**3.2.1 The salt-forming resolving agents**

#### **3.2.1.1 Structurally similar resolving agents**

Comparison of the results of the enantiomeric enrichments mentioned above let us to conclude that the difference between the enantiomeric excesses of the crystalline and the liquid phases is the greatest when the starting enantiomeric purity is near 50% (ee0 ≈ 50%). In the cases of ten separtions made by crystallization this enrichment (Δee = eecryst-eeliq) was

Selective precipitation of (*R>S*)-*N*-propionyl-phenylalanine (**PPA**) was effectuated starting from the aqueous solution of its sodium salt. Addition of less than equimolar amount of hydrochloric acid resulted in the crystallization of the excess of (*R*)-**PPA** to such an extent, that in certain cases the usually remaining racemic composition become unbalanced, too

Essentially the same phenomenon was observed at the fractionated precipitation of the

In light of these experimental data it should be mentioned, that during the purification of enantiomeric mixtures of *N*-acyl-aminoacids not only the suitable methods but also the subtituent on the molecule skeleton (acyl group) may determine the productivity of

The similarities between the enriching processes starting from enantiomeric mixtures or diastereoisomeric mixtures were already discussed above at the presentation of the solvent free methods. It is an interesting question: whether the widely used fractionated

The diastereoisomeric systems discussed bellow contain salts, molecular complexes and

Comparison of the results of the enantiomeric enrichments mentioned above let us to conclude that the difference between the enantiomeric excesses of the crystalline and the liquid phases is the greatest when the starting enantiomeric purity is near 50% (ee0 ≈ 50%). In the cases of ten separtions made by crystallization this enrichment (Δee = eecryst-eeliq) was

precipitation from a solvent will change this trend at the diastereoisomeric mixtures?

aqueous sol. addition of non eq. aqueous HCl sol.

enantiomeric mixture of (*R>S*)-*N*-propionyl-phenylglycine (**PPG**).

aqueous sol.

addition of non eq. aqueous HCl sol.

eesolid% eeliquid%

eesolid% eeliquid%

24.8 -4.3 24.3 12.4 83.3

17 -3.6 -4.8 1.2 0.9 -0.4 16 76

2.5 47.7 39.0 88.7 99.9

ee0%

ee0%

(therefore the (S) enantiomer is enriched in the filtrate)

COONa

NHCOCH2CH3

(*R*>*S*)-**PPA**

(*R*>*S*)-**PPG**

enrichment.

COONa

**3.2 Crystallization of diastereoisomers** 

**3.2.1 The salt-forming resolving agents 3.2.1.1 Structurally similar resolving agents** 

coordinative complexes as well.

NHCOCH2CH3

between 34-95%, (on the average 60%), while the average of the six greatest difference was 70%. This means that the purification of enantiomers by crystallization is a wery fruitful route (independently from the method applied (from melt to using a solvent) to enantiopure materials.

Another approach is the resolution of an enantiomeric mixture with a structurally similar resolving agent. It is the situation when one of the enantiomers of the racemic compound is transformed (with a minimal chemical transformation) into a reagent able to form diastereoisomeric salt with the initial racemic compound. It can be done if the aminoacid is *N*-acylated and one of the pure enantiomers is esterified, or if its carboxylic group is transformed into an amide, or changed with methyl group, respectively.

For example, the racemic *N*-acetyl-phenylglycine (**AcPG**) can be reacted with methyl (*S)* phenylglycinate (**MePG**). The resolution can be treated as a recrystallization of a quasi enantiomeric mixture with *ee0=*50% from water when the less soluble diastereoisomeric salt (namely, the heterochiral quasi-racemic mixture) crystallized containing (*R)*-**AcPG** in good enantiomeric excess (*ee*: 79%).

If the substituents of the compounds would be removed (Ac from **AcPG** → R and S and Me from **MePG** → S), the composition of the mixture would be

$$\mathbf{1R + 3S \equiv RS + SS}$$

Namely, the starting mixture would be an "enantiomeric mixture with ee= 50%". Therefore the above shown crystalline diastereoisomeric salt is a quasi-racemate.

The situation was almost the same when the *N*-acetyl-phenylalanine (**AcPA**) was reacted with methyl (*S)*-phenylalaninate (**MePA**) according to the preceding resolution. The obtained crystalline diastereoisomeric salt also will be a quasi-racemate, but its enantiomeric excess (ee: 93%) was even higher than that of the former case.

It was also observed, that the average *ee* and F values of diastereoisomeric salts obtained at the resolutions (in number 28 resolutions) of racemic *N*-acetyl-phenylalanine and phenylglycine (**AcPA** and **AcPG**) with structurally similar bases (resolving agents) correspond to the eutectic composition (*eeE* value) of the adequate racemate (in these cases the enantiomers of the racemic compounds form racemates). Consequently, if the resolution of a racemic compound is accomplished with structurally similar resolving agents (in water) the purity of the enantiomer obtained from the crystalline diastereoisomeric salt corresponds to the biner phase diagram of the enatiomeric mixture or converge to the

Separation of the Mixtures of Chiral Compounds by Crystallization 21

crystallized and the other isomer remains in the solution as a salt of the applied achiral additive. This method not only economizes a half part of the resolving agent, but the

Hereafter some examples of the above mentiuoned procedures, used in the pharmaceutical

The racemic intermediate of chloramphenicol (**AD.**HCl) is obtained as a hydrochloride salt during the synthesis, and it is dissolved in water (the free base is low-soluble in water). In this case the resolving agent (O,O'-dibenzoyl-(*R,R*)-tartaric acid *N,N*-dimethylamide (**DBTADA**)) is practically insoluble in water but its ammonium salt is well soluble and it can be prepared easily. The two aqueus solutions are mixed so that the molar ratio between the racemic compound and the resolving agent sould become 1/0.5. The diastereoisomeric salt containing the desired enantiomer (*R,R*)-**AD** crystallizes while the hydrochloride salt of

water

The next example one is of the first resolutions of a racemic intermediate of prostaglandines. During the synthesis it is isolated as the water-soluble sodium salt ((*R,S*)-*cis***-Na**). Half an equivalent amount of (*R*)-1-phenylethylamine (**PEA**) neutralized with hydrochlorid acid is added to the aqueous solution of (*R,S*)-*cis***-Na**. The (*S*)-**cis**.(*R*)-**PEA** crystallizes while (*R*)-*cis***-**

water

It may happen frequently, that only one enantiomer of the resolving agent is available, or one of the two enantiomers is significantly cheaper. For this reason mainly (*R,R*)-tartaric acid is used industrially among of tartaric acid enantiomers and their derivatives. It is the case, for example, at the resolution of a racemic intermediate (**AN**) of α-methyl-**DOPA**. The resolution of racemic **AN** is performed in the aqueous solution of its hydrochloric acid salt

(*R,R*)-**AD**.(*R,R*)-**DBTADA**

crystallization (*S*)-*cis .*(*R*)-**PEA** <sup>+</sup> (*R*)-*cis-*Na

crystalline solid in solution

crystalline solid in solution

+ (*S,S*)-**AD**.HCl

efficiency of resolution can also be improved.

industry, will be presented.

(*S,S*)-**AD** remains in solution.

