Novel anthracycline-spacer-β-glucuronide, -β-glucoside, and -β-galactoside prodrugs for application in selective chemotherapy

Article abstract of DOI:10.1016/S0968-0896(99)00095-4

A series of anthracycline prodrugs containing an immolative spacer was synthesized for application in selective chemotherapy. The prodrugs having the general structure anthracycline-spacer-β-glycoside were designed to be activated by β-glucuronidase or β-galactosidase. Prodrugs with -chloro, -bromo or -n-hexyl substituents on the spacer were synthesized as well as prodrugs containing a -β-glucuronyl, -β-glucosyl or -β-galactosyl carbamate specifier. The key step in the synthesis of all prodrugs is the highly β-diastereoselective addition reaction of the anomeric hydroxyl of a glycosyl donor to a spacer isocyanate resulting in the respective β-glycosyl carbamate pro-moieties. The resulting protected pro-moieties were coupled to an anthracycline. Prodrugs were evaluated with respect to activation rate by the appropriate enzyme and additionally, their IC₅₀ values were determined. Optimal prodrugs in this study were at least 100- to 200-fold less toxic than their corresponding drug in vitro and were activated to the parent drug in a half-life time of approximately 2h. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00095-4

Bioorganic & Medicinal Chemistry 7 (1999) 1597±1610
Novel Anthracycline-spacer-ꢀ-glucuronide, -ꢀ-glucoside, and -ꢀ-
galactoside Prodrugs for Application in Selective Chemotherapy
Ruben G. G. Leenders, a,y Eric W. P. Damen, a Edward J. A. Bijsterveld, a
Hans W. Scheeren, a,* Pieter H. J. Houba, b Ida H. van der Meulen-Muileman, b
Epie Boven b and Hidde J. Haisma b
aDepartment of Organic Chemistry, NSR-Center for Molecular Structure, Design and Synthesis, University of Nijmegen, Toernooiveld,
6525 ED Nijmegen, The Netherlands
bDepartment of Medical Oncology, Academic Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
Received 7 December 1998; accepted 8 February 1999
AbstractÐA series of anthracycline prodrugs containing an immolative spacer was synthesized for application in selective che-
motherapy. The prodrugs having the general structure anthracycline-spacer-b-glycoside were designed to be activated by b-glucur-
onidase or b-galactosidase. Prodrugs with -chloro, -bromo or -n-hexyl substituents on the spacer were synthesized as well as
prodrugs containing a -b-glucuronyl, -b-glucosyl or -b-galactosyl carbamate speci®er. The key step in the synthesis of all prodrugs is
the highly b-diastereoselective addition reaction of the anomeric hydroxyl of a glycosyl donor to a spacer isocyanate resulting in the
respective b-glycosyl carbamate pro-moieties. The resulting protected pro-moieties were coupled to an anthracycline. Prodrugs were
evaluated with respect to activation rate by the appropriate enzyme and additionally, their IC50 values were determined. Optimal
prodrugs in this study were at least 100- to 200-fold less toxic than their corresponding drug in vitro and were activated to the
parent drug in a half-life time of approximately 2 h. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
an enzyme. When the prodrug-activating enzyme is tar-
geted to the tumor site using a monoclonal antibody
(mAb), this is called ADEPT (Antibody Directed
Enzyme Prodrug Therapy).8 In this strategy to activate
the prodrug, an enzyme is covalently linked to a mAb
and the mAb-enzyme conjugate is administered ®rst.
After this conjugate has been situated into the tumor
tissue the relatively non-toxic prodrug is administered in
the second step. The prodrug is converted to the parent
drug by the targeted enzyme. When an endogenous
enzyme that is present at an elevated level at the tumor
site is activating the prodrug, this is referred to as
monotherapy.9 For example b-glucuronidase is found to
be present at highly elevated concentrations in necrotic
tumor tissue.10,11 In this monotherapy only the prodrug
is administered. Several approaches toward the synth-
esis and use of enzyme-activated prodrugs have been
described.12±14 Often, the use of such prodrugs is limited
because of a too slow activation rate by the concomitant
enzyme,15 a premature prodrug activation by endo-
genous enzymes,16±18 or a too high cytotoxicity of the
prodrug.19
Since the discovery of doxorubicin DOX (Chart 1) in
the 1960s and the assessment of its remarkable activity
in the treatment of cancer,1 more than 2000 analogues
of this compound have been synthesized and evaluated
for anti-tumor activity. Despite great e€orts put into the
development of anthracyclines with a higher therapeutic
index, no major breakthroughs have been made in this
®eld and doxorubicin remains a frontline cytostatic
agent.2±4 The aggressive nature of cytotoxic chemicals
employed in the treatment of cancer, however, raises the
need for developing more selective therapies5,6 to treat
this class of disease, especially in the case of anthracy-
clines where side e€ects such as myelosuppression and
cumulative and irreversible cardiotoxicity occur.7
promising approach to increase selectivity is to use a
relatively nontoxic prodrug that is selectively activated
to the parent cytostatic at the tumor site by the action of
A
Key words: Anthracyclines; prodrugs; ADEPT; b-glucuronidase.
* Corresponding author. Tel.: +31-024-365-2331; fax: +31-024-365-
2929; e-mail: jsch@sci.kun.nl
Present address: MercaChem, Toernooiveld 100, 6503 CB Nijmegen,
The Netherlands.
Our research20±25 toward the preparation of enzyme-
activated prodrugs has focused on using a prodrug in
y
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00095-4

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R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
Scheme 1.
derivative DAU-1B and b-galactoside36 derivative
DAU-1C, which were designed to be activated by
human b-glucosidase and human b-galactosidase,
respectively. These prodrugs possess a reduced polarity
compared with the more polar glucuronide type pro-
drugs in order to retard their excretion. (2) For the same
reason, prodrug DAU-2A was designed to have a
lipophylic C6-tail on the benzylic carbon of the spacer.
(3) The ortho analogue of DAU-1A, prodrug DAU-3A
(Chart 3). (4) Compounds DAU-4A±7A possess a
chlorine or bromine atom on the aromatic ring of the
spacer. In addition to being electron withdrawing and,
hence, facilitating enzymatic hydrolysis of the b-glucur-
onyl group, bromo- and chloro-aryl groups are known
to have a high serum albumin binding potency,37 which
may retard prodrug excretion. Initially prodrugs of
daunorubicin were synthesized as this drug is more
readily available and easier to handle than doxorubicin.
Finally doxorubicin analogue DOX-1A was synthesized
as doxorubicin is clinically more relevant in the treat-
ment of solid tumor types than daunorubicin. As a pri-
mary evaluation of their biological activity the prodrugs
have been tested in vitro.
Chart 1.
combination with
a human enzyme to minimize
immuno-response26 in cases where a mAb-enzyme con-
jugate is used to activate a prodrug. Prior studies
showed that a b-glucuronyl27±32 carbamate group is
useful for prodrug synthesis when attached to a drug via
a spacer.22±24 Prodrug A (Chart 1) having a b-glucur-
onyl carbamate moiety attached without spacer to the
30-amino group of daunorubicin was activated too
slowly by b-glucuronidase to be suitable for use in
selective chemotherapy.21
The group of prodrugs described in this paper that
are designed to be activated analogously to DAU-1A33
are outlined in Scheme 1. Upon enzymatic hydrolysis of
the glucuronyl group of the prodrug DAU-1A and loss
of a molecule of CO2, the resulting para-amino (or
ortho-amino) benzyloxycarbonyl residue of drug-spacer
molecule 2 splits o€ spontaneously by a 1±6 (or 1±4 in
case of DAU-3A, Chart 3) electron shift process34,35 and
loss of an additional molecule of CO2 to yield the free
drug 3.
Here we present the chemical synthesis of DAU-1A and
the nine derivatives thereof shown in Chart 2, which
have the general structure of anthracycline-spacer-spe-
ci®er. A primary in vitro evaluation of these compounds
as prodrugs is reported. A previous paper24 describes
the biological properties of prodrug DAU-1A. It was
found that DAU-1A is activated by b-glucuronidase at
an acceptable rate to be useful for selective chemother-
apy. The prodrugs described in Chart 2 are designed to
study the e€ect of structural variations in the pro-moi-
ety on enzyme activation, cytotoxicity, and pharmaco-
kinetics. Within this group we prepared: (1) b-glucoside
Chart 2.

R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
1599
can be converted into a hydroxymethyl group by
deprotection, reduction or oxidation, respectively. Fol-
lowing these pathways, pro-moieties F can either be
prepared from carboxylic acids A, aniline-hydro-
chlorides B or commercially available isocyanates C.
In case of the synthesis of DAU-1A to 1C, DOX-1A
and DAU-2A and 3A (Scheme 3) the desired spacer-
isocyanates were generated from the respective spacer-
carboxylic acids in situ. Employing the Curtius rear-
rangement using diphenyl phosphoryl azide,39 spacer-
acyl azides were formed from carboxylic acids. Heating
of the acyl azides resulted in the formation of the
respective spacer-isocyanates 10 and 11. Following the
procedure described earlier by us38 glycosyl carbamates
12 and 13 were prepared from the respective carboxylic
acids 8 and 9 in a one-pot procedure. The protected pro-
moieties 12 and 13 were treated with acetic acid to
remove the silyl protective group on the benzyl alcohol
moiety. The resulting intermediates 14 and 15 were
coupled to daunorubicin or doxorubicin making use of
di-N,N0-succinimidyl carbonate40 or succinimidyl chloro-
formate leading to the protected prodrugs DAU-16a±c,
DAU-17a and DOX-16a. The ortho substituted prodrug
DAU-3A (Chart 3) was synthesized analogously to
DAU-1A (Scheme 3) starting from the silyl-protected o-
hydroxymethyl benzoic acid 19 (Chart 3). The starting
material for prodrug DAU-2A possessing a hexyl sub-
stituent on the spacer, compound 9, was easily obtained
by following Scheme 4. Because of the larger steric
demand of the additional hexyl group, the coupling of
15 to daunorubicin was accomplished using N-succini-
midyl chloroformate which is more reactive than di-
N,N0-succinimidyl carbonate.
Chart 3.
Chemistry
Considering the multiple functionalities present in dau-
norubicin and doxorubicin, their ease of oxidation and
especially the vulnerability of the glycosidic bond con-
necting the anthracyclinone aglycon and daunosamine
sugar, the synthesis of target compounds requires a
subtle approach. Preferably, the anthracycline is
involved in the synthesis sequence in one of the ®nal
reaction steps to prevent side reactions of the anthracy-
cline part.
The general strategy is depicted in Scheme 2. The
designed prodrugs presented in Chart 2 can be prepared
by coupling the benzyl alcohol group of pro-moieties F
to the anthracycline 30-amino group. For the prepara-
tion of compound F via masked intermediates E, in
routes a and b (Scheme 2), introduction of a b-glycosyl
carbamate group to the spacer can be accomplished by
the addition reaction of the anomerically unprotected
glycosyl donor D to spacer isocyanate C. This is the key
step in the synthesis of all prodrugs and when per-
formed in toluene and triethylamine as the catalyst,
leads to the respective spacer-b-glycosyl carbamates E
in a very high b-diastereoselectivity.38 Depending on the
availability of the starting material for A and B, the
hydroxymethyl precursor was either a protected hydroxy-
methyl group, an ester, or a methyl group. These units
Alternatively, compounds DAU-4A to DAU-7A (Scheme
5) having a chlorine or bromine atom on the spacer, were
synthesized starting from substituted41 anilines 23, toluic
isocyanates 24 or terephthalic acid derivatives 28,
depending on the availability of the starting material.
Scheme 2.

