WO2016020603A1

WO2016020603A1 - Industrial process for the synthesis of calixarene compounds

Info

Publication number: WO2016020603A1
Application number: PCT/FR2015/052084
Authority: WO
Inventors: Julien DAUVERGNE
Original assignee: Calixar
Priority date: 2014-08-04
Filing date: 2015-07-28
Publication date: 2016-02-11
Also published as: FR3024448A1; FR3024448B1

Abstract

Process for producing a calix-[z]-arene in which: - a calix-[z]-arene in which R¹=R²=R³=R⁴=X¹=X²=X³=X⁴=H is supplied, - step (a): - this calix-[z]-arene is treated with an alkylating agent transmitting the R⁴ residue, so as to obtain a reaction mixture comprising the monoalkylated calix-[z]-arene in which R¹=R²=R³=R⁴=X¹=X²=X³=X⁴=H, - this reaction mixture is subjected to a separation that is sufficient to remove most of the unreacted alkylating agent; - step (b): this reaction mixture is subjected to a reaction which substitutes each of the positions X¹=X²=X³= H with a tertiary amine (R⁵)₂N-CH₂- in the presence of a secondary amine (R⁵)₂NH so as to form a reaction mixture comprising a calix-[z]-arene in which R¹= R² = R³ = H, X¹ = X² = X³ = -CH₂-N(R⁵)₂, X⁴ = H; - step (c): - this reaction mixture is treated with a second alkylating agent, - this reaction mixture is subjected to a cyanation so as to form a reaction mixture comprising a calix-[z]-arene in which R¹ = R² = R³ = H, X¹ = X² = X³ = -CH₂-CN, X⁴ = H; - the calix-[z]-arene in which R¹ = R² = R³ = H, X¹ = X² = X³ = -CH₂-CN, X⁴ = H is isolated; - step (d): said calix-[z]-arene is subjected to a basic hydrolysis so as to obtain the target calix-[z]-arene.

Description

INDUSTRIAL PROCESS FOR THE SYNTHESIS OF COMPOUNDS

calixarene

TECHNICAL FIELD OF THE INVENTION The invention relates to the field of organic chemistry, namely the synthesis of calixarenic compounds. More particularly, it relates to a novel process for synthesizing calixarenic compounds which can be industrially implemented for larger scale synthesis.

STATE OF THE ART

Document US 201 1/0144314 discloses the calixarenic compounds described by formula (I) below:

where p is 1, 3, 5 or 6; where R ¹ , R ² , R ³ and R ⁴ may be an H atom or an alkyl chain (linear or branched) from C 1 to C ₁₂ ; where X ¹ , X ² and X ³ may be a group - (CH ₂ ) _m - CO ₂ Na (where m is an integer from 0 to 10).

Some molecules of this family of calix- [z] -arenes (where z denotes the number of benzene rings) form well-defined micelles in aqueous solution under certain conditions when for example: n = 4, R ¹ , R ² , R ³ = H, R ⁴ = heptyl chain or higher and m = 1. These micelles formed from anionic calixarenic compounds can complex various hydrophobic molecules. Many publications explore the formation of micelles by calixarenic compounds and the possible technical applications of this phenomenon, for example DNA encapsulation [Rodik, RV et al., "Virus-Sized DNA Nanoparticles for Gene Delivery Based on Micelles of Cationic Calixarenes ", Chem. Eur. J. 17, 5526-5538 (201 1)]. Thus, the patent applications EP 2 307 1 16 A1 and US 201 1/0144314 (National Center for Scientific Research and University Claude Bernard of Lyon) describe the use of certain calixarenic compounds of the type C4Cn_CO2Na of general formula (II) as detergents for the extraction, solubilization and stabilization of membrane proteins in aqueous solution.

where R is an alkyl chain of formula C _n H ₂ n ₊ 1 and n an integer between 1 and 16 representing the number of carbon constituting the alkyl chain Membrane proteins are targets of interest for the development of new drugs. The study of their structure, and in particular their crystallization, requires the temporary maintenance of these amphiphilic macromolecules in an aqueous system without altering their quaternary structure. The use of other calix- [z] -arenes for this purpose is described in WO 2010/1 16055 (National Center for Scientific Research).

In comparison with commercial detergents (see, for example, RMGaravito and S. Ferguson-Miller, "Detergents as Tools in Membrane Bioechmistry", published in J. Biol Chem 2001, vol.276, p.32403-32406, see M. Caffrey, "Membrane Protein Crystallization", published in J. Structural Biology, vol.142 (2003), p.108-132, see G. Privé, "Detergents for the stabilization and crystallization of membrane proteins", published in Methods, vol. .41, p.388-397 (2007)), the calixarenic compounds make it possible to maintain the extracted membrane proteins in aqueous solution in a structural state close to their native state. The formed micelles reconstitute around proteins a structural environment similar to that of the cell membrane. The use of calixarenic detergents allows the crystallization of membrane proteins in a native state and the structural study by X-ray diffraction techniques.

However, the resolution of membrane protein structures by crystallography remains a long and tedious process. The necessary precondition is the availability of a crystal; this work often requires numerous crystallization tests. It is therefore desirable to have calixarenic detergents at low cost, in greater quantity and with a better level of purity to make the experiments more reliable. Currently, the known synthetic processes of these detergents (see for example: K. Suwinska et al., "Trianionic calix [4] arene monoalkyl derivatives: synthesis, solid-state structures and self-assembly properties", published in New J. Chem., Vol.32, p.1988-1998 (2008), CDGutsche et al., "Calixarenes, 4. The synthesis, characterization, and properties of the calixarenes from p-tert-butylphenol", J.Am.Chem.Soc , 1981, 103, p.3782-3792; CDGutsche. & JAlevine, "Calixarenes." 6. Synthesis of a functionalizable calix- [4] -arene in a conformational rigid cone conformation ", J.Am.Chem.Soc, 1982, 104, p.2652-2653) involve several inappropriate steps to perform a large-scale production because of the low kinetics of reaction, the difficulties of solution of the intermediates or the difficulties of treatment of the reaction mediums (formation of foams, demixing delicate phases, ...). The synthesis also involves expensive and / or toxic reagents.

