A convenient protocol for selective cleavage of 2-hydroxy acid amides. Application to semisynthesis of the cyclic heptapeptide aza HUN-7293.

Article abstract of DOI:10.1021/jo025979g

A two-step protocol for the first chemoselective cleavage of 2-hydroxy acid amides has been developed. Mesylation of the model substrate 2-(hydroxypropionylamino)-4-methylpentanoic acid methyl ester (11) followed by treatment with N-ethylthiourea (13) all

Full text of DOI:10.1021/jo025979g

A Con ven ien t P r otocol for Selective Clea va ge of 2-Hyd r oxy Acid
Am id es. Ap p lica tion to Sem isyn th esis of th e Cyclic Hep ta p ep tid e
Aza HUN-7293
Erwin P. Schreiner,* Michael Kern, and Andrea Steck
Novartis Forschungsinstitut, Brunner Strasse 59, A-1235 Vienna, Austria
erwin.schreiner@pharma.novartis.com
Received May 28, 2002
A two-step protocol for the first chemoselective cleavage of 2-hydroxy acid amides has been
developed. Mesylation of the model substrate 2-(hydroxypropionylamino)-4-methylpentanoic acid
methyl ester (11) followed by treatment with N-ethylthiourea (13) allows cleavage of 2-hydroxy
acid amides under smooth conditions. Successful application of this methodology to the open-chain
transesterification product 15 (methylester) of the cyclic heptadepsipeptide HUN-7293, a potent
inhibitor of inducible cell adhesion molecule expression, delivered the corresponding hexapeptide
18 with unprotected N-terminus in 70-75% yield. This result demonstrates that the protocol
developed even works in the presence of an ester and several methylated and unmethylated amide
bonds. Finally, a sequence of ligation of methyl D-dehydroglutaminate (20) to the C-terminus of
the saponification product 21, followed by the degradation protocol and ring closure, allowed
chemical “point mutation” at the DGCN site affording the aza analogue of HUN-7293 (24) in 15%
overall yield. To the best of our knowledge this is the first report on chemoselective cleavage of
2-hydroxy acid amides.
In tr od u ction
The cyclic heptadepsipeptide HUN-7293 (1) is a potent
inhibitor of the expression of the adhesion molecules
ICAM-1 and VCAM-1.1 This cyclic heptadepsipeptide
composed of six L-amino and one D-hydroxy acid (Figure
1) was first isolated from a fungal broth during a screen
for potent inhibitors of inducible cell adhesion molecule
expression in 1992. Independently, the identical natural
product was isolated by a J apanese group from a different
fungal strain screening for anti-HIV compounds.2 Re-
cently, the total synthesis of HUN-7293 has been achieved
by D. Boger and co-workers in collaboration with Novar-
tis chemists.3
To gain insight into the structural requirements for
its unique biological activity, the three-dimensional
structure of 1 has been determined, both in solution by
NMR spectroscopy and in single crystals by X-ray dif-
fraction.4 These investigations revealed that the ester
bond exists in the trans-configuration in solution and in
the crystalline state. Therefore, we assumed that re-
placement of the ester moiety by an amide should be
tolerated without changing the overall conformation of
the molecule. From the viewpoint of a medicinal chemist
F IGURE 1. Structure of HUN-7293 (1).
there are three major reasons to move from a cyclic
depsipeptide to a cyclic peptide: (a) an amide bond is
more convenient to form than an ester bond; (b) an amide
bond is chemically and metabolically more stable than
an ester bond; and (c) the palette of commercially
available optically pure amino acids is much broader than
that for 2-hydroxy acids. So far, ester/amide exchange
for such macrocyclic molecules has been realized only by
total synthesis as demonstrated for Destruxin,5 Leuala-
cin,6 and, recently by the Boger group, also HUN-7293.7
We considered the multitude of steps for the synthesis
of the unnatural amino acids PrLEU and MTO and a
series of critical coupling cycles as a hurdle for a potential
(1) Foster, C. A.; Dreyfuss, M.; Mandak, B.; Meingassner, J . G.;
Naegeli, H.-U.; Nussbaumer, A.; Oberer, L.; Scheel, G.; Swoboda, E.-
M. J . Dermatol. 1994, 21, 847.
(2) Itazaki, H.; Fujiwara, T.; Sato, A.; Kawamura, Y.; Matsumoto,
K. J pn Patent, Kokai Tokkyo Koho 07109299, 1995, Appl. 93/255592,
Oct 13, 1993; Chem. Abstr. 1995, 123, 110270.
(3) Boger, D. L.; Keim, H.; Oberhauser, B.; Schreiner, E. P.; Foster,
C. A. J . Am. Chem. Soc. 1999, 121, 6197.
(5) Cavelier, F.; J acquier, R.; Mercadier, J .-L.; Verducci, J . Tetra-
hedron 1996, 52, 6173.
