C20′ urea derivatives of vinca alkaloids

Abstract

A vinca alkaloid compound that is substituted at the 20-position with a urea or thiourea group is disclosed. The urea's proximal nitrogen atom bonded to the 20-position carbon atom is secondary, whereas the distal nitrogen atom can be unsubstituted only when the compound contains an optionally present 10-fluoro substituent, and is otherwise preferably mono- or di-substituted. Methods of preparing the compounds are disclosed as are compositions for their use and methods of treatment using a compound.

Claims

1. A vinca alkaloid compound or pharmaceutically acceptable salt thereof that is substituted at the 20-position with a urea or thiourea group in which a proximal nitrogen atom that is directly bonded to the 20-carbon atom is secondary and a distal nitrogen atom that is unsubstituted, or contains one or two substituents, wherein said one or two substituents are independently selected from the group consisting of a) a straight or branched chain hydrocarbyl group that has 1 to about 6 carbon atoms that is free of quaternary carbon atoms, b) an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur, said ring structure being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, c) an aralkyl or heteroaralkyl group containing 5 or 6 ring atoms of which up to three ring atoms can independently be nitrogen, oxygen or sulfur and 1-3 carbons in the alkyl portion, said ring structure being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, and d) two substituents on the distal nitrogen atom together with that distal nitrogen atom form a single 5- or 6-membered ring or a fused ring system containing two rings, each of which can contain a 5- or 6-members and can also contain one or two additional ring hetero atoms that can independently be nitrogen, oxygen or sulfur said ring or ring system being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, said vinca alkaloid compound optionally containing a 10-fluoro substituent and having a distal nitrogen atom that is unsubstituted only when said 10-fluoro substituent is present.

2. The vinca alkaloid compound or salt thereof according to claim 1 that is a 20-substituted vinblastine, vincristine or vindesine.

3. The vinca alkaloid compound or salt thereof according to claim 1 whose 20-substituent is a urea.

4. The vinca alkaloid compound or salt thereof according to claim 3 whose distal urea nitrogen atom contains one substituent.

5. The vinca alkaloid compound or salt thereof according to claim 4 whose one substituent is a straight or branched chain hydrocarbyl group that has 1 to about 6 carbon atoms.

6. The vinca alkaloid compound or salt thereof according to claim 4 whose one substituent is an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur.

7. The vinca alkaloid compound or salt thereof according to claim 6 whose one substituent is an aliphatic carbocyclic ring structure containing one ring containing 3-6 carbons.

8. The vinca alkaloid compound or salt thereof according to claim 6 whose one substituent is an aromatic ring structure that contains one or two rings.

9. The vinca alkaloid compound or salt thereof according to claim 8 whose aromatic ring structure contains a single substituent group.

10. The vinca alkaloid compound or salt thereof according to claim 3, wherein two substituents on the distal urea nitrogen atom together with that distal nitrogen atom form a single 5- or 6-membered ring or a fused ring system containing two rings, each of which can contain a 5- or 6-members and can also contain one or two additional ring hetero atoms that can independently be nitrogen, oxygen or sulfur, said ring or ring system being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof.

11. The vinca alkaloid compound or salt thereof according to claim 10, wherein said two substituents on the distal urea nitrogen atom together with that distal nitrogen atom form a 6-membered ring.

12. The vinca alkaloid compound or salt thereof according to claim 10, wherein said two substituents on the distal urea nitrogen atom together with that distal nitrogen atom form a 5-membered ring.

13. The vinca alkaloid compound or salt thereof according to claim 10, wherein said two substituents on the distal urea nitrogen atom together with that distal nitrogen atom form a fused ring system containing two rings, each of which can contain a 5- or 6-members and can also contain one or two additional ring hetero atoms that can independently be nitrogen, oxygen or sulfur.

14. The vinca alkaloid compound or salt thereof according to claim 1 that corresponds in structure to Formula I below: TABLE-US-00016 I R.sup.1 R.sup.2 R.sup.3 Vinblastine (1) CH.sub.3 Vincristine (2) Vindesine (1a) CH.sub.3 OH where Z is H or F, Y is O or S, and R.sup.4 and R.sup.5 are independently selected from the group consisting of hydrido, or a) a straight or branched chain hydrocarbyl group that has 1 to about 6 carbon atoms that is free of tertiary or quaternary carbon atoms, b) an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur, said ring structure being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, c) an aralkyl or heteroaralkyl group containing 5 or 6 ring atoms of which up to three ring atoms can independently be nitrogen, oxygen or sulfur and 1-3 carbons in the alkyl portion, said ring structure being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, and d) two substituents on the distal nitrogen atom together with that distal nitrogen atom form a single 5- or 6-membered ring or a fused ring system containing two rings, each of which can contain a 5- or 6-members and can also contain one or two additional ring hetero atoms that can independently be nitrogen, oxygen or sulfur said ring or ring system being optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, with the proviso that R.sup.4 and R.sup.5 are only both hydrido when Z is F.

15. The vinca alkaloid compound or salt thereof according to claim 14, wherein Z is F.

16. The vinca alkaloid compound or salt thereof according to claim 15 that corresponds in structure to the structural formula below. ##STR00060##

17. A pharmaceutical composition that comprises a cancerous cell proliferation-inhibiting amount of a 20-urea-substituted vinca alkaloid compound of claim 1 or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically acceptable carrier.

18. A method of inhibiting the growth of cancerous cells that comprises contacting said cancerous cells with a cancerous cell proliferation-inhibiting amount of a 20-urea- or thiourea-substituted vinca alkaloid compound of claim 1 or a pharmaceutically acceptable salt thereof.

19. The method according to claim 18, wherein said cancerous cells are contacted a plurality of times.

20. The method according to claim 18, wherein said cancerous cells are contacted in vitro.

21. The method according to claim 18, wherein said contacted cancerous cells are leukemia cells.

22. The method according to claim 18, wherein said contacted cancerous cells are carcinoma cells.

23. The method according to claim 22, wherein said contacted carcinoma cells are resistant to vinblastine.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention contemplates a 20-urea- or thiourea-substituted vinca alkaloid compound or pharmaceutically acceptable salt of such a compound. More particularly, a contemplated vinca alkaloid compound that is substituted at the 20-position with a urea or thiourea group whose proximal nitrogen atom that is directly bonded to the 20 carbon atom is secondary (has one hydrogen atom bonded to it) and whose distal nitrogen is contains one, and more preferably contains two non-hydrido substituents (the R.sup.4 and R.sup.5 groups in the formula below) when the vinca alkaloid compound does not contain a 10-fluoro group. A preferred vinca alkaloid compound is a 20-substituted vinblastine, vincristine or vindesine that can optionally be further substituted at the 10-position with a fluoro group and corresponds in structure to Formula I below:

(2) TABLE-US-00004 I R.sup.1 R.sup.2 R.sup.3 Vinblastine (1) CH.sub.3 0 Vincristine (2) Vindesine (1a) CH.sub.3 OH
where Z is H or F, and R.sup.4 and R.sup.5 are discussed hereinafter.

(3) The R.sup.4 and R.sup.5 substituents are independently selected from the group consisting of hydrido, a) a straight or branched chain or cyclic hydrocarbyl group that has 1-6 carbon atoms, and preferably 2-6 carbon atoms, that is free of tertiary or quaternary carbon atoms, b) an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur. A third substituent, c) is an aralkyl or heteroaralkyl group containing 5 or 6 ring atoms of which up to three ring atoms can independently be nitrogen, oxygen or sulfur and contains 1-3 carbons in the alkyl portion. Alternatively, d) two substituents bonded to the distal nitrogen atom together with that distal nitrogen atom form a single 5- or 6-membered aliphatic or aromatic ring or a fused ring system containing two rings each of which contains 5- or b-ring atoms. At least one of the R.sup.4 and R.sup.5 substituents of a)-d) above is other than hydrido, and more preferably, both R.sup.4 and R.sup.5 substituents are other that hydrido when Z is H. When Z is F, both of R.sup.4 and R.sup.5 can be H, but it is preferred that only one be H and more preferred that both of R.sup.4 and R.sup.5 be other than H.

(4) The single or fused ring system of d) can contain one or two additional hetero atoms that can independently be nitrogen, oxygen or sulfur. Each of the rings or ring systems of b), c) and d) above can optionally be substituted with 1, 2 or 3 substituents selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof.

(5) Examining the preferred one or more preferred two distal nitrogen atom substituents (R.sup.4 and R.sup.5 groups) more closely, both of R.sup.4 and R.sup.5 can be hydrido only when Z is F, but preferably, at least one of R.sup.4 and R.sup.5 is other than hydrido, and more preferably both of R.sup.4 and R.sup.5 are other than hydrido. One preferred R.sup.4 and/or R.sup.5 substituent is a straight or branched chain hydrocarbyl group that has 1-6 carbon atoms, and preferably 2-6 carbon atoms, that is free of tertiary or quaternary carbon atoms. Exemplary hydrocarbyl substituent groups have been generally discussed previously. This hydrocarbyl group excludes those substituents that contain a tertiary carbon atom as in a t-butyl group [(CH.sub.3).sub.3C] or a quaternary carbon as is present in a neo-pentyl group [(CH.sub.3).sub.3CH.sub.2].

(6) A second group of distal nitrogen substituents (R.sup.4 and/or R.sup.5) is an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur. That ring structure is optionally substituted with 1, 2 or 3 substituents (ring substituents) that are themselves selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof. Illustrative ring structures include phenyl, biphenyl, naphthyl, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclodecyl, pyridyl, furanyl, purinyl, isoquinolinyl, tetrahydroisoindanyl and the like that are discussed hereinbefore.

(7) A contemplated aromatic or aliphatic carbocyclic or heterocyclic ring structure R.sup.4 and/or R.sup.5 substituent can contain 1-3 of its own substituents but preferably contains only one such as a halogen like chloro or fluoro, a C.sub.1-C.sub.6-hydrocarbyl group such as methyl, a C.sub.1-C.sub.6-hydrocarbyloxy group such as methoxy, a perfluoro-C.sub.1-C.sub.6-hydrocarbyl group such as trifluoromethyl or a perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy group such as pentafluoroethoxy. When more than one substituent is present on a ring structure substituent, those multiple substituents need not be the same group.

(8) One preferred nitrogen substituent contains one ring that contains 3-6 carbon atoms in the ring. Another preferred substituent is an aromatic ring structure that contains one or two rings. One preferred aromatic ring contains one substituent.

(9) Another contemplated group of distal nitrogen atom R.sup.4 and/or R.sup.5 substituents is an aralkyl or heteroaralkyl group containing 5 or 6 ring atoms of which up to three ring atoms can independently be nitrogen, oxygen or sulfur and 1-3 carbons in the alkyl portion. The aromatic ring portion of a contemplated ring structure is optionally substituted with 1, 2 or 3 substituents that are selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof, as are discussed above. Zero or one ring substituent other than hydrogen is preferred.

(10) Illustrative 5- or 6-membered aromatic and heteroaromatic ring substituents were also discussed previously. The alkyl portion of an aralkyl or heteroaralkyl group can contain 1 to 3 carbon atoms. Illustrative non-ring-substituted aralkyl groups include benzyl and phenethyl groups, whereas illustrative heteroaralkyl groups include methylpyridyl and ethylimidazolyl.

(11) A distal urea nitrogen atom can also form a ring structure together with the two R.sup.4 and R.sup.5 substituent groups. That nitrogen-containing ring structure can contain a single 5- or 6-membered ring or contain a fused two ring structure in which the rings are both 5-membered, or both 6-membered, or in which one ring is 5-membered and the other is 6-membered. A contemplated ring structure can also contain one or two additional hetero atoms (non-carbon atoms) in the ring(s) that can be independently nitrogen, oxygen or sulfur. This nitrogen-containing ring is preferably a 6-membered ring or a 5-/6-membered fused ring system. 20-Urea- or thiourea-substituted vinblastine compounds are illustrated hereinafter that contain distal nitrogen atoms that are present in 5-, 6-, 6/6- and 5/6-membered rings.

(12) These nitrogen-containing rings can be aromatic as in the case of an 1-imidazyl, 1-pyrazolyl, 1-(1,2,4-triazolyl), 2-o-isoxazinyl or a 2-(1,3,2-dioxazolyl) group, but are more usually aliphatic such as 1-piperidinyl, 1-pyrrolidinyl, 1-piperazinyl, 1-morpholinyl, 1-thiomorpholinyl, isoquinolinyl, tetrahydroisoindolinyl and the like. It is noted that a NR.sup.4R.sup.5 cyclic substituent that contains a free hydrogen atom such as that of a secondary nitrogen (NH) present on the 4-position nitrogen of piperazine ring are blocked as with a t-Boc, F-moc, acetyl, C.sub.1-C.sub.6-hydrocarbyl such as methyl, or other group during synthesis and thereafter because of the reactivity with an isocyanate or isothiocyanate reagent.

(13) A urea nitrogen atom-containing ring structure is optionally substituted with 1, 2 or 3 substituents that are selected from the group consisting of C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, phenyl, halogen, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, nitro and mixtures thereof.

(14) A phenyl substituent bonded to a piperidinyl group or the aromatic portion of a ring structure formed by a NR.sup.4R.sup.5 cyclic structure can contain 1-3 substituents independently selected from the group consisting C.sub.1-C.sub.6-hydrocarbyl, C.sub.1-C.sub.6-hydrocarbyloxy, perfluoro-C.sub.1-C.sub.6-hydrocarbyl, perfluoro-C.sub.1-C.sub.6-hydrocarbyloxy, halogen (fluoro, chloro or bromo) and nitro,

(15) Another aspect of the invention is a doubly substituted vinca alkaloid compound. This compound contains a 20-urea or thiourea group as discussed above, and also contains an added fluoro (F) substituent at the 10-position of the molecule. The preparation of different fluoroinated vinca alkaloids was discussed in Va et al., J Am Chem Soc 2010 132:8489-8495 and in WO 2011/103007 published on 25 Aug. 2011, but that synthesis can also be used herein. As is seen from the data that follows, the presence of both the 20-urea substitution and the 10-fluoro substitution in a vinblastine molecule provided growth inhibitory activity against each of the cancer cell lines examined that was greater than the inhibitory activity of either substitution alone.

(16) Pharmaceutical Composition and Methods

(17) A contemplated 20-urea- or thiourea-substituted vinca alkaloid compound can also be used in the manufacture of a medicament (pharmaceutical composition) that is useful at least for inhibiting the proliferation (growth) of hematologic cancer cells such as leukemia or lymphoma cells, as well as cells of carcinomas, sarcomas, melanomas, neuromas and the like. A contemplated compound, medicament or pharmaceutical composition containing the same inhibits that growth by contacting those cancerous cells in vitro, or in vivo as in a subject in need thereof, as is a parent compound. When so used, pharmaceutically acceptable salts, buffers and the like are present that collectively are referred to as pharmaceutically acceptable diluents as compared to those that can be present in a composition that is not intended for pharmaceutical use, as in an in vitro assay.

(18) A compound of the invention can be provided for use by itself, or as a pharmaceutically acceptable salt. The contemplated compounds are amines. Parental vinblastine has reported pKa values of 5.4 and 7.4, whereas vincristine has reported pKa values of 6.04 and 7.67. [The Merck Index, 13.sup.th ed. Merck & Co., Whitehouse Station, N.J., 2001, pages 1778-1779.] Both compounds are sold commercially as their sulfate salts. Vindesine is reported to have pka values of 6.04 and 7.67 [The Merck Index, 12.sup.th ed., Merck and Co., Whitehouse Station, N.J., 1996, page 1704]. Vindesine is also commercially available as the sulfate salt.

(19) Exemplary salts useful for a contemplated compound include but are not limited to the following: sulfate, hydrochloride, hydro bromides, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate.

(20) The reader is directed to Berge, J. Pharm. Sci. 1977 68(1):1-19 for lists of commonly used pharmaceutically acceptable acids and bases that form pharmaceutically acceptable salts with pharmaceutical compounds.

(21) In some cases, the salts can also be used as an aid in the isolation, purification or resolution of the compounds of this invention. In such uses, the acid used and the salt prepared need not be pharmaceutically acceptable.

(22) As is seen from the data that follow, a contemplated compound is active in in vitro assay studies at nanomolar to micromolar amounts. When used in an assay such as an in vitro assay, a contemplated compound is present in the composition in an amount that is sufficient to provide a concentration of about 0.5 nM to about 1000 nM, preferably about 1 nM to about 50 nM to a contact cells to be assayed.

