Synthesis and biological evaluation of novel prodrugs of anthracyclines for selective activation by the tumor-associated protease plasmin

Article abstract of DOI:10.1021/jm9910472

New prodrugs of daunorubicin and doxorubicin designed for selective activation by the serine protease plasmin are described. The low toxic prodrugs 3, 4, and 5 are converted to the corresponding cytotoxic drugs upon proteolysis by the tumor-associated protease plasmin. Application of a self- eliminating spacer was essential for enzyme activation. A prodrug containing a chloro-substituted spacer was synthesized with the aim of enhancing the rate of conversion by plasmin. All prodrugs were highly stable in buffer solution and in serum and on the average 15-fold less cytotoxic than the parent drugs in seven human tumor cell lines. A marked in vitro selectivity was demonstrated by incubation of the doxorubicin prodrugs with a plasmin generating MCF-7 breast cancer cell line transfected with urokinase-type plasminogen activator (u-PA) in comparison with the nontransfected nonplasmin generating cell line. Prodrugs 4 and 5 showed the same cytotoxic effect as the free parent drug doxorubicin in the u-PA transfected cells, indicating complete conversion of the prodrug by plasmin. Addition of the plasmin inhibitor Trasylol drastically increased the ID₅₀ values in the u-PA transfected MCF-7 cells for both prodrugs 4 and 5.

Full text of DOI:10.1021/jm9910472

J . Med. Chem. 1999, 42, 5277-5283
5277
Syn th esis a n d Biologica l Eva lu a tion of Novel P r od r u gs of An th r a cyclin es for
Selective Activa tion by th e Tu m or -Associa ted P r otea se P la sm in
Franciscus M. H. de Groot,† Anton C. W. de Bart,‡ J an H. Verheijen,‡ and Hans W. Scheeren*,†
Department of Organic Chemistry, NSR-Center for Molecular Structure, Design and Synthesis, University of Nijmegen,
Toernooiveld 1, 6525 ED Nijmegen, The Netherlands, and TNO Prevention and Health, Division of Vascular and
Connective Tissue Research, Gaubius Laboratorium, Zernikedreef 9, 2333 CK, Leiden, The Netherlands
Received August 5, 1999
New prodrugs of daunorubicin and doxorubicin designed for selective activation by the serine
protease plasmin are described. The low toxic prodrugs 3, 4, and 5 are converted to the
corresponding cytotoxic drugs upon proteolysis by the tumor-associated protease plasmin.
Application of a self-eliminating spacer was essential for enzyme activation. A prodrug
containing a chloro-substituted spacer was synthesized with the aim of enhancing the rate of
conversion by plasmin. All prodrugs were highly stable in buffer solution and in serum and on
the average 15-fold less cytotoxic than the parent drugs in seven human tumor cell lines. A
marked in vitro selectivity was demonstrated by incubation of the doxorubicin prodrugs with
a plasmin generating MCF-7 breast cancer cell line transfected with urokinase-type plasminogen
activator (u-PA) in comparison with the nontransfected nonplasmin generating cell line.
Prodrugs 4 and 5 showed the same cytotoxic effect as the free parent drug doxorubicin in the
u-PA transfected cells, indicating complete conversion of the prodrug by plasmin. Addition of
the plasmin inhibitor Trasylol drastically increased the ID50 values in the u-PA transfected
MCF-7 cells for both prodrugs 4 and 5.
Ch a r t 1
In tr od u ction
Chemotherapeutic anticancer agents show significant
toxicity toward proliferating normal cells. The lack of
selectivity leads to severe side effects, which is still a
serious drawback in conventional cancer chemotherapy.
A promising approach to increase selectivity is to exploit
the existence of tumor-associated enzymes that are able
to activate a pharmacologically inactive prodrug to the
corresponding active parent drug in the vicinity of the
tumor. A high concentration of a toxic anticancer agent
at the tumor site can be generated via this concept of
“monotherapy”. An attractive feature of prodrug mono-
therapy, unlike “antibody-directed enzyme prodrug
therapy”,1 is that no antibody-enzyme conjugates are
needed, which renders it relatively straightforward and
commercially more interesting.2
In preceding investigations by us and by others it was
shown that “prodrug monotherapy” works well with
anthracycline prodrugs that are activated by â-glucu-
ronidase.3,4 â-Glucuronidase can be found in elevated
concentrations in necrotic areas of tumor tissue.5 The
promising in vivo results that we obtained with the
involved anthracycline prodrugs have stimulated us to
develop new anthracycline prodrugs that are to be
activated by the tumor-associated protease plasmin.
Anthracyclines such as daunorubicin (1) and doxorubi-
cin (2) (Chart 1) are still widely used cytotoxic agents
in cancer chemotherapy due to their very broad activity
spectrum and their high degree of efficacy against many
human tumors.7
ing activity and its involvement in tumor growth, most
likely by its participation in growth factor activation and
angiogenesis.8-11 Active plasmin catalyzes the break-
down of extracellular matrix proteins and, thus, con-
tributes to migration, invasion, and metastasis of tumor
cells.12,13 In the body plasmin is predominantly present
in its inactive pro-enzyme form plasminogen. Active
plasmin is formed locally at or near the surface of tumor
cells by urokinase-type plasminogen activator (u-PA),
produced by the cancer and/or stroma cells.14,15 Plasmin
activity is kept localized because cell-bound urokinase
can activate cell-bound plasminogen into active plasmin,
which stays cell-bound. Active urokinase and active
plasmin do not occur in the blood circulation since they
are very rapidly inhibited by inhibitors such as PAI-1
and R2-antiplasmin, respectively. Many tumor cell lines
and tumors have a significantly higher u-PA level than
that of their normal counterparts,16 and u-PA was
shown to be correlated with invasive behavior and to
be a strong prognostic factor for reduced survival and
increased relapse in many types of tumors.17-20 Plasmin
is a very promising enzyme for exploitation in a tumor-
specific prodrug approach because the proteolytically
active form is localized at tumor level.
