The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater target efficacy

Article abstract of DOI:10.1021/jm0101996

Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin- like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27 for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

Full text of DOI:10.1021/jm0101996

4216
J . Med. Chem. 2001, 44, 4216-4224
Th e Syn th esis of a P r od r u g of Doxor u bicin Design ed to P r ovid e Red u ced
System ic Toxicity a n d Gr ea ter Ta r get Effica cy
Victor M. Garsky,*,† Patricia K. Lumma,† Dong-Mei Feng,† J enny Wai,† Harri G. Ramjit,† Mohinder K. Sardana,‡
Allen Oliff,§,| Raymond E. J ones,§ Deborah DeFeo-J ones,§ and Roger M. Freidinger†
Departments of Medicinal Chemistry, Biological Chemistry, and Cancer Research, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received May 4, 2001
Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited
because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The admin-
istration of a prodrug of doxorubicin, designed to permit selective activation by the tumor,
would reduce general systemic exposure to the active drug and would thereby increase the
therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like
activity that is a member of the kallikrein gene family. PSA’s putative physiological role is the
liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins
I and II. Serum PSA levels have been found to correlate well with the number of malignant
prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should
in principle produce high localized concentrations of the cytotoxic agent at the tumor site while
limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human
semenogelin were constructed. Systematic modification of the amino acid residues flanking
the primary cleavage site led to the synthesis of a series of short peptides which were efficiently
hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series
of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs
for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-
Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and
biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate
PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse
xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its
MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and
tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27
for further study to assess its ability to inhibit human prostate cancer cell growth and
tumorigenesis.
In tr od u ction
drug and would thereby increase the therapeutic index.
This strategy in turn will permit delivery of an increased
amount of active drug to the target site, thereby
increasing efficacy.
Prostate cancer is the second leading cause of cancer
mortality in males. It is estimated that 37 000 men died
of prostate carcinoma in the United States in 1999.1
While cancer that is confined to the prostate can be
treated with surgery or radiation, for metastatic disease
the prognosis is poor. There is currently no effective
therapy that significantly extends survival for men with
androgen refractory prostate cancer, although palliative
therapy is commonly employed.2
The current therapy for metastatic prostate cancer,
a combination of mitoxantrone and prednisone, provides
response rates of 20 to 30%.3 Doxorubicin (I) has limited
utility in prostate cancer due to systemic toxicities,
consisting primarily of cardiotoxicity and myelosuppres-
sion.4 The administration of a prodrug of doxorubicin,
designed to permit selective activation by the tumor,
would reduce general systemic exposure to the active
The concept of using a prodrug to achieve targeted
delivery rather than oral bioavailability was, to our
knowledge, first employed with anticancer drugs and
later also with other medications. We, as well as others,
have considered using the enzyme prostate specific
antigen (PSA) to convert an inactive doxorubicin pro-
drug into the active drug at the site of the tumor.5,6,7
PSA is a serine protease with chymotrypsin-like
activity that is a member of the kallikrein gene family.8,9
PSA’s putative physiological role is the liquefaction of
semen by virtue of its ability to cleave the seminal fluid
proteins semenogelins I and II.10 Clinically PSA is best
known as a serological marker for the presence of
prostate cancer. Serum PSA levels have been found to
correlate well with the number of malignant prostate
cells, and higher levels of PSA are indicative of meta-
static disease.11 Importantly, PSA which is secreted into
systemic circulation lacks enzymatic activity because it
forms a complex with R1-antichymotrypsin and R2-
macroglobulin.12,13 This loss of enzymatic activity does
* Merck Research Laboratories, WP14-3, West Point, PA 19486.
Phone: 215-652-3110. Fax: 215-652-3971. E-mail: vic_garsky@
merck.com.
† Department of Medicinal Chemistry.
‡ Department of Biological Chemistry.
§ Department of Cancer Research.
| Current address: GlaxoSmithKline, King of Prussia, PA 19406.
10.1021/jm0101996 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/31/2001

Synthesis of a Doxorubicin Prodrug
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4217
Ta ble 1. Synthetic Peptides and Analogues from Cleavage
not, however, affect the utility of complexed PSA as a
serological marker for prostate cancer since the immu-
noreactivity is not lost. The use of a prodrug which is
cleaved by the enzyme PSA should in principle produce
high localized concentration of the cytotoxic agent at the
tumor site while limiting systemic exposure to the active
drug.
Cleavage maps following PSA treatment of human
semenogelin I have been constructed by us as well as
others.14 Isolation of the cleavage fragments allowed us
to determine that the most efficient cleavage occurs
between Gln349 and Ser350 in semenogelin I. Systematic
modification of the amino acid residues flanking this
cleavage site led to the synthesis of a series of short
peptides which were efficiently hydrolyzed by PSA.
Subsequent coupling of selected peptides through their
C-terminal carboxyl group to the amino group of doxo-
rubicin provided a series of doxorubicin-peptide conju-
gates which were evaluated in vitro and in vivo as
targeted prodrugs for PSA secreting tumor cells. On the
basis of these studies, we selected Glutaryl-Hyp-Ala-
Ser-Chg-Gln-Ser-Leu-Dox, 27, for further study to as-
sess its ability to inhibit human prostate cancer cell
growth and tumorigenesis.
Site-1 (CS-1) of Semenogelin Ia
PSA
T1/2
sequences
(min)
semenogelin I (1-462)
<10
25
1
H-Ser-Gln-Lys-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ser-Ser-
Thr-Glu-Glu-Arg-Arg-Leu-His-Tyr-Gly-Glu-Asn-Gly-OH
H-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ser-Ser-Thr-Glu-OH
H-Ser-Tyr-Gln -Ser -Ser-Ser-Thr-Glu-OH
2
3
4
5
6
7
8
9
105
>180
>240
H-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ser-OH
Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ser-Ser-Thr-Glu-NH2 >240
Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ala-Ser-Thr-Glu-NH2
Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ser-Lys-Thr-Glu-NH2
Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln -Ser -Ala-Lys-Thr-Glu-NH2 125
Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Tyr -Ser -Ser-Ser-Thr-Glu-NH2 150
145
110
10 Ac-Ala-Asn-Lys-Ala-Ser-Tyr-Gln -Ser -Ser-Ser-Thr-Glu-NH2 105
11 Ac-Ala-Asn-Lys-Ala-Ser-Tyr-Gln -Ser -Ala-Ser-Thr-Glu-NH2
50
a
PSA cleavage site between the two amino acids is indicated
in bold. Underlined amino acids indicate position of substitution.
