WO2009034464A2 - Indole related compounds with physiological activity - Google Patents

Abstract

The invention relates to compounds of Formula I or Ia having dynamin protein inhibitory activity. Compounds embodied by the invention have application in inhibiting cellular endocytosis and cytokinesis, and may be used in the prophylaxis or treatment of various diseases and conditions involving endocytosis or cell proliferation, such as seizures, epilepsy and cancer. Particularly preferred compounds provided by Formula I and Ia are indoles.

Description

INDOLE RELATED COMPOUNDS WITH PHYSIOLOGICAL ACTIVITY

FIELD OF THE INVENTION

The present invention relates to compounds having therapeutic applications and methods for use of the compounds.

BACKGROUND OF THE INVENTION

Endocytosis is the process whereby mammalian cells take up extracellular material and recycle their surface membranes by the formation of numerous membrane vesicles at the plasma membrane. Whilst there are numerous endocytic pathways that have been characterised two are biochemically well-characterised. The first is the rapid synaptic vesicle endocytosis (SVE) that follows vesicle exocytosis in nerve terminals. SVE operates primarily to retrieve empty SVs for later refilling for the next round of synaptic transmission. It requires the GTPase activity of dynamin I. The second is clathrin- mediated endocytosis (CME) which internalises ligand-bound cell surface receptors via clathrin-coated pits in all cells. CME requires the GTPase activity of dynamin II. It also acts as the main entry point for plasma membrane components (such as the receptor- ligand complexes and membrane lipids) or for extracellular fluid into cells.

There are three major dynamin isoforms, with dynamin I being found primarily in neurons, dynamin II being ubiquitously expressed and dynamin IH in neurons and testes. All three dynamins share four domains: a GTPase (required for vesicle fission), pleckstrin homology (PH) (targeting domain and potentially a GTPase inhibitory module), a GTPase effector domain (GED, which controls dynamin self-assembly into rings), and a proline- rich domain (PRD) (which interacts with proteins containing an SH3 domain, and is the site for dynamin phosphorylation in vivo). Each dynamin plays a role in a variety of human pathological conditions such as Alzheimer's disease, Huntington's disease, Stiff- person syndrome, Lewy body dementias, and Niemann-Pick type C diseaseJ^M] More recently mutations in dynamin II have been implicated in the etiology of Charcot-Marie- Tooth syndrome, a demyelinating condition and one of the most common known genetic disorders affecting ~1 in 3000^[5] as well as centronuclear myopathy. ^[6] Viruses, toxins and symbiotic microorganisms also utilise endocytic pathways to gain entry into cells.^[7] Neurotoxins botulinum and tetanus inhibit transmitter release from synapses causing two severe neuroparalytic diseases, tetanus and botulism. Their action is dependent on their internalisation via endocytosis into nerve terminals.^¹ A broad variety of low-specificity endocytosis inhibitors are known to exist: cationic amphiphilic drugs (eg chlorpromazine), concanavalin A, phenylarsine oxide, dansylcadaverine, intracellular potassium depletion, intracellular acidification and decreasing cell culture medium temperature to 4°C. Judicious use of these non-specific inhibitors has contributed considerably to a better understanding of endocytosis. For example, the entry of human polyomavirus, JCV, into cultured cells (causing progressive multifocal leukoencephalopathy) is prevented by chlorpromazine.

SUMMARY OF THE INVENTION

hi an aspect of the invention there is provided a compound of Formula 1, or a physiologically acceptable salt or prodrug thereof, wherein:

Formula I and ring A is an aryl ring, a C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, a heterocycloalkyl or heterocycloalkenyl, the heterocycloalkyl or heterocycloalkenyl having 4 to 6 ring atoms, or a heteroaryl with 5 to 6 ring atoms, where ring A is optionally substituted; ring B is an aryl ring, a C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, a heterocycloalkyl or heterocycloalkenyl, the heterocycloalkyl or heterocycloalkenyl having 4 to 6 ring atoms, or a heteroaryl with 5 to 6 ring atoms;

Ri is alkyl, alkenyl, alkylcarboxy, alkylthiocarboxy, alkylaryl, alkoxy, alkoxy- alkyl, aryl, aryloxy, aryloxyalkyl, alkoxyalkyl, alkylamino, alkenylamino, aminoalkyl, aminoalkenyl, aminoaryl, arylamino, alkylaminoaryl, alkenylaminoaryl, arylaminoalkyl, arylaminoalkenyl, carbocyclic, heterocyclic, alkyl C₃-C₆ cycloalkyl amino, alkyl C₃-C₆ cycloalkenyl amino, alkenyl C₃-C₆ cycloalkyl amino, alkenyl C₃-C₆ cycloalkenyl amino, alkyl C₃-C₆ heterocycloalkyl amino, alkenyl C₃-C₆ heterocycloalkyl amino, alkyl C₃-C₆ heterocycloalkenyl amino, alkenyl C₃-C₆ heterocycloalkenyl amino or an alkylNR group;

R₃ is a C₁-C₄ alkylNR group, C₁-C₄ alkylNR group, lower alkyl, lower alkenyl, aryloxy-lower alkyl, lower alkoxy-lower alkyl, or R₃'-(Z)_n, where R₃' is a lower alkyl, lower alkenyl, aryloxy-lower alkyl, or lower alkoxy-lower alkyl;

NR is N(CH₃ )₂, NCH(CH₃ )₂, an amide, or an N containing heterocyclic ring with 5 to 6 ring members;

Z is a lower alkenyl, cyclo-lower alkenyl, aryl lower alkenyl, lower alkyl, aryloxy- lower alkyl, or lower alkoxy-lower alkyl or alkylamino, alkenylamino, aminoalkyl, aminoalkenyl, aminoaryl, arylamino, alkylaminoaryl, alkenylaminoaryl, arylaminoalkyl, arylaminoalkenyl, carbocyclic, heterocyclic, alkyl C₃-C₆ cycloalkyl amino, alkyl C₃-C₆ cycloalkenyl amino, alkenyl C₃-C₆ cycloalkyl amino, alkenyl C₃-C₆ cycloalkenyl amino, alkyl C₃-C₆ heterocycloalkyl amino, alkenyl C₃-C₆ heterocycloalkyl amino, alkyl C₃-C₆ heterocycloalkenyl amino, alkenyl C₃-C₆ heterocycloalkenyl amino or an alkylNR group; and n is an integer of from 1 to 12.

In another aspect of the invention there is provided a compound of Formula Ia, or a physiologically acceptable salt or prodrug thereof, wherein:

Formula Ia where R₁ and R₃ are as defined in Formula I; and

R₂ is H, lower alkyl, lower alkenyl, aryloxy-loweralkyl, loweralkoxy-lower alkyl, or R₂'-(Z)_n, where R₂'is a lower alkyl, lower alkenyl, aryloxy-lower alkyl, lower alkoxy-lower alkyl, or forms a 5 or 6 membered ring with R₁ optionally including N; and

Z and n are as defined in Formula I. A compound of Formula I or Ia may inhibit dynamin protein activity and so have application in inhibiting cellular endocytosis, and the prophylaxis or treatment of diseases or conditions responsive to inhibition of endocytosis. Diseases and disorders responsive to inhibition of endocytosis include neurological related diseases and conditions including epilepsy. Moreover, the inventors have previously found that Dynamin II has a role in cytokinesis. Hence, inhibition of dynamin activity as described herein may also have application in the treatment of diseases and conditions involving cellular proliferation including cancer.

Hence, in another aspect of the invention there is provided a method for inhibiting a dynamin protein, comprising contacting the dynamin protein with a compound of Formula I or Ia, or a physiologically acceptable salt or prodrug thereof. In another aspect of the invention there is provided a method for inhibiting endocytosis in a cell, comprising treating the cell with an effective amount of a compound of Formula I or Ia, or a physiologically acceptable salt or prodrug thereof.

In another aspect there is provided a method for the prophylaxis or treatment of a disease or condition in a mammal, comprising administering to the mammal an effective amount of a compound of Formula I or Ia, or a physiologically acceptable salt or prodrug thereof, the disease or condition being selected from the group consisting of cell proliferative diseases and conditions, neuropathic pain, epilepsy, seizures, psychotic disorders, psychosis, β-amyloid associated diseases, aberrant up-regulated neuronal excitation, and diseases and conditions mediated by, or associated with, synaptic vesicle endocytosis, synaptic signal transmission, or cell vesicle trafficking.

In another aspect of the invention there is provided a pharmaceutical composition comprising a compound of Formula I or Ia, or a physiologically acceptable salt or prodrug thereof, together with a pharmaceutically acceptable carrier.

The term "dynamin protein" is to be taken to encompass members of the family of dynamin-related GTPases, including classical dynamins (dynamin I, dynamin II, and dynamin III), active fragments of dynamin retaining GTPase activity, modified forms of dynamin, GTPase domains of dynamin, dynamin-like proteins, OPAl, Mx proteins, mitofusins and guanylate-binding proteins/alastins. ^ Generally, the dynamin protein will be dynamin I or dynamin II. A compound of Formula I or Ia may inhibit the GTPase activity of the dynamin protein.

The mammal can be any mammal treatable by a method embodied by the invention, including but not limited to members of the rodent, canine, feline, equine, ovine and primate animal families. In at least some embodiments, the mammal is a human being.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of this application. Throughout this specification the word "comprise", or variations such as

"comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers, integers or steps.

The features and advantages of the invention will become further apparent from the following detailed description of embodiments thereof together with the accompanying drawings.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 highlights the basic design approaches used in developing indole based libraries of dynamin GTPase inhibitors.

Figure 2 is a double reciprocal plot showing the kinetics of a compound embodied by the invention against increasing concentrations of GTP in the inhibition of dynamin GTPase activity. Figure 3 shows indole compounds 25, 33, 34-1 and 35 block internalization of Tf-

TxR in COS-7 cells. A. COS-7 cells were preincubated with vehicle only and then incubated with Tf-TxR (Al) for 15 min at 37°C, acid washed, fixed and internalised Tf- TxR was detected by fluorescence microscopy. Nuclei were stained with DAPI to show the position of the cells (A2). The combined image (A3) shows the position of the cell nucleus and the internalised Tf-TxR (red). B - E. As for A with the preincubation of (B) 30 μM indole 25 for 10 minute prior to the addition of Tf-TxR; (C) 30 μM 33 for 10 minute prior to the addition of Tf-TxR; (D) 30 μM indole 34-1 for 10 minute prior to the addition of Tf-TxR; and (E) 30 μM 35 for 10 minute prior to the addition of Tf-TxR. Figure 4 shows dynamin inhibitors cause cytokinesis failure in HeLa cells.

Figure 5 shows indole compound 34-2 causes cell death following cytokinesis failure. HeLa cells were synchronised at the G2/M boundary with the Cdkl inhibitor, RO-3306 and then released into mitosis. Immediately following RO-3306 wash-out, cells were treated with the indicated dynamin inhibitor (10 μM) and monitored by time-lapse microscopy. The graph illustrates the percentage of those cells failing cytokinesis that either underwent cell death or were viable by the end of the experiment (20 h post-release from RO-3306; n>50 per sample).

Figure 6 shows indole compound 34-2 does not induce cell death in two non- tumourigenic cell lines: hTERT breast epithelial cells (hTERT) and human foreskin fibroblast (HFF) cells. Asynchronously growing HeLa, hTERT and HFF cells were treated with MiTMAB or 34-2 (10 μM) and immediately commenced monitoring by time- lapse microscopy. Shown are representative microscopy images of the final frame of each time-lapse movie, correlating to time-point 20 h. 34-2 causes cell death in the human cervical carcinoma cell line, HeLa, and not in the non-tumourigenic cells, hTERT and HFF.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In this specification, the term "alkyl" used either alone or in a compound word such as alkylaryl refers to a straight chain, branched or mono- or polycyclic alkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, ώo-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, amyl, iso-amyl, sec-amyl, 1 ,2-dimethylpropyl, 1,1- dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1 -dimethylbutyl, 2,2-dimethylbutyl, 3 ,3 -dimethylbutyl, 1 ,2-dimethylbutyl,

1 ,3 -dimethylbutyl, 1 ,2,2-trimethylpropyl, 1 , 1 ,2-trimethylpropyl. Examples of cyclo alkyls include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term "alkyl sulphone" refers to a sulphone group including 2 alkyl substituents on the sulphone S atom where the alkyl groups may be the same or different. The term "alkoxy" refers to an alkyl group with an oxygen radical substituent.

Examples include methoxy, ethoxy, n-propoxy, n-butoxy, and tert-butoxy. The term "alkenyl" refers to a straight chain, branched or cyclic alkenyl with one or more double bonds. Examples of alkenyl include vinyl, allyl, 1 -methylvinyl, butenyl, wo-butenyl, and 3-methyl-2-butenyl.

The term "aryl" used either alone or in compound words such as "alkylaryl", refers to a single, polynuclear, conjugated or fused aromatic hydrocarbon or aromatic heterocyclic ring system. Examples of aryl include phenyl and naphthyl. When the aryl comprises a heterocyclic aromatic ring system, the aromatic heterocyclic ring system can contain one or more heteroatoms independently selected from N, O and S and will normally contain 5 or 6 ring members. The term "heteroaryl" refers to a heterocyclic aromatic ring system. Heterocyclic, heteroaryl, carbocyclic, aryl and other ring systems useful in a compound of Formula I will normally have from 3 to 7 ring atoms unless specified otherwise, and may contain one or more double bonds. These rings may contain one or more substituents as described above. Heterocyclic groups that may be utilized in embodiments of the invention include pyrazinium, imadazolyl, pyranyl, thiopyranyl, morpholinyl, isobenzylfuranyl, furanyl, chromenyl, pyrrolinium,

2H-pyrrolyl, pyrazolinium, pyridinium, pyridazinium, indolizinyl, isoindolyl, JH-indolyl, indolyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, pthalazinyl, naphthyridinyl quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, thienyl, thiophenyl, maleimidyl, thiazolo, aminothiazolo, benzothienyl, and isosteres of the foregoing. The heterocyclic, heteroaryl, carbocyclic, aryl, and other ring systems can be substituted (e.g., they may have one or more substituents listed above) or be unsubstituted.