The plasmin system is recognized to play a key role
in tumor invasion and metastasis by its matrix degrad-
* To whom correspondence should be addressed.
† University of Nijmegen.
‡ TNO Prevention and Health.
10.1021/jm9910472 CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/25/1999

5278 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Brief Articles
Sch em e 1
Sch em e 2
Deprotection of the Boc groups and the tert-butyl ester
under acidic conditions and following protection of both
amines using Aloc-ONSu under basic conditions yielded
the doubly protected tripeptide 13.
The chloro-substituted aminobenzyl alcohol was not
commercially available, and therefore, it was synthe-
sized starting from 4-amino-2-chloro-benzonitrile 14
(Scheme 2).
The cyano functionality was hydrolyzed to the corre-
sponding carboxylic acid 15 by heating under reflux in
50% potassium hydroxide. Treatment of amino acid 15
with 2,2-dimethoxypropane in the presence of hydro-
chloric acid gave the methylester 16, which on reduction
with DIBALH gave the desired amino alcohol 17.
To synthesize anthracycline prodrugs 3-5, 4-ami-
nobenzyl alcohol 18 and the chloro-substituted spacer
17 were linked to the protected tripeptide 13 using
isobutyl chloroformate as a coupling agent (Scheme 3).
Several coupling agents, i.e., 2-ethoxy-1-ethoxycar-
bonyl-1,2-dihydroquinoline (EEDQ) were also tried, but
the highest yields were obtained with the chloroformate.
The benzylic alcohols 19 and 20 were converted subse-
quently into the activated analogues 21 and 22 by
reacting them with 4-nitrophenyl chloroformate. Several
coupling methods were tried, but the best results were
obtained via this route. For the construction of the
tripartate anthracycline prodrugs, the activated 4-ni-
trophenyl carbonates were reacted with daunorubicin
and doxorubicin to yield protected prodrugs 23, 24, and
25. In the final step the Aloc proctecting groups were
removed smoothly with a palladium catalyst in the
presence of morpholine. Treatment with hydrochloric
acid provided the corresponding prodrugs 3, 4, and 5
as their double ammonium salt.
The first anthracycline prodrugs that were designed
for specific activation by plasmin were reported by
Katzenellenbogen et al.21,22 These bipartate anthracy-
cline prodrugs consisted of a tripeptide coupled to the
anthracycline amino function. A major limitation of
these prodrugs was that they were very inefficiently
activated by plasmin. There was most likely too much
steric hindrance for the protease to cleave the tripeptide.
In a recent report, plasmin served as the target enzyme
for tripartate prodrugs of alkylating agents that con-
sisted of tripeptide specifiers coupled to the drug via a
cyclization spacer.23,24
Recently, peptide conjugates of anthracyclines were
reported which contained a monoclonal antibody that
was needed for tumor specificity.25 In these conjugates,
dipeptides were applied for activation by lysosomal
enzymes (in particular cathepsin B, C, or D are men-
tioned) after internalization by the cell. For optimization
of cathepsin B cleavage after internalization, some
model prodrugs containing several tripeptides were
tested for their half-lives.26 These prodrugs were claimed
to be stable extracellularly.27
The prodrugs described in this paper are designed for
extracellular activation by plasmin. The synthesized
tripartate prodrugs contain a self-immolative 1,6-
elimination spacer28 placed between the tripeptide and
the drug to make the proteolytic cleavage possible.
Prodrug cytotoxicity was determined in seven human
tumor cell lines. To obtain ultimate proof of in vitro
selectivity, the prodrugs were tested in a MCF-7 breast
cancer cell line that was transfected with u-PA and in
MCF-7 control cells.
Biologica l Da ta
Prodrugs 3, 4, and 5 were highly stable in a Tris
buffer solution (pH 7.3). No degradation products were
detected after 3 days of incubation, and all prodrugs
remained completely intact upon incubation in bovine
serum for 3 days.
In contrast to plasmin prodrugs of anthracyclines
without a self-immolative spacer,21 prodrugs 3, 4, and
5 were indeed converted to yield the corresponding
anthracyclines upon incubation with human plasmin.
To determine the kinetics of enzymatic hydrolysis,
prodrugs 3, 4, and 5 were incubated at a concentration
of 100 µM with 50 µg/mL human plasmin in Tris buffer
solution. The half-lives of enzymatic hydrolysis for
prodrugs 3, 4, and 5 were 14, 19, and 7 min, respec-
tively, and they were determined using an analytical
RP-HPLC column. As was expected, the chloro-substi-
tuted prodrug 5 showed a clearly enhanced rate of
Ch em istr y
The prodrugs of the present paper were prepared by
attaching the drug moiety to the spacer end of the
tripeptide-spacer moieties. Doubly protected tripeptides
13 were synthesized via well-established peptide chem-
istry (Scheme 1).
