Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors

Article abstract of DOI:10.1016/j.chembiol.2018.07.008

Aldose reductase (AKR1B1), the key enzyme of the polyol pathway, plays a crucial role in the development of long-term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has fai

Full text of DOI:10.1016/j.chembiol.2018.07.008

Brief Communication
Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as
Aldose Reductase Differential Inhibitors
Graphical Abstract
Authors
Francesco Balestri, Luca Quattrini,
Vito Coviello, ..., Antonella Del Corso,
Umberto Mura, Concettina La Motta
Correspondence
concettina.lamotta@unipi.it
In Brief
In this work, Balestri and co-authors
provide the evidence that
pyrazolopyrimidine derivatives are able to
inhibit differently the catalytic activity of
aldose reductase, depending on the
presence of specific substrates. This
finding opens up intriguing opportunities
to target long-term diabetic
complications while preserving the
detoxifying role of the enzyme.
Highlights
d
d
d
Pyrazolopyrimidines behave as aldose reductase differential
inhibitors
They inhibit L-idose and GS-HNE reduction more efficiently
than HNE reduction
They may be useful candidates for a novel approach to treat
diabetes complications
Balestri et al., 2018, Cell Chemical Biology 25, 1–5
November 15, 2018 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2018.07.008

Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
Cell Chemical Biology
Brief Communication
Acid Derivatives of Pyrazolo[1,5-a]pyrimidine
as Aldose Reductase Differential Inhibitors
Francesco Balestri,2,3 Luca Quattrini,1,3 Vito Coviello,1,4 Stefania Sartini,1 Federico Da Settimo,1 Mario Cappiello,2
Roberta Moschini,2 Antonella Del Corso,2 Umberto Mura,2 and Concettina La Motta1,5,
*
1
`
Dipartimento di Farmacia, Universita di Pisa, Via Bonanno 6, Pisa 56126, Italy
Dipartimento di Biologia, Unita di Biochimica, Universita di Pisa, Via L. Ghini 13, Pisa 56126, Italy
2
`
`
3These authors contributed equally
4Present address: V.C. Pietrasanta Pharma S.p.A., Via S. Francesco, 67, Viareggio, LU 55049, Italy
5Lead Contact
*Correspondence: concettina.lamotta@unipi.it
https://doi.org/10.1016/j.chembiol.2018.07.008
SUMMARY
sults in clinical trials with humans or even leading to adverse side
effects. These latter, in particular, have been recently correlated
Aldose reductase (AKR1B1), the key enzyme of the with the complex physiological role played by the enzyme (Alex-
iou et al., 2009).
In fact besides ruling the polyol pathway, AKR1B1 takes part
polyol pathway, plays a crucial role in the develop-
ment of long-term complications affecting diabetic
patients. Nevertheless, the expedience of inhibiting
this enzyme to treat diabetic complications has
failed, due to the emergence of side effects from
compounds under development. Actually AKR1B1
is a Janus-faced enzyme which, besides ruling the
polyol pathway, takes part in the antioxidant defense
to the antioxidant defense mechanisms of the body, being highly
efficient in reducing toxic aldehydes, including 4-hydroxy-2,3-
nonenal (HNE), arising in large quantities from pathological con-
ditions connected with oxidative stress (Srivastava et al., 1999).
At the same time, AKR1B1 catalyzes the reduction of glutathione
conjugates of unsaturated aldehydes, showing in most cases a
catalytic efficiency higher than that displayed against the parent
free aldehyde. In this regard, of special interest is the case of the
3-glutathionyl-4-hydroxynonanal (GS-HNE), whose reduction
mechanism of the body. In this work we report the ev-
idence that a class of compounds, characterized by a
pyrazolo[1,5-a]pyrimidine core and an ionizable frag- product 3-glutathionyl-dihydroxynonane (GSDHN) was shown
to be responsible for cytotoxicity through activation of the nu-
clear factor kB signaling pathways, thus further muddling the
ment, modulates differently the catalytic activity of
the enzyme, depending on the presence of specific
substrates such as sugar, toxic aldehydes, and
glutathione conjugates of toxic aldehydes. The study
stands out as a systematic attempt to generate
aldose reductase differential inhibitors (ARDIs) in-
tended to target long-term diabetic complications
while leaving unaltered the detoxifying role of the
physiological role of this enzyme (Ramana and Srivastava,
2010; Ramana, 2011).
Due to this Janus-faced nature, the expedience of inhibiting
AKR1B1 to prevent and treat long-term diabetic complications
has been debated for a long time and remains controversial.
To overcome the deadlock, we recently launched the concept
of aldose reductase differential inhibitors (ARDIs) as a novel
enzyme.
class of compounds able to selectively inhibit the catalytic activ-
ity of the enzyme depending on the specific substrates to be
transformed (Del Corso et al., 2013).