(23) A contemplated pharmaceutical composition contains a cancerous cell proliferation-inhibiting amount of a contemplated 20-urea- or thiourea-substituted vinca alkaloid compound or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically (pharmaceutically) acceptable carrier. That amount is typically about the same amount to a little less than the amount of a parental vinca alkaloid used to treat the same cancer. Such a composition can be administered to mammalian cells in vitro as in a cell culture to contact those cells, or the cells can be contacted in vivo as in a living, host mammal in need.

(24) More usually, anti-neoplastic drugs such as a 20-substituted vinca alkaloid contemplated here are administered parenterally in vivo in a weight amount per square meter of the recipient's body surface area (bsa). For adults, this amount is typically about 1 to about 20 mg/m.sup.2 bsa, and about one-half those amounts for children, with an amount being chosen so that the maximal amount does not cause leukopenia. Children weighing about 10 kg or less are typically dosed at about 0.05 mg/kg.

(25) For example, vinblastine sulfate is typically administered to adults at 3.7 mg/m.sup.2 bsa for the first dose, 5.5 mg/m.sup.2 bsa for the second weekly dose, 7.4 mg/m.sup.2 bsa for the third weekly dose, 9.25 mg/m.sup.2 bsa for the fourth weekly dose and 11.1 mg/m.sup.2 bsa for the fifth weekly dose. Dosages typically do not exceed 18.5 mg/m.sup.2 bsa, and should not be increased if the white-cell count falls to approximately 3000 cells/mm.sup.3. Usual dosages for adults are about 5.5 to 7.4 mg/m.sup.2 bsa. Dosages of a contemplated 20-position urea- or thiourea-substituted vinca alkaloid compound or its pharmaceutically acceptable salt typically do not exceed those of the parent compound and can be less.

(26) A contemplated composition is typically administered in vivo to a subject in need thereof a plurality of times within one month, such as weekly, and can be administered over a period of several months to several years. More usually, a contemplated composition is administered a plurality of times over a course of treatment.

(27) In usual practice, a contemplated 20-urea- or thiourea-substituted vinca alkaloid compound is administered to treat the same disease state in the same amount and at the same intervals as is a parental, 20-hydroxy-vinca alkaloid. A contemplated 20-urea- or thiourea-substituted vinca alkaloid can be utilized as a first course of treatment, and is preferably administered if there is relapse after a first or later course of treatment, particularly where multiple drug resistance is shown or suspected (indicated).

(28) A contemplated pharmaceutical composition can be administered orally (perorally) or parenterally, which is preferred, in a formulation containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous (which is most preferred), intramuscular, intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.; 1975 and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.

(29) Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules. The amount of a contemplated compound in a solid dosage form is as discussed previously, an amount sufficient to provide a concentration of about 0.5 nM to about 1000 nM, preferably about 1 nM to about 50 nM, in the serum or blood plasma. A solid dosage form can also be administered a plurality of times during a one week time period.

(30) In such solid dosage forms, a compound of this invention is ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the compounds can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings.

(31) A contemplated pharmaceutical composition is preferably adapted for parenteral administration. Thus, a pharmaceutical composition is preferably in liquid form when administered, and most preferably, the liquid is an aqueous liquid, although other liquids are contemplated as discussed below, and a presently most preferred composition is an injectable preparation.

(32) Thus, injectable preparations, for example, sterile injectable aqueous or oleaginous solutions or suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline.

(33) Other liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration. Sterile water solutions of a 20-urea- or thiourea-substituted vinca alkaloid active component or sterile solution of the active component in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. In some aspects, a contemplated 20-urea- or thiourea-substituted vinca alkaloid is provided as a dry powder that is to be dissolved in an appropriate liquid medium such as sodium chloride for injection prior to use.

(34) In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of an injectable composition. Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.

(35) Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.

(36) A mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a contemplated compound is administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.

(37) Where an in vitro assay is contemplated, a sample to be assayed such as cells and tissue can be used. These in vitro compositions typically contain the water, sodium or potassium chloride, and one or more buffer salts such as and acetate and phosphate salts, Hepes or the like, a metal ion chelator such as EDTA that are buffered to a desired pH value such as pH 4.0-8.5, preferably about pH 7.2-7.4, depending on the assay to be performed, as is well known.

(38) Preferably, the pharmaceutical composition is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active compound. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, in vials or ampules.

(39) In another preferred embodiment, a contemplated 20-urea- or thiourea-substituted vinca alkaloid is administered with one or more other anti-neoplastic compounds. Such joint therapy is well known in the art, with other drugs such as cisplatin, 5-fluorouracil and the like being co-administered. That co-administration is usually physically separate administrations of each compound that are timed so that the two or more active agents can act in concert.

(40) Results and Discussion

(41) Chemistry.

(42) The targeted vinblastine C20-urea and -thiourea analogues were prepared by two methods (Scheme 1). The first method (Method 1) involved

(43) ##STR00025##
treating the recently accessible 20-aminovinblastine (7) [Leggans et al., Org. Lett. 2012, 14:1428-1431] with available isocyanates to provide the corresponding ureas. In the instances when the isocyanates were not readily available, Method 2 was used. This method entailed treating 20-aminovinblastine (7) with p-nitrophenyl-chloroformate to provide the activated carbamate 13, which was then treated with a series of amines to provide the additional C20-urea analogues.

(44) The vinblastine C20-thiourea analogues were prepared also using two complementary methods (Scheme 2). Thus, treatment of 20-aminovinblastine (7) with a small series of commercially available

(45) ##STR00026##
isothiocyanates (Method 3) versus isocyanates provided the corresponding C20-thiourea analogues. Alternatively, treatment of 20-isothiocyano-vinblastine (5) [Leggans et al., Org. Lett. 2012 14:1428-1431], made from the reaction of 20-aminovinblastine (7) and carbon disulfide (82%) or that is available upon direct Fe(III)/NaBH.sub.4-mediated functionalization of anhydrovinblastine (KSCN) [Leggans et al., Org. Lett. 2012 14:1428-1431] with available amines (Method 4) also provided a set of C20-thiourea vinblastine analogues.

(46) A series of N,N-disubstituted C20-urea and -thiourea vinblastine analogues were also prepared using Methods 2 and 4 and commercially available N,N-disubstituted amines. Additionally, the carbamate analogue 45 was synthesized for direct comparison with what proved to be the potent C20-urea and -thiourea derivatives. Thus, vinblastine (1) was treated with the N,N-dimethylcarbamyl chloride to provide the C20-carbamate 45 (below).

(47) ##STR00027##
Biological Activity.

(48) The C20-substituted vinblastine analogues were examined for cell growth inhibitory activity against the HCT116 (human colon cancer), and HCT116/VM46 (resistant human colon cancer) tumor cell lines, the latter of which exhibits resistance (100-fold) to vinblastine through overexpression of Pgp. [Lampidis et al., Biochemistry 1997 36:2679-2685; Perego et al., Cancer Res. 2001 61:6034-6037.] As reported [Leggans et al., Org. Lett. 2012 14:1428-1431], the unsubstituted urea 11 on which the studies are based approached but did not match the potency of vinblastine. The results of the examination of the systematically varied monosubstituted C20-urea derivatives prepared herein are summarized in the Table below alongside those of vinblastine (1) and the unsubstituted urea (11) of Leggans et al., Org. Lett. 2012 14:1428-1431.

(49) The cell growth inhibition activity against the L1210 (mouse leukemia) cancerous cell line was also measured and the results were qualitatively and quantitatively (IC.sub.50) nearly identical to those observed with the HCT116 cancerous carcinoma cell line in that IC.sub.50 values were obtained for the urea derivatives that matched or exceeded the activity of vinblastine itself. Results are shown below.

(50) TABLE-US-00005 IC.sub.50 (nM) Compound L1210 HCT116 HCT116/VM46 Vinblastine (1) 6.0 6.8 600 R = H (11) 40 7.5 4400 Alkyl R = methyl (14) 5.7 0.82 530 R = ethyl (15) 2.1 0.73 90 R = n-propyl (16) 6.0 2.7 221 R = i-propyl (17) 5.5 5.7 430 R = cyclopropyl (18) 3.9 0.73 85 R = n-butyl (19) 5.7 4.6 270 R = t-butyl (20) 40 20 670 R = cyclohexyl (21) 5.8 5.4 450 R = 2-hydroxyethyl (22) 48 8.8 >1000 Aryl R = C.sub.6H.sub.5 (23) 6.5 5.1 390 R = p-C.sub.6H.sub.4F (24) 5.7 3.9 400 R = p-C.sub.6H.sub.4Cl (25) 6.2 6.7 590 R = p-C.sub.6H.sub.4CH.sub.3 (26) 4.5 4.8 330 R = p-C.sub.6H.sub.4CF.sub.3 (27) 7.3 7.1 610 R = p-C.sub.6H.sub.4OCH.sub.3 (28) 4.9 2.0 230 R = m-C.sub.6H.sub.4OCH.sub.3 (29) 5.4 0.77 80 R = o-C.sub.6H.sub.4OCH.sub.3 (30) 4.8 0.77 65 R = CH.sub.2C.sub.6H.sub.5 (31) 6.4 7.3 740 R = CH.sub.2CH.sub.2C.sub.6H.sub.5 (32) 6.3 6.3 590 R = CH.sub.2(2-pyridyl) (33) 24 5.6 670 R = CH.sub.2(2-furyl) (34) 5.4 5.1 530

(51) Terminal N-alkyl substituents were not only well tolerated, but provided significant enhancements in activity, improving on the potency of 11 and providing derivatives that substantially surpass that of vinblastine itself. Most notable of these are the urea derivatives 14-18, bearing small N-alkyl substituents. Some of those compounds exhibited IC.sub.50 values of 700-800 pM against HCT116, improving activity against HCT116 about 10-fold relative to 11, and substantially surpassing the potency of vinblastine itself (about 10-fold).

(52) Even the larger N-alkyl derivatives 19 and 21 matched or slightly surpassed the activity of vinblastine and only 20, bearing the large t-butyl substituent, experienced a small, but surprisingly modest loss in activity given expectations. Introduction of a polar group that can serve as either a H-bond donor or acceptor on the alkyl substituent in 22 maintained the activity observed with other small alkyl substituents in the HCT116 cell line; however, it had a deleterious effect in the potency against the resistant cell line (IC.sub.50>1000 nM, HCT116/VM46), similar to that seen in the parent urea 11.

(53) The examination of the monosubstituted C20-urea derivatives bearing N-aryl substituents (23-30) proved even more unexpected. All exhibited cell growth inhibitory activity at levels exceeding the parent urea 11, matching or surpassing the potency of vinblastine itself. Electron-withdrawing or electron-donating substituents on the parent N-phenyl urea 23 are well tolerated.

(54) Although no strongly polar substituents were examined, it is notable that the p-methoxy substituent proved to be among the best of the p-substituents. As a result, the impact of a m- and o-methoxy phenyl substituent was also examined. Significantly, 29 and 30 bearing N-(m-methoxyphenyl) and N-(o-methoxyphenyl) urea substituents respectively, exhibited exceptional activity, substantially exceeding the potency of vinblastine nearly 10-fold (IC.sub.50 770 pM vs 6.8 nM, HCT116) and displaying uniquely potent activity against the vinblastine-resistant HCT116 cell line (IC.sub.50=80 and 65 nM, HCT116/VM46). This activity along with that of 15 and 18 represents a 10-fold improvement over vinblastine.

(55) Placement of a one or two methylene spacer between the phenyl ring and urea nitrogen (31 and 32) maintained activity with both derivatives matching the activity of both 23 and vinblastine itself. Replacement of the phenyl ring in 31 with a heteroaromatic ring (furan or pyridine) provided 33 and 34, which displayed comparable activity to the parent 31, matching or slightly exceeding the potency of vinblastine.

(56) All these observations are unexpected given the apparent steric constraints of the tubulin binding site surrounding the vinblastine C20-center observed in the x-ray crystal structure of a tubulin bound complex. [Gigant et al., Nature 2005 435:519-522.] To further probe just how much space may be available to a C20-derivative, the rigid N-biphenyl urea 35 was prepared and examined. Remarkably, it displayed cell growth inhibitory activity at a level indistinguishable from vinblastine, below.

(57) TABLE-US-00006 IC.sub.50 (nM) Compound HCT116 HCT116/VM46 1 6.8 600 35 6.9 470

(58) This result indicates that sterically demanding modifications to such C20-urea derivatives are likely even beyond those probed herein. As a result and in addition to improvements in potency, this may be a superb site for modulating the physical and chemical properties of the drug that impact additional features including Pgp efflux [Hitchcock, J. Med. Chem. 2012 55:4877-4895], in vivo drug distribution, selective cellular uptake, and metabolism.

(59) A series of thiourea derivatives was also examined as shown below. Although the unsubstituted thiourea derivative 12 approached the activity of the corresponding urea 11 in the original studies (IC.sub.50=7.7 vs 7.5 nM, HCT116), the monosubstituted N-alkyl or N-aryl derivatives 36 and 37 proved to be 3- to 4-fold less active than the corresponding ureas 21 and 23 (HCT116). However, it is notable that the activity difference between sensitive and vinblastine-resistant cell lines diminished in this thiourea series (15- to 25-fold vs 100-fold), suggesting they may be transported by Pgp somewhat less effectively. [Hitchcock, J. Med. Chem. 2012 55:4877-4895]

(60) TABLE-US-00007 0 IC.sub.50 (nM) Compound HCT116 HCT116/VM46 Vinblastine 6.8 600 R = H (12) 7.7 2000 R = C.sub.6H.sub.11 (36) 20 520 R = C.sub.6H.sub.6 (37) 40 650 R = CH.sub.2CH.sub.2(4-FC.sub.6H.sub.4) (38) 60 750

(61) A small, but important series of N,N-disubstituted ureas and thioureas was also examined in order to establish whether the terminal urea nitrogen could be fully substituted or whether the derivatives require or benefit from the presence of an NH H-bond donor, below. Remarkably, all the N,N-disubstituted ureas exhibited potent cell growth inhibitory activity matching or surpassing the activity of vinblastine and indicating that a terminal H-bond donor site is not important to their functional activity.

(62) However, both 39 and 40 were less active than the corresponding monosubstituted urea derivatives 14 and 15. Ureas with cyclic substituents on the terminal nitrogen, 41 and 42, exhibited a similar potency to the acyclic derivatives 39 and 40. An analogous observation was made with the N,N-dimethyl thiourea derivative 43, which approached the potency of vinblastine but exhibited activity slightly lower than the corresponding N,N-dimethyl urea 38.

(63) Interestingly, and like the thiourea derivatives 36 and 37, Compound 43 and especially the urea derivatives 39 and 42 exhibited a diminished activity difference between sensitive and vinblastine-resistant HCT116 cell lines (10-fold vs 100-fold). Comparison of the N,N-dimethyl urea or thiourea 39 and 43 with the N,N-dimethylcarbamate 44 (>1000-fold less active) clearly illustrates the distinction and importance of the C20-amine versus C20-alcohol functionalization, suggesting the H-donor capabilities of the former may be important.

(64) TABLE-US-00008 IC50 (nM).sup.a HCT116/ Compound L1210 HCT116 VM46 Vinblastine (1) 6.0 6.8 600 X = NH, Y = O A = NH.sub.2 (11) 40 7.5 4400 A = N(CH.sub.3 ).sub.2 (39) 5.9 2.8 80 A = N(CH.sub.2CH.sub.3).sub.2 (40) ND 6.7 450 5.3 4.5 360 5.5 3.9 50 0.7 0.72 50 ND 55 ND 2.1 0.88 50 7.7 3.4 710 0.52 0.52 8.4 5.3 3.1 55 0 0.62 0.56 8.7 0.51 0.60 7.5 0.61 0.69 8.7 X = NH, Y = S A = NH.sub.2 (12) ND 7.7 2000 A = N(CH.sub.3).sub.2 (43) ND 8.7 250 X = O, Y = O A = N(CH.sub.3).sub.2 (44) ND 4700 9100 .sup.aL1210 (murine leukemia cell line). HCT116 (human colon cancer cell line). HCT116/VM46 (resistant human colon cancer cell line, Pgp over-expression). Avg IC.sub.50 (4-16 determinations, SD = 10%). ND = Not Done.

