Chemistry and biology of cylindrols: Novel inhibitors of Ras farnesyl-protein transferase from Cylindrocarpon lucidum

Article abstract of DOI:10.1021/jo961074p

Farnesyl-protein transferase (FPTase) is an enzyme responsible for the farnesylation of Ras protein. Farnesylation is required for cell-transforming activity in several tumor-types, and therefore, inhibition of FPTase activity may be a potential target for anticancer drugs. Our continued search for novel inhibitors led to the isolation of a number of bicyclic resorcinaldehyde cyclohexanone derivatives named here cylindrols A₁ to A₄, cylindrols B and B₁, and a number of known compounds, from Cylindrocarpon lucidum. The compounds were isolated by bioassay-guided separation using Sephadex LH-20, silica gel, and reverse phase HPLC. Structures were elucidated by extensive application of 2D NMR and X-ray crystallography. The determination of absolute stereochemistry was accomplished by CD measurements. Chemical transformations of the most abundant compound resulted in a number of key derivatives which were critical for the evaluation of structure activity relationship. These compounds are members of ascochlorin family and showed a wide range of inhibitory activity (0.7 μM to >140 μM) against FPTase. The FPTase activity was noncompetitive with respect to both substrates. Isolation, structures, chemical transformations, and FPTase activity are discussed in detail.

Full text of DOI:10.1021/jo961074p

J . Org. Chem. 1996, 61, 7727-7737
7727
Ch em istr y a n d Biology of Cylin d r ols: Novel In h ibitor s of Ra s
F a r n esyl-P r otein Tr a n sfer a se fr om Cylin d r oca r pon lu cid u m
Sheo B. Singh,* Richard G. Ball, Gerald F. Bills, Carmen Cascales,† J ackson B. Gibbs,‡
Michael A. Goetz, Karst Hoogsteen, Rosalind G. J enkins, J errold M. Liesch,
Russell B. Lingham, Keith C. Silverman, and Deborah L. Zink
Merck Research Laboratories, P.O. Box 2000 Rahway, New J ersey 07065 and West Point, Pennsylvania
19486 and Centro de Investigacio´n Ba´sica, Merck Sharp & Dohme de Espan˜a S.A., J osefa Valca´rcel 38,
28027, Madrid, Spain
Received J une 7, 1996X
Farnesyl-protein transferase (FPTase) is an enzyme responsible for the farnesylation of Ras protein.
Farnesylation is required for cell-transforming activity in several tumor-types, and therefore,
inhibition of FPTase activity may be a potential target for anticancer drugs. Our continued search
for novel inhibitors led to the isolation of a number of bicyclic resorcinaldehyde cyclohexanone
derivatives named here cylindrols A1 to A4, cylindrols B and B1, and a number of known compounds,
from Cylindrocarpon lucidum. The compounds were isolated by bioassay-guided separation using
Sephadex LH-20, silica gel, and reverse phase HPLC. Structures were elucidated by extensive
application of 2D NMR and X-ray crystallography. The determination of absolute stereochemistry
was accomplished by CD measurements. Chemical transformations of the most abundant compound
resulted in a number of key derivatives which were critical for the evaluation of structure activity
relationship. These compounds are members of ascochlorin family and showed a wide range of
inhibitory activity (0.7 µM to >140 µM) against FPTase. The FPTase activity was noncompetitive
with respect to both substrates. Isolation, structures, chemical transformations, and FPTase activity
are discussed in detail.
Ras (p21) protein, a guanine nucleotide (GTP) binding
protein, is a product of the ras oncogene and plays an
important role in signal transduction and regulation of
cell proliferation. It also helps link cell-surface growth
factor receptors to intracellular pathways which are
responsible for regulating cell growth.1 Mutated forms
of the Ras protein can lead to unregulated cell transfor-
mation and are associated with a number of human
cancers.2 Association of the Ras protein to the inner
surface of the cell membrane is a key requirement for
both regulated and unregulated cell transformation. A
series of post-translational modifications are essential for
the cell membrane association of Ras protein. One key
modification is farnesylation of Ras by farnesyl-protein
transferase (FPTase) on the C-terminus cysteine residue
of the CAAX motif.3 Subsequent events include pro-
teolytic cleavage of the AAX tripeptide and esterification
of the newly freed cysteine carboxy group. Selective
inhibition of FPTase represents an indirect (by the way
of blocking the association of mutated Ras to the cell
membrane) but attractive target toward the development
of chemotherapeutic agents for pancreatic and colon
carcinomas where mutated forms of ras are common.4
FPTase inhibitors have been shown to block the growth
of tumors in a nude mouse explant model and this has
validated the approach.5
The search for inhibitors of FPTase, based on either
natural products or the CAAX tetrapeptide, has intensi-
fied. The natural product inhibitors reported to date fall
into three main classes: (a) Inhibitors that are competi-
tive with farnesyl diphosphate (FDP), including cha-
etomellic acids,6 actinoplanic acids,7 and manumycin
analogs;8 (b) inhibitors that are competitive with Ras-
peptide such as the pepticinnamins;9 and (c) those
(4) (a) Gibbs, J . B.; Oliff, A.; Kohl, N. E. Cell 1994, 77, 175. (b) Gibbs,
J . B. Cell 1991, 65, 1. (c) Buss, J . E.; Marsters, J . C. Chem. Biol. 1995,
2, 787.
(5) (a) Kohl, N. E.; Mosser, S. D.; deSolms, J . S.; Giuliani, E. A.;
Pompliano, D. L.; Graham, S. L.; Smith, R. L.; Scolnick, E. M.; Oliff,
A.; Gibbs, J . B. Science 1993, 260, 1934. (b) J ames, G. J .; Goldstein, J .
L.; Brown, M. S.; Rawson, T. E.; Somers, T. C.; McDowell, R. S.;
Crowley, C. W.; Lucas, B. K.; Levinson, A. D.; Marsters, J . C. Science
1993, 260, 1937. (c) Nude mouse study by Kohl, N. E.; Wilson, F. R.;
Mosser, S. D.; Giuliani, E.; deSolms, S. J .; Conner, M. W.; Anthony,
N. J .; Holtz, W. J .; Gomez, R. P.; Lee, T.-J .; Smith, R. L.; Graham, S.
L.; Hartman, G. D.; Gibbs, J . B.; Oliff, A. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 9141.
(6) (a) Singh, S. B.; Zink, D. L.; Liesch, J . M.; Goetz, M. A.; J enkins,
R. G.; Nallin-Omstead, M.; Silverman, K. C.; Bills, G. F.; Mosley, R.
T.; Gibbs, J . B.; Albers-Schonberg, G.; Lingham, R. B. Tetrahedron
1993, 49, 5917. (b) Reference 3e. (c) Singh, S. B. Tetrahedron Lett. 1993,
34, 6521. (d) Lingham, R. B.; Silverman, K. C.; Bills, G. F.; Cascales,
C.; Sanchez, M.; J enkins, R. G.; Gartner, S. E.; Martin, I.; Diez, M. T.;
Pelaez, F.; Mochales, S.; Kong, Y.-L.; Burg, R. W.; Meinz, M. S.; Huang,
L.; Nallin-Omstead, M.; Mosser, S. D.; Schaber, M. D.; Omer, C. A.;
Pompliano, D. L.; Gibbs, J . B.; Singh, S. B. Appl. Microbiol. Biotech.
1993, 40, 370.
(7) (a) Singh, S. B.; Liesch, J . M.; Lingham, R. B.; Goetz, M. A.;
Gibbs, J . B. J . Am. Chem. Soc. 1994, 116, 11606. (b) Singh, S. B.;
Liesch, J . M.; Lingham, R. B.; Silverman, K. C.; Sigmund, J . M.; Goetz,
M. A. J . Org. Chem. 1995, 60, 7896.
(8) (a) Hara, M.; Akasaka, K.; Akinaga, S.; Okabe, M.; Nakano, H.;
Gomez, R.; Wood, D.; Uh, M.; Tamanoi, F. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 2281. (b) Tamanoi, F. Trend. Biochem. Sci. (TIBS) 1993, 18,
349.
(9) Shiomi, K.; Yang, H.; Inokoshi, J .; Van Der Pyl, D.; Nakagawa,
A.; Takeshima, H.; Omura, S. J . Antibiot. 1993, 46, 229.
* Author to whom correspondence should be addressed at mail code
RY80Y-340.
† Merck Sharp & Dohme de Espan˜a S.A.
‡ Merck Research Laboratories, West Point, PA.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
(1) Barbacid, M. Annu. Rev. Biochem. 1987, 56, 779.
(2) Rodenhuis, S. Semin. Cancer Biol. 1992, 3, 241.
(3) (a) Reiss, Y.; Goldstein, J . L.; Seabra, M. C.; Casey, P. J .; Brown,
M. S. Cells 1990, 62, 81. (b) Schaber, M. D.; O’Hara, M. B.; Garsky, V.
M.; Mosser, S. D.; Bergstrom, J . D.; Moores, S. L.; Marshall, M. S.;
Friedman, P. A.; Dixon, R. A. F.; Gibbs, J . B. J . Biol. Chem. 1990,
265, 14701. (c) Manne, V.; Roberts, D.; Tobin, A.; O’Rourke, E.;
DeVirgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, H.-
F.; Barbacid, M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7541. (d) Der,
C. J .; Cox, A. D. Cancer Cells 1991, 3, 331. (d) Gibbs, J . B. Semin.
Can. Biol. 1992, 3, 383. (e) Gibbs, J . B.; Pompliano, D. L.; Mosser, S.
D.; Rands, E.; Lingham, R. B.; Singh, S. B.; Scolnick, E. M.; Kohl, N.
E.; Oliff, A. J . Biol. Chem. 1993, 268, 7617. (f) Casey, P. J .; Seabra, M.
C. J . Biol. Chem. 1996, 271, 5289.
S0022-3263(96)01074-2 CCC: $12.00 © 1996 American Chemical Society

7728 J . Org. Chem., Vol. 61, No. 22, 1996
Singh et al.
inhibitors that are either not competitive with either of
the FPTase substrates or their mechanism of inhibition
is not yet known; this class of inhibitors includes fusi-
dienol,10 preussomerins,11 gliotoxin,12 and 10′-desmethox-
ystreptonigrin.13 We have continued screening for novel
inhibitors of FPTase and recently reported14 yet another
class of novel inhibitor, cylindrol A (1a ), from Cylindro-
carpon lucidum (Ascomycotina, Hypocreales). Cylindrol
A is a novel bicyclic compound with a resorcinaldehyde
group linked to a cyclohexanone unit by a five-carbon
chain. We wish to describe bioassay-guided (bovine
FPTase15 ) isolation,16 structure elucidation including
determination of absolute stereochemistry, chemical
modification, and structure-activity relationship of sev-
eral novel cylindrols (1a -e, 2a ,b) and other related (1f,
2-12) compounds. These compounds are members of
ascochlorin family.
F igu r e 1. COSY (s) and HMBC (nJ CH ) 7 Hz) correlations
of Cylindrol A1 (1b).
compounds were present in widely differing amounts: the
most abundant compound was 1f (∼300 mg/L) while
compound 3 was found at less than 1 mg/L. Amounts of
cylindrols A-A4 and B, B1 (1a -e, 2a ,b) ranged from 2
mg to 80 mg/L of broth.
