Novel substituted N-benzyl(oxotriazinoindole) inhibitors of aldose reductase exploiting ALR2 unoccupied interactive pocket

Article abstract of DOI:10.1016/j.bmc.2020.115885

Recently we have developed novel oxotriazinoindole inhibitors (OTIs) of aldose reductase (ALR2), characterized by high efficacy and selectivity. Herein we describe novel OTI derivatives design of which is based on implementation of additional intermolecular interactions within an unoccupied pocket of the ALR2 enzyme. Four novel derivatives, OTI-(7–10), of the previously developed N-benzyl(oxotriazinoindole) inhibitor OTI-6 were synthetized and screened. All of them revealed 2 to 6 times higher ALR2 inhibitory efficacy when compared to their non-substituted lead compound OTI-6. Moreover, the most efficient ALR2 inhibitor OTI-7 (IC₅₀ = 76 nM) possesses remarkably high inhibition selectivity (S_F ≥ 1300) in relation to structurally related aldehyde reductase (ALR1). Derivatives OTI-(8–10) bearing the substituents –CONH₂, –COOH and –CH₂OH, possess 2–3 times lower inhibitory efficacy compared to OTI-7, but better than the reference inhibitor OTI-6. Desolvation penalty is suggested as a possible factor responsible for the drop in ALR2 inhibitory efficacy observed for derivatives OTI-(8–10) in comparison to OTI-7.

Full text of DOI:10.1016/j.bmc.2020.115885

Journal Pre-proofs
Novel substituted N-benzyl(oxotriazinoindole) inhibitors of aldose reductase
exploiting ALR2 unoccupied interactive pocket
Matú š Hlavá č, Lucia Ková čiková, Marta Šoltésová Prnová, Gabriela
Addová, Gilles Hanquet, Milan Štefek, Andrej Bohá č
PII:
S0968-0896(20)30715-X
https://doi.org/10.1016/j.bmc.2020.115885
BMC 115885
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 August 2020
12 November 2020
16 November 2020
Please cite this article as: M. Hlavá č, L. Ková čiková, M. Šoltésová Prnová, G. Addová, G. Hanquet, M. Štefek,
A. Bohá č, Novel substituted N-benzyl(oxotriazinoindole) inhibitors of aldose reductase exploiting ALR2
unoccupied interactive pocket, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2020.115885
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Novel substituted N-benzyl(oxotriazinoindole) inhibitors of aldose reductase
exploiting ALR2 unoccupied interactive pocket
Matúš Hlaváč1*, Lucia Kováčiková2, Marta Šoltésová Prnová2, Gabriela Addová1, Gilles
Hanquet3, Milan Štefek2, Andrej Boháč1,4
1 Department of Organic Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova
6, 842 15 Bratislava, Slovakia
2 Institute of Experimental Pharmacology and Toxicology, CEM, SAS, Dúbravská cesta 9, 841 04 Bratislava,
Slovakia
3 Université de Strasbourg, Université de Haute-Alsace, CNRS, UMR 7042-LIMA, ECPM, 25 rue Becquerel,
67087 Strasbourg, France
4 Biomagi, Ltd., Mamateyova 26, 851 04 Bratislava, Slovakia, biomagibiomagi@gmail.com
* corresponding author: E-mail: hlavac.matus91@gmail.com
Abstract
Recently we have developed novel oxotriazinoindole inhibitors (OTIs) of aldose reductase
(ALR2), characterized by high efficacy and selectivity. Herein we describe novel OTI
derivatives design of which is based on implementation of additional intermolecular
interactions within an unoccupied pocket of the ALR2 enzyme. Four novel derivatives, OTI-
(7-10), of the previously developed N-benzyl(oxotriazinoindole) inhibitor OTI-6 were
synthetized and screened. All of them revealed 2 to 6 times higher ALR2 inhibitory efficacy
when compared to their non-substituted lead compound OTI-6. Moreover, the most efficient
ALR2 inhibitor OTI-7 (IC50 = 76 nM) possesses remarkably high inhibition selectivity (SF ≥
1300) in relation to structurally related aldehyde reductase (ALR1). Derivatives OTI-(8-10)
bearing the substituents -CONH2, -COOH and -CH2OH, possess 2-3 times lower inhibitory
efficacy compared to OTI-7, but better than the reference inhibitor OTI-6. Desolvation penalty
is suggested as a possible factor responsible for the drop in ALR2 inhibitory efficacy observed
for derivatives OTI-(8-10) in comparison to OTI-7.
Keywords: aldose reductase (ALR2); interactive pocket; N-benzyl(oxotriazinoindole) ALR2
inhibitors; desolvation penalty
1

1. Introduction
Diabetes mellitus (DM), a wide-spread human disorder, has been recognized as a main cause
of health problems such as blindness, neuronal diseases, hearth and kidney failures.1 One of the
most important therapeutic targets, implicated in late diabetic complications, is the polyol
pathway. This pathway involves two steps, where D-glucose is converted to D-fructose via D-
sorbitol. (Fig. 1) In the first step, aldose reductase (ALR2) catalyses reduction of D-glucose to
D-sorbitol while NADPH is oxidized to NADP+.2 Deficiency of NADPH hampers regeneration
of glutathione (GSH) from its dimer (GSSG), causing high excess of intracellular reactive
oxygen species (ROS) in various tissues including the heart, neurons, eyes and kidneys.3 In
addition, high concentration of highly polar D-sorbitol results in osmotic imbalances and cell
swelling.4 D-Fructose, formed in the second step of the polyol pathway, causes excessive
glycation of proteins and lipids and enhances formation of advanced glycation and lipoxidation
end products (AGEs and ALEs), also involved in inflammation and oxidative stress. According
to all these facts, inhibition of ALR2 activity can indirectly suppress above mentioned
pathological imbalances and slow down late diabetic complications.
(ALR2)
*
* competitive reactions
Figure 1. An illustration of the polyol pathway and its implication in the diabetic complications.
Reduction of the carbonyl functional group of lipid peroxidation products, namely 4-
hydroxynonenal (HNE), and its glutathione adduct (GS-HNE) by ALR2 may generate
intermediates contributing to activation of pro-inflammatory signaling. It points to aldose
reductase inhibitors (ARIs) as promising agents in therapy of chronic inflammatory disorders
and even several types of tumors related to chronic inflammation.5-9 Moreover, interventions
2

with ARIs may have potential in attenuation of pathological consequences of several genetic
metabolic disorders including galactosemia and sorbitol dehydrogenase deficiency.10
Over the last 40 years, numerous ARIs have been developed, but only epalrestat as ARI
is therapeutically available in Japan, China and India.11 Most of structurally different ARIs have
been refused in clinical trials mainly for their low in vivo efficacies, pharmacokinetic drawbacks
and/or side effects. The largest class of ARIs are carboxylic acids where carboxylate anion
moiety occupies an anion binding pocket of the active site of ALR2. Štefek et al. developed the
carboxymethylated
thioxotriazinoindole
cemtirestat
(CMTI),
2-(3-thioxo-2H-
[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetic acid, as a novel lead-like candidate with excellent
ALR2 inhibitory efficacy, selectivity and antioxidant activity.12 Very recently, the inhibition
efficacy and selectivity of CMTI was improved by isosteric replacement of sulphur with
oxygen, which led to the discovery of the oxotriazinoindole inhibitor OTI, 2-(3-oxo-2H-
[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetic acid, with markedly increased inhibition
selectivity.13 In this study, we aimed to exploit a previously unoccupied ALR2 pocket
consisting of Trp219, Ala299 and Leu301 (PDB: 4QX4). In order to utilize this pocket to ensure
additional interactions novel N-benzyl(oxotriazinoindole) derivatives were proposed. Besides
the leading unsubstituted N-benzyl derivative OTI-6 previously described by us,13 herein we
proposed, synthesized and screened its substituted derivatives OTI-(7-10), containing different
functional groups in an ortho position on a benzyl aromatic ring. (Scheme 1)
Leu301
Trp219
Trp219
Z
Ala299
N
N
N
N
O
O
N
N
N
N
HO
HO
Z
CN (OTI-7)
76
nM
: -
-
-
-
IC50
=
=
=
=
O
O
CONH (OTI-8)
234
139
241
IC50
IC50
IC50
nM
nM
nM
2
OTI-6
COOH (OTI-9)
CH OH (OTI-10)
2
434
nM (ALR2)
IC50
:
Scheme 1. Design of novel benzyl derivatives OTI-(7-10) from OTI-6.
2. Results and discussion
In this study, we aimed to explore an unoccupied ALR2 pocket in close proximity to the triazine
ring of CMTI inhibitor (PDB: 4QX4). This pocket contains important amino acid residues e.g.
Trp219, Ala299 and Leu301. In order to utilize this pocket for additional ligand-enzyme
3

