Intramolecular hydrogen bonding as a determinant of the inhibitory

potency of N-unsubstituted imidazole derivatives towards

mammalian hemoproteinsw

Lionel Perrin,aFrancois Andre,

aCaroline Aninat,

bRemy Ricoux,

c

Jean-Pierre Mahy,*cNing Shangguan,

dMadeleine M. Joullie

d

and Marcel Delaforgez*a

Received 8th October 2008, Accepted 27th October 2008

First published as an Advance Article on the web 27th November 2008

DOI: 10.1039/b817743k

Enzymes involved in the mammalian microsomal metabolism of drugs are, in numerous cases,

inhibited by compounds bearing an imidazolyl scaffold. However, the inhibition potency is highly

dependent upon the accessibility of the imidazolyl nitrogen lone pair. In order to highlight some

structural parameters of inhibitors that control this phenomenon, a series of compounds

containing a nitrogen unsubstituted imidazolyl moiety with varying degrees of nitrogen lone pair

accessibility was tested on human and rat hepatic cytochromes P450 and microperoxidase 8, an

enzymatically active peptide derived from cytochrome c. In each case, we have shown that the

accessibility of the imidazole lone pair determined the extent of inhibition. Nitrogen accessibility

was tuned not only by varying the steric hindrance in the vicinity of the imidazolyl ring but also

by modifying its surrounding hydrogen bonding network. Compounds in which there exists

intramolecular hydrogen bonding between the imidazole moiety and an H-bond acceptor, such as

an appropriately positioned amide carbonyl group, demonstrated enhanced inhibitory effects.

Conversely, imidazole moieties which are in proximity to H-bond donors, such as an amide NH

group, displayed reduced potency. This trend was observed in cyclo-peptide derivatives in which

the intramolecular H-bond network was adjusted through the modification of the stereochemistry

of a dehydrohistidine residue. It was observed that (Z)-isomers weakly bind heme, whereas

(E)-isomers demonstrated higher degrees of metal binding. Therefore, enzymatic inhibition of

heme-containing proteins by compounds bearing a dehydrohistidine motif seems to be closely

related to its stereochemistry and hydrogen binding propensity. At neutral pH, these differences in

binding affinities can be confidently attributed to the ambident H-bond properties of imidazole

nitrogen atoms. This structure-activity relationship may be of use for the design of novel

imidazolyl compounds as new P450 inhibitors or drug candidates.

Introduction

Metalloproteins represent an important class of enzymes

which are involved in a variety of biochemical processes

such as peroxidase, catalase, monooxygenase and reduc-

tase activities, and oxygen transport and storage. These

functions are related to the presence of a metal center tightly

bound to amino acids such as methionine, cysteine or histidine.1

In the case of hemoproteins, four N-pyrrolyl ligands coordi-

nate the iron in the plane of the porphyrin ring, a fifth axial

position is coordinated by a histidine, methionine or cysteine

residue, and the sixth by an electron rich molecule such as

dioxygen, cyanide, amine, nitroso derivatives or water. In

certain cases, an external molecule containing an electron rich

functional group may serve as a metal ligand, leading to

inhibitory effects. The extent to which such molecules possess

an affinity for complexation to the heme iron determines the

degree of inhibition.2,3

Considering the binding features of iron in hemoproteins, as

well as the importance of their biological functions, these

proteins are relevant targets for studying inhibitory effects.

In particular, cytochromes P450 are highly attractive since a

large panel of isoforms is able to recognize a wide variety of

substrates. Nitrogen rich compounds, such as imidazole

derivatives, represent a large class of cytochrome P450 rever-

sible inhibitors.2–5 Their inhibitory potency comes from

their ability to form stable Fe(II)-N-imidazole complexes.6

aURA 2096 du CNRS, Institut de Biologie et Technologies de Saclay,Commissariat a l’Energie Atomique, Batiment 528, CEA-Saclay,91191 Gif-sur-Yvette, France. E-mail: marcel.delaforge@cea.fr;Fax: +33 1 69 08 87 17; Tel: +33 1 69 08 68 39

b INSERM U620; Universite de Rennes 1, IFR 140,2 Avenue du Pr. Leon Bernard, 35043 Rennes, France

c Laboratoire de Chimie Bioorganique et Bioinorganique, CNRS,Institut de Chimie Moleculaire et des Materiaux d’Orsay, UMR8182, Batiment 420, Universite Paris-Sud XI, 91405 Orsay, France.E-mail: jpmahy@icmo.u-psud.fr; Fax: +33 1 69 15 72 81;Tel: +33 1 69 15 74 21

dDepartment of Chemistry, University of Pennsylvania, 231 South34th Street, Philadelphia, Pennsylvania 19104-6323, USA

w Electronic supplementary information (ESI) available: HPLCseparation and MS-MS spectra of roquefortine and its metabolites.See DOI: 10.1039/b817743kz Marcel Delaforge, CEA-Saclay, iBiTec-S SB2SM, CNRSURA2096, bat. 528, 91191 Gif-sur-Yvette, France.

