NO311004B1 - Use of benzaldehyde derivatives for the preparation of a therapeutic agent for the treatment of microbial infections - Google Patents
Use of benzaldehyde derivatives for the preparation of a therapeutic agent for the treatment of microbial infections Download PDFInfo
- Publication number
- NO311004B1 NO311004B1 NO20001449A NO20001449A NO311004B1 NO 311004 B1 NO311004 B1 NO 311004B1 NO 20001449 A NO20001449 A NO 20001449A NO 20001449 A NO20001449 A NO 20001449A NO 311004 B1 NO311004 B1 NO 311004B1
- Authority
- NO
- Norway
- Prior art keywords
- glucopyranose
- methanol
- compound
- deoxy
- benzylidene
- Prior art date
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- 238000011282 treatment Methods 0.000 title claims description 15
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- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
- WFHFGDIKIXDWNX-ZQOBQRRWSA-N β-cyclodextrin-benzaldehyde Chemical compound O=CC1=CC=CC=C1.OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WFHFGDIKIXDWNX-ZQOBQRRWSA-N 0.000 description 1
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Description
Denne oppfinnelsen vedrører anvendelse av benzaldehydderivater for fremstilling av terapeutiske midler til forebygging og/eller behandling av infeksjoner av virus, protozoer, sopp og andre mikroorganismer ved hjelp av modulering av immunsystemet. This invention relates to the use of benzaldehyde derivatives for the production of therapeutic agents for the prevention and/or treatment of infections by viruses, protozoa, fungi and other microorganisms by means of modulation of the immune system.
Aldehyder reagerer med en rekke O-, S- eller N-holdige nukleofile grupper som hydroksylgrupper, sulfhydrylgrupper og aminogrupper under dannelse av karbonylholdige kondensasjonsprodukter som acetaler, merkaptaler, aminaler etc. Med primære aminer vil reaksjonen imidliertid normalt føre til en addisjonsforbindelse i form av en Schiff-base (et imin). Det er velkjent at in vivo Schiff-basedannelse er involvert I biokjemiske nøkkelprosesser som transaminering, dekarbbksylering og andre aminosyremodifiserende reaksjoner som katalyseres ved hjelp av pyridoksalfosfat, virkningen av aldolase på fruktosedifosfat i glykolysen og kondensasjonen av retinal med rodopsin i synsprosessen. Det er også kjent at signalisering gjennom membraner innebærer karbonylkondensasjonsreaksjoner, f.eks. når det skal settes i gang en respons fra immunsystemet. Aldehydes react with a number of O-, S- or N-containing nucleophilic groups such as hydroxyl groups, sulfhydryl groups and amino groups to form carbonyl-containing condensation products such as acetals, mercaptals, aminals etc. With primary amines, the reaction will normally lead to an addition compound in the form of a Schiff base (an imine). It is well known that in vivo Schiff base formation is involved in key biochemical processes such as transamination, decarboxylation and other amino acid modifying reactions catalyzed by pyridoxal phosphate, the action of aldolase on fructose diphosphate in glycolysis and the condensation of retinal with rhodopsin in the vision process. It is also known that signaling through membranes involves carbonyl condensation reactions, e.g. when a response from the immune system is to be initiated.
Dannelsen av iminer skjer ved en totrinnsmekanisme. Ved å tilsette en amino-nukleofil til karbonylgruppen dannes mellomproduktet karbinolamin (aminohydrin), deretter følger et dehydreringstrinn ved dannelsen av dobbeltbindingen C=N. Begge trinnene er reversible, men begunstiges ved forskjellige pH-verdier. Derfor går reaksjonen etter en karakteristisk klokkeformet pH-/hastighetsprofil hvor den høyeste totale reaksjonshastigheten opptrer ved moderat pH. The formation of imines occurs by a two-step mechanism. By adding an amino nucleophile to the carbonyl group, the intermediate carbinolamine (aminohydrin) is formed, then a dehydration step follows in the formation of the double bond C=N. Both steps are reversible, but are favored at different pH values. Therefore, the reaction follows a characteristic bell-shaped pH/rate profile where the highest overall reaction rate occurs at moderate pH.
Imidlertid vet man at Schiff-baser lett dannes også ved fysiologiske betingelser, og mange karbonylkondensasjonsreaksjoner er vel kjent in vivo (E.Schauenstein et. al., Aldehydes in Biological Systems, London, Pion Ltd., 1977). However, it is known that Schiff bases are easily formed also under physiological conditions, and many carbonyl condensation reactions are well known in vivo (E.Schauenstein et. al., Aldehydes in Biological Systems, London, Pion Ltd., 1977).
Schiff-basen er gjerne selv et reaktivt molekyl, og deltar ofte i ytterligere reaksjoner som fører til addisjon av nukleofile enheter til dobbeltbindingen. For visse svovelholdige aminer, spesielt aminosyrene cystein og metionin og for glutation, vil Schiff-basen som dannes først kunne gjennomgå reversibel intern ringdannelse hvor sulfhydrylgruppen adderes til iminet under dannelse av tiazolidinkarboksylat (M.Friedman, The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins, Oxford, Pergamon Press, 1973). The Schiff base is often itself a reactive molecule, and often participates in further reactions that lead to the addition of nucleophilic units to the double bond. For certain sulphur-containing amines, especially the amino acids cysteine and methionine and for glutathione, the Schiff base that is first formed will be able to undergo reversible internal ring formation where the sulfhydryl group is added to the imine to form thiazolidine carboxylate (M.Friedman, The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins, Oxford, Pergamon Press, 1973).
Indikasjoner på reaksjoner mellom karbonylforbindelser og frie aminogrupper i proteiner under dannelse av reversible Schiff-baser ble rapportert av G.E.Means og R.E.Feeney (Chemical Modification of Proteins, s. 125 -138, San Francisco, Holden-Day, 1971). Aromatiske aldehyder er generelt mer reaktive enn mettede alifatiske aldehyder, og kan danne Schiff-baser selv uten at man fjerner vann under reaksjonen. (R.W.Layer, Chem. Rev. 63(1963), 489-510). Dette er viktig når man tar i betraktning dannelse av Schiff-baser under fysiolpgiske betingelser. Med hemoglobin som kilde for aminogrupper har Zaugg et. al. (J. Biol. Chem. 252 Indications of reactions between carbonyl compounds and free amino groups in proteins to form reversible Schiff bases were reported by G.E.Means and R.E.Feeney (Chemical Modification of Proteins, pp. 125-138, San Francisco, Holden-Day, 1971). Aromatic aldehydes are generally more reactive than saturated aliphatic aldehydes, and can form Schiff bases even without removing water during the reaction. (R.W.Layer, Chem. Rev. 63(1963), 489-510). This is important when considering the formation of Schiff bases under physiological conditions. With hemoglobin as a source of amino groups, Zaugg et. eel. (J. Biol. Chem. 252
(1977), 8542 - 8548) vist at aromatiske aldehyder er to til tre ganger så reaktive som alifatiske aldehyder for dannelse av Schiff-baser. En forklaring på den begrensede reaktiviteten til alkanalene kunne være at det i vannløsning ved nøytral pH kreves et meget stort overskudd av fritt aldehyd for å forskyve likevekten i retning av Schiff-basedannelse (E.Schauenstein et. al., Aldehydes in Biological Systems, London, Pion Ltd., 1977). Benzaldehyd og salicylaldehyd danner lett Schiff-baseiminer med membran-aminogrupper, og det er målt høye likevektskonstanter for reaksjonen av benzaldehyd med aminer (J.J. Pesek og J.H.Frost, Org. Magnet. Res. 8 (1976), 173 -176, J.N.Williams Jr. og R.M.Jacobs, Biochim. Biophys. Acta., 754 (1968), 323 - 331). Med salicylaldehyd kan iminet stabiliseres ytterligere ved hydrogenbinding mellom det enslige elektronparet i iminnitrogenet og orto-hydroksylgruppen (G.E.Means og R.E.Feeney, Chemical Modification of Proteins, s. 125 -138, San Francisco, Holden-Day, 1971, J.M.Dornish og E.O.Pettersen, Biochem. Pharmac. 33(1990), 309-318). (1977), 8542 - 8548) showed that aromatic aldehydes are two to three times as reactive as aliphatic aldehydes for the formation of Schiff bases. An explanation for the limited reactivity of the alkanal channels could be that in water solution at neutral pH a very large excess of free aldehyde is required to shift the equilibrium in the direction of Schiff base formation (E.Schauenstein et. al., Aldehydes in Biological Systems, London , Pion Ltd., 1977). Benzaldehyde and salicylaldehyde readily form Schiff base imines with membrane amino groups, and high equilibrium constants have been measured for the reaction of benzaldehyde with amines (J.J. Pesek and J.H.Frost, Org. Magnet. Res. 8 (1976), 173 -176, J.N.Williams Jr . and R. M. Jacobs, Biochim. Biophys. Acta., 754 (1968), 323-331). With salicylaldehyde, the imine can be further stabilized by hydrogen bonding between the lone pair of electrons in the imine nitrogen and the ortho-hydroxyl group (G.E.Means and R.E.Feeney, Chemical Modification of Proteins, pp. 125 -138, San Francisco, Holden-Day, 1971, J.M. Dornish and E.O.Pettersen , Biochem. Pharmac. 33(1990), 309-318).
Vi har tidligere vist ved å fotografere reaksjoner mellom radioaktivt merkede reagenser at benzaldehydet ikke går inn i cellene, men bindes til cellemembranen (Dornish, J.M. og Pettersen, E.O., Cancer Letters 29(1985), 235-243). Dette stemmer overens med en tidligere undersøkelse som viser at benzaldehydet reagerte med membranproteinene til E. coli (K.Sakaguchi et. al., Agric. Biol. Chem. 43 (1979), 1775-1777). Det ble også funnet at både pyridoksal og pyridoksal-5-fosfat beskytter cellene mot det cytotoksiske anticancer-medikamentet cis-DDP. Cis-DDP virker på kjernen inne i cellen. Selv om pyridoksalet i prinsippet kunne trenge gjennom den lipofile cellemembranen er dette umulig for pyridoksal-5-fosfat på grunn av den ioniske fosfatgruppen. Pyridoksal-5-fosfatet må derfor utøve beskyttelsesvirkningen utenfor cellemembranen. En samtidig observasjon av en forskyvning i spektrografisk absorbans for pyridoksal-5-fosfat til kortere bølgelengder samsvarer med dannelse av Schiff-base-addisjonsforbindelser mellom aldehydet og aminogrupper i cellemembranene (J.M.Dornish og E.O.Pettersen, Cancer Lett. 29, (1985), 235 - 243). We have previously shown by photographing reactions between radioactively labeled reagents that the benzaldehyde does not enter the cells, but binds to the cell membrane (Dornish, J.M. and Pettersen, E.O., Cancer Letters 29(1985), 235-243). This agrees with a previous study showing that the benzaldehyde reacted with the membrane proteins of E. coli (K.Sakaguchi et. al., Agric. Biol. Chem. 43 (1979), 1775-1777). It was also found that both pyridoxal and pyridoxal-5-phosphate protect the cells against the cytotoxic anticancer drug cis-DDP. Cis-DDP acts on the nucleus inside the cell. Although pyridoxal could in principle penetrate the lipophilic cell membrane, this is impossible for pyridoxal-5-phosphate due to the ionic phosphate group. The pyridoxal-5-phosphate must therefore exert its protective effect outside the cell membrane. A simultaneous observation of a shift in spectrographic absorbance for pyridoxal-5-phosphate to shorter wavelengths is consistent with the formation of Schiff base addition compounds between the aldehyde and amino groups in the cell membranes (J.M. Dornish and E.O. Pettersen, Cancer Lett. 29, (1985), 235 - 243).
Disse funnene tyder på at aldehyder bindes til aminer og andre nukleofile enheter på cellemembranen ved å danne Schiff-baser og andre kondensasjonsprodukter. Det er kjent at celler stimuleres til vekst av en kaskade av hendelser som virker fra utsiden av cellemembranen. På samme måte kan derivatene i den foreliggende patentsøknaden virke ved å danne addisjonsforbindelser med ligander på cellemembranen og gi opphav til impulser inne i cellen som har betydning for cellevekstparametre som proteinsyntese og mitose, og på uttrykking av immunrespons og tumorundertrykkende gener. Siden kondensasjonsreaksjonene er reversible kan celleeffektene moduleres ved en likevektforskyvning som involverer de molekylene som bindes sammen. At det finnes dynamiske likevekter på kjemisk nivå stemmer overens med den reversible og ikke-giftige virkningsmåten til benzaldehydderivatene. These findings suggest that aldehydes bind to amines and other nucleophilic units on the cell membrane by forming Schiff bases and other condensation products. It is known that cells are stimulated to grow by a cascade of events that act from outside the cell membrane. In the same way, the derivatives in the present patent application can act by forming addition compounds with ligands on the cell membrane and giving rise to impulses inside the cell that have an impact on cell growth parameters such as protein synthesis and mitosis, and on the expression of immune response and tumor suppressor genes. Since the condensation reactions are reversible, the cell effects can be modulated by an equilibrium shift involving the molecules that are bound together. That there are dynamic equilibria at the chemical level is consistent with the reversible and non-toxic mode of action of the benzaldehyde derivatives.
Immunsystemet er nøye konstruert for å identifisere og eliminere alle substanser som kjennes som fremmede, enten det stammer fra en bakterie-, virus- eller protozoinfeksjon eller fra abnorme celler som kreftceller. For å kunne gi en spesifikk respons overfor det store utvalget av biologisk variasjon som de invaderende substansene representerer, må immunsystemet være svært allsidig. Imidlertid kan overstimulering av dette finstemte systemet føre til forskjellige allergi- og betennelsesreaksjoner og til autoimmunsykdommer. Å modulere immunsystemet, enten ved å opp- eller nedregulere en spesifikk respons, er derfor en stor terapeutisk utfordring. The immune system is carefully designed to identify and eliminate all substances that are perceived as foreign, whether they originate from a bacterial, viral or protozoan infection or from abnormal cells such as cancer cells. In order to be able to provide a specific response to the large range of biological variation that the invading substances represent, the immune system must be very versatile. However, overstimulation of this fine-tuned system can lead to various allergic and inflammatory reactions and to autoimmune diseases. Modulating the immune system, either by up- or down-regulating a specific response, is therefore a major therapeutic challenge.
Ved en immunologisk gjenkjennelsesprosess sperres et fragment av et fremmed protein inne i kløften i et MHC-klasse ll-protein på overflaten av en antigenpresenterende celle (APC). Til dette MHC-antistoffkomplekset bindes også reseptoren for en T-hjelpercelle. For å aktivere en T-hjelpercelle trengs minst to signaler. Det primære signalet gis av antigenet selv, via MHC-klasse ll-komplekset og forsterkes av CD4-koreseptorene. Det andre signalet kan fås fra et spesifikt signaliseringsmolekyl som er bundet i plasmamembranen på overflaten av APC-cellen. Et tilsvarende koreseptorprotein befinner seg på overflaten av T-hjelpercellen. Begge signalene er nødvendige for å aktivisere T-cellene. Når de aktiviseres vil de stimulere sin egen formering ved å skille ut interleukinbaserte vekstfaktorer og syntetisere tilsvarende reseptorer på overflaten. Bindingen av interleukinet til disse reseptorene stimulerer så T-cellene direkte til å formere seg. In an immunological recognition process, a fragment of a foreign protein is trapped within the cleft of an MHC class II protein on the surface of an antigen-presenting cell (APC). The receptor for a T-helper cell is also bound to this MHC-antibody complex. To activate a T-helper cell, at least two signals are needed. The primary signal is given by the antigen itself, via the MHC class II complex and is amplified by the CD4 co-receptors. The second signal can be obtained from a specific signaling molecule that is bound in the plasma membrane on the surface of the APC cell. A corresponding coreceptor protein is located on the surface of the T helper cell. Both signals are necessary to activate the T cells. When they are activated, they will stimulate their own reproduction by secreting interleukin-based growth factors and synthesizing corresponding receptors on the surface. The binding of the interleukin to these receptors then directly stimulates the T cells to multiply.
