USOO8993622B2

(12) United States Patent (10) Patent No.: US 8,993,622 B2 Wadell et al. (45) Date of Patent: Mar. 31, 2015

(54) ANTIVIRAL COMPOUNDS (56) References Cited (75) Inventors: Göran Wadell, Umea (SE); Karin U.S. PATENT DOCUMENTS Edlund, Umea (SE); Marten Strand, 2006/0035245 A1 2/2006 Ason et al. Umeå (SE); Emma Andersson, Umeå 2007.00999 19 A1* 5, 2007 Rana ...... 514,237.5 (SE); Christopher Oberg, Umea (SE); Mikael Elofsson, Umea (SE); Ya-Fang FOREIGN PATENT DOCUMENTS Mei, Umea (SE) JP 11-171848 6, 1999 (73) Assignee: Eirium AB, Umea (SE) W wo.80% 7.58 (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Dörwald, F. Zaragoza. (Side Reactions in Organic Synthesis: A Guide to Successful Synthesis Design, Weinheim: Wiley-VCH (21) Appl. No.: 13/703,481 Verlag GmbH & Co. KGaA. 2005, Preface)." Jordan, V.C. (Nature Reviews: Drug Discovery, 2, 2003, 205-213).* JP 11171848 STN Registry record for CAS 228580-61-8 issued Jun. (22) PCT Fled: Jun. 13, 2011 29, 1999.* Abdel-Monem and Abdel-Hafez, "An efficient, convenient synthesis (86). PCT No.: PCT/SE2O11AOSO724 of novel medium-sized 13H-Dibenzod.h 1,3,7oxadiazecine-8, 14-dione macrollides as anticipated antineoplastic agents. Biorg, and S371 (c)(1), Med. Chem., 2002, 10:2297-2302. (2), (4) Date: Apr. 9, 2013 Allard et al., “Rapidpid typing of human adenoviruses byyag a general PCR combined with restriction endonuclease analysis.” Journal Clinical (87) PCT Pub. No.: WO2011/155898 Microbiology, 2001, 39:498-505. Andersson et al., “Small-molecule screening using a whole-cell viral PCT Pub. Date: Dec. 15, 2011 replication reporter gene aSSay identifies 1-2- (benzoylamino)benzoylamino-benzoic acid as a novel (65) Prior Publication Data antiadenoviral compound.' Antimicrobial agents and chemother, 2010, 54(9):3871-3877. US 2013/0210915A1 Aug. 15, 2013 Baldwin et al., “Outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation. Bone (30) Foreign Application Priority Data Marrow Transplantation, 2000, 26:1333-1338. CA—Abstract SU323402, dated Jun. 22, 1970, Bolotin and Brudz, Jun. 11, 2010 (SE) ...... SE1000623-7 "Esters oftosylanthranoyllanthranilic acid.” copyright 2013, 1 page. Jun. 11, 2010 (SE) ...... SE1000624-5 Deng et al., “Dynamic receptor-based pharmacophore model devel opment and its application in designing novel HIV-1 integrase inhibi tors. J. Med. Chem., 2005, 48:1496-1505. (51) Int. Cl. DiGrandiet al., “Thiourea inhibitors of herpesviruses. Part3. Inhibi AOIN37/2 (2006.01) tors of varicella Zoster virus.” Bioorgan. And Med. Chem. Letters, AOIN 37/44 (2006.01) 2004, 14:4157-4160. A6 IK3I/24 (2006.01) Hashimoto, “New methods and reagents in organic synthesis.” 14.1 A A 6LX3L/95 (2006.01) simple efficeitn preparation of methyl esters with C07C 229/00 (2006.01) trimethylsilyldiazomethane (TMSCHN2) and its application to gas CD7C 23.3/8 (2006.01) chromatographis analysis of fatty acids, Chem. Pharm. Bull. 1981, A6 IK3I/67 (2006.01) R E. iruses in the i ised host.” Clini 3. 37, 3.08: cal1ernozer, Microbiology Adenoviruses Reviews, in1992, Une Immunocompromised5:262-274. nost. Utint A6 IK3I/245 (2006.015 (Continued) C07C 237/40 (2006.01) C07C 237/42 (2006.01) C07C3II/08 (2006.01) Primary Examiner James D. Anderson C07C3II/2 (2006.01) Assistant Examiner — William Lee (52) U.S. Cl (74) Attorney, Agent, or Firm — Fish & Richardson P.C. CPC ...... C07C233/81 (2013.01); A61 K3I/I67 (2013.01); A61 K3I/18 (2013.01); A61 K (57) ABSTRACT 31/196 (2013.01); A61 K3I/245 (2013.01); C07C 237/40 (2013.01); C07C 237/42 The present invention provides eW antiviral compounds and (2013.01); C07C3II/08 (2013.01); C07C pharmacological compositions comprising these CW CO 31 1/21 (2013.01); C07C 2101/14 (2013.01) pounds and their use in the prophylaxis, prevention and treat USPC ...... 514/539.514/563: 560/48. 562,457 ment ofOT viralV1ral infections,1n Iecuons, particularlypart1cularly adenoV1rusad andd herpherpes (58) Field ofOSSO Classification SSea h virus infections. None See application file for complete search history. 13 Claims, 5 Drawing Sheets US 8,993,622 B2 Page 2

(56) References Cited STN International Registry File, RN:905111-57-1, RN:905111-47 9, copyright 2013, 3 pages. OTHER PUBLICATIONS Wadell et al., “Adenoviruses.” p. 970-982 in: Murray et al. (eds.), Manual of Clinical Microbiology, 1999, 7th ed. ASM Press. Wadell, “Molecular epidemiology of human adenoviruses. Current Huth et al., “NMR-driven discovery of benzoylanthranilic acid topics in microbiology and immunology," 1984, 110:16 pages. inhibitors of far upstream element binding protein binding to the International Search Report and Written Opinion in International human oncogene c-myc promoter.” J. Med. Chem., 2004, 47:4851 Application No. PCT/SE2011/050724, report completed Sep. 30, 4857. 2011, 5 pages. Izzedine et al., “Antiviral Drug-Induced Nephrotoxicity.” American International Preliminary Report on Patentability in International Application No. PCT/SE2011/050724, issued Dec. 14, 2012, 9 Journal of Kidney Diseases, 2005, 45:804-817. pageS. Janner et al., “Fatal adenovirus infection in a child with acquired Adenoviridae, Wikipedia, the free encyclopedia, posted on or before immunodeficiency syndrome.” Pediatr. Infect. Dis. J., 1990, 9:434 Feb. 5, 2004, retrieved Jul. 24, 2014. http://en.wikipedia.org/wiki/ 436. Adenoviridae, 8 pages. Kojaoghlanian et al., “The impact of adenovirus infection on the Amide, Wikipedia, the free encyclopedia, posted on or before Aug. immunocompromised host.” Rey Med Virol., 2003, 13:155-71. 14, 2001, retrieved Jul. 9, 2014, http://en.wikipedia.org/wiki/Amide, Kumar and Mukerjee, “Condensation of 2-Methyl-3, 1-benzoxazin 6 pages. 4-one with Schiff Bases: Simultaneous introduction of arylidene and Coxsackievirus, Wikipedia, the free encyclopedia, posted on or amine Moieties.” Indian J. Chem., 1982, 21B:24-26. before Aug. 22, 2003, retrieved Jul. 24, 2014. http://en.wikipedia.org/ Lee and Ahn, "Reactions of amides with potassium permanganate in wiki/Coxsackievirus, 4 pages. neutral aqueous solution.” Journal of Organic Chemistry, 1989, DNA Virus, Wikipedia, the free encyclopedia, posted on or before 54:3744-3747. Nov. 5, 2001, retrieved Jul. 24, 2014. http://en.wikipedia.org/wiki/ Leruez-Ville et al., “Description of an adenovirus A31 outbreak in a DNA , 17 pages. paediatric haematology unit.” Bone Marrow Transplant, 2006, Herpes Simplex Virus, Wikipedia, the free encyclopedia, posted on or 38:23-28. before Aug. 3, 2002, retrieved Jul. 24, 2014. http://en.wikipedia.org/ Mei et al., “Two Closely Related Adenovirus Genome Types with wiki/Herpes simplex virus, 12 pages. Kidney or Respiratory Tract Tropism Differ in Their Binding to Reduction of Nitro Compounds, Wikipedia, the free encyclopedia, Epithelial Cells of Various Origins.” Virology, 1998, 240:254-266. posted on or before Nov. 24, 2006, retrieved Jul. 9, 2014. http://en. Morfin et al., “In vitro susceptibility of adenovirus to antiviral drugs wikipedia.org/wiki/Reduction of nitro compounds, 4 pages. is species-dependent.” Antiviral Therapy, 2005, 10:225-229. RNA Virus, Wikipedia, the free encyclopedia, posted on or before, Nair et al., “Reaction of anthranilic acid with mesyl chloride.” Indian retrieved Jul. 24, 2014, http://en.wikipedia.org/wiki/RNA virus, 9 Journal of Chemistry, Section B: Organic Chemistry Including pageS. Medicinal Chemistry, 1979, 17B:276-277. Section 12.4: Esters, UC-Davis CHEMWiki, retrieved on Jul. 10, Sandberg et al., "Replication-Competent Adl lip Vector (RCAdl lp) 2014, http://chemwiki.ucdavis.edu/Organic Chemistry/Organic Efficiently Transduces and Replicates in Hormone-Refractory Meta Chemistry With a Biological Emphasis, Chapter 12%3A static Prostate Cancer Cells.” Human Gene Therapy, 2009, 20:361 Acyl Substitution reactions/Section 12.4%3A Esters, 6 pages. 373. Trimethylsilyldiazomethane, Wikipedia the free encyclopedia, Segerman et al., “There are two different species Badenovirus recep posted on or before Nov. 10, 2005, retrieved Jul. 24, 2014. http://en. tors: shBAR, common to species B1 and B2 adenoviruses, and shB2AR, wikipedia.org/wiki/Trimethylsilyldiazomethane, 3 pages. exclusively used by species B2 adenoviruses,” Journal of Virology, 2003, 77:1157-1162. * cited by examiner U.S. Patent Mar. 31, 2015 Sheet 1 of 5 US 8,993,622 B2

