US008377639B2

(12) United States Patent (10) Patent No.: US 8,377,639 B2 Ryder (45) Date of Patent: Feb. 19, 2013

(54) COMPOUNDS FOR MODULATING RNA (56) References Cited BINDING PROTEINS AND USES THEREFOR |U.S. PATENT DOCUMENTS (75) Inventor: Sean Ryder, West Boylston, MA (US) 3,953,492 A * 4/1976 Mrozik ...... 558/6 (73) Assignee: University of Massachusetts, Boston, FOREIGN PATENT DOCUMENTS WO WO86/03941 A1 7/1986 MA (US) WO WO02/083629 A1 10/2002 WO WO2004/046095 A1 6/2004 (*) Notice: Subject to any disclaimer, the term of this WO WO2006/110762 A2 10/2006 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 29 days. Massacret et al., Palladium(0)-Catalyzed Asymmetric Synthesis of (21) Appl. No.: 12/823,902 1,2,3,4-Tetrahydro-2-vinylquinoxalines, Eur, J. Org. Chem. 1999, 129-134.” Seftel et al., Comparison of Piperazine and Tetramisole in Treatment (22) Filed: Jun. 25, 2010 of , Brit. med. J., 1968, 4, 93-95.” International Search Report and Written Opinion issued in PCT/ (65) Prior Publication Data US2010/040035 on Jun. 29, 2011. |US 2011/0065704 A1 Mar 17, 2011 Mueller, Joachim et al., “Thioureides of 2-(phenoxymethyl)benzoic acid 4-R substituted: A novel class of anti-parasitic compounds”, Parasitology International, 2009, 58(2), pp. 128-135. (Available online Dec. 25, 2008). Related U.S. Application Data (60) Provisional application No. 61/220,985, filed on Jun. * cited by examiner 26, 2009. Primary Examiner – Jim Ketter Assistant Examiner – Reza Ghafoorian (51) Int. Cl. (74) Attorney, Agent, or Firm – McCarter & English, LLP; CI2O I/68 (2006.01) Elizabeth A. Hanley, Esq.; Jill Mello A ().IN 4 I./06 (2006.01) A0IN 43/653 (2006.01) (57) ABSTRACT A6 IK 31/18 (2006.01) The invention relates to compositions and methods for inhib A6 IK 31/41.96 (2006.01) iting RNA binding proteins (e.g., MEX-3, MEX-5 and POS (52) U.S. Cl...... 435/6.1: 514/383: 514/604: 548/263.6; 1), as well as methods for treating and preventing disorders 548/264.8: 564/89; 56.4/92; 564/152; 564/191 associated with parasitic infections and inflammatory disor (58) Field of Classification Search ...... None ders. See application file for complete search history. 28 Claims, No Drawings US 8,377,639 B2 1 2 COMPOUNDS FOR MODULATING RNA pounds that inhibit the RNA-binding activity of MEX-3, BINDING PROTEINS AND USES THEREFOR MEX-5 and/or POS-1, RNA binding proteins which are required for early embryogenesis in parasitic worms. The RELATED APPLICATIONS identified compounds represent a novel class of anti-parasitic agents that specifically target embryos. This application claims priority to U.S. Provisional Patent Application No. 61/220,985, filed Jun. 26, 2009. The entire Accordingly, in one aspect, the invention pertains, at least contents of the foregoing application are hereby incorporated in part, to method for treating or preventing a parasitic asso in their entirety. ciated state in a subject comprising administering to the sub ject an effective amount of an RNA binding modulatory com GOVERNMENT INTERESTS 10 pound (e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf This invention was made with government support under or a compound of Table 1 or 2), such that the parasitic asso grant nos. 1R21NS059380 and 3R21NS059380-01S1 ciated state is treated or prevented. In one embodiment, the awarded by the National Institutes of Health. The government 15 parasitic associated state is a parasitic or re-infes has certain rights in the invention. tation. In another embodiment, the parasitic associated state SUBMISSION ON COMPACT DISC is a disease caused by a parasitic infestation. In one embodiment, the invention pertains, at least in part, This application incorporates by reference the ASCII text to a method for treating or preventing a parasitic infestation in file identified by the name SequenceListing.txt, containing 20 a subject infested with or at risk for infestation with parasites 1.49 KB of data, created on Nov. 12, 2010 and filed in com by administering to the subject a therapeutically or pesticid puter-readable format (CRF). ally effective amount of an RNA binding modulatory com pound, e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, BACKGROUND OF THE INVENTION III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf According to the World Health Organization (WHO), more 25 or a compound of Table 1 or 2, such that the parasitic infes than one-third of the world’s population, approximately 2 tation is treated or prevented. billion people, is infested with helminths. In 1999, the WHO In another embodiment, the invention pertains, at least in estimated that and soil-based helminths rep part, to a method for protection of plants from a parasitic resented more than 40% of the disease burden due to all infestation by administering to the plants a pesticidally effec tropical disease, excluding malaria. While most parasitic 30 tive amount of an RNA binding modulatory compound, e.g., are preventable and treatable, the effects of an a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, infestation can be chronic and long-term and may eventually VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a com cause death. For example, a person who has endured persis pound of Table 1 or 2, such that the plants are protected. tent and heavy infestations is likely to suffer from anemia, In yet another embodiment, the invention pertains, at least malnutrition and chronic irreversible diseases, such as liver 35 in part, to a method for inhibiting parasitic embryogenesis in , cancer of the bladder and kidney failure. These para a parasite or in a subject suffering from a parasitic infection sites also affect livestock, which can facilitate infestation in by administering to the parasite or subject suffering from the humans by causing contamination via soil or food supplies. parasitic infection a therapeutically or pesticidally effective In addition to the health risk posed to humans by parasites, amount of an RNA binding modulatory compound, e.g., a plants are highly susceptible to parasitic infestations. Effects 40 compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, of damage to plants include stunting, chlorosis, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a com nutrient deficiencies, wilting, root abnormalities and reduced pound of Table 1 or 2, such that embryogenesis is inhibited. yield. In a further embodiment, the invention pertains, at least in While there are a number of antihelminthic treatments part, to a method for reducing parasitic burden in soil, in currently available, some scientists are concerned that the 45 plants or in an suffering from a parasitic infection by parasites will develop resistance to these treatments, espe administering to the soil, plants or animal atherapeutically or cially in developing countries where people are repeatedly pesticidally effective amount of an RNA binding modulatory infected with helminths and receive multiple doses of anti compound, e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, helminthic drugs. In fact, resistance to antihelminthic drugs IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, has been observed in livestock due to frequent and repeated 50 VIIf or a compound of Table 1 or 2, such that the parasitic treatments. Moreover, rates of re-infection by helminths are burden is reduced. very high due to the number and the durability of infective In another embodiment, the invention pertains, at least in eggs on surfaces and in soil. While infection is active, some part, to a method for treating or preventing a disease caused parasitic nematode species (e.g., ) by a parasitic infestation in a subject by administering to the release an estimated 100,000 new embryos per adult female 55 subject a therapeutically effective amount of an RNA binding per day. Currently available anti-helminthics work by para modulatory compound, e.g., a compound of formula I, Ia, Ib, lyzing the infectious nematode through blocking ion channels II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, and receptors and do not inactivate the embryos that cause VIId, VIIe, VIIf or a compound of Table 1 or 2, such that the re-infection. Therefore, it is advantageous to develop new disease is treated or prevented. therapies for the treatment and prevention of parasitic infes 60 In one embodiment, the invention pertains, at least in part, tations and, in particular, therapies that limit the possibility of to a method for treating or preventing an inflammatory disor parasitic re-infection, in both plants and , der in a subject by administering to the subject a therapeuti cally effective amount of an RNA binding modulatory com SUMMARY OF THE INVENTION pound, e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, 65 III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf The present invention is based, at least in part, on the or a compound of Table 1 or 2, such that the inflammatory development and use of screening assays to identify com disorder is treated. US 8,377,639 B2 3 4 In one embodiment, the subject is a plant. In one embodi In one aspect, the present invention provides methods of ment, the subject is an animal. In one embodiment, the subject identifying a compound useful in modulating embryogenesis. is a human. The methods include providing an indicator composition In one embodiment, the parasite is present in a subject. In comprising an RNA-binding protein and an RNA molecule one embodiment, the parasite is a helminth, e.g., a cestode, a 5 comprising an RNA-binding protein recognition element; trematode and a nematode. In one embodiment, the helminth contacting the indicator composition with each member of a is a nematode. library of test compounds underconditions which allow bind In one embodiment, the RNA binding modulatory protein ing of the RNA-binding protein to the RNA molecule com modulates the RNA binding activity of an RNA binding pro prising an RNA-binding protein recognition element to form tein. In one embodiment, the RNA binding protein is required 10 a complex; and detecting the formation of a complex of the for embryogenesis. In one embodiment, the RNA binding RNA-binding protein and the RNA molecule comprising an protein comprises a CCCH zinc finger motif. In one embodi RNA-binding protein recognition element, wherein the abil ment, the RNA binding protein comprises a KH domain. In ity of the compound to modulate interaction of the RNA one embodiment, the RNA binding protein is selected from 15 binding protein and the RNA molecule comprising an RNA the group consisting of MEX-5, POS-1 and MEX-3, or a binding protein recognition element is indicated by a homolog thereof. In one embodiment, the RNA binding pro modulation of complex formation in the presence of the com tein is MEX-5 or a homolog thereof. In one embodiment, the pound as compared to the amount of complex formed in the RNA binding protein is MEX-3 or a homolog thereof. In one absence of the compound, thereby identifying a compound embodiment, the RNA binding protein is POS-1 or a homolog 20 useful in modulating embryogenesis. thereof. In another aspect, the present invention provides methods In another aspect, the invention pertains, at least in part, to of identifying a compound useful for treating a subject with a a pharmaceutical composition comprising a therapeutically parasitic-associated state. The methods include providing an effective amount of an RNA binding modulatory compound, indicator composition comprising an RNA-binding protein e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, 25 and an RNA molecule comprising an RNA-binding protein VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a recognition element; contacting the indicator composition compound of Table 1 or 2, and a pharmaceutically acceptable with each member of a library of test compounds under con carrier. ditions which allow binding of the RNA-binding protein to In another aspect, the invention pertains, at least in a part, the RNA molecule comprising an RNA-binding protein rec to a composition comprising a pesticidally effective amount 30 ognition element to form a complex; and detecting the for of an RNA binding modulatory compound, e.g., a compound mation of a complex of the RNA-binding protein and the offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, VIIe, VIId, VIIe, VIIfor a compound of Table 1 or RNA molecule comprising an RNA-binding protein recogni 2, and an agronomically acceptable carrier. tion element, wherein the ability of the compound to modu In yet another aspect, the present invention provides meth 35 late interaction of the RNA-binding protein and the RNA ods of identifying a compound useful in modulating a bio molecule comprising an RNA-binding protein recognition logical activity of an RNA-binding protein. The methods element is indicated by modulation of complex formation in include providing an indicator composition comprising an the presence of the compound as compared to the amount of RNA-binding protein and an RNA molecule comprising an complex formed in the absence of the compound, thereby RNA-binding protein recognition element; contacting the 40 identifying a compound useful for treating a subject with a indicator composition with each member of a library of test parasitic-associated state. compounds; determining the effect of the compound on a The biological activity of the RNA-binding protein may be biological activity of the RNA-binding protein; and selecting determined by measuring the interaction of the RNA-binding a compound that modulates the biological activity of the protein and an RNA-binding protein recognition element. RNA-binding protein as compared to an appropriate control, 45 Alternatively, the biological activity of the RNA-binding pro thereby identifying a compound useful in modulating a bio tein may be determined by determining the ability of the logical activity of an RNA-binding protein. compound to modulate a biological activity selected from the In another aspect, the invention provides methods of iden group consisting of anterior patterning, germ cell totipotency, tifying a compound useful in modulating a biological activity development of the intestine, development of germline blas of an RNA-binding protein. The methods include providing 50 tomeres, development of pharyngeal tissue, expression and/ an indicator composition comprising an RNA-binding pro or activity of PAL-1, NOS-2, APX-1 protein, and GLP-1. tein and an RNA molecule comprising an RNA-binding pro The indicator composition may be a cell that expresses the tein recognition element; contacting the indicator composi RNA-binding protein or a cell-free composition. tion with each member of a library of test compounds under In one embodiment, the RNA-binding protein comprises a conditions which allow binding of the RNA-binding protein 55 CCCH-type tandem zinc finger. In one embodiment, the to the RNA molecule comprising an RNA-binding protein RNA-binding protein comprising the CCCH-type tandem recognition element to form a complex; and detecting the zinc finger is POS-1. formation of a complex of the RNA-binding protein and the In another embodiment, the RNA-binding protein com RNA molecule comprising an RNA-binding protein recogni prises a KH domain. In one embodiment, the RNA-binding tion element, wherein the ability of the compound to modu 60 protein comprising the KH domain is MEX-3. late interaction of the RNA-binding protein and the RNA In one embodiment, the RNA-binding protein recog molecule comprising an RNA-binding protein recognition nition element comprises the consensus sequence UA(U2_s) element is indicated by a modulation of complex formation in RD(N1 s)G. In another embodiment, the RNA-binding pro the presence of the compound as compared to the formation tein recognition element comprises the consensus sequence of complex in the absence of the compound, thereby identi 65 DKAG(No 3)UHUA. fying a compound useful in modulating a biological activity Detecting the formation of a complex of the RNA-binding of an RNA-binding protein. protein and the RNA molecule may be determined by a gel US 8,377,639 B2 5 6 shift assay or a fluorescence polarization assay. In one In various embodiments, the method includes treating a embodiment, the RNA-binding protein recognition element parasitic infestation in a subject infested with parasites, pro is fluorescently labeled. tecting plants from a parasitic infestation, inhibiting embryo The methods of the invention may further comprise deter genesis in a parasite or in a subject suffering from a parasitic mining the effect of the test compound on a parasitic-associ 5 infestation, reducing parasitic burden in soil, in plants or in a ated state in a non-human animal, comprising administering mammal suffering from a parasitic infection, treating or pre the test compound to the animal and determining the effect of venting an inflammatory disorder and treating a disease in a test compound on the parasitic-associated state in the pres mammal caused by the growth and replication of a parasite. ence and absence of the test compound. Determining the In one embodiment, the invention pertains to a method of effect of the test compound on a parasitic-associated state 10 treating a parasitic infestation in a subject infested with para may be determined by measuring an immune response in the sites by administering to the subject a therapeutically or pes non-human animal. ticidally effective amount of an RNA binding modulatory In one embodiment, the test compound increases the for compound, e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, mation or stability of the complex. In another embodiment, 15 IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, the test compound decreases the formation or stability of the VIIf or a compound of Table 1 or 2, such that the subject is complex. treated. In another aspect, the invention provides compounds iden The term “subject” includes animals (e.g., vertebrates, tified according to the method of the invention which may be amphibians, fish, mammals, non-human animals), including in the form of a composition. 20 humans, that are capable of suffering from a parasitic infes One aspect of the invention provides to methods of inhib tation, an inflammatory disorder (e.g., rheumatoid arthritis, iting embryogenesis in a parasite comprising contacting the psoriasis and multiple sclerosis) or a disease in a mammal parasite with a compound identified according to the methods caused by a parasitic infection (e.g., ). Subjects of the invention. also include primates, such as chimpanzees, monkeys and the In another aspect, the invention provides methods of treat 25 like. In one embodiment of the invention, the subject is suf ing a parasite-associated state in a subject, comprising admin fering from a parasitic infestation or infection, e.g., a helm istering a composition comprising a compound identified in inth infestation. In one embodiment, the subject is at risk for the methods of the invention to the subject, thereby treating a a parasitic infection, e.g., has been exposed to a parasite e.g., parasite-associated state in the subject. a helminth. 30 The term “subject” also includes agriculturally productive DETAILED DESCRIPTION OF THE INVENTION livestock, for example, cattle, , goats, horses, pigs, don keys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese The invention pertains generally to a method for treating or and bees; and domestic pets, for example, dogs, cats, caged preventing a parasitic associated state in a subject comprising and aquarium fish, and also so-called test animals, for administering to the subject an effective amount of an RNA 35 example, hamsters, pigs, rats and mice. binding modulatory compound (e.g., a compound of formula The term “subject” also includes plants. The term “plant” I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, includes all plants and plant parts, for example, all plants and VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2), such plant populations, including wild plants or crop plants (in that the parasitic associated state is treated or prevented. cluding naturally occurring crop plants). Crop plants may be The term “parasitic associated state” includes those dis 40 plants that can be obtained by conventional plant breeding eases and disorders associated with parasites. In one embodi and optimization methods or by biotechnological and recom ment, the parasiticassociated state is a parasiticinfestation. In binant methods or by combinations of these methods, includ one embodiment, the parasitic associated state is a disease ing transgenic plants. Examples of plants include, but are not caused by a parasitic infestation. In one embodiment, the limited to the following plant cultivars: cereals (wheat, barley, parasitic associated state is a parasitic re-infestation. 45 rye, oats, rice, maize, sorghum and related species); beet As used herein, the term “RNA binding modulatory com (sugar beet and fodder beet); pome, stone and berry fruit pound” includes those compounds that are capable of modu (apples, pears, plums, peaches, almonds, cherries, strawber lating the activity of an RNA-binding protein (e.g., an RNA ries, raspberries and blackberries); legumes (beans, lentils, binding protein required for embryogenesis in a parasitic peas, soya); oil crops (rape, mustard, poppy, olives, sunflow worm), e.g., the binding of an RNA-binding protein to a target 50 ers, coconut, castor oil, cocoa, peanut); cucumber plants RNA (e.g., an RNA molecule comprising an RNA-binding (squashes, cucumber, melons); citrus fruits (oranges, lemons, recognition element of the RNA-binding protein). In one grapefruits, mandarines); vegetables (spinach, lettuce, embodiment, the RNA binding modulatory compound is a asparagus, cabbage varieties, carrots, onions, tomatoes, pota compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, toes, paprika); laurels (avocado, cinnamonium, camphor) and VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a com 55 plants such as tobacco, cotton, nuts, corn, coffee, aubergines, pound of Table 1 or 2. In one embodiment, the RNA binding sugar cane, tea, pepper, vines, hops, grapes, bananas and modulatory compound includes compounds that are capable natural rubber plants, as well as ornamental plants. In one of decreasing, inhibiting or preventing the activity of an embodiment, the plant is suffering from a parasitic infesta RNA-binding protein, e.g., the binding of an RNA-binding tion. In another embodiment, the plant is at risk of suffering protein to a target RNA (e.g., an RNA molecule comprising 60 from a parasitic infestation. an RNA-binding recognition element of the RNA-binding Plant parts also include all parts and organs of plants above protein). In another embodiment, the RNA binding modula and below the ground, such as shoot, leaf, flower and root, tory compound includes compounds that are capable of examples of which include, for example, leaves, needles, increasing, augmenting or enhancing the activity of an RNA stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers binding protein, e.g., the binding of an RNA-binding protein 65 and rhizomes. The plant parts also include harvested material, to a target RNA (e.g., an RNA molecule comprising an RNA and vegetative and generative propagation material, for binding recognition element of the RNA-binding protein). example cuttings, tubers, rhizomes, offsets and seeds. US 8,377,639 B2 7 8 The language “subject” also includes soil. The term “soil” re-infestation” or “parasitic re-infection” is intended to includes the soil used in planting any plants or plant parts as include the re-occurrence of a parasitic infestation in a sub described above. The term “soil” also includes that soil that ject. has not yet been planted with any plants or plant parts. In one Examples of trematodes include, for example, Schisto embodiment, the soil suffers from a parasitic infestation. 5 soma spp. (e.g., bovis, Schistosoma curassoni, As used herein, the term “treating” may resultin prevention Schistosoma edwardiense, Schistosoma guineensis, Schisto of the disease or condition, cure of the disease or condition, a soma haematobium, Schistosoma hippotami, Schistosoma decrease in the type or number of symptoms associated with incognitum, Schistosoma indicum, Schistosoma intercala tum, , Schistosoma lieperi, Schisto the condition, either in the long term or short term (i.e., 10 soma malayenesis, , Schistosoma mar amelioration of the condition) or simply a transient beneficial grebowiei, Schistosoma mattheei, , effect to the subject. Schistosoma ovuncatum, Schistosoma nasale, Schistosoma The terms “treating” and “treatment” used in the context of rodhaini, Schistosoma sinesium, Schistosoma spindale, an animal include the administration of a therapeutically Schistosoma sinensium), regenti, Clonorchis effective amount of a RNA binding modulatory compound, 15 sinensis, , Dicrocoelium hospes, e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, , Fascioloides magna, Fasciola giganta, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a Fasciola jacksoni, Metorchis conjunctus, Metorchis albidus, compound of Table 1 or 2, to treat the animal for a parasitic Protofasciola robusta, Parafasciolopsis fasciomorphae, associated state, e.g., parasitic infestation, parasitic embryo , , Opisthorchis genesis, an inflammatory disease (e.g., rheumatoid arthritis, 20 guayaquilensis, , and psoriasis and multiple sclerosis) or a disease in an animal buski). caused by a parasitic infection (e.g., helminthiasis). Examples of cestodes include, for example, cyclophillidea The terms “preventing” and “prevention” used in the con (e.g., Dipylidium caninum, Taenia crassiceps, Taenia hydati text of an animal include the administration of a therapeuti gena, Taenia multiceps, Taenia pisiformis, Taenia serialis, cally or prophylactically effective amount of a RNA binding 25 Taenia taeniaeformis, , Echinococ modulatory compound, e.g., compound offormula I, Ia, Ib, II, cus multilocularis, Echinococcus shiquicus, Echinococcus IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, oligarthrus, Echinococcus vogeli, Echinococcus ortleppi, VIIe, VIIfor a compound of Table 1 or 2, to prevent a parasitic Echinococcus equinus, , , associated state, e.g., parasitic infestation, parasitic embryo , ) and pseudophyl genesis, an inflammatory disease (e.g., rheumatoid arthritis, 30 lidea (e.g., latum, Diphyllobothrium paci psoriasis and multiple sclerosis) or a disease in an animal ficum, Diphyllobothrium cordatum, Diphyllobothrium ursi, caused by a parasitic infection (e.g., helminthiasis), from Diphyllobothrium dendriticum, Diphyllobothrium lanceola occurring. tum, Diphyllobothrium dalliae, Diphyllobothrium yonagoen The terms “treating” and “treatment” used in the context of sis, Diphyllobothrium mansonoides, Spirometra erina plants include the administration to a plant or to soil a pesti 35 ceieuropaei, Spirometra mansonoides). cidally effective amount of a RNA binding modulatory com Examples of include, for example, Dracuncu pound, e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, lus medinensis, , , IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or perstans, Mansonella ozzardi, Mansonella streptocera, a compound of Table 1 or 2, to treat the plant or soil for a , , Acanthocheilonema parasitic associated state, e.g., parasitic infestation, parasitic 40 viteae, , Brugia pahangi, , Cer embryogenesis or reduction of the parasitic burden of the copithifilaria johnstoni, Dipetalonema reconditum, Dipetal plant or soil. onema repens, Dirofilaria immitis, Dirofilaria repens, Dirofi The terms “preventing” and “prevention” used in the con laria tenuis, Dirofilaria ursi, Elaeophora abramovi, text of plants include the administration of a pesticidally Elaeophora bohmi, Elaeophora elaphi, Elaeophora poeli, effective amount of a RNA binding modulatory compound, 45 Elaeophora sagitta, , Foleyella fur e.g., compound of formula I, Ia, Ib, II, IIa, IIc, IIb, III, IV, V, cata, Litomosa westi, Litomosoides brasiliensis, Lito VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a mosoides sigmodontis, Litomosoides wilsoni, Ochoterenella compound of Table 1 or 2, to a plant or to soil to prevent a digiticauda, Onchocerca gibsoni, Onchocerca gutturosa, parasitic associated state, e.g., parasitic infestation or para Onchocerca volvulus, Piratuba digiticauda, Sarconema sitic embryogenesis. 50 eurycerca, Waltonella flexicauda, , The phrases “parasitic infestation” and “parasitic infec Wuchereria kalimantani, binucleatum, Gna tion”, used interchangeable herein, are intended to include the thostoma doloresi, , Gnathostoma presence of parasitic organisms or embryos within the subject lamothei, Gnathostoma malaysiae, Gnathostoma nipponi (e.g., mammal, plant or soil). The term “parasite” includes an cum, , Gnathostoma turgidum, animal or plant that lives in or on a subject (e.g., a 55 Ancylostoma brazilienese, , Ancylos mammal, plant or soil). In one embodiment, the parasite is a toma ceylanicum, , Ancylostoma plu helminth. The term “helminth” includes eukaryotic, worm ridenatum, , , like parasites that live inside their hosts. Examples of helm Angiostrongylus cantonensis, Mermis nigrescens, Trichuris inths include, but are not limited to, (e.g., plathy trichiura, , Caenorhabditis elegans, helminths) for example, trematodes (e.g., flukes) and 60 Strongyloides stercoralis, Micronema (Halicephalobus) cestodes (e.g., tapeworms); thorny-headed worms (e.g., acan delatrix, Haemonchus contortus, Ostertagia sp., Nematodi thocephalans); and roundworms (e.g., nematodes). In one rus sp., Nippostrongylus brasiliensis, Heligmosomoides embodiment, the parasitic infestation is a helminth infesta polygyrus, Dictyocaulus viviparous, , tion. In one embodiment, the parasitic infestation is a nema sp., Enterobius sp., sp., Ascaris lumbri tode infestation. In one embodiment, the helminth or parasitic 65 coides, Ascaris suum, Anisakis pegreffi, Anisakis physeteris, worm is an intestinal parasite, e.g., a parasitic worm that lives Anisakis schupakovi, Anisakis simplex, Anisakis typical, inside the digestive tract. As used herein, the phrase “parasitic Anisakis ziphidarum, Toxocara cati, Baylisacaris procyonis, US 8,377,639 B2 10 Baylisacaris melis, Baylisacaris transfuga, Baylisacaris The language “pesticidally effective amount” of the com columnaris, Baylisacaris devosi, Baylisacaris laevis, pound includes that amount necessary or sufficient to treat, Strongyloides stercoralis, Enterobius vermicularis, Entero prevent or ameliorate a disease or disorder (e.g., parasitic bius anthropopitheci, Enterobius gregorii, Trichinella spira associated state, e.g., parasitic infestation) in a plant or soil lis, , Trichocephalus trichiuris, Capillaria (e.g., plant or soil suffering from or at risk for a parasitic philippinensis, Belonolaimus anama, Belonolaimus euthy associated state, e.g., parasitic infestation). The language chilus, Belonolaimus gracilis, Belonolaimus jara, Belonolai “pesticially effective amount” of the compound also includes mus lineatus, Belonolaimus lolii, Belonolaimus longicauda that amount necessary to reduce the parasitic burden in the tus, Belonolaimus maritimus, Belonolaimus nortoni, soil or plant and the amount necessary to inhibit parasitic Criconemoides sp., Helicotylenchus digonicus, Helicotylen 10 chus labiodiscinus, Helicotylenchus leiocephalus, Helicoty embryogenesis in a parasite or in a plant or in the soil com lenchus platyurus, Helicotylenchus pseudorobustus, Het prising a parasitic infestation. The pesticidally effective erodera zeae, Hoplolaimus tylenchiformis, Hoplolaimus amount can vary depending on such factors as the type of galeatus, Hoplolaimus columbus, Xiphinema americanumv, plant, the type of parasitic infestation, the extent of the para Longidorus elongatus, Longidorus breviannulatus, Meloid 15 sitic infestation, etc. One of ordinary skill in the art would be ogyne chitwoodi, Meloidogyne graminis, Meloidogyne able to study the aforementioned factors and make the deter hapla, Meloidogyne mayaguenesis, Meloidogyne partityla, mination regarding the pesticidally effective amount of the Pratylenchus agilis, Pratylenchus alleni, Pratylenchus cof: compounds without undue experimentation. feae, Pratylenchus convallariae, Pratylenchus crenatus, In another embodiment, the invention pertains, at least in Pratylenchus flakkensis, Pratylenchus hexincisus, Pratylen 20 part, to a method for protection of plants from a parasitic chus loosi, Pratylenchus penetrans, Pratylenchus infestation by administering to the plants a pesticidally effec pseudopratensis, Pratylenchus scribneri, Pratylenchus thor tive amount of aan RNA binding modulatory compound, e.g., nei, Trichodorus obtusus, Trichodorus proximus, Tylencho compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, rhynchus cylindricus, Tylenchorhynchus hordei, Tylencho VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf, or a com rhynchus nudus, Tylenchorhynchus robustus, Globodera 25 pound of Table 1 or 2, such that the plants are protected. pallida, Globodera rostochiensis, Globodera achilleae, Glo The language “protect” and “protection” includes shield bodera artemisiae, Globodera chaubattia, Globodera ing, guarding or preventing plants from damage by a parasitic hypolysi, Globodera leptonepia, Globodera millefolii, Glo infestation in or on the plant or in the soil surrounding the bodera mirabilis, Globodera pseudorostochiensis, Glo plant. The protection can occur by application of a an RNA bodera tabacum solanacearum, Globodera tabacum 30 binding modulatory compound, e.g., compound offormula I, tabacum, Globodera tabacum virginae, Globodera Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, zelandica, Ditylenchus destructor, Heterodera glycines, Het VIIc, VIId, VIIe, VIIfor a compound of Table 1 or 2, to the erodera schachtii, Nacobbus aberrrans, Criconemella inusi plant itself or to the soil surrounding the plant. tatus, Bursaphelenchus xylophilis, Radopholus simila, Roty In yet another embodiment, the invention pertains, at least lenchulus reniformis, Tylenchulus semipenetrans, 35 in part, to a method for modulating, e.g., inhibiting, embryo Belonolaimus longicaudatus, Macroposthonia curvata, genesis, e.g., in a parasite or in a subject suffering from a Macroposthonia discus, Macroposthonia annulata, Macro parasitic infection, by contacting the parasite or administer posthonia rustica, Macroposthonia sphaerocephalus, ing to the subject suffering from a parasitic infection a thera Macroposthonia xenoplax, Aphelen.choides besseyi, Aph peutically effective amount of an RNA binding modulatory elenchoides bicaudatus, Aphelen.choides centralis, Aphelen 40 compound, e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, choides clarus, Aphelen.choides confusus, Aphelen.choides IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, dactylocercus, Aphelen.choides obtusus, Aphelen.choides VIIfor a compound of Table 1 or 2, such that embryogenesis parietinus, Aphelen.choides pusillus, Aphelen.choides sac is inhibited. The parasite may be located externally from a chari, Aphelen.choides vigor and Ditylenchus dipsaci. In one plant (e.g., on a plant or in the soil) or may be located within embodiment, the nematode is C. elegans. 45 the plant or mammal itself. The language “therapeutically effective amount” of the As used herein, the various forms of the term “modulate” compound includes that amount necessary or sufficient to include stimulation (e.g., increasing or upregulating a par treat, prevent or ameliorate a disease or disorder (e.g., a para ticular response oractivity) and inhibition (e.g., decreasing or sitic associated state, e.g., parasitic infestation) in a subject downregulating a particular response or activity). (e.g., a subject suffering from or at risk for a parasitic infes 50 The terms “inhibit” and “inhibiting” refer to decreasing or tation, e.g., infestation by a helminth). A therapeutically downregulating a particular response or activity. The terms effective amount of the compound includes that amount nec “inhibit” and “inhibiting” include, for example, the suppres essary or sufficient to treat, prevent or ameliorate in a subject sion or amelioration of embryogenesis, e.g., parasitic a parasitic associated state, an inflammatory disorder (e.g., embryogenesis. The terms “inhibit” and “inhibiting” also rheumatoid arthritis, psoriasis or multiple sclerosis) or a dis 55 include, for example, the downmodulating or blocking of the ease in a subject caused by a parasitic infestation (e.g., hel interaction between an RNA binding protein and a target minthiasis). The language “therapeutically effective amount” RNA. of the compound also includes that amount necessary to The language “embryogenesis” includes the process by reduce the parasitic burden in a subject and the amount nec which an embryo, e.g., a parasitic embryo, is formed and essary to inhibit parasitic embryogenesis in a parasite or in a 60 develops, e.g., in a parasite. Without being bound by theory, in subject suffering from a parasitic infestation. The therapeu one embodiment, the compounds of formula I, Ia, Ib, II, IIa, tically effective amount can vary depending on such factors as IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, the size and weight of the subject, the type of illness, etc. One VIIe, VIIf or a compound of Table 1 or 2 modulate, e.g., of ordinary skill in the art would be able to study the afore inhibit, the binding of RNA binding proteins required in the mentioned factors and make the determination regarding the 65 early stages of embryogenesis to a target RNA of the RNA effective amount of the compounds without undue experi binding protein. In one embodiment, the RNA binding pro mentation. tein is MEX-5, POS-1 or MEX-3. US 8,377,639 B2 11 12 The term “RNA binding protein,” as used herein, refers to VIId, VIIe, VIIfora compound of Table 1 or 2, modulate (e.g., a protein that selectively or specifically binds to RNA. In one inhibit) the RNA binding activity of MEX-5, but do not sub embodiment, the RNA binding protein binds selectively to stantially inhibit the RNA binding activity of MEX-3 or POS single-stranded RNA. In one embodiment, the RNA binding 1. In another embodiment, the RNA binding modulatory protein binds selectively to double-stranded RNA. An RNA compounds of the invention, e.g., the compounds of formula binding protein of the present invention selectively or specifi I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, cally binds to a target RNA, e.g., RNA that comprises an RNA VIIc, VIId, VIIe, VIIfora compound of Table 1 or 2, modulate binding recognition element for the RNA binding protein. (e.g., inhibit) the RNA binding activity of MEX-3, but do not In one embodiment, the RNA binding protein is required substantially inhibit the RNA binding activity of MEX-5 or for embryogenesis, e.g., parasitic embryogenesis (e.g., helm 10 POS-1. In yet another embodiment, the RNA binding modu inth embryogenesis). In one embodiment, the RNA binding latory compounds of the invention, e.g., the compounds of protein is a helminth RNA binding protein, e.g., a nematode formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, RNA binding protein, a trematode RNA binding protein or a VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or cestode RNA binding protein. In one embodiment, the RNA 2, modulate (e.g., inhibit) the RNA binding activity of POS-1, binding protein is a mammalian RNA binding protein. In one 15 but do not substantially inhibit the RNA binding activity of embodiment, the RNA binding protein is a mammalian RNA MEX-3 or MEX-5. In a further embodiment, the RNA bind binding protein and is not required for embryogenesis. In one ing modulatory compounds of the invention, e.g., the com embodiment of the invention, the RNA binding protein com pounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, prises a CCCH Zinc finger motif. Examples of RNA binding VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of proteins that comprise a CCCH Zinc finger motif include, but 20 Table 1 or 2 modulate (e.g., inhibit) the RNA binding activity are not limited to, MEX-5, POS-1 and MEX-6 or a homolog of two of MEX-3, MEX-5 and POS-1, without substantially thereof. In another embodiment, the RNA binding protein modulating (e.g., inhibiting) the RNA binding activity of the comprises a KH domain. Examples of RNA binding proteins third protein. In one embodiment, the RNA binding modula that comprise a KH domain include, but are not limited to, tory compounds of the invention, e.g., the compounds of MEX-3 and GLD-1, or a homolog thereof. 25 formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, The term “MEX-5” includes a cytoplasmic RNA binding VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or protein that contains two CCCH zinc finger motifs that func 2, modulate (e.g., inhibit) the RNA binding activity of MEX tions along with the similar CCCH zinc finger protein MEX 3, MEX-5 and POS-1. In one embodiment, the RNA binding 6, and is necessary for transducing polarity cues and estab modulatory compounds of the invention, e.g., the compounds lishing soma/germline asymmetry in the early embryo. In 30 offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, regulating the soma/germline asymmetry, MEX-5 activates VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or the SOCS-box protein ZIF-1, which functions as partofan E3 2, modulate (e.g., inhibit) the RNA binding activity of para ubiquitin ligase complex that degrades germ plasm proteins sitic MEX-3, MEX-5 and/or POS-1 without modulating (e.g., in somatic blastomers, resulting in reduced expression of inhibiting) the RNA binding of the mammalian (e.g., human) germline proteins in germline blastomers. MEX-5 is 35 homologs of MEX-3, MEX-5 and/or POS-1. expressed at uniform levels in both oocytes and newly fertil In one embodiment, an RNA binding modulatory com ized eggs (see Schubert C. M. et al., Molecular Cell (2000) pound of the invention, e.g., a compound of formula I, Ia, Ib, 5:671-682; Hunter, C. et al. Development (2002) 129:747 II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, 759; Cuenca, A. A. et al. (2003) Development 130:1255 VIId, VIIe, VIIf or a compound of Table 1 or 2, modulates, 1265; Lin Developmental Biology (2003)258:226-239; DeR 40 e.g., inhibits, the RNA binding activity of an RNA binding enzo C. et al., (2003) Nature 424,685-689; Nishi et al. (2008) protein (e.g., MEX-3, MEX-5 and/or POS-1) by at least about Development 135:687-697). 75%, by about 76%, by about 77%, by about 78%, by about The term “POS-1” includes a CCCH-type zinc finger pro 79%, by about 80%, by about 81%, by about 82%, by about tein that is necessary for the proper fate specification of germ 83%, by about 84%, by about 85%, by about 86%, by about cells, intestine, pharynx and hypodermis. POS-1 is also 45 87%, by about 88%, by about 89%, by about 90%, by about required in posterior blastomers for positive regulation of 91%, by about 92%, by about 93%, by about 94%, by about apx-1 mRNA translation and negative regulation of glp-1 95%, by about 96%, by about 97%, by about 98%, by about mRNA translation by direct binding to the spatial control 99% or by about 100%. In one embodiment, an RNA binding region in the glp-1 mRNA 3’ UTR. POS-1 is first found in low modulatory compound of the invention, e.g., a compound of levels in one-cell embryos and in high levels in germline 50 formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, blastomeres, where it disappears after the P4 division. POS-1 VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or colocalizes with cytoplasmic and perinuclear P granules in 2, modulates, e.g., inhibits, the RNA binding activity of an the germline blastomeres P1, P2, P3 and P4 (see Kohara, Y, et RNA binding protein (e.g., MEX-3, MEX-5 and/or POS-1) al. (1999) Development 126:1–11; Oguro, K. et al. (2003) by about 25%, by about 30%, by about 35%, by about 45%, Development 130:2495-2503). 55 by about 50%, by about 55%, by about 60%, by about 65%, The term “MEX-3” includes a KH domain-containing by about 70%, by about 71%, by about 72%, by about 73%, or RNA binding protein that is necessary for specifying the by about 74%. In a preferred embodiment, an RNA binding identities of the anterior AB blastomer and its descendent, as modulatory compound of the invention, e.g., a compound of well as for the identity of the P3 blastomer and proper segre formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, gation of the germline P granules. MEX-3 is found uniformly 60 VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or in the cytoplasm of oocytes and one-cell stage embryos and 2, modulates, e.g., inhibits, the RNA binding activity of an becomes more abundant in AB and its daughters at the two RNA binding protein (e.g., MEX-3, MEX-5 and/or POS-1) and four cell stages (see Bowerman et al. (1996) Cell 87:205 by at least about 75%. 216). In a further embodiment, the invention pertains, at least in In one embodiment, the RNA binding modulatory com 65 part to a method for reducing parasitic burden in soil, in plants pounds of the invention, e.g., the compounds of formula I, Ia, or in a subject suffering from a parasitic infection by admin Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, VIIc, istering a therapeutically or pesticidally effective amount of US 8,377,639 B2 13 14 an RNA binding modulatory compound, e.g., a compound of In one embodiment, the disease caused by a parasitic infes formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, tation is helminthiasis. The term “helminthiasis” includes VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or diseases caused by the infestation of a mammal by the a 2, such that the parasitic burden is reduced. helminth (e.g., a plathyhelminth, for example, trematodes and The term “parasitic burden” includes the amount (number) cestodes; thorny-headed worms, for example, acanthoceph of parasites and/or parasite eggs present in a given sample. alans; and roundworms, for example, nematodes). Examples Appropriate samples include, but are not limited to, soil of heminthiasis include, but are not limited to, schistosomia samples, plant samples (e.g., from roots, leaves, stems and the sis, swimmer’s , , , paragonimi like) and biological samples (e.g., , blood, tissue, fecal asis, , , , , 10 , , , dracuncu matter and the like). In one embodiment, upon administration liasis, , loa loa , , of an RNA binding modulatory compound, e.g., a compound , , , cutaneous offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, migrans, , , ascariasis, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or anisakiasis, viceral larva migrans, , strongyloidi 2, to the subject, the parasitic burden in that subject is reduced 15 asis, enterobiasis, , , , whip by about 10%, by about 11%, by about 12%, by about 13%, worm and . by about 14%, by about 15%, by about 16%, by about 17%, The articles “a” and “an” are used herein to refer to one or by about 18%, by about 19%, by about 20%, by about 21%, to more than one (i.e. to at leastone) of the grammatical object by about 22%, by about 23%, by about 24%, by about 25%, of the article. By way of example, “an element” means one by about 26%, by about 27%, by about 28%, by about 29%, 20 element or more than one element. by about 30%, by about 31%, by about 32%, by about 33%, As used herein, the term “compound” or “test compound” by about 34%, by about 35%, by about 36%, by about 37%, includes any agent, e.g., nucleic acid molecules, antisense by about 38%, by about 39%, by about 40%, by about 41%, nucleic acid molecule, peptide, peptidomimetic, small mol by about 32%, by about 43%, by about 44%, by about 45%, ecule, or other drug, which binds to an RNA-binding protein, by about 46%, by about 47%, by about 48%, by about 49%, 25 modulates, e.g., inhibits, interaction of an RNA-binding pro 50%, by about 51%, by about 52%, by about 53%, by about tein and an RNA molecule comprising an RNA-binding pro 54%, by about 55%, by about 56%, by about 57%, by about tein recognition element, and/or has a stimulatory or inhibi 58%, by about 59%, by about 60%, by about 61%, by about tory effect on, for example, RNA-binding protein expression 62%, by about 63%, by about 64%, by about 65%, by about or activity, binding affinity or stability. 66%, by about 67%, by about 68%, by about 69%, by about 30 For screening assays of the invention, preferably, the “test 70%, by about 71%, by about 72%, by about 73%, by about compound or agent” screened includes molecules that are not 74%, by about 75%, by about 76%, by about 77%, by about known in the art to modulate activity of an RNA-binding 78%, by about 79%, by about 80%, by about 81%, by about protein and/or expression as described herein. Preferably, a 82%, by about 83%, by about 84%, by about 85%, by about plurality of agents are tested using the instant methods. 86%, by about 87%, by about 88%, by about 89%, by about 35 The term “library of test compounds” is intended to refer to 90%, by about 91%, by about 92%, by about 93%, by about a panel comprising a multiplicity of test compounds. 94%, by about 95%, by about 96%, by about 97%, by about In one embodiment, the agent or test compound is a com 98%, by about 99% or by about 100%. pound that directly interacts with the RNA-binding protein or In one embodiment, the invention pertains, at least in part, directly interacts with a molecule with which the RNA-bind to a method for treating or preventing an autoimmune disor 40 ing protein interacts (e.g., a compound that inhibits or stimu der or inflammatory disorder in a subject by administering to lates the interaction between the RNA-binding protein and the subject a therapeutically effective amount of an RNA the RNA-binding protein target molecule, e.g., an RNA mol binding modulatory compound, e.g., a compound of formula ecule comprising an RNA-binding protein recognition ele I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, ment). In another embodiment, the compound is one that VIIc, VIId, VIIe, VIIfor a compound of Table 1 or 2, such that 45 indirectly modulates expression and/or activity of an RNA the inflammatory disorder is treated. binding protein, e.g., by modulating the activity of a molecule The term “inflammatory disorder” includes those disorders that is upstream or downstream of the RNA-binding protein that are associated with acute or chronic inflammation. in a signal transduction pathway involving the RNA-binding Examples of inflammatory disorders include, but are not lim protein. Such compounds can be identified using screening ited to, allergic reactions, autoimmune disorders (e.g., mul 50 assays that select for such compounds, as described in detail tiple sclerosis, diabetes, e.g., insulin dependent diabetes mel below. litus, chronic obstructive pulmonary disease, lupus, The term “interact” as used herein is meant to include endometriosis, myasthenia gravis, psoriasis, psoriatic arthri detectable interactions between molecules, such as can be tis), cancer, atherosclerosis, ischemic heart disease, asthma, detected using, for example, a gel shift assay, a fluorescence autoimmune diseases, chronic inflammation, chronic pros 55 polarization assay, a yeast two hybrid assay, and coimmuno tatitis, glomerulonephritis, inflammatory bowel disease, pel precipitation. The term interact is also meant to include vic inflammatory disease, e.g., Crohn’s disease, reperfusion “binding” interactions between molecules. Interactions may injury, rheumatoid arthritis, ankylosing spondylitis, trans be protein-protein or protein-nucleic acid in nature. plant rejection and vasculitis. As used herein, the term “contacting” (e.g., contacting a In another embodiment, the invention pertains, at least in 60 cell with a compound) is intended to include incubating the part, to a method for treating a disease caused by a parasitic compound and the cell together in vitro (e.g., adding the infestation in a mammal by administering to the mammal a compound to cells in culture) or administering the compound therapeutically effective amount of an RNA binding modu to a subject such that the compound and cells of the subject latory compound, e.g., a compound offormula I, Ia, Ib, II, IIa, are contacted in vivo. IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, 65 As used herein, the term “indicator composition” refers to VIIe, VIIfor a compound of Table 1 or 2, such that the disease a composition that includes a protein of interest (e.g., an is treated. RNA-binding protein), for example, a cell that naturally US 8,377,639 B2 15 16 expresses the protein, a cell that has been engineered to -continued express the protein by introducing an expression encoding the protein into the cell, or a cell free composition that contains the protein (e.g., purified naturally-occurring protein or recombinantly-engineered protein). 5 As used herein, the term “cell free composition” refers to an isolated composition which does not contain intact cells. Examples of cell free compositions include cell extracts and º,0/O compositions containing isolated proteins. 10 Cl In one embodiment, small molecules can be used as test compounds. The term “small molecule” is a term of the art and includes molecules that are less thanabout 7500, less than about 5000, less than about 1000 molecular weight or less O 15 º/ HO than about 500 molecular weight. In one embodiment, small O OH molecules do not exclusively comprise peptide bonds. In C| another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic 20 molecules (e.g., Cane et al. 1998. Science 282:63), and natu 3 , and ral product extract libraries. In another embodiment, the com pounds are small, organic non-peptidic compounds. In a fur O OH ther embodiment, a small molecule is not biosynthetic. For 25 example, a small molecule is preferably not itself the product O of transcription or translation. I. RNA Binding Modulatory Compounds sºs, H In one embodiment, the RNA binding modulatory com pound is a compound of formula I; 30

