An image contrast agent selectively activated by prostate specific antigen

Article abstract of DOI:10.1016/j.bmc.2005.08.015

A family of image contrast agent conjugates designed to undergo enzymatic activation has been synthesized. The agents underwent activation both with enzymatically active prostate specific antigen (PSA) and α-chymotrypsin, releasing free fluorophore via cl

Full text of DOI:10.1016/j.bmc.2005.08.015

Bioorganic & Medicinal Chemistry 14 (2006) 418–425
An image contrast agent selectively activated by prostate
specific antigen
Graham B. Jones,a,* Curtis F. Crasto,a Jude E. Mathews,a Longfei Xie,a
Miguel O. Mitchell,a Ahmed El-Shafey,a Anthony V. DÕAmicob and Glenn J. Bubleyc
aBioorganic and Medicinal Chemistry Laboratories, Department of Chemistry and Chemical Biology, Northeastern University,
Boston, MA 02115, USA
bDana Farber Cancer Institute and Brigham and Womens Hospital, Harvard Medical School, Boston, MA 02215, USA
cBeth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
Received 5 July 2005; revised 2 August 2005; accepted 9 August 2005
Available online 26 September 2005
Abstract—A family of image contrast agent conjugates designed to undergo enzymatic activation has been synthesized. The agents
underwent activation both with enzymatically active prostate specific antigen (PSA) and a-chymotrypsin, releasing free fluorophore
via cleavage of a three-component system. A hexapeptide derivative showed exclusive activation by PSA and constitutes a method
for tracking PSA activity in vitro.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tease; however, PSA in serum (but not in the prostatic
microenvironment) is rapidly inactivated by binding to
serum proteins.7 An attractive possibility, therefore,
would be the design of an imaging system, which ex-
ploits the enzymatic efficiency of PSA in the prostatic
microenvironment. Our strategy was to conjugate a pro-
teinogenic PSA substrate to a masked fluorophore via
an inert spacer/linker group, such that the free fluores-
cent molecule is liberated on proteolysis (Scheme 1).8
Despite improvements in local therapy and increased
awareness, prostate cancer continues to be second only
to lung cancer as a cause for cancer deaths in men.1 Pri-
or investigations show that the presence of prostatecto-
my Gleason grade P4 in the radical prostatectomy
specimen is the most important predictor of progression
following surgery.2 Unfortunately, the transrectal ultra-
sound guided sextant sampling of the prostate is subject
to sampling error, and therefore biopsy Gleason grade
will underestimate prostatectomy Gleason grade 4 or 5
disease in as many as 40% of men with clinically local-
ized disease.3 Therefore, an imaging method capable of
identifying Gleason grade P4 disease within the pros-
tate gland could provide the basis for patient selection
for more aggressive initial therapeutic approaches.4 A
number of image contrast enhancing agents have been
studied for use in conjunction with ultrasound methods
of detection.5 However, immunohistochemical studies
have also shown that Gleason grade bears an inverse
correlation with the concentration of enzymatically ac-
tive prostate specific antigen (PSA).6 PSA is a serine pro-
1.1. Choice of linker
Our preferred choice for the inert linker was the p-amino-
benzyl alcohol pioneered by Katzenellenbogen and co-
workers.9 This allows coupling of peptide based enzyme
substrates through the N terminus, with the alcohol group
incorporated into a carbamate which masks the amino
containing molecule targeted for delivery. Selective enzy-
matic hydrolysis of the amide group results in (a) genera-
tion of an exomethylene iminium ion which is then
captured by water to regenerate the free linker (b) con-
comitant expulsion of CO2, rendering the reactions essen-
tially irreversible and (c) expulsion of the free amine as
shown in Scheme 2.9 The key is to harness amino contain-
ing substrates in the system where differences in the chem-
istry between the carbamate and amino form are
pronounced, and we have previously employed this
method for enzyme mediated cytotoxin release.10
Keywords: Enzyme substrates; PSA; Linker; Imaging agents; Chemical
synthesis; Prodrugs; Coumarin; Hexapeptides.
*
Corresponding author. Tel.: +1 61 73 73 86 19; fax: +1 61 73 73 87
95; e-mail: gr.jones@neu.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.015

G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
419
PSA
Cleavage Site
spontaneous
release
LINKER
peptide sequence
(recognized by PSA)
LINKER
fluorophore
LINKER
fluorophore
fluorophore
Scheme 1. Three component system for PSA activated image contrast agent.
inert
fluorophore
O
O
R
R
PSA or
R
O
CO2H
O
N
H
O
N
H
+
R
LNCaP
sonicates
NH2
H2N
N
H
NH2
free
fluorophore
PABA
CO2
H2N R
Scheme 2. PSA mediated release of Tyr-linker conjugates.
Anilino containing fluorophores were deemed excellent
substrates for this system, as discernible differences in
UV and fluorescence characteristics would be expected
for the free aniline as opposed to the linked carbamate
form.
