Tricyclic indole and dihydroindole derivatives as new inhibitors of soluble guanylate cyclase

Article abstract of DOI:10.1016/j.bmcl.2009.06.047

The synthesis of new tricyclic fused indole and dihydroindole derivatives and preliminary results from their in vitro inhibitory activity against soluble guanylate cyclase (sGC) are presented.

Full text of DOI:10.1016/j.bmcl.2009.06.047

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4810–4813
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tricyclic indole and dihydroindole derivatives as new inhibitors of soluble
guanylate cyclase
Katerina Spyridonidou a, Manolis Fousteris a, Marazioti Antonia b, Athanasia Chatzianastasiou a,
Andreas Papapetropoulos b, Sotiris Nikolaropoulos a,
*
a Laboratory of Medicinal Chemistry, Department of Pharmacy, School of Health Sciences, University of Patras, 26500, Patras, Greece
b Laboratory of Molecular Pharmacology, Department of Pharmacy, School of Health Sciences, University of Patras, 26500, Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of new tricyclic fused indole and dihydroindole derivatives and preliminary results from
their in vitro inhibitory activity against soluble guanylate cyclase (sGC) are presented.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 30 April 2009
Revised 7 June 2009
Accepted 11 June 2009
Available online 14 June 2009
Keywords:
Soluble guanylate cyclase
cGMP
Indole derivatives
ODQ analogues
Soluble guanylate cyclase (sGC) is an enzyme that catalyzes the
conversion of guanosine 50-triphospate (GTP) to the second mes-
senger guanosine 30,50-cyclic monophosphate (cGMP). sGC carries
a prosthetic heme groups that serves as a nitric oxide (NO) sensor,
which activates sGC activity by up to 400-fold. Increased levels of
cGMP have been shown to lead to smooth muscle relaxation, inhi-
bition of platelet aggregation, anti-apoptotic and anti-inflamma-
tory effects.1 On the other hand, overactivation of the sGC/cGMP
signaling pathway has been shown to occur in several pathological
conditions such as during sepsis2 and neurodegenerative disorders.
Compounds that inhibit the activity of sGC could serve as potential
therapeutic agents as well as chemical biology tools that will aid in
unravelling the role of sGC-regulated pathways in vivo.
Several compounds have been described in the literature as
inhibitors of the sGC. Among them, methylene blue3 and
LY835834 (Fig. 1) are not direct sGC inhibitors, but rather block
cGMP formation by generating superoxide anion radicals that
deactivate NO.5 Despite their extensive use in plethora of research
studies, the off-target effects of these compounds6–8 limit their
pharmacological value in the in vivo evaluation of the sGC activity.
More specific inhibition of sGC has been obtained by other inhibi-
tors like 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ)9 and
its 8-bromo analogue NS2028.10 ODQ acts as an oxidant of the
heme group, as it has been revealed from Raman spectroscopy,11
resulting in the desensitization of the sGC towards the activating
action of NO. Although ODQ does not affect the activity of related
cyclases (for example the particulate guanylate cyclase),9 its bind-
ing affinity to the heme moiety may contribute to the unspecific
interaction with other hemoproteins in vivo, leading to decreased
enzyme selectivity and undesirable side effects.
In the context of the above mentioned data, it is evident that the
development of new inhibitors with increased potency and selec-
tivity against sCG remains a challenge. Additionally, sGC inhibitors
might be useful therapeutically especially in disease states associ-
ated with hypotension.
O
H
N
N
N
N
S
N
Cl
O
Methylene Blue
LY 83583
O
O
N
N
O
N
O
N
Br
N
O
* Corresponding author. Tel.: +30 2610969326/2610997723; fax: +30
2610992776.
ODQ
NS-2028
E-mail address: snikolar@upatras.gr (S. Nikolaropoulos).
Figure 1. Inhibitors of soluble guanylate cyclase.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.047

