Analogues of (-)Picropodophyllin, Synthesis And Uses Thereof
Compounds, compositions, methods of making, and methods of using analogues of (−)-picropodophyllin, as well as a transgenic animal model and its use for identifying anticancer agents.
This application is a continuation of International Application PCT/US09/033,416, filed Feb. 6, 2009 and also claims priority to U.S. Provisional Patent Application Ser. No. 61/027,459, filed Feb. 9, 2008 and U.S. Provisional Patent Application Ser. No. 61/122,945, filed Dec. 16, 2008, the contents of which are hereby incorporated in their entireties herein.
GRANT INFORMATIONThe subject matter of this application was developed at least in part under National Institutes of Health Grant No. NIH/PO1CA097403, so that the United States Government has certain rights herein.
SEQUENCE LISTINGThe specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Feb. 20, 2009. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as “0700503731.TXT,” is 1,3661 bytes and was created on Feb. 20, 2009. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.
1. INTRODUCTIONThe invention relates to (−)-picropodophyllin analogues, methods of synthesizing said analogues and uses thereof. In certain non-limiting embodiments, the invention provides for methods of treating cancers using (−)-picropodophyllin analogues, including basal-like cancers of the breast.
2. BACKGROUND OF THE INVENTIONThe compound (−)-picropodophyllin binds tightly to the human Insulin-like Growth Factor I Receptor Kinase (IGF1 RK) domain and inhibits its kinase activity. Such binding has the effect of inhibiting the anti-apoptotic signal pathways that are associated with IGF1 RK activity. This inhibition tends to decrease cancer cell growth and may also render cancer cells more susceptible to chemotherapy. Importantly, the compound, (−)-picropodophyllin, shows exquisite selectivity for the IGF1 RK over the structurally highly homologous Insulin Receptor Kinase (IRK) domain, the inhibition of which would give an undesired diabetes-type phenotype. This selectivity could lead to a new class of chemotherapeutic drugs.
At present, there is no drug on the market that acts upon the IGF1 RK. Development of such a drug is, therefore, an emerging macromolecular target of great current interest in the pharmaceutical community.
Human breast cancers have been molecularly classified by gene expression profiling into three major subtypes: luminal, ERBB2+ and basal-like. The luminal cancers are estrogen receptor-positive (ER+), whereas the cancers of the other two classes, which either overexpress ERBB2(HER2/NEU) or exhibit phenotypic features of basal/myoepithelial cells, are ER-negative (Perou et al., Nature (2000), 406, 747-52; Sorlie et al., Proc. Natl. Acad. Sci. U.S.A. (2001), 98, 10869-74; Sorlie et al., Proc. Natl. Acad. Sci. U.S.A. (2003), 100, 8418-23). Basal-like cancers also lack progesterone receptor (PR) and ERBB2 (ER-/PR-/ERBB2-; “triple negative breast cancers”), but frequently express EGFR and basal markers, such as cytokeratins 5/6 and/or 14 and p63 (Nielsen et al., Clin. Cancer Res. (2004), 10, 5367-74). The basal-like class (15-20% of all breast cancers; recently reviewed by Yehiely et al., Trends Mol. Med. (2006), 12, 537-44; Finnegan and Carey, Future Oncol. (2007), 3, 55-63; Da Silva et al., J. Clin. Pathol. (2007), 60, 1328-32) includes high proportions of BRCA1-associated and also medullary and metaplastic subtypes. The latter group is heterogeneous and consists of squamous and spindle cell carcinomas and other forms. Basal cancers appear to have extremely bad prognosis (Sorlie et al., Proc. Natl. Acad. Sci. U.S.A. (2001), 98, 10869-74), especially in the early years of follow-up after diagnosis and treatment, although this was not clearly evident in some patient cohorts (Chin et al., Cancer Cell (2006), 10, 529-41). In addition, these aggressive cancers pose a serious problem to targeted therapies, considering that the use of antiestrogens in combination with an anti-ERBB2 antibody (trastuzumab) is not an option, while there is no clear choice for chemotherapeutic intervention (see, for example, Cleator et al., Lancet Oncol. (2007), 8, 235-44; Carey et al, Clin. Cancer Res. (2007), 13, 2329-34).
Interestingly, an association between basal cancers and Kras amplification is now emerging. Ras-family members, apparently serving overlapping but also distinct cellular functions, act as molecular switches in signaling pathways regulating proliferation or apoptosis, and become constitutively active and, thus, oncogenic by mutation (usually at codons 12, 13 or 61). Although Kras is overall the most frequently mutated isoform, there is tissue-specificity and variable incidence of mutations in Ras tumorigenic action, as different human tumors harbor different mutant family members. In contrast to pancreatic ductal adenocarcinoma, in which Kras mutations (most commonly in codon 12) can be found at frequencies as high as 90% (Almoguera et al., Cell (1988), 53, 549-54), the incidence of Kras mutations in breast cancer appears to be low (˜7% on average; reported frequencies of 1/40, 1/25, 4/61 and 3/10; Rochlitz et al., Cancer Res. (1989), 49, 357-360; Prosperi et al., Cancer Lett. (1990), 51, 169-74) Myaikas, et al. Biochem. Biophys. Res. Commun (1998), 251, 609-612; Chen, et al., Cancer Lett., (2005), 229, 115-22). In human breast cancer cell lines, however, the observed frequency was higher (˜13%; 5/40; Hollestelle et al., Mol. Cancer. Res. (2007), 5, 195-201). Signaling elicited by non-mutated, but overexpressed Ras can also collaborate with other deregulated pathways in tumor progression and invasion (Clark and Der, Breast Cancer Res. Treat. (1995), 35, 133-44). For example, in comparison with control breast tissue, 11 of 20 breast cancers exhibited a 2- to 6-fold increase in enzymatically measured Ras activation (relative amount of GTP-bound form; von Lintig et al., Breast Cancer Res. Treat. (2000), 62, 51-62), while Western analysis indicated that in ˜70% of primary breast cancer specimens (n=132) the level of Ras was higher than in normal breast tissue (Dati et al., Int. J. Cancer (1991), 47, 833-38). Notably, in 56% (9/16) of examined basal-like human breast cancers identified by expression profiling, the Kras locus was amplified and, thus, overexpressed (Herschkowitz et al., Genome Biol. (2007), 8, R76).
The IGF signaling system, which is the major determinant of mammalian organismal growth (Efstratiadis A Int. J. Dev. Biol. (1998), 42:955-976), has also been implicated in the pathogenesis of various human cancers (Pollak M N, et al., Nat. Rev. Cancer (2004), 4:505-518.), including breast tumors (Sachdev D and Yee D, J. Mammary Gland Biol Neoplasia (2006), 11:27-39). A seminal observation, in this regard, was that cells lacking Igf1r, the tyrosine kinase receptor mediating the effects of insulin-like growth factors, cannot be transformed by any one of several tested oncoproteins (Sell C, et al. Proc. Natl. Acad. Sci. USA (1993), 90:11217-11221; Sell C, et al. Mol. Cell. Biol. (1994), 14: 3604-3612; Baserga R, Expert Opin. Ther. Targets (2005), 9:753-768). Signaling through Igf1r does not appear to be an oncogenic component per se, but a crucial prerequisite for tumorigenesis because, among other actions such as the promotion of cellular proliferation by stimulation of the Ras/MAPK/ERK pathway, it exerts strong PI3 kinase-dependent and independent antiapoptotic effects that are necessary for tumor growth (Baserga R, Expert Opin. Ther. Targets (2005), 9:753-768). Moreover, the IGF system appears to be involved in resistance to certain anticancer regimes (Ryan P D and Goss P E, Oncologist (2008), 13:16-24). On the basis of these considerations, potential therapeutic approaches for cancer treatment involving blocking of IGF signaling with small molecules or antibodies are currently under development (Sachdev D and Yee D, J. Mammary Gland Biol Neoplasia (2006), 11:27-39; Baserga R, Expert Opin. Ther. Targets (2005), 9:753-768; Ryan P D and Goss P E. Oncologist (2008), 13:16-24; Garcia-Echeverria C. IDrugs (2006), 9:415-419; Hartog H, et al., Eur. J. Cancer (2007) 43:1895-1904).
3. SUMMARY OF THE INVENTIONThe present invention provides for compounds, compositions, methods of making, and methods of using analogues of (−)-picropodophyllin, as well as a transgenic animal model and its use for identifying anticancer agents. It is based, at least in part, on the discoveries that (i) Igf1r was overexpressed in mammary tumors advantageously induced extremely rapidly by oncogenic Kras in a mouse model, and (ii) breast-specific genetic ablation of Igf1r expression demonstrated that the cognate signal transduction pathway is causally involved in tumorigenesis.
In particular non-limiting embodiments, the present invention provides for methods of treating a cancer in a subject comprising administering, to the subject, an effective amount of an analogue of (−)-picropodophyllin. In particular, non-limiting embodiments, the cancer cells demonstrate a mutation in Kras. In a specific, non-limiting embodiment, the cancer is a basal-like cancer of the breast.
The invention provides inhibitors of IGF1RK as well as methods of synthesizing the inhibitors and using them therapeutically. As therapeutics, these small molecules have advantages over antibodies, including circumventing possible immune responses associated with the latter, as well as permitting oral administration and potentially increasing biological half-life. Production cost is also likely to be much lower.
An aspect of the invention is directed to analogues and a method for the synthesis of analogues of the natural product (−)-picropodophyllin with control of both absolute and relative stereochemistry. Absolute and relative stereochemistry are important for biological activity in lignans.
Thus, (−)-podophyllotoxin, the C-2-epimer of (−)-picropodophyllin, features a structurally distinct trans-fused lactone in the D-ring. (−)-Podophyllotoxin acts as an anti-mitiotic and does so by binding tightly to tubulin, thereby preventing its polymerization in spindle formation. The structure of the (−)-podophyllotoxin-tubulin complex has been established by x-ray crystallography.
If one inverts the C-4 center one arrives at the family of epipodophyllotoxins [i.e. epimers at both C-2 and C-4 of (−)-picropodophyllin], including etoposide, teniposide, and TOP-53. These compounds act by binding to the enzyme topoisomerase II and inhibiting the relegation step along the enzymatic reaction coordinate. This leads to a build up of a covalent enzyme-DNA complex that serves as a cellular signal, triggering apoptosis.
Thus, while members of all three of these lignan families are of interest as potential medicinal agents, if one wishes to develop chemotherapeutics that target the IGF1 RK, control of stereochemistry is important, and represents an important feature of this invention.
The natural product (−)-picropodophyllin is shown above as 1.
Aspects of the present invention are directed to analogues of (−)-picropodophyllin having modified E-rings as shown below in formula I.
Many types of substitutents are available for ring E due to the late addition of ring E during synthesis of the compounds.
X, X′, Y, Y′ and Z are ring E substituents and X, X′, Y, Y′ and Z may be independently hydrogen; deuterium; tritium; a C1-C8 saturated or unsaturated, alkyl or cycloalkyl group; a hydroxyl group; an ether-protected hydroxyl group bearing a C1-C8 saturated or unsaturated alkyl or cyclic alkyl group; a carboxylate ester-protected hydroxyl group derived from a C1-C8 saturated or unsaturated, cyclic or acyclic, carboxylic acid; a hydroxyl group protected as a phosphate mono-, di- or triester, the di-, or triester having C1-C4 saturated or unsaturated alkyl group(s); a C1-C8 alkoxy, a C1-C4 alkoxy, a phosphonate mono- or diester-protected hydroxyl group derived from a C1-C8 saturated or unsaturated, cyclic or acyclic, phosphonic acid wherein the diester also contains a C1-C8 saturated or unsaturated alkyl group; a phosphinate ester-protected hydroxyl group derived from a phosphinic acid bearing two C1-C8 saturated or unsaturated, cyclic or acyclic, alkyl groups; a hydroxyl group protected as a sulfate mono- or diester bearing a C1-C4 saturated or unsaturated alkyl group; a hydroxyl group protected as a sulfonate ester derived from a sulfonic acid bearing a C1-C8 saturated or unsaturated, cyclic or acyclic, alkyl group; an amino group; a primary or secondary amine bearing 1 to 2 C1-C8 saturated or unsaturated alkyl group(s), respectively; a carboxamide-protected, unsubstituted or primary amine bearing a C1-C4 saturated or unsaturated alkyl group; an amino group derived from a C1-C8 saturated or unsaturated, cyclic or acyclic, carboxylic acid; a carboxylic acid; a carboxylate ester bearing a C1-C4 saturated or unsaturated alkyl group; a phosphonic acid; a phosphonate mono- or diester bearing 1 to 2 C1-C4 saturated or unsaturated alkyl group(s), respectively; a phosphinic acid having a C1-C8 saturated or unsaturated, cyclic or acyclic, alkyl group or ester bearing a C1-C4 saturated or unsaturated alkyl group; a formyl group; an acetyl group; a benzoyl group; a carboxamide group derived from ammonia or from a primary or secondary amine bearing 1 to 2 C1-C4 saturated or unsaturated alkyl group(s), respectively; a sulfhydryl group; a thioether bearing a C1-C8 saturated or unsaturated, cyclic or acyclic, alkyl group; a sulfonic acid, a sulfonate ester bearing a C1-C4 saturated or unsaturated alkyl group; an alkylsulfonyl group bearing a C1-C8 saturated or unsaturated, cyclic or acyclic, alkyl group; a phenylsulfonyl group; a sulfoxide bearing a C1-C8 saturated or unsaturated, cyclic or acyclic, alkyl group; a phenylsulfoxide; a phenylseleno group; a phenylselenoxide; an azide; a halogen; a cyano group; a nitro group; a nitroso group; a diazonium group; or a trifluoromethyl group with the proviso that when X and X′ are H, Y, Y′, and Z cannot all be methoxy.