OH

OH

(*S,S*)-**AD**.HCl

(*R,R*)-**AD**.HCl

**Na** remains in the solution.

CH2COONa

CH2COONa

(*S*)-*cis-*Na

(*R*)-*cis-*Na

NH2.HCl

OH

+

(*R,R*)-**DBTADA**.NH3

NH2.HCl <sup>+</sup>

(*R*)-**PEA**

CON(CH3) PhOCO <sup>2</sup>

H3N.HOOC OCOPh

NH2.HCl

OH

O2N

OH

OH

O2N

(experimental) eutectic composition. In other words, the characteristics of the resolved compounds strongly influence or even determinate the composition of the diastereoisomeric salts crystallized during the resolutions.36

In five cases among the resolutions of six racemic compounds with structurally similar resolving agents the crystallized diastereoisomeric salts have shown quasi-racemate behaviour (thus these were quasi-resolutions). At the same time in one case, during the resolution of *N*-formyl-phenylalanine (**FoPA**), crystallization of a quasi-homochiral diastereoisomer was observed. The observed quasi-racemate/quasi-conglomerate = 5/1 ratio has also been in acccordance with the earlier described data on the similar ratio of the racemates and conglomerates among the true enantiomeric mixtures,37

On the basis of the above discussed experimental data we concluded, that the behaviour of diastereoisomers follows the behaviour of their constituent enantiomeric mixtures. We have also observed that the similarity between the molecular structures of a racemic compound and a resolving agent has a positive effect on the enantiomeric separation. It also may be established, that a "derivative resolving agent" (use of optically active derivatives of the racemate as resolving agent) would be the optimal agent for separation of any racemic compound, but the best derivative should be found on experimental way.

## **3.2.1.2 Resolving agents with diverse molecular structures**

According to the previously mentioned, the appropriate resolving agents for a racemic compound should be found among the structurally similar compounds because of the higher probability of the formation of well-fitting associates (salts or complexes) from that type of resolving agents and one of the isomers of racemic compound.

Of course, such a well-fitting chiral compound may be found among several other compounds, as well. They are the well-known and traditional resolving agents. Such a universal resolving agent is the O,O'-dibenzoyl-(*R,R*)-tartaric acid (**DBTA**) (mentioned several times previously). Presumably the above discussed findings, that the efficiencies of resolutions using structurally similar resolving agents may be determined by the behaviour of the enantiomeric mixtures of the racemic compound, may also be expanded on that diastereoisomeric salts forming resolutions where the resolving agent has "non-similar" molecule structure (particularly in cases of crystallizations from water).

In the simplest cases the generally used methods follow the first ever resolution by salt formation accomplished by Pasteur (fractionated precipitation method). In this case the racemic compound and the resolving agent are dissolved in a solvent, in a molar equivalent amount. The less soluble diastereoisomeric salt crystallizes (during cooling) and it can be separated from the other diastereoisomeric salt (remained in solution) by filtration. The enantiomeric mixtures are isolated from the diastereoisomers and they are enriched to the desired *ee* values by applying one of the previously discussed methods.

It has to be mentioned, that the enantiomeric mixture found in the crystalline diastereoisomer is usually much purer than that is isolated from the filtrate.

Pope and Peachey38 have been recognized that if the half of the resolving agent necessary for the better soluble diasteroisomer is replaced by an achiral reagent (having the same chemical character as it is for the resolving agent) the less soluble diastereoisomer will be

(experimental) eutectic composition. In other words, the characteristics of the resolved compounds strongly influence or even determinate the composition of the diastereoisomeric

In five cases among the resolutions of six racemic compounds with structurally similar resolving agents the crystallized diastereoisomeric salts have shown quasi-racemate behaviour (thus these were quasi-resolutions). At the same time in one case, during the resolution of *N*-formyl-phenylalanine (**FoPA**), crystallization of a quasi-homochiral diastereoisomer was observed. The observed quasi-racemate/quasi-conglomerate = 5/1 ratio has also been in acccordance with the earlier described data on the similar ratio of the

On the basis of the above discussed experimental data we concluded, that the behaviour of diastereoisomers follows the behaviour of their constituent enantiomeric mixtures. We have also observed that the similarity between the molecular structures of a racemic compound and a resolving agent has a positive effect on the enantiomeric separation. It also may be established, that a "derivative resolving agent" (use of optically active derivatives of the racemate as resolving agent) would be the optimal agent for separation of any racemic

According to the previously mentioned, the appropriate resolving agents for a racemic compound should be found among the structurally similar compounds because of the higher probability of the formation of well-fitting associates (salts or complexes) from that

Of course, such a well-fitting chiral compound may be found among several other compounds, as well. They are the well-known and traditional resolving agents. Such a universal resolving agent is the O,O'-dibenzoyl-(*R,R*)-tartaric acid (**DBTA**) (mentioned several times previously). Presumably the above discussed findings, that the efficiencies of resolutions using structurally similar resolving agents may be determined by the behaviour of the enantiomeric mixtures of the racemic compound, may also be expanded on that diastereoisomeric salts forming resolutions where the resolving agent has "non-similar"

In the simplest cases the generally used methods follow the first ever resolution by salt formation accomplished by Pasteur (fractionated precipitation method). In this case the racemic compound and the resolving agent are dissolved in a solvent, in a molar equivalent amount. The less soluble diastereoisomeric salt crystallizes (during cooling) and it can be separated from the other diastereoisomeric salt (remained in solution) by filtration. The enantiomeric mixtures are isolated from the diastereoisomers and they are enriched to the

It has to be mentioned, that the enantiomeric mixture found in the crystalline

Pope and Peachey38 have been recognized that if the half of the resolving agent necessary for the better soluble diasteroisomer is replaced by an achiral reagent (having the same chemical character as it is for the resolving agent) the less soluble diastereoisomer will be

racemates and conglomerates among the true enantiomeric mixtures,37

compound, but the best derivative should be found on experimental way.

type of resolving agents and one of the isomers of racemic compound.

molecule structure (particularly in cases of crystallizations from water).

desired *ee* values by applying one of the previously discussed methods.

diastereoisomer is usually much purer than that is isolated from the filtrate.

**3.2.1.2 Resolving agents with diverse molecular structures** 

salts crystallized during the resolutions.36

crystallized and the other isomer remains in the solution as a salt of the applied achiral additive. This method not only economizes a half part of the resolving agent, but the efficiency of resolution can also be improved.

Hereafter some examples of the above mentiuoned procedures, used in the pharmaceutical industry, will be presented.

The racemic intermediate of chloramphenicol (**AD.**HCl) is obtained as a hydrochloride salt during the synthesis, and it is dissolved in water (the free base is low-soluble in water). In this case the resolving agent (O,O'-dibenzoyl-(*R,R*)-tartaric acid *N,N*-dimethylamide (**DBTADA**)) is practically insoluble in water but its ammonium salt is well soluble and it can be prepared easily. The two aqueus solutions are mixed so that the molar ratio between the racemic compound and the resolving agent sould become 1/0.5. The diastereoisomeric salt containing the desired enantiomer (*R,R*)-**AD** crystallizes while the hydrochloride salt of (*S,S*)-**AD** remains in solution.

The next example one is of the first resolutions of a racemic intermediate of prostaglandines. During the synthesis it is isolated as the water-soluble sodium salt ((*R,S*)-*cis***-Na**). Half an equivalent amount of (*R*)-1-phenylethylamine (**PEA**) neutralized with hydrochlorid acid is added to the aqueous solution of (*R,S*)-*cis***-Na**. The (*S*)-**cis**.(*R*)-**PEA** crystallizes while (*R*)-*cis***-Na** remains in the solution.

