Bis-pyrrolyl-tetrazolyl derivatives as hybrid polar compounds: A case of lipophilic functional bioisosterism with bis-acetamides

Article abstract of DOI:10.1016/j.ejmech.2012.01.041

Based on previous studies on bis-acetamides that act as hybrid polar compounds to induce leukemia cell differentiation, an attempt was made to bioisosterically replace the amide moiety with the lipophilic non-classical bioisostere tetrazole. A pyrrole group was also included in the molecule in order to retain the hydrogen bond donor capability. Thus, by linking the two polar ring systems with a highly lipophilic methylene chain compounds 2-4 were synthesized and assessed for their anti-proliferative activity in combination with their ability to induce murine erythroleukemia (MEL) cell differentiation. Furthermore, an initial investigation of the structure-activity relation points for the active compound 3 was undertaken by synthesizing compound 5 (a p-xylene analog) and compound 8 (a methylamidopyrrolyl analog). All compounds caused a dose-dependent inhibition of MEL cell growth but to a different extent. Compound 3 (1,6-bis[5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl]hexane) promoted erythroid differentiation in a fifty-fold lower concentration than hexamethylenebisacetamide (HMBA). Though induction of differentiation was to a lesser extent than HMBA, it caused accumulation of 80% Hb-producing cells as compared to that produced by HMBA, leading to differentiation-depended cell growth inhibition equal to that of HMBA after 96 h in culture. Compound 3 represents a potent inducer of hemoglobin gene activation in leukemic cells.

Full text of DOI:10.1016/j.ejmech.2012.01.041

European Journal of Medicinal Chemistry 50 (2012) 75e80
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Bis-pyrrolyl-tetrazolyl derivatives as hybrid polar compounds: A case of lipophilic
functional bioisosterism with bis-acetamides
Maria Chatzopoulou a, Ioannis D. Bonovolias b, Ioannis Nicolaou a, Vassilis J. Demopoulos a,
,
Ioannis S. Vizirianakis b, Asterios S. Tsiftsoglou b
*
a Laboratory of Pharmaceutical Chemistry, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki (A.U.TH.), 54124 Thessaloniki, Greece
b Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki (A.U.TH.), 54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on previous studies on bis-acetamides that act as hybrid polar compounds to induce leukemia cell
differentiation, an attempt was made to bioisosterically replace the amide moiety with the lipophilic non-
classical bioisostere tetrazole. A pyrrole group was also included in the molecule in order to retain the
hydrogen bond donor capability. Thus, by linking the two polar ring systems with a highly lipophilic
methylene chain compounds 2e4 were synthesized and assessed for their anti-proliferative activity in
combination with their ability to induce murine erythroleukemia (MEL) cell differentiation. Furthermore,
an initial investigation of the structureeactivity relation points for the active compound 3 was undertaken
by synthesizing compound 5 (a p-xylene analog) and compound 8 (a methylamidopyrrolyl analog). All
compounds caused a dose-dependent inhibition of MEL cell growth but to a different extent. Compound 3
(1,6-bis[5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl]hexane) promoted erythroid differentiation in a fifty-fold
lower concentration than hexamethylenebisacetamide (HMBA). Though induction of differentiation was
to a lesser extent than HMBA, it caused accumulation of 80% Hb-producing cells as compared to that
produced by HMBA, leading to differentiation-depended cell growth inhibition equal to that of HMBA after
96 h in culture. Compound 3 represents a potent inducer of hemoglobin gene activation in leukemic cells.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 8 June 2011
Received in revised form
18 January 2012
Accepted 18 January 2012
Available online 2 February 2012
Keywords:
Pyrrolyl-tetrazolyl derivatives
Hybrid polar compounds
Inducers of differentiation
MEL cells
1. Introduction
The discovery over the past several years that leukemic cells can
be induced to differentiate into post-mitotic cells, unable to support
Human leukemias are considered as clonal hematopoietic
disorders that are characterized by uncontrolled cell growth, failure
of differentiation and acquired loss of cell death. Evidence indicates
that leukemias are likely to be initiated by transformed hemato-
poietic stem cells (HSCs) [so called leukemia initiating cells (LICs) or
leukemia stem cells (LSCs)] or early uncommitted multipotent
progenitors being genetically or epigenetically aberrant [1e5].
