Design, synthesis and antiproliferative properties of oligomers with chromophore units linked by amide backbones

Article abstract of DOI:10.1016/j.bmcl.2008.02.045

The preparation of oligomers made up of several chromophore units as compounds with potential fluorescent and antiproliferative properties is described. Specifically, chromophore units with protected-amino groups and one carboxylic group are described, to

Full text of DOI:10.1016/j.bmcl.2008.02.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2440–2444
Design, synthesis and antiproliferative properties of oligomers
with chromophore units linked by amide backbones
a
b,c,e
d
d,f
´
´
Isabel Navarro, Juan Aymamı,
Josep Farrera-Sinfreu, Anna Avino,
˜
a
a
g
g
`
`
´
´
´
Natalia G. Beteta, Sonia Varon, Ricardo Perez-Tomas, Wilmar Castillo-Avila,
Ramon Eritja,b,c,e, Fernando Albericio
and Miriam Royo
b,c,h,
a,c,
*
*
*
aCombinatorial Chemistry Unit, Barcelona Scientific Park (PCB), Josep Samitier 1-5, E-08028 Barcelona, Spain
bInstitute for Research in Biomedicine (IRB Barcelona), Josep Samitier 1-5, E-08028 Barcelona, Spain
cNetworking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Josep Samitier 1-5, E-08028 Barcelona, Spain
dCrystax Pharmaceuticals S.L., Josep Samitier 1-5, E-08028 Barcelona, Spain
eInstitute for Advanced Chemistry of Catalonia (IQAC), Spanish Research Council (CSIC), Jordi Girona 18-26,
E-08034 Barcelona, Spain
f
`
Department d’Enginyeria Quı´mica, Universitat Politecnica de Catalunya (UPC), Diagonal 647, E-08028 Barcelona, Spain
gDepartment of Pathology and Experimental Therapeutics, Cancer Cell Biology Research Group, University of Barcelona (UB),
Feixa llarga, E-08907 L’Hospitalet de Llobregat, Barcelona, Spain
hDepartment of Organic Chemistry, University of Barcelona (UB), Martı´ i Franque´s 1, E-08028 Barcelona, Spain
Received 30 January 2008; revised 14 February 2008; accepted 17 February 2008
Available online 5 March 2008
Abstract—The preparation of oligomers made up of several chromophore units as compounds with potential fluorescent and anti-
proliferative properties is described. Specifically, chromophore units with protected-amino groups and one carboxylic group are
described, together with methods to assemble these units using peptide chemistry. Some of these compounds have antiproliferative
activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Interest in new DNA-intercalating drugs has led to the
development of protocols for the rapid synthesis of com-
pounds carrying several chromophore units. In the pre-
vious paper we described the preparation of oligomers
with phosphodiester bonds using the phosphoramidite
method and solid-phase synthesis protocols. Here we de-
scribe the synthesis of oligomers carrying amide bonds.
viously for the synthesis of conformational constrained
peptide nucleic acids.1,2 Polyproline oligomers form a
rigid backbone that is suitable for drug delivery.3,4 The
2-aminoethylglycine backbone is more flexible and has
been used for the design of peptide nucleic acids
(PNA) DNA backbone mimetics.5,6 The synthesis of
several functionalized PNA oligomers containing
7
anthracene, flavin,8 pyrene,9 and naphthalimide10 has
For the synthesis of these polymers, three trifunctional
scaffolds were chosen: 4-aminoproline, 2-aminoethylgly-
cine, and ornithine. All three have two amino groups
and one carboxylic acid function. This distribution al-
lows both the growth of a polyamide skeleton and the
incorporation of the DNA-intercalating unit on the
other amino group if suitable amino-protecting groups
are used. c-Aminoproline backbones had been used pre-
led to compounds with improved DNA binding proper-
ties. Using similar procedures, ornithine has also been
used as a DNA mimetic; deoxyribose has been replaced
with a poly-ornithine chain.11
For the synthesis of 4-aminoproline polymers the back-
bone was grown on solid-phase, and then the intercalat-
ing agent was assembled on solid support. This strategy
is more convenient for the rapid synthesis of large li-
braries, as it is unnecessary to construct each monomer
with its intercalating agent. The synthesis protocols were
based on a previous study.12 Thus, the assembly of
4-aminoproline oligomers was carried out using the
Keywords: Chromophore; Fluorescence; Peptides; Peptide nucleic acids;
Ornithine; 4-Aminoproline; Solid-phase synthesis; Antiproliferative.
