data for
Hz, 1H), 3.50 (br s, 1H), 3.74 (ddd, J = 11.8, 4.9, 2.4 Hz, 1H),
5.08 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 16.2, 17.3, 17.9,
24.8, 25.9, 26.8, 39.7, 39.9, 41.7, 43.6, 61.6, 123.4, 124.4, 131.7,
136.1; [R]D -19.2 (c 1.42, CHCl3).
(2R,3S)-trans-3-[(tert-Butyldimethylsilyl)oxy]methyl-2-((E)-
4,8-dimethyl-3,7-nonadienyl)-2-methylaziridine (6-OTBDMS).
The following procedure is based on that reported by Koohang
et al.20 A solution of the (-)-aziridino alcohol ((-)-9-OH,
0.510 g, 2.15 mmol) in DMF (1.1 mL) was stirred and cooled
at 0 ꢀC under N2, while imidazole (0.374 g, 5.49 mmol) and
TBDMSCl (0.392 g, 2.60 mmol) were added. The ice bath was
removed, and the solution was allowed to stir for 30 min after
which time EtOAc (10 mL) and H2O (10 mL) were added. The
aqueous layer was extracted with EtOAc (3 ꢀ 10 mL). The organic
layers were combined, dried (MgSO4), and concentrated under
reduced pressure to give a yellow oil. Purification by flash column
chromatography (10% MeOH/CH2Cl2) afforded 0.327 g (62%) of
silyl ether, 9-OTBDMS, and 150 mg of recovered (-)-9-OH. Data
for 9-OTBDMS: 1H NMR (400 MHz, CDCl3) δ 0.06 (s, 3H), 0.07
(s, 3H), 0.90 (s, 9H), 1.18 (s, 3H), 1.33 (ddd, J = 13.5, 9.4, 6.6 Hz,
1H), 1.53 (m, 1H), 1.59 (s, 3H), 1.60 (s, 3H), 1.67 (d, J = 1.1 Hz,
3H), 2.03 (m, 7H), 3.57 (dd, J = 10.9, 6.4 Hz, 1H), 3.73 (ddd, J =
10.9, 5.4, 1.1 Hz, 1H), 5.11 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ -5.1, -4.9, 16.1, 17.4, 17.9, 18.5, 24.9, 25.9, 26.13, 26.9, 39.9,
41.7, 43.2, 63.3, 123.8, 124.5, 131.6, 135.8.
1
primary silyl ether 11-OTBDMS: H NMR (500 MHz, CDCl3)
δ 0.09 (s, 3H), 0.10 (s, 3H), 0.91 (s, 9H), 1.32 (s, 3H), 1.46 (ddd,
J = 14.1, 11.1, 6.0 Hz, 1H), 1.60 (s, 3H), 1.62 (s, 3H), 1.63 (m, 1H),
1.68 (d, J= 1.1 Hz, 3H), 1.98 (m, 2H), 2.08 (m, 4H), 2.77 (br s, 1H),
3.56 (ABX, JAX = 3.2 Hz, JBX = 8.1 Hz, 1H), 3.61 (ABX, JAB
=
9.6 Hz, JBX = 8.1 Hz, 1H), 3.74 (ABX, JAB = 9.6 Hz, JAX = 3.2
Hz, 1H), 5.10 (m, 2H); 13C NMR (126 MHz, CDCl3) δ -5.2,
16.2, 17.9, 18.4, 19.4, 22.3, 25.9, 26.0, 26.9, 36.8, 39.9, 62.0, 65.1,
75.9, 123.6, 124.5, 131.7, 136.1; IR (neat) νmax 2105 (N3), 3593
(OH) cm-1
.
(2S,3S)-3-Azido-1-(tert-butyldimethylsilyloxy)-3,7,11-tri-
methyl-(E)-6,10-dodecadien -2-yl Methanesulfonate (12). The
following procedure was based on that reported by Crossland.46
A solution of the primary silyl ether 11-OTBDMS (3.13 g,
7.91 mmol) and Et3N (2.54 g, 25.32 mmol) in CH2Cl2 (30 mL)
was stirred and cooled at 0 ꢀC under N2 as neat MsCl (2.66 g,
23.7 mmol) was added dropwise over 10 min. After 1 h, ice-cold
H2O (10 mL) was added, and the aqueous phase was extracted with
Et2O (3 ꢀ 30 mL). The organic extracts were combined, dried
(MgSO4), and concentrated under reduced pressure. Purification
by flash column chromatography (5% EtOAc/hexanes) afforded
(2R,3S)-trans-2-((E)-4,8-Dimethyl-3,7-nonadienyl)-1-((E)-6,
10-dimethyl-5,9-undecadienyl)-3-[(tert-butyldimethylsilyl)oxy]-
methyl-2-methylaziridine (18). The following procedure is based on
that reported by Koohang et al.20 A solution of bishomogeranic
acid (14b, 110 mg, 0.523 mmol) and pyridine (46 μL, 45 mg,
0.646 mmol) in benzene (4.5 mL) was stirred and cooled at 0 ꢀC
under N2 as oxalyl chloride (50 μL, 73 mg, 0.573 mmol) was
added dropwise. The suspension was allowed to warm to rt, and
stirring was continued until no bubbles were observed (ca.
