Synthesis and evaluation of RGD peptidomimetics aimed at surface bioderivatization of polymer substrates

BIOORGANIC &

MEDICINAL

CHEMISTRY

Bioorganic & Medicinal Chemistry 6 (1998) 1577±1595

Synthesis and Evaluation of RGD Peptidomimetics Aimed at

Surface Bioderivatization of Polymer Substrates

Thierry Boxus,^aRoland Touillaux,^bGeorges Dive^c

and Jacqueline Marchand-Brynaert^a,*

a

Á

Â

Laboratoire de Chimie Organique de Synthese, Universite catholique de Louvain, Departement de Chimie, Batiment Lavoisier,

Ã

Place L. Pasteur 1, B-1348 Louvain-la-Neuve, Belgium

Â

b

Laboratoire de Chimie Physique et Cristallographie, Universite catholique de Louvain, Departement de Chimie, Batiment Lavoisier,

Ã

Place L. Pasteur 1, B-1348 Louvain-la-Neuve, Belgium

Á

c

Â Â

Ã

Centre d'Ingenierie des Proteines, Universite de Liege, Batiment de Chimie B6, B-4000 Sart-Tilman, Belgium

Received 22 January 1998; accepted 7 April 1998

AbstractÐSeveral RGD peptidomimetics have been prepared, in a convergent way, from the common ortho-amino-

tyrosine template (O-substituted with an anchorage-arm or a methyl group, and aN-substituted with a ¯uorine tag for

XPS analysis), and various o-aminoacid derivatives. The most ¯exible compounds have shown a biological activity

similar to that of the peptide reference (RGDS) in the platelet aggregation test. The compound 16a could be ®tted (by

modelisation) with DMP 728 and c(RGDfV), two cyclic peptides that are good ligands of integrins. The compound 16b

has been covalently ®xed on the surface of a poly(ethylene terephthalate) membrane used as support for mammalian

Introduction

Accordingly, our active biocompatibilization strategy of

polymeric substrates relies on the covalent ®xation of

small molecules acting as integrin ligands onto the

material surface.

The use of synthetic polymers in medical engineering

and biotechnological applications has increased con-

siderably in recent years.^1,2For instance, polymer

membranes are developed as supports for in vitro culti-

vations of mammalian cells used as models of biological

barriers to investigate transport of nutrients or phar-

macological agents.^3±5Our interest in this ®eld led us to

perform surface modi®cations of the supports in view to

improve the cellular adhesion and growth.⁶A ®rst

approach consisted in the introduction of functional

motifs susceptible to increase the surface hydrophilicity

and charge.^7±13The chemically modi®ed surfaces were

further biocompatibilized by the covalent coupling of

extracellular matrix (ECM) proteins.^6,14Our current

purpose is to replace proteins by stable synthetic biologi-

cal signals that would similarly promote cell anchorage.

Integrins¹⁵are heterodimeric transmembrane glyco-

proteins involved in cell±cell and cell±matrix inter-

actions;¹⁶these cell surface receptors interact with the

cytoskeleton and play a crucial role in the signal trans-

duction processes.¹⁷Most of integrins bind to adhesive

proteins displaying the Arg-Gly-Asp (RGD) sequence.¹⁸

Platelets aggregation is mediated by the a_IIbb₃integrin,

which natural ligand is the blood protein ®brinogen.¹⁹

The related integrin a_vb₃of endothelial cells has been

recognized as an important mediator of cellular adhesion

and migration in the angiogenesis process;²⁰in this case,

the natural ligand is the ECM protein vitronectin. A lot

of ¯exible linear peptides containing the RGD sequence

bind to integrins, but non speci®cally.²¹On the other

hand, cyclic RGD peptides appear more selective

towards either a_IIbb₃or a_vb₃integrins.²²

Key words: RGD (Arg-Gly-Asp); peptidomimetic; integrin

ligand; poly(ethylene terephthalate) surface; substratum bio-

derivatization.

*Corresponding author. Fax: +32-10-474168;

E-mail: marchand@chor.ucl.ac.be

Over the last 10 years, peptidomimetics^23±25of the RGD

sequence have been developed as potential drugs,

PII: S0968-0896(98)00083-2

1578

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

mainly in the ®eld of orally available anticoagulants

(a_IIbb₃antagonists) for the treatment of thrombo-

embolic diseases.²⁶Very recently, the search of a_vb₃

antagonists has emerged in order to treat cancers²⁷and

other disorders in which neovascularization plays a cri-

tical role.²⁸

We planned to functionalize our cell culture substrates

with RGD peptidomimetics. In the previous literature,

the grafting of RGD peptides was reported to sig-

ni®cantly improve the cellular adhesion on various

polymeric supports.^29±31However the eect of non-

peptide mimics was never considered, though such syn-

thetic signals should be more stable under biological

¯uids, storage and sterilization conditions.

Figure 1. The tyrosine (X=H) template and its arrangement.

hydroxyl function can be used to anchor a second arm

needed for immobilizing the molecule onto polymer

substrates; (d) nitration of the aromatic ring, followed

by reduction, oers the anchorage point of the third

arm mimicking the R (Arg) residue; the distance separ-

ating the acidic and basic residues of the pharmaco-

phore should be within 10±20 A.^27,43,47

In this paper we report the preparation of several RGD

peptidomimetics based on the ortho-amino-tyrosine

template, and their evaluation in the classical platelet

aggregation test. One molecule (16b) equipped with an

anchorage-arm has been covalently ®xed on a poly

(ethylene terephthalate) (PET) microporous membrane

used as cell cultivation support.

The N-tri¯uoroacetyl group was initially chosen as

¯uorine tag for the XPS analysis of the surface modi®ed

polymer membranes. Therefore, in our synthetic plan,

we have avoided using protective groups removable

under basic conditions that could cleave our spectro-

scopic label; accordingly, we played exclusively with the

classical tertio-butyl (^tBu), tertio-butoxycarbonyl (Boc),

and benzyloxycarbonyl (Cbz) groups.

Results and Discussion

Synthesis

Several series of RGD peptidomimetics based on var-

ious rigid scaolds^32,33have been proposed as anti-

platelet agents; for instance, molecules were constructed

from benzodiazepine,³⁴isoquinolone,³⁵isoxazoline,³⁶

pyrazolopiperazinone,³⁷or 3-(hydroxymethyl) benz-

amide³⁸moieties. The situation appears totally dierent

in the case of RGD mimics designed to promote adhe-

sion phenomena; to our knowledge, only four types of

molecules have been very recently disclosed, based on p-

hydroxybenzamide,²⁷piperazine,³⁹carbohydrate,⁴⁰or

benzodiazepine^27,41templates.

(l)-Tyrosine tertio-butylester 1 was reacted with neat

tri¯uoroacetic anhydride to give N-tri¯uoroacetyl-(l)-

tyrosine tertio-butylester. This intermediate was directly

treated with a solution of nitric acid in acetic acid at low

temperature to furnish the mono-nitration product 2

isolated by column-chromatography (Scheme 1).

For chemoselectivity reasons, the O-alkylation of the

phenol ring has to be performed before the reduction of

the nitro group, and the subsequent functionalization of

the resulting aniline. This Williamson reaction, planned

to introduce the anchorage-arm for immobilization onto

polymer membranes, was ®rst examined with methyl-

iodide. Thus, 2 was reacted with methyliodide, in

re¯uxing acetonitrile, in the presence of powdered

potassium carbonate and a crown ether as catalyst. The

product 3a, contaminated with a small amount of N-

methylation product of the tri¯uoroacetamide residue,

was puri®ed by preparative medium pressure liquid

chromatography (MPLC) in 72% yield. The N-protected

anchorage-arm, N-(Boc)-3-bromopropylamine, could

be similarly coupled to 2, giving 3b in 78% yield after

puri®cation (Scheme 1).

When starting this work, without reliable guide in hand,

we decided to examine relatively ¯exible structures

derived from (l)-tyrosine. The utilization of this tem-

plate has been well exempli®ed by the Merck's scien-

tists;^42,43one compound (Aggrastat², MK-383) is in

phase III clinical trials.⁴⁴The advantages of the tyrosine

template and the designed structural features of our

RGD peptidomimetics are summarized in the Figure 1:

(a) the aromatic template is commercially available and

already equipped with the ®rst arm mimicking the d

(Asp) residue; (b) the a-amino function, that has to be

masked with a lipophilic group,⁴³can be advanta-

geously used for the ®xation of a ¯uorine tag in view of

the X-ray photoelectron spectroscopic (XPS) analysis of

the ®nal polymer substrates;^45,46(c) the aromatic

The aromatic ring functionalization with various arms

mimicking the basic residue of Arg was systematically

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1579

Scheme 1. Synthesis of peptidomimetics (®rst family). Reagents and conditons: (i) (CF₃CO)₂O, 0^ꢀC, 2 h; (ii) HNO₃-HOAc, 10 ^ꢀC, 1 h;

(iii) a. CH₃I, CH₃CN, K₂CO₃, re¯ux, 48 h: b. BocNH-(CH₂)₃-Br, CH₃CN, K₂CO₃, re¯ux, 24 h; (iv) H₂, PtO₂, EtOH, 4±12h, 20 ^ꢀC; (v)

acid chloride, pyridine, CH₂Cl₂, 17 h, 20 ^ꢀC; then column chromatography; (vi) Pd-C, EtOAc, 20 ^ꢀC, 18 h; (vii) CF₃CO₂H, 2 h, 20 ^ꢀC.

investigated, using the precursor 3a in which the

anchorage-arm is replaced with a simple methyl group.

Reduction of 3a by catalytic hydrogenation over plati-

nium oxide furnished the key-intermediate aniline 4a

which was directly used without puri®cation. The reac-

tivity of this aniline in the peptide coupling reaction was

examined, under various experimental conditions, using

N-(Cbz)-5-aminovaleric acid as partner. Low yields of the

anilide 5a were obtained when the acid was activated in

situ with diisopropylcarbodiimide/dimethylaminopyridine,

dicyclohexyl-carbodiimide/N-hydroxysuccinimide, or

O-benzotriazol-1-yl-N,N,N⁰,N⁰-tetramethyluroniumhexa-

¯uorophosphate (HBTU). The best results were obtained

when preforming the acid chloride with thionyl chloride

or 1-chloro-N,N, 2-trimethylpropenylamine;⁴⁸reaction

of N-(Cbz)-5-aminovaleryl chloride with 4a and tri-

ethylamine in dichloromethane at room temperature

gave 45% yield of the coupling product 5a after pur-

i®cation by column-chromatography. This strategy was

thus selected for the coupling of N-(Cbz)-6-amino-

caproic acid, N-(Cbz)-isonipecotic acid and N-(Cbz)-

nipecotic acid. Reaction of the corresponding pre-

formed acid chlorides with 4a led, respectively, to the

anilides 6a, 7a, and 8a in 35±55% yields (Scheme 1).

Hydrogenolysis of the Cbz protective group of 5a gave

the free amine 11a (tBu ester) which was submitted to

various guanidylation conditions. Unfortunately, all

attempts to react 11a with unprotected guanidylation

reagents (i.e. aminoiminomethane sulfonic acid⁴⁹and

1H-pyrazole-1-carboxamidine)⁵⁰and with the protected

reagent 17, N,N⁰-ditertio-butoxycarbonyl-3,5-dimethyl-

pyrazole-1-carboxamidine,⁵¹failed. Therefore, we con-

sidered a more convergent route towards 9a (or 15a)

and 10a (or 16a), using N-guanidyl-aminoacid deriva-

tives as partners in the peptide coupling with the aniline

4a. The required acid chlorides 20 were obtained

according to the Scheme 2.

1580

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

d (J ꢁ 8.7 Hz); (b) the proton of the tyrosine a-CH

group is a doublet of doublet at 4.6±4.8 d (J ꢁ 5 and

9.5 Hz); (c) the protons of the tyrosine b-CH₂group

appear to be non-equivalent, giving a ABX pattern

centred at 2.95±3.00 d and 3.25±3.30 d (JAB ꢁ 14 Hz).

In the ¹³C NMR spectra (D₂O), (a) the carbon atom of

the CF₃XPS tag is visible at 118 ppm (Q); (b) the three

substituted carbon atoms of the tyrosine aromatic ring

give lines at 153 ppm, 131 ppm and 127 ppm, corre-

sponding to carbons linked to oxygen, nitrogen and

carbon respectively; (c) the a and b carbons of the ali-

phatic tyrosine chain appear at 57 ppm and 38 ppm,

respectively.

In another set of experiments, we considered the meta-

tri¯uoromethyl-benzenesulfonyl group (XPS tag) for

masking the a-amino function of the tyrosine template.

Due to the high acidity of the sulfonamide proton, the

presence of this function is not compatible with the

phenol ring etheri®cation under the Williamson condi-

tions. Accordingly, the sulfonylation of the a-amino

function was performed after the tyrosine O-alkylation

step. Thus, compound 3a (see Scheme 1) was submitted

Scheme 2. Synthesis of acid chlorides. Reagents and condi-

tions: (i) PhCH₂OH, pTos-OH, benzene, re¯ux, 3 h; (ii) Et₃N,

CH₂Cl₂, 20 h, 20 ^ꢀC; (iii) H₂, Pd-C, EtOH:EtOAc (1:1), 18 h,

20 ^ꢀC; (iv) SOCl₂, CH₂Cl₂, 1 h, 30 min, re¯ux; (v) pridine,

CH₂Cl₂, 20 ^ꢀC.

5-Aminovaleric acid (a, n=4) and 6-aminocaproic acid

(b, n=5) were esteri®ed with benzyl alcohol under stan-

dard conditions (18a,b), then reacted with N,N⁰-ditertio-

butoxycarbonyl-3,5-dimethylpyrazole-1-carboxamidine

17⁵¹and triethylamine in dichloromethane. After pre-

parative MPLC, compounds 19a,b were quantitatively

obtained. Cleavage of the benzyl ester by hydro-

genolysis, followed by reaction with thionyl chloride

gave the acid chlorides 20a,b, which were not puri®ed

(Scheme 2). Reaction of 20b (n=5) with the aniline key-

intermediate 4a in the presence of pyridine furnished the

coupling product 10a in 35% yield after puri®cation by

column-chromatography (Scheme 1). The lower homo-

logue 9a could not be obtained by similarly treating 4a

with the valeric derivative 20a (n=4); in the presence of

pyridine, the acid chloride 20a cyclized intramolecularly

very rapidly (21, Scheme 2). Thus, in our hands, the

peptidomimetic 15a (after deprotection of 9a) was not

accessible. Treatment of 10a with tri¯uoroacetic acid

produced the fully deprotected guanidyl derivative 16a.

