Amidrazone and amidoxime inhibitors of squalene hopene cyclase

Article abstract of DOI:10.1021/jo990237h

The cyclization of squalene catalyzed by the enzyme squalene-hopene cyclase (SHC) leads to the hopanoid family of pentacyclic triterpenes, which are widely found in bacteria as membrane constituents. SHC mediates a cascade of regio- and stereoselective cyclizations that has triggered considerable interest in understanding the enzyme's mechanism of action. This paper reports synthetic studies leading to the preparation of trienylamidrazone 8, trienylamidoxime 9, and tetraenylamidoxime 10 corresponding to a partially cyclized squalene chain. All three compounds displayed significant levels of inhibition when assayed against SHC, with 9 being more active than 8, and amidoxime 10 being the most potent. Detailed profiles of their inhibition kinetics are also presented.

Full text of DOI:10.1021/jo990237h

J . Org. Chem. 1999, 64, 5441-5446
5441
Am id r a zon e a n d Am id oxim e In h ibitor s of Squ a len e Hop en e
Cycla se
Bruce Ganem,*,† Yaohua Dong,† Yi Feng Zheng,‡ and Glenn D. Prestwich‡
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University,
Ithaca, New York 14853-1301, and Department of Medicinal Chemistry, The University of Utah,
30 South 2000 East, Room 201, Salt Lake City, Utah 84112-5820
Received February 8, 1999
The cyclization of squalene catalyzed by the enzyme squalene-hopene cyclase (SHC) leads to the
hopanoid family of pentacyclic triterpenes, which are widely found in bacteria as membrane
constituents. SHC mediates a cascade of regio- and stereoselective cyclizations that has triggered
considerable interest in understanding the enzyme’s mechanism of action. This paper reports
synthetic studies leading to the preparation of trienylamidrazone 8, trienylamidoxime 9, and
tetraenylamidoxime 10 corresponding to a partially cyclized squalene chain. All three compounds
displayed significant levels of inhibition when assayed against SHC, with 9 being more active than
8, and amidoxime 10 being the most potent. Detailed profiles of their inhibition kinetics are also
presented.
Sch em e 1
In tr od u ction
The cyclization of squalene 1 (Scheme 1) catalyzed by
the enzyme squalene-hopene cyclase (SHC) leads to the
hopanoid family of pentacyclic triterpenes.1,2 These tri-
terpenes are widely found in bacteria as membrane
constituents, where they exert many of the same stabiliz-
ing effects as membrane sterols.3 Like the enzyme
oxidosqualene-lanosterol cyclase (OSLC),1 which pro-
motes the conversion of squalene oxide into lanosterol
and other sterols, SHC mediates a cascade of regio- and
stereoselective cyclizations that has triggered consider-
able interest in understanding the enzyme’s mechanism
of action.
In the SHC-catalyzed process, 1 is thought to bind to
the enzyme in the all-chair conformation shown. Cycliza-
tion is initiated by protonation of the terminal alkene
(Scheme 1) and proceeds through a series of alkene-
cation additions leading to the pentacyclic cation 2, the
precursor of hop-22,29-ene 3 and hopan-22-ol 4. A recent
2.9 Å resolution X-ray crystal structure4 of SHC isolated
from Alicyclobacillus acidocaldarius revealed a relatively
nonpolar active site lined by aromatic amino acid resi-
dues that may play a role in stabilizing the various
carbocationic intermediates propagated along the squalene
backbone during cyclization.
inhibit OSLC.5,6 Several aza-substituted mono-, bi-, and
tricyclic structures representing partially cyclized sterol
intermediates were evaluated as inhibitors of OSLC, the
most potent of which displayed IC50 values in the single-
digit micromolar range. However, the design and syn-
thesis of specific SHC inhibitors has not been reported.
As part of our interest in the mechanism of enzyme-
catalyzed glycoside hydrolysis, we have described several
aza analogues of monosaccharide aldonolactams that are
The putative involvement of transient carbocations in
the cyclization of oxidosqualene has triggered interest in
the synthesis of polyisoprenylated cation mimics that
† Cornell University.
(5) For nitrogen-containing monocyclic inhibitors of oxidosqualene
cyclase, see: (a) Taton, M.; Benveniste, P.; Rahier, A.; J ohnson, W. S.;
Liu, H. T.; Sudhakar, A. R. Biochemistry 1992, 31, 7892-7898. (b)
Dodd, D. S.; Oehlschlager, A. C. J . Org. Chem. 1992, 57, 2794-2803.
(c) Rahier, A.; Taton, M.; Beneviste, P.; Place, P.; Anding, C. Phy-
tochemistry 1985, 24, 1223-1232. (d) Rahier, A.; Narula, A. S.;
Beneviste, P.; Schmitt, P. Biochem. Biophys. Res. Commun. 1980, 92,
20-25.
‡ The University of Utah.
(1) Abe, I.; Rohmer, M.; Prestwich, G. D. Chem. Rev. 1993, 93, 2189-
2206.
(2) Abe, I.; Prestwich, G. D. In Comprehensive Natural Products
Chemistry; Barton, D. H. R., Nakanishi, K., Eds.; 1998; Vol. 2; in press.
(3) Ourisson, G.; Rohmer, M.; Poralla, K. Annu. Rev. Microbiol. 1987,
41, 301-333.
(4) Wendt, K. U.; Poralla, K.; Schulz, G. E. Science 1997, 277, 1811-
1815.
(6) Review: Abe, A.; Tomesch, J . C.; Wattanasin, S.; Prestwich, G.
