Water soluble prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one

Article abstract of DOI:10.1016/S0968-0896(02)00514-X

Fourteen prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one (1) were prepared to improve its water solubility and potency. These prodrugs include α-amino acid (1a-1h), aliphatic amino acid (1i-1l), phosphoramidate (1m), and phosphate (1n) derivatives. All of the prodrugs showed improved water solubility. A number of the amino acid prodrugs (1a, 1b, 1d-1f, 1h, 1j, and 1k) exhibited more potent antitumor activity compared to the parent compound (1). The phosphate prodrug 1n also offered a potent antitumor activity, but the phosphoramidate 1m did not show any antitumor activity in vivo. None of the prodrugs exhibited significant toxicities in mice. These results indicate that the design and preparation of the amino acid prodrugs (1a, 1b, 1d-1f, 1h, 1j, and 1k) and phosphate prodrug (1n) are beneficial for enhancing the antitumor activity of 1. The similar approaches may be used to improve water solubility and bioactivity of other poorly soluble aromatic amines.

Full text of DOI:10.1016/S0968-0896(02)00514-X

Bioorganic & Medicinal Chemistry 11 (2003) 1021–1029
Water Soluble Prodrugs of the Antitumor Agent 3-[(3-Amino-4-
methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one
Nguyen-Hai Nam,a,*,y Yong Kim,a Young-Jae You,a Dong-Ho Hong,a,b
Hwan-Mook Kimb and Byung-Zun Ahna,*
aCollege of Pharmacy, Chungnam National University, Taejon 305-764, South Korea
bResearch Institute of Biosciences and Biotechnology, Taejon 305-600, South Korea
Received 30 April 2002; accepted 28 September 2002
Abstract—Fourteen prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-
one (1) were prepared to improve its water solubility and potency. These prodrugs include a-amino acid (1a–1h), aliphatic amino
acid (1i–1l), phosphoramidate (1m), and phosphate (1n) derivatives. All of the prodrugs showed improved water solubility. A
number of the amino acid prodrugs (1a, 1b, 1d–1f, 1h, 1j, and 1k) exhibited more potent antitumor activity compared to the parent
compound (1). The phosphate prodrug 1n also offered a potent antitumor activity, but the phosphoramidate 1m did not show any
antitumor activity in vivo. None of the prodrugs exhibited significant toxicities in mice. These results indicate that the design and
preparation of the amino acid prodrugs (1a, 1b, 1d–1f, 1h, 1j, and 1k) and phosphate prodrug (1n) are beneficial for enhancing the
antitumor activity of 1. The similar approaches may be used to improve water solubility and bioactivity of other poorly soluble
aromatic amines.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
respectively, at 25 ꢀC. A maximum dose of 40mg/kg/day
of 1 in the form of hydrochloride salt was possible with
1 1
Previously, we have reported an antitumor agent 3-[(3-
amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclo-
pent-2-ene-1-one (1, Fig. 1), a novel analogue of com-
bretastatin A-4 (CA-4, Fig. 1).1 Compound 1 exhibited
very potent cytotoxicity against various tumor cell lines
with IC50 values at low nanomolar range (18.2–22.2
nM). However, only modest antitumor activity was
observed in vivo when 1 was administered ip to BDF1
mice bearing Lewis lung carcinoma (3LL) cells at
40mg/kg/day (the tumor mass inhibition rate of
compound 1 was 59% compared to 71% produced by
etoposide, a clinical anticancer drug which was used as a
positive control). The unexpected low antitumor activity
was thought to be due to its low bioavailability, which
was caused, at least in part, by its poor aqueous solubi-
lity. It has been shown that compound 1 and its hydro-
chloride salt had water solubilities of 0.8 and 2.1 mg/mL,
the use of media such as Tween 80or Cremophor
.
Noteworthy however, at this dose the compound
showed no sign of toxicity in the treated mice. Thus, the
improvement of the water solubility is a strategy to
increase administration dosage and to improve
therapeutic values of this antitumor agent.
Introduction of an ionizable pro-moiety into the struc-
ture of compounds is one common approach to improve
the water solubility. For example, amino acid prodrugs
have frequently been investigated for alcohol drugs.2À6
Amide prodrugs in general have been used limitedly due
to their relative stability in vivo. Activated amide pro-
drugs such as amides of a-amino acids, however, have
favorable water solubility and generate a parent drug(s)
in vivo.2,3 The bioconversion of those amide prodrugs
is due to the strong electron-withdrawing effects of the
a-protonated amino group, which activates the amide
linkage towards hydrolysis, and partly (or pre-
dominantly) due to intramolecular catalysis or assis-
tance by the neighboring amino group, the so-called
folding effects.2 This observation led us to design and
synthesize the amino acid prodrugs 1a–h (Fig. 1,
*Corresponding author. Tel.: +82-42-821-5923; fax: +82-42-823-
6566; e-mail: ahnbj@cnu.ac.kr
yPresent address: Department of Biomedical Sciences, College of
Pharmacy, University of Rhode Island, RI 02881, USA.
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00514-X

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N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
Scheme 1). Compound 1h in particular can be viewed
either as a water soluble prodrug of 1 or a hybrid of
compound 1 and melphalan, a drug currently used in
cancer chemotherapy.
folding effects in the bioconversion process of 1i–k, and
the electron-withdrawing effects of the a-protonated
amino group in 1a–h. By incorporation of the phenyl
group between the amide moiety and the amino group
compound 1l has no a-amino group, thus the electron-
withdrawing effects should be absent. In addition, the
intramolecular catalytic reaction of the amino group is
no longer possible for sterical reasons. Compound 1l is
expected to have a similar water solubility with that of
1i–k since the amino group is not directly attached to
the phenyl ring but through a methylene group.
The folding effect toward the bioconversion of certain
aliphatic amino acid prodrugs has also been observed.2
Based on this observation, three aliphatic amino acid
prodrugs (1i–k) with varying chains were designed and
synthesized (Fig. 1, Scheme 2). The prodrug 1l was
synthesized to examine the importance of both the
Figure 1. Structures of 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one (1), combretastatin A-4 (CA-4), and
designed prodrugs.