N-Deprotection of the aminocaproic derivative 6a and

(iso)nipecotic derivatives 7a and 8a by hydrogenolysis,

followed by treatment with tri¯uoroacetic acid to cleave

the tertio-butyl ester, gave the unprotected compounds

12a, 13a and 14a, respectively (Scheme 1).

All the compounds 6±8, 10, 12±14, and 16 were fully

characterized by the usual spectroscopic methods (see

Experimental). The typical features in the ¹H NMR

spectra of the tested compounds (unprotected deriva-

tives in D₂O) are as follows: (a) the three aromatic pro-

tons of the tyrosine backbone give respectively a ®ne

doublet at 7.3±7.5 d (J ꢁ 2 Hz), a doublet of doublet at

7.1±7.2 d (J ꢁ 2 Hz and 8.7 Hz) and a doublet at 7.0±7.1

Scheme 3. Synthesis of peptidomimetics (second family).

Reagents and conditions: (i) K₂CO₃, MeOH:H₂O (1:1), 17 h,

20 ^ꢀC; (ii) Et₃N, CH₂Cl₂, 20 h, 20 ^ꢀC; (iii) H₂, PtO₂, MeOH,

17 h 20 ^ꢀC; (iv) acid chloride, pyridine, CH₂Cl₂, 20 h, 20 ^ꢀC;

then column chromatography; (v) H₂, Pd-C, EtOH:EtOAc

(1:1), 18 h, 20 ^ꢀC; (vi) CF₃CO₂H, 2 h 30 min, 20 ^ꢀC.

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1581

to basic hydrolysis to furnish the free amine 22 which

was directly treated with meta-tri¯uoromethyl-benzene-

sulfonyl chloride and triethylamine; the sulfonamide 23

was puri®ed by ¯ash-chromatography with medium

yields (Scheme 3). Reduction of the aromatic nitro

function by hydrogenation over platinium oxide gave

the aniline key-intermediate 24. This aniline was cou-

pled with N-(Cbz)-6-aminocaproyl chloride and with

(N,N⁰-diterbutoxycarbonyl)-6-guanidino-caproyl chlor-

ide 20b as previously described; the corresponding ani-

lides 25 and 26 were obtained in 48% and 24% yield

after ¯ash-chromatography. The ®nal deprotections

were realized by hydrogenolysis followed by treatment

with tri¯uoroacetic acid to give 27, and by treatment

with tri¯uoroacetic acid to give 28 (Scheme 3). The

NMR characteristics of compounds 25±28 are similar

to those of the previous series bearing the tri¯uoro-

acetamide group (see Experimental): the proton of the

a-CH group gives a doublet of doublet at 4 d; the carbon

of the CF₃XPS tag is visible at 124 ppm (Q).

tion of the ester, guanidino and amino functions was

realized, as usual, by treatment with tri¯uoroacetic acid

at room temperature; compound 16b was quantitatively

recovered (Scheme 1). In the ¹H NMR spectrum, the

three methylene protons of the anchorage-arm give

multiplets at 2.13 d, 3.16 d and 4.16 d; the corresponding

¹³C NMR lines are found at 28.9 ppm, 39.6 ppm and

68.5 ppm.

Biological evaluation (in solution)

The in vitro activity of the compounds listed in Table 1

was assayed in a standard test of platelet aggregation

inhibition.⁵²Human platelet rich plasma (PRP) was

freshly prepared and platelet aggregation was measured

by recording the velocity of light transmission change

with an aggregometer. Serial dilutions of the inhibitors

were added, and aggregation was induced by addition of

adenosine diphosphate (ADP). Inhibition of platelet

aggregation was determined by comparison of light

transmittance values for the control (absence of inhi-

bitor) and the samples. The IC₅₀was determined as the

concentration necessary to inhibit the change in light

transmittance by 50%. We used the tetrapeptide RGDS

(Arg-Gly-Asp-Ser) as active reference compound: it

inhibits ADP-mediated platelet aggregation with a IC₅₀

value of about 100 mM.

At last, we prepared a peptidomimetic fully equipped

for the anchorage onto polymer substrates. Reduction

of the aromatic nitro function of compound 3b (bear-

ing the N-protected aminopropyl arm) by catalytic

hydrogenation over platinium oxide gave the key-

intermediate aniline 4b (Scheme 1). Coupling with the

N-protected 6-guanidino-caproyl chloride 20b in the

presence of pyridine furnished the anilide 10b in 51%

yield after ¯ash-chromatography. Complete deprotec-

We found that the 6-guanidino- (entries 1±3) and the 6-

aminocaproic derivatives (entry 5) are inhibitors, while

Table 1. Biological evaluation in solution

Molecules

Entry

Compound

IC₅₀(mM) (reference)^a

1

2

3

4

5

6

7

COCF₃

Me

16a

465 (129)

16b

28

320 (68)

85 (68)

SO₂PhCF₃

COCF₃

Me

12a

27

unsoluble

125 (100)

>1000

SO₂PhCF₃

COCF₃

13a

14a

COCF₃

>1000

^aIC₅₀value (mM) of the RGDS tetrapeptide (Arg-Gly-Asp-Ser).

1582

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

the (iso)nipecotic derivatives are inactive (entries 6 and

7). In the more active compounds, the a-amino function

of the tyrosine template was masked with a hydrophobic

arylsulfonyl group (entries 3 and 5). The presence of the

anchorage-arm (for surface immobilization) did not

perturb the activity (entry 2). The levels of activities

were similar to that of the reference peptide (RGDS).

DMP 728 (Fig. 2) is a potent and selective a_IIbb₃integ-

rin antagonist,^53,54while the cyclo (Arg-Gly-Asp-D-

Phe-Val) (c[RGDfV]) is a potent and selective a_vb₃

integrin antagonist (Fig. 3).^55,56

The ¯exible RGDS tetrapeptide can adopt many con-

formations allowing its binding to many integrins,^18,21

even though with moderate anity. We could expect a

similar behaviour in the case of the ¯exible peptido-

mimetic 16b, for which we have recorded a signi®cant

anity toward the a_IIbb₃integrin (®brinogen receptor).

The same level of activity toward the a_vb₃integrin

(vitronectin receptor) should be sucient to promote

cell adhesion after immobilization of 16b on the surface

of culture substrates.

Modelisation

Two cyclic RGD peptides have been chosen as reference

compounds in view to evaluate the conformational

arrangement of the selected peptidomimetic 16a

(anchorage-arm replaced with a methyl group). The

Figure 3. Cyclopeptide c(RGDfV).

Figure 2. Cyclopeptide DMP728.

Figure 4. Peptidomimetic 16a.

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1583

The references and compound 16a (Fig. 4) were fully

optimized at the approximate quantum chemistry level

AM1; the molecules were studied as isolated neutral

entities, and not as zwitterionic forms, in order to avoid

internal self-folded conformations.

with heavy ions (Ar⁺⁹) accelerated in a cyclotron, fol-

lowed by immersion into an appropriate solution of

reagents which preferentially etches the tracks, leading

to the creation of pores.⁵⁸The etching treatment of the

PET creates functionalities (chain-endings) on the

membrane open surface that could be used for immobi-

lizing molecules of interest.

As previously pointed out in the literature,^27,41the

overall length of c[RGDfV] is shorter comparatively to

the a_IIbb₃ligands, such as DMP 728. Indeed, the structure

is stabilized by two hydrogen-bonds between the

C=O. . .N-H group of Gly and the adjacent amide

bonds (distances of 2.216 A and 2.568 A, respectively)

(Fig. 3). With its ¯exible side-chains, the potential

ligand 16a displays terminal functions that can be fairly

®tted onto the corresponding carboxyl- and guanidyl

groups of both references (Figs 5 and 6).

The surface displayed functions of PET membranes are

carboxyl- and hydroxyl end-groups (Scheme 4). We

have already demonstrated that the amount of carboxyl

groups could be signi®cantly increased by a surface

oxidative treatment (KMnO₄in 1.2 N H₂SO₄) which

transforms native hydroxyl chain-ends into new car-

boxyl endings^7,9; the resulting membrane was called

PET-CO₂H (Scheme 4).

The surface reactivity of the PET-CO₂H membrane was

3

Surface chemistry

assayed by the covalent coupling of H-lysine followed

Poly(ethylene terephthalate) (PET) is a synthetic aro-

matic polyester widely used as a biomaterial for making

non-resorbable sutures, or performing prosthetic repla-

cements in orthopedic surgery (tendons, ligaments,

facial implants). Actually, the most successful medical

applications of PET are in the area of cardiovascular

surgery, including prosthetic heart valves and vascular

grafts of large to medium diameter (Dacron).^1,2Mem-

branes made from PET ®lms are also appropriate sub-

strates for cultivating mammalian cells.^4,6In our

laboratory, we use microporous membranes obtained by

a track-etching process which allows the preparation of

`capillary-pore' membranes⁵⁷with very uniform, nearly

perfectly round cylindrical pores. Such membranes are

made from homogeneous 10±20 mm thick polymer ®lm

precursors in two steps, consisting of bombardment

by liquid scintillation counting (LSC) of the sample

associated radioactivity.^7,9For that purpose, the labeling

reaction was conducted under mild conditions (water

solution, near the physiological pH, room temperature)

mimicking at best the conditions to be encountered in the

covalent coupling of the peptidomimetic molecule 16b.

Thus, the PET membrane was activated by treatm_ꢀent

with water soluble carbodiimide (0.1% WSC, 1 h, 20 C,

pH 3.5) and then incubated with ³H-lysine (10 ³M

ꢀ

solution, 2 h, 20 C, pH 8); we found a value of about

35 pmol/cm²(open surface) of ®xed label (corrected

value, obtained by subtracting the adsorption contribu-

tion, see Experimental). From a previously established

model of the PET surface⁷, we calculated that this value

corresponds to the functionalization of about 1.2% of

the monomer units (see Experimental).

Figure 5. Superposition of 16a and DMP728.

Figure 6. Superposition of 16a and c(RGDfV).

1584

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

Scheme 4. Covalent coupling of 16a onto the PET membrane surface.

The RGD peptidomimetic 16b was similarly coupled to

the PET-CO₂H membrane activated by the pre-treat-

ment with WSC (Scheme 4). A blank sample was pre-

substrate, displaying about 1% of synthetic `RGD-like'

signals (i.e. about 30 pmol/cm²) should be a good can-

didate to promote the adhesion of anchorage-dependent

cells.

pared by immersing

a non-activated PET-CO₂H

membrane into the solution of 16b in phosphate buer

(0.062%, or ꢁ 10 ³M). The X-ray photoelectron spec-

troscopy (XPS) of the blank sample did not show the

presence of ¯uorine atoms on the surface (thus, no

detectable adsorption). However, the activated mem-

brane ®xed (most probably by covalent grafting) the

RGD peptidomimetic as revealed by the presence of

0.24% of ¯uorine atoms (XPS analysis) in the atomic

composition of the sample surface (sampling depth of

about 50 A). From the experimental F/C Â 100 atomic

ratio of 0.339, we calculated (see Experimental) that

about 1.1% of the surface monomer units have ®xed the

biological signal. This value is in good agreement with

the theoretical value based on the surface radiolabeling

Conclusion

Several RGD peptidomimetics have been constructed

from the ortho-amino-tyrosine template. The synthetic

strategy allowed to equip the structures with an anchorage-

arm and a ¯uorine tag, as required for the surface

immoblilization on polymer substrates, and the sub-

sequent quanti®cation of the amount of ®xed biological

signals by X-ray photoelectron spectroscopy.

The most ¯exible molecule 16 has been selected for

coupling on the poly(ethylene terephthalate) membrane

currently used as cell culture support. In solution, 16a,b

exhibited moderate activities, in the platelet aggregation

test, that range the compounds at the level of the tetra-

peptide RGDS; the presence of an anchorage-arm did

assay (1.2%). According to Massia and Hubbell,^59,60

a

surface concentration of about 10 fmol/cm²of grafted

natural peptides is large enough to improve the cell-

adhesive properties of a biomaterial. Thus our PET

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1585

not perturb the biological response. The structure 16a

could be fairly ®tted with two cyclic RGD peptides

which are representative ligands of the ®brogen- and

vitronectin receptors, respectively.

aqueous acetic acid (1%, 300 mL). The column-

chromatographies (under normal pressure) were carried

out with Merck silica gel 60 of 70±230 mesh ASTM, and

the ¯ash-chromatographies, with Merck silica gel 60 of

230±400 mesh ASTM. The MPLC puri®cations were

realized on a Prochrom equipment, with silica gel of 400

mesh ASTM, under a pressure of 40 bar and a ¯ow of

160 mL/min.

Using the wet-chemistry technique, we enriched the PET

membrane surface with carboxyl functions. Their acti-

vation with WSC allowed to ®x the peptidomimetic 16b

in good yield, as controlled by XPS: almost all the

reactive CO₂H chain-endings assayed by radiolabeling

and LSC have quenched the biological signal.

The melting points were determined with an Electro-

thermal microscope and are uncorrected. The rotations

( 0.1^ꢀ) were determined on a Perkin±Elmer 241 MC

polarimeter. The IR spectra were taken with a Perkin±

Elmer 600 instrument or with a Bio-Rad FTS 135

instrument, and calibrated with polystyrene (1601 cm ¹).

To our knowledge, the present work is the ®rst report

dealing with the covalent coupling of RGD peptidomi-

metics on the surface of PET membrane. The perfor-

mances of such a modi®ed support are currently

examined in our laboratory by culturing dierent mam-

malian cell lines.

