Stereoselective synthesis of E-64 and related cysteine proteases inhibitors from 2,3-epoxyamides

Article abstract of DOI:10.1016/j.bmc.2004.12.018

The stereoselective synthesis of cathepsin inhibitors from indoline type epoxyamides is described. The use of this type of epoxyamides permitted the preparation of E-64 and CA-074 related compounds depending on the order in which the key steps, the oxidat

Full text of DOI:10.1016/j.bmc.2004.12.018

Bioorganic & Medicinal Chemistry 13 (2005) 1691–1705
Stereoselective synthesis of E-64 and related cysteine
proteases inhibitors from 2,3-epoxyamides
*
Francisco Sarabia, Antonio Sanchez-Ruiz and Samy Chammaa
´
´ ´ ´
Departamento de Bioquımica, Biologıa Molecular y Quımica Organica, Facultad de Ciencias, Universidad de Malaga,
´
´
29071 Malaga, Spain
´
Received 28 September 2004;revised 2 December 2004;accepted 6 December 2004
Available online 12 January 2005
Abstract—The stereoselective synthesis of cathepsin inhibitors from indoline type epoxyamides is described. The use of this type of
epoxyamides permitted the preparation of E-64 and CA-074 related compounds depending on the order in which the key steps, the
oxidation of indoline amides to indole amides and oxidative acetal cleavage were undertaken. By taking advantage of the facile sub-
stitution of the indole of the corresponding indole epoxyamides, with various nucleophiles, we were able to prepare different epox-
ysuccinic acids derivatives as potential cathepsin inhibitors.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors, with more potency and selectivity, and conse-
quently potential therapeutical applications.4a–n In fact,
E-64 (1) represents one of the compounds with the most
complete and extensive structure–activity relationship
studies, including rational design based on computa-
tional studies in pharmaceutical research. Associated
with these studies, several syntheses of E-64, as well as
analogues thereof, have been described in the literature.5
In contrast to the nonselective inhibition displayed by E-
64 against cathepsins, CA-074 (8) and related com-
pounds display specific inhibition against cathepsin B.6
The biological activity displayed by certain natural and
synthetic compounds against cysteine-proteases, a large
family of enzymes involved in the degradation of pro-
teins, is due to the alkylation of the Cys-25 residue, an
essential amino acid for their proteolytic action. Among
the various electrophilic groups present in cysteine pro-
tease inhibitors, the oxirane ring represents one of the
most common and efficient groups. Thus, compounds
such as the natural E-64 (1)1 and synthetic related prod-
ucts 2–6,2 as well as the designed compounds CA-074
(8), CA-030 (9) and CA-028 (10)3 lead the list of these
representative bioactive compounds (Figure 1). Particu-
larly striking are the IC50 values for E-64c (2), CA-074
(8) and CA-030 (9) against cathepsin B of 3.36, 2.24
and 2.28 nM, respectively, or the value of E-64c (2)
against cathepsin L of 0.09 nM. The recognition of E-
64 (1) as an inhibitor of the cathepsins, proteases
involved in degenerative diseases, including muscular
distrophy, rheumatoid arthritis and metastasis, made it
one of the most attractive targets in the pharmaceutical
industry. The discovery prompted intense research
activity directed towards the design of novel cathepsin
The stereoselective synthesis of epoxyamides via reac-
tion of chiral aldehydes with stabilized sulfur ylides
has occupied a central position in our research during
the last years,7 finding interesting synthetic applications
in the field of carbohydrates.8 In particular, the high ste-
reofacial selectivity showed by 2,3-O-isopropylidene-D-
glyceraldehyde 12 in its reactions with sulfur ylides pre-
sented an intriguing synthetic value with numerous
applications in the synthesis of natural products.9 In
fact, we envisioned the corresponding epoxyamide 11
as a useful and common precursor for E-64 (1), CA-
074 (8) and related compounds. The reason for using
the indoline derived amide is justified by its facile oxida-
tion to the corresponding indole,10 which is amenable to
the attack of nucleophiles as a mild method of amide
hydrolysis.11 This is in contrast to conventional amides,
which require harsh conditions for hydrolysis, which
could affect the integrity of the oxirane ring contained
Keywords: E-64;CA-074;Cysteine proteases inhibitors;Cathepsins;
Sulfur ylides;Epoxyamides;Stereoselective synthesis.
*
Corresponding author. Tel.: +34 952134258;fax: +34 952131941;
e-mail: frsarabia@uma.es
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.018

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F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
Scheme 1. General approach to the synthesis of E-64 (1), CA-074 (8)
and related compounds from a common intermediate, epoxyamide
(11).
Figure 1. Structures of E-64 (1), CA-074 (8) and related cathepsin
inhibitors.
all the targeted epoxy peptides depicted in Figure 1.
Thus, 11 was prepared by reaction of aldehyde 12 with
the sulfur ylide generated in situ from the sulfonium salt
13 in a 62% yield and high stereoselectivity of 90:10. Fol-
lowing the synthetic route towards E-64 (1), epoxyamide
11 was treated with Amberlyst-15, to obtain diol 14.
This compound was then subjected to the action of
in these molecules. In addition, compound 11 represents
an excellent candidate for the synthesis of a large variety
of analogues of the targeted epoxy-peptides. The possi-
bility of changing the order of the key steps, acetal
hydrolysis and oxidative cleavage of the resulting diol
(strategy 1), or indoline oxidation and hydrolysis (strat-
egy 2), would permit the synthesis of these bioactive
compound E-64 (a and b) or CA-074 (b and a) as well
as related compounds (Scheme 1).
12
NaIO4/SiO2 and the resulting aldehyde 15 was treated
with Oxoneꢁ,13 to afford acid 16 in a 59% overall yield.
The synthesis of the peptidic fragment that contains the
guanidino group was achieved by starting from 1,4-
butanediamine 17, which was monoprotected with the
di-Boc guanidine moiety 1814 to furnish 19. This deriva-
tive was coupled with the Z protected L-leucine deriva-
tive 20, to obtain amide 21. Deprotection of the Z
amino group contained in 21 was done by catalytic
hydrogenation with ammonium formate,15 to obtain
compound 22. In a similar way, the Z-peptide derivative
26 was prepared starting with the monoprotection of 17
with Z–Cl to obtain 23,16 coupling with the Boc-deriva-
tive of L-leucine 24 and final Boc cleavage with trifluoro-
acetic acid (Scheme 2).
The present paper reports the results obtained in pursuit
of the synthesis of these interesting compounds utilizing
sulfur ylide chemistry for the stereoselective synthesis of
the epoxides contained in these cysteine-protease inhib-
itors. Comparatively, our synthetic strategy offers some
advantages with respect to other syntheses described in
the literature, many of which are based on the use of
(+)- or (ꢀ)-diethyl tartrates as starting materials. Thus,
our methodology permits a selective construction of
both types of cysteine proteases inhibitors from a com-
mon precursor, compound 11, taking the advantage of
the asymmetric architecture contained in this com-
pound. In addition, 11 represents an excellent building
block for the preparation of a wide variety of potential
epoxysuccinyl-type cysteine proteases inhibitors in terms
of structural diversity.
With the key fragments 16, 22 and 26 in hand, we pro-
ceeded with the coupling as described in Scheme 3.
Thus, coupling was achieved by the action of the cou-
pling reagent BOP. Other coupling reagents such as
HBTU or the more conventional DCC or EDCI in the
presence of HOBt furnished either decomposition or
poor yields of the resulting epoxyamides 27 and 28, in
contrast to the above mentioned BOP method,17 which
provided 27 and 28, in yields of 58% and 62%, respec-
tively. The advanced precursor for E-64 (1), compound
2. Results and discussion
According to the general synthetic scheme, epoxyamide
11 represented the key compound for the preparation of

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1693
Scheme 3. Reagents and conditions: (a) 1.5 equiv 22, 1.0 equiv
DIPEA, 1.0 equiv BOP, DMF, 25 ꢂC, 8 h, 58% for 27;1.7 equiv 26,
2.0 equiv DIPEA, 1.2 equiv BOP, DCM, 25 ꢂC, 8 h, 62% for 28. (b)
See text for conditions.
reaction. For this reason, different conditions of temper-
ature (25 ꢂC, reflux), solvents (benzene, toluene, methyl-
ene chloride, tetrahydrofuran) and reagents (p-
chloroanil,11a TPAP, TEMPO-Oxone, CAN, ammo-
nium molibdate, among others) were investigated,
unfortunately with similar disappointing results or with
the recovery of starting material in the cases when mild
conditions were used. Therefore, after extensive experi-
mentation with this reaction without obtaining the de-
sired indole 29, it was clear that the transformation of
the indoline amide to the corresponding indole might
be undertaken earlier in the synthetic sequence, prior
to the incorporation of the guanidino group. However,
when 28 was subjected to the oxidation with DDQ in
benzene, no reaction occurred, with only starting mate-
rial being recovered. Similar observations were obtained
in methylene chloride, THF and toluene.
Scheme 2. Reagents and conditions: (a) 1.0 equiv 13, 1.0 equiv NaOH,
CH2Cl2/H2O, 0 ꢂC, 2 h, 62%. (b) Amberlyst-15, MeOH, reflux, 1 h,
91%. (c) 1.5 equiv NaIO4 supported on SiO2, CH2Cl2, 25 ꢂC, 2 h, 86%.
(d) 1.0 equiv of Oxone, 25 ꢂC, DMF, 1 h, 75%. (e) 0.77 equiv 18, THF/
H2O, 50 ꢂC, 1 h, 99% for 19. (f) 1.5 equiv Z–Cl, 2.0 equiv p-TsOH,
H2O/EtOH, 25 ꢂC, 1 h, 40% for 23. (g) 1.6 equiv of Z-Leu-H 20,
1.4 equiv EDCI, CH2Cl2, 25 ꢂC, 8 h, 72% for 21 overall for two steps.
(h) 1.5 equiv of 24, 1.5 equiv EDCI, CH2Cl2, 25 ꢂC, 8 h, 65% for 25. (i)
20.0 equiv NH4HCO2, Pd/C, EtOH, 25 ꢂC, 1.5 h, 75% for 22. (j)
30.0 equiv TFA, CH2Cl2, 25 ꢂC, 99%. BOP = (benzotriazol-1-yloxy)
tris-(dimethylamino)phosphonium hexafluoro-phosphate. DIPEA =
diisopropylethylamine.
In the light of these discouraging results, we decided to
achieve this oxidation with the starting epoxyamide 11
to obtain the corresponding indole 31 by treatment with
DDQ. We expected that the resulting indole amide
would be stable enough to permit the subsequent syn-
thetic steps to be carried out, enabling us to carry this
functional group through until the end of the synthesis.
Thus, we proceeded with the same synthetic pathway as
described in Schemes 2 and 3 for the indoline derivatives
to obtain in good yield the desired E-64 derivative 29
through the intermediates diol 32, aldehyde 33 and acid
34. Finally, the indole hydrolysis by treatment with lith-
ium hydroxide, followed by Boc cleavage, furnished E-
64 (1) as the trifluoroacetate salt, whose physical and
27, was then subjected to oxidation with DDQ in order
to obtain the readily hydrolyzed indole 29, which was to
be submitted to the sequential treatment of LiOH, fol-
lowed by TFA, to afford the targeted E-64. However,
the oxidation of 27 with DDQ was completely unsuc-
cessful, producing decomposition of the indoline amide
27. This discouraging result was attributed to the chem-
ical susceptibility of the Boc-groups and oxirane ring to
the resulting phenols produced during the oxidation

