Approaches to Soft Drug Analogues of Dihydrofolate ...uu.diva-portal.org/smash/get/diva2:167816/FULLTEXT01.pdf · Graffner Nordberg, M. 2001. Approaches to Soft Drug Analogues of

Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 252

_____________________________ _____________________________

Approaches to Soft Drug Analogues of Dihydrofolate

Reductase Inhibitors

Design and Synthesis

BY

MALIN GRAFFNER NORDBERG

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in OrganicPharmaceutical Chemistry presented at Uppsala University in 2001

ABSTRACT

Graffner Nordberg, M. 2001. Approaches to Soft Drug Analogues of Dihydrofolate ReductaseInhibitors. Design and Synthesis. Acta Universitatis Upsaliensis. Comprehensive Summaries ofUppsala Dissertations from the Faculty of Pharmacy 252. 75 pp. Uppsala. ISBN 91-554-5017-2.

The main objective of the research described in this thesis has been the design and synthesis ofinhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration anddevoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key elementin the treatment of many diseases, including cancer, bacterial and protozoal infections, and alsoopportunistic infections associated with AIDS (Pneumocystis carinii pneumonia, PCP). Recentresearch indicates that the enzyme also is involved in various autoimmune diseases, e.g.,rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates havebeen developed to date although problems remain with toxicity and selectivity, e.g., the well-established, classical antifolate methotrexate exerts a high activity but also high toxicity. The newantifolates described herein were designed to retain the pharmacophore of methotrexate, butencompassing an ester group, so that they also would serve as substrates for the endogenoushydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples ofsoft drugs because they in a controlled fashion would be rapidly and predictably metabolized tonon-toxic metabolites after having exerted their biological effect at the site of administration.

A preliminary screening of a large series of simpler aromatic esters as model compounds in abiological assay consisting of esterases from different sources was performed. The structuralfeatures of the least reactive ester were substituted for the methyleneamino bridge in methotrexateto produce analogues that were chemically stable but potential substrates for DHFR as well as forthe esterases.

The new inhibitor showed desirable activity towards rat liver DHFR, being only eight timesless potent then methotrexate. Furthermore, the derived metabolites were found to be poorsubstrates for the same enzyme. The new compound showed good activity in a mice colitis modelin vivo, but a pharmacokinetic study revealed that the half-life of the new compound was similarto methotrexate. A series of compounds characterized by a high lipophilicity and thus expected toprovide better esterase substrates were designed and synthesized. One of these analogues inwhich three methoxy groups were substituted for the glutamic residue of methotrexate exhibitedfavorable pharmacokinetics. This compound is structurally similar to another potent DHFRinhibitor, trimetrexate, used in the therapy of PCP (vide supra). The new inhibitor that undergoesa fast metabolism in vivo is suitable as a model to further investigate the soft drug concept.

Malin Graffner Nordberg, Organic Pharmaceutical Chemistry, Department of MedicinalChemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden

Malin Graffner Nordberg 2001

ISSN 0282-7484ISBN 91-554-5017-2

Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001

Till Micke, Albert och Cecilia

ABBREVIATIONS

AICAR 5-aminoimidazole-4-carboxamide ribonucleotideAIDS acquired immune deficiency syndromeDCC N,N'-dicyclohexylcarbodiimideDHFR dihydrofolate reductaseDIPEA N,N-diisopropylethylamineDMAc N,N-dimethylacetamideDMAP 4-dimethylaminopyridineDME dimethoxyethaneDMF N,N-dimethylformamidedppp 1,3-bis(diphenylphosphino)propaneFA folic acid (FH2 and FH4, respectively, represent the reduced analogues of FA)FDA US food and drug administrationFEP free energy perturbationFPGS folylpolyglutamate synthetaseGAR glycinamide ribonucleotideIBD inflammatory bowel diseaseIC50 inhibitor concentration at 50% inhibition of the enzymei.p. intraperitonealIS inflammation scorei.v. intravenousKi inhibitory constantLIE linear interaction energyMD molecular dynamicsMPO myeloperoxidaseMTX methotrexateNADPH nicotinamide adenine dinucleotide phosphate, reduced formNMR nuclear magnetic resonanceNSAIDs non-steroid anti-inflammatory drugsPCP Pneumocystis carinii pneumoniaPLE pig liver esterasep.o. per oral (via mouth)PTX piritreximPyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphateRA rheumatoid arthritisRFC reduced folate carrierSAR structure-activity relationshipsT½ half-lifeTBAF tetrabutylammonium fluorideTFA trifluoroacetic acidTHF tetrahydrofuranTLC thin-layer chromatographyTMP trimethoprimTMQ trimetrexateTS thymidylate synthetase

LIST OF PAPERS

This thesis is based on the following papers, which will be referred to by their Romannumerals in the text.

I. Graffner-Nordberg, M.; Sjödin, K.; Tunek, A.; and Hallberg, A. Synthesis andEnzymatic Hydrolysis of Esters, Constituting Simple Models of Soft Drugs. Chem.Pharm. Bull. 1998, 46, 591-601.

II. Marelius, J.; Graffner-Nordberg, M.; Hansson, T.; Hallberg, A.; and Åqvist, J.Computation of Affinity and Selectivity: Binding of 2,4-Diaminopteridine and 2,4-Diaminoquinazoline Inhibitors to Dihydrofolate Reductases, J. Comput. Aided Mol.Des. 1998, 12, 119-131.

III. Graffner-Nordberg, M.; Kolmodin, K.; Åqvist, J.; Queener, S F.; and Hallberg, A.Design, Synthesis, Computational Prediction and Biological Evaluation of Ester SoftDrugs as Inhibitors of Dihydrofolate Reductase from Pneumocystis carinii. J. Med.Chem., accepted after minor revision.

IV. Graffner-Nordberg, M.; Kolmodin, K.; Åqvist, J.; Queener, S F.; and Hallberg, A.Design, Synthesis, and Computational Activity Prediction of Ester Soft Drugs asInhibitors of Dihydrofolate Reductase from Pneumocystis carinii. In manuscript.

V. Graffner-Nordberg, M.; Karlsson, A.; Brattsand, R.; Mellgård, B.; and Hallberg, A.Design and Synthesis of Dihydrofolate Reductase Inhibitors Encompassing a BridgingEster Group for Biological Evaluation in a Mouse Colitis Model In Vivo. Inmanuscript.

VI. Graffner-Nordberg, M.; Marelius, J.; Ohlsson, S.; Persson, Å.; Swedberg, G.;Andersson, P.; Andersson, S E.; Åqvist, J.; and Hallberg, A. ComputationalPredictions of Binding Affinities to Dihydrofolate Reductase: Synthesis andBiological Evaluation of Methotrexate Analogues, J. Med. Chem. 2000, 21, 3852-3861.

Reprints were made with kind permission from the publishers.

CONTENTS

1 INTRODUCTION 9

1.1 THE SOFT DRUG CONCEPT 101.2 THE ESTERASES 121.3 DIHYDROFOLATE REDUCTASE (DHFR) 141.4 AN OVERVIEW OF THE FOLATE PATHWAY 151.5 INHIBITORS OF DIHYDROFOLATE REDUCTASE 19

1.5.1 Classical DHFR Inhibitors 191.5.1.1 Methotrexate 19

1.5.2 Non-classical DHFR Inhibitors 201.6 DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY 22

1.6.1 Pneumocystis carinii Pneumonia 221.6.2 Inflammatory Bowel Diseases 231.6.3 Rheumatoid Arthritis 24

2 AIMS OF THE PRESENT STUDY 26

3 ASPECTS OF DRUG-ENZYME INTERACTIONS 27

3.1 METHOTREXATE 273.1.1 Structure-Activity Relationships 28

3.2 NON-CLASSICAL INHIBITORS TOWARDS PNEUMOCYSTIS CARINII 293.2.1 Structure-Activity Relationships 30

4 OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES 31

4.1 METHOTREXATE 314.2 NON-CLASSICAL INHIBITORS 33

4.2.1 Trimetrexate 344.2.2 Piritrexim 34

5 DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR 36

5.1 SYNTHESIS OF SIMPLE AROMATIC ESTER-CONTAINING MODEL COMPOUNDS 365.2 SYNTHESIS OF SOFT DRUG DERIVATIVES OF NON-CLASSICAL ANTIFOLATES 395.3 SYNTHESIS OF NEW DERIVATIVES OF CLASSICAL ANTIFOLATES 465.4 APPLYING SOLID PHASE CHEMISTRY TO THE SYNTHESIS OF CLASSICAL DHFR

INHIBITORS 48

6 BIOLOGICAL RESULTS 51

6.1 ENZYMATIC HYDROLYSIS IN VITRO 516.2 INHIBITORY ACTIVITIES OF NEW INHIBITORS OF DHFR 52

6.2.1 Inhibitory Activities and Selectivities of New Lipophilic Soft DrugInhibitors for pcDHFR 53

6.2.2 Inhibitory Activities of New Classical Inhibitors of Mammalian DHFR 556.3 EFFECT OF A NEW POTENT INHIBITOR OF DHFR IN A MOUSE COLITIS MODEL

IN VIVO 566.4 EVALUATION OF A NEW POTENT DHFR INHIBITOR IN A RAT ARTHRITIS MODEL

IN VIVO 57

7 MOLECULAR DYNAMICS 58

8 PHARMACOKINETICS 62

9 CONCLUDING REMARKS 65

10 SUMMARY 66

11 ACKNOWLEDGEMENTS 67

12 REFERENCES 69

INTRODUCTION – THE SOFT DRUG CONCEPT

- 9 -9

1 INTRODUCTION

The ultimate goal of modern drug discovery is to identify a therapeutic agent that iseffective against a disease. Although the process of drug discovery is a complex issue, it maybe divided into three main steps: i) development of relevant biological system for testing ofthe compounds in vitro and in vivo; ii) identification of “lead” compounds for concept test inthe biological assays; iii) optimization of the “lead” structure to enhance the selectivity ratio,toxicity profile or pharmacokinetics, and ultimately furnish a candidate drug suitable forappropriate in vivo studies and further clinical evaluation. An example of the drug designprocess is depicted in Figure 1.

The project described herein mainly reports the design and synthesis of new ligandsfor the enzyme dihydrofolate reductase (DHFR). However, since all areas presented in italicswithin Figure 1 have been integral parts of this project, it can be described as having had aninterdisciplinary character.

Crystal Structure of the Receptor

Computational Analysis

Ligand Design

Organic Synthesis of New Ligands

Animal StudiesPharmacokineticsMetabolismToxicityEfficacy

Biological Testing

Candidate Drug

Crystallographic Analysis of Ligand-Receptor Complex

Clinical Studies

Figure 1. A schematic picture of the structure-based drug design process.


- 10 -10

1.1 The Soft Drug Concept

Due to increased molecular knowledge regarding endogenous receptors or enzymes,the discovery of new drugs with high potencies and selectivities is facilitated. However, sincethe development of new drugs is an expensive process the toxicology profile must be takeninto account at an early stage of the drug development (see Figure 1). Thus, a highly potentmolecule may be rejected at a late stage of the drug discovery process because of unavoidableand unpredictable side-effects. In order to successfully design safer drugs with increasedtherapeutic ratios, i.e. with large differences between the therapeutic dose and doses causingundesired side-effects, a relatively new approach called soft drug design can be used (Figure2). Herein, the primary goal is to separate the desired local activity from systemic toxicity.

soft drug inactive metabolites

Figure 2. A schematic picture of a soft drug. The active part is drawn in gray.

Soft drugs are active analogues of already known therapeutic agents that undergo fastand predictable deactivation after having exerted a therapeutic effect.1,2 The resultingmetabolites formed after deactivation must have no intrinsic activity of their own in additionto being non-toxic to the host. Hence, it is important to control the process of metabolism.However, this necessitates detailed knowledge of the structural requirements for thedeactivation process to occur. The idea behind the soft drug concept is to build a fragmentinto the molecule of choice that turns the new molecule into a compound that can function asa substrate for the metabolizing enzymes, but still has a sustained activity on its originaltarget. Most drug metabolism is mediated by the cytochrome P450 system, which exhibits alarge inter-individual variability and is subject to inhibition and induction.3 Additionally, thisenzymatic system is often associated with the formation of undesired toxic, active, or high-energy intermediates. When more readily predictable and thus controllable metabolism ispreferred the esterases, an important class of hydrolytic enzymes, are an attractive target.Consequently, if the incorporated fragment contains an ester group potentially it could bereadily deactivated by esterase enzymes. This group of enzymes will be described morethoroughly in Section 1.2.

The newly designed soft drug analogue should be an isosteric/isoelectronic analogueof the parent drug. Thus, it must exhibit e.g., similar physicochemical and steric properties to


- 11 -11

the lead compound. In general, soft drugs are mainly for topical use and are applied oradministered near the site of action thereby having predominantly local effects.

The soft drug concept was first introduced by Nicholas Bodor in 1977.1 Recently, hereported an updated review of successful soft drug design.2 Herein, the soft drug concept hasbeen divided into four major approaches;

1. A soft analogue is an active analogue of a known drug that is close in structure to thelead compound. The metabolically sensitive fragment is incorporated into themolecule of interest to allow for a fast, enzymatically controllable deactivation afterhaving exerted its biological effect.

2. An inactive metabolite-based soft drug is also the active compound, but here themetabolite is already known to be inactive. It is designed by converting this metaboliteinto an isosteric/isoelectronic analogue of the original drug in such way that afterhaving exerted its therapeutic effect it will undergo rapid metabolism, generating theoriginal non-toxic metabolite.

3. Active metabolite-based soft drugs refer to metabolic products of a drug having thesame affinity as the parent drug. For example, the active metabolite is in the highestoxidation-state that still retains activity and is deactivated by one-step oxidation to thefinal inactive metabolite. Hence, if both the hydroxyalkyl- and the aldehyde-form areactive, but not the carboxy-analogue, the one most suitable for soft drug design wouldbe the aldehyde.

4. Pro-soft drugs are (inactive) prodrugs of a soft drug of any of the above mentionedclasses. The pro-soft drugs are transformed through enzymatic processes into theactive soft drug, which is subsequently deactivated by metabolism.

The two most often used strategies in the soft drug design context are the softanalogue approach and the inactive metabolite-based soft drug.2

When describing soft drugs, it is also important to mention the term prodrug. Theprodrug methodology was introduced 19584 and represents a pharmacologically inactivecompound that is metabolized to the active drug in vivo (Figure 3).5-7 The drug derivativemust be converted rapidly, or at a controlled rate into the active therapeutic agent in vivothrough a metabolic biotransformation. There are numerous reasons why one might utilize aprodrug strategy in drug design.5,8 Having an active drug but with poor physicochemicalproperties, the bioavailability may be low and hence no effect might be observed. Theseproblems could originate from poor diffusion into the cell, poor solubility, or unfavorable


- 12 -12

pharmacokinetics. In order to circumvent these problems the molecule must be transformed tomake it more available to produce its biological effect. The main approach for prodrug designis to convert carboxylic acids into esters or amides, thus enhancing the lipophilicity of thedrug molecule. Most often it is the endogenous enzymes that are involved in the activation ofprodrugs with esterases as key enzymes. Consequently, the same enzymatic systems involvedin the activation of prodrugs can be used to deactivate soft drugs. Actually, one can alsodesign a pro-soft drug (vide supra), but the opposite is not possible.

prodrug

Figure 3. A schematic picture of a prodrug. The active part is drawn in gray. The white moiety is an inactivecarrier-molecule.

1.2 The Esterases

As described earlier in Section 1.1 prodrug and soft drug design often relies onenzymatic hydrolysis for drug activation, and deactivation, respectively. With biologicallybased drug design, it has become an important issue to consider the conversion site in thebody, the corresponding enzymes and their activities. An enzyme can be defined as a‘molecular machine’, which modulates reactions within the body.

As in the case of soft drug design, insertion of ester bonds susceptible to enzymaticcleavage by the esterases comprises one approach to make the action of a drug more restrictedto the site of application. A wide distribution of the enzymes in mammalian tissues has beenobserved. Hydrolases (e.g., esterases, lipases, peptidases, glycosidases, amido- andaminohydrolases, pyrophosphatases etc.) are enzymes well suited to drug inactivation sincethey are ubiquitously distributed. Hydrolases catalyze the addition of a molecule of water to avariety of functional groups (Figure 4). The hydrolases vary between species andindividuals9,10 and the same substrate is often hydrolyzed by more than one enzyme. Thehydrolases play an important role in the detoxification process and a decrease in enzymeactivity potentiates the toxicity of certain compounds.

Figure 4. General formula describing ester hydrolysis.

R OR'

OH2O

R OH

OR'OH+

INTRODUCTION – THE ESTERASES

- 13 -13

The carboxyl esterases (EC 3.1.1) play the most important role in the hydrolyticbiotransformation hydrolysis of a variety of ester-containing molecules.11 They are widelydistributed in the tissues and blood9,12,13 and microsomal liver has shown to be the richestsource.13-16 The carboxylic esterases exhibit broad and overlapping substrate specificity17 andcatalyze the hydrolysis of ester bonds in a variety of esters such as aliphatic and aromaticcarboxylic esters, thioesters, phosphates, sulfuric esters, etc.9,13-16 Numerous studies havedemonstrated that a variety of xenobiotics∗ are metabolized by these carboxyl esterases.Probably the most relevant esterases for detoxications in humans are the liver carboxylesterases.14,18

Esterase activity depends on the substrate but it also varies quite strongly betweenspecies.9-11,17,19,20 In fact, rodents (rats, guinea pigs) tend to metabolize ester-containing drugsmore rapidly than humans.2,21 In vitro hydrolytic half-lives, T½, (see Chapter 8) measured inrat blood were often found to be orders of magnitude lower than those measured in humanblood.22,23 Hence, the stability of acyloxyalkyl type esters usually increases in the rat < rabbit< dog < human order,23,24 but there might be variabilities. Besides the usual problems relatedto the extrapolation results of animal test to man, this is one of the additional aspect that cancomplicate early drug evaluations.25,26

Figure 5. A proposed mechanism11,27 for the hydrolysis by the carboxyl esterases. Ser203, Glu335, and His448serve as a catalytic triad and Gly123-Gly124 as part of an oxyanion hole.