25 min). The crude acid chloride was transferred immediately
via syringe to a rapidly stirring solution of 9-OTBDMS (183 mg,
0.521 mmol) obtained from the previous step and Et3N (218 μL,
158 mg, 1.56 mmol) in Et2O (4.5 mL) at 0 ꢀC under N2. After
40 min, H2O (10 mL) was added, and the product was extracted
with EtOAc (3 ꢀ 20 mL). The organic layers were combined,
dried (MgSO4), and concentrated by rotary evaporation with
the water bath maintained at 30 ꢀC. The crude N-acylaziridine
(288 mg) was immediately taken on to the next step.
(-)-(2R,3S)-trans-3-((E)-4,8-Dimethyl-3,7-nonadienyl)-1-((E)-
6,10-dimethyl-5,9-undecadienyl)-3-methylaziridine-2-methanol
(6-OH). The following procedure is based on that reported by
Koohang et al.20 A solution of LiAlH4 (202 mg, 5.32 mmol) in Et2O
(18 mL) was stirred and cooled at 0 ꢀC under N2 as the crude chiral
N-acylaziridine (288 mg, 0.529 mmol) in Et2O (5 mL) was added
dropwise via syringe. The pale gray suspension was heated at reflux
for 48 h, cooled to 0ꢀC, and stirred as sequential dropwise additions
of H2O (0.202 mL), 15% NaOH (0.202 mL), and H2O (0.606 mL)
were performed. After 2 h, the white solid was filtered and washed
with EtOAc (3 ꢀ 10 mL). The filtrates were combined, dried
(MgSO4), and concentrated under reduced pressure to give a pale
yellow oil. Purification by flash column chromatography (50%
EtOAc/hexanes) afforded 69 mg (32%) of the N-alkylaziridino
alcohol as a colorless oil, 21 mg (17%) of the NH aziridino alcohol
(9-OH), and 28 mg (27%) of trishomogeraniol. Data for (-)-6-OH:
1H NMR (500 MHz, CDCl3) δ 1.17 (s, 3H), 1.40 (m, 3H), 1.55 (m,
4H), 1.58 (s, 3H), 1.59 (s, 6H), 1.60 (s, 3H), 1.67 (s, 6H), 2.05 (m,
12H), 2.36 (dt, J = 11.8, 7.7 Hz, 1H), 2.69 (dt, J = 11.8, 7.7 Hz,
1H), 3.53 (dd, J = 11.4, 6.0 Hz, 1H), 3.69 (dd, J = 11.4, 6.0 Hz,
1H), 5.10 (m, 4H); 13C NMR (126 MHz, CDCl3) δ 16.2, 17.9, 19.1,
25.4, 25.9, 26.8, 26.9, 27.8, 28.0, 30.2, 32.80, 39.9, 39.9, 43.5, 50.7,
52.1, 60.1, 123.6, 124.4, 124.5, 124.6, 131.5, 131.7, 135.5, 136.0; [R]D
-3.67 (c 0.85, CHCl3). The spectral data and assignments corres-
pond to those reported in the literature for the racemic form.20
1
2.95 g (78%) of azido mesylate 12 as a colorless oil: H NMR
(500 MHz, CDCl3) δ 0.09 (s, 3H), 0.10 (s, 3H), 0.90 (s, 9H), 1.40 (s,
3H), 1.59 (m, 1H), 1.60 (s, 3H), 1.62 (s, 3H), 1.64 (m, 1H), 1.67 (d,
J = 1.1 Hz, 3H), 1.97 (m, 2H), 2.10 (m, 4H), 3.15 (s, 3H), 3.85
(ABX, JAB = 11.8 Hz, JBX = 7.7 Hz, 1H), 3.94 (ABX, JAB = 11.8
Hz, JAX = 2.8 Hz, 1H), 4.57 (ABX, JAX = 2.8 Hz, JBX = 7.7 Hz,
1H), 5.08 (m, 2H); 13C NMR (126 MHz, CDCl3) δ-5.2, 16.2, 17.9,
18.6, 20.0, 22.3, 25.9, 26.1, 26.8, 37.2, 39.3, 39.9, 62.8, 64.5, 88.1,
122.9, 124.4, 131.7, 136.6; IR (neat) νmax 2108 (N3) cm-1
.
