Straightforward and effective synthesis of γ-aminobutyric acid transporter subtype 2-selective acyl-substituted azaspiro[4.5]decanes

Article abstract of DOI:10.1016/j.bmcl.2015.11.100

Supply of major metabolites such as γ-aminobutyric acid (GABA), β-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of no

Full text of DOI:10.1016/j.bmcl.2015.11.100

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Straightforward and effective synthesis of c-aminobutyric acid
transporter subtype 2-selective acyl-substituted azaspiro[4.5]
decanes
a
a
b
b
b
c
Xiaofeng Ma , Hodney Lubin , Enik o} Ioja , Orsolya Kékesi , Ágnes Simon , Ágota Apáti ,
d
b
b,
⇑
a,
⇑
Tamás I. Orbán , László Héja , Julianna Kardos , István E. Markó
a
Organic and Medicinal Chemistry Laboratories, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
b
c
Functional Pharmacology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary
Laboratory of Molecular Cell Biology, Institute of Enzimology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary
Biomembrane Research Group, Institute of Enzimology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Supply of major metabolites such as c-aminobutyric acid (GABA), b-alanine and taurine is an essential
instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs.
This background motivates the synthesis of novel classes of compounds regulating their selective trans-
Received 3 October 2015
Revised 26 November 2015
Accepted 27 November 2015
Available online xxxx
port through various fluid-organ barriers via the low-affinity
c-aminobutyric acid (GABA) transporter
subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures
and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport
activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane
scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of
the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the
discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Azaspiro[4.5]decane scaffold
c
-Aminobutyric acid (GABA)
GABA transporter subtype GAT2 (slc6a13)
-Amino acid selectivity
x
Impairment of
c-aminobutyric acid (GABA) signalling has been
GAT2-mediated uptake of d-amino levulinic acid (d-ALA) in the
implicated in disease states associated with asthma, chronic
brain leads to accumulation of fluorescent porphyrins in malignant
1
0
obstructive pulmonary disease and cystic fibrosis or acute lung
gliomas helping surgery. At present, therefore, the recognition of
small molecules regulating GAT2 function in these organs is of
great (patho)physiological and pharmacological relevance.
1
,2
injury suggesting that novel approaches to prevent the loss or
even to facilitate GABA signalling are needed. A promising way
to achieve this goal is to inhibit GABA transporters and conse-
quently increasing extracellular GABA level.
In addition to the presence of GAT2 (slc6a13) in Homo sapiens,
Rattus norvegicus (rat) and Mus musculus (mouse) mentioned
above, similar function also appears in domestic animals such as
Bos taurus (cow), Gallus gallus (chicken), Apis mellifera (honeybee)
and in model organisms Danoi rerio (zebrafish), Drosophila melano-
gaster (fruitfly), Caenorhabditis elegans, Bacillus subtili, Halobac-
terium salinarum, and in pathogens Anopheles gambiae,
Localization of the GABA transporter subtype GAT2 (solute car-
rier family 6 member 13 gene: slc6a13; hGAT2, rGAT2, mGAT3) in
3
airway epithelium and smooth muscle suggests a role for GABA
signalling. By performing guanidinoacetic acid (GAA) or taurine
(
Tau) uptake in the sinusoidal membrane of periportal hepato-
1
1
cytes, GAT2 does also regulate creatine or taurocholate biosynthe-
Staphylococcus aureus, Helicobacter pylori. Abundance of GAT2
function across organisms signifies the importance of the develop-
ment of GAT2 subtype-selective inhibitors.
4
,5
ses in the liver. GAT2 in the basolateral membranes of proximal
tubules in the renal cortex.5 may possibly be associated with
GABA-induced natriuresis and blood pressure reduction. Efflux of
,6
Design and synthesis of a variety of small molecule scaffolds
inhibiting GABA transport through the neuronal GAT1 (slc6a1;
hGAT1, rGAT1, mGAT1) and glial GAT3 (slc6a11; hGAT3, rGAT3,
mGAT4) subtypes, that control synaptic and extrasynaptic GABA
5
8
,7
,9
Tau from the brain through GAT2 localised in brain blood vessels
could occur under injurious depolarizing conditions.
levels, has successfully led to the discovery of potent GAT1- and
⇑
Corresponding authors.
E-mail addresses: kardos.julianna@ttk.mta.hu (J. Kardos), istvan.marko@
12
GAT3-selective inhibitors.
