Scalable synthesis of an integrin-binding peptide mimetic for biomedical applications

Article abstract of DOI:10.1016/j.tet.2012.09.002

A scalable, solution-phase synthesis of the selectively protected non-peptide RGD (arginine-glycine-aspartic acid) mimetic 6 is described. This synthesis serves as an alternative to the previously described solid-phase synthesis of this compound, thereby making this important integrin-binding mimetic readily accessible. The free carboxylic acid of 6 was conjugated to a protected diamine, followed by global deprotection to give a derivative 27, suitable for immobilization onto amine-reactive surfaces. The RGD mimetic 28 demonstrated superior biological activity in comparison to a native linear RGD peptide and the semi-synthetic cyclic cRGDfK peptide in a cell attachment inhibition assay.

Full text of DOI:10.1016/j.tet.2012.09.002

Tetrahedron 68 (2012) 9448e9455
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Scalable synthesis of an integrin-binding peptide mimetic for biomedical
applications
,
b
,
a
,
,
,b
Andrew G. Riches a , Teresa Cablewski b, Veronica Glattauer a b, Helmut Thissen a
,
*
Laurence Meagher a
,
b
a CSIRO Materials Science and Engineering, Bayview Avenue, Clayton 3168 VIC, Australia
b CRC for Polymers, 8 Redwood Drive, Notting Hill 3168 VIC, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2012
Received in revised form 16 August 2012
Accepted 3 September 2012
Available online 7 September 2012
A scalable, solution-phase synthesis of the selectively protected non-peptide RGD (arginineeglycine-
easpartic acid) mimetic 6 is described. This synthesis serves as an alternative to the previously described
solid-phase synthesis of this compound, thereby making this important integrin-binding mimetic readily
accessible. The free carboxylic acid of 6 was conjugated to a protected diamine, followed by global de-
protection to give a derivative 27, suitable for immobilization onto amine-reactive surfaces. The RGD
mimetic 28 demonstrated superior biological activity in comparison to a native linear RGD peptide and
the semi-synthetic cyclic cRGDfK peptide in a cell attachment inhibition assay.
Keywords:
RGD mimetic
Integrin
Ó 2012 Elsevier Ltd. All rights reserved.
Biomaterials
1. Introduction
been shown to elicit biospecific attachment and survival of cells.7
Small cyclic peptides are more stable in vivo than their linear
The ability to control cell-material surface interactions effec-
tively is essential to the development of new and improved bio-
materials and biomedical devices for in vitro and in vivo
applications such as diagnostics, drug delivery, implantable de-
vices, and regenerative medicine.1 The immobilization of bioactive
molecules on material surfaces that enhance cell attachment is of
particular interest. Traditional surface modification methods,
which rely on the non-specific adsorption of proteins to mediate
cell attachment are being replaced by improved methods, which
display bioactive molecules, which are recognized by cell-surface
receptors.2 Here, the most important example is the recognition of
extracellular matrix (ECM) proteins by the heterodimeric cell-
surface receptors known as integrins.3 These adhesive in-
teractions are mediated by small integrin-binding motifs within
ECM proteins, of which the arginineeglycineeaspartic acid (RGD)
motif is the most widely studied.4 The modification of biomaterial
surfaces with small RGD peptides overcomes some of the disad-
vantages of immobilizing whole ECM proteins, which include
control of orientation, immunogenicity, instability toward enzy-
matic degradation, sterilization conditions, and risk of pathogen
transmission.5,6 For example, the presentation of short linear RGD
peptides on self-assembled monolayers of oligoethylene glycol has
counterparts and optimization of their selectivity for the
avb3
integrin versus other integrins such as IIb 3 (platelet fibrinogen
a
b
receptor) led to the development of potent and selective
avb3
binding compounds such as cRGDfV8 (f¼
D
-phenylalanine) 1 and the
antiangiogenic agent Cilengitide 29 (Fig. 1). Immobilization of the
cyclic pentapeptide cRGDfK 3, through linkers attached to the ly-
sine -amino group, onto poly(methyl methacrylate) substrates,
3
resulted in materials which showed improved osteoblast adhesion
in vitro and enhanced bone tissue ingrowth into a porous implant
in an in vivo rabbit model.10 Non-peptide mimetics are an attractive
alternative to peptides due to their greater stability and lower cost
of preparation on large scales. This is reflected by the effort that
various groups have put into development of non-peptide RGD
mimetics.11e17 Kessler et al. designed a series of
avb3-selective non-
peptide RGD mimetics18 in which the central glycine residue of the
peptide was replaced by azaglycine (i.e., nitrogen replaces the
a
carbon of glycine).19 The affinity for
avb3 integrin of mimetic 4
(IC50¼0.1 nM) compares favorably with that of 1 (IC50¼2 nM),18
2
(IC50¼0.6 nM),9a and 3 (IC50¼4.2 nM).8b Linker-equipped mimetic 5
was immobilized onto titanium surfaces via the thiol group to give
materials which demonstrated improved cell adhesion and
proliferation.20
The excellent activity and selectivity profile shown by surface-
bound compound 5, together with its non-peptidic nature,
prompted us to undertake studies toward the surface immobili-
zation of this pharmacophore. Kessler prepared compound 5 by
* Corresponding author. Tel.: þ61 3 9545 2540; fax: þ61 3 9545 2446; e-mail
address: andrew.riches@csiro.au (A.G. Riches).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.002

A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
9449
Fig. 1. Cyclic RGD peptides (1e3), non-peptide RGD mimetic (4), and mimetic equipped with a tether for surface immobilization (5).
coupling amino-functionalized linkers to the carboxylic acid group
2. Results and discussion
of the triprotected mimetic 6 (Scheme 1).20 Acid 6 was prepared by
a solid-phase strategy in which 4-formylbenzoic acid was con-
verted to the Fmoc-protected b-amino-tert-butyl ester and linked
The resin used in the solid-phase synthesis was initially
replaced by a methyl group, with the expectation that the methyl
ester could be saponified following assembly of the protected mi-
metic without harm to the tert-butyl ester and the N-Boc groups
(Scheme 2). Thus methyl 4-formylbenzoate 7 was reacted with
ammonium acetate (2 equiv) and mono-tert-butyl malonate 821 in
to the trityl chloride polystyrene (TCP) resin through the aryl
carboxylic acid. Following Fmoc cleavage, Fmoc-protected azagly-
cine and amino benzoate fragments were successively added and
deprotected before finally installing the protected guanidino
group. Compound 6 was then released from the resin under mild
acidic conditions (hexafluoroisopropanol in dichloromethane),
which left the sensitive tert-butyl ester and N-Boc protecting
groups intact. We required multi-gram quantities of carboxylic
acid 6 for immobilization onto biomaterials. Due to the technical
difficulty and expense involved in scaling up solid-phase chemis-
try, we sought to develop a scalable, solution-phase route to
compound 6.
refluxing ethanol to give a mixture of desired
b-amino ester 9
(minor) and cinnamate ester 10 (major).22 Pure racemic23 amino
ester 9 was readily isolated in 23% yield by acidebase extraction.
