Discovery of [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl] acetic acids as highly potent and selective inhibitors of aldose reductase for treatment of chronic diabetic complications

Article abstract of DOI:10.1016/j.bmcl.2009.02.037

Efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel series of highly potent and selective [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl] acetic acid aldose reductase inhibitors. The lead candidate, [6-methyl-3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyri din-1-yl]acetic acid example 16, inhibits aldose reductase with an IC50 of 8 nM, while being inactive against aldehyde reductase (IC50 > 100 μM), a related enzyme involved in the detoxification of reactive aldehydes.

Full text of DOI:10.1016/j.bmcl.2009.02.037

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2006–2008
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]
pyridin-1-yl]acetic acids as highly potent and selective inhibitors of aldose
reductase for treatment of chronic diabetic complications
a
a
a
b
Michael C. Van Zandt a, , Brian Doan , Diane R. Sawicki , Janet Sredy , Alberto D. Podjarny
*
a The Institute for Diabetes Discovery, LLC, 23 Business Park Drive, Branford, CT 06405, USA
b Département de Biologie Structurale et Génomique, IGBMC, CNRS, INSERM, UDS, 1 rue Laurent Fries, BP 10142, 67404 Illkirch CEDEX, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel
series of highly potent and selective [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-
1-yl]acetic acid aldose reductase inhibitors. The lead candidate, [6-methyl-3-(4,5,7-trifluoro-ben-
zothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl]acetic acid example 16, inhibits aldose reductase with
Received 26 November 2008
Revised 5 February 2009
Accepted 9 February 2009
Available online 12 February 2009
an IC50 of 8 nM, while being inactive against aldehyde reductase (IC50 > 100
involved in the detoxification of reactive aldehydes.
lM), a related enzyme
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Aldose reductase
Aldehyde reductase
Diabetes
Chronic diabetic complications which include blindness, renal
failure, neuropathy, limb amputation, myocardial infarction and
stroke arise from elevated levels of glucose in tissues such as the
nerve, kidney, retina and lens. Although glucose is preferentially
metabolized through the glycolytic pathway, during conditions of
hyperglycemia, as observed in diabetes mellitus, elevated blood
glucose levels saturate the normal pathways of glucose metabo-
lism and a dramatic increase in flux through the polyol pathway
results (Scheme 1).1 Glucose entering the polyol pathway is re-
duced to sorbitol by aldose reductase (ALR2) and NADPH. Sorbitol
is subsequently oxidized to fructose by sorbitol dehydrogenase and
NAD+. This increased flux through the polyol pathway results in a
reduced ratio of NADPH to NADP+, and an increased ratio of NADH
to NAD+. These changes, which alter the reduction potential of the
cell are collectively termed oxidative stress. The impact of oxida-
tive stress has been clearly demonstrated.2 It is linked to depleted
intracellular levels of reduced glutathione, increased non-enzy-
matic glycation and activation of protein kinase C.
3-deoxyglucosone and methyl glyoxal to their corresponding nonre-
active alcohols.6 These aldehydes, which are involved in the forma-
tion of various protein cross-links and other advanced glycation end
products (AGEs), are formed as degradation products from glucose
and fructose.7 During conditions of prolonged hyperglycemia, high
concentrations of these aldehydes are formed resulting in the devel-
opment of chronic diabetic complications and accelerated aging.8
As a member of the aldo-ketoreductase super family, ALR1 shares
greater than 85% sequence homology with ALR2. Specific amino acid
changes in the C-terminal loop, a section of the protein lining the
active site cleft known as the specificity pocket, are primarily
responsible for the various substrate and inhibitor specificities.9
Our work in this area has focused on identifying new carboxylic
acid-based templates with significantly improved oral efficacy and
selectivity relative to aldehyde reductase. Following the identifica-
tion and clinical development of lidorestat (1, Fig. 1),10 continued
optimization of this series has resulted in the discovery of a novel
series of [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-
b]pyridin-1-yl]acetic acids.
The target compounds were synthesized using the method illus-
trated in Scheme 2. The synthesis of [6-ethyl-3-(4,5,7-trifluoro-ben-
zothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl]acetic acid (15) is
described as an example. Here, acylation of 2-amino-6-ethylpyri-
dine 2 with pivaloyl chloride gives the corresponding amide 3 in
54% yield after recrystallization. Subsequent treatment with 2 equiv
of tert-butyl lithium followed by alkylation with methyl iodide gives
3-methyl substituted pyridine 4. Retreatment with tert-butyl
lithium provides the bis-anion which, after quenching with dimeth-
Although many potent aldose reductase inhibitors (ARIs) have
been identified and developed, none are currently marketed for
worldwide use.3 Many of these candidates failed to gain acceptance
due to an inadequate therapeutic index.4 In some cases this toxicity
likely resulted from a lack of selectivity relative to aldehyde reduc-
tase (ALR1). The physiological importance of ALR1 has been clearly
demonstrated.5 It converts highly reactive 2-oxoaldehydes like
* Corresponding author. Tel.: +1 203 315 5951; fax: +1 203 488 3042.
E-mail address: michael.vanzandt@ipd-discovery.com (M.C. Van Zandt).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.037

