Synthesis and binding affinity studies of muscarinic receptor antagonists related to dehydrohimbacine

The intramolecular Diels − Alder reaction of bromo-substituted nonatrienoate 24 leads to a mixture of the anti -adducts 25a and 25b , in which the trans -fused lactone 25b , the result of an expected exo -addition, predominates (6.2:1 stereoselectivity). Further introduction of a 2,6-trans - disubstituted piperidine ring ( 26 and 27 ) via palladium catalyzed cross-coupling reactions (Stille and Sonogashira) affords intermediates that are easily transformed into dehydrohimbacine derivatives 14a , 14b , 15a


Introduction
Since memorable times natural products with characteristic biological activities have constituted attractive targets for total synthesis, especially when their structures offer some challenging features with regard to connectivity, functionality and/or stereochemistry.In this context recently there has been interest in the synthesis of a series of piperidine alkaloids 1-5 (Figure 1), several of which have been isolated from the bark of Galbulimima baccata, a species belonging to the magnolia family found in New Guinea and North Queensland. 1 The structure of (+)-himbacine (1), 2 the most representative member of the family, was determined in 1962 via an X-ray diffraction study of the corresponding hydrobromic salt.Medicinal interest in himbacine originates from the finding that it is a potent muscarinic receptor antagonist that displays selectivity for the M 2 receptor. 4Already in 1914 Dale proposed that acetylcholine (ACh) acts through two major receptor subtypes defined as nicotinic and muscarinic. 5These receptors differ in location, function, structure and receptor-effector coupling mechanisms.Whereas nicotinic receptors are ligand-gated cation channels, muscarinic receptors are members of a superfamily, also including e.g. the opsins, which transmit their signal through GTP-binding proteins.This receptor family is structurally characterized by seven hydrophobic regions which span the membrane. 6n the late 1900s, five different subtypes m 1 to m 5 of the muscarinic receptor were identified using molecular biology techniques. 7Their expression in cultured cells devoid of any endogenous muscarinic receptors led to the pharmacological and biological characterization of the five subtypes M 1 -M 5 . 8The pharmacological properties of the cloned receptors are, as a rule, in excellent agreement with the binding as well as with the functional properties of the naturally expressed receptors M 1 -M 4 .These are distributed throughout the body where they regulate vital functions in both the central and autonomic nervous system.The M 2 and M 3 receptors are widely expressed in peripheral tissues, where the effector organs include heart (M 2 ), smooth muscle and exocrine glands (M 3 ).All five muscarinic receptor mRNAs have been identified in the central nervous system by in situ hybridization. 9he muscarinic receptor subtypes M 1 -M 5 can further be divided into two categories, both on the basis of their sequence homology and of their signaling mechanism.The M 1 , M 3 , M 5 form a first homologous group that is known to activate phospholipase C, which results in increased intracellular Ca 2+ concentrations, and cellular excitation.The M 2 , M 4 receptors form a second homologous group that inhibits adenylate cyclase, closes Ca 2+ and open K + channels, resulting in the hyperpolarization of the membrane and inhibitory signaling.
The age-related neurodegenerative diseases, such as Alzheimer's disease, have been associated with loss of cholinergic innervation and reduced levels of synaptic acetylcholine in several brain areas. 10The cholinergic approach to the treatment of the disease is directed towards the enhancement of ACh levels.Whereas this goal can in principle be achieved by inhibitors of cholinesterase, 11 the use of postsynaptic M 1 agonists has also been proposed, 12 especially since the M 1 subtype is almost absent in peripheral effector organs and the number of postsynaptic receptors seems not to be altered.An alternative strategy focuses on the blocking of the presynaptic M 2 receptor subtypes, activation of which inhibits the release of ACh. 13 Obviously, in the latter case M 2 antagonists with M 1 /M 2 selectivity are required.
Several antagonists have been useful in the classification of muscarinic receptor subtypes. 14Next to pirenzepine whose selective M 1 antagonistic activity was discovered in the early 1980s, 15 others have included 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladifenidol (HHSiD), gallamine, the benzodiazepinone derivative AF-DX 116 and himbacine. 16nterestingly, it has also been demonstrated that the blocking action of gallamine and himbacine on the heart differed from competitive inhibition, but could be rationalized by an allosteric mechanism. 17n general the known muscarinic agonists and antagonists are not very selective. 18Himbacine however has been reported to bind to M 2 receptors with K d values of 4.6-10 nM and with a 10-20 fold selectivity against the M 1 receptor.The full selectivity pattern M 1 -M 4 of himbacine in binding studies with rat brain receptor subtypes has been reported.