The term "aryloxy" refers to an aryl group with an oxygen radical substituent. Examples include phenoxy.

The term "carbocyclic" refers to optionally substituted cycloalkyl and cycloalkenyl groups. Cycloalkyl and cycloalkenyl groups of compounds embodied by the invention will normally have 3 to 6 ring atoms.

The term "lower alkyl" encompasses an optionally substituted C₁-C₆ branched or unbranched alkyl chain. Similarly, the term "lower alkenyl" encompasses an optionally substituted C₁-C₆ alkenyl group with at least one double bond. The term "carboxy lower alkenyl" refers to a lower alkenyl substituted with a carboxy group.

The term "heterocycloalkyl" refers to a ring group with no double bonds in its ring and including one or more ring atoms. The term "heterocycloalkenyl" refers to a ring group with one or more double bonds in its ring and including one or more heteroatoms.

The term "hydroxy lower alkenyl" refers to a lower alkenyl substituted with a hydroxy group. The term "amido lower alkenyl" refers to a lower alkenyl substituted with an amido group.

The term "mono or di-halo lower alkenyl" refers to a lower alkenyl substituted with 1 or 2 halo atoms which may be the same or different.

The term "amino lower alkenyl" refers to a lower alkenyl substituted with an amino group.

The term "imido lower alkylene" refers to a lower alkenyl substituted with an imido group.

Substituted alkylenes such as those described above will typically be monosubstituted. Reference to a "C₄ or higher alkyl" or a "C₄ or higher alkenyl" in the context of compounds of Formula I is meant that the alkyl or alkenyl can have 4 or more carbon atoms in a continuous chain.

When Ri is alkyl, alkenyl, alkylcarboxy, alkylaryl, or alkoxy, the alkyl, alkenyl, alkylcarboxy, alkylaryl, or alkoxy can have a Ci-C₁₈ alkyl or alkenyl chain. The Ci-Ci₈ chain may be straight or branched.

In at least some embodiments, Ri is an alkylamino or alkenylamino, wherein the alkylamino or alkenylamino is optionally substituted. Most usually, Ri is a substituted akylamino or alkenylamino.

Typically, the alkyl or alkenyl group of the alkylamino or alkenylamino is a CpC₆ alkyl or alkenyl, usually a Ci-C₄ alkyl or alkenyl, and most usually, a C₂-C₄ alkyl or C₂-C₄ alkenyl.

Typically, the amino group of the alkylamino or alkenylamino is substituted with at least one substituent independently selected from the group consisting of alkyl, alkenyl, aryl, akylaryl, alkenylaryl, alkylarylalkyl, alkylarylalkenyl, an alkyl or alkenyl carbocyclic or heterocyclic group having 5 to 7 ring members and optionally including one or more double bonds, and the heterocyclic group having one or more heteroatoms independently selected from O, N, S and P,, where the alkyl, alkenyl, aryl, alkylaryl, alkenylaryl, alkylarylalkyl, alkylarylalkenyl, cycoalkyl, cycloalkenyl, and the alkyl or alkenyl carbocyclic or heterocyclic group are optionally substituted, such as by alkyl (e.g., Ci-C₈ alkyl, C₁-C₆ alkyl, C₁-C₄ alkyl, or Ci-C₂ alkyl), alkenyl (e.g., C₂-C₈ alkenyl, C₂-C₆ alkenyl, C₂-C₄ alkenyl, or C₂ alkenyl), aryl (e.g., phenyl), alkylaryl (e.g., benzyl), or alkenylaryl (e.g., C₂ alkenyl-phenyl). Typically, the substituent of the amino group of the alkyl or alkenyl is selected from the group consisting of C₅ - Ci₆ alkyl, Cs-Ci₆ alkenyl, C₅-Ci₆ alkylaryl, Cs-Ci₆ alkenylaryl, C₅-Ci₆ alkylarylalkyl, C₅-Ci₆ alkylarylalkenyl, and a C₅-Ci₆ alkyl carbocyclic or heterocyclic group, and a C₅-Ci₆ alkenyl carbocyclic or heterocyclic group.

Usually, the substituent of the amino group is selected from the group consisting OfC₆ - Ci₄ alkyl, C₆_C_]6 alkenyl, C₆-Ci₄ alkylaryl, C₆-Ci₄ alkenylaryl, C₆-Ci₄ alkylarylalkyl, C₆-Ci₄ alkylarylalkenyl, and a C₆-Ci₄ alkyl carbocyclic or heterocyclic group, and a C₆-Ci₄ alkenyl carbocyclic or heterocyclic group and most usually, C₈ - Ci₂ alkyl, C₈-Ci₂ alkenyl, C₈-Ci₂ alkylaryl, C₈-Ci₂ alkenylaryl, C₈-Ci₂ alkylarylalkyl, C₈-Ci₂ alkylarylalkenyl, and a C₈-Ci₂ alkyl carbocyclic or heterocyclic group, and a C₈-Ci₂ alkenyl carbocyclic or heterocyclic group.

In at least some embodiments, Ri of Formula I or Ia is an alkylamino or alkenylamino of Formula II as follows:

Formula II and

V is N, NH, C or CH;

W is an N, NH, CH or CH₂ bonded to ring B;

Y is H, cyano, nitro, NH, amino, oxo, halo, hydroxy, sulfhydryl, carboxy, thiocarboxy, S, SO₃H, or an optionally substituted Ci-C₃ alkyl or Ci-C₃ alkenyl;

R₄ is CH₂R₅, CXR₅ or CHX¹R₅; X is O, S or NH;

X' is cyano, nitro, amino, halo, hydroxy, sulfhydryl, carboxy, thiocarboxy, SO₃H, SO₂R⁶, SO₂NH₂, SO₂NHR⁶, SO₂NHR⁶ ₂, or an optionally substituted Ci-C₃ alkyl or Ci-C₃ alkenyl; and R₅ is H, amino, CHO, nitro, aldehyde, carboxy, thiocarboxy, or an optionally substituted group selected from a C₄ or higher alkyl, a C₄ or higher alkenyl, alkylcarboxy, alkylthiocarboxy, alkenylcarboxy, alkenylthiocarboxy, alkylaryl, alkenylaryl, alkoxy, alkylcycloheteroalkyl, alkylcycloheteroalkenyl, alkenylcycloheteroalkyl, alkenylcycloheteroalkenyl, aryl, alkylamino, alkenylamino, aminoalkyl, aminoalkenyl, aminoaryl, aminocycloalkyl, aminocycloalkenyl, aminoheterocycloalkyl, aminoheterocycloalkenyl, arylamino, alkylaminoaryl, alkenylaminoaryl, arylaminoalkyl, arylaminoalkenyl, a carbocyclic and a heterocyclic group, or is an alkylNR group where NR is N(CH₃)₂ or an optionally substituted N containing heterocyclic ring with 5 or 6 ring members; and

R⁶ is independently an optionally substituted C₁-C₃ alkyl or a C_2-C₃ alkenyl.

Typically, V is C or CH;

W is CH or CH₂;

Y is cyano or amino; R₄ is CH₂R₅, CXR₅ or CHX¹R₅;

X is O or S, and most usually, O; X' is cyano, amino or Ci-C₃ alkyl or Ci-C₃ alkenyl; and R₅ is an optionally substituted group selected from a C₄ or higher alkyl, a C₄ or higher alkenyl, alkylaryl, alkenylaryl, aryl, alkylamino, alkenylamino, aminoalkyl, aminoalkenyl, aminoaryl, aminocycloalkyl, aminocycloalkenyl, aminoheterocycloalkyl, aminoheterocycloalkenyl, arylamino, alkylaminoaryl, alkenylaminoaryl, arylaminoalkyl, arylaminoalkenyl, a carbocyclic and a heterocyclic group, or is an alkylNR group where NR is N(CH₃)₂ or an optionally substituted N containing heterocyclic ring with 5 or 6 ring members; and Usually, V is C or CH;

W is CH;

Y is cyano; R₄ is CXR₅;

X is O or S, and more typically, O; and R₅ is an optionally substituted group selected from a C₄ or higher alkyl, a C₄ or higher alkenyl, alkylaryl, alkenylaryl, alkylamino, and alkenylamino, or is an alkylNR group where NR is N(CH₃)₂ or an optionally substituted N containing heterocyclic ring with 5 or 6 ring members. Most usually, R₅ of a compound of formula II is a alkylamino or alkenylamino. Generally, R₅ is a substituted alkenylamino. The alkylamino or alkenylamino can be susbtituted as described above.

The Z group of a compound of Formula I or Iacan be optionally substituted. For example, Z can be substituted though not exclusively, with one or more substituents selected from hydroxy, carboxy, halo, amino, amido, and imido. In at least some embodiments, Z can be selected from the group consisting of a carboxy cyclo-lower alkenyl, a hydroxy lower alkenyl, an amido lower alkenyl, a mono or di-halo lower alkenyl, amino lower alkenyl, or imido lower alkenyl. The amine substituent of the amino lower alkenyl can be substituted with 1 or 2 lower alkyl groups. When the amide group is substituted with 2 lower alkyl groups, the amino substituent is a di-lower alkyl amino group, which may be incorporated into a 5, 6, 7 or 8 membered ring, which in some embodiement may contain additional hetero atoms chosen from N, O, S and P.

When Ri₁R₃, R₅ or Z is an alkylNR group, the alkyl moiety of the group will typically independently be a C₁-C₄ alkyl and most usually, a C₂-C₄ alkyl.

When R₁, R₃, R₅ or Z is an alkylNR group and the NR group is an amide, the amide can for example be NC=OCH(Ci-C₂), where the Cl or C2 group is independently selected . Most usually, the amide is -NC=OCH(CH₃)₂.

When the NR group of an alkylNR substituent of compound of Formula I or Ia comprises an N containing heterocyclic ring (e.g., when R₃ is alkylNR), the ring may include one or more further heteroatoms selected from O, N, S or P. Suitable heterocyclic rings include those exemplified above, such as pyrazinium, imadazolyl, pyranyl, morpholinyl, pyridinium, maleimydyl, quinolizinyl, furanyl and the like. The heterocyclic ring can be an aryl group. Moreover, the heterocyclic ring may be optionally substituted with one or more substituents. The substituent group(s) can for example be selected from the group consisting of halo, NH, cyano, nitro, amino, hydroxy, alkyl (e.g., Ci-Ci₈ alkyl), alkyloxy, carboxy, thiocarboxy, guanidine, sulfonylamide (and derivatives thereof), and bioisosteres of the foregoing.

When R₂ of a compound of Formula I or Ia is R₂'-(Z)_n, n will typically be an integer of from 1 to 4, more usually an integer of from 1 to 3, or an integer of from 1 to 2.

R₂ will generally be H, lower alky, or lower alkenyl. Typically, R₂ is H or a Ci-C₃ alkyl, Most usually, R₂ is H or CH₃. When R₃ of a compound of Formula I or Ia is R₃ '(Z)_n, n will typically be an integer of from 1 to 6, more usually an integer of from 1 to 4, or an integer of from 1 to 3, and most usually, an integer of 1 to 2.

R₃ will generally be a C₁-C₄ alkylNR group, a lower alkyl, lower alkenyl, aryloxy- lower alkyl, or lower alkoxy-lower alkyl. Typically, R₃ will be a C₁-C₃ alkylNR where NR is N(CH₃)₂ or NCH(CH₃)₂ or usually, a C₂-C₃ alkylNR group where NR is N(CH₃)₂.

A halo atom will generally be selected from I, Br, Cl and F, and most usually, Br and Cl.

In some embodiments, R₁ of a compound of Formula I or Ia may have the following structure:

where X is selected from O and S, and R₅ is as described above. In some embodiments, R₁ of a compound of Formula I or Ia may have the following structure:

wherein X is selected from O and S, X' is selected from NH, O and S, and R₅ is as described above. In some embodiments, R₁ of a compound of Formula I or Ia may have the following structure:

H

> R₅ wherein X is selected from Ci, O and S, and R₅ is as described above. In some embodiments, Ri of a compound of Formula I may have the following structure:

wherein X is selected from C₁, O and S, and R₅ is as described above, and R₆ and R₇ are independently as for R₅.

In at least some embodiments, ring A and ring B of a compound of Formula I or Ia can each independently be a heterocyclic ring containing from 1 to 2 heteroatom ring members selected from the group consisting of O, N and S, or a heteroaryl containing 1 to 2 heteroatom ring members selected from the group consisting of O, S and N. Most usually, the heteroatom(s) of the heterocyclic or heteroaryl ring will be N atom(s).

Moreover, ring A can possess any number of optimal substituents. Examples include but are not limited to hydrogen, hydroxy, halo, lower alkyl, lower alkyl sulfone, hydroxy-lower alkyl, perfluro lower alkyl, lower alkoxy, mono- or di-lower alkyl amino, or a ring system such as a cycloalkyl, cycloalkenyl, cyclo heteroalkyl, cyclo heteroalkenyl, aryl, or heteroaryl group. Examples of perfluoro lower alkyl groups include trifluromethyl and heptafluoropropyl. Ring A substituents may also be selected from the group consisting of NH, cyano, nitro, amino, hydroxy, carboxy, thiocarboxy, guanidine, sulfonylamide (and derivatives thereof), and bioisosteres of the foregoing, heterocyclic groups, and aryl such phenyl and benzyl. In still further embodiments, a heterocyclic groups, heteroaryl or aryl ring of 5 or 6 ring atoms can be fused to ring A. Examples of the resulting fused ring system include naphthyl groups. By "bioisosteres" is meant physiologically acceptable isosteres. Typically, ring A of a compound of Formula I is an optionally substituted phenyl group.

A compound of Formula I or Ia can be a compound of Formula III as follows:

R¹

ΓATVR²

N

R³

Formula III where A, R₁, R₂ and R₃ are as described above for Formula I and Ia.

In at least some embodiments of a compound of Formula I, Ia or III, ring A is an aryl, heteroaryl or heterocyclic ring, the aryl, heteroaryl or heterocyclic ring being optionally substituted and having 5 or 6 ring atoms. Typically, ring B will be an N containing heteroalkenyl with 5 or 6 ring members, and most usually, an N containing heteroalkenyl with 5 ring members.