(-)-(2R,3S)-trans-3-((E)-4,8-Dimethyl-3,7-nonadienyl)-3-me-
thylaziridine-2-methanol (9-OH). The following procedure is
based on those reported by Forestier et al.47 and Davis et al.23
A suspension of LiAlH4 (602 mg, 15.6 mmol) in Et2O (20 mL)
was stirred and cooled at 0 ꢀC under N2 as a solution of the azido
mesylate 13 (2.95 g, 6.23 mmol) in Et2O (10 mL) was added
dropwise over 2 min via syringe. After 4 h, the gray suspension
was cooled to 0 ꢀC and stirred as H2O (0.602 mL), 15% NaOH
(0.602 mL), and H2O (1.8 mL) were added sequentially with
occasional additions of Et2O to maintain the volume at ca. 3 mL.
After 2 h, the white solid was filtered and washed with EtOAc
(4 ꢀ 5 mL). The filtrates were combined, dried (MgSO4), and
concentrated under reduced pressure. Purification by flash
column chromatography (10% MeOH/CH2Cl2) afforded 1.04 g
(70%) of (-)-9-OH as a pale, yellow oil: 1H NMR (500 MHz,
CDCl3) δ 1.17 (s, 3H), 1.34 (m, 1H), 1.58 (s, 3H), 1.58 (m, 1H),
1.60 (s, 3H), 1.66 (s, 3H), 2.05 (m, 7H), 3.48 (dd, J = 11.8, 4.3
(45) Fujii, N.; Nakai, K.; Habashita, Y.; Hotta, Y.; Tamamura, H.;
Otaka, A.; Ibuka, T. Chem. Pharm. Bull. 1994, 42, 2241–2250.
(46) Crossland, R. K.; Servis, K. L. J. Org. Chem. 1970, 35, 3195–3196.
(47) Forestier, M. A.; Ayi, A. I.; Condom, R.; Boyde, B. P.; Conlin, J. N.;
Selway, J.; Challand, R.; Guedj, R. Nucleosides Nucleotides 1993, 12, 915–
924.
4776 J. Org. Chem. Vol. 75, No. 14, 2010

Koohang et al.
JOCArticle
(2R,3S)-trans-3-((E)-4,8-Dimethyl-3,7-nonadienyl)-1-((E)-6,
10-dimethyl-5,9-undecadienyl)-3-methylaziridine-2-methanol
Methanesulfonate (6-OMs). The following procedure is based
on ones reported by Koohang et al.20 and by Crossland and
Servis.46 A solution of 6-OH (46 mg, 0.111 mmol) obtained from
the previous step and Et3N (23 μL, 17 mg, 0.166 mmol) in CH2-
Cl2 (1.2 mL) was stirred at -20 ꢀC under N2 as methanesulfonyl
chloride (10 μL, 15 mg, 0.133 mmol) was added dropwise via
syringe. After 30 min, satd NaHCO3 (1 mL) was added, and the
solution was stirred vigorously for 5 min. The organic phase was
removed, and the aqueous phase was extracted with EtOAc (3 ꢀ
4 mL), dried (MgSO4), and concentrated under reduced pressure
to give 52 mg of a yellow oil. Purification by flash column
chromatography (50% EtOAc/hexanes) gave 46 mg (86%) of
the mesylate as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 1.18
(s, 3H), 1.43 (m, 7H), 1.59 (s, 3H), 1.60 (s, 6H), 1.61 (s, 3H), 1.67
(s, 3H), 1.97-2.22 (m, 12H), 2.31 (dt, J = 11.8, 7.6 Hz, 1H), 2.65
(dt, J = 11.6, 7.4 Hz, 1H), 3.02 (s, 3H), 4.21 (m, 2H), 5.10 (m,
4H). The spectral data correspond to those reported in the
literature for the racemic form.20
Hz), 2.48-2.63 (m, 1H), 3.95-4.17 (app 12 line ABX, assumed
2H), 5.08 (br t, 2H, J = ca. 7 Hz), 5.14 (br t, 2H, J = ca. 7.5 Hz).