(6) Hu, M.-K.; Yang, F.-C.; Chou, C.-C.; Yen, M.-H. Bioorg. Med.
Chem. Lett. 1999, 9, 563.
(4) Hommel, U.; Weber, H.-P.; Oberer, L.; Naegeli, H. U.; Ober-
hauser, B.; Foster, C. A. FEBS Lett. 1996, 379, 69.
(7) Boger, D. L.; Chen, Y.; Foster, C. A. Bioorg. Med. Chem. Lett.
2000, 10, 1741.
10.1021/jo025979g CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/31/2002
J . Org. Chem. 2002, 67, 8299-8304
8299

Schreiner et al.
SCHEME 1
chemical development as a drug. Therefore, in contrast
to Boger’s total synthesis, we planned a semisynthetic
approach starting from the readily available fermentation
product. Our strategy envisaged chemical “point muta-
tions” at the DGCN site involving cleavage of the lactone
followed by degradation of the 2-hydroxy acid unit,
incorporation of a new amino acid, and final ring closure
forming an amide bond. Although in the past a series of
cyclic depsipeptides showing interesting biological activi-
ties such as the antifungal aureobasidin A,8 the antiin-
flammatory salinamides A and B,9 the neurokinin an-
tagonist Sch 217048,10 the insecticide Destruxine,11 and
the calcium blocker leualacin12 were investigated, no
general methodology for the selective cleavage of 2-hy-
droxy acid amides, comparable to the Edman protocol for
peptides, has been developed. We now report the first
protocol for selective cleavage of 2-hydroxy acid amides
in the presence of several methylated and unmethylated
amides as well as an ester bond.
Resu lts a n d Discu ssion
Mod el Rea ction s. On the basis of simple model
substrates two potential approaches were evaluated.
Method A is founded on a report13 on the cyclization of
the thiosemicarbazones of sodium pyruvic acid esters and
subsequent ester cleavage. First, coupling of sodium
pyruvate (2) and D,L-Val-OMe (3) in the presence of EDC
afforded our model substrate N-(2-oxopropanoyl)leucine
methyl ester (4). Indeed, heating of 4 in the presence of
thiosemicarbazone 5 in EtOH at 80 °C resulted in the
formation of the thiosemicarbazide (E)-6 as well as
cleavage products 7 and 8 (Scheme 1). This result
indicated that under these conditions only the (Z)-isomer
(Z)-6 spontaneously cyclized, whereas isomerization of
(E)-6 to (Z)-6, necessary to obtain quantitative conver-
sion, did not occur.
SCHEME 2
Method B takes advantage of a chemical principle
developed for the selective cleavage of chloroacetate
amides, wherein the chloride is first replaced by a
thiourea moiety forming an intermediate thiouronium
salt, which subsequently cyclizes and liberates the amine.14
Coupling of sodium D-lactate (9) and D-Leu-OMe (10)
provided the hydroxyl group of N-(R)-lactyl-(S)-leucine
methylester (11), which was first transformed into the
mesylate 12. In a second step crude 12 was treated with
N-ethylthiourea (13) in EtOH at 80 °C to first undergo
nucleophilic substitution of the mesylate, forming analo-
gously the intermediate thiouronium salt, and then to
cyclize liberating 14 (43%) and the amine 10 in 49% yield
(Scheme 2).
Ap p lica tion to HUN-7293. Both sequences were
applied to the open-chained methylester 15,15 which is
(8) Ikai, K.; Takesako, K.; Shiomi, K.; Moriguchi, M.; Umeda, Y.;
Yamamoto, J .; Kato, I.; Naganawa, H. J . Antibiotics 1991, 44, 925.
(9) Trischman, J . A.; Tapiolas, D. M.; J ensen, P. R.; Dwight, R.;
Fenical, W.; McKee, T. C.; Ireland, C. M.; Stout, T. J .; Clardy, J . J .
Am. Chem. Soc. 1994, 116, 757.
(10) Hegde, V. R.; Puar, M. S.; Chan, T. M.; Dai, P.; Das, P. R.; Patel,
M. J . Org. Chem. 1998, 63, 9584.
(11) Steiner, J . R.; Barnes, C. L. Int. J . Pept. Protein Res. 1988, 31,
212.
(12) Hamano, K.; Kinoshita, M.; Furuya, K.; Miyamoto, M.; Taka-
matsu, Y.; Hemmi, A.; Tanzawa, K. J . Antibiot. 1992, 45 (8), C-4.
(13) J ust, G.; Kim, S. Can. J . Chem. 1977, 55, 427.