INTRODUCTION
This approach, which may be in principle adopted for any
enzyme able to act on different substrates (Cappiello et al.,
Aldose reductase (AKR1B1), the first enzyme of the so-called 2014), appears truly tailored for AKR1B1 which, besides being
polyol pathway, is acknowledged as the critical checkpoint for an aspecific enzyme, is also not permissive (Balestri
the main pathological changes affecting over time the nervous, et al., 2017).
renal, cardiovascular, and ocular systems of diabetic patients.
Differently from ARIs developed until now, characterized by
Accordingly, its inhibition has been pursued over decades as a high but unspecific binding affinity against the target enzyme,
useful therapeutic strategy to prevent the onset of diabetic com- ARDIs should show a differential intrasite binding affinity, being
plications (Del Corso et al., 2008).
able to inhibit the catalytic activity of AKR1B1 against glucose
However, despite the efforts to obtain effective aldose reduc- and GS-alkanals while leaving unaltered, or, in any case less
tase inhibitors (ARIs) (Grewal et al., 2016), a true and wide- affected, the reductive activity of the enzyme toward toxic hydro-
spread ARI therapy is not yet an established fact, and epalre- phobic aldehydes. Accordingly, using an ARDI it is possible in
stat is the only agent successfully marketed in Japan, India, principle to prevent hyperglycemia-induced cell injury without
and China for the treatment of diabetic neuropathy (Li affecting the antioxidant defense.
et al., 2016).
When considering differential inhibition, the choice of the sub-
Products that appear to be promising during preclinical inves- strate to be used for in vitro studies is not a trivial task. In this re-
tigations often fail to proceed any further, showing uncertain re- gard, on the bases of the peculiar nonhyperbolic inhibition
Cell Chemical Biology 25, 1–5, November 15, 2018 ª 2018 Elsevier Ltd.
1

Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
compounds were firstly carried out exploiting L-idose as the sub-
strate, chosen as a viable alternative to the physiologically rele-
vant D-glucose. Actually although similar in terms of structural
rearrangement, the C-5 epimer of D-glucose turns out to be
more useful to investigate the activity of the enzyme, as it
offers a significantly higher concentration of the free aldehyde
form when compared with the parent compound (Balestri
et al., 2015b).
While 5a exhibited an IC50 value of approximately 0.5 mM and
5b and 5c were devoid of any appreciable inhibitory action on
hAKR1B1 (IC50 > 1 mM), the IC50 values of all the other molecules
in inhibiting L-idose reduction were of the same order of magni-
Figure 1. Differential Inhibition of Pyrazolo[1,5-a]pyrimidine Deriva-
tude, ranging from approximately 60 mM to 200 mM. Afterward,
tives, Epalrestat and Sorbinil, on hAKR1B1
focusing on the most active compounds (5a,d–j,l, Figure S1),
4-Substituted-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine derivatives, 5a,d–j,l,
the same assays were repeated using HNE as the substrate to
investigate their ARDI efficacy. The resulting differential inhibi-
tory activity between L-idose and HNE reduction (differential in-
hibition [DI], in percent) is shown in Figure 1.
were tested as differential inhibitors at the concentration of 70 mM using either
0.8 mM L-idose or 0.04 mM HNE as the substrates, in the presence of 8 mU of
purified hAKR1B1. Bars (differential inhibition) indicate the difference between
the percentage inhibition observed using L-idose as the substrate and the
percentage inhibition using HNE as the substrate. Values represent the mean
from at least three independent measurements, and error bars (if not visible are
within the symbol size) refer to the SD of the mean. Epalrestat (epa) and sorbinil
(sorb), adopted as negative controls differential inhibitors, were used at a con-
centration of 0.1 mM and 1.0 mM, respectively.
It is noteworthy that 5a, which inspired the ARDI library being a
promising differential inhibitor when tested on the bovine lens
enzyme (Del Corso et al., 2013), in the case of the human recom-
binant enzyme used in the present study showed a reduced
inhibitory activity and did not exhibit any differential inhibitory ac-
tion between L-idose and HNE reduction. Although disap-
pointing at first glance, the observed activity is not a peculiar
event, it being well documented that AKR1B1 from different
sources is possibly differently targeted by inhibitory molecules,
with the human placental enzyme appearing less susceptible
to inhibition (Kador et al., 1980, 1985; Morjana and Flynn,
1989; Lee et al., 2010).
exerted on the enzyme by aldose hemiacetals (Balestri et al.,
2015a), D,L-glyceraldehyde, usually adopted for inhibition
studies, becomes rather inadequate as a mimicking aldose sub-
strate. Thus when the aim of the test is to evaluate differential in-
hibition, different physiological substrates such as glucose or
structurally related molecules (Balestri et al., 2015b), HNE, and
GS-HNE, chosen as examples of lipid aldehydes and gluta-
thionyl-aldehyde adducts metabolized by the enzyme, respec-
tively, should be used.
