Synthesis of RGD analogs as potential vectors for targeted drug delivery

Article abstract of DOI:10.1006/bioo.2001.1227

RGD analogs bind to integrin receptors with high affinity and therefore have the potential to be used as vectors for the targeted delivery of pharmaceutical agents to designated sites. Critical to this application is the ability to synthesize RGD analogs

Full text of DOI:10.1006/bioo.2001.1227

Bioorganic Chemistry 29, 357–379 (2001)
doi:10.1006/bioo.2001.1227, available online at http://www.idealibrary.com on
Synthesis of RGD Analogs as Potential Vectors for
Targeted Drug Delivery
Ji Jiang,* Wei Wang,* David C. Sane,†,1 and Binghe Wang*,1
*Department of Chemistry, North Carolina State University, Raleigh, North Carolina, 27695-8204;
and †Section of Cardiology, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157-1045
Received August 15, 2001
RGD analogs bind to integrin receptors with high affinity and therefore have the potential
to be used as vectors for the targeted delivery of pharmaceutical agents to designated sites.
Critical to this application is the ability to synthesize RGD analogs with different side chain
functional groups that allow for the ready tethering of pharmaceutical agents without sacrificing
their affinity for the target receptor significantly. A series of RGD analogs intended to be used
as delivery vectors of pharmaceutical agents were prepared and evaluated for their ability to
inhibit platelet aggregation by binding to glycoprotein IIb/IIIa. Among them, compound 11
showed the lowest IC50 against platelets activated by ADP. It was found that such RGD analogs
could tolerate side chain modification fairly well with various functional groups attached such
as amide, amine, ester, protected amine and poly(ethylene glycol). The fact that the compound
with a side chain modification of poly(ethylene glycol) retained high affinity for glycoprotein
IIb/IIIa (IC50 150 nM) suggests the feasibility of tethering fairly large pharmaceutical agents
to such RGD analogs without significant sacrifice of their affinity to the intended receptor.
᭧ 2001 Elsevier Science (USA)
INTRODUCTION
The tripeptide sequence Arginine–Glycine–Aspartic (RGD) acid is a universal
recognition motif through which cells interact with extracellular proteins (1). The
integrin family of receptors is composed of over 20 permutations of alpha and beta
subunits that together form transmembrane cell surface receptors (2,3). Many integrins
recognize the RGD sequence, although other peptide sequences are also targets for
these receptors. Integrin receptor–ligand interactions “integrate” the cell cytoplasm
with the extracellular environment and are involved in a variety of disease processes
including thrombosis, tumor growth and metastases and inflammatory states (2,3).
The multiple roles of integrins have led to the development of RGD peptides or
peptide mimetics to block integrin-mediated activities. The greatest success in this
arena has been the development of inhibitors of ␣IIb␤3 (or GP IIb/IIIa), the fibrinogen
receptor on platelets. Three agents are now approved for the treatment of acute
1 To whom correspondence should be addressed. David C. Sane: Fax: (336) 716-9188; e-mail: dsane@
wfubmc.edu. Binghe Wang: Fax: (919) 515-3757; e-mail: binghe wang@ncsu.edu.
357
0045-2068/01 $35.00
᭧ 2001 Elsevier Science (USA)
All rights reserved.

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JIANG ET AL.
coronary ischemic syndromes and/or for use during percutaneous coronary interven-
tions (4). One of these (abciximab) is a chimeric humanized antibody Fab fragment
to GP IIb/IIIa. Eptifibatide is a cyclical L-homoarginine–glycine–aspartic acid-con-
taining peptide and tirofiban (MK-383) is a peptidomimetic (5).
The success of these agents and the high levels of platelet surface expression of
the fibrinogen receptor have prompted studies that target GP IIb/IIIa for other goals.
The ability of RGD peptides to serve as a thrombus imaging agent has been tested
in early studies. RGD peptides, coupled to iron oxide particles or technetium-99m,
allows for thrombus imaging by magnetic resonance (6) or nuclear scintigraphy (7),
respectively. Furthermore, red blood cells conjugated with RGD ligands have been
envisioned as potential substitutes for platelets (8), thereby overcoming the limitations
of platelet transfusion therapy. Other integrins have also been the target of RGD-
based strategies. Radiolabeled RGD peptides, directed at the vitronectin receptor
(␣v␤3) have been used to image tumors or tumor neovasculature in animal models
(9–11). RGD peptides that bind to ␣v␤3 have also been used to transiently increase
the permeability of tumor vasculature, thereby allowing a higher fractional uptake of
a radiolabeled monoclonal antibody directed at a tumor (12). RGD peptides conjugated
with polylysine have been designed as delivery vehicles for enhancing the efficiency
of transfecting cells with recombinant DNA (13–15). Finally, RGD peptides have
been used in tissue engineering applications to covalently modify biomaterial surfaces,
thereby enhancing cell attachment (16–20).
These and other applications suggest that RGD-based imaging and targeting strate-
gies will be used in a variety of clinical arenas in which integrin receptors play a
dominant role. Although RGD peptides have comprised the bulk of the earliest effort,
future work is likely to focus on peptidomimetics, which have several advantages
over peptide-based approaches. As an example, the arginine residue in short peptides
is known to be a target of cellular methylases, with the resultant methylarginyl group
lacking binding activity for some integrins (21). This modification could limit long-
term applications of peptide-containing compounds.
Imaging or targeting strategies that are based on RGD peptidomimetic compounds
will likely require that an appropriate functional group be incorporated for tethering
of the active agent. As a first step towards our goal of designing integrin-targeting
compounds, we have synthesized a series of 10 analogs of MK-383, a GP IIb/IIIa
inhibitor (22), and the affinities of these analogs for GP IIb/IIIa have been tested by
determining their abilities to inhibit platelet aggregation. The analogs differ in con-
taining either amide, amine, ester, protected amine, or polyethylene glycol-containing
side chains of differing sizes. It was found that these functional groups could be
introduced while retaining platelet inhibitory IC50s in the nanomolar range. These
analogs represent novel base compounds for designing targeting or imaging strategies
directed at the integrin GP IIb/IIIa and set the stage for a general integrin targeting
strategy using one or more of these functional groups.
EXPERIMENTAL
1
General methods. H NMR and 13C NMR spectra were recorded at 300 MHz.
Mass spectral analyses were conducted by the Mass Spectrometry Laboratory for
Biotechnology at North Carolina State University. Elemental analyses were performed

RGD ANALOGS FOR DRUG DELIVERY
359
by Atlantic Microlab Inc. UV spectra were recorded on Shimadzu 1601 UV-Visible
spectrophotometer. Column chromatography was performed using silica gel (230–400
mesh) from Aldrich. Chromatotron plates were made using silica gel (Merck, TLC
grade 7749, with gypsum binder and fluorescent indicator) from Aldrich. Unless
stated otherwise all starting materials were commercially available and all solvents
and reagents were used without purification with the exceptions of THF and CH2Cl2.
THF was distilled from sodium/benzophenone. CH2Cl2 was previously dried over
CaCl2 and then distilled from CaH2.
Affinity of modified RGD analogs (2–11) toward GPIIb/IIIa as determined by
platelet aggregation (25). After informed consent, blood was obtained from healthy
donors taking no medications. Platelet rich plasma (PRP) was prepared by centrifuging
the blood at 200g for 10 min. After removing the PRP, the sample was centrifuged
again at 1200g for 10 min to obtain platelet-poor plasma that was used as the optical
blank. The platelet aggregation assay is based on the fact that the platelet suspension
scatters light and that increased transmission of light occurs after aggregation. Since
interference with light transmission depends more on the number of particles rather
than the size of particles, platelet aggregation results in an increase in light transmis-
sion. Aggregation assays were performed under standard conditions using a Chrono-
Log Lumi-Aggregometer (Havertown, PA), at 37ЊC with a magnetic stirbar (rate 1000
rpm) with monitoring for 5 min after the addition of agonist. Platelet aggregation
was stimulated by ADP at 20 ␮M final concentration. The IC50 of the compound
was obtained by determining the concentration that reduced the maximum slope
(percentage light transmittance/min) of aggregation by 50%.
4-(4-Pyridinyl) butyl chloride (13) (24). 4-Picoline (16.763 g, 0.180 mol) was
dissolved in 82 ml of dry THF and the solution was cooled to Ϫ78ЊC with an acetone-
dry ice bath. n-Butyllithium (79.19 ml, 0.198 mol) was added slowly while keeping
the internal temperature below Ϫ50ЊC. The addition took over 1.5 h, giving an orange
solution with precipitates. The reaction was allowed to warm to room temperature
and then stirred at 40–45ЊC for 2 h. THF (82 ml) was added to dissolve the 4-
picolyllithium slurry to give a deep orange solution. The solution was cooled down
to 0ЊC and carefully added into the solution of 1-bromo-3-chloropropane (18.69 ml,
0.189 mol) in THF (30 ml) at Ϫ78ЊC. During the addition, the temperature was kept
below Ϫ65ЊC. The reaction was allowed to gradually warm up to 0ЊC and then worked
up by adding 150 ml H2O. A yellow solution was obtained. The organic layer was
separated and the aqueous layer was extracted with 2 ϫ 50 ml EtOAc. All the organic
layers were combined and dried over MgSO4. Then it was concentrated and the
residue was purified with flash chromatography using hexanes/ethyl acetate (1:1.5)
1
to give an orange oil 13 (28.0089 g, 92%): H-NMR (300 MHz, CDCl3) ␦ 8.50
(d, 2H, J ϭ 4.86 Hz), 7.13 (d, 2H, J ϭ 4.95 Hz), 3.56 (t, 2H, J ϭ 5.19 Hz), 2.65
(t, 2H, J ϭ 3.3 Hz).
4-(4-Piperidinyl) butyl chloride (14). The reaction flask was flushed with Ar first.
Then acetic acid (7.5 ml), platinum(IV) oxide (0.75 g), and the solution of 13 (15 g,
0.088 mol) in acetic acid (7.5ml) were added sequentially. The black suspension was
subjected to 60 psi H2 in a Parr instrument for 72 h at room temperature. The catalyst
was filtered off and the filtrate was concentrated to afford compound 14 as an acetate

