Design, synthesis and evaluation of a series of novel fumagillin analogues

Article abstract of DOI:10.1016/j.bmc.2003.08.031

A series of fumagillin analogues targeted at understanding tolerability of MetAP2 toward substitution at C4 and C6 were synthesized. Initially, the C6 side chain was maintained as cinnamoyl ester and C4 was modified. It was concluded that replacing the na

Full text of DOI:10.1016/j.bmc.2003.08.031

Bioorganic & Medicinal Chemistry 11 (2003) 5051–5058
Design, Synthesis and Evaluation of a Series of Novel Fumagillin
Analogues
Maria Fardis,a,* Hyung-Jung Pyun,a James Tario,a Haolun Jin,a Choung U. Kim,a
Judy Ruckman,b Yun Lin,c Louis Greenc and Brian Hicked
aDepartment of Medicinal Chemistry, Gilead, 333 Lakeside Dr., Foster City, CA 94404, USA
bCBR International Corp., 2905 Wilderness Place, Suite 202, Boulder, CO 80301, USA
cReplidyne Inc., 1450 Infinite Dr., Louisville, CO 80027, USA
dSomaLogic Inc., 1775 38th Street, Boulder, CO 80301, USA
Received 16 April 2003; revised 16 April 2003; accepted 26 August 2003
Abstract—A series of fumagillin analogues targeted at understanding tolerability of MetAP2 toward substitution at C4 and C6 were
synthesized. Initially, the C6 side chain was maintained as cinnamoyl ester and C4 was modified. It was concluded that replacing
the natural C4 of fumagillin with a benzyl oxime at C4 resulted in moderate loss of activity toward binding to MetAP2. Placement
of a primary or secondary carbamate at C6 did not improve the potency of compounds toward inhibition of MetAP2. However, the
inhibitory activity against MetAP2 was gained back by placing polar groups such as piperazinyl carbamate at C6. Small alkyl
substituents on the amine of piperazinyl carbamate were well tolerated.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
under basic conditions. Subsequently, a number of
analogues of fumagillol were prepared4 including CKD-
7315 and TNP 470 which was advanced to phase III
clinical trials. Ovalicin, the oxidation product of fuma-
gillol, also shows potent antiangiogenic activity.6 It was
proposed that fumagillin analogues inhibit angiogenesis
by covalently binding to His-231 of the enzyme,
methionine aminopeptidase type 2 (MetAP2).7,8 During
protein translation, a methionine (or N-formyl methio-
nine) is installed at the N-terminus of most proteins.
Removal of the terminal methionine as a part of the
translocation process, is performed by methionine
amino peptidases. MetAP2 cleaves the initiator methio-
nine from specific proteins that are essential for cell
cycle progression in endothelial cells.9 Therefore, inhi-
bition of MetAP2 may result in cell cycle arrest and
subsequent apoptosis.10
Angiogenesis, the process by which the tumor vascu-
larizes, is essential for tumor growth and metastasis.1,2
Endothelial cells respond to angiogenic signals pro-
duced by tumors by proliferating and migrating to
neighboring tissues. After undergoing differentiation,
the endothelial cells generate the inner lining of blood
vessels that provide oxygen and nutrients to the growing
tumor. Therefore, inhibition of endothelial cell growth
can block angiogenesis and prevent tumor growth. This
approach to cancer treatment is orthogonal to the con-
ventional chemotherapy, in which the tumor is directly
targeted, and therefore may benefit from lower toxicity
and drug resistance. Compounds with
mechanism of action may also be used for other angio-
genesis-dependent diseases such as rheumatoid arthritis.
a similar
Fumagillin, a natural product from Aspergillus fumiga-
tus was discovered serendipitously and found to inhibit
angiogenesis by blocking endothelial cell growth (Fig.
1).3 Fumagillin is rapidly hydrolyzed to fumagillol
A high resolution crystal structure of fumagillin bound
MetAP2 confirmed ring opening of the spirocyclic epox-
ide by His-231 and subsequent covalent binding to
MetAP2.11 From the crystal structure it is evident that the
substituent at C6 of fumagillin is exposed to the solvent.
Angiogenic activity of fumagillin analogues are asso-
ciated with mild cytotoxicity.12 The goal of our program
*Corresponding author. Fax: +1-650-522-5899; e-mail: mfardis@
gilead.com
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.031

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M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
Figure 1.
was to explore the tolerability of MetAP2 toward inhi-
bitors with lower cytotoxicity and better solubility. We
envisioned modifying the two epoxides which we hypo-
thesized to be associated with the observed cytotoxicity
of the compounds. Since the spiroepoxide of the fuma-
gillin family was shown to be essential for MetAP2
binding,13 we decided to initially keep this moiety intact.
It was reported that the chlorohydrin analogue of the
fumagillin spiroepoxide is as active as the parent com-
pound.14 Hence, a few chlorohydrins were also synthe-
sized and evaluated. Previously reported SAR studies of
fumagillin analogues showed that the exocyclic epoxide
was not essential.13 Other studies suggested the need for
the planar geometry at the 10 position of the molecule.15
Taken together, we decided to replace the exocyclic
epoxide with an oxime functionality which would pro-
vide the desired geometry, substitution pattern, and
easy access to further analogues. A number of C4 oxime
analogues were synthesized and evaluated.16 Oximes
such as compounds 5 and 6 carrying a benzyl sub-
stituent, proved to be an effective isostere for the nat-
ural C4 side chain (Fig. 2). In this paper, we describe the
relationship between modification at C4 and C6 posi-
tions of fumagillin analogues. We found that C4 modi-
fications lead to changes in SAR of the compound. A
series of analogues with C6 carbamates were prepared
that demonstrated equipotent enzymatic activity to
TNP-470.
times resulted in lower overall yields. Therefore, the
reactions were generally halted after 15 h. In some
cases, when the amount of the chlorohydrin was insig-
nificant, the cinnamoyl compound was treated with
LiCl and HOAc to provide the desired chlorohydrin
quantitatively.
