Correlation of antifungal activity of selecteda-substitutedacetophenones with their keto–enol tautomerization energy

A.M. Rodrıgueza,* , F.A. Gianninia, F.D. Suvirea, H.A. Baldonia, R. Furlanb,S.A. Zacchinob, G. Bekec, P. Matyusc, R.D. Enriza, I.G. Csizmadiad

aDepartamento de Quı´mica, Universidad Nacional de San Luis, Chacabuco 917, 5700 San Luis, ArgentinabDepartamento de Farmacognosia, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad de Rosario, 7000 Rosario, Argentina

cInstitute of Organic Chemistry, Semmelweis University of Medicine, H-1092, Budapest, HungarydDepartment of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6

Abstract

The antifungal activity, measured as minimum inhibitory concentration (MIC) of selecteda-substituted acetophenones,showed a linear correlation with the computed enolization energies (DEenolization). This suggested that keto–enol tautomerizationplays a significant role in the action mechanism of thesea-substituted acetophenones. In order to corroborate this conclusion,two related compounds were synthesized which could not undergo keto–enol tautomerization. Their bioassay proved them tobe completely inactive.q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Antifungal activity; a-Substituted acetophenones; Conformational analysis; Hartree–Fock and DFT computations; Keto–enoltautomerization

1. Introduction

The therapy of fungal infections, particularly thosein immune-compromised individuals, is an increas-ingly difficult medical problem [1,2]. Fungi thatonce were innocuous, merely interfering with thefreshness of one’s bread or asymptomatically colonis-ing most human gastrointestinal tracts, are now oftenthe cause of life-threatening infections in immune-compromised patients [3].

Some forms of dermatomycoses are the cause of agreat mortality in immunocompromised patients, such

as those receiving antineoplasic chemotherapy, organtransplants or suffering from AIDS. Thus, organ andbone-marrow transplantation, cytotoxic chemother-apy for malignancy and the use of highly potent anti-bacterial agents were all being used with increasingfrequency with the result that there was a slow butsure increase in the numbers of reported systemicfungal infections [4,5]. In addition, because of thedifficulties in diagnosing invasive fungal infections,the existence of a systemic fungal infection wasoften not proven until autopsy.

The polyene antifungals, such asamphotericin Bstill remain the standard of therapy because of itsbroad spectrum and antifungal activity. However,due to the various side effects associated withampho-tericin B, including toxicity, the azole antifungalssuch asfluconazoleand itraconazoleare now mostwidely used in the treatment of antifungal disease.

Journal of Molecular Structure (Theochem) 504 (2000) 35–50

0166-1280/00/$ - see front matterq 2000 Elsevier Science B.V. All rights reserved.PII: S0166-1280(00)00353-5

www.elsevier.nl/locate/theochem

* Corresponding author.E-mail addresses:amrodri@unsl.edu.ar (A.M. Rodrı´guez),

fsuvire@unsl.edu.ar (F.D. Suvire), hbaldoni@unsl.edu.ar(H.A. Baldoni), szaabgil@citynet.net.ar (S.A. Zacchino),matypet@szerves.sote.hu (P. Ma´tyus), icsizmad@alchemy.chem.utoronto.ca (I.G. Csizmadia).

There have been an increasing number of reports onthe development of resistance to some of azole anti-fungals. Thus, there is a need for novel therapies forserious fungal diseases as well as for the managementof the legions of topical fungal infections [6].

In the course of our screening program for antifun-gal activity, we reported [7] that 8.O.40 neolignansposses a moderate but significant antifungal activityagainst dermatophytes, a type of fungi which charac-teristically infect the keratinized areas of human skin.We also performed a systematic study of antifungal

properties of their phenylpropanoid moieties andstructurally related compounds [8]. We found thatsome arylpropanoids posses strong antifungal effectsagainst dermatophyte fungi displaying an antifungalbehaviour similar or better than the currently used anti-fungal agents such asamphotericin Bor ketoconazole.

