WO2016022956A1

WO2016022956A1 - Asymmetric bisaminoquinolines and bisaminoquinolines with varied linkers as autophagy inhibitors for cancer and other therapy

Info

Publication number: WO2016022956A1
Application number: PCT/US2015/044282
Authority: WO
Inventors: Ravi K. AMARAVADI; Jeffrey Winkler
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2014-08-08
Filing date: 2015-08-07
Publication date: 2016-02-11
Also published as: EP3848355A1; ES2881048T3; EP3177595A4; US20220064118A1; EP3177595B1; EP3177595A1; US20190248747A1; US11001558B2; US10221140B2; US11639335B2; US20170275252A1

Abstract

The invention provides novel asymmetric and symmetric bisaminoquinolmes and related compounds, methods of treatment and syntheses. The novel compounds exhibit effective anticancer activity and are useful in the treatment of a variety of autophagy-related disorders.

Description

Asymmetric Bisaminoquinolines and Bisaminoquinolines with Varied Linkers as Autophagy Inhibitors for Cancer and Other Therapy

RELATED APPLICATIONS AND GOVERNMENT INTEREST

This application claims the benefit of priority of United States provisional application serial number US62/034,897, filed August 8, 2014, of identical title, the entire contents of which are incorporated by reference herein.

The invention in this patent application was made with government support under Grant No. P01-CA114046 awarded by the National Cancer Institute. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention provides novel asymmetric and symmetric bisaminoquinolines and related compounds, methods of treatment and syntheses. The novel compounds exhibit unexpected anticancer activity and are useful in the treatment of a variety of autophagy- related disorders.

BACKGROUND AND DESCRIPTION OF THE INVENTION

Autophagy consists of the sequestration of organelles and proteins in autophagic vesicles (AV) and degradation of this cargo through lysosomal fusion (1). Autophagy allows tumor cells to survive metabolic and therapeutic stresses (2-5). Multiple publications indicate therapy-induced autophagy is a key resistance mechanism to many anti-cancer agents.

Chloroquine (CQ) derivatives block autophagy by inhibiting the lysosome (3,6,7). Based on these findings, clinical trials combining cancer therapies with hydroxychloroquine (HCQ), (which is safer than CQ to dose escalate) have been launched. Preliminary results indicate these combinations have activity (8-13), but it is still unclear if this activity is consistently due to the addition of HCQ. High micromolar concentrations of HCQ are required to inhibit autophagy. While there is some pharmacodynamic evidence of autophagy inhibition with HCQ in cancer patients, it is inconsistent because adequate concentrations are not achieved in all patients. There is an unmet need to develop more potent inhibitors of autophagy. The design and synthesis of dimeric analogs of CQ, that exploit the thermodynamic advantages imparted by polyvalency (14, 15), has been a subject of intensive study for over 10 years (16- 18). An early report by Vennerstrom (17) described the synthesis of heteroalkane-bridged bisquinolines as potential antimalarials, but none of the compounds had sufficient

antimalarial activity to warrant further investigation. Subsequently, Sergheraert (16) reported that tetraquinolines, i.e., dimers of bisquinolines, afforded potent antimalarials, confirming the possibility that the application of the polyvalency strategy could afford increased potency, at least with respect to antimalarial activity.

More recently, Lee (19) has described the potentiation of AKT inhibitors by fluorinated quinoline analogs. Solomon (20) has reported the preparation of "repositioned" chloroquine dimers, based on the use of a piperazine connector. These results suggest that these chloroquine analogs could serve as bases for the development of a new group of effective cancer chemotherapeutics. We have examined the application of the strategy of polyvalency (14, 15) to the synthesis of novel autophagy inhibitors by preparing a dimeric chloroquine (LysOl, Figure 11 or 12), from commercially available materials. We have recently reported a series of BAIs that potently inhibit autophagy and impair tumor growth in vivo (21). The structural motifs that are necessary for improved autophagy inhibition compared to CQ include the presence of two aminoquinoline rings and a triamine linker, as shown in the lead compound, 1 (Lys 01) which is a 10-fold more potent autophagy inhibitor than HCQ. Compared to HCQ, Lys 05, a water soluble salt of LysOl, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice developed Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1{22), providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single agent antitumor activity is observed without toxicity in mice bearing xenograft tumors treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer. However, while Lys05 is 10-fold more potent than HCQ in in vitro autophagy assays, it is cytotoxic only at micromolar concentrations in most cancer cells.

In the present invention, we demonstrate the preparation and the unexpected biological activity of asymmetric bisaminoquinolines and related compounds via changing the linker length and/or disrupting the symmetry of the previously employed linkers. We describe unexpected increase in anti-cancer properties and capacity for autophagy inhibition of bivalent aminoquinolines when linker length is changed substantially and/or asymmetrically from LysOl .

SUMMARY OF THE INVENTION

Herein we demonstrate the preparation and the unexpected biological activity of asymmetric bis-4-aminoquinolines and related compounds by changing the linker. We describe the unexpected increase in anti-cancer properties of bivalent 4-aminoquinolines when linker length is changed substantially and asymmetrically from the parent LysOl scaffold. IA:

IA

wherein R¹ and R¹ are each independently H, F, CI, Br, I, CN, N0₂, optionally substituted Ci-C₆ alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups, preferably a CF₃ group), optionally substituted 0-Ci-C₆ alkyl (preferably OCH₃), optionally substituted C₂-C₇ acyl (preferably acetyl), optionally substituted -(NH)- acyl, or optionally substituted C₂-C₇ ester (oxycarbonyl ester or carboxyester, preferably carboxyester) (each of the aforementioned groups, when substituted, is preferably substituted with one or two hydroxyl groups, one two or three halo groups or a methyl group)ss;

R and R' are each independently H, a Q-Q optionally substituted alkyl group, a CrC₇ (preferably C₂-C₇) optionally substituted acyl group , a C₂-C₇ optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

R" is H, an amine protecting group (preferably benzoyl or benzyl), Cy¹ or (C=0)_z-G, where Cy¹ is an optionally substituted cycloalkyl, aryl or heteroaryl group, G is an optionally substituted C₀-C₁₂ (preferably Ci-C₈, often C]-C₃ alkyl) alkyl, alkene or alkynyl group (wherein optional substiuents include a Cj-Ci₂ (preferably C]-Cg₎ alkyl, alkene or alkynyl group substituted by (N— R^J)-( Ci-C₈ alkyl, alkene or alkynyl group)_z-(Cy²)_x, where R^J is H or a Ci-C₈ alkyl, alkene or alkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is an optionally substituted aryl or heteroaryl group (most preferably benzyl or quinoline);

R^M is independently at each occurrence H, F, CI, Br, I, an optionally substituted -C^ (preferably Q-Cg) alkyl, alkene, alkynyl or alkoxy group (preferably including a CF₃ group); Each p in linker group L^1A is independently 1-10 (often 1, 2, 3, 4, 5, 6, 7 or 8 or more often 1, 2 or 3) and optionally (preferably) each p in a molecule is different;

and the pharmaceutically acceptable salts, enantiomers, diastereomers, solvates and polymorphs thereof, wherein the length of designated linker group L^1A may vary, e.g. as illustrated in the compounds exemplified herein.

In another preferred embodiment, the invention provides a compound of Formula IB:

IB

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof, wherein the substituents are the same as defined for Formula IA; and p and p' in linker group L are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, often 1 , 2, 3, 4, 5, or 6 and preferably, at least one of p or p' is different.

In another preferred embodiment, the invention provides a compound of Formula IC:

IC

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof, wherein the substituents are the same as defined for Formula IA above, and each p in linker group L is independently 1-10 (often 1, 2, 3, 4, 5, 6, 7 or 8 or more often 1, 2 or 3) and optionally (preferably) each p in a molecule is different

In another referred embodiment, the invention provides a compound of Formula ID:

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof, wherein the substituents are the same as defined for Formula IA above and linker group L^1D is an optionally substituted alkylene group containing from 1-20, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 methylene groups, wherein one or more methylene groups in linker group L is preferably substituted with one or two hydroxyl groups, one or two halo groups or one or two Ci-C₃ alkyl groups.

In still another embodiment, the present invention relates to compounds according to the chemical structure IE:

IE

Wherein R¹ and R¹ are each independently H, halo (F, CI, Br or I), CN, N0₂, optionally substituted Ci-C₆ alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally substituted O-Q-Ce alkyl (preferably, OCH₃), optionally substituted C2-C7 acyl (preferably acetyl), optionally substituted -(NH)-acyl, or optionally substituted C₂-C₇ ester (oxycarbonyl ester or carboxyester, preferably carboxyester);

R^M is independently at each occurrence H, F, CI, Br, I, an optionally substituted Q-C12 (preferably Ci-Cg) alkyl, alkene, alkynyl or alkoxy group (preferably including a CF₃ group); R and R' are each independently H, a Ci-C₆ optionally substituted alkyl group, a C1-G7

(preferably C₂-C₇) optionally substituted acyl group, a C₂-C₇ optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

Linker group L^1E is a '

group (either A or A' may be bonded to either of the two amine groups in compound I) wherein at least one of the CH₂ groups in L is optionally substituted with an optionally substituted Q-Q alkyl group including an optionally substituted cyclic alkyl group which, when substituted, are preferably substituted with one or two hydroxyl groups, one two or three halo groups (preferably CI or F) or one or two C!-C₃ alkyl groups (preferably methyl), or at least two methylene groups in linker group L (preferably two adjacent methylene groups) are substituted with alkylene groups to form an optionally substituted C₃-C₈

(preferably a C₅-C₆) cycloalkyl group (ring), which itself, when substituted, is preferably substituted with one or two hydroxyl groups, one two or three halo groups (preferably F or CI) or one or two Q-C₃ alkyl groups (preferably methyl), or linker group L is a

(poly)ethylene glycol group having from 1 to 20 (preferably 2 to 15, 3 to 10 or 4, 5, 6, 7, 8, 9, or 10) ethylene glycol units, each (poly)ethylene glycol group being optionally substituted at one or both of its distal ends with an A group, or linker group L is a linear group containing from 1 to 5 non-contiguous amine groups N-R", each amine group being separated from an adjacent amine group by an alkylene group containing from 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2, 3, 4, 5 or 6) methylene groups or an ethylene glycol containing group having from 1 to 6 (preferably 2 to 6, 3 to 6 or 4, 5 or 6) ethylene glycol units;

X is absent, (CH₂)j, O, S or N-R";

Y is absent, CH₂, O, CH₂0 or N-R" and Y' is absent CH₂, O, OCH₂ or N-R", with the proviso that when one or more of X, Y and Y' is present, each of X and Y, X and Y' or Y and Y', when present, forms a bond;

R" is H or an optionally substituted Ci-C₆ (preferably C[-C₃) alkyl group or an amine protecting group (preferably H, Me, benzoyl or benzyl);

each j is independently 1, 2, 3, 4, 5 or 6 (preferably 1 or 2);

each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 with the proviso that when n is 0, X is (C¾)_j where j is at least 1 and at least one CH₂ group is optionally substituted, preferably with a C!-C3 alkyl group which itself is optionally substituted with one or two hydroxyl groups or one two or three halo groups;

A is absent, C=0 or (CH₂)_j and A' is C=0 or (CH₂)_j wherein at least one CH₂ group in A or A' is optionally substituted, preferably with a C]-C₃ alkyl group which is itself optionally substituted, preferably with one or two hydroxyl groups or one, two or three halo groups; Z is O or N-R^z;

R^z is H or an optionally substituted C1-C3 alkyl group,

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvent or polymorph thereof.

In certain preferred embodiments of the invention, at least one (preferably two) R^M is other than H, especially when R¹ and R¹ are H. In other preferred aspects of the invention, R¹ and R¹ are each independently H, a halo group (most often CI or F), a nitro group or a trifluoromethyl group, most often a chloro or fluoro group. R and R' are preferably each independently H or methyl, preferably H and L is preferably an alkylene group containing from 6 to 20 methylene groups (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19 or 20), a (poly)ethylene glycol group having from 1 to 20 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 2 to 15, 3 to 10 or 4, 5, 6, 7, 8, 9, or 10) ethylene glycol units, each (poly)ethylene glycol group being optionally substituted at one or both of its distal ends with an A group, or L is a linear group containing from 1 to 5 non-contiguous amine groups N-R", each amine group being separated from an adjacent amine group by an alkylene group containing from 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2, 3 or 4) methylene groups or an ethylene glycol containing group having from 1 to 6 (preferably 2 to 6, 3 to 6 or 4, 5 or 6) ethylene glycol units. R" is preferably H, methyl or an amine protecting group (preferably a benzyl or benzoyl group which can be removed to introduce other groups on the amine group after deprotection).

In certain embodiments, one or more amine group of L is optionally substituted with one or two 7-substituted-4-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group through an alkylene group and the 7-position of each quinolinyl group is optionally substituted, preferably with a R¹ and/or R¹ group as broadly described for generic structure I above, or one or more amines of L is substituted with an alkyl group which itself is further optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, a monoalkyl (CrC₆, preferably C_rC₃) amine or a dialkyl (C]-C₆, preferably CrC₃) amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with R¹ and/or R¹ as broadly described for generic structure I above, and each of said alkoxy groups (e.g. methoxy or ethoxy) is optionally further substituted with a C₁-C3 alkosssxy group, preferably a methoxy group, thus forming a diether substituent.