It may happen frequently, that only one enantiomer of the resolving agent is available, or one of the two enantiomers is significantly cheaper. For this reason mainly (*R,R*)-tartaric acid is used industrially among of tartaric acid enantiomers and their derivatives. It is the case, for example, at the resolution of a racemic intermediate (**AN**) of α-methyl-**DOPA**. The resolution of racemic **AN** is performed in the aqueous solution of its hydrochloric acid salt

Separation of the Mixtures of Chiral Compounds by Crystallization 23

In the following example three kind of resolution of racemic ephedrine (**EPh**) via diastereoisomeric crystallization will be presented, as before.40 The favourable resolving agent is a chiral diester of phosphoric acid containing 2-chloro-phenyl substituent (**CPH**), while the structurally similar phosphoric acid containing unsubstituted phenyl group (**PH**)

The above mentioned observation, namely a part of the resolving agent can be replaced by a structurally related compound which is unsuitable for separation of the enantiomers means in general, that the presence of a structurally similar but achiral reagent may also improve

For example, when *N*-acetyl-phenylalanine (**AcPA**) is resolved with half an equivalent amount of (*R*)-phenylglycine amide (**PGA**) in presence of sodium hydroxide both the enantiomeric purity and the productivity were lower than when instead of sodium hydroxide half an equivalent amount of benzylamine (**BA**) was applied (**BA** has a more related structure with the resolving agent than sodium hydroxide). Its presence improved significantly the crystallization of (*S*)-**AcPA.**(*R*)-**PGA** diastereoisomeric salt, as well as the

+ NaOH (*S*)-**AcPA.**(*R*)-**PGA** <sup>+</sup>

(*S*)-**AcPA.**(*R*)-**PGA**

ee: 100%, F: 0.81

ee: 74%, F: 0.42

(*R*)-**AcPA.** Na

+ (*R*)-**AcPA. BA**

in solution crystalline

crystalline in solution

NH2

**BA**

The use of an achiral compound with related molecular skeleton may also be advantageous, if its structure is related with structure of the racemic compound. For example, if the resolution of racemic **AcPA** is carried out using (*R*)-**PEA,** the crystallized diastereoisomeric salt contains almost racemic **AcPA**, but addition of the achiral analogous phenoxy-acetic acid (**POAA**) to the racemic compound, result in a very good enantiodiscrimination. The achiral additive crystallizes quickly together with resolving agent and, during the long

**3.2.1.4 Mixture of a resolving agent and a structurally related achiral molecule** 

is an unsuitable resolving agent.

the results of a resolution.

efficiency of the resolution

NHCOCH3 COOH

NHCOCH3 COOH

+

NH2

(*R*)-**PGA**

CONH2

+

standing, the **POAA** is exchanged for (*S*)-**AcPA** enantiomer in the crystal.

(*S*)-**AcPA**

(*R*)-**AcPA**

(**AN**.HCl) by half an equivalent amount of mono sodium salt of (*R*,*R*)-tartaric acid (**TA**.Na). The crystalline salt of undesired (*R*)**-AN**.**TA** precipitates. After filtration of the diastereoisomeric salt the pure (*S*)-**AN**.HCl can be crystallized from the mother liquor by salting out (addition of sodium chloride into the filtrate) while the racemic **AN.**HCl remains in solution. Since the enantiomeric excess crystallized from the filtrate we can speak about conglomerate behaviour. However, the compound may be a racemate forming material, if the purity of the (*S>R*)-**AN.**HCl enantiomeric mixture is higher than the eutectic composition (eeeu for **AN**.HCl).

#### **3.2.1.3 Mixture resolving agents**

The so called "Dutch resolution" 39 utilize the recognition whereas a part of the original resolving agent can be replaced by another chiral compound with similar chemical character and under such circumstances (when the resolving agent really is a mixture of structurally related molecules) a diastereoisomer (containing usually an enantiomer of the racemate in very high purity) crystallizes from the supersaturated solution. The advantage of this resolution method is that the efficiency may often be superior to the use of any of the resolving agent components alone, and sometimes it works even if one of the reagents alone is unsuitable to form crystallizing diastereoisomeric salt.

(**AN**.HCl) by half an equivalent amount of mono sodium salt of (*R*,*R*)-tartaric acid (**TA**.Na). The crystalline salt of undesired (*R*)**-AN**.**TA** precipitates. After filtration of the diastereoisomeric salt the pure (*S*)-**AN**.HCl can be crystallized from the mother liquor by salting out (addition of sodium chloride into the filtrate) while the racemic **AN.**HCl remains in solution. Since the enantiomeric excess crystallized from the filtrate we can speak about conglomerate behaviour. However, the compound may be a racemate forming material, if the purity of the (*S>R*)-**AN.**HCl enantiomeric mixture is higher than the eutectic

water

The so called "Dutch resolution" 39 utilize the recognition whereas a part of the original resolving agent can be replaced by another chiral compound with similar chemical character and under such circumstances (when the resolving agent really is a mixture of structurally related molecules) a diastereoisomer (containing usually an enantiomer of the racemate in very high purity) crystallizes from the supersaturated solution. The advantage of this resolution method is that the efficiency may often be superior to the use of any of the resolving agent components alone, and sometimes it works even if one of the reagents alone

> O O P O OH

> > **PH**

Cl

0.5 **PH** + 0.5 **CPH**

O O P O OH

**CPH**

(*R*)-**AN**.(*R,R*)-**TA** +

crystalline in solution

(1*R*,2*S*)-**EPh.CPH** in crystalline

(1*R*,2*S*)-**EPh.PH.CPH** in crystalline

(*S*)-**AN**.HCl

(*R,S*)-**AN**.HCl (*S*)-**AN**.HCl in solution crystalline

+ NaCl

F= 0

F=0.76

F=0.81

composition (eeeu for **AN**.HCl).

NH2.HCl H3C

**3.2.1.3 Mixture resolving agents** 

+

is unsuitable to form crystallizing diastereoisomeric salt.

+

+

+

(*R,R*)-**TA**.Na

HO COOH

NaOOC OH

NH2 H .HCl 3C

CH3O

CH3O

CH3O CN

(*R*)-**AN**.HCl

OH

(1*S*,2*R*)-**EPh**

OH

(1*R*,2*S*)-**EPh**

NHCH3

NHCH3

CH3O CN

(*S*)-**AN**.HCl

In the following example three kind of resolution of racemic ephedrine (**EPh**) via diastereoisomeric crystallization will be presented, as before.40 The favourable resolving agent is a chiral diester of phosphoric acid containing 2-chloro-phenyl substituent (**CPH**), while the structurally similar phosphoric acid containing unsubstituted phenyl group (**PH**) is an unsuitable resolving agent.

#### **3.2.1.4 Mixture of a resolving agent and a structurally related achiral molecule**

The above mentioned observation, namely a part of the resolving agent can be replaced by a structurally related compound which is unsuitable for separation of the enantiomers means in general, that the presence of a structurally similar but achiral reagent may also improve the results of a resolution.

