Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 252

______

Approaches to Soft Drug Analogues of Dihydrofolate Reductase Inhibitors

Design and Synthesis

BY

MALIN GRAFFNER NORDBERG

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Organic Pharmaceutical Chemistry presented at Uppsala University in 2001

ABSTRACT

Graffner Nordberg, M. 2001. Approaches to Soft Drug Analogues of Dihydrofolate Reductase Inhibitors. Design and Synthesis. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala 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 of inhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration and devoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infections, and also opportunistic infections associated with AIDS (Pneumocystis carinii pneumonia, PCP). Recent research indicates that the enzyme also is involved in various autoimmune diseases, e.g., rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates have been 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 new antifolates described herein were designed to retain the pharmacophore of methotrexate, but encompassing an ester group, so that they also would serve as substrates for the endogenous hydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples of soft drugs because they in a controlled fashion would be rapidly and predictably metabolized to non-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 a biological assay consisting of esterases from different sources was performed. The structural features of the least reactive ester were substituted for the methyleneamino bridge in methotrexate to produce analogues that were chemically stable but potential substrates for DHFR as well as for the esterases. The new inhibitor showed desirable activity towards rat liver DHFR, being only eight times less potent then methotrexate. Furthermore, the derived metabolites were found to be poor substrates for the same enzyme. The new compound showed good activity in a mice colitis model in vivo, but a pharmacokinetic study revealed that the half-life of the new compound was similar to methotrexate. A series of compounds characterized by a high lipophilicity and thus expected to provide better esterase substrates were designed and synthesized. One of these analogues in which three methoxy groups were substituted for the glutamic residue of methotrexate exhibited favorable pharmacokinetics. This compound is structurally similar to another potent DHFR inhibitor, trimetrexate, used in the therapy of PCP (vide supra). The new inhibitor that undergoes a fast metabolism in vivo is suitable as a model to further investigate the soft drug concept.

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

 Malin Graffner Nordberg 2001

ISSN 0282-7484 ISBN 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 ribonucleotide AIDS acquired immune deficiency syndrome DCC N,N'-dicyclohexylcarbodiimide DHFR dihydrofolate reductase DIPEA N,N-diisopropylethylamine DMAc N,N-dimethylacetamide DMAP 4-dimethylaminopyridine DME dimethoxyethane DMF N,N-dimethylformamide dppp 1,3-bis(diphenylphosphino)propane FA folic acid (FH2 and FH4, respectively, represent the reduced analogues of FA) FDA US food and drug administration FEP free energy perturbation FPGS folylpolyglutamate synthetase GAR glycinamide ribonucleotide IBD inflammatory bowel disease IC50 inhibitor concentration at 50% inhibition of the enzyme i.p. intraperitoneal IS inflammation score i.v. intravenous Ki inhibitory constant LIE linear interaction energy MD molecular dynamics MPO myeloperoxidase MTX methotrexate NADPH nicotinamide adenine dinucleotide phosphate, reduced form NMR nuclear magnetic resonance NSAIDs non-steroid anti-inflammatory drugs PCP Pneumocystis carinii pneumonia PLE pig liver esterase p.o. per oral (via mouth) PTX piritrexim PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RA rheumatoid arthritis RFC reduced folate carrier SAR structure-activity relationships T½ half-life TBAF tetrabutylammonium fluoride TFA trifluoroacetic acid THF tetrahydrofuran TLC thin-layer chromatography TMP trimethoprim TMQ trimetrexate TS thymidylate synthetase LIST OF PAPERS

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

I. Graffner-Nordberg, M.; Sjödin, K.; Tunek, A.; and Hallberg, A. Synthesis and Enzymatic 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 Soft Drugs 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 as Inhibitors 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 Bridging Ester Group for Biological Evaluation in a Mouse Colitis Model In Vivo. In manuscript.

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

Reprints were made with kind permission from the publishers.

CONTENTS

1INTRODUCTION 9

1.1 THE SOFT DRUG CONCEPT 10 1.2 THE ESTERASES 12 1.3 DIHYDROFOLATE REDUCTASE (DHFR) 14 1.4 AN OVERVIEW OF THE FOLATE PATHWAY 15 1.5 INHIBITORS OF DIHYDROFOLATE REDUCTASE 19 1.5.1 Classical DHFR Inhibitors 19 1.5.1.1 Methotrexate 19 1.5.2 Non-classical DHFR Inhibitors 20 1.6 DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY 22 1.6.1 Pneumocystis carinii Pneumonia 22 1.6.2 Inflammatory Bowel Diseases 23 1.6.3 Rheumatoid Arthritis 24

2AIMS OF THE PRESENT STUDY 26

3ASPECTS OF DRUG-ENZYME INTERACTIONS 27

3.1 METHOTREXATE 27 3.1.1 Structure-Activity Relationships 28 3.2 NON-CLASSICAL INHIBITORS TOWARDS PNEUMOCYSTIS CARINII 29 3.2.1 Structure-Activity Relationships 30

4OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES 31

4.1 METHOTREXATE 31 4.2 NON-CLASSICAL INHIBITORS 33 4.2.1 Trimetrexate 34 4.2.2 Piritrexim 34

5DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR 36

5.1 SYNTHESIS OF SIMPLE AROMATIC ESTER-CONTAINING MODEL COMPOUNDS 36 5.2 SYNTHESIS OF SOFT DRUG DERIVATIVES OF NON-CLASSICAL ANTIFOLATES 39 5.3 SYNTHESIS OF NEW DERIVATIVES OF CLASSICAL ANTIFOLATES 46 5.4 APPLYING SOLID PHASE CHEMISTRY TO THE SYNTHESIS OF CLASSICAL DHFR INHIBITORS 48

6BIOLOGICAL RESULTS 51

6.1 ENZYMATIC HYDROLYSIS IN VITRO 51 6.2 INHIBITORY ACTIVITIES OF NEW INHIBITORS OF DHFR 52 6.2.1 Inhibitory Activities and Selectivities of New Lipophilic Soft Drug Inhibitors for pcDHFR 53 6.2.2 Inhibitory Activities of New Classical Inhibitors of Mammalian DHFR 55 6.3 EFFECT OF A NEW POTENT INHIBITOR OF DHFR IN A MOUSE COLITIS MODEL IN VIVO 56 6.4 EVALUATION OF A NEW POTENT DHFR INHIBITOR IN A RAT ARTHRITIS MODEL IN VIVO 57

7MOLECULAR DYNAMICS 58

8PHARMACOKINETICS 62

9CONCLUDING REMARKS 65

10 SUMMARY 66

11 ACKNOWLEDGEMENTS 67

12 REFERENCES 69 INTRODUCTION – THE SOFT DRUG CONCEPT

1 INTRODUCTION

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

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

Crystal Structure of the Receptor

Computational Analysis Crystallographic Analysis of Ligand-Receptor Complex

Ligand Design Biological Testing

Animal Studies Pharmacokinetics Candidate Drug Organic Synthesis of New Ligands Metabolism Toxicity Efficacy

Clinical Studies

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

- 9 - INTRODUCTION – THE SOFT DRUG CONCEPT

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, since the development of new drugs is an expensive process the toxicology profile must be taken into account at an early stage of the drug development (see Figure 1). Thus, a highly potent molecule may be rejected at a late stage of the drug discovery process because of unavoidable and unpredictable side-effects. In order to successfully design safer drugs with increased therapeutic ratios, i.e. with large differences between the therapeutic dose and doses causing undesired side-effects, a relatively new approach called soft drug design can be used (Figure 2). 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 fast and predictable deactivation after having exerted a therapeutic effect.1,2 The resulting metabolites formed after deactivation must have no intrinsic activity of their own in addition to 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 the deactivation process to occur. The idea behind the soft drug concept is to build a fragment into the molecule of choice that turns the new molecule into a compound that can function as a substrate for the metabolizing enzymes, but still has a sustained activity on its original target. Most drug metabolism is mediated by the cytochrome P450 system, which exhibits a large inter-individual variability and is subject to inhibition and induction.3 Additionally, this enzymatic system is often associated with the formation of undesired toxic, active, or high- energy intermediates. When more readily predictable and thus controllable metabolism is preferred the esterases, an important class of hydrolytic enzymes, are an attractive target. Consequently, if the incorporated fragment contains an ester group potentially it could be readily deactivated by esterase enzymes. This group of enzymes will be described more thoroughly in Section 1.2.

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

- 10 - INTRODUCTION – THE SOFT DRUG CONCEPT the lead compound. In general, soft drugs are mainly for topical use and are applied or administered near the site of action thereby having predominantly local effects.

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

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

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

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

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

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

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

- 11 - INTRODUCTION – THE SOFT DRUG CONCEPT pharmacokinetics. In order to circumvent these problems the molecule must be transformed to make it more available to produce its biological effect. The main approach for prodrug design is to convert carboxylic acids into esters or amides, thus enhancing the lipophilicity of the drug molecule. Most often it is the endogenous enzymes that are involved in the activation of prodrugs with esterases as key enzymes. Consequently, the same enzymatic systems involved in the activation of prodrugs can be used to deactivate soft drugs. Actually, one can also design 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 inactive carrier-molecule.

1.2 The Esterases

As described earlier in Section 1.1 prodrug and soft drug design often relies on enzymatic hydrolysis for drug activation, and deactivation, respectively. With biologically based drug design, it has become an important issue to consider the conversion site in the body, 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 enzymatic cleavage by the esterases comprises one approach to make the action of a drug more restricted to the site of application. A wide distribution of the enzymes in mammalian tissues has been observed. Hydrolases (e.g., esterases, lipases, peptidases, glycosidases, amido- and aminohydrolases, pyrophosphatases etc.) are enzymes well suited to drug inactivation since they are ubiquitously distributed. Hydrolases catalyze the addition of a molecule of water to a variety of functional groups (Figure 4). The hydrolases vary between species and individuals9,10 and the same substrate is often hydrolyzed by more than one enzyme. The hydrolases play an important role in the detoxification process and a decrease in enzyme activity potentiates the toxicity of certain compounds.

O O H2O R' H + R'OH RO RO

Figure 4. General formula describing ester hydrolysis.

- 12 - INTRODUCTION – THE ESTERASES

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

Esterase activity depends on the substrate but it also varies quite strongly between species.9-11,17,19,20 In fact, rodents (rats, guinea pigs) tend to metabolize ester-containing drugs 2,21 more rapidly than humans. In vitro hydrolytic half-lives, T½, (see Chapter 8) measured in rat blood were often found to be orders of magnitude lower than those measured in human blood.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 related to the extrapolation results of animal test to man, this is one of the additional aspect that can complicate early drug evaluations.25,26

Glu335 Glu335 Glu335

Ser203 His448 His448 Ser203 His448 Ser203

O O O O H N N O H N N H O H N N O H O O O O H O H R R' nucleophilic O O R' R Gly124 O R attack acyl-enzyme H R' complex tetrahedral Gly123 intermediate

Glu335 Glu335 Glu335

R'OH His448 His448 Ser203 Ser203 His448 Ser203 O O O O H N N O H N N O O H N N O O O H O H R O H2O H R O H H tetrahedral O R nucleophilic intermediate OH attack

Figure 5. A proposed mechanism11,27 for the hydrolysis by the carboxyl esterases. Ser203, Glu335, and His448 serve 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 naturally occur on earth. - 13 - INTRODUCTION – THE ESTERASES

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 and shape to the substrate molecule. The chemical change can occur only when the substrate is anchored in the active site of the enzyme. The enzymatic binding sites are usually composed of a combination of both polar and non-polar amino acids. The carboxyl esterases belong to the group of serine proteases (acetylcholinesterase, butyrylcholinesterase, cholesterol esterase) and the mechanism for ester hydrolysis have been described in an active site consisting of a catalytic triad with Ser203, Glu335, and His448 (Figure 5).11,27 The amino acid sequence 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 to undergo a nucleophilic attack by the serine oxygen, which in turn is more nucleophilic due to the assistance of the basic nitrogen in histidine. The acylated Ser203 and Gly123-Gly124 together form a so-called oxyanion hole where weak hydrogen bonds stabilize the tetrahedral adduct. A less accessible carbonyl oxygen is more difficult to stabilize by hydrogen bonds in the 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 vitro 28 human blood enzymatic hydrolysis, thus increasing T½. Several models of active sites of hydrolytic enzymes have been proposed with the aim of correlating substrate specificity to the structural feature of substrates.29

In addition to the fact that the biological activity is considered to be a function of the chemical structure the physicochemical properties also play an important role. Structure- activity relationships (SAR) have been developed where a set of physicochemical properties of a group of congeners is found to explain variations in biological activity. The effect of structure on the enzymatic half-life has been investigated in a large number of prodrug and soft drug series. Several attempts to establish some kind of quantitative structure-metabolism relationships (QSMR) looking at the enzymatic half-lives of prodrugs and soft drugs have been 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 in biochemistry and medicinal chemistry. DHFR functions as a catalyst for the reduction of dihydrofolate to tetrahydrofolate. Reduced folates are carriers of one-carbon fragments, hence they are important cofactors in the biosynthesis of nucleic acids and amino acids. The inhibition of DHFR leads to partial depletion of intracellular reduced folates with subsequent limitation of cell growth.41 A more illustrative description of the folate pathway will be discussed in Section 1.4. DHFRs isolated from different species are relatively small proteins

- 14 - INTRODUCTION – DIHYDROFOLATE REDUCTASE (DHFR)

(18-22 kDa), which are easily available. DHFR has attracted attention of protein chemists as a model for the study of enzyme structure/function relationships. The species-differences among 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 are trimethoprim (Figure 6a) and pyrimethamine (PTM, Figure 6b), which are used in therapy for their antibacterial and antiprotozoal properties, respectively.

NH Cl 2 NH OCH 2 N 3 N H2N N OCH3 H2N N Et OCH3

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

Compounds that inhibit DHFR exhibit an important role in clinical medicine, as exemplified 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 in asthma;49 TMP in bacterial diseases;50 and of PTM in protozoal diseases.51,52 Lately, a new generation of potent lipophilic DHFR inhibitors such as trimetrexate (TMQ, Figure 11) and piritrexim (PTX, Figure 12) have shown antineoplastic53 and most importantly, antiprotozoal54 activities.

The enzymatic reduction involved in the inhibition of DHFR is a random process in which either the substrate (dihydrofolate) or the cofactor NADPH, forms a binary complex with 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. A review 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 been effective in the inhibition of DHFR, depending upon the source of the enzyme and type of inhibitors. 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 mammalian metabolism.56 The reactions of folate metabolism are interconnected, and the inhibitors discussed 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 building blocks involved in DNA synthesis as well as of important amino acids.

- 15 - INTRODUCTION – AN OVERVIEW OF THE FOLATE PATHWAY

O COOH

O N α COOH γ N H HN N H H2N NN

Figure 7. Folic acid (FA)

Folic acid (FA,) is a water-soluble B vitamin that plays a crucial role in the biosynthesis of DNA. The vitamin consists of 2-amino-4-oxopteridine with a side-chain incorporating both p-aminobenzoic acid and glutamic acid. Humans cannot synthesize folic acid by themselves, and thus depend on a variety of dietary sources for the vitamin. Food 5 folates exist mainly as 5-methyltetrahydrofolate (N -methyl-FH4) and 5-formyl- 5 57 tetrahydrofolate (N -formyl-FH4) (Figure 9). The active form of FA is its reduced tetrahydroform (FH4), which is formed after reduction of dihydrofolic acid by DHFR (Figure 8). FH4 functions as the ‘acceptor’ of single-carbon units and the tetrahydrofolate is subsequently transformed enzymatically from the appropriate cofactor to precursor molecules that lead to the synthesis of purine and pyrimidine (e.g., thymine) nucleotides necessary for DNA, 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 the 5 subsequent reactions, using different derivatives of FH4 in one-carbon transfer reactions. N - Formyl-FH4 (folinic acid, leucovorin) is taken up actively into the cell by a reduced folate 10 carrier (RFC) system where it is converted to N -formyl-FH4. In another enzymatic pathway, 5 5 5 N -formyl-FH4 can, after transport into the cell, be converted to N -methyl-FH4. N -methyl- FH4 may also be taken up actively and form fresh supplies of FH4. The same entrance system is used by MTX and as these reduced tetrahydrofolates although the latter seem to have a relatively low affinity for RFC in comparison to MTX.58

O COOH O COOH + O N COOH NADPH NADP O N COOH N H N H HN N HN N H H dihydrofolate H2N NN H N NN reductase 2 FA H FH2

inhibition by MTX, TMP, TMQ, PTX

dihydrofolate O COOH NADPH reductase O H N COOH N H + HN N NADP Figure 9 H H2N NN H FH4 inhibition by MTX, TMP, TMQ, PTX

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

- 16 - INTRODUCTION – AN OVERVIEW OF THE FOLATE PATHWAY

10 N -formyl-FH4 is responsible for the donation of single carbon groups in the de novo biosynthesis of purine nucleotides in the reactions catalyzed by the enzymes glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) 10 transformylases. As well as being used directly in purine synthesis, N -formyl-FH4 is also 5 10 5 10 converted, via N ,N -methenyl-FH4, to N ,N -methylene-FH4. The latter is converted, first to dihydrofolate (FH2) by the action of thymidylate synthetase (TS) and then to tetrahydrofolate 5 10 (FH4) by DHFR. N ,N -methylene-FH4 donates a methyl group to the 5-position of the pyrimidine ring of deoxyuridylate (dUMP) in the reaction catalyzed by TS forming thymine, 5 10 which is used for the DNAsynthesis. Thus, N ,N -methylene-FH4 is the only de novo source of cellular thymidylate. Inhibition of TS results in a so-called thymine-less death, which leads 5 10 to disruption of the synthesis of dividing cells. During this process, N ,N -methylene-FH4 is oxidized to dihydrofolate and must be converted back to its tetrahydroform by DHFR in order to maintain the reduced folate pool.