360
JIANG ET AL.
1
salt (100%): H-NMR (300 MHz, CDCl3) ␦ 3.54 (t, 2H, J ϭ 6.30 Hz), 3.39 (d, 2H,
J ϭ 11.7 Hz), 2.84 (t, 2H, J ϭ 12.2 Hz), 1.74–1.87 (m, 4H), 1.31–1.49 (m, 7H).
4-(4-Chloro-butyl)-piperidine-1-carboxylic acid benzyl ester (15). The acetate salt
of compound 14 (5 g) was dissolved in 200 ml EtOAc. This organic solution was
washed with 15% aqueous K2CO3 solution (2 ϫ 50 ml). The organic layer was dried
over MgSO4 and concentrated to give a yellow oil (3.5389 g, 21.20 mmol). This oil
was dried on the oil pump for 0.5 h and then dissolved in acetonitrile (90 ml). Pyridine
(4.88 ml, 60.32 mmol) and benzyl chloroformate (4.5 ml, 30.16 mmol) were added
sequentially into the solution at 0ЊC. The reaction was stirred at 0ЊC for 0.5 h and
then at room temperature for 24 h. The solution was concentrated and the residue
was dissolved in EtOAc (300 ml). The organic solution was washed with 1N HCl
(3 ϫ 50 ml), brine (50 ml) and dried over MgSO4. EtOAc was then evaporated to
give a yellow oil. This yellow oil was chromatographed on a silica gel column with
hexanes/EtOAc (5:1) to give a colorless oil 32 (4.4253 g, 71%): 1H-NMR (300 MHz,
CDCl3) ␦ 7.26–7.36 (m, 5H), 5.12 (s, 2H), 4.11–4.17 (m, 2H), 3.54 (t, 2H, J ϭ 6.6
Hz), 2.72–2.79 (m, 2H), 1.66–1.81 (m, 4H), 1.09–1.51 (m, 7H); 13C-NMR (300
MHz, CDCl3) ␦ 154.8, 136.8, 128.1, 127.5, 127.4, 66.5, 44.6, 43.9, 35.4, 32.4, 31.7,
23.6; MS/FAB m/z (MH+) Calcd for C17H24O2NCl 310.16, found 310.2. Anal. Calcd
for C17H24O2NCl: C, 65.90; H, 7.81; N, 4.52. Found: C, 65.65; H, 7.92; N, 4.38.
4-{4-[4-(2-tert-Butoxycarbonylamino-2-carboxy-ethyl)-phenoxy]-butyl}-piperi-
dine-1-carboxylic acid benzyl ester (16) (22). Under the protection of Ar, 60% disperse
NaH was washed with hexanes four times to give a pale powder. The powder was
dried on oil pump for 0.5 h. Then it was added into DMF (4.5 ml). N-Boc-tyrosine
(3.6288 g, 12.9 mmol) was also dissolved in DMF (15 ml) and was added dropwise
to the suspension of NaH (928.8 mg, 38.7 mmol) over 0.5 h at 0ЊC. The suspension
was stirred for 1 h at 0ЊC. Then compound 15 (4.0000 g, 12.9 mmol) in 15 ml DMF
was added into the suspension at 0ЊC over 0.5 h, followed by the addition of Kl
(0.4283 g, 2.58 mmol). The reaction was stirred at 0ЊC for 1 h and then at 40–45ЊC
for 43 h to give a dark solution with white precipitates. DMF was removed and
EtOAc (150 ml) was added into the residue. The organic solution was acidified to
pH 4 with 10% KHSO4 (30 ml). The organic layer was separated and the aqueous
layer was extracted again with EtOAc (100 ml). The combined organic layers were
washed with brine (2 ϫ 20 ml), dried over MgSO4, and concentrated to an oil. The
crude product was chromatographed on a silica gel column with hexanes/EtOAc (2:1)
1
to give compound 16 (5.8390 g, 82%): H-NMR (300 MHz, CDCl3), ␦ 7.30–7.37
(m, 5H), 7.08 (d, 2H, J ϭ 9.0 Hz), 6.82 (d, 2H, J ϭ 8.1 Hz), 5.12 (s, 2H), 4.90–4.95
(m, 1H), 4.50–4.60 (m, 1H), 4.11–4.17 (m, 2H), 3.94 (t, 2H, J ϭ 6.2 Hz), 3.00–3.15
(m, 2H), 2.65–2.80 (m, 2H), 1.65–1.78 (m, 4H), 1.07–1.50 (m, 7H), 1,42 (s, 9H);
13C-NMR (300 MHz, CDCl3) ␦ 175.7, 158.4, 155.7, 137.1, 130.6, 128.7, 128.1, 127.9,
114.8, 80.4, 67.9, 67.3, 54.6, 44.5, 37.1, 36.3, 36.0, 32.3, 29.6, 28.5, 23.3; MS/FAB
m/z (MH+) calcd for C31H42O7N2 555.3, found 555.4. Anal. calcd for C31H42O7N2:
C, 67.13; H, 7.63; N, 5.05. Found: C, 66.87; H, 7.72; N, 4.92.
4-{4-[4-(2-Benzyloxycarbonyl-2-tert-butoxycarbonylamino-ethyl)-phenoxy]-bu-
tyl}-piperidine-1-carboxylic acid benzyl ester (17). 1,3-Dicyclohexylcarbodiimide
(0.4667 g, 2.26 mmol) was added to the solution of 16 (0.9625 g, 1.74 mmol) in
CH2Cl2 (1 ml) at 0ЊC. The reaction was stirred for 10 min with white precipitates

RGD ANALOGS FOR DRUG DELIVERY
361
formed. Then benzyl alcohol (0.4328 g, 4.00 mmol) and 4-dimethylaminopyridine
(0.0425 g, 0.35 mmol) were added sequentially. The solution was stirred at 0ЊC for
1.5 h, then at room temperature for 4.5 h. The resulted suspension was put in the
freezer overnight. The white precipitates that formed were filtered off and the filtrate
was concentrated. The residue was dissolved in EtOAc (25 ml) and the organic
solution was washed with 1N HCl (2 ϫ 10 ml), saturated NaHCO3 (2 ϫ 10 ml) and
brine (10 ml). The organic layer was dried over MgSO4 and then concentrated to
give an oil. The crude oil was chromatographed on a silica gel column with hexanes/
1
EtOAc (1.5:1) to give compound 17 (0.9167 g, 80%): H-NMR (300 MHz, CDCl3),
␦ 7.28–7.35 (m, 10H), 6.93 (d, 2H, J ϭ 8.1 Hz), 6.74 (d, 2H, J ϭ 8.2 Hz), 5.07–5.15
(m, 4H), 4.90–5.00 (m, 1H), 4.50–4.60 (m, 1H), 4.10–4.30 (m, 2H), 3.90 (t, 2H,
J ϭ 6.2 Hz), 3.02 (d, 2H, J ϭ 5.0 Hz), 2.76 (m, 2H), 1.64–1.77 (m, 4H), 1.01–1.47
(m, 7H), 1.41 (s, 9H); 13C-NMR (300 MHz, CDCl3) ␦ 172.0, 158.3, 155.5, 155.3,
137.2, 135.4, 130.5, 128.7, 128.6, 128.5, 128.1, 128.0, 127.8, 114.7, 80.0, 67.9, 67.2,
67.1, 54.7, 44.4, 37.6, 36.4, 36.0, 32.9, 32.3, 29.6, 28.5, 23.3; MS/FAB m/z (MH+)
calcd for C38H48O7N2 645.4, found 645.5. Anal. calcd for C38H48O7N2: C, 70.78; H,
7.50; N, 4.34. Found: C, 70.60; H, 7.56; N, 4.42.
4-(4-{4-[2-Benzyloxycarbonyl-2-(4-carboxy-benzenesulfonylamino)-ethyl]-phe-
noxy}-butyl)-piperidine-1-carboxylic acid benzyl ester (18). Compound 17 (111.5
mg, 0.17 mmol) was dissolved in 25% TFA solution in CH2Cl2 (6.0 ml) and stirred
at room temperature for 1 h. The solution was concentrated and the residue was
dissolved in 3.0 ml CH2Cl2. TEA (1.0 ml) was added and the resulted solution was
stirred for 5 min. Then it was concentrated and dried on oil pump for 0.5 h. The
residue was dissolved in 1.5 ml CH3CN and 1 mL H2O. To this solution, K2CO3
(143.5 mg, 1.04 mmol) was added. Then 4-(chlorosulfonic)benzoic acid (76.5 mg,
0.35 mmol) was added in portions at 0ЊC over 15 min. After the addition, the solution
was stirred at 0ЊC for 0.5 h and at room temperature for 1 h. Another one equivalent
of 4-(chlorosulfonic)benzoic acid (38.2 mg, 0.17 mmol) was added in portions at
0ЊC. The reaction was stirred at 0ЊC for 0.5 h and at room temperature for another
3.5 h. The solution was acidified to pH 1 with 1 N HCl. Then it was concentrated
and the residue was dissolved in 100 ml EtOAc. The organic solution was washed
with 1N HCl (20 ml) and brine (20 ml). The organic layer was dried over MgSO4
and concentrated. The crude product was purified on chromatotron with hexanes/
EtOAc (3:1) to give an oil 18 (60.7 mg, 48.4%): 1H-NMR (300 MHz, CDCl3) ␦ 8.08
(d, 2H, J ϭ 8.2 Hz), 7.78 (d, 2H, J ϭ 8.3 Hz), 7.17–7.35 (m, 10H), 5.23 (d, 1H,
J ϭ 9.3 Hz), 5.13 (s, 2H), 4.93 (s, 2H), 4.15–4.27 (m, 3H), 3.89 (t, 2H, J ϭ 6.2 Hz),
2.97–3.00 (m, 2H), 2.70–2.90 (m, 2H), 1.50–1.77 (m, 4H), 1.15–1.75 (m, 7H); 13C-
NMR (300 MHz, CDCl3) ␦ 170.9, 158.5, 155.7, 144.5, 137.0, 135.3, 134.7, 133.4,
130.9, 130.6, 128.8, 128.6, 128.1, 128.0, 127.3, 126.5, 114.7, 67.9, 67.7, 67.3, 57.2,
44.5, 38.7, 36.3, 36.0, 32.2, 29.6, 23.3; HRMS/FAB m/z (MH+) calcd for C40H44O9N2S
729.2876, found 729.2832.
4-(4-{4-[2-Benzyloxycarbonyl-2-(4-pentanoyl-benzenesulfonylamino)-ethyl]-phe-
noxy}-butyl)-piperidine-1-carboxylic acid benzyl ester (19). Compound 18 (28.0 mg,
0.04 mmol) was dissolved in 1.0 ml CH2Cl2. EDC (8.0 mg, 0.04 mmol) was added
and the reaction was stirred for 10 min to give a cloudy solution. Then HOBT (5.2
mg, 0.04 mmol) was added and the reaction was stirred for 30 min. n-Butyl amine