Activity of the C6 cinnamoyl analogues were evaluated
against MetAP217 in vitro. Cell based assay monitoring
inhibition of proliferation of human umbilical vein
endothelial cells (HUVEC) was performed using the
SRB dye (Table 1).18 Removal of the exocyclic olefin
seems to have a large impact in activity in the cell based
assay, but minor changes were observed in the in vitro
enzyme assay. It was also apparent that substitution of
the oxime for the olefin is an acceptable modification as
observed by the similar activity numbers for compounds
3 and 5.
The C4 diene fumagillol 10, precursor to compound 3,
was prepared by a four-step synthesis from fumagillol
(Scheme 2).16 It was found that conversion of fumagillol
to the diene 10 is best achieved by opening the spiroep-
oxide before the reduction of the exocyclic epoxide to
avoid generating a mixture of olefins in the reaction.
Closure of the chlorohydrin to the epoxide proceeds
well to generate compound 10. Removal of the acetate
group was accomplished using the standard K2CO3 and
MeOH conditions. Formation of 3 and 4 was achieved
using the EDC, DMAP conditions as before.
Results and Discussion
Generation of the benzyl oxime precursor to 5 was per-
formed starting from compound 10.16 Formation of the
cinnamoyl 5 and the subsequent chlorohydrin 6 was
achieved utilizing the peptide coupling method.
We initially prepared a series of analogues carrying the
cinnamoyl group at C6, since CKD-731 (1) was repor-
ted to be one of the most potent inhibitors of MetAP2.5
Maintaining the cinnamoyl group at C6 allowed us to
better compare the analogues with various C4 groups.
A second series of compounds were prepared with the aim
of improving solubility of the benzyloxime analogues
In order to examine the effect of replacing the exocyclic
epoxide with the oxime moiety, compounds 3 and 4
were prepared in which the epoxide was changed to the
olefin (Fig. 2). The purpose of this series was to learn
the effect of modification of C10 from sp3 hybridization,
as in compound 1, to sp2, as in compound 3. We then
explored the effect of replacing the C4 olefin in com-
pound 3 with the C4 oxime of 5. Simultaneously,
modification of the spirocyclic epoxide to the chlorohy-
drin was evaluated.
Table 1. Activity of a series of cinnamoyl derivatives of fumagillin in
enzymatic and cell-based assaysa
Compound
MetAP2IC50 (nM)
HUVEC assay EC50 (nM)
Fumagillin
Fumagillol
0.63
230
0.82
49
TNP470
1 (CKD 731)
0.43
0.96
15.7
1.67
54.4
1.6
0.52
2.6
4.1
170
310
110
160
2
3
4
5
6
Compound 1 was prepared by a peptide coupling strat-
egy using EDC and DMAP (Scheme 1). A small
amount of the chlorohydrin side product was isolated
due to the chloride ion present in EDC. Longer reaction
161
aValues are average of duplicate of triplicate data.

M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
5053
Figure 2.
Scheme 1.
Scheme 2.
(Fig. 3). Considering the encouraging results obtained
from the benzyloxime modifications, we kept the C4
substitution as the oxime moiety while we changed the
C6 group. We initially explored the carbamate group as
the linkage at C6. It was found that N-methyl and N,N-
desired product (Scheme 3). The carbonate 16 was
purified and could be stored for a few months.
Activity of the C6 piperazinyl benzyloximes were very
promising and resembled the parent fumagillin and
TNP-470 (Table 2). Comparing compounds 11 and 12
demonstrated the importance of the basic amine present
in the molecule. Some tolerance around the 30 amine
was observed as is apparent from compounds 14 and 15.
A small improvement of activity was observed for the
cyclic amine 13 over the acyclic one, 14, as evident from
the enzymatic and cell-based assays. Solubility of com-
pound 13 was measured at 144 mM which is an
improvement over CKD-732,19 a highly soluble analo-
gue of fumagillin. In-house measurement of solubility of
CKD-732 was about 120 mM.
dimethyl
carbamates
were
moderately
active
(EC50=570 nM and 550 nM in that order). To improve
the activity, we decided to attach a polar group carrying
a nitrogen, such as a piperazinyl onto our benzyloxime
compounds. N-methyl, ethyl, and benzyl piperazinyl
analogues were synthesized to develop an SAR around
the C6 position of the molecule. In order to examine
the effect of the 30 amine, the carbon analogue 12 was
also prepared. Importance of the cyclic structure was
evaluated by preparing the N,N-dimethyl ethylene
compound 14.
Formation of the carbamate bond at C6 was attempted
under different conditions. Synthesis of intermediate p-
nitrophenyl carbonate gave the highest yield of the
Ovalicin, a natural product structurally related to
fumagillin, has also shown inhibition of MetAP2 activ-
ity.20 Therefore, analogues of fumogillol with an sp2

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M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
Figure 3.
Scheme 3.
Table 2. Activity of
fumagillin in enzymatic and cell-based assays
a
series of the piperazinyl derivatives of
Table 3. Activity of a series of the ovalicin analogues in enzymatic
and cell-based assays
Compound
MetAP2IC50
(nM)
HUVEC Assay
EC50 (nM)
Compound
MetAP2IC50
(nM)
HUVEC Assay
EC50 (nM)
Fumagillin
Fumagillol
TNP470
11
12
13
14
15
0.63
230
0.82
49
0.52
0.80
4.8
0.90
3.2
12
Fumagillin
Fumagillol
0.63
230
0.35
5.4
0.51
50
43
0.82
49
0.46
0.64
36
0.43
0.85
0.55
0.77
0.89
0.95
17
18
19
20
21
15
21
from the exocyclic epoxide to the E-oxime was toler-
ated, although activity of these compounds was slightly
decreased. Therefore, further analogues with the benzy-
loxime at the C4 position were of interest.
center at C6 were of interest. Also, since the C6 position
of fumagillol seems to be exposed to the solvent, as
shown by crystallography,11 we decided to exploit this
position further. Fumaginone 17 was synthesized by
oxidation of fumagillol using PCC (Fig. 4). Presence of
the C6 ketone provides a handle for further modifi-
cation. Furthermore, analogues carrying C4 benzylox-
ime and the corresponding chlorohydrins were prepared
by the same oxidation method from the benzyloxime
alcohol starting material and evaluated. Formation of
oxime from ketone 19 using 50% aqueous NH2OH and
AcOH went smoothly to provide compound 21.
