US 2003O157197A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0157197 A1 OKUNUI et al. (43) Pub. Date: Aug. 21, 2003 (54) PLANTDERVED ANTI-PARASITIC AND (21) Appl. No.: 09/428,203 ANTIFUNGAL COMPOUNDS AND METHODS OF EXTRACTING THE (22) Filed: Oct. 27, 1999 COMPOUNDS Related U.S. Application Data (76) Inventors: CHRISTOPHERO. OKUNJI, SILVER SPRING, MD (US); (60) Provisional application No. 60/105,888, filed on Oct. MAURICE M. IWU, SILVER 27, 1998. SPRING, MD (US); JOAN E. JACKSON, ROCKVILLE, MD (US); JOHN D. TALLY JR., Publication Classification WASHINGTON, DC (US); CYRUS BACCHI, EAST NORTH POINT, NY (51) Int. Cl." ..................................................... A61K 35/78 (US); JOHNSON F. AYAFOR, DSCHANG (CM) (52) U.S. Cl. .............................................................. 424/725 Correspondence Address: ELIZABETH A, ARWINE USAMRMC (57) ABSTRACT FORT DETRICK BUILDING 521 Provided are biologically active extracts from Aframomum FREDERICK, MD 21701 (US) aulocacarpus, Aframomum danellii, Dracaena arborea, (*) Notice: This is a publication of a continued pros Eupatorium Odoratum, GloSSOcalyx brevipes and Napoleo ecution application (CPA) filed under 37 naea imperialis which are Suitable for use in treating fungal CFR 1.53(d). and protozoa diseases. Patent Application Publication Aug. 21, 2003 Sheet 1 of 21 US 2003/0157197 A1 Fig. 1 ANTIFUNGAL ACTIVITY OF Labda-8 (l7) l2-diene-l5, 16 - dia (AD-1) AGAINST Cladosporium cuculiner inun AT DIFFERENT CONCENTRATIONS Patent Application Publication Aug. 21, 2003 Sheet 2 of 21 US 2003/0157197 A1 Fig. 2 SCHEME FOR THE ISOLATION OF ANTIFUNGAL CONSTITUENTS OF Afra Inonum danielli i K. Schum (Fam. Zingibe raceae) Pet ... ether extract of Rhizome (3g) cc "Flash" silica gel 60 l, MeOH in CHCl l-2 (-) Fr. --3 (620mg) (+) Fr 4-7 (+) Fr. 8-ll ( - ) (2 x 200mg) Lobar Silica gel 60 Pet. ether : EtOAC (6 : l) Fr. 1.6-l9 (+) Fr. 26-36 (-) (l77mg) Lobar Silica gel Lobar Silica gel Pet. ether EtOAC (6 : ) Pet. ether : EtOAC (6 : . ) Tol. EtOAC (95:5) Tol: EtOAC (95 : 5) AD-li (93 mg) (+) AD-2 (23 mg) (-) + active - inactive Patent Application Publication Aug. 21, 2003 Sheet 3 of 21 US 2003/0157197 A1 undance TIC: AF-2. O --- 796 i.e. 07: SOOOOOO l 4. OO 6. O. O. 1800 undance TIC: AF-29 - 8. D ... 4 e -- 07 1.2e -- 07 le + O. BOOOOOO 6 OOOOOO 4 OOOOOO 2OOOOOO O itne--> S. OO 0.00 S.O.O. -- 2O. OO Fig 2B. TIC: AF-29-18.) 5000000 fine --> 00 12.00 l.00 400 15 OO 600 l7.00 18. oo 19 OO 2000 21.00 Figure 2C Patent Application Publication Aug. 21, 2003. Sheet 4 of 21 US 2003/0157197 A1 L.(L) chagasi, MHOM/BR/84/BA-3 MM Medium 96 hrs.A. danielli Rhizome Pet. ether Ext. Fr2(9-18). (50mg/ml) 0.6% DMSO Final Concentration 25000 20000 sK 15000 R s S.s 0000 S S. Patent Application Publication Aug. 21, 2003 Sheet 5 of 21 US 2003/0157197 A1 L. (L.) chagasi, MHOM/BR/84/BA-13 MM Medium 96 hrs. Aframomum danielli Stem Pet, ether Extract. 