Synthesis of derivatives of the keto-pyrrolyl-difluorophenol scaffold: Some structural aspects for aldose reductase inhibitory activity and selectivity

Article abstract of DOI:10.1016/j.bmc.2012.12.015

Seven novel ARIs (3a-c, 4a-c and 5) were synthesized with the implementation of an optimized and, partially, selective synthetic procedure, via a Friedel-Crafts acylation reaction. The synthesized ARIs have values of IC₅₀^ALR2 ranging

Full text of DOI:10.1016/j.bmc.2012.12.015

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of derivatives of the keto-pyrrolyl-difluorophenol scaffold:
Some structural aspects for aldose reductase inhibitory activity
and selectivity
⇑
Eleni Kotsampasakou, Vassilis J. Demopoulos
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven novel ARIs (3a–c, 4a–c and 5) were synthesized with the implementation of an optimized and, par-
Received 31 October 2012
Revised 11 December 2012
Accepted 13 December 2012
Available online xxxx
tially, selective synthetic procedure, via a Friedel–Crafts acylation reaction. The synthesized ARIs have
ALR2
values of IC50
ranging from 0.19 lM (in case of compound 3b) to 2.3 lM (in case of compound 4a),
while the values of selectivity index towards ALR1 range from 1 (in case of compound 3b) to 238 (in case
of compound 3a). Finally, we found out that the presence of an additional (secondary) aromatic area is
not a prerequisite feature for ARI activity.
Keywords:
ARI
Ó 2012 Elsevier Ltd. All rights reserved.
Aldose reductase
Aldehyde reductase
Diabetes mellitus
Long-term diabetes complications
Friedel–Crafts acylation reaction
1. Introduction
a cytosolic enzyme, member of the aldo-ketoreductase superfam-
ily, which under normal conditions is responsible for a series of
Diabetes mellitus is a complex metabolic disorder that is charac-
terized by abnormal metabolism of carbohydrates, proteins and lip-
ids, as a result of lack of insulin production or insulin resistance.1
During the 20th century diabetes has acquired an epidemic preva-
lence and from the beginning of the 21st century it is developing to
a pandemic phenomenon. In 2011, 366 million people were af-
fected from the disease, while by 2030 the number is estimated
to reach 522 million patients, with frequency of prevalence 7.7%.2
In addition, diabetes is not exclusively a disease of the developed
world, since it appears in very high rates in some developing coun-
tries of Asia and Latin America.3 Diabetes is closely associated with
high morbidity and mortality due to its chronic complications that
develop over the years because of insufficient glycemic control.4
Among these chronic complications are angiopathy and cardiovas-
cular diseases, neuropathy, nephropathy, retinopathy and cataract.5
The enzyme that is considered responsible for diabetes’ chronic
complications is aldose reductase (ALR2, AR, AKR1B1, EC 1.1.1.21),
physiologic functions.6 ALR2 is the first enzyme of the polyol
pathway, which converts glucose into sorbitol using NADPH as a
co-factor. Sorbitol is slowly converted into fructose by the second
enzyme of the pathway, sorbitol dehydrogenase (SDH), which uses
NAD+ as a co-factor.7
ALR2 has a low affinity to glucose and the polyol pathway is in-
volved only to a minor degree in the metabolism of glucose. How-
ever, in case of hyperglycemia, glucose is rapidly converted by
ALR2 to sorbitol, which cannot easily cross membranes and accu-
mulates into cells, causing cell and tissue disfunction.8,9 Addition-
ally, the exhaustion of NADPH and the disturbance of NADH/NAD+
ratio can cause oxidative stress, which is also related to diabetic
chronic complications.1,9–11 Furthermore, fructose, via oxidation,
gives intermediates that can lead to advanced glycation end prod-
ucts (AGEs), which are also considered responsible for diabetes’
chronic complications.10,12 Finally, recent studies have shown that
ALR2 is related to ischemic and inflammatory pathologic condi-
tions,5,13,14 as well as to some particular types of cancer.5,15
All the above evidence point out that ALR2 is an important
pharmacological target and its inhibition could be the key for the
treatment of many pathological conditions. Of course, this means
that there is a continuously rising need for new and effective
ALR2 inhibitors (ARIs), since today only epalrestat is on the market
and only in Japan. Even though many active ARIs have been
Abbreviations: ARI(s), aldose reductase inhibitor(s); ALR2/AR, aldose reductase;
ALR1, aldehyde reductase; SDH, sorbitol dehydrogenase; AGEs, advanced glycation
end products; DBN, 1,5-diazabicyclo[4.3.0]non-5-ene; SI, selectivity index; TLC, thin
layer chromatography; DMSO, dimethyl sulfoxide.