T1/2) time required for PSA to hydrolyze 50% of compound at a
molar ratio of 1:100, respectively. The structure of semenogelin I
is presented in the Supporting Information figure.
also appears to be a preference for Ser or Gln at the P1′
site (N-terminal) at all the major cleavage points. PSA’s
selective hydrolysis on the C-terminal side of Gln
appears to be unique among known proteases. Impor-
tantly this selective Gln cleavage represents an advan-
tage toward the development of PSA selective prodrugs
in that nonspecific proteolysis would be minimized. On
the basis of these considerations, a series of peptides
was prepared whose sequences flanked CS-1. The pep-
tides were tested as substrates for PSA to determine
their relative efficiencies for hydrolysis by PSA (Table
1). The results are expressed as the time required for
PSA to hydrolyze 50% of the peptide (T1/2). Human
semenogelin I was shown to have a T1/2 of less than 10
min. The 25-mer, 1, which represents P10-P15′ from
CS-1 demonstrates significant cleavage with a 25 min
T1/2. Truncation to the 12-mer, 2, increases the T1/2 to
105 min. Further deletion to the 8-mer, 3, and the
9-mer, 4, dramatically increases the T1/2 to greater than
180 and 240 min, respectively. Anticipating that ulti-
mately blocked C and N termini would be desirable in
the context of a peptide conjugate (to minimize nonspe-
cific exopeptidase hydrolysis), the 12-mer, sequence 2,
was prepared as the N-acetyl C-terminal amide 5. The
T1/2 for 5 was >240 min which represented a less
effective substrate relative to the unblocked 2. Never-
theless we chose this structure, 5, as a starting point
for the development of improved substrates.
Resu lts a n d Discu ssion
Design of P r od r u g, 27. The initial steps in design-
ing a potentially useful doxorubicin prodrug were
2-fold: first, to identify a functionality on doxorubicin
where conjugation with a peptide fragment would yield
a biologically poorly active compound, and second, to
identify a peptide substrate that, when conjugated to
doxorubicin, would be converted by PSA into the active
drug, doxorubicin, in high yield. The coupling of Ac-Ala-
Ala-Ala-OH to form an amide, 14, with the aminogly-
coside of doxorubicin abolished doxorubicin’s cytotoxic
activity on PSA secreting (LNCaP) and PSA nonsecret-
ing (DuPRO) cells (Table 3). This observation achieved
our first objective in that attachment of a peptide to the
aminoglycoside of doxorubicin rendered doxorubicin
inactive to these prostate cancer cell lines. The second
design issue would be addressed with the discovery of
an appropriate peptide substrate which would be linked
to doxorubicin. As will become apparent from the
discussion that follows, the selection of the peptide
fragment to be coupled to doxorubicin was far from
trivial.
The amino acid sequence of human semenogelin I, the
physiological substrate for PSA, was determined in our
laboratories (see Methods section) as well as in others.10
Recombinant human semenogelin I was prepared and
digested with a highly purified preparation of human
PSA. The proteolytic digestion fragments were isolated
via acrylamide gel electrophoresis and their structures
determined by sequencing from the amino terminus.
Five unique amino termini were identified and are
indicated as CS-1 (cleavage site 1) through CS-5 (Sup-
porting Information). The cleavage sites are numerically
ranked according to the number of nanomoles of peptide
produced during a 5 min cleavage reaction, with CS-1
representing the most abundant species found. Se-
menogelin I was shown to be primarily cleaved at the
C-terminus of Gln (CS-1, CS-3) and to a lesser degree
at the C-terminus of Ser and Tyr (CS-2, CS-5). There
A series of analogues based on 5 was prepared in
which libraries at P1′, P2′, P3′ and P4′ (1L-4L) were
prepared by the simultaneous incorporation of an
equimolar mixture of 19 common natural amino acids
in their protected forms (Table 2). Synthetic details and
data providing evidence for the generation of 19 com-
ponent equimolar peptide libraries were presented in a
previous report.15 The four individual libraries were
subjected to PSA digestion for 60 min. Following quench-
ing, the digests were evaluated by automated Edman
sequence analysis. Because only those peptides which
are cleaved by PSA would give products with a free
amino terminus, we were able to determine which
residues were most susceptible to PSA hydrolysis.
The preferred amino acid in P1′ proved to be Ser, it
was gratifying to find, the same as in the native
sequence, 1L. At P2′, Ala and Tyr were preferred over

4218 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Garsky et al.
Ta ble 2. Preferred Substitution Based on PSA Digestion of 19
Component P1′-P4′ Peptide Libraries
Syn th esis of Con ju ga tes
Peptide doxorubicin conjugates were prepared based
on, in part, structural data derived from the above
studies. Synthesis of the peptide-doxorubicin conjugates
utilized a combination of solid-phase and solution
chemistries, Scheme 1. The peptides were prepared by
solid-phase synthesis using a Boc strategy on 4-(oxym-
ethyl)-phenylacetamidomethyl (PAM) resin. The cleav-
age of the peptides from the resin and removal of the
side chain protecting groups were carried out in anhy-
drous HF. Where Orn, Lys, or glutaric acid were
present, Fmoc was used for side chain protection of the
amino group and OFm (fluorenylmethyl ester) on the
carboxyl of glutaric acid, respectively. Both groups are
stable to HF. The peptides were used without purifica-
tion in coupling of the C-terminal carboxyl to the
doxorubicin daunosamine nitrogen using a variety of
coupling agents (see Methods). Following coupling, those
compounds which required removal of the Fmoc or OFm
protecting group were treated with 20% piperidine in
DMF at 0 °C. The peptide conjugates were purified
using reverse phase high-performance liquid chroma-
tography (RP-HPLC) with a Waters C18 Delta Pak (15
µm, 100 Å) column. Identity and purity of the final
products were determined by amino acid analysis,
HPLC, and FAB mass spectroscopy.
preferred
sequencea
substitnb
1L Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln-(19Xaa)-
Ser-Ser-Thr-Glu-NH2
P1′ ) Ser
2L Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln-Ser-(19Xaa)-
P2′ ) Ala,
Tyr
Ser-Thr-Glu-NH2
3L Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln-Ser-Ser-(19Xaa)- P3′ ) Lys
Thr-Glu-NH2
4L Ac-Ala-Asn-Lys-Ile-Ser-Tyr-Gln-Ser-Ser-Ser-
P4′ ) Leu,
Ile
(19Xaa)-Glu-NH2
a
19Xaa ) equimolar mixture of each of the following amino
acids: Ala,Arg,Asn,Asp,Gln,Glu,Gly,His,Ile,Leu,Lys,Met,Phe,Pro,
Ser,Thr,Trp,Tyr,Val. As determined by automated N-terminal
b
sequence analysis following PSA hydrolysis.
the native Ser, 2L. Lys at P3′ was better relative to Ser,
3L, and at P4′ Leu and Ile were preferred over native
Thr, 4L. During the analysis of these libraries we only
observed the formation of cleavage products related to
the hydrolysis following Gln at P1. This observation
further confirmed the selectivity of PSA for Gln. The
singly prepared 12-mer, 6, in which Ala substitutes for
Ser at P2′, exhibited an improved T1/2 of 145 min.