interactions, novel N-benzyl(oxotriazinoindole) derivatives OTI-(7-10) have been designed
and developed.
Predictions and interaction diagrams of OTI-(7-10)
Proposed derivatives OTI-(7-10) have been docked into the ALR2 protein conformer taken
from the complex PDB: 4QX4. As expected, proposed derivatives possess additional H-bonds
in the ALR2 unoccupied lipophilic pocket with Ala299 or Leu301 from backbone and two π-π
interactions of OTI´s benzyl moiety with Trp219. Furthermore, the other important interactions
e.g. of an acetate group in an anion binding pocket are crucial for a high ALR2 activity of all
OTI derivatives (ionic interactions: with His110 and Lys77 and H-bonds with His110, Tyr48
and Trp111). Predicted interactions of OTI-(7-10) inhibitors are depicted in the diagrams (Fig.
2) as well as in the Supporting information of this paper.
NH2
CN2.4 A
Ala299 (HB, NH)
2.1 A
Leu301 (HB, NH)
4.8 A
4.6 A
3.9 A
4.1 A
O
Trp219 (PIPI)
Leu300 (HB, NH)
Trp219 (PIPI)
Trp219 (PIPI)
Trp219 (PIPI)
Leu300 (HB, NH)
N
N
2.2 A
N
N
O 2.0 A
O
N
N
4.9 A
Trp20 (PIPI)
4.9 A
Val47 (VdW)
4.9 A
Trp20 (PIPI)
5.0 A
Val47 (VdW)
N
N
5.0 A 5.2 A
O
O
1.9 A
5.0 A
Trp111 (HB, NH from indol)
Trp20 (PIPI)
1.9 A
Trp20 (PIPI)
Trp111 (HB,NH from indol)
O
2.3 A
2.0 A
2.1 A
O
5.4 A
His110 (IONIC, HB, HN+ from imidazol)
2.3 A
5.5 A
His110 (IONIC, HB, HN+ from imidazol)
Tyr48 (HB, OH)
Tyr48 (HB, OH)
Lys77 (IONIC)
Lys77 (IONIC)
OTI-8 (6)
OTI-7 (5)
O
Ala299 (HB, NH)
2.4 A
2.0 A
4.4 A
4.0 A
4.8 A
Leu301 (HB, NH)
3.8 A
O
OH
Trp219 (PIPI)
Trp219 (PIPI)
Leu300 (HB, NH)
1.9 A
Trp219 (PIPI)
Trp219 (PIPI)
N
N
N
N
Leu300 (HB, NH)
4. 6 A
O
O
1.8 A
N
N
4.9 A
Trp20 (PIPI)
4.8 A
Val47 (VdW)
Trp20 (PIPI)
N
N
5.2 A
O
5.0 A
1.9 A
Trp111 (HB,NH from indol)
Trp20 (PIPI)
O
2.1 A
O
O
His110 (IONIC, HB, HN+ from imidazol)
5.5 A
2.1 A
Tyr48 (HB, OH)
2.3 A
2.2 A
Tyr48 (HB, OH)
Lys77 (IONIC)
5.4 A
Lys77 (IONIC) His110 (IONIC, HB, HN+ from imidazol)
solvent
accessible
part
OTI-9 (7)
OTI-10 (13)
Figure 2. Predicted OTI-(7-10) interactions in a binding site of ALR2 enzyme (PDB: 4QX4).
Synthesis of OTI-(7-10)
Compound 1 (OTI)13 represented a joint starting material for synthesis of all proposed
inhibitors OTI-(7-10). In the first step, OTI underwent acidic esterification by EtOH (abs) in
4

the presence of cat. amount of H2SO4 (conc). Obtained ester 2 was alkylated to 4 by CaH2 in
DMF, followed by addition of 2-(bromomethyl)benzonitrile 3 at 80 °C. Obtained N-(2-
cyanobenzyl) derivative 4 was used as an intermediate for the synthesis of all proposed
compounds OTI-(7-9). Hydrolysis of an ester group of 4 by aq solution of NaOH in a mixture
of MeOH / H2O (1 / 1) at reflux within 30 min provided the N-(2-cyanobenzyl)acetate OTI-7.
Partial hydrolysis of N-(2-cyanobenzyl)ester 4 with TFA / H2SO4 (conc) (4 / 1) at 70 oC within
6 h afforded N-(2-carbamoylbenzy)acetate OTI-8. Biscarboxylic acid OTI-9 was prepared by
complete hydrolysis of 4 in a mixture of H2O / H2SO4 (conc) (1 / 1) at 100 °C within 8 days.
(Scheme 2)
CN
CN
N
N
NH
N
N
N
NH
N
N
N
O
O
O
O
a
b
c
N
N
N
N
90 %
N
75 %
N
74 %
HO
O
HO
1 (OTI)
O
O
O
O
O
5 (OTI-7)
2
4
O
O
OH
NH2
N
N
N
N
O
O
e
N
d
N
N
N
41 %
77 %
HO
HO
O
O
6 (OTI-8)
7 (OTI-9)
Scheme 2. Preparation of OTI-(7-9) from OTI.
Reagents and conditions: (a) EtOH (abs), cat H2SO4 (conc), reflux, 6 h; (b) (1) CaH2 (1.19 mol eq), DMF (abs),
80 °C, 30 min, (2) 2-(bromomethyl)benzonitrile (3) (1.19 mol eq), from rt to 80 °C, 1 h; (c) NaOH (2.00 mol eq),
MeOH / H2O (1 / 1) reflux, 30 min; (d) TFA / H2SO4 (conc) (4 / 1), 70 °C, 6 h; (e) H2O / H2SO4 (conc) (1 / 1), 100
°C, 8 d.
Synthesis
of
hydroxymethyl
derivative
OTI-10
required
THP
protected
(bromomethyl)phenylmethanol 11, which was prepared in three steps starting from phthalic
anhydride (8). Compound 8 was reduced to diol 9 by LiAlH4 in THF (abs) at 0 °C to rt for 16
h in 94 % yield. Afterwards, treatment of diol 9 by conc HBr (48 %) in toluene at 70 °C within
20 min provided product 10 and traces of bis-brominated side product. After FLC separation,
required compound 10 was obtained in 56 % yield. Finally, O-protected compound 11 was
obtained in 89 % yield by reaction of 3,4-dihydropyrane (DHP) in a presence of cat amount of
pyridinium p-toulenesulfonate (PPTS) in refluxing DCM within 30 min. Afterwards, previously
5

prepared ester 2 was alkylated with 11 by CaH2 in DMF at 80 °C within 30 min to perform an
intermediate 12. Compound 12 was hydrolysed to desired N-(2-hydroxymethylbenzyl)
derivative OTI-10 by aq HCl in dioxane at 90 °C within 8 h in 66 % yield. (Scheme 3)
O
a
b
c
OH
OH
OTHP
Br
OH
Br
O
94 %
56 %
89 %
O
8
9
10
11
OH
OTHP
NH
N
N
N
N
N
N
N
N
O
O
O
d
e
N
N
N
82 %
66 %
O
HO
O
O
O
O
2
12
13 (OTI-10)
Scheme 3. Preparation of hydroxymethyl derivative OTI-10.
Reagents and conditions: (a) LiAlH4 (2.00 mol eq), THF (abs), from 0 °C to rt, 16 h; (b) HBr (48 %) (1.12 mol
eq), toluene, 70 °C, 20 min; (c) 3,4-dihydropyran (DHP) (1.16 mol eq), PPTS (pyridinium p-toulenesulfonate)
(0.10 mol eq), DCM (abs), reflux, 30 min; (d) (1) CaH2 (1.19 mol eq), DMF (abs), 80 °C, 30 min, (2) 11 (1.47 mol
eq), from rt to 80 °C, 1 h; (e) HCl (conc) / H2O (1 / 10), dioxane, 90 °C, 8 h.
Determination of enzyme inhibitory activities
Inhibitors OTI-(7-10) were tested for their ability to inhibit the reduction of D,L-
glyceraldehyde using ALR2 isolated from the rat eye lenses. Unsubstituted benzyl analogue
OTI-6 was used as a reference inhibitor. To assess selectivity we used a structurally related
detoxification enzyme (an antitarget), aldehyde reductase (ALR1) isolated from the rat
kidneys.13
6