Attempts to define the regioisomer of oxime 21 by
NMR were unsuccessful. No NOE’s were detected
between oxime OH and any of the ring protons. In
other similar ring systems though, E oxime was reported
as the dominant product.21 Activity of this class
of compounds is shown in Table 3. The following
modifications did not improve the activity of the parent
compound.
Overall, it appears that the exocyclic olefin of fumagillin
can be replaced with other functional groups. The
activity of the compounds may drop slightly, but further
SARs at the C6 position can result in restoration of the
activity. Furthermore, solubility of this class of com-
pounds can be improved by substituting more polar
functional groups at the solvent exposed C6 position.
Experimental
All reactions involving moisture sensitive reagents were
carried out under dry nitrogen in oven dried apparati.
THF was distilled over sodium and benzophenone.
Other solvents were reagent grade quality and from
Aldrich Sureseal bottles and were used as received. The
separations using column chromatography were per-
formed using Merck Silica Gel 230–400 mesh. TLC was
performed with Merck silica gel 60 F254 precoated plates
and the products were visualized with UV and phos-
phomolibdic acid as purchased from Aldrich and dilu-
ted with EtOH.
Conclusions
A series of compounds, 1–6, aimed at understanding the
effect of the exocyclic epoxide of fumagillin were pre-
pared. These compounds carry the cinnamoyl moiety at
C6 and were prepared to compare the effect of modifi-
cations at C4. It was demonstrated that C4 alteration
NMR spectra were acquired using CDCl3 filtered
through basic alumina, unless otherwise noted, using

M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
5055
Figure 4.
300 or 500 MHz Varian instruments. The 13C NMR
spectra were recorded at 75 MHz. Chemical shifts are
reported in ppm with residual CHCl3 (7.26 ppm) and
CDCl3 (77 ppm) as references. Mass spectra were
obtained with a Finnigan MAT LCQ machine under
electrospray ionization conditions.
117.9, 129.7, 134.6, 140.0, 144.9, 153.2, 166.1; EI MS
(m/z) 539.2 [MH+], 561.2 [M+Na].
3-(3,4,5-Trimethoxy-phenyl)-acrylic acid 4-(1,5-dime-
thyl-hexa-1,4-dienyl)-5-methoxy-1-oxa-spiro[2.5] oct-6-yl
ester (3). Under a N2 atmosphere, a mixture of 3,4,5-
trimethoxycinnamic acid (43 mg, 0.18 mmol), EDC
(34.5 mg, 0.18 mmol), and DMAP (22 mg, 0.18 mmol)
was stirred in 1.2 mL of DMF for 10 min. Fumagillol
diene 10b (33 mg, 0.12 mmol) was added to the yellow
solution and the mixture was stirred over night. The
reaction mixture was diluted with EtOAc and the
organic layer was washed with H2O, NH4Cl, and
NaHCO3, dried (MgSO4) and concentrated. Flash
chromatography (30% EtOAc–hexane) yielded 29 mg
(99% based on the recovered starting materials) of pro-
duct as a white solid as well as 18 mg of the starting
3-(3,4,5-Trimethoxy-phenyl)-acrylic acid 5-methoxy-4-[2
-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro
[2.5]oct-6-yl ester (2) and 3-(3,4,5-trimethoxy-phenyl)-
acrylic acid 4-chloromethyl-4-hydroxy-2-methoxy-3-[2-
methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-cyclohexyl ester
(2). Under a N2 atmosphere, a mixture of 3,4,5-tri-
methoxycinnamic acid (84.3 mg, 0.354 mmol), EDC (68
mg, 0.354 mmol), and DMAP (43.2 mg, 0.345 mmol)
was stirred in 3.5 mL of DMF for 10 min. Fumagillol
(100 mg, 0.345 mmol) was added to the yellow solution
and the mixture was stirred over night. The reaction
mixture was diluted with EtOAc and the organic layer
was washed with H2O, NH4Cl, and NaHCO3, dried
(MgSO4) and concentrated. Flash chromatography (30–
40% EtOAc–hexane) yielded 60 mg (34%) of product 1
as well as 8 mg (5%) of the chlorohydrin 2. Data for
1
materials back. H NMR (300 MHz, CDCl3) d 1.05–
1.17 (m, 1H), 1.61 (s, 3H), 1.62 (s, 3H), 1.68 (s, 3H),
1.84–1.98 (m, 1H), 2.03–2.14 (m, 1H), 2.15–2.28 (m,
1H), 2.49 (d, 1H, J=5 Hz), 2.62–2.82 (m, 3H), 2.96 (d,
1H, J=11 Hz), 3.38 (s, 3H), 3.65 (dd, 1H, J=11, 3 Hz),
3.89 (s, 3H), 3.90 (s, 6H), 5.09 (t, 1H, J=7 Hz), 5.26 (t,
1H, J=7 Hz), 5.72 (br s, 1H), 6.42 (d, 1H, J=16 Hz),
6.76 (s, 2H), 7.61 (d, 1H, J=16 Hz); 13C NMR
(75 MHz, CDCl3) d 13.7, 17.5, 25.4, 26.7, 28.5, 29.5,
49.1, 51.2, 56.0, 56.8, 60.4, 60.7, 66.8, 78.8, 105.0, 117.5,
122.8, 129.4, 129.8, 130.9, 131.4, 140.0, 144.6, 153.2,
166.3.
