Patent Application
20090170890
Those of skill in the art have recognized that it would be desirable to identify agents that inhibit more than one specific viral agent, so that different viral infections could be inhibited with a single agent or family of agents. By the same token, it would be desirable to identify such as “pan viral” agent that did not put survival pressure on the virus itself, so as to avoid being be defeated by the frequent mutations exhibited by the viral population. Until now, given the extreme variety of viral infectious modes and characteristics across the various viral families, it has been difficult to establish a common therapy. One pathway, apparently mediated by TSG101, is the subject of ongoing studies. Inhibition via this pathway, however, is primarily based on the generation of selective antibodies, and no specific agent or composition has been identified to interfere with these pathways on a commercial basis. These efforts are discussed, inter alia, in U.S. Pat. Nos. 6,835,816 and 6,248,523.
Recently, a family of compounds were identified as having implications for anti-bacterial activity, and in particular, anti-botulism activity. These compounds are identified in U.S. patent application Ser. Nos. 11/464,001 and 11/464,007, both of which are incorporated by reference herein in their entireties. The compounds, per se, were originally identified in a screen for compounds having anti-tumor activity. It would be truly unusual if such compounds exhibited anti-viral activity as well as anti-bacterial activity. Recently, as described in 60/884,928, also incorporated by reference herein, two related compounds were indicated to have some ability to protect mice from challenge from Ebola virus. In the United States of America, and much of the rest of the world Ebola is a viral threat only in terms of a weapon deliverable, as in “germ warfare.” The treatment of other virus infections by a limited class of therapeutic agents is yet to be established.
1. Field of the Invention
The present invention relates to a set of compositions comprising a family of small molecules that are further effective at preventing, treating, and/or managing viral infection caused by a variety of virus types. The present invention further relates to methods of administration of compositions of the present invention to a patient, human or animal experiencing or at risk of viral infection as well as methods for screening additional compounds to identify related inhibitors that are further effective at inhibiting viral infection. This invention also offers a possible fundamental biological activity pathway for the inhibition of viral infection, by identifying a possible mode of action (MOA) of the studied compounds, and others, as selective Caspase inhibitors.
2. Related Art
Viruses have long been known to be the causative agent in a wide variety of human and animal infectious diseases associated with human and animal morbidity and mortality. Many different viral pathogens have consistently caused debilitating or fatal diseases in humans and animals (e.g., influenza, etc.) while others are emerging or re-emerging (e.g., HIV, West Nile virus, SARS, etc.).
Efforts to treat or prevent viral infection in both animals and humans may be generally classified into two broad categories: vaccines and antiviral drugs. Vaccines generally work by priming the immune system of an individual through administration of an immunogen. The immunogen is typically a killed virus, an attenuated virus, or a viral subunit that is incapable of causing infection but is sufficient to trigger an immune response. Since the immunogen resembles the live virus targeted by the vaccination, the immune system is able to readily identify and eliminate the virus during early stages of actual infection. When available, vaccines are very effective at immunizing individuals against particular viruses that cause disease.
However, vaccines are often limited in that they are generally only effective in immunizing individuals prior to infection (i.e., they are ineffective as a means for treating infected individuals that may or may not yet be experiencing symptoms of disease). Furthermore, vaccines are often ineffective in vaccinating individuals against viruses that are highly mutable since these viruses are able to evade any immunity generated by vaccination.
Research has also focused on developing antiviral medications as a means for treating individuals who are already infected as well as treating or preventing viral disease where vaccination methods are seen as unavailable or unlikely. Such approaches toward developing antiviral medications have generally sought to identify molecules or drugs that interfere with the basic mechanisms or steps of viral infection, through what is called “rational drug design.” Alternatively, antiviral drugs, such as interferons or antibodies, may instead be designed to broadly stimulate the immune system against a range of pathogens.
In general, viruses proceed through a series of steps akin to the following during their normal infection cycle: (1) attachment (i.e., specific binding between viral capsid or coat proteins and receptors on the host cell surface), (2) penetration (i.e., entry into the host cell generally through endocytosis or membrane fusion), (3) uncoating (i.e., digesting or degrading the viral coat to allow the contents and viral genome to be released into the cell), (4) replication
and assembly (i.e., the synthesis of new viral proteins and DNA/RNA, including intermediates, necessary to form new virus particles), (5) maturation (i.e., post-translational modification and processing to form mature virus particles), and (6) release or budding (i.e., freeing the virus particles to infect new host cells). However, not all viruses proceed through all of these steps in the manner summarized. For example, HIV undergoes maturation after being released from the host cell.
Despite noted success in the design and development of novel antiviral drugs in recent years, including the development of protease inhibitors, existing therapies are limited in terms of the number and breadth of viruses that they may be used to treat. In addition, many strains of viruses have become resistant to antiviral drugs as a result of mutation of their viral genomes. Accordingly, there continues to be a need in the art for the development of new classes of antiviral drugs to treat or prevent viral disease, especially those that show promise against a variety of virus types. There also continues to be a need in the art for the development of novel antiviral drugs that are effective against highly mutable viruses that are generally capable of evading treatment via existing vaccines and drugs.
Subsequent to the effective filing date of this application, a broadened description of the effectiveness of the family of compounds addressed in provisional U.S. Patent Application 60/884,928 was presented in U.S. patent application Ser. No. 12/013,640. Applicants have further expanded the range of activities, mechanisms and family of compounds that offer activity against one or more viruses and viral families. At the time of filing this Application, the assignee of this application, Functional Genetics, Inc. is the exclusive licensee of U.S. patent application Ser. No. 12/013,640.
The compounds identified as effective against viral infection in mammals are drawn from the broad class of compounds previously identified in U.S. Ser. No. 60/884,928 and Ser. No. 12/013,640. As set forth in the detailed description of this invention, additional information has been developed that allows identification of a more limited family based not only on structural similarity, but on activity. Activity of compounds is demonstrated herein by both in vitro methods, largely reflected as cell based assays, and in vivo experimentation, largely involving challenge based methodologies. Tolerance for these molecules, as well as general levels of effectiveness are also given.
Specifically, the compounds of the present invention are drawn from the family of compounds described by the following structural formula:
where Y is
Wherein
n is 1 or 2;
X1, X2, X3, X4, X5 and X6 are each independently N, S, O, SO2, CR7 or NR8 and at least one of X1, X2 is N, S, O, SO2 or NR8;
L is a linker which may be a direct bond or
where Z is an optionally substituted alkyl, alkenyl, dialkenyl, trialkenyl, or aryl, or C(O)NH; and
R1, R2, R3, R4, R5, R6 and R7 are each independently hydrogen, amino, amine with stabilized carbocations, carboxyl, optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryoxy, cycloakloxy, heteroaryloxy, aloxycarbonyl, alkylamino, carbornoyl, alkylaminocarbonyl, alkysulfhydryl, alkylhydroxymate;
R8 is hydrogen, OH, a halogen, or an optionally substituted alkyl;
In some embodiments, at least one of R1, R2, R3, or R4 is hydrogen, amidine, 2-imidazoline, amino, guanidine, methyl, aminomethyl-hydroxamine, or methylamine-guanidine. In some embodiments, R5 is hydrogen, amidine, 2-imidazoline, amino, guanidine, methyl, aminomethyl-hydroxamine, methylamine-guanidine, 4-oxy-benzamidine, 1H-indole-6-caboxamidine, or 1H-indole-5-carboxamidine. In some embodiments, R6 is hydrogen, amidine, benzamidine, benzimidazoline, imidazoline, guanidine, imidazole, oxazole, benzofuran-2-yl-imidazoline, benzofuran-2-yl-amidine, benzofuran-2-yl-guanidine, benzothiophene-2-yl-imidazoline, benzothiophene-2-yl-amidine, benzene-2-yl-amidine, benzofuran-2-yl-imidazole, or benzofuran-2-yl-oxazole. In some embodiments, at least one of X1 or X2 is N, NH, S, O, SO2, CH, C—CH3, C-phenyl, N-ethanol, N-chloroethyl, C-amino, C-(2-indole-6-imidazoline), C-(2-indole-6-amidine), C-(2-indole-5-imidazoline), or C-(2-indole-5-amidine). In some embodiments, at least one of X3, X4, X5, or X6 is N, NH, S, O, SO2 or CH. In some embodiments, least one of R1, R2, R3, R4, R5, R6, or R7 is —H, —CH3, —NH2,
In some embodiments, R5 is
In some embodiments, R6 is
In some embodiments, R7 is —H. —CH3, —NH2,
In some embodiments. R8 is —H, —(CH2)2OH, or —(CH2)2Cl. In some embodiments, L is a direct bond,
In some embodiments, the compound has the following structural formula:
Compounds of particular interest include NSC compounds 369723, 294199, 306365, 300510, 294206 and 294202, as well as Compounds 50410 and 50413. Collectively, these, and the compounds set forth in
This application involves extensive data, as is appropriate for a patent application directed to a family of compounds that exhibit activity against multiple families of viruses. The application bears a distinct organization. Below are described a number of Figures that provide certain structural information, followed by activity information for each of the three most tested compounds. The detailed description of this application parallels these Figures, devoting attention to the class collectively, and then the studied performance of each of three different active compounds. Following that, common activity of the compounds in terms of fundamental biological pathways, as well as routes of administration and effectiveness, are presented. Thereafter the claims are presented.