1

The H and ¹³C NMR spectra were recorded on Varian

Gemini 300 (at 300 MHz for proton and 75 MHz for

carbon), or Bruker AM-500 spectrometers (at 500 MHz

for proton and 125 MHz for carbon); the chemical shifts

are reported in ppm (d) down®eld from tetramethyl-

silane (internal standard), or sodium 2,2-dimethyl-2-

silapentane-5-sulfonate (DSS) for the spectra recorded

in D₂O. The atom numbering used for the description of

the spectra is shown in the Figure 7; the attributions

were established by selective decoupling experiments.

The mass spectra were obtained on a Finnigan-MAT

TSQ-70 instrument at 70 eV (electronic impact (EI)

mode), or with a Xenon ION TECH 8 KV (fast atom

bombardment (FAB) mode). The microanalyses were

performed at the Christopher Ingold Laboratories of the

University College, London. The HRMS were performed

at the University of Liege (Belgium) on a VG-AutoSpec-Q

equipment (Fisons Instruments, Manchester).

Experimental

Synthesis

The reagents (analytical grade) were purchased from

Acros, Aldrich, or Fluka. The solvents were distilled,

after drying as follows: acetonitrile, dichloromethane,

dimethoxyethane, triethylamine and pyridine, over

calcium hydride; tetrahydrofurane, over sodium; acetone,

over drierite.

The thin-layer chromatographies were carried out on

silica gel 60 plates F254 (Merck, 0.2 mm thick); visuali-

zation was eected with UV light, iodine vapor, a spray

of ninhydrin in ethanol or a spray of potassium

permanganate (3 g) and potassium carbonate (20 g) in

N-(Tri¯uoroacetyl)-ortho-nitro-l-tyrosine terbutylester (2).

l-Tyrosine t-butylester 1 (478 mg, 2.013 mmol) was

Figure 7. Atoms numbering used in the NMR spectra description.

1586

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

ꢀ

dissolved, at 0 C, in tri¯uoroacetic anhydride (1.5 mL,

2.23 g, 10.6 mmol, 5.27 equiv). After 30 min of stirring at

room temperature, the mixture was concentrated under

vacuum. The residue was dissolved in acetic acid (15 mL)

and treated with concentrated nitric acid (115 mL,

2.4 mmol, 1.2 equiv) diluted in acetic acid (15 mL); the

solution of HNO₃was added dropwise, and the mixture

(C-a), 113.74 (C-6), 115.42 (CF3), 126.34 (C-3), 127.41

(C-4), 135.96 (C-5), 139.23 (C-2), 152.24 (C-1), 156.48

(COCF₃), 168.51 (C-10); MS(FAB^-) m/e 391 (M-1), 335

(M-tBu); Anal. calcd for C₁₆H₁₉F₃N₂O₆(392.23): C, 48.98;

H, 4.88; N, 7.14. Found: C, 48.91; H, 4.94; N, 6.77.

N-(Tri¯uoroacetyl)-O-[N-(terbutoxycarbonyl)-3-amino-

propyl]-ortho-nitro-L-tyrosine terbutylester (3b). N-(Ter-

butoxycarbonyl)-3-bromopropylamine was obtained

by protection of 3-bromopropylamine as usual: a solu-

tion of 3-bromopropylamine hydrobromide (4.97 g,

22.28 mmol) in terbutanol±water (100±130 mL) was

treated with diterbutyl dicarbonate (5.27 mL, 5.01 g,

22.28 mmol, 1 equiv) and sodium hydroxide (1.87 g,

ꢀ

was maintained at 10±14 C during the addition. After

75 min of reaction (TLC control), the crude mixture

was poured onto ice (400 g). The yellow precipitate was

®ltered o. The aqueous phase was extracted with ethyl

acetate (4Â150 mL). The organic phases were dried over

MgSO₄, concentrated under vacuum and puri®ed by

column chromatography on silica gel, with CH₂Cl₂as

eluent, to _ꢀgive 0.307 g (yield: 41%) of 2: R_f=0.6; mp

85.4±87.6 C; IR (KBr) n 3358, 1755, 1709, 1550,

ꢀ

46.89 mmol, 2.1 equiv) during 16 h at 20 C. The mix-

ture was extracted with pentane; the organic phase was

washed with 10% NaHCO₃and water, dried over

MgSO₄and concentrated under vacuum to give 4.51 g

(yield: 85%) of N-Boc-3-bromopropylamine: mp 33±

1161 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.48 (s, 9H,

1

H-b), 3.13 (dd, J=5.2 and 14.2 Hz, 1H, H-7), 3.25 (dd,

J=5.9 and 14.2 Hz, CDCl₃1H, H-7⁰), 4.70 (m, 1H, H-

8), 6.96 (d, J=6.4 Hz, 1H, NH-9), 7.11 (d, J=9.0 Hz,

1H, H-6), 7.36 (dd, J=9.0 and 2.1 Hz, 1H, H-5), 7.89 (d,

J=2.1 Hz, 1H, H-3), 10.5 (br s, 1H, OH); ¹³C NMR

(CDCl₃, 125 MHz) d 27.86 (C-b), 35.87 (C-7), 53.70 (C-

8), 84.40 (C-a), 115.35 (CF₃), 120.25 (C-6), 125.24 (C-3),

127.38 (C-4), 133.17 (C-2), 138.55 (C-5), 154.26 (C-1),

156.50 (COCF₃), 168.51 (C-10); MS(EI) m/e 378; Anal.

calcd for C₁₅H₁₇F₃N₂O₆(378.3): C, 46.62; H, 4.52; N,

7.4. Found: C, 47.7; H, 4.42; N, 7.1.

ꢀ

1

34 C; H NMR (CDCl₃, 500 MHz) d 1.38 (s, 9H), 1.99

(m, 2H), 3.21 (m, 2H), 3.38 (t, 2H), 4.83 (m, NH); ¹³C

NMR (CDCl₃, 125 MHz) d 28.15, 30.61, 32.52, 38.75,

79.09, 155.78; MS(EI) m/e 239, 237.

To a solution of 2 (371 mg, 0.979 mmol) in CH₃CN

(35 mL), under argon atmosphere, were added N-Boc-3-

bromopropylamine (233 mg, 0.976 mmol,

1 equiv),

potassium carbonate (149 mg, 1.077 mmol) and [18-c-6]

crown-ether (0.269 mg). The mixture was re¯uxed,

under stirring, during 22 h (TLC control), and then

worked up as described for 3a. Flash chromatography

on silica gel (hexane:ethyl acetate, 4:1) furnished_ꢀ343 mg

of 3b (yield: 78%): R_f=0.1; mp 104.7±106.2 C; IR

(CDCl₃) n 3423 (NH), 1719 (br, CO), 1534, 1262,

N - (Tri¯uoroacetyl) - O - methyl - ortho - nitro - L - tyrosine

terbutylester (3a). The reaction was conducted under

argon atmosphere. To

a solution of 2 (727 mg,

1.92 mmol) in acetonitrile (150 mL) were added iodo-

methane (0.140 mL, 312 mg, 2.248 mmol, 1.17 equiv),

potassium carbonate (268 mg, 1.94 mmol) and [18-c-

6]crown-ether (54 mg, 0.203 mmol, 0.105 equiv). The

mixture was re¯uxed, under stirring, during 48 h (TLC

control). CH₃CN was removed under vacuum; the residue

was dissolved in CH₂Cl₂(100 mL) and washed with water

(3Â20 mL). Drying over MgSO₄, concentration and

¯ash chromatography on silica gel (hexane:isopropanol,

9:1) gave 698 mg of 3a contaminated with the methyl-

ation product of the tri¯uoroacetamide fraction (¹H

NMR: 2.91 d (s, 3H); ¹³C NMR: 32.99 ppm; MS

(FAB⁺) m/e 407). The product was further puri®ed by

preparative MPLC on reverse phase (Novapak 4 m;

methanol:water, 3:2; 1 mL/min) to furnish 548 mg (yield:

1

1169 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.43 (s, 9H,

H-g), 1.48 (s, 9H, H-b), 2.05 (m, 2H, H-b), 3.14 (dd,

J=5.2 and 14.2 Hz, 1H, H-7), 3.25 (dd, J=5.9 and

14.2 Hz, 1H, H-7⁰), 3.36 (m, 2H, H-c), 4.16 (t, J=7.3 Hz,

2H, H-a), 4.69 (m, 1H, H-8), 5.06 (br s, NH-d), 6.94 (d,

J=7.3 Hz, NH-9), 7.03 (d, J=8.6 Hz, 1H, H-6), 7.30

(dd, J=2.1 and 8.6 Hz, 1H, H-5), 7.67 (d, J=2.1 Hz,

1H, H-3); ¹³C NMR (CDCl₃, 125 MHz) d 27.85 (C-g),

28.25 (C-b), 29.00 (C-b), 35.66 (C-7), 37.90 (C-c), 53.71

(C-8), 67.87 (C-a), 79.06 (C-f), 84.40 (C-a), 114.50 (C-6),

115.40 (CF₃), 126.50 (C-3), 127.40 (C-4), 135.16 (C-5),

139.06 (C-2), 151.57 (C-1), 156.05 (c-e), 156.40

(COCF₃), 168.50 (C-10); Anal. calcd for C₂₃H₃₂F₃N₃O₈

(535.51): C, 51.58; H, 6.02; N, 7.84. Found: C, 51.60; H,

6.02; N, 7.78.

72%) of 3a: R_f(hexane:iPrOH, 9:1)=0.4; IR (KBr) n

3345, 2980, 1726, 1625, 1531 cm

1

;

¹H NMR (CDCl₃,

500 MHz) d 1.46 (s, 9H, H-b), 3.13 (dd, J=5.2 and

14.2 Hz, 1H, H-7), 3.23 (dd, J=6.4 and 14.2 Hz, 1H, H-

7⁰), 3.94 (s, 3H, H-a), 4.68 (m, 1H, H-8), 6.98 (d,

J=7.3 Hz, 1H, NH-9), 7.03 (d, J=8.6 Hz, 1H, H-6),

7.31 (dd, J=2.1 and 8.6 Hz, 1H, H-5), 7.64 (d,

J=2.1 Hz, 1H, H-3); ¹³C NMR (CDCl₃, 125 MHz) d

27.78 (C-b), 35.76 (C-7), 53.77 (C-8), 56.45 (C-a), 84.25

N-(Tri¯uoroacetyl)-O-methyl-ortho-amino-L -tyrosine

terbutylester (4a). A solution of 3a (690 mg, 1.759 mmol)

in methanol (12 mL) containing platinium IV oxide

(40 mg, 0.177 mmol, 1 equiv) was placed in a Parr ¯ask

and shaked under hydrogen atmosphere (p=40 psi)

during 18 h at room temperature. After ®ltration and

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1587

evaporation of the methanol, the residue was dissolved

in ethyl acetate, dried over MgSO₄and concentrated

under vacuum to give 558 mg of crude aniline 4a (yield:

88%): R_f(hexane:ethyl acetate, 7:3)=0.3; IR (CHCl₃) n

3368, 3314, 2981, 2927, 1717, 1615, 1501, 1432,

2H, H-5 + H-6), 6.83 (s, 1H, NH-9), 7.71 (s, 1H, NH-

11), 8.25 (s, 1H, H-3), 8.31 (m, 1H, NH-18), 11.50 (s,

1H, NH-20); ¹³C NMR (CDCl₃, 125 MHz) d 25.15 (C-

14), 26.29 (C-15), 27.81 (c-b), 27.95 (C-26), 28.18 (C-23),

28.66 (C-16), 36.71 (C-7), 37.63 (C-13), 40.61 (C-17),

53.87 (C-8), 55.61 (C-a), 79.06 (C-22), 82.93 (C-25),

83.49 (C-a), 109.75 (C-6), 115.52 (CF₃), 120.52 (C-3),

124.00 (C-5), 127.44 (C-4), 127.73 (C-2), 146.80 (C-1),

153.21 (C-24), 156.01 (C-21), 156.40 (COCF₃), 163.51

(C-19), 168.94 (C-10), 170.61 (C-12); Anal. calcd for

C₃₃H₅₀F₃N₅O₉(717.78): C, 55.22; H, 7.02; N, 9.75.

Found: C, 56.23; H, 7.31; N, 9.13.

1

1362 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.46 (s, 9H,

H-b), 3.04 (m, 2H, H-7), 3.83 (s, 3H, H-a), 4.66 (dt,

J=6.1, 6.1 and 7.7 Hz, 1H, H-8), 6.45 (m, 2H, H-3 +

H-5), 6.68 (d, J=8.6 Hz, 1H, H-6), 6.76 (d, J=7.7 Hz,

NH-9); ¹³C NMR (CDCl₃, 125 MHz) d 27.86 (C-b),

36.47 (C-7), 53.83 (C-8), 55.40 (C-a), 83.22 (C-a), 110.28

(C-6), 115.52 (CF₃), 115.66 (C-3), 119.05 (C-5), 127.20

(C-4), 136.19 (C-2), 146.57 (C-1), 156.40 (COCF₃),

168.98 (C-10); MS (FAB⁺) m/e 363 (M+1), 262, 307,

289.

N-(Tri¯uoroacetyl)-O-[N-terbutoxycarbonyl)-3-amino-

propyl]-ortho-[6-(N, N⁰-diterbutoxycarbonyl)guanidino-

caproyl]-amino-L-tyrosine terbutylester (10b). To

a

N-(Tri¯uoroacetyl)-O-[N-(terbutoxycarbonyl)-3-amino-

propyl]-ortho-amino-L-tyrosine terbutylester (4b).

solution of 4a (874 mg, 1.728 mmol) and pyridine

(411 mg, 5.192 mmol, 3 equiv) in CH₂Cl₂(10 mL), was

added the acid chloride 20b (742 mg, 1.893 mmol, 1.09

equiv) in CH₂Cl₂(10 mL). The mixture was stirred for

15 h at room temperature, under molecular sieves (4 A).

