CHAPTER 8
Synthesis of glycoconjugates and pseudoglycoconjugates as a potential
antitumor agents
8.1. Introduction
Most of the conventional anticancer agents rely on the rapid proliferation of tumour cells. However the low specificity of this approach leads to low therapeutic indices and consequently to undesirable side effects.
Moreover, tumours often show hypoxic regions in which the rate of cell proliferation is slow, thus reducing the treatment efficacy.
Cancer cells are characterized by an altered metabolism compared to normal cells which allows tumours to satisfy increased metabolic demands and adapt to environmental changes. The exploitment of such metabolic alterations could constitute a valid strategy to achieve a selective effect against cancer cells over normal cells, thus giving the opportunity to reduce negative side effects and to obtain a more effective treatment.
8.2. Tumor hypoxia
As a solid tumor grows, existing vasculature is not able to supply nutrients, oxygen and growth factors due to the higher rate of cancer cell proliferation. As a result, cancer cells induce the expression of proangiogenic factors to promote tumour vascularization. However, the newly formed vasculature is irregular and malformed, thus resulting in transient changes in oxygenation, which in addition to diffusion-limited oxygen supply is responsible for tumour hypoxia.1 In fact, median oxygen levels in human tumour cells are generally much lower than those in normal tissues from which the tumours originate, with some regions presenting very low levels.
Hypoxia poses the basis of tumour resistance to cancer treatment, which results in: • radiotherapy-resistance;
• chemotherapy-resistance;
• obstacle to effective surgical removal of tumours, since most of the metastatic and aggressive phenotypes are associated to hypoxic cancer cells.
Tumour hypoxia exerts a physiological selective pressure for cell growth, thus promoting the selection of cells with malignant features and metastatic potential.1 In addition to hypoxia-induced adaptations, tumours undergo more complex metabolic transformations allowing cancer cells to sompensate their increased metabolic needs and adapt to environmental changes.
8.3. Cancer associated metabolic alterations
Among the most important hallmarks of cancer cells there are metabolic alterations that support their rapid proliferation rate, such as dependency on aerobic glycolysis for energy production and anabolic reactions, as well as a higher glucose uptake.
Normal cells generally rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy from glucose, whereas most cancer cells instead rely on glycolysis, uncoupled from OXPHOS. At this purpose, Otto Warburg had indicates glycolysis as the major anaerobic glucose metabolism within tumor cells (the Warburg effect). This is demonstred by:
1) a higher consumption of glucose due to the lower efficiency in energy production by glycolysis,
2) increased extracellular acidosis due to the high production of lactic acid and other acidic species.
This metabolic change ensures an adequate and rapid supply of energy and biosynthetic intermediates from glucose, and thus high vitality, even in the absence of sufficient levels of oxygen in hypoxic regions of cancer tissues.2
8.4. The glycolytic process
During glycolysis, glucose is subjected to a series of biochemical transformations devoted to demolishing its structure with the production of energy (ATP), and each step is catalyzed by specific enzymes (Figure 8.1). In normal cells, the glycolytic process is mostly coupled to OXPHOS, so pyruvate enters mitochondria and undergoes oxidative transformation into acetyl-CoA, which then enters the tricarboxylic acid (TCA) cycle and eventually produces CO2 together with a considerable amount of ATP. Under certain conditions, however, especially under oxygen deprivation, OXPHOS cannot take place, so pyruvate is instead converted into lactate by lactate dehydrogenase (LDH). This final step is fundamental because it allows regeneration of the oxidized co-factor NAD+ which is required for the regular progress of glycolysis (see conversion of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate; Figure 8.1) even when there is not enough oxygen to promote NADH re-oxidation.3
The “anaerobic” glycolytic pathway is much less efficient than OXPHOS in producing energy, as only two molecules of ATP are produced by each glucose molecule, versus the ~36 ATP units usually produced by the TCA cycle. However, glycolysis generates ATP more rapidly than OXPHOS, and this offers a selective advantage for rapidly growing tumor cells.
and this offers a selective advantage for rapidly growing tumor cells.
In most cancer cells, especially the most aggressive pheno-types, there is a substantial uncoupling of glycolysis from OXPHOS with consequent production of high levels of lactate (Warburg effect). This metabolic modification gives the tumor an evolutionary advantage, consisting of an adaptation to the more-or-less transient hypoxic conditions that occur during disease progression. This metabolic preference is also shown by a markedly higher uptake of glucose by cancer cells through transmembrane glucose transporters (GLUT), in order to feed the greater demand for energy and anabolites by rap-idly growing cells and to compensate for the low efficiency of the glycolytic process. Diagnostic use of the radiolabeled
glu-cose analogue [18F]fluorodeoxyglucose (FDG) constitutes an
ex-perimental confirmation of the selective high uptake of
glu-cose in invasive tumors.[13]Owing to this feature, it is clear that
any enzyme or transporter that promotes the glycolytic flux may be considered a potential target for blocking tumor pro-gression.
Lactate: not just a by-product of glycolysis
The end product of glycolysis, lactate, is produced in large excess in tumors. It is not merely a discharge product; on the contrary, it actively contributes to many aspects that promote tumor invasiveness, proliferation, and survival. In fact, the extent of lactate accumulation in primary tumors was found to
be inversely correlated with patient survival in many cases.[14]
First of all, the active secretion of lactic acid outside tumor cells contributes significantly to the acidification of the extra-cellular milieu, in addition to other mechanisms that promote
tumor acidosis.[15]This renders the environment around tumor
tissues more suitable for colonization and invasion by cancer cells. Moreover, lactate also actively stimulates tumor cell mi-gration by activation of b1-integrins, and angiogenesis,
follow-ing a stimulation of VEGF production in endothelial cells.[16]
Furthermore, extracellular lactic acid was found to inhibit the ability of the immune system to eradicate aberrant cells, thus
contributing to the immune escape phenomenon.[17]Finally,
in-creased survival of cancer cells to radiotherapies and to several chemotherapeutic drugs is supported by the general antioxi-dant properties of lactate, which inhibits the cytotoxic actions caused by reactive oxygen species (ROS) generated during
these treatments.[18]
One striking aspect concerning lactate in cancer tissues is its role in a particular cell–cell shuttle system, also known as the
“lactate shuttles”.[19]Tumor cells are normally highly
heteroge-Carlotta Granchi completed her gradu-ate studies in Chemistry and Pharma-ceutical Technology in 2007 and re-ceived her PhD in Medicinal Chemistry in 2011 at the University of Pisa (Italy). During her doctorate studies, she spent a period in 2009 conducting re-search with Paul J. Hergenrother in the Department of Chemistry at the Uni-versity of Illinois at Urbana-Champaign (USA). She is currently a postdoctoral research fellow under the supervision
of Filippo Minutolo in the Department of Pharmaceutical Sciences at the University of Pisa. Her research is focused on the design and synthesis of small molecules that are able to interfere with the spe-cific metabolism of invasive tumors.
Filippo Minutolo studied Chemistry and Pharmaceutical Technology at the University of Pisa (Italy). In 1993, he re-ceived an ENI fellowship to attend a tri-ennial graduate school at the Scuola Normale Superiore in Pisa, including a visiting research period (1994–1995) in the group of Ben L. Feringa at the University of Groningen (Netherlands). In 1996 he received his PhD in Chemis-try and then took a postdoctoral ap-pointment (1997–1999) at the
Universi-ty of Illinois at Urbana-Champaign (USA), where he worked in the research group of John A. Katzenellenbogen. In 2000 he became a researcher at the University of Pisa, and since 2006 he has held an associate professorship in Medicinal Chemistry. His main re-search interests include drug discovery in the fields of anticancer agents and nuclear receptor ligands.
Figure 1. Glucose metabolism through the glycolytic flux. GLUT, glucose transporter; HK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phos-phofructokinase; ALD, aldolase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PDK, pyruvate dehy-drogenase kinase; MCT, monocarboxylate transporters.
1320 www.chemmedchem.org ! 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2012, 7, 1318 – 1350
Figure 8.1. Glucose metabolism through the glycolytic flux. GLUT, glucose transporter; HK, hexokinase;
GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; ALD, aldolase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; MCT, monocarboxylate transporters.
