Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation

Article abstract of DOI:10.1016/S0968-0896(00)00157-7

Novel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in order to confirm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was condensed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was confirmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS₃ and SW1116 tumor cell lines, although the efficacy was different in individual cells. DU-257 was then conjugated through the linker to KM231 monoclonal antibody specifically reactive to GD3 antigen which was shown to be expressed on the surface of many malignant tumors such as SW1116. The conjugate retained its binding specificity for SW1116 cell with a similar activity with KM231. Furthermore, the conjugate showed significant growth inhibition on SW1116 cell at a concentration of 75 μg/mL while no effect on antigen negative cell, HeLaS₃. These results suggest that the conjugate retained its anti-tumor effect only when it bound on and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLe^a expression. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00157-7

Bioorganic & Medicinal Chemistry 8 (2000) 2175±2184
Synthesis of a Novel Duocarmycin Derivative DU-257 and its
Application to Immunoconjugate Using Poly(ethylene glycol)-
dipeptidyl Linker Capable of Tumor Speci®c Activation
Toshiyuki Suzawa, Satoru Nagamura, Hiromitsu Saito, So Ohta, Nobuo Hanai
and Motoo Yamasaki*
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, 3-6-6, Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
Received 21 February 2000; accepted 6 June 2000
AbstractÐNovel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in
order to con®rm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, l-alanyl-
l-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its
property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was con-
densed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model
enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC
analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was
con®rmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS3 and
SW1116 tumor cell lines, although the ecacy was di€erent in individual cells. DU-257 was then conjugated through the linker to
KM231 monoclonal antibody speci®cally reactive to GD3 antigen which was shown to be expressed on the surface of many
malignant tumors such as SW1116. The conjugate retained its binding speci®city for SW1116 cell with a similar activity with
KM231. Furthermore, the conjugate showed signi®cant growth inhibition on SW1116 cell at a concentration of 75 mg/mL while no
e€ect on antigen negative cell, HeLaS3. These results suggest that the conjugate retained its anti-tumor e€ect only when it bound on
and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to
immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLea
expression. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
size and poor distribution to target sites have been major
problems to be overcome.
Duocarmycins are unique anti-tumor antibiotics isolated
from the culture broth of Streptmyces sp.1,2 and a series of
duocarmycin derivatives has been synthesized.3,4 One of
their potent active metabolites is DU-86 which possesses
two segments represented as segment A for DNA alky-
lating ability and segment B for noncovalent binding to
DNA (Scheme 1).5,6 Based on its potency and ecacy,
DU-86 derivative has been applied for clinical investiga-
tions. However, in the most duocarmycin derivatives
their marginal activity against human solid tumors and
their insolubility in an aqueous solution dissuaded us
from considering them for further evaluations. In cancer
chemotherapy using anti-tumor agents including duo-
carmycins, side e€ects due to toxicity on normal tissues,
rapid elimination from the body due to lower molecular
In order to enhance ecacy and reduce side e€ects of
these anti-tumor agents, monoclonal antibody (mAb) has
been demonstrated to be a promising carrier to deliver
them to speci®c tumor tissues.7 9 Anti-tumor agents,10 12
toxin13 and toxic protein14-conjugated mAbs have been
extensively studied. Although such e€orts have been
made for many years, clear treatment e€ect and clinical
application were not still gained. The reasons con-
sidered were toxicity of conjugated agents and immu-
noreaction against conjugate as well as poor speci®city
and anity of mAb. Furthermore, even though con-
jugate is prepared to maintain its speci®city and anity,
cytotoxic potency may be a€ected because covalently
linked drugs are structurally altered by the coupling
chemistry, and their original state may not be restored
even after cleavage from the antibody. Therefore, one of
the most important points for the preparation of desired
*Corresponding author. Tel.: +81 42 725 2555; fax: +81 42 726 8330;
e-mail: myamasaki@kyowa.co.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00157-7

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T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
Scheme 1. Synthetic route to DU-257.
immunoconjugate seems to control the release of the
agents at desired tumor sites.15,16 On the basis of linker
technology, such conjugate is being prepared in recent
years. The linker which can be cleaved to release toxin
when conjugate was internalized into acidic compartment
in tumor cells,11 and peptidyl linker which can be recog-
nized by tumor speci®c proteases to release anti-tumor
agent from polymer prodrugs17 have been reported.
Therefore, combination of PEG with peptide as a linker
between anti-tumor agent and mAb may be desirable
for the preparation of immunoconjugate. From this
point of view, we have designed PEG linker combined
with dipeptide and showed enzymatic release of segment
B of duocarmycin as a model agent.23 It has been sug-
gested that tumor speci®c digestion of the linker and
release of bioactive agents may be carried out only when
the conjugate is delivered to the desired sites, while
stable and non-toxic during circulation in the body. One
of the purposes of this report is to provide a novel potent
duocarmycin derivative carrying an amino residue, to
which mAb can be conjugated via the linker, since pre-
paration of immunoconjugate with anti-tumor agent
has been restricted to particular compounds such as
adriamycin10 12,17 and mitomycin.24 We describe here
the synthesis of a novel duocarmycin derivative DU-257
and the preparation of its immunoconjugate with KM231
mAb25 to evaluate their biological activities based on the
concept of the PEG-dipeptidyl linker system. KM231 is a
On the other hand, poly(ethylene glycol) (PEG), non-
toxic, amphiphilic and non-immunogenic polymer, has
been intensively studied as a modi®cation agent of proteins
for the last two decades.18 20 Modi®cation of proteins with
PEG results in prolonged half-life in blood stream,
reduced antigenicity and immunogenicity, increased pro-
tease resistance, and enhanced solubility in both aqueous
and organic solutions. Furthermore, PEG has not only
been used as such modi®cation agent but also applied to
a hydrophilic linker among bioactive substances such as
hydrophobic antibiotics21 and di€erent proteins.22

T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
2177
mouse monoclonal antibody speci®cally reactive to sia-
lyl lewis a (sLea) carbohydrate antigen, which has been
shown to be overexpressed on the cell surface of various
tumor cells26 including adenocarcinoma such as gastric
cancer, pancreatic cancer and hepatocarcinoma, and
therefore this mAb seems to be a suitable model as a
tumor speci®c carrier of a novel duocarmycin derivative.