gates, such derivatives would also be substrates of a-
chymotrypsin, an important and well-studied enzyme.8
Accordingly, a dummy substrate 4 was prepared to as-
sess proof-of-principle for the release methodology and
to perfect coupling chemistry (Scheme 3). Boc tyrosine
was converted to amide 2 and then the carbamate pre-
cursor was assembled by preparation of the 4-nitrophen-
yl carbonate 3. Nucleophilic displacement gave only
moderate yields of the Boc carbamate when using close
stoichiometry (36% with 4:1 ratio), although near quan-
titative yields could be obtained when using large excess-
es of amine (>10 equiv). With the substrate in hand,
enzymatic release was tracked using both a-chymotrypsin
1.2. Choice of enzyme substrate
Though a number of high-affinity peptide substrates for
PSA have been identified, we initially wished to provide
proof-of-principle with a minimal substrate and selected
tyrosine conjugates for examination. In addition to
precedent for PSA mediated hydrolysis of tyrosyl conju-
OH
OH
OH
p-nitrophenyl
EDC, HOBT
PABA
THF, rt, 20 h
chloroformate
K2 CO3
THF, rt,
NO2
O
O
O
OH
O
N
H
O
O
N
OH 79%
72%
H
Bo cNH
Bo cNH
Bo cNH
3
2
1
1. diethanolamine, DMF, rt, 36%
2. TFA, rt, 99%
OH
OH
OH
O
PSA
OH
OH
OH
N
O
O
O
HN
+
+
OH
OH
N
H
α -chymo
NH2
NH2
NH2
4
Scheme 3. Proof-of-principle: enzymatic release of amine from a p-aminobenzyl carbamate-amino acid conjugate.
NH
O
2
CH
O
3
COOH
H C
3
H N
2
O
H N
2
O
NH
O
7-AMC
Rhodamine 110
disperse orange 11

420
G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
Table 1. Enzyme mediated release of chromophoresd
a
Entry
Substrate
UV kmax conj.
UV kmax free
a-Chymotrypsin
PSAa
Plasmin
Trypsin
1
2
3
4
5
4
7
n/a
300
n/a
497
15
9
5
4
—
—
—
—
0
—
—
—
—
0
11
15
17
328b
285
352c
486
24
18
0
10
6
328b
352c
4.8
a mM/h/mg fluorophore released.
b Fluorescence emission kmax 397.
c Fluorescence emission kmax 435.
d Substrates and controls were incubated for 24–120 h at 37 ꢁC. Specific activity was determined on the basis of mM released fluorophore per unit
time per unit mass of enzyme.
and PSA. Substantial release was observed within 12 h
in both cases (Table 1) and despite numerous attempts
only tyrosine, 4-aminobenzyl alcohol, and diethanola-
mine were detected, suggesting the intermediate anilino
carbamate has a short half-life.
to unmask the Boc group resulted in decomposition of
the molecule, rendering desired target 7 unisolable.
Remedy was found using the alternate bis-alloc sub-
strate 6, which was prepared from commercially avail-
able building block 5 under analogous conditions
(Scheme 4). Nucleophilic displacement followed by
unmasking using Pd chemistry11 allowed isolation of
the hydrochloride salt 7 in good yield, and the product
was freely soluble in assay buffer media.
1.3. Choice of fluorophores
With proof-of-concept for release of amino containing
prodrugs established, we turned our attention to selec-
tion of appropriate fluorophores. For our initial studies
three readily available fluorophore dyes were selected—
aminomethyl coumarin (7-AMC), disperse orange 11,
and rhodamine 110—on the basis that their UV charac-
teristics are all greatly influenced by the electron-donat-
ing capacity of the anilino nitrogen group.
With the alloc route in hand, the coumarin analog 11
was next prepared. This involved coupling of the p-
aminobeznyl amide prepared in Scheme 4 (8) with isocy-
anate 9 to give the masked analog 10 (Scheme 5). The
alloc derivative underwent similarly clean deprotection
to give 11 on workup, the product also soluble in buffer
media. Finally, hoping to exploit the benefits of intra-
molecular hydrogen bonding, the anthraquinone conju-
gate 15 was assembled (Scheme 6). This necessitated
selective removal of the quinone carbonyl group of
aminomethylanthraquinone 12 with dithionite to form
Commencing with the previously available carbonate 3,
coupling with free rhodamine gave the corresponding
adduct cleanly, albeit in low yield. However, all attempts
1. EDCI / HOBT
NO2
OAlloc
O
THF, rt, 20h
p-aminobenzyl
alcohol, 70%
OH
COOH
NH
OAlloc
1. rhodamine 110,
Py, 80oC, 48h, 29%
O
O
O
O
O
O
N
H
O
O
2. Pd(PPh3)4, morpholine
then HCl,
MeOH, 75%
2. p-nitrophenyl
chloroformate
Py, THF, rt 99%
OH
N
H
N
H
+
AllocNH
AllocNH
Cl-
7
NH3
5
6
Scheme 4. Preparation of rhodamine conjugate via alloc protected building block.
CH3
CH3
O
OAlloc
OAlloc
9
O
OCN
O
O
O
O
N
H
O
O
OH
PhCH 3 ∆, 2h, 98%
N
H
N
H
AllocNH
OH
10
AllocNH
8
CH3
O
1. Pd(PPh3)4 , morpholine,
DMF-THF
O
O
O
N
H
O
2. HCl, MeOH
99%
N
H
11
+
NH3
Cl-
Scheme 5. Preparation of tyrosyl aminomethylcoumarin conjugate.

G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
421
O
O
NH2
NH2
1. ClCOCl
CH3
CH3
Na2 S2 O4
DMAP, THF
DM F, H2 O
2. 8, DMAP
THF 83%
70%
13
O
12
OAlloc
OH
O
Pd(PPh 3 )4, morpholine
O
NH O
O
O
O
H3C
HCl, MeOH
90%
O
NH O
O
O
H3C
N
H
N
H
+
AllocNH
Cl-
NH3
15
14
Scheme 6. Preparation of tyrosyl aminomethylanthraquinone conjugate.
required carbamoyl building block 13. Conversion to
the intermediate p-nitrophenyl carbamate was ineffi-
cient, giving a complex mixture which allowed only
low recovered yields of the subsequently coupled prod-
uct 14. However, reaction with phosgene followed by
coupling with 8 gave alloc derivative 14 directly, reoxi-
dation taking place during workup and with yields of
up to 80% attainable on scale-up. Finally, unmasking
allowed isolation of the hydrochloride salt of anthraqui-
none substrate 15 in good yield.
