Synthesis and binding properties of photoactivable biotin-conjugated verapamil derivatives for the study of P-170 glycoprotein

Article abstract of DOI:10.1016/S0968-0896(99)00104-2

The design and synthesis of two photoactivable biotin-labeled analogues of verapamil (6 and 7) is reported. Preliminary evaluation of the biological profile of 6 (EDP 137) and 7 (EDP 141) shows that they have comparable affinities to that of verapamil for

Full text of DOI:10.1016/S0968-0896(99)00104-2

Bioorganic & Medicinal Chemistry 7 (1999) 1873±1880
Synthesis and Binding Properties of Photoactivable
Biotin-Conjugated Verapamil Derivatives for the Study of
P-170 Glycoprotein
Elisabetta Teodori, a Dominique Ettori, b Arlette Garnier-Suillerot, b Fulvio Gualtieri, a,*
Dina Manetti, a Maria Novella Romanelli a and Serena Scapecchi a
aDipartimento di Scienze Farmaceutiche, Universita' di Firenze, via G. Capponi 9, 50121 Firenze, Italy
Â
Laboratoire de Physicochimie Biomoleculaire et Cellulaire (LPBC, UPRES-A 7033), Universite Paris Nord, 74 rue Marcel Cachin,
93017 Bobigny, France
b
Received 8 December 1998; accepted 11 March 1999
AbstractÐThe design and synthesis of two photoactivable biotin-labeled analogues of verapamil (6 and 7) is reported. Preliminary
evaluation of the biological pro®le of 6 (EDP 137) and 7 (EDP 141) shows that they have comparable anities to that of verapamil
for P-170, the protein responsible for multidrug resistance (MDR). Since both appear to bind irreversibly to the protein and the
presence of biotin in their structure makes them easily detectable by avidin, they promise to be of great help in studying the protein
and its mechanism of action. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
putative transmembrane domains and two nucleoside
binding sites12 whose structure at 2.5 nm has been
recently reported.13 P-170 is overexpressed in cancer
cells showing MDR. It is known that cells exhibiting
MDR accumulate a lower intracellular concentration of
drug and that this e€ect is associated with accelerated
e‚ux of anti tumor agents by P-glycoprotein which
pumps out the drug reducing its cellular concentration
below cytotoxic levels.1±3 In fact, while being present in
many tissues, P-170 generally shows high levels in
malignant cells and increasing amounts of it are found
in patients with increasing resistance to chemotherapy,
so that a signi®cant correlation between P-170 expres-
sion and MDR, both in vitro and in vivo, can be detec-
ted.14 As a consequence, P-170 has become the target of
many studies aimed to identify drugs able to block its
extrusive activity and restore the initial response of
tumor cells to chemotherapy.15±17
A 170 KDa glycoprotein, known as P-170 (also referred
to as Pg-170, GP-170, or Pgp) and a 190 KDa protein
called MRP1 (MDR-related protein) are known to play
a critical role in the acquired resistance of cancer to
chemotherapy, known as multidrug resistance (MDR).1±3
Both belong to the super family of the ATP-binding
cassette (ABC) of transport proteins4 and operate as
extrusion pumps, even though their mechanisms of
action seem di€erent.5 Analogous systems of drug
extrusion have been found in microorganisms6,7 and it is
becoming more and more evident that the multidrug
e‚ux systems encountered in prokaryotic cells are very
similar to those observed in eukaryotic cells.8 Moreover,
in addition to their role in cancer cell resistance, these
proteins seem to have a physiological function as well,
since they are expressed also in non cancer tissues: P-170
has been reported to be an important element of the
blood±brain barrier (BBB),9 of the intestinal epithe-
lium10 and, together with other ATP-dependent trans-
porters, in the hepatobiliary secretion.11
The role played by P-170 in physiology, pathology and
pharmacology has stimulated studies aimed to clarify its
structure, functions, and mechanism of action. In partic-
ular, knowledge of the structure of the binding site of
substrates and modulators of the glycoprotein will be
invaluable not only for an understanding of how drugs
interact with it, but also to the design of better and
more speci®c inhibitors. It has been suggested that P-170
antagonists fall into two groups: those that are trans-
ported themselves by the protein and those that are not.18
The best known of these proteins is P-170 which is a
membrane-associated 1280 aa protein composed of 12
Key words: Verapamil analogues; biotin-labeled compounds; multi-
drug resistance; P-glycoprotein; photoanity labeling.
* Corresponding author. Tel.: +39-55-275-7295; fax: +39-55-240776;
e-mail: gualtieri@farm®.scifarm.uni®.it
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00104-2

1874
E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
In general however, the mechanism of action of P-170
and the binding mode of its inhibitors19 are poorly
understood and many things remain to be clari®ed.
Toward this aim, potent and selective ligands of the
protein, in particular those that can be labeled and can
establish covalent bonds with the molecule, are necessary.
First, we designed a molecule where the biotin moiety
was linked through an amide bond to the quaternary
carbon atom of the N-desmethyl derivative of ver-
apamil. As a matter of fact, while the nitrile group is
considered critical for calcium activity of verapamil,30
its role in P-170 protein binding is less well established.
In any case, an analogous p-azido-benzoylderivative of
nitrile-reduced verapamil (3) is an ecient photoanity
labeler of P-170.31 Moreover, N-desmethylderivatives
are, in our experience, less potent as calcium antagonists
but more potent as MDR reverters.32 However, this
molecule could not be obtained since the biotinyl group
also binds to the nitrogen to give compound 4. As a
consequence, we turned to the synthesis of the corre-
sponding N-methyl derivative 5.
