HK1232898B - Antifungal agents and uses thereof - Google Patents

Description

Antifungal agents and their applications

This application is a divisional application of the invention patent application with application date of March 2, 2012, application number 201280021321.9, and invention name “Antifungal Agents and Their Applications”.

Background of the Invention

The present invention relates to the field of treatment of fungal infections.

The need for new antifungal therapies is significant and particularly critical in medicine. Immunocompromised patients present perhaps the greatest challenge to contemporary healthcare delivery. The frequency of fungal infections in these patients has increased significantly over the past 30 years (Herbrecht, Eur. J. Haematol., 56:12, 1996; Cox et al., Curr. Opin. Infect. Dis., 6:422, 1993; Fox, ASM News, 59:515, 1993). Deep-seated mycoses are increasingly observed in patients undergoing organ transplants and those receiving aggressive cancer chemotherapy (Alexander et al., Drugs, 54:657, 1997). The most common pathogens associated with invasive fungal infections are the opportunistic yeast Candida albicans and the filamentous fungus Aspergillus fumigatus (Bow, Br. J. Haeinatol., 101:1, 1998; Wamock, J. Antimicrob. Chemother., 41:95, 1998). An estimated 200,000 patients acquire nosocomial fungal infections each year (Beck-Sague et al., J. Infect. Dis., 167:1247, 1993). The advent of acquired immunodeficiency syndrome (AIDS) has also contributed to the increased number of fungal infections, with virtually all patients contracting some form of mycoses during the course of their illness (Alexander et al., Drugs, 54:657, 1997; Hood et al., J. Antimicrob. Chemother., 37:71, 1996). The most common organisms encountered by these patients are Cryptococcus neoformans , Pneumocystis carinii , and C. albicans (HIV/AIDS Surveillance Report, 1996, 7(2), Year-End Edition; Polis, MA et al., AIDS: Biology, Diagnosis, Treatment and Prevention, Fourth Edition, 1997). New opportunistic fungal pathogens such as Penicillium marneffei , C. krusei , C. glabrata , Histoplasma capsulatum , and Coccidioides immitis have been frequently reported in immunocompromised patients worldwide.

The development of antifungal therapeutic regimens has been an ongoing challenge. Currently available drugs for the treatment of fungal infections include amphotericin B, a macrolide polyene that interacts with fungal membrane sterols; flucytosine, a fluoropyrimidine that interferes with fungal protein and DNA biosynthesis; and various azoles (e.g., ketoconazole, itraconazole, and fluconazole), which inhibit fungal membrane sterol biosynthesis (Alexander et al., Drugs, 54:657, 1997). Even though amphotericin B has a broad spectrum of activity and is considered the "gold standard" of antifungal therapy, its use is limited by infusion-related reactions and nephrotoxicity (Wamock, J. Antimicrob. Chemother., 41:95, 1998). Flucytosine's use is also limited by the development of resistant microorganisms and its narrow spectrum of activity. The widespread use of azoles is causing the emergence of drug-resistant strains of Candida spp. in the clinic. Due to problems associated with current treatments, there is an ongoing search for new treatments.

When the echinocandin caspofungin was approved for marketing in 2001, it represented the first new class of antifungal agents approved in a decade. Since then, two other echinocandin antifungals, anidulafungin and micafungin, have been approved for marketing in various ways. Each of these compounds works by inhibiting β-1,3-glucan synthase, a key enzyme in the synthesis of glucans in the cell walls of many fungi. All three drugs are semi-synthesized using natural products obtained through fermentation as starting materials.

The echinocandins are a broad group of antifungal agents that typically include a cyclic hexapeptide and a lipophilic tail attached to the hexapeptide core via an amide bond. Although many echinocandins are natural products, all clinically relevant members of this class are semi-synthetic derivatives. Although naturally occurring echinocandins have some degree of antifungal activity, they are not suitable as therapeutic agents, primarily due to poor water solubility, insufficient efficacy, and/or hemolysis. Approved echinocandins are products of intense effort to produce derivatives that maintain or improve the inhibitory effect on glucan synthase but do not cause hemolysis. As therapeutic agents, they are attractive compounds in terms of their systemic half-life, large therapeutic window, safety profile, and relative lack of interaction with other drugs. Unfortunately, the poor water solubility and poor intestinal absorption of these compounds have been attributed to their delivery via intravenous infusion. Although patients receiving these drugs often present with serious infections while hospitalized, the ability to transition patients from intravenous delivery while hospitalized to oral delivery at home would be highly desirable, especially given that treatment regimens typically last longer than 14 days. Additionally, oral echinocandins could expand the use of this drug class to include patients presenting with milder fungal infections.

SUMMARY OF THE INVENTION

The present invention features derivatives of echinocandin antifungal agents that may have increased water solubility. More particularly, the present invention features echinocandin compounds that have been modified so that they exhibit (i) activity against one or more fungal species or genera; (ii) increased water solubility and/or amphiphilicity; (iii) an increased therapeutic index; (iv) suitability for topical administration; (v) suitability for intravenous administration; (vi) an increased volume of distribution; and/or (vii) an increased elimination half-life.

The present invention is characterized by compounds of formula (I):

In formula (I), R ¹ is O(CH ₂ CH ₂ O) _n CH ₂ CH ₂ X _{1 , O ( CH 2 CH 2 CH 2 O ) n CH 2 CH 2 X 1 , NHCH 2 CH 2 , NH ( CH 2 CH 2 O} ) _{p CH 2 CH 2 X 3 , NH ( CH 2 CH 2 CH 2 O ) p CH 2 CH 2} CH{CH ₂ [OCH ₂ (CH ₂ ) _c ] _d X ₅ } ₂ , NH(CH ₂ CH ₂ NH) _r CH ₂ CH ₂ X ₅ , NHCH ₂ (CH ₂ ) _q X ₆ or OCH ₂ (CH ₂ ) _q X ₆ ; ^RT is n-pentyl, sec-pentyl or isopentyl; X ₁ is NH ₂ , NHR ^A1 , NR ^{A1 RA2} , NR ^{A1 RA2 RA3} or NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₂ is OH, OR ^B1 or OCH ₂ (CH ₂ ) _v Z ₁ ; X ₃ is NH ₂ , NHR ^C1 , NR ^{C1 RC2} , or NR ^{C1 RC2 RC3} or NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₄ is NR ^{D1 RD2 RD3} or NHCH ₂ (CH ₂ ) _v Z ₁ ; each X ₅ is independently selected from OH, OR ^E1 , NH ₂ , NHR ^E1 , NR ^E1 R ^E2 , NR ^E1 R ^E2 R ^E3 , OCH ₂ (CH ₂ ) _v Z ₁ and NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₆ is selected from NR ^F1 R ^F2 R ^F3 or Z ₁ ; a is an integer of 1-2; b is an integer of 0-3 (e.g., 0, 1, 2, or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2, or 3); n is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); m is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); p is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); r is an integer of 1-5 (e.g., ¹ , 2, 3, 4, or 5); q is an integer of 1-3 (e.g., 1, ² , or 3); v is an integer of 1-3 (e.g., 1, ² , or 3); ^RA1 , ^RA2 , ^RA3 , ^RB1 , ^RC1 , ^RC2 , RC3, ^RD1 , ^RD2 , ^RD3 , ER1, ^ER2 , ER3, ^RF1 , RR Each of ^F2 _and ^RF3 is independently selected _{from CH3} , _CH2CH3 , _{CH2CH2CH3 and} CH( _CH3 ) ₂ ; _Z1 is selected from:

and each of R ^1A , R ^2A , R ^3A , R ^4A , R ^5A , R ^6A , R ^7A , R ^8A , R ^9A , R ^10A , R ^11A , R ^12A , R ^13A , R ^14A , R ^15A , R ^16A , R ^17A , R ^18A , R ^19A , R ^20A , R ^21A and R ^22A is independently selected from H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ , or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of formula (I) is further described by formula (Ia):

In (Ia), R ¹ is O(CH ₂ CH ₂ O) _n CH ₂ CH ₂ X _{1 , O ( CH 2 CH 2 CH 2 O ) n CH 2 CH 2 X 1 , NHCH 2 CH 2 , NH ( CH 2 CH 2 O} ) _{p CH 2 CH 2 X 3 , NH ( CH 2 CH 2 CH 2 O ) p CH 2 CH 2} CH{CH ₂ [OCH ₂ (CH ₂ ) _c ] _d X ₅ } ₂ , NH(CH ₂ CH ₂ NH) _r CH ₂ CH ₂ X ₅ , NHCH ₂ (CH ₂ ) _q X ₆ or OCH ₂ (CH ₂ ) _q X ₆ ; ^RT is n-pentyl, sec-pentyl or isopentyl; X ₁ is NH ₂ , ^{NHR A1} , NR ^A1 RA2 or NR ^{A1 RA2 RA3} ; X ₂ is OH or OR ^B1 ; X ₃ is NH ₂ , NHR ^C1 , NR ^{C1 RC2} or NR ^{C1 RC2 RC3} ; X ₄ is NR ^{D1 RD2 RD3} ; each X ₅ is independently selected from OH, OR ^E1 , NH ₂ , NHR ^E1 , NR ^{E1 RE2} and NR ^{E1 RE2 RE3} ; X ₆ is selected from NR ^{F1 RF2 RF3} ; a is an integer of 1-2; b is an integer of 0-3 (e.g., 0, 1, 2, or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2, or 3); n is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); m is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); p is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); r is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); q is an integer of 1-3 (e.g., 1, 2, or 3); and each of ^RA1 , ^RA2 , RA3, RB1, ^RC1 , RC2, RC3, ^RD1 , ^RD2 , ^RD3 , RE1, ^RE2 , ^ER3 , RF1, RF2, and RF3 is independently selected from CH3, CH4, CH5, CH6, ^CH7 , ^CH8 , CH9, CH10, ^CH11 , CH12, CH13, RD1, ^RD2 , ^RD3 , RE1, RE2, ER3, ^RF1 , ^RF2 , and ^RF3 is independently selected from CH3, _CH4 , CH5 ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ , or a pharmaceutically acceptable salt thereof. In specific embodiments of the compounds of Formula (I) and (Ia), one of X ₁ , X ₃ , X ₄ and X ₅ is selected from N(CH ₃ ) ₃ ⁺ and N(CH ₂ CH ₃ ) ₃ ⁺ . In certain embodiments of the compounds of Formula (I) and (Ia), R ¹ is NHCH[CH ₂ CH ₂ N(CH ₃ ) ₃ ⁺ ] ₂ , NHCH ₂ CH ₂ OCH[CH ₂ CH ₂ N(CH ₃ ) ₃ ⁺ ] ₂ or NHCH ₂ CH ₂ OCH[CH ₂ CH ₂ N(CH ₃ ) ₃ ⁺ ][CH ₂ CH ₂ OCH ₂ CH ₂ OH].