In this respect it is well conceivable that, considering the pecu-
liar structural restrictions required for a molecule to act as an
ARDI, differences in DI susceptibility between different enzyme
forms have certainly more chances to take place. In any case,
moving from 5a, suitable decoration of the pyrazolopyrimidine
scaffold made it possible to recover ARDI features, as shown
in Figure 1.
Screening an in-house collection of previously developed
compounds, characterized by different but customary pharma-
cophoric elements for AKR1B1 molecular recognition such as
the hydantoin ring (Da Settimo et al., 2005a, 2005b), the phenolic
fragment (La Motta et al., 2007), and the carboxylic moiety (Da
Settimo et al., 2001, 2005a, 2005b; La Motta et al., 2008; Cosco-
nati et al., 2009), we identified 7-oxo-4,7-dihydropyrazolo[1,5-a]
pyrimidine-6-carboxylic acid 5a (Figure 1) as a promising ARDI,
being able to intervene in glucose and GS-HNE reduction, cata-
lyzed by AKR1B1 from bovine lens, more efficiently than in HNE
reduction (Del Corso et al., 2013).
In particular, the presence of a p-chloro atom on the 4-benzyl
ring leads to an appreciable result and 5e, showing a DI value of
16%, turns out to be the best differential inhibitor of the whole se-
ries. Changing the less bulky, more electron-attracting chloro
atom into the bulkier, less electron-attracting bromine one, as
in 5f, halved the DI value (DI: 8.44%), and the insertion of the
even bulkier trifluoromethyl group carried out the negative trend,
as 5g showed a DI value of 2.6%. Finally, 5h, showing a DI value
of 1.4% and featuring the ortho-fluoro para-bromo substitution
pattern typical of known ARIs such as ponalrestat, ranirestat,
and minalrestat, turned out to be the least effective among this
subseries.
With this in mind, and aiming to develop a class of effective
ARDIs, we undertook the synthesis of a number of pyrazolopyr-
imidine derivatives and investigated their functional efficacy in
L-idose, HNE, and GS-HNE reduction catalyzed by the human
recombinant enzyme, hAKR1B1.
Insertion of the electron-donating methyl group in the same
position of the 4-benzyl ring retains a significant differential pro-
file of inhibition (5i, DI: 11.0%), and the same was also true when
RESULTS AND DISCUSSION
Moving from the lead compound 5a (Figure S1), identified as a the pendant phenyl ring was replaced by the bulkier naphthalene
promising ARDI toward the bovine lens enzyme (Del Corso core (5j, DI: 11.3%). On the contrary, the presence of a methy-
et al., 2013), we synthesized a number of parent derivatives char- lene spacer between the main pyrazolopyrimidine lipophilic
acterized by suitable substituents in position 4 of the heterocy- area and the additional aromatic ring in position 4, as in 5l, turned
clic core, to verify the influence of electron withdrawal, electron the ARDI profile upside down, as the compound rather preferen-
release, and steric bulk on the differential intrasite binding affinity tially blocks hAKR1B1, even though slightly, when processing
of the compounds. In vitro inhibitory tests on the synthesized HNE instead of L-idose (DI: ꢀ4.03%).
2
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Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
A
B
C
Figure 2. Inhibition Effectiveness of Compounds 5e, 5g, Epalrestat, and Sorbinil on the hAKR1B1-Catalyzed Reduction of the Following Sub-
strates: L-idose (0.6 mM, Diamonds), HNE (0.04 mM, Circles), and GS-HNE (0.04 mM, Triangles)
(A) The enzyme activity in the presence of compound 5e was determined at the indicated inhibitor concentrations. Values represent the mean from at least three
independent measurements, and error bars (if not visible are within the symbol size) refer to the SD of the mean.
(B) The enzyme activity in the presence of compound 5g was determined at the indicated inhibitor concentrations. Values represent the mean from at least three
independent measurements, and error bars (if not visible are within the symbol size) refer to the SD of the mean.
(C) The enzyme activity in the presence of epalrestat (open symbols) and sorbinil (closed symbols) was determined at the indicated inhibitor concentrations.
Values represent the mean from at least three independent measurements, and error bars (if not visible are within the symbol size) refer to the SD of the mean.