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JIANG ET AL.
(7 ␮m, 0.07 mmol) and DMAP (1.0 mg, 0.008 mmol) were added sequentially at
0ЊC. The solution was stirred at 0ЊC for 0.5 h and then at room temperature for 12
h. The precipitates were filtered off and the filtrate was concentrated. The residue
was dissolved in 20 ml EtOAc. The organic solution was washed with 10% citric
acid (2 ϫ 5 ml), saturated Na2CO3 (2 ϫ 5 ml), and brine (5 ml). Then the organic
layer was dried over MgSO4 and concentrated. The crude product was purified on
1
chromatotron with hexanes/EtOAc (1:1) to give compound 19 (30.0 mg, 99%): H-
NMR (300 MHz, CDCl3) ␦ 7.71–7.78 (m, 4H), 7.15–7.35 (m, 10H), 6.87 (d, 2H,
J ϭ 8.5 Hz), 6.68 (d, 2H, J ϭ 8.4 Hz), 6.14–6.18 (m, 1H), 5.29 (d, 1H, J ϭ 9.2
Hz), 5.12 (s, 2H), 4.87–4.95 (m, 2H), 4.11–4.22 (m, 3H), 3.89 (t, 2H, J ϭ 6.4 Hz),
3.46 (q, 2H, J ϭ 6.9 Hz), 2.97 (d, 2H, J ϭ 5.2 Hz), 2.72–2.80 (m, 2H), 1.05–1.78
(m, 15H), 0.97 (t, 3H, J ϭ 7.3 Hz); 13C-NMR (300 MHz, CDCl3) ␦ 170.7, 166.1,
158.6, 155.6, 142.4, 139.0, 134.2, 134.8, 131.0, 130.7, 128.9, 128.7, 128.1, 128.1,
127.8, 127.6, 126.7, 126.6, 114.8, 68.1, 67.8, 67.2, 57.0, 44.5, 40.3, 38.8, 36.4, 36.1,
32.3, 31.9, 29.6, 23.4, 20.4, 14.0; HRMS/FAB m/z (MH+) calcd for C44H53O8N3S
784.3632, found 784.3668. Anal. calcd for C44H53O8N3S: C, 67.41; H, 6.81; N, 5.36.
Found: C, 67.68; H, 7.06; N, 5.18.
2-(4-Pentanoyl-benzenesulfonylamino)-3-[4-(4-piperidin-4-yl-butoxy)-phenyl]-
propionic acid (2). Compound 19 (30.0 mg, 0.04 mmol) was dissolved in CH3OH
(2.0 ml) and DMF (0.4 ml). This clear solution was flushed with Ar for 10 min and
then 10% Pd/C (4.4 mg) was added. The black suspension was flushed with Ar for
10 min and then it was subjected to hydrogen balloon at room temperature for 1 h.
The catalyst was filtered off and the filtrate was concentrated to give 2 (20.0 mg,
1
100%): H-NMR (300 MHz, CD3OD) ␦ 7.76–7.85 (dd, 4H, J ϭ 8.7 Hz), 7.07 (d,
2H, J ϭ 8.1 Hz), 6.70 (d, 2H, J ϭ 8.7 Hz), 3.95 (t, 2H, J ϭ 6.3 Hz), 3.79 (m, 1H),
3.38 (t, 2H, J ϭ 7.3 Hz), 2.83–2.96 (m, 4H), 1.28–1.94 (m, 15H), 0.97 (t, 3H, J ϭ
7.2 Hz); 13C-NMR (300 MHz, CDCl3) ␦ 169.0, 159.1, 144.4, 139.4, 131.8, 130.9,
129.0, 128.3, 115.4, 68.9, 61.1, 56.0, 45.4, 41.1, 39.8, 36.7, 32.6, 30.4, 30.1, 24.0,
21.3, 14.4; HRMS/FAB m/z (MH+) calcd for C29H41N3O6S 560.2794, found 560.2830.
4-(4-Chloro-butyl)-piperidine-1-carboxylic acid tert-butyl ester (20). Compound
14 was dissolved in EtOAc and the organic solution was washed with 15% aqueous
K2CO3 solution to give a pale yellow oil in a similar manner as that for 15. This oil
(0.7320 g, 4.29 mmol) was dissolved in CH2Cl2 (10 ml) and TEA (0.90 ml, 6.44
mmol) was added. At 0ЊC di-tert-butyl dicarbonate (1.1230 g, 4.29 mmol) was added.
The reaction was allowed to warm up to room temperature and stirred for 20 h. The
solution was concentrated and the residue was dissolved in EtOAc. The organic
solution was washed with brine twice, dried over MgSO4 and concentrated. The crude
product was purified on a silica gel column with hexanes/EtOAc (5:1) to give a pale
1
yellow oil 20 (0.9483 g, 80%): H-NMR (300 MHz, CDCl3) ␦ 4.05–4.09 (bd, 2H),
3.54 (t, 2H, J ϭ 6.7 Hz), 2.66 (t, 2H, J ϭ 12.4 Hz), 1.72–1.79 (m, 2H), 1.61–1.68
(m, 2H), 1.45 (s, 9H), 1.21–1.30 (m, 2H), 1.04–1.15 (m, 2H); 13C-NMR (300 MHz,
CDCl3) ␦ 155.0, 79.0, 45.0, 44.0, 35.8, 35.7, 32.7, 28.4, 27.4, 24.0; MS/FAB m/z
(MH+) calcd for C14H26CINO2 276.2, found 276.2. Anal. calcd for C14H26CINO2: C,
60.96; H, 9.50; N, 5.08. Found: C, 61.08; H, 9.52; N, 4.99.
4-{4-[4-(2-Benzyloxycarbonylamino-2-tert-butoxycarbonyl-ethyl)-phenoxy]-butyl}-
piperidine-1-carboxylic acid tert-butyl ester (21). NaH (0.27 g, 6.67 mmol, 60%

RGD ANALOGS FOR DRUG DELIVERY
363
dispersion in mineral oil) was placed in a 100 ml dry flask, then washed with 5 ml
hexanes. Dry DMF (2 ml) was added into the above flask, then the flask was cooled
to 0ЊC with an ice-bath. A solution of Boc-N-tyrosine t-butyl ester (2.20 g, 5.63
mmol) in dry DMF was added dropwise into the suspension. After the reaction mixture
was stirred at 0ЊC for an additional hour, a solution of Boc-N-chloride (1.55 g, 5.63
mmol) was added following by addition of KI (0.187 g, 1.13 mmol). The reaction
mixture was continued stirring at RT for 26 h. The solvent was evaporated under
reduced pressure to give an oil, which was dissolved in 180 mL of ethyl acetate. The
organic layer was washed with 1 N HCl aqueous solution (2 ϫ 35 ml) and brine (35
ml), dried (MgSO4), and concentrated to give an oil. The residue was purified on
silica gel using ethyl acetate/hexanes (1/5) to afford a colorless oil (2.47 g, 72%).
1H-NMR (CDCl3) ␦ 7.34 (s, 5H), 7.03 (d, 2H, J ϭ 8.4 Hz), 6.78 (d, 2H, J ϭ 8.4
Hz), 5.20 (d, 1H, J ϭ 7.9 Hz), 5.10 (s, 2H), 4.49 (dd, 1H, J ϭ 6.1, 13.5 Hz), 4.09
(m, 2H), 3.91 (t, 2H, J ϭ 6.3 Hz), 3.01 (d, 2H, J ϭ 5.5 Hz), 2.67 (brt, 2H, J ϭ 12.3
Hz), 1.61–1.80 (m, 4H), 1.52 (m, 2H), 1.45 (s, 9H), 1.41 (s, 9H), 1.24–1.33 (m, 3H),
1.01–1.15 (2H, m). 13C-NMR (CDCl3) ␦ 170.8, 158.3, 155.8, 155.0, 136.6, 130.6,
129.6, 128.2, 128.0, 114.6, 82.3, 79.3, 67.9, 66.9, 55.5, 44.2, 37.6, 36.4, 36.1, 32.3,
29.6, 28.6, 28.1, 23.3. MS/FAB m/z (MH+) calcd for C35H50N2O7 611.4, found 611.4.
Anal. calcd for C35H50N2O7: C, 68.83; H, 8.25; N, 4.59. Found: C, 68.81; H, 8.35;
H, 4.61.
4-{4-[4-(2-Amino-2-tert-butoxycarbonyl-ethyl)-phenoxy]-butyl}-piperidine-1-
carboxylic acid tert-butyl ester (22). A solution of 21 (1.25 g, 2.05 mmol) in 40 ml
of methanol in the presence of 10% Pd-C (100 mg) was hydrogenated at hydrogen
atmosphere for 7 h. The catalyst was filtered out and washed with methanol (2 ϫ 8
ml). The solvent was evaporated to give a slightly gray solid (0.939 g, 96%). The
1
product was used directly for the next step reaction without purification. H NMR
(CDCl3) ␦ 7.11 (d, 2H, J ϭ 8.4 Hz), 6.82 (d, 2H, J ϭ 8.4 Hz), 4.08 (brs, 2H), 3.93
(t, 2H, J ϭ 6.3 Hz), 3.56 (dd, 1H, J ϭ 5.5, 7.5 Hz), 3.48 (brs, 2H), 3.97 (dd, 1H,
J ϭ 5.5, 13.7 Hz), 2.77 (dd, 1H, J ϭ 7.5, 13.7 Hz), 2.66 (brt, 2H, J ϭ 12.3 Hz),
1.75 (m, 2H), 1.60–1.71 (m, 3H), 1.45 (s, 9H), 1.44 (s, 9H), 1.23–1.38 (m, 4H), 1.07
(m, 2H). 13C NMR (CDCl3) ␦ 174.6, 158.1, 155.1, 130.5, 129.6, 114.6, 81.3, 79.3,
68.0, 56.6, 44.3, 40.5, 36.4, 36.2, 32.4, 29.7, 28.7, 28.2, 23.4.
4-(4-{4-[2-tert-Butoxycarbonyl-2-(4-carboxy-benzenesulfonylamino)-ethyl]-
phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (23). To a solution of
compound 22 (218 mg, 0.46 mmol) and K2CO3 (381 mg, 2.76 mmol) in 4 ml of
CH3CN and 2 ml of H2O at 0ЊC was added 4-(chlorosulfonyl)benzoic acid (202 mg,
0.92 mmol) in portions during 25 min. The reaction mixture was stirred at 0ЊC for
1 h, keeping pH Ͼ 9. An additional of K2CO3 (127 mg, 0.92 mmol) was added, then
4-(chlorosulfonyl)benzoic acid (101 mg, 0.46 mmol) was added in portions during
15 min, keeping pH Ͼ 9. The mixture was stirred at 0ЊC for 1.5 h, then 4-(chlorosulfo-
nyl)benzoic acid (101 mg, 0.46 mmol) was added in portions during 1 h. The reaction
mixture was continued stirring at 10–20ЊC for 2 h. The reaction was acidified to pH
1 using 1 N HCl. The reaction mixture was extracted with ethyl acetate (2 ϫ 60 ml).
The combined organic fractions were dried over anhydrous MgSO4, and concentrated
under reduced pressure to give an oil. The residue was purified on a chromatatron
(1 mm) with MeOH and CH2Cl2 containing 0.1% HOAc) to afford a white solid (167