K. Spyridonidou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4810–4813
4811
Scheme 1. Reagents and conditions: (a) glyoxal monoxime, Zn powder, AcOH, reflux, overnight, 20%; (b) t-BuOK, 18-crown-6, EOM-Cl or SEM-Cl, THF, 0 °C, 0.5 h, 87%; (c)
HCOOEt, NaH, benzene, rt, overnight, 71–79%.
We aimed to synthesize sGC inhibitors with potential in vitro
and in vivo activity by rationally designing and synthesizing a ser-
N
N
O
O
a
b
c
d
d
ies of new derivatives bearing the basic skeleton I. Principal struc-
tural feature of these new derivatives is an indole or
a
N
N
N
N
N
dihydroindole core fused with a five-membered heterocyclic ring
resulting in the formation of a tricyclic skeleton. We considered
this scaffold, either dihydrogenated or fully aromatized in the in-
dole part, promising for the development of potential sCG inhibi-
tors, as it presents molecular and structural similarity with
already known inhibitors of sGC, like ODQ and NS-2028. Despite
that the indole and the dihydroindole nucleus constitute essential
structural components of many bioactive compounds, to the best
of our knowledge similar derivatives have never been studied as
inhibitors of sGC.
5
8
OEt
OEt
OEt
OEt
N
N
N
N
4
6
9
OEt
We herein describe the synthesis and preliminary results con-
cerning the in vitro evaluation of these compounds.
Ph
Ph
N
N
N
N
d
Het
N
7
10
OEt
N
R
Scheme 2. Reagents and conditions: (a) H2NOHꢀHCl, EtOH, 0 °C, 1 h, 75%; (b)
CH3NHNH2, EtOH, 0 °C, 1 h, 72%; (c) PhNHNH2, EtOH, 0 °C, 1 h, 46%; (d) DDQ, 1,4-
dioxane, 1 h, rt, 58–87%.
I
R = alkyl or H
Het = 5-membered heterocylic ring
shift relatively upfield (5.83 d) in compound 7 compared to that
of compound 6 (6.32 d) as a consequence of its interference with
the shielding cone of phenyl ring. Subsequently, the derivatives
5, 6 and 7 were dehydrogenated on treatment with 2,3-dichloro-
5,6-dicyanobenzoquinone (DDQ) in dioxane to the corresponding
fused tricyclic indole derivatives 8, 9 and 10. In an effort to gain ac-
cess to the N-unprotected derivatives and investigate their possible
inhibitory activity against sGC, we decided to remove the EOM pro-
tective group. We chose the derivative 10 as model compound for
initial trial experiments. Extensive attempts to cleave the EOM
group using HCl in dioxane, or HCl in EtOH under various condi-
tions proved unsuccessful resulting either in recovery or in decom-
position of the starting material 10.
The above-mentioned results prompted us to exchange the
EOM moiety with an alternative alkyloxymethyl one which would
be susceptible to hydrolysis under milder conditions. We consid-
ered the employment of the 2-(trimethylsilyl)ethoxymethyl
(SEM) group for the N-pyrrole protection of the 4-keto-tetrahydro-
indole 2. Treatment of 2 with SEM-Cl under the phase transfer con-
ditions described above afforded the N-SEM protected
tetrahydroindolone 11 in excellent yields (87%) (Scheme 1). Subse-
quently, base-catalyzed formylation gave the 5-hydroxymethylene
derivative 12. Upon condensation of the latter with appropriate re-
agents the corresponding tricyclic fused dihydroindole derivatives
13–15 (Scheme 3) were obtained while dehydrogenation with DDQ
afforded the indole derivatives 16–18. Cleavage of the SEM protec-
tive group was accomplished upon treatment with BF3ꢀEt2O in
dichloromethane which generated the corresponding N-hydroxy-
methyl-intermediates. The crude mixture of them was further
Attempting to establish a simple and versatile synthetic meth-
odology for the synthesis of the new tricyclic indole and dihydro-
indole
derivatives,
we
used
a
5-hydroxymethylene
tetrahydroindolone, like 4 (Scheme 1) as a key intermediate mole-
cule which would enable us to construct various fused tricyclic sys-
tems upon condensation with suitable reagents. Preparation of 4
was achieved from the commercially available cyclohexane-1,3-
dione 1 following a three step procedure. Particularly, condensa-
tion of 1 with glyoxal-monoxime12 in the presence of zinc powder
in glacial acetic acid afforded the corresponding 4-keto-tetrahydr-
oindole 2.13 Based on a previous successfully applied procedure for
the protection of the pyrrole nitrogen of a 7-keto-tetrahydroindole
derivative under phase transfer conditions,14 treatment of 2 with
chloromethyl ethyl ether (EOM-Cl) in the presence of potassium
tert-butoxide (t-BuOK) and 18-crown-6 in tetrahydrofuran affor-
ded the N-EOM protected tetrahydroindolone 3 in very good yields
(87%). The latter was formylated with ethyl formate in the pres-
ence of sodium hydride affording the 5-hydroxymethylene deriva-
tive 4.
Condensation of 4 with hydroxylamine hydrochloride under
mild conditions led to the formation of the 4,5-dihydro-isoxazol-
o[5,4-e]indole 5 (Scheme 2) the structure of which has been con-
firmed.15,16 The 4,5-dihydro-pyrrolo[2,3-g]indazole derivatives 6
and 7 were obtained upon condensation of 4 with methylhydr-
azine and phenylhydrazine, respectively. Confirmation of the
structures is consistent with literature data16,17 and additionally
is supported by the 1H NMR signal for 8-H which is observed to

