Functionally selective dopamine D2/D3 receptor agonists comprising an enyne moiety

Article abstract of DOI:10.1021/jm400520c

Dopaminergics of types 1 and 2 incorporating a conjugated enyne as an atypical catechol-simulating moiety were synthesized in enantiomerically pure form and investigated for their metabolic stability. Radioligand binding studies indicated high affinity to D₂-like receptors. The test compounds were evaluated for their ability to differentially activate distinct signaling pathways. Measurement of D_2L- and D_2S-mediated [ ³⁵S]GTPγS incorporation in the presence of coexpressed Gα_o and Gα_i subunits showed significantly biased receptor activation for several test compounds. Thus, the 2-azaindolylcarboxamide (S)-2a exhibited substantial functional selectivity for D_2S-promoted G_o activation over G_i coupling. The most significant bias was determined for the triazolylalkoxy-substituted benzamide (S)-2c that displayed higher potency for G_o activation than for G_i coupling at the D_2L subtype. Functional selectivity for β-arrestin recruitment over G_i activation was observed for the biphenylcarboxamide (R)-1 and the 2-benzothiophenylcarboxamide (S)-2d, whereas the 2-substituted azaindole (S)-2a preferred β-arrestin recruitment compared to G_o coupling.

Full text of DOI:10.1021/jm400520c

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Article
Functionally Selective Dopamine D2/D3
Receptor Agonists Comprising an Enyne Moiety
Christine Hiller, Ralf C. Kling, Frank W Heinemann, Karsten Meyer, Harald Hübner, and Peter Gmeiner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400520c • Publication Date (Web): 03 Jun 2013
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Functionally Selective Dopamine D2/D3 Receptor
Agonists Comprising an Enyne Moiety
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Christine Hiller,1 Ralf C. Kling,1 Frank W. Heinemann,2 Karsten Meyer,2 Harald Hübner,1 Peter
Gmeiner1*
1Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich
Alexander University, Schuhstraße 19, 91052 Erlangen, Germany.
2Department of Chemistry and Pharmacy, Inorganic Chemistry, Friedrich Alexander University,
Egerlandstraße 1, 91058 Erlangen, Germany.
RECEIVED DATE (to be automatically inserted)
TITLE RUNNING HEAD: Functionally Selective Enynes
KEYWORDS: GPCR, dopamine receptor, functional selectivity, ligand bias, G protein, βꢀ
arrestin
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Abstract:
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Dopaminergics of type 1 and 2 incorporating a conjugated enyne as an atypical catecholꢀ
simulating moiety were synthesized in enantiomerically pure form and investigated for their
metabolic stability. Radioligand binding studies indicated high affinity to D2ꢀlike receptors. The
test compounds were evaluated for their ability to differentially activate distinct signaling
pathways. Measurement of D2Lꢀ and D2Sꢀmediated [35S]GTPγS incorporation in the presence of
coꢀexpressed Gαo and Gαi subunits showed significantly biased receptor activation for several
test compounds. Thus, the 2ꢀazaindolyl carboxamide (S)ꢀ2a exhibited substantial functional
selectivity for D2Sꢀpromoted Go activation over Gi coupling. The most significant bias was
determined for the triazolylalkoxyꢀsubstituted benzamide (S)ꢀ2c that displayed higher potency
for Go activation than for Gi coupling at the D2L subtype. Functional selectivity for βꢀarrestin
recruitment over Gi activation was observed for the biphenylcarboxamide (R)ꢀ1 and the 2ꢀ
benzothiophenyl carboxamide (S)ꢀ2d whereas the 2ꢀsubstituted azaindole (S)ꢀ2a preferred βꢀ
arrestin recruitment compared to Go coupling.
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Introduction
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Agonistꢀinduced coupling of one type of receptor to multiple signaling pathways is a general
property of G proteinꢀcoupled receptors (GPCRs).1 Functional selectivity explains the capacity
of a ligand to preferentially activate coupling of a GPCR with a subset of signal transducers.
Thus, ligandꢀGPCR interactions can promote an individual pattern of G proteinꢀdependent and G
proteinꢀindependent signaling. Employing the angiotensin II receptor as a model system,
pioneering work of R. Lefkowitz et al. showed that G proteinꢀ and βꢀarrestinꢀmediated pathways
can be pharmacologically modulated independently with functionally selective ligands.2, 3
Excellent recent studies provided structural insights into the phenomenon of biased signaling.4, 5
Ligandꢀinduced bias between G protein coupling and βꢀarrestin recruitment was also reported for
the dopaminergic drug aripiprazole and structurally related analogs.6ꢀ8
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Dopamine D2 and D3 receptors are known as valuable targets for the treatment of neurological
and psychiatric disorders involving Parkinson's disease, dyskinesia, restless legs syndrome and
schizophrenia.9ꢀ13 Whereas highly selective coupling to Gαo was demonstrated for the D3
subtype, promiscuity in G protein coupling was shown for D2 existing in the splice variants D2long
(D2L) and D2short (D2S).14, 15 Besides agonistꢀmediated inhibition of calcium channels, Go
signaling activates other intracellular effectors like potassium channels and MAPꢀkinases but
does not affect intracellular cAMP levels.16, 17 In contrast, coupling to Gi subtypes led to
inhibition of adenylyl cyclase and ERK activation.18ꢀ21 Coꢀexpression of D2 with the G proteins
Go and Gi indicated the formation of ligand – specific ternary complexes.15, 22 Because novel
types of agonists will have the potential to control dopamineꢀmediated pharmacology in a unique
way, our very recent efforts focused on the identification and optimization of functionally
selective dopaminergics. Starting from phenylpiperazine derivatives, which are known as
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privileged structures for dopamine receptor antagonists,23 we developed the functionally biased
dopamine D2 receptor ligand Nꢀ[3ꢀ[4ꢀ(2,3ꢀdihydrobenzofuranꢀ7ꢀyl)piperazinꢀ1ꢀ
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yl]propyl]pyrazolo[1,5ꢀa]pyridineꢀ3ꢀcarboxamide (FAUC350), which behaves as an antagonist
in the inhibition of cAMP accumulation and as a partial agonist in the stimulation of ERK1/2
phosphorylation.24 As a complement to these studies, we intended to investigate a family of
compounds with a dopamine agonist pharmacophore for their ability to differentiate between Goꢀ
and Giꢀmediated signaling. Our initial investigations using agonists with classical catecholꢀ
simulating functional groups indicated only a weak ability to differentiate between Goꢀ and Giꢀ
induced D2 receptor activation in a GTPγS assay (see, Supporting Information). Thus, we
intended to build our work on atypical bioisosteres such as conjugated enynes.25ꢀ29 Because we
were concerned that diasteric Go/Gi preferences of enantiomers might make it difficult to
interpret our data correctly, an chirospecific synthetic approach was chosen. In this study, we
present chemical synthesis and in vitro biological evaluations of dopaminergic enynes of type 1
and 2 (Figure 1) in enantiomerically pure form.
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Figure 1. Chiral resolution and structural modifications leading to enantiomerically pure target
compounds of type 1 and 2.
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Results and Discussion
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Synthesis. To investigate enantiospecific ligandꢀreceptor interactions of dopaminergic
alkynylcyclohexenylamines, a collection of derivatives was synthesized in enantiomerically pure
form by functionalization of the monopropylamines (R)ꢀ6 and (S)ꢀ6 (Scheme 1) that contain an
asymmetric carbon in the cyclohexene ring as a source of chirality. Since our previously
described reaction sequence for the preparation of racemic alkynylcyclohexenylamines29 was not
suitable to access secondary amines of type 6, an alternative pathway was elaborated. Our
synthesis started from the 1,4ꢀcyclohexanedione monoethylene acetal, which was converted to
the tertiary alcohol 3 by Grignard reaction. Subsequent hydrolysis of the acetal protecting group
and reductive amination of the resulting ketone 4 with propylamine and sodium
triacetoxyborohydride afforded the aminoalcohol 5, which was dehydrated using a reaction
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mixture of PPh3, I2 and imidazole to give the racemic secondary amine 6 in excellent overall
31
yield (52%).30,
To obtain the pure enantiomers (R)ꢀ6 and (S)ꢀ6, chiral resolution was
accomplished by preparative HPLC employing a polysaccharideꢀbased column (Chiralpak® ASꢀ
H) resulting in ee values greater 99%. For an unambiguous assignment of the absolute
configuration, we intended to obtain a high resolution crystal structure. Because attempts to grow
crystals of the pure enantiomers suitable for Xꢀray analysis failed, derivatization by acylation
with 3,5ꢀdinitrobenzoyl chloride was conducted to yield the readily crystallizable amide (S)ꢀ7
from the homochiral secondary amine.