Although leukemias, to some extent, are treatable diseases with
conventional chemotherapeutic agents (antimetabolites, mitotic
blockers, antitumor antibodies, alkylating agents and others), quite
often chemotherapy fails due to cellular heterogeneity and acquired
drug resistance developed during the course of the treatment.
malignant growth with the use of structurally diversified agents,
led to the so called “Differentiation Therapy of Cancer” [6e11]. This
approach has been shown promising [6], since promyelocytic
leukemia (PML) patients nowadays are successfully treated with
differentiating agents, like all-trans retinoic acid, As2O3, and
histone deacetylase inhibitors (HDACIs) that promote granulocytic
differentiation and cell growth inhibition [12e18].
Retrospectively, in the search for inducers of leukemia cells’
differentiation a variety of different classes were explored and
among them the class of hybrid polar compounds (HPCs, formerly
known as polar apolar inducers) appears to be the most efficacious.
In this class, DMSO and hexamethylenebisacetamide (HMBA)
constitute the prototypes, which are strong inducers of differenti-
ation in several transformed cell lines [7]. The key structural
characteristics in HPCs are two highly polar groups that are con-
nected by an apolar methylene chain whose optimal length was
found to be 5e7 carbon atoms [8].
Abbreviations: HDACIs, histone deacetylase inhibitors; HMBA, hexamethylene-
bisacetamide; HPCs, hybrid polar compounds; HSCs, hematopoietic stem cells; LICs,
leukemia initiating cells; LSCs, leukemia stem cells; MEL, murine erythroleukemia;
PML, promyelocytic leukemia.
Most recently, we have proposed the development of novel
agents, that is, agents to be able to reduce cell growth by inducing
* Corresponding author. Tel.: þ30 2310 997631; fax: þ30 2310 997618.
E-mail address: tsif@pharm.auth.gr (A.S. Tsiftsoglou).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.041

76
M. Chatzopoulou et al. / European Journal of Medicinal Chemistry 50 (2012) 75e80
Fig. 1. Chemical structures of the synthesized compounds.
leukemic cell differentiation [19]. The central objective of our
rationale was to develop agents that can be able to act at relatively
low concentrations (<1 mM) and promote terminal maturation and
growth arrest in the majority, if not all leukemic cells.
two tautomeric forms, 2H-tetrazole being the most stable [26]. The
majority of the nucleophilic alkylation reactions result in mixtures
of N1- and N2-alkyl isomers [27], but the regio-selectivity observed
in the present work, is presumably due to the introduced stereo-
requirements of the utilized phase transfer catalyst [28]. The
proposed reaction’s regio-selectivity was established by 13C NMR
analysis. The chemical shifts of the tetrazolyl C-5 in compounds
2e5 ranged from 160.85 to 161.55 and compares favorably to
previously reported values for N2-substituted tetrazoles [29].
Compound 8 was synthesized from 3 in three steps (Scheme 2).
It involved an initial FriedeleCrafts acylation, followed by a halo-
form reaction [25] and a dehydrative amidation of the formed
carboxylic acid [30,31].
Under this framework, a series of 1H-pyrrol-1-yl-2H-tetrazol-5-
yl-derivatives were synthesized as HPCs (compounds 2e5 and 8,
Fig. 1) and assessed for their biological activity as inducers of
differentiation and inhibitors of cell growth. The design was based
on a putative bioisosteric replacement of the acetamide moiety of
HMBA, with that of the more lipophilic pyrrolyl-tetrazolyl struc-
ture. Tetrazoles can broadly act as non-classical bioisosteres of the
amide moiety [20], while the C-2-H of the pyrrole ring is envisioned
to participate in a hydrogen bond as a donor like the NH group of
HMBA [21e23].
