UNIVERSITÀ DI PISA
Dipartimento di Farmacia
Corso di Laurea Specialistica in
Chimica e Tecnologia Farmaceutiche
Tesi di Laurea
:
SYNTHESIS OF INDOLE-BASED INHIBITORS OF
LACTATE DEHYDROGENASE
Relatori: Prof. Filippo Minutolo
Dott.ssa Reshma Rani
Candidata: Francesca Gado (N° MATRICOLA 454834)
Settore Scientifico Disciplinare: CHIM-08
ANNO ACCADEMICO 2013 – 2014
“Il contenuto di questa tesi di laurea è strettamente riservato, essendo presenti argomenti tutelati dalla legge come segreti. Pertanto tutti coloro che ne prendono conoscenza sono soggetti all’obbligo, sanzionato anche penalmente dagli articoli 325 e 623 del codice penale, di non divulgare e di non utilizzare le informazioni acquisite.”
“Il contenuto di questa tesi di laurea è strettamente riservato, essendo presenti
argomenti tutelati dalla legge come segreti. Pertanto tutti coloro che ne prendono
conoscenza sono soggetti all’obbligo, sanzionato anche penalmente dagli articoli 325
e 623 del codice penale, di non divulgare e di non utilizzare le informazioni acquisite.”
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Contents
1 General Introduction 1
1.1 The Carbohydrates Metabolism . . . 1
1.2 Glycolysis . . . 2
1.3 The three catabolic fates of pyruvate . . . 6
1.3.1 Aerobic Conditions . . . 7
1.3.2 Anaerobic Conditions . . . 8
1.4 Pasteur Eect . . . 9
1.5 Cancer . . . 10
1.5.1 Hypoxia . . . 10
1.5.2 Hypoxia and the treatment of cancer . . . 12
1.5.3 Warburg Eect . . . 13
1.5.4 Aerobic glycolysis . . . 16
1.5.5 Extracellular pH . . . 18
1.5.6 The importance of combination therapies . . . 20
1.5.7 Lactate dehydrogenase (LDH) . . . 21
2 The most important inhibitors of glucose metabolism 26 2.1 Antitumoral agents acting on glycolysis . . . 26
2.2 LDH inhibitors . . . 29
3 Introduction to the experimental section 37 3.1 Indoles . . . 37
3.2 Structural design . . . 40
3.3 Enzymatic Assays . . . 49
3.4 Conclusions . . . 50
4 Experimental section 52 4.1 Materials and methods . . . 52
4.2 Procedures . . . 53
4.2.1 Synthesis compound 39 . . . 53
4.2.2 Synthesis compound 40 . . . 54
4.2.3 Synthesis compound 41 . . . 55
4.2.4 Synthesis compound 43a . . . 57
4.2.5 Synthesis compound 43b . . . 58
4.2.6 Synthesis compound 43c . . . 58
4.2.7 Synthesis compound 41a . . . 59
4.2.8 Synthesis compound 41b . . . 60
4.2.9 Synthesis compound 41c . . . 60
4.2.10 Synthesis compound 42a . . . 61
4.2.11 Synthesis compound 42c . . . 62
4.2.12 Synthesis compound 44a . . . 63
4.2.13 Synthesis compound 44b . . . 64
4.2.14 Synthesis compound 44c . . . 64
4.2.15 Synthesis compound 45a . . . 65
4.2.16 Synthesis compound 45b . . . 66
4.2.17 Synthesis compound 45c . . . 66
4.2.18 Synthesis compound 46a . . . 67
4.2.19 Synthesis compound 46c . . . 68
4.2.20 Synthesis compound 47a . . . 69
4.2.21 Synthesis compound 47b . . . 70
4.2.22 Synthesis compound 47c . . . 70
4.2.23 Synthesis compound 48a . . . 71
4.2.24 Synthesis compound 48b . . . 73
4.2.26 Synthesis compound 49 . . . 74 4.2.27 Synthesis compound 50 . . . 75 4.2.28 Synthesis compound 52 . . . 76 4.2.29 Synthesis compound 53 . . . 77 4.2.30 Synthesis compound 54 . . . 78 4.2.31 Synthesis compound 55 . . . 81 Bibliography 82
Chapter 1
General Introduction
1.1 The Carbohydrates Metabolism
Glucose, aldohexose monosaccharide, enters the cell thanks to the transporter GLUT 1 and here it is phosphorylated to glucose-6-phosphate by hexokinase (HE) thus pre-venting the glucose for leaving the cell again as there are no carriers in the membrane for phosphorylated sugars.
O H OH H OH H OH H OH CH2OH O H OH H OH H OH H OH CH2OPO3 2-Mg2+ ATP ADP Hexokinase Glucose Glucose-6-phosphate
Figure 1.1: Glucose Phosphorylation
At this point, glucose-6-phosphate can encounter dierent destinations:
1. it can be oxidized in the pentose phosphate pathway to produce ribose-5-phosphate required for the synthesis of nucleic acids and coenzymes;
2. it can be stored in the form of glycogen;
3. it can be released back into the bloodstream to maintain normal blood glucose rates;
Most of the cells exploit the glucose precisely for this latter purpose (4) in a process known as cellular respiration. It takes place in 3 main phases: in the rst phase glucose is demolished glycolytically to pyruvate which is subsequently oxidized to acetyl coenzyme A (acetyl CoA) that, in the second phase, enters the Krebs cycle in which it is oxidized by enzymes to CO2; the released energy is stored in the form of NADH or
FADH2, the reduced forms of the electron carriers NAD+ and FAD. In the third phase
of respiration they are re-oxidized at the level of the inner mitochondrial membrane where they release their electrons to the protein complexes in the respiratory chain. The transfer of these electrons from one complex to another is a result of the passage of some protons from the matrix to the intermembrane space and it is this proton gradient that allows the formation of ATP by ATP synthase. This latter process is called oxidative phosphorylation. All this happens only under aerobic conditions thanks to which, from an intial molecule of glucose, it is possible to obtain, with the oxidative phosphorylation, as much as 32 molecules of ATP[1].
1.2 Glycolysis
Glycolysis, the process of demolition of glucose up to two molecules of pyruvate, in-cludes 10 stages, 5 of which make up the preparatory phase and 5 the energy recovery phase. The rst stage requires the investment of two ATP molecules and leads to the breaking of the hexose into two molecules of triose phosphate. The rst reaction con-cerns the phosphorylation on the C6 of glucose to glucose-6-phosphate. It is irreversible
and is catalyzed by hexokinase.
O H OH H OH H OH H OH CH2OH O H OH H OH H OH H OH CH2OPO3 2-Mg2+ ATP ADP Hexokinase Glucose Glucose-6-phosphate
Figure 1.2: Phosphorylation of glucose to glucose-6-phosphate
expenditure by the cell. The hexokinase needs ions Mg2+, required for its catalytic
activity, because they protect the negative charges of the phosphate groups making the phosphorus atom more accessible for the nucleophilic attack of the hydroxyl of the glucose. The glucose-6-phosphate is converted into fructose-6-phosphate with an isomerization catalyzed by phosphohexose isomerase.
O H OH H OH H OH H OH CH2OPO3 2-OH CH2OH CH2OPO3 2-OH H H OH O Mg2+ Phosphoexose isomerase Glucose-6-phosphate Fructose-6-phosphate
Figure 1.3: Conversion of glucose-6-phosphate to fructose-6-phosphate
This rearrangement is necessary for the following two steps in which the fructose-6-phosphate is rst phosphorylated and then split into two dierent triose phosphate: glyceraldehyde-3-phosphate and the dihydroxyacetonphosphate. In the rst phospho-rilation phase, fructose-6-phosphate is converted into fructose-1, 6-bisphosphate by phosphofructokinase 1. The enzyme has also a regulatory function: its activity in-creases when the amount of cellular ATP dein-creases or with the backlog of ADP and AMP (especially the latter). On the contrary, its activity decreases with high levels of ATP. OH CH2OH H CH2OPO3 2-OH H H OH O OH CH2OPO3 2-H CH2OPO3 2-OH H H OH O Mg2+ ATP ADP Phosphofructokinase 1 Fructose-6-phosphate Fructose-1,6-bisfosfato
Figure 1.4: Conversion of glucose-6-phosphate to fructose-1,6-bisphosphate Even in this case, the phosphate group is transferred from ATP and so there is the consumption of the second molecule of ATP. As for the hexokinase also the PFK-1 re-quires, for its catalytic activity, ions Mg2+. This irreversible reaction is also considered
to be the rst commandreaction of glycolysis, as both glucose-6-phosphate to fructose-6-phosphate may also have other metabolic destinies, while fructose-1, 6 -bisphosphate
is an exclusive intermediate of glycolysis. Fructose-1,6-bisphosphate, with a reversible reaction, is split into glyceraldehyde-3-phosphate and dihydroxyacetonphosphate. The enzyme, in this case, is named from the type of reaction that catalyzes, that is an aldol and so, it is called aldolase.