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O 10 20 30 A02 (uM) US 8,993,622 B2 1. 2 ANTIVIRAL COMPOUNDS 2000. Outcome and clinical course of 100 patients with aden Ovirus infection following bone marrow transplantation. CROSS-REFERENCE TO RELATED Bone Marrow Transplantation 26:1333-1338). APPLICATIONS A number of different Ads have been isolated from immu nocompromised patients, most frequently from species A, B, This application is a National Stage application under 35 or C (Kojaoghlanian et al. 2003. The impact of adenovirus U.S.C. S371 of International Application No. PCT/SE2011/ infection on the immunocompromised host. Rev Med Virol 050724, having an International Filing Date of Jun. 13, 2011, 13:155-71). Species B serotypes are predominantly associ which claims the benefit of Swedish Application Serial No. ated with renal syndromes and species C serotypes are usu SE1000623-7, filed Jun. 11, 2010 and Swedish Application 10 ally associated with hepatitis. In recent years, infections with Serial No. SE1000624-5, filed Jun. 11, 2010. The disclosure Ad Serotype 31 (species A) have been increasingly reported of the prior applications are considered part of (and are incor and they often occur in patients with infections involving porated by reference in) the disclosure of this application. multiple Ad serotypes, occasionally with lethal outcome (Leruez-Ville et al. 2006. Description of an adenovirus A31 FIELD OF THE INVENTION 15 outbreak in a paediatric haematology unit. Bone Marrow Transplant 38:23-28.). The present invention relates to new antiviral compounds There are no approved specific antiviral compounds for and pharmacological compositions and their use in the pro treatment of Ad infections available today. Established anti phylaxis, prevention and treatment of viral infections, par viral drugs including cidofovir, ribavirin, and ganciclovir ticularly adenovirus and herpes virus infections. have been tested for anti-adenoviral activity both in in vitro experiments and in the clinical setting. The clinical efficacy is BACKGROUND TO THE INVENTION inconclusive, since varying results have been reported for the drugs. Of the approved drugs, Cidofovir appears to be most Adenovirus infections are widespread in Society and are effective against Ads (Morfin et al. 2005. In vitro susceptibil occasionally associated with severe, but rarely with life 25 ity of adenovirus to antiviral drugs is species-dependent. threatening, disease in otherwise healthy individuals. In con Antiviral Therapy 10:225-229). However, cidofovir is asso trast, adenovirus infections present a real threat to immuno ciated with nephrotoxicity and acute renal failure (IZZedline et compromised individuals and can result in disseminated and al. 2005. Antiviral Drug-Induced Nephrotoxicity. American fatal disease. The number of patients undergoing immuno Journal of Kidney Diseases 45:804-817). The need for new Suppressive therapy for Solid organ or hematopoietic stem 30 anti-adenoviral Substances is clearly increasing due to the cell transplantation is steadily increasing, as is the number of large number of immuno-compromised patients undergoing AIDS patients, and this makes the problem of adenovirus transplantations, and also patients suffering from AIDS or infections even more urgent to solve. There is no formally with genetic immunodeficiencies. approved treatment of adenovirus infections today, and exist Herpes viruses are divided into alpha, beta and gamma ing antiviral agents evaluated for their anti-adenoviral effect 35 herpes viruses. All studied herpes viruses enter a latent state give inconsistent results. after the primary infection. This means that they later in life Human adenoviruses are very common pathogens and can be activated and cause morbidity and in immunocompro comprise at least 51 different serotypes; together, these form mized persons even mortality. Herpes simplex virus (HSV)1 six different species, A-F. Adenoviruses (Ads) are associated & 2 and varicellae virus are the members of alpha herpes with a wide variety of clinical symptoms in humans. Such as 40 viruses. The U-L23 gene of HSV encodes a nucleosidekinase upper respiratory illness, acute respiratory disease, gastroen also called thymidine kinase. This gene product is targeted by teritis, hemorrhagic cystitis, and even keratoconjunctivitis acyclovir and its derivatives. This is an effective therapeutic (Wadell, G. 1984. Molecular epidemiology of human aden drug against HSV 1 & 2 and it has also been used against Oviruses. Current topics in microbiology and immunology Varicella-Zoster (VZ) infections in adults and immunocom 110:191-220. Wadell et al. 1999. Adenoviruses, p. 970-982. 45 promized children and adults. There is however a need for In. Murray et al. (eds.), Manual of Clinical Microbiology, 7th new alternative antiviral drugs that can be used on acyclovir ed. ASM Press.). resistant strains and viruses that do not express a thymidine These infections can result in severe disease, although an kinase. Ad infection is most commonly self-limited in otherwise Screening of large compound collections with purified pro healthy individuals. The problem is much more pronounced 50 tein or whole cell-based assays, i.e. high-throughput screen in immunocompromised individuals. This group is steadily ing (HTS), is a common method to identify biologically growing as a result of increasing numbers of AIDS patients active compounds. Cell-based approaches are commonly and patients undergoing immunosuppressive therapy for more labor-intensive but have the benefit of a wider screening Solid organ or hematopoietic stem cell transplantation, and without the limitation of having a preconceived idea of the also because of increased Survival times of these patients. 55 mechanism of action. We have developed a unique whole-cell Immunocompromised individuals are at high risk of devel reporter gene assay based on a GFP-expressing replication oping disseminated disease and multiple organ failure, and an competent Ad vector (Sandberg et al. 2009. Replication Ad infection can become a serious life-threatening disease Competent Ad11p Vector (RCAd11p) Efficiently Transduces (Janney et al. 1990. Fatal adenovirus infection in a child with and Replicates in Hormone-Refractory Metastatic Prostate acquired immunodeficiency syndrome. Pediatr. Infect. Dis. J. 60 Cancer Cells. Human Gene Therapy 20:361-373). The assay 9:434-436). In immunocompromised children, Ads are an can identify compounds that directly or indirectly affectaden important cause of disease and case fatality rates of above Oviral protein expression. This assay was used to Screen 50% have been reported (Hierholzer 1992. Adenoviruses in approximately 9,800 compounds, resulting in a number of the immunocompromised host. Clinical Microbiology compounds that have an inhibitory effect on Ads without Reviews 5:262-274). In pediatric bone marrow transplant 65 killing the host cells. The inhibitory effect was ascertained at (BMT) recipients the incidence of Ad infection is substan four different stages of the viral replication cycle. Here, we tially higher than in adult BMT recipients (Baldwin et al. describe the screening method and report on a novel inhibi US 8,993,622 B2 3 4 tors of Ad replication that is effective on Ad types represent each Re is independently selected from the group consisting ing the six species of human adenoviruses as well as on of hydrogen, —Csalkyl, -Calkenyl, —C-alkynyl, Herpes simplex virus 1 (HSV-1). —(CH2)COH; and US 2006/0035245 describes a method for identifying anti n is 0, 1, 2, 3, 4, or 5: HIV agents comprising for the use in the prophylaxis, prevention and/or treatment of i) determining the effect of the agent on the activity of Tn5 viral diseases. transponase, and Preferably one of X and X is —CO. NH-. ii) determining if the agent can inhibit HIV integrase. The Preferably both X and X are -CO. NH-. compound 2-2-(Benzoylamino)benzoylamino-benzoic Preferably A is selected from phenyl, naphthyl, and het acid (herein referred to as compound A02) was found to 10 eroaryl, unsubstituted or substituted with one to three sub inhibit Tn5 transponase, but failed to inhibit HIV integrase. stituents independently selected from halogen, oxo, nitro, hydroxy, —NRR, -CN. —CF, —OCF, —Calkyl, DESCRIPTION OF THE INVENTION —C alkenyl, -C alkynyl, -C alkyloxy; In one embodiment the present invention provides phar Preferably R is selected from —COH, and —CO, maceutical compositions comprising a compound of the gen 15 Calkyl. eral formula (I), (II) or (III) and pharmaceutical acceptable Preferred compounds are salts thereof: 2-2-(Benzoylamino)benzoylamino-benzoic acid—Com pound A02 2-2-(4-Methyl-benzoylamino)benzoylamino-benzoic acid—Compound 12c A Formula (I) 2-3-(Benzoylamino)benzoylamino-benzamide-Com Yx R pound 12d 2-3-(2-Methyl-benzoylamino)benzoylamino-benzoic acid—Compound 12e R.-- || || --Rs 25 2-2-(4-Methoxy-benzoylamino)benzoylamino-benzoic N-xr r 2 acid—Compound 12f R3 R4 4-Methyl-N-(2-(2-methylphenyl)carbamoylphenyl-ben Formula (II) Zamide Compound 12g R Ethyl 2-3-(benzoylamino)benzoylaminobenzoate Com 30 pound 7c 1 Sen-S1s 2-3-(Benzoylamino)benzoylaminobenzoic acid—Com pound 8c "NS-X | 21 HR, Ethyl 2-4-(benzoylamino)benzoylaminobenzoate Com R3 R4 pound 7d Formula (III) 35 2-4-(Benzoylamino)benzoylaminobenzoic acid—Com R pound 8d R 2-Benzoylamino-N-phenyl-benzamide Compound 11 2-2-(Acetylamino)benzoylaminobenzoic acid—Com Y pound 17a A | | | R. 40 2-2-(Methanesulfonylamino)benzoylaminobenzoic acid— s rR y Compound 17b 3 2-2-(p-Toluenesulfonylamino)benzoylaminobenzoic acid—Compound 17c wherein X is selected from —CO. NH , —CO. NH 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid— CH —SO. NH , SO NH , 45 Compound 17d X is selected from —CO. NH , —CH2—CO NH, 2-2-(Cyclohexanecarboxylamino)benzoylaminobenzoic CO. NH-CH , -SO. NH-, acid—Compound 17e A is selected from the group consisting of hydrogen, 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic —Csalkyl, -C-salkenyl, -Casalkynyl, -(CH),C,cy acid Compound 17f cloalkyl, -(CH2)Coheterocycloalkyl, -(CH2)phenyl, 50 2-2-(Benzylamino)benzoylaminobenzoic acid—Com —(CH2), naphthyl, -(CH2),aryl, -(CH2), heteroaryl, pound 17g —(CH2)NRR, -(CH2)N(R)COCsalkyl, -(CH2), 2-2-(2-Fluorobenzoylamino)-benzoylaminobenzoic COH, -(CH2)COCsalkyl, acid Compound 17h wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, 2-2-(3-Fluorobenzoylamino)-benzoylaminobenzoic aryl, phenyl, naphthyl, heteroaryl, and (CH) are unsubsti 55 acid Compound 17i tuted or substituted with one to three substituents indepen 2-2-(4-Fluorobenzoylamino)-benzoylaminobenzoic dently selected from halogen, oxo, nitro, hydroxy, —NRR, acid—Compound 17 —CN, —CF, —OCF. —Calkyl, -C alkenyl, —C- 2-2-(Phenylacetylamino)benzoylaminobenzoic acid— alkynyl. —C alkyloxy; Compound 17k R is selected from hydrogen, halogen, nitro, hydroxy, 60 Ethyl 2-2-(benzoylamino)benzoylaminobenzoate Com —COH, -COC-alkyl, -Calkyl, -C alkenyl, pound 23a —C alkynyl, -C alkyloxy, —CN, —CF, -OCF, tet Ethyl 2-2-(acetylamino)benzoylaminobenzoate Com razol-5-yl, CONRR: pound 22a R. R. R. and Rs are each independently selected from Ethyl 2-2-(trimethylacetylamino)benzoylaminoben hydrogen, halogen, oxo, nitro, hydroxy, —NRR, -CN. 65 Zoate—Compound 24a —CF, —OCF, —C alkyl, -C alkenyl, -C alky Ethyl 2-2-(cyclohexanecarboxylamino)benzoylamino nyl, -C alkyloxy; benzoate-Compound 25a US 8,993,622 B2 5 6 Ethyl 2-2-(phenylacetylamino)benzoylaminobenzoate— ing Enterovirus infections, Reovirus infections including Compound 26a Rotavirus infections. Togavirus infections including Rubivi Ethyl 2-2-(4-carboxybutanoylamido)benzoylaminoben rus infections. Retrovirus infections including lentivirus Zoate—Compound 27a infections, such as HIV infections. Ethyl 2-2-(methanesulfonylamino)benzoylaminoben Preferably the viral disease can be selected from Adenovi Zoate—Compound 29a rus infections, Herpesvirus infections, Papillomavirus infec Ethyl 2-2-(p-toluenesulfonylamino)benzoylaminoben tions, Parvovirus infections, Polyomavirus infections, Poxvi Zoate Compound 30a rus infections, Arbovirus infection, Arenavirus infections, Ethyl 2-2-(benzylamino)benzoylaminobenzoate Com Astrovirus infections, Birnavirus infections, Bunyavirus pound 40a 10 infections, Calicivirus infections, Coronavirus infections, 2-2-(Benzoylamino)benzoylaminobenzoic acid—Com Flavivirus infections, Hantavirus infections, Hepatitis virus pound 23b infections, including Hepatitis A, Hepatitis B. Hepatitis C, 2-2-(Acetylamino)benzoylaminobenzoic acid—Com and Hepatitis D. Mononegavirus infections, including Filovi pound 22b rus infections, Paramyxovirus infections, and Rhabdovirus 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid— 15 infections, Nidovirales Infections, Orthomyxoviridae infec Compound 24b tions including influenza virus infections, Picornavirus infec 2-2-(Cyclohexanecarboxylamino)benzoylaminobenzoic tions including Enterovirus infections, Reovirus infections acid Compound 25b including Rotavirus infections. Togavirus infections includ 2-2-(Phenylacetylamino)benzoylaminobenzoic acid— ing Rubivirus infections. Compound 26b More preferably the viral disease is an Adenovirus infec 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic tion, a Herpes virus infection or a Picornavirus infection. The acid Compound 27b herpes virus infections can be caused by HSV-1, HSV-2 and/ 2-2-(Methanesulfonylamino)benzoylaminobenzoic acid— or varicella Zoster virus. Compound 29b Most preferably the viral disease is an infection caused by 2-2-(p-Toluenesulfonylamino)benzoylaminobenzoic 25 an Adenovirus. acid Compound 30b In another embodiment the present invention provides new 2-2-(Benzylamino)benzoylaminobenzoic acid—Com compounds of the general formula (I), (II) or (III) and phar pound 40b maceutical acceptable salts thereof: 2-2-(benzoylamino)benzoylaminophenylacetic acid— Compound 23 30 2-2-(benzoylamino)phenylacetylaminobenzoic acid— Compound 28 A. Formula (I) 2-2-(2-fluorobenzoylamino)-benzoylamino-4-chloroben Yx R Zoic acid—Compound 35a 2-2-(2-fluorobenzoylamino)benzoylamino-5-chloroben 35 4N1 n-1s zoic acid Compound 35b 2-2-(2-fluorobenzoylamino)-benzoylamino-4-methoxy R N- HR: benzoic acid Compound 35c R 2-2-(2-fluorobenzoylamino)-benzoylamino-5-methoxy 3 R4 Formula (II) benzoic acid Compound 35d 40 R 2-2-(2-fluorobenzoylamino)-benzoylamino-4,5-difluo robenzoic acid Compound 35e 2-4-chloro-2-(2-fluorobenzoylamino)-benzoylaminoben R- HR zoic acid Compound 35f -*.2 irN-x r -4 5 2-5-chloro-2-(2-fluorobenzoylamino)-benzoylaminoben 45 R Zoic acid—Compound 35g 3 R4 Formula (III) 2-4-methoxy-2-(2-fluorobenzoylamino)-benzoylamino R benzoic acid Compound 35h R2 2-5-methoxy-2-(2-fluorobenzoylamino)-benzoylamino benzoic acid Compound 35i 50 e r X N 2-4,5-difluoro-2-(2-fluorobenzoylamino)-benzoylamino HR benzoic acid Compound 35 An X - R4 ué 5 The viral disease can be selected from infections caused by 3 RNA virus and/or DNA virus. Preferably the viral disease is an infection caused by DNA virus. 55 wherein X is selected from —CO. NH , —CO. NH The viral disease can be selected from Adenovirus infec CH , —SO. NH , SO. , —NH , tions, Herpesvirus infections, Papillomavirus infections, Par X is selected from —CO. NH , —CH2—CO NH, vovirus infections, Polyomavirus infections, Poxvirus infec CO. NH-CH , -SO. NH-, tions, Arbovirus infection, Arenavirus infections, Astrovirus A is selected from the group consisting of hydrogen, infections, Birnavirus infections, Bunyavirus infections, 60 —Cisalkyl, -C2-salkenyl, -C2-salkynyl, -(CH2)C-7cy Calicivirus infections, Coronavirus infections, Flavivirus cloalkyl, -(CH2)Cheterocycloalkyl, -(CH), phenyl, infections, Hantavirus infections, Hepatitis virus infections, (CH2), naphthyl, -(CH2)aryl, -(CH2), heteroaryl, including Hepatitis A, Hepatitis B, Hepatitis C, and Hepatitis —(CH),NRR —(CH),NRRCOCsalkyl, -(CH), D. Mononegavirus infections, including Filovirus infections, COH, -(CH2)COCsalkyl, Paramyxovirus infections, and Rhabdovirus infections, 65 wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, Nidovirales Infections, Orthomyxoviridae infections includ aryl, phenyl, naphthyl, heteroaryl, and (CH) are unsubsti ing influenza virus infections, Picornavirus infections includ tuted or substituted with one to three substituents indepen US 8,993,622 B2 7 8 dently selected from halogen, oxo, nitro, hydroxy, —NRR, Ethyl 2-2-(phenylacetylamino)benzoylaminobenzoate— —CN. —CF, —OCF. —Calkyl, -C alkenyl, —C. Compound 26a alkynyl. —C alkyloxy; Ethyl 2-2-(4-carboxybutanoylamido)benzoylaminoben R is selected from hydrogen, halogen, nitro, hydroxy, Zoate—Compound 27a —COH, -COC-alkyl, -Calkyl, -C alkenyl, Ethyl 2-2-(methanesulfonylamino)benzoylaminoben —C alkynyl, -C alkyloxy, —CN, —CF, -OCF, tet Zoate—Compound 29a razol-5-yl, -CONRR: Ethyl 2-2-(p-toluenesulfonylamino)benzoylaminoben R. R. R. and Rs are each independently selected from Zoate Compound 30a hydrogen, halogen, oxo, nitro, hydroxy, —NRR —CN. Ethyl 2-2-(benzylamino)benzoylaminobenzoate Com —CF, —OCF, —C alkyl, -C alkenyl, -C alky 10 pound 40a nyl, -C alkyloxy, 2-2-(Benzoylamino)benzoylaminobenzoic acid—Com each Re is independently selected from the group consisting pound 23b of hydrogen, —Csalkyl, —C-salkenyl, —C-salkynyl, 2-2-(Acetylamino)benzoylaminobenzoic acid—Com —(CH),COH; and pound 22b n is 0, 1, 2, 3, 4, or 5: 15 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid— further provided that when X and X are —CO. NH and Compound 24b R is —COOH then A is not phenyl, 3-methylphenyl-, 4-me 2-2-(Cyclohexanecarboxylamino)benzoylaminobenzoic thylphenyl, or 4-methoxyphenyl-, and when X and X are acid Compound 25b —CO. NH and R is methyl then A is not3-methylphenyl. 