Bicyclic melatonin receptor agonists containing a ring-junction nitrogen: Synthesis, biological evaluation, and molecular modeling of the putative bioactive conformation

Article abstract of DOI:10.1016/j.bmc.2005.10.042

Employing 1,3-dipolar cycloaddition for the synthesis of the 7a-azaindole nucleus, analogues of melatonin have been synthesized and tested against human and amphibian melatonin receptors. Introducing a phenyl substituent in position 2 of the heterocyclic

Full text of DOI:10.1016/j.bmc.2005.10.042

Bioorganic & Medicinal Chemistry 14 (2006) 1949–1958
Bicyclic melatonin receptor agonists containing
a ring-junction nitrogen: Synthesis, biological evaluation,
and molecular modeling of the putative bioactive conformation
Jan Elsner,a Frank Boeckler,a Kathryn Davidson,b David Sugdenb and Peter Gmeinera,*
aDepartment of Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University,
Schuhstraße 19, D-91052 Erlangen, Germany
bDivision of Reproductive Health, Endocrinology and Development, School of Biomedical Sciences,
Hodgkin Building, KingÕs College London, GuyÕs Campus, London SE1 1UL, UK
Received 25 August 2005; revised 19 October 2005; accepted 25 October 2005
Available online 15 November 2005
Abstract—Employing 1,3-dipolar cycloaddition for the synthesis of the 7a-azaindole nucleus, analogues of melatonin have been syn-
thesized and tested against human and amphibian melatonin receptors. Introducing a phenyl substituent in position 2 of the het-
erocyclic moiety significantly increased binding affinity to both the MT1 and MT2 receptors. Shifting the methoxy group from
position 5 to 2 of the 7a-azaindole ring led to a substantial reduction of MT1 binding when MT2 recognition was maintained.
We theoretically investigated the hypothesis whether the 2-methoxy function of the azamelatonin analogue 27 is able to mimic
the 5-methoxy group of the neurohormone by directing its 2-methoxy function toward the methoxy binding site. DFT calculations
and experimental binding differences of analogue compounds indicate that the energy gained by forming the methoxy-specific
hydrogen-bond interaction should exceed the energy required for adopting an alternative conformation.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
domains connected by intra- and extracellular hydro-
philic loops. The MT1 and MT2 melatonin receptor
subtypes4 are present in humans and other mammals,
and their activation leads to the inhibition of adenylyl
cyclase through activation of a Gi protein.5,6 An addi-
tional subtype (termed Mel1c receptor), not detected in
mammals, has been cloned from chicken, Xenopus,
and zebrafish.7 In mammals, melatonin receptors are
expressed in the brain, with considerable variation in
location and density of expression between species,
and also in some peripheral organs.8 The MT1 subtype
is present within the pars tuberalis of the pituitary and
the hypothalamic SCN, while the MT2 subtype is mainly
expressed in the retina. This diversity and the differences
in tissue distribution suggest different functional physio-
logical roles for each receptor subtype. For example,
there is evidence suggesting that selective MT2 receptor
agonists should be particularly useful for the treatment
of sleep and chronobiotic disorders, including jet lag
and work shift syndrome.9 Therefore, current research
goals include the design of subtype-selective melatonin
receptor agonists and antagonists, which recently led
to a binding hypothesis for known MT2 selective
antagonists.10
In recent years, the pineal hormone melatonin, which
acts on specific G-protein coupled receptors, has become
the focus of considerable interest for its implication in a
wide variety of functions involving the circadian, visual,
neuroendocrine, reproductive, cerebrovascular, and im-
mune systems.1 There is also a growing literature on
the anti-oxidant actions of melatonin. The synthesis of
melatonin and its secretion at night from the pineal
gland are controlled by a circadian clock within the
hypothalamic suprachiasmatic nuclei (SCN)2 and are
synchronized by environmental light.3 Melatonin there-
fore has a chronobiotic function in that it works to alter
the timing of physiological and behavioral processes
through its action. Cloning studies identified three
G-protein coupled receptors. Like all GPCRs, melato-
nin receptors have seven hydrophobic regions that are
thought to form a-helical transmembrane (TM)
Keywords: Melatonin receptor agonist; Bioactive conformation; Sub-
type selectivity; 7a-Azaindole.
*
Corresponding author. Tel.: +49 9131 8529383; fax: +49 9131
8522585; e-mail: gmeiner@pharmazie.uni-erlangen.de
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.042

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J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
The ability of melatonin to alter circadian rhythm syn-
chronization implies its potential use in the treatment
of conditions associated with problems in this area.
However, the use of melatonin as a drug is limited by
its short biological half-life (15–20 min) and its poor
bioavailability. In order to circumvent these problems,
several potent ligands have been designed by employing
bioisosteric replacement of the indole moiety by naph-
thalene and benzofuran analogues of types 111 and 2,12
respectively (Chart 1). The naphthalene derivative
agomelatine (1, R1 = Me) is currently in phase III
clinical trials as a new antidepressant agent.13
2. Chemistry
As an extension of our previous efforts,24 the 5-methoxy
substituted target derivatives 17–22 were prepared
according to the route shown in Scheme 1. Taking advan-
tage of a recently reported highly efficient synthesis of
O-(2,4-dinitrophenyl)hydroxylamine (NH2-ODNP) by
Legault and Charette,25 we were able to prepare the
N-aminopyridinium salt 4 derived from 4-methoxypyri-
dine in 86% yield, thus, avoiding drawbacks exhibited
by the widely used N-aminating reagents hydroxyl-
amine-O-sulfonic acid26 and O-mesitylenesulfonylhydr-
oxylamine.27 1,3-Dipolar cycloaddition of 4 with methyl
propiolate and ethyl phenylpropiolate, respectively, un-
der oxidative conditions, followed by hydrolysis and
decarboxylation of the respective ester intermediates 5
and 6, furnished the pyrazolo[1,5-a]pyridine derivatives
7 and 8 in good yields. Subsequent formylation under
Vilsmeier–Haack conditions gave the aldehydes 9 and
10, respectively, followed by a mild Knoevenagel conden-
sation with nitromethane at room temperature, which
resulted in the formation of nitroalkenes 11 and 12. After
all attempts of direct reduction with LiAlH4 provided
only very modest yields of the product, reduction of the
a,b-unsaturated nitroalkenes to the desired saturated
amines 15 and 16 was performed with NaBH4, followed
by reduction with tin powder in HOAc. Finally, the eth-
ylamino functions were acylated with the appropriate
anhydrides to furnish the target compounds 17–22.
