Kalahide Kahalalide F, of formula I below, may be isolated from a sacoglossan. The compound may be used in the manufacture of pharmaceutical compositions or in the treatment of tumors or viral conditions ##STR00001##

PTO Wrapper PDF
Dossier Espace Google
Patent
   RE39496
Priority
Feb 03 1993
Filed
Aug 14 2003
Issued
Feb 27 2007
Expiry
Feb 03 2014
Inventors
Scheuer, A…
Assg.orig
PharmaMar,…
Assg.curr
PharmaMar,…
Entity
Large
Referenced by
6
References
2
Maint.:
all paid
EXPERIMENTAL
1. A substially Substantially pure compound Kahalalide F compound, said compound hag having a molecular formula of c75H124N14O16, and consisting of five valines, two isoleucines, two threonines, ornithine, dehydroaminobutynic dehydroaminobutyric acid, proline, phenylalanine and 5-methylhexanoic acid; said compound further exhibiting the following physical and chemical properties: [α]D-8° (c 4.32, MeOH); 1H NMR (500 MHz, TFA/DMF); amino acid unit, δ (carbon position, mult, J): Val-1 4.16 (2, t, J=9.0 Hz), 7.11 (NH on 2, d, J=8,9 8.9 Hz), 1.77 (3, m), 0.95 (4, m), 0.95 (5, m), Dhb 9.20 (NH on 2, s), 6.48 (3, q, J=6.9 Hz), 1.43 (4, d, J=6.6 Hz); Phe 4.68 (2, q, J=6.6 Hz), 8.62 (NH on 2, d, J=6.6 Hz), 3.23 (3, dd, J=13.7, 7.2 Hz), 3.00 (3, dd, J=13.7, 9.0 Hz), 7.32 (5, d, J=7.2 Hz), 7.28 (6, t, J=7.5 Hz), 7.21 (7, t, J=7.2 Hz); Vol-2 Val-2 4.36 (2, m), 7.82 (NH on 2, d, J=6.6 Hz), 2.12 (3, m), 0.85 (4, m), 0.77 (5, d, J=6.6 Hz); Ileu-1 4.53 (2, m), 8.38 (NH on 2, d, J=9.6 Hz), 1.98 (3, m), 0.92 (4, d, J=6.6 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, t, J=7.2 Hz); Thr-1 4.63 (2, t, J=9.3 Hz), 8.12 (HN NH on 2, d, J=5.7), 5.07 (3, dq, J=9.6, 6.0 Hz), 1.18 (4, d, J=6.3 Hz); Ileu-2 4.52 (2, m), 7.72 (NH on 2, d, J=8.4 Hz), 1.88 (3, m), 0.88 (4, d, J=6.3 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, d t, J=7.2 Hz) Orn 4.48 (2, m), 7.92 (NH on 2, d, J=7.8 Hz), 1.76 (3, m), 1.83 (4, m), 3.10 (5, p, J=5.1 Hz); Pro 4.42 (2, m), 2.12 (3, m), 1.97 (3, m), 2.02 (4, m), 1.88 (4, m), 3.75 (5, m), 3.68 (5, m); Val-3 4.41 (2, m), 7.90 (NH on 2, d, J=7.2 Hz), 2.12 (3, m), 0.95 (4, m), 0.85 (5, m); Val-4 4.34 (2, m), 7.68 (NH on 2, d, J=8.1 Hz), 2.17 (3, m), 0.95 (4, m), 0.90 (5, m); Thr-2 4.46 (2, m), 7.77 (NH on 2, d, J=8.1 Hz), 4.21 (3, dq, J=6.3, 3.6 Hz), 1.12 (4, d, J=6.6 Hz); Val-5 4.32 (2, m), 7.85 (HN NH on 2, d, J=8.1 Hz), 7.82 (NH on (second conformation), d, J=8.1 Hz), 2.14 (3, m), 0.95 (4, m), 0.90 (5, m); 5-MeHex 2.26 (2, m), 1.60 (3, m), 1.20 (4, m), 1.55 (5, m), 0.87 (6, d, J=7.2 Hz), 0.87 (7, d, J=72 7.2 Hz); 5-MeHex 2.29 (2, m), 1.65 (3, m), 1.40 (3, m), 1.13 (4, m), 1.35 (5, m), 0.90 (6, m), 0.90 (7, m); 13c NMR (125 MHz TFA/DMF): amino acid unit, δ (carbon position); Val-1 170.40 (1), 60.31 (2), 30.75 (3), 19.58 (4), 18.76 (5); Dhb 164.54 (1), 130.30 (2), 131.26 (3), 126.6 12.68 (4); Phe 171.31 (1), 56.27 (2), 36.79 (3), 138.23 (4), 129.86 (5), 128.77 (6), 126.98 (7); Val-2 172.94 (1), 58.57 (2), 32.38 (3), 18.92 (4), 17.60 (5); Ileu-1 171.87 (1), 57.48 (2), 38.78 (3), 14.56 (4), 26.78 (5), 11.67 (6); Thr-1 169.68 (1), 57.37 (2), 71.05 (3), 17.34 (4); Ileu-2 171.92 (1), 57.29 (2), 38.01 (3), 14.78 (4), 26.55 (5), 11.63 (6); Orn 172.01 (1), 52.87 (2), 29.63 (3), 24.39 (4), 40.05 (5); Pro 172.55 (1), 60.23 (2), 29.58 (3), 25.38 (4) 48.03 (5); Val-3 171.28 (1), 57.57 (2), 30.54 (3), 19.61 (4), 18.80 (5); Val-4 171.83 (1), 59.10 (2), 31.26 (3), 19.45 (4), 18.08 (5); thr-2 Thr-2 170.97 (1), 58.89 (2), 67.36 (3), 19.66 (4); Val-5 172.67 (1), 59.64 (2), 30.66 (3), 19.61 (4), 18.43 (5); 5-MeHex 173.83 (1), 36.28 (2), 23.99 (3), 38.96 (4), 28.10 (5), 22.54 (6), 22.50 (7); 5-MeHex (second conformation) 174.08 (1), 33.86 (2), 32.84 (3), 29.75 (4), 34.54 (5), 19.51 (6), 11.20 (7); IR neat (NaCl): 3287 (s, br), 2964 (s, br), 1646 (s), 1528 (s), 1464 (s), 1388 (m), 1228 (m), cm−1; mass spectrum HRFAB m/z (fragment, %) 1477.9408 (M++1, 85); UV (MeOH): λmax 204 (89,630) nm.
3. A pharmaceutical compostion composition comprising a pharmaceutical carrier or diluent and the substantially pure compound Kahalalide F, said compoumd compound having a molecular formula of c75H124N14O16, and conng consisting of five valines, two isoleucines, two threonines, ornithine, dehydroamiobutyric dehydroaminobutyric acid, proline, phenylalanine and 5-methylhexoic 5-methylhexanoic acid; said compound further exhibiting the following physical and chemical properties:
[α]D-8° (c 4.32, MEOH); 1H NMR (500 MHz, TFA/DMF); amino acid unit, δ (carbon position, mult, J): Val-1 4.16 (2, t, J=9.0 Hz), 7.11 (NH on 2, d, J=8,9 8.9 Hz), 1.77 (3, m), 0.95 (4, m), 0.95 (5, ma m); Dhb 9.20 (NH on 2, s), 6.48 (3, q, J=6.9 Hz), 1.43 (4, d, J=6.6 Hz); Phe 4.68 (2, q, J=6.6 Hz), 8.62 (NH on 2, d, J=6.6 Hz), 3.23 (3, dd, J=13.7, 7.2 Hz), 3.00 (3, dd, J=13.7, 9.0 Hz), 7.32 (5, d, J=7.2 Hz), 7.28 (6, t, J=7.5 Hz), 7.21 (7, t, J=7.2 Hz); Val-2 4.36 (2, m), 7.82 (NH on 2, d, J=6.6 Hz), 2.12 (3, m), 0.85 (4, m), 0.77 (5, d, J=6.6 Hz); Ileu-1 Ileu-1 4.53 (2, m), 8.38 (NH on 2, d, J=9.6 Hz), 1.98 (3, m), 0.92 (4, d, J=6.6 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, t, J=7.2 Hz); Thr-1 4.63 (2, t, J=9.3 Hz), 8.12 (NH on 2, d, J=5.7), 5.07 (3, dq, J=9.6, 6.0 Hz), 1.18 (4, d, J=6.3 Hz); Ileu-2 4.52 (2, m), 7.72 (NH on 2, d, J=8.4 Hz), 1.88 (3, m), 0.88 (4, d, J=6.3 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, d t, J=7.2 Hz) Orn 4.48 (2, m), 7.92 (NH on 2, d, J=7.8 Hz), 1.76 (3, m), 1.83 (4, m), 3.10 (5, p, J=5.1 Hz); Pro 4.42 (2, m), 2.12 (3, m), 1.97 (3, m), 2.02 (4, m), 1.88 (4, m), 3.75 (5, m), 3.68 (5, m); Val-3 4.41 (2, m), 7.90 (NH on 2, d, J=7.2 Hz), 2.12 (3, m), 0.95 (4, m), 0.85 (5, m); Val-4 4.34 (2, m), 7.68 (NH on 2, d, J=8.1 Hz), 2.17 (3, m), 0.95 (4, m), 0.90 (5, m); Thr-2 4.46 (2, m), 7.77 (NH on 2, d, J=8.1Hz), 4.21 (3, dq, J=6.3, 3.6 Hz), 1.12 (4, d, J=6.6 Hz); Val-5 4.32 (2, m), 7.85, (NH on 2, d, J=8.1 Hz), 7.82 (NH on (second conformation), d, J=8.1 Hz), 2.14 (3, m), 0.95 (4, m), 0.90 (5, m); 5MeHex 2.26 (2, m), 1.60 (3, m), 1.20 (4, m), 1.55 (5, m), 0.87 (6, d, J=7.2 Hz), 0.87 (7, d, J=7.2 Hz); 5-MeHex 2.29 (2, m), 1.65 (3, m), 1.40 (3, m), 1.13 (4, m), 1.35 (5, m), 0.90 (6, m), 0.90 (7, m); 13c NMR (125 MHz TFA/DMF): amino acid unit, δ (carbon position); Val-1 170.40 (1), 60.31 (2), 30.75 (3), 19.58 (4), 18.76 (5); Dhb 164.54 (1), 130.30 (2), 131.26 (3), 12.68 (4); Phe 171.31 (1), 56-27 56.27 (2), 36.79 (3), 138.23 (4), 129.86 (5), 128.77 (6), 126.98 (7); Val-2 172.94 (1), 58.27 (2), 32.38 (3), 18.92 (4), 17.60 (5); Ileu-1 171.87 (1), 57.48 (2), 38.78 (3), 14.56 (4), 26.78 (5), 11.67 (6); Thr-1 169.68 (1), 57.37 (2), 71.05 (3), 17.34 (4); Ileu-2 171.92 (1), 57.29 (2), 38.01 (3), 14.78 (4), 26.55 (5), 11.63 (6); Orn 172.01 (1), 52.87 (2), 29.63 (3), 24.39 (4), 40.05 (5); Pro 172.35 172.55 (1), 60.23 (2), 29.58 (3), 25.38 (4), 48.03 (5); Val-3 171.28 (1), 57.57 (2), 30.54 (3), 19.61 (4), 18.80 (5); Val-4 171.83 (1), 59.10 (2), 31.26 (3), 19.45 (4), 18.08 (5); Thr-2 170.97 (1), 58.89 (2), 67.36 (3), 19.66 (4); Val-5 172.67 (1), 59.64 (2), 30.66 (3), 19.61 (4), 18.43 (5); 5-MeHex 173.83 (1), 36.28 (2), 23.99 (3), 38.96 (4), 28.10 (5), 22.54 (6), 22.50 (7); 5-MeHex (second conformation) 174.08 (1), 33.86 (2), 32.84 (3), 29.75 (4), 34.54 (5), 19.51 (6), 11.20 (7); IR neat (NaCl): 3287 (s, br), 2964 (s, br), 1646 (s), 1528 (s), 1464 (s), 1388 (m), 1228 (m), cm−1; mass spectrum HRFAB m/z (fragment, %) 1477.9408 (M++1, 85); UV (MeOH): λmax 204 (89,630) nm.
5. A method of treating fungal infections in mammals comprising administering to a patient in need of such treatment, an amount of the substantially puxe compound pure Kahalalide F compound or a pharmaceutically acceptable salt thereof, sufficient to slow or stop the growth of the fungal infection; said compound having a molecula molecular formula of c75H124N14O16, and consisting of five valines, two isoleucines, two theonines threonines, ornithine, dehydroaminobutiric dehydroaminobutyric acid, proline, phenylalanie phenylalanine and 5-methylhexanoic acid; said compound further exhibiting the folowing following physical and chemical properties:
[α]D-8° (c 4.32, MEOH); 1H NMR (500 MHz, TFA/DMF); amino acid unit, δ (carbon position, mult, J): Val-1 4.16 (2, t, J=9.0 Hz), 7.11 (NH on 2, d, J=8,9 8.9 Hz), 1.77 (3, m), 0.95 (4, m), 0.95 (5, ma m); Dhb 9.20 (NH on 2, s), 6.48 (3, q, J=6.9 Hz), 1.43 (4, d, J=6.6 Hz); Phe 4.68 (2, q, J=6.6 Hz), 8.62 (NH on 2, d, J=6.6 Hz), 3.23 (3, dd, J=13.7, 7.2 Hz), 3.00 (3, dd, J=13.7, 9.0 Hz), 7.32 (5, d, J=7.2 Hz), 7.28 (6, t, J=7.5 Hz), 7.21 (7, t, J=7.2 Hz); Val-2 4.36 (2, m), 7.82 (NH on 2, d, J=6.6 Hz), 2.12 (3, m), 0.85 (4, m), 0.77 (5, d, J=6.6 Hz); Ilue-1 Ileu-1 4.53 (2, m), 8.38 (NH on 2, d, J=9.6 Hz), 1.98 (3, m), 0.92 (4, d, J=6.6 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, t, J=7.2 Hz); Thr-1 4.63 (2, t, J=9.3 Hz), 8.12 (NH on 2, d, J=5.7), 5.07 (3, dq, J=9.6, 6.0 Hz), 1.18 (4, d, J=6.3 Hz); Ileu-2 4.52 (2, m), 7.72 (NH on 2, d, J=8.4 Hz), 