OH CH2OPO3 2-H CH2OPO3 2-OH H H OH O Aldolase O H CH2OPO3 2-HO + O CH2OPO32 -HOH2C Fructose-1,6,bisphosphate Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate
Figure 1.5: Cleavage of fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetonphosphate
Only glyceraldehyde-3-phosphate can directly continue glycolysis; the other prod-uct, the dihydroxyacetonphosphate, has to be rst converted reversibly in glyceraldehyde-3-phosphate by triosephosphate isomerase.
O H
CH2OPO3
2-HO O
HOH2C CH2OPO32- Triosephosphate
isomerase Dihydroxyacetone
phosphate
Glyceraldehyde-3-phosphate
Figure 1.6: Conversion of dihydroxyacetone phosphate in glyceraldehyde-3-phosphate This reaction is the last of the preparatory phase of glycolysis. The initial molecule of glucose has been converted to 2 glyceraldehyde-3phosphate with the consumption of 2 molecules of ATP. The phase of energy recovery of glycolysis begins with an oxida-tion of the carbonyl group of glyceraldehyde-3-phosphate to give, not a free carboxyl group, but a particular type of anhydride bond, acyl phosphate, with a high standard hydrolysis energy. O H CH2OPO3 2-HO + P O O O OH NAD+ NADH + H+ O O32-PO CH2OPO3 2-HO
Glyceraldehyde-3 Phosphate 1,3-Bisphosphoglycerate phosphate
Glyceraldehyde-3-phosphate dehidrogenase
Unlike what was seen in the rst stage of glycolysis, the phosphate is an inorganic phosphate and is not derived from ATP hydrolysis. In the seventh reaction of glycolysis there is the transfer of the high-energy phosphate group of 1,3-bisphosphoglycerate to ADP by the phosphoglicero kinase, an enzyme that takes its name from the opposite reaction as it catalyzes the reaction in both directions.
N N NH2 N N H CH2 H OH OH H O H O P O -O O P O -O O -+ CH2OPO3 2-HC OH C O O P O O -O -1,3-biphosphoglycerate ADP Phosphoglycerate kinase Mg2+ CH2OPO3 2-HC OH C O O + N N NH2 N N H CH2 H OH OH H O H O P O -O O P O -O O O P O -O O -3-phosphoglycerate ATP
Figure 1.8: Conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate
The ninth step is an exchange between the reversible phosphate group C2 and C3 of the glycerate catalyzed by phosphoglicero mutase. The reaction requires Mg2+ as
cofactor. CH2OPO3 2-HC OH C O O 3-phosphoglycerate CH2OH HC OPO3 2-C O O 2-phosphoglycerate Mg2+ Phosphoglicero mutase
Figure 1.9: Conversion of 3-phosphoglycerate to 2-phosphoglycerate
The 2-phosphoglycerate dehydrates to phosphoenolpyruvate. The reaction is re-versible and is catalyzed by enolase obtaining a compound with a high transfer poten-tial of the phosphate group.
CH2OH HC OPO3 2-C O O 2-phosphoglycerate CH2 C OPO3 2-C O O Phosphoenolpyruvate H2O Enolase
Figure 1.10: Dehydration of 2-phosphoglycerate to phosphoenolpyruvate
The nal reaction of glycolysis provides, in fact, a transfer of a phosphate group from phosphoenolpyruvate to ADP, catalyzed by the enzyme pyruvate kinase which requires K+, Mg2+ or Mn2+ as cofactors. The pyruvate appears rst in its enol form,
then it tautomerizes in the more stable ketonic form.
CH2 C OPO3 2-C O O Phosphoenolpyruvate CH3 C O C O O Pyruvate +
ATP
+ADP
Mg2+, K+ Pyruvate kinaseFigure 1.11: Conversion of phosphoenolpyruvate to pyruvate
Since from one molecule of glucose we got 2 glyceraldehyde-3-phosphate, in this second phase we obtained 4 ATP molecules and 2NADH. However, considering that 2 ATP were consumed during the rst phase, the net balance is 2 ATP and 2 NADH[1].
1.3 The three catabolic fates of pyruvate
1.3.1 Aerobic Conditions
1. Pyruvate resulting from glycolysis, with an oxidative decarboxylation, is oxidized by a complex, known as the pyruvate dehydrogenase complex, formed by 3 enzymes and by 5 cofactors, to produce acetyl-CoA and CO2. At this point, the Acetyl-CoA
formed enters the Krebs cycle consisting of 8 reactions in which the oxidation energy is stored in the form of reduced coenzymes NADH and FADH2.
Figure 1.13: Krebs Cycle
The NADH and FADH2, arising from both processes, head at the level of the
mitochondrial inner membrane electrons transport chain in which they release their electrons that are transferred through 5 protein complexes to the last acceptor, oxygen. The transfer of electrons takes place in conjunction with the passage of ions H+ from
the matrix to the intermembrane space and it is precisely the formation of this proton gradient that is necessary for the synthesis of ATP by ATP synthase[1].
Figure 1.14: Cellular respiration in aerobic conditions
1.3.2 Anaerobic Conditions
1. In terms of the absence or de ciency of oxygen, the cell cannot take advantage of the electrons transport chain and oxidative phosphorylation to produce ATP and to reoxidize the NADH and FADH2 as the last of the electrons' acceptor,
oxygen, is missing. The lack of regeneration of NAD+would leave the cell without
the electrons acceptor required to oxidize glyceraldehyde-3-phosphate , and this would prevent the smooth running of glycolysis . In these conditions , therefore, the cell resorts to lactic fermentation in which pyruvate is reduced to lactate with the simultaneous reoxidation of NADH to NAD+ by the enzyme lactate
dehydrogenase (LDH ) . There are some tissues and cell types (eg erythrocytes) that convert glucose to lactate even in aerobic conditions. For example during an intense and prolonged eort of the skeletal muscle, ATP demand is high but the availability of O2 is poor , the muscle uses its reserve of glucose (glycogen) for
the production of ATP by lactic fermentation with lactate as nal product. The lactate can therefore reach high levels initially in the muscle, causing pain and cramps and, subsequently, in the blood so, in the recovery phase, it is transported to the liver where it is rst converted to pyruvate (Cory cycle) and nally into
glucose again (gluconeogenesis)[1]. C C O O O CH3 C CH O O CH3 HO Pyruvate Lactate Lactate dehydrogenase NADH+H+ NAD+
Figure 1.15: Lactic Fermentation
2. The yeasts and other microorganisms ferment glucose to ethanol and CO2 with
a process known as alcoholic fermentation. It consists of two stages: in the rst stage there is no redox reaction but a simple decarboxylation of pyruvate to acetaldehyde catalyzed by pyruvate decarboxylase that requires Mg2+ as cofactor
and tiaminapirofosfato (TPP) as coenzyme; The second stage is, instead, a redox reaction catalyzed by alcohol dehydrogenase in which acetaldehyde is reduced to ethanol and NADH is oxidized to NAD +[1].
C C O O O CH3 Pyruvate Pyruvate decarboxylase CO2 TPP, Mg2+ C H3C H O Acetaldeyde Alcohol Dehydrogenase NADH+H+ NAD+ OH CH2 CH3 Ethanol
Figure 1.16: Alcoholic Fermentation
1.4 Pasteur Eect
The transition from an aerobic metabolism to an anaerobic one is scienti cally called Pasteur Eect . Its name derives from the well-known chemist and microbiologist Louis Pasteur who, while studying yeast cultures , observed an increase of more than 10times the consumption of glucose when the cells passed from aerobic conditions to anaerobic conditions. If we calculate the yield in ATP from a molecule of glucose in aerobic conditions we get 32 ATP because from glycolysis we get 2 ATP and 2 NADH , from oxidative decarboxylation 2 NADH , from Krebs cycle 6 NADH and 2 FADH2.
for each FADH2 1,5. The total number of ATP molecules we get is 32. In anaerobic conditions , however, the total yield for each glucose molecule is constituted only by the 2 ATP molecules obtained by glycolysis . From this we can see that the cell under anaerobic conditions requires a glucose amount 32
2 =16 times higher than the one in the
presence of oxygen. Pasteur observed , in fact , that if he led oxygen into an anaerobic suspension of cells which used glucose at high speed through the glycolysis , the rate of metabolism of glucose decreased sharply. The increase in yield of ATP per unit of glucose, compared to the one in hypoxic conditions, determines the increase of the ratio ATP / ADP and this leads to a decrease in the activity of PFK - 1 and, consequently, of glycolysis. We have also to consider that metabolites such as citrate, produced in the Krebs cycle, if it is present in high amounts, comes from the mitochondria and acts as a negative allosteric factor of PFK - 1 determining, also, a decrease in its activity[2].