In compounds 2e4 the lipophilic methylene chain varied
between 5 and 7 carbon atoms [8], while in the most active
template 3 further modifications were made by either drastically
changing the spatial disposition of the pyrrolyl-tetrazolyl groups
with the inclusion of a p-xylene chain (i.e. compound 5) or by
introducing functional residues which could participate in addi-
tional hydrogen-bond interactions such as the methylamido side-
groups (i.e. compound 8).
2.2. Evaluation of biological activity
The widely used model system of cultured murine eryth-
roleukemia (MEL or Friend) cells [32,33] was employed to assess
the biological activity of the synthesized compounds. Such an
approach was based on observations over the past several years
that chemical inducers were able to selectively restrict proliferation
of leukemic cells in vitro (committed cells along the erythrocytic
pathway) through a highly coordinated process regulating gene
expression. In particular, cells committed to erythroid differentia-
tion produce vast amounts of hemoglobin (Hb) (an authentic
erythroid biomarker) and undergo a programmed loss of prolifer-
ative capacity restricted to only a few divisions, due to their irre-
versible growth arrest in G1/G0 phase of cell cycle (differentiation-
dependent growth arrest) [10]. For the evaluation, each agent was
dissolved in DMSO and tested in varying concentrations in a way to
estimate the growth inhibition potential, as well as to determine
the optimum inducing concentration for each compound being
2. Results and discussion
2.1. Chemistry
Compound 1 (Scheme 1) was synthesized by adapting a modi-
fied ClausoneKaas reaction [24,25]. Phase transfer catalyzed
condensation between the anion of 1 and
a,u-dibromoalkanes or
1,4-bis(bromomethyl)benzene provided the bis-pyrrolyl-tetrazolyl
derivatives 2e5 in good yields (Scheme 1). Tetrazole can exist in
Scheme 1. Reagents and reaction conditions: (a) 4-chloropyridine hydrochloride, 1,4-dioxane, reflux 3 h; (b)
tributylammonium chloride, THF, reflux 22 h.
a,u-dibromoalkane or 1,4-bis(bromomethyl)benzene, benzyl-

M. Chatzopoulou et al. / European Journal of Medicinal Chemistry 50 (2012) 75e80
77
Scheme 2. Reagents and conditions: (a) AlCl3, acetic anhydride, rt 2 h; (b) NaOBr, 1,4-dioxane/H2O, rt 2 h; (c) CDI, CH3NH2, THF, rt overnight.
able to promote erythroid differentiation (Figs. 2 and 3). In all
studies reported, however, the concentration of the solvent DMSO
kept way below the 0.5% v/v that does not affect cell growth and
differentiation of MEL cells in culture. HMBA, a well known potent
inducer of MEL cell differentiation and growth inhibitor, was used
as a positive control. Just to mention and in order to facilitate data
comparison between synthesized agents with that of HMBA, only
the optimum inducing concentration (5 mM) of HMBA was used in
these studies. Indeed, at very low concentrations (<0.1 mM), HMBA
exhibited no substantial effect on growth and differentiation of
MEL cells as previously shown [34].
induction of differentiation, instead of being just a direct cytotoxic
effect exhibited by that compound (differentiation-dependent
growth inhibition).
