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Journal of Chromatography A, 1151 (2007) 197202

Applications of counter-current chromatography in organicsynthesis purification of heterocyclic derivatives of lapachol

Raphael S.F. Silva, Gilda G. Leitao, Thiago B. Brum, Ana Paula G. Lobato,Maria do Carmo F.R. Pinto, Antonio V. Pinto

Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro,

Bloco H, CCS, Ilha do Fund ao 21.941-590, Rio de Janeiro, RJ, Brazil

Available online 21 March 2007

Abstract

This work describes the application of counter-current chromatography (CCC) as a useful, fast and economic alternative for the isolation andpurification of heterocyclic derivatives from lapachol and -lapachone, two naturally occurring compounds fromTabebuia species, and nor--

lapachone, a synthetic congener of lapachol. The discussed data comprise four examples of purification of synthetic reactions with different solvent

systemsthe mixture of the oxazole and the imidazole from -lapachone; the quinoxaline from nor--lapachone; and the purification of the

N-oxides from the quinoxaline and the phenazine from nor--lapachone from their respective not fully reacted substrates by means of aqueous

reversed- and normal-phase elution modes and non-aqueous solvent systems. Traditional purification of these reaction products by silica gel column

chromatography demanded a large amount of solvent and time and, in some cases, serious degradation of the products occurred, leading to low

yield of the reaction. High-speed counter-current chromatography (HSCCC) was used as an alternative to optimize the process and raise the yield

of the reactions.

2007 Elsevier B.V. All rights reserved.

Keywords: Lapachol;-Lapachone; nor--Lapachone; Phenazines; Quinoxalines; Heterocycles; Counter-current chromatography

1. Introduction

Lapachol (1) and -lapachone (2) are natural products iso-

lated from the bark of plants of the genus Tabebuia, especially

Tabebuia avellanedae, known in Brazil by the name of Ipe

or Ipe roxo[1,2].These naphtoquinones present an array of

important biological activities such as antitumor and antimi-

crobial properties [36]. Samuel Hooker, at the end of the

19th century described the conversion of lapachol into -

lapachone and nor--lapachone (3), a semi-synthetic congener

of-lapachone,Fig. 1[7,8].Taking into account that quinones

can be easily converted to heterocyclic systems such as imida-

zoles, oxazoles, phenazines and quinoxalines, among others[9]

anddue to their high toxicity to mammalcells [10], ourgroup has

been developing heterocyclic derivatives from these quinones in

order to find less toxic and more bioactive compounds.

Counter-current chromatography (CCC) is a form of

liquidliquid chromatography, which does not use a solid sup-

Corresponding author. Tel.: +55 21 25626795.

port, there can be no loss of compounds and their chemical

structure is better maintained[11].This form of chromatogra-

phy is based on the partition of solutes between two immiscible

liquid phases and compounds are separated according to their

distribution constants KD expressed as the ratio of their con-

centration in the stationary phase to their concentration in the

mobile phase[12].CCC is particularly useful in the preparative

range (mg to g) and the time required for preparative separation

is no more than a few hours[13].

Compared to the application of CCC on phytochemical work

relatively little attention has been given to the use of this tech-

nique on the separation of products from organic synthesis

[1317]. The first papers describing the application of CCC

for the separation and purification of organic synthetic mixtures

were those on the purification of synthetic peptides [14]and on

the purification of catecholamines[13].

Next we discuss the application of high-speed counter-

current chromatography (HSCCC) as an useful alternative

for the isolation and purification of heterocyclic compounds

from synthetic originthe mixture of 4 and 5, respectively

the oxazole and the imidazole from -lapachone, the quinox-

0021-9673/$ see front matter 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.chroma.2007.03.066
http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.chroma.2007.03.066http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.chroma.2007.03.066


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198 R.S.F. Silva et al. / J. Chromatogr. A 1151 (2007) 197202

