CN1100302A - Inhibition and treatment of infection by enveloped virus with calix(n)arene compounds - Google Patents

Inhibition and treatment of infection by enveloped virus with calix(n)arene compounds Download PDF

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CN1100302A
CN1100302A CN94101193A CN94101193A CN1100302A CN 1100302 A CN1100302 A CN 1100302A CN 94101193 A CN94101193 A CN 94101193A CN 94101193 A CN94101193 A CN 94101193A CN 1100302 A CN1100302 A CN 1100302A
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virus
hsv
compound
calix
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黄国茂
漆又毛
刘素莹
蔡惠林
陈正
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Genelabs Technologies Inc
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Genelabs Technologies Inc
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    • C07C309/07—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton
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    • C07C309/11—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton containing etherified hydroxy groups bound to the carbon skeleton with the oxygen atom of at least one of the etherified hydroxy groups further bound to a carbon atom of a six-membered aromatic ring
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    • C07C309/41—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
    • C07C309/43—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton having at least one of the sulfo groups bound to a carbon atom of a six-membered aromatic ring being part of a condensed ring system
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    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/57—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing carboxyl groups bound to the carbon skeleton
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    • C07C309/63—Esters of sulfonic acids
    • C07C309/72—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/73—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton to carbon atoms of non-condensed six-membered aromatic rings
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    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/16—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C317/22—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
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    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/105—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic
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    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
    • C07F9/3804—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se) not used, see subgroups
    • C07F9/3834—Aromatic acids (P-C aromatic linkage)
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
    • C07F9/3804—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se) not used, see subgroups
    • C07F9/3882—Arylalkanephosphonic acids

Abstract

A method for inhibiting cell infection by an enveloped virus, by administering to an infection site, a therapeutically effective amount of a calix(n) arene compound derivatized, at its ring positions meta to the bridge attachments to the ring, with polar substituent having a terminal carboxylate, phosphate, or sulfonate groups, including esters and amides which are cleavable (in vivo). The compound may be administered orally, or topically, e.g., for treatment of herpes virus. The invention also includes a method of inhibiting infection by sexually transmitted enveloped viruses, by topically administering to an area of likely sexual contact a composition containing a prophylactically effective amount of a macrocyclic compound such as described above.

Description

Inhibition and treatment of infection by enveloped virus with calix(n)arene compounds
The present invention relates to suppress method, relate in particular to calix (n) arene compounds (Calix(n) the arene Compounds that uses the present invention's definition by the caused cell infection of enveloped virus) suppress the method for its infection; Aspect relevant, the present invention relates to suppress the method for the infection of coated virus propagated through sexual intercourse.
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The challenge that people face is, need develop virus disease effectively treatment and prophylactic agent, do not produce serious adverse so that suppress viral process, and preferably do not produce the resistance of virus.Since virus duplicate composition that need to use host cell, therefore treat viral infection infected host cell is caused death by suppressing duplicating of virus.It is desirable to before the virus attack host cell, should make virus destroyed or lose activity in the host.This can be finished with achievement in various degree by host's immune system, but should mechanism require more early stage immunoreation, and the early immune reaction can be set up by previous infection or vaccination.In addition, many viruses [for example herpes simplex virus (HSV)] can be avoided host's immune system effectively, and the known immune system (Gottlieb) that can damage the host of at least a virus-human immunodeficiency virus (HIV).
Nucleoside analog is the antiviral drugs of at present extensive use, the effect of this types of drug is duplicating of break virus, this can finish via two kinds of approach: 1) suppress needed enzyme in the nucleic acid process, 2) or produce defective viral genome, premature termination for example by causing duplicating.For example acycloguanosine is a purine analogue, can be used for treating multiple virus disease, comprise herpes simplex virus-1(HSV-1) and herpes simplex virus-2(HSV-2), acycloguanosine can suppress duplicating of virus aspect important several, comprise the thymidine kinase and the archaeal dna polymerase that suppress virus, and dna single chain extension (Elion).Ribavirin is another purine analogue, is a kind of medicine that treatment respiratory syncytial virus (RSV) infects that is specifically designed to.This chemical compound can reduce the GTP level of cell, can stop several to rely on the viral process (Smith) of GTP.Azidothymidine AZT (Azidothymidine; AZT) for being widely used in most at present the medicine that treatment HIV infects, azidothymidine AZT is a kind of thymidine analog, and it is effective especially that it is used for anti-human reverse transcript virus.Archaeal dna polymerase (reverse transcriptase) with the dependenc RNA of AZT blocking virus has high affinity, but its also can block human DNA polymerase simultaneously, thereby causes DNA end stopping of chain (Mitsuya).
Other nucleic acid analog comprises: ganciclovir, vidarabine, idoxuridine, trifluorothymidine and phosphine formates (foscarnet) (a kind of inorganic phosphate analog).As mentioned before, all these medicines virus replication all capable of blocking destroys that the host of political party duplicates and/or the ability of dna replication dna process (referring to, Martin for example) and also have simultaneously.
Based on to viral infection and the understanding of duplicating mechanism, derive another kind of selectable drug therapy, this comprises that attempting blocking virus enters host cell, or changes the compound and immunomodulating of protein synthesis, viral DNA/RNA in host's ribosome.Interferon is a kind of glycoprotein, and they have multiple effect (comprise and strengthen certain specific immunoreation and direct antiviral effect).It is more effective when this types of drug is used for prevention infection than being used for the treatment of the infection of having determined; these medicines can cause the reaction that some are bad usually simultaneously, comprise acute serious discomfort, bone marrow depression, virus resistance and cause host immune system that interferon is produced reaction.
Treating with " antisense (anti-sense) " polymer of nucleic acid is a kind of method, and specific in the method viral genome is the target of selecting.This kind therapy provides the means with height discriminative power, and is therefore pre-in respect of lower side effect; Will face following difficulty but it is used for the treatment of, for example, how screen target, how its be introduced in cell, and how accurately judge and suppress the required dose of each strand generation.Can be attached to and can disturb duplicating that the synthetic medicine of host's ribonucleoprotein matter can blocking virus.These medicines comprise toxin ricin, different phytoprotein (for example pokeweed antiviral protein matter, α-Zhou Qujunsu, and other low-molecular-weight chemical compound).The shortcoming of using these medicines is that they lack selectivity.In treatment HIV infects, developed another therapy, be used for reverse transcription disease toxenzyme-reverse transcriptase at uniqueness specially.Non-retrovirus system can't produce or use this kind enzyme, but if there is not the existence of this kind enzyme, HIV virus just can't be duplicated.In some instances, can provide more drug therapy if can further understand in depth to the configuration aspects of virus replication mechanism.Some virus (comprising orthomyxovirus, Paramyxo virus, herpesvirus, togavirus, and retrovirus) structurally contains a peplos that surrounds virus coat and nucleic acid.When host cell suffers infection of coated virus, the plasma membrane of host cell can change, and contains the protein that is partly with viral password so that comprise, when the nucleoprotein core of the virus of assembling in host cell leaves host cell, it is by the plasma membrane peplos of modified, thereby forms peplos.When the host is infected by the virus, because said structure is very unique for host cell, therefore can make a distinction with normal cell, it can be used as another target in the treatment.
The present invention includes the method for treatment by infection of coated virus.This method comprise will the treatment effective dose deutero-calix (n) aromatic hydrocarbons [calix(n) arene] the compounds position that is used to infect, the position is the polar substituent with terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group between the bridged bond that is connected to ring on this chemical compound ring position.
In a general embodiment, calix (n) aromatic hydrocarbons has following general formula:
Figure 941011933_IMG6
N=4～10 wherein, R 1Be OH, O=, alkyl or aryl ether, ester, acid or its mixture,
R 2For having the polar substituent of terminal carboxylic acid derivates (carboxylate), phosphonate derivative (phosphonate), sulfinate derivant (sulfinate) or sulfonic acid (sulfonate) group, comprise that it can dissociated esters and amide-type,
R 4For＞CHR ", 〉=CR " or its mixture, R here " are hydrogen or carboxylic acid derivates group.
R 1Be preferably OH, perhaps R in the partial oxidation form of this chemical compound 1For OH and=coalition of O.
In one specific embodiment, The compounds of this invention has the following formula structure, n=4,6 or 8 wherein,
R 1For OH or=O, or be its mixture,
R 2Define the same,
R 4For＞CH 2Or 〉=CH, or be its mixture.
In a more particular embodiment, R 2Be (CH 2) mR 2', m=1～3 here, R 2' be sulfonic acid group SO 3R or SO 2NRR ', respectively do for oneself hydrogen or low alkyl group of R and R ' here.
In another embodiment, R 2Be (CH 2) mR 2', m=1～3 here, R 2' sulfinate derivant group SO 2R or S(=O) NRR ', respectively do for oneself hydrogen or low alkyl group of R and R ' here.
In yet another embodiment, R 2Be (CH 2) m-R 2', m=0～3 here, R 2' be carboxylic acid derivates group CO 2R or C(O) NRR ', respectively do for oneself hydrogen or low alkyl group of R and R ' here.
In another embodiment, R 2Be (CH 2) m-R 2', m=0～3 here, R 2' be phosphonate derivative group PO(OR) 2, PO(OH) (OR), PO(OR) (NRR ') or PO(NRR ') 2, respectively do for oneself hydrogen or low alkyl group of R and R ' here.
On the other hand, the present invention relates to new calix (n) arene compounds of the above type, and they has the end group of sulfinate derivant, sulfonic acid, phosphonate derivative and carboxylic acid derivates.
In one embodiment, described new chemical compound comprises having above oxidation or calix (n) aromatic hydrocarbons of partial oxidation, wherein R by structure 1For OH ,=O, alkyl or aryl ether, ester, acid or its mixture, at least 1 R in calix (n) aromatic compound 1Group is=O; R 2For having the polar substituent of terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group; And R 4For＞CH 2Or 〉=CH or its mixture.In preferred embodiments, R 1For OH or=O, and in calix (n) aromatic compound at least 1 R 1Group is=O.
In another embodiment, new chemical compound comprises calix (n) aromatic hydrocarbons with above formula, n=4～10 wherein, R 1For OH ,=O, alkyl or aryl ether, ester, acid or its mixture, R 2For having the polar substituent of terminal carboxylic acid derivates or sulfinate derivant group, and R 4For＞CH 2Or 〉=CH or its mixture.
The compounds of this invention can be taken orally and treats human immunodeficiency virus (HIV), breathes syncytial virus (RSV) or herpes simplex virus (HSV-1 or HSV-2).
The compounds of this invention can be breathed syncytial virus through the inhalation treatment, and topical treatment HSV-1 or HSV-2.Other administering mode is just like intravenous administration.
The invention still further relates to and use calix (n) arene compounds and antiviral nucleoside analogue prescription treatment viral infection.Described calix (n) aromatic hydrocarbons and nucleoside analog can be mixed with ointment for topical treatment, for example treatment is because HSV-1 or the caused damage of HSV-2.In addition, The compounds of this invention can be mixed with liquid agent or tablet treatment whole body for oral administration viral infection, or is mixed with pharmaceutical solutions for the whole body administration.
On the other hand, the present invention also comprises the method for the infection of coated virus that suppresses do as one likes friendship propagation.In the method, the macrocyclic compound that prevents to go up effective dose is used for possible property contact site partly.Described macrocyclic compound is made up of the aromatic ring subunit that the bridged bond with connecting ring links to each other, and the result forms the macro ring skeleton of the continuous chain formation of the atom that links to each other, and contains the negative charge substituent group on the non-skeletal atom of aryl subunit.This negative charge substituent group preferably includes sulfonic acid group, sulfinate derivant group, carboxylic acid derivates group or phosphonate derivative group.In one embodiment, described chemical compound comprises the negative charge substituent group of amide or ester-formin, and they can be dissociated into the negative charge form in vivo.
The ring subunit of macrocyclic compound is preferably included in 3 and 6 naphthalene subunits that sulfinate derivant group or sulfonic acid group are arranged, and/or the substituent phenyl subunit of negative charge is arranged at 4, wherein between 5 ring carbon locations of 7 ring carbon locations of naphthalene or 2 ring carbon atom positions of phenyl and adjacent naphthyl or adjacent phenyl, bridged bond is arranged.
In a general embodiment, described macrocyclic compound contains at least four naphthalene subunits, each naphthalene subunit has polar group at 1 and 8, at 3 and 6 sulfinate derivant or sulfonic acid group are arranged, and between 7 ring carbon locations of 2 ring carbon locations and adjacent subunits of a subunit, bridged bond is arranged.Its universal architecture formula of a preferred chemical compound of the type is as follows:
Figure 941011933_IMG7
N=4,6 or 8 wherein,
R 1For OH ,=O, alkyl or aryl ether, ester, acid or its mixture,
R 2Be sulfonic acid group or sulfinate derivant group, R 4For＞CHR ", 〉=CR " or its mixture, R here " are hydrogen or carboxylic acid derivates group.
One than specific embodiment in, R 1For OH ,=O or its mixture, R 2Be sulfonic acid group, R 4For＞CH 2Or 〉=CH or its mixture.
In another general embodiment, described macrocyclic compound contains at least four phenyl subunits, the phenyl subunit has the negative charge substituent group at 4, and between 5 ring carbon locations of 2 of phenyl subunit ring carbon locations and adjacent phenyl subunit bridged bond is arranged.The universal architecture formula of a preferred compound of the type is as follows:
Figure 941011933_IMG8
Wherein n, R 1, R 2And R 4The same.
In a more particular embodiment, R 2Be (CH 2) mR 2', m=0～3 here, R 2' be the sulfonic acid group.
In another more particular embodiment, R 2Be (CH 2) mR 2', m=0～3 here, R 2' be the sulfinate derivant group.
In another more particular embodiment, R 2Be (CH 2) mR 2', m=0～3 here, R 2' be the carboxylic acid derivates group.
In another more particular embodiment, R 2Be (CH 2) mR 2', m=0～3 here, R 2' be the phosphonate derivative group.
The invention still further relates to the method for set of applications compound, said composition contains aforesaid macrocyclic compound and antiviral nucleoside analogs.
The present composition can be between topical inhibition partner the propagation of viral infection.Described compositions can be example gel lubricant, suppository, and said composition contains the carrier that delivers macrocyclic compound, and in addition, the anti-infective preventive means is made on the surface that this chemical compound can also be used for physical barrier type utensil.
On the other hand, the present invention also comprises the lubricationg jelly compositions that is used to suppress the infection of coated virus through spreading through sex intercourse.This gel combination comprises lubricationg jelly carrier and the aforesaid macrocyclic compound that is dissolved in this carrier.
Another aspect the present invention includes the physical barrier type utensil that is used in combination with above-mentioned macrocyclic compound, is used to suppress do as one likes and propagates caused infection of coated virus.In one embodiment, above-mentioned utensil has the lubricationg jelly that contains the above macrocyclic compound.
The compounds of this invention can effectively prevent the enveloped virus that do as one likes is propagated, comprise hepatitis δ virus (HDV), hepatitis b virus (HBV), hepatitis C virus (HCV), human papillomavirus, herpes simplex virus (HSV-1 and HSV-2), HIV-1 and HIV-2, HTLV-I and HTLV-II.
Following relevant of the present invention being described in detail and accompanying drawing of research, above-mentioned purpose and the feature with other of the present invention will become more obvious.
Fig. 1 represents that of the present invention is the general structural formula of the macrocyclic compound of subunit with the naphthalene.
Fig. 2 A and 2B have represented the form of non-oxide (2A) and the partial oxidation (2B) of the listed chemical compound of Fig. 1, n=4 wherein, and subunit is a chromotropic acid.
Fig. 3 A and 3B represent two logical methods of a macrocyclic compound shown in the composite diagram 2A.
Fig. 4 A and 4B represent to have non-oxide (4A) of blended phenyl and sulfonated naphthalene subunit and the macrocyclic compound of partial oxidation (4B).
Fig. 5 represents the sulfonic acid substituent group on the macro ring chromotropic acid is changed into the reaction of glycyl sulfonamide and sulfuryl amine group.
Fig. 6 represents to contain the reaction equation that sulfonic acid group in the macrocyclic compound of chromotropic acid subunit changes into sulphite or sulfinic acid methyl ester or aryl ester.
Fig. 7 represents to be used for the general structural formula of the macrocyclic compound of being made up of phenyl of the present invention.
The non-oxidised form of Fig. 8 presentation graphs 7 structural formulas, n=4 wherein, subunit is a p-sulfonic acid phenol.
Fig. 9 A and 9B represent the logical method of the non-oxide and partial oxidation form of composite diagram 8 chemical compounds.
Figure 10 represents to replace with acetyl group the reaction equation of the ring hydroxyl on Fig. 8 chemical compound.
Figure 11 is illustrated in the reaction that on the macrocyclic compound that phenyl is a subunit sulfonic acid substituent group is changed into the glycyl sulfoamido.
Figure 12 represents to prepare the reaction equation as the listed similar macrocyclic compound of Fig. 8, but this chemical compound has the bridged bond that contains carboxylic acid.
Figure 13 represents to replace with hydroxy-acid group the reaction equation of hydroxyl in Fig. 8 chemical compound.
Figure 14 represents the reaction equation by right-tert-butyl group precursor preparation calix (n) aromatic hydrocarbons.
Figure 15 represents to prepare the reaction equation of calix (n) aromatic hydrocarbons with right-carboxyl substituent.
Figure 16 represents to prepare through the reaction equation of methene key connection carboxyl substituent to calix (n) aromatic hydrocarbons of para-position.
Figure 17 represents to prepare the reaction equation of similar calix (n) aromatic hydrocarbons as shown in figure 16, but wherein carboxyl substituent is to be connected in the para-position through the ethylidene key.
Figure 18 represents that preparation has the reaction equation of the substituent calix of right-phosphonic acids (ester) (n) aromatic hydrocarbons.
Figure 19 represents that preparation connects the reaction equation of phosphonic acids (ester) substituent group to calix (n) aromatic hydrocarbons of para-position (C-4) through methylene.
Figure 20 represents to prepare the reaction equation of right-2-bromoethyl-O-tosyl-calix (n) aromatic hydrocarbons, and right-the 2-bromoethyl-O-tosyl-calix (n) aromatic hydrocarbons can be used as the precursor of preparation other calixs (n) arene derivatives.
Figure 21 represents to use the preparation of preparation bromoethyl-calix shown in Figure 20 (n) aromatic hydrocarbons to have through the reaction equation of ethylidene connection phosphonic acids (ester) base to calix (n) aromatic hydrocarbons of C-4 position.
Figure 22 represents to prepare through the reaction equation of methylene connection sulfonic group to calix (n) arene derivatives of C-4 position.
Figure 23 represents that preparation connects the reaction equation of sulfonic acid (salt) base to calix (n) arene derivatives of C-4 position through ethylidene.
Figure 24 represents to prepare at methylene bridged bond (R among Fig. 7 in order to introduce other substituent groups on the methylene bridged bond 4) on have the reaction equation of calix (n) aromatic hydrocarbons of chlorine atom.
Figure 25 represents to prepare the reaction equation of similar calix (n) aromatic hydrocarbons shown in Figure 12, and initial substance is the precursor of the cyclisation of Figure 24.
Figure 26 represents to prepare the reaction equation that has carboxymethyl to be connected to calix (n) aromatic hydrocarbons (structural formula L VIII) on the bridged bond methylene, and uses the logical method that the organic copper silicate reagent prepares various calixs (n) aromatic hydrocarbons (structural formula VX) that has the R group through selecting on the methylene bridged bond.
Figure 27 represents that preparation is connected with the reaction equation of various calixs (n) aromatic hydrocarbons of 3-sulfonyl propoxyl group on the different rings position of calix (n) aromatic hydrocarbons.
Figure 28 represents to prepare the reaction equation by phenyl and the mutual alternative macrocyclic compound of naphthyl ring.
Figure 29 A and 29B are for cycle compound KY-1(29A) and KY-42(29B) drug dose be the HSV of function viral discharge curve figure.
Figure 30 represents that compound K Y-1 is right 3The bonded inhibitory action of the HSV-1 of H labelling and cell.
Figure 31 represents the inhibition curve chart that HSV-1 virus plaque forms, (1) when virus and (hollow square) before compound K Y-1 contacts, (2) virus and (closed square) after chemical compound contact, (3) viral and chemical compound period of contact (solid garden circle) is cultivated with the Vero cell.
Figure 32 A represents the situation of the proteinic SDS-PAGE autoradiogram of HSV-1 situation: A band for there being mercaptoethanol to exist; The situation that the B band exists for no mercaptoethanol; And the situation of the proteinic SDS-PAGE autoradiogram of HSV-2 situation: C band for there being mercaptoethanol to exist; The situation that the D band exists for no mercaptoethanol; The KY-1 that all has labelled with radioisotope, the E band is the proteinic situation of dye marker.
Figure 32 B represents the SDS-PAGE autoradiogram situation of the KY-1 chemical compound of labelled with radioisotope; A band and B band are this chemical compound and the bonded situation of glycoprotein gd; C band and D band are this chemical compound and the bonded situation of glycoprotein gB; E band and F band are this chemical compound and the bonded situation of glycoprotein gC.
Figure 33 (A-D) is illustrated in rabbit and goes up with eyepiece and apply after the HSV-1, and the effect curve figure of topical application chemical compound Y-1: Figure 33 A represents the effect to epidermis injury; Figure 33 B represents the effect to conjunctivitis; Figure 33 C represents iritic effect; Figure 33 D represents the effect to the substrate disease.
Figure 34 A represents HSV-1(34A respectively with 34B) with HSV-2(34B) the viral decline situation that must measure: the situation the when cell that the circle expression of solid garden is infected contacts separately with the chemical compound Y-1 of increase concentration; Situation when the cell that the circle expression of hollow garden is infected contacts separately with the acycloguanosine that increases concentration; Closed square is represented the situation of concentration (acycloguanosine+25 μ g/ml chemical compound Y-1) when contacting of the cell that infects and increase; Solid Sui Yuan represents the situation of concentration (acycloguanosine+50 μ g/ml chemical compound Y-1) when contacting of the cell that infects and increase.
1. definition
Except as otherwise noted, defined term has following meaning among the present invention.
" enveloped virus " means the virus that contains protein virus coating of surrounding virus coat. Described enveloped virus comprises: orthomyxovirus and paramyxoviridae, herpesviral, togavirus and retrovirus. When cell suffered infection of coated virus, the plasma membrane of host cell can change and contain part with the protein of viral password, when the nuclear of the virus of assembling in host cell During protein core leaving from host cell, the virus protein meeting is by the plasma membrane coating after modifying, thereby the formation peplos.
" enveloped virus that spreads through sex intercourse " is the known or doubtful enveloped virus of transmission through sex that is. Example comprises particularly: δ, B, hepatitis C virus (HDV, HBV, HCV), papillomavirus, herpes simplex virus 1 and 2(HSV-1 and HSV-2), HIV-1(is also referred to as the HTLV-III), HIV-2, HTLV-I and HTLV-II.
" aryl rings " subunit is to contain the monocycle of at least one aromatic rings (6 π (Pi) electronics namely must be arranged to be five yuan or hexatomic ring of armaticity) or the ring structure that condenses. Example comprises benzene, naphthalene and the ring structure that condenses, and for example naphthane, and heterocycle structure comprises the ring structure that condenses, for example quinoline, isoquinolin and indoles.
" macrocyclic compound that is made up of the aromatic ring subunit " is the cyclic compound that becomes loop chain to form by the annular atoms that connects on the aromatic ring subunit.