The data presented thus far, revealed that compound 3 exerts
concentration- and time-dependent growth inhibitory effect, while
it exhibited substantial differentiation inducing capacity (Figs. 3 and
4B and C). For this reason, this compound was further evaluated for
its potential to activate
to
b
major globin gene expression in comparison
b
-actin gene. RT-PCR analysis indicated that compound 3 induced
indeed transcription of bmajor globin gene in levels comparable to
that seen with HMBA (Fig. 5) and again in a fifty-fold lower
From data illustrated in Figs. 2 and 3, it is apparent that all
compounds caused a dose-dependent inhibition of MEL cell growth
after 48 h in culture, but to a different extent varying from 30% to
concentration. It is known that
regulated upon erythropoiesis and used as a marker for the
assessment of MEL cell differentiation, whereas -actin gene is
a housekeeping gene, encoding a stable gene product, the -actin,
and it is used as control in gene expression studies. It should be
noted that -actin gene expression levels exhibited a similar pattern
b
major globin gene is developmentally
b
80% at concentrations from 0.01
and 8 were found to induce erythroid differentiation in 5%e12% of
cells at 100 M after 96 h in culture. Interestingly, compound 3
mM to 100
mM. Compounds 2, 4, 5
b
m
b
exhibited a minimal effect as growth inhibitor (Fig. 2), but attained
the ability to promote differentiation close to 30% of MEL cells
(Fig. 3). Such behavior was similar to that of the known inducer
HMBA and this effect on cell proliferation was more prominent
after 96 h when differentiating cells accumulate in increasing
numbers (Fig. 4B). A more detailed analysis shown in Fig. 4A indi-
cated that compound 3 inhibited cell growth in a time-dependent
manner by more than 75% inhibition after 96 h in culture. To this
end, its inducing capacity has led to Hb accumulation (an internal
marker of erythroid maturation as previously mentioned) at a level
close to that observed by HMBA (Fig. 4C). The latter, suggests that
the observed growth arrest of MEL cells is attributed to the
in all three MEL cell cultures employed, as expected. These data are
consistent with the results illustrated in Fig. 4B and C where
a substantial accumulation of benzidine-positive (Bzþ) cells and
production of Hb were observed.
3. Conclusion
In this work a bioisosteric replacement of the acetamide moiety
of hybrid polar compounds that act as differentiation inducers with
the more lipophilic pyrrollyl-tetrazolyl scaffold was shown. Among
the synthesized compounds, 3 was shown to inhibit cell growth in
100
80
60
40
20
0
150
2
3
4
5
8
100
50
2
3
4
5
8
0
0.01
HMBA
0.1
1
10
100
1000
Compound
Concentration (µM)
Fig. 3. Percentage of differentiated MEL cells (benzidine-positive cells) after treatment
with 100 M of the synthesized compounds for 96 h. It should be noted that the data of
the optimum differentiation concentration (5 mM) of HMBA is included as a positive
control.
Fig. 2. Dose-dependent changes in cell growth of MEL cells after treatment with
varying concentrations of the synthesized compounds for 48 h. Cell growth is
expressed as percentage of that exhibited by untreated (control) MEL cells.
m

78
M. Chatzopoulou et al. / European Journal of Medicinal Chemistry 50 (2012) 75e80
spectra were recorded on a Bruker AW80 at 80 MHz or a Bruker AM
300 at 300 MHz with internal TMS standard. 13C NMR spectra were
recorded on a Bruker AM 300 at 75.5 MHz. Chemical shifts ( ) are
d
quoted in parts per million (ppm) and are referenced to the residual
solvent peak. Coupling values (J) are given in hertz (Hz). Spin
multiplicities are given as s (singlet), d (doublet), t (triplet), m
(multiplet). Elemental analyses were performed with
a Per-
kineElmer 2400 CHN analyzer in the Department of Organic
Chemistry, School of Chemistry, Aristotle University of Thessaloniki,
Greece, or for compound 6 at Galbraith Laboratories, Inc., Knoxville,
TN. LC-MS analysis was performed on a Shimadzu LC-MS 2010EV
system with SPD-20A UV/Vis Detector (pro-ACTINA S.A., Athens,
Greece). Analysis using Electron Spray Ionization (ESI) interface
was performed in a reverse phase C-18 column (Thermo Scientific
BDS Hypersil, 250 mm ꢀ 4.6 mm, particle size 5
phase consisted of water: methanol (4:6, isocratic mode) at a flow
rate of 0.5 mL minꢁ1. Injection volume was 10
L and product
mM). The mobile
m
detected at 222, 254 nm. The mass detector was operated in the
positive and negative mode with nitrogen as the nebulizer gas at
a flow rate of 1.5 L minꢁ1. Mass spectra were recorded from 50 to
1000 Da. Melting points are uncorrected and were determined in
open glass capillaries using a Mel-Temp II apparatus. Flash column
chromatography was carried out with Merck silica gel 60
(230e400 Mesh ASTM). TLC was run with Fluka Silica gel/TLC-
cards. All solvents used for column chromatography were
routinely distilled prior to use. Petroleum ether refers to the frac-
tion with bp 40e60 ꢂC unless stated otherwise.