FA O COOH

FH2 N COOH R = H

direct intake direct intake O H N R methionine HN N H homocysteine H2N NN H FH4 O CH3 O CHO N R N R HN N HN N H H H2N NN H2N NN H 5 serine H 5 N -methyl-FH4 N -formyl-FH4 = leucovorin

glycine R N O O H N N R HN HN N CHO H2N NN H2N NN H H R N5,N10-methylene-FH 10 4 O N N -formyl-FH4 dUMP N α HN AICAR TNF inhibition by MTXG thymidylate adenosine N synthetase IL-1 GAR transformylase dTMP H2N NN fGAR H FH tra (purine C-8) fAICAR 2 5 10 nsfo N ,N -methenyl-FH4 rmy (purine C-2) lase inhibition by MTXGN

FH4 DNA synthesis RNA, DNA

dihydrofolate reductase

cell division NADPH NADP+

Figure 9. The folic acid pathway.

Intracellularly, folic acid and derived coenzymes also have the ability to be converted to polyglutamated derivatives with additional glutamic acid residues linked by amide bonds by the enzyme folylpolyglutamate synthetase (FPGS). This is an important mechanism for trapping ‘classical’ (for an explanation, see Section 1.5) folates and antifolates within the cell, - 17 - INTRODUCTION – AN OVERVIEW OF THE FOLATE PATHWAY 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 molecules to the pteridine ring of MTX.60,61 This polyglutamation reaction has several biologically important 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 of the molecule a greater intracellular retention is enabled with decreased cellular efflux and trapping of the drug within the cell, with prolonged drug action as a consequence.62 The polyglutamates appear to be somewhat more efficient substrates for DHFR than the 63,64 monoglutamated counterparts. MTX-polyglutamates (MTXGN) are direct inhibitors of DHFR and are thus less reversible inhibitors than MTX itself.61 Tissues with high FPGS activity, such as the liver, accumulate and retain the polyglutamated MTX for prolonged periods of time. Hence, this increased concentration of polyglutamated of MTXGN is responsible for the hepatotoxicity observed after chronic administration of the drug. Compared to monoglutamated drugs, the polyglutamates also have increased affinity to other folate-dependent enzymes such as TS,65,66 and the transformylases of glycinamide ribonucleotide (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 in inhibition of purine synthesis. As a consequence, the polyglutamation of MTX plays an essential role in the development of side effects. A new hypothesis about the mechanism of action of MTX suggests that by inhibiting AICAR transformylase, MTX causes the accumulation of AICAR. This inhibition might result in the subsequent increase of the production of the anti-inflammatory autocoid adenosine,69 an endogenous anti-inflammatory cytokine. This hypothesis could be one of the explanations for the anti-inflammatory properties that are seen in therapy with MTX in autoimmune disorders where much lower concentrations are needed for the effect compared to doses that generate effects through direct inhibition of DHFR. Nevertheless, a recent publication reports that treatment with MTX in inflammatory 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 that polyglutamation of the antifolate is not required for these functions.71

The administration of exogenous reduced folates, such as leucovorin, effectively prevents MTX cytotoxicity in mammalian cells (Figure 9). The amount of leucovorin required to prevent severe clinical toxicity in high-dose MTX chemotherapy regimens is directly proportional to the amount of MTX circulating in plasma.61 Leucovorin does not compete with MTX for binding to DHFR but directly overcomes blockade of MTX of the enzymes by increasing the intracellular pools of FH4 and thereby rescues cells from death. Although effective, this regimen offers a very expensive therapy.

- 18 - INTRODUCTION - INHIBITORS OF DIHYDROFOLATE REDUCTASE

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 in the molecule and thus closely resemble folic acid itself. MTX (Figure 10) is the most well known drug among the ‘classical’ antifolates. Compounds classified as ‘non-classical’ inhibitors of DHFR do not posses the p-aminobenzoylglutamic acid side-chain but rather have a 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}glutamic acid, Figure 10) was first synthesized in 194972 and is today, together with the antibacterial drug TMP (Figure 6a), the DHFR inhibitor most often used in clinic. The most common use of MTX is as an anticancer drug, but lately the drug also is considered to have anti- inflammatory and immunosuppressive properties with accompanied activity against autoimmune disorders (see Section 1.3).

O COOH

NH N COOH 2 H N N N

CH3 H2N N N

Figure 10. Methotrexate (MTX)

MTX serves as an antimetabolite, which means that it has a similar structure to that of a cell metabolite, resulting in a compound with a biological activity that is antagonistic to that of 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 the pteridine 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 of MTX compared to the natural substrate (see Section 3.1).

MTX is a competitive and reversible inhibitor of DHFR that binds tightly to the hydrophobic folate-binding pocket of the enzyme. The drug can be competitively displaced by increased intracellular concentrations of the natural enzyme substrate dihydrofolate. The affinity of MTX for DHFR increases considerably in the presence of the cofactor NADPH. - 19 - INTRODUCTION - INHIBITORS OF DIHYDROFOLATE REDUCTASE

MTX inhibits the synthesis of metabolites involved in one-carbon-unit transfer reactions such as the biosynthesis of the important nucleotides (see Section 1.4). As a result of this process, the synthesis of DNA is disrupted. MTX exists as a highly polar dianion at physiological pH and enters the cells by the energy-dependent RFC system, the major transport system in human tissue of similar compounds. Inside the cell the molecule is polyglutamated, which leads to altered characteristics (Section 1.4). The polyglutamated derivatives of MTX have been postulated to be the probable cause of the anti-inflammatory properties 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 of apoptosis (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 their clinical efficacy. Resistance has been attributed to several different mechanisms including a reduced level of cellular uptake of the drug,44 and to an increase in enzyme levels involved in the folic acid biochemistry. As mentioned above, MTX is transported into cells by the carrier transport system used for the active transport of reduced folates. Consequently, a poor ability to transport the drug into the cell can be one source of natural resistance associated with the drug.75,76 Major limitations, besides the development of resistance with MTX treatment, are bone 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 periodic administration of leucovorin (see Section 1.4), enabling the blockade of tetrahydrofolic acid production to be by-passed (leucovorin ‘rescue’ procedure).

The clinically useful properties of MTX have stimulated the quest for a variety of new analogues with modifications within the molecule, without interfering too much with the pharmacophore of the original drug (Section 3.1). Thus, research is enduring for a more selective 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 the mechanisms 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 of toxicity due to the lack of selectivity (Section 3.2).

- 20 - INTRODUCTION - INHIBITORS OF DIHYDROFOLATE REDUCTASE

OCH3 OCH3 NH2 CH3

N N OCH3 H H2N N

Figure 11. Trimetrexate (TMQ)

TMQ77,78 (Figure 11) is a potent inhibitor of DHFR. It is a ‘non-classical’, lipophilic quinazoline derivative, originally developed as an anticancer agent.79-81 The drug lacks the glutamic acid side-chain, which is the characteristic feature of the ‘classical’ antifolate. Consequently, it cannot be polyglutamated by FPGS and thus does not subsequently undergo retention 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, this occurs with for example, the opportunistic pathogen P. carinii (this is a non-energy dependent process), with subsequent inhibition of the cellular enzyme and thus growth. It is important to mention in this context is that this characteristic also enables the drug to penetrate mammalian cells. TMQ has been approved recently by the FDA for the use in the treatment of Pneumocystis carinii pneumonia82 (PCP, see Section 1.6.1). The administration of TMQ, in combination with leucovorin, is regarded as an good alternative in the treatment of PCP in AIDS patients.54 TMQ therapy is currently recommended for patients with PCP who are either 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 a lipophilic antifolate from the new generation of DHFR inhibitors, originally developed as an anticancer drug.84,85 PTX has also been studied in the treatment of psoriasis.85-87

NH2 CH3 OCH3 N

H2N NN OCH3

Figure 12. Piritrexim (PTX)

- 21 - INTRODUCTION - DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY

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 of response with older antifungal agents. However, more recently it has been definitively characterized as a fungus. The primary route for infection is postulated to be via the airborne route.88 In healthy children by the age of four, 75-90% experience a respiratory infection with P. 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 immune response is compromised. PCP is characterized by dyspnea, a non-productive cough, and fever. The initial step in the pathogenesis is the attachment of inhaled organisms to type I pneumocytes in the alveoli. After proliferation of the organism the pneumocytes are damaged and 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 due to 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 picture was taken with permission from the Atlas website of CarloDenegriFoundation.91

PCP is one of the most common life-threatening diseases of people suffering from AIDS.92-96 Encouragingly, the highly active anti-retroviral therapy (HAART) and prophylaxis in patients predisposed to developing PCP, has caused a large decline in the incidence of the disease in patients with AIDS.97-99 In addition to AIDS, cancer chemotherapy100 and organ transplantation101 also increases the risk of developing the infection.

Currently, first line therapy mainly consists of TMP (Figure 6a) in combination with a sulfonamide (co-trimoxazole).92,97,102,103 Inhaled aerosolized pentamidine104 has also found a place in prophylaxis.105-108 The new generation of DHFR inhibitors; TMQ (Figure 11), as well as PTX (Figure 12), are now used in the clinic as second-line therapies for moderate to severe PCP.82 As mentioned in Section 1.5.2, TMQ and PTX are both potent inhibitors of DHFR - 22 - INTRODUCTION - DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY from P. carinii. Nevertheless, they are not selective, which means that they inhibit the mammalian enzyme even more efficiently.109,110 Due to their systemic host toxicity, both drugs 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 cause inflammation or ulceration in the small and large intestines (Figure 14). Mainly, IBD is classified as either ulcerative colitis, or Crohn’s disease. Ulcerative colitis (UC) causes ulceration 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 gastrointestinal tract, 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 one theory postulates that a virus or a bacterium affects the body’s immune system triggering a sustained a sustained inflammation reaction in the intestinal wall.114 All medical or surgically induced remissions of the disease are most often followed by relapses. However, several medications that are effective in the short-term do not result in sustained remission, and conversely, 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 a growing number of new agents. Systemic administration, as well as topical treatment, with glucocorticoids produces systemic side effects, including adrenosuppression, immuno- suppression, and bone resorption. Budesonide was developed as a corticosteroid with high topical anti-inflammatory activity and with low systemic activity due to rapid hepatic metabolism.115 As a consequence, it follows the criteria for the soft drug concept. Budesonide has found to be as active as conventional corticosteroids (e.g., prednisolone) in UC and the active form of CD.116

The small intestines The large intestines (also called colon)

Rectum

Figure 14. The intestines.

- 23 - INTRODUCTION - DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY

Since the first report in 198945 MTX has drawn attention as a potential treatment for patients suffering from refractory Crohn’s disease.117 Folic acid, which is effective in decreasing side-effects without affecting the drug efficacy can be given systematically during therapy.118 MTX has recently also been proven to be effective in the long-term treatment of IBD with only moderate side-effects.119,120 As in the case of rheumatoid arthritis (Section 1.6.3), antibodies against TNFα (infliximab) have recently become an effective maintenance therapy.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 the disease larger joints such as the hips and knees become affected. The disease is progressive but with a large inter-individual variation in the course. A relapse-remission pattern is common. 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 of the body. The probable cause of rheumatoid arthritis is a pathological reaction of the immune system. 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, for example, 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 the synovium is transformed into an aggressive pannus that grows into, and destroys cartilage and underlying bone, hence causing loss of joint function. There is an abundance of inflammatory cells in the pannus. The relative importance of these cells for the disease progression is under debate. TNF-α is a cytokine that mainly is produced from the pannus cells of macrophage lineage. Recently, promising results have been observed with monoclonal antibodies against

- 24 - INTRODUCTION - DISEASES WITH POTENTIAL USE OF DHFR INHIBITORS IN THERAPY

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

Traditional pharmacological treatment of RA is based on NSAIDs, which mainly provide 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 NSAIDs and glucocorticoids. During the 1980’s, a low weekly dosage of MTX became an established method for the therapy of RA.46,122 In the 1990’s it has gradually been accepted as the leading DMARD, being one of the most potent anti-rheumatic drugs,123 especially in the severe cases of RA. The effectiveness of weekly dosing indicates that the drug is trapped intracellularly due to polyglutamation (Section 1.4). The mechanism of action of MTX in RA is not completely known. The fact that folate supplementation (leucovorin, Section 1.4) administered to MTX-treated RA patients reduces toxicity in a 1:1 ratio without affecting efficacy 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, is involved in the anti-arthritic effect. In treatment with MTX the levels of AICAR rise with a corresponding increase in the concentration of adenosine (Section 1.4). Nevertheless, in one study arthritic rats were treated with different antagonists of adenosine in combination with MTX. Surprisingly, the antagonists enhanced the anti-arthritic effects of MTX,124 a result which does not support “the adenosine hypothesis”. Still, it is possible that AICAR might be involved in the anti-arthritic effect.

- 25 - AIMS OF THE PRESENT STUDY

2 AIMS OF THE PRESENT STUDY

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

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

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

• To assess the impact of the ester group on bioactivity. Thus, to design and synthesize DHFR 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 hydrolysis by esterases as potential soft drugs. The compounds should exhibit favorable bioactivities after local therapy in animal models.

- 26 - ASPECTS OF DRUG-ENZYME INTERACTIONS

3 ASPECTS OF DRUG-ENZYME INTERACTIONS

3.1 Methotrexate

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

MTX and the natural substrate, FA, exhibit close similarities in structure (cf. Figure 10 and ). 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 enzyme resulted in more extensive hydrogen bonding, thereby leading to a higher affinity of MTX to DHFR as compared to FA. This result was later confirmed by NMR spectroscopy studies.129 1 The most important feature for binding of the antifolates is the N and the adjacent 2-NH2, which is also the strongest basic center. The generated interaction consists of ionic bonding of 1 the cation of N /2-NH2 to Glu30 (Figure 16).

O COO

Glu30 O N COO a) O H N H N N O H H N NN H H

OCOO α N γCOO Glu30 7 9 H b) 8 6 O N N10 H 1 N CH3 O N 5 H 2 4 H N2 N3 N4 HH

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

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

- 27 - ASPECTS OF DRUG-ENZYME INTERACTIONS

NH2. The nicotinamide portion of the coenzyme NADPH lies sufficiently close to the pteridine 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-glutamate in 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 closely similar when different species are compared, indicating a high degree of functional homology at critical binding sites of DHFR. Although the amino acid sequence of rat liver DHFR is not yet 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 residues involved in binding are conserved throughout.