In a further embodiment, the thiophenyl moiety is carbonyl substituted with, for example, alkoxy (e.g., ethoxy) and alkyl 35 (e.g., methyl). In one embodiment, the thiophenyl moiety is

40 ~. O S wherein R", Rº, Rº and Rº are each independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, car bonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, acyl or a heterocyclic moiety; or R' and Rº and/or R and R* together 45 with the nitrogen atom to which they are attached are linked º to form a 3-9-membered carbocyclic or heterocyclic ring or 5-9-membered aryl ring; and pharmaceutically acceptable ) salts thereof. In one embodiment, R* and R* are each hydrogen and Rº is 50 aryl, for example, phenyl or a thiophenyl moiety. In another embodiment, the phenyl is a mono- or di-substituted In one embodiment, R* is carbonyl substituted with an aryl phenyl, substituted with, for example, carboxylate, moiety, such as phenyl (e.g., mono-, di- or tri-substituted), for halogen (e.g., chlorine), alkyl (e.g., methyl) or amino (e.g., example, phenyl substituted with halogen (e.g., chlorine, bro carbonylamino, for example, alkylcarbonylamino, such as 55 mine or iodine), alkoxy (e.g., methoxy or ethoxy), nitro, aryl CH3(CH2)3CONH=). In yet another embodiment, the phe (e.g., phenyl) or alkyl (e.g., methyl). In another embodiment, nyl is selected from the group consisting of: the phenyl is selected from the group consisting of:

60 NO2 HO HO

65 US 8,377,639 B2 17 18 -continued

^oCl 2 °s. 10 In yet another embodiment, R* is carbonyl, for example, carbonyl substituted with alkyl (e.g., isopropyl). In one embodiment, R' and R are eachalkyl, for example, /o 2^ 15 aryl substituted alkyl, such as benzyl. Br 2 2 In a further embodiment, Rº is hydrogen and R* is carbo nyl, for example, carbonyl substituted with aryl, such as phe nyl (e.g., -NO2 substituted phenyl, for example, I 2 20 C|

Cl Cl No 25 Cl NO2)

30 /oNO2 and 45.Cl - or a furanyl moiety, such as a furanyl moiety substituted with halogen (e.g., bromine), for example, In yet another embodiment, R*is carbonyl substituted with an aryl moiety, such as naphthyl, for example, an alkoxy (e.g., methoxy) substituted naphthyl. In one embodiment, the napthyl moiety is /

N 40 In yet another embodiment, R* is a benzo[d]imidazole moiety, for example, 1-alkyl-1H-benzo[d]imidazole, such as 1-propyl-1H-benzo[d]imidazole, 1-butyl-1H-benzo[d]imi 45 dazole or 1-benzyl-1H-benzo[d]imidazole. In anotherembodiment, R' is hydrogen and R is aryl (e.g., a diphenylquinoxalinyl moiety, for example, In one embodiment, R* is carbonyl substituted, for example, with an aryl moiety, such as a heteroaryl moiety, 50 such as, a furanyl or benzothiophenyl moiety. In another embodiment, the furanyl moiety is substituted with aryl, for example, phenyl, such as phenyl substituted with halogen (e.g., chlorine). In yet another embodiment, the furanyl moi ety is 55

O 60 In one embodiment, Rº and R* together with the nitrogen | / CI. atom to which they are attached are linked to form a 3-9 membered heterocyclic ring, such as a piperazinyl ring, for example, a piperazinyl ring substituted with a carbonyl moi In a further embodiment, the benzothiophenyl moiety is sub 65 ety (e.g., carbonyl substituted with alkoxy, such as carbonyl stituted with halogen (e.g., chlorine). In yet another embodi substituted with ethoxy). In another embodiment, the piper ment, the benzothiophenyl moiety is azinyl ring is US 8,377,639 B2 19 20 -continued

Áº 5 2 ~ Cy º O 10 C|

In a further embodiment, Rº is sulfonyl (e.g., tosylate). In yet another embodiment, R* is alkyl, for example, aryl 2 ~0/O 2 substituted alkyl, such as an oxydibenzene moiety, including, 15 OH but not limited to, C|

20

/Cro O OH 25 In another embodiment, the RNA binding modulatory O compound of formula I is a compound of formula Ia: sºs, H (Ia) 30 S O isSºse sºlº \oo and O 35 wherein R* and R* are each independently hydrogen or alkyl; and R* and R* are each independently alkyl oraryl, and phar 40 maceutically acceptable salts thereof. In one embodiment, R* and R* are each hydrogen and R” is aryl, such as phenyl or a thiophenyl moiety. In another embodiment, the phenyl is a mono- or di-substituted phenyl, substituted with, for example, carboxylate, halogen (e.g., 45 chlorine), alkyl (e.g., methyl) or amino (e.g., amino substi tuted with carbonyl, which may be further substituted with alkyl, for example, CH3(CH2)3CONH=). In a further In one embodiment, R* is an aryl moiety, for example, embodiment, the thiophenyl moiety may be substituted with 50 phenyl (e.g., a mono-, di- or tri-substituted), such as phenyl carbonyl (e.g., carbonyl substituted with alkoxy, such as car substituted with halogen (e.g., chlorine, bromine or iodine), bonyl substituted with ethoxy) and alkyl (e.g., methyl). alkoxy (e.g., methoxy or ethoxy), nitro, aryl (e.g., phenyl) or In another embodiment, Rº and Rºº are each alkyl, for alkyl (e.g., methyl). example, alkyl substituted with aryl (e.g., phenyl), such as benzyl. 55 In another embodiment, R* is aryl, for example, naphthyl, In yet another embodiment, Rºº is selected from the group such as a naphthyl substituted with alkoxy (e.g., methoxy). consisting of: In yet another embodiment, R* is an aryl moiety, such as a heteroaryl moiety, for example, a furanyl or benzothiophenyl moiety. In another embodiment, the furanyl moiety is substi 60 tuted with aryl, for example, phenyl, such as phenyl substi tuted with halogen (e.g., chlorine). In a further embodiment, HO HO the benzothiophenyl moiety is substituted with halogen (e.g., chlorine).

65 In yet another embodiment, R* is alkyl (e.g., isopropyl). In one embodiment, R* is selected from the group con sisting of: US 8,377,639 B2 21 22

(Ib) S NO 2 R& Js R4b 5 > r

wherein 10 R” and R* are each hydrogen; 22. ' R” is aryl or sulfonyl; and R” is alkyl or aryl; or Rºº and Rºº together with the nitro gen to which they are attached are linked to form a 3-9 membered heterocyclic or 5-9-membered aryl ring; and phar ^O 15 maceutically acceptable salts thereof. In one embodiment, Rº and Rºº are each hydrogen, Rº" is a sulfonyl (e.g., aryl substituted sulfonyl, for example, tosy late), and Rºº is aryl (e.g., an oxydibenzene moiety, for example, o2 S 20

Br

25

In another embodiment, R* and Rºº are each hydrogen, 30 R” is aryl (e.g., phenyl) and R* is aryl, for example, het too Cl /or eroaryl, such as a benzo[d]imidazole moiety (e.g., 1-alkyl 1H-benzo[d]imidazole, for example, 1-propyl-1H-benzo[d] imidazole, 1-butyl-1H-benzo[d]imidazole or 1-benzyl-1H benzo[d]imidazole). 35 In a further embodiment, Rº is hydrogen, Rº" is aryl, for example, a diphenylquinoxalinyl moiety /oCl /oNO 2 40

45 Cl (e.g., )

50 and Rºº and Rºº together with the nitrogen to which they are Cl, attached are linked to form a 3-9 membered heterocyclic ring, for example, a piperazinyl ring, such as a piperazinyl ring substituted with carbonyl (e.g., carbonyl substituted with alkoxy, for example, carbonyl substituted with ethoxy). 55 In yet another embodiment, the compound offormula I is a compound selected from the group consisting of:

S 2 /o NO2, O 60 and A- - *C. ºs,S O 65 In one embodiment, the RNA binding modulatory com H *O. I, pound of formula I is a compound of formula Ib:

US 8,377,639 B2 25 26 -continued -continued

S O

N DJsN - 5 H H | Cl, 2 / S N OH S. 10 HN N N H S O Cl * 2^ 15 S 2 OH

—N O 20 / S S O Js and N N 2^ H H 25

—/ O O 2

S O O S

Js 30 //& ~>N J N O

NO2,

35 and pharmaceutically acceptable salts thereof. In another embodiment, the RNA binding modulatory compound is a compound of formula II: S O 40

N h ~ O. O. (II) O / \/ R5 45 SN2 Br, ! R6

\ 50 H H N wherein N N Š R° and R7 are each hydrogen, hydroxyl, alkyl, alkenyl, N alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy, S 55 aryloxy, carbonyloxy, acyl or a heterocyclic moiety; R" is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, S amino, sulfone, carbonyl, carboxylate, alkoxy, aryloxy, car Js 2 bonyloxy, halogen, acyl, oximyl, hydrazinyl, -NO2, —CN, N NH a heterocyclic moiety or thioether; and pharmaceutically H * 60 acceptable salts thereof. N*NN In one embodiment, R7 is aryl, forexample, phenyl, such as phenyl substituted with halogen (e.g., bromine); Rº is hydro gen and R is alkyl, such as aminocarbonylalkyl, which may 65 be substituted, for example, aryl (e.g., phenyl, including, but not limited to, phenyl substituted with aminocarbonyl. In one embodiment, Rº is US 8,377,639 B2 27 28

SS ׺r 5 Ár | 2 2 Ár[X 2 ! | / and N~9 10 | y In another embodiment, R’ is hydrogen; R is aryl, for example, phenyl (e.g., disubstituted phenyl), for example, phenyl substituted with hydroxyl, halogen (e.g., chlorine), alkyl (e.g., methyl) or amino (e.g., aryl substituted sulfony 15 R*isamino, forexample, amino substituted with sulfonyl, for lamino, for example, phenyl substituted sulfonylamino). In example, sulfonyl substituted with aryl in which the aryl may one embodiment, R is selected from the group consisting of: be substituted with halogen (e.g., bromine); aryl, for example, phenyl, such as trisubstituted phenyl substituted with halogen (e.g., chlorine) and hydroxyl; heteroaryl, for example, thiaz OH 20 olyl, which may be substituted with aryl, such as phenyl, for example, phenyl substituted with hydroxyl, alkyl (e.g., methyl) or halogen (e.g., fluorine). In another embodiment, R" is selected from the group consisting of: 25 HO Cl OH OH

HO 30 N `y. | N NH OH and CI. S F HNs 20 C| o” S 35 NH N º, | N NH 2 and C| OH 40 S Br In a further embodiment, Rº is —NO, or hydrogen. In yet another embodiment, R7 is hydrogen; R is aryl, for example, heteroaryl, such as an oxazolyl moiety 45 o=y-sº - O O

50 ºrN In yet another embodiment, R7 is hydrogen; R is aryl, for example, heteroaryl (e.g., isoxazolyl, thiazolyl or thiadiaz olyl, such as

55 and Rº is halogen (e.g., chlorine). In a further embodiment, R7 is hydrogen; R is alkyl, for example, heteroaryl substituted alkyl in which the heteroaryl 60 may be a benzo[d]imidazole moiety, (e.g., a 1|H]-benzo[d] imidazole) and Rº is alkyl (e.g., methyl) or halogen (e.g., and Rº is amino, for example, carbonylamino, such as carbo chlorine). nylamino substituted with alkyl. In one embodiment, the In yet another embodiment, R7 is hydrogen; R is het 65 alkyl is C1-C, alkyl that may be substituted with thiol, for eroaryl, for example, isoxazolyl, thiazolyl, thiadiazolyl or example heteroaryl (e.g., benzothiazolyl or triazinyl) substi pyrimidinyl such as tuted thiol: aryloxy, for example, phenoxy, such as di- or US 8,377,639 B2 29 30 trisubstituted phenoxy substituted with alkyl (e.g., methyl) or -continued halogen (e.g., bromine); a heterocyclic moiety (e.g., quinazolinonyl) or aryl, for example, phenyl, such as phenyl substituted with alkyl (e.g., methyl). In yet another embodiment, R7 is hydrogen; R is aryl, for example heteroaryl (e.g., isoxazolyl, thiazolyl or thiadiaz olyl, such as

10

15

and Rº is amino, for example carbonylamino, such as carbo 20 nylamino substituted with aryl, for example, phenyl (e.g., mono- or di-substituted phenyl), such as phenyl substituted with alkyl (e.g., t-butyl), alkoxy (e.g., methoxy or n-butoxy) or halogen (e.g., chlorine or bromine). 25 In another embodiment, R’ is hydrogen; R is aryl, for example heteroaryl (e.g., isoxazolyl, thiazolyl or thiadiaz olyl, such as 30

Br

and Rº is amino, for example, carbonylamino, such as carbo 40 nylamino substituted with a heterocyclic moiety (e.g., chromene or pyrrolidinone). In a further embodiment, Rº is selected from the group consisting of: 45

55

60 O O No 2 N y 2 65 O In one embodiment, the RNA binding modulatory com pound of formula II is a compound of formula IIa. US 8,377,639 B2 31 32

O. O. (IIa)

\%^. N ...” R5a k R6a wherein 10 R” is alkyl or aryl; R” is –NO2, alkyl, halogen or hydrogen; and and Rºº is halogen (e.g., chlorine). R” is hydrogen or aryl, and pharmaceutically acceptable 15 In a further embodiment, R7° is hydrogen; Rºº is alkyl, for salts thereof. example, heteroaryl substituted alkyl in which the heteroaryl In one embodiment, R'" is aryl, for example, phenyl, such may be a benzo[d]imidazole moiety, (e.g., a 1|H]-benzo[d] as phenyl substituted with halogen (e.g., bromine); Rºº is imidazole) and Rºº is alkyl (e.g., methyl) or halogen (e.g., hydrogen and Rºº is alkyl, such as, aminocarbonylalkyl, chlorine). which may be substituted, for example, with aryl (e.g., phe 20 In another embodiment, the RNA binding modulatory nyl, including, but not limited to, phenyl substituted with aminocarbonyl). In one embodiment, Rº is compound of formula II is a compound of formula IIb.

25 (IIb) O /* \ / R5b HN ~ i 2° R& H 30 > H

wherein In another embodiment, R7° is hydrogen; Rºº is aryl, for 35 R” is aryl; and example, phenyl (e.g., disubstituted phenyl), for example, phenyl substituted with hydroxyl, halogen (e.g., chlorine), R* is sulfonyl or aryl, and pharmaceutically acceptable alkyl (e.g., methyl) or amino (e.g., aryl substituted sulfony salts thereof. lamino, for example, phenyl substituted sulfonylamino). In 40 In yet another embodiment, Rºº is aryl, such as heteroaryl, one embodiment, Rºº is selected from the group consisting for example, isoxazolyl, thiazolyl, thiadiazolyl or pyrimidi of: nyl, such as

45

OH and OH 50 HNs 2° and 2.

55

OH R* is sulfonyl, for example, sulfonyl substituted with aryl in 60 which the aryl may be substituted with halogen (e.g., bro mine); or aryl, for example, phenyl, such as trisubstituted CI. phenyl substituted with halogen (e.g., chlorine) and hydroxyl; heteroaryl, for example, thiazolyl, which may be substituted with aryl such as phenyl, for example, substituted with In a further embodiment, Rºº is –NO, or hydrogen. 65 hydroxyl, alkyl (e.g., methyl) or halogen (e.g., fluorine). In In yet another embodiment, R7°is hydrogen; Rºº is aryl, for another embodiment, Rºº is selected from the group consist example, heteroaryl, such as an oxazolyl moiety ing of: US 8,377,639 B2 33 34 In yet another embodiment, Rºº is aryl, for example het O eroaryl (e.g., isoxazolyl, thiazolyl or thiadiazolyl, such as

HO 5 N

S 10 /o/r) F Cl N | / ); N

| N 2 and 15 S Cl OH Br and Rºº is aryl for example, phenyl (e.g., mono- or di-substi tuted phenyl), such as phenyl substituted with alkyl (e.g., 20 t-butyl), alkoxy (e.g., methoxy or n-butoxy) or halogen (e.g., chlorine or bromine). In another embodiment, Rºº is aryl, for example heteroaryl (e.g., isoxazolyl, thiazolyl or thiadiazolyl, such as 25

In one embodiment, the RNA binding modulatory com pound of formula II is a compound of formula IIc. Ár S 30 [2. N| ~N/ and (IIc) --O O. O. N ); \/S Rºc. O ^. N …” 35 /

H R6c Js N | | H " and Rºº is a heterocyclic moiety (e.g., chromenyl or pyrroli dinonyl). In a further embodiment, Rº is selected from the group wherein consisting of: R* is aryl; and 45 R* is alkyl, aryl or a heterocyclic moiety, and pharmaceu tically acceptable salts thereof. In yet another embodiment, Rºº is aryl, for example het O O eroaryl (e.g., isoxazolyl, thiazolyl or thiadiazolyl, such as 50 N

| 22. Cl 2 N O

55 N 2 SN } N º and Rºº is alkyl, for example, C-C, alkyl that may be substi 60 tuted with thiol, such as heteroaryl (e.g., benzothiazolyl or O O triazinyl) substituted thiol: aryloxy, for example, phenoxy, such as di- or trisubstituted phenoxy substituted with alkyl No 2^ 3 (e.g., methyl) or halogen (e.g., bromine); a heterocyclic moi 65 ety (e.g., quinazolinonyl)oraryl, for example, phenyl, such as phenyl substituted with alkyl (e.g., methyl). US 8,377,639 B2 35 36 -continued -continued

ºn , ~yN *o, e. O O 15 H Br Br No 2 N -( O ST N O // N

2 2 20 O× H --

O2N

O

25 £S. 2 and ./ } º OH 30 NH O 2^ S N2. Sr. 35 S N

NH O ES =O

~~ In one embodiment, the compound offormula II is selected 40 Tr) 2 from the group consisting of:

60 O H

O H 65

US 8,377,639 B2 39 40 -continued In one embodiment, the compound is selected from the group consisting of:

HN | | /-(-)-- O \--(-)O \. Br NO2 and pharmaceutically acceptable salts thereof. In yet another embodiment, the RNA binding modulatory 15 O O | | compound is a compound of formula III: S—NH HN–S 2 | | O O (III) R10 20 O O | | *-i-N 25 R13 R12 wherein R*, R*, R” and R'' are each independently hydrogen, 30 hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfone, car O i-sº *— O and bonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, halogen, olºO O acyl, oximyl, hydrazinyl, -NO2, —CN, a heterocyclic moi ety or thioether; 35

O O R*, R*, R* and R'' are each independently hydrogen, | | hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, car F -- *— F bonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, acyl, halo 40 O O gen or a heterocyclic moiety; or R'" and Rºº and/or R'" and R” together with the nitrogenatom to which they are attached and pharmaceutically acceptable salts thereof. are linked to form a 3-9-membered heterocyclic ring or 5-9 aryl ring; and pharmaceutically acceptable salts thereof. In a further embodiment, the RNA binding modulatory 45 compound is a compound of formula IV: In another embodiment, Rº and R'" are each hydrogen; R* and R'' are eacharyl, for example, phenyl, such as unsub stituted phenyl: R*, R*, and R'' are each hydrogen and R'" is (IV) —NO2. In another embodiment, Rº and R'" are each hydrogen; 50 R*, R” and R'' are each hydrogen; R is alkyl (e.g., methyl) or hydrogen; and R* and R'' are each aryl, for example, phenyl, such as phenyl substituted with alkoxy (e.g., meth oxy) or halogen (e.g., fluorine). In one embodiment, the phe 55 nyl is wherein R” and Rºº are each independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, car boxylate, alkoxy, aryloxy, carbonyloxy, acyl or a heterocyclic 60 moiety; or R' and Rº and/or R* and Rt together with the nitrogen atom to which they are attached are linked to form a 3-9-membered heterocyclic or 5-9-membered heteroaryl ring; and R” is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, In yet another embodiment, R* and R* are each hydro 65 amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, car gen; R* and R'' are each alkyl (e.g., ethyl) and Rº, Rº, R'" bonyloxy, acyl, halogen or a heterocyclic moiety; and phar and R'' are each hydrogen. maceutically acceptable salts thereof. US 8,377,639 B2 41 42 In one embodiment, R” is hydrogen: R* is aryl, for example, heteroaryl, such as thiophenyl, benzothiazolyl or phthalazinyl C Br

OH OH (e.g., Cl Or ) N 10 S or phenyl, for example, phenyl substituted with alkoxy (e.g., S ethoxy). | / 2 Or In yet another embodiment, the RNA binding modulatory (e.g., 15 compound of formula III is selected from the group consist 29 | `ss ing9. of: 2 20 NH ) O }/ N and Rºº is- acyl (–COCHA), alkyl, for example, alkyl substi-. 25 /O Q tuted with a heterocyclic moiety (e.g., tetrahydrofuranyl), ŽSs such as O H 2 30 | N HN Xºry S O & D 2

35 or aryl, such as naphthyl, for example, naphthyl substituted O with halogen (e.g., chlorine) and hydroxyl | NH S - | 2 O 40 O2N O ( ) Cl OH NO2 45 Br (e.g., OH). Cl OH, £Ž 50 // SN In one embodiment, R” is hydrogen: R* is aryl, for O

example, phenyl (e.g., mono- or disubstituted phenyl), for example, phenyl substituted with —NO2, alkyl (e.g., methyl) or aminocarbonyl (e.g., -CONH2), such as 55

O

60 Or H2N ;

NO2

65 and Rºº is aryl, for example, naphthyl, such naphthyl substi tuted with halogen (e.g., chlorine or bromine) and hydroxyl and pharmaceutically acceptable salt thereof. US 8,377,639 B2 43 44 In one embodiment, the RNA binding modulatory com stituted with aryl (e.g., phenyl, including disubstituted phe pound is a compound of formula V. nyl, which may be substituted with halogen (e.g., chlorine)). In one embodiment, the carbonyloxy is

(V)

10 ~~ CI.

15

20 In one embodiment, the compound offormula V is selected wherein from the group consisting of: R*, R*, R* and Rºº are each independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfone, car bonyl, carboxylate, carbonyloxy, alkoxy, aryloxy, halogen, acyl, oximyl, hydrazinyl, -NO2, —CN, a heterocyclic moi 25 ety or thioether; R” is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, car bonyloxy, acyl or a heterocyclic moiety; and 30 R* is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, car bonyloxy, acyl or a heterocyclic moiety; and pharmaceuti cally acceptable salts thereof. In another embodiment, R*, R* and R* are hydrogen; 35 and R* is hydrogen and R* and R* are each halogen (e.g., chlorine). In yet another embodiment, Rºº, Rºº, Rºº, Rºº and Rºº are hydrogen; and R* is carboxy, for example, carboxy substi 40 tuted with aryl, such as naphthyl or phenyl (e.g., phenyl substituted with alkoxycarbonyl, for example, methoxycar bonyl or ethoxycarbonyl). In one embodiment, the carboxy is 45

50

55

O O

º Os Or 60

65 In yet another embodiment, Rºº, Rºº, Rºº, Rºº and Rºº are hydrogen; and R* is carbonyloxy, such as carbonyloxy sub US 8,377,639 B2 45 46 -continued O Cl H O s N

C| and In another embodiment, Rº", Rºº and Rºº are each hydro 10 gen and Rºº and Rº' are linked to form an aryl moiety, for HN O example, phenyl (e.g., tri-substituted phenyl), for example, phenyl substituted with —NO2, hydroxyl and alkyl, which may be substituted with a heterocyclic moiety, amino or aryl. O O H In one embodiment, the heterocyclic moiety is morpholine or 15 pyrrolidine, which may be substituted with alkyl (e.g., Cl C| methyl). In one embodiment, the amino is substituted with carbonyl, which may be further substituted with alkyl (e.g., methyl, ethyl, n-propyl or n-butyl). In one embodiment, the aryl is furanyl or phenyl, for example, phenyl substituted with 20 halogen (e.g., chlorine), hydroxyl oralkoxy (e.g., methoxy or HN O alkoxy). In one embodiment, Rºº and Rº' are linked to form a ring selected from the group consisting of

25 º O, NO2 2 and pharmaceutically acceptable salts thereof. J S In another embodiment, the RNA binding modulatory H compound is a compound of formula VI: 30 S 2 º 2 NO2 OH 2. O O (VI) R29 OH - 35 NO2 S S R27ºr §2. R3 l H 40 2 ºrs wherein OH O R*7, Rºº, Rºº, Rºº and Rº' are each independently hydro gen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, carbonyloxy, alkoxy, aryloxy, halo o” gen, acyl, oximyl, hydrazinyl, -NO2, —CN, a heterocyclic 45 moiety or thioether; or Rº" and Rºº or Rºº and Rºº or Rºº and °s R” or Rºº and Rº' together with the carbon atoms to which NO2 they are attached are linked to form a 4-9 carbocyclic, het S OH erocyclic or aryl ring; and pharmaceutically acceptable salts 50 thereof. In one embodiment, Rº" and Rºº are linked to forman aryl 2 o°S, moiety (e.g., phenyl); R* is hydrogen; Rº" is aryl, for example, furanyl 55 OH º O

O (e.g., / ); 60 NO2 S and Rºº is carbonyl, for example, carbonyl substituted with 2 N amino, which may in turn be substituted with aryl, for 65 example, phenyl, such as phenyl substituted with halogen OH (e.g., chlorine). In one embodiment, Rº is US 8,377,639 B2 47 48 -continued NO2 R30a (VIa) O S ~~~ and S 2 NH §2. R3 la OH Cl OH 10 wherein NO2 R* and Rº" are each –NO, or alkyl; and pharmaceuti O - cally acceptable salts thereof. In one embodiment, Rº" is NO, and Rº" is alkyl, which S ~~~ 15 may be substituted with a heterocyclic moiety (e.g., morpho 2 NH line). i. another embodiment, Rº" is —NO, and Rº" is alkyl, OH Cl for example, alkyl substituted with a heterocyclic moiety, amino or aryl. In one embodiment, the heterocyclic moiety is 20 pyrrolidine, which may be substituted with alkyl (e.g., methyl). In one embodiment, the amino is substituted with carbonyl, which may be further substituted with alkyl (e.g., methyl, ethyl, n-propyl or n-butyl). In one embodiment, the 25 aryl is furanyl or phenyl, for example, phenyl substituted with In yet another embodiment, Rºº, Rº and Rºº are each halogen (e.g., chlorine), hydroxyl oralkoxy (e.g., methoxy or hydrogen and Rº' and Rº' are each substituted with carbonyl, alkoxy). In one embodiment, Rº" is selected from the group for example, carbonyl substituted with amino, which may be consisting of substituted with aryl, such as thiadiazolyl (e.g., thiadiazolyl substituted with alkyl, for example, methyl or n-propyl) or 30 benzothiazolyl. In one embodiment, Rº" and Rº' are

O S 35

O

40

N O \ X 45 X- -

In a further embodiment, Rº", Rº and Rºº are each hydro 50 gen and Rºº and Rº' are each carbonyl, for example, carbonyl substituted with amino, which may be further substituted with alkyl (e.g., alkyl substituted with aryl, for example, phenyl, such as phenyl substituted with alkoxy (e.g., methoxy). In one embodiment, Rºº and Rº' are each 55

60

65 In one embodiment, the RNA binding modulatory com pound of formula VI is a compound of formula VIa: US 8,377,639 B2 49 50 In yet another embodiment, the RNA binding modulatory -continued compound of formula VI is a compound of formula VIb: NO2 S (VIb) 22 N

OH

10 wherein R” and Rº" are each carbonyl; and pharmaceutically acceptable salts thereof. 15 NO2 In one embodiment, the carbonyl is substituted with amino, which may be substituted with aryl, such as thiadiazolyl (e.g., S thiadiazolyl substituted with alkyl, for example, methyl or 20 22 n-propyl) or benzothiazolyl. In one embodiment, the carbo N nyl is substituted with OH

25

DryNSN: zº O

30 2.

