WO2020223403A1

WO2020223403A1 - Anticancer smac derivatives

Info

Publication number: WO2020223403A1
Application number: PCT/US2020/030548
Authority: WO
Inventors: Ewa D. MICEWICZ; Piotr P. RUCHALA
Original assignee: The Regents Of The University Of California
Priority date: 2019-04-29
Filing date: 2020-04-29
Publication date: 2020-11-05

Abstract

Embodiments provide for compositions and methods concerning lipid modification of Smacs for use in treating cancer.

Description

DESCRIPTION

ANTICANCER SMAC DERIVATIVES BACKGROUND OF THE INVENTION

[0001]. This application claims the benefit of U.S. Provisional Patent Application No. 62/840,231, filed April 29, 2019, which is expressly incorporated by reference herein in its entirety.

1. Field of the Invention

[0002]. This disclosure concerns cell biology, oncology and medicine. More specifically, methods and compositions are provided relating to SMAC derivatives with one or more lipids attached for the treatment of pre-cancer and cancer.

2. Description of Related Art

[0003]. Apoptosis (programmed cell death, PCD) functions as an important mechanism controlling homeostasis, normal development, host defense, suppression of oncogenesis, and its dysfunctional regulation is associated with a variety of human pathologies, including cancer, inflammation and neurodegeneration. Inhibitors of Apoptosis Proteins (IAPs) are key regulators of apoptosis. They contain one or more of Baculovirus IAP. Repeat (BIR) domains, which are approximately 70 amino acid long structural motifs primarily responsible for the anti-apoptotic activity of IAPs. Specifically, they bind and inhibit various caspases, enzymes belonging to cysteine-aspartyl proteases family, which are crucial for the apoptotic process. An apoptotic signaling is in turn regulated by the second mitochondria derived activator of caspases (Smac), also called a direct IAP binding protein with low pi (DIABLO), which has been identified as an endogenous proapoptotic antagonist of IAP proteins. After its release from the mitochondria into cytosol, and subsequent processing by proteases (removal of 55 N- terminal residues), a mature form of Smac effectively antagonizes XIAP, cIAPl and cIAP2 proteins 22-26 promoting programmed cell death. Specifically, N-terminal tetrapeptide AVPI of mature Smac (so called binding motif), is responsible for proapoptotic effects of protein.

[0004]. Targeting IAP proteins represents a promising therapeutic approach in cancer treatment. Various monomeric and dimeric Smac analogues have been already synthesized and tested, including clinical trials (see review Nat Rev Drug Discov, 2012; 11(2): 109-24; for Smac dimers see J Med Chem, 2012;55(1): 106-14; J Med Chem, 2011;54,3306; J Med Chem, 2012; 55,106; J. Med. Chem 2013; 56, 3969; and Bioorg Med Chem Lett, 2014; 24(6): 1452-1457, which are hereby incorporated by reference). Recently bivalent Smac analogues containing two AVPI mimics tethered with a linker and capable of binding to both BIR2 and BIR3 XIAP domains became the focus of researchers due to their high potency.

SUMMARY OF THE DISCLOSURE

[0005]. A small group of lipid-conjugated Smac mimetics was synthesized to probe the influence of the position of lipidation on overall anti-cancer activity. Specifically, compounds were modified with lipid(s) in position 2, 3 and C-terminus. The resulting mini library was screened extensively in vitro against a total number of 50 diverse cancer cell lines revealing that both the position of lipidation as well as the type of lipid, influence their anti-cancer activity and cancer type specificity. Moreover, when used in combination therapy with inhibitor of menin-MLLl protein interactions, position 2 modified analog SM2 showed strong synergistic anti-cancer properties. Lipid-conjugated analogs SM2 and SM6 showed favorable pharmacokinetics and in vivo activity while administered subcutaneously in the preclinical mouse model. Embodiments provide for compositions and methods concerning lipid modification of Smacs for use in treating cancer.

[0006]. In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of a compound of formula I, II, or III

wherein X is SH, SL, or SO2L, Y is SH, SL, or SO2L, Z is SH, SL, or SO2L, n = 0 or 1, R is SH, SL, or O-phenyl-L, and L is an alkyl group having 8 to 20 carbons, or a salt, prodrug, enantiomer, or diastereomer thereof. In some embodiments, the compound of formula I, II, or III comprises at least one L group. In some aspects, L is a linear C18 alkyl chain. In other aspects, L is a linear C15 alkyl chain. Some aspects are directed towards delaying the growth of a tumor comprising administering to the subject an effective amount of a compound of formula I, II, or III, or a salt, prodrug, enantiomer, or diastereomer thereof. Embodiments include methods for extending the life of a cancer patient, inhibiting metastasis of tumor cells, reducing growth of a tumor, killing or inhibiting the growth of cells, killing or inhibiting the growth of cancer cells, or treating cancer comprising administering to the subject an effective amount of a compound of formula I, II, or III, or a salt, prodrug, enantiomer, or diastereomer thereof. In some aspects, at least one of X, Y, Z or R is not hydrogen.

[0007]. In some embodiments, a compound of formula I, II, or III is administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostaticaly, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, orally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via localized perfusion, bathing target cells directly, or any combination thereof. In some aspects, the cancer is one of breast cancer, liver cancer, leukemia, lymphoma, melanoma, osteosarcoma, pancreatic cancer, prostate cancer, colon cancer, and head & neck cancer.

[0008]. In some aspects, a method of treating cancer in a subject, extending the life of a cancer patient, inhibiting metastasis of tumor cells, reducing growth of a tumor, killing or inhibiting the growth of cells, killing or inhibiting the growth of cancer cells, or binding to IAPs comprises administering to the subject an effective amount of at least one of the following compounds

[0009]. Some aspects are directed towards a method of killing or inhibiting the growth of cells comprising contacting the cells with a composition comprising an amount of a compound of formula I, II, or III, or a salt, prodrug, enantiomer, or diastereomer thereof effective to kill or inhibit the growth of the cells. In some embodiments, the cells are in a patient’s body. In some aspects, the cells are cancer cells. In some aspects, the cancer cells are in a tumor. In certain aspects, the cancer cells are breast cancer, liver cancer, leukemia, lymphoma, melanoma, osteosarcoma, pancreatic cancer, prostate cancer, colon cancer, or head & neck cancer cells. In some embodiments, the compound of formula I, II, or III comprises at least one L group. In some aspects, L is a linear C18 alkyl chain. In other aspects, L is a linear C15 alkyl chain.