In the state of the art, one of the critical points concerns the alkylation step (a) of the process whose synthesis efficiency is poor because of the difficulties of separation of the product of interest in the reaction mixture which contains molecules. polarity very close.

While the purification by chromatography of a mixture of molecules difficult to separate is possible for small quantities of product by laboratory techniques, it represents a very significant cost factor on a larger scale.

Given the increasing demand for calixarenic compounds, and knowing that the separation techniques currently used for the preparation of small quantities can hardly be scaled up, it is necessary to have a less expensive preparation process for these molecules. calixarenics with good purity.

The present invention seeks to solve this problem. It presents a novel process suitable for larger scale production (typically greater than 20 g of final product) of known calixarenic compounds at an acceptable cost.

Object of the invention

A first object of the invention is a method of manufacturing a calix- [z] -arene (called "calix- [z] -arene target") structure

where: X ¹ = X ² = X ³ = -CH 2 -CO 2 C (with X = H or Na or K),

X ⁴ = H,

R ¹ = R ² = R ³ = H,

R ⁴ = alkyl and preferably linear C 1 to C ₂₄ alkyl (and even more preferably C ₂ to C ₆ ),

p is an integer between 1 and 5, preferably odd, and preferably equal to 1 or 3, knowing that z is equal to (3 + p),

in which process:

an "initial" calix- [z] -arene is supplied with R ¹ = R ² = R ³ = R ⁴ = H and X ¹ = X ² = X ³ = X ⁴ = H,

in a first step (a):

said initial calix- [z] -arene is treated with an excess of a first alkylating agent

capable of transmitting the radical R ⁴ , in the presence of a weak base which is preferably a hydrogen carbonate of an alkaline element, and preferably NaHCO ₃ , to obtain a first reaction mixture (called "reaction mixture of the step (a) ) ") Having the so-called" monoalkyl "calix- [z] -arene with R ¹ = R ² = R ³ = H and X ¹ = X ² = X ³ = X ⁴ = H, and R ⁴ is the introduced alkyl radical by said first alkylating agent,

said reaction mixture of step (a) is subjected to separation sufficient to remove most of the unreacted alkylating agent;

but insufficient to isolate said monoalkylated calix- [z] -arene from the other calix- [z] -arenes present in said reaction mixture of step (a), the result of this separation being called the "reaction mixture of the step (a) summarily purified "; in a second step (b), said reaction mixture of step (a) summarily purified is subjected to a reaction (typically a Mannich reaction) capable of substituting each of the positions X ¹ = X ² = X ³ = H by a tertiary amine of the type (R ⁵ ) ₂ N-CH ₂ - (where R ⁵ is an alkyl group, and preferably methyl (more preferred) or ethyl), in an acid medium in the presence of an aldehyde (preferably formaldehyde) and an amine (preferably secondary (R ⁵ ) ₂ NH (where R ⁵ is an alkyl group, and preferably methyl (more preferred) or ethyl)), to form a second reaction mixture (called "reaction mixture"). 'step (b)') having a calix- [z] -arene with R ¹ = R ² = R ³ = H and X ¹ = X ² = X ³ = -CH ₂ -N (R ⁵ ) ₂ and where R ⁴ is the alkyl group introduced in step (a) by said first alkylating agent, and X ⁴ = H;

in a third step (c):

said reaction mixture of step (b) is treated firstly with a second alkylating agent, preferably methyl iodide,

and then said reaction mixture is subjected to a cyanation reaction to form a third reaction mixture (called "reaction mixture of step (c)") comprising a calix- [z] -arene with R ¹ = R ² = R ³ = H and X ¹ = X ² = X ³ = -CH ₂ -CN and where R ⁴ is the alkyl group introduced in step (a) by said alkylating agent, and X ⁴ = H;

said reaction mixture of step (c) is subjected to separation to isolate the calix- [z] -arene with R ¹ = R ² = R ³ = H and X ¹ = X ² = X ³ = -CH ₂ -CN and wherein R ⁴ is the alkyl group introduced in step (a) by said first alkylating agent, and X ⁴ = H;

in a fourth step (d), said calix- [z] -arene isolated in step (c) is subjected to basic hydrolysis to obtain the target calix- [z] -arene with X = H.

In a fifth step (e), said target calix- [z] -arene is converted with X = H into the carboxylate salt, which is soluble in water: thus an aqueous solution of said calix- [z] - target arene likely to be used directly for the extraction and solubilization of membrane proteins.

According to an advantageous embodiment, the amounts of reactants of said secondary amine of the type (R ⁵ ) ₂ NH and of said aldehyde are determined from a ¹ H NMR spectrum of the reaction mixture of step (a) summarily purified.

In step (b), said secondary amine is advantageously diethylamine, and said aldehyde is advantageously formaldehyde. In another advantageous embodiment, which can be combined with the above, in step (b), an amount of acetic acid corresponding to 8 to 12 equivalents of reagent per reactive position of calix [z] arene is used to acidify the mixture. medium, and an amount of dimethylamine corresponding to 2 to 4 equivalents of reagent per calix [z] arene reactive position, and an amount of formaldehyde corresponding to 2 to 4 equivalents of reagent per calix [z] arene reactive position.

Preferably, in step (a), an excess of said first alkylating agent is used, and preferably at least 1, 3 and preferably at least 1.4 equivalents of said first alkylating agent are used.

In another advantageous embodiment, which can be combined with the preceding, in step (c) after the addition of said second alkylating agent is allowed to react at a temperature between 50 ° C and 100 ° C, preferably between 65 ° C. and 95 ° C. (more preferably still between 70 ° C. and 90 ° C.) for a duration of at least 12 hours, preferably of at least 24 hours and even more preferably of at least 36 hours. h.

Advantageously, in step (c), an amount of the second alkylating agent corresponding to 1.0 to 1.2 equivalents per tertiary amine function and / or a cyanide of an alkaline element with a quantity of cyanide is used. corresponding to 1.5 to 2.5 equivalents per tertiary amine function. In step (a), said summarily purified reaction mixture advantageously comprises less than 5 mol%, preferably less than 2 mol%, and still more preferably less than 0.5 mol%, of said first alkylating agent.