Replacement of Ser at P3′ with Lys, 7, reduced the T1/2
further to 110 min. However, double substitution with
Ala at P2′ and Lys at P3′, 8, did not provide an additive
effect to PSA hydrolysis with 8 showing a 125 min T1/2
.
PSA hydrolysis of four peptide libraries (P1′-P4′) fol-
lowed by automated Edman sequence analysis allowed
us to efficiently determine the effect of point substitution
relative to cleavage rates. However, a similar approach
was not as readily accessible for evaluating P1-P4
substitution since PSA hydrolysis would not generate
a unique set of amino terminal hydrolytic products. To
determine which amino acids, within the context of the
12-mer substrate, 5, most contributed to improved PSA
cleavage, we prepared single point substitution ana-
logues of 5 for P1 through P4 positions using the
Advanced ChemTech (Louisville, KY) 396 automated
multiple peptide synthesizer. The same set of 19 amino
acids was introduced, using the manufacturer’s Fmoc
chemistry protocols, individually in P1 through P4.
Following isolation from the resin, the 76 peptides,
without further purification, were treated with PSA to
determine their relative rates of cleavage (see Experi-
mental Section for T1/2 determinations). From this study
we determined in a qualitative assessment that the
preferred amino acids (based on T1/2 values) at P1 are
Gln, Tyr, Phe, and Asn; at P2 they are Tyr and Leu; at
P3 Ser and Lys; and at P4 Ala, Ser, and Thr. Confirma-
tion of these results was provided in part by purification
of selected peptides and determination of their T1/2. Tyr
replacement at P1 provided 9, with an improved T1/2 of
150 min, while Ala at P4 produced a compound 10, with
a T1/2 of 105 min (Table 1). By combining the T1/2
enhancing properties of Ala replacement at P2′ (8) and
Ala at P4 (10) a substrate, 11, was synthesized which
had a dramatically improved T1/2 of 50 min (Table 1).
As in the peptide libraries described above, only PSA
cleavage products ascribed to the P1-P1′ site were
observed. These results demonstrate that peptide sub-
strates with enhanced PSA selective cleavage rates
relative to a native sequence can be prepared using a
systematic approach of amino acid substitution.
Biologica l Resu lts
Cytotoxic activities of the peptide-doxorubicin conju-
gates were evaluated in cell culture. Both PSA produc-
ing, LNCaP, and PSA nonproducing, DuPRO, human
prostate cancer cell lines were used for analysis. Table
3 summarizes the results of these assays. EC50 values
report the compound dose required to kill 50% of the
tumor cells. Doxorubicin (Dox) and leucyldoxorubicin
(Leu-Dox), 13, represent the active cytotoxic species
generated from the peptide-doxorubicin conjugates fol-
lowing PSA cleavage of the Gln-Ser (P1-P1′) bond with
subsequent hydrolysis within the protease rich environ-
ment of the prostate cancer cells (data not shown). PSA
T
1/2 cleavage times for the conjugates are also presented
in Table 3.
Leu-Dox, 13, has been shown to be cytotoxic in a
number of cancer cell lines, and its pharmacokinetics
have been studied in clinical trials.16,17 Our previous
report as well as data presented herein (Table 3) show
that Leu-Dox, 13, is also cytotoxic to prostate cancer
cell lines.7 From our peptide library studies we deter-
mined that Leu was a preferred amino acid replacement
at P4′ (Table 2). In light of these observations, that is,
Leu-Dox’s cytotoxicity to prostate cancer cells and its
preference in the context of a peptide substate for PSA,
we concluded that Leu is an appropriate doxorubicin
linker in an extended peptide substrate. Coupling of the
aminoglycoside of doxorubicin to the peptide sequence,
Ac-AlaAsnLys(Nꢀ-Fmoc)AlaSerTyrGlnSerSerSerLeu-
OH (15a ), followed by removal of the Nꢀ-Fmoc group
provided the conjugate 15 (Table 3). The data show that
15 has a T1/2 of 30 min which represents a significant
improvement relative to the unconjugated peptide sub-
strates, 2-11 (Table 1), the best of which gave a T1/2 of
50 min (11). In cell culture, selective cytotoxic activity
of 15 for PSA-secreting vs non-PSA-secreting cells was
demonstrated (Table 3). The EC50 for LNCaP cells,

Synthesis of a Doxorubicin Prodrug
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4219
Sch em e 1
Ta ble 3. Biological Data
in vitro
EC50 (µM)
LNCaPb DuPROc reduction* wgt reduc.** (µmol/kg) (µmol/kg)
in vivo
a
T1/2
min
% PSAd
% tumord
dose
MTDf
doxorubicin-conjugate structure
doxorubicin (Dox)
0.7
1.8
16
6
2.8
2.8
12 H-Ser-Leu-Doxg
13 H-Leu-Dox
14 Ac-Ala-Ala-Ala-Dox
6.7
>100
7.0
14.0
>100
117
10
ND
ND
ND
87
90
93
ND
70
22
95
ND
96
94
95
(5)e
7.6
7.6
15 Ac-Ala-Asn-Lys-Ala-Ser-Tyr-Gln-Ser-Ser-Ser-Leu-Dox
16 Ac-Ala-Asn-Lys-Ala-Ser-Tyr-Gln-Ser-Ala-Ser-Leu-Dox
17 Ac-Ala-Asn-Lys-Ala-Ser-Tyr-Gln-Ser-Ser-Leu-Dox
18 Ac-Lys-Tyr-Gln-Ser-Ser-Ser-Leu-Dox
19 Ac-Orn-Tyr-Gln-Ser-Ser-Ser-Leu-Dox
20 Ac-Lys-Ala-Ser-Tyr-Gln-Ser-Leu-Dox
21 Ac-Orn-Ala-Ser-Tyr-Gln-Ser-Leu-Dox
22 Ac-Orn-Ala-Ser-Tyr-D-Gln-Ser-Leu-Dox
23 Ac-Lys-Ala-Ser-Chg-Gln-Ser-Leu-Dox
24 Ac-Pro-Ala-Ser-Chg-Gln-Ser-Leu-Dox
25 Ac-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox
26 Succinyl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox
27 Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox
30
30
100
60
120
30
12.5 >100
6.3 >100
6.9 >100
7.0 >100
81
70
80
36.6
29.0
29.3
>36.6
32.6
33.0
4.8
100
40
>1200
7.3 >100
57
26
73
14.8
18.9
22.4
29.7
>45.3
29.9
85
>100
4.5 >100
7.4 >100
4.5 >100
10
45
20
55
81
72
87
22.7
21.7
21.5
22.7
28.9
28.6
5.7
5.0 >100
100
30
a
b
T1/2 ) time required for PSA to hydrolyze 50% of compound at a molar ratio of 1:100, respectively. LNCaP: PSA secreting human
prostate cancer cell line. c DuPRO: nonsecreting PSA human prostate cancer cell line. % PSA and % tumor weight reduction relative
d
to vehicle control following intraperitoneal injection (ip) of conjugate (dose) for 5 consecutive days to nude mice bearing LNCaP tumor
xenografts. e 5% increase in tumor weight (P ) 0.925). f MTD, defined as maximum nonlethal drug dose which could be administered (ip)
g
to nontumor bearing nude mice for 5 consecutive days. Compound was not tested due to its insolubility. *P (values): Dox (0.508); 13
(0.360); 17 (0.009); 18 (0.007); 19 (0.001); 21 (0.016); 22 (0.112); 23 (0.001); 25 (<0.001); 26 (<0.001); 27 (<0.001). **P (values): Dox
(0.868); 13 (0.925); 17 (0.006); 18 (0.002); 19 (0.002); 21 (0.006); 22 (0.518); 23 (0.016); 25 (0.001); 26 (<0.001); 27 (<0.001).