50 mg/ml), 0.6% DMSO Final Concentration 40000 - TEST CONTROL 30000 S. 20000 & &X s & 8: s 8 &: s : 10000 3. s 14C-SUBSTRATES p. (LLé-El c Patent Application Publication Aug. 21, 2003 Sheet 6 of 21 US 2003/0157197 A1 Molecular Representation of Labda-8 (17), 2-diene-15, 16- dia (Diterpene Dialdehyde) space-filling model total electron density Surface MEP Onto total electron density surface LUMO Surface isopotential surface at - A - A 1998am. Isopotential surface at - a- a -a -- a a - -5.0 kcal/mol FIG. a 3 Patent Application Publication Aug. 21, 2003 Sheet 7 of 21 US 2003/0157197 A1 L(L) chagasi, MHOM. IBA-13 MM Medium 'Scià, s. erratt 96 hrs. NI(EtOAc) isola. om Napoleonaea imperialis ten cyni e SatomiSO Fiat Cescentration (50mg/ml) 0.6% DMSO Final Concentration 48086 rest control 30000 i. 20000 0000 0000 0000 fi, 2 A c-suBSAtts 67, 9 B c-SUBSTRATES L. (L.) chagasi, MHOM/BR84 BA-13 MM Medium L.(L.) chagasi, MHOM/BR184BA-13 MM Medium 96hrs. NI-Glyco Fraction from Napoleonaea, imperialis 96hrs. NI-Glyco Fraction from Napoleonaea. imperialis (50mg/ml) 0.6% DMSO Final Concentration (50mg/ml) 0.6% DMSO Final Concentration 30000- 500 25000 400 20000 300 S000 200 0000 S000 100 Figure 3 Test results from Napoleonaea imperialis. At 50 g/ml suppression of parasite catabolism of 8 of C-Substrates occurred Patent Application Publication Aug. 21, 2003 Sheet 8 of 21 US 2003/0157197 A1 L. (L.) chagasi, MHOM/BR/84/b., 13 MM Medium 96 hrs. Non-sap isolated from Napoleonaea imperialis L.(L) chagasi, MHOM/BR/84/8A-13 MM Medium (50mg/ml) 0.6% DMSO Final Concentration 96(50ngful) hrs. NI-3 0.6% solated DMSO from Final Napoleonaea Concentration imperials 0000 2SOOO 30000 20000 g O C 15000 so 5 O 3 0000 s -- O000 S000 fy, E. a?. ZZ; ?y 7 0 F - -WY (N- s *C-SUBSTRATES l4c-SUBSTRATES L. (L.) chagasi, MHOM/BR/84/BA-13 MM Medium L(L.) chagasi, MHOM/BR/84/BA-3MM Medium 96 hrs. N1-4 isolated from Napoleonaea imperialis 96 hrs. NI-5 isolated from Napoleonaea imperialis (50mg/ml) 0.6% DMSO Final Concentration (50mg/ml) 0.6% DMSO Final Concentration 30000-3 l l 20000 3 2 s g10000 2: O t o e r O e a s : f9.3 G 4C-SUBSTRATES fy,? H CSUBSTRATES Figure 3 Test results from Napoleonaea imperialis. - At 50 g/ml suppression of parasite catabolism of 8 of C-Substrates occurred Patent Application Publication Aug. 21, 2003 Sheet 9 of 21 US 2003/0157197 A1 Data File C: WHPCHEMV l\DATAWOO50396C. D Sample Name: EOO 60. 69 O.O- 4 O 3 O: 70 atter ACN long C18 column ... On l/rain DAD 254 Acq. Method RDBAYM Seq. Line : - Acq. Operator : dgbg Wial : 100 W Inj : - Sample Name : EOO 60/45 In Volume : 10 Ll Analysis Method : C:\HPCHEMV1\METHODSVIRDBAY.M (modified after loading) OADA, Sig=254,4 Ref=450,80 of OOSQ396C,D A. 8 8 Figure 3. Liquid Chromatographic Separation of Eupatorium odoratum Antileishmania. Fraction EO-O 60-04. Patent Application Publication Aug. 21, 2003 Sheet 10 of 21 US 2003/0157197A1 ... Until ad Rate Oa t d - - - - - - - - - - - - - - - - - - - - - - - - - . 2 O C) d 3 VEHICLE CONTROLS (RED & BLUE & LT. GREEN) V) al O O D O 9 CS Car 4 its "1" list-so-scists: griss-to-a. st: -5-3-3, 3-tit, , , 2.5 UG/ML "N" CRUDE PLANT EXTRACT (PURPLE & AQUA) il- a-...