This metabolic preference is also shown by a markedly higher uptake of glucose by cancer cells through transmembrane glucose transporters (GLUT), in order to feed the greater demand for energy and anabolites by rapidly growing cells and to compensate for the low efficiency of the glycolytic process. Owing to this feature, it is clear that any enzyme or transporter that promotes the glycolytic flux may be considered a potential target for blocking tumor progression.4
Figure 8.2. Oxidative phosphorylation, anaerobic glycolysis and aerobic glycolysis.
Increased aerobic glycolysis may be related to primary defects in oxidative phosphorylation, but it is surely connected to the activation of hypoxia-inducible factor-1-α (HIF-1α), which mediates a pleiotropic response to hypoxia by modulating glycolysis, tissue remodeling, fat metabolism, angiogenesis, erythropoiesis, proliferation, immortalization and pH regulation.
Metastasis and angiogenesis seem to be strictly related to hypoxia and enhanced glycolysis, thus leading some authors to associate the “glycolytic phenotype” with transition from pre-malignant lesions to invasive cancer (metastatic phenotypes).5
Among the most important metabolic alterations in cancer metabolism, hypoxia-inducible factor plays a fundamental coordinating role. HIF transactivates expression of several genes, such as glucose transporters GLUT1 and GLUT3, hexokinases 1 and 2 (HK1, HK2), lactate dehydrogenase A (LDH-A), lactate-extruding monocarboxylate transporter 4 (MCT4), pyruvate dehydrogenase kinase 1 (PDK1), carbonic anhydrases 9 and 12 (CA9, CA12), Na+/H+ transporter (NHE1), choline kinase (ChoK),2 lysyl oxidase (LOX), and vascular endothelial growth factor (VEGF), which promotes angiogenesis. In addition to an increased angiogenesis, HIF promotes the glycolytic pathway and an optimization of sugar metabolism. Pharmacological inhibitors of the enzymes responsible for lactate production and/or proton extrusion may produce an antimetastatic effect, in addition to the reduction of tumour growth.
To conclude, mutations in oncogenic and tumor suppressor patways allow cancer cells to a metabolic reprogramming which is essential to support cell proliferation.4 The peculiar cancer cells metabolism provides good chances to find suitable targets for selective cancer treatment.
8.5. Structure and function of human lactate dehydrogenase
Lactate dehydrogenases are a family of 2-hydroxyacid oxidoreductases diffused in almost all animal tissues, in microorganisms, in yeasts and plants, which catalyze the NAD(H)-dependent interconversion of pyruvate into lactate (Figure 8.3).
Figure 8.3. Reaction catalyzed by LDH.
Human lactate dehydrogenases (hLDHs) constitute a family of tetrameric isozymes composed of combinations of three different subunits: M-type (or A), H-type (or B) and C-type (exclusively expressed in male gender), encoded by different genes, respectively ldh-a, ldh-b, ldh-c. The polypeptide subunits combine to form three homotetramers, C4 (LDH-C4 or LDHX), H4 (LDH1) and M4 (LDH5), and three hybrid tetramers, H3M (LDH2), H2M2 (LDH3), and HM3 (LDH4).6
tissues, the lactate shuttle acts as both a metabolic fuel and a signaling molecule, positioning lactate at the intersection of key processes in cancer progression, such as tumor metabolism and angiogenesis. In addition to the intracellular lactate-shuttle it has been reported a cell-cell lactate shuttle, which is regulated by LDH-dependent conversion of lactate into pyruvate (and back), and the transport of lactate into and out of cells through specific monocarboxylate transporters (MCTs). In tumors, MCT4 is largely involved in hypoxia-driven lactate release, whereas the uptake of lactate into both tumor cells and tumor endothelial cells occurs via MCT1, as shown in Figure 8.4.7
neous in their oxygen and lactate content, and can be roughly classified into two categories: “normoxic/oxidative”, which are closer to blood vessels, or “hypoxic/glycolytic”, which are fur-ther away from the vascular network (Figure 2). These two cell
types establish a symbiotic cell–cell shuttling of lactate; this also occurs normally in skeletal muscle and brain tissue, con-sisting of the production of lactate by the glycolytic cell and
its uptake and use by the oxidative counterpart.[20]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 final conversion of pyruvate to lactate is cat-alyzed by lactate dehydrogenase 5 (LDH5). MCT4 then extrudes lactic acid from hypoxic cells into the extracellular milieu. Lac-tate then functions as a metabolic fuel in oxidative tumor cells, where it is taken up through MCT1 and oxidized to pyruvate by LDH1, thus entering the TCA cycle in mitochondria with
production of energy and CO2. These observations support the
central role played by lactate in tumor function. Therefore, ef-fectors responsible for its production (LDH5), cell extrusion (MCT4), cell uptake (MCT1), or use (LDH1) constitute further potential targets for anticancer drugs.
HIF-1-induced changes in glycolysis
The vast majority of human cancers overexpress several glycol-ysis-related genes, leading to the Warburg effect. Hypoxia-in-ducible factor 1 (HIF-1) is a key player in the promotion of this phenomenon shown by aggressive tumors. This factor is con-stituted by two subunits: HIF-1a and HIF-1b; whereas the b su-bunit is constitutively nuclear, HIF-1a is unstable under nor-moxic conditions because it is rapidly hydroxylated by en-zymes that belong to the family of prolylhydroxylases (PHDs), provided there is sufficient oxygen to support this process. Once hydroxylated, HIF-1a is conjugated to the von Hippel– Landau (VHL) protein, then poly-ubiquitinated and eventually
degraded by the proteasome.[21]Under hypoxic conditions the
PHD-initiated inactivation of HIF-1a does not take place, and this subunit migrates into the cell nucleus where it binds to
ally active HIF-1, which activates the transcription of a series of
genes.[22, 23]The most significant, though not exclusive, target
gene products of HIF-1 that are involved in the promotion of the glycolytic flux are: glucose transporters 1 and 3 (GLUT1, GLUT3), hexokinases 1 and 2 (HK1, HK2), phosphofructokinase 1 (PFK1) and 2 (PFK2, in particular PFKFB3), aldolases A and C (ALDA, ALDC), phosphoglycerate kinase 1 (PGK1), enolase 1 (ENO1), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinases 1 and, most likely, 2 (PDK1, PDK2), lactate dehydrogen-ase 5 (LDH5), and monocarboxylate transporter 4 (MCT4). The HIF-1-induced overexpression of GLUT1 and GLUT3 strongly supports the remarkably higher glucose uptake found in tumor cells, in response to their increased energy and anabo-lite demands, as well as to the lower efficiency of the glycolytic process leading to lactate. Enhanced transcription of enzymes HK1–2, PFK1–2, ALDA–C, PGK1, ENO1, and PKM2 contributes directly to the enhancement of the glycolytic rate from glucose to pyruvate. The role played by PDK1 and PDK2 is to inhibit pyruvate dehydrogenase (PDH), an enzyme that promotes the oxidation of pyruvate to acetyl-CoA in the mitochondria, thus introducing it into the TCA cycle. Therefore, HIF-1 activates the expression of these PDKs, so that pyruvate is precluded from entering the final OXPHOS process, and simultaneously pro-motes the expression of LDH5, which instead converts pyru-vate to lactate. The picture is completed with the enhanced production of MCT4, which is responsible for the extrusion of lactic acid from the cell. These HIF-1-linked proteins are all po-tential targets for antiglycolytic cancer agents, the inhibition of which should lead to selective damage to invasive tumor cells, where HIF-1-promoted gene transcription is more relevant, with fewer side effects expected in normal cells. Nevertheless, other targets involved in the glycolytic flux should also be con-sidered for the development of potential antitumor drugs, as all the effectors of glycolysis were generally found to be over-expressed upon HIF-1 intervention; all of these are discussed in the following sections.