Recently [9], we reported that keto–enol tautomer-ization may be one of the mechanisms of antifungalactivity for these compounds. Although we used rela-tively low level theory in that exploratory study, aninteresting correlative trend was observed when

A.M. Rodrı´guez et al. / Journal of Molecular Structure (Theochem) 504 (2000) 35–5036

Fig. 1. Conformational potential energy curves ofa-substituted acetophenones obtained from RHF/3-21G relaxed single scan computations.Torsional anglex2 measures the rotation about the aliphatic C–C bond. [Code: 1 propiophenone; 2a-fluoroacetophenone; 3 acetophenone; 4a-chloro acetophenone (semi rigid scan withx1 � 0��:

ln[MIC] (Minimal Inhibitory Concentration)values were plotted against computed molecularproperties.

We here report an ab initio study on the keto andenol forms of selected antifungals and the correlationbetween MIC values with their computed enolizationenergies (DEenolization).

To obtain further insights into the role of the keto–enol forms of these compounds, acting as antifungal

agents, we have synthetized two molecules which arenot able to undergo a keto–enol enolization and theirMICs were also evaluated.

2. Computational methods

Ab initio Hartree–Fock optimizations have beencarried out using thegaussian 94 program system

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Fig. 2. Conformational potential energy curves of the enol forms of selecteda-substituted acetophenones obtained from RHF/3-21G relaxedsingle scan computations. Torsional anglex1 measures the phenyl rotation. [Code: 1a-fluoroacetophenone-enol form (cis-endoconformation);2 acetophenone-enol form (endoconformation)].

Scheme 1.

[10]. Two basis set: 3-21G and 6-31G(d,p) wereemployed.

Density functional [B3LYP/6-31G(d,p)] singlepoint computations, using the RHF/6-31G(d,p)geometries, were also performed.

3. Results and discussion

3.1. Molecular conformations

In calculating the enolization energy, first of allone must optimize the structures for both keto andenol forms. However, initially we have to find outhow many possible structures each componentmay have. Potential energy curves (PEC) havebeen obtained via one-dimensional (1D)-scans forsome of the ketoforms (Fig. 1) and a small selec-tion of the enol forms (Fig. 2). According toFig. 1, the keto forms may occur in thesynform �x2 � 0� and in a rotated form which goesbeyond thegauche arrangement. In factx2 may

assume a range of values around 1208 somethinglike 120^ 308 (Scheme 1).

In contrast, Fig. 2 indicates one unique minimumwhich occurs four times on the PEC. Each of theserepresents a minor deviation (less than 158) fromcoplanarity.

x2 < 0^ 158 and x2 < 180^ 158

However, in principle, each of the keto forms can beconverted to either one of the four possible enol forms(Scheme 2).

This led to twice as many enol structures than ketostructures to be optimized. The optimized keto struc-tures are presented in Table 1 and the optimized enolstructures are summarized in Table 2.

3.2. Stabilization energies

Since the effect of Cl, F and Me appears to beappreciably different it seemed desirable to calculatethe stabilization or destabilization energies for the Cl,F and Me substituted compounds with respect to H, or

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Scheme 2.

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Table 1Optimized structures and energies of acetophenone (H, H, H)a-fluoroacetophenone (F, H, H) propiophenone (Me, H, H) anda-cloroacetophenone (Cl, H, H)

Substituents Initial Final RHF/3-21G Final 6-31G(d,p)

RHF B3LYP

x1 x2 x1 x2 Energy DE (kcal/mol) x1 x2 Energy DE (kcal/mol) Energy DE (kcal/mol)

H, H, H 0 0 20.02 0.02 2380.3405371 0.00 0.00 0.00 2382.4902384 0.00 2384.9057535 0.060 120 0.0 119.89 2380.3405369 0.00 0.00 120.162382.4902383 0.00 2384.9058439 0.000 2120 0.0 2119.89 2380.3405369 0.00 0.00 2120.16 2382.4902383 0.00 2384.9058439 0.00

