Application of a new methodology for the synthesis and resolution of a β -(thien-2-yl) proline with the 7-azanorbornane skeleton

The enantiomers of a heavily constrained proline derivative, methyl (1 S ,2 R ,4 R )- and (1 R ,2 S ,4 S )- 2-(thien-2-yl)-7-azabicyclo [2.2.1]heptane-1-carboxylate, have been obtained separately by resolution of the racemic mixture using chiral high performance liquid chromatography. This is a particular example of the application of our new methodology, which takes advantage of the efficiency of chromatographic resolution techniques for the preparation of both enantiomers of a broad variety of proline– α -amino acid chimeras with a 7-azanorbornane skeleton.


Introduction
The introduction of conformational constraints constitutes one of the most promising approaches to the construction of peptide analogues of pharmaceutical interest. 1Furthermore, it has become a useful tool for the elucidation of biologically active conformations 1a,2 by providing information about the spatial requirements for optimal interaction with receptors. 2,3everal β-substituted prolines (Figure 1) have been synthesized as amino acid chimeras in which the functional groups of the amino acid side-chain are combined with the conformational restrictions characteristic of the cyclic amino acid residue. 4Replacement of the natural amino acids in peptides with such proline-α-amino acid chimeras has led to better understanding of the bioactive conformations of cholecystokinin, 5 angiotensin II, 6 bradykinin, 7 and opioid peptides. 8,9][8][9] The proline analogues which contain a 7-azabicyclo[2.2.1]heptane structure, Rb 7 Pro, constitute a distinct class of amino acids, owing to their extra conformational restriction (Figure 1).Hence, the benefits of the parent compound (R = H), as a replacement for proline in a boroarginine thrombin inhibitor, 11 and as starting material in the synthesis of a new class of HIV-1 protease inhibitor, 12 have already been proved.Moreover, we have already described the synthesis of a proline-L-phenylalanine chimera analogue in enantiomerically pure form, methyl (1S,2S,4R)-N-benzoyl-2-phenyl-7-azabicyclo[2.2.1]heptane-1-carboxylate, and its incorporation into a model dipeptide, Bz-Phb 7 Pro-L-Phe-NH i Pr. 13 The X-ray diffraction analysis on this dipeptide 14 revealed that, in the solid state, this constrained peptide containing the azanorbornane proline adopted a type I β-turn whereas the analogous dipeptide sequence incorporating L-proline has been shown to accommodate a βII-turn disposition.On the other hand, the increasing interest in these nitrogen-containing, heavily constrained heterocyclic amino acids is, to a large extent, caused by the current extensive use of a simple proline as an asymmetric catalyst in a variety of reactions, such as aldol, Mannich, Michael, Diels-Alder, and the α-oxidation of aldehydes. 15e now report the synthesis of a heavily constrained proline derivative, methyl N-benzoyl-2exo-(thien-2-yl)-7-azabicyclo[2.2.1]heptane-1-carboxylate, and the resolution of its racemic mixture using chiral high performance liquid chromatography.

Results and Discussion
At present, there are few examples of the asymmetric synthesis of prolines with a 7azanorbornane skeleton, 16,17 the literature reports only two resolution procedures to obtain the two enantiomers of a 7-azanorbornane proline separately.One of them consists in the formation of diastereomers and subsequent separation by crystallization, which involves the introduction of additional synthetic steps to the pathway towards the final products. 18On the other hand, we have already developed a highly efficient and simple resolution method for an azanorbornane proline-phenylalanine chimera analogue, methyl N-benzoyl-2-exo-phenyl-7-azabicyclo-[2.2.1]heptane-1-carboxylate, by chiral high performance liquid chromatography (HPLC). 13herefore, we first embarked on the preparation of a racemic mixture of methyl N-benzoyl-2exo-(thien-2-yl)-7-azabicyclo[2.2.1]heptane-1-carboxylate.Its synthesis was performed by applying the methodology that we had developed previously. 13,17,19,20Our overall strategy is based on building the 7-azabicyclo[2.2.1]heptane skeleton by intramolecular nucleophilic displacement of a 4-aminocyclohexanol derivative, proceeding from the cycloadducts provided by the Diels-Alder reaction of C-4 unsaturated 5(4H)-oxazolones and Danishefsky's diene.