In further aspects, X, X′, Y, Y′ and Z are independently hydrogen, a C1-C8 alkyl or C1-C8 alkoxy group. In further aspects, X, X′, Y, Y′ and Z are hydrogen, methyl, or methoxy with the proviso that when X and X′ are H, Y, Y′, and Z cannot all be methoxy.
In further aspects, the present invention is directed to (−)-picropodophyllin analogues with modified E-rings as shown below.
Wherein
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- (1) X, X′, Y, Y′═H, and Z═OCH3 (compound 19, below)
- (2) X, X′, Y′, Z═H, and Y═OCH3;
- (3) X, X′, Y, Z═H, and Y′═OCH3 (compound 18, below)
- (4) X, X′, Y′═H, and Y, Z═OCH3;
- (5) X, X′, Y═H, and Y′, Z═OCH3 (compound 20, below)
- (6) X, X′, Z═H, and Y, Y′═OCH3 (compound 21, below)
- (7) X, X′, Y, Y′═H, and Z═CH3
- (8) X, X′, Y, Y′═H, and Z═CH3, C(4)-epimer-4-epi-picropodophyllin analogue)
The present invention is also directed to enantiomerically enriched compounds which do not occur naturally and are not readily available from (−)-picropodophyllin. Enantiomerically enriched means that the enantiomeric ratio is at least 95:5, preferably at least 97:3, prior to recrystallization.
The present compounds may be prepared by first preparing a compound of formula (a) by any suitable means such as starting from readily available piperonal, utilizing (i) bromination, (ii) acetalization and (iii) installation of a hydroxymethyl group (halogen/metal exchange and aryllithium trapping with paraformaldehyde).
In the presence of acetic acid, formula (a) cyclizes and, upon losing two molecules of MeOH, transiently provides the highly reactive isobenzofuran (b), which reacts, in situ, via a Diels-Alder reaction with DMAD (dimethyl acetylenedicarboxylate) to form Diels-Alder adduct (c).
Diels-Alder adduct (c) can be selectively hydrogenated, reduced to the diol with LiAlH4 and acetylated to provide meso diacetate (d).
The achiral and symmetrical system bearing all carbons of the target cyclolignan A-D rings is efficiently desymmetrized with porcine pancreatic lipase (PPL) to produce (e) in synthetically useful enantiopurity level.
The compound of formula (e) is subjected to silylation, deacetylation, and oxidation to produce a compound of formula (f) without loss of optical activity. Silylation occurs with a silylating agent, R9X, including but not limited to TIPSCl (triisopropylsilyl chloride), TBDMSCl (tert-butyldimethylsilyl chloride), and TBDPSCl (tert-butyldiphenylsilyl chloride), in the presence of an appropriate base such as imidazole or NEt3. Deacetylation is carried out under standard conditions (e.g. Et2O, NaHCO3, or K2CO3, MeOH or Na, MeOH or NH3, MeOH.) The aldehyde is then oxidized under mild two-electron oxidation conditions (e.g. Swern or Moffatt oxidation (DMSO as oxidant), Ley oxidation (TPAP=tetrapropylammonium perrhuthenate as oxidant) or Dess-Martin oxidation (Dess-Martin periodinane as oxidant.))
R9 is a silyl protecting group such as a triisopropylsilyl-protecting group.
The compound of formula (f) is converted to a compound of formula (g) by retro-Michael ring opening and protection of the C4—OH followed by aldehyde oxidation. The ring opening occurs under typical Michael addition conditions (such as NaOMe and MeOH). The protection step must be carried out under neutral to basic conditions (to avoid aromatization). Use of a silyl (such as TES=triethylsilyl)ether protecting group for the C-4 hydroxyl is an advantageous feature of the present invention. This sets the stage for an efficient desilylative lactonization at the close of the synthesis, unveiling the (−)-picropodophyllin core in a single operation, immediately following the stereocontrolled installation of ring E.
Aldehyde oxidation proceeds smoothly under Lindgren conditions (NaClO2 as oxidant) to give (g). The efficient retro-Michael ring-opening of (f) unveils the (methylenedioxy)cinnamyl system as the vehicle for late installation of ring E.
R10 is a C4 protecting group such as a triethylsilyl-protecting group.
The compound of formula (g) is converted to a compound of formula (h) by transformation of the carboxylic acid into an acyl oxazolidinone functionality. This requires carboxyl activation (e.g. with carbonyl diimidazole) and then condensation with a metalated oxazolidinone to give (h).
The E ring is introduced to the compound of formula (h) to form a compound of formula (i) by CuI-mediated conjugate addition of RMgBr at a temperature of −10 to 10° C.
The compound of formula (i) is converted to a compound of formula (j) by desilylative lactonization by heating with a fluoride source and cyclization to produce the corresponding lactone;
Non-limiting examples of such syntheses are provided in Section 6, Example 1, below, the details of which are incorporated by reference in their entirety into this Section 5.
Further aspects of the invention are directed to (−)-picropodophyllin analogues as shown below.
-
- wherein X, X′, Y, Y′, and Z are defined as above; R1 may be oxo, —OH, —OCH3, —OC2H5, —OC3H7, —OC4H9, —OCH2CH═CH2, —OCH2Ph, —OCH2CH2NH2, —OCOH, OCOCH3, —OCH2OH, OC2H4OH, —OC3H6OH, —OC4H8OH, or a glycoside. In specific, non-limiting embodiments, R1 may be oxo, —OH, —OCH3, —OC2H5, —OC3H7, —OC4H9, —OCH2CH═CH2, —OCH2Ph, —OCH2CH2NH2, —OCOH, OCOCH3, —OCH2OH, OC2H4OH, —OC3H6OH, —OC4H8OH, or a glycoside and X, X′, Y, Y′ and Z are selected from the group consisting of (1) X, X′, Y, Y′═H, and Z═OCH3; (2) X, X′, Y′, Z═H, and Y═OCH3; (3) X, X′, Y, Z═H, and Y′═OCH3; (4) X, X′, Y′═H, and Y, Z═OCH3; (5) X, X′, Y═H, and Y′, Z═OCH3; (6) X, X′, Z═H, and Y, Y′═OCH3; X, X′, Y, Y′═H, and Z═CH3 and (8) X, X′, Y, Y′═H, and Z═CH3, C(4)-epimer-4-epi-picropodophyllin analogue).
-
- wherein X, X′, Y, Y′, and Z are defined as above; R1 and R2 may be independently oxo, —OH, —OCH3, —OC2H5, —OC3H7, —OC4H9, —OCH2CH═CH2, —OCH2Ph, —OCH2CH2NH2, —OCOH, OCOCH3, —OCH2OH, OC2H4OH, —OC3H6OH, —OC4H8OH, or a glycoside. In specific, non-limiting embodiments, R1 and R2 may be independently oxo, —OH, —OCH3, —OC2H5, —OC3H7, —OC4H9, —OCH2CH═CH2, —OCH2Ph, —OCH2CH2NH2, —OCOH, OCOCH3, —OCH2OH, OC2H4OH, —OC3H6OH, —OC4H8OH, or a glycoside, and X, X′, Y, Y′ and Z are selected from the group consisting of (1) X, X′, Y, Y′═H, and Z═OCH3; (2) X, X′, Y′, Z═H, and Y═OCH3; (3) X, X′, Y, Z═H, and Y′═OCH3; (4) X, X′, Y′═H, and Y, Z═OCH3; (5) X, X′, Y═H, and Y′, Z═OCH3; (6) X, X′, Z═H, and Y, Y′═OCH3; X, X′, Y, Y′═H, and Z═CH3 and (8) X, X′, Y, Y′═H, and Z═CH3, C(4)-epimer-4-epi-picropodophyllin analogue).
Examples of suitable R1 and R2 substituents include the substituents disclosed in U.S. Pat. Nos. 5,132,322; 5,300,500; 5,332,811; 5,541,223; and 6,051,721 which are each incorporated herein by reference.
Preferred, non-limiting embodiments of the invention include 5′-Didemethoxy-(−) -picropodophyllin (Compound 18), 3′,5′-Didemethoxy-(−)-picropodophyllin (Compound 19), 5′-Demethoxy-(−)-picropodophyllin (Compound 20), and 4′-Demethoxy-(−)-picropodophyllin (Compound 21), the syntheses of which are described in the working examples.
The effects of the compounds described above on activities of IGF1R can be determined in in vitro and in vivo assays, as described in the Examples below. Example 2 describes a method of monitoring the effect of compounds on cell growth. Inhibition of kinase activity of the IGF1R can be measured by any means known in the art. The kinase domain of the receptor is available from Upstate Biotechnology.
In one embodiment, a cell line is used to assay the effects of these compounds on activities of IGF1R, for example, a murine breast cancer cell line derived from Kras* mice. In one embodiment, the cell line is placed in a multi-well-plate, including, but not limited to a 24-well plate, and a 96-well plate. In one embodiment, cells contained in a subset of wells are treated with a substrate, for example, a substrate used in a colorimetric metabolic assay that reflects cell density, for example, MTT (Thiazol Blue Tetrazolium), and cells in another subset of wells are treated with an IGF1R inhibitor, for example, cyclolignan picropodophyllin (PPP), or the compound(s). The effectiveness of these compounds on activities of IGF1R is compared with that of a known IGF1R inhibitor, for example, PPP.
The Kras* mouse is a suitable in vivo model in which to test the effect of IGF1RK inhibitors on tumor growth (see Examples 3 and 4). The Kras* mouse overexpresses a Kras2 gene carrying the oncogenic substitution G12D. The gene has been knocked-in into the highly and ubiquitously expressed locus Eef1a. Cre-mediated removal of a STOP sequence residing upstream from the Kras* cDNA allows for its tissue-specific expression. Mice in which the oncogene is activated in the mammary epithelium by use of the WAPcre line develop tumors very rapidly (as early as 2 days upon parturition; T50=9 days). Moreover, these tumors display high heterogeneity in histopathology. Four major patterns of invasive carcinoma were detected (well differentiated glandular adenocarcinoma (Dunn A and B Tumors), pale cell carcinoma, squamous cell carcinoma and spindle cell (sarcomatous) carcinoma). In addition, histopathological characterization of the tumors showed that, with the exception of the glandular adenocarcinoma, these tumors express basal markers and share great similarities in expression profiles with human and mouse basal-like breast cancers. Expression profile analysis and immunophenotyping showed that the IGF1R was significantly overexpressed in the Kras*-induced neoplasms. This is believed to be the first description of a mouse model in which a malignant neoplasm is induced by a single tumorigenic stimulus in one step without a requirement for secondary oncogenic events, as evidenced by the rapidity of tumor development. This unexpected observation can be attributed to the abnormally high expression levels of a powerful oncogene.
Accordingly, in one set of embodiments, the present invention provides for a system and a method for producing a transgenic animal model of a human tumor, wherein a tumor in the transgenic animal is induced by expression of an oncogene operably linked to a Eef1a (for Eukaryotic translation Elongation Factor 1 Alpha 1) promoter (e.g., the endogenous promoter), and expression of the oncogene is triggered by excision of a “stop” (transcription termination) signal by Cre-mediated recombination. The oncogene may be introduced into the Eef1a1 locus by “knockin” technology into an area of the gene such that, after transcription, it will be translated. In one embodiment, a cDNA encoding the oncogene is inserted into an intron as part of a construct having a splice acceptor site (i) downstream of a “floxed” stop signal and (ii) upstream of the cDNA coding sequence (to prevent the construct sequence being spliced out from the mRNA precursor and lost during mRNA maturation); preferably, the construct is inserted into the first intron. However, the present invention also envisions inserting a construct comprising the cDNA encoding the oncogene into an exon of the Eef1a locus, although this would be less desirable. In various embodiments of the invention, the oncogene may be any oncogene known in the art, including but not limited to Kras and activating mutations thereof (e.g., at codons 12, 13, or 61, e.g., G12D), Hras, c-myc, Her2/neu, src, Wnt 1, PI3 kinase, etc.