Str u ctu r e Elu cid a tion
Cylin d r ol A1 (1b). Cylindrol A1 is structurally very
closely related to cylindrol A (1a ) whose structure has
been determined14 by a single crystal X-ray crystal-
lography. Electron impact (EI) mass spectral analysis
of cylindrol A1 gave a molecular ion at m/z 430 which
upon high resolution measurement (430.2470) gave a
molecular formula C25H34O6, and this was supported by
the 13C NMR spectrum of cylindrol A1. The molecular
formula of 1b suggested that it has nine degrees of
unsaturation. The infrared spectrum of 1b displayed
absorption bands for hydroxy (3274 cm-1), ester (1731
cm-1), cyclic ketone (1708 cm-1), and conjugated carbonyl
(1628 cm-1) groups. The absorption bands for ester and
ketones were partially fused. The UV spectrum gave
absorption bands at 221 and 295 nm indicating a high
degree of conjugation. 13C NMR analysis (Table 1) of
cylindrol A1 in CDCl3 displayed 25 carbons. The APT
spectrum revealed the presence of the following types of
carbon: six methyls, four methylenes, an oxygenated
methine, two aliphatic methines, an aliphatic quaternary,
two olefinic/aromatic methines, six olefinic/aromatic qua-
ternaries, an ester carbonyl, an aldehyde, and a cyclic
ketone. The 13C NMR shifts were assigned using an
HMQC experiment.
The 1H NMR (400 MHz) analysis (Table 2) of 1b in
CDCl3 exhibited two methyl doublets (δ 0.83 and 0.97)
with J ) 6.6 Hz each connected to two methines (δ 2.56
and 1.94) and an angular methyl singlet. The remaining
three methyl groups were singlets and were sufficiently
deshielded to be either aromatic, olefinic, or acetate. One
of the two olefinic protons appeared as a singlet while
the second appeared as a triplet. The most deshielded
proton appeared at δ 12.67 that could be exchanged,
albeit slowly, with deuterium upon shaking with D2O,
indicating a strong chelation possibly with a peri-carbo-
nyl. Analysis of a 2D 1H-1H COSY spectrum of cylindrol
A1 gave four partial fragments, as shown by bold lines
(Figure 1), establishing structural fragments C9-C11-
C23, C12-C13, C21-C15-C16-C17, and C19-C22. An
HMBC experiment was used to assemble these fragments
into cylindrol A1 and correlations are illustrated by
arrows in Figure 1. The aldehyde proton (H-8, δ 10.05)
gave strong two and three bond HMBC correlations to
C-1 (δ 113.24) and C-2 (δ 163.77). The chelated hydroxy
proton (C2-OH, δ 12.67) produced correlations to C-1, C-2,
and C-3 (δ 111.43). The aromatic methyl group (H-7)
Isola tion
The fermentation broth of C. lucidum (MF 5710, ATCC
74261) was extracted with methyl ethyl ketone and
chromatographed on Sephadex LH-20 in size exclusion
mode using methanol as the eluent. The FPTase active
components were eluted after one half column volume
and were combined to give six successive fractions A-F.
Cylindrols A-A4 and B, B1 (1a -e, 2a ,b), compounds 1f,
2-4 and cylindrocarpol 5 were isolated from these
fractions by crystallization, reverse phase HPLC, and
normal phase silica gel chromatography or combinations
of both (see Experimental Section for details). These
(10) Singh, S. B.; J ones, E. T.; Goetz, M. A.; Bills, G. F.; Nallin-
Omstead, M.; J enkins, R. G.; Lingham, R. B.; Silverman, K. C.; Gibbs,
J . B. Tetrahedron Lett. 1994, 27, 4693.
(11) Singh, S. B.; Zink, D. L.; Liesch, J . M.; Ball, R. G.; Goetz, M.
A.; Bolessa, E. A.; Giacobbe, R. A.; Silverman, K. C.; Bills, G. F.; Pelaez,
F.; Cascales, C.; Gibbs, J . B.; Lingham, R. B. J . Org. Chem. 1994, 59,
6296.
(12) Van Der Pyl, D.; Inokoshi, J .; Shiomi, K.; Yang, H.; Takeshima,
H.; Omura, S. J . Antibiot. 1992, 45, 1802.
(13) Liu, W. C.; Barbacid, M.; Bulgar, M.; Clarck, J . M.; Crosswell,
A. R.; Dean, L.; Doyle, T. W.; Fernandes, P. B.; Huang, S.; Manne, V.;
Pirnik, D. M.; Wells, J . S.; Meyers, E. J . Antibiot. 1992, 45, 454.
(14) Singh, S. B.; Zink, D. L.; Bills, G. F.; J enkins, R. G.; Silverman,
K. C.; Lingham, R. B. Tetrahedron Lett. 1995, 36, 4935.
(15) Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands, E.; O’Hara,
M. B.; Garsky, V. M.; Marshall, M. S.; Pompliano, D. L.; Gibbs, J . B.
J . Biol. Chem. 1991, 266, 14603.
(16) Singh, S. B.; Bills, G. F.; Lingham, R. B.; Martin, I.; Silverman,
K. C.; Smith, J . L. US Patent No. 5,420,334A, 1995.

Novel Inhibitors of Ras Farnesyl-Protein Transferase
J . Org. Chem., Vol. 61, No. 22, 1996 7729
Ta ble 1. 13C NMR Assign m en t of Cylin d r ols in CDCl3
a
position
1b
1d
1e
1f
2a
2b
5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1′
2′
3′
4′
5′
113.24
163.77
111.43
161.74
110.52
142.03
17.94
192.93
20.74
125.49
136.20
76.27
39.53
44.08
36.67
31.12
41.44
213.82
50.45
15.42
15.50
8.02
113.29
163.75
111.36
161.60
110.57
142.01
17.92
192.93
20.72
125.17
136.63
75.90
39.55
44.08
36.70
31.10
41.46
213.25
50.40
15.37
15.57
7.92
113.14*
162.25
113.32*
156.25
113.51*
137.87
14.41
193.20
21.53
124.86
135.38
75.54
39.58
44.04
36.59
31.14
41.44
213.25
50.40
15.37
15.57
7.92
113.25
163.71
111.92
162.26
110.66
141.93
17.97
192.94
21.24
121.31
138.49
32.67
35.64
43.56
36.11
30.97
41.55
214.48
50.52
15.33
15.63
7.57
113.39
163.76
111.87
161.59
110.48
142.03
17.96
192.97
21.32
127.51
135.15
132.86
136.17
48.51
40.82
31.12
41.56
211.50
53.57
10.34
16.29
8.89
113.46
163.77
111.81
161.34
110.41
142.09
17.96
192.99
21.32
128.24
134.74
133.84
134.76
47.19
45.32
73.62
45.59
207.80
53.80
11.50
12.71
8.84
113.10
163.65
112.35
162.58
110.69
141.67
17.95
192.33
21.05
122.08
137.45
39.24
25.55
124.70
135.02
35.54
29.33
78.26
73.43
26.39*
23.37*
15.95**
15.99**
-
11.80
170.51
21.33
-
-
-
11.87
172.55
43.81
25.70
22.32
22.37
11.87
172.17
43.80
25.69
22.25
22.31
16.48
-
12.71
-
12.43
170.09
21.02
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
a
* and ** chemical shifts in same column may be interchanged.
Ta ble 2. 1H-NMR Assign m en t (δ H, m u lt, J ) of Cylin d r ols in CDCl3
no.
1b
1c
1d
1e
1f
2a
2b
5
5
7
8
9
6.18, s
2.46, s
10.05, s
6.18, s
2.47, s
10.06, s
6.17, s
2.47, s
10.06, s
-
6.23, s
6.21, s
6.20, s
2.50, s
10.09, s
3.50, d, 7.5
6.20, s
2.47, s
10.04, s
3.35, dd, 6.4, 2.4
2.56, s
10.13, s
2.48, d, 0.6 2.49, s
10.07, s 10.08, s
3.38, d, 6.9 3.50, t, 7.2
3.35, dd, 14.7, 6.9 3.32, dd, 15.6, 7.2 3.32, dd, 15, 3.9
3.41, dd, 14.7, 6.9 3.39, dd, 15.6, 7.2 3.41, dd, 15, 7.2
3.34, dd, 14,7.2
3.38, dd, 14.4, 7.6
5.54, t, 7.2
10
12
13
5.60, t, 6.9
5.59, brt, 6.9
5.59, t, 7.2
5.28, qt,
6.9, 1.2
2.00, m,
1.83, m
1.56, dd, 15.6, 4.5 1.56, dd, 15.6, 4.5 1.54, dd, 15.6, 3.6 1.50, dd, 15.9, 4.2 1.40, m
5.52, t, 6.9
5.54, t, 7.5
5.18, qt, 1.6, 7.0
2.09, m
5.34, dd, 7.5, 3.9 5.36, dd, 8, 4
5.34, dd, 7.8, 3.6 5.35, dd, 7.6, 4
5.92, d, 16.2 5.94, d, 15.9
5.41, d, 16.2 5.36, d, 16.2
2.10, m
1.84, dd, 16, 7.5
-
1.94, m
1.55, m
1.79, m
1.85, dd, 15.6, 7.2 1.85, dd, 15.6, 7.5 1.81, dd, 15.6, 7.8 1.40, m
14
15
16
-
-
-
-
-
-
5.13, qt, 1.2, 6.8
-
1.95, m
1.53, m
1.78, m
1.95, m
1.54, m
1.79, m
2.18, m
2.25, m
1.94, m
1.53, m
1.80, m
2.19, m
2.25, m
1.98, m
1.60, m
1.80, m
2.34, m
1.80, m
1.63, m
2.00, m
4.90, dt, 11, 5.7 2.12, m; 2.07, m
17
2.18, dd, 13.6, 7.2 2.20, m
2.40, m
2.41, dd, 13, 6.7 1.45, m
2.88, dd, 13.5,
2.27, ddd,
13.2,5,2
-
2.56, q, 6.3
0.56, s
0.97, d, 6.6
0.83, d, 6.6
1.83, s
2.05, s
-
2.30, m
1.62, m
5.7
18
19
20
21
22
23
2′
3′
4′
5′
-
-
-
-
-
-
3.39, dd, 10.4, 2.0
2.56, q, 7.2
0.57, s
2.56, q, 6.3
0.56, s
2.52, q, 6.6
0.52, s
2.45, q, 6.5 2.40, q, 6.5 2.47, q, 6.6
-
0.57, s
0.71, s
0.74,s
1.18*, s
1.22*, s
1.58, d, 1.2
1.75, d, 1.2
-
0.97, d, 6.9
0.84, d, 6.3
1.81, s
0.97, d, 6.6
0.84, d, 6.6
1.81, s
0.94, d, 6.9
0.79, d, 6.9
1.79, s
0.88, d, 6.8 0.84, d, 6.6 0.87, d, 6.9
0.91, d, 6.6 0.82, d, 6.9 0.87, d, 6.9
1.83, d, 1.2 1.93, s
1.93, s
2.07, s
-
-
-
2.22, m
1.64, m
0.96, t, 6.9
-
12.69, s
6.06, brs
2.12, d, 6.3
2.10, m
0.93, d, 6.3
0.93, d, 6.3
12.68, s
2.13, d, 6.6
2.08, m
0.91, d, 6.3
0.91, d, 6.3
12.66, s
-
-
-
-
-
-
-
-
-
-
-
-
-
2-OH 12.67, s
4-OH 6.51, s
11.71, s
6.54, brs
12.71, s
6.06, brs
12.71, s
5.80, brs
12.71, s
7.38, brs
6.07, brs
6.30, brs
showed correlations to three carbons C-6 (δ 142.03), C-1,
and methine carbon C-5 (δ 110.52). The aromatic proton
(H-5) furnished strong correlations through three bonds
to the aromatic methyl group (δ 17.94), C-1, and C-3, and
a weak correlation via two bonds to C-4 (δ 161.74). The
latter two carbons and C-2 were strongly correlated to
both benzylic protons (H-9). These correlations fully
established the substitution pattern around the aromatic
ring and indicated that the side chain was attached, at
C3, between m-diphenol.
correlations from the protons of three methyl groups (H-
20, H-21, and H-22) and from H-17 to the respective
carbons. The ketone group was placed at C-18 based on
the deshielding of H-17 and H-19 and the correlation of
C-22 methyl protons to the carbonyl carbon (δ 213.82).