1
compound 1: H NMR (300 MHz, CDCl3) d 1.05–1.17
(m, 1H), 1.24 (s, 3H), 1.66 (s, 3H), 1.75 (s, 3H), 1.87–
1.98 (m, 1H), 2.07 (d, 1H, J=11 Hz), 2.08–2.25 (m, 3H),
2.32–2.44 (m, 1H), 2.57 (d, 1H, J=4 Hz), 2.63 (t, 1H,
J=6 Hz), 3.02 (d, 1H, J=4 Hz), 3.46 (s, 3H), 3.72 (dd,
1H, J=11, 2 Hz), 3.90 (s, 3H), 3.91 (s, 6H), 5.22 (br t,
1H, J=8 Hz), 5.73 (d, 1H, J=2 Hz), 6.42 (d, 1H, J=16
Hz), 6.76 (s, 2H), 7.59 (d, 1H, J=16 Hz); 13C NMR
(75 MHz, CDCl3) d 13.6, 17.8, 25.4, 25.5, 27.1, 29.1,
48.1, 50.7, 55.9, 56.4, 58.4, 59.3, 60.7, 60.9, 60.3, 78.9,
105.0, 117.6, 118.4, 129.7, 134.7, 139.8, 144.5, 153.1,
166.3. Data for compounds 2: 1H NMR (300 MHz,
CDCl3) d 1.07–1.17 (m, 1H), 1.26 (s, 3H), 1.38–1.48 (m,
1H), 1.67 (s, 3H), 1. 74 (s, 3H), 1.85–1.92 (m, 1H), 2.08–
2.13 (m, 2H), 2.41–2.53 (m, 1H), 2.56 (d, 1H, J=11 Hz),
2.00 (t, 1H, J=7 Hz), 3.34 (s, 3H), 3.34–3.42 (m, 1H),
3.52 (d, 1H, J=11 Hz), 3.90 (s, 3H), 3.91 (s, 6H), 3.88–
3.93 (m, 1H), 4.24 (br s, 1H), 5.20 (br t, 1H, J=7 Hz),
5.60 (d, 1H, J=3 Hz), 6.38 (d, 1H, J=16 Hz), 6.77 (s,
2H), 7.64 (d, 1H, J=16 Hz); 13C NMR (75 MHz,
CDCl3) d 13.9, 17.7, 23.3, 25.6, 27.2, 29.5, 43.0, 50.3,
56.0, 56.4, 60.8, 62.1, 65.7, 75.9, 78.4, 105.1, 117.2,
3-(3,4,5-Trimethoxy-phenyl)-acrylic acid 4-chloromethyl-
3-(1,5-dimethyl-hexa-1,4-dienyl)-4-hydroxy-2-methoxy-
cyclohexyl ester (4). A solution of compound 3 (6.0 mg,
0.012 mmol) in 0.3 mL of THF was stirred with 2.1 mg
(0.051 mmol) of LiCl and Acetic acid (2.9 mL, 0.051
mmol) for 1 day when the starting materials had been
completely consumed as judged by TLC. The reaction
mixture was worked up by removal of THF, followed
by addition of EtOAc. The organic layer was washed
with H2O, NH4Cl, and NaHCO3, dried (MgSO4) and
concentrated. Flash chromatography (30% EtOAc–
hexane) yielded 6.5 mg (100%) of the product as a film.
1H NMR (300 MHz, CDCl3) d 1.07–1.17 (m, 1H), 1.34–
1.46 (m, 1H), 1.64 (s, 3H), 1.70 (s, 3H), 1.77 (s, 3H),
1.92-2.05 (m, 2H), 2.63 (d, 1H, J=11 Hz), 2.79 (ddd,
2H, J=7, 7, 8 Hz), 3.37 (s, 3H), 3.45 (d, 1H, J=11 Hz),
3.60–3.79 (m, 1H), 3.86–3.93 (m, 1H), 3.90 (s, 3H), 3.91

5056
M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
(s, 6H), 4.70 (br s, 1H), 5.12 (t, 1H, J=7 Hz), 5.30 (t,
1H, J=7 Hz), 5.65 (br s, 1H), 6.40 (d, 1H, J=16 Hz),
6.76 (s, 2H), 7.61 (d, 1H, J=16 Hz); EI MS (m/z) 523.2
[MH+], 545.2 [M+Na].
3.49 (d, 1H, J=11.1 Hz), 3.80 (d, 1H, J=11.1 Hz), 3.90
(br, 1H), 4.22 (m, 1H), 5.19 (t, 1H, J=6.6 Hz).
Acetic acid 4-chloromethyl-4-hydroxy-2-methoxy-3-[2-
methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-cyclohexyl ester
(8). A solution of 7.75 g (24.3 mmol) of compound 7
and 5.94 g (48.6 mmol) of DMAP in 150 mL of CH2Cl2
was stirred at 0 ꢀC as 3.45 mL (36.5 mmol) of acetic
anhydride was added. After 1 h at 0 ꢀC, the solution was
diluted with water and the product was extracted with
ethyl acetate. The extracts were washed with water and
brine, dried (MgSO4), and concentrated. The product
was purified by column chromatography to obtain 7.85
g (86% from fumagillol) of the title compound. 1H
NMR (300 MHz, CDCl3) d 1.40 (br d, 1H, J=13.2 Hz),
1.50 (s, 3H), 1.67 (s, 3H), 1.75 (s, 3H), 1.75–1.83 (m,
2H), 1.95–2.03 (m, 1H), 2.13 (s, 3H), 2.13–2.20 (m, 1H),
2.41–2.51 (m, 2H), 2.97 (t, 1H, J=6.6 Hz), 3.27 (br,
1H), 3.30 (s, 3H), 3.51 (d, 1H, J=10.8 Hz), 3.86 (d, 1H,
J=10.8 Hz), 4.14 (br, 1H), 5.19 (br t, 1H, J=7.5 Hz),
5.47 (m, 1H).
3-(3,4,5-Trimethoxy-phenyl)-acrylic acid 4-(1-benzyloxy-
imino-ethyl)-5-methoxy-1-oxa-spiro[2.5]oct-6-yl ester (5).
