Nonpeptide integrin antagonists: RGD mimetics incorporating substituted azabicycloalkanes as amino acid replacements

Article abstract of DOI:10.1002/ejoc.200600840

Azabicyclo[4.3.0]alkanes appropriately substituted on both cycles have been synthesized as potential scaffold mimics of the RGD signaling motif of integrin. Two sets of functionalized azabicycloalkanes have been examined. In vitro assays established that

Full text of DOI:10.1002/ejoc.200600840

FULL PAPER
DOI: 10.1002/ejoc.200600840
Nonpeptide Integrin Antagonists: RGD Mimetics Incorporating Substituted
Azabicycloalkanes as Amino Acid Replacements
Leonardo Manzoni,*[a] Michele Bassanini,[b] Laura Belvisi,[b] Ilaria Motto,[b]
Carlo Scolastico,[b] Massimo Castorina,[c] and Claudio Pisano[c]
Keywords: Angiogenesis / Antitumor agent / Lactams / Azabicycloalkanes / RGD mimetics
Azabicyclo[4.3.0]alkanes appropriately substituted on both
cycles have been synthesized as potential scaffold mimics of
the RGD signaling motif of integrin. Two sets of function-
alized azabicycloalkanes have been examined. In vitro as-
says established that 21 has a good affinity specifically for
αvβ3.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
In this paper we report the synthesis of new substituted
bicyclic lactams (Figure 1) as nonpeptide scaffolds mimick-
ing the RGD sequence.
Introduction
The integrins are a class of cell surface adhesion proteins
that play important roles in cell–cell and cell–matrix inter-
actions,[1] being involved in diverse physiological processes.
In particular, the αvβ3 and αvβ5 subgroups of integrins are
expressed in various cell types, such as endothelial cells,
melanoma, platelets, osteoclasts, and smooth muscle cells.
Furthermore, they also play important roles in angiogenesis
and in tumor cell migration by interacting with vitronectin
on the extracellular matrix, mainly through the recognition
of the tripeptide sequence RGD (Arg-Gly-Asp).[2]
This sequence has been used as a lead for the develop-
ment of different integrin antagonists. The RGD sequence
was first incorporated into various linear and cyclic pep-
tides.[3] During our studies on integrin inhibitors, we re-
ported active and selective small-molecule antagonists of
the αvβ3 receptor in the forms of the cyclic pentapeptides
c(-RGDTemplate-), where “Template” represents azabicy-
clo[x.3.0]alkane amino acids.[4]
Recently, research has been focused on the development
of selective nonpeptide αvβ3 integrin antagonists,[5] because
of the enhanced metabolic stability, bioavailability, and bio-
logical absorption of peptidomimetic compounds.
Figure 1. Library of potential functionalized bicyclic lactam mim-
ics of the RGD sequence.
These lactams are substituted on both cyclic compo-
nents, one bearing an analogue of the Arg side chain and
the other bearing an analogue of the Asp side chain. The
occurrence of both guanidinium and carboxylate groups in
these nonpeptide molecules is an essential element for mim-
icking of the Arg and Asp side chains, respectively, of RGD.
Although many reports on the synthesis of bicyclic lac-
tams can be found in the literature,[6] only a few describe
the preparation of azabicyclo[x.y.0]alkanes bearing func-
tionalized side chains.[7] In the course of our studies of pep-
tide secondary structure mimics we have already synthe-
sized functionalized bicyclic lactams by different strate-
gies,[8] and in particular we have synthesized functionalized
[a] C.N.R. – Istituto di Scienze e Tecnologie Molecolari (ISTM),
c/o Centro Interdisciplinare Studi bio-molecolari e applicazioni
Industriali (CISI),
Via Fantoli 16/15, 20138 Milano, Italy
Fax: +39-02-50320945
E-mail: Leonardo.manzoni@istm.cnr.it
[b] Dipartimento di Chimica Organica e Industriale and Centro
Interdisciplinare Studi bio-molecolari e applicazioni Industriali
(CISI), Università degli Studi di Milano,
Via Venezian 21, 20133 Milano, Italy
[c] R&D Sigma-Tau S.p.A.,
Via Pontina Km 30.400, 00040 Pomezia, Italy
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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L. Manzoni et al.
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6,5-fused bicyclic lactams by a Horner–Emmons-based quence to give compounds 3 and 4. The bis-Cbz carbox-
strategy.[9]
amidine 12 was chosen as the electrophilic guanidation
Here we report the use of these bicyclic lactam deriva- reagent for this transformation.[10] The newly introduced
tives for the preparation of integrin inhibitors. All the non- guanidyl group mimics the arginine side chain.
peptide RGD mimics synthesized have been tested by an in
Removal of the nitrogen protecting group was achieved
vitro integrin-binding assay. The results showed 21 to be a by treatment with HClO4 in tBuOAc as solvent; these con-
good-affinity specific ligand for αvβ3 integrin.
ditions enabled us to remove the Boc moiety, leaving the
tert-butyl ester as the carboxylic protecting group. The re-
sulting free amines 5 and 6 were coupled with the appropri-
ate carboxyl derivative to give compounds 7–11 after benzyl
and Cbz removal by hydrogenolysis on Pd/C.
With the aim of expanding the library of compounds and
studying the influence of the positions of the pharmaco-
phoric groups (guanidyl and carboxyl groups) in the re-
cognition site of the receptor, we decided to synthesize a
second series of RGD mimics, and so we changed the
pharmacophoric group on the proline moiety, introducing
a carboxyl instead of a guanidyl group in position 7 of the
scaffold. The presence of the other pharmacophoric group,
necessary for the binding with the receptor, was ensured by
condensation with an arginine (Scheme 2).
Results and Discussion
The synthetic approach is illustrated in Scheme 1. The
known bicyclic lactams 1[9] (3S) or 2 (3R) were each sub-
jected to a one-step reduction/guanidinylation reaction se-
Scheme 2. Reagents and conditions: a) PDC, DMF, (15, 76%; 16,
74%). b) 1. CsCO3, MeOH, H2O, 2. BnBr, DMF (17, 92%; 18,
94%). c) HClO4, tBuOAc. d) Z-Arg(Z)2-OH, DIC, HOBT, THF.
e) H2, Pd/C, MeOH, (21, 43% over three steps; 22; 46% over three
steps).
Scheme 1. Reagents and conditions: a) Ph3P, H2O, THF, 12, (3,
76%; 4, 89%). b) HClO4, tBuOAc. c) Monobenzyl phthalate,
EDC·HCl, DMAP, HOBT, collidine, THF, 65%. d) Monobenzyl
malonate, EDC·HCl, DMAP, HOBT, collidine, THF. e) Monoben-
zyl N-acetyl-aspartate, EDC·HCl, DMAP, HOBT, THF. f) H2, Pd/
C, MeOH, (7, 41% over two steps; 8; 63% over three steps; 9,
33% over three steps; 10, 66% over three steps; 11, 67% over three
steps).