before23 and segment A of DU-86 was condensed to the
resulting segment B, followed by hydrogenation reaction
to form compound 8 with 6 steps.
Yields of the compounds in each step were almost rea-
sonable except for the ®nal step, by which azido group
of compound 7 was converted to amino group. Although
compound 7 was unstable under the normal reaction
conditions, compound 8 could be prepared as a yellowish
powder with approximately 70±90% purity by controlling
reaction conditions such as temperature, solvent and
reaction time. The stable reaction conditions avoiding
generation of side product and the e€ective puri®cation
conditions to gain quantitative and reproducible yield
for compound 8 up to 90% purity are still under
investigation. Typical HPLC pro®le of the resulting
compound 8 was illustrated in Fig. 1, in which a slight
amount of side product, the structure of which was deter-
mined as elsewhere, was observed. Retention time of the
desired product was at 30.6 min. Condensation of DU-
257 to the PEG-dipeptidyl linker followed by conjugation
to mAb was carried out using DU-257 of ca. 70% purity
without further puri®cation.
Results
Synthesis of DU-257
Synthetic procedure of DU-257 was illustrated in
Scheme 1. Azido group was introduced into 4-hydroxyl
position of segment B via oxyethylene spacer as designed
Condensation of DU-257 to the linker and preparation of
its mAb conjugate
Condensation of DU-257 to the PEG-dipeptidyl linker
and following preparation of mAb conjugate were carried
out by the procedure shown in Scheme 2. Compound 8
with approximately 70% purity was used for preparation
of DU-257-linker moiety without further puri®cation.
Condensation reaction of DU-257 to compound 11, N-
hydroxysuccinimide (NHS) activated form prepared
using N, N0-dicyclohexylcarbodiimide (DCC) from com-
pound 10, underwent quantitatively to yield compound
12. Benzyl protecting group of PEG terminal was sub-
jected to deprotection just before conjugation to mAb.
Figure 1. Typical elution pro®le of DU-257 determined by reversed-
phase HPLC analysis. An aliquot of compound 8 diluted with a
mobile phase (50mM phosphate bu€er, pH 5.9) was injected to the
reversed-phase column.
Conjugation of compound 13 to mAb was performed
via NHS active ester as described before23 and 30
equivalent of compound 14 relative to one mAb molecule
Scheme 2. Synthetic route to the conjugate.

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T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
was used for the reaction. There were no signi®cant
aggregation and polymerization fractions when puri®ed
by gel ®ltration chromatography, although the yield
(45.1%) was slightly low. This may be probably because
KM231 antibody used for conjugation reaction contained
a slight amount of aggregate and the preparation scale
was somewhat smaller than that performed before.23
The resulting conjugate was prepared as a colorless
clear solution. Change in binding anity and precipitate
formation were not observed for the conjugate during
storage for more than one month at 4 ꢀC. The substitu-
tion ratio of conjugated DU-257 per single mAb was
determined to be approximately 2 using the data of Fig.
3(d) based on the method described before.23
Comparison of DU-257 with DU-86 in their
physicochemical properties
Physicochemical property of DU-257 was evaluated
using DU-257 which was further puri®ed by silica gel
chromatography up to 95% purity. Puri®ed DU-257
and DU-86 were analyzed by reversed-phase HPLC as
shown in Fig. 2(a). Purity of DU-257 was ca. 98%.
Retention time of DU-257 shifted to 30.6 min relative to
that of DU-86 (34.0 min), indicating that introduction
of amino group into segment B increased hydrophilicity
of the compound.
Figure 3. Release of DU-257 from PEG-dipeptidyl linker. A portion
of the solution containing the compound 12 (0.1 mg/mL, 200 mL) was
analyzed by reversed-phase HPLC before (a) and after (b) incubation
with 100 mL of thermolysin for overnight at 37 ꢀC. For complete
digestion, the enzyme/substrate ratio of 1/10 was employed. The
resulting main compound after thermolysin treatment was separated
and re-analyzed by reversed-phase HPLC to con®rm the purity and
elution pro®le of valine-conjugated DU-257 moiety (0.20 mg) (c).
Compound 15 (15 mg of the conjugate) was subjected to thermolysin
treatment in the same conditions for overnight and the portion of the
resulting mixture containing 12 mg of the conjugate was injected to
the column (d). In contrast to the other ®gure, disodium hydro-
genphosphate-citric acid bu€er (50 mM, pH 4.8) was used as mobile
phase.