The yield of 9 was improved to 64% by using 20 equiv of ammonium
acetate and conducting the reaction in methanol containing 10%
acetic acid at the lower temperature of 60 ꢀC over a longer time
(64 h). Lower reaction temperature (50 ꢀC) resulted in incomplete
decarboxylation of the intermediate Mannich adduct. The Fmoc
Scheme 1. Solid-phase synthesis of triprotected carboxylic acid 6 (adapted from Ref. 20).

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A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
Scheme 2. Preparation of RGD mimetic, using a methyl ester to protect the aryl carboxylic acid.
protecting group used in the solid-phase synthesis was replaced by
Cbz protection of the azaglycine in order to avoid non-volatile
Fmoc-derived by-products. Thus reaction of benzyl carbazate 11
with an equimolar amount of N,N0-carbonyldiimidazole (CDI), fol-
lowed by treatment of the resulting acyl imidazole in situ with the
of a Cbz group from the azaglycine portion of the system (cf. 12/13
in Scheme 2) and the azaglycine fragment was therefore in-
corporated in Boc-protected form by treatment of tert-butyl car-
bazate 20 with CDI, followed by 19 to give compound 21. Attempts
to selectively cleave the N-Boc group in the presence of the tert-
butyl ester using either trifluoroacetic acid in dichloromethane31,32
or anhydrous hydrochloric acid in various solvents33 were un-
successful. However, exposure of 21 to 3 equiv of methanesulfonic
acid in tert-butyl acetate/dichloromethane for 2 h at room tem-
perature gave the required semicarbazide 22 in 74% yield.34 Careful
monitoring of this reaction was necessary, as the product 22 slowly
underwent further cyclization to 23. The 1H NMR spectrum of 23
showed the absence of the tert-butyl ester and the presence of two
non-equivalent methylene protons. Coupling of semicarbazide 22
with carboxylic acid 14 gave 24 in 79% yield after chromatographic
purification. Hydrogenolysis of 24 efficiently gave carboxylic acid 6,
spectroscopic data for which is reported here for the first time.
In order to prepare an amine-functionalized peptidomimetic,
carboxylic acid 6 was coupled with mono-N-Boc-propane-1,3-
diamine 25 to give tetraprotected conjugate 26 in 75% yield after
purification by silica gel chromatography. Global deprotection of 26
using trifluoroacetic acid gave the highly polar, water-soluble bis-
trifluoroacetate salt 27 in 96% yield after purification by reverse-
phase chromatography. Studies in which 27 and related com-
pounds are being used to functionalize surfaces are currently
underway.
b-amino ester 9 gave compound 12. Hydrogenolysis of the Cbz
group of 12 under flow conditions using a ThalesNano H-CubeÔ
gave pure semicarbazide 13 in excellent yield. In contrast, con-
ventional batch hydrogenolysis (1 atm H2 over PdeC) of 12, gave
additional side products within 2 h, the amount of which increased
as reaction time increased.24 Coupling of 13 with N,N0-di-Boc-3-
guanidinobenzoic acid 1414 gave 15. Selective cleavage of the
methyl ester of 15 proved to be problematic. Treatment of 15 with
lithium hydroxide hydrolyzed the ester, but also cleaved one of the
Boc groups to give 16 in 41% yield.25 Milder treatment of 15 with aq
potassium carbonate in methanol at 45 ꢀC for 6 h cleaved the Boc
group in preference to the methyl ester to give 17 (68%) as the major
product. Other hydrolytic methods such as barium hydroxide in
methanol,26,27 and nucleophilic methods including lithium iodide
in refluxing pyridine,28 sodium cyanide in DMSO,29 and 4-
aminothiophenol/cesium carbonate30 all failed to yield 6. While
compound 16 is a potential substrate for amide coupling, the low
isolated yield of 16 prompted the redesign of the protection
strategy.
Benzyl ester 18 was identified as the starting material for
a modified approach to 6 shown in Scheme 3, as benzyl esters
undergo hydrogenolytic cleavage under conditions which should
not affect the remaining protecting groups. Under the conditions
previously developed for the preparation of methyl ester 9, con-
version of 18 to 19 was accompanied by significant trans-
esterification of the benzoate ester with the methanol solvent to
give an inseparable mixture of 9 and 19. The use of acetic acid alone
as the solvent for this reaction gave, after extractive workup, pure
benzyl ester 19 in 39% yield. The remaining non-acid extractable
fraction consisted of a complex mixture, which was not further
characterized. The benzyl ester precluded hydrogenolytic removal
While compound 15 did not serve as a precursor to 6, as origi-
nally planned, it afforded an opportunity to prepare an RGD mi-
metic suitable for testing in a cell attachment inhibition assay. Thus
treatment of 15 with trifluoroacetic acid to cleave the tert-butyl
ester and both N-Boc groups was followed by conversion of the
resulting trifluoroacetate salt to the corresponding hydrochloride
salt 28 (Scheme 4). The RGD mimetic 28 was assayed for inhibition
of L929 fibroblast attachment to vitronectin-coated polystyrene
plates. Compound 28 showed superior inhibition of cell attach-
ment, i.e., effectively blocked the integrin receptor’s ability to bind

A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
9451
Scheme 3. Preparation of RGD mimetic 27, equipped with amine-bearing spacer.
and did not inhibit cell attachment. Also included in Fig. 2 are
DMSO controls where DMSO only was present at the same con-
centration as in the solutions of compound 28, cRGDfK 3, linear
GRGDS, and cRADfK. Thus inhibition of cell attachment could be
attributed directly to the listed compounds and not to the presence
of small DMSO concentrations, which could cause cell death.