364
JIANG ET AL.
1
mg, 55%). H NMR (CDCl3) ␦ 14.26 (brs, 1H), 8.12 (d, 2H, J ϭ 8.3 Hz), 7.82 (d,
2H, J ϭ 8.3 Hz), 7.01 (d, 2H, J ϭ 8.4 Hz), 6.73 (d, 2H, J ϭ 8.4 Hz), 5.73 (d, 1H,
J ϭ 9.2 Hz), 4.09 (dd, 2H, J ϭ 5.9, 15.1 Hz), 3.89 (t, 2H, J ϭ 6.2 Hz), 2.95 (m,
2H), 2.69 (brs, 2H, J ϭ 12.2 Hz), 1.65–1.79 (m, 4H), 1.46 (s, 9H), 1.30–1.54 (m,
5H), 1.25 (s, 9H), 1.02–1.18 (m, 2H). 13C NMR (CDCl3) ␦ 170.3, 169.5, 158.4,
155.3, 144.8, 133.3, 130.7, 127.3, 127.1, 114.6, 83.1, 79.8, 67.9, 57.6, 44.3, 38.7,
36.3, 36.0, 32.3, 29.5, 28.6, 27.9. MS/FAB m/z (MH+) calcd for C34H48N2O9S 661.3,
found 661.3. Anal. calcd for C34H48N2O9S: C, 61.80; H, 7.32; N, 4.24. Found: C,
61.92; H, 7.31; H, 4.05.
Protected RGD analog 24. To a solution of 23 (22 mg, 0.033 mmol) in 1 ml of
dry CH2Cl2 at 0ЊC under argon atmosphere was added DCC (7.5 mg, 0.036 mmol).
After stirring for 1–2 min, HOBt (5 mg, 0.036 mmol) was added. After 10 min, 3-
(dimethylamino)propylamine (4 ␮l, 0.033 mmol) and DMAP (5 mg, 0.036 mmol)
were added. The reaction was stirred at 0ЊC for 1.5 h and then RT for 3 h. The white
precipitate was filtered out and the solvent was removed under reduced pressure to
give an oil. The residue was redissolved in 80 ml of ethyl acetate, then was washed
with water (2 ϫ 15 ml), saturated NaHCO3 (15 ml), and brine (15 ml), dried over
anhydrous MgSO4, and concentrated to give an oil. The oil was purified on a chromata-
tron (1 mm) with MeOH and CH2Cl2 (1/50 then 1/30 with 0.1% TEA) to afford an
oil (18 mg, 72%). 1H NMR (CDCl3) ␦ 8.91 (brs, 1H), 7.87 (d, 2H, J ϭ 8.3 Hz), 7.82
(d, 2H, J ϭ 8.3 Hz), 7.02 (d, 2H, J ϭ 8.4 Hz), 6.76 (d, 2H, J ϭ 8.4 Hz), 4.06 (m,
2H), 3.91 (t, 2H, J ϭ 6.3 Hz), 3.57 (m, 2H), 2.95–3.03 (m, 4H), 2.67 (m, 2H), 2.60
(t, 2H, J ϭ 5.7 Hz), 2.36 (s, 6H), 1.64–1.84 (m, 6H), 1.45 (s, 9H), 1.39–1.51 (m,
2H), 1.25–1.34 (m, 3H), 1.22 (s, 9H), 1.04–1.11 (m, 2H). 13C NMR (CDCl3) ␦ 169.9,
165.9, 158.4, 155.0, 142.5, 138.0, 130.8, 128.2, 127.4, 127.1, 114.6, 82.9, 79.3, 68.0,
57.3, 56.7, 46.0, 43.8, 38.7, 38.1, 36.4, 36.1, 32.3, 29.8, 29.6, 28.6, 27.9, 24.4. HRMS/
FAB m/z (MH+) calcd for C39H60N4O8S 745.4210, found 745.4228.
RGD analog (3). Compound 24 (13 mg, 0.017 mmol) was treated with 40% TFA
in dry CH2Cl2 (2 ml) at RT for 2.5 h. The solvents were evaporated in vacuo to afford
1
an oil (12 mg, 86%). H NMR (CDCl3) ␦ 7.90 (d, 2H, J ϭ 8.0 Hz), 7.80 (d, 2H,
J ϭ 8.0 Hz), 7.07 (d, 2H, J ϭ 8.0 Hz), 6.75 (d, 2H, J ϭ 8.0 Hz), 4.13 (m, 1H), 3.94
(t, 2H, J ϭ 5.9 Hz), 3.50 (t, 2H, J ϭ 6.5 Hz), 3.36 (m, 4H), 3.16 (t, 2H, J ϭ 7.6
Hz), 3.04 (m, 1H), 2.91 (m, 1H), 2.87 (s, 6H), 2.07 (m, 2H), 1.94 (m, 2H), 1.73–1.79
(m, 3H), 1.51 (m, 2H), 1.38 (m, 2H), 0.88 (m, 2H). 13C NMR (CDCl3) ␦ 174.2, 169.4,
159.6, 145.4, 138.7, 131.6, 129.8, 129.1, 128.3, 115.6, 68.9, 59.2, 56.9, 45.5, 43.7,
39.3, 37.9, 36.9, 35.0, 30.5, 30.2, 26.3, 24.1.
Protected RGD analog (25). To a solution of 23 (130 mg, 0.197 mmol) in 0.5 ml
of dry CH2Cl2 at 0ЊC under argon atmosphere was added EDC (41 mg, 0.216 mmol).
After stirring for 1–2 min, HOBt (40 mg, 0.294 mmol) was added. After 10 min,
1,4-bis(3-aminopropyl)piperazine (5 ␮l, 0.024 mmol) and DMAP (26 mg, 0.216
mmol) were added. After stirring for 10 min, 10 ␮l (0.050 mmol) of 1,4-bis(3-
aminopropyl)piperazine was added. After 30 min, an additional of 1,4-bis(3-aminopro-
pyl)piperazine (5 ␮l, 0.024 mmol) was added. The reaction was continued stirring
for 1 h at 0ЊC and RT for 4 h. The solvent was removed under reduced pressure to
give an oil, which was redissolved in 80 ml of ethyl acetate, washed with saturated
NaHCO3 (2 ϫ 15 ml), dried over anhydrous MgSO4, and concentrated to give an

RGD ANALOGS FOR DRUG DELIVERY
365
oil. The oil was purified on a chromatatron (1 mm) with MeOH and CH2Cl2 (1/30
then 1/20) to afford a white solid (105 mg, 72%). 1H NMR (CDCl3) ␦ 8.23 (brs, 2H),
7.87 (d, 4H, J ϭ 8.2 Hz), 7.81 (d, 4H, J ϭ 8.2 Hz), 7.00 (d, 4H, J ϭ 8.1 Hz), 6.74
(d, 4H, J ϭ 8.1 Hz), 5.59 (brs, 2H), 5.30 (s, 2H), 4.07 (t, 4H, J ϭ 5.4 Hz), 3.89 (t,
4H, J ϭ 5.7 Hz), 3.53 (m, 4H), 2.93 (m, 4H), 2.50–2.70 (m, 12H), 1.64–1.76 (m,
12H), 1.37–1.53 (m, 6H), 1.45 (s, 18H), 1.24 (s, 18H), 1.22–1.32 (m, 6H), 1.01–1.14
(m, 4H). 13C NMR (CDCl3) ␦ 170.0, 165.9, 158.4, 155.0, 142.5, 138.9, 130.7, 128.0,
127.3, 127.1, 114.6, 83.0, 80.0, 68.0, 58.0, 57.4, 52.9, 44.2, 40.6, 38.8, 36.4, 36.1,
32.3, 30.0, 28.6, 27.9, 24.3, 23.2. HRMS/FAB m/z (MH+) calcd for C78H116N8O16S2
1,485.80, found 1,485.80.
RGD analog (4). Compound 25 (25 mg, 0.017 mmol) was treated with 40% TFA
in CH2Cl2 (3 ml) at RT under argon for 4 h. The solvents were evaporated under
1
reduced pressure to give a solid (27 mg, 98%). H NMR (CDCl3) ␦ 7.87 (d, 4H,
J ϭ 8.3 Hz), 7.75 (d, 4H, J ϭ 8.3 Hz), 7.03 (d, 4H, J ϭ 8.6 Hz), 6.72 (d, 4H,
J ϭ 8.6 Hz), 4.03 (dd, 2H, J ϭ 5.4, 8.4 Hz), 3.93 (t, 4H, J ϭ 6.2 Hz), 3.50 (t, 4H,
J ϭ 6.5 Hz), 3.31–3.40 (m, 12H), 3.12 (t, 4H, J ϭ 7.2 Hz), 2.90–3.03 (m, 6H), 2.78
(dd, 2H, J ϭ 8.6, 13.8 Hz), 1.93–2.05 (m, 8H), 1.76 (m, 4H), 1.48–1.63 (m, 6H),
1.29–1.42 (m, 8H). 13C NMR (CDCl3) ␦ 174.3, 169.2, 159.6, 145.3, 138.8, 131.6,
129.8, 129.0, 128.3, 115.6, 68.9, 59.3, 55.9, 51.4, 45.5, 39.2, 38.4, 36.9, 35.0, 30.5,
30.2, 26.1, 24.1. HRMS/FAB m/z (MH+) calcd for C60H84N8O12S2 1173.5728,
found 1173.5723.
Protected RGD analog (26). To a solution of 18 (42 mg, 0.058 mmol) in 2 ml of
dry CH2Cl2 at 0ЊC under argon atmosphere was added EDC (12 mg, 0.0634 mmol).
After stirring for 1–2 min, HOBt (12 mg, 0.0634 mmol) was added. After 30 min,
poly(ethylene glycol (5 ␮l, 0.019 mmol) and DMAP (8 mg, 0.0634 mmol) were
added. After the reaction was stirred at 0ЊC for 50 min, an additional of poly(ethylene
glycol) (2.7 ␮l, 0.01 mmol) was added. The reaction was continued stirring at 0ЊC
for 1.5 h then RT for 4 h. The solvent was removed under reduced pressure to give
an oil. The oil was purified on a chromatatron (1 mm) with MeOH and CH2Cl2 (1/
1
20) to afford an oil (41 mg, 82%). H NMR (CDCl3) ␦ 8.06 (d, 4H, J ϭ 8.0 Hz),
7.89 (d, 4H, J ϭ 8.0 Hz), 7.13–7.37 (m, 20H), 6.88 (d, 4H, J ϭ 8.4 Hz), 6.69 (d,
4H, J ϭ 8.4 Hz), 5.25 (m, 2H), 5.12 (s, 4H), 4.89 (s, 4H), 4.50 (brs, 4H), 4.23 (m,
6H), 3.88 (t, 4H, J ϭ 6.3 Hz), 3.64–3.68 (m, 22H), 2.99 (d, 4H, J ϭ 5.6 Hz), 2.76
(brt, 4H, J ϭ 11.2 Hz), 1.10–1.77 (m, 22H).
RGD analog (5). A solution of 26 (40 mg, 0.023 mmol) in 2 ml of methanol in
the presence of 10% Pd-C (20 mg) was hydrogenated at hydrogen atmosphere for
5.5 h. The catalyst was filtered out and washed with methanol (2 ϫ 4 ml). The solvent
1
was evaporated to give an oil (28 mg, 95%). H NMR (CD3OD) 8.01 (d, 4H, J ϭ
8.0 Hz), 7.74 (4H, J ϭ 8.0 Hz), 7.01 (d, 4H, J ϭ 8.4 Hz), 6.68 (d, 4H, J ϭ 8.4 Hz),
4.50 (brs, 4H), 4.20 (m, 6H), 3.86–3.96 (m, 10H), 3.64–3.70 (m, 16H), 2.65–3.18
(m, 8H), 1.10–1.92 (m, 22H). HRMS/FAB m/z (MH+) calcd for C30H44N4O6S
589.3060, found 589.3073.
(2-Chlorosulfonyl-ethyl)-carbamic acid benzyl ester (29) (26–30). Taurine (1.4600
g, 11.70 mmol) was dissolved in 1 N NaOH (11.7 ml, 11.70 mmol). Benzyl chloro-
formate (2.0 ml, 14 mmol) and 1 N NaOH (14.0 ml, 14 mmol) were added simultane-
ously at 0ЊC. The reaction was stirred at 0ЊC for 0.5 h and at room temperature for