Scheme 1
Then, the Diels-Alder reaction of Danishefsky's diene and the oxazolone 1Z was tested; after 2 days' refluxing in toluene complete conversion of the oxazolone was achieved.Hydrolysis of the adduct mixture gave a mixture of two compounds, rac-2, proceeding from the adducts resulting from the endo-and exo-attacks of the diene, in 92% combined yield (Scheme 2).Subsequent elimination of the methoxy group with oxazolone-ring-opening, by treatment of the hydrolyzed adducts with DBU in methanol, led to rac-3 in 68% yield.
Next, a number of reagents and conditions was tested for hydrogenating the double bond of the enone rac-3.A homogeneous procedure, using Wilkinson's catalyst, did not give complete conversion, even after prolonged reaction (9 days).However, the use of zinc in acetic acid led to the desired hydrogenated compound rac-4 in low yield (50%).Finally, heterogeneous hydrogenation using 20% Pd(OH) 2 /C in dichloromethane and slight heating (30ºC), proved to be very efficient, affording the ketone rac-4 in nearly quantitative yield.This compound, rac-4, is the precursor of the trans-aminocyclohexanol derivative needed to achieve our goal.
The preparation of the desired 4-hydroxyamino-derivative involved conversion of the ketone function in the cyclohexanone ring into a hydroxy group.Moreover, for the subsequent cyclization, the hydroxy and benzamido groups on the cyclohexane skeleton were required to adopt axial positions, which requires the synthesis of the kinetically controlled alcohol.Several studies have demonstrated that the use of bulky reducing agents at low temperatures produces stereoselective processes that provide preferentially the kinetically controlled product.Accordingly, we decided to use lithium tri-sec-butylborohydride (L-Selectride ® ), which offers highly favorable formation of axial alcohols.Thus, reduction of the cyclohexanone derivative rac-4 with L-Selectride ® in THF at -78ºC gave the axial-and equatorial-alcohols, rac-5 (stereoselectivity ratio: 77/23), in 96% combined yield.The ratio of stereoisomers in the crude reaction mixture was determined by integration of the 1 H-NMR signals of the two isomers, at 3.05 ppm (ddd, 1H, J = 14.0 Hz, J = 3.7 Hz, J = 3.7 Hz) and 3.35 ppm (ddd, 1H, J = 14.3 Hz, J = 3.7 Hz, J = 3.7 Hz).Initially, the compound showing a signal at 3.05 ppm was assumed to be the axial isomer, rac-5a; subsequent cyclization of its mesyl derivative, rac-6a, confirmed this.The mixture of the alcohols rac-5 was transformed cleanly into a mixture of the corresponding methanesulfonate derivatives, rac-6a and 6b, in 96% yield by treatment with methanesulfonyl chloride in triethylamine.Furthermore, this methanesulfonate mixture was purified by column chromatography and provided pure samples of both isomers, which were fully characterized.
The cyclization of 4-aminocyclohexanol derivatives has become one of the most widely reported procedures for creating 7-azabicyclo[2.2.1]heptane rings. 22Base-promoted internal nucleophilic displacement of the methanesulfonate group had already been achieved by treatment with potassium tert-butoxide in THF.The same reaction conditions were applied to the mixture of methanesulfonates rac-6.Finally, the separation of the product rac-7 from the non-cyclizable methanesulfonate rac-6b was easily achieved by column chromatography, which provided methyl N-benzoyl-2-exo-(thien-2-yl)-7-azabicyclo[2.2.1]heptane-1-carboxylate (rac-7) in excellent yield (75% from a 77/23 mixture of rac-6a and 6b).In this way, the synthesis was accomplished on a multi-gram scale and ca.1.5 g of a racemic mixture of rac-7 could be obtained from oxazolone 1Z in seven steps and 43% overall yield.