Glycolytic Effectors as Potential Targets in Cancer Therapy
Glucose transporters
The entry of glucose into the cell occurs by facilitated diffusion and depends mainly on glucose transporters (GLUTs). There are three GLUT classes with tissue-specific distribution and distinct affinity for glucose and other carbohydrates. Class 1 comprises four members, GLUT1–GLUT4, whose preferential substrate is glucose, whereas classes 2 (GLUT5) and 3 (GLUT6, 8, 10, HMIT) are more selective for other sugars. All these classes share a common tertiary structure, characterized by 12 transmem-brane domains, in which the sequence of residues is highly conserved. In particular, human class 1 GLUTs are 48–63 %
identical, and have been extensively characterized.[24] GLUTs
are widely overexpressed in cancer cells with respect to normal tissues, especially in high proliferative and malignant tumors, contributing to the high glycolytic flux observed in
Figure 2. Roles of lactate in the symbiotic model of intercellular shuttle be-tween “glycolytic” and “oxidative” tumor cells.
MED
Counteracting Tumor Glycolysis
Figure 8.4. Roles of lactate in the symbiotic model of intercellular shuttle between “glycolytic” and
“oxidative” tumor cells.
8.6. Catalytic mechanism of hLDH
The catalytic mechanism of pyruvate reduction to lactate performed by LDH consists in: 1. initial binding of NADH;
2. pyruvate binding;
3. ternary complex LDH-NADH-pyruvate undergoes a rate-limiting conformational change, which leads to a desolvated ternary complex consequent to the closure of the substrate-specificity loop;8
4. catalytic residues in the active site, such as Arg109, are brought in proximity to the substrate chemical bonds that will be altered in the reaction.
The detailed catalytic mechanism consists in the Arg109-mediated polarization of the pyruvate ketone group, which promotes the direct hydride transfer from NADH to the substrate. Besides the catalytic residues highly conserved in all hLDHs, such as Arg109, Asp168 and His195, other key residues are involved in substrate discrimination, such as Arg171, Gln102, and Thr246. The residues Gln102, Thr246 and Arg109 are fundamental for pyruvate recognition, by enclosing the methyl chain of the native substrate, which is oriented towards these three residues.9 The catalytic mechanism involves the stereospecific hydride transfer from the C(4) carbon of the dihydronicotinamide group of NADH to the pyruvate ketone functionality and the proton donation
to the carbonyl oxygen of pyruvate from the catalytic diad Asp168/His195, thus producing lactate. The suitable orientation of the substrate is ensured by His195, thanks to its imidazole ring that is a proton/donor acceptor, and its protonated/cationic form is stabilized by the Asp168 side chain. The amino acid Arg171 acts to anchor the substrate by a strong two point interaction between its basic chain and the pyruvate carboxylate group, whereas the hydrophobic chain of Ile250 creates a suitable environment to accommodate NADH nicotinamide ring (Figure 8.5).10
Figure 8.5. Schematic representation of the LDH catalysis mechanism.10 8.7. Human LDH isoform 5 as anticancer target
Lactate dehydrogenase is a key enzyme in tumour metabolism, since it strongly depends on the glycolytic pathway to produce energy. This is confirmed by the higher amounts of lactate production among cancer cells compared to normal cells.
Hypoxic tumours show an overexpression of LDH5, whereas it does not recover a critical role in normal cells, since they mainly produce energy from mitochondrial activity, that is, the Krebs cycle, electron transport chain and oxidative phosphorylation (OXPHOS).
Regarding the possible side effects resulting from LDH5 inhibition, this isoform seems to be a “safe target” in anticancer therapy.
To sum up, the advantages of targeting LDH as anticancer therapy are:
1. innovative approach, compared to conventional chemotherapeutic agents;
8.8. Inhibitors of hLDH
In literature are reported only few inhibitors of LDH, such as Oxamate (OXM),11 gossypol, a natural polyphenol dialdehyde extracted from cotton seeds, which is also highly cytotoxic and promiscuous,10 azole derivatives (HICA, HTCA)12 and FX-1113 with a not sufficient and encouraging inhibition activity of LDH (Scheme 8.1).
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8.9. N-hydroxyindole-derived compounds as LDH5 inhibitors
In the attempt to find selective inhibitors of lactate dehydrogenase isoform 5, the research group of Prof. Minutolo14 have designed a series of N-hydroxyindole-2-carboxylates, which represent the starting point for the structural design of the new final glycoconjugate products synthesized in the present work.
Regarding the structure of active site of LDH-A subunit of hLDH5, it is located in a deep position within the protein and its accessibility is narrow, as seen from the X-ray crystal structure. This cavity is rich in polar and cationic residues, such as arginines, which are important to interact with the substrate during the catalytic activity, and confirm the key structural requirements of a carboxylate and a hydroxy group in adjacent positions as substrate-like pharmacophoric motif to ensure strong interactions between the inhibitors and the enzyme.
Among this class of N-hydrox-indole-2-carboxylates the most active compounds are reported in Figures 8.6 and 8.7.
Enzyme kinetic experiments show that these NHI-based inhibitors are competitive with respect to both NADH and pyruvate, and they display a high degree of isoform selectivity for LDH5 over LDH1. The results of the enzymatic assays have been confirmed by modeling studies, showing strong interactions of key residues in the active site of LDH for the most active compounds. These compounds occupy the substrate-binding pocket with their “OH/COOH” polar terminal and, at the same time, the cofactor binding pocket with their aromatic moiety, showing Ki values in the micromolar range.
Compounds 5- or 6-phenyl substituted (Figure 8.6) and the 4-trifluoromethyl-6-phenyl-derivative NHI 8.1 (Figure 8.7), utilized for glycoconjugate synthesis are able to inhibit cancer cells proliferation, especially under hypoxic conditions. Their mechanism of action is supported by the reduction of lactate production in HeLa cells upon exposure to these compounds and the docking
modeling studies highlight the interactions between these compounds and the target enzyme in the active site. ! " # $%%& %& Figure 8.6. Phenyl-substituted NHIs.
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Figure 8.7. 4-trifluoromethyl-6-phenyl-NHI-2-carboxylate (NHI) 8.1.
It was also made an appropriate docking analysis and the obtained results show that NHI 8.1 may occupy the whole substrate pocket and, in part, the cofactor pocket of LDH-A, are in good agreement with the experimental enzyme inhibition data, which indicate that its inhibition is competitive with both pyruvate and NADH.14
Figure 8.8. Docking modeling studies for NHI 8.1.
8.10. General structural requirements
should not be too small, since reduced molecular dimensions do the guarantee selectivity, as it has been demonstrated with the smallest LDH inhibitors, such as oxamate and azole-derivatives. However, some important structural requirements that are common to the majority of the most active compounds among the classes above presented are the presence of a carboxylate group in adjacent position to an hydroxyl or carbonyl group, thus mimicking the natural substrates of lactate dehydrogenases (Figure 8.9).10
Figure 8.9. Generic structural features for the two main classes (lactate-like and pyruvate-like) of LDH
inhibitors.10
The attempt to obtain a selective inhibition of hLDH5 isoform as anticancer strategy must take into account these fundamental structural requirements, in order to achieve a satisfactory degree of interaction between the drug candidate and the target, but this must be connected with the research of suitable structures that would not lead to undesirable side effects due to non-specific interactions. In this context, it is important to obtain a selective action on the muscle isoform of LDH (hLDH5), which is over-expressed in tumours, and to guarantee a selective action that would not lead to non-specific interactions with other biological targets.
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Scheme 8.2. NHIs and their glycoconjugates.
antiproliferative activity on several tumor cell lines and a competitive behavior both with respect to the NADH and the pyruvate. One of the most attractive feature of these molecules was also the high selectivity towards the isoform A with respect to the isoform B. The cellular assays had then confirmed that these compounds can stop the proliferation of cancer cells, especially in hypoxia conditions. Their action mechanism, inhibition of LDH-A, had been effectively confirmed by the reduction of lactate production in HeLa cells exposed of these inhibitors.
A very interesting strategy, now little used, consists of the conjugation of carbohydrates, such as glucose and mannose, to molecules with cytotoxic action, in order to fully exploit the Warburg effect, as therapeutic approach. The resulting glycoconjugates should be avidly catched by tumor cells carachterized by an high level of glycolytic activity.