4.1.2. 5-(1H-Pyrrol-1-yl)-1H-tetrazole (1)
Prepared as described previously by Pegklidou et al. [35].
4.1.3. General method for the preparation of dimers (2e5)
The appropriate
benzene (1 mmol) and
a
,
u
-dibromoalkane or 1,4-bis(bromomethyl)
catalytic amount of benzyl-
Fig. 4. Kinetic analysis of time-dependent effect of compound 3 (0.1 mM) on cell
growth (A), erythroid differentiation (B) and Hb production (C) in MEL cells as
compared to untreated (control) and HMBA-treated (5 mM) cultures.
a
tributylammonium chloride were added to a stirred and under
nitrogen atmosphere solution of 1 (3 mmol) in dry THF (30 mL). The
mixture was cooled to 0 ꢂC and 3 mmol of NaH (60% dispersion in
mineral oil) were added. The resulting mixture was refluxed for
22 h. After this period, it was poured into a stirred ice cold mixture
of dichloromethane (50 mL) and 5% HCl (50 mL). The two phases
were separated and the aqueous phase was extracted with
dichloromethane (2 ꢀ 50 mL). The combined organic extracts were
washed with 10% NaHCO3 (2 ꢀ 50 mL), brine and dried over
anhydrous Na2SO4. The solvents were evaporated under reduced
pressure and the residue was flash chromatographed with a suit-
able mixture of petroleum ether/ethyl acetate [10:1e5:1], followed
by recrystallization from dichloromethane/petroleum ether or
petroleum ether (bp. 80e110 ꢂC).
a time- and concentration-dependent manner and to promote
erythroid differentiation. Though promotion of differentiation was
to a lesser extent than that promoted by HMBA, 3 caused Hb
accumulation to a level close to that observed by HMBA, thus
leading to inhibition of cell growth equal to that of HMBA and in
a fifty-fold lower concentration.
4. Experimental section
4.1. Chemistry
4.1.3.1. 1,5-Bis[5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl]pentane
(2).
4.1.1. General methods
White solid (64%); mp 85 ꢂC; 1H NMR (CDCl3):
d
1.20e2.33 (m, 6H,
All reagents were purchased from SigmaeAldrich and used
without further purification. UVevis spectra were obtained on
a Shimadzu UV-1700 PharmSpec (UVprobe Ver. 2.21). IR spectra
were taken with a Hitachi U-2001 spectrophotometer. 1H NMR
CH2CH2CH2CH2CH2), 4.56 (t, 4H, tetrazolyl-CH2, J ¼ 4.0 Hz),
6.24e6.52 (m, 4H, pyrrolyl-3,4H), 7.32e7.60 (m, 4H, pyrrolyl-2,5H);
13C NMR (CDCl3):
d 22.4, 27.5, 52.2, 111.0, 118.3, 160.9; Anal. Calcd
Fig. 5. Assessment of bmajor globin and
b-actin gene levels in control and agent-treated MEL cells by RT-PCR. Concentrations of compounds were as shown under Fig. 4.

M. Chatzopoulou et al. / European Journal of Medicinal Chemistry 50 (2012) 75e80
79
for C15H18N10: C, 53.24; H, 5.36; N, 41.39. Found: C, 53.29; H, 5.24;
N, 41.52.