148 | Metallomics, 2009, 1, 148–156 This journal is �c The Royal Society of Chemistry 2009

PAPER www.rsc.org/metallomics | Metallomics

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Numerous imidazole derivatives were investigated for their

ability to inhibit hemoproteins such as cytochromes P450,2,4–9

cyclo-oxygenases10 and NO-synthases.11 They are also known

and used for their potent inhibition of lanosterol demethylase

activity in fungi.7,8 In addition, this inhibitory action has

been fruitfully exploited to reduce the rate of metabolism of

certain drugs. For example, it has been demonstrated that

co-administration of ketoconazole with cyclosporine A9 or

ixabepilone12 limits their metabolism and enhances their

therapeutic effects. Finally, another example of heme-

imidazole complexes is found in the case of monooxygenase

inhibition by histamine, a natural derivative of histidine.13,14

It has been demonstrated that the stability of the reversible

imidazole–iron interaction depends upon the accessibility of

imidazolyl nitrogen atoms as well as the hydrophobicity of its

surrounding substituents.15–18 Among the imidazole-based

inhibitors, most of the molecules reported have one of their

two nitrogen atoms substituted.15 In this case, the inhibition

potency is enhanced by the coordination of the electron lone

pair of the remaining nitrogen of the imidazole ring. This effect

has been demonstrated, experimentally and theoretically, in

the case of the binding ofN-substituted imidazoles to the heme

of P450cam.19

We have previously demonstrated that small peptides, and

particularly cyclodipeptides, are recognized, and in some cases

metabolized, by hepatic cytochromes P450.20,21 In the present

study, we describe our efforts to demonstrate the influence of

the chemical environment of imidazole derivatives on their

P450 inhibition. In particular, since it is clear that the strength

of the coordination of the imidazole nitrogen on the iron atom

is related to the availability of its doublet, we examine the

influence of its involvement in a H-bond with a neighboring

hydrogen atom, which is supposed to decrease its availability

and its ability to bind to the iron atom. To this end, a

determination of the preferred mode of interaction between

a series of dehydrohistidine-based molecules (Fig. 1) and

various P450 isoforms has been performed. In this series of

compounds, firstly a group of molecules which cannot estab-

lish any intramolecular hydrogen bonds was studied. This

subset includes (E)-urocanic acid, its methyl ester (1) and

a-bromo derivative (4), His-Phe and N-benzylimidazole. A

second series of compounds which potentially develop intra-

molecular hydrogen bonds consists of roquefortine, iso-

roquefortine, phenylahistin, BS176, c-(Phe-His), c-(Phe-DHis),

(Z)-urocanic acid, its cis methyl ester (2) and a-bromo deri-

vatives (5), as well as the saturated derivative 3. In the latter

group, two types of behavior may occur: one of the two

nitrogens of the imidazole ring may act either as a hydrogen

bond donor or acceptor. An example of the first case is found

in roquefortine, whereas an example of the second case is

illustrated by isoroquefortine.

These differences in H-bonding properties between

roquefortine and isoroquefortine are supported by NMR

analysis (M Joullie, personal observations). In roquefortine,

hydrogen bonding between the NH-group of imidazole

and the oxygen of the neighboring carbonyl group of the

diketopiperazine has been suggested,21 whereas intramolecular

hydrogen bonding between the sp2 nitrogen¼N– of the imidazole

group and the NH-amide group of the diketopiperazine (Fig. 1)

has been proposed for isoroquefortine.22 In terms of binding

affinities, we have previously demonstrated the inhibitory

action of roquefortine towards cytochromes P45021 and

attributed this inhibitory action to the binding of the basic

sp2 nitrogen lone pair of roquefortine to the heme iron atom

(see mechanism in Fig. 2).

