Structure-activity relationships and optimisation of the selective MDR modulator 2-(3,4-dimethoxyphenyl)-5-(9-fluorenylamino)-2-(methylethyl) pentanenitrile and its N-methyl derivative

Article abstract of DOI:10.1016/S0968-0896(01)00191-2

Several ring-substituted derivatives of previously studied MDR inhibitors 2-(3,4-dimethoxyphenyl)-5-(9-fluorenylamino)-2-(methylethyl)pentanenitrile and 2-(3,4-dimethoxyphenyl)-5-[(9-fluorenyl)-N-methylamino]-2-(methylethyl) pentanenitrile have been synthesised and studied with the aim of optimising activity and selectivity. The results show that MDR inhibition is scarcely sensitive to modulation of the electronic properties of the fluorene ring. Even if dramatic improvement was not obtained, one of the compounds (2) showed improved potency and selectivity with respect to the leads and appears to be a better candidate for drug development.

Full text of DOI:10.1016/S0968-0896(01)00191-2

Bioorganic & Medicinal Chemistry9 (2001) 2673–2682
Structure–Activity Relationships and Optimisation of the Selective
MDR Modulator 2-(3,4-Dimethoxyphenyl)-5-(9-fluorenylamino)-
2-(methylethyl) Pentanenitrile and Its N-Methyl Derivative
Silvia Dei,a Elisabetta Teodori,a Arlette Garnier-Suillerot,b Fulvio Gualtieri,a,*
Serena Scapecchi,a Roberta Budriesic and Alberto Chiarinic
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Firenze, Via Gino Capponi 9, 50121 Firenze, Italy
Laboratoire de Physicochimie Biomoleculaire et Cellulaire, ESA CNRS 7033, Universite Paris Nord,
b
´
´
74 rue MarcelCachin, 93017 Bobigny, France
`
Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
c
Received 12 February2001; accepted 23 May2001
Abstract—Several ring-substituted derivatives of previously studied MDR inhibitors 2-(3,4-dimethoxyphenyl)-5-(9-fluorenyl-
amino)-2-(methylethyl)pentanenitrile and 2-(3,4-dimethoxyphenyl)-5-[(9-fluorenyl)-N-methylamino]-2-(methylethyl)pentanenitrile
have been synthesised and studied with the aim of optimising activity and selectivity. The results show that MDR inhibition is
scarcelysensitive to modulation of the electronic properties of the fluorene ring. Even if dramatic improvement was not obtained,
one of the compounds (2) showed improved potencyand selectivitywith respect to the leads and appears to be a better candidate
for drug development. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
extruded, the mechanism of action of these transporters
is still controversial. As a matter of fact, the dominant
‘drug pump’ model has been questioned and, at the
moment, the exact molecular mechanism for P-gp’s
The resistance of human malignancyto chemothera-
peutic agents remains a major obstacle to efficient can-
cer therapy. Multidrug resistance (MDR) is a kind of
resistance of cancer cells to multiple classes of che-
motherapic drugs that can be structurallyand mechan-
9,10
function is far from being clearlyunderstood.
Even if the mechanism of action of transporter proteins
such as P-gp is still obscure, it was found that some
drugs (first generation chemosensitizers) are able to
block their functions.11 This finding was soon con-
sidered a promising approach to overcome MDR in
cancer therapyand has paved the wayto the design and
synthesis of a variety of new molecules (second genera-
tion of chemosensitizers).12,13 Hopefully, these com-
pounds can maintain the MDR-reversal activitywhile
lacking the side effects associated with the first genera-
tion, which are often known drugs and used for other
purposes.
isticallyunrelated. Classic MDR2 is concerned with
1
altered membrane transport, which results in lower cell
concentrations of cytotoxic drug and is related to the
over expression of a varietyof proteins that act as ATP-
dependent extrusion pumps. P-glycoprotein (P-gp)3,4
and MRP15 are the most important and widelystudied
members of the familythat belong to the ABC super-
familyof transporters. It is important to notice that,
besides their role in cancer cell resistance, these proteins
seem to have multiple physiological functions as well,6,7
since theyare expressed also in manyimportant non
tumoral tissues and similar transporting proteins of the
ABC superfamilyare largelypresent in procariotic organ-
isms.8 Due to the unprecedented varietyof substrates
Recently, we reported the MDR-modulating activity of
compounds
2-(3,4-dimethoxyphenyl)-5-(9-fluorenyl-
amino)-2-(methylethyl)pentanenitrile (SC11, labelled as
5 in the reference) and 2-(3,4-dimethoxyphenyl)-5-[(9-
fluorenyl) - N - methylamino] - 2 - (methylethyl)pentane -
nitrile (SC17, labelled as 12 in the reference)14 (Chart 1)
*Corresponding author. Tel.: +39-55-275-7295; fax: +39-55-240776;
e-mail: fulvio.gualtieri@unifi.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00191-2

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S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
that showed interesting potencyand selectivi.ty
Compound SC11 is potent reverter of MDR
with the suitable fluorenone (Scheme 1). In fact, in pre-
vious studies, we observed that functionalization of
fluorene in position 9 could give instable derivatives
that turn rapidlyinto fluorenone. So the high stability
of 9-fluorenone and the commercial availabilityof many
substituted fluorenones prompted us to look for syn-
thetic pathways that involve these intermediates. 2-F-,
2-Br-, 2,7-dibromo-, 2-NO2–, 2-OH– and 2-N(CH3)2–9-
fluorenones are commerciallyavailable; 3,4-dimethoxy-
9-fluorenone was synthesised according to Ladd.15 2-
Methoxy-9-fluorenone 23 was obtained, as previously
a
([I]0.5=0.25 mM) but maintains some negative chrono-
tropic activity(EC 30=0.65 mM), compound SC17 is less
potent ([I]0.5=1.0 mM) but does not show anyresidual
cardiovascular action. [I]0.5 and EC30 are defined in the
pharmacological part. Since potencyand side effects are
two major issues in MDR-inhibiting activity, we have
begun a studyaimed at optimising the pharmacological
properties of our leads through modulation of the elec-
tronic properties of the fluorene nucleus. Therefore, we
have synthesised the series of ring substituted analogues
shown in Chart 1 and tested them as MDR modulators.