4812
K. Spyridonidou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4810–4813
N
N
N
N
O
O
O
e
X
a
b
c
d
N
N
N
N
H
N
N
13
16
19
OCH2CH2Si(CH3)3
OCH2CH2Si(CH3)3
N
N
N
N
N
d
e
12
N
H
14
17
20
OCH2CH2Si(CH3)3
OCH2CH2Si(CH3)3
Ph
Ph
Ph
N
N
N
N
N
N
d
e
N
N
H
15
18
21
OCH2CH2Si(CH3)3
OCH2CH2Si(CH3)3
Scheme 3. Reagents and conditions: (a) H2NOHꢀHCl, EtOH, 0 °C, 1 h, 80%; (b) CH3NHNH2, EtOH, 0 °C, 1 h, 63%; (c) PhNHNH2, EtOH, 0 °C, 1 h, 40%; (d) DDQ, 1,4-dioxane, 1 h, rt,
63–75%; (e) (i) BF3ꢀEt2O, DCM, 0 °C to rt, 1 h; (ii) Triton B, acetonitrile, 0 °C, 4 h, 26–37%.
treated with Triton B in acetonitrile effecting the loss of formalde-
hyde and affording the N-unprotected products 20–21 in moderate
yields (26–37%) over two steps. Unfortunately, 16 proved extre-
mely labile under deprotection conditions leading to decomposi-
tion upon treatment with BF3ꢀEt2O.
duced SNP-stimulated cGMP accumulation in a concentration-
dependent manner that was maximal at 100 M.
To test the ability of compound 8 (which had shown activity in
preliminary screening tests) throughout a broader concentration
range, RASMCs were pretreated for 30 min with 0.1, 1, 10 or
l
To study whether the synthesized compounds are effective sGC
inhibitors, RASMCs were pretreated for 30 min with two different
100
lV of compound 8 and then exposed to the nitric oxide donor
SNP (10
lV). The response to SNP was reduced in a concentration-
concentrations (1 and 100
and then exposed to the nitric oxide donor SNP (10
treated with SNP alone exhibited a substantial increase in cGMP
levels (Fig. 2). The response to SNP was reduced in cells exposed
to the newly synthesized compounds to varying degrees. Pretreat-
ment with compound 5 only inhibited SNP-induced cGMP forma-
l
V) of each of the tested compound
V). RASMC
dependent manner in cells exposed to the derivative 8 (Fig. 3). Both
compounds 7 and 8 were more efficacious than compounds 5 and
6, as pretreatment with 7 and 8 led to the greatest amount of inhi-
bition of cGMP formation.
To study the selectivity of the tested compounds on sGC, we
examined the effect of one of the synthesized compounds (8) that
was most effective on sGC-stimulated cGMP formation. Exposure
l
tion at the highest concentration tested. Compound
6 was
effective in attenuating NO-induced cGMP formation and was
more potent than 5 as it inhibited cGMP at a lower concentration;
it should be noted that higher concentrations of this compound
were not as effective in inhibiting cGMP formation. Derivative 7 re-
of RASMC to ANF (1 lV) increased their cGMP content through
activation of pGC; as expected although ANF-induced cGMP levels
were lower than what was observed for SNP (Fig. 4). The response
to ANF was unaffected by the addition of 100 lV of 8 (Fig. 4). It
125
2000
100
1500
*
75
50
25
0
**
*
**
**
1000
500
0
**
**
µM
10
100
10
1
0.1
100
1
10
100
µM
100
1
1
SNP + 5
SNP + 6
SNP + 7
SNP + 8
Figure 2. Effect of newly synthesized compounds on SNP-induced cGMP produc-
tion. Confluent RASMCs were incubated with the tested compounds (1 and 100 V)
for 30 min. Cells were washed twice with HBSS and then treated with basal or SNP
(10 V) in the presence of IBMX (1 mM) for 15 min. cGMP was extracted and
Figure 3. Effect of compound
RASMCs were incubated with the tested compound (0.1, 1, 10 and 100
30 min. Cells were washed twice with HBSS and then treated with vehicle or SNP
(10 V) in the presence of IBMX (1 mM) for 15 min. cGMP was extracted and
8
on SNP-induced cGMP production. Confluent
l
lV) for
l
l
measured as described in supplementary data. Data are means SEM; n = 8 wells.
measured as described in supplementary data. Data are means SEM; n = 8–16
**P < 0.01, *P < 0.05 compared to SNP.
wells. **P < 0.01, *P < 0.05 compared to SNP.

K. Spyridonidou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4810–4813
4813
mization studies and pharmacological investigations are in
progress.
100
75
50
25
0
**
**
Acknowledgments
This Letter is part of the 03ED375 research project, imple-
mented within the framework of the ‘Reinforcement Programme
of Human Research Manpower’ (PENED) and co-financed by Na-
tional and Community Funds (20% from the Greek Ministry of
Development-General Secretariat of Research and Technology
and 80% from E.U.-European Social Fund).
Basal
ANF
ANF + 8
8
Supplementary data
Figure 4. Effect of compound 8 on pGC. Confluent RASMCs were incubated with 8
(100 V) for 30 min. Cells were washed twice with HBSS and then treated with
basal or ANF (1 V) in the presence of IBMX (1 mM) for 15 min. cGMP was
extracted and measured as described in supplementary data. Data are mean-
SEM; n = 4 wells. **P < 0.01 compared to Basal.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.047.
l
l
s
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L
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In conclusion, following a simple and versatile synthetic meth-
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inhibitors. Preliminary results from in vitro studies demonstrate
the potential of these compounds as candidate sCG inhibitors. Spe-
cifically, the isoxazolo[5,4-e]indole derivative 8 represents a prom-
ising hit compound of the series with inhibitory activity in the
micromolar range and selectivity for sGC. Further structural opti-
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