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F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
spectroscopic properties were identical to a sample of
natural compound.1,18 In a similar way, a series of E-
64 analogues were prepared by reaction of the indole
epoxyamide 29 with various amines (allylamine, benzyl-
amine and n-propylamine) to obtain the corresponding
N-allyl, N-benzyl and N-n-propylamides 36, 37 and 38,
respectively, which were transformed into the trifluorac-
etate salts 39, 40 and 41 after the N-Boc protecting
groups cleavage by treatment with TFA (Scheme 4).
ent nucleophiles including hydroxyl anion, ethoxide,
allylamine, propylamine, isoamylamine or ammonia, to
afford the compounds 2–7, respectively, which represent
some of the most active cathepsin inhibitors described
to date.2 In addition, the reaction of 45 with more compli-
cated nucleophiles offers the opportunity of constructing
a wide variety of novel and more complex cathepsin B
inhibitors related to E-64c (2) (Scheme 5).
Finally, the preparation of the epoxy-peptide CA-074 (8)
was accomplished following the synthetic pathway out-
lined in Scheme 1, using the epoxyamide 11 as the source
of the enantiomerically pure oxirane ring contained in all
these products. In this series of compounds, however, it
was necessary to reverse the order of the two key steps,
oxidation of the indoline amide to indole, followed by
hydrolysis of the indole to the acid and then coupling
to the peptidic residue. Finally, elaboration of the acetal
The synthesis of the E-64c related compounds required the
preparation of the isoamyl amide of L-leucine, derivative
42, which after treatment with TFA to yield the deprotec-
ted amine derivative 43 was reacted with epoxy-acid 16 in
the presence of BOP to obtain the indoline epoxyamide
44 in 60% yield. In this case, the oxidation to the indole
45 was carried out without difficulty by reaction with
DDQ in refluxing benzene. The resulting indole 45 repre-
sents a key common precursor for the preparation of a
familyofE-64crelatedcompoundsbyreactionwithdiffer-
Scheme 5. Reagents and conditions: (a) 1.5 equiv isoamylamine,
1.2 equiv EDCI, CH2Cl2, 25 ꢂC, 4 h, 75%. (b) 22.0 equiv TFA,
CH2Cl2, 25 ꢂC, 40 min, 95%. (c) 1.0 equiv 16, 1.2 equiv 43, 1.2 equiv
BOP, 1.1 equiv DIPEA, DMF, 25 ꢂC, 1 h, 60%. (d) 2.0 equiv DDQ,
C6H6, 80 ꢂC, 72 h, 77%. (e) i. 2.0 equiv LiOH, THF/H2O, 0 ꢂC, 0.5 h,
82% for 2;ii. 2.2 equiv allylamine, CH 2Cl2, 25 ꢂC, 1 h, 99% for 4;iii.
1.1 equiv propylamine, CH2Cl2, 25 ꢂC, 1.5 h, 99% for 5;iv. 1.1 equiv
Scheme 4. Reagents and conditions: (a) 2.0 equiv DDQ, C6H6, 80 ꢂC,
8 h, 92%. (b) Amberlyst-15, MeOH, reflux, 1 h, 95%. (c) 1.5 equiv
NaIO4 supported on SiO2, CH2Cl2, 25 ꢂC, 1 h, 90%. (d) 1.2 equiv of
Oxone, 25 ꢂC, DMF, 1 h, 75%. (e) 2.0 equiv 34, 1.0 equiv 22, 1.0 equiv
DIPEA, 1.0 equiv BOP, DMF, 25 ꢂC, 1 h, 51%. (f) i. 2.0 equiv LiOH,
THF/H2O, 0 ꢂC, 5.0 min, 99% for 35;ii. 2.2 equiv amine, 0.1 equiv
LiCN, CH2Cl2, 25 ꢂC, 1 h, 49% for 36;76% for 37;59% for 38. (g)
22.0 equiv TFA, neat, 25 ꢂC, 1 h, quantitatives for E-64 (1), 39, 40 and
41. DDQ = 2,3-dichloro-5,6-dicyanobenzoquinone.
isoamylamine, CH2Cl2, 35 ꢂC, 8 h, 99% for 6;v. 1.1 equiv NH
,
3(aq)
CH2Cl2, 25 ꢂC, 8 h, 99% for 7. (f) 1.2 equiv EtOH, 1.5 equiv EDCI,
1.5 equiv 4-DMAP, DMF, 25 ꢂC, 4 h, 30% for 3.

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1695
side chain would lead to the desired compound 8. Thus,
acid 4619 was obtained in good yields from the indole 31.
Simultaneously, dipeptide 49 was prepared by coupling
of the L-proline derivative 48 with the Boc L-isoleucine
derivative, promoted by EDCI, followed by Boc depro-
tection. The peptidic bond formation between the acid
46 with the dipeptide 50 was achieved by treatment with
the coupling reagent BOP, to obtain 51 in a 75% yield.
This coupling product was then subjected to the action
of p-toluenesulfonic acid, followed by NaIO4/SiO2 treat-
ment to obtain aldehyde 53, through the diol 52. After
various attempts towards the oxidation of 53 to the acid
54 by methods such as PDC in DMF, the best results
were obtained with m-CPBA,20 providing the acid 54 in
73% yield after purification by flash column chromato-
graphy. With the acid 54 prepared, the reaction with n-
propylamine in the presence of BOP provided amide
55. Finally, the oxidation of indoline amide 55 to the in-
dole 56 with DDQ was achieved, followed by hydrolysis
with lithium hydroxide in THF/H2O to provide the
cathepsin B inhibitor CA-074 (8)21 (Schemes 6 and 7).
This synthetic strategy could be similarly applied for
the synthesis of other CA-074 (8) related compounds
such as CA-030 (9) and CA-028 (10).
3. Conclusions
The stereoselective synthesis of epoxides and their subse-
quent ring opening constitute a powerful synthetic tool
for the preparation of a diverse set of interesting bioac-
tive compounds. In the present paper we have described
the preparation of natural and synthetic cysteine prote-
ases inhibitors, preserving the integrity of the oxirane
ring, which was diastereoselectively prepared via the
reaction of amide-stabilized sulfur ylides with chiral
aldehydes. An interesting contribution is also the use
of epoxyamides derived from indoline as precursors to
the corresponding indoles, which represents a suitable
functional group for (1) the facile transformation to
the corresponding acid, and (2) the facile nucleophilic
displacement by various nucleophiles with the potential
for constructing a library of new and potentially biolog-
ically active inhibitors with applications in medicine.
Finally, the work serves as a basis for the future prepa-
ration of a library using the epoxyamide 11 as a
template. The extension of this chemistry to the solid
phase and the library design of inhibitors are areas that
we are currently investigating.
Scheme 6. Reagents and conditions: (a) 2.0 equiv LiOH, THF/H2O,
0 ꢂC, 10 min, 75%. (b) 65.0 equiv TFA, CH2Cl2, 25 ꢂC, 0.5 h, 85%. (c)
1.0 equiv 48, 1.5 equiv Boc-Ile-H, 1.5 equiv EDCI, 0.85 equiv HOBt,
CH2Cl2, 25 ꢂC, 8 h, 75%. (d) 130.0 equiv TFA, CH2Cl2, 25 ꢂC, 0.5 h,
94%. (e) 1.0 equiv 46, 1.1 equiv 50, 1.2 equiv BOP, 1.0 equiv DIPEA,
DMF, 25 ꢂC, 8 h, 75%. (f) 1.0 equiv TsOH, MeOH, 25 ꢂC, 8 h, 75%.
(g) 2.6 equiv NaIO4 on SiO2, CH2Cl2, 25 ꢂC, 1 h, 99%. (h) 3.0 equiv m-
CPBA, THF, 25 ꢂC, 3 days, 73%. (i) 1.2 equiv n-PrNH2, 1.2 equiv
BOP, 1.2 equiv DIPEA, DMF, 25 ꢂC, 8 h, 90%.
otherwise stated. All solutions used in workup proce-
dures were saturated unless otherwise noted. All reagents
were purchased at highest commercial quality and used
without further purification unless otherwise stated.
All reactions were monitored by thin-layer chromato-
graphy carried out on 0.25 mm E. Merck silica gel plates
(60F-254) using UV light as visualizing agent and 7%
ethanolic phosphomolybdic acid or p-anisaldehyde solu-
tion and heat as developing agents. E. Merck silica gel
(60, particle size 0.040–0.063 mm) was used for flash col-
umn chromatography. Preparative thin-layer chroma-
tography (PTLC) separations were carried out on 0.25,
0.50 or 1 mm E. Merck silica gel plates (60F-254).
4. Experimental
4.1. General techniques
All reactions were carried out under an argon atmo-
sphere with dry, freshly distilled solvents under anhy-
drous conditions, unless otherwise noted. Tetra-
hydrofuran (THF) and ethyl ether (ether) were distilled
from sodium-benzophenone, and methylene chloride
(CH2Cl2), benzene (PhH), and toluene from calcium hy-
dride. Yields refer to chromatographically and spectro-
scopically (1H NMR) homogeneous materials, unless
NMR spectra were recorded on a Bruker Avance-400
instrument and calibrated using residual undeuterated
solvent as an internal reference. The following

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F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
reduced pressure. The resulting crude was purified by
flash column chromatography (silica gel, 40% AcOEt
in hexanes) to obtain pure epoxyamide 11 (2.3 g, 62%)
as a yellow solid: Rf = 0.38 (silica gel, 40% AcOEt in
hexanes);[ a]D ꢀ3.42 (c 0.39, CH2Cl2); 1H NMR
(CDCl3, 400 MHz) d 1.36 (s, 3H, C(CH3)2), 1.44 (s,
3H, C(CH3)2), 3.23–3.27 (m, 2H, CH2CH2N–), 3.31
(dd, J = 2.2, 5.9 Hz, 1H, CH(O)CH), 3.61 (d,
J = 2.2 Hz, 1H, CH(O)CH), 3.95–4.02 (m, 2H), 4.16–
4.32 (m, 3H), 7.02–7.06 (m, 1H, Ph), 7.18–7.21 (m,
2H, Ph), 8.17 (d, J = 8.1 Hz, 1H, Ph); 13C NMR
(CDCl3, 100 MHz) d 142.4, 130.9, 127.7, 124.6, 124.5,
117.3, 110.2, 75.1, 67.2, 58.1, 52.7, 47.2, 28.2, 26.6,
25.1. MS: 290 (100, M+H+), 289 (73;M +), 232 (93),
190 (31), 146 (53), 119 (60), 101 (37), 59 (95);FAB
HRMS m/e 289.1310, M+ calcd for C16H19NO4
289.1314.
4.4. Diol 14. Hydrolysis of epoxyamide 11
A
solution of epoxyamide 11 (1.0 g, 3.46 mmol,
1.0 equiv) in MeOH (30 mL) was treated with Amber-
lyst-15 (3 g) in large excess and refluxed for 1 h until
depletion of starting acetal (TLC with AcOEt 100%).
Then, the suspension was filtered through a Celite pad
and the solvent evaporated, yielding diol 14 (788 mg,
91%) of crude product, as a brown foamy solid, that
was used directly in the next step without further
purification.
Scheme 7. Reagents and conditions: (a) 4.0 equiv DDQ, C6H6, 80 ꢂC,
16 h, 88%. (b) 4.0 equiv LiOH, THF/H2O, 0 ꢂC, 2 h, 60%.
abbreviations were used to explain the multiplicities: s,
singlet;d, doublet;t, triplet;q, quartet;m, multiplet;
band, several overlapping signals;b, broad. Optical
rotations were recorded on a Perkin–Elmer 241 polarim-
eter. High resolution mass spectra (HRMS) were re-
corded on a Kratos MS 80 RFA mass spectrometer
under fast atom bombardment (FAB) conditions.
4.5. Aldehyde 15. Treatment of diol 14 with silica
supported sodium periodate
To a suspension of silica gel (6.4 g) in DCM (50 mL) was
added an aqueous solution of NaIO4 (6.4 mL, 0.65 M,
4.16 mmol, 1.5 equiv) dropwise to form a flaky suspen-
sion. Then, a solution of diol 14 (788 mg, 2.72 mmol,
1.0 equiv) in DCM (6.4 mL) was added. The reaction
was monitored by TLC (silica gel, AcOEt) and after
completion (ca. 1 h), the suspension was filtered through
a fritted glass funnel, and the solid was washed twice
with DCM. The filtrate was evaporated under reduced
pressure and the resulting crude corresponded to alde-
hyde 15 (500 mg, 86%), practically pure by NMR, not
requiring further purification: Rf = 0.50 (silica gel,
4.2. Sulfonium salt 13. Treatment of indoline 2-chloro-
acetamide with methyl sulfide
A suspension of indoline 2-chloroacetamide (5.5 g,
28.13 mmol, 1.0 equiv) in dimethyl sulfide (30 mL) was
heated at 60 ꢂC for 1 week in a pressure sealed-tube.
After that time, the mixture was filtered and the white
solid washed with acetone, to obtain sulfonium salt 12
(5.1 g, 75%) as a white solid. 1H NMR (CDCl3,
1
200 MHz)
d
3.14 (s, 6H, SMe2), 3.19 (m, 2H,
AcOEt); H NMR (CDCl3, 200 MHz) d 3.23 (m, 2H,
CH2CH2N–), 4.07–4.22 (m, 2H, CH2CH2N–), 5.27 (s,
2H, CH2SMe2), 6.99–7.08 (m, 1H, Ph), 7.12–7.19 (m,
2H, Ph), 7.97 (d, J = 8.0 Hz, 1H, Ph).
CH2CH2N–), 3.71 (dd, J = 1.8, 5.8 Hz, 1H, CH(O)CH-
CHO), 3.89 (d, J = 1.8 Hz, 1H, CH(O)CHCHO), 4.04–
4.33 (m, 2H, CH2CH2N), 7.03–7.11 (m, 1H, Ph), 7.17–
7.23 (m, 2H, Ph), 8.15 (d, J = 8.8 Hz, 1H, Ph), 9.19 (d,
J = 6.1 Hz, 1H, CHO).
4.3. Epoxyamide 11. Reaction of 2,3-O-isopropylidene-D-
glyceraldehyde 12 with sulfur ylide derived from sulfonium
salt 13
4.6. Epoxyacid 16. Treatment of aldehyde 15 with Oxone
To a solution of 2,3-O-isopropylidene-D-glyceraldehyde
12 (1.68 g, 12.9 mmol, 1.0 equiv) and sulfonium salt 13
(3.31 g, 12.9 mmol, 1.0 equiv) in DCM was added an
aqueous solution of NaOH (1.29 mL, 40% w/v,
12.9 mmol, 1.0 equiv) at 0 ꢂC. The mixture was vigor-
ously stirred at this temperature, and after 2 h, the reac-
tion mixture was diluted with water and the organic
layer separated, washed with water (twice), brine (twice),
dried over anhydrous MgSO4 and concentrated under
A solution of aldehyde 15 (500 mg, 2.3 mmol, 1.0 equiv)
in DMF (2.3 mL) was treated with Oxone (1.42 g,
2.3 mmol, 1.0 equiv) at room temperature. After 1 h,
the reaction was complete and, then, AcOEt (15 mL)
was added and the solution was washed with water
(3 · 5 mL). The organic layer was separated, dried over
anhydrous MgSO4, filtered and the solvent was evapo-
rated to obtain epoxyacid 16 (402 mg, 75%) as a white
solid, which did not require further purification:

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1697
Rf = 0.19 (silica gel, AcOEt); 1H NMR (CDCl3,
200 MHz) d 3.22–3.30 (m, 2H, CH2CH2N–), 3.85 (d,
J = 1.8 Hz, 1H, CH(O)CHCO2H), 3.93 (d, J = 1.8 Hz,
1H, CH(O)CHCO2H), 4.15–4.40 (m, 2H, CH2CH2N–),
7.03–7.11 (m, 1H, Ph), 7.16–7.28 (m, 2H, Ph), 8.15 (d,
J = 7.9 Hz, 1H, Ph).
neous suspension, a solution of ammonium formate
(25% (w/v), 1.1 mL, 4.36 mmol, 20 equiv) was added at
25 ꢂC. After 1.5 h, the reaction was complete as deter-
mined by TLC (silica gel, 40% AcOEt in hexanes). The
suspension was filtered through a Celite pad and the vol-
atiles were evaporated. The crude mixture was parti-
tioned between AcOEt and brine;the organic layer
was separated and washed twice with brine, dried over
anhydrous MgSO4, filtered and concentrated, to furnish
compound 22 (138 mg, 75%), as a white solid, which did
not require further purification.
4.7. N,N0-Bis-Boc-agmatine 19. Coupling of N,N0-bis-
Boc-methylisothiourea 18 with 1,4-butanediamine 17
To a solution of 1,4-butanediamine 17 (0.18 mL,
1.79 mmol, 1.0 equiv) in a mixture of THF (2.63 mL)
and water (0.12 mL) was added a solution of N,N0-bis-
Boc-methylisothiourea 18 (0.2 g, 0.69 mmol, 0.77 equiv)
in THF (1.7 mL) dropwise at 25 ꢂC. After addition, the
reaction was heated at 50 ꢂC for 1 h, and then, the sol-
vent was evaporated under vacuum. The resulting crude
was partitioned between CHCl3 and saturated aqueous
solution of NaHCO3. The organic extracts were dried
over anhydrous MgSO4, filtered and concentrated under
reduced pressure, to furnish the agmatine derivative 1914
(230 mg, 99%) as a cloudy oil, which was used without
4.10. Amine 23. Monoprotection of 1,4-butanediamine
with CbzCl
1,4-Butanediamine 17 (171 mg, 1.7 mmol, 1.0 equiv) was
dissolved in water (0.33 mL) and bromocresol green was
added as indicator. p-Toluenesulfonic acid (0.586 g in
0.66 mL of water, 3.4 mmol, 2.0 equiv) was added until
colour changed (yellow), and a few drops of KOAc buf-
fer were added to reach the equivalence point (yellowish
green). Then, the mixture was diluted with EtOH
(0.92 mL) and a solution of Z–Cl (255.7 mg, 1.5 mmol,
1.5 equiv) in dimethoxy methane (0.33 mL) was added
dropwise until colour of the solution changed (yellow).
Then, buffer was carefully added again to reach the
equivalence point, but careful not to increase the pH
over the range 3.7–3.8 (yellowish green colour);in this
case, large amounts of diprotected amine is formed. This
procedure was repeated iteratively until all the Z–Cl was
added. The pH is maintained at 3.7 for 1 h, and after
this time, the volatile components were evaporated
and the resulting crude was diluted with water. The
diprotected amine precipitated and it was filtered off
and washed with water. The mother liquors were
washed with benzene and then basified with 40% (w/v)
NaOH, followed by further extraction with benzene
(2 · 5 mL). The organic extracts were washed with brine
and dried with MgSO4. After evaporating the solvent, a
cloudy oil was obtained, which corresponded to
the Z-monoprotected diamine 2316 (187 mg, 40%). This
oil was subjected to coupling with Boc-Leu-OH without
further purification. 1H NMR (CDCl3, 200 MHz) d
1.30–1.60 (m, 4H, 2 · CH2), 2.59–2.79 (m, 2H,
CH2NH2), 2.97–3.39 (m, 2H, CH2NHZ), 4.95–5.09 (m,
2H, CH2Ph), 7.11–7.38 (m, 5H, Ph).
1
further purification in the next step: H NMR (CDCl3,
200 MHz) d 1.30–1.70 (m, 22H), 3.20 (m, 2H), 2.77 (t,
J = 6.6 Hz, 2H), 3.38 (t, J = 6.6 Hz, 2H), 8.31 (bs, 1H),
11.47 (s, 1H).
4.8. Leucine derivative 21. Coupling of Z-leucine 20 with
the agmatine derivative 19
To a solution of Z-Leu-OH 20 (300 mg, 1.13 mmol,
1.0 equiv) in anhydrous DCM (10 mL) was added a
solution of di-Boc-Agm 19 (230 mg, 0.69 mmol,
0.63 equiv) in DCM (10 mL) prior to the addition of
EDCI (200 mg, 1.04 mmol, 1.4 equiv) at 25 ꢂC. The
reaction mixture was stirred overnight and, after that
time, the reaction was worked up by successive washings
with water, saturated aqueous Na2CO3, aqueous solu-
tion of citric acid and brine. The organic layer was then
separated, dried over anhydrous MgSO4, filtered and the
filtrate was concentrated under reduced pressure. The
crude was purified by flash column chromatography (sil-
ica gel, 40% AcOEt in hexanes), to afford pure coupling
product 21 (291 mg, 72% over two steps) as a white so-
lid: Rf = 0.46 (silica gel, 40% AcOEt in hexanes); 1H
NMR (CDCl3, 400 MHz)
d 0.89–0.94 (m, 6H,
CH(CH3)2), 1.46 (s, 18H, 2 · C(CH3)3), 1.53–1.47 (m,
4.11. Leucine derivative 25. Coupling between Boc-Leu 24
and Z-butanediamine 23
6H, 3 · CH2), 1.61–1.64 (m, 1H, CH(CH3)2), 3.21–3.29
(m,
2H,
CH2NHC(O)),
3.29–3.36
(m,
2H,
CH2NHC(@NBoc)), 4.11–4.14 (m, 1H, CHNHZ), 5.06
(m, 2H, OCH2Ph), 5.36 (d, J = 7.5 Hz, 1H, NHZ),
6.42 (bs, 1H, NHLeu), 7.31 (m, 5H, Ph), 8.32 (m, 1H,
Boc-NH); 13C NMR (CDCl3, 100 MHz) d 172.3,
163.4, 156.2, 153.3, 128.5, 128.2, 128.1, 128.0, 83.2,
79.4, 67.0, 66.9, 53.6, 52.3, 41.6, 41.4, 40.3, 39.1, 28.3,
28.0, 26.6, 26.4, 24.7, 22.9, 22.8, 22.0, 21.8.
Boc-Leu-OH 24 (293 mg, 1.27 mmol, 1.5 equiv) was dis-
solved in DCM (10 mL) under Ar atmosphere, and a
solution of Z-butanediamine 23 (187 mg, 0.84 mmol,
1.0 equiv) in 10 mL of DCM was added. When homoge-
neous, EDCI (241 mg, 1.26 mmol, 1.5 equiv) was added,
and the solution was stirred overnight at room temper-
ature. After that time, the reaction was worked up by
washing with water (2 · 10 mL) and brine (2 · 10 mL).
After drying with MgSO4 and solvent evaporation, the
crude was purified by flash column chromatography (sil-
ica gel, 30% AcOEt in hexanes), obtaining pure coupling
product 25 (209 mg, 65% over two steps) as a white
4.9. Leucine derivative 22. Z-Cleavage of 21
To
a solution of Z-Leu-diBoc-Agm 21 (50 mg,
0.087 mmol, 1.0 equiv) in degassed EtOH (7.4 mL) was
added Pd–C (96 mg), and after formation of a homoge-
1
solid: Rf = 0.47 (silica gel, 30% AcOEt in hexanes); H