In certain embodiments of the invention, R¹ and R¹ are each independently H, a halo group, a nitro group or a trifluoromethyl group, preferably a chloro or fluoro group. R and R' are preferably each independently H or a C C₃ optionally substituted alkyl group itself preferably substituted with at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine (where one or both alkyl groups is itself further optionally substituted with a dialkyl amine or an amine substituted with one or two (preferably one) 7-substituted-4- quinolinyl group(s) where the amine group is bonded to the 4-position of the quinolinyl group), an alkoxy group (e.g. methoxy or ethoxy) which may be further substituted with an alkoxy group, preferably a methoxy group (thus forming a diether substituent).

In a particularly preferred embodiment, the invention provides compounds of the formulae IIA, IIB, IIC and IID, and the pharmaceutically acceptable salts, enantiomers, diastereomers, solvates or polymorphs thereof:

Lys-21: X = C1 where X is a halogen (preferably F or CI), or

a pharmaceutically acceptable salt thereof;

IIB:

Lys-25: X = CI; where X is a halogen (preferably F or CI), or

a pharmaceutically acceptable salt thereof;

IIC:

Lys-26: X = CI where X is a halogen (preferably F or CI), or

a pharmaceutically acceptable salt thereof; and

Lys- 74: X=C1 where X is a halogen (preferably F or CI), or

a pharmaceutically acceptable salt thereof. Further preferred compounds according to the present invention include those which are presented or identified in any of figures 10-13 or the various schemes as presented herein.

In other embodiments, the invention provides methods of treatment and

pharmaceutical compositions which use therapeutically-effective amounts of compounds of Formulae IA, IB, IC, ID, IE, IIA, IIB and IIC, the other compounds described herein (e.g. the compounds of Tables 1-2 and Figures 10-13) and the pharmaceutically acceptable salts, enanfiomers, diastereomers, solvents or polymorphs thereof, to treat cancer and other autophagy-related disorders.

As described further hereinafter, compounds of the invention exhibit an unexpectedly high level of cytotoxicity in a variety of cancer cells and evidence effective autophagy inhibition at surprising low doses, indicating their effectiveness in the treatment of broad spectrum of autophagy-related disorders.

These and other aspects of the invention are illustrated further in the Detailed

Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the cytotoxicity of dimeric CQs with longer linkers. (A) IC50 of selected LysOl derivatives (complete dataset in table 1) with longer linkers in a 72 hour alamar blue viability assay. A375 P cells seeded in a 384 well format were treated with a 10 point dilution of concentrations (0.01 nM-lOuM) (B) Colony formation assay in soft agar. Cells from the indicated melanoma (A375, WM983B, SKMEL5) and pancreatic cancer (PANCl, CAPANl) cell lines were plated in soft agar. Medium with compound was changed every 48 hours. Crystal violet staining was used to visualize formed colonies after 2 weeks.

Figure 2 shows the effects of assymetry and longer linker length on the cytotoxicity of dimeric chloroquines. A375P melanoma cells were plated in a 384 well format and treated with increasing concentrations of LysOl derivatives. After 72 hours the cells were analyzed by alamar blue. Complete data is in Table 1, Figure 10. (A) IC50 according to symmetry of linker (B) IC50 by linker length.

Figure 3 shows subcellular localization of dimeric chloroquines with varied linkers. (A) Lys21 was tagged with a fluorescent dye Cy3 (B) A375P melanoma cells stained with lysotracker (light grey), and co-treated with Lys21-Cy3. Lighter color in third frame indicates colocalization.

Figure 4 shows autophagy modulation of dimeric chloroquines with longer linkers in melanoma cell lines. LC3B and actin immunoblotting in 3 different melanoma cell lines. Each compound was administered at 1 uM concentrations for 24 hours. Quantification ofLC3II/actin indicates all dimeric CQs with longer linker produced higher levels of

LC3II/actin compared with Lys05 (dotted line).

Figure 5 shows high throughput phenotypic screen of autophagy inhibition for LysOl derivatives. A375PmCherry-eGFP-LC3 cells were seeded in 384 well plates. Doxorubicin, HCQ , CQ and Lys01-Lys27 (in 0.1% DMSO) were administered through robotic assisted pin tool transfer at concentrations of 0-1 ΟμΜ. Images were obtained by the Operetta high- content fluorescent microscopy. Image analysis using Harmony software reports fraction of nucleated cells with ratio of > 3.5 in cells treated with 10 μΜ compound.

Figure 6 shows high throughput fluorescence microscopy images of A375P mCherry-eGFP- LC3 melanoma cells treated with indicated chloroquine derivatives.

Figure 7 shows histograms of green/red ratio in LysOl -derivative treated A375PmCherry- GFP-LC3 cells.

Figure 8 shows flow cytometry quantification of reactive oxygen species (ROS), autophagy inhibition and apoptosis associated with dimeric CQs with longer linkers. (A) ROS reflected by DHR_123 fluorescence in A375 cells after 24 hours of treatment with luM compound (B) Autophagy inhibition reflected by green/red ratio of A375 mCherry-egfpLC3 cells treated with luM indicated compound for 24 hours.(C) Apoptosis reflected by annexin V detection in A375 cells treated with luM compound at the 24 hours. (D) Apoptosis reflected by annexin V detection in A375 cells treated with indicatd compounds at 3 uM for 24 or 48 hours. PLX: PLX4720; TRA: trametinib. Figure 9 shows the effects of dimeric CQ's with longer linker on growth and invasion in a 3D tissue like culture model. A375P mCherry eGFP-LC3 cells grown into 3 dimensional spheroids and implanted in collagen. After 72 hours of treatment with 3uM of indicated compound, cells were counterstained with DAPI. (A) fluorescence microscopy was used to visualize autophagy inhibition (bright areas) , inhibition of invasion into the collagen and (B) quantification of the DAPI fluorescence indicative of cell death. * p<0.05

Figure 10, Table 1 shows the results of cytotoxicity experiments using compounds according to the present invention. LN229 glioma cells, and A375P melanoma cells were plated in a 384 well format and compounds CQ, HCQ, and Lys01-Lys41 and 72-75 (IUPAC names and chemical structures provided in Figures 11 and 12) were delivered in concentrations between 0.01-10 micromolar using robotic assisted dispenser. After 72 hours incubation at 37 degrees, Alamar blue was applied and viability was determined using absorbance. Absorbance was normalized to DMSO control, and a log IC50 was estimated using Graphpad Prism software..

Figure 11, Table 2 provides the IUPAC names for the compounds which are set forth in attached figure 12.

Figure 12 shows representative chemical structures of compounds according to the present invention.

Figure 13 shows the chemical structures of a number of novel bisaminoquinolines according to the present invention with various halo-substituents (F, CI, Br) on the 7-position of the quinoline moiety.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification to describe the present invention. Where a term is not specifically defined herein, that term shall be understood to be used in a manner consistent with its use by those of ordinary skill in the art. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges that may independently be included in the smaller ranges are also

encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. In instances where a substituent is a possibility in one or more Markush groups, it is understood that only those substituents which form stable bonds are to be used.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those

described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set out below.

The term "patient" or "subject" is used throughout the specification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a human patient of either or both genders.

The term "effective" is used herein, unless otherwise indicated, to describe an amount of a compound or component which, when used within the context of its use, produces or effects an intended result, whether that result relates to the prophylaxis and/or therapy of an infection and/or disease state or as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms (including the term "therapeutically effective") which are otherwise described or used in the present application.

The term "compound" is used herein to describe any specific compound or bioactive agent disclosed herein, including any and all stereoisomers (including diasteromers), individual optical isomers (enantiomers) or racemic mixtures, pharmaceutically acceptable salts and prodrug forms. The term compound herein refers to stable compounds. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein. It is understood that the choice of substituents or bonds within a Markush or other group of substituents or bonds is provided to form a stable compound from those choices within that Markush or other group. In compounds according to the present invention, R¹ and R^M or R¹ and R^M do not together form a cyclic ring.

The term "bioactive agent" refers to any biologically active compound or drug which may be formulated for use in the present invention. Exemplary bioactive agents include the compounds according to the present invention which are used to inhibit autophagy and to treat cancer as well as other compounds or agents which are otherwise described herein.

The terms "treat", "treating", and "treatment", are used synonymously to refer to any action providing a benefit to a patient at risk for or afflicted with a disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc.

Treatment, as used herein, encompasses both prophylactic and therapeutic treatment, principally of cancer. Compounds according to the present invention can, for example, be administered prophylactically to a mammal in advance of the occurrence of disease to reduce the likelihood of that disease. Prophylactic administration is effective to reduce or decrease the likelihood of the subsequent occurrence of disease in the mammal, or decrease the severity of disease that subsequently occurs, especially including metastasis of cancer.

Alternatively, compounds according to the present invention can, for example, be

administered therapeutically to a mammal that is already afflicted by disease. In one embodiment of therapeutic administration, administration of the present compounds is effective to eliminate the disease and produce a remission or substantially eliminate the likelihood of metastasis of a cancer. Administration of the compounds according to the present invention is effective to decrease the severity of the disease or lengthen the lifespan of the mammal so afflicted, in the case of cancer.

The term "pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

The term "inhibit" as used herein refers to the partial or complete elimination of a potential effect, while inhibitors are compounds that have the ability to inhibit.

The term "prevention" when used in context shall mean "reducing the likelihood" or preventing a disease, condition or disease state from occurring as a consequence of administration or concurrent administration of one or more compounds or compositions according to the present invention, alone or in combination with another agent. It is noted that prophylaxis will rarely be 100% effective; consequently the terms prevention and reducing the likelihood are used to denote the fact that within a given population of patients or subjects, administration with compounds according to the present invention will reduce the likelihood or inhibit a particular condition or disease state (in particular, the worsening of a disease state such as the growth or metastasis of cancer) or other accepted indicators of disease progression from occurring.

The term "autophagy" or "autophagocytosis" is used to describe a catabolic process in cells which involves the degradation of a cell's own components through lysosomes.

Autophagy is a highly regulated process of biological systems that plays a normal part in cell growth development and homeostasis.helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which a cell allocates nutrients from unnecessary processes to more-essential processes.

A number of autophagic processes occur in nature, all of which have the degradation of intracellular components via the lysosome as a common feature. A well-known mechanism of autophagy involves the formation of a membrane around a targeted region of a cell, separating the contents from the rest of the cytoplasm. The resultant vesicle then fuses with a lysosome which subsequently degrades the contents.

An "autophagy-related disorder" includes diseases, disease states and/or conditions which benefit from the inhibition of autophagy, including, but not limited to, cancer

(including the metastasis of cancer), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

The term "cancer" shall refer to a proliferation of tumor cells having the unique trait of loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis. As used herein, neoplasms include, without limitation, morphological irregularities in cells in tissue of a subject or host, as well as pathologic proliferation of cells in tissue of a subject, as compared with normal proliferation in the same type of tissue. Additionally, neoplasms include benign tumors and malignant tumors (e.g., colon tumors) that are either invasive or noninvasive. Malignant neoplasms are distinguished from benign neoplasms in that the former show a greater degree of dysplasia, or loss of differentiation and orientation of cells, and have the properties of invasion and metastasis. The term cancer also within context, includes drug resistant cancers, including multiple drug resistant cancers, metastatic cancers and/or recurrent cancers. Examples of neoplasms or neoplasias from which the target cell of the present invention may be derived include, without limitation, carcinomas (e.g., squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), particularly those of the bladder, bone, bowel, breast, cervix, colon (colorectal), esophagus, head, kidney, liver, lung,

nasopharyngeal, neck, thyroid, ovary, pancreas, prostate, and stomach; leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia; benign and malignant lymphomas, particularly Burkitt's lymphoma, Non- Hodgkin's lymphoma and B-cell lymphoma; benign and malignant melanomas;

myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumors of the central nervous system (e.g., gliomas, astrocytomas,

oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer (e.g., small cell lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, including metastatic pleural mesothelioma small cell lung cancer and non-small cell lung cancer), ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma; mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas, among others. It is noted that certain epithelial tumors including ovarian, breast, colon, head and neck, medulloblastoma and B-cell lymphoma, among others are shown to exhibit increased autophagy and are principal target cancers for compounds and therapies according to the present invention.

The term "additional anti-cancer agent" is used to describe an additional compound which may be coadministered with one or more compounds of the present invention in the treatment of cancer. Such agents include, for example, everolimus, trabectedin, abraxane, I LK 286, AV-299, DN-101 , pazopanib, GSK690693, RTA 744. ON 0910.Na, AZD 6244 (ARRY- 142886), AMN-1.07, TKI-258, GSK461364, AZD 1 152. enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a P13 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint- 1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd21 71 , batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 1 1 1 , 131-I-TM-601 . ALT- 1 10, BIO 140, CC 8490, cilengitide, gimatecan. IL13-PE38QQR, INO 1001 , IPdRi KRX- 0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 31 1 , romidepsin, ADS- 100380. sunitinib, 5 -fluoro uracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal, doxorubicin, 5'-deoxy-5-ffuorouridine, vincristine, temozolomide, ZK-304709, selicielib; PD0325901 , AZD-6244, capecitabine, L- Glutamic acid, N -[4-[2-(2-am.ino-4,7-dihydro-4-oxo-l H - py.rrolo[2,3- d Jpyrimidin-5- yl )ethyl] benzoyl] -, di sodium salt, heptahydrate, camptothecin. PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazoie, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-lCl 1 , CHIR-258,); 3-[5- (methylsulfonylpiperadinemethyl)- mdolylj-qumolone, vatalanib, AG-013736. AVE-0005, the acetate salt of [D- Ser(Bu t ) 6 ,Azgly 10 ] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t )-Leu- Arg-Pro- Azgly-NH ₂ acetate [C59H8₄r½Oi₄ -(C₂H₄0₂)x where x = 1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,

hydroxyprogesterone caproate. megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate. CP-724714; TA -165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS- 214662, tipiiarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU 11248, sorafenib, KRN951 , aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate,

cyproterone,

dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine. fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide. imatinib. leuprolide, levamisole, lomustine,

mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291 , squalamine, endostatin, SU5416, SU6668, EMD121974, mterleukin-12, IM862, angiostatin, vitaxin, droloxifeiie, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA- 923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE- 424, HMR- 3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O- (2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001 , ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L- 779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,

hydrocortisone, interleukin-1 1 , dexrazoxane, alemtuzumab, all-transretinoic acid,

ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard,

methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, , diphenhydramine, hydroxyzine,

metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib among others.