In yet other embodiments, the compound of formula (I) is further described by formula (Ib):

In formula (Ib), R ¹ is O(CH ₂ CH ₂ O) _n CH ₂ CH ₂ X _{1 , O ( CH 2 CH 2 CH 2 O ) n CH 2 CH 2 X 1 , NHCH 2 CH 2 , NH ( CH 2 CH 2 O} ) _{p CH 2 CH 2 X 3 , NH ( CH 2 CH 2 CH 2 O ) p CH 2 CH 2} CH{CH ₂ [OCH ₂ (CH ₂ ) _c ] _d X ₅ } ₂ , NH(CH ₂ CH ₂ NH) _r CH ₂ CH ₂ X ₅ , NHCH ₂ (CH ₂ ) _q X ₆ or OCH ₂ (CH ₂ ) _q X ₆ ; ^RT is n-pentyl, sec-pentyl or isopentyl; X ₁ is NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₂ is OCH ₂ (CH ₂ ) _v Z ₁ ; X ₃ is NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₄ is NHCH ₂ (CH ₂ ) _v Z ₁ ; each X ₅ is independently selected from OCH ₂ (CH ₂ ) _v Z ₁ and NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₆ is Z ₁ a is an integer of 1-2; b is an integer of 0-3 (e.g., 0, 1, 2, or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2, or 3); n is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); m is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); p is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); r is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); q is an integer of 1-3 (e.g., 1, 2, or 3); v is an integer of 1-3 (e.g., 1, 2, or 3); _Z is selected from:

and each of R ^1A , R ^2A , R ^3A , R ^4A , R ^5A , R ^6A , R ^7A , R ^8A , R ^9A , R ^10A , R ^11A , R ^12A , R ^13A , R ^14A , R ^15A , R ^16A , R ^17A , R ^18A , R ^19A , R ^20A , R ^21A and R ^22A is independently selected from H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ , or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the compounds of formula (I), (Ia) and (Ib), the compounds are further described by one of the following formulae:

wherein ^R1 and ^RT are as described above.

The present invention is also characterized by compounds of formula (II):

_{In the formula} (II), R ² is NH(CH ₂ CH ₂ O) _{s CH 2 CH 2 X 8 , NH ( CH 2 CH 2 CH 2 O ) s CH 2 CH 2} X ₉ } ₂ , O[CH ₂ (CH ₂ ) _a O] _b CH{ ^CH ₂ [OCH _{2 (} CH ₂ ) _c ] _d X ₉ } ₂ ^, NHCH ₂ (CH ₂ ⁾ _u X ₁₀ or OCH _{2 (} CH ₂ ⁾ _u ^X ₁₀ ^; R ^G3 , OCH ₂ (CH ₂ ) _w Z ₂ or NHCH ₂ (CH ₂ ) _v Z ₂ ; each X ₉ is independently selected from OH, OR ^H1 , NHR ^H1 , NR ^H1 R ^H2 , NR ^H1 R ^H2 R ^H3 , OCH ₂ (CH ₂ ) _w Z ₂ and NHCH ₂ (CH ₂ ) _v Z ₂ ; X ₁₀ is selected from NR ^I1 R ^I2 R ^I3 or Z ₂ ; a is an integer of 1-2; b is an integer of 0-3 (e.g., 0, 1, 2 or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2 or 3); s is an integer of 1-5 (e.g., 1, 2, 3, 4 or 5); t is an integer of 1-5 (e.g., 1, 2, 3, 4 or 5); u is an integer of 1-3 (e.g., 1, 2 or 3); ^RG1 Each of ^RG2 , ^RG3 , ^RH1 , ^RH2 , ^RH3 , ^RI1 , ^RI2 and ^RI3 is independently selected from _CH3 , _CH2CH3 , _CH2CH2CH3 and CH( _CH3 ) ₂ ; w is an integer from 1 to 3 (e.g. _, 1, ₂ or 3); _Z2 is selected _from

and each of R ^1A , R ^2A , R ^3A , R ^4A , R ^5A , R ^6A , R ^7A , R ^8A , R ^9A , R ^10A , R ^11A , R ^12A , R ^13A , R ^14A , R ^15A , R ^16A , R ^17A , R ^18A , R ^19A , R ^20A , R ^21A and R ^22A is independently selected from H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ , or a pharmaceutically acceptable salt thereof.

In certain embodiments, compounds of formula (II) are further described by formula (IIa):

_{In the formula} (IIa), R ² is NH(CH ₂ CH ₂ O) _{s CH 2 CH 2} X _{8 , NH ( CH 2 CH 2 CH 2 O ) s CH 2 CH 2 d X 9} ^} ₂ ^, O[CH ₂ ^{( CH} _{2 ) a O ] b} ^CH _{{ CH 2 [ OCH 2 ( CH 2 ) c ] d} ^G2 R ^G3 ; each X ₉ is independently selected from OH, OR ^H1 , NHR ^H1 , NR ^H1 R ^H2 and NR ^H1 R ^H2 R ^H3 ; X ₁₀ is selected from NR ^I1 R ^I2 R ^I3 ; a is an integer of 1-2; b is an integer of 0-3 (e.g., 0, 1, 2 or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2 or 3); s is an integer of 1-5 (e.g., 1, 2, 3, 4 or 5); t is an integer of 1-5 (e.g., ¹ , ² , 3, 4 or 5); u is an integer of 1-3 (e.g., 1, 2 or 3); and each of RG1, ^RG2 , ^RG3 , ^RH1 , ^RH2 , RH3, ^RI1 , ^RI2 and ^RI3 is independently selected from CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ , or a pharmaceutically acceptable salt thereof. In specific embodiments of the compounds of formula (II) and (IIa), one of X ₈ and X ₉ is selected from N(CH ₃ ) ₃ ⁺ and N(CH ₂ CH ₃ ) ₃ ⁺ . In certain embodiments of the compounds of formula (II) and (IIa), R ² is NHCH[CH ₂ CH ₂ N(CH ₃ ) ₃ ⁺ ] ₂ , NHCH ₂ CH ₂ OCH[CH ₂ CH ₂ N(CH ₃ ) ₃ ⁺ ] ₂ , or NHCH ₂ CH ₂ OCH[CH ₂ CH ₂ N(CH ₃ ) ₃ ⁺ ][CH ₂ CH ₂ OCH ₂ CH ₂ OH].

In yet other embodiments, compounds of formula (I) are further described by formula (IIb):

_{In the formula} (IIb), R ² is NH(CH ₂ CH ₂ O) _{s CH 2 CH 2} X _{8 , NH ( CH 2 CH 2 CH 2 O ) s CH 2 CH 2 d X 9 } 2 , O [} CH _{2 ( CH 2 ) a O ] b CH { CH 2 [ OCH 2 ( CH 2 ) c ] d} ;Each X ₅ is independently selected _{from OCH2} ( _CH2 ) _wZ2 and NHCH2( _CH2 ) _vZ2 ; _X10 is _Z2 ; a is an integer _{of 1-2} ; b is an integer of 0-3 (e.g., 0, 1, 2 or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2 or 3); s is an integer of 1-5 (e.g., 1, 2, 3, 4 or 5); t is an integer of 1-5 (e.g., 1, 2, 3, 4 or 5); u is an integer of 1-3 (e.g., 1, 2 or 3); w is an integer of 1-3 (e.g., 1, 2 or 3); _Z2 is selected from

and each of R ^1A , R ^2A , R ^3A , R ^4A , R ^5A , R ^6A , R ^7A , R ^8A , R ^9A , R ^10A , R ^11A , R ^12A , R ^13A , R ^14A , R ^15A , R ^16A , R ^17A , R ^18A , R ^19A , R ^20A , R ^21A and R ^22A is independently selected from H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ , or a pharmaceutically acceptable salt thereof.

In a specific embodiment of the compounds of formula (II), (IIa) and (IIb), the compounds are further described by one of the following formulae:

wherein ^R2 is as described above.