The process according to the invention is advantageously used for the preparation of target calix- [z] - arenes characterized by z = 4, and in which R ⁴ is a linear C ₂ to C 16 and preferably C ₄ to C ₁ alkyl radical. ₆ .

figures

FIG. 1 shows a typical ¹ H NMR spectrum (200 MHz) in CDCI ₃ at a concentration of 40 mg / ml of the mixture recovered after purification during the step of alkylation (a) of H4C40H with iododecane, containing the unreacted H4C40H compound, the alkylated products MonoC4C10 and BisC4C10.

FIG. 2 shows the ¹ H NMR spectrum (200 MHz) of the Cyano-C4C10 compound in solution in the CDCl ₃ obtained with the synthesis method set up on a mixture of compounds during steps (b) and (c). FIG. 3 shows the ¹ H NMR spectrum (200 MHz) of the C4C10 ₂ CO ₂ H product in solution in CDCl ₃ after purification on a C18 HPLC preparative column. Figure 4 shows the chromatogram of the product C4C10_CO ₂ Na after basification of the carboxylic acid form at pH 8 and lyophilization of the solution.

Description

We give here a general description of the process according to the invention by using a very simple example, namely the preparation of calixarenic compounds of the C4Cn_CO2Na type whose structure corresponds to the molecule (II) with n = 1 in which X ¹ , X ² and X ³ are -CH ₂ CO ₂ Na groups, wherein R ¹ , R ² and R ³ are OH groups, and wherein R ⁴ is an alkyl chain. In a second section, we describe the methodology for the characterization of a mixture of products by ¹ H NMR during the alkylation step (a) and the use of this mixture of compounds to simplify the procedure for synthesizing the compounds. C4Cn_C02Na. Finally, we will present a general procedure for the synthesis of compounds.

Calixarenic molecules are conventionally designated in different ways; one of the main information is the number of phenolic cycles. Thus we speak of calix- [z] -arenes, where z denotes the number of phenolic units with their bridge - CH ₂ -. We use here the abbreviated nomenclature C4Cn_C02Na to designate the calixarenic molecules: the code "C4" designates a calixarenic molecule composed of four phenolic units, the code "Cn" designates the number of carbons n on the aliphatic chain C _n H _{2n + 1} on the lower ring and the code C02Na indicates the presence of carboxylate groups -CH ₂ -C0 ₂ ^" on the upper ring in the form of the sodium salt.

1. General presentation of the process according to the invention

According to the invention, the synthesis of calixarenic detergents is carried out according to the reaction sequence described below from the raw material 1 called H4C40H:

4, Cyano-C4Cn

The process for synthesizing detergents C4Cn_CO2Na is carried out starting from compound 1 H4C40H, the preparation of which, in a manner known to those skilled in the art, in 2 steps (see CD Gutsche et al, "Calixarenes". p-tert-butylcalix [4] arene, J. Org Chem, (1986) vol 51, pp 742-745, CD Gutsche et al, "Calixarenes 12: The synthesis of functionalized calixarenes", Tetrahedron, (1986) Vol 42: 1633-1640). The synthesis of this compound 1 is now well controlled on a large scale (> 300 g) and does not require purification steps by flash chromatography but can be done simply by precipitation / crystallization processes.

According to the state of the art, the selective introduction of an alkyl chain on one of the phenol functions of the compound (1) is carried out with an excess of RI alkyl iodide in the presence of cesium fluoride (see: LC Groenen et al., "Synthesis of monoalkylated calix [4] arenes via direct alkylation." Tetrahedron, vol 47, pp. 8379-8384 (1991), C. Mbemba et al., "Calix [n] arenes as components for the Construction of micellar Systems: synthesis and self-assembly properties of 5, 11, 17-Tris [(dimethylamino) methyl] -25- monoalkoxy-26,27,28-trihydroxycalix [4] arene derivatives. ", J Incl Phenom Macrocycl Chem 61: 29-40 (2008).

The process of the present invention improves the state of the art by: (i) replacing CsF with inexpensive and nontoxic sodium hydrogencarbonate NaHCO ₃ , (ii) reducing the number of equivalents of the alkylating agent (preferably an iodoalkane RI) of 10 to 1.5 and (iii) extending the reaction time to 5 days. according to According to the invention, the alkyl iodide reagent can be replaced by another alkylating agent, and in particular by the corresponding alkyl bromide.

The Applicant has found that the use of an alkali metal hydrogencarbonate (preferred: Na, also conceivable: K or Cs) promotes the formation of a monoalkylated calixarene (MonoC4Cn) target at the expense of bisalkylated calixarene (BisC4Cn), while a carbonate (such as Na ₂ CO ₃ ) leads to a higher level of bisalkylated calixarene.

The alkylation step (step (a)) is represented by reaction scheme 1 below. It leads to a product mixture composed of unreacted starting material H4C40H (1), and MonoC4Cn and BisC4Cn alkylation products.

At this stage, a conversion level of approximately 90-95% of the compound H4C40H and a synthesis yield of 70-90% for the compound MonoC4Cn and 5-10% for the compound BisC4Cn are typically obtained.

Reaction Scheme 1: Step (a)

Alkylation of Compound (1) with RI Alkyl Iodide

H4C40H unreacted

BisC4Cn where R is an alkyl chain and n is an integer from n = 1 to 16 corresponding to the number of carbon atoms constituting the linear aliphatic chain R.

This step (a), even carried out under the improved conditions according to the invention, leads to a mixture of different calixarenic molecules, such as the method according to the state of the art. These calixarenic molecules have very similar polarities but different from the polarity of the excess alkylating agent. Indeed, the calixarenic molecules of this mixture (H4C40H, MonoC4Cn (2) and BisC4Cn) have thin-layer chromatography (TLC) very close Rf frontal reports, which complicates their separation by flash chromatography. By way of example, the alkylation reaction with iodoheptane leads to the calixarenic compounds H4C40H, MonoC4C7 and BisC4C7 which respectively have the frontal ratios 0.505, 0.536 and 0.67 on TLC with the eluent CH ₂ Cl ₂ / Cyclohexane (1: 1 ).

One of the key ideas of the present invention is to refrain from isolating the target molecule (2) from the mixture of calixarenic molecules, but to use for the next step of the synthesis directly the mixture of calixarenic molecules resulting from the step d alkylation (step (a) after removing the remainder of the alkylating agent which had been used in excess.