På 1980-tallet ble det kjent at et syklodekstrin-benzaldehydbasert inklusjonskompleks kunne stimulere immunsystemet ved å styrke de lymfokinaktiverte drepercellene i en musemodell (Y. Kuroki et al., J. Cancer Res. Clin. Oncol. 117, (1991), 109-114). In wrro-studier har senere avslørt naturen til de kjemiske reaksjonene ved APC-donor-/T-cellereseptor-interaksjonsstedene som er ansvarlige for det andre stimulerende signalet, og at disse reaksjonene har form av karbonyl-aminokondensasjoner (Schiff-basedannelse). Videre kan disse interaksjonene imiteres av syntetiske kjemiske enheter. Disse oppdagelsene åpner for nye terapeutiske muligheter for kunstig modulering av immunsystemet. I WO 94/07479 kreves patent for bruk av visse aldehyder og ketoner som danner Schiff-baser og hydrazoner med aminogrupper på T-celleoverflaten. I EP 0609606 A2 er den foretrukne immunstimulerende substansen 4-(2-formyl-3-hydroksyfenoksymetyI)benzosyre (Tucaresol), en forbindelse som opprinnelig var konstruert for å kurere sigdcelleanemi. Denne substansen gis oralt og er systemisk biotilgjengelig. Potensialet til Tucaresol for å kurere et antall sykdommer som f.eks. bakterie-, virus- og protozoinfeksjoner, autoimmunsykdommer og kreft undersøkes nå (H.Chen og J.Rhodes, J. Mol. Med. (1996) 74:497-504) og kombinasjonsstrategier hvor Tucaresol gis sammen med en vaksine for å kurere kronisk hepatitt B, HIV og malignt melanom er under utvikling. In the 1980s, it was known that a cyclodextrin-benzaldehyde-based inclusion complex could stimulate the immune system by enhancing the lymphokine-activated killer cells in a mouse model (Y. Kuroki et al., J. Cancer Res. Clin. Oncol. 117, (1991), 109- 114). In wrro studies have subsequently revealed the nature of the chemical reactions at the APC donor/T cell receptor interaction sites responsible for the second stimulatory signal, and that these reactions take the form of carbonyl-amino condensations (Schiff base formation). Furthermore, these interactions can be imitated by synthetic chemical entities. These discoveries open up new therapeutic possibilities for artificial modulation of the immune system. WO 94/07479 claims a patent for the use of certain aldehydes and ketones which form Schiff bases and hydrazones with amino groups on the T-cell surface. In EP 0609606 A2, the preferred immunostimulatory substance is 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid (Tucaresol), a compound originally designed to cure sickle cell anemia. This substance is given orally and is systemically bioavailable. The potential of Tucaresol to cure a number of diseases such as bacterial, viral and protozoan infections, autoimmune diseases and cancer are now being investigated (H.Chen and J.Rhodes, J. Mol. Med. (1996) 74:497-504) and combination strategies where Tucaresol is given together with a vaccine to cure chronic hepatitis B, HIV and malignant melanoma are under development.
Måling av immunparametre in vitro og undersøkelser av virkningene in vivo resulterte i en klokkeformet dose-/responsprofil (H.Chen og J.Rhodes, J. Mol. Med. (1996) 74:497-504). Dette dose-/responsforholdet, som ellers er noe uvanlig, kan begrunnes ved å anta at en høy konsentrasjon av aldehydmedikamentet fører til at de ko-stimulerende ligandene som trengs for effektiv binding av APC til T-cellen mettes med medikamentmolekyler slik at virkningen svekkes. En dose som er nok til å danne en dynamisk likevekt som bidrar til stimulering uten å blokkere bindingen mellom cellene ser ut til å være optimal. Measurement of immune parameters in vitro and studies of effects in vivo resulted in a bell-shaped dose/response profile (H.Chen and J.Rhodes, J. Mol. Med. (1996) 74:497-504). This dose/response relationship, which is otherwise somewhat unusual, can be justified by assuming that a high concentration of the aldehyde drug leads to the co-stimulatory ligands needed for effective binding of the APC to the T cell being saturated with drug molecules so that the effect is weakened. A dose sufficient to form a dynamic equilibrium conducive to stimulation without blocking the binding between cells appears to be optimal.
Generelt er aldehyder i seg selv ustabile overfor oksidasjon. 4-(2-formyl-3-hydroksyfenoksymetyl)benzosyre (Tucaresol) som presenteres i EP-0609606 er betydelig mer aktiv in vivo enn in vitro. Årsaken til dette kan være at medikamentet oksideres i vannløsning in vitro (H.Chen og J.Rhodes, J. Mol. Med. (1996) 74:497-504). Mange aldehyder er for reaktive til å gis som aldehyder, og selv om benzaldehyd har vist seg å være et aktivt anticancer-medikament in vitro, er det svært irriterende og uegnet for direkte bruk in vivo. I et biologisk system vil karbonylgruppen i aldehydet reagere raskt med nukleofile grupper som finnes i store mengder i alle kroppsvæsker. Disse uønskede sidereaksjonene kan føre til rask metabolisering av medikamentet og vanskeligheter med å kontrollere konsentrasjonen i serum av det aktive medikamentet. Å kontrollere medikamentet på cellenivået innen et smalt konsentrasjonsvindu er avgjørende for å oppnå en effektiv immunmodulering. Tucaresol gis oralt som et ubeskyttet aldehyd, og det kan være grunn til å mistenke at medikamentet kan være utsatt for oksidasjon og at farmakokinetikken kan være vanskelig å kontrollere. In general, aldehydes themselves are unstable to oxidation. 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid (Tucaresol) presented in EP-0609606 is significantly more active in vivo than in vitro. The reason for this may be that the drug is oxidized in aqueous solution in vitro (H.Chen and J.Rhodes, J. Mol. Med. (1996) 74:497-504). Many aldehydes are too reactive to be given as aldehydes, and although benzaldehyde has been shown to be an active anticancer drug in vitro, it is highly irritating and unsuitable for direct use in vivo. In a biological system, the carbonyl group in the aldehyde will react rapidly with nucleophilic groups that are present in large quantities in all body fluids. These unwanted side reactions can lead to rapid metabolism of the drug and difficulties in controlling the serum concentration of the active drug. Controlling the drug at the cellular level within a narrow concentration window is essential to achieve effective immunomodulation. Tucaresol is given orally as an unprotected aldehyde, and there may be reason to suspect that the drug may be subject to oxidation and that the pharmacokinetics may be difficult to control.
Benzaldehydderivatene 4,6-benzyliden-D-glukose og den deutererte analogen (forbindelse 1 og 2) har vist seg å ha høy biotilgjengelighet enten de gis intravenøst eller peroralt. Biotilgjengeligheten for BALB-mus målt som konsentrasjon i serum etter oral tilførsel av forbindelse 2 var 93-99% (C.B. Dunsaed, J.M. Dornish og E.O. Pettersen, Cancer Chemother. Pharmacol. (1995) 35: 464-470). Dessuten kan glukosegruppen ha affinitet til reseptorer på celleoverflaten slik at medikamentet blir mer tilgjengelig på cellenivået. Det frie aldehydet kan lett frigjøres ved hydrolyse av acetalet slik at karbonylgruppen blir tilgjengelig for Schiff-basedannelse med målligandene. The benzaldehyde derivatives 4,6-benzylidene-D-glucose and the deuterated analogue (compounds 1 and 2) have been shown to have high bioavailability whether given intravenously or orally. The bioavailability for BALB mice measured as concentration in serum after oral administration of compound 2 was 93-99% (C.B. Dunsaed, J.M. Dornish and E.O. Pettersen, Cancer Chemother. Pharmacol. (1995) 35: 464-470). In addition, the glucose group can have an affinity for receptors on the cell surface so that the drug becomes more available at the cell level. The free aldehyde can be easily released by hydrolysis of the acetal so that the carbonyl group becomes available for Schiff base formation with the target ligands.
I den foreliggende patentsøknaden er aldehydene derivatiserte med biologisk akseptable karbohydrater som glukose, galaktose og andre og danner acetaler med dem. Sukkergruppen vil dermed gi bidrag til å øke stabiliteten og forbedre biotilgjengeligheten av aldehydfunksjonen for målcellene. Dette fører overraskende nok til karbonylkondensasjonsreaksjonene blir mer effektive og farmakokinetikken lettere kontrollerbar ved bruk av forbindelsene våre sammenliknet med tidligere kjente forbindelser. In the present patent application, the aldehydes are derivatized with biologically acceptable carbohydrates such as glucose, galactose and others and form acetals with them. The sugar group will thus contribute to increasing the stability and improving the bioavailability of the aldehyde function for the target cells. This surprisingly leads to the carbonyl condensation reactions becoming more efficient and the pharmacokinetics easier to control when using our compounds compared to previously known compounds.
For å sammenlikne forbindelse 2 med Tucaresol ble proteinsyntese og inaktivering av celler målt ved like store konsentrasjoner av de to medikamentene. Som det kan ses fra fig. 1, ble det påvist at forbindelse 2 var mer effektiv enn Tucaresol med hensyn til begge de målte parametrene. To compare compound 2 with Tucaresol, protein synthesis and inactivation of cells were measured at equal concentrations of the two drugs. As can be seen from fig. 1, it was demonstrated that compound 2 was more effective than Tucaresol with respect to both measured parameters.
Den immunstimulerende virkningen av forbindelsene ifølge oppfinnelsen kan også brukes ved behandling av visse virussykdommer i kombinasjon med annen behandling mot virus som f.eks. virusmedikamenter eller vaksiner. Mange virustyper vil etter den første infeksjonen inkorporere seg i cellekjernen og er inaktive i lang tid. Onkogene virus som hepatitt B og C, visse retrovirus og visse papillomvirus kan føre til utvikling av kreft. I disse latente periodene er det svært vanskelig å kurere virusinfeksjonen. Men slike virus vil ofte bli aktivert av immunrespons slik at de går ut i blodet, og i dette trinnet er det mulig å bli kvitt virusinfeksjonen. Benzaldehyd-derivatenes evne til å utløse immunresponsen kan brukes i kombinasjon med virusmidler eller vaksiner til å utvikle en behandling for disse sykdommene. The immunostimulating effect of the compounds according to the invention can also be used in the treatment of certain viral diseases in combination with other treatment against viruses such as e.g. antiviral drugs or vaccines. Many types of virus will, after the first infection, incorporate themselves into the cell nucleus and are inactive for a long time. Oncogenic viruses such as hepatitis B and C, certain retroviruses and certain papillomaviruses can lead to the development of cancer. During these latent periods, it is very difficult to cure the viral infection. But such viruses will often be activated by the immune response so that they enter the blood, and in this step it is possible to get rid of the viral infection. The benzaldehyde derivatives' ability to trigger the immune response can be used in combination with antiviral agents or vaccines to develop a treatment for these diseases.
Det er et hovedformål med oppfinnelsen å tilveiebringe forbindelser for forebygging og/eller behandling av sykdommer som er relatert til immunsystemet. It is a main purpose of the invention to provide compounds for the prevention and/or treatment of diseases related to the immune system.
Et annet formål med oppfinnelsen er å tilveiebringe forbindelser som er i stand til å forsterke immunresponsen for å gjøre det mulig å bekjempe infeksjonssykdommer som forårsakes av virus, bakterier, sopp og andre mikroorganismer. Another object of the invention is to provide compounds which are able to enhance the immune response to make it possible to fight infectious diseases caused by viruses, bacteria, fungi and other microorganisms.
Et tredje formål med oppfinnelsen er å tilveiebringe forbindelser for forebygging eller behandling av sykdommer som er relatert til immunproblemer, uten at forbindelsene har giftige bivirkninger. A third object of the invention is to provide compounds for the prevention or treatment of diseases related to immune problems, without the compounds having toxic side effects.
Disse og andre formål med oppfinnelsen oppnås ved de vedlagte patentkravene. These and other objects of the invention are achieved by the attached patent claims.
Forbindelsene som anvendes i henhold til den foreliggende oppfinnelsen har en generell formel (I): The compounds used according to the present invention have a general formula (I):
hvor L er H eller D; where L is H or D;
Ar er fenyl eller mono- eller di- substituert fenyl, hvor substituenten (e) er valgt fra gruppen bestående av alkyl med 1-6 karbonatomer, CF3, fluor, klor, nitro, cyano, OR<1>, SR<1>, N(R<1>)2 eller COOR<1> hvor R<1> er H, CF3 eller alkyl med 1-6 karbonatomer, eller OC(0)R<2>, CA(OR<2>)2, CA[OC(0)R<2>]2, NHC(0)R<2> eller N[C(0)R<2>]2 der A er H eller D og R2 er CF3 eller alkyl med 1-6 karbonatomer, eller C(0)R3 der R3 er H, D, CF3 eller alkyl med 1-6 karbonatomer; Ar is phenyl or mono- or di-substituted phenyl, where the substituent (e) is selected from the group consisting of alkyl with 1-6 carbon atoms, CF3, fluorine, chlorine, nitro, cyano, OR<1>, SR<1>, N(R<1>)2 or COOR<1> where R<1> is H, CF3 or alkyl with 1-6 carbon atoms, or OC(0)R<2>, CA(OR<2>)2, CA [OC(0)R<2>]2, NHC(0)R<2> or N[C(0)R<2>]2 where A is H or D and R2 is CF3 or alkyl with 1-6 carbon atoms , or C(O)R3 where R3 is H, D, CF3 or alkyl of 1-6 carbon atoms;
Y er valgt fra atomene eller gruppen bestående av H, D, fluor, klor, nitro, OR<1>, OC(0)R<2>, N(R<1>)2, NHC(0)R<2> eller N[C(0)R<2>]2 hvor R1 og R2 er som definert ovenfor; Y is selected from the atoms or group consisting of H, D, fluorine, chlorine, nitro, OR<1>, OC(0)R<2>, N(R<1>)2, NHC(0)R<2> or N[C(0)R<2>]2 where R1 and R2 are as defined above;
R er H, CF3 eller alkyl med 1-6 karbonatomer; R is H, CF3 or alkyl of 1-6 carbon atoms;
eller et farmasøytisk akseptabelt salt derav. or a pharmaceutically acceptable salt thereof.
Det er underforstått at enhver stereoisomer i henhold til formel (I) omfattes av den foreliggende oppfinnelsen. It is understood that any stereoisomer according to formula (I) is encompassed by the present invention.
Detaljert beskrivelse av oppfinnelsen Detailed description of the invention
Oppfinnelsen beskrives videre nedenfor med eksempler og vedlagte figurer. The invention is further described below with examples and attached figures.
Beskrivelse av figurene Description of the figures
Fia. 1: Hastigheten av proteinsyntesen i forhold til ubehandlet kontrollgruppe av NHIK 3025-celler behandlet med forbindelse 2 (■) eller Tucaresol (A) i 1 time ved 37°C. Hastigheten av proteinsyntesen ble målt som mengden av [<3>H]-valin som ble inkorporert i løpet av den første timen etter starten av medikament-behandlingen. Proteinsyntesehastigheten ble målt relativt til den totale mengden av protein i cellene. Dataene er representative for et eksperiment utført i kvadruplikat. Standardavvikene vises hvis de er større enn symbolene. Fig. 2: Perifere mononukleære blodceller og superantigen i Ex Vivo 10-medium ble utsatt for enten benzaldehyd, deuterert benzaldehyd, forbindelse 2 eller zilascorb(<2>H). Formeringen av perifere mononukleære blodceller ble målt som inkorporering av tritiert tymidin ved de forskjellige medikamentkonsentrasjonene. Fig. 3: NMRI-mus ble infisert intraperitonealt med miltinvaderende Friend-erytroleukemivirus. Infiserte og uinfiserte mus ble behandlet intraperitonealt daglig med 5 mg/kg av enten forbindelse 2 eller forbindelse 5. Etter 19 dagers behandling ble milten dissekert ut og veid. Fia. 1: The rate of protein synthesis relative to untreated control group of NHIK 3025 cells treated with compound 2 (■) or Tucaresol (A) for 1 hour at 37°C. The rate of protein synthesis was measured as the amount of [<3>H]-valine incorporated during the first hour after the start of drug treatment. The rate of protein synthesis was measured relative to the total amount of protein in the cells. The data are representative of an experiment performed in quadruplicate. The standard deviations are shown if they are larger than the symbols. Fig. 2: Peripheral blood mononuclear cells and superantigen in Ex Vivo 10 medium were exposed to either benzaldehyde, deuterated benzaldehyde, compound 2 or zilascorb(<2>H). The proliferation of peripheral blood mononuclear cells was measured as incorporation of tritiated thymidine at the different drug concentrations. Fig. 3: NMRI mice were infected intraperitoneally with spleen-invading Friend erythroleukemia virus. Infected and uninfected mice were treated intraperitoneally daily with 5 mg/kg of either compound 2 or compound 5. After 19 days of treatment, the spleen was dissected out and weighed.