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F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
NMR (CDCl3, 200 MHz)
d
0.84–0.97 (m, 6H,
DDQ (165 mg, 0.72 mmol, 6.0 equiv) and the mixture
CH(CH3)2), 1.39 (s, 9H, C(CH3)3), 1.39–1.71 (m, 7H,
3 · CH2, CH(CH3)2), 3.07–3.29 (m, 5H), 5.06 (s, 2H,
CH2Ph), 6.50 (bs, 1H, NHZ), 7.31 (m, 5H, Ph).
was refluxed overnight. After work up, a complex mix-
ture of decomposition products was formed. Under
other conditions such as various solvents (THF, DCM
and toluene), and oxidizing reagents (MnO2, Oxone,
TPAP/NMO, Pd–C, NBS/NaHCO3 (aq)) and tempera-
tures, the results were similarly unsuccessful, with the
decomposition or recovery of 27.
4.12. Leucine derivative 26. Boc cleavage of 25
Compound 25 (184.3 mg, 0.42 mmol, 1.0 equiv) was dis-
solved in 6 mL of anhydrous DCM, and TFA (0.98 mL,
30 equiv) was added. When complete (TLC AcOEt–
hexane (30:70)), the reaction mixture was treated with
saturated aqueous Na2CO3 solution until a pH of 9
was achieved. Then, it was extracted with DCM
(2 · 15 mL), the extracts dried with MgSO4 and evapo-
rated, obtaining the leucine derivative 26 (142 mg,
4.15. Epoxyamide 28. Coupling between epoxyacid 16 and
amine 26
Epoxyacid 16 (198 mg, 0.85 mmol, 1.0 equiv) was dis-
solved in 10 mL of anhydrous DCM. HunigÕs base
¨
(0.24 mg, 1.87 mmol, 2.0 equiv), and after 5 min, amino
compound 26 (500 mg, 1.48 mmol, 1.7 equiv) in 10 mL
of anhydrous DCM were added at room temperature.
When homogeneous, BOP (455.6 mg, 1.08 mmol,
1.2 equiv) was added and the mixture was stirred over-
night. The reaction was worked up by washing with
water (3 · 10 mL), drying with MgSO4 and evaporation
under reduced pressure. The resulting crude was washed
with ether and filtered, to afford 28 (289.5 mg, 62%) as a
white solid: Rf = 0.54 (silica gel, AcOEt); 1H NMR
(CDCl3, 200 MHz) d 0.83–0.97 (m, 6H, CH(CH3)2),
1.32–1.71 (m, 7H, 3 · CH2, CH(CH3)2), 3.07–3.32 (m,
6H, 3 · CH2), 3.72 (d, J = 1.8 Hz, 1H, CH(O)CH),
3.80 (d, J = 1.8 Hz, 1H, CH(O)CH), 4.14 (t,
J = 7.3 Hz, 1H), 4.39 (m, 1H, CHNH), 5.05 (s, 2H,
CH2Ph), 6.57 (bm, 1H, NH), 6.87–7.80 (m, 3H, Ph),
7.28 (m, 5H, Ph), 8.07 (d, J = 7.3 Hz, 1H, Ph).
1
99%) as a colourless oil: H NMR (CDCl3, 200 MHz)
d 0.88–0.95 (m, 6H, CH(CH3)2), 1.20–1.44 (m, 1H,
CH(CH3)2), 1.44–1.58 (m, 4H, 2 · CH2), 1.59–1.75 (m,
2H, CH2), 3.09–3.31 (m, 4H, 2 · CH2), 3.38 (dd,
J = 2.4, 9.2 Hz, 1H, CHNH2), 4.89 (bt, J = 5.5 Hz, 1H,
NHZ), 5.07 (s, 2H, CH2Ph), 7.27–7.45 (m, 5H, Ph).
4.13. Epoxyamide 27. Coupling of epoxyacid 16 with
amine 22
A solution of epoxyacid 16 (49 mg, 0.21 mmol,
1.0 equiv) in anhydrous DMF (3.0 mL) was subjected
to the action of HunigÕs base (36.2 lL, 0.21 mmol,
¨
1.0 equiv) at 25 ꢂC, and after 5 min at this temperature,
a
solution of amino compound 22 (137.5 mg,
0.31 mmol, 1.5 equiv) in anhydrous DMF (3.0 mL)
was added at 25 ꢂC. After stirring for 15 min, BOP
(88 mg, 2.1 mmol, 1.0 equiv) was added in one portion
and the reaction mixture was stirred overnight. After
this time, the mixture was diluted with ether, and the
resulting solution was washed with saturated aqueous
NH4Cl solution (3 · 10 mL). The aqueous phase was ex-
tracted with ether (2 · 5 mL) and the combined organic
layers were separated, dried over anhydrous MgSO4, fil-
tered and concentrated under reduced pressure. Purifica-
tion by flash column chromatography (silica gel, AcOEt
(60 ! 80%) in hexanes) afforded the coupling product
27 (82.3 mg, 58%) as a pale brown solid: Rf = 0.76 (silica
gel, 70% AcOEt in hexanes); 1H NMR (CDCl3,
400 MHz) d 0.91–0.93 (m, 6H, CH(CH3)2), 1.47 (s,
9H, C(CH3)3), 1.48 (m, 2H, CH2CH(CH3)2), 1.48 (s,
9H, C(CH3)3), 1.54–1.73 (m, 5H, 2 · CH2, CH(CH3)2),
3.25 (m, 3H), 3.32–3.42 (m, 3H), 3.66 (d, J = 1.6 Hz,
1H, CH(O)CH), 3.76 (d, J = 1.6 Hz, CH(O)CH), 4.19–
4.25 (m, 2H, CH2CH2N–), 4.37–4.43 (m, 1H, CHNH),
6.41–6.43 (bs, 1H, NH), 6.76 (d, J = 8.6 Hz, 1H, NH),
7.03–7.07 (m, 1H, Ph), 7.17–7.20 (m, 2H, Ph), 8.10 (d,
J = 8.1 Hz, 1H, Ph), 8.33–8.35 (m, 1H, NH); 13C
NMR (CDCl3, 100 MHz) d 171.1, 166.4, 163.4, 162.5,
156.2, 153.2, 142.1, 131.1, 127.6, 124.8, 124.6, 117.3,
83.3, 79.4, 54.0, 53.5, 51.6, 47.2, 41.3, 40.3, 39.2, 28.2,
28.0, 26.6, 26.4, 24.9, 22.8, 22.2.
4.16. Oxidative treatment of 28 with DDQ
Indoline amide 28 (20 mg, 0.036 mmol, 1.0 equiv) was
dissolved in 10 mL of dry benzene and DDQ (33 mg,
0.14 mmol, 4.0 equiv) was added. Then, the reaction
mixture was heated under reflux overnight. After work
up, no oxidation had taken affect. Other solvents tested
were THF, toluene and DCM, and no reaction was sim-
ilarly observed with the recovery of starting indoline
amide 28.
4.17. Indole epoxyamide 31. Oxidation of the indoline
amide 11 with DDQ
A solution of epoxyamide 11 (200 mg, 0.69 mmol,
1.0 equiv) in anhydrous benzene (9.0 mL) was treated
with DDQ (314.2 mg, 1.38 mmol, 2.0 equiv). The reac-
tion mixture was heated overnight, after which, diethyl
ether (20 mL) was added, and the organic layer was
washed extensively with saturated aqueous NaHCO3
solution until a constant, pale red colour was achieved.
The organic phase was separated, dried (MgSO4), fil-
tered and concentrated, to yield a flaky brown solid that,
which after purification by flash column chromatogra-
phy (silica gel, 20% AcOEt in hexanes), provided pure
indole-amide 31 (178 mg, 90%) as a white foamy solid:
Rf = 0.33 (silica gel, 20% AcOEt in hexanes);[ a]D
1
4.14. Oxidation of epoxydiamide 27
+30.1 (c 0.4, CH2Cl2); H NMR (CDCl3, 400 MHz) d
1.38 (s, 3H, C(CH3)2), 1.47 (s, 3H, C(CH3)2), 3.39 (dd,
J = 1.6, 5.4 Hz, 1H, CH(O)CH), 4.01–4.10 (m, 3H,
CH(O)CH, –OCHCH2O–), 4.22 (dd, J = 2.2, 5.9 Hz,
A solution of coupling product 27 (83 mg, 0.12 mmol,
1.0 equiv) in dry benzene (10 mL) was treated with

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1699
1H, –OCHCH2O–), 6.70 (d, J = 3.8 Hz, 1H,
CH@CHN), 7.27–7.37 (m, 1H, Ph), 7.34–7.37 (m, 1H,
Ph), 7.56 (d, J = 8.1 Hz, 1H, CH@CHN), 7.66 (d,
J = 3.8 Hz, 1H, Ph), 8.40 (d, J = 8.6 Hz, 1H, Ph); 13C
NMR (CDCl3, 100 MHz) d 164.9, 135.5, 130.2, 125.5,
124.4, 123.7, 121.0, 116.5, 110.64, 74.8, 67.0, 58.5,
52.8, 26.6, 24.9;MS: 288 (34;M+H +), 287 (22;M +),
230 (68), 188 (43), 144 (27), 118 (77), 101 (33), 59
13C NMR (CDCl3, 100 MHz) d 170.9, 165.6, 163.1,
158.1, 156.3, 152.4, 130.2, 125.7, 124.7, 123.4, 121.1,
116.5, 111.2, 83.2, 79.5, 54.4, 53.7, 51.6, 41.4, 40.3,
39.3, 28.3, 28.0, 26.7, 26.2, 24.9, 22.8, 22.3.
4.22. E-64 (1). Hydrolysis of 29
Indole-amide 29 (50.0 mg, 0.076 mmol, 1.0 equiv) was
dissolved in THF (2 mL), and LiOH (1.52 mL, 0.1 M,
2.0 equiv) was added dropwise at 0 ꢂC. After addition,
TLC (silica gel, 40% AcOEt in hexanes) revealed that
the reaction was complete in 5 min. The work up was
carried out by extracting the aqueous phase with AcOEt
(2 · 3 mL), the aqueous extracts were acidified with sat-
urated aqueous citric acid solution until pH 3 and reex-
tracted with AcOEt (3 · 5 mL). Combined organic
layers were dried with MgSO4, filtered and concen-
trated, to obtain crude acid 35, which was subjected to
the action of TFA at 25 ꢂC. After 1 h, the crude mixture
was concentrated under reduced pressure to obtain the
trifluoracetate salt of E-64 (1) (36 mg, 99%) whose spec-
troscopic and physical properties were identical to an
authentic sample of natural E-64 (1)1: 1H NMR
(DMSO-d6, 400 MHz) d 0.84 (d, J = 6.5 Hz, 3H,
CH(CH3)2), 0.88 (d, J = 6.5 Hz, 3H, CH(CH3)2), 1.37–
1.59 (m, 7H, 3 · CH2, CH(CH3)2), 3.00–3.12 (m, 4H,
2 · CH2NH), 3.45 (d, J = 2.2 Hz, 1H, CH(O)CH), 3.66
(d, J = 2.2 Hz, 1H, CH(O)CH), 4.23–4.32 (m, 1H,
CHNH), 7.56 (bs, 1H, NH), 8.14 (bt, J = 5.7 Hz, 1H,
NH), 8.60 (bd, J = 8.1 Hz, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz) d 171.3, 168.9, 165.0, 156.7,
51.3, 41.1, 40.4, 38.1, 26.3, 25.9, 24.3, 22.9, 21.7.
(100);FAB HRMS
C16H17NO4 287.1157.
m/e 287.1154, calcd for
4.18. Diol 32. Hydrolysis of epoxyamide 31
A solution of epoxyamide 31 (500 mg, 1.74 mmol,
1.0 equiv) in MeOH (10 mL) was treated with Amber-
lyst-15 (500 mg) according to the procedure described
above for 11 to obtain diol 32 (18 mg, 95%) of crude
product, as a brown foamy solid, that was used directly
in the next step without further purification [Rf = 0.46
(silica gel, AcOEt)].
4.19. Aldehyde 33. Treatment of diol 32 with silica
supported sodium periodate
Aldehyde 33 (16 mg, 90%) was prepared from diol 32
(18 mg, 0.073 mmol) by treatment with NaIO4
(0.15 mL, 0.65 M, 0.09 mmol, 1.23 equiv) supported on
silica gel (146 mg) according to the same procedure de-
scribed above for the preparation of 15 [Rf = 0.68 (silica
gel, AcOEt)].
4.20. Epoxyacid 34. Treatment of aldehyde 33 with
Oxone
4.23. Reaction of indole epoxyamide 29 with amines.
General procedure
The oxidation of aldehyde 33 (16 mg, 0.074 mmol,
1.0 equiv) was carried out exactly as described for 15
above, to yield epoxyacid 34 (17 mg, 75%) as a brown
solid, which did not require further purification: Rf =
Indole-amide 29 (17 mg, 0.026 mmol, 1.0 equiv) was dis-
solved in DCM (0.5 mL), and amine (2.0 equiv) was
added at 25 ꢂC, followed by the addition of LiCN
(0.1 equiv). After completion of the reaction (ca. 1 h),
the crude mixture was diluted with EtOAc and washes
with water and brine. The organic layer was separated,
dried (MgSO4) and concentrated under reduced pres-
sure. The resulting crude was purified by flash column
chromatography (silica gel, 50 ! 80% AcOEt in hex-
anes), providing pure epoxyamides 36 (7.6 mg, 49%),
37 (12.7 mg, 76%) and 38 (9.1 mg, 59%), as colourless
oils. [36]: Rf = 0.48 (silica gel, AcOEt); 1H NMR (CDCl3,
200 MHz) d 0.87–0.98 (m, 6H, CH(CH3)2), 1.48 (s, 18H,
2 · C(CH3)3), 1.48–1.64 (m, 7H, 3 · CH2, CH(CH3)2),
3.21–3.45 (m, 4H, 2 · CH2NH), 3.49 (d, J = 1.8 Hz,
1H, CH(O)CH), 3.51 (d, J = 1.8 Hz, CH(O)CH), 3.81–
3.92 (m, 2H, NHCH2CH@CH2), 4.32–4.54 (m, 1H,
CHNH), 5.09–5.21 (m, 2H, NHCH2CH@CH2), 5.67–
5.92 (m, 1H, NHCH2CH@ CH2), 6.17–6.31 (bs, 1H,
NH), 6.40–6.54 (bs, 1H, NH), 6.69 (bd, J = 8.6 Hz, 1H,
NH), 8.27–8.41 (m, 1H, NH), 11.46 (m, 1H, BocNH);
13C NMR (CDCl3, 50 MHz) d 171.1, 165.6, 165.5,
163.4, 161.1, 156.3, 153.3, 133.1, 117.2, 83.2, 79.5, 54.8,
54.7, 51.3, 41.4, 40.3, 39.3, 28.3, 28.1, 26.7, 26.3, 24.8,
22.9, 22.1. [37]: Rf = 0.52 (silica gel, 50% AcOEt in hex-
anes); 1H NMR (CDCl3, 100 MHz) d 0.87–0.96 (m,
6H, CH(CH3)2), 1.47 (s, 18H, 2 · C(CH3)3), 1.47–1.72
1
0.54 (silica gel, AcOEt); H NMR (CDCl3, 200 MHz) d
3.86 (d, J = 1.8 Hz, 1H, CH(O)CHCO2H), 4.31 (d,
J = 1.8 Hz, 1H, CH(O)CHCO2H), 6.71 (d, J = 3.7 Hz,
1H, CH@CHN), 7.27–7.37 (m, 3H, Ph), 7.53–7.64 (m,
2H, Ph, CH@CHN), 8.40 (d, J = 7.3 Hz, 1H, Ph).
4.21. Epoxyamide 29. Coupling of epoxyacid 34 with
amine 22
A solution of epoxyacid 34 (186 mg, 0.81 mmol,
2.0 equiv) in anhydrous DMF (2.0 mL) was reacted with
amine 22 (160.0 mg, 0.36 mmol, 1.0 equiv) according to
the same procedure as described above for the coupling
of 16 and 22, to afford, after similar processing, pure
coupling product 29 (305.7 mg, 51%) as a pale yellow
1
solid: Rf = 0.53 (silica gel, 50% AcOEt in hexanes); H
NMR (CDCl3, 400 MHz)
d 0.86–1.00 (m, 6H,
CH(CH3)2), 1.46 (s, 9H, C(CH3)3), 1.48 (s, 9H,
C(CH3)3), 1.48–1.88 (m, 7H, 3 · CH2, CH(CH3)2),
3.20–3.47 (m, 4H, 2 · CH2NH), 3.88 (d, J = 1.8 Hz,
1H, CH(O)CH), 4.13 (d, J = 1.8 Hz, CH(O)CH), 4.37–
4.54 (m, 1H, CHNH), 6.52 (bt, J = 6.1 Hz, 1H, NH),
6.71 (d, J = 3.7 Hz, 1H, CH@CHN), 6.93 (bd,
J = 9.2 Hz, 1H, NH), 7.27–7.37 (m, 2H, Ph), 7.53–7.64
(m, 2H, CH@CHN, Ph), 8.40 (d, J = 6.7 Hz, 1H, Ph);