∗ A completely synthetic chemical compound (i.e., a drug, pesticide, or carcinogen) which does not naturallyoccur on earth.

NNH

Ser203His448

Glu335

O

Ser203His448

Glu335

ROR'

OO

RO

R' Gly124

Gly123

R'OH

H2O

OHO O NNHO O H

H

H

NNH

Ser203His448

Glu335

O OO

RO

HO

R'

NNH

Ser203His448

Glu335

O OO

RO

HO

NNHO O H

O

O RO

H

Ser203His448

Glu335

NNHO OO

H

Ser203His448

Glu335

R OH

Otetrahedral

intermediate

acyl-enzymecomplextetrahedral

intermediate

nucleophilicattack

nucleophilicattack

H

INTRODUCTION – THE ESTERASES

- 14 -14

Substrates or inhibitors act at a particular site on the enzyme known as the active site.The active site (also known as the catalytic site) of the enzyme is complementary in size andshape to the substrate molecule. The chemical change can occur only when the substrate isanchored in the active site of the enzyme. The enzymatic binding sites are usually composedof a combination of both polar and non-polar amino acids. The carboxyl esterases belong tothe group of serine proteases (acetylcholinesterase, butyrylcholinesterase, cholesterolesterase) and the mechanism for ester hydrolysis have been described in an active siteconsisting of a catalytic triad with Ser203, Glu335, and His448 (Figure 5).11,27 The amino acidsequence at the catalytic triad is thought to be highly conserved among the serine proteases,although not completely identical.11 The carbonyl group of the ester functionality is prone toundergo a nucleophilic attack by the serine oxygen, which in turn is more nucleophilic due tothe assistance of the basic nitrogen in histidine. The acylated Ser203 and Gly123-Gly124together form a so-called oxyanion hole where weak hydrogen bonds stabilize the tetrahedraladduct. A less accessible carbonyl oxygen is more difficult to stabilize by hydrogen bonds inthe oxyanion hole. Hence, the steric hindrance around the sp2 oxygen and charge on the sp2

carbon of the ester moiety seem to have the most important influences on the rate of in vitrohuman blood enzymatic hydrolysis, thus increasing T½.28 Several models of active sites ofhydrolytic enzymes have been proposed with the aim of correlating substrate specificity to thestructural feature of substrates.29

In addition to the fact that the biological activity is considered to be a function of thechemical structure the physicochemical properties also play an important role. Structure-activity relationships (SAR) have been developed where a set of physicochemical propertiesof a group of congeners is found to explain variations in biological activity. The effect ofstructure on the enzymatic half-life has been investigated in a large number of prodrug andsoft drug series. Several attempts to establish some kind of quantitative structure-metabolismrelationships (QSMR) looking at the enzymatic half-lives of prodrugs and soft drugs havebeen reported.22,23,28,30-40

1.3 Dihydrofolate Reductase (DHFR)

Dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate-NADP+ oxidoreductase; EC 1.5.1.3) is an enzyme of pivotal importance inbiochemistry and medicinal chemistry. DHFR functions as a catalyst for the reduction ofdihydrofolate to tetrahydrofolate. Reduced folates are carriers of one-carbon fragments, hencethey are important cofactors in the biosynthesis of nucleic acids and amino acids. Theinhibition of DHFR leads to partial depletion of intracellular reduced folates with subsequentlimitation of cell growth.41 A more illustrative description of the folate pathway will bediscussed in Section 1.4. DHFRs isolated from different species are relatively small proteins

INTRODUCTION – DIHYDROFOLATE REDUCTASE (DHFR)

- 15 -15

(18-22 kDa), which are easily available. DHFR has attracted attention of protein chemists as amodel for the study of enzyme structure/function relationships. The species-differencesamong the DHFRs,42 have been used to discover compounds with particular selectivity, e.g.,that are lethal to bacteria but relatively harmless to mammals.43 Such selective inhibitors aretrimethoprim (Figure 6a) and pyrimethamine (PTM, Figure 6b), which are used in therapy fortheir antibacterial and antiprotozoal properties, respectively.

Figure 6. a) trimethoprim (TMP) b) pyrimethamine (PTM)

Compounds that inhibit DHFR exhibit an important role in clinical medicine, asexemplified by the use of; methotrexate (MTX, Figure 10) in neoplastic diseases,44

inflammatory bowel diseases45 and rheumatoid arthritis,46 as well as in psoriasis47,48 and inasthma;49 TMP in bacterial diseases;50 and of PTM in protozoal diseases.51,52 Lately, a newgeneration of potent lipophilic DHFR inhibitors such as trimetrexate (TMQ, Figure 11) andpiritrexim (PTX, Figure 12) have shown antineoplastic53 and most importantly,antiprotozoal54 activities.

The enzymatic reduction involved in the inhibition of DHFR is a random process inwhich either the substrate (dihydrofolate) or the cofactor NADPH, forms a binary complexwith the enzyme, with subsequent rapid binding of the inhibitor, to form a ternary complex(Section 3.1). A tremendous amount of work has been done regarding inhibitors of DHFR. Areview on the QSARs of DHFR inhibitors has covered the literature extensively until 1984.55

The essence of the work is that all steric, electronic, and hydrophobic parameters have beeneffective in the inhibition of DHFR, depending upon the source of the enzyme and type ofinhibitors. A brief summary of the structure-activity relationships is presented in Section 3.

1.4 An Overview of the Folate Pathway

Folate coenzymes are involved with about 20 enzymatic reactions in mammalianmetabolism.56 The reactions of folate metabolism are interconnected, and the inhibitorsdiscussed within this thesis may also disturb metabolism at sites other than those designated.An intact enzyme pathway is necessary to maintain de novo synthesis of the essential buildingblocks involved in DNA synthesis as well as of important amino acids.

N

N

OCH3

OCH3OCH3

NH2

H2NN

N

NH2

H2N Et

Cl

INTRODUCTION – AN OVERVIEW OF THE FOLATE PATHWAY

- 16 -16

Figure 7. Folic acid (FA)

Folic acid (FA,) is a water-soluble B vitamin that plays a crucial role in thebiosynthesis of DNA. The vitamin consists of 2-amino-4-oxopteridine with a side-chainincorporating both p-aminobenzoic acid and glutamic acid. Humans cannot synthesize folicacid by themselves, and thus depend on a variety of dietary sources for the vitamin. Foodfolates exist mainly as 5-methyltetrahydrofolate (N5-methyl-FH4) and 5-formyl-tetrahydrofolate (N5-formyl-FH4) (Figure 9).57 The active form of FA is its reducedtetrahydroform (FH4), which is formed after reduction of dihydrofolic acid by DHFR (Figure8). FH4 functions as the ‘acceptor’ of single-carbon units and the tetrahydrofolate issubsequently transformed enzymatically from the appropriate cofactor to precursor moleculesthat lead to the synthesis of purine and pyrimidine (e.g., thymine) nucleotides necessary forDNA, and also to the synthesis important amino acids, e.g., methionine (Figure 9).

An overview relating to the biochemistry of the folates will be described (Figure 9).Once FA has been reduced to FH4 (Figure 8), a myriad of enzymes is involved in thesubsequent reactions, using different derivatives of FH4 in one-carbon transfer reactions. N5-Formyl-FH4 (folinic acid, leucovorin) is taken up actively into the cell by a reduced folatecarrier (RFC) system where it is converted to N10-formyl-FH4. In another enzymatic pathway,N5-formyl-FH4 can, after transport into the cell, be converted to N5-methyl-FH4. N5-methyl-FH4 may also be taken up actively and form fresh supplies of FH4. The same entrance systemis used by MTX and as these reduced tetrahydrofolates although the latter seem to have arelatively low affinity for RFC in comparison to MTX.58

Figure 8. Sites of action of antifolates, e.g., MTX, in the reduction of folic acid to its tetrahydroforms.

HN

N N

NO

H2N

NH

NH

COOH

O COOH

γα

HN

N N

NO

H2N

NH

NH

COOH

O COOH

NADPH NADP+

HN

N NH

HN

O

H2N

NH

NH

COOH

O COOH

FA

FH4

HN

N NH

NO

H2N

NH

NH

COOH

O COOH

FH2

NADP+

NADPHdihydrofolatereductase

dihydrofolatereductase

inhibition by MTX, TMP, TMQ, PTX

inhibition by MTX, TMP, TMQ, PTX

Figure 9


- 17 -17

N10-formyl-FH4 is responsible for the donation of single carbon groups in the de novobiosynthesis of purine nucleotides in the reactions catalyzed by the enzymes glycinamideribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR)transformylases. As well as being used directly in purine synthesis, N10-formyl-FH4 is alsoconverted, via N5,N10-methenyl-FH4, to N5,N10-methylene-FH4. The latter is converted, first todihydrofolate (FH2) by the action of thymidylate synthetase (TS) and then to tetrahydrofolate(FH4) by DHFR. N5,N10-methylene-FH4 donates a methyl group to the 5-position of thepyrimidine ring of deoxyuridylate (dUMP) in the reaction catalyzed by TS forming thymine,which is used for the DNAsynthesis. Thus, N5,N10-methylene-FH4 is the only de novo sourceof cellular thymidylate. Inhibition of TS results in a so-called thymine-less death, which leadsto disruption of the synthesis of dividing cells. During this process, N5,N10-methylene-FH4 isoxidized to dihydrofolate and must be converted back to its tetrahydroform by DHFR in orderto maintain the reduced folate pool.

Figure 9. The folic acid pathway.

Intracellularly, folic acid and derived coenzymes also have the ability to be convertedto polyglutamated derivatives with additional glutamic acid residues linked by amide bondsby the enzyme folylpolyglutamate synthetase (FPGS). This is an important mechanism fortrapping ‘classical’ (for an explanation, see Section 1.5) folates and antifolates within the cell,

NH

COOH

O COOHFA

HN

N NH

NO

H2N

NH

RCHO

FH2

FH4

HN

N NH

HN

O

H2N

NH

R

adenosine

HN

N NH

HN

O

H2N

NR

CHO

AICAR

fAICAR (purine C-2)

FH4

HN

N NH

NO

H2N

NH

RCH3

HN

N NH

NO

H2N

NR

HN

N NH

NO

H2N

NR

NADP+NADPH

FH2

N5,N10-methylene-FH4

N5,N10-methenyl-FH4

N10-formyl-FH4

N5-formyl-FH4= leucovorin

N5-methyl-FH4

DNA synthesis

cell division

GAR

(purine C-8)

transformylasethymidylatesynthetase

direct intakedirect intake

dUMP

dTMP

serine

glycine

RNA, DNA

TNFαIL-1

inhibition by MTXGN

inhibition by MTXGN

transformylase

dihydrofolate reductase

fGAR

R =

methionine

homocysteine


- 18 -18

thus maintaining high intracellular concentrations.59 The attachments are performed on the γ-carboxyl group of the glutamate residue. FPGS is able to attach up to six glutamate moleculesto the pteridine ring of MTX.60,61 This polyglutamation reaction has several biologicallyimportant consequences. By polyglutamation the hydrophilicity of the molecule is enhanced(due to a high increase in negative carboxylate groups) and together with the increased size ofthe molecule a greater intracellular retention is enabled with decreased cellular efflux andtrapping of the drug within the cell, with prolonged drug action as a consequence.62 Thepolyglutamates appear to be somewhat more efficient substrates for DHFR than themonoglutamated counterparts.63,64 MTX-polyglutamates (MTXGN) are direct inhibitors ofDHFR and are thus less reversible inhibitors than MTX itself.61 Tissues with high FPGSactivity, such as the liver, accumulate and retain the polyglutamated MTX for prolongedperiods of time. Hence, this increased concentration of polyglutamated of MTXGN isresponsible for the hepatotoxicity observed after chronic administration of the drug.Compared to monoglutamated drugs, the polyglutamates also have increased affinity to otherfolate-dependent enzymes such as TS,65,66 and the transformylases of glycinamideribonucleotide (GAR), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)67,68

(Figure 9). AICAR is directly involved in de novo purine synthesis and its inhibition results ininhibition of purine synthesis. As a consequence, the polyglutamation of MTX plays anessential role in the development of side effects. A new hypothesis about the mechanism ofaction of MTX suggests that by inhibiting AICAR transformylase, MTX causes theaccumulation of AICAR. This inhibition might result in the subsequent increase of theproduction of the anti-inflammatory autocoid adenosine,69 an endogenous anti-inflammatorycytokine. This hypothesis could be one of the explanations for the anti-inflammatoryproperties that are seen in therapy with MTX in autoimmune disorders where much lowerconcentrations are needed for the effect compared to doses that generate effects through directinhibition of DHFR. Nevertheless, a recent publication reports that treatment with MTX ininflammatory bowel diseases (IBD) does not cause elevated concentrations of adenosine,neither in plasma nor at the site of the disease.70 However, recent attempts with non-polyglutamated analogues have indicated a release of adenosine, suggesting thatpolyglutamation of the antifolate is not required for these functions.71

The administration of exogenous reduced folates, such as leucovorin, effectivelyprevents MTX cytotoxicity in mammalian cells (Figure 9). The amount of leucovorin requiredto prevent severe clinical toxicity in high-dose MTX chemotherapy regimens is directlyproportional to the amount of MTX circulating in plasma.61 Leucovorin does not competewith MTX for binding to DHFR but directly overcomes blockade of MTX of the enzymes byincreasing the intracellular pools of FH4 and thereby rescues cells from death. Althougheffective, this regimen offers a very expensive therapy.

INTRODUCTION - INHIBITORS OF DIHYDROFOLATE REDUCTASE

- 19 -19

1.5 Inhibitors of Dihydrofolate Reductase

Inhibitors of DHFR are classified as either ‘classical’ or ‘non-classical’ antifolates.The ‘classical’ antifolates are characterized by a p-aminobenzoylglutamic acid side-chain inthe molecule and thus closely resemble folic acid itself. MTX (Figure 10) is the most wellknown drug among the ‘classical’ antifolates. Compounds classified as ‘non-classical’inhibitors of DHFR do not posses the p-aminobenzoylglutamic acid side-chain but rather havea lipophilic side-chain.

1.5.1 Classical DHFR Inhibitors

1.5.1.1 Methotrexate

MTX (N-{4[[(2,4-diamino-6-pteridyl)methyl]-N10-methylamino]benzoyl}glutamicacid, Figure 10) was first synthesized in 194972 and is today, together with the antibacterialdrug TMP (Figure 6a), the DHFR inhibitor most often used in clinic. The most common useof MTX is as an anticancer drug, but lately the drug also is considered to have anti-inflammatory and immunosuppressive properties with accompanied activity againstautoimmune disorders (see Section 1.3).

Figure 10. Methotrexate (MTX)

MTX serves as an antimetabolite, which means that it has a similar structure to that ofa cell metabolite, resulting in a compound with a biological activity that is antagonistic to thatof the metabolite, which in this case is folic acid. MTX differs from the essential vitamin,folic acid, by the substitution of an amino group for an hydroxyl at the 4-position of thepteridine ring. This minor structural alteration transforms the normal substrate into a tight-binding inhibitor of DHFR. X-ray studies provided evidence for a different binding mode ofMTX compared to the natural substrate (see Section 3.1).

MTX is a competitive and reversible inhibitor of DHFR that binds tightly to thehydrophobic folate-binding pocket of the enzyme. The drug can be competitively displacedby increased intracellular concentrations of the natural enzyme substrate dihydrofolate. Theaffinity of MTX for DHFR increases considerably in the presence of the cofactor NADPH.

NH

O COOH

COOH

NNH2

N

NH2

N

NNCH3


- 20 -20

MTX inhibits the synthesis of metabolites involved in one-carbon-unit transferreactions such as the biosynthesis of the important nucleotides (see Section 1.4). As a result ofthis process, the synthesis of DNA is disrupted. MTX exists as a highly polar dianion atphysiological pH and enters the cells by the energy-dependent RFC system, the majortransport system in human tissue of similar compounds. Inside the cell the molecule ispolyglutamated, which leads to altered characteristics (Section 1.4). The polyglutamatedderivatives of MTX have been postulated to be the probable cause of the anti-inflammatoryproperties associated with MTX due to its inhibition of AICAR transformylase (Section 1.4).The immunosuppressive activity seen in MTX treatment may be due to the induction ofapoptosis (i.e., programmed cell death) of activated lymphocytes, e.g., the T-cells.73,74

Intrinsic and acquired resistance to MTX and other antifolate analogues limits theirclinical efficacy. Resistance has been attributed to several different mechanisms including areduced level of cellular uptake of the drug,44 and to an increase in enzyme levels involved inthe folic acid biochemistry. As mentioned above, MTX is transported into cells by the carriertransport system used for the active transport of reduced folates. Consequently, a poor abilityto transport the drug into the cell can be one source of natural resistance associated with thedrug.75,76 Major limitations, besides the development of resistance with MTX treatment, arebone marrow toxicity, gastrointestinal ulceration, and kidney and liver damage. In high doses,given intermittently, the adverse effect on the bone-marrow is relieved by the periodicadministration of leucovorin (see Section 1.4), enabling the blockade of tetrahydrofolic acidproduction to be by-passed (leucovorin ‘rescue’ procedure).