Classical powerful ARIs such as epalrestat and sorbinil were response for the two classical ARIs are also reported. The
also tested as ARDIs. However, as reported in Figure 1, their dif- emerging IC50 values related to L-idose, HNE, and GS-HNE
ferential inhibitory ability appears negligible. This result clearly reduction (Table 1) are consistent with a differential inhibitory ac-
demonstrates that structural requirements defined to date to tion exerted only by 5e.
obtain potent ARIs are not helpful in ARDI design and develop-
ment, and novel pharmacophoric elements must be unveiled to undertook a detailed kinetic analysis of the inhibition action of 5e,
obtain effective and potent differential derivatives. using as negative controls for differential action 5g, displaying
To unveil the pattern of interaction with the target enzyme, we
The inhibitory profile of the most effective ARDI (5e) and of a an IC50 rather comparable with that of 5e, and the powerful
compound (5g), displaying a comparable inhibitory power but ARIs epalrestat and sorbinil. Results reported in Figures S1–S4
a rather modest differential inhibitory ability, was investigated allow determination of the inhibition models of action and the
further. Besides being differentially active between L-idose and relative apparent dissociation constants for the binary enzyme/
HNE reduction, 5e turned out to exert its inhibitory action also to- inhibitor complexes (KiðappÞ) and for the ternary enzyme/sub-
ward the reduction of GS-HNE, thus proving to fulfill thoroughly strate/inhibitor complexes (Ki0ðappÞ) with respect to the different
the functional requirements of an ARDI. The inhibition curves substrates.
referring to the three substrates, reported in Figure 2A, show
For both inhibitors 5e and 5g, there appears to be significant
that the reduction of L-idose and GS-HNE is similarly affected preferential targeting of the free enzyme (Ki0ðappÞ=KiðappÞ>1) to an
by the inhibitor and that the effect is more pronounced than extent that the inhibitory action comes very close to competitive
that on HNE reduction. This is not the case for 5g (Figure 2B), inhibition, as for 5e acting on GS-HNE reduction and 5g acting
which inhibits AKR1B1 irrespective of the used substrate. The on HNE reduction. The measured KiðappÞ values (Table 2), espe-
lack of any differential inhibitory action exerted by epalrestat cially for 5e, appear affected by the nature of the substrate un-
and sorbinil toward the reduction of all three adopted substrates dergoing reduction. In this regard, 5e seems to target the free
is again clearly evident in Figure 2C, where the dose/effect enzyme more efficiently on L-idose and GS-HNE reduction,
approximately 2-fold and 4-fold, respectively, with respect to
the HNE reduction, evidence that may be invoked as the basis
of the observed differential inhibitory action of this compound.
Table 1. Comparison of IC50 Values of 5e, 5g, Sorbinil, and
Epalrestat for the AKR1B1-Dependent Reduction of Different
On the contrary, the apparent Ki values of 5g are rather similar
Substrates
for all of the three tested substrates. The same occurs for epalre-
Substratesa
stat and sorbinil, which display a mixed type of inhibition and an
Inhibitors
5eb
L-idose
HNE
GS-HNE
uncompetitive type of inhibition, respectively. As shown in Fig-
ures S3 and S4, the targeting of these powerful inhibitors to
hAKR1B1 appears to occur irrespective of the substrate under-
going reduction.
157 47
129 42
0.90 0.18
0.12 0.03
403 100
140 24
0.90 0.18
0.16 0.03
157 55
5g
161 80
Sorbinil
Epalrestat
1.47 0.28
0.14 0.04
It is important to point out that the absolute values of the inhi-
bition constants reported in Table 2, for 5e and 5g, necessarily
obtained in the presence of DMSO, must be corrected taking
into account the DI displayed by DMSO on the L-idose and
HNE reduction catalyzed by hAKR1B1 (Misuri et al., 2017). In
particular, the apparent kinetic constants for the tested com-
pounds should be divided for a factor generally defined as
IC50 values (mM) are expressed as the mean SD of three independent
measurements.
aAssay conditions were those described in Figure 2.
bCompound with the most significant IC50 difference between the reduc-
tion of HNE and the reduction of L-idose and GS-HNE (p < 0.0001, one-
way ANOVA, GraphPad Prism 6.0).
Cell Chemical Biology 25, 1–5, November 15, 2018
3

Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
Table 2. Inhibition Kinetic Parameters Related to Compounds 5e, 5g, Epalrestat, and Sorbinil, as Emerging from the Kinetic Analysis of
Figures S1–S4
Compound 5e
Ki0ðappÞ
Compound 5g
Ki0ðappÞ
Epalrestat
Ki0ðappÞ 3 102
Sorbinil
Ki0ðappÞ 3 102
Substrate
L-idose
HNE
KiðappÞ
Ki0/Ki
4.6
KiðappÞ
95
Ki0/Ki
1.8
KiðappÞ 3 102
13 0.3
Ki0/Ki
1.72
1.62
2.36
568 132
619 22
124
8
170 10
3
6
8
0.4
10 1.4
0.5
50 1.5
269 15
70 16
2.3
2,984 1,559
387 103
108
27.6
2.9
17 3.9
78 2.6
GS-HNE
1,475 500
21.0
132 12
8
19 0.5
118 22
Inhibition constants (mM) are expressed as the mean SD of three independent measurements.