1600
R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
Scheme 3.
carbonate, promoieties 27 were coupled to dauno-
rubicin. As isocyanate 24g was not commercially avail-
able, the corresponding promoiety 27g was synthesized
from the bromo terephthalic acid derivative 28 accord-
ing to the one-pot modi®ed Curtius procedure outlined
above for Scheme 3. The monoester 28 was prepared
from the corresponding dicarboxylic acid by esteri®ca-
tion using DCC and obtained from the mixture of pro-
ducts by selective crystallization. Removal of the allyl
protecting group of 30 and reduction of the resulting
carboxylic acid to an alcohol group using BH3-THF
resulted in promoiety 27g.
Deprotection of the carbohydrate promoieties of all
protected prodrugs DAU-16a±c, DAU-17a, DOX-16a
and DAU-31d±i was accomplished using lithium hy-
droxide in MeOH/H2O/THF at 0ꢀC. After neutraliza-
tion of the basic solution with ion exchange resin and
conversion of the glucuronide carboxylic acid group to
the sodium salt using sodium bicarbonate, the polar
prodrugs were puri®ed on a reversed phase C18 column.
The product fraction was lyophilized to yield the pure
prodrugs as red ¯u€y solids.
Scheme 4.
After the addition of glucuronyl donor 18a to iso-
cyanates 24d±f, h and i, benzylic bromination of the
respectively obtained b-glycosyl carbamates 25 and
hydrolysis of the resulting bromides 26, promoieties
27d±f, h and i were obtained. Using di-succinimido

R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
1601
Scheme 5.
In the case of prodrug DOX-1A, however, during the
deprotection of the glucuronyl moiety extensive side
product formation took place due to the base lability of
doxorubicin.42 For this deprotection procedure a max-
imum yield of 37% was attained whereas for the dau-
norubicin prodrug DAU-1A the deprotection was
accomplished in 81%. Deprotection of the prodrug
precursors DAU-31d±i having the electronegative sub-
stituent on the 2-position of the aromatic ring of the
spacer led to extensive decomposition. In case of both
DAU-4A and 6A (X= 2-Cl and -2-Br, respectively),
about 35% yield was attained. Deprotection of
DAU-31h (X= 2-NO2) did not lead to the desired
prodrug DAU-8A, but in nearly quantitative yield
methyl carbamate 32 (Chart 4) was formed. Deprotec-
tion of the prodrug precursors DAU-31e,g having
the electronegative substituent on the 3-position of
the aromatic ring was accomplished in satisfactory
yields in case of DAU-5A (67%) and DAU-7A (50%).
Deprotection of DAU-31i (X=-3-NO2) led to extensive
Chart 4.
decomposition and multiple side-product formation and
only a small amount of DAU-9A was obtained in an
impure state. So it seems that electron-withdrawing
substituents in the aromatic ring of the spacer which
should facilitate hydrolysis of the glucuronide group

1602
R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
also promote base mediated decomposition of the glu-
curonyl carbamate part. Especially electron-withdrawing
substituents in the 2-position of the aromatic ring seem
to make the glucuronyl carbamate function more sus-
ceptible for nucleophillic attack.43
human ovarian cancer cells and these were compared
with the IC50 values of the respective parent drugs (see
Table 1). The b-glucuronide type prodrugs in entries 1,
2, 6 and 8 proved to be 85 to 200 times less toxic than
the parent drug and for that reason seem very apt as
prodrugs to be activated by b-glucuronidase.
Biological Evaluation of the Prodrugs
Conclusions
From all prodrugs synthesized, in vitro activation rates
by the relevant enzyme have been determined. For pro-
drugs with favorable in vitro activation rates, in vitro
antiproliferative e€ects were determined. The chemical
stability of prodrugs DAU-1A and DOX-1A was greater
than 95% over a 24 h incubation period in either PBS/
BSA and human serum both at 37ꢀC.
A ¯exible convergent synthesis has been developed for
the anthracycline prodrugs collected in Chart 2. In this
synthesis (generally outlined in Scheme 2) speci®er,
spacer and drug can be varied independently.
Enzymatic activation rates determined for all prodrugs
showed that (1) the b-glucuronide based prodrugs seem
better substrates than the b-glucoside and b-galactoside
prodrugs, (2) prodrugs containing a 1,6-elimination
spacer showed faster hydrolysis than prodrugs with a
1,4-elimination spacer, (3) chlorine or bromine sub-
stituents at the aromatic ring of the spacer do not have a
profound e€ect on the enzymatic activation rate. A
small accelerating e€ect was found on the enzymatic
activation rate for prodrug DAU-5A having a 3-chlorine
substituted spacer when compared with DAU-1A.
Prodrug activation rate
For the determination of the activation half-lives, the
prodrugs were incubated with the concordant enzyme44
at clinically achievable concentrations (100 mM). All
prodrugs of daunorubicin and doxorubicin possessing
the b-glucuronyl carbamate promoiety were activated by
b-glucuronidase at approximately the same rate. Inter-
mediate drug-spacer molecules were not detected by
HPLC. Half-lives between 2 and 3 h were recorded (see
Table 1). The glucosyl and galactosyl based prodrugs,
however, were activated at a much slower rate than the
glucuronyl based prodrugs. Among the derivatives hav-
ing an electron-withdrawing group on the spacer, only
DAU-5A (entry 8, Table 1) displayed a slightly higher
activation rate than the unsubstituted prodrug DAU-1A
(entry 1). Because the synthesis of DAU-1A is less com-
plicated than that of DAU-5A, DAU-1A was selected to
be further evaluated for use in ADEPT. For the same
reason, DOX-1A was selected for further evaluation.
Unfortunately it was not possible to prepare prodrugs
with stronger electron-withdrawing substituents, for
example, a nitro-substituted spacer (DAU-8A and -9A)
for which a higher enzymatic activation rate than DAU-
1A are expected. Because of extensive decomposition
during the ®nal deprotection step these prodrugs were
not obtained.
DAU-1A and DOX-1A have shown promising results in
monotherapy experiments using mice bearing human
tumor xenografts. These results are published separately.25
Of these, prodrug DOX-1A has been selected for further
evaluation in a phase I trial in cancer patients.
Antiproliferative e€ects
The in vitro antiproliferative e€ects of prodrugs with a
favorable activation rate were determined in OVCAR-3
Experimental
Biological evaluation
Prodrug stability. The plasma stability of DAU-1A and
DOX-1A was determined as described before24 in a
0.1% (w/v) BSA/PBS solution of pH 7.4 as well as in
human serum, both over a 24-h period at 37ꢀC. `Half-
life' in this context refers to release of daunorubicin or
doxorubicin from the tested prodrug.
Table 1. Enzyme activation half-lives and antiproliferative e€ects
Entry
Compound
Activation t1=2 (min)
IC50 (mM)a
1
2
3
4
5
6
7
8
DAU-1A
DOX-1A
DAU-1B
DAU-1C
DAU-2A
DAU-3A
DAU-4A
DAU-5A
DAU-6A
DAU-7A
Daunorubicin
Doxorubicin
135b
170b
300c
400c
950b
125b
150b
90b
10
10
1.5
2.5
n.d.
11
n.d.
8.5
Prodrug activation half-lives. Half-lives of enzyme
hydrolysis were determined by incubating 100 mM pro-
drug at pH 6.845 with 0.03 U/mL of human b-glucur-
onidase46 or 0.3 U/mL bovine liver b-galactosidase44
(purchased from Sigma, G1875) at 37ꢀC. Samples were
prepared and analyzed on reversed phase SiO2±C18
HPLC as described.24
9
150b
170b
Ð
n.d.
n.d.
0.1
10
11
12
Ð
0.05
a
Inhibition concentration [(pro) drug concentrations that gave 50%
growth inhibition in OVCAR-3 cells when compared with control cell
growth].
Antiproliferative e€ects. The antiproliferative e€ects of
daunorubicin, doxorubicin and of all 10 prodrugs on
OVCAR-3 cells were determined by measuring cell
growth with a protein dye stain.15 Cells were harvested
100 mM Prodrug, 0.03 U/mL human b-glucuronidase, pH 6.8, 37ꢀC.
b
c
100 mM Prodrug., 0.3 U/mL bovine liver b-galactosidase,44 pH 6.8,
37ꢀC.