These include GAT1-selective
13
conformationally restricted GABA bioisosters Tiagabine, SK&F
uclouvain.be (I.E. Markó).
http://dx.doi.org/10.1016/j.bmcl.2015.11.100
0
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
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2
X. Ma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
8
(
9976-A, CI-966, NNC-711and GAT3-selective non-bioisosteric5-
GAT2 as a selective and low-affinity subtype. Some of the GAT2
inhibitors showed novel scaffolds, rather than those of other GAT
transport inhibitors disclosed so far. The similarity of substrate
thiophen-2-yl)indoline-2,3-diones.1 Conformational restriction
4
7
of GABA via bicyclo[3.1.0]hexane backbone has provided the most
potent and selective inhibitor of the peripheral betain-GABA
transporter BGT1 (slc6a12; hGAT4, rGAT4, mGAT2).15
binding crevices of peripheral GAT2 and neuronal GAT3 monomers
17
in the occluded substrate binding conformation, however, sug-
According to the homology modelling studies of Schlessinger
et al. GAT2 is avery selective transporter, compared to other neuro-
transmitter transporters.7 Selectivity of GAT2 towards some
gests that GAT2 versus GAT3 selectivity may perhaps better be
achieved by considering the size and the charge-configuration of
molecules fitting additional GAT2 conformations as suggested.7
Great variety of 3D size and charge-configuration of spirocyclic
ring systems may offer a novel scaffold providing GAT2 subtype-
selective inhibitors. Living cells synthesize spirocyclic ring systems
such as ubiquitous bioactive [5,5]-, [5,6]-, and [6,6]-spiroacetals
and alkaloids (for example stepharine, annosqualine, securinine,
viroallosecurinine, halichlorine, pinnaic acids, pinnatoxin A, nor-
x
-amino acids such as b-alanine (b-Ala), Tau, 3-amino-1-propane-
0
sulfonic acid (homoTau), GAA, d-ALA,
c-amino-b-(4 -chlorophenil)-
butyric acid ((R)-baclofen) qualifies GAT2 as a versatile target,
possibly retaining distinguishable binding/transport mode of inter-
action with different substrates/inhibitors. Molecular docking of
more than 500,000 compounds along with uptake data of hits
concluded that selective GAT2 inhibitors exert their effect at high
concentrations (0.5–5 mM), except for the endogenous substrates
zoanthamine, histrionicotoxins, rhynchophylline, spirotryprostatin
A, erythraline) containing diverse spirocyclic cores.1
8,19
The
GABA and b-Ala that are characterised by K
m
values of 0.02 mM
identification of key enzymes dihydroxy ketone and spiroacetal
and 0.03 mM, respectively.7 These findings classify peripheral
,16
synthases
biosynthetic routes to get spirocyclic drug leads. Spirocyclic ring
19
producing reveromycin
A
has inspired novel
2
0
2
1,22
systems are increasingly used in drug discovery.
Leading
scaffolds include derivatives of various spirobicyclic amines such
n-asazaspiro[3.5]nonane (n = 5, 6, 7), 7-azaspiro[4.4]nonane
Table 1
Synthesis of spiro-bicyclic compounds 13a–13g
(
Spiraprilat), n-azaspiro[4.5]decane (Buspiron (n = 8), Enilospirone
O
(n = 4)), 1,3-diazaspiro[4.4]nonane (Irbesartan), 3,8-diazaspiro
NH
2
NH
NH
LiAlH
4
O
(t -BuO)
60 °C
6%
2
+
THF
ref lux
60%
1
O
2
Table 2
4
1
O
O
Synthesis of spiro-bicyclic compounds 12b and 13h–m
O
NuH
TEA
N
NH
N
+
Cl
Cl
O
O
N
O
N
CH Cl2
2
MeOH, r.t.
overnight
60%-93%
Nu
O
O
O
0
°C to r.t.
NuH
TEA
CH Cl
NH
85%
+
Cl
Cl
2
THF, r.t.
overnight
82%-89%
1
2a
13a-13g
2
2
Nu
0
°C to r.t.