1.5 Cancer
1.5.1 Hypoxia
A common feature of growing malignant tumors is constituted by the presence of hypoxic areas. It has been seen, in fact, that tissue hypoxia develops immediately at the beginning, when the tumor mass has a diameter of only a few millimeters. This is due to a lack of regulation in cell growth which involves, in solid tumors, an increase in volume of the cell mass that can compress and occlude the surrounding blood vessels, reducing blood ow to the cells themselves. Moreover, because of the high proliferation rate, many cells grow distant from blood vessels. In the study of tumors, hypoxia has therefore received considerable attention in virtue of the fact that it seems to be a correlation between tumor hypoxia, metastasis and patiens' life expectancy[3]. Hypoxia activates a transcription factor complex that controls the adaptation of cells to low oxygen, termed Hypoxia-inducible factor (HIF). HIF-1 consists of an oxygen sensitive α subunit and of a β subunit, constitutively nuclear. L'HIF-1α is unstable under normoxic conditions and it is rapidly hydroxylated by prolyl hydroxylases (PHD). Once hydroxylated, HIF-1α is conjugated to the Von Hippel-Landau (VHL) protein,
poly-ubiquinated and eventually degraded by the proteasome.
Figure 1.17: HIF regulation
Under hypoxic conditions the PHD-initiated inactivation of HIF-1α cannot take place beacuse the oxygen, necessary for the hydroxylation, is missing. Consequently, this subunit migrates into the cell nucleus where it interacts with the β subunit forming an heterodimer which binds to DNA, in the hypoxia-responsive elements (HREs) in promoters of target genes making use of co-activators, such as p-300/CBP, for a full transcription. In this way it is possible to obtain an increase in trascription of genes coding for erythropoietin, for growth factors such as VEGF and angiopoietin-2 which have a crucial role in angiogenesis, for nitrous oxide synthase (NOS) which is also important in angiogenesis and vasodilation, for glycolytic enzymes and for the pyruvate dehydrogenase kinase (PDK) that inactivates the pyruvate dehydrogenase complex which, converting pyruvate into citrate, allows the Krebs cycle to start[4, 5, 6].
1.5.2 Hypoxia and the treatment of cancer
Tissues, possessing low levels of oxygen, show resistance to radiotherapy and this is one of the principal clinical problems that compromises hypoxic cancer treatment. The mechanism linking hypoxia to tumour radioresistance is generally known as the oxygen enhancement eect. In fact DNA damage is usually provoked by direct ionization from ionizing radiations or is induced by oxygen radicals, such as hydroxyl radical, superox-ide anion etc. In the presence of oxygen, DNA double-strand breaks cannot be repaired because O2 reacts with the broken DNA forming stable organic peroxides which are
hardly repaired by the cell, leading to fatal chromosome aberrations. Oxygen xes the DNA damage, making it permanent. On the contrary, in the absence of oxygen, the damage is more easily repaired: the broken DNA can be restored to its original form, thanks to reparative processes based upon reductions by -SH containing intracellular components. This mechanism explains why tumors with low levels of oxygen show a reduced eect on response to radiotherapy, compared with normal oxygenated tissues. Hypoxic cells have also other characteristics that make them resistant to conventional anticancer therapies:
Because of their distance from blood vessels, they are not su ciently reached by most anticancer drugs;
Hypoxia selects cells that up-regulate genes encoding for proteins involved in drug resistance. Consequently, at present, there are not adequate anticancer drugs able to kill hypoxic cells;
Hypoxic cancer cells are associated with a more metastatic and aggressive behav-ior, which predisposes to the formation of metastases, that compromise curability of tumours by surgery.
Since hypoxia has a negative impact on cancer treatment and prognosis, the unique features of hypoxic tumours, that do not occur in healthy tissues, such as the metabolic switch associated to the Warburg eect(1.5.3), could be exploited in cancer-selective therapy [7].
1.5.3 Warburg Eect
Well over a century ago, Pasteur rst noted that the absence of oxygen resulted in the inhibition of oxidative phosphorylation (OXPHOS) and a switch to glycolysis for ATP generation (Pasteur eect). More than 80 years ago, the renowned biochemist Otto Warburg found that tumor cells, unlike their normal counterparts, utilized glycolysis instead of mitochondrial oxidative phosphorylation for glucose metabolism even when in oxygenrich conditions (Warburg eect)[6].
For unicellular organisms such as microbes, there is evolutionary pressure to reproduce as quickly as possible when nutrients are available. Their metabolic control systems have evolved to sense an adequate supply of nutrients and channel the requisite carbon, nitrogen, and free energy into generating the building blocks needed to produce a new cell. When nutrients are scarce, the cells cease biomass production and adapt metabolism to extract the maximum free energy from available resources to survive the starvation period.
Figure 1.18: Comparison between the metabolism of unicellular and multicellular organisms[8].
In multicellular organisms, most cells are exposed to a constant supply of nutrients. Organism survival requires control systems that prevent aberrant individual cell pro-liferation when nutrient availability exceeds the levels needed to support cell division. Uncontrolled proliferation is prevented because mammalian cells do not normally take
up nutrients from their environment unless stimulated to do so by growth factors; in multicellular organisms they are the ones which stimulate the cells to multiply and which prevent, therefore, an uncontrolled proliferation. Cancer cells overcome this growth factors dependence by acquiring genetic mutations that activate the uptake and the nutrients' metabolism ensuring cells' survival and the necessary fuel for their growth. As a consequence of these mutations, the uptake of nutrients, in particular glucose, can meet or exceed the bioenergetic demands of cell growth and proliferation. Nevertheless, microbes and cells from multicellular organisms have similar metabolic phenotypes under similar environmental conditions as, in both cases, during prolifer-ation, the glucose is rst metabolized through glycolysis to produce ethanol, lactate and other organic acids such as butyrate and acetate. On the contrary, when there are no nutrients (for microorganisms) or stimuli by growth factors (for multicellular organisms), we have an oxidative metabolism[8].
Obviously, in cancer cells the production and the excretion of lactate is much more evident than in normal cells and this observation led Otto Warburg to declare cancer cells metabolized glucose in a totally dierent way from the one of the cells of healthy tissues.
In fact, while in healthy cells, according to Pasteur, an increase in the lactate pro-duction was observed only in anaerobic conditions, in cancer cells it happened also when oxygen was present. Warburg de ned this phenomenon aerobic glycolysis. Warburg speculated that in tumor cells there was an impaired mitochondrial function and suggested that it could contribute to tumorigenesis. He stated: Just as there are many remote causes of plague, heat, insects, rats, but only one common cause, the plague bacillus, there are a great many remote causes of cancer-tar, rays, arsenic, pressure, urethane but there is only one common cause into which all other causes of cancer merge, the irreversible injuring of respiration [9]. Recent studies suggest that mutations aecting mitochondrial DNA (mtDNA) or enzymes of the TCA cycle might contribute to the Warburg eect as they could render the mtDNA encoded components of the respiratory chain complexes defective. However there is no direct evidence that these mutations are su cient for tumorigenesis or that respiration is, in fact, less active in cancer cells than in normal cells.
Nowadays it is known there are, in addition to the beforementioned HIF, several onco-genes implicated in the Warburg eect[5]:
AKT which mobilizes glucose transporters to the cell surface to enhance glucose uptake and activates hexokinase 2 (HK2) to phosphorylate and trap intracellular glucose.
The Myc which activates virtually all glycolytic enzyme genes and directly binds numerous glycolytic genes, including those encoding HK2, enolase, and LDHA. (5). Elevated and sustained activation of MYC, however, is tightly associated with increased mitochondrial reactive oxygen species, which may cause mtDNA mutations that in turn contribute to dysfunctional mitochondria
p53, which is frequently mutated in human cancers, also stimulates mitochondrial respiration by directly transactivating the SCO2 gene for synthesis of cytochrome c oxidase 2.