[0010]. Some embodiments are directed towards a compound of formula I, II, or III

wherein X is SH, SL, or SO2L, Y is SH, SL, or SO2L, Z is SH, SL, or SO2L, n = 0 or 1, R is

SH, SL or O-phenyl-L, and L is an alkyl group having 8 to 20 carbons, or a salt, prodrug, enantiomer, or diastereomer thereof. In some embodiments, the compound of formula I, II, or III comprises at least one L group. In some aspects, L is a linear C18 alkyl chain. In other aspects, L is a linear C15 alkyl chain. In some aspects, at least one of X, Y, Z or R is not hydrogen.

[0011]. In some embodiments, a compound of formula I, II, or III is any one of the following compounds

[0012]. Any of the compounds described here may be in a pharmaceutical composition as an active pharmaceutical ingredient (API). It may be the primary or sole API or other agents may be included in the pharmaceutical composition. Moreover, any pharmaceutical composition or compound disclosed herein may be used in any therapeutic method disclosed herein.

[0013]. Other Smac compounds, compositions, formulations and methods of making or using Smac compounds, including combinations with Smac compounds, can be found in PCT Patent Publication No. WO 2017/044592, entitled Lipid-Conjugated Anticancer Smac Analogs, by Piotr Ruchala el al., and to PCT Patent Publication No. WO 2013/173755, entitled Modification of Peptides using a Bis(Thio ether )Arylbridge Approach, by Piotr Ruchala el al., which are incorporated by reference in their entireties.

[0014]. Certain embodiments are directed to pharmaceutical compositions comprising any of the compounds or Smac derivatives disclosed herein, or a pharmaceutically acceptable salt, prodrug, enantiomer, or diastereomer thereof, and an excipient. The compositions may be administered in any appropriate manner. In some embodiments, the composition is administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostaticaly, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, orally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via localized perfusion, bathing target cells directly, or any combination thereof. In some embodiments, the administration is topical.

[0015]. Methods may involve administering a composition containing about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,

0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,

2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,

4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,

7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,

9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,

245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,

340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441,

450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600,

610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700,

4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 nanograms (ng), micrograms (meg), milligrams (mg), or grams of an Smac derivative, or any range derivable therein.

[0016]. Alternatively, embodiments may involve providing or administering to the patient or to cells or tissue of the patient about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,

1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,

3.7 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,

5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,

7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,

10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,

18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,

98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,

185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,

280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370,

375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500,

510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660,

670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820,

825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975,

980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000,

8000, 9000, 10000 nanograms (ng), micrograms (meg), milligrams (mg), or grams of Smac derivative, or any range derivable therein, in one dose or collectively in multiple doses. In some embodiments, the composition comprises between about 0.1 ng and about 2.0 g of Smac derivative.

[0017]. Alternatively, the composition may have a concentration of Smac derivative that is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,

1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,

3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,

7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,

9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0,

16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,

67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,

260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,

355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470,

475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625,

630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780,

790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940,

950, 960, 970, 975, 980, 990, 1000 micrograms/ml or mg/ml, or any range derivable therein.

[0018]. If a liquid, gel, or semi-solid composition, the volume of the composition that is administered to the patient may be about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,

98, 99, 100 microliters (pi) or milliliters (ml), or any range derivable therein. In certain embodiments, the patient is administered up to about 10 ml of the composition.

[0019]. The amount of Smac derivative that is administered or taken by the patient may be based on the patient’s weight (in kilograms). Therefore, in some embodiments, the patient is administered or takes a dose or multiple doses amounting to about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,

0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,

5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,

7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,

9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,

15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,

250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,

345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450,

460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610,

620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770,

775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925,

930, 940, 950, 960, 970, 975, 980, 990, 1000 micrograms/kilogram (kg) or mg/kg, or any range derivable therein.

[0020]. The composition may be administered to (or taken by) the patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, or any range derivable therein, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or any range derivable therein. It is specifically contemplated that the composition may be administered once daily, twice daily, three times daily, four times daily, five times daily, or six times daily (or any range derivable therein) and/or as needed to the patient. Alternatively, the composition may be administered every 2, 4, 6, 8, 12 or 24 hours (or any range derivable therein) to or by the patient.

[0021]. “Treatment” or“treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease. [0022]. “Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,”“cancerous,”“cell proliferative disorder,”“proliferative disorder,” and“tumor” are not mutually exclusive as referred to herein.

[0023]. The cancers amendable for treatment by the present invention include, but are not limited to, melanoma, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include breast cancer, colon cancer, rectal cancer, colorectal cancer, kidney or renal cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, squamous cell cancer ( e.g . epithelial squamous cell cancer), cervical cancer, ovarian cancer, prostate cancer, liver cancer, bladder cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, thecomas, arrhenoblastomas, hepatoma, hematologic malignancies including non-Hodgkins lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial or uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma, salivary gland carcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi's sarcoma, melanoma, skin carcinomas, Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. The cancerous conditions amendible for treatment of the invention include metastatic cancers.