2-2-(Phenylacetylamino)benzoylaminobenzoic acid— Preferably both X and X are -CO. NH-. Compound 26b Preferably A is selected from phenyl, naphthyl, and het 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic eroaryl, unsubstituted or substituted with one to three sub acid Compound 27b stituents independently selected from halogen, oxo, nitro, 2-2-(Methanesulfonylamino)benzoylaminobenzoic acid— hydroxy, —N(R), —CN, —CF, —OCF. —Calkyl, Compound 29b —C alkenyl, -C alkynyl, -C alkyloxy; 25 2-2-(p-Toluenesulfonylamino)benzoylaminobenzoic Preferably R is selected from —COH, —COC-alkyl, acid Compound 30b Most preferably the compounds are selected from the 2-2-(Benzylamino)benzoylaminobenzoic acid—Com group consisting of pound 40b Ethyl 2-3-(benzoylamino)benzoylaminobenzoate Com 2-2-(benzoylamino)benzoylaminophenylacetic acid— pound 7c 30 Compound 23 2-3-(Benzoylamino)benzoylaminobenzoic acid—Com 2-2-(benzoylamino)phenylacetylaminobenzoic acid— pound 8c Compound 28 Ethyl 2-4-(benzoylamino)benzoylaminobenzoate Com 2-2-(2-fluorobenzoylamino)-benzoylamino-4-chloroben pound 7d Zoic acid—Compound 35a 2-4-(Benzoylamino)benzoylaminobenzoic acid—Com 35 2-2-(2-fluorobenzoylamino)benzoylamino-5-chloroben pound 8d Zoic acid Compound 35b 2-Benzoylamino-N-phenyl-benzamide Compound 11 2-2-(2-fluorobenzoylamino)-benzoylamino-4-methoxy 2-2-(Acetylamino)benzoylaminobenzoic acid—Com benzoic acid Compound 35c pound 17a 2-2-(2-fluorobenzoylamino)-benzoylamino-5-methoxy 2-2-(Methanesulfonylamino)benzoylaminobenzoic acid— 40 benzoic acid Compound 35d Compound 17b 2-2-(2-fluorobenzoylamino)-benzoylamino-4,5-difluo 2-2-(p-Toluenesulfonylamino)benzoylaminobenzoic robenzoic acid Compound 35e acid—Compound 17c 2-4-chloro-2-(2-fluorobenzoylamino)-benzoylaminoben 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid— Zoic acid Compound 35f Compound 17d 45 2-5-chloro-2-(2-fluorobenzoylamino)-benzoylaminoben 2-2-(Cyclohexanecarboxylamino)benzoylaminobenzoic Zoic acid—Compound 35g acid—Compound 17e 2-4-methoxy-2-(2-fluorobenzoylamino)-benzoylamino 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic benzoic acid Compound 35h acid Compound 17f 2-5-methoxy-2-(2-fluorobenzoylamino)-benzoylamino 2-2-(Benzylamino)benzoylaminobenzoic acid—Com 50 benzoic acid Compound 35i pound 17g 2-4,5-difluoro-2-(2-fluorobenzoylamino)-benzoylamino 2-2-(2-Fluorobenzoylamino)-benzoylaminobenzoic benzoic acid—Compound 35. acid Compound 17h In another embodiment the present invention provides 2-2-(3-Fluorobenzoylamino)-benzoylaminobenzoic pharmaceutical compositions comprising a compound acid Compound 17i 55 according to the invention. 2-2-(4-Fluorobenzoylamino)-benzoylaminobenzoic In another embodiment the present invention provides acid—Compound 17 pharmaceutical compositions comprising a compound 2-2-(Phenylacetylamino)benzoylaminobenzoic acid— according to the invention for the use in the prophylaxis, Compound 17k prevention and/or treatment of viral diseases. Ethyl 2-2-(benzoylamino)benzoylaminobenzoate Com 60 The viral disease can be selected from infections caused by pound 23a RNA virus and DNA virus. Preferably the viral disease is an Ethyl 2-2-(acetylamino)benzoylaminobenzoate Com infection caused by DNA virus. pound 22a The viral disease can be selected from Adenovirus infec Ethyl 2-2-(trimethylacetylamino)benzoylaminoben tions, Herpesvirus infections, Papillomavirus infections, Par Zoate—Compound 24a 65 vovirus infections, Polyomavirus infections, Poxvirus infec Ethyl 2-2-(cyclohexanecarboxylamino)benzoylamino tions, Arbovirus infection, Arenavirus infections, Astrovirus benzoate—Compound 25a infections, Birnavirus infections, Bunyavirus infections, US 8,993,622 B2 10 Calicivirus infections, Coronavirus infections, Flavivirus tions including influenza virus infections, Picornavirus infec infections, Hantavirus infections, Hepatitis virus infections, tions including Enterovirus infections, Reovirus infections including Hepatitis A, Hepatitis B, Hepatitis C, and Hepatitis including Rotavirus infections, Togavirus infections includ D. Mononegavirus infections, including Filovirus infections, ing Rubivirus infections. Paramyxovirus infections, and Rhabdovirus infections, More preferably the viral disease is an Adenovirus infec Nidovirales Infections, Orthomyxoviridae infections includ tion, a Herpes virus infection or a Picornavirus infection. The ing influenza virus infections, Picornavirus infections includ herpes virus infections can be caused by HSV-1, HSV-2 and/ ing Enterovirus infections, Reovirus infections including or varicella Zoster virus. Rotavirus infections, Togavirus infections including Rubivi Most preferably the viral disease is an infection caused by rus infections. Retrovirus infections including lentivirus 10 an Adenovirus. infections, such as HIV infections, Preferably the viral disease can be selected from Adenovi DESCRIPTION OF DRAWINGS rus infections, Herpesvirus infections, Papillomavirus infec tions, Parvovirus infections, Polyomavirus infections, Poxvi FIG.1. Distribution of the inhibitory effect and the toxicity rus infections, Arbovirus infection, Arenavirus infections, 15 of the 24 compounds verified as Ad inhibitors at 25uM. Astrovirus infections, Birnavirus infections, Bunyavirus FIG. 2. Dose-response for A02 inhibition of GFP expres infections, Calicivirus infections, Coronavirus infections, sion from the RCAd11pGFP vector in K562 cells. Fluores Flavivirus infections, Hantavirus infections, Hepatitis virus cence intensity was measured after 24 h incubation with infections, including Hepatitis A, Hepatitis B. Hepatitis C, compound A02 and vector. and Hepatitis D. Mononegavirus infections, including Filovi FIG.3. Titration of the effect of A02 on Ad5, Ad11p and the rus infections, Paramyxovirus infections, and Rhabdovirus toxic effect in A549 cells following 24h incubation with virus infections, Nidovirales Infections, Orthomyxoviridae infec and/or compound A02. ICs for Ad5 and Ad 11p was 3.7 LM tions including influenza virus infections, Picornavirus infec and 2.9 uM, respectively. CCs for compound A02 in A549 tions including Enterovirus infections, Reovirus infections cells was 199 uM. ICs is the concentration at which the Ad including Rotavirus infections, Togavirus infections includ 25 replication is inhibited by 50% as determined by qPCR and ing Rubivirus infections. CCs is the concentration at which the cytotoxicity is 50%, More preferably the viral disease is an Adenovirus infec i.e. 50% of the cells are viable, as determined by the XTT tion, a Herpes virus infection or a Picornavirus infection. The assay. Error bars represent the standard deviation of the herpes virus infections can be caused by HSV-1, HSV-2 and/ means from three independent duplicate experiments. or varicella Zoster virus. 30 FIG. 4. A. Binding of S-labelled Ad5 to A549 cells in the Most preferably the viral disease is an infection caused by presence of A02. Error bars represent the standard deviation an Adenovirus. of the means from three independent duplicate experiments. In another embodiment the present invention provides B. Flowcytometry assay detecting Ad5 (grey bars) and Ad 11 methods for the prophylaxis, prevention and/or treatment of a (checkered bars) hexon protein following 24 h incubation viral disease, the methods comprising administering a com 35 with virus and compound A02. Error bars represent the stan pound according to the invention to a subject in need of Such dard deviation of the means from two independent duplicate treatment. experiments. The viral disease can be selected from infections caused by FIG. 5. Dose-response for A02 inhibition of herpes sim RNA virus and DNA virus. Preferably the viral disease is an plex 1 replication in GMK cells as determined by qPCR. infection caused by DNA virus. 40 The viral disease can be selected from Adenovirus infec DEFINITIONS tions, Herpesvirus infections, Papillomavirus infections, Par vovirus infections, Polyomavirus infections, Poxvirus infec As used herein, alkyl means an alkyl group being straight tions, Arbovirus infection, Arenavirus infections, Astrovirus or branched. By —Csalkyl is meant an alkyl group having infections, Birnavirus infections, Bunyavirus infections, 45 from 1 to 8 carbon atoms. Examples include methyl, ethyl, Calicivirus infections, Coronavirus infections, Flavivirus propyl, isopropyl, butyl, hexyl, heptyl, octyl, and the like. The infections, Hantavirus infections, Hepatitis virus infections, alkyl groups may be unsubstituted or Substituted. including Hepatitis A, Hepatitis B, Hepatitis C, and Hepatitis As used herein, alkyloxy means an alkyloxy group being D. Mononegavirus infections, including Filovirus infections, straight or branched. By —C alkyloxy is meant an alkyloxy Paramyxovirus infections, and Rhabdovirus infections, 50 group having from 1 to 6 carbon atoms. Examples include Nidovirales Infections, Orthomyxoviridae infections includ methoxy, ethoxy, propoxy, isopropoxy, butoxy, hexoxy, and ing influenza virus infections, Picornavirus infections includ the like. The alkoxy groups may be unsubstituted or substi ing Enterovirus infections, Reovirus infections including tuted. Rotavirus infections, Togavirus infections including Rubivi As used herein, alkenyl means an alkenyl group being rus infections. Retrovirus infections including lentivirus 55 straight or branched. By —Cs alkenyl is meant an alkenyl infections, such as HIV infections, group having from 2 to 8 carbon atoms. Examples include Preferably the viral disease can be selected from Adenovi ethenyl, propenyl, isopropenyl, butenyl, hexenyl, heptyl, hep rus infections, Herpesvirus infections, Papillomavirus infec tenyl, octenyl, and the like. The alkenyl groups may be unsub tions, Parvovirus infections, Polyomavirus infections, Poxvi stituted or substituted. rus infections, Arbovirus infection, Arenavirus infections, 60 As used herein, alkynyl means an alkynyl group being Astrovirus infections, Birnavirus infections, Bunyavirus straight or branched. By —Cs alkynyl is meant an alkynyl infections, Calicivirus infections, Coronavirus infections, group having from 2 to 8 carbon atoms. Examples include Flavivirus infections, Hantavirus infections, Hepatitis virus ethynyl, propynyl, butynyl, hexynyl, heptynyl, octynyl, and infections, including Hepatitis A, Hepatitis B. Hepatitis C, the like. The alkynyl groups may be unsubstituted or substi and Hepatitis D. Mononegavirus infections, including Filovi 65 tuted. rus infections, Paramyxovirus infections, and Rhabdovirus As used herein, C-cycloalkyl means a cycloalkyl having infections, Nidovirales Infections, Orthomyxoviridae infec from 3 to 7 carbon atoms. Examples include cyclopropyl. US 8,993,622 B2 11 12 cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the intrathoracially, intravenously, epidurally, intrathecally, like. The cycloalkyl groups may be unsubstituted or Substi intracerebroVentricularly and by injection into the joints. tuted. In one embodiment of the present invention, the route of The aryl moieties described here, either alone or with vari administration may be oral, intravenous or intramuscular. ous substituents, contain from 6 to 15 carbon atoms and 5 The dosage will depend on the route of administration, the include phenyl, 1-naphthalenyl and 2-maphthalenyl. severity of the disease, age and weight of the patient and other The heteroaryl moieties described here, either alone or factors normally considered by the attending physician, when determining the individual regimen and dosage level at the with various substituents, contain from 3 to 15 carbonatoms most appropriate for a particular patient. and include furans, thiophenes, indoles, furyl, pyridyl, thie For preparing pharmaceutical compositions from the com nyl, tryptophane and the like. 10 pounds of the present invention, inert, pharmaceutically As used herein, the term “halogen denotes a fluoro, acceptable carriers can be either solid or liquid. Solid form chloro, bromo, or iodo group. preparations include powders, tablets, dispersable granules, The term tetrazol-5-yl includes 1H-tetrazol-5-yl, 2H-tetra capsules, cachets, and Suppositories. Zol-5-yl, and 5H-tetrazol-5-yl. 15 A solid carrier can be one or more Substances, which may As used herein, when two or more groups are used in also act as diluents, flavouring agents, solubilizers, lubri connection with each other, it means that each group is Sub cants, Suspending agents, binders, or tablet disintegrating stituted by the immediately preceding group. For instance, agents; it can also be an encapsulating material. trifluoromethylphenyl means a phenyl group Substituted by a In powders, the carrier is a finely divided solid, which is in trifluoromethyl group. 2O mixture with the finely divided compound of the present As used herein, the terms prevent or prevention and pro invention, or the active component. In tablets, the active com phylaxis are given their ordinary meaning and thus means the ponent is mixed with the carrier having the necessary binding avoidance or alleviation of the serious consequences of a properties in Suitable proportions and compacted in the shape disease or a side-effect by early detection. and size desired. As used herein, the term “mammal” means a human oran 25 For preparing Suppository compositions, a low-melting animal Such as monkeys, primates, dogs, cats, horses, cows, wax Such as a mixture of fatty acid glycerides and cocoa etc. butter is first melted and the active ingredient is dispersed As used herein, the single enantiomers, racemic mixtures therein by, for example, stirring. The molten homogenous and unequal mixtures of two enantiomers are within the scope mixture is then poured into conveniently sized moulds and of the invention, where such isomers exist. It should be under- 30 allowed to cool and solidify. stood that all the diastereomeric forms possible (pure enanti Suitable carriers are magnesium carbonate, magnesium omers, racemic mixtures and unequal mixtures of two or Stearate, talc, lactose, sugar, pectin, dextrin, starch, methyl more diastereomers), tautomers, and atropisomers are within cellulose, sodium carboxymethyl cellulose, a low-melting the scope of the invention. wax, cocoa butter, and the like. As used herein, the term “pharmaceutically acceptable 35 The term composition is also intended to include the for salts' includes acid addition salts and base addition salts. mulation of the active component with encapsulating material Such salts may be formed by conventional means, for as a carrier providing a capsule in which the active component example by reaction of a free acid or a free base form of a (with or without other carriers) is surrounded by a carrier compound of the invention with one or more equivalents of an which is thus in association with it. Similarly, cachets are appropriate acid or base, optionally in a solvent, or in a 40 included. medium in which the salt is insoluble, followed by removal of Tablets, powders, cachets, and capsules can be used as said solvent, or said medium, using standard techniques (e.g. Solid dosage forms suitable for oral administration. in vacuo or by freeze-drying). Salts may also be prepared by Liquid form compositions include solutions, Suspensions, exchanging a counter-ion of a compound of the invention in and emulsions. For example, Sterile water or propylene glycol the form of a salt with another counter-ion using a Suitable ion 45 Solutions of the active compounds may be liquid preparations exchange resin. Suitable for parenteral administration. Liquid compositions Suitable acids are non-toxic and include eg, but are not can also be formulated in solution in aqueous polyethylene limited to, hydrochloric acid, hydrobromic acid, hydroiodic glycol Solution. acid, Sulphuric acid, nitric acid, acetic acid, citric acid, ascor Aqueous solutions for oral administration can be prepared bic acid, lactic acid, malic acid, and tartaric acid. Suitable 50 by dissolving the active component in water and adding Suit bases are non-toxic and include eg, but are not limited to, able colorants, flavouring agents, stabilizers, and thickening Sodium hydroxide, potassium hydroxide, lithium hydroxide, agents as desired. Aqueous solutions for oral use can be made ammonia, methylamine, dimethylamine, trimethylamine, by dispersing the finely divided active component in water and triethylamine. together with a viscous material Such as natural synthetic In the context of the present specification, the term “treat- 55 gums, resins, methyl cellulose, sodium carboxymethyl cellu ment also includes “prophylaxis' unless there are specific lose, and other Suspending agents known to the pharmaceu indications to the contrary. The term “treatment within the tical formulation art. context of the present invention further encompasses to Depending on the mode of administration, the pharmaceu administer an effective amount of a compound of the present tical composition will according to one embodiment of the invention, to mitigate either a pre-existing disease state, acute 60 present invention include 0.05% to 99% weight (percent by or chronic, or a recurring condition. This definition also weight), according to an alternative embodiment from 0.10 to encompasses prophylactic therapies for prevention of recur 50% weight, of the compound of the present invention, all ring condition and continued therapy for chronic disorders. percentages by weight being based on total composition. The compounds of the present invention may be adminis A therapeutically effective amount for the practice of the tered in the form of a conventional pharmaceutical composi- 65 present invention may be determined, by the use of known tion by any route including orally, intramuscularly, Subcuta criteria including the age, weight and response of the indi neously, topically, intranasally, intraperitoneally, vidual patient, and interpreted within the context of the dis US 8,993,622 B2 13 14 ease which is being treated or which is being prevented, by analysis was carried out at 212 nm and mass spectra were one of ordinary skills in the art. recorded by detecting negative (EST) molecular ions with an The above-mentioned Subject-matter for a pharmaceutical electro-spray Waters Micromass