The clinically useful properties of MTX have stimulated the quest for a variety of newanalogues with modifications within the molecule, without interfering too much with thepharmacophore of the original drug (Section 3.1). Thus, research is enduring for a moreselective drug, most importantly, without the severe side-effects often associated with MTX.

1.5.2 Non-classical DHFR Inhibitors

New, more lipophilic antifolates have been developed in an attempt to circumvent themechanisms of resistance, such as decreased active transport, decreased polyglutamation,DHFR mutations etc. These modified antifolates differ from the traditional ‘classical’analogues by increased potency, greater lipid solubility, or improved cellular uptake.Although being very effective as inhibitors, problems still remain with respect to the issue oftoxicity due to the lack of selectivity (Section 3.2).


- 21 -21

Figure 11. Trimetrexate (TMQ)

TMQ77,78 (Figure 11) is a potent inhibitor of DHFR. It is a ‘non-classical’, lipophilicquinazoline derivative, originally developed as an anticancer agent.79-81 The drug lacks theglutamic acid side-chain, which is the characteristic feature of the ‘classical’ antifolate.Consequently, it cannot be polyglutamated by FPGS and thus does not subsequently undergoretention within the cell for prolonged periods of time, as compared to the ‘classical’analogues. Because of its lipophilicity, TMQ readily enter the cells by passive diffusion, thisoccurs with for example, the opportunistic pathogen P. carinii (this is a non-energy dependentprocess), with subsequent inhibition of the cellular enzyme and thus growth. It is important tomention in this context is that this characteristic also enables the drug to penetrate mammaliancells. TMQ has been approved recently by the FDA for the use in the treatment ofPneumocystis carinii pneumonia82 (PCP, see Section 1.6.1). The administration of TMQ, incombination with leucovorin, is regarded as an good alternative in the treatment of PCP inAIDS patients.54 TMQ therapy is currently recommended for patients with PCP who areeither unable to tolerate, or are resistant to first-line therapy with trimethoprim-sulfamethoxazole (co-trimoxazole).

Another potent inhibitor of DHFR is PTX83 (Figure 12), which like TMQ, is alipophilic antifolate from the new generation of DHFR inhibitors, originally developed as ananticancer drug.84,85 PTX has also been studied in the treatment of psoriasis.85-87

Figure 12. Piritrexim (PTX)

N

N N

NH2

H2N

CH3 OCH3

OCH3

N

N

NH

OCH3OCH3

OCH3

NH2

H2N

CH3

INTRODUCTION - DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY

- 22 -22

1.6 Diseases with Potential Use of DHFR Inhibitors in Therapy

1.6.1 Pneumocystis carinii Pneumonia

Before the late 80’s P. carinii was classified as a protozoan, due to the lack ofresponse with older antifungal agents. However, more recently it has been definitivelycharacterized as a fungus. The primary route for infection is postulated to be via the airborneroute.88 In healthy children by the age of four, 75-90% experience a respiratory infection withP. carinii.89 Studies have shown that the infection is almost universal among healthy adults,90

although P. carinii pneumonia (PCP) does not develop and become severe until the immuneresponse is compromised. PCP is characterized by dyspnea, a non-productive cough, andfever. The initial step in the pathogenesis is the attachment of inhaled organisms to type Ipneumocytes in the alveoli. After proliferation of the organism the pneumocytes are damagedand the alveolar spaces subsequently become filled with a foamy exudate consisting of P.carinii, degenerated cells, and host proteins. A typical X-ray picture of an impaired lung dueto PCP infection is illustrated in Figure 13.

Figure 13. A typical X-ray picture of infected lungs (white parts) from a patient suffering from PCP. The picturewas taken with permission from the Atlas website of CarloDenegriFoundation.91

PCP is one of the most common life-threatening diseases of people suffering fromAIDS.92-96 Encouragingly, the highly active anti-retroviral therapy (HAART) and prophylaxisin patients predisposed to developing PCP, has caused a large decline in the incidence of thedisease in patients with AIDS.97-99 In addition to AIDS, cancer chemotherapy100 and organtransplantation101 also increases the risk of developing the infection.

Currently, first line therapy mainly consists of TMP (Figure 6a) in combination with asulfonamide (co-trimoxazole).92,97,102,103 Inhaled aerosolized pentamidine104 has also found aplace in prophylaxis.105-108 The new generation of DHFR inhibitors; TMQ (Figure 11), as wellas PTX (Figure 12), are now used in the clinic as second-line therapies for moderate to severePCP.82 As mentioned in Section 1.5.2, TMQ and PTX are both potent inhibitors of DHFR


- 23 -23

from P. carinii. Nevertheless, they are not selective, which means that they inhibit themammalian enzyme even more efficiently.109,110 Due to their systemic host toxicity, bothdrugs require an expensive co-therapy with leucovorin (Section 1.4).54,109,111-113

1.6.2 Inflammatory Bowel Diseases

Inflammatory bowel disease (IBD) includes a group of chronic disorders that causeinflammation or ulceration in the small and large intestines (Figure 14). Mainly, IBD isclassified as either ulcerative colitis, or Crohn’s disease. Ulcerative colitis (UC) causesulceration and inflammation of the inner lining of the colon (large intestines) and rectum,while Crohn’s disease (CD) is an inflammation that can extend through all the gastrointestinaltract, that is, from mouth to anus. Both diseases are associated with abdominal pain, diarrhea,rectal bleeding, weight loss, and fever. The etiology of the disease is still unclear, but onetheory postulates that a virus or a bacterium affects the body’s immune system triggering asustained a sustained inflammation reaction in the intestinal wall.114 All medical or surgicallyinduced remissions of the disease are most often followed by relapses. However, severalmedications that are effective in the short-term do not result in sustained remission, andconversely, agents used for maintenance may have minimal effects on the active disease.Pharmacological treatments for IBD consists of corticosteroids; aminosalicylates, e.g.,sulfasalazine, mesalazine; antibiotics; different immunomodulators, such as MTX; and agrowing number of new agents. Systemic administration, as well as topical treatment, withglucocorticoids produces systemic side effects, including adrenosuppression, immuno-suppression, and bone resorption. Budesonide was developed as a corticosteroid with hightopical anti-inflammatory activity and with low systemic activity due to rapid hepaticmetabolism.115 As a consequence, it follows the criteria for the soft drug concept. Budesonidehas found to be as active as conventional corticosteroids (e.g., prednisolone) in UC and theactive form of CD.116

Figure 14. The intestines.

The large intestines(also called colon)

The small intestines

Rectum


- 24 -24

Since the first report in 198945 MTX has drawn attention as a potential treatment forpatients suffering from refractory Crohn’s disease.117 Folic acid, which is effective indecreasing side-effects without affecting the drug efficacy can be given systematically duringtherapy.118 MTX has recently also been proven to be effective in the long-term treatment ofIBD with only moderate side-effects.119,120 As in the case of rheumatoid arthritis (Section1.6.3), antibodies against TNFα (infliximab) have recently become an effective maintenancetherapy.121

1.6.3 Rheumatoid Arthritis

Rheumatoid arthritis is a systemic disease, which predominantly affects the joints.Typically, it starts as a symmetrical engagement of finger joints. Later in the course of thedisease larger joints such as the hips and knees become affected. The disease is progressivebut with a large inter-individual variation in the course. A relapse-remission pattern iscommon. The disease is characterized by inflammation of the synovial tissues, joint pain,stiffness, swelling, and with loss of function in the joints, but it can also affects other parts ofthe body. The probable cause of rheumatoid arthritis is a pathological reaction of the immunesystem. One hypothesis is that it is an autoimmune disease, but this has yet to be established.Nevertheless, it is postulated that the malfunction can be triggered by various conditions, forexample, genetic disposition and infections.

Figure 15. a) A normal, healthy joint. b) A joint affected by rheumatoid arthritis.

The arthritic process starts as an inflammatory reaction in the synovial tissue. Later thesynovium is transformed into an aggressive pannus that grows into, and destroys cartilage andunderlying bone, hence causing loss of joint function. There is an abundance of inflammatorycells in the pannus. The relative importance of these cells for the disease progression is underdebate. TNF-α is a cytokine that mainly is produced from the pannus cells of macrophagelineage. Recently, promising results have been observed with monoclonal antibodies against


- 25 -25

TNF-α (infliximab). Infliximab effectively and rapidly neutralizes TNF-α but also reducesthe secretion of pro-inflammatory substances and thus leads to improvements in humans withactive RA. Interestingly, a recent clinical study was published with MTX and infliximabwhere the combination revealed a synergistic effect. Apparently, the combination can exhibitfavorable activity against the arthritis.117

Traditional pharmacological treatment of RA is based on NSAIDs, which mainlyprovide symptomatic relief, glucocorticoids, and disease modifying anti-rheumatic drugs(DMARDs), which have the ability to eventually slow down the progression of the disease.DMARDs include the gold salts (aurothiomalate), hydroxychloroquine, sulfasalazine or D-penicillamine, or MTX, respectively, all of which can help to reduce the need for NSAIDsand glucocorticoids. During the 1980’s, a low weekly dosage of MTX became an establishedmethod for the therapy of RA.46,122 In the 1990’s it has gradually been accepted as the leadingDMARD, being one of the most potent anti-rheumatic drugs,123 especially in the severe casesof RA. The effectiveness of weekly dosing indicates that the drug is trapped intracellularlydue to polyglutamation (Section 1.4). The mechanism of action of MTX in RA is notcompletely known. The fact that folate supplementation (leucovorin, Section 1.4)administered to MTX-treated RA patients reduces toxicity in a 1:1 ratio without affectingefficacy speaks against the theory that MTX-induced inhibition of DHFR is directly involved.One proposed mechanism of action is that adenosine, with its anti-inflammatory properties, isinvolved in the anti-arthritic effect. In treatment with MTX the levels of AICAR rise with acorresponding increase in the concentration of adenosine (Section 1.4). Nevertheless, in onestudy arthritic rats were treated with different antagonists of adenosine in combination withMTX. Surprisingly, the antagonists enhanced the anti-arthritic effects of MTX,124 a resultwhich does not support “the adenosine hypothesis”. Still, it is possible that AICAR might beinvolved in the anti-arthritic effect.

AIMS OF THE PRESENT STUDY

- 26 -26

2 AIMS OF THE PRESENT STUDY

The purpose of this project was to design and synthesize safer analogues of DHFRinhibitors for potential use against Pneumocystis carinii pneumonia (PCP) and inflammatorybowel diseases (IBD). The new inhibitors were intended for local therapy. We planned toexploit the soft drug concept and thus minimize the adverse systemic effects often associatedwith DHFR inhibitors used in clinic (e.g., MTX). The specific objectives of this thesis havebeen:

• To identify the non-pharmacophore element(s) in the core structure of clinicallyestablished antifolates.

• To replace the selected non-pharmacophore element(s) with ester functionalities, andto probe the impact of the surrounding molecular fragments on the rate of enzymaticester hydrolysis.

• To assess the impact of the ester group on bioactivity. Thus, to design and synthesizeDHFR inhibitors that contain a metabolically stable ester group but still:- exhibit an inhibitory effect of DHFR in vitro.- exhibit a pharmacokinetic profile in vivo similar to the parent compounds used in

clinic.

• To design and synthesize ester analogues of DHFR inhibitors susceptible to hydrolysisby esterases as potential soft drugs. The compounds should exhibit favorablebioactivities after local therapy in animal models.

ASPECTS OF DRUG-ENZYME INTERACTIONS

- 27 -27

b)

3 ASPECTS OF DRUG-ENZYME INTERACTIONS

3.1 Methotrexate

The binding of MTX to DHFR is considered to be a complex interaction where theinitial ternary complex of MTX, NADPH, and human DHFR undergo some kind ofisomerization or conformational change, resulting in an even tighter binding.125,126 This effectmakes the determination of Ki-values complicated, providing values with a degree ofuncertainty. Most inhibitory activities of the antifolates are obtained by ‘simple’determination of IC50-values, which makes it difficult to compare the results to those obtainedby other laboratories.

MTX and the natural substrate, FA, exhibit close similarities in structure (cf. Figure 10and ). Early crystallographic studies of human DHFR127,128 revealed that by making a 180°rotation about the C6-C9 bond, the interactions with the important amino acids in the enzymeresulted in more extensive hydrogen bonding, thereby leading to a higher affinity of MTX toDHFR as compared to FA. This result was later confirmed by NMR spectroscopy studies.129

The most important feature for binding of the antifolates is the N1 and the adjacent 2-NH2,which is also the strongest basic center. The generated interaction consists of ionic bonding ofthe cation of N1/2-NH2 to Glu30 (Figure 16).

Figure 16. Proposed interaction with Glu30 in the active site of human DHFR of a) dihydrofolate, andb) MTX, illustrated by the bond rotation of almost 180° around C6-C9.

Examination of the DHFR-MTX complex by X-ray diffraction evidence thepyrimidine ring to be situated in a lipophilic cavity, which would, by decreasing the dielectricconstant and absenting competing water molecules, increase the basic strength at N1/C-2, 2-

N

N NH

NO

H

NH

H

O

ONH

NH

COO

O COO

O

O

N

NH

COO

O COO

CH3

Glu30

Glu30

1

2

34

5

67

89

10

α

γ

2 4

N

N

NN

NN

H

HH

H H

a)


- 28 -28

NH2. The nicotinamide portion of the coenzyme NADPH lies sufficiently close to thepteridine ring to facilitate transfer of a hydride anion to C-6 from the pyridine nucleus.Inhibition of DHFR by MTX is moderately stereospecific, since the isomer with L-glutamatein the side chain is approximately 10-fold more potent than the corresponding D-analogue.130

The amino acids associated with binding of representative inhibitors are identical or closelysimilar when different species are compared, indicating a high degree of functional homologyat critical binding sites of DHFR. Although the amino acid sequence of rat liver DHFR is notyet available, it is most often used as the mammalian standard for inhibition studies.However, a recent homology study, soon to be published,131 demonstrated that the residuesinvolved in binding are conserved throughout.

3.1.1 Structure-Activity Relationships

Several reviews have been published regarding the structure-activity relationshipsamong the antifolates.55,132-134 MTX has been divided into seven regions as illustrated inFigure 17.134

Figure 17. Seven regions of MTX that have been subjected to structure modifications.134

The major changes in Region A include the substitution of the nitrogen atoms in the 1-and 3-positions with carbon atoms. Amines in the 2- and 4-positions are required forinhibitory activity against DHFR. Alkylation of the amino groups leads to a reduction of theinhibitory potencies.135 Region B, comprising the pyrazine moiety, displays a larger toleranceto alterations. The ring can be modified by replacement of the nitrogens at positions 5 and/ or8 with carbon to give either retention or improvement in activity.136,137 Similar substitutionshave rendered several interesting compounds, e.g., the 2,4-diaminoquinazoline analogues.Region C, usually referred as the ‘bridge region’, is a part of the molecule that is tolerant tosignificant structural changes. However, it is important not to alter the position of side-chainattachment (6-position). Hence, substitution in the 7-position is detrimental for binding.138

Accordingly, the main metabolite from MTX, 7-OH-MTX, is inactive as inhibitor. The

N

N N

NN

NH2

H2NMe

NH

COOH

O COOH

A B C D

F

E G

12

3 4 56

78

9 10

3'2'

6'5'


- 29 -29

‘bridge region’ between the C-6 of the pteridine ring and the benzoate moiety may be alteredconsiderably in terms of heteroatom substitutions. At this position, extra methylene-spacers,and nitrogens, respectively, have been introduced without loss of activity. Also, interchangesof C9 and N10, and replacement of N10 by sulfur and oxygen have been carried out. As analternative to the methyl group on N10, other carbon-containing groups with different chainlengths and sizes have been explored. Replacement of the N10 by a simple CH2 or otherbranched chains containing carbon, has also produced analogues of interest. Region Dconsists of a phenyl moiety, but different heterocyclic rings have been introduced heresuccessfully with respect to inhibitory activity towards the enzyme. The phenyl moiety hasbeen halogenated in the 3’- and 5’-positions affording more lipophilic compounds comparedto MTX. Modifications in Region E, comprising the amide bond between the pteroyl moietyand the glutamic acid portion, have most often resulted in compounds of no interest. Changesto the α-carboxyl group in Region F showed this group to be critical for binding to DHFR. Inaddition, an acidic functionality at this position seems to be essential in vitro as well as invivo. The α-carboxyl group is also important for active transport across the cell membrane,leading to subsequent γ-polyglutamation of the molecule. Having a glutamic acid residue ineither the ortho- or meta- positions rendered compounds inactive as substrates for FPGS. Thechirality of the α-center of the glutamic acid residue is L. The D-isomer of MTX isapproximately 10-fold less potent than the L-glutamate counterpart.130 Also, the D-isomer ofMTX has been reported to be biologically inactive,130,139 which mainly is attributed as aconsequence of its poor affinity for the active transport mechanism necessary for penetrationinto the cell (Section 1.4). Finally, the remaining glutamic acid portion in Region G seems totolerate structural changes. Modifications have consisted of, for example, insertion of an extramethylene group into the side-chain, as well as esterification and amidation of the γ-COOHgroup.