Washing with 1.5 N HCl (2Â20 mL), 10% NaHCO₃

(2Â20 mL), and brine (2Â20 mL), drying over MgSO₄

and concentration gave crude 10b which was ¯ash-

chromatographed (silica gel, hexane:isopropanol, 9:1) to

A

solution of 3b (162 mg, 0.303 mmol) in methanol

(10 mL) containing PtO₂(4.4 mg, 0.019 mmol, 0.06

equiv) was hydr_ꢀogenated (Parr apparatus, p=50 psi)

during 5 h at 20 C. After ®ltration and concentration,

the residue was dissolved in chloroform and dried over

MgSO₄. Evaporation under vacuum furnished 125 mg

of crude aniline 4b (yield: 82%): ¹H NMR (CDCl₃,

500 MHz) d 1.43 (s, 9H, H-g), 1.46 (s, 9H, H-b), 1.99 (m,

2H, H-b), 3.03 (m, 2H, H-7), 3.34 (m, 2H, H-c), 4.02 (t,

J=7.3 Hz, 2H, H-a), 4.66 (m, 1H, H-8), 4.73 (br s, NH-

d), 6.40 (m, 2H, H-3 + H-5), 6.70 (d, J=8.6 Hz, 1H, H-

6), 6.77 (br s, NH-9); ¹³C NMR (CDCl₃, 125 MHz) d

27.88 (C-b), 28.28 (C-g), 29.65 (C-b), 36.46 (C-7), 37.83

(C-c), 53.81 (C-8), 65.96 (C-a), 78.80 (C-f), 83.25 (C-a),

111.48 (C-6), 115.40 (CF₃), 115.80 (C-3), 119.05 (C-5),

127.50 (C-4), 136.40 (C-2), 145.64 (C-1), 155.88 (C-e),

156.40 (COCF₃), 168.97 (C-10).

1

furnish 750 mg of 10b (yield: 51%): R_f=0.3; H NMR

(CDCl₃, 500 MHz) d 1.41 (s, 9H, H-g), 1.44 (m, 2H, H-

15), 1.47 (s, 9H, H-b), 1.49 (s, 9H, H-23), 1.50 (s, 9H, H-

26), 1.62 (m, 2H, H-16), 1.76 (m, 2H, H-14), 1.97 (m,

2H, H-b), 2.46 (t, J=7.3 Hz, 2H, H-13), 3.05 (dd, J=6.0

and 14.2 Hz, 1H, H-7), 3.16 (dd, J=5.2 and 14.2 Hz,

1H, H-7⁰), 3.35 (m, 2H, H-c), 3.41 (m, 2H, H-17), 4.07

(t, J=7.0 Hz, 2H, H-a), 4.68 (m, 1H, H-8), 4.74 (m, 1H,

NH-d), 6.75 (dd, J=2.1 and 9.0 Hz, 1H, H-5), 6.79 (d,

J=9.0 Hz, 1H, H-6), 6.87 (d, J=7.8 Hz, 1H, NH-9),

8.17 (s, 1H, NH-11), 8.22 (d, J=2.1 Hz, 1H, H-3),

8.31(m, 1H, NH-18), 11.50 (s, 1H, NH-20); ¹³C NMR

(CDCl₃, 125 MHz) d 25.24 (C-14), 26.36 (C-15), 27.80

(C-b), 27.93 (C-26), 28.20 (C-23 and C-g), 28.75 (C-16),

29.58 (C-b), 36.68 (C-7), 36.86 (C-c), 37.37 (C-13), 40.65

(C-17), 53.87 (C-8), 65.23 (C-a), 79.05 (C-22), 79.28 (C-

f), 82.89 (C-25), 83.45 (C-a), 111.18 (C-6), 115.52 (CF₃),

121.20 (C-3), 123.99 (C-5), 127.58 (C-4), 128.31 (C-2),

146.10 (C-1), 153.18 (C-24), 155.98 (C-21 and C-e),

156.30 (COCF₃), 163.50 (C-19), 168.92 (C-10), 171.11

(C-12); Anal. calcd for C₄₀H₆₃F₃N₆O₁₁(860.96): C,

55.8; H, 7.37; N, 9.76. Found: C, 55.64; H, 7.06; N,

9.06.

N-(Tri¯uoroacetyl)-O-methyl-ortho-[6-(N,N⁰-diterbutoxy-

carbonyl)guanidino-caproyl]-amino-L-tyrosine terbutylester

(10a). To a solution of 4a (514 mg, 1.419 mmol) in

CH₂Cl₂(10 mL) were added, successively, the acid

chloride 20b (612 mg, 1.561 mmol, 1.1 equiv) and pyr-

idine (0.130 mL, 127 mg, 1.607 mmol, 1.13 equiv) in

CH₂Cl₂(10 mL). The mixture was stirred during 20 h at

room temperature. Washing with 1.5 N HCl (2Â20 mL),

10% NaHCO₃(2Â20 mL), and water (2Â20 mL), drying

over MgSO₄and concentration gave crude 10a which

was puri®ed by column-chromatography on silica gel

(hexane:ether, 1:1) t_ꢀo furnish 355 mg of 10a (yield:

35%): mp 53.7±54.6 C; IR (KBr) n 3338, 2980, 2935,

1724, 1641, 1619, 1535, 1483, 1420, 1369, 1334,

N-(Tri¯uoroacetyl)-O-methyl-ortho-[6-(benzyloxycarbonyl)-

amino-caproyl]-amino-L-tyrosine terbutylester (6a). N-

(Benzyloxycarbonyl)-6-aminocaproyl chloride was pre-

pared from N-(benzyloxycarbonyl)-6-aminocaproic acid

(644 mg, 2.43 mmol) in CH₂Cl₂(25 mL) treated with

thionyl chloride (0.9 mL, 1.47 g, 12.3 mmol, 5 equiv) for

2 h at re¯ux; concentration under high vacuum gave the

1

1228 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.44 (m, 2H,

H-15), 1.46 (s, 9H, H-b), 1.48 (s, 9H, H-23), 1.49 (s, 9H,

H-26), 1.62 (m, 2H, H-16), 1.74 (m, 2H, H-14), 2.38 (t,

J=7.3 Hz, 2H, H-13), 3.05 (dd, J=6.0 and 14.2 Hz, 1H,

H-7), 3.16 (dd, J=6.0 and 14.2 Hz, 1H, H-7⁰), 3.41 (m,

2H, H-17), 3.85 (s, 3H, H-a), 4.67 (m, 1H, H-8), 6.77 (m,

1588

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

crude acid chloride (625 mg, 91% yield). To a solution

of 4a (464 mg, 1.28 mmol) in CH₂Cl₂(4 mL) were added

(CDCl₃, 125 MHz) d 27.66 (C-b), 28.22 (C-14), 28.36 (C-

14⁰), 36.56 (C-7), 43.08 (C-15), 44.01 (C-13), 53.87 (C-8),

55.51 (C-a), 66.92 (C-18), 83.32 (C-a), 109.67 (C-6),

115.44 (CF₃), 120.50 (C-3), 124.16 (C-5), 127.33 (C-2),

127.48 (C-4), 127.64 (C-21), 127.77 (C-22), 128.25 (C-

20), 136.51 (C-19), 146.83 (C-1), 154.96 (C-17), 156.26

(COCF₃), 168.87 (C-10), 171.91 (C-12); HRMS

(FAB⁺): calcd for C₃₀H₃₆F₃N₃O₇: 607.2505. Found:

607.2528.

successively

N-(benzyloxycarbonyl)-6-aminocaproyl

chloride (400 mg, 1.41 mmol, 1.1 equiv) and pyridine

(303 mg, 3.83 mmol, 4 equiv) in _ꢀCH₂Cl₂(4 mL). The

mixture was stirred for 20 h at 20 C, then washed twice

with 1.5 N HCl, 10% NaHCO₃and water. Drying

(MgSO₄), concentration and ¯ash-chromatography

(CHCl₃) gave 196 mg of 6a (yield: 25%): R_f=0.2; IR

(CHCl₃) n 3315, 2933, 1718, 1654, 1596, 1560, 1535,

1

1483 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.39 (m, 2H,

N-(Tri¯uoroacetyl)-O-methyl-ortho-[N-(benzyloxycarbon-

yl)-nipecotyl]-amino-L-tyrosine terbutylester (8a). N-

(Benzyloxycarbonyl)-nipecotyl chloride (335 mg, 95%

yield) was prepared, as above, from N-(benzyloxy-

carbonyl)-nipecotic acid (330 mg, 1.25 mmol) and SOCl₂

(750 mg, 6.3 mmol, 5 equiv). To a solution of 4a

(384 mg, 1.06 mmol) and pyridine (0.1 mL, 98 mg,

1.236 mmol, 1.17 equiv) in CH₂Cl₂(10 mL), was added

N-(benzyloxycarbonyl)-nipecotyl chloride (335 mg,

1.19 mmol, 1.12 equiv) in CH₂Cl₂(10 mL). The mixture

H-15), 1.46 (s, 9H, H-b), 1.54 (m, 2H, H-16), 1.73 (m,

2H, H-14), 2.36 (t, J=7.3 Hz, 2H, H-13), 3.04 (dd,

J=6.1 and 14.0 Hz, 1H, H-7), 3.16 (dd, J=5.8 and

14.0 Hz, 1H, H-7⁰), 3.19 (m, 2H, H-17), 3.85 (s, 3H, H-

a), 4.68 (dt, J=6.1, 5.8 and 7.3 Hz, 1H, H-8), 4.84 (m,

1H, NH-18), 5.08 (s, 2H, H-21), 6.78 (m, 2H, H-5 + H-

6), 6.89 (d, J=7.3 Hz, 1H, NH-9), 7.27±7.37 (m, 5H,

Ph), 7.71 (s, 1H, NH-11), 8.25 (s, 1H, H-3); ¹³C NMR

(CDCl₃, 125 MHz) d 25.02 (C-14), 26.11 (C-15), 27.78

(c-b), 29.58 (C-16), 36.70 (C-7), 36.61 (C-13), 40.72 (C-

17), 53.87 (C-8), 55.58 (C-a), 66.42 (C-21), 83.43 (C-a),

109.74 (C-6), 115.42 (CF₃), 120.48 (C-3), 124.02 (C-5),

127.46 (C-4), 127.66 (C-2), 127.90 (C-24 + C-25),

128.35 (C-23), 136.51 (C-22), 146.71 (C-1), 156.26

(C-19), 156.4 (COCF₃), 168.95 (C-10), 170.61 (C-12);

MS (FAB⁺) m/e 510 (M+1-Boc), 446, 136, 95, 91, 83,

69.

ꢀ

was stirred for 17 h at 20 C (TLC control), then

worked-up as above. Flash-chromatography (silica gel,

CH₂Cl_ꢀ₂:EtOAc, 9:1) gave 318 mg of 8a (yield: 49%): mp

50±51 C; IR (®lm) n 3417, 2944, 2865, 1718, 1685,

1

1597, 1536, 1482, 1433, 1370, 1259, 1154 cm

;

¹H

NMR (CDCl₃, 500 MHz) d 1.46 (s, 9H, H-b), 1.52 (m,

1H, H-17), 1.76 (m, 1H, H-18), 1.80 (m, 1H, H-17⁰), 2.02

(m, 1H, H-18⁰), 2.48 (m, 1H, H-13), 2.90 (m, 1H, H-16),

3.04 (dd, J=6.1 and 14.0 Hz, 1H, H-7), 3.05 (m, 0.5 H,

H-14), 3.15 (m, 0.5 H, H-14), 3.16 (dd, J=5.2 and

14.0 Hz, 1H, H-7⁰), 3.84 (s, 3H, H-a), 4.10 (m, 1H, H-

16⁰), 4.21 (m, 0.5 H, H-14⁰), 4.31 (m, 0.5 H, H-14⁰), 4.67

(m, 1H, H-8), 5.14 (s, 2H, H-20), 6.78 (m, 2H, H-5 + H-

6), 6.81 (d, J=7.9 Hz, 1H, NH-9), 7.28±7.38 (m, 5H,

Ph), 7.81 (s, 1H, NH-11), 8.23 (m, 1H, H-3); ¹³C NMR

(CDCl₃, 125 MHz) d 24.18 (C-17), 27.65 (C-18), 27.82

(C-b), 36.78 (C-7), 44.14 (C-16), 44.52 (C-13), 46.36 (C-

14), 53.90 (C-8), 55.63 (C-a), 67.12 (C-20), 83.56 (C-a),

109.81 (C-6), 115.44 (CF₃), 120.68 (C-3), 124.36 (C-5),

127.40 (C-4), 127.46 (C-2), 127.80 (C-23), 127.94 (C-24),

128.41 (C-22), 136.59 (C-21), 146.97 (C-1), 155.19 (C-

19), 156.40 (COCF₃), 168.95 (C-10), 170.85 (C-12); MS

(FAB⁺) m/e 608.5; Anal. calcd for C₃₀H₃₆F₃N₃O₇

(607.62): C, 59.3; H, 5.97; N, 6.91. Found: C, 58.72; H,

5.98; N, 6.64.

N-(Tri¯uoroacetyl)-O-methyl-ortho-[N-(benzyloxycarbon-

yl)-isonipecotyl]-amino-L-tyrosine terbutylester (7a). N-

(Benzyloxycarbonyl)-isonipecotyl chloride was prepared

from N-(benzyloxycarbonyl)-isonipecotic acid (331 mg,

1.257 mmol) in CH₂Cl₂(25 mL) treated with SOCl₂

(0.49 mL, 800 mg, 6.72 mmol, 5.3 equiv) for 1 h 30 min

at re¯ux; concentration under high vacuum gave the

crude acid chloride (343 mg, 97%). To a solution of 4a

(404 mg, 1.115 mmol) in CH₂Cl₂(10 mL) were added

successively N-(benzyloxycarbonyl)-isonipecotyl chlor-

ide (343 mg, 1.218 mmol, 1.09 equiv) in CH₂Cl₂(10 mL),

and pyridine (0.1 mL, 98 mg, 1.236 mmol, 1.11 equiv).