Fig.1. General syntheticscheme. 1 = lapachol;2 = -lapachone;3 = nor--lapachone;4 = imidazolefrom-lapachone;5 = oxazole from-lapachone;6 = quinoxaline

from nor--lapachone;7 = phenazine from nor--lapachone;8 =N-oxide from6;9 =N-oxide from7. Reagents used in the reactions: a = H2SO4; b = H2O2, NaCO3,

THF, reflux 2 h; c = CuSO4, NaCO3, THF, reflux 2 h; d = NH4OAc, benzaldehyde, AcOH, reflux, 2 h; e = ethylenediamnine, 24 h; f = o-phenylenediamine, AcOH,

reflux, 2 h; g= m-chloroperbenzoic acid, CH2Cl2, 2h.

aline from nor--lapachone, 6, and the purification of the

N-oxides 8, from 6 and 9 from 7 (Fig. 1) by means of aque-

ous reverse and normal elution modes and non-aqueous solvent

systems. In the first two examples traditional purification of

these reaction products by silica gel column chromatography

demanded a large amount of solvent and time and HSCCC

was used as an alternative to optimize the process and raise

the yield of the involved chemical reactions. In the last exam-

ple, CCC was used since traditional purification on silica

gel caused serious degradation of products, leading to low

yield.

2. Experimental

2.1. Synthesis

Lapachol was extracted according to extraction procedure

described in the literature[1,2]. -Lapachone, nor--lapachone

and the phenazine 7 were prepared according to literature data

[7,8].

The imidazole and the oxazole from -lapachone (4 and

5, respectively) were prepared by the reaction of-lapachone

(228 mg, 0.9 mmol) with benzaldehyde (20 ml, 1.1 mmol) in the

presence of ammonium acetate (300 mg,4 mmol) andacetic acid

(20 ml) as solvent under reflux according to Pinto and coworkers

[18].

The quinoxaline 6 was synthesized by dissolving nor--

lapachone (176 mg, 0.77 mmol) in ethylenediamine (10 ml)

under stirring for 24 h at room temperature.

TheN-oxide 8 wasprepared by thereactionof thequinoxaline

6(70 mg, 0.28 mmol) andm-choloroperbenzoic acid, MCPBA,

(144 mg, 0.8 mmol) in CH2Cl2 (10 ml) under stirring for 24 h

at room temperature. The N-oxide9 was prepared as described

for 8, with 47 mg (0.15 mmol) of7 and 80 mg (0.45 mmol) of

MCPBA.

All compounds were identified by 1H and 13C NMR spec-

troscopy using a Varian spectrometer (Varian, Palo Alto, CA,

USA) model Gemini 200, at 200 and 50 MHz, respectively.

2.2. Sample preparation

2.2.1. Mixture of oxazole and imidazole

Ice was added to the crude reaction mixture containing 4

and 5. The formed precipitate was filtered under vacuum and

dissolved in the solvent system for the CCC separation.

2.2.2. Quinoxaline sample

The reaction mixture containing 6 and other by-products was

dissolved in water and extracted with ethyl acetate. The organic

phase was evaporated and the solid residue was dissolved in the

planned solvent system for the CCC separation.


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R.S.F. Silva et al. / J. Chromatogr. A 1151 (20 07) 197202 199

2.2.3. N-oxides sample

A 20% sodium carbonate solution was added to the reac-

tion mixture containing the N-oxide and the original not fully

reacted substrate. The biphasic system was stirred for 24 h at

room temperature for the extraction of the residual MCPBA

from the organic phase. After separation of the two phases, the

organic phase was evaporated and the solid residue dissolved on

the CCC solvent system.

2.3. Choice of the solvent system

Small amounts of each of the samples described above were

dissolved in separate test tubes containing the solvent systems

to be tested. The test tubes were shaken and the compounds

allowed to partition between the two phases. Equal aliquots of

each phase were spotted beside each other separately on TLC

plates, developed with hexane/EtOAc 30%. The results were

visualized under UV light (264 and 365 nm).

