Controlling Residual Arylboronic Acids as Potential Genotoxic Impurities in APIs

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Arylboronic acids, but not the corresponding deboronated arenes, recently have been found to be weakly mutagenic in microbial assays [1].  Hence arylboronic acids may be considered potentially genotoxic impurities, and controlling the levels of residual arylboronic acids in APIs could become a regulatory requirement.  The issues should be decided by toxicology studies for the specific arylboronic acids in question.

Several approaches have been successful in removing boronic acids.  Diethanolaminomethyl polystyrene (DEAM-PS) [2],[3] and immobilized catechol [4] have been used to scavenge boronic acids.  Complex formation with diethanolamine may solubilize residual boronic acids in mother liquors.  Since arylboronic acids ionize similarly to phenols, basic washes of an API solution may remove arylboronic acids.  A selective crystallization can purge an arylboronic acid from the API.

The best means to control residual aryl boronic acids in APIs at the ppm level may be to decompose them through deboronation.  Sterically hindered, electron-rich aryl boronates and heteroaromatic boronates are especially prone to deboronation [5],[6],[7].  Deboronation of a hindered difluorobenzeneboronic acid was accelerated in the presence of Pd, and increased in the presence of H2O [8].  Kuivila and co-workers found that protodeboronation of 2,6-dimethoxybenzeneboronic acid was slowest about pH 5, and rapid under more acidic or basic conditions [9].  Butters and co-workers found that protodeboronation occurred for an arylboronate anion, but not for the corresponding arylboronic acid [10].  Amgen researchers found that deboronation of an ortho-substituted arylboronic acid was fastest in the presence of K2CO3 [11].  The Snieckus group found that deboronation occurred readily when pinacol was added to 4-pyridylboronic acid [12].  Percec and co-workers found that deboronation of neopentylglycol boronates, especially an ortho-substituted arylboronic acid ester, was catalyzed by nickel species[13].  Kuivila and co-workers found that CuCl2 catalyzed the deboronation of 2,6-dimethoxybenzeneboronic acid and other arylboronic acids, with formation of the corresponding aryl chlorides [14].  Unfortunately, adding reagents to a reaction mixture increases the burdens of analysis and impurity removal, but additives such as these may accelerate deboronation in difficult cases.  Simply extending the reaction conditions, which are generally basic for efficient Suzuki coupling, or heating with some amount of aqueous hydroxide are probably the preferred treatments to decompose an arylboronic acid.  By knowing the kinetics of the decomposition of the arylboronic acid it may be possible to show by QbD that analyses for the residual arylboronic acid in an API are not necessary.

[1] O’Donovan, M. R.; Mee, C. D.; Fenner, S.; Teasdale, A.; Phillips, D. H. Mutat. Res.: Genet. Toxicol. Environ. Mutagen. 2011, 724(1-2), 1.

[2] Antonow, D.; Cooper, N.; Howard, P. W.; Thurston, D. E. J. Comb. Chem. 2007, 9, 437.

[3] Hall, D. G.; Tailor, J.; Gravel, M. Angew. Chem. Int. Ed. 1999, 38, 3064.

[4] Yang, W.; Gao, X.; Springsteen, G.; Wang, B. Tetrahedron Lett. 2002, 43, 6339.

[5] Goodson, F. E.; Wallow, T. I.; Novak, B. M. Organic Syntheses; Vol. 74; Smith, A. B., III, Ed.; Organic Syntheses, Inc.; 1997; p. 64.

[6] Baudoin, O.; Cesario, M.; Guénard, D.; Guéritte, F. J. Org. Chem. 2002, 67, 1199.

[7] Dai, Q.; Xu, D.; Lim, K.; Harvey, R. G. J. Org. Chem. 2007, 72, 4856.

[8] Cammidge, A. N.; Crépy, K. V. L. J. Org. Chem. 2003, 68, 6832.

[9] Kuivila, H. G.; Reuwer, J. F., Jr.; Mangranite, J. A. Can. J. Chem. 1963, 41, 3081.

[10] Butters, M.; Harvey, J. N.; Jover, J.; Lennox, A. J. J.; Lloyd-Jones, G. C.; Murray, P. M.Angew. Chem. Int. Ed. 2010, 49, 5156.

[11] Achmatowicz, M.; Thiel, O. R.; Wheeler, P.; Bernard, C.; Huang, J.; Larsen, R. D.; Faul, M. M.J. Org. Chem. 2009, 74, 795.