Most usually, the phenyl is unsubstituted. In particularly preferred embodiments, ring A and ring B form an indole group.

The R₁ to R₅ groups of a compound of Formula I or Ia may independently be optionally substituted unless otherwise specified herein. The substituent(s) may for example be independently selected from amino, amido, imido, carboxy, thiocarboxy, hydroxy, cyano, nitro, halo, oxy, sulphone, lower alkyl, lower alkenyl, lower alkoxy, lower alkoxy-loweralkyl, aryloxy-lower alkyl, and aryl, heteroaryl and heterocyclic groups.

Substituents OfR₁ include substituted or unsubstituted heteroaryl and heterocyclic groups with 5 or 6 ring atoms. Examples include substituted or unsubstituted pyrrolinium, furanyl, maleimidyl and thiopheneyl. Particularly, preferred such heteroaryl and heterocyclic groups include those substituted with an alkyl, alkenyl, aryl, heteroaryl or heterocyclic group, such as lower alkyl, lower alkenyl, phenyl, furanyl and indolyl. A compound of Formula I can for instance be a bismaleimideindolyl.

Examples of indolyl compounds of Formula I, Ia and III are illustrated below in Table 1. IC₅₀ values are shown.

Table 1: Structures of indole related inhibitors of dynamin

Endocytosis is a major contributor or direct cause of diverse human diseases. A list of vesicle trafficking-specific diseases has been published, see for example Aridor and Hannan 2000, Traffic l:836-851^[10] and Aridor and Hannan 2002, Traffic 3:781- 790 the contents of which are incorporated herein by reference in their entirety. Accordingly, methods of the invention may for instance be useful in the prophylaxis or treatment of cancers, ophthalmo logic disease, immunodeficiency diseases, gastrointestinal diseases, viral and bacterial infections, other pathogenic infections, neurodegenerative, neurological, and kidney diseases and conditions, and other disorders that involve endocytosis, or which are otherwise sensitive to inhibition of endocytosis or dynamin.

For example, it is known that human polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy, a fatal central nervous system (CNS) demyelinating disease and its entry to neurons is blocked by endocytosis inhibitors such as chlorpromazine. Similarly, infection by HIV, influenza virus and adeno-associated virus is by endocytosis or is sensitive to its inhibitors.

Clathrin-mediated endocytosis (CME) is important for many cell trafficking and cell signaling pathways. Hence, inhibition of dynamin mediated CME can affect cellular processes and finds application in the treatment of diseases and conditions responsive to such inhibition. For example, growth factor receptors (e.g. EGF-R) require dynamin for internalisation and maintenance of cellular activities from signalling to cell growth. Blocking endocytosis with dynamin constructs prevents cell proliferation in many of these examples and provides evidence of anti-cancer activity of dynamin II (the non- neuronal form) inhibitors. Dent's disease (polycystic kidney disease) also involves endocytosis of ClC-5 chloride channel and endocytosis blockers prevent its internalisation.

Dynamin is central to all endocytic trafficking from the cell surface, and also plays a role in trafficking or fission events from the Golgi apparatus, endosomes and mitochondria. Several neurodegenerative diseases are associated with these trafficking pathways. Two are implicated in generation of β-amyloid, namely the endocytic and the secretory pathways (Aridor & Hannan 2000)^[10^. In the brain, disease and conditions in which endocytosis plays a role include Alzheimer's disease, Huntington's disease (HD), stiff-person syndrome, Lewy body dementias, and Niemann-Pick type C disease.

In Alzheimer's disease β-amyloid precursor protein (APP) is internalized from axonal cell surfaces in clathrin-coated vesicles and sorted away from recycling synaptic vesicles, and transported to endosomes and the cell soma. The endosome is the first compartment along the dynamin-dependent endocytic pathway after internalization of APP or ApoE and endosomal alterations are evident in pyramidal neurons in Alzheimer brain. Endocytic pathway activation is prominent in APP processing and β-amyloid formation and is an early feature of neurons in vulnerable regions of the brain in sporadic Alzheimer's disease.

Huntington's disease (HD) is a neurodegenerative disorder principally affecting striatal neurons, yet the mutated gene product huntingtin is not brain-specific. Huntingtin interacts strongly with members of the Huntingtin-interacting protein 1 (HIPl) family. The huntingtin-HIPl interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. Loss of normal huntingtin-HIPl interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain. HIPl is a fundamental component of the dynamin-mediated endocytic machinery. Hence, numerous reports have linked the neurological defects in HD to endocytosis abnormalities (Aridor & Hannan, 2000)^[10].

Another example is the presynaptic synuclein protein which is a prime candidate for contributing to Lewy body diseases, including Parkinson's disease, Lewy body dementia and a Lewy body variant of AD. Exogenous synuclein causes neuronal cell death due to its endocytosis and formation of intracytoplasmic inclusions. Cell death and α-synuclein aggregates are direct consequences of its endocytosis in human neuroblastoma cells.

Endocytosis has also been implicated in epilepsy. For example, mice with targeted disruption of either of two endocytic proteins synaptojanin (SJ) or amphiphysin have reduced SVE and die from random seizures throughout their lives indicating a role in neuronal excitability and a link to epilepsy. Dynamin I and II levels are 3 -fold elevated in the brains of rats after pilocarpine induced epilepsy, a model for temporal lobe epilepsy^[24].

Human epilepsies are heterogeneous conditions, broadly subdivided as genetic or acquired. Whatever the origin, seizures are associated with a massive burst of synaptic transmission. The genes identified to cause epilepsies mostly encode ion channels gated by voltage or neurotransmitters that are highly enriched in nerve terminals and controls their excitability. All these genes directly or indirectly regulate synaptic transmission. Similarly, one common feature of all anti-epileptic drugs (AEDs) is that they reduce synaptic transmission. Synaptic transmission is the release of neurotransmitter by exocytosis from their storage compartments, synaptic vesicles (SVs), in presynaptic nerve terminals. It is controlled by the electrical properties of neurons and chemical transmission across the synapse. The nerve terminal is an electrically excitable structure which maintains a net negative membrane potential so that depolarization can activate voltage-sensitive Ca²⁺ channels to trigger exocytosis. Epilepsy results in the sustained and uncontrolled exocytosis from these terminals.

Currently available epilepsy therapy is unsatisfactory in many patients. Approximately 50% fail to have their seizures controlled by the first medication prescribed and ~30% will continue to have seizures despite trials of multiple drugs. Most AEDs have been discovered using similar traditional methodologies - screening of compounds in animal models of acute provoked seizures. Possibly as a result, most AEDs have similar principal mechanisms of action, such as inhibiting voltage gated sodium channels and/or enhancing GAB Aergic activity. A rare example of a new AED acting via a novel mechanism is levetiracetam (Keppra™) which inhibits the putative SV refilling protein SV2A and inhibits exocytosis^[25>26].

The massive burst of exocytosis that occurs during an epileptic seizure cannot be sustained for more than 1 minute without compensation by synaptic vesicle endocytosis (SVE). SVE occurs after exocytosis and retrieves the empty S Vs for reuse. SVE is the mechanism used to replenish SV supply. There are only -200 SVs in a nerve terminal, enough storage for less than half a minute of maximal synaptic transmission. Thus sustained synaptic transmission (as occurs in a seizure) is a cycle of exocytosis and endocytosis of SVs — the SV cycle. Considerable evidence links most elements of the SV cycle directly or indirectly to epilepsy. Some SV cycle genes cause epilepsy in humans (e.g., synapsin), or epileptic-like seizures when genetically knocked-out in animals (e.g., SV2, amphiphysin or synaptojanin).

A ubiquitous mechanism to stop a seizure is to block synaptic mechanisms that are associated with pathologically sustained neuronal burst firing. A reduction in synaptic transmission is the common feature of all anti-epileptic drugs (AEDs). For most AEDs the mechanistic basis of this reduction is uncertain. Inhibition of SVE by blocking dynamin leads to an activity-dependent run-down in synaptic transmission. The unique aspect of this discovery is the lack of effect on acute or brief bursts of synaptic transmission - being inhibited only after high or prolonged stimulation. Inhibition of key SVE proteins reduces the availability of SVs to sustain or propagate a seizure.

A recent report showed that SVE is the rate limiting step in synaptic transmission after a prolonged stimulation time^[27]. In particular, inhibition of dynamin binding to syndapin with a peptide-based inhibitor (AA peptide) produced an activity-dependent run-down in synaptic transmission while an inactive control "EE peptide" had no effect. The AA peptide had no effect on synaptic transmission for 45 s, but after this delay it became progressively more inhibited. This is direct evidence in a mammalian cell that inhibition of SVE produces an activity- dependent run-down in synaptic transmission, the desired outcome of a novel AED. Targeting components of the SVE pathway with inhibitors exposes multiple novel points of intervention not targeted by any existing AED.

Targeting dynamin has a number of advantages over standard therapy. IN particular, conventional AEDs reduce synaptic transmission at all times, but SVE inhibitors only exert effects at high frequency or after sustained stimulation that is, under conditions associated with a seizure. Inhibiting SVE may also limit the effect to overactive neurons and thus have reduced side-effects by allowing physiological neurotransmission to occur unimpeded.

Endocytic pathways are also utilized by viruses, toxins and symbiotic microorganisms to gain entry into cells. For instance, botulism neurotoxins and tetanus neurotoxin are bacterial proteins that inhibit transmitter release at distinct synapses and cause two severe neuroparalytic diseases, tetanus and botulism. Their action is dependent on their internalisation via endocytosis into nerve terminals. Hence targeting endocytosis with inhibitors has application as a clinically useful strategy. Moreover, it has previously been found that dynamin II is recruited to the midbody of cells during cytokinesis and inhibition of activity of the protein can inhibit cell division as described in Applicant's co-pending International Patent Application No. PCT/AU2008/000203, the contents of which are incorporated herein by reference in its entirety. Traditionally, mitosis has served as a successful anti-tumour target for the treatment of this disease. Drugs that arrest cells in mitosis, known as anti-mitotics, are common treatments for a variety of human tumours, including breast, ovarian and non- small-cell lung cancer. Many of these drugs currently used in the clinic, such as the taxanes (e.g., paclitaxel/taxol) and the vinca alkaloids (e.g., vincristine), inhibit the cell cycle by reducing microtubule dynamics, keeping the spindle assembly checkpoint (S AC) in an active state. Sustained SAC activation is often followed by cell death. However, since microtubules are not only essential for mitosis, but also required for other critical physiological functions, such as intracellular transport and organelle positioning, microtubule inhibitors act on both proliferating and post-mitotic cells and exhibit microtubule-dependent side effects, including peripheral neuropathy. Thus, agents that target mitosis via a novel mechanism of action and with greater specificity toward tumors are particularly desirable for the treatment of human neoplasm. The clinical success of the small molecule protein kinase inhibitor Gleevec for chronic myelogenous leukemiahas resulted in a surge of interest in small molecule inhibitors as highly effective anti-cancer agents. Recent drug developmental focus is on the essential mitotic serine/ threonine protein kinases, Aurora A, Aurora B and the PoIo- like kinase, Plkl, as potential anti-cancer drug targets (Taylor et al. 2008). These protein kinases are essential for mitotic progression, with roles in mitotic entry, spindle assembly, centrosome maturation, chromosome alignment and cytokinesis. Small molecule inhibitors have also been developed to target the kinesin motor protein (KSP) (Jackson et al. 2007), which is required to establish mitotic-spindle bipolarity. These new anti-mitotic inhibitors are very effective at preventing the proliferation of most tumour cells in vitro. Thus, many are currently being extensively studied in pre-clinical or phase I/II clinical trials.

The cellular response to cell-cycle arrest by such inhibitors is varied and includes apoptosis, mitotic catastrophe, mitotic slippage, senescence and reversible mitotic arrest, depending on what cell line and/or inhibitor is studied. For example, aurora kinase inhibitors AZDl 152^[28] and CCT129202^[29] inhibit proliferation by causing polyploidy, leading to apoptosis. The KSP inhibitor, KSP-IA, causes mitotic arrest, followed by mitotic slippage and cell death in HCTl 16 colon carcinoma cells, whereas cell death does not proceed mitotic arrest in HT29 colon carcinoma cells treated with the same inhibitor^. Nevertheless, the exquisite selectivity for mitosis and the distinct ways in which these new agents interfere with mitosis provides two advantages as non- proliferating cells would not be adversely affected by these drugs, namely that they i) overcome limitations of current tubulin-targeted anti-mitotic drugs, and ii) expand the scope of clinical efficacy established by those drugs. For example, proliferating cells treated with the aurora kinase inhibitor ZM447439 undergo cell death, whereas non- proliferating cells are not affected^. Since most cells in the body do not rapidly proliferate in comparison to tumour cells, mitotic inhibitors can have a broader therapeutic index than non-specific cytotoxic agents, such as alkylating agents like cyclophosphamide, that act in a non cell cycle-specific manner, and various studies on animals have been reported^[32>33].

Cytokinesis in animal cells requires two distinct stages, 1) membrane ingression, and 2) membrane abscission. Ingression involves membrane constriction between segregated chromosomes via an actin-myosin II ring to form an intracellular bridge between nascent daughter cells. Abscission is the final step of cytokinesis involving formation of a central γ-tubulin midbody ring (MR) within the intracellular bridge prior to final separation into two daughter cells.