Table 1. Results of in vitro enzyme assays of the novel ALR2 inhibitors OTI-(7-10) in
comparison with the unsubstituted parent compound OTI-6 and previously developed
inhibitors Cemtirestat and Epalrestat.
O
O
Z:
CN
NH2
OH
OH
Z
Epalrestatb
0.227 ± 0.019
n.d.
compound
OTI-6
OTI-7 (5)
OTI-8 (6)
Cemtirestata
OTI-9 (7)
OTI-10 (13)
N
N
N
O
ALR2
IC50 [µM]
0.116 ± 0.008
35.0 ± 1.6
0.139 ± 0.005 0.244 ± 0.003
0.076 ± 0.006 0.236 ± 0.005
0.434 ± 0.014
38 ± 5
N
ALR1
I (%, 100 µM)
HHO
51 ± 1
50 ± 2
62 ± 1
33 ± 5
ALR1
IC50 [µM]
O
n.d.
302
> 100
> 100
> 424
59 ± 6
424
> 100
> 410
> 100
> 230
n.d.
n.d.
OTI-(6-10)
SF
> 1 333
(selectivity factor)
aHlaváč et al. (2020),13 bMajekova et al. (2017) (measured in 1 % DMSO),26 n.d.-not determined. Results are
means ± SD from minimum of three independent repetitions. I is the % of enzyme inhibition observed at 100 μM
inhibitor concentration. Selectivity factor: SF = IC50 (ALR1) / IC50 (ALR2).
According to the results we found out that all substituted N-(benzyl) derivatives OTI-(7-10)
exhibited from 2 to 6-fold better inhibitory efficacy than unsubstituted analogue OTI-6. In
addition, they also revealed low inhibition of ALR1 antitarget, which resulted from a good to
an excellent enzyme selectivity. (Table 1) Obtained results confirmed proposed additional
interactions of substituted N-(benzyl) derivatives OTI-(7-10) within the interactive pocket of
ALR2. The best ALR2 inhibition (IC50 = 76 nM) and selectivity relative to ALR1 (SF > 1333)
was obtained for OTI-7 derivative containing a N-(2-cyanobenzyl) group. Predicted binding
position of OTI-7 in an active site of ALR2 shows a H-bond of a cyano group with Ala299
from backbone (2.4 Å). Moreover, the aromatic ring of a benzyl moiety forms two π-π
interactions with Trp219 (3.9 and 4.6 Å). (Fig. 3)
In Silico
2.4 A
Ala299 (HB, NH)
CN
4.6 A
3.9 A
Trp219 (PIPI)
Trp219 (PIPI)
Leu300 (HB, NH)
N
N
2.2 A
Trp219
O
Val47
N
4.9 A
4.9 A
Val47 (VdW)
Leu300
N
Trp20 (PIPI)
5.0 A 5.2 A
Trp20
O
Trp111 (HB, NH from indol)
Trp20 (PIPI)
1.9 A
Tyr48
His110
Ala299
O
2.0 A
5.4 A
His110 (IONIC, HB, HN+ from imidazol)
2.3 A
Tyr48 (HB, OH)
Trp111
Lys77 (IONIC)
solvent
accessible
part
OTI-7 (5)
Lys77
Figure 3. Predicted pose and interactions of OTI-7 (5) in a binding site of an ALR2 (PDB: 4QX4).
7

In spite of predicted similar positions and interactions of the derivatives OTI-10 and OTI-7,
OTI-10 exhibited about 3-times lower inhibitory activity characterized with IC50 = 241 nM.
The additional reason of lower activity of OTI-10 could be desolvation and rotation penalty
caused by a -CH2OH group. Desolvation penalty effect has been a subject of our recent
publication.14 Derivatives OTI-(8,9) (-CONH2, -COOH, resp.) revealed 2 to 3-fold lower
inhibitory activity in comparison to OTI-7 (-CN). Besides desolvation penalty, the lower
activity of a carbamoyl derivative OTI-8 could be also caused by conformational penalty of -
CONH2 group. Symmetry of delocalized carboxylate group in OTI-9 (-COO-) exhibited better
inhibition activity than OTI-8 (-CONH2) and OTI-10 (-CH2OH). In addition, outer part of
a studied pocket and a benzyl group are well water accessible (Fig. 4) and hence a benzyl group
could provide two conformers. A first conformer allows formation of the predicted H-bond with
an orientation of a polar group inside the pocket and a second conformer prefers orientation
toward the solvent. Consequently, very polar groups would not be oriented inside the pocket,
where the predicted H-bonds could be formed, but owing to solvation they would remain
oriented toward a water environment out of the pocket. Therefore significant H-bond with
Ala299 or Leu301 would not be formed as was predicted and thus the least solvated derivative
OTI-7 revealed the highest inhibitory activity.
Figure 4. Solvents Accessible Surface of the unsubstituted benzyl derivative OTI-6. Blue colour indicates water
accessible part.
In order to determine drug-like parameters, molecular obesity indices for compounds OTI-(6-
10) were calculated (Table 2). The parameters take into account size, inhibitory efficacy and
lipophilicity.
8

Table 2. Physicochemical properties and molecular obesity indices calculated for compounds
OTI-(6-10).
Compound
MW
pKab
pIC50 LogPa LogDa LEc
BEId LLEe LELPf TPSAg
60-140h
90.01
<500h
334.33
359.34
377.35
<5h
2.04
1.61
1.05
1.39
1.31
>0.3h >14.7h >3.8i
<7.5h
5.72
4.37
3.18
4.05
3.82
OTI6
OTI7
OTI8
OTI9
OTI10
4.14
4.14
4.14
6.36
7.12
6.63
-0.98
-1.24
-1.25
-2.50
-1.53
0.36 19.05 4.32
0.37 19.83 5.51
0.33 17.58 5.57
0.34 18.14 5.47
0.34 18.17 5.30
113.8
133.1
378.34 4.14/4.15* 6.86
364.35 4.14 6.61
127.31
110.24
b
aCalculated with MedChem Designer™ Calculated with Pallas 3.112, log D represents the logarithm of the
c
distribution ratio in octanol-buffer [pH 7] Ligand efficiency, LE = -1.4log(IC50)/N, N: number of heavy atoms
dBinding efficiency index, BEI = pIC50/MW eLipophilic ligand efficiency, LLE = pIC50 - logP fLigand efficiency-
g
dependent lipophilicity, LELP = log P/LE. Topological polar surface area, TPSA calculated with MedChem
Designer™ hOptimal drug values.15-17 iMean value for successful lead.17 *BnCOOH.
Calculated values of the ligand efficacy (LE) and the binding efficiency index (BEI) for all
inhibitors OTI-(6-10) were found in the range of optimal values (>0.3 and >14.7, resp). In
addition, all novel compounds exhibited favourable lipophilic ligand efficiency parameter
(LLE) values above the limit 3.8, important for a successful lead. All compounds OTI-(6-10)
showed ligand-efficiency-dependent lipophilicity (LELP) in recommended range (<7.5), which
implies good drug-likeness of all developed inhibitors. Moreover, all compounds have
appropriate topological polar surface area (TPSA) (60–140 Å2) predicting good oral absorbtion.
Based on the results from screening and the SAR conclusions, we have proposed other
appropriate N-benzyl(oxotriazinoindole) analogues shown in Fig. 5. These derivatives should
preserve similar interactions as the parent compounds within a studied ALR2 pocket and they
should have lower desolvation and/or conformational penalties. Acetyl derivative OTI-8a (an
analogue to amide derivative OTI-8) possesses lower polarity and solvation. Methyl ester OTI-
9a is a less polar analogue to carboxylic acid OTI-9. Besides its lower solvation, it could also
possess better penetration through the cell membrane in contrast to OTI-9. In addition, nitro
derivative OTI-9b should be less solvated because of its lower polarity and with no
conformational penalty considering its symmetric delocalized structure (Fig. 5) although -NO2
group is less favorable in drug development due its potential toxicity. More detailed predicted
interaction diagrams of newly proposed analogues OTI-(8a-9a,b) are described in the
Supporting information of this paper.
9