Compounds 15 and 16 were prepared from the known
alcohols 13 and 14 by oxidation with PDC. The newly in-
troduced carboxylic group mimics the aspartyl side chain.
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Protection of the carboxyl groups of 15 and 16 as benzyl Computational Studies
derivatives was achieved by treatment of their preformed
To interpret the experimental results on a structural ba-
sis, molecular docking studies were performed for the com-
pounds 7–11 and 21–22.
The protein binding site was derived from the X-ray crys-
tal structure of the extracellular segment of integrin αvβ3 in
complexation with the cyclic pentapeptide ligand
EMD 121974.[11] For each compound the global minimum
energy conformer derived from an MC/EM[12] conforma-
tional search was selected as the representative starting con-
formation for docking studies.
cesium salts with benzyl bromide, and the nitrogen protect-
ing groups were then removed by treatment with HClO4 to
give compounds 19 and 20, which were coupled with suit-
ably protected arginine components with the aid of DIC
and HOBT as condensing agents to give, after hydro-
genolysis, compounds 21 and 22.
Biological Results
The RGD mimics 7–11 and 21–22 were examined in vitro
The crystal structure of the peptide/integrin complex
for their abilities to compete with [125I]-echistatin for bind- provides the actual conformation of EMD 121974 bound
ing to the purified αvβ3 and αvβ5 receptors (Table 1). It had to the αvβ3 integrin active site and can serve as a basis for
previously been demonstrated that both purified and mem- interpretation of the general mode of interaction of inte-
brane-bound integrins αvβ3 and αvβ5, which had very high grins with other RGD mimics. Examination of the three-
affinity for echistatin, could be inhibited efficiently by linear dimensional structure of the cyclic pentapeptide ligand
and cyclic RGD-containing peptides. Affinities of com- EMD 121974 bound to the αvβ3 integrin receptor (Protein
pounds c(RGDfV), EMD 121974, and ST1646 for the αvβ3 Data Bank entry code = 1L5G[11]) reveals a conformation
and αvβ5 integrins were also determined, for reference, in characterized by an inverse γ-turn with Asp at position
the same assays. Of the seven mimics tested, compound 21 (i+1) and by a distorted βIIЈ-turn with Gly and Asp at the
showed the highest affinity towards αvβ3 integrin.
(i+1)- and (i+2)-positions, respectively. A distance of 8.9 Å
Table 1. Inhibition of 125I-Echistatin binding to αvβ3 and αvβ5 receptors.[a]
[a] IC50 values were calculated as the concentration of compound required for 50% inhibition of echistatin binding as estimated by the
Allfit program. All values are the means (Ϯ standard deviation) of triplicate determinations.
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L. Manzoni et al.
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Figure 2. Top-ranking binding modes of compound 21 (gray ball and stick representation) with the crystal structure of the extracellular
αVβ3 integrin domain overlaid on the bound conformation of EMD 121974 (gray tube representation). Selected integrin residues involved
in the interactions with EMD 121974 are shown in black (tube representation). The Mn2+ ion at MIDAS is shown as a black CPK
sphere. Non-polar hydrogens were removed for clarity.
between the Asp and Arg Cβ atoms and an almost extended
Docking calculations for compound 21 produced top-
conformation of the RGD sequence are observed in this ranked poses in which the important ionic interactions were
pentapeptide bound conformation (Figure 2). The most im- conserved but the stabilizing hydrogen bonds with Arg216
portant interactions involve the positively charged Arg gua- chain β were loosened. Moreover, as the cleft in which the
nidinium group of the ligand and the negatively charged ligands bind is rather shallow, compound 21 shows an alter-
side chains of Asp150 and Asp218 in the α subunit, to- native binding mode, differing in the orientation of the car-
gether with one of the Asp carboxylate oxygens of the li- boxy group, so that it maintains the ionic interaction with
gand and the metal cation in the metal-ion-dependent ad- Ca2+ (average distance 2.4 Å) but not the stabilizing hydro-
hesion site (MIDAS) region of the β subunit. Further stabi- gen bond with Asn215 chain β (Figure 2).
lization could occur through hydrogen bonds between the
For compound 22, analogously to 21, docking calcula-
backbone NH of the Asp residue and the backbone car- tions showed that the inversion of stereochemistry at C-3
bonyl oxygen of Arg216 in the β subunit, as well as between again causes the loss of the electrostatic clamp-like func-
the other Asp carboxylate oxygen and the backbone amide tion.
hydrogen of Asn215 in the β subunit. Moreover, the central
In summary, if it is assumed that the X-ray pose de-
Gly residue is in close contact with the integrin surface.
scribes the best interaction mode with the αvβ3 integrin re-
Starting from this X-ray complex, structural models for ceptor, the models constructed by docking studies for the
the interactions of the selected compounds with the ligand- interaction of ligands 7–11 and 21–22 with αvβ3 integrin
binding site of the αvβ3 integrin receptor were generated confirmed that the bicyclic conformations of these ligands
by automated computational docking by use of the Glide enable them to fit properly in the shallow cleft of the recep-
program,[13] after removal of the peptide ligand. Automated tor, but that the short chain lengths (for compounds 7–11
docking calculations of the compounds 7–11 produced top- and 22) or different orientations of the carboxy groups of
ranked poses conserving only one of the important ionic the ligands in the binding site (for compound 21) may par-
interactions due to the short chain lengths.
tially abolish or modify the polar interactions and the H-
As the guanidine and carboxy groups of the ligands are bonding network governing the recognition process, re-
essential for binding to the integrin subunits α and β, sulting in a reduction in activity.
respectively, acting like an electrostatic clamp in attaching
to charged regions of the protein,[14,15] this could explain for the αVβ3 integrin is about two orders of magnitude
the activity data for ligands 7–11. lower than the affinity of the best cyclic RGD ligand
The binding affinity of the best peptide mimetic – 21 –
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Nonpeptide Integrin Antagonists
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buffer [20 m Tris-HCl (pH 7.4), 150 m NaCl, 2 m CaCl2, 1 m
MgCl2, and 1 m MnCl2]. Aliquots of the diluted receptors
(100 µL per well) were added to 96-well microtiter plates, which
were incubated overnight at 4 °C. The coating solution was re-
moved by aspiration, and blocking solution [coating buffer contain-
ing 1% bovine serum albumin (BSA), 200 µL] was added to the
wells, which were incubated for an additional 2 h at room tempera-
ture. After incubation, the plates were rinsed with blocking solution
(200 µL, 3ϫ) and incubated with the appropriate radiolabeled li-
gand for 3 h at room temperature. [125I]Echistatin (Amersham
(ST1646). The binding properties of the nonpeptide RGD
mimics based on azabicycloalkane scaffolds and described
here thus need to be improved in order to develop nanomo-
lar ligands useful for biomedical applications.