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Scheme 1.a
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O
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O
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HO
HO
O
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c
O
N
NO2
NH
NH
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for
NO2
(S)-6
HO
(S)-7
(S)-6
+
(R)-6
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a Reagents and conditions: (a) ethynylmagnesium bromide, THF, 0 °C to rt, 2 h (90%); (b) 80%
HCOOH, CuSO4, rt, 16 h (79%); (c) propylamine, NaBH(OAc)3, AcOH, DCM, 0 °C to rt, 4 h
(80%); (d) PPh3, I2, imidazole, DCM, 16 h (94%); (e) chiral resolution by preparative HPLC
using Chiralpak ASꢀH column; (f) 3,5ꢀdinitrobenzoyl chloride, Et3N, DCM, 0 °C, 4 h (86%).
Xꢀray diffraction analysis of the benzamide (S)ꢀ7 (Figure 2) revealed the presence of two
independent molecules in the asymmetric unit. Both molecules clearly exhibit (S)ꢀconfiguration
at the stereogenic center but different conformation of the dinitrophenyl moiety (cf. Figs. S1 and
S2, supporting information). The crystal structure further indicated a halfꢀchair conformation of
the cyclohexene ring with an equatorially positioned carboxamide group for both of the
independent molecules.
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Figure 2. Thermal ellipsoid representation of the molecular structure of one out of two
independent molecules of the 3,5ꢀdinitrobenzamide (S)ꢀ7, determined by Xꢀray crystal structure
analysis (50% probability ellipsoids, hydrogen atoms drawn as spheres of arbitrary size).
The test compounds of type 1 and 2 were synthesized by reductive alkylation of the enantiopure
intermediates (R)ꢀ6 and (S)ꢀ6 with the Nꢀ(4ꢀhydroxybutyl)aryl carboxamides 8–13 (Scheme 2).
In detail, the starting compounds 8–13 were prepared by Nꢀacylation of 4ꢀaminobutanol with
commercially available biphenylꢀ4ꢀcarbonyl chloride or by CDIꢀmediated amidation of 4ꢀ
aminobutanol with pyrazolo[1,5ꢀa]pyridinyl, benzo[b]thiophenyl and ferrocenyl carboxylic acids
as well as the recently described 4ꢀ[3ꢀ(1ꢀbutylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)propoxy]ꢀ3ꢀmethoxybenzoic
acid.32 Subsequent oxidation of the hydroxyl group using pyridine sulfur trioxide furnished the
corresponding aldehydes,33 which were used without further purification to reductively alkylate
the secondary amines (R)ꢀ and (S)ꢀ6 in presence of sodium triacetoxyborohydride to afford the
test compounds (R)ꢀ/(S)ꢀ1 and (R)-/(S)ꢀ2aꢀ2e.
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Scheme 2.a
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a Reagents and conditions: (a) aqueous NaOH, biphenylꢀ4ꢀcarbonyl chloride, DCM, 0 °C to rt, 1
h or ArꢀCOOH, CDI, DMF, 50 °, 2 h, after cooling to rt: addition of 4ꢀaminobutanol, 2 h (58ꢀ
94%); (b) pyridine sulfur trioxide, Et3N, DCM/DMSO, 0 °C to rt, 1 h; (c) (R)ꢀ or (S)ꢀ6,
NaBH(OAc)3, DCM, rt, 3ꢀ5 h (36ꢀ71%).
Biological Investigations.
Studies on Metabolic Stability.
Dopamine receptor agonists including ropinirole (4ꢀ[2ꢀ(dipropylamino)ethyl]ꢀ1,3ꢀdihydroꢀ2Hꢀ
indolꢀ2ꢀone) and rotigotine ((S)ꢀ2ꢀ(NꢀpropylꢀNꢀ2ꢀthienylethylamino)ꢀ5ꢀhydroxytetralin), which
have been developed for the treatment of Parkinson’s disease, are known to be extensively
metabolized.34, 35 Due to a substantial firstꢀpass effect, rotigotine displays an oral bioavailability
of only 0.5%.36 Metabolism studies using isolated rat liver perfusion technique detected 15
different metabolites, whereas Nꢀdealkylation and hydroxylation were identified as major
metabolic pathways.37 Hence, we were intrigued by the question whether our newly developed
enyne derivatives display stability against oxidative biotransformation reactions. Due to its
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substantial metabolism, rotigotine was chosen as a positive control to validate the assay.
Interestingly, in vitro metabolism studies using rat liver microsomes revealed superior stability
for the alkynylcyclohexenylamine 1 (used in racemic form) when compared to the reference
agent rotigotine. After 15 min, only 41.8% of rotigotine remained unchanged, whereas the
enzymatic biotransformation of the enyne bearing dopaminergic 1 proceeded significantly slower
(Figure 3). More than 70% of the starting material could still be detected after an incubation time
of 60 min. Thus, the metabolism profile of our alkynylcyclohexenylamines is comparable to the
approved drug pramipexole, which is known to be cleared largely unchanged.38
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Figure 3. Oxidative metabolism profile of rotigotine and the biphenylcarboxamide 1.Results as
mean percentages of nonꢀmetabolized parent compound. Incubations were performed using 0.05
mg/mL of rotigotine or 1, pooled rat liver microsomes (1.0 mg/mL) in the presence of 1 mM
NADPH at 37 °C for 60 min. Aliquots were drawn after predetermined time intervals of 0, 15,
30, 45 and 60 min. All incubations were performed in duplicate.
LC/MS analysis of the slowly formed metabolites revealed Nꢀdepropylation as major metabolic
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pathway to give the derivative 14 (Figure 4).34,
Besides Nꢀdepropylation, the
biphenylcarboxamide 1 showed additional enzyme catalyzed cleavage of the ethynylcyclohexene
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residue resulting in the secondary amine 15 and oxidation of the ethynyl substituent to yield the
carboxylic acid 16.
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Figure 4. Biotransformation pathway of the biphenylcarboxamide 1.