The connectivity of the aromatic and the cyclohexanone
ring through a five-carbon chain and acetate substitution
in the chain was accomplished from HMBC correlations
as follows: the correlations from benzylic protons to C-10
and C-11; H-23 to oxygenated methine carbon (C-12);
angular methyl protons to the C-13; and H-13 to C-12
and C-14 (fragment previously derived from COSY)
The substitution pattern in the cyclohexanone ring of
cylindrol A1 was similarly established using HMBC

7730 J . Org. Chem., Vol. 61, No. 22, 1996
Singh et al.
established the chain connections at C3 and C14. Ad-
ditionally, the HMBC correlations of H-12 to the acetate
carbonyl at δ 170.51 confirmed the presence of the acetate
group at C12. This structural assignment was supported
by mass spectral fragmentation of cylindrol A. Mass
spectral fragmentation and absolute stereochemical as-
signments have been grouped together and are discussed
under a separate heading (vide infra).
F igu r e 2. COSY (s) and selected HMBC (nJ XH ) 7 Hz)
correlations of Cylindrocarpol (5).
Cylin d r ol A2 (1c). EIMS analysis of 1c gave a
molecular ion at m/z 458.2668 which corresponded to a
molecular formula of C27H38O6, i.e. up by one methylene
unit from cylindrol A and two methylene units from A1.
4.90). This obvious acetate group (IR: 1740 cm-1) was
placed in the cyclohexanone ring at C-16 by analysis of
2D COSY spectrum. The C-16 proton appeared as a
doublet of a triplets (J ) 11 Hz, 5.7 Hz) at δ 4.90. The
presence of the triplet with a large coupling constant (J
) 11) is a clear indication that H-16 is axially oriented
and that the coupling is due to the H-15 and H-17 axial
protons. Therefore, the acetate group must be equatorial,
thus establishing the R stereochemistry at C-16 (see later
for the â isomer).
1
Comparison of the H NMR spectrum of 1c with that of
1a and 1b suggested that the extra methylene group was
present in the C12 side chain. This observation was
1
confirmed by 2D H-1H COSY and TOCSY spectrum of
cylindrol A2. The EI mass spectral fragmentation was
identical to that of other non chlorinated cylindrols and
is discussed later.
Cylin d r ol A3 (1d ). The high resolution EI mass
spectral analysis of cylindrol A3 gave a molecular formula
of C28H40O6 (472.2824, M+), suggesting the presence of
two additional methylene units. 1H NMR spectrum
(Table 1) and 13C NMR (Table 2) of cylindrol A3 was
generally similar to that of cylindrols A and A1 except
for the presence of a two methyl doublet at δ 0.93 and
the absence of the acetate methyl (1b) and the methyl
triplet (1a ). 2D 1H-1H COSY and TOCSY spectral
analysis of cylindrol A3 revealed that the two new methyl
groups were connected to a methine (δ 2.10, m), con-
nected in turn to a methylene (d, δ 2.12) linked to a
carbonyl group. This connectivity established a isopen-
tanoyl side chain at C12.
Cylin d r ol A4 (1e). Mass spectral analysis of cylindrol
A4, like other cylindrols, gave a molecular formula of
C28H39ClO6 (506.2463, M+). That A4 is a C-5 chloro-A3
was established by comparison of the molecular formula
1
and H and 13C NMR spectra (Table 1 and 2) of cylindrol
A3 and A4. Chlorine-induced shifts of >3 ppm were
observed for several of the aromatic carbons (C-4, C-5,
and C-6 and the aromatic CH3) in the 13C NMR spectrum
of 1e. This was further substantiated by the comparison
of mass spectral fragmentation (vide infra) and the
presence of a chloride band (732 and 713 cm-1) in the
infrared spectrum.
A number of bicyclic compounds (1f, 2c, 3) related to
cylindrols and monocyclic compounds (4, 5) have also
been isolated from extracts of C. lucidum. Cylindrocarpol
(5), a novel acyclic sesquiterpenoid resorcinaldehyde, was
isolated from the polar fractions. HREIMS of 5 revealed
a molecular formula C23H34O5. The structure of cylin-
drocarpol was elucidated by 2D NMR spectra including
HMBC experiment (Figure 2). The 1H and 13C NMR
assignments are summarized in Table 1 and 2.
Compounds 1f (LL-Z1272ꢀ) and 2c (LL-Z1272ú) were
originally isolated from a Fusarium species by Lederle
group17 and later from Nectria coccinea18 and Verticillium
species.19 Compound 3 was prepared17 from 2c and was
recently reported from Verticillium19 as a natural prod-
uct. The monocyclic compounds 4a (colletorin B) and 4b
(5-chlorocolletorin B) have been reported from Cepha-
losporium diospyr20 and Colletotrichum nicotianae,21
respectively. The identity of these compounds were
Cylin d r ol B (2a ). Molecular formula of C23H30O4
(MW 370) was derived from the high resolution mass
spectral analysis of cylindrol B and this was supported
by 13C NMR spectrum (Table 1). Comparison of the NMR
spectra of the previously described cylindrols with that
of 2a suggested the absence of the acyl group at C12 and
presence of an additional olefin at C12-C13. The
structure of cylindrol B (2a ) was verified by 2D NMR
1
using the methods described for 1b, and the 13C and H
NMR assignments are listed in Tables 1 and 2. The
chemical shift of C-14 methyl group was influenced by
12,13
∆
and was shifted upfield in the 13C NMR spectrum.
The large scalar coupling between H12-H13 (J ) 16 Hz)
revealed a E olefin geometry. Deacylation of series A
cylindrols could easily produce cylindrol B.
Cylin d r ol B1 (2b). Mass and 13C NMR spectral
analysis of cylindrol B1 gave a molecular formula C25H32O6.
(17) Ellestad, G. A.; Evans, R. H., J r.; Kunstmann, M. P. Tetrahe-
dron 1969, 25, 1323.
1
Most of the 13C and H NMR spectrum (Table 1) of 2b
(18) Aldridge, D. C.; Borrow, A.; Foster, R. G.; Large, M. S.; Spencer,
H.; Turner, W. B. J . Chem. Soc., Perkin Trans. 1 1972, 2136.
(19) Takamatsu, S.; Rho, M.-C.; Masuma, R.; Hayashi, M.; Ko-
miyama, K.; Tanaka, H.; Omura, S. Chem. Pharm. Bull. 1994, 42, 953.
(20) Kawagishi, H.; Sato, H.; Sakamura, S.; Kobayashi, K.; Ui, T.
Agric. Biol. Chem. 1984, 48, 1903.
was identical to the spectrum of 2a except for the
presence of an ester carbonyl (δ 170.09), an extra methyl
group (δ 21.02; 2.07), and replacement of one of the
methylene groups with a oxymethine carbon (δ 73.62;

Novel Inhibitors of Ras Farnesyl-Protein Transferase
J . Org. Chem., Vol. 61, No. 22, 1996 7731
F igu r e 3. Mass spectral fragmentations of cylindrols as exemplified by fragmentation of Cylindrol A.
established by comparison of spectroscopic data with the
literature data. Whenever good literature spectroscopic
data was not available, the structure was verified by
extensive 2D NMR spectroscopic analysis. These com-
pounds contributed significantly toward the evaluation
of the structure-activity relationship.
another band at 242 nm (∆ꢀ ) +2.03) presumably due
to the additional chiral center at C-12, thus establishing
identical stereochemistry to the cyclohexanone part of the
molecule in 1a . Since the relative stereochemistry of all
the stereocenters were established by X-ray analysis,22
the absolute stereochemistry of cylindrol A was assigned
as 12R,14S,15R,19R. The stereochemistry of the novel
cylindrol A1 to A4 (1b-e) were accordingly assigned as
12R,14S,15R,19R by comparison of their NMR spectral
data and specific rotation values. The stereochemistries
of cylindrols B and B1 were similarly assigned as
14R,15R,19R, and 14S15S16S,19R. These cylindrols
represent novel members of the ascochlorin family.24
Ch em ica l Mod ifica tion s of 1f. The availability of a
number of natural products gave us an entry into the
FPTase structure-activity relationship (SAR) in the
series (vide infra). To further this and address certain
key issues, a number of semisynthetic modifications were
carried out on the most abundant compound, 1f.