[0024]. The terms“inhibit,”“inhibiting,” and“inhibition,” (and grammatical equivalents) are used according to their plain and ordinary meaning in the area of medicine and biology. In the context of a physiological phenomena, e.g., a symptom, in an untreated subject relative to a treated subject, these terms mean to limit, prevent, or block a biological/chemical reaction to achieve a reduction in the quantity and/or magnitude of the physiological phenomena in the treated subject as compared to a differentially treated subject (such as an untreated subject or a subject treated with a different dosage or mode of administration) by any amount that is detectable and/or recognized as clinically relevant by any medically trained personnel. In some embodiments, the quantity and/or magnitude of the physiological phenomena in the treated subject is about, at least about, or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (or any range derivable therein) lower than the quantity and/or magnitude of the physiological phenomena in the differentially treated subject. Alternatively, in other embodiments, the quantity and/or magnitude of the physiological phenomena in the treated subject is about, at least about, or at most about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0,

14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0 times (or any range derivable therein) lower than the quantity and/or magnitude of the physiological phenomena in the differentially treated subject.

[0025]. “Effective amount” or“therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease. In some embodiments, the subject is administered at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg (or any range derivable therein).

[0026]. “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.

[0027]. “Pharmaceutically acceptable salts” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylicacids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphor sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0028]. Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0029]. The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.”

[0030]. The words“comprising” (and any form of comprising, such as“comprise” and “comprises”),“having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“includes” and“include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0031]. The compositions and methods for their use can“comprise,”“consist essentially of,” or“consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods“consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0032]. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. Moreover, any embodiment disclosed in the paper or supplemental material disclosed herein may be implemented as a composition or method of treating disclosed herein. This includes being used in the context of treating cancer or precancer.

[0033]. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Note that simply because a particular compound is ascribed to one particular generic formula doesn’t mean that it cannot also belong to another generic formula.

BRIEF DESCRIPTION OF THE FIGURES

[0034]. FIGS. 1A-1B Examples of cell viability curves obtained for KOPN-8 mixed lineage leukemia cell line treated with (FIG. 1A) compounds lipidated in position 2 (Mi l) and C- terminus (SM2, SM3), and (FIG. IB) compounds lipidated in position 3 (SM4-SM7).

[0035]. FIG. 2 Synthesis of C-terminally lipidated Smac derivatives.

[0036]. FIG. 3 Synthesis of Smac analogs lipidated at position 3. Conditions: (a) BHA or DPE A/T CTU/NMM/DMS 0/75 °C/10 min/MW; (b) (1) 4M HC1 in l,4-dioxane/30 min; (2) Boc-(F)-tertFeu-OH/TCTU/NMM/DMSO/75 °C/10 min/MW; (c) (1) 4M HC1 in 1,4- dioxane/30 min (2) Boc-N-Me-(F)-Ala-OH/TCTU/NMM/DMSO/75 °C/10 min/MW; (d) Tos- Cl/Py/0 °C r.t./48 h; (e) (1) 3-pentadecylphenol/BuOH/TMG/48h/90 °C; (2) TFA/30 min; (f) (1) l-hexadecanethiol/K₂C0 /NMP/72 h/90 °C; (2) TFA/30 min.

[0037]. FIG. 4 Synthesis of MEV2 analog.

[0038]. FIGS. 5A-5B Cell growth inhibition of various cancer cell lines induced by analogs

SM1-SM3 and Mi l.

[0039]. FIG. 6 Cell growth inhibition of various cancer cell lines induced by analogs SM4- SM7.

[0040]. FIG. 7 Cell growth inhibition of various cancer cell lines induced by analogs MEV1, MEV2, SM2 and equimolar mixture of SM2 and MEV2.

[0041]. FIGS. 8A-8B Apoptotic effects of selected compounds measured by flow cytometry in annexin V/PI assay (FIG. 8A), and corresponding annexin V+/PI+ double positive population values (FIG. 8B). KOPN-8 mixed lineage leukemia cells were treated at 10 mM concentrations with lapidated compounds MEV2, SM2 and equimolar mixture of MEV2 and SM2.

[0042]. FIG. 9 Dose response curves depicting increase in enzymatic activity of caspases- 3/7 and -9 in MDAMB-231 and KOPN-8 cells treated with peptides Mi l, SM2, SM6, and MEV2 at various concentrations.

[0043]. FIG. 10A-10C PK and in vivo experiments. Plasma levels after subcutaneous single dose administration of (FIG. 10A) SM2 and (FIG. 10B) SM6 at 10 mg/kg dose. Anticancer effects of SM2 and SM6 in a xenograft mouse model (FIG. IOC).

[0044]. FIG. 11 Tumor growth delay values obtained for SM2 and SM6 analogues. DETAILED DESCRIPTION

[0045]. The present invention overcomes the deficiencies of the prior art by providing compositions that kill or inhibit the growth of cells comprising contacting the cells with a composition comprising an amount of a compound of formula I, II, or III. The compounds disclosed herein may be used in a method to treat cancer in a subject. When used in a method to treat cancer, the compounds disclosed herein may be used to delay the growth of a tumor.

I. Chemical Definitions

[0046]. The term“alkyl” includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom-substituted alkyl, heteroatom-unsubstituted C_n-alkyl, and heteroatom-substituted C_n-alkyl. In certain embodiments, alkyl groups of 1 to 20 carbon atoms are contemplated. The term“heteroatom- unsubstituted C_n-alkyl” refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The groups, -CH (Me), -CH2CH3 (Et), -CH2CH2CH3 (n- Pr), -CH(CH )₂ (iso- Pr), -CH(CH₂)₂ (cyclopropyl), -CH₂CH₂CH₂CH_{3 (}n-Bu), -CH(CH )CH₂CH (sec-butyl), -CH₂CH(CH )₂ (zso-butyl), -C(CH₃)₃ (ieri-butyl), -CH₂C(CH₃)₃ (neo-pentyl), cyclobutyl, cyclopentyl, and cyclohexyl, are all non-limiting examples of heteroatom-unsubstituted alkyl groups. The term “heteroatom-substituted C_n-alkyl” refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The following groups are all non-limiting examples of heteroatom-substituted alkyl groups: trifluoromethyl, -CH₂F, -CH₂CI, -CH₂Br, -CH₂OH, -CH₂OCH₃, -CH₂OCH₂CF₃, -CH₂0C(0)CH , -CH2NH2, -CH2NHCH3, -CH₂N(CH )2, -CH2CH2CI, -CH2CH2OH, CH₂CH₂0C(0)CH , -CH₂CH₂NHC02C(CH3)3, and -CH₂Si(CH )3.