3%
6
Entry
1
NuH
Product
Yield (%)
93
1
12b
13h-13m
H
N
O
Entry
NuH
Product
Yield (%)
N
N
N
H
N
O
O
O
O
13a
N
1
89
H
N
O
O
O
N
N
N
N
N
13h
2
3
60
89
H
N
O
N
13b
H
N
2
3
85
88
N
O
13i
13j
H
N
O
N
O
13c
H
N
O
N
N
C
2
EtO C
4
5
76
75
O
H
N
O
N
EtO
2
O
1
3d
H
O
N
2
EtO C
4
N
83
N
N
C
2
MeO C
EtO C
2
13k
MeO
2
H
N
O
N
1
3e
3f
O
O
H
N
O
N
N
5
6
88
82
N
N
6
7
89
79
N
N
13l
1
H
N
O
N
H
N
O
N
H
N
H
NH 13g
NH 13m
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X. Ma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 3
O
O
O
Synthesis of spiro-bicyclic compounds 12c and 13n–p
OAc
O
Zn
O
NH NO , TFAA
N
H
4
3
OMe
O
OMe
AcOH:EtOH
(V:V = 2:1)
75%
Cl
Nu
NO
2
41%
O
7
8
10
Boc
O
O
O
O
Cl
H
N
Cl
N
AlCl
3
O
O
O
NuH
O
O
O
O
O
N
N
O
TEA
4% 2 steps
O
O
O
O
O
6
6
Br
Br
Cl
O
2
0
O
1
2c
13n-13p
N
O
N
N
H
4
9%
69%
O
O
Entry
1
Nu
Product
Yield (%)
75
11d
1
0
12d
Br
H
N
O
O
Figure 3. Synthesis of spiro-bicyclic compounds 10, 11d and 12d.
N
O
O
N
N
N
O
O
13n
[
4.5]decane (Fenspiride), 1,3,8-triazaspiro[4.5]decane (Fluspir-
H
N
O
O
22,23
ilene).
These developments together with GABA uptake inhibi-
2
4
N
O
tory activity of 2-azaspiro[4.5]decane-6-carboxylates highlight
azaspiro[4.5]decane derivatives as a promising starting scaffold
for synthesis and evaluation of GAT subtype selectivity.
2
3
78
70
N
H
O
NH
13o
H
N
O
O
The aza-spirobicyclic subunits, core skeletons of the compounds
tested during this study have been synthesized according to three
different, though complementary routes. Our approach towards
the amino-bicycle 2 is based upon the tert-butyl peroxide-induced
radical cyclization between cyclohexylamine and methyl acrylate
N
O
2
EtO C
O
2
EtO C
1
3p
2
5
to form the spirobicyclic amide 1 which can be further reduced
to the desired amine 2 in the presence of LiAlH (Table 1). Access
4
to the more substituted bicyclic structures, including the corre-
sponding [4,4,1] spiro derivatives, required a completely different
strategy. For that purpose, we set upon a method pioneered in
our laboratory and involving the coupling of tert-butyl 2-((tert-
butyldimethylsilyl)oxy)-1H-pyrrole-1-carboxylate (TBSOP)26
3
O
O
N
2
7
O
with functional orthoester 4 in the presence of ZnCl
2
as catalyst.
O
Pyridine
CH CN
°C to r.t.
Br
NH
Br
Under these conditions, the iodine-containing bicycle 5 is pro-
duced in good yields. Treatment of 5 with t-BuOK induces smooth
cyclization, affording the desired unsaturated amide 6. Whilst this
approach generates highly functionalized scaffolds, a simpler
+
Br
3
0
8
9%
1
11a
O
O
O
access to the corresponding [5,4,1] tricycles, such as 10, was also
O
Pyridine
CH CN
°C to r.t.
Cl
Br
28
NH
Cl
N
sought after. Hence, nitration of enol ester 7 provided the a-nitro
+
Cl
3
keto-ester 8 in reasonable yields. Chemoselective reduction of the
nitro-group, using zinc in acetic acid, yielded amine 929, which
underwent smooth intramolecular cyclization to keto-amide 10.
The spirocyclic scaffolds (1, 2, 6, 10), prepared according to these
three strategies, can be further transformed, leading to a variety
of other spirobicyclic derivatives. For example, reaction of the
nitrogen atom with acyl chlorides afforded the corresponding
amides 11, whilst addition of 3-chloro-propionyl chloride pro-
duced the unsaturated amides 12. Finally, Aza-Michael addition
between various amines and the unsaturated amides 12 delivered
a series of spirobicyclic compounds 13 (Tables 1–3).