This invention is described by reference to drawings and figures set forth in the application as originally filed, discussed hereinbelow. The drawings are briefly described below:
The same type of information is given in
Protection against Marburg virus challenge, in vivo, is set forth in
Like 723, Compound 365 has been tested in vivo as well as in vitro.
Compounds 723 and 365 are both balanced four ring structures. The effective FGI compounds also include active three ring compounds.
Compound 57 demonstrates the reduction in viral titer obtained against cowpox using Compound 510 in a cell based assay.
Compound 58 shows, by protein blot, graph and table, the results from using 5110 to treat Hepatitis C Virus, part of a family of Flavivruses that present both health and bioterrorism threats around the world.
In a Vero Cell assay, compound 510 proved effective against West Nile Virus, as shown in
In
In
In
As with other FGI-103 compounds studied, as shown in
The invention of this application finds applicability in the treatment of viral infection. While there is a vast collection of identified viral threats for both animals and humans that have yet to be catalogued and tested, the compounds of FGI-103 have shown themselves to be active in treating a wide range of viruses. These compounds have demonstrated 100% survival when used to treat, either therapeutically or prophylactically, mice challenged by Ebola virus. To the best of the inventors' knowledge, this has not been demonstrated by any agent to date. Ebola is just one of many “bioterrorism” viral threats that the United States government, and others, have identified as posing a potential weapon. The same compounds have simultaneously demonstrated effectiveness against viruses that may be difficult to vaccinate against, such as influenza. The Flu Vaccine works poorly in the elderly and other immune-compromised individuals who are at particular risk from the constantly mutating strains of influenza. To date, no strain has shown the ability to escape the protection conferred by the compounds of the FGI-103 family.
The effective compounds of the invention are not so limited, however. Having been demonstrated effective against viruses in both in vitro and in vivo tests, the compounds of this invention have also been demonstrated as effective in treating animal viruses, such as PRRS Virus and Bovine Corona virus, as well as other viruses, which pose both worldwide health problems and bioterrorism threats, such as SARS and Dengue viruses.
If one of skill in the art takes the compounds of
Y—X—Y1,
with the proviso that one of Y or Y1 may not be present,
Wherein Y and Y1 are each, independently, an alkyl group of 1-6 carbon atoms which may be straight, branched or cyclic, saturated, partially unsaturated or aryl and wherein each of Y and Y1 present comprises two nitrogen atoms bearing one or two hydrogen atoms, with the proviso that up to one of said nitrogen atoms may constitute a ring atom in the event Y is cyclic and bear no hydrogen atoms,
And wherein X is comprised of 3 or 4 ring moieties Z, wherein each Z is independently 5 or 6 atoms and may be saturated, unsaturated or aryl, any 2, 3 or 4 Z may be fused with an adjacent Z, wherein any unfused ring moieties Z are bound to at least one other ring moiety Z by a single bond, or an ethyl or propyl group which may bear an oxygen or nitrogen atom bound by a single or double bond, and wherein said X comprises 1-5 hetero atoms, each independently O, N or S, in place of a ring or ethyl or propyl carbon atom, and wherein said compound may further bear up to
One or two derivatizing moieties selected from the group consisting of hydroxyl (—OH), amino (NH2), amido, chloro, fluoro or other halogen, alkoxy (—OR), aryloxy (—OAr), trialkylammonium (—NR3+), alkylamido (—NHCOR, —NRCOR′), arylamido (—NHCOAr, —NRCOAr, —NArCOAr), arylcarbamoyl (—NHCOOAr, —NRCOOAr), alkylcarbamoyl (—NHCOOR, —NRCOOR′), cyano (—CN), nitro (—NO2), ester (—COOR, —COOAr), or alkyl halo, wherein, each case, said alkyl, R or AR group is from 1-6 carbon atoms.
Medical and scientific terms used herein are used in their standard and ordinary meaning whenever possible. The term “treating” as used herein means preventing, limiting or slowing infection by a virus. Thus, a host may be treated by administration of an FGI-103 compound where that host is given the FGI-103 compound before entering a danger area where the likelihood of infection by a virus is high. This type of treatment has particular application for servicemen and support personnel being deployed into areas where a virus against which there is little natural protection, such as Ebola, is known to be prevalent, or in the event of pandemic infection, such as that presented by influenza, where prophylactic protection is critical.
A host, such as a mammal (representative mammals include humans, commercial domestic animals such as horses, pigs, cows, sheep, goats and the like, animals of value in assays and discovery, including mice, rats, rabbits and their counterparts, as well as non-commercial animals like dogs, cats, monkeys and related domestic animals. It is also possible to treat viral infections in wild animal populations, such as elk, deer, wolves, bison and the like using the compounds of this invention) may be treated by having his virus “cured”—that is, no viral infection remaining in the host's body—or having the virus inhibited, that is, infectious particle generation reduced to a lower level such that the body's own natural resources can catch up with viral propagation and overwhelm the viral invader. In another form of treatment, the administered compound may slow the disease or extend survivorship in time to bring other treatments into play.
Administration may be through any of a variety of routes. Conventional routes of administration include IV, IM and IP. Data suggests that compounds properly derivatized may be administered orally, as shown in
Although presented below are three separate intensive case studies as Examples, it is worth looking at the FGI compounds as a whole. Comparative testing between FGI compounds against a selection of viral agents is set forth in
Another important aspect of the results demonstrated at this early date for the FGI 103 compounds is that they have been shown to be effective not only through in vitro cell based assays, but in vivo as well. It is rare that a class of compounds holds its demonstrated in vitro activity in against a wide variety of viral agents, including RNA virus and DNA viruses, but retain that activity when tested in vivo. Bear in mind that each of the FGI103 compounds structurally presented in
These results clearly suggest that the FGI-103 compounds will have activity against a large number of viruses including Group I viruses (as demonstrated by activity against pox viruses); Group IV viruses (as demonstrated by activity against bovine corona viruses); Group V (represented by Ebola virus) and other families of viruses as represented by the viruses tested as presented in the Figures of this application.
Initial results, together with information on the mode of action of these compounds, clearly indicates that these compounds will have activity against other groups of viruses based on the broad-spectrum activity associated with targeting of the host. These would include viruses in Groups II, III, VI and VII.
Group I: viruses possess double-stranded DNA and include such virus families as Herpesviridae (examples like HSV1 (oral herpes), HSV2 (genital herpes), VZV (chickenpox), EBV (Epstein-Barr virus), CMV (Cytomegalovirus)), Poxyiridae (smallpox) and many tailed bacteriophages. The mimivirus was also placed into this group.
Group II: viruses possess single-stranded DNA and include such virus families as Parvoviridae and the important bacteriophage M13.
RNA viruses
Group III: viruses possess double-stranded RNA genomes, e.g. rotavirus. These genomes are always segmented.
Group IV: viruses possess positive-sense single-stranded RNA genomes. Many well known viruses are found in this group, including the picornaviruses (which is a family of viruses that includes well-known viruses like Hepatitis A virus, enteroviruses, rhinoviruses, poliovirus, and foot-and-mouth virus), SARS virus, hepatitis C virus, yellow fever virus, and rubella virus.
Group V: viruses possess negative-sense single-stranded RNA genomes. The deadly Ebola and Marburg viruses are well known members of this group, along with influenza virus, measles, mumps and rabies.
Group VI: viruses possess single-stranded RNA genomes and replicate using reverse transcriptase. The retroviruses are included in this group, of which HIV is a member.
Group VII: viruses possess double-stranded DNA genomes and replicate using reverse transcriptase. The hepatitis B virus can be found in this group.
We have discovered that many of the host mechanisms necessary for viral propagation utilize targets that are highly conserved among mammalian or eukaryotic species. Consequently, these compounds could have application for human or veterinary viral diseases. These viral diseases could include but are not limited to PRRS virus, porcine or bovine circo viruses, porcine or bovine corona viruses, porcine or bovine RSV, porcine, bovine or avian influenza, EIAV, bluetongue, or foot and mouth diseases (FMD) viruses.
