US 20180185408A1 as) United States a2) Patent Application Publication 0) Pub. No.: US 2018/0185408 A1 Liu et al. (43) Pub. Date: Jul. 5, 2018 (54) ANTIFUNGAL NYLON-3 POLYMERS AND (52) U.S. Cl. COMBINATION THERAPY USING THEM TO CPC wee AGIK 31/785 (2013.01); AGIP 31/10 TREAT FUNGAL INFECTIONS (2018.01) (71) Applicant: Wisconsin Alumni Research (57) ABSTRACT Foundation, Madison, WI (US) A method and corresponding pharmaceutical composition ; — . ; for inhibiting the growth of fungi. The method uses an (72) Inventors: Runhui Liu, Shanghai (CN): Bernard antifungal composition containing as the active ingredient Gellman Madison, WI (U8); Fane one or more nylon-3 copolymers having a formula: ellman, Madison, ; Fang Yun Lim, Madison, WI (US); Leslie Anne Rank, Madison, WI (US); Nancy 0 P. Keller, Madison, WI (US); Jin Woo Bok, Madison, WI (US); Christina M. A NH Hull, Middleton, WI (US); Naomi Marie Walsh, Madison, WI (US); R! 22 Mingwei Huang, Madison, WI (US) R / H3N (21) Appl. No.: 15/850,416 ® (22) Filed: Dec. 21, 2017 or a salt thereof, Related U.S. Application Data wher -adependently hvd C.-C alkyl eac is independently hydrogen or C,-C,-alkyl; (60) Provisional application No. 62/437,151, filed on Dec. each R!, R3, R*, R°, and R®° are each independently 21, 2016. selected from the group consisting of hydrogen or sae : . substituted or unsubstituted C,-C,-alkyl; Publication Classification each R? is C,-C-alkylene; (51) Int. CL “A” is hydrogen or an amino-protecting group; A6IK 31/785 (2006.01) “B” is hydroxyl or a carboxy-protecting group; and A6IP 31/10 (2006.01) “xX,” “Y,” and “Z” are positive numbers. 9 oO 9 p cl 9 Q —~ HN + HN {Bu 5 mol % N s ys LiN(YMS), R THE, rm temp tBu G, 15 BocHN Deprotect . (4) (4) pMM prM MM:TM 40 mol % 60 mol % (heterochiral) Patent Application Publication Cl tBu 5 mol % Jul. > LiN(TMS), THF, rm temp 5,2018 tBu BocHN Deprotect (+) (+) Sheet BMM BTM MM:TM 40 mol % 60 mol % (heterochiral) 1 of 4 FIG. 1 US 2018/0185408 Al Patent Application Publication Jul. 5,2018 GA) oe OH Sheet 72°01 2 50 of 4 Fibroblasts Macrophages Fikroblasta Macrophages ARBCSs (NIH 373) {RAW 264.7} {NIH 373) [RAW 264.7) US 2018/0185408 FIG. 2A FIG. 2B Al Patent Application M 40:80 remolysis Profile ws Publication Jul. 5, 2018 Sheet ATrery 3 SL } of 4 US 2018/0185408 aniimicronial concentration, ug/ml Al Patent Application Publication Jul. 5, 2018 Sheet 4 of 4 US 2018/0185408 Al uM 360.3 BM 3.0 MM:T™ 4A 4B uM FIG. FIG. 0.36 Concentration MM:TM: (Control 0 Cancentration Pot | © in as Mm ac _ a {uO} SAPD O 1-9 LG} UIAOID OOH US 2018/0185408 Al Jul. 5, 2018 ANTIFUNGAL NYLON-3 POLYMERS AND field. See, for example, Seebach et al. (1996) Helv. Chim. COMBINATION THERAPY USING THEM TO Acta. 79:913-941; and Seebach et al. (1996) Helv. Chim. TREAT FUNGAL INFECTIONS Acta. 79:2043-2066. In the first of these two papers Seebach et al. describe the synthesis and characterization of a B-hexa- CROSS-REFERENCE TO RELATED peptide, namely (H-B-HVal-B-HAla-fB-HLeu) 2-OH. Inter- APPLICATIONS estingly, this paper specifically notes that prior art reports on the structure of B-peptides have been contradictory and [0001] Priority is hereby claimed to provisional applica- tion Ser. No. 62/437,151, filed Dec. 21, 2016, which is “partially controversial.” In the second paper, Seebach et al. explore the secondary structure of the above-noted (6 -hexa- incorporated herein by reference. peptide and the effects of residue variation on the secondary FEDERAL FUNDING STATEMENT structure. [0006] Dado and Gellman (1994) J. Am. Chem. Soc. [0002] This invention was made with government support 116:1054-1062 describe intramolecular hydrogen bonding under GM093265 and Al065728 awarded by the National in derivatives of B-alanine and y-amino butyric acid. This Institutes of Health. The government has certain rights in the paper postulates that B-peptides will fold in manners similar invention. to a-amino acid polymers if intramolecular hydrogen bond- ing between nearest neighbor amide groups on the polymer FIELD OF THE INVENTION backbone is not favored. [0003] Disclosed are nylon-3 copolymers that are broadly [0007] Suhara et al. (1996) Tetrahedron Lett. 37(10):1575- effective to inhibit the growth of fungi, while displaying 1578 report a polysaccharide analog of a B-peptide in which only mild to moderate toxicity against mammalian host cells D-glycocylamine derivatives are linked to each other via a (including human host cells). Thus, the present disclosure is C-1 B-carboxylate and a C-2 a-amino group. This class of directed to these nylon-3 copolymers and their use, alone compounds has been given the trivial name “carbopeptoids.” and in combination with other antifungal agents, to prevent [0008] Regarding methods to generate combinatorial and to inhibit fungal infections. libraries, several reviews are available. See, for instance, Ellman (1996) Acc. Chem. Res. 29:132-143 and Lam et al. BACKGROUND (1997) Chem. Rev. 97:411-448. [0004] Many naturally occurring, biologically active com- [0009] In the recent patent literature relating to B-poly- pounds are proteins or peptides based upon a-amino acids peptides, see, for example, U.S. published patent applica- (i.e., sequences of a-amino acids in which the a-carboxyl tions 2008/0166388, titled “Beta-Peptides with Antifungal group of one amino acid is joined by an amide bond to the Activity”; 2008/0058548, titled Concise Beta2-Amino Acid a-amino group of the adjacent amino acid). In recent years Synthesis via Organocatalytic Aminomethylation”; 2007/ an approach to the discovery of new pharmaceutically active 0154882, titled “Beta-polypeptides that inhibit cytomegalo- drugs has been to synthesize libraries of peptides and then to virus infection”; 2007/0123709, titled ‘““Beta-amino acids”; assay for compounds within the library which have a desired and 2007/0087404, titled “Poly-beta-peptides from func- activity, such as a desired binding activity. However, tionalized beta-lactam monomers and antibacterial compo- q-amino acid peptides are not altogether satisfactory for sitions containing same.” See also U.S. published patent pharmaceutical uses, in particular because they are often application 2003/0212250, titled “Peptides.” poorly absorbed and subject to proteolytic degradation in [0010] Invasive fungal disease in the US is associated with vivo. a mortality rate that often rises beyond 50 percent, empha- [0005] Much work on B-amino acids and peptides synthe- sizing the need for improved treatment strategies. (Brown G sized from 8-amino acids has been reported in the scientific D, Denning D W, Gow N A R, Levitz S M, Netea M G, and patent literature. See, for example, the work performed White T C. 2012. Hidden killers: human fungal infections. by a group led by current co-inventor Samuel H. Gellman, Sci Trans] Med 4:165rv13.) Current therapeutics are limited, including: “Application of Microwave Irradiation to the and many antifungal drugs lack efficacy or are toxic to Synthesis of 14-helical Beta-Peptides,” Murray & Gell- humans. (Butts A, Krysan D J. 2012. Antifungal Drug man,” Organic Letters (2005) 7(8), 1517-1520; “Synthesis Discovery: Something Old and Something New. PLOS of 2,2-Disubstituted Pyrrolidine-4-carboxylic Acid Deriva- Pathog 8:e1002870. Roemer T, Krysan D J. 2014. Antifun- tives and Their Incorporation into Beta-Peptide Oligomers,” gal drug development: challenges, unmet clinical needs, and Huck & Gellman, J Org. Chem. (2005) 70(9), 3353-62; new approaches. Cold Spring Harb Perspect Med 4.) Fur- “Effects of Conformational Stability and Geometry of thermore, increasing antifungal drug resistance has been Guanidinium Display on Cell Entry by Beta-Peptides,” observed. (Polvi E J, Li X, O’Meara T R, Leach M D, Potocky, Menon, & Gellman, Journal of the American Cowen L E. 2015. Opportunistic yeast pathogens: reservoirs, Chemical Society (2005) 127(11):3686-7; “Residue require- virulence mechanisms, and therapeutic strategies. Cell Mol ments for helical folding in short alpha/beta-peptides: crys- Life Sci CMLS 72:2261-2287. Verweij P E, Snelders EF, tallographic characterization of the 11-helix in an optimized Kema G H J, Mellado E, Melchers W J G. 2009. Azole sequence,” Schmitt, Choi, Guzei, & Gellman, Journal of the resistance in Aspergillus fumigatus: a side-effect of environ- American Chemical Society (2005), 127(38), 13130-1 and mental fungicide use? Lancet Infect Dis 9:789-795. Sangui- “Efficient synthesis of a beta-peptide combinatorial library netti M, Posteraro B, Lass-Flérl C. 2015. Antifungal drug with microwave irradiation,” Murray, Faroogi, Sadowsky, resistance among Candida species: mechanisms and clinical Scalf, Freund, Smith, Chen, & Gellman, Journal of the impact. Mycoses 58 Suppl 2:2-13. Shah D N, Yau R, Lasco American Chemical Society (2005), 127(38), 13271-80. TM, Weston J, Salazar M, Palmer H R, Garey K W. 2012. Another group, led by Dieter Seebach in Zurich, Switzer- Impact of Prior Inappropriate Fluconazole Dosing on Iso- land, has also published extensively in the beta-polypeptide lation of Fluconazole-Nonsusceptible Candida Species in US 2018/0185408 Al Jul. 5, 2018 Hospitalized Patients with Candidemia. Antimicrob Agents Acid-Activated Antimicrobial Random Copolymers: A Chemother 56:3239-3243.) The need for the development of Mechanism-Guided Design of Antimicrobial Peptide Mim- new antifungal drugs is obvious; however, only one new ics. Macromolecules 46:3959-3964. Sambhy V, Peterson B class of drugs (echinocandins), has been licensed within the R, Sen A. 2008. Antibacterial and Hemolytic Activities of past 15 years. (Ostrosky-Zeichner L, Casadevall A, Galgiani Pyridinium Polymers as a Function of the Spatial Relation- JN, Odds F C, Rex J H.