The most potent compounds were then tested for cardio-
vascular action to check their selectivity.
16
described byKruber, from the commerciallyavailable
hydroxy derivative, but O-alkylation was not performed
with dimethylsulfate, as described in the reference, but
rather with CH3I in KOH, according to a general pro-
17
cedure described byJohnstone.
Since this reaction is
verysimple and has an almost quantitative yield, we
used the same pathways to also obtain the 3- and 4-
methoxy-9-fluorenones 24 and 25, that had previously
been synthesized by more complicated routes.18,19 Thus,
starting from the commerciallyavailable 4-amino-9-
Chemistry
Secondaryamines were obtained byreaction of 5-amino-
2-(3,4-dimethoxyphenyl)-2-(methylethyl)pentanenitrile14
Chart 1.
Scheme 1. (a) Titanium (IV) isopropoxide; (b) NaBH3CN; (c) HCHO/HCOOH.

S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
2675
Scheme 2. HCl 12 N/H2O/NaNO2 at 100 ꢀC; (b) CH3I/DMSO/KOH.
Table 1. Chemical and physical characteristics and MDR modulating activity of compounds 1–11
Mp (ꢀC)c
sis
MDR activityAnaly
d
N
R
Yield (%)a
Eluentb
[I]0.5 (mM) (SEM)
amax
KI (mM) (SEM)
1
2
3
4
5
6
7
8
2-F
2-Br
2,7-Br
2-NO2
2-OH
2-N(CH3)2
2-OCH3
4-OCH3
3-OCH3
3,4-OCH3
2-NH2
H
48.2
63.8
36.9
37.0
60.5
43.8
54.8
50.9
51.1
66.2
54.8
A
B
A
B
203–207
179–181
51–53a
208–211
95–100
90–93e
78–80e
110–112e
121–123
86–90e
229–230
—
0.30 (0.06)
0.25 (0.05)
0.50 (0.1)
0.30 (0.06)
0.40 (0.08)
0.40 (0.08)
0.44 (0.09)
0.20 (0.04)
1.10 (0.2)
0.38 (0.07)
0.50 (0.1)
0.25 (0.05)
1.6 (0.3)
0.75
0.65
0.53
0.80
0.80
0.66
0.75
0.87
0.90
1
0.1 (0.02)
0.08 (0.01)
0.17 (0.4)
0.10 (0.01)
0.1 (0.02)
0.15 (0.02)
0.26 (0.05)
0.12 (0.03)
0.36 (0.05)
0.15 (0.04)
0.15 (0.02)
0.15 (0.04)
1.0 (0.1)
C
29H32ClFN2O2
C29H32BrClN2O2
C29H30Br2N2O2
C29H32ClN3O4
C29H33ClN3O4
C33H39N3O6
C32H36N2O7
C32H36N2O7
C30H35ClN2O3
C33H35N2O8
C29H34ClN3O2
—
C
A
A
C
C
C
—
—
—
9
10
11
SC11
VRP
0.90
0.80
0.80
—
—
—
aAs free base.
bA=CHCl3 99/MeOH 01; B=CH2Cl2 99/MeOH 01; C=CH2Cl2 98/MeOH 02.
cAs hydrochloride, unless otherwise stated. Recrystallization solvent: absolute ethanol+petroleum ether.
dAll compounds have been analyzed for C, H, N after vacuum drying at a temperature below the melting point; the results obtained range with-
1
inꢁ0.4% of the theoretical values. IR and H NMR spectra are consistent with the proposed structures.
eAs oxalate.
fluorenone, the corresponding 4-hydroxy derivative was
obtained20 that was then methylated with CH3I. In the
same waythe 3-methoxyintermediate was also synthe-
sised (Scheme 2).
In the case of 2-NH2 fluorenyl derivatives 11 and 22, it
was necessaryto protect the 2-NH function of the
2
commerciallyavailable fluorenone in advance. We per-
formed the protection both with di-t-butyldicarbonate
(BOC) and with dibenzyldicarbonate (CBZ) (Scheme 3).
The protected derivatives 26 and 27 were then reacted with
5-amino-2-(3,4-dimethoxyphenyl)-2-(methylethyl)penta-
nenitrile14 as previouslydescribed; from the resulting
BOC-protected amine 28 the secondaryamine 11 was
The secondaryamines 1–10 were obtained, according to
the Mattson procedure,21 byreductive alkylation of the
5-amino-2-(3,4-dimethoxyphenyl)-2-(methylethyl)penta-
nenitrile,14 with the substituted 9-fluorenone, using tita-
nium (IV) isopropoxide as Lewis acid catalist and
NaBH3CN as reducing agent (Scheme 1; yields are
reported in Table 1). This reaction always gave accep-
table to good yields. Other procedures such as the one-
pot formation and reduction with NaBH3CN of the
Schiff bases in presence of molecular sieves, or the tra-
ditional synthesis of the Schiff bases with a Dean–Stark
trap in presence of p-toluenesulfonic acid and sub-
sequent reduction with NaBH4, gave the worst results.
easilyobtained. Methlyation of
28, on the contrary,
gave poor results: in this case reductive methylation by
HCOOH/HCHO could not be used because of the
acidic conditions that deprotect and methylate the 2-
NH2 fluorenyl function. On the other hand, use of
HCHO and sodium cyanoborohydride gave 30, that
after deprotection by acidic hydrolysis gave 22, onlyin
a very small yield (19%). Then for the synthesis of the
N-methyl derivative 22 we performed the same reaction
pathwayon the CBZ-protected derivatives. Compound
29 was easilymethylated with HCOOH/HCHO, and the
resulting product was deprotected by acidic hydrolysis
to give 22 in good yields.