3.1.1 Structure-Activity Relationships

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

F O COOH 6' 5' NH2 N COOH 9 4 5 10 H 3 N 2' N 6 N 3' 7 2 Me H2N NN1 8

AB C D EG

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 for inhibitory activity against DHFR. Alkylation of the amino groups leads to a reduction of the inhibitory potencies.135 Region B, comprising the pyrazine moiety, displays a larger tolerance to alterations. The ring can be modified by replacement of the nitrogens at positions 5 and/ or 8 with carbon to give either retention or improvement in activity.136,137 Similar substitutions have 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 to significant structural changes. However, it is important not to alter the position of side-chain attachment (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 - 28 - ASPECTS OF DRUG-ENZYME INTERACTIONS

‘bridge region’ between the C-6 of the pteridine ring and the benzoate moiety may be altered considerably in terms of heteroatom substitutions. At this position, extra methylene-spacers, and nitrogens, respectively, have been introduced without loss of activity. Also, interchanges of C9 and N10, and replacement of N10 by sulfur and oxygen have been carried out. As an alternative to the methyl group on N10, other carbon-containing groups with different chain 10 lengths and sizes have been explored. Replacement of the N by a simple CH2 or other branched chains containing carbon, has also produced analogues of interest. Region D consists of a phenyl moiety, but different heterocyclic rings have been introduced here successfully with respect to inhibitory activity towards the enzyme. The phenyl moiety has been halogenated in the 3’- and 5’-positions affording more lipophilic compounds compared to MTX. Modifications in Region E, comprising the amide bond between the pteroyl moiety and the glutamic acid portion, have most often resulted in compounds of no interest. Changes to the α-carboxyl group in Region F showed this group to be critical for binding to DHFR. In addition, an acidic functionality at this position seems to be essential in vitro as well as in vivo. 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 in either the ortho- or meta- positions rendered compounds inactive as substrates for FPGS. The chirality of the α-center of the glutamic acid residue is L. The D-isomer of MTX is approximately 10-fold less potent than the L-glutamate counterpart.130 Also, the D-isomer of MTX has been reported to be biologically inactive,130,139 which mainly is attributed as a consequence of its poor affinity for the active transport mechanism necessary for penetration into the cell (Section 1.4). Finally, the remaining glutamic acid portion in Region G seems to tolerate structural changes. Modifications have consisted of, for example, insertion of an extra methylene group into the side-chain, as well as esterification and amidation of the γ-COOH group.

3.2 Non-classical Inhibitors towards Pneumocystis carinii

Based on published sequence alignments,140,141 P. carinii DHFR appears to be very similar 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-binding pocket, there are only six non-identical residues. Comparing the volumes of the two active sites of both enzymes, reveals that the binding region for P. carinii is slightly smaller than the active site of the human enzyme, but as there are no other major differences this makes specific design very complex.144 Thus, producing inhibitors with selectivity for P. carinii is a challenging task because of the great similarities between the fungal and the mammalian enzyme.

- 29 - ASPECTS OF DRUG-ENZYME INTERACTIONS

3.2.1 Structure-Activity Relationships

The high potencies of the new ‘non-classical’ DHFR inhibitors provide evidence that the 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 mammalian enzyme110 (Table 2). Consequently, with the exception of TMP and PTM, the main drawback with 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 ratio above 10, especially accompanied by a good potency. As a consequence it is difficult to draw conclusions regarding the SAR for selectivity towards the fungus. A number of lipophilic fused 2,4-diaminopyrimidine ring system related to TMQ and PTX have been examined. This includes 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 compared to 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 exhibit a selectivity ratio of 26 for pcDHFR versus rlDHFR, although with a potency in the micromolar 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 been concentrated on finding new selective analogues towards the fungal enzyme by trying to explore favorable interactions with the amino acids of the lipophilic cavity of the enzyme, that is, substitution in, or of the phenyl ring. As an example, naphthyl analogues (replacement of the phenyl moiety) have been developed with high potency against DHFR.150 A small series of 2,4-diaminoquinazolines, encompassing 9-phenanthryl, 9-anthryl and 9-flourenyl has been synthesized with high potencies.151 Also, several other series of compounds with a small group in the 5-position in the heterocyclic core (i.e., 5-CH3, 5-Cl) have been synthesized.150,152-154 Although, these 5-substituted analogues in general are more potent against pcDHFR than the unsubstituted analogues, they are even more potent against the rat enzyme.155,156 Nevertheless, one new inhibitor within a large series of pteridine analogues was recently published with a ratio of selectivity of 21 in favor of P. carinii in addition to high potency (Figure 18).157 The compound in Figure 18 consists of a dibenz[b,f]azepine instead of the phenyl ring. The IC50-value against the fungal enzyme was 0.21 µM and it was remarkable that the corresponding 10,11-dihydroanalogue exerted about 10 times lower potency (IC50: 1.4 µM), emphasizing the difficulties in achieving selectivity, and in the fine- tuning of ligands towards pcDHFR.

- 30 - ASPECTS OF DRUG-ENZYME INTERACTIONS

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

NH2 N N N

H2N NN

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 the synthesis of MTX, the most established DHFR inhibitor used in the clinic. In Section 4.2 the synthesis 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 For more references, see the following reference,158 and references therein. The methods used can be divided into three main groups as described below.

NH2 NH N 2 I O COOH H2N N NH2 + NH2 N COOH N H N N Br O

CH3 Br H2N NN MTX + O COOH

N COOH H C H 3 N H

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

- 31 - OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES

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 in a complex reaction mixture with MTX isolated in less than 5% yield. An alternative route employing a 1,1,3-trihaloacetone, which is condensed with compound I at pH 5 to afford a mixture of the 6- and 7-isomers of the halomethylpteridine, but the method was accompanied by significant difficulties in separating the generated mixtures (Figure 20).166 It was later found that when using 1,1-dichloroacetone at pH 3.5-5.0 only the 6-methylpteridine was formed,167 which upon bromination afforded the 2,4-diamino-6-bromomethylpteridine II.

O COOH

NH2 N COOH NH2 NH2 N H N NH2 N N N Br N I CH 3 H N NN H N NNH H2N NN MTX 2 II 2 2

NH2 N N OH

NN H2N III

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

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

Method B: Another more efficient route for the synthesis of the substituted pteridines was 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 for the cyclization (Figure 21). The reaction conditions readily permit reactions at the 6-position while allowing for the preparation of several kinds of MTX analogues possessing lipophilic side-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. - 32 - OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES

O COOH O COOR

NH2 N COOH N COOR N H NC N H N N N

CH3 CH3 H2N NN MTX H2NN VI

O COOR

N COOR NC N H NC N N X

CH3 H2NN V H2NN X = Br, Cl O O IV

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 catalytic reduction cyclizes in situ providing the 7,8-dihydropteridinederivative IX (Figure 22). Permanganate oxidation furnishes the heteroaromatic compound, X,177,178 which after ester hydrolysis is coupled with L-glutamic acid to ultimately provide MTX.179

OCOOH O NH NH2 N COOH 2 OR N H N N N N N CH R = Me or Et CH3 H N NN 3 H2N NN MTX 2 X

O O OH NH OR NH2 OR 2 N N NO2 N N N N

CH3 R = Me or Et CH3 R = Me or Et H2N NN H2N NN H IX H VIII

O

NH2 OH OR NO2 N N N + CH R = Me or Et H N N Cl H N 3 2 2 VII

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. - 33 - OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES

4.2.1 Trimetrexate

The original synthesis of the quinazoline derivative TMQ78 was a modification of the early synthesis of unsubstituted phenyl analogues previously described by others.180 The synthesis starts with 6-chloro-o-toluonitrile XI,181 which was subjected to fuming nitric acid affording 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 carbonate resulting in the 2,4-diaminoquinazoline derivative XIII, which is further reduced to provide 2,4,6-triaminoquinazoline (XIV). The 6-amino group in XIV is successfully diazotized under Sandmeyer-type conditions without interfering with the other amino groups, using an acidic solution of sodium nitrite in the presence of cuprous cyanide. A reductive amination of the 2,4-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 of about 30% (Figure 23).

OCH3

OCH3 NH2 CH3 NH2 CH3 CN N N OCH3 N H H N N H N 2 TMQ 2 N XV

NH2 CH3 NH2 CH3 NH2 NO N N 2

H2N N H N N XIV 2 XIII

CH3 CH3 NC NO2 NC

Cl Cl XII XI

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.

COCH3 COCH3 H3CO H3CO H3CO CHO CO2C2H5 CO2C2H5

OCH3 OCH3 OCH3 XVIII XVII XVI

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

- 34 - OVERVIEW OF THE SYNTHESIS OF ANTIFOLATES

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 the acrylate XVII. The acrylate is further hydrogenated under pressure over 5% Pd/C furnishing XVIII. Condensation of XVIII with 2,4,6-triaminopyrimidine, XIX, provides the 7-oxo compound XX. Chlorination of the oxo-function is accomplished with a Vilsmeier-Haack reaction using thionyl chloride in DMF. Compound XXI is further dechlorinated under basic conditions over hydrogen with 5% Pd/C as catalyst to afford PTX.

OCH OCH NH2 CH3 3 NH2 CH3 3 N N

H2N N N H2N N N Cl XXI PTX OCH3 OCH3 OCH NH2 CH3 3 NH2 N N + XVIII H2N N N O H2N N NH2 H OCH3 XX XIX

Figure 25. A retrosynthetic analysis of PTX.

Recently, an alternative route for the synthesis of PTX was reported (Figure 26).183 In this route 4-(2,5-dimethoxyphenyl)-2-butanone XXII146 reacts with DMF and phosphorous oxychloride providing a E/Z-mixture of 3-chloro-2-(2,5-dimethoxybenzyl)crotonaldehyde XXIII. When the mixture (XXIII) is reacted with cyanoacetamide (XXIV) the adduct cyclizes to the pyridone XXV. Compound XXV is transformed to the chloride XXVI, using DMF and thionyl chloride, followed by subsequent condensation with guanidine to yield PTX.

OCH OCH OCH NH2 CH3 3 CH3 3 CH3 3 NC NC N

H2N N N Cl N O N Na OCH H OCH PTX XXVI 3 XXV 3

Cl OCH3 O OCH3 CN Me Me + H2N O O H OCH3 OCH3 XXIV XXIII XXII

Figure 26. A retrosynthetic analysis of PTX.

- 35 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

5 DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

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

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

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

X Et N, diethyl ether ROH + R'COCl 3

O O X = 14X = O O O 2 O 5 O O O O 3 O O 6

Scheme 1

Later on, attempts to improve the yields in some of the reactions were made. Among them, the Mitsunobu reaction184 was adopted for the synthesis of 3. The reaction was useful in this case. Also, the synthesis of 6 was attempted using a ‘Schotten-Baumann-like’ reaction with 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 product at all. For the syntheses of the carboxylic acids 7 and 9, 4-formylbenzoic acid was employed as 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 were prepared according to the same procedure using 4-hydroxybenzaldehyde as the precursor. The subsequent oxidations of the aldehydes were conducted with sodium chlorite, using 2-methyl- 2-butene as scavenger185-187 (Scheme 2).

- 36 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

. NaClO , NaH PO .. 1H O, X 2 2 4 2 X tert-butyl alcohol, 2-methyl-2-butene, r.t. 87-95 % CHO COOH

O O 7 X = O 9 O O O O 8 10 O Scheme 2

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

For the preparation of 11, the synthetic route in Scheme 2 could not be used, and therefore another reaction sequence outlined in Scheme 3 and 4 was considered starting from commercially available methyl (4-hydroxy)phenylacetate. The triflate 13 was prepared in 94% yield using triflic anhydride and pyridine.188 The methyl ester in 13 was hydrolyzed with trifluoroacetic acid (TFA)189,190 to afford the carboxylic acid 14 in a yield of 92%. Lithium iodide in DMF191 was also tried for the demethylation, but no product was formed under these conditions.

O O Me (CF3SO2)2O, Me O pyridine, CH Cl , 0 °C O OH 2 2 OTf 13

HO O TFA, reflux benzyl bromide, Bn O K2CO3, acetone, reflux O OTf OTf 14 15 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%. A transesterification of 13 with benzyl alcohol and DMAP in refluxing toluene, affording compound 15 in one step gave no advantage over the preferred route (60% vs. 87% yield). A palladium-mediated carbonylation of compound 15 was accomplished with 2- trimethylsilylethanol in DMF using palladium acetate [Pd(OAc)2] and dppp to furnish the diester 16 in 79% yield (Scheme 4). Debenzylation of 16 with hydrogen over palladium on charcoal and subsequent reaction of 17 with oxalyl chloride and 2-naphthol accomplished 18 in 51% over three steps. Finally, 18 was transformed to 11 by selective ester cleavage, using

- 37 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR trimethylsilyl chloride (TMSCl) and sodium iodide (NaI) in high yield (94%). Deprotection of the 2-(trimethylsilyl)ethyl group of 18 with TBAF could not be used due to a fast hydrolysis of the ester with this reagent, demonstrating the susceptibility of the 2-naphthylester group.

CO (1 atm), Pd(OAc) , dppp, O 2 O Bn 2-(trimethylsilyl)ethanol, Et3N, DMF, 80 °C Bn O O O OTf 79% SiMe 15 16 3 O

HO H2 (1 atm), 10% Pd/C, THF 1) oxalyl chloride, toluene O O 2) 2-naphthol, Et N, diethyl ether 83% SiMe3 3 17 O 62%

O O NaI, TMSCl, CH3CN, reflux O O 94% O SiMe3 COOH 18 11 O

Scheme 4

The synthesis of the carboxylic acid 12 is outlined in Scheme 5. An initial attempt using tert-butyldimethylsilyl as a protecting group of the carboxylic acid function in 19 was made, 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 was shown to be too unstable for isolation in satisfactory yields. Instead a reaction of compound 19 with 2-(trimethylsilyl)ethanol with DCC, providing 20, was followed by a direct esterification with 2-naphthol and potassium carbonate affording ester 21 in 88% yield over two steps. In contrast to the deprotection of 18, the target ester 12 could be achieved in this system with TBAF furnishing the ester in 71% yield.

O 2-(trimethylsilyl)ethanol, DCC, 2-naphthoic acid, BrCH2 COOH BrCH2 DMAP, CH2Cl2, r.t. O K2CO3, acetone, r.t. 19 20 SiMe3 O O

O TBAF, O O 1M in THF, r.t. 21 SiMe3 12 COOH O

Scheme 5

The target esters mentioned above, were all tested in vitro in esterase assays to give an indication 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 - 38 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR ester for further incorporation into a heterocyclic core more structurally similar to other inhibitors 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 different esterases in vitro (Section 6.1) we selected the ester group anticipated to be the most stable towards hydrolysis (compounds 2 and 3, respectively) to ensure that we would not initially observe ester hydrolysis due to chemical instability.

Commercially available 2,4-diamino-6-nitroquinazoline was reduced under 4 atmospheres (60 psi) of hydrogen over 10% palladium on charcoal using a Parr apparatus forming 2,4,6-triaminoquinazoline180 (22a) in a quantitative yield (Scheme 6). According to the same reference, tin(II)chloride in concentrated hydrochloric acid could be used in the same transformation. However, the conditions were found to be unreliable with appreciable amounts of hydrolyzed product detected (Scheme 6). By virtue of the fact that the free electron pair on the exocyclic nitrogen in the 4-position of the 2,4-diaminoquinazoline has the capability to be delocalized into the ring system this position is very susceptible towards nucleophilic attack. Another approach for the reduction of aromatic nitro groups using sodium 192 borohydride (NaBH4) and 2 M CuSO4 in ethanol did not work at all on this substrate.

O NH2 NH2 NH2 NO2 NH2 HN SnCl2, N H2, 10% Pd/C, N conc. HCl AcOH, DMF, 4 atm H2N N H2N N H2N N + 22a NH2 NH N 2

H2N N 22a Scheme 6

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

- 39 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

NH2 NH2 NH CN 2 N N NaNO2, 2 M HCl, KCN, CuSO , 0 °C H2N N 4 H2N N 22a 23a

NH2 CHO 75% formic acid, reflux N

H2N N 24a

Scheme 7

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

NH2 NHCOC(CH3)3 CHO CHO N pivalic anhydride, DMF N 80 °C H2N N (H3C)3COCHN N 24a 25

NaClO2, NaBH4 ... NaH2PO4 1H2O NHPiv NHPiv CH OH COOH N 2 N

PivHN N PivHN N 28 26

Scheme 8

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

- 40 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

NHPiv O NH2 O ... 1) oxalyl chloride N O NH3, N O 26 2) Et3N, benzyl alcohol dioxane PivHN N H2N N 27 30a

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 to concurrent ester cleavage at a stage where small amounts of the monopivaloyl-protected amine still remained. The yields varied between 40-60% in this last step.