ZG 2000 instrument. The composition comprising a compound according to the present same LC-MS system was also used for purification with a invention is applied analogously for a pharmaceutical com preparativeXTerra R. Prep MSCs 5um 19-x50-mm column position comprising a combination according to the present and an HO/acetonitrile eluent system. H and 'C NMR invention. spectra were recorded in DMSO-d (residual DMSO-ds, Another object of the present invention is a compound as 8, 2.50 ppm and DMSO-d 8–39.51 ppm as internal stan disclosed above for use in medicine. dards) using a Bruker DRX-400 spectrometer. Another object of the present invention is a pharmaceutical 10 formulation comprising a compound as disclosed above in Screening. admixture with pharmaceutically acceptable adjuvants, dilu The screening assay is based on GFP expression from the ents and/or carriers. RCAd11pGFP vector in a K562 cell system. The 9,800 com EXAMPLES 15 pounds were screened for their ability to inhibit emitted fluo rescence and hence expression of the adenoviral genome. To Example 1 be considered as a potential hit, the compound had to decrease the intensity of fluorescence by more than 80% and kill no Screening for Inhibition of Viral Replication more than 50% of the cells. The primary hits of the screening procedure were 408 distinct compounds that showed proper Viruses and Vector The RCAd11 p.GFP vector used in this study is a replica ties of inhibition of RCAd11pGFP expression in K562 cells, tion-competent Advector (Sandberg et al. 2009. Replication representing a hit rate of around 4%. None of the compounds Competent Ad11p Vector (RCAd11p) Efficiently Transduces selected for further study were autofluorescent. and Replicates in Hormone-Refractory Metastatic Prostate 25 Validation of Hits. Cancer Cells. Human Gene Therapy 20:361-373). Ads used in this study were Ad5 (strain F2853-5b), Ad11p To verify the hits and to exclude false positives, the com (p-prototype, strain Slobitski), AdA (strain RI-67), Ad31 pounds were serially diluted in 7 steps for dose-response (strain 1315/63), Ad37 (strain 1477), and Ad41 (strain Tak). analysis using the screening assay. Compounds that exhibited The viruses were propagated in A549 cells and purified on a 30 at least 40% inhibition of fluorescence intensity at 25uM or discontinuous CsCl gradient as described previously (Mei et that had very low toxicity were considered verified hits. al. 1998. Two Closely Related Adenovirus Genome Types with Twenty-four compounds met these criteria. The distribution Kidney or Respiratory Tract Tropism Differ in Their Binding of fluorescence inhibition and cellular toxicity detected in the to Epithelial Cells of Various Origins. Virology 240:254-266). screening process of these 24 hits is Summarized in FIG. 1. The virion band was collected and density was measured on a 35 refractometer. Virions were desalted on a NAP-10 column One of the most efficient and least toxic compounds was A02, (GE healthcare, Buckinghamshire, UK) and eluted with 1.5 2-2-(benzoylamino)benzoylamino-benzoic acid. This ml 10 mM PBS. Virion concentration was determined by compound was evaluated further as a potential drug candi spectrophotometry; 1 OD unit (ODo-ODo) corresponds date. Serial dilution of compound A02 in the screening setup to 280 ug virions or 10' virus particles/ml. The identity of the 40 with RCAd11pGFP in K562 cells showed a clear dose adenovirus types was assessed according to their DNA response, with a 50% inhibitory concentration (ICs) of 28.6 restriction patterns. uM (FIG. 2). Cell Lines In the process of verifying the identity of the inhibitory A549 cells (oat cell carcinoma from the human lung; alveo compounds, combined analysis by liquid chromatography lar basal epithelial cells) were grown in Dulbecco's modified 45 Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, Mo.) and mass spectrometry of the purchased compounds was containing 0.75 g/l NaHCO, 20 mM HEPES (EuroClone, performed. It turned out that the A02 solution in fact con Milan, Italy), penicillin G (100 IU/ml) and streptomycinsul tained three different molecules. The three components were fate (100 lug/ml) combined (1xPEST) (Gibco, Carlsbad, separated by liquid chromatography and their structures were Calif.), and 5% fetal bovine serum (FBS) (Gibco) at 37° C. 50 confirmed by mass spectrometry and NMR spectroscopy. K562 is a non-adherent human erythroleukemia cell line. Compound A01 FSU (Foreskin Umea) is a diploid fibroblast cell line. K562 and FSU cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 0.75 g/l NaHCO, 20 mM HEPES (Euro Clone), 1xPEST (Gibco) and 5% fetal bovine serum (FBS) 55 (Gibco) at 37° C. Compounds NH O The compound collection screened was supplied Chem Bridge (San Diego, Calif.) and consisted of 9,800 low OH molecular weight organic compounds. The compounds were 60 dissolved in DMSO in 5 mM stock solutions and stored in 96-well plates sealed with heat-sealing films at room tem perature in the dark in a controlled dry atmosphere. Com pounds were analyzed by combined liquid chromatography For compound A01, the data were in agreement with those mass spectrometry (LC-MS) using a Waters HPLC system 65 published (Lee and Ahn 1989. Reactions of amides with equipped with an XTerra(RMSCs 5um 4.6-x50-mm column potassium permanganate in neutral aqueous solution. Jour and an HO/acetonitrile/formic acid eluent system using UV nal of Organic Chemistry. 54:3744-3747). US 8,993,622 B2 15 16 Compound A02 nuclease analysis. Journal Clinical Microbiology 39:498 505). Briefly, quantitative real-time PCR was carried out using a degenerate primer pair, Kadgen l (forward)—Kadgen 2 (reverse) (5'-CWT ACA TGCACA TCKCSGG-3 SEQ ID NO:1, and 5'-CRC GGG CRA AYT GCA CCA G-3' NH O SEQID NO:2, respectively); DNA technology A/S, Aarhus, Denmark). This primer pair is specific for the conserved region of the Ad hexon gene and can detect all human Ads. NH O Different FAM-TAMRA probes were used to quantitate Ads 10 from different species: AdB1B2 (5'-6-FAM-AGG ATG CTT OH CGG AGT ACC TGA GTC CGG-TAMRA-3 SEQ ID NO:3) for Ad11p (11) and AdC (5'-6-FAM-AGG ACGCCT CGG AGT ACC TGA GCC CCG-TAMRA-3 SEQ ID NO:4) for Ad5 (all from Applied Biosystems, Cheshire, UK). For Ads from species A, D, E and F the probe AdDF (5'-6- Analytical data for compound A02. "H NMR (400 MHz, 15 FAM-CCG GGCTCAGGTACT CCG AGG CGT CCT-3' DMSO-d): 8 7.25 (t, J=7.4 Hz, 1H), 7.37 (t, J–7.4 Hz, 1H), SEQ ID NO:5) was used (Applied Biosystems). Standard 7.60-7.71 (m, 5H), 7.97-8.01 (m,3H), 8.07 (dd, J=1.4 Hz, 7.8 curves ranging from 5 to 5x10 genome copies were gener HZ, 1H),8.53 (d. J=8.1 Hz, 1H), 8.60 (d. J=8.3 Hz, 1H), 11.87 ated by serial dilution of known amounts of full-length Ad5 or (s, 1H), 12.75 (brs, 1H), 13.80 (brs, 1H); C (100 MHz, Ad11 DNA. The Ad5 DNA standard was used for the Ad DF DMSO-d): & 117.8, 120.8, 1219, 123.0, 123.5, 123.8, 127.1, 2O probe System. The amplification was performed in a 25-yul 127.9, 128.9, 131.2, 132.0, 132.7, 134.1, 134.5, 139.0, 140.3, reaction mixture containing the following: 10ll Ad5 standard 1648, 167.0, 169.8. LCMS (m/z): M-H calcd for DNA or Ad11 standard DNA or 10ul DNA from samples, 2.5 CHNO), 359.10. found, 359.48. ul 10xTaq buffer, 5ul 25 mM MgCl2, 2.0 ul 2.5 mM dNTPs, Compound A03 1.0 ul 25uMKadgen1, 1.0 ul 25uMKadgen2, 0.29 Jul 15uM 25 probe AdB1B2 or probe AdDF or 1.0 ul 5uM probe AdC, 0.2 ul Ampli Taq Gold polymerase at 5 U?ul, 0.25 ul Amperase O uracil N-glycosylase (UNG), and 2.76 ul H2O for Ad 11p and 2.05 ul HO for Ad5 (Applied Biosystems, Roche Molecular Systems, Branchburg, N.J.). The program for the real-time 30 PCR was: 2 min at 50° C. to activate UNG, followed by C O amplification and quantitation (10 min at 95°C. and 40 cycles NH O of 15 sec at 95°C., 1 min at 60° C.). The efficiency of the real-time PCR assay was the same for both probe systems used (data not shown). To standardize the number of aden Oviral genome copies per cell, real-time PCR analysis was 35 performed on the same samples using the cellular RNase Pas C O a reference gene. The TaqMan(R) Rnase P Detection Reagents kit (20x mix containing primers and a FAM/TAMRA probe) (Applied Biosystems, Foster City, Calif.) was used for the s analysis. The PCR reaction mixture was otherwise the same 40 as with Ad primers and probes. Real-time PCR was per Analytical data for compound A03. "H NMR (400 MHz, formed in an ABI PRISM 7700 Sequence Detector (Applied DMSO-d): 8 7.21 (t, J=7.5 Hz, 1H), 7.34-7.43 (m, 2H), Biosystems) and analyzed with Sequence Detector V 1.7a 7.50-7.54 (m, 2H), 7.59-7.62 (m, 2H), 7.65-7.71 (m, 2H), software. 7.91-799 (m, 4H), 8.05 (d. J=7.7 Hz, 1H), 8.29 (d. J=8.1 Hz, The effect of the three molecules on the replication of Ad5 1H), 8.49 (d. J=8.2 Hz, 1H), 8.57 (d. J=8.2 Hz, 1H), 11.66 (s, 45 in A549 cells was assessed in a QPCR assay. A significant 1H), 11.76 (s, 1H), 12.62 (brs, 1H), 13.80 (brs, 1H). LCMS inhibitory effect on Ad5 replication could only be observed (m/z): M-H"I calcd for CHNOs), 478.14. found, for the original compound, A02. Neither the smaller (A01) 478.45. nor the larger (A03) molecule showed any anti-adenoviral Quantitative Real-Time PCR effect. Approximately 1x10 A549 cells were seeded in 24-well 50 Binding Experiments plates (Nunc) on the day before infection. On the day of A549 cells were washed twice and detached from the cul infection, the cells in one well were counted to establish the ture flask with 0.05% EDTA in PBS, resuspended in culture amount of virions to be added. The growth medium was medium, and allowed to recover for 1 h at 37° C. The cell removed and compound and virus were added simulta Suspension was centrifuged at room temperature at 450 g for neously to the cells in 700 ul DMEM with 0.75 g/l NaHCO, 5 min and resuspended in PBS containing 1% FBS and 0.01% 20 mM HEPES, 1xPEST, and 1% FBS. Compounds were NaN (PBS-FBS-NaNs); 200,000 cells per well were dis added in concentrations ranging from 0.5uM to 15uM. The pensed in a 96-well microtiter plate (Nunc). The plate was final concentration of DMSO was less than 1% in all samples. placed on ice and the compound was added to final concen One pg of Ad virions was added per cell. The plate was trations of 5 uMand 15uM. The final concentration of DMSO incubated at 37° C. in 5% CO, and 24 h after infection the was less than 1% in all samples. Five pg. 5-labeled Ad5 cells were harvested, washed once, and resuspended in PBS. 60 virions (with labeling done as described previously by Seg DNA was prepared from the samples using the QIAamp DNA erman et al. 2003 (Journal of Virology 77:1157-1162)) were Blood Mini Kit (QIAGEN, Solna, Sweden) according to the added per cell and the plate was incubated on ice on a rocking manufacturers instructions. The design of primers and platform for 1 h. Following incubation the cells were washed probes for analysis of various Ad types representing different three times with PBS-FBS-NaN, pelleted by centrifugation adenovirus species with quantitative PCR has been described 65 at 800g for 5 min at 4°C. and resuspended in 100 ul PBS. The previously (Allard et al. 2001. Rapid typing of human aden Suspension was transferred to Scintillation tubes containing 2 oviruses by a general PCR combined with restriction endo ml scintillation liquid (Wallac OptiPhase HiSafe 3, Perki US 8,993,622 B2 17 18 nElmer) and cell-associated radioactivity was measured as able; thus, Ad5 could not be used for screening. K562 cells are counts per minute (cpm) using a liquid Scintillation counter not permissive for Ad5 infection and the cell line of choice for (Wallac 1409). verification was A549. Inhibition of Wild-Type Ad5 and Ad 11 p. The discovery of more than one molecule in the most The antiviral potency of compound A02 in the A549 cell promising hit illustrates the necessity for quality control and system was assessed by measuring the effect on newly syn thorough validation to Verify that hits found in a screening thesized viral genomes of Ad5 and Ad 11p by the QPCR assay. campaign represent homogenous preparations of the correct Titration resulted in comparable ICso values of 3.7 LM and molecule, with the desired biological activity. In this particu 2.9M for Ad5 and Ad 11p, respectively (FIG. 3) Inhibition of lar case, the finding provided an opportunity for a preliminary wild-type Ad5 and Ad11p is substantially more efficient than 10 analysis of the structure-activity relationship. The anti-aden inhibition of the viral vector in K562 and A549 (compare oviral effect of A02 only, but not the analogs A01 and A03 has FIGS. 1, 2, and 3). been verified in a number of assays. There appears to be a size Effect on Binding of Ad5 to A549 Cells restriction for the compound to exert its inhibitory effect. A binding assay using isotope-labeled virions was used to Since neither the smaller analog A01 nor the larger A03 address whether the compound would prevent viral adhesion 15 analog has inhibitory effects, it is tempting to speculate that to host cells. At 15uM, compound A02 has no effect on Ad5 there may be a pocket in the target protein into which A02 fits, binding to the surface of A549 cells (FIG. 4A). To further where A01 is too small to cover the required site and A03 is verify inhibition of viral replication, the effect on the expres too bulky to fit. sion of the most abundant viral structural protein (hexon) in Considering the fact that DNA replication of all Ad types A549 cells was studied by FACS analysis. The results showed tested was inhibited by A02, although not with the same that expression of Ad5 and Ad 11p hexon protein is inhibited efficiency, inhibition by this compound appears to be general by compound A02 in a dose-dependent manner (FIG. 4B). for human Ads (Table 1) Inhibition of Ad31 is especially Toxicity. important, since this is one of the most threatening adenovirus Toxicity of A02 in the A549 cell system was also analyzed types, which can infect immunocompromised individuals in by titration, giving a 50% cytotoxic concentration (CCs) of general and pediatric transplant recipients in particular. 199 uM (FIG. 3). This can then be combined to give a selec 25 tivity index (SI-CCs/ICs) of 54 and 68 for Ad5 and Ad 11p. Example 3 respectively. Effect on Different Adenovirus Species. Inhibition of Herpes Virus With the clear-cut effect of A02 on both Ad5 (species C) and Ad 11p (species B) verified at several levels of the infec 30 Quantitative Real-Time PCR for Herpes Simplex Quantifica tion cycle, we performed an analysis to ascertain whether A02 tion could also affect Ads of other species (3). The results are Same procedure as for qPCR for adenovirus quantification summarized in Table 1. DNA replication of all Ads tested is was followed with the exception of a few steps. Namely, inhibited by compound A02 in a dose-dependent way. A02 7x10" green monkey kidney (GMK) cells were seeded in appears to have a general effect on Ads from all species. 35 24-well plate the day before infection. Seventy-five virus particles of HSV-1 (stock 1351-95 P13) were added per cell. TABLE 1. After 24h incubation with the compound A02 at 37°C. in 5% QPCR determination of DNA replication inhibition in A549 CO, the cells were harvested and DNA was prepared from cells for representative Ads from all Species 200 uL of the total 500 uL well-volume to include the DNA of 40 possible released HSV-1 particles. The primers used in the % inhibition of qPCR assay were: Rune 1 (sense position 137680-137697 DNA replication (SD in HSV-1 genome) (5'-GGCCTG GCTATCCGGAGA-3'- SEQ ID NO:6) and Rune 2 (antisense position 137742 Ad type (species) AO25 M AO2 15 M 137726 in HSV-1 genome) (5'-GCG CAG AGA CAT CGC Ad31 (A) 86 (11.8) 98 (0.8) GA-3 SEQ ID NO:7), the final concentration in the reac Ad11p (B2) 79 (4.4) 79 (3.1) 45 tion mixture of the primers were 300 nM. As in the adenovirus Ad5p (C) 72 (6.2) 86 (5.5) quantification a FAM-TAMRA labeled probe was used; Rune Ad37 (D) 86 (3.3) 94 (1.8) 3 (5-CAGCAC ACG ACTTGGCGTTCT GTGT-3 SEQ Ad4 (E) 85 (4.2) 96 (1.2) ID NO:8) to a final concentration of 175 nM in the reaction Ad41 (F) 80 (9.3) 88 (5.4) mixture. 50 Results Discussion The inhibitory effect of A02 on a herpes simplex 1 repli Screening-based strategies are well-suited for identifica cation in GMK cells is shown in FIG. 5. The inhibitory effect tion of compounds with potential anti-adenoviral activity. of A02 on herpes simplex 1 is about the same as observed with The presently used assay is based on a replication competent the representatives of all the six human adenovirus species. Ad11p vector. The GFP gene is located in the E1 region of the Ad11p genome and detection of fluorescence by GFP expres 55 Example 4 sion is directly correlated to Ad11p genome expression. This assay, developed for anti-adenoviral screening, is versatile Synthesis of New Antiviral Compounds due to its robustness, its simplicity and the direct measure ment of inhibition of Ad genome expression. K562 cells were A general synthetic route to compounds 8a-d is presented used in the screening assay, since they are Suspension cells 60 in Scheme 1. Activation of acids 4a-c with oxalyl chloride and that are permissive for Ad 11p infection. Any hits found in a coupling to anilines 5a-c gave amides 6a-d. Subsequent fluo screening campaign must, however, be thoroughly verified ride mediated removal of the 9-fluorenylmethocarbonyl since screening can be imprecise in many respects. We (Fmoc) group followed by benzoylation in a one-pot, two decided to concentrate the verification on Ad5 inhibition step procedure afforded esters 7a-din 9-57% yield from 4a-c since other potential anti-adenoviral drugs have been evalu 65 after HPLC purification (Table 1). Portions of the ethyl esters ated on the basis of their effects on species Cadenovirus 7a-d were saponified and the acids 8a-d were isolated in types. There is no replication-competent Ad5 vector avail 81-86% after HPLC purification (Table 2). US 8,993,622 B2 19 20