which secrete PSA, was 7.0 µM while the EC50 for the
non-PSA secreting DuPRO cell line was 117 µM. Leu-
Dox shows comparable cell kill activity against LNCaP
cells, EC50 ) 6.7 µM, with little selectivity vs DuPRO
cells, EC50 ) 14.0 µM. Doxorubicin was also shown to
be minimally selective against the two cell lines. These

4220 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Garsky et al.
results provide clear evidence that one can achieve
selective targeting, in vitro, of PSA secreting human
cancer cells through the use of a peptide-doxorubicin
conjugate.
Our objective was to further show efficacy against
PSA secreting tumors in vivo. Continued SAR studies
focused on the development of peptide-Dox conjugates
having a minimum molecular weight with the following
properties: high selective potency against PSA secreting
cells, a rapid specific rate of hydrolysis by PSA, resis-
tance to nonspecific proteolysis, aqueous solubility, and
efficacy against PSA-secreting tumors.
minimizing this effect have been described.18,19 We were
able to eliminate the histamine effect in the dipeptidyl
P′ series by substituting the basic Lys or Orn at P5 with
the secondary amino acid Pro (24) without compromis-
ing cytotoxic activity (Table 3). However, with the
removal of the positive charge associated with Lys or
Orn, the aqueous solubility of 24 was reduced to the
degree that further development was difficult. While the
introduction of the hydrophilic amino acid Hyp (25)
provided a conjugate with improved solubility, it was
not quite soluble enough for iv formulation. The intro-
duction of a succinyl group at the N-terminus, in place
of the acetyl group (affording 26), was designed to
improve solubility. While good aqueous solubility was
achieved, it resulted also in an increase in the T1/2 from
20 min (25) to 55 min (26). We found, however, that
use of a glutaryl group afforded 27 which displayed both
good solubility and a T1/2 of 30 min.
The property of being a substrate for PSA is a
requirement for antitumor activity of 27 if it functions
by the proposed biological mechanism underlying this
concept. An additional test of this hypothesis was to
prepare a peptide-doxorubicin conjugate that was not
a good substrate for PSA and to test that compound for
antitumor activity against PSA-producing tumors in
vivo. By incorporating the D-stereoisomer of Gln, the
structurally related conjugate, Ac-Orn-Ala-Ser-Tyr-D-
Gln-Ser-Leu-Dox (22), was specifically designed not to
be a substrate for cleavage by PSA. In fact 22 failed to
show any evidence of cleavage on exposure to PSA for
up to 4 h (Table 3). Additionally, when tested in tumor
xenograft studies, 22 was no more effective than doxo-
rubicin against PSA secreting tumors.7
The SAR studies were continued with Ala substitution
for Ser at P2′ to give 16. This analogue, 16, did not
provide an improvement in the T1/2 relative to 15 as did
a comparable double Ala (P4, P2′) replacement in the
peptide, 11 (Table 1). Truncation of a P′ Ser in 15
produced 17 with a 3-fold loss in the T1/2; however, its
selectivity profile against PSA secreting LNCaP cells
remained. Continued deletion studies combined with
point substitution provided two series of 7-mers having
a favorable profile of in vitro cell kill and in vivo
antitumor activity (Table 3). Coincident with the dele-
tion studies, we introduced the unnatural amino acid
cyclohexylglycine, Chg, as a substitution for Tyr at P2.
This substitution was designed to stabilize the conju-
gates to undesired proteolytic hydrolysis by chymot-
rypsin-like proteases in plasma. Gratifyingly the con-
jugates with Chg as a substitution for Tyr maintained
their selective PSA cleavage profile and in some cases
had an improved T1/2 as in the case of 23 vs 20 (Table
3).
The antitumor activity of these conjugates as well as
Leu-Dox and doxorubicin was determined by assessing
the ability of each compound to suppress the growth of
human prostate cancer cell (LNCaP) xenografts in nude
mice. Serum PSA levels and tumor weights were
determined at the completion of the study. Table 3
reports the percent PSA reduction relative to vehicle
control following conjugate treatment. Tumor weight
loss correlates directly with the level of PSA reduction.
Details describing the experimental biology were re-
cently reported (also Table 3 footnotes).7
The first series of 7-mers as represented by 18 and
19 have an extended four amino acid P′ region. The
second series of 7-mers, starting with 20, have only two
amino acids, Ser and Leu, in P′ linking doxorubicin. We
believe that the shorter P′ may, in principle, offer a
biological advantage in that minimal processing post
PSA cleavage is necessary to liberate active cytotoxic
Leu-Dox and doxorubicin from the intermediate H-Ser-
Leu-Dox (12).
Both series of conjugates are potent inhibitors of
tumor growth. However, we discovered that the ex-
tended P′ series (18, 19) produced an unexpected and
undesirable hypotensive effect which was due to hista-
mine release following iv administration in dogs (data
not shown). Interestingly neither doxorubicin nor the
unconjugated peptide, Ac-Lys-Tyr-Gln-Ser-Ser-Ser-Leu-
NH2, displayed the histamine release associated with
the conjugates (18, 19). It has been reported that
peptides with a hydrophobic functionality in combina-
tion with basic amino groups can sometimes cause
histamine release from mast cells, and approaches to
From these studies 27 emerged as the peptide-
doxorubicin conjugate with the best profile of physical
and biological properties. Compound 27 has a greater
than 20-fold selectivity against human prostate PSA-
secreting LNCaP cells relative to the non-PSA-secreting
DuPRO cell line (Table 3). In nude mouse xenograft
studies using LNCaP prostate cancer cells, 27 reduced
PSA levels by 95% and tumor weight by 87% at a dose
below its MTD (Table 3). Both doxorubicin and Leu-Dox
(13) were ineffective in reducing circulating PSA and
tumor burden at their maximum tolerated doses (Table
3).