N-Methyl derivatives 12–21 were obtained from the cor-
responding secondaryamines 1–10 byEschweiler–Clarke
methylation (Scheme 1; yields are reported in Table 2).

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S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
Table 2. Chemical and physical characteristics and MDR activity of compounds 12–22
Mp (ꢀC)c
sis
MDR activityAnaly
d
N
R
Yield (%)a
Eluentb
[I]0.5 (mM) (SEM)
amax
KI (mM) (SEM)
12
13
14
15
16
17
18
19
20
21
22
SC17
VRP
2-F
2-Br
2,7-Br
2-NO2
2-OH
2-N(CH3)2
2-OCH3
4-OCH3
3-OCH3
3,4-OCH3
2-NH2
—
67.3
82.4
65.2
79.0
75.5
61.4
64.6
70.1
86.3
78.7
80.7
A
B
C
A
D
E
243–246
199–200
212–215
149–153
104–106
118–120
58–60e
62–64
57–60
75–78
100–103
—
—
1.2 (0.2)
2.0 (0.4)
0.5 (0.1)
—
3.8 (0.8)
0.60 (0.12)
—
1.2 (0.3)
0.7 (0.14)
0.84 (0.17)
1.0 (0.2)
1.0 (0.2)
1.6 (0.3)
0.80
0.70
0.57
0.40
0.75
0.81
0.41
0.83
0.97
0.86
0.70
0.80
0.80
0.20 (0.03)
0.60 (0.1)
0.46 (0.11)
0.20 (0.05)
1.6 (0.2)
0.50 (0.07)
0.10 (0.03)
0.80 (0.07)
0.28 (0.04)
0.33 (0.07)
0.40 (0.05)
0.60 (0.2)
1.0 (0.1)
C30H34ClFN2O2
C30H34BrClN2O2
C30H33Br2ClN2O2
C30H34ClN3O4
C30H35ClN2O3
C32H40ClN3O2
C33H38N2O7
C31H37ClN2O3
C31H37ClN2O3
C32H39ClN2O4
C30H36ClN3O2
—
F
E
G
E
—
—
—
—
—
aAs free base.
bA=CHCl3 99/MeOH 01, B=CH2Cl2 100, C=hexane 50/Et2O 50, D=CH2Cl2 97/MeOH 03, E=CH2Cl2 98/MeOH 02, F=CH2Cl2 99/MeOH 01,
G=CH2Cl2 95/ethyl acetate 05.
cAs hydrochloride, unless otherwise stated. Recrystallization solvent: absolute ethanol+petroleum ether.
dAll compounds have been analyzed for C, H, N after vacuum drying at a temperature below the melting point; the results obtained range with-
1
inꢁ0.4% of the theoretical values. IR and H NMR spectra are consistent with the proposed structures.
eAs oxalate.
The compounds of this series possess two chiral centers
and therefore are mixtures of racemic diastereoisomers.
However, at this stage of the research, considering also
nuclear concentration of pirarubicin in resistant cells
with a given inhibitor; (iii) [I]0.5 which measures the
potencyof MDR-reverting agent and represents the
concentration of the inhibitor that causes a half-max-
imal increase in nuclear concentration of pirarubicin at
a=0.5; (iv) KI which is the dissociation constant for the
complex formed between P-gp and the inhibitor.
the modest impact of stereochemistryin the MDR
22ꢂ24
activityof verapamil derivatives,
we decided to
neglect this problem, postponing anydecision to the
biological evaluation of the compounds.
In more detail, the inhibition produced maybe expres-
sed bycalculating the fractional activity:
Pharmacology
r ¼ Via=Va
MDR-reverting activity
The abilityof the examined compounds to revert
MDR was evaluated on anthracycline-resistant erythrol-
eukemia K562 cells measuring the uptake of THP-
adriamycin (pirarubicin) by continuous spectrofluoro-
metric monitoring of the decrease of the anthracycline
fluorescence at 590 nm (lex=480 nm) after incubation
with cells, following the protocols reported in pre-
vious papers.14,25 More details are reported in the
Experimental.
where Va and Via stand for the rate of active efflux of the
chemotherapic drug in the absence and presence of
inhibitor, respectively. At low substrate concentrations,
Va can be written as ka.Ci and Via as kai Ci, therefore
fractional activityas
r ¼ kia=ka
Ci is the intracellular free drug concentration and ka and
kia the active efflux coefficient in the absence and pre-
sence of inhibitor, respectively.26The P-gp-mediated
efflux of anthracyclines by verapamil or by its deriva-
tives is non-competitive.25,27 For non-competitive inhi-
bition r can be simplywritten as a function of the
inhibitoryconstant KI and the inhibitor concentration
[I].28
MDR-modulatoryactivityis described by(i)
a, which
represents the ratio of intracellular concentrations in
MDR cells versus drug-sensitive cells of pirarubicin in the
presence of the MDR-reverting agent and varies between 0
(in the absence of the inhibitor) and 1 (when the amount of
pirarubicin in resistant cells is the same as in sensitive cells);
(ii) amax, which expresses the efficacyof MDR-modulator
and is the maximum increase that can be obtained in the
r ¼ KI=ð½IŠ þ KIÞ

S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
2677
Cardiovascular activity
(vasodilator activity) at the concentrations indicated in
the footnotes of Table 3.
Inotropic and chronotropic activities were tested on
guinea pig isolated atria preparations, and vasodilator
activitywas tested on guinea pig aortic strip prepara-
tions following standard procedures, details of which
Results and Discussion
29
have previouslybeen reported.