In order to gain insight into this structure–activity relation-

ship in the case of (iso)roquefortine and to extend it to a series

of analogs, a metabolism and a binding study to P450s has

been undertaken. Isoroquefortine should possess a lower

inhibitory potential than roquefortine due to the reduced

accessibility of its sp2 nitrogen. Similarly, the (Z)-isomer of

urocanic acid and its derivatives 2, 3 and 5 would be expected

to exhibit hydrogen bonding similar to that of roquefortine

(Fig. 1) and should display markedly increased inhibitory

activity relative to its (E)-isomer and its derivatives 1 and 4.

In order to validate this assumption, heme spectral interaction

and inhibitory studies were performed in the presence of

different human isoforms of cytochrome P450 and on rat or

human liver microsomes. Additionally, in order to dis-

criminate between P450 molecular recognition and heme

binding strength, the same compounds were tested on a simple

hemoprotein model, microperoxidase 8 (MP8).23,24

Microperoxidase 8 is obtained by hydrolytic digestion of

cytochrome c. It contains the heme prosthetic group as well

as the amino acid residues 14–21 of horse cytochrome c,

including His 18, whose imidazole group acts as the fifth axial

ligand of the iron. The absence of a heme iron ligand at the

sixth coordination position allows this site to be occupied by a

number of different ligands. In aqueous solution water

occupies the sixth coordination site.23 Due to these structural

characteristics, microperoxidase 8 is considered to be an

effective model for the study of the redox mechanism of

hemoproteins such as cytochromes, hemoglobin, myoglobin

and peroxidases.23

Experimental

Chemicals

Roquefortine, trans-(E)-urocanic acid, N-benzylimidazole,

His-Phe, testosterone, 6b-hydroxy-testosterone, NADPH,

NADP, glucose-6-phosphate (G6P), glucose-6-phosphate

dehydrogenase (G6PDH), dexamethasone (DEX), clofibrate

(CLO), sodium phenobarbital (PB), 3-methylcholanthrene

(3-MC), and microperoxidase 8 (MP8) were obtained from

Sigma Chemical. c-(Phe-His) was obtained from Bachem.,

BS176, compounds 1–5 and isoroquefortine were synthesized

as previously described.22 c-(Phe-DHis) and (�/+)-phenylahistin

were synthesized and kindly provided by Y. Hayashi25 and

cis-(Z)-urocanic acid by H. Morrison.26 All other chemicals were

of the highest quality commercially available.

Microsomal preparations

Human liver samples were kindly supplied by the surgery

service of the Franche Comte Hospital (University of Besancon)

and microsomes were prepared as previously described.27

Bactosomess-expressed P450 3A4, 2E1, 1A2, 2C19, 2C9 and

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2D6 were obtained from Tebu Co. Pooled human liver

microsomes and Supersomess-expressed P450 4A11 were

obtained from Gentest Co (Woburn MA 01801, USA).

Yeast-expressed human P450 1A1 was prepared as previously

described and produced by SPI-Bio (Montigny le Bretonneux,

France).28 Rat microsomes were prepared from a pool of livers

from 4 to 6 animals untreated or treated by the various

cytochrome P450 inducers as previously described.27

Quantification of enzymes in microsomal fractions

Protein content in microsomal suspensions was determined by

the method of Lowry et al. using bovine serum albumin as a

Fig. 1 Chemical structure of the dehydrohistidine derivatives tested. Intramolecular hydrogen bonds are highlighted in bold.

Fig. 2 Effect of the location of intramolecular H-bond in dehydrohistine-based cyclodipeptides.

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standard.29 The P450 concentration was measured as described

by Omura and Sato.30

Substrate binding to P450s microsomal isoforms

and to microperoxidase 8

Substrate binding to cytochrome P450 was studied by

difference visible spectroscopy using 2 mM P450 from rat liver

microsomes or 0.3 mM P450 from human liver microsomes

in 0.1 M phosphate buffer (pH 7.4). Bactosomess and

Supersomess-expressed P450 were suspended in 0.1 M phos-

phate buffer (pH 7.4) to obtain a P450 concentration of

0.13 mM. The solution was equally divided between two

cuvettes (1 mL cuvette for microsomes and 100 mL cuvette

for Bactosomess and Supersomess). Low amounts of human

liver were used due to their high protein concentrations and

turbidity. After recording the baseline, aliquots (0.5–1 mL) ofsubstrate solutions were added to the sample cuvette, and the