366
JIANG ET AL.
2 h. There was some oil formed in the solution. The solution was washed with EtOAc
three times. Then the aqueous layer was concentrated to give a white solid. The white
solid was converted into tetrabutylammonium salt 28 using 40 W % tetrabutylammon-
ium hydroxide.
To this salt 28 (0.50 mmol), 2 ml CH2Cl2, 40 ␮l of DMF and triphosgene (148.3
mg, 0.25 mmol) were added sequentially. The suspension was stirred at room tempera-
ture for 1 h and then concentrated. The residue was eluted through a small silica gel
with EtOAc and the eluant was concentrated to give a yellow oil. This crude product
was purified on chromatotron using hexanes/EtOAc (1:1) to give compound 29 (102.5
1
mg, 74% for 2 steps): H-NMR (300 MHz, CDCl3) ␦ 7.34 (bs, 5H), 5.42 (bs, 1H),
5.12 (bs, 2H), 3.81–3.87 (m, 4H).
Potassium;
4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butane-1-sulfonate
(32)
(31). Phthalimide potassium derivative (1.6300 g, 8.81 mmol) and 1,4-butane sultone
(0.75 ml, 7.34 mmol) were mixed in DMF (20 ml) and stirred at 80ЊC for 2 h. DMF
was removed and the white residue was dissolved in water. The aqueous solution
was washed with EtOAc twice. The aqueous layer was concentrated to give a white
solid 32. This white solid was used to next step directly without further purification:
1H-NMR (300 MHz, D2O) ␦ 7.82–7.87 (m, 4H), 3.73 (t, 2H, J ϭ 5.7 Hz), 2.98 (t,
2H, J ϭ 6.8 Hz), 1.80–1.82 (m, 4H).
(4-Chlorosulfonyl-butyl)-carbamic acid benzyl ester (34) (32,33). Compound 32
(459.2 mmol, 1.43 mmol) was dissolved in concentrated HCl (4.5 ml) and the solution
was stirred at 107ЊC for 19 h. The gray precipitate that formed was filtered off and
the filtrate was washed with EtOAc three times. The aqueous layer was concentrated
to give a yellow solid. This yellow solid was dissolved in 1 N NaOH (3.0 ml). Benzyl
chloroformate (410 ␮l, 2.86 mmol) and 1 N NaOH (3.0 ml) were added simultaneously
at 0ЊC. The reaction was stirred at 0ЊC for 0.5 h and at room temperature for 2 h.
The aqueous solution was washed with EtOAc three times and then concentrated to
1
give a white solid 33: H-NMR (300 MHz, D2O) ␦ 7.47 (s, 5H), 5.16 (s, 2H), 3.20
(t, 2H, J ϭ 6.2 Hz), 2.95 (t, 2H, J ϭ 7.4 Hz), 1.77–1.82 (m, 2H), 1.63–1.68 (m, 2H).
To compound 33 (4.67 mmol), CH2Cl2 (27.0 ml), DMF (460 ␮l) and thionyl
chloride (681 ␮l, 9.33 mmol) were added sequentially. The suspension was stirred
at room temperature for 3 h and then concentrated. The residue was eluted through
a small silica gel plug using EtOAc. The eluant was concentrated and the residue
was purified on chromatotron using hexanes/EtOAc (1:1) to give compound 34 (924.2
1
mg, 68% for 3 steps): H-NMR (300 MHz, CDCl3) ␦ 7.35 (bs, 5H), 5.10 (s, 2H),
4.83 (bs, 1H), 3.71 (t, 2H, 7.5 Hz), 3.23–3.29 (m, 2H), 2.02–2.12 (m, 2H), 1.62–1.76
(m, 2H).
2-(6-Bromo-hexyl)-isoindole-1, 3-dione (35) (33). Phthalimide potassium deriva-
tive (11.9473 g, 64.50 mmol) was mixed with 1,6-dibromohexane (31.4734 g, 129.00
mmol) in DMF (150 ml). The reaction was stirred at room temperature for 18 h.
Acetone (150 ml) was added to precipitate to KBr. The white precipitate was filtered
off and the filtrate was concentrated. The residue was purified on a silica gel column
using a gradient solvent system of hexanes/EtOAc (50:1 to 10:1 and finally to 5:1)
to give a white solid 35 (14.0899 g, 70%): 1H-NMR (300 MHz, CDCl3) ␦ 7.83–7.86
(m, 2H), 7.70–7.72 (m, 2H), 3.69 (t, 2H, J ϭ 7.1 Hz), 3.42 (t, 2H, J ϭ 6.7 Hz),
1.83–1.88 (m, 2H), 1.70–1.72 (m, 2H), 1.37–1.52 (m, 4H).

RGD ANALOGS FOR DRUG DELIVERY
367
(6-Chlorosulfonyl-hexyl)-carbamic acid benzyl ester (37) (34,35). Compound 35
(2.5000 g, 8.06 mmol) and Na2SO3 (2.0329 g, 16.13 mmol) were dissolved in H2O
(48.0 ml) and 95% EtOH (30.0 ml). The reaction was stirred at 95ЊC for 20 h and
then concentrated. The residue was dissolved in H2O and the resulted aqueous solution
was washed with EtOAc three times. The aqueous layer was then concentrated to
give a solid. Concentrated HCl (25.4 ml, 306.28 mmol) was added and the reaction
was stirred at 110ЊC for 18 h. The resulted precipitates were filtered off and the
filtrate was washed with EtOAc three times. The aqueous layer was concentrated to
give a yellow solid. This yellow solid was dissolved in 1 N NaOH (16.1 ml, 16.12
mmol). Benzyl chloroformate (2.9 ml, 20.15 mmol) and 1 N NaOH (20.2 ml, 20.15
mmol) were added simultaneously at 0ЊC. The reaction was stirred at 0ЊC for 0.5 h
and at room temperature for 2 h. The aqueous solution was washed with EtOAc three
1
times and the aqueous layer was concentrated to give compound 36: H-NMR (300
MHz, D2O) ␦ 7.47 (s, 5H), 5.15 (s, 2H), 3.14–3.16 (m, 2H), 2.90–2.95 (m, 2H),
1.55–1.75 (m, 2H), 1.35–1.55 (m, 6H).
To compound 36 (4.03 mmol), CH2Cl2 (40.0 ml), DMF (400 ␮l), and thionyl
chloride (588 ␮l, 8.06 mmol) were added sequentially. The suspension was stirred
at room temperature for 5 h and then concentrated. The residue was eluted through
a small silica gel plug using EtOAc. The eluant was concentrated and the residue
was purified on chromatotron using hexanes/EtOAc (1:1) to give compound 37 (491.3
1
mg, 37% for 4 steps): H-NMR (300 MHz, CDCl3) ␦ 7.36 (bs, 5H), 5.10 (s, 2H),
4.70–4.73 (m, 1H), 3.65 (t, 2H, 7.7 Hz), 3.16–3.24 (m, 2H), 1.99–2.04 (m, 2H),
1.39–1.59 (m, 6H).
4-{4-[4-(2-Benzyloxycarbonylamino-2-carboxy-ethyl)-phenoxy]-butyl}-piperidine-
1-carboxylic acid tert-butyl ester (39) (22). NaH (3.6000 g, 60% disperse) was washed
with hexanes four times to give a gray powder NaH (2.5320g, 105.50 mmol). Anhy-
drous DMF (10 ml) was added. The solution of Z-Tyr-OH (8.8307 g, 28.00 mmol)
in DMF (38.0 ml) was added to the suspension of NaH at 0ЊC over 0.5 h. The mixture
was stirred at 0ЊC for 1 h to give a yellow green solution with sticky residue in it.
The solution of 20 (7.7242 g, 28.0 mmol) in DMF (38 ml) was added into the reaction
at 0ЊC over 0.5 h, followed by the addition of KI (0.9290 g, 5.6 mmol). The reaction
mixture was stirred at 0ЊC for 1 h and at 40–45ЊC for 72 h. DMF was removed and
the residue was dissolved in EtOAc. The organic solution was acidified to pH 1 with
1 N HCl. The aqueous layer was extracted with EtOAc twice. The combined organic
layers were washed with brine, dried over MgSO4, and concentrated to give an oil.
The crude product was purified on a silica gel column with a gradient solvent system
of hexanes/EtOAc/HOAc (4:1:0.1 to 2:1:0.1 and finally to 1:1:0.1) to give compound
1
39 (6.2252 g, 53%): H-NMR (300 MHz, CDCl3) ␦ 7.34 (bs, 5H), 7.05 (d, 2H, J ϭ
8.4 Hz), 6.79 (d, 2H, J ϭ 8.5 Hz), 5.19 (bd, 1H), 5.11 (s, 2H), 4.63–4.66 (bd, 1H),
4.04 (bd, 1H), 3.93 (t, 2H, J ϭ 6.2 Hz), 3.08–3.12 (m, 2H), 2.60–2.70 (m, 2H),
1.62–1.77 (m, 4H), 1.45 (s, 9H), 1.04–1.50 (m, 7H).
4-{4-[4-(2-Benzyloxycarbonylamino-2-methoxycarbonyl-ethyl)-phenoxy]-butyl}-
piperidine-1-carboxylic acid tert-butyl ester (40) (22). Compound 39 (8.1610 g, 14.7
mmol) was dissolved in DMF (57 ml). Cs2CO3 (2.3985 g, 7.4 mmol) was added. The
reaction was stirred at room temperature for 1.5 h, followed by the addition of MeI
(2.0898 g, 14.7 mmol). Then the reaction was stirred at room temperature for 18 h.

368
JIANG ET AL.
The solution was concentrated and the residue was dissolved in EtOAc. The organic
solution was washed with saturated NaHCO3 twice and brine once, dried over MgSO4,
and concentrated to give a clear oil. The crude product was eluted through a silica
gel plug with EtOAc. Then the eluant was concentrated to give product 40 (8.3379
1
g, 100%): H-NMR (300 MHz, CDCl3) ␦ 7.34 (bs, 5H), 6.99 (d, 2H, J ϭ 8.2 Hz),
6.78 (d, 2H, J ϭ 8.2 Hz), 5.14–5.19 (m, 1H), 5.10 (s, 2H), 4.60–4.63 (m, 1H),
4.05–4.07 (m, 2H), 3.91 (t, 2H, J ϭ 6.2 Hz), 3.71 (s, 3H), 3.04 (bs, 2H), 2.66 (bt,
2H, J ϭ 12.7 Hz), 1.63–1.77 (m, 4H), 1.45 (s, 9H), 1.10–1.54 (m, 7H).
4-{4-[4-(2-Amino-2-methoxycarbonyl-ethyl)-phenoxy]-butyl}-piperidine-1-
carboxylic acid tert-butyl ester (41). Compound 40 (786.5 mg, 1.38 mmol) was
dissolved in CH3OH (16 ml). To this solution 10% Pd/C (157.3 mg) was added. The
suspension was flushed with Ar for 10 min and was subjected to hydrogen balloon
for 1 h. The catalyst was filtered off and the filtrate was concentrated to give a clear
1
oil 41 (552.7 mg, 92%): H-NMR (300 MHz, CDCl3) ␦ 7.09 (d, 2H, J ϭ 8.3 Hz),
6.83 (d, 2H, J ϭ 8.3 Hz), 4.05–4.09 (m, 2H), 3.93 (t, 2H, J ϭ 6.3 Hz), 3.72 (s, 3H),
3.03 (dd, 1H, J1 ϭ 13.6 Hz, J2 ϭ 5.0 Hz), 2.82 (dd, 1H, J1 ϭ 13.6 Hz, J2 ϭ 7.7
Hz), 2.66 (bt, 2H, J ϭ 12.3 Hz), 1.64–1.78 (m, 4H), 1.45 (s, 9H), 1.06–1.54 (m, 7H).
4-(4-{4-[2-(2-Benzyloxycarbonylamino-ethanesulfonylamino)-2-methoxycarbonyl-
ethyl]-phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (42) (22). Com-
pound 41 (791.9 mg, 1.82 mmol) was dissolved in EtOAc (20 ml), followed by the
addition of NaHCO3 (612.6 mg, 7.29 mmol). The solution of 29 (757.7 mg, 2.74
mmol) in EtOAc (25 ml) was added and the suspension was stirred at 40–45ЊC for
43 h. The resulted precipitates were filtered off. The filtrate was washed with 1 N
HCl twice and brine once, dried over MgSO4, and concentrated. The crude product
was purified on a silica gel column using a gradient solvent system of hexanes/EtOAc
1
(3:1 to 1:1) to give compound 42 (1.0559 g, 86%): H-NMR (300 MHz, CDCl3) ␦
7.34 (bs, 5H), 7.06 (d, 2H, J ϭ 8.4 Hz), 6.82 (d, 2H, J ϭ 8.4 Hz), 5.36–5.38 (m,
1H), 5.08 (s, 2H), 4.92–4.95 (m, 1H), 4.31–4.36 (m, 1H), 4.05–4.08 (m, 2H), 3.91
(t, 2H, J ϭ 6.3 Hz), 3.77 (s, 3H), 3.52 (t, 2H, J ϭ 5.5 Hz), 3.09 (dd, 1H, J1 ϭ 14.0
Hz, J2 ϭ 5.2 Hz), 2.87–3.03 (m, 3H), 2.66 (t, 2H, J ϭ 12.0 Hz), 1.05–1.77 (m, 11H),
1.45 (s, 9H); 13C-NMR (300 MHz, CDCl3) ␦ 172.1, 158.6, 155.0, 136.5, 130.6, 128.6,
128.3, 128.2, 127.2, 114.9, 79.3, 68.0, 67.0, 57.4, 53.5, 52.9, 44.2, 38.6, 36.4, 36.1,
35.9, 32.3, 29.6, 28.6, 23.3; HRMS/FAB m/z (MH+) calcd for C34H49N3O9S 676.3268,
found 676.3229. Anal. calcd for C34H49N3O9S: C, 60.42; H, 7.31; N, 6.22. Found:
C, 60.51; H, 7.18; N, 6.13.
4-(4-{4-[2-(4-Benzyloxycarbonylamino-butane-1-sulfonylamino)-2-methoxycar-
bonylethyl]-phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (43) (22).
Compound 41 (811.1 mg, 1.87 mmol) was dissolved in EtOAc (23 ml), followed by
the addition of NaHCO3 (627.7 mg, 7.47 mmol). The solution of 34 (924.2 mg, 3.02
mmol) in EtOAc (23 ml) was added and the suspension was stirred at 40–45ЊC for
65 h. The resulted precipitates were filtered off. The filtrate was washed with 1 N
HCl twice and brine once, dried over MgSO4, and concentrated. The crude product
was purified on a silica gel column using a gradient solvent system of hexanes/EtOAc
1
(3:1 to 1:1) to give compound 43 (766.4 mg, 58%): H-NMR (300 MHz, CDCl3) ␦
7.34 (bs, 5H), 7.07 (d, 2H, J ϭ 8.2 Hz), 6.82 (d, 2H, J ϭ 8.3 Hz), 5.08 (s, 2H), 4.97
(d, 2H, J ϭ 9.2 Hz), 4.29–4.31 (m, 1H), 4.06–4.08 (m, 2H), 3.91 (t, 2H, J ϭ 6.2