The MDR-reverting activityof the compounds synthe-
sised is reported in Tables 1 and 2. In Table 3 is repor-
ted the cardiovascular action of the compounds that
have shown the most promising MDR-reverting activ-
ity. In all cases the corresponding values for verapamil
(VRP), SC11 and SC17 were reported as reference.
Potencyof the drugs is defined as EC
(concentration
50
that gives 50% of the maximum negative inotropic
effect), IC50 (concentration that gives 50% of maximum
vasodilator effect), and EC30 (concentration that gives
30% of the maximum negative chronotropic effect).
Activityis defined as the percent decrease in developed
tension on isolated left atrium (negative inotropic
activity), percent decrease in atrial rate of sponta-
neouslybeating isolated right atrium (negative chrono-
tropic activity), and percent inhibition of calcium-
induced contraction on K+-depolarized aortic strips
As discussed in the Introduction, the preceding research
evidenced two leads for further development.
The desmethyl derivative SC11 had a good potency
but also possessed some negative chronotropic activity
that was only10 times lower than that of verapamil.
Scheme 3. (a) Di-tert-butyl dicarbonate, Et3N, dioxane, 100 ꢀC; (b) dibenzyl dicarbonate, NaOH 1 N, dioxane, 65 ꢀC; (c) titanium isopropoxide,
NaBH3CN, 5-amino-2-(3,4-dimethoxyphenyl)-2-(methylethyl)pentanenitrile; (d) CH2O, HCOOH; (e) ethyl acetate, HCl 3 N; (f) CH2O, NaBH3CN;
(g) HBr/AcOH 33%.

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S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
Table 3. Cardiovascular activityof selected compounds
Cardiovascular activity
Negative inotropyNegative chronotropVyasorelaxant activity
N
Ia%a
(ꢁSEM)
EC50 (mM)b
(95% cl)
Ia%c
(ꢁSEM)
EC30 (mM)b
(95% cl)
Ia%d
(ꢁSEM)
IC50 (mM)b
(95% cl)
2
6
8
10
17
SC11
SC17
VRP
41 (2.3)
68 (0.6)
38 (2.2)
80 (3.5)
55 (2.1)
33 (1.2)
36 (2.6)
84f (2.1)
—
4.48 (3.65–5.38)
—
1.75 (1.35–2.28)
2.40 (1.95–2.90)
—
43 (1.8)
85 (2.5)
61 (4.1)
78e (3.2)
38 (2.1)
74 (4.5)
26 (1.4)
94g (3.4)
—
18 (0.7)
30 (1.5)
15 (0.4)
43 (3.8)
5 (0.2)
46 (2.1)
15 (0.9)
95f (1.7)
—
—
—
—
—
—
—
0.97 (0.75–1.26)
1.56 (1.20–2.03)
0.63 (0.48–0.82)
0.65
0.65 (0.57–0.72)
—
0.07 (0.05–0.10)
—
0.61 (0.40–0.80)
0.38 (0.20–0.70)
aDecrease in developed tension in isolated guinea pig left atrium at 5ꢃ10ꢂ5 M, expressed as percent changes from the control (n=5–6). The left atria
were driven at 1 Hz. 5ꢃ10ꢂ5 M gave the maximum effect for most compounds.
bCalculated from log concentration–response curves (Probit analysis by Litchfield and Wilcoxon with n=5–7). When the maximum effect was
<50%, the EC50 ino., EC30 chrono., IC50 values were not calculated.
cDecrease in atrial rate on guinea pig spontaneouslybeating isolated right atrium at 5 ꢃ10ꢂ5 M, expressed as percent changes from the control (n=7–
8). Pretreatement heart rate ranged from 165 to 190 beats/min. 5ꢃ10ꢂ5 M gave the maximum effect for most compounds.
dPercent inhibition of calcium-induced contraction on K+-depolarized guinea pig aortic strip at 5ꢃ10ꢂ5 M (n=5–6). 5ꢃ10ꢂ5 M gave the maximum
effect for most compounds.
eAt 5ꢃ10ꢂ6 M.
fAt 10ꢂ5 M.
gAt 10ꢂ6 M.
Modulation of the electronic properties of the fluorene
moietythrough ring substitution afforded compounds
that, in general, show a potencyin the same range of
that of the lead without impressive improvement (Table
1). The most interesting among them are 2, 6 and 8,
which are almost equivalent to the lead in terms of
potency, efficacy and affinity. On the other hand, as
shown in Table 3, while 6 and 8 maintain the undesired
cardiovascular action of SC11, compound 2 is appar-
entlydevoid of negative chronotropic and inotropic
action and of vasorelaxant activityas well. Therefore, 2
mayrepresent a new lead toward the discoveryof
potent and selective MDR inhibitors. Finally, com-
pound 10, which is slightlyless potent but is able to
completelyrestore the original sensitivityof the cell line
to the chemotherapic (amax=1) is still endowed with
definite cardiovascular action.
between the nature of the substituent and MDR inhibi-
tion. Nevertheless, the discoveryof compound 2 is a
partial success of this research. In fact, even if this com-
pound is less potent than the most interesting product
14
(MM36) described byus previousl,y
it shows good
potency([I] 0.5=0.25 mM) and affinity( KI=0.08 mM) for
the Pgp and is completelydevoid of cardiovascular
action, whereas MM36 maintains some cardiovascular
activity.
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 Nujol mull for
solids and neat for liquids. NMR spectra were recorded
on a Gemini 200 spectrometer. Chromatographic
separations were performed on a silica gel column by
gravitychromatography(Kieselgel 40, 0.063–0.200 mm;
Merck) or flash chromatography(Kieselgel 40, 0.040–
0.063 mm; Merck). Yields are given after purification,
unless otherwise stated. Where analyses are indicated by
symbols, the analytical results are within ꢁ0.4% of the
theoretical values.