the self-immolative linker. As can be seen (Table 1),
though proof-of-concept is established, in the present
examples, chymotrypsin is more effective than PSA at
cleavage. Though this is unsurprising, more complex oli-
gopeptide substrates are known whose specificity for
PSA outranks chymotrypsin significantly, and it was
decided to elaborate the substrate with the most selectiv-
ity and activity (11) to incorporate these recognition ele-
ments. Based on the work of Denmeade, the HSSKL
motif was selected and a synthetic route to a chimera
of 11 investigated.8 After numerous iterations, the most
successful (and economical) route was to couple bis-
Boc-protected HSSKL (16) with 11 via the BOP route,
which, following deprotection gave hexapeptide sub-
strate 17 in good yield (Scheme 7). Enzymatic studies
confirmed the hypothesis, with the substrate completely
selective for PSA, even against an extended panel of en-
zymes (Table 1). Though less active than 11, the selectiv-
ity demonstrated by 17 is noteworthy and is now the
With three substrates in hand, spectroscopic and enzy-
matic studies were conducted to establish proof-of-con-
cept for use as image contrast agents. Enzymatic release
of the fluorophores was probed using fresh, enzymati-
cally active PSA and chymotrypsin, using UV detection
to quantitate (and fluorescence in the case of 11). Re-
lease of fluorophore correlated with release of p-amino-
benzyl alcohol and tyrosine, confirming the function of
NH2
OH
NHBoc
1. BOP, 11
Hunigs base,
THF-DMF (4:1),
1-2 hr
OH
OH
O
O
O
O
O
O
O
H
H
N
H
N
H
H
N
N
N
N
N
N
H
N
H
N
H
N
H
N
H
OH
NH2
O
O
O
O
O
HN
HN
OH
OH
NHBoc
2. CH2Cl 2-TFA (3:1)
25 oC, 3 hr
O
NH
O
16
17
(87%)
O
CH3
PSA
NH2
OH
H2N
H2N
O
O
OH
O
O
O
H
H
N
+
CO2
+
+
N
OH
N
N
H
N
H
N
H
OH
CH3
NH3
O
O
O
HN
OH
18
Scheme 7. Preparation and activation of HSSKLY aminomethylcoumarin conjugates.

422
G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
basis for ongoing development of appropriate in vitro
and in vivo studies. Several improvements to the basic
system can be envisioned, tailored to the desired applica-
tion, based on the synthetic chemistry platform outlined.
For example, linker architecture has been shown to have
a marked impact on substrate half-life in three compo-
nent systems,9,11 suggesting that enhanced activity may
be attainable, e.g., with halogen containing linker moie-
ties. For application with in vivo analysis it will be
necessary to employ fluorophores with spectral charac-
teristics tailored to match imaging devices. Contrast
agents in the near IR range (e.g., the Cy dye family)
may prove desirable,12 in that the conjugated amino
function has a profound influence on its quantum
yield.13 The coupling chemistries described herein for
amino substituted fluorophores offer flexibility toward
this goal, providing the potential for in situ CCD based
near-IR imaging of systemic agents that are locally acti-
vated under in vivo conditions.12 Besides image contrast
agents, the system may also prove promising for the
slow release of prostate specific chemotherapeutics,
where a systemic drug conjugate could be degraded by
PSA in the prostatic microenvironment.14 The results
of these studies will be reported in due course.
in vacuo, the residue was purified by silica gel chroma-
tography (ethyl acetate–hexanes; 2:1 eluent) to give 2,
(216 mg, 78.5%) as a white solid m.p. 142–14 ꢁC (dec);
1H NMR (300 MHz, CDCl3) d 7.45 (d, J = 8.1 Hz,
2H), 7.28 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H),
6.68 (d, J = 8.1 Hz, 2H), 4.55 (s, 2H), 4.33 (m, 1H),
2.78–3.40 (m, 2H), 1.40 (s, 9H); 13C NMR (75 MHz,
CDCl3) d 176.1, 175.9, 160.6, 160.0, 141.5, 141.2,
141.1, 134.4, 131.9, 131.5, 124.6, 124.5, 119.3, 83.8,
67.8, 61.2, 41.9, 31.8; MS (ESI) m/z 387 (M+H)+;
HRMS (ESI) calcd for C21H27N2O5, (M+H)+ m/z
387.1920, found 387.1916.
2.2. Boc-L-tyrosyl 4-(4-nitrophenoxycarboxyl)methylani-
lide (3)
To a solution of alcohol 2 (200 mg, 0.5175 mmol) in
fresh distilled anhydrous THF (10 ml) were added 4-
nitrophenyl chloroformate (110 mg, 0.5434 mmol) and
anhydrous pyridine (44 ll, 0.5434 mmol). The mixture
was stirred at rt for 12 h. The resulting precipitate was
filtered and the filtrate was evaporated. The residue
following evaporation was purified by silica gel
chromatography (ethyl acetate–hexanes; 1:1 eluent) to
give 3, (204.3 mg, 71.7%) as a white solid m.p. 175–
1
In summary, a three-component system comprised of
enzyme substrate, inert linker, and fluorophore has been
designed and activation by chymotrypsin and PSA dem-
onstrated. A hexapeptide chimera of one of these is
selectively activated by PSA, and the results support
application in the in vitro and in vivo evaluation of more
complex functional substrates, for use as image contrast
agents and chemotherapeutics.