Ma ss Sp ectr a l Stu d ies of Cylin d r ols. Due to an
identical mass spectral fragmentation pattern for all the
series A cylindrols, they can be discussed together and
are exemplified by the fragmentation of cylindrol A. The
cleavage of bonds C13-C14 and C9-C10 produced two
major and characteristic fragment ions at m/z 139
(C9H15O) and 165 (C9H9O3) in the EI mass spectrum. The
other major fragment arose from acyl elimination fol-
lowed by rearrangement to give a ion at m/z 370
(C23H30O4). Most of the further fragmentation is derived
from this fragment as illustrated in Figure 3. Cylindrols
A-A3 (1a -d ) produced common fragment ions except for
their molecular ion. Chlorine-containing cylindrol A4 (1e)
differs in its fragmentation only to the extent that all
the fragment ions containing an aromatic ring were
shifted by 34 mmu. The mass spectral fragmentations
of the compounds 1f, 2, and 3 were significantly different
than cylindrols.17
Methylation of a mixture of compounds 1f and 2a with
diazomethane in methylene chloride at 0 °C followed by
purification gave monomethyl ethers 2d and 6b selec-
tively. The same reaction in methanol resulted in a
complex mixture. On the other hand 1f reacted smoothly
with MOMCl to afford bis-MOM ether 6d . Similarly, the
Rela tive a n d Absolu te Ster eoch em istr y of Cylin -
d r ols 1a -e, 2a ,b. Using single-crystal X-ray diffraction
the relative stereochemistries of cylindrol A (1a )14,22b and
compound 1f22ab have been established. The crystals used
for the X-ray analysis were obtained as needles from
acetone-hexane. Therefore, the stereochemistry of these
two compounds became the basis for the determination
of the stereochemistry of other compounds reported here
including the remainder of the novel cylindrols. The
stereochemistry of ascochlorin has been thoroughly elu-
cidated by X-ray analysis of a p-bromosulfonate deriva-
tive.23 The absolute stereochemistry of LL Z1272ꢀ, a
member of the ascochlorin family, was carefully cor-
related to that of ascochlorin by CD methods by Taka-
matsu and co-workers.19 The absolute stereochemistry
of 1f is identical to that of LL-Z1272ꢀ as evidenced by
their identical specific rotation and CD spectrum.17,19 Like
LL-Z1272ꢀ, the CD spectrum of 1f showed a negative
Cotton effect at 287 nm (∆ꢀ ) -1.48). Likewise, the CD
spectrum of 1a showed a negative Cotton band at 286
nm (∆ꢀ ) -1.47) due to the cyclohexanone ring and
(22) (a) Compound 1f, C23H32O4, Mr ) 372.509, monoclinic, P21, a
) 7.7524(8), b ) 12.252(2), c ) 22.020(3) Å, â ) 94.69(1)° V ) 2084.5-
(9) Å3, Z ) 4, Dx ) 1.187 g cm-3, monochromatized radiation λ(Cu KR)
) 1.541838 Å, µ )0.60 mm-1, F(000) ) 808, T ) 294 K. Data were
collected on a Rigaku AFC5 diffractometer to a θ limit of 72.5° which
yielded 4378 unique reflections. The observed reflections, those having
I g 3σ(I), number 3035. The structure was solved by direct methods
(SHELXS-86, Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467-473)
and refined using full-matrix least-squares on F (SDP-PLUS, Structure
Determination Package Version 3, Enraf-Nonius, Delft, 1985). There
are two independent molecules in a unit cell. The final model was
refined using 486 parameters and the observed data. All non-hydrogen
atoms were refined with anisotropic thermal displacements. Hydrogen
atoms are included at their calculated positions and constrained to
ride with their attached atom. The final agreement statistics are: R
) 0.049, wR ) 0.047, S ) 2.23 with (∆/σ)max ) 0.07. The least-squares
weights were defined using 1/σ2(F). The maximum peak height in a
final difference Fourier map is 0.30(4) eÅ-3, and this peak is without
chemical significance. (b) The atomic coordinates for 1a and 1f
structure have been deposited with the Cambridge Crystallographic
Data Centre. The coordinates can be obtained on request from the
Director, Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK..
(23) (a) Nawata, Y.; Ando, K.; Tamura, G.; Arima, K.; Iitaka, Y. J .
Antibiot. 1969, 22, 511. (b) Natawa, Y.; Iitaka, Y. Bull. Chem. Soc.
J pn. 1971, 44, 2652.
(24) (a) Tamura, G. A.; Suzuki, S.; Takasuki, A.; Ando, K.; Arima,
K. J . Antibiot. 1968, 21, 539. (b) Sasaki, H.; Hosokawa, T.; Nawata,
Y.; Ando, K. Agric. Biol. Chem. 1974, 38, 1463 and references cited
therein.
(21) (a) Kosuge, Y.; Suzuki, A.; Tamura, S. Agric. Biol. Chem. 1974,
38, 1265. (b) Saimoto, H.; Ueda, J .; Sashiwa, H.; Shigemasa, Y.;
Hiyama, T. Bull. Chem. Soc. J pn. 1994, 67, 1178.

7732 J . Org. Chem., Vol. 61, No. 22, 1996
Singh et al.
8b via demethylation26 using TMSCl-NaI gave a mixture
of cyclic ethers 11a and iodo derivative 11b. This type
of cyclization process is well documented in the case of
reactions with sulfuric acid.17,18 The lack of a chelated
hydroxy group signal in the 1H NMR spectrum of 11a
led us to suggest cyclization at C2 rather than at C4.
reaction with TBS chloride initially formed a bis-TBS
ether (observed by TLC) but during aqueous work-up it
furnished a mono TBS ether 6c. Acetylation with acetic
anhydride and pyridine gave the somewhat labile diac-
etate 6a . The C-2 acetate was hydrolytically labile and
yielded the C-2 free hydroxy group. This hydrolytic
cleavage is not surprising in view of the chelation of C-2
hydroxy group with the C-1 aldehyde. Catalytic hydro-
genation of 1f using 10% Pd/C in ethyl acetate gave
compound 7 exclusively.
Oxidation of 1f using Wuts27 procedure through a
bisulfite adduct yielded only the diacetate 6a despite the
quantitative formation of the desired bisulfite adduct.
Sodium hypochlorite-H2O2 oxidation28 likewise did not
result in the oxidation of the benzaldehyde group and as
expected gave 8e (illicicolin, LL-Z 1272δ, a compound
that was previously reported as a natural product).17,18
Oxid a tive Stu d ies. We wished to determine the
effect of an acidic group on the SAR and thus attempted
direct oxidations of the aldehyde group. Numerous
methods of direct oxidations were attempted but failed
to yield the desired compound.
Bor oh yd r id e Red u ction s. Reduction of 1f with
sodium borohydride resulted in instantaneous reduction
of both the aldehyde and ketone groups to afford diol 12a .
Reaction of 1f with 4 equivalents of sodium cyanoboro-
hydride in methanol gave a mixture of products 10b, 10c,
12a and 12b. The ratio of these products were dependent
on the quantity of the reducing agent used and the
reaction time. Selectivity was achieved when the reac-
tion was performed in a mixture of THF-methanol (1:1)
with approximately 3 equiv of sodium cyanoborohydride.
This reaction favored the reduction of the aldehyde and
furnished compounds 10b and 10c in a 1:2.5 ratio. The
hydroxy group at C-18 in compounds 12a and 12b was
assigned as â (axial) in view of the lack of any axial-
axial coupling and the presence of equatorial-equatorial
and equitorial-axial coupling (J ) ∼3 Hz) in their 1H
spectrum. These compounds were important for the
structure-activity studies as described below.
F P Ta se Str u ctu r e-Activity Rela tion sh ip . The
compounds presented in the current study were isolated
by bioassay-guided separation using bovine FPTase.15
The IC50 of the natural products and the synthetic
derivatives are listed in Table 3. The most active
compound 1f inhibited the bovine FPTase with an IC50
of 0.7 µM. The other compounds were all significantly
less active than 1f. Acyl substitution at C12 reduced the
activity by 3 to 20 fold. Cylindrol A1 (1b), having a C12
acetate, was the least active among the series A cylin-
drols. The activity improved with the increase in the
length of the ester side chain. The propionate side chain
(1a ) showed the maximal activity. The chloro compound
Oxidation of 1f with chromium trioxide-based reagents
initially did not produce any product, but when an excess
of reagent was used compound 9 was obtained as the only
identifiable product. A similar product was prepared
from ascochlorin by ozonolysis.17 Due to the difficulty
in direct oxidation of the aldehyde group to acid 8a , an
alternative method was chosen to prepare the methyl
ester 8b. The aldehyde group of 1f was indirectly
converted to the ester via manganese dioxide oxidation
of cyanohydrin intermediate followed by in-situ reaction
with methanol.25 A similar reaction with bis-MOM ether
6d gave methyl ester 8c and hydrolyzed product 8d .
Hydrolysis of 8b and 8c turned out to be extremely
difficult due to either hydrolytic inactivity of the ester
or decarboxylation of the resulting carboxyl group. A
number of basic hydrolytic methods including lithium
hydroxide and potassium carbonate failed to give the
carboxyl derivative. Heating of 8b with dilute aqueous
NaOH gave the decarboxy compound 10a in a small
amount, as the only isolable product. The hydrolysis of
(26) (a) Ayer, W. A., McCaskill, R. H. Can. J . Chem. 1981, 59, 2159.
(b) J ung, M. E.; Lyster, M. A. J . Am. Chem. Soc. 1977, 99, 968. (c)
Olah, G. A.; Narang, S. C.; Gupta, B. G. B.; Malhotra, R. J . Org. Chem.
1979, 49, 1247.
(25) Corey, E. J .; Gilman, N. W.; Ganem, B. E. J . Am. Chem. Soc.
1968, 90, 5616.
(27) Wuts, P. G. M.; Bergh, C. L. Tetrahedron Lett. 1986, 27, 3995.
(28) Dalcanale, E.; Montanari, F. J . Org. Chem. 1986, 51, 567.

Novel Inhibitors of Ras Farnesyl-Protein Transferase
J . Org. Chem., Vol. 61, No. 22, 1996 7733
Ta ble 3. Bovin e F P Ta se Activity of Cylin d r ols a n d
compounds have been evaluated for their potential as
inhibitors of FPTase. The structure-activity relationship
indicated that the bicyclic system, aldehyde, and ketone
are all important for biological activity. Small changes
in the molecule had a profound effect on FPTase activity.
The absolute stereochemistry of the compounds is identi-
cal to that of the ascochlorins. The literature ascochlorins
are mostly chlorinated at C5. This has been attributed
to the presence of the chloride ions present in the
production medium of the fermentation.18 Most of our
compounds are not chlorinated since the production
media used for the fermentation of C. lucidum did not
contain any significant amounts of halogen ions. The
absence of halogenated compounds supports this hypoth-
esis.18 In addition to the FPTase inhibitory activity
reported here, a number of different compounds of the
ascochlorin family have previously been shown to inhibit
5R-reductase,19 to be cytotoxic to HeLa cells,30 and to have
hypolipidemic activities.24b,31
An a logs 1-12
compound no.
IC50 (µM)
rel activity
Natural Products
1f
1a
2c
1d
2b
1e
2a
1b
3
0.7
2.2
2.8
4.6
5.6
1.000
0.320
0.250
0.150
0.130
0.110
0.110
0.050
0.047
0.009
0.005
0.004
6.5
6.5
13.0
15.0
77.0
155.0
170.0
5
4b
4a
Derivatives
2.0
6b
6a
7
10a
10b
2d
10c
12a
12b
6c
0.350
0.110
0.040
0.035
0.020
0.020
0.010
0.007
0.005
0.005
0.005
0.003
6.5
18.6
20.0
30.7
31.0
75.0
Exp er im en ta l Section
101.0
>140.0
>140.0
>140.0
>140.0
Gen er a l P r oced u r e. For general experimental procedure
see reference 7. [3H]-farnesyl diphosphate (FPP) was obtained
from New England Nuclear.
8b
9
F er m en ta tion of C. lu cid u m . C. lucidum (MF 5710) was
isolated from dried cow dung near Weed, Lincoln National
Forest, Otero County, New Mexico. Cultures were maintained
as mixtures of spores and hyphae in sterile soil and stored at
4 °C until ready for use. Seed cultures were inoculated by
using a small portion of the preserved soil aseptically trans-
ferred into a 250 mL Erlenmeyer flask containing 50 mL of
seed medium of the following composition (in g/L); corn steep
liquor, 5.0; tomato paste, 40.0; oat flour, 10.0; glucose, 10.0;
and trace elements solution, 10 mL/L (consisting of, in g/L
2c having a ∆12,13 olefin was two fold more potent when
compared to cylindrols B (2a ) and B1 (2b), the latter two
being almost equipotent. Compounds missing the cyclo-
hexanone ring (4, 5) were basically inactive. This result
clearly illustrates that the FPTase inhibitory activity is
not only due to the benzaldehyde unit but is also
modulated by other parts of the bicyclic molecule. Monom-
ethylation (6b) or acetylation (6a ) of 1f reduced the
activity by 3 fold and 8-9 fold, respectively. The oxida-
tion of the aldehyde to a methyl ester (8b) abolished
activity.