Molecular docking studies may assist this purpose
through exploitation of their ability to explain differences
in the biological results for closely related RGD mimetics
on a structural basis. Efforts to explore new functionalized
bicyclic scaffolds and substitution patterns and to develop
higher affinity integrin ligands with the aid of the docking Pharmacia, 0.05 nM and 0.1 nM, respectively) was used for αvβ3
and αvβ5. After incubation, the plates were sealed and counted in
the γ-counter (Packard). Each data point is the result of the average
of triplicate wells, and was analyzed by nonlinear regression analy-
sis with the Allfit program.
protocol as a virtual screening tool continue in our
laboratories.
Experimental Section
Chemistry
Docking Protocol: The automated molecular docking calculations
were carried out by use of the Glide (grid-based ligand docking
with energetics) module implemented in Schrödinger’s FirstDiscov-
ery Suite, version 2.7.[13] Although the protein is required to be
rigid, the program allows torsional flexibility in the ligand. Confor-
mational flexibility is handled in Glide by means of an extensive
conformational search, augmented by a heuristic screen that
rapidly eliminates conformations deemed unsuitable for binding to
a receptor. During the docking process, the bicyclic backbone con-
formation of the ligands was held fixed, whereas the side chain
dihedral angles were free to rotate. The refined poses are scored
with Schrödinger’s proprietary GlideScore scoring function.
GlideScore is based on Chem-Score[16] but includes a steric-clash
term and adds buried polar terms devised by Schrödinger to penal-
ize electrostatic mismatches.
1
General: H and 13C NMR spectra were recorded in CDCl3 solu-
tion as indicated, at 400 and 100.6 MHz, respectively. The chemical
shift values are given in ppm and the coupling constants in Hz.
Thin-layer chromatography (TLC) was carried out with Merck pre-
coated silica gel F-254 plates. Flash chromatography was carried
out with Macherey–Nagel silica gel 60, 230–400 mesh. Solvents
were dried by standard procedures, and reactions requiring anhy-
drous conditions were performed under nitrogen or argon. Elemen-
tal analyses were performed by the staff of the microanalytical lab-
oratory in our department.
General Procedure A. Reduction/Guanidinylation Reaction: Bis-Cbz
carboxamidine 12 (1.1 equiv.), water (6 equiv.), and triphenylphos-
phane (1.1 equiv.) were added to a solution of azide (1 equiv.) in
THF (1.2 mL). The reaction mixture was allowed to stir at room
temperature for 48 h, the solvent was removed under reduced pres-
sure, and the crude product was purified by flash chromatography
(EtOAc/petroleum ether 6:4).
For each ligand, the global minimum energy conformer derived
from an MC/EM[12] (AMBER*,[17] water GB/SA[18]) conforma-
tional search was selected as the representative starting conforma-
tion.
Compound 3 (3S,6R,7S,9S): The compound was obtained as a yel-
low oil by General Procedure A (76%). [α]2D5 °C = –8.16 (c = 1.4,
CHCl3). 1H NMR (CDCl3, 400 MHz): δ = 1.44 (s, 10 H, Boc, H8),
1.46 [s, 10 H, C(CH3)3, H5], 1.72 (m, 2 H, H4, H5Ј), 1.9 (m, 1 H,
H10Ј), 2.15 (m, 2 H, H4, H8Ј), 2.2 (m, 2 H, H7, H10) 3.37 (m, 1 H,
H6), 3.55 (s, 2 H, H11), 4.03 (m, 1 H, H3), 4.31 (m, 1 H, H9), 5.15
(m, 5 H, NHBoc, OCH2Ph), 7.36 (m, 11 H, Ph, NHCbz), 8.5 [s, 1
H, NH(=NCbz)NHCbz] ppm. 13C NMR (CDCl3, 100.6 MHz): δ
= 26.5, 28.1, 28.5, 28.7, 30.8, 34.6, 39.8, 52.4, 58.2, 64.6, 67.6, 68.6,
79.7, 81.8, 128.2, 128.3, 128.6, 128.9, 129.1, 167.7, 171.3 ppm. MS
(FAB): m/z found: 709 [M + H]+ (calcd. for C37H49N5O9: 707.35).
C37H49N5O9 (707.35): calcd. C 62.78, H 6.98, N 9.89; found: C
62.76, H 6.97, N 9.88.
The recently solved crystal structure of the extracellular domain of
the αvβ3 integrin receptor in complexation with EMD 121974 in
the presence of the proadhesive Mn2+ ion (PDB entry code =
1L5G[11]) was used for docking studies. Because the headgroup of
integrin αvβ3 has been identified as the ligand-binding region in the
X-ray structure, the docking was performed only on the globular
head. The protein structure was prepared and optimized by use of
the Schrödinger pprep and impref scripts[13] with replacement of
Mn2+ ions with Ca2+ ions.
The grid-generation step requires mae input files of both ligand
and active site, including hydrogen atoms. The center of the grid-
enclosing box was defined by the centroid of the bound ligand, as
described in the original PDB entry. The enclosing box dimensions,
which are automatically deduced from the ligand size, fit the entire
active site. For the docking step, the size of the bounding box for
placing the ligand center was set to 12 Å. No further modifications
were applied to the default settings. The GlideScore scoring func-
tion was used to select 30 poses for each ligand.[19]
Compound 4 (3R,6R,7S,9S): The compound was obtained as a
white solid by General Procedure A (89%). M.p. 58–59 °C.
1
[α]2D5 °C = –41.4 (c = 0.86, CHCl3). H NMR (CDCl3, 400 MHz): δ
= 1.42 (s, 9 H, Boc), 1.43 [s, 9 H, C(CH3)3], 1.52 (m, 3 H, H10Ј
,
H8Ј, H5Ј), 1.8 (m, 3 H, H4Ј, H5, H10), 2.08 (m, 1 H, H7), 2.35 (m,
1 H, H4) 2.55 (m, 1 H, H8), 3.38 (m, 1 H, H6), 3.55 (s, 2 H, H11),
4.12 (m, 1 H, H3), 4.40 (m, 1 H, H9), 5.16 (2s, 4 H, OCH2Ph), 5.50
(brs, 1 H, NHBoc), 7.35 (m, 10 H, Ph), 8.35 [s, 1 H, NH(=NCbz)-
NHCbz] ppm. 13C NMR (CDCl3, 100.6 MHz): δ = 24.9, 27.0, 28.1,
28.2, 31.3, 34.5, 39.4, 44.2, 50.5, 58.9, 61.8, 67.3, 68.4, 79.7, 81.9,
The Glide program was initially tested for its ability to reproduce
the crystallized binding geometry of EMD 121974. The program
was successful in reproducing the experimentally found binding
mode of this compound, as it corresponds to the best-scored pose.