Allyloxycarbonyl (Aloc) was chosen as protecting
group. In the first step H-Lys(Boc)-OBu (7) was coupled
with Fmoc-Phe-ONSu (6) (Scheme 1). After Fmoc depro-
tection under basic conditions, the dipeptide 9 was
coupled with the third amino acid Boc-D-Ala-ONSu (10).

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5279
Sch em e 3
Ta ble 1. Cytotoxicity (ID50 Valuesa,b (ng/mL)) of Daunorubicin, Doxorubicin, and Prodrugs 3-5 in Various Tumor Cell Lines
MCF-7
EVSA-T
WIDR
IGROV
M19
A498
H226
daunorubicin (1)
doxorubicin (2)
prodrug 3
prodrug 4
prodrug 5
329
10
1214
176
72
48
8
1195
141
248
38
11
2150
413
140
113
60
225
590
224
309
16
1578
437
350
487
90
1158
473
763
243
199
1698
896
1429
a
b
Drug dose that inhibited cell growth by 50% compared to untreated control cultures. SRB cell viability test.
conversion by plasmin, indicating that the electron
withdrawing substituent facilitates the enzymatic cleav-
age by plasmin.
However, in cells cultured with prodrugs 4 or 5 a
much higher cytotoxicity was observed in u-PA produc-
ing MCF-7 cells as compared with control cells (ID50
)
All prodrugs showed a strongly reduced cytotoxicity
in seven well-characterized human tumor cell lines in
comparison with the corresponding parent drugs (Table
1).29
An average 15-fold reduction of cytotoxicity was
observed, the reduction ranging from 2 to 56 times.
These data may suggest that the seven cell cultures
express different concentrations of u-PA or plasmin,
although these levels are expected to be low. Unfortu-
nately, the plasmin generating activity of these cell lines
is unknown so that no clear conclusions can be drawn
with respect to plasmin-dependent hydrolysis of the
prodrugs.
To test the in vitro selectivity in cell lines with well-
defined plasmin generating activity, the prodrugs 4 and
5 and doxorubicin were incubated for 24 h in a MCF-7
breast cancer cell culture that was transfected with
wildtype u-PA and in MCF-7 control cells lacking u-PA.
The parent compound doxorubicin was equally cytotoxic
for both control nontransfected MCF-7 cells and u-PA
producing MCF-7 cells. Addition of the plasmin inhibitor
Trasylol had no effect (Figure 1).
7.8 µM versus 49 µM for prodrug 4 and 4.1 µM versus
57 µM for prodrug 5). Prodrugs 4 and 5 show the same
cytotoxic effect as the free parent drug doxorubicin in
the u-PA transfected cells, indicating complete conver-
sion of the prodrug by plasmin. Furthermore, the
addition of the plasmin inhibitor Trasylol, which had
no effect on doxorubicin treated cells, drastically in-
creased the ID50 values in the u-PA transfected cells for
both prodrugs 4 and 5 (ID50 ) 7.8 µM versus 84 µM for
prodrug 4 and 4.1 µM versus 107 µM for prodrug 5).
Discu ssion
The prodrugs of the present paper show an average
15-fold reduced cytotoxicity in seven human tumor cell
cultures. It may be concluded that the synthesized
prodrugs are being selectively activated upon incubation
with plasmin generating tumor cells. The findings that
prodrugs 4 and 5 show much higher growth inhibition
potency in the u-PA transfected cell line in comparison
with the nontransfected cell line strongly suggest that
it is plasmin activity that is responsible for the observed
cytotoxicity in this assay. The evidence for plasmin-

5280 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Brief Articles
mmol) of Boc-D-Ala-ONSu (10) and 0.634 mL (1.1 equiv) of
triethylamine in dry dichloromethane. The mixture was stirred
for 16 h after which dichloromethane was added. The organic
layer was washed with 10% citric acid, saturated sodium
bicarbonate, and water, dried (Na2SO4), and evaporated. The
product was purified by means of column chromatography
(chloroform-methanol; 20/1) to afford 2.56 g (4.13 mmol, 99%)
of tripeptide 11 as a white foam: mp 59 °C; 1H NMR (300 MHz,
CDCl3) δ 1.25 (d, 3H, J ) 3.2 Hz, CH3-Ala), 1.43 (br s, 27H,
t-Bu), 1.00-1.90 (m, 6H, CH2-Lys), 2.80-3.30 (m, 4H, N-CH2-
Lys and Bn), 4.15 (m, 1H, HR), 4.35 (m, 1H, HR), 4.64 (br d,
1H, J ) 7.1 Hz, HR), 7.15-7.35 (m, 5H, aromatic) ppm; MS
(FAB) m/e 621 (M + H)+, 643 (M + Na)+. Anal. (C32H52N4O8‚
1/2H2O) C, H, N.