D. Nat. Prod. Rep. 1994, 11, 279-302.
10.1021/jo990237h CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/24/1999

5442 J . Org. Chem., Vol. 64, No. 15, 1999
Ganem et al.
Sch em e 2
Sch em e 3a
potent glycosidase inhibitors.7 When protonated, such
amidines, amidrazones, and amidoximes form resonance-
stabilized cations (5-7, Scheme 2) that effectively mimic
both the half-chair conformation and incipient positive
charge of the resonance-stabilized glycosyl cation. With
that same design strategy in mind, we reasoned that
terpenoid-derived amidrazones or amidoximes represent-
ing partially cyclized intermediates might be effective
inhibitors of squalene-cyclizing enzymes. Here, we de-
scribe synthetic studies leading to the preparation of
trienylamidrazone 8, trienylamidoxime 9, and tetraenyl-
amidoxime 10 corresponding to a partially cyclized
squalene chain. All three compounds displayed signifi-
cant levels of inhibition when assayed against SHC, with
9 being more active than 8 and amidoxime 10 being the
most potent. Detailed profiles of their inhibition kinetics
are also presented.
a
(a) n-BuLi, THF, 0 °C; (b) 13, 0 °C; (c) Lawesson’s reagent,
toluene, 50 °C; (d) NH2NH2, CH3OH (for 8); (e) NH2OH, CH3OH
(for 9).
to hydrolysis than amidrazone 8, in accordance with
earlier observations in our laboratory.10 Therefore, the
synthesis of amidoxime 10 embodying the complete,
partially cyclized, squalene framework became our next
objective.
Discu ssion
A synthesis of 10 was developed (Scheme 4) that relied
on an efficient and highly stereoselective assembly of
tetraenyl bromide 24 from the known 4-benzyloxy-1-
butyne 16.11 The route depicted in Scheme 4 is a higher-
yielding modification of a previously published route to
the corresponding tetraenyl iodide 255b in which assembly
of the new trisubstituted alkene by carboalumination has
been replaced with a highly stereoselective reductive
coupling protocol using organocuprates.
Metalation and alkylation of 16 with formaldehyde
afforded propargylic alcohol 17 in good yield. Aluminum
hydride-mediated reductive iodination of this alkyne12
gave Z-iodoalkene 18 as a single stereoisomer. Coupling
of 18 with lithium dimethylcuprate then generated 19.
Treatment of 19 with PBr3 afforded allylic bromide 20,
which was readily converted to phenyl sulfone 21. The
corresponding sulfone-stabilized allylic anion, generated
from 21 using n-BuLi, reacted with farnesyl bromide to
afford the coupled product 22 in 92% yield. Reductive
desulfurization with concomitant debenzylation was
achieved using lithium in ethylamine, giving tetraenol
23. Reaction of 23 with Ph3PBr2 produced the corre-
sponding homoallylic bromide 24, which, when reacted
with dianion 12, formed lactam 26 in 85% yield. Unlike
reactions with 13, the alkylation of homoallylic bromide
24 was accompanied by almost no base-induced HBr
elimination. The remarkable efficiency of this coupling
attests to the pronounced effect of the newly installed,
Retrosynthetic analysis suggested that the desired
target structures might be obtained by direct C-alkylation
of 2-piperidone 11 (Scheme 3) using an appropriate
homoallylic halide. After experimentation with a variety
of dialkylamide bases generated in situ, it was found that
deprotonation of 11 was best achieved directly using
n-butyllithium (n-BuLi, 2.1 equiv, THF, 0 °C) to afford
dianion 12.8 Addition of homofarnesyl bromide 13, pre-
pared according to a literature report,5d to 12 at 0 °C
afforded the desired homofarnesylated lactam 14 in 63%
yield after chromatography. Treatment of 14 with Lawes-
son’s reagent (p-methoxyphenylthionophosphine sulfide
dimer)9 quantitatively produced thionolactam 15. Fol-
lowing experimental protocols developed in our earlier
work on saccharide mimics,10 thionolactam 15 was con-
verted either to amidrazone 8 (concd NH2NH2 in CH3-
OH, 89% yield) or to amidoxime 9 (NH2OH in CH3OH,
95% yield).
In preliminary bioassays of their effect on homoge-
neous, recombinant A. alcidocaldarius SHC, amidrazone
8, and amidoxime 9 displayed significant levels of inhibi-
tory activity. For 8 and 9, IC50 values of 220 and 56 nM,
respectively, were recorded. Besides being the more
potent inhibitor, amidoxime 9 was also more resistant
(7) Ganem, B. Acc. Chem. Res. 1996, 29, 340-347.
(8) Padwa, A.; Harring, S. R.; Semones, M. A. J . Org. Chem. 1998,
63, 44-54.
(9) (a) Pedersen, B. S.; Scheibye, S.; Nilsson, N. H.; Lawesson, S.
O. Bull. Soc. Chim. Belg. 1978, 87, 223-228. (b) Cava, M. P.; Levinson,
M. I. Tetrahedron 1985, 41, 5061-5087.
(11) Kobayashi, Y.; Mizojiri, R.; Ikeda, E. J . Org. Chem. 1996, 61,
5391-5399.
(10) Papandreou, G.; Tong, M. K.; Ganem, B. J . Am. Chem. Soc.
1993, 115, 11682-11690.
(12) Marshall, J . A.; DeHoff, B. S. J . Org. Chem. 1986, 51, 863-
872.