[0047]. The term“alkylthio” includes straight-chain alkylthio, branched-chain alkylthio, cycloalkylthio, cyclic alkylthio, heteroatom-unsubstituted alkylthio, heteroatom-substituted alkylthio, heteroatom-unsubstituted C_n-alkylthio, and heteroatom-substituted C_n-alkylthio. The term“heteroatom-unsubstituted C_n-alkylthio” refers to a group, having the structure -SR, in which R is a heteroatom-unsubstituted C_n-alkyl, as that term is defined above. The group, -SCH3, is an example of a heteroatom-unsubstituted alkylthio group. The term“heteroatom- substituted C_n-alkylthio” refers to a group, having the structure -SR, in which R is a heteroatom-substituted C_n-alkyl, as that term is defined above.

[0048]. Any apparently unfulfilled valency is to be understood to be properly filled by hydrogen atom(s). For example, a compound with a substituent of -O or -N is to be understood to be -OH or -NH2, respectively.

[0049]. Any genus, subgenus, or specific compound discussed herein is specifically contemplated as being excluded from any embodiment described herein.

[0050]. Compounds described herein may be prepared synthetically using conventional organic chemistry methods known to those of skill in the art and/or are commercially available (e.g., ChemBridge Co., San Diego, CA).

[0051]. Compounds employed in methods of the invention may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of the compounds disclosed herein are contemplated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S- or the //-configuration, as defined by the IUPAC 1974 Recommendations. Compounds may be of the D- or L- form, for example. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic form, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.

[0052]. As noted above, compounds of the present invention may exist in prodrug form. As used herein, "prodrug" is intended to include any covalently bonded carriers which release the active parent drug or compounds that are metabolized in vivo to an active drug or other compounds employed in the methods of the invention in vivo when such prodrug is administered to a subject. Since prodmgs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodmgs of compounds of the present invention as well as methods of delivering prodmgs. Prodmgs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.

[0053]. Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively. Other examples include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.

II. Pharmaceutical Formulations and Administration Thereof

A. Pharmaceutical Formulations and Routes of Administration

[0054]. Pharmaceutical compositions are provided herein that comprise an effective amount of one or more substances and/or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0055]. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0056]. The actual dosage amount of a composition administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0057]. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a compound described herein. In other embodiments, the compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[0058]. In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0059]. The substance may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine.

[0060]. In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. It may be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0061]. In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in certain embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0062]. In certain embodiments the substance is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. In certain embodiments, carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

[0063]. In certain embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.

[0064]. Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina, or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

[0065]. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, certain methods of preparation may include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0066]. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein. [0067]. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

2. Combination Therapy

[0068]. The compositions and related methods of the present invention, particularly administration of compounds for killing or inhibiting the growth of cells or treating cancer by administering a compound of Formula I, II, or III, may also be used in combination with the administration of traditional anti-cancer therapies.

Compounds discussed herein may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks. In embodiments where the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the candidate substance. In other aspects, one or more therapeutic agents disclosed herein may be administered or provided within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours,

17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days,

18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any range derivable therein, prior to administering a different glaucoma, anti-proliferative, or anti-metastatic therapeutic. In some embodiments, more than one course of therapy may be employed. It is contemplated that multiple courses may be implemented.

III. Organisms and Cell Source

Methods can involve cells, tissues, or organs involving the heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, artery, cornea, blood, small intestine, large intestine, brain, spinal cord, smooth muscle, skeletal muscle, ovary, testis, uterus, and umbilical cord. [0069]. Moreover, methods can be employed in cells of the following type: platelet, myelocyte, erythrocyte, lymphocyte, adipocyte, fibroblast, epithelial cell, endothelial cell, smooth muscle cell, skeletal muscle cell, endocrine cell, glial cell, neuron, secretory cell, barrier function cell, contractile cell, absorptive cell, mucosal cell, limbus cell (from cornea), stem cell (totipotent, pluripotent or multipotent), unfertilized or fertilized oocyte, or sperm.

IV. Examples

[0070]. Synthesis of SMI and MEV1. Non-lipidated analogs SMI and MEV1 were synthesized as C-terminal cysteamides by the solid phase method using CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), applying 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry and commercially available amino acid derivatives and reagents (EMD Biosciences, San Diego, CA and Chem-Impex International Inc, Wood Dale, IL). Cysteamine 2-Chlorotrityl Resin (EMD Biosciences, San Diego, CA) was used as a solid support. Peptides were cleaved from resin using modified reagent K (TFA 94% (v/v); phenol, 2% (w/v); water, 2% (v/v); TIS, 1% (v/v); EDT, 1% (v/v); 2 h) and precipitated by addition of ice-cold diethyl ether. Reduced peptides were purified by preparative reverse- phase high performance liquid chromatography (RP-HPLC) to >95% homogeneity and their purity evaluated by the electrospray ionization mass spectrometry (ESI-MS) as well as analytical RP-HPLC.

[0071]. Synthesis of MEV2. Compound was obtained in the reaction of MEV 1 with stearyl bromide and TMG (1:2.2:3) in n-buthanol (90 °C/48h). Subsequently reaction mixture was evaporated on rotary evaporator and compound purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) and its purity valuated by the electrospray ionization mass spectrometry (ESI-MS) as well as analytical RP-HPLC.

[0072]. Synthesis of SM2. Compound was obtained in the reaction of SMI with stearyl bromide and TMG (1: 1.2:2) in nbuthanol (90 °C/48h). Subsequently reaction mixture was evaporated on rotary evaporator and compound purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) and its purity evaluated by the electrospray ionization mass spectrometry (ESI-MS) as well as analytical RP-HPLC.