NHPiv O NH2 O

benzoyl chloride, Et3N N O NH , 28 3 N O dioxane PivHN N 29 H2N N 31a

Scheme 10

The probable metabolites (hydrolyzed 30a and 31a, respectively) were also synthesized 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 analogue 25, furnishing 32a and 33a, respectively. With reduction to 33a over-reduction of the alcohol to the 6-methylanalogue was detected when too long reaction times were used.194

NH2 CHO N

H N N 2 24a NaClO2, . NaH2PO4 1H2O NaBH4

NH2 NH2 COOH CH OH N N 2

H2N N H2N N 32a 33a

Scheme 11

Since the idea behind using a protecting group was to allow easy handling without interfering too much with the total yield, we decided to search for other strategies. A control reaction was run confirming that direct esterification of 2,4-diaminoquinazoline-6-methanol, - 41 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

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

NH2 NH2 O X X N Br N OAr + ArCOOH H2N N Y H2N N Y

34a: X, Y = CH X Y Ar b: X = CH; Y = N c: X = CCH ; Y = N 3 31b: CH N phenyl d: X, Y = N 31c: CCH3 N phenyl 31d: N N phenyl 35a: CH CH 2',5'-(OCH3)2-phenyl 36a: CH CH 3',4'-(OCH3)2-phenyl 37a: CH CH 3',5'-(OCH3)2-phenyl 38a: CH CH 2',3',4'-(OCH3)3-phenyl 39a: CH CH 3',4',5'-(OCH3)3-phenyl 40a: CH CH 3',5'-(CH3)2-phenyl 41a: CH CH 1-naphthyl 42a: CH CH 2-naphthyl 43a: CH CH 9-phenantrenyl 44a: CH CH 9-anthracenyl 45a: CH CH 9-fluorenyl 46a: CH CH 2'-benzylphenyl 47a: CH CH 2'-biphenyl 48a: CH CH 4'-biphenyl 49a: CH CH 3'-iodophenyl 50a: CH CH diphenylmethyl 51a: CH CH 2-thienyl 52a: CH CH 3-thienyl 53a: CH CH 2-furyl 54a: CH CH 3-furyl 55a: CH CH CH2Ph

Scheme 12

General methods for the transforming of the alcohol to the corresponding bromide 169 employed in antifolate chemistry were dibromotriphenylphosphorane (Ph3PBr2) in DMAc, 163,195 196 197 HBr in dioxane, PBr3 in THF, or HBr in AcOH, respectively. In my hands, the last procedure provided the best results for the bromination of the alcohols 33a-33d. After completion, 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 of 2,4-diaminoquinazoline-6-methyl acetate, due to its high reactivity of 34a as its hydrobromic salt. Immediate displacement of the bromide with the appropriate carboxylic acid using potassium 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

- 42 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR 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 to stir for longer times did not improve the yields and heating the reaction mixture did not speed up the reaction time. Neither attempts to esterify the bromides 34a-d in the absence of base 198 relying on an SN1-type reaction did work. Literature describes a method for the treatment of compound 34c with triethylamine in order to avoid rapid inactivation of the hydrobromide salt associated with the reactive bromomethyl compound by the benzoate anion. In my hands the triethylammonium salt of 34c was formed according to 1H NMR.

NH2 O R NH2 O Pd(PPh3)2Cl2, Na2CO3, (HO) B N O 2 DME/H O/EtOH (7:3:2), N O + 2 2 min, 140 °C H2N N R H2N N I RR 49a 56: R = H 58a: R = H 57: OCH3 59a: OCH3

Scheme 13

Due to the lack of commercially available 3-phenylbenzoic acids we decided to introduce some palladium chemistry into the synthesis of the phenyl derivatives 58a and 59a starting from the 3’-iodophenyl compound 49a (Scheme 13). The reaction of choice was the Suzuki reaction199,200 as it has become a valuable tool with respect to synthetic chemistry because of its simplicity as a method for the formation of new carbon-carbon bonds and easy and clean work-up. The reaction relies on the reaction between an organoboron nucleophile and an organic halide or pseudohalide (i.e., a group with similar properties to the good leaving group of the halide, e.g., triflates.). Instead of using conventional heating the relatively new methodology associated with microwave chemistry was applied. The heating of the reaction mixture in a focused single-mode oven is of great advantage to the organic chemist because it enables a fast and controlled heating. When applying microwave-promoted heating the reaction 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 49a and phenylboronic acid (56), and 2,6-dimethoxyphenylboronic acid (57),* respectively. Different conditions were explored in order to find the optimal conditions for this specific reaction. 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 provide catalysts for the evaluation. Sodium or cesium carbonate in stock solutions were used as bases, and DME/H2O/EtOH (7:3:2) or DMF as solvents. The most efficient reaction conditions 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, Uppsala University, Sweden. - 43 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR according to LC-MS, whereas after 80 s microwave irradiation at 140 °C LC-MS still showed the 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 was found in the reactions performed in DMF. The temperature profile versus time from the experiment is shown in Figure 27, which demonstrates one of the advantages with this application compared to traditional heating in terms of rapid and effective heating of the reaction medium.

200

150

C) 100 ° ° ° °

50

Temp. ( 0 0 25 50 75 100 125

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-aromatic moieties using slightly modified literature procedures137,153 (Scheme 14). Compounds 60b-c were synthesized by a condensation reaction mediated by hydrochloric acid with malononitrile and triethyl orthoformate, or triethyl orthoacetate, respectively. Dechlorination of 60b-c yielded 61b-c, which upon cyclization with guanidine in refluxing ethanol, afforded the 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 in comparison to 23b (~80%, 2 days) to form the target molecule, presumably due to steric hindrance when performing the ring closure. Treatment with Ni-Al alloy203 or Raney Ni in refluxing formic acid converted the nitriles 23b-c into the aldehydes 24b-c, which were further reduced to the corresponding alcohols 33b-c by NaBH4 in methanol. For the synthesis of 31d commercially available 2,4-diaminopteridine-6-methanol (33d) was used. The alcohols 33b-d were all brominated as described above for 33a to give 34b-d.

- 44 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

R R NH .HCl NC CN NC CN RC(OEt)3 1. C6H5N H , 10% Pd/C, H2NNH2 + 2 2 CH (CN) 2. HCl Et3N, DMF 2 2 H2NClN H2N N 60b: R = H 61b: R = H 60c: R = CH3 61c: R = CH3

NH2 NH2 CHO CH2Br Ni-Al alloy N 1) NaBH4 N 75% formic acid NH2 R 2) 30% HBr i AcOH . HBr H2N NN H N NN CN 2 N 24b 34b

H2N NN NH2 CH3 NH2 CH3 23b: R = H Raney Ni CHO CH Br N 1) NaBH4 N 2 23c: R = CH3 conc. formic acid 2) 30% HBr i AcOH . HBr H2N NN H2N NN 24c 34c

Scheme 14

In order to synthesize inhibitors with a four-atom long central chain three compounds were made. The synthesis of compound 55a is depicted in Scheme 12. Ester 63a, was prepared by diazotization of 2,4,6-triaminoquinazoline 22a, with potassium iodide and sodium nitrite, yielding the iodo-compound 62a.204,205 A palladium-catalyzed Heck reaction206-208 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 accomplished readily using hydrogen over Pd/C. However, an initial attempt with ammonium formate serving as the hydride donor, together with Pd/C in methanol resulted in the methyl ester instead of the expected phenyl ester.

NH2 NH2 NH2 I N NaNO2, HCl, 0 °C N KI H2N N H2N N 22a 62a

NH2 O

phenyl acrylate, Pd(OAc)2 N O P(o-tol) , Et N, 80 °C 63a 3 3 H2N N

NH2 O

10% Pd/C, H2 , DMF N O

H2N N 64a

Scheme 15

- 45 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

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

NH2 NH2

H , Raney Ni, N N 30% formaldehyde, N N 23a 2 H CH aniline NaCNBH3 3 H2N N H2N N 65a 66a

Scheme 16

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

O O Et3N S Cl S OCHO O O 67

O mCPBA, S O COOH DME O 68

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 of exploring 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 - 46 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR derivatives differed from MTX by the replacement of the methyleneamino group with an ester functionality. One of the compounds retained the pteridine core whereas with the other three derivatives this was replaced by the quinazoline. One compound was made with the goal of synthesizing an analogue that was not a substrate for FPGS (Section 1.4). Finally, we also wanted to synthesize an analogue with D-glutamic acid instead of the usual L-glutamic acid present in MTX.

Compounds 70-72210,211 were synthesized as outlined in Scheme 18. The synthetic route starts with the commercially available di-tert-butyl esters of L-and D-glutamic acid hydrochloride, and di-tert-butyl L-α-aminoadipate (69), respectively. Compound 69 was prepared according to a literature procedure.212 The protected amino acid derivatives were all allowed to react with 4-formylbenzoic acid using PyBOP and N,N-diisopropylethylamine or triethylamine, respectively, affording 70-72. The aldehydes 70-72 were oxidized immediately after purification to afford the carboxylic acids 73-75.

OHC t CO2 Bu n=1, L-isomer n=1, D-isomer CO H H NCOtBu 69: n=2, L-isomer 2 2 n 2

PyBOP, DIPEA or Et N O 3 O

H H HO H t t NCO2 Bu NaClO , NCO2 Bu n 2 n t NaH PO .1H O t O CO2 Bu 2 4 2 O CO2 Bu 70: n=1, L-isomer 73: n=1, L-isomer 71: n=1, D-isomer 74: n=1, D-isomer 72: n=2, L-isomer 75: n=2, L-isomer

Scheme 18

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

- 47 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

NH2 O X N Br HO H t NCO2 Bu H2N NX. n HBr t O CO2 Bu 34a: X=CH K2CO3, 73: n=1, L-isomer 34d: X=N DMF alt. DMAc 74: n=1, D-isomer 75: n=2, L-isomer NH2 O X N O H t NCO2 Bu H2N NX n t HCOOH O CO2 Bu 76a: X=CH, n=1, L-isomer alt. TFA 77a: X=CH, n=1, D-isomer 78a: X=CH, n=2, L-isomer 76d: X=N, n=1, L-isomer NH2 O X N O H NCO2H H2N NX n O CO2H

79a: X=CH, n=1, L-isomer 80a: X=CH, n=1, D-isomer 81a: X=CH, n=2, L-isomer 79d: X=N, n=1, L-isomer

Scheme 19

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

Solid phase synthesis was first introduced by Merrifield in the area of peptide synthesis in the early 1960s.213,214 A great advantage of solid phase synthesis over reactions in solution is the highly simplified procedure for product purification at each step of the target compound synthesis. In the ideal case, all employed soluble reagents can be removed completely and conveniently by filtration and washing of the solid resin, whereas the product remains covalently bound to the solid support. After completion of synthesis, the final compound is liberated from the solid support by using a cleaving agent with subsequent isolation of the compound (note: compare this to the unmasking procedure when using protection groups). It is important that the bond between the product and the carrier sufficiently stable towards all reagents and conditions used in the synthesis. The original idea behind solid phase synthesis was to use a solid support consisting of a water-insoluble polystyrene support (PS). Successful improvements within the area have been the development of the TentaGel resin, and the related ArgoGel, that combine the insoluble polystyrene support with a soluble poly(ethylene glycol) (PEG) polymer. TentaGel resin is based on a very small portion of cross-linked 1% divinylbenzene-polystyrene that is extensively grafted with long PEG spacers (50-60 ethylene oxide units). The PEG content is - 48 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR up to 70% of the resin weight. Therefore, the properties of the PEG chains determine the mechanical, physicochemical behavior of the resin. Due to the fact that this long and flexible spacer is well separated from the PS matrix, the reactions performed on TentaGel exhibit more ‘solution-like’ properties as compared to pure polystyrene beads. The polymer must be solvated properly by the solvents of choice and in addition, the resin must swell sufficiently in order to gain access to the internal surface of the beads. Dichloromethane and DMF are the two solvents most often used to achieve a good loading capacity. The attachment of the molecule to the solid phase is achieved through a cleavable linker.215,216 A linker can be described as a bifunctional protecting group. It is attached to the molecule by a bond that is labile to the cleavage conditions. On the other side, the linker is attached to the solid phase through a more stable bond. The ‘Wang’ linker217 was one of the first linkers introduced and is cleaved under acidic conditions, i.e., 50% TFA. At present a wide range of linker systems218 on different supports is commercially available, and these can be cleaved under different conditions and by various reagents.

NH2 X N Br 34a: X = CH 1) tert-butyl alcohol, Cl O HO . 34d: X = N pyridine H2NNX HBr CO2t-Bu 2) KOH O Cl O Cs2CO3, DMF 82

NH2 O NH2 O X X N O TFA N O

NX H2N CO2t-Bu H2N NX CO2H 83a: X = CH 84a: X = CH 83d: X = N 84d: X = N

Scheme 20

Although we had concluded previously that solid phase synthesis could be difficult to perform with the reactions of these ester-containing DHFR inhibitors, we still wanted to elucidate 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 was prepared from terephthaloyl chloride in 53% yield over two steps (Scheme 20). Cesium carbonate was used as base in the couplings of compounds 34a-d, respectively, providing the esters 83a and 83d. Selective hydrolysis of the tert-butoxy group in 83a and 83d by TFA rendered the zwitterionic compounds 84a and 84d. Hence, esterification of the commercially available Fmoc-Glu-OtBu was conducted on ArgoGel with the acid labile Wang-linker as solid support (Scheme 21).

- 49 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

84a or 84d, 1) DCI, 4-pyrrolidinopyridine, CO2t-Bu CO2t-Bu PyBOP, DIPEA, CH2Cl2 FmocHN COOH + O H2NCO2 HO 2) 25% piperidine, DMF DMF Wang-linker + ArgoGel 85 ArgoGel incl. Wang

NH2 O NH2 O X X N O N O H 50% TFA/CH2Cl2 H NCO2 NCO2H H2N NX H2N NX O CO2t-Bu O CO2H ArgoGel incl. Wang 86a: X = CH 79a: X = CH 86d: X = N 79d: X = N Scheme 21

Fmoc-deprotection using 20% piperidine in DMF yielded the polymer supported glutamic acid derivative 85, which was further reacted with compound 84a and 84d, using PyBOP as coupling reagent, providing the amides 86a and 86d. The amide bond formation occurred 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 one step providing the target compounds 79a and 79d, respectively.

In an attempt to introduce the solid phase earlier in the synthetic route compound 85 was allowed to react with 4-formylbenzoic acid and then oxidized to the corresponding carboxylic 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 the resin probably due to large amounts of hydrobromic acid present in the reaction. Neither attempts using large excesses of base nor direct couplings of 2,4-diaminoquinazoline-6- methanol (33a) with the polymersupported benzoic acid derivative 87 were successful. Earlier experience showed the alcohol in 33a to be a very poor nucleophile, which would explain the very low yield in the coupling step according to 13C NMR spectroscopy. A later control reaction was performed to introduce solid phase chemistry directly on TentaGel S OH without using 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 no cleavage of the linker according to 13C NMR spectroscopy.

CO2t-Bu HOOC 1) 4-formylbenzoic acid H H2NCO2 NCO2 2) NaClO , NaH PO . H O 85 2 2 4 2 87 O CO2t-Bu

NH2 O NH2 N O N Br H H N N NCO2 . HBr 2 H2N N 34a O CO2t-Bu

Scheme 22 - 50 - DESIGN AND SYNTHESIS OF NEW INHIBITORS OF DHFR

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

OOMe OOMe

PEGHN O PEGHN O OH O O

NO2 NO2 O NO2

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 in vitro assays for the estimation of the relative rates of hydrolysis in six different systems containing esterases. The systems consisted of pig liver esterase (PLE), cholesterol esterase, human duodenal mucosa, human liver, rat liver, and human leukocytes, respectively. PLE is regarded as a non-specific carboxyl esterase, which also is commercially available, cheap and easy to handle. Cholesterol esterase, being localized in the intestinal lumen, is an enzyme to which every drug taken orally is subjected. Human duodenal mucosa is one of the first barriers 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 to use leukocytes from human blood, which are the potential targets for immunosuppressive drugs. The results are presented in Table 1. To summarize, we conclude that PLE hydrolyze all compounds, though at very different rates. The esters yielding naphthol or phenol after hydrolysis were, not surprisingly, the esters most prone to hydrolysis. Carboxylic acids as there 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 the esterases, which also is in accordance with literature.28 We confirm these results reporting that the 2,4-diaminoquinazoline esters 30a and 31a are poor substrates for the selected enzymes in vitro.

