Salicylic Acid Derivatives Inhibit Oxalate Production in Mouse Hepatocytes with Primary Hyperoxaluria Type 1

Article abstract of DOI:10.1021/acs.jmedchem.8b00399

Primary hyperoxaluria type 1 (PH1) is a rare life-threatening genetic disease related to glyoxylate metabolism and characterized by accumulation of calcium oxalate crystals. Current therapies involve hepatic and/or renal transplantation, procedures that have significant morbidity and mortality and require long-term immunosuppression. Thus, a pharmacological treatment is urgently needed. We introduce here an unprecedented activity of salicylic acid derivatives as agents capable of decreasing oxalate output in hyperoxaluric hepatocytes at the low micromolar range, which means a potential use in the treatment of PH1. Though correlation of this phenotypic activity with glycolate oxidase (GO) inhibition is still to be verified, most of the salicylic acids described here are GO inhibitors with IC₅₀ values down to 3 μM. Binding mode of salicylic acids inside GO has been studied using in silico methods, and preliminary structure-activity relationships have been established. The drug-like structure and ease of synthesis of our compounds make them promising hits for structural optimization.

Full text of DOI:10.1021/acs.jmedchem.8b00399

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Article
Salicylic Acid Derivatives Inhibit Oxalate Production in
Mouse Hepatocytes with Primary Hyperoxaluria Type 1
Maria Dolores Moya-Garzon, Cristina Martin Higueras, Pablo Peñalver, Manuela Romera, Miguel Xavier
Fernandes, Francisco Franco-Montalban, José A. Gómez-Vidal, Eduardo Salido, and Mónica Díaz-Gavilán
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00399 • Publication Date (Web): 20 Jul 2018
Downloaded from http://pubs.acs.org on July 25, 2018
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Salicylic Acid Derivatives Inhibit Oxalate Production in
Mouse Hepatocytes with Primary Hyperoxaluria Type 1.
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María Dolores Moya-Garzón, Cristina Martín Higueras, Pablo Peñalver,
Manuela Romera,
‡#
†
†
‡
Miguel X. Fernandes, Francisco Franco-Montalbán, José A. Gómez-Vidal, Eduardo Salido,*
†
Mónica Díaz-Gavilán* .
†
Departamento de Química Farmacéutica y Orgánica, Universidad de Granada, Campus de Cartuja s/n,
18071 Granada, Spain.
‡
Hospital Universitario de Canarias, Universidad La Laguna & Center for Rare Diseases (CIBERER),
38320 Tenerife, Spain.
ABSTRACT: Primary Hyperoxaluria Type 1 (PH1) is a rare life-threatening genetic disease related to
glyoxylate metabolism and characterized by accumulation of calcium oxalate crystals. Current therapies
involve hepatic and/or renal transplantation, procedures that have significant morbi-mortality and
require long-term immunosuppression. Thus, a pharmacological treatment is urgently needed. We
introduce here an unprecedented activity of salicylic acid derivatives, as agents capable of decreasing
oxalate output in hyperoxaluric hepatocytes at the low micromolar range, which means a potential use in
the treatment of PH1. Though correlation of this phenotypic activity with glycolate oxidase (GO)
inhibition is still to be verified, most of the salicylic acids described here are GO inhibitors with IC50
values down to 3 µM. Binding mode of salicylic acids inside GO has been studied using in silico
methods and preliminary structure-activity relationships have been established. The drug-like structure
and ease of synthesis of our compounds make them promising hits for structural optimization.
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INTRODUCTION
Primary hyperoxaluria type 1 (PH1) is a rare and life-threatening genetic alteration of
glyoxylate metabolism that is due to a deficit of the enzyme alanine-glyoxylate aminotransferase
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(
AGT). This enzymatic defect leads to an accumulation of glyoxylate, which is then converted to
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the end-product oxalate by lactate dehydrogenase (LDH). When oxalate levels exceed renal
excretion capacity, insoluble crystals of calcium oxalate accumulate, first in kidneys and
secondly in extra-renal tissues. No pharmacological treatment addressing the pathogenesis of
PH1 exists currently, although in some PH1 cases, administration of pyridoxine restores AGT
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,3,4
enzymatic activity.
Double kidney-liver transplantation is the only curative option, but
patients must be subjected to extensive immunosuppressive treatments for the rest of their life,
5,6
with a survival rate of 86, 80 and 69 % at 1 year, 5 years and 10 years, respectively. New
molecular strategies aim to avoid the risks and limitations of surgery and immunosuppression in
PH1. In such sense, current research focuses on two main lines. One of them is the recovery of
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AGT activity and the other one the palliative diminution of oxalate production by inhibiting
glyoxylate formation (substrate reduction therapy, SRT) or glyoxylate oxidation to oxalate (LDH
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,9
inhibition). SRT is a useful approach in inborn errors of metabolism characterized by substrate
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0,11
accumulation.
HYPDH) are being explored as targets for SRT in PH1.
which catalyzes oxidation of glycolate to glyoxylate, has recently been validated as a safe and
Enzymes glycolate oxidase (GO) and hydroxyproline dehydrogenase
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1,12,13
(
The FMN-dependent enzyme GO,
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efficient target for SRT in PH1 using genetically modified mice. GO-deficient mice (Hao1 )
-/-
−/−
developed normally without adverse phenotypic effects and double KO mice (Agxt1 Hao1 )
-/-
showed low levels of oxalate excretion compared to hyperoxaluric mice (Agxt1 ). In addition,
i
the small GO inhibitor (GOi) CCPST (Figure 1), with an IC50 = 43.5 µM and K = 20.3 µM
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against purified recombinant mouse GO (mGO), significantly reduced the production of oxalate
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in Agxt1 mouse hepatocytes in vitro [EC50(24 h) = 25.3 µM, (48 h) = 33 µM, (72 h) = 34 µM].
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Oral administration of CCPST to Agxt1 mice produced a 30-50% reduction in urine oxalate.
However treatment with CCPST needs a high dose which could lead to unwanted side effects
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and therefore special emphasis on the need of more potent GOi’s has been made.