D-Ala -P h e-Lys-OH(‚2HCl) (12). Boc-D-Ala-Phe-Lys(Boc)-
OBu (11) (2.56 g, 4.13 mmol) was stirred in a solution of
hydrochloric acid in ethyl acetate (3 M). After 5 h the solvent
was evaporated, and tert-butyl alcohol was added and evapo-
rated twice to remove remaining hydrochloric acid. The
product was freeze-dried in dioxane/water to yield a cream
colored powder, which was used without further purification:
1H NMR (300 MHz, D2O) δ 0.94 (d, 3H, J ) 7.0 Hz, CH3-Ala),
1.10-1.85 (m, 6H, CH2-Lys), 2.75-2.84 (m, 3H, N-CH2-Lys and
Bn), 3.09 (dd, 1H, J ) 5.4 Hz, J ) 14.1 Hz, Bn), 3.54 (q, 1H,
J ) 7.0 Hz, HR), 3.98 (dd, 1H, J ) 5.4 Hz, J ) 7.8 Hz, HR),
4.54 (dd, 1H, J ) 5.4 Hz, J ) 9.8 Hz, HR), 7.10-7.22 (m, 5H,
aromatic) ppm; MS (FAB) m/e 365 (M + H)+.
F igu r e 1. ID50 values (µM) for doxorubicin and prodrugs 4
and 5 in u-PA producing MCF-7 cells and MCF-7 control cells,
with and without the addition of plasmin inhibitor Trasylol.
Control MCF-7 cells (first and second bar) and u-PA trans-
fected cells (third and fourth bar) were cultured with and
without 400 KIE/mL Trasylol in the presence of doxorubicin
(left) or prodrugs 4 (middle) and 5 (right). Surviving cells were
determined after 24 h as described in the Experimental
Section.
Aloc-D-Ala -P h e-Lys(Aloc)-OH (13). To a solution of 706
mg (1.61 mmol) of D-Ala-Phe-Lys-OH (12) in water/acetonitrile
was added triethylamine until a pH of 9-9.5 was reached.
Then a solution of 704 mg (2.2 equiv) of Aloc-ONSu in
acetonitrile was added, and the mixture was kept basic by
adding triethylamine. After the pH of the mixture did not alter
anymore, a 0.5 M solution of hydrochloric acid in ethyl acetate
was added until a pH of 3 was reached. The mixture was
thoroughly extracted with dichloromethane. The organic layer
was washed with water, dried (Na2SO4), and evaporated to
result in the desired product (13) as a cream colored foam (742
mg, 86%): mp 141 °C; 1H NMR (300 MHz, CDCl3) δ 1.10-
1.95 (m, 6H, CH2-Lys), 1.21 (d, 3H, J ) 6.5 Hz, CH3-Ala), 2.90-
3.30 (m, 4H, N-CH2-Lys and Bn), 4.20 (m, 1H, HR), 4.55 (m,
5H, HR and 4 Aloc), 4.76 (br d, 1H, J ) 5.2 Hz, HR), 5.17-
5.31 (m, 4H, Aloc), 5.83-5.92 (m, 2H, Aloc), 7.20-7.28 (m, 5H,
aromatic) ppm; MS (FAB) m/e 533 (M + H)+, 555 (M + Na)+;
Anal. (C26H36N4O8) C, H, N.
mediated prodrug activation is further supported by the
results obtained by incubation with the specific plasmin
inhibitor Trasyslol. As doxorubicin prodrugs 4 and 5
show high in vitro selectivity and potency, further in
vivo evaluation will be performed with these prodrugs.
Exp er im en ta l Section
1
Ch em istr y. H NMR spectra were measured on a Bruker
AM-300 (300 MHz, FT) spectrometer in the given solvent.
Chemical shift values are reported as δ-values in parts per
million in comparison with TMS as an internal standard. The
assignment of protons is partial for the prodrugs. Mass spectra
were obtained with a double-focusing VG 7070E spectrometer.
Elemental analyses were carried out in triplicate on a Carlo
Erba Instruments CHNSO EA 1108 element analyzer and
were within 0.4% of the theoretical values calculated for C,
H, and N. Melting points were measured on a Reichert
Thermopan microscope and are uncorrected. TLC was per-
formed using precoated silica gel (60F254) plates, and spots
were examined with UV light, an ammonium molybdic solu-
tion, or with a Chloro-TDM test. Chromatography was carried
out on Baker silica gel.
Meth yl 4-Am in o-3-ch lor oben zoa te (16). Starting com-
pound 14 (2.32 g, 15.2 mmol) was dissolved in 100 mL of a
50% potassium hydroxide solution and refluxed for 19 h. The
mixture was diluted with 200 mL water and filtrated. The
filtrate was acidified with concentrated hydrochloric acid, and
the mixture was freeze-dried. To the product was added
methanol, and the inorganic salt was filtered. The filtrate was
evaporated to dryness, and 50 mL of dimethoxypropane, 3 mL
of concentrated hydrochloric acid, and 6 mL methanol were
added. The mixture was stirred under refluxing conditions for
6 h and additionally stirred for 18 h. Then, ethyl acetate was
added, and the organic layer was washed with a saturated
sodium bicarbonate solution and brine, dried (Na2SO4), and
evaporated resulting in a brown oil. Upon the addition of
hexane, a solid precipitated. After filtration, product 16 (1.49
g, 53% for two steps) was obtained as a green powder: mp 86
F m oc-P h e-Lys(Boc)-OBu (8). To a solution of 2.50 g of
Fmoc-Phe-ONSu (6) (5.16 mmol) in dry dichloromethane under
an argon atmosphere were added at 0 °C 0.791 mL of
triethylamine (1.1 equiv) and 1.92 g of H-Lys(Boc)-OBu‚HCl
(7, 1.1 equiv). The mixture was stirred at room temperature
for 5 h, then dichloromethane was added, and the organic layer
was washed with 10% citric acid, saturated sodium bicarbonate
and water, dried (Na2SO4), and evaporated. The white solid
(3.08 g, 89%) was used without further purification: mp 93
1
°C; H NMR (300 MHz, CDCl3) δ 1.10-1.90 (m, 24H, 6 CH2-
1
°C; H NMR (300 MHz, CDCl3) δ 3.86 (s, 3H, Me), 4.07 (br s,
Lys and 18 t-Bu), 3.06 (m, 4H, N-CH2-Lys and Bn), 4.19 (t,
1H, J ) 6.8 Hz, Fmoc), 4.25-4.55 (m, 4H, 2 Fmoc and 2 HR),
7.19-7.78 (m, 13H, aromatic) ppm; MS (FAB) m/e 672 (M +
H)+, 694 (M + Na)+; Anal. (C39H49N3O7) C, H, N.