O
O
Studied
derivatives
NH2
OH
OTI-9 (7)
OTI-8 (6)
Ar
N
O
O
O
O
O
Proposed
derivatives
(possessing
lower solvatation
and / or
N
N
O
N
N
HHO
OTI-9b
OTI-9a
OTI-8a
conformation
penatly)
O
Ser302 (HB, -OH)
O
O
Ala299 (HB, NH)
2.2 A
Ala299 (HB, NH)
Leu301 (VdW)
2.5 A
2.2 A
N
O
5.0 A
Interaction
diagrams
means solvent
accessible part
O
3.9 A
O
3.8 A
4.6 A
2.4 A
4.4 A
2.5 A
Leu300 (HB, NH)
Trp219 (PIPI)
Leu300 (HB, NH)
Trp219 (PIPI)
4.2 A
3.8 A
Trp219 (PIPI)
Figure 5. The structures of studied inhibitors OTI-(8,9) and their proposed promising analogues OTI-(8a-9a,b).
Apart from OTI-(8a-9a,b) analogues, we proceeded with a proposal of other N-substituted
analogues possessing lipophilic naphthalene derivatives OTI-(11-13) with favourable
predictions comprising four π-π interactions with Trp219 and Van der Waals interactions with
Ala299 or Leu301. (Fig. 6)
Trp219 (PIPI)
4.6 A
Trp219 (PIPI)
Trp219 (PIPI)
4.7 A
4.2 A
4.7 A
3.6 A
Leu301 (VdW)
3.8 A
5.0 A
4.1 A
Ala299 (VdW)
4.2 A
4.1 A
4.6 A
Trp219 (PIPI)
3.6 A
Trp219 (PIPI)
2.5 A
N
O 4.5 A
Trp219 (PIPI)
N
3.5 A
N
N
1.9 A
N
Leu300 (HB, -NH)
N
Leu300 (HB, -NH)
O
O
N
5.0 A
Trp20 (PIPI)
N
N
4.5 A
N
4.9 A
4.8 A
Trp20 (PIPI)
Trp20 (PIPI)
O
4.6 A
N
5.0 A
N
2.2 A
Trp111 (HB,NH from indol)
5.0 A
Val47 (VdW)
O
5.0 A
4.9 A
O
1.9 A
Trp111 (HB,NH from indol)
2.2 A
Trp111 (HB,NH from indol)
5.0 A
Val47 (VdW)
Trp20 (PIPI)
5.4 A
2.3 A
O
2.1 A
Trp20 (PIPI)
5.3 A
Trp20 (PIPI)
5.3 A
2.2 A
2.2 A
O
O
His110 (IONIC, HB, -HN+ from imidazol)
His110 (IONIC, -HN+ from imidazol))
1.9 A
Tyr48 (HB, OH)
2.0
A
His110 (IONIC, HB, -HN+ from imidazol)
Lys77 (IONIC)
Tyr48 (HB, OH)
Tyr48 (HB, OH)
Lys77 (IONIC)
Lys77 (IONIC)
solvent accessible part
OTI-13
OTI-12
OTI-11
Figure 6. Proposed naphthalene derivatives OTI-(11-13), their predicted poses and interactions.
Other polycyclic derivatives with favourable predictions and lower solvation include OTI-14
and OTI-15 derivatives. They contain 3,4-dihydronaphtalen-2(1H)-one and 2,3-dihydro-1H-
inden-1-one moiety, resp. Besides their π-π interactions with Trp219, they also revealed H-
bond with Ala299 and OTI-15 also with Leu300. (Fig. 7)
10

O
1.9 A
2.5 A
2.4 A
Ala299 (HB, -NH)
Ala299 (HB, -NH)
4.0 A
O
4.9 A
4.0 A
4.9 A
Trp219 (PIPI)
Trp219 (PIPI)
Trp219 (PIPI)
Trp219 (PIPI)
Leu300 (HB, -NH)
2.4 A
N
N
2.6 A
N
N
Leu300 (HB, -NH)
O
O
N
4.7 A
N
4.9 A
4.9 A
Trp20 (PIPI)
Trp20 (PIPI)
N
4.5 A
N
O
4.9 A
2.2 A
Trp111 (HB,NH from indol)
5.0 A
Val47 (VdW)
O
4.9 A
2.2 A
Trp111 (HB,NH from indol)
5.0 A
Val47 (VdW)
Trp20 (PIPI)
5.3 A
2.2 A
Trp20 (PIPI)
5.3 A
O
3.2 A
O
His110 (IONIC, -HN+ from imidazol))
1.9 A
1.9 A
Tyr48 (HB, OH)
His110 (IONIC)
Tyr48 (HB, OH)
Lys77 (IONIC)
Lys77 (IONIC)
solvent
accessible
part
OTI-15
OTI-14
Figure 7. Predicted ALR2 poses and interactions of proposed polycyclic derivatives OTI-14 and OTI-15.
Conclusions
We have developed four novel N-benzyl(oxotriazinoindole) inhibitors OTI-(7-10), possessing
polar functional groups (-CN, -CONH2, -COOH, -CH2OH, resp.) in ortho position on a benzyl
group. They exhibited 2 to 6-fold better inhibitory activity than their N-(benzyl) lead compound
OTI-6. Our findings confirmed expected additional interactions of novel OTI derivatives in a
studied binding pocket of ALR2. Moreover, the novel derivatives also exhibited ALR1
inhibitory activity in high micromolar concentrations, which resulted in good to excellent ALR1
/ ALR2 selectivity for developed compounds OTI-(7-10). The most efficient ALR2 inhibitor
OTI-7 (IC50 = 76 nM) contains a planar and less solvated -CN functional group on a benzyl
ring. Other derivatives OTI-(8-10) (-CONH2, -COOH and -CH2OH, resp.) revealed 2 to 3-fold
lower ALR2 inhibitory activity than OTI-7. The reason of lower activity of derivatives OTI-
(8-10) was assigned to a desolvation and possibly also to a conformational penalty. Considering
these effects, we have proposed novel analogues OTI-(8a-9a,b) which preserve the favorable
interactions of their parent compounds within ALR2 while possessing less polar substituents.
Therefore, improved inhibition efficacies and drug-like properties of the novel derivatives are
expected. Several polycyclic N-substituted derivatives OTI-(11-15) have also been selected due
to their favorable proposed interactions in ALR2 pocket.
Experimental
11

Computer predictions
The structure of hu-ALR2 protein in a complex with NADP+ (PDB: 4QX4)12 was used for our
predictions. The optimized conformers of the proposed ligands were docked into the enzyme-
cofactor complex by UCSF software Dock Blaster methodology.18 The predicted poses of
ligands in an active site of ALR2 enzyme were analysed and used for composition of OTI-(7-
10) intermolecular interactions diagrams. (Fig. 2-3 and 5-6) The protein conformer of a human
recombinant enzyme AKR1B1 from the complex (PDB: 4QX4), possessing two 3E2
(cemtirestat) ligands and NAP (NADP+) cofactor, was used for modelling, grid generation and
docking. The structures of ligands were proposed by molecular modelling in the Discovery
Studio Visualizer software (release 2019) from Biovia.19 The optimal 3D structures of proposed
ligands together with their protomers, tautomers and isomers were generated by a Ligand
Preparation tool of the Maestro software (release 2016) from Schrodinger at software standard
predifined conditions and pH = 7.0 / ± 1.20 The target protein was prepared for docking in a
Protein Preparation Wizard tool of the Maestro software by standard predefined conditions at
pH 7.0, while the present water molecules were removed. The target hydrogens were
minimized, invalid bond order corrected, missing side chains added and detected amino acid
alternative positions selected. Afterwards, a Receptor Glide Grid Generation tool of the Maestro
software was used at standard predefined conditions exploiting binding position of 3E2 402
ligand, while the second 3E2 403 ligand was removed before grid generation. The optimal
conformers of the ligands were docked into the enzyme-cofactor complex by a Glide Docking
tool of the Maestro software at standard predefined conditions in an extra precision (XP) mode
with flexible ligand sampling, rewarding intramolecular hydrogen bonds and enhancing
planarity of conjugated pi groups. Only the best pose for each ligand was recorded. The most
promising ligands were selected based on their score from docking experiments and the results
of their intermolecular interaction analysis with target amino acid residues.
Chemistry
1
Melting points were measured by Barnstead Electrothermal IA9200 and are uncorrected. H-
13
and C-NMR spectra were recorded on Varian Gemini (300 / 600 MHz and 75 / 150 MHz,
resp.), chemical shifts are given in parts per million (ppm), tetramethylsilane was used as an
internal standard. DMSO-d6 and CDCl3 were used as a solvent, unless otherwise specified. IR
spectra were acquired on FT-IR-ATR REACT IR 1000 (ASI Applied Systems) with diamond
probe and MTS detector. Mass spectra were performed on LC-MS (Agilent Technologies 1200
Series equipped with Mass spectrometer Agilent Technologies 6100 Quadrupole LC-MS). The
12