Su m m a r y
The initial steps in designing the potentially useful
doxorubicin prodrug, 27, were 2-fold: first to identify a
functionality of doxorubicin that when coupled to a
peptide fragment would yield a biologically inactive (i.e.,
noncytotoxic) compound, and second to identify a pep-
tide which, when coupled to doxorubicin, would be
converted by PSA into the active drug, doxorubicin. The
coupling of the tripeptide, Ac(Ala)3-OH, with I to form
14 satisfied the first of the above criteria.
The identification of an appropriate substrate resulted
from cleavage maps following PSA treatment of PSA’s
physiological substrate, semenogelin I. The most ef-
ficient cleavage site from recombinant semenogelin I
occurs between Gln349 and Ser350. Systematic modifica-
tion, through the use of chemically synthesized 19
component peptide libraries and point substitution SAR
studies of the amino acid residues flanking this cleavage

Synthesis of a Doxorubicin Prodrug
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4221
ers whose sequence was taken from the published sequence
of human semenogelin I. The semenogelin I cDNA was tagged
with a tubulin epitope (glu-glu-phe) at the 3′ end and placed
in the bacterial expression vector pGEX 2T. Semenogelin I
protein production was induced with the addition of IPTG.
Semenogelin I was purified to greater than 95% homogeneity
using a glutathione affinity column. The eluate was digested
with thrombin to remove the glutathione transferase leader
and the digest purified using an immunoaffinity column
containing the anti-tubulin antibody YL1/2. The recombinant
semenogelin I protein was used as a substrate for PSA that
was purified from human seminal fluid (York Biologicals Inc.,
StonyBrook, NY). The proteolysis reaction contained recom-
binant semenogelin I and PSA in a molar ratio of 100 to 1,
respectively, in 20 mM TRIS‚HCl, 25 mM NaCl, 0.5 mM CaCl2,
at pH 7.2. The proteolysis reaction was incubated for 5 min
and terminated by the addition of gel sample buffer and
heating to 100 °C for 5 min. The proteolytic digest was resolved
by SDS polyacrylamide gel electrophoresis and transferred via
electroblotting to a PVDF membrane. Regions of the PVDF
membrane containing protein were cut out and submitted for
amino acid sequence analysis (W. M. Keck Foundation, Bio-
technology Resource Laboratory, Yale University, New Haven,
CT). Five unique amino temini were identified and are
indicated as CS-1 (cleavage site number 1) through CS-5 in
the Supporting Information figure. The cleavage sites in the
Supporting Information figure are numerically ranked accord-
ing to the number of nanomoles of amino acid residues
generated during the 5 min cleavage reaction, with CS-1
representing the most common species found.
site, led to the design of short peptides which were
efficiently hydrolyzed by PSA.
Doxorubicin-peptide conjugates were prepared based
on, in part, structural data derived from the peptide
optimization studies described above. Synthesis of the
peptide-doxorubicin conjugates utilized a combination
of solid-phase and solution chemistries.
The conjugates were optimized for maximal PSA T1/2
cleavage rates, selectivity for PSA-secreting tumor cells,
and efficacy in nude mouse xenograft studies using PSA-
secreting human LNCaP prostate cancer cells. From
these studies 27 was found to have the most favorable
profile of physical and biological properties.
Compound 27 is effectively digested by the human
PSA enzyme with a T1/2 of 30 min while resisting
digestion by human proteases in whole blood or plasma
(data not shown). Compound 27 exhibits a >20-fold
selectivity for killing PSA-producing prostate cancer
cells versus non-PSA producing cancer cells in cell
culture (Table 3). In nude mouse xenograft studies using
human LNCaP prostate cancer cells that secrete PSA,
27 demonstrates dramatically improved antitumor ac-
tivity compared to doxorubicin or Leu-Dox as measured
by circulating PSA levels and tumor weights (Table 3).7
Additionally, 22, which is a peptide-doxorubicin con-
jugate that is not cleaved by PSA, was evaluated and
found not to exhibit antitumor activity more effective
than free doxorubicin against LNCaP tumors (Table 3).7
The PSA peptide-doxorubicin conjugate, 27, is in-
tended as an intravenous anticancer agent for the
treatment of hormone refractory prostate cancer. Com-
pound 27 is a prostate-targeted prodrug that is itself
less toxic than the parent drug until activated by
proteolytic cleavage to its active moieties, leucyl-doxo-
rubicin (Leu-Dox) and doxorubicin (Dox). The intial step
in this activation is preferentially carried out by the
serine protease PSA which is expressed in prostate
epithelial tissue but is not found at significant levels in
other tissues. Additional data supporting tumor selec-
tive delivery of doxorubicin by the PSA targeted peptide
conjugate, 27, in a nude mouse xenograft model of
human prostate cancer, was recently presented.20 In
theory, 27 should circulate freely in the body and be
preferentially activated at sites of prostate cancer tissue,
including metastatic foci, by PSA. Therefore, 27 should
exhibit better antitumor activity against prostate can-
cers than free doxorubicin without enhanced toxicities.
T
1/2 Deter m in a tion : Tim e to 50% Clea va ge of P ep tid es
a n d P ep tid e-Dox Con ju ga tes by P u r ified P SA. Oligopep-
tides were mixed with PSA at a molar ratio of 100 to 1,
respectively, in 50 mM TRIS‚HCl and 140 mM NaCl at pH
7.4. The reaction was incubated for 0, 30, 60, 120, 180, or 240
min at 37 °C. The reactions were quenched with ZnCl2 (10 mM
final concentration), and the products were analyzed by HPLC
on a Vydac C18 column using a 5% to 50% gradient of 0.1%
aqueous TFA-acetonitrile. The chromatograms were moni-
tored at 210 nM and 479 nM in order to follow the peptide
and doxorubicin components, respectively, of the peptide-
doxorubicin conjugates. The differential area of the substrate
peak versus the area of the untreated prodrug peak was used
as a measurement of peptide hydrolysis. Results are reported
as the time required for PSA to hydrolyze 50% of the initial
substrate.