[0073]. Synthesis of SM3. Compound was obtained by oxidation of SM2 using Oxone® (2 K H S O 5 · K HS O4 · K 2 S O4 , 3 eq.) in methanol/water (9: 1) mixture for 5 hours at room temperature. Subsequently reaction mixture was evaporated on rotary evaporator and compound purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) and its purity evaluated by the electrospray ionization mass spectrometry (EST MS) as well as analytical RP-HPLC.

[0074]. Analogs SM4-SM6. Analogs SM4-SM6 were synthesized as C-terminal benzhydryl-amides (BHA: SM4 & SM6) or 2,2- diphenylethyl-amides (DPEA: SM5 & SM7). Synthesis was carried out in solution according to the reaction sequence depicted in FIG. 3, using if necessary, the CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC) which was operated in manual mode, and applying tert- butoxycarbonyl (Boc) chemistry and standard, commercially available amino acid derivatives and reagents (Chem-Impex International, Inc., Wood Dale, IL). All compounds were purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) and their purity evaluated by the electrospray ionization mass spectrometry (ESI-MS) as well as analytical RP-HPLC.

[0075]. Synthesis of HCl*Hyp-BHA (la) and HCl*Hyp-DPEA (lb). 5 g (21.6 mMoles) of Boc-Hyp-OH and 7.7 g of TCTU were dissolved in anhydrous DMF (50 mL). Subsequently 3.96 g (3.72 mL, 21,6 mMoles) of benzhydrylamine (or 4.26 g, 21.6 mMoles of 2,2- diphenylethylamine) and 4.75 mL of N-methylmorpholine (43.2 mMoles) were added to the solution and mixture subsequently heated in microwave synthesizer (CEM Liberty, CEM Corporation Inc., Matthews, NC) for 10 min at 75 °C. Obtained solutions were diluted with H20 and extracted with diethyl ether (3x). Ether extracts were combined, washed with the brine (3x) and evaporated on rotary evaporator. Subsequently, each compound was treated with 4 M HC1 in 1,4-dioxane (100 mL) for 30 min to remove Boc protecting groups, concentrated on rotary evaporator and crystallized by addition of ice cold diethyl ether giving solid: HCl*Hyp-BHA (yield = 92.9%) or HCl*Hyp-DPEA (yield=89.7%). Compounds were used without further purification.

[0076]. Synthesis of HCl*tert-Leu-Hyp-BHA (2a) and HCl*tert-Leu-Hyp-DPEA (2b).

Boc-(L)-tertLeu-OH was activated with TCTU (1: 1 ratio), NMM (3 eq) in DMSO (30 min, r.t.) and subsequently reacted with either la or lb (1: 1 ratio) in microwave synthesizer (CEM Liberty) for 10 min at 75 °C. Reaction mixture was diluted with H2O and extracted with diethyl ether (3x). Ether extracts were combined, washed with the brine (3x) and concentrated on rotary evaporator. Obtained oily residue was dissolved in minimal amount of ethyl acetate and precipitated by addition of n-hexane (lx) and dried under the vacuum. Subsequently, each compound was treated with 4 M HC1 in 1,4-dioxane for 30 min to remove Boc protecting groups, concentrated on rotary evaporator and crystallized by addition of ice cold diethyl ether giving solid: HCl*/er/-Leu-Hyp-BHA (yield=77.1%), and HCl*tertLeu-Hyp-DPEA (yield 79.2%). Compounds were used without further purification.

[0077]. Synthesis of Boc-NMe-Ala-tertLeu-Hyp-BHA (3a) and Boc-NMe-Ala-tertLeu- Hyp-DPEA (3b). Boc-NMe-(L)-Ala-OH was activated with TCTU (1: 1 ratio), NMM (3 eq) in DMSO (30 min, r.t.) and subsequently reacted with 2a or 2b (1: 1 ratio) in microwave synthesizer (CEM Liberty) for 10 min at 75 °C. Reaction mixture was diluted with H2O and extracted with diethyl ether (3x). Ether extracts were combined, washed with the brine (3x) and concentrated on rotary evaporator. Obtained oily residue was dissolved in minimal amount of ethyl acetate and precipitated by addition of n-hexane (3x) and dried under the vacuum (3a: yield=64.8%, 3b: yield=73.9%). Compounds were used without further purification.

[0078]. Synthesis of Boc-NMe-Ala-tertLeu-^Tos ° Hyp-BHA (4a) and Boc-NMe-Ala- tertLeu-^Tos °Hyp-DPEA (4b). 1 g of 3a (1.68 mMole, or 1.02 g of 3b) was dissolved in 25 mL of dry pyridine and cooled in the ice bath. Subsequently, 1.6 g of p-toluenesulfonyl chloride (5 eq.) was added with vigorous mixing (magnetic stirrer) maintaining temperature of reaction below 5 °C, then reaction was allowed to proceed for additional 48 hours. Subsequently, reaction mixture was diluted with H2O and extracted with dichloromethane (3x). Organic extracts were combined, washed with acidified water (H2O+HCI) (3x), the brine (3x) and concentrated on rotary evaporator. Obtained oily residue was dissolved in ethyl alcohol and lyophilized giving light powder (4a: yield=92.3%, 4b: yield=84.8%). Compounds were used without further purification.

[0079]. Synthesis of SM4. Compound was obtained in the reaction of 4a with 1- hexadecanethiol and K2CO3 (1:3:3) in N-methyl-2-pyrrolidone (90 °C/72h). Subsequently, reaction mixture was diluted with H2O and extracted with dichloromethane (3x). Organic extracts were combined, washed with acidified water (3x), the brine (3x) and evaporated on rotary evaporator. Obtained oily residue was treated with trifluoroacetic acid for 30 min to remove Boc protecting groups, concentrated on rotary evaporator and crystallized by addition of ice cold diethyl ether giving solid residue that was collected by centrifugation, washed with diethyl ether (2x) and dried under the vacuum. Collected crude compound was purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) and its purity evaluated by the electrospray ionization mass spectrometry (ESI-MS) as well as analytical RP- HPLC.