F, H, H 0 0 20.01 0.00 2478.6462023 3.61 20.01 0.03 2481.3276867 1.51 2484.1216953 1.390 120 0.17 151.16 2478.6519571 0.00 29.77 145.36 2481.3300878 0.00 2484.1239057 0.000 2120 20.32 2151.17 2478.6519571 0.00 9.75 2145.35 2481.3300878 0.00 2484.1239049 0.00

Me, H, H 0 0 0.06 0.01 2419.1619506 0.00 20.01 20.01 2421.5284378 0.00 2424.2197666 0.300 120 28.31 96.95 2419.1588230 1.96 21.60 95.90 2421.5259421 1.57 2424.2202416 0.000 2120 8.36 296.97 2419.1588230 1.96 1.62 295.94 2421.5259421 1.57 2424.2202431 0.00

Cl, H, H 0 0 0.00 0.00 2837.0493166 7.19 20.04 0.01 2841.3809852 0.57 2844.4910168 0.400 120 3.07 106.96 2837.0504626 0.00 22.88 109.32 2841.3818903 0.00 2844.4916518 0.000 2120 23.08 2106.96 2837.0504626 0.00 2.88 2109.28 2841.3818902 0.00 2844.4916483 0.00

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Table 2Optimized structures and energies (energies for the most stable conformations are printed in bold) of enol forms

Substituents Initial Final RHF/3-21G Final 6-31G(d,p)

RHF B3LYP

x1 x2 x3 x1 x2 x3 Energy (hartrees) x1 x2 x3 Energy (hartrees) Energy (hartrees)

Exo H, H 15 – 0.0 29.33 – 45.98 2380.3148176 35.81 – 32.04 2382.4602369 2384.8782593165 – 0.0 150.27 – 245.95 2380.3148176 143.95 – 232.0 2382.4602370 2384.8782692

Endo H, H 15 – 180.0 15.24 – 2175.79 2380.3174647 28.43 – 2174.27 2382.4621846 2384.8800810165 – 180.0 164.64 – 175.83 2380.3174648 151.72 – 174.29 2382.4621849 2384.8800657

Cis-exo F, H 15 0.0 0.0 28.17 5.91 60.29 2478.6258297 36.71 4.96 40.59 2481.3002835 2484.0997844165 0.0 0.0 150.85 25.91 260.26 2478.6258296 142.69 24.94 240.64 2481. 3002833 2484.0997896

Me, H 15 0.0 0.0 28.71 4.32 50.94 2419.1385990 35.66 3.97 35.51 2421.5012779 2424.1993554165 0.0 0.0 150.82 24.33 250.99 2419.1385990 143.82 23.97 235.45 2421.5012778 2424.1993603

Cl, H 15 0.0 0.0 33.12 5.55 43.85 2837.0255045 39.42 4.11 28.75 2841.3569068 2844.4709471165 0.0 0.0 146.30 25.58 243.86 2837.0255043 104.01 24.11 228.74 2841. 3569068 2844.4709501

Cis-endo F, H 15 0.0 180.0 16.52 1.91 2177.19 2478.6330071 31.87 2.31 2175.67 2481.3056881 2484.1054342165 0.0 180.0 163.36 21.90 177.19 2478.6330071 148.31 22.32 175.65 2481.3056882 2484.1054387

Me, H 15 0.0 180.0 20.51 3.37 2166.35 2419.1375785 31.25 2.93 2168.37 2421.5006929 2424.1992330165 0.0 180.0 159.35 23.37 166.40 2419.1375785 148.77 22.95 168.36 2421.5006929 2424.1992403