⇑
Corresponding author. Tel.: +30 2310 997626; fax: +30 2310 997852.
E-mail address: vdem@pharm.auth.gr (V.J. Demopoulos).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.015
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E. Kotsampasakou, V. J. Demopoulos / Bioorg. Med. Chem. xxx (2013) xxx–xxx
amount of solvent employed, and the reaction’s time. In all cases,
we utilized two equivalents (in respect to 1) of the acylating chlo-
rides, followed by a final hydrolytic step. The latter was based on
the report24 that a phenol could be esterified during the course
of an AlCl3 catalyzed acylation reaction. Although, a high degree
of selectivity for the pyrrolyl C-b regioisomers 4a–c was attained
(2a–2c/AlCl3 1:1.07), the selectivity for the respective C-
a com-
pounds 3a–c was poor (2a–2c/AlCl3 1:0.54). Furthermore, in the
case of preparing 3b–c it proved to be a sluggish reaction and addi-
tional reagent/catalyst was added, with a prolongation of the reac-
tions’ time. The C-
AlCl3 with BF3ꢀEt2O, while the utilization of the DBN catalyzed
Friedel–Crafts pyrrolyl C-
acylation methodology25 to 1 was
unsuccessful. Finally, an attempt to implement on 1 the reported26
C- aroylation procedure of N-tosylpyrroles by carboxylic acids
a selectivity was not improved by replacing
a
a
and trifluoracetic anhydride, led to the exclusive formation of 5.
This compound, though, was conveniently prepared by reacting 1
with trifluoroacetic anhydride in refluxing 1,2-dichloroethane.
Scheme 1. Synthesis of target compounds 3a–c, 4a–c and
Reagents: (a) 2a–c/AlCl3 (1:1.07 or 1:0.54), ClCH2CH2Cl; (b) H2O/NaOH, 1,4-
5 from phenol 1.
dioxane; (c) (CF3CO)2O, ClCH2CH2Cl.
2.2. In vitro results
synthesized over the years, most of them are derivatives of the car-
boxylic acid or the hydantoin moiety, which have proved to have
either poor bioavailability or serious adverse effects.16
2.2.1. ALR2 inhibitory activity
The evaluation of the ALR2 inhibitory activity was conducted on
partially purified rat lenses ALR2. Human ALR2 and rat ALR2 exhi-
bit 85% sequence homology, while the catalytic active site of both
the enzymes is considered identical.4 ALR2 inhibitory activity is ex-
pressed as IC50 values (Table 1). All the compounds exhibited sub-
micromolar inhibitory activity, except for 4a, which exhibited
micromolar inhibitory activity.
In order to overcome the problematic pharmacokinetic profile
of carboxylic acids, novel bioisosteric approaches have been imple-
mented, as for example with the trifluoromethyl ketone substi-
tuted ARIs.17 In the present work, and in continuation of
searching for new ARI chemotypes,4 we have prepared and
in vitro tested the keto-pyrrolyl-difluorophenol derivatives 3a–c,
4a–c and 5 (Scheme 1). In these molecules 2,6-difluorophenol
was used as a non-classical bioisosteric replacement of the acetic
acid moiety,18 while the pyrrole ring was substituted by different
keto-groups.
According to Table 1, it was found that the most active com-
pound is 3b with an IC50 of 0.19 lM. Compound 4b also shows
quite strong ALR2 inhibition, which justifies our initial idea of
inserting the 4-bromo-2-fluoro-benzoyl group into the pyrrole
ring. Compounds 3a and 3c also show a quite strong inhibitory
activity, which implies that, both the biphenyl and the trifluoro-
methoxy moieties are important for ARI activity, even though they
proved weaker, than in case of 4-bromo-2-fluorobenzoyl group.