*
Corresponding authors. Tel.: +34 934039942; fax: +34 932045904
(R.E.); e-mail: recgma@cid.csic.es
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.045

J. Farrera-Sinfreu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2440–2444
2441
methylbenzhydrylamine (MBHA) resin applying an
Fmoc/Boc hybrid strategy and using trans-c-Fmoc-ami-
no-a-Boc-L-proline [Boc-Amp(Fmoc)-OH] as a building
block. The Fmoc group was used to protect the c-amino
group of each monomer, which thus facilitated the elon-
gation of the backbone. Boc was the semi-permanent
protecting group for the a-amino group through which
the chromophore units were introduced. a-Amino deriv-
atizations were carried out on solid-phase using stan-
dard Boc chemistry protocols. A glycine molecule was
introduced as a spacer between this amino group and
the chromophore moiety. In general, the Fmoc group
was removed using 20% of piperidine in N,N-dimethyl-
formamide (DMF) and Boc-Amp(Fmoc)-OH (5 equiv)
was coupled to the resin using diisopropylcarbodiimide
(DIPCDI, 5 equiv) and 1-hydroxybenzotriazole (HOBt,
5 equiv) as coupling reagents. Boc groups were removed
using 40% trifluoroacetic acid (TFA) in dichlorometh-
ane (DCM), followed by the treatment of the resin with
5% diisopropylethylamine (DIEA) in DCM. Boc-Gly-
OH (5 equiv) was coupled to the resin using DIPCDI
(5 equiv) and HOBt (5 equiv) as coupling reagents.
Carboxyl derivatives (1a–g, 3 equiv) were coupled to
the resin using O-(benzotriazol-1-yl)-N,N,N,N-tetram-
ethyluronium tetrafluoroborate (TBTU, 3 equiv) and
DIEA (6 equiv) as coupling reagents. Acetylations were
carried out using acetic anhydride (Ac2O, 5 equiv) and
DIEA (5 equiv).
the spacer (Boc-Gly-OH) was introduced at the a-amino
positions. After the removal of the Boc groups, carboxyl
derivatives (1a, 1b) were introduced through these gly-
cines, forming the final homopolymers. A range of di-
mers and trimers (Table 1) were synthesized in this way.
Table 1. Oligomers prepared in this study
Compound
MS (expected) MS (found)
Ac-Agr-Agr-NH2
Ac-Qgr-Qgr-NH2
807.8
885.9
808.4
886.5
Ac-Agr-Agr-Agr-NH2
Ac-Qgr-Qgr-Qgr-NH2
Ac-Agr-Qgr-Agr-NH2
Ac-Qgr-Qgr-Agr-NH2
Ac-Qgr-Agr-Agr-NH2
Ac-Qgr-Agr-Qgr-NH2
Ac-Agr-Agr-Qgr-NH2
Ac-Agr-Qgr-Qgr-NH2
Ac-Qgr-r-Agr-r-Pgr-NH2
Ac-Pgr-r-Ogr-r-Pgr-NH2
Ac-Agr-r-Pgr-r-Qgr-NH2
Ac-Fgr-r-Pgr-r-Agr-NH2
Ac-Qgr-r-r-Agr-r-r-Pgr-NH2
Ac-Pgr-r-r-Ogr-r-r-Pgr-NH2
Ac-Agr-r-r-Pgr-r-r-Qgr-NH2
Ac-Fgr-r-r-Pgr-r-r-Agr-NH2
Ac-Ago-Ago-Ago-NH2
Ac-Qgo-Qgo-Qgo-NH2
Ac-Ago-Qgo-Ago-NH2
Ac-Ago-Qgo-Qgo-NH2
Ac-Agp-Agp-NH-C6H12-OH
1182.2
1299.3
1221.3
1260.3
1221.3
1260.3
1221.3
1260.3
1555.6
1559.6
1555.6
1517.6
1864.0
1868.0
1864.0
1826.0
1188.3
1305.4
1227.3
1266.4
885.9
1182.6
1299.7
1222.6
1260.2
1222.6
1261.6
1221.7
1260.6
1557.6
1559.4
1557.1
1519.2
1865.8
1868.6
1865.2
1827.7
1189.0
1305.9
1227.9
1266.9
884.4
The solid-phase procedures depended on whether homo-
polymers or heteropolymers were the objects of synthe-
sis, as described below.