Ligand Binding Experiments. To evaluate receptor binding of the enantiopure test compounds
of type 1 and 2, radioligand displacement assays were conducted. The binding affinity and
selectivity profiles of the target compounds were compared with those of the reference agent
quinpirole (Table 1).41 Binding data were generated by measuring the ability of the test
compounds to compete with [3H]spiperone for the cloned human dopamine receptor subtypes
D2L, D2S, D3, and D4.4 stably expressed in Chinese hamster ovary (CHO) cells. D1 receptor
affinities were determined utilizing porcine striatal membranes and the D1 selective radioligand
7ꢀchloroꢀ3ꢀmethylꢀ1ꢀphenylꢀ1,2,4,5ꢀtetrahydroꢀ3ꢀbenzazepinꢀ8ꢀol ([3H]SCH 23390). Because the
preferred binding of D2 agonists to the ternary complex is known to result in significantly
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different values for Ki high and Ki low, the high affinity binding site of D2L, selectively labeled by
the D2 agonist 2ꢀdipropylaminoꢀ7ꢀhydroxyꢀ1,2,3,4ꢀtetrahydronaphthalene ([3H]7ꢀOHꢀDPAT),
was also subjected to ligand competition experiments. In fact, all test compounds of type 1 and 2
showed high binding affinity resulting in Ki values between 0.53 and 5.5 nM. Eudysmic ratios
for the enantiomers were close to one except for the triazolyl substituted dialkoxybenzamide 2c
that gave a factor of approximately 8 when the (S)ꢀenantiomer showed higher affinity. Our
subsequent investigations were directed to comprehensive evaluation of the enantiomers of (R)ꢀ1
and (S)ꢀ1, which we described in racemic form (racꢀ1, FAUC 460) earlier, involving the above
mentioned monoaminergic GPCRs.29 The data indicate an agreement of the binding profiles and
only minor stereospecific differences between the optical antipodes. Both enantiomers displayed
substantial D3 affinity and selectivity. Formal exchange of the biphenylcarboxamide moiety by a
pyrazolo[1,5ꢀa]pyridineꢀ3ꢀylcarboxamide unit led to more balanced binding profiles for the
heterocyclic bioisosteres (R)ꢀ2b and (S)ꢀ2b. Thus, Ki values between 7.1 and 22 nM were
determined for the D2ꢀlike subtypes D2L, D2S, D3 and D4.4 indicating significant receptor
recognition in low concentration. While the 2ꢀsubstituted isomers (R)ꢀ2a and (S)ꢀ2a and its
benzothiophene analogs (R)ꢀ2d and (S)ꢀ2d displayed D3 preference, the ferrocenylcarboxamides
(R)ꢀ2e and (S)ꢀ2e revealed subnanomolar affinity for the two subtypes D3 and D4.4. The binding
pattern of the dialkoxybenzamides (R)ꢀ2c and (S)ꢀ2c indicated a balanced binding to D2L, D2S
and D3 with Ki values between 1.0 and 8.0 nM. All test compounds of type 1 and 2 showed only
moderate to poor affinity to the porcine receptors of the adrenergic, dopaminergic and
serotonergic subtypes α1, D1 and 5ꢀHT2, respectively. However, Kiꢀvalues in the nanomolar
range were observed for 5ꢀHT1A, when the ferrocene derivatives (R)ꢀ2e and (S)ꢀ2e gave even
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singleꢀdigit values. Worthy of note is also the poor ability of enantiomeric test compounds to
differentially bind the investigated receptors.
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Table 1. Receptor Binding Data of (R)ꢀ/(S)-1 and (R)ꢀ/(S)ꢀ2a–2e Compared to the Reference Compound Quinpirole Utilizing Human
D2L, D2S, D3 and D4.4 Receptors as well as Porcine D1, 5ꢀHT1A, 5ꢀHT2 and α1 Receptors.
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Ki values [nM ± SEM]a
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[³H]SCH23390 [³H]7ꢀOHꢀDPAT
[³H]spiperone
[³H]WAY100635 [³H]ketanserin
[³H]prazosin
pα1
compd
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pD1
D2L
D2S
D3
D4.4
p5ꢀHT1A
6300 ± 1100
140 ± 28b
280 ± 42b
33 ± 10
p5ꢀHT2
25000 ± 9200
190 ± 35b
quinpirole 87000 ± 6000
18 ± 3.2
260 ± 38
11 ± 2.1
14 ± 2.4
63 ± 13
70 ± 18
18 ± 3.1
21 ± 5.5
230 ± 68
280 ± 91
9.3 ± 1.1
14 ± 2.5
8.8 ± 2.2
3.9 ± 1.6
33 ± 9.7
16 ± 2.8
13 ± 2.3
16 ± 3.0
15 ± 3.7
8.5 ± 1.4
5.8 ± 1.1
11 ± 3.2
37 ± 3.5
110 ± 14
14 ± 3.7
22 ± 6.0
59 ± 18
30 ± 8.1
55000 ± 6200
220 ± 14b
(R)-1
(S)-1
300 ± 64b
190 ± 57
0.60 ± 0.27
1.2 ± 0.82
5.5 ± 1.5b
3.9 ± 1.0
0.55 ± 0.13
1.1 ± 0.33
4.2 ± 2.4
0.53 ± 0.13
3.1 ± 1.3
2.0 ± 0.67
1.1 ± 0.55
1.1 ± 0.50
0.12 ± 0.035
0.17 ± 0.036
3.2 ± 0.33
10 ± 1.5
210 ± 35b
130 ± 14b
(R)-2a
(S)-2a
(R)-2b
(S)-2b
(R)-2c
(S)-2c
(R)-2d
(S)-2d
(R)-2e
(S)-2e
25000 ± 2800
45000 ± 6400b
36000 ± 3500b
33000 ± 4900b
18000 ± 6400b
13000 ± 5100b
5500 ± 70b
18000 ± 9200b
35000 ± 8400b
16000 ± 2100
13000 ± 710b
28000 ± 13000b
8500 ± 3500b
2100 ± 210b
1300 ± 570b
5800 ± 520
1100 ± 520b
2900 ± 1900b
730 ± 290
1500 ± 710b
1800 ± 350b
620 ± 160b
170 ± 57b
66 ± 18
150 ± 35
23 ± 2.1
7.1 ± 3.8
7.8 ± 1.8
8.0 ± 0.91
6.0 ± 0.83
32 ± 4.6
22 ± 4.6
14 ± 3.0
14 ± 3.6
8.6 ± 0.66
10 ± 1.6
48 ± 17
4.3 ± 1.7
1.0 ± 0.30
110 ± 17b
160 ± 42b
40 ± 3.5b
65 ± 25b
0.24 ± 0.069 5.3 ± 0.91
0.43 ± 0.17 4.8 ± 1.4
4700 ± 640b
19000 ± 4300
16000 ± 1600
200 ± 21b
0.63 ± 0.15 0.89 ± 0.17
4.0 ± 1.0
1400 ± 230
0.34 ± 0.085 0.73 ± 0.13
3.7 ± 1.4
3500 ± 450
280 ± 73
a Ki values in nM ± standard error of mean derived from 3ꢀ10 experiments each done in triplicate. Ki values ± standard deviation
derived from two individual experiments each done in triplicate.
b
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Functional Experiments. Functional selectivity explains the ability of a ligand to preferentially
activate coupling of a GPCR with a subset of signal transducers. To investigate the capability of
our newly synthesized test compounds to perform balanced or biased receptor activation, the
pertussis toxin (PTX) insensitive G protein subunits Gαo or Gαi42 were coꢀtransfected with the
dopamine receptor isoforms D2L and D2S. We intended to measure D2L and D2S receptorꢀ
mediated incorporation of [35S]GTPγS when our initial investigations were focused on the
characterization of the reference agent quinpirole (Figure 5).
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a) D2short
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-4
quinpirole [logM]
(S)-2a [logM]
(S)-2c [logM]
Figure 5. Doseꢀresponse curves of the agonist stimulated [35S]GTPγS binding at the dopamine
receptor subtypes D2S(a) and D2L (b). Membranes from cells transiently transfected with D2S or
D2L and the PTXꢀinsensitive G proteins Gαo1 or Gαi2 were stimulated with (S)ꢀ2a, (S)ꢀ2c or the
reference agonist quinpirole. The difference between the areas under the curves (AUCs) for the
graphs derived from the D2+Gαo1 (blue) system and the ones displaying D2+Gαi2 (red) is
highlighted in light grey and indicate a selectivity of Gαo1 over Gαi2. The difference of AUCs for
(S)ꢀ2a at D2L (dark grey) displays a selectivity of Gαi2 over Gαo1.
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The doseꢀresponse curves for D2S receptors coꢀtransfected either with the G protein subunit Gαo
or Gαi showed very similar potency and ligand efficacy when receptor activation was promoted
by quinpirole. For coꢀtransfections of D2L with Gαo or Gαi, the same observation was made. In
both cases, the (grey) area indicating the difference between the areas under the curve (AUC) of
the Goꢀmediated response (blue) and the Giꢀmediated activation (red) displays only a minor bias
for one of the coupling opportunities, namely for Go.