same volume of solvent (DMSO) was added to the reference

cuvette. Difference spectra were recorded between 350 and

520 nm at room temperature, with a Perkin Elmer l18 UV

spectrophotometer equipped with a turbidity accessory. Ks

and DODmax were calculated by linear regression from double

reciprocal plots of 1/DOD versus 1/[substrate].31 Micro-

peroxidase 8 preparations at 2 mM were performed in

20 mM phosphate buffer solutions at pH 6, 7.4 or 8.1 containing

20% methanol in order to obtain a homogenous solution.23

Spectral interactions were measured in a differential mode as for

hepatic microsomal preparations. Control measurements were

done in simple beam spectrometry in order to evaluate the

absolute high spin to low spin transition upon addition of

imidazole derivative.23,24

Cytochrome P450 activities

Incubations of human liver microsomal suspensions (1 mg

protein/mL) were done in the presence of 100 mM of testoster-

one and 0.5 mM of NADPH in a 0.1 M phosphate buffer

(pH 7.4). Experiments were performed at 37 1C in the presence

of 50 mM of each tested compound. Incubations were stopped

after 15 min by the addition of an equivalent volume of

acetonitrile. The final mixture was cooled and centrifuged at

10 000 rpm for 10 minutes. HPLC analysis was performed as

previously indicated; testosterone and 6b-hydroxytestosteronewere detected at 254 nm.21

Inhibition of microperoxidase 8 activity

The peroxidase activity of MP8 was assayed by measurement

of the kinetics of oxidation of o-dianisidine by H2O2.32 The

assay system contained o-dianisidine (100 mM) and MP8

(0.22 mM) in phosphate buffer (0.1 M, pH 7.4) at 25 1C in

the presence or absence of the imidazole derivatives. The

reaction was initiated by addition of hydrogen peroxide

(90 mM). The concentration of oxidized o-dianisidine was

determined by the change in absorbance at 500 nm using a

molar extinction coefficient of 6360 M�1 cm�1.

Molecular modeling

Quantum mechanics calculations have been carried out at the

DFT-B3LYP level of theory.33–35 C, H, O and N atoms have

been represented by an all-electron triple-z quality basis set:

6-311G(d,p).36 A relativistic electron core potential and its asso-

ciated basis set have been used for Br.37 A d polarization function

has been added for Br (a = 0.389). All calculations have been

achieved with the Gaussian 98 suite of programs.38 Geometries

have been fully optimized without any symmetry restrictions. For

each molecule, all major conformers have been optimized and

compared. Only the most stable conformers of the configuration

of interest are presented. The nature of the extrema has been

checked by an analytical calculation of the frequencies.

Results

Recognition of imidazole derivatives by P450

Spectral evaluation of the binding of substrates, using human

liver microsomes, revealed a type II interaction for all the

substrates tested except for BS176, and trans-(E)-urocanic acid

(Table 1). This type II interaction, which is characterized by a

spectral absorption around 430 nm, is characteristic of the

formation of a heme–imidazole complex. The dissociation

constants can be calculated for compounds with values bet-

ween 0.4 and 20 mM (Table 1). These affinities are in the same

range as those of N-substituted imidazoles such as miconazole

for which Ks values were determined to be lower than 1 mM.39

The strongest amplitude spectrum is observed with N-benzyl-

imidazole (Fig. 3) for which the two imidazole nitrogen atoms are

chemically non-equivalent and the accessible nitrogen is in its sp2

hybridization state. For all the other compounds measured, the

Table 1 UV spectral interaction of imidazole derivatives with humanliver microsomes

Compound

Spectral interaction% inhibition

DODmax Ks/mMSpectrumtype

of testosteronehydroxylation

Roquefortine 0.024 0.4 II 88Isoroquefortine 0.006 9.0 II 66BS176 n.m. n.m. n.m. o5c-(Phe-His) 0.053 n.m. II 5c-(Phe-DHis) 0.030 n.m. II 50(�)-Phenylahistin 0.011 9.0 II 54trans-(E)-Urocanicacid

n.m. n.m n.m o5

cis-(Z)-Urocanicacid

0.047 20.0 II o5

Compound 1 0.015 1.4 II 19Compound 2 0.018 4.6 II 46Compound 3 0.017 1.4 II 27Compound 4 0.016 1.0 II 19Compound 5 0.019 0.95 II 71His-Phe 0.037 n.m. II 25N-Benzylimidazole 0.084 2.7 II 48

Spectral interactions were measured at room temperature using

1 mg prot/mL (0.3 nmol P450) of a pool of 10 human liver microsomes

in phosphate buffer. Spectrum type II was characterized by a substrate

dependent formation of a peak around 430 nm and a trough at 390 nm

in the native oxidized cytochrome P450 state. DODmax values were

expressed as nmol P450 and Ks values were determined from double

reciprocal plots from spectra obtained using 0.5 to 100 mM substrate.