Inhibitors of Squalene Hopene Cyclase
J . Org. Chem., Vol. 64, No. 15, 1999 5443
Sch em e 4a
F igu r e 1. Lineweaver-Burk plot for amidrazone 8.
a
(a) n-BuLi, -78 °C, then CH2dO; (b) Red-Al, THF, rt, then
N-iodosuccinimide, -78 °C; (c) Me2CuLi, THF, 0 °C; (d) PBr3,
ether, 0 °C; (e) NaSO2Ph, DMF, rt; (f) n-BuLi, -78 °C, then
farnesyl bromide; (g) Li, EtNH2, -78 °C; (h) Ph3P, Br2, pyr-CH2Cl2;
(i) 12, THF, 0 °C; (j) Lawesson’s reagent, toluene, 50 °C; (k)
NH2OH, CH3OH, 50 °C.
Ta ble 1. P oten cy of Am id r a zon e a n d Am id oxim e
In h ibitor s of Recom bin a n t A. Acid oca ld a r iu s SHC
F igu r e 2. Lineweaver-Burk plot for amidoxime 9.
compd
IC50 (nM)
Ki (nM)
compd
IC50 (nM)
Ki (nM)
8
9
10
220
56
30
180
49
90
28
29
30
9.0a
100a
60a
6.6a
NDb
31a
a
b
Data from refs 14 and 16. ND: not determined.
cuprate-derived methyl group in screening the allylic
hydrogens from adventitious base.
Biologica l Resu lts
Amidrazone 8, amidoxime 9, and amidoxime 10 were
designed as transition-state analogues that mimic the
incipient monocyclic cation produced during the SHC-
mediated cyclization of squalene. All three compounds
displayed good to potent levels of inhibition of homoge-
neous, recombinant A. acidocaldarius SHC, with IC50
values ranging from 220 to 30 nM (Table 1). From
analysis of their Lineweaver-Burk plots (Figures 1-3),
it appeared that the three analogues acted as mixed,
noncompetitive inhibitors of SHC, possibly binding in the
nonpolar channel by which squalene enters the active site
within the enzyme’s central cavity.4
The observation that amidrazone 8 is significantly less
potent than amidoximes 9 and 10 is of interest, since the
SHC-mediated cyclization of squalene likely involves a
progression of partially cyclized carbocationic intermedi-
ates. Amidrazones are typically 103 more basic than the
corresponding amidoximes10 and would be mostly proto-
nated at pH 6.0, the pH optimum of SHC at which the
assays reported here are conducted. By contrast, ami-
doximes 9 and 10 would exist predominantly in the
F igu r e 3. Lineweaver-Burk plot for amidoxime 10.
unprotonated form at pH 6.0. The fact that amidoximes
9 and 10 are more potent than amidrazone 8 suggests
that the unprotonated inhibitor binds to SHC.
For comparative purposes, Table 1 also includes data
on the inhibition of SHC by three other inhibitors of
squalene-cyclizing enzymes. Benzophenone derivative 28
(Scheme 5), known as Ro48-8071, is a new, orally active,
extremely potent, nonterpenoid inhibitor of OSLC re-
cently reported by Hoffmann-La Roche.13 The inhibition
of SHC by 28 has been shown to be mixed, noncompeti-
tive.14 Propiophenone 29 is a structurally related non-
(13) Morand, O. H.; Aebi, J . D.; Dehmlow, H.; J i, Y.-H.; Gains, N.;
Lengsfeld, H.; Himber, J . J . Lipid Res. 1997, 38, 373-390.
(14) Abe, I.; Zheng, Y. F.; Prestwich, G. D. Biochemistry 1998, 37,
5779-5784.

5444 J . Org. Chem., Vol. 64, No. 15, 1999
Ganem et al.
toluene (3 mL), and the resulting mixture was heated at 50
°C for 30 min. Water (5 mL) was added, and the mixture was
extracted with ether. The combined extracts were washed with
brine (5 mL), dried over MgSO4, and concentrated. The residue
was taken up in CH2CH2 and stirred for 1 h to decompose the
excess Lawesson’s reagent, which had the same Rf as 15. After
concentration, the residue was purified by chromatography (4:1
petroleum ether/ethyl acetate) to give the trienylthiolactam
15 (52 mg, 98%) as a semisolid: 1H NMR (300 MHz, CDCl3) δ
9.20 (br s, 1 H), 5.20-5.00 (m, 3 H), 4.40-3.17 (m, 2 H), 2.70-
2.57 (m, 1 H), 2.25-2.19 (m, 1 H), 2.19-1.68 (m, 13 H), 1.68-
1.50, 1.65, 1.59 and 1.56 (overlapping m and 3 s, 14 H); 13C
NMR (75 MHz, CDCl3) δ 207.8, 136.2, 135.2, 131.5, 124.7,
124.4, 123.7, 46.3, 45.0, 40.0, 35.4, 27.0, 26.9, 26.0, 25.7, 24.7,
19.4, 18.0, 16.4, 16.3; IR (film) 3190, 2920, 1570 cm-1; FABMS
m/z 334 (M + 1, 100).
