Remarkable drug-release enhancement with an elimination-based AB3 self-immolative dendritic amplifier

Article abstract of DOI:10.1016/j.bmc.2007.03.054

Self-immolative dendritic prodrugs, activated through a single catalytic reaction by a specific enzyme, could offer significant advantages in inhibition of tumor growth relative to monomeric prodrug, especially if the targeted or secreted enzyme exists at

Full text of DOI:10.1016/j.bmc.2007.03.054

Bioorganic & Medicinal Chemistry 15 (2007) 3720–3727
Remarkable drug-release enhancement with an
elimination-based AB3 self-immolative dendritic amplifier
Amit Sagi,a Ehud Segal,b Ronit Satchi-Fainarob,* and Doron Shabata,*
aDepartment of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences,
Tel Aviv University, Tel Aviv 69978, Israel
bDepartment of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
Received 8 February 2007; revised 13 March 2007; accepted 13 March 2007
Available online 21 March 2007
Abstract—Self-immolative dendritic prodrugs, activated through a single catalytic reaction by a specific enzyme, could offer signi-
ficant advantages in inhibition of tumor growth relative to monomeric prodrug, especially if the targeted or secreted enzyme exists at
relatively low levels in the malignant tissue. We have designed and synthesized new AB3 self-immolative dendritic prodrug system
that releases three active drugs by a single cleavage of the enzyme penicillin-G-amidase. The cleavage signal is transferred from the
dendron focal point to its periphery through fast elimination reactions and the design leads to three-fold signal amplification. In cell-
growth inhibition assays, the elimination-based AB3 self-immolative dendritic prodrug was significantly more effective than a cycli-
zation-based AB3 dendritic prodrug.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
meric prodrug derivatized with the anticancer agents
doxorubicin and camptothecin.7 This prodrug made it
possible to release two different chemotherapeutic drugs
simultaneously at the same location. In another report,
we designed and synthesized fully biodegradable dendri-
mers that disassemble through multi-enzymatic trigger-
ing followed by self-immolative chain fragmentation.8
A practical application for such multi-triggered self-
immolative dendrons was recently demonstrated by the
concept of prodrug activation through a molecular OR
logic trigger.9,10
Self-immolative dendrimers are novel class of molecules
that can amplify a single cleavage event received at the
focal point into multiple release of tail groups at
the periphery.1–3 These dendrimers can be used for the
construction of dendritic prodrugs, if a specific trigger
is attached to the focal point and drug molecules are
linked to the periphery.4,5 Self-immolative dendritic pro-
drugs, activated through a single catalytic reaction by a
specific enzyme, could offer significant advantages in
inhibition of tumor growth relative to monomeric pro-
drug, especially if the targeted or secreted enzyme exists
at relatively low levels in the malignant tissue. We have
shown that single-triggered dendritic prodrugs signifi-
cantly inhibit tumoral cell growth compared to classic
monomeric prodrugs.6 The dendritic platform was also
used for the synthesis of a single-triggered hetero-di-
Rapid release of tail-drug units from the dendritic
platform is essential in order to achieve maximal drug
concentration at a specific location. Therefore, self-imm-
olative dendrons with a fast disassembly mechanism
should have a significant advantage in a dendritic pro-
drug system.11 Here we report the design and synthesis
of a fast AB3 self-immolative dendritic prodrug system,
activated through a single enzymatic cleavage by penicil-
lin-G-amidase (PGA).12
Abbreviations: ACN, acetonitrile; DCM, dichloromethane; DIPEA,
diisopropylethyleneamine; DMF, dimethyl formamide; DMAP, dim-
ethylaminopyridine; EtOAc, ethylacetate; Et3N, triethylamine; Hex,
hexane; MeOH, methanol; Mel, melphalan; PNP, p-nitrophenol; PNA,
p-nitroaniline; TBSCl, tert-butyldimethylsillyl chloride; THF, tetrahy-
drofuran; Trp, tryptophan.
2. Results and discussion
Recently, we reported the synthesis and activation of
dendritic prodrug similar to 1.6 This prodrug releases
three molecules of active drug upon single cleavage by
Keywords: Prodrug; Dendrimer; Enzyme; Self-immolative.