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F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
(m, 7H, 3 · CH2, CH(CH3)2), 3.19–3.41 (m, 4H,
2 · CH2NH), 3.50 (d, J = 1.8 Hz, 1H, CH(O)CH), 3.54
(d, J = 1.8 Hz, CH(O)CH), 4.32–4.48 (m, 3H, CHNH,
CH2Ph), 6.30–6.56 (bs, 1H, NH), 6.61–6.75 (bs, 1H,
NH), 7.17–7.32 (m, 5H, Ph), 8.33 (bs, 1H, NH), 11.45
(m, 1H, BocNH); 13C NMR (CDCl3, 50 MHz) d 171.1,
165.7, 159.7, 159.1, 156.3, 153.3, 137.2, 128.8, 127.9,
127.8, 83.2, 79.5, 54.8, 54.7, 52.4, 41.3, 41.0, 40.3, 39.2,
28.3, 28.1, 26.7, 26.3, 24.7, 22.8, 22.1. [38]: Rf = 0.42 (sil-
ica gel, 70% AcOEt in hexanes); 1H NMR (CDCl3,
100 MHz) d 0.82–0.96 (m, 9H, CH(CH3)2CH3), 1.48 (s,
18H, 2 · C(CH3)3), 1.48–1.73 (m, 9H, 4 · CH2,
CH(CH3)2), 3.12–3.44 (m, 6H, 3 · CH2NH), 3.45 (d,
J = 2.4 Hz, 1H, CH(O)CH), 3.48 (d, J = 2.4 Hz,
CH(O)CH), 4.31–4.53 (m, 1H, CHNH), 6.06–6.31 (bs,
1H, NH), 6.38–6.56 (bs, 1H, NH), 6.57–6.69 (bs, 1H,
NH), 8.28–8.41 (bs, 1H, NH), 11.45 (m, 1H, BocNH);
13C NMR (CDCl3, 50 MHz) d 171.2, 165.7, 163.4,
161.1, 156.2, 153.2, 83.2, 79.4, 54.8, 54.7, 41.3, 40.7,
40.3, 39.2, 28.2, 28.0, 26.7, 26.3, 24.7, 22.8, 22.6, 22.1.
with DCM (2 · 15 mL), the extracts combined, dried
(MgSO4), filtered and evaporated, obtaining compound
43 (378.2 mg, 95%) as a yellowish oil: 1H NMR (CDCl3,
200 MHz) d 0.85–0.98 (m, 12H, 2 · CH(CH3)2), 1.27–
1.46 (m, 2H, CH2), 1.49–1.77 (m, 4H, CH2,
2 · CH(CH3)2), 3.15–3.30 (m, 2H, CH2NH–), 3.35 (dd,
J = 3.7, 9.8 Hz, 1H, CHNH–), 7.17 (bs, 1H, NH).
4.27. Epoxyamide 44. Coupling between epoxyacid 16 and
amine 43
Epoxyacid 16 (61.6 mg, 0.26 mmol, 1.0 equiv) was dis-
solved in DMF (2.4 mL);Hu nigÕs base (49 lL,
¨
0.28 mmol, 1.1 equiv) and, after 5 min, 43 (63.4 mg,
1.71 mL of a 0.185 M solution in DMF, 1.2 equiv) were
sequentially added. When the solution was homoge-
neous, BOP (128.6 mg, 0.3 mmol, 1.2 equiv) was added,
and the reaction stirred at room temperature. After 1 h,
analysis by TLC (silica gel, 5% MeOH in AcOEt) indi-
cated the depletion of starting material. The crude mix-
ture was diluted with ether (10 mL) and washed with
saturated aqueous NH4Cl solution (3 · 10 mL). The
aqueous phase was separated and extracted with ether
(2 · 5 mL) and the organic extracts were combined,
dried (MgSO4), filtered and the solvents evaporated.
Purification by flash column chromatography (silica
gel, 60% AcOEt in hexanes), provided epoxyamide 44
(65.4 mg, 60%) as a yellowish solid: Rf = 0.28 (silica
gel, 40% AcOEt in hexanes);[ a]D +7.54 (c 0.17,
CH2Cl2); 1H NMR (CDCl3, 400 MHz) d 0.89 (d,
J = 6.5 Hz, 6H, CH(CH3)2), 0.90–0.93 (m, 6H,
CH(CH3)2), 1.35–1.40 (m, 2H, CH2), 1.49–1.61 (m,
4H, CH2, 2 · CH (CH3)2), 3.15–3.29 (m, 4H), 3.68 (d,
J = 1.6 Hz, 1H, CH(O)CH), 3.78 (d, J = 1.6 Hz, 1H,
CH(O)CH), 4.19–4.25 (m, 2H, indoline CH2N–), 4.36–
4.41 (m, 1H, CHNH–), 6.08 (bm, 1H, CHNH–), 6.87–
6.89 (bm, 1H, CH2NH–), 7.03–7.08 (m, 1H, Ph), 7.16–
7.19 (m, 2H, Ph), 8.12 (d, J = 8.6 Hz, 1H, Ph); 13C
NMR (CDCl3, 100 MHz) d 170.8, 166.5, 142.1, 128.9,
127.7, 124.9, 124.7, 117.4, 54.1, 53.6, 51.6, 47.2, 41.1,
38.2, 38.0, 28.1, 25.8, 24.9, 22.7, 22.4, 22.3;MS: 416
4.24. Treatment of epoxyamides 36–38 with TFA.
General procedure
Indole-amide (0.03 mmol, 1.0 equiv) was treated with
TFA neat at 25 ꢂC for 1 h. After that time, the crude
mixture was concentrated under reduced pressure to
give the corresponding trifluoroacetate salt in quantita-
tive yield.
4.25. Leucine derivative 42. Coupling between isoamyl-
amine and Boc-Leu-OH 24
Compound 24 (613 mg, 2.64 mmol, 1.0 equiv) was dis-
solved in DCM (20 mL) under an Ar atmosphere, and
isoamyl-amine (0.46 mL, 3.96 mmol, 1.5 equiv) was
added. After homogeneization, EDCI (606 mg,
3.17 mmol, 1.2 equiv) was added, and the solution was
stirred at 25 ꢂC for 4 h. After that time, water (10 mL)
was added, and the organic layer separated, washed with
water (2 · 10 mL) and aqueous citric acid (10 mL, 1 M).
The combined organic layers were dried (MgSO4) and
the solvent evaporated, to furnish 42 (597 mg, 75%) as
a white solid, which was used without further purifica-
tion: [a]D ꢀ31.3 (c 0.62, CH2Cl2);[ a]D ꢀ24.8 (c 0.57,
MeOH); 1H NMR (CDCl3, 400 MHz) d 0.86 (d,
J = 6.5 Hz, 6H, isoamyl CH(CH3)2), 0.87–0.89 (m, 6H,
Leu CH(CH3)2), 1.31–1.36 (m, 2H, CH2), 1.39 (s, 9H,
C(CH3)3), 1.41–1.47 (m, 1H), 1.53–1.61 (m, 3H), 3.17–
3.22 (m, 2H, CH2NH–), 4.03 (m, 1H, CHNH–), 5.00
(bm, 1H, NHBoc), 6.34 (bm, 1H, NH); 13C NMR
(CDCl3, 100 MHz) d 172.5, 155.8, 53.0, 41.3, 38.3,
37.7, 28.3, 25.7, 24.7, 22.8, 22.4, 22.3.
+
(74;M+H ), 227 (85), 190 (98), 186 (28), 146 (34), 120
(100), 119 (80), 118 (40), 88 (50), 86 (40);FAB HRMS
m/e 416.2544, M+ calcd for C23H34N3O4 416.2549.
4.28. Indol epoxyamide 45. Oxidation of epoxyamide 44
with DDQ
A
solution of indoline epoxyamide 44 (59.7 mg,
0.14 mmol, 1.0 equiv) in benzene (2 mL) was treated
with DDQ (65.3 mg, 0.29 mmol, 2.0 equiv) under reflux
conditions for 72 h. After that time, the mixture was di-
luted with ether and washed with saturated aqueous
NaHCO3 solution extensively until constant colour of
the organic layer. This phase was dried with MgSO4, fil-
tered and the solvent evaporated to yield compound 45
(45.6 mg, 77%) as a slightly yellow solid, which was used
without purification in the next step: Rf = 0.60 (silica gel,
40% AcOEt in hexanes);[ a]D +46.4 (c 0.075, CH2Cl2);
1H NMR (CDCl3, 400 MHz) d 0.89 (d, J = 6.5 Hz,
6H, CH(CH3)2), 0.93–0.95 (m, 6H, CH(CH3)2), 1.38
(m, 2H, CH2), 1.52–1.71 (m, 4H, CH2, 2 · CH(CH3)2),
3.21–3.29 (m, 2H, CH2NH–), 3.87 (d, J = 2.2 Hz, 1H,
4.26. Leucine derivative 43. Cleavage of the Boc group in
42
Boc-leucine derivative 42 (0.597 g, 1.98 mmol, 1.0 equiv)
was dissolved in DCM (2.2 mL), and TFA (3.3 mL,
wide excess) was added. After 40 min, the reaction
mixture was treated with saturated aqueous Na2CO3
solution until a pH of 9 was achieved with vigorous stir-
ring for 20 min. Then, the aqueous phase was extracted