- 51 - BIOLOGICAL RESULTS - ENZYMATIC HYDROLYSIS IN VITRO

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

Pig Liver Cholesterol Human Human Rat Liver Human Compd No Esterase Esterase Duodenal Liver S9 S9 Leukocyte Mucosa 170 540 39 320 510 0.46 O 1 O (100) (100) (100) (100) (100) (100)

O 100 240 26 170 170 0.14 2 O (60) (43) (68) (54) (34) (31)

O 120 60 4.6 69 150 0.11 O 3 (70) (11) (12) (22) (29) (24)

150 1240 18 160 440 0.65 O 4 O (88) (227) (46) (51) (86) (141)

36 26 0.7 41 46 0.034 O 5 O (23) (4.7) (1.8) (13) (8.9) (7.3)

O 130 340 4.7 220 730 0.26 O 6 (78) (61) (12) (68) (143) (56)

COOH 2.1 0.3 13 100 0.006 O 7 n.q.

O (1.2) (0.6) (3.9) (20) (1.3) COOH O 36 10 0.7 9.1 11 0.014 8 O (21) (1.8) (1.7) (2.8) (2.1) (3.1)

O 0.5 4.1 O 9 n.q. n.q. n.q. n.q. COOH (0.3) (0.9)

2.6 0.2 3.1 290 0.025 O 10 n.q. O COOH (4.0) (1.1) (1.2) (59) (8.0)

8.6 0.4 5.4 0.003 O 11 n.q. n.q. O COOH (13) (0.2) (1.1) (1.0) O 11 13 0.5 11 27 0.12 O 12 COOH (6.4) (2.3) (1.2) (3.4) (5.1) (37)

NH2 O 0.1 2.5 N O 30a n.q. n.q. n.q. n.q. (0.1) (0.4) NH2 N

NH2 O 4.6 0.6 2.4 2.9 N O 31a n.q. n.q. (6.9) (1.5) (0.8) (0.6) NH2 N

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 and VI)

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

- 52 - BIOLOGICAL RESULTS - INHIBITORY ACTIVITIES OF NEW INHIBITORS OF DHFR

6.2.1 Inhibitory Activities and Selectivities of New Lipophilic Soft Drug Inhibitors for pcDHFR (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

NH2 O X N OAr

H2N N Y

IC50 (µM) Compd XY 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

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

- 53 - BIOLOGICAL RESULTS - INHIBITORY ACTIVITIES OF NEW INHIBITORS OF DHFR

Even though extensive research with crystallographic experiments in combination with computer modeling has been performed, very few new lipophilic inhibitors have been reported 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 of examining a) whether the inhibitory activity of DHFR remained after the introduction of the ester function; b) if we could obtain a selectivity for the fungal enzyme. Moreover, we wanted to explore c) the importance of the nitrogen substitution in the right heterocyclic ring for the pharmacophoric element. All the results from the inhibition experiments are presented in Table 2. First, methoxy groups were introduced in different positions in the original phenyl moiety (35a-39a), capable of accepting hydrogen bonds from amino acid residues in the active site of the enzyme. The compounds showed structural resemblance to the new potent antifolates TMQ and PTX (Figure 11 and Figure 12, respectively). All the compounds exhibited fair to good inhibitory activities against the enzymes, but most of all with a preference for rlDHFR. Replacing the methoxy substituents for methyl groups gave no observed difference in potency (cf. 37a and 40a). When examining the difference in the heterocyclic core (31a-d), compound 31c was found to be the best inhibitor within the series and was also associated with the most favorable selectivity. However, the metabolite 33c formed 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 ester moiety was well accepted by DHFR together with the poor inhibitory activity exhibited by the metabolite 33a, we chose the 2,4-diaminoquinazolines. In paper IV we wanted to explore different scaffolds in the right part of the molecule comprising larger groups, as compared to the 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 drug molecules.222 Among the lipophilic ester-containing compounds, the 1-naphthyl ester 41a was found to be the most potent inhibitor of pcDHFR. The fluorenyl compound (45a) exhibited the highest potency of all the inhibitors against the mammalian enzyme, even higher then the reference compounds 65a and 66a (Table 3). The phenantrene analogue 43a displayed the best combination of selectivity (1.4) and inhibitory activity. The 4’-biphenyl (48a) demonstrated the best selectivity ratio within the series. Unfortunately, the compound was associated with a relatively low potency. The 2-thienyl scaffold (51a) exhibited a reasonably good selectivity (0.93) along with rather high potency, which makes this type of compounds interesting for further investigations. Going up higher in length of the middle bridge the potency 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 the mammalian enzyme (46a and 58a). However, the trend seems quite obvious: if one inhibitor shows a good inhibitory activity against one of the enzymes it also follows the same pattern for the other enzyme.

- 54 - BIOLOGICAL RESULTS - INHIBITORY ACTIVITIES OF NEW INHIBITORS OF DHFR

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

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

IC50 (µM)

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

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 compound MTX, and compound 79a was therefore further evaluated in vivo (Section 6.3 and 6.4). The other compounds in the group displayed a 10-fold decrease in potency of rlDHFR. Thus, we conclude that the ester functionality, with an additional atom in the bridge as compared to MTX, 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 Recombinant Human DHFR.

IC50 (µM) Compd rlDHFR rec. hDHFR

79a 0.019 0.12 80a 0.15 0.078

81a 0.16 0.14 79d 0.12 2.1 MTX 0.0025 0.002

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

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

For this project we wished to evaluate compound 79a in an experimental model of IBD. The compound is structurally similar to MTX, which is used in the treatment of IBD due to its anti-inflammatory and immunosuppressive properties (Section 1.6.2). However, the central part of the structure had been changed to an ester functionality, which makes it a substrate for controlled metabolism by the endogenous esterases. As described in Section 6.1 and also depicted in Table 1, two lipophilic inhibitors of DHFR was evaluated in vitro at an early stage of the project for their susceptibility to the esterases in human leukocytes and human duodenal mucosa (compound 30a and 31a). Both compounds were shown to be stable in the system used. Encouraged by the pharmacokinetics in rat in vivo (Section 8) together with the in vitro pharmacokinetics of compound 79a in blood (Section 8), we wanted to elucidate 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-dinitrobenzene sulfonic acid), a model that is inexpensive and easy to develop. The model is a milder variant of the well-known TNBS-model (2,4,6-trinitrobenzene sulfonic acid).223,224 It is extensively used as a screening tool for potential IBD therapies and their mechanism of action. Another well-established chemically-induced model is the DSS-model (dextran sodium sulfate). Yet, the last model suffers from the lack of rapidly proliferating cells (monocytes and macrophages, compared to leukocytes in the case of DNBS). Additionally, the DNBS model shows a ‘Crohn’s like’ pathogenesis in which MTX and budesonide, respectively, tend to show better results as compared to the DSS-model.

140

120

100

Colon weight 80 MPO

IS 60 % of Controls

40

20

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

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

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

The results illustrate that daily administration of compound 79a (15 mg/kg/day) reduces mucosal damage (colon weight) and myeloperoxidase (MPO) activity (a marker of neutrophil infiltration in the gut), as shown in Figure 29. Elevated levels of MPO are recognized in activated immunocells whereas healthy animals show zero-values. The obtained data shows lower concentrations of MPO in treatment of 79a (15 mg/kg/day) as compared to budesonide, and lower concentrations still compared to MTX. Encouragingly, also the inflammation 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 interesting observation with the mice treated with 79a (15 mg/kg/day) was that the animals seemed to be vigorous, eating and drinking as normal. Additionally, they appeared to be in very good condition, 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 this high dose, interfere with the regeneration of the epithelium cells in the intestinal mucosa, and thereby the healing process. In contrast to MTX, compound 79a did not inhibit the cellproliferation from mouse spleen, suggesting different pharmacodynamics of the two compounds.

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

In addition to the evaluation of compound 79a in the mouse colitis model (paper V) it was also examined for its anti-arthritic properties in a rat arthritis model. Evidently, MTX is one of a very few drugs with anti-arthritic properties that work in both animal models and in the clinic. The models thus offer suitable in vivo systems for evaluation of new analogues to MTX. The antigen-induced arthritis model (AIA) in rat225,226 described in paper VI, is characterized 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 Dark Agouti strain animals are sensitized to methylated bovine serum albumin (mBSA). For sensitization mBSA is injected with Freund’s complete adjuvant (Sigma Chemicals Co.). The model is sensitive to oral tolerance, that is, when giving the anti-arthritic compound by oral administration the arthritis is inhibited, indicating a possible connection between the mucosal level in the gut and the antigen-presenting cells in the joint.227 AIA has previously been shown to be an MTX-sensitive model, although the effective dose is about ten times greater in rats and the onset of the anti-arthritic effect of MTX is shorter.124 An alternative to AIA is the well-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 bone erosion, which is generally not the case with RA.225 The latter method seems to be limited to only a few anti-arthritic compounds.229 However, it has proven to be MTX-sensitive, but is mostly associated with a high variability.

When we performed a pharmacokinetic study of compound 79a in the rat the data obtained revealed that it was quite stable in plasma (Section 8). Thereby it could serve as a - 57 - BIOLOGICAL RESULTS - EVALUATION OF A NEW POTENT DHFR INHIBITOR IN A RAT ARTHRITIS MODEL potential candidate for the arthritis assay. The results from the AIA model show that MTX was at least as effective following both p.o. and i.p. administration supporting previous theories about the action of MTX on immunocompetent cells in the mucosa.230,231 Although compound 79a was given in doses 25 times greater than the effective dose of MTX, no effect of the new compound could be detected. The negative results could be a) due to lack of polyglutamation, which seems to be necessary for the effect of MTX, and/or b) that the new compound does not function as a substrate for the same enzymes as MTX (with the exception of 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 affinity of a drug for a specific enzyme or receptor can be of a great value in the drug discovery process (Figure 1). A suitable drug should exhibit a high affinity to its target enzyme/receptor and at the same time should act only at the target enzyme and thereby be selective (see Section 3.2). In addition, selectivity is important when looking at different species, which is the case with antibacterial drugs, for example. Nowadays, computational methods can be employed to investigate these issues and serve as important tools in drug research. Advances in molecular biology and improved methods for the crystallization of proteins provided knowledge about the three dimensional structure of specific proteins. Hence, structure-based methods 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 new series of ligands can be difficult to perform, due to a lack of knowledge about the exact structure of the ligand-enzyme complex. Several methods have been developed in order to approach this problem.232-234 As mentioned earlier (Section 1.3) DHFR is an enzyme, which has been subjected to extensive characterization because of its relatively small size and ease of co-crystallization with new ligands. In this project, we have performed binding affinity predictions based on existing crystal structures of DHFR in complex with new potential inhibitors.

By molecular simulation methods, such as, molecular dynamics (MD), one can sample available conformations of the ligand and the receptor at a desired temperature and thereby calculate energy averages and thermodynamic properties. Free energy perturbation (FEP)235 techniques in combination with thermal averaging using MD allows us to calculate relative binding 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 it requires conformational sampling of hybrid molecules during gradual perturbation from one molecule to the other. However, it is often possible to obtain reliable relative affinities that

- 58 - MOLECULAR DYNAMICS include the effects of solvation and conformational changes. Due to the fact that FEP is a computationally demanding method an alternative method called the linear interaction energy (LIE) method has been developed,236 in which the absolute binding free energies can be estimated. LIE calculates the binding free energy as a linear function of the difference between the “bound” and “free” ligand from the average force field ligand-surrounding interaction energies obtained by MD simulations (Figure 30). The method is similar to FEP in that it relies on thermodynamic cycles and includes thermal conformational sampling. LIE is classified as a semi-empirical method, and is regarded as a less computationally demanding method as compared to FEP since it involves only simulations of two physical states (bound and free ligand) and thereby no transformation processes are needed in the calculations. The idea 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 subsequent quantification of its contribution to the total binding.

∆G inhibitor + enzyme(aq) bind inhibitor - enzyme(aq) Figure 30.

MTX consists of three major components: the pteridine ring, the p-aminobenzoyl group and the glutamic acid residue (Figure 10). The pteridine has been found to be deeply buried in the active site of the enzyme237 and forms more than half of the hydrogen bonds with DHFR. The conformation of the glutamate residue is determined by electrostatic interactions 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. The calculated results were compared to three ‘non-classical’ DHFR-inhibitors. We also tried to predict 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 large fluctuations due to its flexible glutamic acid residue, particularly in the free state, leading to difficulties in obtaining average values with good precision. It seemed to be difficult to handle ligands with multiple ionic groups, particularly if they were located at the ends of flexible chains, as in this case. The energies for the ‘non-classical’ inhibitors converged much more 1 rapidly than for MTX. Calculations on MTX also revealed that the pKa of N is about 5.7, which means that it is not protonated in solution at neutral pH, although it is known to be protonated in the complex with DHFR.238 Energy is thus required for protonation of the pteridines when binding to the enzyme, while the corresponding quinazolines are protonated at the appropriate pH of the solution used in the predictions. Surprisingly, the result also showed very small differences between the predicted binding affinities of compound 66a and the 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 the linker chain between the heterocyclic group and the phenyl ring (65a vs. 66a). A comparison of the quinazoline compounds with and without this methyl group yielded a 2.3 kcal/mol

- 59 - MOLECULAR DYNAMICS difference in favor of the N-methylated inhibitor. The methyl group binds in a hydrophobic pocket and this appears to reduce the flexibility of other parts of the inhibitor, thus favoring binding. The results also show that the compounds lacking the glutamic acid residue are predicted to exhibit a 105-106-fold lower affinity to the human enzyme. All simulations were performed with the co-factor NADPH present.

In paper III and IV it was intended to predict the individual selectivities of a series of similar inhibitors towards DHFR from rat liver and P. carinii, respectively, and further compare them to experimentally obtained results. Sequence analysis and structural alignment of pcDHFR and hDHFR were performed in paper III in order to compare the active sites of the two species. The results show significant differences in only a few amino acid residues close to the active site between the two species, which also is consistent with literature.141,144,157,239,240 Val115 in human DHFR corresponds to Ile123 in pcDHFR, which contributes to a slightly larger binding pocket in the 5-position of the human enzyme. When comparing 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, Phe31 and Asn64 in the human enzyme correspond to Ser24, Ile33, and Phe69 in P. carinii, respectively (Figure 31).

Ile123 Ser24 Val115 Asp21

NH2 O OCH N O 3 Glu32 Glu30 H2N N OCH3 39a OCH3

Ile33 Phe31 Phe69 Asn64

Figure 31. Compound 39a modelled into DHFR of both P. carinii (green) and the human enzyme (red). The picture 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 be attributed to a hydrogen bond formed between one of the methoxy-groups on the phenyl ring and the amide hydrogen of Asn64. Additionally, a simulation of compound 39a in complex with the human Asn64Phe mutant was performed in order to investigate the importance of Asn64. The calculations indicate that this interaction is important for the affinity, but may not - 60 - MOLECULAR DYNAMICS be the only explanation for the observed selectivity for the human enzyme. In hDHFR Phe31 may 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 different species to more bulky, lipophilic groups in different orientations, with varying flexibility. We described 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 empirical method for docking and scoring. The method relies on intermolecular interactions between the ligand and the rigid protein structure to find the optimal geometry for the ligand in the active site. The method then “scores” the strength of the interaction between the protein and the ligand in that binding conformation. The scoring function includes both polar and non- polar interactions, for example, hydrogen bonds, ionic interactions, hydrophobic, and aromatic interactions. Herein, a scoring function was used to determine the rank order of the selectivity indexes, that is, to calculate and compare the score obtained for a ligand when docked into DHFR from both human and P. carinii. The obtained scores did not differ significantly, 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 in inhibitory activities and selectivity were very small for these compounds, which made it difficult to draw clear conclusions. Nevertheless, we can summarize that the experimentally obtained IC50-values, in general with more selectivity against the mammalian enzyme, are consistent with the computationally obtained results.