The most interesting observation was made when
comparing the activities of the reduced compounds (10
and 12). The FPTase inhibitory activity of 1f was
diminished >40 fold by complete reduction of the alde-
hyde to the methyl group (10b), >28 fold by substitution
of aldehyde with a hydrogen (10a ), and was virtually nil
in the case of the hydroxymethyl compound (10c). The
reduction of both aldehyde and ketone (12a and 12b) had
the greatest effect on the activity. It is interesting to note
that the methyl analogs (10b and 12b) are somewhat
more active than their hydroxymethyl (10c and 12a )
counterparts. It is quite clear from this limited study
that both the aldehyde and ketone play a role in the
biological activity of this series of compounds.
.
.
.
.
FeSO4 7H2O, 1.0; MnSO4 4H2O, 1.0; CuCl2 2H2O, 0.025; CaCl2 -
2H2O, 0.1; H3BO3, 0.056; (NH4)6MoO24.4H2O, 0.019; ZnSO4 -
.
7H2O, 0.2; dissolved in 0.6 N HCl). The pH of the medium
was adjusted to 6.8 by addition of NaOH before sterilization.
Seed medium was prepared using distilled water and was
dispensed into Erlenmeyer flasks that were capped with cotton
plugs before being autoclaved at 121 °C for 20 min. Seed
cultures were incubated at 25 °C, on a gyrotory shaker (220
rpm, 5.1 cm throw) for 74 h prior to inoculation of fermentation
flasks.
Fermentations were performed on either solid substrate or
in liquid production media. The solid substrate production
medium was formulated as follows: millet, 15.0 g/250 mL
Erlenmeyer flask to which was added 15 mL containing 0.5 g
yeast extract, 0.1 g of sodium tartrate, 0.5 g of sucrose, 0.5 g
of alfalfa, 0.1 mL of corn oil, and 0.01 g of FeSO4‚7H2O. Solid
substrate production flasks were capped with cotton plugs and
sterilized at 121 °C for 15 min. Immediately prior to inocula-
tion, distilled water (15.0 mL) was added to each flask, the
flasks were resterilized at 121 °C for 20 min and then cooled.
Each production flask was inoculated with 2.0 mL of vegetative
seed growth mixed throughout the solid substrate. The
production flasks were incubated without agitation at 25 °C
for 21 days. Individual fermentation flasks were extracted
with 50 mL of methyl ethyl ketone.
Compound 1f inhibited the reaction of recombinant
human FPTase29 with Ras-CVLS with an IC50 of 0.43 µM.
To determine the mechanism of inhibition of 1f, a more
detailed kinetic analysis was performed. Compound 1f
was a noncompetitive inhibitor of bovine FPTase with
respect to both FPP and the ras peptide (Ras-CVLS)
exhibiting Ki values6d of 1.2 and 1.5 µM, respectively.
The liquid production medium was formulated as follows
(in g/L): sucrose, 80.0; yellow corn meal, 50.0; and yeast
extract, 1.0. This medium was prepared using distilled water;
50 mL of medium was dispensed into 250 mL Erlenmeyer
flasks that were capped with cotton plugs before being auto-
claved at 121 °C for 20 min. Production flasks were inoculated
with 2.0 mL of vegetative seed growth and were incubated at
25 °C, on a gyrotory shaker (220 rpm, 5.1 cm throw) for 21
Con clu sion
We have described a number of novel compounds in
the ascochlorin family isolated from C. lucidum. These
(30) Hayakawa, S.; Minato, H.; Katagiri, K. J . Antibiot. 1971, 24,
654.
(31) Sasaki, H.; Hosokawa, T.; Sawada, M.; Ando, K. J . Antibiot.
1973, 26, 676.
(29) Omer, C. A.; Kral, A. M.; Diel, R. E.; Prendergast, G. C.; Powers,
S.; Allen, C. M.; Gibbs, J . B.; Kohl, N. E. Biochemistry 1993, 32, 5167
and references cited therein.

7734 J . Org. Chem., Vol. 61, No. 22, 1996
Singh et al.
days. At time of harvest, liquid fermentation flasks were
homogenized and pooled.
(m/z): 430.2470 (M+, calcd for C25H34O6: 430.2354), due to
identical values and assignments to 1a , only low resolution
data is listed 430 (1%, M+), 370 (25%), 257 (5%), 231 (100%),
217 (65%), 203 (45%), 177 (17%), 165 (55%), 139 (70%), 97
(90%); 1c: Colorless gum, [R]23D +20.6° (c 0.3, MeOH); IR: νmax
(ZnSe): 3265, 2930, 1731, 1708, 1626, 1493, 1430, 1373, 1307,
1252, 1185, 1104, 1016, 968 cm-1; See Table 2 for 1H NMR
spectral data; HREIMS (m/z): 458.2768 (M+, calcd for
C27H38O6: 458.2667), 458 (<1%, M+), 370 (20%), 257 (3%), 231
(100%), 217 (60%), 203 (45%), 177 (15%), 165 (55%), 139 (70%),
97 (85%); 1d : colorless gum, [R]23D +26.2° (c 0.71, MeOH); UV
(MeOH) λmax 204 (log ꢀ ) 4.26), 222 (4.19), 296 (4.19), 340 (sh)
nm, IR: νmax (ZnSe): 3275, 2962, 1731, 1708, 1626, 1493, 1430,
1372, 1343, 1306, 1252, 1189, 1171, 1104, 1060, 1016, 978
cm-1; NMR spectral data is presented in Tables 1 and 2;
HREIMS (m/z): 472.2824 (M+, calcd for C28H40O6: 472.2824),
472 (<1%, M+), 370 (25%), 257 (5%), 231 (100%), 217 (55%),
203 (35%), 177 (15%), 165 (45%), 139 (65%), 97 (80%); 1e:
Colorless gum, [R]23D +20.9° (c 0.69, MeOH); UV (MeOH) λmax
204 (log ꢀ ) 4.37), 230 (sh), 259 (3.92), 291 (3.81), 348 (4.22)
nm, IR: νmax (ZnSe): 3373, 2960, 1731, 1708, 1626, 1461, 1421,
1375, 1327, 1285, 1248, 1190, 1168, 1111, 1061, 1015, 974, 911,
796, 732, 713 cm-1; For NMR spectral data see Tables 1 and
2; HREIMS (m/z): 506.2463 (M+, calcd for C28H39ClO6:
506.2434), 506 (<1%, M+), 404 (20%), 266 (65%), 251 (40%),
237 (15%), 211 (10%), 199 (30%), 139 (100%), 97 (85%); 1f: mp.
Isola tion of Cylin d r ols 1a -e, 2a ,b) a n d Com p ou n d s 1f,
2-5). Fermentation broth (1.9 L) was extracted with methyl
ethyl ketone (1.0 L) by shaking on a shaker at room temper-
ature for 2 h. Celite was added to the agitated thick mixture
and was filtered using sintered glass funnel. The filtrate was
transferred into a separatory funnel, and methyl ethyl ketone
layer was separated. The aqueous layer was washed with 400
mL of methyl ethyl ketone. The combined methyl ethyl ketone
extract was concentrated to a small volume on a rotary
evaporator under reduced pressure and lyophilized to remove
residual water. The residue thus obtained was suspended in
100 mL of methanol and filtered. The filtrate contained all of
the Ras farnesyl-protein transferase activity. Methanol was
removed from filtrate under reduced pressure to give 6.5 g of
a dark viscous gum.
The crude gum was dissolved in 50 mL of methanol and
was chromatographed over a Sephadex LH-20 (2.0 L) column
in methanol. Elution with methanol at 10 mL/min afforded
the active components after 1350 mL. The active region was
split into six successive fractions (100 mL each) to give:
fraction A (0.53 g), B (0.7 g), C (1.07 g), D (1.62 g), E (0.73 g),
and F (0.14 g).
Fractions A and B (1.23 g) were combined and flash
chromatographed on a silica gel column (2 × 20 cm). Elution
with 10 to 30% ethyl acetate-hexane gave the following
compounds as amorphous solids listed in order of elution: 1e
(83 mg), 1d (139 mg), fraction G (112 mg), and 5 (115 mg).
Fraction C (250 mg) was chromatographed by a reverse
phase HPLC using a Whatman Partisil-10 column (50%
acetonitrile-water for 20 min followed by a gradient to 70%
acetonitrile over 80 min at 10 mL/min). The fractions were
freeze-dried to give amorphous powders of 5 (20.7 mg tR 25.1
min), 2b (6.6 mg, tR 37.1 min), 2a (9.6 mg, tR 41 min), 1f (30.2
mg, tR 43 min), and 2c (11.5 mg, tR 52 min).
Fraction D was crystallized from acetone to give colorless
rosettes of 90% pure 1f (0.34 g) that upon recrystallization
from hot methanol gave colorless needles of pure 1f.
Crystallization of fraction E from methanol gave 1f (240 mg)
and chromatography of the mother liquor on a flash silica gel
column (2 x 20 cm) and elution with 10 to 30% ethyl acetate
in hexane gave amorphous solids of 4b (17.5 mg), and 4a (92.2
mg).
175-77 °C (acetone-hexane), Lit17 mp. 171.5-172.5), [R]23
D
+6.5° (c 0.83, MeOH); lit.17 +6° (c, 0.93, MeOH); IR: νmax
(ZnSe): 3241, 2968, 1694, 1625, 1490, 1428, 1376, 1341, 1306,
1252, 1170, 1103, 1016, 964 cm-1; See Tables 1 and 2 for NMR
spectral data; HREIMS (m/z): 372.2414 (M+, calcd for
C
23H32O4: 372.2300); CD (MeOH) λmax 286 (∆ꢀ ) -1.48); 2a :
amorphous powder, [R]23D -11.9° (c 0.81, MeOH); UV (MeOH)
λmax 223 (log ꢀ ) 4.23), 232 (4.22), 296 (4.09), 340(sh) nm, IR:
νmax (ZnSe): 3258, 2972, 2935, 2876, 1708, 1625, 1490, 1429,
1373, 1306, 1252, 1105, 971, 904, 838, 736 cm-1; For 1H and
13C NMR assignments see Tables 1 and 2; HREIMS (m/z):
370.2161 (M+, calcd for C23H30O4: 370.2144). 2b: amorphous
powder, [R]23 -11° (c 0.35, MeOH); UV (MeOH) λmax 223 (log
D
ꢀ ) 4.31), 233 (4.32), 295 (4.12), 340(sh) nm, IR: νmax (ZnSe):
3314, 2977, 2939, 2876, 1740, 1715, 1626, 1490, 1429, 1371,
1251, 1224, 1104, 1064, 1026, 975, 942, 904, 838, 803, 737
cm-1; For 1H and 13C NMR assignments see Tables 1 and 2;
HREIMS (m/z): 428.2287 (M+, calcd for C25H32O6: 428.2198).