Fremstilling Manufacturing
Som kjent gjennomgår aldehyder syrekatalyserte kondensasjonsreaksjoner med alkoholer under dannelse av acetaler. Samtidig fås vann som et biprodukt. Reaksjonen er reversibel, og i løsning dannes en likevektsblanding av aldehyd/alkohol og acetal/vann. Likevektsposisjonen bestemmes hovedsakelig av reaktiviteten og konsentrasjonen av hvert molekylslag. For å gjøre reaksjonen fullstendig fjerner man gjerne et av produktene (acetal eller vann) fra reaksjonsblandingen. As is known, aldehydes undergo acid-catalyzed condensation reactions with alcohols to form acetals. At the same time, water is obtained as a by-product. The reaction is reversible, and in solution an equilibrium mixture of aldehyde/alcohol and acetal/water is formed. The equilibrium position is mainly determined by the reactivity and concentration of each molecular species. To complete the reaction, one of the products (acetal or water) is removed from the reaction mixture.
I den foreliggende patentsøknaden kondenseres forskjellige sukkere, deoksysukkere og aminosukkere med aldehyder eller aldehydekvivalenter til sukkeracetaler. Spesielt foretrukket er en reacetaliseringsstrategi hvor aldehydet innføres beskyttet som dimetylacetalet i stedet for aldehydet selv. Dermed dannes metanol som biprodukt. Reaksjonsblandingen oppvarmes moderat ved redusert trykk for å fjerne metanolen så snart den dannes. I de fleste tilfellene vil disse reaksjonsbetingelsene lett forskyve likevekten til fordel for acetalet. In the present patent application, various sugars, deoxysugars and aminosugars are condensed with aldehydes or aldehyde equivalents to sugar acetals. Particularly preferred is a reacetalization strategy where the aldehyde is introduced protected as the dimethyl acetal instead of the aldehyde itself. Methanol is thus formed as a by-product. The reaction mixture is moderately heated under reduced pressure to remove the methanol as soon as it is formed. In most cases, these reaction conditions will easily shift the equilibrium in favor of the acetal.
Acetalisering av sukkere vil normalt føre til at det dannes blandinger av regio- og stereoisomerer. Det kan også opptre ringkontraksjoner som fører til blandinger av pyranoser og furanoser og i noen tilfeller dannes di-acetaliseringsprodukter. Hvis man ikke bruker beskyttelsesstrategier vil man derfor ofte få ytterst komplekse reaksjonsblandinger. Imidlertid ble det fremstilt overraskende rene produktfraksjoner etter passende opparbeiding, spesielt ved væskekromatografi. Identifikasjonen av produktene ble gjort ved GC-MS-spektroskopi og forskjellige NMR-teknikker. Acetalization of sugars will normally lead to the formation of mixtures of regio- and stereoisomers. Ring contractions can also occur which lead to mixtures of pyranoses and furanoses and in some cases di-acetalisation products are formed. If you do not use protection strategies, you will therefore often get extremely complex reaction mixtures. However, surprisingly pure product fractions were produced after suitable work-up, especially by liquid chromatography. The identification of the products was done by GC-MS spectroscopy and various NMR techniques.
De spesifikke reaksjonsbetingelsene og hvilke løsningsmidler og katalysatorer som brukes vil i hvert enkelt individuelt tilfelle avhenge av løseligheten og reaktiviteten av reaktantene og av egenskapene til produktet. Katalysatoren kan være en mineralsyre, f.eks. svovelsyre, en organisk syre, f.eks. paratoluensulfonsyre, et surt ionebyttemiddel, f.eks. Amberlyst 15, en elektrofil mineralleire, f.eks. montmorillonitt K-10 eller en resinstøttet supersyre, f.eks. Nafion NR 50. Reaksjonen kan gjerne utføres i et dipolart, aprotisk løsningsmiddel som dimetylformamid, dimetylacetamid, dimetylsulfoksid, N-metylpyrrolidon, dimetoksyetan eller liknende. Paratoluensulfonsyre i dimetylformamid var det foretrukne og mest brukte reaksjonsmediet. The specific reaction conditions and which solvents and catalysts are used will depend in each individual case on the solubility and reactivity of the reactants and on the properties of the product. The catalyst can be a mineral acid, e.g. sulfuric acid, an organic acid, e.g. paratoluenesulfonic acid, an acidic ion exchange agent, e.g. Amberlyst 15, an electrophilic mineral clay, e.g. montmorillonite K-10 or a resin-supported superacid, e.g. Nafion NR 50. The reaction can preferably be carried out in a dipolar, aprotic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, dimethoxyethane or the like. Paratoluenesulfonic acid in dimethylformamide was the preferred and most commonly used reaction medium.
Forbindelsene av formel (I) hvor L er deuterium kan fremstilles som bekrevet ovenfor, men med utgangspunkt i dimetylacetalet av et aldehyd som deutereres i formylposisjonen. Fremstilling av deutero-benzaldehyd kan utføres med en modifisert Rosenmund-reduksjon med D2-gass i et deuterert løsningsmiddel, som beskrevet i EP 0 283 139 B1. Deutererte benzaldehydderivater med substituenter i fenylringen kan fremstilles i henhold til eksemplene som gis i EP 0 493 883 A1 og EP 0 552 880 A1. The compounds of formula (I) where L is deuterium can be prepared as described above, but starting from the dimethyl acetal of an aldehyde which is deuterated in the formyl position. Production of deutero-benzaldehyde can be carried out with a modified Rosenmund reduction with D2 gas in a deuterated solvent, as described in EP 0 283 139 B1. Deuterated benzaldehyde derivatives with substituents in the phenyl ring can be prepared according to the examples given in EP 0 493 883 A1 and EP 0 552 880 A1.
De følgende eksemplene illustrerer hvordan forbindelsene til den foreliggende oppfinnelsen kan fremstilles. The following examples illustrate how the compounds of the present invention can be prepared.
Forbindelse 1:4. 6- O- benzvliden- D- qlukopvranose Compound 1:4. 6- O- benzvlidene- D- qlukopvranose
Denne kjente forbindelsen ble fremstilt som beskrevet for forbindelse 2, med utgangspunkt i udeuterert benzaldehyddimetylacetal. Identiteten ble bekreftet ved <1>H NMR-spektroskopi i DMSO-d6: 5 rel. til TMS: 7,58-7,29 (5H, m, Ar-H), 6,83 (0,4H, d, OH-1-p), 6,60 (0,6H, d, OH-1-a), 5,61 (1H, s+s, acetal-H), 5,25 (0,4H, d, OH_-3-p), 5,21 (0,4H, d, OH-2-P), 5,62 (0,6H, d, OH-3-ct), 5,00 (0,6H, H-1-cc), 4,82 (0,6H, d, OH-2-cc), 4,49 (0,4H, t, H-1-P), 4,18-4,02 (1H, m, H-6'-a+<p>), 3,89-3,77 (0,6H, m, H-5-cc), 3,75-3,57 (1,6H, m, H-6"-a+p og ]H-3-a), 3,45-3,27 (2,5H, m, H-3-p, H-4-a+p, H-5-P og H-2-a) og 3,11-3,00 (0,4H, m, H-2-p). This known compound was prepared as described for compound 2, starting from undeuterated benzaldehyde dimethyl acetal. The identity was confirmed by <1>H NMR spectroscopy in DMSO-d6: 5 rel. to TMS: 7.58-7.29 (5H, m, Ar-H), 6.83 (0.4H, d, OH-1-p), 6.60 (0.6H, d, OH-1 -a), 5.61 (1H, s+s, acetal-H), 5.25 (0.4H, d, OH_-3-p), 5.21 (0.4H, d, OH-2- P), 5.62 (0.6H, d, OH-3-ct), 5.00 (0.6H, H-1-cc), 4.82 (0.6H, d, OH-2-cc ), 4.49 (0.4H, t, H-1-P), 4.18-4.02 (1H, m, H-6'-a+<p>), 3.89-3.77 ( 0.6H, m, H-5-cc), 3.75-3.57 (1.6H, m, H-6"-a+p and ]H-3-a), 3.45-3, 27 (2.5H, m, H-3-p, H-4-a+p, H-5-P and H-2-a) and 3.11-3.00 (0.4H, m, H -2-p).
Forbindelse 2: 4. 6- 0-( benzvliden- di)- D- alukopvranose Compound 2: 4. 6- O-( benzvlidene-di)- D- alucopvranose
Benzaldehyd-di ble fremstilt og konvertert til benzaldehyddimetylacetal-di som beskrevet i EP 0 283 139 B1. Fremstillingen av 4,6-0-(benzyliden-di)-D-glukopyranose er også beskrevet i EP 0 283 139 B1, men denne gangen ble forbindelsen fremstilt etter en alternativ fremgangsmåte hvor høy renhet var prioritert: D(+)-glukose (706 g, 3,92 mol), benzaldehyddimetylacetal-di (571 g, 3,73 mol), tørr DMF (1,68 kg) og paratoluensulfonsyre (4,5 g, 24 mmol) ble blandet i et tørrdestillasjonsapparat koblet til en vakuumpumpe gjennom en kaldreflukskondensator. Den mekanisk rørte blandingen ble oppvarmet til maks. 69°C ved 30 torr for å destillere av metanol og etter 2 timer var det samlet opp 235 g. Reflukskondensatoren ble så stengt av og temperaturen økt til maks. 73°C for å destillere av DMF. Etter ytterligere 2 timer var det samlet opp ytterligere 1385 g og destillasjonen ble avbrutt. Destillasjonsresten ble avkjølt til 40°C og tilsatt isvann (2,9 I) før det var gått 5 min. Temperaturen falt under 0°C og det dannet seg et bunnfall, delvis som store klumper. Blandingen ble overført til et begerglass og tilsatt ytterligere 8-9 I isvann for å få klumpene til å falle fra hverandre og gå i suspensjon. Suspensjonen ble filtrert på to nutch-filtre og man lot de to filterkakene stå over natten på filtrene tilkoblet vannstrålepumpe og begge filterkakene ble tilført N2 gjennom en omvendt trakt. Filterkakene ble spredd ut på to plater og tørket ved 32°C i 20 timer i en vakuumovn. Vakuumet ble først satt til 13 millibar og så regulert ned til 1 millibar. Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1. The preparation of 4,6-0-(benzylidene-di)-D-glucopyranose is also described in EP 0 283 139 B1, but this time the compound was prepared according to an alternative method where high purity was prioritized: D(+)-glucose ( 706 g, 3.92 mol), benzaldehyde dimethyl acetal-di (571 g, 3.73 mol), dry DMF (1.68 kg) and paratoluenesulfonic acid (4.5 g, 24 mmol) were mixed in a dry distillation apparatus connected to a vacuum pump through a cold flux condenser. The mechanically stirred mixture was heated to max. 69°C at 30 torr to distill off methanol and after 2 hours 235 g had been collected. The reflux condenser was then shut off and the temperature increased to max. 73°C to distil off DMF. After a further 2 hours a further 1385 g had been collected and the distillation was stopped. The distillation residue was cooled to 40°C and ice water (2.9 L) was added before 5 min had elapsed. The temperature fell below 0°C and a precipitate formed, partly as large lumps. The mixture was transferred to a beaker and an additional 8-9 L of ice water was added to cause the lumps to fall apart and go into suspension. The suspension was filtered on two nutch filters and the two filter cakes were left overnight on the filters connected to a water jet pump and both filter cakes were supplied with N2 through an inverted funnel. The filter cakes were spread out on two plates and dried at 32°C for 20 hours in a vacuum oven. The vacuum was first set to 13 millibars and then regulated down to 1 millibar.
Råproduktet ble omkrystallisert (for å fjerne dibenzylidenacetaler) og vasket med vann (for å fjerne DMF og glukose) inntil disse forurensningene var eliminert. Følgelig ble råproduktet (500 g) løst i varm dioksan (800 ml) og løsningen overført til kokende kloroform (9 I) gjennom et foldefilter. Løsningen ble avkjølt, først til romtemperatur, så i et isbad over natten. Bunnfallet ble filtrert fra, tørket i 2 timer på filteret (under tilførsel av N2 som beskrevet over) og tørket videre over natten ved 31 °C i vakuum på en rotavapor. Produktet (142 g) ble suspendert i isvann (1 I), filtrert på en nutch (vasking med 200 ml isvann) og tørket på filteret over natten som beskrevet over. Deretter ble det oppmalt, siktet (0,5 mm gitter) og tørket i vakuum i 5 timer ved 31 °C på en rotavapor. Produktet (96 g) ble igjen suspendert i isvann (500 ml), filtrert (vasking med 150 ml isvann) og tørket (7 timer under N2-strøm). Det ble til slutt oppmalt i en morter, siktet (0,5 mm) og tørket i en vakuumovn. The crude product was recrystallized (to remove dibenzylidene acetals) and washed with water (to remove DMF and glucose) until these impurities were eliminated. Accordingly, the crude product (500 g) was dissolved in hot dioxane (800 mL) and the solution transferred to boiling chloroform (9 L) through a pleated filter. The solution was cooled, first to room temperature, then in an ice bath overnight. The precipitate was filtered off, dried for 2 hours on the filter (while supplying N2 as described above) and further dried overnight at 31 °C in vacuum on a rotavapor. The product (142 g) was suspended in ice water (1 L), filtered on a nutch (washing with 200 ml ice water) and dried on the filter overnight as described above. It was then ground, sieved (0.5 mm grid) and dried in vacuum for 5 hours at 31 °C on a rotavapor. The product (96 g) was resuspended in ice water (500 ml), filtered (washing with 150 ml ice water) and dried (7 hours under N 2 flow). It was finally ground in a mortar, sieved (0.5 mm) and dried in a vacuum oven.
Produktet var et hvitt, finfordelt pulver av høy renhet ifølge HPLC-analysen. Utbyttet var 95 g, 10% av teoretisk utbytte. NMR i DMSO-d6 indikerte et a:P anomerforhold på omtrent 7:3. The product was a white, finely divided powder of high purity according to the HPLC analysis. The yield was 95 g, 10% of theoretical yield. NMR in DMSO-d6 indicated an a:P anomer ratio of approximately 7:3.
<1>H og <13>C NMR (DMSO-d6), 8 rel. til TMS: 7,55-7,28 (5.00H, m, Ar-H), 6,85 (0.27H, d, OH-1-<p>), 6,58 (0,71 H, d, OH-1-a), 5,24 (0.27H, d, OH-3-P), 5,19 (0.28H, d, OH-2-P), 5,61 (0,71 H, d, OH-3-a), 4,99 (0.72H, H-1-a), 4,82 (0,71 H, d, OH-2-cc), 4,48 (0.29H, t, H-1-p), 4,20-4,04 (1,04H, m, H-6'-a+P), 3,88-3,73 (0,78H, m, H-5-a), 3,73-3,56 (1.72H, m, H-6"-a+p og H-3-a), 3,46-3,21 (2,61 H, m, H-3-P, <1>H and <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.55-7.28 (5.00H, m, Ar-H), 6.85 (0.27H, d, OH-1-<p>), 6.58 (0.71H, d, OH-1-a), 5.24 (0.27H, d, OH-3-P), 5.19 (0.28H, d, OH-2-P), 5.61 (0.71 H, d, OH-3-a), 4.99 (0.72H, H-1-a), 4.82 (0.71 H, d, OH-2-cc), 4.48 (0.29H, t, H- 1-p), 4.20-4.04 (1.04H, m, H-6'-a+P), 3.88-3.73 (0.78H, m, H-5-a), 3.73-3.56 (1.72H, m, H-6"-a+p and H-3-a), 3.46-3.21 (2.61 H, m, H-3-P,
H-4-a+p, H-5-p og H-2-cc) og 3,09-2,99 (0,28H, m, H.-2-P); 137,881, 128,854, 128,042, 126,435 (Ar-C), 100,462 (acetal-C), 97,642 (C-1-P), 93,211 (C-1-cc), 81,729 (C-4-a), 80,897 (C-4-p), 75,796 (C-2-P), 72,906 (C-2-cc og C-3-P), 69,701 (C-3-a), 68,431 (C-6-a), 68,055 (C-6-P), 65,810 (C-5-p) og 62,032 (C-5-cc). H-4-a+p, H-5-p and H-2-cc) and 3.09-2.99 (0.28H, m, H.-2-P); 137.881, 128.854, 128.042, 126.435 (Ar-C), 100.462 (acetal-C), 97.642 (C-1-P), 93.211 (C-1-cc), 81.729 (C-4-a), 80.897 (C -4-p), 75.796 (C-2-P), 72.906 (C-2-cc and C-3-P), 69.701 (C-3-a), 68.431 (C-6-a), 68.055 ( C-6-P), 65.810 (C-5-p) and 62.032 (C-5-cc).