In non-limiting set of embodiments of the invention, an Eef1a1 cassette may be prepared comprising (from upstream to downstream) (i) a 5′ arm homologous to the Eef1a1 site being targeted; (ii) a splice-acceptor site; (iii) a loxP site; (iv) a transcription termination signal (e.g., a multiple signal serving, before its removal, as a block “STOP” sequence); (v) a loxP site; (vi) a multiple cloning site; (vii) a polyadenylation signal and (viii) a 3′ arm homologous to the Eef1a1 locus targeted, further comprising a nucleic acid encoding a selectable marker (e.g., neo), positioned upstream of the transcription termination signal. Any gene of interest, including but not limited to an oncogene such as Kras or an activated form thereof (designated “Kras*” herein), may be inserted into the multiple cloning site. A non-limiting example of such a construct is shown in
According to the present invention, a first transgenic animal carrying an Eef1a1 cassette described in the preceding paragraph may be mated with a second transgenic animal carrying a transgene comprising a Cre-gene operably linked to a promoter, where the promoter is either active or shows increased activity in a specific tissue, and/or at a specific developmental stage, and or under certain conditions (e.g., during late pregnancy and lactation, or in response to an agent such as tetracycline or tamoxifen). Thus, an initially dormant oncogene present in the Eef1a1 cassette may be conditionally activated. In progeny animals carrying both the Eef1a1 cassette and the Cre transgene, the nature of the Cre-linked promoter determines the site and timing of oncogene expression, as Cre expression results in removal of the stop transcription signal and, consequently, permits transcription of the oncogene. Accordingly, the present invention may be used to produce tumors in specific tissues; a Cre gene operably linked to a pancreas-specific promoter such as Pdx1 may be used to induce pancreatic cancer in a progeny animal; a Cre cDNA operably linked to a colon-specific promoter such as villin may be used to produce a colon cancer in a progeny animal, and so forth. In a specific, non-limiting embodiment to develop an animal model system for breast cancer (luminal type, ERBB2+ type, and (in particular) basal-like), the second transgenic animal may carry a transgene in which a Cre gene is operably linked to a milk whey acidic protein (“Wap”) promoter, so that a selected progeny animal for use as a model of breast cancer carries the Eef1a1 cassette and the Wap-Cre transgenes. Oncogene expression may be induced in such an animal by lactation.
The transgenic animal produced according to the invention may be any non-human species of animal. In a preferred, non-limiting embodiment, the transgenic animal is a mouse.
In a related set of non-limiting embodiments, the present invention provides for a cell line derived from a transgenic animal model, as described above, where the cell line is produced from a cancerous cell of the transgenic animal model in which the oncogene transgene is being expressed. In a specific, non-limiting example, the cell line is prepared from a mammary tumor of a transgenic mouse expressing activated Kras* under transcriptional control of the Eef1a1 promoter, as described in
In a further related set of non-limiting embodiments, the present invention provides for a trans genic animal carrying, in addition to Eef1a1 cassette and promoter/Cre transgenes as described above, a heterozygous mutation of Igf1R.
In various non-limiting embodiments, the present invention provides for use of a transgenic animal model of a human tumor, as described above, to identify an agent useful for the treatment of said human tumor, where the nature of the tumor is determined by the site of expression of the oncogenic transgene. In various non-limiting embodiments, the transgenic animal model may be a model for breast cancer, or for pancreatic cancer, or for colon cancer, or for lung cancer, or for skin cancer, or for prostate cancer. In a specific, non-limiting embodiment, the transgenic animal model is a model of basal cell type breast cancer.
Accordingly, the present invention provides for a method comprising (i) providing a transgenic animal, as described above, carrying a Eef1a1 cassette comprising a Cre-activatable oncogene and a transgene comprising Cre under the control of a promoter permitting conditional activation of Cre expression; (ii) providing a condition which results in the expression of Cre in the animal with consequent expression of the oncogene; (iii) administering, to said animal, a test agent; and (iv) determining the effect of the test agent on the growth and/or histology of a tumor in the tissue in which the oncogene is activated by Cre and expressed, and/or determining clinical markers associated with malignancy and/or determining the survival of the animal, where the ability of the test agent to inhibit growth of the tumor, or increase differentiation in the histology of the tumor, or decrease one or more clinical marker of malignancy, or increase the survival of the animal, indicates that the test agent may be used to treat the human tumor being modeled, and may be advanced to human clinical trials. Step (iv) may be achieved by providing a second transgenic animal essentially genetically identical to the animal of step (i), providing essentially the same condition as provided in step (ii), and then determining tumor growth and/or histology and/or clinical markers associated with malignancy and/or survival in the second animal, which is not administered the test agent and serves as a negative control and comparing the results with those obtained with the transgenic animal that had received the test agent. Multiple animals may be administered different dosages of test agent to evaluate dose/benefit effect. Clinical markers of malignancy include, but are not limited to, weight of the animal, as well as features associated with the tumor resulting from oncogene expression (for example, a mammary tumor is readily palpable). In preferred non-limiting embodiments, the test agent is a picropodophyllin analogue, such as, but not limited to, those described herein, or another test agent which is an Igf1r inhibitor. Analogous experiments may be performed using a cell line prepared from the transgenic animal expressing the oncogene, wherein, after administration of test agent, features of the malignant phenotype, including rate of proliferation, contact inhibition, ability to grow in soft agar, and markers of apoptosis, may be measured (and where preferably these features are compared between cells exposed to the test agent and control cells which are not).
In further non-limiting embodiments, the present invention provides for a method of treating a cancer, e.g. a cancer for which intact signaling of Igf1r is related to tumor development and/or K ras is activated, amplified and/or mutated, comprising administering, to a subject in need of such treatment, an effective amount of a picropodophyllin analogue as described herein. Igf1r-related cancers include, but are not limited to breast cancer, prostate cancer, glioblastoma, colon cancer, liver cancer, and ovarian cancer. Optionally, said subject may further be administered a dose of a second cancer therapeutic agent, for example, but not limited to, an epidermal growth factor receptor inhibitor such as erlotinib, a Raf/neoangiogenesis inhibitor such as sorafenib, or other agent known in the art.
In still further, non-limiting embodiments, the present invention provides for a method of treating basal like breast cancer, comprising administering, to a human subject diagnosed as having basal like breast cancer, an effective amount of picropodophyllin or an analogue thereof, including, but not limited to, a picropodophyllin analogue as set forth herein. Optionally, said subject may further be administered a dose of a second cancer therapeutic agent, for example, but not limited to, an epidermal growth factor receptor inhibitor such as erlotinib, a Raf/neoangiogenesis inhibitor such as sorafenib, or other agent known in the art. In specific, non-limiting examples, said basal like breast cancer has a phenotype which is estrogen receptor negative, progesterone receptor negative, and ERBB2 negative. In further specific, non-limiting examples, said basal-like breast cancer has a phenotype which includes one or more of the following: epidermal growth factor receptor positive, cytokeratin 5 positive, cytokeratin 6 positive cytokeratin 14 positive, and/or p63 positive.
In specific, non-limiting embodiments of the invention, an effective amount of picropodophyllin or an analogue thereof, for use in methods of treatment as described above, may be between about 30 mg/kg and 80 mg/kg, and may be adjusted to optimize effectiveness using standard pharmaceutical techniques. In those specific non-limiting embodiments where erlotinib is added, the dose may be, for example and not by limitation, between about 25 mg/day and 200 mg/day, or between about 50 mg/day and 150 mg/day, for a treatment interval between about 1 day and until treatment is deemed successful or adverse effects prevent its continuation. In specific, non-limiting embodiments, the amount administered results in a concentration local to the cancer of between about 200 nM and about 800 nM. In specific, non-limiting embodiments, the analogue is compound 18 or compound 21 in an amount that, when administered, results in a concentration local to the cancer of between about 200 nM and about 800 nM or about 300-600 nM or about 400-500 nM.
Said picrodophyllin and/or analogue thereof may be administered by any route known in the art, including but not limited to, local application at tumor site or at site of tumor excision, intravenous administration, intraarterial administration, intraperitoneal administration, intrathecal administration, intraventricular administration, intramuscular administration, subcutaneous administration, oral administration, topical administration, etc. In one specific non-limiting embodiment the picropodophyllin analogue is compound 18 herein. In another specific non-limiting embodiment the picropodophyllin analogue is compound 21 herein.
IGF1RK inhibitors of the invention can be formulated as pharmaceuticals using methods well known in the art. Pharmaceutical formulations of the invention typically comprise at least one IGF1 RK inhibitor of the invention mixed with pharmaceutically acceptable carrier. Preferably the solutions are sterile and non-pyrogenic. Compounds of the invention can be formulated for parental administration by a variety of routes, including by mouth, by injection (e.g., by bolus injection), or by infusion.
IGF1 RK inhibitors of the invention can be administered to patient, either alone or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at doses to inhibit cancer cell growth as a single agent. IGF1RK inhibitors of the invention may also make cancer cells more vulnerable to treatment with other agents and therefore can be used to supplement standard chemotherapy regimens, such as “CHOP” therapy (e.g., the use of combination of drugs such as Cyclophosphamide, Doxorubicin Hydrochloride, Oncavin [Vincristine] and Prednisone) for a variety of cancers. Related members of each family may be substituted for an individual component in this combination, and a subset of these agents may also be used. IGF1RK inhibitors of the invention can be administered simultaneously or sequentially with a variety of chemotherapeutic agents, including but not limited to methotrexate, cis-platin and 5-fluorouracil.
For example, IGF1RK inhibitors such as picropodophyllin analogues of the invention can be used in targeted therapy for the following cancers, either alone or in combination with any of the following drugs:
-
- Breast—HERCEPTIN® and/or taxol, epothilone, letrozole (e.g., FEMARA®), anastrozole (e.g., ARIMIDEX), tamoxifen
- Ovarian—taxol
- Prostate—bicalutamide (e.g., Casodex)
- Pancreatic—gemcitabine
- Non-Hodgkins Lymphoma—RITUXAN®
- Multiple Myeloma—bortezomide (e.g., VELCADE®)
- Lung Cancer—topotecan (e.g., HYCAMTIN®), Etoposide, Tarceva (Genentech)
- Testicular—etoposide (e.g., ETOPOPHOS®, VEPESID®)
- Skin—imiquimod (e.g., Aldara)
- Colorectal—capecitabine (e.g., XELODA®), irinotecan e.g., CAMPTO®), Epothilone
- Myeloid Leukemia—GLEEVEC®
The invention will be further described by reference to the following examples. These examples should not be construed in any way as limiting the invention to anything less than that which is disclosed or which could have been obvious to anyone skilled in the art.
6. EXAMPLE 1 SynthesisBromoacetyl 1 was synthesized by bromination (Conrad, P. C. et al. J. Org. Chem., 1987, 52, 586-591) and subsequent acetalization of piperonal (Keay, B. et al. Can. J. Chem. 1983, 61, 1987-1995.)
To a solution of bromoacetal 1 (10.0 g, 36.3 mmol) in THF (100 mL) at −78° C. was added n-BuLi (25.0 mL, 40.6 mmol, 1.6 M in hexanes) dropwise via syringe. The solution was stirred for 1 h at 0° C. and a suspension of paraformaldehyde (3.30 g, 36.7 mmol) in THF (60 mL) was added via cannula. The reaction was warmed to room temperature, stirred for 2 h, and then quenched with H2O/Et2O. The aqueous layer was extracted with Et2O, and the combined organics were dried (MgSO4), filtered, and concentrated. Flash chromatography (50% EtOAc/hexanes) gave 2 (6.75 g, 82%). On a larger scale, 1 (50 g, 182 mmol) gave 2 in 72% yield (29.7 g). 1H NMR. (200 MHz, C6D6) δ 2.47 (t, J=6 Hz, 1H), 2.97 (s, 6H), 4.50 (d, J=6 Hz, 2H), 5.29 (s, 2H), 5.31 (s, 1H), 6.86 (s, 1H), 7.27 (s, 1H); 13C NMR. (125 MHz, C6D6) δ 52.8, 62.6, 101.6, 101.8, 108.4, 110.1, 130.4, 134.8, 147.5, 148.4; HRMS (EI) calculated for C11H14O5 226.0841, observed 226.0843. Anal. Calculated for C11H14O5: C, 58.39; H, 6.24. Found: C, 58.20; H, 6.07.
The substrate 2 (23.7 g, 105 mmol) was dissolved in excess DMAD (251 g, 1.76 mol) and glacial AcOH (23.2 mL, 0.4 mol), and the mixture was stirred for 2 h at 80° C. Excess DMAD was removed by vacuum distillation, and flash chromatography (30% EtOAc/hexanes) gave 3 (56.4 g, 90%) as a yellow solid. On a smaller scale, 2 (5.53 g, 24.1 mmol) gave 3 in 92% yield (13.6 g): mp 117-119° C.; 1H NMR (300 MHz, CDCl3) δ 3.79 (s, 6H), 5.86 (s, 2H), 5.90 (d, 1 Hz, 1H), 5.95 (d, J=1 Hz, 1H), 6.95 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 53.0, 85.7, 102.2, 105.2, 141.4, 146.2, 152.5, 163.5. Anal. Calculated for C15H12O7: C, 59.21; H, 3.98. Found: C, 59.27; H, 4.11.
IBF Diels-Alder product 3 (43.2 g, 0.14 mol) was dissolved in EtOAc (300 mL), and 10% Pd/C (1.5 g) was added. The reaction mixture was hydrogenated at 48 psi for 6 h. The reaction mixture was filtered through Celite and concentrated to give 4 as a white solid: mp 96-99° C.; 1H NMR (300 MHz, CDCl3) δ 3.53 (s, 6H), 3.61 (dd, J=2, 3 Hz, 2H), 5.41 (dd, J=2, 3 Hz, 2H), 5.92 (d, J=2 Hz, 1H), 5.96 (d, J=1 Hz, 1H), 6.82 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 48.3, 52.3, 81.5, 101.9, 103.9, 137.2, 147.5, 170.5. Anal. Calculated for C15H12O7: C, 58.83; H, 4.61. Found: C, 58.90; H, 4.71.