Figure 1.20: Oncogenes involved in the Warburg eect and their sites of action[5].
1.5.4 Aerobic glycolysis
To make the causes of the Warburg eect clearer, initially tumor hypoxia was thought to be responsible for the switch from aerobic metabolism to anaerobic metabolism. Actually, it was, subsequently, observed that cancer cells appeared to use anaerobic metabolism before exposure to hypoxic conditions.
For example, leukemic cells reside within the bloodstream where the oygen tension is higher than the one in cells of most normal tissues. Similarly, lung tumors, arising in the airways, exhibit aerobic glycolysis even though these tumor cells are exposed to oxygen during tumorigenesis. Thus, although tumor hypoxia is clearly an important factor for some aspects of cancer biology, the available evidence suggests that it may not be the determining factor in the switch to aerobic glycolysis.
At rst glance, the positive contribution of aerobic glycolysis to the tness of cancer cells is decidedly obscure. Aerobic glycolysis would seem detrimental because:
it is much less e cient in energy production than is aerobic metabolism. Speci -cally, glycolysis produces only 2 mol ATP/mole of glucose while oxidative metabolism of glucose results in about 36 molATP/mole of glucose.
it increases acid production resulting in a highly acidic extra-cellular environ-ment. This results in local toxicity including cell death and extra-cellular matrix degradation due to release of proteolytic enzymes.
However we have to understand what the metabolic needs of proliferating cells are. To produce two viable daughter cells at mitosis, a proliferating cell must replicate all of its cellular contents. This imposes a large requirement for nucleotides, amino acids, and lipids. During growth, glucose is used to generate biomass as well as produce ATP. Although ATP hydrolysis provides free energy for some of the biochemical reactions re-sponsible for replication of biomass, these reactions have additional requirements. For most mammalian cells in culture, the only two molecules catabolized in appreciable quantities are glucose and glutamine. This means that glucose and glutamine supply most of the carbon, nitrogen, free energy, necessary to support cell growth and divi-sion. From this perspective, it becomes clear that converting all of the glucose to CO2,
via oxidative phosphorylation in the mitochondria to maximize ATP production, runs counter to the needs of a proliferating cell. Tumors can metabolize glucose through the pentose phosphate pathway (PPP) to generate nicotinamide adenine dinucleotide phosphate (NADPH) that ensures the cell's antioxidant defenses against a hostile mi-croenvironment and chemotherapeutic agents[10]. Moreover, NADPH can contribute to fatty acid synthesis. Tumor cells, with their high replication rate, require a larger quantity of ATP and the aerobic glycolysis is the quickest way to obtain it, though the yield is much lower. Furthermore, with aerobic glycolysis, the cell obtain all the neces-sary intermediates for the biosynthesis of molecules for cell proliferation. In addition, the increased glucose uptake also plays a protective role as it protects cancer cells from apoptosis making them independent from growth factors (Fig.21)[8]. Finally, ROS are by-products of oxidative metabolism, therefore the conversion of pyruvate to lactate protects cancer cells from oxidative stress. The mitochondrial oxidative phosphoryla-tion is, in fact, the major source of ROS producphosphoryla-tion. Cells with excess nutrient uptake that have not converted to aerobic glycolysis would be predicted to have increased oxidative phosphorylation and, consequently, ROS. Yeast studies have also demon-strated that oxidative phosphorylation stops during S phase to limit ROS-mediated
Figure 1.21: Decreased metabolism of glucose by tumors as a result of anticancer therapy. These images have been obtained thanks to the PET technique the infusion of FDG, a glucose analog, in a patient with a form of malignant sarcoma (gastrointestinal stromal tumor) before and after therapy with a tyrosine kinase inhibitor (sunitinib). T indicates the tumor before theraphy. After 4 weeks of therapy (right), the tumor shows no uptake of FDG and excess FDG is excreted in the urine, and therefore the kidneys (K) and bladder (B) are also visualized as labeled[8].
DNA damage, underscoring the importance of limiting oxidative phosphorylation and ROS production in proliferating cells[8].
1.5.5 Extracellular pH
To ensure the continuation of glycolysis, lactic acid formed by lactic fermentation, must be removed. Since lactate is a charged molecule, it cannot cross the plasma membrane by simple diusion; therefore, it requires transporters. The monocarboxylate trans-porter family (MCT) represents the main component responsible for lactate extrusion. MCTs are passive lactate-proton symporters; they have 12 transmembrane domains, which locate the N and the C termini within the cytoplasma. MCT isoforms dier mainly in the lenght of the C terminus and in the size of the intracellular loop between transmembrane domains 6 and 7. Among the populated family of the MCTs, isoforms
MCT1 and MCT4 are the best characterized and the most studied proton-dependent trasporters of monocarboxylic acids. MCT4 have the lowest a nity for the lactate but they are the only ones subjected to HIF-1α regulation and, therefore, isoform 4 allows rapid lactate e ux in hypoxic glycolitic cells. Cancer cells have to extrude lactate to avoid an excessive intracellular acidi cation that would lead to cell death. So, they extrude it and, in this way, they are able to maintain the intracellular pH around neu-tral, determining also an acidi cation of the extracellular matrix responsible for local toxicity and degradation of the matrix itself[4].
Figure 1.22: The extra and intracellular pH of some tumors compared with that of the healthy liver and muscle. It is known that the intracellular pH in tumor cells is almost close to the norm. In contrast, the extracellular pH is clearly acid[11].
The excess generation of lactate that accompanies the Warburg eect would ap-pear to be an ine cient use of cellular resources. Each lactate excreted from the cell wastes three carbons that might, otherwise, be utilized for either ATP production or macromolecular precursor biosynthesis. Actually, in proliferating cells, such as tumor cells, lactate is internalized by hepatocytes, through MCT1, where thanks to the Cory cycle, new glucose from lactate is obtained[8]. Besides, recent studies have shown that tumor cells are normally highly heterogeneous in their oxygen and lactate content and that they can be roughly classi ed into two categories: normoxic/oxidative, which are closer to blood vessels, or hypoxic/glycolytic, which are further away from the vascular
network. In general, glucose is actively taken up through the transporter GLUT into less oxygenated cells, which use it in the glycolytic process to produce ATP. The -nal conversion of pyruvate to lactate is catalyzed by lactate dehydrogenase 5 (LDH5). MCT4, then extrudes lactic acid from hypoxic cells into the extracellular milieu where it is taken up through MCT1 by the other type of cells, that is normoxic/oxidative. In these cells lactate is oxidized to pyruvate by LDH1. Pyruvate enters the TCA cycle in mitochondria with production of energy and CO2[4].
Figure 1.23: Lactate extrusion from the cell[6].
1.5.6 The importance of combination therapies
In general, it appears that inhibition of glycolysis alone is not su cient to produce signi cant anti-tumor eects in vivo. This is probably due to the heterogeneity of tumor cells and tumor environments because some of them might still use oxidative metabolism. This could be overcome by using multiple agents that inhibit ATP pro-duction through both aerobic and anaerobic pathways.
However, any therapeutic strategy, including the ones exploiting metabolic pathways, must recognize the remarkable ability of cancer populations to adapt to and overcome
even highly successful regimens. This can be quanti ed by the following equation, which is known as fundamental law of cancer:
dui dt = σi2 ∂Fi ∂ui (1.1) Brie y, this equation demonstrates that the evolution rate of a cancer (dui/dt) is
dependent on the square of the heterogeneity of the population phenotype (σi) and
the selection pressures within the environment (∂Fi/∂ui). Phenotypic heterogeneity
of cancer cells (σi) is typically due to underlying genetic instability and temporal
and spatial variations in environment caused by, for example, disordered blood ow resulting regions of hypoxia and acidosis. This value of σi increases the rate with
which cancer populations evolve and, thus, confers a substantial inherent capacity to evolve and adapt to changing environmental conditions. Moreover, successful therapies that induce apoptosis and necrosis in cells within cancer populations also create a microenvironment with stronger selection pressure, ∂Fi/∂ui, and consequently, with a
stronger evolutionary rate dui/dt. This is particularly true of therapies designed to
inhibit glycolysis since reduction of glycolysis will exacerbate hypoxia in which cells are, and this lead to an increase in the value of σi and so also in the value of dui/dt[11].