2.4. CCC separations

All separations were performed on a P.C. Inc. (Potomac,

MD, USA) counter-current chromatograph equipped with a

multi-layer coil equilibrated by a counterweight. The volume

of the coil was 80 ml. The pump was Model SD-200 Dynamax

Rainin (Greifensee, Switzerland). A Rainin Dynamax FC-1frac-

tion collector was also used. All separations were performed

at 850 rpm, fractions of 4 ml were collected, at a flow rate of

2 ml/min. Fractions were monitored by TLC, visualized under

UV light (365 nm).

2.4.1. Separation of4

and5

by aqueous solventsystemreversed phase

The stationary upper phase of the solvent system hexane

methanolwater 1:2:1 (v/v/v) was pumped into the coil in the

head-to-tail direction. After the coil was filled with station-

ary phase, rotation started and the mobile phase was pumped

until all the excess of stationary phase came out of the coil

(Vm= 12ml,SF= 0.85, noticed when only mobile phase comes

out from the coil). The sample containing only4and5(270mg)

was dissolved in 10 ml of the solvent system and applied in

the equipment. Sixty fractions were collected and rotation was

stopped on tube 36. The imidazole was obtained in a pure form

in tubes 513 (170 mg,KD= 0.45, yielding 54%) and the oxa-

zole was recovered from the stationary phase, in a pure form, onfractions 4854 (80 mg,KD> 1.8, yielding 27%).

2.4.2. Isolation of6by aqueous solvent systemnormal

phase

The stationary lower phase of the solvent system hexane

ethyl acetatemethanolwater 1:1:1:1 (v/v/v/v) was pumped

into the coil in the tail-to-head direction. After the coil was

filled with stationary phase, rotation started and the mobile

phase was pumped untilhydrodynamic equilibrium(Vm= 12ml,

SF= 0.85). The sample (191 mg), dissolved in both mobile and

stationary phases was injected. Seventy fractions (4 ml) were

collected, rotation stopped at tube 40. The quinoxaline, 6, was

recovered in tubes 39 (120 mg,KDapproximately 0.34, yield-

ing 62%).

2.4.3. Separation of6from 8 and7from 9 by non-aqueous

solvent systemsnormal phase

The samples containing 8 and its substrate, 6 (80 mg) and

9 and its substrate, 7, (50 mg) were separately dissolved in

the biphasic solvent system hexaneacetonitrilemethanol 2:2:1(v/v/v) and injected on the coil. In both cases, the upper phase

was the mobile phase (Vm= 20ml,SF= 0.75).

3. Results and discussion

3.1. Separation of the imidazole and the oxazole from

-lapachone by reversed-phase aqueous solvent system

Semi-synthetic naphthoimidazoles from -lapachone (2) are

promising agents for the chemotherapy of Chagass disease due

to their high trypanocidal activities [18]. These naphthoimi-

dazoles are synthesized by the reaction of-lapachone with

aldehydes in the presence of ammonium acetate[18].Howeverthis reaction is not selective, the corresponding oxazoles are

formed together with the naphthoimidazoles. The separation of

4and 5 by column chromatography over silica gel is effective

but a large amount of solvent is consumed and HSCCC was

used as an alternative and more economic method. The selec-

tion of the solvent system by the test tube partitioning test was

based on the polarity of4and5as shown by TLC and the mix-

ture of hexanemethanolwater 1:2:1 was chosen. The lower

aqueous phase was used as mobile phase. The more polar imi-

dazole eluted with the aqueous mobile phase and was obtained

in a pure form with a KD of approximately 0.45 (54% yield-

ing) while the oxazole was recovered from the stationary phase,in a pure form (KD> 1, 27% yielding),Fig. 2. A raise of 5%

in the yielding of the target compound in this reaction (5) was

obtained when HSCCC was used in this purification instead of

silica gel column chromatography (CC). The yieldings of the

oxazole4 are similar for the two methods. In this example we

can see that the replacement of the oxygen atom in the struc-

ture of the oxazole by the NH group in the imidazole leads to

a great difference on the distribution constant of the two com-

pounds. Theimidazole hasa much higheraffinityfor theaqueous

mobile phase (showing a distribution constant lower than 1 in

this solvent system), whereas the oxazole stays retained in the

stationary organic phase. This can be due to hydrogen bonding

between the NH group of the imidazole with the protic solvents

in the aqueous phase. A total of 400 ml of solvent was required

in this process, which lasted 4 h, showing that the separation

of these compounds by HSCCC is a very economic and fast

process.