0
12%
1
11b
O
O
Pyridine
CH CN
NH
O
Br
N
+
Br
3
0
°C to r.t.
1%
7
2
11c
O
O
N
O
O
O
NaI
acetone; reflux
6 h
4%
NaN
DMF; 100 °C
6 h
4%
3
I
Br
N
3
N
N
1
2
1
1
The synthesis of compound library containing 1, 2, 10, 11a–11d,
12a–d, 13a–p, 14, 15, 18 and 19 is outlined in Tables 2 and 3 and
Figs. 1–3. Compounds 1, 2, 12a and 13a–13g were synthesized
according to the pathway described in Table 1. 2 was further
15
14
11a
Figure 1. Synthesis of spiro-bicyclic compounds 11a–11c, 14and 15.
O
O
Boc
N
Boc
N
I
Boc
Boc O
N
O
OEt
TBSOTf
TEA
TBSO
O
O
N
O
tBuOK, THF
53%
O
4
I
O
O
2
ZnCl , DCM
5
3
3
steps
16
6
Boc O
O
O
O
O
H
N
Br
NiCl
2
•6H
2
O
N
AlCl
3
N
O
O
O
NaH
6%
NaBH
4
49%
steps
7
2
1
8
1
7
19
Figure 2. Synthesis of spiro-bicyclic compounds 18 and 19.
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X. Ma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 4
Inhibitory effect of test compounds on cloned GAT1, BGT1, GAT2 and GAT3 GABA transporter subtypes33
*
Compounds
IC50 (lM, mean ± SEM)
GAT1
BGT1
GAT2
GAT3
O
NH
1
2
1
>5000
>5000
>1000
>5000
>5000
>5000
NH
>5000
>1000
>5000
>1000
>5000
>1000
O
NH
0
O
O
O
N
O
1
1
1
1d
2a
2b
>1000
>1000
>5000
>1000
>1000
>5000
>1000
Br
O
N
>5000
3020 ± 457
>1000
CH
2
O
N
O
371 ± 56
103 ± 23
>1000
CH
2
O
N
O
1
1
2c
>1000
>1000
>1000
>1000
>1000
>1000
O
CH
2
O
O
O
N
2d
>1000
O
CH
2
O
O
N
R
*
*
1
1
1a
1b
R = Br
R = Cl
371 ± 56
>1000
103 ± 23
>1000
>1000
>1000
>1000
>1000
1
1
1
3h
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3i
N
3j
N
O
CO Et
2
1
3k
3l
>1000
164 ± 15
>1000
933 ± 104
N
N
N
1
1
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3m
NH
1
1
4
5
R = I
R = N
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3
O
N
R
1
1c
3a
R = Br
233 ± 13
>5000
>5000
>5000
>5000
>5000
>5000
1
N
675 ± 63
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X. Ma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 4 (continued)
*
Compounds
IC50 (lM, mean ± SEM)
GAT1
>5000
>5000
BGT1
GAT2
>5000
>5000
GAT3
>5000
>5000
1
1
3b
3c
N
N
>5000
>5000
O
CO
2
Et
1
1
3d
3e
>5000
>5000
2330 ± 220
3230 ± 280
>5000
N
CO
2
Me
2050 ± 360
2300 ± 330
2860 ± 510
N
N
N
1
1
3f
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3g
NH
O
O
N
O
R
O
1
3n
3o
N
N
O
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1
NH
CO
2
Et
1
1
3p
8
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
N
O
NH
O
O
O
N
1
9
>1000
>1000
>1000
>1000
O
O
Standards
HN
O
2
1
20.9 ± 1.4
>1000
>1000
72.4 ± 4.4
>1000
114.8 ± 8.7
>1000
OH
N
2
2
0.18 ± 0.01
O
N
COOH
N
OMe
O
2
3
199 ± 18
>1000
21.9 ± 1.7
6.3 ± 0.18
OMe
COOH
OMe
*
Number of determinations: 3–11; SEM: standard error of mean; >5000 or >1000 denotes measurements in which 50% inhibition were not achieved at the highest tested
concentrations (5000 M or 1000 M, respectively). Compounds were fully soluble at these concentrations.
l
l
reacted with 3-chloro-propionyl chloride, in the presence of tri-
ethylamine (TEA), leading directly to the unsaturated amide 12a
in 85% yield. Acrylamide 12a was then engaged in a series of
Michael additions, using a variety of amines, including pyrrolidine,
piperidine, morpholine, ethyl piperidine-3-carboxylate, methyl
the analogous series bearing an amide function instead of a basic
tertiary amine. Hence, amide 1 was thus treated with 3-chloro-
propionyl chloride, in the presence of triethylamine affording, as
expected, the
a,b-unsaturated imide 12b. Michael addition of a
variety of amines gave the desired adducts 13h–m (Table 2).