The present invention also features compounds described by formula (III):

In formula (III), R ¹ is O(CH ₂ CH ₂ O) _n CH ₂ CH ₂ X _{1 , O ( CH 2 CH 2 CH 2 O ) n CH 2 CH 2 X 1 , NHCH 2 CH 2 , NH ( CH 2 CH 2 O} ) _{p CH 2 CH 2 X 3 , NH ( CH 2 CH 2 CH 2 O ) p CH 2 CH 2} CH{CH ₂ [OCH ₂ (CH ₂ ) _c ] _d X ₅ } ₂ , NH(CH ₂ CH ₂ NH) _r CH ₂ CH ₂ X ₅ , NHCH ₂ (CH ₂ ) _q X ₆ or OCH ₂ (CH ₂ ) _q X ₆ ; R ² is H, CH ₃ , CH ₂ CH ₂ NH ₂ or CH ₂ C(O)NH ₂ ; ^RT is n-pentyl, sec-pentyl or isopentyl; X ₁ is NH ₂ , NHR ^A1 , NR ^{A1 RA2} , NR ^{A1 RA2 RA3} or NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₂ is OH, OR ^B1 or OCH ₂ (CH ₂ ) _v Z ₁ ; X ₃ is NH ₂ , NHR ^C1 , NR ^{C1 RC2} or NR ^{C1 RC2 RC3} or NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₄ is NR ^D1 R ^D2 R ^D3 or NHCH ₂ (CH ₂ ) _v Z ₁ ; each X ₅ is independently selected from OH, OR ^E1 , NH ₂ , NHR ^E1 , NR ^E1 R ^E2 , NR ^E1 R ^E2 R ^E3 , OCH ₂ (CH ₂ ) _v Z ₁ and NHCH ₂ (CH ₂ ) _v Z ₁ ; X ₆ is selected from NR ^F1 R ^F2 R ^F3 or Z ₁ ; a is an integer of 1-2; b is an integer of 0-3 (e.g., 0, 1, 2, or 3); c is an integer of 1-2; d is an integer of 0-3 (e.g., 0, 1, 2, or 3); n is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); m is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); p is an integer of 1-5 (e.g., 1, 2, 3, 4, or 5); r is an integer of 1-5 (e.g., ¹ , 2, 3, 4, or 5); q is an integer of 1-3 (e.g., 1, ² , or 3); v is an integer of 1-3 (e.g., 1, ² , or 3); ^RA1 , ^RA2 , ^RA3 , ^RB1 , ^RC1 , ^RC2 , RC3, ^RD1 , ^RD2 , ^RD3 , ER1, ^ER2 , ER3, ^RF1 , RR Each of ^F2 _and ^RF3 is independently selected _{from CH3} , _CH2CH3 , _{CH2CH2CH3 and} CH( _CH3 ) ₂ ; _Z1 is selected from:

and each of R ^1A , R ^2A , R ^3A , R ^4A , R ^5A , R ^6A , R ^7A , R ^8A , R ^9A , R ^10A , R ^11A , R ^12A , R ^13A , R ^14A , R ^15A , R ^16A , R ^17A , R ^18A , R ^19A , R ^20A , R ^21A and R ^22A is independently selected from H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ and CH(CH ₃ ) ₂ or a pharmaceutically acceptable salt thereof. In a particular embodiment of the compound of formula (III), one of X ₁ , X ₃ , X ₄ , X ₅ and X ₆ is selected from N(CH ₃ ) ₃ ⁺ and N(CH ₂ CH ₃ ) ₃ ⁺ .

In a specific embodiment of the compound of formula (III), the compound is further described by one of the following formulae:

wherein ^R1 and ^RT are as described above.

Compounds of the present invention include, but are not limited to, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22 and salts thereof.

Compounds of the invention may have increased amphiphilicity; increased aqueous solubility (eg, in 0.1 M acetate buffer at pH 5.6); increased therapeutic index; increased elimination half-life; and/or increased volume of distribution.

The present invention also features a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In a specific embodiment, the pharmaceutical composition comprises an acetate salt or a hydrochloride salt of a compound of the invention.

The pharmaceutical compositions of the present invention may be formulated in unit dosage form, or any other dosage form described herein, for intravenous, topical, or oral administration.

The present invention is further characterized in that it is a method for treating a fungal infection in a patient, the method being carried out by administering a pharmaceutical composition of the present invention to the patient in an amount sufficient to treat the infection. In a specific embodiment, the pharmaceutical composition is administered intravenously or topically. The pharmaceutical composition can be administered to treat a patient's bloodstream infection, tissue infection (e.g., lung, kidney, or liver infection) or any other type of infection described herein. The fungal infection to be treated can be selected from the following infections: scalp ringworm, tinea corporis, tinea pedis, onychomycosis, perionychia (perionychomycosis), psoriasis versicolor, thrush, vaginal candidiasis, respiratory candidiasis, biliary candidiasis, esophageal candidiasis, urinary candidiasis, systemic candidiasis, mucosal and skin candidiasis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis, or chronic sinusitis. In certain embodiments, the infection to be treated is an infection caused by Candida albicans , C. parapsilosis , C. glabrata, C. guilliermondii, C. krusei , C. lusitaniae , C. tropicalis, Aspergillus fumigatus, A. flavus , A. terreus , A. niger , A. candidus , A. clavatus , or A. ochraceus .

The present invention is characterized by a method for preventing fungal infection in a patient, the method being carried out by giving the patient's pharmaceutical composition of the present invention with an amount sufficient to prevent infection. In a specific embodiment, the pharmaceutical composition is administered intravenously at least once in the period of 1-30 days (such as 1, 2, 3, 4 or 5 times in the period of 1-30 days). For example, the method of the present invention can be used to prepare a patient for invasive medical measures (such as, preparing for surgery, such as receiving transplantation, stem cell therapy, transplants, prosthetics, receiving long-term or frequent intravenous catheterization, or receiving treatment in an intensive care unit), in a patient with weakened immunity (such as, suffering from cancer, suffering from HIV/AIDS, or taking an immunosuppressant), or in a patient undergoing long-term antibiotic therapy, for preventive treatment.

In one embodiment of any of the methods of the invention, the pharmaceutical composition comprises Compound 1, or any other compound described herein, or a pharmaceutically acceptable salt thereof.

The invention also features a method of preventing, stabilizing, or inhibiting the growth of or killing fungi by contacting a fungus or a site susceptible to fungal growth with a compound of the invention, or a pharmaceutically acceptable salt thereof.

As used herein, the terms "sufficient amount" and "adequate amount" refer to the amount of a drug required to treat or prevent an infection. A sufficient amount for practicing the present invention to therapeutically treat or prophylactically treat a condition caused or contributed to by an infection will vary depending on the route of administration, the type of infection, and the patient's age, weight, and general health. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "adequate" amount.

By "fungal infection" is meant an invasion of a host by pathogenic fungi. For example, an infection can include an overgrowth of fungi normally present in or on a patient's body, or the growth of fungi not normally present in or on a patient's body. More generally, a fungal infection can be any condition in which the presence of a fungal population is harmful to the host organism. Thus, a patient is "suffering from" a fungal infection when an excessive fungal population is present in or on a patient's body, or when the presence of a fungal population damages the patient's cells or other tissues.

By "increased amphiphilicity" is meant that the compounds of the present invention have increased solubility in both water (in 0.1 M acetate buffer at pH 5.6) and glycerol compared to the parent echinocandin compound (i.e., compounds of formula (I), (Ia) and (Ib) may have increased amphiphilicity compared to anidulafungin; compounds of formula (II), (IIa) and (IIb) may have increased amphiphilicity compared to caspofungin; and compounds of formula (III) may have increased amphiphilicity compared to micafungin).

By "increased elimination half-life" is meant that the compounds of the invention have an increased elimination half-life (e.g., as observed in the PK studies described in Example 24) when administered under the same conditions (e.g., with the same carrier and other inactive excipients and by the same route) compared to the parent echinocandin compound (i.e., the compounds of Formula (I), (Ia), and (Ib) have an increased elimination half-life compared to anidulafungin; the compounds of Formula (II), (IIa), and (IIb) have an increased elimination half-life compared to caspofungin; and the compound of Formula (III) has an increased elimination half-life compared to micafungin). The compounds of the invention may exhibit an elimination half-life that is at least 25%, 50%, 100%, 200%, or 300% longer than the corresponding parent echinocandin compound.

By "increased volume of distribution" is meant that the compounds of the invention may have an increased volume of distribution (e.g., as observed in the PK study described in Example 24) when administered under the same conditions (e.g., with the same carrier and other inactive excipients and by the same route) compared to the parent echinocandin compound (i.e., the compounds of Formula (I), (Ia), and (Ib) may have an increased volume of distribution compared to anidulafungin; the compounds of Formula (II), (IIa), and (IIb) may have an increased volume of distribution compared to caspofungin; and the compound of Formula (III) may have an increased volume of distribution compared to micafungin). The compounds of the invention may exhibit a volume of distribution that is at least 25%, 50%, 100%, 200%, or 300% greater than the corresponding parent echinocandin compound.

By "enhanced therapeutic index" is meant that, compared to the parent echinocandin compound, administration of the compound of the present invention under the same conditions (e.g., with the same carrier and other inactive excipients and by the same route) increases the ratio of the median lethal dose (LD ₅₀ ) to the median effective dose (ED ₅₀ ) (e.g., as observed using a mouse model of infection) (i.e., the compounds of Formula (I), (Ia), and (Ib) may have an increased therapeutic index compared to anidulafungin; the compounds of Formula (II), (IIa), and (IIb) may have an increased therapeutic index compared to caspofungin; and the compound of Formula (III) may have an increased therapeutic index compared to micafungin). The compounds of the present invention may exhibit a therapeutic index that is at least 25%, 50%, 100%, 200%, or 300% greater than the corresponding parent echinocandin compound. For example, the compounds of the present invention may exhibit a prolonged circulating half-life in vivo compared to the parent echinocandin compound, allowing similar efficacy to be achieved at a lower dose.

As used herein, the term "treating" refers to the administration of a pharmaceutical composition for prophylactic and/or therapeutic purposes. For "preventing a disease," it refers to the prophylactic treatment of a subject who is not already suffering from the disease, but who is susceptible to or at risk for the disease. For "treating a disease," or for "curative treatment," it refers to the treatment of a patient already suffering from the disease to improve or stabilize the patient's condition. Thus, in the claims and embodiments, treatment refers to the administration of a pharmaceutical composition to a patient for either therapeutic or prophylactic purposes.