3.2. Isolation of the quinoxaline,6from nor--lapachone

by normal-phase aqueous solvent system

In the course of our studies on chemical reactivity and phar-

macological activities, nor--lapachone (3) and the quinoxaline

derivative (6) were prepared. The aim of this purification was

to obtain a large amount of the quinoxaline, with high purity,


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Fig. 2. TLC (eluted with hexaneethyl acetate 30%) of the separation of the oxazole, 4 and the imidazole, 5 from -lapachone with the solvent system

hexanemethanolwater 1:2:1, aqueous phase as mobile, 2 ml/min, 850 rpm. Rotation stopped at tube 36. Visualization of compounds was made under UV lamp at

365 nm. The numbers below the spots correspond to the fractions collected (4 ml each)RF of5 = 0.45;RF of4 = 0.69.

to be used in further synthesis (it is one of the reagents in

the synthesis below). The solvent system used in this purifi-

cation was hexaneethyl acetatemethanolwater 1:1:1:1 and

the mobile phase, in this case, was the organic phase. This

is a medium polarity solvent system chosen, again, based on

the polarity of the target compound 6. The quinoxaline was

recovered in a pure form (according to NMR spectroscopy) elut-ing with a KD of approximately 0.34,Fig. 3,while secondary

products of the reaction were found in subsequent tubes and

at the stationary phase. The yield of the quinoxaline is 57%

when purified by silica gel column chromatography whereas

in the purification by HSCCC it raises to 62%, which repre-

sents the real yield of the reaction since in HSCCC there is

no solid support and no loss of compounds. This procedure

lasted about 4 h with a solvent consumption of about 300 ml,

which is remarkable when compared to the amount of sol-

vent consumed during purification by column chromatography

(1.4 l).

3.3. Separation of the N-oxides8 and9 from their

corresponding quinoxaline6and phenazine 7from

nor--lapachione by normal-phase non-aqueous solvent

systems

Nor--lapachone(3) canbe converted in various heterocyclic

systems such as quinoxalines and phenazines. Quinoxalines and

phenazines from nor--lapachone can be formed by conden-

sation reactions with either ethylene diamine or o-phenylene

diamine. Phenazines possess relevant chemotherapeutic activ-

ities and due to their structural similarities quinoxalines are

potential chemotherapeutic agents. Both are raw materials for

the synthesis ofN-oxide derivatives, which are potent antitu-

mour agents. These N-oxide derivatives are biotransformed by

reductases to their free radical forms, which are toxic to thetumour cell[19].Fig. 1shows the scheme of preparation of the

twoN-oxides, withm-chloroperbenzoic acid (MCPBA). At the

end of both reactions part of the original quinoxaline/phenazine

still remains unreacted. The separation of the N-oxide product

from the original substrate by silica gel column chromatog-

raphy caused degradation of the compounds (yielding of the

N-oxides around 23%) and CCC appeared to be a suitable tech-

nique to solve this problem. A fist attempt to purify 7 from 9

was made with an aqueous solvent system, in order to avoid

the toxicity of acetonitrile. Using the upper phase of the sol-

vent system hexaneethyl acetatemethanolwater 1:1:1:1 as

mobile phase, the N-oxide eluted together with the original

unreacted phenazine. Test tube experiments were performed in

the search for the best non-aqueous solvent system. The mix-

tures hexaneacetonitrile; hexaneacetonitrilemethanol and

hexaneacetonitrileethyl acetate were tested in various ratios.