179 ꢁC; H NMR (300 MHz CDCl3) d 8.31 (d, J = 8.7
Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.7 Hz,
2H), 7.39 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H),
6.69 (d, J = 8.7 Hz, 2H), 5.24 (s, 2H), 4.35 (m, 1H),
2.8–3.8 (m, 2H), 1.40 (s, 9H); 13C NMR (375 MHz
CDCl3) d 172.0, 164.0, 156.0, 150.4, 140.6, 138.4,
138.2, 135.2, 131.8, 131.8, 130.4, 130.2, 128.8, 127.7,
125.9, 125.1, 120.8, 120.286, 115.6, 115.3, 115.1, 69.0,
57.0, 54.0, 39.0, 27.5; calcd for C28H29N3O9: C,
60.98; H, 5.30; N, 7.62; found: C, 61.31; H, 5.54;
N, 7.26.
2. Experimental procedures
HSSKL conjugates and protected Y derivatives were
supplied by BACHEM. Unless otherwise stated, all
other reagents were purchased from the Aldrich Chem-
2.3. L-Tyrosyl 4-(N,N-bis(2-hydroxyethyl)carbamoyl)-
methylanilide (4)
1
ical Company and used as supplied. H and 13C NMR
A solution of diethanolamine (0.276 g, 2.6 mmol) in
anhydrous DMF (5.0 ml) was added to a solution of
3 (0.32 g, 0.58 mmol) in anhydrous DMF (5.0 ml).
The mixture was stirred at rt for 42 h, and then
quenched with water (50 ml). The mixture was extracted
with ethyl acetate (3 · 30 ml) and the combined
extracts were washed with brine (1 · 15 ml), and then
dried (Na2SO4). The solution was concentrated in
vacuo and the residue was purified by flash chromatog-
raphy (10% methanol in chloroform) to give
Boc-L-tyrosyl 4-(N,N-bis(2-hydroxyethyl)-carbamoyl)
methylanilide (0.163 g, 36%) as a hygroscopic oil. The
entire anilide (0.314 mmol) was treated with trifluoro-
acetic acid (6 ml) under argon, stirred for 12 h, and then
the TFA was removed by aspiration. The resulting gum
was purified by chromatography (1:9 methanol–chloro-
form) to give 4, (166.5 mg, 99%) as a colorless foam;
1H NMR (300 MHz, d6-acetone): d 7.77–6.83 (m,
10H); 5.35 (s, 1H); 4.65 (s, 2H); 3.96 (m, 4H); 3.75
(m, 1H); 3.55 (m, 1H); 3.40 (m, 7H); 3.01 (s, 1H);
2.85 (s, 1H); calcd for C21H27N3O6: C, 60.42; H, 6.52;
N, 10.06; found: C, 60.77; H, 6.73; N, 9.71.
spectra were obtained either on a 300 MHz Varian Mer-
cury, 300 MHz Bruker AC 300, or 500 MHz Varian
Unity machine (CDCl3 unless otherwise stated). Com-
bustion analyses were performed on a Carlo-Erba
EA1108 system at the Northeastern University Micro-
analytical Facility. Mass spectra were conducted at the
University of Illinois, Urbana Champaign. Chromato-
graphic separations were made using E Merck 230–400
mesh 60H silica gel or using a Harrison Research Inc.
radial chromatotron unit.
2.1. Boc-L-tyrosyl 4-(hydroxymethyl)anilide (2)
To
a solution of p-aminobenzyl alcohol (88 mg,
0.712 mmol) in anhydrous THF (10 ml) cooled to 0 ꢁC
were added HOBt (96 mg, 0.712 mmol), Boc tyrosine
(200 mg 0.712 mmol), and then EDC (150 mg,
0.783 mmol). The mixture was stirred at 0 ꢁC for 2 h
and at rt for 12 h. The solution was washed with HCl
(5%, 2 ml), NaHCO3 (5%, 1 ml), and then the organic
extracts were dried over Na2SO4. After condensation

G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
423
2.4. (Bis)alloc tyrosyl 4-(hydroxymethyl)anilide (8)
0.9 Hz, 2H), 4.50–4.70 (m, 3H), 3.11 (s, 2H); 13C
NMR (75 MHz, CDCl3) d 172.5, 170.4, 156.8, 155.8,
154.0, 153.8, 152.8, 152.2, 150.8, 150.2, 149.2, 140.5,
137.5, 135.5, 134.5, 132.8, 132.2, 131.3, 130.6, 129.9,
129.2, 128.9, 127.8, 127.0, 125.2, 124.2, 121.5, 120.5,
119.8, 118.2, 115.8, 114.5, 113.8, 111.9, 108.2 106.5,
101.8, 69.2, 66.8, 66.2, 56.5, 38.2; MS (ESI) m/z 811
(M+H)+, HRMS (ESI) calcd for C45H39N4O11
(M+H)+ m/z 811.2615, found 811.2606.
To a solution of bis-alloc tyrosine (2.26 g, 6.476 mmol)
in anhydrous THF (50 ml) were added 4-aminobenzylal-
cohol (800 mg, 6.476 mmol), EDCI (1.5 g, 7.77 mmol),
and then HOBT (874 mg, 6.476 mmol). The mixture
was stirred at rt for 12 h, and then condensed in vacuo.