2H, NH2), 6.52 (dd, 1H, J ) 2.3 Hz, J ) 8.6 Hz, aromatic),
6.70 (d, 1H, J ) 2.3 Hz, aromatic), 7.78 (d, 1H, J ) 8.5 Hz,
aromatic) ppm; MS (EI) m/e 185 (M+). Anal. (C8H8NO2Cl) C,
H, N.
Boc-D-Ala -P h e-Lys(Boc)-OBu (11). Fmoc-Phe-Lys(Boc)-
OBu (8) (3.08 g, 4.58 mmol) was dissolved in 100 mL of
dioxane/methanol/2 N sodium hydroxide (70/25/5) and stirred
at room temperature for 1 h. The mixture was neutralized with
acetic acid (0.571 mL), and organic solvents were evaporated.
Water and dioxane were added, and the solution was freeze-
dried. Diisopropyl ether was added, and after filtration the
filtrate was evaporated. The product was dissolved in dry
dichloromethane and added at 0 °C to a solution of 1.19 g (4.16
(4-Am in o-3-ch lor op h en yl)m eth a n ol (17). To a solution
of 840 mg (4.53 mmol) of 16 in dry THF was added 7.55 mL of
DIBALH (2.5 equiv, 1.5 M in toluene) under an argon
atmosphere, and the mixture was stirred for 16 h. The solvent
was evaporated, and dichloromethane was added together with
10% citric acid. Then to the water layer was added potassium
hydroxide, and the water layer was extracted with dichlo-
romethane. The organic layer was washed with saturated

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5281
14.5 Hz, 2′), 2.32 (br d, 1H, J ) 14.9 Hz, 8), 2.42 (s, 3H, 14),
sodium bicarbonate and brine, dried (Na2SO4), and evaporated.
The residual oil was purified by means of column chromatog-
raphy (chloroform-methanol; 9/1) to afford 343 mg (48%) of
the amino alcohol (22): mp 99 °C; 1H NMR (300 MHz, CDCl3)
δ 4.61 (s, 2H, Bn), 6.59 (dd, 1H, J ) 2.3 Hz, J ) 8.2 Hz,
aromatic), 6.72 (d, 1H, J ) 2.3 Hz, aromatic), 7.20 (d, 1H, J )
8.2 Hz, aromatic) ppm; MS (EI) m/e 157 (M+). Anal. (C7H8NOCl)
C, H, N.
2.44 (br d, 1H, 8), 2.90-3.35 (m, 6H, Bn Phe and N-CH2
-Lys
and 10), 3.63 (s, 1H, 4′), 3.88 (br d, 1H, J ) 8.2 Hz, 3′), 4.06
(br s, 4H, OMe and 5′), 4.20 (m, 2H, HR), 4.35-4.70 (m, 5H,
HR and 4 Aloc), 4.96 (2 * d, 2H, J ) 11.7 Hz, Bn spacer), 5.05-
5.35 (m, 4H, Aloc), 5.46 (m, 2H, 1′ and 7), 5.60-6.00 (m, 2H,
Aloc), 7.10-7.35 (m, 7H, aromatic), 7.39 (d, 1H, J ) 8.5 Hz,
3), 7.56 (d, 2H, J ) 7.5 Hz, aromatic), 7.78 (t, 1H, J ) 8.2 Hz,
2), 8.02 (d, 1H, J ) 7.6 Hz, 1) ppm; MS (FAB) m/e 1213 (M +
Na)+. Anal. (C61H70N6O19‚21/2H2O) C, H, N.