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In contrast, the ability of the test compounds to differentiate between Goꢀ and Giꢀmediated
receptor activation was considerably more pronounced. Thus, the large area between the doseꢀ
response curves of the 2ꢀazaindolyl substituted test compound (S)ꢀ2a shows that the ligand
efficacy for D2S receptors coꢀtransfected with Gαo is substantially higher than for the D2SꢀGαi
system. Interestingly, an inverse behavior was observed for (S)ꢀ2aꢀpromoted activation of D2L
when the Giꢀinduced response was superior. Besides maximal ligand efficacy of the test
compounds (Emax), functional selectivity can be also induced by different potencies. An
impressive example is the triazolylalkyoxyphenyl substituted test compound (S)ꢀ2c causing
different AUCs by the ability of the test compound to promote Gαo activation at much lower
concentrations than Gαi coupling. This property could be observed for both test systems
expressing D2L and D2S.
Because we thought that comparison of the individual test compounds’ ꢁAUCs with those of our
traditional reference agent quinpirole was a good measure for functional selectivity, we
calculated their relationships by determining the difference of ꢁAUCs for the test compound and
the reference agent and dividing the result by the ꢁAUC value of the reference agent. Thus, a
value of 0 means that the compound has a profile that is identical to quinpirole, positive
functional selectivity indices (FSIs) indicate a bias for Go and negative FSI values implicate Gi
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selectivity. Table 2 shows that all test compounds displayed substantial bias for D2Sꢀpromoted Go
activation with functional selectivity indices between 1.23 and 3.85. Enantiospecificity of the
ligandꢀpromoted signaling bias was low for all enyne derivatives that we investigated, indicating
that the overall shape and the ligand recognition of both enantiomers at the D2S binding site not
only results in a similar ligand affinity but also in a comparable stabilization of individual ternary
complexes.
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Table 2. Intrinsic Activities of (R)ꢀ/(S)ꢀ1 and (R)ꢀ/(S)ꢀ2a-2e Determined at the Dopamine
Receptor Subtypes D2L and D2S by Measuring the Binding of [35S]GTPγS and the Recruitment of
βꢀArrestinꢀ2 After Stimulation of the D2S Receptor.
D2L
D2S
[35S]GTPγS binding
[35S]GTPγS binding
βꢀarrestina
compd
FSIc
ꢀ0.41
2.85
0.93
ꢀ2.37
1.67
1.26
1.78
4.30
ꢀ0.07
ꢀ0.15
ꢀ0.33
0.26
FSIc
2.65
1.73
2.12
2.08
1.23
2.85
Emax
d
b
b
Gαi2 (Emax)b Gαo1 (Emax
)
Gαi2 (Emax)b Gαo1 (Emax
)
(R)-1
(S)-1
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53
75
90
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40
51
57
45e
78e
50
77
53
59
68
87
31
41
71
85
62
62
67
37
63
78
94
103
60
69
81
87
79 ± 6.7
62 ± 7.9
68 ± 4.6
62 ± 4.9
76 ± 4.0
73 ± 6.1
23
(R)-2a
(S)-2a
(R)-2b
(S)-2b
(R)-2c
(S)-2c
(R)-2d
(S)-2d
(R)-2e
(S)-2e
53e
62
24
66e
57
3.38 100 ± 6.5
72
3.85
1.69
1.85
1.69
1.73
99 ± 3.6
51 ± 5.6
57 ± 4.8
89 ± 3.7
96 ± 4.6
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31
71
103e
a D2Sꢀmediated recruitment of βꢀarrestinꢀ2 determined with the PathHunter® Assay; b
maximum stimulation of [35S]GTPγS binding derived from saturation concentrations of the test
compounds from the pooled curve of three to six individual experiments each done in triplicate;
Emax in [%] relative to the effect of the reference agonist quinpirole; c functional selectivity index
(FSI) calculated using the formula (ꢀAUC[test(GαoꢀGαi)]ꢀꢀAUC[ref(Gαoꢀ
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Gαi)])/ꢀAUC[ref(GαoꢀGαi)]; d D2S mediated recruitment of βꢀarrestin in [% ± SEM] analyzed
from 6ꢀ8 individual experiments each done in triplicate at a concentration of 10ꢀ5 M relative to
the effect of the reference agonist quinpirole; e no gain of saturation in [35S]GTPγS binding up to
a concentration of 10ꢀ4 M, maximum effect determined at 10ꢀ4 M.
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For D2Lꢀpromoted activation the variability of the functional selectivity indices was much higher
varying between ꢀ2.37 for the (S)ꢀenantiomer of the 2ꢀsubstituted azaindole 2a and 4.30 for the
triazolylalkoxyꢀsubstituted benzamide (S)ꢀ2c. For D2L, functional selectivity turned out to be
strongly dependent on the nature of the carboxamide substituent. Only six of the twelve
compounds investigated showed FSIs higher than 1 or below ꢀ1. It is interesting of note that the
2ꢀsubstituted azaindole carboxamide 2a showed significant enantiospecific coupling bias when
the (R)ꢀisomer displayed selectivity for Go coupling and the (S)ꢀenantiomer gave a higher
potency for Giꢀpromoted activation. In contrast, preferential Go activation of the biphenyl
carboxamide 1 depends on the (S)ꢀconfiguration at the stereogenic center.
Receptor mediated recruitment of βꢀarrestinꢀ2 was determined utilizing assay technology that is
based on the complementation of enzyme fragments of βꢀgalactosidase (βꢀgal). For this purpose,
HEKꢀ293 cells stably expressing a fusion protein of βꢀarrestin and the Nꢀterminal fragment of βꢀ
gal were transiently transfected with D2S fused to the Cꢀterminal fragment of the enzyme. After
stimulation of the cells with the appropriate agonists, βꢀarrestin recruitment could be monitored
by measuring the occurring chemiluminescence. To investigate whether the synthesized enynes
show a G proteinꢀindependent signaling, we determined the D2Sꢀmediated βꢀarrestin recruitment
at 10 ꢂM for (R)ꢀ/(S)ꢀ1 and (R)ꢀ/(S)ꢀ2a-2e in comparison to the effect of quinpirole. Significant
βꢀarrestin recruitment of 50 to 100% relative to the reference compound quinpirole was
determined for the test compounds of type 1 and 2. Highest Emax values were observed for the
triazolylalkoxyꢀsubstituted benzamide (R)/(S)-2c. Whereas in most cases ligand specific βꢀ
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arrestin recruitment was similar to the intensity of G protein activation, the biphenyl
carboxamide (R)-1 and the 2ꢀbenzothiophenyl carboxamide (R)-2d showed substantial bias for
βꢀarrestin recruitment over Gi activation (Gi : βꢀarrestin = 57ꢀ60%). In contrast, the 2ꢀsubstituted
azaindole (S)ꢀ2a preferred βꢀarrestin recruitment compared to Go coupling (Go : βꢀarrestin = 57ꢀ
60%).
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Ligand receptor interactions
To explore the relationship between functional selectivity and the individual contacts that are
formed between the ligands and the receptor binding site, docking studies were performed. Thus,
we established a homology model of the D2 receptor based on the crystal structure of the D3
receptor43 and compared the binding modes of our reference compound quinpirole with the
functionally selective test compound (S)-2c (Figure 6). Quinpirole and the enyne moiety of (S)-
2c occupy the orthosteric binding pocket formed by residues of transmembrane helices (TMs) 3,
5, 6, 7 and extracellular loop 2 (EL2) showing hydrophobic interactions to V3.33, C3.36, F5.38,
W6.48, F6.51, F6.52, H6.55 and F7.38 as well as I183 and I184 of EL2 (Figure 6, grey sticks).