(14) Steglich, W.; Batz, H.-G. Angew. Chem. 1971, 83, 83.
obtained from 1 by alkaline methanolysis as a 4:1
mixture of the diastereomeres arising from partial race-
mization (∼20%) of MALA.7 For method A, in the first
step we oxidized 15 with Dess-Martin periodinane to
afford ketone 16 in 89% yield. However, in contrast to
(15) Dreyfuss, M. M.; Foster, C. A.; Naegeli, H.-U.; Oberhauser, B.
WO Patent 96,03430 A1, 1996.
8300 J . Org. Chem., Vol. 67, No. 24, 2002

Selective Cleavage of 2-Hydroxy Acid Amides
SCHEME 3
(LiOH, THF/H2O), producing the lithium salt of the
corresponding hydroxy acid 21 (98%), which was a 5:1
mixture of the 7-S and 7-R isomer occurring from partial
racemization.3 Amide bond formation between 20 and the
open-chain hydroxy acid 21 was effected by treatment
with HATU in DMF producing 22 (93%), the substrate
for the degradation sequence. Mesylation of 22 followed
by treatment with N-ethylthiourea (13) gave the desired
heptapeptide 23 in 56% yield over both steps. Finally,
saponification of the methyl ester and macrocyclization
using BOP as the condensing agent in acetonitrile at a
concentration of 6 × 10-4 M gave aza HUN-7293 (24) in
31% yield (15% from 1). This result of the cyclization step
is superior to the total synthesis route7 via ring closure
forming the MLEU3-LEU4 amide bond (22% yield, 5:1
mixture together with its D-MLEU3 isomer). Neither the
product from a potential C21 epimerization in the course
of the cyclization reaction nor the D-MALA7 isomer were
observed.
Comparative NMR spectroscopic investigations of HUN-
7293 (1) and aza HUN-7293 (24) confirmed our working
hypothesis that the exchange of 7-COO by 7-CONH does
not significantly affect the overall conformation. In a
CDCl3 solution both compounds, 1 and 24, exist as one
main conformer (85%) besides traces of two others (about
10 and 5%). For 1 it was reported that conformations in
CDCl3/DMSO and MeOH/H2O are very similar, suggest-
ing that conformations observed in organic solvents can
be assumed to also exist in aqueous solution.4 The
biological profile of 24 was found to be very similar to
that observed for 1; however, IC50 values for inhibition
of the single adhesion molecules were found to be (10-
20)-fold higher.7
the transformation of 4, treatment of 16 with thiosemi-
carbazide (5) did not give detectable amounts of the
desired amine. For method B we found our model reaction
to be more successful. Mesylation of 15 in the presence
of N-methylmorpholine (NMM) and subsequently heating
of crude mesylate 17 with 13 in EtOH at 80 °C for 24 h
(Scheme 3) yielded highly reproducibly the desired amine
18 in 70-75% yield.
In the course of our medicinal chemistry program 18
served as a central building block for the synthesis of a
series of novel cyclic peptides related to HUN-7273 (1)
accessed via coupling with N-protected amino acids and
final ring closure forming an amide bond between MALA7
and the amino acids newly introduced. Corresponding
D-MALA isomers originating from racemization in the
saponification steps were conveniently separated from
the products with natural stereochemistry for MALA7 by
flash chromatography on silica gel, consistently eluting
as less polar byproducts.
Syn th esis of a za HUN-7293. For the semisynthesis
of aza HUN-7293 (24) itself we first formed the bond
between MALA7 and the D-dehydro-Gln moiety followed
by the degradation protocol and ring fusion between
D-dehydro-Gln and PrLEU2 (Scheme 4). Generally, this
route was found to yield less of the corresponding
D-MALA isomers as observed for that via 18. The synthon
for the amino counterpart of (2R)-hydroxy-4-cyanobutyric
acid ((2R)-DGCN) methyl D-dehydroglutaminate 20 was
obtained as the corresponding hydrochloride from the
N-Boc derivative 197 by esterification with CH2N2 in Et2O
and subsequent cleavage of the Boc-protecting group with
hydrochloric acid in CH2Cl2. Starting from 1, the lactone
bond was selectively cleaved under alkaline conditions
Con clu sion
In conclusion, a two-step sequence for selective cleav-
age of 2-hydroxy acid amides in the presence of an ester
and a series of methylated and unmethylated amide
bonds has been developed. Evaluating two potential
approaches, we found that transformation of the hydroxyl
group into a leaving group via mesylation followed by
treatment with N-ethylthiourea at elevated temperature
gave the corresponding amine in good overall yield.