" calix (n) aromatic hydrocarbons " or " calix aromatic compound " refer to the macrocyclic compound of following skeleton structure:
Wherein n is preferably 4-10, and in preferred embodiments, n is 4,6 or 8.
The compound that the calix of partial oxidation (n) aromatic hydrocarbons refers to have the following formula structure, wherein R1At least one is=O in the group. In calix (n) aromatic hydrocarbons of complete oxidation, All R1Group is=O. The structure example of the calix of partial oxidation (n) aromatic hydrocarbons is seen Fig. 9 B. In addition, because each R1Be=O, should think to be connected in R on the same aromatic ring4Group is 〉=the CR form, i.e. this R4Group is that two keys are connected on this aromatic ring, sees Fig. 9 B.
" bridged bond is connected Preordering and puts " refers to 2 and 6 of ring position on each ring of described compound in the calix aromatic compound.
" non-bridged bond position " refers to 1,3,4 and 5 of ring position on each ring of described compound in the calix aromatic compound.
" bridged bond between position ring position " refers to 4 of ring position on each ring of described compound in the calix aromatic compound.
" polar substituent " refers to that Octanol/water Partition Coefficients is less than 1 radicals R.
" polar substituent that terminal carboxylic acid derivates, phosphonate derivative, sulfonic acid or sulfinate derivant are arranged " refers to the R:-CO of following formula2 -Or R '-CO2 -(carboxylic acid derivates) ,-PO3 -Or R '-PO3 -(phosphonate derivative) ,-SO3 -Or R '-SO3 -(sulfonic acid) ,-SO2 -Or R '-SO2 -(sulfinate derivant), R ' is carboxylic acid derivates, phosphonate derivative or the sulfonic acid group straight chain that 1～4 atom is arranged to the phenyl ring of calix aromatic hydrocarbons of effectively join dependency here, one preferably R ' straight chain be (CH2) m ', here m=1～3.
" carboxylic acid derivates " group comprises hydroxy-acid group-CO2 -, carboxylate and carboxylic acid ester and carboxylic acid amides, it can dissociate in vivo. The ester of carboxylic acid has general formula-CO2-R, R is the alkyl of unsubstituted low alkyl group or replacement here, and carboxylic acid amides has general formula CONRR ', NRR ' is secondary amine or tertiary amine here, i.e. respectively do for oneself hydrogen or low alkyl group or unsubstituted low alkyl group of R and R '. If carboxylate or carboxylic acid amides warp respectively in vivo Serum esterase or amidase hydrolysis, carboxylate or carboxylic acid amides can be dissociated into corresponding hydroxy-acid group so.
" phosphonate derivative " group comprises phosphonyl group-PO3 -2, comprise phosphonate, and the phosphonate ester that can dissociate in vivo and phosphonic amide. Phosphonate ester has general formula-PO3RR ', R and R ' are the low alkyl group of low alkyl group or replacement here, phosphonic amide has general formula PO(OR), (NRR '), PO(NRR ')2, respectively do for oneself hydrogen or low alkyl group of R and R ' here. If through serum phosphatase or phosphamidase hydrolysis, phosphonate ester or phosphonic amide can be dissociated into corresponding phosphonyl group so respectively for phosphonate ester or phosphonic amide in vivo.
" sulfonic acid " group comprises sulfonic acid group-SO3 -, comprise sulfonate, and the sulphonic acid ester that can dissociate in vivo and sulfonamide, sulphonic acid ester has general formula-SO3R, R is the low alkyl group of unsubstituted low alkyl group or replacement here, sulfonamide has general formula SO2NRR ', here respectively do for oneself hydrogen or low alkyl group of R and R '. If through serum esterase or sulfamidase hydrolysis, so, sulphonic acid ester or sulfonamide can be dissociated into corresponding sulfonic acid group respectively for sulphonic acid ester or sulfonamide in vivo.
" sulfinate derivant " group comprises sulfinic acid group-SO3 -, comprise sulfinate, and sulfinic acid ester or the sulfenamide that can dissociate in vivo. The sulfinic acid ester has general formula-SO2R, R is the low alkyl group of unsubstituted low alkyl group or replacement here, sulfenamide has general formula SONRR ', here respectively do for oneself hydrogen or low alkyl group of R and R '. If through serum esterase or sulfamidase hydrolysis, sulfinic acid ester or sulfenamide can be dissociated into corresponding sulfinic acid group so respectively for sulfinic acid ester or sulfenamide in vivo.
Term " sulfonic acid ", " sulfinate derivant ", " phosphonate derivative " and " carboxylic acid derivates " comprise corresponding sour form and any salt that pharmaceutically is suitable for thereof.
" negative electrical charge substituting group " refers to substituting group electronegative under physiological situation. Can provide the group example of negative electrical charge to comprise sulfonic acid group (SO3 -), sulfinate derivant group (SO2 -), phosphonate derivative group (PO3 2), carboxylic acid derivates group (CO2 -). The negative electrical charge substituting group has following formula preferably :-X-SO3 -，-X-SO 2 -、-X-PO 3 -Or-X-CO2 -, X is effectively join dependency carboxylic acid derivates group, phosphonate derivative group, sulfinate derivant group and the sulfonic acid group straight chain that 1～4 atom is arranged to the phenyl ring of calix aromatic hydrocarbons here. One preferably chain be (CH2) m, here m=0-3.
" low alkyl group " refers to contain the alkyl of the straight or branched of 1-5 carbon atom.
" low alkyl group of replacement " refers at its carbon atom one or more substituent low alkyl groups are arranged.
II. preparation aryl subunit macrocyclic compound
This part narration is used for the aryl synthesis of large ring compounds of two kinds of fundamental types of the inventive method. The synthetic method of first type of compound (naphthyl is subunit) is seen following A part, and the synthetic method of second type compound (phenyl is subunit) is seen B and C part. From this three parts route of synthesis, people will understand by mixing subunit (for example naphthyl and phenyl subunit) how to prepare macrocyclic compound. Described synthetic method also can be widely used in by the synthetic macrocyclic compound of heterocycle subunit (particularly with sulfonic acid group or the substituent heterocycle subunit of sulfinate derivant group).
The macrocyclic compound of the naphthalene subunit that A. replaces
Fig. 1 has shown the general structure formula that is used for macrocyclic compound of the present invention that is made up of the naphthalene subunit that replaces. Fig. 2 A and 2B have represented respectively the example of the type compound, and (I) is non-oxide form, and (II) is the form of partial oxidation. This compound is the tetramer of chromotropic acid (1,8-dihydroxy-3,6-disulfonic acid naphthalene) subunit, and it is by methylene or methine (＞CH2Or 〉=CH) bridged bond (R4) connect. As shown in the figure, methylene/methine bridged bond and inner annular atoms (at ring position 1,2,7 and 8) have consisted of A continuous chain is connected with R in 1 and 8 position1=OH or=the O polar group. Non-chain atom on the 3-6 position of ring (being positioned at each substituting group of the upper 3-6 position of ring) is connected with R2=sulfonic acid group or sulfinate derivant group substituting group. By H1And C13The feature of partial oxidation structure has been reasoned out in the research of NMR and mass spectral analysis.
In the narration of the discussion of following A-C part and synthetic route, usually only provide the non-oxide subunit form of compound. Should be realized that, the heating with acid situation under, be exposed in the air after, the partially or completely oxidation of described compound, namely shown in Fig. 2 B, described compound contains one or more R1Ketone group (=O), and encircling and relevant bridged bond (R4) between two keys are arranged. It should further be appreciated that same reaction mechanism also is applicable to the partial oxidation form of compound usually, the structure shown in Fig. 2 B for example, or contain other R1The similar structures of=O group, exception be R1Modification reaction is generally optionally modifies R1-OH group keeps corresponding R1=O group is constant.
To find out that below compound preferably contains wherein R1Be the chromotropic acid derivative of polar substituent, polar substituent has for example OH ,=O, CO2H, or ether, thioether, the alkyl or aryl that ester, thioesters connect, or by the combination of above group, for example wherein the OH group in the partial oxidation structure is replaced by an above-mentioned group.
As previously discussed, R2Be sulfonic acid group or sulfinate derivant group.
R 3Be hydrogen atom, do not have electric charge or electronegative substituting group, will limit by activity hereinafter described. R3Be preferably hydrogen.
Also will find out below, connect the R of chromotropic acid derivative subunit4Bridged bond is preferably＞CHR or 〉=CR form (being illustrated in undersaturated bridged bond in the form of partial oxidation), R is the little group of hydrogen or carbon containing here, for example low alkyl group, alkenyl, ketone, hydroxy-acid group or aryl, described bridged bond also can be-CH2NR′CH 2-form, R ' similarly is the little group of H or carbon containing, for example low alkyl group here.
In addition, bridged bond also can be circulus in macrocyclic compound, comprises the aryl rings structure, the big ring of dimerization as shown in Figure 4, or the similar structures that becomes bridge to form by heterocycle (such as pyrans or furan nucleus).
The number of subunit can be 4(such as Fig. 2 A structure) to 8, big ring contains 4,6 or 8 subunits preferably, in the reaction equation of the following stated, formed macrocyclic compound can comprise the mixture that the subunit by different numbers (n) forms, common structure for example, n=4(4 subunit) structure and the structure that contains in addition 6 or 8 subunits.
Below table 1 listed by R1、R 2、R 3And R4The representative macrocyclic compound that substituting group replaces, they have been synthesized and have tested its antiviral activity. Show the KY on left hurdle and the analog title that the Y number refers to respective compound. For example, R wherein1Be OH, R2Be SO2NH 2、R 3Be H, R4For-CH2-compound be called KY-3. Although in table and unlisted, there are wherein one or more R in the state that these compounds all can partial oxidation1Group is=O, and adjacent bridged bond contains double key carbon and is connected on the ring.
Table 1
KY R 1R 2R 3R 4
KY-1 OH SO 3Na H >CH 2
KY-3 OH SO 2NH 2H >CH 2
KY-42 OH SO 3Na H >CHCO 2H
KY-48 OH SO 3Na H >CHCHOHCH 2OH
KY-85 OH SO 3Na OH >CHC 6H 6
KY-97 OH SO 3Na H >CH 2CH=CH 2
KY-110 OH SO 3Na H >CHO(O)CH 3
KY-121 OH SO 2C 6H 3(OH) 2H >CH 2
KY-123 OH SO 2Na H >CH 2
KY-143 OH SO 3Na OH >CH 2
KY-147 OH SO 2NHCH 3H >CH 2
KY-148 OH SO 2NHEt H >CH 2
KY-151 OCH 3SO 3Na H >CH 2
KY-158 OH SO 2CH 3H >CH 2
KY-171 OH SH H >CH 2
KY-175 OH SO 3CH 3H >CH 2
KY-176 OH SO 2NHC 6H 6H >CH 2
KY-193 OH SO 3Na Br >CHBrCH 2Br
KY-194 OH SO 3Na Br >CH 2
KY-270 OCOCH 3SO 3Na H >CH 2
KY-272 OCOCH 3SO 3Na H >CHCO 2H
KY-276 OCOEt SO 3Na H >CH 2
KY-277 COEtCl SO 3Na H >CH 2
KY-280 OCH 3SO 3Na H >CH 2
KY-281 OCOC 3H 7SO 3Na H >CH 2
KY-284 OCH 3SO 3Na H >CHCO 2H
KY-285 OCOCH 3SO 3Na H >CH 2
KY-288 OCOPr SO 3Na H >CH 2
KY-289 OCOC 4H 9SO 3NH 4H >CH 2
KY-290 OCOBu SO 3Na H >H 2
KY-291 OCOC 5H 11SO 3NH 4H >CH 2
KY-293 OCOCH=CHCH 3SO 3NH 4H >CH 2
KY-294 OCO(CH 2) 6CO 2H SO 3NH 4H >CH 2
KY-307 O(CH 2) 5CO 2H SO 3NH 4H >CH 2
KY-346 OH SO 3Na H -CH 2N(CH 3)CH 2
KY-352 OH SO 3NHC 6H 11O 5H >CH 2
KY-357 OH SO 2NHCH 2CO 2Na H >CH 2
KY-359 OH SO 2NHOH H >CH 2
KY-395 OCH 3SO 3Na H -CH 2N(CH 3)CH 2-
KY-397 OCH 3SO 2NH 2H >CH 2
KY-398 OCH 3SO 2NHCH 2CO 2H H >CH 2
KY-399 OCH 3SO 2NHCH 2CO 2H H -CH 2N(CH 3)CH 2-
Y-20 OH SO 3Na H -CH 2C 4H 2OCH 2-
Y-34 OH SO 3Na H -CH 2C 6H 4CH 2
Y-66 OH SO 3Na H >CHCO 2H
KYY-19 OH SO 2NHCH(CH 2) 2(CO 2H) 2H >CH 2
Fig. 3 A and 3B have illustrated two of preparation macro ring chromotropic acid chemical compound synthetic methods preferably.The cyclisation that the described method of Fig. 3 A relates to chromotropic acid derivant (comprising chromotropic acid itself) and aldehyde (RCHO) forms macrocyclic compound, the tetramer as shown in Figure 2, and wherein the chromotropic acid subunit is to connect by the methylene group that R replaces, i.e. R 4For＞CHR(comprise 〉=CR).Shown in synthetic route provide preparation to have the easy method of the macrocyclic compound of various different methylene bridged bond R groups, this method is to carry out cyclization in the presence of formula RCHO aldehyde.
For example, have in order to prepare-CH 2The macrocyclic compound of-bridged bond as KY-1 chemical compound (IV), makes chromotropic acid (III) and formaldehyde reaction.General reaction condition provides in KY-1 synthetic in embodiment 1A.Equally, KY-42 gets (embodiment 1C) by the Acetic acid,oxo-,monohydrate cyclisation; KY-48 makes in the presence of glyceraldehyde; KY-85 makes in the presence of benzaldehyde; KY-97 makes in the presence of acrylic aldehyde; KY-110 makes in the presence of methylglyoxal.People can understand, various other RCHO aldehyde that have little alkyl, alkenyl, acid and other hydrocarbon R group also all are suitable for.In addition, the R bridged bond can be modified behind cyclization again.For example, KY-193 can make by the bromination of KY-97 chemical compound.
In the method shown in Fig. 3 B, the cyclisation of chromotropic acid derivant (III) is by carrying out result generation-CH with the hexamethylenetetramine reaction 2N(CH 3) CH 2The 3-atom chain bridge of-(V).The cyclization of synthetic KY-346 is seen embodiment 1J.-CH 2N(CH 3) CH 2-bridged bond can be modified by known method behind cyclization, generation-CH 2N(R ') CH 2-the bridged bond that replaces of various N-, R ' is various carbonaceous little groups here.In the structure of partial oxidation, some bridged bonds can be=CHN(R ') CH 2-form.
As previously discussed, chemical compound shown in Fig. 4 A is the macro ring naphthalene of typical band shape bridged bond (is the phenyl bridged bond at this).As described in embodiment 3, in the presence of hydrochloric acid, make chromotropic acid and 1, the acetic acid solution of 2-benzene dimethanol reacts, and obtains above-claimed cpd.Similarly method can be used for for example connection chromotropic acid subunits such as furan, pyrans, pyrroles of other ring bridged bond.Fig. 4 A and 4B represent the non-oxidised form (VI) and the partial oxidation form (VIII) of chemical compound.
The synthetic R that has through selecting of known available two logical methods 1, R 2And R 4Substituent macrocyclic compound.One of them method is, the derivant of chromotropic acid is modified after cyclisation again, and the result is through the product of cyclisation or contain R through selecting 1, R 2And R 4Substituent group perhaps contains the substituent substituent group that can easily change into through selecting.This method is by wherein containing SO 2NH 2R 2Substituent KY-3 synthetic and being described in detail seen embodiment 1B for details.This moment, the chromotropic acid (VIII) through cyclisation at first reacted with chlorosulfonic acid, generated corresponding R 2=SO 2The C1 derivant (IX, Fig. 5).Make the reaction of this macrocyclic compound and ammonia then, generate required R 2=SO 2NH 2Derivant (X, Fig. 5).
Can synthesize KY-357(R with similar method 2=SO 2NHCH 2CO 2H), its method be make last subunit and glycine under the alkaline pH condition, react (XI, Fig. 5).
Fig. 6 (cooperating with Fig. 5) illustrates that the sulfonic acid group with the chromotropic acid of cyclisation changes the method for sulfinate (XII) and sulfinic acid methyl ester (XIV) into.As shown above, the first step of entire reaction course relates to generation corresponding sulphonic acid chloride derivant (IX).Handle with sodium sulfite then, generate corresponding sulfinate XII.In the presence of sodium bicarbonate, react then, obtain corresponding sulfinic acid methyl ester (XIV) with dimethyl sulfate.
Equally, have various different R 1Substituent macrocyclic compound can make by modifying chromotropic acid after cyclisation.When synthesizing KY-151, for example can make (R=OCH 3) chromotropic acid of cyclisation presses the described chromotropic acid dimethyl ether that generates cyclisation under alkali condition with the dimethyl sulfate reaction of embodiment 1F.Equally, at preparation KY-307(R 1=O(CH 2) 5CO 2H) time, can under basic reaction conditions, make the chromotropic acid of cyclisation and 6-bromine acid reaction, at first make the chromotropic acid of cyclisation be transformed into the diether of caproic acid.
As another embodiment, at preparation chemical compound KY-272 and KY-294(R wherein for example 1Be the OCOR form) in, can be described by the synthetic KY-285 of embodiment 1J, the macrocyclic compound that forms by the cyclisation chromotropic acid is descended and formula RCOCl acyl chloride reaction in alkali condition.
In second logical method, by the derivatization reaction of naphthalene subunit, the substituent group through selecting is formed on the subunit naphthalene, make the subunit cyclization then, generate required macro ring.For K-175(R 2=SO 3CH 3) synthetic, by the above chromotropic acid and sulfonic acid chloride are reacted, generate corresponding R 2=SO 2The Cl substituent group.Further with NaOCH 3Reaction generates required R 2Substituent group, reaction be described in detail 1H referring to embodiment.
People understand, the synthetic method that generates substituent macrocyclic compound through selecting comprise at first the chromotropic acid derivatization reaction and cyclization after subsequently the derivatization reaction of subunit.For example at preparation KY-397(R 1=OCH 3, R 2=SO 2NH 2) in, at first at R 1The position makes the reaction of chromotropic acid subunit, generates dimethyl ether derivative as previously discussed.Carry out making described chemical compound at R as previously discussed after the cyclization with formaldehyde 2Derive further in the position, with SO 3Group changes required SO into 2NH 2Substituent group.
The above KY chemical compound can easily change various sulfinate derivants, sulfonic acid, its acid and salt into.According to the method for knowing, salt and sour form are exchanged on general cation exchange resin, replace another kind of cation with a kind of cation.Therefore, for example in the presence of ammonium salt (as ammonium chloride),, can obtain the ammonium salt of several KY chemical compounds shown in the table 1 by the proton cation exchange.In addition, make macrocyclic compound and various metal cation (as the cation of Ca, Ba, Pt, Cu, Bi, Ge, Zn, La, Nd, Ni, Hf or Pd) reaction, can obtain the slaine and the metallo-chelate of described macrocyclic compound, wherein metal carries out chelating at the inboard polarity recess of described chemical compound.
As described in embodiment 1A, 1B, 1C and 1J, studied the physical features of several too cycle compounds that the present invention makes with absorption spectrum and mass spectrum and nuclear magnetic resoance spectrum (NMR).Described chemical compound comprises four poly-macrocyclic compound (as shown in Figure 2), or four combinate form formulas account for main mixture.
B. calix (n) aromatic compound
Fig. 7 represents can be used for this type of calix (n) arene compounds general formula of the inventive method.An instantiation compound of the type is referring to Fig. 8, and it is the tetramer (XV) by the phenol p-sulfonic acid subunit of methylene bridged bond connection.As shown in the figure, the methylene bridged bond forms successive chain with " inboard " annular atoms ( ring position 2,1 and 6), and 1 at ring has R simultaneously 1=OH group.Non-chain atom on 4 atoms of ring (3～5 of ring positions are gone up each substituent group) has R 2=sulfonic acid substituent group.
Fig. 9 A row illustrate the logical method that generates calix (n) arene compounds.Precursor shown in the left side (XVI) is tert-butyl group calix (n) aromatic hydrocarbons, and wherein n is the number of phenol subunit (having para-position tert-butyl group substituent group) in this macrocyclic compound, and bridged bond is a methylene.Having on the tert-butyl group calix aromatic hydrocarbons market of 4,6 and 8 subunits is can be available, and calix (n) aromatic hydrocarbons of more subunits and odd number subunit can make by general purification process.
In the sulfonating reaction shown in Fig. 9 A, the tert-butyl group calix aromatic hydrocarbons of subunit number that usually will be through selecting and concentrated sulphuric acid are in 75～85 ℃ of reactions 4～5 hours, so that in fact fully with the sulfonic group replacement 4-position tert-butyl group.Sulfonating reaction be described in detail 2A referring to embodiment.Said method has been used to prepare the macrocyclic compound of n=4 shown in Figure 8, and have 6 with the relevant macrocyclic compound of 8 phenol subunits.
The similar approach of sulfonation calix aromatic hydrocarbons that is used to prepare 1 OH group of partial oxidation is referring to shown in Fig. 9 B.Use concentrated sulphuric acid in more than 100 ℃ the tert-butyl group calix aromatic hydrocarbons initiation material this moment, is better than 150～170 ℃ most and handles.This reaction is sulfonation subunit ring and the inboard OH group of oxidation partly effectively.Shown in Fig. 9 B, partial oxidation reaction can produce conjugated calix (n) aromatic hydrocarbons structure (XV III), and wherein bridged bond provides non-localized electronics.Above-mentioned conjugated structure is colored, and the visualization of coloured product can be used for monitoring the process of oxidation reaction.This reaction be described in detail 2B referring to embodiment.
People understand that required macrocyclic compound also can directly prepare by purgation: under the condition of suitable formation bridged bond, as by the above-mentioned method for preparing naphthalene subunit macrocyclic compound p-sulfonic acid phenol (or its precursor) being reacted.Be described in detail the reaction that contains the macrocyclic compound of carboxylic acid bridge bond group referring to Figure 12 preparation.In the method, press the described similar condition of embodiment 1C, make the reaction of phenol p-sulfonic acid and Acetic acid,oxo-,monohydrate, generate the cyclization structure shown in the XX II.In embodiment 2F, be described in detail the synthetic of XX II.
According to the logical method shown in the II A of top, calix (n) arene compounds of above generation can be carried out structural modification, so that obtain R through selecting 1Group, modified sulfonyl and/or add R 2Group.R 1And R 2Substituent scope is described identical with above-mentioned part II A in fact.Figure 10,11 and 13 has been described in detail the R that changes the macrocyclic compound that has generated 1Or R 4The various reaction methods of group.In Figure 10, sulfonation structure shown in Figure 8 is handled with acetic anhydride, generate O-acetyl group R 1Group.This reaction be described in detail 2C referring to embodiment.
Embodiment 2G has narrated at R 1The position generates the similar reaction method of tosylate.
Figure 11 has been described in detail the generation sulfonamides, as the logical method of the glycyl sulfonamide (X XI) of Fig. 8 chemical compound.Be similar to the described reaction of Fig. 5, make sulfonated phenyl calix (n) aromatic compound (X VII) and chlorosulfonic acid reaction, generate corresponding sulfonic acid chloride analog (XX).Further reaction of amine (as glycine) with through selecting obtains required sulfonamide.Being described in detail of reaction referring to R among the embodiment 2D 2=SO 2NH 2Chemical compound synthetic, and glycyl sulfonamide compounds synthetic among the embodiment 2E.