Recent work by the present inventors has revealed a new molecular signalling pathway that allows mitotic progression to complete abscission, as described in the Applicants co-pending International Patent Application No. PCT/AU2008/000203. In particular, the inventors discovered a signalling pathway localised to two midbody rings (FMRs) that flank the centrally located MR. The endocytic protein, dynamin II (dynll) is phosphorylated by cdkl/cyclin Bl on S764 at the onset of mitosis and then moves to the intracellular bridge where it localises to the FMRs with the Ca²⁺-dependent phosphatase, calcineurin (CaN). An influx of Ca²⁺ at the midbody activates CaN to dephosphorylate dynll, which allows cells to continue progressing through cytokinesis to complete abscission generating two independent daughter cells. Ca²⁺ chelation by EGTA, small molecule CaN inhibitors, or depletion of CaN or dynll, all lead to cytokinesis failure in the abscission stage and an increase in the number of tetraploid cells. Ca²⁺ and CaN have also previously been reported to have multifunctional cellular roles and participate in different mitotic stages earlier than cytokinesis. In contrast, dynll action is exquisitely specific, and it is not required for progression through any other cell cycle phase. Thus, dynll represents a new anti-mitotic anti-cancer drug target. Further, several epidemiological studies have reported a reduced incidence of cancer in schizophrenia patients taking antipsychotic medication (Mortensen 1992). One class of antipsychotic agents is the phenothiazines. In particular, a screen of 297 psychotropic drugs found that such compounds were 18 times more likely to contain anticancer properties than expected from randomly screening compounds'^. Chlorpromazine is the archetypical member of the phenothiazines, and has been reported to inhibit clathrin-dependent endocytosis^³⁵'. Recent data generated (Prof. Phil J. Robinson, Children's Medical Research Institute, Westmead, Sydney, Australia) reveals that chlorpromazine inhibits endocytosis by inhibition of dynll (IC₅₀ = 1.3 ± 0.4 μM), which was also reported in a separate publication^. In addition, chlorpromazine has been reported to cause multinucleation due to cytokinesis failure^³⁷¹. This is consistent with the link between endocytosis and cytokinesis^³⁹'⁴⁰¹. Thus, the anti-cancer properties of chlorpromazine is linked to the disruption of cytokinesis potentially resulting in cell death. Moreover, recent screening of chlorpromazine in a nine cancer cell lines shows it to be a potent inhibitor of cell growth (GI₅₀ of 13-32 μM) (Prof. Phil J. Robinson, Children's Medical Research Institute, Westmead, Sydney, Australia). As dynll plays an essential role in abscission, these findings show that dynamin inhibition is a new mechanism to target cancer cell proliferation. As such, inhibition of dynamin activity can also have application in the prophylaxis or treatment of cell proliferative diseases and conditions including cancers.

Compounds embodied by the invention may also have application in treating neuropathic pain. Neuropathic pain typically develops when peripheral nerves are damaged through surgery (including spine surgery), bone compression in cancer, diabetes, viral infection (including shingles or HIV infection), AIDS, alcoholism, amputation, chemotherapy, facial nerve problems, or multiple sclerosis, and is a major factor causing impaired quality of life for millions of people worldwide. Anti-convulsant drugs such as phenytoin and gabapentin are highly efficacious in treating neuropathic pain. These drugs act through modulation of synaptic vesicle transmission, indicating the potential of the inhibition of synaptic vesicle endocytosis by quaternary salts embodied by the invention in the treatment of this debilitating condition. That is, inhibition of synaptic vesicle endocytosis may halt or limit pain signalling and thereby reduce or ameliorate the sensation of pain experienced. Hence, examples of specific diseases and conditions for which methods of the invention find application for the prophylaxis or treatment of include but are not limited to cell proliferative diseases and conditions, multifocal leukoencephalopathy, polycystic kidney disease, β -amyloid associated diseases, Alzheimer's disease, Huntington's disease, stiff-person syndrome, Lewy body dementias, Parkinson's disease, neurodegenerative disorders, neuropsychiatric disorders, psychotic disorders, psychosis, bipolar disorders, schizophrenia, aberrant up-regulated neuronal excitation, seizures, epilepsy, neuropathic pain, migraine, tetanus, botulism, nerve viral infection, HIV infection, influenza, mucolipidosis, cell proliferative diseases and conditions, neurological, diseases responsive to inhibition of dynamin activity, and diseases and conditions mediated or otherwise associated with cellular endocytosis, synaptic signal transmission, synaptic vesicle endocytosis (e.g., such as epilepsy), or cell vesicle trafficking. Cell proliferative diseases and conditions that may be treated by compounds embodied by the invention include cancer, skin conditions such as psoriasis and scleroderma, benign growths, and cardiovascular diseases and conditions including atherosclerosis. The cancer may for instance be selected from the group consisting of carcinomas, sarcomas, lymphomas, leukaemias, and cancer of the liver, tongue, mouth, oropharynx, nasopharynx, gastrointestinal tract, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin (e.g., melanoma and basil cell carcinoma) amongst others. Suitable pharmaceutically acceptable salts include acid and amino acid addition salts, base addition salts, esters and amides that are within a reasonable benefit/risk ratio, pharmacologically effective and appropriate for contact with animal tissues without undue toxicity, irritation or allergic response. Representative acid addition salts include hydrochloride, sulfate, bisulfate, maleate, fumarate, succinate, tartrate, tosylate, citrate, lactate, phosphate, oxalate and borate salts. Representative base addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides. The salts may include alkali metal and alkali earth cations such a sodium, calcium, magnesium and potassium, as well as ammonium and amine cations. The provision of such salts is well known to the skilled addressee. Suitable pharmaceutical salts are for example exemplified in S. M Berge et al, J. Pharmaceutical Sciences (1997), 66:1-19^[12], the contents of which is incorporated herein in its entirety by cross-reference.

Prodrugs of compounds of the invention include those in which groups selected from carbonates, carbamates, amides and alkyl esters have been covalently linked to free amino, amido, hydroxy or carboxylic groups of the compounds. Suitable prodrugs also include phosphate derivatives such as acids, salts of acids, or esters, joined through a phosphorus-oxygen bond to a free hydroxl or other appropriate group. A prodrug can for example be inactive when administered but undergo in vivo modification into the active compound that binds to dynamin such that the GTPase activity of the protein is inhibited, as a result of cleavage or hydrolysis of bonds or other form of bond modification post administration. The prodrug form of the active compound can have greater cell membrane permeability than the active compound thereby enhancing potency of the active compound. A prodrug can also be designed to minimise premature in vivo hydrolysis of the prodrug external of the cell such that the cell membrane permeability characteristics of the prodrug are maintained for optimum availability to cells and for systemic use of the compound.

Esterified prodrugs may for instance be provided by stirring a compound embodied by the invention with an appropriate anhydride or acid chloride (in molar excess) in a pyridine/N ,N-dimethylformamide (DMF) solution in the presence of a suitable catalyst such as dimethylaminopyridine (DMAP). In some cases, the solution may need to be refluxed to drive the reaction to completion. On completion of the reaction, the esterified product is purified by either recrystallization or by chromatography. Representative esters include C₁-C₇ alkyl, phenyl and phenyl(Ci-₆) alkyl esters. Preferred esters include methyl esters. Examples of suitable prodrug groups are shown in Table 2.

Table 2: Examples of prodrug groups

Compounds embodied by the invention can be administered to an individual in need of such treatment alone or be co-administered with one or more other therapeutic compounds or drugs. For example, compound can be co-administered in combination with drugs conventionally used for treating cancer or neurological diseases and disorders. By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes, wherein the administered drugs have overlapping therapeutic windows. By "sequential" administration is meant one is administered after the other. The compounds will generally be formulated into a pharmaceutical composition comprising the mimetic(s) and a pharmaceutically acceptable carrier. Pharmaceutical compositions include sterile aqueous solutions suitable for injection and sterile powders for the extemporaneous preparation of injectable solutions. Such injectable compositions will be fluid to the extent that syringability exists. Injectable solutions will typically be prepared by incorporating the active(s) in the selected carrier prior to sterilising the solution by filtration. In the case of sterile powders, preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active and any additional desired ingredient from previously sterile filtered solutions thereof. For oral administration, the compound can be formulated into any orally acceptable carrier deemed suitable. In particular, the compound can be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule. Moreover, a compound can be provided in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions or syrups. Compounds as described herein can also be formulated into topically acceptable preparations including creams, lotions or ointments for internal or external application. Topically acceptable compositions can be applied directly to the site of treatment including by way of dressings and the like impregnated with the preparation.

A pharmaceutical composition embodied by the invention can also incorporate one or more preservatives such as parabens, chlorobutanol, phenol, and sorbic acid. In addition, prolonged absorption of the composition may be brought about by the inclusion of agents for delaying absorption such as aluminium monosterate. Tablets, troches, pills, capsules and like can also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatine, a disintegrating agent such as corn starch, potato starch or alginic acid, a lubricant such as magnesium sterate a sweetening agent such as sucrose, lactose or saccharin; and a flavouring agent.

Pharmaceutically acceptable carriers include any suitable conventionally known physiologically acceptable solvents, dispersion media, isotonic preparations and solutions including for instance, physiological saline. Use of such ingredients and media for pharmaceutically active substances is well known. Except insofar as any conventional media or agent is incompatible with the mimetic, use thereof is expressly encompassed. It is particularly preferred to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Compounds embodied by the invention can also be provided in dosage unit form. A dosage unit form as used herein is to be taken to mean physically discrete units, each containing a predetermined quantity of the active calculated to produce a therapeutic or prophylactic effect. When the dosage unit form is a capsule, it can contain the active in a liquid carrier. Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugars or both.

Pharmaceutical compositions embodied by the invention will generally contain at least about 0.1% by weight of the mimetic up to about 80% w/w of the composition. The amount of the compound in the composition will be such that a suitable effective dosage will be delivered to the individual taking into account the proposed mode of administration. Preferred oral compositions will contain between about 0.1 μg and 4000 mg of the mimetic.

The dosage of a compound embodied by the invention will depend on a number of factors including whether the active is to be administered for prophylactic or therapeutic use, the disease or condition for which the active is intended to be administered, the severity of the condition, the age of the individual, and related factors including weight and general health of the individual as may be determined in accordance with accepted medical principles. For instance, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the individual's response. Similarly, frequency of administration can be determined in the same way that is, by continuously monitoring the individual's response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.

Typically, a compound will be administered in accordance with a method embodied by the invention at a dosage up to about 50 mg/kg body weight and preferably, in a range of from about 5 μg/kg to about 100 μg/kg body weight.

Routes of administration include but are not limited to respiritoraly, intravenously, intraperitonealy, subcutaneously, intramuscularly, by infusion, orally, rectally, topically and by implant. With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply the target tissue to be treated. Suitable pharmaceutically acceptable carriers and formulations useful in compositions of the present invention may for instance be found in handbooks and texts well known to the skilled addressee, such as "Remington: The Science and Practice of Pharmacy (Mack Publishing Co., 1995)", the contents of which is incorporated herein in its entirety by reference.

The invention will be further described herein after with reference to non-limiting Examples.

EXAMPLE 1: Development of dynamin GTPase inhibitors

1. Identification of lead compounds

A medicinal chemistry program requires a viable lead compound. Subsequent optimisation of such a lead can facilitate the development of a more selective, potent and less toxic analogue. The most difficult task is often the initial discovery of the lead compound.

Most leads are derived from existing compound libraries, combinatorial libraries or are natural product-derived. In the present study, an additional challenge faced was the identification of a lead compound amenable to rapid synthetic development. Screening a number of compound libraries (data not shown) revealed the structurally interesting bisindolylmaleimides (BIMs) (1) and (2) as -100 μM potent leads. BIMs of this nature are inhibitors of protein kinase C and glycogen synthase kinase 3 ^[40'^41].

IC₅₀ ~ 100 μM IC₅₀ - 100 μM

Bisindolylmaleimides: BIM 1 and 2 lead compounds

While these compounds can be synthesized, their relatively complex structure complicates rapid assembly of a small series of targeted compound libraries (typically 12 - 24 compounds in a single library). As can be seen above, compounds 1 and 2 possess pseudo-symmetry, with each half differing only by the addition of the dimethylaminopropyl side chain at the lower indole nucleus.

2. Synthesis and screening of indole nucleus analogues

To identify a minimal pharmacophore, the possibility of reducing 1 to the bare indole nucleus (see Fig. 1, right hand side) was examined^¹³'.

Using this indole nucleus library 1 analogues were synthesized in a one step procedure commencing with five commercially available indoles (3-7). Deprotonation (NaH, THF, 0⁰C) followed by the addition of a number of a-chloro-ω-N,iV- dimethylamines and heating to reflux gave substituted indoles (8-17) (Scheme 1).

3 R₁ = H; R₂ = H 8 R₁ = H; R₂ = H

4 R₁ — Hj H2 — OHo 10 R₁ = H; R₂ = CH₃

5 R₁ = CHO; R₂ = H 12 R₁ = CHO; R₂ = H

6 R₁ = CH₂CO₂H; R₂ = H 14 R₁ = CH₂CO₂H; R₂ = H

7 R₁ = CH₂CH₂CO₂H; R₂ = H 16 R₁ = CH₂CH₂CO₂H; R₂ = H n = 2

9 R₁ = Hj R₂ = H

11 R₁ = Hi R₂ = CH₃

13 R₁ = CHO; R₂ = H

15 R₁ = CH₂CO₂H; R₂ = H

17 R₁ = CH₂CH₂CO₂H; R₂ = H

Scheme 1 : (i) NaH, THF O⁰C to reflux, 70 h.

Purification via short column chromatography afforded the desired compounds as either viscous oils or solids. Those compounds isolated as solids were recrystallized from the relevant solvents. These compounds were then assayed along with the parent indoles for phospholipid-stimulated dynamin I GTPase inhibitory activity.

Briefly, Dynamin I was purified from sheep brain as previously described^[19]. Dynamin GTPase activity was determined by hydrolysis of GTP by a colorimetric method also described previously^. Specifically, purified dynamin I (20 nM or 200 nM) was incubated in GTPase buffer (10 mM Tris, 10 mM NaCl, 2 mM Mg²⁺, 0.05% Tween 80, pH 7.4, 1 μg/mL leupeptin and 0.1 mM PMSF) and 0.3 mM GTP in the presence of test compound for 12 min at 30⁰C. The final assay volume was 40 μL and was performed in round bottomed 96-well plates. Plates were prepared on ice and plate incubations were performed in a dry heating block with shaking (Eppendorf Thermomixer). Dynamin I GTPase activity was stimulated with the addition of 20 μg/mL L-phosphatidylserine

(prepared by probe sonication). The reaction was terminated with 10 μL 0.5 M EDTA pH 8.0, and the samples are stable for several hours at room temperature. To each well was added 150 μL of Malachite green solution (Malachite green (50 mg), ammonium molybdate tetrahydrate (500 mg) 1 M HCl (50 μL): the solution was passed through 0.45 μm filters and stored in the dark for up to 2 months at room temperature)^²⁰'. Colour was developed for 5 min and was stable for up to 2 hours, and the sample absorbances in each plate were determined on a microplate reader at 650 run. Phosphate release was quantified by comparison with a standard curve of sodium dihydrogen orthophosphate monohydrate (baked at 110°C overnight to dry) from 1 - 100 μM, which was run in each experiment. For some of these studies (as indicated in Table 3-4) the dynamin concentration in the assay was tested at the standard 200 nM as well as being decreased to 20 nM in later work.