For example, when *N*-acetyl-phenylalanine (**AcPA**) is resolved with half an equivalent amount of (*R*)-phenylglycine amide (**PGA**) in presence of sodium hydroxide both the enantiomeric purity and the productivity were lower than when instead of sodium hydroxide half an equivalent amount of benzylamine (**BA**) was applied (**BA** has a more related structure with the resolving agent than sodium hydroxide). Its presence improved significantly the crystallization of (*S*)-**AcPA.**(*R*)-**PGA** diastereoisomeric salt, as well as the efficiency of the resolution

The use of an achiral compound with related molecular skeleton may also be advantageous, if its structure is related with structure of the racemic compound. For example, if the resolution of racemic **AcPA** is carried out using (*R*)-**PEA,** the crystallized diastereoisomeric salt contains almost racemic **AcPA**, but addition of the achiral analogous phenoxy-acetic acid (**POAA**) to the racemic compound, result in a very good enantiodiscrimination. The achiral additive crystallizes quickly together with resolving agent and, during the long standing, the **POAA** is exchanged for (*S*)-**AcPA** enantiomer in the crystal.

Separation of the Mixtures of Chiral Compounds by Crystallization 25

D + H DH + R + R + LH L + H

Where *K*s = salt solubility constant, *K*d = salt dissociation constant, *K*b = base dissociation constant, *K*a = acid dissociation constant, PD and PL are the precipitated diastereoisomeric

The efficiency of the resolution (S = F, see above in point 1)14 can be calculated by means of the next formula, using the known thermodynamic constants data, and the concentrations of the starting compounds (co = concentration of the racemic compound, [RH]o =concentration

0.5coS [ ] <sup>0</sup> ( ) <sup>0</sup>

From the relationship deduced on basis of the model the regularity of several "rule of thumb" based on experimental observations were justified. A fundamental conclusion is that the efficiency of resolution is the function of both the hydrogen ion concentration and

a. With derivation of the equation, describes the thermodynamic equilibrium model, can be justified that in so far as KdL>KdD, the minimum value (Smin) of the S = f(H+) function

<sup>+</sup> <sup>=</sup> that corresponds to the proton concentration provided by hydrolysis of the neutral diastereoisomeric salt; namely that concentrations occur at the equivalent resolution discovered by Pasteur: It menans that this type of resolution results in the smallest separation efficiency. It is worth to mention, that the essential condition, whereas the dissociation constant of the rather soluble salt is bigger, is

*<sup>K</sup> <sup>H</sup> K KK K K K K*

<sup>−</sup> = − ++ + + − −+

+

0.5

*RH RH sL sD*

*b b sL dL sD dD*

*H K K RH c y K K*

Kb Kb

KdD KdL

+

KsD KsL

DL + 2RH DHR LHR

2RH

of resolving agents) and of [H+] = proton concentration, as well.

+

1

*sL sD*

the initial concentration of the resolving agent.

at *H KKb RH*

Ka + 2H

PD PL

solid

liquid

salts, respectively.

#### **3.2.2 Influence of solvents**

In the majority of the above discussed fractionated precipitations or crystallizations of diastereoisomeric salts water was used as a solvent. The experimental observation of decades is that the dissociation in solvent of diastereoisomeric salts and of salts formed with achiral auxiliaries in water or in other protic solvents, is considerably influenced by the proton concentartion (pH value). Naturally this value influences both the quantity and the isomeric purity of the crystallized salt, namely the efficiency of the resolution.41

There are also examples in the literature, when the configuration of the enantiomer in the crystalline diastereoisomeric salt and/or the efficiency of the resolution are determined by the polarity of solvent (the dielectric constant characteristic for this), respectively.

In other cases, the use of solvent mixtures could influence which diastereoisomer can be found in the crystalline phase. In such cases, either the crystallizing diastereoisomer or the one remained in the mother liquor form stable solvate with one of the solvents. After a short summary of the theoretical background, it will be shown via examples, how can we use the influence of pH, solvent polarity, and solvate formation, respectively, to improve the efficiency of enantiomeric separation.

#### **3.2.2.1 The influence of pH**

The pH dependence of resolutions based on fractionated crystallization of diastereoisomeric salts was described by a thermodynamical equilibrium model.33

The equilibrium model of a resolution of DL racemic base with an acidic resolving agent (RH) can be outlined bellow.

NH2

CH3

(*S*)-**AcPA.**(*R*)-**PEA**

crystalline

ee: 5%, F: 0.05

ee: 88%, F: 0.50

(*S*)-**AcPA.**(*R*)-**PEA**

crystalline

(*R*)-**PEA**

NH2

4

2

2

isomeric purity of the crystallized salt, namely the efficiency of the resolution.41

the polarity of solvent (the dielectric constant characteristic for this), respectively.

salts was described by a thermodynamical equilibrium model.33

(*R*)-**PEA**

**POAA**

In the majority of the above discussed fractionated precipitations or crystallizations of diastereoisomeric salts water was used as a solvent. The experimental observation of decades is that the dissociation in solvent of diastereoisomeric salts and of salts formed with achiral auxiliaries in water or in other protic solvents, is considerably influenced by the proton concentartion (pH value). Naturally this value influences both the quantity and the

There are also examples in the literature, when the configuration of the enantiomer in the crystalline diastereoisomeric salt and/or the efficiency of the resolution are determined by

In other cases, the use of solvent mixtures could influence which diastereoisomer can be found in the crystalline phase. In such cases, either the crystallizing diastereoisomer or the one remained in the mother liquor form stable solvate with one of the solvents. After a short summary of the theoretical background, it will be shown via examples, how can we use the influence of pH, solvent polarity, and solvate formation, respectively, to improve the

The pH dependence of resolutions based on fractionated crystallization of diastereoisomeric

The equilibrium model of a resolution of DL racemic base with an acidic resolving agent

CH3

O COOH

NHCOCH3

(*S*)-**AcPA**

**3.2.2 Influence of solvents** 

efficiency of enantiomeric separation.

**3.2.2.1 The influence of pH** 

(RH) can be outlined bellow.

(*R*)-**AcPA**

COOH

NHCOCH3

COOH

Where *K*s = salt solubility constant, *K*d = salt dissociation constant, *K*b = base dissociation constant, *K*a = acid dissociation constant, PD and PL are the precipitated diastereoisomeric salts, respectively.

The efficiency of the resolution (S = F, see above in point 1)14 can be calculated by means of the next formula, using the known thermodynamic constants data, and the concentrations of the starting compounds (co = concentration of the racemic compound, [RH]o =concentration of resolving agents) and of [H+] = proton concentration, as well.

$$0.5 \text{c}\_{o} \text{S} = K\_{s\text{L}} - K\_{s\text{D}} + \left(1 + \frac{K\_{b}}{\left[H^{+}\right]} + \frac{\left[H^{+}\right]}{K\_{RH}} + \frac{K\_{b}}{K\_{RH}}\right) \left(\frac{K\_{s\text{L}}K\_{d\text{L}} - K\_{s\text{D}}K\_{d\text{D}}}{\left[RH\right]\_{0} - 0.5c\_{0}y - \left(K\_{s\text{L}} + K\_{s\text{D}}\right)}\right)$$

From the relationship deduced on basis of the model the regularity of several "rule of thumb" based on experimental observations were justified. A fundamental conclusion is that the efficiency of resolution is the function of both the hydrogen ion concentration and the initial concentration of the resolving agent.

a. With derivation of the equation, describes the thermodynamic equilibrium model, can be justified that in so far as KdL>KdD, the minimum value (Smin) of the S = f(H+) function at *H KKb RH* <sup>+</sup> <sup>=</sup> that corresponds to the proton concentration provided by hydrolysis of the neutral diastereoisomeric salt; namely that concentrations occur at the equivalent resolution discovered by Pasteur: It menans that this type of resolution results in the smallest separation efficiency. It is worth to mention, that the essential condition, whereas the dissociation constant of the rather soluble salt is bigger, is