S N 35

y - - NO2 S 40 2 In another embodiment, the compound of formula VI is N selected from the group consisting of OH NO2 45 S

22 N 50 OH Cl

N NO2 55 NO2

S

2 NH N 60 OH

65 Cl US 8,377,639 B2 51 52 -continued dently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, car bonyloxy, halogen, acyl, oximyl, hydrazinyl, -NO2, —CN, a heterocyclic moiety or thioether; or R* and Rºº together with the atoms to which they are attached are linked to form a 4-12 membered carbocyclic, aryl or heterocyclic ring; or R*, R* or R* and R*, Rºor Rºº together with the atoms S to which they are attached are linked to form a 4-12 mem H | H 10 bered carbocyclic, aryl or heterocyclic ring; or S N 2 N S R* and Rºº together with the atoms to which they are | N | attached are linked to form a 4-12 membered carbocyclic, N_". O O ! / aryl or heterocyclic ring; and A is O, S, NRºº or CR*R*: 15 D is O, S, NR* or CR*R*, S E is O, S, NR* or CR*R*7: O | .< O G is O, S, NR” or CR”R*; and pharmaceutically accept N able salts thereof. N NH HN N In one embodiment, p and q are each a double bond; U is O: sº Se 20 W is CR” and Z is CR”; X is N. Y is N: R* is thiol (e.g., —SH) or aryl, for example, phenyl, such as phenyl substi Cºl -O - tuted with halogen (e.g., bromine) or quinoline, for example, O quinoline substituted with aryl (e.g., phenyl), Rºº is thiol, for example, thiol substituted with aryl (e.g., phenyl, which may HN S 25 be substituted with —NO, and carboxylate) or aryl. In one - embodiment, Rºº is N

HN 30

OH O

°s o’ 35 °s and R* is and pharmaceutically acceptable salts thereof. In yet another embodiment, the RNA binding modulatory 40 compound is a compound of formula VII:

(VII) 45 z-NU Br Alternatively, Rºº is wherein 50 U is NR*, O or S: W is N or CR” when q is a double bond, or W is NR*, CR*R* or C=D when q is a single bond; X is N or CR” when q is a double bond, or X is NR”, CR*R* or C=E when q is a single bond; 55 Y is NR*, CR*R*7 or C=G when p is a single bond, or Y is N or CR” when p is a double bond; Zis NR*, CR*R* or C=A when p is a single bond, or Z is N or CR” when p is a double bond; 60 p and q are each independently a single or double bond; R32, R*, R*, R*, R*, R+7, R52, R° and Rºº are each independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, In another embodiment, Y is CR”; R* and Rºº are each aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, aryl (e.g., phenyl) and Rºº is amino, for example, amino carbonyloxy, acyl or a heterocyclic moiety; 65 substituted with carbonyl, which may be substituted with aryl R*, R*, R36, R37, R3s, Rao, R*, R42, R4s, R*, R*, R*, (e.g., phenyl, for example phenyl substituted with alkyl, such R*, R*, R*, R*, R*, R*, R* and Rºº are each indepen as methyl) In one embodiment, Rº is US 8,377,639 B2 53 54

HO

HO /*C. (e.g., is );

In yet another embodiment, p and q are each a double bond; 10 and Rºº is amino, for example, amino substituted with aryl, U is S; W is CR” and Zis CR*; X is N.Y is N: R* is amino such as, naphthyl or thiol. In one embodiment, Rºº is amino, for example, amino substituted with carbonyl or oximyl. In another embodiment, the oximyl is an aryl substituted oximyl, such as phenyl substituted oximyl in which the phenyl may be further 15 substituted with —NO2. In yet another embodioxy, such as ethoxy) or aryl (e.g., heteroaryl, for example, triazole). Alter natively, Rºº is thiol, for example, thiol substituted with alkyl (e.g., aryl substituted alkyl, such as phenyl substituted alkyl, (e.g., which may be further substituted with halogen, such as chlo 20 rine). In one embodiment, Rºº is In another embodiment, p and q are each a double bond; U is S; W is CR” and Z is CR”; X is CR”;Y is CR”: R* and O R” are eachalkyl (e.g., methyl), Rºº is carbonyl, forexample, N 25 carbonyl substituted with amino (e.g., -NH2): R* is amino, º o” S. for example, amino substituted with carbonyl, such as aryl O substituted carbonyl (e.g., pyrazine substituted carbonyl, for NO2 example, 30

H º N | 2 Or Ns 35

and Rºº is amino (e.g., amino substituted with carbonyl, such Áº, 40 as carbonyl substituted with cyclohexenyl, for example,

In another embodiment, Rº is alkyl, for example, t-butyl O or alkyl substituted with aryl (e.g., phenyl, for example phe nyl substituted with halogen, such as chlorine) or carbonyl 45 (e.g., alkoxy substituted carbonyl, for example, ethoxyl sub A.H stituted carbonyl). Alternatively R* is aryl (e.g., heteroaryl, such as pyridine) or thiol (e.g., -SH). In another embodi O ). ment, Rº is 50 In another embodiment, Rºº is carbonyl, for example, car bonyl substituted with alkoxy (e.g., methoxy); R* is alkyl (e.g., ethyl); and Rºº is aryl, for example, phenyl, such as 55 phenyl substituted with halogen (e.g., chlorine). In one Cl 2 embodiment, Rºº is

SpӼ 60

In yet another embodiment, p and q are each a double bond; U is S; W is CR” and Z is CR”; X is N. Y is CR”: R* is 65 hydrogen; Rºº is aryl, for example, phenyl, such as phenyl substituted with hydroxyl US 8,377,639 B2 55 56 In yet another embodiment, Rºº and R* are linked to join alkyl (e.g., methyl) substituted phenoxy oralkoxy (e.g., meth a ring (e.g., a 6-membered or 12-membered carbocyclic ring), oxy) substituted phenoxy. In another embodiment R^* is a for example, thiol substituted alkyl (e.g., arylalkylthioalkyl, such as phenyl substituted alkylthioalkyl in which the phenyl may be further substituted with halogen, for example chlorine); a heterocy clic substituted alkyl (e.g., alkyl substituted with ºccº 10

In one embodiment, Rº is amino, for example, amino ); substituted with carbonyl (e.g., alkyl substituted carbonyl 15 such as alkyl substituted with carboxylate). In one embodi ment, Rºº is aryl substituted alkyl, for example, phenyl substituted alkyl (e.g., unsubstituted phenyl or phenyl substituted with halo 20 gen, such as chlorine, or phenyl substituted with alkoxy, such O as isopropoxy). Alternatively, Rºº is aryl, for example, H N thiophenyl or phenyl, for example, phenyl substituted with OH. hydroxyl or alkoxy (e.g., methoxy). In another embodiment, R” is selected from the group consisting of O 25 In another embodiment, Rºº is carbonyl, for example, alkoxy (e.g., methoxy) substituted carbonyl.

In another embodiment, Rº and Rºº are linked to form a 30 6-membered heterocyclic ring, for example,

H S N S N 35

In yet another embodiment, p and q are each a double bond; Uis NR*: X is N.Y is N. Wis CR” and Zis CR”: R* is thiol (e.g., -SH); Rºº is hydrogen, alkyl (e.g., methy or alkyl substituted with aryl, for example, phenyl, or alkenyl)oraryl, for example, unsubstituted phenyl or phenyl substituted with alkyl (e.g., methyl) or halogen (e.g., fluorine). In one embodi ment, Rºº is

50

55

In a further embodiment, Rºº is alkyl, for example, alkyl 65 substituted with aryloxy, for example, naphthyloxy or phe noxy, such as halogen (e.g., bromine) substituted phenoxy, US 8,377,639 B2 57 58 -continued OH

OH

HO O

2 HO Or 10 In another embodiment, Rºº and Rºº are linked to form a 6-membered heterocyclic ring S

15 O O

In another embodiment, p and q are each a double bond; U Ås 20 is NR*; W is CR”, X is N.Y is CR”; Zis N: R* is aryl (e.g., (e.g., NH ). phenyl); R* is a heterocyclic moiety

In another embodiment, U is NR**; X is N. Y is N; W is CR”; Z is CR*: R* is hydrogen; Rºº is hydrogen; Rº is 2s amino, for example, amino substituted with carbonyl (e.g., carbonyl substituted with aryl, such as phenyl, which may be substituted with hydroxyl). In yet another embodiment, R* is a heterocyclic moiety 30

O R* is amino, such as, amino substituted with carbonyl (e.g., carbonyl substituted with aryl, for example, phenyl, such as º, 35 phenyl substituted with alkoxy (e.g., methoxy), alkyl (e.g., (e.g., OH Or methyl)) or sulfonyl (e.g., sulfonyl substituted with aryl, for example, phenyl, which may be substituted with alkyl (e.g., O methyl)). In one embodiment, Rºº is 40

Cl - 'V' Ö ). 45 H In another embodiment, U is NR**; W is CR”, X is CR”, Y is CR”; Z is N: R* is hydrogen or alkyl (e.g., alkyl o substituted with carboxylate, for example, 50 —CH2CH2COOH), Rºº is hydrogen or alkyl (e.g., methyl); R” is alkenyl (e.g., alkenyl substituted with a heterocyclic H moiety, for example, No cº J.sº,

55 O In another embodiment, Rºº and Rºº are linked to form a 5 NH or 6-membered heterocyclic ring, for example

- }= NH 60 O ) or aryl (e.g., phenyl or benzothiazolyl); Rºº is aryl, for example, phenyl, such as phenyl substituted with hydroxyl, 65 alkoxy (e.g., methoxy), alkyl (e.g., ethyl) or a heterocyclic moiety. In one embodiment, Rºº is US 8,377,639 B2 59 60 -continued

5 º Or ); Or 10

R” is aryl, such as furanyl or phenyl, for example, phenyl substituted with hydroxyl, halogen (e.g., bromine orchlorine) 15 and alkoxy (e.g., methoxy). In one embodiment, Rºº is

Cl Br 20 & N H

25 . . O OH 2 OH > _^ Or In another embodiment, Rºº is amino (e.g., amino substi OH tuted with carbonyl, for example, aryl substituted carbonyl, such as phenyl substituted carbonyl, including –NO2 substi OH tuted phenyl) or aryl (e.g., phenyl, for example, phenyl sub 30 stituted with carbonyloxy, such as alkyl substituted carbony OH. loxy, including, but not limited to methyl substituted carbonyloxy). In one embodiment, Rºº is

35 In one embodiment, U is NR*, q is a double bond and p is a single bond; W is N; X is CR”;Y is C=G; Z is C=A; A is O, R* is aryl, for example, phenyl substituted with sulfonyl (e.g., amino substituted sulfonyl), carboxylate or halogen 40 O h X, (e.g., chlorine), such as

O Cl 45 In another embodiment, p and q are each a double bond; U is NR*; W is N; X is N;Y is N; Z is CR*: R* is hydrogen; R* is amino, for example, amino substituted with carbonyl (e.g., aryl substituted carbonyl, for example, heteroaryl sub 50 ºO OH stituted carbonyl, such as thiophenyl substituted carbonyl O

HO 55

Or ; C| ). 60 In yet another embodiment, U is NR*; q is a double bond R* is alkyl (e.g., methyl); G is CR”R*, R* is hydrogen and and p is a single bond; W is CR”; X is N;Y is NR* and Z is R* is aryl, for example, phenyl, such as phenyl substituted C=A; A is S. R* is hydrogen; Rºº is hydrogen, alkyl (e.g., with alkoxy (e.g., methoxy or ethoxy) or carbonyloxy, for methyl, ethyl or aryl substituted alkyl, for example, phenyl 65 example, carbonyloxy substituted with aryl (e.g., heteroaryl, substituted alkyl) or aryl (e.g., phenyl, such as phenyl substi for example, thiophenyl or furanyl). In one embodiment, Rºº tuted with alkyl, such as methyl or ethyl, for example, 1S US 8,377,639 B2 61 62 -continued 29

DC x C O O O ~$ O O S 10

ºr, O | Or 29

15 O

O ~s O S ºr, 20 In one embodiment, the RNA binding modulatory com pound of formula VII is a compound of formula VIIa: In yet another embodiment, U is NR*; p and q are each a single bond; W is NR*, X is C=E, Y is C=G; Z is C=A: R* 25 is hydrogen; A and E are O: R* is aryl, for example, phenyl, (VIIa) such as unsubstituted phenyl or phenyl substituted with halo gen (e.g., chlorine or fluorine) or carbonyl, for example, car bonyl substituted with alkoxy (e.g., ethoxy), for example, 30

wherein

O O 35 U° is O or S: Cl F R” is amino, thiol or aryl; and C| Cl R* is aryl, thiol, or alkyl; and pharmaceutically accept able salts thereof. In one embodiment, U* is S. R* is amino, for example, 40 amino substituted with carbonyl or oximyl. In another embodiment, the oximyl is aryl substituted oximyl, such as 2 2 Or phenyl substituted oximyl in which the phenyl may be further Brs substituted with —NO2. In yet another embodiment, the car bonyl may be substituted with carbonyl (e.g., carbonyl sub 45 stituted with alkoxy, such as ethoxy) or aryl (e.g., heteroaryl, for example, triazole). Alternatively, Rº" is thiol, for example, thiol substituted with alkyl (e.g., aryl substituted alkyl, such as phenyl substituted alkyl, which may be further substituted with halogen such as chlorine). In one embodi G is CR”R*: R* is hydrogen; Rºº is aryl, for example, 50 ment, Rº" is phenyl, such as phenyl substituted with alkoxy (e.g., meth oxy) or carbonyloxy (e.g., carbonyloxy substituted with aryl, for example, heteroaryl such as thiophenyl or furanyl). In one NO2 embodiment, Rºº is

65 US 8,377,639 B2 63 64 In another embodiment, R* is alkyl, for example, t-butyl or alkyl substituted with aryl (e.g., phenyl, for example, phe (VIIb) nyl substituted with halogen, such as chlorine) or carbonyl (e.g., alkoxy substituted carbonyl, for example, ethoxy sub stituted carbonyl). Alternatively R* is aryl (e.g., heteroaryl, such as pyridine) or thiol (e.g., -SH). In another embodi ment, Rº is

10 wherein R” is hydrogen, aryl or alkyl, and R” is alkyl or aryl; or Rºº and Rºº" together with the Cl 2 Or 15 atoms to which they are attached are linked to form a 6-mem bered heterocyclic ring; and pharmaceutically acceptable salts thereof. In one embodiment, Rºº is hydrogen, alkyl (e.g., methy or alkyl substituted with aryl, for example, phenyl, oralkenyl) or 20 aryl, for example, unsubstituted phenyl or phenyl substituted In another embodiment, U" is O: R* is thiol (e.g., -SH) with alkyl (e.g., methyl) or halogen (e.g., fluorine). In one or aryl, for example, phenyl, such as phenyl substituted with embodiment, Rº" is halogen (e.g., bromine) or quinoline, for example, quinoline substituted with aryl (e.g., phenyl); Rºº is thiol, for example, 25 thiol substituted with aryl (e.g., phenyl, which may be sub stituted with —NO, and carboxylate) or aryl. In one embodi ment, Rº" is

30

OH ~ 35 and Rºº is 40 In a further embodiment, Rº" is alkyl, for example, alkyl substituted with aryloxy, for example, naphthyloxy or phe noxy, such as halogen (e.g., bromine) substituted phenoxy, alkyl (e.g., methyl) substituted phenoxy oralkoxy (e.g., meth oxy) substituted phenoxy. In another embodiment R^* is a 45 thiol substituted alkyl (e.g., arylalkylthioalkyl, such as phenyl substituted alkylthioalkyl in which the phenyl may be further Br substituted with halogen, for example chlorine); a heterocy clic substituted alkyl (e.g., alkyl substituted with Alternatively, Rºº" is 50

55 );

60 aryl substituted alkyl, for example, phenyl substituted alkyl (e.g., unsubstituted phenyl or phenyl substituted with halo gen, such as chlorine, or phenyl substituted with alkoxy, such as isopropoxy). Alternatively, Rºº is aryl, for example, 65 thiophenyl or phenyl, for example, phenyl substituted with In another embodiment, the RNA binding modulatory hydroxyl or alkoxy (e.g., methoxy). In another embodiment, compound of formula VII is a compound of formula VIIb: R” is selected from the group consisting of

US 8,377,639 B2 67 68 In one embodiment, Rºº is amino (e.g., amino substituted -continued with carbonyl, for example, aryl substituted carbonyl, such as phenyl substituted carbonyl, including –NO2 substituted phenyl) or aryl (e.g., phenyl, for example, phenyl substituted OH with carbonyloxy, such as alkyl substituted carbonyloxy, 5 including, but not limited to methyl substituted carbonyloxy). In one embodiment, Rºº is Or OH. 29 10 In one embodiment, the RNA binding modulatory com cº Cº pound of formula VII is a compound of formula VIIf: O2's

In a further embodiment, the RNA binding modulatory 20 compound of formula VII is a compound of formula VIIe:

(VIIe) 25

30 q' is a single or double bond; wherein Wis NH when q is a single bond; or N when q is a double R* is hydrogen, alkyl or aryl; and bond; R* is aryl; and pharmaceutically acceptable salts thereof. 35 X is C=O is when q is a single bond; or CR” when q is a double bond; In one embodiment, Rºº is hydrogen, alkyl (e.g., methyl, R* is alkyl; ethyl oraryl substituted alkyl, for example, phenyl substituted R" is hydrogen, alkoxy or carbonyloxy; and alkyl) or aryl (e.g., phenyl, for example, phenyl substituted R" is hydrogen or carbonyloxy; and pharmaceutically with alkyl, such as methyl or ethyl, for example, acceptable salts thereof. 40 In one embodiment, q is a double bond, X?is CR”; W is N: R* is alkyl (e.g., methyl); R* is phenyl substituted with sulfonyl (e.g., amino substituted sulfonyl), carboxylate or halogen (e.g., chlorine), such as 45

º Or & 50 R* is aryl such as furanyl or phenyl, for example, phenyl substituted with hydroxyl, halogen (e.g., bromine orchlorine) and alkoxy (e.g., methoxy). In one embodiment, Rºº is 55

Br

60

R" is hydrogen or alkoxy (e.g., methoxy or ethoxy) and OH > OH > R" is carbonyloxy, for example, aryl substituted carbony 65 loxy, such as furanyl or thiophenyl substituted carbonyloxy. In another embodiment, q' is a single bond, Wis NH; X is C=O: R* is phenyl, such as unsubstituted phenyl or phenyl US 8,377,639 B2 69 70 substituted with halogen (e.g., chlorine or fluorine) or carbo -continued nyl, for example, carbonyl substituted with alkoxy (e.g., ethoxy), for example,

S O O C| 10 Cls N

15 F HS Cl Br Sr. | 20 N ` * 2 Or C ;

R" is hydrogen, alkoxy (e.g., methoxy) or carbonyloxy, for 25 Br, example, aryl substituted carbonyloxy, such as furanyl or thiophenyl substituted carbonyloxy; and Rºº' is hydrogen or carbonyloxy, for example, aryl substituted carbonyloxy, ºr. such as furanyl or thiophenyl substituted carbonyloxy. H-4 30 HN In another embodiment, the RNA binding modulatory HO Y- O compound of formula VII is a compound is selected from the group consisting of: sº

Br 35 OH,

N 40 S | Br – -N OH | }– S NO2 N

45 S 2ss as 2*

50

Cl 55 --ºr, NO2 60

65 "Sk US 8,377,639 B2 71 72 -continued -continued

Cl

10 S º 2 ` 29

15

20

25

30

35

40

45

50

55

60

65 US 8,377,639 B2 73 74 -continued -continued

N N NH, 10

O

15

20

25

NH2

O ESEO 30

F

Cl 35 O NNN

O N 40 ^ NH, /TN.

2 45

Br, 50 Cl

O NS NH 55 /TN.

O 60 O

65 US 8,377,639 B2 75 76 -continued -continued OH Cl

OH,

10

15

20

2

25

O O 30

35

O N `NH /TN. 40

45

50 O

55

60

65

US 8,377,639 B2 79 80 -continued -continued O OH O

5 HO O,

S NH 10 \ | O

O 15 N

20 O and

S NH

25 \ / Ho O O

O * HSS R O Sr. /

C O 40 and pharmaceutically acceptable salts thereof. as In yet another embodiment, the RNA binding modulatory compound is a compound of Table 1:

S ºr 50 TABLE 1 N_".