R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
1603
with 0.25% trypsin and 0.2% EDTA in PBS to obtain a
single cell suspension and seeded in 96-wells tissue cul-
ture plates (50,000 cells/mL 100 mL/well, in supple-
mented DMEM, three wells per concentration). After
24 h drug or prodrug was added (100 mL/well in sup-
plemented DMEM) at di€erent concentrations with a
range of three or more logs and the cells were allowed to
proliferate for 72 h. Cells were ®xed with 25% tri-
chloroacetic acid for 1 h at 4ꢀC and washed with water.
After staining the cells with 0.4% sulforhodamine B in
1% (v/v) acetic acid for 15 min at room temperature,
they were washed with 1% acetic acid and air-dried.
The bound dye was solubilized with 10 mM unbu€ered
Tris and the absorbance was read at 492 nm. The
absorbance was linear with cell concentrations from
1000 to 200,000 cells/well. Separate wells were ®xed 24 h
after seeding to subtract background staining. The
antiproliferative e€ects were determined and expressed
as IC50 values which are the (pro)drug concentrations
that gave 50% growth inhibition when compared with
control cell growth.
N-[4-(Hydroxymethyl)phenyl] O-(methyl 2,3,4-tri-O-ace-
tyl-ꢀ-glucuronyl) carbamate (14a). Compound 12a
(1.227 g, 2.055 mmol) was stirred in 120 mL of THF/
H2O/AcOH (1/1/1). The course of the deprotection
reaction was followed by TLC (SiO2, Et2O). After 3 h,
no starting material was detected, the reaction mixture
was diluted with 200 mL of H2O and the THF was
removed by evaporation. The aqueous layer was washed
®ve times with 100 mL portions of CH2Cl2, the organic
extracts were combined and washed with aqueous satu-
rated NaHCO3 until gas evolution ceased. After that the
organic layer was dried with brine and Na2SO4 succes-
sively and evaporated. The resulting foam was sonicated
in i-Pr2O and ®ltered to give 911 mg of 14a, mp 173ꢀC,
92%. 1H NMR (100 MHz, CDCl3) d 1.96 (9H,s), 3.64
(3H,s), 4.13 (1H, d, J=9.3 Hz), 4.61 (2H, s), 5.00±5.35
(3H, m), 5.71 (1H, d, J=7.5 Hz), 6.92 (1H, s), 7.19±7.28
(4H, m).
N-[4-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-
(methyl 2,3,4-tri-O-acetyl ꢀ-glucuronyl) carbamate
(DAU-16a). Compound 14a (600 mg, 1.448 mmol) was
stirred with 369 mg (1.1 equiv) of di-N-N0-succinimidyl
carbonate and 550 mL (2.5 equiv) of i-Pr2NEt in 25mL
of CH2Cl2. After 1.5 h, no starting material was detected
on TLC (SiO2, Et2O) and a solution of 790 mg (1.1
equiv) of DAU-HCl and 550 mL (2.5 equiv) of i-Pr2NEt
in 10 mL of DMF were added. The course of the
reaction was monitored by TLC (SiO2, CH2Cl2/EtOH,
10/1). After all of the active ester starting material had
disappeared, the reaction mixture was diluted with
600 mL of CH2Cl2 and washed with 100 mL portions of
aqueous 0.5 N KHSO4 (3Â), H2O, aqueous saturated
NaHCO3 (2Â), H2O (2Â), and with brine. The organic
layer was dried over Na2SO4 and evaporated. The
resulting red residue was puri®ed twice by means of cir-
cular chromatography using a chromatotron supplied
with a 2 mm silica plate and CH2Cl2/EtOH (10/1 and
30/1, respectively). After evaporation of the eluent, the
resulting red product was sonicated in i-Pr2O and ®l-
trated to yield 1.076 g of DAU-16a, 84%, mp 163±
164ꢀC. 1H NMR (400 MHz, CDCl3) d 1.28 (3H, d,
J=6.6 Hz), 1.78 (1H, dt, J=12.9 Hz, J=3.8 Hz), 1.87
(1H, dd, J=13.3, Hz J=5.8 Hz), 2.05 (9H, s), 2.09 (1H,
dd, J=15.2, Hz J=3.7 Hz), 2.30 (1H, d, J=14.9 Hz),
2.41 (3H, s), 2.88 (1H, d, J=18.8 Hz), 3.20 (1H, d,
J=18.8 Hz), 3.67 (1H, s), 3.72 (3H, s), 3.88 (1H, m),
4.05 (3H, s), 4.22 (1H, d, J=9.8 Hz), 4.15±4.25 (1H, m),
4.48 (1H, s), 4.89 (1H, d, J=12.2 Hz), 4.95 (1H, d,
J=12.2 Hz), 5.15±5.30 (4H, m), 5.38 (1H, t, J=9.3 Hz),
5.47 (1H, d, J=3.1 Hz), 5.77 (1H, d, J=8.0 Hz), 7.16
(1H, s), 7.18 (2H, d, J=8.0 Hz), 7.24 (2H, d, J=8.0 Hz)
7.37 (1H, d, J=8.4 Hz), 7.76 (1H, t, J=8.0 Hz), 8.00
(1H, d, J=7.7 Hz), 13.22 (1H, s), 13.94 (1H, s).
Chemistry
General. Daunorubicin and doxorubicin hydrochlorides
were a generous gift of Pharmachemie bv (Haarlem,
The Netherlands). Chromatotron model 7924-T Harri-
son Research (Palo Alto, CA, USA) equipped with
plates (thickness 2 mm, diameter 8.5 cm) made from
Merck silicagel 60 PF254 which contains gypsum (art.
7749) was used when circular chromatography is indi-
cated. When cation exchange material was indicated,
amberlite resin IR-120 (Na) BDH (Poole, Dorset, UK)
was converted to the H+-form using 1 N HCl prior to
use. Reversed phase chromatography was performed
with a liquid chromatography pump LC-410 (Kontron)
using a pre-packed column (24 cm, diameter 11 mm)
containing octadecylsilane (4063 mm) Merck (Darm-
stadt, Germany). Prior to use the RP-C18 column was
equilibrated with demineralized water. 1H NMR spectra
at 400 MHz were obtained on a Bruker AM-400. Che-
mical shifts are expressed in ppm down®eld from inter-
nal standard Me4Si. Et3N, i-Pr2NEt, CH2Cl2, pyridine
and CCl4 were dried by distillation over CaH2, PhMe
was dried by distillation over sodium and THF by dis-
tillation over LiAlH4. In all cases demineralized H2O
was used. Allyl alcohol was dried by distillation over
Mg (with a catalytic amount of I2 added).
Synthesis of N-[4-(daunorubicin-N-carbonyl oxymethyl)-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-1A)
N-[4-(t-Butyldimethylsilyloxymethyl)phenyl] O-(methyl
2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carbamate (12a). 954 mg
(3.59 mmol) of 847 was stirred with 853 mL (1.1 equiv) of
(PhO)2P(O)N3 and 549 mL (1.1 equiv) of Et3N in 10 mL
of PhMe under an argon atmosphere. After 12 h the
reaction mixture was stirred at 80ꢀC for 3 h. The mix-
ture was cooled to ambient temperature and 719 mg (0.6
equiv) of methyl 2,3,4-tri-O-acetyl glucuronic acid 18a
was added. The reaction was worked-up as previously
described38 to yield 1.227 g of 12a, 95% from 18a. Phy-
sical data are according to the literature.38
N-[4-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-ꢀ-
glucuronyl carbamate sodium salt (DAU-1A). To 1.255 g
(1.193 mmol) of DAU-16a was added 57.3 mL (6 equiv)
of a 0.10 N LiOH solution in MeOH/H2O/THF (2.5/
1.0/0.5), the resulting deep blue solution was stirred at
0ꢀC under an argon atmosphere. Progress of the depro-
tection was monitored on reversed-phase TLC (SiO2±
C18 MeCN/H2O, 1/1). After 2 h of deprotection, the

1604
R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
reaction mixture was diluted with 150 mL of H2O and
neutralized by adding ca. 10 g of amberlite cation
exchange material (H+ form), 10 mL of THF was added
to homogenize the suspension. The amberlite was
removed by ®ltration and ca. 150 mg of NaHCO3 were
added. The MeOH and THF suspended in the water
layer were removed by evaporation and the red aqueous
product solution was transferred to a reversed phase
column packed with RP-C18 material and eluted with
300 mL of H2O to remove the excess of NaHCO3. The
column was successively washed with MeCN/H2O (1/4)
to elute the product and the MeCN was removed by
evaporation. Freeze drying of the aqueous product
solution gave 892 mg of 81%, mp 175ꢀC (dec.). Anal.
calcd for C42H43N2O20Na.4H2O: C, 50.91; H, 5.19; N,
2.83. Found: C, 50.73; H, 4.96; N, 2.94. MS (FAB+)
m/z 942 ([M+1+Na]+), 941 ([M+Na]+), 920 ([M+1
+H]+), 919 ([M+H]+). 1H NMR (400 MHz,
(CD3)2SO)) d 1.11 (3H, d, J=6.6 Hz), 1.47 (1H, dd,
J=12.1 Hz J=3.4 Hz), 1.82 (1H, dt, J=11.5 Hz J=
3.4 Hz), 2.07 (1H, dd, J=14.1 Hz J=6.1 Hz), 2.19 (1H,
dd, J=14.1 Hz, J=3.1 Hz), 2.27 (3H, s), 3.11 (1H, d,
J=18.5 Hz), 3.13 (1H, d, J=18.5 Hz), 3.20±3.65 (4H,
m), 3.71 (1H, m), 3.95 (3H, s), 4.16 (1H, q, J=6.6 Hz),
4.70 (1H, d, J=5.1 Hz), 4.76±5.32 (4H, m), 4.84 (1H, d,
J=12.8 Hz), 4.88 (1H, d, J=12.8 Hz), 4.90 (1H, t, J=
5.1 Hz), 5.20 (1H, d, J=3.0 Hz), 5.28 (1H, d, J=8.2 Hz),
5.52 (1H, s), 6.84 (1H, d, J=8.0 Hz), 7.23 (2H, d, J=
8.4 Hz), 7.43 (2H, d, J=8.4 Hz), 7.60 (1H, dd, J=6.8 Hz
J=3.0 Hz), 7.80±7.90 (2H, m), 9.90 (1H, s) 13.25 (1H,
s), 13.99 (1H, s).
(1H, dd, J=14.2 Hz J=5.6Hz), 2.20 (1H, d, J=11.9 Hz),
2.95 (1H, d, J=18.8 Hz), 3.01 (1H, d, J=18.8 Hz), 3.05±
3.60 (4H, m), 3.71 (1H, m), 3.99 (3H, s), 4.15 (1H, q,
J=6.3 Hz), 4.57 (2H, s), 4.62±4.71 (1H, m), 4.68 (1H, d,
J=4.9 Hz), 4.80±5.30 (4H, m), 4.88 (2H, s), 4.94 (1H, t,
J=4.2 Hz), 5.21 (1H, d, J=2.7 Hz), 5.33 (1H, d, J=
8.0 Hz), 5.46 (1H, s), 6.83 (1H, d, J=8.0 Hz), 7.24 (2H,
d, J=8.1 Hz), 7.43 (2H, d, J=8.1 Hz), 7.65 (1H, t,
J=4.8 Hz), 7.85±7.95 (2H, m), 9.91 (1H, s) 13.28 (1H,
s), 14.03 (1H, s).
Synthesis of N-[4-(daunorubicin-N-carbonyl oxymethyl)
phenyl] O-ꢀ-glucosyl carbamate (DAU-1B)
N-[4-(t-Butyldimethylsilyloxymethyl)phenyl] O-(2,3,4,6-
tetra-O-acetyl-ꢀ-glucosyl) carbamate (12b). Analogously
to the preparation of 12a from 500 mg (1.88 mmol) of 8
and 339 mg (0.5 equiv) of 18b. Compound 12b was iso-
lated in 77% yield (calculated from 18b) (452 mg). Phy-
sical data are according to literature.38
N-[4-(Hydroxymethyl)phenyl] O-(2,3,4,6-tetra-O-acetyl-
ꢀ-glucosyl) carbamate (14b). Analogously to the pre-
paration of 14a from 240 mg (0.394 mmol) of 12b.
Compound 14b was isolated in 68% yield (132 mg) as
1
an oil. H NMR (100 MHz, CDCl3) d 1.91, 1.92, 1.94
and 1.96 (4s, 12H), 3.70±3.85 (1H, m), 3.98 (1H, dd,
J=12.4 Hz, J=2.0 Hz), 4.22 (1H, dd, J=12.4 Hz J=
4.3 Hz), 4.54 (2H, s), 4.90±5.20 (3H, m), 5.62 (1H, d,
J=7.8 Hz), 6.93 (1H, s), 7.15±7.25 (4H, m).
N-[4-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-
(2,3,4,6-tetra-O-acetyl-ꢀ-glucosyl) carbamate (DAU-
16b). Analogously to the preparation of DAU-16a from
80 mg (0.161 mmol) of 14b. Compound DAU-16b was
isolated in 58% yield (97 mg), mp 145±148ꢀC. 1H NMR
(400 MHz, CDCl3) d 1.29 (3H, d, J=6.5 Hz), 1.70±1.95
(3H, m), 2.02, 2.04, 2.06 and 2.07 (4s, 12H), 2.11 (1H,
dd, J=14.8 Hz J=3.9 Hz), 2.31 (1H, d, J=14.8 Hz),
2.41 (3H, s), 2.91 (1H, d, J=18.7 Hz), 3.22 (1H, d, J=
18.7 Hz), 3.65 (1H, m), 3.85±3.95 (2H, m), 4.07 (3H, s),
4.12 (1H, d, J=11.7 Hz), 4.21 (1H, q, J=6.5 Hz), 4.31
(1H, dd, J=11.7 Hz J=4.6 Hz), 4.48 (1H, s), 4.92 (1H,
d, J=12.2 Hz), 4.97 (1H, d, J=12.2 Hz), 5.10±5.35 (5H,
m), 5.48 (1H, d, J=3.3 Hz), 5.75 (1H, d, J=8.1 Hz),
7.10 (1H, s), 7.22 (2H, d, J=7.9Hz), 7.31 (2H, d, J=
7.9 Hz), 7.38 (1H, d, J=8.6Hz), 7.78 (1H, t, J=8.0 Hz),
8.02 (1H, d, J=7.5 Hz), 13.26 (1H, s), 13.96 (1H, s).
Synthesis of N-[4-(doxorubicin-N-carbonyl oxymethyl)
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DOX-1A)
N-[4-(Doxorubicin-N-carbonyl oxymethyl)phenyl] O-(methyl
2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carbamate (DOX-16a).
Analogously to the preparation of DAU-16a from
570 mg (1.18 mmol) of 14a. Compound DOX-16a was
isolated in 69% yield (850 mg), mp 165±167ꢀC. 1H
NMR (400 MHz, CDCl3) d 1.28 (3H, d, J=6.5 Hz),
1.75±1.90 (3H, m), 2.05 (9H, s), 2.14 (1H, dd, J=
14.7 Hz, J=3.9 Hz), 2.32 (1H, d, J=14.7 Hz), 2.97 (1H,
d, J=18.9 Hz), 3.06 (1H, s, 14-OH), 3.24 (1H, d,
J=18.9 Hz), 3.66 (1H, m), 3.72 (3H, s), 3.86 (1H, m),
4.06 (3H, s), 4.13 (1H, q, J=6.5 Hz), 4.22 (1H, d,
J=9.7 Hz), 4.55 (1H, s), 4.75 (2H, s), 4.93 (1H, d,
J=12.4 Hz), 4.96 (1H, d, J=12.4 Hz), 5.15±5.30 (4H,
m), 5.36 (1H, t, J=9.3 Hz), 5.48 (1H, d, J=3.0 Hz), 5.77
(1H, d, J=8.0 Hz), 7.20 (2H, d, J=7.0 Hz), 7.20±7.35
(3H, m), 7.38 (1H, d, J=8.5 Hz), 7.78 (1H, t, J=8.0 Hz),
8.01 (1H, d, J=7.8 Hz), 13.19 (1H, s), 13.93 (1H, s).
N-[4-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-ꢀ-
glucosyl carbamate (DAU-1B). Analogously to the pre-
paration DAU-1A (with the following modi®cations:
After the amberlite was ®ltered o€, no NaHCO3 was
added. Prior to RP-18 column chromatography, the
crude product suspension was homogenized by adding
ca. 10% (v/v) MeCN) from 12.5 mg (11.9 mmol) of
DAU-16b. Prodrug DAU-1B was isolated in 79%
(8.7 mg), mp 182±184ꢀC. Anal. calcd for C42H46
N2O19.2.5H2O: C, 54.37; H, 5.54; N, 3.02. Found: C,
54.45; H, 5.24; N, 2.88. MS (FAB+) m/z 906 ([M+1
+Na]+), 905 ([M+Na]+). 1H NMR (400 MHz, (CD3)2
SO) d 0.06 (s, 4H), 1.13 (3H, d, J=6.5 Hz), 1.47 (1H,
d, J=12.8 Hz), 1.83 (1H, dt, J=12.8 Hz J=3.5 Hz),
N-[4-(Doxorubicin-N-carbonyl oxymethyl)phenyl] O-ꢀ-
glucuronyl carbamate sodium salt (DOX-1A). Analo-
gously to the preparation of DAU-1A from 850 mg
(0.808 mmol) of DOX-16a. Prodrug DOX-1A was iso-
lated in 37% yield (280 mg), mp 191ꢀC (dec.). Anal.
calcd for C42H43N2O21Na.4H2O: C, 50.10; H, 5.11; N,
2.78. Found: C, 50.31; H, 4.81; N, 3.00. MS (FAB+) m/
1
z 958 ([M+1+Na]+), 957 ([M+Na]+). H NMR (400
MHz, (CD3)2SO) d 1.12 (3H, d, J=6.3 Hz), 1.47 (1H, d,
J=12.3 Hz), 1.83 (1H, dt, J=12.9 Hz J=3.5 Hz), 2.12