*
Corresponding authors. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640
9293; e-mail: chdoron@post.tau.ac.il
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.054

A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
3721
PGA (Fig. 1). The disassembly pathway is initiated
by removal of phenylacetic acid, elimination of
azaquinone-methide, and decarboxylation to generate
amine-intermediate 1a. The latter cyclizes to release
dimethylurea derivative and phenol 1c, which rapidly
undergoes triple elimination to release the three drug
units. The rate-determining step was found to be the
cyclization of 1a–1c.
release the three drug units (Fig. 2). The disassembly
of this molecule after the enzymatic cleavage is solely
based on elimination reactions and therefore is expected
to occur very rapidly.
Although molecule like 2a was reported before,2 there is
no published procedure for its synthesis. We developed a
simple and efficient new synthetic pathway for dendritic
compounds like 2, reported here. To compare the disas-
sembly rate of the above dendritic systems, we synthe-
sized AB3 molecules 3 and 4 (Fig. 3). Both molecules
are designed for activation by PGA and have three units
of tryptophan as a model drug. Tryptophan was used
for initial evaluation as it contains a strong UV chromo-
phore, allowing us to monitor the disassembly reaction.
In order to enhance the release-rate of the peripheral
drug units, we sought to design a system that would dis-
assemble without the slow cyclization step. Taking into
consideration this objective, we designed AB3-dendritic
molecule 2. The first step in the reaction is catalytic
cleavage of phenylacetic acid by PGA,13 followed by
elimination of azaquinone-methide and decarboxylation
to release amine-intermediate 2a. This amine intermedi-
ate further disassembles through triple elimination to
The synthesis of compounds 3 and 4 was performed
as presented in Figures 4 and 5. Compound 5 was
Drug
NH
O
O
O
Drug
Enzymatic
cleavage
O
NH
O
N
N
O
O
O
O
O
H O
2
N
H
O
1
Drug
NH
HO
O
CO
2
NH
HO
2
NH
O
Drug
NH
Drug
NH
O
Drug
2
O
O
O
O
Triple
elimination
Drug
NH
O
O
N
NH
NH
Drug
Drug
Drug
NH
2
OH
NH
O
O
O
O
O
O
O
1a
1c
Drug
NH
2
N
N
Drug
NH
Figure 1. Disassembly mechanism of AB3 self-immolative dendritic molecule 1.
Drug
NH
O
O
O
O
Enzymatic
cleavage
Drug
NH
N
H
O
O
O
H O
2
N
H
O
O
2
Drug
NH
HO
O
CO
2
NH
HO
2
Drug
NH
O
NH
Drug
2
O
O
Triple
elimination
Drug
NH
NH
2
NH
NH
Drug
Drug
2
O
O
O
2a
Drug
NH
2
Figure 2. Disassembly mechanism of AB3 self-immolative dendritic molecule 2.

3722
A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
NH
NH
NH
NH
NH
NH
O
HO2C
O
O
HO2C
O
O
O
O
O
NH
O
NH
O
O
N
N
O
O
N
O
O
H
HO2C
O
HO2C
O
O
N
O
O
N
H
H
HO2C
HO2C
3
4
NH
NH
NH
NH
Figure 3. Chemical structures of AB3 self-immolative dendritic molecules with tryptophan tail units and a trigger that is activated by PGA.
O2N
O
O
O
O
O
O2N
O
O
N
Tryptophan
N
O
O
O
3
O
O
O
O
N
H
5
O2N
Figure 4. Chemical synthesis of dendritic molecule 3.
TBSO
TBSO
TBSO
TBSO
TBSO
TBSO
HO
HO
Pd/C
Ammonium formate
Ac O/HNO
2
3
TBSCl
NO
NH
2
2
TBSO
TBSO
TBSO
HO
7
6
8
9
TBSO
TBSO
TBSO
TBSO
11
HO
O
O
TBSO
N
H
Phosgene
NCO
N
O
O
Amberlyst 15
H
TBSO
N
10
12
H
O N
O
O
2
HO
HO
O
O
O
p
-nitrophenyl
N
O
O
chloroformate
Tryptophan
O N
O
N
O
O
H
2
HO
4
H
O
N
13
N
H
O
O
O
14
H
O
O N
2
Figure 5. Chemical synthesis of dendritic molecule 4.
synthesized as previously described.6,14 Three equiva-
lents of tryptophan was reacted with tricarbonate 5 to
generate dendritic molecule 3 (Fig. 4).
acid and acetic anhydride afforded compound 8. The lat-
ter was reduced with palladium on carbon and ammo-
nium formate to give amine 9. Reaction of phosgene
with 9 in situ generated isocyanate 10, which was imme-
diately reacted with alcohol 11 to give compound 12.