The term "unit dosage form" refers to a physically discrete unit suitable for use as a unit dosage, such as a pill, tablet, sugar-coated tablet, hard capsule, or soft capsule, each unit containing a predetermined amount of drug. By "hard capsule" is meant a capsule comprising a cover film forming a two-part, capsule-shaped container capable of carrying a solid or liquid payload of drug and excipients. By "soft capsule" is meant a capsule molded into a single container carrying a liquid or semisolid payload of drug and excipients.

Other features and advantages of the invention will become apparent from the following detailed description, drawings, and claims.

Attached photos

FIG1 is a table of MEC and MIC values for inhibition of Aspergillus spp. obtained using the method described in Example 25.

FIG2 is a table of MIC values obtained at 24 and 48 hours against Candida spp. using the method described in Example 26.

3A and 3B are reverse phase HPLC chromatograms of a mixture of anidulafungin and an isomer of Compound 1 ( FIG. 3A ) and a pure sample of Compound 1 ( FIG. 3B ). The chromatograms were obtained using the method described in Example 30.

Detailed description

The present invention features echinocandin compounds that have been modified so that they exhibit (i) activity against one or more fungal species or genera; (ii) increased water solubility and/or amphiphilicity; (iii) an increased therapeutic index; (iv) amenable to topical administration; (v) amenable to intravenous administration; (vi) an increased volume of distribution; and/or (vii) an increased elimination half-life.

synthesis

The compounds of the present invention include compounds of formula (I), (II) and (III). These compounds can be synthesized, for example, by coupling echinocandins with suitable acyl, alkyl, hydroxyl and/or amino groups, either functionalized or non-functionalized, under standard reaction conditions, as described in the Examples.

Typically, the semi-synthetic echinocandin compounds of the present invention can be prepared by modifying the naturally occurring echinocandin framework. For example, pneumocandin _B0 is prepared by a fermentation reaction; wherein the fermentation and mixing broth produces a mixture of products, which are then separated to produce pneumocandin _B0 , which is used to synthesize caspofungin (see US Pat. No. 6,610,822, which describes the extraction of echinocandin compounds, such as pneumocandin _B0 , WF 11899, and echinocandin B, by performing several extraction methods; and see US Pat. No. 6,610,822, which describes methods for purifying crude extracts).

For the semi-synthetic routes to the compounds of the present invention, the stereochemistry of the compound will be determined by the starting materials. Thus, the stereochemistry of the non-natural echinocandin derivatives will typically have the same stereochemistry as the naturally occurring echinocandin framework from which they are derived (representative stereochemistries are described in the Examples). Thus, any of the following compounds: anidulafungin, caspofungin, or micafungin can be used as starting materials for the synthesis of the compounds of the present invention, which share the same stereochemical configuration for each amino acid residue found in the naturally occurring compound.

Therefore, the echinocandins of the present invention can be derived from cyclic peptide antifungal agents produced by culturing various microorganisms.

The compounds of the present invention can be synthesized, for example, using the methods described in the Examples.

The compounds of the present invention can also be used as starting materials for the synthesis of compounds of formula (Ib) and (IIb). For example, amine-terminated compounds can be used to prepare guanidine derivatives. The conversion of amino groups to guanidine groups can be accomplished using standard synthetic protocols. For example, Mosher has described a general method for preparing mono-substituted guanidines by reacting aminoimidomethanesulfonic acids with amines (Kim, K.; Lin, Y.-T.; Mosher, HS Tetrahedron Lett. 29: 3183, 1988). A convenient method for the guanylation of primary and secondary amines was developed by Bernatowicz using 1H -pyrazole-1-carboxamidine hydrochloride; 1-H-pyrazole-1-(N,N'-bis(tert-butyloxycarbonyl)carboxamidine; or 1-H-pyrazole-1-(N,N'-bis(benzyloxycarbonyl)carboxamidine. These reagents react with amines to give mono-substituted guanidines (see Bernatowicz et al., J. Org. Chem. 57:2497, 1992; and Bernatowicz et al., Tetrahedron Lett. 34:3389, 1993). In addition, thioureas and S-alkyl-isothioureas have been shown to be useful intermediates for the synthesis of substituted guanidines (Poss et al., Tetrahedron Lett. 33 :5933). 1992). For example, compounds of formula (Ib) and (IIb) containing heterocycles can be synthesized by coupling hydroxyalkyl or aminoalkyl substituted heterocycles to the parent echinocandin compound using coupling methods such as those described in the Examples.

Therapies and preparations

The present invention features compositions and methods for treating or preventing diseases or conditions associated with fungal infections (e.g., yeast infections) by administering compounds of the invention. The compounds of the invention can be administered by any suitable route for treating or preventing diseases or conditions associated with fungal infections. The compounds can be administered to humans, domestic pets, livestock, or other animals together with a pharmaceutically acceptable diluent, carrier, or excipient. When administered orally, the compounds can be in unit dosage form or as a liquid oral dosage form. Administration can be topical, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, via suppository, or oral administration.

The therapeutic agent can be in the form of a liquid solution or suspension; in the form of a tablet or capsule, syrup or oral liquid dosage form for oral administration; in the form of a powder, nasal drops for intranasal preparations; formulated as ear drops; as an aerosol, or formulated for topical administration, such as a cream or ointment.

Methods for preparing formulations well known in the art can be found, for example, in "Remington: The Science and Practice of Pharmacy" (20th edition, ed. A.R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, a polyalkylene glycol such as polyethylene glycol, an oil of plant origin, or a hydronaphthalene. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycholate, and deoxycholate, or may be oily solutions for administration as nasal drops, or as a gel. The concentration of the compound in the formulation will vary depending on a variety of factors, including the dose of the drug to be administered and the route of administration.

The compound or combination can optionally be administered as a pharmaceutically acceptable salt, such as an acid addition salt; a metal salt formed by replacing an acidic proton with a metal, such as an alkali metal or alkaline earth metal salt (e.g., sodium, lithium, potassium, magnesium, or calcium salt); or a metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids such as tannic acid and carboxymethylcellulose; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Metal complexes include zinc and iron, among others.

The preparation utilized for oral administration comprises tablet, and it comprises the active component as the mixture with nontoxic pharmaceutically acceptable excipient.These excipients can be for example inert diluent or filler (as, sucrose and sorbitol), lubricant, glidant and anti-binding agent (as, magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talcum powder).The preparation utilized for oral administration can also be unit dosage form, as chewable tablet, tablet, sugar-coated tablet, or capsule (that is, as hard gelatin capsule, active component wherein mixes with inert solid diluent, or as soft gelatin capsule, active component wherein mixes with water or oil medium) provide.

The compounds of the present invention can be formulated with excipients that improve the oral bioavailability of the compounds. For example, the compounds of the present invention can be formulated for oral administration with medium-chain (C8-C12) fatty acids (or pharmaceutically acceptable salts thereof), such as capric acid, caprylic acid, lauric acid, or pharmaceutically acceptable salts thereof, or mixtures thereof. The formulations may optionally include medium-chain (C8-C12) alkyl alcohols among other excipients. Alternatively, the compounds of the present invention can be formulated for oral administration with one or more medium-chain alkyl sugars (e.g., alkyl (C8-C14) β-D-maltoside, alkyl (C8-C14) β-D-glucoside, octyl β-D-maltoside, octyl β-D-maltopyranoside, decyl β-D-maltoside, tetradecyl β-D-maltoside, octyl β-D-glucoside, octyl β-D-glucopyranoside, decyl β-D-glucoside, dodecyl β-D-glucoside, tetradecyl β-D-glucoside) and/or medium-chain sugar esters (e.g., sucrose monocaprate, sucrose monocaprylate, sucrose monolaurate, and sucrose monotetradecanoate).

The formulation can be administered to a patient in a therapeutically effective amount. Typical dosages range from about 0.01 μg/kg to about 800 mg/kg, or about 0.1 mg/kg to about 50 mg/kg of body weight per day. The preferred dosage of the drug to be administered will likely depend on such variables as the type and extent of the disease, the overall health of the particular patient, the specific compound to be administered, the excipients used to formulate the compound, and its route of administration.

The compounds of the present invention can be used to treat, for example, scalp ringworm, body ringworm, tinea pedis, onychomycosis, perionychia, tinea versicolor, oral thrush, vaginal candidiasis, respiratory tract candidiasis, biliary tract candidiasis, esophageal candidiasis, urethral candidiasis, systemic candidiasis, mucosal and cutaneous candidiasis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis and chronic sinusitis.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, prepared, and evaluated, and are intended to be considered purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Analytical HPLC was performed using the following column and conditions: Phenomenex Luna C18(2), 5 μm, 100 Ǻ, 2.0 × 150 mm, 1-99% CH ₃ CN (0.1% TFA) in H ₂ O (0.1% TFA)/15 min. Preparative HPLC was performed using the following column: Waters Nova-Pak HR C18, 6 μm, 60 Ǻ, 19 × 300 mm, CH ₃ CN/H ₂ O various linear gradients and modifiers when necessary, 10 mL/min.

The following abbreviations are used in the following examples: min (minute), hr (hour), mmol (millimole), mL (milliliter), μm (micrometer), Ǻ (angstrom), THF (tetrahydrofuran), DMF (dimethylformamide), TLC (thin layer chromatography), TFA (trifluoroacetic acid), HPLC (high performance liquid chromatography), RP (reverse phase), DIEA (diisopropylethylamine), LC/MS (liquid chromatography/mass spectrometry), _TR (retention time on HPLC), C (Celsius), and FMOC (fluorenylmethoxycarbonyl).

Example 1. Synthesis of Compound 1.

Anidulafungin (5 mg; 0.004 mmol) dissolved in anhydrous DMSO (0.2 mL) was treated with choline chloride (13 mg; 0.093 mmol) and HCl (4 M in 1,4-dioxane; 1.0 μL; 0.004 mmol). The resulting solution was stirred at room temperature for 2 days and heated at 40°C for ~8 hr, then diluted with water and acetonitrile and purified by preparative RP HPLC, eluting with water (0.1% TFA)/CH ₃ CN (0.1% TFA). The product was isolated by lyophilization to afford 2.0 mg of compound 1 as a white solid. HPLC _TR 10.84 min (90%). LC/MS, ESI+ m/z 1225.60 [M] ⁺ .