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1701
CH(O)CH), 4.12 (d, J = 2.2 Hz, 1H, CH(O)CH), 4.38–
4.44 (m, 1H, CHNH–), 5.96 (bs, 1H, CHNH), 6.71 (d,
J = 3.7 Hz, 1H, CH@CHN), 6.87 (bs, 1H, CH2NH),
7.29 (m, 1H, Ph), 7.35 (m, 1H, Ph), 7.55 (m, 2H, Ph,
CH@CHN), 8.35 (d, J = 7.5 Hz, 1H, Ph); 13C NMR
(CDCl3, 100 MHz) d 170.7, 165.9, 165.7, 130.2, 127.9,
125.7, 124.7, 123.4, 121.1, 111.3, 54.4, 53.7, 51.6, 41.3,
38.2, 38.1, 25.8, 24.9, 22.7, 22.4, 22.3;MS: 414 (78;
M+H+), 299 (18), 227 (82), 188 (36), 170 (38), 118
(72), 117 (100), 88 (30), 86 (45);FAB HRMS m/e
414.2397, M+ calcd for C23H32N3O4 414.2392.
3.46 (d, J = 1.8 Hz, 1H, CH(O)CH), 3.68 (d,
J = 1.8Hz, 1H, CH(O)CH), 4.22–4.40 (m, 3H, CHNH,
OCH2CH3), 5.92 (m, 1H, CH2NH), 6.56 (d,
J = 8.6 Hz, 1H, CHNH); 13C NMR (CDCl3,
100 MHz) d 170.7, 166.4, 166.0, 62.3, 53.8, 53.0, 51.3,
41.2, 38.3, 38.0, 25.8, 24.8, 22.8, 22.4, 22.3, 22.2, 14.1.
4.31. Allyl epoxyamide 4. Reaction of indol epoxyamide
45 with allyl-amine
Indole-amide 45 (19.1 mg, 0.046 mmol, 1.0 equiv) was
dissolved in DCM (0.5 mL), and allyl-amine (0.38 mL
of a 0.254 M solution in DCM, 2.2 equiv) was added
at 25 ꢂC. The mixture was stirred overnight at this tem-
perature, and, after that, the crude mixture was concen-
trated under reduced pressure and dried under high
vacuum. The transamidation reaction occurred in quan-
titative yield, providing pure epoxyamide 4 (20 mg, 99%)
as a colourless oil: 1H NMR (CDCl3, 400 MHz) d 0.87–
0.90 (m, 12H, 2 · CH(CH3)2), 1.33–1.39 (m, 2H, CH2),
1.50–1.59 (m, 4H, CH2, 2 · CH (CH3)2), 3.11–3.30 (m,
2H, CH2NH–), 3.54–3.57 (m, 2H, CH(O)CH), 3.83–
3.86 (m, 2H, CH2CH@CH2), 4.50–4.47 (m, 1H,
CHNH), 5.11–5.17 (m, 2H, CH@CH2), 5.72–5.82 (m,
1H, CH@CH2), 6.49 (bs, 1H, CH2NH), 7.22 (bs, 1H,
CHNH); 13C NMR (CDCl3, 100 MHz) d 171.2, 165.9,
165.8, 133.1, 117.1, 54.6, 54.5, 51.5, 41.5, 41.3, 38.1,
37.9, 25.8, 25.7, 22.8, 22.4, 22.3, 22.2.
4.29. E-64c (2). Hydrolysis of 45
Indole-amide 45 (43.6 mg, 0.1 mmol, 1.0 equiv) was dis-
solved in THF (2 mL), and LiOH (2.17 mL, 0.1 M,
2.0 equiv) was added dropwise at 0 ꢂC. After addition,
TLC (silica gel, 40% AcOEt in hexanes) revealed that
the reaction was complete. The work up was carried
out by extracting the aqueous phase with AcOEt
(2 · 3 mL), the aqueous extracts were acidified with
Amberlyst-15 until pH 5 and reextracted with AcOEt
(3 · 5 mL). Combined organic layers were dried with
MgSO4, filtered and concentrated, to obtain pure acid
2 (27 mg, 82%) as a white solid and whose spectroscopic
and physical properties were identical to those reported
in the literature: 1H NMR (DMSO-d6, 400 MHz) d
0.80–0.96 (m, 12H, 2 · CH(CH3)2), 1.27 (q, J=7.0 Hz,
2H, CH2), 1.43–1.63 (m, 4H, CH2, 2 · CH(CH3)2),
3.05 (m, 2H, CH2NH–), 3.45 (d, J = 1.7 Hz, 1H,
CH(O)CH), 3.66 (d, J = 1.7 Hz, 1H, CH(O)CH), 4.29
(m, 1H, CHNH–), 8.02 (t, J = 5.4 Hz, 1H, CH2NH),
8.56 (d, J = 8.4 Hz, 1H, CHNH); 13C NMR (DMSO-
d6, 100 MHz) d 170.9, 168.7, 164.8, 52.6, 51.2, 41.1,
37.9, 36.7, 25.1, 24.3, 22.8, 22.3, 21.7.
4.32. n-Propyl epoxyamide 5. Treatment of indol
epoxyamide 45 with n-propylamine
Indole-amide 45 (21 mg, 0.051 mmol, 1.0 equiv) was dis-
solved in DCM (0.5 mL), and n-propylamine (4.5 lL,
0.056 mmol, 1.1 equiv) in DCM was added at 25 ꢂC.
After stirring for 1.5 h at this temperature, the solvent
was evaporated and the crude product was dried under
high vacuum. The transamidation reaction occurred in
quantitative yield as seen by NMR, without affecting
the oxirane moiety, to afford epoxyamide 5 (20 mg,
4.30. Loxistatine (3). Ethanolysis of 45
Indole-amide 45 (54.2 mg, 0.13 mmol, 1.0 equiv) was
dissolved in freshly distilled dry EtOH (2 mL), and
LiOEt (1.31 mL, 0.1 M in EtOH, prepared by treatment
of dry EtOH with BuLi at 0 ꢂC) was added. After
10 min, the reaction was diluted with AcOEt (5 mL)
and washed with water (2 · 3 mL). This procedure
caused the hydrolysis of the ester (75% of the acid 2
recovered). To achieve the synthesis of this compound,
free acid 2 (31 mg, 0.099 mmol, 1.0 equiv) was dissolved
in DMF (2 mL), and 4-DMAP (18.1 mg, 0.15 mmol,
1.5 equiv) and EDCI (28.0 mg, 0.146 mmol, 1.5 equiv)
were sequentially added. When the mixture was homo-
geneous, dry EtOH (6.85 lL, 0.12 mmol, 1.2 equiv)
was added;the reaction was stirred at room temperature
for 4 h;then, it was worked up by dilution with AcOEt
(10 mL) and washing with water (2 · 5 mL) and satu-
rated aqueous NaHCO3 solution (5 mL). The organic
extracts were dried with MgSO4, filtered and the sol-
vents evaporated, affording pure loxistatine (3) (10 mg,
30%), as a colourless oil, whose spectroscopic and phys-
ical properties were identical to those reported in the lit-
1
99%) as a colourless oil: H NMR (CDCl3, 200 MHz)
d 0.81–0.98 (m, 15H, CH3, 2 · CH(CH3)2), 1.30–1.65
(m, 8H, 3 · CH2, 2 · CH(CH3)2), 3.13–3.29 (m, 4H,
2 · CH2NH–), 3.46 (d, J = 2.4 Hz, 1H, CH(O)CH),
3.47 (d, J = 2.4 Hz, 1H, CH(O)CH), 4.26–4.44 (m, 1H,
CHNH), 5.98–6.23 (bs, 2H, 2 · NH), 6.71–6.76 (bd,
J = 8.6 Hz, 1H, NH); 13C NMR (CDCl3, 50 MHz) d
171.0, 165.8, 165.6, 54.9, 54.7, 51.4, 42.3, 40.8, 38.2,
38.0, 25.8, 24.8, 22.8, 22.6, 22.4, 22.3, 22.2, 11.2;FAB
HRMS m/e 356.2538, M++1 calcd for C18H33N3O4
356.2544.
4.33. Isoamyl epoxyamide 6. Treatment of 45 with
isoamyl amine
Indole-amide 45 (18 mg, 0.043 mmol, 1.0 equiv) was dis-
solved in DCM (0.5 mL), and isoamyl amine (5.45 lL,
0.047 mmol, 1.1 equiv) in DCM was added at room tem-
perature. The reaction mixture was stirred overnight at
35 ꢂC, and was worked up by evaporating the solvent
and drying at high vacuum. The transamidation reac-
tion occurred in quantitative yield as seen by NMR, to
obtain epoxyamide 6 (19 mg, 99%) as a colourless oil:
1
erature: H NMR (CDCl3, 400 MHz) d 0.90–0.95 (m,
12H, 2 · CH(CH3)2), 1.32 (t, J = 7.1 Hz, 3H,
OCH2CH3), 1.39 (q, J = 7.3 Hz, 2H, CH2), 1.47–1.72
(m, 4H, CH2, 2 · CH(CH3)2), 3.24 (m, 2H, CH2NH–),

1702
F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1H NMR (CDCl3, 200 MHz) d 0.82–0.96 (m, 18H,
3 · CH(CH3)2), 1.37 (q, J = 7.3 Hz, 4H, 2 · CH2),
1.46–1.74 (m, 5H, CH2, 3 · CH(CH3)2), 3.12–3.36 (m,
4H, 2 · CH2NH), 3.46 (d, J = 1.8 Hz, 1H, CH(O)CH),
3.47 (d, J = 1.8 Hz, 1H, CH(O)CH), 4.22–4.49 (m, 1H,
CHNH), 6.03–6.10 (m, 2H, 2 · CH2NH), 6.72 (d,
J = 7.9 Hz, 1H, CHNH); 13C NMR (CDCl3, 50 MHz)
d 171.0, 165.8, 165.5, 54.9, 54.7, 51.4, 41.3, 38.2, 38.1,
37.4, 25.8, 25.7, 24.8, 22.8, 22.4, 22.3, 22.2;FAB HRMS
m/e 384.2873, M++1 calcd for C20H37N3O4 384.2857.
solution (2 · 15 mL). After separation of the phases,
the organic layer was dried with MgSO4, filtered and
evaporated. The resulting solid was suspended in a min-
imum volume of ether and filtered through a fritted glass
funnel. This procedure was repeated until no solid re-
mained in the mother liquor, thus obtaining 47 (0.51 g,
82%) as a white solid: Rf = 0.58 (silica gel, 20% AcOEt
in hexanes);[ a]D ꢀ56.9 (c 0.15, CH2Cl2); 1H NMR
(CDCl3, 400 MHz) d 1.31 and 1.43 (2s, 9H, C(CH3)3),
1.73–2.32 (m, 4H, Prol 2 · CH2), 3.12–3.25 (m, 2H, ind-
oline CH2), 3.38–3.54 (m, 1H, Prol CH2N), 3.57–3.72
(m, 1H, Prol CH2N), 3.98–4.09 (m, 1H, indoline
CH2N), 4.11–4.23 and 4.32–4.41 (m, 1H, indoline
CH2N), 4.44 (dd, J = 4.7, 8.1 Hz, 1H, NCHCO), 4.59
(dd, J = 3.4, 8.0 Hz, 1H, NCHCO), 6.95–7.04 (m, 1H,
Ph), 7.13 (m, 2H, Ph), 8.18–8.24 (m, 1H, Ph); 13C
NMR (CDCl3, 100 MHz) d 127.6, 127.36, 124.5, 124.3,
123.7, 123.6, 117.3, 117.1, 58.7, 58.4, 47.5, 46.8, 46.7,
30.4, 29.5, 28.5, 28.2, 28.1, 24.18, 23.7.
4.34. Epoxyamide 7. Reaction of 45 with aqueous
ammonia
Indole-amide 45 (19 mg, 0.045 mmol, 1.0 equiv) was dis-
solved in DCM (0.5 mL), and 30% aqueous ammonia
(2.81 lL, 0.049 mmol, 1.1 equiv) was added at 25 ꢂC.
The mixture was stirred overnight at this temperature,
and was worked up by evaporating the solvent and dry-
ing at high vacuum. The obtained crude corresponded
to epoxyamide 7 (18 mg, 99%), which did not require
4.37. L-Proline derivative 48. Cleavage of the Boc in 47
1
further purification: H NMR (DMSO-d6, 200 MHz) d
0.85–0.93 (m, 12H, 2 · CH(CH3)2), 1.27 (q, J = 7.1 Hz,
2H, CH2), 1.40–1.63 (m, 4H, CH2, 2 · CH(CH3)2),
3.10 (m, 2H, CH2NH), 3.44 (d, J = 1.8 Hz, 1H,
CH(O)CH), 3.61 (d, J = 1.8 Hz, 1H, CH(O)CH), 4.40–
4.55 (m, 1H, CHNH), 7.50 (bs, 1H, NH), 7.77 (bs,
1H, NH), 8.05 (t, J = 5.3 Hz, 1H, NH), 8.56 (d,
J = 7.9 Hz, 1H, NH); 13C NMR (DMSO-d6, 50 MHz)
d 171.0, 168.0, 165.4, 52.6, 52.5, 41.1, 37.9, 36.7, 25.1,
24.3, 22.8, 22.4, 22.3, 21.6.
Compound 47 (0.508 g, 1.6 mmol, 1.0 equiv) was dis-
solved in DCM (10 mL), and TFA (8 mL, wide excess)
was added. After 30 min, analysis by TLC (40% AcOEt
in hexanes) revealed completion of the reaction, so it
was treated with saturated aqueous Na2CO3 solution
under vigorous stirring for 20 min until pH reached 9.
After that time, the mixture was extracted with CHCl3
(2 · 15 mL), the extracts were dried with MgSO4, fil-
tered and evaporated, to obtain 48 (290 mg, 85%) as a
yellowish oil, which was used in the next step without
further purification: [a]D ꢀ79.4 (c 0.17, CH2Cl2); 1H
4.35. Epoxyacid 46. Hydrolysis of indol amide 31
NMR (CDCl3, 400 MHz)
d 1.73–1.94 (m, 3H,
Indole-amide 31 (182 mg, 0.63 mmol, 1.0 equiv) was dis-
solved in THF (5 mL), and LiOH (5.57 mL, 0.1 M in
H2O) was added dropwise at 0 ꢂC. After TLC analysis
(silica gel, 20% AcOEt in hexanes) indicated no presence
of starting material (ca. 5 min), AcOEt (10 mL) was
added and the aqueous phase was treated with Amber-
lyst-15 until a pH of 5 was achieved, and, then, extracted
with AcOEt (3 · 15 mL). The organic extracts were
combined, dried with MgSO4, filtered and concentrated
under reduced pressure, to afford epoxyacid 46 (90 mg,
2 · CH2), 2.16–2.32 (m, 1H, CH2), 2.97 (m, 1H, Prol
CH2N), 3.18 (m, 2H, indoline PhCH2), 3.25 (m, 1H,
Prol CH2N), 3.98–4.07 (m, 2H, indoline CH2N), 4.15
(m, 1H, NCHCO), 7.02 (m, 1H, Ph), 7.18 (m, 2H, Ph),
8.17 (d, J = 8.1 Hz, 1H, Ph); 13C NMR (CDCl3,
100 MHz) d 142.5, 127.6, 124.7, 117.2, 47.4, 30.3, 28.1,
26.2.
4.38. Dipeptide 49. Coupling between of L-proline deriv-
ative 48 with Boc-Ile-OH
1
75%) as a colourless oil: H NMR (CDCl3, 200 MHz)
d 1.34 (s, 3H, C(CH3)2), 1.45 (s, 3H, C(CH3)2), 3.30
(dd, J = 1.8, 3.5 Hz, 1H, CH(O)CHC(@O)), 3.42 (d,
J = 1.8 Hz, 1H, CH(O)CHC(@O)), 3.87–4.05 (m, 2H),
4.10–4.25 (m, 1H); 13C NMR (CDCl3, 50 MHz) d
173.2, 110.1, 74.4, 65.8, 58.1, 51.2, 26.7, 25.0.
A solution of 48 (290 mg, 1.33 mmol, 1.0 equiv) in DCM
(25 mL) was treated with HOBt (150 mg, 1.1 mmol,
0.85 equiv), prior to the addition of Boc-Ile-H
(483.2 mg, 2.0 mmol, 1.5 equiv), followed of EDCI
(385.5 mg, 2.0 mmol, 1.5 equiv) at 25 ꢂC. The reaction
mixture was stirred at this temperature overnight. After
this time, the crude mixture was washed with saturated
aqueous Na2CO3 solution (2 · 15 mL) and water
(15 mL). The organic phase was separated, dried with
MgSO4, filtered and evaporated. Purification by flash
column chromatography (silica gel, 30% AcOEt in hex-
anes) furnished pure 49 (430 mg, 75%) as a white solid:
Rf = 0.52 (silica gel, 30% AcOEt in hexanes);[ a]D ꢀ87.0
4.36. Boc-proline derivative 47. Coupling between Boc-
Pro-H and indoline
To a solution of Boc-Pro-H (0.5 g, 2.32 mmol, 1.0 equiv)
in anhydrous DCM (25 mL) was added indoline (0.23 g,
1.92 mmol, 0.83 equiv) and EDCI (0.67 g, 3.4 mmol,
1.5 equiv) at 25 ꢂC. The reaction mixture was kept at
this temperature until the reaction was complete as
judged by TLC (ca. 4 h). Then, the crude mixture was
diluted with DCM (20 mL) and the resulting organic
solution was washed with saturated aqueous NaHCO3
solution (2 · 15 mL) and saturated aqueous Na2CO3
1
(c 0.2, CH2Cl2); H NMR (CDCl3, 400 MHz) d 0.88 (t,
J = 7.5 Hz, 3H, CH2CH3), 1.00 (d, J = 7.0 Hz, 3H,
CHCH3), 1.04–1.15 (m, 1H, Ile CH2), 1.49–1.61 (m,
1H, Ile CH2), 1.40 (s, 9H, C(CH3)3), 1.70–1.80 (m, 1H,
Ile CH), 1.96–2.07 (m, 2H, Prol CH2), 2.16–2.28 (m,