The term "alkyl" is used herein to refer to a fully saturated monovalent radical containing carbon and hydrogen (up to 10 carbon atoms or as otherwise indicated), and which may be a straight chain, branched or cyclic. Examples of alkyl groups are methyl, ethyl, n- butyl, n-heptyl, isopropyl, 2-methyl propyl, tert-butyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.

The term "substituted" as that term relates to alkyl groups which are described above include one or more functional groups such as lower alkyl groups containing 1-6 carbon atoms which are optionally substituted with 1 or 2 hydroxyl groups or between 1 and 5 (preferably 3-5) fluoro groups, acyl (CrC₆), halogen (F, CI, Br, I, e.g., alkyl halos, e.g., CF₃), amido, hydroxyl, carboxy/carboxylic acid, thioamido, cyano, nitro, alkenyl (C₂-C₆) alkynyl (C₂-C₆), azido, alkoxy (Ci-C₆), (including alkoxy groups which are further substituted with a Cj-C₆ alkoxy group thus producing a diether group), amino, C[-C₆ alkylamino and dialkyl- amino, where the alkyl groups may be optionally substituted with 1 or 2 hydroxyl groups or an amine, aminoalkyl or dialkyl group which itself is substituted one or two alkyl groups or a 7-substituted-4-quinolinyl group, C₂-C₆ acylamino, C₂-C₆ oxyacylester or carboxyester, aryloxy, aryloxy(C₁-C₆)alkyl, carboxamido, thio, C₂-C₆ ether or thioether, a 7-substituted-4- aminoquinolinyl group (or a substitution on an amine group which forms a 7-substituted-4- aminoqunolinyl group) and the like. Preferred substituents on alkyl groups (within context, especially on the amino group of the 7-substituted-4-aminoquinoline) or a linker which contains at least one amine group, include, for example, at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine (where one or both alkyl groups is itself further optionally substituted with a dialkyl amine or an amine substituted with one or two

(preferably one) 7-substituted-4-quinolinyl group(s) where the amine group is bonded to the 4-position of the quinolinyl group) or an alkoxy group (e.g. methoxy or ethoxy) which may be further substituted with an alkoxy group, preferably a methoxy group, thus forming a diether substituent.

The term "aryl" refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl). Other examples include heterocyclic aromatic (heteroaromatic or heteroaryl) ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, in particular, quinoline groups, in particular, 7-substituted-amino quinoline groups, as well as other groups.

The term "substituted" as used in the term "substituted aryl, substituted aromatic, substituted heteroaryl, or substituted heteroaromatic" herein signifies that a substitution on the 7-position of 4-aminoquinoline may be present, said substituents being selected from atoms and groups, which when present enhance the activity of the compound as an inhibitor of autophagy. Examples of substituents that may be present in a substituted aromatic or heteroaromatic group include, but are not limited to, groups such as H, halo (F, CI, Br or I), CN, N0₂, optionally substituted -C6 alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally substituted 0-C]-C₆ alkyl (preferably, OCH₃), optionally substituted C₂-C₇ acyl (preferably acetyl) or optionally substituted C₂-C₇ ester (oxycarbonyl ester or carboxyester, preferably carboxyester);. It is noted that each of the substituents disclosed herein may themselves be substituted.

The term "co-administration" or "adjunct therapy" shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time. Although compounds according to the present invention may be co-administered to a patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, including sequential

administration. Preferably, effective concentrations of all co-administered compounds or compositions are found in the subject at a given time. The term co-administration or adjunct therapy also contemplates other bioactive agents being coadministered with pharmaceutical compositions according to the present invention, especially where a cancer has metastasized or is at risk for metastasis.

The term "amine protecting group" refers to a moiety or group which can be readily removed from a functional group to which the protecting group is attached to allow further reaction. Exemplary amine-protecting groups include carbobenzyloxy (Cbz group, removed by hydrogenolysis), p-Methoxylbenzyl carbon (Moz or MeOZ group, removed by

hydrogenolysis), tert-butyloxycarbonyl (BOC group, removed by concentrated strong acid or by heating at elevated temperatures), 9-Fluorenylmethyloxycarbonyl (FMOC group, removed by weak base, such as piperidine or pyridine), acyl group (acetyl, benzoyl, pivaloyl, by treatment with base), benzyl (Bn groups, removed by hydrogenolysis), carbamate, removed by acid and mild heating, p-methoxybenzyl (PMB, removed by hydrogenolysis), 3,4- dimethoxybenzyl (DMPM, removed by hydrogenolysis), p-methoxyphenyl (PMP group, removed by ammonium cerium IV nitrate or CAN); tosyl (Ts group removed by concentrated acid and reducing agents, other sulfonamides, Mesyl, Nosyl & Nps groups, removed by samarium iodide, tributyl tin hydride.

The term "radiotherapy" or "radiation therapy" is used to describe therapy for cancer which may be used in conjunction with the present compounds. Radiation therapy uses high doses of radiation, such as X-rays, or other energy sources such as radioisotopes (gamma, beta or alpha emitters), to destroy cancer cells. The radiation damages the genetic material of the cells so that they can't grow. Although radiation damages normal cells as well as cancer cells, the normal cells can repair themselves and function, while the cancer cells cannot.

Radiation therapy may be used in combination with the presently claimed compounds, alone or in combination with additional anticancer compounds as otherwise disclosed herein, depending on the cancer to be treated. Radiotherapy therapy is most effective in treating cancers that have not spread outside the area of the original cancer, but it also may be used if the cancer has spread to nearby tissue. Radiotherapy is sometimes used after surgery to destroy any remaining cancer cells and to relieve pain from metastatic cancer.

Pharmaceutical Compositions

Compounds according to the present invention may be readily formulated into pharmaceutical compositions, useful in the inhibition of autophagy in a biological system and/or the inhibition, treatment or prevention of diseases states and/or conditions which benefit from the inhibition of autophagy including cancer (and its metastasis), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus (systemic lupus erythematosus), chronic urticaria and Sjogren's disease. Pharmaceutical compositions comprise an effective amount of one or more compounds according to the present invention in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional agent, in the case of cancer, preferably an anticancer agent as otherwise described herein.

As noted above, the compounds and method of the invention may be used to inhibit autophagy as otherwise described herein, and are useful for the inhibition (including prophylaxis) and/or treatment of cancer and its metastasis, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus (systemic lupus erythematosus), chronic urticaria and Sjogren's disease. The treatment of cancer or malaria are important aspects of the present invention.

In methods according to the present invention, subjects or patients in need are trreated with the present compounds, pharmaceutical compositions in order to inhibit, reduce the likelihood or treat a disease state, condition and/or infection as otherwise described herein. The disease states, conditions and infections treated by the present compounds and compositions are readily recognized and diagnosed by those of ordinary skill in the art and treated by administering to the patient an effective amount of one or more compounds according to the present invention.

Generally, dosages and routes of administration of the compound are determined according to the size and condition of the subject, according to standard pharmaceutical practices. Dose levels employed can vary widely, and can readily be determined by those of skill in the art. Typically, amounts in the milligram up to gram quantities are employed. The composition may be administered to a subject by various routes, e.g. orally, transdermally, perineurally or parenterally, that is, by intravenous, subcutaneous, intraperitoneal, itrathecally or intramuscular injection, among others, including buccal, rectal and transdermal administration. Compositions may also be administered by inhalation to the lungs. Subjects contemplated for treatment according to the method of the invention include humans, companion animals, laboratory animals, and the like.

Formulations containing the compounds according to the present invention may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, capsules, powders, sustained-release formulations, solutions, suspensions, emulsions, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

Pharmaceutical compositions according to the present invention typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, additives and the like. Preferably, the composition isabout 0.1% to about 85%, about 0.5% to about 75% by weight of a compound or compounds of the invention, with the remainder consisting essentially of suitable

pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

Liquid compositions can be prepared by dissolving or dispersing the compounds (about 0.5% to about 20% by weight or more), and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension. For use in oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.

When the composition is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like. In a tablet formulation, the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.

An injectable composition for parenteral administration will typically contain the compound in a suitable i.v. solution, such as sterile physiological salt solution. The composition may also be formulated as a suspension in a lipid or phospholipid, in a liposomal suspension, or in an aqueous emulsion.

Methods for preparing such dosage forms are known or is apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co., 1985). The composition to be administered will contain a quantity of the selected compound in a pharmaceutically effective amount for inhibiting autophagy in a biological system, including a patient or subject according to the present invention.

Method of Treatment

According to one aspect of the invention, a method is provided for treating a mammalian patient or subject to inhibit autophagy in that patient or subject. Compounds according to the present invention described herein may be used to inhibit autophagy in a manner consistent with inhibiting, treating and/or preventing disease states and/or conditions including cancer (including metastasis of cancer), rheumatoid arthritis, malaria,

antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

According to the present invention, in patients or subjects in need thereof, are treated by administering to the patient or subject an effective amount of one or more compounds according to the present invention, optionally in combination with at least one additional bioactive agent useful for treating the same disease state or condition. Compounds according to the present invention may be used to inhibit, reduce the likelihood or treat cancer, including the metastasis of cancer in a patient or subject in need of such treatment. The treatment is useful for any cancer for which inhibition of autophagy represents a favorable result or for which metastasis is a risk element. Therapy with at least one additional anticancer agent as otherwise described herein is also contemplated in the present methods. The numerous cancers which may be treated pursuant to the present method are described hereinabove.

In another aspect the present invention is directed to a method for treating a disease state and/or condition which benefits from the inhibition of autophagy, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjorgen's disease. In this method, a patient or subject in need of treatment is administered an effective amount of a compound as otherwise described herein optionally in combination with a pharmaceutically acceptable carrier, additive or excipient in order to inhibit, treat and/or prevent the above disease states of conditions. In alternative embodiments, at least one additional bioactive agent is coadministered with a compound according to the present invention.

In certain preferred embodiments of the invention, at least one (preferably two) R^M is other than H, especially when R¹ and R¹ are H. In other preferred aspects of the invention, R¹ and R^1' are each independently H, a halo group (most often CI or F), a nitro group or a trifluoromethyl group, most often a chloro or fluoro group. R and R' are preferably each independently H or methyl, preferably H and L is preferably an alkylene group containing from 6 to 20 methylene groups (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19 or 20), a (poly)ethylene glycol group having from 1 to 20 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 2 to 15, 3 to 10 or 4, 5, 6, 7, 8, 9, or 10) ethylene glycol units, each (poly)ethylene glycol group being optionally substituted at one or both of its distal ends with an A group, or L is a linear group containing from 1 to 5 non-contiguous amine groups N-R", each amine group being separated from an adjacent amine group by an alkylene group containing from 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2, 3 or 4) methylene groups or an ethylene glycol containing group having from 1 to 6 (preferably 2 to 6, 3 to 6 or 4, 5 or 6) ethylene glycol units. R" is preferably H, methyl or an amine protecting group (preferably a benzyl or benzoyl group which can be removed to introduce other groups on the amine group after deprotection).

In certain embodiments, one or more amine group of L is optionally substituted with one or two 7-substituted-4-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group through an alkylene group and the 7-position of each quinolinyl group is optionally substituted, preferably with a R¹ and/or R¹ group as broadly described for generic structures IA, IB, IC, ID and IE above, or one or more amines of the linker group is substituted with an alkyl group which itself is further optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, a monoalkyl (C_\-C , preferably C1-C3) amine or a dialkyl (Ci-C₆, preferably Ci-Cs) amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with R¹ and/or R¹ as broadly described for generic structures ΙΑ,ΙΒ, IC, ID and IE above, and each of said alkoxy groups (e.g.

methoxy or ethoxy) is optionally further substituted with a C!-C₃ alkoxy group, preferably a methoxy group, thus forming a diether substituent.

Further preferred methods relate to the use/administration of the compounds according to the present invention which are presented in the various schemes which are presented in the tables and figures presented herein.

In the methods treating or inhibiting cancer or the metastasis of cancer, the

compounds described above may be coadministered with at least one additional anticancer agent including, for example, everolimus, trabectedin, abraxane. I LK 286. AV-299, DN- 101, pazopanib. GSK690693, RTA 744, ON 0910.Na, A7.D 6244 (ARRY- 1 42886). AMN-107, TKI-258, GS 461364, AZD 1 152, enzastaurin. vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK.

inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint- 1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd21 71 , batabulin.

ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene. oblimersen, ticilimumab, ipilimumab, gossypol, Bio 1 1 1 , 1 31 -I-TM-601 , ALT-1 10, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR. INO 1001 , IPdR, KRX-0402. lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 31 1 , romidepsin, ADS- 100380, sunitinib. 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK- 304709, seliciclib; PD0325901 , AZD-6244, capecitabine, L-Glutamic acid, N -[4-[2-(2- amino-4.7-dihydro-4-oxo- l H - pyrrolo[2,3- d ]pyrimidin-5-yl)etliyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC- l ( i 1 , CHIR-258,); 3-[5-(methylsulfonylpiperadinemethyl)- indolylj-quinolone, vatalanib. AG-013736, AVE-0005, the acetate salt of [D- Ser(Bu t ) 6 ,Azgly 10 ] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t )-Leu-Arg-Pro- Azgly-NH ₂ acetate

[C5 H₈4N]₈Oi4 -(C₂H₄0₂) where x = 1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxy progesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-

165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-

166, GW-572016, lonafarnib, BMS-214662, tipifamib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A. FK-228, SU1 1248, sorafenib,

KRN951 , aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette- Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine,

chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine. dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, meehlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate. pentostatin, plicamycin, porfimer. procarbazine, raltitrexed. rituximab.

streptozocin, teniposide, testosterone, thalidomide, thioguanine. thiotepa, tretinoin, vindesine, 1 3-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine.

floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcilriol. valrubicin, mithramycin. vinblastine, vinorelbine, topotecan, razoxin, marimastat. COL-3, neovastat, BMS-275291 , squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA- 923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR- 3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP- 23573, RAD001 , ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684,

LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa- 2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11 , dexrazoxane, alemtuzumab, all- transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine,

hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, ,

diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlo erazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, among others, and mixtures thereof.

In methods involving infections, disease states and/or conditions caused by rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the compounds according to the present invention may be

coadministered with additional agents which are traditionally used in therapy for these disease states and/or conditions.

Examples

The following examples illustrate and describe the present invention but are not intended to limit the invention in any way.

Chemical Methods

Strategy for synthesis of bivalent aminoquinoline autophagy inhibitors with varying linker lengths

The compounds are prepared as previously described (PNAS) by Buchwald-Hartwig coupling of the appropriate spacer molecule diamine with the requisite 4- substitutedquinoline, which are either commercially available or could be readily prepared by standard methods. To establish the role of linker length, and of the asymmetric nature of the linker length of spermidine (3,4) relative to LysOl (2,2), we have prepared a series of new compounds that systematically vary the number of methylenes, i.e., CH2 groups, between the nitrogen atoms of the linker, where x and y = 1 -4 in the formula -HN(CH2)xNR(CH2)yNH-. These compounds are listed in Figure 10, Table 1, Figure 11, Table 2 and Figure 12as Lys 34-41 and Lys 72-75.

In addition, numerous compounds according to the present invention may be readily prepared pursuant to the synthetic methods provided inter alia on pages 21-28 of

international application PCT/US2012/035251 (WO 2012/149186), relevant portions of which are incorporated by reference herein. Additional compounds may be prepared by analogy from the disclosed methods as well as analogously from synthetic procedures which are well known in the art.

Chemistry Experimental Protocol (Exemplary Syntheses) :

4-Bromo-7-chloroquinoline: , J. Org. Chem. 2007 (72) 2232

At 0 °C under an atmosphere of argon, phosphorus tribromide (5.8 mL, 0.079 moles, 1.10 equiv.) was added slowly to a solution of 7-chloro-4-hydroxyquinoline (13.02 g, 0.073 moles, 1.00 equiv.) in anhydrous DMF (150 mL, 0.5 M soln.). The reaction was allowed to warm to rt and followed by TLC. Complete consumption of starting material was observed after 90 minutes stirring. The reaction mixture was poured onto ice and the pH was rendered alkaline using solid sodium bicarbonate. This resulted in a white precipitate. The mixture was then filtered and the resulting solid was dried under vacuum affording an off-white solid (17.30 g, 99 %). The material was recrystallised from ethyl acetate to give white needles (12.20 g, 70 %). R/= 0.70 (hex:EtoAc; 1 :1); Mp = 99 - 101 °C, EtoAc (lit., ^{Eur 1 0rg}- ^chem- ^20024181 103 - 104 ° C, hex).

Lyso-01:^Mc4/"ee

Under argon, a mixture of degassed dioxane and water (10:1; 22 mL, 0.4 M soln.) was added to 4-bromo-7-chloroquinoline (4.78 g, 19.840 mmol, 2.25 equiv.), ΒΓΝΑΡ (0.81 g, 1.302 mmol, 0.15 equiv.), Pd(OAc)₂ (0.16 g, 0.716 mmol, 0.08 equiv.), potassium phosphate tribasic (6.91 1 g, 32.560 mmol, 3.69 equiv.) and N-methylethane-l,2-diamine (0.94 mL, 8.814 mmol, 1.00 equiv.). The reaction was heated to 120 ⁰ C in a sealed tube and heating was maintained for 16 hours. The reaction was then cooled to rt and filtered on a pad of celite washing through with chloroform (3 * 50 mL). The reaction was rendered acidic via the addition of aqueous HCI (1 M, 18 mL, 17.628 mmol, 2 equiv.) resulting in the precipitation of the HCI salt. This precipitate was isolated via filtration and the remaining organic layer was extracted with deionised water (2 ^χ 50 mL). The isolated HCI salt was added to the aqueous washings which was then rendered basic via the addition of ammonium hydroxide (until pH > 9). This now alkaline mixture was extracted with CHC1₃ (3 x 40 mL). Combined chloroform fractions were washed with brine, dried over Na₂S0₄ and filtered. Concentration under reduced pressure resulted in the isolation of pure compound as an orange solid (2.74 g, 71 %). The material was recrystallised from EtOAc, resulting in the isolation of needles (43 % recrystallised yield). R/= 0.70.

A solution of the Lyso-01 (2.68 g, 6.098 mmol, 1.00 equiv.) in MeOH (20 mL) was treated with 1M aqueous HCI (20 mL). The resulting reaction was stirred vigorously at room temperature for 4 hours. The product was insoluble in the reaction media and was collected via filtration. The residue was collected and taken up in excess water. Sonication and heating was necessary to solubilise the product. The aqueous solution was lyophilised to give a white crystalline solid (1.77 g, 53 %). Starting material (0.726 g, 27 %) was recovered from the reaction. Mp = 270 ^°C (decomposition) (Lit., ^PNAS 270 °C); HPLC analysis of Lyso-05 showed > 99% purity.

Lyso-10:

Lyso-1 (0.26 g, 0.60 mmol, 1.00 equiv.) was placed under a blanket of argon. Pyridine (0.5 mL, 5.95 mmol, 10.00 equiv.) followed by acetic anhydride (1.1 mL, 11.90 mmol, 20.00 equiv.) were added and the resulting reaction was heated to 150 °C for 2 hours. Following this the reaction mixture was poured onto a solvent mixture of CHC1₃ (40 mL) and water (40 mL) and agitated vigorously. The resulting layers were allowed to settle and separated. The aqueous layer was further extracted with CHC1₃ (3 ^χ 40 mL). Combined CHC1₃ extracts were washed with brine (120 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded a brown oil (0.12 g, 38 % yield) which was found to be very pure.

Lyso-20:

To a microwave vial was added spermine (0.57 g, 2.83 mmol, 1.00 equiv.), 4-Bromo-7- chloroquinoline (1.32 g, 6.38 mmol, 2.25 equiv.), ΒΓΝΑΡ (0.11 g, 0.17 mmol, 0.06 equiv.), Pd(OAc)₂ (0.02 g, 0.09 mmol, 0.03 equiv.) and K₃P0₄ (1.80 g, 8.50 mmol, 3.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 9 mL, 0.3 M) was added and the reaction was heated to 120 °C for 16 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room temperature. Following this the reaction was filtered on celite, washing through with MeOH (100 mL). The resulting solution was concentrated to give a solid. This solid was washed with CHC1₃ (3 x 30 mL) to remove impurities, affording the product as a yellowish solid (1.09 g, 73 % yield) which was found to be very pure.

L so-21:

To a sealable tube was added spermidine (0.98 g, 6.76 mmol, 1.00 equiv.), 4-Bromo-7- chloroquinoline (3.69 g, 15.21 mmol, 2.25 equiv.), BINAP (0.17 g, 0.27 mmol, 0.04 equiv.), Pd(OAc)₂ (0.03 g, 0.14 mmol, 0.02 equiv.) and K₃P0₄ (4.30 g, 20.28 mmol, 3.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 22.5 mL, 0.3 M) was added and the reaction was heated to 120 °C for 16 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room temperature. Following this the reaction was filtered on celite, washing through with MeOH (100 mL). The resulting solution was concentrated to give a solid. This solid was solubilised in CHC1₃ (60 mL) and 1 M_aq HC1 (60 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 ^χ 60 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- propanol (4:1 ; 3 * 40 mL). These combined extracts were washed with brine (120 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white solid (2.05 g, 65% yield). R/= 0.10 (EtOAc: MeOH: TEA; 80:15:5).

Lyso-25:

Lyso-20 (0.13 g, 0.25 mmol, 1.00 equiv.) was dissolved in DCM (2.5 mL, 0.1 M).

Formaldehyde 37 wt% (0.08 g, 0.98 mmol, 4.00 equiv.), then sodium triacetoxyborohydride (0.21 g, 0.98 mmol, 4.00 equiv.) was added and the reaction was stirred at room temperature for 4 hours. At this point, TLC analysis showed complete consumption of Lyso-20. A 2 M_aq NaOH solution (15 mL) was added to break up borane salts in the reaction mixture. The resulting mixture was stirred at rt for 1 hour before CHC1₃ (20 mL) was added. The resulting biphasic mixture was allowed to settle and separated. The aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_aq HC1 solution (20 mL). The mixture was stirred vigorously at room temperature for 20 minutes, resulting in the formation of a water soluble salt of the desired product. The mixture was allowed to settle and both layers were separated. The aqueous layer was further washed with CHC1₃ (2 ^χ 40 mL) and combined chloroform washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11 , resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- propanol (4:1 ; 3 x 40 mL). These combined extracts were washed with brine (120 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white solid (16.0 mg, 12% yield). R/= 0.10 (EtO Ac :MeOH : TEA; 80: 15:5).

Lyso-26:

Lyso-21 (0.11 g, 0.23 mmol, 1.00 equiv.) was dissolved in DCM (2.5 mL, 0.1 M).

Formaldehyde 37 wt% (0.04 g, 0.46 mmol, 2.00 equiv.), then sodium triacetoxyborohydride (0.19 g, 0.90 mmol, 4.00 equiv.) was added and the reaction was stirred at room temperature for 4 hours. At this point, TLC analysis showed complete consumption of Lyso-21. A 2 M_aq NaOH solution (15 mL) was added to break up borane salts in the reaction mixture. The resulting mixture was stirred at rt for 1 hour before CHC1₃ (20 mL) was added. The resulting biphasic mixture was allowed to settle and separated. The aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_aq HC1 solution (15 mL). The mixture was stirred vigorously at room temperature for 20 minutes, resulting in the formation of a water soluble salt of the desired product. The mixture was allowed to settle and both layers were separated. The aqueous layer was further washed with CHC1₃ (2 15 mL) and combined chloroform washings were discarded at this point. Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHCI3 and i- propanol (4:1; 3 x 15 mL). These combined extracts were washed with brine (60 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white solid (86.0 mg, 79% yield). R/= 0.30 (EtO Ac : MeOH : TEA; 80:15:5).

Ethyl (£)-3-((4-methoxyphenyl)amino)but-2-enoate:

Prepared pursuant to Tetrahedron, 2012, 5522.

6-Methoxy-2-methylquinolin-4-ol:

Prepared pursuant to Euro. J. Med. Chem. 2010, 3803. -Chloro-6-methoxy-2-methylquinoline: . Euro. J. Med. Chem. 2010, 3803

Prepared pursuant to Euro. J. Med. Chem. 2010, 3803.

Lyso-27:

To a 20 mL microwave vial was added 1,9-diamino nonane (0.34 g, 2.18 mmol, 1.00 equiv.), -Chloro-6-methoxy-2-methylquinoline (1.02 g, 4.90 mmol, 2.25 equiv.), BINAP (0.07 g, 0.12 mmol, 0.05 equiv.), Pd(OAc)₂ (0.02 g, 0.07 mmol, 0.03 equiv.) and K₃P0₄ (1.39 g, 6.53 mmol, 3.00 equiv.). The vial was sealed and placed under argon. Fully degasses solvent dioxane:water (10:1, 7 mL, 0.3 M) was added and the reaction was heated to 100 °C for 16 hours. The reaction was followed by TLC and NMR. The reaction was allowed to cool to room temperature and filtered on celite, washing through with MeOH (100 mL). The filtrate was concentrated to give 0.87 g (crude yield 80% ) of an impure brown solid. This material was purified via flash column chromatography (eluent: EtOAc:MeOH:TEA; 90:9:1) to give the product as an off-white solid (0.43 g, 40% yield). R/= 0.25 (EtOAc:MeOH:TEA; 90:9:1).