Several compounds that covalently bind to P-170 pro-
tein have been synthesized and studied;20±26 few of them
are dihydropyridine like 123,25 and verapamil analogues
like 2 and 320,26 but representatives of other chemical
classes have also been proposed.21,22,24 Frequently they
are photoactivable molecules, most of them carrying an
azido group like azidopine 1, one of the most used
compounds,27 and the vast majority are labeled with 3H,
or an equivalent radioisotope.
At the same time, to maintain the nitrile group, we
designed compound 6 and 7 where the biotin moiety is
linked, through a suitable spacer, to the same qua-
ternary carbon atom of verapamil. Since the ortho azido
group of 6 could su€er for steric hindrance problems
to give irreversible binding to the P-170 protein, we
also synthesized compound 7 which carries the azido
group in a less hindered position of the benzene ring.
Chemistry
Radioisotope labeling of suitable molecules has pro-
vided precious tools for the progress of molecular
biology, pharmacology and life sciences in general.
However, these kinds of probes su€er from some
relevant drawbacks such as expensive synthesis, short
half-life, safety, and ®nally, the requirement of special
facilities and disposal methods. To overcome these prob-
lems, biotin-labeling of suitable molecules represents an
alternative approach that has been widely used.28 In
fact, two proteins, avidin (from egg white) and strep-
tavidin (from Streptomices avidini), bind with out-
The synthetic pathway chosen to synthesize compounds
4 and 5 is shown in Scheme 1. 2-(3,4-Dimethoxyphenyl)-
2-isopropylacetonitrile33 was alkylated with 3-bromo-
propanol to give 8 which in turn was reduced with
AlH3. After protection of the amine group with di-tert-
butyl dicarbonate, the resulting compound 10 was
transformed into the methansulfonyl derivative 11. The
compound was smoothly transformed in the amines 12
and 14 and, by cleavage of the protecting group, in the
amines 13 and 15. At this point, while 15 gave the bio-
tinyl compound 5, the corresponding compound 13
reacted with two molecules of succinimido biotinate34 to
give two compounds of identical molecular weight
(measured by FAB) separated by column chromato-
graphy. Most likely, the compounds are the diastereo-
isomers corresponding to the R and S forms of
verapamil, that, unlike the diastereoisomers of com-
pounds 5±7, can be separated. However, no further
studies to establish their absolute con®guration were
carried out due to the lack of anity of both com-
pounds for P-170.
standing anity (Kaꢀ1015
M
1) to biotin and this can
be exploited to construct sensitive methods29 for the
detection of biotin-labeled molecules.
In this paper we report on the design, synthesis and
preliminary evaluation of the anity for P-170 of a few
photoactivable biotin-labeled analogues of verapamil
(4±7) which were designed following our previous work
on compounds able to covalently bind to P-170.26
In Scheme 2 the synthesis of compounds 6 and 7 is
reported. The homoveratryl acetonitrile was alkylated
in two steps to give compound 17, which was trans-
formed in 18 and 19 by reaction with N-methyl-2-(o-
azidophenyl)ethylamine (22) and N-methyl-2-(p-azido-
phenyl)ethylamine (23) whose synthesis is described in
Scheme 3. Finally, reaction with succinimido biotinate
gave compounds 6 and 7.
Compounds 5, 6 and 7 are diastereomeric mixtures of
the R and S forms of verapamil. However, at this step of
the research, no attempt was made to separate the isomers.

E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
1875
Scheme 1. (a) BuLi, BrCH2CH2CH2OH; (b) AlH3; (c) di-tert-butyl dicarbonate; (d) methansulfonyl chloride; (e) 2-(o-azidophenyl)ethylamine40 or
22; (f) HCl; (g) succinimidobiotinate.34
Scheme 2. (a) BuLi, BrCH2CH2CH2OH; (b) LICKOR (BuLi/t-BuOK), BrCH2CH2CH2Cl; (c) 22 or 23; (d) succinimidobiotinate.34
Biology
The overall concentration, Cn, of pirarubicin bound to
the nucleus of drug-resistant cells was determined, at the
steady-state, in the presence of di€erent concentrations
of the drug to be tested. In all cases, Cn increased as the
concentration of inhibitor increased, and this can be
quanti®ed using the following equation:
MDR reverting activity
The anity of the compounds synthesized for P-170
protein was deduced by their ability to revert MDR.
This was evaluated on anthracycline-resistant erythro-
leukemia K 562 cells, measuring the uptake of THP-
adriamicin (pirarubicin) by continuous spectro-
¯uorometric monitoring of the decrease of the ¯uor-
escence signal of the anthracycline at 590 nm (lex=480
nm) after incubation with cells. The decrease of ¯uor-
escence occurring during incubation with cells is due to
quenching after intercalation of antracyclin between the
base-pair of DNA. We have previously shown that this
methodology allows accurate measurement of the con-
centration of anthracyclines bound to the nucleus in
steady-state, their initial rates of uptake and kinetics of
active e‚ux.32,35±39
Â
à Â
Ã
ꢀ ˆ ꢀCn†
ꢀCn† = ꢀCn†
Ro
ꢀCn†
Ri
S
Ro
where ꢁCn† is the overall concentration of pirarubicin
S
bound to the nucleus of sensitive cells and ꢁCn† and
Ro
ꢁCn† are the overall concentrations of pirarubicin
Ri
bound to the nucleus of resistant cells, in the absence
and presence of a concentration [i] of inhibitor, respec-
tively. The value of a represents the fold increase in the
nuclear concentration of pirarubicin in the presence of the
MDR-reverting agent; a varies between 0 (in the absence
of inhibitor) and 1 (when the amount of pirarubicin in

1876
E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
Scheme 3. (a) Tri¯uoroacetic anhydride; (b) CH3I, KOH.