RGD ANALOGS FOR DRUG DELIVERY
369
Hz), 3.75 (s 3H), 3.04–3.14 (m, 3H), 2.93 (dd, 1H, J1 ϭ 14.0 Hz, J2 ϭ 7.4 Hz),
2.62–2.77 (m, 5H), 1.59–1.77 (m, 8H), 1.45 (s, 9H), 1.06–1.59 (m, 7H); 13C-NMR
(300 MHz, CDCl3) ␦ 172.4, 158.6, 156.6, 155.1, 136.7, 130.7, 128.7, 128.3, 127.4,
114.9, 79.4, 68.1, 66.8, 57.5, 53.3, 52.9, 44.5, 40.4, 38.8, 36.4, 36.1, 32.3, 29.6, 28.7,
23.3, 20.9; HRMS/FAB m/z (MH+) calcd for C36H53N3O9S 704.3581, found 704.3610.
Anal. calcd for C36H53N3O9S: C, 61.43; H, 7.59; N, 5.97. Found: C, 61.00; H, 7.37;
N, 5.84.
4-(4-{4-[2-(6-Benzyloxycarbonylamino-hexane-1-sulfonylamino)-2-carboxy-
ethyl]-phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (44). Compound
41 (524.1 mg, 1.21 mmol) was dissolved in EtOAc (10 ml), followed by the addition
of Na2CO3 (511.8 mg, 4.83 mmol). The solution of 37 (491.3 mg, 1.47 mmol) in
EtOAc (20 ml) was added and the suspension was stirred at 40–45 ЊC for 72 h. The
resulted precipitates were filtered off. The filtrate was washed with 1 N HCl twice
and brine once, dried over MgSO4, and concentrated. The crude product was purified
on a silica gel column using a gradient solvent system of hexanes/EtOAc (3:1 to 1:1)
1
to give compound 44 (528.6 mg, 60%): H-NMR (300 MHz, CDCl3) ␦ 7.35 (brs,
5H), 7.06 (d, 2H, J ϭ 8.4 Hz), 6.82 (d, 2H, J ϭ 8.4 Hz), 5.09 (s, 2H), 4.77–4.79
(m, 1H), 4.73 (d, 1H, J ϭ 9.1 Hz), 4.31–4.35 (m, 1H), 3.92 (t, 2H, J ϭ 6.3 Hz),
3.76 (s, 3H), 3.14–3.20 (m, 2H), 3.08 (dd, 1H, J1 ϭ 13.9 Hz, J2 ϭ 5.1 Hz), 2.95
(dd, 1H, J1 ϭ 14.0 Hz, J2 ϭ 7.1 Hz), 2.62–2.78 (m, 4H), 1.06–1.77 (m, 19H), 1.45
(s, 9H), 13C-NMR (300 MHz, CDCl3) ␦ 172.3, 158.7, 156.6, 155.1, 136.8, 130.7,
128.7, 128.3, 127.3, 115.0, 79.4, 68.1, 66.8, 57.4, 53.9, 52.9, 44.3, 41.0, 39.0, 36.5,
36.2, 32.4, 29.8, 29.7, 28.7, 28.0, 26.3, 23.5, 23.4; HRMS/FAB m/z (MH+) calcd for
C38H57N3O9S 732.3894, found 732.3888. Anal. calcd for C38H57N3O9S: C, 62.36; H,
7.85; N, 5.74. Found: C, 62.34; H, 7.78; N, 5.57.
4-(4-{4-[2-(2-Benzyloxycarbonylamino-ethanesulfonylamino)-2-carboxy-ethyl]-
phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (45). Compound 42
(77.4 mg, 0.12 mmol) and LiOH (13.7 mg, 0.57 mmol) were dissolved in 3 ml of
THF/CH3OH/H2O (1:1:1). The reaction was stirred at room temperature for 18 h and
then concentrated. The residue was acidified with 1 N HCl and the aqueous solution
was extracted with EtOAc three times. The combined organic layers were washed
with brine once, dried over MgSO4, and concentrated. The crude product was purified
on a silica gel column with a gradient solvent system of hexanes/EtOAc/HOAc
(2:1:0.1 to 1:1:0.1) to give compound 45 (65.7 mg, 86%): 1H-NMR (300 MHz, CDCl3)
␦ 7.34 (brs, 5H), 7.12 (d, 2H, J ϭ 8.1 Hz), 6.81 (d, 2H, J ϭ 8.1 Hz), 5.43–5.47 (m,
1H), 5.13–5.15 (m, 1H), 5.08 (s, 2H), 4.34–4.41 (m, 1H), 3.99–4.06 (m, 2H), 3.92
(t, 2H, J ϭ 6.2 Hz), 3.45–3.56 (m, 2H), 3.13 (dd, 1H, J1 ϭ 14.1 Hz, J2 ϭ 5.1 Hz),
2.93–3.08 (m, 3H), 2.60–2.70 (m, 2H), 1.61–1.78 (m, 4H), 1.45 (s, 9H), 0.98–1.51
(m, 7H); 13C-NMR (300 MHz, CDCl3) ␦ 174.4, 158.6, 156.8, 155.3, 148.0, 138.6,
136.4, 130.8, 128.7, 128.4. 128.3, 127.5, 114.9, 79.8, 68.0, 67.3, 57.4, 53.3, 44.3,
38.4, 36.3, 36.1, 32.3, 29.6, 28.7, 23.3; HRMS/FAB m/z (MH+) calcd for C33H47N3O9S
662.3111, found 662.3135.
4-(4-{4-[2-(4-Benzyloxycarbonylamino-butane-1-sulfonylamino)-2-carboxy-
ethyl]-phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (46). Compound
43 (684.4 mg, 0.97 mmol) and LiOH (116.53 mg, 4.87 mmol) were dissolved in 24
ml of THF/CH3OH/H2O (1:1:1). The reaction was stirred at room temperature for 18

370
JIANG ET AL.
h and then concentrated. The residue was acidified with 1 N HCl and the aqueous
solution was extracted with EtOAc three times. The combined organic layers were
washed with brine once, dried over MgSO4, and concentrated. The crude product was
purified on a silica gel column with a gradient solvent system of hexanes/EtOAc/
1
HOAc (2:1:0.1 to 1:1:0.1) to give compound 46 (626.7 mg, 93%): H-NMR (300
MHz, CDCl3) ␦ 7.33 (brs, 5H), 7.13 (d, 2H, J ϭ 7.8 Hz), 6.79 (d, 2H, J ϭ 7.5 Hz),
5.15–5.30 (m, 2H), 5.08 (s, 2H), 4.31–4.32 (m, 1H), 4.04–4.07(m, 2H), 3.89 (bs,
2H), 3.09–3.10 (m, 3H), 2.93–2.96 (m, 1H), 2.62–2.75 (m, 4H), 1.04–1.73 (m, 15H),
1.45 (s, 9H); 13C-NMR (300 MHz, CDCl3) ␦ 174.5, 158.5, 156.9, 155.3, 136.6, 130.9,
128.7, 128.3, 127.8, 114.9, 79.7, 68.0, 67.1, 57.4, 53.2, 44.3, 40.5, 38.5, 36.3, 36.1,
32.3, 29.6, 28.7, 23.3, 20.8; HRMS/FAB m/z (MH+) calcd for C35H51N3O9S 690.3424,
found 690.3447. Anal. calcd for C35H51N3O9S: C, 60.94; H, 7.45; N, 6.09. Found:
C, 59.87; H, 7.55; N, 5.66.
4-(4-{4-[2-(6-Benzyloxycarbonylamino-hexane-1-sulfonylamino)-2-carboxy-
ethyl]-phenoxy}-butyl)-piperidine-1-carboxylic acid tert-butyl ester (47). Compound
44 (420.8 mg, 0.57 mmol) and LiOH (68.9 mg, 2.88 mmol) were dissolved in 14.1
ml of THF/CH3OH/H2O (1:1:1). The reaction was stirred at room temperature for 18
h and then concentrated. The residue was acidified with 1 N HCl and the aqueous
solution was extracted with EtOAc three times. The combined organic solutions were
washed with brine once, dried over MgSO4, and concentrated. The crude product was
purified on a silica gel column with a gradient solvent system of hexanes/EtOAc/
1
HOAc (2:1:0.1 to 1:1:0.1) to give compound 47 (358.0 mg, 87%): H-NMR (300
Mhz, CDCl3) ␦ 7.33 (bs, 5H), 7.13 (d, 2H, J ϭ 6.9 Hz), 6.79 (d, 2H, J ϭ 7.5 Hz),
5.14–5.43 (m, 2H), 5.08 (s, 2H), 4.26–4.27 (m, 1H), 4.04–4.07 (m, 2H), 3.89 (bs,
2H), 3.12 (bs, 3H), 2.89–2.90 (m, 1H), 2.62–2.70 (m, 4H), 1.04–1.72 (m, 19H), 1.45
(s, 9H); 13C-NMR (330 MHz, CDCl3) ␦ 176.3, 158.3, 156.9, 155.2, 136.6, 130.7
128.6, 128.1, 114.7, 79.7, 67.9, 66.8, 57.6, 53.6, 44.2, 40.9, 38.4, 36.2, 35.9, 32.2,
29.5, 28.5, 27.6, 26.0, 23.2, 23.0, HRMS/FAB m/z (MH+) calcd for C37H55N3O9S
718.3737, found 718.3726.
2-(2- Benzyloxycarbonylamino-ethanesulfonylamino)-3- [4-(4-piperidin- 4-yl-
butoxy)-phenyl]-propionic acid (6). Compound 45 (300.0 mg, 0.45 mmol) was dis-
solved in CH2Cl2 (6.7 ml). TFA (2.2 ml, 29.03 mmol) in CH2Cl2 (6.7 mL) was added
at 0ЊC. The reaction was stirred at 0 ЊC for 10 min and at room temperature for 1 h.
The solution was concentrated and dried on oil pump to give compound 6 as a TFA
1
salt (357.2 mg, 140%): H-NMR (300 MHz, CD3OD) ␦ 7.32 (brs, 5H), 7.17 (d, 2H,
J ϭ 8.1 Hz), 6.83 (d, 2H, J ϭ 8.1 Hz), 5.09 (s, 2H), 4.17–4.21 (m, 1H), 3.91 (t, 2H,
J ϭ 5.8 Hz), 3.32 (bd, 4H), 3.11 (dd, 1H, J1 ϭ 13.2 Hz, J2 ϭ 3.9 Hz), 2.79–2.93
(m, 5H), 1.85–1.90 (m, 2H), 1.69–1.73 (m, 2H), 1.42–1.51 (m, 3H), 1.30–1.39 (m,
4H); 13C-NMR (300 MHz, CD3OD) ␦ 175.0, 159.7, 158.6, 138.3, 131.8, 130.2, 129.6,
129.2, 128.9, 115.7, 68.9, 67.7, 59.3, 53.7, 45.4, 39.2, 36.8, 34.8, 30.4, 30.1, 24.0;
HRMS/FAB m/z (MH+) calcd for C28H39N3O7S 562.2584, found 562.2612.
2-(4-Benzyloxycarbonylamino-butane-1-sulfonylamino)-3-[4-(4-piperidin-4-yl-
butoxy)-phenyl]-propionic acid (7). Compound 46 (100.0 mg, 0.15 mmol) was dis-
solved in CH2Cl2 (2.1 ml). TFA (715 ␮l, 9.28 mmol) in CH2Cl2 (2.2 ml) was added
at 0ЊC. The reaction was stirred at 0ЊC for 10 min and at room temperature for 1 h.
The solution was concentrated and dried on oil pump to give compound 7 as a TFA