Incubations of 100 mM testosterone were performed in the presence of

50 mM imidazole derivatives.

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protonation state of the two nitrogen atoms generally exchanges

very rapidly, but interaction spectra led to the opposite conclusion.

In the case of stereoisomer couples (roquefortine/isoroquefortine

(Fig. 1) or trans/cis-urocanic acids), there are important dis-

crepancies between the two stereoisomers. Roquefortine contains

an (E)-dehydrohistidine residue and exhibits a stronger affinity as

well as spectral amplitude relative to isoroquefortine. This may be

the consequence of an intramolecular hydrogen bond between the

imidazole NH-group and a carbonyl of the diketopiperazine

moiety. In the case of isoroquefortine, such an intramolecular

hydrogen bond is not possible, and as a consequence, a lower

interaction spectrum was observed with human liver microsomes.

The spin shift constant measured (Ks) for the (E)-isomer

roquefortine was at least 20 times lower than that of its

(Z)-isomer, namely isoroquefortine. This has been confirmed using

liver microsomes from rat treated with various inducers or using

expressed human liver microsomes (Table 2). In addition, only

roquefortine was recognized by all microsomal preparations or

isoforms tested. In humans, isoroquefortine was recognized only

by P450 3A4 and produced a type I spectral signature, with a

minimum around 420 nm, characteristic of the low-spin

hexacoordinate P450 Fe(III)H2O species in the reference cuvette

and a minimum around 390 nm, that is characteristic of

the formation of the high-spin Fe(III)P450 species in the sample

cuvette. Roquefortine had the smallest Ks values for all the

microsomal preparations tested, including human liver or

yeast-expressed cytochrome P450s (Table 2).

Comparison of the interaction mode of (Z)- and (E)-urocanic

acids showed that the (Z)-isomer was the only one recognized

by rat and human liver microsomes (Table 1). In this case, the

spectral interaction mode was interpreted as a type II.

Cytochrome P450 inhibitory effect

Upon incubation using human liver microsomes in the presence

of 50 mM imidazole derivatives and 100 mM testosterone, all the

compounds, except BS176, cis- and trans-urocanic acid, and

c-(Phe-His), exhibited inhibitory properties (Table 1). Observa-

tion of the amplitude of interaction shows that inhibition ranges

are not only related to the presence of the imidazole moiety but

also to the stereochemistry of the double bond. Roquefortine

demonstrated the most potent inhibitory properties whereas

the derivatives of cis urocanic acid (compounds 2, 3 and 5) were

more efficient inhibitors than their trans analogs 1 and 4.

Interaction with microperoxidase 8

Microperoxidase 8 is a simple model of hemoprotein which is

derived from cytochrome c. The binding of imidazole to the

Fig. 3 Spectral interaction of 100 mM N-benzyl imidazole (dashed

line), roquefortine (thin line) or isoroquefortine (bold line) on 2 mMmicroperoxidase 8. Difference spectra were obtained by addition of

100 mM imidazole derivatives to a 2 mM solution of microperoxidase 8

in a 20 mM phosphate buffered solution containing 20% methanol.

Table 2 Spectral interaction of roquefortine and isoroquefortine with rat liver microsomes from variously pretreated rat, human liver microsomesand bactosomess or baculosomess expressed human P450 isoformsa

Isoroquefortine Roquefortine

DODmax Ks/mM Spectrum type DODmax Ks/mM Spectrum type

Human liver microsomes0.006 9.0 II 0.024 0.43 II

Rat liver microsomesUT-rat 0.048 101.0 II 0.058 0.63 IIDEX-rat 0.050 67.0 I DODmax1 = 0.033b Ks1 = 0.17 II