(65) Cell growth inhibition by disubstituted C20 urea analogs was systematically probed, incorporating cyclic amines as the terminal nitrogen (below). Compounds 42 and 53-54 exhibited little or no change in the activity against the sensitive HCT116 cell line, but show a clear trend against the resistant HCT116/VM46 cell line with the incorporation of a polar atom in the six-membered ring having a pronounced negative effect on the activity (CS>O>NMe). After observing this trend, analogs were prepared incorporating additional non-polar functionality on the terminal cyclic amine (Compounds 55-59).

(66) C20-Urea vinblastine analogs in which the terminal nitrogen is allylic (Compound 55) or benzylic (Compounds 57-59) provided a further enhancement in the activity of approximately 10-fold relative to vinblastine and where the resulting activity against the resistant HCT116/VM46 is 80-fold better than vinblastine and 8-fold better than the saturated piperidine-based urea 42. Incorporation of a six-membered cyclic amine with a hydrophobic phenyl substituent that was not benzylic (Compound 55) to the urea nitrogen did not provide the enhanced activity in the HCT116/VM46 cell line observed with the unsaturated piperidine Compound 55 or fused phenyl ring analogs Compounds 57-59. This result suggests that an electronic effect is contributing to the enhanced activity and that it may not simply be the additional van der Waal interactions derived from an added hydrophobic aromatic ring. Addition of a methoxy group to the potent isoindoline (Compound 59) did not further impact the cell growth activity.

(67) Significant in these observations is not only the exceptional activity of the new derivatives, but their reduced differential in activity against the sensitive and resistant tumor cell line (about 13-16-fold versus about 90-fold for Compound 1). Presumably, this indicates that the new derivatives are not as effective substrates for Pgp efflux as vinblastine itself, whereas the more polar analogs Compounds 41 and 54 and especially the unsubstituted urea Compound 11 are effective substrates.

(68) Clearly, the C20-position within vinblastine represents a key site amenable to functionalization capable of simultaneously enhancing potency and presumably decreasing relative Pgp transport central to clinical resistance.

(69) Although less pronounced, but as detailed in the initial report [Leggans et al., Org. Lett. 2012 14:1428-1431] the amide 9 and methyl carbamate 10 were found to be more than 10-fold less active than the urea 11, further highlighting a unique role the urea terminal nitrogen plays in potentiating the activity. The importance of the C20-amine versus alcohol functionalization and distinctions between urea/thiourea versus carbamate/amide derivatives is illustrated in the Table below.

(70) TABLE-US-00009 IC.sub.50 (nM) Compound X Y Z HCT116 1 6.8 39 NH O NMe 2.8 43 NH S NMe 8.7 10 NH O O 75* 9 NH O 90* 44 O O NMe 4700 *Leggans et al., Org. Lett. 2012 14:1428-1431.

(71) As a result and given the size of substituents tolerated, even the intermediate p-nitrophenyl-carbamate 13 used to prepare the ureas herein was tested and proved to be a surprisingly effective agent (IC.sub.50=55 nM, HCT116), matching the activity of the methyl carbamate 10.

(72) Two additional C20-amines (45 and 46) were prepared by reductive amination (H.sub.2CO (5 equiv), NaBH.sub.3CN (20 equiv), THF, 4 hours, 32% (45) NHMe, 33% (46) NMe.sub.2) of 20-aminovinblastine (7) in order to establish the generality of the observations made with the unsubstituted primary amine 7 itself, and the results are shown below. Although the C20-methylamine proved more potent than 7, it was still 10-fold less active than vinblastine and the C20-dimethylamine derivative 46 was the least active of the C20-amines examined, suggesting the importance of a H-bond donor with regard to biological activity.

(73) TABLE-US-00010 IC.sub.50 (nM) Compound HCT116 HCT116/VM46 Vinblastine 6.8 600 R.sup.4 and R.sup.5 = H (7) 600 >10000 R.sup.4 = H R.sup.5 = CH.sub.3 (45) 60 8500 R.sup.4 and R.sup.5 = CH.sub.3 (46) 980 >10000

(74) The 20-(methylamino)vinblastine (45) was enlisted to establish whether the active urea derivatives require or benefit from the H-bond donor site of the derivatized C20-amine. Thus, treatment of 45 with ethyl isocyanate provided the N-methyl urea 47 for comparison with 15. As seen from the data below, there was a 700-fold decrease in activity between 15 and 47 (HCT116), clearly illustrating the importance of a H-bond donor site on the C20-position. This observation clearly suggests that

(75) TABLE-US-00011 IC.sub.50 (nM) Compound HCT116 HCT116/VM46 R = H (15) 0.73 90 R = Me (47) 520 8500
the incorporation of a urea functionality maintains a key H-bond site directly attached to the C20-position and that it best approximates the acidity of the vinblastine C20-alcohol, while permitting for further functionalization on the urea terminal nitrogen that maintains or in many cases improves the activity of the compound.

(76) In other recent work, the incorporation of a fluorine atom at the 10-position provided a potent molecule (48) with an 8-fold improvement in activity over vinblastine itself. [Gotoh et al., ACS Med. Chem. Lett. 2011 2:948-952.] It was of interest to determine whether the incorporation of both the 10-F substituent and a C20-urea would have an additive effect in enhancing the potency of vinblastine. An analogue 49 with both functionalities was prepared and evaluated as shown, below. Only a modest improvement in potency was observed in 49 relative

(77) TABLE-US-00012 IC.sub.50 (nM) Compound HCT116 HCT116/VM46 R = OH, X = H (1) 6.8 600 R = NHCONHEt, X = H (15) 0.73 90 R = OH, X = F (48) 0.80 80 R = NHCONHEt, X = F (49) 0.62 70
to 15 and 48, suggesting that these modifications are not fully additive. Nonetheless, the modified vinblastine 49 is at least 10-fold more potent than vinblastine exhibiting a sub-nanomolar IC.sub.50 for cell growth inhibition (620 pM, HCT116) and a nearly 10-fold improved activity against a vinblastine-resistant cell line (IC.sub.50=70 nM, HCT116/VM46).
Binding to Tubulin.

(78) Given the apparent steric constraints of the tubulin binding site surrounding the vinblastine C20-center [Gigant et al., Nature 2005 435:519-522] and the size of the C20-urea substituents that support and improve on the functional potency of vinblastine itself in the cytotoxic cell growth assays, it was not clear whether these effects could be related to their target (tubulin) binding affinity or derived from their impact on other properties of the molecules (e.g., cell permeability, metabolism, solubility). As a result, two representative C20-urea derivatives 35 and 39 were examined in a well-established tubulin binding assay conducted by measuring the competitive displacement of .sup.3H-vinblastine from porcine tubulin [Owellen et al., Biochem. Pharmacol. 1977 26:1213-1219].

(79) Notably, 35 contains the large biphenyl urea substituent yet matches the functional activity of vinblastine, whereas 39 bears the much smaller N,N-dimethylurea whose functional activity slightly exceeds that of vinblastine (3-fold). Importantly, these binding studies confirmed that 39 binds tubulin with a slightly better affinity than vinblastine and further established that even 35, bearing the large biphenyl substituent, remarkably binds with an affinity matching or even slightly exceeding that of vinblastine.

(80) Thus, the effects of the urea 39 as well as 35 observed in the functional assays correlate directly with their target tubulin binding affinities. These unanticipated observations with 35 highlight that the vinblastine interaction with tubulin surrounding the C20-center is flexible and capable of reorganization to accommodate even a very large substituent. It is notable that this site is adjacent to the nucleotide binding site involving the T5 loop in the N-terminal 1 tubulin nucleotide binding domain and adjacent to the H6 helix and H6-H7 loop that links the nucleotide binding domain to the intermediate domain. It is likely this region is capable of significant reorganization to accommodate the binding of 35 or that the urea substituent may extend into the nucleotide binding site and displace a bound nucleotide.

(81) TABLE-US-00013 % .sup.3H-vinblastine HCT116 Compound remaining bound.sup.a IC.sub.50 (nM) 1 50.0 6.8 39 45.1 3.5 2.8 35 48.5 6.9 6.9 .sup.aCompetitive binding of ligand vs [.sup.3H]VBL (1:1) measuring the remaining bound [.sup.3H]VBL. Average of 3 repeat determinations, normalized to have dpm (25 L VLB + 25 L [.sup.3H]VLB) = 50.0%. 1, X = OH (vinblastine)
Further Tubulin Binding Studies

(82) The C20 urea derivative Compound 58 was examined in a tubulin binding assay conducted by measuring the competitive displacement of .sup.3H-vinblastine from porcine tubulin Illustrated below [Owellen et al., Biochem. Pharmacol. 1977 26:1213-1219]. The binding studies established that Compound 58 binds tubulin with a higher affinity than vinblastine, establishing that its enhanced potency in the cell growth functional assays correlates directly with its target tubulin binding affinity and suggests that the improved intrinsic activity is a direct result of the inhibition of microtubule formation.

(83) Tubulin Binding Properties

(84) TABLE-US-00014 0 % .sup.3H-vinblastine HCT116 Compound remaining bound.sup.a IC.sub.50 (nM) 1 50.0 6.8 58 41.2 0.60 .sup.aCompetitive binding of ligand versus [.sup.3H]VBL (1:1) measuring the remaining bound [.sup.3H]VBL. Average of two repeat determinations, normalized to have dpm (25 L VLB + 25 L [.sup.3H]VLB) = 50.0%. 1, X = OH (vinblastine)

(85) To confirm that the exceptional activity observed in our lab would be observed by others, vinblastine (1) and Compounds 55, 57 and 58 were examined offsite at an independent laboratory in a more comprehensive human tumor 15-cell line panel including cell lines of clinical interest from breast, lung, colon, prostate and ovary tissue (below). Compounds 55, 57 and 58 exhibited remarkable potency against all cell lines examined with the exception of MCF-7, with all three compounds displaying at least a 10-fold improvement in activity over vinblastine (range of 10-200-fold more potent).

(86) Cell Growth Inhibition in 15-Cell Line Panel

(87) TABLE-US-00015 IC.sub.50 (nM) Cell Line.sup.a 1 55 57 58 AU565 4.0 0.15 0.13 0.11 NCI-H520 4.5 0.17 0.14 0.10 HCC1143 3.8 0.13 0.16 0.09 HCC70 3.5 0.21 0.13 0.04 HCT116 6.8 0.22 0.26 0.16 KPL4 2.9 0.06 0.06 0.04 LNCaP-FGC 5.1 0.45 0.02 0.24 LS174T 19.6 0.46 0.30 0.45 MCF-7 >110.sup.b >12.5.sup.b 2.1 >12.5.sup.b MDA-MB-468 4.6 0.40 0.12 0.39 SW403 7.9 0.50 1.2 0.45 T47D 5.0 0.51 0.55 0.41 ZR-75-1 8.0 0.71 0.52 0.45 PA-1 4.6 0.11 0.19 0.11 HCT116/VM46 >110.sup.b 6.4 6.6 3.5 .sup.aCell line identities are provided hereinafter. .sup.bHighest concentration tested.

(88) Compound 57 exhibited exceptional potency against LNCaP-FGC (20 pM) whereas Compound 58 provided the best activity against the resistant HCT116/VM46 cell line (3.5 nM) in this cell line panel and a reduced differential from the sensitive HCT116 cell line of 20-fold. The average IC.sub.50 value for vinblastine in this human tumor cell line panel was 6.1 nM, excluding the two cell lines for which it was inactive, and the comparative average IC.sub.50 values were 310 pM, 200 pM, and 200 pM for Compounds 55, 57 and 58, respectively, representing average enhancements of 30-fold for Compounds 57 and 58 over the activity of vinblastine.

CONCLUSIONS

(89) A remarkable series of previously inaccessible C20-urea derivatives of vinblastine were prepared and found to match or substantially exceed the potency of vinblastine in functional cell-based growth inhibition assays. In addition to defining structural features of the urea required for or potentiating their activity that are directly related to their relative tubulin binding affinity, the studies established an unprecedented steric tolerance for the size of a C20-substituent. A H-bond donor on the C20-position was unequivocally shown to be an important feature of the potent vinblastine analogues. Although this site is known to be critical to the properties of vinblastine and is located deeply embedded in the tubulin bound complex where such substituents would be apparently sterically constrained, the studies revealed that sterically demanding ureas are not only tolerated but that functionalization of this site offers a superb opportunity for enhancing potency as much as 10-fold. In addition to improvements in potency, the C20-site can also be a superb site for modulating the physical and chemical properties of the drug that impact additional features including Pgp efflux, in vivo drug distribution, selective cellular uptake, and metabolism.

(90) A series of disubstituted C20 urea derivatives of vinblastine were prepared and Compounds 55 and 57-59 were found to not only possess extraordinary potency, but to exhibit further improved activity against the Pgp overexpressing vinblastine-resistant HCT116/VM46 cell line, displaying a reduced differential in activity against the sensitive and resistant HCT116 cell line of only 10- to 20-fold (vs ca. 100-fold for vinblastine).

(91) Compound 57 was found to bind tubulin with a higher affinity than vinblastine, confirming that its enhanced potency observed in the cell growth functional assays correlates with its target tubulin binding affinity. Examination of Compounds 55, 57 and 58 in a human tumor 15-cell line panel revealed that these C20 urea analogs are on average 20- to 30-fold more potent than vinblastine across a broad spectrum of clinically relevant human cancer cell lines (range of 10-200-fold more potent), displaying low pM IC50 values (40-450 pM for 58). Clearly, the C20 position within vinblastine represents a key site amenable to functionalization capable of simultaneously improving tubulin binding affinity, substantially enhancing biological potency, and presumably decreasing relative Pgp transport central to clinical resistance.

EXPERIMENTAL SECTION

General Procedures

(92) All commercial reagents were used without further purification unless otherwise noted. THF was distilled prior to use. All reactions were performed in oven-dried (200 C.) glassware and under an inert atmosphere of anhydrous Ar unless otherwise noted.

(93) Column chromatography was performed with silica gel 60. TLC was performed on Whatman silica gel (250 m) F.sub.254 glass plates and spots visualized by UV. PTLC was performed on Whatman silica gel (250 and 500 m) F.sub.254 glass plates.

(94) Optical rotations were determined on a Rudolph Research Analytical Autopol III automatic polarimeter using the sodium D line (=589 nm) at room temperature (23 C.) and are reported as follows: [].sup.D.sub.23, concentration (c=g/100 mL), and solvent. FT-IR spectroscopy was recorded on a Nicolet 380 FT-IR instrument.

(95) .sup.1H NMR was recorded on a Bruker 600 MHz spectrometer. Chemical shifts are reported in ppm from an internal standard of residual CHCl.sub.3 (6 7.26 for .sup.1H). Proton chemical data are reported as follows: chemical shift (6), multiplicity (ovlp=overlapping, br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet), coupling constant, and integration.

(96) High resolution mass spectra were obtained on an Agilent ESI-TOF/MS using Agilent ESI-L low concentration tuning mix as internal high resolution calibration standards. The purity of each tested compound (>95%) was determined on an Agilent 1100 LC/MS instrument using a ZORBAX SB-C18 column (3.5 mm, 4.6 mm50 mm, with a flow rate of 0.75 mL/minute and detection at 220 and 254 nm) with a 10-98% acetonitrile/water/0.1% formic acid gradient (two different gradients).

(97) Cell Line Key

(98) AU565 (Breast, overexpression of her2/neu), NCI-H520 (Lung), HCC1143 (Breast, triple negative), HCC70 (Breast, overexpression of p53), HCT116 (Colon), KPL4 (Breast, overexpression of erbB2), LNCaP-FGC (Prostate), LS174T (Colon, high levels of MUC2 mRNA), MCF-7 (Breast, overexpression of her2/neu), MDA-MB-468 (Breast, triple negative, amplified EGFR), SW403 (Colon, KRAS.sup.G12V mutation), T47D (Breast, mutant p53), ZR-75-1 (Breast, overexpression of her2), PA-1 (Ovary, overexpression of AIB1), HCT116/VM46 (Colon, vinblastine resistant).

(99) General Methods for the Synthesis of Ureas.