The propyl groups of both ligands are enclosed in a pocket comprising residues D3.32, W6.48,
F6.52, Y7.35, F7.38, T7.39 and Y7.43. The ligands exhibit the canonical salt bridge between
their positively charged nitrogen and the carboxylate of D3.32. In contrast to quinpirole, the
functionally selective agonist (S)-2c exhibits additional interactions to residues that are located at
an extended binding pocket closer to the extracellular surface of the receptor and spanned by TM
2, 3, 5, 6, 7 and EL2 (Figure 6, blue sticks). The amide and the methoxyphenyl moieties of (S)-
2c are surrounded by residues V2.61, L2.64, E2.65, C182 (EL2), F3.28, V3.29, Y7.35, S7.36 and
T7.39. This cavity has been shown to be connected to conformational changes upon activation
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and to be involved in functionally selective signaling.44 The triazole moiety of (S)-2c bearing the
terminal butyl chain points into the direction of TM 5, thereby forming a lid over the
extracellular entrance of D2 and establishing interactions to residues I183 and A185 of EL2,
N6.58, I6.59, P7.32 and Y7.35. Recent crystal structures of 5ꢀHT1B45 and 5ꢀHT2B5 receptors
suggested residues homologous to N6.58 and I6.59 of D2 to contribute to adjusting a receptor
conformation that specifically favors binding of one signal transducer over the other. Within the
structure of 5ꢀHT2B, the coꢀcrystallized ligand ergotamine has been shown to behave as a
functionally selective agonist inducing βꢀarrestin signaling to a higher extent than coupling to G
proteins and that this effect is more pronounced compared to smaller ligands like lysergic acid
diethylamide (LSD) and methylergonovine.5 As ergotamine connects both, the orthosteric and an
extended binding pocket, it is capable of stabilizing a distinct receptor conformation that
specifically interferes with G protein binding. Comparable to ergotamine, our functionally
selective D2 agonist (S)-2c occupies the orthosteric and an extended binding pocket (Figure 6,
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Supporting Information), which might be the origin for the preference of receptor conformations
associated with an atypical signal transducing profile.
Figure 6. Ligandꢀreceptor interactions for quinpirole (a) and (S)-2c (b). The backbone of the D2
model is represented as light grey ribbons, quinpirole and (S)-2c are shown as orange and green
sticks, respectively. Amino acids stabilizing both ligands are visualized as grey sticks, whereas
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additional amino acids comprising an extended binding pocket of (S)-2c are highlighted as blue
sticks. The figure was prepared using PyMOL.
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Conclusion
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Functional selectivity explains the capacity of a ligand to preferentially activate coupling of a
GPCR with a subset of signal transducers. Enantiomerically pure dopaminergics of type 1 and 2
incorporating a conjugated enyne as an atypical catecholꢀsimulating moiety were investigated.
Metabolism studies revealed the atypical pharmacophores to be stable against oxidative
biotransformation reactions. Radioligand binding studies indicated substantial affinity to D2ꢀlike
receptors.
The test compounds were studied for their ability to differentially activate distinct signaling
pathways. Measurement of D2Lꢀ and D2Sꢀmediated [35S]GTPγS incorporation in the presence of
coexpressed PTXꢀinsensitive Gαo and Gαi subunits showed significantly biased receptor
activation for several test compounds. Compared to the reference agent quinpirole that displayed
only a minor bias for Go coupling the 2ꢀazaindolyl carboxamide (S)ꢀ2a exhibited substantial
selectivity for D2Sꢀpromoted Go activation over Gi coupling. The highest functional selectivity
was determined for the triazolylalkoxyꢀsubstituted benzamide (S)ꢀ2c that displayed higher
potency for Go activation than for Gi coupling at the D2L subtype. Compared to the reference
compound quinpirole, significant bias for βꢀarrestin recruitment over Gi activation was observed
for the biphenylcarboxamide (R)ꢀ1 and the 2ꢀbenzothiophenyl carboxamide (R)ꢀ2d, whereas the
2ꢀsubstituted azaindole (S)ꢀ2a preferred βꢀarrestin recruitment compared to Go coupling. These
observations demonstrate the feasibility of differentially activating distinct signaling pathways
by biased agonists. Further investigations are needed to determine the consequences of biased
agonism in vivo and to elucidate structural properties that trigger functional selectivity.
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Experimental section
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Chemistry. All reactions were carried out under nitrogen atmosphere. Reagents and dry
solvents were of commercial quality and were used as purchased. MS were run on a JEOL JMSꢀ
GC Mate II spectrometer by EI (70 eV) with solid inlet or a Bruker Esquire 2000 by APC or ES
ionization. HRꢀEIMS was run on a JOEL JMSꢀGC Mate II using PeakꢀMatching (M/ꢁM >
5000). NMR spectra were obtained on a Bruker Avance 360 or a Bruker Avance 600
spectrometer relative to TMS in the solvents indicated (J value in hertz). Melting points were
determined with a MELꢀTEMP II melting point apparatus (Laboratory Devices, USA) in open
capillaries and are given uncorrected. IR spectra were performed on a Jasco FT/IR 410
spectrometer. Purification by flash chromatography was performed using Silica Gel 60; TLC
analyses were performed using Merck 60 F254 aluminum sheets and analyzed by UV light, (254
nm), in the presence of iodine or by spraying with ninhydrin reagent. Analytical HPLC was
performed on Agilent 1100 HPLC systems employing a VWL detector or on Agilent 1200 HPLC
systems using a DAD detector. As column, a ZORBAX ECLIPSE XDBꢀC8 (4.6 mm x 150 mm,
5 ꢃm) was used. HPLC purity was measured using following binary solvent systems: system A,
eluent CH3OH in 0.1% aqueous formic acid, 10% to 100% CH3OH in 15 min, 100% for 6 min,
flow rate 0.5 mL/min, λ 254 nm or 220 nm; system B, eluent CH3CN in 0.1% aqueous formic
acid, 5% to 80% in 18 min, 80% to 95% in 2 min, 95% for 2 min, flow rate 0.5 mL/min, λ 254
nm or 220 nm. The purity of all test compounds and key intermediates was determined to be >
95%. Preparative HPLC was performed on Agilent 1100 HPLC systems employing a VWL
detector.
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(R)-N-[4-[(4-Ethynylcyclohex-3-enyl)propylamino]butyl]biphenyl-4-carboxamide ((R)-1).
To an iceꢀcooled solution of 8 (34.7 mg; 0.13 mmol) in CH2Cl2/DMSO (2 mL/1 mL) was added
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Et3N (72 ꢃL; 0.516 mmol) and SO3ꢄpyridine (102.5 mg; 0.34 mmol). After stirring at room
temperature for 1 h, the mixture was diluted with H2O (10 mL) and extracted with EtOAc (4 x 20
mL). The combined organic layers were dried with MgSO4 and concentrated. The resulting oil
was diluted with CH2Cl2 (5 mL) and slowly added to an iceꢀcooled solution of (R)-6 (7.5 mg;
0.05 mmol) in CH2Cl2 (5 mL). Then, NaBH(OAc)3 (48.6 mg; 0.23 mmol) was added in one
portion at 0 °C. The suspension was allowed to warm to room temperature and stirred for 4 h.
The reaction was terminated by adding saturated aqueous NaHCO3 and the aqueous layer was
extracted with CH2Cl2. The combined organic layers were dried (MgSO4) and the solvent was
removed under reduced pressure. Purification of the resulting residue by HPLC (CH3CN–H2O +
0.1% HCOOH) yielded (R)-1 as white solid (6.6 mg; 35%): analytical data according to the
literature.29 [α]26D = + 38.6° (c 0.4, MeOH). (S)-1 (7.5 mg; 36%) [α]26D = ꢀ 40.4° (c 0.5, MeOH)
was prepared from (S)-6 as described above for (R)-1.