Stability of DU-257 in phosphate bu€er (pH 7.0) was
examined by HPLC. In order to prevent nonspeci®c
adsorption and precipitate formation of DU-86, stability
test was carried out in the presence of 20% acetoni-
trile.27 As shown in Fig. 2(b), change in peak height was
Figure 2. Comparison of DU-257 with DU-86 in terms of reversed-phase HPLC pro®le (a) and stability in aqueous solution (b). Both DU-257 (&)
and DU-86 (*) were dissolved in PBS (pH 7.0) containing 20% acetonitrile and the solutions were incubated at 37 ꢀC. Both solutions containing
0.125 mg of the individual compounds were injected to the column. Elution pro®les of the compounds before incubation (a) and the change in rela-
tive peak height during 24 h incubation (b) were plotted.

T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
2179
almost the same with that observed for DU-86 through-
out the experimental period. Approximately 80% of both
compounds remained even after 24h incubation. These
results indicate that the stability of DU-257 was compar-
able to that of DU-86 when incubated in aqueous solu-
tions.
Enzymatic digestion of PEG-dipetidyl linker
HPLC pro®le for compound 12 was shown in Fig. 3(a).
As expected from the data shown in Fig. 1, compound 12
generated double peaks, because the presence of approxi-
mately 30% of impurity was suggested in compound 8
before condensation with the linker moiety. When the
compound was treated with thermolysin overnight to
complete the digesting reaction, both peaks almost dis-
appeared and new peaks generated at retention times of
31.2 min and 32.2 min, respectively (Fig. 3 (b)). These
newly generated peaks were isolated as shown in Fig. 3(c)
and determined by mass analysis. Indicated molecular
masses were m/z=620 (M+H)+ for the compound
with former retention time and m/z=622 (M+H)+ for
that of the latter one. From these results, the main peak
generated by thermolysin treatment was identi®ed to be
DU-257-valine moiety, and the minor peak seemed to be
hyperhydrated form of this compound. Results of amino
acid composition analysis also suggested these peaks as
valine-conjugated forms.
Figure 4. Concentration dependency of DU-257 and its related com-
pound on growth inhibition of HeLaS3 cell. DU-86 (*) and puri®ed
DU-257 (*) as well as DU-257 enzymatically generated as valine-
conjugated form (&) were subjected to growth inhibition assay using
HeLa S3 cell described as in experimental.
Table 1. Cytotoxic e€ect of DU-257 and its related compounds
Compound
IC50(nM)
When the mAb conjugate was subjected to thermolysin
treatment under the same conditions, similar HPLC pro-
®le with that shown in Fig. 3(b) was observed although
signal intensity was not necessarily high (Fig. 3(d)). No
detectable peaks were observed for the conjugate solution
when the enzyme was not added to the reaction mixture
(data not shown). These data indicate that the conjugate
was also cleaved by the enzyme to release DU-257-
valine moiety even if DU-257 was conjugated to mAb
through the linker.
HeLaS3
SW1116
DU-86
DU-257
H-Val-DU-257
5.2
6.3
6.9
300
280
300
against SW1116 cell was also examined in a similar way
to obtain the data shown in Table 1. Cytotoxic activities
of DU-86 and DU-257 for SW 1116 cell were 40 to 60
times weak as compared to those for HeLaS3 cell.
Cytotoxic activity of DU-257 and its released form from
the linker
Anity and cell speci®city of the conjugate
Cytotoxic activity of DU-257 and enzymatically digested
compound puri®ed by HPLC (H-Val-DU-257, Fig. 3(c))
was examined using HeLaS3 cell. As shown in Fig. 4,
DU-257 signi®cantly inhibited growth of the cell with
dose dependent fashion. Substitution of oxyethylamine
arm at 4-methoxy position of DU-86 did not a€ect the
cytotoxicity of the compound. IC50s were summarized
in Table 1. Interestingly, the released compound from
the linker, l-valine-conjugated form with DU-257, also
exhibited signi®cant cytotoxicity. This means that, even
if the linker was digested speci®cally between amino
acids at tumor sites, the conjugate possibly kill tumor
cells with almost the same ecacy as free DU-257. The
side product generated during preparation of compound
8 and the isolated compound after the treatment with
the enzyme, which is identi®ed to be valine-conjugated
side product, were quite low cytotoxicity for the cells
investigated (data not shown). Consequently, the con-
jugate composed of the side product does not seem to be
responsible for growth inhibition of the target cell. IC50s
We have designed the conjugation reactions so that the
substitution ratio is to be approximately 2 based on the
fact shown previously23 that excessive modi®cation with
the linker resulted in signi®cant loss of binding anity
of the conjugate. As expected, the conjugate retained
similar binding anity with KM231 mAb for SW1116
cell expressing GD3 antigen (Fig. 5(a) and (b)). Conse-
quently, the loss of binding anity was not caused under
the conditions of our conjugate preparation. In contrast,
both conjugate and mAb showed no binding anity for
HeLaS3 cell (Fig. 5(d) and (e)). Thus, anity and spe-
ci®city of the mAb were completely maintained in the
conjugate.