Figure 1. Structures of GO inhibitors CCPST and CDST.
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4
Though the idea of using GOi’s for the treatment of PH1 has been suggested before and
several small-molecule GOi’s have been described, no useful drug has been identified for the
treatment of PH1 patients. Non-favorable physicochemical properties, such as poor aqueous
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solubility contribute to this in molecules such as CDST (Figure 1) (K
i
= 15 nM). Available
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17,
structures of spinach (sGO) and human enzymes (hGO) co-crystallized with inhibitors such
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as CCPST
and CDST
has allowed the establishment of some structure-activity
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relationships.
Known GOi’s bear a polar head with an acidic functionality; the presence
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of a protonated heteroatom located β with respect to the acidic function is beneficial.
Flat,
electron rich fragments stabilize ternary complex enzyme-cofactor-substrate by π-π interactions
with FMN flavin ring, and hydrophobic aliphatic or aromatic groups hang from the polar head.
The polar moiety of these inhibitors must enter into the GO active site while the side chain
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remains in the hydrophobic access channel causing a disorder that prevents its closure.
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Therefore, a two-region binding site is envisaged for GOi’s. According to crystallographic
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data,
CDST and CCPST mimic glyoxylate interactions in the active site of hGO (Figure 2).
Carboxyl groups of CDST, CCPST and glyoxylate overlay interacting with residues Arg167,
Arg263 and Tyr26 while the N atoms at position 3 of the heterocyclic rings of CDST and
CCPST overlay with the keto group in glyoxylate and interact with His260. Thiadiazole ring of
CCPST does not carry a proton and neither does the glyoxylate keto group. Therefore,
interaction of these two molecules would require the protonation of His260 and this would
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contribute to the weaker binding of CCPST compared to CDST. CDST and CCPST also
hydrogen bond to Tyr132 via the ring N2. This group of aminoacids constitutes the first of the
two binding regions for GOi’s. The second binding region establishes hydrophobic interactions
with the side chain of the inhibitor and is constituted by variable aminoacids for each GOi.
Trp110, Leu205, Tyr208 are noteworthy residues interacting with CCPST and CDST. Within
this second binding site, flexible chains such as the one of CDST would help to a better
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accommodation of the inhibitor.
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Figure 2. Interactions of glyoxylate (up), CCPST (center) and CDST (down) in hGO active
site. Obtained from crystallographic data (PDBid: 2RDU; 2W0U; 2RDT from top to bottom).
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Small proline-like inhibitors for HYPDH have also been prepared and their IC50s on
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isolated enzyme found to be around 0.30 mM. No data on cells have yet been published for this
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type of inhibitors.
Apart from GO and HYPDH inhibition using small molecules, development of
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siRNA
and antisense oligonucleotides against these enzymes are currently under
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research. Better ADME properties and an easier delivery into affected cells favors small
molecules over nucleotide molecules. However, it is also possible that synergistic effects could
arise from small drug/siRNA co-administration.
In our search for novel GOi’s as possible drugs for the treatment of PH1, we looked for
structural motifs matching the required characteristics of this type of inhibitors. We envisaged
the salicylic core as a promising candidate as it presents a protonated heteroatom located β
22
relatively to an acidic functionality. Its flat electron-rich ring bears a carboxylic acid that could
reproduce interactions at Arg167, Arg263 and Tyr26 while the protonated oxygen of the
phenolic function could act as hydrogen donor in the interaction with His260. As in the case of
CDST, no energetically costly protonation of His260 would be required. In addition, salicylate
3
0
derivatives are broadly used as therapeutic agents and the preparation of new compounds based
on this structure would easily lead to drug-like hits with good ADME properties. Our
preliminary docking studies for salicylic acid agreed with this presumption, showing favorable
interactions within hGO crystallographic structure and a binding pattern comparable to the one
of CDST and CCPST in the binding regions 1 and 2 (Figure 3). Using the same docking
methodology, interaction figures were obtained for CDST and CCPST agreeing with the
interaction profile already described from crystallographic studies (Figure S1).
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Figure 3. Docked salicylic acid inside hGO active site (PDBid: 2RDT).
We describe the design, synthesis and biological evaluation of salicylic acid derivatives as
novel agents decreasing oxalate output from PH1-mouse primary hepatocytes in a more effective
way than CCPST. The drug-like structures of these compounds make them promising hits for
structural modification. This finding opens the gate for salicylic acid derivatives to be used in the
treatment of PH1. No bibliographic precedent could be found relating salicylate core with this
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kind of biological activity. Therefore a new target for salicylates as well as a novel application
in the decrease of oxalate production is herein introduced.
RESULTS AND DISCUSSION
For all the compounds included in this study, we have evaluated their inhibitory activity on
recombinant mGO as well as their capacity to diminish oxalate production in hyperoxaluric
mouse hepatocyte cultures. In the cell-based assay, glycolate added to the cell culture medium is
metabolized to glyoxylate by GO and this last one oxidized to oxalate mainly by LDH. Excreted
oxalate is quantified from the extracellular medium using a colorimetric assay. When a good
GOi is added, the first step is blocked and a decrease of oxalate release is then observed. We
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have used theoretical partition coefficients (expressed as logPth), corresponding to unionized
species, as an empirical indicator to discard too lipophilic molecules (logP > 5) which could be
retained in the cellular membrane. Favorable logPth values (between 0 and 5) would help good
membrane permeability. Additionaly, we have made in silico calculations about binding mode
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inside the human enzyme. Mouse and human GO proteins share 89.5 % sequence identity. We
have chosen purified mGO as a high-quality tool for the screening of potential inhibitors as
activity results can be safely translated in to a human setting and, at the same time, the
comparability of our findings can be assured when the biological testing is escalated to the
available mouse cellular systems. On the other hand, prediction of the binding mode for each
compound inside hGO has allowed comparison with glyoxylate, CCPST and CDST
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crystallographic data.