Some viruses are causative of more chronic diseases and the morbidity or mortality relates to the presence of virus. These diseases include hepatocellular carcinoma (associated with either HBV or HCV), chronic fatigue syndrome (associated with EBV) and other diseases linked with viral infection. The FIG-103 compounds should be effective in the treatment of these virus-related diseases, as well as the viruses themselves.
The FGI-103 compounds could be used for the treatment or prevention (prophylaxis) of single viral pathogens (e.g., HIV or HBV) or combinations thereof (HIV and HBV). Likewise, these individual or broad-spectrum applications could entail any or all of the virus groups detailed above.
Another method could be the use of the compounds for certain indications associated with one or more viruses in humans and animals. For example, these compounds could be used for the prevention or treatment of respiratory virus infections, which can be caused by one or more of the pathogens from the groups identified above. Likewise, these compounds could have application against one or more blood-borne pathogens (e.g., HIV and/or HBV and HCV).
The compounds could have application for the prevention, treatment or maintenance of acute or chronic viruses in humans. Acute applications include short-term prevention or treatment of viral infection, examples of which include influenza, rotavirus or filovirus infection. Chronic applications could include recurrent outbreaks, such as is observed with genital herpes) or infrequent outbreaks (such as those associated with zoster infection during shingles). Likewise, treatment could be intended over the long term to maintain low levels of viral load for chronic virus infection (e.g., for HIV, HBV or HCV treatment). The assays set forth herein, demonstrating effective protection, prophylactically and/or therapeutically, against a wide number of viruses, both in vitro and in vivo can of course be used to assess the activity of other FGI103-compounds responsive to the chemical structural formula provided.
The various diseases against which this compound has been tested in some format are set forth in
The effectiveness, at nanomolar levels, of 723 in treating Hepatitis B virus (HBV) is set forth in
Dengue fever, and similar viruses such as Hepatitis C virus and West Nile virus, are also treated by administration of 723. The cells used in the assay reflected in
Among the most dramatic and feared of viral invaders, and those most widely identified as bioterrorism threats, are Hemorrhagic Fever viruses, like Ebola and Marburg. In vitro tests against these viruses showed 100% infection inhibition at relatively low dosages. The EC50 value for this member of the FGI-103 compound family is at or below 0.050 uM, a remarkably low dose.
Many compounds shown to be effective against viral challenges in vitro lose potency, or fail to exhibit similar prophylactic or therapeutic efficacy, in vivo. There are a variety of reasons for this—fundamentally, mammalian systems are complicated—no matter what route of administration is used, there are fundamental obstacles to delivering in vivo the same type of anti-viral effectiveness frequently observed in vitro. As demonstrated in
Importantly, as depicted in
As shown in
An important parameter in developing a prophylactic or therapeutic agent for administration to mammals, particularly humans, is the time the agent remains in the blood, frequently measured as serum concentration. The time course for serum concentration for 723, for mice, receiving 10 mg/kg of 723 at time 0 and 12 hours, is set forth in
Agents of the type addressed, that have effectiveness in treating infection in a large number of mammalian hosts against a range of viruses are always desirable. The FGI-103 family of compounds presents just this type of opportunity. It is always most desirable, however, to provide an agent that can be taken orally, and provide protection. This permits more widespread distribution, and greater follow-through—patients are more easily dosed with an oral formulation than with those that require inoculation. To demonstrate the suitability of 723 and other 103 compounds for oral administration, compound 723 was “derivatized” to provide a prodrug that would be digested to provide the active agent, by modifying the amide groups with hydroxy moieties, as shown in
Safety is always a paramount consideration when formulating an agent to be administered to mammals, including humans. CC50 (a measure of a compound's cytotoxicity) is thus a parameter of fundamental importance. As shown in
The compounds of FGI-103 as a group are not all novel compounds, per se. Many have not been prepared in pharmaceutically pure condition, or in formulations suitable for administration (suspensions and solutions in a pharmaceutically acceptable carrier). The use of 723 and other FGI-103 compounds requires a synthesis method that facilitates this kind of pharmaceutical preparation. A suitable synthesis scheme is set forth in
Compound 723 is a four ring compound of the general formula Y—X—Y1 described above, with two fused ring systems. As shown in
Hepatitis C virus is a blood born pathogen for which no known vaccine exists. The effectiveness of 365 against HCV is shown in
Compound 723 was tested against Dengue Fever. This important virus, and potential terror weapon, is also strongly inhibited by Compound 365, at very low levels, as reflected in
Ebola is of course an important virus to be evaluated. Ebola constitutes a potential bioterrorism threat, and is endemic in parts of the world where humans with no native protection may be dispatched. 365 shows powerful inhibition of Ebola virus. As reflected in
As noted with Compound 723, perhaps no virus has received as much attention as HIV in terms of potential treatments and cures. In
The development of anti-viral agents against viruses that infect humans is of paramount importance. Of similar importance is effectiveness against PRRS and similar viruses that destroy billions of dollars of livestock every year.
Compound 365 shows similar efficacy against Rift Valley Fever Virus, providing 100 percent inhibition (zero viral particle release) at safe values far below the CC50 index for this compound. With an EC50 of only 0.37 uM, this agent provides a large safety index of 30 for the treatment of RVFV. This data is reflected in
SARS is a virus that swept through parts of China and Asia, without prevention or treatment other than supportive treatment. A related virus, Bovine Corona Virus, presents a commercial threat. Both are inhibited by 365, without crossing the “Safety Barrier” by 365. While more potent agents are typically less safe, 365, like the other compounds of the FGI-103 family, presents opportunities to treat viruses in humans and commercially important mammals without sacrificing safety.
Like Compound 723, Compound 365 has also been shown to be effective in vivo as well as in vitro. As noted above, a method of therapeutically treating Ebola at this time is unknown. Most “treatment” consists of supportive measures in hopes the body's natural immune systems can address the virus. As shown in
Although 365 did not ultimately improve survival outcomes for mice challenged with pox virus (when administered at day 0, 2 and 5) it did uniformly extend survival times. Extending survival time, as noted above, is of importance in treating agents that have no known inhibitors, providing time for the body's natural defenses to work against the invader. The effectiveness of 365 in extending survival times against pox virus is set forth in
As with 723, 365 is intended for treatment of humans, and commercially or veterinarily important mammals. Accordingly, safety is a paramount concern. As shown in
Like 723, once administered, 365 leaves the serum relatively rapidly, the time course of serum concentration following a 5 mg/kg dose given to BALB/c mice being reflected in
As discussed above, 365 presents strong anti-viral activity as a therapeutic, and prophylactically, against a wide variety of viruses. To provide a source of pharmaceutically pure and acceptable 365, and prepare suitable formulations including pharmaceutically appropriate suspension and solutions, a synthesis scheme must be provided that can be practiced in accordance with GLP. Such a scheme is set forth in
Compound 510, benzenecarboxylmidamide,4,4′-(2,5-thiphenediyl)bis (dihydrochloride as a salt) has a performance so similar to 723 that it is sometimes referred to as FGI's “backup” 103 compound. And yet, as reflected in
As has been previously discussed, Hemorrhagic Fever Viruses like Ebola and Marburg Virus present dangerous opportunities as terror weapons. The provision of an inhibitory agent that is safely tolerated by humans is therefore an important goal. 510, like its sister compounds, provides 100% inhibition of these viruses, as measured in terms of cell survival, reflected in
As shown in testing for 723 and 365, inhibition of pox virus is an important and ordinarily difficult goal. 510 provides strong inhibition of pox virus, with low viral titers on Vero cells at modes dosages. The testing, reflected in
Like 723 and 365, demonstration of in vivo activity paralleling that predicted by in vitro testing is of importance if 510 is going to be an effective anti-viral agent. 510 does indeed give the same sort of in vivo protection against the same virus targets, conferring 100% protection when administered at 10 mg/kg at the time of, and days 2 and 5 following, Marburg Virus challenge. This data, set forth in
Like the other members of FGI-103 compounds, 510 is susceptible of simple synthesis from commonly available starting materials that will make it practicable to provide pharmaceutically pure compound, and pharmaceutically acceptable preparations including solutions and suspensions in a pharmaceutically acceptable carrier. A simple synthesis scheme of this compound is set forth in
It is rare that an initial compound identified by testing and structure, as is the case for the FGI-103 compounds, is the best possible compound of that type. It is frequently the case that derivatization of the starting compound, to embrace small substituent and substitution additions and subtractions, may give improved efficacy in the treatment of one or more viruses in one or more mammalian hosts. Actual examples of derivatization follow. Knowledge of a possible method of action, however, may guide the person skilled in the art in the proper course for derivatization. While the inventors are not bound by this theory, there is increasing evidence that the FGI-103 compounds act by selectively inhibiting certain Caspase enzymes (proteases) that are vital to virus particle assembly—that is, in the conventional life cycle of the virus, intervening late in that cycle, blocking some proteolytic action necessary for the virus to mature or be brought to the cell surface. It is not unreasonable to conclude that a critical digestion step where the virus “hijacks” a protease on the way to the surface is somehow inhibited by the FGI-103 compounds.