Scheme 1. Reagents and conditions: (a) protected amino acid (N-Boc-gly for 1a, N-Boc-l-Ala for 1b, N-Boc-d-Ala for 1c, N-Boc-l-Phe for 1d,
.
N-Fmoc-O-tert-Bu-l-Ser for 1e, N-Boc-S-trityl-l-Cys for 1f, diBoc-l-Lys for 1g, and N-Boc-Melphalan for 1h), DCC, HOBt H2O, DMF; (b) 4 N
HCl-dioxane, rt, 3 h (for 1a–d and 1g–h); piperidine/CH2Cl2 then 4 N HCl-dioxane, rfx, 3 h (for 1e); or 3 N HCl-aq. AcOH, 90 ꢀC, 1.5 h (for 1f).
.
Scheme 2. Reagents and conditions: (a) HOOC-R-NHBoc, DCC, HOBt H2O, DMF (b) 4 N HCl-dioxane, rt, 3 h.

N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
1023
The use of phosphates as an ionizable pro-moiety is
another approach to improve the water solubility. We
initially designed the phosphoramidate 1m as a prodrug
of 1. However, phosphoramidates and phosphate pro-
drugs of steric alcohols often suffer from poor bio-
conversion in vivo.2,3 Prodrugs with various linked
phosphates have been designed to overcome such
hurdles.7À15 Different masked lactone linkers have been
developed for this purpose.10À15 Among these, a tri-
methyl-lock linker (Fig. 1) has been demonstrated to
have favorable pharmacokinetic properties.15 We have
therefore chosen this linker and synthesized a phosphate
prodrug 1n.
[g-aminobutyric acid, 6-aminohexanoic acid (caproic
acid)] and 4-aminomethyl benzoic acid were protected
with Boc by reacting with Boc anhydride [(Boc)2O] in
aq NaOH. The coupling reaction of 1 with protected
amino acids was carried out in the presence of DCC/
.
HOBt H2O as coupling reagents. Removal of the Boc
protecting group was performed by stirring in 4 N HCl-
dioxane to yield the hydrochloride form of 1-amino acid
amides (1a–d, 1g, 1h, and 1i–l) directly. For the synth-
esis of 1e the deprotection step was carried out in
piperidine to remove the Fmoc group followed by
refluxing in 4 N HCl-dioxane to deprotect the hydroxy
group and to yield the hydrochloride salt of 1e directly.
In the case of 1f the coupling product of 1 and N-Boc-S-
trityl-l-Cys was refluxed in HCl/aq AcOH to remove
both the Boc and trityl protecting groups to afford the
hydrochloride salt of 1f.
In this paper, the synthesis and results from the biolo-
gical evaluation of these prodrugs (1a–n) are presented
and discussed.
The synthesis of the phosphate prodrugs 1m and 1n is
shown in Scheme 3. The phosphoramidate 1m was
obtained via three steps. The first step was involved the
phosphorylation of 1 by tetrabenzyl pyrophosphate to
give a 1-dibenzylphosphoramidate intermediate (2). The
intermediate 2 was deprotected with trimethylsilyl
iodide (generated in situ) at room temperature to gen-
erate a debenzylated 1-dihydroxyphosphoramidate (3)
which was reacted with sodium methoxide to give the
disodium phosphoramidate 1m in 45% overall yield.
Reaction of 1 with 3-[20-(dibenzylphosphono)oxy-40,60-
dimethylphenyl)-3,3-dimethylpropionic acid (4)15 in the
Chemistry
The syntheses of amino acid or short-chain aliphatic
amino acid amide hydrochlorides of 1 are shown in
Schemes 1 and 2. The synthetic sequences involved
coupling of 1 with protected amino acids followed by
deprotection step. Commercially available Boc-amino
acids (N-tert-butyloxycarbonylamino acids) were used
including N-Boc-Gly, N-Boc-l-Ala, N-Boc-d-Ala, N-
Boc-l-Phe, N-diBoc-l-Lys, N-Boc-b-Ala. For cysteine
and serine, N-Boc-S-trityl-l-Cys and N-Fmoc-O-tert-
Bu-l-Ser were used. Short-chain aliphatic amino acids
.
presence of DCC/HOBt H2O as coupling reagents gave
Scheme 3. Reagents and conditions: (a) P2O7Bn4, TEA, MeCN, rfx; (b) TMS-Cl, NaI, acetonitrile, rt; (c) THF, MeONa, rt; (d) 3-[20-(dibenzyl-
phosphono)oxy-40,60-dimethylphenyl)-3,3-dimethylpropionic acid (4),15 DCC, HOBt H2O, CH2Cl2.
.

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N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
5. The intermediate 5 was debenzylated by trimethylsilyl
iodide to furnish 6, which was reacted with sodium
methoxide, as described for the case of 1m, to yield 1n in
22% overall yield.
or folding effects of the chained aliphatic amines in 1i–k
might facilitate the bioconversion of these prodrugs. In
attempt to gain some insights into the importance of
these effects, compound 1l was synthesized and eval-
uated. In this compound the amino group is not
attached at the a-position but is linked to an amide
group through a bulky phenyl moiety and thus the
electron-withdrawing effects of the a-protonated amine
group is absent and the folding effects should be
blocked. Compound 1l was found to be rather inactive
(IC50 values of >2000 nM) compared to the parent 1
(18.2–22.2 nM). These results suggest that the prodrugs
1a–k were unlikely cytotoxic per se but were conversed
to the parent 1 under cell culture conditions. Thus, the
electron-withdrawing effects of the a-protonated amine
groups and the folding effects of the chained aliphatic
amines towards the cleavage of the amide linkages
seemed to be important for the bioconversion of these
prodrugs. Exceptionally however, the d-amino acid
prodrug 1c was found to be less active than its corre-
sponding l-amino acid prodrug 1b. It has been shown
previously that prodrugs with d-amino acid(s) are
much more stable to enzymatic hydrolysis than that
with l-amino acid(s).16 This could be the reason for the
low activity observed with 1c. A similar result has also
been observed with other d-amino acid prodrugs.17
All final products were obtained in sufficient purity after
purification through a silica gel column eluting with
7.5–10% MeOH in CH2Cl2 and/or recrystallization
1
from appropriate solvents. H NMR, IR, HR-MS and
elemental analysis were used to confirm the structures of
compounds. IR spectra revealed the peaks at 1640–1660
cmÀ1 range due to the presence of the newly added
amide carbonyl group in addition to the ketone signals
appeared at 1690–1720 cmÀ1 range.