Successful application of this methodology to the semi-
synthesis of aza HUN-7293 (24) demonstrated that
degradation of the 2-hydroxy acid moiety of a cyclodep-
sipeptide followed by incorporation of an amino acid and
final cyclization is an attractive strategy to achieve
backbone ester/amide exchange by a chemical “point
mutation” strategy without multistep total synthesis of
the related cyclic peptide.
Exp er im en ta l Section
Gen er a l. All the reactions were carried out under an argon
atmosphere. 1H NMR was measured at 250, 400, or 500 MHz,
and 13C NMR was taken at 62.9, 100.6, or 125.8 MHz in the
solvent indicated. High-resolution mass spectra were recorded
in the ESI mode. Samples for analytics were additionally
purified by size exclusion chromatography to remove traces
of silica gel. Elemental analyses were performed by Solvias.
(R,S)-3-Meth yl-2-(2-oxop r op ion yla m in o)bu tyr ic a cid
m eth yl ester 4: To a solution of sodium pyruvate (2) (1.1 g,
10 mmol), D,L-valine methylester hydrochloride (3) (1.68 g, 10
mmol), and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
J . Org. Chem, Vol. 67, No. 24, 2002 8301

Schreiner et al.
SCHEME 4a
a
Reagents and conditions: (a) CH2N2, Et2O, rt; (b) HCl, CH2Cl2, rt; (c) LiOH/THF, H2O, rt; (d) HATU/DIEA, DMF, 0 ˚C; (e) Ms-Cl/
NMM, CH2Cl2, rt; (f) 9, EtOH, 80 ˚C; (g) LiOH, THF/H2O; (h) BOP/DIEA, MeCN, rt.
hydrochloride (EDC; and 2.88 g, 10 mmol) in dry DMF (100
mL) at room temperature was added N-methylmorpholine (1.1
mL, 15 mmol) and the reaction mixture was stirred overnight.
The solvent was evaporated and the residue obtained was
dissolved in EtOAc and sequentially washed with 1 M aqueous
HCl, aqueous NaHCO3 solution, and brine, dried over Na2SO4,
and concentrated. Flash chromatography with EtOAc/c-hexane
(1:1) afforded 4 (484 mg, 24%) as a pale yellow oil: 1H NMR
(225 MHz, CDCl3) δ 7.36 (br s, 1H), 4.47 (dd, J ) 9.2 and 5.0
Hz, 1H), 3.75 (s, 3H), 2.47 (s, 3H), 2.22 (m, 1H), 0.93 (d, J )
6.9 Hz, 3H), 0.91 (d, J ) 6.9 Hz, 3H); 13C NMR (62.9 MHz,
CDCl3) δ 196.3, 171.3, 159.9, 57.3, 52.3, 31.3, 24.4, 18.9, 17.7;
HRMS(ESI) calcd for C9H16NO4 [M + H]+ 202.1079, found
202.1075.
mg (17%) of 11 as a colorless oil. 11: 1H NMR (250 MHz,
CDCl3) δ 6.90 (d, J ) 8.2 Hz, 1H), 4.58 (dt, J ) 8.6 and 5.0
Hz, 1H), 4.22 (dq, J ) 6.8 and 5.2 Hz, 1H), 3.70 (s, 3H), 3.11
(d, J ) 5.1 Hz; 1H), 1.67-1.49 (m, 3H), 1.40 (d, J ) 6.8 Hz,
3H), 0.90 (d, J ) 6.2 Hz, 6H); 13C NMR (62.9 MHz, CDCl3) δ
174.8, 173.8, 68.8, 52.7, 50.8, 41.9, 25.3, 23.2, 22.2, 21.5; [R]20
D
-22.3 (c 1, MeOH); HRMS(ESI) calcd for C10H19NO4Na [M +
Na]+ 240.1212, found 240.1210.
Clea va ge of 11 p r od u cin g (S)-Leu -OMe 10 a n d 2-eth yl-
a m in o-5-m eth yl-th ia zol-4-on e 14: To a solution of 7 (100
mg, 0.46 mmol) in CH2Cl2 (5 mL) at 0 °C was added N-
methylmorpholine (126 µL, 1.15 mmol) and Ms-Cl (89 µL,1.15
mmol) and the reaction was allowed to warm to room tem-
perature within 3 h. Conversion was checked by TLC (SiO2,
EtOAc). The solvent was evaporated and the residue was
redissolved in EtOAc and sequentially washed with 1 M
aqueous HCl, aqueous NaHCO3 solution, and brine. After
drying over Na2SO4 the solvent was evaporated delivering
crude 12. A solution of 12 and 13 (240 mg, 2.3 mmol) in 3 mL
of dry EtOH was heated at 80 °C for 24 h. The solvent was
evaporated and the residue obtained purified by chromatog-
raphy with 0-10% methanol in EtOAc affording 31 mg (43%)
of 14 and 33 mg (49%) of the amine 10 as colorless foam. 14:
1H NMR (400 MHz, CDCl3) δ 4.10 (q, J ) 7.3 Hz, 1H), 3.38 (q,
J ) 7.3 Hz, 1H), 1.64 (d, J ) 7.3 Hz, 3H), 1.41 (t, J ) 7.3 Hz,
3H); 13C NMR (62.9 MHz, CDCl3) δ 189.44, 182.04, 49.74,
40.57, 19.43, 14.64. 10 was found to be identical with a sample
obtained by treatment of an aqueous solution of the com-
mercially available (S)-Leu-OMe hydrochloride with Na2CO3
and subsequent extraction with EtOAc.