Separation of the Mixtures of Chiral Compounds by Crystallization 27

The behaviour of racemic compounds may also be influenced with the choice of pH value of the aqueous solution. The isoelectronic point of 2-phenylglycine (**PG**) is at neutral pH value (Ip = 7.0). The free racemic aminoacid with (*S*)-camphorsulfonic acid (**CSA**) forms a well crystallizing salt in water, but the amino acid in the precipitated diastereoisomer have almost racemic composition. When the crystallization is started from an aqueous solution containing half an equivalent amount of **CSA** and equivalent amount of hydrochloric acid (total 50% excess of acid to the PG as base!), the crystalline salt contains (*S*)-2-phenylglycine in high enantiomeric purity (and in good yield), while the almost pure (*R*)-2-phenylglycine

was obtained from filtrate by crystallization of its hydrochloric acid salt.44

CH3 CH3

HCl

The resolution of racemic free phenylalanine (**PA**) was also carried out with high efficiency, if near the half an equivalent amount of resolving agent (O,O'-dibenzoyl-*(R,R)*-tartaric acid (**DBTA**) or the optically active *N*-benzoyl-d-phenylalanine (**BPA**)) was applied in aqueous methanolic solution which contained more than 0.5 equivalen amount of hydrochloric acid,

In the previous chapter the crucial role of the pH value has been shown via examples.

NHSO2 CH3

It was also recognized that tha polarity of the solvents or solvent mixtures (quantified by the dielectric constant of solvents) can also determinate the composition of the crystallized diastereoisomeric salt.4 For example, in the resolution of α-aminocaprolactam (**AC**) with (*S*)- *N*-tosylphenylalanine ((*S*)-**TsPA**) in various solvents the (*S*)-**AC** was predominant when the dielectric constant of the solvent was ε<27 or ε>62. However, the salt of the antipode (*R*)-**AC**

water (*S*)-**PG.CSA** (*R*)-**PG.HCl**

ee > 99%

solvent ε<27 or ε>62

*(S*)-**AK.***(S*)-**TPA**

. crystalline

crystalline

ε>29 or ε<58

solvent

*(S*)-**TPA** *(R*)-**AK.***(S*)-**TPA**

crystalline in solution

+

**CSA**

crystallized when the solvent was in meium polarity (29<ε<58).46,47,48

COOH

COOH

+

**3.2.2.2 Influence of dielectric constant** 

*(R*)-**AK** COOH

+

O

HO3S

NH2

(*S*)-**PG**

NH2

(*R*)-**PG**

too.45

HN

HN

O

O

*(S*)-**AK**

NH2

NH2

realized at the majority of resolutions. (if KdL>KdD is not valid, application of the equivalent amount of resolving agent gives the best result.)

In the light of the above deduction the experimental observation, that the tartaric acid (**TA**, as a dibasic acid) is an efficient resolving agent is not surprising because in the most cases the hemitartarates crystallize and one carboxylic group remains "free". This additional acidic group increases the proton concentration compared to the proton concentration of the solution of a neutral salt (salt of a monobasic acid).

In practice, an excess (10-20%) of the resolving agent used to be added to the racemic compound and, of course, the excess shifts the pH value of the solution as well.

b. Based on the equation of the thermodynamic equilibrium model, the optimum relative concentration of the resolving agent can be determined. It means that the efficiency of S is maximal when the denominator of the second member of multiplication in the equation tends to zero, namely S → Smax if [RH]o = 0.5coy + (KsL + KsD). If the solubility constants of the diastereoisomers are low, and we tend to obtain the highest yield (y ≈ 1.0), then [RH]o ~ 0.5co, consequently using half an equivalent amount of resolving agent will result in the highest efficiency. At the same time it is the theoretical argument of the successfulness of the method disclosed by Pope-Peachey.33

The dramatic influence of the *p*H of the medium was first encountered at the separation of optical isomers of *cis*-permethrinic acid 42 carried out with half an equivalent amount of (*S*)- 2-benzylaminobutanol ((*S*)-**BAB**). The resolution started in the presence of a 25 mol % excess of sodium hydroxide. In such a medium the diastereoisomeric salt containing the almost pure (*S*)-**CPA** crystallized (*ee*: 96%) with a yield of 27% counted to the amount of racemic acid. After removal of the precipitate by filtration, the excess alkali was neutralized with a counted amount of hydrochloric acid. That time the resolving agent remained in the filtrate crystallized with (*R*)-**CPA** enantiomer as diastereoisomeric salt and in the second mother liquor an almost racemic **CPA-Na** salt was found (it can be recycled into the resolution).

The resolution of *trans*-permethrinic acid was carried out in analogue mode.43

In the light of the above deduction the experimental observation, that the tartaric acid (**TA**, as a dibasic acid) is an efficient resolving agent is not surprising because in the most cases the hemitartarates crystallize and one carboxylic group remains "free". This additional acidic group increases the proton concentration compared to the proton concentration of the

In practice, an excess (10-20%) of the resolving agent used to be added to the racemic

b. Based on the equation of the thermodynamic equilibrium model, the optimum relative concentration of the resolving agent can be determined. It means that the efficiency of S is maximal when the denominator of the second member of multiplication in the equation tends to zero, namely S → Smax if [RH]o = 0.5coy + (KsL + KsD). If the solubility constants of the diastereoisomers are low, and we tend to obtain the highest yield (y ≈ 1.0), then [RH]o ~ 0.5co, consequently using half an equivalent amount of resolving agent will result in the highest efficiency. At the same time it is the theoretical argument

The dramatic influence of the *p*H of the medium was first encountered at the separation of optical isomers of *cis*-permethrinic acid 42 carried out with half an equivalent amount of (*S*)- 2-benzylaminobutanol ((*S*)-**BAB**). The resolution started in the presence of a 25 mol % excess of sodium hydroxide. In such a medium the diastereoisomeric salt containing the almost pure (*S*)-**CPA** crystallized (*ee*: 96%) with a yield of 27% counted to the amount of racemic acid. After removal of the precipitate by filtration, the excess alkali was neutralized with a counted amount of hydrochloric acid. That time the resolving agent remained in the filtrate crystallized with (*R*)-**CPA** enantiomer as diastereoisomeric salt and in the second mother liquor an almost racemic **CPA-Na** salt was found (it can be recycled into the resolution).