Biological Results and Discussion
Cytotoxicity of the synthesized prodrugs was deter-
mined in two tumor cell lines including B16 (murine
melanoma) and HCT116 (human colon tumor). The
results are summarized in Table 1.
It has been mentioned earlier that amides in general are
too stable to be effective as prodrugs. However, most of
the amino acid prodrugs of 1 showed potent cytotoxi-
cities in both tumor cell lines. Thus, the prodrugs 1a–k
would be cytotoxic per se or would likely be conversed
to the parent compound. The electron-withdrawing
effects of the a-protonated amine groups present in 1a–h
The decreased activity of the phophoramidate 1m indi-
cates that this prodrug might not cleave under cell cul-
ture conditions or the rate of hydrolysis was too low for
Table 1. Cytotoxicity and antitumor activity of prodrugs 1a–j
Compd
Cytotoxicitya (IC50b nM)
Antitumor activity
B16
HCT116
Dosec
(mg/kg/day)
Body weight
changes (%)
IRd (%)
1a (Gly)
149
157
682
132
67
75
77
32
16076
197
1512
125
91
77
65
47
+5.6
72.2
1b (l-Ala)
1c (d-Ala)
1d (l-Phe)
1e (l-Ser)
1f (l-Cys)
1g (l-Lys)
1h (Melphalan)
1i (b-Ala)
75
75
87
78
81
84
+7.3
+11.4
+7.5
+8.6
+7.5
+11.2
+9.2
+10.1
+10.0
+6.2
66.7
28.4
65.7
77.4
78.2
59.7
76.3
17.0
76.2
73.6
109e
136
67
143
>2000
>2000
84
107
82
151
75
77
1j (4-AA)
1k (6-AA)
1l (Scheme 22)
1m (Scheme 23)
1n (Scheme 23)
1.HCl
Adriamycin
Etoposideh
Melphalani
Vehicle
82
>2000
>2000
89
.5 17.2
40Nt
ntf
84
g
+14.8
+11.4
—
74.5
116
59.0 40+10
22.2
62
nt
nt
36
50
078.6
À6.5
39.7
0
0.2 mL/mice/day
+19.7
aCancer cell lines: B16, murine melanoma; HCT116, colon cancer.
bA sample’s concentration which produces a 50% reduction in cell growth. The values shown were the averages from a triplicate experiment.
cDose equivalent to 60mg/kg/day of 1. HCl.
dInhibiton rate (%)=(1ÀT/C)ꢁ100, calculated on day 15; T=mean tumor volume of the sample-treated group and C=mean tumor volume of the
negative control group; mice were implanted sc with 3LL cells (107 cells/0.2 mL PBS/mice), and the drugs were administered ip in 0.2 mL vehicle
(5% DMSO+20% cremophor/saline).
eEquivalent to 50mg/kg/day of melphalan.
fNot tested.
gInactive (IR<10%).
hEtoposide, used as a positive control, injected on the days 1, 5, and 9 at 36 mg/kg/day.
iMelphalan was given at 50mg/kg/day with the same schedule as prodrugs.

N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
1025
the prodrug to observe any activity. The highly hydro-
philic nature of the prodrug might effectively prevent
the compound from crossing the lipophilic layer of the
cellular membrane, rendering it noncytotoxic. In con-
trast, the phosphate 1n exhibited potent cytotoxicity
against both tumor cell lines, suggesting that the
prodrug was bioconversed to the parent compound 1.
and 78.2%, respectively, vs 72.2% of 1a) despite
the presence of the bulkier a-methylenehydroxy and
a-methylenethiol groups. Thus, the presence of the
a-methylenehydroxy and a-methylenethiol moieties
were favorable for the bioactivity of this prodrug,
probably due to the possible roles of the hydroxy and
thiol groups in the cleavage of the prodrug to the parent
compound. The melphalan prodrug 1h also offered a
good regression of tumor mass with an IR value recor-
ded at 76.3%, higher than that (59%) of 1. When mel-
phalan was administered to one group of mice at 50mg/
kg/day, a dose equivalent to its content in 1h given at
109 mg/kg/day, it showed only moderate activity (IR
value of 39.7%). Thus, the hybrid 1h showed a good
synergistic activity. Moreover, the toxicity of melphalan
was notable, as manifested by the body weight loss of
the melphalan-treated mice (Table 1), while 1h exhibited
no toxicity at the given dose. These results indicate the
benefits of the design of hydrid prodrugs in this way.
To examine whether the prodrugs are conversed to the
parent compound 1 in vivo, the antitumor activities were
determined in BDF1 mice bearing 3LL cells. The phos-
phoramidate 1m was also evaluated as a representative
of the in vitro inactive compounds.
We have previously reported that compound 1 did not
1
manifest significant toxicity in mice at 40mg/kg/day.
Therefore, increase of dosage is possible given that the
pharmacokinetic profile of the compound is improved.
All of the synthesized prodrugs offer substantial
increased water solubility (Table 2), which permit the
dosing up to 100 mg/kg/day. However, preliminary
experiments with 1a indicated that this prodrug showed
signs of toxicity in mice at doses greater than 76 mg/kg/
day, equivalent to 60mg/kg/day of 1. As a result, the
evaluation of this prodrug series was performed at doses
of compounds that are equivalent to 60mg/kg/day of 1,
hypothesizing that 100% bioconversion is achieved in
vivo (Table 1). Etoposide, a drug currently used in can-
cer chemotherapy was administered to one group at 36
mg/kg/day on day 1, 5, 9 and used as a positive control.