course of the reactions was followed by TLC analysis (Merck Silica gel 60 F254). UV lamp
(254 nm) and iodine vapours were used for visualization of TLC spots. Starting chemicals were
purchased from Sigma-Aldrich, Fluorochem, AlfaAesar or Acros vendors. All tested
compounds OTI-(6-10) possess purity over 95 %. Their purity was determined by combustion
analysis, HPLC and melting point. Combustion analysis was measured on vario MICRO cube
(elemental analysis found values for carbon, hydrogen, and nitrogen within 0.4 % of the
calculated values for their molecular formulas). Detailed descriptions of the intermediates and
the final products OTI-(7-10), their predicted poses in ALR2 and spectra figures are deposited
in the Supporting information of this paper.
Ethyl 2-(3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl)acetate
(2)
To a solution of 100 mg (0.41 mmol, 1.00 mol eq) of 1 (OTI) dissolved in 30 ml of EtOH (abs),
1.00 ml of H2SO4 (conc) was added and the reaction stirred at reflux under Ar. After 6 h TLC
analysis confirmed a presence of a new product without any starting compound. The mixture
was cooled down and excess of EtOH evaporated by RVE. Crude product was dissolved in 30
ml of DCM, washed with water (5 x 30 ml) and saturated aq solution of NaHCO3 (3 x 30 ml).
Separated organic layer was dried over Na2SO4, filtered and concentrated under reduced
pressure yielding 85.0 mg (0.31 mmol, 76 %) of ester 2 as an orange solid product. Novelty:
ethyl 2-(3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl)acetate (2) is described in the
literature by its M.P., 1H-NMR,13C-NMR, IR, MS and elemental analysis.13 M.p.: 247.0 - 253.0
°C [DCM], lit.: 248.0 - 252.0 °C [MeOH / DCM].13
1H-NMR
DMSO-d6 (300 MHz)
13.14 (br)
NH
N
N
7.95 (8.0, 1.0)
7.34 (8.0, 7.3, 1.0)
7.58 (8.0, 7.3, 1.0)
O
1.22 (7.1)
N
O
7.56 (8.0, 1.0)
5.03
4.18 (7.1)
O
2
1H NMR (300 MHz, DMSO-d6): δ 13.14 (br s, 1H, -NH-), 7.95 (dd, 1H, J(8,9) = 8.0 Hz, J(7,9)
= 1.0 Hz, H-C(9)), 7.58 (ddd, 1H, J(6,7) = 8.0 Hz, J(7,8) = 7.3 Hz, J(7,9) = 1.0 Hz, H-C(7)),
7.56 (dd, 1H, J(6,7) = 8.0 Hz, J(6,8) = 1.0 Hz, H-C(6)), 7.34 (ddd, 1H, J(8,9) = 8.0 Hz, J(7,8)
= 7.3 Hz, J(6,8) = 1.0 Hz, H-C(8)), 5.03 (s, 2H, NCH2COOEt), 4.18 (q, 2H, J(CH2,CH3) = 7.1
Hz, -OCH2CH3), 1.22 (t, 3H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3).
13

Ethyl
2-[2-(2-cyanobenzyl)-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-
yl]acetate (4)
To a solution of 100.0 mg (0.37 mmol, 1.00 mol eq) 2 in 3 ml DMF (abs), 18.5 mg (0.44 mmol,
1.19 mol eq) of CaH2 was added and the reaction stirred at 80 °C for 30 min. Then, the mixture
was cooled to rt and 86.5 mg (0.44 mmol, 1.19 mol eq) of benzonitrile 3 was added portionwise
and the mixture stirred at 80 °C within 1 h. After consumption of 2 (confirmed by a TLC
analysis) the reaction was cooled down in an ice bath, 10 ml of water added and the reaction
stirred for 10 min. Resulting mixture was extracted with DCM (3 x 15 ml), a combined organic
phase washed with brine (3 x 20 ml), dried over Na2SO4, filtered and concentrated under
reduced pressure. A crude product was purified by FLC (SiO2, Hex / EA = 1 / 5) to give 128
mg (0.33 mmol, 90 %) of 4 as an orange solid material. Novelty: ethyl 2-[2-(2-cyanobenzyl)-
3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl]acetate (4) has not been described in the
literature. M.p.: 175.0 - 180.0 °C [Hex / EA].
1H-NMR
13C-NMR
CDCl3 (600 MHz)
CDCl3 (150 MHz)
a
a
a
b
a
CN
a
a
4 A
2
CN
O
b
a
1
a
a
c
b
N
N
N
a N
a
a
a
2
1
O
a
a
4
a
a
a
b
b
9
8
N
a N
5
7
6
N
O
N
b
a
c
a: 7.98 (7.7, 1.0), 7.72 (7.7, 1.0)
7.52 (8.0, 1.0), 7.14 (8.0, 1.0)
b: 2 x 7.55 (8.0, 7.3, 1.0)
7.41 (8.0, 7.7, 1.0)
7.23 (8.0, 7.7, 1.0)
c: 5.70, 4.89
1.28 (7.1)
O
O
14.2
a: 154.3, 154.1, 143.9, 139.7
133.6, 2 x 133.3,131.5
129.4, 128.6, 123.9, 122.3
118.1, 117.6, 112.6, 110.3
b: 62.5, 54.8, 42.2,
166.7
b
4.24 (7.1)
O
4
4
1H-NMR (600 MHz, CDCl3): δ 7.98, 7.72, 7.52 and 7.14 (4 x dd, 4 x 1H, J(6,7 or 8,9 or A3,A4
or A5,A6) = 7.7 or 8.0 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(6, 9, A3 and A6)), 2 x
7.55, 7.41 and 7.23 (4 x ddd, 4 x 1H, J(7,8 or A4,A5) = 8.0 Hz, J(6,7 or 8,9 or A3,A4 or A5,A6)
= 7.7 or 7.3 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(7, 8, A4 and A5)), 5.70 and 4.89
(2 x s, 2H, -CH2COOEt and -CH2Ar), 4.24 (q, 2H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3), 1.28 (t,
3H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3). 13C-NMR (150 MHz, CDCl3): 166.7 (-COOEt), 154.3,
154.1, 143.9, 139.7, 133.6, 2 x 133.3, 131.5, 129.4, 128.6, 123.9, 122.3, 118.1, 117.6, 112.6,
110.3, 62.5, 54.8, 42.2 and 14.2 (-OCH2CH3). FT-IR (solid, cm-1): 2983 (w), 2931 (w), 2228
(m, -CN), 1738 (s), 1662 (s), 1633 (s), 1600 (s), 1568 (s), 1498 (m), 1478 (s), 1408 (s), 1373
(m), 1314 (w), 1274 (m), 1200 (s), 1131 (w), 1104 (s), 1052 (s), 1015 (m), 955 (m), 936 (m),
874 (w), 786 (m), 755 (s), 730 (m), 697 (m), 670 (m), 557 (m), 486 (w), 465 (w), 430 (s). MS
14

(ESI m/z): 387.0 (95 %) [M-e-]. Anal. calcd for C21H17N5O3 (387.39): C, 65.11; H, 4.42; N,
18.08; Found: C, 64.36; H, 4.49; N, 18.45.
2-(2-(2-Cyanobenzyl)-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl)acetic
acid 5 (OTI-7)
To a solution of 100 mg (0.26 mmol, 1.00 mol eq) ester 4 in 20 ml of MeOH, 20.6 mg (0.52
mmol, 2.00 mol eq) of NaOH in 3 ml water was added and the mixture refluxed for 30 min.
After consumption of starting material 4 (confirmed by TLC analysis) the reaction mixture was
cooled down in an ice bath and acidified with 1 M HCl to pH = 3. After 10 min of stirring,
formed precipitate was filtered off, washed with ice water and dried under reduce pressure to
yield 70.0 mg (0.19 mmol, 75 %) of acid 5 (OTI-7) as a brown solid product. Novelty: 2-[2-(2-
cyanobenzyl)-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl]acetic acid 5 (OTI-7) has
not been described in the literature. M.p.: 280.9 - 284.5 °C [MeOH / H3O+].
1H-NMR
13C-NMR
DMSO-d6 (600 MHz)
DMSO-d6 (150 MHz)
a
b
a
a
4
a
a
2
CN
A
b
1
CN
a
a
a
b
c
a
N
4N
N
a
2
N
Na
1
a N
O
a
b
b
9
a
O
8
7
a
6
a
N
a
a
a
a
N
c
b
a: 7.94 (7.7, 1.0), 7.89 (7.7, 1.0)
7.59 (8.0, 1.0), 7.43 (8.0, 1.0)
b: 7.66 (8.0, 7.7, 1.0)
7.63 (8.0, 7.7, 1.0)
7.51 (8.0, 7.5, 1.0)
7.35 (8.0, 7.5, 1.0)
HO
13.35 (br s)
HO
169.1
a: 154.3, 153.4, 144.6, 140.5
134.0, 133.6, 133.1, 131.7
128.9, 128.7, 123.7, 121.7
117.7, 117.6, 111.8, 111.4
b: 55.2, 42.5
O
O
5 (OTI-7)
5 (OTI-7) c: 5.56, 4.94
1H-NMR (600 MHz, DMSO-d6): δ 13.35 (br s, 1H, -COOH), 7.94, 7.89, 7.59 and 7.43 (4 x dd,
4 x 1H, J(6,7 or 8,9 or A3,A4 or A5,A6) = 8.0 or 7.7 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0
Hz, H-C(6, 9, A3, A6)), 7.66, 7.63, 7.51 and 7.35 (4 x ddd, 4 x 1H, J(7,8 or A4,A5) = 8.0 Hz,
J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.7 or 7.5 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-
C(7, 8, A4, A5)), 5.56 and 4.94 (2 x s, 2 x 2H, -CH2COOH and -CH2Ar). 13C-NMR (150 MHz,
DMSO-d6): 169.1 (-COOH), 154.3, 153.4, 144.6, 140.5, 134.0, 133.6, 133.1, 131.7, 128.9,
128.7, 123.7, 121.7, 117.7, 117.6, 111.8, 111.4, 55.2, 42.5. FT-IR (solid, cm-1): 3300 - 2800
(br, COOH) 2226 (w), 1762 (m), 1626 (m), 1604 (s), 1572 (s), 1502 (m), 1468 (m), 1420 (m),
1381 (m), 1341 (m), 1316 (w), 1278 (m), 1229 (m), 1207 (m), 1162 (w), 1134 (w), 1115 (w),
1084 (w), 1058 (w), 1039 (w), 994 (w), 934 (w), 885 (w), 788 (s), 767 (s), 668 (m), 611 (m),
584 (m), 556 (m), 499 (w), 457 (w), 433 (s). MS (ESI m/z): 358.1 (80 %) [M-H+], 314.0 (100
%) [M-CO2-H+]. Anal. calcd for C19H13N5O3 (359.35): C, 63.51; H, 3.65; N, 19.49 Found: C,
63.30; H, 3.60; N, 19.50.
15