Cl, H 15 0.0 180.0 21.10 2.59 183.73 2837.0326363 32.54 2.22 2175.40 2841.3624615 2844.4765664165 0.0 180.0 158.76 22.59 176.26 2837.0326363 147.50 22.22 175.41 2841.3624615 2844.4765674

Trans-exo F, H 15 180.0 0.0 14.30 2176.89 64.06 2478.6323797 27.81 2174.59 49.39 2481.3011739 2484.1007583165 180.0 0.0 165.33 2183.11 264.06 2478.6323797 151.05 2185.41 249.58 2481. 3011739 2484.1007496

Me, H 15 180.0 0.0 37.32 188.83 46.76 2419.1333054 44.97 2172.08 34.70 2421.4973800 2424.1953093165 180.0 0.0 140.02 2188.85 246.75 2419.1333054 132.82 2187.89 234.72 2421. 4973801 2424.1953078

Cl, H 15 180.0 0.0 34.59 187.73 44.0 2837.0216199 43.91 187.27 32.58 2841.3534008 2844.4670030165 180.0 0.0 142.72 172.23 243.94 2837.0216187 133.62 172.72 232.64 2841. 3534008 2844.4670021

Trans-endo F, H 15 180.0 180.0 0.0 180.0 179.98 2478.6315295 24.09 182.18 2170.79 2481.2994332 2484.0999883165 180.0 180.0 179.99 180.0 179.992478.6315294 155.92 177.83 170.682481.2994331 2484.0999872

Me, H 15 180.0 180.0 35.73 186.27 2172.95 2419.1336182 46.05 185.22 2173.35 2421.4978572 2424.1957525165 180.0 180.0 142.19 173.74 172.912419.1336181 133.97 174.97 173.152421.4978573 2424.1957511

Cl, H 15 180.0 180.0 34.14 185.18 2172.95 2837.0212199 42.56 184.67 2172.37 2841.3528369 2844.4665632165 180.0 180.0 147.54 174.81 172.992837.0212199 135.60 175.36 172.522841.3528370 2844.4665616

in other words with respect to the parent compoundacetophenone.

In order to calculate the stabilization energy, oneneeds to calculate the energy change associated withthe following pair of isodesmic reactions.

The energies of the large molecules are given inTables 1 and 2 and the energies of the small compo-nent molecules summarized in Table 3. The stabiliza-tion energies were calculated for all structures at theRHF/3-21G, RHF/6-31G(d,p) and single pointB3LYP/6-31G(d,p) levels of theory. The data are

tabulated in Tables 4 and 5 for the keto and enolcompounds, respectively. The results are also showngraphically for the keto compounds in Fig. 3. It isinteresting to note that Cl destabilizes the moleculein all conformations at all levels of theory. It is also

interesting to note that the patterns in Fig. 3 are the samefor thegaucheconformation at all levels of theory.

The stabilization energies for theexoenol form aregiven in Fig. 4 and those for theendoenol form arepresented in Fig. 5. The pattern forexo-transis analo-gous toendo-trans. However, theexo-cisandendo-cis

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Table 3Optimized energy values for component molecules

Molecules E (hartrees) RHF/3-21G E (hartrees) RHF/6-31G(d,p) E (hartrees) B3LYP/6-31G(d,p)

CH4 239.9768774 240.2017046 240.5238417CH3–CH3 278.7939477 279.2382349 279.8383837CH3–F 2138.2818931 2139.0397359 2139.7375637CH3–Cl 2496.6894830 2499.097899 2500.1121376CH2yCH2 277.6009881 278.0388400 278.5932816CH2yCH–CH3 2116.4240095 2117.0816140 2117.9158693CH2yCH–F 2175.92056697 2176.8875732 2117.8236401CH2yCH–Cl 2534.3148771 2536.9390787 2538.1894683CH2yCPh(OH)exo 2380.3148176 2382.4602370 2384.8782692endo 2380.3174648 2382.4621849 2384.8800810