[12] Alessi, M.; Larkin, A. L.; Ogilvie, K. A.; Green, L. A.; Lai, S.; Lopez, S.; Snieckus, V. J. Org. Chem. 2007, 72, 1588.

[13] Moldoveanu, C.; Wilson, D. A.; Wilson, C. J.; Leowanawat, P.; Resmerita, A.-M.; Liu, C.; Rosen, B. M.; Percec, J. Org. Chem. 2010, 75, 5438.

[14] Kuivila, H. G.; Reuwer, J. F., Jr.; Mangravite, J. A. J. Am. Chem. Soc. 1964, 86, 2666.


The usefulness of boronic acids and their esters is well documented.

Over the last decade much interest has been shown in the field of boron chemistry particularly in the inorganic aspect. Boronic acids, RB(OH)2, have been one such compound that has attracted a widespread interest over the last decade. They have been involved in various fields, such as sugar chemistry, thermoplastics, enzymes and various other areas in chemistry and biology.

Until now, the synthesis of these compounds has usually involved the reaction of Grignards or organolithium derivatives with trialkyl borates. Although widely applicable, this method normally works well only at low temperatures and is limited to substrates lacking functional groups which could react with the organometallic intermediates.(synthesis will be discussed later)

Figure 1-1: Some common boronic acids.

Boronic esters, RB(OR¢ )2, have also made great strides in research over the last decade. They are being used in macrocyclic chemistry, carbohydrate chemistry, and as molecular recognition compounds.

Figure 1-2: Some common boronic esters.






Before I begin to talk about boronic acids I feel that it is fair to discuss the properties of these compounds first. By doing so, it will be easier to understand why these compounds react in the manner they do.


  • Acid Strength


Phenylboronic acid is about three times as strong as boric acid which is a weak acid. This is in agreement with the substitution effect in acetic acid. Since any effect that causes a reduction in electron density on the proton of the oxygen strengthens the acid, then an effect that increases the electron density on the proton weakens the acid. When a substitution of an ortho hydrogen by an alkyl or phenyl occurs a decrease in the strength of the phenylboronic acid occurs. A meta or para susbstitution on the other hand results in only a slight decrease in strength. When electronegative substituents replace a proton of the oxygen, the strength of the acid increases considerably. This increase occurs in the following manner:

NO2 > F > Cl > Br > COOH


  • Substitution in the Aromatic Nucleus


When treated with a concentrated solution of NaOH, n-Butylboronic acid gives a hydrated sodium salt and a calcium salt. This substitution in the aromatic nucleus forms the structure of the barium salt of p-carboxyphenylboronic acid.

Figure 1-2: Structure of the barium salt of p-carboxyphenylboronic acid


  • Anhydride formation


Another property of boronic acids is that they form anyhydrides easily. Formation of the anhydride is very important in determining the melting-point of the particular boronic acid. This is main reason why the melting point of boronic acids are not frequently misquoted. Phenylboronic acid has a melting point of ~218oC, and was first prepared in 1882. Boronic anhydrides are very easily hydolysed, and readily form esters of the same acid. This is done by heating with an alcohol or phenol.



Figure 1-3: Formation of esters via boronic acids

History of Boronic Acids:

The chemistry of boronic acids is a relatively new topic with respect to chemistry. Early accounts of boronic acid synthesis involved the production of diboronic acids. This synthesis was accomplished by using tetrahydrofuran as the solvent to attain a higher temperature for the completion of metallation, and a Grignard reagent was formed in the process.

Figure 1-4: Formation of diboronic acid.

The Importance of the Grignard reagent:

Formation of the Grignard reagent in the above step is essential to the formation of the diboronic acid. By insertion of the Grignard a polarization occurs causing there to be a slight negative charge on the carbon of the phenyl group and a slight positive charge on the Mg. This causes the phenyl group to act as a nucleophile easily react with (MeO)3B.

Another technique that was used in the early years of boronic acid synthesis also involved the formation of the Grignard reagent. A general procedure was to attach an alkyl group or aryl group to boron by use of a boron trihalide, and a Grignard reagent to supply a hydorcarbon group. Another method that was used in the early years was Khotinsky’s method. This involved the formation of a carbon-boron bond by interaction with an alkyl borate at 0oC.


B(OR)3(in ether) added to ArMgBr ® ArB(OR)2 ® ArB(OH)2

The use of boron triflouride and borontrichloride have also been used to form boronic acids when reacted with Grignard reagents.