Deprotection of the tert-butyldimethylslyl groups of 12
with amberlyst-15 afforded triol 13, which was activated
with 4-nitrophenyl-chloroformate to give tricarbonate
Dendritic molecule 4 was prepared starting from
tri(hydroxymethyl)-1,3,5-benzene 6. Thus, triple protec-
tion with tert-butyldimethylslyl chloride of the hydroxy
groups of 6 gave compound 7 and reaction with nitric

A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
3723
14. The latter was reacted with three equivalents of tryp-
tophan to generate AB3 dendritic molecule 4.
prodrugs 15 and 16 were synthesized with the chemo-
therapeutic drug melphalan as a tail unit and a trigger
that is activated by PGA (Fig. 9). Three equivalents of
melphalan was coupled with tricarbonates 5 and 14 to
afford AB3 self-immolative dendritic prodrugs 15 and
16, respectively.
The disassembly reactions of dendritic molecules 3 and 4
were evaluated in phosphate-buffered saline (PBS, pH
7.4) in presence and absence of PGA. The release of
tryptophan was monitored by a reverse-phase HPLC
at a wavelength of 320 nm. The results are presented
in Figures 6 and 7. No disassembly of either system
was observed in the buffer without PGA (data not
shown). In the presence of PGA, dendritic molecule 3
disassembled to release tryptophan within approxi-
mately four days (Fig. 6), whereas dendritic molecule 4
released its tryptophan tail-units within 40 min (Fig. 7).
In order to evaluate the in vitro antitumor activity of the
prodrugs, compounds 15 and 16 were incubated at var-
ied concentrations with human T-lineage acute lympho-
blastic leukemia MOLT-3 cells in the presence or
absence of 1 lM PGA. The data from the cell prolifera-
tion assays are presented in Figure 10. A colorometric
assay based on the tetrazolium salt XTT was used to
evaluate the cytotoxicity of the compounds.
Under the experiment conditions the enzymatic cleavage
occurs within seconds. Therefore, the observed release
time of the tryptophan is also the actual disappearance
time of the intermediate forms after the enzymatic cleav-
age. This dramatic enhancement of tail-unit release with
the elimination-based system (dendritic molecule 4)
compared to the cyclization-based system (dendritic
molecule 3) is best viewed by superimposition of the
graphs (Fig. 8).
Melphalan prodrugs 15 and 16 exhibited significantly
(about 100-fold) reduced toxicity than free melphalan
in the absence of PGA. XTT cytotoxicity assays showed
a decrease of 17-fold in IC50 for prodrug 15 (100 vs
6 lM with PGA) and 200-fold for prodrug 16 (100 vs
0.5 lM with PGA). In the presence of PGA, prodrug
15 showed some increased cytotoxicity but still notably
less than that of free melphalan (6 and 0.3 lM, respec-
tively). However, when prodrug 16 was activated by
PGA, the cytotoxicity was almost identical to that of
free melphalan (IC50 values of free melphalan and pro-
drug 16 in the presence of PGA were 0.3 and 0.5 lM,
We decided to apply the elimination-based dendritic sys-
tem to the synthesis of an anticancer prodrug and to
evaluate it in a tumor cell cytotoxicity assay. Dendritic
Figure 6. PGA-catalyzed release of tryptophan from dendritic compound 3 (compound 3 [500 lM] in PBS, PGA [1 mg/mL]).
Figure 7. PGA-catalyzed release of tryptophan from dendritic compound 4 (compound 4 [500 lM] in PBS, PGA [1 mg/mL]).

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A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
described here will generate a promising self-immolative
AB3 prodrug that is suitable for site-specific drug
delivery.