Table 4Stabilization energies of keto forms ofa-substituted acetophenones

a-Substituents DEstabil. (kcal/mol) RHF/3-21G RHF/6-31G(d,p) B3LYP/6-31G(d,p)

Syn Gauche Syn Gauche Syn Gauche

F, H, H 20.41 24.02 10.37 21.14 21.39 22.72Me, H, H 22.73 20.76 21.05 10.52 10.33 10.09Cl, H, H 12.40 11.68 13.42 12.85 11.90 11.56

structures behave in a completely different fashion.Undoubtedly the –Cl· ··HO– type interaction is stabi-lizing while the analogous interactions involving Fand Me are destabilizing.

3.3. Keto–enol tautomerization

The enolization energies (DEenolization) computed atseveral levels of theory are summarized in Table 6.

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Table 5Stabilization energies of enol compounds

DEstabil. (kcal/mol)RHF/3-21G

DEstabil. (kcal/mol)RHF/6-31G(d,p)

DEstabil. (kcal/mol)B3LYP/6-31G(d,p)

ExoCis FH 15.38 15.45 15.55

MeH 11.18 12.31 12.08ClH 12.01 12.23 12.19

Trans FH 11.26 14.89 14.94MeH 12.84 13.53 13.48ClH 14.45 14.44 14.67

EndoCis FH 12.53 13.28 13.14

MeH 11.82 12.68 12.15ClH 20.80 20.02 20.19

Trans FH 13.46 17.20 16.55MeH 14.31 14.46 14.34ClH 16.36 16.02 16.08

Fig. 3. Stabilization energy values ofa-substituted acetophenones in theirsyn(s) andgauche(g) forms computed at several levels of theory.

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Fig. 4. Stabilization energy values for the enol forms ofa-substituted acetophenones in theirexoconformations computed at several levels oftheory.

Fig. 5. Stabilization energy values for the enol forms ofa-substituted acetophenones in theirendoconformations computed at several levels oftheory.

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Table 6Enolization energies ofa-substituted acetophenones

Compound RHF/3-21G RHF/6-31G(d,p) B3LYP/6-31G(d,p)

E (hartrees)enol form

E (hartrees)keto forma

DEenoliz.b

(kcal/mol)E (hartrees)enol form

E (hartrees)keto forma

DEenoliz.b

(kcal/mol)E (hartrees)enol form

E (hartrees)keto forma

DEenoliz.b

(kcal/mol)

HH Exo 2380.3148176 2380.3405371 16.14 2382.4602370 2382.4902384 18.83 2384.8782692 2384.9058439 17.30Endo 2380.3174647 2380. 3405371 14.48 2382.4621849 2382.4902384 17.60 2384.8800810 2384.9058439 16.17

FH Cis-exo 2478.6258297 2478.6519571 16.39 2481.3002835 2481.3300878 18.70 2484.0997896 2484.1239057 15.13Cis-endo 2478.6330071 2478.6519571 11.89 2481.3056882 2481.3300878 15.31 2484.1054387 2484.1239057 11.59Trans-exo 2478.6323797 2478.6519571 12.29 2481.3011739 2481.3300878 18.14 2484.1007583 2484.1239057 14.53Trans-endo 2478.6315295 2478.6519571 12.82 2481.2994332 2481.3300878 19.24 2484.0999883 2484.1239057 15.01

MeH Cis-exo 2419.1385990 2419.1619506 14.65 2421.5012779 2421.5284378 17.04 2424.1993603 2424.2202416 13.10Cis-endo 2419.1375785 2419.1619506 15.29 2421.5006929 2421.5284378 17.41 2424.1992403 2424.2202416 13.18Trans-exo 2419.1333054 2419.1619506 17.98 2421.4973801 2421.5284378 19.49 2424.1953093 2424.2202416 15.65Trans-endo 2419.1336182 2419.1619506 17.78 2421.4978573 2421.5284378 19.19 2424.1957525 2424.2202416 15.37