Cell growth inhibition of the conjugate
Growth inhibitiory e€ect of the conjugate on tumor
cells were summarized in Fig. 6. The conjugate exhibited
signi®cant inhibitory e€ect on the growth of SW1116
human colorectal carcinoma cell and the growth of

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T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
Figure 5. Binding of the conjugate to the SW1116 (a±c) and HeLa S3 (d±f) cell lines. Conjugate (a, d) and KM231 mAb (b, e) were reacted to the
individual cells at a concentration of 20 mg/mL and the resulting cells were incubated with FITC-labelled anti-mouse IgG antibody followed by
FACS analysis. The cells in the absence of both conjugate and KM231 were also analyzed as a control (c, f).
agent with tumor-speci®c antibody, the linker of which
is cleaved by tumor expressing protease only after the
conjugate is reached to the target site and then activated
form of anti-tumor agent is released to kill tumor cell.23
Previously, we used segment B of duocarmycin derivatives
as a model compound of anti-tumor agent to show the
preparation of the conjugate and enzyme-speci®c diges-
tion of the linker.23 In this report, in order to demonstrate
the principle of the linker system and tumor-speci®c e-
cacy of the immunoconjugate, a novel duocarmycin deri-
vative DU-257 was designed to possess an amino residue
capable of its conjugation to the linker. The mechanism
of duocarmycin derivatives on killing tumor cells has been
recently investigated5 and cancer chemotherapy using
duocarmycins has been suggested to overcome multi-drug
resistance,28 which most hydrophobic anti-tumor agents
encountered in tumor chemotherapy. This report is the
Figure 6. Cell growth inhibition e€ect of the conjugate on HeLa S3
and SW1116 cell lines. The conjugate (&) and KM231 mAb alone (&)
were reacted to the individual cells at a concentration of 75 mg/mL and
the growth of these resulting cells was compared to that of untreated
cells. Each point was measured in triplicate.
®rst approach using a biologically active duocarmycin
derivative for the development of immunoconjugate.
Various duocarmycin derivatives have been reported,1 4
and their cytotoxicities depend largely upon the struc-
more than 20% of the cell were inhibited at 75 mg/mL of
the conjugate, the concentration of which corresponds
to approximately 1 mM of DU-257 calculated from the
substitution ratio. However, no e€ect of the conjugate
was observed for the growth of HeLaS3 cell. When the
mAb alone was added, an inhibitory e€ect was slightly
observed only on the growth of SW1116 cells, but this
e€ect seemed to be nonspeci®c. Enhancement of SW1116
cell speci®c growth inhibitory e€ect of the conjugate was
thus con®rmed in this experiment.
ture of the individual segments.3,4 Considering the level
of potency, stability and material supply, DU-86 was
selected to design DU-257, and synthetic route of DU-
257 was established with a reasonable yield as shown in
Scheme 1. Introduction of the oxyethylamine arm into
DU-86 to form DU-257 retained its cytotoxicity with-
out loss of potency (Fig. 4, Table 1) and the stability in
aqueous solution was maintained (Fig. 2). Additionally,
DU-257 enhanced its hydrophilicity estimated from the
pro®le of reversed-phase HPLC analysis, probably due
to the introduction of amino residue within the molecule.
One of the problems to be solved for the development of
duocarmycin derivatives as preferable anti-tumor agents
has been their hydrophobic properties. Increased
hydrophilicity of DU-257 will be preferable like DU-86
analogue, KW-21896,29 and these properties of DU-257
Discussion
We have proposed a novel immunoconjugate system using
PEG-dipeptidyl linker for the conjugation of anti-tumor

T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
2181
seem to be enough for further evaluation as an anti-
tumor agent.
times lower than that for HeLaS3 cell in our conditions,
even though circumvention of multi-drug resistance has
been suggested for duocarmycin derivatives.28 As sLea
antigen expressed on SW1116 cell was estimated to be
approximately 1.9Â106,37 the cell seems to be one of the
feasible targets for con®rming the principle of the linker
system and evaluating the ability of the conjugate. The
conjugate almost completely abolished its cytotoxicity
against HeLaS3 cell (Fig. 6), indicating that DU-257
was bound stably to the conjugate and none of the
e€ects were expressed to antigen negative cells; i.e. the
linker was not digested at non-target cells and undesired
side e€ects were supposed to be diminished. However, on
the basis of the fact that inhibition of SW1116 cell
growth was speci®cally enhanced at the concentration
of 75 mg/mL, it is concluded that cytotoxic activity was
expressed only when i) the conjugate bound speci®cally
to SW1116 cell, and simultaneously, ii) the linker was
speci®cally digested at the cell. Our results suggest that
SW1116 cell possesses linker-digesting activity to release
active form of DU-257 moiety. When the cell is treated
with DU-257 or H-Val-DU-257 at a concentration of 1
mM as free drug, ca. 80% of cell growth inhibition
should be anticipated (Table 1). Considering the
potency of conjugated DU-257 shown in Fig. 6, at least
10±20% of the conjugate was estimated to be digested
under the experimental conditions. The ecacy of the
conjugate seems to depend greatly upon digestion ecacy
of the linker peptide as well as sensitivity of target cell
against anti-tumor agent. The amount of the conjugate
prepared in this study was not enough for complete eva-
luation of the ecacy especially at higher concentra-
tions. In order to enhance anti-tumor e€ect, further
study should be required to increase the concentration of
the conjugate and/or to prepare the conjugate in which
substitution ratio of DU-257 in one mAb molecule is
increased.38 Additionally, selection of linker peptide must
be essential for increasing the potency of the conjugate.