More specifically, according to the invention, it is sufficient at this stage of a summary separation which is not intended to obtain the compound of interest MonoC4Cn (2) perfectly pure but quickly remove the alkylating agent on a gel of silica eluting with cyclohexane and then isolating all the fractions containing the product of interest after elution with a CH ₂ Cl ₂ / cyclohexane (1: 1) eluent, even if these fractions are impure and contaminated by the product. starting material H4C40H (1) unreacted or the product BisC4Cn. This mixture of compounds will be advantageously separated at a later stage of the synthesis when the products formed of this mixture according to the reaction sequence will have a much better difference in polarity between them.

The determination of the mass composition of the mixture can be carried out by NMR (Proton Magnetic Resonance) spectrometry analysis of the proton. This will be described in greater detail below.

Thus, according to the invention, it is precisely determined at the alkylation stage the molar proportions of the compounds constituting the mixture to adapt accordingly the amounts of reagents of the multi-step synthesis process with this mixture instead of a pure compound.

The functionalization of the upper part of the calixarenic crown follows the same synthetic methodology as the state of the art developed by Gutsche et al.

The next step (b) shown in Reaction Scheme 2 below is a Mannich reaction which involves introducing dimethylamino methyl (DMAM) functions on the aromatic positions of the calix- [4] -arene as a function of the phenol functions. This reaction is carried out on the mixture obtained in step (a) after having separated from the excess of RI and isolated mainly MonoC4Cn product.

Reaction Scheme 2: Step (b)

Mannich reaction on the mixture obtained in step (a) after removal of the alkylkation agent

2, MonoC4Cn

3, DMAM-MonoC4Cn

MACRs-BisC4Cn

According to the invention, the molar amount of reactive positions (ie the number of carbons at the para position of the unsubstituted, free phenolic functions) of the mixture obtained in step (a) is firstly determined in order to adjust the amounts of reagents. This calculation is made from the molar proportions of the mixture of step (a) calculated by ¹ H NMR analysis and will be explained in greater detail below.

At this stage of the process, a mass of quantitative product is formed relative to the proportions of the mixture engaged during the reaction. By way of example, a mixture containing 3.3 g of H4C40H (M = 424.5 g / mol, 7.73 mmol), 33.2 g of MonoC4C10 (M = 564.8 g / mol, 58.56 mmol) and 4.96 g of BisC4C10 (M = 705.0 g / mol; 7.04 mmol) will drive at a mass of approximately 54 g, ie 5.0 g of DMAM-C40H (M = 652.9 g / mol), 43.1 g of DMAM-MonoC4C10 (M = 819.2 g / mol) and 5.9 g of DMAM-BisC4C10 (M = 819.2) g / mol).

According to the invention, given the quantitative transformation yield of each of the species of the mixture, the reaction mixture obtained after treatment is directly engaged without purification phase in the cyanation step (c) which is represented in reaction scheme 3 below. -from below. It integrates two separate reactions, performed successively in the same container (approach called in English "one-pot reaction").

Reaction Scheme 3: Step (c)

Cyanation of the mixture obtained from step (b)

3, DMAM-MonoC4Cn ⁴ <Cyano-MonoC4Cn

DMAM-BisC4Cn Cyano-BisC4Cn

The process of this step uses one equivalent of Mel iodomethane per tertiary amine function Me ₂ NCH ₂ - present on the upper ring of the calix- [4] -arene, then two equivalents of cyanide ions per quaternary ammonium function Me ₃ N ⁺ CH ₂ - formed in situ in the reaction medium.

The present inventors have found that it is at this stage of the multi-step synthesis that the reaction mixture (Cyano-C40H, Cyano-MonoC4Cn and Cyano-BisC4Cn) is the most easy to separate by chromatographic techniques, thanks to the difference in polarity of the compounds following the introduction of the nitrile functions on the upper crown of the calixarenes. According to this process, 15-20 g of the compound (4) Cyano-MonoC4Cn can be effectively isolated according to the length of the aliphatic chain and with an excellent purity (> 95%) despite the reaction of a mixture of compounds when steps (b) and (c).

The compound (4) purified Cyano-MonoC4Cn then undergoes a hydrolysis reaction in a basic medium with a solution of hydroxide ions (NaOH or KOH) whose concentration is typically between 1 M and 3M. This step called here step (d), known as such, is represented by reaction scheme 4 below.

Reaction Scheme 4: Step (d)

Basic hydrolysis of Cyano-MonoC4Cn

4, Cyano-C4n5, C4n_C02H

Purification of the carboxylic acid (5) C4Cn_CO2H on a preparative HPLC column makes it possible to obtain a sample with a purity greater than or equal to 97% after concentration of the pure fractions and freeze-drying. The intermediate calixarene triacid (5) C4Cn_CO2H corresponding to the free acid is not usable in the state for membrane protein extraction tests. In this form, the compound is insoluble in water because of the carboxylic acid functions which give it properties comparable to a fatty acid. To make it soluble, it is necessary to deprotonate the carboxylic acid functions so as to condition the compound in its conjugated basic form (that is to say Na or K carboxylate salt) which is responsible for its detergency and will enable it to form micelles in aqueous solution.

Thus, the last step of the process referred to herein as step (e) shown in Scheme 5 is to convert the carboxylic acid C4Cn_CO2H to the sodium carboxylate salt C4Cn_CO2Na and then lyophilize the adjusted solution to pH 8. Reaction Scheme 5: Step (e)

Transformation of carboxylic acid C4Cn CO2H to sodium carboxylate salt 6

C4Cn C02Na

5, C4- C02H6, C4nCO2Na

The sodium carboxylate salt C4Cn_CO2Na is soluble in water up to 40% w / v. The lyophilized product is stored in the absence of moisture and can be used as such for the preparation of solutions for the extraction / purification of membrane proteins or any other application in biochemistry requiring the use of a detergent.

The process according to the invention can be used for the synthesis of compounds of calix- [z] -arenes type whose lower ring contains an ether group (-OR) and three hydroxyl groups. The method can be used for calix- [z] -arenes with z between 4 and 8, but gives the best results for z = 4; for higher values (eg z = 6) the yield of the monoalkylated form decreases and the process forms larger amounts of bisalkylated products.