Caspases have traditionally been studied in connection with their role as effectors of apoptosis and inflammation. However, recent evidence has also pointed to additional roles for caspases in other cellular processes, such as cellular proliferation and cell-cycle progression. See, e.g., Los M., et al., Caspases: more than just killers? Trends Immunol. 22(1): 31-4 (2001); Algeciras-Schimnich A, et al., Apoptosis-independent functions of killer caspases. Curr Opin Cell Biol. 14(6): 721-6 (2002); Launay S., et al., Vital functions for lethal caspases. Oncogene 24(33): 5137-48 (2005. Current research at FGI, described herein as a basis in part for the present invention, suggests yet another role for caspases, wherein the protease activity of caspases is utilized by viruses during their infection cycle. Indeed, caspase activity appears to be critical or essential for viral infection by a variety of virus types in light of evidence, described herein, showing that FGI-103 caspase inhibitors interfere with and/or block the ability of viruses to successfully infect host cells. Therefore, it is generally advanced herein that various compositions and methods for inhibiting caspases may be effective in preventing, treating, and/or managing viral infection and disease.
Many of the FGI-103 compounds tested selectively inhibit caspases 2, 3 and 8, and to a lesser degree, Caspase 1. Collective values for this inhibition as a family are set forth in the graph of
The profile for Caspase inhibition by FGI 510 is specifically given in
The timing of intervention by FGI-103 compounds is reinforced by the data obtained from the MDCK cell assay that is reflected in the data set forth in
As noted above, given the identification of a particular method of action, and desired intervention (selective Caspase inhibition to prophylacitcally and/or therapeutically treat virus infections in mammals) it maybe become desirable to derivatize one or more core molecules of the FGI-103 Compound family, in order to improve efficacy, improve safety or tolerability, to tailor a compound to specific situations, modes of administration or viruses, etc. Derivatization, per se, is a well established methodology known to those of skill in the art. To further demonstrate how an FGI-103 compound may be derivatized however, Compound 365 was subjected to a series of derivatizations, as described below. These are offered not to identify compounds of improved or selective activity, but rather to demonstrate the susceptibility of the FGI-103 compounds to derivatization.
Synthetic Routes for 306365 (BG9), and its Derivatives (BG11, BG15, BG16, BG17, BG20)
Instrumentation
IR: Thermo-Scientific Nicolet 6700 FT-IR Diamond Crystal.
NMR: 1H and 13C NMR spectra were recorded on a
Varian Gemini-200 spectrometer (at 200 and 50 MHz, respectively), and
Bruker Ultrashield Advance III spectrometer (at 500 and 125 MHz, respectively), in the indicated solvent using TMS or TMS-Na salt as internal standard.
Chemical shifts are expressed in ppm (ä) values and coupling constants (J) in Hz.
ESI MS spectra of the synthesized compounds were recorded on an Agilent Technologies 6210 Time-of-Flight LC/MS instrument in the positive ion mode using CH3CN/H2O=1/1 with 0.2% HCOOH as the carrying solvent solution. The samples were dissolved in pure acetonitrile (HPLC grade). The selected values were as follows: capillary voltage 4 kV; gas temperature 350° C.; drying gas 12 l min-1; nebuliser pressure 45 psig; fragmentator voltage 70 V.
General Methods
All steps were performed at least in duplicate. The target compounds were characterized by IR, 1H and 13C NMR, HPLC-ESIMS spectra (bases and salts). Melting points of the salts were =>280° C. All salts were water soluble.
Synthesis of BG9
A suspension of 1,5-diaminonaphthalene 1 (5.0 g, 31.6 mmol), ethyl acetoacetate 2 (40 mL, 313.5 mmol) and p-TsOH (2.38 mg) in 250 mL of anhydrous ethanol was heated to reflux in a two-neck round bottom flask equipped with Dean-Stark trap filled molecular sieves (4 Å). After heating for 4 hours in an oil bath at a temperature of 120° C. with stirring, the mixture was cooled to room temperature and evaporated to dryness (upon cooling precipitation occurs). The crude product was triturated with ethanol, and precipitate was collected on Büchner funnel and dried under reduced pressure to give BG5 (2-butenoic acid, 3-[[5-[[(E or Z)-3-ethoxy-1-methyl-3-oxo-1-propenyl]amino]-1-naphthalenyl]amino]-, ethyl ester, (E or Z)-) as the product with a yield of 11.39 g (94%). IR of BG5 (ATR): 3442, 3253, 2977, 2923, 1652, 1606, 1506, 1440, 1267, 1158, 787 cm−1. A mixture of BG5 (2.00 g, 5.23 mmol) and Eaton's reagent (5.5 mL, 29.1 mmol, commercial, Aldrich) was stirred at 90° C. for 5 hours. The reaction mixture was cooled to 5° C. in an ice bath and carefully poured onto saturated ice-cold Na2CO3 solution (31 g Na2CO3/100 mL H2O). The solid was filtered, filtrate was washed with water (3×50 mL), EtOH (10 mL) and dried under reduced pressure at 50° C. to give BG6 (quino[8,7-h]quinoline-1,7-dione, 4,10-dihydro-3,9-dimethyl-) as a product with a yield of 1.4276 g (94%). IR of BG6 (ATR): 3227, 3169, 3059, 2978, 1630, 1601, 1546, 1520, 1492, 1434, 1354, 1211, 1153, 1029, 843 cm−1. 1H NMR of BG6 (200 MHz, TFAd): 9.15 (d, J=9.6 Hz, 2H), 8.87 (d, J=9.6 Hz, 2H), 7.58 (s, 2H), 3.15 (s, 6H) ppm. 13C NMR of BG6 (50 MHz, TFAd): 172.18, 139.66, 130.28, 127.79, 125.15, 123.15, 121.93, 111.70, 22.27 ppm. A suspension of BG6 (1.37 mg, 4.73 mmol) in POCl3 (28.38 mL, 309.10 mmol) was heated at 90° C. for 6 hours, and continued at 120° C. for 14 hours. Upon cooling to room temperature, the mixture was slowly poured onto ice/water mixture and concentrated aqueous NH3 was added until pH 9. Formed precipitate was filtered and washed well with H2O and EtOH. The product BG7 (quino[8,7-h]quinoline, 1,7-dichloro-3,9-dimethyl-) was dried under reduced pressure at 60° C. with give a yield of 1.4442 g (93%). IR of BG7 (ATR): 2922, 1582, 1481, 1432, 1359, 1083, 1027, 877, 849, 829, 749 cm−1. 1H NMR of BG7 (200 MHz, TFAd): 9.30 (d, J=9.6 Hz, 2H), 8.78 (d, J=9.6 Hz, 2H), 8.18 (s, 2H), 3.10 (s, 6H) ppm. 13C NMR of BG7 (50 MHz, TFAd): 162.18, 158.98, 138.44, 129.65, 129.52, 128.30, 127.52, 126.19, 22.45 ppm. A suspension of BG7 (160 mg, 0.49 mmol) in 6 mL of Me2N(CH2)3NH2 was stirred at reflux under Ar for 48 h. Then the mixture was poured onto 40 mL of ice-cold water. The obtained solid was filtered, and the cake was washed with MeOH (5 mL) and dried at 45° C. under reduced pressure to give BG8 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[3-(dimethylamino)propyl]-3,9-dimethyl-) with a yield of 579.4 mg (86%). M.p.=257-260° C.; IR of BG8 (ATR): 3334, 2950, 2860, 2824, 2772, 1586, 1535, 1353, 1175, 1100, 1022, 840, 815, 760 cm−1. 1H NMR of BG8 (500 MHz, TFAd): 9.00 (d, J=9.5 Hz, 2H), 8.57 (d, J=9.5 Hz, 2H), 7.07 (s, 2H), 3.99-3.97 (m, 4H), 3.67-3.64 (m, 4H), 3.22 (s, 12H), 3.06 (s, 6H), 2.62-2.57 (m, 4H) ppm. 13C NMR of BG8 (125 MHz, TFAd): 158.13, 157.80, 137.58, 127.06, 122.40, 121.99, 103.02, 58.21, 45.36, 42.43, 25.41, 21.57 ppm. ESI-HRMS (m/z) of BG8: [M+2H]2+ 230.16557 (error 1.72 ppm), [M+H]+ 459.32310 (error 0.07 ppm). BG8 (300 mg, 0.65 mmol) was suspended in 10 mL of MeOH and the reaction mixture was vigorously stirred. 15 mL of 8.6 M EtOH/HCl solution was added and stirring was continued for 48 hours at room temperature. The solvent was removed under reduced pressure, and obtained solid was suspended in 21 mL of 96% EtOH, filtered, and the cake was washed with 9 mL of 96% EtOH. Upon drying at 45° C. under reduced pressure BG9 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[3-(dimethylamino)propyl]-3,9-dimethyl-, tetrachloride) was obtained (342.6 mg; 87%). BG9: Mp=>280° C.; IR (ATR): 3354, 3210, 2964, 2681, 2477, 1633, 1608, 1565, 1469, 1439, 1356, 1041, 830, 745 cm−1; 1H NMR (500 MHz, CD3OD): 8.99 (d, J=9.5 Hz, 2H), 8.66 (d, J=9.6 Hz, 2H), 7.16 (s, 2H), 3.83-3.79 (m, 4H), 3.44-3.40 (m, 4H), 2.98 (s, 12H), 2.94 (s, 6H) ppm; 13C NMR (125 MHz, CD3OD): 157.03, 156.76, 137.09, 126.26, 122.57, 121.64, 116.73, 103.00, 56.57, 43.77, 42.02, 24.75, 21.12 ppm.