resistant cells is the same as in sensitive cells). The
potency of the compounds in reverting MDR is expres-
sed by [i]0.5 which represents the concentration of the
inhibitor that causes a half-maximal increase in cellular
pirarubicin (a=0.5), while its ecacy is expresed as
shown that both compounds are able to bind irreversibly
to the protein, as indicated by the substantial reduction
of P-170 protein functionality. In fact, under irradiating
conditions that do not damage the cells and after accurate
washing of unbound 6 and 7, the amount of pirarubicin
found in the cell nucleus was de®nitly increased.
a
max, the maximum increase that can be obtained in the
concentration of pirarubicin in resistant cells with a
given inhibitor.
Work is in progress to establish the eligibility of 6 and 7
as biotin-labeled photoactivable ligands of P-170 pro-
tein; the results will be referred in due time.
Photolabeling
Irradiation was performed on resistant cells in the pres-
ence of the photolabelers, under conditions that did
not damage the cells.26 The level of pirarubicin incor-
porated into the nucleus after irradiation and accurate
washing of unbound photolabeler, compared to that of
sensible cells, gives a measure of P-170 functionality and
consequently of the irreversible binding.
Experimental
Chemistry
All melting points were taken on a Buchi apparatus and
are uncorrected. Infrared spectra were recorded with a
Perkin±Elmer 681 spectrophotometer in a Nujol mull
for solids and neat for liquids. NMR spectra were
recorded on a Gemini 200 spectrometer. Chromato-
graphic separations were performed on a silica gel column
by gravity chromatography (Kieselgel 40, 0.063±0.200
mm, Merck) or ¯ash chromatography (Kieselgel 40,
0.040±0.063 mm, Merck). Yields are given after puri-
®cation, unless otherwise stated. Where analyses are
indicated by symbols, the analytical results are within
‹0.4% of the theoretical values (Table 2). CF-FAB
spectrometric measurements in positive ion mode were
carried out using a 7070-E mass spectrometer (VG
Analitical, Manchester, UK).
Results and Discussion
Compounds 4 and 5 did not show anity for P-170
protein, as is shown by the fact that they do not reverse
MDR in anthracycline-resistant erythroleukemia K 562
cells (Table 1). This result seems to suggest that, besides
calcium antagonistic activity, the ciano group, at least in
this case, is critical also for binding to P-170. This is
con®rmed by the fact that compounds 6 and 7 do show
MDR reverting activity (Table 1), being able to restore
the concentration of pirarubicin in resistant cells with
identical ecacy with respect to verapamil (amax=0.8
for 6 and 7; amax=0.7 for verapamil) at a slightly higher
concentration ([i]0.5=10 mM for both 6 and 7; ver-
apamil, [i]0.5=1.6 mM). As a consequence, it can be
reasonably accepted that both compounds show anity
for P-170 glycoprotein and can be used to label the
protein itself.
Table 2. Elemental analyses
Calculated
H%
Found
H%
Compd
C%
N%
C%
N%
4a
4b
5
6
7
60.17
60.17
63.12
62.00
62.00
69.27
68.28
66.10
57.48
66.24
67.72
66.75
68.29
76.79
61.62
66.48
66.48
61.33
61.33
7.24
7.24
7.73
7.00
7.00
8.37
9.69
9.26
8.13
8.26
8.31
8.42
8.50
8.45
7.13
7.38
7.38
6.88
6.88
14.36
14.36
14.73
14.47
14.47
5.05
4.98
3.67
3.05
13.32
16.46
12.98
15.93
6.89
60.34
60.46
63.34
62.26
62.33
68.98
68.45
66.34
57.68
66.56
67.99
66.51
68.44
76.98
61.45
66.23
66.74
60.95
61.54
7.41
6.95
7.41
7.34
7.29
8.71
9.40
8.96
8.44
8.03
8.68
8.13
8.68
8.61
7.41
7.53
7.09
7.12
7.03
14.09
14.18
14.36
14.14
14.76
4.89
5.23
3.98
3.33
13.06
16.13
13.22
16.21
6.55
Moreover, preliminary irradiation experiments, performed
according to the protocol described previously,26 have
8
9
Table 1. MDR reverting activity of compounds 4±7 compared with
verapamila
10
11
12
13
14
15
16
17
18
19
22
23
N
amax
[i]0.5(mM) (a=0.5) (M‹SEM)
4
5
6
n.a.
n.a.
0.8
0.8
0.7
n.a.
n.a.
10‹2
10‹2
1.6‹0.3
4.49
4.61
7
Verapamil
15.51
15.51
31.80
31.80
15.84
15.37
31.56
31.98
a
Evaluated on erthroleukemia K 562 cells line (see Experimental).
n.a.=not active.