Gen er a l Meth od s for Syn th esizin g P ep tid es a n d P r o-
tected P ep tid e In ter m ed ia tes. All of the peptides were
synthesized by Merrifield solid-phase peptide synthesis using
a double-coupling protocol on the Model 430A Applied Biosys-
tems peptide synthesizer (Foster City, CA). The resin for the
synthesis of carboxy terminal peptide amides was p-methyl-
benzhydrylamine (MBHA) and 4-(oxymethyl)-phenylacetami-
domethyl (PAM) for carboxy terminal peptides (purchased from
ABI). NR-Boc-protected amino acids of the L-configuration and
reagents were supplied by the manufacturer. Protected NR-
Boc amino acids of the D-configuration as well as NR-Fmoc
amino acids were purchased from Bachem or prepared by
standard methods. Side-chain protection was Arg (Tos), Asp
(OcHex), Glu(OcHex), His(Bom), Lys(Cl-Z), Orn(Cl-Z), Ser(Bzl),
Thr(Bzl), Tyr(Br-Z). Amino acids were coupled in NMP as a
solvent using dicyclohexylcarbodiimide (DCC) and hydroxy-
benzotriazole (HOBT). ABI software version 1.40 for HOBT/
NMP cycles was used starting with 0.5 mmol of resin and a
4-fold excess of activated protected amino acid. Deprotection
and removal of the peptides from the resin support were
effected using anhydrous HF with 10% anisole at 0° for 1 h.21
The Fmoc and fluorenyl methyl ester (OFm) protecting groups
were not removed by HF. Peptides were purified by prepara-
tive high-pressure liquid chromatography (HPLC) (Separations
Technology) on reverse-phase radial compression C18 columns
(Waters, Delta-Pak, 15 µm, 100 Å, 5 × 30 cm). A step gradient
(5%A to 50%A) (100 mL increments) was generated from 1 L
each of successively increasing concentrations of mobile phase
Exp er im en ta l Section
Abbr evia tion s. Abbreviations for common amino acids are
in accordance with recommendations of IUPAC. Other ab-
breviations: Boc, tert-butyloxycarbonyl; Bom, benzyloxym-
ethyl; Br-Z, 2-bromobenzyloxycarbonyl; Bzl, benzyl; Chg,
L-cyclohexylglycine; Cl-Z, 2-chlorobenzyloxycarbonyl; DCC,
dicyclohexylcarbodiimide; DIEA, N,N-diisopropylethylamine;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPPA,
diphenylphosphoryl azide; Fmoc, fluorenylmethoxycarbonyl;
HBTU, O-benzotriazole N′,N′,N′,N′-tetramethyluronium hexa-
fluorophosphate; HOBT, N-hydroxybenzotriazole; Hyp, trans-
4-hydroxy-L-proline; NMP, N-methylpyrrolidone; OcHex, O-
cyclohexyl; OFm, fluorenylmethyl; Orn, L-ornithine; PAM,
4-(oxymethyl)-phenylacetamidomethyl; TEA, triethylamine;
TFA, trifluoroacetic acid; Tos, tosyl.
P r ep a r a tion of Sem en ogelin I a n d th e Deter m in a tion
of P SA Clea va ge Sites. Semenogelin I cDNA was cloned from
total mRNA that had been isolated from human prostate tissue
(CLONTECH, Palo Alto, CA) using deoxyoligonucleotide prim-

4222 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Garsky et al.
(solvent A, 0.1% trifluoroacetic acid/H2O; solvent B, acetoni-
trile/0.1% trifluoroacetic acid). A flow rate of 80 mL/min was
used to elute the peptides. Detection was performed by
monitoring the UV absorbance at 214 nm. Homogeneous
product fractions (>98% pure) were pooled and freeze-dried.
The homogeneity of the peptides was demonstrated by analyti-
cal HPLC using a Vydac 15 cm, 218TP5415, C18 column.
Identity was confirmed by amino acid compositional analysis
following HCl hydrolysis (results not shown) and mass spectral
analysis.
Ma ss Sp ectr a l An a lysis. Mass measurements of the
compounds were conducted on one of the following three
instruments: VG Autospec -Q by use of electrospray (ES) or
fast atom bombardment (FAB) ionization; VG ZAB-HF by use
of FAB; or the Bruker BioApex 7T FT/ICR/MS by use of ES.
Unless otherwise noted, the m/z values are reported as [M+H]+
(Supporting Information table).
Syn th esis of P ep tid es 1, 2, 3, 4. The title compounds were
individually prepared by solid-phase methods as outlined
above, starting with 0.5 mmol of C-terminal N-Boc-protected
amino acid PAM resin. The subsequent protected amino acids
were coupled using standard ABI protocols as described above.
Following completion of the assembly of the resin bound
peptides, the N-terminal Boc-protecting groups were removed
using the TFA deblocking cycle. Cleavage and deprotection of
the peptides were effected using the following standard HF
procedure. To the dried resin was added anisole (1 mL/g resin)
as a scavenger. The vessel was cooled with liquid nitrogen and
evacuated. Anhydrous liquid HF was distilled into the reaction
(9 mL/g of resin) following which the mixture was stirred for
1 h at 0 °C. The HF was removed by vacuum distillation. The
residue was triturated with ethyl ether, filtered, and washed
3× with additional ether. The peptide was extracted with 10%
acetic acid 3× (15 mL/g of resin) and the filtrate lyophilized
to give the desired crude product. Purification of the peptides
was achieved by preparative RP-HPLC as described above. A
sample of crude peptide (400-500 mg) was dissolved in 50 mL
of 5% acetic acid and subsequently pumped onto a preparative
HPLC column which had been equilibrated in 5% starting
buffer (additional details in general methods above). Homo-
geneity of the peptides was demonstrated by analytical RP-
HPLC. See Supporting Information table for analytical data
for the purified peptides.
Syn th esis of P ep tid es 5, 6, 7, 8, 9, 10, 11. Starting with
0.5 mmol of MBHA resin to provide the final product in the
C-terminal amide form, the title compounds were prepared as
reported above. The N-terminal acetyl group was introduced
by coupling acetic acid, 2 mmol (4 equiv). Cleavage and
deprotection of the resin bound peptides were effected using
HF as described above. Lyophilized crude peptides were
purified by preparative HPLC as described above.
Syn th esis of P ep tid e Libr a r ies 1L, 2L, 3L, 4L. The
mixed peptide libraries were prepared and characterized as
described in a previous report.15 In brief, the primary protected
dodecapeptide sequences were assembled on 0.5 mmol MBHA
resin using the ABI synthesizer. For the mixed position, Xaa,
a manual coupling was carried out with one equivalent of an
equimolar mixture of 19 Boc-protected amino acids (Table 2)
which was dissolved in NMP and coupled using 1.05 equiv
(0.525 mmol) of HBTU and HOBT and 2.1 equiv of DIEA. The
reactions were allowed to proceed to completion (24 h) as
monitored by ninhydrin. Deprotection and cleavage were
performed with anhydrous HF as described above using 10%
p-cresol/p-thiocresol, 1:1 (v/v), as scavengers. After removal of
the HF and scavengers in vacuo, the free peptides were
precipitated with diethyl ether, filtered, and extracted with
50% aqueous acetic acid, diluted with H2O, and lyophilized
providing the 19-component peptide libraries.
were as described in 19. Purification conditions were as
described for example 18.