The prodrugs were reconstituted in 0.2 mL vehicle (5%
DMSO+20% Cremophor1/saline) and dosed from day
1 and every other day until day 11 (totals six injections).
The results summarized in Table 1 demonstrate that
most of the amino acid prodrugs showed more potent
antitumor activity than the parent compound 1 in
BDF1 mice bearing 3LL cells. Careful analyses of the
data indicated that the antitumor activity, in general,
seemed to correlate to the cytotoxicity. The glycine
prodrug 1a showed a higher inhibition rate (IR) value
(72.2%) than the l-alanine prodrug of 1 (1b, IR value
of 66.7%), suggesting that the presence of the a-methyl
group might somewhat lower the rate of the amide
linkage hydrolysis. The d-alanine prodrug 1c,
which was much less active in vitro compared to other
l-amino acid prodrugs, was found to have a marginal
antitumor activity (IR value, 28.4%). Compounds 1e
and 1f, the serine and cystein prodrugs of 1, exhibited
more potent activities than that of 1a (IR values of 77.4
Among three amino acid prodrugs containing aliphatic
chains, for example, 1i–k, the most potent activity was
observed with 1j. 1k was found to be slightly less potent
than 1j, while 1i showed only marginal activity. These
results suggest that the folding effects of the terminal
amino group might play an important role towards the
amide hydrolysis of these prodrugs, as the formation of
the more stable five-membered ring intermediate under
the amino-mediated intramolecular cyclative cleavage of
1j was more favorable, compared to the constrained
four- or less stable seven-membered ring intermediates
in the cases of 1i and 1k, respectively (Fig. 2). Hence, 1j
was likely converted to the parent drug 1 at a faster rate
than 1i and 1k. The inactivity of the phosphoramidate
1m which was also inactive in vitro, suggests that this
type of prodrugs is stable in vivo, and thus the proposed
cleavage of this prodrug catalyzed by phosphoramidases
as shown in Figure 3 unlikely occurred fast enough for
the prodrug to be effective. Meanwhile, compound 1n
exhibited good antitumor activity with an IR value of
74.5%. Previously, Nicolaou et al.15 have shown that
trimethyl-lock linked phosphate prodrugs of amines
which have structural similarity with prodrug 1n,
were effectively cleaved in the presence of alkaline
Table 2. Water solubility of the prodrugs 1a–n
Prodrug
Solubility
(mg/mL)
Prodrug
Solubility
(mg/mL)
1a (Gly)
>10a
6.65
6.43
5.94
6.75
4.68
>10a
1h (Melphalan)
1i (b-Ala)
1j (4-AA)
4.67
7.12
6.81
5.27
1b (l-Ala)
1c (d-Ala)
1d (l-Phe)
1e (l-Ser)
1f (l-Cys)
1g (l-Lys)
1k (6-AA)
1l (Scheme 2)
1m (Scheme 3)
1n (Scheme 3)
5.12
>10a
>10a
aDue to limited amounts of the prodrugs, no further effort was made
to measure the maximum solubility of these compounds.
Figure 2. Possible pathways for generation of 1 from prodrugs 1i–k
(D, a parent drug).

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N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
Figure 3. Possible pathways for generation of 1 from prodrugs 1l and 1m.
phosphatases (AP), regardless of the nature of amine
parent drugs. Alkaline phosphates are widely dis-
tributed in a variety of tissues such as liver, kidney
tubules, and intestinal epithelium. Though we have not
performed experiments to confirm the cleavage
mechanism for the prodrug, the results indicate that the
in vivo cleavage of 1n likely occurred as depicted in
Figure 3.
ing points were determined using an electrothermal
IA6304 open capillary melting point apparatus and are
uncorrected. High-resolution mass spectra (HRMS)
were measured on JEOL JMS-HX110/HX110 instru-
ment. Syntheses of compounds and biological proce-
dures are described as follow. For convenient follow-up,
the compounds are described in the same order they
appeared in Schemes 1–3.
In summary, we have reported in this paper the synth-
esis and biological evaluation of a series of amino acid
prodrugs and phosphate prodrugs of the antitumor
1-Gly. HCl (1a). All glasswares were flame dried, and
the reaction was carried out under nitrogen atmosphere.
agent
3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-tri-
.
HOBt H2O (207 mg, 1.53 mmol) and DCC (271 mg, 1.2
methoxyphenyl)cyclopent-2-ene-1-one (1). Most of the
amino acid prodrugs exhibited more potent antitumor
activity compared to the parent compound. The phos-
phate prodrug 1n showed a potent antitumor activity.
None of the prodrugs showed significant toxicity as
manifested by no body weight loss of prodrugs-treated
mice. These results indicate that the design and pre-
paration of the amino acid prodrugs and phosphate
prodrug like 1n are beneficial for enhancing the anti-
tumor activity of 1. Similar strategies are possible for
other aromatic amine drugs, which are often poorly
soluble in aqueous systems.
mmol) were added to a solution of 1 (369 mg, 1 mmol)
in anhydrous DMF (5 mL) at 0 ꢀC. The reaction mix-
ture was kept under continuous stirring for 15 min. N-
Boc-Gly (209 mg, 1.2 mmol) in the solid form was then
added at once, and the reaction mixture was stirred for
additional 12 h while it was gradually allowed to reach
ambient temperature. The solvent was then removed
under reduced pressure. The crude 1-N-Boc-Gly was
purified on a silica gel column using 33% ethyl acetate
in hexane. 1-N-Boc-Gly was directly dissolved in 4 N
HCl-dioxane (10mL) and stirred at room temperature
for 3 h. After removal of solvent, the residue was pur-
ified by silica gel column chromatography eluting with
7.5% MeOH in CH2Cl2 to furnish 355 mg of 1a. Yield:
76%; mp 145 ꢀC; IR (KBr) 3441 (NH), 1690(CO
Experimental
1
ketone), 1664 (CO amide) cmÀ1; H NMR (DMSO-d6,
300 MHz) d 2.58–2.66 (2H, m), 2.75–2.87 (2H, m), 3.74
(6H, s), 3.80(2H, br), 3.81 (3H, s), 3.89 (3H, s), 6.54
(2H, s), 6.67 (1H, d, J=8.12 Hz), 6.71 (1H, dd, J=8.12,
2.00 Hz), 6.78 (1H, d, J=2.00 Hz). Anal. calcd for
C23H27ClN2O6: C, 59.67; H, 5.88; N, 6.05. Found C,
59.84; H, 6.01; N, 6.26.