As previously shown for the indole nucleus, introducing
a phenyl group in position 2 as well as lengthening the
N-acyl side chain by up to two carbon atoms are both
effective ways to increase the binding affinity at both
subtypes.14 Based on extended SAR data of a series of
tryptamines, it has been concluded that the 5-methoxy
group is not an essential requirement for biological
activity; however, it clearly shows major interactions
with a specific binding site in the melatonin receptor
for a broad variety of ligands.15 Moreover, it has been
reported that appropriate substituents ortho to the ethy-
lamido side chain in naphthalenic melatonin analogues
modulate the binding affinity to the melatonin recep-
tor.16 Thus, we were further interested in the effects of
ÔswitchingÕ the methoxy group from position 5 of the
pyrazolo[1,5-a]pyridine scaffold to position 2. Recently,
we have been able to demonstrate by in vitro17–19 and
in vivo20,21 studies in the field of dopamine receptor li-
gands that the pyrazolo[1,5-a]pyridine moiety is a valu-
able heterocyclic bioisostere of indoles. Therefore, we
were intrigued to determine whether this finding holds
true for other GPCRs like the melatonin receptors as
well. A further aim of this study was to increase or mod-
ify both, the binding affinity toward the two receptor
subtypes and the receptor activation. Taking advantage
of the beneficial pharmacokinetics of the pyrazolo[1,5-
a]pyridine bearing antiallergic agent ibudilast22 and the
antiplatelet agent KC-764,23 we herein report the syn-
thesis of azamelatonins of type 3, their binding proper-
ties at the recombinant MT1 and MT2 subtypes
determined in radioligand competition experiments
and their intrinsic activity using the pigment aggregation
assay on a clonal Xenopus melanophore cell line.
NO2
OMe
R1
MeO
MeO
a
d
R2
R2
N
N
N
N
N
+
5: R1 = COOMe, R2 = H
6: R1 = COOEt, R2 = Ph
7: R1 = R2 = H
8: R1 = H, R2 = Ph
9: R1 = CHO, R2 = H
10: R1 = CHO, R2 = Ph
NH2
11: R2 = H
12: R2 = Ph
ODNP
b
c
b
c
4
e
O
NO2
R2
NH2
HN
R3
MeO
f
MeO
g
MeO
R2
R2
N
N
N
N
N
N
17: R2 = H, R3 = Me
18: R2 = H, R3 = Et
19: R2 = H, R3 = Pr
15: R2 = H
16: R2 = Ph
13: R2 = H
14: R2 = Ph
R3
O
O
O
20: R2 = Ph, R3 = Me
21: R2 = Ph, R3 = Et
22: R2 = Ph, R3 = Pr
X
X
X
X
N
N
N
O
R2
H
Scheme 1. Reagents and conditions: (a) methyl propiolate or ethyl
phenylpropiolate, K2CO3, air-O2, DMF, rt, 16 h (43% for 5; 35% for
6); (b) H2SO4 (v/v 50%), 80 ꢁC (3 h, 83% for 7; 16 h, 80% for 8); (c)
POCl3, DMF, rt, 1 h (91% for 9; 94% for 10); (d) CH3NO2,
CH3NH3Cl, CH3CO2K, MeOH, rt (3 days, 84% for 11; 10 days,
74% for 12); (e) NaBH4, MeOH, rt, 50 min (78% for 13; 75% for 14);
(f) Sn, HOAc, EtOH, rt, 3 days (60% for 15; 59% for 16); (g)
(R3CO)2O, Et3N, THF, 0 ꢁC to rt, 3 h (89% for 17; 85% for 18; 82%
for 19; 84% for 20; 87% for 21; 84% for 22).
melatonin
(R1 = Me)
1
2
3
O
R2 = H, Ph, OMe
R3 = OMe, H
R1 = Me, Et, Pr
X =
N
R1
H
Chart 1.

J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
1951
O
cells. Ligand efficacy of the test compounds was assessed
in a well-established model system of melatonin action,
the pigment aggregation response of Xenopus laevis
melanophores.31,32
H
a
OMe
OMe
N
N
N
N
b
23
24
The results of both tests as well as MT1/MT2 selectivity
ratios of the new compounds are reported in Table 1.
The analysis demonstrated that all synthesized com-
pounds were full agonists in the functional assay. Fur-
ther examination of the results, especially with respect
to the modifications at the C-2 and C-5 positions and
in the acyl side chain, can be summarized to the follow-
ing points: (1) As exemplified by the analogue 17 the pyr-
azolo[1,5-a]pyridine moiety is capable of serving as an
useful bioisostere for the melatonin indole nucleus, thus,
corroborating the previously reported insight that the
proton-donor NH indole may not be essential for the
anchoring of a ligand to the melatonin receptor.33,34
The reduced affinity of 17 when compared to the natural
neurohormone might be due to the polarization of the
aromatic system that is caused by the additional nitrogen
atom. (2) The positive effect of a 2-phenyl substitution al-
ready described for melatonin itself and some ana-
logues14 is also observed in our series and affects both
MT1 and MT2 subtypes, leading to the agonists 20–22,
which were almost equipotent to melatonin in the X. lae-
vis melanophore assay. This increase in potency can be
ascribed to both the increased population of the active
conformation for binding to the melatonin receptor
and to the presence of an auxiliary binding site around
the C-2 position,35 which obviously exist in both receptor
subtypes. For comparison, the affinity and efficacy of
previously reported 2-phenyl melatonin analogues14
were investigated in both hMT1 and hMT2 receptors as
well as in the X. laevis melanophore aggregation model.
The results obtained in both test systems for 20–22 are
very similar to their respective melatonin-derived
homologues, which leads us to the presumption that
the mutual structural elements are ÔprivilegedÕ for sub-
type-unselective MT receptor recognition and activation.
Pr
HN
NH2
NO2
O
d
c
OMe
OMe
OMe
N
N
N
N
N
N
27
26
25
Scheme 2. Reagents and conditions: (a) POCl3, DMF, rt, 1 h (98%);
(b) CH3NO2, HOAc, NH4OAc, ultrasound, 22 ꢁC, 6 h (54%); (c)
LiAlH4, THF, 0 ꢁC to rt, 2 h (45%); (d) butyric anhydride, Et3N, THF,
0 ꢁC to rt, 3 h (72%).
The 2-methoxy substituted target derivative 27 was pre-
pared as outlined in Scheme 2, starting from 2-meth-
oxypyrazolo[1,5-a]pyridine (23) that was prepared
according to the method described by Ochi et al.28 After
formylation furnished the aldehyde 24, application of an
ultrasound promoted Knoevenagel condensation29 with
nitromethane gave the nitroalkene 25 in moderate yield.
Subsequent reduction with LiAlH4 and acylation with
butyric anhydride led to the target molecule 27.
3. Results and discussion
3.1. Receptor binding and functional assay
The binding affinity of melatonin and its 7a-aza
analogues (17–22 and 27) was determined in competi-
tion radioligand binding assays using 2-[125I]-iodomela-
tonin as described previously30 employing recombinant
human MT1 and MT2 subtypes expressed in NIH 3T3
Table 1. Binding affinity of melatonin and the pyrazolo[1,5-a]pyridines 17–22 and 27 on human MT1 and MT2 receptors and their agonist activity in
the Xenopus laevis melanophore assay
O
HN
R3
R4
R2
N
N
Compound
R2
R3
R4
Receptor binding (Ki, nM)a
Selectivity (MT1/MT2)
Xenopus melanophores (EC50, nM)b
hMT1
hMT2
Melatonin
0.45 0.05
12.3 3.7
14.5 1.9
7.61 0.45
0.58 0.04
1.86 0.09
1.37 0.10
118 8.2
0.30 0.02
4.01 0.09
2.84 0.06
0.52 0.12
0.40 0.03
0.38 0.07
0.39 0.10
1.55 0.51
1.5
3.1
5.1
14.6
1.5
4.9
3.5
76
0.02 0.003
10.2 0.29
9.06 0.85
1.81 0.04
0.09 0.006
0.06 0.001
0.03 0.001
253 6.9
17
18
19
20
21
22
27
H
Me
Et
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
Pr
Ph
Ph
Ph
OMe
Me
Et
Pr
Pr
a Ki values are means SEM of three concentration–response curves.
b EC50 values are means SEM of three concentration–response curves.