A mixture of 3,4,5-trimethoxycinnamic acid (29.3 mg,
0.123 mmol), EDC (24.0 mg, 0.123 mmol), DMAP (15.0
mg, 0.123 mmol), and 4-benzyloxime fumagillol16 (25.0
mg, 0.082 mmol) in 0.82 mL of DMF was stirred for 1
day. The reaction mixture was diluted with EtOAc and
washed with H2O, NH4Cl, and NaHCO3, dried
(MgSO4) and concentrated. Flash chromatography
(30% EtOAc–hexane) yielded 24.7 mg (85% based on
the recovered starting materials) of the product as a film
as well as 8 mg of the starting material. 1H NMR
(300 MHz, CDCl3) d 1.10–1.28 (m, 1H), 1.86 (s, 3H),
1.94 (br d, 1H, J=13 Hz), 2.05–2.16 (m, 1H), 2.23 (dt,
1H, J=5, 14 Hz), 2.52 (d, 1H, J=4 Hz), 2.56 (d, 1H,
J=4 Hz), 3.29 (d, 1H, J=11 Hz), 3.39 (s, 3H), 3.75 (dd,
1H, J=3, 11 Hz), 3.90 (s, 3H), 3.91 (s, 6H), 5.11 (d, 1H,
J=13 Hz), 5.16 (d, 1H, J=13 Hz), 5.78 (br s, 1H), 6.42
(d, 1H, J=16 Hz), 6.77 (s, 2H), 7.27–7.40 (m, 5H), 7.62
(d, 1H, J=16 Hz).
Acetic acid 4-chloromethyl-3-(1,5-dimethyl-hexa-1,4-die-
nyl)-4-hydroxy-2-methoxy-cyclohexyl ester (9). Under a
N2 atmosphere, to a solution of 7.70 g (19.4 mmol) of
WCl6 in 60 mL of THF at ꢁ78 ꢀC was added 27 mL
(38.8 mmol) of 1.43 M n-BuLi in hexane over 15 min.
After 15 min at ꢁ78 ꢀC, the solution was stirred at rt for
35 min and a solution of 2.26 g (6.26 mmol) of com-
pound 8 in 20 mL of THF was added over 10 min. After
1 h, 1.5 M sodium tartrate (80 mL) and 2.0 M NaOH
(80 mL) were added to the reaction mixture and the
product was extracted with ether. The extracts were
washed with a mixture of 100 mL of 1.5 M sodium tar-
trate and 100 mL of 2 M NaOH, saturated aq NaHCO3,
and water. The residue was purified by column chro-
matography (25% EtOAc–hexane) to yield 1.84 g (86%)
of the product as a clear oil. 1H NMR (300 MHz,
CDCl3) d 1.62 (br s, 1H), 1.63 (s, 3H), 1.70 (s, 3H), 1.73
(s, 3H), 1.68–1.79 (m, 2H), 1.75–1.83 (m, 2H), 2.10 (s,
3H), 2.51 (d, 1H, J=11.1 Hz), 2.68–2.87 (m, 2H), 3.31
(s, 3H), 3.41 (d, 1H, J=11.1 Hz), 3.65 (m, 2H), 5.11 (br
t, 1H, J=7.1 Hz), 5.25 (br t, 1H, J=6.2 Hz), 5.54 (m,
1H).
3-(3,4,5-Trimethoxy-phenyl)-acrylic acid 3-(1-benzylox-
yimino-ethyl)-4-chloromethyl-4-hydroxy -2-methoxy-
cyclohexyl ester (6). A solution of compound 5 (5.3 mg,
0.010 mmol) in 0.10 mL of THF was stirred with 5.0 mg
(0.12 mmol) of LiCl and acetic acid (7.0 mL, 0.12 mmol)
for 1 day when the starting materials had been com-
pletely consumed as judged by TLC. The reaction mix-
ture was worked up by removal of THF, followed by
addition of EtOAc. The organic layer was washed with
H2O, NH4Cl, and NaHCO3, dried (MgSO4) and con-
centrated to give 5.7 mg (100%) of the product as a film.
1H NMR (300 MHz, CDCl3) d 1.17–1.29 (m, 1H), 1.33–
1.45 (m, 1H), 1.90–2.07 (m, 2H), 2.07 (s, 3H), 3.05 (d,
1H, J=11 Hz), 3.16–3.27 (m, 2H), 3.23 (s, 3H), 3.56
(dd, 1H, J=3, 11 Hz), 3.89 (s, 3H), 3.91 (s, 6H), 4.62 (br
s, 1H), 5.08 (d, 1H, J=12 Hz), 5.15 (d, 1H, J=12 Hz),
5.53 (br s, 1H), 6.39 (d, 1H, J=16 Hz), 6.77 (s, 2H),
7.27–1.43 (m, 5H), 7.63 (d, 1H, J=16 Hz); EI MS (m/z)
562.2 [MH+], 584.2 [M+Na].