Surprisingly, the privileged scaffold of biphenyl in case of C-b sub-
stitution at the pyrrole ring attributed much lower inhibitory activ-
The biphenyl ring system, present in compounds 3a and 4a, is
considered as a privileged scaffold, providing affinity, due to its ri-
gid aromatic structure, which adapts well to protein hydrophobic
pockets, where
p-stacking with phenylalanine and tyrosine are
commonly observed.19
The 4-bromo-2-fluoro-phenyl moiety, present in compounds 3b
and 4b, has been shown to be correlated with ALR2 inhibition,
since known ARIs contain this group (e.g., minalrestat6 and ranire-
stat20). Our intention was to introduce it as a 4-bromo-2-fluoro-
benzoyl substructure, which could be easily inserted into the
pyrrole ring via a Friedel–Crafts acylation reaction.
ity, than in case of C-
a substitution. Generally, in this series of
compounds, C- substitution at the pyrrole ring gives more active
a
ALR2 inhibitors. Finally, the strong inhibitory potency of com-
pound 5 indicates that the presence of an additional (secondary)
aromatic area is not a prerequisite feature for ARI activity, as it
was believed before.27
The trifluoromethoxy moiety, present in compounds 3c and 4c, is
considered not just a bioisosteric replacement of a methoxy group,
since it attains vertical spatial disposition, it has a special electronic
distribution, and the C–O bond acquires a character between a single
and a double. Overall, it has a unique hydrogen bonding ability,
critical to putative ligand–enzyme interactions.21,22
The trifluoro-acetyl group, present in compound 5, was selected
in an effort to minimize lipophilicity, while enhancing metabolic
stability and retaining the capacity of hydrogen bond formation.23
2.2.2. ALR1 inhibitory activity
Aldehyde reductase (EC 1.1.1.2, ALR1, AKR1A1) is a cytosolic en-
zyme, member of the aldoketoredouctase superfamily, like aldose
reductase (ALR2). It also catalyzes several physiological functions,
among them, detoxification of toxic aldehydes.28–30 Furthermore,
the two enzymes share a high level of sequence (ꢁ65%) and struc-
tural homology,6 as well as physical and chemical properties.31
This has as a result the increased tendency of many substrates or
ligands to bind with both ALR1 and ALR2.28,32 The inhibition of
both the enzymes is thought to lead to toxic phenomena,6 due to
the resulting insufficient detoxification capacity of the living cell.
However, it has been indicated that there are other available en-
zymes for the aldehydes’ detoxification, in case of parallel inhibi-
tion of ALR2 and ALR1,7 but this matter needs further investigation.
In the present study, ALR1 inhibitory activity was evaluated
on partially purified ALR1 obtained from rat’s kidney. It is
expressed as IC50 values accompanied with the selectivity index
2. Results and discussion
2.1. Chemistry
In a previous study4 in our laboratory we have utilized Friedel–
Crafts aroylations on 2,6-difluoro-4-(1H-pyrrol-1-yl)phenyl
arylcarboxylates in order to prepare C-
a or C-b substituted keto-
pyrrolyl-difluorophenol derivatives. In the present work we
targeted to control the selectivity of the Friedel–Crafts aroylation
on the phenol 1, without esterifying it. Thus, we systematically
altered (Scheme 1) the equivalents (in respect to 1) of AlCl3, the
(SI = IC50ALR1/IC50ALR2) and the % inhibition of ALR1 at 10ꢂ5
(Table 1). From the obtained results we can see that almost all of
our compounds present a relatively high selectivity, except for
M
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E. Kotsampasakou, V. J. Demopoulos / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Table 1
The IC50 values for ALR2 and ALR1, the% inhibition of ALR1 at 10ꢂ5 M and the selectivity index for compounds 3a–c, 4a–c and 5
ALR2
ALR1
Compound
IC50
SEMa
(l
M)
IC50
121.4c
SEMa
(
lM)
% Inhibition ALR1 at 10ꢂ5
M
Selectivity indexb (SI)
3a
4a
3b
4b
3c
4c
5
0.51 0.030
2.3 0.026
0.19 0.003
0.40 0.009
0.52 0.005
0.75 0.003
0.93 0.012
0.25 0.01
—
5.85
72.85
96.8
75.3
30.85
38.05
26.5
—
238
1.87
1
14.08
31.15
17.52
20.3
—
5.63 0.026
0.19 0.0014
4.31 0.305
16.2d
13.14d
18.87d
Sorbinil
Valproic acid
—
56.1 1.56
—
—
a
b
c
n = 3.