Forbindelse 3: 4, 6- O- benzvliden- D- qalaktopvranose Compound 3: 4, 6- O- benzvlidene- D- qalactopvranose
D(+)-galaktose (15,0 g, 0,083 mol) og tørr DMF (80 ml) ble blandet under omrøring ved 50°C i et destillasjonsapparat. Suspensjonen som dannet seg ble tilsatt benzaldehyddimetylacetal (12,2 g, 0,083 mmol) og paratoluensulfonsyre (0,14 g) og metanol/DMF ble sakte destillert av ved hjelp av en vannstrålepumpe. Etter 3 timer var det meste av galaktosen brukt opp og det resterende DMF fjernet på en rotavapor koblet til en vakuumpumpe. Destillasjonsresten, en nokså seig sirupsaktig masse, ble renset på en Lobar C RP-8-kolonne med metanol/vann (1:1) som eluent. Produktfraksjonene ble frysetørket. D(+)-galactose (15.0 g, 0.083 mol) and dry DMF (80 mL) were mixed with stirring at 50°C in a still. To the resulting suspension was added benzaldehyde dimethyl acetal (12.2 g, 0.083 mmol) and paratoluenesulfonic acid (0.14 g) and methanol/DMF was slowly distilled off using a water jet pump. After 3 hours, most of the galactose was used up and the remaining DMF was removed on a rotavapor connected to a vacuum pump. The distillation residue, a rather tough syrupy mass, was purified on a Lobar C RP-8 column with methanol/water (1:1) as eluent. The product fractions were freeze-dried.
Gasskromatografi for TMS-derivatene viste at produktet hovedsakelig besto av to isomerer. På basis av <1>H-, <13>C-, COSY-, DEPT- og C-H-korrelasjons-NMR-spektra ble produktet identifisert som a- og P-anomerene av tittelforbindelsen. Gas chromatography for the TMS derivatives showed that the product consisted mainly of two isomers. On the basis of <1>H, <13>C, COSY, DEPT and C-H correlation NMR spectra, the product was identified as the α and β anomers of the title compound.
<1>H og <13>C NMR (DMSO-d6), 8 rel. til TMS: 7,49-7,27 (5H, m, Ar-H), 5,57 (1H, s, acetal-H), 5,22 (0,5H, d, H-1-a), 4,56 (0,5H, d, H-1-P), 4,23+4,18 (0,5H+0,5H, d+d, H-4-oc+P), 4,14-3,98 (2H, m, H-6-cc+P), 3,94-3,79 (1,5H, m, H-2-ct, H-3-cc og H-5-a), 3,69-3,49 (1,5H, m, H-2-p, H-3-P og H-5-P); 137,422, 129,981 128,902 126,639 og 126,590 (Ar-C), 101,325 (acetal-C), 96,540 (C-1-P), 93,161 (C-1-a), 76,581 (C-4-a), 76,093 (C-4-P), 71,889 + 71,802 (C-2-P+C-3-P), 69,404 (C-6-a), 69,182 (C-6-P), 68,566 + 68.057 (C-2-cc + C-3-P), 66,579 (C-5-P) og 62,886 (C-5-cc). <1>H and <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.49-7.27 (5H, m, Ar-H), 5.57 (1H, s, acetal-H), 5.22 (0.5H, d, H-1-a), 4.56 (0.5H, d, H-1-P), 4.23+4.18 (0.5H+0.5H, d+d, H-4-oc+P), 4.14- 3.98 (2H, m, H-6-cc+P), 3.94-3.79 (1.5H, m, H-2-ct, H-3-cc and H-5-a), 3.69-3.49 (1.5H, m, H-2-p, H-3-P and H-5-P); 137.422, 129.981 128.902 126.639 and 126.590 (Ar-C), 101.325 (acetal-C), 96.540 (C-1-P), 93.161 (C-1-a), 76.581 (C-4-a), 76.093 (C -4-P), 71.889 + 71.802 (C-2-P+C-3-P), 69.404 (C-6-a), 69.182 (C-6-P), 68.566 + 68.057 (C-2-cc + C-3-P), 66.579 (C-5-P) and 62.886 (C-5-cc).
Forbindelse 4: metvl- 4. 6- 0- benzvliden- a- D- mannopyranosid Compound 4: metvl- 4. 6- 0- benzvlidene- a- D- mannopyranoside
Metykx-mannopyranosid (18,1 g, 0,093 mol), benzaldehyddimetylacetal (21,0 g, 0,138 mol) og tørr DMF ble blandet under omrøring ved 50-55°C i et destillasjonsapparat. Det ble tilsatt paratoluensulfonsyre (ca. 0,1 g) og 10 min. senere ble det koblet til en vannstrålepumpe for å destillere av metanol. Etter 4 timer ble reaksjonsblandingen inndampet til et hvitt fast stoff. Inndampingsresten ble vasket med dibutyleter, filtrert og filterkaken ble løst i acetonitril. Det begynte å danne seg et bunnfall og blandingen ble satt i kjøleskap i 5 dager. Deretter ble bunnfallet filtrert fra og filtratet inndampet. Inndampingsresten ble renset på en Lobar C RP-8-kolonne med 30% acetonitril i vann som eluent. Produktfraksjoner fra 4 separate synteser ble frysetørket og kombinert. Methylx-mannopyranoside (18.1 g, 0.093 mol), benzaldehyde dimethyl acetal (21.0 g, 0.138 mol) and dry DMF were mixed with stirring at 50-55°C in a still. Paratoluenesulfonic acid (approx. 0.1 g) was added and 10 min. later it was connected to a water jet pump to distill off methanol. After 4 hours, the reaction mixture was evaporated to a white solid. The evaporation residue was washed with dibutyl ether, filtered and the filter cake was dissolved in acetonitrile. A precipitate began to form and the mixture was refrigerated for 5 days. The precipitate was then filtered off and the filtrate evaporated. The evaporation residue was purified on a Lobar C RP-8 column with 30% acetonitrile in water as eluent. Product fractions from 4 separate syntheses were freeze-dried and combined.
Gasskromatografisk analyse av TMS-derivatene indikerte at produktet besto av 95 arealprosent monoacetaler. Monoacetalene besto for sin del av 4 topper som kunne integreres til henholdsvis 0,4, 3,2, 94,1 og 2,4 areal-%. På basis av <1>H-, <13>C-, COSY-, DEPT- og C-H-korrelasjons NMR-spektra sammen med gasskromatografi og MS-spektroskopi ble det dominerende molekylslaget identifisert som tittelforbindelsen. Gas chromatographic analysis of the TMS derivatives indicated that the product consisted of 95 area percent monoacetals. The monoacetals for their part consisted of 4 peaks which could be integrated to 0.4, 3.2, 94.1 and 2.4 area-% respectively. On the basis of <1>H, <13>C, COSY, DEPT and C-H correlation NMR spectra together with gas chromatography and MS spectroscopy, the dominant molecular species was identified as the title compound.
<1>H og <13>C NMR (aceton-d6), 8 rel. til TMS: 7,54-7,30 (5H, m, Ar-H), 5,60 (1H, s, acetal-H), 4,71 (1H, s, H-1), 4,34 (1H, bred s, OH), 4,22-4,02 (2H, m+bred s, IH-6'+OH), 3,94-3,82 (3H, m, H-2, H-3 og H-4), 3,80-3,60 (2H, m, H-5+H-6") og 3,39 (3H, s, CH3); 139,264, 129,396, 128,662 og 127,211 (Ar-C), 102,825 (C-1), 102,468 (acetal-C), 79,888 (C-4), 72,090 (C-3), 69,308 (C-6), 69,127 (C-2), 64,363 (C-5) og 54,921 (CH3). <1>H and <13>C NMR (acetone-d6), 8 rel. to TMS: 7.54-7.30 (5H, m, Ar-H), 5.60 (1H, s, acetal-H), 4.71 (1H, s, H-1), 4.34 ( 1H, broad s, OH), 4.22-4.02 (2H, m+broad s, IH-6'+OH), 3.94-3.82 (3H, m, H-2, H-3 and H-4), 3.80-3.60 (2H, m, H-5+H-6") and 3.39 (3H, s, CH3); 139.264, 129.396, 128.662 and 127.211 (Ar-C ), 102.825 (C-1), 102.468 (acetal-C), 79.888 (C-4), 72.090 (C-3), 69.308 (C-6), 69.127 (C-2), 64.363 (C-5) and 54.921 (CH 3 ).
Forbindelse 5:4. 6-( benzvliden- di)- 2- deoksv- D- alukopvranose Connection 5:4. 6-( benzvlidene-di)- 2- deoxyv- D- alucopvranose
Benzaldehyd-di ble fremstilt og konvertert til benzaldehyddimetylacetal-di som beskrevet i EP 0 283 139 B1. Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.
2-deoksy-D-glukose (10 g, 60,9 mmol), tørr DMF (35 ml), 2-deoxy-D-glucose (10 g, 60.9 mmol), dry DMF (35 mL),
benzaldehyddimetylacetal-di (11,7 g, 76,4 mmol) og paratoluensulfonsyre (70 mg, 0,37 mmol) ble blandet under N2 til en hvit grøt. Ved oppvarming til 45-50°C ble det dannet en fargeløs løsning i løpet av en halv time. En vakuumpumpe ble tilkoblet for å fjerne metanol gjennom en avkjølt kolonne (for å hindre tap av benzaldehyddimetylacetal-di). Trykket ble regulert trinnvis ned fra 70 millibar til 20-30 millibar i løpet av 4,5 timer og temperaturen ble opprettholdt på 40-45°C. Deretter ble destillasjonen avbrutt, apparatet ombygd uten kolonnen og DMF ble fjernet ved kortbanedestillasjon ved 50-55°C, maks. vakuum. Resten var en svakt gul sirupsaktig masse. benzaldehyde dimethyl acetal-di (11.7 g, 76.4 mmol) and paratoluenesulfonic acid (70 mg, 0.37 mmol) were mixed under N 2 to a white slurry. On heating to 45-50°C, a colorless solution was formed within half an hour. A vacuum pump was connected to remove methanol through a cooled column (to prevent loss of benzaldehyde dimethyl acetal-di). The pressure was regulated stepwise down from 70 millibars to 20-30 millibars during 4.5 hours and the temperature was maintained at 40-45°C. The distillation was then interrupted, the apparatus rebuilt without the column and the DMF was removed by short path distillation at 50-55°C, max. vacuum. The rest was a pale yellow syrupy mass.
1/4 av sirupsmassen ble løst i svakt alkalisk (NaHCOa) metanol/vann 60/40 og renset på en Merck LiChroprep RP-8 omvendt fase-kolonne med metanol/vann 60/40 som eluent. Produktf raksjoner ble konsentrert for å fjerne metanol og frysetørket til et hvitt, ullent fast stoff. Produkter fra fire separate synteser ble kombinert til et utbytte på 3,5 g, 23% av det teoretiske. 1/4 of the syrup mass was dissolved in weakly alkaline (NaHCOa) methanol/water 60/40 and purified on a Merck LiChroprep RP-8 reverse phase column with methanol/water 60/40 as eluent. Product fractions were concentrated to remove methanol and freeze-dried to a white, off-white solid. Products from four separate syntheses were combined in a yield of 3.5 g, 23% of theory.
Gasskromatografisk analyse av TMS-derivatene og NMR-spektroskopi viste at produktet besto av en 1:1 blanding av a- og (3-anomerene. Gas chromatographic analysis of the TMS derivatives and NMR spectroscopy showed that the product consisted of a 1:1 mixture of the α- and (3-anomers.
<1>H og <13>C NMR (DMSO-d6), 6 (ppm) rel. til TMS: 7,52-7,28 (m, 5H, Ar-H I + II), 6,9-6,65 (bred s, 1/2 H, OH-1 II), 6,55-6,32 (bred s, 1/2 H, OH-1 I), 5,25-5,12 (m, 1 H, OH-3 II og H-1 I), 5,12-5,0 (d, 1/2 H, OH-3 II), 4,84-4,73 (dd, 1/2 H, H-1 II), 4,20-4,02 (m, 1H, H-6 l+ll), 3,98-3,73 (m, 1H, H-3 I og H-5 I), 3,73-3,58 (m, 1,5H, H-6' l+ll og H-3 II), 3,42-3,18 (2,5 H, H-4 l+ll og H-5 II og hbO), 2,10-1,86 (m, 1H, H-2 l+ll) og 1,62-1,34 (m, 1H, H-2' l+ll); 137,979, 137,926, 128,841, 128,036 og 126,432 (Ar-C l+ll), 101,5-100,0 (acetal-C l+ll), 94,057 og 91,424 (C-1 l+ll), 83,916 og 83,093 (C-4 l+ll), 68,374 og 68,119 (C-6 l+ll), 66,889, 66,092, 64,174 og 62,604 (C-3 l+ll og C-5 l+ll) og 41,932 og 40,051 (C-2 l+ll). <1>H and <13>C NMR (DMSO-d6), 6 (ppm) rel. to TMS: 7.52-7.28 (m, 5H, Ar-H I + II), 6.9-6.65 (broad s, 1/2 H, OH-1 II), 6.55-6 .32 (broad s, 1/2 H, OH-1 I), 5.25-5.12 (m, 1 H, OH-3 II and H-1 I), 5.12-5.0 (d , 1/2 H, OH-3 II), 4.84-4.73 (dd, 1/2 H, H-1 II), 4.20-4.02 (m, 1H, H-6 l+ ll), 3.98-3.73 (m, 1H, H-3 I and H-5 I), 3.73-3.58 (m, 1.5H, H-6' 1+ll and H- 3 II), 3.42-3.18 (2.5 H, H-4 l+ll and H-5 II and hbO), 2.10-1.86 (m, 1H, H-2 l+ll ) and 1.62-1.34 (m, 1H, H-2' 1+11); 137.979, 137.926, 128.841, 128.036 and 126.432 (Ar-C l+ll), 101.5-100.0 (acetal-C l+ll), 94.057 and 91.424 (C-1 l+ll), 83.916 and 83.093 ( C-4 l+ll), 68.374 and 68.119 (C-6 l+ll), 66.889, 66.092, 64.174 and 62.604 (C-3 l+ll and C-5 l+ll) and 41.932 and 40.051 (C-2 l+ll).