Figure 13 represents basic substitution reaction: R 1=OH is by R 1The non-proprietary general synthetic method that=carbon-based group replaces.In embodiment 2H, be described in detail this reaction method: by actor (R 1=OH, R 2=the tert-butyl group, R 4=CH 2, n=4) obtain a kind of intermediate (R 1=CN, R 2=the tert-butyl group, R 4=CH 2, n=4).And then further change, obtain product (R 1=CO 2H, R 2=SO 3H, R 4=CH 2, n=4).
People understand, at R 1The substituent change of position can be carried out on the OH position in the macrocyclic compound (structure shown in Fig. 9 B) of partial oxidation selectively.Promptly to the ring OH group complete=O group that is that special reaction will keep, the result contained=the mixing R of O group 1Group.
R 3Be generally hydrogen, but can be uncharged or electronegative substituent group, be similar to described R with top II A 3Group.
The R that connects chromotropic acid derivant subunit 4Bridged bond preferably＞CHR or 〉=CR, R is hydrogen or the little group that contains carbon low alkyl group, alkenyl, ketone or hydroxy-acid group as previously discussed here, or aryl, perhaps R 4For-CH 2NR ' CH 2-, R ' similarly is hydrogen or little group such as the low alkyl group that contains carbon here.In addition, be similar to dimerization macrocyclic compound shown in Figure 4, the bridged bond in macrocyclic compound can be a ring structure, comprises aromatic ring structure.
As previously discussed, the number of subunit can be from 4(Fig. 8 structure for example) between 8, change, the macrocyclic compound that contains 4,6 and 8 subunits is better.In the described below reaction equation, the macrocyclic compound of generation can comprise the mixture with different subunit number (n) chemical compounds, for example main n=4 structure (4 subunits) and the other mixture that 5～8 subunit structures are formed that contains.
Being synthesized and having tested representative calix (n) arene compounds of its antiviral activity is by R in the following table 2 1, R 2And R 4The chemical compound that replaces.The same with table 1, KY in table 2 left-hand column and Y numeral are meant the similar title of respective compound.The 2nd hurdle of this table is illustrated in R 1The chemical compound position partial oxidation and that saturated and unsaturated bridged bond mesomethylene carbon group is arranged.
Table 2
Compound R 1R 2R 4n
Y-1 OH SO 3-CH 2- 8
KY-226 O/OH SO 3-CH 2/=CH- 8
Y-49 OH SO 3-CH 2- 4
KY-225 O/OH SO 3-CH 2/=CH- 4
Y-77 OH SO 3-CH 26
Y-48 O/OH SO 3-CH 2/=CH- 6
KY-268 O/OH SO 3-CH 2/=CH- 3
KY-269 O/CO 2CH 3SO 3-CH 2/=CH- 4
KY-271 O/CO 2CH 3SO 3-CH 2/=CH- 3
Y-78 O/OH SO 2NH 2-CH 2- 8
Y-100 O/OH SO 2OCH 3-CH 2- 8
By the method for knowing,, or in the presence of suitable salt, react by in acid, reacting according to the above, can be easily change the R moiety combinations thing of chemical compound shown in the table 2 and the above chemical compound into various sulfonic acid or sulfonate.
C. the macrocyclic compound that has sulfonic acid, sulfinate derivant, phosphonate derivative and carboxylic acid derivates group
According to the present invention, it is calix (n) arene compounds that a class can be useful on the chemical compound that suppresses infection of coated virus, wherein connect bridged bond ring position between the position, promptly have substituent R 24 (Fig. 7), replace by the electronegative substituent group that has terminal sulfonic acid, sulfinate derivant, phosphonate derivative or carboxylic acid derivates group.
The method of calix (n) aromatic compound that preparation wherein has the sulfonic acid group at ring 4 bit strips is included in the chemical compound that ring 1 bit strip has different substituents referring to embodiment 2A, 2B and 2C.The chemical compound that has sulfuryl amine group comprises that its end has the chemical compound of carboxyl respectively referring to embodiment 2D and 2E.
Figure 14 represents tert-butyl group calix (n) aromatic hydrocarbons (XX V) is changed into the method for unsubstituted calix (n) aromatic hydrocarbons (XX VI), and compounds X X VI can be used as more following synthetic initial substances.This reaction be described in detail 2J referring to embodiment.
Figure 15 represents to have the method that calix (n) aromatic hydrocarbons (XX VII) to acetyl group changes calix (n) aromatic hydrocarbons (XX VIII) that has carboxyl into.Be described in detail 2K referring to embodiment.
Shown in Figure 16 in order to obtain to carboxy methylation compound (XXX I), with the compounds X X VI that obtains above by with dimethylamine and formaldehyde reaction, make it change (dimethylamino) methyl compound (XX IX) accordingly into.Change this compounds X X IX into corresponding cyano methyl chemical compound (XXX) then, XX heats in acid with this compounds X, changes required carboxy methylation compound (XXX I) into.Be described in detail 2L referring to embodiment.
Figure 17 represents the synthetic method of carboxyethyl calix (n) aromatic hydrocarbons (XXX III).The intermediate X X IX that top (Figure 16) obtained successively with the sodium salt reaction of Mel and diethylmalonate, obtain diethyl malonyl methyl compound (XXX II).In acid, heat, obtain required compound XXX III.Be described in detail 2M referring to embodiment.
Figure 18 represents synthetic method to phosphonic acids (ester) calix (n) aromatic hydrocarbons (XXX VI).In the method, the compounds X X VI that obtains is above carried out iodate, with the reaction of diethyl phosphinate, obtain diethyl phosphonate chemical compound (XXX V) then.In acid, reflux, obtain required compounds X XX VI.Be described in detail 2N referring to embodiment.
Figure 19 represents the synthetic method of (phosphonomethyl) calix (n) aromatic hydrocarbons (XXX IX).As shown in figure 19, the compounds X X VI that obtains is above carried out chloromethylation, get compounds X XX VII, with the triethyl phosphorite reaction, get diethyl phosphonyl ester compounds (XXX VIII) again.In acid, heat, obtain required (phosphonomethyl) chemical compound and be described in detail 2O referring to embodiment.What must understand is that the chloromethyl intermediate also can be used for synthetic sulfonyl methyl calix (n) aromatic hydrocarbons analog.
Figure 20 represents to be used to prepare right-2-bromoethyl chemical compound synthetic of phosphonoethyl or sulfonyl ethyl calix (n) aromatic hydrocarbons, is described in detail the 2P referring to embodiment.With reference to Figure 20, the compounds X X VI that makes is above carried out allylation on phenolic hydroxyl group, obtain compounds X L, heat then, obtain rearrangement product XLI.Carry out tosylation with the hydroxyl on the protection phenyl, obtain compounds X L II, make it change III into again hydroxyethyl derivative XL.Further with triphenylphosphine dibromide reaction, obtain required to bromoethyl compounds X L IV.
With being similar to above-mentioned reaction sequence shown in Figure 19, will be used for synthetic to bromoethyl calix (n) aromatic hydrocarbons XL IV to phosphonoethyl compounds X L VI.Reaction equation shown in Figure 21 be described in detail 2Q referring to embodiment.
As shown in figure 22, more than the intermediate of Ying Yonging also can be used for synthesizing sulfonyl methyl calix (n) aromatic hydrocarbons XL VII to chloromethyl calix (n) aromatic hydrocarbons (compounds X XX VII), is described in detail the 2R referring to embodiment.
Equally, as shown in figure 23, recited above right-2-bromoethyl intermediate X L IV can be used for syntheticly to sulfonyl ethyl calix (n) aromatic hydrocarbons XL IX, is described in detail the 2S referring to embodiment.
People understand that when needs, the method by method shown in Figure 6 and embodiment 1D exemplify can change the sulfonic acid group into the sulfinate derivant group.
Foregoing synthetic method can be used for preparing position (C between bridged bond 4Ring position) the substituent calix of negative charge (n) aromatic compound is arranged, described negative charge substituent group end has sulfonic acid, sulfinate derivant, phosphonate derivative and carboxylic acid derivates group.Listed synthetic method has shown that acidic group is directly connected to ring and upward or by alkyl bond (as methyl key and ethyl key) is connected.Can understand how to prepare by above discussion people by be connected to the acid-based compound on the ring than long alkyl group.As previously discussed, acidic group can also be changed into corresponding salt.
People also can understand the various esters and the amide of the terminal acidic group that how to prepare macrocyclic compound of the present invention in (for example calix aromatic compound).Described derivant can be used as " prodrug ", and wherein ester or amide change corresponding electronegative acidic group into by enzymatic hydrolysis (as passing through esterase) in vivo.
In general, the ester of carboxylic acid and sulfonic acid can esterification routinely make, and wherein for example acid is transformed into acyl chlorides, then with alcohol (as alkylol) reaction.The amide of carboxylic acid or sulfonic acid can and make similarly by the reaction of acyl chlorides and amine (as alkylamine) equally.Ester comprises aryl and low alkyl group (as the normal-butyl alkyl) carbonic ester preferably.Amide comprises the amide of low alkyl group preferably.
Can change phosphonic acids calix (n) aromatic hydrocarbons into corresponding ester or amide according to the phosphonic acids esterification of routine or the method for amidation process equally.Referring to Figure 21, narrate above the method for one of preparation diethyl phosphonic ester.
Except C in calix (n) aromatic hydrocarbon ring 4The position has outside the polar substituent, and the present invention also imagines to make at other ring positions and calix (n) aromatic hydrocarbons that replaces in the bridged bond position of macrocyclic compound and is applied to the inventive method.For example, C 3And/or C 5Ring position can replace with halogen (as F or C1).Relevant as previously discussed several naphthalene nucleus macro ring, at " inboard " ring position (at the C of calix (n) aromatic hydrocarbons 1Ring position) replacement of carrying out and antiviral activity adapt, and same, as previously discussed, the replacement and the antiviral activity that carry out in the bridged bond position of naphthalene nucleus macro ring adapt.
Figure 24 and 25 expressions are connected the method for carboxyl to the methylene bridged bond of calix (n) aromatic hydrocarbons.In the method, will carry out acetylation to the hydroxyl of tert-butyl group calix (n) aromatic hydrocarbons XX V, and on the methylene bridged bond of product L, carry out oxidation, get ketone bridged bond compound L I.Use sodium borohydride reduction, then react, obtain chlorating compound L III on the methylene bridged bond with thionyl chloride.Be described in detail referring to embodiment 2T and 2U.
Referring to Figure 25, the compound L III is carried out cyaniding, then with acid reaction, on the methylene bridged bond, generate carboxyl.Compound L V and aluminum chloride reaction with obtaining make it slough the tert-butyl group, and the result obtains calix (n) the aromatic hydrocarbons LVI of carboxylic acid bridged bond.In addition, as previously discussed, by with sulfuric acid reaction, make compound L V at C 4Ring position carries out sulfonation, and people understand that similarly method can be used for generating accordingly to phosphonic acids or to carboxylic acid calix (n) aromatic hydrocarbons, but bridged bond carboxyl and/or ring hydroxyl begin and need protect.
By method shown in Figure 26, shown in Figure 26 left side with suitable cuprate reagent, use above-claimed cpd L III and can make various bridged bond substituent groups, the reaction shown in Figure 26 the right is represented how calix (n) aromatic hydrocarbons L III to be changed into has carboxymethyl to be connected to chemical compound on the methylene bridged bond.Being described in detail of this reaction referring to embodiment 2W and 2X.Final reacting product (L VIII) can be carried out the para-position sulfonation, perhaps derive from para-position with other acid groups as previously discussed.
Figure 27 represents to relate to calix (n) aromatic hydrocarbons and propane-1, the various derivation reactions of 3-inkstone.As shown in the figure, this reaction can encircle addition alkyl sulfonic acid on the hydroxy position effectively.This method provides the preparation ring to connect another approach of calix (n) aromatic hydrocarbons of sulfonic acid group, and the reaction condition of giving an example is referring to embodiment 2P and 2Z.
At last, Figure 28 represents to prepare the method that contains the mixing macrocyclic compound that replaces phenyl and naphthyl.This macrocyclic compound describes in detail in embodiment 3B.
III. suppress viral infection
This part test contains the inhibitory action of the compositions of macrocyclic compound to various infection of coated virus.Herpesvirus, herpes simplex virus-1(HSV-1) are arranged the enveloped virus of test and herpes simplex virus-2(HSV-2), they are distrand DNA virus (Roizman); Human immunodeficiency virus (HIV) (RNA retrovirus) (Popovic; Barre-Simoussi); And influenza A and B virus and respiratory syncytial virus (RSV), they are RNA viruses (Chanock).
In order to compare, also tested non-enveloped virus through selecting, they comprise adenovirus (distrand DNA virus) (Rowe; Hilleman); And rhinovirus (single-stranded RNA virus) (Dick).The inhibition of viral infection is normally measured with the situation of detectable cytopathic effect in the cultured cell that is suppressed at infection.HSV-1 and the HSV-2 inhibitory action in cultured cell is also with the combining of following inhibition virus and the cell that can infect, and the viral plaque in the cell that infected of inhibition forms and represents.
In addition, as described in embodiment 4, use lineup's cell line, tested the toxicity of many representative aryl macrocyclic compound (comprising chemical compound shown in table 1 and the table 2) pair cell strains in cell culture.Briefly, be that KY-or Y-chemical compound through selecting are added in the cell culture with ultimate density 5,10,25,50 or 100 μ g/ml.3 days after scouring cells to be removing medicine, and with vital staining agent dyeing, measure the percent of dead cell in each culture.IC 50Drug level (promptly being the drug level numerical value that makes cell death 50%), KY-143 and KY-163 are 50 μ g/ml, the IC of the every other KY chemical compound that is tried 50Be 100 μ g/ml or bigger.Molecular weight is that 1404 its drug level of daltonian KY-1 are 100 μ g/ml, is equivalent to about 66 μ M.
A. suppressing HSV infects: naphthalene subunit chemical compound
With the cytopathic effect (CPE) that contains in table 1 and 2 in the HSV infection cell that compositions test that a chemical compound constitutes is suppressed at cultivation.In embodiment 5 reported method, with HSV-1 or HSV-2 vero cells infection, and it is grown in culture medium, significantly as seen until cytopathic effect.Cell forms uniform monolayer fibroblast shape cell under the situation about infecting not having.Infect with HSV, cytopathic effect is characterized in that the cell agglutination and the molten born of the same parents that infected are followed in obviously activity of circle cell in the suspension after 24 hours after 24～72 hours.
In the medicine inhibitory action of embodiment 5 report, cell contact with HSV-1 or HSV-2 virus, the while contacts with aryl macrocyclic compound through selection, and the final drug level of chemical compound is 10 μ g/ml.Check cytopathic effect after 24 hours.Do not observe tangible cytopathic effect yet if drug level is 10 μ g/ml, some chemical compounds repeat test with the drug level of 20 μ g/ml so.
Below table 3 listed 50 naphthylene macrocyclic compound that are used for this test.Symbol "+" in the 2nd hurdle represents that this chemical compound is effective to suppressing cytopathic effect when 10 or 20 μ g/ml.Symbol "-" is illustrated under the 10 or 20 μ g/ml dosage and observes cytopathic effect (CPE).
Table 3
CPE HSV-1 HSV-2
Chemical compound 10,20 μ g/ml IC 50(μ g/ml) IC 50(μ g/ml)
KY-1 + 2.7 1.7
KY-3 + 2.4 2.5
KY-42 + 1 3
KY-48 - N 1N
KY-85 - N N
KY-97 + N N
KY-110 - N N
KY-121 + 1.5 1.8
KY-123 + 1.5 1.5
KY-129 + 1 1
KY-143 - N N
KY-147 - N N
KY-148 - N N
KY-151 + 1.25 1.8
KY-158 - N N
KY-171 + 2.5 3
KY-175 - N N
KY-176 N N N
KY-193 GC N N
KY-194 + 1 1
KY-280 + 2 2
KY-272 + N N
KY-276 + 1.3 1.2
KY-277 + 1 1.2
KY-280 + 1.1 1
KY-281 + 0.5 1.5
KY-284 + 1 1.6
KY-285 + 1 1.5
KY-286 + 2 2
KY-288 + 1.7 2
KY-289 + 2.2 1.7
KY-290 + 1.2 1.3
KY-291 + 1.4 2
KY-293 + 1.9 2.7
KY-294 + 1 2.2
KY-301 + 1 1
KY-307 + .8 2
KY-308 + .9 1.2
KY-345 + 5 6.7
KY-346 + 4.4 6.2
Table 3
CPE HSV-1 HSV-2
Chemical compound 10,20 μ g/ml IC 50(μ g/ml) IC 50(μ g/ml)
KY-352 + 3.4 4.1
KY-357 + 4 3.3
KY-359 + 5.75 4.2
KY-376 + 2.7 1
KY-395 - N 9
Y-4 + 5.5 6.4
Y-14 + 2.5 3.5
Y-20 + 5 3.2
Y-34 + 2.5 2
Y-66 + N N
1N is illustrated in the Cmax of test and does not see inhibition CPE, perhaps Yu Ce IC 50Do not see effective inhibitory action.
As described in embodiment 6, further reduce the activity of its anti-HSV of test check with the chemical compound in the table 3 with plaque.After overnight incubation, make Vero cell and 0.625～10 μ g/mlKY chemical compound serial dilution liquid and HSV-1 or HSV-2 virus contact 2 hours., so that after removing medicine and extracellular virus cell was further cultivated 2 days in washing, dyeing then and calculating plaque forms.The plaque that generates is recorded percent inhibition divided by infected and untreated whole plaques.(represent to measure the concentration (IC that produces 50% plaque minimizing required compound with the contrast percentage rate by the mass action curve that suppresses plaque 50).In table 3 right hand column, listed the IC that infects by HSV-1 and HSV-2 50Value.
Compound structure shown in the reference table 1, following R group can be considered to the activity (not seeing to protect cell to exempt from cytopathic effect) that provides low under 10～20 μ g/ml dosage: big side chain in the methylene bridged bond among KY-48, KY-49 and the KY-110; The R of OH among the KY-143 3Group, R among KY-147 and the KY-148 2The sulfonamide that has nonpolar alkyl on the position; R among KY-158 and the KY-175 2The sulfinic acid ester or the sulphonic acid ester that have nonpolar alkyl on the position; Trimethylamine bridged bond and R among the KY-395 1The substituent combination of methyl ether on the position." GC " symbol of KY-193 means and has formed some giant cells, and this shows partial inhibition.
Check under 10～20 μ g/ml dosage to have the chemical compound that suppresses cytopathic effect (CPE) fully below, following R group is considered to group preferably.
R 1OH is contained in the position, comprises the combination of OH and=O group; Alkyl and aryl ester comprise the combination of described ester and=O; Alkyl ether comprises the combination of described ether and=O.
At R 2Suitable group has sulfonic acid or sulfonate on the position, and sulfinic acid and salt thereof have the polarity amido (as NH 2, NHOH, N-glycoside (KY-352) and amino acids) sulfonamide.
R 3On the position, group is hydrogen or bromine preferably.
Suitable bridge bond group is to replace and unsubstituted methylene, and wherein the R group is not big alkyl, is preferably hydroxy-acid group.
Select the guideline of R group to be further: at least 1 its ED in 2 HSV tests 50The chemical compound of value≤1 μ g/ml should have following 1 R group, it is characterized in that:
The R of chemical compound 1Group is lower alkyl ether or ester, or contain terminal carboxylic acid group normally have most active, especially at other R of chemical compound 1Have=the O moiety combinations on the position.
R 2Group is sulfonic acid or sulfonate or the sulfonamide that has terminal carboxylic acid.This feature shows R 2The acid groups of position helps increasing active.
R 4Bridged bond is methylene or the methylene that has hydroxy-acid group.
In the embodiment 7 described viral inhibition tests, evaluation selected naphthalene subunit chemical compound when the drug level through selecting is up to 10 μ g/ml suppresses the ability that HSV-1 and HSV-2 virus must be measured.Briefly, be that the heLa cells of cultivation is contacted with virus with the KY chemical compound of serial dilution, make its growth 24 hours, freeze/thaw is 3 times then, so that separating viral particles.Make the Vero cell infection, press the plaque number of the viral lysate of embodiment 6 described inspection serial dilutions.With the drug level is function, and with the viral decline drawing that must measure, the curve chart that Figure 29 A and 29B are respectively compound K Y-1 and KY-2 depends on medicine and virus, and the viral decline that must measure relevant with dosage is between about 3～5 orders of magnitude.Suppress the viral degree that must measure normally HSV-1 greater than HSV-2.Can be observed similar result with several other KY chemical compounds.
B. suppress the HSV activity: calix (n) arene compounds
Several calixs (n) arene compounds listed with above table 2 carries out the active research of similar anti-HSV, the results are shown in Table 4.By the 2nd hurdle of table 4 as can be seen, the chemical compound in 10～20 all tests of μ g/ml concentration can suppress CPE.Reduce test-compound in the test at plaque and the inhibitory action of HSV-1 and HSV-2 is listed in (IC in the column on the right of this table 50Represent with μ g/ml).
Table 4
CPE HSV-1 HSV-2
Chemical compound 10,20 μ g/ml IC 50IC 50
(μg/ml) (μg/ml)
Y-1 + 7 7.2
KY-226 + 1.6 3
KY-49 + 5 10
KY-225 + 1.8 1.8
KY-77 + 8 11
Y-48 + 1.5 1
KY-268 + 4.4 1.5
KY-269 + 2.4 2.3
KY-271 + 4.2 2.4
The high activity of observed benzene subunit chemical compound and the high activity of naphthalene subunit chemical compound are more or less the same for example poor about 1～3 μ g/ml IC 50Value.
Y-48(n=6) and the R of partial oxidation Y-225(n=4) all arranged the most activated chemical compound Y-226(n=8), 1The OH/=O group, the relative non-oxide congener of the chemical compound of various piece oxidation is compared higher activity in fact.
Specific activity is slightly lower mutually for the n=3 compound K Y-268 of partial oxidation and its n=4,6 and 8 congener.In non-oxide chemical compound, specific activity is slightly higher mutually with the n=6 chemical compound for its corresponding n=4 of n=8 chemical compound Y-1.
At R 1Add acetyl group on the position, what the activity of the chemical compound of partial oxidation almost do not produce and changes, and this is consistent with the result that naphthalene subunit chemical compound obtains, at R 1The activity that the adding Arrcostab obtains on the position is equally matched with the analog of partial oxidation.
As in benzene subunit chemical compound, selecting the active general guide principle of optimization compound, use the above identical rule of discussing usually.Therefore, work as R 2Group is during with electronegative group (as sulfonic acid) terminating, and is so pre-in respect of the highest activity.More in general, the present invention's imagination will be with R 2Group is that calix (n) arene compounds of the polar substituent of sulfonic acid, phosphonic acids or hydroxy-acid group (comprise the ester or the amide of described acid, they can be dissociated into corresponding acid by hydrolysis in vivo) terminating is used for the treatment of infection of coated virus.To being used to suppress the infection of the enveloped virus that do as one likes propagates, the present invention imagines and uses its R 2Group is the electronegative substituent macrocyclic compound with sulfonic acid, sulfinate derivant, phosphonate derivative or the terminating of carboxylic acid derivates group.