Our work on salicylates began with the screening of a small group of salicylic-related
compounds (1-17, Figure 4) for a preliminary outlook on their GO inhibitory activity and their
-/-
capacity to reduce oxalate production in Agxt1 mouse primary hepatocytes. The group included
simple salicylic acids (1-9), benzofused derivatives (10-16) and the structuraly related
chloroquinoline (17), which were chosen according to structural diversity criteria. These
aromatic systems aim at exploring different types of interaction at the enzymatic active site.
Differently sized, rigid or flexible, aliphatic, aromatic and heteroaromatic side chains with polar
and hydrophobic functionalities were searched to explore interaction modes at the access
channel. Except for 16, all of them were obtained from commercial sources.
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Figure 4. Structures of salicylate-related compounds selected for a preliminary screening on
primary and secondary assays.
Salicylic acid (1) showed a low inhibitory effect (less than 30 %) at 25 µM over mGO
catalytic activity, lower than the activity found for CCPST (34.8 %) (Table 1). Neither halogen
substitutions on the phenyl ring (2, 3) nor chloride-quinoline (17) showed inhibitory increases
compared to compound 1. Addition of an amine group on C5 (4) instead of C4 (5) did indeed
yield higher inhibition of the enzymatic activity (71.9 ± 3.2 % at 25 µM), with an IC of 7.8 ±
50
-/-
1.2 µM, but did not decrease oxalate release in Agxt1 cells. In turn, 5-acetyl (6) and polar 5-
glutamine (7) substitutions did not improve GO inhibition compared to 1. Insufficient
hydrophobic interactions may be the reason for the low GO inhibitory activity observed for these
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compounds. Compounds 8 and 9 (diflunisal) present hydrophobic side chains, a key feature for
GO inhibition. Compound 9 still presented an inhibition percentage lower than 30 %, but
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compound 8 inhibited 82.8 ± 5.6 % GO enzymatic activity at 25 µM with an IC50 of 34.5 ± 1.2
µM. However, even with a favorable logPth value, it was neither efficient decreasing oxalate
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Table 1. Biological data of the first set of compounds and CCPST and in silico prediction of
partition coefficient (logPth). Biological determinations: (i) Enzymatic inhibition on purified
mouse glycolate oxidase (mGO), assays either at single concentration (25 µM) and IC
calculation after fitting dose-response curves. (ii) Excreted oxalate in Agxt1 mouse primary
cultured hepatocytes, compared to control (relative oxalate).
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
mGO
(%)
2
Relative
oxalate
Compound
IC50 (µM)
R
b
logPth
c
CCPST
34.8 ± 4.8
18.5 ± 13.7
20.8 ± 4.4
13.5 ± 4.6
71.9 ± 3.2
14.9 ± 7.9
22.1 ± 1.0
7.8 ± 2.8
43.5 ± 1.1
n/a
0.63 ± 0.16
n/a
1
2
3
4
5
6
7
8
9
n/a
n/a
n/a
1.46
1.98
1.60
0.68
0.68
0.77
0.42
0.29
3.42
3.25
1.00
5.93
4.87
4.62
6.17
0.72
2.53
n/a
n/a
7.8 ± 1.2
n/a
0.93
0.88
0.88 ± 0.02
n/a
n/a
n/a
n/a
n/a
82.8 ± 5.6
27.9 ± 0.6
42.3 ± 1.1
96.3 ± 0.5
75.3 ± 2.6
75.1 ± 2.3
1.5 ± 2.1
34.5 ± 1.2
n/a
0.88 ± 0.18
0.88 ± 0.08
0.99
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
39.9 ± 1.1
2.9 ± 1.1
11.1 ± 1.1
n/a
0.94
0.97
0.98
0.77 ± 0.01
0.99 ± 0.01
1.11 ± 0.03
n/a
n/a
47.4 ± 2.3
26.2 ± 1.6
24.8 ± 5.9
n/a
0.91 ± 0.04
n/a
n/a
n/a
n/a
a
Percentage of inhibition of purified mGO at 25 µM of the drug, after 1 min.
Oxalate output tested at 24h post-treatment with 10 µM of the drug in Agxt1
b
-/-
mouse primary hepatocytes cultured with 5 mM glycolate, in 6-well plates.
Data are represented as mean ± SD of n = 3 replicates. One-way ANOVA
c
statistical analysis, p<0.05 was considered statistically significant. Data at 12.5
-
/-
µM in Agxt1 mouse primary hepatocytes. IC : Calculated on mouse
5
0
2
glycolate oxidase in saturated conditions of glycolate (22.2 mM). R : IC50
curve-fitted score. n/a: Not assessed.
A total of seven commercial benzofused salicylates (naphthylic acids) with different
substitution patterns were included in the preliminary screening (10-16, Figure 4). The additional
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benzene ring was thought to possibly reinforce the binding of the molecule to the target via
hydrophobic interactions. The increase in the hydrophobicity consequent to the adition of the
fused benzene is in fact reflected in higher logPth values (Table 1). The simplest of these
molecules carried a bromine group bound to the naphthalene core (10), while more complex
structures had an electron-rich linker (azo group, sulfur or oxygen atom) to aromatic cycles
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9
0
(
tetrazole, benzene or naphthalene). 4-Substituted 1-hydroxy-2-naphthylic acids with flexible
ether or thioether linkers, 11 and 12, presented good inhibition percentages on isolated mGO
96.3 ± 0.5 and 75.3 ± 2.6 %, respectively) (Table 1). Compounds 13-15 are 4-arylazo-3-
(
hydroxy-2-naphthylic acids with rigid azo linkers; 13 and 14 bear polar sulfonic functionalities
on the aromatic side chain while 15 contains lipophilic halogens. Within this group, compound
13 yielded a 75.1 ± 2.3 % mGO inhibition at 25 µM, the activity of 15 remained close to 50 %,
while 14 dramatically lost inhibition activity. Finally, compound 10, with no extended side
chain, showed an inhibitory activity below 50 %. Replacing the bromine atom on the C7 of 10
with an aromatic side chain (16), using Suzuki-Miyaura cross-coupling, led to lower inhibitory
activity. From this first set of compounds, we found the naphthyl derivative 11 to be the best
GOi (IC = 2.9 ± 1.1 µM), which also resulted in the most efficient hit in vitro, but still limited
5
0
to only 23 % reduction of excreted oxalate at 10 µM.