Based on the results in paper II, we went further with the FEP method in paper VI for the calculations of the relative binding free energies of new ‘classical’ antifolates compared to MTX. The results from the FEP calculations with the 2,4-diaminoquinazoline compound 79a predicted the compound to have only a 10-fold lower affinity than MTX for the human enzyme. The calculated Ki-values of the inhibitors were compared with experimental data obtained from an inhibition assay performed in-house using recombinant human DHFR. The results obtained confirmed the calculated ranking of their inhibitory potencies, but showed a larger difference between MTX and compound 79d (the pteridine derivative most structurally similar to MTX) than predicted. However, additional experiments on rat liver DHFR ∗ performed by Queener showed IC50-values that were more consistent with the calculated ratios. The explanation for the poor IC50-value for compound 79d, could be explained by the demand for energy upon protonation before binding to the enzyme (vide supra). The binding of 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. - 61 - MOLECULAR DYNAMICS

Figure 32. Compound 79a (yellow) and MTX (green) docked into the active site of the human enzyme illustrating important 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 quantitatively the uptake of drugs by the body, their biotransformation, distribution, and elimination from the body’. Some of the important parameters are described below:

1) Absorption of a drug can be defined as the passage from its site of administration into the circulation. Drugs can be administered by enteral or parenteral route. With parenteral administration, e.g., intravenous or intramuscular routes, the gastrointestinal tracts are bypassed. 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 gaining access to the systemic circulation. The bioavailability of a drug is the fraction of the administered dose, which reaches the systemic circulation. After intravenous administration, the bioavailability is by definition 100%; for the other routes of administration, the bioavailability varies between zero and 100%. Bioavailability is influenced by several factors, such as drug characteristics (e.g., solubility, particle size, logD), formulation, changes at the site of administration (e.g., pH, blood flow), and the route of administration. Both the gastrointestinal tract wall and the liver are capable of metabolizing drugs. This ‘first pass effect’, 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 variable bioavailability. Another route of administration is inhalation, where the treatment can be both

- 62 - PHARMACOKINETICS for local and systemic use. It shows a rapid absorption, but the total dose absorbed is usually variable 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. time curve usually represents distribution to extravascular tissues.241

3) The half-life, T½, of a drug is characterized by two independent pharmacokinetic parameters – 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 the time point at which the concentration falls by one-half. Elimination of a drug covers both excretion (i.e., elimination of unchanged drug from the body), and biotransformation (metabolism). Renal excretion and hepatic biotransformation are the major routes of drug elimination and the primary pharmacokinetic parameter describing this elimination is clearance. Clearance is defined as the volume of blood or plasma, which is cleared of the drug per unit of time, most often expressed as mL min-1. For example the hepatic clearance of a drug is defined as the volume of blood perfused in the liver that is cleared of the drug per unit of 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 in the plasma/ blood or predominantly distributed into extravascular tissues. Thus it depends on plasma 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, we decided to substitute into the phenyl ring to enable additional investigations in an in vivo screening assay. In paper III and VI we evaluated the in vivo pharmacokinetics after oral and intravenous administration in male SD rats of the even more lipophilic ester 39a and its polar analogue 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 administration site. Plasma was analyzed for content of the compounds, as well as their metabolites formed after enzymatic hydrolysis, using the LC-MS/MS technique. We found the methotrexate analogue 79a to be relatively inert in vivo displaying similar kinetics to MTX after intravenous administration. The clearance was estimated to be 15 mL/min/kg (MTX: 11 mL/min/kg) and the terminal T½ to be 1h (MTX: 0.9 h). The volume of distribution at steady state 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 the results, we conclude that compound 79a has pharmacokinetics similar to MTX. The obtained data prompted us to believe that compound 79a is sufficiently metabolically stable in vivo to retain the desired bioactivity.

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10000

1000 nmol/L)

100

10 Concentration (

1 0 20 40 60 80 100 120 Time (min) Time (min)

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

In contrast to 79a, the data obtained from the more lipophilic analogue 39a demonstrates much faster pharmacokinetics, which is also more in accordance with the soft drug concept (Figure 33). The mean concentration versus time data of compound 39a was fitted 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 to contribute, each with 50% of the total area under the curve. However, after longer infusion times with 39a the terminal post-infusion phase will become the dominating phase. The very degree of clearance, approximately 200 mL/min/kg, is in close proximity to the cardiac output for the rat242 reflecting a high propensity of the drug to undergo a fast metabolism in vivo. The high clearance could be explained by extensive extra-hepatic metabolism, which is in accordance with the high esterase activity in different tissues in addition to the liver.19 It is important to mention in this context, compounds 39a and 79a also have been evaluated in rat blood in vitro in a pilot study. In this study, no hydrolysis was detected after 45 min. The experiments were run both in the presence of, and in the absence of a non-specific esterase inhibitor (phenylmethylsulfonyl fluoride, PMSF).

When the animals were subjected to an oral dose of compound 39a we were unable to detect any traces of the ester in plasma two minutes. However, the metabolite 33a was detected immediately, following both p.o. and i.p. administration. Based on these findings, we wish to iterate that compound 39a is a potentially soft drug candidate, used for topical application.

∗ The analyses were performed with the computer program WinNonlin* (ver. 1.5, Pharsight Corporation) using the weight 1/(ypred2). The goodness of fit was evaluated by the Akaike Information Criterion (AIC) and visual inspection of residual plots.

- 64 - CONCLUDING REMARKS

9 CONCLUDING REMARKS

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

• Fourteen aromatic model esters were evaluated as esterase substrates in vitro, in esterase systems comprising commercially available esterases and cell/ tissue systems, for example, human leukocytes and human intestinal mucosa homogenates. As expected, the lipophilic esters were good substrates for the esterases. Incorporating more polar features into the molecules made the compounds poor substrates for the esterases. 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 good inhibitory 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 poor inhibitors of the mammalian enzyme, which is a pre-requisite for the soft drug concept.

• One of the esters (compound 39a) with structurally similarity to TMQ, used in the treatment 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 high plasma clearance. This suggests that soft drug analogues of antifolates may be obtainable. These analogues are intended for topical administration in animal model of PCP.

NH2 O OCH N O 3

H2N N OCH3 OCH 39a 3

• In spite of the fact that we used molecular modeling in the design process, we were not successful in the identification of new inhibitors with selectivity for P. carinii DHFR. 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

- 65 - CONCLUDING REMARKS

respect to rat liver DHFR. The compound was stable towards enzymatic hydrolysis as

deduced by the relatively long plasma half-life of the compound (T½: 0.9 h) and was used for topical treatment in an experimental model of IBD. The compound exhibited good anti-inflammatory properties in a colitis model in mice, with even better results than with established drugs. Compound 79a was further evaluated in an arthritic model in rat, but with unsatisfactory results.

NH2 O

N O H N COOH H2N N 79a O COOH

10 SUMMARY

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

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

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

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

- 66 - ACKNOWLEDGEMENTS

11 ACKNOWLEDGEMENTS

This work was carried out at Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Faculty of Pharmacy, Uppsala University. The Swedish Academy of Pharmaceutical Science is acknowledged for allowing me to attend international scientific meetings and national courses. Acta Chemica Scandinavica for making it possible to accompany national conferences. Financial support was obtained from the Swedish Foundation for Strategic Research (SSF). I would like to express my deepest thanks to all friends and colleagues who have been there for me during the years: Professor Anders Hallberg, my supervisor and friend, for your support and never-ending encouragement. 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 at the department. Docent Anette M. Johansson, my former co-advisor, for friendly and inspiring guidance and for constructive criticism of the thesis. Thank you also for always listening and being there when I needed in the very beginning of the project. Professor Kristina Luthman for your strong enthusiasm in the teaching context. Also, for nice discussions during the years. Docent Uno Svensson, for your inherent expertise in medicinal chemistry and also your patience with all the amount of teaching. Docent Anders Tunek for your belief in the ideas of soft drugs in the early stages of the project. My co-authors Drs Björn Mellgård, Sven E. Andersson, Göte Swedberg, and also Åsa Persson, Sofie Ohlson, and Karin Sjödin. Professor Johan Åqvist, Dr John Marelius and Karin Kolmodin for good collaboration and for being patient with my weak understanding in computational chemistry. Professor Sherry F. Queener for generously providing us with pharmacological data concerning the inhibitory activities of dihydrofolate reductase. Docents Eva Åkerblom and Gunnar Lindeberg for introducing me to the field of solid phase chemistry. 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 there when you are needed. Good luck in the future! Lotta Wahlberg for doing an excellent job and for nice discussions. Marianne Åström for skillful secretarial assistance and for always being willing to help. Karin Olsson for excellent cleaning of all the glassware. The staff at the BMC library for all help with articles during the years. Drs Johan Hultén and Mats Larhed, for constructive criticism of the thesis.

- 67 - ACKNOWLEDGEMENTS

Dr Nick Bonham, for constructive criticism and excellent linguistic revision of the thesis. All linguistically mistakes are my own last desperate changes and are definitely not his responsibility. 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 Lena Ripa, Annika Jenmalm-Jensen, and Charlotta Liljebris for nice discussions concerning everything. 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 Petra Johannesson. The former members of B5:305a, especially Drs Martin Hedberg, Ulf Appelberg, Berit Backlund-Höök, Patrizia Caldirola, Johan Hultén, Neeraj Garg, Nick Bonham, and Tero Linnanen. Erika Larsson, Katarina Sandberg and Matts Balgård, for contributing with skilful technical assistance 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 BMC very enjoyable. The students that I have been teaching during the years, for making graduate work even more enjoyable 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 familjen Grüwenz och även familjen Wulff. Tack även till personalen på Pilfinkens och Lingonbackens montessoriförskolor. Mina svärföräldrar; Anita och Bosse för att ni har försökt vara förstående under den långa tiden som doktorand. Ett speciellt tack till dig Anita, för all hjälp det sista halvåret med barnen. 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. Tack Jocke 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 jag får 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 chansen att “komma igång” igen?!

- 68 - REFERENCES

12 REFERENCES

1. Bodor, N. In Design of Biopharmaceutical Properties Through Prodrugs and Analogs; Roche, E. B., Ed.; Acad. Pharmaceut. Sci.: Washington, DC, 1977, pp 98-135. 2. For a review, see: Bodor, N.; Buchwald, P. Med. Res. Rev. 2000, 20, 58-101. 3. Gillette, J. R. Drug Metab. Rev. 1979, 10, 59-87. 4. Albert, A. Nature 1958, 182, 421-423. 5. Bundgaard, H. (Ed.) Design of Prodrugs. Elsevier: Amsterdam, 1985. 6. Bodor, N.; Kaminski, J. J. Annu. Rep. Med. Chem. 1987, 22, 303-313. 7. Stella, V. J. Adv. Drug Deliv. Rev. 1996, 19, 111-114. 8. Silverman, R. B. In The Organic Chemistry of Drug Design and Drug Action; Academic Press: San Diego, 1992; pp 353-401. 9. Heymann, E. In Metabolic Basis of Detoxication. 1; Jakoby, W. B.; Bend, J. R.; Caldwell, J., Eds.; Academic: New York, 1982, pp 229-245. 10. Williams, F. M. Clin. Pharmacokinet. 1985, 10, 392-403. 11. For a review, see: Satoh, T.; Hosokawa, M. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 257-288. 12. Heymann, E. In Enzymatic Basis of Detoxication. 2; Jakoby, W. B., Ed.; Academic: New York, 1980, pp 291-323. 13. Satoh, T. In Reviews in Biochemical Toxicology. 8; Bend, J. R.; Hodgson, B. E.; Philpot, R. M., Eds.; Elsevier: New York, 1987, pp 155-181. 14. Junge, W.; Krisch, K. CRC Crit. Rev. Toxicol. 1975, 3, 371-435. 15. Buch, H.; Buzello, W.; Heymann, E.; Krisch, K. Biochem. Pharmacol. 1969, 18, 801-811. 16. Fonnum, F.; Sterri, S. H.; Aas, P.; Johnsen, H. Fundam. Appl. Toxicol. 1985, 5, S29-S38. 17. Krisch, K. In The Enzymes. 5; Boyer, P. D., Ed.; Academic Press: New York, 1971, pp 43-69. 18. Junge, W.; Heymann, E.; Krisch, K.; Hollandt, H. Arch. Biochem. Biophys. 1974, 165, 749-763. 19. Leinweber, F. J. Drug Metab. Rev. 1987, 18, 379-439. 20. Walker, C. H.; Mackness, M. I. Biochem. Pharmacol. 1983, 32, 3265-3269. 21. Morgan, E. W.; Yan, B.; Greenway, D.; Petersen, D. R.; Parkinson, A. Arch. Biochem. Biophys. 1994, 315, 495-512. 22. Quon, C. Y.; Stampfli, H. F. Drug Metab. Dispos. Biol. Fate Chem. 1985, 13, 420-424. 23. Quon, C. Y.; Mai, K.; Patil, G.; Stampfli, H. F. Drug Metab. Dispos. Biol. Fate Chem. 1988, 16, 425- 428. 24. Minagawa, T.; Kohno, Y.; Suwa, T.; Tsuji, A. Biochem. Pharmacol. 1995, 49, 1361-1365. 25. Chappell, W. R.; Mordenti, J. Adv. Drug Res. 1991, 20, 1-116. 26. Campbell, D. B. Ann. N. Y. Acad. Sci. 1996, 801, 116-135. 27. Cygler, M.; Schrag, J. D.; Sussman, J. L.; Harel, M.; Silman, I.; Gentry, M. K.; Doctor, B. P. Protein Sci. 1993, 2, 366-382. 28. Durrer, A.; Wernly-Chung, G. N.; Boss, G.; Testa, B. Xenobiotica 1992, 22, 273-282. 29. Tafi, A.; van Almsick, A.; Corelli, F.; Crusco, M.; Laumen, K. E.; Schneider, M. P.; Botta, M. J. Org. Chem. 2000, 65, 3659-3665, and references therein. 30. Durrer, A.; Walther, B.; Racciatti, A.; Boss, G.; Testa, B. Pharm. Res. 1991, 8, 832-839. 31. Hattori, K.; Kamio, M.; Nakajima, E.; Oshima, T.; Satoh, T.; Kitagawa, H. Biochem. Pharmacol. 1981, 30, 2051-2056. 32. Kawaguchi, T.; Suzuki, Y.; Nakahara, Y.; Nambu, N.; Nagai, T. Chem. Pharm. Bull. (Tokyo) 1985, 33, 301-307. 33. Maksay, G.; Otvos, L. Drug Metab. Rev. 1983, 14, 1165-1192. 34. van de Waterbeemd, H.; Testa, B.; Marrel, C.; Cooper, D. R.; Jenner, P.; Marsden, C. D. Drug Des. Deliv. 1987, 2, 135-143. 35. Anderson, B. D.; Conradi, R. A.; Spilman, C. H.; Forbes, A. D. J. Pharm. Sci. 1985, 74, 382-387. 36. Feldman, P. L.; James, M. K.; Brackeen, M. F.; Bilotta, J. M.; Schuster, S. V.; Lahey, A. P.; Lutz, M. W.; Johnson, M. R.; Leighton, H. J. J. Med. Chem. 1991, 34, 2202-2208. 37. Nielsen, N. M.; Bundgaard, H. J. Med. Chem. 1989, 32, 727-734. - 69 - REFERENCES