Gen er a l P r oced u r e for th e Cou p lin g of Sp a cer s 17 a n d
18 t o t h e P r ot ect ed Tr ip ep t id e 13. Aloc-D-Ala -P h e-
Lys(Aloc)-P AB-OH (19). A solution of 730 mg of 13 (1.37
mmol) was dissolved in dry THF under an argon atmosphere
and cooled to -40 °C. N-Methylmorpholine (166 µL, 1.1 equiv)
and isobutylchloroformate (196 µL, 1.1 equiv) were added. The
reaction mixture was stirred for 3 h at a temperature below
-30 °C. A solution of 4-aminobenzyl alcohol (203 mg, 1.2 equiv)
and N-methylmorpholine (181 µL, 1.2 equiv) in dry THF was
added dropwise to the reaction mixture. After 2 h, THF was
evaporated and dichloromethane was added. The organic layer
was washed with saturated sodium bicarbonate, a 0.5 N
potassium bisulfate solution, and brine, dried (Na2SO4), and
evaporated. The residual pale yellow solid was purified by
means of column chromatography (chloroform-methanol; 9:1)
to afford 812 mg (93%) of the desired product 19 as a cream
colored powder: mp 156 °C; 1H NMR (300 MHz, DMSO-d6) δ
0.96 (d, 3H, J ) 7.1 Hz, CH3-Ala), 1.10-1.85 (m, 6H, CH2-
Lys), 2.77 (dd, 1H, J ) 10.2 Hz, J ) 13.6 Hz, Bn Phe), 2.97
(br d, 2H, J ) 5.9 Hz, N-CH2-Lys), 3.09 (br d, 1H, J ) 10.4
Hz, Bn Phe), 4.00 (t, 1H, J ) 7.1 Hz, HR), 4.20-4.60 (m, 8H,
2 HR and 4 Aloc and CH2-OH), 5.00-5.35 (m, 4H, Aloc), 5.76-
5.95 (m, 2H, Aloc), 7.05-7.30 (m, 7H, aromatic), 7.41 (d, 1H,
J ) 7.0 Hz, NH), 7.56 (d, 2H, J ) 8.4 Hz, aromatic), 8.12 (d,
1H, J ) 7.7 Hz, NH), 8.18 (d, 1H, J ) 8.1 Hz, NH), 9.80 (s,
1H, NH anilide) ppm; MS (FAB) m/e 638 (M + H)+, 660 (M +
Na)+. Anal. (C33H43N5O8‚1/2H2O) C, H, N.
Gen er a l P r oced u r e for th e Dep r otection of P r otected
P r od r u gs 3-5. D-Ala -P h e-Lys-P ABC-DAU(‚2HCl) (3). To
a solution of 19 mg (0.016 mmol) of protected prodrug 23 in
dry THF/dichloromethane under an argon atmosphere was
added morpholine (14 µL, 10 equiv) together with a catalytic
amount of Pd(PPh3)4. The reaction mixture was stirred for 1
h in the dark. The red precipitate was collected by means of
centrifugation. Ethyl acetate was added, and the mixture was
acidified using 0.5 mL of 1 M hydrochloric acid/ethyl acetate.
The precipitate was collected by means of centrifugation and
washed several times with ethyl acetate. tert-Butyl alcohol was
added and evaporated, and the resulting red film was freeze-
dried in water yielding 18 mg (100%) of daunorubicin prodrug
1
3: mp 128 °C; H NMR (300 MHz, CDCl3/CD3OD) δ 1.20 (d,
3H, J ) 6.9 Hz, sugar CH3), 1.28 (d, 3H, J ) 6.6 Hz, CH3-
Ala), 1.25-2.00 (m, 8H, CH2-Lys and 2′), 2.09 (dd, 1H, J )
6.1 Hz, 8), 2.33 (br d, 1H, J ) 15.2 Hz, 8), 2.42 (s, 3H, 14),
2.89-3.02 (m, 3H, N-CH2-Lys and 10), 3.19-3.38 (m, 3H, Bn
Phe and 10), 3.62 (s, 1H, 4′), 3.87 (m, 1H, 3′), 4.05 (s, 3H, OMe),
4.18 (m, 2H, 5′ and HR), 4.52 (br d, 1H, J ) 6.6 Hz, HR), 4.65
(m, 1H, HR), 4.95 (2 * d, 2H, J ) 12.2 Hz, Bn spacer), 5.24 (br
s, 1H, 1′), 5.48 (br s, 1H, 7), 7.05-7.35 (m, 7H, aromatic), 7.39
(d, 1H, J ) 8.4 Hz, 3), 7.52 (d, 2H, J ) 8.2 Hz, aromatic), 7.77
(t, 1H, J ) 8.1 Hz, 2), 8.01 (d, 1H, J ) 7.7 Hz, 1) ppm; MS
(FAB) m/e 1023 (M + H)+, 1045 (M + Na)+. Anal. (C53H62N6-
O15‚6.4HCl) C, H, N.
Gen er a l P r oced u r e for th e Activa tion of Ben zylic
Alcoh ols 19 a n d 20 w ith 4-Nitr op h en yl Ch lor ofor m a te.
Aloc-D-Ala -P h e-Lys(Aloc)-P ABC-P NP (21). To a solution
of 384 mg (0.602 mmol) of 19 in dry THF/dichloromethane
under an argon atmosphere were added 4-nitrophenylchloro-
formate (182 mg, 1.5 equiv) and dry pyridine (73 µL, 1.5 equiv).
The mixture was stirred at room temperature for 48 h, and
then ethyl acetate was added. The organic layer was washed
with 10% citric acid, brine, and water, dried (Na2SO4), and
evaporated yielding a yellow solid. The product was purified
by means of column chromatography (dichloromethane-
methanol; 30:1) to afford 324 mg (67%) of carbonate 21: 1H
NMR (300 MHz, CDCl3/CD3OD) δ 1.21 (d, 3H, J ) 7.1 Hz,
CH3-Ala), 1.25-2.05 (m, 6H, CH2-Lys), 2.95 (br d, 1H, J ) 9.7
Hz, Bn Phe), 3.13 (br t, 2H, J ) 5.6 Hz, N-CH2-Lys), 3.27 (dd,
1H, J ) 4.2 Hz, J ) 16.2 Hz, Bn Phe), 4.08 (q, 1H, J ) 7.1 Hz,
HR), 4.25 (dd, 1H, HR), 4.30-4.65 (m, 5H, HR and 4 Aloc),
5.04-5.35 (m, 4H, Aloc), 5.26 (s, 2H, CH2-OH), 5.65-6.00 (m,
2H, Aloc), 7.10-7.35 (m, 5H, aromatic), 7.39-7.43 (2 * d, 4H,
aromatic), 7.71 (d, 2H, J ) 8.3 Hz, aromatic), 8.28 (d, 2H, J )
9.1 Hz, aromatic) ppm; MS (FAB) m/e 803 (M + H)+, 825 (M
+ Na)+.