The residue was dissolved in ethyl acetate (30 ml) and
the solution was washed with HCl (5%, 2 ml), NaHCO3
(5%, 1 ml), and then the organic extracts were dried over
Na2SO4. After condensation in vacuo, the residue was
purified by silica gel chromatography (ethyl acetate–hex-
anes; 2:1 eluent) to give the title compound (2.09 g, 71%)
2.7. L-Tyrosyl rhodamine (7)
A catalytic amount of Pd(PPh3)4 (ꢀ1 mg) and morphol-
ine (0.5 ml) was added to a degassed solution of bis(al-
loc)-L-tyrosyl rhodamine (14.7 mg, 0.0182 mmol) in
anhydrous THF (5 ml). The solution was stirred under
argon for 48 h, and then the resulting red precipitate
was collected by centrifugation, and washed with ethyl
acetate (2 · 5 ml). The solid was then exposed to a solu-
tion of hydrochloric acid (0.5 M, 2 ml) in ethyl acetate
(5 ml) followed by methanol (1 ml). The resulting (yel-
low) solution was condensed in vacuo solution and the
residue was purified by silica gel chromatography (ethyl
acetate–methanol; 10:1 eluent) to give 6, (8.7 mg, 74.7%)
as a yellow solid m.p. 213–216 ꢁC; 1H NMR (300 MHz,
CD3OD) d 8.38 (d, J = 5.1 Hz, 1H), 8.23 (s, 1H), 7.80–
7.92 (m, 1H), 7.50-7.70 (m, 4H), 7.30–7.48 (m, 2H),
7.20–7.26 (m, 2H), 7.15 (d, J = 5.1 Hz, 1H), 6.90–7.10
(m, 2H), 6.75 (d, J = 5.7 Hz, 1H), 6.66 (t, J = 5.1 Hz,
1H), 5.22 (s, 2H), 4.23 (m, 1H), 2.84–3.28 (m, 2H); 13C
NMR (75 MHz, CD3OD) d 176.2, 171.5, 161.0, 142.0,
141.8, 137.0, 136.7, 136.5, 135.9, 135.8, 135.6, 135.5,
135.4, 134.9, 134.5, 134.3, 134.2, 132.9, 132.8, 131.4,
128.8, 124.9, 121.4, 121.1, 120.9, 119.5, 108.2, 70.8,
61.1, 40.7; MS (ESI) m/z 643 (M+HÀ2HCl)+, HRMS
(ESI) calcd for C37H31N4O7 (M+HÀ2HCl)+ m/z
643.2193, found 643.2215.
1
as a pale solid m.p. 177–119 ꢁC; H NMR (300 MHz,
CDCl3) d 7.45 (d, J = 8.7 Hz, 2H), 7.27–7.32 (m, 4H),
7.08 (d, J = 8.7 Hz, 2H), 5.80–6.10 (m, 2H), 5.13–5.42
(m, 4H), 4.70 (dd, 1H, J = 5.7, 1.5 Hz), 4.55 (s, 2H),
4.49 (m, 1H), 2.90–3.20 (m, 2H); 13C NMR (75 MHz,
CDCl3) d 172.0, 157.0, 154.0, 150.0, 138.0, 137.0,
135.0, 133.0, 132.0, 130.3, 127.4, 121.0, 120.0, 118.0,
117.0, 69.0, 65.0, 63.0, 57.0, 38.0; MS (ESI) m/z 455
(M+H)+; HRMS (ESI) calcd for C24H27N2O7 (M+H)+
m/z 455.1818, found 455.1827.
2.5. Bis (alloc)-L-tyrosyl 4-(4-nitrophenoxycarboxyl)-
methylanilide (6)
To a solution of 8 (100 mg, 0.22 mmol) THF (5 ml) were
added 4-nitrophenyl chloroformate (57 mg, 0.28 mmol)
and anhydrous pyridine (23 ll, 0.28 mmol). The mixture
was stirred at rt for 12 h. then the formed precipitate
was filtered, and the filtrate was recovered and evaporat-
ed. This residue was purified by silica gel chromatogra-
phy (ethyl acetate–hexanes; 1:2 eluent) to give 6
(143.3 mg, 98.8%) as a white solid m.p. 87–90 ꢁC (dec);
1H NMR (300 MHz, CDCl3) d 8.25 (d, J = 9.0 Hz,
2H), 7.30–7.44 (m, 6H), 7.23 (d, J = 9.0 Hz, 2H), 7.10
(d, J = 8.7 Hz, 2 H), 5.78–6.09 (m, 2H), 5.65 (d,
J = 5.7 Hz, 1H), 5.10–5.50 (m, 5H), 4.72 (d,
J = 5.7 Hz, 2H), 4.53–4.60 (m, 3H), 3.14 (s, 1H), 3.11
(s, 1H); 13C NMR (75 MHz, CDCl3) d 170.2, 156.1,
155.8, 155.1, 153.8, 152.7, 150.5, 145.8, 137.9, 134.3,
132.4, 131.2, 130.7, 130.6, 129.9, 126.9, 126.4, 125.5,
122.5, 122.0, 121.7, 120.7, 119.8, 118.5, 116.1, 70.9,
69.5, 66.5, 55.5, 37.5; MS (ESI) m/z 620 (M+H)+;
HRMS (ESI) calcd for C31H30N3O11 (M+H)+ m/z
620.1880, found 620.1892.
2.8. (Bis)alloc tyrosyl 4-(hydroxymethyl)anilide coumarin
conjugate (10)
A solution of (bis)alloc tyrosyl 4-(hydroxymethyl)anilide
(8, 0.100 g, 0.22 mmol) and coumarin isocyanate (9,
66 mg, 0.33 mmol) in toluene (10 ml) was refluxed for
2 h. The solution was condensed in vacuo and the resi-
due was crystallized from CH2Cl2–MeOH:Et2O to give
1
10 (138 mg, 98%) as a white solid m.p. 139–142 ꢁC; H
NMR 1H NMR (CD3OD, 500 MHz) d 7.45 (d, 2H,
J = 10 Hz), 7.41–7.36 (m, 3H), 7.28-7.24 (m, 3H),
7.18–7.14 (m, 3H), 7.02–6.98 (m, 3H), 6.09 (d, 1H,
J = 1.5 Hz), 5.94-5.86 (m, 1H), 5.82–5.74 (m, 1H),
5.36–5.31 (m, 1H), 5.26–5.23 (m, 1H), 5.18 (d, 1H,
J = 17.5 Hz), 5.12–5.07 (m, 3H), 4.65–4.63 (m, 2H),
4.45–4.44 (m, 2H), 4.39 (t, 1H, J = 7 Hz), 3.53 (br s,
3H), 3.04 (dd, 1H, J = 13.7, 7 Hz), 2.96 (dd, 1H,
J = 13.7, 7 Hz); MS (ESI) m/z 656 (M+H)+.