Ac-Agp-Agp-Agp-NH-C6H12-OH 1249.3
Ac-Qgp-Qgp-NH-C6H12-OH 964.0
1246.4
962.6
4-Aminoproline backbone: Agr (Acridine), Qgr (indoloquinoline), Fgr
(fluorenyl), Pgr (2-phenylquinoline), Ogr (chromene); ornithine back-
bone: Ago (acridine), Qgo (indoloquinoline); 2-aminoethylglycine
backbone: Agp (acridine), Qgp (indoloquinoline), Ac: acetyl; r:
Na-acetyl-c-aminoproline.
Protocol I, for homopolymers (see Scheme 1). The
c-peptide backbone was synthesized first by repetitive
couplings of Boc-Amp(Fmoc)-OH using Fmoc chemis-
try and, after the removal of Boc protecting groups,
C6H12: n-hexyl.
O
O
a)
Fmoc
Fmoc
Fmoc
b)
H2N
N
H
N
H
N
H
N
H
N
N
H
MBHA resin
Boc
n
n
c)
O
O
O
O
O
N
H
N
H
NH2
N
H
N
H
N
H
N
N
O
d)
e)
N
O
O
n
n
n
NH
NH
O
NH
O
O
R
R
R
1a
1b
N
N
N
H
COOH
COOH
Scheme 1. Solid-phase synthesis of homo-oligomers. Reagents and conditions: (a) i—piperidine-DMF (2:3), 30 min; ii—(2S,4R)-Boc-Amp(Fmoc)-
OH/DIPCDI/HOBt (3 equiv, 3 equiv, 3 equiv) in DMF, iii—repeat steps i and ii n times; (b) i—TFA-DCM (4:6), 30 min. ii—DIEA-DCM (1:9),
1 min. (c) i—Boc-Gly-OH/DIPCDI/HOBt (3 equiv, 3 equiv, 3 equiv) in DMF; ii—TFA-DCM (4:6), 30 min. iii—DIEA-DCM (1:9), 1 min. iv—R-
COOH (1a or 1b)/TBTU/DIEA (3 equiv, 3 equiv, 6 equiv) in DMF. (d) i—Piperidine-DMF (2:3), 30 min; ii—Ac2O/DIEA (5 equiv, 10 equiv) in
DMF, 2 h. (e) Anhydrous HF.

2442
J. Farrera-Sinfreu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2440–2444
Protocol II, for heteropolymers (see Scheme 2). In this
procedure, the fluorophore was introduced sequentially
after each aminoproline coupling. Thus, Boc-Amp(F-
moc)-OH was attached to the MBHA resin, the Boc
protecting group was removed and the spacer (Boc-
Gly-OH) was coupled. After the removal of the
Boc group the fluorophore was introduced using the
coupling agents mentioned below. This cycle gave
the first aminoproline functionalized with the first fluo-
rophore. Then, the Fmoc group was removed, and the
sequence was repeated, obtaining an aminoproline
dimer with two different fluorophores. Repetitions of
these steps allowed the synthesis of several dimers and
trimers (Table 1).
positions of the proline that did not have a fluorophore
were acetylated. Thus, the a-amino positions that were
maintained without monomer were acetylated after the
Boc group removal.