Solid-Phase Receptor-Binding Assay: The receptor-binding assays 128.1, 128.2, 128.6, 128.7, 128.9, 129.0, 134.6, 136.8, 154.1, 156.2,
were performed as described previously.[20,21] Purified receptors
αvβ3 and αvβ5 (Chemicon International Inc., Temecula, CA) were
diluted to 500 ngmL–1 and 1000 ng per well, respectively, in coating
163.2, 163.8, 168.7, 170.9 ppm. MS (FAB): m/z found: 709 [M +
H]+ (calcd. for C37H49N5O9: 707.35). C37H49N5O9 (707.35): calcd.
C 62.78, H 6.98, N 9.89; found: C 62.75, H 6.96, N 9.89.
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General Procedure B. Cleavage of the Boc Protecting Group: HClO4
(70% in water, 3 equiv.) was added at 0 °C to a solution of com-
pound 3 or 4 (1 equiv.) in tBuOAc (0.03 ). The reaction mixture
was then allowed to warm to room temperature. When the reaction
was complete (TLC), triethylamine was added and after 30 min the
aqueous phase was extracted with EtOAc. The collected organic
phases were dried with Na2SO4 and filtered, and the solvent was
evaporated under reduced pressure. The crude product was carried
over to the next reaction without further purification.
Compound 8 (3S,6R,7S,9S): The compound was obtained as a
white solid by General Procedure C (63% over two steps).
Protected Product: Yellow oil, flash chromatography: EtOAc/petro-
leum ether, 7:3. [α]2D5 °C = –4.4 (c = 0.34, CHCl3). 1H NMR (CDCl3,
400 MHz): δ = 1.30 (m, 2 H, H5Ј, H10Ј), 1.48 [s, 9 H, C(CH3)3],
1.60 (m, 4 H, H8Ј, H10, H4Ј, H7), 2.20 (m, 1 H, H5), 2.60 (m, 2 H,
H4, H8), 3.40 (m, 3 H, H12, H6), 3.55 (s, 2 H, H11), 4.40 (m, 2 H,
H9, H3), 5.18 (m, 6 H, OCH2Ph), 7.30–7.45 (m, 16 H, Ph,
NHC=O), 8.35 [m, 1 H, NH(=NCbz)NHCbz] ppm. HETCOR
NMR (CDCl3, 400 MHz): δ = 28.5, 30.0, 40.0, 43.0, 44.0, 52.0,
57.5, 65.0, 68.0, 129.0 ppm. MS (FAB): m/z found: 784 [M + H]+
(calcd. for C42H49N5O10: 783.35). C42H49N5O10 (783.35): calcd. C
64.35, H 6.30, N 8.93; found: C 64.30, H 6.28, N 8.91.
Compound 5 (3S,6R,7S,9S): The compound was obtained as a col-
orless oil by General Procedure B.
Compound 6 (3R,6R,7S,9S): The compound was obtained as a
white solid by General Procedure B.
Deprotected Product: [α]2D5 °C = –44.4 (c = 1.57, MeOH). H NMR
1
(D2O, 400 MHz): δ = 1.39 [s, 9 H, C(CH3)3], 1.50 (m, 3 H, H8Ј,
H10Ј, H5), 1.85 (m, 3 H, H4Ј, H7, H10), 2.20 (m, 2 H, H4, H5), 2.55
(m, 1 H, H8), 3.20 (m, 4 H, H12, H11), 3.41 (m, 1 H, H6), 4.30 (dd,
J = 8.9, J = 8.9 Hz, 1 H, H9),4.40 (m, 1 H, H3) ppm. HETCOR
NMR (D2O, 400 MHz): δ = 26.0, 27.5, 29.5, 34.0, 40.0, 43.0, 45.5,
50.5, 60.0, 65.0 ppm. MS (FAB): m/z found: 426 [M + H]+ (calcd.
for C19H31N5O6: 425.23). C19H31N5O6 (425.23): calcd. C 53.63, H
7.34, N 16.46; found: C 53.60, H 7.32, N 16.42.
Compound 7 (3S,6R,7S,9S): EDC (0.075 g, 0.376 mmol), HOBT
(0.0376 mmol), and collidine (22.7 µL, 0.171 mmol) were added un-
der nitrogen to a solution of monobenzyl phthalate (0.963 g,
0.376 mmol) in dry THF (3.5 mL). The solution was stirred for
10 min, and a solution of 5 (0.104 g, 0.171 mmol) in dry THF
(2 mL) was added by cannula, followed by a catalytic quantity of
DMAP. After 12 h the solvent was evaporated under reduced pres-
sure and the crude product was purified by flash chromatography
(EtOAc/petroleum ether, 7:3) to give the protected compound as a
yellow oil. [α]2D5 °C = –9.75 (c = 0.83, CHCl3). 1H NMR (CDCl3,
400 MHz): δ = 1.40 [s, 9 H, C(CH3)3], 1.60, 1.80 (m, 5 H, H8, H4Ј,
H10Ј, H5), 2.05 (m, 1 H, H10), 2.15 (m, 1 H, H7), 2.65 (m, 1 H, H8)
2.75 (m, 1 H, H4), 3.40 (m, 1 H, H6), 3.52 (m, 2 H, H11), 4.25 (m,
1 H, H3), 4.38 (m, 1 H, H9), 5.15 (m, 6 H, OCH2Ph), 6.52 (s, 1 H,
NHC=O), 7.20–7.35 (m, 19 H, Ph), 8.35 [s, 1 H, NH(=NCbz)-
NHCbz] ppm. HETCOR NMR (CDCl3, 400 MHz): δ = 24.0, 25.5,
27.0, 31.0, 34.0, 40.0, 44.0, 58.5, 64.5, 68.0, 130.0 ppm. MS (FAB):
m/z found: 846 [M + H]+ (calcd. for C47H51N5O10: 845.36).
C47H51N5O10 (845.36): calcd. C 66.73, H 6.08, N 8.28; found: C
66.70, H 6.05, N 8.25.
Compound 9 (3R,6R,7S,9S): The compound was obtained as a
white solid by General Procedure C (33% over two steps).
Protected Product: Flash chromatography: toluene/acetone, 85:5.