Two alternative synthetic schemes are provided below.

Anidulafungin (3.00 g; 2.63 mmol) was suspended in dry THF (5 ml) and treated with phenylboronic acid (386 mg; 3.17 mol). The mixture was stirred until all solids dissolved (~30 min), then stirred for an additional 30 min. The THF was removed in vacuo at room temperature. The residue was redissolved in THF and concentrated to dryness, then suspended in dry _CH₃CN and concentrated to dryness to remove the water. The resulting solid was mixed with N,N-dimethylethanolamine hydrochloride (9.25 g; 73.6 mmol) in dry DMSO (10 mL) until dissolved. The resulting clear, viscous solution was treated with 4M HCl in dioxane (0.33 mL). The solution was stirred at room temperature for 3 days. The reaction was diluted with water (10 mL), and the resulting solution was then slowly added to a 300 mL aqueous solution of sodium bicarbonate (12.4 g; 2 eq. vs. amine hydrochloride) with vigorous stirring. The resulting flocculent precipitate was isolated by centrifugation. The solid was then triturated with 200 mL of water to yield a clear, homogeneous suspension, which was isolated by centrifugation. The solid was dissolved in DMSO (15.0 mL) and treated with DIEA (0.460 mL; 2.64 mmol), followed by _CH₃I (0.250 mL; 4.02 mmol), and the solution was stirred at room temperature for 20 min. Water (7 mL), methanol (7 mL), and acetic acid (0.300 mL) were added sequentially. The resulting solution was further diluted with water (7 mL) and purified by preparative RP HPLC, eluting with CH₃CN/aqueous 0.05 M ammonium acetate, pH 5.0. Fractions of interest were combined and concentrated in vacuo at 25°C, then lyophilized to yield 2.33 g of compound 1 as a white solid. HPLC _TR 10.84 min (>98%). LC/MS, ESI+/- m/z 1225.60 [M] ⁺ .

Anidulafungin (0.052 g; 0.046 mmol) was suspended in dry THF (~2 mL) and treated with phenylboronic acid (7 mg; 0.057 mol). The mixture was stirred until all solids dissolved (~30 min), then stirred for an additional 30 min. The THF was removed in vacuo at room temperature. The residue was redissolved in THF and concentrated to dryness, then suspended in dry _CH₃CN and concentrated to dryness to remove the water. The resulting solid was suspended in 20% TFA/ _CH₃CN (2.5 mL) at 0°C, treated with choline chloride (0.406 g; 2.9 mmol) and allowed to warm to room temperature. The solution was stirred at room temperature for 3 hours, then at 5°C overnight. The reaction was concentrated in vacuo at room temperature, then diluted with methanol and water and purified by preparative RP HPLC, eluting with _CH₃CN /aqueous 0.05 M ammonium acetate, pH 5.0. The fractions of interest were combined and concentrated in vacuo at 25°C, then lyophilized to afford 37 mg of compound 1 as a white solid. HPLC _TR 10.84 min (98%). LC/MS, ESI+/- m/z 1225.60 [M] ⁺ .

Example 2. Synthesis of Compound 2.

N,N-Dimethylethanolamine (9.9 μL; 0.100 mmol) in anhydrous DMF was treated with HCl (4 M in 1,4-dioxane; 26.0 μL; 0.104 mmol). Anidulafungin (5 mg; 0.004 mmol) was added, and the resulting solution was stirred at room temperature for 1 day and at 40°C for 3 days. The reaction was then diluted with water and acetonitrile and purified by preparative RP HPLC, eluting with water (0.1% TFA)/CH ₃ CN (0.1% TFA). The product was isolated by lyophilization to yield 2.1 mg of compound 2 as a white solid. HPLC _TR 10.94 min (86%). LC/MS, ESI+ m/z 1211.58 [M+H] ⁺ .

Example 3. Synthesis of Compound 3.

Anidulafungin (5 mg; 0.004 mmol) was mixed with N-methyl-2-aminoethanol hydrochloride (0.1 g; 0.9 mmol). Anhydrous DMSO (0.1 mL) was added, and the resulting solution was treated with HCl (4M in 1,4-dioxane; 1.0 μL; 0.004 mmol) and stirred at room temperature for 4 days. The reaction was then diluted with water and purified by preparative RP HPLC, eluting with water (0.1% TFA)/CH ₃ CN (0.1% TFA). The product was isolated by lyophilization to yield 3.1 mg of compound 3 as a white solid. HPLC _TR 10.98 min (98%). LC/MS, ESI+ m/z 1197.57 [M+H] ⁺ .

Example 4. Synthesis of Compound 4.

Ethanolamine (6.0 μL; 0.10 mmol) in anhydrous DMF was treated with HCl (4 M in 1,4-dioxane; 26.0 μL; 0.104 mmol). Anidulafungin (6.4 mg; 0.0056 mmol) was added to give a clear solution, which was stirred at room temperature for 16 days. The reaction was then diluted with water and acetonitrile and purified by preparative RP HPLC, eluting with water (0.1% TFA)/CH ₃ CN (0.1% TFA). The product was isolated by lyophilization to yield 2.8 mg of compound 4 as a white solid. HPLC _TR 10.90 min (95%). LC/MS, ESI+ m/z 1183.55 [M+H] ⁺ .

Example 5. Synthesis of Compound 5.

Anidulafungin (20 mg; 0.018 mmol) and 2-(2-aminoethoxy)ethanol hydrochloride (51 mg; 0.36 mmol) were dissolved in anhydrous DMSO (0.7 mL) and treated with HCl (4 M in 1,4-dioxane; 4.0 μL; 0.016 mmol). The resulting solution was stirred at room temperature for 4 days, then diluted with water and purified by preparative RP HPLC, eluting with water (0.1% TFA)/CH ₃ CN (0.1% TFA). The product was isolated by lyophilization to afford 13 mg of compound 5 as a white solid. HPLC _TR 10.82 min (>99%). LC/MS, ESI+/- m/z 1227.6 [M+H] ⁺ , 1225.6 [MH] ^- .

Example 6. Synthesis of Compound 6.

The 2-(2-aminoethoxy)-ethylhemiaminal ether of anidulafungin trifluoroacetate (18 mg; 0.013 mmol) was dissolved in anhydrous THF and concentrated to dryness at <30°C. The solid residue was dissolved in DMSO and treated with DIEA (9 μL; 0.052 mmol) followed by _CH₃I (2.5 μL; 0.040 mmol). The resulting solution was stirred at room temperature overnight and then treated with additional _CH₃I (1 μL; 0.016 mmol) and stirred for 2 more hours. The solution was then diluted with water and methanol and purified by preparative RP HPLC, eluting with water (0.1% TFA)/ _CH₃CN (0.1% TFA). The product was isolated by lyophilization to yield 9 mg of compound 6 as a white solid. HPLC T _R 10.92 min (>99%). LC/MS, ESI+/-m/z 1269.6 [M] ⁺ , 1267.6 [M-2H] ^- .

Example 7. Synthesis of Compound 7.

2-(2-Aminoethyl)-aminoethanol (139 mg; 1.33 mmol) in 0.5 mL of DMSO was treated with HCl (4 M in dioxane; 0.670 mL; 2.68 mmol) to give a biphasic mixture. Anidulafungin (35 mg; 0.031 mmol) was added, followed by an additional 0.5 mL of DMSO. The mixture was heated to 40°C to give a clear solution, which was heated at 35°C overnight. The solution was then diluted with water and methanol and purified by preparative RP HPLC, eluting with water (0.1% TFA)/ _CH3CN (0.1% TFA). The product was isolated by lyophilization to give 11 mg of compound 7 as a white solid. HPLC _TR 10.28 min (>95%). LC/MS, ESI+/- m/z 1226.6 [M] ⁺ , 1224.6 [M-2H] ^- .

Example 8. Synthesis of Compound 8.

Anidulafungin (450 mg; 0.395 mmol) was resuspended in acetonitrile (20 mL) and concentrated to dryness. The sample was then dissolved in 20 mL of anhydrous THF and concentrated to ~10 mL. Under an argon atmosphere, the solution was treated with phenylboronic acid (58 mg; 0.48 mmol) followed by activation of 3Å molecular sieves. The mixture was stirred slowly overnight, and the supernatant was transferred to a dry flask containing 2 x 5 mL THF rinses. The THF solution was concentrated to dryness, and the solid residue was suspended in 40 mL of dry _CH₃CN . The suspension was concentrated to 20 mL and cooled to -10°C under an argon atmosphere. It was then treated with 4-methoxythiophenol (75 μL; 0.47 mmol) and then TFA (2.4 mL). The resulting mixture was stirred at -15°C overnight and then quenched at -10°C by the slow addition of water until a flocculent precipitate formed. The mixture was stirred at 0°C for 30 minutes and then separated. Additional water was added to the suspension, producing additional precipitation. The precipitates were dried under vacuum, then combined and triturated with _Et₂O . The solid was isolated and triturated a second time with _Et₂O . The isolated solid was then dried under vacuum overnight to yield 393 mg of anidulafungin hemiaminealdehyde-(4-methoxy)phenyl sulfide as a white powder. HPLC _TR 13.3 min (88%). LC/MS, ESI+/- m/z 1187.60 [M+H] ⁺ , 1185.58 [MH] ^- .