All c-aminoproline oligomers were finally treated with
20% piperidine solution to remove the N-terminal Fmoc
group, acetylated at this position, and treated with
anhydrous HF to obtain the desired products. Products
were finally purified by a reverse-phase semi-preparative
HPLC (Fig. 1).
Ornithine polymers (Table 1) were synthesized following
the same synthetic strategies used in the synthesis of c-
aminoproline oligomers, just replacing the Amp scaffold
by Boc-L-Orn(Fmoc)-OH. Backbone elongation was
carried out through the d-amino function using Fmoc
chemistry and monomers were introduced in the a-ami-
no group using Boc chemistry and using glycine as a
spacer.
Protocol III. This protocol was applied for those poly-
mers that did not have monomer at all the a-amino
proline positions. These compounds were treated as
heteropolymers. They were synthesized using protocol
II (see Scheme 2), with the difference that those a-amino
O
Fmoc
N
H
N
H
a)
b)
N
O
H2N
Fmoc
O
N
H
N
H
N
NH
O
Boc
MBHA resin
Fmoc
R1
c), a)
O
O
O
O
N
H
N
N
H
N
N
Fmoc
H
N
H
N
H
N
O
O
N
Boc
N
H
b)
O
NH
NH
O
O
NH
R2
R1
O
d)
R1
O
O
O
NH2
N
N
H
N
N
H
O
O
NH
R2
NH
O
O
R1, R2 = Chromophore
R1
1a
1e
1b
N
N
N
N
H
COOH
COOH
COOH
1f
1g
O
O
COOH
COOH
Scheme 2. Example of the solid-phase synthesis of a heterodimer. Reagents and conditions: (a) (2S,4R)-Boc-Amp(Fmoc)-OH/DIPCDI/HOBt
(3 equiv, 3 equiv, 3 equiv) in DMF. (b) i—TFA-DCM (4:6), 30 min. ii—DIEA-DCM (1:9), 1 min; iii—Boc-Gly-OH/DIPCDI/HOBt (3 equiv, 3 equiv,
3 equiv) in DMF; iv—TFA-DCM (4:6), 30 min. v— DIEA-DCM (1:9), 1 min. vi—Rn-COOH (1a or 1b or 1e or 1f or 1g)/TBTU/DIEA (3 equiv, 3
equiv, 6 equiv) in DMF. (c) Piperidine-DMF (2:3), 30 min. (d) i—Piperidine-DMF (2:3), 30 min; ii—Ac2O/DIEA (5 equiv, 10 equiv) in DMF, 2 h;
iii—anhydrous HF.

J. Farrera-Sinfreu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2440–2444
2443
Table 2. Antiproliferative activity
Ac-Fgr-r-Pgr-r-Agr-NH2
Compound
IC50 (lM)
Jurkat
clon E6-1
IC50 (lM)
GLC-4S
IC50 (lM)
NIH-3T3
1c
1c, methyl ester
1d
n.a.
n.a.
12
n.a.
n.a.
n.a.
n.a.
135
54
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
376
n.a.
n.a.
n.a.
426
n.a.
357
263
227
1d, methyl ester
Ac-Agr-Agr-NH2
Ac-Qgr-Qgr-NH2
Ac-Agr-Qgn-NH2
Ac-Qgr-Agr-NH2
Ac-Agr-Agr-Agr-NH2
Ac-Qgr-Qgr-Qgr-NH2
Ac-Agr-Agr-Qgr-NH2
Ac-Agr-Qgr-Agr-NH2
Ac-Qgr-Agr-Agr-NH2
Ac-Qgr-Agr-Qgr-NH2
Ac-Agr-Qgr-Qgr-NH2
8
149
37
92
125
165
134
n.a.
174
91
120
n.a.
130
86
0
2
4
6
8
10
12
14
Figure 1. HPLC chromatogram of trimer Ac-Fgr-r-Pgr-r-Agr-NH2.