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1703
2H, Prol CH2), 3.13–3.28 (m, 2H, PhCH2), 3.70–3.78
(m, 1H, Prol CH2N), 3.85–3.90 (m, 1H, Pro CH2N),
4.01–4.09 (m, 1H, indoline CH2N), 4.41–4.48 (m, 1H,
indoline CH2N), 4.29 (dd, J = 8.6, 9.7 Hz, 1H, Ile
CHNH), 4.76 (dd, J = 4.8, 7.5 Hz, 1H, Pro CHN),
5.10 (d, J = 9.7 Hz, 1H, NHBoc), 6.97 (m, 1H, Ph),
7.14 (m, 2H, Ph), 8.15 (d, J = 8.6 Hz, 1H, Ph); 13C
NMR (CDCl3, 100 MHz) d 171.2, 169.7, 155.6, 142.8,
131.0, 127.2, 124.3, 123.6, 117.1, 58.5, 56.1, 47.6, 37.7,
28.6, 28.2, 27.9, 24.9, 24.1, 15.2, 10.9;MS: 430
(M+H+) (32), 374 (12), 356 (14), 331 (23), 330 (96),
312 (19), 311 (53), 255 (62), 237 (14), 217 (25), 211
(51), 130 (23), 120 (36), 119 (34), 118 (20), 86 (23), 70
(100), 57 (35);FAB HRMS m/e 430.2694, M+ calcd
for C24H36N3O4 430.2705.
1H, Ile CH2), 1.79–1.85 (m, 1H, CHCH3), 1.95–2.04
(m, 2H, Pro CH2), 2.17–2.26 (m, 2H, Pro CH2), 3.09
(dd, J = 1.6, 4.8 Hz, 1H, CH(O)CHC(@O)), 3.17–3.24
(m, 2H, PhCH2), 3.32 (d, J = 1.6 Hz, 1H,
CH(O)CHC(@O)), 3.74–3.92 (m, 3H, Pro CH2N,
–OCH2CH), 4.01–4.09 (m, 3H, indoline CH2N,
–OCH2CH), 4.39–4.46 (m, 1H, indoline CH2N), 4.59
(dd, J = 8.1, 9.1 Hz, 1H, Ile CHNH), 4.75 (dd, J = 4.8,
7.0 Hz, 1H, Pro CHN), 6.65 (d, J = 9.1 Hz, 1H, Ile
CHNH), 6.98 (m, 1H, Ph), 7.14 (m, 2H, Ph), 8.15 (d,
J = 8.1 Hz, 1H, Ph); 13C NMR (CDCl3, 100 MHz) d
169.9, 167.3, 127.4, 124.5, 123.8, 117.3, 105.4, 74.3,
65.9, 58.8, 58.6, 54.2, 53.2, 47.7, 37.7, 28.8, 28.1, 26.4,
25.2, 25.0, 24.4, 15.3, 11.0;MS: 500 (78;M+H +), 484
(13), 381 (87), 217 (100), 120 (33), 70 (79), 59 (33);
FAB HRMS m/e 500.2754, M+ calcd for C27H38N3O6
500.2760.
4.39. Dipeptide 50. Cleavage of the Boc group in 49
Compound 49 (430 mg, 1.0 mmol, 1.0 equiv) was dis-
solved in DCM (10 mL), and TFA (10 mL, wide excess)
was added. After 30 min, analysis by TLC revealed com-
pletion of the reaction, so it was treated with saturated
aqueous Na2CO3 solution until a pH of 9 was achieved.
The mixture was extracted with DCM (2 · 15 mL), the
extracts were combined, dried with MgSO4, filtered
and evaporated, to obtain deprotected dipeptide 50
(307.9 mg, 94%) as a white solid: 1H NMR (CDCl3,
400 MHz) d 0.86 (t, J = 7.5 Hz, 3H, CH2CH3), 0.97 (d,
J = 7.0 Hz, 3H, CHCH3), 1.02–1.13 (m, 1H, Ile CH2),
1.48–1.60 (m, 1H, Ile CH2), 1.64–1.74 (m, 1H, CHCH3),
1.91–2.02 (m, 2H, Pro CH2), 2.14–2.24 (m, 2H, Pro
CH2), 3.11–3.27 (m, 2H, PhCH2), 3.39 (d, J = 6.5 Hz,
1H, Ile CHNH), 3.63–3.72 (m, 2H, Pro CH2N), 3.98–
4.07 (m, 1H, indoline CH2N), 4.39–4.45 (m, 1H, indo-
line CH2N), 4.74 (dd, J = 4.8, 7.5 Hz, 1H, Pro CHN),
6.96 (m, 1H, Ph), 7.09–7.13 (m, 2H, Ph), 8.09 (d,
J = 8.1 Hz, 1H, Ph); 13C NMR (CDCl3, 100 MHz) d
127.3, 124.4, 123.8, 117.2, 58.8, 57.2, 47.7, 47.4, 38.6,
28.0, 25.0, 23.7, 15.6, 11.0.
4.41. Diol 52. Acidic treatment of acetal 51
Epoxypeptide 51 (257 mg, 0.51 mmol, 1.0 equiv) was
dissolved in MeOH (25 mL), and of p-toluenesulfonic
acid (98 mg, 0.51 mmol, 1.0 equiv) was added. After stir-
ring overnight at 25 ꢂC, the reaction mixture was
washed with saturated aqueous NaHCO3 solution
(10 mL) and the aqueous phase extracted with AcOEt
(2 · 20 mL). The combined organic layers were dried
(MgSO4), filtered and concentrated, to furnish diol 52
(151.3 mg, 75%) as a white solid: Rf = 0.34 (silica gel,
1
5% MeOH in AcOEt);[ a]D ꢀ81.7 (c 0.23, CH2Cl2); H
NMR (CD3OD, 400 MHz) d 0.92 (t, J = 7.5 Hz, 3H,
CH2CH3), 1.05 (d, J = 7.0 Hz, 3H, CHCH3), 1.13–1.22
(m, 1H, Ile CH2), 1.56–1.63 (m, 1H, Ile CH2), 1.86–
1.93 (m, 1H, CHCH3), 1.97–2.07 (m, 2H, Pro CH2),
2.16–2.22 (m, 1H, Pro CH2), 2.33–2.39 (m, 1H, Pro
CH2), 3.17 (dd, J = 2.2, 4.3 Hz, 1H, CH(O)CHC(@O)),
3.24 (m, 2H, PhCH2), 3.52 (d, J = 2.2 Hz, 1H,
CH(O)CHC(@O)), 3.58–3.67 (m, 3H, HOCH2CH),
3.74–3.79 (m, 1H, Pro CH2N), 3.99–4.05 (m, 1H, Pro
CH2N), 4.14–4.20 (m, 1H, indoline CH2N), 4.36–4.43
(m, 1H, indoline CH2N), 4.57 (d, J = 8.6 Hz, 1H, Ile
CHNH), 4.78 (dd, J = 5.6, 7.8 Hz, 1H, Pro CHN),
7.03 (m, 1H, Ph), 7.14 (m, 1H, Ph), 7.23 (d,
J = 7.0 Hz, 1H, Ph), 8.08 (d, J = 8.1 Hz, 1H, Ph); 13C
NMR (CDCl3, 100 MHz) d 172.1, 171.7, 170.8, 144.1,
133.4, 128.2, 125.9, 125.3, 118.2, 71.2, 64.6, 60.8, 60.4,
59.9, 56.7, 56.5, 53.0, 49.1, 38.1, 30.0, 29.1, 26.2, 25.8,
15.2, 11.2;MS: 460 (9;M+H +), 341 (25), 281 (17), 217
(90), 119 (25), 86 (12), 70 (100);FAB HRMS m/e
460.2440, M+ calcd for C24H34N3O6 460.2447.
4.40. Epoxypeptide 51. Coupling of epoxyacid 46 and
dipeptide 50
Epoxyacid 46 (166 mg, 0.88 mmol, 1.0 equiv) was dis-
solved in DMF (3 mL) and treated with HunigÕs base
¨
(110 mg, 0.88 mmol, 1.0 equiv) at room temperature.
After 5 min, a solution of dipeptide 50 (320 mg,
0.97 mmol, 1.1 equiv) in DMF (3 mL) was added to
the reaction mixture, followed by addition of BOP
(507 mg, 1.2 mmol, 1.4 equiv). The resulting reaction
mixture was stirred overnight at this temperature. After
that time, ether (15 mL) was added and the organic solu-
tion was washed with saturated aqueous NH4Cl solution
(3 · 10 mL), and water (10 mL). The combined organic
layers were dried with MgSO4, filtered and concentrated
under vacuum. Purification by flash column chromato-
graphy (silica gel, 75% AcOEt in hexanes), provided
epoxypeptide 51 (330 mg, 75%) as a white solid:
Rf = 0.26 (silica gel, 75% AcOEt in hexanes);[ a]D
4.42. Epoxyaldehyde 53. Oxidative cleavage of diol 52
SiO2 (700 mg) was suspended in DCM (6 mL), and
NaIO4 (0.7 mL, 0.65 M in water, 0.45 mmol) was added
dropwise to form a flaky suspension. Then, a solution of
diol 52 (80 mg, 0.17 mmol, 1.0 equiv) in DCM (6 mL)
was added dropwise at 25 ꢂC. After 1 h at this tempera-
ture, the crude mixture was filtered through a fritted
glass funnel, removing the silica, and the solid was
washed twice with DCM. The liquid phase was concen-
trated until dryness to obtain a crude that corresponded
to aldehyde 53 (74 mg, 99%), as a white solid, practically
1
ꢀ67.5 (c 0.14, CH2Cl2); H NMR (CDCl3, 400 MHz)
d 0.86 (t, J = 7.5 Hz, 3H, CH2CH3), 0.99 (d,
J = 6.5 Hz, 3H, CHCH3), 1.05 (m, 1H, Ile CH2), 1.34
(s, 3H, C(CH3)2), 1.42 (s, 3H, C(CH3)2), 1.42–1.49 (m,

