Design, synthesis and preliminary pharmacological evaluation of rigid analogues of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP)

Some frozen analogues of 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and of 1-(3-pyridyl)piperazine have been synthesized and tested on rat cerebral cortex by means of binding studies. Among the synthesized substances, only compound 2c was found to displace [ 3 H]- cytisine from the nicotinic binding sites on rat cerebral cortex. Some possible explanations for the inactivity of the other compounds are given.


Introduction
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which modulate synaptic transmission.They are formed by five subunits arranged to form a pore, through which cations cross the cellular membrane.To date, 17 different subunits have been identified in vertebrate species, and according to the subunit composition, location, and sensitivity to αbungarotoxin (αBTX), they can be classified into different groups: 1) muscle-type receptors, found at the skeletal neuromuscular junction and in the electric organs of Torpedo fishes, formed by four different subunits (α1, β1, γ, δ or α1, β1, ε, δ); 2 αBTX-insensitive neuronal receptors, found in the peripheral and central nervous systems, formed by different combinations of α 2-6 and β 2-4 subunits; 3) α BTX-sensitive neuronal receptors, found in the CNS, containing α7-10 subunits.According to a recent classification, the subtypes formed by the most recently identified subunits (α9 and α10), which are expressed primarily in sensory epithelia, form a separate group. 1 There is great interest in nAChRs, since they seem to be involved in several physiological functions, such as synaptic transmission, modulation of presynaptic transmitter release, cognitive processes and control of movement in normal subjects, as well as in several pathological processes.In fact, dysfunction of nAChR has been linked to a number of human diseases such as depression, schizophrenia, Alzheimer's and Parkinson's diseases, Tourette's syndrome. 24][5] Other therapeutically important applications of nicotinic ligands are the treatment of nicotine addiction and the management of pain. 66][17][18][19][20] The nicotinic pharmacophore is formed by two groups: an H-bond acceptor atom, usually a pyridyl nitrogen or a carbonyl oxygen, and a positive nitrogen, which can be protonated or quaternarized; the proposed distance between these two groups ranges from 4.5 Å 15 to 5.5 Å. 16,21 As a part of our research in the field of nicotinic ligands, we have recently reported the synthesis and pharmacological evaluation of a series of analogues of 1,1-dimethyl-4phenylpiperazinium iodide (DMPP) 22,23 (general formula A, Figure 1), A well-known nicotinic agonist which, lacking the H-bond acceptor group, does not fit the proposed nicotinic pharmacophore.DMPP is reported to bind to the central nicotinic receptor with K i values ranging from 57 nM 24 to 250 nM; 22 it has been shown 23  substituents on the phenyl ring greatly improved affinity but in general, derivatives with a permanent positive charge showed higher potency than their tertiary amino analogues.However, by introducing an H-bond forming group (NH 2 , F, OCOMe, NO 2 ) in position 3 on the phenyl ring, or by replacing the phenyl ring with N-containing heterocycles, compounds were synthesized endowed with good affinity for the nicotinic receptor also as uncharged amines.
Since the possibility to cross the blood brain barrier is a crucial feature for drugs in the treatment of CNS pathologies, we decided to focus our attention on the uncharged 1-(3pyridyl)lpiperazines 1a and 1b (Figure 1), endowed with good affinity (K i 90 nM).In these molecules, as well as in DMPP, the rotation around the arylpiperazine bond is free; this observation prompted us to evaluate the effect on affinity, and eventually selectivity, of reducing the conformational freedom of this part of the molecule.Therefore, the compounds of general formula B (Figure 1) were designed, in which the two cycles (aromatic and piperazine rings) are connected through a suitable spacer (Z) that fixes their relative orientation.
This modification of the structure of DMPP and analogues could also help to find the bioactive conformation of this class of molecules.In fact, the conformational analysis of these molecules using different computational methods ends up with different low-energy conformations.As pointed out by Dijsktra, 25 the quantum mechanical semiempirical program AM1 yields a conformation of DMPP similar to that found in the crystal structure, 26 with the dihedral angle (C 2' -C 1' -N-lp, Figure 2) at 120°.This minimum energy conformation has been explained by the presence of two opposite factors: the possibility of conjugation between the nitrogen lone pair and the π orbitals, and the steric hindrance between the aromatic ring and the methylene groups of the piperazine ring. 25,26ther computational methods produce different results: ab initio (HF) gives a conformation in which the two rings, phenyl and piperazine, are orthogonal (τ 0°), while molecular mechanics Compounds of general formula B, with Z being only a one-carbon unit, should mimic the τ 90° conformation, while the orthogonal conformation could be approached with Z being a longer saturated alkyl chain.
Methylation of 2a was first attempted by reacting the secondary amine with formaldehyde and formic acid, but only decomposition of the starting material was observed.Treatment with paraformaldehyde in a Parr apparatus under hydrogen pressure and in the presence of Pd/C, according to Abreo, 21 gave the expected product 2b 28 in very low yield (10%), while MeI in DMF gave slightly better results (31% yield).Finally, treatment of 2a with benzyl chloroformate gave 7, which was then reduced with LiAlH 4 affording 2b with good yield.Compounds 2a and 2b were successfully reduced with NaBH 3 CN in acetic acid 29 obtaining the corresponding 1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles 4a 30 and 4b.Methiodides 2c and 4c were then obtained by treatment with MeI in ether.

Scheme 2
Therefore, we decided to reduce 11 in two steps (scheme 3): the reaction with Raney/Ni gave the lactam 13 which was reduced with LiAlH 4 to 3a, however without substantial improvement in the yields.To obtain the tertiary amine 3b, 3a was treated with benzyl chloroformate to give 14 which was then reduced with LiAlH 4 obtaining 3b.Subsequent methylation with MeI failed to give the desired methiodide 3c, and 15 was obtained as the only product.