As can be seen from Table 3, six members of Library 1 are modest inhibitors of dynamin I GTPase activity, with 16 and 17 having IC₅₀ values of 131±5 μM and 143 ±7 μM being most active. Compounds 8-11 were also active but with low potency, with all found to have approximate IC₅o's of =300 μM. Whilst the parent BIMs are PKC inhibitors, the analogues were not analysed for PKC inhibition and indeed, there is no evidence to suggest that such simplified analogues would retain such ability^[14].

Of the Library 1 analogues, only those containing an N2-Alkyl-N(CH₃)₂ were active. No inhibition was noted in the absence of these groups (data not shown), suggesting that it, along with the indole group of compounds 1 and 2, is important for activity. Examination of 8-17 also highlighted additional functionality at C2 increasing activity, with a minimal chain length or a H-bonding group mimicking the imide moiety being desirable for activity. Table 3: Library 1 - Inhibition of dynamin 1 GTPase activity by substituted indoles

8 - 17.

IC₅₀

Compound Ri R₂ R₃ (μM)^fl

8 H H ~300^b

9 H H I ~300^b

10 H CH₃ ~300^b

11 H CH₃ ~300^b

O

13 x\ CH₃

14 *Λ» H -

O

15 H -

" IC₅₀ determinations are the average of at least three separate experiments, each in duplicate, dynamin concentration was 200 nM. *~ 50% inhibition at 300 μM drug concentrations, full IC₅₀ determination not conducted; ^c "-" no inhibition at 300 μM drug concentration.

Given the apparent desirability for an alkyl-N(CH₃)₂ at the indole N and additional functionality at C3, this finding was explored via further simplification or mimicking of the malimide functionality of the compounds 1 and 2. Analogues 12 - 17 posses functionality amenable to rapid synthetic modifications, presenting simple means for exploration of the available chemical space at C3 either via simple peptide coupling approaches (-CO₂H, 14-17); or via Knovenagel approaches (-CHO; 12 and 13). The Knovenagel approach^{[15 18]} was selected for the development of a second generation indole library as dynamin I GTPase inhibitors. Synthesis of Library 2 utilized a previously reported family of cyanoamides (18a- h)^[17'¹⁸l Knovenagel condensations of 18a-h with 12 and 13 at ethanol reflux in the presence of catalytic quantities of piperidine afforded the desired adducts 19-34 in good to excellent yields (Scheme 2). Simple recrystallisation from ethanol or aetonitrile gave samples of sufficient purity for biological evaluation.

12 R, * CHiCH₂N(CX₃)-, 18a h 19 - 34

13 R_< = CHa(CH₂JyN(CHj)J 3 ^ PhCH₂ fcrfl^as sβe Tabie 2 b - P-OCH₃PnCH₂ d - eihyl e - pro?>yf

Scheme 2: Knovenagel condensations: (/) piperidine (cat), EtOH reflux, 3 h.

Table 4: Library 2 - Inhibition of dynamin 1 GTPase by substituted indoles 19- 34

22 CH₃ C

I -CH₂CH₃

23 CH₃ -(CH₂)₂CH₃ C

24 CH₃ -(CH₂)₃CH₃ -300*

25 CH₃ -(CH₂)₅CH₃ 59±3 7.7±2.0 26 ^CH3

-(CH₂)₇CH₃ 38±2 9.1±1.7

27 CH₃ 3 V ⁵^r- Q 201±15 Ar

28 CH₃ l I 222±22

29 CH₃ \ ¹-X] -300*

30 CH₃ *^v^y -CH₂CH₃

31 CH₃ *^Y -(CH₂)₂CH₃

32 CH₃ -(CH₂)₃CH₃

33 CH₃ ^V~^^N- .(CH₂)₅CH₃ 102±9 6.6±1.5

34-1 CH₃ ^!cvγ -(CH₂)₇CH₃ 22±4 3.3±0.8

34-2 -(CH₂)₇CH₃ 1.30±0.3

^αIC₅o determinations are the average of at least three separate experiments, each in duplicate, dynamin concentration was 200 nM; ^b~ 50% inhibition at 300 μM drug concentrations, full IC₅₀ determination not conducted; ^c "-" no inhibition at 300 μM drug concentration; d dynamin was assayed at 20 nM.

Library 2 delivered eleven active compounds (Table 3) with introduction of an aromatic ring, 19, 20, 27, 28 returning IC50 values below 300 μM, but were less potent than either 16 or 17. Regardless, the presence of an aromatic ring imparts more activity than the corresponding cyclohexyl analogues 21 and 29 with ICso's ~300 μM. The introduction of long alkyl chains has a marked effect on potency with C6 alkyl chains, 25 and 33, as potent or more potent than the lead compounds (ICso's 59±3 and 102±9 μM respectively); and C8 alkyl chains, 26 and 34, 2-5 times more potent than the original lead compounds (ICso's 38±2 and 22±4 μM respectively). As before, there is no clear preference for the N,N-dimethylethyl side chain over the N,N-dimethylpropyl side chain. Taking the most active compound as 34-1 the R₂ methyl was removed, affording a 1 times more potent analogue, 34-2, with an IC₅₀ for dynamin inhibition of 1.3 μM.

Given this trend in increased activity with increased chain length we then synthesized the corresponding analogues with ClO, Cl 2 and C14 alkyl chains (35 - 40). These analogues were tested for their ability to inhibit dynamin I GTPase activity (Table 5). All ClO - C14 analogues, 35 - 40, returned inhibitory values better than the lead compounds (Library 3, Table 5), but did not offer any significant improvement over analogues 25, 26, 33 and 34, suggesting that the optimal chain length is C8.

Throughout the development of more potent analogues it was assumed the dimethylamino-alkyl moiety was important for activity. To confirm this, analogues of 19 - 26 lacking the N,N-dimethylaminoethyl or the N,N-dimethylaminopropyl moieties were synthesized from 2-methylindole-3-carboxaldehyde giving 41 - 50 (Scheme T). This group of analogues (Library 3) was then tested for its dynamin I GTPase inhibition.

Table 5: Inhibition of dynamin 1 GTPase by substituted indoles 35 - 50

Compound R₂ R₃ R₅ IC₅₀

(μM)^fl

35 CH₃ -(CH₂)₉CH₃ 28±4

36 CH₃ -(CH₂)! !CH₃ 32±3

37 CH₃ ^Y (CH₂)₁₃CH₃ 38±1

38 CH₃ -(CH₂)₉CH₃ 30±8

39 CH₃ -(CH₂)_nCH₃ 42±10

40 CH₃ -(CHz)₁₃CH_{3 5}2±8

I

41 CH₃ H -CH₂CH₃ _b

42 CH₃ H b

44 CH₃ H b

^*Ό

45 CH₃ H -(CH₂)₂CH₃ b

46 CH₃ H -(CH₂)₅CH₃ b

47 CH₃ H -(CH₂)₇CH₃ b

48 CH₃ H -(CH₂)₉CH₃ b

49 CH₃ H -(CH₂)_πCH₃ b

50 CH₃ H -(CH₂)₁₃CH₃ b " IC_5O determinations are the average of at least three separate experiments, each in duplicate, dynamin was 200 nM; b "-" no inhibition at 300 μM drug concentration.

As can be seen from Table 5, none of analogues 41 - 50 lacking the N₁N- dimethylaminoalkyl group display any dynamin I GTPase activity inhibition at the highest drug concentrations evaluated (300 μM, Table 5). As such the activity of analogues 25, 26, and 33 - 40 is not solely due to the presence of long alkyl chains, but rather to a combination of this and the N,N-dimethylaminoalkyl moiety. This result confirms the importance of the dimethylamino functional group, as also observed in the other libraries assayed. Hence, using a number of small targeted compound libraries, potent indole- based dynamin inhibitors were found and structure activity relationships of the compounds identified.

Since the BIMs are competitive inhibitors of ATP binding to PKC, the kinetics of inhibition of dynamin I by 34-1 was determined (see Fig. 2). Double-reciprocal plots show that 34-1 is non-competitive with respect to GTP. This indicates that 34-1 binds dynamin I at an allosteric site rather than the GTP binding site, and so alters the conformation of the GTP binding site.

3. Discussion

Deconstruction of the lead bisindolylmaleimide compounds 1 and 2 allowed the design and synthesis of a series of small targeted libraries to explore the structural requirements for inhibition of dynamin 1 GTPase activity by these analogues. Modifications developed herein allowed synthetically simple approaches to new inhibitors of dynamin displaying greater potency (about 100-fold) than the original lead compounds. One of the inhibitors, 34-1, was a non-competitive inhibitor with respect to GTP, a relatively rare mode of inhibitor action that reduces activity without altering GTP affinity. This is suggestive of binding at an allosteric site. EXAMPLE 2: Dynamin GTPase inhibitors block cell-based endocytosis

1. Inhibition of clathrin-mediated endocytosis (CME)

The ability of four of the potent indole analogues developed in Example 1 to block clathrin-mediated endocytosis (CME) in cells was assessed by examining their effect on on the internalization of transferrin-Texas Red (Tf-TxR) (which binds to the transferrin receptor) using previously established approaches in COS-7 cells^. After 15 min of internalization under control conditions (DMSO treatment) the cells showed considerable endocytosis of Tf-TxR (Fig. 3A) in the periphery and perinuclear region of the cell, which is typical of Tf localization in early and recycling endosomes. After 10 min preincubation in the presence of each indole 26, 33, 34-1 and 35 at 30 μM, CME was greatly reduced (Fig. 3B-E). It is clear that 26, 33, 34-1 and 35 differ in their ability to block CME, as evidenced by the very low, but observable, levels of Tf-TxR apparent (observed as white or bright shade inside each cell in column 1 ; cell positions are marked by DAPI stained nuclei in column 2 and 3) with 26 (Fig. 3Bl), 33 (Fig. 3Cl) and 35 (Fig. 3El). With 34-1 (which is simply labled 34 in Fig. 3) the complete lack of white/colour that is normally _ associated with Tf-TxR uptake was noted, with the image being completely black representing a complete block of endocytosis. Tf-TxR CME mediated internalization was completely blocked at 100 μM with all 4 compounds (data not shown). Importantly cell morphology was unaffected by the drug treatment even after 30 min exposure (data not shown) indicating that there was no membrane disruption.

The effect of indoles 26, 33, 34-1 and 35 in COS-7 or U2OS cells was then quantified using a previously reported automated quantitative CME assay based on endocytosis of EGF-A488. The IC₅₀ for inhibition of EGF CME by 26 in COS-7 cells was 17.3±0.63 μM (Table 5), which compares favourably with its IC₅₀ for dynamin inhibition in vitro, 9.05±l .68 μM. Identical trends were observed for indoles 33, 34-1 and 35 returning in-cell ICs₀(_CME)S of 15±0.63; 10.7±0.61; and 23.1±0.79 μM versus in vitro dynamin ICsoμ,, _vl(r0)S 6.6±1.52; 3.3±0.75; and 10.1 μM respectively. The overall rank order of potency for CME inhibition closely matches the rank order of potency for inhibition of dynamin' s GTPase activity, suggesting that the mechanism of inhibition is via dynamin. Indole 33 also potently inhibited endocytosis of transferrin (Tf) in another cell line U2OS (Table 6), indicating that endocytosis inhibition is likely to be widespread in different cell types. The data shown in Table 6 is the mean fluorescence as a percent of control cells (triplicate determinations on approximately 1,200 cells each) ±SEM. The results are representative of three independent experiments.

Table 6: IC₅₀ for in vitro inhibition of dynl and endocytosis block (CME) of EGF in COS-7 or Tf in U2OS cells

2. Methods - Texas red-Tfn uptake COS-7 or U2OS cells were cultured in DMEM supplemented with 10% FCS at

37°C under 5% CO2 in a humidified incubator. Tf and EGF uptake were analysed as follows. Cells were plated on glass coverslips to 60% confluency. The cells were serum- starved overnight (16 h) in DMEM minus FCS. Cells were then incubated with various indoles (usually 30 μM) or vehicle for 10 or 15 min prior to addition of 5 μg/ml Tf-TxR for 15 min at 37°C. Cell surface-bound Tf was removed by incubating the cells in an ice cold acid wash solution (0.2 M acetic acid + 0.5 M NaCl, pH 2.8) for 15 min. Cells were immediately fixed with 4% PFA for 10 min then washed 3 times with PBS. Nuclei were stained using DAPI. Coverslips were mounted using mounting medium containing DABCO and the fluorescence was monitored using a Leica DMLB bright field microscope and SPOT digital camera. Quantitative analysis of the inhibition of EGF endocytosis in COS-7 or U2OS cells was performed on large numbers of cells by an automated process. Cells were grown in poly-D-lysine- coated 96 well plates and pre- incubated with varying concentrations of drugs for 15 minutes prior to addition of 1 μg/ml EGF-A488 for 10 min at 37°C. All conditions were carried out in triplicate. Cells were washed twice with ice-cold PBS and subjected to an acid wash (0.2 M acetic acid, 0.5 M NaCl, pH 2.8) at 4°C for 10 min to remove surface bound EGF. Cells were washed again with PBS before fixation in 4% PFA (pH 7.4). Nuclei were stained with DAPI. Blue (DAPI) images were collected automatically using an Olympus 1X81 epifluorescence microscope. Nine images were collected from each well, averaging 40-50 cells per image. The average integrated intensity of EGF-A488 signal per cell was calculated for each well using Metamorph (Molecular Devices, Sunnyvale, CA), and the data expressed as a percentage of control cells (vehicle treated). The average number of cells for each data point was -1,200. IC₅₀ values were calculated using Graphpad (Prism) and data was expressed as mean ± 95% confidence interval (CI) for 3 wells and ~ 1,200 cells.