2.6. Bis(alloc) L-tyrosyl rhodamine
Carbonate 6 (69 mg, 0.112 mmol) and rhodamine 110
(40 mg 0.112 mmol) were dissolved in anhydrous pyri-
dine (10 ml) and the solution was stirred at 80 ꢁC for 2
days. The solution was evaporated to dryness and the
residue was purified by silica gel chromatography (ethyl
acetate–hexanes; 2:1 eluent) to give the title compound
(26 mg, 28.6%) as a yellow oil together with recovered
rhodamine (16 mg); 1H NMR (300 MHz, CDCl3) d
8.34 (s, 1 H), 7.98 (d, J = 7.2 Hz, 2 H), 7.50–7.70 (m,
5H), 7.00-7.40 (m, 8H), 6.68(d, J = 8.4 Hz, 1H), 6.64
(d, J = 8.7 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 6.43 (s,
1H), 6.20 (d, J = 8.1 Hz, 1H), 5.70–6.12 (m, 2H), 5.12–
5.50 (m, 4H), 5.06 (s, 2H, CH2), 4.72 (dd, J = 5.7,
2.9. L-Tyrosyl 4-(hydroxymethyl)anilide coumarin conju-
gate (11)
A solution of 10 (50 mg, 76.5 mmol) in DMF–THF
(1:1, 4 ml) was treated with morpholine (1 ml) and
Pd(Ph3)4 (10 mg). The mixture was stirred at rt for

424
G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
1 h and then concentrated in vacuo. The residue was
purified by silica gel chromatography (CH2Cl2–
MeOH–Et3N; 93:5:2 eluent) to give the title com-
pound (0.041 g, 99%) as a white solid m.p. 183–
185 ꢁC;. 1H NMR (CD3OD, 500 MHz) d 7.57 (s,
1H), 7.58–7.50 (m, 3H), 7.39–7.35 (m, 3H), 7.03 (d,
J = 8.0 Hz, 2H), 6.72 (d, J = 8.0 Hz, 2H), 6.15 (br s,
1H), 5.14 (s, 2H), 4.69 (br s, 5H, exch.), 3.60 (dd,
J = 5.5, 8.0 Hz, 1H), 3.02 (dd, J = 5.5, 14.0 Hz, 1H),
2.75 (dd, J = 5.5, 14.0 Hz, 1H), 2.42 (s, 3H); 13C
2.12. L-Tyrosysl-disperse orange carbamate (15)
A catalytic amount of Pd(PPh3)4 and of morpholine
(50 ll) was added to a degassed solution of 14 (38 mg,
0.053 mmol) in anhydrous THF (5 ml). The solution
was stirred under argon for 48 h and the resulting red
precipitate was recovered by centrifugation and washed
with ethyl acetate (10 ml). The solid was dissolved in
methanol (1 ml), the solution was condensed in vacuo,
and then the residue was purified via silica gel chroma-
tography (ethyl acetate–methanol; 10:1 eluent) to give
NMR (CD3OD, 75 MHz)
d 170.0, 156.1, 153.9,
1
153.5, 153.4, 149.6, 142.4, 137.2, 134.2, 132.0, 131.8,
130.6, 130.0, 128.5, 124.9, 120.7, 120.0, 118.9, 117.2,
114.7, 114.5, 111.7, 105.3, 68.8, 56.3, 37.6, 17.9;
HRMS calcd for C27H26N3O6(MH+) m/z 488.1822,
found 488.1825.
15 (26.1 mg, 90%) as a red solid m.p. 221–224 ꢁC; H
NMR (300 MHz, CD3OD) d 8.19–8.30 (m, 2H), 8.10–
8.15 (m, 1H), 7.82–7.86 (m, 2H), 7.64 (d, 1H,
J = 8.1Hz), 7.54 (d, 2H, J = 8.4Hz), 7.41 (d, 2H,
J = 8.1Hz), 7.10–7.18 (m, 2H), 6.86–6.92 (m, 1H),
6.76(d, 2H, J = 8.4Hz), 5.18 (s, 2H), 4.20 (m, 1H), 3.0–
3.4 (m, 2H), 2.40 (s, 3H); MS (MALDI) m/z (M+Na)+
572.86; calcd for C32H27N3O6: C, 69.93; H, 4.95; N,
7.65; found: C, 70.31; H, 5.06; N, 7.33.
2.10. De-oxy disperse orange 13
Sodium dithionite (14 g, 80.41 mmol) was added to a sus-
pension of the disperse orange 11 (12, 2 g, 8.439 mmol) in
DMF (100 ml), and water (100 ml) and the mixture was
heated to 90 ꢁC over 30 min. The mixture was stirred for
48 h and then cooled to rt The resulting yellow precipi-
tate was filtered and then purified by silica gel chroma-
tography (hexanes–ethyl acetate; 1:1 eluent) to give 13
(1.08 g, 58%) as a yellow solid m.p. 103–105 ꢁC; 1H
NMR (300 MHz, CDCl3) d 8.30 (dd, 1H, J = 8.4Hz,
0.9Hz), 7.78 (d, 1H, J = 7.5Hz), 7.54–7.59 (m, 1H),
7.45 (d, 2H, J = 6.9Hz), 7.16 (d, 1H, J = 8.1Hz), 3.87
(s, 2H), 3.76 (s, 2H), 2.22 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 184.7, 141.8, 139.8, 132.7, 132.0, 130.7, 129.2,
126.0, 127.6, 127.2, 126.8, 124.4, 117.8, 28.4, 18.3; MS
(ESI) m/z 224.2 (M+H)+; HRMS (ESI) calcd for
C15H16NO (M+H)+ m/z 224.1075, found 224.1075.