DODmax2 = 0.208 Ks2 = 2.353MC-rat 0.050 6.0 II 0.077 0.69 IICLO-rat 0.0084 26.3 II 0.067 0.46 IIPB-rat n.m. n.m. I or II 0.082 2.55 IIP450 isoforms1A2 n.m. n.m. n.m. 0.0105 0.91 II2E1 n.m. n.m. n.m. n.m. n.m. n.m.3A4 0.010 1.6 I 0.010 1.60 II2C9 n.m. n.m. n.m. 0.010 0.83 II2C19 n.m. n.m. n.m. 0.011 0.71 II4A11 n.m. n.m. n.m. n.m. n.m. n.m.2D6 n.m. n.m. n.m. 0.007 4.80 II

a Difference spectra were obtained by addition of increasing amounts of substrate to microsomal suspension containing 2 mM P450 from rats,

either untreated (UT-rat) or treated with 3-methylcholanthrene (3MC-rat), dexamethasone (DEX-rat), phenobarbital (PB-rat), or clofibrate

(CLO-rat), 0.13 mMP450 from yeast-expressed human P450 or 0.3 mMP450 from human liver microsomes. Spectrum type I was characterized by a

substrate dependent peak formation at 390 nm and a through at 420 nm and type II by a peak around 430 nm and a trough at 390 nm in the native

oxidized cytochrome P450 states. DODmax values and Ks values are means of two to four measurements. b In a few cases, two values were obtained

from a biphasic curve; n.m. not measurable; n.d. not determined.

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heme functionality of microperoxidase 8 has already been

reported and discussed.23,24 N-Benzylimidazole was used as

a reference compound for the formation of a low spin

N-imidazole–iron complex (absorption at 409 nm) from the

high spin aqua-complex (broad absorption peak around

394 nm) (Fig. 3). In differential mode visible spectroscopy,

all the tested compounds except BS176 and phenylahistin gave

a 394 to 409 nm shift (Table 3). This spectral shift was

concentration-dependent. His-Phe, c-(Phe-His) and com-

pound 3, which have mobility between the amide function

and the imidazole moiety and can form an intramolecular

CQO� � �HN(Im) bond (Fig. 2), gave similar high spin to low

spin transition ratios with respect to N-benzylimidazole,

whereas c-(Phe-DHis), which forms an intramolecular

NH� � �N(Im) bond, exhibited lower interaction spectrum.

(Z)- and (E)-urocanic acid and compounds 4 and 5 showed

similar spectral transitions of approximately 50% of that of

N-benzylimidazole. The strongest differences between E and

Z configurations were observed in the roquefortine series.

Roquefortine led to around 60% transition spectra whereas

isoroquefortine showed around 5% (pH 7.4). Similarly the

cis-urocanic methyl ester 2 led to a 4 times stronger inter-

action spectrum than its trans isomer 1. BS176, which is a

N-substituted isoroquefortine, was unable to significantly

modify the microperoxidase 8 spin state.

The concentration of imidazole–iron complexes may be

related to the decrease of the peroxidase activity, since

N-benzylimidazole, His-Phe, c-(Phe-His), compound 3,

roquefortine and (Z)-urocanic acid inhibited more than 60%

of the initial rate of o-dianisidine peroxidation. Isoroquefortine,

c-(Phe-DHis) and (E)-urocanic acid and its derivatives 1, 2, 4

and 5 showed either no or low inhibition of such activity

(Table 3). BS176, which is unable to form a significant

concentration of microperoxidase complex, is still able to

inhibit microperoxidase activity. We have observed that this

compound can be oxidized under such conditions, it could

then act as a competitive inhibitor for the oxidation of

ortho-dianisidine.

Cytochrome P450 metabolism of imidazole derivatives

Liver microsomes from control or dexamethasone pretreated

rats in combination with a NADPH-generating system were

used for the metabolism study. Under these conditions, only

substituted cyclodipeptides were metabolized. Isoroquefortine,

and phenylahistin, were significantly metabolized (turnover

values being around 2 nmol metabolized min�1 nmol P450�1),

whereas c-(Phe-His), c-(Phe-DHis) and His-Phe were not.

Metabolism of urocanic acid derivatives was not studied.

Using human liver microsomes, more than 60% of

isoroquefortine was metabolized in three major mono-

hydroxylated metabolites, the main one being hydroxylated

on the isoprenyl function (see figures A and B in the ESIw)whereas roquefortine yielded almost no metabolite (see figure

A in ESIw). This isoroquefortine metabolism is only observed

in the presence of NADPH and was inhibited by ketoconazole,

a classical P450 3A inhibitor. Other P450 inhibitors such as

furafylline (1A2), quinidine (2D6) and sulfaphenazole (2C9)

had no significant effects on the formation of metabolites.