Forbindelse 6: 4, 6- Q-( 4- karbometoksvbenzvliden)- D- qlukopvranose Compound 6: 4, 6- Q-( 4- carbomethoxybenzvlidene)- D- qulcopvranose
Metyl-4-formylbenzoat (100 g, 0,609 mol), metanol (91,5 g, 2,86 mol), trimetylortoformiat (71 g, 0,67 mol) og konsentrert saltsyre (165 jllI) ble blandet til en grøt i en 500 ml trehalset kolbe. Grøten ble omdannet til en svakt gul løsning på få minutter og temperaturen økte spontant fra 15°C til 30°C. Etter 15 minutters omrøring ble reaksjonsblandingen refluksert ved 58°C i 25 minutter til og så nedkjølt til 10°C (isvann). Det ble laget en alkalisk løsning ved å løse KOH (8,3 g) i metanol (53 ml) og 7 ml av løsningen ble tilsatt til reaksjonsblandingen. Etter 25 minutters omrøring ved 10°C ble reaktoren ombygd for kortbanedestillasjon og alle flyktige stoffer fjernet i vakuum (vannstrålepumpe). Destillasjonen fortsatte deretter med en vakuumpumpe til det var igjen en fargeløs olje ved 112-114°C/0,5 millibar. Oljen omdannet seg til et fargeløst fast stoff med smeltepunkt 32-33°C, og ble identifisert ved NMR som metyl-4-formylbenzoatdimetylacetal. Utbyttet var 108,75 g, 85% av det teoretiske. Methyl-4-formylbenzoate (100 g, 0.609 mol), methanol (91.5 g, 2.86 mol), trimethyl orthoformate (71 g, 0.67 mol) and concentrated hydrochloric acid (165 µL) were mixed to a slurry in a 500 ml wooden flask. The porridge was converted to a slightly yellow solution in a few minutes and the temperature increased spontaneously from 15°C to 30°C. After stirring for 15 minutes, the reaction mixture was refluxed at 58°C for another 25 minutes and then cooled to 10°C (ice water). An alkaline solution was made by dissolving KOH (8.3 g) in methanol (53 mL) and 7 mL of the solution was added to the reaction mixture. After 25 minutes of stirring at 10°C, the reactor was rebuilt for short path distillation and all volatile substances removed in vacuum (water jet pump). Distillation was then continued with a vacuum pump until a colorless oil was left at 112-114°C/0.5 millibar. The oil turned into a colorless solid with a melting point of 32-33°C, and was identified by NMR as methyl-4-formylbenzoate dimethyl acetal. The yield was 108.75 g, 85% of the theoretical.
D(+)-glukose (8,0 g, 44,4 mmol), tørr DMF (25 ml), metyl-4-formylbenzoatdimetylacetal (10,4 g, 49,5 mmol) og paratoluensulfonsyre ble blandet ved 50°C under N2 til en hvit suspensjon. Apparatet var koblet til en vakuumpumpe gjennom en vertikal kondensator og fordampningen av metanol startet ved 80-100 millibar, 55°C. Vakuumet ble gradvis økt til 40 millibar mens temperaturen ble holdt på 55-60°C. Reaksjonsblandingen ble gradvis klar og apparaturen ble ombygd for kortbanedestillasjon av DMF. Destillasjonsresten var en svakt gul sirupsmasse. D(+)-glucose (8.0 g, 44.4 mmol), dry DMF (25 mL), methyl 4-formylbenzoate dimethyl acetal (10.4 g, 49.5 mmol) and paratoluenesulfonic acid were mixed at 50°C under N2 to a white suspension. The apparatus was connected to a vacuum pump through a vertical condenser and the evaporation of methanol started at 80-100 millibar, 55°C. The vacuum was gradually increased to 40 millibars while the temperature was maintained at 55-60°C. The reaction mixture gradually became clear and the apparatus was rebuilt for short path distillation of DMF. The distillation residue was a pale yellow syrupy mass.
Sirupsmassen ble løst i en varm løsning av 100 mg NaHC03 i 20 ml metanol og 8 ml vann og felt ved tilsetning av 100 ml etylacetat. Bunnfallet ble isolert fra modervæsken ved filtrering, vasket med kaldt vann (4 x 15-20 ml) og overført til en rotayaporkolbe. Fuktigheten ble fjernet ved å tilsette etylacetat og inndampe to ganger. Produktet ble endelig tørket under høyt vakuum. Det ble filtrert av mer bunnfall fra modervæsken og vasket og tørket til nok en produktporsjon. De to porsjonene ble slått sammen til 1,94 g rent produkt, 13% av det teoretiske utbyttet. The syrup mass was dissolved in a warm solution of 100 mg of NaHCO 3 in 20 ml of methanol and 8 ml of water and filtered by adding 100 ml of ethyl acetate. The precipitate was isolated from the mother liquor by filtration, washed with cold water (4 x 15-20 ml) and transferred to a rotayapore flask. The moisture was removed by adding ethyl acetate and evaporating twice. The product was finally dried under high vacuum. It was filtered off more sediment from the mother liquor and washed and dried for another product portion. The two portions were combined to give 1.94 g of pure product, 13% of the theoretical yield.
Gasskromatografisk analyse av TMS-derivatene viste to isomerer i forholdet 2:1. Gas chromatographic analysis of the TMS derivatives showed two isomers in the ratio 2:1.
<1>H- og <13>C NMR (DMSO-d6), 8 (ppm) rel. til TMS: 7,99 og 7,61 (dd, 2+2H, furfuryl-H), 6,87 (d, 0.67H OH-1 II), 6,59 (d, 0.28H, OH-1 I), 5,68 (s+s, 1H, acetal-H l+ll), 5,29 (d, 0.68H, OH-3 II), 5,21 (d, 0.67H, OH-2 II), 5,16 (d, 0,31 H, OH-3 I), 5,00 (t, 0.30H, H-1 I), 4,85 (d, 0,28H, OH-2 I), 4,48 (t, 0.73H, H-1 II), 4,25-4,08 (m, 1.14H, H-6), 3,95-3,77 (m, 3.43H, OCH3 og H-5 I), 3,78-3,59 (m, 1,40H, H-3 I og H-6'), 3,49-3,23 (m, 3.59H, H-4 I og II, H-5 II, H-2 I og H-3 II), 3,10-2,98 (m, 0.72H, H-2 II); 165,978, 142,553, 142,553, 129,959, 129,067, 126,763, 99,982, 99,812, 97,661, 93,238, 81,794, 81,794, 80,963, 75,808, 72,902, 69,658, 68,487, 68,110, 65,714,61,952 og 52,253. <1>H- and <13>C NMR (DMSO-d6), 8 (ppm) rel. to TMS: 7.99 and 7.61 (dd, 2+2H, furfuryl-H), 6.87 (d, 0.67H OH-1 II), 6.59 (d, 0.28H, OH-1 I) , 5.68 (s+s, 1H, acetal-H l+ll), 5.29 (d, 0.68H, OH-3 II), 5.21 (d, 0.67H, OH-2 II), 5 .16 (d, 0.31 H, OH-3 I), 5.00 (t, 0.30H, H-1 I), 4.85 (d, 0.28H, OH-2 I), 4.48 (t, 0.73H, H-1 II), 4.25-4.08 (m, 1.14H, H-6), 3.95-3.77 (m, 3.43H, OCH3 and H-5 I) , 3.78-3.59 (m, 1.40H, H-3 I and H-6'), 3.49-3.23 (m, 3.59H, H-4 I and II, H-5 II , H-2 I and H-3 II), 3.10-2.98 (m, 0.72H, H-2 II); 165,978, 142.553, 142553, 129,959, 129,067, 126,763, 99,982, 99,812, 97,661, 93,238, 81,794, 81,794, 80,965.95.9.9.9.CLIBRATION, 75, 75, 75, 75, 75, 75, 75.
Forbindelse 7: 4, 6- 0- benzvliden- 2- deoksv- D- qlukopyranose Compound 7: 4, 6- 0- benzvlidene- 2- deoxv- D- qlucopyranose
12-deoksy-D-glukose (10,0 g, 60,9 mmol), tørr DMF (34 ml), benzaldehyddimetylacetal (11,6 g, 76,2 mmol) og paratoluensulfonsyre (70 mg, 0,37 mmol) ble blandet under N2 til en hvit grøt. Reaksjonsblandingen ble omrørt ved romtemperatur i 30 minutter og ved oppvarming til 45-50°C ble den faste substansen gradvis løst. Det ble koblet til en vakuumpumpe for å fjerne metanol gjennom en avkjølt kolonne (for å forhindre tap av benzaldehyddimetylacetal) og reaksjonen fortsatte i 4,5 timer. Deretter ble destillasjonen avbrutt, kolonnen fjernet og DMF destillert fra ved kortbanedestillasjon ved 50-55°C, maksimalt vakuum. Destillasjonsresten var en svakt gul sirupsmasse. 12-deoxy-D-glucose (10.0 g, 60.9 mmol), dry DMF (34 mL), benzaldehyde dimethyl acetal (11.6 g, 76.2 mmol) and paratoluenesulfonic acid (70 mg, 0.37 mmol) were mixed under N2 to a white mush. The reaction mixture was stirred at room temperature for 30 minutes and by heating to 45-50°C the solid substance was gradually dissolved. A vacuum pump was connected to remove methanol through a cooled column (to prevent loss of benzaldehyde dimethyl acetal) and the reaction continued for 4.5 hours. The distillation was then interrupted, the column removed and DMF distilled from by short path distillation at 50-55°C, maximum vacuum. The distillation residue was a pale yellow syrupy mass.
Sirupsmassen ble løst i svakt alkalisk (NaHC03) metanol/vann 60/40 og renset på en Merck LiChroprep RP-8 omvendt fase-kolonne med metanol/vann 60/40 som eluent. Produktfraksjonene ble konsentrert for å fjerne metanol og frysetørket til et hvitt, ullent fast stoff. Produktene fra fire separate synteser ble kombinert til 3,18 g, 21% av det teoretiske utbyttet. The syrup mass was dissolved in weakly alkaline (NaHCO 3 ) methanol/water 60/40 and purified on a Merck LiChroprep RP-8 reverse phase column with methanol/water 60/40 as eluent. The product fractions were concentrated to remove methanol and freeze-dried to a white, woolly solid. The products from four separate syntheses were combined to give 3.18 g, 21% of the theoretical yield.
Gasskromatografisk analyse av TMS-derivatene og NMR-spektroskopi viste at produktet besto av en 1:1 blanding av a- og p-anomerene. Gas chromatographic analysis of the TMS derivatives and NMR spectroscopy showed that the product consisted of a 1:1 mixture of the α- and β-anomers.
<1>H og <13>C NMR (DMSO-d6), 8 (ppm) rel. til TMS: 7,52-7,30 (m, 5H, Ar-H I + II), 6,85-6,68 (bred s, 1/2 H, OH-1 II), 6,50-6,35 (bred s, 1/2 H, OH-1 I), 5,61 (s+s, 1H, acetal-H l+ll), 5,23-5,12 (m, 1 H, OH-3 II og H-1 I), 5,12-5,02 (d, 1/2 H, OH-3 II), 4,84-4,74 (dd, 1/2 H, H-1 II), 4,20-4,04 (m, 1H, H-6 l+ll), 3,98-3,74 (m, 1H, H-3 I og H-5 I), 3,74-3,57 (m, 1,5H, H-6' l+ll og H-3 II), 3,42-3,18 (2,5 H, H-4 l+ll og H-5 II og HaO), 2,08-1,88 (m, 1H, H-2 l+ll) og 1,62-1,32 (m, 1H, H-2' l+ll). <1>H and <13>C NMR (DMSO-d6), 8 (ppm) rel. to TMS: 7.52-7.30 (m, 5H, Ar-H I + II), 6.85-6.68 (broad s, 1/2 H, OH-1 II), 6.50-6 .35 (broad s, 1/2 H, OH-1 I), 5.61 (s+s, 1H, acetal-H l+ll), 5.23-5.12 (m, 1 H, OH- 3 II and H-1 I), 5.12-5.02 (d, 1/2 H, OH-3 II), 4.84-4.74 (dd, 1/2 H, H-1 II) , 4.20-4.04 (m, 1H, H-6 1+11), 3.98-3.74 (m, 1H, H-3 I and H-5 I), 3.74-3, 57 (m, 1.5H, H-6' 1+ll and H-3 II), 3.42-3.18 (2.5 H, H-4 1+ll and H-5 II and HaO), 2.08-1.88 (m, 1H, H-2 1+ll) and 1.62-1.32 (m, 1H, H-2' 1+ll).
Forbindelse 8: 2- acetamido- 4, 6- 0- benzvliden- di- 2- deoksv- D- qlukopyranose Compound 8: 2- acetamido- 4, 6- 0- benzvlidene- di- 2- deoxv- D- qlucopyranose
Benzaldehyd-di ble fremstilt og konvertert til benzaldehyddimetylacetal-di som beskrevet i EP 0 283 139 B1. Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.
Benzaldehyddimetylacetal-di (8,7 g, 56,8 mmol), N-acetyl-D-glukosamin (10,0 g, 45,2 mmol), tørr DMF (30 ml) og paratoluensulfonsyre (88 mg, 0,46 mmol) ble blandet under N2 til en hvit suspensjon. Reaksjonsblandingen ble omrørt ved 50°C i 45 minutter, så ble det tilkoblet en vakuumpumpe gjennom en vertikal kondensator og reaksjonen fortsatte i 2 timer ved 55°C/60-70 mbar. Apparaturen ble ombygd for å fjerne DMF ved kortbanedestillasjon og destillasjonen fortsatte ved 55-60°C, maks. vakuum i 1 time til. Destillasjonsresten var en gulhvit, myk fast substans. Benzaldehyde dimethyl acetal-di (8.7 g, 56.8 mmol), N-acetyl-D-glucosamine (10.0 g, 45.2 mmol), dry DMF (30 mL) and paratoluenesulfonic acid (88 mg, 0.46 mmol ) was mixed under N2 to a white suspension. The reaction mixture was stirred at 50°C for 45 minutes, then a vacuum pump was connected through a vertical condenser and the reaction continued for 2 hours at 55°C/60-70 mbar. The apparatus was rebuilt to remove DMF by short path distillation and the distillation continued at 55-60°C, max. vacuum for 1 more hour. The distillation residue was a yellow-white, soft solid.
Det ble laget en løsning ved å blande NaHC03 (150 mg) i 30 ml metanol/vann (3:2) og destillasjonsresten ble nøytralisert ved å tilsette løsningen. Dette resulterte i en kremaktig grøt som ble filtrert, vasket 2-3 ganger med en 1% NaHC03-løsning og flere ganger med eter. Produktet ble funnet å være tilstrekkelig rent ved analyse (GC) og tørket i vakuum. Utbyttet var 8,8 g, 63% av det teoretiske. A solution was made by mixing NaHCO 3 (150 mg) in 30 ml methanol/water (3:2) and the distillation residue was neutralized by adding the solution. This resulted in a creamy slurry which was filtered, washed 2-3 times with a 1% NaHCO 3 solution and several times with ether. The product was found to be sufficiently pure by analysis (GC) and dried in vacuo. The yield was 8.8 g, 63% of the theoretical.
Gasskromatografisk analyse av TMS-derivatene indikerte en 1:1 isomerblanding. NMR-spektroskopi i DMSO-d6-løsning identifiserte produktet som en 3:1 anomerblanding. Gas chromatographic analysis of the TMS derivatives indicated a 1:1 isomeric mixture. NMR spectroscopy in DMSO-d6 solution identified the product as a 3:1 anomeric mixture.
<1>H og <13>C NMR (DMSO-d6), 8 rel. til TMS: 7,83 (s+s, 1H, NH), 7,51-7,28 (m, 6H, Ar-H), 7,0-6,2 (bred s, 1H, OH-1), 5,65-5,05 (bred s, 1 H, OH-3), 4,99 (d, 1H, H-1 I), 4,61 (d, 0,3H, H-1 II), 4,21-4,03, 3,92-3,67 og 3,51-3,22 (m, 6H, H-2, H-3, H-4, H-5 og H-6), og 1,85 (s+s, 3H, CH3); 169,452 (C=0), 137,818, 128,869,128,035 og 126,438 (Ar-C), 100,505 (Acetal-C), 96,056, 91,500, 82,471, 81,505, 70,549, 68,300, 67,961, 67,218, 65,906, 62,123, 58,038, 54,790 (sukker-C) og 23,123 og 22,674 (CH3). <1>H and <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.83 (s+s, 1H, NH), 7.51-7.28 (m, 6H, Ar-H), 7.0-6.2 (broad s, 1H, OH-1) , 5.65-5.05 (broad s, 1 H, OH-3), 4.99 (d, 1H, H-1 I), 4.61 (d, 0.3H, H-1 II), 4.21-4.03, 3.92-3.67 and 3.51-3.22 (m, 6H, H-2, H-3, H-4, H-5 and H-6), and 1.85 (s+s, 3H, CH3); 169,452 (C = 0), 137,818, 128,869,128,035 and 126,438 (AR-C), 100,505 (Acetal-c), 96,056, 91,500, 82,471, 81,505, 70.10541, 68,11, 68,1, 67.91, 67.91, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67.9. -C) and 23.123 and 22.674 (CH 3 ).