1704
F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
pure by NMR, not requiring further purification:
Rf = 0.56 (silica gel, 5% MeOH in AcOEt);[ a]D ꢀ99.9
phy (silica gel, AcOEt) afforded pure amide 55
(55.2 mg, 90%) as a pale brown solid: Rf = 0.39 (silica
gel, AcOEt); H NMR (CD3OD, 400 MHz) d 0.92 (t,
1
1
(c 0.11, CH2Cl2); H NMR (CDCl3, 400 MHz) d 0.87
(t, J = 7.0 Hz, 3H, CH2CH3), 0.98–1.07 (m, 1H, Ile
CH2), 1.00 (d, J = 6.5 Hz, 3H, CHCH3), 1.40–1.50 (m,
1H, Ile CH2), 1.81–1.89 (m, 1H, CHCH3), 1.97–2.06
(m, 2H, Pro CH2), 2.20–2.28 (m, 2H, Pro CH2), 3.16–
3.27 (m, 2H, PhCH2), 3.40 (dd, J = 2.2, 5.9 Hz, 1H,
J = 7.4 Hz, 3H, Ile CH2CH3), 0.93 (t, J = 7.0 Hz, 3H,
CH2CH2CH3), 1.05 (d, J = 6.8 Hz, 3H, CHCH3), 1.15–
1.24 (m, 1H, Ile CH2), 1.46–1.58 (sext, J = 7.0 Hz, 2H,
CH2CH2CH3), 1.56–1.65 (m, 1H, Ile CH2), 1.84–1.96
(m, 1H, CHCH3), 1.95–2.07 (m, 2H, Pro CH2), 2.12–
2.24 (m, 1H, Pro CH2), 2.30–2.43 (m, 1H, Pro CH2),
3.17 (t, J = 7.0 Hz, 2H, CH2CH2CH3), 3.21–3.26 (m,
2H, PhCH2), 3.52 (d, J = 1.6 Hz, 1H, CH(O)CH), 3.62
(d, J = 1.6 Hz, 1H, CH(O)CH), 3.71–3.79 (m, 1H, Pro
CH2N), 3.98–4.05 (m, 1H, Pro CH2N), 4.13–4.20 (m,
1H, indoline CH2N), 4.35–4.43 (m, 1H, indoline
CH2N), 4.57 (d, J = 8.7 Hz, 1H, Ile CHNH), 4.76 (dd,
J = 5.3, 7.8 Hz, 1H, Pro CHN), 7.02 (m, 1H, Ph), 7.14
(m, 1H, Ph), 7.23 (d, J = 7.3 Hz, 1H, Ph), 8.08 (d,
J = 8.1 Hz, 1H, Ph); 13C NMR (CD3OD, 100 MHz) d
172.3, 172.2, 169.1, 169.0, 144.5, 133.8, 128.6, 126.3,
125.7, 118.6, 61.2, 57.2, 55.2, 54.8, 42.6, 38.4, 30.4,
29.5, 26.6, 26.3, 24.0, 16.0, 12.1, 11.5.
CH(O)CHC(@O)),
3.69
(d,
J = 2.2 Hz,
1H,
CH(O)CHC(@O)), 3.75–3.90 (m, 2H, Pro CH2N),
4.02–4.08 (m, 1H, indoline CH2N), 4.38–4.45 (m, 1H,
indoline CH2N), 4.63 (dd, J = 7.0, 9.1 Hz, 1H, Ile
CHNH), 4.75 (dd, J = 4.8, 7.5 Hz, 1H, Pro CHN),
6.72 (d, J = 9.1 Hz, 1H, Ile CHNH), 6.97–7.01 (m, 1H,
Ph), 7.15 (m, 2H, Ph), 8.15 (d, J = 8.1 Hz, 1H, Ph),
8.99 (d, J = 5.9 Hz, 1H, CHO); 13C NMR (CDCl3,
100 MHz) d 194.5, 169.7, 169.5, 165.2, 142.8, 131.1,
127.5, 124.5, 123.9, 117.3, 58.8, 58.3, 54.5, 52.6, 47.8,
47.7, 37.8, 28.8, 28.1, 25.0, 24.3, 15.4, 10.9;MS: 428
(100;M+H +), 356 (20), 309 (19), 239 (21), 237 (26),
217 (28), 196 (33), 120 (37), 119 (20), 118 (18), 73
(100), 70 (64);FAB HRMS m/e 428.2173, M+ calcd
for C23H30N3O5 429.2185.
4.45. Indole epoxypeptide 56. Oxidation of indoline
epoxypeptide 55 with DDQ
4.43. Epoxyacid 54. Oxidation of the aldehyde 53
To a solution of epoxypeptide 55 (114 mg, 0.23 mmol,
1.0 equiv) in benzene (10 mL) was added DDQ
(214 mg, 0.94 mmol, 4.0 equiv), and the solution was
heated at reflux for 16 h under Ar atmosphere. After
that time, ether (15 mL) was added and the organic
phase was washed extensively with saturated aqueous
NaHCO3 solution until no colour changes of the organic
layer were apparent. The organic extracts were dried
with MgSO4 and the solvent evaporated, to afford in-
dole amide 56 (100 mg, 88%) as a brown solid, which
was used in the next step without further purification:
Epoxyaldehyde 53 (74 mg, 0.174 mmol, 1.0 equiv) was
dissolved in THF (5 mL), and m-CPBA (136 mg,
0.79 mmol, 3.0 equiv) was added. The reaction was stir-
red at room temperature for 3 days, after which, the sol-
vent was evaporated and the crude was subjected to
purification by flash column chromatography (silica
gel, 30% MeOH in AcOEt ! MeOH) to obtain epoxy-
acid 54 (56 mg, 73%) as a colourless oil: 1H NMR
(CD3OD, 400 MHz)
d
0.89 (t, J = 7.5 Hz, 3H,
CH2CH3), 1.02 (d, J = 7.0 Hz, 3H, CHCH3), 1.10–1.19
(m, 1H, Ile CH2), 1.53–1.64 (m, 1H, Ile CH2), 1.91 (m,
1H, CHCH3), 2.00 (m, 2H, Pro CH2), 2.13–2.20 (m,
1H, Pro CH2), 2.31–2.38 (m, 1H, Pro CH2), 3.21–3.25
(m, 2H, PhCH2), 3.38 (d, J = 1.6 Hz, 1H, CH(O)CH),
3.54 (d, J = 1.6 Hz, 1H, CH(O)CH), 3.73–3.78 (m, 1H,
Pro CH2N), 3.98–4.05 (m, 1H, Pro CH2N), 4.13–4.19
(m, 1H, indoline CH2N), 4.36–4.43 (m, 1H, indoline
CH2N), 4.58 (d, J = 9.1 Hz, 1H, Ile CHNH), 4.77 (dd,
J = 4.8, 7.0 Hz, 1H, Pro CHN), 7.02 (m, 1H, Ph), 7.14
(m, 1H, Ph), 7.22 (d, J = 7.0 Hz, 1H, Ph), 8.08 (d,
J = 8.1 Hz, 1H, Ph); 13C NMR (CD3OD, 100 MHz) d
172.4, 172.2, 170.2, 144.5, 133.8, 128.6, 126.3, 125.7,
118.6, 61.6, 57.0, 56.1, 54.5, 38.4, 30.38, 29.5, 26.6,
26.3, 16.1, 11.5.
1
Rf = 0.55 (silica gel, 30% AcOEt in hexanes); H NMR
(CDCl3, 400 MHz) d 0.85–0.89 (m, 6H, 2 · CH3), 1.05
(d, J = 7.0 Hz, 1H, CHCH3), 1.45–1.51 (m, 2H,
CH2CH2CH3), 1.81–1.89 (m, 1H, CHCH3), 2.05–2.22
(m, 3H, Pro CH2CH2), 2.34–2.40 (m, 1H, Pro CH2),
3.15–3.20 (m, 2H, CH2CH2CH3), 3.42 (d, J = 2.2 Hz,
1H, CH(O)CH), 3.49 (d, J = 2.2 Hz, 1H, CH(O)CH),
3.79–3.85 (m, 1H, Pro CH2N), 3.90–3.96 (m, 1H, Pro
CH2N), 4.64 (dd, J = 7.5, 9.1 Hz, 1H, Ile CHNH),
5.29 (dd, J = 4.3, 8.6 Hz, 1H, Pro CHN), 6.09 (t,
J = 5.6 Hz, 1H, NHn-Pr), 6.66 (d, J = 3.7 Hz, 1H,
CH@CHN), 6.71 (d, J = 9.1 Hz, 1H, Ile CHNH),
7.25–7.27 (m, 1H, Ph), 7.30–7.34 (m, 1H, Ph), 7.51–
7.55 (m, 2H, Ph, CH@CHN), 8.40 (d, J = 8.1 Hz, 1H,
Ph); 13C NMR (CDCl3, 100 MHz) d 170.4, 169.7,
169.6, 165.8, 135.8, 130.2, 125.1, 124.0, 123.9, 120.8,
116.7, 109.8, 59.1, 54.8, 54.7, 54.5, 47.6, 40.7, 37.5,
29.5, 24.9, 24.4, 22.5, 15.2, 11.1, 10.8.
4.44. Epoxypeptide 55. Coupling of epoxyacid 54 with n-
propylamine
To a solution of 54 (54 mg, 0.12 mmol, 1.0 equiv) in
DCM (3 mL) was added HunigÕs base (24 lL,
4.46. CA-074 (8). Hydrolysis of 56
¨
n-propylamine
0.14 mmol,
1.2 equiv),
(12 lL,
0.14 mmol, 1.2 equiv) and BOP (64.3 mg, 0.15 mmol,
1.2 equiv) at 25 ꢂC. After stirring overnight at this tem-
perature, the reaction was worked up by diluting with
DCM and washing with water and a solution of citric
acid. The organic extracts were dried with MgSO4 and
evaporated. Purification by flash column chromatogra-
Indole-amide 56 (21 mg, 0.044 mmol, 1.0 equiv) was dis-
solved in THF (1 mL), and LiOH (0.871 mL, 0.1 M in
H2O, 2.0 equiv) was added dropwise at 0 ꢂC. After
30 min, the aqueous solution was extracted with AcOEt
and the aqueous phase was treated with Amberlyst-15
until a pH of 5 was achieved, and then extracted with

F. Sarabia et al. / Bioorg. Med. Chem. 13 (2005) 1691–1705
1705
Asgian, J. L.;James, K. E.;Li, Z. Z.;Carter, W.;Barrett,
A. J.;Mikolajczyk, J.;Salvesen, G. S.;Powers, J. C.
Med. Chem. 2002, 45, 4958–4960;(d) Greenbaum, D.;
Medzihradsky, K. F.;Burlingame, A.;Bogyo, M. Chem.
Biol. 2000, 7, 569–581.
AcOEt (3 · 5 mL). After drying with MgSO4 and sol-
vent evaporation, epoxyacid 8 (9.65 mg, 60%) was ob-
tained as a white solid.21
J.
6. Powers, J. C.;Asgian, J. L.;Ekici, O. D.;James, K. E.
Chem. Rev. 2002, 102, 4639–4750.
Acknowledgements
´
´
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7. (a) Lopez Herrera, F. J.;Sarabia Garcı a, F. R.;Pino
Gonzalez, M. S. Recent Res. Devel. Org. Chem. 2000,
´
This work was financially supported by Fundacion
Ramon Areces, the Direccion General de Investigacion y
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4, 465–490;(b) Lo pez Herrera, F. J.;Sarabia Garcı a, F.;
Pedraza Cebrian, G. M.;Pino Gonza lez, M. S. Tetrahe-
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dron Lett. 1999, 40, 1379–1380;(c) Lo pez-Herrera, F. J.;
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44, 8353–8356;(d) Assiego, C.;Pino Gonza lez, M. S.;
Lopez-Herrera, F. J. Tetrahedron Lett. 2004, 45, 2611–
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2613;(e) Martı n-Ortiz, L.;Chammaa, S.;Pino-Gonza lez,
M. S.;Sa nchez-Ruiz, A.;Garcı a-Castro, M.;Assiego, C.;
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´
Cientıfica Tecnica (ref. BQU2001-1576) and the Direc-
cion General de Universidades e Investigacion, Consejerıa
´
´
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de Educacion y Ciencia, Junta de Andalucıa (FQM 0158).
We thank Dr. J. I. Trujillo (St Louis, USA) for assis-
tance in the preparation of this manuscript. We thank
´
´
´
Unidad de Espectroscopıa de Masas de la Universidad
de Sevilla for exact mass spectroscopic assistance.
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Sarabia, F. Tetrahedron Lett. 2004, 45, 9069–9072.
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