Leu -Dox, 13. Fmoc-Leu (182 mg, 0.515 mmol) was dissolved
in DMSO (6 mL) to which was added HOBT (1.70 mL of a 1
M solution in NMP) followed by DCC (1.55 mL of a 1 M
solution in NMP). The solution was stirred at room termpera-
ture for 30 min followed by the addition of 200 mg of
doxorubicin hydrochloride (0.35 mmol) dissolved in DMSO (3
mL). The solution was cooled to 5 °C and the pH adjusted with
DIEA (2M in NMP) to ∼7 using indicator strips (pH 0-14,
EM Science). After being stirred for 18 h at 5-10 °C the
reaction was transferred to a 200 mL round-bottomed flask,
cooled to 0 °C, and to it added 4 mL of piperidine for the
deblocking of the Fmoc protecting group. The reaction was
evaporated in vacuo and the oil dissolved in 200 mL of a buffer
consisting of 80% ammonium acetate (0.1% in H2O), (A) and
20% acetonitrile (B). The solution containing the product was
purified by RP-HPLC on a C18 radial compression column
(Waters, Delta Pak, 300 Å, 15 µm, 5 × 30 cm) which had been
equilibrated in 80:20 (A:B). A step gradient was generated (100
mL increments) from 1 L each of successively increasing (5%)
concentrations of mobile phase (20% to 40%). A flow rate of
80 mL/min was used to elute the product. The elution was
monitored at 260 nm. The pure fractions were pooled and
freeze-dried. The dried product was dissolved in 200 mL of
1% acetic acid and freeze-dried again to remove possible traces
of ammonium acetate. Purity of the product was checked by
analytical RP-HPLC and determined to be >98%.
Ac-Ala -Ala -Ala -Dox, 14. Ac-Ala-Ala-Ala-OH (Sigma) (4.6
mg, 12.3 µmol) and doxorubicin hydrochloride (9.7 mg, 16.8
µmol) were dissolved in DMSO (1 mL). To the stirred solution
was added 11.4 mg HOBT (84 µmol) and 31.9 mg (84 µmol)
HBTU in DMSO (1 mL) followed by 8.6 µL (89 µmol) of DIEA.
The solution was stirred at ambient temperature overnight.
The reaction was found to have gone to completion as
determined by analytical RP-HPLC (conditions found in
footnote c, Supporting Information table). The reaction was
diluted with a 1% acetic acid aqueous solution (8 mL) and
purified by semipreparative RP-HPLC (C18 Vydac, 218TP
1022), elution at 8 mL/min, with a linear gradient of 0.1%
aqueous TFA-acetonitrile from 90:10 to 10:90 over 60 min
with detection at 214 nm. Fractions with the desired purity
(>98%) were pooled and lyophilized.
Ac -Ala -As n -L y s -Ala -S e r -T y r -G ln -S e r -S e r -S e r -L e u -
Dox, 15. The primary peptide sequence was prepared on a
0.5 mmol scale as described above. Lysine was introduced as
Boc-Lys (Fmoc)-OH. Following HF cleavage and ether precipi-
tation, the protected peptide intermediate was extracted with
DMSO (3 × 50 mL) and the DMSO removed in vacuo. The
residue was triturated with H2O, filtered and dried in vacuo
to provide Ac-Ala-Asn-Lys (Fmoc)-Ala-Ser-Tyr-Gln-Ser-Ser-
Ser-Leu-OH (15a ). Compound 15a was used without purifica-
tion by dissolving 113 mg (0.080 mmol) in DMSO (2 mL), and
to the solution was added HOBT (3.3 equiv, 0.264 mL of a 1
M solution in NMP) followed by DCC (3.3 equiv, 0.264 mL of
a 1 M solution in NMP). After the mixture stirred for 30 min,
doxorubicin hydrochloride (46.4 mg, 0.08 mmol) in DMSO (0.8
mL) was added followed by the addition of DIEA to a pH of
∼7 (pH indicator paper). After the reaction mixture was stirred
for 18 h at 5-10 °C, the Fmoc protecting group was removed
at 0 °C by the addition of 3 mL of piperidine. Following 1 min
at 0 °C the reaction was evaporated in vacuo, the resulting
residue was suspended in 400 mL of aqueous NH4HCO3 (0.1%)
and filtered, and the filtrate was purified as described above
by preparative HPLC on a Delta Pak C18 column which had
been equilibrated with 100% A buffer (0.1% NH4HCO3). A step
gradient was generated (100 mL increments) from 1 L each of
successively increasing (5%) concentrations of mobile phase
(B ) acetonitrile). Pure fractions (>98%) were pooled and
lyophilized. The dried product was dissolved in 1% acetic acid
and relyophilized.
H-Ser -Leu -Dox, 12. Synthesis of the titled compound was
from the dipeptide intermediate, Fmoc-Ser-Leu-OH (12a ),
which was prepared by solid-phase chemistry as described in
example 15. Fmoc-Ser(tBu) was coupled to H-Leu-PAM resin
followed by HF cleavage to provide, after workup, 12a .
Reaction conditions for the coupling of 12a with doxorubicin
Ac -Ala -As n -L y s -Ala -S e r -T y r -G ln -S e r -Ala -S e r -L e u -
Dox, 16. Synthesis of the intermediate, Ac-Ala-Asn-Lys(Fmoc)-

Synthesis of a Doxorubicin Prodrug
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4223
Ala-Ser-Tyr-Gln-Ser-Ala-Ser-Leu-OH (16a ), and the titled
compound was as described for 15.
Ac-Ala -Asn -Lys-Ala -Ser -Tyr -Gln -Ser -Ser -Leu -Dox, 17.
Synthesis of the intermediate, Ac-Ala-Asn-Lys(Fmoc)-Ala-Ser-
Tyr-Gln-Ser-Ser-Leu-OH (17a ), and the titled compound was
as described for 15.
synthesis as described above. The introduction of the succinyl
group was via coupling of the monofluorenylmethylester of
succinic acid. Introduction of doxorubicin was as described for
example 19. Removal of the fluorenylmethyl protecting group
was achieved by treatment with piperidine as described in
example 15. Purification by preparative HPLC was as de-
scribed for example 18.