Forbindelse 9: 2- acetamido- 2- deoksv- 4. 6- 0-( 3- nitrobenzvliden)- D- glukopvranose Compound 9: 2- acetamido- 2- deoxy- 4. 6- O-( 3- nitrobenzvlidene)- D- glucopvranose
3-nitrobenzaldehyd (100 g, 0,66 mol), metanol (99 g, 3,1 mol), trimetylortoformiat (77,3 g, 0,73 mol) og konsentrert saltsyre (165 ul) ble blandet i en 500 ml trehalset kolbe til en gul grøt som forvandlet seg til en løsning i etter 5 minutter. Reaksjonsblandingen ble refluksert av ~50°C i 15 minutter og avkjølt til 10°C med isvann. Reaksjonen ble stanset ved å tilsette 6,6 ml av en løsning som var laget ved å løse 2,5 g KOH i 16 ml metanol. Omrøringen fortsatte i 15 minutter og reaktoren ble ombygd for kortbane-vakuumdestillasjon. Flyktige substanser (CH3OH + HCOOCH3) ble destillert fra med vannstrålepumpe, destillasjonen ble avbrutt og en vakuumpumpe tilkoblet. Deretter fortsatte destillasjonen og en gul olje ble destillert fra ved 93 - 97,5°C/20 mbar. Ved NMR ble oljen funnet å være 3-nitrobenzaldehyddimetylacetal av høy renhet. Utbyttet var 127 g, 97,6% av det teoretiske. 3-Nitrobenzaldehyde (100 g, 0.66 mol), methanol (99 g, 3.1 mol), trimethyl orthoformate (77.3 g, 0.73 mol), and concentrated hydrochloric acid (165 µl) were mixed in a 500 mL three-necked flask to a yellow mush which turned into a solution in after 5 minutes. The reaction mixture was refluxed at ∼50°C for 15 min and cooled to 10°C with ice water. The reaction was quenched by adding 6.6 ml of a solution made by dissolving 2.5 g of KOH in 16 ml of methanol. Stirring was continued for 15 minutes and the reactor was converted for short path vacuum distillation. Volatile substances (CH3OH + HCOOCH3) were distilled off with a water jet pump, the distillation was interrupted and a vacuum pump connected. Distillation then continued and a yellow oil was distilled from at 93 - 97.5°C/20 mbar. By NMR, the oil was found to be 3-nitrobenzaldehyde dimethyl acetal of high purity. The yield was 127 g, 97.6% of the theoretical.
3-nitrobenzaldehyddimetylacetal (5,5 g, 0,028 mol), N-acetyl-D-glukosamin (5,0 g, 0,023 mol), paratoluensulfonsyre (50 mg, 0,263 mmol) og tørr DMF (15 ml) ble blandet under omrøring ved 50°C til en svakt gul suspensjon. Etter 1/2 time ble det tilkoblet en vakuumpumpe og reaksjonen fortsatte ved 56°C/50 mbar i 11 timer. Reaksjonsblandingen ble inndampet og inndampingsresten fordelt mellom et lite volum svakt alkalisk (NaHC03) vann og kloroform. Vannfasen (som dannet en osteaktig suspensjon) ble ekstrahert to ganger med kloroform og filtrert og vasket flere ganger med vann og eter. Produktet ble tørket i vakuum til et svakt brunlig pulver. Utbyttet var 570 mg, 7% av det teoretiske. 3-Nitrobenzaldehyde dimethyl acetal (5.5 g, 0.028 mol), N-acetyl-D-glucosamine (5.0 g, 0.023 mol), paratoluenesulfonic acid (50 mg, 0.263 mmol) and dry DMF (15 mL) were mixed with stirring at 50°C to a pale yellow suspension. After 1/2 hour a vacuum pump was connected and the reaction continued at 56°C/50 mbar for 11 hours. The reaction mixture was evaporated and the evaporation residue partitioned between a small volume of weakly alkaline (NaHCO 3 ) water and chloroform. The aqueous phase (which formed a cheesy suspension) was extracted twice with chloroform and filtered and washed several times with water and ether. The product was dried in vacuo to a slightly brownish powder. The yield was 570 mg, 7% of the theoretical.
Gasskromatografisk analyse av TMS-derivatene indikerte at produktet besto av to isomerer i et 2:1 forhold. NMR-spektroskopi identifiserte produktet som en blanding av a- og p-anomerer. <1>H og 1<3>C NMR (DMSO-d6), 8 rel. til TMS: 8,4-8,1 (m, 2H, Ar-H), 8,05-7,79 (m, 2H, Ar-H), 7,79-7,60 (t, 1H, NH), 6,80 (d, 1H, OH-1), 5,81 (s+s, 1H, acetal-H), 5,30 og 5,18 (s+s, 1/2 H + 1/2 H, H-1), 4,30-4,10, 3,93-3,3 (m, 6H, H-2 - H-6), og 1,85 (d, 3H, CH3); 169,331 (C=0), 147,490, 139,544, 133,018, 129,862, 123,732, 120,884 (Ar-C), 99,000, 98,806 (acetal-C), 95,924, 91,406, 82,433, 81,454, 70,320, 68,240, 67,907, 67,020, 65,574, 61,833, 57,875, 54,609 (sukker-C) 23,014 og 22,557 (CH3). Gas chromatographic analysis of the TMS derivatives indicated that the product consisted of two isomers in a 2:1 ratio. NMR spectroscopy identified the product as a mixture of α- and β-anomers. <1>H and 1<3>C NMR (DMSO-d6), 8 rel. to TMS: 8.4-8.1 (m, 2H, Ar-H), 8.05-7.79 (m, 2H, Ar-H), 7.79-7.60 (t, 1H, NH ), 6.80 (d, 1H, OH-1), 5.81 (s+s, 1H, acetal-H), 5.30 and 5.18 (s+s, 1/2 H + 1/2 H, H-1), 4.30-4.10, 3.93-3.3 (m, 6H, H-2 - H-6), and 1.85 (d, 3H, CH3); 169,331 (C=0), 147,490, 139,544, 133,018, 129,862, 123,732, 120,884 (Ar-C), 99,000, 98,806 (acetal-C), 95,924, 91,406, 82,433, 81,454, 70,320, 68,240, 67,907, 67,020, 65,574 , 61.833, 57.875, 54.609 (sugar-C) 23.014 and 22.557 (CH3).
Forbindelse 10: 4, 6- 0-( benzvliden- di)- D- qalaktopvranose Compound 10: 4, 6- O-( benzvlidene-di)- D- qalactopvranose
Benzaldehyd-di ble fremstilt og konvertert til benzaldehyddimetylacetal-di som beskrevet i EP 0 283 139 B1. Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.
D(+)-galaktose (15,0 g, 0,0833 mol) og tørr DMF (80 ml) ble omrørt i et destillasjonsapparat ved 45°C. Det ble tilsatt benzaldehyddimetylacetal-di (12,8 g, 0,0836 mol) og paratoluensulfonsyre (0,14 g) og metanol og DMF sakte destillert av i vakuum (vannstråle). Etter 3 timer ble det montert en vakuumpumpe og det resterende DMF ble destillert fra. Porsjoner av destillasjonsresten ble løst i metanol/vann (1:1) som inneholdt NaHC03 (11 mg/ml) og renset på en Lobar C RP-8-kolonne med metanol/vann (1:1) som eluent. Produktfraksjoner fra 7 individuelle synteser ble frysetørket og slått sammen til et hvitt, ullent produkt. Utbyttet var 6,62 g, 30% av det teoretiske. D(+)-galactose (15.0 g, 0.0833 mol) and dry DMF (80 mL) were stirred in a still at 45°C. Benzaldehyde dimethyl acetal-di (12.8 g, 0.0836 mol) and paratoluenesulfonic acid (0.14 g) were added and methanol and DMF slowly distilled off in vacuo (water jet). After 3 hours a vacuum pump was fitted and the remaining DMF was distilled off. Aliquots of the distillation residue were dissolved in methanol/water (1:1) containing NaHCO 3 (11 mg/ml) and purified on a Lobar C RP-8 column with methanol/water (1:1) as eluent. Product fractions from 7 individual syntheses were freeze-dried and pooled into a white, woolly product. The yield was 6.62 g, 30% of the theoretical.
Gasskromatografisk og NMR-analyse viste at produktet besto av en 1:1 anomerblanding. <1>H og 13C NMR (DMSO-d6), 8 rel. til TMS: 7,52-7,30 (m, 5H, Ar-H), 6,62 (0,5H, d, OH-1-P), 6,32 (0.5H, d, OH-1-a), 5,05 (0.5H, t, H-1-a), 4,85+4,69+4,49 (1H+0,5H+0,5H, m+d+d, OH-2+OH-3), 4,35 (0,5H, t, H-1-P), 4,12-3,92+3,81 -3,71 +3,69-3,59+3,49-3,39+3,39-3,28 (3H+1 H+0.5H+1 H+2H, m+m+m+m+m, H-2-H-6+H20); 138,753, 138,690, 128,607, 128,319,127,913 og 126,3 (Ar-C), 99,345 (acetal-C), 97,307 og 93,178 (C-1), 76,738 og 76,158 (C-4), 72,101, 71,605, 68,947, 68,862, 68,486 og 67,730 (C-2, C-3 og C-6) og 65,829 og 62,068 (C-5). Gas chromatographic and NMR analysis showed that the product consisted of a 1:1 anomeric mixture. <1>H and 13C NMR (DMSO-d6), 8 rel. to TMS: 7.52-7.30 (m, 5H, Ar-H), 6.62 (0.5H, d, OH-1-P), 6.32 (0.5H, d, OH-1- a), 5.05 (0.5H, t, H-1-a), 4.85+4.69+4.49 (1H+0.5H+0.5H, m+d+d, OH-2 +OH-3), 4.35 (0.5H, t, H-1-P), 4.12-3.92+3.81 -3.71 +3.69-3.59+3.49 -3.39+3.39-3.28 (3H+1H+0.5H+1H+2H, m+m+m+m+m, H-2-H-6+H2O); 138.753, 138.690, 128.607, 128.319, 127.913 and 126.3 (Ar-C), 99.345 (acetal-C), 97.307 and 93.178 (C-1), 76.738 and 76.158 (C-4), 72.101, 71.68, 68.97, 68.97 68.486 and 67.730 (C-2, C-3 and C-6) and 65.829 and 62.068 (C-5).
Forbindelse 11: 4. 6- 0-( benzvliden- di)- D- mannopvranose Compound 11: 4.6-0-(benzvlidene-di)-D-mannopvranose
Benzaldehyd-di ble fremstilt og konvertert til benzaldehyddimetylacetal-di som beskrevet i EP 0 283 139 B1. Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.
D(+)-mannose (15,0 g, 0,0833 mol) og tørr DMF (70 ml) ble omrørt i en destillasjonsapparata ved 40°C. Benzylidendimetylacetal-di og paratoluensulfonsyre (0,14 g) ble tilsatt og det dannet seg en klar løsning. Det ble tilkoblet en vakuumpumpe og metanol og DMF ble sakte destillert fra ved 70-20 mbar, 45-50°C. Etter 3 timer ble det resterende DMF destillert av ved maks. vakuum, slik at det ble igjen en svakt gul sirupsmasse. D(+)-mannose (15.0 g, 0.0833 mol) and dry DMF (70 mL) were stirred in a still at 40°C. Benzylidenedimethylacetal-di and paratoluenesulfonic acid (0.14 g) were added and a clear solution formed. A vacuum pump was connected and methanol and DMF were slowly distilled from at 70-20 mbar, 45-50°C. After 3 hours, the remaining DMF was distilled off at max. vacuum, so that a faint yellow syrupy mass remained.
Resten ble vasket flere ganger med eter for å fjerne lipofile substanser. Noen porsjoner av råproduktet ble løst i svakt alkalisk (NaHCOa) metanol/vann (3:2) og renset på en Lobar C RP-8-kolonne med metanol/vann (3:2) som eluent. gasskromatografi av TMS-derivatene indikerte at produktet besto av 4 isomerer i forholdet 10:3:1:4. Det ble re-eluert med metanol/vann (1:4) til et hvitt, ullent produkt som besto av bare to isomerer i forholdet 70/30, ifølge gasskromatografisk analyse. Utbyttet var 1,42 g, 6,4% av det teoretiske. The residue was washed several times with ether to remove lipophilic substances. Some portions of the crude product were dissolved in weakly alkaline (NaHCO 3 ) methanol/water (3:2) and purified on a Lobar C RP-8 column with methanol/water (3:2) as eluent. gas chromatography of the TMS derivatives indicated that the product consisted of 4 isomers in the ratio 10:3:1:4. It was re-eluted with methanol/water (1:4) to give a white, woolly product consisting of only two isomers in the ratio 70/30, according to gas chromatographic analysis. The yield was 1.42 g, 6.4% of the theoretical.
På basis av 1H-, <13>C-, COSY-, DEPT- og C-H-korrelasjons-NMR ble den kjemiske strukturen bekreftet og a- og p-anomerene funnet å nå en likevekt ved et 1:8-forhold. On the basis of 1H, <13>C, COSY, DEPT and C-H correlation NMR, the chemical structure was confirmed and the α- and β-anomers were found to reach an equilibrium at a 1:8 ratio.
<1>H og <13>C NMR (DMSO-d6) av den dominerende isomeren, 8 rel. til TMS: 7,5-7,28 (m, 5H, Ar-H), 6,56 (d, 1H, OH-1), 5,0-4,85 (m, 3H, OH-2 og OH-3), 4,10-4,02 (m, 1H, H-6), 3,63-3,40 (m, 5H, H-2, H-3, H-4, H-5 og H-6'); 138,045, 128,825, 128,029, 126,438 (Ar-C), 100,802 (Acetal-C), 95,233 (C-1), 78,967 (C-4), 72,032 (C-3), 68,317 (C-6), 67,252 (C-2), 63,495 (C-5). <1>H and <13>C NMR (DMSO-d6) of the dominant isomer, 8 rel. to TMS: 7.5-7.28 (m, 5H, Ar-H), 6.56 (d, 1H, OH-1), 5.0-4.85 (m, 3H, OH-2 and OH -3), 4.10-4.02 (m, 1H, H-6), 3.63-3.40 (m, 5H, H-2, H-3, H-4, H-5 and H -6'); 138.045, 128.825, 128.029, 126.438 (Ar-C), 100.802 (Acetal-C), 95.233 (C-1), 78.967 (C-4), 72.032 (C-3), 68.317 (C-6), 67.252 ( C-2), 63.495 (C-5).
Forbindelse 12: 2- acetamido- 4, 6- 0- benzvliclen- 2- cleoksv- a- D- qalaktopvranose Compound 12: 2- acetamido- 4, 6- 0- benzvliclen- 2- cleoxv- a- D- qalactopvranose
Benzaldehyddimetylacetal (2,0 ml, 14 mmol) og deretter paratoluensulfonsyre-monohydrat (15 mg) ble tilsatt til en omrørt suspensjon av N-acetyl-D-galaktosamin (1,50 g, 6,77 mmol) i acetonitril (37 ml). Reaksjonsblandingen ble så senket ned i et varmt (60°C) oljebad og omrørt under nitrogen i 3 timer, mens man observerte at det dannet seg et tykt hvitt bunnfall. Reaksjonsblandingen ble så filtrert og det faste stoffet vasket med kald diklormetan (omtrent 2 ml), deretter fortsatte man med sugefiltrering under nitrogenstrøm. Det hvite pulveret ble så plassert i et forhåndsveid glass og satt under vakuum (0,06 mbar) i 72 timer slik at man fikk det rene, ønskede produktet som bare a-isomeren (1,74 g, 83%). Benzaldehyde dimethyl acetal (2.0 mL, 14 mmol) followed by paratoluenesulfonic acid monohydrate (15 mg) was added to a stirred suspension of N-acetyl-D-galactosamine (1.50 g, 6.77 mmol) in acetonitrile (37 mL) . The reaction mixture was then immersed in a hot (60°C) oil bath and stirred under nitrogen for 3 hours, while a thick white precipitate was observed to form. The reaction mixture was then filtered and the solid washed with cold dichloromethane (about 2 mL), then continued with suction filtration under nitrogen flow. The white powder was then placed in a preweighed glass and placed under vacuum (0.06 mbar) for 72 hours to give the pure desired product as only the α-isomer (1.74 g, 83%).