After describing the process in its entirety, we describe some critical aspects.

2. Detailed description of certain critical aspects of the process according to the invention

2.1 Isolation and Quantitative Characterization of the monoalkylated product (2) MonoC4Cn of the mixture resulting from step (a)

As indicated previously, instead of isolating the perfectly pure intermediate (2) MonoC4Cn from the reaction mixture resulting from step (a) from the other calixarenic compounds, namely the unreacted starting material (1) H4C40H and of the secondary product BisC4Cn resulting from a double alkylation of the lower crown of the calix- [4] -arene, it is sufficient in the process according to the invention essentially to eliminate the excess of the alkylating agent (which is preferably an iodoalkane).

Indeed, the separation of said reaction mixture can be done only by flash chromatography on silica gel and it is found that the calixarenic compounds H4C40H, Mono-C4Cn and BisC4Cn of the mixture have very similar polarities according to the R _F frontal ratios observed on a TLC plate; because of this, the separation is very complicated, especially in large quantities. On the other hand, the alkylating agent (in this case R1) has a polarity that is very different from that of the calixarenic molecules. At this stage of the process, a brief and rapid separation by flash chromatography on silica gel using a high flow rate but a low separation power is sufficient to remove the alkylating agent but insufficient to separate the calixarenic molecules of the mixture. . Nevertheless, such a process is capable of treating large quantities of reaction mixture and isolating a product mixture enriched with the desired product; and therefore fully compatible with the restrictions of industrial production of C4Cn_CO2Na calixarene detergents given the increasing demand for the product needed to purify the membrane proteins and lead to their crystallization. According to one embodiment, this separation can be carried out by flash chromatography on silica gel using silica as a stationary phase and a CH ₂ Cl ₂ / cyclohexane mixture as a solvent.

By way of example, it is possible to proceed as follows: The reaction crude resulting from stage (a) after treatment (dry residue) is solubilized with the minimum of CH ₂ Cl ₂ and then adsorbed on an amount of silica equivalent to the mass of said dry residue (solid deposit). The solid deposit is transferred to an empty column with a nominal capacity of 120 g and purified in one go with an Interchim brand PuriFlash ® 4100 automated flash chromatography device.

By way of example, it is possible to use a Chromabond® IR-Si column of 800 g of irregular silica conditioned with cyclohexane to purify a reaction crude of 50-60 g. The solid deposit is chromatographed at an elution rate of 160 mL / min a CH ₂ Cl ₂ / cyclohexane gradient (0: 10 → 4: 6). Each fraction is analyzed by TLC with a CH ₂ Cl ₂ / cyclohexane eluent (1: 1) so as to collect all the fractions (even impurities) containing the monoalkylated (2) MonoC4Cn calixarenic product. In the eluent CH ₂ Cl ₂ / Cyclohexane (1: 1), the product typically has a R _F value which varies from 0.35 (for an alkyl chain R = methyl) to about 0.63 (R = hexadecyl). ). Advantageously, a more polar eluent is used for the elution of shorter chain compounds so as to obtain a R _F around 0.4-0.5. After concentration of the solvent, pooled fractions are obtained a solid composed of a mixture of calixarenic molecules MonoC4Cn, BisC4Cn and H4C40H. The numerical mass y of solid will be used in the following section to calculate the mass proportions of the mixture.

2.2 Analysis of the mixture obtained at the end of the alkylation step (a) by spectroscopy

Proton NMR

The mixture obtained after purification in the alkylation step (a) is dissolved in CDCl ₃ (20 mg per 500 μl of deuterated solvent) and then analyzed by ¹ H NMR spectrometry.

FIG. 1 shows a typical ¹ H NMR spectrum of the mixture from which the molar ratios of the molecules can be precisely characterized from the integrations of the phenol functions of the calixarenic molecules which will present different chemical shifts.

The structural conformations adopted by the calixarenic compounds in the CDCl ₃ of the mixture after purification in the H4C40H alkylation step are shown below.

H4C40H conformation "cone"

BisC4Cn

The compound H4C40H displays on the spectrum a singlet at 10.3 ppm corresponding to 4H. The compound MonoC4Cn exclusively adopts a cone conformation and displays on the spectrum 2 singlets at 9.6 and 9.9 ppm respectively corresponding to 2H and 1H, that is to say protons H _b and H _a (see Figure 1). Finally, the compound BisC4Cn displays on the spectrum 3 singlets at 8.1, 8.3 and 9 ppm, all corresponding to 2H, but different conformations of the molecule, namely the 1, 3-alternation conformations, cone and 1, 2-alternating. The proportions for each of the conformations may vary according to the length of the aliphatic chain R grafted on the calix- [4] -arene.

Thus, from the integrations of these signals and the weighing mass weighed therein, the masses of each of the compounds in the mixture and their amounts of material can be calculated using the mathematical development below.

In the following, the index 0 refers to the unalkylated product H4C40H, the index 1 to the monoalkylated product MonoC4Cn and the index 2 to the bisalkylated product BisC4Cn. The notation i means "integration". To determine the proportions of the compounds in the mixture, the integrations of the peaks mentioned above are used on the ¹ H NMR spectrum. The presence of traces of solvent is neglected; the mass of these traces is negligible compared to the total mass of calixarenic products.

For mathematical resolution, it is necessary to modify the integration values according to the number of protons associated with the signal and also to add the integrations of the conformations corresponding to the same molecule. For the compound H4C40H, the modified integration i ₀ is equal to the integration of the singlet at 10.3 ppm divided by 4. For the compound MonoC4Cn, the modified integration is directly equal to the integration of the singlet at 9.9 ppm without modification of the value. For the compound BisC4Cn, the modified integration i ₂ is equal to the sum divided by 2 of the singlet integrations located 9.1, 8.3 and 8.2 ppm.

The calculation method used depends on the number of calixarenic compounds recovered in the mixture after purification during the alkylation step. Indeed, in some cases, it will be possible to separate during purification by flash chromatography BisC4Cn bisalkylation compound.

In the case of a mixture of two compounds (in the example presented below: the compounds H4C40H and MonoC4Cn), we have ax (equation I), where x is the ratio of the modified integrations i ₀ / ii and mo + m ^ y (equation II), where y is the mass of the isolated mixture.