[0080]. Synthesis of SM5. Compound was synthesized from 4b using SM4 protocol.

[0081]. Synthesis of SM6. Compound was obtained in the reaction of 4a with 3- pentadecylphenol and 1,1,3,3-tetramethylguanidine (1:3:3) in n-butanol (90 °C/72h). Subsequently, reaction mixture was diluted with H2O and extracted with dichloromethane (3x). Organic extracts were combined, washed with acidified water (3x), the brine (3x) and evaporated on rotary evaporator. Obtained oily residue was treated with trifluoroacetic acid for 30 min to remove Boc protecting groups, concentrated on rotary evaporator and crystallized by addition of ice cold diethyl ether giving solid residue that was collected by centrifugation, washed with diethyl ether (2x) and dried under the vacuum. Collected crude compound was purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) and its purity evaluated by the electrospray ionization mass spectrometry (ESI-MS) as well as analytical RP-HPLC.

[0082]. Synthesis of SM7. Compound was synthesized from 4b using SM6 protocol.

[0083]. Analytical RP-HPLC. Analytical RP-HPLC was performed on a Varian ProStar

210 HPLC system equipped with ProStar 325 Dual Wavelength UV-Vis detector with the wavelengths set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA). Mobile phases consisted of solvent A, 0.1% trifluoroacetic acid (TLA) in water, and solvent B, 0.1% TLA in the mixture of 12% of acetonitrile and 88% of isopropanol (vol/vol). Analyses of peptides were performed with an analytical reversed phase Cortecs® C18+ 2.1x50 mm column (Waters Corp., Milford, MA), applying linear gradient of solvent B from 0 to 100% over 30 min (flow rate: 0.2 ml/min).

[0084]. LC-MS analysis. LC-MS analysis was performed on a Thermo Scientific™ LTQ Orbitrap XL LC-MS system equipped with Thermo Scientific™ Dionex™ UltiMate 3000 HPLC. Mobile phases consisted of solvent A, 0.1% formic acid (LA) in water, and solvent B, 0.1% LA in the mixture of 12% of acetonitrile and 88% of isopropanol (vol/vol). Analyses of peptides were performed with an analytical reversed phase Cortecs® C 18+ 2.1 x50 mm column (Waters Corp., Milford, MA), applying linear gradient of solvent B from 0 to 100% over 30 min (flow rate: 0.2 ml/min).

[0085]. Cell growth inhibition assay. Experiments were carried out using PrestoBlue™ Cell Viability Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's protocol. Briefly, specific cancer cells were plated in a 96- well plate at a density of 5x10³ cells/well in a total volume of 50 pi of culture media, and treated with various concentrations of tested peptides (50 mΐ of 0-200 mM peptides in culture media). The cells' viability was assessed after 48 h by fluorescence measurement (Ex/Em:560/590, incubation time 30 min) employing the SpectraMAX M2 microplate reader (Molecular Devices, Sunnyvale, CA). All experiments were carried out in triplicate.

[0086]. Enzymatic activity of caspases-3/7 and -9. Enzymatic activity of caspases-3/7 and -9 was measured using commercially available Caspase-Glo® 3/7 and Caspase-Glo® 9 assays (Promega Corp., Madison, WI) utilizing manufacturers protocols. Briefly, MDA-MB-231 (or KOPN-8) cells were plated in a white-walled 96-well plate at a density of 5xl0³ cells/well in a total volume of 50 pi of culture media and treated with various concentrations of tested peptides (50 mΐ of 0-100 mM peptides in culture media) for 24 hours. Subsequently, 100 mΐ of appropriate Caspase-Glo® reagent was added to each well and cells incubated for additional 60 min. Luminescence values were determined employing the SpectraMAX M2 microplate reader (Molecular Devices, Sunnyvale, CA). All experiments were carried out in triplicate.

[0087]. Annexin V-propidium iodide (PI) flow cytometry apoptosis assay. Annexin V- propidium iodide (PI) flow cytometry apoptosis assay was measured using commercially available FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) utilizing manufacturers protocols. Briefly, KOPN-8 mixed lineage leukemia cells were treated at 10 mM concentrations with lipidated compounds MEV2, SM2 and equimolar mixture of MEV2 and SM2. Cell samples were collected in indicated time points, stained with FITC-annexin V and propidium iodide (PI) and assayed using the BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA). The results were analyzed using FlowJo software.

[0088]. Pharmacokinetic (PK) studies. C57BL/6 mice were weighted and individually dosed with SM2 or SM6 subcutaneously at 10 mg/kg dose. Subsequently small samples of blood were collected at the indicated timepoints and centrifuged (3000 rpm/10 min). Obtained plasma samples were transferred into the 0.5 mL centrifuge tubes and immediately diluted with 4 volumes of a DMSO/ACN/IPA mixture (1: 1: 1) containing 0.1% of TFA. Subsequently samples were centrifuged at 13000 rpm for 10 min and obtained supernatants analyzed using the Agilent 6460 Triple Quadmpole LC/MS System (Agilent Technologies, Santa Clara, CA) with N-methylated Mi l (Ml l-Me) as an internal standard.

[0089]. Animal experiments. All animal experiments were approved by the UCLA Animal Care and Use Committee (ARC# 1999- 173 -23) and conformed to local and national guidelines. Each group consisted of 8 experimental animals. Subcutaneous engraftment model experiments were carried out using BALB/SCID gnotobiotic mice (8 weeks old, females) which were obtained from the UCLA AALAC- accredited Department of Radiation Oncology Facility and subcutaneously injected with 2.0xl0⁶ cells of human breast cancer line (MDA- MB-361, leg). After 3 weeks, palpable tumors of approximately 5 mm diameter appeared and treatment was initiated. In general, each animal received subcutaneously a total of 10 doses of the compound in 2% Cremophor EL (Sigma- Aldrich, St Louis, MO) at indicated doses on days 1-5 and 8-12. Control animals were injected with vehicle. Tumor size was assessed with calliper and its volume calculated using formula: V=L*W2/2 (V=tumor volume, L=length, W=width, L>W) and animals sacrificed as necessary according to the UCLA Animal Care guidelines.