HO 55 HN

HO O O

S 60 HO NH O | / NH HN }=s O NH HO 2 US 8,377,639 B2 81 82 TABLE 1-continued TABLE 1-continued

10

15

OH

20

25

30

35

40

45

50

55

65 US 8,377,639 B2 83 84 TABLE 1-continued In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbonatoms in its backbone (e.g., C1-Ca for straight chain, Cs-Co for branched chain). Likewise, cycloalkyls may have from 3-8 carbon atoms in their ring 5 structure. The term “C1-C, includes alkyl groups containing 1 to 6 carbon atoms. Moreover, the term alkyl includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on 10 one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, aryl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, -COOH, alkyl 15 carbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, * O, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbo nyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, O O amino (including alkyl amino, dialkylamino, arylamino, dia rylamino, and alkylarylamino), acylamino (including alkyl 20 carbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxy N 2 N late, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfona H mido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alky laryl, or an aromatic or heteroaromatic moiety. Cycloalkyls 25 can be further substituted, e.g., with the substituents described above. An “alkylaryl” oran “arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl” also includes the side chains of natural and N unnatural amino acids. HS—K 30 The term “aryl” includes groups, e.g., 5- and 6-membered N and single-ring aromatic groups, that may include from zero to N four heteroatoms, for example, benzene, phenyl, pyrrole, O OH furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, 35 pyridazine, and pyrimidine, and the like. Furthermore, the term “aryl” includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylene dioxyphenyl, quinoline, isoquinoline, napthridine, indole, 40 benzofuran, purine, benzofuran, deazapurine, or indolizine. O N O Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles,” “heteroaryls” or “heteroaromatics.” The aromatic ring can be substituted at Cl C| one or more ring positions with such substituents as described 45 above, as for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, aryl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, -COOH, alkyl carbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl, and pharmaceutically acceptable salts thereof. alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aryla 50 lkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbo In one embodiment, the compound is not abamectin, prazi nyl, alkylthiocarbonyl, phosphate, phosphonato, phosphi quantel, albendazole, , mebendazole, nato, cyano, amino (including alkyl amino, dialkylamino, niclosamide, , Suramin, thiabendazole, pyrantel arylamino, diarylamino, and alkylarylamino), acylamino (in pamoate, levamisole, triclabendazole, flubendazole, fen cluding alkylcarbonylamino, arylcarbonylamino, carbamoyl bendazole, octadepsipeptide, oxamniquine, metrifonate, 55 and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, bithionol, niridazole, stibophen, ciclobendazole, oxantel, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, pyrvinium, bephenium, desapidin or dichlorophen. sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocy The term “alkyl” includes saturated aliphatic groups, clyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl including straight-chain alkyl groups (e.g., methyl, ethyl, pro groups can also be fused or bridged with alicyclic or hetero pyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), 60 cyclic rings which are not aromatic so as to form a polycycle branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, (e.g., tetralin). The term heteroaryl includes unsaturated etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclobutyl, cyclic compounds such as azirine, oxirene, dithiete, pyrro cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl sub line, pyrrole, furan, dihydrofuran, dihydrothiophene, stituted cycloalkyl groups, and cycloalkyl substituted alkyl thiophene, pyrazole, imidazole, oxazole, thiazole, isothiaz groups. The term alkyl further includes alkyl groups that may 65 ole, 12,2,3-triazole, 1,2,4, triazole, dithiazole, tetrazole, pyri include oxygen, nitrogen, sulfur or phosphorous atoms dine, pyran, pyrimidine, pyran, thiapyrane, diazine, thiazine, replacing one or more carbons of the hydrocarbon backbone. dioxine, triazine and tetrazene. US 8,377,639 B2 85 86 The term “heterocyclic moiety” includes saturated cyclic described above, but which contain at least one triple bond. moieties having a closed ring of atoms in which at least one For example, the term “alkynyl” includes straight-chain alky atom is not a carbon. As used herein, heterocyclic moieties do nyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexy not include heteroaryl moieties, in which the closed ring of nyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched atoms is both heterocyclic and aromatic and/or unsaturated. 5 chain alkynyl groups, and cycloalkyl or cycloalkenyl Examples of heterocyclic moieties include aziridine, ethyl substituted alkynyl groups. The term “alkynyl” further ene oxide, thiirane, dioxirane, azetidine, oxetane, thietane, includes alkynyl groups which include oxygen, nitrogen, sul dioxetane, dithietane, pyrrolidine, tetrahydrofuran, tetrahy fur or phosphorous atoms replacing one or more carbons of drothiophene, imidazolidine, oxazolidine, thiazolidine, diox the hydrocarbon backbone. In certain embodiments, a olane, dithiolane, piperidine, tetrahydropyran, thiane, pip 10 straight chain or branched chain alkynyl group has 6 or fewer erzine, pxazine, dithiane, dioxane and trioxane. carbon atoms in its backbone (e.g., C2-Ca for straight chain, The term “heterocyclic moiety” includes both “unsubsti Cs-Co for branched chain). The term C2-Co includes alkynyl tuted heterocyclic moieties” and “substituted heterocyclic groups containing 2 to 6 carbon atoms. moieties,” the latter of which includes moieties having sub Moreover, the term “alkynyl” includes both “unsubstituted stituents replacing a hydrogen on one or more of the atoms on 15 alkynyls” and “substituted alkynyls,” the latter of which the closed ring. Such substituents can include, for example, refers to alkynyl moieties having substituents replacing a alkyl, alkenyl, alkynyl, halogens, hydroxyl, aryl alkylcarbo hydrogen on one or more carbons of the hydrocarbon back nyloxy, arylcarbonyloxy, alkoxycarbonyl oxy, aryloxycarbo bone. Such substituents can include, for example, alkyl, alk nyloxy, -COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbo enyl, alkynyl, halogens, hydroxyl, aryl, alkylcarbonyloxy, nyl, aminocarbonyl, alkylaminocarbonyl, 20 arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phos —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, ami phate, phosphonato, phosphinato, cyano, amino (including nocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkyl amino, dialkylamino, arylamino, diarylamino, and alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phos alkylarylamino), acylamino (including alkylcarbonylamino, phinato, cyano, amino (including alkyl amino, dialkylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, 25 arylamino, diarylamino, and alkylarylamino), acylamino (in sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, cluding alkylcarbonylamino, arylcarbonylamino, carbamoyl alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trif and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, luoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aro thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, matic or heteroaromatic moiety. sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocy The term “alkenyl” includes unsaturated aliphatic groups 30 clyl, alkylaryl, or an aromatic or heteroaromatic moiety. analogous in length and possible substitution to the alkyls The term “acyl” includes compounds and moieties which described above, but that contain at least one double bond. For contain the acyl radical (CH3CO-). It also includes substi example, the term “alkenyl” includes straight-chain alkenyl tuted acyl moieties. The term “substituted acyl” includes acyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, groups where one or more of the hydrogenatoms are replaced heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain 35 by for example, alkyl, alkenyl, alkynyl, halogens, hydroxyl, alkenyl groups, cycloalkenyl (alicyclic) groups (cycloprope aryl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbony nyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohepte loxy, aryloxycarbonyloxy, -COOH, alkylcarbonyl, arylcar nyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl bonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, groups, and cycloalkyl or cycloalkenyl substituted alkenyl dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phos groups. The term “alkenyl” further includes alkenyl groups 40 phate, phosphonato, phosphinato, cyano, amino (including which include oxygen, nitrogen, sulfur orphosphorous atoms alkyl amino, dialkylamino, arylamino, diarylamino, and replacing one or more carbons of the hydrocarbon backbone. alkylarylamino), acylamino (including alkylcarbonylamino, In certain embodiments, a straight chain or branched chain arylcarbonylamino, carbamoyl and ureido), amidino, imino, alkenyl group has 6 or fewer carbon atoms in its backbone sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, (e.g., C2-Ca or straight chain, Cs-Co for branched chain). 45 alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trif Likewise, cycloalkenyl groups may have from 3-8 carbon luoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aro atoms in their ring structure. The term C2-Co includes alkenyl matic or heteroaromatic moiety. groups containing 2 to 6 carbon atoms. The term “acylamino” includes moieties wherein: an acyl Moreover, the term “alkenyl” includes both “unsubstituted moiety is bonded to an amino group. For example, the term alkenyls” and “substituted alkenyls,” the latter of which refers 50 includes alkylcarbonylamino, arylcarbonylamino, carbamoyl to alkenyl moieties having substituents replacing a hydrogen and ureido groups. on one or more carbons of the hydrocarbon backbone. Such The terms “alkoxyalkyl,” “alkylaminoalkyl” and “thio substituents can include, for example, alkyl, alkenyl, alkynyl, alkoxyalkyl” include alkyl groups, as described above, which halogens, hydroxyl, aryl alkylcarbonyloxy, arylcarbonyloxy, further include oxygen, nitrogen or sulfur atoms replacing alkoxycarbonyl oxy, aryloxycarbonyloxy, -COOH, alkyl 55 one or more carbons of the hydrocarbon backbone, e.g., oxy carbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, gen, nitrogen or sulfur atoms. alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbo The term “alkoxy” includes substituted and unsubstituted nyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, alkyl, alkenyl, and alkynyl groups covalently linked to an amino (including alkyl amino, dialkylamino, arylamino, dia oxygen atom. Examples of alkoxy groups include methoxy, rylamino, and alkylarylamino), acylamino (including alkyl 60 ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. carbonylamino, arylcarbonylamino, carbamoyl and ureido), Examples of substituted alkoxy groups include haloge amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxy nated alkoxy groups. The alkoxy groups can be substituted late, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfona with groups such as alkyl, alkenyl, alkynyl, halogen, mido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alky hydroxyl, aryl, alkylcarbonyloxy, arylcarbonyloxy, alkoxy laryl, or an aromatic or heteroaromatic moiety. 65 carbonyloxy, aryloxycarbonyloxy, -COOH, alkylcarbonyl, The term “alkynyl” includes unsaturated aliphatic groups arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylami analogous in length and possible substitution to the alkyls nocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, US 8,377,639 B2 87 88 alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino The term “thioether” includes compounds and moieties (including alkyl amino, dialkylamino, arylamino, diary which contain a sulfuratom bonded to two different carbon or lamino, and alkylarylamino), acylamino (including alkylcar hetero atoms. Examples of thioethers include, but are not bonylamino, arylcarbonylamino, carbamoyl and ureido), limited to, alkthioalkyls, alkthioalkenyls, and alkthioalky amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxy nyls. The term “alkthioalkyls” include compounds with an late, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfona alkyl, alkenyl, oralkynyl group bonded to a sulfur atom which mido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alky is bonded to an alkyl group. Similarly, the term “alkthioalk laryl, oran aromatic or heteroaromatic moieties. Examples of enyls” and “alkthioalkynyl” refer to compounds or moieties halogen substituted alkoxy groups include, but are not limited in which an alkyl, alkenyl or alkynyl group is bonded to a 10 sulfur atom that is covalently bonded to an alkenyl or alkynyl to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chlo group, respectively. romethoxy, dichloromethoxy, trichloromethoxy, etc. The term “sulfonyl” includes moieties containing a sulfo The term “amine” or “amino” includes compounds where nyl functional group (e.g., SO2) attached to two carbons via a a nitrogen atom is covalently bonded to at least one carbon or covalent bond to the sulfur atom of the sulfonyl functional heteroatom. The term includes “alkyl amino” which com 15 group. prises groups and compounds wherein: the nitrogen is bound The term “hydroxyl” or “hydroxyl” includes groups with to at least one additional alkyl group. The term “dialkyl an –OH or –OT. amino” includes groups wherein: the nitrogen atom is bound The term “halogen” includes fluorine, bromine, chlorine, to at least two additional alkyl groups. The term “arylamino” iodine, etc. and “diarylamino” include groups in which the nitrogen is 20 The term “heteroatom” includes atoms of any element bound to at least one or two aryl groups, respectively. The other than carbon or hydrogen. Preferred heteroatoms are term “alkylarylamino,” “alkylaminoaryl” or “arylami nitrogen, oxygen, sulfur and phosphorus. noalkyl” refers to an amino group which is bound to at least II. Screening Assays one alkyl group and at least one aryl group. The term “alka The invention provides methods (also referred to herein as minoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound 25 “screening assays”) for identifying modulators, i.e., candi to a nitrogen atom which is also bound to an alkyl group. date or test compounds or agents (e.g., peptidomimetics, The term “amide,” “amido” or “aminocarbonyl” includes small molecules or other drugs) which modulate, for example compounds or moieties which contain a nitrogen atom which one or more biological activities of an RNA-binding protein, is bound to the carbon of a carbonyl or a thiocarbonyl group. e.g., the ability to 1) interact, e.g., bind, e.g., form a complex, The term includes “alkaminocarbonyl” or “alkylaminocarbo 30 with an RNA molecule, e.g., an RNA molecule comprising an nyl” groups which include alkyl, alkenyl, aryl or alkynyl RNA-binding recognition element of the RNA-binding pro groups bound to an amino group bound to a carbonyl group. tein, 2) modulate embryogenesis, 3) modulate cell viability, It includes arylaminocarbonyl and arylcarbonylamino 4) modulate anterior patterning, 5) modulate germ cell toti groups, which include aryl or heteroaryl moieties bound to an potency, 6) modulate development of the intestine, 7) modu amino group that is bound to the carbon of a carbonyl or 35 late development of germline blastomeres, 8) modulate thiocarbonyl group. The terms “alkylaminocarbonyl,” “alk development of pharyngeal tissue, 9) modulate expression enylaminocarbonyl,” “alkynylaminocarbonyl,” “arylami and/or activity of a gene known to be directly or indirectly nocarbonyl,” “alkylcarbonylamino,” “alkenyl carbony regulated by the RNA-binding protein, e.g., PAL-1, NOS-2, lamino,” “alkynylcarbonylamino,” and “arylcarbonylamino” APX-1 protein, and GLD-1, 10) modulate tubercle forma are included in term “amide.” Amides also include urea 40 tion, or for testing or optimizing the activity of such agents. groups (aminocarbonylamino) and carbamates (oxycarbony The assays can be used to identify agents that modulate the lamino). function of an RNA-binding protein or a molecule in a signal The term “carbonyl” or “carboxy” includes compounds transduction pathway involving the RNA-binding protein. and moieties which contain a carbon connected with a double The function of an RNA-binding protein can be affected at bond to an oxygen atom. The carbonyl can be further substi 45 any level, including transcription, protein expression, protein tuted with any moiety which allows the compounds of the localization, and/or cellular activity. The subject assays can invention to perform its intended function. For example, car also be used to identify, e.g., agents that alter the interaction of bonyl moieties may be substituted with alkyls, alkenyls, alky an RNA-binding protein with a binding partner, e.g., an RNA nyls, aryls, alkoxy, aminos, etc. Examples of moieties which molecule comprising an RNA-binding protein recognition contain a carbonyl include aldehydes, ketones, carboxylic 50 element, or modulate, e.g., inhibit, the stability of such inter acids, amides, esters, anhydrides, etc. The term “carboxy” action. further includes the structure of —COOH and –COOT. The subject screening assays can measure the activity of an The term “oximyl” includes compounds and moieties that RNA-binding protein directly (e.g., formation of a complex contain a carbon connected with a double bond to a nitrogen with an RNA molecule comprising an RNA-binding protein atom, which is, in turn connected to a hydroxyl or an alkoxyl 55 recognition element), or can measure a downstream event group. The term “hydrazinyl" includes compounds and moi controlled by modulation of the RNA-binding protein (e.g., eties that contain a carbon connected with a double bond to a embryogenesis, cell differentiation, expression and/or activ nitrogenatom, which is, in turn, connected to an amino group. ity of a gene known to be directly or indirectly regulated by The term “thiocarbonyl” or “thiocarboxy” includes com the RNA-binding protein, e.g., PAL-1, NOS-2, APX-1 pro pounds and moieties which contain a carbon connected with 60 tein, and GLD-1). a double bond to a sulfur atom. The subject screening assays employ indicator composi The term “ether” includes compounds or moieties which tions. These indicator compositions comprise the compo contain an oxygen bonded to two different carbon atoms or ments required for performing an assay that detects and/or heteroatoms. For example, the term includes “alkoxyalkyl” measures a particular event. The indicator compositions of which refers to an alkyl, alkenyl, or alkynyl group covalently 65 the invention provide a reference readout and changes in the bonded to an oxygen atom which is covalently bonded to readout can be monitored in the presence of one or more test another alkyl group. compounds. A difference in the readout in the presence and US 8,377,639 B2 89 90 the absence of the compound indicates that the test compound evaluate the efficacy of treatment of nematode parasitism is a modulator of the molecule(s) present in the indicator with an RNA binding modulatory compound identified composition. herein is described, for example, in Lok J. B. (“Strongyloides The indicator composition used in the screening assay can stercoralis: a model for translational research on parasitic be a cell that expresses an RNA-binding protein and/or an nematode biology” 2007 Wormbook, ed. The C. elegans RNA molecule comprising an RNA-binding protein recogni Research Community, WormEook, doi/10.1895/worm tion element. For example, a cell that naturally expresses or, book.1.134.1, at www.wormbook.org), the entire contents of more preferably, a cell that has been engineered to express the which is incorporated herein by reference. S. stercoralis is a protein and/or the RNA molecule comprising an RNA-bind significant pathogen of humans and can be maintained in ing protein recognition element by introducing into the cellan 10 laboratory dogs and gerbils. Examples of animal models that expression vector encoding the protein may be used. The cell may be used to determine the efficacy of treatment of nema may be a helminth cell, a plant cell, a yeast cell, a bacterial tode parasitism with an RNA binding modulatory compound cell, or a mammalian cell, e.g., a human cell. Alternatively, identified herein are described, for example, in Camberis, M. the indicator composition can be a cell-free composition that et al., (Animal Model of Nippostrongylus brasiliensis and includes the protein and/or the RNA molecule comprising an 15 Heligmosomoides polygyrus, 2003 Current Protocols in RNA-binding protein recognition element (e.g., a cell extract Immunology 19.12.1-19.12.27), the entire contents of which or a composition that includes e.g., either purified natural or is incorporated herein by reference. recombinant protein and/or RNA). In one embodiment, the screening assays of the invention The indicator compositions used in the screening assays of are high throughput or ultra high throughput (e.g., Fernandes, the invention can be a cell that expresses an RNA-binding 20 P. B., Curropin Chem. Biol. 1998.2:597; Sundberg, SA, Curr protein or biologically active fragment thereof, e.g., a frag Opin Biotechnol. 2000, 11:47). ment of the protein that interacts, e.g., binds, to an RNA Exemplary cell based and cell free assays of the invention binding protein recognition element, e.g., a fragment com are described in more detail below. prising a CCCH-type tandem zinc finger or a KH domain. Cell Based Assays In another embodiment, the indicator composition com 25 The indicator compositions of the invention may be cells prises more than one polypeptide. For example, in one that express an RNA-binding protein and/or an RNA mol embodiment the subject assays are performed in the presence ecule comprising an RNA-binding protein recognition ele of more than one RNA-binding protein, e.g., MEX-5, GLP-1, ment. For example, a cell that naturally expresses endogenous NOS-2, MEX-6, POS-1, MEX-3. PAL-1. It will be under polypeptide and/or RNA molecule, or, more preferably, a cell stood that in addition to the recited proteins, e.g., helminth 30 that has been engineered to express one or more exogenous proteins, e.g., nematode proteins, suitable proteins for use in polypeptides and/or RNA molecules, e.g., by introducing into the methods of the invention include plant and mammalian the cell an expression vector encoding the protein may be homologues of such proteins, e.g., TTP, the mammalian used in a cell based assay. homologue of MEX-5. One of ordinary skill in the art can The cells used in the instant assays can be eukaryotic or identify such proteins based on sequence and/or database 35 prokaryotic in origin. For example, in one embodiment, the and/or homology searching and analyses. cell is a bacterial cell. In one embodiment, the cell is a helm Compounds that modulate the expression and/or activity of inth, e.g., nematode, cell. In another embodiment, the cell is a an RNA-binding protein, identified using the assays plant cell. In another embodiment, the cell is a fungal cell, described herein can be useful for treating a subject that e.g., a yeast cell. In another embodiment, the cell is a verte would benefit from the modulation of expression and/or 40 brate cell, e.g., an avian or a mammalian cell (e.g., a murine activity of the RNA-binding protein, e.g., a subject with a cell, rat cell, rhesus monkey, or a human cell). In another parasitic associate state. embodiment, the cell is a human cell. In one embodiment, secondary assays can be used to con In another embodiment, cells for use in the methods of the firm that the modulating agent affects the RNA-binding pro invention are derived from a cell line, preferably one which tein molecule in a specific manner. For example, compounds 45 expresses low levels of endogenous RNA-binding protein identified in a primary screening assay can be used in a and/or an RNA molecule comprising an RNA-binding pro secondary, tertiary, etc. screening assay to determine whether tein recognition element and is then engineered to express the compound affects an RNA-binding protein-related activ recombinant protein and/or RNA. ity as described herein. Accordingly, in another aspect, the Recombinant expression vectors that may be used for invention pertains to a combination of two or more of the 50 expression of polypeptides are known in the art. For example, assays described herein. For example, a modulating agent can the cDNA is first introduced into a recombinant expression be identified using a cell-based or a cell-free assay, e.g., to vector using standard molecular biology techniques. A cljRNA detect an interaction, e.g., formation of a complex, and the can be obtained, for example, by amplification using the ability of the agent to modulate the activity of the RNA polymerase chain reaction (PCR) or by screening an appro binding protein or a molecule involved in a signal transduc 55 priate cINA library. tion pathway involving the RNA-binding protein can be con When used in mammalian cells, the expression vector’s firmed using a biological read-out to measure, e.g., control functions are often provided by viral regulatory ele embryogenesis, an immune response, e.g., cytokine produc ments. For example, commonly used promoters are derived tion, in vitro or in vivo. from polyoma virus, adenovirus, cytomegalovirus and Sim Moreover, a modulator of an RNA-binding protein expres 60 ian Virus 40. Non-limiting examples of mammalian expres sion and/or activity identified as described herein (e.g., a sion vectors include pGDM8 (Seed, B., (1987) Nature 329: small molecule) may be used in an animal model and/or plant 840) and pmT2PC (Kaufman, et al. (1987), EMBO J. 6:187– model to determine the efficacy, toxicity, or side effects of 195). A variety of mammalian expression vectors carrying treatment with such a modulator. Alternatively, a modulator different regulatory sequences are commercially available. identified as described herein may be used in an animal model 65 Vector DNA may be introduced into cells via conventional to determine the mechanism of action of such a modulator. An transfection techniques. As used herein, the various forms of example of a nematode parasite model that may be used to the term “transfection” are intended to refer to a variety of US 8,377,639 B2 91 92 art-recognized techniques for introducing foreign nucleic In another embodiment, protein expression may be mea acid (e.g., DNA) into host cells, including calcium phosphate sured. For example, standard techniques such as Western co-precipitation, DEAE-dextran-mediated transfection, lipo blotting or in situ detection can be used. fection, or electroporation. Suitable methods for transfecting In one embodiment a downstream effect of modulation of host cells can be found in Sambrook, et al. (Molecular Clon an RNA-binding protein, e.g., the effect of a compound on ing: A Laboratory Manual, 2nd Edition, Cold Spring Harbor cell viability and/or embryogenesis, and/or tubercle forma Laboratory press (1989)), and other laboratory manuals. tion in a plant, may be used as an indicator of modulation of For stable transfection of cells, it is known that, depending the activity of an RNA-binding proteinby, for example, moni upon the expression vector and transfection technique used, toring directly (e.g. by microscopic examination of the cells), only a small fraction of cells may integrate the foreign DNA 10 or indirectly, e.g., by monitoring one or more markers of, for into their genome. In order to identify and select these inte example, embryogenesis. grants, a gene that encodes a selectable marker (e.g., resis Standard methods for detecting mRNA of interest, such as tance to antibiotics) is generally introduced into the host cells reverse transcription-polymerase chain reaction (RT-PCR) along with the gene of interest. Preferred selectable markers 15 and Northern blotting, are known in the art. Standard methods include those which conferresistance to drugs, such as G418, for detecting protein secretionin culture supernatants, such as hygromycin and methotrexate. Nucleic acid encoding a enzyme linked immunosorbent assays (ELISA), are also selectable marker can be introduced into a host cell on a known in the art. Proteins can also be detected using antibod separate vector from that encoding an RNA-binding protein ies, e.g., in an immunoprecipitation reaction or for and/or an RNA molecule comprising an RNA-binding pro 20 and FACS analysis. tein recognition element, on the same vector. Cells stably The ability of the test compound to modulate an RNA transfected with the introduced nucleic acid can be identified binding molecule interaction with a target molecule can also by drug selection (e.g., cells that have incorporated the select be determined. For example, in one embodiment, the interac able marker gene will survive, while the other cells die). tion of an RNA-binding molecule and an RNA molecule In one embodiment, within the expression vector coding 25 comprising an RNA-binding protein recognition element can sequences are operatively linked to regulatory sequences that be measured as described in, for example, Pagano, et al. allow for constitutive expression of the molecule in the indi (2007).J. Biol. Chem. 282:8883 and Farley, etal. (2008) RNA cator cell (e.g., viral regulatory sequences, such as a cytome 14; 2685. In certain embodiments of the invention, the RNA galovirus promoter/enhancer, may be used). Use of a recom binding protein recognition element comprises the consensus 30 sequence UA(U2-s)RD(N1 s)G. Instill other embodiments of binant expression vector that allows for constitutive the invention, the RNA-binding protein recognition element expression of the genes in the indicator cell is preferred for comprises the consensus sequence DKAG(No 3)|UHUA. In identification of compounds that enhance or inhibit the activ one embodiment, the RNA-binding protein recognition ele ity of the molecule. In an alternative embodiment, within the ment, i.e., DKAG(No 3)UHUA, binds with MEX-3. In expression vector the coding sequences are operatively linked 35 another embodiment, the RNA-binding protein recognition to regulatory sequences of the endogenous gene (i.e., the element, i.e., UA(U2_s)RD(N1 s)G, binds with POS-1. promoter regulatory region derived from the endogenous Determining the ability of the test compound to modulate, gene). Use of a recombinant expression vector in which for example, an RNA-binding molecule, binding to a target expression is controlled by the endogenous regulatory molecule (e.g., a target RNA molecule, e.g., an RNA-binding sequences is preferred for identification of compounds that 40 protein recognition element) can also be accomplished, for enhance or inhibit the transcriptional expression of the mol example, by determining the ability of the molecules to be ecule. coimmunoprecipitated or by coupling the target molecule For example, an indicator cell can be transfected with an with a radioisotope or enzymatic label such that binding of the expression vector comprising an RNA-binding protein, or target molecule to an RNA-binding molecule or an RNA biologically active fragment thereof, incubated in the pres 45 binding molecule-interacting polypeptide can be determined, ence and in the absence of a test compound, and the effect of e.g., by detecting the labeled target molecule in a complex. the compound on the expression and/or activity of the mol Alternatively, for example, an RNA-binding molecule can be ecule or on a biological response regulated by the RNA coupled with a radioisotope or enzymatic label to monitor the binding protein, e.g., an RNA-binding protein-related activ ability of a test compound to modulate an RNA-binding mol ity, can be determined. The biological activities of an RNA 50 ecule binding to a target molecule in a complex. binding protein include activities determined in vivo, or in Determining the ability of the test compound to interact vitro, according to standard techniques. Activity can be a with an RNA-binding molecule can be accomplished, for direct activity, such as an association with a target molecule example, by coupling the compound with a radioisotope or (e.g., an RNA molecule comprising an RNA-binding protein enzymatic label such that interaction of the compound can be recognition element). Alternatively, activity may be an indi 55 determined by detecting the labeled compound in a complex. rect activity, such as, for example, a cellular signaling activity For example, targets can be labeled with 125I, 35S, 14C, or occurring downstream of the interaction of the protein with a 3H, either directly or indirectly, and the radioisotope detected target molecule or a biological effect occurring as a result of by direct counting of radioemmission or by scintillation the signaling cascade triggered by that interaction, such as counting. Alternatively, compounds can be labeled, e.g., with, embryogenesis and/or cell differentiation. 60 for example, horseradish peroxidase, alkaline phosphatase, Compounds that modulate RNA-binding protein produc or luciferase, and the enzymatic label detected by determina tion, expression and/or activity of may be identified using tion of conversion of an appropriate substrate to product. various “read-outs.” In another embodiment, fluorescence technologies can be For example, in one embodiment, gene expression of an used, e.g., fluorescence polarization, time-resolved fluores RNA-binding protein can be measured. In another embodi 65 cence, and fluorescence resonance energy transfer (Selvin, P ment, expression of a gene controlled by an RNA-binding R, Nat. Struct. Biol. 20007:730; Hertzberg R P and Pope AJ, protein can be measured. Curr Opin Chem Biol. 2000 4:445). US 8,377,639 B2 93 94 It is also within the scope of this invention to determine the binding protein are identified based on their ability to modu ability of a compound to interact with an RNA-binding pro late the interaction of an RNA-binding protein with a target tein without the labeling of any of the interactants. For molecule to which the RNA-binding protein binds, e.g., RNA example, a microphysiometer may be used to detect the inter molecule comprising an RNA-binding protein recognition action of a compound with a an RNA-binding protein without element. Suitable assays are known in the art that allow for the the labeling of either the compound or the molecule (McCo detection of protein-protein interactions (e.g., immunopre nnell, H. M., et al. (1992) Science 257:1906-1912). As used cipitations and the like) or that allow for the detection of herein, a “microphysiometer” (e.g., Cytosensor) is an analyti interactions between a DNA or RNA binding protein and a cal instrument that measures the rate at which a cell acidifies target DNA or RNA sequence (e.g., electrophoretic mobility its environment using a light-addressable potentiometric sen 10 shift assays, DNAse I footprinting assays and the like). By sor (LAPS). Changes in this acidification rate may be used as performing such assays in the presence and absence of test an indicator of the interaction between compounds. compounds, these assays may be used to identify compounds In yet another aspect of the invention, an RNA-binding that modulate (e.g., inhibit or enhance) the interaction of an protein or fragments thereof may be used as “bait protein” RNA-binding protein with a target molecule. e.g., in a two-hybrid assay or three-hybrid assay (see, e.g., 15 In the methods of the invention for identifying test com U.S. Pat. No. 5,283,317; Zervos, et al. (1993) Cell 72:223 pounds that modulate an interaction between an RNA-bind 232; Madura, et al. (11993).J. Biol. Chem. 268:12046-12054; ing protein and a target molecule, the complete RNA-binding Bartel, et al. (1993) Biotechniques 14:920–924; Iwabuchi, et protein may be used in the method, or, alternatively, only al. (1993) Oncogene 8: 1693–1696; and Brent WO94/10300), portions of the protein may be used. For example, an isolated to identify other proteins, which bind to or interact with an 20 CCH-type tandem zinger finder domain or a KH domain may RNA-binding protein (“binding proteins” or “bp”) and are be used. An assay may be used to identify test compounds that involved in an RNA-binding protein activity. Such proteins either stimulate or inhibit the interaction between the an are also likely to be involved in the propagation of signals by RNA-binding protein and a target molecule. A test compound the RNA-binding protein. The two-hybrid system is based on that stimulates the interaction between the protein and a target the modular nature of most transcription factors, which con 25 molecule is identified based upon its ability to increase the sist of separable DNA-binding and activation domains. degree of interaction as compared to the degree of interaction Briefly, the assay utilizes two different DNA constructs. In in the absence of the test compound and such a compound one construct, the gene that codes foran RNA-binding protein would be expected to, e.g., increase, the activity of an RNA is fused to a gene encoding the DNA binding domain of a binding protein in the cell. A test compound that inhibits the known transcription factor (e.g., GAL-4). In the other con 30 interaction between the protein and a target molecule is iden struct, a DNA sequence, from a library of DNA sequences, tified based upon its ability to decrease the degree of interac that encodes an unidentified protein (“prey” or “sample”) is tion between the protein and a target molecule as compared to fused to a gene that codes for the activation domain of the the degree of interaction in the absence of the compound and known transcription factor. If the “bait” and the “prey” pro such a compound would be expected to, e.g., decrease, RNA teins are able to interact, in vivo, forming an RNA-binding 35 binding protein activity. protein-dependent complex, the DNA-binding and activation In one embodiment, the amount of binding of an RNA domains of the transcription factor are brought into close binding protein to an RNA molecule comprising an RNA proximity. This proximity allows transcription of a reporter binding protein recognition element in the presence of the test gene (e.g., Lac.7) which is operably linked to a transcriptional compound is greater than the amount of binding in the regulatory site responsive to the transcription factor. Expres 40 absence of the test compound, in which case the test com sion of the reporter gene can be detected and cell colonies poundis identified as a compound that enhances binding of an containing the functional transcription factor can be isolated RNA-binding protein to an RNA molecule comprising an and used to obtain the cloned gene which encodes the protein RNA-binding protein recognition element. In another which interacts with the RNA-binding protein. embodiment, the amount of binding of the RNA-binding Cell-Free Assays 45 protein to an RNA molecule comprising an RNA-binding Alternatively, the indicator composition can be a cell-free protein recognition element in the presence of the test com composition that includes an RNA-binding protein, e.g., a pound is less than the amount of binding of an RNA-binding cell extract from a cell expressing the protein or a composi protein to an RNA molecule comprising an RNA-binding tion that includes purified either natural or recombinant pro protein recognition element in the absence of the test com tein. 50 pound, in which case the test compound is identified as a In one embodiment, the indicator composition is a cell free compound that inhibits binding of an RNA-binding protein to composition. Polypeptides expressed by recombinant meth an RNA molecule comprising an RNA-binding protein rec ods in a host cells or culture medium can be isolated from the ognition element. host cells, or cell culture medium using standard methods for For example, interaction, e.g., formation of a complex, of protein purification. For example, ion-exchange chromatog 55 the test compound to an RNA-binding protein can be deter raphy, gel filtration chromatography, ultrafiltration, electro mined either directly or indirectly as described above. Deter phoresis, and immunoaffinity purification with mining the ability of an RNA-binding protein to interact with may be used to produce a purified or semi-purified protein a test compound can also be accomplished using a technology that may be used in a cell free composition. Alternatively, a such as real-time Biomolecular Interaction Analysis (BIA) lysate or an extract of cells expressing the protein of interest 60 (Sjolander, S, and Urbaniczky, C. (1991) Anal. Chem. can be prepared for use as cell-free composition. Cell extracts 63:2338-2345; Szabo, et al. (1995) Curr. Opin. Struct. Biol. with the appropriate post-translation modifications of pro 5:699-705). As used herein, “BIA” is a technology for study teins can be prepared using commercially available resources ing biospecific interactions in real time, without labeling any found at, for example Promega, Inc. of the interactants (e.g., BLAcore). Changes in the optical In one embodiment, compounds that specifically modulate 65 phenomenon of surface plasmon resonance (SPR) may be an activity of an RNA-binding protein may be identified. For used as an indication of real-time reactions between biologi example, compounds that modulate an activity of an RNA cal molecules. US 8,377,639 B2 95 96 In one embodiment of the above assay methods, it may be 37:2678) oligocarbamates (Cho, et al. (1993). Science. 