To a solution of the dimethyl ester 4 (25.0 g, 82 mmol) in Et2O (500 mL) at 0° C. was carefully added LiAlH4 (6.2 g, 0.16 mol), and the resulting reaction mixture was refluxed for 1 day. Quenching was carried out by the sequential careful addition of H2O (6 mL; 30 min stirring), 15% NaOH (6 mL, 30 min stirring), and H2O (19 mL; 30 min stirring). The mixture was neutralized with 1 N HCl solution (100 mL), followed by the addition of H2O (2.5 L) and extraction with EtOAc (6.5 L). The organics were dried (Na2SO4) and concentrated to give 5 as a white solid (16.8 g, 82%): mp 177-179° C.; 1H NMR (500 MHz, CD3OD) δ 2.65-2.68 (ddd, J=4, 6, 9 Hz, 2H), 2.77 (dd, J=9, 10 Hz, 2H), 3.15 (dd, J=6, 10 Hz, 2H), 5.24 (d, J=4 Hz, 2H), 5.90 (d, J=1 Hz, 1H), 5.94 (d, J=1 Hz, 1H), 6.85 (s, 2H); 13C NMR (125 MHz, C5D5N) δ 44.6, 60.1, 82.4, 101.6, 103.6, 138.2, 146.7. Anal. Calculated for C13H14O5: C, 62.39; H, 5.64. Found: C, 62.26; H, 5.59.
To a solution of 5 (57 g, 0.23 mol) and DMAP (1.4 g, 11.4 mmol) in pyridine (750 mL) at −10° c. was added Ac2O (69.9 g, 0.68 mol). The mixture was stirred for 16 h at room temperature and EtOAc was added. The organic phase was washed with saturated NaHCO3 solution, 1 N HCl, and CuSO4 (aqueous, saturated). Drying (MgSO4) and concentration provided 6 (76 g, 100%): mp 120-122° C.; 1H NMR (300 MHz, CDCl3) δ 2.05 (s, 6H), 2.82-2.85 (m, 2H), 3.24 (dd, J=10, 11 Hz, 2H), 3.75 (dd, J=6, 11 Hz, 2H), 5.26 (d, J=4 Hz, 2H), 5.94 (d, J=1 Hz, 1H), 5.99 (d, J=1 Hz, 1H), 6.76 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 21.5, 40.6, 63.1, 82.2, 102.1, 103.8, 136.7, 147.6, 171.2; Anal. Calculated for C17H18O7: C, 61.07; H, 5.43. Found: C, 61.20; H, 5.61.
A 5 L RB flask was charged with PPL (263 g, crude, Sigma) and buffer solution (50 mM KPO4, pH 7.8, 3.5 L). Diacetate 6 (20.0 g, 59.8 mmol) in DMSO (380 mL) was added via a sidearm while stirring with a mechanical stirrer. The reaction was quenched with 4 L of EtOAc after 2.5 h at room temperature. Following centrifugation to remove insoluble material, the organic layer was separated and washed with water. The organics were dried over MgSO4 and concentrated. Flash chromatography (50-80% EtOAc/hexane) gave in the following order 6 (4.1 g, 21%); 7 [11.5 g, 66%; (83% based on recovered 6)], and diol 5 (0.7 g, 5%). The monoacetate 7 was determined to be 95% ee by examination of the 1H NMR spectrum of its derivative Mosher ester: mp 131-134° C.; 1H NMR (360 MHz, CDCl3) δ1.43-1.58 (br. s, 1H), 2.06 (s, 3H), 2.77-2.84 (ddd, J=6, 9, 13 Hz, 2H), 2.87 (dd, J=9, 10 Hz, 1H), 3.23 (dd, J=10, 11 Hz, 1H), 3.28 (dd, J=6, 10 Hz, 1H), 3.76 (dd, J=6, 11 Hz, 1H), 5.25 (d, J=4 Hz, 1H), 5.32 (d, J=4 Hz, 1H), 5.94 (d, J=1 Hz, 1H), 5.98 (d, J=2 Hz, 1H), 6.75 (s, 1H), 6.83 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 21.5, 40.3, 43.8, 61.0, 63.4, 82.3, 82.4, 102.0, 103.7, 103.8, 136.7, 137.2, 147.3, 147.4, 171.5; [α]24D=+52.6° (c 0.6, CHCl3); HRMS (FAB, 3-NBA) calculated for C15H16O6 292.0947 [M+], observed 292.0952. Anal. Calculated for C15H16O6: C, 61.64; H, 5.52. Found: C, 61.72; H, 5.65.
To a solution of 7 (14.5 g, 49.6 mmol) and imidazole (7.4 g, 0.11 mol) in DMF (150 mL) at 0° C. was added of TIPSCI (11.7 mL, 54.6 mmol) and the mixture was stirred for 7 h at room temperature. Et2O was added and the mixture was washed with saturated aqueous NaHCO3 and water. The organic phase was dried over MgSO4, filtered and concentrated to give 7 as an oil (22.3 g, 100%). 1H NMR (300 MHz, CDCl3) 0 1.03 (d, J=3 Hz, 18H), 1.03-1.22 (m, 3H), 2.06 (s, 3H), 2.762.77 (m, 3H), 3.14 (apt 1, J=10 Hz, 1H), 3.40-3.43 (m, 1H), 3.78 (dd, J=5, 11 Hz, 1H), 5.24 (d, J=4 Hz, 1H), 5.33 (d, J=3 Hz, 1H), 5.94 (d, J=1 Hz, 1H), 5.97 (d, J=1 Hz, 1H), 6.74 (s, 1H), 6.83 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 12.5, 18.6, 21.5, 40.2, 44.3, 61.8, 63.3, 82.3, 82.9, 101.8, 103.6, 104.1, 136.9, 137.5, 147.1, 147.2, 171.3; [α]24D=+3.0° (c 0.9, CHCl); HRMS (FAB, 3-NBA, LiI) calculated for C24H36O6SiLi 455.2442, observed 455.2443.
To a solution of acetate 8 (7.1 g, 15.8 mmol) in methanol (60 mL) was added K2CO3 (438 mg, 3.17 mmol). The resulting suspension was stirred for 1.5 h at room temperature, Dowex 50×8 resin (H+ form, 900 mg) was then added, and stirring was continued for 30 min. The solution was filtered, concentrated and the residue purified by flash chromatography (30% EtOAc in hexanes) to yield alcohol 9 (6.43 g, 100%). 1H NMR (300 MHz, CDCl3) δ 0.98-1.07 (m, 21H), 2.79-2.85 (m, 2H), 2.97-3.11 (m, 2H), 3.17 (dd, J=5, 11 Hz, 1H), 3.31 (dd, J=6, 10 Hz, 1H), 5.19 (d, J=4 Hz, 1H), 5.20 (d, J=5 Hz, 1H), 5.96 (s, 2H), 6.73 (s, 1H), 6.76 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 12.4, 18.6, 44.4, 44.5, 61.1, 62.1, 82.0, 82.1, 101.9, 103.3, 103.4, 137.3, 137.6, 147.0, 147.1; IR (ATR) 3419 cm−1; [α]24D=−26.8° (c 1.3, CHCl3); HRMS (FAB, 3-NBA, LiI) calculated for C22H34O5SiLi 413.2336, observed 413.2341.
To a solution of oxalyl chloride (29 mL of a 2.0 M solution in CH2Cl2, 58.1 mmol) at −78° C. was added a solution of DMSO (4.86 mL, 32.6 mmol) in CH2Cl2 (20 mL) via cannula. After 10 min of stirring at −78° C., a solution of 9 (13.9 g, 34.2 mmol) in CH2Cl2 (20 mL) was added dropwise via cannula. After an additional 30 min at −78° C., a solution of NEt3 (16.2 mL, 116 mmol) in CH2Cl2 (14 mL) was added. The resulting reaction was allowed to warm to −40° C. and kept there for 2 h. EhO (500 mL) was then added at −40° C., and the reaction mixture was allowed to warm to room temperature. The mixture was washed with H2O, aqueous NI—LiCl, and brine. The organics were dried (MgSO4), filtered, and concentrated to give 10 (14.3 g, 100%). 1H NMR (300 MHz, CDCl3) 80.97-1.20 (m, 21H), 2.99-3.12 (m, 2H), 3.22 (ddd, J=3, 5, 8 Hz, 1H), 3.46-3.51 (m, 1H), 5.38 (s, 1H), 5.40 (s, 1H), 5.96 (d, J=1 Hz, 1H), 5.98 (d, J=1 Hz, 1H), 6.84 (s, 1H), 6.85 (s, 1H), 9.07 (d, J=3 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 12.6, 18.6, 47.3, 54.2, 62.8, 81.0, 82.7, 102.0, 103.9, 104.0, 136.9, 137.5, 147.4, 147.5, 202.5; [α]24D=−26.3° (c 0.8, CHCl3); HRMS (FAB, 3-NBA, NaI) calculated for C22H32O5SiNa 427.1917, observed 427.1925.
To a solution of aldehyde 10 (10.5 g, 26.0 mmol) in MeOH (500 mL) was added NaOMe (1.40 g, 26 mmol) and the mixture was heated to reflux for 5 h. H2O (245 mL) was then added, and CO2 was bubbled through the solution until the pH reached 8 (pH paper). MeOH was removed in vacuo, and the resulting aqueous layer was extracted with CH2Cl2. The combined organics were dried (MgSO4), filtered, and evaporated to provide 11 as a white solid (9.50 g, 90%): mp 87-89° C.; 1H NMR (500 MHz, CDCl3) δ 0.93-1.03 (m, 21H), 1.74 (d, J=5 Hz, 1H), 3.17 (app t, J=10 Hz, 1H), 3.34 (ddd, J=2, 4, 6 Hz, 1H), 3.80 (dd, J=4, 10 Hz, 1H), 4.97 (app t, J=Hz, 1H), 6.01 (s, 1H), 6.02 (s, 1H), 6.83 (s, 1H), 6.93 (s, 1H), 7.24 (s, 1H), 9.61 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 12.5, 18.5, 43.4, 62.7, 70.0, 102.4, 109.8, 110.9, 125.3, 133.8, 136.0, 145.6, 148.8, 150.7, 192.9; IR (ATR) 3395, 1674, 1645 cm−I; [α]24D+82.0° (c 1.0, CHCl3); HRMS (FAB, 3-NBA) calculated for C22H33O5Si [(M+H)+] 405.2097, observed 405.2096. Anal. Calculated for C22H32O5Si: C, 65.31; H, 7.97. Found: C, 65.45; H, 7.98.
To a solution of aldehyde 11 (900 mg, 2.25 mmol) in DMF (20 mL) was added TESCI (715 JtL, 3.38 mmol) and imidazole (460 mg, 6.75 mmol) at 0° C. and the reaction mixture was allowed to warm to r.t. overnight. Water was added and the product was extracted with Et20, the organic layer dried over MgSO4 and evaporated. The residue was then dissolved in t-BuOH (40 mL) and 2-methyl-2-butene (12 mL). A solution of NaClO2 (1.81 g, 20 mmol) and NaH2PO4 (1.93 g) in water (20 mL) was added and the mixture was stirred overnight. The product was extracted with Et2O, dried over MgSO4, concentrated and purified by silica gel column chromatography (10-30% EtOAc in hexanes) to give the acid 12 as a white powder (1.02 g, 85% over 2 steps). 1H NMR (400 MHz, CDCl3) 00.65 (q, J=8.0 Hz, 6H), 0.94 (t, J=8.0 Hz, 9H), 1.06 (m, 21H), 3.05, (apt t, J=10 Hz, 1H), 3.34 (ddd, J=10.0, 4.5, 1.6 Hz, 1H), 3.77 (dd, J=10.0, 4.5 Hz, 1H), 5.03 (s, 1H), 6.03 (s, 2H), 6.81 (d, J=4.8 Hz, 2H), 7.67 (s, 1H).
To a solution of acid 12 (940 mg, 1.76 mmol) in THF (20 mL) at room temperature was added carbonyl diimidazole (428 mg, 2.64 mmol). The reaction mixture was stirred overnight, water was added and the product was extracted with Et2O. The organic phase was dried over MgSO4 and evaporated to provide the crude acyl imidazolide, which was used in the next step without further purification. To a deoxygenated solution of 2-oxazolidinone (306 mg, 3.52 mmol) in THF (10 mL) at 78° C. was added n-BuLi (1.65 mL of a 1.6 M solution in hexanes, 2.64 mmol). After 1 h, a solution of acylimidazolide (1.06 g, 1.76 mmol) in THF (8 mL) was added dropwise at −78° C. and the reaction mixture was allowed to stir for 5 h at this temperature. The reaction was quenched with water and extracted with Et2O. The organic layer was dried (MgS04), concentrated and the residue purified by column chromatography on silica gel (hexanes/EtOAc/Et3N, 90:10:05 to 70:30:0.5) to yield 13 (650 mg, 62%). 1H NMR (400 MHz, CDCl3) J 0.65 (q, J=8.0 Hz, 6H), 0.94 (t, J=8.0 Hz, 9H), 1.03 (m, 21H), 3.17, (apt t, J=10 Hz, 1H), 3.38 (ddd, J=10.0, 6.0, 1.6 Hz, 1H), 3.71 (dd, J=10.0, 6.0 Hz, 1H), 4.00 (m, 1H), 4.16 (apt q, J=9.0 Hz, 1H), 4.49 (m, 2H), 4.99 (s, 1H), 6.00 (s, 1H), 6.74 (s, 1H), 6.79 (s, 1H), 7.13 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 5.0, 6.8, 11.9, 18.0, 44.1, 45.8, 61.8, 62.1, 68.5, 76.7, 77.1, 77.4, 101.4, 109.2, 110.3, 125.0, 126.3, 131.4, 137.2, 147.5, 148.8, 153.5, 169.7.