Sch em e 5
Tr ien yla m id r a zon e Acetic Acid Sa lt (8). Anhydrous
hydrazine (0.1 mL, 3.2 mmol) was added to a solution of 15
(21 mg, 0.06 mmol) in anhydrous CH3OH (1 mL) at 0 °C. The
resulting mixture was kept at 4 °C for 12 h. The solvent was
removed in vacuo, and the residue was purified by chroma-
tography with CH3CN/H2O/HOAc (94:5:1 to 91:8:1). After
concentration of the fractions, the residue was dissolved in
CH3OH (5 mL) and treated with decolorizing carbon for 30
min at room temperature. After filtration and concentration,
trienylamidrazone acetic acid salt 8 (22 mg, 89%) was obtained
as a pale yellow oil: 1H NMR (300 MHz, D2O) δ 5.12-4.92
(m, 3 H), 3.48-3.20 (m, 2 H), 2.70-2.56 (m, 1 H), 2.10-1.60,
1.85, 1.56, 1.54, 1.49 and 1.49 (overlapping m and 5 s, 32 H);
13C NMR (75 MHz, D2O) δ 177.9, 164.2, 133.3, 131.7, 127.8,
121.7, 121.6, 120.4, 38.1, 36.9, 30.8, 29.6, 24.0, 22.6, 22.2, 20.3,
19.5, 15.2, 14.5, 12.9; IR (film) 3200, 2920, 1685, 1620, 1450
cm-1; FABMS m/z 332 (M+, 100).
Tr ien yla m id oxim e (9). A solution of anhydrous hydroxyl-
amine (1 M, 0.63 mL, 0.6 mmol) in CH3OH was added to a
solution of 15 (21 mg, 0.06 mmol) in CH3OH (1 mL), and the
resulting mixture was heated at 50 °C for 15 h. The solvent
was removed in vacuo, and the residue was chromatographed
with a gradient 49:1 to 19:1 CH2Cl2/CH3OH as the eluent to
give trienylamidoxime 9 (20 mg, 95%) as an oil: 1H NMR (300
MHz, CDCl3) δ 5.31 (br s, 1 H), 5.17-5.00 (m, 3 H), 3.18 (br t,
2 H, J ) 5.6 Hz), 2.58-2.44 (m, 1 H), 2.12-1.72 (m, 13 H),
1.72-1.36, 1.67, 1.58 (overlapping m and 2 s, 15 H); 13C NMR
(75 MHz, CDCl3) δ 155.9, 135.4, 134.9, 131.2, 124.4, 124.2,
123.9, 41.8, 39.7, 35.3, 32.2, 26.8, 26.7, 26.3, 25.7, 25.4, 21.4,
17.7, 16.1, 16.0; IR (film) 3400, 3200, 2920, 2850, 1650, 1450
cm-1; FABMS m/z 333 (M + 1, 100).
terpenoid inhibitor known as BIBX79.15 The terpenoid
inhibitor S-18 30 is the most potent of a series of thia-
substituted analogues of 2,3-oxidosqualene.16
In summary, we have demonstrated that amidrazone
and amidoxime mimics of a monocyclic intermediate in
the enzyme-catalyzed cyclization of squalene are highly
effective inhibitors of SHC. Moreover, the compounds
reported herein represent the first designed transition-
state analogues in the study of SHC inhibition. The
inhibition data for amidoximes 9 (IC50 ) 30 nM, Ki ) 90
nM) and 10 (IC50 ) 56 nM, Ki ) 49 nM) indicate biological
activity comparable to 18-thia-2,3-oxidosqualene (IC50
)
60 nM, Ki ) 31 nM) and only slightly weaker than Ro48-
8071 (IC50 ) 9 nM, Ki ) 6.6 nM), the best inhibitors of
SHC known to date.
Exp er im en ta l Section 17
5-Ben zyloxy-2-p en tyn -1-ol (17). A solution of n-butyl-
lithium (1.55 M, 9.0 mL, 14.0 mmol) in hexane was added
dropwise to a solution of 4-benzyloxybutyne (2.13 g, 13.3 mmol)
in dry THF (40 mL). The yellow solution was stirred at -78
°C for 2 h and then treated with dry paraformaldehyde (600
mg, 20 mmol) at once. The resulting mixture was allowed to
warm slowly to room temperature and then stirred for 12 h.
Saturated aqueous NH4Cl (20 mL) was added, and the layers
were separated. The aqueous layer was extracted with ether.
The combined organic layers were dried over MgSO4, concen-
trated, and chromatographed with a gradient 9:1 to 4:1 to 7:3
petroleum ether/ethyl acetate as the eluent to give 17 (2.1 g,
83%) as a pale liquid: 1H NMR (300 MHz, CDCl3) δ 7.42-
7.21 (m, 5 H), 4.54 (s, 2 H), 4.20 (dt, 2 H, J ) 5.9, 2.1 Hz), 3.57
(t, 2 H, J ) 7.0 Hz), 2.51 (tt, 2 H, J ) 7.0, 2.1 Hz), 2.33 (t, 1
H, J ) 5.9 Hz); 13C NMR (75 MHz, CDCl3) δ 128.2, 128.7,
128.0, 83.2, 79.9, 73.2, 68.5, 51.4, 20.4; IR (film) 3400, 3070,
2925, 2870, 2230, 1475, 1450, 1350 cm-1; FABMS m/z 195 (M
+ 2H2, 20), 135 (C6H5CH2OC2H4+, 67).