2.13. HSSKLY-tyrosysl-coumarin carbamate conjugate
(17)
Pentapeptide NH2-HSSKL-CO2H (10 mg, 17.6 lmol),
Boc anhydride (8.86 mg, 40 lmol), and Et3N (16 ll,
106 lmol) were stirred at 40 ꢁC for 12 h in DMF
(2 ml). The solution was concentrated in vacuo, the res-
idue was redissolved in DMF–THF (1:1, 10 ml), and
then 11 (9 mg, 20.2 lmol), BOP (10 mg, 22 lmol), and
Hunigs base (8 ll, 44 lmol). The mixture was stirred
at rt for 12 h. The solution was concentrated in vacuo
and the residue was dissolved in MeOH (2 ml) and the
solution passed through a silica gel plug (6% MeOH,
94% CH2Cl2 eluent). Removal of eluents and concentra-
tion in vacuo gave the Boc-protected derivative of 17
1
2.11. Disperse orange-4 bis-alloc-L-tyrosyl carbamate
(14)
(21.8 mg, 87%) as a colorless gum; H NMR (CDCl3,
300 MHz) d 8.41 (m, 1H), 7.62 (s, 1H), 7.65–7.59 (m,
3H), 7.55 (s, 1H), 7.44–7.31 (m, 3H), 7.11 (m, 2H),
6.85 (m, 2H), 6.25 (s, 1H), 5.19 (s, 2H), 5.10 (br s,
13H), 4.65–4.58 (m, 2H), 4.51–4.49 (m, 2H), 4.38 (m,
1H), 4.11–3.96 (m, 2H), 3.97–3.84 (m, 2H), 3.66 (m,
1H), 3.57–3.41 (m, 2H), 3.13 (m, 1H), 2.99 (m, 2H),
2.82 (m, 1H), 2.41 (s, 3H), 2.01–1.87 (m, 1H), 1.83–
1.64 (m, 6H), 1.57-1.46 (m, 2H), 1.35 (s, 18H), 0.99–
0.94 (m, 6H); (ESI MS 1239 = M+). A portion of this
product (14 mg, 11.1 lmol) was treated with a solution
of TFA (25% in CH2Cl2, 3 ml), stirred at rt for 3 h,
and then condensed in vacuo. The residue was dissolved
in DMF (1 ml) and the solution was passed through a
short plug of silica gel. The eluents were condensed in
vacuo and the residue was washed with CH2Cl2
(3 · 1 ml) then hexanes (3 · 1 ml) to give 17 (11.4 mg,
A solution of phosgene (20%) in toluene (1.8 ml,
3.60 mmol) and DMAP (482 mg, 3.60 mmol) were
added to
a
degassed solution of 13 (400 mg,
1.79 mmol) in THF (20 ml). The solution was stirred
at rt for 24 h and the excess phosgene was removed
with an argon sweep. 8 (750 mg, 1.79 mmol) was then
added and the solution was stirred at rt for 24 h. The
mixture was filtered and the filtrate was condensed in
vacuo. This residue was purified via silica gel chroma-
tography (hexanes–ethyl acetate; 1:1) to give 14
(145 mg, 22%) as a yellow oil; 1H NMR (300 MHz,
CDCl3) d 10.0 (s, 1H), 8.20–8.30 (m, 2H), 8.10 (d,
1H, J = 7.5 Hz), 7.70–7.80 (m, 4H), 7.63 (d, 1H,
J = 7.5 Hz), 7.30-7.50 (m, 4H), 7.24 (d, 1H,
J = 7.2 Hz), 7.11 (d, 2H, J = 8.1z), 5.80–6.20 (m,
2H), 5.00–5.50 (m, 6H), 4.72 (d, 2H, J = 5.7Hz),
4.40–4.60 (m, 3H), 3.10–3.20 (m, 2H), 2.41 (s, 3H);
13C NMR (75 MHz, CDCl3) d 186.9, 182.7, 169.3,
154.2, 153.7, 150.7, 150.7, 150.5, 142.5, 138.6, 134.5,
134.4, 133.0, 132.7, 132.5, 132.4, 131.3, 130.6, 129.3,
127.6, 127.1, 126.3, 124.8, 124.4, 121.7, 120.4, 119.8,
118.5, 69.4, 67.3, 66.5, 57.2, 38.0, 20.2; MS (MALDI)
m/z (M+Na)+ 740.84. Also isolated was recovered 8
(639 mg) [yield 83% based on 8].