87
129
112
103
169
82
For the solid-phase synthesis of 2-aminoethylglycine
backbones, the scaffolds containing several intercalating
agents were prepared in solution (see Scheme 3). These
aminoethylglycine derivatives were prepared from the
N-Boc-2-aminoethylglycine methyl ester, which was re-
acted with the carboxyl derivatives of acridine (1c) and
indoloquinoline (1d) using DIPCDI and hidroxy-3,4-
dihydro-4-oxo-1,2,3-benzotriazine (HOOBt) as coupling
reagents, yielding compounds 2c–d. Hydrolysis of the
resulting compounds with aq NaOH in dioxane (1:1)
gave the desired derivatives 3c–d. Oligomers with these
N-aminoethylglycine scaffolds were then synthesized
on polyethyleneglycol-polystyrene supports with the 6-
aminohexylsuccinyl linker.13 Standard solid-phase pep-
tide synthesis protocols were used. The synthesis cycle
consisted of two reactions, removal of the Boc group
using 50% TFA in DCM and 5% cresol as a scavenger,
followed by the coupling of the appropriate monomer (5
molar excess) using [O-(7-azabenzotriazol-1-yl]-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU, 4.5
molar excess) and DIEA (15 molar excess) as coupling
reagents. After the assembly of the sequence, the result-
ing supports were treated with conc. aqueous ammonia
for 2 h at 55 °C to yield the desired unprotected oligo-
mers (Table 1).
151
n.a., not active; n.d, not determined.
The antiproliferative activity of the new compounds
having the aminoprolyl backbones were analyzed on
two human cancer cell lines, Jurkat clon E6-1 and
GLC-4S, and one mouse fibroblast cell line, NIH-3T3.
MTT assay was performed to determine IC50 values. Re-
sults are shown in Table 2. All compounds had a mod-
erate antiproliferative activity in cancer cell lines (IC50
37–170 lM), whereas IC50 values for NIH-3T3 were
very high (IC50 > 227 lM), suggesting a low toxicity to
non-tumoral cells. As comparison the carboxyl deriva-
tive 1c and its methyl ester were not active in these con-
ditions whereas the carboxyl derivative 1d and its methyl
ester were only active on Jurkat clon E6-1 cell lines.
In summary, we report an efficient solid-phase synthesis
of several oligomers carrying fused heterocyclic deriva-
tives linked through amide bonds. c-Aminoproline,
2-aminoethylglycine, and ornithine backbones were
studied. All oligomers were obtained rapidly and in high
yields, suitable for the preparation of a wide range of
these derivatives. Preliminary antiproliferative assays
showed moderate but selective growth-inhibitory prop-
erties. The methods described here can be used in the
search for more active compounds and for the introduc-
tion of fluorescent compounds into synthetic peptides.
The excitation and emission wavelengths of the new
compounds were measured (see Supporting informa-
tion). Optimal excitation wavelengths ranged from 248
to 360 nm and the corresponding emission wavelengths
ranged from 396 to 505 nm. The indoloquinoline (Qgr)
derivatives with c-aminoproline backbone had the
higher emission wavelengths.
Acknowledgments
This study was supported by the OTRI (PTR), Crystax
Pharmaceuticals S.L., the Spanish Ministry of Educa-
tion (BFU2004-02048, CTQ2005-00315, BFU2007-
63287), the Generalitat de Catalunya (2005/SGR/
00693), and the Instituto de Salud Carlos III (CIBER-
BNN).
R
O
O
O
H
N
a)
Boc
Boc
N
N
H
OCH3
N
H
OCH3
b)
1c
1d
N
N
R
O
O
N
H
Boc
N
Supplementary data
N
H
OH
CONHCH2COOH
CONHCH2COOH
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.02.045.
Scheme 3. Synthesis of 2-aminoethylglycine derivatives. Reagents: (a)
R-COOH (1c or 1d)/DIPCDI/HOOBt/N-ethylmorpholine, (b) NaOH
(water/ethanol).

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J. Farrera-Sinfreu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2440–2444
7. Balasundaram, G.; Takahashi, T.; Ueno, A.; Mihara, H.
Bioorg. Med. Chem. 2001, 9, 1115.
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