1.88 (3, m), 0.88 (4, d, J=6.3 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, d t, J=7.2 Hz) Orn 4.48 (2, m), 7.92 (NH on 2, d, J=7.8 Hz), 1.76 (3, m), 1.83 (4, m), 3.10 (5, p, J=5.1 Hz); Pro 4.42 (2, m), 2.12 (3, m), 1.97 (3, m), 2.02 (4, m), 1.88 (4, m), 3.75 (5, m), 3.68 (5, m); Val-3 4.41 (2, m), 7.90 (NH on 2, d, J=7.2 Hz), 2.12 (3, m), 0.95 (4, m), 0.85 (5, m); Val-4 4.34 (2, m), 7.68 (NH on 2, d, J=8.1 Hz), 2.17 (3, m), 0.95 (4, m), 0.90 (5, m); Thr-2 4.46 (2, m), 7.77 (NH on 2, d, J=8.1), 4.21 (3, dq, J=6.3, 3.6 Hz), 1.12 (4, d, J=6.6 Hz); Val-5 4.32 (2, m), 7.85, (NH on 2, d, J=8.1 Hz), 7.82 (NH on (second conformation), d, J=8.1 Hz), 2.14 (3, m), 0.95 (4, m), 0.90 (5, m); 5MeHex 2.26 (2, m), 1.60 (3, m), 1.20 (4, m), 1.55 (5, m), 0.87 (6, d, J=7.2 Hz), 0.87 (7, d, J=7.2 Hz); 5MeHex 2.29 (2, m), 1.65 (3, m), 1.40 (3, m) 1.13 (4, m), 1.35 (5, m), 0.90 (6, m), 0.90 (7, m); 13c NMR (125 MHz, TFA/DMF): amino acid unit, 6 (carbon position); Val-1 170.40 (1), 60.31 (2), 30.75 (3), 19.58 (4), 18.76 (5); Dhb 164.54 (1), 130.30 (2), 131.26 (3), 12.68 (4); Phe 171.31 (1), 56-27 56.27 (2), 36.79 (3), 138.23 (4), 129.86 (5), 128.77 (6), 126.98 (7); Val-2 172.94 (1), 58.27 (2), 32.38 (3), 18.92 (4), 17.60 (5); Ileu-1 171.87 (1), 57.48 (2), 38.78 (3), 14.56 (4), 26.78 (5), 11.67 (6); Thr-1 169.68 (1), 57.37 (2), 71.05 (3), 17.34 (4); Ileu-2 171.92 (1), 57.29 (2), 38.01 (3), 14.78 (4), 26.55 (5), 11.63 (6); Orn 172.01 (1), 52.87 (2), 29.63 (3), 24.39 (4), 40.05 (5); Pro 172.35 172.55 (1), 60.23 (2), 29.58 (3), 25.38 (4), 48.03 (5); Val-3 171.28 (1), 57.57 (2), 30.54 (3), 19.61 (4), 18.80 (5); Val-4 171.83 (1), 59.10 (2), 31.26 (3), 19.45 (4), 18.08 (5); Thr-2 170.97 (1), 58.89 (2), 67.36 (3), 19.66 (4); Val-5 172.67 (1), 59.64 (2), 30.66 (3), 19.61 (4), 18.43 (5); 5-MeHex 173.83 (1), 36.28 (2), 23.99 (3), 38.96 (4), 28.10 (5), 22.54 (6), 22.50 (7); 5-MeHex (second conformation) 174.08 (1), 33.86 (2), 32.84 (3), 29.75 (4), 34.54 (5), 19.51 (6), 11.20 (7); IR neat (NaCl): 3287 (s, br), 2964 (s, br), 1646 (s), 1528 (s), 1464 (s), 1388 (m), 1228 (m), cm−1; mass spectrum HRFAB m/z (fragment, %) 1477.9408 (M++1, 85); UV (MeOH): λmax 204 (89,630) nm.
2. The compound Kahalalide F compound of claim 1, which further has the folowing following non-stereospecific structure: ##STR00004##
4. The pharmaceutical composition of claim 3, wherein the compound Kahalalide F compound further has the folowing non-streospecific following non-stereospecific structure ##STR00005##
6. The method of treatment of claim 5, wherein compound Kahalalide F has the following non-stereospecific structure: ##STR00006##
7. The method of claim 5, wherein the fungal infection is caused by Aspergillus oryzae.
8. The method of claim 5, wherein the fungal infection is caused by Penicillium notatum.
9. The method of claim 5, wherein the fungal infection is caused by Trichophyton mentagrophy.
10. The method of claim 5, wherein the fungal infection is caused by Candida albicans.