Predicted binding modes show that compounds 1-17 reproduce with only slight differences
the interactions of glyoxylate, CCPST and CDST in the active site of hGO (Table S1)
(
representative 2D-interaction diagrams for group 1-17 are shown in Figure 5; 2D-interaction
diagrams for the whole group 1-17 in Figure S2). Electrostatic and/or hydrogen bond interactions
with Arg167 and Arg263 can be established through both carboxy and hydroxy groups of the β-
hydroxybenzoic fragment of 1-17. Salicylic acids 1-9 also establish hydrogen bond interactions
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with His260 through the heteroatoms in the salicylic head although they lack interaction with
Tyr26 (except 6 and 7). Contrary to what had been initially envisaged, interactions at His260 are
established, like glyoxylate and CCPST, by hydrogen donation from the aminoacid. Except 6 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
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0
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0
1
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4
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7
8
9
0
9
, they all interact with Tyr132 but, differently to CDST and CCPST, they do it by hydrophobic
bonding. Quinoline 17 followed a similar interaction pattern than compounds 1-9 in this binding
region 1.
A different binding pattern was found for β-hydroxy naphthylic derivatives (10-16) in this
first binding region (Table S1). While interactions at Arg167 and Arg263 were conserved,
interaction with His260 could not be observed for these compounds. However, a hydrogen bond
interaction with Tyr26, which was not observed in 1-9 or quinoline 17, was found to be
established now (except for compound 12), happening through the β-OH and/or the carboxy
group of the salicylic head. In all cases, Tyr26 appears to behave as hydrogen donor. Compound
11 only interacts with Tyr26 through the carboxylate group, remaining the β-OH functionality
free for interaction with Tyr132 (Figure 5). This interaction βOH-Tyr132 is not observed in any
other naphthylic or salicylic derivative and it happens by hydrogen donation from the β-hydroxy
group of 11. It is remarkable as 11 was also the most potent mGOi within this first group of
compounds (1-17). Compounds 10, 13 and 16 interact with Tyr132 by hydrophobic while 15
interacts via π-π stacking interactions. Compounds 12 and 14 lack any type of interaction with
Tyr132 (Figure 5).
About interactions in the binding region 2, compounds 1-7 and 10, lacking a hydrophobic
tail establish hydrophobic interactions with Trp110, through the salicylic aromatic ring (ring A),
except for compound 6 that uses the acetyl group for this interaction. Interestingly, when a
second aromatic ring is bonded to the salicylic head, interaction with Trp110 normally happens
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6
through the ring in the side chain (ring B), except for compound 15, which presents no
interaction with Trp110 (Figure 5). Bonding to this aminoacid happens by means of π-π stacking
in all the compounds that present ring B (8, 11-14) except in 9 and 16, that interact via
hydrophobic forces. Only compounds with a side chain pending from the β-hydroxybenzoic
fragment interact at Leu205 and Tyr208 (1-6 vs 7-16, Table S1). Quinoline 17 is the only
exception able to interact at Leu205 without presenting a hydrophobic side chain.
0
1
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4
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7
8
9
0
The choice of testing 2-hydroxynaphthoic acids vs single-ring salicylic acids was aimed at
establishing novel hydrophobic interactions that would eventually increase the binding affinity of
the molecules. Prediction models showed naphthoic derivatives 12, 13, 15 and 16 could establish
hydrophobic (12, 13, 16) or π-π stacking (15) interactions through the extended aromatic system
(
Figure 5). These interactions could produce a slight increase of the GO inhibitory activity (12,
3 or 15 vs 14 or 17, Table 1) but are not essential (12 vs 11, Table 1). Furthermore, they are not
1
related to a capacity of decreasing oxalate release. On the other hand, the presence of the
condensed aromatic system is detrimental for the aqueous solubility of the compounds and
complicates the synthetic process. Thus, we decided to concentrate our efforts on the structural
optimization of simple salicylic acids. Predicted binding modes for group 1-17 indicated that
aromatic rings, either electron-rich or electron-poor carbocycles or heterocycles, attached to the
salicylic head reinforce binding to hGO by interaction at the binding region 2. Both, aryl and
heteroaryl rings have led to mGO inhibitors such as the furylsalicylate 8 (IC = 34.5 ± 1.2 µM),
5
0
or the naphthoic acids 11 and 12 with nitrophenyl and phenyltetrazole side chains (IC50 = 2.9 ±
.1 and 11.1 ± 1.1 µM, respectively) (Table 1). Taking all this into account, we focused on the
preparation of salicylic acids bearing aromatic heterocycles and carbocycles as hydrophobic tails
heteroaryl and biphenyl analogues, respectively).
1
(
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9
0
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3
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7
8
9
0
8
11
1
2
15
Figure 5. 2D Diagrams of interactions between compounds 8, 11, 12, 15 and human glycolate
oxidase residues (purple: basic, cyan: polar uncharged, green: non-polar). Grey shadow
represents interaction with solvent. Hie260: His260 protonated on N
Schrödinger)].