[α]2D5 °C = –10.5 (c = 1, CHCl3). H NMR (CDCl3, 400 MHz): δ =
1
1.49 [s, 13 H, H4Ј, H8Ј, H5Ј, H10Ј, C(CH3)3], 1.70 (m, 2 H, H10,
H7), 2.15 (m, 1 H, H5), 2.60 (m, 2 H, H4, H8), 3.40 (m, 3 H, H12,
H6), 3.50 (m, 2 H, H11), 4.35 (m, 2 H, H9, H3), 5.15 (m, 6 H,
OCH2Ph), 7.35 (m, 16 H, Ph, NHC=O), 8.35 [m,
1 H,
NH(=NCbz)NHCbz] ppm. HETCOR NMR (CDCl3, 400 MHz):
δ = 27.0, 28.0, 40.0, 42.0, 51.5, 58.5, 68.0, 129.0 ppm. MS (FAB):
m/z found: 784 [M + H]+ (calcd. for C42H49N5O10: 783.35).
C42H49N5O10 (783.35): calcd. C 64.35, H 6.30, N 8.93; found: C
64.37, H 6.30, N 8.94.
A catalytic amount of Pd/C was added to a solution of the pro-
tected compound (0.101 g, 0.170 mmol) in MeOH (5 mL). The re-
action mixture was hydrogenated at 1 atm for 4 h, the catalyst was
then removed by filtration through a Celite pad, and the solvent
was removed under reduced pressure to give 7 as a white solid in
41% yield over three steps. [α]2D5 °C = –41.8 (c = 0.32, H2O). 1H
NMR (D2O, 400 MHz): δ = 1.40 [s, 9 H, C(CH3)3], 1.50 (m, 3 H,
H8, H10Ј, H7), 1.85 (m, 3 H, H5Ј, H4Ј, H10), 2.25 (m, 2 H, H5, H4),
2.55 (m, 1 H, H8), 3.20 (m, 2 H, H11), 3.45 (m, 1 H, H6), 4.34 (dd,
J = 8.9, J = 8.9 Hz, 1 H, H9), 4.55 (m, 1 H, H3), 7.5 (m, 4 H,
Ph) ppm. HETCOR NMR (D2O, 400 MHz): δ = 26.5, 27.5, 29.5,
34.0, 40.0, 43.0, 51.0, 60.0, 66.0, 130.0 ppm. MS (FAB): m/z found:
Deprotected Product: [α]2D5 °C = –50.8 (c = 0.92, MeOH). H NMR
1
(D2O, 400 MHz): δ = 1.48 [s, 9 H, C(CH3)3], 1.57 (m, 3 H, H8Ј,
H10Ј, H5Ј), 1.90 (m, 3 H, H4Ј, H5, H10), 2.22 (m, 2 H, H4, H7), 2.60
(m, 1 H, H8), 3.25 (m, 2 H, H11), 3.41 (m, 1 H, H12, H6), 4.32 (dd,
J = 8.9, J = 8.9 Hz, 1 H, H9),4.42 (m, 1 H, H3) ppm. HETCOR
NMR (D2O, 400 MHz): δ = 26.0, 27.0, 33.5, 38.5, 42.0, 42.5, 50.9,
59.9, 64.5 ppm. MS (FAB): m/z found: 426 [M + H]+ (calcd. for
C19H31N5O6: 425.23). C19H31N5O6 (425.23): calcd. C 53.63, H
7.34, N 16.46; found: C 53.65, H 7.33, N 16.43.
General Procedure D. Coupling with Monobenzyl N-Acetyl-aspart-
488 [M + H]+ (calcd. for C24H33N5O6: 487.24). C24H33N5O6 ate and Hydrogenolysis: DIC (1.1 equiv.) and HOBT (0.1 equiv.)
(487.24): calcd. C 59.12, H 6.82, N 14.36; found: C 59.10, H 6.80,
N 14.37.
were added under nitrogen to a solution of monobenzyl N-acetyl-
aspartate (1 equiv.) in dry THF (0.1 ). The solution was stirred
for 10 min, and a solution of either 5 or 6 (1 equiv.) in dry THF
(0.5 ) was then added by cannula. After 4 h the solvent was evapo-
rated under reduced pressure and the crude product was purified
by flash chromatography.
General Procedure C. Coupling with the Monobenzyl Malonate and
Hydrogenolysis: EDC (2 equiv.), HOBT (1 equiv.), and collidine
(1 equiv.) were added under nitrogen to a solution of monobenzyl
malonate (2.2 equiv.) in dry THF (0.1 ). The solution was stirred
for 10 min, and a solution of 5 or 6 (1 equiv.) in dry THF (0.1 )
was then added by cannula, followed by a catalytic quantity of
DMAP. After 12 h the solvent was evaporated under reduced pres-
sure, and the crude product was purified by flash chromatography.
A catalytic amount of Pd/C was added to a solution of the pro-
tected compound (1 equiv.) in MeOH (0.1 ). The reaction mixture
was hydrogenated at 1 atm for 4 h, the catalyst was then removed
by filtration through a Celite pad, and the solvent was removed
under reduced pressure to give the desired product.
A catalytic amount of Pd/C was added to a solution of the pro-
tected compound (1 equiv.) in MeOH (0.1 ). The reaction mixture
was hydrogenated at 1 atm for 4 h, the catalyst was then removed
by filtration through Celite pad, and the solvent was removed un-
der reduced pressure to give the desired product.
Compound 10 (3S,6R,7S,9S): The compound was obtained as a
white solid by General Procedure C (66% over two steps).
Protected Product: Yellow oil, flash chromatography: toluene/ace-
tone, 6:4. [α]2D5 °C = –15.6 (c = 0.93, CHCl3). 1H NMR (CDCl3,
1314
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1309–1317

Nonpeptide Integrin Antagonists
FULL PAPER
400 MHz): δ = 1.42 [s, 9 H, C(CH3)3], 1.51 (m, 3 H, H10, H4Ј, H5), filtered, and the solvent was evaporated under reduced pressure.
1.78 (m, 3 H, H10, H5, H8Ј), 2.0 (s, 3 H, CH3C=O), 2.18 (m, 1 H, The crude product was purified by flash chromatography.