This invention is concerned with a cytotoxic and antiviral compound isolated from the sacoglossan, Elysia rafescens rufescens.

According to the invention there is provided, a the new compound, the peptide, Kahalalide F, of the formula I: ##STR00002##

The antitumor activities of this compound has been determined “in vitro” in cell cultures of human lung carcinoma A-549 and human colon carcinoma HT-29. The procedure was carried out using the metnhodology methodology described by Raymond J. Bergeron et al. Biochem. Bioph. Res. Comm. 1984, 121(3), 848-854 and by Alan C. Schroeder et al. J. Med. Chem. 1981, 24 1078-1083.

The antiviral activities of this compound the compound Kahalalide F have also been determined “in vitro” against HSV (Herpes simplex virus) and VSV (Vesicular stomatitis virus). The methodology used to carry out this determination is described by Raymond J. Bergeron et al. Biochem. Bioph. Res. Comm. 1984, 121(3), 848-854 and by Alan C. Schroeder et al. J. Med. Chem. 1981, 24 1078-1083.

Therefore, the present invention also provides a method of treating any mammal affected by a malignant tumor sensitive to compounds above described, which comprises administering to the affected individual a therapeutically effective amount of these compounds or a pharmaceutical composition thereof; and a method of treating viral infections in mammals, comprising administering to a patient in need of such treatment, an antiviral effective amount of the compounds described in the present invention.

The present invention also relates to pharmaceutical preparations which contain as active ingredient these compounds, or a pharmaceutically acceptable acid addition salt thereof, as well as the process for its preparation.

Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) suitable composition for oral, topical or parenteral administration, and they may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when administered parenterally.

The correct dosage of a pharmaceutical composition of these compounds this compound will vary according to the particular formulation, the mode of application and particular site, host and tumor being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of disease shall be taken in account. Administration can be carried out continuously or periodically within the maximum tolerated dose.

Kahalalide F was isolated from the sacoglossan, Elysia rufescens Elysia rufescens (family Plakobranchidae, order Sacoglossa), collected near Black point Point, Oahu. This animal varies in size between 1 and 4 cm; it is dark red-brown in color with light-colored spots. There is orange fringing of the parapodia, which have very small dark green spots from sequestered chloroplasts. Elysia rufescens feeds on the delicate, feather-like green alga Bryopsis Bryopsis sp. 1 Kahalalide F can also be isolated from this alga. Two hundred animals were collected over the period of several weeks during the spring, of 1991 and extracted with EtOH. The extracts were then chromatographed by silica gel flash chromatography (hexane, hexane/EtOAc (1:1), EtOAc, EtOAc (1:1) EtOAc/MeOH (1:1), MeOH and MeOH/HOAc (98:2)). The peptides were eluted with EtOAc/MeOH (1:1). Final purification was accomplished by repeated HPLC (RP C18) using MeCN/H2O with 0.1% TFA (70-45% H2O). ##STR00003##

The structures of the peptides were elucidated by 2D NMR experiments (HMQC, HMBC, TOCSY, COSY and ROESY).

Kahalalide F was isolated as a white amorphous powder in 0.02% 0.01% yield. A molecular formula of C75H124N14O16 was deduced from detailed analyses of the 13C and 1H NMR spectra and the high resolution FAB mass spectrum. The 14 substructures in this compound arise from five valines, two isoleucines, two threonines, ornithine, dehydroaminobutyric acid. , proline, phenilalanine phenylalanine and 5-methythexanoic 5-methylhexanoic acid (5-MeHex). Kahalalide F is the largest peptide in this series of compounds.

EXPERIMENTAL

General Considerations

Optical rotations were measured on a Jasco DIP-370 digital polarimeter. Infrared spectra were recorded on a Nicolet MX-5 FTIR spectrometer. Gas chromatography was accomplished using a Hewlett-Packard Model 5890 instrument. Mass spectra were measured on a VG-70SE magnetic sector mass spectrometer. NMR spectra were measured on a General Electric QE-300 or a GN OMEGA 500 instrument. 1H NMR chemical shifts are reported in ppm with the chemical shift of the residual protons of the solvent used as internal standards. 13C NMR chemical shifts are reported in ppm by using the natural abundance 13C of the solvent as an internal standard. Ultraviolet spectra were recorded on a Hewlett-Packard Model 8452A diode array spectrophotometer. All solvents were distilled from glass before use.