CH2CH2CH2CH2CH2CH2), 4.67 (t, 4H, tetrazolyl-CH2, J ¼ 3.04 Hz),
6.23e6.81 (m, 4H, pyrrolyl-3,4H), 6.99e7.29 (m, 4H, pyrrolyl-2,5H).
Crude 7 and 295 mg (1.81 mmol) 1,10-carbonyldiimidazole (CDI)
were refluxed in 4.2 mL dry THF under a nitrogen atmosphere for
2 h. The mixture was cooled at rt,1.7 mL of a solution of 2 M CH3NH2
in THF was added and the resulting mixture was stirred overnight at
rt and under a nitrogen atmosphere. The reaction mixture was
poured into water, and extracted with AcOEt (2 ꢀ 40 mL). The
organic extracts were washed with 1 M HCl, brine, dried over
anhydrous Na2SO4, were concentrated under reduced pressure and
recrystallized from EtOH/H2O (177 mg, 38%); mp 173e174 ꢂC; IR
4.1.3.2. 1,6-Bis[5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl]hexane
White solid (72%); mp 108 ꢂC; UV (EtOH absolute) lmax: 230.6 εmax
11 120; 1H NMR (CDCl3):
1.21e1.75(m, 4H, CH2CH2CH2CH2CH2CH2),
(3).
:
d
1.84e2.36 (m, 4H, CH2CH2CH2CH2CH2CH2), 4.55(t, 4Htetrazolyl-CH2,
J ¼ 6.6 Hz), 6.27e6.52 (m, 4H, pyrrolyl-3,4H), 7.34e7.60 (m, 4H,
pyrrolyl-2,5H); 13C NMR (CDCl3):
d 25.5, 28.7, 53.1, 111.7, 118.9, 161.5;
m/z[ESIpositive]353(MþH)þ;Anal. CalcdforC16H20N10:C,54.53;H,
5.72; N, 39.75. Found: C, 54.38; H, 5.45; N, 39.73.
(nujol): 3313 (NeH), 1643 (C]O) cmꢁ1
;
1H NMR (CDCl3):
4.1.3.3. 1,7-Bis[5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl]heptane
(4).
d
1.44e1.51 (m, 4H, CH2CH2CH2CH2CH2CH2), 2.03e2.14 (m, 4H,
White solid (68%); mp 60 ꢂC; 1H NMR (CDCl3):
d
1.11e1.55 (m, 6H,
CH2CH2CH2CH2CH2CH2), 2.89 (s, 6H, CH3), 4.64 (t, 4H, tetrazolyl-
CH2, J ¼ 7.0 Hz), 6.30e6.35 (m, 2H, pyrrolyl-4H), 6.47 (br s, 2H, NH),
6.74e6.80 (m, 2H, pyrrolyl-3H), 7.15e7.28 (m, 2H, pyrrolyl-5H); LC-
MS purity 98.1% (222 nm) and 97.1% (254 nm); m/z [ESI positive] 467
(M þ H)þ, 489 (M þ Na)þ m/z [ESI negative] 465 (M ꢁ H)ꢁ.
CH2CH2CH2CH2CH2CH2CH2), 1.79e2.25 (m, 4H, CH2CH2CH2CH2
CH2CH2CH2), 4.55 (t, 4H, 4H tetrazolyl-CH2, J ¼ 8.0 Hz), 6.27e6.49
(m, 4H, pyrrolyl-3,4H), 7.35e7.58 (m, 4H, pyrrolyl-2,5H); 13C NMR
(CDCl3):
d 25.9, 28.0, 28.8, 53.2, 111.7, 118.9, 161.5; Anal. Calcd for
C17H22N.100.06 hexane: C, 56.11; H, 6.20; N, 37.69. Found: C, 56.47;
H, 6.06; N, 37.82.