3.2 Non-classical Inhibitors towards Pneumocystis carinii

Based on published sequence alignments,140,141 P. carinii DHFR appears to be verysimilar to vertebrate DHFR. Also, high-resolution crystal structures of DHFRs from human127

and P. carinii142,143 show only minor differences. Within the active site of the folate-bindingpocket, there are only six non-identical residues. Comparing the volumes of the two activesites of both enzymes, reveals that the binding region for P. carinii is slightly smaller than theactive site of the human enzyme, but as there are no other major differences this makesspecific design very complex.144 Thus, producing inhibitors with selectivity for P. carinii is achallenging task because of the great similarities between the fungal and the mammalianenzyme.


- 30 -30

3.2.1 Structure-Activity Relationships

The high potencies of the new ‘non-classical’ DHFR inhibitors provide evidence thatthe p-aminobenzoylglutamic acid side chain in MTX is not essential for binding to DHFR.Both TMQ and PTX are much more potent inhibitors than TMP against the mammalianenzyme110 (Table 2). Consequently, with the exception of TMP and PTM, the main drawbackwith these new potent antifolates is their lack of selectivity against DHFR derived from P.carinii. Unfortunately, almost all the recently-developed inhibitors seem to follow this trend.

It is definitely a challenging task to discover new inhibitors with a selectivity ratioabove 10, especially accompanied by a good potency. As a consequence it is difficult to drawconclusions regarding the SAR for selectivity towards the fungus. A number of lipophilicfused 2,4-diaminopyrimidine ring system related to TMQ and PTX have been examined. Thisincludes the 2,4-diaminopteridines, the 2,4-diaminoquinazolines, the 2,4-diaminopyrido-[2,3-d]pyrimidines (5-deazapteridines), and the 2,4-diaminopyrido[3,2-d]pyrimidines (8-deazapteridines).145 Other fused bicyclic ring systems, such as 2,4-diaminothieno[2,3-d]-pyrimidines,146 2,4-diaminopyrrolo[2,3-d]pyrimidines,147 and 2,4-diaminofuro[2,3-d]-pyrimidines148 have been designed, but they all exhibited relatively poor activity as comparedto TMQ and PTX. In general, the most selective inhibitors have been found in the 2,4-diaminopteridine series, for example, a 6-CH2SPh analogue has been demonstrated to exhibita selectivity ratio of 26 for pcDHFR versus rlDHFR, although with a potency in themicromolar range.110 Among the fused analogues, 2,4-diamino-5-[(2’-napthylthio)-methyl]furo[2,3-d]pyrimidine and 2,4-diamino-5-[(2’-phenylanilino)methyl]furo[2,3-d]pyrimidine, displayed selectivity ratios of around 18.149 Other attempts have mainly beenconcentrated on finding new selective analogues towards the fungal enzyme by trying toexplore favorable interactions with the amino acids of the lipophilic cavity of the enzyme, thatis, substitution in, or of the phenyl ring. As an example, naphthyl analogues (replacement ofthe phenyl moiety) have been developed with high potency against DHFR.150 A small seriesof 2,4-diaminoquinazolines, encompassing 9-phenanthryl, 9-anthryl and 9-flourenyl has beensynthesized with high potencies.151 Also, several other series of compounds with a smallgroup in the 5-position in the heterocyclic core (i.e., 5-CH3, 5-Cl) have beensynthesized.150,152-154 Although, these 5-substituted analogues in general are more potentagainst pcDHFR than the unsubstituted analogues, they are even more potent against the ratenzyme.155,156 Nevertheless, one new inhibitor within a large series of pteridine analogues wasrecently published with a ratio of selectivity of 21 in favor of P. carinii in addition to highpotency (Figure 18).157 The compound in Figure 18 consists of a dibenz[b,f]azepine instead ofthe phenyl ring. The IC50-value against the fungal enzyme was 0.21 µM and it wasremarkable that the corresponding 10,11-dihydroanalogue exerted about 10 times lowerpotency (IC50: 1.4 µM), emphasizing the difficulties in achieving selectivity, and in the fine-tuning of ligands towards pcDHFR.


- 31 -31

It was suggested that the compound is thought to be selective because of its capabilityto adopt a favorable ‘puckered’ orientation of the seven-membered ring in thedibenz[b,f]azepine moiety. According to the results of modeling this allows the compound tofit more comfortably into the active site of pcDHFR than into the active site of hDHFR.

Figure 18.

4 OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES

A number of reviews covering the syntheses of ‘classical’ DHFR inhibitors and the‘non-classical’ antifolates, have been published.77,134,145,158-160 I will initially focus on thesynthesis of MTX, the most established DHFR inhibitor used in the clinic. In Section 4.2 thesynthesis of the new lipophilic antifolates TMQ and PTX will be surveyed.

4.1 Methotrexate

Syntheses of MTX have been reported independently by several groups.77,137,161-163 Formore references, see the following reference,158 and references therein. The methods used canbe divided into three main groups as described below.

Figure 19. A retrosynthetic analysis of the first synthesis of MTX.

N

N N

NNH2

H2N

NCH3

NH

O

COOH

COOH

MTX

Br OBr

N

N

NH2

NH

COOH

NH

H3C

O COOH

I

+

+

NH2

NH2

H2N

N

N N

NNH2

H2N

N

OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES

- 32 -32

Method A: The first synthesis of MTX72 was an adaption of the Waller method164,165

used for the early preparation of folic acid, and involves a ‘one-pot’ condensation of 2,4,5,6-tetraaminopyrimidine I, 2,3-dibromopropionaldehyde, and the sodium salt of N-p-methylaminobenzoyl L-glutamic acid in aqueous solution (Figure 19). This method resulted ina complex reaction mixture with MTX isolated in less than 5% yield. An alternative routeemploying a 1,1,3-trihaloacetone, which is condensed with compound I at pH 5 to afford amixture of the 6- and 7-isomers of the halomethylpteridine, but the method was accompaniedby significant difficulties in separating the generated mixtures (Figure 20).166 It was laterfound that when using 1,1-dichloroacetone at pH 3.5-5.0 only the 6-methylpteridine wasformed,167 which upon bromination afforded the 2,4-diamino-6-bromomethylpteridine II.

Figure 20. A retrosynthetic analysis of MTX following Method A.

Piper and Montgomery168,169 reported the preparation of the hydroxymethylpteridineanalogue III, by a condensation reaction of 2,4,5,6-tetraaminopyrimidine (I) and 1,3-dihydroxyacetone (Figure 20). Bromination, using dibromotriphenylphosphorane in DMAcprovides the 6-bromomethylpteridine II. All reactions described above are variants of theoriginal Isay synthesis,170,171 which relies on the condensation of, in this case I, with anunsymmetrical dicarbonyl compound. This route suffers from the drawback that thecondensation mainly leads to a mixture of the 6- and 7-isomers.172

Method B: Another more efficient route for the synthesis of the substituted pteridineswas described in 1973.173,174 Subsequently, this route has been called the Taylor synthesis and,in contrast to Method A, utilizes a pyrazine precursor rather then a pyrimidine precursor forthe cyclization (Figure 21). The reaction conditions readily permit reactions at the 6-positionwhile allowing for the preparation of several kinds of MTX analogues possessing lipophilicside-chains in addition to a variety of other amino acids. Also, the replacement of the N10-atoms by other heteroatoms can be achieved readily. Following this route, the pyrazine N-oxide IV is reacted with the side chain to give 2-amino-3-cyano-6-chloromethylpyrazine N-oxide V.175,176 Deoxygenation of V with triethyl phosphite provides VI, which after a base-catalyzed cyclization with guanidine, affords MTX.

N

N N

NNH2

H2N

NCH3

NH

O

COOH

COOH

MTX

N

N N

NNH2

H2N

Br

II

N

N N

NNH2

H2N

OH

III

N

N NH2

NH2

NH2

H2NI


- 33 -33

Figure 21. A retrosynthetic analysis of MTX following Method B.

Method C: A third approach for the synthesis of MTX involves 2,4-diamino-6-chloro-5-nitropyrimidine and the oxime, VII, affording the intermediate, VIII, which upon catalyticreduction cyclizes in situ providing the 7,8-dihydropteridinederivative IX (Figure 22).Permanganate oxidation furnishes the heteroaromatic compound, X,177,178 which after esterhydrolysis is coupled with L-glutamic acid to ultimately provide MTX.179

Figure 22. A retrosynthetic analysis of MTX following Method C.

4.2 Non-classical Inhibitors

Methods used for the synthesis of TMQ, and PTX, respectively, are described below.

N

N N

NNH2

H2N

NCH3

NH

O

COOH

COOH

MTX

H2N N

NNCH3

NH

O

COORNC

COOR

O

V

H2N N

NNCH3

NH

O

COORNC

COOR

H2N N

NNC X

O IV

VI

X = Br, Cl

N

N NH

NNH2

H2N

NCH3

OR

O

N

N N

NNH2

H2N

NCH3

NH

O COOH

COOH

MTX

N

N N

NNH2

H2N

NCH3

OR

O

N

N NH

NH2

H2N

NCH3

OR

O

NOH

NO2

X

H2N

NNCH3

OR

O

OH

N

N

NO2

NH2

H2N ClVII

VIIIIX

R = Me or Et

R = Me or Et R = Me or Et

+ R = Me or Et


- 34 -34

4.2.1 Trimetrexate

The original synthesis of the quinazoline derivative TMQ78 was a modification of theearly synthesis of unsubstituted phenyl analogues previously described by others.180 Thesynthesis starts with 6-chloro-o-toluonitrile XI,181 which was subjected to fuming nitric acidaffording 6-chloro-3-nitro-o-toluonitrile XII, as the major product in a 3:1 mixture with 6-chloro-5-nitro-o-toluonitrile. The resulting mixture is condensed with guanidine carbonateresulting in the 2,4-diaminoquinazoline derivative XIII, which is further reduced to provide2,4,6-triaminoquinazoline (XIV). The 6-amino group in XIV is successfully diazotized underSandmeyer-type conditions without interfering with the other amino groups, using an acidicsolution of sodium nitrite in the presence of cuprous cyanide. A reductive amination of the2,4-diamino-6-quinazolinecarbonitrile XV over Raney Ni under a high pressure of hydrogen(50 psi) in the presence of 3,4,5-trimethoxyaniline finally furnishes TMQ in a total yield ofabout 30% (Figure 23).

Figure 23. A retrosynthetic analysis of TMQ.

4.2.2 Piritrexim

PTQ is a pyrido[2,3-d]pyrimidine derivative, which also is called, 5-deaza-pteridine.83,182 Its retrosynthesis is depicted in Figure 24 and Figure 25.

Figure 24. A retrosynthetic analysis of an intermediate in the synthesis of PTX.

N

N

NH2

H2N

NH

OCH3

OCH3

OCH3

CH3

N

N

NH2

H2N

NH2

CH3

CH3

NC

Cl

NO2

CH3

NC

ClXII

XIV

TMQ

XI

N

N

NH2

H2N

NO2

CH3

N

N

NH2

H2N

CNCH3

XIII

XV

H3CO

OCH3

COCH3

CO2C2H5

H3CO

OCH3

COCH3

CO2C2H5

XVIII XVII

H3CO

OCH3

CHO

XVI


- 35 -35

In Figure 24, the synthesis of the non-heterocycle XVIII starts with a ‘Knoevenagel-type’ reaction of 2’,5’-dimethoxybenzaldehyde XVI with ethyl acetoacetate affording theacrylate XVII. The acrylate is further hydrogenated under pressure over 5% Pd/C furnishingXVIII. Condensation of XVIII with 2,4,6-triaminopyrimidine, XIX, provides the 7-oxocompound XX. Chlorination of the oxo-function is accomplished with a Vilsmeier-Haackreaction using thionyl chloride in DMF. Compound XXI is further dechlorinated under basicconditions over hydrogen with 5% Pd/C as catalyst to afford PTX.

Figure 25. A retrosynthetic analysis of PTX.

Recently, an alternative route for the synthesis of PTX was reported (Figure 26).183 Inthis route 4-(2,5-dimethoxyphenyl)-2-butanone XXII146 reacts with DMF and phosphorousoxychloride providing a E/Z-mixture of 3-chloro-2-(2,5-dimethoxybenzyl)crotonaldehydeXXIII. When the mixture (XXIII) is reacted with cyanoacetamide (XXIV) the adductcyclizes to the pyridone XXV. Compound XXV is transformed to the chloride XXVI, usingDMF and thionyl chloride, followed by subsequent condensation with guanidine to yieldPTX.


N

N N

NH2

H2N

CH3 OCH3

OCH3

N

N NH

NH2

H2N

CH3

O

OCH3

OCH3XX

PTX

N

N N

NH2

H2N

CH3

Cl

N

N

NH2

H2N NH2

XIX

OCH3

OCH3

XXI

XVIII+

N

N N

NH2

H2N

CH3 OCH3

NaPTX

H2N

CN

O

XXIV

OCH3

OCH3

O

Me

H

Cl

XXIII

Cl N

CH3NC

XXVI

OCH3

OCH3

OCH3

OCH3

O

Me

XXII

NC

O NH

CH3

XXV

OCH3

OCH3

+

DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

- 36 -36

5 DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

5.1 Synthesis of Simple Aromatic Ester-containing Model Compounds (PaperI)

Before starting the synthesis of the ester-containing DHFR inhibitors, we decided tosynthesize simple aromatic esters as model compounds. The compounds were designed inorder to evaluate the influence of the substitution pattern around the ester functionality oncleavage by different esterases. The new compounds comprised a naphthalene and a phenylgroup linked together by a two or three atoms long ester bridge.

The synthesis of compounds 1-6 was performed by esterification of the alcohol, thatis, phenol, 2-naphthol, benzyl alcohol, and 2-naphthalenemethanol, respectively, with theappropriate acid chlorides using triethylamine as base (Scheme 1).

Scheme 1

Later on, attempts to improve the yields in some of the reactions were made. Amongthem, the Mitsunobu reaction184 was adopted for the synthesis of 3. The reaction was useful inthis case. Also, the synthesis of 6 was attempted using a ‘Schotten-Baumann-like’ reactionwith 5% sodium hydroxide and the acid chloride afforded the phenol ester in 35% yield.Other conditions, e.g., pyridine in diethyl ether and sodium hydride in THF gave no productat all.

For the syntheses of the carboxylic acids 7 and 9, 4-formylbenzoic acid was employedas starting material, which after conversion to the acid chloride, was esterified as for 1-6,using 2-naphthol or 2-naphthalenemethanol, respectively. The carboxylic acids 8 and 10 wereprepared according to the same procedure using 4-hydroxybenzaldehyde as the precursor. Thesubsequent oxidations of the aldehydes were conducted with sodium chlorite, using 2-methyl-2-butene as scavenger185-187 (Scheme 2).

X

O

O

O

O

O

O

O

OX =

O

O

OO

X =

Et3N, diethyl etherROH + R'COCl

1 4

5

3

2

6


- 37 -37

Scheme 2

Attempts to introduce DCC, a common coupling reagent for esters, for theesterification of the aldehyde precursor to 10 were made, but unfortunately the route wasunproductive with these substrates.

For the preparation of 11, the synthetic route in Scheme 2 could not be used, andtherefore another reaction sequence outlined in Scheme 3 and 4 was considered starting fromcommercially available methyl (4-hydroxy)phenylacetate. The triflate 13 was prepared in94% yield using triflic anhydride and pyridine.188 The methyl ester in 13 was hydrolyzed withtrifluoroacetic acid (TFA)189,190 to afford the carboxylic acid 14 in a yield of 92%. Lithiumiodide in DMF191 was also tried for the demethylation, but no product was formed under theseconditions.

Scheme 3

The carboxylic acid group of compound 14 was protected by benzylation through SN2-type esterification with benzyl bromide in refluxing acetone yielding 15 in 95%. Atransesterification of 13 with benzyl alcohol and DMAP in refluxing toluene, affordingcompound 15 in one step gave no advantage over the preferred route (60% vs. 87% yield). Apalladium-mediated carbonylation of compound 15 was accomplished with 2-trimethylsilylethanol in DMF using palladium acetate [Pd(OAc)2] and dppp to furnish thediester 16 in 79% yield (Scheme 4). Debenzylation of 16 with hydrogen over palladium oncharcoal and subsequent reaction of 17 with oxalyl chloride and 2-naphthol accomplished 18in 51% over three steps. Finally, 18 was transformed to 11 by selective ester cleavage, using

X

CHO

O

O

O

O

X

COOH

O

O

O

O

tert-butyl alcohol, 2-methyl-2-butene, r.t. NaClO2, NaH2PO4

1H2O,

87-95 %

X =

8

7 9

10

....

MeO

OHO

HO

OOTf

MeO

OOTf

OTf

BnO

O

(CF3SO2)2O, pyridine, CH2Cl2, 0 °C

TFA, reflux benzyl bromide,K2CO3, acetone, reflux

13

14 15


- 38 -38

trimethylsilyl chloride (TMSCl) and sodium iodide (NaI) in high yield (94%). Deprotection ofthe 2-(trimethylsilyl)ethyl group of 18 with TBAF could not be used due to a fast hydrolysisof the ester with this reagent, demonstrating the susceptibility of the 2-naphthylester group.

Scheme 4

The synthesis of the carboxylic acid 12 is outlined in Scheme 5. An initial attemptusing tert-butyldimethylsilyl as a protecting group of the carboxylic acid function in 19 wasmade, but the product seemed to be unstable during work-up. Using an alternative procedure,the carboxylic acid function in 4-bromomethyl benzoic acid 19 was esterified with 3,4-dihydro-2H-pyran in the presence of p-toluenesulfonic acid, but here also the product wasshown to be too unstable for isolation in satisfactory yields. Instead a reaction of compound19 with 2-(trimethylsilyl)ethanol with DCC, providing 20, was followed by a directesterification with 2-naphthol and potassium carbonate affording ester 21 in 88% yield overtwo steps. In contrast to the deprotection of 18, the target ester 12 could be achieved in thissystem with TBAF furnishing the ester in 71% yield.