Synthesis of BG11
A suspension of 1,5-diaminonaphthalene 1 (5.0 g, 31.6 mmol), ethyl acetoacetate 2 (40 mL, 313.5 mmol) and p-TsOH (2.38 mg) in 250 mL of anhydrous ethanol was heated to reflux in a two-neck round bottom flask equipped with Dean-Stark trap filled molecular sieves (4 Å). After heating for 4 hours in an oil bath at a temperature of 120° C. with stirring, the mixture was cooled to room temperature and evaporated to dryness (upon cooling precipitation occurs). The crude product was triturated with ethanol, and precipitate was collected on Büchner funnel and dried under reduced pressure to give BG5 (2-butenoic acid, 3-[[5-[[(E or Z)-3-ethoxy-1-methyl-3-oxo-1-propenyl]amino]-1-naphthalenyl]amino]-, ethyl ester, (E or Z)-) as the product with a yield of 11.39 g (94%). IR of BG5 (ATR): 3442, 3253, 2977, 2923, 1652, 1606, 1506, 1440, 1267, 1158, 787 cm−1. A mixture of BG5 (2.00 g, 5.23 mmol) and Eaton's reagent (5.5 mL, 29.1 mmol, commercial, Aldrich) was stirred at 90° C. for 5 hours. The reaction mixture was cooled to 5° C. in an ice bath and carefully poured onto saturated ice-cold Na2CO3 solution (31 g Na2CO3/100 mL H2O). The solid was filtered, filtrate was washed with water (3×50 mL), EtOH (10 mL) and dried under reduced pressure at 50° C. to give BG6 (quino[8,7-h]quinoline-1,7-dione, 4,10-dihydro-3,9-dimethyl-) as a product with a yield of 1.4276 g (94%). IR of BG6 (ATR): 3227, 3169, 3059, 2978, 1630, 1601, 1546, 1520, 1492, 1434, 1354, 1211, 1153, 1029, 843 cm−1. 1H NMR of BG6 (200 MHz, TFAd): 9.15 (d, J=9.6 Hz, 2H), 8.87 (d, J=9.6 Hz, 2H), 7.58 (s, 2H), 3.15 (s, 6H) ppm. 13C NMR of BG6 (50 MHz, TFAd): 172.18, 139.66, 130.28, 127.79, 125.15, 123.15, 121.93, 111.70, 22.27 ppm. A suspension of BG6 (1.37 mg, 4.73 mmol) in POCl3 (28.38 mL, 309.10 mmol) was heated at 90° C. for 6 hours, and continued at 120° C. for 14 hours. Upon cooling to room temperature, the mixture was slowly poured onto ice/water mixture and concentrated aqueous NH3 was added until pH 9. Formed precipitate was filtered and washed well with H2O and EtOH. The product BG7 (quino[8,7-h]quinoline, 1,7-dichloro-3,9-dimethyl-) was dried under reduced pressure at 60° C. with give a yield of 1.4442 g (93%). IR of BG7 (ATR): 2922, 1582, 1481, 1432, 1359, 1083, 1027, 877, 849, 829, 749 cm−1. 1H NMR of BG7 (200 MHz, TFAd): 9.30 (d, J=9.6 Hz, 2H), 8.78 (d, J=9.6 Hz, 2H), 8.18 (s, 2H), 3.10 (s, 6H) ppm. 13C NMR of BG7 (50 MHz, TFAd): 162.18, 158.98, 138.44, 129.65, 129.52, 128.30, 127.52, 126.19, 22.45 ppm. A suspension of BG7 (160 mg, 0.49 mmol) in 6 mL of Et2N(CH2)3NH2 was stirred at 150° C. under argon gas for 56 hours. Then the mixture was poured onto 40 mL of ice-cold water. The obtained solid was filtered, the cake was washed with MeOH (5 mL) and dried at 45° C. under reduced pressure to give BG10 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[3-(diethylamino)propyl]-3,9-dimethyl-) with a yield of 208.4 mg (83%). Mp.=192-195° C.
IR of BG10 (ATR): 3232, 2966, 2929, 2818, 1594, 1530, 1353, 1064, 1025,831, 750 cm−1. 1H NMR of BG10 (200 MHz, CD3CO2D): 9.05 (d, J=9.4 Hz, 2H), 8.59 (d, J=9.4 Hz, 2H), 7.01 (s, 2H), 3.91-3.84 (m, 4H), 3.51-3.18 (m, 8H), 2.90 (s, 6H), 2.45-2.27 (m, 4H), 1.32 (t, J=7.1 Hz, 12H) ppm. 13C NMR of BG10 (50 MHz, CD3CO2D): 156.64, 156.12, 136.57, 125.95, 122.08, 121.53, 116.36, 102.33, 50.46, 47.64, 41.81, 23.60, 8.62 ppm. ESI-HRMS (m/z) of BG10: [M+2H]2+ 258.19756 (error 2.08 ppm), [M+H]+ 515.38770 (error 0.36 ppm). BG10 (300 mg, 0.58 mmol) was suspended in 10 mL of MeOH and the reaction mixture was vigorously stirred. 15 mL of 8.6 M EtOH/HCl solution was added and stirring was continued for 48 hours at room temperature. The solvent was removed under reduced pressure, and obtained solid was suspended in 21 mL of 96% EtOH, filtered, and the cake was washed with 9 mL of 96% EtOH. Upon drying at 45° C. under reduced pressure BG11 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[3-(diethylamino)propyl]-3,9-dimethyl-, tetrahydrochloride) was obtained (303.5 mg; 79%). BG11: Mp=>280° C.; IR (ATR): 3164, 2949, 2575, 2478, 1632, 1610, 1560, 1468, 1439, 1347, 1030, 821, 750 cm−1; 1H NMR (500 MHz, CD3OD): 9.07 (d, J=9.5 Hz, 2H), 8.74 (d, J=9.5 Hz, 2H), 7.20 (s, 2H), 3.83-3.80 (m, 4H), 3.40-3.38 (m, 4H), 2.96 (s, 12H), 2.35-2.29 (m, 4H), 1.38 (t, J=7.2 Hz, 12H) ppm; 13C NMR (125 MHz, CD3OD): 157.20, 156.75, 137.19, 126.41, 122.66, 121.72, 116.87, 103.02, 50.98, 48.71, 24.24, 21.03, 9.36 ppm.