Common chemicals and solvents were purchased from
commercial sources. Solvents were distilled before use.
Protected amino acids were purchased from either
Sigma Aldrich Co. Ltd. (USA) or Fluka Co. Ltd.
(USA). All 1H NMR spectra were recorded using a
Varian-Gemini 300 (300 MHz) spectrometer with tetra-
methylsilane as the internal standard. Chemical shifts
are reported in ppm (d) and coupling constants are
reported in Hz. Infrared spectra were measured using
KBr plates on a Perkin-Elmer 1600 series FTIR. Melt-
1-L-Ala. HCl (1b). This compound was synthesized
according to the same procedure described for 1a using
N-Boc-l-Ala instead of N-Boc-Gly. Yield: 68%; mp
178 ꢀC; IR (KBr) 3405 (NH), 1715 (CO ketone), 1676

N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
1027
1
(CO amide) cmÀ1; H NMR (DMSO-d6, 300 MHz) d
1-L-Lys. 2HCl (1g). This compound was synthesized by
the same procedure described for 1a using Na, Ne-
diBoc-l-Lys instead of N-Boc-Gly. Yield: 68%; mp
123 ꢀC; IR (KBr) 3460(NH), 1691 (CO ketone), 1665
1.58 (3H, d, J=7.20Hz), 2.66–2.82 (2H, m), 2.87–3.01
(2H, m), 3.78 (6H, s), 3.81 (3H, s), 3.87 (3H, s), 4.53
(1H, q, J=7.20Hz), 6.51 (2H, s), 6.70(1H, d, J=9.50
Hz), 6.76 (1H, dd, J=9.50, 2.10 Hz), 6.85 (1H, d,
J=2.00 Hz). Anal. calcd for C24H29ClN2O6: C, 60.44;
H, 6.13; N, 5.87. Found C, 60.02; H, 6.18; N, 6.11.
1
(CO amide) cmÀ1; H NMR (DMSO-d6, 300 MHz) d
1.35–1.40(2H, m), 1.67–1.82 (2H, m), 1.88–1.95 (2H,
m), 2.58–2.71 (2H, m), 2.88–3.02 (4H, m), 3.76 (6H, s),
3.81 (3H, s), 3.89 (3H, s), 4.09–4.13 (1H, m), 6.67 (2H,
s), 6.69 (1H, d, J=8.02 Hz), 6.75 (1H, d, J=2.11 Hz),
6.89 (1H, dd, J=8.02, 2.11 Hz). Anal. calcd for
C27H37Cl2N3O6: C, 56.84; H, 6.54; N, 7.37. Found C,
57.02; H, 6.55; N, 7.18.
1-D-Ala. HCl (1c). This compound was synthesized by
the same procedure described for 1a using N-Boc-d-Ala
instead of N-Boc-Gly. Yield: 69%; mp 139 ꢀC; IR (KBr)
3376 (NH), 1703 (CO ketone), 1655 (CO amide) cmÀ1
;
1H NMR (DMSO-d6, 300 MHz) d 1.62 (3H, d, J=7.08
Hz), 2.64–2.80(2H, m), 2.89–3.03 (2H, m), 3.81 (3H, s),
3.84 (6H, s), 3.88 (3H, s), 4.33 (1H, q, J=7.09 Hz), 6.57
(2H, s), 6.71 (1H, dd, J=7.78, 1.98 Hz), 6.74 (1H, d,
J=7.78 Hz), 6.82 (1H, d, J=1.99 Hz). Anal. calcd for
C24H29ClN2O6: C, 60.44; H, 6.13; N, 5.87. Found C,
60.24; H, 6.28; N, 6.09.
1-L-p-[bis(2-chloroethyl)]amino-Phe. 2HCl (1h). This
compound was synthesized by the same procedure as
described for 1a using N-Boc-l-p-[bis(2-chloro-
ethyl)]amino-Phe (N-Boc-Melphalan) instead of N-Boc-
Gly. Yield: 48%; mp 172 ꢀC; 1H NMR (DMSO-d6,
300 MHz) d 2.55–2.69 (2H, m), 2.78–2.96 (2H, m), 3.60–
6.68 (8H, m), 3.74 (6H, s), 3.81 (3H, s), 3.87 (3H, s), 5.06
(1H, m), 6.08 (1H, br), 6.54 (2H, s), 6.66 (1H, d, J=8.22
Hz), 6.72 (1H, dd, J=8.22, 2.07 Hz), 6.88 (1H, d,
J=2.07 Hz), 8.25 (1H, s). Anal. calcd for
C34H41Cl4N3O6: C, 55.94; H, 5.66; N, 5.76. Found C,
56.05; H, 5.49; N, 5.48.
1-L-Phe. HCl (1d). This compound was synthesized by
the same procedure described for 1a using N-Boc-l-Phe
instead of N-Boc-Gly. Yield: 78%; mp 173 ꢀC; IR (KBr)
3445 (NH), 1693 (CO ketone), 1667 (CO amide) cmÀ1
;
1H NMR (DMSO-d6, 300 MHz) d 2.61–2.75 (2H, m),
2.87–3.00 (3H, m), 3.25–3.51 (2H, m), 3.82 (3H, s), 3.87
(6H, s), 3.91 (3H, s), 4.63 (1H, m), 6.49 (2H, s), 6.66
(1H, d, J=8.01 Hz), 6.71 (1H, dd, J=8.01, 2.05 Hz),
6.78 (1H, d, J=2.05 Hz), 7.01–7.35 (5H, m). Anal. calcd
for C30H33ClN2O6: C, 65.15; H, 6.01; N, 5.07. Found C,
64.99; H, 6.13; N, 5.26.