E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
1877
2-(3,4-Dimethoxyphenyl)-5-hydroxy-2-isopropylpentane-
nitrile (8). 3.5 mL (5.64 mmol) of BuLi (1.6 M in hex-
ane) were added to a solution of 0.8 mL (5.64 mmol)
of i-Pr2NH in 10 mL of anhyd THF at 0ꢂC. After
10 min the mixture was cooled to 20ꢂC and 0.5 g
(2.28 mmol) of 2-(3,4-dimethoxyphenyl)-2-isopropyl-
acetonitrile33 were added. Then the mixture was cooled
to 50ꢂC and 0.2 mL (2.28 mmol) of 3-bromo-1-propanol
was added. The temperature was kept at 50ꢂC for 2 h
and then allowed to return to room temperature. The
mixture was treated with NH4Cl and extracted with ether.
After drying with Na2SO4, the solvent was removed
under reduced pressure and the residue puri®ed by ¯ash
cromatography using ethylacetate:cyclohexane (80:20)
as eluting system. Title compound 8 (0.5 g, 80% yield)
20ꢂC, a solution of 0.10 mL (1.29 mmol) of methan-
sulfonyl chloride in 3 mL of anhydrous ethyl ether were
added. The mixture was left for 1.5 h at 20ꢂC, then
®ltered and the solvent was removed under reduced
pressure. Title compound 11 (0.47 g, 94% yield) was
obtained as a thick oil. IR (neat) n 3400 (NH), 1740 and
1700 (CO) cm 1. 1H NMR (CDCl3) d 0.78 and 0.82 (2d,
J=6.6 Hz, 6H, 2CH3), 1.41 (s, 9H, (CH3)3), 1.62±1.92
(m, 5H, 2CH2 and CH), 3.05 (s, 3H, CH3), 3.49±3.75
(m, 2H, CH2N), 3.88 (s, 6H, 2OCH3), 4.22 (t, J=5.6 Hz,
2H, CH2), 6.72±6.88 (s, 3H, aromatics) ppm. Anal.
(C22H37NO7S) C, H, N.
N1-t-Butoxycarbonyl-2-(3,4-dimethoxyphenyl)-2-isopropyl-
N5-[2-(o-azido-phenethyl)] 1,5-pentyldiamine (12). 0.40 g
(0.87 mmol) of 11 were mixed with 0.28 g (1.74 mmol) of
2-(o-azidophenyl)ethylamine40 with 1 mL of anhydrous
CH3CN. The mixture was heated at 60ꢂC for 7 h. After
cooling, CHCl3 was added and the organic layer washed
with NaOH 10%. After drying with Na2SO4, the sol-
vent was removed under reduced pressure and the resi-
due puri®ed by column cromatography using CHCl3:
MeOH (9:1) as eluting system. Title compound 12
(0.15 g, 32% yield) was obtained as a thick oil. IR (neat)
was obtained as a thick oil. IR (neat) n 3550 (OH), 2240
(CN) cm
1
.
1H NMR (CDCl3) d 0.79 and 0.82 (2d,
J=6.6 Hz, 6H, 2CH3), 1.50±1.72 (m, 2H, CH and OH),
1.84±2.31 (m, 4H, 2CH2), 3.59 (t, J=5.6 Hz, 2H, CH2O),
3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.81±6.97 (m,
3H, aromatics) ppm. Anal. (C16H23NO3) C, H, N.
5-Amino-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-pentanol
(9). To a solution of 0.07 g (1.8 mmol) of LiAlH4 and
0.24 g (1.8 mmol) of AlCl3 in dry ether, 0.5 g (1.8 mmol)
of 8, dissolved in anhyd ethyl ether, were added. The
mixture was left at room temperature for 16 h, then
5 mL of H2O and 2 mL of H2SO4 were added and the
mixture was extracted with ethyl ether. The aqueous
solution was made basic with NaOH and extracted with
ethyl ether. After drying with Na2SO4, the solvent was
removed under reduced pressure and the residue puri-
®ed by column cromatography using CHCl3:MeOH
(9:1) as eluting system. Title compound 9 (0.38 g, 75%
yield) was obtained as a thick oil. IR (neat) n 3500
1
n 3370 (NH), 2120 (N3), 1700 (CO) cm 1. H NMR
(CDCl3) d 0.76 and 0.78 (2d, J=7.2 Hz, 6H, 2CH3),
1.39 (s, 9H, 3CH3), 1.40±1.58 (m, 1H, CH), 1.71±1.89
(m, 4H, 2CH2), 2.62±2.75 (m, 2H, CH2), 2.84 (bs, 4H,
2CH2), 3.41±3.75 (m, 2H, CH2), 3.85 (s, 6H, 2OCH3),
6.78 (s, 3H, aromatics), 6.95±7.32 (m, 4H, aromatics)
ppm. Anal. (C29H43N5O4) C, H, N.
2-(3,4-Dimethoxyphenyl)-2-isopropyl-N5-[2-(o-azido-
phenethyl)]-1,5-pentyl-diamine (13). 5 mL of HCl 6 N
were added to a solution of 0.15 g (0.28 mmol) of 12 in
1 mL of ethyl acetate. The mixture was left for 12 h at
room temperature, then treated with a 10% solution of
NaOH, extracted with ethyl acetate and dried with
Na2SO4. Removal of the solvent gave 0.11 g of 13 as an
oil (92% yield). IR (neat) n 3300±3450 (NH and NH2),
2120 (N3) cm 1. 1H NMR (CDCl3) d 0.72 and 0.78 (2d,
J=6.8 Hz, 6H, 2CH3), 1.31±1.55 (m, H, CH), 1.74±1.95
(m, 6H, CH2, NH2 and CH2), 2.59±2.73 (m, 2H, CH2),
1
(OH), 3400 (NH2) cm 1. H NMR (CDCl3) d 0.74 and
0.76 (2d, J=5.6 Hz, 6H, 2CH3), 1.42±1.58 (m, 1H, CH),
1.78±1.98 (m, 4H, CH2CH2), 2.05 (bs, 2H, NH2), 3.03
and 3.15 (2d, Jgem=12.4 Hz, 2H, CH2NH2), 3.62 (t,
J=5.6 Hz, 2H, CH2O), 3.85 (s, 6H, 2OCH3), 6.81 (s,
3H, aromatics) ppm. Anal. (C16H27NO3) C, H, N.