ClH Cis-exo 2837.0255045 2837.0504626 15.66 2841.3569068 2841.3818903 15.68 2844.4709501 2844.4916518 12.99Cis-endo 2837.0326363 2837.050462 11.19 2841.3624615 2841.3818903 12.19 2844.4765674 2844.4916518 9.47Trans-exo 2837.0216199 2837.0504626 18.10 2841.3534008 2841.3818903 17.88 2844.4670030 2844.4916518 15.47Trans-endo 2837.0212199 2837.0504626 18.35 2841.3528370 2841.3818903 18.23 2844.4665632 2844.4916518 15.74

a The energy of the global minimum conformation is used.b DE� Enol energy2Keto energy (smallest values in a family are printed in bold).

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45Table 7Minimum inhibitory concentration (MIC) values for selecteda-substituted acetophenones using four different fungi

Compounda DEenoliz.b Microsporum gypseumA Trichophyton mentagrophytesB Trichophyton rubrumC Microsporum canisD

MICc ln[MIC] MIC c ln[MIC] MIC c ln[MIC] MIC ln[MIC]

IHHH 14.48 50 3.91 50 3.91 50 3.91 50 3.91MeHH 14.65 60 4.09 60 4.09 50 3.91 60 4.25ClHH 11.19 12.5 2.53 12.5 2.53 10 2.30 3.12 1.14

IIHHH 50 3.91 50 3.91 50 3.91 50 3.91MeHH 60 4.09 60 4.09 50 3.91 60 4.25ClHH 12.5 2.53 12.5 2.53 10 2.30 3.12 1.14

IIIHHH 40 3.69 25 3.22 40 3.69 50 3.91MeHH 25 3.22 25 3.22 50 3.91 40 3.69ClHH 10 2.30 10 2.30 6 1.79 3 1.10

IVHHH 6.25 1.83 3 1.10 6.25 1.83 3 1.10MeHH 20 3.00 6.25 1.83 20 3.00 20 3.00ClHH 10 1.10 3 1.10 1.5 0.41 3 1.10

a X may be H, Me and Cl.b Smallest values found (cf. bold figures in Table 6).c In mg/ml.

The lowest values of the several structures computedare given as bold numbers.

Table 7 presents the MIC for four families (I –IV ) ofcompounds using four different fungi (A–D). In Fig. 6,the various MIC values presented in clusters for theelectron donating Me group and the electron withdraw-ing Cl in comparison to the reference substituent: H.

The Me substituent compounds are more sensitiveto the structure of the aromatic moiety and they alsohave varying effects on different fungi. The leastselective and therefore most effective compoundsturned out to be those with chlorine substitution.

The ln[MIC] values were plotted against theDEenolizationvalues for each of the four fungi (Fig. 7A–C) behave quite similarly but the fourth oneMicro-sporum canis(D) shown a great sensitivity towards

the chloro compound (Fig. 7D). The characteristics ofthe fitted straight lines are summarized in Table 8. Thephysical meaning of the slope and intercepts are given interms of characteristic MIC values in the last twocolumns of Table 8. The exponential of the slope (em)gives the change in MIC value for 1 kcal/mol change inenolization energy. [MIC]0 is the MIC value a particularcompound may have withDEenolization� 0:0 (i.e. whenthe enol and keto forms occur in equal concentration).

4. Experimental section

4.1. Synthesis

Scheme 3 summarizes the synthesis of certainselected compounds.

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Fig. 6. Clusters of MIC values for three substituents of variable electron donating and withdrawing ability.

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Fig. 7. Correlation of MIC values ofa-substituted acetophenones with energy of enolization using four different fungi. (A:Microsporum gypseum; B: Trichophyton mentagro-phytes; C: Trichophyton rubrum; D: Microsporun canis). Data points were computed at the RHF/3-21G level of theory.