1-ꢀ-Ala. HCl (1i). This compound was synthesized by
the same procedure as described for 1a using N-Boc-b-
Ala instead of N-Boc-Gly. Yield: 88%; 1H NMR
(DMSO-d6, 300 MHz) d 2.59–2.68 (2H, m), 2.77–3.02
(4H, m), 3.74 (6H, s), 3.86 (3H, s), 3.89 (3H, s), 3.99
(2H, t, J=6.91 Hz), 6.51 (2H, s), 6.65 (1H, d, J=8.17
Hz), 6.69 (1H, dd, J=8.17, 2.11 Hz), 6.75 (1H, d,
J=2.11 Hz). Anal. calcd for C24H29ClN2O6: C, 60.44;
H, 6.13; N, 5.87. Found C, 60.18; H, 6.21; N, 5.98.
1-L-Ser. HCl (1e). 1-N-Fmoc-O-tert-Bu-l-Ser was syn-
thesized by the same procedure described for 1a using
N-Fmoc-O-trityl-l-Ser instead of N-Boc-Gly. The Fmoc
group was removed by stirring 1-N-Fmoc-O-tert-Bu-l-
Ser in piperidine/CH2Cl2 for 3 h. Removal of the O-tert-
Bu group was achieved by refluxing 1-O-tert-Bu-l-Ser
in 4 N HCl-dioxane for 3 h. 1-l-Ser. HCl (1e) was pur-
ified as described for 1a. Yield: 52%; mp 157 ꢀC; IR
(KBr) 3510(OH), 3454 (NH), 1700(CO ketone), 1668
1-4-Aminobutyric amide. HCl (1j). This compound was
synthesized by the same procedure described for 1a
using 4-(N-Boc-amino)butyric acid instead of N-Boc-
Gly. Yield: 80%; 1H NMR (DMSO-d6, 300 MHz) d
1.51–2.23 (2H, m), 2.62–2.76 (2H, m), 2.78–3.05 (4H,
m), 3.73 (6H, s), 3.87 (3H, s), 3.89 (3H, s), 4.12 (2H, t,
J=7.08 Hz), 6.55 (2H, s), 6.67–6.69 (2H, m), 6.78 (1H,
d, J=2.15 Hz). Anal. calcd for C25H31ClN2O6: C,
61.16; H, 6.36; N, 7.22. Found C, 60.98; H, 6.32; N,
7.18.
(CO amide) cmÀ1 1H NMR (DMSO-d6, 90MHz) d
;
2.61–2.69 (2H, m), 2.77–2.93 (2H, m), 3.73 (6H, s), 3.81
(3H, s), 3.89 (3H, s), 3.90–3.98 (2H, m), 4.12–4.21 (1H,
m), 6.66 (2H, s), 6.78 (1H, d, J=2.10Hz), 6.81–6.99
(2H, m). Anal. calcd for C24H29ClN2O7: C, 58.48; H,
5.93; N, 5.68. Found C, 58.27; H, 6.11; N, 5.61.
4-(N-Boc-Amino)butyric acid was obtained by the fol-
lowing procedure: 4-aminobutyric acid (1.03 g, 10
mmol) was dissolved in 20mL dioxane, 10mL water,
and 10mL NaOH solution (1 N aq) at 0 ꢀC. To this
solution was added 2.4 g (Boc)2O (11 mmol). The
resulting mixture was stirred for 30min and con-
centrated in vacuo to 15 mL. The pH of the mixture was
adjusted to 3–4 using HCl (1 N aq) and extracted with
ethyl acetate twice (20mL each). The organic layers
were combined, dried over sodium sulfate and solvent
was removed at reduced pressure using evaporator. The
residue was dried and used directly without any further
purification.
1-L-Cys. HCl (1f). 1-N-Boc-S-trityl-l-Cys was synthe-
sized by the same procedure described for 1a using N-
Boc-S-trityl-l-Cys instead of N-Boc-Gly. Concomitant
deprotection of N-Boc and S-trityl groups was achieved
by refluxing 1-N-Boc-S-trityl-l-Cys in 3 N HCl-AcOH
for 3 h. Purification method of 1-l-Cys. HCl (1f) was
the same as described for 1a. Yield: 47%; mp 138 ꢀC; IR
(KBr) 3450(NH), 1697 (CO ketone), 1671 (CO amide)
1
cmÀ1; H NMR (DMSO-d6, 300 MHz) ꢀ 2.63–2.75 (2H,
m), 2.79–3.02 (2H, m), 3.74 (6H, s), 3.82 (3H, s), 3.88
(3H, s), 3.94–4.01 (2H, m), 4.10–4.18 (1H, m), 6.55 (2H,
s), 6.70(1H, d, J=8.00 Hz), 6.74 (1H, dd, J=8.00, 2.01
Hz), 6.84 (1H, d, J=2.01 Hz). Anal. calcd for
C24H29ClN2O6S: C, 56.63H, 5.74; N, 5.50. Found C,
56.71; H, 5.82; N, 5.49.
1-6-Aminohexanoic amide. HCl (1k). This compound
was synthesized by the same procedure as described for

1028
N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
1a using 6-(N-Boc-amino)hexanoic acid (N-Boc-caproic
acid) instead of N-Boc-Gly. Yield: 78%; 1H NMR
(DMSO-d6, 300 MHz) d 1.42–2.47 (6H, m), 2.64–2.77
(2H, m), 2.80–3.00 (2H, m), 3.05–3.14 (2H, m), 3.75
(6H, s), 3.87 (3H, s), 3.89 (3H, s), 4.17 (2H, t, J=7.41
Hz), 6.52 (2H, s), 6.65 (1H, dd, J=7.88, 2.31 Hz), 6.72–
6.83 (2H, m). Anal. calcd for C27H35ClN2O6: C, 62.48;
H, 6.80; N, 5.40. Found C, 62.29; H, 6.87; N, 5.78.