DU-257 was then condensed to the linker moiety to
form compound 12 and evaluated its release using ther-
molysin as a model enzyme to con®rm whether the
digested compound is biologically active or not. Only if
the linker was cleaved around the tumor site by tumor-
speci®c proteases to release biologically active form of
DU-257, anti-tumor e€ect will be anticipated. The
results suggest that DU-257 was released almost com-
pletely as a conjugated form with valine from the linker
consisting of alanyl-valiyl sequence after the treatment
of thermolysin. This ®nding was agreed with the data
shown previously.23 Digestion ecacy by thermolysin
seemed to be very high in the case of alanyl-valyl linker.
It should be noted that this digested compound had a
similar cytotoxic activity with unconjugated DU-257
(Fig. 4, Table 1). Consequently, DU-257 moiety seems
to be released as an active form whenever the target
tumor cells express thermolysin-like enzyme. In fact,
elevated expression of thermolysin-like enzymes has been
reported in many tumors30 32 and existence of digesting
activity of the alanyl-valyl linker in cell homogenate
from melanoma cell line was also reported.23 It is well
known that many tumor cells overexpress speci®c pro-
teases such as cathepsins33 and matrix metallopro-
teases.34 Such enzymes may also have an ability to digest
the linker peptide and must be available as candidate
activators of the conjugate, although further analysis
including cell speci®city, digestion ecacy and cleavage
site within the linker should be required.
In the preparation steps of immunoconjugate, dena-
turation and precipitation of mAb should be generally
avoided.35 Both in the preparation steps and during
storage, loss of speci®city and anity of the conjugate
(Fig. 5) as well as generation of precipitates were not
observed. PEG-based linker seems to stabilize the con-
jugate. Hydrophobic property of anti-tumor agent some-
times results in such undesired phenomenon and has been
required to be overcome for many years. Improvement of
the solubility is important for the preparation of e€ective
and stable immunoconjugate. Furthermore, PEG-linker
seems to be responsible for exposing the dipeptide portion
to the target enzyme since release of DU-257 was retained
e€ectively even if the linker was conjugated to the anti-
body. DU-257 moiety was released when the conjugate
was subjected to the reaction with thermolysin (Fig. 3(d)),
and at least 2 molecules of DU-257 moiety could be
released from single conjugate as an active form. These
indications suggest some advantages of the PEG-dipepti-
dyl linker system for application to immunoconjugate.
Conclusion
We demonstrated here the preparation of a novel anti-
tumor agent DU-257 capable of conjugation to antibody
via a PEG-dipeptidyl linker. DU-257 seemed to be one
of the promising candidates for the preparation of
immunoconjugate because similar potency and stability
with DU-86, and increased hydrophilic property have
been retained. The basic concept of our linker system
has been con®rmed by demonstrating enzymatic release
of DU-257 moiety and cytotoxic activity of digested
compound using DU-257 conjugated to PEG-dipeptidyl
linker. Finally, we tried to apply the conjugate for the
therapy of malignant colorectal cancer using SW1116
cell as a model in vitro and demonstrated the conjugate
to be activated speci®cally at target cell, although some
improvements including peptide sequence and the
extent of substitution will be suggested in future study.
In summary, DU-257 described here will be one of the
candidates for novel anti-tumor agent and its conjugated
form with KM231 antibody via PEG-dipeptidyl linker
will also contribute to increase the treatment ecacy for
cancer patient bearing sLea positive tumor.
Cyototoxic e€ect of the conjugate was evaluated using
SW1116 cell from human colorectal adenocarcinoma
origin, which is known to be invasive malignant cancer.
Most of the known hydrophobic anti-tumor agents can
not be available due to multi-drug resistance36 and
e€ective therapeutics still have not been established for
the treatment of such cancers. One can easily recognize the
malignancy of SW1116 cell, because the potency of DU-
257-related compounds for SW1116 cell was 40 to 60

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T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
Experimental
3.80 (s, 3H, 2-COOCH3), 3.32 (t, J=5.2 Hz, 2H,
BrCH2).
Materials
PEG-dicarboxylic acid (average molecular weight 600)
and protected amino acid were purchased from Fluka
Biochemicals and Kokusan Chemicals, Co., respectively.
Thermolysin (EC 3.4.24.4, 9390 pU/mg) was the pro-
duct of Daiwa Kasei K.K. (Osaka, Japan). DU-8639 and
segment A of duocarmycin (compound 6)3 were prepared
as described previously. KM231 mouse IgG1 mAb was
supplied in our laboratory.25,26 Other compounds were of
the highest pure grade commercially available.
Compound 3. Compound 2 (94 mg, 0.26 mmol) was dis-
solved in 9.5 mL of DMF, and sodium azide (85 mg,
1.3 mmol) was then added, followed by stirring at room
temperature for 25 h. Appropriate amounts of ethyl
acetate and a phosphate bu€er (pH 7) were added to the
resulting solution to extract the ethyl acetate layer.
After the organic layer was dried over anhydrous
sodium sulfate, the solvent was removed under reduced
pressure to give 79 mg of compound 3 (yield 95%). SIMS
m/z: 321 (M+H)+; 1H NMR (100 MHz, CDCl3): d 9.27
(brs, 1H, 1-NH), 7.10 (s, 1H, H-3), 6.80 (s, 1H, H-4), 4.24
(t, J=5.2 Hz, 2H, OCH2), 4.00 (s, 3H, 7-OCH3), 3.91 (s,
3H, 5-OCH3), 3.82 (s, 3H, COOCH3), 3.43 (t, J=5.2 Hz,
2H, N3CH2).