5-Ben zyloxy-(Z)-3-iod o-2-p en ten -1-ol (18). A solution of
Red-Al (3.33 M, 4.7 mL, 15.6 mmol) in toluene was added
dropwise to a solution of 17 (1.78 g, 9.4 mmol) in dry THF (25
mL). After being stirred at room temperature for 12 h, the
reaction mixture was then cooled to -78 °C and treated with
a solution of N-iodosuccinimide (3.79 g, 16.9 mmol) in dry THF
(15 mL). The resulting mixture was stirred at -78 °C for 30
min and then allowed to warm to 0 °C. Saturated aqueous
Rochelle’s salt (50 mL) was added, followed by saturated
Tr ien ylla cta m (14). A solution of n-butyllithium (1.5 M,
1.27 mL, 1.9 mmol) in hexane was added dropwise to a solution
of δ-valerolactam 11 (90 mg, 0.9 mmol, 0.2 M) in dry THF (3
mL) at 0 °C. After the mixture was stirred at 0 °C for 2 h,
homofarnesyl bromide 13 (0.29 mL, 1.0 mmol) was added neat
to the dianion solution. The resulting mixture was stirred at
0 °C for 3 h and then quenched with saturated aqueous NH4-
Cl (10 mL). The mixture was extracted with ether, and the
extracts were dried over MgSO4, concentrated, and purified
by chromatography (49:1 CH2Cl2/CH3OH) to give the lactam
14 (182 mg, 63%) as a colorless oil: 1H NMR (300 MHz, CDCl3)
δ 6.62 (br s, 1 H), 5.20-5.00 (m, 3 H), 3.37-3.17 (m, 2 H),
2.30-2.17 (m, 1 H), 2.17-1.75 (m, 13 H), 1.75-1.37, 1.65, 1.57
and 1.56 (overlapping m and 3 s, 15 H); 13C NMR (75 MHz,
CDCl3) δ 175.3, 135.5, 134.8, 131.1, 124.3, 124.1, 123.7, 42.3,
40.4, 39.6, 31.5, 26.7, 26.6, 26.0, 25.6, 25.2, 21.3, 17.6, 16.0,
15.9; IR (film) 3300, 3200, 2920, 1670 cm-1; FABMS m/z 318
(M + 1, 100).
Tr ien ylth ion ola cta m (15). Lawesson’s reagent (65 mg,
0.16 mmol) was added to a solution of trienyllactam 14 in dry
(15) Mark, M.; Muller, P.; Mainer, R.; Eisele, B. J . Lipid Res. 1996,
37, 148-158.
(16) Zheng, Y. F.; Abe, I.; Prestwich, G. D. Biochemistry 1998, 37,
5981-5987.
(17) General procedures: King, S. B.; Ganem, B. J . Am. Chem. Soc.
1994, 116, 562-570.

Inhibitors of Squalene Hopene Cyclase
J . Org. Chem., Vol. 64, No. 15, 1999 5445
aqueous sodium thiosulfate (50 mL), and the aqueous layer
was extracted with ether. The combined organic extracts were
dried over MgSO4, concentrated, and chromatographed using
4:1 petroleum ether/ethyl acetate to give 18 (2.57 g, 86%) as a
were dried over MgSO4, concentrated, and chromatographed
with 9:1 petroleum ether/ethyl acetate to give 22 (1.87 g, 92%)
1
as an oil: H NMR (300 MHz, CDCl3) δ 7.81 (d, 2 H, J ) 7.8
Hz), 7.55 (t, 1 H, J ) 7.0 Hz), 7.41 (t, 2 H, J ) 7.8 Hz), 7.38-
7.22 (m, 5 H), 5.16-4.89 (m, 4 H), 4.46 (s, 2 H), 3.72 (td, 1 H,
J ) 10.5, 3.2 Hz), 3.40 (t, 2 H, J ) 7.0 Hz), 2.87 (ddd, 1 H, J
) 14.0, 7.0, 3.2 Hz), 2.33 (ddd, 1 H, J ) 14.0, 7.0, 3.2 Hz), 2.26
(t, 2 H, J ) 7.0 Hz), 2.12-1.85 (m, 8 H), 1.66 (s, 3 H), 1.58 &
1.55 (overlapping 2 s, 9 H), 1.21 (s, 3 H); 13C NMR (75 MHz,
CDCl3) δ 142.2, 138.7, 138.2, 137.9, 135.1, 133.3, 131.2, 129.1,
128.6, 128.3, 127.5, 124.2, 123.8, 118.7, 118.4, 72.8, 68.4, 64.7,
39.6, 26.7, 26.5, 26.4, 25.6, 17.6, 16.8, 16.3, 15.9; IR (film) 3080,
3030, 2920, 2860, 1670, 1580 cm-1; FDMS m/z 534 (M+, 100).
Tetr a en ol (23). Ethylamine (30 mL) was condensed at -78
°C to a flask containing 22 (1.02 g, 1.9 mmol) in dry THF (5
mL). Lithium wire (135 mg, 19.0 mmol) was rinsed with
hexane, cut into small pieces, and added under argon. The
reaction mixture turned deep blue. After 30 min at -78 °C,
solid NH4Cl was added to the reaction mixture until the blue
color disappeared. The mixture was then diluted with ether
(20 mL) and water (30 mL), and the aqueous layer was
extracted with ether. The combined extracts were dried over
MgSO4, concentrated, and chromatographed with 9:1 petro-
leum ether/ethyl acetate to give alcohol 23 (540 mg, 93%) as a
colorless liquid: 1H NMR (300 MHz, CDCl3) δ 5.30-5.17 and
5.17-5.00 (2 m, 4 H), 3.62 (br t, 2 H, J ) 6.2 Hz), 2.23 (t, 2 H,
J ) 6.4 Hz), 2.15-1.90 (m, 12 H), 1.66 (s, 3 H), 1.62 (s, 3 H),
1.58 (s, 9 H), 1.52 (br s, 1 H); 13C NMR (75 MHz, CDCl3) δ
135.8, 135.2, 131.6. 131.5, 128.2, 124.7, 124.5, 124.2, 60.2, 43.0,
40.0, 28.5, 28.3, 27.0, 26.9, 25.9, 17.9, 16.3, 16.2, 16.0; IR (film)
3350, 2920, 1660, 1440, 1380 cm-1; FDMS m/z 304 (M+, 100).