ꢀ

The mixture was stirred for 16 h at 20 C (TLC control),

then washed with 1 N HCl (3Â10 mL), 10% NaHCO₃

(3Â10 mL), and brine (3Â10 mL). Drying (MgSO₄),

concentration and ¯ash-chromatography (silica gel,

hexane-isopropanol, 10:1) gave 380 mg of 7a (yield:

ꢀ

56%): R_f=0.1; mp 62.3±63.5 C; IR (KBr) n 3422,

3309, 3083, 2944, 1717, 1696, 1675, 1597, 1536,

N-(Tri¯uoroacetyl)-O-methyl-ortho-(6-guanidino-caproyl)-

amino-(L)-tyrosine (16a). The Boc-protected precursor

10a (76.5 mg, 0.106 mmol) was dissolved in tri¯uoro-

1

1369 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.46 (s, 9H,

H-b), 1.74 (m, 2H, H-14), 1.90 (m, 2H, H-14⁰), 2.43 (m,

1H, H-13), 2.88 (m, 2H, H-15), 3.03 (dd, J=6.1 and

14.0 Hz, 1H, H-7), 3.16 (dd, J=5.2 and 14.0 Hz, 1H, H-

7⁰), 3.85 (s, 3H, H-a), 4.23 (m, 2H, H-15⁰), 4.67 (m, 1H,

H-8), 5.13 (s, 2H, H-18), 6.78 (m, 2H, H-5 + H-6), 7.03

(d, J=7.6 Hz, 1H, NH-9), 7.27±7.37 (m, 5H, Ph), 7.81

(s, 1H, NH-11), 8.25 (d, J=2.1 Hz, 1H, H-3); ¹³C NMR

acetic acid (1.7 mL) and left for 2.5 h at 20 C. After

ꢀ

evaporation under vacuum, the residue was dissolved in

water and washed with chloroform. The aqueous phase

was freeze_ꢀ-dried to give 58 mg of 16a (yield: 86%): mp

95.5±96.5 C; IR (KBr) n 3416, 1664, 1542, 1206,

1138 cm ¹; ¹H NMR (D₂O, 500 MHz) d 1.44 (m, 2H, H-

15), 1.64 (m, 2H, H-16), 1.73 (m, 2H, H-14), 2.47 (t,

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1589

J=7.3 Hz, 2H, H-13), 2.98 (dd, J=9.5 and 14.0 Hz, 1H,

H-7), 3.20 (t, J=7.3 Hz, 2H, H-17), 3.30 (dd, J=5.2 and

14.0 Hz, 1H, H-7⁰), 3.85 (s, 3H, H-a), 4.68 (dd, J=9.5

and 5.2 Hz, 1H, H-8), 7.06 (d, J=8.6 Hz, 1H, H-6),

7.15 (dd, J=2.1 and 8.6 Hz, 1H, H-5), 7.44 (d,

J=2.1 Hz, 1H, H-3); ¹³C NMR (D₂O, 125 MHz) d 27.45

(C-14), 27.80 (C-15), 30.14 (C-16), 38.44 (C-13),

38.54 (C-7), 43.58 (C-17), 57.79 (C-8), 58.55 (C-a),

114,82 (C-6), 118.37 (CF₃), 127,71 (C-4), 128.04 (C-3),

130.41 (C-5), 131.58 (C-2), 153.40 (C-1), 159.33 (C-19),

160.85 (COCF₃), 176.83 (C-12), 178.51 (C-10); HRMS

(FAB⁺): calcd for C₂₁H₂₇F₆N₅O₇: 462.1964. Found:

462.1971.

28.28 (C-16), 37.23 (C-13), 37.32 (C-7), 40.57 (C-17),

55.93 (C-8), 56.33 (C-a), 111.77 (C-6), 117.34 (CF3),

124.41 (C-3), 126.89 (C-5), 127.99 (C-4), 130.15 (C-2),

150.63 (C-1), 158.71 (COCF₃), 173.98 (C-10), 174.37 (C-

12); HRMS (FAB⁺): calcd for C₁₈H₂₄F₃N₃O₅:

420.1746. Found: 420.1749.

N-(Tri¯uoroacetyl)-O-methyl-ortho-(isonipecotyl)-amino-

L-tyrosine (13a). A solution of 7a (130 mg, 0.214 mmol)

in EtOAc:EtOH (1:1, 10 mL) was placed in a Parr ¯ask

and hydrogenated (pH₂=40 psi) in the presence of Pal-

ladium (10% on C; 23 mg, 0.021 mmol), during 18 h

under vigorous shaking. After ®ltration and concentra-

tion under vacuum, the residue (93 mg) was dissolved in

ꢀ

N-(Tri¯uoroacetyl)-O-(3-aminopropyl)-ortho-(6-guanidino-

caproyl)-amino-L-tyrosine (16b). The Boc-protected

precursor 10b (750 mg, 0.871 mmol) was _ꢀdissolved in

CF₃CO₂H (13.5 mL) and left for 2 h at 20 C. Work up

CF₃CO₂H (3 mL) and left for 2.5 h at 20 C. Workup as

above gave 100 mg (Yield: 100%) of 13a: ¹H NMR

(D₂O, 500 MHz) d 1.94 (m, 2H, H-14), 2.16 (m, 2H, H-

14⁰), 2.85 (tt, J=3.4 and 11.9 Hz, 1H, H-13), 2.97 (dd,

J=9.5 and 14. Hz, 1H, H-7), 3.11 (m, 2H, H-15), 3.29

(dd, J=5.2 and 14 Hz, 1H, H-7⁰), 3.53 (m, 2H, H-15⁰),

3.83 (s, 3H, H-a), 4.75 (m, 1H, H-8), 7.03 (d, J=8.8 Hz,

1H, H-6), 7.13 (dd, J=2.1 and 8.8 Hz, 1H, H-5), 7.44 (d,

J=2.1 Hz, 1H, H-3); ¹³C NMR (D₂O, 125 MHz) ppm

27.55 (C-14), 27.63 (C-14⁰), 38.20 (C-7), 42.64 (C-13),

45.61 (C-15), 57.00 (C-8), 58.52 (C-a), 114.74 (C-6),

118.11 (CF₃), 127.53 (C-4), 127.76 (C-3), 130.39 (C-5),

131.17 (C-2), 153.30 (C-1), 160.96 (COCF₃), 175.84 (C-

12), 177.66 (C-10); HRMS: calcd for C₁₈H₂₂F₃N₃O₅:

418.1590. Found: 418.1603.

as above gave 616 mg of 16b (yield: 97%): [a]_d+2.5^ꢀ

20

(c 1, H₂O); ^1HNMR (D₂O, 500 MHz) d 1.44 (m, 2H, H-

15), 1.63 (m, 2H, H-16), 1.71 (m, 2H, H-14), 2.13 (m,

2H, H-b), 2.45 (t, J=7.3 Hz, 2H, H-13), 2.96 (m, 1H, H-

7), 3.16 (m, 2H, H-c), 3.20 (t, J=7.3 Hz, 2H, H-17), 3.28

(m, 1H, H-7⁰), 4.16 (t, J=7.3 Hz, 2H, H-a), 4.68 (m, 1H,

H-8), 7.04 (d, J=8.6 Hz, 1H, H-6), 7.16 (m, 1H, H-5),

7.27 (m, 1H, H-3); ¹³C NMR (D₂O, 125 MHz) d 27.45

(C-14), 27.77 (C-15), 28.92 (C-b), 30.06 (C-16), 38.28 (C-

7 + C-13), 39.65 (C-c), 43.45 (C-17), 57.62 (C-8), 68.46

(C-a), 115.89 (C-6), 118.37 (CF₃), 127.50 (C-4), 129.22

(C-3), 130.96 (C-5), 132.01 (C-2), 153.00 (C-1), 159.24

(C-19), 160.84 (COCF₃), 176.61 (C-12), 178.50 (C-10);

HRMS (FAB⁺): calcd for C₂₁H₃₂F₃N₆O₅: 505.2386.

Found: 505.2385.

N-(Tri¯uoroacetyl)-O-methyl-ortho-(nipecotyl))-amino-L-

tyrosine (14a). A solution of 8a (160 mg, 0.263 mmol) in

EtOAc:-EtOH (1:1, 10 mL) was placed in a Parr ¯ask

and hydrogenated (pH₂=40 psi) in the presence of Pal-

ladium (10% on C; 28 mg, 0.026 mmol), under vigorous

shaking, during 18 h. After ®ltration and concentration,

the residue (111 mg) was dissolved in CF₃CO₂H

N-(Tri¯uoroacetyl)-O-methyl-ortho-(6-amino-caproyl)-

amino-L-tyrosine (12a).

A solution of 6a (55 mg,

0.09 mmol) in EtOAc (5 mL)) was placed in a Parr ¯ask

and hydrogenated (pH₂=40 psi) in the presence of

Palladium (10% on C, 2 mg, 0.21 equiv). The mixture

was vigorously shaked during 72 h (fresh catalyst was

added three times). After ®ltration and concentration,

the residue was dissolved in CHCl₃, dried over MgSO₄

and concentrated under vacuum. The residue was dis-

ꢀ

(3.5 mL) and left for 2.5 h at 20 C. Work up as above

gave 120 mg (yield: 100%) of 14a: ¹H NMR (D₂O,

500 MHz) d 1.85 (m, 2H, H-17 + H-18), 1.99 (m, 1H,

H-17⁰), 2.15 (m, 1H, H-18⁰), 2.97 (m, 1H, H-7), 3.03 (m,

1H, H-13), 3.11 (m, 1H, H-16), 3.29 (m, 2H, H-16⁰+

H-14), 3.29 (m, 1H, H-7⁰), 3.45 (m, 1H, H-14⁰), 3.83 (s,

3H, H-a), 4.75 (m, 1H, H-8), 7.03 (d, J=8.6 Hz, 1H, H-

6), 7.13 (dd, J=2.1 and 8.6 Hz, 1H, H-5), 7.45 (d,

J=2.1 Hz, 1H, H-3); ¹³C NMR (D₂O, 125 MHz) d 23.07

(C-17), 28.25±28.30 (C-18), 38.07±38.13 (C-7), 41.76 (C-

13), 46.41 (C-16), 47.22 (C-14), 56.91 (C-8), 58.52 (C-a),

114.76 (C-6), 118.13 (CF₃), 127.32 (C-4), 127.78±127.86

(C-3), 130.50±130.56 (C-5), 131.14 (C-2), 153.30±153.38

(C-1), 160.96 (COCF₃), 175.78 (C-12), 176.11 (C-10);

HRMS: calcd for C₁₈H₂₂F₃N₃O₅: 418.1590. Found:

418.1587.

ꢀ

solved in CF₃CO₂H (2 mL) and left for 2.5 h at 20 C.

After evaporation, the residue was dissolved in water

and extracted with CHCl₃. The aqueous phase was

freeze-dried to give 40 mg of 12a (yield: 100%): IR

(CD₃OD) n 3419, 2947, 2361, 1683, 1541, 1490, 1207,

1

1143, 1029 cm ¹; H NMR (CD₃OD, 500 MHz) d 1.47

(m, 2H, H-15), 1.71 (m, 4H, H-14 + H-16), 2.46 (t,

J=7.3 Hz, 2H, H-13), 2.93 (t, J=7.3 Hz, 2H, H-17),

2.96 (dd, J=9.5 and 14.2 Hz, 1H, H-7), 3.23 (dd, J=5.2

and 14.2 Hz, 1H, H-7⁰), 3.84 (s, 3H, H-a), 4.61 (m, 1H,

H-8), 6.91 (d, J=8.6 Hz, 3H, H-6), 6.96 (dd, J=2.1 and

8.6 Hz, 1H, H-5), 7.84 (d, J=2.1 Hz, 1H, H-3); ¹³C

NMR (CD₃OD, 125 MHz) d 26.16 (C-14), 26.88 (C-15),

O-Methyl-ortho-nitro-L-tyrosine terbutylester (22). To a

solution of 3a (2.27 g, 5.785 mmol) in MeOH:H₂O (1:1,

1590

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

100 mL), was added K₂CO₃(4.207 g, 30.14 mmol). The

ꢀ

under vacuum gave 1.074 g of crude 23 (Yield: 91%):

ꢀ

mixture was stirred during 17 h at 20 C, then con-

mp 40±41 C; IR (CHCl₃) n 3275, 2980, 1734, 1617,

centrated under vacuum. Toluene was added and dis-

tilled. The residue was dissolved in CH₂Cl₂and washed

with water (3Â). The organic phase was dried (MgSO₄),

and concentrated under vacuum to give the crude amine

22 (1.504 g, 88% yield): R_f(SiO₂; CH₂Cl₂:EtOAc,

9:1)=0.1; IR (CHCl₃) n 3383, 2977, 2934, 1727, 1623,

1531, 1506, 1457, 1358, 1289, 1260, 1154, 1090,

1517, 1438, 1327, 1280, 1232, 1161, 1132, 1105 cm ¹; ¹H

NMR (CDCl₃, 300 MHz) d 1.24 (s, 9H, H-b), 2.90 (m,

2H, H-7), 3.80 (s, 3H, H-a), 4.06 (m, 1H, H-8), 5.44 (d,

1H, NH-9), 6.52 (d, 1H, H-5), 6.63 (d + s, 2H, H-6 +

H-3), 7.58 (t, 1H, H-y), 7.76 (d, 1H, H-x), 7.93 (d, 1H,

H-z), 8.04 (s, 1H, H-v); ¹³C NMR (CDCl₃, 75 MHz) d

27.59 (C-b), 38.58 (C-7), 55.44 (C-a), 57.17 (C-8), 82.69

(C-a), 110.37 (C-6), 117.32 (C-3), 120.82 (C-5), 122.9

(CF₃), 124.04 (C-v), 127.53 (C-4), 129.0 (C-x), 129.64

(C-y), 130.42 (C-z), 131.4 (C-w), 133.56 (C-2), 141.3 (C-

u), 147.13 (C-1), 169.61 (C-10); MS (FAB⁺) m/e 475

(M+1), 474, 419, 401, 373.