(100) Method 1:

(101) A solution of 20-aminovinblastine (7, 3.5 mg, 0.004 mmol) in THF (3 mL) was treated with an isocyanate (0.008 mmol). The reaction mixture was stirred for 2 hours at 25 C. and then was quenched with the addition of distilled H.sub.2O (3 mL). The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2 and the combined organic extracts were washed with saturated aqueous NaCl (3 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. Preparative thin layer chromatography (PTLC; SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided the urea (15, 19-21, 23-32, and 34-35); yields (35-98%). Isocyanates used include: ethyl isocyanate, n-butyl isocyanate, t-butyl isocyanate, cyclohexyl isocyanate, phenyl isocyanate, benzyl isocyanate, phenethyl isocyanate, o-methoxyphenyl isocyanate, m-methoxyphenyl isocyanate, p-methoxyphenyl isocyanate, p-fluoro-phenyl isocyanate, p-chlorophenyl isocyanate, p-tolyl isocyanate, p-trifluoromethylphenyl isocyanate, furfuryl isocyanate and p-biphenyl isocyanate.

(102) Method 2:

(103) A solution of 20-aminovinblastine (7, 5.7 mg, 0.007 mmol) in THF (3 mL) was treated with 4-nitrophenyl chloroformate (2.1 mg, 0.011 mmol, 1.5 equiv). The reaction mixture was stirred for 4 hours at 25 C. and then was concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH=95:5) provided 13 (4.6 mg, 67%, off white solid). A solution of 13 (4.0 mg, 0.004 mmol) in THF (3 mL) was treated with an amine (0.008 mmol). The reaction mixture was stirred for 1 hour at 25 C. and then was quenched with the addition of distilled H.sub.2O (3 mL). The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, and the combined organic extracts were washed with saturated aqueous NaCl (3 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided the urea (14, 16-18, 22, 33, 39-42 and 52-59); yields (40-99%). The amines used include: methylamine, propylamine, isopropylamine, cyclopropylamine, dimethylamine, diethylamine, 2-(aminomethyl)pyridine, morpholine, piperidine, ethanolamine, pyrrolidine, N-methylpiperazine, tetrahydropyridine, 4-phenylpiperidine, 1,2,3,4-tetrahydroisoquinoline, isoindoline and 4-methoxyisoindoline.

(104) General Methods for the Synthesis of Thioureas.

(105) Method 3:

(106) A solution of 20-aminovinblastine (7, 8.0 mg, 0.010 mmol) in THF (4 mL) was treated with an isothiocyanate (0.031 mmol). The reaction mixture was stirred for 2 hours at 25 C. and then was quenched with the addition of distilled H.sub.2O (3 mL). The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, and the combined organic extracts were washed with saturated aqueous NaCl (3 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided the thiourea (37); yields (70%). The isothiocyanate used: phenylisothiocyanate.

(107) Method 4:

(108) A solution of 20-aminovinblastine (7, 8.2 mg, 0.010 mmol) in THF (3 mL) was treated with carbon disulfide (915 L, 15 mmol). The reaction mixture was stirred for 13 hours and then was quenched with the addition of distilled H.sub.2O (5 mL). The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, and washed with saturated aqueous NaCl (2 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided 5 (7.0 mg, 82%, white solid). [Leggans et al., Org. Lett. 2012 14:1428-1431.]

(109) A solution of 20-isothiocyanovinblastine (5, 6.0 mg, 0.007 mmol) in THF (3 mL) was treated with an amine (0.011 mmol). The reaction mixture was stirred for 1 hour at 25 C. and then was quenched with the addition of distilled H.sub.2O (3 mL). The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, and the combined extracts were washed with saturated aqueous NaCl (3 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided the thiourea (36, 38, and 43); yields (26-92%). The amines used include: cyclohexylamine, 4-fluorophenethylamine and dimethylamine.

(110) General Method for the Synthesis of Ureas with N,N-Disubstituted Distal Amino Groups

(111) A solution of 20-aminovinblastine.sup.1 (5.7 mg, 0.007 mmol) in THF (3 mL) was treated with 4-nitrophenyl chloroformate (2.1 mg, 0.011 mmol, 1.5 equiv) and triethylamine (10 L, 0.07 mmol, 10 equiv). The reaction mixture was stirred at 25 C. until consumption of 20-aminovinblastine was observed by LCMS (typically 4 hours) and then the secondary amine was added (0.07 mmol). The reaction mixture was stirred at 25 C. until the reaction was complete by LCMS (typically 3-4 h) and then concentrated under a stream of N.sub.2. PTLC (SiO.sub.2, EtOAc:MeOH=94:6) provided the urea (Compounds 52-59); yields (34-79%).

(112) Physical Data for Specific Compounds

(113) ##STR00052##

(114) Compound (R=20-NHCO.sub.2C.sub.6H.sub.4NO.sub.2) 13

(115) Yield: 67%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.87 (br s, 1H), 8.11 (d, J=9.2 Hz, 2H), 8.04 (br s, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.18-7.14 (m, 1H), 7.14-7.08 (m, 2H), 6.76 (d, J=9.1 Hz, 2H), 6.63 (s, 1H), 6.11 (s, 1H), 5.85 (dd, J=10.0, 3.9 Hz, 1H), 5.48 (s, 1H), 5.30 (d, J=9.8 Hz, 1H), 4.00 (t, J=13.2 Hz 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.72 (s, 1H), 3.63 (s, 3H), 3.60-3.55 (m, 4H), 3.43 (d, J=13.6 Hz, 1H), 3.37 (dd, J=15.9, 4.5 Hz, 1H), 3.35-3.26 (m, 2H), 3.20-3.13 (m, 2H), 2.98 (d, J=13.8 Hz, 1H), 2.85-2.77 (m, 2H), 2.70 (s, 3H), 2.46-2.42 (m, 1H), 2.35 (d, J=11.9 Hz, 1H), 2.24 (d, J=13.0 Hz, 1H), 2.20-2.13 (m, 1H), 2.11 (s, 3H), 1.85-1.76 (m, 2H), 1.64 (d, J=14.8 Hz, 1H), 1.47-1.40 (m, 2H), 1.37-1.29 (m, 2H), 0.96 (t, J=7.4 Hz, 3H), 0.83 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3467, 2924, 1736, 1218 cm.sup.1; HRESI-TOF m/z 975.4492 (C.sub.53H.sub.62N.sub.6O.sub.12+H.sup.+, required 975.4498); [].sub.D.sup.2313 (c 0.1, CHCl.sub.3).

(116) Compound (R=20-NHCONHCH.sub.3) 14

(117) Yield: 85%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 7.97 (br s, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.19-7.13 (m, 1H), 7.13-7.07 (m, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.0, 4.4 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=10.7 Hz, 2H), 4.54 (br s, 1H), 4.33 (br s, 1H), 3.84 (t, J=13.2 Hz 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.39-3.36 (m, 2H), 3.32-3.21 (m, 3H), 3.20-3.10 (m, 2H), 2.86 (d, J=4.8 Hz, 3H), 2.83 (d, J=16.6 Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.58 (d, J=13.8 Hz, 1H), 2.45 (dd, J=16.9, 10.5 Hz, 1H), 2.38 (d, J=12.8 Hz, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 1.85-1.74 (m, 4H), 1.69 (d, J=13.7 Hz, 2H), 1.44-1.30 (m, 2H), 0.82 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3539, 2870, 1721, 1554, 1461, 1223, 1039, 712 cm.sup.1; HRESI-TOF m/z 867.4631 (C.sub.53H.sub.62N.sub.6O.sub.12+H.sup.+, required 867.4651); [].sub.D.sup.231.5 (c 0.05, CHCl.sub.3).

(118) Compound (R=20-NHCONHCH.sub.2CH.sub.3) 15

(119) Yield: 95%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 7.98 (br s, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.19-7.14 (m, 1H), 7.14-7.07 (m, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=9.9, 4.0 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=9.8 Hz, 1H), 4.59 (br s, 1H), 4.29 (br s, 1H), 3.83 (t, J=12.0 Hz, 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.37 (d, J=15.0 Hz, 2H), 3.34-3.19 (m, 4H), 3.18-3.13 (m, 2H), 2.83 (d, J=16.0 Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.57 (d, J=13.1 Hz, 1H), 2.47-2.43 (m, 1H), 2.38 (d, J=13.4 Hz, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 1.86-1.73 (m, 4H), 1.68 (d, J=14.3 Hz, 2H), 1.41-1.38 (m, 4H), 1.19 (t, J=7.2 Hz, 3H), 0.82 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3463, 2921, 1739, 1500, 1459, 1228, 1039, 739 cm.sup.1; HRESI-TOF m/z 881.4797 (C.sub.49H.sub.64N.sub.6O.sub.9+H.sup.+, required 881.4808); [].sub.D.sup.23+5.4 (c 0.08, CHCl.sub.3).

(120) Compound (R=20-NHCONHCH.sub.2CH.sub.2CH.sub.3) 16

(121) Yield: 40%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 7.98 (br s, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.18-7.14 (m, 1H), 7.14-7.08 (m, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.1, 4.3 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=9.8 Hz, 1H), 4.63 (br s, 1H), 4.28 (br s, 1H), 3.83 (t, J=12.0 Hz, 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.37 (dd, J=15.9, 4.7 Hz, 2H), 3.34-3.27 (m, 2H), 3.27-3.11 (m, 4H), 2.83 (d, J=16.2 Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.57 (d, J=13.8 Hz, 1H), 2.47-2.42 (m, 1H), 2.38 (d, J=13.1 Hz, 1H), 2.24-2.15 (m, 2H), 2.09 (s, 3H), 1.87-1.75 (m, 4H), 1.60-1.52 (m, 6H), 1.40-1.34 (m, 2H), 0.95 (t, J=7.4 Hz, 3H), 0.82 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3436, 2952, 1739, 1547, 1459, 1243, 1032, 755 cm.sup.1; HRESI-TOF m/z 895.4953 (C.sub.50H.sub.66N.sub.6O.sub.9+H.sup.+, required 895.4964); [].sub.D.sup.23+4.0 (c 0.05, CHCl.sub.3).

(122) Compound (R=20-NHCONHCH(CH.sub.3).sub.2) 17

(123) Yield: 59%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 8.00 (br s, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.17-7.08 (m, 3H), 6.65 (s, 1H), 6.09 (s, 1H), 5.87 (dd, J=10.1, 3.7 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=9.3 Hz, 1H), 4.29 (br s, 1H), 4.14 (br s, 1H), 3.96-3.91 (m, 1H), 3.81-3.77 (m, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.73-3.71 (m, 1H), 3.60 (s, 3H), 3.37 (d, J=14.8 Hz, 2H), 3.34-3.26 (m, 2H), 3.23 (t, J=11.9 Hz, 1H), 3.18-3.11 (m, 2H), 2.83 (d, J=16.0 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.56 (d, J=13.6 Hz, 1H), 2.47-2.43 (m, 1H), 2.36 (d, J=14.4 Hz, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 1.86-1.73 (m, 4H), 1.47-1.42 (m, 1H), 1.37-1.29 (m, 3H), 1.20 (dd, J=6.4, 2.5 Hz, 6H), 0.82 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3471, 3323, 2923, 1741, 1459, 1239, 1041 cm.sup.1; HRESI-TOF m/z 917.4761 (C.sub.50H.sub.66N.sub.6O.sub.9+Na.sup.+, required 917.4783); [].sub.D.sup.23+6.1 (c 0.1, CHCl.sub.3).

(124) Compound (R=20-NHCONHCH(CH.sub.2).sub.2) 18

(125) Yield: 70%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.02 (br s, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.18-7.13 (m, 1H), 7.13-7.07 (m, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J=10.2, 4.4 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=6.6 Hz, 1H), 5.17 (s, 1H), 4.66 (s, 1H), 3.91 (t, J=13.8 Hz, 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.56 (s, 3H), 3.41-3.13 (m, 6H), 3.08-3.00 (m, 2H), 2.83 (d, J=16.2 Hz, 1H), 2.77 (br s, 1H), 2.71 (s, 3H), 2.70-2.68 (m, 1H), 2.67 (s, 1H), 2.63 (d, J=13.5 Hz, 1H), 2.47-2.43 (m, 1H), 2.38 (d, J=12.1 Hz, 1H), 2.24 (d, J=14.2 Hz, 1H), 2.19 (dd, J=12.9, 7.7 Hz, 1H), 2.15 (s, 1H), 2.11 (s, 3H), 1.86-1.70 (m, 4H), 1.52-1.49 (m, 2H), 0.81 (t, J=7.3 Hz, 3H), 0.76 (t, J=7.3 Hz, 3H), 0.70-0.69 (m, 2H), 0.50-0.48 (m, 2H); IR (film) .sub.max 3629, 2952, 1740, 1506, 1458, 1245, 998, 748 cm.sup.1; HRESI-TOF m/z 893.4792 (C.sub.50H.sub.64N.sub.6O.sub.9+H.sup.+, required 893.4808); [].sub.D.sup.23+19 (c 0.09, CHCl.sub.3).

(126) Compound (R=20-NHCONHCH.sub.2CH.sub.2CH.sub.2CH.sub.3) 19

(127) Yield: 45%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.85 (br s, 1H), 7.98 (br s, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.18-7.13 (m, 1H), 7.13-7.07 (m, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.2, 4.4 Hz, 1H), 5.46 (s, 1H), 5.31 (d, J=11.1 Hz, 1H), 4.56 (br s, 1H), 4.27 (br s, 1H), 3.80 (s, J=1.6 Hz, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.37 (dd, J=15.9, 4.7 Hz, 2H), 3.31-3.20 (m, 4H), 3.15 (br s, 2H), 3.09 (dd, J=14.5, 7.2 Hz, 4H), 2.83 (d, J=16.2 Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.55 (d, J=13.8 Hz, 1H), 2.47-2.43 (m, 1H), 2.38 (d, J=12.1 Hz, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 2.07 (s, 1H), 1.86-1.72 (m, 4H), 1.56-1.49 (m, 2H), 1.38-1.33 (m, 4H), 0.93 (t, J=7.3 Hz, 3H), 0.82 (t, J=7.3 Hz, 3H), 0.77 (t, J=7.3 Hz, 3H); IR (film) .sub.max 3544, 2952, 1736, 1501, 1458, 1240, 1030, 761 cm.sup.1; HRESI-TOF m/z 909.5106 (C.sub.51H.sub.68N.sub.6O.sub.9+H.sup.+, required 909.5121); [].sub.D.sup.23+14 (c 0.06, CHCl.sub.3).

(128) Compound (R=20-NHCONHC(CH.sub.3).sub.3) 20

(129) Yield: 45%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 7.99 (br s, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.19-7.14 (m, 1H), 7.13-7.07 (m, 2H), 6.65 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.1, 3.9 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=10.2 Hz, 1H), 4.54 (br s, 1H), 4.10 (br s, 1H), 3.83 (t, J=14.1 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.74 (s, 1H), 3.60 (s, 3H), 3.40-3.22 (m, 5H), 3.17-3.12 (m, 2H), 2.71 (s, 3H), 2.68 (s, 1H), 2.54 (d, J=13.6 Hz, 1H), 2.47-2.42 (m, 1H), 2.35 (d, J=13.4 Hz, 1H), 2.21-2.14 (m, 2H), 2.11 (s, 3H), 1.87-1.75 (m, 2H), 1.73-1.62 (m, 4H), 1.49-1.41 (m, 2H), 1.38 (s, 9H), 1.37-1.31 (m, 2H), 0.82 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3457, 2941, 1741, 1556, 1458, 1244, 1036, 741 cm.sup.1; HRESI-TOF m/z 909.5128 (C.sub.51H.sub.68N.sub.6O.sub.9+H.sup.+, required 909.5121); [].sub.D.sup.23+3.0 (c 0.1, CHCl.sub.3).

(130) Compound (R=20-NHCONHC.sub.6H.sub.11) 21

(131) Yield: 51%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 7.99 (br s, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.20-7.14 (m, 1H), 7.14-7.07 (m, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.3, 4.1 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=9.3 Hz, 1H), 4.53 (br s, 1H), 4.22 (br s, 1H), 4.02 (d, J=9.0 Hz, 1H), 3.83 (t, J=12.9 Hz, 1H), 3.79 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.60 (s, 3H), 3.50-3.45 (m, 1H), 3.39-3.21 (m, 3H), 3.16-3.12 (m, 2H), 2.83 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.55 (d, J=13.5 Hz, 1H), 2.47-2.42 (m, 1H), 2.37 (d, J=12.7 Hz, 1H), 2.21-2.14 (m, 2H), 2.11 (s, 3H), 2.04-1.90 (m, 2H), 1.86-1.74 (m, 4H), 1.64-1.59 (m, 2H), 1.37-1.31 (m, 6H), 1.20-1.02 (m, 6H), 0.82 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3460, 2919, 1738, 1556, 1459, 1242, 1033, 752 cm.sup.1; HRESI-TOF m/z 935.5276 (C.sub.53H.sub.70N.sub.6O.sub.9+H.sup.+, required 935.5277); [].sub.D.sup.23+3.8 (c 0.09, CHCl.sub.3).