Synthesis of BG15
A suspension of 1,5-diaminonaphthalene 1 (5.0 g, 31.6 mmol), ethyl acetoacetate 2 (40 mL, 313.5 mmol) and p-TsOH (2.38 mg) in 250 mL of anhydrous ethanol was heated to reflux in a two-neck round bottom flask equipped with Dean-Stark trap filled molecular sieves (4 Å). After heating for 4 hours in an oil bath at a temperature of 120° C. with stirring, the mixture was cooled to room temperature and evaporated to dryness (upon cooling precipitation occurs). The crude product was triturated with ethanol, and precipitate was collected on Büchner funnel and dried under reduced pressure to give BG5 (2-butenoic acid, 3-[[5-[[(E or Z)-3-ethoxy-1-methyl-3-oxo-1-propenyl]amino]-1-naphthalenyl]amino]-, ethyl ester, (E or Z)-) as the product with a yield of 11.39 g (94%). IR of BG5 (ATR): 3442, 3253, 2977, 2923, 1652, 1606, 1506, 1440, 1267, 1158, 787 cm−1. A mixture of BG5 (2.00 g, 5.23 mmol) and Eaton's reagent (5.5 mL, 29.1 mmol, commercial, Aldrich) was stirred at 90° C. for 5 hours. The reaction mixture was cooled to 5° C. in an ice bath and carefully poured onto saturated ice-cold Na2CO3 solution (31 g Na2CO3/100 mL H2O). The solid was filtered, filtrate was washed with water (3×50 mL), EtOH (10 mL) and dried under reduced pressure at 50° C. to give BG6 (quino[8,7-h]quinoline-1,7-dione, 4,10-dihydro-3,9-dimethyl as a product with a yield of 1.4276 g (94%). IR of BG6 (ATR): 3227, 3169, 3059, 2978, 1630, 1601, 1546, 1520, 1492, 1434, 1354, 1211, 1153, 1029, 843 cm−1. 1H NMR of BG6 (200 MHz, TFAd): 9.15 (d, J=9.6 Hz, 2H), 8.87 (d, J=9.6 Hz, 2H), 7.58 (s, 2H), 3.15 (s, 6H) ppm. 13C NMR of BG6 (50 MHz, TFAd): 172.18, 139.66, 130.28, 127.79, 125.15, 123.15, 121.93, 111.70, 22.27 ppm. A suspension of BG6 (1.37 mg, 4.73 mmol) in POCl3 (28.38 mL, 309.10 mmol) was heated at 90° C. for 6 hours, and continued at 120° C. for 14 hours. Upon cooling to room temperature, the mixture was slowly poured onto ice/water mixture and concentrated aqueous NH3 was added until pH 9. Formed precipitate was filtered and washed well with H2O and EtOH. The product BG7 (quino[8,7-h]quinoline, 1,7-dichloro-3,9-dimethyl-) was dried under reduced pressure at 60° C. with give a yield of 1.4442 g (93%). IR of BG7 (ATR): 2922, 1582, 1481, 1432, 1359, 1083, 1027, 877, 849, 829, 749 cm−1. 1H NMR of BG7 (200 MHz, TFAd): 9.30 (d, J=9.6 Hz, 2H), 8.78 (d, J=9.6 Hz, 2H), 8.18 (s, 2H), 3.10 (s, 6H) ppm. 13C NMR of BG7 (50 MHz, TFAd): 162.18, 158.98, 138.44, 129.65, 129.52, 128.30, 127.52, 126.19, 22.45 ppm. A suspension of BG7 (200 mg, 0.61 mmol) in 3 mL of 2-piperidin-1-yl-ethylamine was stirred at 170-180° C. under argon gas for 10 hours. Then the mixture was poured onto 40 mL of ice-cold water. The obtained solid was filtered, the cake was washed with 5 mL of MeOH and dried at 45° C. under reduced pressure to give BG12 in a yield of 255 mg (82%). Mp=306-310° C.; IR of BG12 (ATR): 3376, 2925, 2841, 2767, 1594, 1580, 1465, 1123, 1038, 750 cm−1. ESI-HRMS (m/z) of BG12: [M+2H]2+ 256.18169 (error 3.38 ppm), [M+H]+ 511.35584 (error 2.87 ppm). BG12 (390 mg, 0.76 mmol) was suspended in 10 mL of MeOH and the reaction mixture was vigorously stirred. 15 mL of 8.6 M EtOH/HCl solution was added and stirring was continued for 48 hours at room temperature. The solvent was removed under reduced pressure, and obtained solid was suspended in 21 mL of 96% EtOH, filtered, and the cake was washed with 9 mL of 96% EtOH. Upon drying at 45° C. under reduced pressure BG15 (quino[8,7-h]quinoline-1,7-diamine, 3,9-dimethyl-N1,N7-bis[2-(1-piperidinyl)ethyl]-, tetrahydrochloride) was obtained (341.6 mg; 68%). BG15: Mp=>280° C.; IR (ATR): 3345, 3224, 2943, 2644, 1634, 1608, 1564, 1438, 1360, 831, 744 cm−1; 1H NMR (500 MHz, D2O): 8.86 (d, J=9.5 Hz, 2H), 8.48 (d, J=9.5 Hz, 2H), 7.19 (s, 2H), 4.29-4.24 (m, 4H), 3.83 (bd, J=12 Hz, 4H), 3.74-3.70 (m, 4H), 3.27-3.21 (m, 4H), 3.02 (s, 6H), 2.15-2.10 (m, 4H), 2.01-1.92 (m, 6H), 1.59-1.52 (m, 2H) ppm; 13C NMR (125 MHz, D2O): 158.28, 158.21, 138.14, 127.37, 123.45, 122.95, 118.13, 104.39, 56.63, 40.54, 25.57, 23.81, 23.03 ppm.
Synthesis of BG16
A suspension of 1,5-diaminonaphthalene 1 (5.0 g, 31.6 mmol), ethyl acetoacetate 2 (40 mL, 313.5 mmol) and p-TsOH (2.38 mg) in 250 mL of anhydrous ethanol was heated to reflux in a two-neck round bottom flask equipped with Dean-Stark trap filled molecular sieves (4 Å). After heating for 4 hours in an oil bath at a temperature of 120° C. with stirring, the mixture was cooled to room temperature and evaporated to dryness (upon cooling precipitation occurs). The crude product was triturated with ethanol, and precipitate was collected on Büchner funnel and dried under reduced pressure to give BG5 (2-butenoic acid, 3-[[5-[[(E or Z)-3-ethoxy-1-methyl-3-oxo-1-propenyl]amino]-1-naphthalenyl]amino]-, ethyl ester, (E or Z)-) as the product with a yield of 11.39 g (94%). IR of BG5 (ATR): 3442, 3253, 2977, 2923, 1652, 1606, 1506, 1440, 1267, 1158, 787 cm−1. A mixture of BG5 (2.00 g, 5.23 mmol) and Eaton's reagent (5.5 mL, 29.1 mmol, commercial, Aldrich) was stirred at 90° C. for 5 hours. The reaction mixture was cooled to 5° C. in an ice bath and carefully poured onto saturated ice-cold Na2CO3 solution (31 g Na2CO3/100 mL H2O). The solid was filtered, filtrate was washed with water (3×50 mL), EtOH (10 mL) and dried under reduced pressure at 50° C. to give BG6 (quino[8,7-h]quinoline-1,7-dione, 4,10-dihydro-3,9-dimethyl-) as a product with a yield of 1.4276 g (94%). IR of BG6 (ATR): 3227, 3169, 3059, 2978, 1630, 1601, 1546, 1520, 1492, 1434, 1354, 1211, 1153, 1029, 843 cm−1. 1H NMR of BG6 (200 MHz, TFAd): 9.15 (d, J=9.6 Hz, 2H), 8.87 (d, J=9.6 Hz, 2H), 7.58 (s, 2H), 3.15 (s, 6H) ppm. 13C NMR of BG6 (50 MHz, TFAd): 172.18, 139.66, 130.28, 127.79, 125.15, 123.15, 121.93, 111.70, 22.27 ppm. A suspension of BG6 (1.37 mg, 4.73 mmol) in POCl3 (28.38 mL, 309.10 mmol) was heated at 90° C. for 6 hours, and continued at 120° C. for 14 hours. Upon cooling to room temperature, the mixture was slowly poured onto ice/water mixture and concentrated aqueous NH3 was added until pH 9. Formed precipitate was filtered and washed well with H2O and EtOH. The product BG7 (quino[8,7-h]quinoline, 1,7-dichloro-3,9-dimethyl-) was dried under reduced pressure at 60° C. with give a yield of 1.4442 g (93%). IR of BG7 (ATR): 2922, 1582, 1481, 1432, 1359, 1083, 1027, 877, 849, 829, 749 cm−1. 1H NMR of BG7 (200 MHz, TFAd): 9.30 (d, J=9.6 Hz, 2H), 8.78 (d, J=9.6 Hz, 2H), 8.18 (s, 2H), 3.10 (s, 6H) ppm. 13C NMR of BG7 (50 MHz, TFAd): 162.18, 158.98, 138.44, 129.65, 129.52, 128.30, 127.52, 126.19, 22.45 ppm. A suspension of BG7 (200 mg, 0.61 mmol) in 2-piperidin-1-yl-propylamine was stirred at 170-180° C. under argon gas for 10 hours. Then the mixture was poured onto 40 mL of ice-cold water. The obtained solid was filtered, the cake was washed with 5 mL of MeOH and dried at 45° C. under reduced pressure to give BG13 (quino[8,7-h]quinoline-1,7-diamine, 3,9-dimethyl-N1,N7-bis[3-(1-piperidinyl)propyl]-) with a yield 293 mg (89%). Mp.=304-307° C.