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (21 mg; 0.017 mmol) was dissolved in 0.1 mL of 1-methyl-2-aminoethanol, resulting in a clear solution that was blanketed with argon and heated at 60°C for 3 hours, then stirred at room temperature overnight. The reaction was diluted with methanol, acidified by the addition of TFA, further diluted with water, and purified by preparative RP HPLC using _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 22 mg of compound 8 as a white solid. HPLC _TR 11.15 min (84%). LC/MS, ESI+/- m/z 1197.57 [M+H] ⁺ , 1219.55 [M+Na]+, 1195.56 [MH] ^- .

Example 9. Synthesis of Compound 9.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (20 mg; 0.016 mmol) was dissolved in 0.1 mL of 1-methyl-2-(2-aminoethoxy)ethanol, resulting in a clear solution that was blanketed with argon and heated at 60°C for 8 hours, then stirred at room temperature overnight. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RPHPLC using _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 15 mg of compound 9 as a white solid. HPLC _TR 11.18 min (86%). LC/MS, ESI+/- m/z 1241.60 [M+H] ⁺ , 1239.59 [MH] ^- .

Example 10. Synthesis of Compound 10.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (21 mg; 0.017 mmol) was dissolved in 0.1 mL of mPEG ₄ -NH ₂ , resulting in a clear solution that was blanketed with argon and heated at 50°C overnight, then at 65°C for 2 hours. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RP HPLC eluting with CH ₃ CN/H ₂ O and 0.1% TFA. The purified product was isolated by lyophilization to afford 13 mg of compound 10 as a white solid. _{HPLC TR} 11.26 min (88%). LC/MS, ESI+/- m/z 1329.65 [M+H] ⁺ , 1351.63 [M+Na] + , 1327.64 [MH] ^- .

Example 11. Synthesis of Compound 11.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (20 mg; 0.016 mmol) was dissolved in 0.3 mL of 2-(2-aminoethoxy)ethanol, resulting in a clear solution that was blanketed with argon and stirred overnight at room temperature, then heated at 60°C for 1 hour. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RP HPLC using _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 15 mg of compound 11 as a white solid. HPLC _TR 10.83 min (94%). LC/MS, ESI+/- m/z 1227.58 [M+H] ⁺ , 1225.57 [MH] ^- .

Example 12. Synthesis of Compound 12.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (19 mg; 0.015 mmol) was dissolved in 0.1 mL of 2-(2-aminoethoxy)-ethylamine, resulting in a clear solution that was blanketed with argon and heated at 60°C for 1.5 hr. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RP HPLC eluting with _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 11 mg of compound 12 as a white solid. HPLC _TR 10.06 min (94%). LC/MS, ESI+/- m/z 1226.60 [M+H] ⁺ , 1225.59 [MH] ^- .

Example 13. Synthesis of Compound 13.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (20 mg; 0.016 mmol) was dissolved in 0.1 mL of diamine, resulting in a clear solution. The solution was blanketed with argon and heated at 60°C for 1.5 hours, then allowed to stir overnight at room temperature. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RP HPLC using _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 11 mg of compound 13 as a white solid. HPLC _TR 10.06 min (92%). LC/MS, ESI+/- m/z 1270.62 [M+H] ⁺ , 1268.61 [MH] ^- .

Example 14. Synthesis of Compound 14.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (21 mg; 0.017 mmol) was dissolved in 0.1 mL of mono-BOC-NH-PEG ₄ -NH ₂ , resulting in a clear solution that was blanketed with argon and heated at 50°C until the product:starting material ratio was ~1:1. The reaction was treated with TFA (4 mL), stirred at room temperature for ~30 min, concentrated in vacuo, then diluted with water and purified by preparative RP HPLC eluting with CH ₃ CN/H ₂ O and 0.1% TFA. The purified product was isolated by lyophilization to afford 5 mg of compound 14 as a white solid. HPLC _TR 10.13 min (76%). LC/MS, ESI+/- m/z 1314.65 [M+H] ⁺ ,1312.64 [MH] ^- .

Example 15. Synthesis of Compound 15.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (49 mg; 0.039 mmol) suspended in 0.5 mL of _CH₃CN was treated with 0.3 mL of ethylenediamine at 0°C to give a clear solution, which was allowed to return to room temperature and stirred for 4 hours. The solution was diluted with 0.5 mL of methanol and 2 mL of water and acidified with TFA. The solution was further diluted with 2 mL each of methanol and water and then purified by preparative RPHPLC using _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 30 mg of compound 15 as a white solid. HPLC _TR 10.13 min (88%). LC/MS, ESI+/- m/z 1182.57 [M+H] ⁺ , 1180.56 [MH] ^- .

Example 16. Synthesis of Compound 16.

N,N-Dimethylaminoethyl anidulafungin aminal: Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (150 mg; 0.119 mmol) was dissolved in 0.5 mL of N,N-dimethylethylenediamine to give a pale yellow solution, which was heated at 45°C for 18 hours. The reaction was diluted with 80 mL of _Et₂O to precipitate the product. The collected solid was triturated with _Et₂O , then isolated and dried under vacuum overnight to yield 153 mg of N,N-dimethylaminoethyl anidulafungin aminal as an off-white solid. HPLC _TR 10.37 min (76%). LC/MS, ESI+/- m/z 1210.60 [M+H] ⁺ , 1208.60 [MH] ^- .

N,N-Dimethylaminoethyl anidulafungin aminal (153 mg; ≤ 0.119 mmol) in anhydrous DMSO was treated with _CH₃I (8 μL; 0.129 mmol) and stirred at room temperature overnight. The reaction was treated with additional _CH₃I (3 μL; 0.048 mmol) and DIEA (7 μL; 0.040 mmol) and stirred at room temperature for an additional 2 hours. The reaction was acidified with ~5 μL of TFA, diluted with water and methanol, and purified by preparative RP HPLC eluting with _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to yield 64 mg of compound 16 as a white solid. HPLC T _R 10.36 min (92%). LC/MS,ESI+/- m/z 1224.61 [M] ⁺ , 1222.61 [M-2H] ^- , 1268.61 [M+formate] ^- .

Example 17. Synthesis of Compound 17.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (82 mg; 0.065 mmol) was dissolved in 0.3 mL of diethylenetriamine, yielding a clear solution blanketed with argon and heated at 60°C for 45 min. The cooled solution was diluted with ether to precipitate the product, and the supernatant was removed. The solid was concentrated in vacuo, then dissolved in 1 mL of methanol and 1 mL of water and acidified with TFA. The resulting solution was purified by preparative RP HPLC, eluting with _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to yield 44 mg of compound 17 as a white solid. HPLC _TR 9.77 min (84%). LC/MS, ESI+/- m/z 1225.61 [M+H] ⁺ , 1223.61 [MH] ^- .

Example 18. Synthesis of Compound 18.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (19 mg; 0.015 mmol) was dissolved in 0.3 mL of N-(2-aminoethyl)-2-aminoethanol, resulting in a clear solution that was blanketed with argon and stirred overnight at room temperature, then heated at 60°C for 1.5 hours. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RPHPLC using _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 10 mg of compound 18 as a white solid. HPLC _TR 10.09 min (90%). LC/MS, ESI+/- m/z 1226.60 [M+H] ⁺ , 1225.59 [MH] ^- .

Example 19. Synthesis of Compound 19.

Pneumocandin _B0 hemiamine aldehyde-(4-methoxy)phenyl sulfide: Pneumocandin _B0 (509 mg; 0.48 mmol) was suspended twice in _CH3CN (25 mL) and concentrated in vacuo at room temperature to remove water. The sample was then suspended in anhydrous THF (25 mL) and concentrated to dryness. The residue was dissolved in anhydrous THF (25 mL) and concentrated to 10 mL at room temperature. The resulting suspension was treated with phenylboronic acid (69 mg; 0.57 mmol) and stirred at room temperature until all solids dissolved, resulting in a slightly cloudy solution. The solution was concentrated to dryness, and the remaining solids were suspended in _CH3CN (25 mL). The resulting suspension was concentrated to ~10 mL, cooled to -10°C under dry argon, and treated with 4-methoxythiophenol (72 μL), followed by the dropwise addition of trifluoroacetic acid (1.4 mL). The mixture was stirred at -15 to -20°C under argon for 2 days, then brought to 0°C and treated dropwise with 25 mL of water, yielding a white suspension. This was stirred at 0°C for 20 minutes and then separated. The solid was triturated and resuspended in 20 mL of 25% _CH₃CN / _H₂O , then separated and dried under vacuum. The resulting solid was triturated with diethyl ether. The supernatant was removed, and the solid was dried under vacuum to yield 430 mg of the title compound as a dense white powder. HPLC purification to 89%, UV det. at 216-224 nm; LC/MS ESI +/- m/z 1187.59 [M+H] ⁺ (calcd. 1187.59), 1185.58 [MH] ^- (calcd. 1185.58), 1045.55 [M-MeOPhSH] ^- (calcd. 1045.55).

Pneumocandin B _0- hemiaminealdehyde-(4-methoxy)phenylsulfideamine: Pneumocandin B _0- hemiaminealdehyde-(4-methoxy)phenylsulfideamine (533 mg; 0.45 mmol) suspended in anhydrous THF (15 mL) was treated with phenylboronic acid (67 mg; 0.55 mmol) and then treated with 3 Å molecular sieves (~15 Å beads). The mixture was stirred at room temperature overnight under argon to give a clear solution, which was concentrated to ~7 mL in a vented flask under a stream of dry argon. The solution was then transferred to a dry flask, and the molecular sieves were rinsed with 2 Å THF to give a reaction volume of ~10 mL. The solution was cooled to 0°C under argon and treated with BH ₃ -DMS (2.0 M in THF; 1.4 mL; 2.8 mmol) and stirred at 0°C. After 2 hr, anhydrous THF (2 mL) was added to dissolve the formed gel, followed by additional _BH₃ -DMS (0.5 mL; 1.0 mmol). The mixture was maintained at 0°C for over 1 hour and then stirred at room temperature for ~3 hr before being cooled to 0°C and quenched by the slow addition of 1 M HCl (1.3 mL). The quenched reaction was stored at -20°C overnight and then concentrated to ~3 mL. The resulting mixture was diluted with methanol and water (~1:1) to 20 mL and then purified by preparative RP HPLC eluting with 10-70% _CH₃CN / _H₂O (0.1% TFA). Fractions of interest were combined and lyophilized to yield 245 mg (42% TY) of the title compound as a bright white solid. HPLC purity: 94%, UV det. at 216-224 nm; LC/MS ESI +/- m/z 1173.61 [M+H] ⁺ (calcd. 1173.61), 1031.57 [M-MeOPhSH] ^- (calcd. 1031.57).