R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
1605
2.08 (1H, dd, J=14.4 Hz, J=5.4 Hz), 2.20 (1H, d,
J=14.4 Hz), 2.27 (3H, s), 2.93 (2H, s), 3.00±3.80 (10H,
m), 3.97 (3H, s), 4.17 (1H, q, J=6.5 Hz), 4.87 (2H, s),
4.92 (1H, t, J=4.4 Hz), 5.21 (1H, d, J=2.6 Hz), 5.35
(1H, d, J=8.2 Hz), 6.83 (1H, d, J=8.0 Hz), 7.24 (2H, d,
J=8.3 Hz), 7.42 (2H, d, J=8.3 Hz), 7.62 (1H, dd,
J=6.2 Hz J=3.5 Hz), 7.85±7.90 (2H, m), 9.88 (1H, s)
13.26 (1H, s), 14.00 (1H, s).
3.9 Hz), 4.63 (1H, d, J=5.0 Hz), 5.68 (1H, d, J=5.5 Hz),
4.86 (2H, s), 4.92 (1H, t, J=4.3 Hz), 5.04 (1H, d, J=
4.6 Hz), 5.20 (1H, d, J=2.6 Hz), 5.30 (1H, d, J=8.0 Hz),
5.51 (1H, s), 6.82 (1H, d, J=8.0 Hz), 7.23 (2H, d, J=
8.4 Hz), 7.421 (2H, d, J=8.3 Hz), 7.62 (1H, t, J=7.6 Hz),
7.85±7.90 (2H, m), 9.83 (1H, s) 13.24 (1H, s), 14.00
(1H, s).
Synthesis of N-[4-(daunorubicin-N-carbonyl-1-oxyheptyl)-
phenyl]O-ꢀ-glucuronyl carbamate sodium salt (DAU-2A)
Synthesis of N-[4-(daunorubicin-N-carbonyl oxymethyl)
phenyl] O-ꢀ-galactosyl carbamate (DAU-1C)
4-(1-Hydroxyheptyl) bromobenzene (21 (Scheme 4)).
0.78 g (0.9 equiv) of Mg turnings, a crystal of I2 and
30 mL of THF were brought into a three-necked ¯ask
connected with a dropping funnel and a re¯ux con-
denser under an argon atmosphere. From 6.26 g (=1
equiv) of n-hexyl bromide in 10 mL of THF in the
dropping funnel, approximately one third was added to
the reaction vessel. The Grignard reagent was agitated
and the rest of the n-hexyl bromide solution was added.
When the magnesium had almost disappeared, the
reaction mixture was cooled to 10ꢀC and 7.0 g (1.0
equiv) of 4-bromobenzaldehyde 20 in 10 mL of THF
were added slowly. Stirring was continued for 10 min at
10ꢀC and the reaction mixture was quenched on 20 g
of ice, 10 mL of 15% H2SO4 and the reaction mixture
was extracted with 350 mL portions of Et2O. The
organic layer was washed with brine and dried over
Na2SO4. Puri®cation by column chromatography (SiO2-
N-[4-(t-Butyldimethylsilyloxymethyl)phenyl] O-(2,3,4,6-
tetra-O-acetyl-ꢀ-galactosyl) carbamate (12c). Analo-
gously to the preparation of 12a from 400 mg
(1.50 mmol) of 8. Compound 12c was isolated in 57%
yield (261 mg) (calculated from 18c). Physical data are
according to literature.38
N-[4-(Hydroxymethyl)phenyl] O-(2,3,4,6-tetra-O-acetyl-
ꢀ-galactosyl) carbamate (14c). Analogously to the pre-
paration of 14a from 225 mg (0.368 mmol) of 12c.
Compound 14c was isolated in quantitative yield
(183 mg) as an oil. 1H NMR (100 MHz, CDCl3) d 1.92,
1.95, 1.99 and 2.04 (4s, 12H), 3.90±4.20 (3H, m), 4.55
(2H, s), 5.05 (1H, dd, J=10.1 Hz, J=3.3 Hz), 5.27 (1H,
t, J=7.9 Hz), 5.37 (1H, d, J=2.9 Hz), 5.64 (1H, d, J=
7.8 Hz), 7.20 (2H, d, J=10.1 Hz), 7.30 (2H, d, J=
10.1 Hz), 7.65 (1H, s).
1
n-hexane) yielded 2.9 g, 34%, of 21 as an oil. H NMR
N-[4-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-
(2,3,4,6-tetra-O-acetyl-ꢀ-galactosyl) carbamate (DAU-
16c). Analogously to the preparation of DAU-16A
from 179 mg (0.36 mmol) of 14c. Compound DAU-16c
was isolated in 45% yield (171 mg), mp 170±173ꢀC. 1H
NMR (400 MHz, CDCl3) d 1.29 (3H, d, J=6.5 Hz), 1.77
(1H, s), 1.78 (1H, dt, J=13.1 Hz, J=4.0 Hz), 1.88 (1H,
dd, J=13.2 Hz, J=5.0 Hz), 2.00, 2.03, 2.05 and 2.17 (4s,
12H), 4.11 (1H, dd, J=14.8 Hz, J=4.0 Hz), 2.31 (1H, d,
J=14.9 Hz), 2.42 (3H, s), 2.91 (1H, d, J=18.8 Hz), 3.22
(1H, d, J=18.8 Hz), 3.67 (1H, d, J=5.9 Hz), 3.91 (1H,
bs), 4.07 (3H, s), 4.05±4.25 (4H, m), 4.49 (1H, s), 4.92
(1H, d, J=12.3 Hz), 4.98 (1H, d, J=12.3 Hz), 5.12 (1H,
dd, J=10.4 Hz J=3.1 Hz), 5.19 (1H, d, J=8.32 Hz),
5.25 (1H, s), 5.35 (1H, t, J=9.4 Hz), 5.4 (1H, d, J=
3.1 Hz), 5.49 (1H, d, J=3.4 Hz), 5.71 (1H, d, J=8.3Hz),
7.14 (1H, s), 7.22 (2H, d, J=7.9 Hz), 7.31 (2H, d, J=
7.9 Hz), 7.38 (1H, d, J=8.5 Hz), 7.78 (1H, t, J=8.1 Hz),
8.02 (1H, d, J=7.7 Hz), 13.25 (1H, s), 13.96 (1H, s).
(100 MHz, CDCl3) d 0.86 (3H, t, J=5.6 Hz), 1.10±1.70
(10H, m), 2.35 (1H, bs), 4.58 (1H, t, J=6.3 Hz), 7.17
(2H, d, J=8.3 Hz), 7.45 (2H, d, J=8.3 Hz).
4-(1-t-Butyldimethylsilyloxyheptyl) bromobenzene (22).
655 mg (2.42 mmol) of 21, 247 mg (1.5 equiv) of imida-
zole, 1.46 g (4.0 equiv) of TBDMS-Cl and a catalytic
amount of DMAP were dissolved in 20 mL of CH2Cl2
and stirred for 3 days under an argon atmosphere. After
that, the reaction mixture was diluted with 200 mL of
Et2O, washed with 100 mL portions of, respectively,
aqueous 0.5 N NaHSO4, aqueous saturated NaHCO3
and brine. The organic layer was dried over NaSO4 and
evaporated. The product was puri®ed by means of col-
umn chromatography (SiO2, n-hexane) to give 602 mg
1
of 22, 65%, as an oil. H NMR (100 MHz, CDCl3) d
0.14 (3H, s), 0.02 (3H, s), 0.67 (9H, s), 0.70±1.75 (13H,
m), 4.58 (1H, t, Alk1-H, J=5.8 Hz), 7.15 (2H, d, J=
8.5 Hz), 7.42 (2H, d, J=8.4 Hz).
N-[4-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-ꢀ-
galactosyl carbamate (DAU-1C). Analogously to the
preparation of DAU-1B from 62 mg (59.1 mmol) of
DAU-16c. Prodrug DAU-1C was isolated in 77% yield
(40 mg), mp 206±210ꢀC. Anal. calcd for C42H46
N2O19.2H2O: C, 54.90; H, 5.48; N, 3.05. Found: C,
54.90; H, 5.44; N, 3.10. MS (FAB+) m/z 906 ([M+1
+Na]+), 905 ([M+Na]+). 1H NMR (400 MHz, (CD3)2
SO) d 1.11 (3H, d, J=6.4 Hz), 1.46 (1H, dd, J=12.8 Hz
J=3.8 Hz), 1.82 (1H, dt, J=12.8 Hz J=3.5 Hz), 2.08
(1H, dd, J=14.1 Hz, J=5.7Hz), 2.19 (1H, d, J=14.7 Hz),
2.25 (3H, s), 2.93 (2H, t, J=19.8 Hz), 3.30±3.75 (8H, m),
3.97 (3H, s), 4.16 (1H, q, J=6.6 Hz), 4.50 (1H, d, J=
4-(1-t-Butyldimethylsilyloxyheptyl) benzoic acid (9). To
1.1 mL (1.6 equiv) of a 1.6 N n-BuLi solution and 1 mL
of THF in a 3-necked ¯ask connected with a dropping
funnel under an argon atmosphere, 437 mg (=1.0 equiv)
of 22 dissolved in 10 mL of THF were added slowly at
78ꢀC. The reaction mixture was stirred at 50ꢀC for
15 min and carefully added to a few grams of solid CO2
in 10 mL of THF under an argon atmosphere. The
reaction mixture was quenched with ice and 20 mL of
aqueous 0.5 N KHSO4 were added. The product was
extracted with three 100 mL portions of Et2O and the
combined organic layer was washed with brine and
dried over Na2SO4. The product was puri®ed by means