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(R)-N-[4-[(4-Ethynylcyclohex-3-enyl)propylamino]butyl]pyrazolo[1,5-a]pyridine-2-
carboxamide ((R)-2a). Compound (R)-2a was prepared according to the protocol of (R)-1 using
a solution of 9 (45.4 mg; 0.19 mmol) in CH2Cl2/DMSO (2 mL/1 mL), Et3N (0.14 mL; 0.98
mmol) and SO3ꢄpyridine (237.8 mg; 0.78 mmol) as well as (R)-6 (15.9 mg; 0.1 mmol) and
NaBH(OAc)3 (51.4 mg; 0.24 mmol). The crude compound was purified by flash chromatography
(CH2Cl2 + 1–2% MeOH) to afford (R)-2a as yellowish oil (4.4 mg; 12%). IR: 3307, 2931, 1664,
1552, 1513, 1475, 1326, 1257, 1076, 804, 769, 740 cmꢀ1. 1HꢀNMR (600 MHz, CDCl3) δ 0.86 (t,
J = 7.4 Hz, 3 H), 1.40ꢀ1.70 (m, 7 H), 1.84ꢀ1.89 (m, 1 H), 2.04ꢀ2.12 (m, 1 H), 2.18ꢀ2.30 (m, 3 H),
2.37ꢀ2.54 (m, 4 H), 2.77ꢀ2.84 (m, 2 H), 3.47ꢀ3.51 (m, 2 H), 6.13ꢀ6.16 (m, 1H), 6.85 (ddd, J =
1.4, 6.9, 6.9 Hz, 1 H), 7.05 (s, 1 H), 7.14 (ddd, J = 1.1, 6.7, 8.9 Hz, 1 H), 7.17ꢀ7.21 (m, 1 H),
7.58 (ddd, J = 1.2, 1.2, 8.9 Hz, 1 H), 8.37 (ddd, J = 1.1, 2.1, 7.1 Hz, 1 H). 13CꢀNMR (360 MHz,
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CDCl3) δ 11.82, 21.82, 24.82, 26.10, 27.54, 28.19, 30.09, 39.12, 50.02, 52.40, 55.43, 74.86,
84.94, 97.88, 113.53, 119.24, 119.59, 123.63, 128.38, 135.51, 141.34, 148.03, 162.11. HRꢀEIMS
calcd 378.2420; found 378.2419. [α]27D = + 29.4° (c 0.2, CHCl3). (S)-2a (6.8mg; 16%) [α]27D = ꢀ
29.6° (c 0.5, CHCl3) was prepared from (S)-6 (18.4 mg; 0.11 mmol) as described above for (R)-
2a.
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(R)-N-[4-[(4-Ethynylcyclohex-3-enyl)propylamino]butyl]pyrazolo[1,5-a]pyridine-3-
carboxamide ((R)-2b). Compound (R)-2b was prepared according to the protocol of (R)-1 using
a solution of 10 (124.6 mg; 0.53 mmol) in CH2Cl2/DMSO (2 mL/1 mL), Et3N (0.37 mL; 2.67
mmol) and SO3ꢄpyridine (654.1 mg; 2.14 mmol) as well as (R)-6 (28.9 mg; 0.18 mmol) and
NaBH(OAc)3 (93.7 mg; 0.44 mmol). The crude compound was purified by HPLC (MeOH–H2O
+ 0.1% HCOOH) to afford (R)-2b as yellowish oil (5.4 mg; 8%). IR: 3308, 3094, 3026, 2930,
2862, 2806, 2092, 1637, 1555, 1531, 1468, 1442, 1367, 1334, 1273, 1247, 1218, 1175, 1151,
1123, 1078, 1011, 975, 919, 891, 828 cmꢀ1. 1HꢀNMR (360 MHz, CDCl3) δ 0.85 (t, J = 7.3 Hz, 3
H), 1.38ꢀ1.70 (m, 7 H), 1.81ꢀ1.88 (m, 1 H), 2.00ꢀ2.12 (m, 1 H), 2.15ꢀ2.29 (m, 3 H), 2.36ꢀ2.43 (m,
2 H), 2.49 (ddd, J = 1.9, 7.1, 7.1 Hz, 2 H), 2.75ꢀ2.83 (m, 2 H), 3.48 (ddd, J = 5.8, 7.0, 7.0 Hz, 2
H), 6.02ꢀ6.08 (m, 1H), 6.12ꢀ6.16 (m, 1 H), 6.91 (ddd, J = 1.5, 6.9, 6.9 Hz, 1 H), 7.34 (ddd, J =
1.1, 6.8, 8.9 Hz, 1 H), 8.12 (s, 1 H), 8.31 (ddd, J = 1.2, 1.2, 8.9 Hz, 1 H), 8.48 (ddd, J = 1.1, 1.1,
13
7.0 Hz, 1 H). CꢀNMR (600 MHz, CDCl3) δ 11.87, 22.05, 24.97, 26.51, 27.82, 28.28, 30.16,
39.33, 50.04, 52.54, 55.36, 74.82, 85.00, 106.95, 113.56, 119.60, 119.65, 126.31, 128.79, 135.63,
140.14, 140.62, 163.26. HRꢀEIMS calcd 378.2420; found 378.2420. [α]27 = + 24.2° (c 0.3,
D
CHCl3). (S)-2b (20.6mg; 39%) [α]27 = ꢀ 24.9° (c 1.7, CHCl3) was prepared from (S)-6 (22.5
D
mg; 0.14 mmol) as described above for (R)-2b.
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(R)-4-[3-(1-Butyl-1H-1,2,3-triazol-4-yl)propoxy]-N-[4-[(6-ethynylcyclohex-3-
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6
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8
enyl)propylamino]butyl]-3-methoxybenzamide ((R)-2c). Compound (R)-2c was prepared
according to the protocol of (R)-1 using a solution of 11 (74.8 mg; 0.19 mmol) in CH2Cl2/DMSO
(2 mL/1 mL), Et3N (0.13 mL; 0.93 mmol) and SO3ꢄpyridine (226.2 mg; 0.74 mmol) as well as
(R)-6 (12 mg; 0.074 mmol) and NaBH(OAc)3 (47 mg; 0.22 mmol). The crude compound was
purified by flash chromatography (CH2Cl2 + 1% MeOH) to afford (R)-2c as yellowish oil (25.6
mg; 63%). IR: 3286, 2933, 2108, 1637, 1581, 1544, 1506, 1465, 1268, 1224, 1130, 1029, 761
cmꢀ1. 1HꢀNMR (360 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 3 H), 0.94 (t, J = 7.3 Hz, 3 H), 1.20ꢀ1.38
(m, 2 H), 1.44ꢀ1.71 (m, 7 H), 1.81ꢀ1.97 (m, 3 H), 2.08ꢀ2.33 (m, 6 H), 2.43ꢀ2.64 (m, 4 H), 2.81 (s,
1 H), 2.85ꢀ2.97 (m, 3 H) 3.44ꢀ3.49 (m, 2 H), 3.92 (s, 3 H), 4.11 (t, J = 6.4 Hz, 2 H), 4.31 (t, J =
7.3 Hz, 2 H), 6.10ꢀ6.15 (m, 1 H), 6.53 (s, 1 H), 6.85 (d, J = 8.4 Hz, 1 H), 7.25ꢀ7.29 (m, 1 H),
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7.30 (s, 1 H), 7.46 (d, J = 2.0 Hz, 1 H). CꢀNMR (600 MHz, CD3OD) δ 11.84, 13.43, 19.71,
21.83, 22.09, 24.89, 26.23, 27.53, 28.18, 28.67, 30.11, 32.29, 39., 49.90, 50.03, 52.47, 55.50,
56.13, 68.04, 74.94, 84.89, 111.21, 111.88, 119.11, 119.67, 120.80, 127.60, 135.37, 147.00,
149.37 151.08, 167.12. HRꢀEIMS calcd 549.3679; found 549.3681. [α]27 = + 18.9° (c 2.2,
D
CHCl3). (S)-2c (27.1mg; 64%) [α]27D = ꢀ 18.5° (c 1.3, CHCl3) was prepared from (S)-6 (12.6 mg;
0.08 mmol) as described above for (R)-2c.