2c: 1H NMR (CDCl3) δ: 0.74 (3H, s), 0.87 (3H, d, J ) 6.6 Hz),
0.84 (3H, d, J ) 6.6 Hz), 1.80 (1H, m), 1.92 (3H, s), 2.00 (1H,
m), 2.06 (3H, s), 2.40 (1H, dd, J ) 12.6, 6.3 Hz), 2.49 (1H, m),
2.61 (3H, s), 2.87 (1H, dd, J ) 13.5, 5.7 Hz), 3.54 (2H, d, J )
7.5 Hz), 4.90 (1H, dd, J ) 5.7, 11.1 Hz), 5.32 (1H, J ) 16.2
Hz), 5.5 (1H, t, J ) 6.9 Hz), 5.92 (1H, d, J ) 15.9 Hz), 6.39
(1H, brs), 10.15 (1H, s), 12.71 (1H, s), 13C NMR (CDCl3) δ: 8.83,
11.51, 12.43, 12.61, 14.46, 21.02, 22.23, 45.36, 45.54, 47.20,
53.85, 73.64, 111.50, 113.15, 113.67, 128.26, 133.76, 134.20
(2C), 137.82, 156.11, 162.20, 170.05, 193.20, 207.81; HREIMS
Fraction F (130 mg) was chromatographed on a 50 mL silica
gel column and eluted with 5 to 15% acetone in hexane to give
chromatographically homogeneous 4b (12.4 mg), 4a (27.7 mg),
and fraction H (3.3 mg). The latter fraction was chromato-
graphed on a Whatman Partisil-10 22 × 250 mm HPLC
column and eluted with a 60 min gradient of 50 to 70%
acetonitrile-water at a flow rate of 10 mL/min. The fractions
eluted at 30 min yielded amorphous solid of 3 (2.0 mg) after
lyophilization.
Fraction G (112 mg) was rechromatographed on a reverse
phase preparative Whatman Partisil 10 (C-18) HPLC column
(22 × 250 mm) eluted initially for 10 min with 50% acetonitrile
in water followed by a gradient to 60% acetonitrile over 60
min at a flow rate of 10 mL/min. Lyophilization afforded 1b
(11.6 mg, tR 30 min), 1a (22.5 mg, tR35 min), 1c (4.4 mg, tR 42
min), and 1d (5.4 mg, tR 48.7 min) all as powders.
1
(m/z): 462.1723 (M+, calcd for C25H31ClO6: 462.1803). 3: H
NMR (CDCl3) δ: 0.80 (3H, s), 0.95 (3H, d, J ) 6.8 Hz), 0.97
(3H, d, J ) 7.6 Hz), 1.93 (3H, brs), 2.45 (1H, q, J ) 6.8 Hz),
2.60 (3H, s), 2.63 (1H, m), 3.54 (2H, d, J ) 7.2 Hz), 5.42 (1H,
d, J ) 16 Hz), 5.54 (1H, brt, J ) 7.2 Hz), 5.98 (1H, d, J ) 16
Hz), 5.99 (1H, d, J ) 10 Hz), 6.36 (1H, brs), 6.55 (1H, dd, J )
10, 2 Hz), 10.15 (1H, s), 11.71 (1H, brs); HREIMS (m/z):
402.1701 (M+, calcd for C23H27ClO4: 402.1598). 4a : 1H NMR
(CDCl3) δ: 1.59 (3H, s), 1.67 (3H, s), 1.81 (3H, s), 2.08 (4H, m),
2.49 (3H, s), 3.40 (2H, d, J ) 7.2 Hz), 5.05 (1H, m), 5.26 (1H,
t, J ) 7.2 Hz), 6.21 (1H, s), 6.26 (1H, brs), 10.07 (1H, s), 12.75
(1H, s), C-13 NMR (CDCl3) δ: 16.22, 17.69, 17.96, 25.65, 26.33,
39.70, 110.87, 111.56, 113.22, 120.95, 123.68, 132.10, 139.76,
139.76, 141.97, 162.65, 163.61, 192.96; EIMS (m/z): 288 (M+).
4b: 1H NMR (CDCl3) δ: 1.57 (3H, s), 1.65 (3H, s), 1.79 (3H, s),
2.03 (4H, m), 2.60 (3H, s), 3.40 (2H, d, J ) 7.2 Hz), 5.06 (1H,
m), 5.23 (1H, qt, J ) 7.2, 1.2 Hz), 6.41 (1H, brs), 10.14 (1H, s),
12.69 (1H, s), 13C NMR (CDCl3) δ: 14.42, 16.17, 17.65, 22.02,
25.63, 26.60, 39.75, 113.61, 114.40, 120.70, 124.17, 131.45,
137.00, 137.58, 156.43, 162.18, 193.19; HREIMS (m/z): 322.1345
(M+, calcd for C18H23ClO3: 322.1336). Cylin d r oca r p ol (5):
Colorless gum, [R]23D -11.7° (c 0.35, MeOH); UV (MeOH) λmax
P h ysica l P r op er ties. 1a : For other physical data see
reference 14. HREIMS (m/z): 444.2614 (<1%, M+, calcd for
C26H36O6: 444.2510), 370.2115 (93%, M-C2H5CO2H, calcd
for C23H30O4: 370.2144), 257.1173 (15%, calcd for C16H17O3:
257.1178), 231.1042 (99%, calcd for C14H15O3: 231.1021),
217.0838 (93%, calcd for C13H13O3: 217.0864), 214.0967 (72%,
calcd for C14H14O2: 214.0993), 203.0744 (98%, calcd for
C12H11O3: 203.0708), 177.0561 (35%, calcd for C10H9O3:
177.0552), 139.1135 (72%, calcd for C9H15O: 139.1123), 97.0646
(50%, calcd for C6H9O: 97.0653), CD (MeOH) λmax 242 (∆ꢀ )
+2.03), 286 (∆ꢀ ) -1.47); 1b: Rods from acetone-hexane, mp.
157-58 °C, [R]23 +23.65° (c 0.52, MeOH); UV (MeOH) λmax
D
204 (log ꢀ ) 4.19), 223 (4.15), 296 (4.16), 340 (sh) nm, IR: νmax
(ZnSe): 3274, 2971, 1731, 1708, 1626, 1493, 1430, 1372, 1342,
1307, 1284, 1250, 1174, 1104, 1059, 1033, 1017, 913, cm-1
;
NMR spectral data is presented in Tables 1 and 2; HREIMS

Novel Inhibitors of Ras Farnesyl-Protein Transferase
J . Org. Chem., Vol. 61, No. 22, 1996 7735
205 (log ꢀ ) 4.34), 295 (4.21), 340(sh) nm, IR: νmax (ZnSe):
3349, 2925, 1625, 1490, 1430, 1371, 1305, 1284, 1254, 1166,
1105, 1058, 1034, 1011 cm-1; for NMR spectral data see Tables
1 and 2; HREIMS (m/z): 390.2416 (M+, calcd for C23H34O5:
390.2660).
acetate-hexane to give 6d as a colorless gum (145 mg).1H
NMR (CDCl3) δ: 0.54 (3H, s), 0.85 (3H, d, J ) 6.6 Hz), 0.88
(3H, d, J ) 6.6 Hz), 1.35 (2H, m), 1.60 (2H, m), 1.78 (3H, s),
1.80 (1H, m), 1.95 (2H, m), 2.31 (2H, m), 2.45 (1H, q, J ) 6.6
Hz), 2.56 (3H, s), 3.46 (2H, d, J ) 6.9 Hz), 3.45 (3H, s), 3.57
(3H, s), 5.00 (2H, s), 5.15(1H, t, J ) 6.6 Hz), 5.24 (2H, s), 6.74
(1H, s), 10.36 (1H, s); HREIMS (m/z): 460.2816 (M+, calcd for
C27H40O6: 460.2825).
Hyd r ogen a tion of 1f. Palladium/carbon (10%, 10 mg) was
added to a solution of 1f (40 mg) in ethyl acetate (1.5 mL).
The mixture was evacuated under vacuum and flushed with
hydrogen from a balloon. This process was repeated three
times and finally the stirred reaction mixture was connected
to a hydrogen-filled balloon and was allowed to continue
overnight. After completion of the reaction, catalyst was
removed by filtration through Celite, and the product was
purified on a 1 × 10 cm silica gel column. Elution with 20%
ethyl acetate-hexane yielded diastereomeric mixture of 7 (20
mg) as an amorphous powder. 1H NMR (CDCl3) δ: 0.57 (3H,
s), 0.87, 0.91 (3H, d, J ) 6.6 Hz), 0.88, 0.90 (3H, d, J ) 6.9
Hz), 1.00 (3H, d, J ) 6.0 Hz), 1.21-1.36 (5H, m), 1.41 (1H,
m), 1.61 (2H, m), 1.84 (1H, m), 2.00 (1H, m), 2.09 (3H, s), 2.19
(3H, s), 2.34 (2H, m), 2.47 (1H, brq, J ) ∼5 Hz), 2.54-2.70
(2H, m), 4.70 (2H, brs, 2 x OH), 6.25 (1H, s); HREIMS (m/z):
360.2624 (M+, calcd for C23H36O3: 360.2664).
Ch r om iu m Tr ioxid e Oxid a tion of 1f. To a cooled (0 °C)
solution of 1f (10 mg) in acetone (0.5 mL) was added J ones
reagent (20 µL, ∼2 equiv). The solution was stirred at 0 °C
for 30 min followed by room temperature for 4 h. No reaction
was observed (TLC and HPLC); therefore, additional amounts
of the J ones reagent was added. The starting compound was
now consumed and a product was formed. 2-Propanol was
added to quench the reagent, and the product was taken up
in ethyl acetate, washed with water, and dried over sodium
sulfate. (Another reaction with 10 mg of 1f was performed in
identical conditions using CrO3 in acetic acid as a reagent, and
this gave the same product. Both reactions were combined
after workup and chromatographed together.) Solvent was
removed under reduced pressure, and product was purified
on a small silica gel column. Elution of the column with 20%
ethyl acetate-hexane gave chromatographically homogeneous
gum of 9 (5.8 mg). 1H NMR (CDCl3) δ: 0.63 (3H, s), 0.89 (3H,
d, J ) 6.6 Hz), 0.92 (3H, d, J ) 6.6 Hz), 1.54-1.70 (3H, m),
1.83-1.91 (2H, s), 2.17 (3H, s), 2.26-2.50 (5H, m).
Mn O2 Oxid a tion of Cya n oh yd r in of 1f. To a solution of
1f (200 mg, 0.54 mmol) in methanol (8 mL) were added
manganese dioxide (936 mg, 20 equiv) and potassium cyanide
(338 mg, 10 equiv) followed by acetic acid (0.31 mL, 10 equiv),
and the mixture was stirred at room temperature under
nitrogen overnight. Progress of the reaction was slow, there-
fore, an additional 2 equiv of potassium cyanide and acetic
acid was added, and the mixture was heated at 70 °C for 6 h
whereupon most of the starting compound was consumed and
a major and few minor products were formed (silica gel, TLC,
hexane-ethyl acetate, 7:3). After completion of the reaction,
the mixture was filtered through Celite, and the filtrate was
diluted with 50 mL of ethyl acetate and washed with water,
dried over sodium sulfate, and evaporated under reduced
pressure. The product mixture was chromatographed over a
silica gel column eluted with 20% ethyl acetate-hexane to give
methyl ester 8b (60 mg) as an amorphous powder. 1H NMR
(CDCl3) δ: 0.57 (3H, s), 0.88 (3H, d, J ) 6.6 Hz), 0.91 (3H, d,
J ) 6.6 Hz), 1.30-1.65 (4H, m), 1.84 (3H, s), 1.75-2.1 (3H,
m), 2.32 (2H, m), 2.45 (1H, q, J ) 6.9 Hz), 2.46 (3H, s), 3.42
(2H, d, J ) 7.2 Hz), 3.92 (3H, s), 5.30 (1H, t, J ) 6.6 Hz), 5.66
(1H, brs, OH), 6.23 (1H, s), 12.00 (1H, brs, OH); 13C NMR
(CDCl3) δ: 7.6, 15.12, 15.37, 16.52, 22.20, 24.02, 31.09, 32.83,
35.83, 36.28, 41.63, 43.59, 50.58, 51.73, 105.5, 111.34, 111.70,
121.84, 138.29, 140.88, 159.17, 162.79, 173.0, 213.0; HREIMS
(m/z): 402.2423 (M+, calcd for C24H34O5: 402.2406).