5-(t-Butoxycarbonyl)amino-4-(3,4-dimethoxyphenyl)-4-
isopropyl-1-pentanol (10). To a solution of 0.38 g
(1.35 mmol) of 9 in 5 mL of THF, 0.3 mL (2.10 mmol)
of Et3N and 0.36 g (1.65 mmol) of di-tert-butyl dicar-
bonate were added. The mixture was left at room
temperature for 16 h then 20 mL of ethyl acetate were
added and the organic layer washed with water. After
drying with Na2SO4, the solvent was removed under
reduced pressure. Title compound 10 (0.42 g, 81%
yield) was obtained as a thick oil. IR (neat) n 3460
2.72±2.85 (m, 4H, 2CH2), 2.95 and 3.17 (2d, Jgem
=
12.4 Hz, 2H, CH2NH2), 3.85 (s, 6H, 2OCH3), 6.79 (s,
3H, aromatics), 7.02-7.36 (m, 4H, aromatics) ppm.
Anal. (C24H35N5O2) C, H, N.
N1,N5-Dibiotinyl-2-(3,4-dimethoxyphenyl)-2-isopropyl-N5-
[2-(o-azidophenethyl)]-1,5-pentyldiamine (4a, 4b). 0.11 g
(0.26 mmol) of 13 and 0.18 g (0.52 mmol) of succinimido
biotinate34 were dissolved in 0.5 mL of anhydrous
DMSO. After 12 h at room temperature CHCl3 was
added and the mixture was washed with water. After
drying with Na2SO4 the solvent was removed under
reduced pressure and the obtained solid was puri®ed
by column chromatography using CHCl3:abs. EtOH:
petroleum ether:NH4OH 30% (340:65:60:8) as eluting
system.
1
(OH), 3380 (NH), 1740 and 1700 (CO) cm 1. H NMR
(CDCl3) d 0.76 and 0.78 (2d, J=5.6 Hz, 6H, 2CH3),
1.50 (s, 9H, (CH3)3), 1.58±1.82 (m, 5H, 2CH2 and CH),
3.49±3.75 (m, 4H, CH2O and CH2N), 3.85 (s, 6H,
2OCH3), 6.80 (s, 3H, aromatics) ppm. Anal. (C21H35
NO5) C, H, N.
1-[5-(t-Butoxycarbonyl)amino-4-(3,4-dimethoxyphenyl)-
4-isopropylpentyl] methansulfonate (11). To a solution
of 0.42 g (1.10 mmol) of 10 and 0.18 mL (1.29 mmol)
of dry Et3N in 20 mL anhyd ethyl ether cooled to
The ®rst fraction (Rf 0.58) was 4a (50 mg, 22% yield).
Mp 170±172ꢂC. IR (nujol) n 3250 (NH), 2120 (N3),
1
1710 (CO), 1660 (CO) cm 1. H NMR (CDCl3) d 0.74

1878
E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
and 0.80 (2d, J=6.8 Hz, 6H, 2CH3), 1.29±1.91 (m, 17H,
8CH2 and CH), 2.08±2.47 (m, 4H, 2CH2), 2.70±3.22 (m,
10H, 4CH2+2CH), 3.28±3.55 (m, 4H, 2CH2), 3.87 (s,
3H, OCH3), 3.89 (s, 3H, OCH3), 4.25±4.35 (m, 2H,
2CH), 4.38±4.53 (m, 2H, 2CH), 6.79 (s, 3H, aromatics),
7.02±7.36 (m, 4H, aromatics) ppm. (M+H+) 878. Anal.
(C44H63N9O6S2) C, H, N.
(3,4-dimethoxyphenyl)acetonitrile (0.50 g, 2.82 mmol),
title compound 16 (0.47 g, 70% yield) was obtained as a
1
thick oil. IR (neat) n 3500 (OH), 2240 (CN) cm 1. H
NMR (CDCl3) d 1.64 (bs, 1H, OH), 1.66±1.82 (m, 2H,
CH2), 1.83±2.08 (m, 2H, CH2), 3.68 (t, J=6.8 Hz, 2H,
CH2), 3.80 (t, J=7.2 Hz, 1H, CH), 3.87 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 6.81±6.88 (m, 3H, aromatics) ppm.
Anal. (C13H17NO3) C, H, N.
The second fraction (Rf 0.47) was 4b (60 mg, 26% yield).