Scheme 5

The target esters mentioned above, were all tested in vitro in esterase assays to give anindication of a ranking order (Section 6.1). We suspected that esters 1-2 and 4-5, respectively,could be easily hydrolyzed and we decided to take the structural features of the least reactive

O

O O

OSiMe3

OTf

BnO

O

OSiMe3

O

HO

O

OSiMe3

O

BnO

O

O

COOHO

79%

83%62%

94%

CO (1 atm), Pd(OAc)2, dppp, 2-(trimethylsilyl)ethanol, Et3N, DMF, 80 °C

H2 (1 atm), 10% Pd/C, THF 1) oxalyl chloride, toluene

2) 2-naphthol, Et3N, diethyl ether

NaI, TMSCl, CH3CN, reflux

1118

17

1615

BrCH2 COOH

OO

SiMe3

O

O

BrCH2O

SiMe3

O

COOH

O

O

TBAF, 1M in THF, r.t.

2-(trimethylsilyl)ethanol, DCC, DMAP, CH2Cl2, r.t.

2-naphthoic acid,K2CO3, acetone, r.t.

19 20

1221


- 39 -39

ester for further incorporation into a heterocyclic core more structurally similar to otherinhibitors of DHFR.

5.2 Synthesis of Soft Drug Derivatives of Non-classical Antifolates (Papers I,

III and IV)

After we had evaluated compounds 1-12 for their susceptibility towards differentesterases in vitro (Section 6.1) we selected the ester group anticipated to be the most stabletowards hydrolysis (compounds 2 and 3, respectively) to ensure that we would not initiallyobserve ester hydrolysis due to chemical instability.

Commercially available 2,4-diamino-6-nitroquinazoline was reduced under 4atmospheres (60 psi) of hydrogen over 10% palladium on charcoal using a Parr apparatusforming 2,4,6-triaminoquinazoline180 (22a) in a quantitative yield (Scheme 6). According tothe same reference, tin(II)chloride in concentrated hydrochloric acid could be used in thesame transformation. However, the conditions were found to be unreliable with appreciableamounts of hydrolyzed product detected (Scheme 6). By virtue of the fact that the freeelectron pair on the exocyclic nitrogen in the 4-position of the 2,4-diaminoquinazoline has thecapability to be delocalized into the ring system this position is very susceptible towardsnucleophilic attack. Another approach for the reduction of aromatic nitro groups using sodiumborohydride (NaBH4) and 2 M CuSO4 in ethanol192 did not work at all on this substrate.

Scheme 6

The amino group in the 6-position of 22a was then subjected to a selectivediazotization under Sandmeyer-type conditions with cuprous cyanide forming 2,4-diamino-6-cyanoquinazoline180 (23a) in high yield. The nitrile was further converted to thecorresponding aldehyde (24a) in refluxing formic acid with Ni-Al alloy serving as catalyst(Scheme 7).

N

N

NO2

NH2

H2N

N

N

NH2

NH2

H2N

H2, 10% Pd/C,AcOH, DMF, 4 atm

N

N

NH2

NH2

H2N

HN

N

NH2

O

H2N

+

SnCl2, conc. HCl

22a

22a


- 40 -40

Scheme 7

Before proceeding, we assumed that the exocyclic amino groups in 24a would beprotected due to possible competing reactions in ensuing steps. Screening of a series ofprotection groups, i.e., trifluoroacetic anhydride, di-tert-butyl dicarbonate, trityl chloride,acetic anhydride, diethyl pyrocarbonate, and allyloxybenzotriazol carbonate finally revealedthat only the pivaloyl group provided suitable protection of the amino functions in thesubsequent transformations (Scheme 8). Actually, none of the other protecting groupsreagents were prone to reaction with the substrate according to TLC, indicating a very poorreactivity of the 2,4-diamino groups. Despite reports that direct pivaloylation of similarcompounds containing formyl groups is not suitable,193 we succeeded with this one stepreaction furnishing compound 25 in good yields, although the yields varied significantlybetween different batches.

Scheme 8

Oxidation of 25 to the carboxylic acid 26, conversion to the acid chloride andtreatment with benzyl alcohol provided 27 in 56% yield (Scheme 9), while reduction of 25with sodium borohydride gave 28, which after subsequent reaction with benzoyl chloride andtriethylamine furnished the ester 29 in 90% yield (Scheme 10).

N

N

NH2

NH2

H2N

N

N

CHONH2

H2N

N

N

CNNH2

H2N

NaNO2, 2 M HCl,KCN, CuSO4, 0 °C

75% formic acid, reflux

22a 23a

24a

N

N

CHONH2

H2N

N

N

CH2OHNHPiv

PivHN

NaBH4

N

N

CHONHCOC(CH3)3

(H3C)3COCHN

N

N

COOHNHPiv

PivHN

26

pivalic anhydride, DMF

80 °C

24a

28

25

....NaClO2, NaH2PO4 1H2O


- 41 -41

Scheme 9

Deprotection of 27 and 29 was conducted using aqueous ammonia in dioxane,eventually delivering 30a and 31a, respectively. However, I terminated the reactions due toconcurrent ester cleavage at a stage where small amounts of the monopivaloyl-protectedamine still remained. The yields varied between 40-60% in this last step.

Scheme 10

The probable metabolites (hydrolyzed 30a and 31a, respectively) were alsosynthesized because of the importance of evaluating their inhibitory activities towards DHFR(Section 6.2.1). The aldehyde 24a was subjected to oxidation and reduction, respectively(Scheme 11), according to the same methods as described above for the diprotected analogue25, furnishing 32a and 33a, respectively. With reduction to 33a over-reduction of the alcoholto the 6-methylanalogue was detected when too long reaction times were used.194

Scheme 11

Since the idea behind using a protecting group was to allow easy handling withoutinterfering too much with the total yield, we decided to search for other strategies. A controlreaction was run confirming that direct esterification of 2,4-diaminoquinazoline-6-methanol,

N

N

NHPiv

PivHN

O N

N

NH2

H2N

ONH3, dioxane

28

O O

benzoyl chloride, Et3N

31a29

N

N

NHPiv

PivHN

O

O

N

N

NH2

H2N

O

O

NH3, dioxane

26 1) oxalyl chloride2) Et3N, benzyl alcohol

....

30a27

NaBH4

N

N

CHONH2

H2N

N

N

CH2OHNH2

H2N

N

N

COOHNH2

H2N

24a

32a 33a

NaClO2, NaH2PO4 1H2O.


- 42 -42

33a, with benzoyl chloride gave no product of interest according to TLC. Other attempts withDCC, or Mitsunobu conditions184 gave no positive results verifying the poor nucleophilicityof the alcohol. By virtue of the fact that literature describes the synthesis of different aminesin similar compounds using SN2 conditions, we found that the esterification could be achievedby reacting the bromomethyl compound 34a with the appropriate carboxylic acid usingpotassium carbonate in DMF. This procedure allowed a large series of compounds inacceptable yields to be made (Scheme 12).

Scheme 12

General methods for the transforming of the alcohol to the corresponding bromideemployed in antifolate chemistry were dibromotriphenylphosphorane (Ph3PBr2) in DMAc,169

HBr in dioxane,163,195 PBr3 in THF,196 or HBr in AcOH,197 respectively. In my hands, the lastprocedure provided the best results for the bromination of the alcohols 33a-33d. Aftercompletion, the reaction mixture was triturated with cold diethyl ether to cause precipitation.This method was preferable to simple evaporation in vacuo to avoid the easy formation of2,4-diaminoquinazoline-6-methyl acetate, due to its high reactivity of 34a as its hydrobromicsalt. Immediate displacement of the bromide with the appropriate carboxylic acid usingpotassium carbonate or cesium carbonate as bases in DMF, DMSO or DMAc, respectively,afforded the esters 31b-d and 35a-55a in various yields. The most convenient conditions were

N

N

Y

XNH2

H2N

BrArCOOH

CH CCH3N CH CH CH CH CHCHCHCHCHCHCHCHCHCHCHCHCHCHCHCHCH

NN N CH CHCH CH CHCHCHCHCHCHCHCHCHCHCHCHCHCHCHCHCH

N

N

Y

XNH2

H2N

O Ar

O

+

a: X, Y = CHb: X = CH; Y = Nc: X = CCH3; Y = Nd: X, Y = N

31b:31c: 31d: 35a: 36a: 37a: 38a:39a:40a:41a:42a:43a:44a:45a:46a:47a:48a:49a:50a:51a:52a:53a:54a:55a:

phenylphenylphenyl2',5'-(OCH3)2-phenyl3',4'-(OCH3)2-phenyl3',5'-(OCH3)2-phenyl2',3',4'-(OCH3)3-phenyl3',4',5'-(OCH3)3-phenyl3',5'-(CH3)2-phenyl1-naphthyl2-naphthyl9-phenantrenyl9-anthracenyl9-fluorenyl2'-benzylphenyl2'-biphenyl4'-biphenyl3'-iodophenyldiphenylmethyl2-thienyl3-thienyl2-furyl3-furylCH2Ph

34 X ArY


- 43 -43

found to be potassium carbonate in DMF. The syntheses of the precursors of the non-quinazoline compounds are described later in this section. Allowing the reaction mixtures tostir for longer times did not improve the yields and heating the reaction mixture did not speedup the reaction time. Neither attempts to esterify the bromides 34a-d in the absence of baserelying on an SN1-type reaction did work. Literature describes198 a method for the treatmentof compound 34c with triethylamine in order to avoid rapid inactivation of the hydrobromidesalt associated with the reactive bromomethyl compound by the benzoate anion. In my handsthe triethylammonium salt of 34c was formed according to 1H NMR.

Scheme 13

Due to the lack of commercially available 3-phenylbenzoic acids we decided tointroduce some palladium chemistry into the synthesis of the phenyl derivatives 58a and 59astarting from the 3’-iodophenyl compound 49a (Scheme 13). The reaction of choice was theSuzuki reaction199,200 as it has become a valuable tool with respect to synthetic chemistrybecause of its simplicity as a method for the formation of new carbon-carbon bonds and easyand clean work-up. The reaction relies on the reaction between an organoboron nucleophileand an organic halide or pseudohalide (i.e., a group with similar properties to the good leavinggroup of the halide, e.g., triflates.). Instead of using conventional heating the relatively newmethodology associated with microwave chemistry was applied. The heating of the reactionmixture in a focused single-mode oven is of great advantage to the organic chemist because itenables a fast and controlled heating. When applying microwave-promoted heating thereaction times can decrease from several hours to only a few minutes.201 The Suzuki-couplings were conducted in a Smith SynthesizerTM single mode microwave cavity with 49aand phenylboronic acid (56), and 2,6-dimethoxyphenylboronic acid (57),* respectively.Different conditions were explored in order to find the optimal conditions for this specificreaction. The Smith reaction kit consisting of Pd(PPh3)2Cl2, Pd(OAc)2, and Hermanns catalyst(trans-di-µ-acetobis[2-(di-o-tolylphosphino)benzyl]dipalladium(II)), was used to providecatalysts for the evaluation. Sodium or cesium carbonate in stock solutions were used asbases, and DME/H2O/EtOH (7:3:2) or DMF as solvents. The most efficient reactionconditions for these substrates were Pd(PPh3)2Cl2 and sodium carbonate in DME/H2O/EtOH(7:3:2).202 After 120 s at 140 °C full conversion of the starting material (49a) occurred * 2,6-Dimethoxyphenylboronic acid was a generous gift from Pär Holmberg, Dept. of Org. Pharm. Chem., BMC, UppsalaUniversity, Sweden.

N

N

NH2

H2N

(HO)2B

N

N

NH2

H2N

O

O

+

Pd(PPh3)2Cl2, Na2CO3,DME/H2O/EtOH (7:3:2),

2 min, 140 °C

R

R

R R56: R = H57: OCH3

58a: R = H59a: OCH3

O

O

I49a


- 44 -44

according to LC-MS, whereas after 80 s microwave irradiation at 140 °C LC-MS still showedthe presence of the starting iodo-compound (49a). Unfortunately, hydrolysis products (33a)were observed by LC-MS and this could be one explanation for the moderate isolated yields(around 40%) compared to similar couplings with other substrates. Only starting material wasfound in the reactions performed in DMF. The temperature profile versus time from theexperiment is shown in Figure 27, which demonstrates one of the advantages with thisapplication compared to traditional heating in terms of rapid and effective heating of thereaction medium.

Time (s)

Figure 27. The temperature profile vs. time in the synthesis of 58a and 59a.

In the synthesis of the esters 31b-c, the precursors were prepared from non-aromaticmoieties using slightly modified literature procedures137,153 (Scheme 14). Compounds 60b-cwere synthesized by a condensation reaction mediated by hydrochloric acid withmalononitrile and triethyl orthoformate, or triethyl orthoacetate, respectively. Dechlorinationof 60b-c yielded 61b-c, which upon cyclization with guanidine in refluxing ethanol, affordedthe two substituted 2,4-diaminopyrido[2,3-d]pyrimidines 23b-c, respectively. Compound 23c(~60%, 10 days) was obtained in a lower yield and also needed longer a reaction time incomparison to 23b (~80%, 2 days) to form the target molecule, presumably due to sterichindrance when performing the ring closure. Treatment with Ni-Al alloy203 or Raney Ni inrefluxing formic acid converted the nitriles 23b-c into the aldehydes 24b-c, which werefurther reduced to the corresponding alcohols 33b-c by NaBH4 in methanol. For the synthesisof 31d commercially available 2,4-diaminopteridine-6-methanol (33d) was used. Thealcohols 33b-d were all brominated as described above for 33a to give 34b-d.

0

50

100

150

200

0 25 50 75 100 125

Tem

p. ( °° °°

C)


- 45 -45

Scheme 14

In order to synthesize inhibitors with a four-atom long central chain three compoundswere made. The synthesis of compound 55a is depicted in Scheme 12. Ester 63a, wasprepared by diazotization of 2,4,6-triaminoquinazoline 22a, with potassium iodide andsodium nitrite, yielding the iodo-compound 62a.204,205 A palladium-catalyzed Heckreaction206-208 of 62a with phenyl acrylate provided the α,β-unsaturated ester 63a in 40%yield (Scheme 15). Subsequent reduction of the double bond to furnish 64a was accomplishedreadily using hydrogen over Pd/C. However, an initial attempt with ammonium formateserving as the hydride donor, together with Pd/C in methanol resulted in the methyl esterinstead of the expected phenyl ester.

Scheme 15

RC(OEt)3 H2N NH2

NHHCl

2 CH2(CN)2

N

N N

CNNH2

H2N

R

N

CNR

NC

H2N Cl

N

N N

CHONH2

H2N

N

CNR

NC

H2N

R = HR = CH3

R = H R = CH3

R = HR = CH3

+1. C6H5N

2. HClH2, 10% Pd/C,

Et3N, DMF

Ni-Al alloy75% formic acid

N

N N

CH2BrNH2

H2N

HBr

HBr

N

N N

NH2

H2N

CHOCH3

N

N N

NH2

H2N

CH2BrCH3

Raney Niconc. formic acid

24b

23c:23b:

61c:61b:

60c:60b:

24c

1) NaBH4

2) 30% HBr i AcOH

1) NaBH4

2) 30% HBr i AcOH

34b

34c

.

.

.

N

N

NH2

H2N

I

N

N

NH2

H2N

O

O

phenyl acrylate, Pd(OAc)2P(o-tol)3, Et3N, 80 °C

N

N

NH2

H2N

O

O

10% Pd/C, H2 , DMF

N

N

NH2

H2N

NH2 NaNO2, HCl, 0 KI

°C

22a 62a

63a

64a


- 46 -46

To explore the influence of the ester moiety in terms of inhibitory activity we felt itnecessary to synthesize two reference compounds, 65a and 66a, both lacking the esterfunction (Scheme 16). A subsequent reductive amination of 23a was accomplished byhydrogenation over Raney Ni at atmospheric pressure with aniline affording 65a.180 A finalreductive methylation using 37% formaldehyde and sodium cyanoborohydride at pH 2-3provided the N10-methylated compound 66a in 89% yield.

Scheme 16

We also considered the introduction of solid phase chemistry (see Section 5.4 for amore detailed description) to build a combinatorial library consisting of lipophilic 2,4-diaminoquinazoline esters. We implemented this idea by introduction of a polystyrene resinwith sulfonyl chloride as the linker on polymeric support (Scheme 17). Reaction of thesulfonyl chloride with 4-hydroxybenzaldehyde in the presence of triethylamine afforded thealdehyde 67. Subsequent oxidation with sodium chlorite to afford 68 was not successful andinstead m-chloroperbenzoic acid (mCPBA) was used with satisfactory results. Before a furthercoupling reaction was performed a control experiment with 31a was run in order to find theoptimal conditions for cleavage of the final product from the resin. Triethylamine, formicacid, Pd(OAc)2, and dppp in DMF at 140 °C, were tried,209 but unfortunately only 2,4-diamino-6-methylquinazoline was detected by 13C NMR spectroscopy, a fact that prompted usto stop using this method.