Equation (I) can be written: (m ₀ .M ₁ ) / (Mo.m ₁ ) = x then in the form mo = (xm ₁ .Mo) / M ₁ (equation III).

By combining (II) and (III), we can write: m ₁ + [(xM ₀ ) / M ₁ ] .m ₁ = y (equation IV). From equation (IV), the value of the term mi is isolated and calculated and rm ₀ is calculated from equation (II) and the calculated value ΓΤΗ.

In the case of a mixture of three compounds (in the example presented below: the compounds H4C40H, MonoC4Cn and BisC4Cn), the following equations are used to calculate the term rm ₀ (taken by default in the example): no n ^ x (Ι '), where x is the ratio of the modified integrations i ₀ / ii

η ₀ / η ₂ = χ '(II'), where x 'is the ratio of the modified integrations io / i ₂

mo + m ₁ + m ₂ = y (ΙΙΓ), where y is the mass of the isolated mixture.

For the material quantity ratios, the numerator is given the same indices so that the term m having the index of the numerators (in the example here 0) can be calculated thereafter.

The equation (Ι ') can be written: (m ₀ .M ₁ ) / (Mo.m ₁ ) = x then in the form m ₁ = M ₁ /(Mo.x).m ₀ (IV), and the equation (II ') can be written: (mo.M ₂ ) / (Mo.m ₂ ) = x' then in the form m ₂ = M ₂ /(Mo.x').m ₀ (V ).

By combining the equations (ΙΙΙ '), (IV) and (V), we can write: mo + [M ₁ /(Mo.x)].mo+[M ₂ /(Mo.x')].m ₀ = y (VI '). From equation (VI '), the value of the term rm ₀ is isolated and calculated _.

As before, one calculates mi or m ₂ then uses the equation (ΙΙΙ ') to determine the missing term.

In this way, one can determine both the masses of the compounds present in the mixture as well as their amounts of material. Calculation of the quantities of reagents to be used during the Mannich reaction in step (b)

During the Mannich reaction, the amounts of Me ₂ NH, formaldehyde and acetic acid reagents are adjusted accordingly according to the composition of the mixture H4C40H, MonoC4Cn and BisC4Cn. To do this, the number of equivalents of reagents is adapted to the quantity of reactive position material n _R on the calixarene molecules. These correspond to the para positions of the unsubstituted phenolic functions. This amount of material can be calculated as follows: n _R = (4-n ₀₎ + (3-n ₁₎ + ₍₂ 2.n)

with n ₀ , the amount of H4C40H material in the mixture

n-ι, the amount of MonoC4Cn material in the mixture

n ₂ , the amount of BisC4Cn material in the mixture

From this total number of moles of reactive positions n _R in the mixture, the reaction medium is preferably added in the order indicated, while stirring:

A quantity of material of glacial acetic acid 100% equivalent to 10. n _R, 10 reactive equivalents per reactive position calix- [4] -arene,

A quantity of material dimethylamine 40% w / v in water equivalent to 3-n _R, or 3 reactive equivalents per reactive position calix- [4] -arene,

A quantity of material of formaldehyde 35% w / v in water equivalent to 3-n _R, or 3 reactive equivalents per reactive position calix- [4] -arene. The solution content of Me ₂ NH and HCHO reagents must be taken into consideration when calculating volumes. The reaction is carried out for several days at room temperature in an inert medium to ensure the complete transformation of the reactive positions and the introduction of tertiary amine functions. After treatment of the reaction medium, a quantitative mass is recovered with respect to the composition of the starting mixture.

2.3 Catalytic reaction in step (c) and separation of intermediate CvanoC4Cn

For carrying out this step, the values of the composition of the mixture employed during the Mannich reaction in step (b) are used by considering the quantitative transformation for each calixarenic compound in the mixture. The dried solid residue from step (b) after treatment is dissolved in DMSO at a concentration of 0.125 M (ie 8 ml / mmol of calixarene molecules). The in situ formation of the quaternary ammonium functions is carried out by the addition over a period of 15 minutes of an amount of iodomethane material Mel equivalent to n _R (ie one equivalent of Mel per tertiary amine function) and then the heating at 40.degree. ° C of the reaction medium with stirring to fully solubilize the solid. Once only the complete dissolution of the solid, a quantity of cyanide ions NaCN equivalent to 2.n _R is added at once to the reaction medium.

As indicated above, according to an essential aspect of the invention, the normal phase separation of the mixture of calixarenic products by flash chromatography is most effective during step (c) of cyanation. At this point, the difference in polarities between the calixarenic molecules is better following the introduction of the polar nitrile functions. This feature is used to purify 15-20 g batches of the Cyano-C4Cn compound (4) in a preparative manner rather than at any other stage of the process.

The PuriFlash ® 4100 automated flash chromatography system at Interchim can be used with a Chromabond ® IR-SI column of 1600g of irregular silica packaged in CH ₂ Cl ₂ to purify a reaction crude of 40-50 g. The solid deposit is chromatographed at an elution rate of 160 ml / min with an AcOEt / cyclohexane gradient adapted according to the length n of the aliphatic chain on the calix- [4] - arene (n <7, 0: 10 → 35 : 65 → 1: 1, n≥10, 0: 10 → 4: 6 → 6: 4).

The yield of the reaction should be 45-60%. Compound (4) Cyano-C4Cn is perfectly characterized by ¹ H NMR as well as electrospray ionization mass spectrometry and positive mode detection. According to the drying time and the power of the vacuum applied to dry the pure product (4) Cyano-C4Cn, it can be found in inclusion complex with a molecule of acetonitrile which is detected on the NMR spectrum ¹ H of the compound (4) in solution in CDCl ₃ according to the singlet at 2.1 ppm.