(R,S)-3-Meth yl-2-(2-E-(N-th iocar bon ylam in o)h ydr azon o-
p r op ion yla m in o)bu tyr ic a cid m eth yl ester (E)-6: A solu-
tion of 4 (90 mg, 0.45 mmol) and 5 (50 mg, 0.55 mmol) in
ethanol (6 mL) was stirred for 6 h at 80 °C. The solvent was
evaporated and the residue purified by chromatography with
EtOAc/MeOH (0-20% MeOH), delivering 61 mg (50%) of E-6
and 27 mg (42%) of 8 as white solids as well as 23 mg (39%)
of Val-OMe 7 as a colorless oil. E-6: 1H NMR (225 MHz,
CDCl3) δ 9.14 (br s, 1H), 7.94 (br s, 1H), 7.77 (d, J ) 9.1, 1H),
7.45 (br s, 1H), 4.51 (dd, J ) 9.1 and 6.6 Hz, 1H), 3.70 (s, 3H),
2.26 (m, 1 H), 2.11 (s, 3H), 0.91 (d, J ) 6.8 Hz, 3H), 0.90 (d, J
) 6.8 Hz, 3H); 13C NMR (62.9 MHz, CDCl3) δ 179.4, 173.0,
163.4, 142.8, 57.8, 52.1, 31.0, 19.1, 18.4, 10.9; HRMS(ESI) calcd
for C10H19N4O3S [M + H]+ 275.1178, found 275.1179. 7 was
found to be identical with a commercial sample.
(S)-2-((R)-2-Hydr oxypr opion ylam in o)-4-m eth ylpen tan o-
ic a cid m eth yl ester 11: To a solution of (S)-Leu-OMe (1.78
g, 9.8 mmol) and lithium (R)-lactate (9) (1.0 g, 9.8 mmol) in
EtOAc/DMF (6:1, 60 mL) at 0 °C was added propanphosphonic
acid anhydride (50% in EtOAc, 6 mL, 20 mmol) and DIEA (5.14
mL, 30 mmol). The reaction mixture was allowed to warm to
room temperature overnight. The solvent was evaporated and
the residue obtained was redissolved in EtOAc and sequen-
tially washed with 1 M aqueous HCl, aqueous NaHCO3
solution, and brine, dried over Na2SO4, and concentrated.
Flash chromatography with EtOAc/c-hexane (1:2) afforded 364
N-[(2S,4R)-2-[[N-[N-[N-[(2S,4R)-2-[N-[(R)-2-Hyd r oxy-4-
cya n obu ta n oyl]a m in o]-4-m eth ylh ep ta n oyl]-N-m eth yl-L-
le u cin yl]-L-le u cin yl]-N 1′-m e t h oxy-N -m e t h yl-L-t r yp t o-
p h a n yl]a m in o]-4-m eth ylh ep ta n oyl]-N-m eth yl-L-a la n in e
m eth yl ester 15: To a solution of 1 (30 g, 30.6 mmol) in dry
methanol (450 mL) was added sodium hydride (40 mg, 1.67
mmol) with stirring at room temperature until no starting
material could be observed on TLC. Then the reaction mixture
was neutralized with solid CO2 and the solvent was evapo-
rated. After the residual methanol was removed by codistil-
lation with toluene the material was used for the next step.