3. Conclusion
Scheme 4. Preparation of RGD mimetic 28.
This study successfully developed a scalable, solution-phase
synthesis of Kessler’s triprotected RGD peptide mimetic 6. Two
approaches using different protecting group strategies were eval-
uated, whereby the carboxylic acid was protected as either a methyl
or a benzyl ester. The methyl ester route was compromised by the
loss of an N-Boc group during the final ester cleavage step. In
contrast, the benzyl ester was selectively removed at the final step
to ultimately deliver a scalable synthesis of carboxylic acid 6 in six
steps from 4-formylbenzoic acid. Conjugation of 6 with protected
diamine linker 25 was followed by global deprotection to yield
amine-functionalized mimetic 27, which is particularly suitable for
covalent attachment to surfaces equipped with amine-reactive
groups. RGD mimetic ester 28 was prepared by trifluoroacetic
acid deprotection of 15 and shown to have activity comparable to
cyclic peptide 3 in a cell adhesion inhibition assay. It is anticipated
that biomaterials modified with immobilizable RGD peptide mi-
metics such as 27 will exhibit desirable properties in regard to the
modulation of cellular responses. By allowing cost-effective access
to larger quantities of this type of surface-immobilizable RGD mi-
metic than have hitherto been available, the synthetic route de-
scribed in this study will contribute to further expanding the
applications of peptide mimetics in biomedical applications.
to the epitope on vitronectin (EC50 4.5
m
M) when compared to both
cRGDfK 3 (EC50 92
mM) and linear GRGDS (EC50 240 mM) (Fig. 2). As
expected, the linear GRGDS peptide8a bound to and blocked the
integrin receptor less effectively than the cRGDfK 3 peptide. The
non-binding cyclic peptide cRADfK35 served as a negative control
4. Experimental section
4.1. Synthesis
€
4.1.1. General. Melting points were determined on Buchi B-545
digital melting point apparatus and are uncorrected. Infrared
spectra were determined on a Nicolet 6700 FTIR instrument using
Fig. 2. Inhibition of L929 fibroblast attachment by mimetic 28 in comparison to 1 and
linear RGD peptide.

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A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
attenuated total reflectance (ATR) on a diamond crystal. The 1H
NMR spectra were recorded at 200 MHz (Bruker AC200SX),
400 MHz (Bruker Av400) or 500 MHz (Bruker DRX500). 13C NMR
spectra were recorded at 50, 100 or 125.7 MHz on the same in-
struments. The 19F NMR spectra were recorded at 188.3 or 376 MHz
DMF (5 mL) was added over 5 min. Stirring was continued at
ꢃ10 ꢀC for 15 min, then a solution of amine 9 (2.80 g, 10.0 mmol) in
DMF (15 mL) was added over for 10 min and the mixture was
allowed to warm to room temperature overnight. The mixture was
diluted with water (100 mL) and extracted twice with 1:1 EtOAc/
P.S. (1 M aq citric acid (10 mL) was added to break initial emulsion).
The combined organic phase was washed with water (ꢂ2), brine,
dried (Na2SO4), and evaporated. The crude product was chroma-
tographed through a short column of SiO2, eluting with 40/60%
EtOAc in P.S. The major fraction gave essentially pure 12 (2.29 g,
49%) as a colorless foam, mp 135e6 ꢀC; Rf (60% EtOAc in P.S.) 0.28;
on the same instruments. Chemical shifts (d) are measured in parts
per million using known solvent chemical shifts as an internal
standard. CDCl3 was used as solvent unless otherwise stated. Low-
resolution electron impact (EI) positive ion mass spectra were run
on a ThermoQuest MAT95XL mass spectrometer using an ioniza-
tion energy of 70 eV. High-resolution EI mass spectra were ob-
tained with a resolution of 5000e10,000 using PFK as the reference
compound. Low-resolution positive and negative ion electrospray
(ES) mass spectra were acquired either with a Shimadzu LCMS-
2010EV mass spectrometer using a cone voltage of 50 V, the
source was maintained at 80 ꢀC using methanol containing 0.1%
formic acid as solvent at a flow rate of 0.1 mL/min, or with a VG
Platform mass spectrometer using a cone voltage of 50 or 30 V, the
source was maintained at 80 ꢀC using methanol solvent with a flow
rate of 0.04 mL/min. High-resolution positive ion electrospray mass
spectra were acquired either in house with a Micromass Q-TOF II
mass spectrometer using a cone voltage of 35 V and a capillary
voltage of 3.0 kV and the sample introduced by direct infusion at
nmax 3317, 2979, 1714, 1664 (sh), 1612, 1532 cmꢃ1
;
dH 7.93 (J¼8.3 Hz,
d, 2H), 7.38e7.30 (m, 7H), 6.60 (br d, J¼8.6 Hz, 1H), 6.57 (br s, 1H),
6.37 (br s, 1H), 5.29e5.24 (m, 1H), 5.16 (AB quartet, JAB¼12.1 Hz,
2H), 3.88 (s, 3H), 2.76e2.72 (m, 2H), 1.28 (s, 9H); dC 170.3, 166.8,
158.0, 157.1, 146.4, 135.5, 129.7, 129.0, 128.5, 128.3, 128.1, 126.2, 81.5,
67.8, 52.0, 50.3, 41.4, 27.8; m/z (ESþ) 494 (100%, MNaþ), 438 (41);
HRMS (ES): MNaþ, found 494.1911. C24H29N3NaO7 requires
494.1903.