Two hundred sacoglossans (Elysia rufescens), were collected at Black Point, O'ahu during April and May 1992 1991, and extracted 3 times with EtOH. Spring appears to be the time of year Elysia rufescens is in greatest abundance at Black Point. The combined extracts were then chromatographed using silica gel flash chromatography (hexane, hexane/EtOAc (1:1), EtOAc, EtOAc/MeOH (1:1), MeOH, MeOH/HOAc (98:2)). The depsipeptides were found in the EtOAc/MeOH (1:1) fraction. Repeated HPLC RP18 RP C18 MeCN/H2O/TFA (55/45/1 30/70/1)—MeCN/H2O/TFA ((30/70/1 55/45/1) yielded six the new depsipeptides.

KAHALALDDE F KAHALALIDE F

Final purification was accomplished by HPLC on RP18 RP C18 MeCN/H2O/TFA (55/45/1). Physical data: [α]D-8° [α]D-8° (c 4.32, MeOH); 1H NMR (500 MHz, TFA/DMF); amino acid unit, δ (carbon position, mult, J): Val-1 4.16 (2, t, J=9.0 Hz), 7.11 (NH on 2, d, J=8.9 Hz), 1.77 (3, m), 0.95 (4, m), 0.95 (5, m); Dhb 9.20 (NH on 2, s), 6.48 (3, q, J=6.9 Hz), 1.43 (4, d, J=6.6 Hz); Phe 4.68 (2, q, J=6.6 Hz), 8.62 (NH on 2, d, J=6.6 Hz), 3.23 (3, dd, J=13.7, 7.2 Hz), 3.00 (3, dd, J=13.7, 9.0 Hz), 7.32 (5, d, J=7.2 Hz), 7.28 (6, t, J=7.5 Hz), 7.21 (7, t, J=7.2 Hz); Val-2 4.36 (2, m), 7.82 (NH on 2, d, J=6.6 Hz), 2.12 (3, m), 0.85 (4, m), 0.77 (5, d, J=6.6 Hz); Ileu-1 4.53 (2, m), 8.38 (NH on 2, d, J=9.6 Hz), 1.98 (3, m), 0.92 (4, d, J=6.6 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, t, J=7.2 Hz); Thr-1 4.63 (2, t, J=9.3 Hz), 8.12 (NH on 2, d, J=5.7), 5.07 (3, dq, J=9.6, 6.0 Hz), 1.18 (4, d, J=6.3 Hz); Ileu-2 4.52 (2, m), 7.72 (NH on 2, d, J=8.4 Hz), 1.88 (3, m), 0.88 (4, d, J=6.3 Hz), 1.40 (5, m), 1.13 (5, m), 0.88 (6, d t, J=7.2 Hz); Orn 4.48 (2, m), 7.92 (NH on 2, d, J=7.8 Hz), 1.76 (3, m), 1.83 (4, m), 3.10 (5, p, J=5.1 Hz); Pro 4.42 (2, m), 2.12 (3, m), 1.97 (3, m), 2.02 (4, m), 1.88 (4, m), 3.75 (5, m), 3.68 (5, m); Val-3 4.41 (2, m), 7.90 (NH on 2, d, J=7.2 Hz), 2.12 (3, m), 0.95 (4, m), 0.85 (5, m); Val-4 4.34 (2, m), 7.68 (NH on 2, d, J=8.1 Hz), 2.17 (3, m), 0.95 (4, m), 0.90 (5, m); Thr-2 4.46 (2, m), 7.77 (NH on 2, d, J=8.1), 4.21 (3, dq, J=6.3, 3.6 Hz), 1.12 (4, d, J=6.6 Hz); Val-5 4.32 (2, m), 7.85, (NH on 2, d, J=8.1 Hz), 7.82 (NH on (second conformation) d, J=8.1 Hz), 2.14 (3, m), 0.95 (4, m), 0.90 (5, m); 5-MeHex 2.26 (2, m), 1.60 (3, m), 1.20 (4, m), 1.55 (5, m), 0.87 (6, d, J=7.2 Hz), 0.87 (7, d, J=7.2 Hz); 5-MeHex 2.29 (2, m), 1.65 (3, m), 1.40 (3, m), 1.13 (4, m), 1.35 (5, m), 0.90 (6, m), 0.90 (7, m) ; 13C NMR (125 MHz TFA/DMF): amino acid unit, δ (carbon position); Val-1 70.40 170.40 (1), 60.31 (2), 30.75 (3), 19.58 (4), 18.76 (5); Dhb 164.54 (1), 130.30 (2), 131.26 (3), 12.68 (4); Phe 171.31 (1), 56.27 (2), 36.79 (3), 138.23 (4), 129.86 (5), 128.77 (6), 126.98 (7); Val-2 172-94 172.94 (1), 58.57 (2), 32.38 (3), 18.92 (4), 17.60 (5); Ileu-1 171.87 (1), 57.48 (2), 38.78 (3), 14.56 (4), 26.78 (5), 11.67 (6); Thr-1 169.68 (1), 57.37 (2), 71.05 (3), 17.34 (4); Ileu-2 171.92 (1), 57.29 (2), 38.01 (3), 14.78 (4), 26.55 (5), 11.63 (6); Orn 172.01 (1), 52.87 (2), 29.63 (3), 24.39 (4), 40.05 (5); Pro 172.55 (1), 60.23 (2), 29.58 (3), 25.38 (4), 48.03 (5); Val-3 171.28 (1), 57.57 (2), 30.54 (3), 19.61 (4), 18.80 (5); Val-4 171.83 (1), 59.10 (2), 31.26 (3), 19.45 (4), 18.08 (5); Thr-2 170.97 (1), 58.89 (2), 67.36 (3), 19.66 (4); Val-5 172.67 (1), 59.64 (2), 30.66 (3), 19.61 (4), 18.43 (5), 5-MeHex 173.83 (1), 36.28 (2), 23.99 (3), 38.96 (4), 28.10 (5), 22.54 (6), 22.50 (7); 5-MeHex (second conformation) 174.08 (1), 33.86 (2), 32.84 (3), 29.75 (4), 34.54 (5), 19.51 (6), 11.20 (7) ; IR neat (NaCl): 3287 (s, br), 2964 (s, br), 1646 (s), 1528 (s), 1465 (s), 1388 (m), 1228 (m), cm−1; mass spectrum HRFAB m/z (fragment, %) 1477.9408 (M++1, 85) (calcd for C75H125N14O16: 1477.9398); UV (MeOH): λmax 204 (89,630) nm.