8302 J . Org. Chem., Vol. 67, No. 24, 2002

Selective Cleavage of 2-Hydroxy Acid Amides
N-[(2S,4R)-2-[[N-[N-[N-[(2S,4R)-2-[N-[(2-oxo-4-cya n obu -
ta n oyl]a m in o]-4-m eth ylh ep ta n oyl]-N-m eth yl-L-leu cin yl]-
L-leu cin yl]-N1′-m eth oxy-N-m eth yl-L-tr yptoph an yl]am in o]-
4-m eth ylh ep ta n oyl]-N-m eth yl-L-a la n in e m eth yl ester 16:
A solution of 15 (200 mg, 0.2 mmol), Dess-Martin periodinane
(102 mg, 0.24 mmol), and pyridine (33 µL, 0.4 mmol) in CH2-
Cl2 (20 mL) was stirred at room temperature for 6 h. Then a
saturated Na2S2O3 solution (1 mL) and a 5% aqueous NaHCO3
solution (5 mL) were added and the mixture was stirred for
an additional 30 min. After dilution with CH2Cl2 the organic
layer was washed with 0.1 M aqueous HCl, saturated NaHCO3
solution, and brine. Purification by size exclusion on Sephadex
LH-20 with MeOH gave 180 mg (89%) of 16. 1H NMR (500
MHz, CDCl3, only assigned signals of the major conformer) δ
8.40 (d, J ) 8.0 Hz, 1 H), 7.55 (d, J ) 7.9 Hz, 1 H), 7.48 (d, J
) 8.9 Hz, 1H), 7.41 (d, J ) 8.2 Hz, 1H), 7.36 (t, J ) 7.3 Hz,
1H), 7.14 (t, J ) 7.9 Hz, 1H), 7.00 (s, 1H), 6.28 (d, J ) 6.3 Hz,
1H), 5.43 (q, J ) 7.3 Hz, 1H), 5.02 (m, 2H), 4.90-4.78 (m, 2H),
4.16 (m, 1H), 4.05 (s, 3H), 3.74 (s, 3H), 3.40-3.23 (m, 3H), 3.16
(m, 1H), 2.98 (s, 3H), 2.95 (s, 3H), 2.94 (s, 3H), 2.64 (m, 1H),
1.47 (d, J ) 7.4 Hz, 3H), 0.44 (d, J ) 6.6 Hz, 3H), -0.09 (d, J
product was dissolved in CH2Cl2 (50 mL) and a 1 M solution
of diazomethane in Et2O was added until the solution became
slightly yellow. The solvent was again evaporated and the
residue obtained was purified by chromatography with EtOAc/
c-hexane (1:2) affording 1.52 g (77%) of (R)-2-(dimethylethyl-
oxycarbonylamino)-4-cyanobutyric acid methyl ester as a color-
less oil.
To a solution of (R)-2-(dimethylethyloxycarbonylamino)-4-
cyanobutyric acid methyl ester (163 mg, 0.67 mmol) in dioxane
(3 mL) was added 32% aqueous HCl (0.1 mL) and the solution
was stirred at room temperature for 5 h. Evaporation of the
solvent gave 100 mg (84%) of 20: 1H NMR (500 MHz, CD3-
OD) δ 4.78 (br s, 3H), 4.19 (t, J ) 6.5 Hz, 1H), 3.90 (s, 3H),
2.78 (m, 2H), 2.31 (m, 2H); 13C NMR (125.8 MHz, CD3OD) δ
160.34, 110.09, 44.59, 43.40, 18.16, 4.88; HRMS(ESI) calcd for
C6H11N2O2 [M + H]+ 143.0821, found 143.0820; [R]20 -32.6
D
(c 1, MeOH).
(2R)-N-[N-[(2S,4R)-2-[[N-[N-[N-[(2S,4R)-2-[N-[(R)-2-Hy-
d r oxy-4-cya n ob u t a n oyl]a m in o]-4-m et h ylh ep t a n oyl]-N-
m et h yl-L-leu cin yl]-L-leu cin yl]-N1′-m et h oxy-N-m et h yl-L-
t r yp t op h a n yl]a m in o]-4-m et h ylh ep t a n oyl]-N-m et h yl-L-
a la n in yl]a m in o-4-cya n obu ta n oic a cid m eth yl ester 22:
To a solution of 20 (239 mg, 1.34 mmol), 21 (597 mg, 0.6 mmol),
and HATU (570 mg, 1.5 mmol) in DMF (10 mL) at 0 °C was
added DIEA (0.48 mL, 2.8 mmol) with stirring for 2 h. The
solvent was evaporated and the residue was dissolved in
EtOAc and sequentially washed with 1 M aqueous HCl,
aqueous NaHCO3 solution, and brine. After drying over
anhydrous Na2SO4 the solvent was removed and the crude
product purified by chromatography with EtOAc/c-hexane/
MeOH (4:8:1). Yield: 625 mg (93%) as a colorless oil. 1H NMR
) 6.6 Hz, 3H), -0.48 (ddd, J ) 13.8, 10.9 and 2.9 Hz, 1H); 13
C
NMR (125.8 MHz, CDCl3, only assigned signals of the major
conformer) δ 194.52, 173.39, 173.28, 172.74, 172.43, 170.33,
170.02, 159.19, 132.77, 124.13, 122.93, 122.55, 121.82, 120.15,
119.28, 118.65, 108.67, 107.15, 65.94, 61.55, 54.25, 52.53,
52.18, 48.39, 47.27, 40.53, 38.52, 37.77, 37.72, 33.64, 31.25,
31.15, 30.48, 29.57, 24.13, 23.83, 23.16, 14.79, 11.55; [R]20
D
-127.2 (c 1, MeOH). Anal. Calcd for C54H86N8O10: C, 64.39;
H, 8.61; N, 11.12. Found: C, 64.12; H, 8.63; N, 11.13.