4.1.4. (ꢁ)-Methyl-(10-{[hydrazino]carbonyl}-amino)-tert-butyl-4-
propanoyl-benzoate 13. The Cbz protected semicarbazide 12
(201 mg, 0.426 mmol) was dissolved in ethanol (20 mL) and hy-
drogenated using a ThalesNano H-CubeÔ (PdeC CatCartÔ, 1 bar
H2, 50 ꢀC, 1 mL/min). The effluent was evaporated to give 13
(139 mg, 97%) as a colorless solid, which was used without further
purification, mp 149e52 ꢀC; nmax 3357, 2978, 1716, 1668, 1611, 1521,
a rate of 2 mL/min using NaI as an internal calibrant, or by Chem-
icalAnalysis Pty Ltd. with an Agilent HPLC system, series 1100,
Agilent LC/MSD TOF detector, using 0.1% formic acid in methanol as
solvent at a flow rate of 0.3 mL/min and calibrating the spectrum
using an Agilent TOF reference mass solution kit containing the
reference ions of purine and HP-0921 (hexakis(1H,1H,3H-tetra-
fluoropropoxy)phosphazine). Thin layer chromatography (TLC) was
performed on Merck pre-coated 0.25 mm silica F254 aluminum-
backed plates (#5554) or RP-18 F254 plates (#5559). Column
chromatography was performed using Merck (#9385, 230e400
mesh) silica gel 60, while radial chromatography was performed
using a Harrison Research Chromatotron (#7924 T). Reverse-phase
column chromatography was performed using Chromatarex C18
silica (Fuji Silysia Chemical Ltd). All anhydrous reactions were
performed under a dry argon atmosphere. All extracts were dried
over anhydrous magnesium sulfate unless otherwise stated. All
inorganic solutions are aqueous unless otherwise specified. Anhy-
drous solvents were dried using a Pure Process Technology Glass
Contour Solvent Purification System. Petroleum spirit (P.S.) refers to
the fraction of bp 40e60 ꢀC.
1436 cmꢃ1
;
dH 8.01 (d, J¼8.2 Hz, 2H), 7.43 (d, J¼8.2 Hz, 2H), 7.14 (br
d, J¼8.3 Hz, 1H), 5.96 (br s, 1H), 5.37e5.32 (m, 1H), 3.93 (s, 3H), 3.78
(br s, 2H), 2.86e2.77 (m, 2H), 1.35 (s, 9H); dC 170.1, 166.7, 159.1,
146.9, 129.8, 129.1, 126.3, 81.4, 52.0, 49.9, 41.9, 27.9; m/z (ES) 360
(100%, MNaþ), 338 (8, MHþ), 304 (32); HRMS (ES): [2M]Naþ, found
697.3206. C32H46N6NaO10 requires 697.3173.
4.1.5. 3-{[Bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}-
benzoic
acid
14. A mixture of 3-aminobenzoic acid
(4.31 g, 31.4 mmol), N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-
carboxamidine (4.88 g, 15.7 mmol), triethylamine (4.77 g,
47.2 mmol), and methanol (60 mL) was heated at 40 ꢀC for 20 h. The
mixture was evaporated and the residue taken into ethyl acetate
and washed with 1 M aq HCl, then brine, dried (MgSO4), and
evaporated. The crude product was chromatographed through
a short column of SiO2 (20/40% EtOAc in P.S.) to give 1425 (5.55 g,
93%) as a colorless solid, mp decomp. >240 ꢀC; Rf (40% EtOAc in P.S.)
4.1.2. (ꢁ)-Benzyl-(10-amino)-tert-butyl-4-propanoyl-benzoate
9. Acetic acid (25 mL) was added to a mixture of methyl 4-
formylbenzoate 7 (9.57 g, 58.3 mmol), ammonium acetate (89.9 g,
1170 mmol), mono-tert-butyl malonate 821 (18.7 g, 117 mmol), and
methanol (120 mL) and the mixture heated to 60 ꢀC for 64 h. The
methanol was evaporated and the residue partitioned between
EtOAc and satd NaHCO3. The organic phase was extracted with 1 M
aq citric acid (ꢂ3). The aqueous phase was made basic to approx-
imately pH 9 with NaHCO3 and extracted with EtOAc (ꢂ3). The
combined extracts were washed with brine, dried (MgSO4), and
evaporated to give 9 (10.4 g, 64%) as a pale-yellow syrup, which
crystallized on standing, mp 63e5 ꢀC; nmax 2978, 1716, 1611,
0.42; nmax 2980, 1719, 1695 (sh), 1640, 1575 (sh), 1408 cmꢃ1
; dH
11.58 (br s, 1H), 10.44 (br s, 1H), 8.12e8.06 (m, 2H), 7.83 (dt, J¼7.8,
1.2 Hz, 1H), 7.44 (t, J¼8.0 Hz, 1H), 1.51 (s, 18H); dC 171.4, 163.5 (br),
154.0, 153.5 (br), 137.3, 130.4, 129.4, 128.2, 126.7, 124.0, 84.2 (br),
80.2 (br), 28.3; m/z (ES) 402 (100%, MNaþ), 380 (23, MHþ), 346 (37),
268 (43).
4.1.6. (ꢁ)-Methyl-(10-{[(N0-di-tert-butoxycarbonyl-guanidino-
benzoyl)hydrazino]carbonyl}-amino)-tert-butyl-4-propanoyl-benzo-
ate 15. The benzoic acid 14 (113 mg, 0.300 mmol) was dissolved in
dry DMF (3 mL) and HBTU (136 mg, 0.360 mmol) was added, fol-
lowed by collidine (364 mg, 3.00 mmol). After stirring at room
temperature for 15 min, the semicarbazide 13 (100 mg,
0.300 mmol) was added and stirring continued with TLC monitor-
ing. After 2 h the mixture was poured into 1 M aq citric acid and the
resulting precipitate collected. Chromatography through a short
silica column (40/80% ethyl acetate in P.S.) gave 15 (186 mg, 89%)
as a colorless foam, mp decomp. >143 ꢀC; Rf (60% EtOAc in P.S.)