*R*>*S*

crystal filtrate

crystal filtrate

+ 0.23 HCl

COONa

0.27 *(S*)*-***CPA**-(*S*)-**BAB**

COO

*0.23 (R*)*-***CPA**.(*S*)-**BAB**

Ph

Ph COO

NH2

OH

NH2

OH

+ 0.23

Cl Cl N. HCl OH

Ph

Cl Cl

compound and, of course, the excess shifts the pH value of the solution as well.

of the successfulness of the method disclosed by Pope-Peachey.33

+ + NaOH

0.5 0.25

(*S*)-**BAB.**HCl

Ph

*(R,S)-***CPA.**Na

Cl Cl

COONa

N. HCl OH

Cl Cl

*(R,S)-***CPA.**Na

COONa

The resolution of *trans*-permethrinic acid was carried out in analogue mode.43

Cl Cl

equivalent amount of resolving agent gives the best result.)

solution of a neutral salt (salt of a monobasic acid).

realized at the majority of resolutions. (if KdL>KdD is not valid, application of the

The behaviour of racemic compounds may also be influenced with the choice of pH value of the aqueous solution. The isoelectronic point of 2-phenylglycine (**PG**) is at neutral pH value (Ip = 7.0). The free racemic aminoacid with (*S*)-camphorsulfonic acid (**CSA**) forms a well crystallizing salt in water, but the amino acid in the precipitated diastereoisomer have almost racemic composition. When the crystallization is started from an aqueous solution containing half an equivalent amount of **CSA** and equivalent amount of hydrochloric acid (total 50% excess of acid to the PG as base!), the crystalline salt contains (*S*)-2-phenylglycine in high enantiomeric purity (and in good yield), while the almost pure (*R*)-2-phenylglycine was obtained from filtrate by crystallization of its hydrochloric acid salt.44

The resolution of racemic free phenylalanine (**PA**) was also carried out with high efficiency, if near the half an equivalent amount of resolving agent (O,O'-dibenzoyl-*(R,R)*-tartaric acid (**DBTA**) or the optically active *N*-benzoyl-d-phenylalanine (**BPA**)) was applied in aqueous methanolic solution which contained more than 0.5 equivalen amount of hydrochloric acid, too.45

## **3.2.2.2 Influence of dielectric constant**

In the previous chapter the crucial role of the pH value has been shown via examples.

It was also recognized that tha polarity of the solvents or solvent mixtures (quantified by the dielectric constant of solvents) can also determinate the composition of the crystallized diastereoisomeric salt.4 For example, in the resolution of α-aminocaprolactam (**AC**) with (*S*)- *N*-tosylphenylalanine ((*S*)-**TsPA**) in various solvents the (*S*)-**AC** was predominant when the dielectric constant of the solvent was ε<27 or ε>62. However, the salt of the antipode (*R*)-**AC** crystallized when the solvent was in meium polarity (29<ε<58).46,47,48

Separation of the Mixtures of Chiral Compounds by Crystallization 29

It can be frequent observed at the fractionated crystallization of diastereomeric salts that the crystalline diastereomer forms solvate with the solvent. As an example can be mentioned the first ever resolution by salt formation accomplished by Pasteur wher the "d-quinotoxined-tartrate" obtained was a hexahydrate.54 Of course it can be found numerous similar

It is not rare the phenomenon that the selective solvate formation of diastereomers gives a much better separation or the solvate formation is essential for successful resolution. It is true because only in this case crystallizes the diastereomer. E.g. in methanol *trans*-(*R*) chrysanthemic acid (*trans*-(*R*)-**CHRA**) forms with (*R,R*)-2-*N,N*-dimethlyamino-1,3 propanediol [(*R,R*)-**DMAD**] crystals containing the solvent. If the resolution was carried out in another solvent (e.g. in methyl isobutyl ether) best results were obtained if some methanol was added, because it permitted the precipitation of the methanol solvate (is

crystalline

*(R*)-**AML.***(R,R)***-TA.**(CH3)2NCOCH3 crystalline

*(S*)-**AML.***(R,R)***-TA.**CH3SOCH3 crystalline

**3.2.2.4 Presence of solvates and compounds with similar effect** 

crystallized the (*R*)-*CHRA.*(*R,R*)-**DMAD.**CH3OH).17

N

CH3 (*R*)-**CHRA**

H3C CH3

(*R,R*)-**DMAD**

+

(*R,R*)-**TA**

HO COOH

HOOC OH

OHOH

<sup>+</sup> CH3

**3.2.2.4.1 When the separation of diastereomers is possible just from solvate forming** 

The separation of enantiomers of racemic amlodipine (**AML**) with (*R,R*)-tartaric acid by fractionated crystallization of diastereomers from the common solvents was without success, but in **DMSO**, however, the salt of (*S*)-**AML** crystallizes as a **DMSO** solvate.56 Even more surprisingly, in *N,N*-dimethyl-acetamide it is the solvate of the (*R*)-**AML** salt which

CH3O

H3C <sup>S</sup> CH3 O

N O

H3C H3C

CH3

CH3

+ CH (*R*)-**CHRA.**(*R,R*)-**DMAD.**CH3OH 3OH

examples in the literature.55

CH3 CH3

CH3 CH3

crystallizes in good yield and high purity.57

COOH

COOH

(*S*)-**CHRA**

N H

H3COOC COOC2H5

N H

H3COOC COOC2H5

Cl

Cl

H3C <sup>O</sup> NH2 (*S*)-**AML**

H3C <sup>O</sup> NH2 (*R*)-**AML**

H3C

H3C

CH3

CH3

**solvent** 

That observation can be generalized: systematic variation of the solvent, the diastereoisomer containing the desired enantiomer can be brought to crystallization. (Using the adequate solvent one of the diastereoisomers is crystallized from the mixture of diastereoisomers, and the other one can be crystallized from another solvent.)

For example, if the resolution of flumequine intermediate (**FTHQ)** is resolved with O,O'-dipara-toluoyl-(*R,R*)-tartaric acid ((*R,R*)-**DPTTA)** in presence of acetic acid the (*R*)-**FTHQ** enantiomer crystallizes in the salt, while the (*R*)-**FTHQ** enantiomer forms a better crystallizing salt when isopropyl alcohol is used as a solvent.49

#### **3.2.2.3 Resolution in two immiscible solvents**

One variant of the resolution methods is that salt formation is carried out in a system of two immiscible solvents (e.g. water and dichloromethane). In this case the more stable diastereomeric salt separates, while the free enantiomer dissolves in one of the phases (usually the organic one).50,51

This manner was used at the resolution of the anxiolytic tofisopam (**TOF**) with half equivalent of (*R,R*)-**DBTA.** It was accomplished in a water−chloroform system. The diastereomeric salt containing the (*R*)*-***TOF** enantiomer was crystallized from the biphase solvent, while free (*S*)-**TOF** was recoverable from organic phase.52,53

That observation can be generalized: systematic variation of the solvent, the diastereoisomer containing the desired enantiomer can be brought to crystallization. (Using the adequate solvent one of the diastereoisomers is crystallized from the mixture of diastereoisomers, and

For example, if the resolution of flumequine intermediate (**FTHQ)** is resolved with O,O'-dipara-toluoyl-(*R,R*)-tartaric acid ((*R,R*)-**DPTTA)** in presence of acetic acid the (*R*)-**FTHQ** enantiomer crystallizes in the salt, while the (*R*)-**FTHQ** enantiomer forms a better

COOH

COOH

(*R,R*)-**DPTTA**

OCOPhCH3

One variant of the resolution methods is that salt formation is carried out in a system of two immiscible solvents (e.g. water and dichloromethane). In this case the more stable diastereomeric salt separates, while the free enantiomer dissolves in one of the phases

This manner was used at the resolution of the anxiolytic tofisopam (**TOF**) with half equivalent of (*R,R*)-**DBTA.** It was accomplished in a water−chloroform system. The diastereomeric salt containing the (*R*)*-***TOF** enantiomer was crystallized from the biphase

(*R,R*)-**DBTA**

PhOCO HOOC

<sup>+</sup> CHCl3

COOH

H2O

(*R*)-**TOF.**(*R,R*)-**DBTA** crystalline

CH3COOH

H3C CH3 OH

*(R*)-**FTHQ.**(*R,R*)-**DPTTA** crystalline

*(S*)-**FTHQ.**(*R,R*)-**DPTTA** crystalline

the other one can be crystallized from another solvent.)