1.5.7 Lactate dehydrogenase (LDH)
In hypoxic conditions, OXPHOS is not active because of the absence of su cient levels of oxygen so, pyruvate must be converted to lactate by LDH to allow the continuation of glycolysis. Consequently, it is evident that LDH covers a central position in the metabolic reprogramming of tumor cells, playing a key role in the maintenance of altered glycolytic metabolism and permitting survival of tumor cells when glycolysis represents the only energetic source[12]. Lactate dehydrogenase is a tetrameric enzyme with 5 isoforms composed of dierent associations of two kinds of subunits, the M (muscle) and H (heart) types, encoded by two dierent genes, LDH-A and LDH-B, respectively. These isoforms can be:
LDH-A e LDH-B;
Heterotetramers: LDH2 (M1H3), LDH3 (M2H2) e LDH4 (M3H1);
Figure 1.24: The two homotetramers (LDH-1 and LDH-5) and the three heterote-tramers (LDH-2, LDH-3 and LDH-4) are schematically represented[7].
The greater the number of H subunits an LDH contains, the lower its ability to catalyze the reaction from pyruvate to lactate. Therefore LDH5, composed of four M subunits, possesses the highest e ciency in converting pyruvate to lactate under anaer-obic conditions, such as in skeletal muscle, liver, and also hypoxic tumors. On the other hand, LDH1, composed of four H subunits, possesses higher a nity for lactate, and it is primarily involved in the conversion of lactate to pyruvate in aerobic tissues such as heart, kidney, spleen, and brain, as well as in some oxygenated tumor portions[4].
Recently, it has been observed that hLDH5 (human LDH5) is subject to post-transcriptional regulation by acetylation at lysine 5, which leads to a decrease of the enzymatic activ-ity. In fact, once the acetylated protein is recognized by the HSC70 chaperone, it is delivered to lysosomes for degradation. On the contrary, Lys5 acetylation of this en-zyme was found to be signi cantly reduced in human pancreatic cancer specimens, thus promoting a higher hLDH5 activity in these tissues[12]. Studies have shown that can-cer cells have a higher mitochondrial membrane potential (∆ψm). It was demonstrated
that the increased lactate production in the cells, the decrease in oxidative phosphory-lation and increased mitochondrial potential are related phenomena. In fact, a fraction of the proton mitochondrial potential, usually used to produce ATP, is dissipated in the tumor cells and increases in them ∆ψm. Studies were conducted on breast cancers cell
lines Neu 4145, because they possess high sensitivity to treatment with the 2-deoxy-glucose (an antimetabolite) and a high LDH activity. In these cells shRNA (RNA fragments that reduce the expression of the target gene) were used; they mitigated the activity of the enzyme in a dierent way.
Figure 1.25: A-Proliferation in tumor cells in comparison with the healthy lines EpH4 and NMuMg in the presence of increasing concentrations of 2-deoxyglucose; B-Comparison between the activity of LDH in healthy cells and cancer cells[13].
Figure 1.26: LDH decrease after the introduction of sh-RNA; LDH-A activity decrease after the introduction of shRNA[13].
From the images we can see how, by reducing the expression of LDH-A, the speed of cell proliferation decreases in both normoxic and hypoxic conditions. To ensure that it depends on LDH-A, cDNA (recombinant DNA) has been introduced in cells, restoring the enzyme. We can see from the images, how the proliferation rate has increased again both in the presence and absence of oxygen, demonstrating the close correlation between the latter and the LDH-A.
Figure 1.27: In both images we can observ how the introduction of the cDNA in cell lines L2-5.c15 leads to an increase in the proliferation rate in both hypoxic conditions and normoxic[13].
Figure 1.28: A- LDH increase after introduction of the cDNA; B-Quanti cation of ATP levels during cell growth in the presence and in the absence of oxygen; C-EPH-4 Cell proliferation curve [13].
Studies, therefore, show that the LDH-A has a great importance in regulating the speed of proliferation and in cancer cells it proved to be overexpressed. Individuals, who have a congenital lack of the enzyme, generally, do not show any symptoms; only in cases of intense physical exercise do they undergo myoglobinuria[13]. So, it seems clear, that drugs, which are able to inhibit LDH, might have a good anticancer acivity and, at the same time, also a good tolerability for patients and they are the object of this thesis.
The accepted catalytic mechanism of LDH is rather simple and starts with the initial binding of NADH to the enzyme followed by binding of the pyruvate. Then, the LDH-NADHpyruvate ternary complex undergoes a rate-limiting conformational change, in which a substrate speci city loop closes to form a desolvated ternary complex, princi-pally in order to bring the catalytical residue Arg109 into the active site where it polar-izes the ketone functionality of pyruvate, thus promoting hydride and proton transfer to the substrate. Apart from the catalytic residues Arg109, Asp168, and His195, which are highly conserved in all LDHs, other amino acids are also involved in substrate discrimination and recognition, such as Gln102, Arg171, and Thr246, together with Arg109. For example, Gln102, Arg109 and Thr246 are implicated in pyruvate recogni-tion by enclosing the methyl side chain of the native substrate, which is in fact oriented towards these residues. The catalytic mechanism involves a direct and stereospeci c transfer of a hydride ion (H−) from the C
4 carbon of the dihydronicotinamide ring
(H+) to the carbonyl oxygen of pyruvate from the catalytic diad Asp1684/His195,
nally producing lactate. The imidazole ring of His195 has the function of proton donor/acceptor in this reaction and it also orients the substrate in the proper position for its interaction with the C4 of NADH. The other aminoacid of the catalytic diad,
Asp168, interacts with His195 by stabilizing the protonated/cationic form of its imi-dazole ring through an H-bond between the side chain carboxylic group of Asp168 and imidazole ring of His195. The function of the last catalytic residue Arg171 is to x the substrate through a strong two-point interaction between its side chain and the carboxylate of pyruvate. It is important to note that Ile250 with its hydrophobic side chain provides an environment suitable for the nicotinamide ring of NADH [7].
Figure 1.29: Schematic representation of the LDH catalysis mechanism. LDH active site bound to both substrate pyruvate and cofactor NADH[7].
Chapter 2
The most important inhibitors of
glucose metabolism
2.1 Antitumoral agents acting on glycolysis
Cancer cells, as we have already discussed, switch to aerobic glycolysis and are able to survive in hypoxic environment. It may be possible, however, to take advantage of this peculiar metabolic feature of cancer cells for selective anticancer therapy. Up to now, many synthetic and natural inhibitors of variuos stages of glycolysis have been identi ed. GLUT and MCTs inhibitors have been also studied. However, there are only a few molecules in preclinincal studies or clinical trials that are known to exploit, or interfere with, the increased glycolytic process of invasive tumors[4]:
Lonidamine (1): it is a direct inhibitor of HK, the enzyme that catalyzes the phosphorylation of the 6-position of glucose. It is one of the most studied and e cient HK inhibitors. It possesses an indazole scaold, with a carboxylate at the 3-position and a 2,4-dichlorobenzyl substituent on N1. Lonidamine proved to be an e cient anti-proliferative agent even against some resistant breast cancer cells, and its mechanism of action, implying a decrease in glucose use and of lactate/ATP production, was con rmed. It has completed a phase 3 trial, but its e cacy was undermined by pancreatic and hepatic toxicity[4].
N N OH O Cl Cl 1 Figure 2.1: Lonidamine (1)
2-deoxy-D-glucose (2), HK inhibitor, is a glucose analogue in which the hydroxy group at position 2 is replaced by hydrogen. This compound was found to inhibit hexokinase by competition with glucose (ki=0.25). It showed promising results
in a phase 1 trial, but its action on hypoxic tumors was not satisfactory[4].
O HO HO OH 2 OH Figure 2.2: 2-deoxy-glucose (2)
Dichloroacetate (3), analogue of pyruvate, is a Pyruvate dehydrogenase kinase inhibitor, whose ability to decrease lactate production was already well-known for its approved clinical use in the treatment of hereditary lactic acidosis and of genetic mitochondrial diseases in humans. Because mitochondria in tumors are hyperpolarized relative to those in healthy cells, and this condition is related to re-sistance to apoptosis, it was demonstrated that this state can be reversed by DCA (Dichloroacetate). This molecule caused an enhanced e ux of pro-apoptotic fac-tors from mitochondria and increased ROS production, thus inducing apoptosis in cancer cells, without aecting mitochondria of noncancerous cells.
DCA was reported to show even better activity against cancer models in vivo than in vitro, and this evidence promoted the start of several clinical trials[4].