1436 cmꢃ1
;
dH 8.02 (d, J¼8.3 Hz, 2H), 7.46 (d, J¼8.3 Hz, 2H), 4.45 (t,
J¼6.8 Hz, 1H), 3.93 (s, 3H), 2.61 (d, J¼6.8 Hz, 2H), 1.43 (s, 9H); dC
170.9, 166.8, 149.8, 129.8, 129.1, 126.3, 80.9, 52.5, 52.0, 45.0, 28.0; m/
z (ES) 581 (8%, [2M]Naþ), 302 (100); HRMS (ES): [2M]Naþ, found
581.2834. C30H42N2NaO8 requires 581.2839.
4.1.3. (ꢁ)-Methyl-(10-{[benzyloxyhydrazino]carbonyl}-amino)-tert-
butyl-4-propanoyl-benzoate 12. A solution of 1,10-carbon-
yldiimidazole (1.78 g, 11.0 mmol) in DMF (10 mL) was cooled to
ꢃ10 ꢀC and a solution of benzyl carbazate 11 (1.66 g, 10.0 mmol) in
0.41; nmax 3256, 2979, 1719, 1628, 1568 cmꢃ1
; dH [MeOH-d4]
7.97e7.94 (m, 3H), 7.81 (d, J¼7.9 Hz, 1H), 7.67 (d, J¼7.9 Hz, 1H),
7.50e7.41 (m, 3H), 5.30 (t, J¼6.7 Hz, 1H), 3.89 (s, 3H), 2.85e2.75 (m,
2H), 1.51 (br s, 18H), 1.34 (s, 9H); dC [MeOH-d4] 171.6, 169.4, 168.3,

A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
9453
160.1, 155.3, 148.7, 138.3, 134.3, 130.8, 130.2, 127.9, 125.6, 123.5, 82.9
(br), 82.4, 52.7, 52.0, 42.8, 28.5, 28.4; m/z (ES) 721 (100%, MNaþ),
699 (25, MHþ); HRMS (ES): MNaþ, found 721.3185. C34H46N6NaO10
requires 721.3173.
extracts were made basic to pH 9 by addition of concentrated aq
ammonium hydroxide then extracted with ethyl acetate (ꢂ3). The
combined extracts were washed with brine, dried (Na2SO4), and
evaporated to give the
b
-amino ester 19 (11.8 g, 39%) as a pale-
yellow oil; nmax 2978, 1715, 1610 cmꢃ1
;
dH 8.06 (d, J¼8.4 Hz, 2H),
4.1.7. (ꢁ)-(10-{[(N0-tert-Butoxycarbonyl-guanidinobenzoyl)hydra-
zino]carbonyl}-amino)-tert-butyl-4-propanoyl-benzoic acid 16. The
methyl ester 15 (210 mg, 0.301 mmol) was dissolved in a mixture of
methanol (2 mL), THF (2 mL) and water (1 mL) and lithium hy-
droxide monohydrate (50 mg, 1.19 mmol) was added. The mixture
was stirred at room temperature for 18 h then acetic acid (1 mL)
was added and the mixture evaporated (bath temperature ꢄrt). The
residue was subjected to reverse-phase chromatography through
a column of C18 silica; 30/70% MeOH in H2O (0.1% TFA). The major
fraction gave 16 as a colorless oil (72 mg, 41%); Rf [RP-18 plate, 50%
MeOH in H2O (0.1% TFA)] 0.24; nmax 3249 (br), 2983,1665,1585 (sh)
7.49e7.45 (m, 4H), 7.39e7.29 (m, 3H), 5.38 (s, 2H), 4,45 (t, J¼6.8 Hz,
1H), 2.60 (d, J¼6.8 Hz, 2H), 1.44 (s, 9H); dC 171.1, 166.4, 150.3, 136.2,
130.2, 129.3, 128.8, 128.4, 128.3, 126.6, 81.2, 66.8, 52.8, 45.3, 28.3; m/
z (ES) 378 (27%, MNaþ); HRMS (ES): MNaþ, found 378.1693;
C21H25NNaO4 requires 378.1681.
4.1.11. (ꢁ)-Benzyl-(10-{tert-butoxycarbonyl[hydrazino]carbonyl}-
amino)-tert-butyl-4-propanoyl-benzoate 21. A solution of carbon-
yldiimidazole (6.40 g, 39.5 mmol) in dry DMF (40 mL) was cooled
to ꢃ10 ꢀC and a solution of tert-butyl carbazate 20 (4.74 g,
35.9 mmol) was added over 10 min. The mixture was stirred at
ꢃ10 ꢀC for 15 min and then a solution of the amino ester 19 (12.8 g,
35.9 mmol) in DMF (20 mL) was added over 10 min. The mixture
was then allowed to stir at room temperature for 18 h. Aq citric acid
(1 M) was added to give pH <5 and the mixture extracted twice
with 1:1 EtOAc/P.S. The combined organic extracts were washed
successively with water (ꢂ2), 1 M aq citric acid and brine, then
dried (Na2SO4) and evaporated to give 21 (16.8 g, 91%) as a colorless
foam, mp 61e3 ꢀC; nmax 3326, 2978, 1717, 1669 (sh), 1611,
cmꢃ1
;
dH [MeOH-d4] 7.99 (d, J¼8.2 Hz, 2H), 7.94 (d, J¼7.8 Hz, 1H),
7.86 (br s,1H), 7.64 (t, J¼7.8 Hz,1H), 7.58 (br d, J¼8.2 Hz,1H), 7.49 (d,
J¼8.2 Hz, 2H), 5.29 (t, J¼6.8 Hz, 1H), 2.87e2.76 (m, 2H), 1.58 (s, 9H),
1.36 (s, 9H); dC [MeOH-d4] 171.8, 169.7, 168.9, 160.1, 155.8, 153.6,
148.6, 136.0, 135.2, 131.8, 131.1, 129.0, 127.9, 127.0, 86.4, 82.6, 52.3,
43.0, 28.4, 28.3; dF [MeOH-d4] 77.84 (s); m/z (ES) 607 (MNaþ), 585
(MHþ); HRMS (ES): MHþ, found 585.2672. C28H37N6O8 requires
585.2667.
1536 cmꢃ1
;
dH 7.98 (d, J¼8.4 Hz, 2H), 7.43e7.30 (m, 7H), 6.71 (br d,
4.1.8. (ꢁ)-Methyl-(10-{[(N0-tert-butoxycarbonyl-guanidinobenzoyl)-
hydrazino]carbonyl}-amino)-tert-butyl-4-propanoyl-benzoate
17. The methyl ester 15 (38 mg, 0.0544 mmol) was dissolved in
J¼8.6 Hz, 1H), 6.53 (br s, 1H), 6.45 (br s, 1H), 5.32 (s, 2H), 5.31e5.26
(m, 1H), 2.81e2.70 (m, 2H), 1.44 (s, 9H), 1.27 (s, 9H); dC 170.5, 166.4,
158.1, 156.0, 146.8, 136.3, 130.1, 129.3, 128.8, 128.4, 128.3, 126.5, 82.3,
81.8, 66.8, 50.2, 41.4, 28.3, 28.1; m/z (ES) 536 (100%, MNaþ); HRMS
(ES): MNaþ, found 536.2375. C27H35N3NaO7 requires 536.2373.
