Descripciones textuales de los Casos de Uso del Sistema

The organic synthesis of all the member of Eltex family exploited as pivotal compound the (R)-N-Boc pipecolic acid, enantiopure commercially available. Eltex compounds included esters and amides that were obtained using different alcohols or amines to functionalize the pipecolic acid, while the final coupling with phenylglyoxylic acid was the same for all the derivatives.

Synthesis of Elte421(1) and ElteN378 (5) were straightforward, since no stereochemical control was required, while a continuous monitoring of the stereochemistry at position 3 during the entire process of R-Elte421 and S-Elte421 made theirs synthesis more challenging.

Esters

The first inhibitor we synthesized was a 1:1 disteroisomeric mixture at carbon 3, named Elte421(1) (Scheme 3.1).

Scheme 3.1. Synthesis of Elte421 (1)

The first step of the procedure simultaneously introduced an alkyl chain on 3-phenylpropionaldheyde (6) and reduced it, after treatment with a stoichiometric amount of propylmagnesium chloride 2M in ether at -78°C to have (3R/S)-1-phenylhexan-3-ol (7). The obtained alcohol, racemic at position 3, underwent a first coupling with (R)-N-Boc pipecolic acid (2 equiv.), using diisopropylcarbodiimide

196 (DIC, 2 equiv.) as coupling agent and 4-dimethylaminopyridine (DMAP) in catalytic amount (0.1 equiv.). Besides flash chromatography on silica gel, the purification of (8) required several washing with a saturated solution of NH4Cl to get rid of diisopropylurea (DCU), a water soluble side-product of DIC. After the removal of Boc protecting group with a 99% TFA solution in DCM, achieved in almost quantitative yield, the free amine of (9) underwent the final coupling with phenylglyoxylic acid. In the first attempt, DIC-DMAP protocol, above described, was exploited but the final compound was obtained in low yield since the presence of urea was detected and purification of (1) to get rid of that was not successful. Phenylglyoxylic acid was, thus, activated to the correspondent chloride (10) with an excess of thionyl chloride and coupled with (3R/S)-1-phenylhexan-3-yl piperidine-2-carboxylate (9) in the presence of TEA (2 equiv.) to give Elte421 (1) as pure final product in 68 % yield. The ¹H NMR spectrum showed the presence of two diatereoisomers in 1:1 ratio (d1 and d2) and two rotamers (r1 and r2, trans/cis ratio of the order of 4:1) with different patter of signals (Figure 3.11)

Figure 3.11. ¹H NMR, 400 MHz spectrum of the Elte421 (1) in DMSO at 27°C (region of CH3 group) In the region of CH3 group, for instance, two doublets of triplets (corresponding to trans and cis ω rotamers of the mixture of the diastereoisomers) were observed, whose integrals yield the intensity ratio 3:1,indicating that the trans-cis ration in DMSO is identical for the diastereoisomers. Due to the high barriers, the cis-trans isomerization in Elte421 followed, in standard conditions, a kinetics that is similar to that of the cis-trans isomerization in proline (i.e. cis-trans swaps in the order of seconds per molecule). In order to test the intrasolute force field, the cis-trans equilibrium of R-Elte421 and S-Elte421 in DMSO has been also evaluated by REM simulations, yielding trans/cis ratio of the order of 4:1, in reasonable agreement with the 3:1 ratio of the experimental data (data not shown).

197 Figure 3.12. ¹H NMR, 400 MHz spectrum of the Elte421 (1) in DMSO at: a. 30°C, b. 70°C, c. 99°C.

A variable temperature NMR study has been conducted on Elte421. Increasing the temperature during the acquisition of ¹H spectra reduced the energy barrier between the rotamers, promoting the cis-trans interconversion. A clear evidence of this phenomenon was the overlapping of doublets of triplets of CH3 terminal group at high temperature, ending in a loss of multiplicity (Figure 3.12).In the ¹³C NMR spectrum the total number of signals was higher than expected, due the partial superimposition of the carbon chemical shifts of the two rotamers for each diastereoisomer.

To confirm the hypotheses derived from theoretical calculations and elucidate the contribute of the single diasteroisomer on binding affinity, we decided to undertake the enantioselective synthesis of R-Elte421 and S-R-Elte421. Using boranes with the appropriate absolute stereochemistry, we successfully synthesized alcohols (S)-7 and (R)-7 as enantiopure compounds, crucial intermediates to obtain diasteroisomers (2) and (3), separately (Scheme 3.2 and 3.3).