Mp 178±180ꢂC. IR (nujol) n 3250 (NH), 2120 (N3), 1710
2-(3,4-Dimethoxyphenyl)-2-(3-chloropropyl)-5-hydroxy-
valeronitrile (17). 0.5 g (2.12 mmol) of 16 were added to
a solution of 2.5 mL (4.25 mmol) of BuLi (1.6 M in
hexane) and 0.5 g (4.25 mmol) of potassium tert-but-
oxide in 10 mL of dry THF at 70ꢂC. After 1 h, 0.5 mL
(2.28 mmol) of 1-bromo-3-chloropropane were added
and the mixture was allowed to return to room tem-
perature. The mixture was then treated with NH4Cl and
extracted with ether. After drying with Na2SO4, the
solvent was removed under reduced pressure and the
residue puri®ed by ¯ash cromatography using ethyl-
acetate:cyclohexane (80:20) as eluting system. Title
compound 17 (0.29 g, 44% yield) was obtained as a
1
(CO) 1660 (CO) cm 1. H NMR (CDCl3) d 0.70 and
0.78 (2d, J=6.8 Hz, 6H, 2CH3), 1.28±1.89 (m, 17H,
8CH2 and CH), 2.05±2.39 (m, 4H, 2CH2), 2.61±3.18 (m,
10H, 4CH2+2CH), 3.21±3.53 (m, 4H, 2CH2), 3.85 (s,
3H, OCH3), 3.87 (s, 3H, OCH3), 4.19±4.35 (m, 2H,
2CH), 4.38±4.51 (m, 2H, 2CH), 6.79 (s, 3H, aromatics),
7.02±7.36 (m, 4H, aromatics) ppm. (M+H+) 878. Anal.
(C44H63N9 O6S2) C, H, N.
N1-t-Butoxycarbonyl-2-(3,4-dimethoxyphenyl)-2-isopropyl-
N5-[2-(o-azido-phenethyl)]-N5-methyl-1,5-pentyldiamine
(14). Following the procedure described for 12, starting
from 11 (0.32 g, 0.70 mmol) and 0.25 g (1.42 mmol) of
22, title compound 14 (0.17 g, 45% yield) was obtained
1
thick oil. IR (neat) n 3500 (OH), 2240 (CN) cm 1. H
NMR (CDCl3) d 1.28±1.80 (m, 4H, 2CH2), 1.85 (bs, 1H,
OH), 1.87±2.18 (m, 4H, 2CH2), 3.45 (t, J=5.6 Hz, 2H,
CH2Cl), 3.58 (t, J=5.6 Hz, 2H, CH2O), 3.87 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 6.78±6.97 (m, 3H, aro-
matics) ppm. Anal. (C16H22ClNO3) C, H, N.
1
as an oil. IR (neat) n 2120 (N3), 1700 (CO) cm 1. H
NMR (CDCl3) d 0.78 and 0.81 (2d, J=7.2 Hz, 6H,
2CH3), 1.40 (s, 9H, 3CH3), 1.40±1.58 (m, 2H, CH2),
1.71±1.89 (m, 3H, CH2 and CH), 2.31 (s, 3H, NCH3),
2.35±2.85 (m, 6H, 3CH2), 3.51±3.82 (m, 2H, CH2), 3.85
(s, 6H, 2OCH3), 6.78 (s, 3H, aromatics), 6.98±7.30 (m,
4H, aromatics) ppm. Anal. (C30H45N5O4) C, H, N.
2-(3,4-Dimethoxyphenyl)-2-[3-(1-hydroxy)propyl]-5-[2-
(o-azidophenethyl) methylamino]valeronitrile (18). Fol-
lowing the procedure described for 12, starting from 17
(0.29 g, 0.93 mmol) and 22 (0.17 g, 0.96 mmol), title
compound 18 (0.14 g, 33% yield) was obtained as a
2-(3,4-Dimethoxyphenyl)-2-isopropyl-N5-[2-(o-azido-
phenethyl)]-N5-methyl-1,5-pentyldiamine (15). Following
the procedure described for 13, starting from 14 (0.15 g,
0.28 mmol). Title compound 15 (0.12 g, 97% yield) was
thick oil. IR (neat) n 3500 (OH), 2240 (CN), 2120 (N3)
cm
1
.
1H NMR (CDCl3) d 1.21±1.48 (m, 2H, CH2),
obtained as a thick oil. IR (neat) n 3450 (NH2), 2120
(N3) cm
1.56±1.78 (m, 2H, CH2), 1.88±2.08 (m, 4H, 2CH2), 2.22
(s, 3H, NCH3), 2.35±2.58 (m, 4H, 2CH2), 2.63±2.76 (m,
2H, CH2), 3.58 (t, J=5.6 Hz, 2H, CH2O), 3.86 and 3.87
(2s, 6H, 2OCH3), 6.78±6.99 (m, 3H, aromatics), 7.00±7.28
(m, 4H, aromatics) ppm. Anal. (C25H33N5O3) C, H, N.
1
.
1H NMR (CDCl3) d 0.72 and 0.78 (2d,
J=6.8 Hz, 6H, 2CH3), 1.25±1.65 (m, 3H, CH and NH2),
1.74±1.98 (m, 4H, 2CH2), 2.29 (s, 3H, NCH3), 2.38±2.69
(m, 4H, 2CH2), 2.65±2.78 (m, 2H, CH2), 2.99 and 3.09
(2d, Jgem=12.4 Hz, 2H, CH2NH2), 3.85 (s, 6H,
2OCH3), 6.80 (s, 3H, aromatics), 6.98±7.28 (m, 4H,
aromatics) ppm. Anal. (C25H37N5O2) C, H, N.