[0090]. Screen of SMAC mimetics. Preliminary screen of new Smac mimetics was carried out exclusively in vitro using growth inhibition assay (PrestoBlue™, Invitrogen, Carlsbad, CA) and various cancer cell lines. This approach provides more reliable data than pure biophysical method(s) (e.g. measurement of binding affinity to BIR2/BIR3 XIAP domain) as it takes into account not only binding potency but also cell permeability, stability in the cell’s microenvironment, the compounds’ solubility, etc. Obtained results are summarized in FIGS. 6 and 7 and an example of cell growth curves is presented in FIG. 1. Initially compounds SM1- SM7 and Ml 151 (orally active Smac mimetic lipidated in position 2) were tested against a set of 20 diverse cancer cell lines including: breast cancer, liver cancer, leukemia, lymphoma, melanoma, prostate cancer, colon cancer and head & neck cancer (for full list see FIG. 6) which were arbitrarily selected. Analysis of obtained results suggested that the C-terminal lipidation strategy produced analogs with greater therapeutic potential which in turn prompted us to test compounds SM1-SM3 against additional cancer cell lines (in total, 50 cancer cell lines were tested, see Table 1 for complete list). Orally available analog Mi l was also tested in this additional set. Generally, obtained results suggest that the position of lipidation as well as the type of lipid, influence bioactivity. In most cancer cell lines, the highest bioactivity was observed for analogs SM2/SM3 (C-terminal lipidation), followed by SM6/SM7 (position 3 lipidation with 3-pentadecylphenoxy-moiety) which were slightly more potent than position 2 modified analog Mi l. In the case of 3-pentadecylphenoxy-lipid-modified compounds, C- terminal benzhydryl- amide (BHA) seems to be preferred moiety (SM6) over C-terminal 2,2- diphenylethyl- amide (DPEA) (SM7). Oxidation of a thioether group to the corresponding sulfone (SM2 versus SM3) also affects bioactivity but observed effects seem to depend on the type of cancer cell line. Specifically, in most tested breast cancer cell lines improvement in bioactivity was observed due to oxidation, and a reversed trend was present in leukemia and the majority of prostate cancer cell lines, with limited influence observed for pancreatic and head & neck cancer cell lines. Lipid-conjugation in position 4 (SM2 versus SMI) generally appears to be beneficial although a reverse effect was also observed in the majority of prostate cancer and some lymphoma cell lines. Similar results were also observed before for position 2 lipidation. The in vitro results for various Smac mimetics showed low nanomolar range bioactivity (16: ICso=0.9 ±0.2 nM; 24: IC₅o= 1.2+0.3 nM; 13: IC₅o= 3.4+0.6 nM).

[0091]. Analysis of the data revealed also that analog SM2 exhibits significant bioactivity against various leukemia cell lines, including mixed lineage leukemia (MLL) cell lines KOPN- 8 and Molt-4. Since acute leukemias with translocations of the MLL gene constitute about 5% to 10% of acute leukemias in adults and 70% of acute leukemias in infants and remain mostly incurable diseases, effects of SM2 in shows in combination therapy with the inhibitor of menin-MLLl protein interactions, (such interactions are crucial for leukemogenesis in the case of MLL78) were examined.

[0092]. An analog MEV2 was used, which is modified/doublelipidated derivative of the previously described compound MCP-179. Results are summarized in FIG. 7. In this case, selected leukemia and lymphoma cell lines were treated with MEV 1 (non-lipidated precursor), MEV2, SM2 and an equimolar mixture of MEV2 and SM2. Findings show that indeed in many cases using the Smac/menin-MLLl inhibitor combination therapy may be beneficial (KOPN- 8, MV-4-11, Nalm-6, SEM, CEM-R, CEM-TL, THP-1, TF-1), however results vary depending on the type of cancer. To confirm those results, an additional flow cytometry apoptosis assay was performed on the KOPN-8 leukemia cell line which has shown promising results in the preliminary cell growth inhibition assay. The KOPN-8 cells were treated with either 1 mM or 10 mM concentrations of SM2, MEV2, or an equimolar mixture of SM2 and MEV2, cultured for a specific period of time and subsequently stained with the annexin V and propidium iodide (PI). Obtained time course samples were assayed using the BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA). Results (FIG. 8) confirmed that indeed there are strong synergistic effects of lapidated Smac/menin-MLLl inhibitor combination therapy since an equimolar mixture of SM2 & MEV2 promoted markedly higher levels of apoptosis measured as annexin V+/PI+ double positive population in all time points (FIG. 8B).

[0093]. To assess whether observed bioactivity of lipidated Smac mimetics is indeed due to an increase in apoptosis, enzymatic activity of caspases-3/7 and -9 were measured in a metastatic breast cancer cell line, MDA-MB-231 and the MLL rearranged leukemia cell line, KOPN-8, that were treated with various concentrations (0-50 pM) of analogs: Mi l, MEV2, SM2 and SM6. Interestingly, in the case of MDA-MB-231 cells, only caspase-3/7 seem to be selectively affected by the treatment with lipidated Smac analogs (FIG. 9), regardless of the position of lipidation resulting in a -6-10 fold increase in enzymatic activity. The most robust response was generated by position 3 lipidated compound SM6 (-9.3 fold increase). Those results are in line with previous findings which reported the same caspase-3/7 specificity for both monomeric and dimeric position 2 lipidated Smacs. Moreover, potent effects observed for all lipid modified Smac derivatives are still approximately 30% less effective than previously described tail-to-tail dimer, SMAC17-2X47. In addition, menin-MLLl inhibitor MEV2 had no effect on the promotion of apoptosis in the MDA-MB-231 cancer cells. However, in the KOPN-8 mixed lineage leukemia cell line only MEV2 shows any significant increase in enzymatic activity of caspase-3/7 (~3.6x) which perhaps is not surprising since MEV2 was designed to interfere with menin-MLLl protein interactions. Similarly to Smacs, MEV2 does not affect caspase-9 driven apoptosis. In the same system lipidated Smacs exhibit very limited effects (SM2: -1.5 and SM6: -1.4 fold increase, Mi l: inactive).