Br om otetr a en e (24). To a solution of Ph3P (642 mg, 2.4
mmol) in dry CH2Cl2 (10 mL) under argon at 0 °C was added
bromine (0.13 mL, 2.4 mmol) dropwise until an orange color
persisted, and then a few crystals of Ph3P were added to
discharge the excess bromine. Pyridine (0.25 mL, 3.1 mmol)
was added followed by the addition of alcohol 23 (620 mg, 2.0
mmol) in dry CH2Cl2 (5 mL). After the mixture was stirred at
room temperature for 4 h, the solvent was removed in vacuo,
and the solid residue was diluted with hexane (20 mL) and
filtered through a pad of Celite. The solid was rinsed well with
hexane. The combined extracts were dried over sodium sulfate,
concentrated, and chromatographed with hexane as the eluent
to give bromide 24 (653 mg, 87%) as a colorless liquid: 1H
NMR (300 MHz, CDCl3) δ 5.28-5.17 and 5.17-5.00 (2 m, 4
H), 3.41 (t, 2 H, J ) 7.5 Hz), 2.52 (t, 2 H, J ) 7.5 Hz), 2.13-
1.90 (m, 12 H), 1.67 (s, 3 H), 1.61 and 1.59 (overlapping 2 s,
12 H); 13C NMR (75 MHz, CDCl3) δ 135.7, 135.2, 132.2, 131.5,
128.0, 124.7, 124.5, 124.2, 43.2, 40.0, 32.0, 28.5, 28.2, 27.0, 26.9,
1
yellow oil: H NMR (300 MHz, CDCl3) δ 7.42-7.21 (m, 5 H),
5.93 (t, 1 H, J ) 5.6 Hz), 4.52 (s, 2 H), 4.16 (br t, 2 H, J ) 4.8
Hz), 3.61 (t, 2 H, J ) 6.4 Hz), 2.78 (t, 2 H, J ) 6.4 Hz), 2.02
(br t, 1 H, J ) 4.8 Hz); 13C NMR (75 MHz, CDCl3) δ 138.3,
136.2, 128.7, 128.0, 105.3, 73.4, 68.9, 67.5, 45.5; IR (film) 3400,
2820, 1680, 1450, 1350 cm-1; FABMS m/z 211 (M - C6H5CH2O,
3), 135 (C6H5CH2OC2H4+, 42).
5-Ben zyloxy-(E)-3-m eth yl-2-p en ten -1-ol (19). A solution
of methyllithium (1.4 M, 54.4 mL, 76.1 mmol) in ether was
added to a suspension of freshly prepared CuI (7.54 g, 39.6
mmol) in dry THF (50 mL) at 0 °C dropwise. A solution of vinyl
iodide 18 (2.42 g, 7.6 mmol) in dry THF (10 mL) was added by
cannula to the solution of lithium dimethylcuprate generated
above. After being stirred at 0 °C for 12 h, the reaction mixture
was poured into ice-cold saturated aqueous NH4Cl (50 mL)
covered with ether (25 mL). The organic layer was washed with
3% aqueous NH4OH until clear, and the combined aqueous
layers were extracted with ether. The extracts were dried over
MgSO4, concentrated, and then chromatographed with 7:3
petroleum ether/ethyl acetate to give 19 (1.44 g, 92%) as a
colorless oil: 1H NMR (300 MHz, CDCl3) δ 7.42-7.21 (m, 5
H), 5.46 (tq, 1 H, J ) 7.0 Hz), 4.50 (s, 2 H), 4.14 (d, 2 H, J )
7.0 Hz), 3.56 (t, 2 H, J ) 6.7 Hz), 2.34 (t, 2 H, J ) 6.7 Hz),
1.68 (s, 3 H), 1.40 (br s, 1 H); 13C NMR (75 MHz, CDCl3) δ
138.3, 136.5, 128.3, 127.6, 127.5, 125.1, 72.8, 68.5, 59.2, 39.4,
16.4; IR (film) 3400, 2810, 1670, 1450, 1350 cm-1; FABMS m/z
189 (M + 1 - H2O, 100).
5-Ben zyloxy-(E)-1-br om o-3-m eth yl-2-p en ten e (20). A
solution of phosphorus tribromide (0.25 mL, 2.7 mmol) in dry
ether (2 mL) was added dropwise to a solution of alcohol 19
(1.24 g, 6.0 mmol) in dry ether (20 mL) at 0 °C. After the
solution was stirred at 0 °C for 10 min, the ice bath was
removed and stirring continued for another 10 min. The
reaction mixture was quenched with brine (30 mL). The
aqueous layer was extracted with ether, and the combined
ether layers were washed with saturated aqueous sodium
bicarbonate then brine until neutral. The organic layer was
dried over MgSO4 and concentrated to give bromide 20 (1.46
g, 91%) as a pale yellow liquid used directly for next step
without further purification: 1H NMR (300 MHz, CDCl3) δ
7.42-7.21 (m, 5 H), 5.59 (tt, 1 H, J ) 8.1, 1.1 Hz), 4.50 (s, 2
H), 4.01 (d, 2 H, J ) 8.1 Hz), 3.56 (t, 2 H, J ) 6.7 Hz), 1.74 (s,
3 H); 13C NMR (75 MHz, CDCl3) δ 140.6, 138.3, 128.3, 127.6,
127.5, 122.0, 72.8, 68.3, 39.5, 29.2, 16.2; IR (film) 2920, 2820,
1670, 1450 cm-1; FDMS m/z 189 (M+ - Br, 100).