Typical Procedure for Introduction of E Rings (14-17)
To a suspension of CuCN (297 mg, 3.30 mmol) in THF (8 mL) at 5° C. was added 3.3 mL of a solution of the desired mono or di-methoxy phenylmagnesium bromide (1M in THF, 3.30 mmol). The resulting mixture was stirred for 30 min and a solution of Michael acceptor 13 (250 mg, 0.41 mmol) in THF (6 mL) was added dropwise at the same temperature. After 1.5 h at 5° C., a saturated aqueous solution of NH4Cl was added and the product was extracted with Et2O, the organic phase dried over MgSO4 and evaporated. The product was filtered through a short silica gel column (hexanes/EtOAc 80:20) and used directly for the next step.
Typical Procedure for Desilylative Lactonization
To a solution of precursor 14-17 (0.21 mmol) in THF was added TBAF (1.0 M solution in THF, 4 eq.). The mixture was refluxed for 30 minutes and allowed to cool to r.t. A saturated solution of NH4Cl was added and the product was extracted with Et2O, the organic layer dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (Hexanes/EtOAc, 7:3).
From Michael acceptor 13 (0.23 mmol, 140 mg) 55 mg of 18 (68% over 2 steps) were obtained as a white powder. 1H NMR (400 MHz, DMSO-d6) 02.50 (m, 1H), 3.38 (m hidden under water peak, 1H), 3.76 (s, 3H), 3.95 (d, J=7.2 Hz, 1H), 4.36 (dd, J=1004, 604 Hz, 1H), 4040 (dd, J=9.2, 604 Hz, 1H), 4049 (dd, J=9.2, 1.6 Hz, 1H), 5.92 (s, 2H), 5.94 (s, 1H), 5.97 (d, J=604 Hz, 1H), 6.84-6.89 (m, 3H), 7.07 (s, 1H), 7.32 (t, J=8.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 43.2, 4304, 44.3, 55.5, 67.7, 69.8, 101.2, 105.0, 107.7, 11204, 11504, 121.8, 130.1, 131.8, 135.3, 145.1, 146.2, 146.2, 159.9, 17804; HRMS (FAB, 3-NBA) calculated for C20H18O6 354.1103, observed 354.1100.
From Michael acceptor 13 (0.23 mmol, 139 mg) 50 mg of 19 (63% over 2 steps) were obtained as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 2.49 (app. q, J=7.0 Hz, 1H), 3.29 (dd, J=9.6, 7.8 Hz, 1H), 3.78 (s, 3H), 3.92 (d, J=7.8 Hz, 1H), 4.36 (dd, J=10.0, 6.4 Hz, 1H), 4.39 (dd, J=9.2, 6.4 Hz, 1H), 4.49 (dd, J=9.2, 1.2 Hz, 1H), 5.91 (s, 2H), 5.92 (s, 1H), 5.95 (d, J=6.4 Hz, 1H), 6.96 (d, J=8.8 Hz, 1H), 7.06 (s, 1H), 7.19 (d, J=8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 42.6, 43.3, 44.6, 55.5, 67.7, 69.7, 101.2, 104.9, 107.7, 114.4, 130.6, 132.4, 135.2, 135.4, 146.1, 146.1, 158.4, 178.5; HRMS (FAB, 3-NBA) calculated for C20H18O6 354.1103, observed 354.1113.
From Michael acceptor 13 (0.26 mmol, 160 mg) 40 mg of 20 (41% over 2 steps) were obtained as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 2.49 (m, 1H), 3.37 (dd, J=9.6, 8.0 Hz, 1H), 3.73 (s, 3H), 3.78 (s, 3H), 3.89 (d, J=8.0 Hz, 1H), 4.35 (dd, J=10.0, 6.4 Hz, 1H), 4.40 (dd, J=8.8, 6.4 Hz, 1H), 4.50 (d, J=8.8 Hz, 1H), 5.91 (d, J=2.0 Hz, 2H), 5.96 (s, 1H), 5.97 (d, J=5.6 Hz, 1H), 6.79 (d, J=8.0 Hz, 1H), 6.87 (s, 1H), 6.96 (d, J=8.0 Hz, 1H), 7.06 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 43.1, 43.4, 44.3, 55.9, 67.7, 69.7, 101.1, 104.9, 107.7, 112.2, 113.0, 121.7, 132.4, 135.4, 135.5, 146.1, 146.1, 148.0, 149.2, 178.5; HRMS (F AB, 3-NBA) calculated for C20H18O6 384.1209, observed 384.1213.
From Michael acceptor 13 (0.25 mmol, 150 mg) 48 mg of 21 (50% over 2 steps) were obtained as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 2.49 (m, 1H), 3.38 (dd, J=9.2, 8.0 Hz, 1H), 3.74 (s, 6H), 3.91 (d, J=7.2 Hz, 1H), 4.34 (dd, J=9.60, 6.4 Hz, 1H), 4.40 (dd, J=9.0, 6.4 Hz, 1H), 4.50 (d, J=9.0 Hz, 1H), 5.92 (s, 2H), 5.96 (d, J=6.0 Hz, 1H), 6.00 (s, 1H), 6.45 (s, 3H), 7.06 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 43.1, 43.5, 44.1, 55.6, 67.7, 69.8, 98.8, 101.2, 105.0, 107.7, 131.6, 135.3, 145.8, 146.2, 161.0, 178.4; HRMS (FAB, 3NBA) calculated for C20H18O6 384.1209, observed 384.1221.
7. EXAMPLE 2 In Vitro Testing of Inhibition of Cell GrowthA murine breast cancer cell line derived from Kras* mice was used to assay the effect of IGF1RK inhibitors. Approximately 30,000 cells/well were plated in a 24-well plate which corresponds to 20% confluence. The next day (Day 0 for the experiment) two of wells were treated with MTT (Thiazol Blue Tetrazolium), a substrate used in a colorimetric metabolic assay that reflects cell density. The cells in the rest of the wells were treated with DMSO (PPP solvent), PPP or a derivative of PPP at 500 nM concentration. Forty-eight hours later the duplicate wells were assayed. The effectiveness of each of the PPP derivatives was compared with that of the original PPP.
The five compounds listed in Table 1 were tested. The results of the experiments with compounds 3 and 6 are shown in Table 2.
Compound 18 appeared to be as effective as PPP at inhibiting growth of the Kras* breast cancer cell line. By varying the concentration of compound 18, we obtained a preliminary estimate of ˜400 nM for IC50 value, which very similar to the behavior seen with PPP itself on this test cell line. Moreover, the 4-methoxy compound (19) shows essentially no activity under exactly the same conditions. Such a dramatic SAR (Structure Activity Relationship) is quite remarkable.
8. EXAMPLE 3 In Vivo Testing 8.1. Materials and Methods MiceIn addition to the mice conditionally expressing oncogenic Kras (for details see EXAMPLE 5, Materials and Methods), three mouse strains were used which have been described previously; two cre-expressing strains, Hs-cre1 and Wapcre/+ (Dietrich P, et al., Mamm. Genome (2000), 11:196-205; Ludwig T, et al., Oncogene (2001), 20:3937-3948), and Igf1rflox/flox mice (Dietrich P, et al., Mamm. Genome (2000), 11:196-205). Molecular, histological, microarray and other analyses were performed as described in EXAMPLE 5, Materials and Methods and Table 9.
Drug TreatmentsDetails of the preclinical trial using picropodophyllin synthesized as described (Buchardt O, et al. J. Pharm. Sci. (1986), 75:1076-1080) and Erlotinib (purchased from Hwasun Biotechnology Co) are described in Results. The drugs were dissolved in DMSO and cremophor (9:1) and injected intraperitoneally. To calculate tumor volumes, the formula for a prolate spheroid (π/6×a×b2 or ˜a×b2/2, where a and b are the major and minor axis, respectively) was used. The lengths of axes were determined microscopically from sections of tumor nodules using a computer assisted morphometry system (SpotAdvanced VS. 4.0.1, Nikon Eclipse E400). For xenograft experiments, 5×106 MDA-MB-231 cells were injected bilaterally into the fat pads of mammary glands 3 and 4 of female SCID/NOD mice. At ten days post injection, when tumors were readily palpable, the mice were randomly divided into two groups of five mice each and received the same treatment (vehicle or PPP) described above for the Kras* mice for 3 weeks. For in vitro experiments, MDA-MB-231 cells were grown in DMEM supplemented with 10% fetal bovine serum. Equal numbers of cells were seeded in multiple wells of 24-well plates at low density (≦20% confluence at day 0) and either DMSO or PPP dissolved in DMSO was added to the medium (final concentrations: DMSO 01%, PPP 500 nM). Cell viability (duplicates) was measured on days 2, 4 and 6 by using the Thiazolyl Blue Tetrazolium Bromide (MTT) colorimetric assay (Mosmann T, J. Immunol. Methods (1983), 65:55-63).
8.2. Results and Discussion Tumor Development in Mice Expressing Oncogenic Kras*A suitable mouse model has been identified to evaluate Igf1r as a potential therapeutic target in the context of our research program aiming to generate mouse tumors by design using a variant of a genetic scheme involving cre/loxP recombination (Politi K, et al., Oncogene (2004), 23:1558-1565). Depending on the tissue-specificity of the promoter driving cre expression, tumors develop at chosen anatomical sites of progeny derived by mating Cre-producers with mice carrying a dormant oncogenic transgene that becomes functional after excision of a foxed DNA segment blocking its expression.
For development of a targeting cassette for high-level expression of any chosen cDNA introduced into a defined genomic site by knock-in, the Eef1a1 locus (encoding a translation elongation factor) was used as a recipient site for transgenic knock-in of various sequences, including a constitutively active oncogenic Kras cDNA [Kras 4B(G12D); Kras*]. Eef1a1 is strongly transcribed (Soares et al., Proc. Natl. Acad. Sci. U.S.A. (1994), 9, 9228-32) and yields an abundant protein product (˜2% of the total cell protein; Codeelis, Trends Biochem. Sci. (1995), 20, 169-70). In the Eef1a1 targeting cassette (
Ubiquitous activation of Kras* expression by removing the foxed block using a cre transgene transcribed in 2-cell stage embryos (Dietrich et al., Mammalian Genome (2000), 11:196-205) caused embryonic lethality (not shown). On the other hand, crosses of Kras* mice with partners expressing Cre in particular tissues resulted in tumor development in the pancreas, prostate, skin, intestine and the hematopoietic system.
To activate expression of Kras* in mammary glands, a transgenic line (Ludwig et al., Oncogene (2001), 20, 3937-48) carrying cre inserted into the Wap locus, encoding a milk protein, which is specifically transcribed in alveolar and ductal mammary epithelial cells during late pregnancy and throughout lactation (Robinson et al., Development (1995), 121, 2079-90) was used. Unexpectedly, lactating females with an Eef1a1-Kras*/Wapcre genotype (n=28) developed palpable multifocal, fully invasive tumors extremely rapidly. Specifically, these malignant breast carcinomas appeared within a period of 2 days to ˜2 months after the first delivery of pups with a median time of tumor-free survival (T50) of only 9 days (
Female mice developing tumors were sacrificed when moribund within a period of 9 days to ˜3 months (this brief time of observation only rarely permitted the detection of lung metastases). In all examined cases (n=37), the Kras*-induced carcinomas involved most or all mammary glands and were either multifocal or consisted of large masses generated by coalescence of smaller components. The tumors were histologically heterogeneous and four coexistent types of invasive carcinomas were identified at variable proportions: adenocarcinomas (Dunn Type A/B tumors), and pale, squamous and spindle cell (sarcomatous) carcinomas [PCC, SCC and SRC;
Overall, however, the slow-growing adenocarcinomas, which exhibited a very low proliferation index (˜10-20% of the corresponding value for any other component), were on average the smallest tumor constituent (14% of the total tumor mass; ˜40-50% of the size of any other form; Table 3).