In this way, if the carbohydrate uptake is not disturbed by sugar conjugation with the cytotoxic agent, the glycoconjugate will be introduced very quickly and effectively into the cancer cells. This has been previously demonstrated with glucoconjugates of known anticancer agents such as nitrogenous mustard.15
On this basis, we thought that the glycoconjugation of glucose with LDH5 glycolytic process inhibitors, such as NHIs, could be an important element of synergy to oppose the Warburg effect. Recently, in view of these considerations, it was synthesized the glucoconjugate (+)-8.3 of methyl ester 8.2, in order to match the inhibitory effect on LDH5 with a high cellular uptake by GLUT transporters (Scheme 8.2).
These studies have indicated that all compounds are good inhibitors of LDH5 with a competitive mechanism against NADH, showing inhibition values (Ki) in the low micromolar range, despite the glycoconjugate (+)-8.3 is less potent than derivatives 8.1 and 8.2. On the contrary, cytotoxic studies on a cell line of breast cancer (MCF-7) showed that (+)-8.3 is much more effective than its not conjugate analogues.
In these experiments, it was demonstrated that glucoconjugated (+)-8.3 showed a very effective and dose dependent reduction of lactic acid production of cervical cancer cells (HeLa), while the compound 8.1 has proved to be less potent in this assay. Futhermore, compound 8.1, despite being a potent inhibitor of LDH5, has showed cell permeability problems that reduce its effectiveness in cellular assays (vide infra).
Ester 8.2 combines a good inhibitory action on the enzyme, with a good permeability by passive diffusion across the cell membrane that maintains good efficacy in assays of cells. Finally, the glucose derivative (+)-8.3, although it is a less potent inhibitor on the isolated enzyme, thanks to the use of systems of active transport (GLUT) in tumor cells, dispayed the best cytotoxic activity of
8.11. Synthesis of β-gulo and α-mannoglycoconjugates
Actually, compound 8.2 is the first LDH-inhibitor designed to exploit as tumor cells avidity for glucose, such as their high glycolytic activity, manifested by the over-expression of LDH5. Indeed, this compound showed an increased cytotoxicity against tumor cells and greater cellular permeability compared to its not-conjugated analogues.
Therefore, we have developed synthetic pathways to change the sugar portion connected to the NHI inhibitor 8.2 in order to improve cellular uptake and enhance the inhibition potency toward the enzyme.
For these reason, our attention was directed to the synthesis of new glycoconjugates (+)-8.4 and (+)-8.5 in which the inhibitor NHI 8.2 was linked to different monosaccharides, such as D-mannose or D-gulose (Scheme 8.3), considering that these sugars, particularly the first one, is overexpressed on many tumor lesions.
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Scheme 8.3. NHI glycoconjugates.
Theoretically, these new glycoconjugates could be synthesized using traditional glycosyl donors, for example D-mannose, which could be obtained in a simple way, but the complete α- or β-stereoselectivity in the glycosylation process with D-mannose and D-gulose, respectively, was certainly a critical point in the synthetic pathway.
Recently, a new glycosylation process was developed in our laboratory, by means of glycal derived vinyl epoxides 1.1α and 1.1β, bearing at C(6) a benzyloxy functionality, in a regio- and stereoselective fashion, as described in Chapter 1 (Scheme 8.4).
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Scheme 8.4. Glycal derived vinyl epoxides 1.1α and 1.1β.
For the mannoglycoside (+)-8.4, we first performed a simple and not expensive synthetic strategy consisting in the glycosilation reaction of NHI 8.2, in the presence of TMSOTf as the catalyst, by
the α-manno trichloroacetimidate (-)-5.1. Likewise, we thought interesting to repeat the same glycosylation by means of vinyl epoxide 1.1α and then to use also vinyl epoxide 1.1β, as the glycosyl donor, to obtain both glycoconjugates (+)-8.4 and (+)-8.5.
The vinyl epoxides 1.1α and 1.1β cannot be isolated, because unstable and therefore they must be prepared only in situ by cyclization under basic conditions with t-BuOK of their ultimate precursors, the corresponding trans hydroxy mesylates 1.3α and 1.3β, which are obtained from the commercially available tri-O-acetyl-D-glucal (+)-3.6, through consolidated procedures developed in our laboratory and showed in Chapter 1.16
Following a typical glycosylation reaction, epoxide 1.1β was treated with a small amont of indole derivative 8.2 (the glycosyl acceptor, 1.1 equiv). 1H NMR analysis of the crude reaction product showed the presence of the 2,3-unsaturated-β-O-glycoside 8.6, as the only reaction product. In this way, our original glycosylation process, which uses the vinyl epoxide 1.1β as the glycosyl donor, turned out to be completely 1,4-regio- and β-stereoselective with the exclusive formation of the corresponding syn-1,4-addition product (the coordination product) also with this particular type of
O-nucleophile. After purification by flash chromatography, β-O-glycoside 8.6 was obtained pure
with good yield (68%) (Scheme 8.5).
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Scheme 8.5. Glycosylation of NHI 8.2 by vinyl epoxide 1.1β.
Likewise, the application of the same protocol to diastereisomeric vinyl epoxide 1.1α led to 2,3-unsaturated-α-O-glycoside (-)-8.7 (the corresponding coordination product) as the only reaction product, in a completely 1,4-regio- and α-stereoselective glycosylation process (Scheme 8.6).
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functionalization of the double bond present in O-glycosides 8.6 and (-)-8.7 and the debenzylation, by hydrogenolysis, of the C(6)-OBn group. For this reason, before prosecuting in the synthesis, we thought opportune to verify the stability of the glycosidic bond present in 8.6 and (-)-8.7 to the hydrogenolysis conditions. In this way, the β-glycoconjugate 8.6 was subjected to catalytic hydrogenation reaction by using 5% Pd/C in anhydrous AcOEt. Unlikely, after a very short time (20 minutes), the crude reaction product subjected to preparative TLC, showed only products deriving from the hydrogenolysis of the N-O bond present in the starting glycoside: the deoxy gulopyranose 8.8α,β (as a mixture of α- and β-anomers) and the indole derivative 8.9 (1H NMR) (Scheme 8.7). ! "#! !"# $! ! % &'( )* +,!!& ! "#! !"!!$" $! !$ $ % &'( )* +,!!& -$.// 01/)23& 45!67 !"%
Scheme 8.7. Catalityc hydrogenation (Pd/C) of 2,3-unsaturated-β-O-glycoside 8.6.
As this negative result could be due to the allylic nature of the glycosidic bond present in β-O-glycoside 8.6, we thought to functionalize the double bond present in 8.6 and (-)-8.7 before the hydrogenolysis step. For this reason, glycoconjugates 8.6 and (-)-8.7 were dihydroxylated by catalytic OsO4/NMO protocol in t-BuOH, with the exclusive obtainment of the corresponding, fully hydroxylated β- and α-O-glycosides 8.10 and (+)-8.11, with 62% and 68% yield, respectively (Scheme 8.8).17 ! ! "#! "#! !"# $%&%!"$ ! '! '! ! ( )*+ ,-./!!) ( )*+ ,-./!!) ! ! "#! "#! !"%& $0&%!"%% ! '! '! ! ( )*+ ,-./!!) ( )*+ ,-./!!) !1!23(.! 45) 678 !1!23(.! 45) 698 !' !' !' !'
Scheme 8.8. Catalytic dihydroxylation of glycosides 8.6 and (-)-8.7.
It should be emphasized that, as previously found in our laboratory on structurally related systems,17 the dihydroxylation reactions of 8.6 and (-)-8.7 proceed in a completely stereoselective
fashion, by an exclusive attack of the electrophile on the α-face in the case of 8.6 and β-face in the case of (-)-8.7. Reasonably, the observed complete α- or β-face selectivity can be attributed to the steric effects by the two allyl substituents, both α- or β-oriented, present at the C(1) and C(4) carbons of the unsaturated system of 8.6 and (-)-8.7.