261: desirable to immobilize either an RNA-binding protein or an 11303), and hydantoins (DeWitt, et al. supra). An approach RNA molecule comprising an RNA-binding protein recogni for the synthesis of molecular libraries of small organic mol tion element for example, to facilitate separation of com ecules with a diversity of 104-105 as been described (Carell, plexed from uncomplexed forms of one or both of the mol et al. (1994). Angew. Chem. Int. Ed. Engl. 33:2059; Carell, et ecules, or to accommodate automation of the assay. Binding al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061). to a surface can be accomplished in any vessel suitable for The compounds of the present invention can be obtained containing the reactants. Examples of Such vessels include using any of the numerous approaches in combinatorial microtitre plates, test tubes, and micro-centrifuge tubes. In library methods known in the art, including: biological librar one embodiment, a fusion protein can be provided in which a 10 domain that allows one or both of the proteins to be bound to ies; spatially addressable parallel solid phase or solution a matrix is added to one or more of the molecules. For phase libraries, synthetic library methods requiring deconvo example, glutathione-S-transferase fusion proteins or glu lution, the ‘one-bead one-compound’ library method, and tathione-S-transferase/targetfusion proteins can be adsorbed synthetic library methods using affinity chromatography onto glutathione sepharose beads (Sigma Chemical, St. 15 selection. The biological library approach is limited to pep Louis, Mo.) or glutathione derivatized microtitre plates, tide libraries, while the other four approaches are applicable which are then combined with the test compound or the test to peptide, non-peptide oligomer or small molecule libraries compound and either the non-adsorbed target protein or of compounds (Lam, K. S. (1997) Anticancer Drug Des. RNA-binding protein, and the mixture incubated under con 12:145). Other exemplary methods for the synthesis of ditions conducive to complex formation (e.g., at physiologi 20 molecular libraries can be found in the art, for example in: cal conditions for salt and pH). Following incubation, the Erb, et al. (1994). Proc. Natl. Acad. Sci., USA 91:11422-; beads or microtitre plate wells are washed to remove any Horwell, et al. (1996) Immunopharmacology 33:68-; and in unbound components, the matrix is immobilized in the case Gallop, et al. (1994); J. Med. Chem. 37:1233. of beads, and complex formation is determined either directly Exemplary compounds which can be screened for activity or indirectly, for example, as described above. Alternatively, 25 include, but are not limited to, peptides, nucleic acids, carbo the complexes can be dissociated from the matrix, and the hydrates, small organic molecules, and natural product level of binding or activity determined using standard tech extract libraries. niques. Candidate/test compounds include, for example, 1) pep Other techniques for immobilizing proteins on matrices tides such as soluble peptides, including Ig-tailed fusion pep can also be used in the screening assays of the invention. For 30 tides and members of random peptide libraries (see, e.g., example, proteins may be immobilized utilizing conjugation Lam, K. S., et al. (1991) Nature 354:82–84; Houghten, R., et of biotin and streptavidin. Biotinylated protein or target mol al. (1991) Nature 354:84-86) and combinatorial chemistry ecules can be prepared from biotin-NHS(N-hydroxy-succin derived molecular libraries made of D- and/or L-configura imide) using techniques known in the art (e.g., biotinylation tion amino acids; 2) phosphopeptides (e.g., members of ran kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the 35 dom and partially degenerate, directed phosphopeptide wells of streptavidin-coated 96 well plates (Pierce Chemical). libraries, see, e.g., Songyang, Z., et al. (1993) Cell 72:767 Alternatively, antibodies which are reactive with protein or 778); 3) antibodies (e.g., antibodies (e.g., intracellular, poly target molecules but which do not interfere with binding of clonal, monoclonal, humanized, anti-idiotypic, chimeric, and the protein to its target molecule can be derivatized to the single chain antibodies as well as Fab, F(ab')2, Fab expression wells of the plate, and unbound target or SLIM protein is 40 library fragments, and -binding fragments of antibod trapped in the wells by conjugation. Methods for ies); 4) small organic and inorganic molecules (e.g., mol detecting such complexes, in addition to those described ecules obtained from combinatorial and natural product above for the GST-immobilized complexes, include immu libraries); 5) enzymes (e.g., endoribonucleases, hydrolases, nodetection of complexes using antibodies reactive with an nucleases, proteases, synthatases, isomerases, polymerases, RNA-binding protein. 45 kinases, phosphatases, oxido-reductases and ATPases), and Test Compounds 6) mutant forms of molecules. A variety of test compounds can be evaluated using the The test compounds of the present invention can be screening assays described herein. The term “test compound” obtained using any of the numerous approaches in combina includes any reagent or test agent which is employed in the torial library methods known in the art, including: biological assays of the invention and assayed. More than one com 50 libraries; spatially addressable parallel solid phase or Solu pound, e.g., a plurality of compounds, can be tested at the tion phase libraries; synthetic library methods requiring same time in a screening assay. The term “screening assay” deconvolution; the ‘one-bead one-compound’ library preferably refers to assays which test the ability of a plurality method; and synthetic library methods using affinity chroma of compounds to influence the readout of choice rather than to tography selection. The biological library approach is limited tests which test the ability of one compound to influence a 55 to peptide libraries, while the other four approaches are appli readout. Preferably, the subject assays identify compounds cable to peptide, non-peptide oligomer or small molecule not previously known to have the effect that is being screened libraries of compounds (Lam, K. S. (1997) Anticancer Drug for. In one embodiment, high throughput screening may be Des. 12:145). used to assay for the activity of a compound. Examples of methods for the synthesis of molecular librar In certain embodiments, the compounds to be tested can be 60 ies can be found in the art, for example in: DeWitt, et al. derived from libraries (i.e., are members of a library of com (1993) Proc. Natl. Acad. Sci., U.S.A. 90:6909; Erb, et al. pounds). While the use of libraries of peptides is well estab (1994) Proc. Natl. Acad. Sci., USA 91:11422, Zuckermann, lished in the art, new techniques have been developed which et al. (1994) J. Med. Chem. 37:2678; Cho, et al. (1993) have allowed the production of mixtures of other compounds, Science 261:1303; Carrell, et al. (1994) Angew. Chem. Int. Such as benzodiazepines (Bunin, et al. (1992). J. Am. Chem. 65 Ed. Engl. 33:2059; Carell, et al. (1994)Angew. Chem. Int. Ed. Soc. 114:10987; DeWitt et al. (1993). Proc. Natl. Acad. Sci., Engl. 33:2061; and Gallop, et al. (1994) J. Med. Chem. USA 90:6909) peptoids (Zuckerman. (1994). J. Med. Chem. 37: 1233. US 8,377,639 B2 97 98 Libraries of compounds can be presented in solution (e.g., expel parasitic worms from the body of a subject. In one Houghten (1992) Biotechniques 13:412–421), or on beads embodiment, the anti-helmintic agent is a vermifuge agent (Lam (1991) Nature 354:82–84), chips (Fodor (1993) Nature (e.g., a compound that stuns the parasitic worm prior to or 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), substantially at the same time as the expelling). In another spores (Ladner U.S. Pat. No. 409), plasmids (Cull, et al. embodiment, the anti-helmintic agent is a vermicide (e.g., a (1992) Proc. Natl. Acad. Sci., USA 89:1865-1869) or phage compound that kills the parasitic worm prior to or substan (Scott and Smith (1990) Science 249:386-390; Devlin (1990) tially at the same time as the expelling). Examples of anti Science 249:404–406; Cwirla, et al. (1990) Proc. Natl. Acad. helmintic agents include, but are not limited to, abamectin Sci., USA87:6378-6382; Felici(1991).J. Mol. Biol. 222:301 (e.g., Affirm(R, Agri-Mekº, Avermectinº, Avid R, Ver 310; Ladner supra.). 10 Compounds identified in the subject screening assays may timec R or Zephyr'R), praziquantel (e.g., Biltricide R), be used, e.g., in methods of modulating embryogenesis, a albendazole (e.g., Albenza R, Eskazole(R) or Zentel®), dieth parasitic associated state. It will be understood that it may be ylcarbamazine (e.g., Hetrazan(R), Carbilazine.R., Caricide R, desirable to formulate such compound(s) as pharmaceutical Cypiprº, Ethodryl(R), Notezine R, Spatonin R, Filaribits R or compositions (described supra) prior to contacting them with 15 Banocide Forte R), mebendazole (e.g., Ovex R, VermoxR or cells. AntioxR), niclosamide (e.g., Niclocideº), ivermectin (e.g., Once a test compound is identified that directly or indi Stromectol R, Mectizan R or Ivexterm(R), suramin (e.g., Ger rectly modulates, e.g., inhibits, an RNA-binding protein manin R), thiabendazole (e.g., Mintezolº, Tresaderm(R or activity by one of the variety of methods described herein, the Arbotect(R), pyrantel pamoate, levamisole (e.g., Ergami selected test compound (or “compound of interest”) can then 20 solº), triclabendazole, (e.g., Fasonex R) flubendazole (e.g., be further evaluated for its effect on cells, for example by Flutelmium(R, Flubenol R, Biovermin(R) or Flumoxal R), fen contacting the compound of interest with cells either in vivo bendazole (e.g., PanacurºR), Safe-Guard(R) or Panacur Rab (e.g., by administering the compound of interest to a subject) bit R), octadepsipeptide (e.g., emodepside), piperazine or ex vivo (e.g., by isolating cells from the subject and con derivatives, amino acetonitrile derivatives (e.g., monepantel, tacting the isolated cells with the compound of interest or, 25 Zolvix(R), oxamniquine (e.g., Vansil R or Mansil R), metri alternatively, by contacting the compound of interest with a fonate (e.g., trichlorfon), bithionol, niridazole (e.g., Ambil cell line) and determining the effect of the compound of gar?), stibophen, ciclobendazole, oxantel, pyrvinium, interest on the cells, as compared to an appropriate control bephenium, desapidin and dichlorophen. One of skill in the (such as untreated cells or cells treated with a control com art using conventional medical diagnoses would be able to pound, or carrier, that does not modulate the biological 30 response). determine the appropriate anti-helmintic agent to administer The instant invention also pertains to compounds identified in combination with the RNA binding modulatory com in the subject screening assays. pound, e.g., the compounds of a compound of formula I, Ia, III. Pharmaceutical Compositions Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, VIIc, The invention also pertains to pharmaceutical composi 35 VIId, VIIe, VIIfor a compound of Table 1 or 2. tions comprising a therapeutically effective amount of an The RNA binding modulatory compounds, e.g., com RNA binding modulatory compound, e.g., a compound of pounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or Table 1 or 2, that are basic in nature are capable of forming a 2, and a pharmaceutically acceptable carrier. Each of these 40 wide variety of pharmaceutically acceptable salts with vari compounds may be used alone or in combination as a part of ous inorganic and organic acids. The acids that may be used to a pharmaceutical composition of the invention. prepare pharmaceutically acceptable acid addition salts of the In one embodiment, the RNA binding modulatory com compounds disclosed herein that are basic in nature are those pound, e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, that form non-toxic acid addition salts, i.e., salts containing IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or 45 pharmaceutically acceptable anions, such as the hydrochlo a compound of Table 1 or 2, is administered in combination ride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, with an additional agent, e.g., an anti-helminthic agent. The phosphate, acid phosphate, isonicotinate, acetate, lactate, language “in combination with’” an additional agent, e.g., an salicylate, citrate, acid citrate, tartrate, pantothenate, bitar antihelminthic agent, includes co-administration of the com trate, ascorbate, succinate, maleate, gentisinate, fumarate, pound with an additional agent, e.g., an antihelminthic agent, 50 gluconate, glucaronate, saccharate, formate, benzoate, administration of the compound first, followed by adminis glutamate, methanesulfonate, ethanesulfonate, benzene tration of an additional agent, e.g., an antihelminthic agent, sulfonate, p-toluenesulfonate and palmoate (i.e., 1,1'-meth and administration of an additional agent, e.g., antihelminthic ylene-bis-(2-hydroxy-3-naphthoate)] salts. Although such agent first, followed by administration of the compound. The salts must be pharmaceutically acceptable for administration compound can be administered substantially at the same time 55 to a subject, e.g., a mammal, it is often desirable in practice to as the additional agent, e.g., antihelminthic agent, or at sub initially isolate the compound from the reaction mixture as a stantially different times as the additional agent, e.g., antihel pharmaceutically unacceptable salt and then simply convert minthic agent. Optimal administration rates for a given pro the latterback to the free base compound by treatment with an tocol of administration of the compound and/or the additional alkaline reagent and subsequently convert the latter free base agent, e.g., antihelminthic agent, can be readily ascertained 60 to a pharmaceutically acceptable acid addition salt. The acid by those skilled in the art using conventional dosage determi addition salts of the base compounds are readily prepared by nation tests conducted with regard to the specific compounds treating the base compound with a substantially equivalent being utilized, the particular compositions formulated, the amount of the chosen mineral or organic acid in an aqueous mode of application, the particular site of administration and solvent medium or in a suitable organic solvent, such as the like. 65 methanol or ethanol. Upon careful evaporation of the solvent, The phrases “anti-helmintic agent” and “anti-helminthic the desired solid salt is readily obtained. The preparation of agent,” used interchangeably herein, include compounds that other compounds not specifically described in the experimen US 8,377,639 B2 99 100 tal section can be accomplished using combinations of the stracheal, subcutaneous, subcuticular, intraarticular, subcap described reactions that will be apparent to those skilled in the sular, Subarachnoid, intraspinal and intrasternal injection and art. infusion. The RNA binding modulatory compounds, e.g., com The phrases “systemic administration,” “administered sys pounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, temically,” “peripheral administration” and “administered VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of peripherally,” as used herein, includes the administration of Table 1 or 2, that are acidic in nature are capable of forming a the RNA binding modulatory compound, e.g., compound of wide variety of pharmaceutically acceptable base salts. The formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, chemical bases that may be used as reagents to prepare phar VIIa, VIIb, VIIe, VIId, VIIe, VIIfor a compound of Table 1 or maceutically acceptable base salts of those compounds that 10 2, other than directly into the , such are acidic in nature are those that form non-toxic base salts that it enters the subject’s system and, thus, is subject to with such compounds. Such non-toxic base salts include, but metabolism and other like processes, for example, subcuta are not limited to those derived from such pharmaceutically neous administration. acceptable cations such as alkali metal cations (e.g., potas In some methods, the compositions of the invention can be sium and sodium) and alkaline earth metal cations (e.g., cal 15 topically administered to any epithelial surface. An “epithe cium and magnesium), ammonium or water-soluble amine lial surface” includes an area of tissue that covers external addition salts such as N-methylglucamine-(meglumine), and surfaces of a body, or which lines hollow structures including, the lower alkanolamnionium and other base salts of pharma but not limited to, cutaneous and mucosal surfaces. Such ceutically acceptable organic amines. The pharmaceutically epithelial surfaces include oral, pharyngeal, esophageal, pul acceptable base addition salts of the compounds that are 20 monary, ocular, aural, nasal, buccal, lingual, vaginal, cervical, acidicinnature may be formed with pharmaceutically accept genitourinary, alimentary, and anorectal surfaces. able cations by conventional methods. Thus, these salts may Compositions can be formulated in a variety of conven be readily prepared by treating the compounds disclosed tional forms employed for topical administration. These herein with an aqueous solution of the desired pharmaceuti include, for example, semi-solid and liquid dosage forms, cally acceptable cation and evaporating the resulting solution 25 such as liquid solutions or suspensions, suppositories, to dryness, preferably under reduced pressure. Alternatively, douches, enemas, gels, creams, emulsions, lotions, slurries, a lower alkyl alcohol solution of the compounds of the inven powders, sprays, lipsticks, foams, pastes, toothpastes, oint tion may be mixed with an alkoxide of the desired metal and ments, salves, balms, douches, drops, troches, chewing gums, the solution subsequently evaporated to dryness. lozenges, mouthwashes, rinses. The term “pharmaceutically acceptable carrier” includes 30 Conventionally used carriers for topical applications any carrier that is suitable for administration to a mammal. include pectin, gelatin and derivatives thereof, polylactic acid These compositions may also contain adjuvants such as or polyglycolic acid polymers or copolymers thereof, cellu preservatives, wetting agents, emulsifying agents and dis lose derivatives such as methyl cellulose, carboxymethyl cel persing agents. Prevention of the action of microbes may be lulose, or oxidized cellulose, guar gum, acacia gum, karaya ensured by the inclusion of various antibacterial and antifun 35 gum, tragacanth gum, bentonite, agar, carbomer, bladder gal agents, for example, paraben, chlorobutanol, phenol sor wrack, ceratonia, dextran and derivatives thereof, ghatti gum, bic acid, and the like. It may also be desirable to include hectorite, ispaghula husk, polyvinypyrrolidone, silica and isotonic agents, such as sugars, sodium chloride, and the like derivatives thereof, xanthan gum, kaolin, talc, starch and into the compositions. In addition, prolonged absorption of derivatives thereof, paraffin, water, vegetable and animal oils, the injectable pharmaceutical form may be brought about by 40 polyethylene, polyethylene oxide, polyethylene glycol, the inclusion of agents which delay absorption such as alu polypropylene glycol, glycerol, ethanol, propanol, propylene minum monostearate and gelatin. In some cases, in order to glycol (glycols, alcohols), fixed oils, sodium, potassium, alu prolong the effect of a drug, it is desirable to slow the absorp minum, magnesium or calcium salts (such as chloride, car tion of the drug from subcutaneous or intramuscular injec bonate, bicarbonate, citrate, gluconate, lactate, acetate, glu tion. This may be accomplished by the use of a liquid suspen 45 ceptate or tartrate). sion of crystalline or amorphous material having poor water Standard composition strategies for topical agents can be solubility. The rate of absorption of the drug then depends applied to the RNA binding modulatory compounds of the upon its rate of dissolution which, in turn, may depend upon invention or a pharmaceutically acceptable salt thereof in crystal size and crystalline form. Alternatively, delayed order to enhance the persistence and residence time of the absorption of a parenterally-administered drug form is 50 drug, and to improve the prophylactic efficacy achieved. accomplished by dissolving or suspending the drug in an oil For topical application to be used in the lower intestinal vehicle. tractor vaginally, a rectal suppository, a suitable enema, a gel, Pharmaceutical compositions of the present invention may an ointment, a solution, a suspension or an insert can be used. be administered to a subject, e.g., a non-human animal or a Topical transdermal patches may also be used. Transdermal human, orally, parenterally, topically, rectally, nasally, intra 55 patches have the added advantage of providing controlled vaginally or intracisternally. They are, of course, given by delivery of the compositions of the invention to the body. forms suitable for each administration route. For example, Such dosage forms can be made by dissolving or dispersing they are administered in tablets or capsule form, by injection, the agent in the proper medium. inhalation, eye lotion, ointment, etc., administration by injec Compositions of the invention can be administered in the tion, infusion orinhalation; topical by lotion or ointment; and 60 form of suppositories for rectal or vaginal administration. rectal or vaginal suppositories. These can be prepared by mixing the agent with a suitable The phrases “parenteral administration” and “adminis non-irritating carrier which is solid at room temperature but tered parenterally” as used herein include modes of adminis liquid at rectal temperature and therefore will melt in the tration other than enteral and topical administration, usually rectum or vagina to release the drug. Such materials include by injection, and includes, without limitation, intravenous, 65 cocoa butter, beeswax, polyethylene glycols, a suppository intramuscular, intraarterial, intrathecal, intracapsular, wax or a salicylate that is solid at room temperature, but liquid intraorbital, intracardiac, intradermal, intraperitoneal, tran at body temperature and, therefore, will melt in the rectum or US 8,377,639 B2 101 102 vaginal cavity and release the active agent. Compositions ing agents may also be added. Tablets, and other solid dosage which are suitable for vaginal administration also include forms, such as dragees, capsules, pills and granules, may be pessaries, tampons, creams, gels, pastes, foams, films, or scored or prepared with coatings and shells, such as enteric spray compositions containing such carriers as are known in coatings and other coatings well known in the pharmaceuti the art to be appropriate. The carrier employed in the phar cal-formulating art. They may also be formulated so as to maceutical compositions of the invention should be compat provide slow or controlled release of the active ingredient ible with vaginal administration. therein using, for example, hydroxypropylmethyl cellulose in For ophthalmic applications, the pharmaceutical composi varying proportions to provide the desired release profile, tions can be formulated as micronized suspensions in iso other polymer matrices, liposomes and/or microspheres. tonic, pH adjusted sterile saline, or, preferably, as solutions in 10 They may be sterilized by, for example, filtration through a isotonic, pH adjusted sterile saline, either with or without a bacteria-retaining filter, or by incorporating sterilizing agents preservative such as benzylalkonium chloride. Alternatively, in the form of sterile solid compositions which can be dis for ophthalmicuses, the compositions can beformulated in an solved in sterile water, or some other sterile injectable ointment such as petrolatum. Exemplary ophthalmic compo medium immediately before use. These compositions may sitions include eye ointments, powders, solutions and the like. 15 also optionally contain opacifying agents and may be of a Powders and sprays can contain, in addition to the com composition that they release the RNA binding modulatory pound of the invention, carriers such as lactose, talc, alumi compound, e.g., compound offormula I, Ia, Ib, II, IIa, IIb, IIc, num hydroxide, calcium silicates and polyamide powder, or III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf mixtures of these substances. Sprays can additionally contain or a compound of Table 1 or 2, only, or preferentially, in a customary propellants, such as chlorofluorohydrocarbons 20 certain portion of the gastrointestinal tract, optionally, in a and volatile unsubstituted hydrocarbons, such as butane and delayed manner. Examples of embedding compositions propane. which can be used include polymeric substances and waxes. Ordinarily, an aqueous aerosol is made by formulating an The active ingredient can also be in micro-encapsulated form, aqueous solution or suspension of an RNA binding modula if appropriate, with one or more of the above-described tory compound, e.g., compound of formula I, Ia, Ib, II, IIa, 25 excipients. Liquid dosage forms for oral administration IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, include pharmaceutically acceptable emulsions, microemul VIIe, VIIf or a compound of Table 1 or 2, together with sions, solutions, suspensions, syrups and elixirs. In addition conventional pharmaceutically acceptable carriers and stabi to the active ingredient, the liquid dosage forms may contain lizers. The carriers and stabilizers vary with the requirements inert diluents commonly used in the art, such as, for example, of the particular compound, but typically include nonionic 30 water or other solvents, solubilizing agents and emulsifiers, surfactants (e.g., Tweens, Pluronics, polyethylene glycol and such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, the like), proteins like serum albumin, sorbitan esters, oleic ethyl acetate, benzyl alcohol, benzyl benzoate, propylene acid, lecithin, amino acids such as glycine, buffers, salts, glycol, 1,3-butylene glycol, oils (in particular, cottonseed, sugars or sugar alcohols. Aerosols generally are prepared groundnut, corn, germ, olive, castor and sesame oils), glyc from isotonic solutions. Generation of the aerosol or any 35 erol, tetrahydrofuryl alcohol, polyethylene glycols and fatty other means of delivery of the present invention may be acid esters of sorbitan, and mixtures thereof. accomplished by any of the methods known in the art. For Besides inert diluents, the oral compositions can also example, in the case of aerosol delivery, the compound is include adjuvants such as wetting agents, emulsifying and supplied in a finely divided form along with any suitable suspending agents, sweetening, flavoring, coloring, perfum carrier with a propellant. 40 ing and preservative agents. Liquefied propellants are typically gases at ambient con Suspensions, in addition to compounds of the invention, ditions and are condensed underpressure. The propellant may may contain suspending agents as, for example, ethoxylated be any acceptable and known in the art including propane and isostearyl alcohols, polyoxyethylene sorbitol and sorbitan butane, or other lower alkanes, such as those of up to 5 esters, microcrystalline cellulose, aluminum metahydroxide, carbons. The composition is held within a container with an 45 bentonite, agar-agar and tragacanth, and mixtures thereof. appropriate propellant and valve, and maintained at elevated Sterile injectable forms of the compositions of this inven pressure until released by action of the valve. tion can be aqueous or oleaginous suspension. These suspen Compositions of the invention can also be orally adminis sions may be formulated according to techniques known in tered in any orally-acceptable dosage form including, but not the art using suitable dispersing or wetting agents and sus limited to, capsules, cachets, pills, tablets, lozenges (using a 50 pending agents. flavored basis, usually sucrose and acacia or tragacanth), Wetting agents, emulsifiers and lubricants, such as sodium powders, granules, or as a solution or a suspension in an lauryl sulfate and magnesium stearate, as well as coloring aqueous or non-aqueous liquid, or as an oil-in-water or water agents, release agents, coating agents, sweetening, flavoring in-oil liquid emulsion, or as an elixir or syrup, or as pastilles and perfuming agents, preservatives and antioxidants can (using an inert base, such as gelatin and glycerin, or sucrose 55 also be present in the compositions. The sterile injectable and acacia) and/or as mouth washes and the like, each con preparation may also be a sterile injectable solution or sus taining a predetermined amount of sucrose octasulfate and/or pension in a nontoxic parenterally-acceptable diluent or sol antibiotic or contraceptive agent(s) as an active ingredient. A vent, for example, as a solution in 1,3-butanediol. Among the compound described herein may also be administered as a acceptable vehicles and solvents that may be employed are bolus, electuary or paste. In the case of tablets for oral use, 60 water, Ringer’s solution and isotonic sodium chloride solu carriers which are commonly used include lactose and corn tion. In addition, sterile, fixed oils are conventionally starch. Lubricating agents, such as magnesium stearate, are employed as a solvent or suspending medium. For this pur also typically added. For oral administration in a capsule pose, any bland fixed oil may be employed including syn form, useful diluents include lactose and dried corn starch. thetic mono- or di-glycerides. Fatty acids, such as oleic acid When aqueous suspensions are required for oral use, the 65 and its glyceride derivatives are useful in the preparation of active ingredient is combined with emulsifying and suspend injectables, as are natural pharmaceutically-acceptable oils, ing agents. If desired, certain sweetening, flavoring or color such as olive oil or castor oil, especially in their polyoxyethy US 8,377,639 B2 103 104 lated versions. These oil solutions or suspensions may also 2, may be formulated as animal feeds, animal feed premixes, contain a long-chain alcohol diluent or dispersant. The RNA animal feed concentrates, pills, pastes, suspensions, solu binding modulatory compound, e.g., compound of formula I, tions, gels, tablets, boluses and capsules. In addition, the an Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, RNA binding modulatory compounds, e.g., compounds of VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, or a 5 formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, pharmaceutically acceptable salt thereof will represent some VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or percentage of the total dose in other dosage forms in a mate 2, may be administered to the animals in their drinking water. rial forming a combination product, including liquid solu For oral administration, the dosage form chosen should tions or suspensions, suppositories, douches, enemas, gels, provide the animal with about 0.01 mg/kg to 100 mg/kg of creams, emulsions, lotions slurries, soaps, shampoos, deter 10 animal body weight per day of the RNA binding modulatory gents, powders, sprays, lipsticks, foams, pastes, toothpastes, compound, e.g., compounds of formula I, Ia, Ib, II, IIa, IIb, ointments, salves, balms, douches, drops, troches, lozenges, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, mouthwashes, rinses and others. VIIf or a compound of Table 1 or 2. For parenteral adminis In one embodiment, the compounds of the invention may tration, the dosage form chosen should provide the animal be administered prophylactically. For prophylactic applica 15 with about 0.01 mg/kg to 100 mg/kg of animal body weight tions, the pharmaceutical composition of the invention can be per day of the RNA binding modulatory compound, e.g., applied prior to potential infection. The timing of application compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, prior to potential infection can be optimized to maximize the VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a com prophylactic effectiveness of the compound. The timing of pound of Table 1 or 2. application will vary depending on the mode of administra 20 The RNA binding modulatory compounds, e.g., com tion, doses, the stability and effectiveness of composition, the pounds of formula, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, frequency of the dosage, e.g., single application or multiple VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of dosage. One skilled in the art will be able to determine the Table 1 or 2, may also be applied topically to the animals in most appropriate time interval required to maximize prophy the form of dips, dusts, collars, medallions, sprays and pour lactic effectiveness of the compound. 25 on formulations. For topical application, dips and sprays usu An RNA binding modulatory compound, e.g., compound ally contain about 0.5 ppm to 5,000 ppm or about 1 ppm to offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, 3,000 ppm of the RNA binding modulatory compound, e.g., VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, 2, when present in a composition will generally be present in VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a com an amount from about 0.000001% to about 100%, more pref 30 pound of Table 1 or 2. In addition, the RNA binding modu erably from about 0.001% to about 50%, and most preferably latory compounds, e.g., compounds offormula I, Ia, Ib, II, IIa, from about 0.01% to about 25% of total weight. IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, For compositions of the present invention comprising a VIIe, VIIf or a compound of Table 1 or 2, may be formulated carrier, the composition comprises, for example, from about as eartags for animals, particularly quadrupeds such as cattle 1% to about 99%, preferably from about 50% to about 99%, 35 and sheep. and most preferably from about 75% to about 99% by weight Pesticidal Compositions of at least one carrier. The invention also pertains to pesticidal compositions Also, the separate components of the compositions of the comprising a pesticidally effective amount of an RNA bind invention may be preblended or each component may be ing modulatory compound, e.g., compound of formula I, Ia, added separately to the same environment according to a 40 Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, VIIc, predetermined dosage for the purpose of achieving the VIId, VIIe, VIIf or a compound of Table 1 or 2, and an desired concentration level of the treatment components and agronomically acceptable carrier. Each of these compounds so long as the components eventually come into intimate may be used alone or in combination as a part of a pesticidal admixture with each other. Further, the present invention may composition of the invention. be administered or delivered on a continuous or intermittent 45 In one embodiment, the RNA binding modulatory com basis. pound, e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, The RNA binding modulatory compounds, e.g., com IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or pounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, a compound of Table 1 or 2, is administered in combination VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of with an agricultural agent. The language “in combination Table 1 or 2, may be used in the veterinary sector in any 50 with’ an agricultural agent includes co-administration of the technique routine to one of skill in the art, including, for compound and with an agricultural agent, administration of example, oral administration, parenterally (e.g., intraruminal, the compound first, followed by administration of an agricul intramuscular, intravenous or subcutaneous injection) or by tural agent, and administration of an agricultural agent first, transdermal methods. The RNA binding modulatory com followed by administration of the compound. The compound pounds, e.g., compounds of formula I, Ia, Ib, II, IIa, IIb, IIc, 55 can be administered substantially at the same time as the III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf agricultural agent or at substantially different times as the or a compound of Table 1 or 2 may also be dispersed or agricultural agent. Optimal administration rates for a given dissolved in a pharmaceutically acceptable carrier for injec protocol of administration of the compound and/or the agri tion or transdermal application. Alternatively, the RNA bind cultural agent can be readily ascertained by those skilled in ing modulatory compounds, e.g., compounds offormula I, Ia, 60 the art using conventional determination tests conducted with Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, VIIa, VIIb, VIIc, regard to the specific compounds being utilized, the particular VIId, VIIe, VIIf or a compound of Table 1 or 2, may be compositions formulated, the mode of application, the par formulated into an implant for subcutaneous administration. ticular site of administration and the like. For oral administration to warm-blooded animals, the The term “agricultural agent” includes, for example, insec RNA binding modulatory compounds, e.g., compounds of 65 ticides, attractants, sterilizing agents, bactericides, acari formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, cides, nematicides, fungicides, growth-regulating sub VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or stances, fertilizers or herbicides, such as aldimorph,