+

**3.2.2.3 Resolution in two immiscible solvents** 

N H

N H

*(S*)-**FTHQ**

*(R*)-**FTHQ**

CH3

(usually the organic one).50,51

CH3

F

F

crystallizing salt when isopropyl alcohol is used as a solvent.49

H3CPhOOC

solvent, while free (*S*)-**TOF** was recoverable from organic phase.52,53

(*S*)-**TOF** OCOPh

N N

N N

CH3 CH3

CH3 CH3

(*R*)-**TOF**

H3C H3C

H3C H3C

#### **3.2.2.4 Presence of solvates and compounds with similar effect**

It can be frequent observed at the fractionated crystallization of diastereomeric salts that the crystalline diastereomer forms solvate with the solvent. As an example can be mentioned the first ever resolution by salt formation accomplished by Pasteur wher the "d-quinotoxined-tartrate" obtained was a hexahydrate.54 Of course it can be found numerous similar examples in the literature.55

It is not rare the phenomenon that the selective solvate formation of diastereomers gives a much better separation or the solvate formation is essential for successful resolution. It is true because only in this case crystallizes the diastereomer. E.g. in methanol *trans*-(*R*) chrysanthemic acid (*trans*-(*R*)-**CHRA**) forms with (*R,R*)-2-*N,N*-dimethlyamino-1,3 propanediol [(*R,R*)-**DMAD**] crystals containing the solvent. If the resolution was carried out in another solvent (e.g. in methyl isobutyl ether) best results were obtained if some methanol was added, because it permitted the precipitation of the methanol solvate (is crystallized the (*R*)-*CHRA.*(*R,R*)-**DMAD.**CH3OH).17

#### **3.2.2.4.1 When the separation of diastereomers is possible just from solvate forming solvent**

The separation of enantiomers of racemic amlodipine (**AML**) with (*R,R*)-tartaric acid by fractionated crystallization of diastereomers from the common solvents was without success, but in **DMSO**, however, the salt of (*S*)-**AML** crystallizes as a **DMSO** solvate.56 Even more surprisingly, in *N,N*-dimethyl-acetamide it is the solvate of the (*R*)-**AML** salt which crystallizes in good yield and high purity.57

Separation of the Mixtures of Chiral Compounds by Crystallization 31

From the crystalline diastereomer could be isolated the L-**MEN** with a good enantiomeric

The widely applied **TADDOL** (**TAD**) was also a suitable reagent of several diastereomeric molecular complex, for example enantiomers of phospholene oxides (**PHO**) could be obtained with high optical purity after the fractionated crystallization and decomposition of

> 1.dissolution 2.crystallization 3.filtration

It is well known that the coordanative complex of several metal is well crystallize. The calcium salt of dibenzoyltartaric acid (**DBTAC**), was used be quite a few for separation of diastereomeric coordinative complex by fractionated crystallization. For example the separation of derivatives of phospholene oxides (previously mentioned) was carried out

> 1.dissolution 2.crystallization 3.filtration

Although the methods based on fractionated crystallization suggest that during crystallization the less soluble diastereomer is crystallized, but at the enantiomeric separations realized by fractionated crystallization, or precipitation, respectively, was observed in some cases that instead of expected racemate behaviour was crystallized the

(*S*)-**PPHO.**(*R,R*)-**TAD** + (*S*)-**PPHO** crystalline in solution

(*S*)-**PPHO.**(*R,R*)-**DBTAC** + (*S*)-**PPHO**

crystalline in solution

purity (ee: 83%).

P O

(*R*)-**PPHO**

P O

(*S*)-**PPHO**

using (**DBTAC**).9

+

**3.2.5 Time of crystallization** 

(*R,R*)-**DBTAC**

HOOC OCOPh

PhOCO COOCa1/2

P O (*S*)-**PPHO**

P O (*R*)-**PPHO**

the precipiated diastereomers.8

<sup>+</sup> <sup>O</sup>

O

Ph

Ph

(*R,R*)-**TAD**

**3.2.4 Resolving agents forming coordinative complexes** 

Ph OH

Ph OH

#### **3.2.2.4.2 Presence of achiral compound**

It is well known separation of diastereomers when the enantiomeric purity reachable using a resolving agent may be significantly improved if the crystallization of diastereomeric salt is promoted by crystallization of an achiral compound having analogue molecular structure with a part of the resolving agent molecules.

This phenomenon was observed at the resolution of racemic α-phenylethylamine (**PEA**) using (*R*)-*N*-(1-phenyl-ethyl)-glutaric acid ((*R*)-**PEGA**) as resolvong agent. From acetone was crystallized diastereomeric salt containing (*S*)-**PEA** with ee: 58%. If is added an equivalent of carbamide to the mixture the enantiomeric excess of the isolated (*S*)-**PEA** was 90%**.**<sup>58</sup>

#### **3.2.3 Resolving agents forming diastereomer complexes**

The difference betwee, solubility of diastereomeric salts, their solvent deppendence justify that although the ions of salt estabilish strong interactions, neverthless the formation of secondary interactions between the compounds containing these ions (formation of diastereomeric molecular complexes) is the reason of differential solubility of diastereomeric salts in the respective solvent. Naturally, it is not by chance that the diastereomeric salt of dibenzoyltartaric acid (**DBTA**) are well separable and it is a very suitable resolving agent for a wide range of racemic basis. It means that the enantiomers of racemic compounds which do not contain either acidic or basic functional groups form diastereomerically releted molecular complexes with **DBTA**. These complexes can be separeted by fractionated crystallization. Such several alcohols with various molecular structures could be resolved, e.g. the resolution of racemic menthol (**MEN**) was carried out with (*R,R*)-**DBTA** in solution, too. In hexane insoluble reagent contacting the dissolved racemic menthol forms crystalline diastereomeric molecular complex.59 (as was shown earlier at the crystallization of diastereomers from melt).

From the crystalline diastereomer could be isolated the L-**MEN** with a good enantiomeric purity (ee: 83%).

The widely applied **TADDOL** (**TAD**) was also a suitable reagent of several diastereomeric molecular complex, for example enantiomers of phospholene oxides (**PHO**) could be obtained with high optical purity after the fractionated crystallization and decomposition of the precipiated diastereomers.8

(*S*)-**PPHO**

30 Advances in Crystallization Processes

It is well known separation of diastereomers when the enantiomeric purity reachable using a resolving agent may be significantly improved if the crystallization of diastereomeric salt is promoted by crystallization of an achiral compound having analogue molecular structure

This phenomenon was observed at the resolution of racemic α-phenylethylamine (**PEA**) using (*R*)-*N*-(1-phenyl-ethyl)-glutaric acid ((*R*)-**PEGA**) as resolvong agent. From acetone was crystallized diastereomeric salt containing (*S*)-**PEA** with ee: 58%. If is added an equivalent of

carbamide to the mixture the enantiomeric excess of the isolated (*S*)-**PEA** was 90%**.**<sup>58</sup>

O

complex.59 (as was shown earlier at the crystallization of diastereomers from melt).