38. Nielsen, N. M.; Bundgaard, H. Int. J. Pharm. 1987, 39, 75-85. 39. Bundgaard, H.; Buur, A.; Chang, S. C.; Lee, V. H. L. Int. J. Pharm. 1988, 46, 77-88. 40. Altomare, C.; Carotti, A.; Cellamare, S.; Ferappi, M.; Cagiano, R.; Renna, G. Int. J. Pharm. 1988, 48, 91-102. 41. Roth, B. Fed. Proc. 1986, 45, 2765-2772. 42. Falco, E. A.; Hitchings, G. H.; Russell, P. B.; VanderWert, H. Nature 1949, 164, 107. 43. Burchall, J. J.; Hitchings, G. H. Mol. Pharmacol. 1965, 1, 126-136. 44. Jolivet, J.; Cowan, K. H.; Curt, G. A.; Clendeninn, N. J.; Chabner, B. A. N. Engl. J. Med. 1983, 309, 1094-1104. 45. Kozarek, R. A.; Patterson, D. J.; Gelfand, M. D.; Botoman, V. A.; Ball, T. J.; Wilske, K. R. Ann. Intern. Med. 1989, 110, 353-356. 46. Weinblatt, M. E.; Coblyn, J. S.; Fox, D. A.; Fraser, P. A.; Holdsworth, D. E.; Glass, D. N.; Trentham, D. E. N. Engl. J. Med. 1985, 312, 818-822. 47. Weinstein, G. D.; Frost, P. Arch. Dermatol. 1971, 103, 33-38. 48. Abu-Shakra, M.; Gladman, D. D.; Thorne, J. C.; Long, J.; Gough, J.; Farewell, V. T. J. Rheumatol. 1995, 22, 241-245. 49. Mullarkey, M. F.; Blumenstein, B. A.; Andrade, W. P.; Bailey, G. A.; Olason, I.; Wetzel, C. E. N. Engl. J. Med. 1988, 318, 603-607. 50. Green, E.; Demos, C. H. In Folate Antagonists as Therapeutic Agents. 2; Sirotnak, F. M.; Burchall, J. J.; Ensminger, W. B.; Montgomery, J. A., Eds.; Academic Press: Orlando, 1984, pp 191-249. 51. Ferone, R.; Burchall, J. J.; Hitchings, G. H. Mol. Pharmacol. 1969, 5, 49-59. 52. Kan, S. C.; Siddiqui, W. A. J. Protozool. 1979, 26, 660-664. 53. Maroun, J. Semin. Oncol. 1988, 15, 17-21. 54. Allegra, C. J.; Chabner, B. A.; Tuazon, C. U.; Ogata Arakaki, D.; Baird, B.; Drake, J. C.; Simmons, J. T.; Lack, E. E.; Shelhamer, J. H.; Balis, F.; Walker, R.; Kovacs, J. A.; Clifford Lane, H.; Masur, H. N. Engl. J. Med. 1987, 317, 978-985. 55. Blaney, J. M.; Hansch, C.; Silipo, C.; Vittoria, A. Chem. Rev. 1984, 84, 333-407. 56. Kisliuk, R. L. In Antifolate Drugs in Cancer Therapy. Jackman, A., Ed.; Human Press: Totowa, 1999, pp 13-36. 57. Ratanasthien, K.; Blair, J. A.; Leeming, R. J.; Cooke, W. T.; Melikian, V. J. Clin. Pathol. 1977, 30, 438-448. 58. Grem, J. L.; Takimoto, C. H.; Multani, P.; Chu, E.; Ryan, D.; Chabner, B. A.; Allegra, C. J.; Johnston, P. G. In Cancer Chemotherapy and Biological Response Modifiers; Pinedo, H. M.; Longo, D. L.; Chabner, B. A., Eds.; Elsevier Science 1999, pp 1-38. 59. Barredo, J.; Moran, R. G. Mol. Pharmacol. 1992, 42, 687-694. 60. Johnson, T. B.; Nair, M. G.; Galivan, J. Cancer Res. 1988, 48, 2426-2431. 61. Allegra, C. J. In Cancer Chemotherapy: Principles and Practice. Chabner, B.; Collins, J., Eds.; Lippincott: Philadelphia, 1990, pp 110-153. 62. Balinska, M.; Galivan, J.; Coward, J. K. Cancer Res. 1981, 41, 2751-2756. 63. Coward, J. K.; Parameswaran, K. N.; Cashmore, A. R.; Bertino, J. R. Biochemistry 1974, 13, 3899- 3903. 64. Kumar, P.; Kisliuk, R. L.; Gaumont, Y.; Freisheim, J. H.; Nair, M. G. Biochem. Pharmacol. 1989, 38, 541-543. 65. Kisliuk, R. L.; Gaumont, Y.; Baugh, C. M.; Galivan, J.; Maley, G. F.; Maley, F. In Chemistry and Biology of Pteridines. Kisliuk, R. L.; Brown, G. M., Eds.; Elsevier North-Holland: New York, 1979, pp 261. 66. Allegra, C. J.; Chabner, B. A.; Tuazon, C. U.; Ogata Arakaki, D.; Baird, B.; Drake, J. C.; Masur, H. Semin. Oncol. 1988, 15, 46-49. 67. Allegra, C. J.; Drake, J. C.; Jolivet, J.; Chabner, B. A. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 4881- 4885. 68. Baggott, J. E.; Vaughn, W. H.; Hudson, B. B. Biochem. J. 1986, 236, 193-200. 69. Cronstein, B. N.; Naime, D.; Ostad, E. J. Clin. Invest. 1993, 92, 2675-2682.

- 70 - REFERENCES

70. Egan, L. J.; Sandborn, W. J.; Mays, D. C.; Tremaine, W. J.; Lipsky, J. J. Inflamm. Bowel Dis. 1999, 5, 167-173. 71. Urakawa, K.; Mihara, M.; Suzuki, T.; Kawamura, A.; Akamatsu, K.; Takeda, Y.; Kamatani, N. Immunopharmacology 2000, 48, 137-144. 72. Seeger, D. R.; Cosulich, D. B.; Smith Jr, J. M.; Hultquist, M. E. J. Am. Chem. Soc. 1949, 71, 1753- 1758. 73. Genestier, L.; Paillot, R.; Fournel, S.; Ferraro, C.; Miossec, P.; Revillard, J. P. J. Clin. Invest. 1998, 102, 322-328. 74. Genestier, L.; Paillot, R.; Quemeneur, L.; Izeradjene, K.; Revillard, J. P. Immunopharmacology 2000, 47, 247-257. 75. Bertino, J. R.; Mini, E.; Sobrero, A.; Moroson, B. A.; Love, T.; Jastreboff, M.; Carmen, M.; Srimatkandada, S.; Dube, S. K. In Advances in Enzyme Regulation. 24; Weber, G., Ed.; Pergamon: New York, 1985, pp 3-12. 76. Bertino, J. R.; Srimatkandada, S.; Carman, M. D.; Mini, E.; Jastreboff, M.; Moroson, B. A.; Dube, S. K. In Modern Trends in Human Leukemia VI; Neth, R.; Gallo, R. C.; Greaves, M. F.; Janka, G., Eds.; Springer-Verlag: Berlin, 1985. 77. Elslager, E. F.; Davoll, J. In Lectures in Heterocyclic Chemistry. 2, Castle, R. N.; Townsend, L. B., Eds.; Hetero Corp. Orem: UT, 1974, pp S97-S133. 78. Elslager, E. F.; Johnson, J. L.; Werbel, L. M. J. Med. Chem. 1983, 26, 1753-1760. 79. Bertino, J. R.; Sawacki, W. L.; Moroson, B. A.; Cashmore, A. R.; Elslager, E. F. Biochem. Pharmacol. 1979, 28, 1983-1987. 80. Weir, E. C.; Cashmore, A. R.; Dreyer, R. N.; Graham, M. L.; Hsiao, N.; Moroson, B. A.; Sawacki, W. L.; Bertino, J. R. Cancer Res. 1982, 42, 1696-1702. 81. Lin, J. T.; Cashmore, A. R.; Baker, M.; Dreyer, R. N.; Ernstoff, M.; Marsh, J. C.; Bertino, J. R.; Whitfield, L. R.; Delap, R.; Grillo-Lopez, A. Cancer Res. 1987, 47, 609-616. 82. Am. J. Hosp. Pharm. 1994, 51, 591-592. 83. Grivsky, E. M.; Lee, S.; Sigel, C. W.; Duch, D. S.; Nichol, C. A. J. Med. Chem. 1980, 23, 327-329. 84. Duch, D. S.; Edelstein, M. P.; Bowers, S. W.; Nichol, C. A. Cancer Res. 1982, 42, 3987-3994. 85. Kovacs, J. A.; Allegra, C. J.; Swan, J. C.; Drake, J. C.; Parrillo, J. E.; Chabner, B. A.; Masur, H. Antimicrob. Agents Chemother. 1988, 32, 430-433. 86. Perkins, W.; Williams, R. E.; Vestey, J. P.; Tidman, M. J.; Layton, A. M.; Cunliffe, W. J.; Saihan, E. M.; Klaber, M. R.; Manna, V. K.; Baker, H.; Mackie, R. M. Br. J. Dermatol. 1993, 129, 584-589. 87. Guzzo, C.; Benik, K.; Lazarus, G.; Johnson, J.; Weinstein, G. Arch. Dermatol. 1991, 127, 511-514. 88. Hughes, W. T.; Bartley, D. L.; Smith, B. M. J. Infect. Dis. 1983, 147, 595. 89. Volk, W. A.; Benjamin, D. C.; Kadner, R. J.; Parsons, J. T. In Essentials of Medical Microbiology; J. B. Lippincott Company: Philadelphia, 1986; pp 744-770. 90. Peglow, S. L.; Smulian, A. G.; Linke, M. J.; Pogue, C. L.; Nurre, S.; Crisler, J.; Phair, J.; Gold, J. W.; Armstrong, D.; Walzer, P. D. J. Infect. Dis. 1990, 161, 296-306. 91. www.cdfound.to.it/HTML/lung.htm. 92. Miller, R. F.; Mitchell, D. M. Thorax 1995, 50, 191-200. 93. McKenzie, R.; Travis, W. D.; Dolan, S. A. Medicine (Baltimore) 1991, 70, 326-343. 94. Mills, J. Rev. Infect. Dis. 1986, 8, 1001-1011. 95. Kovacs, J. A.; Hiemenz, J. W.; Macher, A. M.; Stover, D.; Murray, H. W.; Shelhamer, J.; Lane, H. C.; Urmacher, C.; Honig, C.; Longo, D. L.; Parker, M. M.; Natanson, C.; Parillo, J. E.; Fauci, A. S.; Pizzo, P. A.; Masur, H. Ann. Intern. Med. 1984, 100, 663-671. 96. Glatt, A. E.; Chirgwin, K. Arch. Intern. Med. 1990, 150, 271-279. 97. Schliep, T. C.; Yarrish, R. L. Semin. Respir. Infect. 1999, 14, 333-343. 98. Kaplan, J. E.; Hanson, D.; Dworkin, M. S.; Frederick, T.; Bertolli, J.; Lindegren, M. L.; Holmberg, S.; Jones, J. L. Clin. Infect. Dis. 2000, 30 Suppl 1, S5-14. 99. Arozullah, A. M.; Yarnold, P. R.; Weinstein, R. A.; Nwadiaro, N.; McIlraith, T. B.; Chmiel, J. S.; Sipler, A. M.; Chan, C.; Goetz, M. B.; Schwartz, D. N.; Bennett, C. L. Am. J. Respir. Crit. Care Med. 2000, 161, 1081-1086. 100. Sparano, J. A.; Sara, C. Curr. Opin. Oncol. 1996, 8, 392-399. - 71 - REFERENCES

101. Kontoyannis, D. P.; Rubin, R. H. Infect. Dis. Clin. North Am. 1995, 9, 811-822. 102. Fischl, M. A.; Dickinson, G. M.; La Voie, L. J. Am. Med. Assoc. 1988, 105, 45-48. 103. Walker, R. E.; Masur, H. In Pneumocystis carinii Pneumonia, Walzer, P. D., Ed.; Marcel Dekker, Inc.: New York, 1994, pp 439-466. 104. Montgomery, A. B.; Debs, R. J.; Luce, J. M.; Corkery, K. J.; Turner, J.; Brunette, E. N.; Lin, E. T.; Hopewell, P. C. Lancet 1987, 480-483. 105. Golden, J. A.; Chernoff, D.; Hollander, H.; Feigal, D.; Conte, J. E. Lancet 1989, 1, 654-657. 106. Monk, J. P.; Benfield, P. Drugs 1990, 39, 741-756. 107. Leoung, G. S.; Feigal, D. W.; Montgomery, A. B.; Corkery, K.; Wardlaw, L.; Adams, M.; Busch, D.; Cordon, S.; Jacobson, M. A.; Volberding, P. A.; Abrams, D. N. Engl. J. Med. 1990, 323, 769-775. 108. Hirschel, B.; Lazzarin, A.; Chopard, P. N. Engl. J. Med. 1991, 324, 1079-1083. 109. Allegra, C. J.; Kovacs, J. A.; Drake, J. C.; Swan, J. C.; Chabner, B. A.; Masur, H. J. Exp. Med. 1987, 165, 926-931. 110. Broughton, M. C.; Queener, S. F. Antimicrob. Agents Chemother. 1991, 35, 1348-1355. 111. Sattler, F. R.; Allegra, C. J.; Verdegem, T. D. J. Infect. Dis. 1990, 161, 91-96. 112. Masur, H.; Polis, M. A.; Tuazon, C. V.; Ogota, A. D.; Kovacs, J. A.; Katz, D.; Hilt, D.; Simmons, T.; Feuerstein, I.; Lundgren, B.; Lane, H. C.; Chabner, B. A.; Allegra, C. J. J. Infect. Dis. 1993, 167, 1422- 1426. 113. Falloon, J.; Allegra, C. J.; Kovacs, J.; O'Neill, D.; Ogata-Arakaki, D.; Feuerstein, I.; Polis, M.; Davey, R.; Lane, H. C.; LaFon, S.; Rogers, M.; Zunich, K.; Turlo, J.; Tuazon, C.; Parenti, D.; Simon, G.; Masur, H. Clin. Res. 1990, 38, 361A. 114. Heuschkel, R. B. Curr. Opin. Gastroenterol. 2000, 16, 565-570. 115. Dahlberg, E.; Thalen, A.; Brattsand, R.; Gustafsson, J. A.; Johansson, U.; Roempke, K.; Saartok, T. Mol. Pharmacol. 1984, 25, 70-78. 116. Thiesen, A.; Thomson, A. B. Aliment. Pharmacol. Ther. 1996, 10, 487-496. 117. Maini, R.; St Clair, E. W.; Breedveld, F.; Furst, D.; Kalden, J.; Weisman, M.; Smolen, J.; Emery, P.; Harriman, G.; Feldmann, M.; Lipsky, P. Lancet 1999, 354, 1932-1939. 118. Morgan, S. L.; Baggott, J. E.; Vaughn, W. H.; Austin, J. S.; Veitch, T. A.; Lee, J. Y.; Koopman, W. J.; Krumdieck, C. L.; Alarcon, G. S. Ann. Intern. Med. 1994, 121, 833-841. 119. Feagan, B. G.; Fedorak, R. N.; Irvine, E. J.; Wild, G.; Sutherland, L.; Steinhart, A. H.; Greenberg, G. R.; Koval, J.; Wong, C. J.; Hopkins, M.; Hanauer, S. B.; McDonald, J. W. N. Engl. J. Med. 2000, 342, 1627-1632. 120. Lémann, M.; Zenjari, T.; Bouhnik, Y.; Cosnes, J.; Mesnard, B.; Rambaud, J. C.; Modigliani, R.; Cortot, A.; Colombel, J. F. Am. J. Gastroenterol. 2000, 95, 1730-1734. 121. Rutgeerts, P.; D'Haens, G.; Targan, S.; Vasiliauskas, E.; Hanauer, S. B.; Present, D. H.; Mayer, L.; Van Hogezand, R. A.; Braakman, T.; DeWoody, K. L.; Schaible, T. F.; Van Deventer, S. J. Gastroenterology 1999, 117, 761-769. 122. Kremer, J. M.; Phelps, C. T. Arthritis Rheum. 1992, 35, 138-145. 123. For a review, see: Bannwarth, B.; Labat, L.; Moride, Y.; Schaeverbeke, T. Drugs 1994, 47, 25-50. 124. Andersson, S. E.; Johansson, L. -H.; Lexmüller, K.; Ekström, G. M. Eur. J. Pharm. Sci. 2000, 9, 333- 343. 125. Appleman, J. R.; Beard, W. A.; Delcamp, T. J.; Prendergast, N. J.; Freisheim, J. H.; Blakley, R. L. J. Biol. Chem. 1988, 263, 10304-10313. 126. Blakley, R. L.; Cocco, L. Biochemistry 1985, 24, 4772-4777. 127. Davies, J. F.; Delcamp, T. J.; Prendergast, N. J.; Ashford, V. A.; Freisheim, J. H.; Kraut, J. Biochemistry 1990, 29, 9467-9479. 128. Oefner, C.; D'Arcy, A.; Winkler, F. K. Eur. J. Biochem. 1988, 174, 377-385. 129. Stockman, B. J.; Nirmala, N. R.; Wagner, G.; Delcamp, T. J.; DeYarman, M. T.; Freisheim, J. H. FEBS Lett. 1991, 283, 267-269. 130. Cramer, S. M.; Schornagel, J. H.; Kalghatgi, K. K.; Bertino, J. R.; Horvath, C. Cancer Res. 1984, 44, 1843-1846. 131. Cody, V. Personal Communication.