The Dunn adenocarcinomas were well-differentiated microacinar structures (Dunn Type A) or occasionally solid nests without glandular differentiation (Dunn Type B) and corresponded morphologically to tumor types induced by the mouse mammary tumor virus (MMTV; Hoeber-Harper, New York), (1959) pp. 38-84; Sass B and Dunn T B, J Natl Cancer Inst (1979); 62:1287-1293). On average, they were the smallest and slowest-growing tumor constituents (Table 3). The PCC, which consisted of large, lightly-staining (“pale”) cells, were also adenocarcinomas, but exhibited in some areas signs of keratinization. Morphologically, they are indistinguishable from a rare tumor type that appears specifically in the GR strain of mice carrying an active endogenous MMTV provirus (Mtv2) on chromosome 18. However, immunophenotypic differences between the Kras*- and MMTV-induced PCC were identified. Clear evidence of keratinization (squamous metaplasia) was seen in SCC, whereas the spindle-cell tumors exhibited sarcomatous metaplasia. The microacinar (Dunn A), pale and squamous cell tumors were correlated with the presence of corresponding forms of carcinoma in situ (CIS; also referred to in mice as “mammary intraepithelial neoplasm”, MIN; Cardiff R D et al., The Mouse in Biomedical Research. Volume II: Diseases, Second ed., eds. Fox J G et al., (Elsevier, New York), (2007), pp. 581-622;
One type of carcinoma in situ (small-cell, low nuclear grade CIS) exhibited a pattern similar to that of lobular neoplasia in human CIS. It was composed of small cells filling lobules, but never extralobular ducts, and could be discriminated from the cytologically and immunophenotypically identical invasive microacinar carcinoma solely on the basis of preservation of a myoepithelial layer. A second CIS type, also retaining a discontinuous layer of myoepithelial (p63-positive) cells, formed multiple cellular layers in ducts, and was also present in lobuloalveolar units, where it filled alveolar spaces in the form of solid nests or nodules. This high-grade CIS was found either in isolation or adjacent to an invasive component and consisted of large cells indistinguishable in appearance and immunostaining characteristics from those seen in PCC. Interestingly, in several independent cases, conglomerates of large ductal CIS cells were observed budding from a suprabasal location outward to acquire a position between the (still intact) luminal epithelial layer and the overlying myoepithelial layer (at least in the human mammary epithelium, multilineage progenitor cells are thought to reside in a suprabasal position). A third type of CIS was composed of squamous cells and was the only one exhibiting foci of sarcomatous metaplasia. Apparently, this CIS was giving rise to invasive squamous cell carcinoma quite early, as it was observed less frequently than the other forms.
To assess the origin, relationships and signaling characteristics of the carcinomas by immunophenotyping, an extensive panel of markers was used (
The results indicated that the cancer forms induced by Kras* could be classified into two groups differing in the cell-of-origin. Whereas expression of exclusively luminal epithelial cell markers (such as keratin 18; Krt18) was detected in small-cell CIS and Dunn A/B adenocarcinomas, the large-cell CIS and the pale, squamous and sarcomatous carcinomas exhibited positive immunostaining both for luminal markers (Krt18) and for basal cell markers (Krt5, Krt14, p63), and also for presumptive stem cell markers. Apparently, the Dunn adenocarcinomas, which are also hormone receptor-positive (ER+/PR+), are luminal-type neoplasms, presumably derived from differentiated luminal epithelial cells, whereas the ER−/PR− pale, squamous and sarcomatous tumors are basal-like cancers derived from undifferentiated, bipotential precursor cells and not from myoepithelial cells, as they do not co-express p63 and smooth muscle actin (SMA), which are the functional markers of myoepithelial differentiation. This interpretation is also strongly supported by expression profiling data.
On the other hand, a potential relationship between the three basal forms is not a straightforward issue. For example, it was considered that the PCC and the metaplastic and more extensively keratinized squamous cell carcinomas (SCC), which are often found intimately interspersed, might be related and could represent two extreme forms of a spectrum of adenosquamous carcinomas. However, it may be more likely that these two tumor forms originate from different cell precursors, as suggested from the identification of distinct CISs and the results of immunophenotyping. In regard to the spindle-cell (sarcomatous) carcinomas (SRC), it is important to note that they have lost the epithelial marker E-cadherin, which is present in PCC and SCC, but strongly express vimentin, a mesenchymal cell marker. Nevertheless, as in other cases of histopathologically heterogeneous mammary tumors, the SRC presumably are epithelial and not mesenchymal tumors that could be derived from an adenosquamous component by EMT (epithelial-mesenchymal transition).
The term “precursor cell” is used herein to refer to the cell-of-origin of the basal-like carcinomas that have been identified in these experiments. It is notable that the PCC expresses Gata3, a luminal cell marker for mature mammary epithelium, which is absent from SCC. This difference may by indicative of distinct cells of origin, but it cannot be interpreted as suggesting that PCC could be derived from terminally differentiated luminal cells, as Gata3 is expressed in precursor cells of mammary primordia. Differences in the expression of particular markers, such as CK5/14 and p63, also exist between SRC and the other two basal-like components (PCC and SCC). Nevertheless, the overall similarities between the three basal forms, including the expression of putative stem cell markers and particular signaling components validated by molecular data, permit a clear-cut demarcation from the luminal Dunn adenocarcinomas.
“Stem cell markers” should not be confused with “markers of stemness”, i.e. cellular constituents that bestow stem cell behavior (self-renewal capacity and potential to generate differentiated derivatives) and/or are uniquely expressed in stem cells. In fact, with the apparent exception of the Lgr5 gene, markers of stemness are generally unavailable. Instead, “stem cell markers” correspond to an “expression signature” (i.e. a combinatorial, rather than exclusive expression of some set of cellular elements) capable of discriminating a cell population enriched in operationally defined stem cells. Analysis of the distribution of the CD29 (integrin β1; Itgb1) and CD49f (integrin α6; Itga6) by using double immunofluorescence showed that both of these markers were co-localized in the basement membrane of the myoepithelial layer while, in addition, Itgb1 was present in the basolateral, but not the luminal aspect of luminal cells. This distribution was maintained in microacinar adenocarcinomas, but only for Itgb1, whereas the basal-like forms had altered patterns. Thus, all SCC cells exhibited intense labeling for both markers co-localized circumferentially, while the intensity of signal in PCC and SRC was variable and did not involve all cells. An additional marker that was examined was Sca1, which was found to be present in PCC and SCC, but not in SRC or Dunn tumors.
Of the three major molecularly classified subtypes of human breast cancer (Perou C M, et al. Nature (2000), 406:747-752; Sortie T, et al. Proc Natl Acad Sci USA (2001), 98:10869-10874; Sortie T, et al. Proc Natl Acad Sci USA (2003), 100:8418-8423), luminal cancers are estrogen receptor-positive (ER+), whereas the other two classes are ER-negative and either overexpress ERBB2 (ERBB2+) or exhibit phenotypic features of basal/myoepithelial cells (basal-like cancers). The latter also lack progesterone receptor (PR) and ERBB2 (“triple negative breast cancers”; see Da Silva L, et al., J. Clin. Pathol. (2007), 60: 1328-1332; Reis-Filho J S and Tutt A N, Histopathology (2008), 52:108-118), but frequently express EGFR and basal markers, such as cytokeratins (CK) 5/6 and/or 14 and p63 (Nielsen T O, et al., Clin. Cancer Res. (2004), 10:5367-5374.). The basal-like group (15-20% of all breast cancers), which is quite heterogeneous, includes high proportions of BRCA1-associated and also medullary and metaplastic (squamous, spindle cell and other) subtypes. Interestingly, KRAS amplification was detected in 56% (9/16) of examined basal like human breast cancers (Herschkowitz J. et al., Genome Biol. (2007), 8:R76).
On the basis of their distinct features, the Kras*-induced ER−/PR− pale, squamous and sarcomatous mouse carcinomas, which are immunopositive for both luminal (CK18) and basal cell markers (CK5, CK14, p63, and rarely smooth muscle actin), and also for presumptive stem-cell markers (Table 4), are analogous to some of the forms of human basal-like cancers. It is likely that these basal-like murine tumors are derived from undifferentiated, bipotential precursor cells and not from myoepithelial cells (this hypothesis concerning “cells-of-origin” is discussed in detail in EXAMPLE 5). Consistent with this view is the fact that the Kras* activating cre is embedded in the Wap locus that is not expressed in fully differentiated myopithelial cells.
A morphological similarity between mouse pale cell carcinoma and a type of human basal-like breast cancer that was also correlated with KRAS copy gain, was observed. In a collection of human breast cancer specimens (n=94), 17 samples (18%) were found to be basal-like (triple-negative and positive for CK5/6), while 77 (82%) were non-basal (R. Parsons and H. H., unpublished). Analysis of the 17 basal cancers for amplification of the KRAS locus by using CGH showed that 5 of the specimens scored positive, while further analysis of a subset by using FISH identified a sixth positive sample. The corresponding KRAS amplification frequency in the non-basal samples detected by CGH was 4/77 (˜5% vs. ˜35%, 6/17; P=0.002, Fisher's exact test). Three of the basal-like specimens with amplified KRAS displayed medullary features (large tumor nodules with pushing, rather than infiltrative borders, composed of large cells with irregular, sometimes bizarre nuclei growing in a syncytial fashion) but none of them met all of the criteria for classical medullary carcinoma classification (they are referred to here as “atypical medullary breast cancer”, AMBC; Fadare O and Tavassoli F A, Adv. Anat. Pathol. (2007), 14:358-373). Interestingly, two of these AMBCs, contained abundant large cells with pale or clear cytoplasm and exhibited a strong resemblance to the histomorphological signature of the PCC observed in our mouse model. To validate this correlation, an available set of triple-negative AMBCs (n=8) was examined and it was observed that most of them (7/8) were at least focally comparable to the PCC in Kras* mice (
To complement the morphological information described above, the expression profiles of normal postinvolutional mammary glands (n=5) and Kras*-induced carcinomas (n=14) were examined by microarray analysis, and it was found that they were readily discriminated by unsupervised hierarchical clustering (
Comparisons of the profiling results with lists of basal and luminal markers chosen for classification of human breast cancers (Sorlie et al., BMC Genomics (2006), 7:127; Herschkowitz et al., Genome Biol. (2007), 8, R76) and also with datasets of upregulated and downregulated genes in basal and non-basal breast cancers (Farmer et al., Oncogene (2005), 24, 4660-71; Richardson et al., Cancer Cell (2006), 9, 121-32) showed unequivocally that the Kras*-induced tumors are basal-like carcinomas, in agreement with the histological evidence. In fact, the null hypothesis that there is no statistical difference in the representation of basal and luminal markers in the groups of upregulated and downregulated genes in Kras* tumors was overwhelmingly rejected (Table 5). In addition, consistent with the hypothesis that the basal-like Kras* cancers evolve from precursor cells of the mammary epithelium, the data showed that the pattern of overexpressed genes in the tumors resembled much more the profile of a mammary cell population enriched in stem cells than that of another population consisting predominantly of luminal cells (Stingl et al., Nature (2006), 439, 993-97; high statistical significance; Table 5). Not unexpectedly, there was a high degree of similarity between the profiles of Kras*-induced mouse lung (Sweet-Cordero et al., Nature Genet. (2005), 37, 48-55) and mammary tumors (Table 5). Finally, comparisons of the microarray results with those for other mouse mammary tumors supported strongly the view that Kras* deregulates to a much larger extent all major signaling pathways (Tables 6-8).
One of the most interesting changes detected in Kras*-induced mammary cancers by RNA profiling was an increased expression of Igf1r, which was confirmed by Northern analysis. This prompted an investigation about the potential involvement of IGF signaling in the development of mammary tumors in this mouse model.
A compelling reason for asking this question is evidence in the prior art suggesting that cells lacking Igf1r cannot be transformed by any one of several tested oncoproteins (Baserga, Expert Opin. Ther. Targets (2005), 9, 753-68). Apparently, Igf1r-mediated signaling is not an oncogenic component per se, but a crucial prerequisite for oncogenesis considering that, among other effects, it provides a necessary and strong antiapoptotic signal for tumor growth. The Igf1r gene is neither amplified nor obligatorily overexpressed in human breast cancer. Thus, despite reported cases of overexpression, it is not uncommon to detect Igf1r mRNA levels that are equal or even lower in cancer cells than in normal breast tissue (Chong et al., Anticancer Res. (2006), 26(1A), 167-73). Nevertheless, it is interesting that 36% of examined basal-like breast cancers (n=64) were shown by immunohistochemistry to be Igf1r-positive (Lerma et al., Mod. Pathol. (2007), 20, 1200-07).
To examine the impact of the absence of Igf1r on the development of mammary tumors induced by Kras* overexpression, tumorigenic progression was compared between animals carrying the oncogenic transgene in a background either wild-type for Igf1r (Eef1a1-Kras*/Wapcre mice serving as controls) or possessing one or two floxed Igf1r alleles that could be conditionally ablated (Eef1a1-Kras*/Wapere/Igf1rfl/+ and Eef1a1-Kras*/Wapcre/Igf1rfl/fl genotypes; n=11 and n=14, respectively).