1952
J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
CF3
(3) At each of the cloned receptor subtypes, the effect of
lengthening the N-acyl side chain from one carbon atom
to three carbons was different. While the binding affini-
ties at the MT2 receptor increased on going from acetyl
to butanoyl as the acylating group, no such consistent ef-
fect could be observed at the MT1 receptor. (4) As shown
by compound 27, shifting the methoxy group from posi-
tion 5 to 2 of the pyrazolo[1,5-a]pyridine moiety can
modulate the affinity and selectivity of these ligands,
resulting in a Ki value of 1.55 nM at the MT2 receptor
and a MT1/MT2 selectivity ratio of 76. Taking advantage
of the results derived from the 5-methoxy series, we
decided to prepare the butanoyl derivative 27 to gain
maximum binding at the MT2 receptor. Interestingly,
the comparison of the obtained data for compounds 19
and 27 reveals that switching the 5-methoxy group to po-
sition 2 has a much greater effect on potency in the func-
tional pigment aggregation assay in melanophores and
on binding affinity to the MT1 receptor subtype than it
does on binding affinity to the MT2 receptor subtype.
One possible explanation is that both the Mel1c receptor
of lower vertebrates, which mediates pigment aggrega-
tion in Xenopus melanophores,36 and the human MT1
receptor subtype may be more reliant on hydrogen bond-
ing to the 5-methoxy oxygen than the human MT2 recep-
tor subtype. We have made similar observations for
melatonin and N-acetyltryptamine, where upon removal
of the 5-methoxy group the affinity is clearly more
adversely affected at the MT1 (ꢀ1550-fold decrease)
and the Mel1c subtypes (ꢀ1380-fold decrease) than at
the MT2 subtype (ꢀ187-fold decrease).37
O
H
N
CH3O
N
CH3
28
Chart 2.
conformer, which is expected to direct its 2-methoxy
group conformations in favor of the abs toward the
abs, was chosen from a similar DISCO run (1 acceptor
atom, 1 donor site, 1 donor atom, 1 acceptor site, and
2 hydrophobic centroids). Subsequently, both conform-
ers of 27 were subjected to a series of DFT calculations40
(see Table 2) giving more reliable, relaxed structures. As
we had no indications for the correct conformation of
the propyl side chain from the structural template 28,
we only included a methyl side chain for both conforma-
tions in our quantum chemical calculations. The final
conformers derived from these calculations were again
submitted to a DISCO alignment yielding the final phar-
macophoric superpositions as depicted in Figure 1. For
the abs aligned conformer (Fig. 1A), a six point Ôphar-
macophore modelÕ with an rmsd fit of 0.238 was re-
trieved, while for the mbs aligned conformer (Fig. 1B),
a nine-point Ôpharmacophore modelÕ with an rmsd fit
of 1.312 was obtained. Within the DFT calculations,
increasing basis set levels show a narrowing gap between
both aligned conformers, which directs the 2-methoxy
group toward the auxiliary binding site. At the highest
level of calculation an energy difference of 1.54 kcal/
mol was found, which we try to evaluate subsequently
considering the relative binding contributions of the
auxiliary and methoxy binding site. Due to the funda-
mental thermodynamic relationship between the differ-
ence in the free energy of binding DDG and the
difference in the logarithmic inhibition constants DpKi
given by the equation
3.2. Theoretical investigations
Based on the high MT2 affinity of 27, we were intrigued
by the question of how 27 is able to compensate the
omission of the 5-methoxy group, which is usually
found to have a strong adverse effect as stated before.
In principle, two different explanations are conceivable:
Instead of the 5-methoxy, the 2-methoxy group could
bind to the specific Ômethoxy binding siteÕ (mbs) or the
2-methoxy group could bind to an Ôauxiliary binding
siteÕ (abs) close to position 2, which is known to accept
even phenyl moieties of ligands such as 20–22. These
two states resemble a discrimination of two different
binding situations for a naphthyl ligand postulated by
Langlois et al.16 In order to investigate, whether 27 is
able to adopt a conformation allowing it to mimic the
regular interaction of the missing 5-methoxy group, we
performed an extensive conformational sampling of 27
using SYBYL6.938 multisearch (10,000 searches yielding
203 unique conformational clusters) and the implement-
ed TRIPOS force field. The most favorable conformer,
which is able to interact with the mbs, was selected by
a DISCO pharmacophore search (>1 acceptor atoms,
>1 donor sites, 1 donor atom, 1 acceptor site, and 2
hydrophobic centroids), using the crystal structure
(CSD-ID of the (R)-enantiomer: SEGVIJ) of the high
affinity rigidized tricyclic ligand 28 (Chart 2) after inver-
sion to the (S)-enantiomer as a structural template.39
The inversion was performed since this configuration
proved to show a higher MT receptor binding in the cor-
responding series. For comparison, the most reasonable
DDG ¼ RT ꢁ ln 10 ꢁ DpKi;
we can assess the relative binding contribution of the
mbs, for instance, from the already cited example of
melatonin and N-acetyltryptamine.37 While the loss of
the 5-methoxy group in N-acetyltryptamine decreases
its binding affinity at the MT1 receptor by ꢀ1550-fold
equaling a reduction of the binding energy by about
4.4 kcal/mol, the MT2 receptor seems to be less sensitive
to the absence of this interaction, showing a ꢀ187-fold
attenuated receptor binding, which equals a reduction
in binding energy of about 3.1 kcal/mol (Fig. 2). On
the other hand, we can estimate from the introduction
of a phenyl moiety in position 2 of melatonin, leading
to a ꢀ3.5- and 3.2-fold increase of affinity at MT1 and
MT2 receptors, respectively,37 that additional binding
interactions in the magnitude of 0.7 kcal/mol are formed
between the substituent and the abs in both subtypes
and that the abs tolerates large substituents up to the
size of a phenyl moiety very well. Taking also a series
of 2-halo analogues into consideration, we can conclude

J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
1953
Table 2. Quantum chemical calculations on the abs and mbs aligned conformations of 27
Method/basis
Calcd type
E [hartree]
DE [kcal/mol] mbs ꢂ abs
Abs aligned
Mbs aligned
B3LYP/3-21G
B3LYP/6-31G(d)
OPT
OPT
OPT
SP
ꢂ776.706687984
ꢂ781.005469098
ꢂ781.222977630
ꢂ781.265971776
ꢂ776.700447881
ꢂ781.002460477
ꢂ781.220374865
ꢂ781.263512658
+3.92
+1.88
+1.63
+1.54
B3LYP/6-311+G(d,p)
B3LYP/6-311++G(2df,p)
Figure 1. Comparison of the two different final DISCO pharmacophore models, involving the auxiliary binding site (abs) oriented conformation (A)
and the methoxy binding site (mbs) oriented conformation (B) of 27 (carbons colored in black) fitted on the rigidized tricyclic subtype-unselective
agonist 28 (carbons colored in gray). For both models, specific acceptor and donor binding sites (as and ds) adjacent to the amidoethane side chain
have been included leading to 6 and 9 pharmacophoric points (with an rmsd fit of 0.238 and 1.312) satisfied in models A and B, respectively. The
picture was prepared with VMD1.8.2.