ALR2
Defined as IC50ALR1/IC50
.
Extrapolated from the % inhibition at 5 ꢃ 10ꢂ5 M (=20.6).
d
Extrapolated from the % inhibition at 10ꢂ5 M.
3b and 4a, which have none and low selectivity respective. The
most selective compound proved to be 3a, which implies that
Perkin–Elmer 2400 CHN analyzer (in the Department of
Organic Chemistry, School of Chemistry, Aristotle University of
Thessaloniki).
the biphenyl group attributes selectivity, but only in case of C-
a
substitution at the pyrrole ring as the C-b substituted analogue
(4a) showed very low selectivity.
4.2. Chemistry
3. Conclusions
4.2.1. Preparation of 4-bromo-2-fluorobenzoyl chloride (2b)
In
a mixture of 4-bromo-2-fluorobenzoic acid (944 mg,
In the present study seven novel ARIs (3a–c, 4a–c and 5) were
synthesized with the implementation of an optimized and, par-
tially, selective synthetic procedure. The synthesized ARIs were
4.31 mmol) in CH2Cl2 (3.8 mL), oxalyl chloride (1095 mg,
8.62 mmol) in CH2Cl2 (3.7 mL) was added and the mixture was
stirred at room temperature and under a nitrogen atmosphere
biologically evaluated for ALR2, as well as ALR1 inhibitory activi-
for 1 h. Then DMF (14 lL) was added to the mixture and stirring
ALR2
ties and SIs were calculated. The estimated values of IC50
ran-
was continued at room temperature, under a nitrogen atmosphere
for 24 h. The mixture was evaporated under reduced pressure and
the product [IR (neat) 1773, 1734 cmꢂ1] was used as an acylating
agent in the following reactions without further purification, con-
sidering it being 80% pure.
ged from 0.19 M (in case of compound 3b) to 2.3 lM (in case of
l
compound 4a), while the calculated values of SI ranged from 1
(in case of compound 3b) to 238 (in case of compound 3a). From
the used substituents, the 4-bromo-2-fluorobenzoyl moiety proved
to attribute the strongest ALR2 inhibitory activity, since com-
pounds 3b (IC50ALR2 = 0.19 M) and 4b (IC50ALR2 = 0.40
l lM) are
4.2.2. General procedure for selective Friedel–Crafts aroylation
of phenol 1 with AlCl3 as Lewis acid in C-b position of the pyrrole
ring. Preparation of (1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-
3-yl)(aroyl)methanones (4a–c)
the most active ARIs synthesized in this study. In case of SI, we can-
not derive to a definite conclusion, since the 4-bromo-2-fluor-
obenzoyl moiety in case of C-
a substitution at the pyrrole ring
provides no selectivity, while in case of C-b substitution the SI is
To a mixture of anhydrous AlCl3 (164 mg, 1.231 mmol) in ethyl-
ene dichloride (10 mL) the appropriate arroyl chloride 2a–c
(1.149 mmol) was added and stirred at room temperature and
under a nitrogen atmosphere. After 10 min, a solution of the 2,
6-difluoro-4-(1H-pyrrol-1-yl)phenol (1)18 (100 mg, 0.5128 mmol)
in ethylene dichloride (5 mL) was added and the resulting mixture
was stirred for 48 h under a nitrogen atmosphere. The reaction was
quenched with ice and water and the product was extracted with
CHCl3 (+10% EtOH) (3 ꢃ 20 mL). The combined organic extracts
were washed with a saturated NaCl solution, dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The residue was
dissolved in 1,4-dioxane (14 mL) and to this a solution of aqueous
5% NaOH solution (14 mL) was added. The mixture was stirred at
room temperature and under nitrogen atmosphere for 24 h.