Additional potential application for these single-trig-
gered dendritic systems could be in the amplification
of signals in diagnostic assays. For example, chromo-
genic amines can be used as tail units. The amine is col-
orless when attached to the dendritic platform, but upon
release a distinct new chromophore can be detected by
spectrophotometry.18 Introduction of phenylacetamide
as a trigger will generate a molecular sensor for the
enzyme PGA. A single cleavage by PGA will result in
release of three reporter units and generation of a strong
chromophore. There are several examples for reporter
molecules with primary or secondary amine groups that
can generate UV, UV–vis, or fluorescence signals.
Figure 8. PGA-catalyzed release of tryptophan from dendritic com-
pound 3 (purple) with t1/2 of 1400 min versus release from dendritic
compound 4 (red) with t1/2 of 10 min.
respectively). Interestingly, in the absence of PGA both
prodrugs were relatively not toxic even at very high
doses of 100 lM. No toxicity was observed for the plat-
form building blocks within the activity range of mel-
phalan or melphalan prodrug.6
3. Conclusions
In summary, we have designed and synthesized new
AB3 self-immolative dendritic prodrug system that
releases three active drugs upon a single cleavage by
the model enzyme PGA. The cleavage signal is trans-
ferred from the dendron focal point to its periphery
through fast elimination reactions and is amplified
three-fold. The elimination-based AB3 dendritic
prodrug showed significant enhancement of drug release
in comparison to a cyclization-based AB3 dendritic
prodrug. This difference was noticeably reflected in a
cytotoxicity assay. Our new trimeric prodrug system
could offer significant advantages in inhibition of
tumor growth relative to regular monomeric prodrugs,
especially if the targeted or secreted enzyme exists at
relatively low levels in the malignant tissue.
Site-specific drug delivery via a prodrug approach has
generated considerable interest for enhancing the
potency and for diminishing the side effects of a drug.15
Rapid release of chemotherapeutic drugs from a
prodrug system is important if a high concentration of
active drug is needed at the tumor site. The described
elimination-based AB3 self-immolative dendritic
prodrug system had a rapid activation pathway. Fur-
thermore, a single cleavage by PGA was efficiently
amplified to release three active drug molecules. The
results shown in this study provide a proof of concept
for this amplifying approach. Similar dendritic prodrugs
with triggers that are activated by specific endogenous
tumoral enzymes could be applied for selective chemo-
therapy. For example, legumain, a recently identified
lysosomal protease, is a promising candidate target for
prodrug therapy since it is overexpressed in the majority
of human solid tumors.16 Legumain promotes cell
migration and its overexpression is associated with
enhanced tissue invasion and metastasis. The enzyme
cleaves the amide linkage of the tri-peptide Asn-Ala-
Ala.17 Linking this peptide, instead of the PGA
substrate, to the elimination-based dendritic system
4. Experimental
4.1. General
All reactions requiring anhydrous conditions were per-
formed under an Ar or N2 atmosphere. Chemicals and
solvents were either A.R. grade or purified by standard
techniques. Thin layer chromatography (TLC) was per-
formed on silica gel plates Merck 60 F254; compounds
Cl
Cl
N
N
Cl
NH
O
Cl
NH
O
HO2C
CO2H
O
O
HO2C
CO2H
O
O
O
O
NH
O
N
NH
N
N
O
O
O
O
O
Cl
H
N
O
O
O
Cl
O
N
N
N
O
O
H
H
HO2C
15
NH
Cl
HO2C
16
NH
Cl
Cl
N
Cl
N
Cl
Cl
Figure 9. Chemical structures of AB3 self-immolative dendritic prodrugs with melphalan tail units and a trigger that is activated by PGA.

A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
3725
100
90
80
70
60
50
40
30
20
10
0
Melphalan
prodrug 15
prodrug 15+PGA
prodrug 16
prodrug 16+PGA
1
10
100
1000
10000
100000
Melphalan-equivalent concentration [Log nM]
Figure 10. Growth inhibition assay of leukemia MOLT-3 cell line with dendritic prodrug 15 or 16 in the presence or absence of PGA. Cells were
incubated for 72 h. Full blue squares represent melphalan, full green triangles prodrug 15, empty green triangles prodrug 15 with PGA, full red circles
prodrug 16, empty red circles prodrug 16 with PGA. Symbols represent mean SD.