It was observed that, in contrast to cancer manifestation after the first birth with a T50 of 9 days in control mice, ablation of both foxed Igf1r alleles in experimental animals resulted in tumor development only after three pregnancies, while the latency increased dramatically (11-fold; T50=101 days; P<0.0001, log-rank test;
Comparison of the expression profiles of Kras* cancers developing in the presence or absence of Igf1r signaling revealed, among other effects (Table 9), significant differences in transcript levels for Egf ligands that were confirmed by Northern analysis. Specifically, with intact Igf1r, there was >100-fold increase over normal in the amount of steady-state mRNA for Hbegf present in the tumors, while the levels of overexpressed transcripts for Areg, Ereg and Tgfa were less dramatic (˜4-, 15- and 6-fold, respectively). Interestingly, a similar overexpression of Egf ligands was observed in an HrasG12V breast cancer model (Sarkisian C J, et al. Nat. Cell Biol. (2007), 9:493-505), implying a more general feedback loop involving Ras protein function. It was found that elimination of Igf1r only slightly affected the overexpression of Areg, but resulted in the reduction of the Ereg and Tgfa transcripts to almost normal levels, while the previously enormous amount of Hbegf mRNA was reduced approximately by half. In contrast, Igf1 and Igf2 transcripts encoding IGF ligands were virtually absent from the tumors and Igf1 and Igf2 polypeptides were below detection limits by immunohistochemistry (Table 4) indicating absence of IGF autocrine/paracrine signaling cues. Accordingly, unless it is eventually found that Igf1r-mediated signaling is triggered by EGF ligands acting through non-canonical IGF1R-EGFR heterodimers (Riedemann, J., et al., Biochem. Biophys. Res. Commun. (2007), 355, 707-714), it is likely that the IGF functions are served in the Kras* mammary cancers by endocrine action of IGF1 circulating in serum. Assuming this to be the case, it appears that IGF signaling potentiates Erbb-mediated activities by upregulating Egf ligands through an unknown mechanism, which could be transcriptional and could involve Ap1 sites present in the promoter regions of some of these ligands (Ornskov D, et al., Biochem. Biophys. Res. Commun. (2007), 354:885-891). Interestingly, Fosl1, an Ap1 component, is highly overexpressed in the Kras* tumors (
The strong genetic evidence for an Igf1r role in mammary tumorigenesis at least in the examined model is significant in the context of efforts to develop therapeutic approaches for treating breast cancer by blocking of IGF signaling. This could turn out to be significant for basal-like carcinomas, which have poor prognosis (Sorlie T, et al. Proc. Natl. Acad. Sci. USA (2001), 98:10869-10874) and pose a serious problem to targeted therapies (Cleator S, et al., Lancet Oncol. (2007). 8:235-244; Carey L A, et al., Clin. Cancer Res. (2007), 13:2329-2334), considering that the use of antiestrogens in combination with trastuzumab (anti-ERBB2 antibody) is not an option, while there is no clear choice for chemotherapy. Therefore, the Kras* model was used in a preclinical study testing the efficacy of the cyclolignan picropodophyllin (PPP), which has recently emerged as a potent, nontoxic and highly specific Igf1r inhibitor (Girnita, et. al., Cancer Res. (2004), 64, 236-42). Although the molecular mechanism of PPP action is still unknown, its inhibitory effects appear to be exerted by abrogation of Igf1r phosphorylation and promotion of its degradation, while the homologous insulin receptor is not affected (Girnita, et. al., Cancer Res. (2004), 64, 236-42; Vasilcanu, et al., Oncogene, (2008), 27: 1629-1638). Cell lines of Igf1r null fibroblasts are apparently insensitive to PPP, whereas the drug reduces the viability of cancer cell lines and causes tumor regression in mouse xenografts of multiple myeloma (Menu et al., Int. J. Cancer (2007), 121, 1857-61) and uveal melanoma (Girnita et al., Clin. Cancer Res. (2006), 12, 1383-91). Therefore, the Kras* model described herein was used for testing the potential therapeutic effects of PPP on breast cancer. For this purpose, PPP was administered either alone or in combination with erlotinib, an Egfr inhibitor (Yang S X, et al., Clin. Cancer Res. (2005), 11: 6226-6232), taking into consideration the overexpression of Erbb ligands and Igf1r/Egfr signaling relationships described above.
Mice at a progressed stage of tumorigenesis bearing at least one readily palpable tumor were injected intraperitoneally once daily either with vehicle or with PPP and Erlotinib, alone or in combination, at doses of 30 mg/kg and 50 mg/kg, respectively, for a period of 3 weeks, considering that the weight of some tumors in the controls could reach or exceed ˜1 g by that time. At the end of treatment, we measured tumor growth relative to control values by calculating tumor mass, and we did not attempt sequential measurements using a caliper because a pilot study indicated that they were inaccurate. First, the tumors developing in each gland tended to be multifocal and uneven, and progressively coalesced into larger masses precluding reliable evaluation. In addition, the treatment resulted in extensive tumor necrosis and fibrosis detectable only histologically, which would have artificially inflated macroscopic measurements.
All glands carrying tumors in treated and control animals were analyzed, and for statistical evaluation, it was taken into account that the cancers exhibited pronounced size heterogeneity (see
Not unexpectedly, vehicle administration did not alter the histopathological or immunophenotypic profile of tumors, whereas the specimens of mice treated either with PPP or with a PPP/Erlotinib combination displayed a marked reduction or even absence of the pale and squamous cell components accompanied by extensive keratinization and vacuolation (
To ascertain whether the reduction in tumor volume was a consequence of decreased proliferation or increased cell death, proliferation indices were determined, and as a measure of apoptosis, the expression of activated caspase-3 after only 3 days of PPP or PPP plus Erlotinib treatment, i.e. prior to a drastic decrease of the pale and squamous cell components was examined. It was observed that, while proliferation was still at control levels, the numbers of caspase-3-positive cells in the microacinar and non-glandular components were 2- and 9-fold higher, respectively, than in controls (
To evaluate IGF as a drug-target in human cells, the receptor in MDA-MB-231 mammary cancer cells, which possess a KrasG13D mutant gene (Kozma S C, et al., Nucleic Acids Res. (1987), 15:5963-5971; Hollestelle A, et al., Mol Cancer Res. (2007), 5:195-201) and share similarities in transcriptional profile with basal-like mammary tumor cell line (Charafe-Jauffret E, et al., Oncogene (2006), 25:2273-2284) was targeted. Pharmacological inhibition using PPP drastically reduced the in vitro viability of MDA-MB-231 cells (
A comparison between the mouse mammary tumors induced by K-ras* activation and human breast cancer is shown in
It has been shown that overexpression of oncogenic Kras* in mouse mammary glands leads to rapid development of histopathologically heterogeneous malignant tumors predominantly simulating human basal-like breast cancers, but also including a luminal type. Although the incidence of KRAS mutations in human breast cancer is not very high, it is still appreciable (˜7% in tumors and ˜13% in cancer cell lines; see Hollestelle A, et al., Mol. Cancer. Res. (2007), 5:195-201; Malaney S and Daly R J, J. Mammary Gland Biol. Neoplasia (2001), 6:101-113). Moreover, in ˜70% of primary breast cancers, the level of RAS is higher than that in normal tissue (Dati C, et al. Int. J. Cancer (1991). 47:833-838). Such elevated RAS activity is apparently required even for mammary carcinogenesis induced by RAS mutations (Sarkisian C J, et al. Nat. Cell Biol. (2007), 9:493-505). Clearly, regardless of cause (mutation and/or overexpression), the important element contributing to oncogenesis is the perturbation of the Ras pathway, which can be dissected genetically by mouse modeling. The diversity of cancerous forms and especially the rapidity of tumor manifestation increases further the utility of the mouse model that have been described here, which can be used advantageously after appropriate genetic testing for preclinical evaluation of treatment regimes, as exemplified by our results.
Seemingly, the single-step tumorigenesis that has been observed does not conform to the widely accepted multi-hit model of carcinogenesis (Vogelstein B and Kinzler K W, Trends Genet. (1993), 9:138-141; Hanahan D and Weinberg R A, Cell (2000), 100:57-70). However, from the standpoint that cancer is a disease of malfunctioning cell signaling, all cases of tumor development can be viewed, regardless of timing, as variants of a more general hypothesis positing that contributing “hits” correspond to recruitment and combinatorial engagement of deregulated pathways predominantly involved in apoptosis and growth control. Apparently, in the case of our model, highly overexpressed, constitutively active Kras* can elicit synergism of downstream pathways that are simultaneously deregulated to a degree sufficient for rapid development of invasive cancer. Analogous inverse reciprocity between Kras* expression levels and tumor latency in other mouse models has been observed other than the extremely rapid development of carcinomas of the skin and the oral mucosa induced by Kras* observed by others (Vitale-Cross L, et al., Cancer Res. (2004), 64:8804-8807). It remains to be seen whether, by exceeding normally affordable limits, oncogene overexpression overrides homeostatic capabilities and/or if the excessive deregulation that it causes permits novel and abnormal signaling interactions.
An additional open question is why the constitutively acting oncogenic Kras*, which has ceased to respond to upstream effectors in signaling relays and has presumably acquired autonomy in deregulating signaling, is not refractory to the silencing of Igf1r. Ras proteins control proliferation through the Raf→MEK→Erk pathway, but also interact directly with the p110 catalytic subunit of the PI3K complex, thus affecting anti-apoptosis. It is notable, in this regard, that loss-of-function missense mutations in the Ras binding domain of p110 inhibit almost completely Kras and Hras oncogenicity in mouse models of lung and skin tumors, respectively (Gupta S, et al. Cell (2007), 129:957-968). The mechanistic details in this case (involving a different tissue) are unclear. However, a testable hypothesis is that, without the crucial participation of Igf1r signaling that exerts both PI3K-dependent and PI3K-independent anti-apoptotic effects, the direct activation of the PI3K pathway by Kras* is, despite its overexpression, inadequate for attaining a level of anti-apoptosis able of promoting oncogenicity. It is noted that the results of a previous study (Cristofanelli et al. Oncogene (2000), 19:3245-3255) showing that 32D cells could become tumorigenic by the combined action of Hras and Irs1 (a downstream effector of Igf1r), but not by either one of these components acting alone, could also be interpreted as indicating a collaboration between the Erk and PI3K pathways. Perhaps, among other effects, Kras* triggers in this case the operation of a positive feedback loop that enhances its action through the upregulation of Igf1r expression which, in turn, could amplify proliferative and anti-apoptotic signaling by increasing the expression of Egf ligands. The downregulation of such ligands by genetic inactivation of Igf1r could explain why PPP is more effective than Erlotinib in the treatment of Kras*-induced tumors. Presumably, pharmacological inhibition of Egfr alone does not attenuate tumor growth sufficiently because the Egf ligands are still expressed at high levels and can function through other receptors of the family (the most likely candidates are Erbb2:Erbb3 heterodimers). In contrast, in addition to the direct pharmacological inhibition of Igf1r activity by PPP, there is an indirect effect on Egf ligand downregulation preventing the robust formation of homo or heterodimers between the Erbb receptor family members.
9. EXAMPLE 4 In Vivo TestingMice were generated that carried the Ef1/Kras* oncogenic mutation together with one or two alleles of the IGF1R locus conditionally knocked out (Xuan et al., J. Clin. Invest. 2002, 110, 1011-19) and the WAPcre transgene (which would activate the oncogene and ablate the IGF1R gene in the same cells during pregnancy/lactation). Parous mice were observed for the development of invasive tumors and compared the tumor latency with control females that carried only the oncogene and the WAPcre gene. In such mice, IGF1R inactivation has a profound effect on tumor latency, even in mice where only one of the two alleles is ablated.
To test the effect of IGF1RK inhibitors on tumor growth, parous Ef1/Kras*; WAPcre mice are monitored for tumor development every day upon parturition and examined with palpation for tumor development. Once tumors are readily palpable (1-2 mm diameter), the tumor size and body weight of the mice are documented. Mice are then treated with either vehicle or an IGF1RK inhibitor. Typically, mice are injected daily with 100-150 μl of a solution containing 90% DMSO and 10% of an emulsifier (Cremophor EL, Sigma). In this solution, for example, PPP is completely soluble at a concentration of 6 mg/ml.
After 21 days of treatment, the mice are sacrificed, weighed, and the initial tumors measured individually. In addition, tumors which were not palpable at the time of initial palpation, but had grown during the therapy period, are also isolated. In an experiment comparing vehicle treatment with PPP, the total tumor volume per mouse in the vehicle group was 653±219 mm3 (number of mice: 7, total mammary gland affected: 21). Mice treated with PPP showed a much slower tumor growth with a total tumor volume per mouse of 54±28 mm3 (p=0.05) at the end of the study.
10. EXAMPLE 5 10.1. Materials and Methods MiceKnock-in transgenic mice conditionally expressing oncogenic Kras were generated by standard procedures using targeted W9.5 ES cells. The targeting vector was prepared using the Eef1a1 cassette described in
Standard protocols were followed for Southern, Northern and Western analyses. The probes used were a 616 bp XbaI/SacII fragment immediately upstream from the 5′ arm (Southern) and the Kras* cDNA described above (Northern). The anti-Kras-specific antibody used for Western analysis was sc-521 (Santa Cruz Biotechnology). To examine the effects of dominant-negative IGF1R, plasmid 486Stop (D'Ambrosio C, et al., Cancer Res. (1996), 56:4013-4020; kindly provided by Dr. R. Baserga, Thomas Jefferson University, Philadelphia) or control pcDNA3 plasmid were introduced into MDA-MB-231 cells grown in DMEM supplemented with 10% fetal bovine serum by using a nucleofector (Amaxa Biosystems). RNA interference experiments with MDA-MB-231 cells were performed as described (Rochester M A, et al., Cancer Gene Ther. (2005), 12:90-100) using the siRNA duplex R4 [sense strand 5′CAAUGAGUACAACUACCGCTT3′ (SEQ ID NO: 1); antisense strand 5′GCGGUAGUUGUACUCAUUGTT3′ (SEQ ID NO: 2)], which targets the human IGF1R mRNA at nucleotides 639-657, and a scrambled control duplex [Scr4; sense strand 5′GUCACACCGAUAAGUCACATT3′ (SEQ ID NO: 3); antisense strand 5′UGUGACUUAUCGGUGUGACTT3′ (SEQ ID NO: 4)].