tion of 27 (Fig. 1A), evaluation of the assessable relative
binding contributions of both abs and mbs (Fig. 2) indi-
cates that the mbs aligned conformer of 27 (Fig. 1B)
should yield more favorable interactions. Summing up
the previously estimated interaction energies, we have
gathered evidence that the methoxy-mbs interaction
energy exceeds the typical abs-interaction energy and
the calculated energy effort for adopting the mbs-aligned
conformation (Fig. 1B) by about 2.2 and 0.9 kcal/mol for
MT1 and MT2, respectively (Fig. 2). Although such a
purely ligand-based relative comparison of binding affin-
ities can of course not be regarded as a final proof for this
conclusion, it casts a new light on the relative energy dif-
ference of both conformers and provides the opportunity
to rank this conformational energy versus interaction
energies with the receptor. In the more probable mbs
binding hypothesis, MT2 selectivity of 27 could be based
Figure 2. Estimation of the more favorable conformer of 27 based on
the energy contents of its receptor interactions and conformational
upon different capabilities of MT1 and MT2 to adapt
either to the slight displacement of the side chain–amide
strain. The values shown in this figure are absolute numbers of relative
interaction energies given in kilo calories per mole, which
teraction patterns of the heteroaromatic moiety.
interactions or to the steric requirements and altered p-in-
were deduced from characteristic binding differences of melatonin,
N-acetyltryptamine, and 2-substituted melatonin derivatives37 or
obtained by quantum chemical calculations.
4. Conclusion
that the order of magnitude of the interactions with the
abs is quite consistent for 2-substituents with different
sizes and hydrophobicities.37 Furthermore, none of
these substituents in position 2 is capable of inducing
MT1/MT2 selectivity. Thus, one possible explanation
for the selectivity-inducing properties of the 2-methoxy
substituent in 27 could be a putative subtype-specific
interaction with significant energy content between the
methoxy group and the MT2-abs, which should not be
present in MT1. Except for this hypothesis involving
such an MT2 specific interaction partner in the abs,
which is then likely to favor the abs aligned conforma-
In conclusion, we were able to show that the pyrazol-
o[1,5-a]pyridine moiety can serve as a useful bioisostere
of indole in the search for melatonin receptor agonists.
In general, most of the structure–affinity relationships
previously described for indole-derived melatonin ana-
logues37 are found to be consistent with our series, espe-
cially the effects exhibited by lengthening of the N-acyl
side chain and introduction of a 2-phenyl substituent.
However, we found that by shifting the 5-methoxy
group into position 2 of the aromatic core and, at the
same time, keeping the N-butanoyl side chain, a MT2
selective agonist with MT1/MT2 selectivity ratio of 76

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J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
and good binding affinity (1.55 nM) can be produced.
This may provide a starting point for the development
of additional selective agonists, which are useful tools
in defining the physiological roles of the receptor sub-
types. Furthermore, considering the indole ring of mela-
tonin as the structural site of catabolic inactivation,41 it
could be assumed that the reported pyrazolo[1,5-a]pyri-
dines are metabolically more stable when compared with
melatonin, as indicated by the pharmacokinetics of the
pyrazolo[1,5-a]pyridine bearing antiallergic agent ibudi-
last22 and the antiplatelet agent KC-764.23
5.3. Ethyl 5-methoxy-2-phenylpyrazolo[1,5-a]pyridine-3-
carboxylate (6)
This compound was prepared as described for 5 using 4
(3 g; 9.7 mmol) and K2CO3 (1.9 g; 13.7 mmol) in DMF
(21 ml) and ethyl phenylpropiolate (1.8 g; 10.3 mmol) to
afford 6 (1 g, 35%) as a light yellow oil. IR 1702, 1683,
1
1289, 1050 cmꢂ1; H NMR d 1.26 (t, J = 7.1 Hz, 3H),
3.95 (s, 3H), 4.28 (q, J = 7.1 Hz, 2H), 6.63 (dd,
J = 7.5 Hz, 2.8 Hz, 1H), 7.45–7.41 (m, 3H), 7.53 (br d,
J = 2.8 Hz, 1H), 7.72–7.75 (m, 2H), 8.33 (br d,
J = 7.5 Hz, 1H); EIMS 296 (M+); Anal. (C17H16N2O3):
C, H, N.
5. Experimental
5.4. 5-Methoxypyrazolo[1,5-a]pyridine (7)
All reactions were carried out under nitrogen atmo-
sphere, except 1.3-dipolar cycloadditions and ester
hydrolyses. Solvents were purified and dried by standard
procedures. All reagents were of commercial quality and
used as purchased. MS were run on a Finnegan MAT
TSQ 70 spectrometer by EI (70 eV) with solid inlet.
A suspension of 5 (5 g; 24 mmol) in 50% H2SO4 (40 ml)
was heated at 80 ꢁC for 3 h. After cooling to rt and then
to 0 ꢁC, the solution was neutralized with a 5 N NaOH
solution, extracted with EtO2, extract dried (MgSO4),
and evaporated. The residue was purified by flash chro-
matography (hexane–EtOAc, 8:2) to give 7 (3 g, 83%) as
1
The H NMR spectra were obtained on a Bruker AM
360 (360 MHz) spectrometer, if not otherwise stated in
CDCl3 relative to TMS. IR spectra were performed on
a Jasco FT/IR 410 spectrometer. Purification by chro-
matography was performed using Silica Gel 60, TLC
analyses were performed using Merck 60 F254 aluminum
sheets and analyzed by UV light (254 nm) or in the pres-
ence of iodine. CHN elementary analyses were per-
formed at the Department of Organic Chemistry of
the Friedrich Alexander University.
a colorless oil. IR 2937, 1646, 1340, 1024 cmꢂ1 1H
;
NMR d 3.84 (s, 3H), 6.32 (br d, J = 2.5 Hz, 1H), 6.44
(dd, J = 7.5 Hz, 2.8 Hz, 1H), 6.74 (d, J = 2.8 Hz, 1H),
7.85 (d, J = 2.5 Hz, 1H), 8.28 (br d, J = 7.5 Hz, 1H);
EIMS 148 (M+); Anal. (C8H8N2O): C, H, N.