Acetic acid 4-(1,5-dimethyl-hexa-1,4-dienyl)-5-methoxy-
1-oxa-spiro[2.5]oct-6-yl ester (10). To a stirring solution
of 5.11 g (14.8 mmol) of 9 in THF was added 15.6 mL
(15.6 mmol) of 1 M potassium tert-butoxide solution in
THF at 0 ꢀC. After 1 h, an additional 0.75 mL (0.75
mmol) of 1 M potassium tert-butoxide solution was
added and the resulting solution was stirred for 5 min at
0 ꢀC. The solution was diluted with saturated aq
NaHCO3 and the product was extracted with ethyl ace-
tate. The combined extracts were washed with water
and saturated aq NaCl, dried (MgSO4), and con-
centrated. The product was purified by chromatography
to afford the title compound as a white solid (3.51 g,
1-Chloromethyl-3-methoxy-2-[2-methyl-3-(3-methyl-but-
2-enyl)-oxiranyl]-cyclohexane-1,4-diol (7). A mixture of
7.13 g (25.2 mmol) of fumagillol and 4.40 g (103.7
mmol) of LiCl in 70 mL of THF was stirred at 0 ꢀC, as 7
mL (122.3 mmol) of acetic acid was added. After 10
min, the cold bath was removed and the mixture was
stirred at ambient temperature for 15 h. The reaction
mixture was diluted with water and saturated aq
NaHCO3 and the product was extracted with ethyl ace-
tate. The organic extracts were washed with water, dried
(MgSO4), and concentrated to obtain 7.75 g (96%) of
the crude title compound as an oil. The crude product
1
1
was used in the next reaction without purification. H
77%). H NMR (300 MHz, CDCl3) d 1.15 (ddd, 1H,
NMR (300 MHz, CDCl3) d 1.33 (br d, 1H, J=13.8 Hz),
1.47 (s, 3H), 1.66 (s, 3H), 1.74 (s, 3H), 1.68–1.87 (m,
2H), 2.05–2.23 (m, 3H), 2.32–2.49 (m, 2H), 2.98 (t, 1H,
J=6.0 Hz), 3.26 (br d, 1H, J=10.2 Hz), 3.33 (s, 3H),
J=11.1, 3.9, 3.0 Hz), 1.59 (s, 3H), 1.62 (s, 3H), 1.69 (s,
3H), 1.56–1.75 (m, 1H), 1.79–2.00 (m, 1H), 2.12 (s, 3H),
2.17 (td, 1H, J=13.5, 4.8 Hz), 2.47 (d, 1H, J=5.1 Hz),
2.70 (d, 1H, J=5.1 Hz), 2.63–2.82 (m, 2H), 2.87 (d, 1H,

M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
5057
J=11.1 Hz), 3.36 (s, 3H), 3.58 (dd, 1H, J=11.1, 2.7
Hz), 5.09 (br t, 1H, J=7.1 Hz), 5.23 (br t, 1H, J=7.1
Hz), 5.63 (m, 1H).
1H), 1.72 (s, 3H), 1.82–1.95 (m, 1H), 1.97–2.05 (m, 1H),
2.15 (s, 6H), 2.27–2.37 (m, 2H), 3.08 (br s, 3H), 3.25 (s,
3H), 3.35–3.48 (m, 2H), 3.66 (br d, 1H, J=12 Hz), 5.03
(s, 2H), 5.09 (br s, 1H), 5.30–5.39 (m, 1H), 7.25–7.38 (m,
5H); EI MS (m/z) 420.3 [MH+], 442.3 [M+Na].
4-Methyl-piperazine-1-carboxylic acid 4-(1-benzyloxyi-
mino-ethyl)-5-methoxy-1-oxa-spiro[2.5]oct-6-yl ester
(11). Under a N2 atmosphere, a solution of compound
16 (16.5 mg, 0.035 mmol), 1-methylpiperazine (7.03 mg,
0.070 mmol) and DMAP (1.3 mg, 0.011 mmol) in 0.350
mL of dry CH2Cl2 was stirred for 1.5 h. The reaction
mixture was worked up by diluting with CH2Cl2 and
washing the organic layer with H2O. Removal of the
solvent followed by flash chromatography (0–15%
MeOH–EtOAc) gave 10.4 mg (69%) of the product as a
4-Benzyl-piperazine-1-carboxylic acid 4-(1-benzyloxyi-
mino-ethyl)-5-methoxy-1-oxa-spiro[2.5]oct-6-yl ester
(15). Preparation was same as for compound 11. 1H
NMR (300 MHz, CDCl3) d 1.16 (br d, 1H, J=14 Hz),
1.79–1.93 (m, 1H), 1.83 (s, 3H), 1.97–2.10 (m, 1H), 2.14
(dd, 1H, J=14, 4 Hz), 2.44 (br s, 4H), 2.49 (d, 1H, J=4
Hz), 2.54 (d, 1H, J=4 Hz), 3. 09 (d, 1H, J=11 Hz), 3.38
(s, 3H), 3.53 (br s, 6H), 3.67 (dd, 1H, J=11, 3 Hz), 5.09
(d, 1H, J=12 Hz), 5.15 (d, 1H, J=12 Hz), 5.57 (br s,
1H), 7.25–7.36 (m, 10H); 13C NMR (75 MHz, CDCl3) d
12.2, 25.5, 28.1, 43.6, 46.4, 51.1, 52.5, 56.7, 60.0, 62.8,
66.9, 75.1, 78.3, 127.0, 127.4, 127.5, 128.1, 128.9, 137.5,
138.0, 154.6, 156.3; EI MS (m/z) 508.2 [MH+], 530.2
[M+Na].
1
white solid. H NMR (300 MHz, CDCl3) d 1.13–1.25
(m, 1H), 1.83–1.92 (m, 1H), 1.84 (s, 3H), 1.98–2.19 (m,
2H), 2.13 (s, 3H), 2.40 (br s, 4H), 2.49 (d, 1H, J=4 Hz),
2.55 (d, 1H, J=4 Hz), 3.10 (d, 1H, J=11 Hz), 3.38 (s,
3H), 3.53 (br s, 4H), 3.68 (dd, 1H, J=11, 3 Hz), 5.09 (d,
1H, J=12 Hz), 5.15 (d, 1H, J=12 Hz), 5.58 (br s, 1H),
7.25–7.38 (m, 5H); EI MS (m/z) 432.2 [MH+], 454.2
[M+Na].
Carbonic acid 4-(1-benzyloxyimino-ethyl)-5-methoxy-1-
oxa-spiro[2.5]oct-6-yl ester 4-nitro-phenyl ester (16).