BF3(Cl,Br) + RMgBr ® RBF2(Cl,Br) ® RB(OH)2+ 2HF(Cl,Br)

Modern day Synthesis and important reactions of Boronic Acids:


Today there are numerous ways to synthesize boronic acids, and modifications continue to appear. One such technique involves the preparation of aryl boronic acid by heating a mixture of trialkyl borate with magnesium and the aryl bromide. The reaction requires heat which starts the reaction at ~150oC and by ~230oC the magnesium disappears after heating it for 2-3 hours under reflux. After the usual procedure of hydolysis the phenyl boronic acid was produced and isolated. The yield attained was between 33-44 %. The advantage of using bromobenzene over chlorobenzene is that you don’t have to heat the mixture as long. Otherwise, the same results are obtained.

Figure 1-5: The Cowie reaction used to synthesize aryl boronic acids.

Tri-n-butyl borate even reacts with dibromobenzene to produce a diboronic acid. However, this method is not preferred because the yield is substantially lower at 16%. On the otherhand, p-Dichlorobenzene responded poorly to the same reaction.

Figure 1-6: Synthesis of diboronic acid via dibromobenzene.


As was mentioned earlier when attempting to synthesize boronic acids, anhydrides can be easily formed. One such example occurs when attempting to prepare o-phenyldiboronic acid via the above method. A phenylboronic anhydride is produced with a mixture of olefins. This result is unexpected since hydrolysis of the later formed trimer will produce a phenylboronic acid. The exact mechanism of this reaction is unknown, but the most probable one is given below.





Figure 1-7: Probable mechanism of phenylboronic anhydride synthesis.


There are numerous other ways to synthesize boronic acids. As a result there are numerous groups of boronic acids. The first group which will discussed are the alpha-Aminoboronic acids. Although there are several examples of aminomethylboronate syntheses, the most important is probably the boronate amino acid isotere. This complex is a useful inhibitor of the serine chymotrypsin . Thus, it has triggered a great interest in both synthetic and medicinal use.




Figure 1-8: Formation of alpha-Aminoboronic acid

Another group of boronic acids that have vastly been studied are the alpha-haloboronic acids. These are produced by hydolysis of halomethyl boranes.


Figure 1-9: Formation of alpha-haloboronic acids

The aryl boronic acids undergo protodeboronation on treatment with carboxylic acids, such as HCO2H. The reaction proceeds via the formation of a six-membered cyclic transition state.



Figure 1-10: The protodebromonation of aryl boronic acids.


Synthetic routes to boronic acid synthesis:


There are six general synthetic routes to boronic acid synthesis. The first used, and in many ways the least likely, was the slow partial oxidation of the spontaneously inflammable trialkylboranes followed by the hydrolysis of the ester.


Et3B + O2 ® EtB(OEt)2 ® EtB(OH)2


Ph3B + O2 ® PhB(OPh)2 ® PhB(OH)2

The ready availability of many trialkylboranes makes this an attractive route but it is still relatively used very little. Dialkyl borinic acids can also be oxidized to produce boronic acids.


R2B(OH) + 1/2O2 ® RB(OH)2 + R(OH)

A second route to produce boronic acids involves the acid hydolysis of trialkyl- or triaryl-boranes. This method involves the change of these substituted boranes to borinic, boronic, and boric acids respectively.



R3B ® R2B(OH) ® RB(OH)2® B(OH)3

The third synthetic route is via halogenation and subsequent hydrolysis. While the appropriate alkyldihalogenborane intermediates can be prepared via the fourth route; the reaction of organometallics on boron trihalides.

Br2 H2O

Bu3B ® BuBBr2 + Bu2BBr ® BuB(OH)2 + Bu2B(OH)

I2 H2O air

R3B ® R2BI ® R2B(OH) ® RB(OH)2


BCl3 + Ar2Hg ® ArHgCl + ArBCl2 ® ArB(OH)2


R3B + BX3 ® RBX2 + R2BX® RB(OH)2+ R2B(OH)

By far the most widely used method for preparing boronic acids is the reaction of aryl or alkyl Grignard reagents on organo-orthoborates or boron halides. The preferred starting materials include B(OMe)3, B(OBu)3 and BF3.

500C H3O+

B(OR)3 + ArMgX ® [ArB(OR)3MgX] ® ArB(OH)2

Organolithium compounds have also been used to synthesize boronic acids.