(132) Compound (R=20-NHCONHCH.sub.2CH.sub.2OH) 22

(133) Yield: 60%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.78 (br s, 1H), 7.99 (br s, 1H), 7.52 (d, J=7.8 Hz, 1H), 7.19-7.09 (m, 3H), 6.69 (s, 1H), 6.08 (s, 1H), 5.89-5.85 (m, 1H), 5.48 (s, 1H), 5.31 (d, J=11.8 Hz, 1H), 4.59 (br s, 1H), 3.83-3.72 (m, 4H), 3.80 (s, 3H), 3.79 (s, 3H), 3.76 (s, 1H), 3.57 (s, 3H), 3.57-3.54 (m, 1H), 3.42-3.37 (m, 3H), 3.34-3.29 (m, 1H), 3.26-3.17 (m, 4H), 3.08-3.03 (m, 1H), 2.87 (br s, 1H), 2.82 (d, J=16.0 Hz, 1H), 2.72 (s, 3H), 2.68 (s, 1H), 2.60-2.42 (m, 1H), 2.33-2.17 (m, 1H), 2.14-2.09 (m, 1H), 2.11 (s, 3H), 1.97-1.77 (m, 2H), 1.75-1.64 (m, 3H), 1.50-1.45 (m, 1H), 1.38-1.34 (m, 2H), 1.16 (dd, J=14.3, 4.6 Hz, 2H), 0.83-0.81 (m, 3H), 0.77 (t, J=7.1 Hz, 3H); IR (film) .sub.max 3399, 2927, 1738, 1502, 1458, 1232, 1040 cm.sup.1; HRESI-TOF m/z 897.4753 (C.sub.49H.sub.64N.sub.6O.sub.10+H.sup.+, required 897.4756); [].sub.D.sup.2314 (c 0.2, CHCl.sub.3).

(134) Compound (R=20-NHCONHC.sub.6H.sub.5) 23

(135) Yield: 87%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) .sup.1H NMR (600 MHz, CDCl.sub.3) 9.87 (br s, 1H), 7.98 (br s, 1H), 7.47 (d, J=8.1 Hz, 1H), 7.45 (d, J=8.0 Hz, 2H), 7.31 (t, J=7.8 Hz, 2H), 7.16-7.14 (m, 1H), 7.13-7.05 (m, 3H), 6.62 (s, 1H), 6.09 (s, 1H), 5.85 (dd, J=10.3, 4.6 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=9.4 Hz, 1H), 4.80 (s, 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.39-3.35 (m, 2H), 3.32-3.28 (m, 1H), 3.22-3.18 (m, 1H), 3.10 (s, 3H), 2.82 (d, J=16.3 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.59 (d, J=13.8 Hz, 1H), 2.47-2.42 (m, 1H), 2.36-2.28 (m, 2H), 2.25-2.13 (m, 2H), 2.11 (s, 3H), 2.04 (s, 1H), 1.85-1.76 (m, 3H), 1.71-1.65 (m, 2H), 1.64-1.54 (m, 2H), 1.24-1.20 (m, 2H), 0.82 (t, J=7.4 Hz, 3H), 0.80 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3468, 2940, 1742, 1501, 1460, 1237, 1031, 760 cm.sup.1; HRESI-TOF m/z 929.4807 (C.sub.53H.sub.64N.sub.6O.sub.9+H.sup.+, required 929.4807); [].sub.D.sup.23+16 (c 0.9, CHCl.sub.3).

(136) Compound (R=20-NHCONH(4-fluorophenyl)) 24

(137) Yield: 47%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.81 (br s, 1H), 7.97 (br s, 1H), 7.49 (d, J=8.1 Hz, 1H), 7.42 (dd, J=8.8, 4.8 Hz, 2H), 7.18-7.14 (m, 1H), 7.12-7.08 (m, 2H), 7.01 (t, J=8.6 Hz, 2H), 6.63 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.2, 4.5 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=12.1 Hz, 1H), 4.71 (br s, 1H), 3.80 (s, 6H), 3.75 (s, 1H), 3.61 (s, 3H), 3.40-3.36 (m, 2H), 3.32-3.28 (m, 1H), 3.21-3.06 (m, 4H), 2.82 (d, J=16.5 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.59 (d, J=13.7 Hz, 1H), 2.47-2.42 (m, 1H), 2.34 (d, J=12.7 Hz, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 1.85-1.75 (m, 4H), 1.59-1.50 (m, 4H), 1.23-1.21 (m, 2H), 0.82 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3490, 2921, 1745, 1509, 1455, 1228, 1043, 736 cm.sup.1; HRESI-TOF m/z 947.4726 (C.sub.53H.sub.63FN.sub.6O.sub.9+H.sup.+, required 947.4713); [].sub.D.sup.234.4 (c 0.08, CHCl.sub.3).

(138) Compound (R=20-NHCONH(4-chlorophenyl)) 25

(139) Yield: 63%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.80 (br s, 1H), 7.95 (br s, 1H), 7.40 (d, J=7.9 Hz, 2H), 7.38 (d, J=8.1 Hz, 1H), 7.19-7.07 (m, 5H), 6.65 (s, 1H), 6.08 (s, 1H), 5.86 (dd, J=10.2, 4.5 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=12.1 Hz, 1H), 4.81 (br s, 1H), 3.79 (s, 6H), 3.75 (s, 1H), 3.60 (s, 3H), 3.36 (d, J=15.7 Hz, 2H), 3.31-3.26 (m, 2H), 3.22-3.15 (m, 2H), 3.01-2.91 (m, 1H), 2.82 (d, J=15.7 Hz, 1H), 2.72 (s, 3H), 2.67 (s, 1H), 2.61-2.55 (m, 2H), 2.49-2.43 (m, 2H), 2.21-2.14 (m, 2H), 2.10 (s, 3H), 1.83-1.74 (s, 4H) 1.36-1.27 (m, 4H), 1.16-1.04 (m, 2H), 0.82 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3376, 2923, 1736, 1493, 1455, 1240, 1090, 765 cm.sup.1; HRESI-TOF m/z 963.4407 (C.sub.53H.sub.63ClN.sub.6O.sub.9+H.sup.+, required 963.4418); [].sub.D.sup.23+21 (c 0.07, CHCl.sub.3).

(140) Compound (R=20-NHCONH(4-methylphenyl)) 26

(141) Yield: 40%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 7.99 (br s, 1H), 7.47 (d, J=7.9 Hz, 1H), 7.33 (d, J=8.1 Hz, 2H), 7.18-7.07 (m, 3H), 6.61 (s, 1H), 6.08 (s, 1H), 5.85 (d, J=5.9 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.3 Hz, 1H), 4.74 (br s, 1H), 4.30 (br s, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.67-3.62 (m, 3H), 3.61 (s, 3H), 3.40-3.34 (m, 2H), 3.31-3.27 (m, 1H), 3.20-3.16 (m, 1H), 2.81 (d, J=15.7 Hz, 1H), 2.71 (s, 3H), 2.66 (s, 1H), 2.58 (d, J=13.3 Hz, 1H), 2.46-2.41 (m, 1H), 2.32 (s, 3H), 2.24-2.14 (m, 2H), 2.11 (s, 3H), 2.03 (s, 1H), 1.87 (d, J=13.8 Hz, 2H), 1.84-1.76 (m, 2H), 1.63-1.53 (m, 4H), 1.38-1.34 (m, 2H), 1.21 (dd, J=14.5, 5.7 Hz, 2H), 0.81 (t, J=7.6 Hz, 3H), 0.78 (t, J=7.6 Hz, 3H); IR (film) .sub.max 3369, 2912, 1722, 1507, 1445, 1230, 1017, 729 cm.sup.1; HRESI-TOF m/z 943.4949 (C.sub.54H.sub.66N.sub.6O.sub.9+H.sup.+, required 943.4964); [].sub.D.sup.23+36 (c 0.1, CHCl.sub.3).

(142) Compound (R=20-NHCONH(4-trifluoro-methylphenyl)) 27

(143) Yield: 60%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.82 (br s, 1H), 7.94 (br s, 1H), 7.58 (d, J=8.6 Hz, 2H), 7.51 (d, J=8.6 Hz, 2H), 7.48 (d, J=7.9 Hz, 1H), 7.18-7.14 (m, 1H), 7.13-7.07 (m, 2H), 6.65 (s, 1H), 6.09 (s, 1H), 5.87 (dd, J=10.0, 4.2 Hz, 1H), 5.46 (s, 1H), 5.31 (d, J=10.3 Hz, 1H), 4.99 (br s, 1H), 3.90-3.84 (m, 2H), 3.80 (s, 6H), 3.75 (s, 1H), 3.67-3.63 (m, 1H), 3.61 (s, 3H), 3.40-3.36 (m, 2H), 3.33-3.29 (m, 1H), 3.27-3.20 (m, 1H), 3.13-3.10 (m, 1H), 2.83 (d, J=15.8 Hz, 1H), 2.72 (s, 3H), 2.68 (d, J=11.9 Hz, 1H), 2.60 (d, J=13.8 Hz, 1H), 2.48-2.43 (m, 1H), 2.40 (d, J=13.3 Hz, 1H), 2.33-2.24 (m, 2H), 2.20-2.15 (m, 1H), 2.10 (s, 3H), 2.01 (s, 1H), 1.90-1.87 (m, 2H), 1.84-1.75 (m, 2H), 1.66 (d, J=14.9 Hz, 2H), 1.40-1.32 (m, 2H), 0.82 (t, J=7.2 Hz, 6H); IR (film) .sub.max 3334, 2931, 1716, 1537, 1472, 1232, 1023, 745 cm.sup.1; HRESI-TOF m/z 997.4685 (C.sub.54H.sub.63F.sub.3N.sub.6O.sub.9+H.sup.+, required 997.4681); [].sub.D.sup.2318 (c 0.04, CHCl.sub.3).

(144) Compound (R=20-NHCONH(4-methoxyphenyl)) 28

(145) Yield: 36%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.80 (br s, 1H), 8.01 (br s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.24-7.20 (m, 1H), 7.18-7.09 (m, 2H), 6.93 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.9 Hz, 2H), 6.39 (s, 1H), 6.34 (s, 1H), 6.08 (s, 1H), 5.88 (dd, J=10.0, 4.7 Hz, 1H), 5.41 (s, 1H), 5.30 (d, J=10.1 Hz, 1H), 4.60 (d, J=12.0 Hz, 1H), 4.04 (t, J=12.0 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.77 (s, 1H), 3.66 (s, 3H), 3.65-3.58 (m, 2H), 3.52-3.48 (m, 1H), 3.38-3.33 (m, 2H), 3.31-3.27 (m, 1H), 3.02-2.93 (m, 2H), 2.86-2.79 (m, 2H), 2.73 (s, 3H), 2.66 (s, 1H), 2.48-2.41 (m, 1H), 2.18-2.14 (m, 2H), 2.11 (s, 3H), 2.04-1.97 (m, 1H), 1.83-1.74 (m, 2H), 1.35-1.28 (m, 2H), 0.87 (t, J=7.3 Hz, 3H), 0.79 (t, J=7.3 Hz, 3H); IR (film) .sub.max 3447, 2899, 1742, 1507, 1459, 1234, 1036, 736 cm.sup.1; HRESI-TOF m/z 959.4917 (C.sub.54H.sub.66N.sub.6O.sub.10+H.sup.+, required 959.4913); [].sub.D.sup.239.4 (c 0.04, CHCl.sub.3).

(146) Compound (R=20-NHCONH(3-methoxyphenyl)) 29

(147) Yield: 50%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.82 (br s, 1H), 7.96 (br s, 1H), 7.47 (d, J=7.9 Hz, 1H), 7.19 (t, J=8.1 Hz, 1H), 7.16-7.13 (m, 1H), 7.12-7.06 (m, 3H), 6.99 (d, J=8.6 Hz, 1H), 6.62 (s, 1H), 6.61 (d, J=8.1 Hz, 1H), 6.08 (s, 1H), 5.85 (dd, J=10.2, 4.6 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J=9.9 Hz, 1H), 4.82 (s, 1H), 3.79 (s, 6H), 3.78 (s, 3H), 3.74 (s, 1H), 3.66-3.62 (m, 3H), 3.59 (s, 3H), 3.38-3.35 (m, 2H), 3.31-3.27 (m, 1H), 3.22-3.18 (m, 1H), 3.15-3.10 (m, 1H), 3.03 (d, J=13.3 Hz, 2H), 2.82 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.66 (s, 1H), 2.58 (d, J=13.8 Hz, 1H), 2.47-2.40 (m, 1H), 2.34 (d, J=13.0 Hz, 1H), 2.22-2.14 (m, 2H), 2.10 (s, 3H), 1.84-1.76 (m, 3H), 1.75-1.69 (m, 2H), 1.38-1.31 (m, 2H), 1.23-1.21 (m, 1H), 0.81 (ovlp t, J=7.4 Hz, 3H), 0.80 (ovlp t, J=7.4 Hz, 3H); IR (film) .sub.max 3483, 2985, 1745, 1501, 1454, 1228, 1033, 760 cm.sup.1; HRESI-TOF m/z 959.4905 (C.sub.54H.sub.66N.sub.6O.sub.10+H.sup.+, required 959.4913); [].sub.D.sup.2319 (c 0.05, CHCl.sub.3).

(148) Compound (R=20-NHCONH(2-methoxyphenyl)) 30

(149) Yield: 35%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.85 (br s, 1H), 8.07 (d, J=9.1 Hz, 1H), 8.01 (br s, 1H), 7.49 (d, J=7.9 Hz, 1H), 7.17-7.12 (m, 1H), 7.13-7.07 (m, 2H), 7.05-6.95 (m, 2H), 6.88-6.86 (m, 1H), 6.64 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J=10.0, 4.7 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.1 Hz, 1H), 5.13-5.10 (m, 1H), 4.76 (br s, 1H), 4.57 (br s, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.80 (s, 3H), 3.74 (s, 1H), 3.56 (s, 3H), 3.40-3.36 (m, 2H), 3.34-3.20 (m, 2H), 3.14-3.07 (m, 2H), 2.71 (s, 3H), 2.67 (s, 1H), 2.60 (d, J=13.7 Hz, 1H), 2.50-2.42 (m, 1H), 2.39 (d, J=12.9 Hz, 1H), 2.27 (d, J=13.7 Hz, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 2.09-1.99 (m, 2H), 1.84-1.76 (m, 4H), 1.50-1.39 (m, 2H), 1.38-1.28 (m, 2H), 0.81 (ovlp t, J=7.4 Hz, 3H), 0.80 (ovlp t, J=7.4 Hz, 3H); IR (film) .sub.max 3451, 2919, 1730, 1531, 1461, 1257, 952, 721 cm.sup.1; HRESI-TOF m/z 959.4909 (C.sub.54H.sub.66N.sub.6O.sub.10+H.sup.+, required 959.4913); [].sub.D.sup.23+24 (c 0.03, CHCl.sub.3).

(150) Compound (R=20-NHCONHCH.sub.2C.sub.6H.sub.5) 31

(151) Yield: 96%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 7.98 (br s, 1H), 7.47 (d, J=8.1 Hz, 1H), 7.45 (d, J=8.0 Hz, 2H), 7.31 (t, J=7.8 Hz, 2H), 7.17-7.08 (m, 4H), 6.62 (s, 1H), 6.09 (s, 1H), 5.85 (dd, J=10.3, 4.6 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=9.4 Hz, 1H), 4.68 (s, 1H), 4.48-4.31 (m, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.60 (s, 3H), 3.38-3.33 (m, 2H), 3.31-3.26 (m, 1H), 3.22-3.18 (m, 1H), 3.10 (s, 3H), 2.83 (d, J 16.3 Hz, 1H), 2.70 (s, 3H), 2.67 (s, 1H), 2.59 (d, J=13.8 Hz, 1H), 2.47-2.42 (m, 1H), 2.36-2.28 (m, 2H), 2.25-2.13 (m, 2H), 2.11 (s, 3H), 2.04 (s, 1H), 1.83-1.71 (m, 3H), 1.71-1.65 (m, 2H), 1.64-1.54 (m, 2H), 1.24-1.20 (m, 2H), 0.83 (t, J=7.4 Hz, 3H), 0.80 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3454, 2992, 1737, 1501, 1459, 1231, 1033, 729 cm.sup.1; HRESI-TOF m/z 943.4950 (C.sub.54H.sub.66N.sub.6O.sub.9+H.sup.+, required 943.4964); [].sub.D.sup.23+29 (c 0.3, CHCl.sub.3).