Scheme 1. Synthesis of esters 7a-d and acids 8a-d.

(COCl), pyridine fuely O freyalO "a O 1 GREN (Sl NH O OH 1N 21 Xslt 1n 2 21 21 O 4a-c 5a-c 6a-d 2. BzCl,St. pyridine Sl O O O 1 r " Co., O 21 prl21 OH 21 C2 8a-d 7a-d

25 TABLE 2 TABLE 2-continued Inhibition of adenovirus replication with Inhibition of adenovirus replication with Synthesized compounds. synthesized compounds. 30 Cpd Structure R Inh.*

11 O 18 Cpd Structure R Inh.* NH O 7a. O Ethyl O 35 8a. H 8 N H NH O O NH OR *08.% inhibition-- of Ad5 at 15 M. 40 To validate if the carboxylic acid moiety of the lead com pound A01 was critical for biological activity, reference com 7b O Ethyl 8 pound 11 without the carboxylic acid was synthesized 8b H 8 (Scheme 2). The route was essentially as described in Scheme C NH O as reagent1, but we (benzotriazol-1-yloxy)tris(dimethylamino) were more successful when using the coupling phos NH phonium hexafluorophosphate (BOP) in the first coupling as OR compared to using oxalyl chloride, and 10 was isolated in 28% yield. O 50 Scheme 2. Synthesis of compound 11. 7c O Ethyl 25 8c H H 39 FmocHN O N NH O 55 OH O BOP, DIPEA, DMF OR -- HN 9 4a 7d O Ethyl 15 60 FmocHN O 8d H 47 1. TBAF, THF NH O N 2. BZC., pyridine H HN OR 65 O 10 US 8,993,622 B2 21 22 -continued All compounds were screened for inhibition of intracellu O laradenoviral replication essentially as described in Example 1. Compound A02 contains a right hand ring with ortho NH O connected Substituents and a central ring with ortho con nected substituents as well. Moving to carboxylic acid moiety to meta (8a) and para (8b) position resulted in a sharp N decrease in activity. Changing the Substitution pattern on the central ring to meta (8c) and para (8d) resulted in reduced 11 activity, although less dramatic as compared to 8a and 8b. 10 This result indicates that there seem to be more room for Esters 7a-d, acids 8a-d, and 11 were purified by normal changes in the left hand part of the molecule. The wiggle phase column chromatography followed by reversed-phase room seen in the left hand portion of the molecule is further HPLC to give the compounds in >95% purity according to the evident since A02, 12c, and 12f show virtually identical HPLC UV trace. 15 activities, as do 12e and 8c (Table 2 and 3). Thus, a second Biology and Structure-Activity Relationships generation of compounds where the left hand ring is varied has been designed, synthesized and evaluation is underway. Based on lead compound A02, compounds 7a-d, 8a-d, and 11 (Scheme 1, Table 2) were designed and synthesized to The ortho, ortho substituent pattern thus appears favourable study the importance of the substituent pattern and if the for the (benzoylamino)benzoylamino-benzoic acid class of carboxylic acid is required for activity. In addition a set of inhibitors of adenovirus replication. commercially available analogs, compounds 12c-g, were The esters 7a-d corresponding to the acids 8a-d were included in the assay as potential prodrugs and/or to probe the acquired and tested (Table 3). importance of the carboxylic acid. A comparison of the ester/ TABLE 3 acid pairs 7c/8c and 7d/8d showed that the acids were more 25 active than the corresponding esters (Table 1). The ester 7c Inhibition of adenovirus Ad5 replication with inhibits replication to Some extent but the corresponding commercial compounds. amide 12d is less active indicating that the presence of a % Inh. 96 Inh. carboxylic acid in the ortho position is beneficial but not at 20 at 5 critical (Table 2 and 3). The preference for acid 8c appears not Cpd Structure M M 30 to be a steric effect since 12d and 8c will occupy very similar volumes. This is further evident when comparing A02 with 12c O 98.5 89.2 8a, 8b. 11, and 12g where it even seems worse to have a carboxylate in the meta and para position (8a and 8b) than none at all (11) or a methyl group in the ortho position (12g) C. O 35 (Table 2 and 3). H Typical Procedure for the Synthesis of Compounds 6a-d 's O OH (Scheme 3). 12d O 44.2 51.1 Ethyl H N 40 3-2-amino-(N-Fmoc)benzoylamino)-benzoate NH O Compound 6a O NH2

45 Scheme 3 12e O 55.8 44 O H N NH O HN O 1N OH 50

5b 12f O 96.5 25.2 FmocHN O O O 55 OH O N H O OH 4a FmocHN O 12g O 22.2 24.5 60 O

65 C- r 6a

US 8,993,622 B2 27 28 Benzoylation of Benzamide Compound 11. sphere before being diluted with DCM and washed with HCl (aq., 1M), NaHCO (aq., sat. 2x) and brine. The organic FmocHN O phase was dried over NaSO, filtered, and concentrated. The crude was purified by column chromatography (SiO, tolu ene:acetone 100:0->98:2) to give compound 20 in quant yield (2.24 g). 1H-NMR (400 MHz: CDC1): 8 11.60 (s, 1H), 8.82 (d. 10 O J=8.4 Hz, 1H), 8.12-8.08 (m, 2H), 7.75-7.71 (m, 2H), 7.66 10 7.61 (m, 2H), 7.18 (ddd, J=7.9, 7.3, 1.1 Hz, 1H), 4.36 (q, J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz, 3H). NH O Ethyl 2-(2-aminobenzoylamino)-benzoate Compound 21 N 15 NO O 11 TBAF (0.35 mL, 0.345 mmol) was added to a stirred solu NH O tion of Fmoc-protected 10 (100 mg, 0.23 mmol) in THF (3 mL) at rit. The reaction was stirred o.n. before a pyridine (70 uL, 0.917 mmol) and BZC1 (80 uL, 0.689 mmol) was added. r -e- After 3 h, the reaction was concentrated and purified by column chromatography (SiO, toluene:acetone 100:0->95: 25 2O 5) to give benzoylated 11 in 87% yield. Benzoylated 11 was NH2 O further purified by RP-HPLC before biological testing. 1H-NMR (400 MHz: Pyr): & 12.56 (s, 1H), 11.42 (s, 1H), 9.19 (d. J=8.4 Hz, 1H), 8.32-8.27 (m, 2H), 8.10 (dd, J=7.9, 1.3 HZ, 1H), 8.04-7.99 (m, 2H), 7.52-7.37 (m, 7H), 7.22-7.17 30 (pyr-d5 overlap, 2H), 7.03 (td, J=7.6, 1.1 Hz, 1H). 1N 13C-NMR (100 MHz: Pyr): & 168.97 (s, 1C), 165.34 (s, 1C), 140.67 (s, 1C), 139.29 (s.1C), 135.56 (s, 1C), 132.81 (s, C. 21 1C), 132.09 (s.1C), 129.22 (s, 1C), 129.16 (s, 1C), 129.09 (s. 1C), 127.70 (s, 1C), 125.04 (s, 1C), 122.80 (s, 1C), 122.33 (s, 35 1C), 121.78 (s, 1C), 121.39 (s, 1C). Nitro compound 20 (2.24g, 7.14 mmol) and Pd/C (220 mg) ESIMS m/z calcd for CHNO, 315.11. found: was dissolved in EtOAc (50 mL) and MeOH (20 mL) and 315.52. stirred vigorously under H atmosphere (1 atm). After 5h, the Pd/C was filtered off over a pad of celite and florisil to leave Ethyl 40 pure aniline compound 21 in quant. yield (1.98 g) over two 2-(2-nitrobenzoylamino)-benzoate—Compound 20 steps. 1H-NMR (400 MHz: CDC1): 8 11.86 (s, 1H), 8.83 (d. NO O NH2 O J=8.5 Hz, 1H), 8.10 (dd, J=8.0, 1.5 Hz, 1H), 7.74 (dd, J=8.0, 45 1.4 Hz, 1H), 7.59 (ddd, J=8.5, 7.3, 1.4 Hz, 1H), 7.29-7.25 (m, 1H), 7.14-7.10 (ddd, J=8.3, 7.4, 1.1 Hz, 1H), 6.78 (ddd, J=8.0, 7.2, 1.0 Hz, 1H), 6.72 (dd, J=8.2, 0.8 Hz, 1H), 5.76 (s. 2H), s s1\ Her 4.42 (q, J=7.1 Hz, 2H), 1.44 (t, J=7.1 Hz, 3H). NO O Typical Procedure for the Synthesis of Amides 22a-27a (Scheme 6). Ethyl 2-2-(benzoylamino)benzoylaminobenzoate— Compound 23a

r Scheme 6 2O NH2 O A catalytic amount of DMF was added to a stirred solution of 2-nitrobenzoic acid (2.30 g, 13.8 mmol) and oxalyl chlo 60 NH O ride (6.0 mL, 69 mmol) in 1,2-dichloroethane (70 mL) at r.t. and under nitrogen flow. After 1 h, the reaction was concen trated, re-dissolved in 1,2-dichloroethane and concentrated again. The crude was immediately dissolved in 1,2-dichloro ethane (25 mL) before ethyl 2-aminobenzoate (1.00 mL, 6.76 65 mmol) and pyridine (1.70 mL. 21.0 mmol) was added. The 21 reaction was stirred 45 min at r.t. and under nitrogen atmo US 8,993,622 B2 29 30 -continued -continued O O NAS NH O MYNH O 5 O NH O C O so-1N 1N