1606
R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
of column chromatography (SiO2, CH2Cl2/EtOH, 10/1)
to yield 179 mg, 45%, of 9 as an oil. 1H NMR (100 MHz,
CDCl3) d 0.14 (3H, s), 0.03 (3H, s), 0.88 (9H, s), 0.70±
1.70 (13H, m), 4.69 (1H, t, J=5.5 Hz), 7.37 (2H, d,
J=8.1 Hz), 8.04 (2H, d, J=8.1 Hz).
52.73; H, 5.69; N, 2.70. MS (FAB+) m/z 1026 ([M+1
+Na]+), 1025 ([M+Na]+), 1004 ([M+1+H]+), 1003
([M+H]+). Because of chirality of the spacer ArCÃH
(alkyl)(O)-, a diastereomeric mixture was formed in a
ratio of 1/0.2, determined by dividing the integrals of
the clear singlets of the respective ArNH- signals found
1
N-[4-(1-t-Butyldimethylsilyloxyheptyl)phenyl] O-(methyl
2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carbamate (13a). Ana-
logously to the preparation of 12a from 189 mg
(0.539 mmol) of 9 and 135 mg (0.75 equiv) of 18a. After
column chromatography (SiO2, Et2O/n-hexane, 1/1)
compound 13a was isolated in 73% (201 mg) (calculated
from 18a) as an oil. Due to the racemic starting material,
a diastereomeric mixture of 13a must be formed, con-
versely, this was not observed in the proton NMR spec-
trum. 1H NMR (100 MHz, CDCl3) d 0.23 (3H, s), 0.07
(3H, s, 0.59 (9H, s), 0.52±1.63 (13H, m), 1.98 (bs, 9H),
3.66 (3H, s), 4.15 (1H, d, J=9.4 Hz), 4.51 (1H, t, J=
6.3 Hz), 5.10±5.45 (3H, m,), 5.73 (1H, d, J=7.6 Hz), 6.95
(1H, bs), 7.13 (2H, d, J=8.7 Hz), 7.03 (2H, d, J=8.7).
at 9.78 and 9.84 ppm in the proton-NMR spectrum. H
NMR (400 MHz, (CD3)2SO) d 0.76 and 0.81 (2H, t,
J=6.8 Hz resp. 6.7 Hz), 1.05±1.25 (13H, m), 1.39 and
1.46 (1H, dd, J=12.3 Hz, J=4.1 Hz resp. 12.3 and 4.0
Hz), 1.79 (1H, dt, J=12.9 Hz, J=3.9 Hz), 2.08 (1H, m),
2.15 (1H, d, J=13.4 Hz), 2.23 (3H, s), 2.90 (1H, d,
J=18.4 Hz), 2.97 (1H, d, J=18.4 Hz), 3.00±3.45 (4H,
m), 3.65 (1H, m), 3.97 and 3.98 (s), 4.12 (1H, q, J=
6.2 Hz), 4.66 and 4.68 (1H, d, J=5.4 Hz resp.
J=5.5 Hz), 4.91 (1H, t, J=4.3 Hz), 5.00 (1H, t, J=
4.7 Hz), 5.18 and 4.21 (1H, d, J=1.6 Hz resp J=
2.4 Hz), 5.10±5.15 (1H, m), 5.25 (1H, d, J=8.0 Hz), 5.50
(1H, s), 6.72 (1H, d, J=7.8 Hz), 7.16 and 7.20 (2H, d,
J=8.5 Hz resp. J=8.5 Hz), 7.37 and 7.41 (2H, d, J=
8.2 Hz resp. J=8.3 Hz), 7.63 (1H, m), 7.85±7.95 (2H,
m), 9.78 and 9.84 (1H, s), 13.19 (1H, s), 14.01 (1H, s).
N-[4-(1-Hydroxyheptyl)phenyl] O-(methyl 2,3,4-tri-O-
acetyl-ꢀ-glucuronyl) carbamate (15a). Analogously to
the preparation of 14a from 226 mg (0.331 mmol) of
13a. Compound 15a was isolated in 92% yield (173 mg)
as an oil. 1H NMR (100 MHz, CDCl3) d 0.80±1.90
(13H, m), 2.05 (9H, s), 3.73 (3H, s), 4.19 (1H, d, J=
8.9 Hz), 4.63 (1H, t, J=6.8 Hz), 5.15±5.30 (3H, m), 5.76
(1H, d, J=7.4 Hz), 7.12 (1H, bs), 7.22 (4H, bs).
Synthesis of N-[2-(daunorubicin-N-carbonyl oxymethyl)-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-3A)
N-[2-(t-Butyldimethylsilyloxymethyl)phenyl] O-(methyl
2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carbamate (ortho-12a).
Analogously to the preparation of 12a from 400 mg
(1.50 mmol) of 1948 (Chart 3). Compound ortho-12a was
isolated in 69% yield (310 mg) (calculated from 18a),
mp 92±94ꢀC. 1H NMR (100 MHz, CDCl3) d 0.00 (3H, s,
SiMeaMeb-), 0.05 (3H, s), 0.83 (9H, s), 1.96 (9H, s), 3.66
(3H, s), 4.16 (1H, d, J=9.4 Hz), 4.60 (1H, d, J=18.1 Hz),
4.72 (1H, d, J=18.1 Hz), 5.01±5.32 (3H, m), 5.76 (1H,
d, J=7.8Hz), 6.90±7.35 (3H, m), 7.90 (1H, d, J=8.0 Hz),
8.61 (1H, s).
N-[4-(Daunorubicin-N-carbonyl-(1-oxyheptyl))phenyl] O-
(methyl
2,3,4-tri-O-acetyl-ꢀ-glucuronyl)
carbamate
(DAU-17a). 173 mg (0.305 mmol) of 15a, 109 mg (2.0
equiv) of N-succinimidyl chloroformate and 74 mL (3.0
equiv) of pyridine in 10 mL of CH2Cl2 were stirred for
2 h at room temperature under an argon atmosphere. A
solution of 431 mg (2.5 equiv) of DAU-HCl, 333 mL of i-
Pr2NEt (6.0 equiv) in 10 mL of DMF was added and
stirring was continued for 18 h. The product was pur-
i®ed as described for DAU-16a to give 122 mg, 36% of
DAU-17a, mp 71ꢀC. Because of chirality of the spacer -
ArCÃH(alkyl)(O)-, a diastereomeric mixture was formed
in a ratio of 1/1, determined by dividing the integrals of
the clear singlets of the respective ArNH-signals found
N-[2-(Hydroxymethyl)phenyl] O-(methyl 2,3,4-tri-O-ace-
tyl-ꢀ-glucuronyl) carbamate (ortho-14a). Analogously
to the preparation of 14a, from 310 mg (0.52 mmol) of
ortho-12a. Compound ortho-14a was isolated in 68%
yield (171 mg), mp 132ꢀC. 1H NMR (100 MHz, CDCl3)
d 1.96 (9H, s), 3.64 (3H, s), 4.11 (1H, d, J=9.4 Hz), 4.59
(2H, s), 5.09±5.21 (3H, m), 5.74 (1H, d, J=7.5 Hz),
6.95±7.30 (3H, m), 7.70 (1H, d, J=7.7 Hz), 8.15 (1H, s).
1
at 6.77 and 6.86 ppm. H NMR (400 MHz, CDCl3) d
0.80±1.90 (18H, m), 2.00±2.05 (9H, m), 2.09 (1H, dd,
J=15.2 Hz J=3.7 Hz), 2.30 (1H, d, J=14.9 Hz), 2.40
(3H, bs), 2.88 (1H, d, J=18.8 Hz), 3.20 (1H, d, J=
18.8 Hz), 3.65±3.90 (5H, m), 4.06 and 4.09 (3H, s), 4.22
(1H, bd, J=9.8 Hz), 4.15±4.25 (1H, m), 4.48 (1H, s),
4.90 (7H, m), 5.79 (1H, bd, J=8.0 Hz), 6.77 and 6.86
(1H, s), 7.15±7.25 (4H, m), 7.38 (1H, bd, J=9.4 Hz),
7.77 (bt, 1H, J=9.0 Hz), 8.02 (1H, bd, J=7.5 Hz), 13.26
and 13.28 (1H, s), 13.95 and 13.98 (1H, s).
N-[2-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-
(methyl 2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carbamate (ortho-
DAU-16a). 100 mg (0.213 mmol) of ortho-14a was stir-
red with 43 mg (1.1 equiv) of succinimidyl chloro-
formate and 21 mL (1.05 equiv) of pyridine in 10 mL of
CH2Cl2 and coupled to 144 mg (1.2 equiv) of DAU-HCl
using 73 mL (2 equiv) of i-Pr2NEt and 10 mL of DMF
(analogously to DAU-16a). 62% (133 mg) of ortho-
DAU-16a was obtained, mp 151±153ꢀC. 1H NMR
(400 MHz, CDCl3) d 1.30 (3H, d, J=6.5 Hz), 1.80±1.85
(3H, m) 2.00, 2.04 and 2.05 (3s, 9H), 2.16 (1H, dd,
J=14.8 Hz, J=4.1 Hz), 2.33 (1H, d, J=15.2 Hz), 2.43
(3H, s), 2.95 (1H, d, J=18.9 Hz), 3.23 (1H, d, J=18.9 Hz),
3.67 (1H, d, J=6.7 Hz), 3.72 (3H, s), 3.90 (1H, m), 4.07
(3H, s), 4.20±4.25 (2H, m), 4.46 (1H, s), 4.96 (1H, d,
J=12.6 Hz), 5.07 (1H, d, J=12.6 Hz), 5.16 (1H, t,
N-[4-(Daunorubicin-N-carbonyl-(1-oxyheptyl))phenyl] O-
ꢀ-glucuronyl carbamate sodium salt (DAU-2A). Analo-
gously to the preparation of DAU-1A (except that the
product was eluted from the reversed-phase column
with CH3CN/H2O, 1/2) from 60 mg (0.054 mmol) of
DAU-17a. Prodrug DAU-2A was isolated in 39% yield
(21 mg), mp 193ꢀC (dec.). Anal. calcd for C48H55N2
O20Na.5H2O: C, 52.75; H, 5.99; N, 2.56. Found: C,