3. Discussion The design and synthesis of a series of targeted libraries of indole-related dynamin

I GTPase inhibitors has lead to molecules with potent in-cell activity against clathrin- mediated endocytosis. Four of the most potent indole analogues blocked endocytosis of EGF or transferrin in two distinct cell lines. The correlation of their in vitro and in-cell activities suggests they inhibit CME via inhibition of dynamin.

EXAMPLE 3: Anti-cancer activity of compounds of Formula I

Studies were undertaken to evaluate the anti-cancer activity of indole-related compounds embodied by the invention.

1. Materials and Methods 1.1 Cell culture

HeLa cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and grown at 37°C in a humidified 5% CO2 atmosphere. Human foreskin fibroblasts (HFF) and human TERT-immortalized breast epithelial (hTERT) cell lines were maintained in MCDB Medium 170 (Gibco). 1.2 In vitro growth inhibition assay

Growth inhibitory assays were carried out as described previously ^[21>22]. Cells in logarithmic growth were transferred to 96- well plates. Cytotoxicity was determined by plating cells in duplicate in 100 μl medium at a density of 2500 cells/well for HT29 (human colon adenocarcinoma), H460 (human lung carcinoma), A431 (human squamous cell carcinoma), and Dul45 (human prostate carcinoma) cells , 3000 cells/well for SW480 (human colon adenocarcinoma), 3500 cells/well for MCF7 (human breast adenocarcinoma), BE2-C (human neuroblastoma) and SJ-G2 (human glioblastoma), and 2000 cells/well for A2780 (human ovarian cancer). On day 0, (24 h after plating) when the cells were in logarithmic growth, 100 μl medium with or without the test agent (indole compounds 25, 26, 33, 34-1, 34-2 or 35) was added to each well. After 72 h drug exposure growth inhibitory effects were evaluated using an MTT (3-[4,5-dimethyltiazol- 2-yl] 2,5-diphenyl-tetrazolium bromide) assay^[23] and absorbance read at 540 nm. The IC₅₀ was the drug concentration at which cell growth is 50% inhibited based on the difference between the optical density values on day 0 and those at the end of drug exposure. Each experiment was conducted in triplicate.

1.3 Immunofluorescence microscopy

Cells were fixed in ice-cold 100% methanol for 10 min at -20⁰C, and then blocked in 3% bovine serum albumin/PBS for 45 min before the primary antibody, mouse anti-a- tubulin (clone DMlA; Sigma) was applied. Donkey anti-mouse Texas Red dye- conjugated AffiniPure secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) were then applied. Cell nuclei were counterstained with DAPI (4', 6'-diamidino-2- phenylindole) (Sigma). Cells were washed three times with PBS between each step except for after blocking. Cells were viewed and scored for multinucleation with a fluorescence microscope (Olympus).

1.4 Time-lapse microscopy analysis

HeLa cells were seeded into 6-well trays at 60% confluency and then synchronised at the G2/M border by treatment with the selective cdkl small-molecule inhibitor, RO- 3306 (9 μM) for at least 18 h. Cells were allowed to progress through mitosis following RO-3306 wash-out. Immediately following release into the cell cycle, the indicated dynamin inhibitor was added and cells were viewed with an Olympus 1X80 inverted microscope and a time-lapse series was acquired using a fully motorized stage, 10x objective, and Metamorph software using the Time-lapse modules. Temperature was controlled in a humidified atmosphere with 5% CO2. Imaging was performed for 20 hours with a lapse time of 10 minutes.

As hTERT and HFF cells are not able to be synchronised with RO-3306, asynchronously growing cells were treated with the indicated dynamin inhibitor, and the effect of these inhibitors was compared in different cell types. Immediately upon addition of the dynamin inhibitor, cells were viewed with an Olympus 1X80 inverted microscope and a time-lapse series was acquired as described above.

2. Indole analogues induce multinucleation in cells

The finding that DynII is required for successful completion of mitosis in a mammalian cell as described in Applicant's co-pending International Patent Application No. PCT/AU2008/000203 extends previous invertebrate studies^³'⁴⁴¹. Moreover, depletion of DynII by RNAi induces multinucleation, and a small molecule dynamin inhibitor (MiTMAB) ^⁴⁵'⁴²^ induces multinucleation in HeLa cells as shown in Fig. 4. In brief, HeLa cells were synchronised at the G2/M boundary with the Cdkl inhibitor, RO-3306. Immediately following RO-3306 wash-out, cells were treated with the indicated dynamin inhibitor for 6 h. Cells were fixed, stained for γ-tubulin and scored for multinucleation which is an index of cytokinesis failure. Neither the DMSO vehicle, nor analogs of MiTMAB that do not inhibit dynamin GTPase activity caused multinucleation (data not shown). An increase in the number of multinucleated HeLa cells was observed following a short treatment with all seven Indole compounds tested (Fig. 4). Indole 34-2 was the most effective at causing multinucleation (Fig. 4), showing that dynamin inhibitors in the Indole class cause cytokinesis failure due to inhibition of dynll GTPase activity.

3. Indoles induce multinucleation at the ingression and/or abscission stage of cytokinesis

Time-lapse microscopy was employed to define the point of cytokinesis failure induced by indoles in HeLa cells which were synchronised at the G2/M boundary with RO-3306 (Vassilev et al. 2006). Immediately following release into the cell cycle, cells were treated with the indicated dynamin inhibitor and observed by time-lapse microscopy for 20 h. This analysis is more accurate than fixed cell analysis as it allows individual cells to be monitored throughout a mitotic division thereby observing its individual fate. A dramatic effect of the Indole compounds was revealed. Of those cells that entered mitosis, between 38-92% of cells failed cytokinesis, resulting in multinucleation (data not shown). 34-2 was the most effective at inducing multinucleation in this assay. Of those 34-2 treated cells that entered mitosis, 92.8% failed cytokinesis. This was significantly more potent than MiTMAB, which induced multinucleation in 57.9% of cells that entered mitosis (data not shown). Time-lapse analysis of the cell cycle revealed that the point of failure for all Indole compounds tested, except for 34-2, is the abscission stage. This was revealed as cells remained connected to each other via an intracellular bridge post- ingression for a prolonged period of time prior to abscission or generation of a multinucleated cell. MiTMAB treated HeLa cells also failed cytokinesis at the abscission stage. This is consistent with our discovery of a role for dynll in membrane abscission to successfully complete cytokinesis. No effect on progression through any other stage of mitosis was observed in cells treated with these dynamin inhibitors.

In contrast to the other indoles tested, 34-2 has a dual mode-of-action. Of those cells that became multinucleated, 32% failed cytokinesis due to an inability to complete abscission in an analogous manner to cells treated with the other Indole compounds. However the other 68% of 34-2 treated multinucleated cells failed cytokinesis due to a failure in the membrane ingression stage. This provides a potential explanation why 34-2 is more effective at causing cytokinesis failure (Fig. 4). Overall, collectively, the results show that indoles caused cytokinesis failure.

4. Indole analogues inhibit growth in a range of cancer cell lines

The growth inhibitory properties of dynamin inhibitory indoles was evaluated. Nine cancer cell lines derived from different tissues were incubated for 72 hr with each dynamin inhibitor (100 μM). At 72 h after drug exposure cell growth was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. Growth of all cell lines tested was completely inhibited by all six indole compounds tested (data not shown). This was analogous to another dynamin inhibitor, MiTMAB. Next, the GI₅₀, (i.e., the concentration that inhibits cell growth by 50% for these indoles) was determined.. The GI₅₀ ranged from 3.4-36.7 μM (Table 7) showing the indoles effectively inhibit growth in a diverse range of cancer cell lines. The data shown in Table 7 are means ± SEM for 3 independent experiments.

Table 7: Growth inhibition (MTT assay) induced by indole related compounds in nine cancer cell lines after 72 hour exposure

5. Compound 34-2 causes cell death following cytokinesis failure

A key characteristic of the new anti-mitotic anti-cancer agents currently being developed, such as the Aurora kinase inhibitor AZDl 152^[26], are that they cause cell death following failure of a specific mitotic event. Therefore, indole-based dynamin inhibitors described herein were tested for anti-proliferative properties by inducing cell death specifically following cytokinesis failure. Time-lapse analysis was employed to monitor the fate of individual cells following a failed mitotic division. Compound 34-2 induced cell death specifically following cytokinesis failure at the 20 hr time point measured (Fig. 5). The most striking observation was that among the 34-2-treated cells which showed failed cytokinesis 100% of them resulted in cell death. MiTMAB was also very effective at causing cell death (88.8%) specifically following formation of a multinucleated cell. The other indole compounds tested did not cause cell death within 20 hr and the resulting multinucleated cells remained viable for the duration of the experiment (Fig. 5) (although they did cause cell death after 72 hours exposure (Table 6)). Thus, the anti-proliferative property of 34-2 is due to its ability to induce cell death specifically following cytokinesis failure.

6. Compound 34-2 does not induce cell death in non-tumourigenic cell lines

A study was undertaken to evaluate whether there may be a preference for the dynamin inhibitors described herein to induce cell death in cancer cells over other proliferative cells. Such a characteristic would suggest that the drugs may specifically target cancer cells. In the present study, effect of 34-2 compared to MiTMAB in non- tumorigenic cells was assessed. Time-lapse video analysis of culture cells treated with the inhibitors revealed that 34-2 (10 μM) did not induce cell death in human TERT- immortalised breast epithelial cells (hTERT) or in human foreskin fibroblasts (HFF) during the 20 h treatment period (Fig. 6). Note that the cells retain their normal shape and morphology, rather than rounding up. This contrasts with findings in the HeLa cervical carcinoma cell line (Fig. 6). MiTMAB (10 μM) showed no selectivity between non- cancer and cancer cell lines, inducing cell death in hTERT and HFF cells as effectively as in HeLa cells. These data suggest that 34-2 preferentially induces cell death in cancer cells.

EXAMPLE 3: Anti-seizure activity of compounds of Formula I

Since the indoles inhibit the activity of dynl which is required for synaptic vesicle endocytosis we reasoned that these compounds would reduce seizure activity in animal models of seizure. 1. Seizure models

The Anticonvulsant Screening Program (ASP) at the National Institutes of Health (NIH) (Bethesda, Maryland, United States) undertakes preclinical anticonvulsant screening of efficacy and toxicity in three industry-standard models of acute animal seizure. The initial ASP screens are for acute models of seizure, which utilize non- epileptic animals that are induced to have seizures by application of an electrical or chemical stimulus. The extent that a test drug increases the threshold to evoke seizures is a measure of its "anti-epileptic" effect. The animals do not have spontaneous seizures and are therefore not truly "epileptic", yet these models have good predictive powers. The three models used are i) scMet (subcutaneous injection of pentylenetetrazol), i) MES (maximal electroshock; 60 Hz), and ii) 6 Hz test (minimal clonic seizure; 6 Hz)^[46]

The first two of these are the "classical" models and are effective at predicting drug effects against generalised tonic-clonic seizures in humans, with sodium channel inhibition believed to be particularly involved in this effect. In contrast, the 6 Hz test is believed to identify a different spectrum of anti-epileptic effect and potentially compounds with novel mechanisms of action. The new generation AED, levetiracetam, is highly effective in this test but not against the first two^[46l

2. Activity of indoles 25, 33 and 35 in seizure models Three indoles, 25, 33, 35, were evaluated in the three seizure animal seizure models described in Example 3.1 above. All were negative in the scMet and MES screens, yet were without toxicity to the animals.

The same three compounds evaluated in the 6 Hz model at 100 mg/kg were found to each have similar low levels of protection, without toxicity. Although the response was low in each case (1/4 mice were protected), the low protection level occurred for all three compounds and occurred over multiple time points after administration from 0.25 to 4 hrs. The fact the responses appear at more than one time point indicates there is a level of consistent seizure protection being exhibited. Convincing protection was demonstrated when consideration is given the structural similarities of the compounds and the fact that they possessed low level activity across multiple time points.

When compound 33 was tested in the 6 Hz model at 300 mg/kg full protection was noted 1 hr post treatment in 4/4 mice (Table 7). The results show that these compounds have anti-seizure activity in rodent models. Table 7: Anticonvulsant evaluation (6 Hz, mice) of indole 33

Add ID: Indole 33

Solvent Code: MC

Solvent Prep: M&P, SB

Route Code: IP

Current (mA): 32

Time to Peak Effect

Time (Hours) 0.25 0.5 1.0

Dose #

Test (mg/kg) Dths N/F C N/F C N/F C

6Hz 300 0/4 1/4 I₅ 4/4 15

N/F = number of animals protected from seizure over the number tested C: ^l5 Minimal motor impairment detected in 1 animal

3. Discussion

The three indole compounds tested were effective in the 6 Hz test with no toxicity, but were not effective in the ScMET or MES tests. This spectrum of effects is indicative of a non-traditional mechanism of anti-convulsant action. The indoles offer protection in the 6 Hz animal models, without effect in the MES or scMET tests. This is the same profile that lead to the development of levetiracetam as an AED with a truly novel molecular target, and suggests a spectrum of efficacy that differs from conventional AEDs. It is, therefore concluded that the data for the acute seizure model validates the efficacy of the compounds tested, and dynamin and endocytosis as a novel AED target. EXAMPLE 4: Synthesis of compounds

4. Chemistry

4.1 General methods THF and ether were freshly distilled from sodium-benzophenone. Flash chromatography was carried out using silica gel 200-400 mesh (60 A). ¹H and ¹³CNMR were recorded at 300 MHz and 75 MHz respectively using a Bruker Avance 300 MHz spectrometer in CDCl₃ and DMSO-d₆. GCMS was performed using a Shimadzu GCMS- QP2100. The instrument uses a quadrupole mass spectrometer and detects samples via electron impact ionization (EI). The spectra were run on the VG Autospec-oa-tof tandem high resolution mass spectrometer using CI (chemical ionization), with methane as the carrier gas and PFK (perfluorokerosene) as the reference. All samples returned satisfactory analyses.