US 8,377,639 B2 107 108 cadusafos, carbaryl, carbofuran, carbophenothion, carbosul carbamate, N-(3,4,4-trifluoro-1-oxo-3-butenyl)-glycine, fan, cartap, chloethocarb, chlorethoxyfos, chlorfenapyr, chlo N-(4-chlorophenyl)-3-[4-(difluoromethoxy)phenyl]-4,5-di rfenvinphos, chlorfluazuron, chlormephos, chlorpyrifos, hydro-4-phenyl-1H-pyrazole-1-carboxamide, N-[(2-chloro chlorpyrifos M, chlovaporthrin, cis-resmethrin, cisper 5-thiazolyl)methyl]-N'-methyl-N'-nitro-guanidine, N-me methrin, clocythrin, cloethocarb, clofentezine, cyanophos, thyl-N'-(1-methyl-2-propenyl)-1,2 cycloprene, cycloprothrin, cyfluthrin, cyhalothrin, cyhexatin, hydrazinedicarbothioamide, N-methyl-N'-2-propenyl-1,2 cypermethrin, cyromazine, deltamethrin, demeton M, deme hydrazinedicarbothioamide and O,O-diethyl 2 ton S, demeton-S-methyl, diafenthiuron, diazinon, dichlor (dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate. vos, diflubenzuron, dimethoate, dimethylvinphos, diofeno Treatment of the plants and soil with the RNA binding lan, disulfoton, docusat-sodium, dofenapyn, effusilanate, 10 emamectin, empenthrin, endosulfan, Entomopfthora spp., modulatory compounds, e.g., compounds offormula I, Ia, Ib, esfenvalerate, ethiofencarb, ethion, ethoprophos, etofemprox, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, etoxazole, etrimfos, fenamiphos, fenazaquin, fenbutatin VIId, VIIe, VIIfora compound of Table 1 or 2, may be carried oxide, fenitrothion, fenothiocarb, fenoxacrim, fenoxycarb, out directly or by allowing the compounds to act on the fempropathrin, fenpyrad, fenpyrithrin, fempyroximate, fenval 15 surroundings, environment or storage space by the customary erate, fipronil, fluazinam, fluazuron, flubrocythrinate, flucy treatment methods, for example by immersion, spraying, cloxuron, flucythrinate, flufenoxuron, flutenzine, fluvalinate, evaporation, fogging, scattering, painting on and, in the case fonophos, fosmethilan, fosthiazate, fubfemprox, furathiocarb, of propagation material, in particular in the case of seeds, also granulosis viruses, halofenozide, HCH, heptenophos, by applying one or more coats. hexaflumuron, hexythiazox, hydroprene, imidacloprid, isa 20 Depending on the plant species or plant cultivars, their zofos, isofenphos, isoxathion, ivermectin, nuclear polyhedro location and growth conditions (soils, climate, vegetation sis viruses, lambda-cyhalothrin, lufenuron, malathion, period, diet), the treatment according to the invention may mecarbam, metaldehyde, methamidophos, Metharhizium also result in superadditive (“synergistic”) effects. Thus, for anisopliae, Metharhizium flavoviride, methidathion, methio example, reduced application rates and/or a widening of the carb, methomyl, methoxyfenozide, metolcarb, metoxadiaz 25 activity spectrum and/or an increase in the activity of the one, mevinphos, milbemectin, monocrotophos, naled, niten substances and compositions to be used, better plant growth, pyram, nithiazine, novaluron, omethoate, oxamyl, increased tolerance to high or low temperatures, increased oxydemethon M, Paecilomyces fumosoroseus, parathion A, tolerance to drought or to water or soil salt content, increased parathion M, , phenthoate, phorate, phosalone, flowering performance, easier harvesting, accelerated matu phosmet, phosphamidon, phoxim, pirimicarb, pirimiphos A, 30 pirimiphos M, profenofos, promecarb, propoxur, prothiofos, ration, higher harvest yields, better quality and/or a higher prothoate, pymetrozine, pyraclofos, pyresmethrin, pyre nutritional value of the harvested products, better storage thrum, pyridaben, pyridathion, pyrimidifen, pyriproxyfen, stability and/or processability of the harvested products that quinalphos, ribavirin, salithion, sebufos, silafluofen, spi exceed the effects which were actually to be expected may OCCUI?. nosad, sulfotep, sulprofos, tau-fluvalinate, tebufenozide, 35 tebufenpyrad, tebupirimiphos, teflubenzuron, tefluthrin, The RNA binding modulatory compounds, e.g., com temephos, temivinphos, terbufos, tetrachlorvinphos, theta pounds of I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, cypermethrin, thiamethoxam, thiapronil, thiatriphos, thiocy VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or clam hydrogen oxalate, thiodicarb, thiofanox, thuringiensin, 2, may used in unchanged form or together with an agronomi tralocythrin, tralomethrin, triaratheme, triazamate, triazo 40 cally acceptable carrier. The term “agronomically acceptable phos, triazuron, trichlophenidine, trichlorfon, triflumuron, trimethacarb, vamidothion, vaniliprole, Verticillium lecanii, carrier” includes any carrier suitable for administration to a YI 5302, zeta-cypermethrin, zolaprofos, (1R-cis)-[5-(phe plant or soil, for example, customary excipients in formula nylmethyl)-3-furanyl]-methyl-3-[(dihydro-2-oxo-3(2H) tion techniques, such as solutions (e.g., directly sprayable or furanylidene)-methyl]-2,2-dimethylcyclopropanecarboxy 45 dilutable solutions), emulsions, (e.g., emulsion concentrates late, (3-phenoxyphenyl)-methyl-2,2,3,3 and diluted emulsions), wettable powders, suspensions, tetramethylcyclopropanecarboxylate, 1-[(2-chloro-5 soluble powders, powders, dusts, pastes, soluble powders, thiazolyl)methyl]tetrahydro-3,5-dimethyl-N-nitro-1,3,5 granules, suspension-emulsion concentrates, encapsulation triazine-2(1H)-imine, 2-(2-chloro-6-fluorophenyl)-4-[4-(1, into polymeric materials, coatable pastes, natural and syn 1-dimethylethyl)phenyl]-4,5-dihydro-oxazole, 50 2-(acetyloxy)-3-dodecyl-1,4-naphthalenedione, 2-chloro-N thetic materials impregnated with active compound and [[[4-(1-phenylethoxy)-phenyl]-amino]-carbonyl]-benza microencapsulations in polymeric substances. These formu mide, 2-chloro-N-[[[4-(2,2-dichloro-1,1-difluoroethoxy) lations are produced in a known manner, for example by phenyl]-amino]-carbonyl]-benzamide, 3-methylphenyl mixing the compounds with agronomically acceptable car propylcarbamate, 4-[4-(4-ethoxyphenyl)-4-methylpentyl)-1 55 rier, such as liquid solvents or solid carriers, optionally with fluoro-2-phenoxy-benzene, 4-chloro-2-(1,1-dimethylethyl) 5-[[2-(2,6-dimethyl-4-phenoxyphenoxy)ethyl]thio]-3(2H) the use of surfactants, including emulsifiers, dispersants, and/ pyridazinone, 4-chloro-2-(2-chloro-2-methylpropyl)-5-[(6 foam-formers. iodo-3-pyridinyl)methoxy]-3(2H)-pyridazinone, 4-chloro-5 If the agronomically acceptable carrier is water, it may also [(6-chloro-3-pyridinyl)methoxy]-2-(3,4-dichlorophenyl)-3 60 possible to employ, for example, organic solvents as auxiliary (2H)-pyridazinone, Bacillus thuringiensis strain EG-2348, solvents. Suitable liquid solvents include, for example, aro [2-benzoyl-1-(1,1-dimethylethyl)-hydrazinobenzoic acid, matics (e.g., xylene, toluene and alkylnaphthalenes); chlori 2)-dimethyl-3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4,5] nated aromatics or chlorinated aliphatic hydrocarbons (e.g., dec-3-en-4-ylbutanoate, [3-[(6-chloro-3-pyridinyl)methyl] chlorobenzenes, chloroethylenes and methylene chloride); 2-thiazolidinyldenel-cyanamide, dihydro-2-(nitromethyl 65 aliphatic hydrocarbons (e.g., cyclohexane); paraffins (e.g., ene)-2H-1,3-thiazine-3(4H)-carboxaldehyde, ethyl(2-[[1,6 petroleum fractions, mineral and vegetable oils); alcohols dihydro-6-oxo-1-(phenylmethyl)-4-pyridazinyl]oxy]ethyl] (e.g., butanol or glycol and also their ethers and esters); US 8,377,639 B2 109 110 ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl The contents of all references, patent applications and pat ketone and cyclohexanone) and strongly polar solvents (e.g., ents, cited throughout this application are hereby expressly dimethylformamide and dimethyl sulphoxide). incorporated by reference. Each reference disclosed herein is Suitable solid agronomically acceptable carriers include, incorporated by reference herein in its entirety. Any patent for example, ammonium salts and ground natural minerals application to which this application claims priority is also (e.g., kaolins, clays, talc, chalk, quartz, attapulgite, montmo incorporated by reference herein in its entirety. rillonite and diatomaceous earth); ground synthetic minerals (e.g., highly disperse silica, alumina and silicates); crushed EXEMPLIFICATION OF THE INVENTION and fractionated natural rocks (e.g., calcite, marble, pumice, sepiolite and dolomite); synthetic granules of inorganic and 10 Example 1 organic meals; granules of organic material (e.g., sawdust, Identification of Modulators of RNA Binding coconut shells, maize cobs and tobacco stalks); Proteins MEX-5, POS-1 and MEX-3 Suitable emulsifiers and foam-formers include, for example, nonionic and anionic emulsifiers (e.g., polyoxyeth 15 Expression and Purification of Recombinant MEX-5, POS-1, ylene fatty acid esters, polyoxyethylene fatty alcohol ethers, and MEX-3 for example, alkylaryl polyglycol ethers, alkylsulphonates, MEX-5: The expression and purification of recombinant alkyl sulphates and arylsulphonates) protein hydrolysates. MEX-5 protein has previously been described in Pagano, J. Suitable dispersants include, for example, lignin-sulphite M., Farley, B. M., McCoig, L. M. and Ryder, S. P. (2007) waste liquors and methylcellulose. 20 “Molecular basis of RNA recognition by the embryonic Tackifiers such as carboxymethylcellulose and natural and polarity determinant MEX-5;”JBiol Chem; 282,8883-8894. synthetic polymers in the form of powders, granules or lati The fragment of mex-5 containing the TZF domain (amino ces, such as gum arabic, polyvinyl alcohol and polyvinyl acids 236-350) was amplified from a commercially available acetate, as well as natural phospholipids, such as cephalins ORFeome clone (Open Biosystems) and sub-cloned into the and lecithins, and synthetic phospholipids, can be used in the 25 vectorpMal-c (NEB), which encodes maltose binding protein formulations. Other additives may include, for example, min as an N-terminal fusion. This construct is termed pMal eral and vegetable oils. MEX-5 (236-350). Colorants such as inorganic pigments, for example, iron MEX-5 is expressed in Escherchia. coli JM109 liquid cul oxide, titanium oxide and Prussian Blue, and organic dye tures grown at 37° C. Cultures were induced during mid log stuffs, such as alizarin dyestuffs, azo dyestuffs and metal 30 phase by the addition of 0.1 mM isopropyl 1-thio-fl-D-galac phthalocyanine dyestuffs, and trace nutrients such as salts of topyranoside (IPTG) and allowed to grow for three hours. An iron, manganese, boron, copper, cobalt, molybdenum and amount of 100 pum zinc acetate (final concentration) was zinc may also be included in the agronomically acceptable added at the time of induction. The pelleted cells were resus carrier. pended in lysis buffer (50 mM Tris pH 8.8, 200 mM. NaCl, 2 The pesticidal compositions may be administered to the 35 mM DTT, EDTA free protease inhibitor tablet (Roche), 100 plantor soil by any techniques known in the art, including, for puM Zn(OAc)2) and lysed by sonication. The soluble protein example, spraying, atomizing, dusting, scattering, coating or was purified in three steps using standard chromatography pouring. One of skill in the art would be able to determine the methods: (1) amylose affinity (NEB), (2) Hi-TRAP-Q (GE appropriate technique for administration without undue Healthcare), and (3) Hi-TRAP-S (GE Healthcare). The Q experimentation according the specific pest to be combated, 40 buffer was composed of 50 mM Tris, pH 8.8, 20 mM-1500 the specific chemical composition and formulation of the mM NaCl, 2 mM DTT, and 100 puM Zn(OAc). The S buffer compound being employed, the method of applying the com was composed of 50 mM MOPS, pH 6.0, 20 mM-1500 mM pound/formulation, and the locus of treatment. NaCl, 2 mM DTT, 100 puM Zn(OAc). The pure fractions, In one embodiment, the pesticidal compositions compris determined by coomassie-stained SDS-PAGE, were dialyzed ing an RNA binding modulatory compound, e.g., compound 45 into storage buffer (20 mM Tris, pH 8.0, 25 mM NaCl, 100 offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI,VIa, VIb, VII, puM Zn(OAc)2, 2 mM DTT). The protein concentration was VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or determined by measuring absorbance at 280 nm and the cal 2, may be administered by foliar application. In another culated extinction coefficient. The protein was stored at 50 embodiment, the pesticidal composition may also reach the puM concentration at 4°C. plants through the root system via the soil (systemic action) 50 POS-1: The expression and purification of recombinant by drenching the locus of the plant with a liquid preparation or POS-1 protein has been previously described in Farley, B. M., by incorporating the substances into the soil in solid form, Pagano, J. M. and Ryder, S. P. (2008) “RNA target specificity e.g., in the form of granules (soil application). In rice culti of the embryonic cell fate determinant POS-1” RNA, 14, vations, these granules may be dispensed over the flooded 2685–2697. The sequence encoding amino acids 80-180 of paddy field. The pesticidal compositions of the invention may 55 POS-1 was amplified from the corresponding ORFeome also be applied to tubers or seed grain, for example, by soak (Open Biosystems) clone via PCR and cloned in frame into ing, spraying or drenching the seed grain or tubers in a liquid the multiple cloning site of pHMTc, a derivative of pVal-c2x pesticidal composition or by coating the tubers or seed grain (NEB) that includes an N-terminal 6-his tag and a TEV pro with a solid pesticidal composition. tease site after MBP. The pesticidal compositions disclosed herein generally 60 The protein was expressed from this construct in E. coli comprise between 0.1 and 95% by weight of active com strain BL21 (DE3) Gold (Stratagene). The protein expression pound, preferably between 0.5 and 90%. Favorable applica was induced by addition of 1 mM IPTG and 100 puM tion rates are, in general, 1 g to 2 kg of active substance (AS) Zn(OAc), at mid-log phase. Cells were induced for three per hectare (ha), for example, 10 g to 1 kg AS/ha or 20 g to 600 hours prior to harvesting and the cells were lysed and purified gAS/ha. For application of tubers or seed grain, dosages of 10 65 using an amylose column (NEB). The eluate was passed mg to 1 g active substance per kg of seed grain or tubers may through a HiTrap SP column, and the collected flow through be used. was purified over a HiTrap Q (GE Healthcare) column. The