Then, the mixture was concentrated to half of its volume, water
(30 mL) was added and the mixture was acidified under cooling
(ice bath) with aqueous solution of HCl (10%). Afterwards, the mix-
ture was extracted with EtOAc (3 ꢃ 20 mL) and the combined or-
ganic extracts were washed with saturated NaCl solution, dried
over anhydrous Na2SO4 and evaporated under reduced pressure.
The residue was dissolved in a mixture of petroleum ether/EtOAc
(1:1 in case of 4a–b and 2:1 in case of 4c) and was washed with
a phosphate buffer (Na2HPO4–KH2PO4) pH 8. After it was ascer-
tained with TLC that the acid of the used aroyl chloride has been
fully removed, the combined organic extracts were washed with
saturated NaCl solution, dried over anhydrous Na2SO4 and
good (14.1). On the contrary, the 4-biphenylcarbonyl moiety pro-
vides a very high SI in case of C-a substitution (238), while in case
of C-b substitution the SI is low (1.87). Finally, we found out that
the presence of an additional (secondary) aromatic area is not a
prerequisite feature for ARI activity, since compound 5 has an
ALR2
IC50
value of 0.93 lM.
4. Experimental section
4.1. General notes
All reagents were purchased from Sigma–Aldrich and used
without further purification, except for the solvents for flash chro-
matography and recrystallization, which were distilled. [1,10-
Biphenyl]-4-carbonyl chloride (2a) was obtained from Aldrich
(product number: 161144) and 4-(trifluoromethoxy)benzoyl chlo-
ride (2c) was obtained from Fluka (product number: 91754). IR
spectra were taken with a Perkin–Elmer FT–IR System Spectrum
BX. 1H NMR spectra were recorded on a Bruker AM 300 at
300 MHz, using TMS as the internal standard. UV–vis measure-
ments were taken with UV/vis Spectrometer Lamda 20. Melting
points are uncorrected and were determined in open glass capillar-
ies using a Mel-Temp II apparatus. Flash column chromatography
was carried out with Merck Silica Gel 9385. TLC was run with Fluka
Silica gel/TLC-cards. Elemental analyses were performed by
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4
E. Kotsampasakou, V. J. Demopoulos / Bioorg. Med. Chem. xxx (2013) xxx–xxx
evaporated under reduced pressure. The residue was flash chro-
matographed with a mixture of petroleum ether/EtOAc 6:1. Ana-
lytical samples were obtained by recrystallization from CH2Cl2/
petroleum ether (4a and 4c), or CH2Cl2 (4b).
under a nitrogen atmosphere. After 10 min, a solution of the 2,6-di-
fluoro-4-(1H-pyrrol-1-yl)phenol (1)17 (200 mg, 1.024 mmol) in
ethylene dichloride (5 mL) was added and the resulted mixture
was stirred for 24 h under a nitrogen atmosphere. At this point,
TLC analysis showed that the reaction was not completed. Thus,
a solution of crude chloride 2b, (estimated as being 273 mg,
1.149 mmol) in ethylene dichloride (7.5 mL) was added, followed
by anhydrous AlCl3 (82 mg, 0.615 mmol). The resulting mixture
was stirred at room temperature for another 24 h under a nitrogen
atmosphere. Afterwards, the mixture was processed as in Sec-
tion 4.2.2. The residue was flash chromatographed with a mixture
of petroleum ether/EtOAc 10:1–8:1. Analytical samples were ob-
tained by recrystallization from CH2Cl2/petroleum ether in case
of 3b and from CH2Cl2 in case of 4b.
4.2.2.1. [1,10-Biphenyl]-4-yl(1-(3,5-difluoro-4-hydroxyphenyl)-
1H-pyrrol-3-yl)methanone (4a).
above general procedure (104 mg, 54%). Mp 205–207 °C; IR (KBr)
3064, 1592 cmꢂ1 1H NMR (CDCl3 + DMSO-d6) d 7.31–7.37 (m,
Prepared according to the
;
1H), 7.40–7.45 (m, 2H), 7.48–7.50 (m, 1H), 7.59–7.68 (m, 4H), 7.9
(d, J = 8.3 Hz, 2H), 9.55 (br s, 1H). Anal. Calcd for C23H15F2NO2: C,
73.59, H, 4.03, N, 3.73. Found: C, 73.39, H, 4.09, N, 3.63.