Tumor cell line
HeLaS3 (human cervix epitheloid cancer) and SW1116
(human colorectal carcinoma cell) were obtained as
previously indicated.3,25
Compound 4. Compound 3 (79 mg, 0.25 mmol) was dis-
solved in a mixture of tetrahydrofurane (THF) (8 mL)
and water (10 mL), and then 1N sodium hydroxide
(2.5 mL) was added, followed by stirring at room tem-
perature for 3.5 h. The reaction mixture was made acidic
by addition of 1N HCl, and extracted using chloroform.
The chloroform layer was washed with brine, dried over
anhydrous sodium sulfate, and the solvent was removed
to give 75 mg of compound 4 (yield 98%). SIMS m/z: 307
(M+H)+; 1H NMR (100 MHz, CDCl3): d 9.40 (brs, 1H,
1-NH), 7.20 (s, 1H, H-3), 6.81 (s, 1H, H-4), 4.25 (t,
J=5.2 Hz, 2H, OCH2), 3.98 (s, 3H, 7-OCH3), 3.90 (s,
3H, 5-OCH3), 3.42 (t, J=5.2 Hz, 2H, N3CH2).
General procedures
TLC was run on glass-backed silica gel plates (Kieselgel 60
F254, Merck & Co., Inc.) using various solvents as indi-
cated. Protein concentration was determined by absor-
bance at 280 nm (1 mg/mL=1.4 absorbance units).40 1H
NMR spectra were recorded on JEOL FX100 or Bruker
AM-500. Chemical shifts were reported in parts per mil-
lion (ppm) down®eld from tetramethylsilane. Mass spec-
tra were measured with a Hitachi B-80 or a Shimadzu QP-
1000 spectrometer. Amino acid analysis was performed by
acid degradation of the compound followed by HPLC
analysis of phenylisothiocyanate derivatization of resulting
amino acids.37
Compound 5. Compound 4 (75 mg, 0.25 mmol) was dis-
solved in 7 mL of methylene chloride and DCC (103 mg,
0.5 mmol) was added, followed by stirring under ice
cooling for 1 h. To the resulting solution were added p-
nitrophenol (70 mg, 0.5 mmol) and dimethylaminopyr-
idine (61 mg, 0.5 mmol), followed by stirring for 80 min.
The insoluble matter was ®ltered o€, and 0.5N HCl was
added to the ®ltrate, followed by extraction with
chloroform. The organic layer was washed with a satu-
rated sodium hydrogencarbonate solution and brine,
and then dried over anhydrous sodium sulfate. After the
solvent was evaporated o€, compound 5 was recrys-
tallyzed from ethanol to give 72 mg of compound 5 (yield
HPLC
HPLC was performed at room temperature on TRI-
ROTAR SR HPLC system (Japan Spectroscopic Co.)
equipped with a UNISIL PACK 5C18-150A reversed-
phase column (GL Sciences, Inc.). Absorbance was
monitored at 330 nm. Solvent was 50 mM phosphate
bu€er (pH 5.9) with an acetonitrile linear gradient from
10% to 70% concentration at a ¯ow rate of 0.7 mL/min.
Alternatively, 50mM disodium hydrogenphosphate-citric
acid bu€er (pH 4.8) was also used as a mobile phase.
1
69%). SIMS m/z: 428 (M+H)+; H NMR (100 MHz,
Compound 2. In 5 mL of DMF was dissolved compound
1 (100 mg, 0.4 mmol) prepared as described previously,23
and anhydrous potassium carbonate (275 mg, 2 mmol)
was added. To the resulting solution was added dropwise
1,2-dibromoethane (173 mL, 2.0 mmol), followed by
stirring under nitrogen atmosphere at room temperature
for 19 h. After addition of a phosphate bu€er (pH 7),
the mixture was extracted with ethyl acetate. The organic
layer was washed with brine, and then dried over anhy-
drous sodium sulfate. After the solvent was removed
under reduced pressure, the residue was puri®ed using
20 mL of silica gel using a hexane-ethyl acetate mixture
(2:1). The solvent was evaporated o€ to give 94 mg of
compound 2 (yield: 65%). SIMS m/z: 359 (M+H)+; 1H
NMR (100 MHz, CDCl3): d 9.23 (brs, 1H, 1-NH), 7.10
(s, 1H, H-3), 6.82 (s, 1H, H-4), 4.38 (t, J=5.2 Hz, 2H,
OCH2), 4.00 (s, 3H, 7-OCH3), 3.91 (s, 3H, 5-OCH3),
CDCl3): d 9.08 (brs, 1H, 1-NH), 8.26 (d, J=8.98 Hz, 2H,
C6H4NO2), 7.40 (d, J=8.98 Hz, 2H, C6H4NO2), 7.21 (s,
1H, H-3), 6.81 (s, 1H, H-4), 4.10 (t, J=5.4 Hz, 2H,
OCH2), 3.96 (s, 3H, H-7), 3.81 (s, 3H, H-5), 3.46 (t,
J=5.4 Hz, 2H, N3CH2).