R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
1607
J=8.0 Hz), 5.25±5.35 (4H, m), 5.50 (1H, m), 5.80 (1H,
d, J=7.5), 7.08 (1H, t, J=7.4 Hz), 7.26 (1H, d, J=
8.8 Hz), 7.32 (1H, t, J=7.7 Hz), 7.39 (1H, d, J=8.5 Hz),
7.78 (1H, t, J=8.1 Hz), 7.84 (1H, m), 8.03 (1H, d,
J=7.7 Hz), 8.20 (1H, s), 13.28 (1H, s), 13.97 (1H, s)
evaporated and dried under reduced pressure (0.1 mm
Hg) to give the crude 26d which was used in the next
step without further puri®cation. H NMR (100 MHz,
CDCl3) d 2.05 (9H, s), 3.75 (3H, s), 4.25 (1H, d, J=
9.2 Hz), 4.42 (2H, s), 5.10±5.45 (3H, m), 5.85 (1H, d,
J=7.6 Hz), 7.10±7.50 (3H, m), 8.09 (1H, d, J=8.4 Hz).
1
N-[2-(Daunorubicin-N-carbonyl oxymethyl)phenyl] O-ꢀ-
glucuronyl carbamate sodium salt (DAU-3A). Analo-
gously to the preparation of DAU-1A from 20 mg
(19 mmol) of ortho-DAU-16a. Prodrug DAU-3A was
isolated in 69% yield (12.2 mg), mp 171ꢀC (dec). Anal.
calcd for C42H43N2O20Na.3.5H2O: C, 51.38; H, 5.13;
N, 2.85. Found: C, 51.36; H, 4.86; N, 2.69. MS
(FAB+) m/z 942 ([M+1+Na]+), 941 ([M+Na]+), 920
([M+1+H]+), 919 ([M+H]+). 1H NMR (400 MHz,
(CD3)2SO) d 1.12 (3H, d, J=6.4 Hz), 1.46 (1H, dd,
J=12.6 Hz), 1.85 (1H, dt, J=12.6 Hz, J=3.5 Hz), 2.11
(1H, dd, J=14.4 Hz, J=5.8 Hz), 2.20 (1H, d, J=
14.4 Hz), 2.26 (3H, s), 2.93 (1H, d, J=18.2 Hz), 2.99
(1H, d, J=18.2 Hz), 3.15±3.65 (4H, m), 3.72 (1H, m),
3.99 (3H, s), 4.17 (1H, q, J=6.4 Hz), 4.70±4.90 (2H, m),
4.94 (1H, t, J=4.4 Hz), 4.98 (2H, s), 5.20±5.25 (2H, m),
5.22 (1H, d, J=3.1 Hz), 5.32 (1H, d, J=5.5 Hz), 5.34
(1H, d, J=8.0 Hz), 5.55 (1H, s), 7.03 (1H, d, J=8.0 Hz),
7.15 (1H, t, J=7.3), 7.27 (1H, t, J=7.7), 7.33 (1H, d,
J=7.6 Hz), 7.40 (1H, d, J=7.8 Hz), 7.66 (1H, dd,
J=5.9 Hz J=3.9 Hz), 7.88±7.94 (2H, m), 9.25 (1H, s)
13.29 (1H, s), 14.04 (1H, s).
N-[4-(Hydroxymethyl) 2-chlorophenyl] O-(methyl 2,3,4-
tri-O-acetyl-ꢀ-glucuronyl) carbamate (27d). The crude
26d was dissolved in 10 mL of acetone and 10 mL (3
equiv) of an aqueous 0.2 N AgNO3 solution were
added. After 2 days, the mixture was ®ltered and eva-
porated. The resulting oil was redissolved in CH2Cl2
and washed with saturated aqueous NaHCO3 (2Â),
with brine and dried over Na2SO4. After the CH2Cl2
had been evaporated, 27d was crystallized from Et2O to
give 175 mg, 52%, from 25d, as white crystals. 1H NMR
(100 MHz, CDCl3) d 1.98 (9H, s), 3.67 (3H, s), 4.16 (1H,
d, J=9.3 Hz), 4.56 (2H, s), 5.00±5.40 (3H, m), 5.75 (1H,
d, J=7.5 Hz), 7.17 (1H, d, J=8.4 Hz), 7.31 (2H, bs),
7.37 (1H, d, J=8.4 Hz).
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 2-chloro-
phenyl] O-(methyl 2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carb-
amate (DAU-31d). Analogously to the preparation of
DAU-16a from 45 mg (0.87 mmol) of 27d. Compound
DAU-31d was isolated in 28% yield (26 mg), mp 150±
155ꢀC. 1H NMR (400 MHz, CDCl3) d 1.29 (3H, d,
J=6.6 Hz), 1.74 (1H, dt, J=13.1 Hz, J=4.1 Hz), 1.90
(1H, dd, J=13.1 Hz J=4.8 Hz), 2.05 (9H, s), 2.12 (1H,
dd, J=14.9 Hz, J=4.1 Hz), 2.32 (1H, d, J=14.8 Hz),
2.41 (3H, s), 2.95 (1H, d, J=18.8 Hz), 3.25 (1H, d,
J=18.8 Hz), 3.65 (1H, s), 3.73 (3H, s), 3.87 (1H, m),
4.08 (3H, s), 4.21 (1H, d, J=9.5 Hz), 4.20±4.25 (1H, m),
4.44 (1H, s), 4.95 (1H, d), 5.10±5.30 (4H, m), 5.35 (1H,
t, J=8.9 Hz), 5.49 (1H, d, J=3.7 Hz), 5.81 (1H, d,
J=7.9 Hz), 7.21 (1H, d, J=8.3 Hz), 7.26 and 7.34 (2s,
2H), 7.40 (1H, d, J=8.4 Hz), 7.79 (1H, t, J=8.1 Hz),
8.00±8.10 (2H, m), 13.29 (1H, s), 13.99 (1H, s).
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 2-chloro-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-4A)
N-(4-Methyl 2-chlorophenyl) O-(methyl 2,3,4-tri-O-ace-
tyl-ꢀ-glucuronyl) carbamate (25d). 4-Methyl-2-chloro-
phenyl isocyanate 24d was prepared from 500 mg of 4-
methyl-2-chloroaniline-HCl 23d by re¯uxing it for 2 h
with 339 mL (1.0 equiv) of diphosgene in 20 mL of PhMe
under an argon atmosphere. The reaction mixture was
allowed to cool to ambient temperature and 1.5 mL (ca.
4 equiv) of Et3N were added to combine with the liber-
ated HCl. 469 mg (0.5 equiv) of 18a were added. After
0.5 h, 18a had disappeared and the reaction mixture was
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 2-chloro-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-
4A). Analogously to the preparation of DAU-1A from
26 mg (0.024 mmol) of DAU-31d. Prodrug DAU-4A was
isolated in 37% (8.5 mg), mp 175±179ꢀC. Anal. calcd for
C42H42N2O20ClNa.5H2O: C, 48.38; H, 5.02; N, 2.69.
Found: C, 48.22; H, 4.70; N, 3.08. MS (FAB+) m/z 975
([M+Na]+), 955 ([M+2+H]+), 954 ([M+1+H]+),
1
taken to dryness (100% b-isomer by H NMR of the
crude reaction mixture) dissolved in Et2O and washed
with 5% aqueous KHSO4, saturated aqueous NaHCO3,
brine, dried over Na2SO4 and evaporated. The crude
product was puri®ed by column chromatography (SiO2,
Et2O/n-hexane, 1/1, to elute the apolar symmetrical
urethane and Et2O to elute the product) and the pro-
duct fraction was crystallized from i-Pr2O to give
645 mg, 92%, of 25d as white crystals, mp 142±143ꢀC.
1H NMR (100 MHz, CDCl3) d 2.05 (9H, s), 2.29 (3H, s),
3.74 (3H, s), 4.26 (1H, d, J=9.5 Hz), 5.10±5.55 (3H, m),
5.86 (1H, d, J=7.4 Hz), 7.07 (1H, d, J=8.3 Hz), 7.10±
7.30 (2H, m,), 7.92 (1H, d, J=8.3 Hz).
1
953 ([M+H]+). H NMR (400 MHz, (CD3)2SO) d 1.12
(3H, d, J=6.4 Hz), 1.48 (1H, d, J=12.6), 1.85 (1H, t,
J=13.4 Hz), 2.05 (2H, m), 2.25 (3H, s), 2.90 (6H, m),
3.73 (1H, m), 3.98 (3H, s), 4.16 (1H, m), 4.73 (1H, d,
J=5.1 Hz), 4.85 (2H, m), 5.00 (1H, d, J=5.2 Hz), 5.23
(1H, bs), 5.27 (1H, d, J=7.9 Hz), 5.53 (1H, s), 7.27 (1H,
d, J=7.3 Hz), 7.44 (1H, s,), 7.57 (1H, d, J=8.12 Hz),
7.64 (1H, m), 7.85±8.00 (2H, m), 9.22 (1H, s).
N-[4-(Bromomethyl) 2-chlorophenyl] O-(methyl 2,3,4-tri-
O-acetyl-ꢀ-glucuronyl) carbamate (26d). Compound
25d (325 mg, 0.65 mmol) was dissolved in 10 mL of CCl4
and 127 mg (1.1 equiv) of NBS and a catalytic amount
of AIBN was added. The solution was radiated for 1 h
using a 250 W lamp and the temperature raised to
re¯ux. After cooling, the reaction mixture was ®ltered,
Synthesis of N-[4-(daunorubicin-N-carbonyl oxymethyl)
3-chlorophenyl] O-ꢀ-glucuronyl carbamate sodium salt
(DAU-5A)
N-(4-Methyl 3-chlorophenyl) O-(methyl 2,3,4-tri-O-acetyl-
ꢀ-glucuronyl) carbamate (25e). 200 mg (1.19 mmol) of