(152) Compound (R=20-NHCONHCH.sub.2CH.sub.2C.sub.6H.sub.5) 32

(153) Yield: 98%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 7.97 (br s, 1H), 7.40 (d, J=8.1 Hz, 1H), 7.31 (t, J=7.8 Hz, 2H), 7.17-7.08 (m, 6H), 6.39 (s, 1H), 6.07 (s, 1H), 5.89 (dd, J 10.3, 4.6 Hz, 1H), 5.39 (s, 1H), 5.32 (d, J=9.4 Hz, 1H), 4.52 (s, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.74 (s, 1H), 3.65 (s, 3H), 3.64-3.63 (m, 2H), 3.49-3.43 (m, 1H), 3.37-3.32 (m, 1H), 3.31-3.19 (m, 1H), 3.10 (s, 3H), 2.99 (d, J=15 Hz, 1H), 2.95-2.89 (m, 2H), 2.72 (s, 3H), 2.67 (s, 1H), 2.58-2.50 (m, 1H), 2.45-2.37 (m, 1H), 2.40-2.30 (m, 2H), 2.25-2.13 (m, 2H), 2.11 (s, 3H), 2.00-1.94 (m, 1H), 1.84-1.70 (m, 4H), 1.71-1.65 (m, 2H), 1.64-1.54 (m, 2H), 1.34-1.28 (m, 2H), 0.87 (t, J=7.4 Hz, 3H), 0.83 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3448, 2927, 1742, 1555, 1461, 1236, 1038, 749 cm.sup.1; HRESI-TOF m/z 957.5106 (C.sub.55H.sub.68N.sub.6O.sub.9+H.sup.+, required 957.5121); [].sub.D.sup.2317 (c 0.06, CHCl.sub.3).

(154) Compound (R=20-NHCONHCH.sub.2(2-pyridyl)) 33

(155) Yield: 79%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 8.52 (s, 1H), 8.04 (br s, 1H), 7.66 (t, J=8.1 Hz, 1H), 7.48 (d, J=7.9 Hz, 2H), 7.39 (d, J=7.7 Hz, 1H), 7.18-7.08 (m, 3H), 6.67 (s, 1H), 6.09 (s, 1H), 5.87-5.83 (m, 1H), 5.54 (br s, 1H), 5.47 (s, 1H), 5.30 (d, J=10.4 Hz, 1H), 4.62-4.52 (m, 2H), 3.87-3.83 (m, 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.57 (s, 3H), 3.39-3.28 (m, 3H), 3.23-3.17 (m, 1H), 3.10-3.02 (m, 1H), 2.83 (d, J=15.0 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.57 (d, J=13.8 Hz, 1H), 2.48 (q, J=9.5 Hz, 1H), 2.35 (d, J=14.6 Hz, 1H), 2.28 (d, J=9.8 Hz, 1H), 2.21-2.16 (m, 1H), 2.11 (s, 3H), 1.99 (s, 1H), 1.45-1.32 (m, 1H), 1.86-1.70 (m, 8H), 1.27-1.22 (m, 2H), 0.80 (t, J=6.8 Hz, 3H), 0.73 (t, J=7.1 Hz, 3H); IR (film) .sub.max 3375, 2925, 1737, 1503, 1459, 1230, 1039 cm.sup.1; HRESI-TOF m/z 944.4913 (C.sub.53H.sub.65N.sub.7O.sub.9+H.sup.+, required 944.4916); [].sub.D.sup.23+2.7 (c 0.2, CHCl.sub.3).

(156) Compound (R=20-NHCONHCH.sub.2(2-furyl)) 34

(157) Yield: 96%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 7.98 (br s, 1H), 7.50 (d, J=8.2 Hz, 1H), 7.35 (s, 1H), 7.17-7.08 (m, 3H), 6.66 (s, 1H), 6.31 (d, J=9.0 Hz, 2H), 6.21 (br s, 1H) 6.09 (s, 1H), 5.86 (dd, J=9.8, 4.7 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.0 Hz, 1H), 4.40-4.37 (m, 2H), 3.80 (s, 6H), 3.74 (s, 1H), 3.59 (s, 3H), 3.40-3.28 (m, 2H), 3.26-3.20 (m, 1H), 3.22-3.18 (m, 1H), 3.10-3.00 (m, 3H), 2.83 (d, J=17.3 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.55 (d, J=14.2 Hz, 1H), 2.47-2.42 (m, 1H), 2.35 (d, J=12.8 Hz, 1H), 2.25-2.15 (m, 1H), 2.11 (s, 3H), 2.02 (s, 1H), 1.85-1.76 (m, 3H), 1.71-1.65 (m, 4H), 1.37-1.20 (m, 4H), 0.81 (t, J=7.4 Hz, 3H), 0.72 (t, J=7.5 Hz, 3H); IR (film) .sub.max 3388, 2925, 1739, 1504, 1459, 1230, 1039 cm.sup.1; HRESI-TOF m/z 933.4736 (C.sub.52H.sub.64N.sub.6O.sub.10+H.sup.+, required 933.4756); [].sub.D.sup.23+11 (c 0.1, CHCl.sub.3).

(158) Compound (R=20-NHCONH(4-biphenyl)) 35

(159) Yield: 44%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.82 (br s, 1H), 7.98 (br s, 1H), 7.58-7.51 (m, 6H), 7.45 (d, J=8.0 Hz, 1H), 7.41 (t, J=7.6 Hz, 2H), 7.31 (t, J=7.4 Hz, 1H), 7.17-7.13 (m, 1H), 7.13-7.06 (m, 2H), 6.64 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.4, 4.8 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=10.2 Hz, 1H), 4.82 (br s, 1H), 3.82 (t, J=13.8 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.75 (s, 1H), 3.61 (s, 3H), 3.39-3.36 (m, 2H), 3.32-3.28 (m, 1H), 3.19 (t, J=11.4 Hz, 1H), 3.14-3.06 (m, 1H), 3.02 (d, J=12.5 Hz, 2H), 2.82 (d, J=15.7 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.60 (d, J=13.8 Hz, 2H), 2.47-2.42 (m, 1H), 2.35 (d, J=13.0 Hz, 1H), 2.23 (d, J=14.2 Hz, 1H), 2.21-2.14 (m, 1H), 2.11 (s, 3H), 1.82-1.77 (m, 4H), 1.73-1.65 (m, 4H), 1.37-1.34 (m, 1H), 0.82 (t, J=7.4 Hz, 6H); IR (film) .sub.max 3444, 2967, 1735, 1523, 1459, 1240, 1039, 744 cm.sup.1; HRESI-TOF m/z 1005.5122 (C.sub.59H.sub.68N.sub.6O.sub.9+H.sup.+, required 1005.5121); [.sub.a].sub.D.sup.238.8 (c 0.1, CHCl.sub.3).

(160) Compound (R=20-NHCSNHC.sub.6H.sub.11) 36

(161) Yield: 26%, Method 4. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.85 (br s, 1H), 8.03 (br s, 1H), 7.51 (d, J=7.7 Hz, 1H), 7.16-7.14 (m, J=7.0 Hz, 1H), 7.12-7.06 (m, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.84 (dd, J=10.1, 4.1 Hz, 1H), 5.47 (s, 2H), 5.35-5.33 (m, 1H), 5.29 (d, J=9.1 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.73 (s, 1H), 3.67-3.63 (m, 2H), 3.60 (s, 3H), 3.44 (s, 1H), 3.39-3.33 (m, 2H), 3.32-3.26 (m, 2H), 3.24-3.14 (s, 2H), 3.09 (q, J=7.7 Hz, 1H), 2.81 (d, J=14.7 Hz, 1H), 2.70 (s, 3H), 2.65 (s, 1H), 2.62-2.60 (m, 1H), 2.47-2.42 (m, 1H), 2.38-2.26 (m, 3H), 2.24-2.14 (m, 3H), 2.10 (s, 3H), 2.08-1.97 (m, 5H), 1.83-1.66 (m, 4H), 1.44-1.36 (m, 4H), 1.20-1.06 (m, 2H), 0.87 (t, J=6.9 Hz, 3H), 0.81 (t, J=6.9 Hz, 3H); IR (film) .sub.max 3467, 2912, 1727, 1506, 1456, 1230, 1087, 774 cm.sup.1; HRESI-TOF m/z 951.5051 (C.sub.53H.sub.70N.sub.6O.sub.8S+H.sup.+, required 951.5048); [].sub.D.sup.23+9.2 (c 0.2, CHCl.sub.3).

(162) Compound (R=20-NHCSNHC.sub.6H.sub.5) 37

(163) Yield: 70%, Method 3. .sup.1H NMR (500 MHz, CDCl.sub.3) 9.82 (br s, 1H), 8.09 (br s, 1H), 7.54 (m, 4H), 7.49 (d, J=8.0 Hz, 1H), 7.40 (t, J=7.2 Hz, 1H), 7.24-7.19 (m, 1H), 7.19-7.14 (m, 2H), 6.60 (s, 1H), 6.25 (s, 1H), 6.18 (s, 1H), 5.92 (dd, J=10.0, 4.3 Hz, 1H), 5.56 (s, 1H), 5.37 (d, J=10.0 Hz, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.80 (s, 1H), 3.70 (s, 3H), 3.49-3.32 (m, 4H), 3.17-3.07 (m, 2H), 2.94-2.80 (m, 3H), 2.78 (s, 3H), 2.65-3.62 (s, 1H), 2.52-2.43 (m, 1H), 2.33-2.22 (m, 4H), 2.19 (s, 3H), 1.94-1.80 (m, 2H), 1.44-1.37 (m, 3H), 1.14 (d, J=9.2 Hz, 1H), 0.88 (t, J=6.3 Hz, 6H); IR (film) .sub.max 3449, 2930, 1737, 1498, 1458, 1231, 1039, 751 cm.sup.1; HRESI-TOF m/z 945.4573 (C.sub.53H.sub.64N.sub.6O.sub.8S+H.sup.+, required 945.4579); [].sub.D.sup.23+8.1 (c 0.2, CHCl.sub.3).

(164) Compound (R=20-NHCSNHCH.sub.2CH.sub.2 (4-fluorophenyl)) 38

(165) Yield: 91%, Method 4. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 8.01 (br s, 1H), 7.51 (d, J=8.8 Hz, 1H), 7.26-7.24 (m, 2H), 7.17-7.08 (m, 3H), 6.97-6.94 (m, 2H), 6.64 (s, 1H), 6.12 (s, 1H), 5.85 (dd, J=10.1, 3.4 Hz, 1H), 5.75 (br s, 1H), 5.48 (s, 1H), 5.30 (d, J=10.2 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.77-3.74 (m, 3H), 3.61 (s, 3H), 3.60 (s, 1H), 3.42-3.35 (m, 2H), 3.32-3.28 (m, 1H), 3.25-3.10 (m, 3H), 3.02-2.97 (m, 1H), 2.96-2.91 (m, 1H), 2.83-2.80 (m, 1H), 2.72 (s, 3H), 2.66 (s, 1H), 2.55 (d, J=13.7 Hz, 1H), 2.47-2.42 (m, 1H), 2.32 (d, J=13.8 Hz, 1H), 2.22-2.17 (m, 2H), 2.11 (s, 3H), 1.84-1.65 (m, 4H), 1.54-1.44 (m, 2H), 1.37-1.22 (m, 5H), 0.82 (t, J=7.2 Hz, 3H), 0.74 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3468, 2961, 1737, 1507, 1461, 1226, 1038 cm.sup.1; HRESI-TOF m/z 991.4790 (C.sub.55H.sub.67N.sub.6O.sub.8S+H.sup.+, required 991.4798); [].sub.D.sup.23+6.6 (c 0.2, CHCl.sub.3).

(166) Compound (R=20-NHCON(CH.sub.3).sub.2) 39

(167) Yield: 99%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 7.98 (br s, 1H), 7.48 (d, J=8.2 Hz, 1H), 7.17-7.13 (m, 1H), 7.12-7.07 (m, 2H), 6.62 (s, 1H), 6.09 (s, 1H), 5.86 (dd, J=10.0, 5.1 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J=10.3 Hz, 1H), 4.68 (s, 1H), 3.90 (t, J=14.7 Hz, 1H), 3.79 (s, 3H), 3.79 (s, 3H), 3.73 (s, 1H), 3.58 (s, 3H), 3.38-3.35 (m, 2H), 3.31-3.27 (m, 2H), 3.23-3.19 (m, 2H), 3.17-3.12 (m, 2H), 3.05 (s, 6H), 2.82 (d, J=16.0 Hz, 1H), 2.70 (s, 3H), 2.67 (s, 1H), 2.58 (d, J=13.9 Hz, 1H), 2.47-2.42 (m, 1H), 2.38 (d, J=13.1 Hz, 1H), 2.23-2.14 (m, 2H), 2.10 (s, 1H), 1.87-1.74 (m, 4H), 1.69 (d, J=13.9 Hz, 2H), 1.40-1.33 (m, 4H), 0.81 (t, J=7.4 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3472, 2955, 1723, 1500, 1459, 1258, 1038, 777 cm.sup.1; HRESI-TOF m/z 881.4790 (C.sub.49H.sub.64N.sub.6O.sub.9+H.sup.+, required 881.4808); [].sub.D.sup.2350 (c 0.04, CHCl.sub.3).

(168) Compound (R=20-NHCON(CH.sub.2CH.sub.3).sub.2) 40

(169) Yield: 88%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.87 (br s, 1H), 8.02 (br s, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.18-7.13 (m, 1H), 7.13-7.07 (m, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J=10.2, 4.1 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.2 Hz, 1H), 4.35 (br s, 1H), 3.85 (t, J=12.7 Hz, 2H), 3.79 (s, 6H), 3.74 (s, 1H), 3.59 (s, 3H), 3.51-3.45 (m, 1H), 3.40-3.20 (m, 3H), 3.17-3.12 (m, 1H), 2.83 (d, J=16.2 Hz, 1H), 2.71 (s, 7H), 2.58 (d, J=13.7 Hz, 1H), 2.47-2.42 (m, 1H), 2.39 (d, J=12.7 Hz, 1H), 2.23 (d, J=14.4 Hz, 1H), 2.20-2.14 (m, 1H), 2.11 (s, 3H), 2.08 (s, 1H), 1.88-1.67 (m, 4H), 1.37-1.32 (m, 1H), 1.29-1.25 (m, 4H), 1.23 (t, J=7.1 Hz, 6H), 0.81 (t, J=7.3 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3486, 2940, 1741, 1520, 1458, 1233, 1042, 709 cm.sup.1; HRESI-TOF m/z 909.5130 (C.sub.51H.sub.68N.sub.6O.sub.9+H.sup.+, required 909.5121); [].sub.D.sup.2338 (c 0.08, CHCl.sub.3).

(170) Compound (R=20-NHCO-(morpholine)) 41

(171) Yield: 99%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 8.06 (s, 1H), 7.99 (br s, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.19-7.14 (m, 1H), 7.13-7.07 (m, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J=10.1, 4.1 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.1 Hz, 1H), 4.48 (s, 1H), 3.85 (d, J=10.9 Hz, 3H), 3.80 (s, 3H), 3.80 (s, 3H), 3.79-3.77 (m, 1H), 3.77-3.64 (m, 11H), 3.62-3.56 (m, 4H), 3.48-3.44 (m, 1H), 3.38-3.36 (m, 1H), 3.33-3.05 (m, 2H), 2.83 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.60 (d, J=13.6 Hz, 1H), 2.47-2.42 (m, 1H), 2.40 (d, J=12.5 Hz, 1H), 2.29-2.22 (m, 2H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 1.90-1.74 (m, 2H), 1.70-1.67 (m, 2H), 1.37-1.32 (m, 2H), 0.81 (t, J=7.3 Hz, 3H), 0.76 (t, J=7.5 Hz, 3H); IR (film) .sub.max 3448, 2919, 1740, 1538, 1449, 1247, 1032, 711 cm.sup.1; HRESI-TOF m/z 923.4897 (C.sub.51H.sub.66N.sub.6O.sub.10+H.sup.+, required 923.4913); [].sub.D.sup.238.0 (c 0.1, CHCl.sub.3).