B Expression and Purification of AR
B Protein Concentration Determination
B Inhibition Assay
f1 + ð½DMSOꢁ=KDMSOÞg, where [DMSO] refers to the concentra-
tion of DMSO present in the assay and KDMSO refers to the spe-
cific dissociation constants measured for DMSO either acting as
competitive inhibitor of L-idose reduction or acting as a mixed-
type inhibitor of the HNE reduction. Thus, for L-idose reduction,
only the KiðappÞ value will be reduced by factor of 1.85, while the
KiðappÞ and Ki0ðappÞ values measured when HNE was used as sub-
strate will be reduced by a factor of 1.75 and 1.53, respectively.
No correction is required for the apparent inhibition constant
referring to GS-HNE reduction, DMSO being ineffective on the
reduction of this substrate (Misuri et al., 2017). With such an
approach, the competitive inhibitory efficiency of 5e toward
L-idose and GS-HNE reduction appears to be expressed by
essentially the same Ki value (67 4 mM and 70 16 mM, respec-
tively) which is less than one-half of the Ki value measured for
HNE reduction.
B Statistical Analysis
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures, one scheme, and two tables
and can be found with this article online at https://doi.org/10.1016/j.
chembiol.2018.07.008.
ACKNOWLEDGMENTS
The authors acknowledge Regione Toscana for the financial support (IDARA
Project, DD650/2014).
AUTHOR CONTRIBUTIONS
F.B. conducted the kinetic characterization study. R.M. and F.B. expressed
and purified the target protein. M.C. conceived and applied
a kinetic
SIGNIFICANCE
models-based approach for validation of ARDI effectiveness. L.Q., V.C., and
S.S. conceived and conducted the chemical synthesis of the inhibitors.
A.D.C., U.M., F.D.S., and C.L.M. devised the work project. A.D.C. and
C.L.M. wrote the paper. All authors analyzed and discussed the achieved re-
sults and reviewed the manuscript.
In this work we presented a series of pyrazolo[1,5-a]pyrimi-
dine derivatives, developed as aldose reductase differential
inhibitors. Among the synthesized compounds, derivative
5e, 4-(4-chlorobenzyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]py-
DECLARATION OF INTERESTS
rimidine-6-carboxylic acid, emerged as
a viable lead,
proving to exert its inhibition toward the catalytic activity
of hAKR1B1 with a preferential action against both L-idose
and GS-HNE with respect to HNE. It clearly represents an
original compound which, differently from traditional aldose
reductase inhibitors (ARIs) developed to date, is intended to
target long-term diabetic complications while leaving unal-
tered the detoxifying role of the enzyme. Further studies in
animal models will prove its robustness as a prototypical
drug candidate, exploitable to prevent the onset and pro-
gression of hyperglycemia-induced complication without
the emergence of the adverse side effects commonly shown
by known ARIs.
The authors declare no competing interests.
Received: November 7, 2017
Revised: April 20, 2018
Accepted: July 23, 2018
Published: August 16, 2018
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Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
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reductase inhibitors. Bioorg. Med. Chem. 13, 491–499.
Misuri, L., Cappiello, M., Balestri, F., Moschini, R., Barracco, V., Mura, U., and
Del-Corso, A. (2017). The use of dimethylsulfoxide as a solvent in enzyme in-
hibition studies: the case of aldose reductase. J. Enzyme Inhib. Med. Chem.
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Da Settimo, F., Primofiore, G., La Motta, C., Sartini, S., Taliani, S., Simorini, F.,
Marini, A.M., Lavecchia, A., Novellino, E., and Boldrini, E. (2005b). Naphtho
[1,2-d]isothiazole acetic acid derivatives as a novel class of selective aldose
reductase inhibitors. J. Med. Chem. 48, 6897–6907.
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muscle: purification, substrate specificity, immunological characterization
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La Motta, C., Da Settimo, F., and Mura, U. (2013). A new approach to control
the enigmatic activity of aldose reductase. PLoS One 8, e74076.