[0094]. To characterize further the most promising lipidated analogs SM2 and SM6, preliminary pharmacokinetic (PK) studies were performed on the mouse model. Experimental animals were individually weighed and subsequently received a single subcutaneous (SC) dose (10 mg/kg) of each compound. Blood samples were collected at specified time points via retro- orbital bleeding and analyzed using the Agilent 6460 Triple Quadmpole LC/MS System (Agilent Technologies, Santa Clara, CA). For analog SM2 observed plasma half-life (t^1/2) is -28.8+1.0 h and for the compound SM6 is t^1/2~39.9+1.0 h (FIG. 10). Those figures are significantly higher than previously observed values for position 2 lipidated monomeric analog Mi l (t^1/2~2.2 h), and also its dimeric counterpart D7 (t^1/2~2.8 h)51.

[0095]. The utility of analogs SM2 and SM6 was tested further in the preclinical subcutaneous engraftment mouse model in vivo. Both analogs were administered subcutaneously at 10 mg/kg or 20 mg/kg doses (2x5 injections) in 2% Cremophor EL (Sigma- Aldrich, St Louis, MO). The treatment of the experimental, cancer-bearing animals with both SM2 and SM6 resulted in a dose dependent anticancer response (FIG. 11). The treatment of animals with 10 doses of both lipidated Smacs at the escalating dosage from 10 to 20 mg/kg showed progressively longer tumor growth delay values (FIG. 12) reaching -8.0 and -8.9 days of delay for SM2 and SM6 respectively at the 20 mg/kg dose. These results are slightly lower than values previously reported for position 2 lipidated analog Mi l which exhibited -11.0 days of tumor growth delay at 15 mg/kg dose. Moreover, lapidated Smac derivatives are significantly less active than previously described dimeric derivatives, including SMAC17-2X with reported tumor growth delay values: -10.2 days at 2.5 mg/kg.

[0096]. In conclusion, a novel family of monomeric anticancer Smac mimetics lipidated in positions 3 and C-terminus, was synthesized, and characterized in vitro and in vivo. An extensive screen for anticancer activity against various human cancer cell lines was performed revealing the role of the position of lipidation in overall anti-cancer activity and cancer type specificity. Selected analogs, SM2 and SM6, were characterized further in murine model showing favorable pharmacokinetics, and in vivo efficacy. Moreover, SM2 showed strong synergistic effects when used in combination with inhibitor of menin-MLLl protein interactions. Collectively, the findings demonstrate that lipid modification of Smac mimetics is a viable approach in the development of novel anticancer candidates.

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Claims

1. A method of killing or inhibiting the growth of cells comprising contacting the cells with a composition comprising an amount of a compound of formula I, II, or III effective to kill or inhibit the growth of the cells

wherein X is SH, SL, or SO2L;

Y is SH, SL, or S0₂L;

Z is SH, SL, or SO2L;

n = 0 or 1 ;

R is SH, SL or O-phenyl-L; and

L is an alkyl group having 8 to 20 carbons; or a salt, prodrug, enantiomer, or diastereomer thereof.

2. The method of claim 1, wherein the cells are in a patient’s body.

3. The method of claim 1 or 2, wherein the cells are cancer cells.

4. The method of any of claims 1 to 3, wherein the cancer cells are breast cancer, liver cancer, leukemia, lymphoma, melanoma, osteosarcoma, pancreatic cancer, prostate cancer, colon cancer, or head & neck cancer cells.

5. The method of any of claims 1 to 4, wherein the cancer cells are in a tumor.

6. The method of any of claims 1 to 5, wherein the compound of formula I, II, or III comprises at least one L group.

7. The method of any of claim 1 to 6, wherein L is a linear C 18 alkyl chain.

8. The method of any of claims 1 to 6, wherein L is a linear C15 alkyl chain.

9. A method of treating cancer in a subject comprising administering to the subject an effective amount of a compound of formula I, II, or III,

wherein X is SH, SL, or SO2L;

Y is SH, SL, or S0₂L;

Z is SH, SL, or SO2L;

n = 0 or 1 ;

R is SH, SL or O-phenyl-L; and

10. The method of claim 9, wherein treating cancer is further defined as delaying the growth of a tumor.

11. The method of claim 9 or 10, wherein the compound of formula I, II, or III is administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostaticaly, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, orally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via localized perfusion, bathing target cells directly, or any combination thereof.

12. The method of any of claims 9 to 11, wherein the cancer is one of breast cancer, liver cancer, leukemia, lymphoma, melanoma, osteosarcoma, pancreatic cancer, prostate cancer, colon cancer, and head & neck cancer.

13. The method of any of claims 9 to 12, wherein the compound of formula I, II, or III comprises at least one L group.

14. The method of any of claims 9 to 13, wherein L is a linear C18 alkyl chain.

15. The method of any of claims 9 to 13, wherein L is a linear C 15 alkyl chain.

16. A compound of formula I, II, or III,

wherein X is SH, SL, or SO2L;

Y is SH, SL, or S0₂L;

Z is SH, SL, or SO2L;

n = 0 or 1 ;

R is SH, SL or O-phenyl-L; and

17. The compound of claim 16, wherein the compound of formula I, II, or III comprises at least one L group.

18. The compound of claim 16 or 17, wherein L is a linear C18 alkyl chain.

19. The compound of claim 16 or 17, wherein L is a linear C15 alkyl chain.

20. The compound of any of claims 16 to 19, wherein the compound is one of

21. The method of any one of claims 1 to 15, wherein the compound is at least one of