(172) Compound (R=20-NHCO-(piperidine)) 42

(173) Yield: 34%, Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.85 (br s, 1H), 8.02 (br s, 1H), 7.50 (d, J=7.4 Hz, 1H), 7.17-7.14 (m, 1H), 7.11-7.09 (m, 2H), 6.59 (s, 1H), 6.09 (s, 1H), 5.84 (dd, J=10.1, 4.1 Hz, 1H), 5.45 (s, 1H), 5.29 (d, J=10.4 Hz, 1H), 4.45 (s, 1H), 3.82 (t, J=14.7 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.73 (s, 1H), 3.59 (s, 3H), 3.52-3.48 (m, 2H), 3.43-3.40 (m, 2H), 3.38-3.34 (m, 2H), 3.31-3.26 (m, 4H), 3.18-3.12 (m, 2H), 2.82 (d, J=16.6 Hz, 1H), 2.70 (s, 3H), 2.66 (s, 1H), 2.47-2.38 (m, 4H), 2.28-2.20 (m, 2H), 2.18-2.14 (m, 2H), 2.10 (s, 3H), 2.07 (s, 1H), 1.90-1.76 (m, 4H), 1.69 (s, 1H), 1.35-1.27 (m, 5H), 0.80 (t, J=7.3 Hz, 3H), 0.75 (t, J=7.3 Hz, 3H); IR (film) .sub.max 3458, 2958, 1791, 1509, 1466, 1251, 1061, 737 cm.sup.1; HRESI-TOF m/z 921.5103 (C.sub.52H.sub.68N.sub.6O.sub.9+H.sup.+, required 921.5121); [].sub.D.sup.23 9.2 (c 0.02, CHCl.sub.3).

(174) Compound (R=20-NHCSN(CH.sub.3).sub.2) 43

(175) Yield: 71%, Method 4. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 7.98 (br s, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.18-7.13 (m, 1H), 7.12-7.08 (m, 2H), 6.64 (d, J=3.9 Hz, 1H), 6.10 (s, 1H), 5.86 (dd, J=10.0, 5.1 Hz, 1H), 5.54 (s, 1H), 5.47 (s, 1H), 5.31 (d, J=10.0 Hz, 1H), 4.70-4.67 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.75 (s, 1H), 3.59 (s, 3H), 3.44 (s, 6H), 3.38 (d, J=12.2 Hz, 2H), 3.31 (d, J=4.7 Hz, 1H), 3.27-3.15 (m, 3H), 3.14-3.03 (m, 2H), 2.83 (d, J=15.2 Hz, 1H), 2.72 (s, 3H), 2.70 (s, 1H), 2.67 (s, 1H), 2.54 (d, J=13.7 Hz, 1H), 2.48-2.43 (m, 1H), 2.38 (d, J=14.1 Hz, 1H), 2.23-2.16 (m, 2H), 2.11 (s, 3H), 1.96 (d, J=15.2 Hz, 1H), 1.85-1.74 (m, 2H), 1.38-1.29 (m, 4H), 0.81 (t, J=7.4 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3458, 2963, 1728, 1536, 1474, 1235, 1061, 737 cm.sup.1; HRESI-TOF m/z 897.4559 (C.sub.49H.sub.64N.sub.6O.sub.8S+H.sup.+, required 897.4579); [].sub.D.sup.2356 (c 0.07, CHCl.sub.3).

(176) Compound (R=20-OCON(CH.sub.3).sub.2) 44

(177) Yield: 88%. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.02 (br s, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.17-7.12 (m, 1H), 7.12-7.07 (m, 2H), 6.61 (s, 1H), 6.09 (s, 1H), 5.84 (dd, J=10.2, 4.1 Hz, 1H), 5.46 (s, 1H), 5.29 (d, J=10.0 Hz, 1H), 4.34 (s, 1H), 3.84 (t, J=12.7 Hz, 1H), 3.79 (s, 6H), 3.74 (s, 1H), 3.58 (s, 3H), 3.47 (dd, J=14.6, 7.2 Hz, 2H), 3.40-3.19 (m, 4H), 3.16-3.12 (m, 2H), 2.82 (d, J=16.2 Hz, 1H), 2.70 (s, 9H), 2.66 (s, 1H), 2.57 (d, J=13.7 Hz, 1H), 2.46-2.41 (m, 1H), 2.38 (d, J=12.7 Hz, 1H), 2.22 (d, J=14.4 Hz, 1H), 2.20-2.13 (m, 1H), 2.10 (s, 3H), 2.07 (s, 1H), 1.86-1.76 (m, 2H), 1.72 (d, J=14.5 Hz, 2H), 1.38-1.30 (m, 2H), 0.80 (t, J=7.3 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3472, 2969, 1731, 1539, 1459, 1224, 1048, 740 cm.sup.1; HRESI-TOF m/z 882.4642 (C.sub.49H.sub.63N.sub.5O.sub.10+H.sup.+, required 882.4648); [].sub.D.sup.2364 (c 0.03, CHCl.sub.3).

(178) Compound (R=20-NHCH.sub.3) 45

(179) A solution 20-aminovinblastine (8.8 mg, 0.011 mmol) in THF (3 mL) was treated with a 37% formaldehyde in water solution (4 L, 0.05 mmol). The reaction mixture was stirred for 4 h at 25 C. and then was treated with sodium cyanoborohydride (12 mg, 0.20 mmol). The reaction mixture was stirred for 1 h at 25 C. and then was quenched with distilled H.sub.2O (3 mL). The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, and the combined organic extracts were washed with saturated aqueous NaCl (3 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided 45 (2.8 mg, 32%, off white solid) and 46 (2.9 mg, 33%, off white solid). For 45: .sup.1H NMR (600 MHz, CDCl.sub.3) 9.87 (br s, 1H), 8.01 (br s, 1H), 7.51 (d, J=7.5 Hz, 1H), 7.18-7.14 (m, 1H), 7.11-7.08 (m, 2H), 6.58 (br s, 1H), 6.09 (s, 1H), 5.85 (dd, J=9.9, 4.0 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J=9.8 Hz, 1H), 4.07-3.91 (m, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.73 (s, 1H), 3.61 (s, 3H), 3.36 (dd, J=16.3, 4.6 Hz, 1H), 3.31-3.26 (m, 1H), 3.12-3.04 (m, 2H), 2.83 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 3H), 2.46-2.41 (m, 1H), 2.37-2.31 (m, 1H), 2.31-2.20 (m, 1H), 2.17-2.13 (m, 1H), 2.11 (s, 3H), 1.87-1.73 (m, 2H), 1.18-1.08 (m, 2H), 0.81 (t, J=7.4 Hz, 3H), 0.76 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3487, 2953, 1729, 1505, 1461, 1239, 1037, 735 cm.sup.1; HRESI-TOF m/z 824.4576 (C.sub.47H.sub.61N.sub.5O.sub.8+H.sup.+, required 824.4593); [].sub.D.sup.23114 (c 0.03, CHCl.sub.3).

(180) Compound (R=20-N(CH.sub.3).sub.2) 46

(181) .sup.1H NMR (600 MHz, CDCl.sub.3) 9.89 (br s, 1H), 7.98 (br s, 1H), 7.52 (d, J=7.7 Hz, 1H), 7.17-7.11 (m, 3H), 6.57 (br s, 1H), 6.10 (s, 1H), 5.85 (dd, J=9.5, 4.3 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J=10.1 Hz, 1H), 3.97-3.92 (m, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.73 (s, 1H), 3.61 (s, 3H), 3.38-3.27 (m, 4H), 3.20 (d, J=13.8 Hz, 1H), 3.17-3.12 (m, 1H), 2.84 (d, J=16.2 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 3H), 2.46-2.41 (m, 1H), 2.38 (s, 6H), 2.31-2.29 (m, 1H), 2.18-2.14 (m, 1H), 2.11 (s, 3H), 1.87-1.73 (m, 6H), 1.49-1.42 (m, 2H), 1.36-1.31 (m, 2H), 0.83-0.78 (m, 6H); IR (film) .sub.max 3404, 2951, 1734, 1501, 1459, 1227, 1037, 731 cm.sup.1; HRESI-TOF m/z 838.4752 (C.sub.48H.sub.63N.sub.5O.sub.8+H.sup.+, required 838.4749); [].sub.D.sup.23+16 (c 0.01, CHCl.sub.3).

(182) Compound (R=20-NMeCONHCH.sub.2CH.sub.3) 47

(183) Yield: 95%, Method 1. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.83 (br s, 1H), 7.95 (br s, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.16-7.09 (m, 3H), 6.66 (s, 1H), 6.08 (s, 1H), 5.86 (dd, J=9.9, 3.9 Hz, 1H), 5.48 (s, 1H), 5.31 (d, J=10.6 Hz, 1H), 4.54 (br s, 1H), 4.29 (br s, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.74 (s, 1H), 3.70-3.65 (m, 1H), 3.57 (s, 3H), 3.43-3.37 (m, 2H), 3.33-3.18 (m, 5H),), 3.05-3.02 (m, 2H), 2.98 (br s, 3H), 2.82 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.56 (d, J=14.2 Hz, 1H), 2.47-2.43 (m, 1H), 2.36 (d, J=11.6 Hz, 1H), 2.24-2.16 (m, 2H), 2.11 (s, 3H), 1.87-1.75 (m, 3H), 1.38-1.32 (m, 5H), 1.19 (t, J=7.2 Hz, 3H), 0.83 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.3 Hz, 3H); IR (film) .sub.max 3462, 3400, 2926, 1735, 1504, 1460, 1243, 1039 cm.sup.1; HRESI-TOF m/z 895.4956 (C.sub.50H.sub.66N.sub.6O.sub.9H.sup.+, required 895.4964); [].sub.D.sup.23+17 (c 0.1, CHCl.sub.3).

(184) Compound 49

(185) Iron(III) chloride hexahydrate (55 mg, 0.21 mmol) was added to a solution of ()-vindoline (19 mg, 0.041 mmol) and 10-fluorocatharanthine [Gotoh et al., ACS Med. Chem. Lett. 2011, 2:948-952] (15 mg, 0.041 mmol) in CF.sub.3CH.sub.2OH (0.1 mL), aqueous 0.1 N HCl (1.0 mL) and H.sub.2O (1.0 mL) at 23 C. under Ar. The reaction mixture was stirred for 2 hours at 23 C. Meanwhile, in a separate flask, a mixture of iron(III) oxalate hexahydrate (198 mg, 0.41 mmol) in degassed H.sub.2O (40 mL) was cooled to 0 C. and placed under Ar. CsN.sub.3 (215 mg, 1.23 mmol) was added to the mixture at 0 C., followed by the vindoline coupling solution and NaBH.sub.4 (31 mg, 0.81 mmol) in H.sub.2O (1 mL). The resulting mixture was stirred for 30 minutes before being quenched by addition of 28-30% aqueous NH.sub.4OH (4 mL).

(186) The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, the organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3N=97:3:3) provided 10-fluoro-20-azidovinblastine (50, 2.4 mg, 7%, white solid), and 10-fluoro-20-azidoleurosidine (6.0 mg, 17%, white solid). A solution of compound 50 (2.4 mg, 0.0028 mmol) in THF/H.sub.2O (1/1 mL) was treated with CoCl.sub.2.6H.sub.2O (35 mg, 0.15 mmol) followed by NaBH.sub.4 (17 mg, 0.45 mmol). The reaction mixture was stirred for 2 hours before being quenched with the addition of saturated NaHCO.sub.3 (1 mL).

(187) The mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, and washed with saturated aqueous NaCl (1 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure. PTLC (SiO.sub.2, EtOAc:MeOH=90:10) provided 10-fluoro-20-aminovinblastine (51, 1.6 mg, 70%, white solid). A solution 51 (1.6 mg, 0.0019 mmol) in THF (3 mL) was treated with ethyl isocyanate (2 L 0.025 mmol). The reaction mixture was stirred for 4 hours at 25 C. and then was concentrated under reduced pressure. PTLC (SiO.sub.2, CH.sub.2Cl.sub.2:MeOH=92:8) provided 49 (1.0 mg, 59%, white solid).

(188) 50: .sup.1H NMR (600 MHz, CDCl.sub.3) 9.76 (br s, 1H), 8.00 (br s, 1H), 7.39 (dd, J=8.8, 5.1 Hz, 1H), 6.86-6.82 (m, 1H), 6.76 (dd, J=9.6, 2.4 Hz, 1H), 6.56 (s, 1H), 6.10 (s, 1H), 5.86 (dd, J=10.3, 4.6 Hz, 1H), 5.48 (s, 1H), 5.29 (d, J=10.2 Hz, 1H), 3.93 (t, J=14.1 Hz, 1H), 3.82-3.76 (m, 1H), 3.80 (s, 6H), 3.72 (s, 1H), 3.63 (s, 3H), 3.40-3.33 (m, 2H), 3.30-3.25 (m, 2H), 3.13-3.07 (m, 1H), 2.95 (d, J=14.4 Hz, 1H), 2.83-2.73 (m, 3H), 2.69 (s, 3H), 2.66 (s, 1H), 2.62 (s, 1H), 2.43-2.40 (m, 2H), 2.25 (d, J=13.9 Hz, 1H), 2.18-2.14 (m, 1H), 2.10 (s, 3H), 2.07 (s, 1H), 1.85-1.77 (m, 2H), 1.63-1.52 (m, 2H), 1.47-1.41 (m, 2H), 0.93 (t, J=7.5 Hz, 3H), 0.78 (t, J 7.4 Hz, 3H); IR (film) .sub.max 2925, 2109, 1734, 1614, 1460, 1228, 1038 cm.sup.1; HRESI-TOF m/z 854.4218 (C.sub.46H.sub.56FN.sub.7O.sub.8+H.sup.+, required 854.4247); [].sub.D.sup.236.0 (c 0.08, CHCl.sub.3).

(189) 51: .sup.1H NMR (600 MHz, CDCl.sub.3) 9.81 (br s, 1H), 8.00 (br s, 1H), 7.40-7.37 (m, 1H), 6.89-6.85 (m, 1H), 6.78 (d, J=9.2 Hz, 1H), 6.51 (s, 1H), 6.10 (s, 1H), 5.89 (dd, J=10.1, 4.0 Hz, 1H), 5.45 (s, 1H), 5.31 (d, J=10.1 Hz, 1H), 3.92 (t, J=14 Hz, 1H), 3.82-3.77 (m, 1H), 3.81 (s, 6H), 3.74 (s, 1H), 3.63 (s, 3H), 3.38 (d, J=16.0, 4.7 Hz, 2H), 3.32-3.27 (m, 1H), 3.10-3.05 (m, 2H), 2.84 (d, J=15.7 Hz, 1H), 2.75-2.68 (m, 3H), 2.71 (s, 3H), 2.64 (s, 1H), 2.50-2.45 (m, 1H), 2.17 (d, J=3.4 Hz, 1H), 2.11 (s, 3H), 2.08 (s, 1H), 2.05 (s, 1H), 1.87-1.75 (s, 4H), 1.42-1.15 (m, 6H), 0.88 (t, J=7.4 Hz, 3H), 0.78 (t, J=7.3 Hz, 3H); HRESI-TOF m/z 828.4311 (C.sub.46H.sub.58FN.sub.5O.sub.8+H.sup.+, required 828.4311); [].sub.D.sup.2323 (c 0.10, CHCl.sub.3).

(190) 49: .sup.1H NMR (600 MHz, CDCl.sub.3) 9.77 (br s, 1H), 7.95 (br s, 1H), 7.39 (dd, J=8.8, 5.2 Hz, 1H), 6.86 (t, J=9.4 Hz, 1H), 6.77 (d, J=9.3 Hz, 1H), 6.58 (s, 1H), 6.09 (s, 1H), 5.88 (dd, J=9.8, 4.6 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=11.4 Hz, 1H), 4.41 (br s, 1H), 4.21 (br s, 1H), 3.84-3.77 (m, 2H), 3.80 (s, 6H), 3.75 (s, 1H), 3.61 (s, 3H), 3.40 (dd, J=17.0, 5.2 Hz, 2H), 3.35-3.19 (m, 4H), 3.16-3.08 (m, 2H), 2.82 (d, J=14.9 Hz, 1H), 2.71 (s, 3H), 2.65 (s, 1H), 2.57 (d, J=14.1 Hz, 1H), 2.47-2.43 (m, 1H), 2.39 (d, J=12.8 Hz, 1H), 2.22-2.17 (m, 2H), 2.11 (s, 3H), 1.87-1.75 (m, 4H), 1.69 (d, J=14.6 Hz, 2H), 1.43-1.38 (m, 3H), 1.19 (t, J=7.1 Hz, 3H), 0.79 (t, J=7.4 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); HRESI-TOF m/z 899.4713 (C.sub.49H.sub.63FN.sub.6O.sub.9+H.sup.+, required 899.4713).