IR (ATR): 3263, 2920, 2850, 2804, 1581, 1526, 1468, 1435, 1116, 1027, 751 cm1
ESI-HRMS (m/z): [M+2H]2+ 270.19601 (error 1.73 ppm), [M+H]+ 539.38481 (error 1.61 ppm). BG13 (450 mg, 0.83 mmol) was suspended in MeOH (10 mL) and the reaction mixture was vigorously stirred. EtOH/HCl solution (8.6M) was added and stirring was continued for 48 hours at room temperature. The solvent was removed under reduced pressure, and obtained solid was suspended in 96% EtOH (21 mL), filtered, and the cake was washed with 96% EtOH (9 mL). Upon drying at 45° C. under reduced pressure BG16 (quino[8,7-h]quinoline-1,7-diamine, 3,9-dimethyl-N1,N7-bis[3-(1-piperidinyl)propyl]-, tetrahydrochloride) was obtained (509.1 mg; 89%). BG16: Mp=>280° C.; IR (ATR): 3361, 3182, 2944, 2536, 1636, 1606, 1562, 1438, 1354, 835, 745 cm−1; 1H NMR (500 MHz, CD3OD): 9.11 (d, J=8.5 Hz, 2H), 8.77 (d, J=8.5 Hz, 2H), 7.18 (s, 2H), 3.83-3.79 (m, 4H), 3.62 (bd, J=12 Hz, 4H), 3.37-3.30 (m, 4H), 3.07-2.95 (m, 4H), 2.95 (s, 6H), 2.39-2.32 (m, 4H), 2.00-1.84 (m, 10H), 1.59-1.51 (m, 2H) ppm; 13C NMR (125 MHz, CD3OD): 157.29, 156.73, 137.27, 126.55, 122.71, 121.69, 116.96, 55.75, 54.64, 42.12, 24.43, 24.12, 22.86, 20.97 ppm.
Synthesis of BG17
A suspension of 1,5-diaminonaphthalene 1 (5.0 g, 31.6 mmol), ethyl acetoacetate 2 (40 mL, 313.5 mmol) and p-TsOH (2.38 mg) in 250 mL of anhydrous ethanol was heated to reflux in a two-neck round bottom flask equipped with Dean-Stark trap filled molecular sieves (4 Å). After heating for 4 hours in an oil bath at a temperature of 120° C. with stirring, the mixture was cooled to room temperature and evaporated to dryness (upon cooling precipitation occurs). The crude product was triturated with ethanol, and precipitate was collected on Büchner funnel and dried under reduced pressure to give BG5 (2-butenoic acid, 3-[[5-[[(E or Z)-3-ethoxy-1-methyl-3-oxo-1-propenyl]amino]-1-naphthalenyl]amino]-, ethyl ester, (E or Z)-) as the product with a yield of 11.39 g (94%). IR of BG5 (ATR): 3442, 3253, 2977, 2923, 1652, 1606, 1506, 1440, 1267, 1158, 787 cm−1. A mixture of BG5 (2.00 g, 5.23 mmol) and Eaton's reagent (5.5 mL, 29.1 mmol, commercial, Aldrich) was stirred at 90° C. for 5 hours. The reaction mixture was cooled to 5° C. in an ice bath and carefully poured onto saturated ice-cold Na2CO3 solution (31 g Na2CO3/100 mL H2O). The solid was filtered, filtrate was washed with water (3×50 mL), EtOH (10 mL) and dried under reduced pressure at 50° C. to give BG6 (quino[8,7-h]quinoline-1,7-dione, 4,10-dihydro-3,9-dimethyl-) as a product with a yield of 1.4276 g (94%). IR of BG6 (ATR): 3227, 3169, 3059, 2978, 1630, 1601, 1546, 1520, 1492, 1434, 1354, 1211, 1153, 1029, 843 cm−1. 1H NMR of BG6 (200 MHz, TFAd): 9.15 (d, J=9.6 Hz, 2H), 8.87 (d, J=9.6 Hz, 2H), 7.58 (s, 2H), 3.15 (s, 6H) ppm. 13C NMR of BG6 (50 MHz, TFAd): 172.18, 139.66, 130.28, 127.79, 125.15, 123.15, 121.93, 111.70, 22.27 ppm. A suspension of BG6 (1.37 mg, 4.73 mmol) in POCl3 (28.38 mL, 309.10 mmol) was heated at 90° C. for 6 hours, and continued at 120° C. for 14 hours. Upon cooling to room temperature, the mixture was slowly poured onto ice/water mixture and concentrated aqueous NH3 was added until pH 9. Formed precipitate was filtered and washed well with H2O and EtOH. The product BG7 (quino[8,7-h]quinoline, 1,7-dichloro-3,9-dimethyl-) was dried under reduced pressure at 60° C. with give a yield of 1.4442 g (93%). IR of BG7 (ATR): 2922, 1582, 1481, 1432, 1359, 1083, 1027, 877, 849, 829, 749 cm−1. 1H NMR of BG7 (200 MHz, TFAd): 9.30 (d, J=9.6 Hz, 2H), 8.78 (d, J=9.6 Hz, 2H), 8.18 (s, 2H), 3.10 (s, 6H) ppm. 13C NMR of BG7 (50 MHz, TFAd): 162.18, 158.98, 138.44, 129.65, 129.52, 128.30, 127.52, 126.19, 22.45 ppm. A suspension of BG7 (300 mg, 0.92 mmol) in 11 mL of Et2N(CH2)2NH2 was stirred at reflux under argon gas for 45 hours. Then the mixture was poured onto ice-cold water (40 mL). The obtained solid was filtered, the cake was washed with MeOH (5 mL) and dried at 45° C. under reduced pressure to give BG14 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[2-(diethylamino)ethyl]-3,9-dimethyl-) with a yield of 268 mg (90%). Mp.=242-246° C.; IR of BG14 (ATR): 3376, 2973, 2842, 1594, 1578, 1518, 1360, 1334, 1024, 828, 751 cm−1. 1H NMR of BG14 (200 MHz, TFAd): 8.73 (d, J=8.8 Hz, 2H), 8.24 (d, J=8.8 Hz, 2H), 6.81 (s, 2H), 4.05 (bs, 4H), 3.52 (bs, 4H), 3.24 (bs, 8H), 2.73 (s, 6H), 1.32 (bs, 12H) ppm. 13C NMR of BG14 (50 MHz, TFAd): 159.19, 158.70, 138.18, 127.68, 123.06, 118.40, 104.01, 53.14, 50.76, 40.95, 22.21, 9.54 ppm. ESI-HRMS (m/z) of BG14: [M+2H]2+ 244.18208 (error 5.13 ppm), [M+H]+ 487.35548 (error 2.28 ppm). BG14 (223 mg, 0.46 mmol) was suspended in MeOH (10 mL) and the reaction mixture was vigorously stirred. EtOH/HCl solution (8.6M) was added and stirring was continued for 48 hours at room temperature. The solvent was removed under reduced pressure, and obtained solid was suspended in 96% EtOH (21 mL), filtered, and the cake was washed with 96% EtOH (9 mL). Upon drying at 45° C. under reduced pressure BG17 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[2-(diethylamino)ethyl]-3,9-dimethyl-, tetrahydrochloride) was obtained (176 mg; 61%). BG17: Mp=>280° C.; IR (ATR): 3384, 3188, 3066, 2663, 1635, 1610, 1556, 1441, 1348, 1071, 830, 744 cm−1; 1H NMR (500 MHz, CD3OD): 9.14 (d, J=9.0 Hz, 2H), 8.85 (d, J=9.0 Hz, 2H), 7.30 (s, 2H), 4.18-4.14 (m, 4H), 3.68-3.63 (m, 4H), 3.46-3.38 (m, 8H), 2.97 (s, 6H), 1.44-1.40 (m, 12H) ppm; 13C NMR (125 MHz, CD3OD): 157.37, 157.22, 137.49, 126.68, 123.01, 121.88, 117.29, 103.41, 51.11, 39.68, 21.05, 9.16 ppm.