methanol (2 mL) and 1 M aq K2CO3 (60 mL) was added. The mixture
was heated to 45 ꢀC for 6 h, then evaporated. The residue was di-
luted with 1 M aq HCl and extracted with EtOAc (ꢂ3). The com-
bined organic phase was washed with brine, dried (MgSO4), and
evaporated. Chromatography of the crude product through a short
silica column [5/10% MeOH in CH2Cl2 (1% HOAc)] gave 17 (22 mg,
68%) as a colorless foam; Rf [5% MeOH in CH2Cl2 (1% HOAc)] 0.20;
4.1.12. (ꢁ)-Benzyl-(10-{[hydrazino]carbonyl}-amino)-tert-butyl-4-
propanoyl-benzoate 22. The N-Boc-semicarbazide 21 (34.4 g,
67.0 mmol) was dissolved in dry dichloromethane (60 mL) and tert-
butyl acetate (180 mL) was added. The mixture was cooled in an
ice-water bath and methanesulfonic acid (19.5 g, 203 mmol) was
added. After 5 min, the mixture was removed from the cold bath
and allowed stir at room temperature for 2 h, after which time
reaction was shown to be complete by TLC analysis. The mixture
was diluted with an equal volume of P.S. and extracted with 1 M aq
citric acid (ꢂ3). The combined citric acid extracts were washed with
1:1 EtOAc/PS and then cooled in ice as the pH was adjusted to ca. 10
by addition of concentrated aq ammonium hydroxide. The mixture
was then extracted with dichloromethane (ꢂ3). The combined
dichloromethane extracts were washed with brine, dried (Na2SO4),
and evaporated to give semicarbazide 22 (20.60 g, 74%) as a white
nmax 3294, 2979, 1721, 1651, 1585 cmꢃ1 1H NMR [MeOH-d4]
; d 7.95
(d, J¼8.3 Hz, 2H), 7.72 (s, 1H), 7.67 (d, J¼7.4 Hz, 1H), 7.48 (d,
J¼8.3 Hz, 2H), 7.46e7.38 (m, 2H), 5.28 (t, J¼6.7 Hz, 1H), 3.88 (s, 3H),
2.86e2.75 (m, 2H), 1.48 (s, 9H), 1.33 (s, 9H); dC [MeOH-d4] 171.8,
169.6, 168.4, 160.2, 159.4, 157.7, 148.8, 139.8, 135.2, 131.1, 130.8,
130.4, 129.2, 128.0, 126.2, 124.7, 82.5, 82.4, 52.8, 52.2, 42.9, 28.6,
28.4; m/z (ES) 621 (100%, MNaþ), 599 (34, MHþ); HRMS (ES): MHþ,
found 599.2822. C29H39N6O8 requires 599.2829.
4.1.9. Benzyl 4-formylbenzoate 18. 4-Carboxybenzaldehyde (15.2 g,
101.2 mmol) was dissolved in dry DMF (100 mL) and Cs2CO3 (32.8 g,
101 mmol) was added, followed by benzyl bromide (15.7 g,
91.8 mmol). The mixture was stirred at room temperature for 3 h
then filtered. The filtrate was diluted with 20% EtOAc in P.S. and
washed successively with water (ꢂ2), satd NaHCO3, and brine then
dried (MgSO4) and evaporated to give benzyl 4-formylbenzoate
1836 (21.3 g, 97%) as a colorless solid. This aldehyde oxidized on
storage and was used immediately without further purification; dH
10.12 (s, 1H), 8.26 (d, J¼8.3 Hz, 2H), 7.97 (d, J¼8.3 Hz, 2H), 7.50e7.37
(m, 5H), 5.42 (s, 2H); dC 191.8, 165.6, 139.4, 135.7, 135.3, 130.5, 129.7,
128.9, 128.7, 128.5, 67.5.
solid, mp 135e7 ꢀC; nmax 3358, 2978, 1717,1675, 1611, 1523 cmꢃ1
; dH
8.01 (d, J¼8.4 Hz, 2H), 7.43e7.30 (m, 7H), 7.08 (br d, J¼8.6 Hz, 1H),
6.05 (br s, 1H), 5.33 (s, 2H), 5.32e5.26 (m, 1H), 3.77 (br s, 2H),
2.82e2.72 (m, 2H), 1.31 (s, 9H); dC 170.3, 166.4, 159.7, 147.4, 136.3,
130.1, 129.3, 128.7, 128.4, 128.3, 126.5, 81.6, 66.8, 50.1, 42.1, 28.1; m/z
(ES) 436 (100%, MNaþ), 380 (25); HRMS (ES): MNaþ, found
436.1845. C22H27N3NaO5 requires 436.1848.
4.1.13. (ꢁ)-Benzyl 4-(1-amino-2,6-dioxohexahydropyrimidin-4-yl)-
benzoate 23. Boc-protected semicarbazide 21 (1.03 g, 2.01 mmol)
t
was treated with MeSO3H and BuOAc as above except that the
4.1.10. (ꢁ)-Benzyl-(10-amino)-tert-butyl-4-propanoyl-benzoate
19. A mixture of 4-carboxybenzyl benzaldehyde 18 (20.3 g,
84.5 mmol), mono-tert-butyl malonate ammonium salt 823 (29.9 g,
169 mmol), ammonium acetate (97.7 g, 1270 mmol), and acetic acid
(150 mL) was heated to 60 ꢀC for 24 h. The mixture was cooled and
poured into ice-water. The pH was adjusted to ca. 9 by addition of
concentrated aq ammonium hydroxide and extracted with EtOAc
(ꢂ2). The organic extracts were diluted with an equal volume of P.S.
and extracted with 1 M aq citric acid (ꢂ3). The combined citric acid
reaction mixture was allowed stir at room temperature for 11 days
before workup. The crude product was purified by radial chroma-
tography (5% MeOH in EtOAc) to give the cyclized compound 23
(289 mg, 42%) as a colorless solid, mp 58e59 ꢀC; Rf (5% MeOH in
EtOAc) 0.38; nmax 3442, 2977, 2938, 2878, 1652, 1437 cmꢃ1
; dH 8.00
(d, J¼8.3 Hz, 2H), 7.41e7.24 (m, 7H), 6.86 (br s, 1H), 5.29 (s, 2H),
4.72e4.62 (m, 1H), 4.45 (br s, 2H), 2.91 (dd, J¼16.6, 4.8 Hz, 1H), 2.69
(dd, J¼16.6, 9.6 Hz, 1H); dC 166.0, 165.8, 153.7, 143.7, 135.9, 131.0,
130.9, 128.9, 128.6, 128.4, 126.3, 67.2, 50.9, 39.5; m/z (EI) 339 (47%,

9454
A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
M
þ.), 254 (100), 240 (61),147 (52),131 (47), 91 (90); HRMS (EI): Mþ.
