Efficient synthesis of kainic acid analogues

The present paper deals with an improved synthesis of two molecular hybrids of AMPA and KA, compounds CIP-A and CIP-B , and their transformation into CIOP-A and CIOP-B , the corresponding amido derivatives. Exploiting the continuous-flow technology, a significant improvement in the synthesis of the glutamate agonists CIP-A and CIP-B was accomplished, in terms of overall yield, time, and excess of ethyl chlorooximinoacetate. Moreover, we find out the HPLC conditions suitable to separate, at a preparative level, the three intermediates formed in the 1,3-dipolar cycloaddition step.

As expected, compound CIP-A turned out to be a potent agonist at both AMPA and KA receptors without any activity at NMDA receptors. 8,9The eutomer of CIP-A was characterized by the S configuration at the amino acidic stereogenic center.3][14][15] In parallel, we carried out a detailed investigation on the structure-activity relationships of these rigidified aspartate-glutamate analogues to find out selective iGluR ligands.The bicyclic structure was either simplified into monocyclic derivatives or was conserved and the chain connecting the amino acidic moiety and the distal acidic group was elongated or shortened.7][18][19][20][21][22][23][24][25][26][27][28][29][30] In the planned derivatives, we always preserved the aminoacid moiety because the X-ray structural analyses of the iGluRs bilobular ligand-binding core in complex with agonists or antagonists evidenced the crucial role played by such a group. 31In a recent paper, 32 it was described a series of derivatives generated by the incorporation of the structural elements of both kainic acid and neodysiherbaine A (neoDH), two naturally occurring pro-convulsant agents.Surprisingly, some of them, e.g.derivative IKM-159, are characterized by the presence in their structure of the -carboxylate amido group (Figure 2) and are provided with a potent AMPA-selective antagonistic activity. 32n this ground, we designed the corresponding analogues of CIP-A and CIP-B in order to test their pharmacological profile and their selectivity versus AMPA receptor subtypes.
The present paper deals with an improved synthesis of CIP-A and CIP-B and their transformation into CIOP-A and CIOP-B, the corresponding amido derivatives (Figure 2).

Results and Discussion
According to a literature report, 9 the synthesis of CIP-A and CIP-B, as pure enantiomers, has been accomplished as depicted in Scheme 1.
The major drawbacks associated to the described procedure 9 are: i) the low reactivity of dipolarophile 1 and ii) the failure to separate cycloadduct 3 from 4 by column chromatography.For such a reason, the mixture was transformed into the corresponding secondary amines 5 and 6 which were separated by column chromatography and then reconverted into the N-Boc derivatives 3 and 4, respectively.
In order to overcome the first drawback, we decided to investigate the feasibility of the cycloaddition reaction with the flow technology by using a procedure previously applied to similar pericyclic reactions. 33,34As shown in scheme 2, an ethyl acetate solution of N-Boc-3,4dehydro-L-proline methyl ester 1, prepared under flow conditions as previously reported, 35 and ethyl chlorooximinoacetate were mixed and delivered to a glass column filled with solid K2CO3 heated at 90 °C.The desired cycloadducts were obtained in good yield (70%) in only 10 min and a slight excess of chloroxime.This result represents a significant improvement over the above described conventional methodology because the reaction time was considerably reduced (from 6 days to 10 min) and the overall yield improved (from 56% to 70%).
ARKAT-USA, Inc Scheme 1. Previously reported synthesis of CIP-A and CIP-B. 9Scheme 2. The cycloaddition reaction in a continuous flow reactor.
The second drawback, due to the incapability to split with a silica gel column chromatography the mixture of cycloadducts 2, 3, and 4, was explored by making use of a preparative HPLC.After a substantial number of attempts, we found out the conditions suitable to separate the cycloadducts at a preparative level.An excellent separation (see experimental section) was obtained with the amylose tris-(5-chloro-2-methyl-phenyl-carbamate) stationary phase, allowing us to collect a substantial amount of each stereoisomer.The cycloaddition step yielded intermediates 2:3:4 in the ratio 40:14:46.
Cycloadducts 2 and 4 were then oxidized with a catalytic amount of hydrated ruthenium (IV) oxide and a 10% aqueous solution of sodium periodate in a biphasic system water/ethyl acetate to give intermediate 7 and 8, respectively.Their stereochemistry was secured by 1 H NMR. As a matter of fact, derivative 7 shows proton 3a at 4.53 as a doublet (J = 9.3 Hz), whereas the same proton in derivative 8 resonates at 4.10 as a doublet of doublet (J = 1.3, 10.4 Hz).Intermediates 7 and 8 were treated with an aqueous 6N HCl solution, according to the procedure described for the synthesis of IKM 159 (Scheme 3). 36Scheme 3. Reagents and conditions: a: RuO2*H2O, NaIO4, H2O/AcOEt; b: HCl 6N, Δ.
Unfortunately, we obtained a 1:2 unsplitable mixture of CIOP-B and 9 or CIOP-A and The hydroxynitrile side-products 9 and 10 derived from the low stability of the isoxazoline ring under aqueous acidic conditions.Therefore, we decided to remove the nitrogen protecting group by treating derivatives 7 and 8 with an excess of trifluoroacetic acid; amides 11 and 12 were obtained in good yield (Scheme 4).Final compounds CIOP-B and CIOP-A were obtained, in quantitative yield, through the alkaline hydrolysis of the two ester groups under mild conditions, i.e. 2.2 equivalent of an aqueous K2CO3 solution.Using harsher basic condition, such as an aqueous NaOH solution, we observed a substantial degradation of the cycloadducts.

Conclusions
We have accomplished a significant improvement in the synthesis of glutamate agonists CIP-A and CIP-B in terms of overall yield, time, and excess of ethyl chlorooximinoacetate. Furthermore, we find out the HPLC conditions suitable to separate, at a preparative level, the three cycloadducts 2, 3, and 4. In such a way, we avoided the lengthy procedure based on the transformation of the unsplittable mixture of derivative 3 and 4 into the corresponding secondary amines 5 and 6, their separation by a silica gel column chromatography and then their reconvertion into the N-Boc derivatives 3 and 4. Furthermore, the two regioisomers 2 and 4 were transformed into final derivatives CIOP-B and CIOP-A, respectively, via an oxidation step, removal of the N-Boc protecting group and a careful hydrolysis of the two ester groups.The biological results of the novel derivatives CIOP-A and CIOP-B will challenge the involvement of the amino acid moiety in determining the binding to the active site of the ionotropic glutamate receptors.These data will be reported in due course.

Figure 2 .
Figure 2. Structures of model and target compounds.