5.5. 5-Methoxy-2-phenylpyrazolo[1,5-a]pyridine (8)
This compound was prepared as described for 7 but
heating for 16 h using 6 (1 g; 3.4 mmol) in 50% H2SO4
(10 ml) to afford 8 (605 mg; 80%) as a colorless oil after
purification by flash chromatography (hexane–EtOAc,
5.1. N-Amino-(4-methoxy)pyridinium 2,4-dinitropheno-
late (4)
1
8:2). IR 2928, 1649, 1420, 1028 cmꢂ1; H NMR d 3.85
A mixture of 4-methoxypyridine (1.5 g; 14 mmol) and O-
(2,4-dinitrophenyl)hydroxylamine25 (3.1 g; 15.1 mmol) in
MeCN (9 ml) was stirred at 40 ꢁC for 24 h. After the addi-
tion of Et2O, the resulting yellow-orange solid was collect-
ed and dried yielding 4 (3.7 g; 86%). Mp 139 ꢁC; IR 3197,
3095, 1536, 1507, 1257, 738 cmꢂ1; 1H NMR (CD3)2SO d
4.01 (s, 3H), 6.35 (d, J = 9.8 Hz, 1H), 7.52–7.56 (m, 2H),
7.76 (br s, 2H), 7.80 (dd, J = 9.8 Hz, 3.2 Hz, 1H), 8.60
(d, J = 3.2 Hz), 8.66–8.69 (m, 2H); EIMS 184 (M+)–
C6H4N2O5, 124 (M+)–C6H9N2O.
(s, 3H), 6.43 (dd, J = 7.5 Hz, 2.8 Hz, 1H), 6.61 (br s,
1H), 6.72 (br d, J = 2.8 Hz), 7.33–7.46 (m, 3H), 7.92–
7.94 (m, 2H), 8.29 (br d, J = 7.5 Hz, 1H); EIMS 224
(M+); Anal. (C14H12N2O): C, H, N.
5.6. 5-Methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (9)
To a solution of POCl3 (1.6 g; 10.4 mmol) in DMF
(4 ml), 7 (500 mg; 3.4 mmol) dissolved in DMF (1 ml)
was slowly added. The mixture was stirred at rt for 1 h
and after cooling to 0 ꢁC it was diluted with H2O, made
alkaline with 2 N NaOH, and extracted with CHCl3.
After drying (MgSO4), the extract was evaporated and
the residue was purified by flash chromatography (hex-
ane–EtOAc, 1:1) to afford 9 (541 mg; 91%) as a white
5.2. Methyl 5-methoxypyrazolo[1,5-a]pyridine-3-carbox-
ylate (5)
Methyl propiolate (945 mg; 11.2 mmol) was added drop-
wise to a mixture of 4 (3.1 g; 10.2 mmol), K2CO3 (2 g;
14.5 mmol), and DMF (22 ml) and the reaction mixture
was stirred vigorously at rt for 24 h. The suspension was
filteredover Celiteand the filtrate evaporated. The residue
was dissolved in EtO2, solution was washed three times
with water, dried (MgSO4), and evaporated. The crude
product was purified by flash chromatography (hexane–
EtOAc, 9:1) to afford 5 (903 mg; 43%) as a light yellow
solid. Mp 93 ꢁC; IR 1662, 1280, 1201, 831 cmꢂ1
;
1H
NMR d 3.96 (s, 3H), 6.72 (dd, J = 7.5 Hz, 2.8 Hz, 1H),
7.59 (d, J = 2.8 Hz, 1H), 8.27 (s, 1H), 8.37 (d,
J = 7.5 Hz, 1H), 9.96 (s, 1H); EIMS 176 (M+); Anal.
(C9H8N2O2): C, H, N.
5.7. 5-Methoxy-2-phenylpyrazolo[1,5-a]pyridine-3-carb-
aldehyde (10)
1
oil. IR 1699, 1536, 1278, 1051, 773 cmꢂ1; H NMR d
3.90 (s, 3H), 3.93 (s, 3H), 6.62 (dd, J = 7.5 Hz, 2.8 Hz,
1H), 7.42 (dd, J = 2.8 Hz, 0.7 Hz, 1H), 8.28 (s, 1H), 8.32
(dd, J = 7.5 Hz, 0.7 Hz, 1H); EIMS 206 (M+); Anal.
(C10H10N2O3): C, H, N.
This compound was prepared as described for 9 using 8
(440 mg; 2 mmol) and POCl3 (919 mg; 6 mmol) in DMF
(2.5 ml) to afford 10 (463 mg; 94%) as a white solid after
purification by flash chromatography (hexane–EtOAc,

J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
1955
1:1). Mp 92 ꢁC; IR 1735, 1437, 1243, 700 cmꢂ1
NMR d 3.98 (s, 3H), 6.74 (dd, J = 7.5 Hz, 2.8 Hz, 1H),
7.50–7.56 (m, 3H), 7.74–7.77 (m, 3H), 8.39 (br d,
J = 7.5 Hz, 1H), 10.04 (s, 1H); EIMS 252 (M+); Anal.
(C15H12N2O2): C, H, N.
;
1H
(20 mg; 0.53 mmol), yielding 14 (38 mg; 75%) after puri-
fication by flash chromatography (hexane–EtOAc, 9:1)
as light yellow crystals. Mp 152 ꢁC; IR 1646, 1543,
1459, 1428, 1021, 700 cmꢂ1
;
1H NMR d 3.57 (t,
J = 7.5 Hz, 2H), 3.90 (s, 3H), 4.47 (t, J = 7.5 Hz, 2H),
6.48 (dd, J = 7.5 Hz, 2.5 Hz, 1H), 6.65 (d, J = 2.5 Hz,
1H), 7.40–7.51 (m, 3H), 7.64–7.67 (m, 2H), 8.26 (d,
5.8. 5-Methoxy-3-(2-nitroethenyl)-pyrazolo[1,5-a]pyri-
dine (11)
J = 7.5 Hz,
1H);
(C16H15N3O3): C, H, N.
EIMS
297
(M+);
Anal.
To a solution of 9 (400 mg, 2.3 mmol) in MeOH (4 ml)
were added nitromethane (150 mg; 2.5 mmol), methyl-
amine hydrochloride (57.5 mg; 0.85 mmol), and potassi-
um acetate (57.5 mg; 0.59 mmol), and the reaction
mixture was stirred at rt for 3 days. The precipitate was
collected, washed with a small amount of MeOH and hex-
ane, and dried to afford 11 (418 mg; 84%) as maroon
prisms. Mp 157 ꢁC; IR 1646, 1538, 1490, 1344, 1214,
5.12. 2-(5-Methoxypyrazolo[1,5-a]pyridine-3-yl)-ethylamine
(15)
To a solution of 13 (250 mg; 1.1 mmol) in EtOH (12 ml),
tin powder (0.3 g) and HOAc (3 ml) were added and the
mixture was stirred for 3 days at rt. The solid particles
were filtered off and the filtrate evaporated. The residue
was diluted with H2O, made alkaline with 2 N NaOH,
and extracted five times with CHCl3. The combined ex-
tracts were washed with saturated sodium chloride solu-
tion, dried (MgSO4), concentrated, and purified by flash
chromatography (CH2Cl2–MeOH, 8:2) to give 15
(130 mg; 60%) as a colorless oil. IR 2935, 1649, 1559,
1
833 cmꢂ1; H NMR (CD3)2SO d 3.96 (s, 3H), 6.82 (dd,
J = 7.5 Hz, 2.5 Hz, 1H), 7.64 (br d, J = 2.5 Hz, 1H),
8.10 (d, J = 13.1 Hz, 1H), 8.48 (d, J = 13.1 Hz, 1H), 8.60
(s, 1H), 8.71 (d, J = 7.5 Hz, 1H); EIMS 219 (M+); Anal.