23a 29a Pyridine (43 uL, 0.54 mmol) was added to a stirred solution Pyridine (43 uL, 0.54 mmol) was added to a stirred solution 15 of aniline 21 (50 mg, 0.18 mmol) in DCM (0.9 mL) at room of aniline 21 (50 mg, 0.18 mmol) in DCM (0.9 mL) at room temperature under nitrogen atmosphere. After 5 min, meth temperature under nitrogen atmosphere. After 5 min, ben anesulfonyl chloride 9 (29.9 uL, 0.198 mmol) was added and Zoylchloride 3 (22.5 LL, 0.198 mmol) was added and the the reaction was stirred overnight before being diluted with reaction was stirred for 2 h before being diluted with DCM DCM and washed with HCl (3x, aq, 1M), NaHCO (aq, sat), and washed with HCl (3x, aq, 1M), NaHCO (aq, sat), and and brine. The organic phase was dried over NaSO con brine. The organic phase was dried over Na2SO, concen centrated and purified by column chromatography (toluene: trated and purified by column chromatography (toluene:ac acetone 100:0->98:2) to give methanesulfonamide 29a in etone 100:0->98:2) to give benzamide 23a in 62% yield (36 60% yield (38 mg). mg). 25 Ethyl 2-2-(p-Toluenesulfonylamino)benzoylamino Ethyl 2-2-(Acetylamino)benzoylaminobenzoate— benzoate Compound 30a Compound 22a Procedure for the Synthesis of Benzylamide 4.0a (Scheme 8). 30 Ethyl Ethyl 2-2-(benzylamino)benzoylaminoben 2-2-(Trimethylacetylamino)benzoylaminobenzoate Zoate Compound 40a Compound 24a

35 Ethyl 2-2-(Cyclohexanecarboxylamino)benzoyl Scheme 8 aminobenzoate Compound 25a NH2 O

Ethyl 2-2-(Phenylacetylamino)benzoylaminoben 40 Zoate Compound 26a Cul Ethyl 2-2-(4-Carboxybutanoylamido)benzoylami nobenzoate Compound 27a 45 r 1 Typical Procedure for the Synthesis of Sulfonamides 29a-30a (Scheme 7). O O 50 Ethyl 2-2-(methanesulfonylamino)benzoylamino benzoate Compound 29a C O

55 r 40a Scheme 7 NH2 O DIPEA (61 uL, 0.54 mmol) was added to a stirred solution of aniline 1 (50 mg, 0.18 mmol) in DCM (0.9 mL) at room NH O -e- 60 temperature under nitrogen atmosphere. After 5 min, benzyl bromide 11 (23 uL, 0.198 mmol) was added and the reaction mixture was heated in microwave at 80° C. for 20 min, then 1N 100° C. for 45 min more before being diluted with DCM and washed with HCl (3x, aq, 1M), NaHCO (aq, sat), and brine. 65 The organic phase was dried over Na2SO, concentrated and 21 purified by column chromatography (toluene: acetone 100: 0->98:2) to give benzylamine 4.0a in 45% yield (30 mg).

US 8,993,622 B2 33 34 2-2-(p-Toluenesulfonylamino)benzoylaminobenzoic 'C NMR (360 MHz, Pyridine-d5): 8 172.7, 169.2, 151.1, acid Compound 30b 143.0, 140.1, 135.1, 134.2, 133.8, 132.4, 129.2, 128.8, 127.8, 127.6, 122.7, 120.7, 118.3, 116.3, 115.9, 113.0, 47.4 ESIMS m/z calcd for CHNO" 347.13. found "H NMR (500 MHz, Pyridine-dis): 8 13.15 (s, 1H), 11.33 347.22. (s, 1H), 9.02 (d. J=8.5 Hz, 1H), 8.50 (dd, J=1.4, 8.0 Hz, 1H), 5 8.04 (d. J=8.2 Hz, 1H), 7.98 (dd, J=0.9, 7.8 Hz, 1H), 7.93 (d. Example 5 J=8.2 Hz, 2H), 7.62-7.65 (m, 1H), 7.40-743 (m, 1H), 7.22 7.26 (m, 1H), 7.04 (t, J=7.0 Hz, 1H), 7.00 (d. J=8.1 Hz, 2H), Synthesis of Second Generation of Antiviral Compounds 1.95 (s, 3H). 10 'C NMR (500 MHz, Pyridine-dis): 8 1722, 1672, 143.9, Based on the first generation inhibitors we concluded that 1414, 139.4, 136.8, 135.6, 133.8, 132.8, 132.0, 129.8, 127.9, the ortho, ortho substituent pattern was optimal, that the free 127.5, 124.5, 124.0, 122.6, 120.4, 118.6, 20.8. acid was beneficial and that it seemed to be room for variation ESIMS m/z calcd for CHNOSI" 411.09. found in the N-terminal. Consequently, compounds 17a-k (Table 4) 411.14. 15 with variation in the N-terminal were designed and synthe sized (Scheme 10). A more efficient and high-yielding route 2-2-(Benzylamino)benzoylaminobenzoic than the previous (Scheme 1) was envisioned using the nitro acid Compound 40b group of 13 as protected aniline. Gratifyingly, coupling of 13 with 5a followed by hydrogenation gave 15 quantitatively. HNMR (360 MHz, Pyridine-d5): 8 13.13 (s1H),921 (d. Compound 15 was subsequently reacted with a few select J=8.4 Hz, 1H), 8.50 (dd, J=1.4, 7.9 Hz, 1H), 8.15 (d. J=7.9 Hz, activated acids, Sulfonyl chlorides. Succinic anhydride, and 1H), 7.56-7.60 (m, 1H), 7.48 (d. J=7.4 Hz, 2H), 7.15-7.34 (m, benzyl bromide to give 16a-k. Hydrolysis concluded the route 5H), 6.80 (d. J=8.4 Hz, 1H), 6.64-6.66 (m. 1H), 4.46 (s. 2H). to give 17a-k (Scheme 10, Table 4).

Scheme 10. Synthesis of acids 17a-k. NO O NH2 O (COCl), DMF (cat.), OH 1N CHCICH2Cl, pyr He

13 5a NH2 O NO O H2, Pd/C NH O NH O EtOAC:MeOH -- 1N 1N

15 14 RCOC or RSOC or BnBr

R RS YNH O NH O NaOH (aq), pyr NH O NH O 1S OH

16a-k 17a-k

Compounds 23 and 28 were synthesized to probe the (as sumed) binding pocket further (Scheme 11 and 12). 23 and 28 60 each contains an additional methylene group compared to the parent 1. The added methylene group gives an overall slightly longer inhibitor and separates the amide-carboxylic acid in 23 and amide-amide in 28, groups that are commonly participat ing in directed hydrogen bonding to receptors. Apart from 65 using phenylacetate building blocks 18 and 24 respectively, the route to 23 and 28 follows suite with the one presented in Scheme 6.

US 8,993,622 B2

Scheme 12. Synthesis of acid 28. O OEt O OEt NO BOP, DIPEA, NO H2, Pd/C OH HN - N - HerEtOAC:MeOH

O O

24 5a 25 O OEt NH2 H N

O

26 BZCl, pyridine, CHCl2 O O

O OH O OEt NH NaOH (2M), NH pyridine H N

O O 28 27

Biology—Second Generation Inhibitors TABLE 4-continued Most of the second generation inhibitors displayed lower 35 activity than A02 (Table 4). However, compound 17h showed Inhibition of adenovirus replication with statistically significantly higher activity than A02, and com synthesized compounds. pound 17 was of similar potency. Noteworthy is that com- %. Inh. at % Inh. at pounds 17e and 17g that exhibit similarities with A02 show Cpd Structure 15 M 5 M diminished but not abolished activity while the other tested 40 compounds showed much lower activities. 17b n; O 23 - 12 O

TABLE 4 # NH NH O Inhibition of adenovirus replication with 45 synthesized compounds. OH

%. Inh. at % Inh. at Cpd Structure 15 M 5 M 17C 835 224 AO2 O 96+ 1. 89 + 5 50 O A. Sa NH O A NH O O NH O NH O 55 OH OH

17a. O 24 - 14 4-11 17 O 52 18 457

l NH O 60 NH O

NH O NH O

OH OH 65 US 8,993,622 B2 39 40 TABLE 4-continued TABLE 4-continued Inhibition of adenovirus replication with Synthesized compounds. Inhibition of adenovirus replication with synthesized compounds. %. Inh. at % Inh. at 5 Cpd Structure 15 M 5 M %. Inh. at % Inh. at 0. ... a o ill. 8 17e O 55 11 201 Cpd Structure 15 M 5 M

NH O 10 17k O 21 - 12 32 19 NH O NH O OH NH O 15 OH

17f O O O HO ~s O 2O NH O

OH NH O

25 NH OH

-- 17g O O 79 S 44 - 17 O NH O 30

OH 28 O O NH O OH 35 H N

17 F O 96 --- 1 96 --- 1 O

NH O 40 NH O OH Example 6

45 Synthesis of Third Generation of Antiviral Compounds

17 O O 41 - 32 F NH O 50 A further round of optimization (third generation inhibi tors) was performed around 17h, the previous best compound NH O from the second generation. Here, the central and C-terminal OH rings were decorated with electron donating (OMe), interme 55 diate (Cl), and electron withdrawing (F) Substituents. The synthetic route (Scheme 15) followed the one outlined previ ously in Scheme 3. Where amino benzoate esters were not commercially available, a methyl esterification of the corre 17 O 951-- 7610 sponding commercial amino benzoic acid was conveniently NH O 60 carried out using trimethylsilyldiazomethane (Hashimoto, Chem. Pharm. Bull. 1981, 29(5), 1475-1478). Substantial amounts of bi-products were seen after the hydrogenolysis of OH 65 the 4,5-difluoro-2-nitrobenzamide, nucleophilic aromatic Substitution products may be a likely cause. In all, compounds 35a-i were synthesized (Table 5). US 8,993,622 B2

Scheme 15. Synthesis of acids 35a-i. NH2 O

OH

R R 29a-e TMSCHN, DCM:MeOH

NO O NO O NH2 O R (COCl)2. O1 DMF (cat.), NH O H2, Pd/C OH CHCICH2Cl, pyr EtOAe:MeOH -e- -e- R R4 O1 R4 2 R3 R3 R R2 5a, 31a-e 13, 30a-e R = Me or Et R 32a

NH2 O

NH O

R R4 O1 R3 R R 33a

2-fluorobenzoic acid, (COCl), DMF (cat.), CHCICH2Cl, pyr F O F O

NH O NH O

NH O NaOH (2M), pyr NH O se R R4 OH R4 O1 R3 R3 R2 R R R 35a R - R4 = H, F, Cl, OMe

60 For the four chlorinated compounds 32a, 32b, 32f, and 32g, conditions (see example in Scheme 5) gave enough desired reduction with hydrogen gas over palladium on charcoal product to move forward, although the method is less than resulted in the corresponding undesired des-chloro com ideal. For nitro compounds 32a and 32b with a methyl ester pound. A small screen to find conditions that avoided reduc 65 rather than an ethyl ester, no desired compound could be ing the chloro substitutent was carried out: NaBH4/Pd/C: isolated. Rather a product where the methyl ester was cleaved SnCl2-2H2O, and FeCl3/C/hydrazine. FeCl3/C/hydrazine was isolated as the major product, presumably due to the US 8,993,622 B2 hydrazine. Starting with the corresponding ethyl ester instead TABLE 5-continued did give enough desired product to obtain 35a and 35b, although in poor yields. Inhibition of adenovirus replication with synthesized compounds. 5

Scheme 10. Example of nitro reduction of %. Inh. at 15 %. Inh. at chloro-substituted compounds 32a, 32b, 32f, and 32g. Cpd Structure M 5 M

NO O 10 35d F O 783 28 - 20 FeCl, C hydrazine -e- C. O 48%3. NH O NH C 1N COOH 15

32f OMe NH2 O 20 35e F O O O NH O NH O

C O NH 25 COOH

33f F F Biology. Third Generation Inhibitors 30 35f F O 782 57 S TABLE 5 NH O Inhibition of adenovirus replication with synthesized compounds. 35 NH COOH %. Inh. at 15 % Inh. at C Cpd Structure M 5 M

35a F O 61 - O O 40 35g F O 91 - 2 89 2 NH O NH O NH COOH NH 45 COOH C C 35b F O O O 3S F O 89 6 923 NH O 50 NH O NH COOH NH COOH 55 MeO