<1>H NMR 8H (300 MHz, d6-DMSO) 1,83 (3H, s, CH3), 3,80-4,17 (6H, m, H-2, H-3, H-4, H-5 og H-6), 4,65 (1H, d, OH-3), 5,06 (1H, t, H-1), 5,59 (1H, s, ArCH), 6,52 (1H, d, OH-1), 7,33-7,55 (5H, m, ArH) og 7,69 (1H, d, NH);13C NMR 8C{<1>H} (75 MHz, d6-DMSO) 23 (CH3), 50, 62, 65, 69 og 76 (C-2, C-3, C-4, C-5, C-6), 91 (C-1), 100 (ArCH), 126, 128,128 og 138 (arom. C) og 170 (C=0). <1>H NMR 8H (300 MHz, d6-DMSO) 1.83 (3H, s, CH3), 3.80-4.17 (6H, m, H-2, H-3, H-4, H -5 and H-6), 4.65 (1H, d, OH-3), 5.06 (1H, t, H-1), 5.59 (1H, s, ArCH), 6.52 (1H , d, OH-1), 7.33-7.55 (5H, m, ArH) and 7.69 (1H, d, NH); 13C NMR 8C{<1>H} (75 MHz, d6-DMSO ) 23 (CH3), 50, 62, 65, 69 and 76 (C-2, C-3, C-4, C-5, C-6), 91 (C-1), 100 (ArCH), 126 , 128,128 and 138 (arom. C) and 170 (C=0).
Forbindelse 13: 4. 6- 0-( 3- nitrobenzen)- D- alukopvranose Compound 13: 4. 6- O-(3- nitrobenzene)- D- alucopvranose
3-nitrobenzaldehyddimetylacetal ble fremstilt som beskrevet for forbindelse 9. 3-Nitrobenzaldehyde dimethyl acetal was prepared as described for compound 9.
3-nitrobenzaldehyddimetylacetal (21,9 g, 0,11 mol), D(+)-glukose (16,0 g, 0,09 mol), paratoluensulfonsyre (100 mg, 0,5 mmol) og tørr DMF (50 ml) ble blandet under N2 og omrørt ved 58°C i 25 minutter. Det ble tilkoblet en vakuumpumpe og metanol og DMF ble sakte destillert av gjennom en avkjølt kolonne ved 55-60°C, 30-40 mbar i løpet av 4 timer og 15 minutter. Apparaturen ble ombygd for å fjerne det meste av DMF ved kortbanedestillasjon og destillasjonen fortsatte i 1,5 time til. Destillasjonsresten var en svakt gul sirupsmasse. 3-nitrobenzaldehyde dimethyl acetal (21.9 g, 0.11 mol), D(+)-glucose (16.0 g, 0.09 mol), paratoluenesulfonic acid (100 mg, 0.5 mmol) and dry DMF (50 mL) was mixed under N2 and stirred at 58°C for 25 min. A vacuum pump was connected and methanol and DMF were slowly distilled off through a cooled column at 55-60°C, 30-40 mbar over 4 hours and 15 minutes. The apparatus was rebuilt to remove most of the DMF by short path distillation and the distillation continued for another 1.5 hours. The distillation residue was a pale yellow syrupy mass.
Sirupsmassen ble løst i svakt alkalisk (NaHC03) metanol/vann 60:40 og renset på en Lobar C RP-8-kolonne med metanol/vann 60:40 som eluent. Produktfraksjonene ble inndampet (for å fjerne metanol), frysetørket og slått sammen til 5 g av et hvitt, ullent fast stoff. Produktet ble renset en gang til med metanol/vann 60:40 som eluent for å få tittelforbindelsen tilstrekkelig ren. Utbyttet var 3,3 g, 12% av det teoretiske. Gasskromatografi indikerte at produktet besto av to isomerer i et 70:30-forhold. The syrup mass was dissolved in weakly alkaline (NaHCO 3 ) methanol/water 60:40 and purified on a Lobar C RP-8 column with methanol/water 60:40 as eluent. The product fractions were evaporated (to remove methanol), freeze-dried and combined to give 5 g of a white, woolly solid. The product was purified once more with methanol/water 60:40 as eluent to obtain the title compound sufficiently pure. The yield was 3.3 g, 12% of the theoretical. Gas chromatography indicated that the product consisted of two isomers in a 70:30 ratio.
<1>H NMR (DMSO-d6), 5 rel. til TMS: 8,33-7,63 (5H, m, Ar-H), 6,89+6,60 (1H, d+d, OH-1-l+ll), 5,78 (1H, s+s, acetal-H-i+ii), 5,34 (0.65H, d, OH-3-II), 5,57+5,71 (1,12H, d+d, OH-2-II + OH-3-I), 4,99 (0.56H, m, H-1-I), 4,88 (0.32H, OH-2-I), 4,49 (0.74H, m, H-1-II), 4,28-4,12 (1H, m, H-6'-l+ll), 3,85-3,53 (2.27H, m, H-3-I, H-5-I og H-6"-l+ll), 3,49-3,32 (2.58H, m, H-2-I, H-3-II, H-4-I+II og H-5-II) og 3,12-2,98 (0.85H, m, H-2-II). <1>H NMR (DMSO-d6), 5 rel. to TMS: 8.33-7.63 (5H, m, Ar-H), 6.89+6.60 (1H, d+d, OH-1-l+ll), 5.78 (1H, s +s, acetal-H-i+ii), 5.34 (0.65H, d, OH-3-II), 5.57+5.71 (1.12H, d+d, OH-2-II + OH-3-I), 4.99 (0.56H, m, H-1-I), 4.88 (0.32H, OH-2-I), 4.49 (0.74H, m, H-1- II), 4.28-4.12 (1H, m, H-6'-1+11), 3.85-3.53 (2.27H, m, H-3-I, H-5-I and H-6"-l+ll), 3.49-3.32 (2.58H, m, H-2-I, H-3-II, H-4-I+II and H-5-II) and 3.12-2.98 (0.85H, m, H-2-II).
Forbindelse 14: 4, 6- 0-( 2- hvdroksvbenzvliden)- D- qlukopvranose Compound 14: 4, 6- 0-( 2- hydroxybenzvlidene)- D- qulkopvranose
Salicylaldehyd (74,4 g, 0,609 mol), metanol (91,5 g, 2,86 mol), trimetylortoformiat (71 g, 0,67 mol) og konsentrert saltsyre (165 blandes i en 500 ml trehalset kolbe. Reaksjonsblandingen omrøres ved romtemperatur i 15 minutter og reflukseres i ytterligere 25 minutter. Etter avkjøling (isvann) tilsettes en alkalisk løsning som lages ved å løse KOH (1,1 g) i metanol (7 ml) og omrøringen fortsetter i 25 minutter. Alle flyktige stoffer fjernes (vannstrålepumpe) og det dannede salicylaldehyddimetylacetalet destilleres i vakuum (vakuumpumpe). D(+)-glukose (8,0 g, 44,4 mmol), tørr DMF (25 ml), salicylaldehyddimetylacetal (8,33 g, 49,5 mmol) og paratoluensulfonsyre blandes under N2 ved 50°C. Apparaturen tilkobles til en vakuumpumpe og fordampingen av metanol starter. Temperaturen opprettholdes ved 55-60°C og vakuumet reguleres gradvis ned for å gjøre reaksjonen fullstendig. Apparaturen ombygges for kortbanedestillasjon for å fjerne DMF. Det dannes en sirupsaktig rest. Salicylaldehyde (74.4 g, 0.609 mol), methanol (91.5 g, 2.86 mol), trimethyl orthoformate (71 g, 0.67 mol) and concentrated hydrochloric acid (165) are mixed in a 500 ml three-necked flask. The reaction mixture is stirred at room temperature for 15 minutes and refluxed for another 25 minutes. After cooling (ice water), an alkaline solution prepared by dissolving KOH (1.1 g) in methanol (7 ml) is added and stirring is continued for 25 minutes. All volatiles are removed ( water jet pump) and the formed salicylaldehyde dimethyl acetal is distilled in vacuo (vacuum pump). D(+)-glucose (8.0 g, 44.4 mmol), dry DMF (25 ml), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol begins. The temperature is maintained at 55-60°C and the vacuum is gradually regulated down to complete the reaction. The apparatus is converted for short path distillation to remove DMF. a syrupy residue is formed.
Resten løses i svakt alkalisk (NaHCOa) metanol/vann og renses på en Lobar RP-8-kolonne med metanol/vann som eluent. Produktfraksjonene frysetørkes og slås sammen. Identiteten bekreftes ved NMR-analyse. The residue is dissolved in weakly alkaline (NaHCOa) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.
Forbindelse 15: 2- deoksv- 4. 6- 0-( 2- hvdroksvbenzvliden)- D- qlukopvranose Salicylaldehyddimetylacetal fremstilles som beskrevet for forbindelse 14. Compound 15: 2-deoxy-4.6-0-(2-hydroxybenzvlidene)-D-qlucopvranose Salicylaldehyde dimethyl acetal is prepared as described for compound 14.
2-deoksy-D-glukose (7,3 g, 44,5 mmol), tørr DMF (25 ml), 2-deoxy-D-glucose (7.3 g, 44.5 mmol), dry DMF (25 mL),
salicylaldehyddimetylacetal (8,33 g, 49,5 mmol) og paratoluensulfonsyre blandes under N2 ved 50°C. Apparaturen kobles til en vakuumpumpe og fordampingen av metanol starter. Temperaturen holdes ved like på 55-60°C og vakuumet reguleres gradvis ned for å drive reaksjonen til ende. Apparaturen ombygges for kortbanedestillasjon for å fjerne DMF. Det dannes en sirupsaktig rest. salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.
Resten løses i svakt alkalisk (NaHCC«3) metanol/vann og renses på en Lobar RP-8-kolonne med metanol/vann som eluent. Produktfraksjonene frysetørkes og slås sammen. Identiteten bekreftes ved NMR-analyse. The residue is dissolved in weakly alkaline (NaHCC«3) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.
Forbindelse 16: 2- acetamido- 2- deoksv- 4. 6- Q-( 2- hvdroksvbenzvliden)-D- glukopvranose Compound 16: 2-acetamido-2-deoxy-4.6-Q-(2-hydroxybenzvlidene)-D- glucopvranose
Salicylaldehyddimetylacetal fremstilles som beskrevet for forbindelse 14. Salicylaldehyde dimethyl acetal is prepared as described for compound 14.
Salicylaldehyddimetylacetal (9,5 g, 56,5 mmol), N-acetyl-D-glukosamin (10,0 g, 45,2 mmol), tørr DMF (30 ml) og paratoluensulfonsyre (88 mg, 0,46 mmol) blandes under N2. Reaksjonsblandingen omrøres ved 50°C ved redusert trykk for å drive reaksjonen til ende. Apparaturen ombygges for å fjerne. DMF ved kortbanedestillasjon og destillasjonen fortsettes ved 55-60°C, maks. vakuum. Mix salicylaldehyde dimethyl acetal (9.5 g, 56.5 mmol), N-acetyl-D-glucosamine (10.0 g, 45.2 mmol), dry DMF (30 mL) and paratoluenesulfonic acid (88 mg, 0.46 mmol) under N2. The reaction mixture is stirred at 50°C under reduced pressure to drive the reaction to completion. The apparatus is rebuilt to remove. DMF by short path distillation and the distillation is continued at 55-60°C, max. vacuum.
Resten nøytraliseres ved å tilsette metanol/vann som inneholder litt NaHC03 og renses på en Lobar RP-8-kolonne med metanol/vann som eluent. Identiteten bekreftes ved NMR-spektroskopi. The residue is neutralized by adding methanol/water containing a little NaHCO 3 and purified on a Lobar RP-8 column with methanol/water as eluent. The identity is confirmed by NMR spectroscopy.
Forbindelse 17: 4, 6- 0-( 2- hvdroksvbenzvliden)- D- qalaktopvranose Compound 17: 4, 6- O-( 2- hydroxybenzvlidene)- D- qalactopvranose
D(+)-gaiaktose (8,0 g, 44,4 mmol), tørr DMF (25 ml), salicylaldehyddimetylacetal (8,33 g, 49,5 mmol) og paratoluensulfonsyre blandes under N2 ved 50°C. Apparaturen kobles til en vakuumpumpe og fordampningen av metanol starter. Temperaturen holdes ved like ved 55-60°C og vakuumet reguleres gradvis ned for å drive reaksjonen til ende. Apparaturen ombygges for kortbanedestillasjon for å fjerne DMF. Det dannes en sirupsaktig rest. D(+)-gaiactose (8.0 g, 44.4 mmol), dry DMF (25 mL), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.
Resten løses i svakt alkalisk (NaHC03) metanol/vann og renses på en Lobar RP-8-kolonne med metanol/vann som eluent. Produktfraksjonene frysetørkes og slås sammen. Identiteten bekreftes ved NMR-analyse. The residue is dissolved in weakly alkaline (NaHCO 3 ) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.
Forbindelse 18: 2- deoksv- 4. 6- 0-( 2- hvdroksvbenzvliden)- D- qalaktopyranose Salicylaldehyddimetylacetal fremstilles som beskrevet for forbindelse 14. Compound 18: 2-deoxy-4.6-0-(2-hydroxybenzvlidene)-D-galactopyranose Salicylaldehyde dimethyl acetal is prepared as described for compound 14.
2-deoksy-D-galaktose (7,3 g, 44,5 mmol), tørr DMF (25 ml), 2-deoxy-D-galactose (7.3 g, 44.5 mmol), dry DMF (25 mL),
salicylaldehyddimetylacetal (8,33 g, 49,5 mmol) og paratoluensulfonsyre blandes under N2 ved 50°C. Apparaturen kobles til en vakuumpumpe og fordampningen av metanol starter. Temperaturen holdes ved like ved 55-60°C og vakuumet reguleres gradvis ned for å drive reaksjonen til ende. Apparaturen ombygges for kortbanedestillasjon for å fjerne DMF. Det dannes en sirupsaktig rest. salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.
Resten løses i svakt alkalisk (NaHCOs) metanol/vann og renses på en Lobar RP-8-kolonne med metanol/vann som eluent. Produktfraksjonene frysetørkes og slås sammen. Identiteten bekreftes ved NMR-analyse. The residue is dissolved in weakly alkaline (NaHCO 3 ) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.
Forbindelse 19: 2- acetamido- 2- deoksv- 4. 6-( 2- hvdroksvbenzvliden)-D- qalaktopvranose Compound 19: 2- acetamido- 2- deoxy- 4. 6-( 2- hydroxybenzvlidene)-D- chalactopvranose
Salicylaldehyddimetylacetal fremstilles som beskrevet for forbindelse 14. Salicylaldehyde dimethyl acetal is prepared as described for compound 14.
N-acetyl-D-galaktosamin (10,0 g, 45,2 mmol), tørr DMF (25 ml), salicylaldehyddimetylacetal (8,33 g, 49,5 mmol) og paratoluensulfonsyre blandes under N2 ved 50°C. Apparaturen kobles til en vakuumpumpe og fordampningen av metanol starter. Temperaturen holdes ved like ved 55-60°C og vakuumet reguleres gradvis ned for å drive reaksjonen til ende. Apparaturen ombygges for kortbanedestillasjon for å fjerne DMF. Det dannes en destillasjonsrest. N-Acetyl-D-galactosamine (10.0 g, 45.2 mmol), dry DMF (25 mL), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A distillation residue is formed.
Det lages en svakt alkalisk løsning ved å blande NaHC03 i metanol/vann og destillasjonsresten nøytraliseres ved å tilsette denne løsningen. Grøten som dannes filtreres og vaskes med 1% NaHC03-løsning og vann. Produktet analyseres ved gasskromatografi og omkrystalliseres hvis nødvendig. Produktet tørkes i vakuum når det er tilstrekkelig rent. A weakly alkaline solution is made by mixing NaHCO3 in methanol/water and the distillation residue is neutralized by adding this solution. The slurry that forms is filtered and washed with 1% NaHCO 3 solution and water. The product is analyzed by gas chromatography and recrystallized if necessary. The product is dried in a vacuum when it is sufficiently clean.
Identiteten bekreftes ved NMR-analyse. The identity is confirmed by NMR analysis.
Forbindelse 20: 4. 6- 2mvdroksvbenzvliden)- D- mannopyranose Salicylaldehyddimetylacetal fremstilles som beskrevet for forbindelse 14. Compound 20: 4. 6- 2-hydroxybenzvlidene)-D-mannopyranose Salicylaldehyde dimethyl acetal is prepared as described for compound 14.