,
evaporated. The residue was partitioned between CH2Cl2 and H2O.
found 339.1203. C18H17N3O4 requires 339.1214.
The aqueous phase was washed successively with CH2Cl2 and
EtOAc then freeze-dried to give 27 as a colorless syrup (404 mg,
96%); Rf [RP-18 plate, 20% CH3CN in H2O (0.1% TFA)] 0.47; nmax 3070,
4.1.14. (ꢁ)-10-{[(N0-Di-tert-butoxycarbonyl-guanidinobenzoyl)hydra-
zino]carbonyl}-amino-tert-butyl-4-propanoyl-benzoic acid 24. The
aryl carboxylic acid 14 (3.03 g, 7.99 mmol) was dissolved in dry DMF
(25 mL), and HBTU (3.64 g, 9.60 mmol) and collidine (9.68 g,
79.9 mmol) were added, followed by the semicarbazide 22 (3.30 g,
7.99 mmol). The mixture was stirred at room temperature for 4 h,
then partitioned between 1 M aq HCl and EtOAc. The combined
organic phase was washed with water (ꢂ3), brine, dried (MgSO4),
and evaporated. The crude product was chromatographed through
a short silica column [20/60% EtOAc in P.S. (1% HOAc)]. The major
fraction gave the title compound 24 (4.89 g, 79%) as a colorless
foam; Rf (60% EtOAc in P.S.) 0.31; nmax 3255, 2980, 1717, 1627,
2456, 1661, 1581 cmꢃ1
; dH [MeOH-d4] 7.85e7.78 (m, 4H), 7.58 (t,
J¼7.9 Hz, 1H), 7.52e7.46 (m, 3H), 5.30 (t, J¼6.5 Hz, 1H), 3.49 (t,
J¼6.5 Hz, 2H), 2.99 (t, J¼7.3 Hz, 2H), 2.93e2.82 (m, 2H), 1.99e1.92
(m, 2H); dC [MeOH-d4] 173.1, 169.2, 167.9, 161.1 (q, J¼37.1 Hz), 158.9,
156.7, 146.3, 135.5, 134.1, 132.8, 130.2, 128.6, 127.4, 126.5, 126.2,
124.2, 116.6 (q, J¼202.0 Hz), 50.6, 40.3, 37.1, 36.3, 27.5; dF [MeOH-
d4] ꢃ77.4; m/z (ES) 485 (100%, MHþ); HRMS (ES): MHþ, found
485.2260. C22H28N8O5 requires 485.2261.
4.1.18. (ꢁ)-3-{[(N0-3-Guanidinobenzoyl)hydrazino]carbonyl}-amino-
3-(4-carboxamidophenyl)propionic acid hydrochloride salt 28. The
methyl ester 15 (388 mg, 0.555 mmol) was treated with a mixture of
TFA/CH2Cl2/iPr3SiH/H2O (96:96:2:2) (5 mL) at room temperature for
3 h and then evaporated. The crude product was purified by reverse-
phase chromatography through a short column of C18 silica (5/40%
CH3CN in H2O, 0.1% CF3CO2H). The major fraction was collected and
evaporated, and theresiduedissolvedin2 M HCland evaporated(ꢂ2)
to give 28 (178 mg, 69%) as a colorless solid, mp decomp. >226 ꢀC; Rf
[RP-18 plate, 40% CH3CN in H2O (0.1% TFA)] 0.22; nmax 3318, 3198,
1569 cmꢃ1
; dH 11.63 (br s, 1H); 10.34 (br s, 1H), 9.15 (br s, 1H),
7.95e7.81 (m, 5H), 7.52 (d, J¼7.7 Hz, 1H), 7.44e7.23 (m, 8H), 6.79 (br
d, J¼8.1 Hz, 1H), 5.32 (s, 2H), 5.28e5.21 (m, 1H), 2.76e2.62 (m, 2H),
1.54 (br s, 9H), 1.47 (br s, 9H), 1.24 (s, 9H); dC 170.2, 166.6, 166.2,
163.3,157.8,154.0,153.2,146.8,137.0,136.2,132.4,130.0,129.3,129.0,
128.6, 128.2, 128.1, 126.6, 124.1, 121.9, 84.0, 81.4, 80.1, 66.6, 50.7, 41.6,
28.2, 27.9; m/z (ES) 797 (100%, MNaþ); HRMS (ES): MNaþ, found
797.3471. C40H50N6NaO10 requires 797.3486.
1670,1580 cmꢃ1
; dH [DMSO-d6] 10.25 (s,1H),10.14 (s,1H), 8.19 (s,1H),
4.1.15. (ꢁ)-10-{[(N0-Di-tert-butoxycarbonyl-guanidinobenzoyl)hy-
drazino]carbonyl}-amino-tert-butyl-4-propanoyl-benzoic acid 6. A
solution of the benzyl ester 24 (8.02 g, 10.4 mmol) in abs ethanol
(200 mL) was hydrogenated over 10% PdeC (448 mg), at 1 atm H2
for 2 h. Evaporation gave 6 as a colorless syrup (6.86 g, 97%), which
retained traces of ethanol even after prolonged drying in vacuo; Rf
7.91 (d, J¼8.3 Hz, 2H), 7.77 (d, J¼7.8 Hz,1H), 7.71 (s,1H), 7.66e7.48 (m,
7H), 7.41 (d, J¼7.9 Hz, 1H), 7.19 (d, J¼8.5 Hz, 1H), 5.18e5.11 (m, 1H),
3.84(s, 3H), 2.83e2.71 (m, 2H); dC [DMSO-d6] 171.8,166.1,165.6,157.3,
155.9, 148.6, 135.5, 134.1, 129.8, 129.1, 128.2, 127.4, 126.8, 125.3, 123.2,
52.1, 50.0; m/z (ES) 443 (100%, MHþ), 194 (84); HRMS (ES): MHþ,
found 443.1690. C20H23N6O6 requires 443.1674.