Ɛ
[Maestro software
(
Synthesis of salicylic acid derivatives. Most of our final compounds were prepared, in
moderate to high yields, following an easy one-step procedure based on Suzuki-Miyaura cross-
coupling, starting with commercial aryl halides (18) and boronic acids (19) (Scheme 1). Three
different standard methods were followed depending on easiness of purification and yield
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5
5
5
5
5
5
5
5
6
criteria. These methods differ in the catalytic system, reaction solvent and the use of
conventional or microwave heating. Palladium acetate was used to catalyze this process, either in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
®
complex with triphenylphosphine or microencapsulated in polyurea (PdEnCat ). The use of
®
microencapsulated palladium acetate (PdEnCat ) was preferred due to its economic and
environmental advantages derived from its easy removal/reuse and the environmentally friendly
34
®
ligand-free solvent system. PdEnCat reactions were developed in an ethanol/water mixture
while soluble palladium acetate was used in dimethylformamide/water systems. In all reactions
self-coupling processes were minimized by displacement of oxygen with argon in the reaction
system.
a
Scheme 1. Synthesis of aryl salicylates by Suzuki-Miyaura cross-coupling
R2
R2
1
R OOC
1
R OOC
5
+
(R O)2B Ar
Ar
R3-4
R3
R4
18
19
20-55
a
Reagents and conditions: Method A: i) K
HCl 10 %, H O, 0 ºC. Method B: i) K CO
ii) HCl 10 %, H O, 0 ºC. Method C: i) K
microwave; ii) HCl 10 %, H O, 0 ºC. Detailed structures of 18 and 19 in Table S2.
2
CO
3
, Pd(OAc)
2
, PPh
3
, DMF/H
2
O (1/1), 100 ºC; ii)
O (1/1), 100 ºC;
O (1/1), 100 ºC
®
2
2
3
, PdEnCat 30 0.4 mmol/g, EtOH/H
2
2
2
CO
3
, Pd(OAc)
2
, PPh
3
, DMF/H
2
2
1 3 2
When 2-hydroxyesters were used as starting materials type 18 (R = -CH , R = -OH) (e.g.
3
4
methyl 5-iodosalicylate 18a: R = -H, R = -I), hydrolysis of the ester functionalities happened in
the mildly basic aqueous medium of the Suzuki reaction. This way, the final coupled free
carboxylic acids were obtained in a two-step (coupling-hydrolysis) one-pot procedure. However,
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2
3 3
when 2-alkoxybenzoates were used as starting materials type 18 (R = -CH , R = -OCH ), ester
hydrolysis did not happen in the Suzuki coupling conditions. These results can be explained by
means of a neighbor group participation process. Basic hydrolysis of salicylate esters seems to be
mediated by the neighboring free hydroxy group, probably by hydrogen bridging to the basic
carbonyl group. This intramolecular bond is not possible in the case of 2-alkoxybenzoates and
thus the hydrolysis of this type of esters requires more drastic conditions.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
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5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reaction yields expressed in this work refer to purified products and have been calculated
considering only product presenting more than 95 % purity in HPLC. For that reason, some
yields fall below 50 %. Purification at the low scale used in this work was the major issue found
in the synthetic protocol of our compounds, due to low solubility in organic solvents and high
affinity for polar stationary phases of chromatographic systems. Eventual large-scale production
however, could allow the use of non-chromatographic purification methods and the consequent
increase of yield. In this work, further reaction optimization studies were not carried out as the
most active compounds in terms of mGO inhibition and oxalate decrease were obtained in
satisfactory yields, ranging between 72-100 %.
Heteroaryl analogues. Taking 8 (Figure 4) as the reference heteroaryl compound, we started
making minor structural modifications to explore the influence of each structural fragment on the
biological activity of this compound. In order to determine to what extent is the salicylic head
necessary for the GO inhibitory activity of 8, we synthesized the non-salicylic derivatives 20-25
3
5
36
(
Figure 6), i) with no phenolic or carboxy functionalities (20 , 21 ); ii) with decreased or null
hydrogen donor capacity at C2 (22-23); iii) unable to ionize at physiological pH (24); iv) with
increased hydrogen donor capacity on the phenolic group (25).
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compounds 20-25 were prepared according to the general procedure indicated in Scheme 1.
Under these conditions ester 24, lacking a free neighboring hydroxy group (Figure 6), did not
undergo hydrolysis. Compound 23 (Figure 6) was prepared from 24 by treatment with sodium
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
hydroxide.
Figure 6. Structures of the non-salicylic compounds tested. In brackets: isolated yields in
Suzuki-Miyaura coupling (method A: 21-25; method C: 20).
Like before, the biological activity of 20-25 on recombinant mGO and on hyperoxaluric mouse
hepatocytes was evaluated (Table 2) and binding modes (Table S3, Figures 7 and S3) were
calculated. Unfavorable negative values of logPth were found for some of the compounds in this
set (Table 2).
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Table 2. Biological data of non-salicylic compounds 20-24 and 25, and in silico prediction of
partition coefficient (logPth). Comparison with CCPST and 8. Biological determinations: (i)
-
Enzymatic inhibition on purified mouse glycolate oxidase (mGO); (ii) Excreted oxalate in Agxt1
/-
mouse primary cultured hepatocytes, compared to control (relative oxalate).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
mGO
%)
Relative
oxalate
Compound
b
logPth
(
c
CCPST
8
34.8 ± 4.8
82.8 ± 5.6
22.7 ± 8.7
24.2 ± 5.4
0
0.63 ± 0.16
n/a
0.88 ± 0.18
0.86 ± 0.11
0.32 ± 0.06*
0.98 ± 0.23
0.82 ± 0.04
0.90 ± 0.10
0.86 ± 0.04
0.29
0.00
0.01
-0.54
-0.26
-0.22
-2.24
2
0
21
2
2
2
2
2
3
4
5
0
17.5 ± 4.8
5.9 ± 3.1
a
Percentage of inhibition of purified mGO at 25 µM of the drug, after 1 min.
b
Oxalate output decrease tested at 24h post-treatment with 10 µM of the drug
-/-
in Agxt1 mouse primary hepatocytes cultured with 5 mM glycolate, in 6-well
plates. Data are represented as mean ± SD of n = 3 replicates. One-way
ANOVA statistical analysis, *p<0.05 was considered statistical significant.
c
-/-
Data at 12.5 µM in Agxt1 mouse primary hepatocytes. N/A: Not assessed.
None of the compounds 20-25 presented satisfactory activities against purified mGO
Table 2).