1
99%) as a pale white solid m.p. 168–173 ꢁC (dec); H
NMR (CD3OD, 500 MHz) d 8.36 (m, 1 H), 7.58 (s,
1H), 7.59–7.51 (m, 3H), 7.49 (s, 1H), 7.41–7.36 (m,
3H), 7.05 (d, J = 8.0 Hz, 2H), 6.71 (d, J = 8.0 Hz, 2H),
6.17 (br s, 1H), 5.13 (s, 2H), 5.05 (br s, 15 exch. H),
4.61–4.55 (m, 2H), 4.47–4.41 (m, 2H), 4.30 (dd, 1H,
J = 5, 7 Hz), 4.03–3.91 (m, 2H), 3.92–3.81 (m, 2H),
3.63 (dd, J = 5.5, 8.0 Hz, 1H), 3.51–3.36 (m, 2H), 3.05
(dd, J = 5.5, 14.0 Hz, 1H), 2.95 (t, 2H, J = 8 Hz), 2.77
(dd, J = 5.5, 14.0 Hz, 1H, CH), 2.40 (s, 3H, CH3),

G. B. Jones et al. / Bioorg. Med. Chem. 14 (2006) 418–425
425
1.98–1.85 (m, 1H), 1.80–1.62 (m, 6H), 1.55–1.48 (m,
Acknowledgments
2H), 0.98 (d, 3H, J = 6 Hz), 0.92 (d, 3H, J = 6.5 Hz);
13C NMR (CD3OD, 75 MHz) d 174.5, 172.8, 172.0,
171.0, 170.0, 167.8, 162.2, 161.8, 156.1, 153.9, 153.5,
153.4, 149.6, 142.4, 137.3, 135.0, 134.2, 132.0, 131.8,
130.6, 130.0, 128.5, 126.7, 124.9, 120.7, 120.0, 118.9,
118.9, 117.2, 115.0, 114.7, 114.5, 111.7, 105.3, 68.8,
61.5, 56.3, 56.1, 55.8, 53.2, 52.0, 51.0, 40.3, 39.3, 37.6,
31.1, 26.7, 26.5, 24.8, 22.3, 22.1, 20.6, 17.9; ESI-MS
1040.14 (M+); C51H65N11O13 req. C, 58.89; H, 6.30; N,
14.81; found: C, 59.21; H, 6.55; N, 14.59.
We thank the Department of Defense (DAMD-17-02-1-
0254) and the Prostate Cancer Foundation for financial
support of this work.
References and notes
1. Parker, S. L.; Tong, T.; Bolden, S. Ca. Cancer J. Clin.
1996, 46, 5.
2. Bagshaw, M. A.; Cox, R. S.; Hancock, S. L. J. Urol. 1994,
152, 1781.
2.14. General procedure for enzymatic assays
3. Bostwick, D. G. Am. J. Surg. Path. 1994, 18, 796.
4. DÕAmico, A. V.; Debruyne, F.; Huland, H.; Richie, J. P.
Prostate 1999, 41, 208.
5. Watanabe, M. Nippon Rinsho 1998, 56, 1040; Ragde, H.;
Kenny, G. M.; Murphy, G. P. Prostate 1977, 32, 279;
Bogers, H. A.; Sedelaar, J. P.; Beerlage, H. P. Urology
1999, 54, 97.
6. Nadji, M.; Tabei, S. Z.; Castro, A.; Chu, T. M.; Murphy,
G. P.; Wang, M. C.; Morales, A. R. Cancer 1981, 48, 1229.
7. Lilja, H.; Abrahamsson, P.; Lundwall, P.-A. J. Biol.
Chem. 1989, 264, 1894.
8. Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A.
V.; Isaacs, J. T. Cancer Res. 1998, 58, 2537.
9. Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A.
J. Med. Chem. 1981, 24, 479.
10. Jones, G. B.; Mitchell, M. O.; Weinberg, J. S.; DÕAmico,
A. V.; Bubley, G. A. Bioorg. Med. Chem. Lett. 2000, 10,
1987.
11. de Groot, F. M. H.; Loos, W. J.; Koekkoek, R.; van
Berkom, L. W. A.; Busscher, G. F.; Seelen, A. E.;
Albrecht, C.; de Brujin, P.; Scheeren, H. W. J. Org.
Chem. 2001, 66, 8815.
12. Weissleder, R.; Tung, C. H.; Mahmood, U.; Bogdanov,
A., Jr. Nat. Biotechnol. 1999, 17, 375.
13. Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis,
C. J.; Waggoner, A. S. Bioconj. Chem. 1993, 4, 105.
14. Vaage, J.; Barbera, E. Am. Assoc. Cancer Res. 1994, 35,
2481.
In duplicate, PSA (Cortex Biochem), 20 ll, 0.5 mg/ml,
pH 7.4, 0.01 M phosphate-buffered saline (PBS) or
a-chymotrypsin (Sigma, TLCK-treated, 20 ll, 0.5 mg/
ml, pH 7.4, 0.01 M PBS), 160 ll PBS, and 20 ll sub-
strate (1.0 mg/ml; 1:1, EtOH–H2O) were incubated
for 24 h at 37 ꢁC. Duplicate control reactions contain-
ing PBS (180 ll) and substrate (20 ll) solutions were
incubated under identical conditions. The two cuvettes
were placed in a Varian (Cary WinUV) spectropho-
tometer for 24 h and kinetics were recorded. To deter-
mine the release of free fluorophore and linker,
substrates and controls were also incubated at 37 ꢁC
for 24–120 h and then reactions were terminated by
addition of cold trichloroacetic acid (10% v/v). En-
zyme was removed by centrifugation (700g, 30 min,
4 ꢁC) using Ultrafree-MC tubes (Millipore), and peak
areas for control and reaction mixtures were deter-
mined by HPLC. Conditions: C-18 reverse-phase col-
umn (15 cm); mobile phase A 99% H2O + 1% TEA;
mobile phase B 99% methanol + 1% TEA; linear gra-
dient 10–80%; flow rate 1 ml/min; monitoring wave-
length = 253 nm. Specific activity (mmol/h/mg) was
determined as: [concn] released substrate (mmol/
ml) · total vol. of the assay (ml)/time of the reaction
(h) · [concn] enzyme mg/ml · vol enzyme (ml).