2.4 Purification on a preparative HPLC column of the compound (5) C4Cn CO2H and conditioning in the form of sodium carboxylate salt

In comparison with the state of the art, the present invention establishes the purification of the carboxylic acid (5) C4Cn_CO2H on a preparative column HPLC UPTISPHERE STRATEGY C18-3 Interchim (250 mm x 50 mm, particle size 10 μΜ) of C18 grafted silica stationary phase using the PuriFlash ® 4100 system (Interchim ®) and allows samples to be obtained with a purity greater than or equal to 97% after concentration of the pure fractions and lyophilization (see Figure 4). In step (d) after the treatment of the reaction medium, a 50% w / v solution of the residue in MeOH is produced which is purified in several injection runs with the eluent couple ACN HPLC grade (A). and H ₂ 0 MilliQ (B) and the method below:

o Elution gradient:

^■ X% of A for 14 minutes

^■ X at 100% of A in 1 minute

^■ 100% of A for 5 minutes

^■ 100 to X% of A in 1 minute

- X% of A for 5 minutes n = 1, X = 40; n = 3, X = 63; n = 5, X = 68; n = 7, X = 73; n = 9, X = 80; n = 10, X = 85; n = 12, X = 92 o Injection approximately 9 ml of a 50% w / v solution of reaction crude

injection valve with a 10 mL loop

o 230 nm detection

o Flow rate 120 mL / min

o Collector: 32 mL fractions, tubes

For each run of injection, each fraction of the main peak is analyzed by HPLC on an Interchim ® US10C183-250 / 046 analytical column with the following method: A: ACN + 0.1% TFA, B: H ₂ 0 + 0.1% TFA, isocratic elution X% of A; injection 50μί flow rate 1 mL / min, thermostatic column 25 ° C, detection 230 nm, run 30 min.

Fractions ≥ 97% purity by self-integration are pooled and concentrated to remove the solvent. The remaining suspension is lyophilized and gives a fine white powder perfectly characterized by ¹ H NMR (see FIG. 4). Assays were conducted in solution in d ₆ -DMSO C4Cn_C02H for compounds having an alkyl chain n <5 and in CDCl ₃ for those having an alkyl chain n> 5. The yield of the reaction is 70-85%.

3. Example of a complete procedure of the process

To carry out step (a), 30 g (70.7 mmol) of the compound (1) were solubilized in 100 ml of DMF and 6.3 g (1.0 equivalent equivalents) were added at 40 ° C. with stirring. of NaHCO ₃ under protective gas, then 1.5 equivalents of the alkyl iodide were added. It was allowed to react for 5 days and then poured into water at room temperature. The aqueous phase was extracted with CH ₂ Cl ₂ and the organic phases were washed with water and brine. The organic phases are combined and then dried over MgSO ₄ . The suspension is filtered and the cake washed with CH ₂ Cl ₂ . The filtrate is concentrated on the evaporator until an oily residue is obtained which can form a yellow paste.

To carry out step (b), the residue obtained at the end of step (a) was solubilized in CH ₂ Cl ₂ and added silica for chromatography; the removal of the alkylating agent by chromatography was carried out as described above in section 2.1. Then a ¹ H NMR spectrum of the isolated mixture was recorded; Figure 1 shows such a spectrum for R = C ₀ H ₂₁ . From this spectrum, the masses of calixarenic compounds were calculated present in the mixture and their amounts of material as described above in section 2.2. The amounts of reagents for the reaction were then calculated as described above in section 2.3 and then glacial acetic acid and the other reactants were added as described above. It was allowed to react for 5 days and the solvent evaporated. Then a solution of 10% Na ₂ CO ₃ (15 mL / mmol of product) was added very slowly with stirring. The aqueous phase was extracted with CH ₂ Cl ₂ . This organic phase was dried then filtered and the solvent was evaporated. The product has been kept away from moisture or reused immediately in step (c). To carry out step (c), a quantity of DMSO equivalent to 8 ml / mmol of product was added and bubbling argon in the reaction medium. The equivalent amount of iodomethane was added, as described above in section 2.4, the temperature was raised to 40 ° C to obtain a homogeneous solution, then the NaCN solution was added; the solution faded and became yellow-orange. The temperature was raised to 80 ° C and allowed to react for 2 days at this temperature. The reaction medium is cooled and then poured into an aqueous phase comprising a volume of water of about five times the volume of DMSO and a volume of 2N HCl equivalent to about twice the equivalent of NaCN. Precautions well known to those skilled in the art have been taken because of the release of very toxic HCN. The aqueous phase was extracted with CH ₂ Cl ₂ and these organic phases were washed with water and brine. The combined organic phases were dried over MgSO ₄ and filtered and the solvent was evaporated; a brown-brown solid was obtained. This solid was dissolved in CH ₂ Cl ₂ and then silica was added and the mixture purified as described above in section 2.5 to obtain a pure fraction of compound (4). Figure 2 shows the ¹ H NMR spectrum of this compound.

To perform step (d), the compound (4) was dissolved in ethanol (7 mL / mmol of product) and a solution of potassium hydroxide (3.5 mL / mmol of product corresponding to 12 equivalents) was added. The reaction mixture was heated for 2 to 3 days at 110 ° C and then poured into a mixture of ice and 1N HCl corresponding to about 2 equivalents of potassium hydroxide. It was stirred and extracted with CH ₂ Cl ₂ ; the CH ₂ CI ₂ phases were dried and the solvent was evaporated. For purification, the solid was dissolved in methanol. The purification was carried out on a preparative HPLC column of Uptisphere Strategy C18-3 type from Interchim (250 mm × 50 mm, 10 μηη particle size) with the PURIFLASH 4100 system from Interchim. Finally, a fine white powder (yield of the order of 70 to 85%) was obtained which was characterized by ¹ H NMR, mass spectrometry and HPLC. Figure 3 shows this spectrum for the compound called C4C10_CO2H with R4 = decyl. This synthesis was carried out with different aliphatic chains R, and here we indicate the values of R _f of the compounds C4n_CO2H (compound 5) in the eluent methanol / CH ₂ Cl ₂ (3: 97) + 1% acetic acid (v / v) depending on the length n of the aliphatic chain of the rest R:

n = 1, R _f = 0.098; n = 3, R _f = 0.105; n = 5, R _f = 0.118; n = 7, R _f = 0.118; n = 8, R _f = 0.138; n = 9, R _f = 0.151; n = 10, R _f = 0.145; n = 11, R _f = 0.151; n = 12, R _f = 0.151; n = 16, R _f = 0.151.