Histological Analysis, Immunophenotyping, Flow Cytometry and FISHMouse tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The primary antibodies and the conditions used for immunohistochemistry are listed in Table 9. Semiquantitative scoring (− to +++) was according to the system used for HercepTest™ (Dako), with − representing no staining, + weak staining in >15% of the cells, ++ strong focal staining in <15-20% of the cells, and +++ strong staining in >15-20% of the cells. Immunofluorescence was performed on paraffin sections by standard procedures. For flow cytometry, single cell suspensions from mammary glands and tumors prepared as described (Stingl J, et al. Nature (2006), 439: 993-997) were processed using the EasySep kit (StemCell Technologies) and analyzed on a FACS Aria instrument (Becton Dickinson). For fluorescence in situ hybridization analysis we used three BAC clones (Invitrogen; RP11-157L06, RP11-583L24 and RP11-707G18) spanning the human chromosome 12p12.1 region containing the KRAS locus. BAC DNA was labeled by nick-translation using spectrum red dUTP fluorochrome (Abbott Molecular). A Spectrum green-labeled centromeric 12 (CEP 12) probe (Abbott Molecular) was used to enumerate chromosome 12. FISH was performed by standard methods and hybridization signals were scored on at least 100 interphase nuclei on DAPI-stained slides. The human breast cancer specimens analyzed histologically or by FISH were obtained from Surgical Pathology files of the College of Physicians, Columbia University, following approval of the Institutional Review Board.
Microarray AnalysisFor expression profiling, total RNA was extracted from mammary tumors of Kras*/WAP-cre bitransgenic or age-matched postinvolutional female mice. The samples were profiled individually. For each assay, 5-8 μg of total RNA were used to generate biotinylated cRNA that was fragmented (15 μg) and then hybridized to Mouse Genome 430 2.0 Array DNA Chips (Affymetrix). The microarrays were scanned (Affymetrix Scanner) and expression values for the genes were determined using Affymetrix GeneChip Operating Software 1.2. Filtering and further analysis using the raw data was performed with GeneSpring 6.0 software. A ratio of ≧2 (experimental/control for “upregulated and control/experimental for “downregulated” entries) was used as a widely accepted cut-off point indicative of a significant difference in transcript levels. An unsupervised clustering dendrogram was generated by using Cygwin software. Literature data compared with our results (see Table 5) were filtered to select and use only markers represented in the Affymetrix 430 2.0 chip, which also corresponded to known genes (unknown ESTs were ignored) and were at least 2-fold upregulated or downregulated in comparison with corresponding controls. For comparative identification of signaling molecules (see Tables 6 and Table 7), key-members of all major signaling pathways were listed in Table 8) and they were selected in a non-redundant fashion from the following databases: KEGG (www.genome.ad.jp/kegg/kegg2.html), BioCarta (cgap.nci.nih.gov/Pathways/BioCarta_Pathways), Cancer Cell Map (cancer.cellmap.org/cellmap), Pathway-Express (vortex.cs.wayne.edu/ontoexpress) and Wikipathways (www.wikipathways.org).
Statistical AnalysisValues were expressed as mean±S.E.M. Results were considered as statistically significant if P<0.05. For analysis of survival curves, a log-rank test was used. Differences between groups were examined using Student's t test. The null hypothesis that there is no marker difference in pairwise comparisons between microarray datasets was evaluated using Fisher's exact test.
10.2. Results and DiscussionExample 3 has shown that the Kras*-induced murine mammary carcinomas are histopathologically heterogeneous and include, in addition to three basal-like components, a luminal type. On the basis of morphological and molecular criteria, an advanced hypothesis is that the cells-of-origin of the basal-like carcinomas are epithelial progenitors. This is consistent with observations indicating that populations of flow-sorted CD24+/CD29hi or CD24+/CD49fhi cells (depleted of hematopoietic contaminants; Lin), which were enriched in mammary gland-forming stem cells defined operationally by cleared fat pad transplantation assays (Shackleton M, et al., Nature (2006), 439:84-88; Stingl J, et al., Nature (2006), 439: 993-997), exhibit phenotypic similarities with basal-like tumors (Asselin-Labat M L, et al., J. Natl. Cancer Inst. (2006), 98:1011-1014). Interestingly, comparative FACS analysis of cell suspensions isolated from Kras*-induced tumors (n=3) and normal mammary glands of age-matched parous females (n=5) showed an increase in the number of CD24+/CD49fhi neoplastic cells (
Since the exact stages in the developmental progression from stem cells to differentiated luminal and myoepithelial cells remain largely undefined, a likely scenario accounting for the Kras* tumor heterogeneity in conjunction with the hypothesis described above, is parallel transformation of three different cell types. There are three reasons for such a scenario. First, the originator of the Dunn A/B tumors is a fully committed luminal-type cell that can still respond to oncogenic stimuli without becoming apoptotic. Second, the Wap gene, and therefore cre, is expressed in bipotent alveolar progenitor cells of virgin and parous mice (Booth B W, et al., J. Cell Physiol. (2007), 212:729-736; Matulka L A, et al., Dev. Biol. (2007), 303:29-44), and also that a significant expansion of the Cd24+/Cd49high compartment containing mammary bipotent progenitors/stem cells (Shackleton M, et al., Nature (2006), 439:84-88) occurs in Kras* mice, it is speculated that the squamous cell carcinoma probably originates from such cell precursors. Squamous metaplasia has been widely reported in mouse models of breast cancer, including MMTV-Wnt mice, in which progenitor cells are thought to be the target of oncogenic transformation (Li Y, et al., Proc. Natl. Acad. Sci. USA (2003), 100:15853-15858). In the context of this scheme, to account for Gata3 expression and the presence of a distinct CIS, it is proposed that a third cell of-origin, also bearing the CD24 and CD49f markers, gives rise to pale cell carcinoma.
11. EXAMPLE 6 Efficacy of Compounds 18 and 21 to Ameliorate Kras*-Induced Mammary CarcinomasThe efficacy of compounds 18 “(NB3)” and 21 “(NB6)” to ameliorate Kras*-induced mouse mammary carcinomas was tested. While testing NB3 and NB6 (4 mice each), 2 controls were added (2 tumor-bearing mice treated with vehicle). The control data were pooled with the previous, and the new results are listed in Table 10:
If, on the basis of the new control data, the previous “% tumor volume in comparison with the control” for PPP (7.3%) is normalized, it becomes 6.5%, i.e. NB3 has the same potency as PPP. Importantly, however, NB6 (compound 21) appears to be 7 times more potent.
While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention.
Various publications are cited above, the contents of which are hereby incorporated by reference in their entireties.
Claims
1. A compound of formula I: wherein X, X′, Y, Y′ and Z are independently H, OCH3, OCH2CH3, O-i-CH2CH2CH3, O-n-CH2CH2CH3, SCH3, SCH2CH3, S-i-CH2CH2CH3, S-n-CH2CH2CH3, S(O)CH3, S(O)CH2CH3, S(O)-i-CH2CH2CH3, S(O)-n-CH2CH2CH3, S(O)2CH3, S(O)2CH2CH3, S(O)2-i-CH2CH2CH3, S(O)2-n-CH2CH2CH3, CN, SCN, OCN, N3, halogen, CH3, CH2CH3, i-CH2CH2CH3, n-CH2CH2CH3, CF3, CCH, CCCH3, CH═CH2, Ph, NH2, NHCH3, N(CH3)2, NHCH2CH3, N(CH2CH3)2, NO, NO2, CHO, COCH3, COCH2CH3, CO2H, CO2CH3, CO2CH2CH3, OC(O)H, OC(O)CH3, OSO3H, OSO3CH3, OSO3CH2CH3, OPO3H2, OPO3HCH3, OPO3HCH2CH3, OPO3(CH3)2, OPO3(CH2CH3)2, P(O)(OH)2, P(O)(OCH3)(OH), P(O)(OCH2CH3)(OH), P(O)(OCH3)2, or P(O)(OCH2CH3)2 with the proviso that when X and X′ are H, Y, Y′, and Z cannot all be methoxy and that when X′ is H, and Y, Y′, and Z are methoxy, X cannot be a halogen.
2. The compound of claim 1 wherein X, X′, Y, Y′ and Z are independently H, OCH3, OCH2CH3, SCH3, SCH2CH3, S(O)CH3, S(O)CH2CH3, S(O)2CH3, CN, halogen, CH3, CH2CH3, CF3, N(CH3)2, NHCH2CH3, N(CH2CH3)2, NO, NO2, CHO, CO2CH3, or CO2CH2CH3.
3. The compound of claim 1 wherein X, X′, Y, Y′ and Z are independently H, OCH3, OCH2CH3, CN, halogen, CH3, CH2CH3, CF3, CHO, CO2CH3, or CO2CH2CH3.
4. The compound of claim 1 wherein X, X′, Y, Y′ and Z are independently H, CH3, OCH3, CN, halogen, CF3, or SCH3.
5. The compound of claim 1 wherein X, X′, Y, Y′ and Z are independently H, OCH3, OCH2CH3, SCH3, SCH2CH3, S(O)CH3, S(O)CH2CH3, S(O)2CH3, CN, CH3, CH2CH3, CF3, N(CH3)2, NHCH2CH3, N(CH2CH3)2, NO, NO2, CHO, CO2CH3, or CO2CH2CH3.
6. The compound of claim 1 wherein X, X′, Y, Y′ and Z are independently H, OCH3, OCH2CH3, CN, CH3, CH2CH3, CF3, CHO, CO2CH3, or CO2CH2CH3.
7. The compound of claim 1 wherein X, X′, Y, Y′ and Z are independently H, CH3, OCH3, CN, CF3, or SCH3.
8. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier thereof.
9. A compound of formula II: wherein X, X′, Y, Y′ and Z are independently hydrogen, a C1-C8 alkyl, or C1-C8 alkoxy group with the proviso that when X and X′ are H, Y, Y′, and Z cannot all be OCH3; and
- R1 may be oxo, —OH, —OCH3, —OC2H5, —OC3H7, —OC4H9, —OCH2CH═CH2, —OCH2Ph, —OCH2CH2NH2, —OCOH, —OCOCH3, —OCH2OH, —OC2H4OH, —OC3H6OH, —OC4H8OH, or a glycoside.
10. The compound of claim 9 wherein X, X′, Y, Y′ and Z are hydrogen, methyl, or methoxy.
11. The compound of claim 9 wherein
- X, X′, Y, Y′═H, and Z═OCH3;
- X, X′, Y′, Z═H, and Y═OCH3;
- X, X′, Y′═H, and Y, Z═OCH3;
- X, X′, Z═H, and Y, Y′═OCH3; or
- X, X′, Y, Y′═H, and Z═CH3.
12. A pharmaceutical composition comprising a compound of claim 9 and a pharmaceutically acceptable carrier thereof.
13. A method of preparing a compound of claim 1 comprising the steps of: cyclizing a compound of formula (a) to form isobenzofuran (b), and reacting (b) in situ, via a Diels-Alder reaction to form Diels-Alder adduct (c) selecting hydrogenating Diels-Alder adduct (c), reducing to a diol and acetylating to form meso diacetate (d) and desymmetrizing (d) with porcine pancreatic lipase (PPL) to produce (e), subjecting (e) to silylation, deacetylation, and oxidation to produce a compound of formula (f) wherein R9 is a silyl protecting group such as a triisopropylsilyl-protecting group, converting the compound of formula (f) to a compound of formula (g) with a complementary silyl protecting group by retro-Michael ring opening and protection of the C4OH under neutral to basic conditions, followed by aldehyde oxidation, subjecting the compound of formula f to aldehyde oxidation under Lindgren conditions to form a compound of formula (g), wherein R10 is a C4 protecting group such as a triethylsilyl-protecting group, converting the compound of formula (g) to a compound of formula (h) by transformation of the carboxylic acid into an acyl oxazolidinone functionality using carboxyl activation and then condensation with a metalated oxazolidinone, introducing aromatic ring R to the compound of formula (h) to form a compound of formula (I) by CuI-mediated conjugate addition of RMgBr at a temperature of −10 to 10° C., converting the compound of formula (I) to a compound of formula (j) by desilylative lactonization by heating with a fluoride source and cyclization to produce the corresponding lactone
14. The compound of claim 1 wherein the halogen is selected from fluorine, chlorine, bromine, and iodine.
15. The compound of claim 1 wherein the halogen is selected from fluorine and iodine.
16. The compound of claim 1 wherein the halogen is fluorine.
17. The compound of claim 2 wherein the halogen is selected from fluorine, chlorine, bromine, and iodine.
18. The compound of claim 2 wherein the halogen is selected from fluorine and iodine.
19. The compound of claim 2 wherein the halogen is fluorine.
Type: Application
Filed: Jul 10, 2014
Publication Date: Jan 29, 2015
Inventors: David B. Berkowitz (Lincoln, NE), Sylvain Broussy (Lincoln, NE)
Application Number: 14/327,617
International Classification: C07D 493/04 (20060101);