The experimental values indicate the need to maintain the carboxylic acid and the free
phenol in any derivative prepared from 8 in order to preserve the inhibitory activity against mGO
8 vs 20, 21, 23 and 24); they also suggest the irreplaceability of the phenolic group by a benzylic
(
(
hydroxy group (8 vs. 22). These data mean the carboxy and the aromatic β-OH groups are
playing essential roles in the interaction with the enzyme. From the experimental data it would
be reasonable to think the β-OH is behaving as a hydrogen donor (8 vs 22 and 23). In this line,
compound 25, in which an electron-withdrawing nitro group is situated para with respect to the
phenolic OH, would be expected to be a better inhibitor. However, it resulted in a drastic loss of
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2
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2
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5
6
GO inhibition activity (Table 2). Binding mode predictions for 8 (Figure 5) did not show
interaction through the β-OH. Analysis of the 2D-interaction diagram for 22, 23 and 25 show
that the benzylic alcohol in 22 behaves as a hydrogen acceptor from Tyr132 (Table S3, Figure
S3). When this benzylic hydroxy group is substituted by a methoxy group (23), the hydrogen
bond with Tyr132 disappears and instead the methyl group appears involved in hydrophobic
interactions with the same aminoacid (Table S3, Figure S3). In the case of compound 25 (Figure
0
1
2
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6
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8
9
0
1
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1
2
3
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5
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8
9
0
7
), the presence of the nitro group displaces the molecule in the active center and avoids the
essential interactions at Arg167 and Arg263 and the interaction with His260, all of which are
established by the carboxy groups of 8, 22 and 23. Thus according to the predicted binding
mode, the β-OH group of 8 would not participate in hydrogen bonding to the enzyme while the
benzylic alcohol in 22 and the methoxy group in 23 would interact with the enzyme via hydrogen
bond or hydrophobic forces. The better mGO inhibitory activity found for 8 vs 22 and 23 could
then be due to the less bulky group at the β position, which allows a better accommodation of the
molecule inside the active site of mGO, rather than to hydrogen donation through the phenolic
functionality. However, the presence of the β-OH is needed as its removal leads to a loss of
inhibitory activity (8 vs 20) (Table 2). 2D-Interaction diagram for compound 20 (Table S3,
Figure S3) suggests a lack of interaction with His260 and Tyr26 that could be the reason for the
absence of activity of this compound. Thus, a suitably sized substituent situated β relatively to
the carboxylic acid in ring A, seems to be necessary to allow the right disposition of the essential
interacting groups of inhibitor and enzyme. It could not be clarified at this point whether
interaction through that β-substituent could improve the potency of the inhibitors.
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21
25
Figure 7. 2D Diagrams of interactions between compounds 21 and 25 and human glycolate
oxidase residues (purple: basic, cyan: polar uncharged, green: non-polar). Grey shadow
Ɛ
represents interaction with solvent. Hie260: His260 protonated on N [Maestro software
(
Schrödinger)].
Compounds 21 and 24 have no free carboxylic acid in the polar head; in 21 it has been
removed while in 24 it has been esterified into a methylcarboxylate. Thus, these two molecules
are expected to interact poorly at Arg167 and Arg263. The binding mode predictions show the
two molecules turn around in the binding site of hGO, drawing the formylfuran moiety towards
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the binding region 1 and leaving the polar head orientated towards the binding region 2. With
this disposition, 21 uses the formyl group in the side chain to interact with both Arg167 and Arg
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263 as hydrogen acceptor. Also, 21 interacts as hydrogen acceptor from Tyr26 through both, the
formyl and the furan oxygen atoms while lacks interaction with His260 (Table S3, Figure 7).
Formyl group of compound 24 binds to His260 and Tyr132 acting as acceptor in hydrogen
bonds. No interaction between 24 and Arg167 or Arg 263 was predicted (Table S3, Figure S3).
Both, 21 and 24 establish π-π stacking bonds to Trp110 (Table S3).
In the binding region 2, compounds 20-25 interact with Trp110 and Tyr208, but only some
of them do the same with Leu205 (Table S3, Figure S3).
Compound 21 was the only one in this group (20-25) to produce oxalate decrease on PH1-
mouse primary hepatocytes (Table 2). Compound 21 resulted more effective than CCPST at the
cellular level, lowering oxalate output to 32 % at 10 µM on cell-based assays, without cytotoxic
effects. However, 21 is not a potent GOi. This finding suggests compound 21 may exert its
biological effect by interaction with an alternative biological target, a possibility that deserves
further investigation. Possible interferences of 21 in the biological methods were investigated
and discarded (see Assay interference test in Experimental Section and Tables S4-S5).
Once the need to maintain the salicylic polar head to achieve GO inhibition was confirmed,
we explored modifications of the side chain. First was moving it to the C5 position of the
salicylic head, the para position with respect to the phenol function (C5 isomers). Compound
35
2
6 (Figure 8), C5 isomer of 8, was prepared as described in Scheme 1 using 18a and 5-formyl-
5
2
-furanboronic acid (19a: Ar = 2-furyl, R = -H) (Table S2) as starting materials (method B,
®
quantitative yield). The use of PdEnCat allowed a drastic increase of the reaction yield vs non-
encapsulated palladium acetate.
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0
Figure 8. Structures of the heteroarylsalicylates tested. In brackets: Isolated yields in Suzuki-
Miyaura coupling. Method A: 33-38 and 40. Method B: 26, 31 and 32. Method C: 27, 28, 39 and
41-44. For compounds 39, 41-44, yields are referred to their isolation as potassium carboxylates.