HPLC resolution of rac-7
Once the synthesis of the racemic compound rac-7 was achieved, we undertook the preparation of the enantiomerically pure form by HPLC resolution using a chiral stationary phase.Specifically, a non-commercial polysaccharide-derived support consisting of mixed 10undecenoate/3,5-dimethylphenylcarbamate of cellulose covalently attached to allylsilica gel was used. 23The excellent chiral discrimination exhibited by this stationary phase towards a variety of compounds, together with its high chemical stability, make it especially suitable for resolutions on a preparative scale.We had already shown the efficacy of this methodology for resolving racemic mixtures of constrained phenylalanine analogues. 13,24he resolution of rac-7 was initially investigated on an analytical scale (eluent: 95/5 nhexane/2-propanol, k' 1 = 1.81, α = 1.58,R s = 3.70).From these satisfactory results, the optimal separation conditions on the preparative scale were determined by adding a certain amount of chloroform to the eluent to enhance the solubility of the compound.
Finally, the separation was performed using a 93/2/5 mixture of n-hexane/chloroform/2propanol as the eluent (flow-rate 18 mL min -1 ) on a 150×20 mm ID column filled with the 10undecenoate/3,5-dimethylphenylcarbamate of cellulose bonded on allylsilica gel (see Figure 2).Under these conditions, working with the repetitive injection mode (see Figure 3), HPLC resolution of rac-7 (1 g) in chloroform (5 mL) was achieved, with successive injections of 200 µL.A total of 27 injections was required, with one injection being performed every 11 min.The optical purity of the resolved enantiomers was assessed at the analytical level (Figure 4).To summarize, the resolution of 1g of rac-7 finally resulted in 492 mg of the optically pure first-eluted enantiomer and 395 mg of the second-eluted enantiomer in 99.4% enantiomeric purity.

Assignment of absolute configurations
Initially the assignment of absolute configuration was tried by X-ray diffraction analysis of a single crystal obtained from a solution of one of the diastereomeric dipeptides resulting from coupling of the racemic mixture with an L-phenylalanine derivative, in the way we have already described for the proline-phenylalanine chimera analogue. 13Thus, saponification of rac-7 using potassium hydroxide in methanol led to the corresponding acid, and subsequent coupling of this racemic mixture with enantiomerically pure L-phenylalanine isopropylamide, using the reagent designed by Castro et al., (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), gave a mixture of two diastereomeric dipeptide derivatives.After complete separation of the dipeptides by column chromatography, both compounds were obtained individually.However, all attempts to obtain a crystal suitable for single-crystal X-ray analysis, from either of the two resulting diastereomeric dipeptides, failed.
We then decided to assign the absolute configuration by transformation of the enantiomeric β-(thien-2-yl) derivatives into the corresponding proline-aspartic acid chimeras (Scheme 3).Oxidative cleavage of the thienyl substituent on the azabicyclic ring of the first-and the secondly-eluting enantiomers of 7 proceeded in 57% yield, and led to the proline-aspartic acid chimera analogues (1S,2R,4R)-8 and (1R,2S,4S)-8, respectively.The establishment of the absolute configuration for each enantiomer of 8 was carried out by determination of the specific rotations and comparison with the corresponding data for the same compounds characterized previously (Scheme 3). 13In this way, the first-eluted enantiomer could be identified as that having the (1S,2R,4R) configuration, and the second one as (1R,2S,4S).

Conclusions
A versatile and efficient methodology has been applied for the synthesis of a new chimera, a combination of α-amino acids and a proline constrained by a 7-azanorbornane skeleton.Cycloaddition of (Z)-2-phenyl-4-(thien-2-ylmethylene)-5(4H)-oxazolone and Danishefsky's diene followed by manipulation of the resulting adducts has led to the synthesis of a racemic mixture of methyl N-benzoyl-2-exo-(thien-2-yl)-7-azabicyclo[2.2.1]heptane-1-carboxylate.Furthermore, the high efficacy of the HPLC resolution of this compound has provided the two enantiomers of this constrained proline.This proline is a very special kind of amino acid where the rigidity provided by the azabicyclic skeleton is combined with the presence of a βsubstituent, which mimics the α-amino acid side-chain.These proline-α-amino acid chimeras are new surrogates to be incorporated into peptides whose structural and biological properties shed light upon the nature of the effects induced by this type of conformational restriction and the influence of the absolute configurations of the stereogenic centers.Their use in the area of catalysis, as replacements for proline, could result in fascinating applications for these heavily constrained amino acids.

Figure 1 .
Figure 1.Structure of proline and some constrained analogues.

Figure 3 .
Figure 3. HPLC resolution of rac-7 on preparative scale by repetitive injection mode.