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Cell Chemical Biology 25, 1–5, November 15, 2018
5

Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Bacterial and Virus Strains
E. coli XL-1 Blue competent cells
E. coli BL21(DE3)pLysS cells
Chemicals, Peptides, and Recombinant Proteins
D,L-Dithiothreitol
SOURCE
IDENTIFIER
Agilent Technologies
Promega
CAT# 200249
CAT# L1195
Sigma-Aldrich
CAT# D0632
CAT# G5001
CAT# D2650
CAT# NN10871
CAT# MI04205
N/A
D,L-glyceraldehyde
Sigma-Aldrich
Dimethylsulfoxide
Sigma-Aldrich
NADPH
Carbosynth
L-idose
Carbosynth
HNE
Moschini et al., 2015
Cayman Chemicals
Sigma-Aldrich
GSHNE
CAT# 10627
CAT# T9424
CAT# 18080044
CAT# 27079804
CAT# A2153
TRIꢀReagent
SuperScriptꢁ III Reverse Transcriptase
T. aquaticus DNA polymerase
Bovine serum albumin
Critical Commercial Assays
QIAEX II Extraction kit
Plasmid MiniPrep Kit
Oligonucleotides
Thermo Fisher Scientific
GE Healthcare
Sigma-Aldrich
QIAGEN
CAT# 20021
EuroClone
CAT# EMR500050
Primer for hAKR1B1: 5’-CAT ATG GCA
Balestri et al., 2015b
Balestri et al., 2015b
N/A
N/A
AGC CGT CTC CTG CTC AA-3’
Primer for hAKR1B1: 5’-GAA TTC TCA AAA
CTC TTC ATG GAA-3’
Recombinant DNA
pGEM plasmid
Promega
CAT# A3600
Software and Algorithms
GraphPad Instat version 6.0
GraphPad
RRID: SCR_002798
Other
Matrex Orange A
Millipore
CAT# 19211
Ultracelꢀ 10 kDa ultrafiltration membranes
Diethylaminoethyl cellulose DE-52
Sephadex G75
Merck-Millipore
Whatman GE HealthCare
GE Healthcare
CAT# PLGC02510
CAT# 4057-050
CAT# 17005001
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Concettina
La Motta (concettina.lamotta@unipi.it).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
E. coli XL-1 Blue competent cells and E. coli BL21(DE3)pLysS cells were cultured in Luria Bertani (LB) broth at 37ꢂC.
METHODS DETAILS
Chemistry Reagents and Experimentation
3-Aminopyrazolo, diethyl 2-(ethoxymethylene)malonate, and the suitably substituted alkyl halides, used to obtain the target inhibi-
tors, were from Alfa Aesar, Aldrich and Fluka. The carboxylic acid derivatives, 5a-l, were synthesized as outlined in Scheme S1.
e1 Cell Chemical Biology 25, 1–5.e1–e3, November 15, 2018

Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
¨
Melting points were determined using a Reichert Kofler hot-stage apparatus and are uncorrected. Routine nuclear magnetic reso-
nance spectra were recorded in DMSO-d6 solution on a Varian Gemini 200 spectrometer, operating at 200 MHz, and on a Bruker 400
spectrometer, operating at 400 MHz. Evaporation was performed in vacuo (rotary evaporator). Analytical TLC was carried out on
Merck 0.2 mm precoated silica gel aluminium sheets (60 F-254). Purity of the target inhibitors, 5a-l, was determined by HPLC anal-
ysis, using a Merck Hitachi D-7000 liquid chromatograph (UV detection at 242 nm) and a Discovery C18 column (250 mm x 4.6 mm,
5 mm, Supelco), with a gradient of 40% water and 60% methanol and a flow rate of 1.4 mL/min. All the compounds showed percent
purity values R95%.
Synthesis of Ethyl 7-Oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate, 3
The commercially available 3-aminopyrazole 1 (1.00 mmol) was dissolved in 10 mL of glacial acetic acid. Diethyl ethoxymethylene-
malonate 2 (0.24 mL, 1.20 mmol) was then added and the solution was refluxed under stirring for 2 hours (TLC analysis). After cooling
at room temperature, the reaction mixture was treated with crushed ice and the white solid that precipitated was collected by filtra-
tion, washed with water and purified by recrystallization from MeOH (yield: 95%). P.f.: 295ꢂC. 1H-NMR (d, DMSO-d6): 1.30 (t, 3H), 4.00
(s, 1H), 4.20 (q, 2H), 6.33 (d, 1H), 7.50 (d, 1H), 8.33 (s, 1H).