(191) Compound (R=20-NHCO-pyrrolidine) 52

(192) Yield: 79%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.00 (br s, 1H), 7.50 (d, J=7.9 Hz, 1H), 7.16-7.14 (m, 1H), 7.11-7.09 (m, 2H), 6.62 (s, 1H), 6.11 (s, 1H), 5.85 (dd, J=10.1, 4.2 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.1 Hz, 1H), 4.25 (s, 1H), 3.89 (q, J=12.0, 10.3 Hz 1H), 3.80 (s, 3H), 3.80 (s, 3H), 3.74 (s, 1H), 3.59 (s, 3H), 3.56-3.52 (m, 2H), 3.49-3.45 (m, 2H), 3.38-3.35 (m, 2H), 3.30 (td, J=9.4, 4.7 Hz, 1H), 3.27-3.13 (m, 4H), 3.07-3.00 (m, 2H), 2.83 (d, J=16.0 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.59 (d, J=13.8 Hz, 1H), 2.49-2.39 (m, 2H), 2.23 (d, J=15.3 Hz, 1H), 2.20-2.15 (m, 1H), 2.11 (s, 3H), 1.97-1.93 (m, 4H), 1.87-1.77 (m, 2H), 1.70 (d, J=14.6 Hz, 1H), 1.38-1.32 (m, 4H), 0.81 (t, J=7.3 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3461, 2927, 1737, 1227 cm.sup.1; HRESI-TOF m/z 907.4953 (C.sub.51H.sub.66N.sub.6O.sub.9+H.sup.+, required 907.4964); [].sub.D.sup.238 (c 0.2, CHCl.sub.3).

(193) Compound (R=20-NHCO-thiomorpholine) 53

(194) Yield: 34%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.84 (br s, 1H), 8.01 (br s, 1H), 7.51 (d, J=7.9 Hz, 1H), 7.18-7.15 (m, 1H), 7.13-7.10 (m, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J=10.5, 4.6 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.4 Hz, 1H), 4.42 (s, 1H), 3.88-3.85 (m, 1H), 3.80 (s, 3H), 3.80 (s, 3H), 3.74 (s, 1H), 3.60 (s, 3H), 3.39-3.35 (m, 2H), 3.30 (td, J=9.4, 4.6 Hz, 1H), 3.24-3.19 (m, 3H), 3.13-3.11 (m, 1H), 2.83 (d, J=16.0 Hz, 1H), 2.71 (s, 3H), 2.69-2.68 (m, 4H), 2.67 (s, 1H), 2.60 (d, J=13.8 Hz, 1H), 2.48-2.43 (m, 1H), 2.39 (d, J=13.4 Hz, 1H), 2.22-2.15 (m, 2H), 2.11 (s, 3H), 1.84-1.78 (m, 4H), 1.75-1.72 (m, 3H), 1.36-1.33 (m, 4H), 1.29-1.26 (m, 2H), 0.81 (t, J=7.4 Hz, 3H), 0.74 (t, J 7.4 Hz, 3H); IR (film) .sub.max 3468, 2924, 1735, 1502, 1458, 1293, 1227 cm.sup.1; HRESI-TOF m/z 939.4683 (C.sub.51H.sub.66N.sub.6O.sub.9S+H.sup.+, required 939.4685); [].sub.D.sup.2358 (c 0.05, CHCl.sub.3).

(195) Compound (R=20-NHCON-methylpiperazine) 54

(196) Yield: 77%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.85 (br s, 1H), 8.00 (br s, 1H), 7.51 (d, J=7.9 Hz, 1H), 7.17-7.15 (m, 1H), 7.12-7.09 (m, 2H), 6.62 (s, 1H), 6.09 (s, 1H), 5.85 (dd, J=10.2, 4.3 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.4 Hz, 1H), 4.46 (s, 1H), 3.85 (t, J=12.9 Hz, 1H), 3.80 (s, 3H), 3.80 (s, 3H), 3.74 (s, 1H), 3.65-3.62 (m, 2H), 3.60 (s, 3H), 3.51-3.48 (m, 2H), 3.39-3.34 (m, 2H), 3.30 (td, J=9.5, 4.8 Hz, 1H), 3.23 (d, J=11.8 Hz, 2H), 3.15-3.12 (m, 2H), 2.84-2.80 (m, 7H), 2.71 (s, 3H), 2.67 (s, 1H), 2.58 (d, J=13.8 Hz, 1H), 2.47-2.37 (m, 6H), 2.29 (s, 3H), 2.21-2.15 (m, 2H), 2.11 (s, 3H), 1.92-1.88 (m, 1H), 1.84-1.81 (m, 1H), 1.66 (d, J=14.4 Hz, 1H), 0.81 (t, J=7.4 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3465, 2932, 1736, 1503, 1458, 1227, 1038 cm.sup.1; HRESI-TOF m/z 936.5226 (C.sub.52H.sub.69N.sub.7O.sub.9+H.sup.+, required 936.5229); [.sub.a].sub.D.sup.2315 (c 0.2, CHCl.sub.3).

(197) Compound (R=20-NHCO-1,2,5,6-tetrahydropyridine) 55

(198) Yield: 71%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.01 (br s, 1H), 7.50 (d, J=7.9 Hz, 1H), 7.17-7.14 (m, 1H), 7.11-7.08 (m, 2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.87-5.84 (m, 1H), 5.85 (dd, J=10.2, 3.9 Hz, 1H), 5.73 (d, J=10.1 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.2 Hz, 1H), 4.46 (br s, 1H), 4.09-4.01 (m, 2H), 3.89-3.84 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.75-3.71 (m, 2H), 3.74 (s, 1H), 3.60 (s, 3H), 3.50-3.46 (m, 1H), 3.39-3.34 (m, 2H), 3.30 (td, J=9.4, 4.8 Hz, 1H), 3.22 (dd, J=11.9, 5.6 Hz, 1H), 3.13 (d, J=12.5 Hz, 2H), 2.83 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.59 (d, J=13.7 Hz, 1H), 2.47-2.42 (m, 1H), 2.40 (d, J=13.5 Hz, 1H), 2.24-2.15 (m, 4H), 2.11 (s, 3H), 1.89-1.69 (m, 4H), 1.35-1.25 (m, 5H), 0.81 (t, J=7.4 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3452, 2926, 1737, 1503, 1458, 1230, 1040 cm.sup.1; HRESI-TOF m/z 919.4936 (C.sub.52H.sub.66N.sub.6O.sub.9+H.sup.+, required 919.4964); [].sub.D.sup.23136 (c 0.03, CHCl.sub.3).

(199) Compound (R=20-NHCO-4-phenylpiperidine) 56

(200) Yield: 73%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.01 (br s, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.24-7.10 (m, 8H), 6.62 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J=10.0, 4.3 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.1 Hz, 1H), 4.55 (br s, 1H), 4.33 (d, J=13.5 Hz, 1H), 4.26 (d, J=13.0 Hz, 1H), 3.87-3.83 (m, 1H), 3.81-3.77 (m, 1H), 3.80 (s, 6H), 3.74 (s, 1H), 3.56 (s, 3H), 3.39-3.35 (m, 2H), 3.31-3.25 (m, 2H), 3.20-3.16 (m, 2H), 3.04-2.95 (m, 2H), 2.83 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.61 (d, J=13.9 Hz, 1H), 2.47-2.40 (m, 2H), 2.25 (d, J 14.2 Hz, 1H), 2.20-2.15 (m, 1H), 2.11 (s, 3H), 1.94-1.87 (m, 3H), 1.84-1.72 (m, 5H), 1.37-1.22 (m, 6H), 0.81 (t, J=7.4 Hz, 3H), 0.78 (t, J=7.4 Hz, 3H); IR (film) .sub.max 3442, 2922, 1738, 1503, 1233, 1038 cm.sup.1; HRESI-TOF m/z 997.5432 (C.sub.58H.sub.72N.sub.6O.sub.9+H.sup.+, required 997.5433); [].sub.D.sup.2322 (c 0.05, CHCl.sub.3).

(201) Compound (R=20-NHCO-1,2,3,4-tetrahydroisoquinoline) 57

(202) Yield: 58%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.00 (br s, 1H), 7.48 (d, J=8.0 Hz, 1H), 7.17-7.13 (m, 5H), 7.11-7.09 (m, 2H), 6.63 (s, 1H), 6.11 (s, 1H), 5.85 (dd, J=10.1, 4.1 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J=10.2 Hz, 1H), 4.77-4.71 (m, 2H), 4.57 (s, 1H), 3.94-3.89 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.74 (s, 1H), 3.74-3.71 (m, 2H), 3.52 (s, 3H), 3.40-3.36 (m, 2H), 3.30 (td, J=9.5, 4.8 Hz, 1H), 3.24-3.20 (m, 2H), 3.14-3.10 (m, 2H), 2.95-2.94 (m, 2H), 2.83 (d, J=16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.62 (d, J=13.8 Hz, 1H), 2.47-2.41 (m, 1H), 2.40 (d, J=13.3 Hz, 1H), 2.27 (d, J=14.2 Hz, 1H), 2.21-2.16 (m, 1H), 2.11 (s, 3H), 2.05 (s, 1H), 1.84-1.74 (m, 4H), 1.39-1.25 (m, 4H), 0.81 (t, J=7.4 Hz, 3H), 0.75 (t, J=7.5 Hz, 3H); IR (film) .sub.max 2922, 2852, 1737, 1617, 1459, 1227, 1039 cm.sup.1; HRESI-TOF m/z 969.5102 (C.sub.56H.sub.68N.sub.6O.sub.9+H.sup.+, required 969.5120); [].sub.D.sup.23+25 (c 0.07, CHCl.sub.8).

(203) Compound (R=20-NHCO-isoindoline) 58

(204) Yield: 45%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 7.99 (br s, 1H), 7.47 (d, J=7.9 Hz, 1H), 7.33-7.31 (m, 2H), 7.29-7.26 (m, 2H), 7.15-7.12 (m, 1H), 7.10-7.07 (m, 2H), 6.62 (s, 1H), 6.11 (s, 1H), 5.85 (dd, J=10.4, 4.7 Hz, 1H), 5.47 (s, 1H), 5.29 (d, J=10.6 Hz, 1H), 4.92-4.85 (m, 4H), 4.41 (br s, 1H), 3.95 (t, J=13.6 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.75 (s, 1H), 3.51 (s, 3H), 3.41-3.36 (m, 2H), 3.32-3.16 (m, 5H), 2.83 (d, J=16.1 Hz, 1H), 2.72 (s, 3H), 2.66 (s, 1H), 2.46-2.43 (m, 2H), 2.33-2.29 (m, 1H), 2.21-2.16 (m, 2H), 2.11 (s, 3H), 2.05 (s, 1H), 1.85-1.71 (m, 4H), 1.35-1.25 (m, 4H), 0.80 (t, J=7.3 Hz, 6H); IR (film) .sub.max 3468, 2928, 1737, 1500, 1460, 1228, 1040 cm.sup.1; HRESI-TOF m/z 955.4952 (C.sub.55H.sub.66N.sub.6O.sub.9+H.sup.+, required 955.4964); [].sub.D.sup.2356 (c 0.03, CHCl.sub.8).

(205) Compound (R=20-NHCO-5-methoxy-isoindoline) 59

(206) Yield: 64%; Method 2. .sup.1H NMR (600 MHz, CDCl.sub.3) 9.86 (br s, 1H), 8.00 (br s, 1H), 7.48 (d, J=8.0 Hz, 1H), 7.20 (d, J=8.4 Hz, 1H), 7.15-7.12 (m, 1H), 7.10-7.07 (m, 2H), 6.85-6.82 (m, 2H), 6.63 (s, 1H), 6.11 (s, 1H), 5.85 (dd, J=10.2, 4.5 Hz, 1H), 5.47 (s, 1H), 5.29 (d, J=10.1 Hz, 1H), 4.88-4.78 (m, 4H), 4.39 (br s, 1H), 3.97-3.92 (m, 1H), 3.87 (d, J 7.3 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 6H), 3.74 (s, 1H), 3.50 (s, 3H), 3.42-3.23 (m, 5H), 3.18-3.15 (m, 2H), 2.71 (s, 3H), 2.66 (s, 1H), 2.63 (d, J=13.7 Hz, 1H), 2.47-2.39 (m, 2H), 2.30-2.27 (m, 1H), 2.21-2.16 (m, 2H), 2.11 (s, 3H), 1.85-1.76 (m, 6H), 1.35-1.28 (m, 2H), 0.80 (t, J=7.3 Hz, 3H), 0.79 (t, J 7.3 Hz, 3H); IR (film) .sub.max 3461, 2926, 1737, 1617, 1460, 1236, 1035, 741 cm.sup.1; HRESI-TOF m/z 985.5061 (C.sub.56H.sub.68N.sub.6O.sub.10+H.sup.+, required 985.5069); [].sub.D.sup.2327 (c 0.2, CHCl.sub.3).

(207) Cell Growth Inhibition Assay.

(208) Compounds were tested for their cell growth inhibition of L1210 (ATCC #CCL-219, mouse lymphocytic leukemia, see Supporting Information) cells, HCT116 (ATCC #CCL-247, human colorectal carcinoma) cells, and HCT116/VM46 (a vinblastine-resistant strain of HCT116) cells in culture. A population of cells (>110.sup.6 cells/mL as determined with a hemocytometer) was diluted with an appropriate amount of Dulbecco-modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) to a final concentration of 30,000 cells/mL.

(209) To each well of a 96-well plate (Corning Costar), 100 L of the cell-media solution was added with a multichannel pipette. The cultures were incubated at 37 C. in an atmosphere of 5% CO.sub.2 and 95% humidified air for 24 hours. The test compounds were added to the plate as follows: test substances were diluted in DMSO to a concentration of 1 mM and 10-fold serial dilutions were performed on a separate 96-well plate. Fresh culture medium (100 L) was added to each well of cells to constitute 200 L of medium per well followed by 2 L of each test agent. Compounds were tested in duplicate (n=2-8 times) at six concentrations between 0-1,000 nM or 0-10,000 nM. Following addition, cultures were incubated for an additional 72 hours.

(210) A phosphatase assay was used to establish the IC.sub.50 values as follows: the media in each cell was removed and 100 L of phosphatase solution (100 mg phosphatase substrate in 30 mL 0.1 M NaOAc, pH 5.5, 0.1% Triton X-100 buffer) was added to each well. The plates were incubated at 37 C. for either 5 minutes (L1210) or 15 minutes (HCT116 and HCT116/VM46). After the given incubation time, 50 L of 0.1 N NaOH was added to each well and the absorption at 405 nm was determined using a 96 well plate reader. As the absorption is directly proportional to the number of living cells, the IC.sub.50 values were calculated and the reported values represent of the average of 4-16 determinations (SD10%).

(211) Tubulin Binding Competition Assay.

(212) The competitive displacement of .sup.3H-vinblastine (obtained from Moravek Biochemicals, Inc.) from purified porcine tubulin (tubulin and general tubulin buffer obtained from Cytoskeleton, Inc.) was measured using a procedure previously described. [Owellen et al., Biochem. Pharmacol. 1977 26:1213-1219.] As described, a 100 L sample of tubulin solution diluted with 850 L buffer was incubated with 25 L of 7.210.sup.5 M .sup.3H-vinblastine for 15 minutes at 37 C., after which 25 L of 7.210.sup.5 M unlabeled alkaloid was added and incubation continued for 60 minutes. Tubulin-bound .sup.3H-vinblastine was adsorbed onto DEAE filter paper and counted directly. Millipore Steriflip filter units were used to wash the filter paper with buffer under mild suction.

(213) Each of the patents, patent applications and articles cited herein is incorporated by reference.

(214) The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.