Described chemical compound also comprises the electronegative ester or the amide that can be dissociated into acid groups in vivo by hydrolysis.The ester of various sulfonic acid, phosphonic acids and carboxylic acid and amide have shown in vivo and have been dissociated into corresponding acid (Svensson, 1988,1991 through hydrolysis; Stella; Bundgaard), described ester and amide comprise the ester and the amide of low alkyl group.The method of the various instantiation compounds of preparation the type is narrated in above-mentioned part II.
As with as described in top II B and the II C, have terminal acidic group (maybe can be dissociated into the group of terminal acidic group) calix (n) arene compounds and can put at other ring positions and bridge location and replace.One preferably embodiment be R 1Substituent group is the OH group, if perhaps this chemical compound is a partial oxidation, and R so 1Substituent group is OH and the combination of=OH group.
C. the comparison of anti--HSV chemical compound
KY-1 is compared the inhibitory action of persister HSV-1 and HSV-2 and the antiviral agent of several HSV of being used for the treatment of infection.The chemical compound that is put to the test is nucleoside analog acycloguanosine (ACV), ganciclovir(DHPG), Phosphonium formic acid (PFA) and phosphoryl methoxy base ethyl adenine (PMEA).Measure the viral inhibition that must measure as stated above: wild type or persister HSV-1 or HSV-2 with being connected of antiviral compound infect hela cell in the presence of the diluent through what select, and use the hela cell lysate vero cells infection of serial dilution as previously discussed.Inhibiting being described in detail referring to embodiment 8.
Table 5 has been listed ID 90Concentration (this concentration suppresses 90% virus and must measure).KOS(HSV-1) and 333(HSV-2) be wild-type virus; KOS(PMEA) and KOS(PFA) ' be drug resistance HSV-1 strain with the sudden change of DNA polymerase.333(DHPG) strain is the drug resistance HSV-2 strain with thymidine enzyme mutant.Except DHPG and PMEA as persister HSV-2 inhibitor as persister HSV-1 inhibitor, when measuring with the viral drug level that must measure of needs inhibition, compare with wild type strain HSV-1 or HSV-2, all nucleoside analogs to the activity of persister at least about low 20 times.On the contrary, to persister HSV-1 and HSV-2, the aryl macrocyclic compound demonstrates with anti-wild type strain same in fact effectively active.
Table 5
Medicine (the ID of test 90) #
Strain/medicine KY-1 ACV DHPG PFA PMEA
The virus thing is selected (uM) (uM) (uM) (uM) of mutational site (ug/ml)
HSV-1 KOS None 1.9 14 2 180 100
KOS(PMEA)' DNA pol 2.6 380 NT 3000 >2000
KOS(PFA)' DNA pol 4.3 100 1 >1000 >1000
HSV-2 333 None 3.2 ≈10 2 150 155
333(DHPG) TK 3.7 >100 215 NT 120
The data of table 5 show, under the effectively similar drug level of anti-wild-type virus, aryl macrocyclic compound overriding resistance strain HSV is effective, on the contrary, except DHPG as HSV-1 strain inhibitor, 2 persisters all demonstrate significant resistance to ACV, DHPG, PFA and PMEA, and this needs the high ID of several times by suppressing virus 90Drug level is confirmed.
D. suppress RSV and influenza A virus infectivity
With the test of the representative macrocyclic compound of table 1 through cultivating behind influenza A virus (strain of A/ Taiwan) or the RSV viral infection MDCK or the HEp2 cell in suppress cytopathic effect.In suppressing the infective method of influenza A virus, with this viral infection mdck cell, and this cell is grown in culture medium, until obviously seeing cytopathic effect.Do not having under the situation about infecting, cell forms the fibroblast-like cellular layer of uniform monolayer.Use viral infection, cytopathic effect is characterised in that observes cell agglutination.As described in embodiment 9 and 11, for each test-compound, with being added in the cultured cells when the viral infection of the drug level of 0.1,1,10,25 and 100 μ g/ml.After 24 hours,, measure the cell agglutination percentage rate according to the percentage ratio of cell granulations coagulation cell total in certain area of visual field.With the drug level is function, draws with suppressing coagulation, lowers 50% effective dose in the coagulation cell percentage ratio of measuring with respect to contrast (not treated with medicaments) so that measure.The ED that records 50Be worth face table 6 as follows.
Be determined at the ED that suppresses RSV cytopathic effect (cell agglutination) in the HEp2 cell with similar method 50, it the results are shown in Table 6.Be described in detail referring to embodiment 9.
Table 6
ED 50(μg/ml)
Chemical compound influenza A RSV
(Taiwan)
KY-1 >188 0.19
KY-3 6 0.75
KY-42 94 1.50
KY-47 94 >250
KY-85 >250 1
KY-97 5 0.5
KY-110 >250 4
KY-123 31.3 1
KY-151 >94 1.5
KY-193 5.0 0.8
KY-194 7.9 0.5
In general, compare with influenza A/ Taiwan virus, RSV is more responsive significantly for the inhibitory action of chemical compound.At R 2Sulfonamide (the SO that has polarity amine on the position 2NH 2), and bridge bond group through selecting is arranged, have the highest IAV activity.Except that compound K Y-47, all chemical compounds all have higher anti-RSV activity.
E. suppress the HIV infectivity: naphthalene subunit chemical compound
It is described to press embodiment 12, tests it with the representative macrocyclic compound of table 1 and suppresses two HTLV-III strain (HTLV-III BWith the strain of RF-II) one of the cytopathic effect of the cell that infects.Briefly be to use the HTLV-III 8Or the long-term cell that infects of RF-II HIV strain cultivates in the presence of the KY chemical compound through selecting of serial dilution, and then with indicator cell co-cultivation.The situation that syncytial virus generates is examined under a microscope record.Two HIV strains are produced valid density (μ the g/ml) (ED that suppresses the syncytial virus generation fully 100) list in table 7." N " is not meant and carries out this virus test with this chemical compound.
Table 7
Suppressing syncytium forms
HIV-HXB HIV-RF-II
Compd E D 100ED 100
KY-1 8 N
KY-3 16 N
KY-42 8 N
KY-48 250 N
KY-85 32 N
KY-97 32 N
KY-110 63 N
KY-121 16 16
KY-123 16 16
KY-129 16 8
KY-143 250 125
KY-147 250 250
KY-148 250 N
KY-151 32 125
KY-158 500 7500
KY-171 125 250
KY-175 63 250
KY-176 125 250
KY-193 63 500
KY-194 63 125
KY-270 16 32
KY-272 63 250
KY-276 16 32
KY-277 16 32
KY-280 16 32
KY-281 16 32
KY-284 16 32
KY-285 16 32
KY-286 16 32
KY-288 8 16
KY-289 16 32
KY-290 16 32
KY-291 16 32
KY-293 16 63
KY-294 16 16
KY-301 8 8
KY-307 8 32
KY-308 8 32
KY-345 63 125
KY-346 16 32
Table 7
Suppressing syncytium forms
HIV-HXB HIV-RF-II
Compd E D 100ED 100
KY-352 32 125
KY-357 32 63
KY-359 32 63
KY-376 8 16
KY-395
Y-4 8 125
Y-14 16 32
Y-20 4 16
Y-34 N N
Y-66 N N
From The above results as can be seen, general relation is arranged between the antiviral activity to above-mentioned two strain virus, promptly to the HTLV-III BThe compounds effective of strain is also the most effective to the RF-11 strain.
The structure of chemical compound in the reference table 1, following R group are considered to provide inferior suitable activity (to the ED of two strain virus 50Value 〉=63 μ g/ml): the big side chain of methylene bridged bond among the KY-48; The methyl ketone group of bridged bond among the KY-110, R among the KY-143 3Hydroxyl; R among KY-147 and the KY-148 2The sulfonamide that has nonpolar alkyl on the position; R among KY-158 and the KY-175 2The sulfinic acid ester or the sulphonic acid ester that have nonpolar alkyl on the position; R among the KY-272 1The combination of methyl ester and acetyl group bridged bond on the position.It is identical that These characteristics reduces active factor with anti-HSV viral infection in fact, promptly shows under 10～20 μ g/ml dosage CPE unrestraint effect.
Equally, the above-mentioned active factor of high anti-HSV that provides is identical with giving the most highly active factor of anti-HIV infectivity usually.These factors comprise: R 1Group on the position is OH, comprise OH and=combination (partial oxidation) of O group; Alkyl and aryl ester comprise the combination of this ester and=O; Alkyl ether comprises the combination of this ether and=O.
R 2On the position preferably group be sulfonic acid or sulfonate, sulfinic acid and its salt and have the polarity amido (as NH 2, NHOH, N-glycoside (KY-352) and aminoacid) and the sulfonamide of sulfonic acid.Especially sulfonic acid, sulfonate and sulfonamide with terminal carboxyl group have very high activity.
R 3Suitable group is a hydrogen on the position, has the activity that OH and Br then give reduction.
Bridged bond preferably replaces and unsubstituted methylene, if bridged bond be＞CHR or 〉=CHR, the R group should not be big alkyl so.
Select the further guideline of R base to be, at least 1 ED during chemical compound is tested two HSV 50Value≤1 μ g/ml, this chemical compound has 1 following radicals, and this group is characterised in that:
R 1Group is lower alkyl ether or ester, perhaps contains the chemical compound of a terminal carboxyl group, and maximum activity is arranged usually, particularly at other R of chemical compound 1Have and=O moiety combinations on the position.
R 2Group is sulfonic acid or sulfonate or the sulfonamide that has terminal carboxylic acid.These characteristics show R 2The acidic group of position helps increasing active.
R 4Bridged bond is methylene or the methylene that has hydroxy-acid group.
The above-mentioned group of R preferably provides to instruct selects R 1～R 4R group on the position is so that the effect of preferred compound.
F. suppress the HIV infectivity: calix (n) aromatic compound
As embodiment 12 with as described in the top, the compound test representative with table 2 suppresses with two kinds of HTLV-III strain (HTLV-III BWith the RF-II) in cytopathic effect in a kind of cell of infection.The IC that HXB and the RS-11 strain of HIV recorded 50Value is listed in following table 8, and unit is μ g/ml.
Table 8
HIV-HXB HIV-RF-11
Compound I C 50IC 50
Y-1 16 N
KY-226 16 250
Y-49 N N
KY-225 32 125
Y-77 N N
Y-48 N N
KY-268 32 32
KY-269 32 32
KY-271 32 63
Interesting is the same analog of Y-1 chemical compound and the corresponding partial oxidation of KY-226() anti-HXB strain has similar activity, but the anti-HSV virus of KY-226 has obviously higher activity.The every other chemical compound that is tried all has the R of partial oxidation 1=O group, and all chemical compounds all have similar activity.
IV. about the characteristic of enveloped virus
This part research suppresses the characteristic of enveloped virus method.The narration of following A part shows, the macrocyclic compound that is used for this method is by working at least in part in conjunction with virus envelope protein selectively, and should combination stops virally attached on the cell that can infect, so suppresses viral infection.Described research is in U.S. Patent Application Serial 647, the 720(date of application 1991,1,29) be described in detail in (United States Patent (USP) 5,196,452 at present), below the B part Study inhibitory action of macrocyclic compound to non-enveloped virus.
A. the mechanism that suppresses of viral infection
First aspect, the Research on ability that stops HSV to be attached to the macrocyclic compound on the cell that can infect sees embodiment 14 for details.Briefly, make the HSV-1 of Vero cell and labelled with radioisotope or HSV-2 under the situation that does not have the KY chemical compound, perhaps contact under the situation that 10 μ g/ml KY-1 chemical compounds exist, as many as was measured viral combination in 4 hours after contact virus.Figure 30 be illustrated in 4 hours culture period virus (labelled with radioisotope) with the bonded curve chart of cell.Under the situation that does not have medicine to exist, bonded viral load stably increases in 2 hours, increases a little at 2-4 hour.On the contrary, under the situation that has medicine to exist, virus and cell combine with 1/2 hour be summit, estimation may effectively stop in this time and virally reached balance with combining of cell.
Second aspect, research is described in detail referring to embodiment 15 to the effect of drug compound before with the HSV-1 infection cell, between infection period or after infecting.In above-mentioned research, make that a concentration chemical compound contacts in the KY-2 compound concentration of cell and a series of increases, measure the degree that infects by the number of after infecting 24 hours, investigating plaque.Before the cell infection between (solid grid), infection period (solid garden) and infect back (hollow grid) treated with medicaments after plaque form situation about reducing and represent with the percentage ratio of matched group, referring to Figure 31.With viral period of contact cell treated with medicaments the time, find out viral inhibition fairly obviously, this show viral inhibition appear at virus combine and enter with permissive cell permissive cell during.
The third aspect, the HSV-1 virus suspension that makes purification was cultivated 1 hour with KY-1 or its sodium salt or contrast solution, and being diluted to sequence drug level then is 10 1～10 -4μ g/ml is described in detail referring to embodiment 16.The viral suspension that adds serial dilution carries out the plaque numeration, measures in duplicate, referring to table 9.Symbol " * " in the table to such an extent as to the expression plaque can not count too much.The result of this research shows, the inhibitory action that the KY chemical compound infects HSV is because (to small part because) medicine combines with HSV is particulate.In addition, be 10 at final drug level -2～10 -4It is much lower that this concentration ratio of μ g/ml(suppresses the required concentration of HSV in the Vero cell culture) situation under can find out viral inhibition completely.Therefore can conclusion, medicine in conjunction with/virally inactivated in fact be irreversible, promptly the dilution for many times effect is irreversible.
Table 9
Plaque number after 10 times of dilutions of KY sequence
The initial virus of chemical compound adds
Amount (PFU/ cell) 1 10 110 210 310 410 5
Contrast 0.3 XX XX XX 50,41 9,40,0
(culture medium is only arranged) 3 XX XX XX XX 38,49 8,4
KY 1 0.3 3,2 0,0 0,0 0,0 0,0 0,0
(10μg/ml) 3 2,2 2,1 17,16 5,2 0,0 0,0
KY 217 0.3 2,8 3,3 0,0 0,0 0,0 0,0
(10μg/ml) 3 X,X X, X 6,0 5,0 0,0 0,0
Fourth aspect, the KY-1 chemical compound of inspection labelled with radioisotope and combine (embodiment 17) of HSV-1 and HSV-2 virus protein.After the chemical compound combination, with SDS-PAGE (SDS-PAGE) fractionated virus protein, and the gel spectrogram is by radiating the photography tracing automatically.Among Figure 32 A, A band and B band is to have mercaptoethanol to have (A band) and do not having mercaptoethanol to have under (B band) the proteic automatic radiation of the HSV-1 bands of a spectrum of taking pictures among the figure, among the figure C be with and D to be be spectrogram like the HSV-2 protide.As shown in the figure, the bands of a spectrum on the right contain the molecular weight sign.As measuring from SDS-PAGE, molecular weight is arranged is 45,66 and about 130 kilodaltons to the main bands of a spectrum of bonded medicine among the HSV-1.The main bands of a spectrum of bonded medicine have similar molecular weight in HSV-2.The bonded main bands of a spectrum of KY shown in Figure 32 B are equivalent to the molecular weight of HSV glycoprotein gd, gB and gC.
B. to the effect of non-enveloped virus
The KY chemical compound suppressed they are non-enveloped virus by rhinovirus and adenovirus 5 and 7() due to the ability of cell infection similarly study.In KY-1(concentration is 1～100 μ g) in the presence of with rhinovirus infection Vero cell (10 5), after 24 hours, check the cytopathic effect of cell at viral infection, show viral infection.Investigate in the arbitrary concentration that is subjected to reagent thing KY, the result shows that cell agglutination does not reduce.
In its concentration range of KY-1(also is 1～100 μ g) in the presence of with adenovirus infection Vero cell, viral infection was checked the cytopathic effect of cell after 24 hours.Arbitrary concentration at the KY-1 medicine is investigated, and the result shows that cell agglutination does not reduce.
Generally speaking, the macrocyclic compound of broad range all is effective inhibitors to the cell infection of every kind of enveloped virus in several enveloped viruses that are studied.Particularly relevant HSV virus has been carried out the research in conjunction with test, and the result shows, the antiviral activity of The compounds of this invention depends on and the combining of peplos composition, suppressed virus conversely again with combining of peplos composition and be attached to the cell that can infect.The infection that The compounds of this invention suppresses non-enveloped virus is invalid significantly, and this is consistent with above-mentioned mechanism.
V. the combination of macrocyclic compound and antiviral nucleoside analogs is to the inhibition of virus
The present invention also comprises the macrocyclic compound that contains the above type and the compositions of antiviral nucleoside analogs.In virus replication or transcription stage, nucleoside analog can suppress duplicating of virus effectively.
Being used for the present invention has with the nucleoside analog that macrocyclic compound makes up:
(1) pyrophosphoric acid analog; as phosphorus formic acid (PFA), phosphine acetic acid (PAA), methyl di 2 ethylhexyl phosphonic acid (MDP), carbonyldiphosphonate (COMDP), phosphono glyoxalic acid (COPAA); with and various halogen and/or methyl substituted derivant, they are inhibitor of viral nucleic acid polymerase.Especially described chemical compound is known to suppress herpesvirus (Blackburn Sidwell) and influenza virus (Sidwell) infection, and can suppress the activity of reverse transcriptase in the retrovirus (as people HIV).
(2) base analogue of Xiu Shiing, as IUDR, trifluorothymidine, AraA and azidothymidine AZT (AZT), didanosine (DDI), D4T, zalcitabine (DDC) and ribavirin.The main anti-herpesvirus of trifluorothymidine, IUDR and AraA be effectively (Nicolson, 1984a, 1984b).Anti-several RNA of ribavirin and DNA viruses are effectively (Sidwell), and AZT anti-HIV with other di-deoxynucleoside analog (as DDI) is effective (Fischl).
(3) sugar analogue of Xiu Shiing; as 5 '-amino-2 '; 5 '-dideoxy 5 '-the N-acyl derivative of ioduria glycosides; 5 '-amino-5 '-sulfamide derivative of deoxyribosylthymine and 2 ' deoxidation-5-ethyl uridnine; and N-acyl derivative, 5 '-sulphuric acid and 5 '-sulfamic acid nucleoside analog such as nucleocidin, adenosine 5 ' sulfamic acid and ribavirin, they mainly act on is Profilin synthetic (Martin).
(4) phosphoric acid analog comprises acyclic nucleoside phosphoric acid class, as acycloguanosine and gangiclovir, and their isosteric phosphoric acid analog.Chemical compound can be used as the viral selective substrate (Galbraith) of virus thymidine kinase described in synthesizing in ribonucleoside triphosphote analog cell.Secondly, the ribonucleoside triphosphote analog can be used as the selective substrate of viral dna polymerase, because described analog does not have the necessary bi-functional of chain elongation, so they can play chain terminating agent (Allen).Described chemical compound shows the effect with anti-following virus: herpesvirus (Collins), comprise HSV-1 and HSV-2, and varicella zoster (VZV) and cytomegalovirus (CMV) are (Smith).
This compounds also comprises the (phosphonomethyl) ether of nucleoside and their no ring analogues, as N-(3-hydroxyl-2-phosphonium mesitoyl methoxy propyl group)-(HPMP-) and N-(2-phosphonium mesitoyl methoxy ethyl) (PME-) heterocyclic base derivant.Described chemical compound is anti-herpesvirus, adenovirus, cytomegalovirus (DeClercq), poxvirus, vaccinia virus and retrovirus specifically.
The ability of viral infection due to the clear described two based compositions inhibition enveloped virus of embodiment 18 invading the exterior, as previously discussed, embodiment 18 has checked with viral must the measuring after the HSV-1 of serial dilution or the HSV-2 granule vero cells infection.
Figure 34 A represent when the cell that infects separately with the solid round spot of macrocyclic compound Y-1(that increases concentration), separately with the acycloguanosine (hollow garden circle) of the concentration that increases concentration, with the acycloguanosine+25 μ g/mlY-1 chemical compounds (solid grid) of increase concentration and with acycloguanosine+50 μ g/ml Y-1 chemical compound (Filled Ellipse) declines that HSV-1 virus must be measured when contacting of increase concentration.Any in two kinds of medicines of application separately, the viral maximum minimizing that must measure is slightly less than 3 logarithms (order of magnitude).
Effect with two Y-1 compound concentrations subject composition.Under the lower concentration of Y-1 chemical compound (25 μ g/ml), the compositions that two kinds of chemical compounds constitute suppresses viral must measuring and surpasses 7 logarithms, promptly is higher than two medicines and uses 10 times of the inhibitory action summation that obtains separately.Under the higher concentration of Y-1 chemical compound, to compare with the summation that macrocyclic compound and acycloguanosine are used the gained effect separately, the compositions inhibitory action of two kinds of chemical compound formations is wanted high several magnitude.Shown in Figure 34 B, the chemical compound of administration combination must be measured to suppress HSV-2 virus, has obtained similar result.
Above-mentioned two kinds of chemical compounds can be mixed with for example tablet, gel, ointment or injection form, preferably are mixed with gel form, and the preferred part by weight of macrocyclic compound and nucleoside analog is 10: 1～1: 1.Figure 34 A and Figure 34 B's is viral that discharge curve shows that when macrocyclic compound and the administration of about 5: 1 composition of proportions compositionss of nucleoside, inhibitory action increases significantly.Macrocyclic compound in the compositions preferably selects anti-target virus (herpesvirus as previously discussed, respiratory syncytial virus or retrovirus) to have suitable activity.Equally, nucleoside analog preferably selects anti-target virus to have active (Martin).
A benefit that merges Pharmaceutical composition is in fact only to need to use two type compounds than low dosage just can reach the selectivity viral inhibition, so just reduced the side effect of combination of Chinese medicine thing, its characteristics are that also it has higher antiviral activity.
VI. set of applications compound treatment viral infection
Therapeutic Method of the present invention is that calix of the present invention (n) arene compounds is delivered medicine to infection site by the infection of coated virus individuality.Compositions of the present invention comprise new calix (n) arene compounds and be applicable to that this chemical compound is oral, topical or non-pharmaceutical carrier through gastrointestinal administration.Compositions can contain calix (n) arene compounds individually, and perhaps also combination has antiviral nucleoside analogs.
The dosage form of compositions should be pharmaceutically effective, can effectively suppress the viral infection of host cell.As previously discussed, chemical compound dosage can effectively suppress the viral infection of cell usually in 1～50 μ g/ml scope.Therefore, for many purposes, effective dose preferably can produce the compound concentration of above-mentioned scope at infection site.For topical, compositions contain 1～5% or above calix (n) aromatic hydrocarbons of 1-5% be suitable.
As with as described in the top, in the compositions that contains calix (n) aromatic hydrocarbons and nucleoside analog, the dosage of comparable in fact one or two chemical compound of composition dosage is lower.
The problem that administration composition need be considered, especially non-through gastrointestinal administration or when oral when medicine, this problem is meant the side effect of whole body.The support of carrying out is of the present invention to be studies show that, calix (n) aromatic compound (particularly its sulfoacid compound) shows blood coagulation resisting function after oral or intravenous administration.The conclusion that people obtain from described research is: administration polycationic compounds such as protamine sulfate (intravenous administration) can stop the blood coagulation resisting function of calix in the blood flow (n) aromatic hydrocarbons effectively.During time of administration protamine is adjusted to the highest blood drug level corresponding to calix (n) aromatic hydrocarbons.By method commonly used, the dosage of protamine is equivalent to the about 1mg of per approximately 100 units heparin anticoagulants, intravenous administration, intravenous administration calix (n) aromatic hydrocarbons simultaneously, perhaps in administration potion protamine in 1～2 hour posterior vein of oral macrocyclic compound.Usually recommend method with the slow infusion of protamine (promptly 10 minutes be no more than 50mg total amount).