Gen er a l P r oced u r e for th e Cou p lin g of Doxor u bicin
a n d Da u n or u bicin to Ca r bon a tes 21 a n d 22. Aloc-D-Ala -
P h e-Lys(Aloc)-P ABC-DAU (23). 4-Nitrophenyl carbonate 21
(51 mg, 0.0635 mmol) and daunorubicin-HCl (37.6 mg, 1.05
equiv) in N-methylpyrrolidinone were treated at room tem-
perature with triethylamine (10.0 µL, 1.05 equiv). The reaction
mixture was stirred in the dark for 16 h and was then diluted
with 10% 2-propanol/ethyl acetate. The organic layer was
washed with brine and water and was dried (Na2SO4). After
evaporation of the solvents, the crude product was purified
by means of circular chromatography using a chromatotron
supplied with a 2 mm silica plate (chloroform-methanol; 60:1
and 20:1 respectively) to yield 73 mg (97%) of 23: mp 119-
125 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.23 (d, 3H, J )
6.6 Hz, sugar CH3), 1.26 (d, 3H, J ) 6.6 Hz, CH3-Ala), 1.25-
2.00 (m, 7H, CH2-Lys and 2′), 2.10 (dd, 1H, J ) 4.1 Hz, J )
Aloc-D-Ala -P h e-Lys(Aloc)-P ABC-DOX (24): yield (76%);
mp 124 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.22 (d, 3H,
J ) 7.2 Hz, sugar CH3), 1.28 (d, 3H, J ) 6.6 Hz, CH3-Ala),
1.25-2.00 (m, 8H, CH2-Lys and 2′), 2.15 (dd, 1H, J ) 3.3 Hz,
J ) 13.9 Hz, 8), 2.35 (br d, 1H, 8), 3.05 (br d, 1H, J ) 18.9 Hz,
10), 2.90-3.35 (m, 5H, Bn Phe and N-CH2-Lys and 10), 3.61
(m, 1H, 4′), 3.73 (m, 1H, 3′), 3.85 (m, 1H, HR), 4.08 (s, 3H,
OMe), 4.07-4.32 (m, 2H, HR and 5′), 4.35-4.70 (m, 5H, HR
and 4 Aloc), 4.76 (s, 2H, 14), 4.97 (m, 2H, Bn spacer), 5.03-
5.38 (m, 4H, Aloc), 5.49 (br s, 1H, 1′), 5.55 (br d, 1H, J ) 8.3
Hz, 7), 5.60-6.00 (m, 2H, Aloc), 7.05-7.35 (m, 7H, aromatic),
7.41 (d, 1H, J ) 8.2 Hz, 3), 7.56 (d, 2H, J ) 7.3 Hz, aromatic),
7.79 (t, 1H, J ) 8.2 Hz, 2), 8.04 (d, 1H, J ) 7.6 Hz, 1) ppm;
MS (FAB) m/e 1230 (M + Na)+. Anal. (C61H70N6O20‚31/2H2O)
calculated C 57.68%, H 6.11%, N 6.62%; measured C 57.77%,
H 5.74%, N 6.19%.
D-Ala -P h e-Lys-P ABC-DOX(‚2HCl) (4): yield (96%); mp
>320 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.21 (d, 3H,
J ) 7.0 Hz, sugar CH3), 1.29 (d, 3H, J ) 6.4 Hz, CH3-Ala),
1.30-2.10 (m, 8H, CH2-Lys and 2′), 2.18 (m, 1H, 8), 2.36 (d,
1H, J ) 14.1 Hz, 8), 2.80-3.40 (m, 6H, Bn Phe and N-CH2-
Lys and 10), 3.62 (br s, 1H, 4′), 3.91 (m, 1H, 3′), 4.00-4.15 (m,
4H, OMe and 5′), 4.21 (br d, 1H, J ) 7.3 Hz, HR), 4.51 (m, 1H,
HR) 4.64 (m, 1H, HR), 4.77 (s, 2H, 14), 4.97 (2 * d, 2H, J )
11.9 Hz, Bn spacer), 5.29 (br s, 1H, 1′), 5.49 (br s, 1H, 7), 7.10-
7.35 (m, 7H, aromatic), 7.44 (d, 1H, J ) 8.3 Hz, 3), 7.54 (d,
2H, J ) 8.4 Hz, aromatic), 7.82 (t, 1H, J ) 8.1 Hz, 2), 8.04 (d,
1H, J ) 7.8 Hz, 1) ppm; MS (FAB) m/e 1039 (M + H)+. Anal.
(C53H62N6O16‚4.7HCl) C, H, N.