Once the double bond was functionalized, the catalytic hydrogenation of the glycoconjugate 8.6, in the presence of 5%Pd/C in absolute EtOH, was repeated but, after 45 minutes, the exclusive reduction of the indole nucleus was again observed with the formation of indole derivative 8.9. At this point, we thought useful to try the mild hydrogenolysis by Pd(OH)2/C, as the catalyst, which had given excellent results on other sensitive systems.18 In a first moment, we decided to evaluate this deprotection protocol with β-O-glycoside (+)-8.3, taken as an appropriate model, since less expensive and, therefore, easily available in our laboratory in a sufficient amount. The hydrogenolysis of (+)-8.3 by H2/10-20%Pd(OH)2/C protocol in 1:1 MeOH/AcOEt mixture gave, once again, after only 30 minutes at room temperature, the reduced indole 8.9 and a mixture of the α- and β-D-glucose 8.12α,β (1H NMR, Scheme 8.9), to indicate that also this procedure could not be applied to glycosides bearing the N-hydroxyindole residue.
! "#$ %& "''() ' ' '* *' *' '* +,-.!"# ! * "#$ %& "''() !"$ ' '* *' *' '* '* , */0 12./230%4+'*-/5" ()'*567'89 !"%&!'"
Scheme 8.9. Hydrogenation of glycoside (+)-8.3 by means of the mild H2-Pd(OH)2/C protocol.
Alternatively to catalytic hydrogenation, an oxidative method of debenzylation was tried with derivative 8.6 by means of DDQ protocol, which was found to be effectively used in the synthesis of natural products.19 In our case, however, the treatment of 8.6 with DDQ in 18:1 CH2Cl2/H2O mixture, also for prolonged periods, was completely ineffectual and the starting reaction compound was recovered unreacted. No positive result was obtained also by application of BCl3 debenzylation protocol20 to glycoside 8.6.
At this point, it appeared clear that an O-benzyl protected glycosyl donor, as 1.1β, could not be used for our purpose. Probably, vinyl epoxide 8.13β, the corresponding free 6-OH analogue, could be more appropriate, because its use, obviously, eliminates the final debenzylation step (Scheme
! ! ! ! "! #$! !"#$! #"#!
Scheme 8.10. Glycosyl donor 1.1β and its corresponding 6-O-debenzylated analogue 8.13β.
The synthesis of vinyl epoxide 8.13β starts from trans diol 3.9, on its own prepared from tri-O-acetyl-D-glucal (+)-3.6, as previously described (see Chapter 3). The treatment of trans diol 3.9 with TBSCl (1.0 equiv) afforded silyl derivative 8.14, as the only reaction product, which was subjected to mesylation by MsCl/Py protocol to give the desired all-protected glycal 8.15. The subsequent desilylation by means of TBAF/THF protocol led to the trans hydroxy mesylate 8.16β. The deprotection of tetrahydropyranyl acetal present in 8.16β was carried out in EtOH in the presence of a catalytic amount of PPTS at 35°C, leading to the trans hydroxy mesylate (+)-8.17, bearing a free hydroxy functionality at C(6) (Scheme 8.11).
!"#$!"# % % &% $"%!! % %'( '(% '(% % %& &% )&*% % %)+, &% )&*% % %)+, -.% )&*% % %& -.% )&*% % %& -.% &% )+,/0 123456708 9-:; <=/ -./0 *>; <=/ )+': )&: <=/ **),?@A%& B<=/ !"& $"%' $"%( $"%#! !"#$$"%) !$+C%D
Scheme 8.11. Synthesis of glycosyl donor 8.13β.
Trans hydroxy mesylate (+)-8.17 is the ultimate precursor of epoxy alcohol 8.13β, which, as usual,
is not stable and can only be prepared in situ by cyclization of trans hydroxy mesylate (+)-8.17 under basic conditions (t-BuOK). Once formed, epoxide 8.13β was immediately treated with NHI 8.2, as the nucleophile (1.5 equiv in CH3CN), to give the desired β-O-glycoside (+)-8.18, which
was purified and characterized. These results indicated that, under these conditions, the new vinyl epoxide 8.13β, as the corresponding 6-OBn substituted analogue 1.1β, turned out to be a regio- and stereoselective glycosyl donor leading to β-O-glycoside (+)-8.18 through a completely 1,4-regio and β-stereoselective process (complete 1,4-syn-stereoselectivity, Scheme 8.12). However, the new procedure was not considered completely suitable to our synthetic requirements, because the yield, due to the complexity of the crude reaction mixture, was too low, with respect to the original protocol based on epoxide 1.1β, (13% and 68% yield with 8.13β and 1.1β, respectively). Probably, the observed low yield is the consequence of the presence, in epoxide 8.13β, of the free primary hydroxy functionality. Actually, the -OH group of another molecule of 8.13β could intermolecularly and, therefore, negatively interfere with the nucleophile-oxirane oxygen coordination which is necessary for the occurrence, in these conditions, of the addition by a weak nucleophile as 8.2. This interference could slow down the addition process to the point that the unstable epoxide decomposes with the formation of a complex reaction mixture, as found (Scheme 8.12). ! ! "! !"#$! ! "! #$%&!"#% '(! !" !&)*!+ ,"-,. ."/ "! ! #$%&!"#! "! ! . ,0 -12 '3!!, !"&
Scheme 8.12. Synthesis of glycoside (+)-8.18.
At this point, all considering, we decided to use the new vinyl epoxide 8.19β in which the primary hydroxy functionality is now protected as tetrahydropyranyl ether (-OTHP), that is with a protective group which can be easily removed by acid hydrolysis. Actually, deprotecting condition of this type appeared to be compatible with the glycosidic bond present in glycoconjugates obtainable by the use of NHI 8.2, as the glycosyl acceptor.
The vinyl epoxide 8.19β, as the previously glycosyl donors 1.1β and 8.13β, was obtained in situ by cyclization with t-BuOK in CH3CN of the corresponding stable precursor, trans hydroxy mesylate 8.16β, and immediately treated with the glycosyl acceptor NHI 8.2 to give, after only 30 minutes at room temperature, the glycosylation product, NHI β-O-D-glycoside 8.20, by the usual complete 1,4-regio- and β-stereoselectivity, with a good yield (77%) after purification by flash chromatography (Scheme 8.13).
! ! "#$! !"#$! ! "#$! !"#%! %&! !# !'()!* +#, +--#. "#$! ! !"&' #! ! -+/, $0 %1!!+
Scheme 8.13. Synthesis of NHI β-O-D-glycoside 8.20.
Functionalization of the double bond present in β-O-D-glycoside 8.20, by OsO4/NMO protocol, afforded the corresponding syn-dihydroxylated product 8.21, in accordance with a complete, sterically favored α-facial stereoselective electrophilic addition (70% yield) (Scheme 8.14, see also Chapter 4).17 ! "#$! !"#$ #! ! % &'( $) *+!!& ! "#$! !"#% #! ! % &'( $) *+!!& !,!-.%*! /0& 123 !# !#
Scheme 8.14. Dihydroxylation by OsO4/NMO protocol of β-O-glycoside 8.20 .
The deprotection of the C(6)-OTHP functionality of 8.21 by using catalytic PPTS in absolute EtOH under controlled temperature at 40°C for 46 h, (Scheme 8.15) afforded the desired β-O-glycoconjugate (+)-8.5 (a NHI β-O-D-gulopyranoside) in good yield (75% yield) after recrystallization. ! "#$! !"#$ #! ! % &'( $) *+!!& ! #! ,-./!"% #! ! % &'( $) *+!!& $$"0123!# 456& !# !# !# !#
Scheme 8.15. Deprotection of C(6)-OTHP functionality in β-O-glycoside 8.21 under acid conditions to
NHI β-O-D-gulopyranoside (+)-8.5.
On the basis of these satisfactory results, we elaborated a synthetic process to obtain the second glycoconjugate of our interest, the α-mannoglycoside (+)-8.4, starting from vinyl epoxide 8.19α, the glycosyl donor diastereoisomer of 8.19α (Scheme 8.16).
! !
! !
"#$! "#$!
!"#$! !"#$"
Scheme 8.16. Diastereoisomeric glycosyl donors 8.19β and 8.19α.