(C10H9N3O3): C, H, N.
1
5.9. 5-Methoxy-3-(2-nitroethenyl)-2-phenylpyrazolo[1,5-
a]pyridine (12)
1244, 1018, 813 cmꢂ1; H NMR d 2.81 (t, J = 6.7 Hz,
2H), 2.98 (t, J = 6.7 Hz, 2H), 3.85 (s, 3H), 6.42 (dd,
J = 7.5 Hz, 2.5 Hz, 1H), 6.65 (d, J = 2.5 Hz, 1H), 7.74
(s, 1H), 8.23 (d, J = 7.5 Hz, 1H); EIMS 191 (M+); Anal.
(C10H13N3O): C, H, N.
This compound was prepared as described for 11 using 10
(463 mg; 1.8 mmol), nitromethane (113 mg; 1.9 mmol),
methylamine hydrochloride (50 mg; 0.74 mmol), and
potassium acetate (50 mg; 0.51 mmol), and the reaction
mixture was stirred at rt for 10 days, yielding 12
(400 mg; 74%) as orange crystals. Mp 166 ꢁC; IR 1740,
5.13. 2-(5-Methoxy-2-phenylpyrazolo[1,5-a]pyridine-3-yl)-
ethylamine (16)
1
1648, 1457, 1316, 1072, 769 cmꢂ1; H NMR d 4.01 (s,
This compound was prepared as described for 15 using 14
(200 mg; 0.67 mmol) in EtOH (6 ml), tin powder
(150 mg), and HOAc (1.5 ml) yielding 16 (108 mg; 60%)
after purification by flash chromatography (CH2Cl2–
MeOH, 8:2) as a colorless oil. IR 2931, 1645, 1249,
3H), 6.73 (dd, J = 7.5 Hz, 2.6 Hz, 1H), 6.92 (d,
J = 2.6 Hz, 1H), 7.50 (d, J = 13.6 Hz, 1H), 7.50–7.56 (m,
3H), 7.64–7.70 (m, 2H), 8.33 (d, J = 13.6 Hz, 1H), 8.42
(d, J = 7.5 Hz, 1H); EIMS 295 (M+); Anal.
(C16H13N3O3): C, H, N.
1
1222, 1022, 700 cmꢂ1; H NMR d 2.91–3.01 (m, 4H),
3.88 (s, 3H), 6.43 (dd, J = 7.5 Hz, 2.7 Hz, 1H), 6.69 (d,
J = 2.7 Hz, 1H), 7.36–7.48 (m, 3H), 7.70–7.73 (m, 2H),
8.26 (d, J = 7.5 Hz, 1H); EIMS 267 (M+); Anal.
(C16H17N3O): C, H, N.
5.10. 5-Methoxy-3-(2-nitroethyl)-pyrazolo[1,5-a]pyridine
(13)
A mixture of 11 (300 mg; 1.4 mmol) and MeOH (14 ml)
was stirred during the portionwise addition (20 min) of
NaBH4 (164.5 mg; 4.4 mmol). The solution was stirred
for 30 min after the addition of NaBH4 was completed,
then adjusted to pH 5 with HOAc, and concentrated.
The residue was diluted with H2O and extracted three
times with CH2Cl2. The extracts were combined, washed
with H2O, dried (MgSO4), concentrated, and purified by
flash chromatography (hexane–EtOAc, 3:1) to give 13
(236 mg; 78%) as light yellow crystals. Mp 140 ꢁC; IR
1650, 1544, 1459, 1238, 1014, 829 cmꢂ1; 1H NMR d 3.41
(t, J = 7.1 Hz, 2H), 3.88 (s, 3H), 4.60 (t, J = 7.1 Hz, 2H),
6.46 (dd, J = 7.5 Hz, 2.5 Hz, 1H), 6.63 (d, J = 2.5 Hz,
1H), 7.74 (s, 1H), 8.24 (br d, J = 7.5 Hz, 1H); EIMS 221
(M+); Anal. (C10H11N3O3): C, H, N.
5.14. N-[2-(5-Methoxypyrazolo[1,5-a]pyridine-3-yl)-ethyl]-
acetamide (17)
To a cooled solution (0 ꢁC) of 15 (18.4 mg, 0.1 mmol)
in THF (1.5 ml) were added Et3N (34 mg; 0.34 mmol)
and acetic anhydride (33.8 mg; 0.34 mmol). The ice
bath was removed and the solution was stirred for
3 h. The solvent was evaporated in vacuo, and the res-
idue was taken up in ethyl acetate and washed with
H2O, saturated aqueous NaHCO3, and brine. The
organic phase was dried (MgSO4), concentrated in vac-
uo, and purified by flash chromatography (CH2Cl2–
MeOH, 95:5) to afford 17 (20 mg; 89%) as colorless
crystals. Mp 122 ꢁC; IR 1650, 1560, 1459, 1241,
808 cmꢂ1
;
1H NMR
d
1.94 (s, 3H), 2.89 (t,
5.11. 5-Methoxy-3-(2-nitroethyl)-2-phenylpyrazolo[1,5-a]
pyridine (14)
J = 6.9 Hz, 2H), 3.51 (dt, J = 6.9 Hz, 6.5 Hz, 2H),
3.86 (s, 3H), 5.56 (br s, 1H), 6.43 (dd, J = 7.5 Hz,
2.5 Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H), 7.71 (s, 1H),
8.23 (br d, J = 7.5 Hz, 1H); EIMS 233 (M+); Anal.
(C12H15N3O2): C, H, N.
This compound was prepared as described for 13 using
12 (50 mg; 0.17 mmol) in MeOH (5 ml) and NaBH4

1956
J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
5.15. N-[2-(5-Methoxypyrazolo[1,5-a]pyridine-3-yl)-ethyl]-
propionamide (18)
J = 7.5 Hz, 1H); EIMS 323 (M+); Anal. (C19H21N3O2):
C, H, N.