Acetyla tion of 1f. To a solution of 1f (20 mg) in pyridine
(0.2 mL) was added acetic anhydride (0.1 mL), and the solution
was stirred at room temperature for 48 h. After completion
of the reaction, pyridine and excess acetic anhydride were
removed under a stream of nitrogen and finally dried under
vacuum. The crude product was purified on a pipette filled
with silica gel and eluted with 20% ethyl acetate in hexane to
give 20 mg of chromatographically homogeneous 6a as an
amorphous powder. 1H NMR (CDCl3) δ: 0.57 (3H, s), 0.87 (3H,
d, J ) 6.6 Hz), 0.90 (3H, d, J ) 6.9 Hz), 1.35 (3H, m), 1.61
(1H, m), 1.75 (3H, s), 1.82 (2H, m), 1.97 (1H, m), 2.30 (3H, s),
2.33 (2H, m), 2.35 (3H, s), 2.43 (1H, q, J ) 6.6 Hz), 2.61 (3H,
s), 3.18 (2H, d, J ) 6.6 Hz), 5.01 (1H, t, J ) 6.6 Hz), 6.91 (1H,
s), 10.27(1H, s); HREIMS (m/z): 413.2354 (M+ - Ac; calcd for
C25H33O5: 413.2328), 371.2247 (M+ - 2 - Ac; calcd for
C23H31O4: 371.2222).
Meth yla tion of 1f a n d 2a . To a cooled (0 °C) solution of
a 2:3 mixture of 1f and 2a in methylene chloride (1 mL) was
added an excess (5 mL) of an ethereal solution of diaz-
omethane, and the solution was stirred overnight at 0 °C.
Reaction was complete, and a polar (SiO2: TLC, hexane-ethyl
acetate, 7:3) product was formed. Solvents were carefully
evaporated under a stream of nitrogen, and the products were
purified on a silica gel column (1 × 10 cm) eluted with 10%
ethyl acetate-hexane. Both compounds were finally separated
by reverse phase HPLC using Whatman ODS-3 (22 × 250 mm)
column. Elution with a 50% to 70% gradient of acetonitrile-
water at 10 mL/min gave 2d (tR 16 min) and 6b (tR 18 min).
The fractions were lyophilized to give an amorphous colorless
powder. 2d : 1H NMR (CDCl3) δ: 0.69 (3H, s), 0.81 (3H, d, J )
6.6 Hz), 0.84 (3H, d, J ) 6.6 Hz), 1.63 (2H, m), 1.90 (3H, s),
1.95 (1H, m), 2.38 (3H, m), 2.56 (3H, s), 3.45 (2H, d, J ) 7.2
Hz), 3.90 (3H, s), 5.34 (1H, d, J ) 15.9 Hz), 5.49 (1H, t, J )
7.2 Hz), 5.89 (1H, d, J ) 15.9 Hz), 6.29 (1H, s), 10.13 (1H, s),
12.41 (1H, s); HREIMS (m/z): 384.2329 (M+, calcd for
C24H32O4: 384.2301); 6b: 1H NMR (CDCl3) δ: 0.55 (3H, s),
0.87 (3H, d, J ) 6.9 Hz), 0.90 (3H, d, J ) 6.6 Hz), 1.38 (2H,
m), 1.63 (1H, m), 1.79 (3H, brs), 1.83 (2H, m), 1.98 (2H, m),
2.31 (2H, m), 2.45 (1H, q, J ) 6.6 Hz), 2.56 (3H, s), 3.30 (2H,
d, J ) 6.6 Hz), 3.89 (3H, s), 5.20(1H, t, J ) 6.6 Hz), 6.28 (1H,
s), 10.12 (1H, s), 12.39 (1H, brs); HREIMS (m/z): 386.2492
(M+, calcd for C24H34O4: 386.2457).
P r ep a r a tion of TBS eth er of 1f. To a solution of 1f (100
mg, 0.27 mmol) in dimethylformamide (1 mL) was added
diisopropylethylamine (0.21 mL, 1.2 mmol) followed by tert-
butyldimethylsilyl chloride (121 mg, 0.8 mmol). The solution
was stirred under nitrogen overnight. Ice-water (50 mL) was
added, and the mixture was stirred for 20 min. The product
was extracted with ethyl acetate (100 mL) and washed
sequentially with water, 10% aqueous citric acid, water, 10%
aqueous sodium bicarbonate, and water. The extract was
dried over sodium sulfate, ethyl acetate was removed under
reduced pressure, and the product was crystallized from
methanol as colorless crystals of 6c (115 mg, 88.5%), 6c: 1H
NMR (CDCl3) δ: 0.29 (6H, s), 0.56 (3H, s), 0.87 (3H, d, J )
6.9 Hz), 0.90 (3H, d, J ) 6.9 Hz), 1.01 (9H, s), 1.30-1.65 (4H,
m), 1.77 (3H, s), 1.82 (1H, m), 1.98 (2H, m), 2.31 (2H, m), 2.44
(1H, q, J ) 6.9 Hz), 2.49 (3H, s), 3.30 (2H, d, J ) 6.9 Hz), 5.19
(1H, t, J ) 6.6 Hz), 6.19 (1H, s), 10.11 (1H, s), 12.47 (1H, brs,
OH), HREIMS (m/z): 486.3151 (M+, calcd for C29H46O4Si:
486.3165).
P r ep a r a tion of Bis-MOM Eth er of 1f. To a solution of
1f (117 mg, 0.32 mmol) in CH2Cl2 (2 mL) was added diisopro-
pylethylamine (0.35 mL, 1.92 mmol) followed by MOM chloride
(0.15 mL, 1.92 mmol), and the solution was stirred at room
temperature under nitrogen for 5 h. After completion of the
reaction, ice was added followed by ethyl acetate (100 mL).
The ethyl acetate layer was washed with water, 10% aqueous
citric acid, and water, dried (Na2SO4), and evaporated under
reduced pressure to give crude product which was chromato-
graphed over a small silica gel column eluted with 5% ethyl
Mn O2 Oxid a tion of Cya n oh yd r in of 6d . Reaction was
repeated as described for the preparation of 8b. 6d (130 mg,
0.28 mmol) in 3 mL of methanol was reacted with MnO2 (486
mg, 20 equiv), KCN (182 mg, 10 equiv), and AcOH (0.13 mL,
8 equiv) for 4 days. Two products were formed and were
purified on preparative TLC using hexane-ethyl acetate (4:1).

7736 J . Org. Chem., Vol. 61, No. 22, 1996
Singh et al.
Bands were eluted (listed in order of their elution) with acetone
to give 8d (5 mg) and 8c (20 mg) both as a colorless gum. 8c:
1H NMR (CDCl3) δ: 0.55 (3H, s), 0.85 (3H, d, J ) 6.6 Hz),
0.88 (3H, d, J ) 6.9 Hz), 1.35 (2H, m), 1.63 (2H, m), 1.77 (3H,
s), 1.82 (1H, m), 1.96 (2H, m), 2.32 (2H, m), 2.44 (1H, q, J )
6.6 Hz), 2.47 and 2.58 (3H, s), 3.35 (2H, brd, J ) 6.9 Hz), 3.44
(3H, s), 3.52 (3H, s), 3.88 (3H, s), 4.95 (2H, s), 5.18 (1H, t, J )
6.6 Hz), 5.18 (2H, s), 6.73 (1H, s); HREIMS (m/z): 490.2925
(M+, calcd for C28H42O7: 490.2930); 8d : 1H NMR (CDCl3) δ:
0.55 (3H, s), 0.86 (3H, d, J ) 6.6 Hz), 0.89 (3H, d, J ) 6.9 Hz),
1.35 (2H, m), 1.63 (2H, m), 1.81 (3H, s), 1.80 (1H, m), 1.97
(2H, m), 2.31 (2H, m), 2.45 (1H, q, J ) 6.6 Hz), 2.50 (3H, s),
3.36 (2H, d, J ) 6.9 Hz), 3.46 (3H, s), 3.92 (3H, s), 5.23 (1H, t,
J ) 6.6 Hz), 5.23 (2H, s), 6.47 (1H, s), 10.50 (1H, brs, OH);
HREIMS (m/z): 446.2715 (M+, calcd for C26H38O6: 446.2668).
Hyd r olysis-Deca r boxyla tion of 8b. To a solution of 8b
(30 mg) in THF (3 mL), methanol (3 mL), and water (4 mL)
was added LiOH (10 mg), and the solution was stirred at room
temperature for 2 h. TLC examination indicated no reaction;
addition of 20 mg of K2CO3 and a further 2 h of stirring did
not promote reaction either. NaOH (4 N, 0.1 mL) was then
added, and the solution was heated at 70 °C for overnight.
The starting material was now consumed, the reaction mixture
was acidified at -78 °C with dilute HCl. The products were
extracted with ethyl acetate (2 × 50 mL), washed with water,
dried (Na2SO4), and evaporated to give a complex mixture of
products. These products were chromatographed on a reverse
phase Whatman C-18 (9.4 × 250 mm) column and eluted with
a gradient of 40 to 60% acetonitrile-water containing 0.2%
TFA. Elution at 4 mL/ min gave 10a (0.7 mg) and a number
of unidentified orange compounds of higher MW (than 8b)
compounds. 10a : 1H NMR (CDCl3) δ: 0.57 (3H, s), 0.88 (3H,
d, J ) 6.9 Hz), 0.91 (3H, d, J ) 6.6 Hz), 1.41 (2H, m), 1.80
(2H, m), 1.84 (3H, s), 1.90 (1H, m), 1.96 (2H, m), 2.22 (3H, s),
2.34 (2H, m), 2.45 (1H, q, J ) 6.6 Hz), 3.39 (2H, d, J ) 6.6
Hz), 4.92 (1H, s), 5.27 (1H, t, J ) 7.2 Hz), 6.24 (2H, s); HREIMS
(m/z): 344.2412 (M+, calcd for C22H32O3: 344.2351).