L so-28:

Lyso-20 (0.32 g, 0.62 mmol, 1.00 equiv.) was suspended in DCM (6 mL, 0.1 M). Freshly distilled benzaldehyde (0.4 mL, 3.69 mmol, 6.00 equiv) then sodium triacetoxyborohydride (1.30 g, 6.15 mmol, 10.00 equiv.) were added and the reaction was stirred at room

temperature for 12 hours, until TLC analysis showed complete consumption of Lyso-20. A 2 Maq NaOH solution (30 mL) was then added to break up borane salts in the reaction mixture. The resulting mixture was stirred at rt for 1 hour before DCM (30 mL) was added. The resulting biphasic mixture was allowed to settle and separated. The aqueous layer was discarded at this point.

To the DCM solution was added 1 M_aq HC1 solution (30 mL). The mixture was stirred vigorously at room temperature for 20 minutes, resulting in the formation of a water soluble salt of the desired product. The mixture was allowed to settle and both layers were separated. The aqueous layer was further washed with CHC1₃ (2 x 30 mL) and combined DCM washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of DCM (3 ^χ 30 mL). These combined extracts were washed with brine (100 mL) and dried oyer Na₂S0₄. Filtration followed by solvent evaporation afforded a yellow oil (0.35 g, 80% crude yield) which contained impurities. This oil was purified via flash column chromatography [gradient: 100% EtOAc (150 mL); then EtOAc:MeOH:TEA; 90:9:1] to give the product as a yellow solid (0.21 g, 49% yield). R/= 0.30 (EtO Ac :MeOH : TEA; 90:9:1).

N- 2-Cyanoethyl)-N-(3-cyanopropyl)-benzylamine:

Prepared pursuant to the procedure of J. Org. Chem. 1980, 1589. -(3-aminobutyl)-N-(3-aminopropyl)(phenylmethyl)amine:

Prepared pursuant to the procedure of J. Org. Chem. 1980, 1589. L so-29:

To a microwave tube was added N-(3-aminobutyl)-N-(3-aminopropyl)(phenylmethyl)amine (96.00 mg, 0.41 mmol, 1.00 equiv.), 4-Bromo-7-chloroquinoline (222.00 mg, 0.92 mmol, 2.25 equiv.), ΒΓΝΑΡ (10.00 mg, 0.02 mmol, 0.04 equiv.), Pd(OAc)₂ (2.00 mg, 0.01 mmol, 0.02 equiv.) and K₃P0 (260.00 mg, 1.22 mmol, 3.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 1.5 mL, 0.3 M) was added and the reaction was heated to 100 °C for 13 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room temperature. Following this the reaction was filtered on celite, washing through with MeOH (20 mL). The resulting solution was concentrated to give a yellow oil. This oil was solubilised in CHC1₃ (20 mL) and 1 M_aq HC1 (20 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHCI₃ (2 ^χ 20 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into CHCI₃ (3 ^χ 20 mL). These combined extracts were washed with brine (60 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a yellow solid (112.00 mg, 49% yield). R_f= 0.60 (EtOAc:MeOH:TEA; 80:15:5).

L so-30:

Under argon, Lyso-20 (0.25 g, 0.47 mmol, 1.00 equiv.) was dissolved in anhydrous pyridine (4.7 mL, 0.1 M) and freshly distilled benzoyl chloride (0.3 mL, 2.37 mmol, 5.00 equiv.) was added. The reaction was stirred at room temperature for 2 hours, until TLC analysis showed complete consumption of Lyso-20. Water (20 mL) and CHCI₃ (20 mL) were then added to the reaction mixture and stirring was maintained for a further 30 minutes. The layers of this biphasic mixture were then separated. Following this the aqueous layer was further extracted with CHCI₃ (2 x 10 mL). At this point the aqueous layer was discarded and the CHCI₃ extracts were combined.

To the CHCI3 solution (containing the product) was added 1 M_aq HC1 (40 mL). The resulting mixture was stirred vigorously at room temperature for 30 minutes, resulting in the formation of the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHCI₃ (2 ^χ 20 mL). These combined organic washings were discarded at this point. Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into CHC1₃ (3 x 15 mL). These combined extracts were washed with brine (60 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white foam (0.13 g, 37% yield). R = 0.40 (EtOAc:MeOH:TEA; 80: 15:5).

L so-31:

Under argon, Lyso-21 (0.21 g, 0.45 mmol, 1.00 equiv.) was dissolved in anhydrous pyridine (4.5 mL, 0.1 M) and freshly distilled benzoyl chloride (0.3 mL, 2.27 mmol, 5.00 equiv.) was added. The reaction was stirred at room temperature for 2 hours, until TLC analysis showed complete consumption of Lyso-21. Water (10 mL) and CHC1₃ (10 mL) were then added to the reaction mixture and stirring was maintained for a further 30 minutes. The layers of this biphasic mixture were then separated. Following this the aqueous layer was further extracted with CHCI₃ (2 x 10 mL). At this point the aqueous layer was discarded and the CHC1₃ extracts were combined.

To the CHCI3 solution (containing the product) was added 1 M_aq HC1 (30 mL). The resulting mixture was stirred vigorously at room temperature for 30 minutes, resulting in the formation of the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHCI3 (2 x 10 mL). These combined organic washings were discarded at this point.

Using ΝΗ4ΌΗ, the pH of the aqueous layer was adjusted to 11 , resulting in the liberation of the free base of the product. The product was extracted into CHC1₃ (3 x 15 mL). These combined extracts were washed with brine (60 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the 0.20 g (77% crude yield) of a red oil, which contained impurities. The material obtained was purified via flash column chromatography (eluent: EtOAc:MeOH:TEA; 84: 15:1) to give the product as an orange solid (0.14 g, 55% yield). R/= 0.45 (EtO Ac : MeOH : TEA; 80:15:5).

N-(fer/-butylcarbonyl)-3-hydroxypropylamine:

I ⁰

O N ^ OH

H

Prepared pursuant to the procedure of J. Med. Chem. 2009, 7029. N- teri-butylcarbonyl)-3-amino-propionaldehyde:

Prepared pursuant to the procedure of J. Med. Chem. 2009, 7029. Compound XXI

Lyso-21 (0.30 g, 0.64 mmol, 1.00 equiv.) was dissolved in DCM (13 mL, 0.05 M). N-{tert- butylcarbonyl)-3-amino-propionaldehyde (0.33 g, 1.92 mmol, 3.00 equiv.), then sodium triacetoxyborohydride (0.73 g, 3.84 mmol, 6.00 equiv.) was added and the reaction was stirred at room temperature for 12 hours. At this point, TLC analysis showed complete consumption of Lyso-21. A 2 M_aq NaOH solution (30 mL) was added to break up borane salts in the reaction mixture. The resulting mixture was stirred at rt for 1 hour before DCM (20 mL) was added. The resulting biphasic mixture was allowed to settle and separated. The aqueous layer was discarded at this point.

To the DCM solution was added 1 M_aq HC1 solution (30 mL). The mixture was stirred vigorously at room temperature for 20 minutes, resulting in the formation of a water soluble salt of the desired product. The mixture was allowed to settle and both layers were separated. The aqueous layer was further washed with DCM (2 x 30 mL) and combined DCM washings were then discarded.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into DCM (3 x 30 mL). These combined extracts were washed with brine (100 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded a white foam which contained some impurities. This foam was purified via flash column chromatography (eluent: gradient, 100% EtOAc→ EtOAc:MeOH:TEA; 80:15:5) to give the product as a white solid (0.23 g, 57% yield). R_f= 0.50 (EtOAc:MeOH:TEA; 80:15:5).

Compound XX2

A solution of the compound from above (Compound XXI) (0.21 g, 0.34 mmol, 1.00 equiv.) in DCM (3.5 mL, 0.1 M) was treated with TFA (2 mL, 26.118 mmol, 77.00 equiv.). The resulting reaction was stirred at room temperature and followed by TLC. After 4 hrs, the reaction was found to have reached completion and was quenched via the addition of saturated NaHC0₃ solution. Once the reaction mixture was neutralised, it was further basified to pH 11 using 2 M_aq NaOH solution. The now alkaline mixture was extracted with a solvent mixture of CHC1₃ and -PrOH (4:1; 3 ^χ 40 mL). Combined organic extracts were washed with brine (100 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a yellow solid (0.13 g, 75% yield). R/= 0.00 (EtO Ac :MeOH : TE A ; 80:15:5).

To a microwave tube was added compound XX2 (133.00 mg, 0.25 mmol, 1.00 equiv.), 4- Bromo-7-chloroquinoline (86.00 mg, 0.35 mmol, 1.40 equiv.), ΒΓΝΑΡ (8.00 mg, 0.01 mmol, 0.05 equiv.), Pd(OAc)₂ (2.00 g, 0.008 mmol, 0.03 equiv.) and K3PO₄ (107.00 g, 0.51 mmol, 2.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 1.0 mL, 0.3 M) was added and the reaction was heated to 120 °C for 13 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room temperature. Following this the reaction was filtered on celite, washing through with MeOH (50 mL). The resulting solution was concentrated to give 223.00 mg of a brown solid. This solid was solubilised in CHC1₃ (30 mL) and 1 M_aq HC1 (30 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 x 30 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and j-PrOH (4:1; 3 x 50 mL). These combined extracts were washed with brine (150 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded 155.00 mg (89% crude yield) of a white foam which contained some starting material. This foam was purified via flash column chromatography (eluent: gradient EtOAc: MEOH: TEA; 90:9:1→

EtOAc:MeOH:TEA; 85: 15:5) to give the product as an orange solid (77.00 mg, 44% yield). R/= 0.60 (EtOAc:MeOH:TEA; 80: 15:5). Compound XX3

Lyso-20 (0.23 g, 0.43 mmol, 1.00 equiv.) was dissolved in DCM (9 mL, 0.05 M). N-{tert- butylcarbonyl)-3-amino-propionaldehyde (0.50 g, 2.86 mmol, 6.00 equiv.), then sodium triacetoxyborohydride (0.91 g, 4.31 mmol, 10.00 equiv.) was added and the reaction was stirred at room temperature for 12 hours. At this point, TLC analysis showed complete consumption of Lyso-20. A 2 M_aq NaOH solution (30 mL) was added to break up borane salts in the reaction mixture. The resulting mixture was stirred at rt for 1 hour before DCM (20 mL) was added. The resulting biphasic mixture was allowed to settle and separated. The aqueous layer was discarded at this point.

To the DCM solution was added 1 M_aq HC1 solution (30 mL). The mixture was stirred vigorously at room temperature for 20 minutes, resulting in the formation of a water soluble salt of the desired product. The mixture was allowed to settle and both layers were separated. The aqueous layer was further washed with DCM (2 30 mL) and combined DCM washings were then discarded.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into DCM (3 ^χ 30 mL). These combined extracts were washed with brine (100 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white foam (0.29 g, 81% yield). R/ = 0.50 (EtO Ac :MeOH : TEA; 80:15:5).

Compound XX4

A solution of Compound XX3 (0.28 g, 0.33 mmol, 1.00 equiv.) in DCM (3.3 mL, 0.1 M) was treated with TFA (2 mL, 26.118 mmol, 79.00 equiv.). The resulting reaction was stirred at room temperature and followed by TLC. After 4 hrs, the reaction was found to have reached completion and was quenched via the addition of saturated NaHC0₃ solution. Once the reaction mixture was neutralised, it was further basified to pH 11 using 2 M_aq NaOH solution. The now alkaline mixture was extracted with a solvent mixture of CHC1₃ and i- PrOH (4:1; 3 ^χ 40 mL). Combined organic extracts were washed with brine (100 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a yellow solid (0.09 g, 42% yield). R = 0.00 (EtOAc:MeOH:TEA; 80:15:5).

L so-33:

To a microwave tube was added compound XX3 (90.00 mg, 0.14 mmol, 1.00 equiv.), 4- Bromo-7-chloroquinoline (48.00 mg, 0.20 mmol, 1.40 equiv.), ΒΓΝΑΡ (4.00 mg, 0.007 mmol, 0.05 equiv.), Pd(OAc)₂ (1.00 mg, 0.004 mmol, 0.03 equiv.) and K3PO4 (60.00 mg, 0.28 mmol, 2.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 0.5 mL, 0.3 M) was added and the reaction was heated to 120 °C for 13 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room temperature. Following this, the reaction was filtered on celite, washing through with MeOH (50 mL). The resulting solution was concentrated to give 139.00 mg of a brown solid. This solid was solubilised in CHC1₃ (30 mL) and 1 M_aq HC1 (30 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 x 30 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- PrOH (4:1; 3 * 50 mL). These combined extracts were washed with brine (150 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded 85.00 mg (63% crude yield) of a brown foam which contained some starting material. This foam was purified via flash column chromatography (eluent: CHCl₃:MeOH:TEA; 85:15:5) to give the product as a white solid (49.00 mg, 36% yield). R/= 0.55 (CHCl₃:MeOH:TEA; 80:15:5). -Bis -cyanopropy -benzylamine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

N /V-Bis(4-aminobutyl)-benzylamine:

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Bis- 4-aminobutyl)-amine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Lyso-38:

To a microwave tube was added bis-(4-aminobutyl)-amine (0.31 g, 1.96 mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (1.06 g, 4.41 mmol, 2.25 equiv.), BINAP (0.09 g, 0.15 mmol, 0.08 equiv.), Pd(OAc)₂ (0.02 g, 0.10 mmol, 0.05 equiv.) and K₃P0₄ (1.90 g, 7.84 mmol, 4.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 6.5 mL, 0.3 M) was added and the reaction was heated to 120 °C for 12 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room

temperature. Following this, the reaction was filtered on celite, washing through with MeOH (50 mL). The resulting solution was concentrated under reduced pressure. This solid was solubilised in DCM (40 mL) and 1 M_aq HQ (40 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 ^χ 40 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 1 1, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- PrOH (4:1 ; 3 40 mL). These combined extracts were washed with brine (120 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white solid

(0.44 g, 46% yield). R/= 0.10 (EtOAc:MeOH:TEA; 80: 15:5).