1608
R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
commercially available 4-methyl-3-chlorophenyl isocya-
nate 24e was dissolved in 10 mL of PhMe under an
argon atmosphere. 199 mg (0.5 equiv) of 18a and one
drop of Et3N were added. The course of the reaction
was followed by means of TLC (SiO2, Et2O). After 18a
had disappeared, the reaction mixture was taken to
dryness (a/b of the crude reaction mixture was 1/10 by
1H NMR) and sonicated in Et2O. The solid material
was removed and the solution was concentrated and
refrigerated overnight. 434 mg, 81%, of 25e was
obtained as white crystals, mp 155ꢀC. The a-isomer
dd, J=12.7 Hz, J=3.2 Hz), 2.08 (1H, dd, J=13.9 Hz
J=5.2 Hz), 2.17 (1H, d, J=13.6 Hz), 2.24 (3H, s), 3.00
(6H, m 40-H), 3.70 (1H, m), 3.96 (3H, s), 4.13 (1H, q,
J=6.5 Hz), 4.69 (1H, d, J=3.9 Hz), 4.94 (2H, s), 5.07
(1H, bs), 5.21 (1H, s), 5.28 (1H, d, J=8.1 Hz), 5.51 (1H,
s), 6.95 (1H, d, J=7.9 Hz), 7.23 (1H, d, J=8.4 Hz),
7.35±7.40 (2H, m), 7.55±7.60 (2H, m), 7.80±7.95 (2H,
m), 10.14 (1H, s).
Synthesis of N-[4-(daunorubicin-N-carbonyl oxymethyl)
2-bromophenyl] O-ꢀ-glucuronyl carbamate sodium salt
(DAU-6A)
1
stayed in solution. H NMR (100 MHz, CDCl3) d 1.99
(9H, s), 2.24 (3H, s, 3.66 (3H, s), 4.16 (1H, d, J=9.4 Hz),
5.00±5.45 (3H, m), 5.71 (1H, d, J=7.6 Hz), 6.90±7.15
(3H, m), 7.39 (1H, s).
N-[4-(Methyl) 2-bromophenyl] O-(methyl 2,3,4-tri-O-
acetyl-ꢀ-glucuronyl) carbamate (25f). Analogously to
the preparation of 25d from 263 mg (1.20 mmol) of 23f.
The crude product was puri®ed by means of column
chromatography (SiO2, Et2O/n-hexane, ꢀ 1/1) to give
N-[4-(Bromomethyl) 3-chlorophenyl] O-(methyl 2,3,4-tri-
O-acetyl ꢀ-glucuronyl) carbamate (26e). Analogously to
the preparation of 26d from 332 mg (0.66 mmol) of 25e.
The crude 26e which was obtained was used in the next
1
310 mg (95% from 18a) of 25f, mp 139 C. H NMR
(100 MHz, CDCl3) d 2.05 (9H, s), 2.30 (3H, s), 3.75 (3H,
s), 4.22 (1H, d, J=9.1 Hz), 5.10±5.45 (3H, m), 5.83 (1H,
d, J=7.8 Hz), 7.15±7.45 (2H, m), 7.35 (1H, d, J=
2.0 Hz) 7.93 (1H, d, J=8.3 Hz).
1
step without further puri®cation. H NMR (100 MHz,
CDCl3) d 1.99 (9H, s), 3.66 (3H, s), 4.20 (1H, d,
J=8.9 Hz), 4.47 (2H, s), 5.05±5.45 (3H, m), 5.71 (1H, d,
J=7.5 Hz), 6.90±7.50 (3H, m), 7.63 (1H, s).
N-[4-(Bromomethyl) 2-bromophenyl] O-(methyl 2,3,4-tri-
O-acetyl-ꢀ-glucuronyl) carbamate (26f). Analogously
to the preparation of 26d from 250 mg (0.458 mmol) of
25f. The crude bromide 26f which was obtained was
N-[4-(Hydroxymethyl) 3-chlorophenyl] O-(methyl 2,3,4-
tri-O-acetyl-ꢀ-glucuronyl) carbamate (27e). Analo-
gously to the preparation of 27d from the crude 26e
obtained as described above. After workup the crude
product was sonicated in i-Pr2O and ®ltered to give 27e
1
used without further puri®cation in the next step. H
NMR (100 MHz, CDCl3) d 2.05 (9H, s), 3.75 (3H, s),
4.23 (1H, d, J=9.1 Hz), 4.42 (3H, s), 5.10±5.45 (3H, m),
5.83 (1H, d, J=7.8 Hz), 7.35 (1H, dd, J=8.4 Hz J=
2.0 Hz), 7.40 (1H, s), 7.59 (1H, d, J=2.0 Hz), 8.09 (1H,
d, J=8.4 Hz).
1
in 76% yield (259 mg) (calculated from 25e). H NMR
(100 MHz, CDCl3) d 1.99 (9H, s), 3.66 (3H, s), 4.16 (1H,
d, J=9.3 Hz), 4.63 (2H, s), 5.00±5.45 (3H, m), 5.69 (1H,
d, J=7.5 Hz), 7.10±745 (3H, m), 7.55 (1H, s).
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 3-chloro-
phenyl] O-(methyl 2,3,4-tri-O-acetyl-ꢀ-glucuronyl) car-
bamate (DAU-31e). Analogously to the preparation of
DAU-16a from 100 mg (0.193 mmol) of 27e. Compound
DAU-31e was obtained in 76% yield (82 mg), mp 154±
158ꢀC. 1H NMR (400 MHz, CDCl3) d 1.29 (3H, d,
J=6.5 Hz), 1.78 (1H, dt, J=13.0 Hz J=3.9 Hz), 1.85±
1.90 (2H, m), 2.05 (9H, s), 2.05±2.15 (1H, m), 2.28 (1H,
d, J=14.9 Hz), 2.42 (3H, s), 2.87 (1H, d, J=19.0 Hz),
3.19 (1H, d, J=18.8 Hz), 3.68 (1H, bs), 3.74 (3H, s),
3.87 (1H, bs), 4.05 (3H, s), 4.15±4.20 (1H, m), 4.24 (1H,
d, J=9.8 Hz), 4.46 (1H, s), 4.97 (1H, d, J=12.7 Hz),
5.01 (1H, d, J=12.7 Hz), 5.12±5.40 (4H, m), 5.48 (1H,
d, J=2.4 Hz), 5.73 (1H, d, J=8.0 Hz), 7.00±7.50 (5H,
m), 7.76 (1H, t, J=8.1 Hz), 8.00 (1H, d, J=8.2 Hz),
13.24 (1H, s), 13.95 (1H, s).
N-[4-(Hydroxymethyl) 2-bromophenyl] O-(methyl 2,3,4-
tri-O-acetyl-ꢀ-glucuronyl) carbamate (27f). Analogously
to the preparation of 27d from 0.458 mmol of crude 26f
described above. Compound 27f was obtained in 80%
(206 mg) (calculated from 25f) as an oil. 1H NMR
(100 MHz, CDCl3) d 2.04 (9H, s), 3.75 (3H, s), 4.22 (1H,
d, J=9.2 Hz), 4.65 (3H, s), 5.20±5.39 (3H, m), 5.83 (1H,
d, J=7.5 Hz), 7.20±7.40 (2H, m), 7.57 (1H, d, J=
1.8 Hz), 8.07 (1H, d, J=8.1 Hz).
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 2-bromo-
phenyl] O-(methyl 2,3,4-tri-O-acetyl-ꢀ-glucuronyl) car-
bamate (DAU-31f). Analogously to the preparation of
DAU-16a from 178 mg (0.317 mmol) of 27f. Compound
DAU-31f was obtained in 14% yield (51 mg), mp 149ꢀC.
1H NMR (400 MHz, CDCl3) d 1.22 (3H, d, J=6.4 Hz),
1.58 (1H, s), 1.70 (1H, dt, J=13.2 Hz, J=4.2 Hz), 1.82
(1H, dd, J=13.3 Hz, J=5.1 Hz), 1.95 (9H, s), 2.04 (1H,
dd, J=14.9 Hz, J=4.1 Hz), 2.24 (1H, d, J=14.4 Hz),
2.34 (3H, s), 2.83 (1H, d, J=18.8 Hz), 3.16 (1H, d,
J=18.8 Hz), 3.59 (1H, m), 3.68 (3H, s), 3.81 (1H, m),
4.01 (3H, s), 4.15 (1H, d, J=9.6 Hz), 4.10±4.20 (1H, m),
4.38 (1H, s), 4.86 (1H, d, J=12.5 Hz), 4.89 (1H, d,
J=12.5 Hz), 5.05±5.25 (4H, m), 5.28 (1H, t, J=9.3 Hz),
5.42 (1H, d, J=3.7 Hz), 5.74 (1H, d, J=8.0 Hz), 7.15±
7.25 (2H, m), 7.32 (1H, d, J=8.6 Hz), 7.41 (1H, s,), 7.71
(1H, t, J=8.0 Hz), 7.96 (1H, d, J=7.6 Hz), 7.90±8.00
(1H, m), 13.19 (1H, s), 13.90 (1H, s).
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 3-chloro-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-
5A). Analogously to the preparation of DAU-1A from
45 mg (42 mmol) of DAU-31e. Prodrug DAU-5A was
obtained in 67%, (27 mg), mp 205ꢀC (dec.). Anal. calcd
for C42H42N2O20ClNa.6H2O: C, 47.53; H, 5.13; N, 2.64.
Found: C, 47.25; H, 4.75; N, 2.64. MS (FAB+) m/z 977
([M+2+Na]+), 976 ([M+1+Na]+), 975 ([M+Na]+),
955 ([M+2+H]+), 954 ([M+1+H]+), 953 ([M+H]+).
1H NMR (400 MHz, (CD3)2SO) d 1.11 (3H, d, J=
6.4 Hz), 1.46 (1H, dd, J=12.0 Hz, J=3.6 Hz), 1.81 (1H,