Therefore at the same time under the situation of administration calix (n) aromatic compound, can regulate the speed of infusion simultaneously, like this so that the infusion velocity of protamine sulfate is no more than 50mg/10 minute.Therefore when the administration The compounds of this invention absorbed in the blood, compositions of the present invention can comprise protamine, and its consumption is for can effectively lowering the blood coagulation resisting function of macrocyclic compound.If the present invention can be lowered the blood coagulation resisting function of macrocyclic compound.If the present composition also contains nucleoside analog and low dosage macrocyclic compound more, because macrocyclic compound is a low dosage, the protamine consumption can reduce or cancel so.
A. injectable compositions
About its effect of compositions of intravenous administration and pharmacokinetics after deliberation.In brief, the macrocyclic compound that is used for type described in this method shows: when intravenous administration, (a) from blood, eliminate (the about 5-8 of t1/2=hour) more lentamente, (b) mainly exist with free state, and the activity that (c) in blood, keeps inhibition virus (as HSV-1, HSV-2, RSV and HIV) to infect.
Injectable compositions comprises that calix (n) aromatic hydrocarbons is in suitable injection solution, as physiological saline solution solution.This solution can contain nucleoside analog and/or protamine in addition.
B. topical composition: treatment genital herpes lesion
For suppressing the viral infection of skin and mucosa, preferably compositions is mixed with ointment.Use topical composition treatment genital herpes lesion and narrate in following research, this research is introduced in embodiment 13 in detail.In brief, make in the female Cavia porcellus transvaginal with HSV-2 and to infect, press after the method inoculation HSV-2 later 6 hours or 48 hours of embodiment 13 topical therapeutic 3 times every day then.Animal groups comprises KY-1 group or the acycloguanosine group in control animal group (not treatment behind the viral infection), placebo group (vehicle treatment), the carrier.Obtain the vaginal secretions assay specimen, by the CPE test evaluation virus activity of standard.The order of severity of (21 days) genitals damage is pressed 0-5 in the initial course of infection +The level record.
After 3～4 days of inoculation HSV-2, the blister damage appears on the skin of external genitalia.Lesion development became the ulcer stage in 7-8 days, healing gradually in 15～21 days.By the lesion development order of severity, be listed in the table 10 with the effect of KY-1 preparation topical therapeutic.Damage AUC with the treatment group day entry of placebo after infecting 6 hours significantly increases (P＜0.05); But compare with untreated matched group, on average the highest damage is kept the score does not have difference.Compare with placebo, treated with 5%KY-1 in back 6 hours in infection, by the AUC value of measuring and on average the highest damage keep the score and show that all degree of injury obviously reduces (P＜0.001), treated with 1%KY-1 in back 6 hours in infection, AUC obviously reduces (P＜0.01), but the average the highest damage no significant difference of keeping the score.
Table 10
Damage is kept the score
Treatment area under curve P value is the highest damage P value of keeping the score on average
Contrast 37.0--3.6---
Placebo+6h 47.0＜0.05 3.9 NS
Placebo+48h 42.8 NS 3.6 NS
KY 5% +6hr 3.8 <0.001 0.8 <0.001
KY 5% +48h 45.7 NS 3.7 NS
KY 1% +6h 30.8 <0.01 2.9 NS
KY 1% +48h 46.6 NS 4.3 NS
ACV 5% +6h 2.7 <0.001 0.6 <0.001
ACV 5% +48h 45.8 NS 3.8 NS
All do not observe skin irritant sign with arbitrary prescription.In whole therapeutic process, it is normal that the genitals outward appearance keeps; Do not observe rubescent or swelling.Cavia porcellus also keeps outward appearance normal and healthy in whole research work.
In another research with above-mentioned Cavia porcellus genitals model, make zoogenetic infection with HSV-2, with KY-1 or Y-1 topical therapeutic, drug level is 2% or 5% then.It is described to press embodiment 13, and animal is 6 or 24 hours begin treatments after infection.To the order of severity of treatment of animals and record infection every day, totally 19 days.List in the table 11 with the effect that KY-1 and Y-1 topical therapeutic infect, and with compare with 5% acycloguanosine (ACV) treatment infection.
The effect that infects with placebo (carrier is only arranged) treatment in this research obviously is worse than not treatment group.Inoculate administration 2% in back 6 hours or 6%Y-1 and carry out Drug therapy, comparing with the placebo treatment group has the animal of damage decreased number, and average damage is kept the score and reduced and the highest damage reduction of keeping the score.And, inoculate after 24 hours with 6%KY-1 or Y-1 or 2%KY-1 prescription and treat, have the animal decreased number of damage, and the damage order of severity reduces.
Table 11
Local KY-1 and the Y-1 of using is to HSV-
The effect of 2 genitals damage
The % damage is kept the score
Treatment group time N animal injury (AUC) average peak
Not medication-9 66.7 9.1 1.3
Placebo 6h 10 100 29.0 3.0
Y-1(2%) 6h 9 66.7 16.9 1.8
Y-1(6%) 6h 10 30 8.1 1.2
Placebo 24h 10 100 22.3 2.7
Y-1(2%) 24h 10 60 22.0 2.3
Y-1(6%) 24h 10 40 14.7 1.8
KY-1(2%) 24h 10 70 16.2 2.2
KY-1(6%) 24h 10 50 17.5 1.7
ACV(5%) 24h 8 37.5 11.7 1.5
C. topical composition: treatment ocular infection
In another embodiment, be suitable for ophthalmic administration, comprise the ointment of calix (n) aromatic compound or suitable solution as cornea surface administration topical composition in order to make chemical compound.Compositions also comprises the effective nucleoside compound of target viral infection.
In embodiment 10 described Therapeutic Method, before with HSV-1 infected rabbits cornea, use the chemical compound Y-1 that changes local dose one by one.The order of severity of epithelial diseases (Figure 33 A), conjunctivitis (Figure 33 B), iritis (Figure 33 C) and substrate inflammation (Figure 33 D) that slit lamp biomicroscope's evaluation of using on the applying clinical records.
Infect after 7 days, observe the epithelial diseases order of severity culminate (Figure 33 A) in the animal groups of placebo treatment.Also obtain the parameter (Figure 33 B-D) of conjunctivitis, iritis and substrate inflammation by this research.
In the evaluation result of all 4 eye infection, the severe infections that Drug therapy produces than placebo treatment will be still less, infects aspect the development reducing ophthalmic diseases that HSV-1 causes, and the Y-1 of various concentration is all effective.With various concentration Y-1 treatments, variant mutually statistically.
Concentration is that 12.5 μ g/50 μ l topical therapeutics are the most effective ophthalmic therapies.Infect back 6 days epithelium disease degrees and alleviate, infect resilience a little in back 7 days.With other 2 Y-1 dosage treatment relatively, this concentration is effectively in the development that reduces the ophthalmic that is caused by HSV-1, and only with the scorching relating to parameters of slight conjunctivitis, iritis and substrate.Higher concentration (18.75 μ g/50 μ l) also is effective to reducing the corneal epithelium advancing of disease that is caused by HSV-1.But, the Y-1 corneal epithelial surface of this concentration and toxic a little to conjunctivitis, iritis and substrate inflammation.This toxicity all increases and obtains proof by infecting after 6 and 7 days the parameter value of all diseases.
Scrape the titre that thing (trial target) obtains virus from inoculating 0,3,5 and 7 day the tear film in back and infecting the epithelium that formed in back 7 days.As described in embodiment 10, reduce and the viral titration of multiple regression analysis mensuration by plaque.In the research of tear film, in all animals of administration local dose Y-1, investigate virus titer and significantly reduce, though do not see difference when maximum dose level (12.5 and 18.7 μ g/50 μ l), this reduction be it seems relevant with dosage.Scraping scraping of getting after 7 days observes virus titer and reduces relevant with dosage in the thing.
According to above-mentioned research, obtain effective dose/scope.The optium concentration of chemical compound is 12.5 μ g/50 μ l in this research.
D. Orally administered composition
Of the present invention studies show that of support of being carried out, about 0.5 hour of oral administration (as gavage) back, the type macrocyclic compound of the present invention of application existed in blood plasma, reached peak value simultaneously at about 2-4 hour.The time of active drug concentration is about 4～18 hours in the blood behind intravenous administration.When this chemical compound of intravenous administration, medicine has the short volume of distribution half-life, this influences drug distribution to body extraorgan lacuna, the short volume of distribution half-life is normally useful to the medicine with anticoagulation side effect, because the concentration of chemical compound in blood flow can more accurately be measured.
VII. as the topical compositions that suppresses infection of coated virus through spreading through sex intercourse
A method of the present invention be with contain as previously discussed macrocyclic compound be used for may the contact of generation property a certain zone of health or several zone.Described zone can comprise the skin surface of anus-genital area and mouth, and the mucosal tissue of vagina, rectum, mouth and throat.Of the present invention studies show that of support of being carried out, above-described macrocyclic compound is suitable for topical on skin and mucosa, and promptly The compounds of this invention can not produce stimulation such as swelling or rubescent.
The compositions of local usefulness can comprise the carrier that pharmaceutically is suitable for that is suitable for topical.Therefore, the present composition can be for example suspendible liquor, solvent agent, ointment, lotion, sexual intercourse lubricant, ointment, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder doses, syrup, face cream agent or lozenge.The method for preparing above-mentioned composition is known in pharmaceuticals industry.Except containing macromolecular compound, the present composition can also comprise the antiviral nucleoside analogs of discussing in the above part of V.
Compositions of the present invention is lubricious gel (sexual intercourse lubricant) preferably in the embodiment at one, and it comprises lubricated gel carrier, and macrocyclic compound of the present invention is dissolved in this carrier.In the sexual intercourse contact, gel carrier partly makes the degree of minimizing that frays, and therefore can reduce enveloped virus enters the probability that damaged tissue enters blood then.As with as described in the IVA of top, macrocyclic compound of the present invention can be combined closely effectively to enveloped virus, and therefore can suppress virus is attached to the cell that can infect.Like this, the macrocyclic compound in the gel just can stop the enveloped virus granule before infection takes place.
The dosage form of compositions is pharmaceutically effective, and promptly this dosage can suppress the infection of the enveloped virus through spreading through sex intercourse effectively.As previously discussed, the chemical compound dosage in 1～75 μ g/ml scope can suppress virus infected cell usually effectively.Therefore, to many purposes, effective dose preferably can produce the dosage of above-mentioned scope compound concentration at infection site.To local application, it is suitable that compositions contains 1～10% macrocyclic compound.Except that containing macrocyclic compound of the present invention, also contain in the compositions of antiviral nucleoside analogue (as described in part of V), in the compositions dosage of chemical compound in fact a chemical compound can be lower, perhaps the dosage of two chemical compounds is all lower.
A. topical compositions: prevent that genital herpes virus from infecting
In embodiment 19, studied the ability that suppresses the cytopathic effect of HSV-1 and HSV-2 infection cell as the chemical compound Y-1 of a lubricating gel agent compositions part in great detail.Gel preparation is by commercial lubricant gel (K-Y Jelly, the Johnson﹠amp that has bought; Johnson) and the Y-1 chemical compound through selecting dosage constitute.In this research, cell is contacted with HSV-1 or HSV-2, be that the Y-1 gel combination of 40 μ g/ml contacts simultaneously with final drug level.Inoculate after 24 hours, check the cytopathic effect (being that garland cells forms) of cell.
The results are shown in table 12.Symbol "+" represents that it is effective that the Y-1 chemical compound of this concentration suppresses cytopathic effect (CPE).Cytopathic effect has been observed in symbol "-" expression.
Table 12
Compositions Y-1 HSV-1 HSV-2
Concentration (KOS) (333)
The 5ug/ml of 5% Y-1--
K-Y gel 10ug/ml+/-+/-
20ug/ml + +
40ug/ml + +
The 5ug/ml of 10% Y-1--
K-Y gel 10ug/ml+/-+/-
20ug/ml + +
40ug/ml + +
The 5ug/ml of 20% Y-1--
K-Y gel 10ug/ml+/-+/-
20ug/ml + +
40ug/ml + +
The independent 5ug/ml of Y-1--
10ug/ml +/- +/-
20ug/ml + +
40ug/ml + +
The above results shows, is about 20 μ g/ml or more than the 20 μ g/ml, the gel compositions that contains chemical compound Y-1 can suppress cell infection in significant effective ground at drug level.
B. suppressing HIV infects
In another research, the lubricating gel compositions of having tested the Y-1 chemical compound that contains various dose suppresses the cytopathic effect of the cell of following viral infection (embodiment 20), and used virus has a kind of in the strain of two kinds of HTLV-III, i.e. HTLV-III BWith the RF-II.Briefly, in containing in the presence of the Y-1 chemical compound gel combination of selection of dilution, will use the HTLV-III BOr the chronically infected cell of RF-II cultivates, and then with indicator cell co-cultivation.The degree that forms with syncytial virus under microscopic examination is kept the score.
The result is as follows shown in the table 13, and wherein "-" expression does not have cell fusion (or stoping fusion fully), and 4+ represents to have and the indistinguishable cell fusion degree of untreated tester.
Table 13
Y-1 20% Y-1 10% Y-1 5% Y-1
The K-Y of the K-Y of the K-Y of ug/ml
Gel gel gel
500 - - -
250 - - -
125 - - -
63 +/- +/- +/-
32 4+ 4+ 4+
16 4+ 4+ 4+
8 4+ 4+ 4+
6 4+ 4+ 4+
4 4+ 4+ 4+
As shown above, cell contacts with the Y-1 chemical compound of concentration greater than about 63 μ g/ml, produces to suppress the cell fusion that HIV causes.This result shows, the Y-1 chemical compound that contains in the gel combination can suppress the cell infection that the HTLV Strain by test causes effectively.
VIII. contraceptive device
On the other hand, the present invention also comprises the physical barrier type utensil with the above type macrocyclic compound and usefulness, is used to suppress the infection of the enveloped virus that do as one likes propagates.In one embodiment, this utensil comprises physical barrier type utensil (for example contraceptive device), and lubricant compositions is coated on this utensil, and described lubricant compositions is to contain macrocyclic compound of the present invention and lubricant gel carrier.
In one embodiment, described parts are condom, and wherein lubricant compositions is coated on the outer surface of this condom.Contraceptive device can also be a kind of condom, and the compositions that wherein contains macrocyclic compound is coated in the internal layer surface of this condom, and purpose is to mix with the seminal fluid of injecting condom.Condom can be (promptly the placing male's penis) that is used for male's general type, perhaps can be to insert type, and for example can be that the women uses.Wherein this contraceptive device comprises as inserting the contraceptive device of vagina before beginning at heterosexual intercourse.
Described utensil can also be cervical cap, barrier film or contraceptive sponge, can be placed near the cervix uteri, and therefore, cervical cap or barrier film can be with the gel that for example contains macrocyclic compound of the present invention or ointment coating or injections.Equally, contraceptive sponge can soak into the solution that contains macrocyclic compound of the present invention, ointment etc.
In the compatibility research of estimating macrocyclic compound of the present invention and latex condom, the K-Y gel mixture of 20%W/W Y-1 chemical compound is imposed on the latex condom inside of several trades mark.50ml garden taper centrifuge tube has inserted each condom to imitate vertical penis, then the opening of each condom dialysis ring seal mouth.Again the condom that seals is placed 37 ℃ of DDWs to keep 24 hours.
After the dialysis, water exists to have determined whether the Y-1 chemical compound with the analysis of HPLC method.In this susceptiveness test (5 μ g/ml), all do not detect the Y-1 chemical compound for arbitrary condom, this shows that macrocyclic compound does not ooze out by the latex condom.Therefore, in order to prevent the propagation of viral infection when the sexual intercourse, macrocyclic compound of the present invention and the coexistence of latex contraceptive device are suitable for.
The following examples are described in detail method and the application in control is infected for preparing macrocyclic compound of the present invention, and the following examples purpose is at length to narrate rather than limit the scope of the invention.
Raw material
All chemical tests are buied by Aldrich Chemical Co. or other commercial companies.
Embodiment 1
Preparation naphthalene macrocyclic compound.
A.KY-1（R 1=OH，R 2=SO 3Na，R 3=H，R 4=＞CH 2）
To 50ml(41mM) add 15ml 37% formaldehyde in chromotropic acid two sodium solutions, mol ratio to the end be 5: 1 formaldehyde: chromotropic acid.Mixture in the flask of close plug in 1 week of stirring at room.Resulting 70ml dark red solution filters under vacuum, and adding 200ml acetonitrile makes and separates out precipitation after the concentrating filter liquor.The product that the filtration collection is separated out is also dry under vacuum.The productive rate of KY-1 is 95%.This chemical compound has following feature:
Fusing point (M.P.)＞300 ℃;
HPLC(CH 3CN/MeOH/H 2O/TFA): 14 ' 48 " single broad peak;
（IR/KBr）=3425（OH），1638（Ar），11811044（SO 3）cm -1
UV（H 2O）：238.0，358.5nm
Molecular weight: through mass spectral analysis is 1505(M+1);
1H NMR(CD 3OD), chemical shift (γ): 5.20 (CH 2), 8.01 (ArH) ppm;
C 13NMR(D 2O), chemical shift (γ): 27.19,120.18121.69,122.06,122.67,133.30,142.97,154.42 and 181ppm.
Elementary analysis: (C 22H 10O 16S 4Na 4) 2* 6H 2O or (C 22H 11O 16S 4Na 4) 2* 5H 2O
Measured value: C 33.17, H 2.54, and Na 11.93;
Value of calculation: C 32.75, H 2.23, and Na 11.41;
C 33.16,H 2.13,Na 11.56。
B.KY-3（R 1=OH，R 2=SO 2NH 2，R 3=H，R 4=-CH 2-）
KY-1(2mM) handle with the 5ml chlorosulfonic acid, mixture stirs half an hour in 50 ℃.The mixture that obtains is added in the 20g trash ice to separate out product, filter and collect and wash with ether.
Crude product is dissolved in the 100ml25% ammonia spirit, and in room temperature reaction 2 hours.Mixture concentrates under vacuum, and remaining grease is dissolved in the low amounts of water and filters.In filtrate, add acetonitrile and separate out product, filter and collect and wash with acetonitrile.This chemical compound has following feature:
Fusing point (M.P.)＞300 ℃;
Mass spectrum: 1452(M-7NH 2);
HPLC(CH 3CN/MeOH/H 2O/TFA): 11 ' 46 is " unimodal;
（IR/KBr）=3430（OH），3187，1686（NH 2）,1637（Ar），1211,1110,1044（SO 3）cm -1
UV（H 2O）∶246nm;
1H NMR(D 2O), chemical shift (γ): 5.15 (CH 2), 7.5-8.2 (ArH) ppm;
Elementary analysis: (C 44H 40O 26S 10N 12Na 4) 16H 2O
Measured value: C 28.62, H 3.93, and N 8.82,
S 17.17，Na 5.44;
Value of calculation: C 28.51, H 3.89, and N 9.07
S 17.28，Na 4.97。
C.KY-42（R 1=OH，R 2=SO 3Na，R 3=H，R 4=＞CHCOOH）
(10mM) places 50ml water with the chromotropic acid disodium, in the 5ml aqueous solution and the 10ml37% mixed in hydrochloric acid of room temperature and Acetic acid,oxo-,monohydrate (10.0mM).Mixture boiled 8 hours, the color of solution becomes kermesinus.Add in the 50ml water solution that obtains and filtration.Concentrated filtrate also adds ethanol, to separate out the KY-42 product.Productive rate is 87%.This chemical compound has following feature:
Fusing point (M.P.)＞300 ℃;
Mass spectrum: 1623(M-3H 2O);
HPLC(CH 3CN/MeOH/H 2O/TFA): 10 ' 36 is " unimodal;
（IR/KBr）=3452（OH），1801，1719（Co），1638（Ar），1206，1050（SO 3）cm -1
UV（H 2O）：238.0，351.5，520nm;
1H NMR(D 2O), chemical shift (γ): 7.10(CHCO 2H), 8.00(ArH) ppm;
C 13NMR(D 2O), chemical shift (γ): 116.04,
118.90,120.94,121.27,122.30,124.30,124.68,
126.60,128.37,136.48,136.71,140.50,143.93,
144.26,145.75,152.01,154.33,156.01,156.67;
Elementary analysis: (C 48H 4O 40S 8Na 8) 44H 2O
Measured value: C 32.74, H2.50;
Value of calculation: C 32.58, H2.71.
D.KY-123（R 1=OH，R 2=SO 2Na，R 3=H，R 4=＞CH 2）
KY-1(2mM) handle with the 5ml chlorosulfonic acid, mixture stirs half an hour in 50 ℃.To separate out product, filter and collect product in the mixture adding 50g trash ice that obtains, wash with ether then.Rough sulfonic acid chloride product is handled with the 4ml aqueous solution of sodium sulfite (20mM).Gradation adds a small amount of 50% sodium hydroxide in the reactant mixture, makes reactant mixture keep alkalescence to keep 2 days.Except that after desolvating, add ethanol to separate out product, add 50% sulfuric acid acidation product, add ethanol subsequently so that separate out sodium sulfate.(1: 2, V/V) required product was separated out in mixing with ether with ethanol.The productive rate of product is 39%.
E.KY-147（R 1=OH，R 2=SO 2NHCH 3，R 3=H，R 4=＞CH 2）
By being reacted, chromotropic acid (disodium salt) and sulfonic acid chloride generate N-methyl chromotropic acid acyl chlorides in the presence of DMF.This reaction was under agitation carried out 4 hours in 80 ℃.Under vacuum, remove and desolvate and excessive thionyl chloride, add ether, collect by filtering then, and wash with ether to separate out the chromotropic acid acyl chlorides.Add crude product in the 20ml methylamine and stirred 2 hours.Remove whole solvents from the material of gained, residue is dissolved in the 200ml cold methanol and filters.In filtrate, add acetonitrile to separate out chromotropic acid sulfonyloxy methyl amine.Productive rate is 56%.
Chromotropic acid sulfonyloxy methyl amine (2mM) is placed 3ml water, and with 1ml37% formaldehyde in 1 week of room temperature reaction.Add acetonitrile to separate out product, filtration is collected product and is washed with acetonitrile.Productive rate is 85%.
F.KY-151（R 1=OCH 3，R 2=SO 3Na，R 3=H，R 4=＞CH 2）
With KY-1(50mM) be dissolved in the 80ml 0.2M sodium hydrate aqueous solution and in 50 ℃ of heating, slowly DIYU added dimethyl sulfate (0.2M) in 1 hour.The restir mixture was at room temperature placed after 2 hours 2 days continuously.In the material of gained, add 100ml saturated nacl aqueous solution and filtration.Precipitation is successively with ethanol, acetonitrile and ether washing.Be dissolved in the 100ml methanol exsiccant material and filtration.Concentrated filtrate also adds ether, to separate out the dimethyl ether disodium of chromotropic acid.
G.KY-158（R 1=OH，R 2=SO 2CH 3，R 3=H，R 4=＞CH 2）
The KY-1 that is obtained by embodiment 1A at first handles with thionyl chloride, obtains the chromotropic acid sulfonic acid chloride.This chemical compound with excessive sodium sulfite reduction, obtains the sulfonate sodium (R of corresponding cyclisation chromotropic acid in the presence of sodium bicarbonate 2=SO 2Na).This sulfonate is pressed the described method of embodiment 1A with dimethyl sulfate and is handled in the presence of sodium bicarbonate.The productive rate of product is about 21%.