Scheme 17

5.3 Synthesis of New Derivatives of Classical Antifolates (Paper V)

Along with the design of the ‘non-classical’ analogues we had the intention ofexploring the tolerability of DHFR towards the ester function with the series of ‘classical’antifolates. Four new ‘classical’ inhibitors (79a-81a and 79d) were synthesized and all the

N

N

NH

NH2

H2N

N

N

N

NH2

H2NCH3

23aH2, Raney Ni,

aniline30% formaldehyde,

NaCNBH3

65a 66a

SO

OCl

Et3N

SO

OO COOH

SO

OO CHO

mCPBA,DME

67

68


- 47 -47

derivatives differed from MTX by the replacement of the methyleneamino group with an esterfunctionality. One of the compounds retained the pteridine core whereas with the other threederivatives this was replaced by the quinazoline. One compound was made with the goal ofsynthesizing an analogue that was not a substrate for FPGS (Section 1.4). Finally, we alsowanted to synthesize an analogue with D-glutamic acid instead of the usual L-glutamic acidpresent in MTX.

Compounds 70-72210,211 were synthesized as outlined in Scheme 18. The syntheticroute starts with the commercially available di-tert-butyl esters of L-and D-glutamic acidhydrochloride, and di-tert-butyl L-α-aminoadipate (69), respectively. Compound 69 wasprepared according to a literature procedure.212 The protected amino acid derivatives were allallowed to react with 4-formylbenzoic acid using PyBOP and N,N-diisopropylethylamine ortriethylamine, respectively, affording 70-72. The aldehydes 70-72 were oxidized immediatelyafter purification to afford the carboxylic acids 73-75.

Scheme 18

The esters 76a-78a and 76d were formed by nucleophilic displacement of theappropriate bromide by using potassium carbonate in anhydrous DMF, or DMAc,respectively, as depicted in Scheme 19. Compound 34d168,169 was used in the synthesis of76d. The overall yield for the last two steps combined varied from 23 to 51%. Final selectivehydrolysis of the tert-butyl esters 76a-78a and 76d, with HCOOH or TFA provided 79a-81aand 79d in good yields (Scheme 19).

OHC

CO2H

CO2tBu

H2N CO2tBu

H HN CO2

tBu

O

O

CO2tBu

HO HN CO2

tBu

O

O

CO2tBu

n=1, L-isomer n=1, D-isomer n=2, L-isomer

70: n=1, L-isomer71: n=1, D-isomer72: n=2, L-isomer


nnnn

nnnn nnnn

69:

PyBOP,DIPEA or Et3N

NaClO2, NaH2PO4 1H2O.


- 48 -48

Scheme 19

5.4 Applying Solid Phase Chemistry to the Synthesis of Classical DHFRInhibitors (Paper VI)

Solid phase synthesis was first introduced by Merrifield in the area of peptidesynthesis in the early 1960s.213,214 A great advantage of solid phase synthesis over reactions insolution is the highly simplified procedure for product purification at each step of the targetcompound synthesis. In the ideal case, all employed soluble reagents can be removedcompletely and conveniently by filtration and washing of the solid resin, whereas the productremains covalently bound to the solid support. After completion of synthesis, the finalcompound is liberated from the solid support by using a cleaving agent with subsequentisolation of the compound (note: compare this to the unmasking procedure when usingprotection groups). It is important that the bond between the product and the carriersufficiently stable towards all reagents and conditions used in the synthesis. The original ideabehind solid phase synthesis was to use a solid support consisting of a water-insolublepolystyrene support (PS). Successful improvements within the area have been thedevelopment of the TentaGel resin, and the related ArgoGel, that combine the insolublepolystyrene support with a soluble poly(ethylene glycol) (PEG) polymer. TentaGel resin isbased on a very small portion of cross-linked 1% divinylbenzene-polystyrene that isextensively grafted with long PEG spacers (50-60 ethylene oxide units). The PEG content is

N

N X

XNH2

H2N

Br HO HN CO2

tBu

O

O

CO2tBu

HBr

N

N X

XNH2

H2N

O HN CO2

tBu

O

O

CO2tBu


76a: X=CH, n=1, L-isomer77a: X=CH, n=1, D-isomer78a: X=CH, n=2, L-isomer76d: X=N, n=1, L-isomer

K2CO3,DMF alt. DMAc

34a: X=CH34d: X=N

n

n

.

CO2H

N

N X

XNH2

H2N

O HN CO2H

O

O

n

79a: X=CH, n=1, L-isomer80a: X=CH, n=1, D-isomer81a: X=CH, n=2, L-isomer79d: X=N, n=1, L-isomer

HCOOHalt. TFA


- 49 -49

up to 70% of the resin weight. Therefore, the properties of the PEG chains determine themechanical, physicochemical behavior of the resin. Due to the fact that this long and flexiblespacer is well separated from the PS matrix, the reactions performed on TentaGel exhibitmore ‘solution-like’ properties as compared to pure polystyrene beads. The polymer must besolvated properly by the solvents of choice and in addition, the resin must swell sufficiently inorder to gain access to the internal surface of the beads. Dichloromethane and DMF are thetwo solvents most often used to achieve a good loading capacity. The attachment of themolecule to the solid phase is achieved through a cleavable linker.215,216 A linker can bedescribed as a bifunctional protecting group. It is attached to the molecule by a bond that islabile to the cleavage conditions. On the other side, the linker is attached to the solid phasethrough a more stable bond. The ‘Wang’ linker217 was one of the first linkers introduced andis cleaved under acidic conditions, i.e., 50% TFA. At present a wide range of linkersystems218 on different supports is commercially available, and these can be cleaved underdifferent conditions and by various reagents.

Scheme 20

Although we had concluded previously that solid phase synthesis could be difficult toperform with the reactions of these ester-containing DHFR inhibitors, we still wanted toelucidate whether this synthetic methodology could be used for the synthesis of 79a and 79d.The reaction route starts following a literature procedure,219 whereas the monoester 82 wasprepared from terephthaloyl chloride in 53% yield over two steps (Scheme 20). Cesiumcarbonate was used as base in the couplings of compounds 34a-d, respectively, providing theesters 83a and 83d. Selective hydrolysis of the tert-butoxy group in 83a and 83d by TFArendered the zwitterionic compounds 84a and 84d. Hence, esterification of the commerciallyavailable Fmoc-Glu-OtBu was conducted on ArgoGel with the acid labile Wang-linker assolid support (Scheme 21).

Cl

Cl O

O

N

N X

XO

NH2

H2N CO2H

O

TFA

CO2t-BuHO

O

N

N X

XO

NH2

H2N CO2t-Bu

O

N

N X

XBr

NH2

H2N HBr

83a: X = CH83d: X = N

1) tert-butyl alcohol, pyridine2) KOH Cs2CO3,

DMF

84a: X = CH84d: X = N

34a: X = CH34d: X = N

82

.


- 50 -50

Scheme 21

Fmoc-deprotection using 20% piperidine in DMF yielded the polymer supportedglutamic acid derivative 85, which was further reacted with compound 84a and 84d, usingPyBOP as coupling reagent, providing the amides 86a and 86d. The amide bond formationoccurred smoothly according to the 13C NMR spectra of 86a and 86d. Hydrolysis in 50%TFA/dichloromethane for 1.5 h cleaved both the tert-butyl ester and the Wang-linker in onestep providing the target compounds 79a and 79d, respectively.

In an attempt to introduce the solid phase earlier in the synthetic route compound 85was allowed to react with 4-formylbenzoic acid and then oxidized to the correspondingcarboxylic acid (87) (Scheme 22). Esterification of this carboxylic acid with 2,4-diamino-6-bromomethylquinazoline 34a was tried, but unfortunately this route lead to cleavage of theresin probably due to large amounts of hydrobromic acid present in the reaction. Neitherattempts using large excesses of base nor direct couplings of 2,4-diaminoquinazoline-6-methanol (33a) with the polymersupported benzoic acid derivative 87 were successful. Earlierexperience showed the alcohol in 33a to be a very poor nucleophile, which would explain thevery low yield in the coupling step according to 13C NMR spectroscopy. A later controlreaction was performed to introduce solid phase chemistry directly on TentaGel S OH withoutusing any attached linker. In this case compound 87 was allowed to react with 2,4-diamino-6-bromomethylpteridine hydrobromide (34d). Here, the esterification worked well with nocleavage of the linker according to 13C NMR spectroscopy.

Scheme 22

FmocHN COOH

CO2t-Bu

OHO

N

N X

XNH2

H2N

O

O

O

HN CO2

CO2t-Bu

50% TFA/CH2Cl2

H2N CO2

CO2t-Bu

N

N X

XNH2

H2N

O

O

O

HN CO2H

CO2H

+

ArgoGel ArgoGel incl. Wang

ArgoGel incl. Wang

Wang-linker +

79a: X = CH79d: X = N

1) DCI, 4-pyrrolidinopyridine, CH2Cl22) 25% piperidine, DMF

84a or 84d,

PyBOP, DIPEA,

DMF85

86a: X = CH86d: X = N

H2N CO2

CO2t-Bu

N

N

NH2

H2NHN CO2

CO2t-BuO

O

O

HN CO2

CO2t-BuO

HOOC

N

N

Br

NH2

H2N HBr

85

1) 4-formylbenzoic acid2) NaClO2, NaH2PO4 H2O 87

34a.

.


- 51 -51

Initially, we employed the photolabile linker, P-linker-2,220 with TentaGel S NH2 assolid support (Figure 28) as an acid/base stable linker. The P-linker-2 required deprotection ofthe tert-butyl ester prior to photolysis, which gave no advantages over the other linker.Furthermore, side-products and liberated polyethylene glycol (PEG) were observed afterphotolysis. Early attempts were also made to accomplish the attachment of the exocyclicamino groups of the 2,4-diaminoquinazolines to different linkers, for example, the photolabilelinker-5220 (Figure 28) but no reaction was observed indicating a very poor nucleophilicity ofthe amines.

Figure 28. a) P-linker-2.220 b) P-linker-5.220

6 BIOLOGICAL RESULTS

6.1 Enzymatic Hydrolysis In Vitro (Paper I)

The synthesized model esters 1-14 described in Section 5.1 were all selected for invitro assays for the estimation of the relative rates of hydrolysis in six different systemscontaining esterases. The systems consisted of pig liver esterase (PLE), cholesterol esterase,human duodenal mucosa, human liver, rat liver, and human leukocytes, respectively. PLE isregarded as a non-specific carboxyl esterase, which also is commercially available, cheap andeasy to handle. Cholesterol esterase, being localized in the intestinal lumen, is an enzyme towhich every drug taken orally is subjected. Human duodenal mucosa is one of the firstbarriers a drug has to penetrate after oral administration. Human and rat liver, respectively,were selected due to their high content of esterases. Finally, we also had the opportunity touse leukocytes from human blood, which are the potential targets for immunosuppressivedrugs. The results are presented in Table 1. To summarize, we conclude that PLE hydrolyzeall compounds, though at very different rates. The esters yielding naphthol or phenol afterhydrolysis were, not surprisingly, the esters most prone to hydrolysis. Carboxylic acids asthere anions are poorly accepted by the esterases since they are products from hydrolysis.35,38

(cf. 1-6 vs. 7-12) Moreover, esters with a high polarity seem to be poorer substrates for theesterases, which also is in accordance with literature.28 We confirm these results reporting thatthe 2,4-diaminoquinazoline esters 30a and 31a are poor substrates for the selected enzymes invitro.

PEGHN O

O OMe

O

NO2

O

ONO2

PEGHN O

O OMe

OH

NO2

BIOLOGICAL RESULTS - ENZYMATIC HYDROLYSIS IN VITRO

- 52 -52

Table 1. Rates of Hydrolysis in Six Different Biological Systems.

a The numbers within brackets are the relative rates of hydrolysis as compared to compound 1 for each system.

6.2 Inhibitory Activities of New Inhibitors of DHFR (Papers III, IV, V andVI)

In this project we first had to examine whether the ester moiety was accepted byDHFR or not. A preliminary experiment on bovine DHFR for compounds 30a and 31aencouraged us to design new inhibitors for further evaluation.

Compd No Pig LiverEsterase

CholesterolEsterase

HumanDuodenalMucosa

HumanLiver S9

Rat LiverS9

HumanLeukocyte

O

O

1 170

(100)

540

(100)

39

(100)

320

(100)

510

(100)

0.46

(100)

O

O2 100

(60)

240

(43)

26

(68)

170

(54)

170

(34)

0.14

(31)

O

O

3 120

(70)

60

(11)

4.6

(12)

69

(22)

150

(29)

0.11

(24)

O

O

4 150

(88)

1240

(227)

18

(46)

160

(51)

440

(86)

0.65

(141)

O

O

5 36

(23)

26

(4.7)

0.7

(1.8)

41

(13)

46

(8.9)

0.034

(7.3)

O

O

6 130

(78)

340

(61)

4.7

(12)

220

(68)

730

(143)

0.26

(56)

O

O

COOH

7 2.1

(1.2)n.q. 0.3

(0.6)

13

(3.9)

100

(20)

0.006

(1.3)

O

OCOOH

8 36

(21)

10

(1.8)

0.7

(1.7)

9.1

(2.8)

11

(2.1)

0.014

(3.1)

O

O

COOH

9 0.5

(0.3)n.q. n.q. n.q. 4.1

(0.9)n.q.

O

OCOOH

10 2.6

(4.0)n.q. 0.2

(1.1)

3.1

(1.2)

290

(59)

0.025

(8.0)

O

OCOOH

11 8.6

(13)n.q. n.q. 0.4

(0.2)

5.4

(1.1)

0.003

(1.0)

O

O

COOH

12 11

(6.4)

13

(2.3)

0.5

(1.2)

11

(3.4)

27

(5.1)

0.12

(37)

N

N

O

ONH2

NH2

30a 0.1

(0.1)n.q. n.q. n.q. 2.5

(0.4)n.q.

N

N

O

ONH2

NH2

31a 4.6

(6.9)n.q. 0.6

(1.5)

2.4

(0.8)

2.9

(0.6)n.q.

BIOLOGICAL RESULTS - INHIBITORY ACTIVITIES OF NEW INHIBITORS OF DHFR

- 53 -53

6.2.1 Inhibitory Activities and Selectivities of New Lipophilic Soft Drug Inhibitors forpcDHFR (Papers III and IV)

Table 2. The IC50-values for a Series of New Lipophilic DHFR-inhibitors Together with the Ratios of Selectivity(rlDHFR/pcDHFR).a

a The method for the inhibitory assay is described elsewhere.110,221

N

N

Y

XNH2

H2N

O Ar

O

IC50 (µµµµM)Compd X Y Ar pcDHFR rlDHFR rlDHFR/pcDHFR

31a CH CH Ph 0.60 0.39 0.64

31b CH N Ph 5.5 3.9 0.72

31c CCH3 N Ph 0.26 0.23 0.91

31d N N Ph 9.8 7.0 0.72

35a CH CH 2’,5’-(OCH3)2Ph 2.4 0.42 0.17

36a CH CH 3’,4’-(OCH3)2Ph 0.51 0.11 0.21

37a CH CH 3’,5’-(OCH3)2Ph 0.46 0.35 0.76

38a CH CH 2’,3’,4’-(OCH3)2Ph 1.4 0.5 0.37

39a CH CH 3’,4’,5’-(OCH3)3Ph 0.56 0.026 0.05

40a CH CH 3’,5’-(CH3)2Ph 0.52 0.19 0.37

41a CH CH 1-naphthyl 0.11 0.093 0.85

42a CH CH 2-naphthyl 0.85 0.43 0.51

43a CH CH 9-anthryl 0.49 0.21 0.43

44a CH CH 9-phenanthryl 0.49 0.67 1.37

45a CH CH 9-fluorenyl 0.22 0.032 0.15

46a CH CH 2’-benzylphenyl 1.4 0.13 0.09

47a CH CH 2’-biphenyl 0.69 0.11 0.16

57a CH CH 3’-biphenyl 0.38 0.11 0.29

49a CH CH 3’-iodophenyl 0.48 0.17 0.35

58a CH CH 3-[2’,6’-(OCH3)2]Ph 1.6 0.73 0.46

48a CH CH 4’-biphenyl 11 19 1.73

50a CH CH CH(Ph)2 5.8 4 0.69

51a CH CH 2-thienyl 0.27 0.25 0.93

52a CH CH 3-thienyl 1.5 0.35 0.23

53a CH CH 2-furyl 4.3 0.78 0.18

54a CH CH 3-furyl 0.98 0.52 0.53

55a CH CH benzyl 11.2 2.34 0.21

TMQ 0.042 0.008 0.19

PTX 0.034 0.044 0.13

TMP 27 121 4.5


- 54 -54

Even though extensive research with crystallographic experiments in combinationwith computer modeling has been performed, very few new lipophilic inhibitors have beenreported where selectivity has been established for pcDHFR in favor of the human enzyme(Section 3.2.1). In paper III we wanted to design a series of lipophilic esters with the aim ofexamining a) whether the inhibitory activity of DHFR remained after the introduction of theester function; b) if we could obtain a selectivity for the fungal enzyme. Moreover, we wantedto explore c) the importance of the nitrogen substitution in the right heterocyclic ring for thepharmacophoric element. All the results from the inhibition experiments are presented inTable 2. First, methoxy groups were introduced in different positions in the original phenylmoiety (35a-39a), capable of accepting hydrogen bonds from amino acid residues in theactive site of the enzyme. The compounds showed structural resemblance to the new potentantifolates TMQ and PTX (Figure 11 and Figure 12, respectively). All the compoundsexhibited fair to good inhibitory activities against the enzymes, but most of all with apreference for rlDHFR. Replacing the methoxy substituents for methyl groups gave noobserved difference in potency (cf. 37a and 40a). When examining the difference in theheterocyclic core (31a-d), compound 31c was found to be the best inhibitor within the seriesand was also associated with the most favorable selectivity. However, the metabolite 33cformed after ester hydrolysis proved to be too potent as an inhibitor of the enzyme (Table 3).All other metabolites were in the micromolar range. After having concluded that the estermoiety was well accepted by DHFR together with the poor inhibitory activity exhibited by themetabolite 33a, we chose the 2,4-diaminoquinazolines. In paper IV we wanted to exploredifferent scaffolds in the right part of the molecule comprising larger groups, as compared tothe phenyl moiety, as well as different heteroaromatics. The substituents in compounds 46a-47a and 50a were chosen because they are privilege structures in already existing drugmolecules.222 Among the lipophilic ester-containing compounds, the 1-naphthyl ester 41a wasfound to be the most potent inhibitor of pcDHFR. The fluorenyl compound (45a) exhibitedthe highest potency of all the inhibitors against the mammalian enzyme, even higher then thereference compounds 65a and 66a (Table 3). The phenantrene analogue 43a displayed thebest combination of selectivity (1.4) and inhibitory activity. The 4’-biphenyl (48a)demonstrated the best selectivity ratio within the series. Unfortunately, the compound wasassociated with a relatively low potency. The 2-thienyl scaffold (51a) exhibited a reasonablygood selectivity (0.93) along with rather high potency, which makes this type of compoundsinteresting for further investigations. Going up higher in length of the middle bridge thepotency dropped (50a, 55a and 64a).