Figure 2
In an early structure-activity relationship study the natural alkaloids (1, 2, 4, 5) and their corresponding dihydro-derivatives were evaluated as antagonists of muscarinic receptors in guinea-pig ileal longitudinal muscle and electrically stimulated left atrium. 20Himbacine was the most potent compound and displayed a 15-fold selectivity for the M 2 receptor.Reduction of the double bond in himbacine or removal of the N-methyl group (cf.himbeline, 2) led to reduced selectivity.The same trend was observed in subsequent structure-activity relationship studies aiming at the development of himbacine analogues with high potency (better than 1 nM) and selectivity (better than 100-fold). 21Structures 6-8 (Figure 2) are representative molecules and their binding affinities at M 2 sites and M 1 /M 2 selectivities are given in Table 1.Compared to himbacine the severely truncated analogue 6 lost both affinity for the M 2 receptor (about 260fold) and selectivity.21a Whereas the replacement of the original tricyclic moiety of himbacine with a dihydroanthracene core as in 7 leads to an increase in affinity at the M 2 receptor subtype, a preferential binding at the M 1 receptor subtype is now observed.21b The importance of the structural integrity of the ABC-ring part, including the heterocyclic ring, for optimal M 2 selectivity was also confirmed by the observation that the removal of the carbonyl oxygen of himbacine affords a molecule (8) with reduced affinity and inversed selectivity.21c Finally, an interesting series of analogues has been developed by Chackalamannil on the basis of their finding that dihydrohimbacine (9) has a potency and selectivity comparable to that of himbacine in cloned human M 1 and M 2 receptors. 22Various N-alkylated derivatives such as 10, in which the nitrogen atom is located at the same relative position to the intact ABC-ring as in himbacine, were prepared.Several of these analogues display a potency comparable to that of himbacine, but again at the expense of selectivity.In essence, so far, analogue development on the basis of the himbacine skeleton has not met the expectations.In contrast, in another structural series, highly active compounds such as 11 (Figure 3) with >100-fold selectivity against M 1 , M 3 , and M 5 receptors have been developed. 23

Results and Discussion
Inspired by the above SAR studies that indicate that the deletion of substantial parts of the skeleton and/or functionality of the natural derivative results in loss of selectivity, we recently became interested in the synthesis of intact stereoisomers of himbacine with focus on the stereogenic centers in the tricyclic system of the molecule. 24Also variations in the geometry of the two carbon interconnecting unit between the two cyclic parts of the molecule were investigated, i.e. the incorporation of an acetylenic and of a (Z)-ethylenic next to the natural (E)ethylenic bridge.Three different isomeric series were synthesized, which, in combination with the three different above mentioned interconnecting units, afforded nine derivatives, including synthetic (+)-himbacine.Interestingly, the binding affinity studies showed that, next to himbacine, the two analogues 12 and 13 (Figure 4) possess a moderate 10-fold selectivity for the M 2 receptor, albeit with reduced potency (Table 1).Analogue 12 is epimeric at C-4 and at C-4a relative to himbacine whereas analogue 13 corresponds to 11,12-dehydrohimbacine.In a further search at optimizing both the potency and M 2 /M 1 selectivity of himbacine-like derivatives we wish to report now on the synthesis of a few stereoisomeric derivatives 14 and 15 characterized by the presence of (i) a double bond at C-4,C-4a and of (ii) an interconnecting two carbon unit of the (E)-ene or yne type, and in which the other structural features of himbacine are kept intact.At the conceptual level, derivatives within the a-configurational series, such as 14a and 15a, were preferred.They respect the structural integrity of the two cyclic parts of himbacine and include a bridging unit, which by its nature imposes an extended geometry to the molecule, presumably a crucial factor in determining the biological activity.For a synthetic chemist, himbacine (1) possesses an attractive structure.Its skeleton consists of a tricyclic ABC-ring system, to which is connected, via a (E)-double bond, the N-methyl piperidine D-ring.The ABC-ring part itself consists of a trans-fused perhydronaphtalene with a cis-fused γ-lactone.The two cyclic parts also display interesting stereochemical patterns: in the ABC-ring part six contiguous stereocenters are present, while the D-ring features a less common 2,6-trans-disubstituted piperidine ring.The first enantioselective total synthesis of himbacine was reported by Hart and Kozikowski in 1995. 25 Other enantioselective syntheses of himbacine have been subsequently reported by Chackalamannil, 26 by Terashima, 27