M. C. Van Zandt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2006–2008
2007
Scheme 1. Polyol pathway.
sodium hydride provides 2-methylbenzthiazole 12. Hydrolysis of
the heterocyclic ring with aqueous sodium hydroxide followed by
acidification with 2 N HCl gives the desired 2-aminothiophenol as
the hydrochloride salt (13). Condensation with 7-azaindole 3-aceto-
nitrile 8 as a melt provides benzothiazole 14 in 86% yield. Finally,
hydrolysis with aq NaOH in ethanol provides the target compound
15 in 5% overall yield (9 linear steps).
F
F
N
S
F
N
O
With lidorestat proceeding to a phase II clinical trial, the major
objective of this program has been to identify a highly potent and
efficacious back-up candidate with increased selectivity. All com-
pounds in the program were initially tested for potency against hu-
man aldose reductase (hALR2) and selectivity relative to human
aldehyde reductase (hALR1). The results from these experiments
are listed in Table 1.
Based on our experience with lidorestat and related carboxylic
acid based aldose reductase inhibitors9, the previously optimized
4,5,7-trifluorobenzthiazole side-chain which interacts directly
with the specificity pocket11 was kept constant while variations
in the indole core were explored.
OH
Figure 1. Lidorestat.
ylformamide and hydrolysis, gives the intermediate aldehyde.
Without purification, cyclization with 4 N hydrochloric acid gives
the desired 7-azaindole 5 in 58% yield (2 steps). Formation of
gramine intermediate 6 with aq dimethylamine and formaldehyde
in acetic acid followed by displacement with potassium cyanide in
acetic acid gives indole-3-acetonitrile derivative 7. Alkylation with
ethyl bromoacetate using sodium hydride in acetonitrile provides
the N-alkylated product (8) in 55% yield. In parallel, the 2-amino-
thiophenol hydrochloride salt 13 is prepared from 2,3,5,6-tetrafluo-
roaniline 9. Acylation with acetic anhydride in pyridine at 120 °C
followed by treatment with P4S10 in benzene conveniently gives
the intermediate thioamide 11. Subsequent cyclization using
As illustrated in Table 1, the 7-aza-analogs are very potent and
selective relative to ALR1. Example (17), along with the 60-methyl
(16) and 60-ethyl (15) substituted compounds have IC50’s of 7, 8
and 12 nM respectively. Activity for ALR1 is minimal with all com-
pounds having IC50’s > 100 lM.
Scheme 2. Reagents and conditions: (a) PivCl, Et3N, CH2Cl2, (54%); (b) (i) t-BuLi, (ii) MeI (69%); (c) (i) t-BuLi, THF; (ii) DMF; (d) 6 N HCl (58% c and d); (e) 40 wt %
dimethylamine, 37 wt % formaldehyde, acetic acid, 100 °C (88%); (f) KCN, aq acetic acid, 110 °C (93%); (g) ethyl bromoacetate, NaH, acetonitrile (55%); (h) Ac2O, pyridine,
120 °C (82%); (i)P4S10, benzene (88%); (j) NaH, toluene (96%); (k) 30% aq NaOH, ethylene glycol, 125 °C (73%); (l) melt, cat. BHT, 120 °C (86%); (m) aq NaOH, ethanol (56%).

2008
M. C. Van Zandt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2006–2008
Table 1
In vitro activity of 7-azaindoleacetic acid ARIs
F
F
N
S
F
N
R
X
O
OH
Ex.
R
X
hALR2a (nM)
HALR1b (nM)
1
17
16
15
H
H
CH
N
N
5
7
8
12
27,000
>100,000c
>100,000
>100,000
CH3
CH2CH3
N
a
b
c
Figure 3.
Recombinant human aldose reductase.
Recombinant human aldehyde reductase.
%Inhibition < 50% @ 100,000 lM.
Although it is not clear what selectivity profile is necessary to
avoid potential aldehyde reductase related toxicity, combining
the 4,5,7-trifluorobenzothiazole with novel heterocycle cores such
as the 7-azaindole, can result is new ARIs with exceptional potency
and selectivity.
Acknowledgments
This work was supported, in part, by the Centre National de la
Recherche Scientifique (CNRS), the Institut National de la Santé
et de la Recherche Médicale and the Hôpital Universitaire de Stras-
bourg (H.U.S.). We thank the personnel of the SBC, and in particular
Andrzej Joachimiak and Ruslan Sanishvili (Nukro), for their help in
data collection.
Supplementary data
Figure 2.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.037.
The use of X-ray crystallography early in the program was
essential. The structures provided a clear understanding of the
important interactions that make up the enzyme-inhibitor com-
plexes. The complexes were obtained using human ALR2 expressed
in Escherichia Coli and crystallized with the oxidized form of the
coenzyme b-NADPH+ at pH 5 and 277 K. Diffraction data for exam-
ple 17 was collected with a laboratory source at a resolution of
1.8 Å. As illustrated in Figure 2, the inhibitor is oriented in the ac-
tive site of ALR2 in a manner such that the hydrophilic carboxylate
head forms tight hydrogen bonds with the OH of Tyr 48 (2.74 Å),
the NE2 of His 110 (2.74 Å) and the NE1 of Trp 111 (2.96 Å). These
hydrogen bonds anchor the inhibitor in an anionic well deep with-
in the enzyme active site. Interactions between the aromatic
hydrophobic side chain of the inhibitor and apolar and aromatic
residues lining the active sites further stabilize the inhibitors.
As this class of inhibitors bind to the ALR2 active site, a confor-
mational change occurs opening a pocket between Trp 111 and Leu
300. The specific opening of the pocket varies to accommodate the
particular inhibitor, producing an ‘induced fit’. Since the residues
lining this pocket are not conserved in ALR1, the interactions in
this pocket are specific for ALR2. This is exemplified in Figure 3,
where the structure of the complex ALR2—example 17 has been
superposed with the structure of ALR1 (magenta). Using this
superposed structure, it is clear that the inhibitor has strong steric
clashes with the ALR1 residues Pro 301 (1.35 Å) and Tyr 116
(1.10 Å). These clashes can explain the strong selectivity of this
class of inhibitors for ALR2 versus ALR1. The structure of example
17 has been deposited in the Protein Data Bank. The PDB code is
3G5E.
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