Compound 7. In a stream of argon, 50% sodium hydride
(3.7 mg, 90 mmol) was dissolved in 1.5 mL of DMF, and
a solution prepared by dissolving compound 6 (18.5 mg,
70 mmol) obtained according to the method described
previously3 in 2.9 mL of DMF was added at 20 ꢀC,
followed by stirring for 3 h. To the resulting solution was
added a solution prepared by dissolving compound 5
(37 mg, 87 mmol) in 6 mL of DMF, and the temperature
of the mixture was allowed to rise from 20 ꢀC to room
temperature, followed by stirring for 24 h. Ethyl acetate
and a phosphate bu€er (pH 7.0) were added to the

T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
2183
resulting mixture, the organic layer was washed with
brine, and dried over anhydrous sodium sulfate. After
the solvent was evaporated o€, the residue was puri®ed
by 10 mL of silica gel using a chloroform:methanol
(100:1) to give 28 mg of compound 7 (yield 57%). SIMS
m/z: 548 (M+H)+; 1H NMR (500 MHz, CDCl3): d
11.32 (brs, 1H, 1-NH), 9.37 (brs, 1H, 10-NH), 7.10 (s,
1H, H-7), 7.06 (s, 1H, H-30), 6.80 (s, 1H, H-40), 4.42 (m,
2H, H-5), 4.10 (t, J=5.4 Hz, 2H, OCH2), 4.02 (s, 3H, 70-
OCH3), 3.88 (s, 3H, 50-OCH3), 3.82 (s, 3H, 3-COOCH3),
3.67 (m, 1H, H-4a), 3.47 (t, J=5.4 Hz, 2H, N3CH2),
2.60 (s, 3H, 2-CH3), 2.37 (dd, J=7.5, 3.4 Hz, 1H, H-4),
1.37 (t, J=4.2 Hz, 1H, H-4).
(OCH2CH2)n), 3.23 (s, 1H, CH(Ala)), 2.23 (brq, J=
6.0 Hz, 1H, CH(Val)), 1.26 (s, 1H, CH(Val)), 1.17 (d,
J=2.8Hz, 3H, CH3(Ala)), 0.89 (q, J=2.5 Hz, 6H,
CH3(Val)), DU-257 moiety; 11.58 (brs, 1H, 1-NH), 9.40
(brs, 1H, 10-NH), 7.12 (s, 1H, H-7), 6.95 (d, J=2.3 Hz,
1H, H-30), 6.81 (s, 1H, H-40), 4.45 (m, 2H, H-5), 4.08 (s,
3H, 70-OCH3), 3.90 (s, 3H, 50-OCH3), 3.88 (t, J=5.2 Hz,
2H, OCH2), 3.82 (s, 3H, 3-COOCH3), 3.67 (m, 1H, H-4a),
3.20 (q, J=6.4 Hz, 2H, CH2), 2.63 (s, 3H, 2-CH3), 2.38
(dd, J=7.5, 3.4 Hz, 1H, H-4), 1.37 (t, J=4.2 Hz, 1H, H-4).
Conjugation of linker-DU-257 moiety to antibody. Com-
pound 12 (0.45 mg, 0.33 mmol) was dissolved in 0.4 mL of
methanol, and 10% palladium carbon catalyst (1 mg)
was added, followed by vigorous stirring in a hydrogen
stream at 15 ꢀC for 5 h. After removal of the catalyst
by ®ltration, the solvent was removed from the ®ltrate
under reduced pressure at a temperature below 0 ꢀC to
obtain 0.14 mg (0.11 mmol) of compound 13. The
resulting compound 13 (0.14 mg) was dissolved in
250 mL of NHS in methylene chloride (0.076 mg/mL),
and 250 mL of DCC in methylene chloride (0.14 mg/mL)
was added, followed by stirring for 2.5 h at 0 ꢀC. The
insoluble matter was ®ltered o€, and the solvent was
removed under reduced pressure to form compound 14.
The residue was dissolved in 36 mL of dimethylsulfoxide,
and 204 mL of phosphate bu€er was added. To the
resulting mixture was added 0.56 mL of KM231 anti-
body (0.99 mg/mL) under ice cooling, followed by gen-
tle stirring at 4 ꢀC for 24 h. The reaction mixture was
then puri®ed by gel ®ltration chromatography using
Superose 12 column (10Â300 mm, Amersham-Pharma-
cia Biotech) equilibrated with phosphate bu€ered saline
(PBS). Antibody fractions were concentrated to give
250 mL of compound 15 (0.5 mg/mL) (yield 45.1%).
Compound 8 (DU-257). Compound 7 (20 mg, 36.6 mmol)
was dissolved in 1.1 mL of a mixture of acetic acid
(0.2 mL) and THF (9.8 mL), and 10% palladium carbon
catalyst (7.3 mg) was added at 10±15 ꢀC, followed by
vigorous stirring in a stream of hydrogen for 80 min
while maintaining the temperature at 10±15 ꢀC. After
the mixture was cooled below 20 ꢀC and the catalyst
was removed by ®ltration, the solvent was evaporated
o€ to give 9 mg (17.3 mmol) of compound 8 (yield 47%).