General Procedure for the Synthesis of Ethyl 4-Substituted-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-
carboxylate Derivatives, 4a-l
The appropriate alkyl halide (1.20 mmol) was added to an ice-cooled solution of ethyl 7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-
carboxylate 3 (1.00 mmol) and K2CO3 (1.20 mmol) in dry DMF. After addition, the reaction mixture was heated at T=90ꢂC under stir-
ring until the disappearance of the starting material (TLC analysis). After cooling at room temperature, the solvent was removed under
reduced pressure and the resulting crude material was treated with crushed ice. The white solid that precipitated was collected by
filtration, washed with water and purified by recrystallization from the suitable solvent (Table S1).
General Procedure for the Synthesis of 4-Substituted-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic Acid
Derivatives, 5a-l
A suspension of ethyl ester, 4a-l (1.00 mmol) in 3 mL of 5% NaOH was heated at T=100ꢂC with stirring until a solution was achieved
(TLC analysis). After cooling at room temperature, the solution was filtered and acidified with concentrated hydrochloric acid under
ice-cooling. The white solid that precipitated was collected by filtration, washed with water and purified by recrystallization from the
suitable solvent (Table S2).
Enzymatic Assay
The activity of human placental aldose reductase (hAKR1B1) was determined at 37ꢂC, following the decrease in absorbance at
340 nm due to NADPH oxidation (ε340: 6.22mM-1 cm-1) (Balestri et al., 2016). The standard assay mixture (700 mL) contained
0.25 M sodium phosphate buffer pH 6.8, 0.4 M ammonium sulfate, 0.5 mM EDTA, 0.18 mM NADPH, and 4.7 mM glyceraldehyde.
The rate of NADPH oxidation in the absence of the substrate was subtracted as a blank. One unit of enzyme activity is the amount
that catalyzes the conversion of 1 mmol of substrate/min in the above assay conditions. The above assay conditions were also adop-
ted when L-idose, HNE or GSHNE (at the indicated concentrations) were used as substrates in inhibition studies.
Expression and Purification of AR
The human recombinant enzyme was expressed in E. coli cells as previously described (Balestri et al., 2015b). Total RNA was ex-
tracted from human placenta with TRIꢀReagent, according to the manufacturer’s protocol. cDNA was prepared from total RNA
by reverse transcription, using 200 units of SuperScriptꢁ III Reverse Transcriptase and 0.5 mg of an oligo-dTprimer in a mixture
(50 mL total volume) containing 0.5 mM of each dNTP, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.1 mg/mL bovine
serum albumin in 50 mM Tris–HCl, pH 8.3. After 60 min at 50ꢂC the incubation mixture was used for PCR amplification or stored at
-20ꢂC. Aliquots of 1 mL of crude cDNA were amplified in a Bio-Rad Gene Cyclerꢁ thermocycler, using 2.5 units of T. aquaticus DNA
polymerase, 1 mM of each dNTP, 1 mM of each PCR primer, 50 mM KCl, 2.5 mM MgCl2 and 0.1 mg/mL BSA in 10 mM Tris–HCl, pH
8.3, containing 0.1% (v/v) Triton X-100. At the 5’ end, we used the specific primer: 5’-CAT ATG GCA AGC CGT CTC CTG CTC AA-3’,
corresponding to the sequence encoding the first six amino acids of the mature protein and containing an Nde I restriction site for
ligation into the expression vector, which at the same time provided the ATG codon for an additional methionine in position 1. At the 3’
end the specific primer: 5’-GAA TTC TCA AAA CTC TTC ATG GAA-3’ encoded the last five amino acids, followed by a stop codon and
an Eco RI restriction site. After an initial denaturation step at 95ꢂC for 5 min, 35 amplification cycles were performed (1 min at 95ꢂC,
30 s at 50ꢂC, 1 min at 72ꢂC) followed by a final step of 7 min at 72ꢂC. An amplification product of about 900 bp was obtained, in agree-
ment with the expected size (948 bp). The crude PCR product was ligated into a pGEM vector without further purification, using a 1:5
(plasmid:insert) molar ratio and incubating the mixture overnight, at room temperature. After transformation of E. coli XL-1 Blue
competent cells with the ligation product, positive colonies were selected by PCR using the plasmid’s primers SP6 and T7 and grown
in LB/ampicillin medium. DNA was extracted using the Plasmid MiniPrep Kit and custom sequenced at Eurofins MWG. pGEM
plasmid containing the appropriate sequence was digested with Nde I and Eco RI restriction enzymes for 2 h at 37ꢂC and the diges-
tion product was separated on agarose gel. The obtained fragment was purified from gel using QIAEX II Extraction kit and ligated into
the expression vector pET30, previously linearized with the same enzymes. The resulting plasmid was sequenced and shown to
encode the mature protein.