5-B e n zy lo x y -(E )-3-m e t h y l-1-p h e n y ls u lfin y l-2-p e n -
ten e (21). Sodium phenylsulfinate (1.34 g, 8.2 mmol) was
added once to a solution of bromide 20 (1.46 g, 5.4 mmol) in
dry DMF (20 mL). After being stirred at room temperature
for 12 h, the reaction mixture was poured into water (25 mL)
and extracted with ether. The combined extracts were washed
with brine (25 mL) once, dried over MgSO4, concentrated, and
chromatographed with a gradient 9:1 to 4:1 petroleum ether/
ethyl acetate to give 21 as a colorless oil: 1H NMR (300 MHz,
CDCl3) δ 7.83 (br d, 2 H, J ) 7.5 Hz), 7.58 (br t, 1 H, J ) 7.0
Hz), 7.45 (br t, 1 H, J ) 7.5 Hz), 7.38-7.20 (m, 5 H), 5.24 (t,
1 H, J ) 8.1 Hz), 3.80 (d, 1 H, J ) 8.1 Hz), 3.46 (t, 2 H, J )
6.7 Hz), 2.29 (t, 2 H, J ) 6.5 Hz), 1.32 (s, 3 H); 13C NMR (75
MHz, CDCl3) δ 143.8, 138.8, 138.5, 133.8, 129.2, 128.8, 128.7,
127.9, 112.3, 73.2, 68.5, 56.3, 39.9, 16.7; IR (film) 3070, 3020,
2910, 2860, 1450, 1300 cm-1; FABMS m/z 331 (M + 1, 100).
Ben zyloxytetr a en e (22). A solution of n-butyllithium
(1.6M, 1.67 mL, 2.7 mmol) in hexane was added dropwise to a
solution of 21 (800 mg, 2.4 mmol) in dry THF (15 mL) at -78
°C. The resulting yellow solution was stirred at -78 °C for 1
h, and then a solution of farnesyl bromide (0.73 mL, 2.7 mmol)
in dry THF (5 mL) was added dropwise to the anion solution
at -78 °C. After 4 h at -78 °C, the reaction mixture was
quenched with CH3OH (1 mL), water (25 mL), and saturated
aqueous NH4Cl (25 mL) and then warmed to 0 °C. The aqueous
layer was extracted with ether, and the combined extracts
26.0, 17.9, 16.3, 16.3, 15.9; IR (film) 2920, 1670, 1450 cm-1
;
FDMS m/z 366 (M+ - 1, 100), 368 (M + 1, 96).
Tetr a en ylla cta m (26). A solution of n-butyllithium (1.6 M,
1.23 mL, 2.0 mmol) in hexane was added dropwise to a solution
of δ-valerolactam 12 (93 mg, 0.94 mmol, 0.2 M) in dry THF (3
mL) at 0 °C. After the solution was stirred at 0 °C for 2 h,
bromide 24 (0.36 mL, 1.0 mmol) was added neat to the dianion
solution generated above. The resulting mixture was stirred
at 0 °C for 2 h and then quenched with saturated aqueous
NH4Cl (10 mL). The mixture was extracted with ether, and
the extracts were dried (MgSO4), concentrated, and purified
by chromatography (99:1 CH2Cl2/CH3OH) to give 26 (305 mg,
85%) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ 6.85 (s,
1 H), 5.18-5.00 (m, 4 H), 3.32-3.16 (m, 2 H), 2.29-1.74 (m,
18 H), 1.74-1.38, 1.64, 1.57 and 1.56 (overlapping m and 3 s,
18 H); 13C NMR (75 MHz, CDCl3) δ 175.7, 135.3, 135.1, 134.9,
131.4, 125.0, 124.7, 124.5, 124.4, 42.5, 40.7, 40.0, 37.2, 30.1,
28.5, 28.5, 27.0, 26.9, 26.3, 25.9, 21.6, 17.9, 16.3, 16.2, 16.2;
IR (film) 3300, 3200, 2920, 2860, 1670 cm-1; FABMS m/z 386
(M + 1, 100).
Tet r a t h iola ct a m (27). Lawesson’s reagent (32 mg, 0.08
mmol) was added to a solution of 26 in dry toluene (2 mL),
and the resulting mixture was heated at 50 °C for 30 min.
Water (5 mL) was added, and the mixture was extracted with
ether. The combined extracts were dried over MgSO4 and
concentrated. The residue was taken in CH2Cl2 and stirred

5446 J . Org. Chem., Vol. 64, No. 15, 1999
Ganem et al.
for 1 h to decompose the excess Lawesson’s reagent. After
concentration, the residue was purified by chromatography
with 4:1 petroleum ether/ethyl acetate to afford 27 (52 mg,
98%) as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 8.92 (br s,
1 H), 5.22-5.02 (m, 4 H), 3.39-3.20 (m, 2 H), 2.70-2.55 (m, 1
H), 2.47-2.32 (m, 1 H), 2.20-1.68 (m, 17 H), 1.68-1.52, 1.66,
1.63 and 1.58 (overlapping m and 3 s, 17 H); 13C NMR (75
MHz, CDCl3) δ 208.3, 135.4, 135.1, 134.6, 131.5, 125.5, 124.7,
124.5, 124.5, 46.0, 45.1, 40.0, 37.2, 33.7, 28.5, 28.5, 27.0, 26.9,
26.0, 24.5, 19.4, 17.9, 16.3, 16.3, 16.1; IR (film) 3190, 2920,
2860, 1670, 1570, 1500 cm-1; FDMS m/z 401 (M+, 100).