H.KY-175（R 1=OH，R 2=SO 3CH 3，R 3=H，R 4=＞CH 2）
Chromotropic acid is at first handled with thionyl chloride, obtains the chromotropic acid sulfonic acid chloride.This chemical compound methanol solution with Feldalat NM in the presence of sodium salt is handled then.This product is pressed the described method of embodiment 1A and is handled, and obtains macrocyclic compound.The productive rate of product is about 29%.
I.KY-285（R 1=OCOCH 3，R 2=SO 3Na，R 3=H，R 4=＞CH 2）
The KY-1(0.66mmol that obtains by embodiment 1A) is dissolved in the 3ml water that contains the 0.1g sodium hydroxide.To wherein adding 1g chloroacetic chloride (13mmol), under agitation reactant liquor spends the night in room temperature reaction.Remove and desolvate, add 25ml ethanol, separate out product.Be dissolved in the methanol crude product and filtration.Filtrate is separated out precipitation, obtain 87% productive rate.
J.KY-346（R 1=OH，R 2=SO 3Na，R 3=H，R 4＝-CH 2-N（CH 3）CH 2）
Under agitation the chromotropic acid disodium salt is dissolved in (concentration is 50mM) in the 80ml water in 50 ℃, becomes clarification, in above-mentioned solution, add hexamethylenetetramine (50mM) at uniform temp then until solution, and continuous stirring 2 hours again.The color transition of this mixture is dark blue color during this period.Mixture was stirring at room 2 days.The dark blue color solution of gained is filtered, and concentrating filter liquor and the flask of packing into evaporation are handled with 200ml methanol subsequently, separate out product KY-346, and the productive rate of KY-346 is 85%.This chemical compound has following feature:
M.P.＞300℃;
HPLC(CH 3CN/MeOH/H 2O/TFA): 13 ' 07 is " unimodal;
（IR/KBr）=3425（OH），1626（Ar），1197，1052（SO 3）cm -1
UV（H 2O）：232.0，377.5nm
Elementary analysis: (C 13H 11O 8NS 2Na 2) 4* 12H 2O
Measured value: C 33.17, H 3.13, and N 2.75;
Value of calculation: C 33.98, H 3.59, and N 2.96.
Molecular weight: through gel filtration is 1668.
Embodiment 2
Preparation calix (n) aromatic compound
A.Y-49（R 1=OH，R 2=SO 3H，R 4=-CH 2-，n=4）
In room temperature, 10g4-tert-butyl group calix (4) aromatic hydrocarbons is handled half an hour with the 200ml concentrated sulphuric acid, place then in 75～85 ℃ of oil baths and reacted 4 hours.When not having water-fast material to be picked, reaction is done.The grease of gained is splashed in the 500g trash ice solution filtration under diminished pressure.From filtrate, remove and anhydrate, in residue, add the 500ml acetonitrile, place 4 hours, filter then and collect and wash with acetonitrile, ethyl acetate and ether to separate out crude product.Output is 8g(73%).With the crude product recrystallization, obtain pure products with methanol-ether or methanol-acetonitrile system.In recrystallization process, also can form single crystal compound.
With the synthetic Y-77(R of similar approach 1=OH, R 3=SO 3H, R 4=-CH 2-, n=6) and Y-1(R 1=OH, R 3=SO 3H, R 4=-CH 2-, n=8).
B.KY-225（R 1=-OH，＝O，R 2=SO 3H，R 4=＞CH 2，≥CH，n=4）
In room temperature, with 1g4-tert-butyl group calix (4) aromatic hydrocarbons and 10ml95～98% sulfuric acid reaction half an hour, then 160 ℃ of reactions 5 minutes.After the mixture cooling of gained, pour in the 100ml trash ice lentamente it and filtration.Solution adds the 300ml acetonitrile through evaporation in residue, separate out a large amount of precipitations, filters the collecting precipitation thing, and washs with acetonitrile.Crude product is dissolved in the 20ml methanol, adds ether and separate out product.Productive rate is 84%.
With the synthetic Y-48(R of similar approach 1=-OH or=O, R 3=SO 2H, R 4=-CH 2-, n=6) and Y-226(R 1=-OH or=O, R 2=SO 3H, R 4=-CH 2-, n=8).
O-acetylate (the R of C.Y-1 1=-OCOCH 3, R 2=SO 3H, R 4=＞CH 2, n=8)
0.75gY-1 stirred in the anhydrous acetic anhydride of 30ml spend the night.Successive reaction is dissolved in the solvent until raw material.After being chilled to room temperature, filtering suspension liquid.Solid washs 2 times and vacuum drying with acetonitrile.This material washs and recrystallization again.
13CNMR(D20, δ): 173.9,151.6,144.1,135.6.130.1,34.2 and 22.4.
D.Y-78（R 1=-OH，R 2=SO 2NH 2，R 4=＞CH 2，n=8）
Under blanket of nitrogen, 1g Y-1 and 20ml chlorosulfonic acid one arise from 60-70 ℃ of heating 1 hour.Grease is poured in the frozen water into filtering precipitate after being chilled to room temperature.Behind the cold water washing precipitate, precipitate is added to 50ml contains in the solution of 5.7g glycine and 2.1g sodium hydroxide, in stirring at room 2 hours.Crude product is dissolved in the 100ml25% ammonia spirit, and in room temperature reaction 2 hours.Mixture concentrates under vacuum, and remaining grease is dissolved in the low amounts of water and filters.In filtrate, add acetonitrile, separate out product, filter and collect and wash with acetonitrile.
Glycyl sulfonamide (the R of E.Y-1 1=-OH, R 2=SO 2NHCH 2CO 2H, R 4=＞CH 2, n=8)
Under blanket of nitrogen, 1g Y-1 and 20ml chlorosulfonic acid were in 60-70 ℃ of heating 1 hour.After being chilled to room temperature, grease is poured in the frozen water, and filtering precipitate.Behind the cold water washing precipitate, precipitate is added to 50ml contains in the solution of 5.7g glycine and 2.1g sodium hydroxide, in stirring at room 2 hours.Remove whole solvents from the material that generates after, residue is dissolved in the 200ml cold methanol and filters.In filtrate, add acetonitrile so that separate out product.
F. the Y-49(R of acetyl group bridging 1=-OH, R 2=SO 3H, R 4=-CHCO 2H-, n=4)
In 100 ℃, the solution reaction in the 30ml18% concentrated hydrochloric acid is 2 hours with 4.3g p-hydroxybenzenyl sulfonate and 9g Acetic acid,oxo-,monohydrate.Behind the product drying under reduced pressure, add 50ml methanol, the insoluble impurity of filtering.In filtrate, add ether, separate out product, filter then and collect and vacuum drying.
Tosylate (the R of G.Y-49 1=-SO 3C 6H 4CH 3, R 2=SO 3H, R 4=＞CHCO 2H, n=4)
Under blanket of nitrogen, in the suspension of 1.06g natrium carbonicum calcinatum, 10ml dimethyl formamide and 0.75g Y-49, add the 1.9g toluene sulfochloride.After backflow was spent the night, the mixture of gained was chilled to room temperature and filters.Filtrate is diluted to separate out crude product with ether.With acetonitrile/ether solvent recrystallization, obtain product.
Carboxylic acid derivates (the R of H.Y-49 1=-CO 2H, R 2=SO 3H, R 4=＞CHCO 2H, n=4)
Under blanket of nitrogen, to with ice-cooled 1.25g2, add the 1.0ml trifluoromethanesulfanhydride anhydride in the 10ml anhydrous methylene chloride solution of 6-di-t-butyl-4-picoline and 0.65g 4-tert-butyl group calix [4] aromatic hydrocarbons, after stirring under the room temperature was spent the night, mixture diluted with the 10ml pentane and filters.Filtrate extracts with ice-cooled 1N sodium hydrate aqueous solution, ice-cooled 1N aqueous hydrochloric acid solution and saturated sodium-chloride water solution successively, with anhydrous sodium sulfate drying, filters and vacuum concentration by silicagel pad.Residue is dissolved in the anhydrous diisopropylethylamine of 10ml, 0.5ml trimethylsilyl cyanide and 20mg four-(triphenyl phasphine) palladic mixture, is chilled to room temperature after blanket of nitrogen flows through night next time again, adds the 50ml ether, and with the suspension filtered that obtains.Filtrate is behind vacuum concentration and silica gel column chromatography (hexane/ethyl acetate eluting), and cyanide intermediate and 10ml concentrated sulphuric acid spend the night with dilution of 10ml water and backflow in 80 ℃ of heating 3 hours.After being chilled to room temperature, the mixture that obtains is joined in 0.5g active carbon and the 50g ice.After the filtration, the filtrate that obtains is concentrated into about 15ml volume under vacuum, and with the solid filtering that obtains.Solid is dissolved in the minimum methanol, separates out precipitation by adding ether.After C18 reversed phase chromatography (methanol eluting) purification obtains product.
Methyl ether (the R of I.Y-1 1=-OMe, R 2=SO 3Na, R 4＞CH 2, n=8)
The mixture of 447mg Y-1,1.53ml 6N sodium hydrate aqueous solution and 9ml dimethyl sulfate was in 60 ℃ of heating 20 hours.The mixture that obtains is splashed in the 100ml dehydrated alcohol that is stirring.Centrifugal 20 minutes of the suspension (9000rpm) that forms is removed supernatant then.Once more the solid that obtains is dissolved in the 6ml water, solution that obtains such as the above-mentioned Ethanol Treatment of using, centrifugal and remove supernatant.Remaining solid obtains the 420mg product through lyophilization.
13CNMR(D20, δ): 161.2,140.9,137.6,129.5,63.6, and 33.5.
J.XXⅥ.（R 1=-OH，R 2=H，R 3=R 5=H，R 4=-CH 2-，n=4）
Press Gutsche, Levine and Sujeeth, 1985 described methods are with 4-tert-butyl group calix (4) aromatic hydrocarbons (XX V; Figure 13) preparation calix (4) aromatic hydrocarbons XX VI.With 5.0g(6.75mmol) hot solution of XX VI in 250ml toluene place flask at the bottom of the three neck gardens of 500ml band mechanical agitator and gas inlet pipe.Solution is chilled to 50-55 ℃, uses 5.0g(37mmol) the aluminum trichloride (anhydrous) processing, and under noble gas, stirred 2 hours in 50-55 ℃.Mixture places ice bath to cool off, and and 125ml 1N hydrochloric acid stirred together 30 minutes, separate organic facies and washing, dry and evaporation, obtain yellow residue.This residue grinds with the 500ml ether, insoluble matter CHCl 3-CH 3The OH recrystallization gets 1.9g(66%) the XX VI, be cream-coloured microcrystal, m.p.313-318 ℃.
K.XXⅧ.（R 1=-OH，R 2=COOH，R 3=R 5=H，R 4=-CH 2-，n=4）
Prepare calix (4) aromatic hydrocarbons XX VIII by Yilmaz and the described method of Vural.With known 1.3g to acetyl group calix (4) aromatic hydrocarbons (XX VII) (Yilmaz and Vural, 1991; No et al., 1986) be dissolved in the 50ml2N sodium hydrate aqueous solution.Add 8g iodine and the solution of 20g potassium iodide in 40ml water, and mixture is stirred.Solution in water-bath warm 1 hour, the filtering iodoform adds the 20g sodium sulfite in filtrate.In filtrate, add concentrated hydrochloric acid then, obtain faint yellow precipitation, leach precipitation again, wash with water and drying.Crude product is dissolved in 10% aqueous solution of sodium bisulfite, and uses charcoal treatment.After the filtration, solution 1N hcl acidifying.Filter and collect the product of separating out,, dry in vacuum desiccator with distilled water wash until no chloride ion, get 1.04g(79%) the XX VIII.M.p.320 ℃ (decomposition).
L.XXⅪ.（R 1=-OH，R 2=-CH 2COOH，R 3=R 5＝H，R 4=-CH 2-，n=4）
Press Gutsche and Nam, 1989 described methods are used the XI to (dimethylamino) methyl-calix (4) aromatic hydrocarbons (XX IX) preparation calix (4) arene derivatives XX.
To 15.9g(39.5mmol) add 45ml acetic acid, 22.5g(0.2mol in the solution of the 360mlTHF of calix (4) aromatic hydrocarbons (XX VI)) 40% dimethylamine agueous solution and 116.2g(0.2mol) 37% formalin.Reactant mixture removes under vacuum and desolvates, and residue is dissolved in the 250ml water in stirring at room 24 hours.Aqueous solution 200ml extracted with diethyl ether 2 times with the neutralization of 10% solution of potassium carbonate, are used the nutsch filter filtering-depositing.Product is dry under vacuum, uses the chloroform recrystallization then, obtains 19.1g(78%) to (dimethylamino) methyl-calix (4) aromatic hydrocarbons XX IX, be the white needles thing.
In the 220ml DMSO solution of 16.3g, add 9.57ml(0.15mol lentamente to (dimethylamino) methyl-calix (4) aromatic hydrocarbons) methyl iodide.Reactant mixture after 30 minutes, adds 15g(0.3mol in stirring at room) NaCN, mixture was in 80 ℃ of heating 2 hours under blanket of nitrogen, and cooling solution is handled with the 1L frozen water then, uses the 2N hcl acidifying, filters and air drying.Crude product CH 3The CN recrystallization obtains 12.8g(88%) to cyanogen methyl calix (4) aromatic hydrocarbons XXX, be faint yellow solid.
In 25ml DMSO and 5ml concentrated hydrochloric acid solution, add 0.5mmol cyanogen methyl-calix (4) aromatic hydrocarbons and backflow are spent the night, after room temperature is with the dilution of 100ml water, filter collecting precipitation, obtain pure XX XI with recrystallizing methanol.
M.XXXⅢ（R 1=-OH，R 2=-CH 2CH 2COOH，R 3=R 5=H，R 4=-CH 2-，n=4）
Press Gutsche and Nam, 1989 described methods, preparation calix (4) arene derivatives XXX III.
To 3.26g(5mmol) (dimethylamino) methyl-calix (4) aromatic hydrocarbons (XX IX; Embodiment L) add 1.90ml(30mmol in the solution of 80ml DMSO) methyl iodide.Mixture stirred after 30 minutes, adding is by 1.20gNa, 7.28g the diethyl malonic acid sodium of diethyl malonate and 28mlEtOH preparation, reactant mixture heated 2 hours in 80 ℃ under blanket of nitrogen, cooling solution then, and pour in the 200ml frozen water, the 2N hcl acidifying used, and, obtain 5.50g(99% by method processing commonly used) to (diethyl malonyl) methyl-calix (4) aromatic hydrocarbons XX XII crude product.Crude product is dissolved in 100ml DMSO and 30ml concentrated hydrochloric acid, and under blanket of nitrogen, was hydrolyzed and decarboxylation in 10 hours in 120 ℃ of heating.Cooling mixture then, and pour in the 500ml frozen water, stirred 10 minutes and filtered, precipitation obtains 2.24g(69% with acetone-re-crystallizing in ethyl acetate) the XXX III, be colourless crystallization.m.p.224℃。
N.XXXⅥ（R 1=-OH，R 2=-PO 3H，R 3=R 5=H，R 4=-CH 2-，n=4）
Adopt the method for Arduini etc. and Hirao etc. to prepare XXX VI derivant.
With calix (4) aromatic hydrocarbons (XX VI) and Hg(OCOCF 3) 2In chloroform, reflux, obtain almost four-(Hg-OCOCF of quantitative yield 3) the calix arene derivatives.Then evaporate chloroform, react in chloroform by calix arene derivatives and iodine and carry out the exchange of metal iodine, obtain right-iodo-calix (4) aromatic hydrocarbons XXX IV, be brown compound, productive rate is 40%.
Under blanket of nitrogen, with HPO(OEt) 2(10mmol), triethylamine (10mmol), Pd(PPh 3) 4(0.3mmol) and the spissated toluene solution of right-iodo-calix (4) aromatic hydrocarbons (1.0mmol) stirred 3 days in 100 ℃.After room temperature is with the dilution of 50ml ether, reactant mixture is filtered, then under fine vacuum (100 ℃, air pressure＜0.1mmHg) concentrate.The concentrated solution of gained obtains pure phosphonic acid diester (XXX V) through the silica gel column chromatography purification, refluxes in 5ml6N hydrochloric acid then and spends the night, and obtains the phosphonic acids product.Remove and to desolvate that (100 ℃, behind the air pressure＜0.1mmHg), the solid recrystallizing methanol obtains pure XXX VI.
O.XXXⅨ.（R 1=-OH，R 2=-CH 2PO 3H，R 3＝R 5＝H，R 4=-CH 2-，n=4）
By described methods such as Almi by chloromethyl-calix (4) aromatic hydrocarbons being prepared calix (4) arene derivatives XL IV.
To in-10 ℃ of refrigerative 1.0g(2.4mmol) calix (4) aromatic hydrocarbons (XX VI) and 14.4g(81mmol) drip 4.7ml(40.3mmol in the solution of the 100ml chloroform of chloromethyl-n-octyl ether) butter of tin, add in about 15 minutes.Remove cooling bath then, reactant mixture remains to whole calix aromatic hydrocarbons initial substances in room temperature and has carried out reacting (after about 50 minutes), with thin layer chromatography (hexane: ethyl acetate=4: 3) identify.Add entry then lentamente, separate biphase.Organic layer distilled water wash 2 times are then through dried over sodium sulfate.Remove sodium sulfate subsequently, evaporating solvent obtains residue, and reuse normal hexane washing is also filtered, and obtains 1.23g(80%) product is to chloromethyl-calix (4) aromatic hydrocarbons XXX VII.
1g(1.6mmol) derivant XXX VII refluxed 6 hours in the 20ml NSC 5284, excessive NSC 5284 is removed in distillation, the solid residue of gained (phosphonic acid diester XXX VIII) under vacuum dry 8 hours.Add the 60ml20% hydrochloric acid solution, with the reaction mixture refluxed that obtains 20 hours, evaporation subsequently removes desolvated, the precipitation of gained is used methanol wash, is washed with chloroform subsequently for the 1st time after filtration, and dry under vacuum, obtain 1.11g(80%) the XXX IX, be white solid.m.p.360℃。
P.XLⅣ.（R 1=-OTs，R 2=-CH 2CH 2Br，R 3=R 5=H，R 4=-CH 2-，n=4）
Press Gutsche, Levine and Sujeeth, 1985; Gutsche, Dhawan etc., 1983 described methods prepare right-2-bromoethyl-calix (4) arene derivatives XLIV.
(a) in the solution of the 100mlTHF of 2.14g calix (4) aromatic hydrocarbons (XX VI) and 10ml DMF, add the 2.0g sodium hydride, add the 28g allyl bromide, bromoallylene subsequently.Mixture was refluxed 1 hour, and THF is removed in evaporation then, and residue distributes between water and chloroform.Chloroform extraction liquid washes with water, dry and evaporation, residue 95% ethyl alcohol recrystallization obtains 2.18g(74%) O-pi-allyl-calix (4) aromatic hydrocarbons XL, be colourless spicule.
(b) in noble gas with 1.66g(2.84mmol) the 25ml N of O-pi-allyl calix (4) aromatic hydrocarbons, N-diethylaniline vlil 2 hours.With the solution cooling, pour in the 250ml ice-water, stir with the 250ml concentrated hydrochloric acid, and filter, obtain crude product, use the isopropyl alcohol crystallization then, obtain 1.22g(74%) right-pi-allyl-calix (4) aromatic hydrocarbons XLI, be cream-coloured spicule, m.p.245～248 ℃.
(c) 2.09g(3.57mmol) solution and the 1.0g(42mmol of the anhydrous THF of 100ml of right-pi-allyl-calix (4) aromatic hydrocarbons) the sodium hydride reaction, subsequently with 4.0g(21mmol) the p-toluenesulfonyl chloride reaction, mixture heated was refluxed 1.5 hours.Evaporation removes desolvates, and obtains light brown oily thing, and this grease is dissolved in the 100ml chloroform, on ice bath, cool off, and with 100ml ice-water treatment.Organic facies drying and evaporation, residue isopropyl alcohol recrystallization obtains 3.41g(79.5%) right-pi-allyl calix (4) aromatic hydrocarbons (XLII) of tosylation.
(d) solution of the 60ml dichloromethane of the 3.50g tosylation right-pi-allyl-calix (4) aromatic hydrocarbons and 40ml methanol cooling in dry ice-propanone is bathed keeps blue color (10-15 minute) with ozonization until it.Become bubble to feed solution nitrogen, add the 2g tetrahydro boron sodium until blue decoloration.Solution is poured in the ice-cooled dilute hydrochloric acid solution in stirring at room 3～4 hours, handles with conventional method, obtains crude product, is the white resinous thing.Obtaining 1.51g(43% with 3: 5 acetone-hexane recrystallization) crystallite is right-2-ethoxy-calix (4) aromatic hydrocarbons phenol oxygen-tosylate (XLIII).
(e) by 6.5g(25mmol) triphenyl phasphine and Br 2The solution-treated of the 50ml acetonitrile of the solution of the 150ml anhydrous acetonitrile of the dibromo triphenyl phasphine (Schaefer etc., 1973) of preparation step (d) product (by the preparation of 3.25g step (c) product).Mixture is in stirring at room 2 hours and filtration, and evaporation removes and desolvates, and obtains the orange of viscosity.This grease stirred 8 hours with 250ml95% ethanol, filtered to collect to obtain 3.10g(78%) right-the 2-bromoethyl-O-tosyl-calix (4) aromatic hydrocarbons XLIV, be white powder.
Q.XLVI（R 1=-OH，R 2=-CH 2CH 2PO 3H，R 3=R 5=H，R 4=-CH 2-，n=4）
With right-2-bromoethyl derivant XLIV(embodiment P) preparation calix (4) aromatic hydrocarbons XLVI, the modification method of its method is served as reasons right-chloromethyl-calix (4) aromatic hydrocarbons (embodiment O) preparation XXXIX.
Under blanket of nitrogen, with the bromide XLIV(1mmol of purification) in 10mlP(OEt) 3The middle backflow spent the night, fine vacuum (100 ℃, remove excessive phosphite ester under 0.1mmHg) after, under blanket of nitrogen, residue (diethyl phosphite XLV) is added in the mixed liquor of 5ml DMSO and 1ml 6N sodium hydroxide, and, slough the toluenesulfonic acid ester group in 100 ℃ of heated overnight.Fine vacuum (100 ℃,＜remove DMSO under 0.1mmHg) after, residue is with the dilution of 25ml hot water, and use the concentrated hydrochloric acid esterification, obtains precipitating through cooling, filters collecting precipitation then.Solid obtains the XLVI of purification with recrystallizing methanol.
R.XLVII.（R 1=-OH，R 2=-CH 2SO 3H，R 3=R 5=H，R 4=-CH 2-，n=4）
In room temperature to right-chloromethyl-calix (4) aromatic hydrocarbons (XX VII, embodiment O; 2.5mmol) 10ml 95% alcoholic acid solution in add sodium sulfite aqueous solution (2M, 11mmol).After backflow is spent the night, desolvate until separating out precipitation through distilling to remove.Filter collecting precipitation, the saturated aqueous sodium chloride washing with cold is suspended in the minimum water then, by the pillar of being filled by Amberlite IR-120 resin and water.To contain ultraviolet is active level part concentrates of product under vacuum, the residue recrystallizing methanol obtains pure XLVII.