(2R, 3S)-6-OPP: 31P NMR (162 MHz, CD3OD) δ -6.0 (d, J=
19.5 Hz, 1P), -5.2 (d, J = 19.5 Hz, 0.97P), -3.9 (s, 1.78 P),
6.2 (s, 0.14 P). The spectral data correspond to those reported in
the literature20 with minor deviations due to solvent differences
(CD3OD vs CD3CN).
Squalene Synthase Assays. The assay procedure was modified
from that of Zhang and co-workers.37 The assay mixture contained
50 mM MOPS (3-(N-morpholino)propanesulfonic acid), pH 7.2,
20 mM MgCl2, 2% (v/v) Tween 80, 1 mM DTT (dithiothreitol),
1 mg/mL of BSA (bovine serum albumin), 1.0 mM NADPH, 0 or
1.5 mM PPi, 100 μM [3H]FPP (7.5 μCi/μmol), 0-300 μM of
inhibitor generally made by dilution of a 3-mM stock solution of
the inhibitor in methanol, and enzyme (0.1 μg/reaction) to a final
volume of 200 μL. The mixture was equilibrated at 30 ꢀC for 5 min
before the addition of the enzyme, and was incubated for an
additional 5 min after the reaction was started by the addition of
the enzyme. The reaction was stopped by the addition of 300 μL of
1:1 (v/v) 40% aq KOH/CH3OH). Sufficient solid NaCl was added
to saturate the reaction mixture, and then 2 mL of ligroine contain-
ing 0.2% squalene was added. The mixture was vortexed vigorously
for 30 s. A 1-mL portion of the organic layer was applied to a 0.5 ꢀ
6 cm alumina column packed in a Pasteur pipet and pre-equili-
brated with 2 mL of ligroine containing 0.2% squalene. The eluent
was collected in a scintillation vial, and 5 mL of Cytoscint scintilla-
tion fluor was added. The sample was analyzed for tritium on a
liquid scintillation spectrometer.
(2R,3S)-trans-[2-((E)-4,8-Dimethyl-3,7-nonadienyl)-1-((E)-6,
10-dimethyl-5,9-undecadienyl)-2-methylaziridin-3-yl]methyl Di-
phosphate, Tris(tetrabutylammonium) Salt (6-OPP). The follow-
ing procedure was based on those reported by Koohang et al.20
and Poulter et al.34 A solution of the enantiopure mesylate 6-
OMs (44 mg, 0.091 mmol) obtained from previous step in dry
˚
acetonitrile (1 mL) containing molecular sieves (3 A, powdered,
activated) was stirred at rt under N2 as a solution of (NBu4)3-
HP2O7 (165 mg, 0.183 mmol) in dry acetonitrile (1 mL) was
added dropwise over 5 min via syringe. After 60 h, the molecular
sieves were filtered and washed with dry acetonitrile (5 mL), and
the filtrate was extracted with hexane (3ꢀ7 mL). The hexane-
soluble extracts were combined and concentrated under reduced
pressure to give ca. 10 mg of recovered starting material.
Concentration of the acetonitrile-soluble extracts under reduced
pressure at <30 ꢀC afforded 103 mg of a mixture of the aziridino
diphosphate (2R,3S)-6-OPP (73%) and (NBu4)3HP2O7 (1:1 by
31P NMR analysis) as a brown viscous oil that was analyzed by
NMR spectroscopy. Some high-field 1H NMR peaks for 6-OPP
were obscured by overlap with the following intense resonances
for the NBu4þ counterions: 1.02 (t, J=7.3 Hz, 12H), 1.41 (sextet,
J=7.6 Hz, 8H), 1.66 (app quintet, J=ca. 7.8 Hz, 8H), 3.20-
Acknowledgment. We thank the National Institutes of
Health for financial support of this work through Grant
Nos. GM 13956 (R.M.C.) and GM 21328 (C.D.P.) and the
National Science Foundation for providing support for the
VOICE NMR facility at the University of Illinois at Urbana-
Champaign.
Supporting Information Available: General experimental
and instrumentation, preparative procedures, and characteriza-
tion data for camphanate derivatives, aziridines, aziridino di-
phosphates, and methanediphosphonates, experimental data
for squalene synthase purification, and reproductions of 1H,
13C, and/or 31P NMR spectra of selected compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
3.27 (m, 4ꢀ 8H); (2S, 3R)-6-OPP: H NMR (400 MHz, CD3-
OD) δ 1.22 (s, 3H) 1.51-1.58 (br m, ca. 4H), 1.59 (br s, 12H),
1.63 - 1.70 (m), 2.05 (m, 12H) 2.16 (app br quintet, 1H, J = 7.6
J. Org. Chem. Vol. 75, No. 14, 2010 4777