1

1019 cm ¹; H NMR (CDCl₃, 500 MHz) d 1.42 (s, 9H,

H-b), 2.83 (dd, J=7.3 and 14.2 Hz, 1H, H-7), 2.98 (dd,

J=5.5 and 14.2 Hz, 1H, H-7⁰), 3.56 (m, 1H, H-8), 3.93

(s, 3H, H-a), 7.01 (d, J=8.6 Hz, 1H, H-6), 7.41 (dd,

J=2.1 and 8.6 Hz, 1H, H-5), 7.72 (d, J=2.1 Hz, 1H, H-3);

¹³C NMR (CDCl₃, 125 MHz) d 27.88 (C-b), 39.48 (C-7),

55.91 (C-8), 56.44 (C-a), 81.49 (C-a), 113.38 (C-6), 126.21

(C-3), 130.07 (C-4), 135.12 (C-5), 139.25 (C-2), 151.69

(C-1), 173.82 (C-10); MS (FAB⁺) m/e 297, 241, 57.

N-(meta-Tri¯uoromethyl-benzenesulfonyl)-O-methyl-ortho-

[6-N,N⁰-(diterbutoxycarbonyl) guanidino-caproyl]-amino-

L-tyrosine terbutylester (26). To

a solution of 24

(229 mg, 0.483 mmol) in CH₂Cl₂(5 mL) were added the

acid chloride 20b (207 mg, 0.528 mmol, 1.09 equiv) and

pyridine (0.117 mL, 114 mg, 1.447 mmol, 3 equiv) in

N-(meta-Tri¯uoromethyl-benzenesulfonyl)-O-methyl-ortho-

nitro-L-tyrosine terbutyl ester (23). To a solution of 22

(1.454 g, 4.91 mmol) in CH₂Cl₂(63 mL), were added

successively Et₃N (0.75 mL, 0.546 mg, 5.397 mmol, 1.1

equiv) and meta-tri¯uoromethyl-benzenesulfonyl chlor-

ide (0.865 mL, 1.32 g, 5.397 mmol, 1.1 equiv). The mix-

ꢀ

CH₂Cl₂(5 mL). The mixture was stirred at 20 C for

20 h, then washed twice with 1.5 N HCl, and water.

Drying (MgSO₄), concentration and ¯ash chromato-

graphy (silica gel, CH₂Cl₂:EtOAc, 9:1) gave 92 mg

ꢀ

ture was stirred at 20 C during 20 h, then washed with

(Yield: 24%) of 26: IR (CDCl₃) n 3332, 2978, 2930,

1

1 N HCl (2Â10 mL) and water (2Â10 mL). After drying

(MgSO₄), concentration and ¯ash-chromatography

(silica gel, CHCl₃), the sulfonamide 23 was recovered

(1.26 g, 51% yield): IR (CDCl₃) n 3650, 3273, 3076,

2982, 2937, 2846, 1733, 1624, 1575, 1534, 1160 cm ¹; ¹H

NMR (CDCl₃, 500 MHz) d 1.22 (s, 9H, H-b), 2.93 (dd,

J=7.3 and 14.2 Hz, 1H, H-7), 3.04 (dd, J=5.9 and

14.2 Hz, 1H, H-7), 3.87 (s, 3H, H-a), 4.05 (m, 1H, H-8),

5.75 (br s, 1H, NH-9), 6.95 (d, J=8.6 Hz, 1H, H-6), 7.36

(dd, J=2.1 and 8.6 Hz, 1H, H-5), 7.58 (d, J=2.1 Hz,

1H, H-3), 7.58 (t, J=8.0 Hz, 1H, H-y), 7.75 (d,

J=8.0 Hz, 1H, H-x), 7.93 (d, J=8.0 Hz, 1H, H-z), 8.00

(s, 1H, H-v); ¹³C NMR (CDCl₃, 125 MHz) d 27.38 (C-

b), 37.68 (C-7), 56.24 (C-a), 56.85 (C-8), 83.43 (C-a),

113.46 (C-6), 122.91 (CF₃), 123.80 (C-v), 126.24 (C-3),

127.48 (C-4), 129.15 (C-x), 129.75 (C-y), 130.22 (C-z),

131.34 (C-w), 135.41 (C-5), 138.85 (C-u), 140.84 (C-2),

151.94 (C-1), 169.14 (C-10); MS (FAB^-) m/e 503 (M±1),

209; Anal. calcd for C₂₁H₂₃F₃N₂O₇S (504.47): C, 49.99;

H, 4.59; N, 5.55; S, 6.35. Found: C, 49.79; H, 4.50; N,

5.28; S, 6.09.

1723, 1641, 1617, 1537, 1481, 1161, 1134 cm

;

¹H

NMR (CDCl₃, 500 MHz) d 1.27 (s, 9H, H-b), 1.44 (m,

2H, H-15), 1.48 (s, 9H, H-23), 1.49 (s, 9H, H-26), 1.62

(m, 2H, H-16), 1.74 (m, 2H, H-14), 2.37 (m, 2H, H-13),

2.86 (dd, J=7.3 and 14.2 Hz, 1H, H-7), 3.00 (dd, J=5.2

and 14.2 Hz, 1H, H-7⁰), 3.41 (m, 2H, H-17), 3.84 (s, 3H,

H-a), 4.06 (m, 1H, H-8), 5.23 (d, J=9.4 Hz, 1H, NH-9),

6.72 (d, J=8.6 Hz, 1H, H-6), 6.82 (dd, J=2.1 and

8.6 Hz, 1H, H-5), 7.55 (dd, J=8.0 Hz, 1H, H-y), 7.67 (s,

1H, NH-11), 7.73 (d, J=8.0 Hz, 1H, H-x), 7.94 (d,

J=8.0 Hz, 1H, H-z), 7.99 (s, 1H, H-v), 8.14 (d,

J=2.1 Hz, 1H, H-3), 8.31 (m, 1H, NH-18), 11.50 (s, 1H,

NH-20); ¹³C NMR (CDCl₃, 125 MHz) d 25.09 (C-14),

26.35 (C-15), 27.58 (C-b), 27.96 (C-26), 28.19 (C-23),

28.69 (C-16), 37.61 (C-13), 38.72 (C-7), 40.65 (C-17),

55.54 (C-a), 57.14 (C-8), 79.14 (C-22), 82.96 (C-25 +

C-a), 109.60 (C-6), 120.50 (C-3), 122.91 (CF₃), 124.12

(C-v), 124.39 (C-5), 127.48 (C-4), 127.56 (C-2), 128.93

(C-x), 129.53 (C-y), 130.51 (C-z), 131.37 (C-w), 141.20

(C-u), 146.65 (C-1), 153.24 (C-24), 156.00 (C-21), 163.47

(C-19), 169.67 (C-10), 170.58 (C-12); MS (FAB^-) m/e

829 (M-1) 828, 711, 209; Anal. calcd for

C₃₈H₅₄F₃N₅O₁₀S (829.92): C, 54.99; H, 6.55 ; N, 8.43.

Found: C, 55.10; H, 6.20; N, 8.16.

N-(meta-Tri¯uoromethyl-benzenesulfonyl)-O-methyl-ortho-

amino-L-tyrosine terbutylester (24). A solution of 23

(1.259 g, 2.495 mmol) in methanol (20 mL) was placed in

a Parr ¯ask and hydrogenatad (pH₂=40 psi) in the pre-

sence of platinium IV oxide (56.7 mg, 0.249 mmol, 0.1

N-(meta-Tri¯uoromethyl-benzene sulfonyl)-O-methyl-ortho-

[6-(benzyloxycarbonyl) amino-caproyl]-amino-L-tyrosine

terbutylester (25). To a solution of 24 (669 mg,

1.409 mmol) in CH₂Cl₂(5 mL), were added N-(benzyl-

oxycarbonyl) aminocaproyl chloride (400 mg, 1.409 mmol,

ꢀ

equiv), under vigorous shaking, during 17 h at 20 C.

After ®ltration and concentration, the residue was dis-

solved in CH₂Cl₂and dried over MgSO4. Concentration

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1591

1 equiv) and pyridine (0.33 mL, 323 mg, 4.08 mmol, 2.89

equiv) in C_ꢀH₂Cl₂(5 mL). The mixture was stirred for

20 h at 20 C, then worked-up as before to furnish

490 mg (yield: 48%) of 25: IR (®lm) n 3370, 2937, 1715,

1698, 1597, 1536, 1162 cm ¹; R_f(SiO₂; CH₂Cl₂:EtOAc,

9:1)=0.1; ¹H NMR (CDCl₃, 500 MHz) d 1.25 (s, 9H, H-

b), 1.37 (m, 2H, H-15), 1.52 (m, 2H, H-16), 1.70 (m, 2H,

H-14), 2.34 (t, J=7.3 Hz, 2H, H-13), 2.82 (dd, J=7.3

and 14.2 Hz, 1H, H-7), 2.97 (dd, J=5.6 and 14.2 Hz,

1H, H-7⁰), 3.18 (m, 2H, H-17), 3.80 (s, 3H, H-a), 4.05

(m, 1H, H-8), 4.96 (m, 1H, NH-18), 5.06 (m, 2H, H-21),

5.61 (d, J=9.5 Hz, 1H, NH-9), 6.68 (d, J=8.6 Hz, 1H,

H-6), 6.80 (m, 1H, H-5), 7.27±7.37 (m, 5H, H-23+H-

24+H-25), 7.51 (dd, J=8.0 Hz, 1H, H-y), 7.67 (s, 1H,

NH-11), 7.70 (d, J=8.0 Hz, 1H, H-x), 7.90 (d,

J=8.0 Hz, 1H, H-z), 7.98 (s, 1H, H-v), 8.14 (d,

J=2.1 Hz, 1H, H-3); ¹³C NMR (CDCl₃, 125 MHz) d

24.88 (C-14), 26.03 (C-15), 27.45 (C-b), 29.48 (C-16),

37.40 (C-13), 38.53 (C-7), 40.65 (C-17), 55.41 (C-a),

57.21 (C-8), 66.29 (C-21), 82.66 (C-a), 109.53 (C-6),

120.42 (C-3), 123.00 (CF₃), 123.89 (C-v), 124.30 (C-5),

127.30 (C-4), 127.58 (C-2), 127.80 (C-24+C-25), 128.25

(C-23), 128.71 (C-x), 129.41 (C-y), 130.35 (C-z), 131.11

(C-w), 136.51 (C-22), 141.23 (C-u), 146.55 (C-1), 156.25

(C-19), 169.68 (C-10), 170.61 (C-12); HRMS: calcd for

C₃₅H₄₂F₃N₃O₈S: 721.2645. Found: 721.2647.

(pH₂=40 psi) in the presence of Palladium catalyst

(10% _ꢀon C, 4.5 mg, 0.042 mmol, 0.27 equiv) during 18 h

at 20 C, under vigorous shaking. Filtration and con-

centration under vacuum gave a residue which was dis-

solved in tri¯uoroacetic acid (6 mL) and left for 2.5 h at

ꢀ

20 C. Work up as before furnished 88 mg (yield: 90%)

7.5^ꢀ(c 0.04;

20

of 27 (hygroscopic solid): [a]_d

H₂O:CH₃CN, 20:80); IR (®lm) n 3423, 2948, 2534, 1674,

1

1655, 1539, 1204, 1136 cm

;

¹H NMR (CD₃OD,

500 MHz) d 1.47 (m, 2H, H-15), 1.72 (m, 4H, H-14+H-

16), 2.46 (t, J=7.3 Hz, 2H, H-13), 2.72 (dd, J=9.5 and

14.0 Hz, 1H, H-7), 2.94 (m, 2H, H-17), 3.00 (dd, J=5.2

and 14.0 Hz, 1H, H-7⁰), 3.81 (s, 3H, H-a), 4.05 (dd,

J=5.2 and 9.5 Hz, 1H, H-8), 6.74 (d, J=8.6 Hz, 1H, H-

6), 6.83 (dd, J=2.1 and 8.6 Hz, 1H, H-5), 7.55 (dd,

J=8.0 Hz, 1H, H-y), 7.74 (d, J=2.1 Hz, 1H, H-3), 7.78

(d, J=8.0 Hz, 1H, H-x), 7.81 (d, J=8.0 Hz, 1H, H-z),

7.91 (s, 1H, H-v); ¹³C NMR (CD₃OD, 125 MHz) d

26.16 (C-14), 26.93 (C-15), 28.31 (C-16), 37.32 (C-13),

39.22 (C-7), 40.57 (C-17), 56.19 (C-a), 59.27 (C-8),

111.55 (C-6), 124.16 (C-3), 124.64 (C-v), 124.90 (CF₃),

126.85 (C-5), 127.89 (C-4), 129.62 (C-x), 129.79 (C-2),

131.02 (C-y), 131.46 (C-z), 131.96 (C-w), 143.82 (C-u),

150.18 (C-1), 174.14 (C-10), 174.34 (C-12); HRMS;

calcd for C₂₃H₂₈F₃N₃O₆S: 532.1729. Found: 532.1743.

Benzyl 6-amino-caproate para-toluene sulfonate (18b). A

mixture of 6-aminocaproic acid (5 g, 38.11 mmol), ben-

zyl alcohol (250 mL) and para-toluene sulfonic acid

(8.38 g, 42.3 mmol, 1.1 equiv) in benzene (500 mL) was

N-(meta-Tri¯uoromethyl-benzenesulfonyl)-O-methyl-ortho-

(6-guanidino-caproyl)-amino-L-tyrosine (28). A solution

of 26 (75 mg, 0.131 mmol)_ꢀin tri¯uoroacetic acid (2 mL)

was left for 2 h at 20 C, then evaporated under

vacuum. The residue was dissolved in water and extrac-

ted with CHCl₃. Freeze-drying of the aqueous phase

gave 120 mg (yield ꢁ 100%) of 28 (hydroscopic white

ꢀ

heated at 100 C for 3 h (azeotropic distillation with a

Dean±Stark equipment). The solution was cooled to

ꢀ

20 C under argon atmosphere. After addition of ether

(500 mL), the product was allowed to crystallize at

1

ꢀ

powder): H NMR (CD₃OD, 500 MHz) d 1.46 (m, 2H,

30 C, during 3 days. Filtration, washing with ether

and drying under vacuum gave 14.38 g (yield: 95%) of

H-15), 1.64 (m, 2H, H-16), 1.74 (m, 2H, H-14), 2.45 (t,

J=7.3 Hz, 2H, H-13), 2.73 (dd, J=9.5 and 14.0 Hz, 1H,

H-7), 3.00 (dd, J=4.6 and 14.0 Hz, 1H, H-7⁰), 3.18 (t,

J=7.3 Hz, 2H, H-17), 3.81 (s, 3H, H-a), 4.04 (m, 1H, H-

8), 6.74 (d, J=8.6 Hz, 1H, H-6), 6.83 (m, 1H, H-5), 7.55

(dd, J=8.0 Hz, 1H, H-y), 7.73 (d, J=2.1 Hz, 1H, H-3),

7.79 (d, J=8.0 Hz, 1H, H-z), 7.81 (d, J=8.0 Hz, 1H, H-

x), 7.91 (s, 1H, H-v); ¹³C NMR (CD₃OD, 125 MHz) d

26.27 (C-14), 27.21 (C-15), 29.60 (C-16), 37.49

(C-13), 39.25 (C-7), 42.33 (C-17), 56.18 (C-a), 59.34 (C-

8), 111.53 (C-6), 124.22 (C-3), 124.62 (C-v), 124.90

(CF₃), 126.91 (C-5), 127.85 (C-4), 129.69 (C-x), 129.80

(C-2), 131.06 (C-y), 131.46 (C-z), 132.02 (C-w), 143.83

(C-u), 150.22 (C-1), 158.68 (C-19), 174.33 (C-12), 174.58

(C-10); HRMS; calcd for C₂₆H₃₁F₆N₅O₈S: 574.1947.