Biological evaluation
Compounds 2-4 were tested in vitro on rat brain homogenates to evaluate their affinity for the central nicotinic receptors, according to a previously reported experimental protocol. 23[ 3 H]-Cytisine was used as radioligand; this compound is reported to label the α4β2 subtype, which is believed to represent up to 90% of the high affinity agonist binding site in the brain. 32 33mong the frozen analogues of DMPP and of 3-pyridylpiperazine (compounds 2a-c, 4a-c, 3a and 3b), only methiodide 2c shows affinity for the central nicotinic receptor with K i 2.02 µM (confidence limits 1.24-3.29 µM); the other compounds do not displace [ 3 H]-cytisine from rat cerebral cortex up to a 100 µM concentration.
As far as the piperazino-indoles are concerned, the lack of affinity of secondary bases (2a and 4a) and tertiary bases (2b and 4b) is not surprising, since also 1-phenyl-4-methylpiperazine (the tertiary base of DMPP) is devoid of affinity. 23On the contrary, it seems that the freezing of the phenylpiperazinium moiety into a tricyclic structure is detrimental for activity, since the methiodide 4c does not interact with the receptor, and the affinity of compound 2c is 8-fold lower than that of DMPP.The lack of affinity of the aza compounds 3a and 3b is also unexpected, since the parent compounds 1a and 1b show K i values in the nanomolar range.Some explanations regarding the inactivity of compounds 3-4 are however possible.The conformation of DMPP and of 1(a,b), which have been constrained, respectively, into the hexahydropyrazino[1,2-a]indole 4c and the tetrahydropyrido[4',3':4,5]pyrrolo[1,2-a]pyrazines 3(a,b), may not be the right one.In this way, in fact, the molecules have been frozen into a pseudoplanar conformation, with a value of the dihedral angle τ of 90° (Figure 2), which may not be the bioactive conformation.In this respect, the synthesis of substances (general formula B, Figure 1) in which the central five-membered ring has been replaced by a larger ring that allows a more orthogonal disposition between the aromatic and the piperazine rings will help to clarify this point.
On the other hand, a limiting factor in the interaction of these molecules with the receptor may be the space available at the binding site.In fact, 2c (K i 2.02 µM) and 4c (Ki > 100 µM), differing for the double/single bond within the indole ring, show a difference in their volume of 4.58 Å 3 .In this respect, it must be noticed that also 1,1,3-trimethyl-4-phenylpiperazinium iodide (the 3-methyl analogue of DMPP) 23 is completely devoid of affinity.In addition, the reduction of the indole double bond, while reducing the surface of the aromatic part, induces a bending in the molecule (Figure 3) which may not be compatible with the space available at the binding site.A further consideration can be made regarding compounds 3a and 3b.One reason for their lack of affinity could be the wrong orientation of the pyridyl nitrogen in the binding site; in fact, in 1,2,3,4-tetrahydropyrido[4',3':4,5]pyrrolo[1,2-a]pyrazines B the pyridyl ring has been constrained into one of the two possible "planar" conformations.In this respect, the synthesis of the isomeric 6,7,8,9-tetrahydropyrido[2',3':4,5]pyrrolo[1,2-a]pyrazines C (Figure 4), which is on the way, may help to clarify this point.
Conclusions Some frozen analogues of 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and of 1-(3pyridyl)piperazine (1a and 1b) have been synthesized and tested on rat cerebral cortex by means of binding studies.Compound 2c shows an 8-fold lower affinity for the nicotinic receptor than the lead compound DMPP; the other compounds do not interact with the nicotinic receptor.The decrease or lack of affinity of these compounds suggests that the structures in which the lead compounds have been frozen do not represent their bioactive conformations, or their volume is not compatible with the space available within the interaction site.The synthesis of other frozen analogues may help to clarify the bioactive conformation of aryl piperazine.

Figure 2 .
Figure 2. Low-energy conformation of DMPP, calculated with different methods.Hydrogen atoms are shown in gray, carbon atoms in green, nitrogen atoms in blue.

Figure 3 .
Figure 3. Minimized conformations of compound 2c (left) and 4c (right).Hydrogen atoms are shown in gray, carbon atoms in green, nitrogen atoms in blue.

Inc Experimental section General Procedures.
27l melting points were taken on a Büchi apparatus and are uncorrected.Infrared spectra were recorded with a Perkin-Elmer 681 spectrophotometer in a Nujol mull for solids and neat for liquids.Unless otherwise stated, NMR spectra were recorded on a Gemini 200 spectrometer.Chromatographic separations were performed on a silica gel column by gravity chromatography (Kieselgel 40, 0.063-0.200mm,Merck)or flash chromatography (Kieselgel 40, 0.040-0.063mm,Merck).Yields are given after purification, unless otherwise stated.Where analyses are indicated by symbols, the analytical results are within ± 0.4% of the theoretical values.27Toa solution of ethyl indole-2carboxylate (1.2 g, 6.34 mmol) in anhydrous DMF (15 mL) potassium tert-butoxide (1.06 g, 9.4 mmol) was added at room temperature.After 40 min, chloroacetonitrile (0.8 mL, 12.6 mmol) was added dropwise and the solution heated at 65 °C for 30 min and left stirring at room temperature for 20 h.Water (20 mL) was then added and all the solvents distilled to give a solid residue that was treated with water and extracted with CH 2 Cl 2 .Drying (Na 2 SO 4 ) and removal of the solvent gave 1 of .41g the title compound (white solid, 97% yield).Mp 98-99 °C.