Based on these results it is difficult to draw any conclusions in terms of selectivity.The fungal enzyme seems to be less tolerant of the more flexible and bulky analogues than themammalian enzyme (46a and 58a). However, the trend seems quite obvious: if one inhibitorshows a good inhibitory activity against one of the enzymes it also follows the same patternfor the other enzyme.


- 55 -55

6.2.2 Inhibitory Activities of New Classical Inhibitors of Mammalian DHFR (Papers V andVI)

Table 3. The IC50-values for the Metabolites 32a, 33a-d, and Some Miscellaneous Compounds.

In papers V and VI we present the IC50-values of a small series of new ester-containing ‘classical’ antifolates on rat liver DHFR as well as on recombinant human DHFR.It is interesting to note the very good inhibitory activity exhibited by compound 79a (19 nM).This new potent antifolate was only eight times less potent then the reference compoundMTX, and compound 79a was therefore further evaluated in vivo (Section 6.3 and 6.4). Theother compounds in the group displayed a 10-fold decrease in potency of rlDHFR. Thus, weconclude that the ester functionality, with an additional atom in the bridge as compared toMTX, does not seem to interfere too much with the inhibitory activity.

Table 4. Inhibitory Concentrations (IC50) of Compounds 79a-81a and 79d Against Rat Liver and RecombinantHuman DHFR.

IC50 (µµµµM)

Compd pcDHFR rlDHFR rlDHFR/pcDHFR

32a >32 >32 ---

33a 52 40 ---

33b >104 >104 ---

33c 10.5 8.5 ---

33d >51 >51 ---

30a 2.5 0.42 0.17

64a 2.1 1.3 0.6

84a 1.0 1.1 1.1

65a 0.98 0.44 0.45

66a 0.096 0.069 0.72

IC50 (µµµµM)

Compd rlDHFR rec. hDHFR

79a 0.019 0.1280a 0.15 0.078

81a 0.16 0.14

79d 0.12 2.1

MTX 0.0025 0.002

BIOLOGICAL RESULTS - EFFECT OF A NEW POTENT INHIBITOR OF DHFR IN A MOUSE COLITIS MODEL

- 56 -56

6.3 Effect of a New Potent Inhibitor of DHFR in a Mouse Colitis Model InVivo (Paper V)

For this project we wished to evaluate compound 79a in an experimental model ofIBD. The compound is structurally similar to MTX, which is used in the treatment of IBD dueto its anti-inflammatory and immunosuppressive properties (Section 1.6.2). However, thecentral part of the structure had been changed to an ester functionality, which makes it asubstrate for controlled metabolism by the endogenous esterases. As described in Section 6.1and also depicted in Table 1, two lipophilic inhibitors of DHFR was evaluated in vitro at anearly stage of the project for their susceptibility to the esterases in human leukocytes andhuman duodenal mucosa (compound 30a and 31a). Both compounds were shown to be stablein the system used. Encouraged by the pharmacokinetics in rat in vivo (Section 8) togetherwith the in vitro pharmacokinetics of compound 79a in blood (Section 8), we wanted toelucidate whether compound 79a could have bioactivity in a mouse colitis model in vivo.

In the study we used the chemically-induced DNBS-model (2,4-dinitrobenzenesulfonic acid), a model that is inexpensive and easy to develop. The model is a milder variantof the well-known TNBS-model (2,4,6-trinitrobenzene sulfonic acid).223,224 It is extensivelyused as a screening tool for potential IBD therapies and their mechanism of action. Anotherwell-established chemically-induced model is the DSS-model (dextran sodium sulfate). Yet,the last model suffers from the lack of rapidly proliferating cells (monocytes andmacrophages, compared to leukocytes in the case of DNBS). Additionally, the DNBS modelshows a ‘Crohn’s like’ pathogenesis in which MTX and budesonide, respectively, tend toshow better results as compared to the DSS-model.

Figure 29. The effects of compound 79a, MTX and budesonide in a mouse colitis model assessed by threeindependent parameters.

0

20

40

60

80

100

120

140

Budesonide, 0.1 µmol/kg MTX, 0.5 mg/kg Compd 79a, 60 mg/kg Compd 79a, 15 mg/kg

% o

f Con

trol

s Colon weight

MPO

IS

BIOLOGICAL RESULTS - EFFECT OF A NEW POTENT INHIBITOR OF DHFR IN A MOUSE COLITIS MODEL

- 57 -57

The results illustrate that daily administration of compound 79a (15 mg/kg/day)reduces mucosal damage (colon weight) and myeloperoxidase (MPO) activity (a marker ofneutrophil infiltration in the gut), as shown in Figure 29. Elevated levels of MPO arerecognized in activated immunocells whereas healthy animals show zero-values. The obtaineddata shows lower concentrations of MPO in treatment of 79a (15 mg/kg/day) as compared tobudesonide, and lower concentrations still compared to MTX. Encouragingly, also theinflammation score (IS) favors the new analogue. However, at greater concentrations of 79a(60 mg/kg/day), the animal suffered from side-effects, such as nausea. An interestingobservation with the mice treated with 79a (15 mg/kg/day) was that the animals seemed to bevigorous, eating and drinking as normal. Additionally, they appeared to be in very goodcondition, which was not the case with the mice treated with budesonide and MTX.Regarding the ‘negative’ effects of the higher dose of 79a, we propose that it might in thishigh dose, interfere with the regeneration of the epithelium cells in the intestinal mucosa, andthereby the healing process. In contrast to MTX, compound 79a did not inhibit thecellproliferation from mouse spleen, suggesting different pharmacodynamics of the twocompounds.

6.4 Evaluation of a New Potent DHFR Inhibitor in a Rat Arthritis Model InVivo (Paper VI)

In addition to the evaluation of compound 79a in the mouse colitis model (paper V) itwas also examined for its anti-arthritic properties in a rat arthritis model. Evidently, MTX isone of a very few drugs with anti-arthritic properties that work in both animal models and inthe clinic. The models thus offer suitable in vivo systems for evaluation of new analogues toMTX. The antigen-induced arthritis model (AIA) in rat225,226 described in paper VI, ischaracterized by a high reproducibility, which permits easy quantification of the response.124

The AIA model does resemble the human disease in several respects. In this model DarkAgouti strain animals are sensitized to methylated bovine serum albumin (mBSA). Forsensitization mBSA is injected with Freund’s complete adjuvant (Sigma Chemicals Co.). Themodel is sensitive to oral tolerance, that is, when giving the anti-arthritic compound by oraladministration the arthritis is inhibited, indicating a possible connection between the mucosallevel in the gut and the antigen-presenting cells in the joint.227 AIA has previously beenshown to be an MTX-sensitive model, although the effective dose is about ten times greater inrats and the onset of the anti-arthritic effect of MTX is shorter.124 An alternative to AIA is thewell-established adjuvant arthritis model in rat228 together with collagen-induced arthritis.However, the first method is regarded as an aggressive method and other effects causes boneerosion, which is generally not the case with RA.225 The latter method seems to be limited toonly a few anti-arthritic compounds.229 However, it has proven to be MTX-sensitive, but ismostly associated with a high variability.

When we performed a pharmacokinetic study of compound 79a in the rat the dataobtained revealed that it was quite stable in plasma (Section 8). Thereby it could serve as a

BIOLOGICAL RESULTS - EVALUATION OF A NEW POTENT DHFR INHIBITOR IN A RAT ARTHRITIS MODEL

- 58 -58

potential candidate for the arthritis assay. The results from the AIA model show that MTXwas at least as effective following both p.o. and i.p. administration supporting previoustheories about the action of MTX on immunocompetent cells in the mucosa.230,231 Althoughcompound 79a was given in doses 25 times greater than the effective dose of MTX, no effectof the new compound could be detected. The negative results could be a) due to lack ofpolyglutamation, which seems to be necessary for the effect of MTX, and/or b) that the newcompound does not function as a substrate for the same enzymes as MTX (with the exceptionof DHFR). Whether compound 79a is a substrate for other enzymes in the folic acid pathway(see Section 1.4) remains an uninvestigated topic.

7 MOLECULAR DYNAMICS (PAPERS II-IV AND VI)

Due to time-consuming work in the lab and thus high costs, prediction of the affinityof a drug for a specific enzyme or receptor can be of a great value in the drug discoveryprocess (Figure 1). A suitable drug should exhibit a high affinity to its target enzyme/receptorand at the same time should act only at the target enzyme and thereby be selective (seeSection 3.2). In addition, selectivity is important when looking at different species, which isthe case with antibacterial drugs, for example. Nowadays, computational methods can beemployed to investigate these issues and serve as important tools in drug research. Advancesin molecular biology and improved methods for the crystallization of proteins providedknowledge about the three dimensional structure of specific proteins. Hence, structure-basedmethods rely on good models of the receptor-ligand structure, with the protein database (pdb)serving as a major source for crystallographic data. However, even when the three-dimensional structure of the target molecule is known, computational predictions with a newseries of ligands can be difficult to perform, due to a lack of knowledge about the exactstructure of the ligand-enzyme complex. Several methods have been developed in order toapproach this problem.232-234 As mentioned earlier (Section 1.3) DHFR is an enzyme, whichhas been subjected to extensive characterization because of its relatively small size and easeof co-crystallization with new ligands. In this project, we have performed binding affinitypredictions based on existing crystal structures of DHFR in complex with new potentialinhibitors.

By molecular simulation methods, such as, molecular dynamics (MD), one can sampleavailable conformations of the ligand and the receptor at a desired temperature and therebycalculate energy averages and thermodynamic properties. Free energy perturbation (FEP)235

techniques in combination with thermal averaging using MD allows us to calculate relativebinding free energies between structurally related compounds, that is, the difference in ∆Gbind

between pairs of ligands. The free energy perturbation method is time consuming because itrequires conformational sampling of hybrid molecules during gradual perturbation from onemolecule to the other. However, it is often possible to obtain reliable relative affinities that

MOLECULAR DYNAMICS

- 59 -59

include the effects of solvation and conformational changes. Due to the fact that FEP is acomputationally demanding method an alternative method called the linear interaction energy(LIE) method has been developed,236 in which the absolute binding free energies can beestimated. LIE calculates the binding free energy as a linear function of the differencebetween the “bound” and “free” ligand from the average force field ligand-surroundinginteraction energies obtained by MD simulations (Figure 30). The method is similar to FEP inthat it relies on thermodynamic cycles and includes thermal conformational sampling. LIE isclassified as a semi-empirical method, and is regarded as a less computationally demandingmethod as compared to FEP since it involves only simulations of two physical states (boundand free ligand) and thereby no transformation processes are needed in the calculations. Theidea behind the method is based on identifying each important receptor-ligand interaction(both electrostatic and non-polar) in a given conformation of the complex with subsequentquantification of its contribution to the total binding.

Figure 30.

MTX consists of three major components: the pteridine ring, the p-aminobenzoylgroup and the glutamic acid residue (Figure 10). The pteridine has been found to be deeplyburied in the active site of the enzyme237 and forms more than half of the hydrogen bondswith DHFR. The conformation of the glutamate residue is determined by electrostaticinteractions of mainly the γ-carboxylate. It is a challenging molecule due to three ionic sites,but also because of its flexibility. In paper II we studied MTX using the LIE approach. Thecalculated results were compared to three ‘non-classical’ DHFR-inhibitors. We also tried topredict the absolute affinities of MTX and the quinazoline analogues 65a-66a (Scheme 16)together with an analogous pteridine derivative. The simulations of MTX exhibited very largefluctuations due to its flexible glutamic acid residue, particularly in the free state, leading todifficulties in obtaining average values with good precision. It seemed to be difficult to handleligands with multiple ionic groups, particularly if they were located at the ends of flexiblechains, as in this case. The energies for the ‘non-classical’ inhibitors converged much morerapidly than for MTX. Calculations on MTX also revealed that the pKa of N1 is about 5.7,which means that it is not protonated in solution at neutral pH, although it is known to beprotonated in the complex with DHFR.238 Energy is thus required for protonation of thepteridines when binding to the enzyme, while the corresponding quinazolines are protonatedat the appropriate pH of the solution used in the predictions. Surprisingly, the result alsoshowed very small differences between the predicted binding affinities of compound 66a andthe corresponding pteridine compound. In all simulations the bound ligands were protonated.A larger difference in binding affinities was found to come from the N-methyl group in thelinker chain between the heterocyclic group and the phenyl ring (65a vs. 66a). A comparisonof the quinazoline compounds with and without this methyl group yielded a 2.3 kcal/mol

inhibitor - enzyme(aq)inhibitor + enzyme(aq)∆Gbind

MOLECULAR DYNAMICS

- 60 -60

difference in favor of the N-methylated inhibitor. The methyl group binds in a hydrophobicpocket and this appears to reduce the flexibility of other parts of the inhibitor, thus favoringbinding. The results also show that the compounds lacking the glutamic acid residue arepredicted to exhibit a 105-106-fold lower affinity to the human enzyme. All simulations wereperformed with the co-factor NADPH present.

In paper III and IV it was intended to predict the individual selectivities of a series ofsimilar inhibitors towards DHFR from rat liver and P. carinii, respectively, and furthercompare them to experimentally obtained results. Sequence analysis and structural alignmentof pcDHFR and hDHFR were performed in paper III in order to compare the active sites ofthe two species. The results show significant differences in only a few amino acid residuesclose to the active site between the two species, which also is consistent withliterature.141,144,157,239,240 Val115 in human DHFR corresponds to Ile123 in pcDHFR, whichcontributes to a slightly larger binding pocket in the 5-position of the human enzyme. Whencomparing the structures of the enzymes in the region where the phenyl ring is positioned,three amino acid residues can be identified that differ between the two species; Asp21, Phe31and Asn64 in the human enzyme correspond to Ser24, Ile33, and Phe69 in P. carinii,respectively (Figure 31).

Figure 31. Compound 39a modelled into DHFR of both P. carinii (green) and the human enzyme (red). Thepicture shows the important interactions with the amino acids that differ between the enzymes.

As deduced by the experimental data from the mammalian enzyme (rlDHFR, Table 2)and also from Figure 31 the greater selectivity towards hDHFR for compound 39a may beattributed to a hydrogen bond formed between one of the methoxy-groups on the phenyl ringand the amide hydrogen of Asn64. Additionally, a simulation of compound 39a in complexwith the human Asn64Phe mutant was performed in order to investigate the importance ofAsn64. The calculations indicate that this interaction is important for the affinity, but may not

N

N

NH2

H2N

O

OOCH3

OCH3OCH339a

Ser24Asp21

Phe69Asn64

Phe31

Glu30

Val115 Ile123

Ile33

Glu32

MOLECULAR DYNAMICS

- 61 -61

be the only explanation for the observed selectivity for the human enzyme. In hDHFR Phe31may contribute to stronger binding by ring stacking with the aromatic group of the inhibitor,an interaction which cannot be accomplished by the corresponding Ile33 in pcDHFR.

In paper IV we wished to explore the tolerance of the enzymes from two differentspecies to more bulky, lipophilic groups in different orientations, with varying flexibility. Wedescribed the individual rankings within a new series of compounds comprising more bulky,lipophilic substituents. The ranking of the different compounds is based on a true empiricalmethod for docking and scoring. The method relies on intermolecular interactions betweenthe ligand and the rigid protein structure to find the optimal geometry for the ligand in theactive site. The method then “scores” the strength of the interaction between the protein andthe ligand in that binding conformation. The scoring function includes both polar and non-polar interactions, for example, hydrogen bonds, ionic interactions, hydrophobic, andaromatic interactions. Herein, a scoring function was used to determine the rank order of theselectivity indexes, that is, to calculate and compare the score obtained for a ligand whendocked into DHFR from both human and P. carinii. The obtained scores did not differsignificantly, which should not be surprising when comparing the IC50-values in Table 2.Also, MD simulations were performed for a set of compounds. However, the differences ininhibitory activities and selectivity were very small for these compounds, which made itdifficult to draw clear conclusions. Nevertheless, we can summarize that the experimentallyobtained IC50-values, in general with more selectivity against the mammalian enzyme, areconsistent with the computationally obtained results.