Lyso-39:

A solution of Lyso-38 (0.13 g, 0.27 mmol, 1.00 equiv.) in DCM (3 mL, 0.1 M) was treated with formaldehyde 37 wt% (0.04 g, 0.55 mmol, 2.00 equiv.) then sodium

triacetoxyborohydride (0.23 g, 1.09 mmol, 4.00 equiv.). The resulting reaction was stirred at room temperature for 6 hours. Following this, 2 M_aq NaOH (15 mL) was added to the reaction mixture to break up any borane salts. This mixture was stirred at room temperature for 1 hour before the product was extracted into CHC1₃ (3 x 15 mL). The basic aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_aq HC1 (40 mL). The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHCI₃ (2 x 15 mL). These combined organic washings were discarded at this point.

Using NHUOH, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- PrOH (4:1; 3 x 15 mL). These combined extracts were washed with brine (50 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white solid (0.08 g, 60% yield). R = 0.30 (EtO Ac : MeOH : TEA; 80:15:5).

N V-Bis(4-cyanobutyl)-benzylamine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

i\yV-Bis(5-aminopentyl)-benzylamme

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Bis-(4-aminopentyl)-amine: . Bioorg. Med. Chem. Lett. 2003. 3267

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267. Lyso-40:

To a microwave tube was added Bis-(4-aminopentyl)-amine (0.40 g, 1.43 mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (0.78 g, 3.23 mmol, 2.25 equiv.), ΒΓΝΑΡ (0.07 g, 0.11 mmol, 0.08 equiv.), Pd(OAc)₂ (0.02 g, 0.07 mmol, 0.05 equiv.) and K₃P0₄ (1.22 g, 5.74 mmol, 4.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10: 1, 5 mL, 0.3 M) was added and the reaction was heated to 120 °C for 12 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room

temperature. Following this, the reaction was filtered on celite, washing through with MeOH (50 mL). The resulting solution was concentrated under reduced pressure. This solid was solubilised in CHC1₃ (20 mL) and 1 M_aq HC1 (20 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 ^χ 20 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and /^'- PrOH (4:1 ; 3 * 30 mL). These combined organic extracts were washed with brine (100 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a yellow solid (0.51 g, 69% yield). R = 0.05 (EtOAc:MeOH:TEA; 84:15:1).

Lyso-41:

A solution of Lyso-40 (0.45 g, 0.88 mmol, 1.00 equiv.) in DCM (9 mL, 0.1 M) was treated with formaldehyde 37 wt% (0.14 g, 1.76 mmol, 2.00 equiv.) then sodium

triacetoxyborohydride (0.74 g, 3.51 mmol, 4.00 equiv.). The resulting reaction was stirred at room temperature for 6 hours. Following this, 2 M_aq NaOH (30 mL) was added to the reaction mixture to break up any borane salts. This mixture was stirred at room temperature for 1 hour before the product was extracted into CHC1₃ (3 x 30 mL). The basic aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_aq HC1 (40 mL). The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 30 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- PrOH (4:1; 3 ^χ 30 mL). These combined extracts were washed with brine (100 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a white solid (0.39 g, 85% yield). R = 0.10 (EtO Ac :MeOH : TEA; 84:15:1).

To a vial was added 4-Chloro-6-methoxy-2-methylquinoline (508.00 mg, 2.45 mmol, 2.40 equiv,), ΒΓΝΑΡ (95.00 mg, 0.15 mmol, 0.15 equiv.), Pd(OAc)₂ (17.00 mg, 0.08 mmol, 0.075 equiv.), finely ground K₃P0₄ (867.00 mg, 4.09 mmol, 4.00 equiv.) and N-(2-aminoethyl)-N- methyl-l,2-ethanediamine (130.0 μί, 1.02 mmol, 1.00 equiv.). The reagents were placed under a blanket of Argon; then a mixture of degassed dioxane and water (4 mL, 10:1) was added. The reaction vial was sealed and heated to 120 ^°C for 12 hours. The reaction was then cooled to room temperature and filtered on a pad of celite; washing with chloroform (3 10 mL). Combined organic layers were acidified to pH 1 using 1M aqueous HC1 solution (4 mL, 4.09 mmol, 4.00 equiv.) and diluted with water (20 mL). The resulting biphasic mixture was separated and the water layer was washed with chloroform (2 15 mL). The pH of the aqueous layer was adjusted to 11 using ammonium hydroxide. The now alkaline mixture was washed with chloroform (3 x 20 mL). These 3 chloroform extracts were combined, washed with brine, dried over Na₂S0₄ and concentrated to give the product as an off-white solid (415.00 mg, 88% yield, > 95% purity). Recrystallisation from EtOAc afforded off-white crystals (0.225 g, 48% recrystallised yield). R = 0.45 (CHCl₃:MeOH:TEA; 85: 10:5); p ^: 205 - 206 °C with decomp. (EtOAc).

A solution of Lyso-42 (1 equiv.) in DCM was treated with a 1 M solution of HCl ether (10 equiv.). HPLC analysis of Lyso-43 showed >99% purtity.

L s 44:

To a vial was added 4-Chloro-6-methoxy-2-methylquinoline (559.00 mg, 2.70 mmol, 2.40 equiv,), BINAP (105.00 mg, 0.17 mmol, 0.15 equiv.), Pd(OAc)₂ (19.00 mg, 0.08 mmol, 0.075 equiv.), finely ground K₃P0₄ (1061.00 mg, 5.00 mmol, 4.00 equiv.) and spermidine (180.0 iL, 1.13 mmol, 1.00 equiv.). The reagents were placed under a blanket of Argon; then a mixture of degassed dioxane and water (4 mL, 10: 1) was added. The reaction vial was sealed and heated to 120 ^°C for 12 hours. The reaction was then cooled to room temperature and filtered on a pad of celite; washing with chloroform (3 x 10 mL). Combined organic layers were acidified to pH 1 using 1M aqueous HCl solution (5 mL, 4.5 mmol, 4.00 equiv.) and diluted with water (20 mL). The resulting biphasic mixture was separated and the water layer was washed with chloroform (2 x 15 mL). The pH of the aqueous layer was adjusted to 11 using ammonium hydroxide. The now alkaline mixture was washed with chloroform (3 ^χ 20 mL). These 3 chloroform extracts were combined, washed with brine, dried over Na₂S0₄ and concentrated to give the product as an off-white solid (549.00 mg, 99% yield, > 90% purity). Recrystallisation from EtOAc afforded off-white crystals (123 mg, 22% recrystallised yield). R/= 0.05 (CHCl₃:MeOH:TEA; 85:10:5); Mp = 198 199 ^°C with decomp.(EtOAc).

A solution of Lyso-44 (1 equiv.) in DCM was treated with a 1 M solution of HCI in diethyl ether (10 equiv.). HPLC of Lyso-45 analysis showed >97% purity.

L s 46:

To a stirred mixture of Lys 44 (272.00 mg, 0.56 mmol, 1.00 equiv.) in formic acid (2.2 mL, 0.25 M) was added 37% aqueous formaldehyde (99.8 mg, 1.23 mmol, 2.20 equiv.). The reaction was heated to 100 ^°C and heating was maintained for 2 hours. TLC analysis revealed that all the starting material had been consumed. The reaction mixture was poured onto ice and basified via the addition of ammonium hydroxide (until pH > 9). The now alkaline mixture was extracted with chloroform (3 x 15 mL). Combined organic extracts were washed with brine and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as an off-white solid (261.00 mg, 93% yield, > 85%» purity). The material was purified via flash column chromatography (gradient: 100% EtOAc→ 84:15:1 ;

EtOAc:MeOH:TEA) to give a pure off-white solid (102.00 mg, 36% yield). R/= 0.15

(CHCl₃:MeOH:TEA; 85:10:5); Mp = 140 - 141 °C. Lys 47:

A solution of Lyso-46 (1 equiv.) in DCM was treated with a 1 M solution of HCI

ether (10 equiv.). HPLC analysis of Lyso-47 showed >99% purity.

Lyso-72:

To a microwave tube was added Bis-(hexamethyl)-triamine (0.73 g, 3.40 mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (1.86 g, 7.66 mmol, 2.25 equiv.), BINAP (0.11 g, 0.17 mmol, 0.05 equiv.), Pd(OAc)₂ (0.02 g, 0.10 mmol, 0.03 equiv.) and K₃P0₄ (2.17 g, 10.21 mmol, 3.00 equiv.). The vial was sealed and placed under argon. A solvent mixture of dioxane and water (10:1, 11 niL, 0.3 M) was added and the reaction was heated to 120 °C for 14 hours. The reaction was followed by TLC and NMR and once complete it was cooled to room temperature. Following this, the reaction was filtered on celite, washing through with MeOH (50 mL). The resulting solution was concentrated under reduced pressure. This solid was solubilised in CHC1₃ (50 mL) and 1 M_aq HCI (50 mL) was added. The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was further washed with CHC1₃ (2 ^χ 50 mL). These combined organic washings were discarded at this point.

Using ΝΗ₄ΌΗ, the pH of the aqueous layer was adjusted to 11, resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- PrOH (4:1; 3 x 40 mL). These combined organic extracts were washed with brine (120 mL) and dried over Na₂S0₄. Filtration followed by solvent evaporation afforded the product as a yellow solid (1.66 g, 90% yield). R/= 0.10 (EtOAc:MeOH:TEA; 80: 15:5); Mp = 100 - 102 °C.

A solution of Lyso-72 (1 equiv.) in DCM was treated with a 1 M solution of HCI

ether (10 equiv.). HPLC analysis of Lyso-73 showed >99% purity.

Lyso-74:

A solution of Lyso-72 (0.93 g, 1.73 mmol, 1.00 equiv.) in THF (17 mL, 0.1 M) was treated with formaldehyde 37 wt% (0.28 g, 3.45 mmol, 2.00 equiv.) then sodium

triacetoxyborohydride (1.46 g, 6.91 mmol, 4.00 equiv.). The resulting reaction was stirred at room temperature for 4 hours. Following this, 2 M_aq NaOH (30 mL) was added to the reaction mixture to break up any borane salts. This mixture was stirred at room temperature for 1 hour before the product was extracted into CHC1₃ (30 mL). The basic aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_aq HCI (30 mL). The mixture was stirred vigorously at room temperature for 30 minutes, affording the corresponding water soluble HCl-salt of the product. The mixture was allowed to settle and the resulting layers were separated. The aqueous layer, containing the product, was then further washed with CHCI₃ (2 x 30 mL). These combined organic washings were discarded at this point.

Using Ν¾ΌΗ, the pH of the aqueous layer was adjusted to 11 , resulting in the liberation of the free base of the product. The product was extracted into a solvent mixture of CHC1₃ and i- PrOH (4:1; 3 ^χ 30 mL). These combined extracts were washed with brine (120 mL) and dried over Na₂S0₄, Filtration followed by solvent evaporation afforded the product as a yellow solid (0.34 g, 36% yield). R/= 0.35 (EtOAc:MeOH:TEA; 80:15:5); Mp = 58 - 60 °C.

Lyso-75:

A solution of Lyso-74 (1 equiv.) in DCM was treated with a 1 M solution of HCI

ether (10 equiv.). HPLC analysis of Lyso-75 showed >99% purity.

Lyso-82:

To a 20 mL microwave vial was added 1,9-diamino nonane (0.27 g, 1.72 mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (1.00 g, 4.12 mmol, 2.40 equiv.), BINAP (0.05 g, 0.09 mmol, 0.05 equiv.), Pd(OAc)₂ (0.01 g, 0.05 mmol, 0.03 equiv.) and K₃P0₄ (1.09 g, 5.15 mmol, 3.00 equiv.). The vial was sealed and placed under argon. Fully degasses solvent dioxane:water (10:1, 6 mL, 0.3 M) was added and the reaction was heated to 120 °C for 12 hours. The reaction was followed by TLC and NMR. The reaction was allowed to cool to room temperature and filtered on celite, washing through with MeOH (100 mL). The filtrate was concentrated to give 1.09 g (crude yield 131%)) of an impure solid. This material was purified via flash column chromatography (eluent: Gradient; 100% EtOAc→ EtOAc:MeOH:TEA; 90:9:1) to give the product as a white solid (0.40 g, 37% yield). R = 0.40

(EtOAc:MeOH:TEA; 90:9:1); Mp = 158 - 160 °C. Lyso-83:

A solution of Lyso-82 (1 equiv.) in DCM was treated with a 1 M solution of HCI

ether (10 equiv.). HPLC analysis of Lyso-83 showed >90% purity.

Results

The present analogs have been designed to test an important factor: the role of the linker between the two chloroquinoline moieties. Toward that end, we have observed that replacement of the linker of LysOl with longer linkers, such as spermine (Lys 20 and 25) and spermidine (Lys21 and 26) lead to significant increases in potency. While the spermine linker introduces an additional nitrogen functionality (and an additional positive charge under physiological conditions), the spermidine linker is more similar to the LysOl linker. However, unlike the LysOl linker, the spermidine linker is not symmetrical. The spermidine linker contains three and four methylenes, respectively, between the three nitrogen atoms of the triamine linker, while the LysOl connector contains two methylenes between each of the three nitrogen atoms.