1.hexane 2.crystallization 3.filtration

N H

CH3

The difference betwee, solubility of diastereomeric salts, their solvent deppendence justify that although the ions of salt estabilish strong interactions, neverthless the formation of secondary interactions between the compounds containing these ions (formation of diastereomeric molecular complexes) is the reason of differential solubility of diastereomeric salts in the respective solvent. Naturally, it is not by chance that the diastereomeric salt of dibenzoyltartaric acid (**DBTA**) are well separable and it is a very suitable resolving agent for a wide range of racemic basis. It means that the enantiomers of racemic compounds which do not contain either acidic or basic functional groups form diastereomerically releted molecular complexes with **DBTA**. These complexes can be separeted by fractionated crystallization. Such several alcohols with various molecular structures could be resolved, e.g. the resolution of racemic menthol (**MEN**) was carried out with (*R,R*)-**DBTA** in solution, too. In hexane insoluble reagent contacting the dissolved racemic menthol forms crystalline diastereomeric molecular

*(S*)-**PEA.**(*R*)-**PEGA**

ee: 58% F: 0.36

acetone *(S*)-**PEA.**(*R*)-**PEGA**

H2N <sup>C</sup> NH2 O

(*1R,2S,5R*)-**MEN.**(*R,R*)-**DBTA** + (*1S,2R,5S*)-**MEN** crystalline in solution

ee: 90% F: 0.49 (*R*)-**PEGA**

acetone

**3.2.2.4.2 Presence of achiral compound** 

with a part of the resolving agent molecules.

+ HOOC

**3.2.3 Resolving agents forming diastereomer complexes** 

CH3

CH3

NH2

NH2

(*S*)-**PEA**

(*R*)-**PEA**

(*1R,2S,5R*)-**MEN**

(*1S,2R,5S*)-**MEN**

+

(*R,R*)-**DBTA**

PhOCO COOH

HOOC OCOPh

#### **3.2.4 Resolving agents forming coordinative complexes**

It is well known that the coordanative complex of several metal is well crystallize. The calcium salt of dibenzoyltartaric acid (**DBTAC**), was used be quite a few for separation of diastereomeric coordinative complex by fractionated crystallization. For example the separation of derivatives of phospholene oxides (previously mentioned) was carried out using (**DBTAC**).9

#### **3.2.5 Time of crystallization**

Although the methods based on fractionated crystallization suggest that during crystallization the less soluble diastereomer is crystallized, but at the enantiomeric separations realized by fractionated crystallization, or precipitation, respectively, was observed in some cases that instead of expected racemate behaviour was crystallized the

Separation of the Mixtures of Chiral Compounds by Crystallization 33

In the most cases it is not necessary to effectuate so quickly the filtration of crystalls, because the stabilition of thermodinamic equilibrium need a longer period. This is so even if the racemic compound of resolving agent is resolved using one of enantiomers of racemic

The resolution of *N*-formyl-phenylalanine (**FoPA**) with (*S*)-phenylethylamine (**PEA**) in water after 2 hours gave diastereomeric salt with high purity, but this enantiomeric excess diminished when the filtration of the precipitated salt was carried out after a longer

> 1.dissolution 2.crystallization 3.filtration

If the reolution of racemic **PEA** is accomplished with (*S*)-**FoPA** (reciprocal resolution), in function of crystallization time practically the same thing was happened as expected on basis of the foregoing, but the results were not identical because at the normal and the

> 1. dissolution 2. crystallization 3. filtration

The behaviour of the enantiomeric mixtures can be interpretated by formation of their homo- and heterochiaral aggregates having diastereomeric property. Due to spontaneous formation of these aggregates the non-racemic mixtures of enantiomers can be enriched by processes required some selective crystallization. The running of biner meltingpoint/composition phase diagrams (conglomerate or racemate), and the kinetic controlled crystallizations (kinetic conglomerates), respectively, determine the fact, that the crystalline phase contains the enantiomer or the racemic proportion. These regularities are also reflected by the running of curves where the enantiomeric purity of the product obtained (ee) during the selective crystallizations and precipitations were plotted against the

At the separation of racemic compounds using another chiral material (namely resolution) the behaviour of enantiomeric mixture forming the racemate determines the efficiency of

+ (*R*)-**FoPA.***(S*)-**PEA**

2 hours 1 week

*(S*)-**PEA.**(*S*)-**FPA**

0.5 hour 3 weeks ee: 78.6% ee: 43.3%

crystalline in solution

+ (*R*)-**PEA.**(*S*)-**FPA**

ee: 90.8% ee: 65.1%

crystalline in solution

(*S*)-**FoPA.***(S*)-**PEA** +

CH3

(*S*)-**PEA**

reciprocal resolutions the systems are in diastereoisomeric relationship.

COOH

NHCHO

(*S*)-**FPA**

NH2

compound.62

standing (1 week).

(*S*)-**FoPA**

CH3

(*S*)-**PEA**

CH3

(*R*)-**PEA**

**4. Conclusions** 

NH2

initial composition (ee0).

+

NH2

(*R*)-**FoPA**

COOH NHCHO

COOH NHCHO

enantiomeric excess (conglomerate like behaviour), namely its faster crystallization determines the separation (kinetic conglomerate).

#### **3.2.5.1 Thermodinamic control**

In general, at the fractionated precipitation if is expected a conglomerate like behaviour on base of binary phase diagram of diasteroemeric mixture, the filtration is efffectuated when the quantity of crystalline precipitate is constant. However if the difference between the crystállization rate of diastereomers is not large enough, can be occured that both diastereomers are crystallized in near similar quantity. In this case we cold not talk about enantiomeric enrichment. If instead of quick filtration is given time for stabilition of thermodynamic equilibrium, in turn is obtain a separation with good result. This can be demonstrate via resolution of tamsulosin intermediate (**TAI**) using as resolving agent **DBTA**  in the mixture of water and ethanol, when at the end of crystallization was not carried out enantiomweric enrichment, but if the crystalline mixture was allowe to stand for 2 days, was reached a very high enantiomeric excess.60

Similar phenomenon was also observed at the resolution of racemic oxirane derivatives, containing two terciary aminogroups.61

#### **3.2.5.2 Kinetic controlled crystallization**

Accordingly to the previously examples would appear to be advantageous that the time of crystallization to be more longer but if one of the diastereomers crystallizes quickly and the other is wrong solvable, then neverthless its opposite is also favourable.

Thus in the resolution of the intermediate of flumequine (**FTHQ)** in ethyl acetate with diparatoluyl-tartaric acid (*R,R*)-**DPTTA,** after a crystallizing of 5 minutes the (*R*)-**FTHQ**.(*R,R*)- **DPTTA** salt predominates, but on standing 3 weeks its antipode (*S*)-**FTHQ**.(*R,R*)-**DPTTA** will be in excess in the crystals.48

In the most cases it is not necessary to effectuate so quickly the filtration of crystalls, because the stabilition of thermodinamic equilibrium need a longer period. This is so even if the racemic compound of resolving agent is resolved using one of enantiomers of racemic compound.62

The resolution of *N*-formyl-phenylalanine (**FoPA**) with (*S*)-phenylethylamine (**PEA**) in water after 2 hours gave diastereomeric salt with high purity, but this enantiomeric excess diminished when the filtration of the precipitated salt was carried out after a longer standing (1 week).

If the reolution of racemic **PEA** is accomplished with (*S*)-**FoPA** (reciprocal resolution), in function of crystallization time practically the same thing was happened as expected on basis of the foregoing, but the results were not identical because at the normal and the reciprocal resolutions the systems are in diastereoisomeric relationship.