Upon evaluation against mGO, 26 resulted in a slightly less potent inhibitor than its C4
isomer (8), but still with an IC50 value lower to the one of CCPST (Table 3). In turn, the in vitro
efficacy on oxalate diminution was remarkably improved, showing an EC50 value of 3.45 µM, 5-
fold better than CCPST (see Calculation of EC50 and cytotoxicity test for 26 and 27 section
below). If compared to CCPST, 26 represents a marked improvement of the phenotypic effect on
cells with only a slight increase of the inhibitory activity on mGO. Using Cornish-Bowden and
Dixon plots we determined that 26 behaves as a competitive inhibitor with an inhibition constant
i
(K ) on mGO of 47.2 μM. As in the case of 21, this discrepancy between the enzymatic potency
of 26 and its efficiency on cells deserves further investigation as it could mean the existence of
alternative target(s) for this compound. The interaction diagram obtained for 26 inside hGO
(Figure S4) indicates this compound binds to Arg167, Arg263 and His260, through the carboxy
and the β-OH groups, and establishes hydrophobic interactions with Tyr132, Trp110, Leu205
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and Tyr 208 through both, the salicylic ring (ring A) and the furan ring (ring B). The formyl
group establishes no interaction to the target and appears solvated. Compared to its C4 isomer, 8,
a more important participation of the β-OH in the attachment of the molecule to the target can be
observed in 26 as it participates as hydrogen acceptor in the binding to His260. The main binding
difference between CCPST and 26 is the lack of interaction of compound 26 at Tyr26 (Table
S7).
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Table 3. Biological data of heteroarylsalicylates 26-44 and in silico prediction of partition
coefficient (logPth). Comparison with 8 and CCPST. Biological determinations: (i) Enzymatic
inhibition on purified mouse glycolate oxidase (mGO), at single concentration and IC50
-/-
calculation. (ii) Excreted oxalate in Agxt1 mouse primary hepatocytes, compared to control
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
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2
2
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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5
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8
9
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4
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8
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(
relative oxalate).
a
mGO
(%)
IC50
(µM)
2
Relative
oxalate
Compound
R
b
logPth
c
CCPST
8
34.8 ± 4.8 43.5 ± 1.1
82.8 ± 5.6 34.5 ± 1.2
42.7 ± 12.8 38.2 ± 1.2
0.63 ± 0.16
n/a
0.29
2.59
-0.29
0.65
-0.11
-0.11
0.13
0.13
0.56
0.56
0.06
0.06
0.99
0.99
0.90
0.90
1.02
1.02
0.34
2.76
0.88
0.81
0.88 ± 0.18
d
2
2
2
2
6
7
8
9
0
d,e
n/r
n/r
0
35.8 ± 6.9 39.3 ± 1.0
0.94
0.96
0.98
0.87 ± 0.15
0.88 ± 0.19
0.88 ± 0.01
0.68 ± 0.14*
n/a
98.0 ± 1.6
85.7 ± 9.3
0
4.6 ± 1.1
7.9 ± 1.1
n/a
30
3
3
1
2
0
n/a
33
22.8 ± 5.6
26.6 ± 1.9
28.7 ± 1.3
n/a
1.02 ± 0.3
0.89 ± 0.16
1.00 ± 0.20
0.93 ± 0.18
0.74 ± 0.16*
0.82 ± 0.01
0.99 ± 0.12
0.94 ± 0.10
0.89 ± 0.14
0.95 ± 0.02
0.93 ± 0.01
3
4
n/a
35
n/a
3
3
6
7
52.1 ± 13.4 9.4 ± 1.3
35.4 ± 7.2 32.5 ± 1.1
30.0 ± 2.4 39.5 ± 1.1
41.4 ± 7.8 21.6 ± 1.3
0.87
0.96
0.91
0.80
38
3
4
4
4
9
0
1
2
25.2 ± 5.0
28.1 ± 9.3
28.7 ± 10.3
16.2 ± 6.2
22.7 ± 2.3
n/a
n/a
n/a
n/a
n/a
43
4
4
0.88 ± 0.03
a
b
Percentage of inhibition of purified mGO at 25 µM, after 1 min. Oxalate output decrease 24h
after treatment with 10 µM of the drug in Agxt1 mouse primary hepatocytes cultured with 5
-/-
mM glycolate, in 6-well plates. Data represented as mean ± SD of n = 3 replicates. One-way
c
ANOVA statistical analysis, *p<0.05 was considered statistical significant. Data at 12.5 µM in
-/-
d
Agxt1 mouse primary hepatocytes. Values below the detection limit of the colorimetric
e
method. No interference due to the low concentration of the drug. IC50: Calculated in saturated
conditions of glycolate (22.2 mM). R : IC50 curve-fitted score. n/a: Not assessed. n/r: Not
2
reliable data due to interference.
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To check the role of the formyl group on 26 and how its suppression or replacement would
affect the biological activity of this molecule, compound 27 (Figure 8) with an unsubstituted
furan side chain, was prepared using the Suzuki protocol (Scheme 1). Contrarily to 26, the use of
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PdEnCat was not effective for the preparation of 27, with most of the starting materials
remaining unreacted after 24 h. Instead, 27 could be obtained in good yield (83 %) by catalysis
using non-encapsulated palladium acetate. Formyl removal led to a 4-fold increase in the mGO
inhibitory activity while the in vitro efficacy (EC50 = 3.56 µM) remained unchanged if compared
to 26 (Table 3). However, these results must be interpreted carefully as compound 27 interferes
in both biological assays, GO-inhibition and oxalate quantification on cells, by inhibiting the
horseradish peroxidase (HRP) that is used in the evaluation protocols (calculated IC50 of 27
against the oxalate determination kit = 18 µM) (see Assay interference test in Experimental
Section and Tables S4-S6). This interference prevents us from giving a faithful value of activity
for this compound against mGO, as to obtain an IC50 value against mGO, concentrations of at
least 40 µM of 27 had to be used, exceeding the IC50 value found for 27 against HRP. For the
i
same reason, calculation of K was not accomplished for compound 27. However, in the case of
oxalate determination on cells, the activity values reported above can be considered indicative as
the effect of the interference is not significant at concentrations up to 5 µM of 27 (Table S6).