Pneumocandin hemiamine aldehyde-(4-methoxy)phenylsulfide amine (20 mg; 0.016 mmol) was dissolved in 2, 2'-(ethylenedioxy)bis(ethylamine) (0.1 mL). The solution was heated at 40°C overnight and then at 60°C for 1.5 hours before being diluted with methanol (0.5 mL) and water (2 mL) and acidified with TFA. The acidified mixture was further diluted with water and methanol and then purified by preparative RP HPLC eluting with water (0.1% TFA)/ _CH3CN (0.1% TFA). The product was isolated by lyophilization to afford 15 mg of compound 19 as a white solid. HPLC _TR 9.22 min (99%). LC/MS, ESI+/- m/z 1181.70 [M+H] ⁺ , 1179.70 [MH] ^- .

Example 20. Synthesis of Compound 20.

Pneumocandin hemiamine aldehyde-(4-methoxy)phenylsulfide amine (17 mg; 0.013 mmol) was dissolved in 2,2'-oxybis(ethylamine) (0.1 mL). The solution was heated at 40°C overnight, then diluted with methanol (0.5 mL) and water (2 mL) and acidified with TFA. The acidified mixture was further diluted with water and methanol, then purified by preparative RP HPLC, eluting with water (0.1% TFA)/ _CH3CN (0.1% TFA). The product was isolated by lyophilization to afford 12 mg of compound 20 as a white solid. HPLC _TR 9.22 min (96.0%). LC/MS, ESI+/- m/z 1137.68 [M+H] ⁺ , 1135.67 [MH] ^- .

Example 21. Synthesis of Compound 21.

Pneumocandin hemiamine aldehyde-(4-methoxy)phenylsulfide amine (19 mg; 0.015 mmol) was dissolved in 2-(aminoethylamino)ethanol (0.1 mL). The solution was heated at 60°C for 2 hours, then diluted with methanol (0.5 mL) and water (2 mL) and acidified with TFA. The acidified mixture was further diluted with water and methanol and then purified by preparative RP HPLC, eluting with water (0.1% TFA)/ _CH3CN (0.1% TFA). The product was isolated by lyophilization to afford 11 mg of compound 21 as a white solid. HPLC _TR 9.34 min (97.7%). LC/MS, ESI+/- m/z 1137.68 [M+H] ⁺ , 1135.67 [MH] ^- .

Example 22. Synthesis of Compound 22.

Pneumocandin hemiamine aldehyde-(4-methoxy)phenylsulfide amine (19 mg; 0.015 mmol) was dissolved in 2-(aminoethylamino)ethanol (0.1 mL). The solution was stirred at room temperature for 1 hr, heated at 60°C for 15 min, diluted with methanol (0.5 mL) and water (2 mL), and acidified with TFA. The acidified mixture was further diluted with water and methanol and then purified by preparative RPHPLC, eluting with water (0.1% TFA)/ _CH3CN (0.1% TFA). The product was isolated by lyophilization to afford 13 mg of compound 22 as a white solid. _{HPLC TR} 9.12 min (93.1%). LC/MS, ESI+/- m/z 1136.69 [M+H] ⁺ , 1134.68 [MH] ^- .

Example 23. In vivo activity following IP administration.

The purpose of these studies was to evaluate the effects of test compounds in a mouse model of candidiasis infection. The amount of test article was not corrected.

Inoculum preparation

Candida albicans ( C. albicans ) R303 strain was transferred from frozen stock onto Sabauroud dextrose agar (SDA) plates and grown at 35°C for ~24 hours. An inoculum was prepared by transferring colonies from the plates to phosphate-buffered saline (PBS) and adjusting the concentration to approximately ¹⁰ CFU/mL using a spectrophotometer. The stock solution was diluted 1:9 to prepare the inoculum. Concentration was verified using a dilution plate count method before each run.

Female CD-1 mice were used in this study. The animals were approximately 7 weeks old and weighed approximately 15-30 g at the start of the study.

Mice were rendered neutropenic by IP injection of cyclophosphamide (150 mg/kg in 10 mL/kg) 4 and 1 days prior to inoculation. Each animal was inoculated with the appropriate concentration by injection of 0.1 mL of inoculum into the tail vein. Test compounds were administered IP 2 hours after infection.

In a typical procedure, kidneys were collected from 4 mice in control group 1 (untreated) 2 hours after infection, and from another 4 mice in control group 2 (untreated) 24 hours after infection. Kidneys were aseptically removed from each mouse and combined in sterile test tubes. Aliquots (2 mL) of sterile PBS were added to each tube and the contents were homogenized using a tissue homogenizer (Polytron 3100). Serial dilutions of the tissue homogenate were performed and 0.1 mL aliquots were spread on SDA plates and incubated overnight at 35°C. CFU/kidney was determined from colony counts. Data were analyzed using either the Tukey-Kramer multiple comparison test or the Dunnett test (GraphPad InStat version 3.06, GraphPad Software (GraphPad Software), San Diego, CA).

The data reported below are the average of 4 mice. Each operation included an independent control and was tabulated independently.

First operation

Table 1

Second operation

Table 2

The third operation

Table 3

Fourth operation

Table 4

Fifth operation

Table 5

.

in conclusion

This mouse model was used as a primary screening tool to test the efficacy of the compounds of the present invention. The mice were made neutropenic to ensure that the observed results were attributable to the test article and to the fact that the mouse immune system had not been inoculated with Candida albicans, an organism known to accumulate in and infect the kidneys.

Kidneys were harvested from infected, but untreated, control mice 2 and 24 hours after infection. Kidneys were then evaluated for fungal load, as measured by the number of colony-forming units (CFU, reported on a logarithmic scale). As expected, untreated mice showed an increase in fungal load from 2 to 24 hours after inoculation with C. albicans .

Kidneys from mice infected with one of the test articles were removed and evaluated 24 hours later, showing that the level of fungal load varied with the test article. The lower the CFU, the more effective the compound was in treating the fungal infection in the kidney.

The best performing compounds are those with the best combination of the following properties: (i) activity (i.e., an inactive compound will not reduce fungal load), (ii) tissue penetration (i.e., a compound that does not enter the kidney will not treat infection there), and (iii) half-life (e.g., a compound with a short half-life may not show therapeutic effect at 24 hours).

Based on these studies, the inventors concluded that: (a) compounds 8, 9, 10, and 14 performed poorly (i.e., did not possess the right combination of properties in this assay to treat C. albicans infection); (b) compounds 2, 3, 11, and 13 performed moderately, demonstrating some ability to control C. albicans infection; and (c) compounds 1, 4, 5, 6, 7, 12, 15, 16, 17, 18, 19, 20, 21, and 22 performed strongly, significantly reducing the levels of C. albicans CFUs found in the kidneys of mice.

Example 24. Pharmacokinetics in Beagle Dogs

The test articles were administered to beagle dogs weighing approximately 6-10 kg. Each test article was administered at 1.4 mg/kg over 1-10 minutes in aqueous saline (with or without 0.5% Tween). Diphenhydramine must be kept on hand to prevent the dogs from exhibiting a histamine response. The dogs were fasted for at least 12 hours prior to each dosing and provided food 4 hours after blood sampling; no water was given 1 hour before and 4 hours after each dosing event. The dose administered to each animal was based on its most recent body weight. The test article was administered as a slow intravenous bolus through a catheter placed in the cephalic vein.

Blood was collected via the jugular vein. All blood samples (~1 mL each) were collected into _K3EDTA tubes. Immediately after blood collection, the samples were inverted for several minutes and kept on wet ice pending centrifugation. Within ~30 minutes of collection, the samples were centrifuged under refrigeration (~5°C at ~2000g for ~10 minutes) to obtain plasma. After separation, the plasma was immediately frozen on dry ice. Plasma samples were stored at approximately -70°C until analysis.

Plasma (100 μL) was precipitated with 400 μL of 0.1% formic acid in acetonitrile containing an internal standard (100 ng/mL pneumocandin). The sample was then blocked and vortexed for approximately 30 seconds, followed by centrifugation at 14,000 rpm for 10 minutes at room temperature. After centrifugation, 200 μL of the supernatant was transferred to a plastic autosampler vial containing 200 μL of 0.1% formic acid in water and vortexed. The sample was then analyzed by LCMS/MS.

All pharmacokinetic analyses were performed using WinNonlin version 4.1 (Pharsight Corp) by noncompartmental analysis. The results are provided in Table 6 below.

Table 6. PK value ¹

The observed half-life of anidulafungin was approximately 12 hours, which is consistent with previously reported values.

Compound 1 was found to have a surprisingly large volume of distribution and a surprisingly long half-life. These pharmacokinetic properties may provide advantages such as reduced dosing, reduced dosing frequency, and/or improved efficacy in the treatment/prevention of certain fungal infections.

The large volume of distribution observed for Compound 1 is consistent with the distribution of this compound into certain tissues, such as the kidney, liver, lung, and/or spleen. The large volume of distribution observed for Compound 1 may have clinical significance for the use of this compound to treat infections located in these tissues.

Example 25. In vitro activity: MEC and MIC values against Aspergillus spp.

The MEC and MIC values (µg/mL) of anidulafungin, caspofungin, amphotericin B, compound 1, and compound 16 against various Aspergillus species in the presence and absence of 50% human serum were obtained as follows.