OH Cl Cl O 3 Figure 2.3: Dichloroacetate (3)
Silybin (also known as silibinin)(4), is a natural avonoid with strong inhibitory eects on the proliferation and survival of various cancer cells through direct interaction with GLUT1 and GLUT4. A phase 2 clinical trial designed to as-sess their eectiveness in patiens with prostate cancer was completed in 2008. A phase 1 trial in patiens with advanced hepatocellular carcinoma is currently recruiting[4]. O HO OH O OH O O OH OCH3 OH 4 Figure 2.4: Sylabin (4)
Glucophosphamide (5) is a glucose-containing cytotoxic agent, whose action is enhanced in cancer cells overexpressing GLUTs. It has completed clinical trials on several tumors and is composed of an alkylating moiety linked to a β-D-glucose unit that takes advantage of the transmembrane glucose transport system, which is greatly increased in malignant phenotypes[4].
O H HO H HO H H OH H P OH N H O Cl HN Cl 5 Figure 2.5: Glucophosphamide (5)
In the table below there are the principles antiglycolytic compounds and more detailed informations about their clinical trials[4].
2.2 LDH inhibitors
Until recently, the only well-characterized and speci c inhibitor of LDH was oxamic acid (6), a small molecule that inhibits both the A and B isoforms of LDH by competing with pyruvic acid, the enzyme's natural substrate. Although it displays good selectivity
HO
OH O
O
6
Figure 2.6: Oxamic acid (6)
for LDH and weak toxicity in healthy animals, oxamic acid has the drawback of poor cellular penetration; as consequence, it was found to inhibit aerobic glycolysis and the proliferation of tumor cells cultured in vitro only at high concentrations, which
cannot be expected to be reached in vivo. However it still considered as the LDH-A reference inhibitor. LDH-Another known LDH-LDH-A inhibitor is gossypol (7), a natural polyphenol dialdehyde extracted from cotton seeds, which is also highly cytotoxic and promiscuous[15, 17]. Gossypol and related compounds are competitive inhibitors of NADH binding to LDH, while oxamic acids are competitive inhibitors of pyruvate binding[16]. O H OH HO HO OH O H OH OH 7 Figure 2.7: Gossypol(7)
Some very small azoles possessing vicinal OH and COOH groups, such as
3-hydroxyisoxazole-4-carboxylic acid (HICA) (8) and 4-hydroxy-1,2,5-thiadiazole-3-carboxylic acid (HTCA) (9) showed IC50 values of 54 and 10 µM, respectively, on LDH-A, and the hydroxyl-carboxylic substitution pattern proved to be essential for their inhibitory activity.
N S N HO COOH N O HO COOH HICA HTCA 8 9
Figure 2.8: 3-hydroyisoxazole-4-carboxilic acid (HICA) (8); 4-hydroxy-1,2,5-thiadiazole-3-carboxylic acid (HTCA) (9)
Over the past years, LDH inhibitors were synthetyzed with a dierent aim, for their antimalarian activity. LDH, in fact, is essential to the metabolism of the Plasmodium Falciparum because it relays on glycolysis only for energy production. So, lactate
de-hydrogenase enzyme from Plasmodium falciparum (pfLDH) has been considered to be a potential molecular drug target for antimalarials. The amino acid sequence of malar-ial lactate dehydrogenase (pfLDH)revealed many residues at the active site that are unique to pfLDH compared to any other known LDH, including human LDH-M and LDH-H. Therefore, pfLDH appears to be an attractive target for development of anti-malarial drugs. In a preliminary study of the inhibition of pfLDH by gossypol-related compounds, derivatives of 8-deoxyhemigossylic acid were shown to be promising as se-lective inhibitors of pfLDH. Among them, there were also compounds highly sese-lective for LDH-A over LDH-B, despite the high sequence homologies of these two human LDHs. One of these compounds, FX11 (10), is a LDH-A inhibitor competitive with the NADH cofactor. It displays a Ki of 8 µM on LDH-A and a > 10-fold selectivity over the other isoform LDH-B. Although FX11 contains a potentially redox-active catechol moiety that may make it unsuitable as a drug, it is an important proof-of-concept for LDH-A inhibition as a tractable anticancer strategy [15, 17].
COOH
OH
OH
10
Figure 2.9: FX11 (10)
More recently, Gallo avin (11) has been identi ed as a novel inhibitor of human LDH5 and LDH1. It was tested in competition with both pyruvate (Ki=5.46 µM
on hLDH5 and Ki=15.1 µM on hLDH1) and NADH (Ki=56.0 µM on hLDH5 and
Ki=23.2 µM on LDH1). The resulting kinetik data, revealed that gallo avin
preferen-tially binds the free enzyme without being fully competitive with neither the substrate or the cofactor. It reduced lactate pruduction, decreased ATP levels, completely block the enzymatic activity of hLDH1 and hLDH5 and inhibit cell growth (60%) an in-duced apoptosis at a concentration of approximately 200 µM. A recent discovery has associated additional pharmacological activities to Gallo avin which is proved to be
able to interfere with the interaction of hLDH5 with ssDNA, consequently blocking RNA synthesis in vitro. Some glycolytic enzymes, such as LDH5 itself, are known to also be ssDNA binding proteins, playing a role in DNA trascription and replication. This interation involves the NADH binding site. In light of this property of gallo avin, its action in cancer cells do not rely exclusively on glycolysis and on LDH enzymatic activity but it can be abscribed to an inhibition of the RNA synthesis, rather than to the impairment of aerobic gycolysis [18].
O OH HO HO O O O OH 11 Figure 2.10: Gallo avin (11)
An other class of compounds acting as inhibitors of LDH is formed by quino-line 3-sulfonamides. GSK researchers developed an assay where recombinant human LDHA or LDHB enzymes catalyzed conversion of lactate to pyruvate, and the level of NADH produced in this reaction was measured through the conversion of resazurin to resoru n by diaphorase. In this way they were able to identify 3-((3-carbamoyl-7-(3, 5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl)amino) benzoic acid as an NADH-competitive LDHA inhibitor. Subsequent lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 to 3 nM, selectivity over LDHB on the order of 10 to 80-fold and without possessing any appreciable activity against a panel of common enzymes, receptors, and ion channels. LDHA inhibition led to a rapid re-duction of glucose uptake and lactate prore-duction and resulted in profound changes in overall metabolism and survival in hepatocellular carcinoma cells. It is notable that researchers were able to achieve LDHA selectivity over LDHB, given that the co-factor pocket where quinoline-3-sulfonamides bind dier only in two conservative changes
(Ala98-Val98 and Ile116-Val117 in LDHA and LDHB, respectively). The crystal struc-tures of compounds bound to LDHA demonstrate binding in the NADH pocket only and these compounds are not competitive versus pyruvate. However these compound have some limitations [19]:
pharmacokinetic properties of these quinoline 3-sulfonamides are unacceptable for in vivo use. In fact the optimization of both potency and selectivity towards this challenging target turned out to be incompatible with oral bioavailability and low in vivo clearance for this chemical class;
these compounds at doses of 10 µM and higher exhibit direct mitochondrial eects that are likely not mediated by LDH inhibition;
Figure 2.11: Structures of the Quinoline 3-sulfonamides inhibitors and their potency on recombinant human LDH enzymes.
An other group is costitued by the Bifunctional inhibitors. This term indicate compounds with a co-factor like portion and a substrate mimic portion which are connected through a linker. In this way they can extended in both substrate and and cofactor-binding pockets, in order to achieve a dual binding in NADH and pyruvate binding pockets of the LDH active site. Among them we consider the bifunctional malonate inhibitors discovered by Astrazeneca because they are some of the most potent LDH inhibitors reported in literature so far. Compound 16, in which the two antipodal portions are linked by a urea group, is the rst compound to be active in the enzymatic inhibition assays with an IC50of 4.2 µM. Moreover this compound displayed
a relevant increase in enzyme-binding a nity when compared with its single fragments, with a Kd of 0.13µM.
S N H N O H N HN O O OH HO O O 16 Figure 2.12: Compound 16
The replacement of urea portion with an amide group in the central linker produced other active inhibitors, such as 19 and 20. Among them S-propyl derivative 20 is the most potent inhibitors of this class with Kd=0.008 µM and a IC50=0.27µM. The
mal-onate derivatives were riported to lack activity in cell-based assays, probably because of their low cell penetration due to the diacid malonic functionality. For this reason dimethyl ester of compound 19 (IC50=0.5µM) was prepared and this displayed a good
antiproliferative activity in cells (IC50=4.8µM) [18].