Molecular modeling

In order to obtain more information concerning the relative

stability of the different tautomers of roquefortine, urocanic

acids and their derivatives, the energy-minimized structures of

these molecules have been determined by quantum mechanics

at the DFT-level. Fig. 4 indicates the most stable isomers of

each protonation state and their relative energy in kcal mol�1.

In the gas phase, the more favorable conformation of iso-

roquefortine is slightly more stable than that of roquefortine

by 2.8 kcal mol�1. This can be explained by a lesser cyclic

constraint in isoroquefortine (H-bond based on a 6 membered

ring) than in roquefortine (H-bond based on a 7-membered

ring). However, considering the precision of the calculation

level, these two isomers have to be considered as isoenergetic.

For both isomers, the more stable tautomer is the one that can

develop an intramolecular hydrogen bond. The loss of this

hydrogen bond leads to structures with increased energies

of 7.3 kcal mol�1 (isoroquefortine) and 12.6 kcal mol�1

(roquefortine). In these two molecules, the imidazole ring

has a different role with respect to the establishment of a

hydrogen bond. In isoquefortine, imidazole acts as an H-bond

acceptor, whereas it acts as a donor in roquefortine. Hence,

roquefortine can act as a Lewis base and efficiently bind a

metal center whereas isoroquefortine cannot.

The same analysis can be performed in the case of urocanic

acids and their methyl ester and a-brominated derivatives.

Whereas the two tautomers of (E)-urocanic acid are iso-

energetic, one isomer of (Z)-urocanic acid is much more stable

than the others. This isomer is characterized by an H-bond

Table 3 UV spectral interaction of imidazole derivatives with micro-peroxidase 8

Compound

Spectral interactiona Inhibition ofperoxidaseactivitydDOD409–394 nm Percentb

N-Benzylimidazole 0.28 100 58Roquefortine 0.17 61 76Isoroquefortine 0.014 5 0BS176 o0.005 n.d.c 60c-(Phe-His) 0.29 103 80c-(Phe-DHis) 0.057 20 0(�)-Phenylahistin n.d.c n.d.c 0trans-(E)-Urocanic acid 0.16 57 18cis-(Z)-Urocanic acid 0.19 68 69Compound 1 0.03 11 25Compound 2 0.12 43 39Compound 3 0.29 105 55Compound 4 0.07 37 30Compound 5 0.12 44 13His-Phe 0.34 121 41

a Difference spectra were obtained by addition of 100 mM imidazole

derivatives to a 2 mM solution of microperoxidase 8 in a 20 mM

phosphate buffered solution containing 20% methanol. b Percentage

of the spectral interaction obtained with N-benzylimidazole which

possesses only one accessible tertiary nitrogen. c n.d. no detectable

peak shift. d Percentage of the initial velocity of peroxidase activity

obtained using 100 mM o-dianisidine as substrate, 90 mM H2O2,

0.22 mM microperoxidase 8, 100 mM imidazole derivatives in 0.1 M

phosphate buffer, pH 7.4 at 25 1C. Peroxidase activity was 23 nmol

o-dianisidine consumed min�1 nmol microperoxidase�1. Values are

the mean of two to four independent determinations.

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between the imidazole (acting as an H-bond donor) and the

carboxylic function (acting as the complementary acceptor).

Hence the imidazole ring of this isomer can behave as a Lewis

base and efficiently bind the iron atom of heme. Noteworthily,

the reverse situation, in which the imidazole ring acts as

an acceptor and the carboxylic as a donor is slightly less

favorable. The same computational study applied to the

urocanic acid methyl esters and the a-brominated derivatives

leads to very similar results. In each case, the Z conformation

in which the imidazole group acts as an H-bond donor and the

carbonyl group as an H-bond acceptor is the most stable

configuration.

Finally, the conformational sampling of the dihydro isomer

3 revealed the absence of an intramolecular H-bond. This

result shows that the energy that could be gained through

H-bonding cannot compensate the cost associated with the

formation of a 7-membered ring.

Discussion

Imidazole derivatives are largely used as hemoprotein inhibi-

tors, and the most effective compounds generally contain

N-substituted imidazoles.15 A few unsubstituted imidazole

derivatives exhibit weak inhibitory effects mainly related to

the rapid proton exchange between the two imidazole nitrogens.

To our knowledge, this report represents the first study

of the hemoprotein inhibitory properties of dehydrohistidine-

based derivatives in which nitrogen atoms are not substituted.