For the synthesis of epoxide 8.19α, trans diol 8.23, corresponding to 6-O-THP-D-gulal, was considered useful, as the starting material. Following the experience acquired on glycal-derived epoxides, trans diol 8.23 was prepared by using a non-coordinating hydroxy ion equivalent as Me3SiO- present in Bu4N+Me3SiO-, a salt prepared by addition of potassium trimethyl silanolate (Me3SiOK) to a solution of tetrabutyl amonium bromide (TBAB) in THF.16d In this way, after cyclization under alkaline conditions (t-BuOK) in anhydrous THF of trans hydroxy mesylate 8.16β to vinyl epoxide 8.19β, the addition of a freshly prepared solution of Bu4N+Me3SiO- in THF affords, through a clean 1,2-addition process, the desired trans diol 8.23. Reasonably, in these reaction conditions, the corresponding O-TMS derivative 8.22, deriving from an anti attack of Me3SiO- constitutes the primary reaction product, only subsequently hydrolyzed to trans diol 8.23 in the aqueous workup. Trans diol 8.23 was treated with TBSCl to give the mono allyl C(3)-O-TBS derivative 8.24 which was mesylated (MsCl/Py) at C(4) to the all-protected glycal derivative 8.25 and, finally, deprotected by TBAF/THF protocol to give trans hydroxy mesylate 8.16α (Scheme 8.17).
! ! "#$! !"#$! ! !# "#$! %&! !"#%! !'()!* +#, +-! !./%0, "#$! #! !"&& ()1-2%0 ,./!' ! !# "#$! #! !"&' ! !"(. "#$! #! !"&( "(.+3 45/678930 :%; <=+ ! !"(. "#$! %&! !"&) %&+3 $> <=+ ! ! "#$! !"#$" !'()!* +#, +-#?! ! !# "#$! %&! !"#%" "(@; "#;
Scheme 8.17. Synthesis of diastereoisomeric vinyl epoxide 8.19α.
The vinyl epoxide 8.19α, having the hydroxy functionality at C(6) protected as -OTHP ether, was, as usual, prepared in situ by cyclization with t-BuOK in CH3CN of trans hydroxy mesylate 8.16α and then treated with glycosyl acceptor NHI 8.2 (Scheme 8.18). Also in this case, as expected, vinyl epoxide 8.19α turned out to be, once again, an excellent glycosyl donor, able to give, in the presence of the glycosyl acceptor NHI 8.2 and after only 30 minutes at room temperature, the corresponding α-glycoconjugate 8.26 with a complete 1,4-regio- and syn-stereoselectivity and with a good yield after purification (58%).
! ! "#$! ! "#$! %&! !# !'()!* +#, +--#. "#$! ! !"#$ #! ! -+/, $0 %1!!+ 234 !"%$! !"%&!
Scheme 8.18. Synthesis of NHI β-O-glycoside 8.26.
Once obtained, the unsaturated α-glycoconjugate 8.26 was dihydroxylated by OsO4/NMO protocol. Also in this case, the syn-stereoselective attack of the electrophile is influenced by the allyl
substituents at C(4) and C(1), now located on the α-face, and therefore it takes place selectively on the opposite β-face to give the corresponding, fully hydroxylated derivative 8.27 (65% yield). The deprotection of the -OTHP moiety present at C(6) of 8.27, carried out in absolute EtOH for 20 h at 40°C in the presence of PPTS, provided the NHI α-O-D-mannopyranoside (+)-8.4 with 80% yield after recrystallization (Scheme 8.19).
! "#$! !"#$ #! ! % &'( $) *+!!& ! "#$! !"#% #! ! % &'( $) *+!!& !,!-.%*! /0& 123 !# !# ! #! 4567!"& #! ! % &'( $) *+!!& $$"8.9:!# -/0& !# !# ;/3
Scheme 8.19. Synthesis of NHI α-O-D-mannopyranoside (+)-8.4.
In this way, and with our complete sactisfaction, we have succeded in the synthesis of the two glycoconjugates (+)-8.4 and (+)-8.5 which were prepared through in a completely stereoselective fashion and with satisfactory yields. Glycoconjugates (+)-8.4 and (+)-8.5 are currently subjected to biological assays in the laboratory of Prof. Adriano Martinelli of our Department and in the laboratory of Prof. Paul Hergenrother at University of Illinois-Urbana (United States).
8.12. Synthesis of carba 1,2-epoxides
The conjugation of the derivative NHI 8.2 with carbohydrate molecules, was designed to amplify the “Warburg effect”. The glycoconjugates would have to be picked up avidly by tumor cells characterized by a high glycolytic activity.
However, the exact mechanism of action of these glycoconjugates has not yet been clarified and, it is not yet clear if these compounds act as prodrugs, after cleavage of the glycosidic bond, or they are active by themselves.
This structural change converts the glycosidic bond (acetal) in ether which is a functionality much more stable and, therefore, more resistant to the hydrolytic action of endogenous enzymes. For the synthesis of pseudoglycosides 8.28 and 8.29, one possible retrosynthetic approach have indicated as plausible precursors, respectively, diastereoisomeric 1,2-epoxides carbapyranose 4.2α and 4.2β, or 4.14α and 4.14β, 6-deoxy-1,2-anhydrous analogues of cyclophellitol, reasonably obtained from olefin 4.1 and 1.19, respectively (Scheme 8.20, see also Chapter 4).
!" !" !"#! !"#$ " !" !" " # $%& '( )*""$ # $%& '( )*""$ "! "! "! "! "+ +" +" " "+ +" +" " "+ +" +" "+ +" +" "! !" !" "! " !"#$%&'($$)*%$ %"#!&'()'*+ %",%!&'()'-. %",&'()'*+ ,",$&'()'-. %"#"&'()'*+ %",%"&'()'-. %",&'()'*+ ,",$&'()'-.
Scheme 8.20. Retrosynthetic approach to carbapyranosides 8.28 and 8.29.
Therfore, the first goal was to synthesize the vinyl carbaepoxides 4.2α and 4.2β21 and among the various approaches available in the literature for their construction, we chose the one that envisages the transformation of the glycal system present in primary alcohol 3.4 into the carbaglycal system present in the corresponding primary alcohol 3.5, by means of the thermal Claisen rearrangement,22 as previously described in Chapter 3 (Scheme 8.21). Then, the carbacycle 3.5 would have been transformed into vinyl carba epoxides 4.2α and 4.2β, by common procedures.
! !"# !"# $! $! "#! "#! !"# !"$
Scheme 8.21. Thermal Claisen rearrangement.
(+)-3.6, following a variant of the synthetic pathway described in Schemes 3.3 and 3.4 of Chapter 3, consisting in the use of O-benzyl ether (-OBn), instead of the previously used O-p-methoxybenzyl ether (-OPMB) functionality in order to protect the hydroxy groups.
Saponification of the tri-O-acetyl-D-glucal (+)-3.6 with MeONa/MeOH gave D-glucal 8.30, which was then protected at the primary hydroxy functionality with the sterically hindered TBSCl, in a 1:12 mixture of DMF/THF to give the -OTBS derivative 8.31.22,23 The –OBn protection of the remaining secondary hydroxy groups was obtained by reaction of trans diol 8.31 with NaH/BnBr/ tetrabutylammonium iodide (TBAI) protocol in THF at 0°C with the formation of the all-protected glycal 8.32, without observing any migration of the silyl group. Deprotection (TBAF/THF) of the primary -OTBS led to primary alchool 3.4 which was oxidized to aldehyde 8.33 by freshly prepared 2-iodoxy benzoic acid (IBX),24 in anhydrous ethyl acetate at 80°C for 3 h. Aldehyde 8.33 is not stable and should be used immediately or stored at low temperature (-78 °C) before subsequent treatment. Aldehyde 8.33 was transformed into allyl vinyl ether 8.34 by means of Wittig reaction, using the phosphorus ylide obtained in situ by reaction between methyl-triphenyl phosphonium iodide (MeI-Ph3P+) and potassium hexamethyldisilazide (KHMDS) in THF (Scheme 8.22). ! ! !"# !$ $! "#! $! "#! !"#$ ! ! !$ !%& '%(! '%(! %&! $! !"#% !"#& ! ! !%& !%& $! ! %&! %&! !"## #"' ! !%& !%& ! %&! %&! !"#' !"#( !%& $! %&! #"( )*!+, )*!$ -../ '%(01 2)34'$3 -../ +,$4%&%5 '%"6 '$3 '%"3 '$3 78/ 6%9 "#!:; -../ -../ <<=>0$>6 ?$)2( '$3 @A/ 20% ABCD0 +,%$B :;!$4'$3 E./ FGHI#")
primary alcohol 3.5.