(2H, m), 3.86 (6H, s), 3.93 (6H, overlap), 6.51 (2H, s),
6.77–6.82 (2H, m) 6.89 (1H, m), 9.82 (2H, br, overlap).
Anal. calcd for C21H24NO8P: C, 56.13; H, 5.38; N, 3.12.
Found C, 56.38; H, 5.28; N, 3.17.
Disodium salt of 3 (1m). Sodium methoxide (104 mg, 2.0
mmol) was added to a solution of 3 (820mg, 1.83
mmol) in MeOH (5 mL). The resulting mixture was
stirred for 12 h at room temperature and concentrated.
The residue was recrystallized from cold acetone/water.
6-(N-Boc-Amino)hexanoic acid (N-Boc-caproic acid)
was obtained by the same procedure as described for
4-(N-Boc-amino)butyric acid.
1
Yield: 75%; H NMR (CD3OD, 300 MHz) d 2.63–2.74
(2H, m), 2.85–3.01 (2H, m), 3.87 (6H, s), 3.91 (3H, s),
3.94 (3H, s), 6.50(2H, s), 6.75 (1H, d, J=9.00 Hz), 6.80
(1H, dd, J=8.89, 1.95 Hz), 6.85 (1H, d, J=1.95 Hz).
1-4-Aminomethylbenzoic amide. HCl (1l). This com-
pound was synthesized by the same procedure as
described for 1a using 4-(N-Boc-amino)methyl benzoic
acid instead of N-Boc-Gly. Yield: 64%; 1H NMR
(DMSO-d6, 300 MHz) d 2.60–2.73 (2H, m), 2.85–2.94
(2H, m), 3.82 (3H, s), 3.87 (6H, s), 3.91 (3H, s), 6.49
(2H, s), 6.67 (1H, d, J=8.11 Hz), 6.73 (1H, dd, J=8.12,
2.13 Hz), 6.79 (1H, d, J=2.05 Hz), 7.03–7.25 (2H, m),
7.66–7.80(2H, m). Anal. calcd for C 29H31ClN2O6: C,
64.62; H, 5.80; N, 5.20. Found C, 64.55; H, 5.87; N,
5.31.
HRMS
C21H22NNa2O8P: 493.0911.
m/z
494.1207
(M+H)+.
calcd
for
Disodium salt of 1-{3-[20-(dihydroxyphosphono)oxy-
40,600-dimethylphenyl]-3,3-dimethylpropionic} amide (1n)
1-{3-[20-(Dibenzyloxyphosphono)oxy-40,60-dimethylphenyl]-
3,3-dimethylpropionic} amide (5). DCC (112.5 mg, 0.50
.
mmol) and HOBt H2O (84 mg, 0.63 mmol) were added
to a solution of 3-[20-(dibenzylphosphono)oxy-40,60-
dimethylphenyl)-3,3-dimethylpropionic acid (4)15 (200
ꢀ
4-(N-Boc-Amino)methyl benzoic acid was obtained by
the same procedure described for 4-(N-Boc-amino)-
butyric acid.
mg, 0.415 mmol) in anhydrous CH2Cl2 (50mL) at 0 C.
The reaction mixture was kept under continuous stir-
ring for 15 min. Compound 1 (139 mg, 0.377 mmol) was
then added at once, and the reaction mixture was stirred
for additional 24 h while it was gradually allowed to
reach ambient temperature. The solvent was removed
under reduced pressure rotary evaporation, and the
desired product was eluted from a silica gel column.
Yield: 52%; 1H NMR (CDCl3, 300 MHz) d 1.67 (6H, s),
2.11 (3H, s), 2.45 (3H, s), 2.68 (2H, s), 2.71–2.82 (2H,
m), 2.89–3.04 (2H, m), 3.78 (3H, s), 3.85 (6H, s), 3.91
(3H, s), 5.12–5.18 (4H, m), 6.49 (2H, s), 6.73 (1H, s),
6.79–7.05 (4H, m), 7.36–7.45 (10H, m). Anal. calcd for
C48H52NO9P: C, 70.49; H, 6.41; N, 1.71. Found C,
70.68; H, 6.59; N, 1.66.
Disodium salt of 1-phosphoramidate (1m)
1-Dibenzyloxyphosphoramidate (2). Compound 1 (1.05
g, 2.84 mmol) was added to 30mL of dry MeCN fol-
lowed by addition of TEA (0.4 mL, 3.10 mmol). The
mixture was heated to reflux for approximately 5 min.
Solid tetrabenzyl pyrophosphate (1.68 g, 3.12 mmol)
was added to the warm reaction mixture. The reaction
mixture was kept under continuous stirring at reflux for
1 h and then allowed to cool to room temperature. To
the reaction milieu was added petroleum ether (30mL),
and the insoluble precipitate was removed by filtration.
After removal of all solvents under reduced pressure, a
residue was eluted from a silica gel column using gra-
dient ethyl acetate in hexane as eluent to give 1.43 g of
2. Yield: 77%; 1H NMR (CDCl3, 300 MHz) d 2.65–2.76
(2H, m), 2.79–3.00 (2H, m), 3.88 (6H, s), 3.91 (3H, s),
3.95 (3H, s), 5.28 (4H, overlap), 6.49 (2H, s), 6.75–6.88
(3H, m), 7.00–7.33 (10H, m). Anal. calcd for
C35H36NO8P: C, 66.76; H, 5.76; N, 2.22. Found C,
66.51; H, 5.85; N, 2.29.
1-{3-[20-(Dihydroxyphosphono)oxy-40,60-dimethylphenyl]-
3,3-dimethylpropionic} amide (6). This compound was
prepared from 5 as described for 3. Yield: 71%; 1H
NMR (CD3OD, 300 MHz) d 1.62 (6H, s), 2.14 (3H, s),
2.40(3H, s), 2.74 (2H, s), 2.75–2.87 (2H, m), 2.91–3.08
(2H, m), 3.81 (3H, s), 3.87 (6H, s), 3.94 (3H, s), 6.51
(2H, s), 6.62 (1H, s), 6.81 (1H, d, J=7.80Hz), 6.89 (1H,
dd, J=7.80, 2.21 Hz), 7.05 (1H, s), 7.07 (1H, d, J=2.21
Hz). 9.64 (2H, br, overlap). Anal. calcd for
C34H40NO9P: C, 64.04; H, 6.32; N, 2.20. Found C,
64.21; H, 6.31; N, 2.28.