Found: 574.1950.

ꢀ

salt 18b: mp 106±107 C; IR (KBr) n 3465, 2943, 2869,

1

3041, 1728, 1626, 1482, 1196, 1142 cm

;

¹H NMR

(CDCl₃, 500 MHz) d 1.19 (m, 2H), 1.48 (m, 4H), 2.20 (t,

J=7.3 Hz, 2H), 2.31 (s, 3H), 2.74 (m, 2H), 5.04 (s, 2H),

7.16 (d, J=8.4 Hz, 2H), 7.27±7.37 (m, 5H), 7.68 (br s,

3H), 7.72 (d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃,

125 MHz) d 21.17, 24.02, 25.65, 26.92, 33.71, 39.55,

66.02, 125.74, 128.04, 128.09, 128.44, 128.97, 135.91,

140.76, 141.02, 173.01; MS (FAB⁺) m/e 222.

N,N⁰-Diterbutoxycarbonyl-3,5-dimethylpyrazole-1-carbox-

amidine (17). To a solution of 3,5-dimethylpyrazole-1-

carboxamidine nitrate (2 g, 9.74 mmol) and di-t-butyl

dicarbonate (11.53 mL, 10.96 g, 48.71 mmol, 5 equiv) in

dry THF (80 mL), was added, under argon atmosphere,

sodium hydride (90% purity, 1.23 g, 48.71 mmol_ꢀ, 5

equiv). The mixture was stirred under re¯ux (80 C)

during 6 h. Ethanol was cautiously added dropwise, and

the solution was concentrated under vacuum. The resi-

due was dissolved in CH₂Cl₂and washed with water.

N-(meta-Tri¯uoromethyl-benzenesulfonyl)-O-methyl-ortho-

(6-amino-caproyl)-amino-l-tyrosine (27). A solution of 25

(112 mg, 0.154 mmol) in EtOH:EtOAc (50:50, 10 mL)

was placed in

a Parr ¯ask and hydrogenated

1592

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

Drying over MgSO₄, concentration and ¯ash chroma-

ꢀ

Biological evaluation (in solution)

tography gave 2.43 g (yield: 73%) of 17: mp 100±101 C;

IR (KBr) n 3322, 3111, 2937, 1761, 1694, 1656 cm ¹; ¹H

NMR (CDCl₃, 500 MHz) d 1.50 (s, 9H), 1.52 (s, 9H), 2.21

(s, 3H), 2.55 (s, 3H), 5.92 (s, 1H), 9.05 (br s, 1H); ¹³C

NMR (CDCl₃, 125 MHz) d 13.40, 15.09, 27.94, 80.49,

82.53, 111.24, 140.37, 144.00, 149.52, 150.32, 157.46; MS

(EI) m/e 338 (M), 282, 254, 238, 182, 165, 138, 96, 57, 43;

Anal. calcd for C₁₆H₂₆N₄O₄(338.40): C, 56.78; H, 7.74;

N, 16.55. Found: C, 56.66; H, 7.76; N, 16.47.

The purity of the pepdidomimetics was controlled by

HPLC before use, with the following conditions: column:

Nucleosil C18, 5 m, 25 cm; temperature: 25 C; eluent:

ꢀ

gradient from 20% CH₃CN+0.015% TFA 80%

H₂O+0.015% TFA to 80% CH₃CN+0.015% TFA

20% H₂O+0.015%; ¯ow rate: 1 mL/min; equipment:

Beckman, System Gold, 126 P Solvent module, 168

Detector (Analis, Belgium). The retention times (area)

were: 16b: t=5.43 min (98.8%); 16a: t=7.53 min

(96.9%); 12a: t=6.88 min (100%); 13a: t=11.53 min

(94.1%); 27: t=8.67 min (98.5%).

Benzyl 6-(N,N⁰-diterbutoxycarbonyl)-guanidino-caproate

(19b). To a solution of 17 (2.5 g, 7.38 mmol) in CH₂Cl₂

(300 mL), were added 18b (3.19 g, 8.12 mmol, 1.1 equiv)

and triethylamine (1.2 mL, 873 mg, 8.63 mmol, 1.16

The phosphate buered saline (PBS) solution (pH 7.3)

was prepared from NaCl (4 g), KCl (0.1 g), KH₂PO₄

(0.1 g) and Na₂HPO₄, 2H₂O (0.71 g) dissolved in water

(HPLC grade, 500 mL). The stock solutions of peptido-

mimetics contained 1 to 5 mg of product per milliliter of

PBS buer; if needed, 0.5% DMSO could be added for

complete dissolution. The tested concentrations were

ꢀ

equiv) The mixture was stirred for 20 h at 20 C, then

washed successively with 1 N HCl, water, 10% NaHCO₃

and water (3Â). Drying (MgSO₄), concentration and

chromatography on preparative MPLC gave 3.40 g

(yield: 98%) of 19b: R_f(SiO₂; hexane:isopropanol,

2:1)=0.2; IR (KBr) n 3328, 2980, 1740, 1728, 1651,

1

6

1627, 1417, 1370, 1362, 1156 cm

;

¹H NMR (CDCl₃,

within 10 to 10 ¹M.

500 MHz) d 1.38 (m, 2H), 1.49 (s, 9H), 1.50 (s, 9H), 1.57

(m, 2H), 1.68 (m, 2H), 2.36 (t, J=7.3 Hz, 2H), 3.39 (m, 2H),

5.11 (s, 2H), 7.20±7.40 (m, 5H), 8.30 (m, 1H), 11.50 (br

s, 1H); ¹³C NMR (CDCl₃, 125 MHz) d 24.51, 26.26, 27.98,

28.20, 28.62, 34.01, 40.58, 66.05, 79.11, 82.93, 128.10,

128.44, 135.95, 153.24, 156.02, 163.54, 173.21; MS (EI)

m/e 463, 351, 290, 202, 188, 161, 158, 145, 144, 130, 117,

108, 65; Anal. calcd for C₂₄H₃₇N₃O₆(463.57): C, 62.18;

H, 8.04; N, 9.06. Found: C, 62.44; H, 8.21; N, 8.96.

Blood was drawn from the antecubital vein of healthy

adult volunteers, who denied taking any medication for

the previous 15 days, into a plastic syringe containing

one part of 3.8% trisodium citrate to nine parts of

blood. Platelet rich plasma (PRP) was prepared by cen-

trifuging the blood at 1500 g for 10 min at room tem-

perature. The PRP was drawn o and the remaining

blood was centrifuged at 6000 g for 20 min at room

temperature to make platelet poor plasma (PPP). The PRP

was adjusted with PPP to a count of 3Â10⁵platelets/mL;

platelet count was measured with a Coulter counter.

400 mL of the PRP preparation and 50 mL of the solution

of peptidomimetic to be_ꢀtested, or saline, were pre-

incubated for 2 min at 37 C in an aggregometer; 50 mL

of 0.047 mM ADP were added, and the aggregation was

monitored during 4 min. Results were calculated as fol-

lows: [observed % aggregation (antagonist)] divided by

[maximum % aggregation (control)] equals the % of

control. The % inhibition=100 % of control. Con-

centration±response curves were constructed and the

IC₅₀were determined as the concentration of antagonist

required to produce 50% inhibition of the response to

the agonist. At least two determinations were made for

each compound and the IC₅₀calculated by ®tting to a

four parameter equation (average standard error of

30%).

6-(N,N⁰-Diterbutoxycarbonyl)-guanidino-caproyl chloride

(20b). A solution of 19b (2.9 g, 6.25 mmol) in EtOH:

EtOAc (1:1; 50 mL), placed in a Parr ¯ask, was hydro-

genated (pH₂=40 psi) in the presence of Palladium

catalyst (10% on C; 102 mg, 0.957 mmol, 0.153 equiv),

ꢀ

during 18 h at 20 C (vigorous shaking). Filtration,

concentration and drying under vacuum gave 2.21 g

(yield: 94%) of 6-(N,N⁰-diterbutoxycarbonyl)-guani-

ꢀ

1

dino-caproic acid: mp 83.1±84.1 C; H NMR (CDCl₃,

500 MHz) d 1.41 (m, 2H), 1.49 (s, 9H), 1.50 (s, 9H), 1.60

(m, 2H), 1.67 (m, 2H), 2.36 (t, J=7.3 Hz, 2H), 3.43 (m,

2H), 8.40 (s, 1H), 11.50 (br s, 1H); ¹³C NMR (CDCl₃,

125 MHz) d 24.15, 26.11, 27.92, 28.12, 28.53, 33.64,

40.72, 79.51, 83.15, 153.12, 155.88, 162.88, 178.57; MS

(EI) m/e 373 (M), 317, 261, 244, 188, 161, 130, 57; Anal.

calcd for C₁₇H₃₁N₃O₆(373.44): C, 54.67; H, 8.36; N,

11.25. Found: C, 54.75; H, 8.11; N, 10.81. A solution of

this acid (0.6 g, 1.6 mmol) and thionyl chloride (0.6 mL,

978 mg, 8.22_ꢀmmol, 5.12 equiv) in CH₂Cl₂(20 mL) was

re¯uxed (50 C) during 1.5 h, then concentrated under

vacuum. The excess of SOCl₂was removed by azeo-

tropic distillation with toluene (3Â) and CHCl₃(3Â).

The acid chloride 20b was dried under high vacuum

(617 mg, 98% yield) and used without puri®cation.

Modelisation

All the degrees of freedom describing the molecular

geometry have been fully optimized at the approximate

quantum chemistry level AM1,⁶¹using the minimization

procedures available in Gaussian suite of programs.⁶²In

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1593

the structures superpositions, the starting point was the

®tting of the respective carboxyl functions.

Activation of PET-CO₂H. The PET-CO₂H disks (1

sample per 20 mL of reactive solution) were immersed

into a solution of 1-(3-dimethylaminopropyl)-3-ethyl-

carbodiimide hydrochloride (water soluble carbodi-

imide=WSC; 0.1 g) in 0.1 N MES buer (100 mL), for

Surface chemistry

ꢀ

The PET microporous membrane was manufactured by

Whatman SA (Louvain-la Neuve, Belgium) by track-

etching treatment of PET Mylar A ®lm (Dupont de

Nemours, Brussels) characterized by a thickness of

12 mm, a density of 1.39 g/cm³(ASTM D 1505-66), a

1 h at 20 C, under shaking. The samples were taken o

the solution and rinsed successively with 0.1 N MES

buer (1Â10 min; 20 mL per sample) and water

(2Â10 min; 20 mL per sample). The activated PET-

CO₂H disks were directly used for the radiolabeling and

the coupling to the peptidomimetic.

ꢀ

melting point of 251 C (ASTM D 3418-82), a M_nof

48,800 and a MW of 88,800. The membrane contained

1.45Â10⁶pores/cm²(apparent surface) of 0.49 mm in

diameter. For the surface modi®cations, disks of 13 mm

in diameter were cut o the membrane; the open surface

of a disk sample is 3.01 cm²(apparent surface and

internal surface of the pores).

Radiolabeling of PET-CO₂H. The activated PET- CO₂H

disks were individually treated in small pyrex tubes

containing 1.5 mL of the _ꢀradioactive lysine* solution

(10 ³M), during 2 h at 20 C, under shaking. The disks

were individually washed with 1.5 mL of phosphate

buer (pH 8, 1Â10 min and 2Â5 min), 1.5 mL of water

(1Â5 min), 1.5 mL of 0.005 M HCl (3Â10 min) and

1.5 mL of water (1Â5 min and 2Â5 min). The samples

were drained over ®lter paper and directly used for the

radioactivity measurement; they were individually

placed in 20 mL polyethylene vials containing 5 mL of

aqualuma cocktail (Lumac, Basel, Switzerland). The

blank samples (references for the counting of the non-

speci®c ®xation or adsorption of the radioactive label)

were prepared according to the previous procedures,

but, in the activation step, the carbodiimide was omitted.

The phosphate buer (pH 8.2) was prepared from

Na₂HPO₄.2H₂O (4.215 g) and NaH₂PO₄.H₂O (0.2065 g)

dissolved in water (250 mL, HPLC grade). The MES

buer (pH 3.5) was obtained from 2-(N-morpholino)-

ethanesulfonic acid (MES, 5.331 g) dissolved in water

(250 mL, HPLC grade). l-[4,5-³H] lysine mono-

hydrochloride in aqueaus solution was purchased from

Amersham (Little Chalfont, UK); the labeling solution

(10 ³M) was prepared as follows: to 250 mL of un-

labeled lysine solution (0.1826 g/10 mL phosphate buer)

were added 187.5 mL of labeled lysine (as=98 Ci/mmol);

this solution was diluted to 25 mL with phosphate buf-

fer. Water (HPLC grade) was obtained with a Milli-Q

system (Millipore, Bedford, MA, USA).