Diastereoisomeric epoxides 4.2α and 4.2β21c were synthesized starting from a common precursor, the endocyclic olefin 4.1, obtained by simple -O-benzylation (NaH/BnBr in DMF) of the primary alcohol 3.5. The treatment of olefin 4.1 with MCPBA in CH2Cl2 afforded an 80:20 mixture of the diastereoisomeric epoxides 4.2α and 4.2β (1H NMR) which were separated by flash chromatography (Scheme 8.23).21c !"# "#! "#! !"# "#! "#! ! !"# "#! "#! ! $ %&'"( &)*&+* !"# !"$! !"$" !"# )! "#! %"& ,-) "#". /%0
Scheme 8.23. Synthesis of diastereisomeric epoxides 4.2α and 4.2β.
Carbapyranose 1,2-epoxides 4.2α and 4.2β having the gluco- and manno-type configuration, respectively (named also α-gluco 4.2α and β-manno 4.2β), were taken into consideration as useful synthetic tools. Actually, epoxides 4.2α and 4.2β had stimulated our attention as useful “carbaglycosyl donor” for the construction of carba O-glycosyl derivatives of N-hydroxy indole 8.2, the glycosyl acceptor, of our interest. This favorable possibility was based on the assumption that these two epoxides could give addition products deriving from nucleophilic attack at C(1), actually, the carba anomeric center, affording corresponding β-carbaglucopyranosides and α−carbamannopyranosides from 4.2α and 4.2β, respectively (Scheme 8.24, where ROH is a generic O-nucleophile). !"# "#! "#! !"# "#! "#! !"# "#! "#! ! ! $ !"# !"$! !"$" !"# "#! "#! !"# "#! "#! $ "%&'()'*+,&-./('#-0123 !%&'()'4'##-./('#-0123 !5 !6 !5 !6 6!5 6!5
Thus, nucleophilic attack of NHI 8.2 to 1,2-epoxides 4.2α and 4.2β could lead, under appropiate reaction conditions, to the desired carba-O-glycosides 8.36 and 8.37, having a β-gluco and α-manno configuration, respectively (Scheme 8.25).
!"# "#! "#! ! !"# "#! "#! ! !"#! !"#" "#! "#! $"%& $"%' ! "#! "#! ! $ %&' () *+!!% $ %&' () *+!!% !"# !, !"# !, $,-$"# $"#
Scheme 8.25. Hypothesized nucleophilic attack of NHI 8.2 to diastereisomeric epoxides 4.2α and 4.2β. The reactivity of these 1,2-epoxides 4.2α and 4.2β in nucleophilic addition reactions had been previously examined, in a comprehensive manner, by Ogawa,21a,b,25 who had used these systems as carbaglycosyl donors for the construction of N- and O-pseudodisaccharides. Commonly, the epoxides were opened by amines and azide ion in uncatalyzed reactions, or by alcohols under Lewis acid catalysis, but more often under basic conditions.
As more accurately discussed in the following Section, the results obtained had indicated that epoxide 4.2β, with β-manno configuration, effectively reacts with O- and N-nucleophiles by exclusive nucleophilic attack at the sterically and electronically favored C(1) oxirane carbon. Corresponding α-O- and α-N-carbamannopyranosides are in this way obtained through a trans diaxial ring opening process (Scheme 8.26, See also Chapter 1).
!"# !"# !"# # $% & ' !"#! !"# !"# !"# & ' #( $% "!"#$%&'()**'+,-./'#0&$
Scheme 8.26. Sterically and electronically favored nucleophilic attack to epoxide 4.2β.
C(1) oxirane carbon, that would be sterically and electronically favored, leads to an unfavorable
trans-diequatorial ring opening (route a), whereas the attack at C(2) oxirane carbon, that would
lead to a trans-diaxial opening (route b) is sterically hindered and electronically unfavorable (Scheme 8.27). !"# !"# !"# # $#% ! " # #!" !"# & & ' ' !"#!$ !"#!$$ #!" !"# !"# & ' !"#$%&! !"#$%#" #% $# !"# !"# !"# #$ #%' & ()&*+,--./0 ()'*+,--./0 !"#
Scheme 8.27. The two regioisomeric routes for nucleophilic attack to epoxide 4.2α.
As a result, epoxide 4.2α, which could reasonably be an effective β-carbaglucosyl donor for the synthesis of glycoconjugates bearing a β-carbaglucosidic bond, is practically and unfortunately (it is often the main product by oxidation of the corresponding olefin) not useful for this purpose. However, as strongly interested in the reaction our O-nucleophile (NIH 8.2) with epoxide 4.2α, we wanted to verify, even if extensively examined by other authors, the regiochemical behaviour of epoxide 4.2α in nucleophilic addition reactions, in the residual hope that the use of NIH 8.2 and/or the contemporary application of particular reaction conditions could make also this oxirane system useful to our synthetic strategy. In particular, we hoped that the use of strongly coordinating reaction conditions, by the presence of an ion such as Li+ (from LiClO4), could modify, at least partially, the conformers population toward the triaxial conformer 4.2α’’, through bidentate chelation by the metal cation (4.2α’’-Li). Subsequent nucleophilic attack on 4.2α’’-Li would necessarily occur at C(1), following a trans diaxial opening process, with the formation of the desired β-O-glycoconjugate 8.36 (Scheme 8.28).
!"# !"# !"# # # #!" !"# #!" $%& '() !"# !"# !"# #) '( #!" !"# #!" #) '( !"#!$ %"&' !"#!$$()* '()*+*'),*%"# -. -. -. -. '() %&#/01
Scheme 8.28. Possible ring opening at C(1) in nucleophilic addition by NHI 8.2 to epoxide 4.2α in the
presence of LiClO4.
However, also under these modified reaction conditions, the desired product was not found, in any case, in the crude reaction mixtures (1H NMR). In many cases, we recovered unreacted both epoxide 4.2α and nucleophile 8.2 (entries 1, 2, 3, 6 and 9, Table 8.1). In some cases, the hydrolysis of the methyl ester group of NHI 8.2 was also observed (entries 4 and 5, Table 8.1). In the presence of Sc(OTf)3 and Cu(OTf)2 (entries 7 and 8, Table 8.1), the formation of the bicyclic compound 8.38, was obtained (vide infra), accompanied by decomposition products of NHI 8.2. When the addition reaction was carried out in the presence of BF3.Et2O (entry 10, Table 8.1) at 0°C, NHI 8.2 and epoxide 4.2α were recovered unreacted and by warming up to room temperature, 1H NMR analysis of the crude reaction product showed the presence of a complex reaction mixture. Often, these reactions have been repeated by changing the ratio between the reactive species: similar, unsatisfacory results were obtained.
Table 8.1. Several attempts to pseudoglycosylation reaction of NHI 8.2 by epoxide 4.2α.
Entry Substrate Nucleophile Reaction conditions
1 4.2α 8.2 K2CO3, acetone, r.t, 5 days
2 4.2α 8.2 K2CO3, CH3CN, 60°C, 5 days
3 4.2α 8.2 t-BuOLi, acetone, r.t., 1 week
4 4.2α 8.2 t-BuOLi, toluene, 80°C, 5 days
5 4.2α 8.2 t-BuOLi, CH3CN, 70°C, 10 days
9 4.2α 8.2 LiClO4, CH3CN, 70°C, 14 days
10 4.2α 8.2 BF3. Et2O, Et2O,0°C→ r.t., 8 h
In view of the discouraging results obtained with epoxide 4.2α, and on the basis of the studies present in the literature, much more promising was the use of epoxide 4.2β for obtaining the α- carbamanno derivative (-)-8.37 (Scheme 8.29). Also in this case, several reaction conditions have been tried, but in Scheme 8.29 we have reported only the positive result.