2-(3,4-Dimethoxyphenyl)-2-[3-(1-biotin)propylester]-5-[2-
(o-azidophenethyl) methylamino]valeronitrile (6). Fol-
lowing the procedure described for 4, starting from 18
(0.07 g, 0.15 mmol), title compound 6 (0.06 g, 60% yield)
was obtained as a thick oil. IR (neat) n 3250 (NH), 2240
(CN), 2120 (N3), 1740 (CO), 1680 (CO) cm 1. 1H NMR
(CDCl3) d 1.21±1.51 (m, 4H, 2CH2), 1.53±1.78 (m, 6H,
3CH2), 1.81±2.08 (m, 4H, 2CH2), 2.22 (s, 3H, NCH3),
2.35±2.58 (m, 5H, 2CH2+CH), 2.63±2.76 (m, 4H,
2CH2), 2.84±2.96 (m, 1H, CH), 3.08±3.19 (m, 1H, CH),
3.87 (s, 6H, 2OCH3), 3.94±4.18 (m, 2H, CH2O), 4.21±
4.32 (m, 1H, CH), 4.45±4.51 (m, 1H, CH), 5.42 (bs, 1H,
NH), 5.82 (bs, 1H, NH), 6.78±6.99 (m, 3H, aromatics),
7.00±7.28 (m, 4H, aromatics) ppm. Anal. (C35H47N7
O5S) C, H, N.
N1-Biotinyl-2-(3,4-dimethoxyphenyl)-2-isopropyl-N5-[2-
(o-azidophenethyl)]-N5-methyl-1,5-pentyldiamine (5).
Following the procedure described for 4, starting from
15 (0.12 g, 0.27 mmol). Title compound 5 (0.08 g, 45%
yield) was obtained as a thick oil. IR (neat) n 3250
1
(NH), 2120 (N3), 1710 (CO) cm 1. H NMR (CDCl3) d
0.74 and 0.78 (2d, J=6.8 Hz, 6H, 2CH3), 1.28±1.89 (m,
11H, 5CH2 and CH), 2.06±2.18 (m, 2H, CH2), 2.30 (s,
3H, NCH3), 2.35±2.85 (m, 10H, 5CH2), 2.98±3.10 (m,
1H, CH), 3.85 (s, 6H, 2OCH3), 4.15±4.28 (m, 1H, CH),
4.39±4.48 (m, 1H, CH), 6.80 (s, 3H, aromatics), 7.02±
7.28 (m, 4H, aromatics) ppm. (M+H+) 666. Anal.
(C35H51N7O4S) C, H, N. The oily product was trans-
formed into the hydrochloride that was recrystallized
from abs ethanol and anhyd ether. Mp 125±127ꢂC.
2-(3,4-Dimethoxyphenyl)-2-[3-(1-hydroxy)propyl]-5-[2-
( p-azidophenethyl) methylamino]valeronitrile (19). Fol-
lowing the procedure described for 12, starting from
17 (0.33 g, 1.06 mmol) and 23 (0.20 g, 1.13 mmol), title
compound 19 (0.18 g, 37% yield) was obtained as a
2-(3,4-Dimethoxyphenyl)-5-hydroxyvaleronitrile (16).
Following the procedure described for 5, starting from

E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
1879
thick oil. IR (neat) n 3500 (OH), 2240 (CN), 2120 (N3)
cm
Biology
1
.
1H NMR (CDCl3) d 1.21±1.48 (m, 2H, CH2),
1.52±1.74 (m, 2H, CH2), 1.83±2.08 (m, 4H, 2CH2), 2.18
(s, 3H, NCH3), 2.35±2.48 (m, 2H, CH2), 2.43±2.55 (m,
2H, CH2), 2.60±2.72 (m, 2H, CH2), 3.58 (t, J=5.6 Hz,
2H, CH2O), 3.86 and 3.87 (2s, 6H, 2OCH3), 6.79±6.98
(m, 5H, aromatics), 7.08±7.17 (m, 2H, aromatics) ppm.
Anal. (C25H33N5O3) C, H, N.
Drugs and chemicals. Puri®ed pirarubicin was provided
by Laboratoire Roger Bellon (France). Concentrations
were determined by diluting stock solutions to approxi-
5
mately 10 M and using e480=11500 M 1 cm 1. Stock
solutions were prepared just before use. Unless other-
wise stated, bu€er solutions were HEPES bu€er con-
taining 5 mM HEPES, 132 mM NaCl, 3.5 mM KCl,
1 mM CaCl2, 5 mM glucose, at pH 7.25.
2-(3,4-Dimethoxyphenyl)-2-[3-(1-biotin)propylester]-5-[2-
( p-azidophenethyl) methylamino]valeronitrile (7). Fol-
lowing the procedure described for 4, starting from 19
(0.18 g, 0.40 mmol), title compound 7 (0.06 g, 22% yield)
was obtained as a thick oil. IR (neat) n 3250 (NH), 2240
(CN), 2120 (N3), 1740 (CO), 1680 (CO) cm 1. 1H NMR
(CDCl3) d 1.28±1.48 (m, 4H, 2CH2), 1.53±1.78 (m, 6H,
3CH2), 1.88±2.08 (m, 4H, 2CH2), 2.19 (s, 3H, NCH3),
2.22±2.38 (m, 5H, 2CH2+CH), 2.58±2.76 (m, 4H,
2CH2), 2.79±2.96 (m, 1H, CH), 3.08±3.19 (m, 1H, CH),
3.88 (s, 6H, 2OCH3), 3.94±4.18 (m, 2H, CH2O), 4.21±
4.32 (m, 1H, CH), 4.48±4.53 (m, 1H, CH), 5.47 (bs, 1H,
NH), 5.78 (bs, 1H, NH); 6.75±6.98 (m, 5H, aromatics),
7.03±7.17 (m, 2H, aromatics) ppm. Anal. (C35H47N7
O5S) C, H, N.