For expression of recombinant protein, the pET-30 vector, containing the sequence encoding hAKR1B1, was used to transform E.
coli BL21(DE3)pLysS cells. Protein expression was induced by adding IPTG to a final concentration of 0.4 mM when the culture had
Cell Chemical Biology 25, 1–5.e1–e3, November 15, 2018 e2

Please cite this article in press as: Balestri et al., Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors, Cell Chemical
Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.008
reached a value of O.D.600=0.8. Cells were grown overnight at 37ꢂC, then harvested by centrifugation and sonicated. The enzyme
was purified from the cell extract by three chromatographic steps. In particular, the cell extract (approximately 0.3 g of proteins)
in 0.1 M sodium phosphate buffer pH 7 containing 2 mM DTT (standard buffer) was applied to a column (30 x 3.5 cm) of DE-52 resin,
previously equilibrated in standard buffer, and eluted by the same buffer at a flow rate of 15 ml/h monitoring on each fraction the
absorbance at 280 nm until the value declined to approximately 0.5. A linear gradient of NaCl ranging from 0 to 120 mM in standard
buffer was then applied allowing the enzyme to elute at approximately 100 mM salt concentration. Active fractions were pooled,
concentrated and dialyzed against standard buffer devoid of DTT on Ultracelꢀ 10 kDa ultrafiltration membrane, and then applied
on a 23x3.5 cm column containing Matrex Orange A resin, previously equilibrated in standard buffer devoid of DTT and eluted in
the same buffer at a flow-rate of 15 ml/h until the absorbance values at 280 nm fit the baseline. The enzyme was then eluted by sup-
plementing the elution buffer of 0.1 mM NADPH. The fractions containing hAR activity were pooled, supplemented of 2 mM DTT and
concentrated on Ultracelꢀ 10 kDa ultrafiltration membrane up to 7 mL.
The sample was then applied on a Sephadex G-75 column (80x2.6 cm) previously equilibrated in standard buffer and eluted at a
flow rate of 15 ml/h.
The fractions exhibiting enzymatic activity were tested for purity by SDS-Page electrophoresis using the silver stain nitrate tech-
nique (Laemmli, 1970; Wray et al., 1981), pooled, and concentrated on a YM10 membrane. The purified human recombinant enzyme
(5.3 U/mg of protein) was stored at -80ꢂC in standard buffer supplemented of 30% (w/v) glycerol. Immediately before use, the enzyme
was extensively dialyzed against standard buffer devoid of DTT.
Protein Concentration Determination
The protein concentration of enzyme preparations was evaluated by the Bradford method (Bradford, 1976).
Inhibition Assay
The sensitivity of AKR1B1 to inhibition was tested in the presence of inhibitors dissolved at proper concentrations in solvent mixture
(40% DMSO/60% methanol). Further dilutions with the solvent mixture were then performed to obtain the desired concentrations of
the inhibitors in order to keep the concentration of DMSO in the assay constant at 0.28 % (v/v). The reaction rate was measured as
described above (see Enzymatic Assays) either in the absence (v0) or in the presence (vi) of different concentrations of inhibitors and
results were expressed as percent of inhibition (i.e. v0-vi/v0). IC50 is defined as the concentration of inhibitor leading to 50% inhibition.
IC50 values were determined by non linear regression analysis by fitting the data to the equation describing one site competition in a
log-dose inhibition curve. The analysis was performed using standard statistical software (GraphPad Instat version 6.0, San Diego,
CA). Each curve was generated using at least five concentrations of inhibitors and each concentration was tested at least in triplicate.
The kinetic characterization of the inhibitors was performed using at least four different inhibitors concentrations and four different
concentrations of L-idose and HNE as substrates; in the case of GSHNE, three different concentrations of this substrate were used. In
all cases, the rate values were reported as the mean standard deviation from at least three independent measurements. Data were
analyzed through Hanes-Woolf plots obtained by plotting [S]/v0 versus [S] (Hanes, 1932). Dissociation constants for the binary
enzyme-inhibitor complex ((app)Ki) and for the ternary enzyme-inhibitor-substrate complex ((app)K’i) were determined from secondary
plots of KM/Vmax versus inhibitor concentration and 1/Vmax versus inhibitor concentration, respectively.
Statistical Analysis
IC50 values and inhibitor dissociation constants (app)Ki and (app)K’i are reported as the mean S.D. (standard deviation) of three in-
dependent determinations. For IC50, each experiment was conducted using at least five concentrations of inhibitors and each con-
centration was tested at least in triplicate. For the determination of inhibition constants, at least four different inhibitors concentrations
and at least three different substrate concentrations were used. The rate values were reported as the mean SD from at least three
independent measurements. The statistical significance was evaluated by One Way ANOVA (GraphPad Instat version 6.0, San
Diego, CA).
e3 Cell Chemical Biology 25, 1–5.e1–e3, November 15, 2018