R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
1609
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 2-bromo-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-
6A). Analogously to the preparation of DAU-1A from
37 mg (33 mmol) of DAU-31f. Prodrug DAU-6A was
obtained in 34% yield (11.2 mg), mp 213ꢀC (dec). Anal.
calcd for C42H42N2O20BrNa.3H2O: C, 47.96; H, 4.60;
N, 2.66. Found: C, 47.75; H, 4.59; N, 3.08. MS (FAB+)
m/z 1021 ([M+2+Na]+), 1019 ([M+Na]+), 999 ([M+2
amount of Pd(PPh3)4 was added. After 15 min 30 had
disappeared on TLC (SiO2, Et2O) and the reaction
mixture was diluted with 100 mL of Et2O and washed
with 100 mL portions of aqueous 0.5 N KHSO4 (3Â)
and with brine. The organic layer was dried over
Na2SO4 and evaporated to obtain 246 mg of the car-
boxylic acid in a quantitative yield, mp 110ꢀC. This was
1
used without further puri®cation in the next step. H
1
+H]+), 997 ([M+H]+). H NMR (400 MHz, (CD3)2
NMR (100 MHz, CDCl3) d 2.04 (9H, s), 3.73 (3H, s),
4.23 (1H, d, J=9.5 Hz), 5.10±5.40 (3H, m), 5.77 (1H, d,
J=7.6 Hz), 7.36 (1H, dd, J=8.6 Hz J=2.2 Hz), 7.56
(1H, s), 7.79 (1H, d, J=2.2 Hz) 7.96 (1H, d, J=8.6 Hz).
SO) d 1.13 (3H, d, J=6.5 Hz), 1.48 (1H, dd, J=12.4 Hz,
J=5.3 Hz), 1.83 (1H, dt, J=13.3 Hz J=3.5 Hz), 2.10±
2.20 (2H, m), 2.26 (3H, s), 2.94 (1H, d, J=18.5 Hz),
3.10±3.65 (5H, m 40-H), 3.73 (1H, m), 3.99 (3H, s), 4.18
(1H, q, J=6.3 Hz), 4.72 (1H, d, J=5.6 Hz), 4.75±5.20
(2H, m), 4.93 (2H, s), 5.22 (1H, d, J=3.5 Hz), 5.27 (1H,
d, J=8.1 Hz), 5.55 (1H, s), 6.98 (1H, d, J=8.0 Hz), 7.31
(1H, d, J=8.4 Hz), 7.52 (1H, d, J=8.2 Hz), 7.61 (1H, s,),
7.67 (1H, t, J=7.8 Hz), 7.90±8.00 (2H, m). 8.31 (1H, s).
N-[4-(Hydroxymethyl) 3-bromophenyl] O-(methyl 2,3,4-
tri-O-acetyl-ꢀ-glucuronyl) carbamate (27g). 153 mg
(0.265 mmol) of the latter carboxylic acid were stirred in
15 mL of THF at room temperature under an argon
atmosphere. 1.33 mL (5.0 equiv) of a 1 M BH3-THF
solution were added slowly over a period of 20 min. The
reaction mixture was stirred overnight at room tem-
perature. When the acid had almost disappeared on
TLC (SiO2, Et2O), the reaction mixture was quenched
with 15 mL of H2O. When gas evolution ceased, the
reaction mixture was diluted with 200 mL of EtOAc and
washed with 100 mL portions of saturated aqueous
NaHCO3 (3Â) and with brine. The organic layer was
dried over Na2SO4 and evaporated to yield 140 mg,
Synthesis of N-[4-(daunorubicin-N-carbonyl oxymethyl)
3-bromophenyl] O-ꢀ-glucuronyl carbamate sodium salt
(DAU-7A)
3-Bromo terephthalic acid allyl ester (28). Bromoter-
ephthalic acid (3.0 g, 12 mmol) was dissolved in 150 mL
of THF under an argon atmosphere and 1.25 mL (1.5
equiv) of AllOH and a catalytic amount of DMAP was
added. After cooling the mixture to 0ꢀC, 2.2 g (1.0
equiv) of DCC were added and the reaction mixture was
stirred at 0ꢀC for 2 h and overnight at room tempera-
ture. The reaction mixture was ®ltrated and evaporated,
the product was puri®ed by means of column chroma-
tography (SiO2, CH2Cl2/EtOH, 10/1) and crystallized
from n-hexane to yield 893 mg, 26%, of 28 as white
1
94%, of the pure 27g as an oil, H NMR (100 MHz,
CDCl3) d 2.00 (9H, s), 3.67 (3H, s), 4.14 (1H, d,
J=9.6 Hz), 4.63 (2H, s), 5.00±5.30 (3H, m), 5.69 (1H, d,
J=7.6 Hz), 7.15±7.40 (3H, m), 7.63 (1H, d, J=2.2 Hz).
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 3-bromo-
phenyl] O-(methyl 2,3,4-tri-O-acetyl-ꢀ-glucuronyl) car-
bamate (DAU-31g). Analogously to the preparation of
DAU-16a from 50 mg (89 mmol) of 27g. Compound
needles, mp 117ꢀC. H NMR (100 MHz, CDCl3) d 4.88
1
(2H, d, J=5.6 Hz), 5.30±5.55 (2H, m), 5.90±6.25 (1H,
m), 7.79 (1H, d, J=8.0 Hz), 8.10 (1H, dd, J=8.0 Hz,
J=1.5 Hz), 8.40 (1H, d, J=1.5 Hz).
DAU-31g was obtained in 56% (56 mg), mp 147ꢀC. H
1
NMR (400 MHz, CDCl3) d 1.13 (3H, d, J=6.5 Hz), 1.62
(1H, s), 1.81 (1H, dt, J=13.2 Hz, J=3.3 Hz), 1.89 (1H,
dd, J=13.2 Hz, J=5.0 Hz), 2.06 (9H, s), 2.09 (1H, dd,
J=14.7 Hz, J=3.8 Hz), 2.30 (1H, d, J=14.7 Hz), 2.42
(3H, s), 2.88 (1H, d, J=18.8 Hz), 3.20 (1H, d, J=18.7 Hz),
3.69 (1H, m), 3.72 (3H, s), 3.90 (1H, m), 4.04 (3H, s),
4.20±4.25 (1H, m), 4.50 (1H, s), 4.95 (1H, d, J=12.8 Hz),
5.00 (1H, d, J=12.8 Hz), 5.15±5.30 (4H, m), 5.40 (1H, t,
J=9.4Hz), 5.47 (1H, d, J=2.7 Hz), 5.75 (1H, d, J=
8.0 Hz), 7.08 and 7.16 (2H, 2d, J=8.2 Hz), 7.36 (1H, d, J=
8.5 Hz), 7.54 and 7.60 (2H, 2s), 7.76 (1H, t, J=8.0 Hz),
7.99 (1H, d, J=7.8 Hz), 13.22 (1H, s), 13.94 (1H, s).
N-[4-(Carboxylic acid allyl ester) 3-bromophenyl] O-
(methyl 2,3,4-tri-O-acetyl-ꢀ-glucuronyl) carbamate (30).
341 mg (1.20 mmol) of 28, 284 mL (1.1 equiv) of (PhO)2-
P(O)N3 and 183 mL (1.1 equiv) of Et3N were stirred
overnight at room temperature under an argon atmo-
sphere in 15 mL of PhMe. Subsequently, the reaction
mixture was stirred at 85ꢀC for 1 h, cooled to ambient
temperature and 197 mg (0.5 equiv) of anomerically
unprotected glucuronic acid 18a were added and the
mixture was treated as for 12a. The crude product was
puri®ed by means of column chromatography (SiO2,
Et2O/n-hexane, 3/1) to give 294 mg, 80%, of 30 as a
white powder, mp 61ꢀC. 1H NMR (100 MHz, CDCl3) d
2.05 (9H, s), 3.72 (3H, s), 4.24 (1H, d, J=9.5 Hz), 4.79
(2H, d, J=5.6 Hz), 5.10±5.50 (5H, m), 5.77 (1H, d, J=
7.6 Hz), 5.70±6.25 (1H, m), 7.34 (1H, dd, J=8.6 Hz, J=
2.2 Hz), 7.58 (1H, s), 7.74 (1H, d, J=2.2 Hz), 7.83 (1H,
d, J=8.6 Hz)
N-[4-(Daunorubicin-N-carbonyl oxymethyl) 3-bromo-
phenyl] O-ꢀ-glucuronyl carbamate sodium salt (DAU-
7A). Analogously to the preparation of DAU-1A from
41.5 mg (37.2 mmol) of DAU-31g. Prodrug DAU-7A was
obtained in 50% yield (18.7 mg), mp 181ꢀC. Anal. calcd
for C42H42N2O20BrNa.5H2O: C, 46.38; H, 4.82; N,
2.58. Found: C, 46.37; H, 4.49; N, 2.68. MS (FAB+)
m/z 1021 ([M+2+Na]+), 1019 ([M+Na]+), 999
1
N-[4-(Carboxylic acid) 3-bromophenyl] O-(methyl 2,3,4-tri-
O-acetyl-ꢀ-glucuronyl) carbamate. 263 mg (0.427 mmol)
of 30, 186 mL (5 equiv) of morpholine in 20 mL of THF
were stirred for 2 h at room temperature while bubbling
argon through the solution. After that, a catalytic
([M+2+H]+), 997 ([M+H]+). H NMR (400 MHz,
(CD3)2SO) d 1.11 (3H, d, J=6.4 Hz), 1.47 (1H, dd, J=
11.6Hz, J=3.1 Hz), 1.82 (1H, dt, J=12.4 Hz, J=3.3 Hz),
2.08 (1H, dd, J=13.4 Hz, J=5.0 Hz), 2.19 (1H, dd, J=
13.4 Hz, J=2.5 Hz), 2.25 (3H, s), 3.05±3.65 (7H, m),

1610
R. G. G. Leenders et al. / Bioorg. Med. Chem. 7 (1999) 1597±1610
3.97 (3H, s), 4.16 (1H, q, J=6.6 Hz), 4.70 (1H, d, J=
5.5 Hz), 4.85±4.95 (1H, m), 4.90 (1H, d, J=13.5 Hz),
4.93 (1H, d, J=13.5 Hz), 5.20±5.25 (2H, m), 5.28 (1H,
d, J=8.0 Hz), 5.54 (1H, s), 6.96 (1H, d, J=7.9 Hz), 7.36
and 7.44 (2H, 2d J=8.4 Hz), 7.63 (1H, t, J=4.5 Hz),
7.77 (1H, s), 7.85±7.90 (2H, m), 10.15 (1H, s), 13.23
(1H, s), 14.01 (1H, s).
J. W.; Pinedo, H. M.; Haisma, H. J. Biochem. Pharmacol.
1996, 52, 455.
25. Houba, P. H. J.; Boven, E.; Erkelens, C. A. M.; Leenders,
R. G. G.; Scheeren, J. W.; Pinedo, H. M.; Haisma, H. J. Br. J.
Cancer 1998, 78, 1600.
26. Sharma, S. K.; Bagshawe, K. D.; Melton, R. G.; Sher-
wood, R. F. Cell. Biophys. 1992, 21, 109.
27. b-Glucuronyl carbamates as prodrugs have not been
reported so far by others whereas several approaches toward
the use of b-glucuronides as prodrugs are described in litera-
ture. For example, see refs 28±32.
Acknowledgements
28. Bosslet, K.; Czech, J.; Ho€mann, D. Cancer Res. 1994, 54,
2151.
29. Jacquesy, J.-C.; Gesson, J.-P.; Monneret, C.; Mondon, M.;
Renoux, B.; Florent, J.-C.; Koch, M.; Tillequin, F.; Sedlacek,
H. H.; Gerken, M.; Kolar, C.; Gaudel, G. Patent, WO 92/
19639, 1992.
We would like to thank Pharmachemie BV Haarlem,
The Netherlands and the Dutch Cancer Foundation,
Nederlandse Kankerbestrijding Koningin Wilhelmina
Fonds, for their ®nancial support.
30. Wang, S. M.; Chern, J. W.; Yeh, M. Y.; Ng, J. C.; Tung,
E.; Ro‚er, S. R. Cancer Res. 1992, 52, 4484.
References and Notes
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Cell Biophysics 1994, 24±25, 185.
32. Schmidt, F.; Florent, J.-C.; Monneret, C.; Straub, R.;
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2. Weiss, R. B. Seminars in Oncology 1992, 670±686.
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4. Priebe, W., Ed. Anthracycline Antibiotics. ACS Symposium
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5. For recent reviews see: Eisenbrand, G.; Lauck-Birkel, S.;
Tang, W.C. Synthesis 1996, 1246: and see ref. 6.
6. Sinhababu, A. K.; Thakker, D. R. Adv. Drug Del. Rev.
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8. For a recent review see: Niculescu-Duvaz, I.; Springer, C. J.
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33. In other publications this prodrug is referred to as ``dau-
norubicin-GA3'' or ``DNR-GA3'' see refs 24,25.
34. For the activation pathway of the prodrugs described in
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35. Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. J.
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36. An a-galacosyl carbamate derivative has already been
described in literature: Azoulay, M.; Florent, J.-C.; Monneret,
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Bosslet, K.; Czech, J.; Ho€man, D. Anti-Cancer Drug Design
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man, W. H.; Anlyan, A. J. J. Biol. Chem. 1947, 169, 449.
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39. Shioiri, T.; Ninomiya, K.; Yamada, S.-I. J. Am. Chem.
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19, 241.
40. Ghosh, A. K.; Duong, T. T.; McKee, S. P.; Thompson, W.
J. Tetrahedron Lett 1992, 33, 2781.
41. For convenience the aromatic ring of the spacer is num-
bered starting on the lowest carbon.
14. Eisenbrand, G., Lauck-Birkel, S., Tang, W. C. Synthesis
1996, 1246.
42. Tong, G. L.; Wu, H. Y.; Smith, T. H.; Henry, D. W. J.
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43. This may also explain the lower yields in the coupling
reaction to the amino-group of daunorubicin of pro-moieties
27d, f and h having electron withdrawing substituents in the 2-
position, (see Experimental). Side products may be formed by
nucleophillic attack of the daunorubicin-30-NH2 on the car-
bamate carbonyl of 27d, f, and h.
44. Bovine liver b-galactosidase was used to determine the
hydrolysis rates of both DAU-1B and DAU-1C. This enzyme
hydrolyses both b-galactosidase and b-glucosidase substrates.
45. pH 6.8 was used for activation rate measurements, re¯ect-
ing the tumor interstitial pH; see: Martin, G. R.; Jain, R. K.
Cancer Res. 1994, 54, 5670.
46. Human b-glucuronidase was produced by a mouse cell line
transfected with human b-glucuronidase gene and secreted in the
culture medium from which it was isolated. See: Houba, P. H. J.;
Boven, E.; Haisma, H. J. Bioconjugate Chem. 1996, 7, 606.
47. Kita, Y.; Akai, S.; Ajimura, N.; Yoshigi, M.; Tsugoshi, T.;
Yasuda, H.; Tamura, Y. J. Org. Chem. 1986, 51, 4150.
48. Compound 19 was prepared from phthalide by ring open-
ing according to Cain, B. F. J. Org. Chem. 1976, 41, 2029,
followed by silylation according to ref 47.
23. De Bont, H. B. A.; Leenders, R. G. G.; Scheeren, J. W.;
Haisma, H. J.; De Vos, D. European Patent, 95203671, 1995.
24. Houba, P. H. J.; Leenders, R. G. G.; Boven, E.; Scheeren,