H7), 2.47 (m, 1 H, H4), 2.61 (m, 1 H, H8), 2.73 (ddd, J = 6.63, J =
Compound 15 (3S,6R,7S,9S): The compound was obtained as a yel-
6.63, J = 16.9 Hz, 1 H, H13Ј), 3.02 (ddd, J = 4.82, J = 4.82, J =
low oil by General Procedure E (flash chromatography: petroleum
16.9 Hz, 1 H, H13), 3.40 (m, 1 H, H6), 3.55 (s, 2 H, H11), 4.32 (m,
ether/EtOAc, 8:2, 76%). [α]2D5 °C = –39.7 (c = 1.0, CHCl3). 1H NMR
2 H, H9, H3), 4.86 (m, 1 H, H12), 5.21, 5.86 (2ϫ s, 7 H, OCH2Ph,
(CDCl3, 400 MHz): δ = 1.45 [s, 9 H, C(CH3)3], 1.48 [s, 9 H,
NH), 6.67 (d, J = 8.55 Hz, 1 H, NHAc), 6.96 (d, J = 6.36 Hz, 1
C(CH3)3], 1.54 (m, 2 H, H8Ј, H5Ј), 1.73 (m, 1 H, H4Ј), 2.15 (m, 2
H, NHC=O), 7.38 (m, 15 H, Ph), 8.46 [s, 1 H, NH(=NCbz)-
H, H5, H7), 2.36 (m, 1 H, H10), 2.54 (m, 2 H, H10, H4), 2.68 (m, 1
NHCbz] ppm. HETCOR NMR (CDCl3, 400 MHz): δ = 27.0, 28.8,
H, H8), 3.41 (m, 1 H, H6), 4.12 (m, 1 H, H3), 4.38 (m, 1 H, H9), 5.43
31.8, 33.0, 35.0, 37.0, 40.5, 44.0, 50.0, 58.5, 65.2 ppm. MS (FAB):
(brd, 1 H, NHBoc) ppm. HETCOR NMR (CDCl3, 100.6 MHz): δ
m/z found: 855 [M + H]+ (calcd. for C45H54N6O11: 854.39).
= 27.0, 29.0, 35.1, 36.0, 42.0, 52.5, 58.5, 64.5 ppm. MS (FAB): m/z
C45H54N6O11 (854.39): calcd. C 63.22, H 6.37, N 9.83; found: C
found: 413 [M + H]+ (calcd. for C20H32N2O7: 412.32). C20H32N2O7
63.18, H 6.35, N 9.82.
(412.32): calcd. C 58.24, H 7.82, N 6.79; found: C 58.27, H 7.80,
Deprotected Product: [α]2D5 °C = –34.9 (c = 1.01, MeOH). H NMR
(D2O, 400 MHz): δ = 1.38 [s, 9 H, C(CH3)3], 1.40 (m, 3 H, H8,
H10, H5), 1.78 (m, 3 H, H4, H5Ј, H10Ј), 1.90 (s, 3 H, CH3CO), 2.08
(m, 2 H, H4Ј, H7), 2.45 (m, 2 H, H13, H8Ј), 2.58 (m, 1 H, H13), 3.15
(m, 2 H, H11), 3.35 (m, 1 H, H6), 4.22 (m, 2 H, H3, H9), 4.48 (m,
1 H, H12) ppm. HETCOR NMR (D2O, 400 MHz): δ = 22.2, 26.0,
27.0, 29.0, 33.8, 34.0, 39.0, 40.0, 43.0, 49.2, 52.5, 59.8, 65.0 ppm.
MS (FAB): m/z found: 497 [M + H]+ (calcd. for C22H36N6O7:
496.26). C22H36N6O7 (496.26): calcd. C 53.21, H 7.31, N 16.92;
found: C 53.2, H 7.30, N 16.91.
N 6.78.
1
Compound 16 (3R,6R,7S,9S): The compound was obtained as a
yellow oil by General Procedure E (flash chromatography: petro-
leum ether/EtOAc, 8:2, 74%). [α]2D5 °C = –44.7 (c = 1.0, CHCl3). 1H
NMR (CDCl3, 400 MHz): δ = 1.46 [s, 9 H, C(CH3)3], 1.48 [s, 9 H,
C(CH3)3], 1.62 (m, 3 H, H4Ј, H8Ј, H5Ј), 2.05 (m, 1 H, H10Ј), 2.20
(m, 1 H, H5), 2.37 (m, 2 H, H4, H7), 2.61 (m, 2 H, H10, H8), 3.44
(m, 1 H, H6), 4.18 (m, 1 H, H3), 4.43 (m, 1 H, H9), 5.65 (brs, 1 H,
NHBoc) ppm. 13C NMR (CDCl3, 100.6 MHz): δ = 21.0, 28.1, 28.4,
29.8, 34.5, 36.0, 42.2, 50.4, 58.7, 78.0, 79.0, 80.0, 82.1, 170.9, 175.9,
176.6 ppm. MS (FAB): m/z found: 413 [M + H]+ (calcd. for
C20H32N2O7: 412.32). C20H32N2O7 (412.32): calcd. C 58.24, H
7.82, N 6.79; found: C 58.22, H 7.79, N 6.80.
Compound 11 (3R,6R,7S,9S): This compound was obtained as a
white solid by General Procedure C (67% over two steps).
Protected Product: White solid, flash chromatography: toluene/ace-
tone, 1:1). [α]2D5 °C = –16.3 (c = 2.37, CHCl3). 1H NMR (CDCl3,
General Procedure F. Esterification with Benzyl Bromide: An aque-
ous solution of CsCO3 (20%) was added to a solution of either 15
400 MHz): δ = 1.40 [s, 9 H, C(CH3)3], 1.50 (m, 4 H, H8Ј, H10Ј
,
H4Ј, H5), 1.85 (m, 1 H, H10), 2.05 (s, 3 H, CH3C=O), 2.10 (m, 1 or 16 (1 equiv.) in a mixture of MeOH/H2O (10:1, 0.1 ) until pH 7
H, H5), 2.30 (m, 2 H, H7, H4), 2.55 (m, 1 H, H8), 2.74 (ddd, J =
5.57, J = 5.57, J = 16.8 Hz, 1 H, H13Ј), 3.03 (ddd, J = 4.66, J =
4.66, J = 17.1 Hz, 1 H, H13), 3.38 (m, 1 H, H6), 3.50 (s, 2 H, H11),
was reached. The solvent was evaporated under reduced pressure,
the residue was dissolved in DMF and evaporated again, and a
solution of benzyl bromide in DMF (0.4 , 1.1 equiv.) was added
4.25 (m, 1 H, H3), 4.41 (dd, J = 8.57, J = 8.57 Hz, 1 H, H9), 4.89 to the remaining cesium salt. After 1 h the solvent was evaporated
(m, 1 H, H12), 5.15, (ms, 6 H, OCH2Ph), 6.80 (m, 1 H, NHAc),
7.30 (m, 17 H, NHAsp, Ph), 8.35 [s, H, NH(=NCbz)-
under reduced pressure, and the residue was then dissolved in
EtOAc and washed with water. The organic phase was dried with
Na2SO4, filtered, and evaporated under reduced pressure, and the
crude product was purified by flash chromatography.