Ac-Lys-Tyr -Gln -Ser -Ser -Ser -Leu -Dox, 18. The interme-
diate for the titled compound, Ac-Lys(Fmoc)-Tyr-Gln-Ser-Ser-
Ser-Leu-OH (18a ), was prepared by automated solid-phase
synthesis as described in example 15. Compound 18a (0.214
mg, 0.2 mmol) was dissolved in 4 mL of DMSO. To the stirred
solution was added 2.64 equiv of HOBT (0.53 mL of a 1 M
solution in NMP) and 2.40 equiv of DCC (0.48 mL of a 1 M
solution in NMP). After 30 min doxorubicin hydrochloride was
added (100 mg, 0.172 mmol) in 2 mL of DMSO followed by
the dropwise addition of DIEA to adjust the pH ∼ 7 (pH
indicator paper). After the mixture was stirred at room
temperature overnight, 1 mL of DMSO was added and the
reaction cooled to 0 °C followed by the addition of piperidine
(3 mL) to remove the Fmoc protecting group. After 1 min the
reaction was evaporated in vacuo. The crude solid was
suspended in 150 mL of a 90:10 (A:B) solution where buffers
A ) 15% acetic acid in H2O and B ) 15% acetic acid in
methanol. Following filtration, the filtrate which contained the
product was applied to a Delta Pak C18 preparative HPLC
column and the product eluted as described earlier using the
above sample dissolution buffers. The column was equilibrated
with 90:10 (A:B) and the product eluted using a step gradient
(100 mL increments) from 1 L of the following successively
increasing concentrations of A:B: 80:20, 70:30, 65:35, 60:40,
55:45, 50:50. Pure (>98%) product fractions were pooled,
concentrated in vacuo, and lyophilized from water.
Ac-Or n -Tyr -Gln -Ser -Ser -Ser -Leu -Dox, 19. Synthesis of
the intermediate, Ac-Orn (Fmoc)-Tyr-Gln-Ser-Ser-Ser-Leu-OH
(19a ) was as described for 15a in example 15. To a stirred
solution of 19a (212 mg, 0.2 mmol) in 8 mL of DMF was added
doxorubicin hydrochloride (100 mg, 0.172 mmol) followed by
the addition of 20 µL of TEA. The solution was cooled to 0 °C
and to it added 24 µL of DPPA (0.55 equiv, 0.11 mmol) followed
by a second addition of DPPA (24 µL) after 5 min. The pH of
the reaction was adjusted to ∼7 (pH indicator paper) with TEA.
After being stirred at 0 °C for 90 min, additional DPPA (24
µL) was added and the pH readjusted to ∼7 with TEA.
Following stirring at 0 °C overnight, DMF (1 mL) was added
followed by 3 mL of piperidine. The removal of the Fmoc
protecting group was allowed to proceed at 0 °C for 1 min and
the reaction concentrated to an oil in vacuo. Purification of
the crude to provide pure (>98%) product, 19, was as described
for 18.
Ac-Lys-Ala -Ser -Tyr -Gln -Ser -Leu -Dox, 20. Synthesis of
the titled compound was as described for 19 from the inter-
mediate Ac-Lys(Fmoc)-Ala-Ser-Tyr-Gln-Ser-Leu-OH (20a ).
Ac-Or n -Ala -Ser -Tyr -Gln -Ser -Leu -Dox, 21. Synthesis of
the titled compound was as described for 19 from the inter-
mediate Ac-Orn(Fmoc)-Ala-Ser-Tyr-Gln-Ser-Leu-OH (21a ).
Ac-Or n -Ala -Ser -Tyr -D-Gln -Ser -Leu -Dox, 22. Synthesis of
the titled compound was as described for 19 from the inter-
mediate Ac-Orn(Fmoc)-Ala-Ser-Tyr-D-Gln-Ser-Leu-OH (22a ).
Ac-Lys-Ala -Ser -Ch g-Gln -Ser -Leu -Dox, 23. Synthesis of
the titled compound was as described for 19 from the inter-
mediate Ac-Lys(Fmoc)-Ala-Ser-Chg-Gln-Ser-Leu-OH (23a ).
Ac-P r o-Ala -Ser -Ch g-Gln -Ser -Leu -Dox, 24. Synthesis of
the titled compound was as described for 19 from the inter-
mediate Ac-Pro-Ala-Ser-Chg-Gln-Ser-Leu-OH (24a ).
Ac-Hyp -Ala -Ser -Ch g-Gln -Ser -Leu -Dox, 25. The inter-
mediate for the titled compound, Ac-Hyp-Ala-Ser-Chg-Gln-Ser-
Leu-OH (25a ), was prepared by solid-phase synthesis as
described above. Boc-Hyp was introduced without protection
of the hydroxyl side chain. Reaction conditions for the coupling
with doxorubicin were as described in example 19. Purification
conditions were as described for example 18.
Glu ta r yl-Hyp -Ala -Ser -Ch g-Gln -Ser -Leu -Dox, 27. The
intermediate for the titled compound, Glutaryl(OFm)-Hyp-Ala-
Ser-Chg-Gln-Ser-Leu-OH (27a ), was prepared by automated
solid-phase synthesis as described in Example 15. The glutaryl
functionality was introduced as the monofluorenylmethylester
of glutaric acid using the usual coupling protocol (general
methods section). HF cleavage and workup was as described
for 15. Compound 27a was used without purification by
dissolving 170 mg (0.16 mmol) in DMF:DMSO, 1:1 (20 mL).
To the stirred solution was added 80 mg of doxorubicin
hydrochloride (0.14 mmol) followed by 50 µL of DIEA (0.28
mmol). The solution was cooled (0 °C) and to it added DPPA,
43 µL (0.20 mmol). After 30 min an additional 43 µL of DPPA
was added and the pH adjusted to ∼7 (pH indicator paper).
After 4 h the reaction was shown to be complete by analytical
HPLC. DMF (10 mL) was added followed by piperidine (3.5
mL). After 1 min at 0 °C the solution was concentrated in
vacuo to an oil. The crude product was triturated with diethyl
ether and filtered. The dried crude was dissolved in 250 mL
of a buffer consisting of 80% ammonium acetate (0.1% in H2O)
(A) and 20% acetonitrile (B). The product was purified by RP-
HPLC on a C18 radial compression column which had been
equilibrated in 80:20 (A:B). A step gradient was generated (100
mL increments) from 1 L each of successively increasing (5%)
concentrations of mobile phase (20% to 40%). A flow rate of
80 mL/min was used to elute the product. Pure fractions were
pooled and lyophilized. The dried product was dissolved in 1%
acetic acid and lyophilized again to remove possible traces of
ammonium acetate. Purity of the product (90 mg, 40% yield
from 27a ) was checked by analytical HPLC and determined
to be >98%.
Su p p or tin g In for m a tion Ava ila ble: Figure containing
amino acid sequence of human semenogelin I and table
containing synthetic peptides and doxorubicin conjugates
analytical data. This material is available free of charge via
the Internet at http://pubs.acs.org.
Ack n ow led gm en t. We thank Dr. J oel R. Huff for
his support and encourgement, Kathleen Haskell, Eliza-
beth McAvoy, and David Kiefer for supporting the cell
culture and mouse tumor xenograft studies, Dr. S.
Gardell for his determinations of histamine levels in rat
plasma, Patricia Bunting and Sylvia Volksdorf for their
determinations of histamine levels in dog plasma, Dr.
Charles W. Ross III for mass spectral analyses, Mei-J y
Tang for amino acid analyses, and J ean Kaysen for
typing the manuscript.
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