Compound SC17 was less potent than SC11, but pre-
sented a fairlygood selectivityas it was devoid of car-
diovascular activity. Therefore, in this case the issue
was that of improving potencywhile maintaining
selectivity. Unfortunately, this goal was not reached,
as ring substitution produced onlya slight improve-
ment in potencyin the series (Table 2). This confirms
our previous finding that N-methyl derivatives have
14,30
reduced MDR-inhibitoryactivit.y
Moreover, the
results concerning cardiovascular activitywere also
disappointing in this series, as one of the most potent
compounds, 17, was endowed with a slight negative
inotropic activity(Table 3) thus less selective than the
lead.
General procedure for the synthesis of fluorene derivatives
(1–10)
A
mixture of 5-amino-2-(3,4-dimethoxyphenyl)-2-
(methylethyl)pentanenitrile14 (300 mg, 1.09 mmol), the
suitable fluorenone (1.09 mmol) and titanium (IV) iso-
propoxide (0.45 mL, 1.36 mmol) was stirred with a dry-
ing tube at room temperature for 1–4 h, depending on
the starting compound. After the suitable time, the IR
spectrum of the mixture showed no ketone band, and
the viscous solution was diluted with abs ethanol
In general, it is apparent that modulation of the elec-
tronic properties of the aromatic moietythrough ring
substitution does not produce anysubstantial variation
in both MDR and cardiovascular activities. Accord-
ingly, we could not evidence any significant correlation

S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
2679
(2 mL). Sodium cyanoborohydride (60 mg, 0.74 mmol)
was added, and the solution was stirred for 20 h. Water
(0.5 mL) was then added, the resulting inorganic pre-
cipitate was filtered and washed with ethanol, and the
filtrate was concentrated in vacuo. The crude product
was dissolved in chloroform, filtered to remove the
solids, washed with a solution of NaHCO3 and water,
dried over Na2SO4 and concentrated in vacuo. The
crude substance was then purified bycolumn chroma-
tographyand transformed into the corresponding
hydrochloride or oxalate. The chemical and physical
characteristics of the compounds 1–10 are reported in
resulting suspension was stirred for 5 min, then 200 mg
(1.02 mmol) of 2-hydroxy-9-fluorenone and 0.13 mL
(2.04 mmol) of iodomethane were added. The mixture
was stirred at rt for 15 min, then was diluted with 20 mL
of water and treated with CH2Cl2 in order to extract the
title compound. The organic layer was washed with
water, dried over Na2SO4 and concentrated in vacuo.
Title compound was obtained in an almost quantitative
yield (230 mg). Mp 77–78 ꢀC (ethanol). IR (neat) n
1720 cmꢂ1 (C¼O). 1H NMR (CDCl3) d 7.55 (d,
J=7.33 Hz, 1H, aromatics); 7.39–7.30 (m, 3H, aro-
matics); 7.19–7.12 (m, 2H, aromatics); 6.94–6.87 (dd,
J=8.25 Hz, J=2.38 Hz, 1H, aromatics); 3.81 (s, 3H,
OCH3).
1
Table 1. Their IR and H NMR spectra are consistent
with the proposed structures.
The spectra of 2-(3,4-dimethoxyphenyl)-5-[9-(2-fluoro)-
fluorenylamino]-2-(methylethyl)pentanenitrile (1) are
reported as an example. IR (neat) n 3370 cmꢂ1 (NH),
2240 cmꢂ1 (CN). 1H NMR (CDCl3) d 7.64–7.50 (m, 3H,
fluorene); 7.40–7.22 (m, 3H, fluorene); 7.10–7.06 (m,
1H, fluorene); 6.87–6.81 (m, 3H, phenyl); 4.83 (s, 1H, 9-
H fluorene); 3.89 (s) and 3.88 (s) (3H, OCH3); 3.87 (s)
and 3.86 (s) (3H, OCH3); 2.34–2.26 (m, 2H, CH2–N);
2.18–2.01 (m, 2H, CH2–C–Ph); 1.79 (bs, 1H, NH); 1.82–
1.72 (m, 1H, C–CHH–C); 1.58–1.38 (m, 1H, C–CHH–
C); 1.18–1.01 (m, 1H, CH3–CH); 1.17 (d) and 1.16 (d)
(J=6.60 Hz, 3H, CH3–CH); 0.78 (d, J=6.60 Hz, 3H,
CH3–CH).
Following the same procedure, 3-methoxy-9-fluorenone
(24) and 4-methoxy-9-fluorenone (25) were obtained,
starting from the corresponding hydroxy derivatives.20
Their IR and H NMR spectra are consistent with the
proposed structure.
1
2 - (N - ter - Butoxycarbonyl) - amino - 9 - fluorenone (26).
0.54 mL (3.84 mmol) of triethylamine and 570 mg
(2.61 mmol) of di-ter-butyldicarbonate (BOC) were
added to a solution of 500 mg (2.56 mmol) of 2-amino-
9-fluorenone in 8 mL of 1,4-dioxane. Reaction was
maintained for 48 h at 100 ꢀC. In anycase (TLC) the
starting fluorenone did not disappear. The solvent was
then removed under reduced pressure and the residue
dissolved in ethyl acetate, and washed with water. After
drying with Na2SO4 the organic layer was concentrated
in vacuo and the residue purified bycolumn chromato-
2-(3,4-Dimethoxyphenyl)-5-[[9-(2,7-dibromo)fluorenyl]-N
- methylamino]-2-(methylethyl)pentanenitrile (14). 90 mg
(0.15 mmol) of 3 were dissolved in 3 mL of abs etha-
nol, and to this solution 3 mL of HCOOH and 3 mL
of HCHO were added. The mixture was refluxed at
80 ꢀC for 2 h and concentrated in vacuo. The residue
was then dissolved in CH2Cl2 and the organic layer
was washed with a saturated solution of Na2CO3 and
with water, dried over Na2SO4 and concentrated in
vacuo. The crude product was then purified bycol-
umn chromatographyusing hexane/ether (50:50) as
eluting system. Compound 14 was obtained as an oil
graphyusing CHCl 100 as the eluent. Title compound
3
was obtained with 41% yield (310 mg). IR (nujol) n
3300 cmꢂ1 (NH); 1735 cmꢂ1 (C¼O); 1700 cmꢂ1
(C¼O). 1H NMR (CDCl3) d 7.64–7.57 (m, 3H, aro-
matics); 7.47–7.41 (m, 3H, aromatics); 7.26–7.21 (m,
1H, aromatics); 1.54 (s, 9H, CH3). Anal. (C18H17NO3)
C, H, N.