C

3Sc F O 95 - 22 79 - 21 35 F O 97 - 1 91 6 60 NH O NH O

NH NH COOH COOH 65 OMe MeO US 8,993,622 B2 45 46 TABLE 5-continued antibody was washed away in PBS and the cells were exam ined in fluorescent microscope. Inhibition of adenovirus replication with Synthesized compounds. Results Inhibition of the RNA-virus, coxsackievirus A24 variant %. Inh. at 15 % Inh. at Cpd Structure M 5 M (CVA24V), part of the picornaviridae family, by compound 35 is summarized in Table 6. The inhibition is relative to the 35 F O 93 - 2 94 3 fluorescence of CVA24v. infected cells alone. No visible fluo NH O rescence was observed at 50 and 25 uM (+++), however at 10 12.5 and 6.25 uM some infected cells were detected (++), NH indicating a dose-dependent inhibition of CVA24V replica COOH F tion by compound 35. F 15 TABLE 6 Inhibition of the RNA-virus, coxsackievirus A24 variant Inhibition of the RNA-Virus, Coxsackievirus Relative Fluorescent Focus Assay Compound 35 inhibition of Approximately 2x10 human corneal epithelial (HCE) COC. CVA24v. cells were plated in 24-well plates the day prior infection. On 50 M ------the day of infection the media was removed and was replaced 25 M ------with media containing 70 virus particles of CVA24V per cell 12.5 M ---- and serial dilutions of compound 35, ranging from 50- to 6.25 M ---- 6.25 uM. After 18 h of incubation at 37° C. in 5% CO, the 25 cells were fixed using ice-cold 100% methanol for 5 min. EC50 and Toxicity of Select Compounds Thereafter, the cells were incubated with a monoclonal Cellular toxicity is an important factor in the development mouse anti-VP1 structural protein antibody (clone 5-D8/1, ofan antiviral agent and something that we have considered in DakoCytomation) diluted 1:200 in PBS, for 1 h in RT. The the evaluation of compounds with anti-adenoviral effect. cells were then washed with PBS and incubated with FITC 30 Compound A02 was selected partly based on the low cellular labeled anti-mouse secondary antibody (DakoCytomation), toxicity. The more efficient 17h is also slightly more toxic diluted 1:100 for 1 h, RT, in the dark. Further, unbound than A02 (Table 7). TABLE 7

Effective concentration and toxicity of selected compounds with inhibitory effect.

Toxicity at Toxicity at Cpd Structure EC50 30 M* 60 M*

AO2 O 3.7 207 293

NH O

NH O

OH

11.4 6 - 10 1113 NH O

NH O

OH US 8,993,622 B2 47 48 TABLE 7-continued

Effective concentration and toxicity of selected compounds with inhibitory effect.

Toxicity at Toxicity at Cpd Structure EC50 30 M* 60 M*

17 F O 2.3 179 29, 12 C O C O OH

17 O 3.5 14 - 10 1010

NH O

NH O

OH

F O 4.14 145-- 25 -- 8

NH O

NH

COOH C

F O O.68 117-- 249-- C O NH

COOH

C

F O 1.5 10 15 1815

NH O

NH

COOH MeO US 8,993,622 B2 49 50 TABLE 7-continued Effective concentration and toxicity of selected compounds with inhibitory effect. Toxicity at Toxicity at Cpd Structure EC50 30 M* 60 M* 35 F O 1.4 1812 28 - 12

NH O

NH

COOH

OMe

35 F O 0.27 6 : 4 169

NH O

NH

COOH F

F

DMSO 2 - 1 122

*% dead cells after 24h at 30 and 60 uM.

Conclusions phase was dried over MgSO filtered, and concentrated. The 2-2-Benzoylamino)benzoylamino-benzoic acid (1) was 35 crude amide was purified by short-pass column chromatog identified as a potent adenoviral compound in a previous raphy (SiO, toluene:acetone98:2->95:5) and taken directly screening campaign. Here, initial attempts at optimizing 1 is to the next reaction. effected by screening three generations of designed and Syn Procedure B: Fmoc Deprotection and In Situ Coupling to an thesized compounds as well as seven commercial, structur Acid Chloride (Exemplified by 11) ally similar compounds. In the first generation, we conclude 40 TBAF (1 M, 0.35 mL, 0.345 mmol) was added to a stirred that the ortho, ortho substituent pattern and the presence of a solution of Fmoc-protected aniline (100 mg, 0.23 mmol) in carboxylic acid in 1 is favourable for this class of compounds THF (3 mL) at r.t. The reaction was stirred o.n. before pyri and that the direction of the amide bonds as in 1 is obligate. dine (70 uL, 0.917 mmol) and BZC (80 uL, 0.689 mmol) was Although there seem to be room for some variability in the added. After 3 h, the reaction was concentrated and purified N-terminal moiety of the compound class, a second set of 45 by column chromatography (SiO, toluene:acetone designed compounds showed that the variability appears lim 100:0->95:5) to give benzoylated product in 87% yield. The ited to substituted benzamides. One such benzamide (17h) shows improved activity over 1. In a third generation, the benzoylated product was further purified by RP-HPLC. substituents on the middle and C-terminal rings were varied Procedure C: Saponification (Exemplified by 8d) resulting in further potent inhibitors with very low cell tox 50 NaOH (aq., 2 M, 0.5 mL) was added to a stirred solution of icity. Compound 35 displayed an excellent EC50-value of the ethyl ester (8.5 mg, 21.9 mmol) in pyridine (2 mL) at r,t. 0.3 uM in this whole-cell assay along with a CC50 of 240 LM After 2 h, the pyridine was removed under reduced pressure, giving a toxicity/activity selectivity index of 890. the residue diluted with EtOAc, and washed with HCl (aq., 1 Synthetic Procedures M). The organic phase was dried over MgSO filtered, and Procedure A: Synthesis of an Acid Chloride and Coupling to 55 concentrated. The crude was purified by semi-preparative an Aniline (Exemplified by 6a) RP-HPLC to give the carboxylic acid product in 85% yield. A catalytic amount of DMF was added to a stirred solution Procedure D: BOP-Activation of a Carboxylic Acid and Cou of Fmoc-aminobenzoic acid (100 mg, 278 umol) and oxalyl pling to an Aniline (Exemplified by 10) chloride (0.49 mL, 5.57 mmol) in 1,2-dichloroethane (3 mL) Aniline (114 ul, 1.25 mmol) was added to Fmoc-ami at r.t. and under nitrogen atmosphere. After 40 min, the reac 60 nobenzoic acid (300 mg, 835 umol), BOP (652 mg, 1.25 tion was concentrated, re-dissolved in 1,2-dichloroethane and mmol), and DIPEA (0.44 mL, 2.50 mmol) in DMF (8 mL) at concentrated again. The crude was immediately dissolved in rt. After 2 h, more aniline (228 ul, 2.50 mmol) was added and 1,2-dichloroethane (4 mL) before pyridine (0.22 mL, 2.78 the reaction was stirred o.n. The reaction was diluted with mmol) and ethyl aminobenzoate (184 mg, 1.11 mmol) was HCl (aq., 1 M, 70 mL) and loaded onto a conditioned C18 added. The reaction was stirred on. at r.t. and under nitrogen 65 SPE-column (10 g). The column was washed with HCl (aq., atmosphere before being diluted with DCM and washed with 1 M, 70 mL) and H2O (140 mL) before being eluted with HCl (aq., 1M), NaHCO (aq., sat.) and brine. The organic MeOH and acetone. The pooled organic fractions were con US 8,993,622 B2 51 52 centrated and purified by column chromatography (SiO, by column chromatography (toluene:acetone 100:0->98:2) toluene:acetone 100:0->95:5->90:10) to give the benzamide to give the aniline 33f in 46% yield (115 mg). in 28% yield. Procedure E: Hydrogenation (Exemplified by 15) 2-2-(Acetylamino)benzoylaminobenzoic The nitro compound (2.24 g, 7.14 mmol) and Pd/C (220 acid—Compound 17a mg) was dissolved in EtOAc (50 mL) and MeOH (20 mL) and stirred vigorously under H atmosphere (1 atm). After 5h, the 17a was synthesized by procedures A, E, F, and C. Pd/C was filtered off over a pad of celite and florisil to leave 1H-NMR (500 MHz: Pyr): 8 13.33 (s, 1H), 11.56 (s, 1H), pure aniline product in quant. yield (1.98 g) over two steps. 10 9.15 (d. J=8.3 Hz, 1H), 8.96 (d. J=8.3 Hz, 1H), 8.54 (dd, Procedure F: Coupling of an Acid Chloride or Sulfonyl Chlo J=7.8, 1.4 Hz, 1H), 8.14 (dd, J=7.9, 1.1 Hz, 1H), 7.64-7.61 ride with an Aniline (Exemplified by 16d) (ddd, J=7.0, 1.5 Hz, 1H), 7.45-7.42 (ddd, J=1.2 Hz, 1H), 7.23 Pyridine (43 uL, 0.54 mmol) was added to a stirred solution (t, J=7.6 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H), 2.20 (s.3H). of the aniline (50 mg, 0.18 mmol) in DCM (0.9 mL) at room 15 13C-NMR (126 MHz: Pyr): 8 172.47, 168.69, 167.89, temperature under nitrogen atmosphere. After 5 min, meth 141.92, 140.74, 133.87, 132.75, 132.23, 128.01, 123.29, anesulfonyl chloride (29.9 uL, 0.198 mmol) was added and 122.10, 121.85, 120.59, 118.94, 25.05. the reaction was stirred over night before being diluted with DCM and washed with HCl (3x, aq, 1M), NaHCO (aq, sat), ESIMS m/z calcd for CHNO" 299.10. found and brine. The organic phase was dried over NaSO, con 299.25. centrated and purified by column chromatography (toluene: 2-2-(Methanesulfonylamino)benzoylaminobenzoic acetone 100:0->98:2) to give the derivatized aniline in 60% acid Compound 17b yield (38 mg). Procedure G: Benzylation of an Aniline (Exemplified by 16i) 17b was synthesized by procedures A, E, F, and C. DIPEA (61 uL, 0.54 mmol) was added to a stirred solution 25 of the aniline (50 mg, 0.18 mmol) in DCM (0.9 mL) at room 1H-NMR (360 MHz: Pyr): 8 13.40 (s, 1H), 9.01 (d. J=8.4 temperature under nitrogen atmosphere. After 5 min, benzyl HZ, 1H), 8.50 (dd, J=7.9, 1.4 Hz, 1H), 8.15 (d. J=7.9 Hz, 1H), bromide 11 (23 uL, 0.198 mmol) was added and the reaction 8.03 (d. J=8.3 Hz, 1H), 7.60 (td, J=7.9, 1.3 Hz, 1H), 7.44 (t, J=7.8 Hz, 1H), 7.23 (t, J–7.7 Hz, 1H), 7.08 (t, J=7.6 Hz, 1H), mixture was heated in microwave at 80° C. for 20 min, then 30 100° C. for 45 min more before being diluted with DCM and 3.31 (s.3H). washed with HCl (3x, aq, 1M), NaHCOs (aq, sat), and brine. 13C-NMR (91 MHz. Pyr): & 174.29, 169.26, 143.36, The organic phase was dried over Na2SO4, concentrated and 142.11, 135.70, 135.07, 133.93, 130.29, 125.65, 124.17, purified by column chromatography (toluene: acetone 100: 122.43, 120.68, 41.70. 0->98:2) to give the benzylated aniline in 45% yield (30 mg). 35 ESIMS m/z calcd for CHNOS' 335.35. found Procedure H: TMSCHN-Mediated Esterification (Exem 335.26. plified by 31e) TMSCHN (1.9 mL, 3.82 mmol. 2.0 M in hexanes) was 2-2-(p-Toluenesulfonylamino)benzoylaminobenzoic added to a stirred solution of the aminobenzoic acid (600 mg. acid—Compound 17c 3.47 mmol) in 20 mL of DCM:MeOH (9:1) under nitrogen 40 atmosphere at room temperature. After 30 min, the reaction 17c was synthesized by procedures A, E, F, and C. mixture was quenched by glacial HOAc before being diluted 1H-NMR (500 MHz: Pyr): 8 13.13 (s, 1H), 9.01 (d. J=8.4 with DCM and washed with NaHCO (aq., sat.). The organic HZ, 1H), 8.48 (dd, J=7.9, 1.4 Hz, 1H), 8.02 (d. J=8.2 Hz, 1H), phase was dried over NaSO, filtered, and concentrated to 45 7.96 (dd, J=7.8, 0.9 HZ, 1H), 7.92 (d. J=8.2 Hz, 2H), 7.64-7.60 give the methylaminobenzoate in 99% yield (644 mg). (ddd, J=8.6, 7.1, 1.5 Hz, 1H), 741-7.38 (ddd, J=8.3, 7.2, 1.1 Procedure I: Acid-Catalyzed Esterification (Exemplified by HZ, 1H), 7.24-7.21 (ddd, J=7.9, 7.1, 0.8 Hz, 1H), 7.03-6.97 31a) (m,3H), 1.95 (s.3H). 1-amino-4-chloro-benzoic acid (500 mg, 2.9 mmol) and 50 13C-NMR (126 MHz: Pyr): 8 172.16, 167.23, 143.91, HSO (conc, 0.5 mL) in EtOH (30 mL) was refluxed for 3 141.39, 139.40, 136.83, 133.79, 132.78, 131.97 129.75, days before being concentrated to half-volume and diluted 127.90, 127.45, 124.52, 124.04, 123.32, 122.57, 120.39, with NaHCO3 (aq, sat) and extracted with DCM (3x) (even 118.61, 20.83. though the reaction had not yet finished). The pooled DCM ESIMS m/z calcd for C. H.N.O.S" 411.09. found fractions was washed with brine, dried over MgSO, concen 55 trated and purified by column chromatography (toluene:ac 411.14. etone 100:0->98:2) to give the ethyl ester in 64% yield (370 2-2-(Trimethylacetylamino)benzoylaminobenzoic mg). acid Compound 17d Procedure J: Nitro-Reduction Using FeCl3/C/Hydrazine 60 (Exemplified by 33f) 17d was synthesized by procedures A, E, F, and C. Hydrazine (0.80 mL, 16.5 mmol) added to a stirred solu 1H-NMR (500 MHz: Pyr): 8 13.34 (s, 1H), 11.94 (s, 1H), tion of nitro compound 32f (275 mg, 0.789 mmol), charcoal 9.14 (dd, J=8.5, 1.1 Hz, 2H), 8.51 (dd, J=7.9, 1.6 Hz, 1H), (50 mg. 4.16 mmol), and FeCl3 (38 mg, 0.23 mmol) in MeOH 8.16 (dd, J=7.9, 1.3 Hz, 1H), 7.62-7.58 (ddd, J=8.7, 7.1, 1.6 (10 mL) at 65° C. under nitrogen atmosphere. The tempera 65 HZ, 1H), 7.45-742 (ddd, J=8.5, 7.2, 1.3 Hz, 1H), 7.23-7.20 ture was raised to reflux and kept there for 3 h before all solids (ddd, J=8.3, 7.3, 1.0 Hz, 1H), 7.05-7.02 (ddd, J=8.3, 7.3, 1.1 were filtered off over celite. The crude material was purified HZ, 1H), 1.39 (s.9H).