Under a N2 atmosphere, a mixture of 4-benzyloxime
fumagillol (56.6 mg, 0.185 mmol), bis(4-nitrophe-
nyl)carbonate (169 mg, 0.555 mmol) and DMAP (68
mg, 0.555 mmol) in 0.10 mL of dry CH2Cl2 was stirred
for 6 h. The reaction mixture was diluted with CH2Cl2
and washed with H2O. The organic layer was dried
(MgSO4) and concentrated. Flash chromatography (10–
25% EtOAc–hexane) yielded 75.3 mg (87%) of the
product as a white foam. 1H NMR (300 MHz, CDCl3) d
1.20–1.30 (m, 1H), 1.86 (s, 3H), 1.91–2.05 (m, 1H),
2.11–2.26 (m, 2H), 2.53 (s, 2H), 3.20 (d, 1H, J=11 Hz),
3.42 (s, 3H), 3. 76 (dd, 1H, J=3, 11 Hz), 5.11 (d, 1H,
J=12 Hz), 5.16 (d, 1H, J=12 Hz), 5.61 (br s, 1H), 7.26–
7.42 (m, 7H), 8.28 (d, 1H, J=7 Hz), 8.29 (d, 1H, J=7
Hz).
4-Methyl-piperidine-1-carboxylic acid 4-(1-benzyloxyi-
mino-ethyl)-5-methoxy-1-oxa-spiro[2.5]oct-6-yl ester
(12). Preparation was same as for compound 11. 1H
NMR (300 MHz, CDCl3) d 0.95 (d, 3H, J=6 Hz), 1.16
(br d, 1H, J=14 Hz), 1.02–1.12 (m, 2H), 1.46–1.70 (m,
3H), 1.79–1.92 (m, 1H), 1.84 (s, 3H), 1.98–2.08 (m, 1H),
2.15 (dt, 1H, J=5, 14 Hz), 2.49 (d, 1H, J=4 Hz), 2.56
(d, 1H, J=4 Hz), 2.67–2.91 (m, 2H), 3.12 (d, 1H, J=11
Hz), 3.38 (s, 3H), 3.67 (dd, 1H, J=3, 11 Hz), 4.04–4.20
(m, 2H), 5.09 (d, 1H, J=12 Hz), 5.15 (d, 1H, J=12 Hz),
5.58 (br s, 1H), 7.26–7.37 (m, 5H); 13C NMR (75 MHz,
CDCl3) d 12.2, 21.6, 25.6, 28.1, 30.7, 33.6, 44.1, 46.4,
51.1, 56.7, 60.0, 66.6, 75.1, 78.4, 127.4, 127.4, 128.1,
138.0, 156.5; EI MS (m/z) 431.3 [MH+], 453.3 [M+Na].
4-Ethyl-piperazine-1-carboxylic acid 4-(1-benzyloxyi-
mino-ethyl)-5-methoxy-1-oxa-spiro[2.5]oct-6-yl ester
(13). Preparation was same as for compound 11. 1H
NMR (300 MHz, CDCl3) d 1.01 (t, 3H, J=7.4 Hz), 1.17
(br d, 1H, J=15.3 Hz), 1.84 (s, 3H), 1.80–1.92 (m, 1H),
2.00–2.09 (m, 1H), 2.13 (td, 1H, J=13.8, 1.8 Hz), 2.40–
2.48 (m, 6H), 2.49 (d, 1H, J=4.2 Hz), 2.56 (d, 1H,
J=4.2 Hz), 3.11 (d, 1H, J=11.1 Hz), 3.38 (s, 3H), 3.53
(m, 4H), 3.68 (dd, 1H, J=11.1, 2.7 Hz), 5.12 (ABqt, 2H,
J=12.0 Hz), 5.58 (br, 1H), 7.29–7.35 (m, 5H); MS (ESI)
446.2 (M+H).
5-Methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxira-
nyl]-1-oxa-spiro[2.5]octan-6-one (17).
A mixture of
fumagillol (37 mg, 0.13 mmol), pyridinium chloro-
chromate (198 mg, 0.917 mmol) and pyridine (0.36 mL,
4.5 mmol) in 2.6 mL dry CH2Cl2 was stirred for 8 h at
room temperature when all of the starting materials
disappeared on TLC. The reaction mixture was loaded
onto a silica gel column and the product was eluted with
0–30% EtOAc–CH2Cl2 to give 25.8 mg (70%) of the
1
product as a clear oil. H NMR (300 MHz, CDCl3) d
1.29 (s, 3H), 1.66 (s, 3H), 1.66–1.75 (m, 1H), 1.75 (s,
3H), 1.88 (d, 1H, J=11 Hz), 2.00–2.22 (m, 2H), 2.40
(ddd, 1H, J=6.5, 7.5, 13.0 Hz), 2.52 (ddd, 1H, J=14.1,
5.4, 3.9), 2.61 (t, 1H, J=6.5 Hz), 2.63–2.72 (m, 1H),
2.73 (d, 1H, J=4.5 Hz), 3.06 (d, 1H, J=4.5 Hz), 3.51 (s,
3H), 4.09 (d, 1H, J=11 Hz), 5.19 (br t, 1H, J=7.5 Hz);
13C NMR (75 MHz, CDCl3) d 13.7, 17.8, 25.5, 27.2,
33.0, 36.6, 51.6, 53.4, 58.4, 58.4, 60.3, 83.0, 118.0, 138.0,
206.9; EI MS (m/z) 281.2 [MH+], 303.2 [M+Na].
(2-Dimethylamino-ethyl)-carbamic acid 4-(1-benzyloxyi-
mino-ethyl)-5-methoxy-1-oxa-spiro[2.5]oct-6-yl ester
(14). Under a N2 atmosphere, a solution of compound
16 (19.3 mg, 0.041 mmol), N,N-dimethylethylenedia-
mine (6.8 mL, 0.062 mmol) and DMAP (1.0 mg, 0.008
mmol) in 0.40 mL of dry CH2Cl2 was stirred for 1.5 h.
The reaction mixture was worked up by diluting with
CH2Cl2 and washing the organic layer with H2O.