To diversify even further our small library of aza-spirobicyclic
bioisosters of GABA, different alpha-halogenated imides bearing a
Cl, Br and I-containing side-chain were synthesized (11a–11c and
14). To generate further diversity through click-chemistry, the
azide adduct 15 was also designed and synthesized (Fig. 1).
1
,2,5,6-tetrahydropyridine-3-carboxylate, diethylamine, piper-
azine, providing in all cases, the desired aza-Michael addition
products (13a–13g) in good to excellent yields (Table 1).
Whilst amine 2 can be easily derivatized by a variety of
electrophiles, we were also interested in testing the activities of
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6
X. Ma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Having successfully prepared these bicyclo-[5,4,1] derivatives,
structural motifs are of great (patho)-physiological and pharmaco-
logical relevance. Synthetic spirocyclicamine therapeutics with a
range of size and charge-separation and biological activity are
increasingly recognised. Based on GABA transport activity of aza-
spiro[4.5]decane carboxylates a new scaffold has been synthesized
by developing three straightforward and efficient complementary
routes. Biological evaluation of the novel scaffold synthesized this
way discovered a subclass of a GAT2-selective compounds with
acyl-substituted azaspiro[4.5]decane core.
we turned our attention to the assembly of the analogous spiro-
bicyclic compounds 12c, 13n–13p, 18 and 19, bearing a smaller
five-membered ring system (Table 3). Reduction of the conjugated
double bond with NaBH
4
in the presence of NiCl
2
ꢀ6H
2
O provided
Boc-protected-amide 17. Finally, deprotection of the Boc group
was accomplished by treatment of 17 with a catalytic amount of
AlCl
3
giving amide 18 in 49% yield over 2 steps.30 N-allylation of
1
1
8, using allyl bromide and NaH in THF, afforded tertiary amide
9 (Fig. 2).
The Boc-containing compound 6 could also be deprotected
Acknowledgements
using AlCl
3
, affording the unsaturated amide 20. Addition of
N, gave directly
3
-chloro-propionyl chloride, in the presence of Et
3
I.E.M., X.M. and H.L. gratefully acknowledge financial support
by the Fonds National pour la Recherche Scientifique (FNRS –
ERA n° R 50.02.11F) and the Université Catholique de Louvain. Á.
A. and T.I.O. thank financial support by the Hungarian Brain
Research Program (KTIA_13_NAP-A-I/6) and Hungarian Scientific
Research Fund (OTKA K112112 grant), respectively. E.I., O.K., L.H.,
Á.S. and J.K. acknowledge financial support by the KMR_12-1-
the Michael acceptor 12c in 64% over two steps. Reaction of 12c
with morpholine, piperidine and ethyl piperidine-3-carboxylate
furnished the adducts 13n–p (Table 3).
The synthesis of the ketone-containing spiro-bicyclic imides 10,
1d and 12d is illustrated in Figure 3. The enol ester31 7 was trans-
1
formed initially into the corresponding
tion with NH NO in the presence of TFAA.
chemoselective reduction of the nitro-group with Zn powder in
a-nitro keto-ester by reac-
28
4
3
A subsequent
2
012-0112 and ERA-Chemistry OTKA 102166 grants.
2
9
EtOH and AcOH ensued, giving the amide 10 in 75% yield. This
keto-spiro lactam was reacted, on the one hand, with 2-bro-
moacetyl bromide to obtain imide 11d and on the other hand with
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.11.
3
3
-chloro-propionyl chloride and Et N to provide the unsaturated
imide 12d (Fig. 3).
1
00.
GABA transporter activity of the synthesized spirobicyclic
derivatives was tested on HEK293 cell lines stably expressing
GAT1, BGT1, GAT2 and GAT3 GABA transporter subtypes. Two of
the tested inhibitors 12a and 13d showed selectivity towards
GAT2 with low affinity (Table 4), alike all of the few GAT2 sub-
type-selective compounds found in a >500,000 entry-containing
References and notes
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