- 72 - REFERENCES

132. Gready, J. In Advances in Pharmacology and Chemotherapy. 17; Academic Press: New York, 1980, pp 37-102. 133. McCormack, J. J. Med. Res. Rev. 1981, 1, 303-331. 134. Rosowsky, A. Prog. Med. Chem. 1989, 26, 1-252. 135. Baker, B. R. Design of Active-Site-Directed Irreversible Enzyme Inhibitors; Wiley: New York, 1967; pp 199. 136. Montgomery, J. A.; Piper, J. R. In Folate Antagonists as Therapeutic Agents; Sirotnak, F. M.; Burchall, J. J.; Ensminger, W. B.; Montgomery, J. A., Eds.; Academic Press: Orlando, 1984, pp 219-260. 137. Piper, J. R.; McCaleb, G. S.; Montgomery, J. A.; Kisliuk, R. L.; Gaumont, Y.; Sirotnak, F. M. J. Med. Chem. 1986, 29, 1080-1086. 138. Rosowsky, A.; Chen, K. K. N. J. Med. Chem. 1974, 17, 1308. 139. McGuire, J. J.; Bolanowska, W. E.; Coward, J. K.; Sherwood, R. F.; Russell, C. A.; Felschow, D. M. Biochem. Pharmacol. 1991, 42, 2400-2403. 140. Blakley, R. L. In Folates and Pterins: Chemistry and Biochemistry of Folates. 1; Blakley, R. L.; Benkovic, S. J., Eds.; Wiley-Interscience: New York, 1984, pp 191-253. 141. Edman, J. C.; Edman, U.; Cao, M.; Lundgren, B.; Kovacs, J. A.; Santi, D. V. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 8625-8629. 142. Cody, V.; Galitsky, N.; Luft, J. R.; Pangborn, W.; Gangjee, A.; Devraj, R.; Queener, S. F.; Blakley, R. L. Acta Crystallogr. D Biol. Crystallogr. 1997, 53, 638-649. 143. Champness, J. N.; Achari, A.; Ballantine, S. P.; Bryant, P. K.; Delves, C. J.; Stammers, D. K. Structure 1994, 2, 915-924. 144. Gschwend, D. A.; Sirawaraporn, W.; Santi, D. V.; Kuntz, I. D. Proteins 1997, 29, 59-67. 145. For a review, see: Gangjee, A.; Elzein, E.; Kothare, M.; Vasudevan, A. Curr. Pharm. Design 1996, 2, 263-280. 146. Rosowsky, A.; Mota, C. E.; Wright, J. E.; Freisheim, J. H.; Heusner, J. J.; McCormack, J. J.; Queener, S. F. J. Med. Chem. 1993, 36, 3103-3112. 147. Gangjee, A.; Mavandadi, F.; Queener, S. F.; McGuire, J. J. J. Med. Chem. 1995, 38, 2158-2165. 148. Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L.; Queener, S. F.; Barrows, L. R. J. Med. Chem. 1994, 37, 1169-1176. 149. Gangjee, A.; Guo, X.; Queener, S. F.; Cody, V.; Galitsky, N.; Luft, J. R.; Pangborn, W. J. Med. Chem. 1998, 41, 1263-1271. 150. Piper, J. R.; Johnson, C. A.; Krauth, C. A.; Carter, R. L.; Hosmer, C. A.; Queener, S. F.; Borotz, S. E.; Pfefferkorn, E. R. J. Med. Chem. 1996, 39, 1271-1280. 151. Schornagel, J. H.; Chang, P. K.; Sciarini, L. J.; Moroson, B. A.; Mini, E.; Cashmore, A. R.; Bertino, J. Biochem. Pharmacol. 1984, 33, 3251-3255. 152. Gangjee, A.; Shi, J.; Queener, S. F.; Barrows, L. R.; Kisliuk, R. L. J. Med. Chem. 1993, 36, 3437-3443. 153. Gangjee, A.; Vasudevan, A.; Queener, S. F.; Kisliuk, R. L. J. Med. Chem. 1995, 38, 1778-1785. 154. Rosowsky, A.; Hynes, J. B.; Queener, S. F. Antimicrob. Agents Chemother. 1995, 39, 79-86. 155. Gangjee, A.; Zhu, Y.; Queener, S. F. J. Med. Chem. 1998, 41, 4533-4541. 156. Gangjee, A.; Vasudevan, A.; Queener, S. F.; Kisliuk, R. L. J. Med. Chem. 1996, 39, 1438-1445. 157. Rosowsky, A.; Cody, V.; Galitsky, N.; Fu, H.; Papoulis, A. T.; Queener, S. F. J. Med. Chem. 1999, 42, 4853-4860. 158. For a review, see: Rahman, L. K.; Chhabra, S. R. Med. Res. Rev. 1988, 8, 95-155. 159. For a review, see: DeGraw, J. I.; Colwell, W. T.; Piper, J. R.; Sirotnak, F. M.; Smith, R. L. Curr. Med. Chem. 1995, 2, 630-653. 160. For a review, see: Roth, B.; Cheng, C. C. Prog. Med. Chem. 1982, 19, 269-331. 161. Temple Jr, C.; Elliott, R. D.; Montgomery, J. A. J. Org. Chem. 1982, 47, 761-764. 162. Taylor, E. C.; Palmer, D. C.; George, T. J.; Fletcher, S. R.; Tseng, C. P.; Harrington, P. J. J. Org. Chem. 1983, 48, 4852-4860. 163. Su, T. -L.; Huang, J. -T.; Burchenal, J. H.; Watanabe, K. A.; Fox, J. J. J. Med. Chem. 1986, 709-715. 164. Angier, R. B.; Boothe, J. H.; Hutchings, B. L.; Mowat, J. H.; Semb, J.; Stokstad, E. L.; SubbaRow, Y.; Waller, C. W.; Cosulich, D. B.; Fahrenbach, M. J.; Hultquist, M. E.; Kuh, E.; Northey, E. H.; Seeger, D. R.; Sickels, J. P.; Smith, J. M. Science 1946, 103, 667-669. - 73 - REFERENCES

165. Waller, C. W.; Hutchings, B. L.; Mowat, J. H.; Stokstad, E. L.; Boothe, J. H.; Angier, R. B.; Semb, J.; SubbaRow, Y.; Cosulich, D. B.; Fahrenbach, M. J.; Hultquist, M. E.; Kuh, E.; Northey, E. H.; Seeger, D. R.; Sickels, J. P.; Smith, J. M. J. Am. Chem. Soc. 1948, 70, 19-22. 166. Hultquist, M. E.; Dreisbach, P. F. U.S. Patent 2,443,165, 1948. 167. Catalucci, E. Offen. 2,741,383. 1979. 168. Piper, J. R.; Montgomery, J. A. J. Heterocycl. Chem. 1974, 11, 279-280. 169. Piper, J. R.; Montgomery, J. A. J. Org. Chem. 1977, 42, 208-211. 170. Isay, O. Chem.Ber. 1906, 39, 250-265. 171. Waring, P.; Armarego, W. L. F. Aust. J. Chem. 1985, 38, 629-631. 172. Baugh, C. M.; Shaw, E. J. Org. Chem. 1964, 29, 3610-3612. 173. Taylor, E. C.; Perlman, K. L.; Kim, Y. -H.; Sword, I. P.; Jacobi, P. A. J. Am. Chem. Soc. 1973, 95, 6413-6418. 174. Taylor, E. C.; Perlman, K. L.; Sword, I. P.; Sequin-Frey, M.; Jacobs, P. A. J. Am. Chem. Soc. 1973, 95, 6407-6412. 175. Taylor, E. C.; Portnoy, R. C. J. Org. Chem. 1973, 38, 806-808. 176. Taylor, E. C.; Kobayashi, T. J. Org. Chem. 1973, 38, 2817-2821. 177. Elliott, R. D.; Temple Jr, C.; Montgomery, J. A. J. Org. Chem. 1970, 35, 1676-1680. 178. Parry, D.; Moreau, J.; Bayle, M. Eur. J. Med. Chem. -Chim. Ther. 1975, 10, 147-153. 179. Nair, M. G. J. Org. Chem. 1985, 50, 1879. 180. Davoll, J.; Johnson, A. M. J. Chem. Soc. 1970, 997-1002. 181. Häring, M. Helv. Chim. Acta 1960, 43, 104-113. 182. Bergstrom, D. E.; Ruth, J. L. J. Am. Chem. Soc. 1976, 98, 1587-1589. 183. Troschütz, R.; Zink, M.; Gnibl, R. J. Heterocycl. Chem. 1999, 36, 703-706. 184. For a review, see: Mitsunobu, O. Synthesis 1981, 1-28. 185. Lindgren, B. O.; Nilsson, T. Acta Chem. Scand. 1973, 27, 888-890. 186. Kraus, G. A.; Taschner, M. J. J. Org. Chem. 1980, 45, 1175-1176. 187. Kraus, G. A.; Roth, B. J. Org. Chem. 1980, 45, 4825-4830. 188. Subramanian, L. R.; Hanack, M.; Chang, L. W. K.; Imhoff, M. A.; Schleyer, P. V. R.; Effenberger, F.; Kurtz, W.; Sgang, P. J.; Dueber, T. E. J. Org. Chem. 1976, 41, 4099-4103. 189. Shieh, W. C.; Carlson, J. A. J. Org. Chem. 1992, 57, 379-381. 190. Tabor, D. C.; Evans Jr, S. A.; Kenan Jr, W. R. Synth. Commun. 1982, 12, 855-864. 191. Dean, P. D. G. J. Chem. Soc. 1965, 6655. 192. Yoo, S. -e.; Lee, S. -h. Synlett 1990, 419-420. 193. Taylor, E. C.; Yoon, C. M. Synth. Commun. 1988, 18, 1187-1191. 194. Taylor, E. C. J. Heterocycl. Chem. 1990, 27, 1-12. 195. Piper, J. R.; Malik, N. D.; Rhee, M. S.; Galivan, J.; Sirotnak, F. M. J. Med. Chem. 1992, 35, 332-337. 196. Srinivasan, A.; Broom, A. D. J. Org. Chem. 1981, 46, 1777-1781. 197. Piper, J. R.; Johnson, C. A.; Maddry, J. A.; Malik, D. N.; McGuire, J. J.; Otter, G. M.; Sirotnak, F. M. J. Med. Chem. 1993, 36, 4161-4171. 198. Gangjee, A.; Vasudevan, A.; Queener, S. F. J. Med. Chem. 1997, 40, 479-485. 199. For a review, see: Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457-2483. 200. For a review, see: Suzuki, A. J. Organomet. Chem. 1999, 576, 147-168. 201. Larhed, M.; Hallberg, A. J. Org. Chem. 1996, 61, 9582-9584. 202. Badone, D.; Baroni, M.; Cardamone, R.; Ielmini, A.; Guzzi, U. J. Org. Chem. 1997, 62, 7170-7173. 203. Piper, J. R.; McCaleb, G. S.; Montgomery, J. A.; Kisliuk, R. L.; Gaumont, Y.; Thorndike, J.; Sirotnak, F. M. J. Med. Chem. 1988, 31, 2164-2169. 204. Harris, N. V.; Smith, C.; Bowden, K. Eur. J. Med. Chem. 1992, 27, 7-18. 205. Harris, N. V.; Smith, C.; Bowden, K. Synlett 1990, 577-578. 206. For a review, see: Beletskaya, I. P.; Cheprakov, A. V. Chem. Rev. 2000, 100, 3009-3066. 207. Bräse, S.; de Meijere, A. In Metal-Catalyzed Cross-coupling Reactions. Diedrich, F.; Stang, P. J., Eds.; Wiley-VCH: Weinheim, 1998. 208. For a review, see: Heck, R. F. Org. React. 1982, 27, 345-390. 209. Jin, S.; Holub, D. P.; Wustrow, D. J. Tetrahedron Lett. 1998, 39, 3651-3654. - 74 - REFERENCES

210. Hynes, J. B.; Yang, Y. C. S.; McCue, G. H.; Benjamin, M. B. Adv. Exp. Med. Biol. 1983, 163, 101-114. 211. Hynes, J. B.; Yang, Y. C. S.; McGill, J. E.; Harmon, S. J.; Washtien, W. L. J. Med. Chem. 1984, 27, 232-235. 212. Bisset, G. M. F.; Bavetsias, V.; Thornton, T. J.; Pawelczak, K.; Calvert, A. H.; Hughes, L. R.; Jackman, A. L. J. Med. Chem. 1994, 37, 3294-3302. 213. Merrifield, R. B. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1962, 21, 412. 214. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. 215. Morphy, J. R. Curr. Opin. Drug Discov. Dev. 1998, 1, 59-65. 216. For a review, see: Blackburn, C.; Albericio, K.; Kates, S. A. Drugs Future 1997, 22, 1007-1025. 217. Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328-1333. 218. For a review, see: James, I. W. Tetrahedron 1999, 55, 4855-4946. 219. Buckle, D. R.; Smith, H. J. Chem. Soc. 1971, 2821-2823. 220. Åkerblom, E. B.; Nygren, A. S.; Agback, K. H. Mol. Divers. 1998, 3, 137-148. 221. Chio, L. -C.; Queener, S. F. Antimicrob. Agents Chemother. 1993, 37, 1914-1923. 222. Hajduk, P. J.; Bures, M.; Praestgaard, J.; Fesik, S. W. J. Med. Chem. 2000, 43, 3443-3447. 223. Morris, G. P.; Beck, P. L.; Herridge, M. S.; Depew, W. T.; Szewczuk, M. R.; Wallace, J. L. Gastroenterology 1989, 96, 795-803. 224. Wallace, J. L.; Le, T.; Carter, L.; Appleyard, C. B.; Beck, P. L. J. Pharmacol. Toxicol. Meth. 1995, 33, 237-239. 225. Carpenter, T. A.; Everett, J. R.; Hall, L. D.; Harper, G. P.; Hodgson, R. J.; James, M. F.; Watson, P. J. Skeletal Radiol. 1994, 23, 429-437. 226. Griffiths, R. J. Agents Actions 1992, 35, 88-95. 227. Ekström, G. M.; Szajewski, K.; Persson, Å. Scand. J. Immunol. 1997, 45, 566. 228. Billingham, M. F. J. Pharmacol. Ther. 1983, 21, 389-428. 229. Smith, R. J.; Sly, L. M. J. Pharmacol. Exp. Ther. 1996, 277, 1801-1813. 230. Kanerud, L.; Engstrom, G. N.; Tarkowski, A. Ann. Rheum. Dis. 1995, 54, 256-262. 231. Kanerud, L.; Scheynius, A.; Hafstrom, I. Arthritis Rheum. 1994, 37, 1138-1145. 232. Hansson, T.; Marelius, J.; Åqvist, J. J. Comput. Aided Mol. Des. 1998, 12, 27-35. 233. Lamb, M. L.; Jorgensen, W. L. Curr. Opin. Chem. Biol. 1997, 1, 449-457. 234. Oprea, T. I.; Marshall, G. R. Persp. Drug Discov. Des. 1998, 9, 35-61. 235. For a review, see: Kollman, P. A. Chem. Rev. 1993, 93, 2395-2417. 236. Åqvist, J.; Medina, C.; Samuelsson, J. E. Protein Eng. 1994, 7, 385-391. 237. Rejto, P. A.; Bouizida, D.; Verkhiver, G. M. Theor. Chem. Acc. 1999, 101, 138-142. 238. Cocco, L.; Roth, B.; Temple, C. J.; Montgomery, J. A.; London, R. E.; Blakley, R. L. Arch. Biochem. Biophys. 1983, 226, 567-577. 239. Queener, S. F. J. Med. Chem. 1995, 38, 4739-4759. 240. Cody, V.; Chan, D.; Galitsky, N.; Rak, D.; Luft, J. R.; Pangborn, W.; Queener, S. F.; Laughton, C.; Stevens, M. F. G. Biochemistry 2000, 39, 3556-3564. 241. Rowland, M.; Tozer, T. N. Clinical Pharmacokinetics: Concept and Applications; 2nd ed.; Lea & Febiger: Philadelphia, 1989. 242. Mostafavi, S. A.; Lewanczuk, R. Z.; Foster, R. T. Biopharm. Drug Dispos. 2000, 21, 121-128.

- 75 - Approaches to Soft Drug Analogues of Dihydrofolate Reductase Inhibitors. Design and Synthesis.

by

Malin Graffner Nordberg

Corrections

Page Location Error Correction Summary 5 List of Papers, Paper III J. Med. Chem., accepted after J. Med. Chem., accepted minor revision 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 OCOOH OCOOH N COOH N COOH R = H R = H

18 Line 12 from top concentration of concentration of MTXGN polyglutamated of MTXGN 21 Line 9 from top in this context is that in this context that 23 Line 13 from bottom sustained a sustained sustained 35 Figure 26 See Figure 26 below 36 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. C6H5N1. C5H5N 46 Scheme 16, second arrow 30% formaldehyde 37% formaldehyde 51 Line 5 from bottom there anions… their anions… 52 Table 1 in footnotes Rates of hydrolysis should be expressed as µM/min 53 Table 2 57a resp. 58a 58a resp. 59a 54 Line 13 from bottom The phenantrene analogue 43a The phenanthrene analogue 57 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…

OCH OCH OCH NH2 CH3 3 CH3 3 CH3 3 NC NC N

H2N N N Cl N O N OCH OCH H OCH PTX 3 XXVI 3 XXV 3

Cl OCH3 O OCH3 CN H3C Me + H2N O O H OCH3 OCH3 XXIV XXIII XXII

Figure 26. A retrosynthetic analysis of PTX.