B(OR)3 + R’Li ® [R’B(OR)3]Li ® R’B(OH)2

The final route to synthesize boronic acids involves the aromatic substitution of an existing boronic acids by conventional organic techniques; this is particularly important since the Grignard or organolithium syntheses just mentioned cannot be carried out in the presence of other reactive functional groups such as nitro, amino, carboxy, sulphono, etc. A typical sequence of reactions is as follows:


Figure 1-11: Formation of boronic acids via aromatic substitution.




Handling, Toxicity, and Analysis of Boronic Acids:

Since there are numerous boronic acids I will analyse each of the above headings in terms of just one boronic acid, namely benzene boronic acid. Benzene boronic acid like most boronic acids are sensitive to oxygen and are susceptible to hydolysis. When handling benzene boronic acid one must adapt to the chemical and physical characteristics of that particular compound.

The toxicity of benzene boronic acid is fairly high. It has an lethal dosage number of 50.(LD 50) That is, 50% of the rats that took a dosage of 740 mg/kg died. The dosage of 740 mg/kg is the approximate amount of benzene boronic acid that causes intake of it to be lethal.

The analysis of volatile boronic acids can be achieved by vapor phase chromatography provided that inert liquid and stationary phases are used, protic sites are converted to trimethylsilyl groups and injection port temperatures are regulated such that thermal degradation of the molecules does not occur. As a general rule, the retention time of boronic acids on a nonpolar column is similar to that of the corresponding hydrocarbon. Analytical determinations of boronic acids depend on complete cleavage of the boron-carbon bond.(cleavage of both if diboronic acid) Cleavage can occur via oxidation, or use of a variety of reagents. These reagents include NaOH, alkaline hydrogen peroxide, sulphuric acid, etc. Also, it appears that some boronic acids can be titrated by NaOH in the presence of mannitol using phenolphthalein as an indicator.


Applications of Boronic Acids and recent research:

Boronic acids have been used for various reasons. Alkyl boronic acids have been used for research and developnment purposes. These include the nonyl and dodecyl boronic acids. These boronic acids are termed as ‘bacteriastatic’, ‘fungistatic’and ‘surface active’. They are used as gasoline additives, polymer components and stabilizers for aromatic amines. These properties are of importance to slime organisms in the manufacturing of paper, and to minimize bacterial damage in paint, leathers, and polymers. They are also used in the dehydrated form, (RBO)3, as potential oil additives for stabilization against oxidation and polymerization. Alkyl boronic acids are also used in motor fuels, particularly in race cars.

Recently, boronic acids have been used as reagents in Suzuki coupling reactions to produce aryl substituted pyrrolo benzodiazepines(PBDs). These compounds have been used as gene targeting vectors and as antitumour and diagnostic agents.


Figure 1-12: Use of boronic acids to produce PBD via Suzuki Coupling.

Tandem Suzuki coupling-cyclisation with 2-Formylthiophene-3-boronic acid, was used to synthesise the thieno [2,3-c]-1,7-naphthyridine ring system.

Palladium catalysed Suzuki-type cross coupling of an iodocyclopropane with Thiophene-3-boronic acid, promoted by cesium fluoride.

Asymmetric boronic acid Mannich reaction of Furan-2-boronic acid, 15297, with an aldehyde in the presence of a chiral amine template.

Figure 1-13: New Boronic acids reactions.




There are numerous ways to synthesize boronic esters. Among these involve b -eliminations. b -eliminations are oxidative processes in which the boron becomes bonded to oxygen or another electronegative element, carbon loses an electronegative ligand at the b -position, and a carbon-carbon p -bond is formed. It is not remarkable that b -eliminations of boron and an electronegative atom occur, but boron is inusual for a relatively metallic element in that b -eliminations tend to be very slow. There are various types of b -eliminations; these includeb -elimination of halides, vinylic chlorides, alkoxides, oxide anions and azides.

Boronic esters can be easily made by the interaction of boronic acids with a hydoxy-compound. In this reaction water is removed as an azeotrope.

RB(OH)2 + 2R’OH ® RB(OR’)2 + 2H2O

Esters of alkyl boronic acids are in most cases more slowly hydrolysed than esters of arylboronic acid, or of boric acid, and indeed di-n-butylboronate. As a consequence they can be isolated from the Grignard system because of this relative stability. As a rule of thumb, boronic esters are far from being hydrolytically stable, although they have good thermal stability.