(60% EtOAc in P.S.) 0.19; nmax 2978, 1715, 1640 cmꢃ1
; dH (MeOH-d4)
8.00e7.96 (m, 3H), 7.80 (br d, J¼8.0 Hz,1H), 7.67 (br d, J¼7.8 Hz,1H),
7.49e7.42 (m, 3H), 5.32e28 (m, 1H), 2.86e2.76 (m, 2H), 1.51 (s,
18H), 1.34 (s, 9H); dC (MeOH-d4) 171.7, 169.7, 169.6, 160.2, 155.4,
148.5, 138.3, 134.5, 131.0, 130.3, 128.0, 127.8, 125.6, 123.6, 83.2(br),
82.4, 52.1, 42.9, 28.5, 28.4; m/z (ES) 707 (MNaþ); HRMS (ES): MNaþ,
found 707.3016. C33H44N6NaO10 requires 707.3017.
4.2. Cell culture
L929 mouse fibroblasts (cell line ATCC-CCL-1, Rockville, MD, USA)
were used to investigate the ability of compound 28 to inhibit cell
adhesion to vitronectin. Cells were cultured in MEM/Glutamax
(Invitrogen)medium containing 10%fetalbovineserumwith 1%non-
essential amino acids at 37 ꢀC, 5% CO2 in air. Cells were collected by
trypsinization (MesencultÒ-ACF Dissociation kit, STEMCELL Tech-
nologies) and then extensively washed three times with serum free
medium. Bovine vitronectin37 was coated onto 96-well polystyrene
4.1.16. (ꢁ)-tert-Butyl-3-{[(N0-3-di-tert-butoxycarbonyl-guanidino-
benzoyl)hydrazino]carbonyl}-amino-3-(4-(3-tert-butox-
ycarbonylaminopropyl)-carboxamidophenyl)propionoate
26. Carboxylic acid 25 (1.04 g, 1.52 mmol) was dissolved in dry
DMF (10 mL) and HBTU (692 mg, 1.82 mmol) was added, followed
by collidine (1.85 g, 15.3 mmol) and then 1-Boc-propane-1,3-
diamine (392 mg, 2.25 mmol). The mixture was stirred at ambi-
ent temperature for 4 h (complete by TLC) and then partitioned
between 1:1 EtOAc/P.S. and 1 M HCl. The combined organic phase
was washed with water (ꢂ3), brine, dried (MgSO4), and evaporated.
The crude product was chromatographed through a short column
of SiO2, eluting with 60/100% EtOAc in P. S. (1% HOAc) to give the
title product 26 as a colorless foam (964 mg, 75%); Rf (99:1 EtOAc/
plates (Nunc, Denmark) at 5 mg/mL in phosphate buffered saline
(PBS) at 4 ꢀC for 16 h. Prior to cell studies, plates were thoroughly
washed three times with PBS and blocked in 1% bovine serum al-
bumin(BSA) in PBS for 1 h. Stock solutionsofcyclic RGDfK 3 (Peptides
International Inc, USA), cyclic RADfK (Peptides International Inc,
USA), GRGDS (GenScript, USA) peptides, and RGD mimetic 28 were
madeupataconcentrationof100 mMinDMSOthendiluted10times
in PBS and subsequently in cell culture media (without serum com-
ponent). Peptides (140 mL, 100 mMe0 nM) were pre-incubated with
cells (140
m
L, 30ꢂ104 cells/mL) in a low binding plate (CorningÒ Ultra
HOAc) 0.56; nmax 3300, 2978, 1719, 1692, 1644, 1539 cmꢃ1
;
dH
low attachment 96 well) at 37 ꢀC for 1 h. Subsequently, 100
mL was
[MeOH-d4] 7.95 (br s, 1H), 7.83e7.76 (3H, m), 7.67 (br d, J¼7.9 Hz,
1H), 7.49e7.42 (m, 3H), 5.29 (t, J¼6.8 Hz, 1H), 3.40 (t, J¼6.8 Hz, 2H),
3.12 (t, J¼6.8 Hz, 2H), 2.86e2.75 (m, 2H), 1.78e1.71 (m, 2H), 1.51 (s,
18H), 1.43 (s, 9H), 1.34 (s, 9H); dC [MeOH-d4] 175.3, 171.8, 170.0,
169.7, 160.2, 158.7, 155.4, 147.1, 138.4, 134.7, 134.6, 130.3, 128.6, 128.1,
127.9, 125.7, 123.7, 83.3(br), 82.5, 80.2, 52.0, 42.9, 38.9, 38.4, 30.9,
28.9, 28.5, 28.4, 20.9; m/z (ES) 863 (100%, MNaþ); HRMS (ES):
MNaþ, found 863.4285. C41H60N8NaO11 requires 863.4279.
transferredtoapreparedvitronectinplateandcellsfurtherincubated
at 37 ꢀC for 90 min. Cell adhesion was quantitated by MTS colori-
metric assay (Promega) and absorbance read in a Biotek reader.
Acknowledgements
We thank Drs. Roger Mulder, Jo Cosgriff, and Carl Braybrook for
assistance with NMR and Mass spectrometry and Penny Bean
(CSIRO) for a gift of bovine vitronectin.
4.1.17. (ꢁ)-3-{[(N0-3-Guanidinobenzoyl)hydrazino]carbonyl}-amino-
3-(4-(3-aminopropyl)-carboxamidophenyl)propionic acid bis-tri-
fluoroacetate salt 27. Compound 26 (494 mg, 0.587 mmol) was
treated with a mixture of TFA/CH2Cl2/iPr3SiH/H2O (96:96:2:2)
(10 mL) at room temperature for 3 h then the mixture was
Supplementary data
1H and 13C NMR spectra for compounds 9, 12e19, 21e24, 6,
26e28. Supplementary data associated with this article can be

A.G. Riches et al. / Tetrahedron 68 (2012) 9448e9455
9455
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Kessler, H. J. Med. Chem. 2001, 1938e1950.
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