!"# "#! "#! ! !"#! "#! $%&%$"%& ! "#! ' ()* +, -.!!( !"# !/ '/01$"#23/-45 67%(89:#%;1</) =>?(1 @1ABCD ;@E
Scheme 8.29. Successful protocol for the synthesis -OBn protected NHI carba-α-O-D-mannopyranoside (-)-8.37.
The nucleophile NHI 8.2 (1.0 equiv) was deprotonated by treatment with 1.0 M LHMDS in THF (0.95 equiv), in order to obtain a more nucleophilic species. 12-Crown-4 was added to the solution (4 equiv) to enhance the nucleophilic properties of 8.2 and the solution was maintained at room temperature for 3 h. Subsequently, epoxide 4.2β (2 equiv) was added and the reaction mixture was warmed up to 70°C for 6 days. The purification on preparative TLC (8:2 hexane/Et2O, 2 runs) gave the desired -OBn protected NHI carba-α-O-D-mannopyranoside 8.37 (45% yield), which had to be deprotected (O-debenzylated) in order to have the final all-deprotected carba analogue 8.29, necessary for testing.
In the first moment, in view of the found incompatibility of the hydrogenolysis reaction with glycoconjugates (+)-8.3 and (+)-8.6, we evaluated several different deprotection protocols, considering the instability of the N-O bond present in (-)-8.37, under reducing conditions (H2/Pd-C). However no satisfactory results were obtained.
The unsuccessful deprotection by BCl3 protocol,20 was particularly significant and prompted us to change, once again, the starting epoxide and the synthetic strategy.
On the basis of the results previous described in Chapter 5, we thought that epoxide tri-O-acetyl derived (+)-4.14β, synthesized starting from tri-O-acetyl carba-D-glucal (-)-1.19 could be the carbaglycosyl donor more appropriate to our synthetic purpose.
At the first moment, we decided to use the racemic (±)-4.14β, because immediately available in a sufficient amount and less expensive to prepare.
!"# "#! "#! ! $±%&!"#!! "#! $±%&$"!% ! "#! ' ()* +, -.!!( !"# !/ '/01$"%23/-45 67&(89:;&<1=/) >?@(1 A1BCDE F7G "#! $±%&$"!& ! "#! ' ()* +, -.!!( !"# !"# /! $±%&$"%' ! /! ' ()* +, -.!!( !/ !/ "#7!2+D ?@( HIG -.!'C -.!/ JIIG
Scheme 8.30. Synthesis of NHI α-O-D-mannopyranoside (±)-8.29.
The reaction of epoxide (±)-4.14β with NHI/LHMDS/12-Crown-4 protocol in THF afforded after 6 days at 70°C a complex reaction mixture, constituted by carba-O-glycoside (±)-8.42 accompanied by partially hydrolized compounds (1H NMR), which was acetylated (Ac2O/Py) to give, as the only reaction product, the desired all-acetyl protected carbaglycoside (±)-8.43. Tetra-O-acetyl-pseudomannoglycoside (±)-8.43 was purified by flash chromatography and recovered in good yield (89%). Saponification under alkaline conditions (MeONa/MeOH) afforded the NHI
carba-α-O-D,L-mannopyrsanoside (±)-8.29, ready to be sent for biologycal essays in the laboratory of Prof. Paul Hergenrother at University of Illinois-Urbana in the United States (Scheme 8.30).
In order to synthesize NHI carba-α-O-D-mannopyrsanoside 8.29, as pure enantiomer, we developed the synthesis of the necessary chiral tri-O-acetyl carba-D-glucal (-)-1.19 (Scheme 8.31), starting from primary alcohol (-)-3.3, on its own was prepared as previously described in this thesis (Scheme 3.3, Chapter 3).
!"#$ "#$! %! !"#$ "#$! &'! !% %! &'! !&' &'! &'! !&' &'! &'! ! !&' &'! &'! ! ( )*+*!"! )(+*#"$% )(+*#"$& )*+*&"&' )(+*$"&$! )(+*$"&$" &',!-". /01 2334 556 1%,17,-%,!8 ,/9: ;<4 &',!-". /01 2334 :+8=$>-?%@*%,! ,+8!*$A!B8$CDECDC
Scheme 8.31. Enantiomeric synthesis of enantiomerically pure epoxide (+)-4.14β.
Primary alcohol (-)-3.3 was acetylated (Ac2O/Py) to all-protected derivative (+)-8.40, then deprotected to trans diol (+)-8.41 by means of DDQ procedure.26 Subsequent peracetylation of
trans diol (+)-8.41 afforded tri-O-acetyl carba-D-glucal (-)-1.19, as a pure enantiomer. At this point, application of NBS-THF/H2O/base protocol, as previously described in Chapter 4, gives a 70:30 mixture of the diastereoisomeric epoxides (+)-4.14β and (+)-4.14α, that are the tri-O-acetyl-substituted epoxides corresponding to the tri-O-benzyl-tri-O-acetyl-substituted epoxides 4.2β and 4.2α, respectively (Scheme 8.31). !±"#!"#$ $%& %&$ %&$ $ !'"#%"%#! %&$ !"#& $ %&$ ( )*+ ,-./$$) $%& $0 (012!"&340.56 78#)9:;<#=2>0* ?@A)2 B2CDEF G8H %&$ !"#$ $ %&$ ( )*+ ,-./$$) $%& $%& 0$ !"&' $ 0$ ( )*+ ,-./$$) $0 $0 %&8$3,E @A) ./$(D ./$0
Scheme 8.32. Enantioselective synthesis of pseudo-α-mannoglycoside 8.29.
Few steps remained for the synthesis of NHI carba-α-O-D-mannopyranoside 8.29: the separationof the necessary epoxide (+)-4.14β from the mixture with the diastereoisomeric epoxide (+)-4.14α
and the subsequent opening reaction with nucleophile NHI 8.2. Unfortunately, due of the prolunging of the previous steps, I was not able to bring the synthesis of carbaglycoside 8.29 to an end. However, the conditions for a positive final result have been arranged.
8.13. Enzymatic and cellular assays of glycoconjugates and carbaglycoconjugate 8.13.1. Enzyme inhibition Table 8.2. Entry Compound hLDH5 NADH Ki (µM) hLDH1 NADH Ki (µM) 1 8.2 5.1 ± 1.1 - 2 (+)-8.3 37.8 ± 0.9 26.9 ± 3.2 3 (+)-8.4 40.2 ± 0.5 34.7 ± 2.3 4 (+)-8.5 34.7 ± 6.8 29.6 ± 2.6 6 (±)-8.29 72% @ 500
The LDH inhibitory activities of β-glucopyranoside (+)-8.3, β-gulopyranoside (+)-8.4, α-mannopyranoside (+)-8.5 and the racemic carba-O-glycoside (±)-8.29, were reported in the following Table 8.2, with respect to the NHI 8.2 aglycone. Enzyme inhibitory activity was determined by the measurement of competition with cofactor NADH for two isoforms hLDH1 and
hLDH5.
Glycoconjugates (+)-8.3, (+)-8.4 and (+)-8.5 showed a modest non-selective inhibition of both isoforms, with Ki values reaching the low micromolar range (Ki 27-40 µM vs NADH entries 2, 3 and 4, Table 8.2), in the conversion of pyruvate to lactate by this enzyme where NADH is converted to NAD+. Moreover inhibition by glycoadducts was weaker that of NHI 8.2, which was found to be a very potent inhibitor (entry 1, Ki = 5.1 µM). Different is the case of the carba-O-glycoside (±)-8.29 that showed an even lower inhibition activity (entry 5, 72% Ki @ 500 µM vs NADH). In any case, only this result is not sufficient to delineate an inhibitory profile of this compound whereby we should repeat in vitro and in vivo cellular assays with the enantiomerically pure α-carbamannopyranoside 8.29.