(R)-N-[4-[(4-Ethynylcyclohex-3-enyl)propylamino]butyl]benzo[b]thiophene-2-
carboxamide (R)-2d. Compound (R)-2d was prepared according to the protocol of (R)-1 using a
solution of 12 (33.9 mg; 0.16 mmol) in CH2Cl2/DMSO (2 mL/1 mL), Et3N (0.11 mL; 0.8 mmol)
and SO3ꢄpyridine (195.6 mg; 0.64 mmol) as well as (R)-6 (13.1 mg; 0.08 mmol) and
NaBH(OAc)3 (42.4 mg; 0.2 mmol). The crude compound was purified by flash chromatography
(CH2Cl2 + 2% MeOH) to afford (R)-2d as colorless oil (20.4 mg; 65%). IR: 3307, 3062, 3026,
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2931, 2868, 2810, 2092, 1626, 1563, 1544, 1509, 1470, 1457, 1432, 1375, 1338, 1298, 1247,
1207, 1179, 1158, 1073, 974, 916, 868, 835 cmꢀ1. 1HꢀNMR (600 MHz, CDCl3) δ 0.86 (t, J = 7.4
Hz, 3 H), 1.41ꢀ1.63 (m, 7 H), 1.84ꢀ1.89 (m, 1 H), 2.05ꢀ2.13 (m, 1 H), 2.18ꢀ2.30 (m, 3 H), 2.38ꢀ
2.55 (m, 4 H), 2.79ꢀ2.85 (m, 2 H), 3.45ꢀ3.51 (m, 2 H), 6.13ꢀ6.15 (m, 1 H), 6.47 (s, 1 H), 7.38ꢀ
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7.43 (m, 2 H), 7.77 (s, 1 H), 7.82ꢀ7.86 (m, 2 H). CꢀNMR (600 MHz, CDCl3) δ 11.85, 21.84,
24.92, 26.21, 27.46, 28.13, 30.12, 40.07, 49.97, 52.52, 55.51, 74.94, 84.89, 119.68, 122.69,
124.87, 124.97, 125.11, 126.22, 135.35, 138.65, 139.13, 140.73, 162.27. HRꢀEIMS calcd
394.2079; found 394.2079. [α]24 = + 35.8° (c 1.0, CHCl3). (S)-2d (23.7 mg; 71%) [α]24 = ꢀ
D
D
33.3° (c 0.7, CHCl3) was prepared from (S)-6 (13.9 mg; 0.085 mmol) as described above for (R)-
2d.
(R)-N-[4-[(4-Ethynylcyclohex-3-enyl)propylamino]butyl]ferrocene carboxamide (R)-2e.
Compound (R)-2e was prepared according to the protocol of (R)-1 using a solution of 13 (33.5
mg; 0.11 mmol) in CH2Cl2/DMSO (2 mL/1 mL), Et3N (80 ꢃL; 0.56 mmol) and SO3ꢄpyridine
(135.7 mg; 0.44 mmol) as well as (R)-6 (13.1 mg; 0.08 mmol) and NaBH(OAc)3 (42.4 mg; 0.2
mmol). The crude compound was purified by flash chromatography (hexane/EtOAc 2:1) to
afford (R)-2e as orange solid (24.3 mg; 68%). Mp 98 °C. IR: 3309, 3093, 2926, 2854, 2093,
1725, 1630, 1541, 1456, 1436, 1377, 1342, 1300, 1219, 1180, 1105, 1075, 1024, 1001, 973, 915,
820 cmꢀ1. 1HꢀNMR (360 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 3 H), 1.38ꢀ1.64 (m, 7 H), 1.81ꢀ1.89
(m, 1 H), 2.00ꢀ2.13 (m, 1 H), 2.16ꢀ2.29 (m, 3 H), 2.36ꢀ2.52 (m, 4 H), 2.73ꢀ2.84 (m, 2 H), 3.35ꢀ
3.41 (m, 2 H), 4.19 (s, 5 H), 4.33 (t, J = 1.9 Hz, 2 H), 4.64 (t, J = 1.9 Hz, 2 H), 5.74 (m, 1 H),
6.13ꢀ6.18 (m, 1 H). 13CꢀNMR (600 MHz, CDCl3) δ 11.88, 22.13, 25.01, 26.53, 27.91, 28.36,
30.18, 39.49, 50.06, 52.51, 55.35, 68.01, 69.69, 70.26, 74.81, 76.49, 85.02, 119.61, 135.69,
170.01. HRꢀEIMS calcd 446.2032; found 446.2031. [α]23D = + 30.8° (c 1.0, CHCl3). (S)-2e (13.8
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mg; 29%) [α]23 = ꢀ 28.4° (c 1.0, CHCl3) was prepared from (S)-6 (26.1 mg; 0.16) as described
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4
D
5
6
above for (R)-2e.
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8-Ethynyl-1,4-dioxaspiro[4.5]decan-8-ol (3). To a diluted 0.5 molar solution of
ethynylmagnesium bromide (154 mL; 52 mmol) in THF (50 mL) a solution of 1,4ꢀ
dioxaspiro[4.5]decanꢀ8ꢀon (5.42 g; 34.7 mmol) in THF (20 mL) was added dropwise at 0 °C.
This reaction mixture was continuously stirred for 2 h at room temperature. After addition of
saturated aqueous NH4Cl the mixture was extracted with Et2O. The combined organic layers
were dried over MgSO4 and concentrated. The residue was purified by flash chromatography
(hexane/EtOAc 3:1) to give 3 as a yellowish oil (5.69 g, 90% yield).46 IR: 3430, 3284, 2956,
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2884, 1702, 1436, 1367, 1253, 1162, 1105, 1033, 973, 773, 665, 549, 516 cmꢀ1. HꢀNMR (600
MHz, CDCl3) δ 1.76ꢀ1.84 (m, 4 H), 1.90ꢀ1.95 (m, 2 H), 1.96ꢀ2.02 (m, 3 H), 2.48 (s, 1 H), 3.95
(s, 4 H). 13CꢀNMR (360 MHz, CDCl3) δ 31.28, 37.04, 64.29, 64.34, 67.22, 72.04, 86.94, 107.83.
HRꢀEIMS calcd 182.0943; found 182.0942.
4-Ethynyl-4-hydroxycyclohexanone (4). A solution of 3 (1.70 g; 9.33 mmol) and CuSO4 (13
mg; 0.5 mol%) in 80% formic acid (6 mL) was stirred at room temperature for 16 h. After
dilution with water, the mixture was alkalized with 6N NaOH and extracted with Et2O. The
combined organic layers were dried over MgSO4 and evaporated. The residue was purified by
flash chromatography (hexane/EtOAc 3:1) to give 4 as a white solid (1.02 g, 79% yield).47 Mp
110 °C. IR: 3333, 3278, 2958, 1685, 1403, 1344, 1266, 1250, 1225, 1186, 1133, 1117, 1078, 953
1
cmꢀ1. HꢀNMR (360 MHz, CDCl3) δ 2.12ꢀ2.31 (m, 5 H), 2.45ꢀ2.61 (m, 5 H). 13CꢀNMR (360
MHz, CDCl3) δ 37.29, 38.77, 66.31, 73.13, 85.82, 209.54. HRꢀEIMS calcd 138.0681; found
138.0682.
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1-Ethynyl-4-propylaminocyclohexan-1-ol (5). To an iceꢀcooled suspension of NaBH(OAc)3
(5.68 g; 26.8 mmol) in CH2Cl2 (40 mL) a solution of 4 (1.48 g; 10.7 mmol) in CH2Cl2 (20 mL),
propylamine (3.5 mL; 42.9 mmol) and glacial acetic acid (0.67 mL; 11.77 mmol) was added
slowly. The mixture was allowed to warm to room temperature and stirred for 4 h. Then, the
reaction was quenched with saturated aqueous NaHCO3 and basified to pH 12 by 6N NaOH. The
aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (MgSO4) and
evaporated under reduced pressure. Purification by flash chromatography (CH2Cl2 + 5% MeOH
saturated with NH3) yielded 5 as a white solid (1.55 g; 80%). Mp 72 °C. IR: 3306, 2936, 2858,
2823, 2676, 2104, 1457, 1371, 1259, 1226, 1132, 1106, 1074, 969, 928, 907, 835 cmꢀ1. 1HꢀNMR
(360 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3 H), 1.41ꢀ1.64 (m, 5 H), 1.70ꢀ1.80 (m, 1 H), 1.89ꢀ2.05
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(m, 4 H), 2.40ꢀ2.61 (m, 4 H). CꢀNMR (360 MHz, CDCl3) δ 11.82, 23.53, 28.00, 30.36, 36.90,
38.46, 49.27, 55.72, 73.02, 86.95. APCIꢀMS m/z 182 [M++1].