LSC counting results (average of ®ve dierent samples

standard deviation): sample: 57.53.8 pmol/cm²of

open surface; blank: 22.92.9 pmol/cm²of open surface;

corrected value: 34.63.3 pmol/cm²of open surface. Since

a PET interface domain of 50 A in depth contains about

2860 pmol of monomer units/cm², the value of 35 pmol/

cm²corresponds to 1.22% of surface derivatization.⁷

Radioactivity measurement: the amount of labeled lysine

®xed on the PET disks was measured by liquid scintil-

lation counting (LSC) of the sample-associated radio-

activity according to references 7±9, using a Tri-Carb

1600 TR liquid scintillation analyser (PACKARD).

Coupling of peptidomimetic to PET-CO₂H. The acti-

vated PET-CO₂H disks were individually treated in

small pyrex tubes containing 1.5 mL of peptidomimetic

solution (30.9 mg of 16b in 50 mL of phosphate buer),

XPS analysis: the surface chemical composition of the

modi®ed PET disks was determined by X-ray photo-

electron spectroscopy according to references 10 and

11 and 45-46, using a SSI-X probe (SSX-100/206)

spectrometer from Fisons (Surface Science Laboratories,

Mountain View, CA).

ꢀ

during 2 h at 20 C, under shaking. The disks were

individually washed as described for the radiolabelling,

then air-dried and storred in the dark.

The blank samples (references for the XPS analysis of

the peptidomimetic adsorption) were prepared accord-

ing to the previous procedures, but, in the activation

step, the carbodiimide was omitted.

PET membrane oxidation. The PET disks (1 sample per

10 mL of reactive solution) were immersed into an acidic

solution of KMnO₄(6 g, in 120 mL of 1.2 N H₂SO₄),

ꢀ

and heated at 60 C during 1 h, under shaking with an

Edmund B hler stirrer (model KL-2). The PET disks

were taken o the solution with tweezers and washed

successively with 6 N HCl (1Â20 min and 2Â5 min;

10 mL per sample) and water (2Â10 min; 10 mL per

sample). The disks were drained over ®lter paper and

air-dried. Oxidized PET is called PET- CO₂H.

XPS analysis results: sample: C_1s, 70.86%; O_1s27.58%;

N_1s, 1.32%; F_1s, 0.24%; blank; C_1s, 70.69%; O_1s, 28.96%;

N_1s, 0.35%; F_1s, 0.00%. F/C Â 100 atomic ratio: sam-

ple: 0.339; blank: 0.000 (no correction needed). Calcu-

lation of the percentage of surface derivatization: we

considered a theoretical monomer unit consisting of

1594

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

[(PET)_x+

(PET RGD

peptidomimetic)_y],

i.e.

15. Cheresh, D. A.; Mecham, R. P.; Integrins, Molecular and

Biological Responses to Extracellular Matrix; Academic Press:

London, 1994.

[(C₁₀H₈O₄)_x+ (C₃₁H₃₆O₉N₆F₃)_y], where x+y=1. Thus,

the F/C atomic ratio is 3x/[31x+10(1-x)]. For F/

C=0.00339 (experimental value), x=0.01079, i.e. 1.08%

of modi®ed units.

16. Hynes, R. O. Cell 1992, 69, 11.

17. Clark, E. A.; Brugge, J. S. Science 1995, 268, 233.

18. Ruoslahti, E. Ann. Rev. Cell. Dev. Biol. 1996, 12, 697.

Acknowledgements

19. Kieer, N.; Phillips, D. R. Ann. Rev. Cell. Dev. Biol. 1990,

6, 329.

This work was generously supported by the Fonds

National de la Recherche Scienti®que (FNRS, Belgium),

the Fonds de Developpement Scienti®que (Universite

catholique de Louvain), and the Fonds pour la for-

mation a la Recherche dans l'Industrie et l'Agriculture

(FRIA, Belgium).

20. Luscinskas, F. W.; Lawler, J. FASEB J. 1994, 8, 929.

21. D'Souza, S. E.; Ginsberg, M. H.; Plow, E. F. Trends Bio-

chem. Sci. 1991, 16, 246.

22. Pfa, M.; Tangemann, K.; Muller, B.; Gurrath, M.; Mul-

ler, G.; Kessler, H.; Timpl, R.; Engel, R. J. Biol. Chem. 1994,

269, 20233.

23. Olson, G. L.; Bolin, D. R.; Bonner, M. P; Bos, M.; Cook,

C. M. J. Med. Chem. 1993, 36, 3039.

The biological evaluations have been performed in the

laboratory of hematology (UCL, Prof. Lavenne). The

XPS analyses have been realized in the laboratory of

chemistry at interfaces (UCL, Prof. P. Rouxhet). The

LSC measurements have been done in the laboratory of

cellular biochemistry (UCL, Prof. Y.-J. Schneider).

24. Marshall, G. R. Tetrahedron 1993, 49, 3547.

25. Giannis, A.; Kolter, T. Angew. Chem. Int. Ed. Engl. 1993,

32, 1244.

26. Ojima, I.; Chakravarty, S.; Dong Q. Bioorg. Med. Chem.

1995, 3, 337.

27. Haubner, R.; Finsinger, D.; Kessler, H; Angew. Chem. Int.

Ed. Engl. 1997, 36, 1375.

References

1. Silver, F. H. Biomaterials, Medical Devices and Tissue Engi-

neering, an Integrated Approach; Chapman and Hall: London,

1994.

28. Giannis, A.; Rubsam, F. Angew. Chem. Int. Ed. Engl. 1997,

36, 588.

29. Pierschbacher, M. D.; Polarek, J. W.; Craig, W. S.;

Tschopp; J. F.; Sipes, N. J.; Harper, J. R. J. Cell. Biochem.

1994, 56, 150.

2. Ratner, B. D.; Homann, A. S; Schoen, F. J; Lemons, J. E.

Biomaterials Science, an Introduction to Materials in Medicine;

Academic: San Diego, 1996.

30. Ito, Y.; Imanishi, Y. Polym. News 1995, 20, 107.

31. Hubbell, J. A. Biotechnology 1995, 13, 565.

32. Samanen, J. Ann. Reports Med. Chem. 1996, 31, 91.

33. Schror, K. Drugs 1995, 50, 7.

3. Gan, L.-S.; Eads, C.; Niederer, T.; Bridgers, A.; Yanni, S.;

Hsyu, P.-H.; Pritchard, F. J.; Thakker, D. Drug Dev. Ind.

Pharm. 1994, 20, 615.

4. Halleux, C.; Schneider, Y.-J. Cell Physiol. 1994, 158, 17.

34. Blackburn, B. K.; Lee, A.; Baier, M.; Kohl, B.; Olivero, A.

G. J. Med. Chem. 1997, 40, 717.

5. Griesser, H. J.; Chatelier, R. C.; Gengenbach T. R.; John-

son, G.; Steele, J. G. J. Biomater. Sci. Polym. Ed. 1994, 5, 531.

35. Fisher, M. J.; Gunn, B.; Harms, C. S.; Kline, A. D.; Mul-

laney, J. F. J. Med. Chem. 1997, 40, 2085.

6. Jaumotte-Thelen, S.; Dozot-Dupont, I.; Marchand-Bry-

naert, J.; Schneider, Y.-J. J. Biomed. Mater. Res. 1996, 32, 569.

36. Xue, C.-B.; Wityak, J.; Sielecki, T. M.; Pinto, D. J.; Batt,

D. G. J. Med. Chem. 1997, 40, 2064.

7. Marchand-Brynaert, J.; Deldime, M.; Dupont, I.; Dewez, J.-

L.; Schneider, Y.-J. J. Colloid Interface Sci. 1995, 173, 236.

37. Askew, B. C.; Bednar, R. A.; Bednar, B.; Claremon, D. A.;

Cook, J. J. J. Med. Chem. 1997, 40, 1779.

8. Mougenot, P.; Koch, M.; Dupont, I.; Schneider, Y.-J.;

Marchand-Brynaert, J. J. Colloid Interface Sci. 1996, 177, 162.

38. Xue, C.-B.; Roderick, J.; Jackson, S.; Rafalski, M.; Rock-

well, A. Bioorg. Med. Chem. 1997, 5, 693.

9. Boxus, T.; Deldime-Rubbens, M.; Mougenot, P.; Schneider,

Y.-J.; Marchand-Brynaert, J. Polym. Adv. Technol. 1996, 7, 589.

39. Corbett, J. W.; Graciana, N. R.; Mousa, S. A.; De Grado,

W. F. Bioorg. Med. Chem. Lett. 1997, 7, 1371.

10. Marchand-Brynaert, J.; Jongen, N.; Dewez, J.-L.; J.

Polym. Sci., Part A: Polym. Chem. 1997, 1227.

40. Nicolaou, K. C.; Trujillo, J. I.; Chibale, K. Tetrahedron

1997, 53, 8751.

11. Noiset, O.; Henneuse, C.; Schneider, Y.-J.; Marchand-

Brynaert, J. Macromolecules 1997, 30, 540.

41. Keenan, R. M.; Miller, W. H.; Kwon, C.; Ali, F. E.; Call-

ahan, J. F. J. Med. Chem. 1997, 40, 2289.

12. Henneuse C.; Goret, B.; Marchand-Brynaert, J. Polymer

1998, 39, 835.

42. Hartman, G. D.; Egbertson, M. S.; Halczenko, W.;

Laswell, W. L.; Duggan, M. E. J. Med Chem. 1992, 35,

4640.

13. Noiset, O.; Schneider, Y.-J.; Marchand-Brynaert, J. J.

Polym. Sci., Part A: Polym. Chem. 1997, 35, 3779.

14. Marchand-Brynaert, J.; Boxus, J.; Deldime-Rubbens, M.;

Henneuse, C.; Jaumotte-Thelen, S.; Mougenot, P.; Noiset, O.;

Schneider, Y.-J. ICPSI-2 Proceedings (International Con-

ference on Polymer-Solid Interface), Namur University Press,

Belgium, 1997.

43. Egbertson, M. S.; Chang, C. T.-C.; Duggan, M. E.; Gould,

R. J.; Halczenko, W. J. Med. Chem. 1994, 37, 2537.

44. Halczenko, W.; Cook, J. J.; Holahan, M. A.; Sitko, G. R.;

Stranieri, M. T. Bioorg. Med. Chem. Lett. 1996, 6, 2771.

T. Boxus et al./Bioorg. Med. Chem. 6 (1998) 1577±1595

1595

45. Deldine, M.; Dewez, J.-L.; Schneider, Y.-J.; Marchand-

Brynaert, J. Appl. Surf. Sci. 1995, 90, 1.

55. Haubner, R.; Gratias, R.; Diefenbach, B.; Godman, S. L.;

Jonczyk, A.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 7461.

46. Mougenot, P.; Marchand-Brynaert, J. Macromolecules

1996, 29, 3552.

56. Wermuth, J.; Goodman, S. L.; Jonczyk, A.; Kessler, H. J.

Am. Chem. Soc. 1997, 119, 1328.

47. Harada, T.; Katada, J.; Tachiki, A.; Asari, T.; Ijima, K.;

Uno, I.; Ojima, I.; Hayashi, Y. Bioorg. Med. Chem. Lett. 1997,

7, 209.

57. Ferain, E.; Legras, R. Nucl. Instrum. Meth. Phys. Res. B.

1994, 84, 331.

58. Legras, R.; Jongen, Y.; European Patent W 087/ 05850, 1986.

48. Ghosez, L.; Marchand-Brynaert, J. In Encyclopedia of

Reagents for Organic Synthesis; Wiley: London, 1995; 2, 1231.

59. Massia, S. P; Hubbell, J. A. Anal. Biochem. 1990, 187, 292.

60. Massia, S. P; Hubbell, J. A. J. Cell. Biol. 1991, 114, 1089.

49. Kim, K.; Lin, Y.; Mosher, H. S. Tetrahedron Lett. 1988,

29, 3183.

61. Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stuart, J. J.

P. J. Am. Chem. Soc. 1985, 107, 3902.

50. Bernatowicz, M. S.; Wu, Y. L.; Matsueda, G.R. J. Org.

Chem. 1992, 57, 2497.

62. Gaussian 94, Revision D.4, Frisch, M. J.; Trucks, G. W.;

Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.; Robb, M. A.;

Cheeseman, J. R.; Keith, T.; Peresson, G. A.; Montgomery, J.

A.; Raghavachari, K.; Al-Laham, M. A.; Zakrzewski, V. G.;

Ortiz, J. V.; Foresman, J. B.; Cioslowski, J.; Stefanov, B. B.;

Nanayakkara, A.; Challacombe, M.; Peng, C. Y.; Ayala, P. Y.;

Chen, W.; Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gom-

perts, R.; Martin, R. L.; Fox, D. J.; Binkley, J. S.; Defrees, D.

J.; Baker, J.; Stewart, J. P.; Head-Gordon, M.; Gonzales, C.;

Pople, J. A., Gaussian, Inc., Pittsburgh PA, 1995.

51. Drake, B.; Patek, M.; Lebl, M. Synthesis 1994, 579.

52. Mousa, S. A.; Bozarth, J. M.; Forsythe, M. S.; Lorelli, W.;

Ramachandran, N.; Jackson, S.; De Grado, W. F.; Reilley, T.

M. Cardiology 1993, 83, 374.

53. Bach II, A. C.; Eyermann, C. J.; Gross, J. D; Bower, M. J.;

Harlow, R. L. J. Am. Chem. Soc. 1994, 116, 3207.

54. Wityak, J.; Sielecki, T. M.; Pinto, D. J.; Emmett, G.; Sze, J.

Y. J. Med. Chem. 1997, 40, 50.

Article Doi

Synthesis and evaluation of RGD peptidomimetics aimed at surface bioderivatization of polymer substrates

DOI: 10.1016/S0968-0896(98)00083-2

Source and publish data:

Authors:

Article abstract of DOI:10.1016/S0968-0896(98)00083-2

Full text of DOI:10.1016/S0968-0896(98)00083-2

Products guided by the article

R&D Labs maybe for 216502-85-1

Relevant to this article

Hot Product