Cytotoxic activity of longer and assymetric linker compounds in cancer cells.

LN229 glioma cells, and A375P melanoma cells were plated in a 384 well format and compounds CQ, HCQ, and LysOl -Lys41 and 72-75 (structures provided in Appendix A, Chemical Structures) were delivered in concentrations between 0.01-10 micromolar using robotic assisted dispenser. After 72 hours incubation at 37 degrees, Alamar blue was applied and viability was determined using absorbance. Absorbance was normalized to DMSO control, and a log IC50 was estimated using Graphpad Prism software (Figure 11, Table 1). LN229 cells produced similar data (data not shown). Selected IC50s of the most promising compounds are presented in Figure 1 A. The increased cytotoxicity of longer linker and assyemtric compounds was confirmed in a 2 week colony formation assay using 5 different cell lines (Figure IB). Analysis of the cytotoxicity findings alone elucidated some key features of potential structure activity relationships. First, substitution of the central nitrogen substitution beyond methyl reduces activity. Second, there was no clear advantage of assymetric versus symmetric linkers (Figure 2A) with regards to cytotoxicity alone. Third, increasing Linker length from Lys05 was associated with increased activity but beyond a certain threshold of linker carbons, there is no increased potency for these compounds in the alamar blue viability assay (Figure 2B).

Longer linker compounds localize to the lysosome.

To confirm that dimeric CQ's with longer linkers retain their presumed lysosomal subcellular localization, Lys21/26 was functionalized into a fluorescent probe by attaching a Cy3 dye to the central nitrogen. This compound referred to as Lys21-Cy3 (Figure 3 A) was used to treat A375 melanoma (Figure 3B). Costaining with lysotracker green and subsequent fluorescent microscopy allowed the visualization of lysosomal localization for the Lys21 derivative. After 48 hours of treatment lysosomal mass had decreased and the red fluorescence was more cytoplasmic indicative of lysosomal rupture and diffusion of intact compound.

Longer linker compounds are associated with more profound autophagy modulation, reactive oxygen species (ROS) and apoptosis

As an initial method of determining the effects of the longer linker compounds on

autophagic vesicle accumulation, a series of most cytotoxic dimeric CQ's were used to treat 3 different cell lines (luM, 24 hours). Cell lysates were collected and immunoblotting against

LC3 and actin as performed (Figure 4). To determine if increased cytotoxicity associated with LysOl derivatives correlated with more potent inhibition of autophagy, LC3 immunoblotting was performed. LC3 is a ubiquitin-like protein which exists as a unconjugated form (LC3I) or conjugated to AV membranes (LC3II)(23). Quantification of LC3II/actin bands

demonstrated that all of the longer linker compounds were superior to Lys05 at modulating

LC3II levels. Although there was some variability across melanoma cell lines, in all cases the longest linker compound Lys75 produced the most consistent and substantial increase in the

LC3II/actin ration. To confirm that LC3II modulation observed was indeed due to the

inhibition of autophagic flux, A375P mCherry-GFP-LC3 cells were used in a high throughput phenotypic screen. In cells expressing this tandem fluorescent reporter construct autophagy induction results in accumulation of red puncta, because the green GFP fluorescence is

partially quenched when autophagic vesicles fuse with the lysosomes. In cells where

autophagic flux is inhibited distally due to lysosomal impairment, the green signal persists, turning puncta into a yellow color. Lys01-Lys27, HCQ, CQ and doxorubicin which serves as a control were administered to cells in concentrations ranging from 0-10 uM using pin wheel transfer, Operetta confocal imaging allowed image capture for high content analysis (Figure 5, Figure 6). Simple analysis of per cell (based on nuclear Hoescht staining) basis

demonstrated that at 10 μΜ concentrations, compounds which had previously been identified as having lower IC50^'in viability experiments also produced green/red fluorescent ratios>

3.5. Histograms of % cells with gree red ratio> 3.5 demonstrates that in some cases lower doses of the compounds also produced this phenotype indicative of effective autophagy

inhibition (Figure 7). Previous studies have demonstrated that CQ derivatives produce ROS in cancer cells. Each of the longer linker LysOl derivative was superior to Lys05 in inducing ROS (Figure 8 A). Lys26 (closely related to Lys21) and Lys75 produced the highest levels of ROS. Flow cytometry was used to quantify the green/red ration indicative of inhibition of autophagic flux in A375P mCHerry-eGFP-LC3 cells treated with longer linker dimeric CQs (Figure 8B). Again, Lys21 and Lys75 were most effective at inhibiting autophagic flux.

Finally, to determine if this inhibition of autophagic flux had a functional consequence of cell death, annexin V which reflects apoptosis was measured in A375 cells treated with

compounds. Smilar to the results observed with autophgic flux, Lys21 and Lys75produced the most apoptosis at the 24 hour timepoint (Figure 8C). To put this degree of apoptosis into clinical context, BRAF mutant melanoma cells were treated with longer linker dimeric CQs or standard of care BRAF, or BRAF (PLX4720) and MEK inhibitor (trametinib). The longer linker LysOl derivatives produced significantly more apoptosis than clinically approved

kinase inhibitors despite equimolar dosing (Figure 8D).

Dimeric CQ's with longer linkers impair growth and invasion of melanoma in a 3 dimensional tissue like culture system.

The 3D spheroid model has been used extensively to mimic the tumor microenvironment (Ma et al Clin Can Research 2011). Autophagy levels are more refelective of in vivo autophagy dynamics in the 3D models compared to traditional two dimensional cell culture. In this format cancer cells are grown as spheroids and then implanted in a collagen matrix. The spheroids thn begin to invade and grow within this tissue microenvironment. Treatment with various longer linker dimeric chloroquines once again demonstrated that Lys75 was the most potent compound at inhibiting growth and invasion of the 3D spheroids (Figure 9A, B). Longer linker compounds

To further investigate the role of linker length with regards to potency we prepare a series of compounds with even longer linkers ( Figure 13 Compounds Lys86-Lys97). We also revisit the effects of quinolone ring substitution in the setting of these longer linked compound (Figure 13).

Summary and Conclusion

Increasing linker length between quinolone rings in dimeric chloroquines significantly improved anti-cancer cytoxicity, ROS production, and autophagy inhibition. Assyemtric

dimeric chloroquines showed similar anticancer cytotoxicity in 72 hour viability assays to comparable symmetric counterparts. However in the case of Lys21, superior autophagy

inhibition and ability to induce apoptosis was observed compared to comparable compounds that were symmetric.

The GI toxicity associated with Paneth cell dysfunction observed at LD30 doses of

Lys05, support the mechanism of action of the present invention, and also suggests that colon cancers, which often share features with Paneth cells, may be a tumor type, among others, that may be particularly sensitive to Lys05 and its optimized derivatives. Additional cancers which represent particularly important targets include melanoma, and non-small cell lung cancer, since melanoma cell lines demonstrated the highest difference in sensitivity to LysOl compared to HCQ, and an EGFR mutated lung cancer cell line demonstrated sensitivity to both HCQ and Lys05. Further mechanistic studies are planned that will potentially identify pharmacodynamics assays that guide drug development. Pharmacokinetic studies are planned in mice to establish initial in vivo profile.

Advantages over other similar technologies: The present invention represents the most potent bisaminoquinoline derivatives reported thus far in cell lines. Other autophagy compounds are not focused on the lysosome as a target and consequently do not evidence the level of activity of compounds according to the present invention. A. References Cited:

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17. Vennerstrom JL, Ager AL, Jr., Dorn A, Andersen SL, Gerena L, Ridley RG, and Milhous WK. Bisquinolines. 2. Antimalarial N,N-bis(7-chloroquinolin-4-yl)heteroalkanediamines. J Med Chem. 1998;41(22):4360-4.

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Claims

What is claimed is:

1. A compound of Formula IA:

wherein R¹ and R¹ are each independently H, F, CI, Br, I, CN, N0₂, optionally substituted C]-C₆ alkyl, optionally substituted 0-d-C₆ alkyl, optionally substituted C₂-C₇ acyl, optionally substituted -(NH)-acyl, or optionally substituted C₂-C₇ ester;

R and R' are each independently H, a Ci-C₆ optionally substituted alkyl group, a C1-C7 optionally substituted acyl group, a C2-C7 optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

R" is H, Cy¹ or (C=0)_z-G, where Cy¹ is an optionally substituted cycloalkyl, aryl or heteroaryl group G is an optionally substituted C0-C12 alkyl, alkene or alkynyl group (wherein optional substiuents include a C1-C12 alkyl, alkene or alkynyl group substituted by (N— R^J)-( C]-C alkyl, alkene or alkynyl group)_z-(Cy²)_x, where R^J is H or a Ci-C₈ alkyl, alkene or alkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is an optionally substituted aryl or heteroaryl group);

R^M is independently at each occurrence H, F, CI, Br, I, an optionally substituted C1-C12 alkyl, alkene, alkynyl or alkoxy group;

Each p in linker group L^1A is independently 1-10;

and the pharmaceutically acceptable salts, enantiomers, diastereomers, solvates and polymorphs thereof.

2. A compound of Formula IB

IB

wherein R¹ and R¹ are each independently H, F, CI, Br, I, CN, N0₂, optionally substituted C]-C₆ alkyl, optionally substituted 0-Ci-C₆ alkyl, optionally substituted C₂-C₇ acyl, an optionally substituted -(NH)-acyl, or optionally substituted C2-C₇ ester;

R and R' are each independently H, a Q-Q optionally substituted alkyl group, a C_!-C₇ optionally substituted acyl group, a C₂-C₇ optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

R" is H, Cy¹ or (C=0)_z-G, where Cy¹ is an optionally substituted cycloalkyl, aryl or heteroaryl group G is an optionally substituted C₀-Ci₂ alkyl, alkene or alkynyl group (wherein optional substiuents include a C1-C12 alkyl, alkene or alkynyl group substituted by (N— ^J)-( C]-C₈ alkyl, alkene or alkynyl group)_z-(Cy²)_x, where R^J is H or a Q-Cg alkyl, alkene or alkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is an optionally substituted aryl or heteroaryl group);

R^M is independently at each occurrence H, F, CI, Br, I, an optionally substituted C₀-C₁₂ alkyl, alkene, alkynyl or alkoxy group;

p and p' in linker L^1B are each independently 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10),

3. A compound of Formula IC :

IC wherein R¹ and R¹ are each independently H, F, CI, Br, I, CN, N0₂, optionally substituted Q-C₆ alkyl, optionally substituted 0-Ci-C₆ alkyl, optionally substituted C₂-C₇ acyl, an optionally substituted -(NH)-acyl, or optionally substituted C₂-C₇ ester;

R and R' are each independently H, a C C₆ optionally substituted alkyl group, a Q-C7 optionally substituted acyl group, a C₂-C₇ optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

R" is H, Cy¹ or (C=0)_z-G, where Cy¹ is an optionally substituted cycloalkyl, aryl or heteroaryl group G is an optionally substituted C₀-Ci₂ alkyl, alkene or alkynyl group (wherein optional substiuents include a C_\-Cn alkyl, alkene or alkynyl group substituted by (N— R^J)-( Ci-C₈ alkyl, alkene or alkynyl group)_z-(Cy²)_x, where R^J is H or a C C₈ alkyl, alkene or alkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is an optionally substituted aryl or heteroaryl group);

R^M is independently at each occurrence H, F, CI, Br, I, an optionally substituted Co-Ci₂ alkyl, alkene, alkynyl or alkoxy group;

Each p in linker group L is independently 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);

and the pharmaceutically acceptable salts, enantiomers, diastereomers, solvatess and polymorphs thereof.