Figure 4
So far the various total synthetic approaches towards himbacine have all involved the use of a Diels-Alder reaction in establishing the central B-ring nucleus of the ABC-ring skeleton. 25,26,27,28Among the six different bond construction sets that one may distinguish following this particular cycloaddition strategy, two sets correspond to the intermolecular reaction mode, one of which has been realized in practice, 27 and the four remaining construction sets correspond to intramolecular versions of this popular construction reaction (IMDA). 29ymptomatic of how pervasive this reaction type has become in terms of cyclic skeleton construction, especially when involving stereochemical constraints, is the observation that the three synthetic approaches that were first reported almost simultaneously in 1995, involved pathway i (Scheme 1). 25,30,31One of the benefits of this particular cycloaddition route resides in the obtainment of the functionalized ABC-ring system as present in himgravine (5), whose selective reduction to himbacine (1) has been reported before. 2 Also, depending on the reaction conditions and the nature of the R group, the stereochemical outcome of the process could be directed towards the himbacine-like configuration.25b A second construction set (ii) was exploited successfully by Chackalamannil in the total syntheses of himbacine and more recently of himandravine. 26,32This cycloaddition whereby the R group already contains the intact D-ring leads exclusively to one stereoisomer in which the γ-lactone is trans-fused, the result of an anti,exo-approach (vide infra).