-a dra'a-a-a-heard its dra-al-rihrilrdrdrdr"thrihrheid its “Ax'"my." akra was ifAt f. '- 25 UG/ML"NI" (DARK GREEN) n-site:-- - - r:-size:----------- first-startisetsers s pa s: rtist-actitizeta-a-actiti. 9 10:0 2:1) 50.9 4): S: :: :(): Chanbir R 1st Fuld TME (HH: MM: SS) Canbit 8 Chatter C fathbir C s t Canber El st Fluid 50 UG/ML"NI" (YELLOW & PINK- SUPERIMPOSED) is Figure 4A Patent Application Publication Aug. 21, 2003 Sheet 11 of 21 US 2003/0157197 A1 as e?o cane Eel F. Ga. He LEISHMANIA (LEISHMANIA) CHAGASl ONTR 5 G/MLA"I" (AGUA) G/MLVNI" (PURPLE) CONTROL (DK. BLUE) . 2:23 2:35:88 287 2:18:88 22:39 25:33 Tine (MMSS Figure 4B Patent Application Publication Aug. 21, 2003 Sheet 12 of 21 US 2003/0157197 A1 (jii is: it at a ii., LESHMANIA (LEISHMANIA) CHAGAS 3VEH. CONTROLS . " " - .2 et 'i's 12.5 UGIML"NI" PURPLE & AQUA iii- t --4----ti-et--th-E-R-birth E- -- a--- is 4...t- : th 1 ti i te: ' ' ' ti A'. -- . is a 16c -is ?ix it is at if... iki -- * : * 'a. it. “ -6. ' ' ' ' ' k. k. 4r 30- 25 UGIML"N." DARKGREEN w 15 -- 50 UGML. "N ----------------------------------------T-6:5): :: :19: 7:2) 5") - YELLOW & PINK-- TIME (HH: MM.SS) -...-- laber E -or-minary won-r--a-- +7 HRS -- - - - - - - - - - - - - - laber - ---------------a-a-a- - - - - - - - - - - - - - - - - - - - - - - - - - - - Chamber ) -----------------------------------------. i- -- chamber 1. ....I.I.I.I.I.I.I.I. 1 hamberints ri: ... ---...--i: - - - Figure 4C Patent Application Publication Aug. 21, 2003 Sheet 13 of 21 US 2003/0157197 A1 A 3NWASROW33 NSC Qin??ag O 1. Patent Application Publication Aug. 21, 2003 Sheet 14 of 21 US 2003/0157197 A1 - 12. L.(L.) chagasi, MHOM/BR/84/BA-3 MM Medium 96 hrs, MeOH extract (EOMeOH) of Eodoratum (50mg/ml) 0.6% oMSO Final Concentration 30000 TEST CONTROL 20000 As 10000 14C-SUBSTRATES Patent Application Publication Aug. 21, 2003 Sheet 15 of 21 US 2003/0157197 A1 3, 1.a L. (L.) chagasi, MHOM/BR/84/BA-13 MM2 Medium 96 hrs.Fraction EOO60-04/BN59168 from Eodoraftint 6% DMSO Final Concentration 15000 TEST E CONTROL 10000 Aa 5000 en 14C-SUBSTRATES Patent Application Publication Aug. 21, 2003 Sheet 16 of 21 US 2003/0157197 A1 s NWSNOLINISO Patent Application Publication Aug. 21, 2003 Sheet 17 of 21 US 2003/0157197 A1 Fig. 6 ABLE - 1 Nulleric Code abbreviations of 'C-substrates used for drug tests * Numeric Commercial Code 14C-Substratest Source 2 L-Arginine (guanidino-14C) A ---- 3. L-Aspartic Acid (4-14C) L-Asparagine (U-14C) L-Glutamic Acid (U-14C) L-Glutamine (U-14C) Glycine (U-14C) L-Isoleucine (U-14C) O L-Leucine (1-14C) 2 L-Methionine (1-14C) l3 L-Ornithine (1-14C) 5 L-Proline (U-14C) 7 Taurine (U-14C) 18 L-Threonine (U-14C) 2O Tyramine (7-14C) 24 L-Fucose (1-14C) 25 D-Galactose (1-14C) 28 D-Mannose (1-14C) 42 Orotic Acid (carboxyl-14C) Nss 44 Succinic Acid (1, 4-l4C) N 46 Na-n-Butyric Acid (1-14C) 49 D-Glucosamine (1-14c) 52 Na-Glycocholic Acid (1-14C) 53 L-Methionine (methyl-14C) 19 Patent Application Publication Aug.