4.2.2.2. (4-Bromo-2-fluorophenyl)(1-(3,5-difluoro-4-hydroxy-
phenyl)-1H-pyrrol-3-yl)methanone (4b).
ing to the above general procedure (39 mg, 49%). Mp 206–207 °C;
IR (KBr) 3165, 1620 cmꢂ1 1H NMR (CDCl3 + DMSO-d6) d 6.70–
6.80 (m, 1H), 6.87–7.04 (m, 3H), 7.32–7.47 (m, 4H), 9.34 (br s,
1H). Anal. Calcd for C17H9BrF3NO2: C, 51.54, H, 2.29, N, 3.54. Found:
C, 51.14, H, 2.34, N, 3.43.
Prepared accord-
4.2.4.1. (4-Bromo-2-fluorophenyl)(1-(3,5-difluoro-4-hydroxy-
;
phenyl)-1H-pyrrol-2-yl)methanone (3b).
to the above procedure (56 mg, 14%). Mp 184–186 °C; IR (KBr)
3087, 1600 cmꢂ1 1H NMR (CDCl3 + DMSO-d6) d 6.19–6.33 (m,
Prepared according
;
1H), 6.66–6.78 (m, 1H), 6.80–6.95 (m, 2H), 6.96–7.06 (m, 1H),
7.25–7.42 (m, 3H), 9.43 (br s, 1H). Anal. Calcd for C17H9BrF3NO2:
C, 51.54, H, 2.29, N, 3.54. Found: C, 51.18, H, 2.37, N, 3.43.
4.2.2.3.
(1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(4-
(trifluoromethoxy) phenyl)methanone (4c). Prepared according
to the above general procedure (109 mg, 55%). Mp 133–134 °C;
4.2.4.2. (4-Bromo-2-fluorophenyl)(1-(3,5-difluoro-4-hydroxy-
IR (KBr) 3143, 1603 cmꢂ1
;
1H NMR (CDCl3) d 6.04 (br s, 1H),
phenyl)-1H-pyrrol-3-yl)methanone (4b).
Prepared according
6.83–6.89 (m, 1H), 7.00–7.10 (m, 3H), 7.35 (d, J = 8.4 Hz, 2H),
7.50–7.56 (m, 1H), 7.95 (d, J = 8.4 Hz, 2H). Anal. Calcd for
to the above procedure (64 mg, 16%).
C
18H10F5NO3. 0.2 CH2Cl2: C, 54.61, H, 2.62, N, 3.50. Found: C,
4.2.5. Friedel-Crafts aroylation of phenol 1 with AlCl3 as Lewis
acid in positions C-a and C-b of the pyrrole ring. Preparation of
(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-yl)(aroyl)-
methanones 3c and 4c
54.73, H, 2.55, N, 3.47.
4.2.3. Friedel–Crafts arroylation of phenol 1 with AlCl3 as Lewis
acid in positions C-
(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-
yl)(aroyl)methanones 3a and 4a
a
and C-b of the pyrrole ring. Preparation of
In a mixture of anhydrous AlCl3 (164 mg, 1.231 mmol) in ethyl-
ene dichloride (10 mL), 4-(trifluoromethoxy)benzoyl chloride (2c)
(546 mg, 0.38 mL, 2.298 mmol) was added and stirred at room
temperature and under a nitrogen atmosphere. After 10 min, a
solution of the 2,6-difluoro-4-(1H-pyrrol-1-yl)phenol (1)18
(200 mg, 1.024 mmol) in ethylene dichloride (5 mL) was added
and the resulted mixture was stirred for 24 h under a nitrogen
atmosphere. At this point, TLC analysis showed that the reaction
was not completed. Thus, 2c (273 mg, 0.19 mL, 1.149 mmol), anhy-
drous AlCl3 (82 mg, 0.615 mmol) and ethylene dichloride (7.5 mL)
were added. The resulted mixture was stirred in room temperature
for another 24 h under a nitrogen atmosphere. Afterwards, the
mixture was processed as in Section 4.2.2. The residue was flash
chromatographed with a mixture of petroleum ether/EtOAc 9:1–
7:1. Analytical samples were obtained by recrystallization from
CH2Cl2/petroleum ether.