(2-Indol-l-yIethyl)dimethylamine (8)

To a cooled solution (0⁰C) of indole (3.0 g, 25.6 mmol) and THF (5OmL) was added slowly NaH (50% dispersion in mineral oil) (2.0 g, 41.7 mmol). This solution was allowed to stir 30min upon which time N,N-dimethylamino-2-chloroethane (3.2 g, 29.75 mmol) was added and solution refluxed for 70 hrs. Solution was allowed to cool and water added (50 mL). The organic layer was then removed and the aqueous layer extracted with ethyl acetate (2 x 5OmL). Combined organic layers were then evaporated under reduced pressure giving orange oil. This was purified via short column chromatography DCMrMeOH (90:10). Combination of the desired fractions and concentration under reduced pressure gave the desired product as yellow oil, 3.50 g (70%).

¹H ΝMR (CDCl₃): 7.71 (dd, J= 8.5 Hz, 1.0 Hz, IH), 7.43 (d, J= 8.4 Hz, IH), 7.30 (dt, J= 7.0 Hz, 1.0 Hz, IH), 7.17 (m, 2H), 6.55 (d, J= 3.5 Hz, IH), 4.25 (t, J= 7.0 Hz, 2H), 2.64 (t, J= 7.0 Hz, 2H), 2.26 (s, 6H);

¹³C ΝMR (CDCl₃): 135.7, 128.0, 127.3, 121.0, 120.3, 119.1, 109.1, 100.4, 56.0, 44.9, 43.8;

HRMS (m/z): [Mf calcd. for C₂H₁₆N₂, 188.1313; found, 188.1319. (3-Indol-l-ylpropyl)dimethylamine (9)

Synthesized using the general procedure as for (8) gave a yellow oil (68%).

¹H NMR (CDCl₃): 7.72 (dd, J= 8.4 Hz, 0.6 Hz, IH), 7.43 (d, J= 8.4 Hz, IH), 7.28 (dt, J= 7.0 Hz, 1.1 Hz, IH), 7.17 (m, 2H), 6.56 (d, J= 3.1 Hz, IH), 4.24 (t, J= 6.9 Hz, 2H), 2.31 (t, J= 7.0 Hz, 2H), 2.26 (s, 6H), 2.00 (quin, J= 6.9 Hz, 2H);

¹³C NMR (CDCl₃): 135.6, 128.1, 127.4, 120.9, 120.4, 118.7, 108.9, 100.5, 55.9, 44.9, 43.5, 27.7;

HRMS (m/z): [M]⁺ calcd. for C₁₃Hi₈N₂, 202.1470; found, 202.1475.

Dimethyl-[2-(2-methylindol-l-yl)ethyllamine (10)

Synthesized using the general procedure as for (8) gave a yellow oil (75%).

¹H NMR (CDCl₃): 7.45 (d, J= 7.8 Hz, IH), 7.37 (d, J= 8.2 Hz, IH), 7.10 (m, 2H), 7.00 (dt, J= 7.8 Hz, 0.9 Hz, IH), 4.15 (t, J= 6.7 Hz, 2H), 2.56 (t, J= 6.7 Hz, 2H), 2.22 (s, 3H), 2.17 (s, 6H); 1³C NMR (75 MHz, CDCl₃): 135.9, 128.1, 126.2, 120.8, 118.4, 118.0, 109.3,

108.6, 58.6, 45.1, 43.2, 9.3;

HRMS (m/z): [M]⁺ calcd. for Ci₃H₁₈N₂, 202.1470; found, 202.1461.

Dimethyl-[3-(2-methylindol-l-yl)propyl]amine (11) Synthesized using the general procedure as for (8) gave a yellow oil (73%).

¹H NMR (CDCl₃): 7.47 (d, J= 7.8 Hz, IH), 7.35 (d, J= 8.2, IH), 7.10 (m, 2H), 7.00 (dt, J= 7.8 Hz, 1.1 Hz, IH), 4.10 (t, J= 7.8 Hz, 2H), 3.35 (t, J= 6.9 Hz, 2H), 2.37 (s, 3H), 2.13 (t, J= 6.9 Hz, 2H), 2.09 (s, 6H), 1.80 (quin, J= 6.9 Hz, 2H);

¹³C NMR (CDCl₃): 135.9, 128.1, 126.0, 120.8, 118.4, 117.9, 110.2, 108.6, 55.6, 45.0, 42.7, 27.8, 9.3;

HRMS (m/z): [M]⁺ calcd. for Ci₄H₂₀N₂, 216.1626; found, 216.1631.

l-(2-Dimethylaminoethyl)-2-methyl-lH-indole-3-carbaldehyde (12)

Synthesized using the general procedure as for (8) gave an off white solid (78%); m.p 50-52⁰C.

¹H NMR (CDCl₃): 10.25 (s, IH), 8.14 (d, J= 8.1 Hz, IH), 7.43 (d, J= 7.5 Hz, IH), 7.24 (m, 2H), 4.29 (t, J= 7.2 Hz, 2H), 2.72 (s, 3H), 2.62 (t, J= 7.2 Hz, 2H), 2.29 (s, 6H); ¹³C NMR (CDCl₃): 184.4, 148.6, 136.0, 125.2, 122.4, 121.9, 119.9, 113.1 108.9, 55.7,

43.9, 40.4, 8.38;

HRMS (m/z): [M]⁺ calcd. for C₁₄H₁₈N₂O, 230.1419; found, 230.1422.

3-[l-(2-Dimethylaminoethyl)-2-methyl-lH-indol-3-yl]propionic acid (16)

Synthesized using the general procedure as for (8) gave a white solid (54%); m.p 68-70 ⁰C.

¹H NMR (CDCl₃): 10.60 (br, IH), 7.48 (d, J= 7.9 Hz, IH), 7.31 (d, J= 8.1 Hz, IH), 7.08 (m, 2H), 6.96 (dt, J= 7.0, 1.1Hz, IH), 4.01 (t, J= 5.9 Hz, 2H), 2.69 (t, J= 7.4 Hz, 2H), 2.43 (t, J= 5.8 Hz, 2H), 2.32 (t, J= 7.3 Hz, 2H), 2.14 (s, 6H), 2.07 (s, 3H) 1.88 (quin, J= 7.4 Hz, 2H);

¹³C NMR (CDCl₃): 172.7, 136.2, 127.0, 122.3, 120.7, 118.1, 118.0, 113.6, 111.2,

61.4, 57.2, 45.2, 33.2, 25.3, 23.9;

HRMS (m/z): [M]⁺ calcd. for C₁₆H₂₂N₂O₂, 274.1681; found, 274.1689.

3-(l-(3-(dimethylamino)propyl)-2-methyl-lH-indol-3-yl)propanoic acid (17)

Synthesized using the general procedure as for (11) gave a white solid (60%); m.p 74-76 ⁰C.

¹H NMR (CDCl₃): 10.61 (br, IH), 7.50 (d, J= 7.9 Hz, IH), 7.31 (d, J= 8.1 Hz, IH), 7.10 (m, 2H), 6.95 (dt, J= 7.1, 1.5 Hz, IH), 4.01 (t, J= 5.9 Hz, 2H), 2.69 (t, J= 7.4 Hz, 2H), 2.38 (t, J= 5.8 Hz, 2H), 2.31 (t, J= 7.3 Hz, 2H), 2.14 (s, 6H), 2.07 (s, 3H) 1.88 (quin, J= 7.4 Hz, 2H), 1.80 (quin, J= 5.8 Hz, 2H);

¹³C NMR (CDCl₃): 173.0, 137.2, 128.0, 122.7, 120.9, 118.3, 118.0, 113.9, 112.3,

61.5, 57.5, 46.1, 33.1, 25.2, 23.9, 22.8; HRMS (m/z): [M]⁺ calcd. for C₁₇H₂₄N₂O₂, 288.1838; found, 288.1845.

N-Benzyl-2-cyano-3-[l-(2-dimethylaminoethyl)-2-methyl-lH-indol-3-yl]acrylamide (19>

1 -(2-Dimethylaminoethyl)-2-methyl-lH-indole-3-carbaldehyde (12) (0.2g, 0.87mmol), N-benzyl-2-cyanoacetamide (0.15g, 0.87 mmol) ethanol (5ml) and piperidine were refluxed for 2hr. After this time water (30 ml) was added to solution. This was then extracted with ethyl acetat (2 x 50 ml). Organic layers were combined, dried over MgSO₄ and concentrated under reduced pressure giving an orange solid. This was recrystallized from MeOH giving an orange solid (82%); m.p 146-148°C.

¹H NMR (DMSO-(I₆): 8.68 (t, J= 5.4 Hz, IH), 8.38 (s, IH), 7.97 (d, J= 7.1 Hz, IH), 7.55 (d, J= 7.1 Hz, IH), 7.12 (m, 8H), 4.42 (d, J= 4.3 Hz, 2H), 4.32 (t, J= 4.5 Hz, 2H), 2.58 (m, 5H), 2.19 (s, 6H);

¹³C NMR (DMSO-d₆): 162.4, 146.1, 144.6, 139.4, 136.9, 128.2, 127.3, 126.7, 124.5, 122.4, 121.2, 121.0 118.3, 110.5, 107.6, 98.2, 57.9, 45.5, 41.1, 40.6, 11.2. HRMS (m/z): [M]⁺ calcd. for C₂₄H₂₆N₄O, 386.2107; found, 386.2112.

2-Cyano-N-(4-methoxybenzyl)-3-[l-(2-dimethylaminoethyl)-2-methyl-lH-indol-3-yl]- N-(4-methoxybenzyl-acryIamide (20)

Synthesized using the general procedure as for (19) gave an orange solid (47%); m.p 165-167°C.

¹H NMR (DMSO-de): 8.33 (s, IH), 8.07 (br, IH), 7.96 (m, 3H), 7.57 (d, J= 7.5 Hz, IH), 7.22 (m, 2H), 7.12 (d, J= 8.7 Hz, 2H), 4.29 (t, J= 7.2 Hz, 2H), 3.84 (s, 3H),

2.72 (s, 3H), 2.62 (t, J= 7.2 Hz, 2H), 2.29 (s, 6H); ¹³C NMR (75 MHz, DMSO-d₆): 162.4,

150.5, 145.5, 144.3, 136.1, 132.8, 125.2, 124.8, 122.3, 121.1, 120.7, 118.7, 114.2, 111.6,

107.6, 97.6, 58.1, 55.7, 43.9, 40.4, 11.4, 8.38; HRMS (m/z): [M]⁺ calcd. for C₂₅H₂₈N₄O₂, 416.2212; found, 416.2219.

2-Cyano-N-cyclohexyl-3-[l-(2-dimethylaminoethyl)-2-methyl-lH-indol-3- yljacrylamide (21)

Synthesized using the general procedure as for (19). Recrystalization from ethanol gave a yellow solid (76%); m.p 150-152 ⁰C. 1H NMR (DMSO-Cl₆): 8.35 (s, IH), 8.07 (br, IH), 7.95 (d, J= 7.3 Hz, IH), 7.60 (d,

J= 7.3 Hz, IH), 7.21 (m, 2H), 4.28 (t, J= 7.2 Hz, 2H), 3.71 (m, IH), 2.75 (s, 3H), 2.60 (t,

J= 7.1 Hz, 2H), 2.31 (s, 6H), 1.91 (m, 2H), 1.35 (m, 2H), 1.20-1.15 (m, 4H);

¹³C NMR (75 MHz, DMSO-d₆): 162.4, 145.6, 144.1, 137.5, 125.5, 122.1 121.1,

120.9, 118.1, 110.3, 108.2, 98.4, 55.6, 48.9, 43.8, 40.3, 25.5, 24.3, 24.1, 10.85; HRMS (m/z): [M]⁺ calcd. for C₂₄H₃₂N₄O, 392.2576; found, 392.2581. 2-Cyano-N-ethyl 3-[l(2-dimethylaminoethyl)-2-methyl-lH4ndolO-yl]aciylamide

(22)

Synthesized using the general procedure as for (19). Recrystalization from ethanol gave a yellow solid (58%); m.p 135-137 ⁰C. 1H NMR (DMSO-de): 8.33 (s, IH), 8.07 (br, IH), 7.96 (d, J= 7.2 Hz, IH), 7.57 (d,

J= 7.5 Hz, IH), 7.22 (m, 2H), 4.29 (t, J= 7.2 Hz, 2H), 3.24 (quin, J= 6.6 Hz, 2H), 2.72 (s, 3H), 2.62 (t, J= 7.2 Hz, 2H), 2.29 (s, 6H), 1.14 (t, J= 7.3 Hz, 3H);

¹³C NMR (DMSO-d₆): 162.2, 145.5, 144.2, 136.8, 124.5, 122.4, 121.1, 120.9, 118.3, 110.4, 107.5, 98.9, 55.7, 34.7, 43.9, 40.4, 13.6, 10.4; HRMS (m/z): [M]⁺ calcd. for C₁₉H₂₄N₄O, 324.1950; found, 324.1959.

2-Cyano-N-propyI-3-[l-(2-dimethylaminoethyl)-2-methyl-lH-indol-3-yl]acrylamide

Synthesized using the general procedure as for (19). Recrystalization from ethanol gave a yellow solid (54%); m.p 136-138 ⁰C. 1H NMR (DMSO-de): 8.08 (s, IH), 7.85 (br, IH), 7.74 (d, J= 6.9 Hz, IH), 7.37 (d,

J= 6.8 Hz, IH), 7.07 (m, 2H), 4.49 (t, J= 5.6 Hz, 2H), 3.49 (q, J= 5.8 Hz, 2H), 2.94 (s, 3H), 2.85 (t, J= 5.6Hz, 2H), 2.60 (s, 6H), 1.99 (quin, J= 6.4 Hz, 2H) 1.42 (t, J= 6.5Hz, 3H);

¹³C NMR (DMSO-d₆): 162.2, 145.5, 144.2, 136.8, 124.5, 122.4, 121.1, 120.9, 118.3, 110.4, 107.5, 98.9, 57.9, 45.4, 41.7, 41.3, 22.2, 11.4, 11.3;

HRMS (m/z): [M]⁺ calcd. for C₂₀H₂₆N₄O, 338.2107; found, 338.2113.