4-Ethynyl-N-propylcyclohex-3-enamine (6). To a solution of 5 (1.32 g; 7.28 mmol), PPh3
(5.73 g; 21.84 mmol) and imidazole (1.05 g; 15.43 mmol) in CH2Cl2 (60 mL) was added
dropwise a solution of I2 (3.81 g; 15 mmol) in CH2Cl2 (40 mL). The mixture was stirred at room
temperature overnight. After addition of saturated aqueous NaHCO3 and basification with 6N
NaOH the organic layer was separated. The aqueous layer was extracted with CH2Cl2. The
combined organic layers were dried (MgSO4) and evaporated. The crude product was purified by
flash chromatography (CH2Cl2 + 3% MeOH saturated with NH3) to give 6 as a yellow oil (1.12
g; 94%). IR: 3310, 3030, 2957, 2926, 2823, 2874, 2093, 1373, 1302, 1226, 1124, 1058, 975, 912,
891, 835 cmꢀ1. 1HꢀNMR (600 MHz, CDCl3) δ 0.94 (t, J = 7.4 Hz, 3 H), 1.46ꢀ1.62 (m, 3 H), 1.91ꢀ
2.05 (m, 2 H), 2.16ꢀ2.48 (m, 3 H), 2.60ꢀ2.69 (m, 2 H), 2.74ꢀ2.85 (m, 2 H), 3.64 (s, 1 H), 6.10ꢀ
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6.14 (m, 1 H). CꢀNMR (360 MHz, CDCl3) δ 11.83, 23.55, 28.34, 28.79, 33.06, 49.04, 52.07,
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6
74.93, 85.01, 119.68, 134.41. APCIꢀMS m/z 164 [M++1].
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The isolation of enantiopure (R)-6 and (S)-6 was accomplished by chiral resolution of racemic 6
employing HPLC separation with a Chiralpak ASꢀH column (1 cm x 25 cm; Chiral Technologies
Europe) at a flow of 4.7 mL/min of CH3CN + 0.1% NHEt2: tR = 4.7 min for (R)-6 and tR = 9.8
min for (S)-6. (R)-6: [α]27D = + 83.9° (c 0.8, CHCl3). (S)-6: [α]27D = ꢀ 83.6° (c 0.8, CHCl3).
(S)-N-(4-Ethynylcyclohex-3-enyl)-3,5-dinitro-N-propylbenzamide ((S)-7). To an iceꢀcooled
solution of (S)-6 (19.4 mg; 0.12 mmol) in CH2Cl2 (6 mL) was added dropwise Et3N (66 ꢃL; 0.48
mmol) and a solution of 3,5ꢀdinitrobenzoyl chloride (32.9 mg; 0.14 mmol) in CH2Cl2 (3 mL).
The reaction mixture was stirred at 0 °C for 4 h. After the addition of saturated NaHCO3
solution, the organic layer was separated and the aqueous layer was extracted with CH2Cl2. The
combined organic layers were dried (MgSO4), evaporated and the residue was purified by flash
chromatography (hexane/EtOAc 20:1 to 10:1). The obtained product was crystallized from a
Et2O/pentane solution to give (S)-7 as yellow crystals (36.6 mg; 86%). IR: 3292, 3101, 2964,
2932, 2875, 2094, 1632, 1590, 1542, 1471, 1422, 1344, 1315, 1187, 1132, 1102, 1079, 1034,
978, 914, 815 cmꢀ1. 1HꢀNMR (600 MHz, CDCl3) δ 0.74 (t, J = 7.4 Hz, 0.9 H), 1.01 (t, J = 7.4 Hz,
2.1 H), 1.51ꢀ1.61 (m, 0.7 H), 1.69ꢀ1.81 (m, 1.4 H), 1.94ꢀ2.02 (m, 1.7 H), 2.06ꢀ2.24 (m, 1.7 H),
2.32ꢀ2.60 (m, 2.6 H), 2.88 (s, 0.7 H), 2.94 (s, 0.3 H), 3.14 (t, J = 8.1 Hz, 0.7 H), 3.28 (ddd, J =
5.2, 11.4, 13.2 Hz, 0.7 H), 3.40 (ddd, J = 5.2, 11.4, 13.2 Hz, 0.7 H), 3.54ꢀ3.60 (m, 0.7 H), 4.40ꢀ
4.46 (m, 0.3 H), 6.05ꢀ6.08 (m, 0.7 H), 6.21ꢀ6.24 (m, 0.3 H), 8.59 (d, J = 2.1 Hz, 1.4 H), 8.63 (d, J
= 2.1 Hz, 0.7 H), 9.18 (t, J = 2.1 Hz, 0.3 H), 9.19 (t, J = 2.1 Hz, 0.7 H); rotamers were observed.
13CꢀNMR (600 MHz, CDCl3) δ 11.24, 11.69, 22.23, 24.24, 25.98, 27.13, 28.77, 28.89, 29.32,
30.00, 44.07, 48.34, 51.55, 54.69, 75.67, 76.35, 83.29, 84.08, 119.09, 119.20, 11.32, 119.56,
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126.62, 126.76, 132.79, 133.84, 139.77, 140.24, 147.88, 148.06, 166.26, 166.76; rotamers were
observed. APCIꢀMS m/z 358.2 [M++1]
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N-[4-(Hydroxybutyl)]biphenyl-4-carboxamide (8)
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To an iceꢀcooled mixture of 4ꢀaminobutanol (100 mg; 0.12 mmol) and aqueous NaOH (10%; 0.4
mL) in CH2Cl2 (1.2 mL) solution of biphenylꢀ4ꢀcarbonyl chloride (230 mg; 1.06 mmol) in
CH2Cl2 (3 mL) was added dropwise, and the reaction was stirred at room temperature for 1 h.
After addition of H2O, the organic layer was separated and the aqueous layer was extracted with
CH2Cl2. The combined organic layers were washed with 2NꢀHCl, H2O and saturated aqueous
NaCl, dried (MgSO4) and evaporated under reduced pressure. The resulting white solid 8 (263.3
mg; 92%) was used without further purification. Analytical data according to the literature.48
N-(4-Hydroxybutyl)pyrazolo[1,5-a]pyridine-2-carboxamide
(9).
A
solution
of
pyrazolo[1,5ꢀa]pyridineꢀ2ꢀcarboxylic acid (121.5 mg; 0.75 mmol) and carbonyldiimidazole
(182.3 mg; 1.12 mmol) in DMF (10 mL) was stirred at 50 °C for 2 h. After cooling to room
temperature and dropwise addition of 4ꢀaminobutanol (0.21 mL; 2.25 mmol), the reaction
mixture was stirred at room temperature for 2 h. Then, saturated aqueous NaCl solution was
added, the aqueous layer was neutralized with aqueous HCl and extracted with CH2Cl2. The
combined organic layers were dried (MgSO4) and evaporated. Purification of the residue by flash
chromatography (CH2Cl2 + 3% MeOH) yielded 9 as a white solid (131.2 mg; 75%). Mp 60 °C.
IR: 3398, 3325, 3090, 3034, 2936, 2866, 1651, 1557, 1514, 1476, 1423, 1327, 1259, 1145, 1052,
1
842, 806 cmꢀ1. HꢀNMR (600 MHz, CDCl3) δ 1.67ꢀ1.78 (m, 4 H), 3.54 (dd, J = 6.9, 13.1 Hz, 2
H), 3.73 (t, J = 6.2 Hz, 2 H), 6.85 (ddd, J = 1.4, 6.9, 6.9 Hz, 1 H), 7.14 (ddd, J = 1.1, 6.6, 8.9 Hz,
1 H), 7.24 (s, 1 H), 7.58 (ddd, J = 1.3, 1.3, 8.9 Hz, 1 H), 8.37 (ddd, J = 1.1, 2.1, 6.9 Hz, 1 H).
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