To a mixture of anhydrous AlCl3 (82 mg, 0.615 mmol) in ethyl-
ene dichloride (5 mL), biphenyl]-4-carbonyl chloride (2a) (249 mg,
1.149 mmol) was added and stirred at room temperature and un-
der a nitrogen atmosphere. After 10 min, a solution of the 2,6-di-
fluoro-4-(1H-pyrrol-1-yl)phenol (1)18 (100 mg, 0.5128 mmol) in
ethylene dichloride (2.5 mL) was added and the resulting mixture
was stirred for 11 h under a nitrogen atmosphere. Afterwards, the
mixture was processed as in Section 4.2.2. The residue was flash
chromatographed with a mixture of petroleum ether/EtOAc 9:1–
7:1. Analytical samples were obtained by recrystallization from
CH2Cl2/petroleum ether.
4.2.3.1. [1,10-Biphenyl]-4-yl(1-(3,5-difluoro-4-hydroxyphenyl)-
1H-pyrrol-2-yl)methanone (3a).
above procedure (65 mg, 34%). Mp 214–216 °C; IR (KBr) 3132,
1597 cmꢂ1 1H NMR (CDCl3 + DMSO-d6) d 6.24–6.33 (m, 1H), 6.80–
6.93 (m, 3H), 6.97–7.05 (m, 1H), 7.32–7.45 (m, 3H), 7.56–7.68 (m,
4H), 7.9 (d, J = 8.1 Hz, 2H), 9.31 (br s, 1H). Anal. Calcd for C23H15F2NO2:
C, 73.59, H, 4.03, N, 3.73. Found: C, 73.04, H, 4.11, N, 3.64.
Prepared according to the
4.2.5.1.
(1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(4-
;
(trifluoromethoxy)phenyl)methanone (3c). Prepared according
to the above procedure (66 mg, 17%). Mp 158.5-160.5 °C; IR (KBr)
3132, 1608 cmꢂ1 1H NMR (CDCl3) d 6.18 (br s, 1H), 6.34–6.43 (m,
;
1H), 6.86–7.02 (m, 3H), 7.08–7.13 (m, 1H), 7.34 (d, J = 8.5 Hz, 2H),
7.96 (d, J = 8.5 Hz 2H). Anal. Calcd for C18H10F5NO3. 0.1 CH2Cl2: C,
55.49, H, 2.62, N, 3.57. Found: C, 55.63, H, 2.62, N, 3.56.
4.2.3.2. [1,10-Biphenyl]-4-yl(1-(3,5-difluoro-4-hydroxyphenyl)-
1H-pyrrol-3-yl)methanone (4a).
Prepared according to the
above procedure (39 mg, 20%).
4.2.5.2.
(1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(4-
(trifluoromethoxy)phenyl)methanone (4c). Prepared according
4.2.4. Friedel–Crafts aroylation of phenol 1 with AlCl3 as Lewis
acid in positions C- and C-b of the pyrrole ring. Preparation of
to the above procedure (84 mg, 21%).
a
(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-yl)(aroyl)methanones
3b and 4b
4.2.6. Preparation of 1-(1-(3,5-difluoro-4-hydroxyphenyl)-1H-
pyrrol-2-yl)-2,2,2-trifluoroethanone (5)
To a solution of crude 4-bromo-2-fluorobenzoyl chloride (2b)
(estimated as being 546 mg, 2.298 mmol, Section 4.2.1.) in ethyl-
ene dichloride (10 mL), anhydrous AlCl3 (164 mg, 1.231 mmol)
was added and the mixture was stirred at room temperature and
In a mixture of ethylene dichloride (2.4 mL) and trifluoroacetic
anhydrite (2.379 g, 1.6 mL, 11.33 mmol), 2,6-difluoro-4-(1H-
pyrrol-1-yl)phenol (1)18 (100 mg, 0.513 mmol) was added and the
mixture was stirred under reflux (bath temperature 65–70 °C)
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E. Kotsampasakou, V. J. Demopoulos / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
and nitrogen atmosphere for 4 h. The reaction was poured into ice
and water and the product was extracted with CH2Cl2 (3 ꢃ 20 mL).