2-[2-(2-Carbamoylbenzyl)-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-
yl]acetic acid 6 (OTI-8)
A solution of 20.0 mg (0.05 mmol, 1.00 mol eq) ester 4 in 1 ml of CF3COOH (TFA) and 0.25
ml of H2SO4 (conc) was stirred at 70 °C for 6 h. After complete consumption of the starting
compound 4 (confirmed by a TLC analysis), the reaction was cooled down and 20 ml of H2O
and 20 ml of brine added, and the mixture extracted with EA (5 x 15 ml). Combined organic
layer was dried over Na2SO4, filtered and concentrated under reduced pressure. A crude product
6 (OTI-8) was purified by trituration with a mixture of Hex / EA to give 15.0 mg (0.04 mmol,
77 %) of acid 6 (OTI-8) as a solid compound. Novelty: 2-[2-(2-carbamoylbenzyl)-3-oxo-2,3-
dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl]acetic acid 6 (OTI-8) has not been described in the
literature. M.p.: 221.3 - 225.1 °C [EA].
1H-NMR
DMSO-d6 (150 MHz)
13C-NMR
DMSO-d6 (600 MHz)
a
O
b
b
O
b
3
3
b
A 2
b
2
A
b
a
1
1
NH2
8.00, 7.53 (2 x br)
NH2
b
a
b
c
c
N
N
N
a
b N
2
b
2
1
1
O
O
b
4N
b
b
4
b
b
9
6
N b
8
7
9
8
7
6
b
N
N
b
a
b
c
a: 170.7, 169.1
c
HO
13.10 (br)
HO
a: 7.95 (7.7, 1.0), 7.58 (7.7, 1.0)
7.54 (7.1, 1.0), 7.03 (7.1, 1.0)
b: 7.62 (8.0, 7.7, 1.0)
7.38 - 7.31 (3H)
b: 154.1, 153.7, 144.7, 136.0
135.5, 132.9, 131.5, 130.4
128.2, 127.5, 127.3, 123.6
121.7, 117.7, 111.7
a
O
O
6 (OTI-8)
6 (OTI-8)
c: 55.2, 42.5
c: 5.60, 4.95
1H-NMR (600 MHz, DMSO-d6): δ 13.10 (br s, 1H, -COOH), 8.00 and 7.53 (2 x br s, 2 x 1H, -
ArCONH2), 7.95, 7.58, 7.54 and 7.03 (4 x dd, 4 x 1H, J(6,7 or 8,9 and A3,A4 or A5,A6) = 7.7 or
7.1 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(6, 9, A3 and A6)), 7.62 and 7.38 - 7.31 (1
x ddd and m, 1H and 3H, J(7,8 or A4,A5) = 8.0 Hz, J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.7 Hz,
J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(7, 8, A4 and A5)), 5.60 and 4.95 (2 x s, 2 x 2H, -
CH2COOH and -CH2Ar). 13C-NMR (150 MHz, DMSO-d6): 170.7, 169.1, 154.1, 153.7, 144.7,
136.0, 135.5, 132.9, 131.5, 130.4, 128.2, 127.5, 127.3, 123.6, 121.7, 117.7, 111.7, 55.2 and
42.5. FT-IR (solid, cm-1): 3357 (m), 3191 (m), 2959 (m), 2928 (m), 2858 (w), 2342 (w), 1736
(s), 1671 (s), 1606 (s), 1578 (s), 1498 (m), 1465 (m), 1413 (m), 1375 (w), 1282 (m), 1199 (s),
1122 (s), 941 (m), 748 (s), 678 (m), 631 (m), 504 (w), 438 (m). MS (ESI m/z): 378.0 (100 %)
[M+H+], 400.1 (20 %) [M+Na+], 777.2 (80 %) [(2 x M) + Na+]. Anal. calcd for C19H15N5O4
(377.36): C, 60.48; H, 4.01; N, 18.56; Found: C, 60.85; H, 4.39; N, 18.75.
16

2-{[5-(Carboxymethyl)-3-oxo-3,5-dihydro-2H-[1,2,4]triazino[5,6-b]indol-2-
yl]methyl}benzoic acid 7 (OTI-9)
A solution of 30.0 mg (0.08 mmol, 1.00 mol eq) of ester 4 in 2 ml of H2SO4 (conc) and 2 ml of
H2O was stirred at 100 °C within 8 d. After complete consumption of starting compound 4 and
its amidic intermediate (confirmed by a TLC analysis), the reaction was cooled down, 20 ml of
H2O added and the resulting mixture extracted with EA (5 x 15 ml). Combined organic layer
was dried over Na2SO4, filtered and concentrated to a half of its volume. Then, the solution was
placed into a refrigerator, where the product 7 (OTI-9) precipitated. After filtration, crude
product was triturated with a mixture of Hex / EA to give 12.0 mg (0.03 mmol, 41 %) of bisacid
7 (OTI-9) as a yellow solid compound. Novelty: 2-{[5-(carboxymethyl)-3-oxo-3,5-dihydro-
2H-[1,2,4]triazino[5,6-b]indol-2 yl]methyl}benzoic acid 7 (OTI-9) has not been described in
the literature. M.p.: 260 - 277 °C (dec) [EA].
1H-NMR
DMSO-d6 (600 MHz)
DMSO-d6 (150 MHz)
13C-NMR
a
b
3
O
O
b
b
b
a
3
5
5
b
A
A
b
1
1
OH 13.15 (br)
b
OH
a
b
c
c
N
N
N
a
b N
2
b
1
2
4
O
1
O
b
4N
b
b
9
6
b
b
N b
8
7
9
8
7
6
b
N
N
b
a
b
c
c
a: 169.1, 168.6
HO
a: 7.96, 7.93, 7.58, 7.00
4x (7.7, 1.0)
b: 7.62, 7.48, 7.41, 7.33
4 x (8.0, 7.7, 1.0)
c: 5.74, 4.95
HO
a
b: 154.2, 153.6, 144.5, 138.3
133.0, 132.7, 131.5, 131.0
130.0, 127.7, 127.3, 123.6
121.6, 117.7, 111.7
13.15 (br)
O
O
7 (OTI-9)
7 (OTI-9)
c: 55.2, 42.5
1H-NMR (600 MHz, DMSO-d6): δ 13.15 (2 x br s, 2 x 1H, 2 x -COOH), 7.96, 7.93, 7.58 and
7.00 (4 x dd, 4 x 1H, J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.7 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6)
= 1.0 Hz, H-C(6, 9, A3 and A6)), 7.62, 7.48, 7.41 and 7.33 (4 x ddd, 4 x 1H, J(7,8 or A4,A5) =
8.0 Hz, J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.7 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-
13
C(7, 8, A4 and A5)), 5.74 and 4.95 (2 x s, 2 x 2H, -CH2COOH and -CH2Ar). C-NMR (150
MHz, DMSO-d6): 169.1, 168.6, 154.2, 153.6, 144.5, 138.3, 133.0, 132.7, 131.5, 131.0, 130.0,
127.7, 127.3, 123.6, 121.6, 117.7, 111.7, 55.2 and 42.5. FT-IR (solid, cm-1): 3000 - 2200 (br, -
COOH) 2822 (m), 2606 (m), 2088 (w), 1725 (m), 1688 (m), 1635 (m), 1602 (s), 1569 (s), 1499
(m), 1467 (m), 1406 (m), 1313 (m), 1225 (s), 1203 (s), 1134 (w), 1107 (m), 1056 (w), 968 (m),
940 (s), 827 (w), 788 (s), 733 (s), 663 (s), 614 (w), 582 (w), 554 (w), 483 (w), 436 (s). MS (ESI
m/z): 377.0 (25 %) [M-H+]-, 365.1 (100 %) [M-CO2-H++CH3OH]-, 333.1 (100 %) [M-CO2-H+],
755 (30 %) [(2 x M)-H+]. Anal. calcd for C19H14N4O5 (378.34): C, 60.32; H, 3.73; N, 14.81;
Found: C, 60.55; H, 4.05; N, 14.97.
17