N-[(2S,4R)-2-[[N-[N-[N-[(2S,4R)-2-a m in o-4-m eth ylh ep -
ta n oyl]-N-m eth yl-L-leu cin yl]-L-leu cin yl]-N1′-m eth oxy-N-
m et h yl-L-t r yp t op h a n yl]a m in o]-4-m et h ylh ep t a n oyl]-L-
m eth yl-L-a la n in e m eth yl ester 18: To a solution of 15 (31
g, 30.6 mmol) in CH2Cl2 (400 mL) at 0 °C was added
N-methylmorpholine (11 mL, 100 mmol) and Ms-Cl (3.16 mL,
40 mmol) and the reaction was allowed to warm to room
temperature within 3 h. Conversion was checked by TLC (SiO2,
EtOAc). For workup EtOAc/c-hexane (1:2, 200 mL) was added
and the solution was washed with 1 M aqueous HCl, saturated
NaHCO3 solution, and brine. After drying over Na2SO4 the
solvent was evaporated affording crude 14. A solution of 14
(35 g) and 9 (25 g, 0.24 mol) in dry EtOH (150 mL) was heated
at 80 °C for 24 h. For workup the solvent was evaporated and
the residue obtained was taken up in toluene (1.5 L). The
solution was extracted with 20% K2HPO4 solution (5 × 200
mL) and dried over Na2SO4. The solvent was removed and the
residue obtained purified by chromatography with EtOAc/c-
hexane/MeOH (6:12:1-2) affording 20.6 g (75%) of 18 as a
(500 MHz, d
6-DMSO, 410 K) δ 7.74 (br s, 1H), 7.61 (d, J ) 8.0
Hz, 1 H), 7.48 (br s, 1H), 7.38 (d, J ) 8.2 Hz, 1H), 7.24 (s, 1H),
7.19 (t, J ) 7.1 Hz, 1H), 7.06 (t, J ) 7.1 Hz, 1H), 5.47 (d, J )
5.6 Hz, 1H), 5.25 (br s, 1H), 4.97 (m, 2H), 4.82 (m, 2H), 4.67
(br s, 1H), 4.43 (m, 1H), 4.03 (s, 3H), 4.02 (q, J ) 6.8 Hz, 1H),
3.67 (s, 3H), 3.30 (dd, J ) 15.3, 7.0 Hz, 1H), 3.01 (dd, J )
15.3, 8.6 Hz, 1H), 2.97 (br s, 3H), 2.92 (r bs, 3H), 2.47 (m, 2H),
2.13 (m, 1H), 2.00 (m, 2H), 1.78 (m, 1H), 1.70-1.15 (series of
multiplets, 27H), 0.88 (m, 24H); HRMS(ESI) calcd for C59H94N10-
O
11Na [M + Na]+ 1141.7001, found 1141.6997; [R]20 -116.2
D
(c 0.5, MeOH). Anal. Calcd for C59H94N10O11: C, 63.30; H, 8.46;
N, 12.51. Found: C, 63.12; H, 8.41; N, 12.50.