2-(N-Benzyloxycarbonyl)-amino-9-fluorenone (27).
A
1
(60 mg, 65.2% yield). IR (neat) n 2240 cmꢂ1 (CN). H
solution of 740 mg (2.58 mmol) of dibenzyldicarbonate
(CBZ) in 2.6 mL of 1,4-dioxane was dropwise added to
a solution of 500 mg (2.56 mmol) of 2-amino-9-fluor-
enone in 5.2 mL of 1,4-dioxane/NaOH 1 N (1:1). The
mixture was kept 36 h at 65 ꢀC. Check of the reaction
(TLC) showed that the starting fluorenone did not
disappear. The solvent was then removed by
reduced pressure, and the resultant residue was dis-
solved in H2SO4 1 N at pH 2 and extracted with
ethyl acetate. The organic layer, after washing with
water and drying with Na2SO4, was concentrated in
vacuo giving a residue that was purified byflash
chromatography (cyclohexane/ethyl acetate 9:1).
510 mg of the title compound were obtained (62.8%
yield). IR (nujol) n 3360 cmꢂ1 (NH); 1710 cmꢂ1 (C¼O);
NMR (CDCl3) d 7.68–7.66 (m, 2H, fluorene); 7.50–
7.47 (m, 4H, fluorene); 6.96–6.82 (m, 3H, phenyl);
4.74 (s, 1H, 9-H fluorene); 3.88 (s, 3H, OCH3); 3.87
(s, 3H, OCH3); 2.46–2.40 (m, 2H, CH2–N); 2.16 (s,
3H, CH3–N); 2.15–2.02 (m, 2H, CH2–C–Ph); 1.94–
1.82 (m, 1H, C—CHH–C); 1.61–1.42 (m, 1H, C–
CHH–C); 1.23 (d, J=6.60 Hz, 3H, CH3–CH); 1.21–
1.11 (m, 1H, CH3–CH); 0.81 (d, J=6.60 Hz, 3H,
CH3–CH).
The oilyproduct was transformed into the hydrochlo-
ride that recrystallized from abs ethanol/anhydrous
ether. Mp 212–215 ꢀC. Anal. (C30H33Br2ClN2O2) C, H, N.
1
Compounds 12, 13 and 15–21 were obtained in the same
way; their chemical and physical characteristics are
1610 cmꢂ1 (C¼O). H NMR (CDCl3) d 7.69–7.27 (m,
11H, aromatics); 7.81–7.66 (m, 1H, aromatic); 2.19 (s,
2H, CH2); 1.58 (bs, 1H, NH). Anal. (C21H15NO3) C, H, N.
1
reported in Table 2 and their IR and H NMR spectra
are consistent with the proposed structures.
2-(3,4-Dimethoxyphenyl)-5-[[9-[2-(N-ter-butoxycarbonyl)-
amino] - fluorenyl] - amino] - 2 - (methylethyl)pentanenitrile
(28). Following the general procedure reported for
2-Methoxy-9-fluorenone (23). 230 mg (4.08 mmol) of
grounded KOH were added to 2 mL of DMSO. The

2680
S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
compounds 1–10, the title compound was synthesized,
using 270 mg (0.98 mmol) of 5-amino-2-(3,4-dimethoxy-
phenyl)-2-(methylethyl)pentanenitrile,14 290 mg (0.98
mmol) of 26, 0.405 mL (1.23 mmol) of titanium (IV)
isopropoxide and 50 mg (0.79 mmol) of sodium cyano-
borohydride. The crude product was purified by column
chromatographyusing CH 2Cl2/MeOH 98:2 as the
eluent. Title compound was obtained as an oil (360 mg,
nenitrile (30). 150 mg of 28 (0.27 mmol) in a mixture of
H2O (1 mL) and AcOH (0.1 mL) were treated with 37%
HCHO (0.2 mL) and NaBH3CN (60 mg, 0.74 mmol).
The stirred solution was treated with additional AcOH
as necessaryto maintain the acidityat approximately
pH 5. The mixture was kept at 80 ꢀC for 2 h, then HCl
2N (0.13 mL) was added, and the mixture was extracted
with EtOAc. The aqueous phase was made basic with
K2CO3 and extracted with chloroform. The organic
layer was dried over Na2SO4 and concentrated in vacuo,
and the residue was chromatographed (CHCl3/hexane/
MeOH 59:40:1) to afford 30 mg of an oil (yield 19.5%).
1H NMR (CDCl3) d 7.63–7.21 (m, 7H, aromatics,
fluorene); 6.97–6.80 (m, 3H, aromatics, phenyl); 4.74 (s,
1H, 9-H fluorene); 3.87 (s, 6H, OCH3); 2.46–1.61 (m,
9H, Ph–C–CH2–CH2–CH2–N and N–CH3); 1.54 (s,
9H, CH3); 1.27–1.16 (m, 1H, CH–CH3); 1.23 (d) and
1.20 (d) (3H, J=6.60 Hz, CH–CH3); 0.80 (d,
J=6.60 Hz, 3H, CH–CH3). Anal. (C35H43N3O4) C, H,
N.