This fact suggests the formyl group in 26 is not essential for achieving oxalate decrease and
therefore this group could be replaced to obtain structurally diverse analogues. The binding
prediction of 27 inside hGO suggests a similar interaction profile to 26, except for an extra
interaction at Tyr26, established by the carboxylate group of 27, and a better hydrophobic
wrapping in the binding region 2 (Figure S4). The equivalent elimination of the formyl group in
the C4 isomer of 26, compound 8, resulted in compound 28. In this case, the removal of the
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formyl group did not mean any improvement of the inhibitory activity against mGO and,
additionaly, 28 is inactive on PH1 mouse primary hepatocytes (Table 3). Comparison of the
predicted binding profiles of 28 and 27 (C4/C5 isomers) shows, as it happened in the pair of
isomers 8/26, a more important binding participation of the β-OH in the case of the C5 isomer
(Table S7, Figure S4). In both C5 compounds, 26 and 27, the β-OH group behaves as hydrogen
acceptor in the binding to His260 (Table S7, Figure S4).
1
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No enzymatic or colorimetric interference was found for compound 26 in any of the
biological assays (see Assay interference test in Experimental Section and Tables S4-S5). This
different behavior of 26 and 27 against HRP could be due to a lower selectivity derived from the
smaller size of 27. At this point we wanted to check whether 26 could be producing 27 after
metabolism, so we could have the same misleading effect on the oxalate decrease results
obtained in vitro for 26. Metabolite profiling of compound 26 using mouse liver microsomes,
showed the formation of two main metabolites corresponding to reduction and oxidation of the
formyl group (29 and 31, Figure 8), meaning 40.1 % and 48.2 % of the total metabolites after 60
min. A minor metabolite could be also observed, accounting for 11.6 % after 60 min (Figure S5).
This metabolite, which presents a different retention time than 27 in LC-MS analysis (Figures
S5-S6), seems to correspond to a formyl group oxidation plus decarboxylation on the salicylic
ring. The samples were analyzed by LC-HRMS in negative ionization mode. Compound 26
showed a high intrinsic clearance with an elimination half-life of 13.3 min while 27 showed a
medium intrinsic clearance with an elimination half-life of 61.9 min (Tables S8 and S9, Figure
S7). No metabolites could be detected for compound 27 after 60 min incubation.
We then proceeded to prepare the main metabolites of 26, structures 29 and 31 (Figure 8),
in order to examine their biological activity and their capacity to produce interferences in the
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biological testing process. The primary alcohol 29 and its C4 isomer 30 (Figure 8), were made by
reduction of the formyl group in 26 or 8 respectively. Both presented satisfactory GO inhibitory
activities (IC50 = 4.6 ± 1.1 µM for 29 and IC50 = 7.9 ± 1.1 µM for 30) but low decrease of oxalate
output in PH1 mouse hepatocytes in culture (Table 3). No interference could be observed for
these compounds (see Assay interference test in Experimental Section and Table S10). Oxidized
derivatives of 26 and 8, carboxylic acids 31 and 32 (Figure 8), were totally inactive as mGOi’s
and therefore interference by inhibition of HRP was discarded. The absence of interference
found for the metabolites of 26 (29 and 31) confirms the activity of this compound. Thus 26 is a
moderate mGOi with a high capacity to decrease oxalate production in hyperoxaluric
hepatocytes. Interestingly, its metabolite 31 presents the same activity profile, with null activity
as mGOi but with capacity to decrease oxalate production in approximately 30 % after 24 h
incubation with cells at 10 µM (Table 3). Thus, in the cell system, metabolism of the aldehyde
26 happens in favor of the less active carboxylic acid 31, causing the time-dependant loss of
activity of 26. A common mechanism of action could be involved in the phenotypic activity of
compounds 26, 27 and 31 on cells, which is higher than their activity as mGOi’s on pure
enzyme. For these compounds, possible interactions with alternative biological targets, different
from GO, should be considered.
0
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Since the orientation of the ring in the binding site can be crucial for a good interaction
with GO, the furan ring was also bound to the salicylic molecule by its carbon atom C3’.
Compounds 33 and 34 (Figure 8), C3’ isomers of 28 and 27 respectively, were prepared by
Suzuki-Miyaura coupling. None of them presented satisfactory mGO inhibitory activity or
capacity to decrease the production of oxalate in cells (Table 3).
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We then replaced the furan ring in 8 and 26 by different nitrogen or sulfur heterocycles.
The latter contains a sulfur atom in a similar position to CCPST. We prepared thiophene (35-42),
pyrazole (43) and pyridine derivatives (44) (Figure 8) according to the general procedure
indicated in Scheme 1. Isosteric change in the formylfuran 8 (C4 isomer) to give the
formylthiophene 35 (C4 isomer) (Figure 8), led to loss of the mGO inhibitory activity and the
1
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38
efficacy on cell-based assays (Table 3). Differently, formylthiophene derivative 36 (C5 isomer)
(
Figure 8) (IC50 = 9.4 ± 1.3 µM) was a 4-fold more potent mGO-inhibitor than its formylfuran
isoster 26 (C5 isomer) (IC50 = 38.2 ± 1.2 µM). Unfortunately, it did not decrease oxalate
excretion in PH1 hepatocytes (Table 3). The same isosteric change in the nonformylated furan 27
3
9
(
C5 isomer) raised thiophene 38 (Figure 8), which showed a huge decrease of efficacy (0.82 ±
.01 vs. 0 relative oxalate) while conserving the same potency as mGOi (IC50 = 39.5 ± 1.1 µM)
Table 3). As we did with the furan derivatives, we checked the importance of the orientation of
0
(
the thiophene by binding it to the polar head by its C3’ carbon atom (compounds 39 and 40,
Figure 8). C3’-Thienyl derivatives did not improve the potency of their C2’ counterparts (37 and
38) (Table 3). Exploration of different substitution on the C2’-bound thiophene ring, such as
acetyl (41) or methyl (42) groups, led to poor mGOi’s without activity on cells (Table 3).
Nitrogen heterocycles pyrazol (43) and pyrimidine (44) produced no improvement in the mGO
inhibitory activity or the oxalate decrease capacity. In summary, although the C5
formylthiophene derivative 36 represented an increase of the mGO inhibitory activity with
respect to its formylfuran isoster 26, any of the other sulphur or nitrogen derivatives 35-44
presented relevant activities on mGO inhibition or any improvement with respect to the furan
analogues. Neither oxalate decrease activities were obtained, pointing out the crucial role of the
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