Rea ction of 8b w ith TMS Iod id e. To a solution of 8b
(4.8 mg, 0.013 mmol) in anhydrous acetonitrile (1 mL) were
added trimethylsilyl chloride (0.026 mL) and sodium iodide
(28 mg), and the solution was heated at 60 °C for 8 h followed
by stirring at room temperature for 4 days. After completion
of the reaction, ethyl acetate (50 mL) was added and the
solution was washed with 5% aqueous sodium thiosulfate (20
mL) and water (20 mL), dried (Na2SO4), and evaporated under
reduced pressure. Products were purified on silica gel plates
(hexane-ethyl acetate, 4:1) and bands were eluted with
acetone to give 11a (0.8 mg) and 11b (2.0 mg) both as
amorphous solids. 11a : 1H NMR (CDCl3) δ: 0.61 (3H, s), 0.84,
0.86, 0.88, 0.92 (6H, d, J ) 6.9 Hz), 1.29 (3H, s), 1.4-1.64 (6H,
m), 1.80 (2H, m), 2.00 (1H, m), 2.34 (2H, m), 2.42 (1H, q, J )
7.2 Hz), 2.45 (3H, s), 2.65 (2H, m), 3.91 (3H, s), 6.19 (1H, s),
HREIMS (m/z): 402.2403 (M+, calcd for C24H34O5: 402.2406);
11b: 1H NMR (CDCl3) δ: 0.63 (3H, s), 0.84, 0.86, 0.93, 0.97
(6H, d, J ) 6.9 Hz), 1.30 (3H, s), 1.4-1.64 (6H, m), 1.82 (2H,
m), 2.00 (1H, m), 2.33 (2H, m), 2.35 (3H, s), 2.45 (1H, q, J )
7.2 Hz), 2.64 (2H, m), 4.82 (1H, brs, OH), 6.36 (1H, s); HREIMS
(m/z): 470.1330 (M+, calcd for C22H31IO3: 470.1277).
water (0.5 mL), and H2O2 (0.01 mL) was added to a solution
of NaClO3 (6 mg, 0.054 mmol) in water (0.5 mL), and the
solution was stirred at room temperature for 4 h. There was
no product formed, and the starting material was intact (TLC,
hexane-ethyl acetate, 7:3); therefore, an additional 3 equiv
of NaClO3 was added and the solution was stirred overnight.
A faster moving product was formed. The reaction mixture
was acidified and extracted with ethyl acetate (100 mL). Ethyl
acetate extract was washed with water, dried (Na2SO4),
evaporated under reduced pressure, and chromatographed
over a small silica gel column eluted with 10% ethyl acetate-
hexane to give a powder of chloro compound 8e which was
identified as illicicolin C by comparison of NMR spectrum19
and mass spectral analysis.
Sod iu m Bor oh yd r id e Red u ction of 1f. To a solution of
1f (40 mg, 0.11 mmol) in a 3:2 mixture of tetrahydrofuran-
methanol (1 mL) was added sodium borohydride (5.2 mg, 0.11
mmol), and the solution was stirred at ambient temperature
for 10 min. The product diol was formed almost instanta-
neously. The reaction mixture was concentrated to dryness,
and ethyl acetate (100 mL) was added. The ethyl acetate
solution was washed with water (2 × 50 mL), dried (Na2SO4),
and evaporated under reduced pressure to give a gummy
residue which was chromatographed on a 1 × 10 cm silica gel
column. Elution of the column with 25% ethyl acetate-hexane
afforded 12a (20 mg) as an amorphous powder. 1H NMR
(CDCl3) δ: 0.81 (3H, d, J ) 6.3 Hz),0.84 (3H, s), 0.95 (3H, d,
J ) 7.5 Hz), 1.30 (3H, m), 1.41-1.63 (5H, m), 1.74-1.91 (2H,
m), 1.83 (3H, s), 2.19 (3H, s), 3.40 (2H, d, J ) 6.9 Hz), 3.84
(1H, dd, J )3.0, 3.0 Hz), 4.84 (2H, brs), 5.27 (1H, t, J ) 6.9
Hz), 5.29 (1H, brs, OH), 6.22 (1H, s), 7.66 (1H, brs, OH), 13C
NMR (CDCl3) δ: 12.40, 15.71, 16.49, 17.28, 19.12, 22.25, 25.51,
32.91, 33.89, 36.18, 36.53, 38.51, 39.48, 60.52, 73.29, 109.40,
112.13, 115.58, 121.51, 134.19, 139.28, 154.70, 155.39; HRE-
IMS (m/z): 376.2580 (M+, calcd for C23H36O4: 376.2613).
Sod iu m Cya n obor oh yd r id e Red u ction of 1f. Sodium
cyanoborohydride (10 mg, 0.16 mmol) was added to a solution
of 1f (16 mg, 0.043 mmol) in methanol (2 mL) and acetic acid
(0.02 mL), and the solution was stirred overnight at room
temperature. After all of the starting material was consumed,
ethyl acetate (100 mL) was added to the reaction mixture,
washed with water (2 × 50 mL), dried (Na2SO4), and evapo-
rated under reduced pressure to give a mixture of four
products. These products were purified on preparative silica
gel plates (250 µm) developed in ethyl acetate-hexane (7:3).
Four bands were eluted with acetone to give 10b (2.1 mg), 12b
(3.3 mg), 10c (1.7 mg), and 12a (2.6 mg) mostly as a gum.
When this reaction was carried out in controlled conditions,
the reduction of the ketone was minimized. Typical experi-
ment: To a solution of 1f (70 mg, 0.19 mmol) in a mixture of
tetrahydrofuran-methanol (1:1, 4 mL) and acetic acid (0.1 mL)
was added sodium cyanoborohydride (11.9 mg, 0.19 mmol), and
the solution was stirred overnight under nitrogen. The
progress of the reaction was extremely slow and most of the
starting material was left intact. Additional amounts of
sodium cyanoborohydride (17.9 mg, 0.28 mmol) were added,
and reaction mixture was stirred for additional 24 h. Two
major products 10b and 10c were formed, and starting
material was consumed. The reaction mixture was worked
up as described in the previous experiment, and products were
purified on a small silica gel column eluted with 10 to 20%
ethyl acetate-hexane to give clean gums of 10b (16 mg) and
10c (40 mg). 10b: 1H NMR (CDCl3) δ: 0.58 (3H, s), 0.88 (3H,
d, J ) 6.9 Hz), 0.92 (3H, d, J ) 6.6 Hz), 1.34-1.46 (2H, m),
1.50-1.69 (2H, m), 1.79-2.10 (3H, m), 1.86 (3H, brs), 2.07 (3H,
s), 2.19 (3H, s), 2.34 (2H, m), 2.45 (1H, q, J ) 6.6 Hz), 2.56
(3H, s), 3.41 (2H, d, J ) 7.5 Hz), 4.68 (1H, brs, OH), 5.08 (1H,
brs, OH), 5.28 (1H, brt, J ) 6.9 Hz), 6.27 (1H, s), HREIMS
(m/z): 358.2473 (M+, calcd for C23H34O3: 358.2508); 10c: 1H
NMR (CDCl3) δ: 0.57 (3H, s), 0.88 (3H, d, J ) 6.6 Hz), 0.91
(3H, d, J ) 6.6 Hz), 1.30-1.65 (4H, m), 1.85 (3H, s), 1.87-2.1
(3H, m), 2.20 (3H, s), 2.32 (2H, m), 2.46 (1H, q, J ) 7.2 Hz),
3.41 (2H, d, J ) 6.9 Hz), 4.88 (2H, brs), 5.05 (1H, brs, OH),
5.30 (1H, t, J ) 6.9 Hz), 6.23 (1H, s), 7.68 (1H, brs, OH);
HREIMS (m/z): 374.2455 (M+, calcd for C23H34O4: 374.2457);
12b: 1H NMR (CDCl3) δ: 0.82 (3H, d, J ) 6.6 Hz), 0.84 (3H,
Oxid a tion of 1f th r ou gh Su lfite Ad d u ct. To a solution
of 1f (32 mg, 0.086 mmol) in ethanol (1 mL) was added an
aqueous solution of sodium hydrogen sulfite (100 mg in 0.5
mL of water, 10 equivalent), and the solution was stirred at
room temperature for 30 min. The adduct formed was filtered.
The filtrate was heated at 60 °C overnight, and further
amounts of precipitate were collected. Combined precipitate
was dried in a vacuum desiccator to give adduct as a colorless
powder (40 mg, 97.7%).
The adduct was dissolved in anhydrous DMSO (0.3 mL),
and acetic anhydride (0.1 mL) was added. The mixture was
stirred overnight which finally gave a homogeneous solution.
The reaction was quenched with aqueous K2CO3. Ethyl
acetate (50 mL) was added and acidified with 4 N aqueous
HCl to pH 2, and the organic solution was washed with water,
dried (Na2SO4), and evaporated to give diacetate 6a .
Rea ction of 1f w ith Na ClO3-H2O2. A solution of 1f (10
mg, 0.027 mmol) in acetonitrile (0.5 mL), NaH2PO4 (10 mg) in

Novel Inhibitors of Ras Farnesyl-Protein Transferase
J . Org. Chem., Vol. 61, No. 22, 1996 7737
s), 0.96 (3H, d, J ) 7.2 Hz), 1.27-1.40 (3H, m), 1.41-1.62 (5H,
m), 1.74-1.91 (2H, m), 1.84 (3H, s), 2.08 (3H, s), 2.19 (3H, s),
3.40 (2H, d, J ) 6.9 Hz), 3.84 (1H, dd, J )2.7, 2.7 Hz), 4.69
(1H, brs, OH), 5.14 (1H, brs, OH), 5.25 (1H, t, J ) 6.9 Hz),
6.23 (1H, s); HREIMS (m/z): 360.2702 (M+, calcd for
C23H36O3: 360.2664).
linear with respect to both substrates, enzyme level, and time.
Less than 10% of the [3H]-FPP was utilized during the reaction
period. Compounds were dissolved in 100% dimethyl sulfoxide
and were diluted twenty fold into the assay.
Ack n ow led gm en t. The authors are thankful to Mr.
Assa y of F a r n esyl-P r otein Tr a n sfer a se. Recombinant
human FPTase and the Ras peptide were prepared as de-
scribed by Omer et al.29 Bovine FPTase assay was performed
as described elsewhere.6d The human transferase assay was
carried out in a volume of 100 µL containing 100 mM N-(2-
hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),
pH 7.4, 5 mM MgCl2, 10 µM ZnCl2, 5 mM dithiothreitol (DTT),
0.1% (w/v) polyethylene glycol 20,000, 50 nM [3H]-FPP, 400
nM Ras-CVLS, and 2 nM FPTase at 31 °C for 20 min.
Reactions were initiated with FPTase and quenched with 0.1
mL of 30% (v/v) trichloroacetic acid in ethanol. Precipitates
were collected onto glass fiber filtermats using a Tomtec Mach
II cell harvestor, washed thoroughly with 100% ethanol, dried,
and counted in an LKB Betaplate counter. The assay was
J ack L. Smith for providing initial mass spectral data.
Su p p or tin g In for m a tion Ava ila ble: 1H (400 or 300 MHz)
and 13C (100 or 75 MHz) NMR spectra of 1a , 1b, 1d , 1f, 2a ,
2b, 5; 1H (400 or 300 MHz) NMR spectra of 1c, 1e, 2d , 6b, 6c,
6d , 7, 8b, 8d , 10a , 10b, and 12a ; a 400 MHz HMBC correlation
of 1b; and ORTEP diagrams of 1a and 1f from single-crystal
X-ray structure determination (30 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O961074P