US 8,993,622 B2 57 58 133.75, 133.58, 132.26, 132.03,131.63 (d.J=2.0 Hz), 128.41, 2-2-(2-Fluorobenzoylamino)-benzoylaminobenzoic 127.88, 124.88 (d.J=3.2 Hz), 124.49, 123.99, 120.52, 119.35, acid 116.57 (d. J=23.1 Hz). 2-2-(3-Fluorobenzoylamino)-benzoylaminobenzoic ESIMS m/z calcd for CHCIFN.O., 411.06. found: acid 411.15. 2-2-(4-Fluorobenzoylamino)-benzoylaminobenzoic acid 2-4-methoxy-2-(2-fluorobenzoylamino)-benzoy Ethyl 2-2-(benzoylamino)benzoylaminobenzoate laminobenzoic acid Compound 35h Ethyl 2-2-(acetylamino)benzoylaminobenzoate Ethyl 2-2-(trimethylacetylamino)benzoylaminoben 10 35h was synthesized by procedures A, E, A, and C. ZOate 1H-NMR (400 MHz, Pyr): 8 13.25 (s, 1H), 12.82 (d. J=4.6 Ethyl 2-2-(cyclohexanecarboxylamino)benzoylamino HZ, 1H), 9.07 (d.J=8.3 Hz, 1H),8.87 (brs, 1H), 8.47 (d. J=7.8 benzoate HZ, 1H), 8.20-8.15 (m, 2H), 7.51 (t, J=7.8 Hz, 1H), 7.47-7.40 Ethyl 2-2-(phenylacetylamino)benzoylaminobenzoate (m. 1H), 7.25-7.15 (m, under Pyr, 3H), 6.73 (dd, J=8.8, 2.5 Ethyl 2-2-(4-carboxybutanoylamido)benzoylaminoben HZ, 1H), 3.72 (s.3H). 15 13C-NMR (100 MHz, Pyr): 8 172.38, 167.60, 163.14, ZOate 162.59 (d. J=2.0 Hz), 16027 (d. J–250.8 Hz), 142.49, 141.86, 2-2-(Benzoylamino)benzoylaminobenzoic acid 133.80, 133.71, 132.06, 131.39 (d. J=1.5 Hz), 129.59, 124.97 2-2-(Acetylamino)benzoylaminobenzoic acid (d. J–3.3 Hz), 122.96, 120.44, 118.64, 116.66 (d. J–23.1 Hz), 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid 114.32, 109.68, 107.16, 55.32. 2-2-(Cyclohexanecarboxylamino)benzoylaminoben ESIMS m/z calcd for C.H.FNOI", 409.38. found: Zoic acid 4O945. 2-2-(Phenylacetylamino)benzoylaminobenzoic acid 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic 2-5-methoxy-2-(2-fluorobenzoylamino)-benzoy acid laminobenzoic acid Compound 35i 25 2-2-(benzoylamino)benzoylaminophenylacetic acid 2-2-(benzoylamino)phenylacetylaminobenzoic acid 35i was synthesized by procedures A, E, A, and C. 2-2-(2-fluorobenzoylamino)-benzoylamino-4-chlo 1H-NMR (360 MHz, Pyr): 8 13.35 (s, 1H), 12.21 (d. J=6.6 robenzoic acid HZ, 1H), 9.15 (d. J=8.3 Hz, 1H), 9.08 (d. J=9.1 Hz, 1H), 8.49 2-2-(2-fluorobenzoylamino)benzoylamino-5-chlo (d. J=7.7 Hz, 1H), 8.27 (t, J–7.5 Hz, 1H), 7.78 (d. J=2.3 Hz, 30 1H), 7.54 (t, J=8.0 Hz, 1H), 7.44-7.38 (m, 1H), 7.23-7.17 (m, robenzoic acid under Pyr, 4H), 3.77 (s.3H). 2-2-(2-fluorobenzoylamino)-benzoylamino-4-methoxy 13C-NMR (91 MHz, Pyr): 8 172.41, 167.30, 161.94, benzoic acid 160.76 (d. J–250.0 Hz), 155.82, 141.79, 133.87, 133.61 (d. 2-2-(2-fluorobenzoylamino)-benzoylamino-5-methoxy benzoic acid J=8.7 Hz), 133.45, 132.16, 131.76, 124.98 (d. J=3.1 Hz), 35 124.43, 124.33, 123.61, 123.30, 120.47, 118.87, 118.67, 2-2-(2-fluorobenzoylamino)-benzoylamino-4,5-difluo 116.66 (d. J=24.1 Hz), 112.61, 55.40. robenzoic acid ESIMS m/z calcd for C.H.FNOI", 409.11. found: 2-4-chloro-2-(2-fluorobenzoylamino)-benzoylamino 4O939. benzoic acid 40 2-5-chloro-2-(2-fluorobenzoylamino)-benzoylamino 2-4,5-difluoro-2-(2-fluorobenzoylamino)-benzoy benzoic acid laminobenzoic acid—Compound 35 2-4-methoxy-2-(2-fluorobenzoylamino)-benzoylamino benzoic acid 35 was synthesized by procedures A, E, A, and C. 2-5-methoxy-2-(2-fluorobenzoylamino)-benzoylamino 1H-NMR (360 MHz: Pyr): & 14.10 (s, 1H), 12.58 (d. J=6.3 45 benzoic acid, and HZ, 1H), 9.13-9.03 (m, 2H), 8.58 (d. J=7.7 Hz, 1H), 8.23 (td, 2-4,5-difluoro-2-(2-fluorobenzoylamino)-benzoy J=7.8, 1.3 Hz, 1H), 8.01 (t, J=9.7 Hz, 1H), 7.49 (t, J=8.1 Hz, laminobenzoic acid 1H), 7.43 (q, J=6.7 Hz, 1H), 7.25-7.13 (m, 3H). or a pharmaceutically acceptable salt thereof. 13C-NMR (91 MHz: Pyr): & 174.51, 165.64, 162.38, 2. A pharmaceutical composition comprising a compound 160.39 (d. J=251.0Hz), 145.5 (dd, J-246.0, 13.8 Hz), 141.35, 50 according to claim 1, and a pharmaceutically acceptable adju 137.97, 137.86, 134.14, 134.05, 132.64, 132.34, 131.79, vant, diluent or carrier. 125.08 (d. J=2.7 Hz, 1C), 123.30, 122.41, 120.23, 119.21, 3. A method for the prophylaxis or treatment of a viral 116.93 (d. J=19.0 Hz), 116.78 (d. J=240 Hz), 111.30 (d. disease, the method comprising administering a therapeuti J=23.6 Hz). cally effective amount of a compound of the general formula ESIMS m/z calcd for CHF.N.O., 413.08. found: 55 (I), (II) or (III) or a pharmaceutically acceptable salt thereofto 413.19. a Subject in need of Such treatment: The invention claimed is: 1. A compound selected from: Ethyl 2-3-(benzoylamino)benzoylaminobenzoate Formula (I) 2-3-(Benzoylamino)benzoylaminobenzoic acid 60 Ethyl 2-4-(benzoylamino)benzoylaminobenzoate 2-2-(Acetylamino)benzoylaminobenzoic acid 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid 2-2-(Cyclohexanecarboxylamino)benzoylaminoben Zoic acid 65 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic acid US 8,993,622 B2 59 60 -continued 2-2-(Benzoylamino)benzoylamino-benzoic acid Formula (II) 2-2-(4-Methyl-benzoylamino)benzoylamino-benzoic R acid X X 2-3-(Benzoylamino)benzoylamino-benzamide 1'na r 2-3-(2-Methyl-benzoylamino)benzoylamino-benzoic R-- -HRs acid 2-2-(4-Methoxy-benzoylamino)benzoylamino-ben R3 R4 Zoic acid Formula (III) 4-Methyl-N-(2-(2-methylphenyl)carbamoylphenyl R 10 benzamide R Ethyl 2-3-(benzoylamino)benzoylaminobenzoate 2X1 X2 N 2-3-(Benzoylamino)benzoylaminobenzoic acid Ethyl 2-4-(benzoylamino)benzoylaminobenzoate 2-4-(Benzoylamino)benzoylaminobenzoic acid Yx IJK R4 O' 15 3 2-Benzoylamino-N-phenyl-benzamide 2-2-(Acetylamino)benzoylaminobenzoic acid wherein X is CO. NH . 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid X is CO. NH , 2-2-(Cyclohexanecarboxylamino)benzoylaminoben A is selected from the group consisting of hydrogen, Zoic acid —Calkyl, -(CH2)C,cycloalkyl, -(CH), phenyl, 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic —(CH2), naphthyl, -(CH), heteroaryl, -(CH), acid COH, and —(CH2)COCsalkyl, 2-2-(2-Fluorobenzoylamino)-benzoylaminobenzoic wherein alkyl, cycloalkyl, phenyl, naphthyl, and heteroaryl acid are unsubstituted or substituted with one to three sub 25 2-2-(3-Fluorobenzoylamino)-benzoylaminobenzoic stituents independently selected from halogen, —C. acid alkyl, and —C alkyloxy; 2-2-(4-Fluorobenzoylamino)-benzoylaminobenzoic R is selected from hydrogen, —COH, -COC-alkyl, acid —Calkyl, tetrazol-5-yl, and —CONRR: 2-2-(Phenylacetylamino)benzoylaminobenzoic acid R. R. R. and Rs are each independently selected from 30 Ethyl 2-2-(benzoylamino)benzoylaminobenzoate hydrogen, halogen, and —C alkyloxy; Ethyl 2-2-(acetylamino)benzoylaminobenzoate each R is independently selected from the group consist Ethyl 2-2-(trimethylacetylamino)benzoylaminoben ing of hydrogen and —Csalkyl; and ZOate n is 0, 1, 2, 3, 4, or 5. Ethyl 2-2-(cyclohexanecarboxylamino)benzoylamino 4. The method according to claim 3 wherein A is selected 35 from phenyl, naphthyl, and heteroaryl, unsubstituted or Sub benzoate stituted with one to three substituents independently selected Ethyl 2-2-(phenylacetylamino)benzoylaminobenzoate from halogen, —Calkyl, and —C alkyloxy. Ethyl 2-2-(4-carboxybutanoylamido)benzoylaminoben 5. The method according to claim 3 wherein R is selected ZOate from —COH, and —COC alkyl. 40 2-2-(Benzoylamino)benzoylaminobenzoic acid 6. The method according to claim 3 wherein the viral 2-2-(Acetylamino)benzoylaminobenzoic acid disease is selected from Adenovirus infections, Herpes virus 2-2-(Trimethylacetylamino)benzoylaminobenzoic acid infections, Papillomavirus infections, Parvovirus infections, 2-2-(Cyclohexanecarboxylamino)benzoylaminoben Polyomavirus infections, Poxvirus infections, Arbovirus Zoic acid infection, Arenavirus infections, Astrovirus infections, Bir 45 2-2-(Phenylacetylamino)benzoylaminobenzoic acid navirus infections, Bunyavirus infections, Calicivirus infec 2-2-(4-Carboxybutanoylamido)benzoylaminobenzoic tions, Coronavirus infections, Flavivirus infections, Hantavi acid rus infections, Hepatitis virus infections, including Hepatitis 2-2-(benzoylamino)benzoylaminophenylacetic acid A, Hepatitis B. Hepatitis C, and Hepatitis D. Mononegavirus 2-2-(benzoylamino)phenylacetylaminobenzoic acid infections, including Filovirus infections, Paramyxovirus 50 2-2-(2-fluorobenzoylamino)-benzoylamino-4-chlo infections, and Rhabdovirus infections, Nidovirales Infec robenzoic acid tions, Orthomyxoviridae infections including influenza virus 2-2-(2-fluorobenzoylamino)benzoylamino-5-chlo infections, Picornavirus infections including Enterovirus robenzoic acid infections, Reovirus infections including Rotavirus infec 2-2-(2-fluorobenzoylamino)-benzoylamino-4-methoxy tions, Retrovirus infections including lentivirus infections, 55 benzoic acid Such as HIV infections, Togavirus infections including 2-2-(2-fluorobenzoylamino)-benzoylamino-5-methoxy Rubivirus infections. benzoic acid 7. The method according to claim 6 wherein the viral 2-2-(2-fluorobenzoylamino)-benzoylamino-4,5-difluo disease is an Adenovirus infection. robenzoic acid 8. The method according to claim 6 wherein the viral 60 2-4-chloro-2-(2-fluorobenzoylamino)-benzoylamino disease is selected from a Herpesvirus infection and a Picorna benzoic acid virus infection. 2-5-chloro-2-(2-fluorobenzoylamino)-benzoylamino 9. The method according to claim 8 wherein the herpes benzoic acid virus infection is caused by HSV-1, HSV-2 and/or varicella 2-4-methoxy-2-(2-fluorobenzoylamino)-benzoylamino Zoster virus. 65 benzoic acid 10. The method according to claim 3 wherein the com 2-5-methoxy-2-(2-fluorobenzoylamino)-benzoylamino pound is selected from benzoic acid and US 8,993,622 B2 61 62 2-4,5-difluoro-2-(2-fluorobenzoylamino)-benzoy laminobenzoic acid or a pharmaceutically acceptable salt thereof. 11. The method according to claim3, wherein A is selected from the group consisting of hydrogen, —Csalkyl, 5 —(CH2)C cycloalkyl, -(CH2)phenyl, -(CH2)COH, and —(CH2)COC-alkyl, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted with one to three substituents independently selected from halogen, —Calkyl, and —C alkyloxy. 10 12. The method according to claim 3, wherein A is phenyl, unsubstituted or substituted with one to three substituents independently selected from halogen, —Calkyl, and —C alkyloxy. 13. The method according to claim 3 wherein n is 0 or 1. 15 k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,993,622 B2 Page 1 of 1 APPLICATIONNO. : 13/703481 DATED : March 31, 2015 INVENTOR(S) : Göran Wadell et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Claims Column 59, Line 31, In Claim 3, before “and” insert ---C1-6 alkyl --.

Signed and Sealed this Thirteenth Day of October, 2015 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office