1
NHCbz] ppm. HETCOR NMR (CDCl3, 400 MHz): δ = 23.6, 23.8,
28.4, 30.1, 31.5, 34.6, 36.4, 39.6, 44.1, 49.8, 59.1, 62.4, 67.2, 67.6,
69.7, 82.2, 128.4, 128.5, 128.7, 128.8, 128.9, 129.1, 129.5, 134.9,
135.8, 137.1, 154.3, 156.4, 157.4, 156.4, 157.4, 168.3, 170.5, 170.8,
171.1, 171.9 ppm. MS (FAB): m/z found: 855 [M + H]+ (calcd. for
C45H54N6O11: 854.39). C45H54N6O11 (854.39): calcd. C 63.22, H
6.37, N 9.83; found: C 63.20, H 6.35, N 9.84.
Compound 17 (3S,6R,7S,9S): The compound was obtained as a
white solid by General Procedure F (flash chromatography: petro-
leum ether/EtOAc, 6:4, 92%). [α]2D5 °C = –23.5 (c = 1.0, CHCl3). 1H
NMR (CDCl3, 400 MHz): δ = 1.46 [s, 9 H, C(CH3)3], 1.48 [s, 9 H,
C(CH3)3], 1.48–1.52 (m, 3 H, H4Ј, H8Ј, H5Ј), 2.05 (m, 1 H, H5),
2.17 (m, 1 H, H7), 2.35 (m, 1 H, H10), 2.56 (m, 2 H, H10Ј, H4), 2.63
(m, 1 H, H8), 3.38 (m, 1 H, H6), 4.05 (m, 1 H, H3), 4.34 (m, 1 H,
Deprotected Product: [α]2D5 °C = –21.8 (c = 0.22, MeOH). H NMR
1
(D2O, 400 MHz): δ = 1.42 [s, 9 H, C(CH3)3], 1.51 (m, 3 H, H8Ј,
H10Ј, H5Ј), 1.85 (m, 3 H, H4, H7, H10), 1.98 (s, 3 H, CH3CO), 2.06 H9), 5.15 (s, 2 H, OCH2Ph), 5.28 (brs, 1 H, NHBoc), 7.40 (m, 5
(m, 2 H, H4, H5), 2.55 (m, 1 H, H8), 2.58 (m, 1 H, H13Ј), 2.66 (m, H, Ph) ppm. HETCOR NMR (CDCl3, 400 MHz): δ = 26.5, 28.7,
1 H, H13), 3.21 (m, 1 H, H4Ј), 3.34 (m, 1 H, H6), 4.32 (m, 2 H, H3, 29.0, 35.5, 36.7, 42.5, 52.8, 58.7, 64.6, 67.5 ppm. MS (FAB): m/z
H9), 4.50 (m, 1 H, H12) ppm. HETCOR NMR (D2O, 400 MHz): δ found: 503 [M + H]+ (calcd. for C27H38N2O7: 502.27). C27H38N2O7
= 22.1, 23.8, 25.0, 28.8, 29.0, 34.5, 39.0, 40.0, 42.5, 49.0, 51.8, 60.0,
65.0 ppm. MS (FAB): m/z found: 497 [M + H]+ (calcd. for
C22H36N6O7: 496.26). C22H36N6O7 (496.26): calcd. C 53.21, H
7.31, N 16.92; found: C 53.19, H 7.32, N 16.90.
(502.27): calcd. C 64.52, H 7.62, N 5.57; found: C 64.49, H 7.60,
N 5.57.
Compound 17 (3R,6R,7S,9S): The compound was obtained as a
yellow oil by General Procedure F (flash chromatography: petro-
leum ether/EtOAc, 6:4, 94%). [α]2D5 °C = –38.1 (c = 1.0, CHCl3). 1H
NMR (CDCl3, 400 MHz): δ = 1.44 [s, 9 H, C(CH3)3], 1.48 [s, 9 H,
C(CH3)3], 1.50–1.72 (m, 3 H, H4Ј, H8Ј, H5Ј), 2.05 (m, 1 H, H5),
2.22 (m, 1 H, H7), 2.36 (m, 2 H, H4, H10), 2.60 (m, 2 H, H10Ј, H8),
3.41 (m, 1 H, H6), 4.10 (m, 1 H, H3), 4.41 (m, 1 H, H9), 5.15 (s, 2
H, OCH2Ph), 5.50 (brs, 1 H, NHBoc), 7.40 (m, 5 H, Ph) ppm. 13C
NMR (CDCl3, 100.6 MHz): δ = 24.7, 26.9, 28.1, 28.5, 34.5, 36.4,
42.5, 50.4, 58.7, 61.3, 65.9, 79.8, 82.0, 128.6, 128.7, 128.9, 135.6,
155.8, 170.8, 171.4 ppm. MS (FAB): m/z found: 503 [M + H]+
General Procedure E. Oxidation: A solution of PDC in DMF (1 ,
4.8 equiv.) was added to a solution of either 13 or 14 (1 equiv.) in
DMF (0.1 ). The reaction mixture was stirred for 12 h, a saturated
solution of NaCl was then added, and the aqueous phase was ex-
tracted with EtOAc. The collected organic layers were evaporated
under reduced pressure, the residue was dissolved in EtOAc and
washed with a saturated solution of NaHCO3, and the aqueous
phase was acidified with a solution of HCl (0.5 ) and then ex-
tracted with EtOAc. The organic phase was dried with Na2SO4 and
Eur. J. Org. Chem. 2007, 1309–1317
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1315

L. Manzoni et al.
FULL PAPER
(calcd. for C27H38N2O7: 502.27). C27H38N2O7 (502.27): calcd. C
64.52, H 7.62, N 5.57; found: C 64.53, H 7.63, N 5.57.
C52H60N6O12: 960.43). C52H60N6O12 (960.43): calcd. C 64.99, H
6.29, N 8.74; found: C 64.97, H 6.28, N 8.75.
1
Deprotected Product: White solid. [α]2D5 °C = –39.3 (c = 1, H2O). H
Compound 19 (3S,6R,7S,9S): The compound was obtained as a yel-
NMR (D2O, 400 MHz): δ = 1.42 [s, 9 H, C(CH3)3], 1.50–1.71 (m,
4 H, H13Ј, H13, H8Ј, H5Ј), 1.85 (m, 2 H, H12Ј, H4Ј), 1.98–2.40 (m, 6
H, H10Ј, H12, H4, H5, H10, H7), 2.52 (m, 1 H, H8), 3.20 (m, 2 H,
H14, H14Ј), 3.38 (m, 1 H, H6), 3.91 (m, 1 H, H11), 4.38 (m, 1 H,
H9), 4.50 (m, 1 H, H3) ppm. HETCOR NMR (D2O, 400 MHz): δ
= 24.0, 27.8, 29.0, 40.0, 41.5, 48.8, 53.8, 60.0, 66.0 ppm. MS (ESI):
m/z found: 469 [M + H]+ (calcd. for C21H36N6O6: 468.27).