Scheme 1
In the present study we wish to elaborate on the construction set shown in Scheme 2. 33 This third pathway bears some analogy with the previous approach since in both cases the intramolecular cycloaddition leads to the simultaneous formation of the A-and B-rings.This novel strategy involves as a key step the cycloaddition of an acrylate derived from a 3,4,5trisubstituted pentadienyl alcohol bearing a stereocenter (i.e., at C-1) in the diene/dienophile tether that is expected to play a stereodirecting role in the process.Following our preliminary report on this work, 33a an almost identical synthetic approach has been disclosed by Sherburn and co-workers.33b The stereochemical outcome of the Diels−Alder reaction is determined by (1) the antior syn-approach of the dienophile relative to the orientation of the methyl group at C-1 on the planar pentadienyl moiety, and by (2) the exoor endo-mode of addition.On one hand the anti-approach should be preferred on the basis of steric considerations, especially when R is voluminous, thus favoring the aand b-configurations; on the other hand the exo-mode of addition is expected here on the basis of a literature precedent dealing with the synthesis of drimane sesquiterpenes. 34Hence at the outset the preferred anti,exo-formation of the b-type stereoisomeric relationship was anticipated.As will be discussed later in more detail a very recent joint synthetic computational investigation into the optimization of the stereoselectivity in the IMDA reaction of pentadienyl acrylates has corroborated this expectation. 35Subsequent isomerization of the trans-fused lactone (b) to the more stable cis-configuration should afford the desired stereochemistry (a) as present in derivatives 14a and 15a.A further strategic choice allowing for the convergent synthesis of both 14 and 15, which only differ in the connecting unit, consists of the incorporation of a vinylic halogen atom (i.e., R = Br), whose replacement using one of the several existing cross-coupling reactions, should allow for the completion of the carbon framework of the molecule possessing the genuine D-ring as in 14 and 15. anti Central in the approach following the above strategy stands alcohol 23, whose synthesis is outlined in Scheme 3.After protection of the hydroxyl group in (S)-ethyl lactate as tertbutyldimethysilyl ether, the resulting ester 16 was reduced with diisobutylaluminum hydride affording aldehyde 17 (80% yield). 36Conversion of the latter using the protocol of Corey−Fuchs 37 gave the known geminal dibromo substituted alkene 18 (84% yield). 38The stereoselective introduction of the cyclohexenyl moiety was then realized via a Stille coupling with stannane 20. 39The latter was obtained in 79% yield by reaction of enol triflate 19 with hexamethylditin (tetrakis(triphenylphosphine)palladium(0), lithium chloride, THF, 60 °C). 40everal studies have recently appeared involving the replacement of one or both halogen atoms in 1,1-dibromo-1-alkenes. 41In particular the stereoselective replacement of the (E)-halogen atom using the Stille reaction has been studied in detail by Shen and Wang.41a Using the same optimized reaction conditions, tris(dibenzylideneacetone)dipalladium(0) and tri-2-furylphosphine in refluxing toluene, vinyl bromide 21 was obtained in 71% yield.Although the (Z)stereochemistry at the trisubstituted double bond was expected, its unambiguous configurational assignment followed from the stereospecific transformation of 21 into C-silylated alcohol 22 after treatment with tert-butyllithium at −78 °C.This rearrangement that follows a metal-halogen exchange implies, of course, a cis-relationship between the halogen atom and the silyloxyethyl group.Desilylation of 21 with tetrabutylammonium fluoride afforded alcohol 23 in 88% yield.The further conversion of 23 into himbacine analogues 14a, 14b, 15a and 15b is shown in Scheme 4. First the alcohol 23 was converted into the corresponding acrylate 24 using acryloyl chloride and diisopropylethylamine (DIPEA) in dichloromethane (77% yield).The Diels−Alder reaction of 24 required high temperatures (185 °C, toluene) and the presence of both an antioxidant (TEMPO) and a base (DIPEA). 42After 24 h a mixture of the two stereoisomeric vinylic bromides 25a and 25b was obtained in 79% yield.The expected anti,exo-adduct (25b) predominated (ratio 25a:25b = 1:6.2).The structural assignment of both adducts follows from the X-ray crystal structure determination of 25b and from the observation that upon basic treatment the trans-fused lactone in 25b is isomerized into the more stable cis-fused adduct 25a.33a In line with this experimental result, force field calculations indicate a relative steric energy difference of 9.9 kJ mol -1 in favour of 25a. 43he transformation of 25a and of 25b into the desired analogues 14a, 15a and 14b, 15b, respectively, involves palladium catalyzed cross-coupling reactions.The Stille reaction between bromides 25a and 25b, and the vinyltributylstannane 26 using tris(dibenzylideneacetone)dipalladium(0) and tri-2-furylphosphine in the presence of DIPEA (toluene, reflux) afforded dienes 28a (80% yield) and 28b (77% yield), respectively.The synthesis of vinylstannane 26 proceeded by reaction of a higher order tributyltincuprate derivative, obtained from copper(I) cyanide, n-butyllithium and tributyltinhydride, 44 with the known terminal alkyne 27 (80% yield). 28The Sonogashira coupling involved reaction of the same bromides 25a and 25b with alkyne 27 (bis(triphenylphosphine)palladium(II) acetate, triethylamine in acetonitrile) and afforded enynes 29a (76% yield) and 29b (73% yield), respectively. 45It is worth noting that the above conditions are the result of thorough experimentation. 46The final conversion of the N-Boc derivatives 28a, 28b, 29a and 29b into the corresponding N-methylated analogues 14a, 14b, 15a, and 15b proceeded in two steps: after acidic removal of the Boc protective group (trifluoroacetic acid, dichloromethane), the resulting crude secondary amines were treated with 37% aqueous formaldehyde and sodium cyanoborohydride in acetonitrile to yield 14a (81% yield), 14b (80% yield), 15a (75% yield) and 15b (78% yield), respectively.
At this point it is interesting to focus in more detail on the stereochemical outcome of the Diels−Alder reaction of the pentadienyl acrylate system as present in precursor 24. 47Very recently a few related cases have been reported in considerable detail.The relevant transformations dealing with C-1 substituted derivatives are depicted in Scheme 5.The reaction of the double activated dienophile substrates was studied by Paddon−Row and Sherburn: 48 (i) the more reactive maleate gave isomers a and b in the same ratio (1:6.1) as observed in the reaction of 24; (ii) the fumarate led to the formation of the three isomers a, b and c with a distinct preference for the anti,exo-adduct b.The cycloaddition of the acrylate studied by White and Snyder (iii) required harsher reaction conditions and led to a mixture of the a, b and c stereoisomers; 49 although the observed stereoselectivity was poor, the anti,exo-adduct b again predominated.Lastly, the cycloaddition of a C-3 bromine substituted pentadienyl acrylate, described by Paddon−Row and Sherburn (iv) leads to the corresponding stereoisomers a and b, with again a clear preference for the anti,exo-adduct b. 35 Interestingly in the previous studies elaborate DFT calculations have corroborated the experimental results.Since the cycloadditions were shown to proceed under kinetic control, the relative energies of the different transition states were calculated.Our result is almost identical to the one observed in (iv).This is not really surprising in view of the presumed determining influence of the steric interference, related to 1,3 A-strain, 50 between the C-1 alkyl and C-3 bromine substituents.The latter substituent has been termed a "steric directing group" by Paddon−Row and Sherburn in this particular context. 35

Binding affinity studies
The binding studies of synthetic (+)-himbacine 1 and of the four derivatives 14a, 14b, 15a and 15b for the four muscarine receptor subtypes were carried out by analysis of competition experiments with [ 3 H]-N-methylscopolamine (NMS) at 25 °C in a 50 mM sodium phosphate/ 2 mM magnesium chloride buffer (pH 7.4). 51The [ 3 H]-NMS saturation curves in each cell line were compatible with recognition of a single receptor subtype.In the present set of experiments, the [ 3 H]-NMS K D values were 0.26, 0.50, 0.42 and 0.18 nM at M 1 , M 2 , M 3 and M 4 muscarinic receptors, respectively.Representative competition curves are included in the supporting information section.The corresponding pK i values are given in Table 2 The results of the binding studies indicate that all analogues possess a lower affinity for each receptor subtype when compared with himbacine.Analogue 15a shows an 18-fold selectivity for the M 2 relative to the M 1 receptor.It is interesting to note that the structure of 15a involves an acetylenic connecting bridge instead of the natural (E)-ethylenic moiety.