D(+)-mannose (8,0 g, 44,4 mmol), tørr DMF (25 ml), salicylaldehyddimetylacetal (8,33 g, 49,5 mmol) og paratoluensulfonsyre blandes under N2 ved 50°C. Apparaturen kobles til en vakuumpumpe og fordampningen av metanol starter. Temperaturen holdes ved like ved 55-60°C og vakuumet reguleres gradvis ned for å drive reaksjonen til ende. Apparaturen ombygges for kortbanedestillasjon for å fjerne DMF. Det dannes en sirupsaktig rest. D(+)-mannose (8.0 g, 44.4 mmol), dry DMF (25 mL), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.
Resten løses i svakt alkalisk (NaHC03) metanol/vann og renses på en Lobar RP-8-kolonne med metanol/vann som eluent. Produktfraksjonene frysetørkes og slås sammen. Identiteten bekreftes ved NMR-analyse. The residue is dissolved in weakly alkaline (NaHCO 3 ) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.
Biologiske eksperimenter Biological experiments
Eksempel 1 Example 1
Proteinsvntese Protein synthesis
Proteinsyntesehastigheten ble beregnet som tidligere beskrevet (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981). Kort sagt ble celleproteinet merket til metningspunktet ved minst 2 dagers preinkubering med [<14>C]-valin av konstant spesifikk radioaktivitet (0,5 Ci/mol). For å holde den spesifikke radioaktiviteten konstant ble det brukt en høy konsentrasjon av valin i mediet (1,0 mM). Ved denne valinkonsentrasjonen vil fortynningen av [<14>C]valin i det intracellulære og det proteolytisk genererte valinet være neglisjerbar (Ronning, O.W., et al., Exp. Cell Res. 123: 63-72, 1979). Proteinsyntesehastigheten ble beregnet utfra inkorporeringen av [<3>H]valin relativt til den totale [<14>C]-radioaktiviteten i proteinet ved begynnelsen av de forskjellige måleperiodene og uttrykt som et prosenttall pr. time (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981). The rate of protein synthesis was calculated as previously described (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981). Briefly, the cell protein was labeled to saturation point by at least 2 days of preincubation with [<14>C]-valine of constant specific radioactivity (0.5 Ci/mol). To keep the specific radioactivity constant, a high concentration of valine was used in the medium (1.0 mM). At this valine concentration, the dilution of [<14>C]valine in the intracellular and the proteolytically generated valine will be negligible (Ronning, O.W., et al., Exp. Cell Res. 123: 63-72, 1979). The protein synthesis rate was calculated from the incorporation of [<3>H]valine relative to the total [<14>C] radioactivity in the protein at the beginning of the different measurement periods and expressed as a percentage per hour (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981).
Man kan se av fig. 1 at forbindelse 2 fører til sterkere hemming av proteinsyntesen enn Tucaresol. It can be seen from fig. 1 that compound 2 leads to stronger inhibition of protein synthesis than Tucaresol.
Eksempel 2 Example 2
Eksperimenter med forbindelse 2 og forbindelse 5 på NMRI- mus infisert med FRI END ervtroleukemivirus ( FLV) Experiments with compound 2 and compound 5 on NMRI mice infected with FRI END ervtroleukemia virus (FLV)
Virus: Eveline-celler ble tilveiebrakt av prof. Gerhard Hunsman, Munchen. Vi har Virus: Eveline cells were provided by Prof. Gerhard Hunsman, Munich. We have
vist at dette viruset, som opprinnelig ble brukt som en kilde for Friend hjelpervirus, inneholder et defekt virus av samme størrelse som det miltfokusdannende viruset (Spleen Focus Forming Virus - SFFV) som gir erytroleukemi hos NMRI-mus etter 4-8 uker. showed that this virus, which was originally used as a source for Friend helper virus, contains a defective virus of the same size as the Spleen Focus Forming Virus (SFFV) which causes erythroleukemia in NMRI mice after 4-8 weeks.
Mus: NMRI-musene kom fra gamle Bomholt Gård i Danmark, og ble innkjøpt via SIFF. Musene ankom 6. mai og var med i eksperimentet fra 11. mai. De ble infisert intraperitonealt med 50 mikroliter supernatant fra Evelin-kultur. Behandlingen ble startet etter 24 timer. Forbindelse 2 og forbindelse 5 ble løst i steril isotonisk glyserolløsning i en konsentrasjon som tilsvarer 5 mg pr. kg ved tilførsel av 50 mikroliter intraperitonealt. Mice: The NMRI mice came from the old Bomholt Gård in Denmark, and were purchased via SIFF. The mice arrived on 6 May and participated in the experiment from 11 May. They were infected intraperitoneally with 50 microliters of Evelin culture supernatant. The treatment was started after 24 hours. Compound 2 and compound 5 were dissolved in sterile isotonic glycerol solution in a concentration corresponding to 5 mg per kg by administration of 50 microliters intraperitoneally.
Eksperimentet ble satt opp som følger: The experiment was set up as follows:
10 mus uinfisert kontroll 10 mice uninfected control
10 mus infisert kontroll 10 mice infected control
5 mus uinfisert, behandlet med forbindelse 2 5 mice uninfected, treated with compound 2
10 mus infisert, behandlet med forbindelse 2 10 mice infected, treated with compound 2
5 mus uinfisert, behandlet med forbindelse 5 5 mice uninfected, treated with compound 5
10 mus infisert, behandlet med forbindelse 5 10 mice infected, treated with compound 5
Musene fikk intraperitoneale injeksjoner en gang om dagen i 19 dager. Fra 1. juni til 16. juni, da de ble avlivet, fikk de ingen behandlinger. Musene ble avlivet 16. juni. Det ble tatt blodprøver (for senere analyse). Milten ble fjernet og veid (se tabell 1 nedenfor). En bit av milten ble frosset i nitrogen for å lage tynne snitt og en bit ble fiksert i formalin. The mice received intraperitoneal injections once a day for 19 days. From June 1 to June 16, when they were euthanized, they received no treatments. The mice were euthanized on 16 June. Blood samples were taken (for later analysis). The spleen was removed and weighed (see Table 1 below). A piece of the spleen was frozen in nitrogen to make thin sections and a piece was fixed in formalin.
Resultatene vises også i fig. 3. The results are also shown in fig. 3.
Som man kan se er det en signifikant forskjell i miltvekten for de infiserte dyrene sammenliknet med de uinfiserte. Miltvekten for de uinfiserte dyrene som behandles med forbindelse 2 eller forbindelse 5 ligger over vekten for de uinfiserte kontrolldyrene, selv om dette ikke er signifikant. Man ser at infiserte dyr som ble behandlet med forbindelse 2 faktisk har en lavere gjennomsnittlig miltvekt enn de uinfiserte dyrene som ble behandlet på samme måte (her går man ut fra at resultatet skyldes et dyr i kontrollgruppen som hadde en forholdsvis stor milt). As can be seen, there is a significant difference in the spleen weight for the infected animals compared to the uninfected ones. The spleen weight of the uninfected animals treated with compound 2 or compound 5 is above the weight of the uninfected control animals, although this is not significant. It is seen that infected animals that were treated with compound 2 actually have a lower average spleen weight than the uninfected animals that were treated in the same way (here it is assumed that the result is due to an animal in the control group that had a relatively large spleen).
En histologisk undersøkelse avslørte at de uinfiserte kontrolldyrene hadde en normal miltanatomi. Alle dyrene i den infiserte ubehandlede gruppen hadde invasjon av patologiske leukemiceller i den røde pulpa. Miltene fra begge de uinfiserte kontrollgruppene som ble behandlet med h.h.v. forbindelse 2 og 5, har forstørrede kimsentre, noe som tolkes som et uttrykk for immunstimulering. Man finner ikke leukemiske endringer i milten for den infiserte gruppen som ble behandlet med forbindelse 2.1 gruppen som ble behandlet med forbindelse 5 hadde dyret med den største milten (308 g) leukemiske endringer, mens alle i den infiserte kontrollgruppen hadde leukemiske endringer. A histological examination revealed that the uninfected control animals had normal spleen anatomy. All the animals in the infected untreated group had invasion of pathological leukemic cells in the red pulp. The spleens from both uninfected control groups that were treated with h.v. compounds 2 and 5, have enlarged germinal centers, which is interpreted as an expression of immune stimulation. No leukemic changes are found in the spleen of the infected group treated with compound 2.1 the group treated with compound 5, the animal with the largest spleen (308 g) had leukemic changes, while all of the infected control group had leukemic changes.
Resultatene er oppmuntrende tatt i betraktning den aggressive karakteren av FLV hos mus og også når man sammenlikner virkningen av medikamentene med virkningene av azidothymin og andre virusbehandlinger. The results are encouraging considering the aggressive nature of FLV in mice and also when comparing the effects of the drugs with the effects of azidothymine and other viral treatments.
Eksempel 3 Example 3
Formering av perifere mononukleære blodceller Proliferation of peripheral blood mononuclear cells
Oppfinnerne utførte et eksperiment hvor perifere mononukleære blodceller ble utsatt for superantigen sammen med benzaldehyd, deuterert benzaldehyd, forbindelse 2 eller zilascorb(<2>H). Superantigen brukes som en meget aktiv standard for formering av T-celler og presenteres til T-celler ved hjelp av antigenpresenterende celler. The inventors performed an experiment in which peripheral blood mononuclear cells were exposed to superantigen together with benzaldehyde, deuterated benzaldehyde, compound 2 or zilascorb(<2>H). Superantigen is used as a highly active standard for the propagation of T cells and is presented to T cells by means of antigen-presenting cells.
Eksperimentet viste (se figur 2) at ved å tilsette benzaldehyd, deuterert benzaldehyd eller forbindelse 2 økte formeringen av perifere mononukleære blodceller signifikant på en klokkeformet doseavhengig måte, mens det ble funnet en svært liten virkning med zilascorb(<2>H). Det at vi var i stand til å øke formeringssignalet fra superantigenet tyder på at forbindelsene, fungerer ved at de medvirker til å stimulere T-cellene. The experiment showed (see Figure 2) that adding benzaldehyde, deuterated benzaldehyde or compound 2 significantly increased the proliferation of peripheral blood mononuclear cells in a bell-shaped dose-dependent manner, while very little effect was found with zilascorb(<2>H). The fact that we were able to increase the propagation signal from the superantigen suggests that the compounds work by helping to stimulate the T cells.
Konklusjoner Conclusions
Benzaldehydderivatene i henhold til denne oppfinnelsen reagerer til Schiff-baser med visse grupper på celleoverflaten, f.eks. frie aminogrupper. Siden mange celleprosesser, som proteinsyntese, cellesyklus, immunrespons etc, kontrolleres av signaler fra celleoverflaten vil disse bindingene endre oppførselen til cellen. Vi har også vist at benzaldehydkompleksene på celleoverflaten endrer adhesjonskarakteristikaene til cellen. Vi har vist at forbindelsene i henhold til denne oppfinnelsen kan være nyttige i nye behandlinger for å bekjempe virusinfeksjoner og muligens også infeksjoner av andre mikroorganismer. The benzaldehyde derivatives according to this invention react to Schiff bases with certain groups on the cell surface, e.g. free amino groups. Since many cell processes, such as protein synthesis, cell cycle, immune response, etc., are controlled by signals from the cell surface, these bonds will change the behavior of the cell. We have also shown that the benzaldehyde complexes on the cell surface change the adhesion characteristics of the cell. We have shown that the compounds of this invention may be useful in new treatments to combat viral infections and possibly also infections by other microorganisms.
Administrering Administration
De terapeutiske midlene som fremstilles ved foreliggende oppfinnelse kan gis ved antiviral behandling. Disse terapeutiske midlene kan også gis som immunmodulatorer. The therapeutic agents produced by the present invention can be given by antiviral treatment. These therapeutic agents can also be given as immunomodulators.
Til dette formålet kan forbindelsene av formel (I) formuleres på en hvilken som helst egnet måte for å gis til en pasient, enten alene eller tilsatt egnede farmasøytiske bærere eller hjelpestoffer. For this purpose, the compounds of formula (I) may be formulated in any suitable manner for administration to a patient, either alone or with the addition of suitable pharmaceutical carriers or excipients.
Det foretrekkes spesielt at formuleringene for systemisk terapi fremstilles enten som orale preparater eller parenterale formuleringer. It is particularly preferred that the formulations for systemic therapy are prepared either as oral preparations or parenteral formulations.
Egnede enterale preparater vil være tabletter, kapsler, f.eks. bløte eller harde gelatinkapsler, korn eller pulvere, geleer, suspensjoner, løsninger eller stikkpiller. Slike preparater vil fremstilles på kjent måte ved å blande en eller flere av forbindelsene av formel (I) med ikke-giftige, inerte faste eller flytende bærere. Suitable enteral preparations will be tablets, capsules, e.g. soft or hard gelatin capsules, granules or powders, gels, suspensions, solutions or suppositories. Such preparations will be prepared in a known manner by mixing one or more of the compounds of formula (I) with non-toxic, inert solid or liquid carriers.
Egnede parenterale preparater av forbindelsene av formel (I) er injeksjons- eller infusjonsløsninger. Suitable parenteral preparations of the compounds of formula (I) are injection or infusion solutions.
Når de gis utvortes kan forbindelsene av formel (I) formuleres som en oppløsning, salve, krem, sirup, tinktur, spray eller liknende som inneholder forbindelsene av When given externally, the compounds of formula (I) can be formulated as a solution, ointment, cream, syrup, tincture, spray or the like containing the compounds of
i formel (I) med tilsetning av ikke-giftige, inerte faste eller flytende bærere som er vanlige i lokale preparater. Det er spesielt godt egnet å bruke en formulering som beskytter den aktive ingrediensen mot luft, vann eller liknende. in formula (I) with the addition of non-toxic, inert solid or liquid carriers common in topical preparations. It is particularly well suited to use a formulation that protects the active ingredient from air, water or the like.
Preparatene kan inneholde inerte eller farmakodynamisk aktive tilsetninger. F.eks. kan tabletter eller granulater inneholde en serie bindemidler, fyllmaterialer, bærersubstanser og/eller fortynningsmidler. Flytende preparater kan for eksempel foreligge i form av en steril løsning. Kapsler kan inneholde et fyllmateriale eller et fortykningsmiddel i tillegg til den aktive ingrediensen. Preparatet kan dessuten også inneholde smaksforbedrende tilsetninger så vel som slike substanser som vanligvis brukes som holdbarhets-, stabilisator-, emulgeringsmidler eller fuktighetsbevarende midler, salter for å variere det osmotiske trykket, buffere og andre tilsetninger. The preparations may contain inert or pharmacodynamically active additives. E.g. tablets or granules may contain a series of binders, fillers, carrier substances and/or diluents. Liquid preparations can, for example, be available in the form of a sterile solution. Capsules may contain a filler or thickener in addition to the active ingredient. Furthermore, the preparation may also contain flavor-enhancing additives as well as such substances that are usually used as preservatives, stabilizers, emulsifiers or humectants, salts to vary the osmotic pressure, buffers and other additives.
Doseringen kan variere i samsvar med sykdommen, bruksmåten og innføringsveien, så vel som pasientens behov. Generelt vil en daglig dose i en systemisk terapi for en gjennomsnittlig voksen pasient være omtrent 0,01-500 mg/kg kroppsvekt en eller to ganger om dagen, fortrinnsvis 0,5-100 mg/kg kroppsvekt en eller to ganger om dagen og helst 1-20 mg/kg vekt en eller to ganger om dagen. The dosage may vary according to the disease, the method of use and the route of administration, as well as the needs of the patient. In general, a daily dose in a systemic therapy for an average adult patient will be about 0.01-500 mg/kg body weight once or twice a day, preferably 0.5-100 mg/kg body weight once or twice a day and preferably 1-20 mg/kg weight once or twice a day.
Hvis man ønsker det kan det farmasøytiske preparatet av forbindelsen av formel (I) inneholde en antioksidant, f.eks. tokoferol, N-metyl-tokoferamin, butylert hydroksyanisol, askorbinsyre eller butylert hydroksytoluen. If desired, the pharmaceutical preparation of the compound of formula (I) may contain an antioxidant, e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.
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