1,2-Phenylenedimethanol (9)
A solution of phthalic anhydride 8 (440 mg, 2.97 mmol, 1.00 mol eq) in 15 ml of THF (abs)
was added dropwise at 0 °C to the stirred suspension of LiAlH4 (226 mg, 5.96 mmol, 2.00 mol
eq) in 20 ml of THF (abs) within 30 min. Then, the mixture was stirred overnight at room
temperature. Afterwards, the reaction was cooled to 0 °C and 10 ml of isopropanol added
slowly, followed by methanol (10 ml) and water (10 ml). Obtained wax-like precipitate was
filtered through Celite and obtained filtrate washed with EA and DCM. Organic solution was
evaporated and aqueous mixture extracted with EA (3x 20 ml) and DCM (3x 10 ml). Combined
organic layer was dried over Na2SO4, filtered and concentrated over reduced pressure totally
yielding 385 mg (2.79 mmol, 94 %) of diol 9 as a colourless oil, which solidified by storing in
a refrigerator. Novelty: 1,2-phenylenedimethanol (9) is described in the literature by its M.P.,
1H-NMR, 13C-NMR, IR and HRMS.21 M.p.: 52.0 - 57.7 °C [EA / DCM], lit: 61 - 63 °C [EA]21.
1H-NMR
CDCl3 (600 MHz)
a
4.73
3
a
2.98 (br)
4
OH
2
1
OH
a : 7.38 - 7.30 (4 H)
9
1H-NMR (600 MHz, CDCl3): δ 7.38 - 7.30 (m, 4H, 2 x H-C(3 and 4)), 4.73 (s, 4H, 2 x -
CH2OH), 2.98 (br s, 2H, -CH2OH).
2-(Bromomethyl)phenyl]methanol (10)
A solution of 105 mg (0.76 mmol, 1.00 mol eq) 1,2-phenylenedimethanol (9) in 20 ml of toluene
was heated to 70 °C. At this temperature, 80 µl (0.85 mmol, 1.12 mol eq) of 48 % solution of
HBr was added dropwise. After 20 min, TLC analysis confirmed a presence of product 10 and
a small amount of probably bis brominated side product. Reaction mixture was cooled down,
neutralized with saturated aq solution of Na2CO3 and extracted with diethyl ether (3 x 15 ml).
Combined organic layer was evaporated and resulted mixture purified by FLC (Hex / EA = 5 /
1) to afford 80.0 mg (0.40 mmol, 56 %) of desired product 10 as a white solid compound.
1
Novelty: 2-(bromomethyl)phenyl]methanol (10) is described in the literature by its M.P., H-
NMR, IR22 and MS spectra.23 M.p.: 61.0 - 63.8 °C [Et2O], lit: 55 °C [hexane].22
18

1H-NMR
CDCl3 (600 MHz)
a
c
b
OH
Br
a: 7.43 (7.5, 1.5)
7.39 (7.5, 1.5)
5
1
2
4
3
b
c
a
b: 7.35 (7.9, 7.5, 1.5)
7.31 (7.9, 7.5, 1.5)
c: 4.86, 4.66
10
1H-NMR (600 MHz, CDCl3): δ 7.43 and 7.39 (2 x dd, 2 x 1H, J(3,4 or 5,6) = 7.5 Hz, J(3,5 or
4,6) = 1.5 Hz, H-C(3 and 6)), 7.35 and 7.31 (2 x ddd, 2 x 1H, J(4,5) = 7.9 Hz, J(3,4 or 5,6) =
7.5 Hz, J(3,5 or 4,6) = 1.5 Hz, H-C(4 and 5)), 4.86 and 4.66 (2 x s, 2 x 2H, -CH2OH and CH2Br).
2-{[2-(Bromomethyl)benzyl]oxy}tetrahydro-2H-pyran (11)
A solution of 245 mg (1.22 mmol, 1.00 mol eq) of arylmethanol 10 in 20 ml of DCM (abs), 130
µl (1.42 mmol, 1.16 mol eq) of 3,4-dihydro-2H-pyran (DHP) and 30.6 mg (0.12 mmol, 0.10
mol eq) of PPTS (pyridinium p-toulenesulfonate), was stirred under Ar for 30 min at reflux.
Subsequent TLC analysis confirmed presence of a new product and traces of two side products.
Then, 20 ml of NaHCO3 (sat aq sol) was added and resulting mixture extracted with DCM (3 x
15 ml). Combined organic layer was dried over Na2SO4, filtered and concentrated under
reduced pressure yielding 310 mg (1.09 mmol, 89 %) of tetrahydropyran 11 as a colourless oily
product. Novelty: 2-{[2-(bromomethyl)benzyl]oxy}tetrahydro-2H-pyran (11) is described in
the literature by its 1H-NMR and crystal property description.24
13C-NMR
1H-NMR
CDCl3 (150 MHz)
CDCl3 (600 MHz)
c
c
c
c
c
c
c
4.94 (12.3)
4.65 (12.3)
3
5
3
5
A
1
a
A
a
b
b
3.94 (11.1, 8.5, 3.0)
3.59 (1H)
1
a
a
b
b
O
O
a
a
5
1
O
99.0 O
5
1
3
Br
3
4.74 (8.5, 3.0)
Br
a
4.68 (10.2)
4.63 (10.2)
a
a: 136.7, 136.2, 130.6, 128.7
128.6, 128.3
b: 66.4, 62.3
a: 7.42 (7.4, 1.9), 7.39 (7.4, 1.9)
b: 7.32 (8.0, 7.4, 1.9)
7.30 (8.0, 7.4, 1.9)
c: 1.92 - 1.52 (6H)
11 c: 31.0, 30.5, 25.4, 19.4
11
1H-NMR (600 MHz, CDCl3): δ 7.42 and 7.39 (2 x dd, 2 x 1H, J(3,4 or 5,6) = 7.4 Hz, J(3,5 or
4,6) = 1.9 Hz, H-C(3 and 6)), 7.32 and 7.30 (2 x ddd, 2 x 1H, J(4,5) = 8.0 Hz, J(3,4 or 5,6) =
7.4 Hz, J(3,5 or 4,6) = 1.9 Hz, H-C(4 and 5)), 4.94 (d, 1H, Jgem =12.3 Hz, -CH2OTHP), 4.74
(dd, 1H, J(A2,A3) = 8.5 Hz, J(A2,A3) = 3.0 Hz, H-CA(2)), 4.68 (d, 1H, Jgem = 10.2 Hz, -CH2Br),
4.65 (d, 1H, Jgem = 12.3 Hz, -CH2OTHP), 4.63 (d, 1H, Jgem = 10.2 Hz, -CH2Br), 3.94 (ddd, 1H,
Jgem = 11.1 Hz, J(A5,A6) = 8.5 Hz, J(A5,A6) = 3.0 Hz, H-CA(6)), 3.59 (m, 1H, H-CA(6)), 1.92 -
13
1.52 (m, 6H, H-CA(3-5)). C-NMR (150 MHz, CDCl3): 136.7, 136.2, 130.6, 128.7, 128.6,
19