Scheme 3.2. Synthesis of R-Elte421 (2)

Allylboration of 3-phenylpropionaldehyde (6) with (+)-β-allyldiisopinocampheylborane in Et2 O-dioxane mixture was led at -100°C for 1 h in freshly distilled diethyl ether. The following oxidation with 10% H2O2 and a 3M NaOH solution at reflux provided (3S)-1-phenylhex-5-en-3-ol ((S)-11) as enantiopure compound, after purification by flash chromatography to get rid of isopinocampheol, side product from allylborane. In the next step a careful use of Pd/C catalyst had to be done. A catalytic amount was strongly suggested since the use of a stoichiometric quantity led to the oxidation of the

198 alcoholic moiety to ketone instead of reducing of the double bond on alkyl chain as desired. This side reaction was already described in literature.³⁶ The stereochemistry at position 3 of (R)-7 was assigned by optical rotatory power in EtOH, comparing it with the value reported in literature.^37,38 While (S)-11 has a S stereochemistry, an inversion of configuration at carbon 3 was observed after the reduction of the alkene. The R configuration was preserved during all the synthetical procedure. The last steps, including an initial coupling with N-Boc pipecolic acid, deprotection and final coupling with phenylglyoxylic acid, were the same reported for the synthesis of Elte421 and allowed to obtain R-Elte421 (2) in a good yield (Scheme 3.2).

Scheme 3.3. Synthesis of S-Elte421 (3)

For the synthesis of S-Elte421 (3), we exploited the procedure described above (Scheme 2), excepted from using the (-)-β-allyldiisopinocamphenylborane instead of the (+)-β-allyldiisopinocamphenylborane to obtain (R)-11 (Scheme 3.3). NMR and IR spectra are comparable to the ones of R-Elte421 (2).

Amides

In order to replace an ester group with an amide at position 3, we converted (3R/S)-1-phenylhexan-3-ol(7) in a primary amine (14), through an intermediate conversion in an azide derivative (13) (Scheme 3.4).

Scheme 3.4. Synthesis of ElteN420 (4)

(3R/S)-1-phenylhexan-3-ol (7) was first activated to the correspondent tosylate (12), using p-tosyl chloride (2 equiv.) in 2 mL of dry pyridine. The ¹H-NMR spectrum of the crude showed the presence

199 of p-tosyl chloride as impurity, not removable by flash chromatography at this stage. The crude was used and the impurity was easily removed in the next step, that provided azide (13), by treatment of the tosylate with NaN3 (8 equiv.) in dry DMF, overnight. The presence of (13) was confirmed from IR spectrum that showed a very strong signal at 2099 cm^-1 corresponding to N3 asymmetrical stretching.

In the next step, all the attempts of reduction, using triphenylphosphine in a 30% THF solution in water, went frustrated.³⁹ Azide (13) was successfully reduced by a catalytic hydrogenation, providing (3R/S)-1-phenylhexan-3-amine (14). The above described procedure closely resembled the one used to obtain ester derivatives, except for the last in which we preferred to use a coupling agent as DIC to insert phenylglyoxylic acid, instead of activating it to the correspondent chloride. The procedure allowed us to obtain ElteN420 (4) as a 1:1 racemic mixture (Scheme 4). The ¹H-NMR spectrum of ElteN420 appeared quite complex due to the presence of two rotamers (1:1.3 ratio), each as 1:1 mixture of diastereoisomers.

Surprisingly, the synthesis of the most potent inhibitor (5) required the easiest syntetic procedure (Scheme 3.5). Once synthesized for the first time ElteN378, different coupling agents and reaction conditions have been tested in order to optimize the procedure, increasing the final yield and shortening the required times, as discussed above.

Scheme 3.5. Synthesis of ElteN378 (5)

First procedure: commercially available 3-phenylpropan-1-amine (16) was activated with 2 equiv. of DIC in dry DCM and reacted with N-Boc pipecolic acid (2 equiv.) and a catalytic amount of DMAP (0.1 equiv.). TLC analysis of the reaction mixture after 16 hours still showed the presence of the starting material (16), while a massive white precipitate of DCU appeared in the flask. The reaction was thus stopped and the crude was purified by flash chromatography and several acid washings to get rid of the DCU. The desired product (17) was isolated in 26% yield, also due to the challenging work-up. N-Boc protecting group of (2S)-tert-butyl 2-(3-phenylpropylcarbamoyl)piperidine-1-carboxylate (17) was carefully removed with an excess of a TFA solution in DCM (5 equiv.), added over a period of 4 hours at 0°C, due to the decomposition of the product caused by a strong acid environment.

Intermediate (18) was directly used without further purification since the ¹H NMR spectrum showed an acceptable purity. The final coupling reaction was realized using the same DIC-DMAP in DCM protocol above described. Again the purification was not straightforward, requiring two flash chromatographes and ElteN378 (5) was obtained in 36% yield.

Optimized procedure: initial amidation was led using HATU, instead of DIC as coupling agent.

Primary amine (16) was reacted with N-Boc pipecolic acid (1 equiv.), in a DIPEA solution in dry DMF. Pipecolic acid was activated with an excess of HATU (6 equiv.). Side products of HATU were easily removed from the crude by flash chromatography to obtain (17) in a very good yield (80%). The following step involving the removal of N-Boc protecting group was achieved as above described and allowed to obtain (18) in 72% yield. Regarding the final amidation, we decided to first activate in a