4.2. Growth inhibition and induction of differentiation of MEL cells
4.1.3.4. 1,4-Bis((5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl)methyl)benzene
4.2.1. Chemicals and cell cultures
(5). White solid (44%). mp 192e193 ꢂC; UV (EtOH absolute) lmax
:
All agents were dissolved in DMSO (SigmaeAldrich) and used at
different concentrations in cell cultures. Attention was paid to keep
the concentration of solvent DMSO below the 0.5% v/v so that it
does not affect cell growth and differentiation. MEL cells were used
throughout this study. These cells were seeded at 1.0 ꢀ 105 cells/mL
and grown in DMEM medium (Gibco) containing 10% v/v FBS (Fetal
Bovine Serum, Gibco) and 1% v/v PS (Penicillin-Streptomycin,
Gibco) at 37 ꢂC with 5% CO2 and humidified atmosphere (w95%).
232.9 εmax: 17 760. 1H NMR (CDCl3-DMSO-d6):
6.34e6.40 (m, 4H, pyrrolyl-3,4H), 7.40e7.48 (m, 8H, pyrrolyl-2,5H
and phenyl-H); 13C NMR (DMSO-d6):
56.6, 112.5, 119.5, 129.3,
d 5.95 (s, 4H, CH2),
d
134.7, 161.6; Anal. Calcd for C18H16N10: C, 58.06; H, 4.33; N, 37.61.
Found: C, 57.96; H, 4.18; N, 37.60.
4.1.4. 1,10-(1,10-(2,20-(Hexane-1,6-diyl)bis(2H-tetrazole-5,2-diyl))
bis(1H-pyrrole-2,1-diyl))diethanone (6)
To a stirred and under a nitrogen atmosphere mixture of
2728 mg (20.46 mmol) anhydrous AlCl3 in 1,2-dichloroethane
(30 mL), 1048 mg (10.26 mmol) of acetic anhydride were added
slowly and over a 15 min period. Subsequently, 609 mg (1.73 mmol)
of 3 were added and the resulting mixture was stirred for 2 h under
a nitrogen atmosphere. The reaction mixture was poured into ice
and water and the product was extracted with CH2Cl2 (2 ꢀ 20 mL).
The combined organic extracts were washed with an aqueous
solution of 10% NaHCO3, brine and dried over anhydrous Na2SO4.
The solvents were evaporated under reduced pressure and the
residue was flash chromatographed with petroleum ether/EtOAc
9:4 to provide an analytical sample of the title compound (709 mg,
94%); mp 63e65 ꢂC; IR (nujol): 1652 cmꢁ1 (C]O); 1H NMR (CDCl3):
4.2.2. Assessment of cell growth, erythroid differentiation and Hb
production
Exponentially growing MEL cells (1.0 ꢀ 105 cells/mL) in culture
with DMEM medium supplemented with 10% v/v FBS and 1% v/v PS,
were incubated with compound 3 (10ꢁ4 M). Also, cell cultures with
no addition (untreated-control) and/or the known inducer HMBA
(5 mM) were served as negative and positive control, respectively.
Cell growth in control and agent-treated MEL cell cultures was
determined at various time intervals by removing samples of cells
and measuring their number using a Neubauer hematocytometer.
Cell growthofagent-treated cellswasexpressed asabsolute valuesor
a percentage of that observed for the control untreated cultures.
Erythroiddifferentiationwasassessed bystainingbenzidine-positive
cells (Bzþ, Hb producing cells) with the use of benzidine-H2O2 dilu-
tion, as earlier described [36,37]. The content of total cellular Hb was
determined spectrophotometrically, as previously described [38].