1
66.4% yield). H NMR (CDCl3) d 7.67–7.21 (m, 7H,
aromatics, fluorene); 6.90–6.72 (m, 3H, aromatics,
phenyl); 4.82 (s, 1H, 9-H fluorene); 3.87 (s, 3H, OCH3);
3.86 (s, 3H, OCH3); 2.31–1.67 (m, 6H, Ph–C–CH2–
CH2–CH2–N); 1.58 (bs, 1H, NH); 1.54 (s, 9H, CH3);
1.15 (d, J=6.60 Hz, 3H, CH–CH3); 1.16–1.03 (m, 1H,
CH–CH3); 0.77 (d, J=6.60 Hz, 3H, CH–CH3). Anal.
(C34H41N3O4) C, H, N.
2-(3,4-Dimethoxyphenyl)-5-[[9-(2-amino)-fluorenyl]-amino]-
2-(methylethyl)pentanenitrile (11). To a solution of
200 mg (0.361 mmol) of 28 in 2 mL of ethyl acetate,
1.55 mL of HCl 3N were added under vigorous stirring.
After 4 days at rt the solution was basified with NaOH
33% and extracted with CHCl3. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The
resulting crude product was purified bycolumn chro-
2-(3,4-Dimethoxyphenyl)-5-[[9-[2-(N-benzyloxycarbonyl)-
N-methylamino]-fluorenyl]-amino]-2-(methylethyl)penta-
nenitrile (31). Following the same procedure as descri-
bed for 14, and using 450 mg (0.78 mmol) of 29, 12 mL
of abs EtOH, 12 mL of HCOOH and 12 mL of HCHO,
680 mg of crude product were obtained, and purified by
matographyusing CH Cl2/MeOH 98:2 as eluting sys-
2
tem. 90 mg of the title compound were obtained (oil,
54.8% yield). IR (neat) n 3470 cmꢂ1, 3395 cmꢂ1
,
column chromatographyusing CH Cl2/MeOH 99:1 as
2
3230 cmꢂ1 (NH); 2260 cmꢂ1 (CN). H NMR (CDCl3) d
7.55–7.14 (m, 7H, aromatics, fluorene); 6.90–6.65 (m,
3H, aromatics, phenyl); 4.77 (s, 1H, 9-H fluorene); 3.88
(s) and 3.87 (s) (3H, OCH3); 3.86 (s), 3.85 (s) (3H,
OCH3); 2.38–1.86 (m, 6H, C–CH2–CH2–CH2–N and
NH2); 1.81–1.66 (m, 1H, C–CHH–C), 1.58–1.40 (m, 1H,
C–CHH–C); 1.15 (d, J=6.60 Hz, 3H, CH–CH3); 1.16–
1.01 (m, 2H, CH–CH3 and NH); 0.78 (d, J=6.60 Hz,
3H, CH–CH3).
the eluent. Title compound was obtained as a yellow oil
1
1
(270 mg). H NMR (CDCl3) d 7.77–7.20 (m, 12H, aro-
matics, fluorene and CBZ); 7.00–6.80 (m, 3H, aro-
matics, phenyl); 5.22 (s, 2H, CH2 CBZ); 4.80 (s, 1H, 9-H
fluorene); 3.86 (s, 3H, OCH3); 3.84 (s, 3H, OCH3); 2.49–
2.40 (m, 2H, CH2–N); 2.19 (s) and 2.15 (s) (3H, N–
CH3); 2.13–1.88 (m, 4H), 1.63–1.42 (m, 1H) (Ph–C–
CH2–CH2 and NH); 1.24–1.12 (m, 1H, CH–CH3); 1.22
(d) and 1.19 (d) (3H, J=6.60 Hz, CH–CH3); 0.82 (d,
J=6.60 Hz, 3H, CH-CH3). Anal. (C38H41N3O4) C, H,
N.
The oilycompound was transformed into the corre-
sponding hydrochloride. Mp 229–230 ꢀC. Anal.
(C29H34ClN3O2) C, H, N.
2-(3,4-Dimethoxyphenyl)-5-[[9-(2-amino)-fluorenyl]-N-me-
thylamino]-2-(methylethyl) pentanenitrile (22).
2-(3,4-Dimethoxyphenyl)-5-[[9-[2-(N-benzyloxycarbonyl)
-amino]-fluorenyl]-amino]-2-(methylethyl)pentanenitrile
(29). Following the general procedure reported for
compounds 1–10, and using 300 mg (1.09 mmol) of
5-amino-2-(3,4-dimethoxyphenyl)-2-(methylethyl)penta-
nenitrile,14 350 mg (1.10 mmol) of 27, 0.45 mL
(1.36 mmol) of titanium (IV) isopropoxide, and 50 mg
(0.79 mmol) of sodium cyanoborohydride, 450 mg of
title compound were obtained, without further purifica-
tion. 1H NMR (CDCl3) d 7.62–7.18 (m, 12H, aromatics,
fluorene and CBZ); 6.94–6.78 (m, 3H, aromatics,
phenyl); 5.22 (s, 2H, CH2 CBZ); 4.80 (s, 1H, 9-H fluor-
ene); 3.87 (s, 3H, OCH3); 3.86 (s, 3H, OCH3); 2.40–1.98
(m, 6H, Ph–C–CH2–CH2–CH2–NH and NH); 1.83–1.68
(m, 1H, C–CHH–C), 1.56–1.32 (m, 1H, C–CHH–C);
1.18 (d, J=6.60 Hz, 3H, CH–CH3); 1.16–1.01 (m, 1H,
CH–CH3); 0.78 (d, J=6.60 Hz, 3H, CH–CH3). Anal.