Amino acid analysis by GC-MS with a Chirasil-Val column indicates that Kahalalide F consists of 2-D-allo-Ileu, L-Orn, L-Phe, D-Pro, L-Thr, D-Allo-Thr, 3 D-Val and 2 L-Val.

TABLE II TABLE I
1H and 13C NMR Data for Kahalalide F (I) KF in DMF/TFA
Amino Acid Carbon 13C, ppma Mult. 1H, ppmb Multiplicity
Valine-1 1 170.4 s (NH) 7.11 d, J=8.9
2 60.3 d 4.16 t, J=9.0
3 30.8 d 1.77 m
4 19.6 q 0.95 m
5 18.8 q 0.95 m
Dehydroamino 1 164.5 s (NH) 9.20 s
butyric acid 2 130.3 s
3 131.3 d 6.48 q, J=6.9
4 12.7 q 1.43 d, J=6.6
Phenylalanine 1 171.3 s (NH) 8.62 d, J=6.6
2 56.3 d 4.68 q, J=6.6
3 36.8 t 3.23 dd, J=13.7,
3.00 7.2
3.00 dd, J=13.7,
9.0
4 138.2 s
5, 5′ 129.9 d 7.32 d, J=7.2
6, 6′ 128.8 d 7.28 t, J=7.5
7 127.0 d 7.21 t, J=7.2
Valine-2 1 172.9 s (NH) 7.82 d, J=6.6
2 58.6 d 4.36 m
3 32.4 d 2.12 m
4 18.0 q 0.85 m
5 17.6 q 0.77 d, J=6.6
Isoleucine-1 1 171.9 s (NH) 8.38 d, J=0.6
2 57.5 d 4.53 m
3 38.8 d 1.98 m
4 14.6 q 0.92 d, J=6.6
5 26.8 t 1.40, 1.13 m, m
6 11.7 q 0.88 t, J=7.2
Threonine-1 1 169.7 s (NH) 8.12 d, J=5.7
2 57.4 d 4.63 t, J=9.3
3 71.1 d 5.07 dq, J=9.6, 6.0
4 17.3 q 1.18 d, J=6.3
Isoleucine-2 1 171.9 s (NH) 7.72 d, J=8.4
2 57.3 d 4.52 m
3 38.0 d 1.88 m
4 14.8 q 0.88 d, J=6.3
5 26.6 t 1.40, 1.13 m, m
6 11.6 q 0.88 t, J=7.2
Ornithine 1 172.0 s (NH) 7.92 d, J=7.8
2 52.9 d 4.48 m
3 29.6 t 1.76 m
4 24.4 t 1.83 m
5 40.1 t 3.10 p, 5.1
Proline 1 172.6 s
2 60.2 d 4.42 m
3 29.6 t 2.12, 1.97 m, m
4 25.4 t 2.02, 1.88 m, m
5 48.0 t 3.75, 3.68 m, m
Valine-3 1 171.3 s (NH) 7.90 d, J=7.2
2 57.6 d 4.41 m
3 30.5 d 2.12 m
4 19.6 q 0.95 m
5 18.8 q 0.85 m
Valine-4 1 171.8 s (NH) 7.68 d, J=8.1
2 59.1 d 4.34 m
3 31.3 d 2.17 m
4 19.5 q 0.95 m
5 18.1 q 0.90 m
Threonine-2 1 171.0 s (NH) 7.77 d, J=8.1
2 58.9 d 4.46 m
3 67.4 d 4.21 dq, J=6.3, 3.6
4 19.7 q 1.12 d, J=6.6
Valine-5 1 172.7 s (NH) 7.85 d, J=8.1
conf. #2 7.85 d, J=7.81
(NH) 7.82
2 59.6 d 4.32 m
3 30.7 d 2.14 m
4 19.6 q 0.95 m
5 18.4 q 0.90 m
5-Methyl- 1 173.8 s
Hexanoic acid 2 36.3 t 2.26 m
3 24.0 t 1.60 m
4 39.0 t 1.20 m
5 28.1 d 1.55 m
6 22.5 q 0.87 d, J=7.2
7 22.5 q 0.87 d, J=7.2
5-Methyl- 1 174.1 s
Hexanoic acid 2 33.9 t 2.29 m
(second 3 32.8 t 1.65, 1.40 m
conformation) 4 29.8 t 1.13 m
5 34.5 d 1.35 m
6 19.5 q 0.90 m
7 11.2 q 0.90 m
aat 125 MHz, DMF signal at 35.2 ppm;
bat 500 MHz, DMF signal at 2.91 ppm.