This compound was prepared as described for 17 using 15
(26.5 mg; 0.14 mmol) in THF (1.5 ml), Et3N (41.2 mg;
0.41 mmol), and propionic anhydride (53.4 mg;
0.41 mmol) yielding 18 (25 mg; 85%) after purification
by flash chromatography (CH2Cl2–MeOH, 95:5) as col-
orless crystals. Mp 109 ꢁC; IR 1647, 1560, 1457, 1244,
5.19. N-[2-(5-Methoxy-2-phenylpyrazolo[1,5-a]pyridine-
3-yl)-ethyl]-butyramide (22)
This compound was prepared as described for 17 using
16 (23 mg; 0.09 mmol) in THF (1.5 ml), Et3N
(33.9 mg; 0.34 mmol), and butyric anhydride (53.8 mg;
0.34 mmol) yielding 22 (24 mg; 84%) after purification
by flash chromatography (CH2Cl2–MeOH, 95:5) as col-
orless crystals. Mp 123 ꢁC; IR 1645, 1457, 1248, 1022,
1
808 cmꢂ1; H NMR d 1.13 (t, J = 7.6 Hz, 3H), 2.16 (q,
J = 7.6 Hz, 2H), 2.92 (t, J = 6.8 Hz, 2H), 3.55 (dt,
J = 6.8 Hz, 6.2 Hz, 2H), 3.86 (s, 3H), 5.50 (br s, 1H),
6.43 (dd, J = 7.5 Hz, 2.7 Hz, 1H), 6.65 (br d, J = 2.7 Hz,
1H), 7.71 (s, 1H), 8.23 (br d, J = 7.5 Hz, 1H); EIMS 247
(M+); Anal. (C13H17N3O2): C, H, N.
1
699 cmꢂ1; H NMR d 0.87 (t, J = 7.4 Hz, 3H), 1.48–
1.59 (m, 2H), 1.96 (t, J = 7.6 Hz, 2H), 3.06 (t,
J = 6.8 Hz, 2H), 3.44 (dt, J = 7.0 Hz, 6.2 Hz, 2H), 3.89
(s, 3H), 5.40 (br s, 1H), 6.46 (dd, J = 7.5 Hz, 2.7 Hz,
1H), 6.66 (br d, J = 2.7 Hz, 1H), 7.37–7.49 (m, 3H),
7.69–7.73 (m, 2H), 8.27 (br d, J = 7.5 Hz, 1H); EIMS
337 (M+); Anal. (C20H23N3O2): C, H, N.
5.16. N-[2-(5-Methoxypyrazolo[1,5-a]pyridine-3-yl)-ethyl]-
butyramide (19)
This compound was prepared as described for 17 using
15 (23 mg; 0.12 mmol) in THF (1.5 ml), Et3N
(47.3 mg; 0.47 mmol), and butyric anhydride (75 mg;
0.47 mmol) yielding 19 (30 mg; 82%) after purification
by flash chromatography (CH2Cl2–MeOH, 95:5) as col-
orless crystals. Mp 102 ꢁC; IR 1649, 1537, 1456, 1246,
811 cmꢂ1; 1H NMR d 0.92 (t, J = 7.4 Hz, 3H), 1.58–1.68
(m, 2H), 2.10 (t, J = 7.6 Hz, 2H), 2.88 (t, J = 6.8 Hz,
2H), 3.52 (dt, J = 6.8 Hz, 6.2 Hz, 2H), 3.86 (s, 3H),
5.51 (br s, 1H), 6.44 (dd, J = 7.5 Hz, 2.7 Hz, 1H), 6.66
(br d, J = 2.7 Hz, 1H), 7.71 (s, 1H), 8.24 (br d,
J = 7.5 Hz, 1H); EIMS 261 (M+); Anal. (C14H19N3O2):
C, H, N.
5.20. 2-Methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde
(24)
This compound was prepared as described for 9 using
2328 (120.5 mg; 0.81 mmol) and POCl3 (383 mg;
2.5 mmol) in DMF (1 ml) to afford 24 (140 mg; 98%)
as a white solid after purification by flash chromatogra-
phy (hexane–EtOAc, 1:1). Mp 85 ꢁC; IR 1654, 1631,
1
1511, 1411, 1064, 759 cmꢂ1; H NMR d 4.13 (s, 3H),
6.94 (ddd, J = 7.1 Hz, 6.7 Hz, 1.4 Hz, 1H), 7.47 (ddd,
J = 8.9 Hz, 7.1 Hz, 1.4 Hz, 1H), 8.12–8.15 (br d,
J = 8.9 Hz, 1H), 8.31–8.34 (br d, J = 6.7 Hz, 1H), 9.92
(s, 1H); EIMS 176 (M+); Anal. (C9H8N2O2): C, H, N.
5.17. N-[2-(5-Methoxy-2-phenylpyrazolo[1,5-a]pyridine-
3-yl)-ethyl]-acetamide (20)
5.21. 2-Methoxy-3-(2-nitroethenyl)-pyrazolo[1,5-a]pyri-
dine (25)
This compound was prepared as described for 17 using 16
(20 mg; 0.08 mmol) in THF (1.5 ml), Et3N (25.6 mg;
0.26 mmol), and acetic anhydride (25.4 mg; 0.26 mmol)
yielding 20 (19 mg; 84%) after purification by flash chro-
matography (CH2Cl2–MeOH, 95:5) as colorless crystals.
A mixture of 24 (100 mg; 0.57 mmol), nitromethane
(417 mg; 6.9 mmol), HOAc (0.1 ml), and ammonium
acetate (94.4 mg; 1.2 mmol) was sonicated at 22 ꢁC for
6 h. After addition of CH2Cl2, the organic phase was
washed with H2O and brine, dried (MgSO4), and con-
centrated. The residue was purified by flash chromatog-
raphy (hexane–EtOAc, 8:2) to give 25 (67 mg; 54%) as a
yellow solid. Mp 120 ꢁC; IR 1639, 1608, 1542, 1461,
1
Mp 134 ꢁC; IR 1645, 1457, 1429, 1248, 700 cmꢂ1; H
NMR d 1.80 (s, 3H), 3.06 (t, J = 6.9 Hz, 2H), 3.43 (dt,
J = 6.9 Hz, 6.5 Hz, 2H), 3.89 (s, 3H), 5.43 (br s, 1H),
6.46 (dd, J = 7.5 Hz, 2.7 Hz, 1H), 6.74 (d, J = 2.7 Hz,
1H), 7.37–7.49 (m, 3H), 7.69–7.72 (m, 2H), 8.27 (br d,
J = 7.5 Hz, 1H); EIMS 309 (M+); Anal. (C18H19N3O2):
C, H, N.
1207, 829 cmꢂ1 1H NMR (CD3)2SO d 4.12 (s, 3H),
;
7.10 (ddd, J = 7.2 Hz, 6.7 Hz, 1.2 Hz, 1H), 7.59 (ddd,
J = 8.9 Hz, 7.2 Hz, 1.2 Hz, 1H), 7.75 (d, J = 13.1 Hz,
1H), 8.11 (d, J = 8.9 Hz, 1H), 8.34 (d, J = 13.1 Hz,
1H), 8.73 (d, J = 6.7 Hz, 1H); EIMS 219 (M+); Anal.
(C10H9N3O3): C, H, N.