When aryl and alkyl groups are of ordinary reactivity then both esterification and hydolysis involves B-O fission, and not C-O fission. The is because(+)-2-methylheptanol is isolated unchanged in rotatory power after being converted into and obatined from the ester of phenylboronic acid. This optical result is confirmed when an (+)-alcohol is reacted with and phenylboron dichloride.

Figure 1-12: Ester preparation via a (+)-alcohol and a phenylboron dichloride.

Cyclic esters are an important part of boronic ester chemistry. One such method to prepare these involves the mixing of dihydric alcohols and phenols. Halogeno-ester preparation involves mutual replacement reactions. These produce both alkyl-and arylboronic halogeno-esters, and the reaction occurs quickly.

n-BuB(OBun)2 + BCl3 ® n-BuBCl(OBun) + BunOBCl2


Figure 1-13: Synthesis of halogeno-esters.


a -amino boronic esters:

Secondary amines react readily with iodomethylboronic esters to produce stable (dialkylamino)methyl boronic esters. These reactions are illustrated by the conversion of pinacol iodomethylboronate to piperidine and triethylamine deritvatives.


Figure 1-14: Synthesis of piperidine and triethyl amine derivatives


Air Oxidation:

Cyclic boronic acids are generally stable in air, and can be stored for a number of days or weeks without protection from atmospheric oxygen. But, this is misguiding because autooxidations can consume the entire sample within a relatively short time. Many accounts have been recorded where people used boronic esters for a number of weeks, but after a few months of storage the entire compound decomposed due to exposure to air by a hole in the bottle. Thus, a rubber septum cap is not sufficient for long-tem storage. Analytical samples of boronic esters should be stored in a screwcap vials. But, should be replaced every year if one intends to store these esters for a long time.

Reactions of boronic esters with lithium compounds:

The reaction of esters with lithium compounds is a complex process which involves several factors to consider. Among these are the choice of chiral diol, protection of the a-halo boronic ester, catalysis of the reaction, etc. When making the choice of the chiral diol, depending on the chiral diol used, there may be two different stereochemical outcomes that may result. One such case is when one uses two different borate complexes. That is, the borate complex derived from pinanediol alkylboronate rearranges stereoselectivity, but the borate complex from the pinanediol(dichloromethyl)borate yields a gross mixture of diastereomeric a-chloro boronic esters.


Figure 1-15: Reactions of esters with Lithium compounds.

Figure 1-16: New Boronic ester reactions.





  • Fieser & Fieser. Reagents for organic synthesis. Wiley-Interscience. New York, 1969.
  • Gerrard,W. The Organic Chemistry of Boron. Academic Press. New


York, 1969.

3. Herbert & Brown. Hydroboration. W.A. Benjamin, INC. New York, 1962.

4. Muetterties, E. The Chemistry of Boron and Its Compounds. John Wiley & Sons, 1967.

  1. Proctor, G. Asymmetric Synthesis. Oxford Science Publications. New York, 1996.



  • Abel et al. Comprehensive organometallic Chemistry II-A review of the literature 1982-1994. Volume 6,7, and 12. Pergamon Press. United Kingdom, 1995.
  • Greenwood N.N. and Earnshaw. Chemistry of the Elements. Pergamon Press. New York, 1984.
  • Katritzky et al. Comprhensive Organic Functional Group Transformations. Volumes 1,2,4. Pergamon Press. Glasgow, 1995.
  • Wilkenson et al. Comprehensive Organometallic Chemistry. Volumes 1,4. Pergamon Press. New York, 1982.


Web Sites:

  4. www.netscape/chemistry/boronic




  • Howard et al. Synthesis of Novel C7-Aryl Substitued Pyrrolo(2,1-C)(1,4)Benodiazepines(PBDs) via Pro-N-10-Troc Protection and Suzuki Coupling. Bioorganic & Medicinal Chemistry Letters 1998, Vol , Iss 2, pp. 3017-3018.
  • Metteson, D. Functional compatibilities in boronic ester chemistry. Journal of Oganometallic Chemistry. Washington, 1999. (web of science)
  • Nishimura et al. Preparation and Thermal Properties of Thermoplastic Poly(vinyl alcohol) Complexes with Boronic Acids. Kyoto, Japan, 1998.




One thought on “Controlling Residual Arylboronic Acids as Potential Genotoxic Impurities in APIs

    June 29, 2015 at 5:45 am

    Reblogged this on ORGANIC CHEMISTRY SELECT.

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