Conclusions
We have developed a concise convergent approach towards the synthesis of 4,4adehydrohimbacine derivatives.The key step in the synthesis involves an intramolecular Diels−Alder reaction of a substituted pentadienyl acrylate (24), which afforded with good stereoselectivity the trans-fused lactone bromide 25b, the adduct resulting from the expected preferred anti,exo-addition.Subsequent attachment of the piperidine D-ring involved Stille (vinylstannane 26) and Sonogashira (alkyne 27) coupling reactions.

Experimental Section
General Procedures.All air sensitive reactions were run under Ar or N 2 atmosphere and reagents were added through septa using oven dried syringes.Et 2 O and THF were distilled from benzophenone ketyl prior to use.N,N-Diisopropylethylamine (DIPEA), Et 3 N, CH 3 CN and HMPA were distilled from CaH 2 and CH 2 Cl 2 was distilled from P 2 O 5 .TLC were run on glass plates pre-coated with silica gel (Merck, 60F254).Column chromatography was performed on silica gel (Merck, 230-400 mesh) and HPLC separations were performed on Bio-Sil D 90-10, 10µm columns (Bio-Rad) of 1 × 25 cm and 2.2 × 25 cm. 1 H NMR chemical shifts are reported in ppm (δ) relative to CDCl 3 (7.26ppm) or C 6 D 6 (7.16 ppm) as an internal reference. 13C NMR chemical shifts are reported in ppm (δ) relative to CDCl 3 (77.16ppm) or C 6 D 6 (128.06 ppm) as an internal reference.Mass spectra were recorded at 70 eV.
[ 3 H]-NMS binding.CHO cell homogenates (at a concentration equivalent to about 0.05 to 0.1 nM muscarinic receptors in the binding assays) were incubated at 25 °C, in the presence of [ 3 H]-NMS and of the indicated concentrations of the unlabelled drugs, in 1.0 mL of a 50 mM sodium phosphate buffer (pH 7.4) enriched with 2 mM MgCl 2 .Non-specific binding was defined as binding in the presence of 10 µM atropine.The tracer concentrations used were 0.25 nM for competition curves with hM 1 , hM 3 or hM 4 receptors, or 0.8 nM for competition curves with hM 2 receptors.To verify the tracer affinities at these receptors, saturation curves were obtained by varying the [ 3 H]-NMS concentration between 0.05 and 3.0 nM.At 25 °C, an incubation period of 2 h for incubations with hM 1 , hM 2 or hM 4 receptors, 4 h for incubations with hM 3 receptors was sufficient to achieve equilibrium tracer binding.The incubations were terminated by filtration over glass microfiber filters C (K-LAB), pre-soaked in 0.01% polyethyleneimine (Sigma, St Louis, FL) to reduce non-specific binding.The filters were rinsed 3 times with 2 mL of ice-cold 50 mM sodium phosphate buffer (pH 7.4) then soaked at least 4 h in "Lumagel Plus" (Lumac Lsc.) and the radioactivity counted in a Packard 1500 Tricarb liquid scintillation analyser.The total tracer binding (in the absence of competitor) was kept below 20% (typically between 10 and 15%) of the radioactivity offered, to avoid alterations of the free tracer concentration with increasing competitor concentrations.Non-specific binding was always below 1% of the radioactivity offered (that is, below 10% of the bound radioactivity).Duplicate determinations were always within 5% (typically 3%) of each other.Data Analysis.All competition curves were performed in duplicate, and repeated twice.The experimental data were analyzed by non-linear curve fitting, using the Graph Pad program.The tracer affinity was determined in the same experiment by saturation curve analysis.The unlabeled drugs K i values were calculated assuming that they inhibited competitively [ 3 H]-NMS binding, using the Cheng and Prusoff equation.

Table 2 .
. K i values (nM) and pK i values (between parentheses) at cloned CHO cells expressing hM 1 , hM 2 , hM 3 , and hM 4 muscarine receptors a a Since the distribution of the K i values is lognormal, pK i values, −log (K i concentration units), are also mentioned; the calculated s.e.m. values vary between ±0.01 and ±0.40.b synthetic (+)himbacine, see reference 24.