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Table 8Characterists of the linear correlation between ln[MIC] values andDEenolization

Fungus RHF/3-21G RHF/6-31G(d,p) B3LYP/6-31G(d,p)

kR2l Slopea

mIntercepb

ln[MIC] 0

em� D[MIC] [MIC] 0 kR2l Slopea

mIntercepb

ln[MIC] 0

em� D[MIC] [MIC] 0 kR2l Slopea

mIntercepb

ln[MIC] 0

em� D[MIC] [MIC] 0

A Microsporumgypseum

1.00 0.44 2.36 1.55 10.59 0.96 0.28 20.88 1.32 0.41 0.70 0.21 0.76 1.23 2.14

B Trichophytonmentagrophytes

1.00 0.44 2.36 1.55 10.59 0.96 0.28 20.88 1.32 0.41 0.70 0.21 0.76 1.23 2.14

C Trichophytonrubrum

1.00 0.48 3.03 1.62 20.70 0.99 0.31 21.48 1.36 0.23 0.79 0.25 0.19 1.28 1.21

D Microsporumcanis

1.00 0.85 28.35 2.34 0.0002 0.98 0.55 25.53 1.73 0.003 0.74 0.43 22.45 1.54 0.09

a Slope� Dln[MIC]/kcal/mol of enolization.b ln[MIC] 0 is the ln[MIC] value whenDEenolization� 0.0 kcal/mol.

Compound3 was prepared through a Grignardreaction [11] with piperonylonitrile (1) (Aldrich)and ethylmagnesium bromide in dry Et2O and subse-quent hydrolysis, followed by a chlorination reactionwith cooper (II) chloride and lithium chloride in DMF[12] (total yield from1: 60%).

Compound6 was prepared through a Grignardreaction [11] with piperonylonitrile (1) (Aldrich)and isopropylmagnesium chloride in dry Et2O andsubsequent hydrolysis, followed by a chlorinationreaction with cooper (II) chloride and lithium chloridein DMF [12] (total yield from1: 40%).

Compound4 was prepared from3 by reaction withenthylene glycol in dry benzene catalyzed by 10-camphorsulfonic acid in a Dean and Stark apparatus(yield 65%).

All compounds were fully characterized by spectro-scopic methods and physical data are available uponrequest.

4.2. Antifungal assays

With the aim to compare the antifungal activityobtained for compounds4 and6 with those recentlyreported for other arylpropanoids, we use the samebioassay reported earlier [9]. Thus the fungistaticactivity of compounds4 and 6 was evaluated withthe agar dilution method by using Saboureaud-Chlor-amphenicol agar for both yeast and dermatophytespecies.

The antifungal agents ketoconazole (Jenssen Phar-maceutica) and amphotericin B (Sigma Chemical Co.)were included in the assay as positive control. Drug-free solution was also used as blank control.

It is interesting to note that both compounds4 and6are devoid of any antifungal activity (Table 9).

5. Conclusions

The linear correlation between ln[MIC] values andDEenolization suggested that keto–enol tautomerizationplays a significant role in the biological action-mechanism of the a-substituted acetophenonesstudied. The experimentally determined negative

A.M. Rodrı´guez et al. / Journal of Molecular Structure (Theochem) 504 (2000) 35–50 49

Scheme 3.

Table 9Minimal inhibitory concentration for synthetic compounds4 and6

Compound [MIC]mg/ml

Structure Number

4 . 200

6 . 200

bioassay of two derivatives, which could not undergoketo–enol tautomerization, corroborated such aconclusion.

Acknowledgements

The research was supported by grants from theUniversidad Nacional de San Luis (UNSL), Fundacio´nAntorchas (Proyecto A-13622/1-49) and the ProjectHUN2/99/OG of the Programa de Cooperacio´n Cien-tıfica SECyT-OMFB. R.D.E. is a carrier researcher ofCONICET. The continuos financial support of theNatural Sciences and Engineering Research Council(NSERC) is gratefully acknowledged.

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