S N H N O H N HN LINKER O OH HO O O Compound Structure IC50 (µM) R1 Linker hLDH5 17 H N O S-propyl 3.1 18 HN S-propyl 0.29 19 Me 0.5 20 S-propyl 0.27
Figure 2.13: Bifunctional malonate inhibitors of AstraZeneca (Maccles eld, UK): in-hibition data un hLDH5.
A similar fragment-based strategy was more recently applied by researchers at ARIAD Pharmaceuticals (MA, USA) and it results in other LDH potent inhibitors. They possess, on one side, a 6-(-3- uorophenyl)nicotinic acid terminal portion which binds in the nicotinamide binding pocket of the enzyme and, on the other side, a second nicotinic acid portion, connected to the rest of the molecule through a thio-arylalkyl chain which instead lies in the adenosine site. Compound 21 proved to be the most active of these inhibitors. Its linker was shortened and substitued with 4 hydroxyl groups in order to achieve additional interaction in the LDH active site, by mimicking the diphosphate portion of the NADH cofactor. It has an IC50=0.12µM. The presence
of the four hydroxyl groups and their chirality was found to be very important. In fact they mediated hydrogen bonds with the protein and their removal or changes in their stereochemestry caused substantial reductions of the inibition potencies of the result-ing compounds. Futhermore the presence of the carboxylic groups at the both ends of the two portions caused a reduced permeability through the cell membrane and, con-sequently, poor results in cellular assays. Therefore, lead compound 21 was modi ed removing one of these groups from either side leading to compounds which displayed a good activity in the enzyme inhibition assays but lower than parent compound 21 [18]. N HOOC S HN O MeO Cl O OH OH OH OH O F N COOH 21 Figure 2.14: Compound 21.
The last class of LDH inhibitors are the dihidropyrimidines. Researchers at Roche (Basal, Switzerland) and at Genentech (CA, USA) discovered new hLDH5 inhibitors possesing a 2-thio-6-oxo-1, 6-dihydropirimidine structure. The parent compound of this class is represented by compound 22 which reported an IC50=8.8µM in the enzymaic
assay on hLDH5. Its pharmacophiric groups are costitued by the sulfonilide substituent on the anilide ring, the cyano group of the central pyrimidine scaold and the p-chloro atom on the pheripherical phenyl substituent; in fact any attemp to modify or remove them led to a loss of inhibition potency. Compound 23 is the most potent inhibitor of this class with an IC50=0.48µM. It is obtained introducing a methyl group on the
aliphatic methylene and this increased the activity by more than ten folds. Such an improvement of inhibition potency was mantained even when additional halogens, such as chlorine or uorine atoms, were added in the phenyl ring in the ortho position with respect to the already present chlorine atom. This compounds exhibited activities in the nanomolar range. Similarly the replacement of the methyl group of 23 with an ethyl substituent led to compound 26 which mantains an almost unchanged inhibitory activity. Unfurtunately, compounds 23-26 did not demonstrate any activity on cell colture experiments, since they ere not able to reduce lactate production even at high concentation (50µM) probably because of their poor cellular permeability and high proteins binding. The eect of chirality on the inhibition potency of these compounds was not further investigated, although the enantiomer included in the complex with the enzyme found in the crystal structure displayed an R-con guration [18].
S H2N O O H N S R1 O N HN O CN R3 R2 Compound Structure IC50(µM )hLDH5 R1 R2 R3 22 H Cl H 8.8 23 Me Cl H 0.48 24 Me Cl Cl 0.75 25 Me Cl F 0.71 26 Et Cl H 0.65
Table 2.1: Genetech (CA,USA) dihydropyrimidine-based inhibitors: activity data on humans LDH5.
Chapter 3
Introduction to the experimental
section
3.1 Indoles
Researchers in our department found new LDH inhibitors. The X-ray crystal structure of the LDH-A subunit of hLDH5 shows that the active site is located in a rather deep position within the protein and accessibility to it is narrow. This cavity normally hosts both the substrate (pyruvate) and the cofactor (NADH). Overall, it is quite polar and rich in cationic residues (arginines). This would explain why the inhibitors so far discovered have carboxylates, closely associated with a hydroxyl or a carbonyl oxygen atom. Thiadiazole derivative HTCA cannot be functionalized further, and any substituent placed in the only free position of isoxazole HICA has aorded inactive compounds. Thus, the before mentioned azoles are not ideal candidates for further optimization. It was important to considered those pharmacological requirement and so they synthesized unusual class of heterocyclic derivatives, the N-hydroxyindoles (NHI), bearing a carboxylic acid group in the 2-position. The NHI scaold has been largely neglected in the design of biorelevant molecules, possibly because of the relative lack of synthetic methods for its assembly. On the contrary, numerous isolated natural products have been discovered to contain NHI portions, such as nocathiacin I, a stable antibiotic.
Compounds 27-37 were thus designed to retain the hydroxyl-carboxylate motifs of other LDH-A inhibitors, with the dierence that their N-OH group is slightly less acidic than typical phenol groups.
R1 R2 R3 N COOH OH 27-37 Compound R1 R2 R3 27 H H H 28 CH3 H H 29 CF3 H H 30 Cl H H 31 Br H H 32 H H H 33 C6H5 H H 34 H C6H5 H 35 H H C6H5 36 CF3 H C6H5 37 H H N N NN H
Figure 3.1: N-Hydroxyindoles (NHI) Designed as LDH-A[15]
It is important to note that even the smallest member of this class (27) showed a modest, but relevant, inhibition of both isoforms. In the 4-chlorosubstituted NHI 30, the introduction of methyl (28), tri uoromethyl (29), or bromo substituents (31) in position 4 decreased the inhibition of LDH-B, whereas the LDH-A inhibition was in some cases increased. When the bromine atom was shifted to the 6 position, the resulting compound (32) proved to be less active than its 4-substituted counterpart 31. They tried to introduce a phenil ring; in compound 33 they obtained poor results but if the phenyl group was present in position 5 (34) or 6 (35), they obtained a 99% and 84% inhibiton respectively. Both compounds showed practically no detectable inhibition of the other isoform (<3%), thus revealing a good level of selectivity. The presence of a 4-CF3 and a 6-phenyl in compound 36 caused a notable 87% LDH-A
inhibition, although a minimal residual activity on LDH-B (11%) could be detected. The replacement of the 6-phenyl group with a COOH-mimicking heteroaryl portion, such as the tetrazole, caused a dramatic decrease in the inhibitory potency of the resulting compound (37) with a poor 11% inhibition on LDHA.
It was con rmed, thus, when the N-OH/COOH pharmacophoric motif is modi ed,
Figure 3.2: Colorimetric measurement of inhibition of the enzymatic activity of LDH-A (gray bars) and LDH-B[15].
the enzyme inhibition potency is negatively aected and that compounds possessing phenyl rings, either at the 5 (34) or at the 6 position (35, 36), are the most potent and selective inhibitors of LDH-A [15].
3.2 Structural design
Accordingly to the previous paragraph (3.1) compound 36 was revealed to be the most promising LDH-A inhibitor. Docking studies show its interactions in the active site of LDH.
Figure 3.3: Compound 21 interactions in the LDH active site.
The carboxylic group of compound 36 shows a strong interaction with Ariginine (R169) and Treonine (T248), whereas the N -hydroxy group shows an H-bond interac-tion with the nitrogen of T248 and a water molecule that mediates the interacinterac-tion of 36 with the catalytic Histidine (H193). An important role of residues R169, T248, and H193 for the ligand interactions has already been highlighted by the analysis of the X-ray structure of the LDH complexed with the natural substrate pyruvate and NADH. In fact, this structure con rms the interactions between the pyruvate carboxylate group with the arginine and threonine residues of the enzyme, as well as that occurring be-tween the carbonyl oxygen atom of the substrate and the abovementioned histidine amino acid [15]. However, docking studies were made also on dierently substituted indoles containing a similar OH/COOH pharmacophoric motif with a good overlap of their carboxy- and hydroxyl-groups to those present in the previously developed NHIs inhibitors. These studies revealed that they maintained the same interactions in the receptor (Figure 3.4-3.5) and they are able to interact in the same way with the
amminoacids of the active site. During my thesis I synthesized variously substitued indoles in particular the N -H, N -Acetyl, N -methyl and N -sulfonyl indoles to check their acivity on LDH-A. The docking analysis of the N -H and N -Me indoles in the enzyme active site display a cavity surrounding the nitrogen atom in position 1, which may host other various substituents of limited size, such as those present in our N-Ac and N-Ms derivatives
Figure 3.4: Interactions of the N-H indole in the active site.