C21H36N6O6 (468.27): calcd. C 53.83, H 7.74, N 17.94; found: C
53.84, H 7.75, N 17.92.
low oil by General Procedure B.
Compound 20 (3R,6R,7S,9S): The compound was obtained as a
white solid by General Procedure B.
General Procedure G. Coupling with Z-Arg(Z)2OH and Hydro-
genolysis: DIC (1.3 equiv.) and HOBT (1.3 equiv.) were added un-
der nitrogen to a solution of Z-Arg(Z)2OH in dry THF (0.1 ,
1.3 equiv.). The solution was stirred for 10 min, and a solution of
either 19 or 20 (1 equiv.) in dry THF (0.5 ) was then added by
cannula. After 12 h the solvent was evaporated under reduced pres-
sure and the crude product was purified by flash chromatography.
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra of all new synthesized compounds.
A catalytic amount of Pd/C was added to a solution of the pro-
tected compound (1 equiv.) in MeOH (0.1 ). The reaction mixture
was hydrogenated at 1 atm for 4 h, the catalyst was then removed
by filtration through a Celite pad, and the solvent was removed Acknowledgments
under reduced pressure to give the desired product.
This work was supported by the Consiglio Nazionale delle Ricer-
che (CNR) and the Ministero dell’Università e della Ricerca
(MUR).
Compound 21 (3S,6R,7S,9S): The compound was obtained as a
white solid by General Procedure G (43% over three steps).
Protected Product: Yellow oil, flash chromatography: EtOAc/petro-
1
leum ether, 1:1. [α]2D5 °C = –8.13 (c = 1, CHCl3). H NMR (CDCl3,
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400 MHz): δ = 1.31–1.50 (m, 3 H, H8Ј, H4Ј, H5Ј), 1.46 [s, 9 H,
C(CH3)3], 1.60–1.82 (m, 4 H, H13, H12), 1.96 (m, 1 H, H5), 2.14
(m, 1 H, H7), 2.23 (m, 1 H, H4), 2.35 (dd, J = 8.7, J = 15.7 Hz, 1
H, H10Ј), 2.51 (dd, J = 5.4, J = 15.7 Hz, 1 H, H10), 2.60 (m, 1 H,
H8), 3.33 (m, 1 H, H6), 3.93 (m, 1 H, H14), 4.06 (m, 1 H, H14Ј),
4.17 (m, 1 H, H3), 4.29 (dd, J = 8.9, J = 8.9 Hz, 1 H, H9), 4.35 (m,
1 H, H11), 5.0–5.30 (m, 8 H, OCH2Ph), 5.88 (brd, 1 H, NH), 6.90
(brd, 1 H, NH), 7.23–7.43 (m, 20 H, Ph) ppm. HETCOR NMR
(CDCl3, 400 MHz): δ = 25.2, 27.0, 28.1, 30.3, 36.5, 42.3, 45.0, 51.5,
54.8, 58.4, 64.1, 67.5, 94.0 ppm. MS (FAB): m/z found: 961 [M +
H]+ (calcd. for C52H60N6O12: 960.43). C52H60N6O12 (960.43):
calcd. C 64.99, H 6.29, N 8.74; found: C 64.98, H 6.30, N 8.73.
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Deprotected Product: White solid. [α]2D5 °C = –87.4 (c = 0.63, H2O).
1H NMR (D2O, 400 MHz): δ = 1.42 [s, 9 H, C(CH3)3], 1.48–1.59
(m, 2 H, H8Ј, H5Ј), 1.63–1.72 (m, 2 H, H13Ј, H13), 1.82–1.95 (m, 3
H, H12, H12Ј, H4), 2.07–2.22 (m, 4 H, H4, H5, H10, H7), 2.23 (m, 1
H, H4), 2.33 (dd, J = 5.6, J = 13.6 Hz, 1 H, H10Ј), 2.54 (m, 1 H,
H8), 3.18 (m, 2 H, H14, H14Ј), 3.40 (m, 1 H, H6), 3.97 (m, 1 H,
H11), 4.36 (dd, J = 8.9, J = 8.9 Hz, 1 H, H9), 4.39 (m, 1 H,
H3) ppm. HETCOR NMR (D2O, 400 MHz): δ = 24.0, 26.7, 26.9,
28.5, 35.0, 39.5, 41.2, 43.0, 50.9, 53.8, 59.9, 65.3 ppm. MS (FAB):
m/z found: 469 [M + H]+ (calcd. for C21H36N6O6: 468.27).
C21H36N6O6 (468.27): calcd. C 53.83, H 7.74, N 17.94; found: C
53.81, H 7.73, N 17.93.
Compound 22 (3R,6R,7S,9S): The compound was obtained as a
white solid by General Procedure G (46% over three steps).
Protected Product: White foam, flash chromatography: EtOAc/pe-
troleum ether, 6:4. [α]2D5 °C = –20.9 (c = 1, CHCl3). 1H NMR
(CDCl3, 400 MHz): δ = 1.46 [s, 9 H, C(CH3)3], 1.50–1.75 (m, 6 H,
H13, H13Ј, H12Ј, H8Ј, H4Ј, H5Ј), 1.84 (m, 1 H, H12), 1.95 (m, 1 H,
H5), 2.18 (m, 1 H, H7), 2.25 (m, 1 H, H4), 2.35 (dd, J = 8.7, J =
15.7 Hz, 1 H, H10Ј), 2.56 (m, 2 H, H8, H10), 2.60 (m, 1 H, H8), 3.38
(m, 1 H, H6), 3.98 (m, 2 H, H14Ј, H14), 4.18 (m, 1 H, H3), 4.30 (m,
1 H, H11), 4.40 (dd, J = 8.4, J = 8.4 Hz, 1 H, H9), 5.00–5.20 (m, 8
H, OCH2Ph), 5.80 (brs, 1 H, NH), 7.05 (brs, 1 H, NH), 7.31–7.43
(m, 20 H, Ph) ppm. HETCOR NMR (CDCl3, 400 MHz): δ = 25.1,
28.3, 30.0, 35.0, 37.0, 42.8, 45.0, 50.0, 55.2, 59.8, 62.2, 68.0,
70.0 ppm. MS (ESI): m/z found: 961 [M + H]+ (calcd. for
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Eur. J. Org. Chem. 2007, 1309–1317

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Received: September 26, 2006
Published Online: January 19, 2007
Eur. J. Org. Chem. 2007, 1309–1317
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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