4. A compound of Formula ID :

wherein R¹ and R¹ are each independently H, F, CI, Br, I, CN, N0₂, optionally substituted Ci-C₆ alkyl, optionally substituted O-Q-Q alkyl, optionally substituted C₂-C₇ acyl, optionally substituted -(NH)-acyl, or optionally substituted C₂-C₇ ester;

R and R' are each independently H, a Ci-C₆ optionally substituted alkyl group, a Ci-C₇ optionally substituted acyl group, a C₂-C₇ optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

R" is H, Cy¹ or (C=0)_z-G, where Cy¹ is an optionally substituted cycloalkyl, aryl or heteroaryl group G is an optionally substituted C₀-Ci₂ alkyl, alkene or alkynyl group (wherein optional substiuents include a Ci-Ci₂ alkyl, alkene or alkynyl group substituted by (N-— R^J)-( Cj-Cg alkyl, alkene or alkynyl group)_z-(Cy²)_x, where R^J is H or a Ci-Cg alkyl, alkene or alkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is an optionally substituted aryl or heteroaryl group);

Linker group L^1D is an optionally substituted alkylene group containing from 1-20 methylene groups;

5. A compound of formula IE:

IE

Wherein R¹ and R¹ are each independently H, halo (F, CI, Br or I), CN, N0₂, optionally substituted C]-C₆ alkyl, optionally substituted 0-Ci-C6 alkyl, optionally substituted -(NH)- acyl, optionally substituted C₂-C₇ acyl or optionally substituted C₂-C₇ ester (oxycarbonyl ester or carboxyester, preferably carboxyester);

R^M is independently at each occurrence H, F, CI, Br, I, an optionally substituted Q-C1₂ (preferably Ci-C₈) alkyl, alkene, alkynyl or alkoxy group;

R and R' are each independently H, a Ci-C6 optionally substituted alkyl group, a Ci-C₇ (preferably C₂-C₇) optionally substituted acyl group, a C₂-C₇ optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R' is bonded);

Linker group L^1E is a ^CH₂Y^X-^Y'CF¾r- ^ _{Qr a} ^CH.-CH,— Z^A' group wherein at least one of the CH₂ groups in L^1E is optionally substituted with an optionally substituted Ci-C₆ alkyl group including a cyclic alkyl group, each of which alkyl groups is optionally substituted, or at least two methylene groups (preferably two adjacent methylene groups) are optionally substituted with alkylene groups to form an optionally substituted C₃-C₈ cycloalkyl group, or linker group L^1E is a (poly)ethylene glycol group having from 1 to 20 ethylene glycol units, each (poly)ethylene glycol group being optionally substituted at one or both of its distal ends with an A group, or L is a linear group containing from 1 to 5 non-contiguous amine groups N-R", each amine group being separated from an adjacent amine group by an alkylene group containing from 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2, 3, 4, 5 or 6) methylene groups or an ethylene glycol containing group having from 1 to 6 (preferably 2 to 6, 3 to 6 or 4, 5 or 6) ethylene glycol units;

X is absent, (CH₂)_j O, S or N-R"; Y is absent, CH₂, O, CH₂0 or N-R" and Y' is absent CH₂, O, OCH₂ or N-R", with the proviso that when one or more of X, Y and Y' is present, each of X and Y, X and Y' or Y and Y', when present, forms a stable bond;

R" is H or an optionally substituted Cj-C₆ alkyl group or an amine protecting group;

each j is independently 1, 2, 3, 4, 5 or 6;

each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 with the proviso that when n is 0, X is (CH₂)j where j is at least 1 and at least one CH₂ group is optionally substituted, preferably with a C1-C3 alkyl group which itself is optionally substituted with one or two hydroxyl groups or one, two, or three halo groups;

A is absent, C=0 or (CH₂)j and A' is C=0 or (CH₂)_j wherein at least one CH₂ group in A or A' is optionally substituted, preferably with a C C3 alkyl group which is itself optionally substituted;

Z is O or N-R^z;

R^z is H or an optionally substituted Cj-C₃ alkyl group,

6. The compound according to claim 5 wherein at least two R^M are other than H.

7. The compound according to claim 5 or 6 wherein R¹ and R¹ are each independently H or a halo group (preferably F or CI).

8. The compound according to any of claims 5-7 wherein L is an alkylene group containing from 6 to 20 methylene groups (C₆-C₂₀ alkylene group).

9. The compound according to any of claims 5-7 wherein L is a (poly)ethylene glycol group having from 1 to 20 ethylene glycol units optionally substituted at one or both distal ends of said (poly)ethylene glycol group with an A group.

10. A compound according to any of claims 5-7 wherein L is a linear group containing from 1 to 5 non-contiguous amine groups N-R", each amine group being separated from an adjacent amine group by an alkylene group containing 1, 2, 3, 4, 5, 6, 7, or 8 methylene groups or an ethylene glycol containing group containing 1, 2, 3, 4, 5 or 6 ethylene glycol units.

11. A compound of formulae IIA, IIB, IIC or IID, and the pharmaceutically acceptable salts, enantiomers, diastereomers, solvates or polymorphs thereof:

IIB:

where X is a halogen, preferably F or CI.

12. A compound according to claim 5 wherein X is CI (Lys-74) or a pharmaceutically acceptable salt thereof.

13. A compound selected from the group consisting of the compounds listed in Figures 10, 11, 12 or 13 herein, and the pharmaceutically acceptable salts, enantiomers, diastereomers, solvates and polymorphs thereof.

14. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound according to any one of claims 1-13 in combination with a pharmaceutically acceptable carrier, additive or excipient.

15. The composition according to claim 14 comprising a compound according to claim 11.

16. The composition according to claim 15 wherein said compound is Lys-21, Lys-25, Lys-2, Lys-75, a mixture thereof or a pharmaceutical acceptable salt(s) thereof.

17. The composition according to any of claims 14-16 further comprising an effective amount of at least one additional anticancer agent.

18. The composition according to claim 17 wherein said anticancer agent is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK- 1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inliibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PT3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint- 1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody or a mixture thereof.

19. The composition according to claim 17 wherein said anticancer agent is selected from the group consisting of everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101 , pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY- 142886), AMN-107, TK1-258, GSK461364, AZD 1 152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin,

ofatumumab. zanolimumab. edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol. Bio 1 11 , 131-I-TM-601 , ALT-1 10, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR. INO 1001 , IPdR, KRX-0402, lucanthone, LY

317615, neuradiab, vitespan. Rta 744, Sdx 102, talampanel, atrasentan, Xr 311 , romidepsin, ADS- 100380, sunitimb, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK- 304709, seliciclib; PD0325901 , AZD-6244. capecitabine, 1, -Glutamic acid, N -[4-[2-(2- amino-4,7-dihydro-4-oxo-l H - pyrrolo[2,3- d ]pyrimidin-5-yl)eihyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-l Cl l , CHlR-258,); 3-[5-(methylsulfonylpiperadinemethyl)- indolylj-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D- Ser(Bu t ) 6 .Azgly 10 ] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t )-Leu-Arg-Pro- Azgly-NH ₂ acetate

[C59¾₄Ni80i₄ -(C₂H402) where x = 1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-

166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A. F -228, SU11248, sorafenib,

chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate. pentostatin, plicamycin. porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol. valrubicin. mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovasta BMS-275291 , squalamine, endostatin, SU5416, SI J 6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA- 923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR- 3339, ZK186619, PT 787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP- 23573, RAD001 , ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa- 2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11 , dexrazoxane, alemtuzumab, all- transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine,

diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib and mixtures thereof.

20. A method of inhibiting autophagy in a biological system in which inhibition of autophagy is desired, said method comprising exposing said biological system to an effective amount of at least one compound according to any of claims 1-13 hereof.

21. A method of inhibiting or treating cancer in a patient in need comprising

administering to said patient an effective amount of at least one composition according to any of claims 14-19.

22. The method according to claim 21 , wherein said cancer is metastatic.

23. The method according to claim 21 , wherein said cancer is recurrent.

24. The method according to claim 21, wherein said cancer is a drug-resistant cancer.

25. A method of reducing the likelihood that cancer will occur in a patient or that a cancer will metastasize in a patient comprising administering at least one composition according to any of claims 14-19, optionally in combination with at least one additional anticancer agent.

26. The method according to any of claims 21-25, wherein said cancer is a carcinoma, cancer of the esophagus, head, kidney, liver, lung, nasopharyngeal, neck, ovary, pancreas, prostate, and stomach; a leukemia, a malignant lymphoma, a malignant melanoma;

myeloproliferative diseases; a sarcoma, a tumor of the central nervous system, a germ-line tumor, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma, or a mixed type of neoplasia.

27. The method according to claim 26, wherein said leukemia is acute myelogenous leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia,

micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia

28. The method according to claim 26, wherein said lymphoma is Burkitt's lymphoma, Non-Hodgkin's lymphoma or B-cell lymphoma.

29. The method according to claim 26, wherein said sarcoma is Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral

neuroepithelioma or synovial sarcoma.

30. The method according to claim 26, wherein said tumor of the central nervous system is a glioma, astrocytoma, oligodendroglioma, ependymoma, gliobastoma, neuroblastoma, ganglioneuroma, ganglioglioma, meduUoblastoma, pineal cell tumor, meningioma, meningeal sarcoma, neurofibroma, or Schwannoma.

31. The method according to claim 26, wherein said germ-line tumor is bowel cancer, breast cancer, prostate cancer, cervical cancer or uterine cancer.

32. The method according to claim 26, wherein said lung cancer is small cell lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, metastatic pleural mesothelioma, small cell lung cancer or non-small cell lung cancer.

33. The method according to claim 26, wherein said mixed neoplasia is carcinosarcoma and Hodgkin's disease and said tumors of mixed origin is Wilms' tumor and

teratocarcinomas .

34. The method according to claim 26, wherein said cancer is ovarian, breast, colon, head and neck, medulloblastoma or B-cell lymphoma,

35. The method according to claim 26, wherein said cancer is melanoma or non-small cell lung cancer.

36. A method of treating a disease state or condition in a patient in need wherein said disease state or condition responds favorably to inhibition of autophagy comprising administering to said patient an effective amount of a compound according to any of claims 1-13 to said patient.

37. The method according to claim 36, wherein said disease state or condition is rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria or Sjogren's disease.

38. The method according to claim 36 or 37, wherein said disease state is malaria.

39. Use of a compound according to any of claims 1-13 in the manufacture of a

medicament for the inhibiton of autophagy in a biological system in which inhibition of autophagy is desired.

40. Use of a compound according to any of claims 1-13 in the manufacture of a

medicament for use in inhibiting or treating cancer in a patient in need, optionally in combination with at least one additional anticancer agent.

41. The use according to claim 40 wherein said cancer is metastatic.

42. The use according to claim 40 wherein said cancer is drug resistant.

43. Use of a compound according to any of claims 1-13 in the manufacture of a

medicament for reducing the likelihood that cancer will occur in a patient or that a cancer will metastasize in a patient, optionally in combination with at least one additional anticancer agent.

44. Use according to any of claims 40-43, wherein said cancer is a carcinoma, cancer of the esophagus, head, kidney, liver, lung, nasopharyngeal, neck, thyroid, ovary, pancreas, prostate, and stomach; a leukemia, a malignant lymphoma, a malignant melanoma;

myeloproliferative diseases; a sarcoma, a tumor of the central nervous system, a germ-line tumor, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma or a mixed type of neoplasia.

45. Use according to claim 44, wherein said leukemia is acute myelogenous leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APL), acute T-cell

lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia,

46. Use according to claim 44, wherein said lymphoma is Burkitt's lymphoma, Non- Hodgkin's lymphoma or B-cell lymphoma.

47. Use according to claim 44, wherein said sarcoma is Ewing's sarcoma,

hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral

neuroepithelioma or synovial sarcoma.

48. Use according to claim 44, wherein said tumor of the central nervous system is a glioma, astrocytoma, oligodendroglioma, ependymoma, gliobastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell tumor, meningioma, meningeal sarcoma, neurofibroma, or Schwannoma.

49. Use according to claim 44, wherein said germ-line tumor is bowel cancer, breast cancer, prostate cancer, cervical cancer or uterine cancer.

50. Use according to claim 44, wherein said lung cancer is small cell lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, metastatic pleural mesothelioma, small cell lung cancer or non-small cell lung cancer.

51. Use according to claim 44, wherein said mixed neoplasia is carcinosarcoma and Hodgkin's disease and said tumors of mixed origin is Wilms' tumor and teratocarcinomas.

52. Use according to claim 44, wherein said cancer is ovarian, breast, colon, head and neck, medulloblastoma or B-cell lymphoma,

53. Use according to claim 44, wherein said cancer is melanoma or non-small cell lung cancer.

54. Use according to claim 52, wherein said cancer is colon cancer.

55. Use according to any of claims 40-54, wherein said additional anticancer agent is selected from the group consisting of everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101 , pazopanib, GSK690693, R I A 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258. GSK461364, AZD 1 152, enzastaurin, vandetanib, ARQ-197, M -0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab. amrubicin. oregovomab, Lep-etu, nolatrexed, azd2 I 71 , batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 11 1 , 13 l-I-TM-601 , ALT- 1 10, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, I O 1001 JPdRj KRX-0402, lucanthone, LY

317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 31 1 , romidepsin, ADS- 100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5 '-deoxy-5 -11 uorouri dine, vincristine, temozolomide, ZK- 304709, seliciclib; PD0325901 , AZD-6244, capecitabine, L-Glutamic acid, N -[4-[2-(2- amino-4,7-dihydro-4-oxo-l I f - pyrrolo[2,3- d jpyrimidi n- 5 -yl)eth ] j benzoy 1 | -. di sodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-lCl 1 , CHIR-258,); 3-[5-(methylsulfonylpiperadinemetliy])- indolylj-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D- Ser(Bu t ) 6 .Azgly 10 ] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t )-Leu-Arg-Pro- Azgly-NH ₂ acetate

[C5 ¾₄Ni₈0i4 -(02Η₄0₂)χ where x = 1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714: TAK- 165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI- 166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib,

K.RN951 , aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette- Gucrin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine,

chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer. procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine.

floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valnibicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291 , squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin- 12. 1M862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA- 923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR- 3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP- 23573, RAD001 , ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa- 2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11 , dexrazoxane, alemtuzumab, all- transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine,

56. Use according to any of claims 40-54, wherein said additional anticancer agent is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PI -1 modulator, a Be 1-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR-TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an A T inhibitor, a JAK/STAT inhibitor, a checkpoint- 1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody or a mixture thereof.

57. Use of a compound according to any of claims 1-13 in the manufacture of a medicament for treating a disease state or condition in a patient in need wherein said disease state or condition responds favorably to inhibition of autophagy.

58. Use according to claim 57, wherein said disease state or condition is rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria or Sjogren's disease.

59. Use according to claim 58, wherein said disease state or condition is malaria.