(C37H39N3O4) C, H, N.
Procedure A. Following the same procedure as
described for 11 and using 30 mg (0.053 mmol) of 30
dissolved in 1 mL of ethyl acetate and 0.46 mL of HCl
3 N, 20 mg of a pure product were obtained (oil, 80.7%
yield).
Procedure B. To 250 mg (0.085 mmol) of 31 dissolved
in 0.5 mL of anhyd CH2Cl2, 0.24 mL of HBr in 33%
acetic acid were added, maintaining the mixture in ice-
bath and with a drying tube. After 3 h, the solvent was
removed byreduced pressure and the residue dissolved
in CH2Cl2. The organic layer was washed with 10%
NaOH and with water, dried over Na2SO4 and con-
centrated in vacuo. 150 mg of a pure product were
1
obtained (oil, yield 68%). H NMR (CDCl3) d 7.55–
7.16 (m, 7H, aromatics, fluorene); 6.95–6.67 (m, 3H,
aromatics, phenyl); 4.79 (s) and 4.71 (s) (1H, 9-H fluor-
ene); 3.88 (s, 3H, OCH3); 3.87 (s, 3H, OCH3); 2.51–2.45
(m, 2H, CH2–N); 2.18 (s), 2.17 (s) (3H, N–CH3); 2.18–
2-(3,4-Dimethoxyphenyl)-5-[[9-[2-(N-ter-butoxycarbonyl)
-amino]-fluorenyl]-N-methylamino]-2-(methylethyl)penta-

S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
2681
1.82 (m, 3H), 1.61–1.48 (m, 1H) (Ph–C–CH2–CH2);
1.30–1.19 (m, 1H, CH–CH3); 1.27 (bs, 2H, NH2); 1.22
(d, J=6.60 Hz, 3H, CH–CH3); 0.81 (d, J=6.60 Hz, 3H,
CH–CH3).
function of time. After about 20 min, the curve F=f(t)
reached a plateau and the fluorescence intensitywas
equal to Fn. The drug-cells system was thus in a steady
state and the overall concentration Cn of drug inter-
calated between the base pairs in the nucleus was
Cn=CT. (F0ꢂFn)/F0. Once the steadystate was reached,
the inhibitor at concentration [i] ([i] was varied from
0.05 to 10 mM), was added and a new steadystate was
reached, the fluorescence intensitybeing Fin. The overall
concentration Cin of drug intercalated between the base
pairs in the nucleus was then Cin=CT. (F0ꢂFni )/F0. An
aliquot of the solution was then taken awayand cell
viabilitywas assessed bytrpyan blue exclusion. Cell
membranes were then permeabilized bythe addition of
0.05% Triton X-100 yielding the equilibrium state
which was characterised bya new value FN of the fluo-
rescence intensity. The overall concentration CN of drug
intercalated between the base pairs in the nucleus was
then CN=CT. (F0ꢂFN)/F0.We checked that tested com-
pounds did not affect the fluorescence of THP-adria-
mycin. a was measured with the following expression:
a=(CinꢂCn)/(CNꢂCn).
The oilyproduct was transformed into the correspond-
ing hydrochloride (abs EtOH/anhyd ether) mp 100–
103 ꢀC. Anal. (C30H36ClN3O2) C, H, N.
Pharmacology
Drugs and chemicals
Purified pirarubicin was provided byLaboratoire Roger
Bellon (France). Concentrations were determined by
diluting the stock solutions to approximately10 ꢂ5 M
and using e480=11 500 Mꢂ1 cmꢂ1. Stock solutions were
prepared just before use. Buffer solutions were HEPES
buffer containing 5 mM HEPES, 132 mM NaCl, 3.5 mM
CaCl2, 5 mM glucose, at pH 7.25.
Cell lines and cultures
K 652 is a human leukemia cell line.31 Cells resistant to
doxorubicin were obtained bycontinuous exposure to
increasing doxorubicin concentrations and were main-
tained in medium containing doxorubicin (400 nM)
until 1–4 weeks before experiments. This subline
expresses a unique membrane glycoprotein with a
molecular weight of 180 000 Da.32 Doxorubicin-sensi-
tive and-resistant erythroleukemia K 562 cells were
grown in suspension in RPMI 1640 (Sigma) medium
supplemented with l-glutamine and 10% FCS at 37 ꢀC
in a humidified atmosphere of 95% air and 5% CO2.
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5
Cultures, initiated at a densityof 10 cells/mL, grew
exponentiallyto 8–10 ꢃ105 cells/mL in 3 days. For the
spectrofluorometric assays, in order to have cells in the
exponential growth phase, culture was initiated at
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Cellular drug accumulation
The uptake of pirarubicin in cells was followed by
monitoring the decrease in the fluorescence signal at
590 nm (lex=480 nm) following the method previously
described.33 Using this method it is possible to accu-
ratelyquantifythe kinetics of the drug uptake bythe
cells and the amount of anthracycline intercalated
between the base pairs in the nucleus at the steadystate,
as incubation of the cells with the drug proceeds with-
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buffer at 37 ꢀC using a circulating thermostated water
bath. Cells, 2ꢃ106, were suspended in 2 mL of glucose
containing HEPES buffer at pH 7.3, under vigorous
stirring; 20 mL of the stock anthracycline solution was
quickly added to this suspension yielding an anthracy-
cline concentration CT equal to 1 mM. The decrease of
the fluorescence intensity F at 590 nm was followed as a
22. Haussermann, K.; Benz, B.; Gekeler, V.; Schumacher, K.;
Eichelbaum, M. Eur. J. Clin. Pharmacol. 1991, 40, 53.

2682
S. Dei et al. / Bioorg. Med. Chem. 9 (2001) 2673–2682
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