SIMS m/z: 521 (M+H)+; 1H NMR (500 MHz, CDCl3):
d 11.58 (brs, 1H, 1-NH), 9.40 (brs, 1H, 10-NH), 7.12 (s,
1H, H-7), 6.95 (d, J=2.3 Hz, 1H, H-30), 6.81 (s, 1H, H-
40), 4.45 (m, 2H, H-5), 4.08 (s, 3H, 70-OCH3), 3.92 (t,
J=5.2 Hz, 2H, OCH2), 3.90 (s, 3H, 50-OCH3), 3.82 (s,
3H, 3-COOCH3), 3.67 (m, 1H, H-4a), 3.20 (q, J=6.4 Hz,
2H, CH2), 2.63 (s, 3H, 2-CH3), 2.38 (dd, J=7.5, 3.4 Hz,
1H, H-4), 1.37 (t, J=4.2 Hz, 1H, H-4).
BzlO-CO-PEG-CO-Ala-Val-OH (10). Benzylated PEG-
dipeptidyl linker (compound 10) was prepared as
described elsewhere.23 The procedure is stated brie¯y;
tert-butyl ester of dipeptides, H-Ala-Val-OtBu, was
condensed to mono-benzyl ester of PEG-diacid using
NHS and DCC, followed by deprotection of tert-butyl
protecting group to form compound 10. TLC (CHCl3/
Digestion of linker with model enzyme. Thermolysin
(0.1 mL) was added to the compound 12 (0.1±0.2 mL,
0.2 mg/mL in PBS) to make a solution, in which ®nal
enzyme-substrate ratio was to be 1/10 so that the linker
could be digested completely. The resulting solution was
incubated at 37 ꢀC and an aliquot of reaction mixture
was analyzed by reversed-phase HPLC. In the case of
the conjugate, an aliquot of the solution containing
15 mg of the conjugate was digested in a similar way and
the portion of the reaction mixture corresponding to
12 mg of the conjugate was injected to the column.
1
CH3OH=10:1) Rf=0.2, H NMR (100 MHz, CDCl3): d
7.36 (5H, m, C6H5), 5.19 (2H, s, CH2), 4.12 (4H, s, OCH2),
3.64 (4nH, brs, (OCH2CH2)n), 3.23(1H, s, CH(Ala)), 2.23
(1H, brq, J=6.0 Hz, CH(Val)), 1.26 (1H, s, CH(Val)), 1.17
(3H, d, J=2.8 Hz, CH3(Ala)), 0.89 (6H, brd, J=2.5 Hz,
CH3(Val)).
BzlO-CO-PEG-CO-Ala-Val-DU-257 (12). Compound
10 (27 mg, 30 mmol) was dissolved in 2.4 mL of methylene
chloride, and then NHS (4.2 mg) and DCC (7.5 mg)
were successively added under ice cooling, followed by
stirring for 2 h. The insoluble matter was ®ltered o€,
and the solvent was removed from the ®ltrate under
reduced pressure. Then the residue was dissolved in
2 mL of pyridine, followed by addition of compound 8
(7.8 mg, 15 mmol) in 1.5 mL of pyridine at 0 ꢀC. The
resulting mixture was stirred for 3 h. After the solvent
was removed under reduced pressure, the residue was
puri®ed using 5 mL of silica gel using 5 mL each of chloro-
form±methanol mixture (100:1, 80:1, 60:1, 40:1, 20:1, 10:1,
5:1) to give 18mg (13 mmol) of desired compound 12 (yield
Flow Cytometry. Binding activities of the conjugates
and uncoupled antibody to target cells were measured
as follows; Samples (10 mg/mL) were added to SW1116
(1Â106 cells), and the mixture was subjected to reaction
for 30 min under ice cooling. The cells were centrifuged
and washed with PBS for three times, followed by removal
of unreactive conjugate. To the resulting mixture was
added 20mL of ¯uorescein isothiocyanate-labelled anti-
mouse IgG antibody (Wako Pure Chemical Industries,
Ltd., 30 times dilution) and the resulting mixture was
subjected to reaction for 30 min. After centrifugation
and washing with PBS were repeated three times, mea-
surement was carried out by ¯uorescence-activated cell
sorter (FACS) analysis using EPICS Elite ¯ow cytometer
(Coulter Corporation, Hialeah, Fla.).
1
86.7%). TLC(CHCl3/CH3OH=10:1) Rf=0.5, H NMR
(500 MHz, CDCl3): ꢀ linker moiety: 7.36 (m, 5H, C6H5),
5.19 (s, 2H, CH2), 4.12 (s, 4H, OCH2), 3.64 (brs, 4nH,

2184
T. Suzawa et al. / Bioorg. Med. Chem. 8 (2000) 2175±2184
Cell growth inhibition assay. Inhibitory e€ect of duo-
carmycin derivatives and the conjugate on cell growth
was examined by using HeLaS3 and SW1116 cell lines.
Each of the cell suspensions was put into a 96-well ¯at plate
in an amount of 50 mL (1Â103 cells/well), and cultured in a
CO2 incubator at 37 ꢀC for 2 h. Then the cells in the
plate were mixed with 50 mL of a solution containing
each compound, followed by further incubation for
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3
68 h. After washing the plate, 20 mL of H-thymidine
(463 kBq/mL) was added to each well and the cells were
incubated for 4 h. Then the cells were harvested to
determine the radioactivity of 3H-thymidine incorpo-
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The cell growth inhibiting activity was calculated
according to the following equations;
Cell Growth Inhibiting Activity ꢀ%† ˆ ‰1 ꢀRadioactivity
of Treated Cells†=ꢀRadioactivity of Control Cells†Š  100
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