3.2.1 Synthetic pathways
The synthesis of indole derivatives that mimics the substrate pyruvate was synthesized according to Figure 3.6. Br COOMe NH2 Br COOMe N H COOMe 38 39 Br N OH COOMe H 40 N OH COOMe H 41 a
(a) BrCH2COOMe, Na2CO3, 80 °C overnight; (b) NaOMe(25%), dry ether, 40 - 42 °C, 3-4 h; (c) unsubstituted
phenylboronic acid, Pd(OAc)2, PPh3, K2CO3, Toulene 110 °C overnight;
b
c
Figure 3.6: Synthetic pathway for indole derivatives
The rst step is to synthesize intermediate 39 by N -alkylation of methyl 2-amino-5-bromobenzoate (38). This intermediate (39) was used for cyclization to aord cyclized product 40 by base-promoted cyclization. This cyclized product (40) was used as starting material for the Suzuki coupling reaction to obtain nal product (41). By following this pathway intermediate 39 and 40 were synthesized in 78% and 60% respectively, and compound 40 was used as starting material for the synthesis of the desired product 41. Unfortunately, Suzuki coupling reaction at the nal step was not a good choice because of many side reactions occurring at this level. In fact, nal product (41) was obtained in a very low yield and decarboxylation was the main side reaction. Although this synthetic pathway was designed for the preparation of a large series of desired compounds, because of the side products, this is not an e cient synthetic pathway. In the light of these results, we followed a dierent reaction pathway for the synthesis of an extended series including compound 41, which is outlined in Figure 3.7. This new synthetic pathway includes four/ ve steps for the synthesis of desired product (41) and its analogues. As shown in the general Figure 3.7, there are four steps in the
preparation of N unsubstituted target molecules and ve steps for preparation of N -methylated (46) and N -acylated target molecules (48).
Br COOMe NH2 Br COOMe NH COOMe COOMe NH COOMe N H OH COOH
(a) BrCH2COOMe, Na2CO3, 80 oC overnight; (b) NaOMe(25%), dry ether, 40-42 oC, 3-4hr; (c) un-/substituted phenylboronic acid, Pd(OAc)2, PPh3, K2CO3, toulene 110 oC overnight; (d) CH3I, CH3CN, Na2CO3, 80 oC overnight; (e) CH3COCl, CH3CN, 0 oC-RT, 2-3hr; (f) DBU, CH3CN, RT; (g) 50% NaOH, ethanol, 40 oC, 20-30min or LiOH 2N, THF/MeOH, RT, 3hr.
COOMe N COOMe COCH3 N H OH COOMe N OH COOMe COCH3 COOMe N COOMe CH3 N OH COOMe CH3 a N OH COOH CH3 Br-CH2-COOMe CH3COCl CH3I c d e f b g g 38 39 41 42 43 44 45 46 47 48 R R R R R R R R b B(OH)2 R
R = H (a), OMe (b), OCF3 (c). R = H (a), OMe (b), OCF3 (c).
R = H (a), OMe (b),
OCF3 (c).
Figure 3.7: Synthesis of N-unsubstituted/-substituted 2-hydroxy-3-carboylate indole derivatives
Some of these steps proved to be critical when speci c substrates were used and, therefore, they required optimization. In general for the preparation of target molecules (41 and 42), the four nal steps are: 1) N -alkylation; 2) Pd-catalyzed cross-coupling (Suzuki); 3) base-catalyzed cyclization; 4) hydrolysis of nal compound. For the prepa-ration of nal compounds, intermediate 43 was prepared with a Pd-catalyzed cross-coupling reaction. Intermediate 39 was prepared by using Na2CO3 as the base under
re uxing for overnight at 80 and by following reaction mechanism mentioned in Figure 3.8.
O OCH3 NH2 Br OMe H2 C Br O O OCH3 N Br H H2 C H OMe O Br O OCH3 N H Br COOMe 39 38
Figure 3.8: Reaction mechanism for N-alkylation.
The desired cyclized product was prepared with sodium methoxide as the base by using intermediate 43 which was synthesized by Pd-catalyzed cross-coupling reactions of intermediate 39 with either substituted or un-substituted phenylboronic acid in presence of K2CO3in dry toluene at 110 in 70-80% yield (Figure 3.7). The cyclization
reaction mechanism is drawn in Figure 3.9:
N H OMe OMe O O OMe H H N H O OMe OMe H O N H O OMe O H2O H N H OH OMe O 41 43 R R R R
Figure 3.9: Reaction mechanism for base-cyclization reaction.
In the last step the hydrolysis of methyl 3-hydroxy-5-phenyl-1H-indole-2-carboxylate was performed by base hydrolysis either by using 50% aqueous NaOH in ethanol at 40 or 2N aqueous LiOH in THF/MeOH at RT, according to the following gure:
N H OH O O CH3 OH N H OH O O CH3 HO N H OH OH O R R R 41 42
Figure 3.10: Hydrolysis of methyl carboxylates
In continuation of preparation of target molecules, N -methyl substituted nal prod-ucts (45, 46) were prepared with ve nal steps which are: 1) N -alkylation; 2)
Pd-catalyzed cross-coupling (Suzuki); 3) N -methylation, 4) base Pd-catalyzed cyclization; 5) hydrolysis of nal compound. Intermediate compounds 39 and 43 were prepared by similar synthetic pathways discussed earlier in this section (Figure 3.7). Interme-diates 44a-c were prepared by the reaction of their respective intermeInterme-diates 43a-c with iodomethane in presence of Na2CO3 at 80 in dry acetonitrile. Optimization
of the yields in the N -methylation step were obtained thanks to the various reaction conditions that were tested (Table 3.1), using the model reaction shown in Figure 3.11.
COOMe N H COOMe COOMe N COOMe CH3 43a 44a Figure 3.11: N-methylation.
No. Reaction condition %yield
1
K2CO3 (2eq.),CH3I (1.5eq.) Only starting
40 material exists Overnight 2 K2CO3 (2eq.), CH3CN,CH3I (1.5eq.) 15% 80 Overnight 3 Na2CO3 (2eq.), CH3CN,CH3I (1.5eq.) 20% 80 Overnight 4
Na2CO3 (2eq.), CH3CN,CH3I (1.5eq.) twice
45% 80
24hr
Table 3.1: Reaction condition.
After this optimization process, the best yields were obtained when 1.5 equivalent of iodomethane was added drop wise in the solution of 43a in dry acetonitrile in the presence of Na2CO3 at 80 . By following similar condition intermediate 44b-c
were synthesized. Desired N-methylated cyclized products 45a-c were prepared in the presence of sodium methoxide as the base via base-promoted cyclization reaction by using intermediate 44a-c, as discussed earlier in this section and reaction mechanism is drawn in Figure 3.12.
N OMe OMe O O OMe H H N O OMe OMe H O N O OMe O H2O H N OH OMe O 45 44 H3C CH3 CH3 CH3 R R R R
Figure 3.12: Reaction mechanism for base-cyclization.
Final carboxylic acid derivatives, 46a-c, were produced by base-promoted hydrol-ysis of compound 45a-c either by using 50% aqueous NaOH in ethanol at 40 or 2N aqueous LiOH in THF/MeOH at RT, according to the mechanism displayed in Figure 3.13. N OH O O CH3 OH N OH O O CH3 HO N OH OH O CH3 CH3 CH3 45 46 R R R
Figure 3.13: Hydrolysis of N-methyl.substituted indole carboxylates.
In addition, the preparation of target N -acetylated cyclized nal products (48a-c) synthetic routes were designed following ve nal steps which are: 1) N -alkylation; 2) Pd-catalyzed cross-coupling (Suzuki); 3) N-acetylation, 4) base catalyzed-cyclization; 5) hydrolysis of nal compound. Intermediate compounds 39 and 43 were prepared by similar synthetic pathway discussed earlier in this section by following Figure 3.7. Intermediates 47a-c were prepared by the reaction of their respective intermediate 43a-c with acetylchloride in presence of K2CO3 from 0 to room temperature in dry
aceto nitrile. The reaction mechanism is displayed in Figure 3.14.
Final N -acetylated cyclized products 48a-c were synthesized by base cyclization reaction of N -acetylated intermediate (47a-c) by using a non nucleophilic base, 1,8-Diazabicycloundec-7-ene (DBU), in dry toluene. The use of anhydrous and non-nucleophilic conditions proved to be essential in the e cient production of 48a-c.