To perform step (e), compound (5) was solubilized in ethanol (6 mL / mmol of product) and neutralized with 1M NaOH solution to pH 7. The ethanol was evaporated and water (about 6 mL / mmol of product) was added, then the NaOH solution was added dropwise, controlling with a pH electrode to adjust the pH of the solution. The total amount of NaOH added was of the order of 3 to 4 equivalents, depending on the compounds as a function of their aliphatic chain R. The solution was filtered, the water removed by evaporating several times with water. ethanol, until a dry product (cream powder) with a yield between 90 and 100% is obtained. The water can also be removed by lyophilization. The purity of the product was monitored by HPLC (greater than 97%). The powder is soluble in water (up to typically 40% by weight) and can be used for biochemical tests. Figure 4 shows the chromatogram of the pure compound called C4C10_CO2Na with R4 = decyl. The characteristics of the main peaks are given below:

Picn ⁰ retention ^T YP ^e width area height height

# [min] [min] [mAUs] [mAU]%

3 5,, 327 BB. 0,, 1928 2. 605.63 1. .68162e-l .640e -3

4 6,, 044 BB 0. ,, 1954 7, 59563 6 · ..35546e-1 7 .696e-3

5. 6. 853 BB. 0-, 3413 14. 93139 6. .1049.1e-1 0. .0151

6 7,, 854 BB 0. .2844 8. 3363.5 3. .91550e-1 ^■ 8 .447e-3

7 8. .94.2 BV 0, .5148 182. 98718 5 _. .09731 0 .. .1854

8 10,, 092 VV 0,, 5428, 83. 14696 ^, 1.85553, 0, 0842.

9 11,, 733 VV 0,, 8200 583. 8: 2581 9.78596 Q, .5916

12 _. , _. 8.5.2 VB ^' 0,, 2.818 22 ^' . 94483 1.03831 0,, 0232

11, 16. 195 BV 0. .7106 9.576: 63e4 2106.31396 97. .0568

12, 514, VB. 0, .8646 44.0. 578.98 7.2541.2 g _, .4.46.4

13 20., 544 BB 0. ..5480 3.3 .. 75030 8. .7 u 42e-1 0. .0: 3 2

14, 21. 973 BV 0- .5734 50. .49228 1.087.99 0. .0.512

23., 56.3 ^' VB 1, .3539 515. 6553.3 4.7: 67: 23 0. .5.225.

27. 27. 958 BBA 1.23.37 878 .. 76929 8.9.1732 o., .8904

Claims

1. Process for producing a calix- [z] -arene (called "calix- [z] -arene target") of structure

where: X ¹ = X ² = X ³ = -CH ₂ -C O ₂ X (with X = H or Na or K),

X ⁴ = H,

R ¹ = R ² = R ³ = H,

in which process:

in a first step (a):

said reaction mixture of step (a) is subjected to separation sufficient to remove most of the unreacted alkylating agent; but insufficient to isolate said monoalkylated calix- [z] -arene from the other calix- [z] -arenes present in said reaction mixture of step (a), the result of this separation being called the "reaction mixture of the step (a) summarily purified ";

in a second step (b), said reaction mixture of step (a) summarily purified is subjected to a reaction capable of substituting each of the positions X ¹ = X ² = X ³ = H by a tertiary amine of type (R ⁵ ) ₂ N-CH ₂ - (where R ⁵ is an alkyl group, and preferably methyl (more preferred) or ethyl), in an acid medium in the presence of an aldehyde (preferably formaldehyde) and an amine (preferably secondary (R ⁵ ) ₂ NH (where R ⁵ is an alkyl group, and preferably methyl (more preferred) or ethyl)), to form a second reaction mixture (called "reaction mixture of step (b)") comprising a calix- [z] - arene with R ¹ = R ² = R ³ = H and X ¹ = X ² = X ³ = -CH ₂ -N (R ⁵ ) ₂ and where R ⁴ is the alkyl group introduced at step (a) by said first alkylating agent, and X ⁴ = H;

in a third step (c):

2. The method of claim 1, wherein is converted in a fifth step (e) said calix- [z] -arene target with X = H carboxylate salt.

3. A process according to claim 1 or 2, wherein the amounts of reagents of said (R ⁵ ) ₂ NH-type secondary amine and said aldehyde are determined from a ¹ H NMR spectrum of the reaction mixture of the step ( a) summarily purified.

The process according to one of claims 1 to 3, wherein in step (b) said secondary amine is diethylamine, and said aldehyde is formaldehyde.

A process according to claim 4 dependent on claim 3, wherein in step (b) is used an amount of acetic acid corresponding to 8 to 12 equivalents of reagent per calix [z] arene reactive position to acidify the medium, and a quantity of dimethylamine corresponding to 2 to 4 equivalents of reagent per calix [z] arene reactive position, and an amount of formaldehyde corresponding to 2 to 4 equivalents of reagent per calix [z] arene reactive position.

Process according to any one of claims 1 to 5, wherein in step (a) an excess of said first alkylating agent is used, and preferably at least 1, 3 and preferably at least 1, 4 is used. equivalents of said first alkylating agent.

Process according to any one of claims 1 to 6, wherein in step (c) after the addition of said second alkylating agent is allowed to react at a temperature between 50 ° C and 100 ° C, preferably between 65 ° C and 95 ° C (even more preferably between 70 ° C and 90 ° C) for a period of at least 12 hours, preferably at least 24 hours and even more preferably at least 36 hours.

Process according to any one of claims 1 to 7, wherein in step (c) an amount of the second alkylating agent corresponding to 1.0 to 1.2 equivalents per tertiary amine function is used.

Process according to any one of claims 1 to 8, wherein in step (c) a cyanide of an alkaline element is used with an amount of cyanide corresponding to 1.5 to 2.5 equivalents per tertiary amine function.

10. Process according to any one of claims 1 to 9, wherein said reaction mixture of step (a) summarily purified comprises less than 5 mol%, preferably less than 2 mol%, and even more preferably less than 0 mol%. 5 mol% of said first alkylating agent.

1 1. Process according to any one of Claims 1 to 10, in which the target calix- [z] -arene is characterized by z = 4, and R ⁴ is a linear C ₂ to C ₁₆ and preferably C ₄ to C ₆ alkyl radical. Ci ₆ .