1-Dihydroxyphosphoramidate (3). A mixture of 2 (1.31
g, 2 mmol) and sodium iodide (1.2 g, 8 mmol) in anhy-
drous acetonitrile (15 mL) was stirred under nitrogen
and trimethylsilyl chloride (1 g, 8 mmol) was added
dropwise. The solution was kept under vigorous stirring
for 3 h and quenched with water. The acidified reaction
mixture was extracted with ethyl acetate three times.
The organic layers were combined and the solvent was
removed under reduced pressure rotary evaporation.
The final solid product was obtained (884 mg) by tri-
turation in petroleum ether. Yield: 88%; 1H NMR
(CD3OD, 300 MHz) d 2.68–2.77 (2H, m), 2.83–3.05
Disodium salt of 1-{3-[20-(dihydroxyphosphono)oxy-40,60-
dimethylphenyl]-3,3-dimethylpropionic} amide (1n). This
compound was prepared from 6 as described for 1m.
1
Yield: 61%; H NMR (CD3OD, 300 MHz) d 1.65 (6H,
s), 2.12 (3H, s), 2.43 (3H, s), 2.73 (2H, s), 2.75–2.84 (2H,
m), 2.92–3.00 (2H, m), 3.82 (3H, s), 3.85 (6H, s), 3.91
(3H, s), 6.50(2H, s), 6.63 (1H, s), 6.79 (1H, d, J=8.10
Hz), 6.87 (1H, dd, J=8.10, 2.25 Hz), 6.93 (1H, d,
J=2.25 Hz), 7.02 (1H, s). HR-MS m/z 682.3811
(M+H)+. calcd for C34H38NNa2O9P: 681.2147.

N.-H. Nam et al. / Bioorg. Med. Chem. 11 (2003) 1021–1029
1029
Cytotoxicity assays
Acknowledgements
Tumor cells were maintained in plastic dishes in RPMI-
1640medium supplemented with 1%0 fetal bovine
serum. On day 0, 180 mL of a tumor cell suspension
(3ꢁ104 cells/mL in culture medium) were seeded in each
well of 96-well plates. The plates were incubated in a
5% CO2 incubator at 37 ꢀC for 24 h and then samples in
20 mL culture medium were added at various con-
centrations. The plates were incubated for another 48 h.
Cytotoxicity was measured by SRB’s method as descri-
bed in literature18 with slight modifications.19,20 The
values shown for these compounds are averages of three
determinations.
We thank Dr. Keykavous Parang (University of Rhode
Island, USA) for reading the manuscript.
References and Notes
1. Nam, N. H.; Kim, Y.; You, Y. J.; Hong, D. H.; Kim,
H. M.; Ahn, B. Z. Bioorg. Med. Chem. Lett. 2002, 12, 1955.
2. Bundgaard, H. In A Textbook of Drug Design and Devel-
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Wright, J. J. K.; Knipe, J.; Rose, W. C.; Casazza, A. M.; Vyas,
D. M. Bioorg. Med. Chem. Lett. 1996, 15, 1837.
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1996, 6, 945.
Water solubility
The solubilities of prodrugs were measured by adding
excess quantities of the prodrugs to a predetermined
volume of distilled water. The resulting mixtures were
stirred at 25 ꢀC for 24 h and the solutions were filtered.
Appropriately diluted filtrates were assayed by HPLC.
No chemical degradation was observed.
11. Wang, B.; Wang, W.; Zhang, H.; Shan, D.; Smith, T. D.
Bioorg. Med. Chem. Lett. 1996, 6, 2823.
Antitumor experiments
12. Wang, B.; Zhang, H.; Zheng, A.; Wang, W. Bioorg. Med.
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Antitumor experiments were carried out as described in
our previous reports.19,20 Briefly, 3LL cells were inocu-
lated sc into BDF1 mice on the day 0(1 ꢁ107 cells/
mouse/0.2 mL PBS). Prodrugs were dissolved in a med-
ium comprising of 5% DMSO and 20% Cremophor1.
Each compound was dosed with six injections from day
1 and every other day. Body weights of mice were
tracked every day and tumor sizes were measured with
calipers from day 9. Tumor volumes were calculated by
the following equation: tumor volume (mm3)=[length
(mm)ꢁwidth (mm)2]/2. The inhibition rate was eval-
uated as (1ÀT/C)ꢁ100% (where T is the mean tumor
volume of the treated group, C is the mean tumor
volume of the control group). Each group consisted of
10mice. Etoposide was administered at day 1, 5, and 9
at 36 mg/kg/day. No death was recorded within the
experimented period.
15. Nicolaou, M. G.; Yuan, C. S.; Borchardt, R. T. J. Org.
Chem. 1996, 61, 8636.
16. Upshall, D. G.; Gouldstone, S. J.; Macey, N.; Maidment,
M. P.; West, S. J.; Yeadon, M. J. Biopharm. Sci. 1990, 1, 111.
17. Ohsumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.;
Morinaga, Y.; Suga, Y.; Nihei, Y.; Ohishi, K.; Akiyama, Y.;
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18. Skehan, P.; Storeng, R.; Scudiero, D.; Monk, A.; Mac-
Mahon, J.; Vistica, D.; Warren, J.; Bokesch, H.; Kenny, S.;
Boyd, M. R. J. Nat. Cancer Inst. 1990, 82, 1107.
19. Nam, N. H.; You, Y. J.; Kim, Y.; Hong, D. H.; Kim, H.
M.; Ahn, B. Z. Phytother. Res. In press.
20. Kim, Y.; Kim, S. B.; You, Y. J.; Ahn, B. Z. Planta Med.
2002, 68, 271.