5.18. N-[2-(5-Methoxy-2-phenylpyrazolo[1,5-a]pyridine-
3-yl)-ethyl]-propionamide (21)
This compound was prepared as described for 17 using 16
(20 mg; 0.08 mmol) in THF (1.5 ml), Et3N (25.6 mg;
0.26 mmol), and propionic anhydride (33.4 mg;
0.26 mmol) yielding 21 (21 mg; 87%) after purification
by flash chromatography (CH2Cl2–MeOH, 95:5) as col-
orless crystals. Mp 129 ꢁC; IR 1645, 1457, 1223, 1022,
5.22. 2-(2-Methoxypyrazolo[1,5-a]pyridine-3-yl)-ethylamine
(26)
A suspension of 25 (40 mg, 0.18 mmol) in THF (2 ml)
was treated with LiAlH4 (1 M in THF; 183 ll;
0.18 mmol) at 0 ꢁC and the reaction mixture was stirred
at rt for 2 h. After the addition of H2O (0.2 ml), the sus-
pension was filtered and washed with EtO2. The filtrate
was dried (MgSO4), evaporated, and the residue was
purified by flash chromatography (CH2Cl2–MeOH,
95:5) to give 26 (16 mg, 45%) as a colorless oil. IR
1
699 cmꢂ1; H NMR d 1.04 (t, J = 7.6 Hz, 3H), 2.01 (q,
J = 7.6 Hz, 2H), 3.07 (t, J = 7.0 Hz, 2H), 3.44 (dt,
J = 7.0 Hz, 6.5 Hz, 2H), 3.89 (s, 3H), 5.41 (br s, 1H),
6.45 (dd, J = 7.5 Hz, 2.7 Hz, 1H), 6.73 (br d, J = 2.7 Hz,
1H), 7.37–7.49 (m, 3H), 7.69–7.73 (m, 2H), 8.26 (br d,

J. Elsner et al. / Bioorg. Med. Chem. 14 (2006) 1949–1958
1957
1
2923, 1641, 1515, 1409, 1342 cmꢂ1; H NMR d 2.73 (t,
J = 6.74, 2H), 2.93 (t, J = 6.74 Hz, 2H), 4.04 (s, 1H),
6.53 (ddd, J = 7.1 Hz, 6.65 Hz, 1.24 Hz, 1H), 7.00
(ddd, J = 8.96 Hz, 6.65 Hz, 1.24 Hz, 1H), 7.23–7.24
(m, 1H), 8.18–8.20 (br d, J = 7.1 Hz, 1H); EIMS 191
(M+); Anal. (C10H13N3O): C, H, N.
The change in distribution of pigment within melano-
phores was quantitated using a Bio-Tek microtitre plate
reader (model EL3115, Anachem, Luton, UK) by mea-
suring the change in absorbance (630 nm) before and
after drug treatment. The fractional change in absor-
bance, 1 ꢂ (Af/Ai), where Ai is the initial absorbance be-
fore drug treatment and Af is the final absorbance, was
calculated to determine agonist activity. To test for
antagonist activity, melatonin (1 nM) was then added
and absorbance was read again after 60 min (Aa).
Antagonist response was calculated as 1 ꢂ (Aa/Ai). All
drugs were freshly prepared from 10 mM stock solutions
in DMSO kept at ꢂ20 ꢁC. The maximal concentration
of solvent was 1% v/v which did not cause pigment redis-
tribution in melanophores.
5.23. N-[2-(2-Methoxypyrazolo[1,5-a]pyridine-3-yl)-ethyl]-
butyramide (27)
This compound was prepared as described for 17 using 26
(18 mg; 0.09 mmol) in THF (1.5 ml), Et3N (37 mg;
0.37 mmol), and butyric anhydride (58.7 mg; 0.37 mmol)
yielding 27 (26 mg; 72%) after purification by flash chro-
matography (CH2Cl2–MeOH, 95:5) as colorless crystals.
Mp 115 ꢁC; IR 1636, 1518, 1409, 1214, 744 cmꢂ1; H
1
NMR d 0.91 (t, J = 7.4 Hz, 3H), 1.56–1.67 (m, 2H), 2.09
(t, J = 7.5 Hz, 2H), 2.80 (t, J = 6.6 Hz, 2H), 3.46 (dt,
J = 6.6 Hz, 5.8 Hz, 2H), 4.05 (s, 3H), 5.70 (br s, 1H),
6.55 (ddd, J = 6.9 Hz, 6.8 Hz, 1.4 Hz, 1H), 7.02 (ddd,
J = 8.9 Hz, 6.8 Hz, 1.1 Hz, 1H), 7.24 (br d, J = 8.9 Hz,
1H), 8.19 (br d, J = 6.9 Hz, 1H); EIMS 261 (M+); Anal.
(C14H19N3O2): C, H, N.
5.26. Data analysis
In receptor binding studies, the four parameter logistic
equation was used for curve-fitting concentration–re-
sponse data to obtain the concentration of compounds
producing 50% inhibition of the specific 2-[125I]-iodom-
elatonin binding (for determination of Ki). The concen-
tration of agonists producing 50% of maximal pigment
aggregation (EC50), or antagonists inhibiting melato-
nin-induced aggregation by 50% (IC50), was determined
in the same way.
5.24. Melatonin receptor binding studies
NIH3T3 cells expressing recombinant human MT1 and
MT2 receptors were cultured, harvested, and lysed as de-
scribed previously.30 Membrane aliquots were stored in
liquid nitrogen until used. For competition assays, 2-
5.27. Modeling
[
125I]-iodomelatonin (70 pM for hMT1; 110 pM for
Conformational sampling of 27 using SYBYL6.9 multi-
search38 and pharmacophore modeling using DISCO38
was performed on a SGI Octane 2 workstation. DFT
calculations were performed on an 8 Intel Xeon proces-
sor Linux cluster using Gaussian98.40
hMT2) was incubated with NIH3T3 cell membranes at
room temperature (ꢀ21 ꢁC) for 90 min in a 96-well
Multiscreen plate with a PVDF membrane (Millipore,
Whatman Ltd, Maidstone, Kent, UK) pre-treated with
2% v/v polyethylenimine. Binding assays were terminat-
ed by the rapid addition of 100 ll ice-cold buffer (50 mM
Tris–HCl, 5 mM MgCl2, pH 7.4) to each well and
immediate vacuum filtration. Each well was then rinsed
with a further 2· 100 ll buffer. Membrane-bound 2-
Acknowledgments
The authors wish to thank Professor Peter J. Garratt for
helpful discussions. This work was supported by the
BMBF and Fonds der Chemischen Industrie, and a
Wellcome Trust grant (#065816) to D.S.
[
125I]-iodomelatonin trapped on each filter was counted
(Cobra II Auto-Gamma, PACKARD). All assays were
done on triplicate homogenate aliquots. Specific binding
was calculated by subtracting non-specific binding
(defined using 1 lM melatonin) from total binding.
Preliminary studies showed that equilibrium is reached
under the binding conditions used, and saturation
experiments gave Kd values close to those reported
previously (hMT1, 81 pM; hMT2, 131 pM).
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