were visualized by irradiation with UV light and/or by
treatment with a solution of phosphomolybdic acid
(25 g), Ce(SO4)2ÆH2O (10 g), concd H2SO4 (60 mL),
and H2O (940 mL), followed by heating. Flash chroma-
tography was performed by using silica gel Merck 60
(particle size 0.040–0.063 mm) and the eluent given in
4.4. Compound 7
Compound 619 (2.95 g, 17.53 mmol) was dissolved in
DMF and cooled to 0 ꢁC. Imidazole (4.77, 70.16 mmol)
and TBSCl (10.57 g, 70.16 mmol) were added. The reac-
tion was allowed to warm to room temperature and was
stirred for additional 3 h. The reaction was monitored
by TLC (EtOAc/Hex 5:95). After completion, the reac-
tion was diluted with EtOAc and washed with NH4Cl
solution. The organic layer was dried over magnesium
sulfate and the solvent was removed under reduced pres-
sure. The crude product was purified by column chro-
matography on silica gel (EtOAc/Hex 5:95) to give
compound 7 (6.09 g, 68%) in the form of colorless oil.
1
parentheses. H NMR spectroscopy was performed by
using a Bruker AMX 200 or 400 instrument. The chem-
ical shifts are expressed in d relative to tetramethylsilane
(TMS) (d = 0 ppm) and the coupling constants J in Hz.
The spectra were recorded in CDCl3 or CD3OD as a sol-
vent at room temp. HR-MS: liquid secondary ionization
(LSI-MS): VG ZAB-ZSE with 3-nitrobenzyl-alcohol
matrix. All reagents, including salts and solvents, were
purchased from Sigma–Aldrich (Milwaukee, MN).
1H NMR (200 MHz, CDCl3): d = 7.12 (3H, s); 4.69 (6H,
s); 0.92 (27H, s); 0.06 (18H, s). 13C NMR (100 MHz,
CDCl3): d = 143.27, 124.34, 67.00, 27.94, 20.39, À3.28.
MS (FAB): m/z: 509.4 [M+H]+.
4.2. Cell culture
Tumor cells (Molt-3 human acute lymphoblastic leuke-
´
mia cells) were a kind gift from Holger Lode (Charlte
Children’s Hospital, Berlin). Cells were cultured in
RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS) at 37 ꢁC with 5% CO2.
4.5. Compound 8
Acetic anhydride (30 mL) was cooled to 5 ꢁC and nitric
acid (2 mL, 71%) was added dropwise. After the addi-
tion was completed, the mixture was stirred for 15 min
at room temperature and then cooled to À20 ꢁC. A solu-
tion of compound 7 (5.85 g, 11.46 mmol) in 10 mL of
acetic anhydride was added dropwise. The reaction mix-
ture was allowed to warm to 0 ꢁC and was stirred for
additional 30 min. After completion, the reaction was
diluted with EtOAc and was washed with NaHCO3
solution followed by brine. The organic layer was dried
over magnesium sulfate and the solvent was removed
under reduced pressure. The crude product was purified
by column chromatography on silica gel (EtOAC/Hex
5:95) to give compound 8 (4.78 g, 75%) in the form of
a yellow oil.
4.3. Compound 3
Compound 5 (20 mg, 0.019 mmol) was dissolved in
DMF. Tryptophan (15 mg, 0.076 mmol) was added, fol-
lowed by the addition of Et3N (150 lL, 1 mmol). The
reaction mixture was stirred overnight and monitored
by TLC (EtOAc/MeOH 9:1 + 1% of acetic acid) and by
HPLC. After completion, the solvent was removed under
reduced pressure and the crude product was purified by
column chromatography on silica gel (EtOAc/MeOH
9:1, 1% of acetic acid) followed by additional purification
using a reverse phase chromatography (HPLC) with C-18
semi-preparative column, to give compound 3 (23 mg,
75%) in the form of a white powder. HRMS (MALDI-
TOF): m/z calcd for C66H65N9O17Na: 1278.4355; found:
1278.4390 [M+Na]+.
1H NMR (200 MHz, CDCl3): d = 7.49 (2H, s); 4.75 (6H,
s); 0.92 (27H, s); 0.06 (18H, s). 13C NMR (100 MHz,

3726
A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
CDCl3): d = 144.64, 141.23, 122.30, 64.97, 61.39, 25.93,
18.36, À5.29. MS (FAB): m/z: 556.4 [M+H]+.