2-Cyano-N-butyl -3-[l-(2-dimethylammoethyl)-2-methyl-lH-indol-3-yl] acrylamide (24) Synthesized using the general procedure as for (19). Recrystalization from ethyl acetate gave a yellow solid (54%); m.p 137-139 ⁰C.

¹H NMR (DMSO-d₆): 8.33 (s, IH), 8.07 (br, IH), 7.96 (d, J= 7.2 Hz, IH), 7.57 (d, J= 7.5 Hz, IH), 7.22 (m, 2H), 4.29 (t, J= 7.2 Hz, 2H), 3.22 (q, J= 6.9 Hz, 2H) 2.72 (s, 3H), 2.62 (t, J= 7.2 Hz, 2H), 2.29 (s, 6H), 1.48 (quin, J= 7.2 Hz, 2H), 1.33 (sept, J= 7.9 Hz, 2H), 0.89 (t, J= 7.2 Hz, 3H);

¹³C NMR (DMSO-d₆): 162.2, 145.5, 144.2, 136.8, 124.5, 122.4, 121.1, 120.9, 118.3, 110.4, 107.5, 98.9, 55.7, 43.9, 40.4, 39.5, 25.4, 19.4, 13.0, 12.2;

HRMS (m/z): [M]⁺ calcd. for C₂iH₂₈N₄O, 352.2263; found, 352.2270. 2-Cyano-3-[l(2-dimethylaminoethyl)-2-methyl-lH-indoI-3-yl] -N-hexylacrylamide

(25)

Synthesized using the general procedure as for (19). Recrystalization from ethyl acetate gave a yellow solid (67%); m.p 119-121 ⁰C. 1H NMR (300 MHz, DMSO-d₆): 8.25 (s, IH), 8.10 (br, IH), 8.00 (d, J= 7.1 Hz,

IH), 7.62 (d, J= 7.1 Hz, IH), 7.20 (m, 2H), 4.32 (t, J= 7.2 Hz, 2H), 3.22 (q, J= 7.2 Hz, 2H), 2.70 (s, 3H), 2.59 (t, J= 7.2 Hz, 2H), 2.30 (s, 6H), 1.46 (quin, J= 7.3 Hz, 2H), 1.27 (m, 6H), 0.85 (t, J= 6.5 Hz, 2H);

¹³C NMR (75 MHz, DMSO-4): 163.1, 145.2, 144.2, 136.2, 125.0, 122.3, 120.9, 19.9, 118.2, 110.4, 106.5, 99.0, 55.8, 44.1, 40.2, 38.7, 28.6, 25.8, 25.2, 21.7, 11.3;

HRMS {m/z)\ [M]⁺ calcd. for C₂₃H₃₂N₄O, 380.2576; found, 380.2578.

2-Cyano-3-[l-(2-dimethylaminoethyl)-2-methyl-lH-indol-3-yl] -N-octylacyrlamide (26) Synthesized using the general procedure as for (19). Recrystalisation from ethylacetate gave a yellow solid (56%); m.p 112-113 ⁰C.

¹H NMR (DMSO-de): 8.30 (s, IH), 8.10 (br, IH), 7.99 (d, J= 7.0 Hz, IH), 7.59 (d, J= 7.5 Hz, IH), 7.20 (m, 2H), 4.28 (t, J= 7.1 Hz, 2H), 3.27 (q, J= 6.4 Hz, 2H), 2.74 (s, 3H), 2.62 (t, J= 7.2 Hz, 2H), 2.30 (s, 6H), 1.50 (quin, J= 6.7 Hz, 2H), 1.31 (m, 12H), 0.92 (t, J= 6.3 Hz, 3H);

¹³C NMR (DMSO-do): 162.1, 144.2, 143.6, 1363, 125.5, 123.3, 121.0, 120.4, 118.3, 110.2, 107.4, 99.0, 56.7, 44.3, 41.4, 40.2, 28.9, 28.5, 26.1, 25.4, 22.1, 12.4;

HRMS (m/z): [M]⁺ calcd. for C₂₅H₃₆N₄O, 408.2889; found, 408.2895.

2-Cyano-3- [ 1 -(3-diπiethylaminop ropyl)-2 methyl- 1 H-indol-3-yl] N-benzyl-acrylamide (27)

Synthesized using the general procedure as for (19) gave an orange solid (64%); m.p 165-168 °C. 1H NMR (DMSO-de): 8.58 (t, J= 5.4 Hz, IH), 8.28 (s, IH), 7.95 (d, J= 7.0 Hz,

IH), 7.64 (d, J= 7.1 Hz, IH), 7.13-7.10 (m, 8H), 4.54 (d, J= 4.4 Hz, 2H), 4.20 (t, J= 7.0 Hz, 2H), 2.72 (s, 3H), 2.17 (t, J= 6.9 Hz, 2H), 2.10 (s, 6H), 1.82 (quin, J= 6.9 Hz, 2H); ¹³C NMR (DMSO-d₆): 163.2, 146.3, 145.0, 140.4, 137.3, 128.1, 126.9, 126.5, 124.5, 122.3, 121.2, 120.8 118.2, 110.4, 108.0, 98.1, 54.3, 44.6, 40.6, 26.8, 9.9; HRMS (m/z): [M]⁺ calcd. for C₂₅H₂₈N₄O, 400.2263; found, 400.2270.

2-Cyano-3-[l-(3-dimethyIaminopropyl)-2-methyl-lH-indol-3-yl]-N-(4- methoxybenzyl)acrylamide (28)

Synthesized using the general procedure as for (19) gave a yellow solid (74%); m.p 179-180 °C.

¹H NMR (DMSO-de): 8.38 (s, IH), 8.11 (br, IH), 7.94 (m, 3H), 7.55 (d, J= 7.5 Hz, IH), 7.22 (m, 2H), 7.11 (d, J= 8.5 Hz, 2H), 4.25 (t, J= 7.5 Hz, 2H), 2.73 (s, 3H), 2.17 (t, J= 6.7 Hz, 2H), 2.15 (s, 6H), 1.85 (quin, J= 6.9 Hz, 2H);

¹³C NMR (DMSOd₆): 162.5, 150.5, 146.5, 144.1, 138.1, 132.3, 127.2, 124.9, 122.3, 121.0, 120.7, 118.5, 114.1, 110.6, 108.3, 97.9, 58.0, 55.3, 44.9, 43.3, 40.2, 26.8, 11.0; HRMS (m/z): [M]⁺ calcd. for C₂₆H₃₀N₄O₂, 430.2369; found, 430.2375.

2-Cyano-3-[l-(3-dimethylaminopropyl)-2-methyl-lH-indol-3-yl]-N- cyclohexylacrylamide (29)

Synthesized using the general procedure as for (19). Recrystalization from ethanol gave a light yellow solid (67%); m.p 163-165 ⁰C.

¹H NMR (DMSO-Cl₆): 8.40 (s, IH), 8.16 (br, IH), 8.02 (d, J= 7.4 Hz, IH), 7.67 (d, J= 7.5 Hz, IH), 7.26 (m, 2H), 4.28 (t, J= 7.0 Hz, 2H), 3.74 (m, IH), 2.72 (s, 3H), 2.20 (t, J= 7.0 Hz, 2H), 2.08 (s, 6H), 1.90 (m, 2H), 1.82 (quin, J= 7.0 Hz, 2H), 1.32 (m, 2H), 1.20-1.15 (m, 4H); 1³C NMR (DMSO-(I₆): 162.2, 145.3, 144.0, 136.1, 125.1, 121.9, 120.8, 120.2,

118.5, 111.0, 107.1, 97.2, 55.4, 48.5, 44.5, 40.2, 31.0, 26.5, 25.0, 24.5, 23.8, 10.0; HRMS (m/z): [M]⁺ calcd. for C₂₅H₃₄N₄O, 406.2733; found, 406.2739.

2-Cyano-3-[l-(3-dimethylaminopropyl)-2-methyl-lH-indol-3-yl]-N-hexylacrylamide (33)

Synthesized using the general procedure as for (19). Recrystalization from ethanol gave a yellow solid (46%); m.p 100-102 ⁰C. ¹H NMR (DMSO-dβ): 8.35 (s, IH), 8.10 (br, IH), 7.98 (d, J= 7.1 Hz, IH), 7.58 (d, J= 7.5 Hz, IH), 7.21 (m, 2H), 4.25 (t, J= 6.2 Hz, 2H), 3.22 (q, J= 7.1 Hz, 2H), 2.58 (s, 3H) 2.20 (t, J= 6.6 Hz, 2H), 2.10 (s, 6H), 1.85 (quin, J= 6.9 Hz, 2H) 1.52 (quin, J= 7.3 Hz, 2H), 1.27 (m, 6H), 0.89 (t, J= 6.4 Hz, 2H); 1³C NMR (DMSO-d₆): 164.4, 147.5, 146.3, 138.0, 127.5, 124.1, 123.1, 121.7,

119.5, 112.2, 108.4, 98.6, 56.3, 45.0, 41.5, 32.0, 28.5, 27.8, 26.8, 26.0, 22.9, 14.1, 11.2, 9.7;

HRMS (m/z): [M]⁺ calcd. for C₂₄H₃₄N₄O, 394.2733; found, 394.2735.

Although the invention has been described with reference to a number of embodiments, it will be appreciated by those skilled in the art that numerous variations and/or modifications may be made. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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Claims

1. A compound of Formula 1 , or a physiologically acceptable salt or prodrug thereof, wherein:

Formula I and ring A is an aryl ring, a C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, a heterocycloalkyl or heterocycloalkenyl, the heterocycloalkyl or heterocycloalkenyl having 4 to 6 ring atoms, or a heteroaryl with 5 to 6 ring atoms, where ring A is optionally substituted; ring B is an aryl ring, a C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, a heterocycloalkyl or heterocycloalkenyl, the heterocycloalkyl or heterocycloalkenyl having 4 to 6 ring atoms, or a heteroaryl with 5 to 6 ring atoms;

R₁ is alkyl, alkenyl, alkylcarboxy, alkylthiocarboxy, alkylaryl, alkoxy, alkoxy- alkyl, aryl, aryloxy, aryloxyalkyl, alkoxyalkyl, alkylamino, alkenylamino, aminoalkyl, aminoalkenyl, aminoaryl, arylamino, alkylaminoaryl, alkenylaminoaryl, arylaminoalkyl, arylaminoalkenyl, carbocyclic, heterocyclic, alkyl C₃-C₆ cycloalkyl amino, alkyl C₃-C₆ cycloalkenyl amino, alkenyl C₃-C₆ cycloalkyl amino, alkenyl C₃-C₆ cycloalkenyl amino, alkyl C₃-C₆ heterocycloalkyl amino, alkenyl C₃-C₆ heterocycloalkyl amino, alkyl C₃-C₆ heterocycloalkenyl amino, alkenyl C₃-C₆ heterocycloalkenyl amino or an alkylNR group;

R₃ is a C₁-C₄ alkylNR group, C₁-C₄ alkylNR group, lower alkyl, lower alkenyl, aryloxy-lower alkyl, lower alkoxy-lower alkyl, or R₃ '-(Z)_n, where R₃' is a lower alkyl, lower alkenyl, aryloxy-lower alkyl, or lower alkoxy-lower alkyl;

NR is N(CH₃)₂, NCH(CH₃)₂, an amide, or an N containing heterocyclic ring with 5 to 6 ring members;

Z is a lower alkenyl, cyclo-lower alkenyl, aryl lower alkenyl, lower alkyl, aryloxy- lower alkyl, or lower alkoxy-lower alkyl or alkylamino, alkenylamino, aminoalkyl, aminoalkenyl, aminoaryl, arylamino, alkylaminoaryl, alkenylaminoaryl, arylaminoalkyl, arylaminoalkenyl, carbocyclic, heterocyclic, alkyl C₃-C₆ cycloalkyl amino, alkyl C₃-C₆ cycloalkenyl amino, alkenyl C₃-C₆ cycloalkyl amino, alkenyl C₃-C₆ cycloalkenyl amino, alkyl C₃-C₆ heterocycloalkyl amino, alkenyl C₃-C₆ heterocycloalkyl amino, alkyl C₃-C₆ heterocycloalkenyl amino, alkenyl C₃-C₆ heterocycloalkenyl amino or an alkylNR group; and n is an integer of from 1 to 12.

2. A compound of Formula Ia, or a physiologically acceptable salt or prodrug thereof, wherein:

Formula Ia where

R₂ is H, lower alkyl, lower alkenyl, aryloxy-loweralkyl, loweralkoxy-lower alkyl, or R₂ '-(Z)_n, where R₂ 'is a lower alkyl, lower alkenyl, aryloxy-lower alkyl, lower alkoxy-lower alkyl, or forms a 5 or 6 membered ring with R₁ optionally including N; and

Z and n are for Formula I.

3. A pharmaceutical composition comprising a compound of Formula I or Ia as defined in claim 1 or 2, or a physiologically acceptable salt or prodrug thereof, together with a pharmaceutically acceptable carrier.

4. A method for inhibiting dynamin protein, comprising contacting a dynamin protein with a compound of Formula I or Ia as defined in claim 1 or 2, or a physiologically acceptable salt or prodrug thereof.

5. A method for inhibiting endocytosis in a cell, comprising treating the cell with an effective amount of a compound of Formula I or Ia as defined in claim 1 or 2, or a physiologically acceptable salt or prodrug thereof.

6. A method for the prophylaxis or treatment of a disease or condition in a mammal, comprising administering to the mammal an effective amount of a compound of Formula I or Ia as defined in claim 1 or 2, or a physiologically acceptable salt or prodrug thereof, the disease or condition being selected from the group consisting of cell proliferative diseases and conditions, neuropathic pain, epilepsy, seizures, psychotic disorders, psychosis, β- amyloid associated diseases, aberrant up-regulated neuronal excitation, and diseases and conditions mediated by, or associated with, synaptic vesicle endocytosis, synaptic signal transmission, or cell vesicle trafficking.

7. A method according to claim 6 wherein the disease or condition is cancer or epilepsy.