S.XLIX.（R 1=-OH，R 2=-CH 2CH 2SO 3H，R 3=R 5=H，R 4=-CH 2-，n=4）
With bromoethyl derivant XLIV(embodiment P) preparation calix (4) arene derivatives XLIX; its method is as follows: the method for sulfonating with embodiment R obtains XLVIII successively; hydrolysing step with embodiment Q is sloughed tosyl, obtains sulfonic acid XLIX with the Amberlite IR-120 step of embodiment R.
T.LII.(R 1=-OAc, R 2=the tert-butyl group, R 3=R 5=H, R 4=-CH(OH)-, n=4)
Use known calix aromatic hydrocarbons LI(Gormer etc. by the following method; 1990:R 1=-OAc, R 2=the tert-butyl group, R 3=R 5=H, R 4=-C(=0)-, n=4) preparation calix aromatic hydrocarbons LII.
With 1.5g(2.3mmol) right-tert-butyl group-calix (4) aromatic hydrocarbons (XXV) reflux in the solution of 37ml acetic anhydride and 0.1ml concentrated sulphuric acid.Reactant mixture is added in the 300ml ice-water, obtains slow crystalline grease.Collect crystalline solid, wash with water for several times, and use the petroleum ether drying, obtain the O-acetyl group-right-tert-butyl group-calix (4) aromatic hydrocarbons (L), be white crystals.
To adding 1.2g(1.5mmol in the flask at the bottom of the three neck gardens that condensing tube, agitator and charging hopper are housed) O-acetyl group-right-tert-butyl group calix (4) aromatic hydrocarbons and 70ml acetic anhydride; 20 ℃ and stir under; to wherein dripping the solution of 3.5g chromium oxide (IV) in 15ml acetic anhydride and 5ml acetic acid mixed liquor; reactant stirred 8 hours in 140 ℃; after the cooling; left standstill 12 hours in the reactant mixture adding 600ml ice-water and with it, collect the yellow mercury oxide that obtains, and wash with water.Obtain the pure ketone group derivative L I(O-acetyl group-right-tert-butyl group-calix (4) aromatic hydrocarbons, R with recrystallizing methanol 4=-C(=O)-) (Gormer etc., 1990).m.p.305℃。
Under room temperature and blanket of nitrogen, the ketone group derivant (1mmol) that preceding step is obtained is dissolved in 10ml invariably in the ethanol, and adds tetrahydro boron sodiums (8mmol) to wherein being divided into some aliquots.Ketone group (is observed 167cm with infrared spectrum after finishing reduction reaction -1The carbonyl spectral line at place disappears and differentiates), drip 1ml acetic acid, the mixture that obtains stirred 1 hour.Fine vacuum (＜0.1mmHg) remove down and desolvate, resulting solid refluxed 20 minutes with 5ml methanol.Except that after desolvating, residue obtains O-acetyl group-right-tert-butyl group product LII that pure hydroxyl methylene bridged bond connects through the silica gel column chromatography purification.
U.LIII.（R 1=-OH，R 2=R 3=R 5=H，R 4=-CH（Cl）-，n=4）
Under blanket of nitrogen, with derivative L II(embodiment T; 0.5mmol) in the 5ml thionyl chloride, reflux.SO 2Effusion stop after, fine vacuum (＜0.1mmHg) down excessive thionyl chloride is removed in distillation.In residue, add 5ml THF, repeat to distill, obtain chlorinated derivative LIII to remove remaining thionyl chloride.
V.LVI.（R 1=-OH，R 2=R 3=R 5=H，R 4=-CH（CO 2H）-，n=4）
(a) under blanket of nitrogen, will contain chlorinated derivative LIII(1mmol) and the reactant mixture of Cyanogran. (1.1mmol) in the 10ml dimethyl sulfoxide in 80 ℃ the heating 6 hours.Mixture is poured in the 50ml ice-water then, uses the 3N hcl acidifying, filters and collects the precipitation that obtains.Filtrate is added in 25ml dimethyl sulfoxide and the 5ml concentrated hydrochloric acid mixed liquor and refluxes and spend the night., filter and collect the precipitation that obtains with the dilution of 100ml water in room temperature, obtain LV with chloroform/recrystallizing methanol.
(b)-tert-butyl group right in order to slough will be joined by the product mark aliquot that preceding step obtains under blanket of nitrogen in the toluene suspension of (60 ℃) aluminum chlorides (10mmol) of 50ml heat.After stirring is spent the night, mixture is chilled to 0 ℃, and drips 100ml1N hydrochloric acid.Add the back and separate organic facies, and vacuum concentration.Obtain pure LVI with chloroform/recrystallizing methanol.
W.LVII.(R 1=-OAc, R 2=the tert-butyl group, R 3=R 5=H, R 4=-CH(CH 2CH=CH 2)-, n=4)
With chlorinated derivative LIII(embodiment U) be dissolved among the minimum THF, mixture splash into stirring, cooling (78 ℃) contain (CH 2CH=CH 2) 2CuLi(0.2M is in THF solution 3mmol).Make suspension be warmed to room temperature then.After stirring was spent the night, suspension was with the mixed liquor extraction of 3: 1 saturated ammonium chlorides of 5ml and saturated ammonia solution.Organic facies is through anhydrous sodium sulfate drying, filtration and vacuum concentration.Residue obtains O-acetyl group-right-tert-butyl group allyl deriv LVII through the silica gel column chromatography purification.
If desired, by following examples b method deacetylate and tert-butyl group partly.
X.LVIII.（R 1=-OH，R 2=R 3=R 5=H，R 4=-CH（CH 2CO 2H）-，n=4）
(a) calix aromatic hydrocarbons (LVII) (1mmol) is placed the 10ml dichloromethane, carry out ozonisation in-78 ℃, become blue color until reactant mixture.Add 2ml formic acid and 1ml hydrogen peroxide then, to room temperature, logical simultaneously nitrogen is washed with the reactant mixture temperature.Mixture refluxes and spends the night, and removes under vacuum then and desolvates, and obtains LVIIa.
(b) will in the mixed liquor of 4ml methanol and 1ml 6N sodium hydroxide, reflux by the O-acetylate (0.2mmol) that preceding step obtains and spend the night, with deacetylate.Except that after desolvating, residue dilutes with 10ml water, and is acidified to pH2 under vacuum.Filter and collect the precipitation that obtains, with chloroform/hexane recrystallization.Method by EXAMPLE V step b is sloughed the tert-butyl group, obtains pure LVIII.
Y.LXII.(R 1=-O(CH 2) 3SO 3Na, R 2=H, low alkyl group, R 3=R 5=H, R 4=-CH 2-, n=4)
Press Shinkai etc., 1989, described method prepares calix (4) aromatic hydrocarbons LXII.
Under blanket of nitrogen in 50 ℃ with calix (4) aromatic hydrocarbons XXVI(1.54mmol) be dissolved among the 100ml THF.The cooling back adds 1.20g sodium hydride (30mmol; 60% oily dispersion liquid), stir the mixture to overflow and stop (about 1 hour) until hydrogen.Drip 2.26g propane-1 then, 3-sulfone (18.5mmol), mixture was in stirring at room 24 hours.Add methanol to decompose remaining sodium hydride, vapourisation under reduced pressure solvent then, residue is dissolved in the 500ml hot water.By the centrifugal insoluble matter of removing.Add sodium acetate then and saltout, product precipitation obtains the LXII(productive rate 10% of purification), m.p.＞300 ℃.
Z.LXIV.（R 1=-O（CH 2） 3SO 3Na，R 2=SO 3Na，R 3=R 5=H，R 4=-CH 2-，n=4）
To 10ml dimethyl sulfoxide, right-sulfonyl-calix (4) aromatic hydrocarbons (XV, Fig. 8; 1mmol) and in the mixture of 1ml 6N sodium hydroxide add propane-1,3-sulfone (9mmol), the reactant mixture that obtains is in 60 ℃ of heated overnight.Vacuum (＜0.1mmHg) remove down desolvate after, solid residue dilutes with minimum water, under agitation splashes in the 100ml ethanol.Filter and collect the precipitation that obtains, the water dilution that reuse is minimum, and splash into once more in the 100ml ethanol.Filter and collect the precipitation that obtains, with methanol/CH 3The CN recrystallization obtains pure LXIV.
Embodiment 3
The macrocyclic compound of preparation bridge joint aryl
A.VII. blended naphthyl/phenyl macro ring
The 30ml 37% salt acid treatment of 22ml of the solution of the 55ml water of 10g chromotropic acid disodium.Add 5gl in this solution, the solution of the 55ml acetic acid of 2-benzene dimethanol should react and reflux 6 hours.The mixture of gained adds the 500ml acetonitrile through filtering the back, to separate out crude product, filters and collects.Crude compound further through LH-20 resin column chromatography purification, is used ethanol elution.
B.LXXV. naphthyl-phenyl macro ring (n=2 naphthyl+2 phenyl)
Method by propositions such as Mendoza prepares blended macro ring LXXV.
Chromotropic acid in the presence of the 5ml concentrated hydrochloric acid (III, Fig. 3 A; 10mmol) with 2,5-dihydroxymethyl-3-tert-butyl phenol (LXXII; 1mmol) in 100 ℃ of heated overnight.Fine vacuum (100 ℃,＜0.1mmHg) remove down desolvate after, residue is dissolved in the minimum water, by the pillar and the eluting of Sephadex LH-20 and water filling.In 2ml concentrated hydrochloric acid and 2,5-dihydroxymethyl-3-tert-butyl phenol (LXXII; 0.6mmol) exist down, contain the separated product (LXXIII of 2 chromotropic acid unit and 1 phenol unit; 0.6mmol) again in 100 ℃ of heated overnight.With Sephadex LH-20 post separated product (LXXIV; 0.05mmol), dry under vacuum, under blanket of nitrogen, in the 1ml concentrated sulphuric acid, heated 6 hours then in 80 ℃.With after the 5ml cold water dilution, use the 100mg charcoal treatment, with the filtration of gained mixture, vacuum (＜remove most of moisture content in the filtrate under 0.1mmHg).Residue is dissolved in the saturated sodium-chloride water solution of heat.Be chilled to 0 ℃ and separate out precipitation.Filtering-depositing is dissolved in the minimum water, by the pillar and the eluting of Amberlite IR-120 and water filling.The effluent that will contain pure products merges, and lyophilization obtains pure LXXV(0.03mmol).
Embodiment 4
The cytotoxicity of proliferative cell
Use the toxicity that lineup's somatic cell system measures medicine, comprise the KB(nasopharyngeal carcinoma cell), HeLaS 3(neck epithelial cancer cells), the PLC(hepatoma carcinoma cell), HepG 2(human hepatoma cell), HepG 2T 14(with the hepatoma carcinoma cell of HBV transfection), WI38(normal human lung fibroblast), the BT549(breast cancer cell) and, the SW480(breast cancer cell) and the A549(lung carcinoma cell).
In each hole of the porous culture plate in 24 holes, add 1ml and contain the RPMI-1640 culture medium of 5% hyclone (FCS) and penicillin/streptomycin (P/S), and inoculate 5 * 10 4Cell.At postvaccinal the 2nd day, one in 50 test compounds that table 3 is listed added in the cell, and concentration is 1～100 μ g/ml.Remove culture medium after 3 days, cell 40% methanol of Commassie Blue and the solution-dyed of 7% acetic acid.Result's II in the above partly discusses.
Embodiment 5
Inhibitory action to the HSV virus activity: cytopathic effect
Containing 7%CO in 37 ℃ 2The damping incubator in the Vero cell is kept in the RPMI-1640 culture medium that contains 5% hyclone, the 100 penicillin/ml of unit and 100 μ g streptomycin/ml.Use the HSV-1(Kos-1 of Strain HSV) and HSV-2(333) strain.
In each hole of 96 hole microdroplet plates, add 0.2ml and contain the RPMI-1640 culture medium of 5%FCS and 0.1% methylcellulose (15cps), and inoculate 1 * 10 5The Vero cell.Cultivation is spent the night, after making cell carry out the logarithmic (log) phase multiplication, culture medium is gone in suction, replace with 100 μ l same medium, this culture medium contains 2%FCS and 50 μ l contrast liquid or drug solution, and 50 μ l HSV-1 or HSV-2 virus liquid, the final concentration of medicine is 10 μ g/ml, and the content of virus is about 3 plaque forming units (PFU)/cell, promptly 6 * 10 5The PFU/ hole.
Cell was in 37 ℃ of cultivations 24 hours, and cytopathic effect can clearly be observed during this period.Do not having under the situation of viral infection, cell forms uniform monolayer fibroblast.Having under the situation of viral infection, cell forms the suspension of garden shape cell, cell agglutination subsequently, and their outward appearance is distinguished by very easy and normal fibroblast.If do not observe the cytopathy effect, with 10 μ g/ml repeated trials.Parallel group of cell of virus inoculation be not as the contrast of Vero cell cytotoxicity test.
Above table 1 listed the structural formula of test compound, table 3 the 2nd row have showed that the protection cell avoids the chemical compound of cytopathic effect (+).
Embodiment 6
Inhibitory action to the HSV virus activity: plaque reduces
The method of Vero cell by embodiment 5 is kept in the RPMI-1640 culture medium that contains 5% hyclone.In 24 well culture plates, add the RPMI-1640 culture medium that 1ml contains 5%FCS and 0.1% methylcellulose (15cps), inoculate 4 * 10 therein 5The Vero cell.Cultivation is spent the night, after making cell carry out the logarithmic (log) phase multiplication, the sucking-off culture medium, the culture medium identical with 100 μ l replaces, this culture medium contains 2%FCS, and it contains 50 μ l contrast liquid or drug solution, and contains 50 μ lHSV-1 or HSV-2 virus liquid, the final concentration of medicine is 0.15,2.5,5,10 or 20 μ g/ml, and the content of virus is about 1 * 10 3PFU/ml, i.e. 50PFU/ hole.
, inhale virus removal and also remove medicine after 2 hours in 37 ℃ of cultivations, cell washs with PBS, adds the RPMI-1640 culture medium that 0.5ml contains 1% methylcellulose (4K cps) of 2%FCS and penicillin/streptomycin (P/S).Remove culture medium after 2 days.Cell dyes with 50% alcoholic solution of 0.8% crystal violet.With the plaque counting that forms, and divided by the percentage ratio of the plaque that forms in the matched group with the calculating inhibition.ED 50Value representation 50% viral plaque is suppressed required drug concentrations, ED 50Supposition is calculated to calculate the linear dose response of inhibitory action viral plaque.The IC that calculates 50Be worth in the above and list in the table 3 and table 4.
Embodiment 7
Inhibitory action to the HSV virus activity: the viral inhibition that must measure
The RPMI-1640 culture medium that 5ml is contained 5%FCS and P/S adds in the 25T flask, and inoculates 1 * 10 6The conspicuous S that draws 3Cell.After 24 hours, the sucking-off culture medium is with 6 * 10 6The serial dilution liquid of PFU HSV-1 or HSV-2 and the KY chemical compound through selecting replaces, and drug level is 10,5,2.5,1.25 and 0.625 μ g/ml.Contain at 2ml in 37 ℃ that growth is after 24 hours among the RPMI-1640 of 2%FCS and P/S, cell is freezing until titrating the time in-70 ℃.In order to discharge virus from cell, with cell freezing/thawing 3 times, and sequence ground carries out 10 times of dilutions.
In each hole of 24 hole porous culture plates, add 1ml and contain the RPMI-1640 culture medium of 5%FCS, P/S and 0.1 methylcellulose (15cps), and inoculate 1 * 10 5The Vero cell.After removing culture medium on the 2nd day, add the virus of 100 μ l10 times serial dilutions, duplicate.After 2 hours, remove virus removal in 37 ℃ of cultivations, the RPMI-1640(that adds 0.5ml methylcellulose (4K cps) contains 2%FCS+P/S) culture medium.Remove culture medium after 2 days.Cell with the plaque counting that forms, calculates titre by extension rate with the 50% alcoholic solution dyeing of 0.8% crystal violet.
Shown in Figure 29 A and the 29B, the viral minimizing that must measure is the function of KY chemical compound (KY-1 and KY-2) concentration.
Embodiment 8
The activity of overriding resistance virus HSV-1 and HSV-2 strain
Use following HSV-1 and HSV-2 Strain: KOS, be wild type HSV-1 virus; KOS(PMEA) and KOS(PFA), be drug resistance HSV-1 virus with dna polymerase mutant; 333, be wild type HSV-2 virus, and 333(DHPG), for having the drug resistance HSV-2 virus of thymidine kinase sudden change.
Press embodiment 7 described methods substantially, check that with in above-mentioned 5 HSV strains each KY-1, acycloguanosine (ACV), DHPG, PFA and PMEA are to the viral inhibition that must measure.In brief, will conspicuous draw S 3Cell inoculation is put in the 25T flask that contains culture medium, and sucking-off culture medium after 24 hours is with 6 * 10 6The HSV strain virus of PFU through selecting and the serial dilution liquid replacement of KY-1, ACV, DHGP, PFA and PMEA.Contain at 2ml in 37 ℃ that growth is after 24 hours among the RPMI-1640 of 2%FCS and penicillin, streptomycin (P/S), cell is freezing until titrating the time in-70 ℃.In order to discharge virus from cell, with cell freezing/thawing 3 times, and sequence ground carries out 10 times of dilutions, and serial dilution liquid is added in the Vero cell culture.After 2 hours, remove virus removal in 37 ℃ of cultivations, add the RPMI-1640 culture medium (containing 2%FCS+P/S) of 0.5ml methylcellulose (4Kcps).Remove culture medium after 2 days.Cell dyes with 50% alcoholic solution of 0.8% crystal violet.With the plaque counting that forms, calculate titre by extension rate.From drug dose reaction assay IC 90Concentration value, IC 90Value representation suppresses 90% virus must measure each required drug level.This value has been listed in the top table 5.
Embodiment 9
Inhibitory action to the RSV virus activity
Evaluate the antiviral activity of KY-and Y-chemical compound in the tissue culture with following test: in the flat tissue culturing plate in 96 holes (Falcon 307), by in the above-mentioned cell toxicity test similarly condition carry out.In this test, the 2%FCS-MEM solution of chemical compound is carried out serial dilution, with 4 parts of test compounds of 2 times of diluent same form (0.05ml/ hole) of sequence.Except above-mentioned one group as antiviral with organize the control wells, in all holes, add 0.05ml and contain about 100 50 3nfective dose (TCID of tissue culture 50) suitable virus.Then in every hole, add about 3 * 10 3HEp2 cell (0.1ml).Comprise in each test: contain antiviral and do not contain the control wells (virus control) of virus or contain culture medium and do not contain viral or antiviral control wells (tissue contrast).Then with the challeng virus back titration.All bread boards place and contain 5%CO 2Incubator in cultivated 5～7 days in 35 ℃.When the virus control hole shows that 70%～100%CPE(comprises syncytium) time, all holes observed.With CPE in the virus control hole relatively, in measuring the metapore of 4 parts of every group of same form after the antiviral ultimate density demonstrations＜50%CPE, calculating medium effective concentration (IC 30).The ED that each test compound calculates 30Value is listed in the table 5.
Embodiment 10
The simple virus of herpes (HSV) activity: the Topically active of the HSV-1 virus of anti-eye culture in the body
Test with New Zealand white rabbit, at virus inoculation preceding 2 days at least, handling made it adapt to various devices and condition in experimental cage in experimental cage.After the laundering period, these animals are made the slit lamp examination of eyes, eliminate the white rabbit of all original anterior ocular segment defectives that just exists.Then, contain minimal essential medium (MEM.Gibco) the 80 μ l of HSV-1 virus McKrae strain for the local inoculation of white rabbit eyes, virus quantity is every milliliter 10 5Plaque forming unit (10 5Pfu/ml); Need 30 seconds of massage eyes after the inoculation, then animal is put back in the experimental cage 1 white rabbit of every cage.
Behind the virus inoculation the 4th of (PI) the day, with examination with slitlamp microscope white rabbit eyes.To the order of severity of cornea epithelial cell, iris and conjunctive disorder, make the classification record to keeping the score of increasing progressively of 4+ with 0+.After the inspection white rabbit is divided into 4 groups, 5 every group all exist cornea, substrate and conjunctival damage.Begin to do the topical therapeutic test after the grouping immediately.The treatment grouping comprises:
The 1st group: 5 of white rabbits, make topical therapeutic with Y-1, drug level is 6.25 μ g/50 μ l, 5 times/day, totally 4 days.
The 2nd group: 5 of white rabbits, make topical therapeutic with Y-1, drug level is 12.5 μ g/50 μ l, 5 times/day, totally 4 days.
The 3rd group: 5 of white rabbits, make topical therapeutic with Y-1, drug level is 18.75 μ g/50 μ l, 5 times/day, totally 4 days.
The 4th group: 5 of white rabbits, with placebo (sterilized water) treatment, 5 times/day, totally 5 days.
To the tolerance studies of Y-1 ascending-dose concentration, be based on its 90% effective dose (ED 90) concentration, this ED 90Concentration be by viral must measure or the cytopathic effect of virus measure determined.It is 1/2ED that the 1st group of white rabbit accepted to contain 6.25 μ g/50 μ l( 90Concentration) the local eye drip treatment of Y-1; The 2nd group of white rabbit accepted to contain 12.5 μ g/50 μ l(and is ED 90Concentration) the local eye drip treatment of Y-1; The 3rd group of white rabbit accepted the i.e. 1.5 times of ED of 18.75 μ g/50 μ l( 90Concentration) Y-1 eye drip treatment.Y-1 dosage all is formulated in the 50 μ l capacity (eye drop of standard), obtains comprising above-mentioned concentration.
Behind the virus inoculation (PI) the 4th day, with 0-19 μ g Y-1(in 50 μ l) the beginning topical therapeutic, treatment lasted till behind the virus inoculation the 7th day.From behind the virus inoculation the 3rd day to the 7th day, all will make ocular slit lamp examination to all animals every day, writes down the viral-induced oculopathy range degree of HSV-1 every day.
In addition, in viral infection the 0th day (before the inoculation), the 3rd day, the 5th day and the 7th day, all animals are taken a sample to check as eye, see the HSV-1 virus that whether has infection.In brief, dab the conjunctival sac of portion and top now, obtain the tear film, and this cotton swab was placed for 10 seconds in animal nose fornix with cotton swab.Respectively this cotton swab is embathed in HankShi buffer saline (NBSS, Gibco laboratory) then,, this virus was adsorbed 5 minutes on the HFF of example cell monolayer by a virus of 50 μ l-HBSS leachate.Cell monolayer behind this viral adsorption adds minimal essential medium (MEM; The Gibco laboratory) make into hydrate, 37 ℃ of cultivations, observe every day, continued for 2 weeks, checks whether occur infecting corresponding to cytopathic effect (HSV CPE) with HSV.Whether make cell blind (order) for the culture that HSV CPE do not occur and pass (generation), be that virus is negative with conclusive evidence.
Animal is killed in work in the 7th day behind the virus inoculation, takes out the cornea epithelium, and HSV virus is cultivated on the HFF cell monolayer and obtained.Whether the cornea epithelium is done to cultivate altogether, and to culture that HSV CPE do not occur make blind passage and cultivate with the light microscopy of counter-rotating every day, be that virus is negative with conclusive evidence.
The clinical effectiveness that is used to do 3 kinds of Y-1 concentration of single pharmaceutical treatment is compared with result with placebo, in the process of making topical therapeutic with each preparation of Y-1 and after treatment finishes, the effect of its Cure Viruses damage is done mutually relatively, and compare with result with placebo treatment, shown in Figure 33 (A-D).