1H NMR (200 MHz, MeOD): d = 7.53–7.29 (11H, m);
5.09 (2H, s); 4.58 (2H, s); 4.54 (4H, s); 3.64 (2H, s).
13C NMR (400 MHz, MeOD): d = 170.86, 155.88,
140.33, 138.34, 135.28, 128.61, 128.08, 126.45, 124.73,
119.62, 66.18, 63.60, 63.43, 60.05, 29.22. MS (FAB):
m/z: 451.2 [M+H]+.
4.6. Compound 9
Compound 8 (5.43 g, 9.76 mmol) was dissolved in a
50:50 THF/MeOH solution. A catalytic amount of pal-
ladium was added to the mixture, followed by the addi-
tion of ammonium formate (1 g, 15.8 mmol). The
reaction mixture was stirred in room temperature for
2.5 h and monitored by TLC (EtOAc/Hex 5:95). After
completion, the salts were filtered out and the solvent
was removed under reduced pressure. The residue was
diluted with EtOAc and washed with brine. The organic
layer was dried over magnesium sulfate, and the crude
product was purified by column chromatography on sil-
ica gel (EtOAc/Hex 5:95) to give compound 9 (4.39 g,
85%) in the form of a yellow oil.
4.9. Compound 14
Compound 13 (236 mg, 0.523 mmol) was dissolved in
dry THF and a catalytic amount of pyridine was added.
The solution was cooled to À20 ꢁC and a solution of
PNP-chloroformate (1.6 g, 7.8 mmol) in dry THF was
added dropwise. The temperature was not allowed to
exceed À10 ꢁC. The mixture was stirred for 6 h and
monitored by HPLC and by TLC (EtOAc/Hex 50:50).
After completion, the reaction mixture was diluted with
EtOAc and washed with saturated NH4Cl solution. The
organic layer was dried over magnesium sulfate and the
solvent was removed under reduced pressure. The crude
product was purified by column chromatography on
silica gel (EtOAc/Hex 50:50) to give compound 14
(395 mg, 80%) in the form of a pale yellow powder.
1H NMR (200 MHz, CDCl3): d = 6.9 (2H, s); 4.66 (6H,
s); 4.56 (2H, s); 0.87 (27H, s); 0.03 (18H, s). 13C NMR
(100 MHz, CDCl3): d = 146.93, 131.40, 128.51, 126.96,
67.02, 66.88, 27.79, 20.15, À3.26. MS (FAB): m/z:
525.3 [M+H]+.
1H NMR (200 MHz, CDCl3): d = 8.26 (6H, m); 7.65
(2H, s); 7.52–7.33 (15H, m); 5.36 (4H, s); 5.27 (2H, s);
5.14 (2H, s); 3.70 (2H, s). 13C NMR (400 MHz, CDCl3):
d = 175.54, 160.46, 160.32, 157.56, 150.53, 143.45,
139.91, 138.37, 136.61, 134.21, 133.79, 132.21, 130.45,
128.91, 124.99, 75.29, 72.87, 72.34, 72.03, 49.05, 35.2.
HRMS (MALDI-TOF): m/z calcd for C46H35N5O18Na:
968.1846; found: 968.1869 [M+Na]+.
4.7. Compound 12
Toluene was heated to reflux (110 ꢁC) and a solution of
20% phosgene in toluene (9.8 mL, 19 mmol) was added.
Then, a solution of compound 9 (1 g, 1.9 mmol) in tolu-
ene was slowly added dropwise with a syringe. The reac-
tion mixture was stirred for 30 min at reflux and
1
monitored by H NMR. After the isocyanate derivative
was observed, the solvent was removed under reduced
pressure. A solution of compound 1114 (596 mg,
2.47 mmol)in DMF, followed by 0.5 mL Et3N, was
added to the isocyanate residue. The reaction mixture
was stirred for 1 h and monitored by TLC (EtOAc/
Hex 30:70). After completion, the solvent was removed
under reduced pressure. The crude product was purified
by column chromatography on silica gel (EtOAc/Hex
20:80) to give compound 12 (751 mg, 50%) in the form
of a white solid.
4.10. Compound 4
Compound 14 (20 mg, 0.021 mmol) was dissolved in
DMF. Tryptophan (17.3 mg, 0.085 mmol) was added,
followed by the addition of Et3N (15 lL, 0.1 mmol).