The test organisms were obtained from the American Type Culture Collection (Manassas, VA). Isolates were maintained at -80°C and then thawed and sub-cultured before testing.

MIC testing was performed according to the procedures described by the Clinical and Laboratory Standards Institute (CLSI). (See Clinical and Laboratory Standards Institute (CLSI) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard, 3rd edition. CLSI document M27-A3 [ISBN 1-56238-666-2]. The assay was performed by Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2008; and Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi (Approved Standard, 2nd ed. CLSI document M38-A2 [ISBN 1-56238-668-9]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2008) and used an automated liquid handler for serial dilutions and liquid transfers. Fill the wells in columns 1-12 of a standard 96-well microdilution plate with 150 μL of DMSO. These will become the 'mother plates' from which 'daughter' or test plates will be prepared. Drug (150 μL, 80× the highest concentration desired in the test plate) is dispensed into the appropriate wells in column 1 of the mother plate and mixed. Serial 1:1 dilutions are prepared through column 11 of the 'mother plate'. The wells in column 12 contain no drug and thus serve as organism growth control wells. Daughter plates are loaded with 185 μL of RPMI or RPMI supplemented with 50% human serum per well. Daughter plates are prepared by transferring 5 μL of drug solution from each well of the mother plate to each corresponding well of each daughter plate.

A standardized inoculum of Aspergillus was prepared according to the CLSI method. 2 mL of 0.85% saline was dispensed onto an agar slant. The suspension was prepared using a swab. After a short period of time to allow the aggregate to settle, a small amount of the suspension was dispensed into RPMI and the suspension adjusted to a turbidity of 0.5 McFarland. Dilutions of each isolate were prepared in RPMI to achieve the cell concentration described in the CLSI method. The inoculum was dispensed into sterile reservoirs divided by length and used to inoculate the plates. 10 μL of the standardized inoculum was transferred to each well of the daughter plate. Thus, each well of the daughter plate ultimately contained 185 μL of broth, 5 μL of drug solution, and 10 μL of inoculum.

Cover the plate with a lid, place it in a plastic bag, and inoculate at 35°C. Incubate the Aspergillus plate for 48 hours before reading. Observe the microplate from the bottom using a plate viewer. For each master plate, inspect an uninoculated solubility control plate for evidence of drug precipitation.

Record both the MIC and the Minimum Effective Concentration (MEC) values. When testing filamentous fungi, the MEC value is particularly applicable to the echinocandins. The MIC value is the amount at which the drug inhibits the growth of the organism. The MEC value is the minimum concentration of the drug that results in the formation of small, round, dense hyphae, compared to the hyphal growth observed in the growth control wells.

MEC values, which are typically significantly different from the MIC values for this class of antifungals, are the measure that should be used to determine the sensitivity of filamentous fungi to echinocandins. Growth of the Aspergillus strain in each well was compared to growth controls at 48 hr.

The MEC and MIC values are provided in Figure 1. This data shows that, relative to anidulafungin, compounds 1 and 16 retain their activity against Aspergillus strains. Therefore, several modifications were made that resulted in prolonged pharmacokinetic effects without limiting activity against Aspergillus spp.

Example 26. In vitro activity: MIC values against Candida spp. at 24 and 48 hours.

The MIC values (µg/mL) of anidulafungin, caspofungin, amphotericin B, compound 1, and compound 16 against various Candida species in the presence and absence of 50% human serum were obtained as follows.

Test organisms were obtained from the American Type Culture Collection (Manassas, VA). Isolates were maintained at -80°C and then thawed and sub-cultured prior to testing.

The MIC test method follows the procedures described by the Clinical and Laboratory Standards Institute and utilizes an automated liquid handler for serial dilution and liquid transfer. Columns 1-12 of a standard 96-well microdilution plate are filled with 150 μL of DMSO. These become the 'mother plates' from which 'daughter' or test plates are prepared. Drug (150 μL, 80× the highest concentration expected in the test plate) is dispensed into the appropriate wells in column 1 of the mother plate and mixed. Serial 1:1 dilutions are prepared through column 11 of the 'mother plate'. The wells in column 12 contain no drug and serve as control wells for organism growth. Each well of the daughter plate is loaded with 185 μL of RPMI or RPMI supplemented with 50% human serum. Daughter plates are prepared by transferring 5 μL of drug solution from each well of the mother plate to each corresponding well of each daughter plate.

Standardized inocula of Candida species were prepared according to the CLSI protocol. For each Candida isolate, colonies were harvested from streak plates and suspended in RPMI. Dilutions were prepared in RPMI for each isolate to achieve the cell concentration described in the CLSI protocol. The inoculum was distributed into sterile, divided-by-length reservoirs and used to inoculate the plates. 10 μL of the standardized inoculum was transferred to each well of the daughter plate. Thus, each well of the daughter plate ultimately contained 185 μL of broth, 5 μL of drug solution, and 10 μL of inoculum.

Cover the plate with a lid, place it in a plastic bag, and inoculate at 35°C. Read the Candida isolates after 24 hours of incubation and again after 48 hours. Observe the microplate from the bottom using a plate viewer. For each master plate, inspect an uninoculated solubility control plate for evidence of drug precipitation.

For Candida species, the minimum inhibitory concentration (MIC) was read according to CLSI guidelines. The MIC was defined as the lowest concentration of the antifungal agent that substantially inhibited the growth of the organism, as observed visually (MIC values are provided in Figure 2).

These data indicate that compounds 1 and 16 retain their activity against Candida strains relative to anidulafungin. Therefore, several modifications were made that resulted in a prolonged pharmacokinetic effect without limiting activity against Candida.

Serum is known to differentially alter the antifungal properties of echinocandin drugs (see Paderu et al., Antimicrob Agents Chemother. 51:2253 (2007)). Compounds 1 and 16 were found to have superior activity against Candida glabrata strains in 50% human serum compared to anidulafungin under these same conditions. This difference in activity may be clinically relevant to the use of these compounds to treat certain bloodstream infections.

Example 27. Amphiphilicity of Compound 1.

The solubility of Compound 1 (acetate salt) was determined in aqueous buffers of various pH values to evaluate the suitability of formulations of this compound in aqueous vehicles for administration by injection (eg, intravenous or intramuscular injection).

The results are provided in Table 7 (below) along with anidulafungin as a comparison.Compound 1 was found to have more restricted aqueous solubility than anidulafungin over a wide pH range.

Table 7

The solubility of Compound 1 (acetate salt) was also determined in non-aqueous solvents to evaluate the solubility of this compound in formulations in non-aqueous vehicles. The results are provided in Table 8 (below).

Table 8

Example 28. Synthesis of Compound 23.

Anidulafungin (20 mg; 0.018 mmol) in _CH₃CN (10 ml) was treated with phenylboronic acid (2.5 mg; 0.021 mmol) and the mixture was stirred for 30 minutes. The resulting solution was concentrated to dryness in vacuo, and the solid was dissolved in a solution of DMSO (0.3 mL) and 1,3-diamino-2-propanol hydrochloride. The mixture was treated with HCl (4 M in dioxane) until acidic on wet pH paper. The resulting solution was heated at 40-45°C for 8 days, then diluted with water and purified by preparative RP HPLC, eluting with water (0.1% TFA)/ _CH₃CN (0.1% TFA). The product was isolated by lyophilization to yield 33 mg of compound 23 as a white solid. HPLC T _R 11.28 min (93%). LC/MS, ESI+/- m/z 1212.58 [M+H] ⁺ ,1210.57 [MH] ^- .

Example 29. Synthesis of Compound 24.

Anidulafungin hemiamine aldehyde-(4-methoxy)phenyl sulfide (20 mg; 0.016 mmol) was mixed with serinol (114 mg) and anhydrous DMSO (15 μL). The mixture was blanketed with argon and heated at 70°C for 2.5 hours. The reaction was diluted with methanol and water, acidified by the addition of TFA, further diluted with water, and purified by preparative RP HPLC eluting with _CH₃CN / _H₂O and 0.1% TFA. The purified product was isolated by lyophilization to afford 13 mg of compound 24 as a white solid. HPLC _TR 10.71 min (94% @ 220 nm). LC/MS, ESI+/- m/z 1213.57 [M+H] ⁺ , 1211.56 [MH] ^- .

Example 30. Separation of isomers.

Compound 1 purified by preparative RP HPLC and eluted with _CH3CN / _H2O and 0.1% TFA was found as a mixture of isomers (see Figures 3A and 3B). The two isomers observed are believed to differ in stereochemistry in which the choline substituent is attached to the anidulafungin starting material (see isomers shown below).

Other implementation plans

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any changes, uses, or adaptations of the invention which generally follow the principles of the invention and include such departures from the disclosure as come within known or customary practice in the art to which the invention pertains and which may be applied to the basic features heretofore described, and which fall within the purview of the following claims.

Other implementations are within the scope of the claims.

This application claims the benefit of U.S. Provisional Application Serial No. 61/448,807, filed March 3, 2011, which is incorporated herein by reference.

Claims (8)

1. A compound selected from compound 5, compound 16 and compound 18, or a pharmaceutically acceptable salt thereof: . 2. Compound 5 or a pharmaceutically acceptable salt thereof, . 3. Compound 16 or a pharmaceutically acceptable salt thereof, . 4. Compound 18 or a pharmaceutically acceptable salt thereof, . 5. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 6. The pharmaceutical composition of claim 5, wherein a pharmaceutically acceptable salt of said compound is an acetate or hydrochloride. 7. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a patient with a fungal infection, wherein the compound is administered to the patient in an amount sufficient to treat the infection. 8. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, stabilizing or inhibiting fungal growth or killing fungi, said use comprising contacting the fungus or a part of the fungus that is susceptible to growth with the compound of claim 1 or a pharmaceutically acceptable salt thereof.