128.3, 99.0 (-OCHO-), 66.4, 62.3, 31.0, 30.5, 25.4 and 19.4. FT-IR (solid, cm-1): 2939 (m),
2867 (w), 1723 (w), 1454 (m), 1385 (m),1260 (w), 1200 (m), 1118 (s), 1076 (m), 1022 (s), 971
(m), 904 (m), 869 (m), 814 (w), 760 (m), 606 (m), 533 (w)
Ethyl 2-{3-oxo-2-{2-{[(tetrahydro-2H-pyran-2-yl)oxy]methyl}benzyl}-2,3-dihydro-
5H-[1,2,4]triazino[5,6-b]indol-5-yl}acetate (12)
To a solution of 200.0 mg (0.74 mmol, 1.00 mol eq) 2 in 5 ml DMF (abs), 37.0 mg (0.88 mmol,
1.19 mol eq) of CaH2 was added and the mixture stirred at 80 °C under Ar within 30 min. Then,
the reaction was cooled to rt, 310 mg (1.09 mmol, 1.47 mol eq) of tetrahydropyran 11 was
added dropwise and the solution was stirred at 80 °C within 1 h under Ar. After consumption
of 2 (confirmed by a TLC analysis) the reaction was cooled down, 10 ml of water added, and
the mixture stirred for 10 min in an ice bath. Resulting mixture was extracted with DCM (3 x
15 ml), washed with brine (3 x 20 ml), dried over Na2SO4, filtered and concentrated under
reduced pressure. A crude product was purified by FLC (SiO2, Hex / EA = 1 / 5) to give 286
mg (0.60 mmol, 82 %) of 12 as red oil. Novelty: ethyl 2-{3-oxo-2-{2-{[(tetrahydro-2H-pyran-
2-yl)oxy]methyl}benzyl}-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl}acetate (12) has not
been described in the literature.
1H-NMR
CDCl3 (600 MHz)
5.06 (12.3)
4.80 (12.3)
a
b
d
d
4
4
2
b
A
d
1
B
2
O
1
a
O
3.95 (11.1, 8.5, 3.0)
3.58 (1H)
c
4.78 (8.5, 3.0)
N
N
a
2
1
O
4
b
b
9
8
7
N
6
a: 7.93 (7.8, 1.0), 7.43 (7.2, 1.0)
7.35 (7.2, 1.0), 7.13 (7.8, 1.0)
b: 7.53 (8.0, 7.2, 1.0)
7.31 (8.0, 7.8, 1.0)
N
a
c
1.28 (7.1)
O
2 x 7.27 (8.0, 7.8, 1.0)
c: 5.62, 4.89
d: 1.90 - 1.50
O
4.24 (7.1)
12
1H-NMR (600 MHz, CDCl3): δ 7.93, 7.43, 7.35 and 7.13 (4 x dd, 4 x 1H, J(6,7 or 8,9 or A3,A4
or A5,A6) = 7.8 and 7.2 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(6, 9, A3 and A6)),
7.53, 7.31, 2 x 7.27 (4 x ddd, 4 x 1H, J(7,8 or A4,A5) = 8.0 Hz, J(6,7 or 8,9 or A3,A4 or A5,A6)
= 7.8 and 7.2 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(7, 8, A4 and A5)), 5.06 (d, 1H,
Jgem = 12.3 Hz, -OCH2Ph), 5.62 and 4.89 (surprisingly 2 x s, 2 x 2H, -CH2COOEt and -CH2Ph),
4.80 (d, 1H, Jgem = 12.3 Hz, -OCH2Ph), 4.78 (dd, 1H, J(B2,B3) = 8.5 Hz and J(B2,B3) = 3.0 Hz,
H-CB(2)), 4.24 (q, 2H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3), 3.95 (ddd, 1H, Jgem = 11.1 Hz,
J(B5,B6) = 8.5 Hz, J(B5B6) = 3.0 Hz, H-CB(6)), 3.58 (m, 1H, H-CB(6)), 1.90 - 1.50 (m, 6H, 3 x
-CH2- from C(B3-B5)), 1.28 (t, 3H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3). Due to moisture
20

instability of the 12, its other spectra and analysis were not recorded and compound 12 was
used directly in the next step.
2-{2-[2-(Hydroxymethyl)benzyl]-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-
5-yl}acetic acid 13 (OTI-10)
To a solution of 50.0 mg (0.11 mmol, 1.00 mol eq) ester 12 in 10 ml of dioxane, 1 ml of H2O and
0.1 ml of HCl (37 % w/w) were added and resulting solution stirred at 90 °C for 8 h. After that,
TLC analysis confirmed presence of a new product without side products. Reaction mixture was
cooled down, 30 ml of water added and extracted with DCM (3 x 15 ml). Combined organic
layer was dried over Na2SO4, filtered and concentrated under reduce pressure yielding 25.0 mg
(0.07 mmol, 66 %) of acid 13 (OTI-10) as a yellow solid compound. Novelty: 2-{2-[2-
(hydroxymethyl)benzyl]-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl}acetic acid 13
(OTI-10) has not been described in the literature. M.p.: 220.0 - 238.0 °C (dec) [DCM].
1H-NMR
13C-NMR
DMSO-d6 (600 MHz)
DMSO-d6 (150 MHz)
a
a
a
b
b
a
c
54A
a
54A
2
2
a
b
1
1
OH
5.23
a
OH
a
b
c
N
N
a
N
a
2
N
1
a
2
O
a
1
a
O
a
4
9
4
8
7
b
9
N
8
5
N
a
a
b
6
5
7
b
N
6
a
N
O
a
a
c
HO
169.1
HO
13.35 (br)
a: 7.94 (7.5, 1.0), 7.59 (7.3, 1.0)
7.43 (7.3, 1.0), 7.02 (7.5, 1.0)
b: 7.62 (8.0, 7.5, 1.0)
7.34 (8.0, 7.3, 1.0)
7.27 (8.0, 7.3, 1.0)
7.19 (8.0, 7.5, 1.0)
O
a: 154.0, 153.9, 144.9, 142.3, 134.8
132.8, 131.5, 128.0, 127.6, 127.5
127.3, 123.6, 121.5, 117.7, 111.8
c: 5.45, 4.92, 4.70
b: 63.3, 53.4, 42.7
OTI-10
OTI-10
1H-NMR (600 MHz, DMSO-d6): δ 13.35 (br s, 1H, -COOH), 7.94, 7.59, 7.43 and 7.02 (4 x dd,
4 x 1H, J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.5 or 7.3 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz,
H-C(6, 9, A3 and A6)), 7.62, 7.34, 7.27 and 7.19 (4 x ddd, 4 x 1H, J(7,8 or A4,A5) = 8.0 Hz, J(6,7
or 8,9 or A3,A4 or A5,A6) = 7.5 or 7.3 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(7, 8, A4
and A5)), 5.45, 4.92 and 4.70 (3 x s, 3 x 2H, -CH2COOEt, -CH2Ph and -CH2OH), 5.23 (s, 1H, -
OH). 13C-NMR (150 MHz, DMSO-d6): 169.1 (-COOH), 154.0, 153.9, 144.9, 142.3, 134.8, 132.8,
131.5, 128.0, 127.6, 127.5, 127.3, 123.6, 121.5, 117.7, 111.8, 63.3, 53.4 and 42.7. FT-IR (solid,
cm-1): 3207 (w), 2922 (w), 2712 (w), 2602 (w), 2522 (w), 2108 (w), 2087 (w), 1728 (s), 1606
(s), 1577 (s), 1502 (m), 1467 (m), 1414 (s), 1374 (m), 1228 (s), 1203 (s), 1116 (w), 1087 (w),
1045 (m), 997 (w), 954 (w), 937 (w), 893 (w), 786 (m), 751 (s), 670 (m), 610 (m), 585 (m), 478
(w), 434 (s). MS (ESI m/z): 363.0 (95 %) [M-H+], 319.1 (95 %) [M-CO2-H+]. Anal. calcd for
C19H16N4O4 (364.36): C, 62.63; H, 4.43; N, 15.38 Found: C, 62.75; H, 4.49; N, 15.55.
21

Animals: Male Wistar rats 8 - 9 weeks old, weighing 200 - 230 g, were used as organ donors.
The animals came from the Breeding Facility of the Institute of Experimental Pharmacology
Dobrá Voda (Slovak Republic). The study was approved by the Ethics Committee of the
Institute and performed in accordance with the Principles of Laboratory Animal Care (NIH
publication 83 - 25, revised 1985) and the Slovak law regulating animal experiments (Decree
289, Part 139, July 9th 2003).
Enzyme assays: Preparation of ALR2 from the rat lens and ALR1 from the rat kidneys was
previously reported.25 Enzyme activities were assayed spectrophotometrically by determining
NADPH consumption at 340 nm and were expressed as decrease of the optical density (O.D.) /
s/mg protein.25 The experimental procedures are in detail described in Supporting information to
this paper.
Acknowledgements
MH and AB equally contributed to writing of this paper. This research was supported by
Biomagi, Ltd. (design of novel OTI inhibitors) and by VEGA 1/0670/18 project (synthesis).
Our gratitude belongs to LK, MSP and MS for biological assays and evaluation of a biological
activity supported by VEGA 2/0005/2018. MH, GH contributed to the development of
synthesis and AG to analysis of prepared compounds. We are grateful to Mgr. Juraj Filo, PhD.,
for the measurement of NMR spectra and Bc. Lenka Soboličová for synthesis of intermediates
and the inhibitor OTI-7. Finally, we would like to thank bilateral project APVV SK-FR-2015-
0014 and COST Action CA15135 MuTaLig for networking and Cotutelle joint PhD French-
Slovak program for MH bilateral study.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version, at http://xxx.
These data include spectra of compounds described in this article together with predicted poses
and interaction analyses for all developed inhibitors. Included are also experimental procedures
comprising isolation of rat kidney ALR1, rat eye lens ALR2 and enzyme activity assays.
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Highlights:
 Four novel aldose reductase inhibitors exploiting an unoccupied enzyme pocket were
developed
 All novel inhibitors revealed higher inhibitory efficacy and selectivity compared to their
lead
 Additional inhibitor-enzyme interactions were suggested to be responsible for the
improved biological activity
 Based on this study, several novel promising derivatives were proposed
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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