The best results were achieved with the solvent system

hexaneacetonitrilemethaol 2:2:1. From a sampleof 80 mg of6

and 8, 40 mg of the unreacted quinoxaline elutedwith an approx-

imate distribution constant of 1.6 and 30 mg of the N-oxide

Fig. 3. TLC (eluted with hexaneethyl acetate 30%) of the isolation of the quinoxaline from nor--lapachone, 6, with the solvent system hexaneethyl

acetatemethanolwater 1:1:1:1, organic phase as mobile, 2 ml/min, 850 rpm. Rotation stopped at tube 40. Visualization of compounds was made under UV

lamp at 365nm. The numbers below the spots correspond to the fractions collected (4 ml each). Subsequent fractions correspond to secondary products of the

reaction.RF of6 = 0.76.


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R.S.F. Silva et al. / J. Chromatogr. A 1151 (20 07) 197202 201

Fig. 4. TLC (eluted with hexaneethyl acetate 30%) of the separation of the quinoxaline from nor--lapachone,6, from itsN-oxide,8, with the solventy system

hexaneacetonitrilemethanol 2:2:1, upper phase as mobile, 2 ml/min, 850 rpm. Rotation stopped at tune 50. Visualization of compounds was made under UV lamp

at 365 nm. The numbers below the spots correspond to the fractions collected (4 ml each)RF of6 = 0.76;RF of8 = 0.33.

Fig. 5. TLC (eluted with hexaneethyl acetate 30%) of the separation of the phenazine from nor--lapachone,7, from its N-oxide,9, with the solventy system

hexaneacetonitrilemethanol 2:2:1, upper phase as mobile, 2 ml/min, 850 rpm. Rotation stopped at tune 28. Visualization of compounds was made under UV lamp

at 365 nm. The numbers below the spots correspond to the fractions collected (4 ml each). S = original sample, spotted at the TLC for comparison with the fractions.

RFof7 = 0.83;RF of9 = 0.75.

was recovered from the stationary phase (KD> 2, 38% yield-

ing),Fig. 4.In the case of the phenazine, a sample of 50 mg was

used and the unreacted starting material (30 mg) eluted with a

KD of approx. 0.95 while the N-oxide was obtained from the

stationary phase (KD> 1.4, 15 mg, 30% yielding),Fig. 5.The

yielding of both N-oxides when purified by HSCCC instead

of silica gel CC is remarkable (from 23% to 38% and 30%

for 7and 9, respectively), showing that HSCCC is a powerful

technique in the purification of labile compounds. Comparing

the KD of compounds 6 and 7 we can see that the quinoxa-

line, 6, which is one benzene ring shorter, has more affinity

to the stationary phase (higher KD) than the phenazine, 7.

The oxidation of the original aza-compounds to their respec-

tive N-oxides caused a significant modification of their KD.

Nevertheless, this modification was not significant enough to

enable the separation of theN-oxides from their unreacted sub-

strates in an aqueous solvent system, where they eluted together

in the organic mobile phase. The retained compounds in the

last fractions in both purification procedures contained impuri-ties from the reactions. A similar non-aqueous solvent system,

heptaneacetonitrilemethanol, was used by Duret et al. [17]

to purify 2-alkylquinolines obtained by liquid combinatorial

synthesis in a CPC equipment.

4. Conclusions

The examples presented in this work demonstrate the versa-

tility of HSCCC in the isolation and purification of compounds

of low and medium polarities and show the potential of this

technique in the synthetic heterocyclic chemistry. The economy

of time and organic solvent is remarkable. In accordance to the

concept of green chemistry the consumption of hazardous sol-

ventsto theenvironment is minimizedwith this technique. These

results open the perspective for a broader use of HSCCC on syn-

thetic chemistry since separation processes can be scaled-up and

used in the pharmaceutical industry.

Acknowledgements

One of us (G.G.L.) is indebted to CNPq for the opportunity

of presenting this work at CCC2006 (NIH, USA). R.S.F.S. is

indebted to CAPES, for a scholarship.

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