RGD ANALOGS FOR DRUG DELIVERY
371
1
salt: H-NMR (300 MHz, CD3OD) ␦ 7.28–7.34 (m, 5H), 7.19 (d, 2H, J ϭ 9.0 Hz),
6.85 (d, 2H, J ϭ 8.1 Hz), 5.06 (s, 2H), 4.13 (dd, 1H, J1 ϭ 9.6 Hz, J2 ϭ 4.5 Hz),
3.94 (t, 2H, J ϭ 5.9 Hz), 3.31–3.36 (m, 2H), 3.13 (dd, 1H, J1 ϭ 13.8 Hz, J2 ϭ 5.1
Hz), 3.03 (t, 2H, J ϭ 6.2 Hz), 2.87–2.96 (m, 2H), 2.78–2.82 (m, 1H), 2.64–2.67 (m,
2H), 1.88–1.93 (m, 2H), 1.72–1.76 (m, 2H), 1.47–1.57 (m, 5H), 1.32–1.45 (m, 6H);
13C-NMR (300 MHz, CD3OD) ␦ 175.2, 159.7, 159.0, 139.0, 131.8, 130.5, 129.6,
129.1, 128.8, 115.7, 68.9, 67.5, 59.5, 54.1, 45.5, 41.3, 39.2, 36.9, 34.9, 30.5, 30.1, 29.6,
24.1, 22.0; HRMS/FAB m/z (MH+) calcd for C30H43N3O7S 590.2900, found 590.2914.
2-(6-Benzyloxycarbonylamino-hexane-1-sulfonylamino)-3-[4-(4-piperidin-4-yl-
butoxy)-phenyl]-propionic acid (8). Compound 47 (78.0 mg, 0.11 mmol) was dis-
solved in CH2Cl2 (1.6 ml). TFA (536 ␮l, 6.96 mmol) in CH2Cl2 (1.6 ml) was added
at 0ЊC. The reaction was stirred at 0ЊC for 10 min and at room temperature for 1 h.
The solution was concentrated and dried on oil pump to give compound 8 as a TFA
1
salt: H-NMR (300 MHz, CD3OD) ␦ 7.28–7.35 (m, 5H), 7.18 (d, 2H, J ϭ 8.7 Hz),
6.85 (d, 2H, J ϭ 8.7 Hz), 5.07 (s, 2H), 4.11 (dd, 1H, J1 ϭ 9.6 Hz, J2 ϭ 4.5 Hz),
3.94 (t, 2H, J ϭ 6.2 Hz), 3.32–3.37 (bd, 2H), 3.07–3.15 (m, 3H), 2.89–2.97 (m,
3H), 2.89–2.97 (m, 2H), 2.77 (dd, 1H, J1 ϭ 13.8 Hz, J2 ϭ 10.2 Hz), 2.62 (t, 2H,
J ϭ 7.4 Hz), 1.89–1.99 (m, 2H), 1.71–1.80 (m, 2H), 1.33–1.59 (m, 11H), 1.20–1.30
(m, 4H); 13C-NMR (300 MHz, CD3OD) ␦ 159.7, 159.0, 138.6, 131.8, 130.6, 129.6,
129.1, 128.9, 115.7, 68.9, 67.4, 59.8, 54.4, 45.4, 41.8, 39.1, 36.9, 34.9, 30.5, 30.2,
29.0, 27.4, 24.5, 24.1; HRMS/FAB m/z (MH+) calcd for C32H47N3O7S 618.3213,
found 618.3248.
2-(2-Amino-esthanesulfonylamino)-3-[4-(4-piperidin-4-yl-butoxy)-phenyl]-
propionic acid (9). Compound 6 (127.3 mg, 0.23 mmol) was dissolved in CH3OH
(2.5 ml) and 3 drops of HOAc were added. Under Ar 10% Pd/C (25.5 mg) was added.
The black suspension was subjected to H2 balloon at room temperature for 1 h. The
catalyst was filtered off and the filtrate was concentrated to give compound 9 as an
acetic acid salt (111.3 mg, 115%): 1H-NMR (300 MHz, CD3OD) ␦ 7.19 (d, 2H, J ϭ
8.1 Hz), 6.86 (d, 2H, J ϭ 8.7 Hz), 4.23–4.26 (m, 1H), 3.96 (t, 2H, J ϭ 6.3 Hz),
3.31–3.38 (m, 2H), 3.12–3.22 (m, 5H), 2.84–2.98 (m, 3H), 2.02 (s, HOAc), 1.91–2.00
(m, 2H), 1.72–1.81 (m, 2H), 1.47–1.60 (m, 3H), 1.28–1.44 (m, 4H); 13C-NMR (300
MHz, CD3OD) ␦ 175.4, 159.7, 131.7, 130.0, 115.7, 68.9, 50.9, 45.4, 39.0, 36.8, 35.6,
34.9, 30.5, 30.1, 24.0; HRMS/FAB m/z (MH+) calcd for C20H33N3O5S 428.2219,
found 428.2234.
2-(4-Amino-butane-1-sulfonylamino)-3-[4-(4-piperidin-4-yl-butoxy)-phenyl]-
propionic acid (10). Compound 7 (85.5 mg, 0.15 mmol) was dissolved in CH3OH
(1.6 ml). Under Ar 10% Pd/C (17.1 mg) was added and the black suspension was
flushed with Ar for 10 min. Then the reaction was subjected to H2 balloon at room
temperature for 1 h. The catalyst was filtered off and the filtrate was concentrated to
give compound 10 as a TFA salt (63.8 mg, 97% for two steps): 1H-NMR (300 MHz,
CD3OD) ␦ 7.19 (d, 2H, J ϭ 7.5 Hz), 6.82 (d, 2H, J ϭ 7.2 Hz), 3.93–3.97 (m, 3H),
3.30–3.35 (m, 2H), 3.03–3.07 (m, 1H), 2.67–2.95 (m, 7H), 1.89–1.94 (m, 2H),
1.67–1.77 (m, 6H), 1.49–1.60 (m, 3H), 1.28–1.44 (m, 4H); 13C-NMR (300 MHz,
CD3OD) ␦ 178.0, 159.4, 131.9, 131.3, 115.5, 68.9, 61.3, 52.7, 45.4, 40.0, 39.9, 36.9,
34.9, 30.5, 30.2, 27.1, 24.0, 21.7; HRMS/FAB m/z (MH+) calcd for C22H37N3O5S
456.2532, found 456.2545.

372
JIANG ET AL.
2-(6-Amino-hexane-1-sulfonylamino)-3-[4-(4-piperidin-4-yl-butoxy)-phenyl]-
propionic acid (11). Compound 8 (85.8 mg, 0.14 mmol) was dissolved in CH3OH
(1.6 ml). Under Ar 10% Pd/C (34.2 mg) was added, followed by the addition of two
drops of HOAc. The black suspension was flushed with Ar for 10 min and then
subjected to H2 balloon at room temperature for 1.5 h. The catalyst was filtered off
and the filtrate was concentrated to give compound 11 as an acetic acid salt (68.7
1
mg, 97% for two steps): H-NMR (300 MHz, CD3OD) ␦ 7.19 (d, 2H, J ϭ 8.1 Hz),
6.83 (d, 2H, J ϭ 8.1 Hz), 3.93–4.01 (m, 3H), 3.30–3.37 (m, 2H), 3.05–3.08 (m,
1H), 2.67–2.97 (m, 7H), 1.99 (s, HOAc), 1.90–1.94 (m, 2H), 1.71–1.80 (m, 2H),
1.44–1.62 (m, 6H), 1.28–1.42 (m, 9H); 13C-NMR (300 MHz, CD3OD) ␦ 159.5, 131.9,
131.1, 115.6, 68.9, 53.7, 45.4, 40.7, 40.0, 36.9, 34.9, 30.5, 30.1, 28.6, 28.2, 26.8,
24.2, 24.0; HRMS/FAB m/z (MH+) calcd for C24H41N3O5S 484.2845, found 484.2851.
RESULTS AND DISCUSSION
The design. As stated earlier, there have been many literature reports of the develop-
ment of RGD analogs as potential GP IIb/IIIa antagonists. One prominent example
is MK-383 (the active ingredient of Tirofiban, Fig. 1), which has been approved by
the U.S. Food and Drug Administration for the treatment of myocardial infarction
and unstable angina pectoris. This RGD analog offers a good lead for the development
of drug delivery vectors to target GP IIb/IIIa. The modification of MK-383 to suit
the need of targeted drug delivery requires the incorporation of a functional group
which (1) can allow for the tethering of a pharmaceutical reagent and (2) would not
affect the binding affinity to the GP IIb/IIIa receptor. For the tethering of a pharmaceuti-
cal reagent to an RGD analog, it is desirable to have a functional group that can be
easily conjugated to a handle on the pharmaceutical agent to be delivered. A carboxyl
group should allow for the tethering of compounds with an amino or hydroxyl group
through amide or ester formation, respectively. On the other hand, an amino group
should allow for the tethering of a compound with a carboxyl group through amide
bond formation. It is known that the terminal carboxyl and amino groups of an RGD
analog are essential for their affinity (22). Modification of these two ionizable function
groups would most likely lead to the abolishment of the biological activity. Therefore,
an additional carboxyl or amino group needs to be introduced for the tethering of the
pharmaceutical agents to be delivered. By examining the structure of MK-383, the
side chain sulfonamide moiety appears to be the only site that could be modified
without reducing its affinity for GP IIb/IIIa. Based on this consideration, we have
designed a series of 10 modified MK-383 analogs (Fig. 2) that have side chains
containing ester, amide, amine, protected amine, and poly(ethylene glycol). These
analogs will help us to examine the effect of side-chain subunits with different
FIG. 1. MK-383 (Tirofiban), 1, IC50 ϭ 11 nM.