Our studies demonstrate that these compounds interact with

the heme iron of cytochrome P450 and microperoxidase 8.

Formation of the iron–imidazole complex is sufficiently stable

to perturb the catalytic cycle of such hemoproteins. Inhibitory

potency is dependent upon the formation of intramolecular

hydrogen bonds between either imidazole nitrogen and

the surrounding CO or NH groups of amide functions.

Comparison of the inhibitory effects between c-(Phe-His)

and c-(Phe-DHis) (Table 3) clearly demonstrates that this

intramolecular interaction is associated with the planarity

imposed on the molecule by the Ca–Cb double bond of the

dehydro-residue. The (E)-configuration allows strong hydrogen

bonding between the diketopiperazine carbonyl and the

imidazole NH group. Therefore, the imidazole sp2 nitrogen

is accessible to bind the heme iron. In contrast, in the

(Z)-configuration, the basic nitrogen atom of imidazole is

inaccessible due to hydrogen bonding with a vicinal NH

group, which lowers the binding potency to the heme iron

atom. This interpretation of the effect of the molecular

environment on the binding of ligand to heme is strongly

supported by quantum mechanical optimized structures of the

different isomers of the molecules herein studied.

Such discrepancies in the affinities of (Z)- and (E)-dehydro-

histidines arise from differences in imidazole nitrogen

accessibility. As indicated by NMR and molecular modeling,

(Z)-isomers possess an imidazole nitrogen doublet, which is

involved in a hydrogen bond with a NH group of the

diketopiperazine (Fig. 2 and 4). This conformation greatly

minimizes the availability of the imidazole nitrogen doublet

for heme iron binding. The accessible nitrogen doublet of the

imidazole shows only a low affinity for iron association.15–18 In

contrast, in (E)-isomers such as roquefortine, the hydrogen

atom of the protonated imidazole nitrogen is bonded to the

oxygen of the diketopiperazine carbonyl group (Fig. 2 and 4),

making the lone pair of the remaining nitrogen atom available

for binding to heme iron.

In general, (Z)-dehydrohistidine isomers are preferentially

formed by dehydration of histidine derivatives.40 In the same

way, natural dehydro-amino acids are predominantly found in

the (Z)-configuration.41–44 Interestingly, some methods for

the preparation of dehydro-diketopiperazines have been

recently patented.45 Alternatively, examples of natural

Fig. 4 Relative energies of tautomers of roquefortine/isoroquefortine and urocanic acids. The relative energies are given with respect to the more

stable isomer and are expressed in kcal mol�1.

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(E)-dehydrohistidine are few, and roquefortine is an exception

that has been of interest due to its antibacterial activities.10

The first total synthesis of roquefortine was recently reported

using a novel elimination strategy.46

In the urocanic acid series, cis isomers exhibit higher

inhibition strength towards P450s and MP8. This result is in

agreement with the structure of the most stable tautomers of

these cis-compounds in which the imidazole ring acts as an

H-bond donor with respect to the intramolecular carbonyl

group. Experimentally, the inhibition potency of these

compounds is reduced by changing the stereochemistry of

the double bond from cis to trans (Z to E). In trans isomers,

no intramolecular H-bond can be developed, hence both

imidazole tautomers are in equilibrium, which results in a

decrease of binding strength. Finally, compound 3 highlights

the sterical constraints induced by a,b-saturation on the

nitrogen lone pair accessibility. Indeed, the planarity of the

dehydro-compounds causes the Ca–H or Cb–H to point

towards a direction similar to that of the imidazole lone pair

and, hence, decreases the binding strength of the molecule

compared to compound 3 in which the planarity is lost.

Our results suggest intriguing possibilities for the develop-

ment of hemoprotein inhibitors, which may be deactivated by

simple structural modification. Such structural modifications

can easily be obtained under light exposure or saturation

reduction. In the case of urocanic acid isomers, further

research is in progress in an attempt to correlate hemoprotein

inhibition properties to in vivo modifications of physiological

parameters and precisely address the impact of the substitution

on the a and/or b position in the dehydro series.

Acknowledgements

Part of this work was supported by the AC 009E Environne-

ment et Sante program of the French Ministere de l’Ecologie,

de l’Energie , du Developpement durable et de l’Amenagement

du Territoire. We also wish to thank Ms S. Blondel for

her excellent technical assistance. The synthetic work was

supported by NSF (CHEM 0515443).

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