The reaction mixture was stirred overnight and moni-
tored by TLC (EtOAc/MeOH 9:1 + 1% of acetic acid)
and by HPLC. After completion, the solvent was removed
under reduced pressure and the crude product was puri-
fied by column chromatography on silica gel (EtOAc/
MeOH 9:1, 1% of acetic acid) followed by additional puri-
fication using a reverse phase HPLC with C-18 semi-pre-
parative column (H2O/CH3CN gradient 10%–100%), to
give compound 4 (18 mg, 75%) in the form of a white
powder. HRMS (MALDI-TOF): m/z calcd for C61H56
N8O15Na: 1163.3647; found: 1163.3757 [M+Na]+.
1H NMR (200 MHz, CDCl3): d = 7.41–7.29 (11H, m);
5.11 (2H, s); 4.71 (2H, s); 4.65 (2H, s); 3.74 (2H, s);
0.94 (27H, s); 0.08 (18H, s). 13C NMR (100 MHz,
CDCl3): d = 170.96, 156.20, 139.49, 138.00, 136.29,
131.44, 131.19, 130.79, 129.65, 126.69, 124.34, 121.59,
68.46, 66.70, 64.87, 46.79, 27.92, 20.21, À3.42. MS
(FAB): m/z: 525.3 [M+H]+.
4.11. Compound 15
4.8. Compound 13
Compound 5 (20 mg, 0.019 mmol) was dissolved in
DMF. Melphalan (15.5 mg, 0.076 mmol) was added,
followed by the addition of Et3N (15 lL, 0.1 mmol).
The reaction mixture was stirred overnight and moni-
tored by TLC (EtOAc/MeOH 9:1 + 1% of acetic acid)
and by HPLC. After completion, the solvent was re-
moved under reduced pressure and the crude product
was purified by column chromatography on silica gel
(EtOAc/MeOH 9:1, 1% of acetic acid) followed by addi-
tional purification using a reverse phase HPLC with
C-18 semi-preparative column (H2O/CH3CN gradient
Compound 12 (286 mg, 0.36 mmol) was dissolved in
MeOH/DCM 1:1 and Amberlyst 15 was added. The
reaction mixture was stirred in room temperature for
2 h and monitored by TLC (EtOAc/Hex 9:1). After
completion, the Amberlyst 15 was filtered out and the
solvent was removed under reduced pressure. The crude
product was purified by column chromatography on sil-
ica gel (EtOAc/Hex 9:1) to give compound 13 (105 mg,
65%) in the form of a white solid.

A. Sagi et al. / Bioorg. Med. Chem. 15 (2007) 3720–3727
3727
10%–100%), to give compound 15 (20 mg, 70%) in the
References and notes
form of a white powder. HRMS (MALDI-TOF): m/z
calcd for C72H83N9O17Cl6Na: 1578.3945; found:
1578.3930 [M+Na]+.
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4.12. Compound 16
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Compound 14 (20 mg, 0.021 mmol) was dissolved in
DMF. Melphalan (17.3 mg, 0.085 mmol) was added,
followed by the addition of Et3N (15 lL, 0.1 mmol).
The reaction mixture was stirred over night and moni-
tored by TLC (EtOAc/MeOH 9:1 + 1% of acetic acid)
and by HPLC. After completion, the solvent was
removed under reduced pressure and the crude product
was purified by column chromatography on silica gel
(EtOAc/MeOH 9:1, 1% of acetic acid) followed by addi-
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phy (HPLC) with C-18 semi-preparative column (H2O/
CH3CN gradient 10%–100%), to give compound 16
(21 mg, 70%) in the form of a white powder. HRMS
(MALDI-TOF): m/z calcd for C67H74N8O15Cl6Na:
1463.3262; found: 1463.3297 [M+Na]+.
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Activation solution (100 lL) was added to 5 mL XTT
reagent. The reaction solutions (50 lL) were added to
each well. The plate was incubated for 2 h, shaken gently
to evenly distribute the dye in the wells. Absorbance was
measured at a wavelength of 450–500 nm. In order to
measure reference absorbance (to measure nonspecific
readings), a wavelength of 630–690 nm was used.