(2R)-N-[N-[(2S,4R)-2-[[N-[N-[N-[(2S,4R)-2-Am in o-4-m e-
th ylh eptan oyl]-N-m eth yl-L-leu cin yl]-L-leu cin yl]-N1′-m eth -
oxy-N-m eth yl-L-tr yptoph an yl]am in o]-4-m eth ylh eptan oyl]-
N-m eth yl-L-alan in yl]am in o-4-cyan obu tan oic acid m eth yl
ester 20: 19 (590 mg, 0.53 mmol) was transformed into 20
according to the procedure described for 18, yielding 300 mg
(56%) of the title compound as a colorless oil. 1H NMR (500
MHz, d6-DMSO, 410 K) δ 7.71 (br s, 1H), 7.58 (d, J ) 8.0 Hz,
1 H), 7.35 (d, J ) 8.2 Hz, 1H), 7.21 (s, 1H), 7.17 (t, J ) 7.6 Hz,
1H), 7.02 (t, J ) 7.5 Hz, 1H), 5.21 (br s, 1H), 4.91 (m, 1H),
4.87 (br s, 1H), 4.78 (m, 2H), 4.64 (br s, 1H), 4.39 (m, 1H),
4.00 (s, 3H), 3.65 (m, 1H), 3.64 (s, 3H), 3.28 (dd, J ) 15.3, 7.0
Hz, 1H), 2.99 (dd, J ) 15.3, 8.5 Hz, 1H), 2.95 (br s, 3H), 2.51
(t, J ) 7.2 Hz, 2H), 2.10 (m, 1H), 1.98 (m, 1H), 1.76-1.12
(series of multiplets, 27H), 0.87 (m, 24H); HRMS (ESI) calcd
1
yellow oil. H NMR (500 MHz, CD3OD, only assigned signals
of the major conformer) δ 7.61 (d, J ) 8.0 Hz, 1 H), 7.45 (d, J
) 8.2 Hz, 1H), 7.25 (t, J ) 7.5 Hz, 1H), 7.23 (s, 1H), 7.11 (t, J
) 7.5 Hz, 1H), 5.19-5.13 (m, 2H), 5.00-4.92 (m, 2H), 4.38 (dt,
J ) 12.1 and 2.8 Hz, 1H), 4.07 (s, 3H), 3.79(dd, J ) 9.2 and
6.7 Hz, 1H), 3.75 (s, 3H), 3.48 (dd, J ) 15.1 and 3.2 Hz, 1H),
3.15 (s, 3H), 2.95 (s, 3H), 2.94 (s, 3H), 0.41 (d, J ) 6.6 Hz,
3H), 0.06 (d, J ) 6.6 Hz, 3H), -0.49 (ddd, J ) 13.8, 10.9 and
2.9 Hz, 1H); 13C NMR (125.8 MHz, CD3OD, only assigned
signals of the major conformer) δ 176.81, 174.92, 170.74,
170.60, 132.84, 124.02, 122.83, 122.68, 120.20, 118.81, 108.64,
107.02, 65.41, 61.73, 53.56, 51.73, 49.37, 49.06, 37.86, 31.32,
29.88, 24.03, 22.31, 18.96, 13.38; HRMS(ESI) calcd for
for C54H90N9O9 [M + H]+ 1008.6862, found 1008.6862; [R]20
D
-119.4 (c 0.33, MeOH). Anal. Calcd for C54H89N9O9: C, 64.32;
H, 8.90; N, 12.50. Found: C, 64.03; H, 8.92; N, 12.46.
Aza -HUN-7293 (24): To a solution of 20 (300 mg, 0.3 mmol)
was added a LiOH solution (0.5 M, 0.7 mL) with stirring
overnight at room temperature. The solvent was removed and
the residue obtained was dissolved in EtOAc and washed with
brine. After drying over anhydrous Na2SO4 the solvent was
evaporated. The crude betain and BOP (531 mg, 1.2 mmol)
were dissolved in dry acetonitrile (500 mL), DIEA (0.21 mL,
1.2 mmol) was added, and the reaction mixture was stirred at
room temperature for 18 h. The solvent was removed and the
residue dissolved in EtOAc (200 mL) and sequentially washed
with 1 M HCl, saturated aqueous NaHCO3 solution, and brine.
C
49H84N7O8 [M + H]+ 898.6381, found 898.6380; [R]20D -121.9
(c 1, MeOH). Anal. Calcd for C49H83N7O8: C, 65.52; H, 9.31;
N, 10.92. Found: C, 65.22; H, 9.34; N, 10.88.
(R)-2-Am in o-4-cya n obu tyr ic a cid m eth yl ester h yd r o-
ch lor id e 20: To a solution of Boc-(D)-Gln-OH 19 (2.0 g, 8.1
mmol) in pyridine (30 mL) was added Ac2O (0.92 mL, 9.7
mmol) with stirring at room temperature overnight. The
solvent was evaporated and the residue was redissolved in
EtOAc and washed with 1 M aqueous HCl and brine. After
drying over Na2SO4 the solvent was removed. The crude
J . Org. Chem, Vol. 67, No. 24, 2002 8303

Schreiner et al.
After drying over Na2SO4 the solvent was removed and the
crude product purified by chromatography with EtOAc/c-
ing of the manuscript.
hexane/MeOH (7:14:1). Yield: 91 mg (31%). [R]20 -151.4 (c
D
Su p p or tin g In for m a tion Ava ila ble: Comparison of the
1H and 13C NMR spectra of 1 and 24; copies of 1H and 13C NMR
spectra of 2, (E)-4, 7, and 10, respectively. This material is
available free of charge via the Internet at http://pubs.acs.org.
0.5, MeOH).
Ack n ow led gm en t. E.P.S. thanks J acques Eustache
for fruitful dicussions. Furthermore, the authors thank
Berndt Oberhauser and Dieter Scholz for critical read-
J O025979G
8304 J . Org. Chem., Vol. 67, No. 24, 2002