Aloc-D-Ala -P h e-Lys(Aloc)-P AB(Cl)-OH (20): yield (55%);
mp 173 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.23 (d, 3H,
J ) 7.1 Hz, CH3-Ala), 1.25-2.00 (m, 6H, CH2-Lys), 2.99 (dd,
1H, J ) 9.2 Hz, J ) 13.8 Hz, Bn Phe), 3.14 (br d, 2H, J ) 5.7
Hz, N-CH2-Lys), 3.25 (dd, 1H, J ) 4.3 Hz, Bn Phe), 4.07 (br d,
1H, J ) 7.0 Hz, HR), 4.30 (dd, 1H, J ) 5.0 Hz, HR), 4.35-4.65
(m, 5H, HR and 4 Aloc), 4.69 (s, 2H, CH2-OH), 5.10-5.31 (m,
4H, Aloc), 5.65-6.00 (m, 2H, Aloc), 7.10-7.32 (m, 5H, aro-
matic), 7.37-7.49 (m, 2H, aromatic), 7.79 (br s, 1H, aromatic)

5282 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Brief Articles
ppm; MS (FAB) m/e 672 (M + H)+, 694 (M + Na)+. Anal.
(C33H41N5O8Cl‚H2O) C, H.
cells in 24 h were cultured in HEPES buffered Dulbecco’s
Modified Eagle’s Medium (DMEM) containing 1 g/L glucose
and Glutamax, supplemented with 10% (v/v) heat-inactivated
fetal calf serum, 100 IU/mL penicillin and 100 µg/mL strep-
tomycin. All cells were grown at 37 °C in a humid atmosphere
with 5% (v/v) CO2. At the beginning of the experiment 5 ×
104 MCF-7 and MCF-7.WT.u-PA cells were seeded into wells
of a 24-well plate. The medium was aspirated the next day
and replaced with 500 µL of fresh medium containing 10%
FCS, 0.15 µM human plasminogen, and (pro)drugs 4, 5, and
doxorubicin in a concentration of 0, 0.1, 0.3, 1, 3, 10, 20, 40,
60, 80, 100, and 150 µM. After 24 h, medium was aspirated
and cells were fixed with 0.5 mL of 2.5% (v/v) glutaraldehyde
and stained with 0.2% (v/v) crystalviolet. The area covered by
cells was determined by video microscope analysis (Optimas
software). Cultures without doxorubicin or analogue were used
as 100% control. From dose-response curves for doxorubicin
and both prodrugs ID50 values were determined, and the
results were averaged from three experiments. The bars in
Figure 1 indicate the standard error of the mean (SEM).
Aloc-D-Ala -P h e-Lys(Aloc)-P ABC(Cl)-P NP (22): yield
1
(96%); H NMR (300 MHz, CDCl3) δ 1.31 (d, 3H, J ) 6.8 Hz,
CH3-Ala), 1.15-2.10 (m, 6H, CH2-Lys), 2.90-3.40 (m, 4H,
N-CH2-Lys and Bn Phe), 4.02 (m, 1H, HR), 4.21 (m, 1H, HR),
4.36 (dd, 1H, J ) 5.5 Hz, J ) 13.1 Hz, HR), 4.45-4.75 (m, 4H,
Aloc), 4.95-5.35 (m, 4H, Aloc), 5.36 (s, 2H, CH2-OH), 5.66 (m,
1H, Aloc), 5.91 (m, 1H, Aloc), 7.10-7.45 (m, 9H, aromatic),
7.61 (br d, 1H, aromatic), 7.97 (br s, 1H, aromatic), 8.27 (d,
2H, J ) 9.1 Hz, aromatic) ppm; MS (FAB) m/e 837 (M + H)+,
859 (M + Na)+. Anal. (C40H44N6O12Cl‚11/4H2O) calculated C
55.94%, H 5.46%, N 9.79%; measured C 56.32%, H 5.42%, N
9.31%.
Aloc-D-Ala -P h e-Lys(Aloc)-P ABC(Cl)-DOX (25): yield
1
(75%); mp 111 °C; H NMR (300 MHz, CDCl3/CD3OD) δ 1.23
(d, 3H, J ) 6.9 Hz, sugar CH3), 1.28 (d, 3H, J ) 6.5 Hz, CH3-
Ala), 1.20-2.05 (m, 8H, CH2-Lys and 2′), 2.14 (dd, 1H, J )
4.1 Hz, J ) 14.9 Hz, 8), 2.35 (br d, 1H, 8), 2.90-3.35 (m, 6H,
Bn Phe and N-CH2-Lys and 10), 3.63 (br s, 1H, 4′), 3.86 (m,
1H, 3′), 4.06 (s, 3H, OMe), 4.00-4.35 (m, 3H, 2 HR and 5′),
4.35-4.75 (m, 5H, HR and 4 Aloc), 4.76 (s, 2H, 14), 5.00-5.38
(m, 6H, Bn spacer and 4 Aloc), 5.48 (br s, 1H, 1′), 5.59 (br d,
1H, J ) 8.5 Hz, 7), 5.55-6.05 (m, 2H, Aloc), 7.05-7.35 (m,
6H, aromatic), 7.28-7.47 (m, 2H, aromatic), 7.70-7.85 (m, 2H,
aromatic), 8.01 (d, 1H, J ) 7.7 Hz, 1) ppm; MS (FAB) m/e 1263
(M + Na)+. Anal. (C61H69N6O20Cl‚4H2O) calculated C, H, N
6.40%; measured C, H, N 5.93%.
Ack n ow led gm en t. We are grateful to Pharma-
chemie BV, Haarlem, for financial support.
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