US 8,377,639 B2 113 114 TABLE 2

Compound Code Structure MEX-5 POS-1 MEX-3

A. O –0.103068 0.0750083

O O.

ºO H O –0.03691.19 0.055 1393

O O,

ºO O –0.0208.829 –0.0247867

O

–0.0158025 –0.0379.222

–0.01.06525 –0.0161771

O O | | — NH HN — S | O US 8,377,639 B2 115 116 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3 F OH –0.007.108 –0.0278922

–0.00640326 0.241935

–0.00335,626 0.01952.17

O

–0.00142058 0.0054316

–0.00094.1405 0.085093 US 8,377,639 B2 117 118 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

K O C| 1.38E-05 0.0581118

O

Cl I IC

O OH

0.01.21396 –0.000.86.3837 N ~N

0.01621.17 0.116787

0.0208867

0.0229711 0.04766.17

0.034.1767 0.0430453 US 8,377,639 B2 119 120 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

H 0.0351486 §ºr { IO N~ o |

C|

0.0351786

HS j-N *S* \ S /

N 0.050021 0.105408 2 N N O O º SH

N S 0.0550846 0.08985.78

0.0621.123

2.

S.

OH

0.0626.293

0.064008 0.0539474 US 8,377,639 B2 122 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3 X | 0.0668833 i-N O2N O ( )()

Cl OH

Y 0.067749 0.0604342

Z 0.0687865 0.126705

AA 0.0713937 0.00702445

AB 0.0730459 0.108744

AC H 0.0758265 N N HO N_{~~ 4O HO & ^. }

AD OH 0.0803921 0.0826922 US 8,377,639 B2 123 124 TABLE 2-continued

Compound Code Structure MEX-5 POS-1 MEX-3

AE 0.0810947 0.0360551 0.1623.54

HO S O

AF 0.081336 0.226509

AG NO2 0.0861502

OH O

AH 0.0875937 0.145547

AI 0.0912883 0.02497.58

AJ 0.0923393 | HN —S

O ( ) | NO2

US 8,377,639 B2 127 128 TABLE 2-continued Compound Code StruCture MEX-5 POS-1 MEX-3

AR 0.129328

AS 0.131352

O

S

AT 0.132089

O Oyjº-o NT N)—( )C O

AU N — NH 0.134432 0.247521

Br AV O S 0.1401.39 0.109312

AW 0.140504

CI)N NH)—º ~ S US 8,377,639 B2 129 130 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

AX N 0.141.245 XN

AY 0.141274 0.180409

HO

0.1435.26 0.2535.27

NF

BA 0.151983 0.221567

BB 0.15.3606

Br

BC 0.154715

BD 0.1547.59

US 8,377,639 B2 133 134 TABLE 2-continued

Compound Code Structure MEX-5 POS-1 MEX-3

BK 0.1835.19

O S /& >N CN O H

BL 0.188022 0.2755.26

N

BM 0.188023 OH

BN 0.1887.11

BO 0.190286

BP 0.19239 0.0454242 HO

US 8,377,639 B2 137 138 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3 BX NO2 0.21 1718 S

22 N N

OH

BY 0.2131.89 0.143999 C. O 0.214246 o2 S

CA 0.21 63.67 0.2301.19

CB 0.21706 0.23927

CC 0.224919

CD OH 0.226379 0.123733

NO2

US 8,377,639 B2 141 142 TABLE 2-continued

Compound Code Structure MEX-5 POS-1 MEX-3

CK O S 0.233621

CL SH 0.235.106

CM N SH 0.2363.12

CN 0.239.373

CO ( ) 0.240608 0.160345 Cl

CP 0.241.146

O J.

Br US 8,377,639 B2 143 144 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

CQ 0.2451 97

O

CR 0.24.654

| S NH O –/ N- N> O O

CS N- NN 0.25 1831

| N}–sº º \

CT C| 0.252156

CU 0.2533:12

CV O 0.255468

0.03984.67 - US 8,377,639 B2 145 146 TABLE 2-continued Compound Code POS-1 MEX-3

CX 0.0430453

CY 0.0434891

OH

CZ 0.0488653

DA 0.0651114

O

DB 0.0689837 O \ O O S 2 N O N OH

C| S2°

DC O NH2 - 0.0749757

H N Br i O ESEC) O

DD 0.0750083

US 8,377,639 B2 149 150 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

DL 0.1431

NH

DM 0.143623

DN 0.166782

N OJ O

DO Br 0.166873

HO O

DP O 0.167907 |

Oi-N NS/ \ US 8,377,639 B2 151 152 TABLE 2-continued

Compound Code Structure MEX-5 POS-1 MEX-3

0.171713

O O

HO NH O HN O HO

DR 0.171815

O

> & Z

O / \,

DS 0.176583

O O | | F O-S—NH HN–SHO F

DT H - 0.177235 O N

O S > CºZºº JC is

Br

DU 0.180302

/ h US 8,377,639 B2 153 154 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

DV HO O 0.184556

O S

DW H 0.189883 >O &O SC N O/ >NH

DX 0.196968

OH N-> § / O O O S

O O \

DY N 0.200285

hX—N *— O O

Cl 0.2013.7 ºrH H O S OH

C| O

EA O 0.203009

EB 0.209605 US 8,377,639 B2 155 156 TABLE 2-continued

Compound Code Structure MEX-5 POS-1 MEX-3 EC y 0.2173.93 N HN– –C–

ED 0.220348

EE 0.2251.08

EF 0.22817.2

EG 0.229412

Cl

EH 0.2301.19

H N N OH

Cl US 8,377,639 B2 157 158 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

EI 0.230.383 Cl

OH

0.233942

HO EK N 0.236507

EL 0.237867

EM 0.241791

EN 0.247036

EO HO 0.253.668

Cl US 8,377,639 B2 159 160 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

EP 0.259513 N

O

O

Cl 0.26035

H H ºr N ºr N O \ O S OH

O

ER NO2 0.261571

ES 0.26453

HO

ET NO2 0.2651.18

NH

OH

EU NO2 0.26601 S

2 NH

OH US 8,377,639 B2 161 162 TABLE 2-continued Compound Code Structure POS-1 MEX-3

EV 0.274806 Br C

º (O

EW O 0.275,235

HN N N O

~s

EX 0.0673989

EY 0.0896497

EZ 0.123959

FA 0.209341 US 8,377,639 B2 163 164 TABLE 2-continued Compound Code Structure MEX-5 POS-1 MEX-3

FB - - 0.258457

Further screenings were performed on select compounds sigmoidal dose response function in order to determine the of the invention, as described above, and the results are shown ICso, the concentration that gives half maximal inhibition. in Table 3. 20 The results are seen in Table 4.

TABLE 3 TABLE 4 Dose Response MEX-5 Dose Response POS-1 Compound (IM) (HM) Gel Shift MEX-5 Gel Shift POS-1 25 Compound (HM) (HM) E 1.5 0.32 BR 39 820 BK 540 ND E 2.0 0.17 DG 160 ND BR 78 150 DD ND 300 DJ 3.5 ND 30 BK 110 ND C 33 7 DG 170 ND BO 75 16 DD ND 260 DJ 3.8 ND C 9.5 5.1 Example 2 35 BO 120 33 Hermaphrodite Worm Reproduction Assay In order to further evaluate the ability of a particular RNA binding modulatory compound identified herein to inhibit 40 Example 5 embryogenesis, e.g., in a nematode, the compounds identified herein are tested in a standard hermaphrodite worm reproduc C. elegans Viability/Sterility Assay tion assays. Larval worms are hatched overnight into medium containing the compound in an agar plate. Then, feeder bac teria is added to each plate and the worms cultured until they 45 In order to determine whether select compounds of the begin to produce eggs. Adult worms are removed or killed. invention inhibited viability of embryos, Caenorhabditis Finally, the ratio of dead eggs to hatchlings is determined by elegans N2 strain worms were cultured on NGM agar plates inspection with a stereomicroscope. using OP50 strain Escherchia coli as food. Young adult worms with a single row of embryos were selected for micro Example 4 50 injection. Each worm was mounted on a dried agarose padded coverslip in halocarbon oil. Varying concentrations of com Dose Response Gel Shift Assays pound, or a DMSO control, were microinjected into each gonad arm of the worm using an inverted with A further dose response assay was performed on select DIC optics. Injected worms were then allowed to recover for compounds of the invention as described below. A sub-satu 55 rating concentration of MEX-5 or POS-1 (120 nM) was 2-3 hours at 15° C. Approximately 10-40 worms were equilibrated with limiting fluorescein labeled RNA (2-3 nM, injected per compound tested. Individual worms that survived TCR2 RNA or MEX-3 UTR fragment RNA respectively) in microinjection were singled out onto new NGM agar plates the presence of varying concentrations of compound. Follow and allowed to recover overnight at 15° C. The plates were ing equilibration, the reactions were loaded onto a 5% slab 60 moved to room temperature the next morning. At the end of polyacrylamide gel and subjected to electrophoresis for about the day, the adult worm was transferred to a new plate. The 1 hour to separate protein-RNA complex from free RNA. The embryos laid on each plate were monitored for hatching and gel was scanned on a FUJIFLA-5000 imager, and the fraction inspected for unusual phenotypes. Nematode fertility was of bound RNA determined by dividing the intensity of the estimated by counting the total number of progeny on both bound RNA by the intensity of the bound RNA plus the free 65 plates. Viability was estimated by monitoring the extent of RNA. The fraction of the compound bound to the protein was hatching. The results of this assay using compound E are plotted as a function of compound concentration and fit to a shown below in Table 5. US 8,377,639 B2 165 166 TABLE 5 seemed to lose fertility over the course of two days. In con trast, 27 worms were injected with DMSO controls, 10 sur vived injection, and they continued to produce eggs after Worm No. Embryos Hatchlings Embryos Hatchlings wards. 1 O 34 O O 2 O 2 O O EQUIVALENTS 3 2 6 O O 4 O 4 O O Those skilled in the art will recognize, or be able to ascer 5 O 67 O O tain using no more than routine experimentation, numerous 6 1 11 O O 10 equivalents to the specific polypeptides, nucleic acids, meth ods, assays and reagents described herein. Such equivalents The results of this assay indicate that compound E may are considered to be within the scope of this invention and are sterilize worms at a stage prior to fertilization. The worms covered by the following claims.

SEQUENCE LISTING

<16 O: NUMBER OF SEQ ID NOS : 5

<210 - SEQ ID NO 1 <211 - LENGTH : 3 O <212 > TYPE : RNA <213 - ORGANISM: Artificial Sequence <22 0 - FEATURE : < 2.23 - OTHER INFORMATION: Synthetic oligonucleotide: MEX-5 binding RNA (TCR2)

<400: SEQUENCE: 1

uuucuuuaua acuuguuaca auuuuugaaa 30

<210 - SEQ ID NO 2 <211 LENGTH: 28 <212 > TYPE : RNA <213 - ORGANISM: Artificial Sequence <22 0 - FEATURE : < 2.23 - OTHER INFORMATION: Synthetic oligonucleotide: POS-1 binding RNA (BMFO 13)

<400: SEQUENCE: 2

aacuaullauu auuuguuaull Cauaullull 28

<210 - SEQ ID NO 3 <211 - LENGTH : 3 O <212 > TYPE : RNA <213 - ORGANISM: Artificial Sequence <22 0 - FEATURE : < 2.23 - OTHER INFORMATION: Synthetic oligonucleotide: MEX-3 binding RNA (SEQ14) <400: SEQUENCE: 3

C??gCaggää gugug Cagag lullllagga C9u 30

<210 - SEQ ID NO 4 <211 - LENGTH : 3 O <212 > TYPE : RNA <213 - ORGANISM: Artificial Sequence <22 0 - FEATURE : < 2.23 - OTHER INFORMATION: Synthetic oligonucleotide: Fluorescent TCR2 RNA <22 0 - FEATURE : <221 NAME/KEY: misc feature < 2.23 - OTHER INFORMATION: FITC label on 3 end

<400: SEQUENCE: 4

uuucuuuaua acuuguuaca auuuuugaaa 30

<210: SEQ ID NO 5 <211 - LENGTH : 28 US 8,377,639 B2 167 168 – continued

< 212 > TYPE : RNA <213: ORGANISM: Artificial Sequence < 22 O > FEATURE : <223 - OTHER INFORMATION: Synthetic oligonucleotide: Fluorescent mex3 RNA < 22 O > FEATURE : <221: NAME/KEY: misc feature < 22.3 × OTHER INFORMATION: FITC label on 3 '' end

< 400 - SEQUENCE: 5 aacuaullauu auuuguuauu Callauuuu 28

The invention claimed is: 15 the RNA binding activity of an RNA binding protein selected 1. A method for treating or preventing a parasitic associated from a group consisting of POS-1 and MEX-5. state in a subject comprising administering to said subject an 8. The method of claim 7, wherein the parasite is present in effective amount of compound E: a subject.

20 9. The method of claim 7 or 8, wherein said parasite is a helminth. 10. The method of claim 9, wherein said helminth is selected from the group consisting of a cestode, a trematode | | and a nematode. 25 /O O-- *—O O\ 11. A method for treating or preventing an inflammatory disorder caused by a parasitic associated state in a subject comprising administering to said subject a therapeutically effective amount of compound E: 30 or a pharmaceutically acceptable salt thereof, such that said parasitic associated state is treated or prevented, wherein compound E modulates the RNA binding activity of an RNA binding protein selected from a group consisting of POS-1 and MEX-5. 35 2. The method of claim 1, wherein said subject is a plant, an | | animal or a human. O i-sº *— O 3. The method of claim 1, wherein said parasitic associated / O O \ state is a parasitic infestation or parasitic re-infestation or a disease caused by a parasitic infestation. 40 4. The method of claim 1, wherein said method includes or a pharmaceutically acceptable salt thereof, such that said protecting plants from a parasitic infestation, inhibiting inflammatory disorder is treated or prevented, wherein com embryogenesis in a parasite or in a subject suffering from a pound E modulates the RNA binding activity of an RNA parasitic infestation, or reducing parasitic burden in soil, in binding protein selected from a group consisting of POS-1 plants or in an animal suffering from a parasitic infection. and MEX-5. 5. The method of any one of claims 1, 2, 3 and 4, wherein 45 said parasite is a helminth. 12. The method of claims 1, 7 and 11, wherein the RNA 6. The method of claim 5, wherein said helminthis selected binding protein is required for embryogenesis. from the group consisting of a cestode, a trematode and a 13. A pharmaceutical composition comprising a therapeu nematode. tically effective amount of compound E: 7. A method for inhibiting embryogenesis in a parasite, 50 comprising contacting said parasite with an effective amount of compound E: 55 | i O i-sº *— O | | / O O \ 60 /O O-- *—O O\ or a pharmaceutically acceptable salt thereof, and a pharma ceutically acceptable carrier, whereincompound E modulates the RNA binding activity of an RNA binding protein selected 65 from a group consisting of POS-1 and MEX-5. or a pharmaceutically acceptable salt thereof, such that said 14. A composition comprising a pesticidally effective embryogenesis is inhibited, wherein compound E modulates amount of compound E: US 8,377,639 B2 169 170 22. The method of claim 21, wherein the parasite is present in a subject. 23. The method of claim 21 or 22, wherein said parasite is a helminth. O O 24. The method of claim 23, wherein said helminth is selected from the group consisting of a cestode, a trematode and a nematode. /º-K)-- | \-HQ)—| \ 25. A method for treating or preventing an inflammatory disorder caused by a parasitic associated state in a subject and an agronomically acceptable carrier, wherein compound 10 comprising administering to said subject a therapeutically E modulates the RNA binding activity of an RNA binding effective amount of compound DI: protein selected from a group consisting of POS-1 and MEX 5. 15. A method for treating or preventing a parasitic associ ated state in a subject comprising administering to said sub 15 ject an effective amount of compound DI: O O | 20 | O O

or pharmaceutically acceptable salt thereof, such that said inflammatory disorder is treated or prevented, wherein com 25 pound DI modulates the RNA binding activity of an RNA binding protein POS-1. 26. The method of claims 15, 21 and 25, wherein the RNA binding protein is required for embryogenesis. or a pharmaceutically acceptable salt thereof, such that said 27. A pharmaceutical composition comprising a therapeu parasitic associate state is treated or prevented, wherein com 30 pound DI modulates the RNA binding activity of an RNA tically effective amount of compound DI: binding protein POS-1. 16. The method of claim 15, wherein said subject is a plant, an animal or a human. 17. The method of claim 15, wherein said parasitic associ 35 ated state is a parasitic infestation or parasitic re-infestation or a disease cause by a parasitic infestation. O O 18. The method of claim 15, wherein said method includes | protecting plants from a parasitic infestation, inhibiting embryogenesis in a parasite or in a subject suffering from a 40 | parasitic infestation, or reducing parasitic burden in soil, in O O plants or in an animal suffering from parasitic infection. 19. The method of any one of claims 15-18, wherein said or pharmaceutically acceptable salt thereof, and a pharma parasite is a helminth. ceutically acceptable carrier, wherein compound DI modu 20. The method of claim 19, wherein said helminth is 45 lates the RNA binding activity of an RNA binding protein selected from the group consisting of a cestode, a trematode POS-1. and a nematode. 28. A composition comprising a pesticidally effective 21. A method for inhibiting embryogenesis in a parasite, amount of compound DI: comprising contacting said parasite with an effective amount of compound DI: 50

O O 55 | | O O

60 and an agronomically acceptable carrier, wherein compound DI modulates the RNA binding activity of an RNA binding or a pharmaceutically acceptable salt thereof, such that said protein POS-1. embryogenesis is inhibited, wherein compound DI modulates binding activity of an RNA binding protein POS-1.