TABLE I TABLE II
In vitro Activity of Kahalalide F KF from Elysia rufescens
Assay (M.I.C. μg/mL)
Cytoxicity μg/mL (IC50)
A-549 2.5
HT-29 0.25-0.5
Antiviral μg/mL (% reduction)
Mv 2 Lu/HSV II 0.5 (95%)
CV-1/HSV-1 >8
BHK/VSV >8
Antifungal 6 mm disk 50 μg/disk
Aspergillus oryzae 19 mm
Penicillium notatum 26 mm
Tricophyton mentagrophy 34 mm
Saccharomyces cerevisiac neg
Candida albicans 16 mm

INVENTORS:

Scheuer, Alice E. D., Hamann, Mark T., Gravalos, Dolores G.

THIS PATENT IS REFERENCED BY THESE PATENTS:
Patent Priority Assignee Title
10538535, Apr 27 2017 PHARMA MAR, S A Antitumoral compounds
11332480, Apr 27 2017 PHARMA MAR, S A Antitumoral compounds
11339180, Apr 27 2017 Pharma Mar, S.A. Antitumoral compounds
11713325, Apr 27 2017 Pharma Mar, S.A. Antitumoral compounds
7482429, Feb 09 2000 PHARMA MAR, S A Kahalalide F and related compounds
7683028, Feb 26 2003 Pharma Mar, S.A.U. Kahalalide compositions
THIS PATENT REFERENCES THESE PATENTS:
Patent Priority Assignee Title
20040214755,
WO2004035613,
ASSIGNMENT RECORDS    Assignment records on the USPTO
/
Executed onAssignorAssigneeConveyanceFrameReelDoc
Aug 14 2003PharmaMar, S.A.(assignment on the face of the patent)
MAINTENANCE FEES AND DATES:    Maintenance records on the USPTO
Date Maintenance Fee Events
Jan 15 2009M1552: Payment of Maintenance Fee, 8th Year, Large Entity.
Feb 17 2010ASPN: Payor Number Assigned.
Feb 17 2010RMPN: Payer Number De-assigned.
Feb 14 2013M1553: Payment of Maintenance Fee, 12th Year, Large Entity.


Date Maintenance Schedule
Feb 27 20104 years fee payment window open
Aug 27 20106 months grace period start (w surcharge)
Feb 27 2011patent expiry (for year 4)
Feb 27 20132 years to revive unintentionally abandoned end. (for year 4)
Feb 27 20148 years fee payment window open
Aug 27 20146 months grace period start (w surcharge)
Feb 27 2015patent expiry (for year 8)
Feb 27 20172 years to revive unintentionally abandoned end. (for year 8)
Feb 27 201812 years fee payment window open
Aug 27 20186 months grace period start (w surcharge)
Feb 27 2019patent expiry (for year 12)
Feb 27 20212 years to revive unintentionally abandoned end. (for year 12)