Based on the results in paper II, we went further with the FEP method in paper VI forthe calculations of the relative binding free energies of new ‘classical’ antifolates compared toMTX. The results from the FEP calculations with the 2,4-diaminoquinazoline compound 79apredicted the compound to have only a 10-fold lower affinity than MTX for the humanenzyme. The calculated Ki-values of the inhibitors were compared with experimental dataobtained from an inhibition assay performed in-house using recombinant human DHFR. Theresults obtained confirmed the calculated ranking of their inhibitory potencies, but showed alarger difference between MTX and compound 79d (the pteridine derivative most structurallysimilar to MTX) than predicted. However, additional experiments on rat liver DHFRperformed by Queener∗ showed IC50-values that were more consistent with the calculatedratios. The explanation for the poor IC50-value for compound 79d, could be explained by thedemand for energy upon protonation before binding to the enzyme (vide supra). The bindingof compound 79a and MTX into the active site of human DHFR is illustrated in Figure 32.

∗ Dept. of Pharmacology and Toxicology, School of Medicine, Indiana University, USA.

MOLECULAR DYNAMICS

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Figure 32. Compound 79a (yellow) and MTX (green) docked into the active site of the human enzyme illustratingimportant interactions with neighbouring amino acid residues. Hydrogen bonds are indicated in gray. The

quinazoline superimposes well on the pteridine moiety from MTX.The ester carbonyl is oriented opposite to N-CH3.

8 PHARMACOKINETICS (PAPERS III AND VI)

The word pharmacokinetics is defined as ‘the science which describes quantitativelythe uptake of drugs by the body, their biotransformation, distribution, and elimination fromthe body’. Some of the important parameters are described below:

1) Absorption of a drug can be defined as the passage from its site of administrationinto the circulation. Drugs can be administered by enteral or parenteral route. With parenteraladministration, e.g., intravenous or intramuscular routes, the gastrointestinal tracts arebypassed. The enteral route occurs through swallowing (oral) or by rectal administration.After absorption from the gastrointestinal tract, the drug has to pass the liver before gainingaccess to the systemic circulation. The bioavailability of a drug is the fraction of theadministered dose, which reaches the systemic circulation. After intravenous administration,the bioavailability is by definition 100%; for the other routes of administration, thebioavailability varies between zero and 100%. Bioavailability is influenced by several factors,such as drug characteristics (e.g., solubility, particle size, logD), formulation, changes at thesite of administration (e.g., pH, blood flow), and the route of administration. Both thegastrointestinal tract wall and the liver are capable of metabolizing drugs. This ‘first passeffect’, can result in an appreciable fraction of the dose not reaching the systemic circulation.Drugs that undergo extensive first pass metabolism often have low and variablebioavailability. Another route of administration is inhalation, where the treatment can be both

PHARMACOKINETICS

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for local and systemic use. It shows a rapid absorption, but the total dose absorbed is usuallyvariable due to concurrent swallowing of the drug.

2) Distribution primarily determines the early rapid decline in plasma concentration.Following an intravenous bolus dose, the initial decline in the blood concentration vs. timecurve usually represents distribution to extravascular tissues.241

3) The half-life, T½, of a drug is characterized by two independent pharmacokineticparameters – a) the clearance of the drug and b) the volume of distribution. In essence, the T½

can be measured by following the plasma concentration of the drug in the body and taking thetime point at which the concentration falls by one-half. Elimination of a drug covers bothexcretion (i.e., elimination of unchanged drug from the body), and biotransformation(metabolism). Renal excretion and hepatic biotransformation are the major routes of drugelimination and the primary pharmacokinetic parameter describing this elimination isclearance. Clearance is defined as the volume of blood or plasma, which is cleared of the drugper unit of time, most often expressed as mL min-1. For example the hepatic clearance of adrug is defined as the volume of blood perfused in the liver that is cleared of the drug per unitof time. Consequently, the hepatic clearance can never be greater than the hepatic blood flow.b) The volume of distribution gives information about whether the drug is located mainly inthe plasma/ blood or predominantly distributed into extravascular tissues. Thus it depends onplasma protein binding, binding in tissues, partition into fatty tissues etc.

Although in Paper I we concluded that the 2,4-diaminoquinazoline esters 30a and 31a,exhibited slow rates of hydrolysis in vitro (Table 1) and were thus poor as soft drugs, wedecided to substitute into the phenyl ring to enable additional investigations in an in vivoscreening assay. In paper III and VI we evaluated the in vivo pharmacokinetics after oral andintravenous administration in male SD rats of the even more lipophilic ester 39a and its polaranalogue 79a, respectively. The intravenous dose was given as a bolus dose into a tail vein.Blood was collected by repeated samplings from the tail vein opposite to the administrationsite. Plasma was analyzed for content of the compounds, as well as their metabolites formedafter enzymatic hydrolysis, using the LC-MS/MS technique. We found the methotrexateanalogue 79a to be relatively inert in vivo displaying similar kinetics to MTX afterintravenous administration. The clearance was estimated to be 15 mL/min/kg (MTX: 11mL/min/kg) and the terminal T½ to be 1h (MTX: 0.9 h). The volume of distribution at steadystate was 0.42 L/kg (MTX: 0.36 L/kg) and the oral bioavailability was calculated to be 3.4%(MTX: 5.8%) revealing a poor absorption from the gastrointestinal tract. As deduced from theresults, we conclude that compound 79a has pharmacokinetics similar to MTX. The obtaineddata prompted us to believe that compound 79a is sufficiently metabolically stable in vivo toretain the desired bioactivity.

PHARMACOKINETICS

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Figure 33. The pharmacokinetics of 39a calculated by fitting a two-compartment model to the meanconcentration vs. time data by non-linear regression analysis.∗∗∗∗

In contrast to 79a, the data obtained from the more lipophilic analogue 39ademonstrates much faster pharmacokinetics, which is also more in accordance with the softdrug concept (Figure 33). The mean concentration versus time data of compound 39a wasfitted to an intravenous two-compartment model. This model estimated the initial (i.e.,distribution) T½ to be 1.2 min and the terminal T½ to be 25 min. Both phases were predicted tocontribute, each with 50% of the total area under the curve. However, after longer infusiontimes with 39a the terminal post-infusion phase will become the dominating phase. The verydegree of clearance, approximately 200 mL/min/kg, is in close proximity to the cardiac outputfor the rat242 reflecting a high propensity of the drug to undergo a fast metabolism in vivo. Thehigh clearance could be explained by extensive extra-hepatic metabolism, which is inaccordance with the high esterase activity in different tissues in addition to the liver.19 It isimportant to mention in this context, compounds 39a and 79a also have been evaluated in ratblood in vitro in a pilot study. In this study, no hydrolysis was detected after 45 min. Theexperiments were run both in the presence of, and in the absence of a non-specific esteraseinhibitor (phenylmethylsulfonyl fluoride, PMSF).

When the animals were subjected to an oral dose of compound 39a we were unable todetect any traces of the ester in plasma two minutes. However, the metabolite 33a wasdetected immediately, following both p.o. and i.p. administration. Based on these findings, wewish to iterate that compound 39a is a potentially soft drug candidate, used for topicalapplication. ∗ The analyses were performed with the computer program WinNonlin* (ver. 1.5, Pharsight Corporation) usingthe weight 1/(ypred2). The goodness of fit was evaluated by the Akaike Information Criterion (AIC) and visualinspection of residual plots.

1

10

100

1000

10000

0 20 40 60 80 100 120

Time (min)Time (min)

Con

cent

ratio

n(n

mol

/L)

CONCLUDING REMARKS

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9 CONCLUDING REMARKS

The aim of this project was to identify and synthesize new soft drug analogues ofdihydrofolate inhibitors. In this thesis the design, synthesis, MD, and ultimately, thebiological evaluation of the new inhibitors have been described. The results are summarizedbelow:

• Fourteen aromatic model esters were evaluated as esterase substrates in vitro, inesterase systems comprising commercially available esterases and cell/ tissue systems,for example, human leukocytes and human intestinal mucosa homogenates. Asexpected, the lipophilic esters were good substrates for the esterases. Incorporatingmore polar features into the molecules made the compounds poor substrates for theesterases. No clear rank order of the different esters in each system was apparent.

• One of the ester functions was selected and incorporated between a 2,4-diaminoquinazoline and a phenyl moiety. The new ester analogue exhibited goodinhibitory activity towards DHFR. We thus demonstrated that the new antifolates,comprising an ester group in the bridge region, are well tolerated by DHFR.Additionally, the metabolites formed after hydrolysis by the esterases, were all poorinhibitors of the mammalian enzyme, which is a pre-requisite for the soft drugconcept.

• One of the esters (compound 39a) with structurally similarity to TMQ, used in thetreatment of PCP, was evaluated for its pharmacokinetic properties in vivo in the rat.The selected ester displayed fast elimination, with short plasma half-life and highplasma clearance. This suggests that soft drug analogues of antifolates may beobtainable. These analogues are intended for topical administration in animal model ofPCP.

• In spite of the fact that we used molecular modeling in the design process, we werenot successful in the identification of new inhibitors with selectivity for P. cariniiDHFR. The compounds were also potent inhibitors of the mammalian DHFR.

• We have synthesized a new ester analogue (compound 79a), structurally similar to the‘classical’ antifolate MTX. The ester was only eight times less potent than MTX with

N

N

NH2

H2N

O

OOCH3

OCH3OCH339a

CONCLUDING REMARKS

- 66 -

respect to rat liver DHFR. The compound was stable towards enzymatic hydrolysis asdeduced by the relatively long plasma half-life of the compound (T½: 0.9 h) and wasused for topical treatment in an experimental model of IBD. The compound exhibitedgood anti-inflammatory properties in a colitis model in mice, with even better resultsthan with established drugs. Compound 79a was further evaluated in an arthriticmodel in rat, but with unsatisfactory results.

10 SUMMARY

I. Retained inhibitory activity of DHFR was achieved after replacement of themethyleneamino-bridge (common amongst the antifolates) with an ester-containing bridge.

II. Ester analogues of DHFR inhibitors, which exhibit either fast or slowhydrolysis can be prepared by appropriate structural modifications.

III. One ester analogue of DHFR inhibitors showed a favorable pharmacokineticprofile in experimental models.

IV. One ester analogue of DHFR inhibitors exerted an anti-inflammatory effect inan experimental colitis model.

HN COOH

O COOH79a

N

N

NH2

H2N

O

O

ACKNOWLEDGEMENTS

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11 ACKNOWLEDGEMENTS

This work was carried out at Organic Pharmaceutical Chemistry, Department ofMedicinal Chemistry, Faculty of Pharmacy, Uppsala University.

The Swedish Academy of Pharmaceutical Science is acknowledged for allowing me to attendinternational scientific meetings and national courses. Acta Chemica Scandinavica for makingit possible to accompany national conferences. Financial support was obtained from theSwedish Foundation for Strategic Research (SSF).

I would like to express my deepest thanks to all friends and colleagues who have been therefor me during the years:

Professor Anders Hallberg, my supervisor and friend, for your support and never-endingencouragement. For enthusiastic guidance through research and your strong belief in science.Also for spreading such a spontaneous and free atmosphere at the department. ☺

Professor Uli Hacksell, for your early support when I applied for working my first summer atthe department.

Docent Anette M. Johansson, my former co-advisor, for friendly and inspiring guidance andfor constructive criticism of the thesis. Thank you also for always listening and being therewhen I needed in the very beginning of the project.

Professor Kristina Luthman for your strong enthusiasm in the teaching context. Also, for nicediscussions during the years.

Docent Uno Svensson, for your inherent expertise in medicinal chemistry and also yourpatience with all the amount of teaching.

Docent Anders Tunek for your belief in the ideas of soft drugs in the early stages of theproject.

My co-authors Drs Björn Mellgård, Sven E. Andersson, Göte Swedberg, and also ÅsaPersson, Sofie Ohlson, and Karin Sjödin.

Professor Johan Åqvist, Dr John Marelius and Karin Kolmodin for good collaboration and forbeing patient with my weak understanding in computational chemistry.

Professor Sherry F. Queener for generously providing us with pharmacological dataconcerning the inhibitory activities of dihydrofolate reductase.

Docents Eva Åkerblom and Gunnar Lindeberg for introducing me to the field of solid phasechemistry.

Dr Hans Ericsson for valuable discussions within the field of pharmacokinetics.

Sorin Srbu, for struggling with our computer network. Keep on going!

Arne Andersson for excellent technical assistance and expertise, and for always being therewhen you are needed. Good luck in the future! Lotta Wahlberg for doing an excellent job andfor nice discussions. Marianne Åström for skillful secretarial assistance and for always beingwilling to help. Karin Olsson for excellent cleaning of all the glassware. The staff at the BMClibrary for all help with articles during the years.

Drs Johan Hultén and Mats Larhed, for constructive criticism of the thesis.

ACKNOWLEDGEMENTS

- 68 -

Dr Nick Bonham, for constructive criticism and excellent linguistic revision of the thesis. Alllinguistically mistakes are my own last desperate changes and are definitely not hisresponsibility. Dr Marc Roddis for your help with the writing of the abstract for the thesis.

Dr Susanna Lindman, Anja Johansson, Karl Vallin and Anna Karlsson, and also, Drs LenaRipa, Annika Jenmalm-Jensen, and Charlotta Liljebris for nice discussions concerningeverything. Thank you also Anja for your constructive comments of the thesis.

My latest co-workers in the lab for pleasant times, especially Dr Mathias Alterman and PetraJohannesson.

The former members of B5:305a, especially Drs Martin Hedberg, Ulf Appelberg, BeritBacklund-Höök, Patrizia Caldirola, Johan Hultén, Neeraj Garg, Nick Bonham, and TeroLinnanen.

Erika Larsson, Katarina Sandberg and Matts Balgård, for contributing with skilful technicalassistance and for your patience with the molecules during summers and diploma work.

The former and present members at the department for making the time I have spent at BMCvery enjoyable.

The students that I have been teaching during the years, for making graduate work even moreenjoyable and for keeping up the theory of basic organic chemistry.

All my friends outside BMC, for good times.

---

Alla som har varit deltagande i barnpassning genom åren. Ett speciellt tack till familjenGrüwenz och även familjen Wulff. Tack även till personalen på Pilfinkens ochLingonbackens montessoriförskolor.

Mina svärföräldrar; Anita och Bosse för att ni har försökt vara förstående under den långatiden som doktorand. Ett speciellt tack till dig Anita, för all hjälp det sista halvåret medbarnen. Till Elsa för att du alltid ser livet med sådan glädje.

Mormor, nu skulle du ha varit stolt över mig!

Min familj; mamma och pappa, för att ni alltid har stöttat mig i mina egna beslut genom åren.Tack för att Ni alltid har gjort vad Ni har tyckt varit bäst för Er själva genom livet med flyttar,jobb och dyl. Av det har jag lärt mig mycket och Ni är mina förebilder i många lägen. TackJocke och Otto, mina bröder, för att Ni har uppfostrat mig som bara bröder kan göra.

Tack älskade Cecilia och Albert, mina underbara barn. Att få känna sig så omtyckt som jagfår av Er är något oerhört stort! Ja Cecilia, nu är “molekylboken” färdig och “profesor Halbej”är nog ganska nöjd. Nu kan jag äntligen sy din fjärilsdräkt (om det fortfarande är aktuellt)!

Till sist: Tack älskade Micke, min bästa vän och oändliga följeslagare. Vad vore allt utan dig,din tro på mig och ditt tålamod (speciellt den sista tiden)? Nu ska vi väl äntligen få chansenatt “komma igång” igen?!

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Approaches to Soft Drug Analogues of Dihydrofolate ReductaseInhibitors. Design and Synthesis.

by

Malin Graffner Nordberg

Corrections

Page Location Error CorrectionSummary 5 List of Papers, Paper III J. Med. Chem., accepted after

minor revisionJ. Med. Chem., accepted

14 Line 15 from top …charge… …partial charge…16 Line 1 from top Folic acid (FA,) Folic acid (FA)16 Line 2 from bottom is used by MTX and as… is used by MTX as…17 Figure 9

NH

COOH

O COOH

R =NH

COOH

O COOH

R =

18 Line 12 from top concentration ofpolyglutamated of MTXGN

concentration of MTXGN

21 Line 9 from top in this context is that in this context that23 Line 13 from bottom sustained a sustained sustained35 Figure 26 See Figure 26 below36 Line 10 from bottom Also, the synthesis of 6… Also, the synthesis of 4…40 Scheme 7, second arrow 75% formic acid, reflux 75% formic acid, Ni-Al alloy,

reflux

45 Scheme 14, first arrow 1. C6H5N 1. C5H5N46 Scheme 16, second arrow 30% formaldehyde 37% formaldehyde51 Line 5 from bottom there anions… their anions…52 Table 1 in footnotes Rates of hydrolysis should be

expressed as µM/min53 Table 2 57a resp. 58a 58a resp. 59a54 Line 13 from bottom The phenantrene analogue 43a The phenanthrene analogue57 Line 7 from bottom …and other effects… …and among other effects…64 Line 8 from top degree of clearance… high value of clearance…64 Line 12 from top …context, compounds…. …context, that compounds….64 Line 2 from bottom …is a potentially soft drug… …is potentially a soft drug…


N

N N

NH2

H2N

CH3 OCH3

OCH3PTX

H2N

CN

O

XXIV

OCH3

OCH3

O H

Cl

XXIII

Cl N

CH3NC

XXVI

OCH3

OCH3

OCH3

OCH3

O

Me

XXII

NC

O NH

CH3

XXV

OCH3

OCH3

+H3C

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Approaches to Soft Drug Analogues of Dihydrofolate ...uu.diva-portal.org/smash/get/diva2:167816/FULLTEXT01.pdf · Graffner Nordberg, M. 2001. Approaches to Soft Drug Analogues of