Cell lines and cultures. K562 is a human leukemia cell
line, established from a patient with a chronic myelo-
geneous leukemia in blast transformation.41 K562 cells
resistant to doxorubicin were obtained by continuous
exposure to increasing doxorubicin concentrations, and
were maintained in medium containing doxorubicin
(400 nM) until 1±4 weeks before experiments. This sub-
line expresses a unique membrane glycoprotein with a
molecular weight of 180,000 daltons.42 Doxorubicin-
sensitive and -resistant erythroleukemia K562 cells were
grown in suspension, in RPMI 1640 (Sigma) medium
supplemented with l-glutamine and 10% FCS at 37ꢂC
in a humidi®ed atmosphere of 95% air and 5% CO2.
Cultures, initiated at a density of 105 cells/mL grew
exponentially to 8±10Â105 cells/mL in 3 days. For the
spectro¯uorometric assays, in order to have cells in the
exponential growth phase, culture was initiated at
5Â105 cells/mL, and cells were used 24 h later, when
the culture had grown to about 8±10Â105 cells/mL.
Cell viability was assessed by trypan blue exclusion.
The cell number was determined by Coulter counter
analysis.
Tri¯uoroacetyl-2-(o-azidophenethyl)amide (20). Tri¯uoro-
acetic anhydride (0.5 mL, 3.8 mmol) was added to
a
solution of 2-(o-azidophenyl)ethylamine40 (0.4 g,
2.5 mmol) in anhyd ethyl ether. After 30 min the sol-
vent was removed and the crude material (0.65 mg) was
used without puri®cation for the following reaction. IR
1
(neat) n 3300 (NH), 2120 (N3), 1700 (CO) cm 1. H
NMR (CDCl3) d 2.86±2.92 (m, 2H, CH2), 3.56±3.63 (m,
2H, CH2), 6.58 (bs, 1H, NH), 7.12±7.41 (m, 4H, aro-
matics) ppm.
Cellular drug accumulation. The uptake of THP-adria-
mycin in cells was followed by monitoring the decrease
in the ¯uorescence signal at 590 nm (lex=480 nM) fol-
lowing the method previously described.43 Using this
method it is possible to accurately quantify the kinetics
of the drug uptake by the cells and the amount of
anthracycline intercalated between the base pairs in the
nucleus at the steady state, as incubation of the cells
with the drug proceeds without compromising cell via-
bility. All experiments were conducted in 1 cm quartz
cuvettes containing 2 mL of bu€er at 37ꢂC. We checked
that tested compounds did not a€ect the ¯uorescence of
THP-adriamycin.
Tri¯uoroacetyl-2-(p-azidophenethyl)amide (21). Following
the procedure described for 20, starting from 2-(p-azi-
dophenyl)ethylamine, title compound 21 was obtained.
1
IR (neat) n 3300 (NH), 2120 (N3), 1700 (CO) cm 1. H
NMR (CDCl3) d 2.83±2.90 (m, 2H, CH2), 3.56±3.63 (m,
2H, CH2), 6.55 (bs, 1H, NH), 7.00 (d, J=8.0 Hz, 2H,
aromatics), 7.17 (d, 2H, aromatics) ppm.
2-(o-Azidophenethyl)methylamine (22). A mixture of
methyl iodide (5.7 g, 0.04 mol), 20 (1.89 g, 0.01 mol), and
dry acetone (50 mL) was heated nearly to re¯ux and
powdered potassium hydroxide (2.24 g, 0.04 mol) was
then added. The reaction mixture was heated under
re¯ux for 5 min, excess of methyl iodide and acetone
was removed at reduced pressure, water (50 mL) was
added, and then the mixture was heated under re¯ux for
10 min to give N-methylamine 22. IR (neat) n 3300
(NH), 2120 (N3) cm 1. 1H NMR (CDCl3) d 2.45 (s, 3H,
CH3), 2.79 (s, 4H, 2CH2), 6.98±7.35 (m, 4H, aromatics)
ppm. Anal. (C9H12N4) C, H, N.
Photolabeling. Ultraviolet irradiation, in the presence of
the photolabeler at concentration of 10 mM, was carried
out using a 125-W lamp (HPW 125 W-TS, Philips) as a
light source. Cells, 106/mL in 2 mL, were placed in a
25 mL cultured ¯ask, and irradiation was carried out at
a distance of 7 cm from the cells for 20 min at 25ꢂC. The
plastic ¯ask allowed transmittance of UV light in the
300±400 nM range, and no additional ®lter was neces-
sary. The intensity, Iio, of the light beam incident just
within the ¯ask was measured using the potassium fer-
rioxalate system as actinometer.44 Ioi was equal to
2.5‹0.5Â1014 quanta/s. To eliminate the unbound
molecules, cells were washed twice in a 25 mL volume of
bu€er.
2-( p-Azidophenethyl)methylamine (23). Following the
procedure described for 22, starting from 21, title com-
pound 23 was obtained. IR (neat) n 3300 (NH), 2120
1
(N3) cm 1. H NMR (CDCl3) d 2.43 (s, 3H, CH3), 2.80
(s, 4H, 2CH2), 6.98 (d, 2H, aromatics), 7.18 (d, J=
8.0 Hz, 2H, aromatics) ppm. Anal. (C9H12N4) C, H, N.

1880
E. Teodori et al. / Bioorg. Med. Chem. 7 (1999) 1873±1880
25. Boer, R.; Dichtl, M.; Borchers, C.; Ulrich, W. R.; Mar-
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