d
1.18e1.61 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.85e2.32, 2.42 (m, 4H,
CH2CH2CH2CH2CH2CH2), (s, 6H, COCH3), 4.64 (t, 4H tetrazolyl-CH2,
J ¼ 4.8 Hz), 6.28e6.53 (m, 2H, pyrrolyl-4H), 7.00e7.31 (m, 4H,
pyrrolyl-3,5H); Anal. Calcd for C20H24N10O2: C, 55.04; H, 5.54; N,
32.09. Found: C, 55.24; H, 5.63; N, 32.39.
4.2.3. RNA extraction and RT-PCR analysis
MEL cells grown in culture, were treated separately with HMBA
(5 mM), compound 3 (10ꢁ4 M) for 0 h, 24 h, 48 h, 72 h and 96 h, as
described under Fig. 4. Untreated parental cells were served as
control. At the end of time intervals, cells were harvested from
culture, washed twice with PBS before the isolation of total cyto-
4.1.5. 1,10-(2,20-(Hexane-1,6-diyl)bis(2H-tetrazole-5,2-diyl))bis(N-
methyl-1H-pyrrole-2-carboxamide) (8)
To a cold (ice bath) solution of 6 (628 mg, 1.44 mmol) in 1,4-
dioxane (18 mL) and H2O (13 mL) a freshly prepared cold solution
of NaOBr [prepared by the addition of (1076 mg, 6.73 mmol) Br2 to
a stirred mixture of 25 mL 12% NaOH and 6.2 mL 1,4-dioxane] was
added dropwise. After 2 h of stirring at rt, acetone (10 mL) was
added and the reaction mixture was acidified with HCl, extracted
with CH2Cl2 (2 ꢀ 50 mL), washed with brine, dried over anhydrous
Na2SO4 and the solvents were removed under reduced pressure.
The residue was dissolved in EtOH, H2O was added and the
precipitated was collected and dried to afford 7 (393 mg) which
was used in the next step without further purification. IR (nujol):
plasmic RNA and processed for RT-PCR analysis. 1
RNA from each sample was then used to assess the steady-state
level of RNA transcripts encoded by bmajor globin and/or
-actin
mg of cytoplasmic
b
genes. The generated DNA products were analyzed electrophoret-
ically in 1% w/v agarose gel, stained with EtBr, visualized under UV
light and the data obtained are shown in Fig. 5.
One step RT-PCR was performed with the RobusT-I RT-PCR kit
(Finnzymes) in 25
mL volume reactions. Total RNA extracted
according to acid guanidinium thiocyanate-phenol-chloroform
extraction method [39] from untreated (control) and agent-
treated MEL cells and indicated in the text, was used as template
3121 (OeH), 1673 (C]O) cmꢁ1
;
1H NMR (CDCl3/DMSO-d6):
d
1.22e1.64 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.82e2.34 (m, 4H,

80
M. Chatzopoulou et al. / European Journal of Medicinal Chemistry 50 (2012) 75e80
ulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia, Nat.
(1
mg/reaction). The RT-PCR running conditions were the following:
Med. 7 (2001) 444e451.
50 ꢂC for 30 min, 94 ꢂC for 2 min, (94 ꢂC for 30 s, 58 ꢂC for 1 min and
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72 ꢂC for 1 min) for 30 cycles and 72 ꢂC for 10 min.
b-actin gene was
used as internal control. The following pairs of primer sequences
were used: bmajor globin, 50-CTGCTGGTTGTCTACCCTTGG-30 (sense)
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and 50-CCTGAAGTTCTCAGGATCCAC-30 (anti-sense);
b
-actin, 50-
TGGAATCCTGTGGCATCCATGAAAC-30 (sense) and 50-TAAAACG-
CAGCTCAGTAACAGTCCG-30 (anti-sense). RT-PCR products were
analyzed by agarose gel (1% w/v) electrophoresis.
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Acknowledgments
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This work was financially supported by the Greek State Schol-
arship Foundation and Aristotle University’s Research Committee
[C.M.]. Acknowledgments are made to Dr. Dionysia Papagianno-
poulou (School of Pharmacy, Aristotle University of Thessaloniki)
for her contribution in obtaining the NMR spectral data and Mrs
Anna Maranti, M.Sc. (pro-ACTINA S.A.) and Dr. Polyxeni Alexiou
(Department of Science, Agricultural University of Athens) in
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