Tetr a en yla m id oxim e (10). A solution of anhydrous hy-
droxylamine (1 M, 0.62 mL, 0.6 mmol) in CH3OH was added
to a solution of tetraenylthiolactam 27 (25 mg, 0.06 mmol) in
anhydrous CH3OH (1 mL), and the resulting mixture was
heated at 50 °C for 15 h. The solvent was removed in vacuo,
and the residue was chromatographed using a gradient 99:1
to 49:1 to 19:1 CH2Cl2/CH3OH to give tetraenylamidoxime 10
(21.5 mg, 86%) as an oil: 1H NMR (300 MHz, CDCl3) δ 5.30
(br s, 1 H), 3.18 (br t, 2 H, J ) 5.7 Hz), 2.28 (heptet, 1 H, J )
4.2 Hz), 2.13-1.77 (m, 17 H), 1.77-1.40, 1.67, 1.58 and 1.58
(overlapping m and 3 s, 18 H); 13C NMR (75 MHz, CDCl3) δ
156.0, 135.1, 134.9, 134.7, 131.2, 124.6, 124.4, 124.3, 41.8, 39.7,
37.0, 35.2, 30.4, 28.3, 28.2, 26.8, 26.7, 26.2, 25.7, 21.3, 17.7,
16.0, 16.0, 15.9; IR (film) 3380, 3240, 2920, 2850, 1650, 1450
cm-1; FABMS m/z 401 (M + 1, 100).
concentrated using a Speed-Vac and subjected to silica gel TLC
(Whatmann LK6D). The TLC plates were developed twice: 5
cm in CHCl3 and then 15 cm in hexane. The Rf values of
squalene, hopene, and hopan-2-ol were 0.45, 0.77, and 0.15,
respectively. The reaction mixtures were then analyzed using
a radio-TLC scanner (Bio-Scan, System 500), which has an
instrumental reproducibility of (5-10% for repeated scans of
the same sample lanes. Quantification of activity was obtained
using only the squalene to hopene (major product) conversion;
the small, variable amount of hopanol was generally in the
baseline noise and was not included in the calculations. The
percentage of activity was plotted against inhibitor concentra-
tion to determine the IC50 value. All assays were carried out
in duplicate.19
In h ibition Kin etics. The experiments were carried out in
duplicate using three concentrations of each inhibitor (8: 0,
186, 372 nM; 9: 0, 55, 82 nM; 10: 0, 42, 84 nM). For each
inhibitor concentration, [14C]squalene was added to give four
substrate concentrations: 1, 1.6, 2.0, and 4.0 µM. The mixtures
were incubated at 60 °C for 30 min and analyzed using a radio-
TLC scanner as described. Reaction velocities (V) obtained at
different substrate concentrations for each inhibitor concentra-
tion were plotted as double reciprocal plots of 1/V versus 1/[S]
as shown in Figures 1-3. The slopes from each Lineweaver-
Burk analysis were replotted against [I] to obtain the enzyme-
inhibitor dissociation constant Ki.18
E n zym e P u r ifica t ion a n d Assa y. The recombinant A.
acidocaldarius SHC was expressed in E. coli and purified
according to the published method.18 The enzyme converted
squalene into a 17:1 mixture of hop-22(29)-ene and hopan-22-
ol and showed an apparent Km ) 1.8 µM and kcat ) 2.4 min-1
in the presence of 0.1% Triton X-100 in the assay mixture.
The reaction mixture contained 100 mM sodium citrate, pH
6.0, 0.1% (v/v) Triton X-100, 5 µM [14C]squalene (7.0 mCi/
mmol), and 0.5 µg of purified recombinant SHC in a final
volume of 1 mL. Incubations were carried out at 60 °C for 30
min, stopped by extraction of 1 mL of CH2Cl2. The extract was
Ack n ow led gm en t. We thank the National Insti-
tutes of Health (GM 35712 to B.G., GM 44836 to G.D.P.)
for generous financial assistance. Support of the Cornell
Nuclear Magnetic Resonance Facility by the NSF (CHE
7904825; PGM 8018643) and NIH (RR02002) is grate-
fully acknowledged. We thank C. Feil and K. Poralla
(Universita¨t Tu¨bingen) for providing the plasmid encod-
ing A. acidocaldarius SHC and A. Perez (University of
Costa Rica) for helpful discussions at Utah.
Su p p or tin g In for m a tion Ava ila ble: 13C NMR spectra for
8-10, 14, 15, 17-24, 26, and 27. This material is available
free of charge via the Internet at http://pubs.acs.org.
(18) Ochs, D.; Tappe, C. H.; Ga¨rtner, P.; Kellner, R.; Poralla, K. Eur.
J . Biochem. 1990, 194, 75-80.
(19) Abe, I.; Dang, T.; Zheng, Y.-F.; Madden, B. A.; Feil, C.; Poralla,
K.; Prestwich, G. D. J . Am. Chem. Soc. 1997, 119, 11333-11334.
J O990237H