Synthesis of BG20
A suspension of 1,5-diaminonaphthalene 1 (5.0 g, 31.6 mmol), ethyl acetoacetate 2 (40 mL, 313.5 mmol) and p-TsOH (2.38 mg) in 250 mL of anhydrous ethanol was heated to reflux in a two-neck round bottom flask equipped with Dean-Stark trap filled molecular sieves (4 Å). After heating for 4 hours in an oil bath at a temperature of 120° C. with stirring, the mixture was cooled to room temperature and evaporated to dryness (upon cooling precipitation occurs). The crude product was triturated with ethanol, and precipitate was collected on Büchner funnel and dried under reduced pressure to give BG5 (2-butenoic acid, 3-[[5-[[(E or Z)-3-ethoxy-1-methyl-3-oxo-1-propenyl]amino]-1-naphthalenyl]amino]-, ethyl ester, (E or Z)-) as the product with a yield of 11.39 g (94%). IR of BG5 (ATR): 3442, 3253, 2977, 2923, 1652, 1606, 1506, 1440, 1267, 1158, 787 cm−1. A mixture of BG5 (2.00 g, 5.23 mmol) and Eaton's reagent (5.5 mL, 29.1 mmol, commercial, Aldrich) was stirred at 90° C. for 5 hours. The reaction mixture was cooled to 5° C. in an ice bath and carefully poured onto saturated ice-cold Na2CO3 solution (31 g Na2CO3/100 mL H2O). The solid was filtered, filtrate was washed with water (3×50 mL), EtOH (10 mL) and dried under reduced pressure at 50° C. to give BG6 (quino[8,7-h]quinoline-1,7-dione, 4,10-dihydro-3,9-dimethyl-) as a product with a yield of 1.4276 g (94%). IR of BG6 (ATR): 3227, 3169, 3059, 2978, 1630, 1601, 1546, 1520, 1492, 1434, 1354, 1211, 1153, 1029, 843 cm−1. 1H NMR of BG6 (200 MHz, TFAd): 9.15 (d, J=9.6 Hz, 2H), 8.87 (d, J=9.6 Hz, 2H), 7.58 (s, 2H), 3.15 (s, 6H) ppm. 13C NMR of BG6 (50 MHz, TFAd): 172.18, 139.66, 130.28, 127.79, 125.15, 123.15, 121.93, 111.70, 22.27 ppm. A suspension of BG6 (1.37 mg, 4.73 mmol) in POCl3 (28.38 mL, 309.10 mmol) was heated at 90° C. for 6 hours, and continued at 120° C. for 14 hours. Upon cooling to room temperature, the mixture was slowly poured onto ice/water mixture and concentrated aqueous NH3 was added until pH 9. Formed precipitate was filtered and washed well with H2O and EtOH. The product BG7 (quino[8,7-h]quinoline, 1,7-dichloro-3,9-dimethyl-) was dried under reduced pressure at 60° C. with give a yield of 1.4442 g (93%). IR of BG7 (ATR): 2922, 1582, 1481, 1432, 1359, 1083, 1027, 877, 849, 829, 749 cm−1. 1H NMR of BG7 (200 MHz, TFAd): 9.30 (d, J=9.6 Hz, 2H), 8.78 (d, J=9.6 Hz, 2H), 8.18 (s, 2H), 3.10 (s, 6H) ppm. 13C NMR of BG7 (50 MHz, TFAd): 162.18, 158.98, 138.44, 129.65, 129.52, 128.30, 127.52, 126.19, 22.45 ppm. A suspension of BG7 (300 mg, 0.92 mmol) in Me2N(CH2)2NH2 was stirred at reflux under argon gas for 7 days. Then the mixture was poured onto ice-cold water (40 mL). The obtained solid was filtered, the cake was washed with MeOH (5 mL), and dried at 45° C. under reduced pressure to give BG18 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[2-(dimethylamino)ethyl]-3,9-dimethyl-) with a yield of 534 mg (81%). Mp.=317-318° C.
IR of BG18 (ATR): 3401, 2944, 2816, 2763, 1598, 1578, 1524, 1458, 1102, 1042, 1023, 812, 787, 747 cm−1. 1H NMR of BG18 (200 MHz, CD3CO2D): 9.10 (d, J=9.6 Hz, 2H), 8.49 (d, J=9.6 Hz, 2H), 7.11 (s, 2H), 4.20 (bs, 4H), 3.71 (bs, 4H), 3.04 (s, 12H), 2.91 (s, 6H) ppm. 13C NMR of BG18 (50 MHz, CD3CO2D): 156.64, 156.60, 136.57, 125.92, 121.97, 116.60, 102.79, 55.85, 43.76, 39.43, 20.81 ppm. ESI-HRMS (m/z): [M+2H]2+ 216.14969 (error 0.78 ppm), [M+H]+ 431.29161 (error 0.37 ppm). BG18 (338 mg, 0.78 mmol) was suspended in MeOH (10 mL) and the reaction mixture was vigorously stirred. EtOH/HCl solution (8.6M) was added and stirring was continued for 48 hours at room temperature. The solvent was removed under reduced pressure, and obtained solid was suspended in 96% EtOH (21 mL), filtered, and the cake was washed with 96% EtOH (9 mL). Upon drying at 45° C. under reduced pressure BG20 (quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[2-(dimethylamino)ethyl]-3,9-dimethyl-, tetrahydrochloride) was obtained (382.9 mg; 85%). BG20: Mp=>280° C.; IR (ATR): 3430, 3361, 3180, 3068, 3029, 2974, 2699, 1634, 1609, 1554, 1445, 1344, 988, 828, 746 cm−1; 1H NMR (200 MHz, D2O): 8.69 (d, J=9.6 Hz, 2H), 8.39 (d, J=9.6 Hz, 2H), 7.24 (s, 2H), 4.34-4.20 (m, 4H), 3.89-3.78 (m, 4H), 3.22 (s, 12H), 3.02 (s, 6H) ppm; 13C NMR (50 MHz, D2O): 158.40, 138.50, 127.77, 123.58, 123.22, 118.57, 104.57, 57.56, 57.14, 46.01, 41.02, 23.00, 21.85, 17.74 ppm.
Compositions of the present invention may further comprise a pharmaceutical composition comprising a therapeutically effective amount of any of the small molecules (or combinations of small molecules) described above together with other materials, such as a suitable carrier, excipient, etc., for administration to a human or animal experiencing a viral infection or at risk of a viral infection. Such pharmaceutical compositions may be either in solid, gel or liquid form and may be administered as appropriate to an individual IV, IM, IP or parenterally, topically, orally, or through mucosal surfaces and routes (including, for example, rectal and vaginal suppositories). The exact dosage corresponding to a therapeutically effective amount will vary from mammal to mammal and virus to virus. The dosage ranges set forth above in specific examples for each of the FGI-103 compounds tested are representative, and provide sufficient information to those of skill in the art, following the assay procedures set forth herein and known to those of skill in the art, to arrive at suitable dosage values for any given virus and mammalian host. Those of skill in the art are well equipped by conventional protocols, given the identification of targets and compounds herein, to identify specific dosages for specific mammals, specific viruses, and specific modes of administration. See, e.g., “Remington: The Science and Practice of Pharmacy,” University of the Sciences in Philadelphia, 21st ed., Mack Publishing Co., (2005), the disclosure of which is hereby incorporated by reference in its entirety.
A “therapeutically effective” amount of the inventive compositions can be determined by prevention or amelioration of viral infection of host cells or viral disease in a patient or animal. It will be understood that, when administered to a human patient, the total daily usage of the agents or composition of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular or physiological response to be achieved; activity of the specific composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the composition; the duration of the treatment; drugs used in combination or coincidental with the composition; and like factors well known in the medical and veterinary arts. For example, it is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
While the present invention has been disclosed with reference to certain embodiments and examples, numerous modification, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.
This application claims benefit of priority to U.S. Provisional Patent Application No. 60/982,227 filed Oct. 24, 2007, which is incorporated by reference in its entirety. This application also incorporates by reference the entire disclosure of U.S. Patent Application Publication WO 2008/089125 A2 and the corresponding U.S. patent application Ser. No. 12/013,640.
Work resulting in the invention disclosed and claimed herein was funded in part by U.S. Government Contract HDTRA1-07-C-0080, and the government may enjoy limited rights therein to certain applications pertaining to hemorrhagic fever virus treatment.
| Number | Date | Country | |
|---|---|---|---|
| 60982227 | Oct 2007 | US |