Removal of the solvent followed by flash chromato-
graphy (0–1% TEA–EtOAc followed by a second col-
umn using 0–10% MeOH–CH2Cl2) gave 11.2 mg (66%)
4-Chloromethyl-4-hydroxy-2-methoxy-3-[2-methyl-3-(3-
methyl-but-2-enyl)-oxiranyl]-cyclohexanone (18). During
the large-scale synthesis of compound 17 starting from
fumagillol (104.7 mg, 0.371 mmol) using the procedure
1
of the product as a white solid. H NMR (300 MHz,
80 ꢀC, DMSO-d6) d 1.09–1.20 (m, 1H), 1.70–1.78 (m,

5058
M. Fardis et al. / Bioorg. Med. Chem. 11 (2003) 5051–5058
described above, a small amount (7.8 mg, 7%) of the
above compound was also obtained in addition to 68%
of 17. Data for 18: H NMR (300 MHz, CDCl3) d 1.50
19.3, 25.5, 27.1, 28.8, 53.7, 53.9, 56.0, 56.6, 59.6, 62.6,
78.9, 118.2, 134.4, 156.0; EI MS (m/z) 296.2 [MH+],
318.3 [M+Na].
1
(s, 3H), 1.68 (s, 3H), 1.76 (s, 3H), 1.89 (ddd, 1H, J=14,
6, 2 Hz), 2.05–2.14 (m, 1H), 2.14–2.23 (m, 1H), 2.28
(ddd, 1H, J=2, 13, 5 Hz), 2.47 (d, 1H, J=12 Hz), 2.42–
2.55 (m, 1H), 2.78 (dt, 1H, J=6, 13 Hz), 3.06 (t, 1H,
J=6 Hz), 3.42 (s, 3H), 3.55 (d, 1H, J=11 Hz), 3.84 (d,
1H, J=12 Hz), 3.91 (d, 1H, J=11 Hz), 4.31 (br s, 1H),
5.19 (br t, 1H, J=7 Hz); 13C NMR (75 MHz, CDCl3) d
17.9, 21.5, 25.8, 27.3, 29.7, 35.1, 35.3, 49.2, 50.3, 59.1,
62.5, 75.7, 82.3, 117.8, 135.1, 208.4; EI MS (m/z) 317.4
[MH+], 339.4 [M+Na].
Acknowledgements
The authors would like to thank Professor Jun Liu for
helpful advice. We would like to thank Cheng Y. Yang
for his work on the stability of the fumagillin analogues.
References and Notes
4 - (1- Benzyloxyimino - ethyl) - 5 - methoxy - 1- oxa - spir-
o[2.5]octan-6-one (19). Preparation was same as for
compound 17 from C-4 benzyloxime fumagillol. 1H
NMR (300 MHz, CDCl3) d 1.76 (ddd, 1H, J=6, 6, 12
Hz), 1.95–2.02 (m, 1H), 1.90 (s, 3H), 2.51–2.63 (m, 2H),
2.70 (s, 2H), 2.92 (d, 1H, J=9 Hz), 3.39 (s, 3H), 4.06 (d,
1H, J=9 Hz), 5.08 (s, 2H), 2.26–7.39 (m, 5H); EI MS
(m/z) 304.3 [MH+], 326.2 [M+Na].
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3-(1-Benzyloxyimino-ethyl)-4-chloromethyl-4-hydroxy-2-
methoxy-cyclohexanone (20). A solution of 4-benzy-
loxime fumaginone 19 (10.2 mg, 0.034 mmol) in 0.33
mL of THF was stirred with 17 mg (0.41 mmol) of
LiCl and HOAc (23 mL, 0.41 mmol) for 1 day at
room temperature when the starting materials was
completely consumed. The reaction mixture was
worked up by diluting with CH2Cl2 and washing the
organic layer with H2O. Removal of the solvent fol-
lowed by flash chromatography (25% EtOAc–hexane)
gave 11.5 mg (100%) of the product as a film. 1H
NMR (300 MHz, CDCl3) d 1.87 (ddd, 1H, J=2, 6,
14 Hz), 2.04 (s, 3H), 2.05–2.11 (m, 1H), 2.31 (ddd, 1H,
J=2, 4, 14 Hz), 2.84 (ddd, 1H, J=6, 14, 14), 2.93 (d,
1H, J=12 Hz), 3.16 (d, 1H, J=11 Hz), 3.20 (d, 1H,
J=11 Hz), 3.32 (s, 3H), 4.02 (d, 1H, J=12 Hz), 4.84 (d,
1H, J=2 Hz), 5.10 (d, 1H, J=12 Hz), 5.17 (d, 1H,
J=12 Hz), 7.26–7.42 (m, 5H); 13C NMR (75 MHz,
CDCl3) d 18.6, 34.0, 35.4, 48.6, 53.3, 59.8, 74.4, 76.0,
85.3, 128.0, 128.1, 128.6, 137.7, 160.6, 207.7; EI MS (m/
z) 346.3 [M+Li].
6. Corey, E. J.; Guzman-Perez, A.; Noe, M. C. J. Am. Chem.
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5-Methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxira-
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aqueous NH2OH and 13 mL of AcOH for 2 h. The
reaction mixture was worked up by diluting with EtOAc
and washing the organic layer with H2O. Removal of
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1
oil. H NMR (300 MHz, CDCl3) d 1.32 (s, 3H), 1.64 (s,
3H), 1.73 (s, 3H), 1.72–1.76 (m, 1H), 1.80–1.88 (m,
2H), 2.17 (ddd, 1H, J=7, 7, 14 Hz), 2.32 (ddd, 1H,
J=7, 7, 14 Hz), 2.43 (dt, 1H, J=17, 6 Hz), 2.66 (d,
1H, J=5 Hz), 2.73-2.79 (m, 2H), 3.02 (dt, 1H, J=17, 6
Hz), 3.28 (s, 3H), 4.04 (d, 1H, J=3 Hz), 5.18 (br t, 1H,
J=7 Hz); 13C NMR (75 MHz, CDCl3) d 15.6, 17.8,