The combined organic extracts were washed with saturated NaCl
solution, dried over anhydrous Na2SO4 and evaporated under re-
duced pressure. The residue was flash chromatographed with a
mixture of petroleum ether/EtOAc 6:1 (87 mg, 58%).
dissolved in DMSO. The final concentration of DMSO in all incuba-
tions was 0.3%. The compounds were tested at five concentrations,
the log (dose)–response curves were then constructed from the
inhibitory data, and the IC50 values were calculated by least-square
analysis of the linear portion of the log (dose) versus response
curves (0.912 < r2 < 0.996). The experiments were performed in
triplicate.
Analytical sample was obtained by recrystallization from
CH2Cl2/petroleum ether. Mp 100–102 °C; IR (KBr) 3345,
1664 cmꢂ1 1H NMR (CDCl3) d 1.7 (br s, 1H), 6.43–6.51 (m, 1H),
;
Supplementary data
6.86–6.98 (m, 2H), 7.11–7.18 (m, 1H), 7.35–7.42 (m, 1H). Anal.
Calcd for C12H6F5NO2: C, 49.50, H, 2.08, N, 4.81. Found: C, 49.19,
H, 2.06, N, .4.68.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.015.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
4.3. Biological evaluation
4.3.1. ALR2 preparation
References and notes
Lenses were quickly removed from Fischer-344 rats of both
sexes following euthanasia. The experiments conform to the law
for the protection of experimental animals (Republic of Greece)
and are registered at the Veterinary Administration of the Republic
of Greece. ALR2 from rat lens was partially purified according to
the reported procedure4,33 as follows: lenses were quickly removed
from rats following euthanasia and stored at ꢂ20 °C until used. The
lenses were homogenized in 5 vol of cold distilled water. The
homogenate was centrifuged at 10,000 g at 0–4 °C for 15 min.
The supernatant was precipitated with saturated ammonium sul-
fate at 40% salt saturation and this solution was centrifuged at
10,000g at 0–4 °C for 15 min. The latter supernatant was either
used directly or stored for maximum 24 h at ꢂ80 °C.
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Kidneys were quickly removed from Fischer-344 rats of both
sexes following euthanasia. The experiments conform to the law
for the protection of experimental animals (Republic of Greece)
and are registered at the Veterinary Administration of the Republic
of Greece. ALR1 from rat kidney was partially purified according to
the reported procedure4,34 as follows: kidneys were homogenized
in a knife homogenizer followed by processing in a glass homoge-
nizer with a teflon pestle in 3 vol of 10 mM sodium phosphate buf-
fer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium
salt, and 2.5 mM b-mercaptoethanol. The homogenate was centri-
fuged at 10,000g at 0–4 °C for 30 min and the supernatant was sub-
jected to ammonium sulfate fractional precipitation at 40%, 50%,
and 75% salt saturation. The pellet obtained from the last step, pos-
sessing ALR1 activity, was redissolved in 10 mM sodium phosphate
buffer, pH 7.2, containing 2.0 mM EDTA dipotassium salt and
2.0 mM b-mercaptoethanol to achieve total protein concentration
of approx. 20 mg/mL. DEAE DE 52 resin was added to the solution
(33 mg/mL) and after gentle mixing for 15 min removed by centri-
fugation. The supernatant containing ALR1 was then stored in
smaller aliquots at ꢂ80 °C. No appreciable contamination by
ALR2 in ALR1 preparations was detected since no activity in terms
of NADPH consumption was observed in the presence of glucose
substrate up to 150 mM.
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4.3.3. Enzyme assays
ALR2 and ALR1 activities, according to the reported procedure,4
were assayed spectrophotometrically by determining NADPH con-
sumption at 340 nm. In order to determine ALR2 inhibitory
activity,
surements took place at 30 °C, whereas ALR1 inhibitory activity
was determined with -glycuronate as a substrate and the mea-
surements took place at 37 °C. Compounds 3a–c, 4a–c and 5 were
D,L-glyceraldehyde was used as a substrate and the mea-
D
Please cite this article in press as: Kotsampasakou, E. ; Demopoulos, V. J. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/
j.bmc.2012.12.015