Embodiment 11
Inhibitory action to influenza A virus activity
Evaluation methodology to KY compounds influenza A virus activity is described with embodiment 9, is not both external to infect mdck cell (kidney cell line) with influenza virus (Taiwan A strain).
Embodiment 12
The inhibitory action of the cell example that human immunodeficiency virus (HIV) is brought out
With people CD 4 +Indicator cells (VB) and chronically infected H 9Cell is kept in the RPMI-1640 culture medium, is containing 7%CO 2The damping incubator in 37 ℃ of cultivations, added 5% hyclone, penicillin 100 units/ml and streptomycin 100 μ g/ml in the culture medium.Used HIV Strain is the HTLV-III BWith the strain of RF-II (from the National Institutes of Health (Bethesda, MD) obtain).
Suppress to measure for making cell fusion, a KY compounds (1mg/ml is in PBS) of selecting for use with garden, one 96 hole at the bottom of culture plate, at 1: 2 and 1: 2 8Between do sequence dilution.KY chemical compound with this dilution moves into another flat culture plate in piece 96 holes then.Again with the chronically infected H of 25 μ g 9Cell (2 * 10 6Individual cell/ml) or 25 μ g add in each hole with the chronically infected cell of HIV virus RF-II strain, cultivate 45 minutes for 37 ℃, and every then hole adds 25 μ l VB cells (about 5 * 10 4Individual cell), this cell and viral chorista are containing 5%CO in the damping incubator 2Air in cultivated 18 hours.The situation that syncytium forms is observed under phase contrast microscope and is kept the score, and record makes syncytium form the KY compound concentrations (ED that suppresses fully 100).The results are shown in Table 7.
Embodiment 13
The therapeutic effect that topical infects genitals HSV
A. virus and virus inoculation
HSV-2 virus MS strain is used for infected animal.Female Hartley strain Cavia porcellus (Charles River raising experiment chamber, Kingston, New York) body weight 250-300g wiped behind the intravaginal secretions 1 hour with cotton swab, was 2.0 * 10 in its amount of intravaginal inoculation HSV-2 virus 5Plaque forming unit.
B. the treatment of Cavia porcellus
In first research, every group of 10 Cavia porcelluss are carried out topical therapeutic, (the 0.1ml medicine is used for intravaginal, 0.1ml medicine is used for skin of external genitalia), from inoculating HSV-2 virus back 6 hours or 48 hours, treat 3 times (per approximately 8 hours once) totally 7 days every day.Do not inoculate yet treatment in the same way of infected animals, be used for mensuration and have or not skin irritation for 3 groups.
In second research, every group of 8-10 animal, 6 hours or 24 hours begin treatments behind virus inoculation, the topical therapeutic preparation of KY-1 with 2% or 6% or Y-1 is treated 3 every day, (shown in table 8B).KY-1 or Y-1 preparing preparation be, this chemical compound is dissolved in 1.5% methocel solution, the final concentration that makes chemical compound be 2% or the 5%(w/w).Control animal (10) is not given any treatment or treats with placebo (1.5% methocel solution).
C. sample collection and virus are measured
In order to measure the influence of Drug therapy to the intravaginal virus replication, behind the HSV virus inoculation the 1st, 3,5,7 day and 10 days, get vaginal secretions with cotton swab, immersion contains in the small test tube of 2.0ml culture medium, after cotton swab in vitro stirs and washes, at-70 ℃ with the freezing preservation of this culture medium, until being used for the HSV-2 titration of virus.After whole sample collections are finished, they are thawed, and do the sequence dilution, with microdroplet cytopathic effect algoscopy, measure the titre of HSV-2 virus with rabbit kidney cell.
D. effect assessment
In order to measure Drug therapy to the formation of external genitalia damage and the influence of expansion, in the overall process of initial infection (19-21 days), the order of severity to the external genitalia damage is kept the score with the 0-5+ classification, damage is kept the score-natural law area under the curve and virus titer-natural law area under a curve, with its damage keep the score peak value and virus titer peak value, treatment not with the placebo treatment animal between or between usefulness placebo treatment and drug-treated animals, compare with Mann-Whitney U extreme difference summation (U range sum) mensuration.The p value is equal to or less than 0.05 expression, and the two has significant difference.This result with reference to table 10 and table 11, is partly discussed in the above-mentioned IV of this paper.
Animal is behind virus inoculation, and whether exist damage and the damage order of severity to keep the score to it every day, continues 19 days (divide by 0-5+ and keep the score).To damage marking system and tabulate, with every day damage keep the score to the time (my god) area under a curve and observed operated scoring peak value represent.11 data that provide are provided.In test, to known antiviral agent acycloguanosine of 8 animals administers (ACV), medicine is mixed with 5% concentration, as positive control.
Embodiment 14
To HSV-1 virus and the bonded inhibitory action of Vero cell
As described in example 5 above, the Vero cell is kept in the RPMI-1640 culture medium.Cell culture is spent the night, carry out after the logarithmic (log) phase multiplication, this culture medium of sucking-off replaces with 100 μ l experiment culture medium, and this culture medium contains 2% hyclone (FCS), by 50 μ l contrast liquid or drug solution, and 50 μ l H 3The HSV-1 of labelling virus liquid is formed, medicine be 10 μ g/ml at last, the content of virus is about 3 plaque forming units (PFU)/each cell, promptly 6 * 10 5The PFU/ hole.Cell with cell sucking-off from suspension, is washed 2 times with PBS cultivate 5,30,60,120 and 240 minutes respectively in the experiment culture medium after, measures the virus quantity (cpm that is combined on the cell 3H).The results are shown in Figure 30, virus is represented with solid round spot in conjunction with contrast among the figure, and medicine suppresses virus in conjunction with representing with solid grid.
Embodiment 15
Medicine contacts the influence that HSV is suppressed with virus
The Vero cell is kept at (as mentioned above) in the RPMI-1640 culture medium.Cell culture is spent the night, after carrying out the logarithmic (log) phase multiplication, this preserves culture medium sucking-off, the culture medium that contains 2% hyclone with 100 μ l replaces, compound K Y-1 is done the sequence dilution, the drug dilution liquid of 50 μ l concentration between 0.625 μ g/ml and 10 μ g/ml is added first group of culture plate, and add 50 μ l HSV-1 viral suspensions simultaneously, make every hole contain 5 * 10 6PFU.Cell was cultivated 2 hours at 37 ℃, used the PBS washed cell then, and measured the number of viral plaque, and is described with embodiment 6.
In second group of cell hole, the medicine with serial dilution adds to cell earlier, adds HSV-1 virus then, and cell was cultivated 2 hours in 37 ℃ in the presence of virus.Washed cell adds viral suspension to remove after the free medicine, and every hole contains 5 * 10 6PFU.This cell was cultivated 2 hours at 37 ℃, use the PBS washed cell then after, measure viral plaque number, method is as described in the embodiment 6.
In the 3rd group of cell hole, 100 μ l viral suspensions are added to cell, every hole contains 5 * 10 6PFU cultivates cell 2 hours at 37 ℃, then with the PBS washed cell to remove unconjugated virus.Compound K Y-1 is done the sequence dilution, and the medicine 100 μ l between 0.625 μ g/ml and 10 μ g/ml add to cell with concentration.Exist under the situation of medicine, cell was cultivated 2 hours at 37 ℃, used the PBS washed cell then, and the above measures the number of viral plaque for another example.
With the viral plaque number of being measured in above-mentioned every kind of drug treating method, represent that with the percent of the matched group virus plaque number of not making drug treating mapping is as Figure 31.Solid round spot represents that cell contacts with virus with medicine simultaneously.Solid grid is illustrated in and adds before the virus, and cell and medicine are done pre-cultivation together, and hollow grid is illustrated in and adds before the medicine, and cell and virus are done pre-cultivation together.
Embodiment 16
The inactivation of the HSV-1 virus that the KY compounds causes
Purified HSV-1 viral suspension is cultivated in containing the RPMI-1640 culture medium of 2%FCS, penicillin and streptomycin (Gibco laboratory).With contrast solution, KY-1 or KY-217 solution add in the viral suspension of five equilibrium, and the final concentration that makes medicine is 10 μ g/ml, and the virion final concentration is 6 * 10 6Or 6 * 10 5PFU/ml.This suspension was cultivated 1 hour at 37 ℃, done the sequence dilution by 10 times of dilution methods then, making last drug level is 10,10 0, 10 -1, 10 -2, 10 -3And 10 -4μ g/ml.Suspension granule with this serial dilution adds to the Vero cell again, cultivates 2 hours (described with embodiment 6), measures the viral plaque of cell after 48 hours.On 2 culture plates, to each virus concentration on every block of plate and each drug level, calculate the number of cell virus plaque, be shown in Table 9.
Embodiment 17
The KY compounds combines with the HSV virus protein
A. compound K Y and HSV virus protein combines
Above-mentioned HSV-1 and HSV-2 viral suspension, concentration all is approximately 5 * 10 7PFU/ml adds 5 * 10 5Cpm 14The compound K Y-1(50 μ g/ml of C labelling), cultivated 2 hours at 37 ℃.Every part of viral suspension is divided into halves, and (SDS) makes solution with 0.5% sodium lauryl sulphate, contains or do not contain 1% mercaptoethanol.Four sample solutions fractionated on 8.5% polyacrylamide gel, the program according to standard develops gel by automatic radiation according to art then.The automatic radiography photo of four samples is seen Figure 32 A, and for HSV-1 virus bands of a spectrum, the A band contains mercaptoethanol, and the B band does not contain mercaptoethanol, and for HSV-2 virus bands of a spectrum, the D band contains mercaptoethanol, and the E band does not contain mercaptoethanol, and the C band is marker protein matter.
B. the evaluation of conjugated protein
As mentioned above, HSV-1 and HSV-2 viral suspension make solution with SDS, and make it fractionated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis technology (SDS-PAGE), each sample is made electrophoretic separation in triplicate, be equivalent to bands of a spectrum D, B and C among Figure 32 B, two clotting glue to every bands of a spectrum are analyzed with immunoblot assay, and method is as follows: the gel that at first makes bands of a spectrum D, B and C respectively with the mouse monoclonal antibody reaction of the anti-HSV viral glycoprotein of specificity gD, gB and gC.These monoclonal antibodies are to obtain from doctor S.Chatterjee of Alabama university place.Then these gels are cultivated with the goat anti-mouse antibody of alkali phosphatase enzyme mark, so that the glycoprotein in every bands of a spectrum of labelling.The glycoprotein that has binding antibody can be identified by the method for standard, promptly by when having nitrogen blue tetrazole and bromine chloro-indole phosphate to exist and H 2O 2The method of reaction is identified.The result is shown in Figure 32 B.
Embodiment 18
Composition of medicine is to the inhibitory action of HSV virus
A. to the inhibitory action of HSV-1 virus
As described in example 7 above, the HSV-1 virion is available from the hela cell that infects, and on the porous culture plate in 24 holes, each hole cultivates 1 * 10 5Vero cell, every hole are added with 1ml RPMI-1640 culture medium, and contain 5%FCS, penicillin and streptomycin and 0.1% methylcellulose (15cps).At second day, after removing culture medium, will be by the virus 100 μ l of 10 times of dilution method serial dilutions, the duplicate adding in the cell hole, in Vero cell culture hole, add (i) more respectively and only add the as many as 50 μ g/ml of GL-288(of a certain specific concentrations), (ii) only add the acycloguanosine (as many as 50 μ g/ml) and the 25 μ g/ml GL-288 of a certain specific concentrations; Perhaps (iv) acycloguanosine of a certain specific concentrations (as many as 50 μ g/ml) and 50 μ g/ml GL-288.
After 37 ℃ are cultivated 2 hours, remove virus, in the cell culture hole, add the RPMI-1640 culture medium methylcellulose (4K cps) that 0.5ml contains 2%FCS and penicillin, streptomycin.After two days, remove this culture medium.50% alcoholic solution pair cell dyeing with 0.8% crystal violet.Count formed viral plaque number, calculate virus titer from extension rate.
Shown in Figure 34 A, the viral minimizing that must measure is the function (increase with drug level, viral must measure minimizing) of compound concentration.
B. to the inhibitory action of HSV-2 virus
As described in example 7 above, the HSV-2 virion is available from the hela cell that infects.With above described in the A, the Vero cell dyes with the virion serial dilution liquid inductance that only is added with GL-288, or dyes with the viral dilution liquid inductance that only is added with acycloguanosine, and perhaps the viral dilution liquid inductance with adding acycloguanosine and GL-288 dyes.After 37 ℃ are cultivated 2 hours, remove viral liquid, in the cell culture hole, add 0.5ml and contain methylcellulose (4K cps) in the RPMI-1640 culture medium of 2%FCS and penicillin, streptomycin.After two days, remove this culture medium, cell dyes with 50% alcoholic solution of 0.8% crystal violet.Count formed viral plaque number, calculate virus titer from extension rate.
Shown in Figure 34 B, the viral minimizing that must measure is the function of compound concentration.
Embodiment 19
Inhibitory action to the HSV virus activity: cytopathic effect
The Vero cell is kept in the RPMI-1640 culture medium that is added with 5% hyclone, penicillin 100 units/ml, streptomycin 100 μ g/ml, is containing 7%CO 2The damping incubator in 37 ℃ of cultivations.Test is HSV-1(Kos-1 with HSV virus) strain and HSV-2(333) strain.
The preparation of chemical compound Y-1 topical preparation is that Y-1 is dissolved in (Johnson ﹠amp in the K-Y gel; Johnson), make 5%, 10%, 15% and the 20%(w/w) drug level.Then, each Y-1 preparation is dissolved in containing the RPMI-1640 culture medium of 2%FCS and dilutes.
On the microdroplet culture plate in one 96 hole, each hole adds 0.2ml and contains 5%FCS and 0.1% methylcellulose (15 cps) and RPMI-1640 culture medium, with 1 * 10 5The Vero cell places each hole to cultivate.Overnight incubation, cell is done after the logarithmic (log) phase multiplication, culture medium is gone in suction, the RPMI-1640 culture medium that contains following ingredients with 100 μ l replaces: 2%FCS, 50 μ l contrast liquid or drug solution and 50 μ l virus HSV-1 or HSV-2, the final concentration of medicine is respectively 5 μ g/ml, 10 μ g/ml, 20 μ g/ml and 40 μ g/ml, the content of virus is approximately the 3PFU/ cell, and promptly 6 * 10 5The PFU/ hole.
Cell was cultivated 24 hours at 37 ℃, at this moment between in, cytopathic effect is obviously as seen.When not being infected by the virus, cell forms uniform fibroblast monolayer.After being infected by the virus, cultured cell becomes the cell suspension of garden shape, cell agglutination subsequently, and its outward appearance is easy to distinguish with normal fibroblast.One group in the parallel work cell of virus inoculation not simultaneously is as the cytotoxicity contrast to the Vero cell.Use the K-Y gel not see any cytotoxicity separately.Result of the test is as shown in table 12, and "+" expression cytopathic effect suppresses fully in the table, and "-" expression cytopathic effect does not suppress entirely.
Embodiment 20
With the Y-1 preparation of K-Y gel preparation inhibitory action to the viral-induced cell fusion of HIV
With people CD 4 +Indicator cells (VB) and chronically infected H 9Cell is kept in the RPMI-1640 culture medium that is added with 5% hyclone, 100 units/ml penicillin and 100 μ g/ml streptomycins, is containing 7%CO 2The damping incubator in 37 ℃ of cultivations.Used HIV virus is the HTLV-III BWith the strain of RF-II, (Bethesda MD) obtains from National Institutes of Health.
The preparation of chemical compound Y-1 topical preparation is that Y-1 is dissolved in K-Y gel (Johnson ﹠amp; Johnson) in, make 5%, 10%, 15% and the 20%(w/w) drug level.Then, each Y-1 preparation is dissolved among the PBS, the final concentration that makes Y-1 is 1mg/ml.
Measure in order to make cell fusion, in one 96 flat culture plate in hole of each Y-1 compositions serial dilution liquid immigration.Add the chronically infected H of 25 μ l to each hole again 9Cell (2 * 10 6Cell/ml), perhaps add 25 μ l by the chronically infected cell of RF-II strain HIV virus was cultivated 45 minutes in 37 ℃.Add 25 μ lVB cells (about 5 * 10 to each hole then 4Cell), and this cell and viral chorista containing 5%CO 2The wet air incubator in, coexistence was cultivated 18 hours.The degree that syncytium forms is observed under phase contrast microscope and is kept the score.Syncytium forms note " 4+ " when suppressing fully.Do not see that syncytium forms note: "-".Result of the test is as shown in table 13.
Although the present invention is described the experimental technique of this chemical compound inhibition virus of chemical compound and application preferably, can carry out various corrections and change in the case of without departing from the present invention.

Claims (34)

1, the method that suppresses the infection of coated virus cell, this method comprises deutero-calix (n) aromatic compound to effective dose on the infection site administering therapeutic, and this chemical compound has the polar substituent of terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group on position ring position between ring position that bridged bond connects.
2, the process of claim 1 wherein that the subunit number in the chemical compound (n) is 4～10.
3, the process of claim 1 wherein that calix (n) aromatic compound is partly oxidized.
4, the process of claim 1 wherein that calix (n) aromatic compound has the general structure of following formula,
N=4,6 or 8 wherein;
R 1For OH or=O or its mixture;
R 2For having the polar substituent of terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group;
R 4For＞CH 2Or 〉=CH, or its mixture.
5, the method for claim 4, wherein R 2Be (CH 2) mR 2', m=1-3 here, R 2' be the sulfonic acid group.
6, the method for claim 5, wherein the sulfonic acid group is SO 3R or SO 2NRR ', R and R ' are low alkyl group.
7, the method for claim 4, wherein R 2Be (CH 2) mR 2', m=1-3 here, R 2' be the sulfinate derivant group.
8, the method for claim 7, wherein the sulfinate derivant group is SO 2R or S(=O) NRR ', R and R ' they are low alkyl group.
9, the method for claim 4, wherein R 2Be (CH 2) m-R 2', m=0-3 here, R 2' be the carboxylic acid derivates group.
10, the method for claim 9, wherein the carboxylic acid derivates group is CO 2R or C(O) NRR ', R and R ' they are low alkyl group here.
11, the method for claim 4, wherein R 2Be (CH 2) m-R 2', m=0-3 here, R 2' be the phosphonate derivative group.
12, the method for claim 11, wherein the phosphonate derivative group is PO(OR) 2, PO(OH) (OR), PO(OR) (NRR '), PO(NRR ') 2, respectively do for oneself H or low alkyl group of R and R ' here.
13, the described method of claim 1 is used for the treatment of HIV, RSV, HSV-1 or HSV-2 viral infection, and wherein chemical compound is an oral administration.
14, the described method of claim 1 is used for the treatment of HIV, RSV, and HSV-1 or HSV-2 viral infection, wherein chemical compound is an intravenous administration.
15, the described method that is used for oral or parenteral administration of claim 1, this method also comprises the protamine sulfate of using effective dose to the receptor, to suppress the blood coagulation resisting function of calix (n) aromatic compound.
16, the method for claim 1, this method also comprise to the receptor uses antiviral nucleoside analogs.
17, the method that suppresses infection of coated virus through spreading through sex intercourse, this method comprises the lubricating gel agent compositions that possible property contact area topical administration is contained the macrocyclic compound that prevents effective dose, this macrocyclic compound is made of the aromatic ring subunit that the bridged bond by connecting ring connects, the result has formed and has constituted the continuous chain of the connection atom of macro ring skeleton, and also contains electronegative substituent group on the non-skeletal atom of this macrocyclic compound aryl subunit.
18, the method for claim 17, wherein compositions is used with physical barrier type utensil.
19, the method for claim 17, wherein chemical compound has following form:
Figure 941011933_IMG3
N=4,6 or 8 wherein;
R 1For OH or=O, alkyl or aryl ether, ester, acid or its mixture;
R 2For having the substituent polar substituent of terminal negative charge, terminal negative charge substituent group is selected from sulfonic acid group, sulfinate derivant group, carboxylic acid derivates group and phosphonate derivative group;
R 4For＞CH 2Or 〉=CH, or its mixture.
20, the method for claim 19, wherein R 2At least one is=O in the group.
21, the method for claim 17, wherein enveloped virus is selected from hepatitis δ virus, hepatitis b virus, hepatitis C virus, human papillomavirus, HSV-1, HSV-2, HIV-1, HIV-2, HTLV-I and HTLV-II.
22, the method for claim 17, wherein said virus are selected from hepatitis δ virus, hepatitis b virus and hepatitis C virus.
23, the method for claim 17, wherein said virus is selected from HSV-1 and HSV-2.
24, the method for claim 17, wherein said virus are selected from HIV-1, HIV-2, HTLV-I and HTLV-II.
25, calix (n) aromatic compound that has the following formula structure,
N=4～10 wherein;
R 1For OH or=O, at least one R in calix (n) aromatic compound here 1Group is=O;
R 2For having the polar substituent of terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group;
R 4For＞CH 2Or 〉=CH, or its mixture.
26, the chemical compound of claim 25, wherein R 2Be (CH 2) mR 2', m=1-3 here, R 2' be SO 3R or SO 2NR ' R ", R wherein, R ' and R " respectively do for oneself H or low alkyl group.
27, the chemical compound of claim 25, wherein R 2Be (CH 2) m-R 2', m=0～3 here, R 2' be CO 2R ' or C(O) NR ' R ", R here, R ' and R " respectively do for oneself H or low alkyl group.
28, the chemical compound of claim 25, wherein R 2Be (CH 2) m-R 2', m=0～3 here, R 2' be PO(OR) 2Or PO(OH) (OR), R is H or low alkyl group here.
29, calix (n) aromatic compound that has the following formula structure,
N=4～10 wherein; R 1Be OH; R 2For having the polar substituent of terminal carboxylic acid derivates or sulfinate derivant group; R 4For＞CH 2Or 〉=CH or its mixture.
30, suppress the pharmaceutical composition of infection of coated virus cell, comprising:
Give deutero-calix (n) aromatic compound of effective dose on the infection site administering therapeutic, this chemical compound has the polar substituent of terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group on position ring position between ring position that bridged bond connects.
31, suppress the Pharmaceutical composition of the infection of coated virus through spreading through sex intercourse, said composition comprises:
Deutero-calix (n) aromatic compound, this chemical compound have the polar substituent of terminal carboxylic acid derivates, phosphonate derivative, sulfinate derivant or sulfonic acid group on position ring position between ring position that bridged bond connects.
32, the compositions of claim 31, wherein enveloped virus is selected from hepatitis δ virus, hepatitis b virus, hepatitis C virus, human papillomavirus, HSV-1, HSV-2, HIV-1, HIV-2, HTLV-I and HTLV-II.
33, be used to suppress the physical barrier type utensil of the infection of coated virus through spreading through sex intercourse, it comprises:
Physical barrier type utensil with by carrier be dissolved in the combination of compositions application that the macrocyclic compound in the carrier forms, described macrocyclic compound is that the aromatic ring subunit that the bridged bond by connecting ring connects constitutes, the result has formed and has constituted the continuous chain of the connection atom of macro ring skeleton, and also contains electronegative substituent group on the non-skeletal atom of this macrocyclic compound aryl subunit.
34, the utensil of claim 33, wherein this utensil is condom, barrier film or cervix uteri sponge.
CN94101193A 1992-08-06 1994-02-03 Inhibition and treatment of infection by enveloped virus with calix(n)arene compounds Pending CN1100302A (en)

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AU4803393A (en) 1994-03-03

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