RGD ANALOGS FOR DRUG DELIVERY
373
FIG. 2. RGD analogs designed.
structural features on their affinity for GP IIb/IIIa. The comparison of compounds 2
and 3 will allow us to examine the effect of an additional positive charge (a protonated
tertiary amine). The comparison of 3 and 4 will allow us to examine the effect of
increased molecular weight since 4 is the dimer of 3. Compound 5 has a poly(ethylene
glycol) unit bridging two RGD analog molecules. The synthesis of this molecule will
allow us to examine the effect of tethering a macromolecule to such a RGD analog
on its affinity to GP IIb/IIIa. The series of compounds 6, 7, and 8 allow for the
examination of the effect of side chain length on their binding affinities. The series
of 9, 10, and 11 serves the same purpose. By comparing the series containing 6, 7,
and 8 and the series containing 9, 10, and 11, we would be able to examine the effect
of masking the positive charge of the protonated amine on the side chain.
Synthesis. The synthesis of the RGD analog backbone started with the alkylation of
4-methylpyridine (12) followed by hydrogenation to give 13 using modified literature
procedures (Scheme 1) (23,24). Different catalysts were examined in order to reduce
the pyridine ring of 13 to a piperidine ring (14). Among them (Pd/C, Pt/C, and PtO2)
only PtO2 worked very well giving 100% yield. Then the secondary amine of 14 was
protected with a Z group using benzyl chloroformate to give compound 15. Reactions
using NaH/DMF as base to couple L-tyrosine and alkyl bromide/chloride have been
reported without racemization (23,24). Using the same conditions, compound 16 was
obtained in 82% yield. The carboxylic acid of 16 was protected as a benzyl ester
(17). After removal of the Boc group, the free amine was coupled to commercially
available 4-(chlorosulfonyl) benzoic acid to give 18 in 49% yield (Scheme 1). In this
reaction a mixed solvent system (CH3CN/H2O) was used in order to get a homogeneous
reaction to improve the yield. Compound 18 was further coupled with n-butyl amine
to give compound 19. Then both protecting groups of compound 19 were cleaved
by hydrogenation to give RGD analog 2. Similar procedures were followed for the
synthesis of RGD analogs 3, 4, and 5 (Scheme 2).
The synthesis of RGD analogs with different alkyl chain linkers (6–11) followed
essentially the same routes (Schemes 3 and 4). The synthesis of the side chain linkers
29, 34, and 37 are illustrated in Scheme 3. Starting with taurine (27), the primary
amino group was first protected with a Z group. This was followed by treatment with

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JIANG ET AL.
SCHEME 1. Synthetic route for RGD analog 2. Conditions: (a)(I) n-BuLi, Ϫ78 ЊC; (II) 40–45 ЊC,
2 h; (III) Br(CH2)3Cl, Ϫ65 ЊC-r.t.; 92%; (b) PtO2, H2, HOAc, 60 psi, 100%; (c) (I) aq. K2CO3; (II) benzyl
chloroformate, pyridine, CH3CN, 87%; (d) N-Boc-L-tyrosine, NaH, DMF, Kl, 82%; (e) benzyl alcohol,
DCC, DMAP, 80%; (f) (I) TFA, CH2Cl2; (II) 4-(chlorosulfonyl) benzoic acid, K2CO3, CH3CN, H2O,
49%; (g) n-butylamine, EDC, HOBT, DMAP, CH2Cl2, 99%; (h) Pd/C, H2, 100%.
triphosgene to give the sulfonyl chloride of the two-carbon linker 29 in 75%. The
synthesis of the four-carbon linker started with the reaction of potassium phthalimide
with sulfonate 31 to give 32. Then the phthalimide protecting group was removed
and replaced with a Z group because of its mild cleavage conditions. Attempts were
made without success to convert 33 to its corresponding sulfonyl chloride (34) using
triphosgene, which worked well with the synthesis of two-carbon linker 29. Then we
studied the reactions using thionyl chloride under various conditions and found that
the best yield was obtained under dilute conditions (2 eq of thionyl chloride in CH2Cl2)
at room temperature giving compound 34 in three steps with 68% overall yield.
Similar strategy was followed for the synthesis of the six-carbon linker 37. First,
potassium phthalimide was reacted with 1,6-dibromohexane to give 35. Then the
phthalimide protecting group was removed and replaced with a Z group to give 36,
which was converted to the corresponding sulfonyl chloride 37 by reacting with
thionyl chloride.
The synthesis of the key intermediate 41 for RGD analogs 6 to 11 was similar to
that of compound 22. As shown in Scheme 5, 41 was obtained in 66% yield starting
from 38. This key intermediate was coupled with the respective side chain linkers
(29, 34, and 37) using NaHCO3 or Na2CO3 in EtOAc to give compounds 42–44. The
yields ranged from 58 to 86%, depending on the activity of different sulfonyl chloride.
It was found that a longer carbon chain between the amino group and the sulfonyl
group resulted in a diminished reactivity of the corresponding sulfonyl chloride. Then
the methyl group, Z group and Boc group were cleaved using standard conditions
(Scheme 5) to give the desired RGD analogs 6 to 11.

RGD ANALOGS FOR DRUG DELIVERY
375
SCHEME 2. Synthesis of RGD analogs 3–5. Conditions: (a) (I) aq. K2CO3; (II) Boc2O, CH2Cl2,
80%; (b) NaH, Kl, DMF, 72%; (c) 10% Pd/C, H2, 96%; (d) 4-(chlorosulfonyl)benzoic acid, K2CO3,
CH3CN, H2O, 55%; (e) DCC, HOBt, DMAP, 72%; (f) 40% TFA; (g) 1,4-bis(3-aminopropyl)piperazine,
DCC, HOBt, DMAP, 72%; (h) 40% TFA, 98%; (i) EDC, HOBt, DMAP, poly(ethylene glycol) (average
MW ca. 300), 82%; (j) H2/Pd-C, 95%.
BIOLOGICAL EVALUATION
Platelet aggregation assays to determine the binding affinity of the modified RGD
analogs (2–11) toward GPIIb/IIIa were conducted using human volunteer blood sam-
ples (25). Platelets were activated with the addition ADP at 20 ␮M concentration.
The concentration of the RGD analog that inhibited 50% of the platelet aggregation
was signified as IC50. The IC50s of the modified RGD analogs are listed in Table 1.
The only structural difference between compounds 2 (210 nM) and 3 (87 nM) is the
added amino group in 3, and yet compound 3 showed much higher affinity toward
GPIIb/IIIa. This indicates that an ionizable amino group on the side chain helps to
improve the binding affinity. Compounds 3 and 4 were designed to examine the effect
of increased molecular weight. The IC50 of analog 4 (37 nM) was less than half of
analog 3 (87 nM), as expected due to the doubling of the molecular weight and the
number of RGD units in 4. This indicates that increasing the molecular weight of such
RGD analogs does not affect their binding to GPIIb/IIIa adversely. The conjugation of

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JIANG ET AL.
SCHEME 3. Synthesis of side chain linkers 29, 34, and 37. Conditions: (f) 1,6-dibromohexane,
DMF, 70%; (g) (I) Na2SO3, EtOH, H2O; (II) conc. HCl; (III) 1 N NaOH, benzyl chloroformate; (h)
SOCl2, CH2Cl2, DMF, 37% for 4 steps.
SCHEME 4. Synthesis of RGD analogs 6–11. Conditions: (a) NaH, Kl, DMF, 20, 72%; (b) (I)
Cs2CO3, DMF; (II) Mel, DMF, 100%; (c) H2, Pd/C, CH3OH, 92%; (d) 29, 34 or 37, NaHCO3 or Na2CO3,
EtOAc, 45 ЊC, 58–86%; (e) LiOH, THF, CH3OH, H2O, 86–93%; (f) 25% TFA, CH2Cl2, DCM; (g) H2,
Pd/C, CH3OH, 100% for two steps.

RGD ANALOGS FOR DRUG DELIVERY
377
TABLE 1
Inhibition of Platelet Aggregation (IC50)
Compound
IC50 (nM)
2
3
210
87
4
37
5
6
7
8
9
10
11
150
180
180
300
100
150
50
a polymer such as poly(ethylene glycol) to the side chain of RGD analog (5) did not
significantly affect the potency either (IC50: 150 nM). This indicates the feasibility
of tethering a fairly large pharmaceutical agent to an RGD analog for delivery purposes
without sacrificing its high affinity for the target receptor. However, it should be
noted that the dimeric RGD analog 5 with a polar poly(ethylene glycol) chain as a
linker does not bind to GPIIb/IIIa as well as the analogous compound (4) with an
alkylamine linker. This further indicates that an ionizable amino group on the side
chain helps to improve the binding affinity. The side chain length also seemed to
affect the affinity of these RGD analogs to GP IIb/IIIa to a degree. For example, with
compounds 6–8, the IC50 ranged from 180 to 300 nM. With compounds 9–11, the
IC50 ranged from 50 to 150 nM. The conversion of the free amines of compounds
9–11 to their protected form 6–8 did result in a noticeable effect on their binding
affinity, again indicating the beneficial effect of the side chain amino group. The most
significant is the comparison of 11 (IC50: 50 nM) with 8 (IC50: 300 nM). However,
it is more important to note that all compounds showed sub-␮M inhibitions of the
GP IIb/IIIa binding. This indicates that in general RGD analog MK-383 tolerates side
chain modification fairly well with various functional groups attached such as amide,
amine, ester, protected amine and poly(ethylene glycol). Such information is very
important for the further design of modified RGD analogs as vector for the delivery
of pharmaceutical reagent.
CONCLUSIONS
A series of RGD analogs have been synthesized as potential delivery vectors of
pharmaceutical agents. Their affinity for the target GPIIb/IIIa receptor has also been
evaluated using platelet aggregation studies. It was found that such RGD analogs
could tolerate side chain modifications fairly well with various functional groups
attached such as amide, amine, ester, protected amine, and poly(ethylene glycol). The
fact that side chain modification with poly(ethylene glycol) was tolerated well (IC50:
150 nM) suggests the feasibility of tethering fairly large pharmaceutical agents to
such RGD analogs without significant sacrifice of their affinity to the intended receptor.

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ACKNOWLEDGMENTS
Financial support from the American Heart Association (#9740117N) and the North Carolina Biotechnol-
ogy Center (98005-ARG-0008) is gratefully acknowledged. Wei Wang acknowledges a Glaxo graduate
fellowship.
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