Patent Application
20100144652
The present invention relates to a composition.
In particular the present invention relates to a composition and to the sequential use of the components of the composition. The present invention also relates to the use of the composition or components thereof in therapy applications.
In 2004, breast and prostate cancer were the third and fourth most common form of cancer, respectively, in Europe (Boyle & Ferlay, 2005). Current treatments for advanced hormone-independent cancer are limited, with resistance and toxicity being common problems with many therapies (Gottesman et al., 2002). There is therefore, a requirement to develop more efficacious therapeutic interventions for treating cancer. Solid tumours are particularly difficult to treat because most conventional chemotherapeutic drugs and radiation therapy only target the rapidly growing peripheral cells, leaving the slower growing tumour cells in the core to survive (Brown & Giaccia, 1998).
Normal, healthy cells undergo aerobic respiration to produce ATP, however, Warburg first demonstrated that even in the presence of oxygen, cancer cells create ATP anaerobically, relying on glucose for glycolysis (Warburg). Hence, inhibiting glycolysis could specifically target cancer cells; the effects on healthy normal cells should be minimal. Blocking glycolysis is possible by using 2-deoxy-D-glucose (2DG), which is structurally related to glucose and therefore, should not be toxic to healthy cells. The glucose molecule has a hydroxy group at its second carbon, however if this is changed to hydrogen, 2-deoxy-D-glucose (2DG) is formed (see
2DG was shown to inhibit growth of adramycin-resistant MCF-7 cells which exhibit an enhanced rate of glycolysis (Kaplan et al., 1990). Several groups have shown 2DG to be an effective anti-tumour compound in vitro and in vivo (Aft et al., 2002; Gupta et al., 2005; Lampidis et al., 2006; Liu et al., 2001; Maschek et al., 2004). However, very few clinical studies have tested 2DG as a single agent therapy and the results of such studies have been disappointing (Landau BR et al., 1958, Kaplin O et al., 1990). In vivo studies have provided strong evidence that using 2DG in combination with existing anti-cancer agents, such as adriamycin or paclitaxel may enhance efficacy (Maschek G et al., 2004). For example, 2DG is currently being used in a Phase 1 trial in combination with the microtubule disruptor Docetaxel in patients with solid tumours (Raez et al., 2005). The microtubule disruptor Docetaxel would attack the rapidly growing cells and the inner core, reliant on glycolysis, could be targeted with 2DG. However, taxanes are toxic, not orally bioavailable and tumours often develop resistance to the taxanes (Polizzi et al, 1999).
The present invention is based on the surprising finding that action against tumours of a combination of a glycolytic inhibitor and a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group is improved compared to the action of the materials alone or compared to what would be expected from the combination.
According to one aspect of the present invention, there is provided a composition comprising (a) a glycolytic inhibitor (b) a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group; wherein (a) and (b) are different.
According to one aspect of the present invention, there is provided a product comprising (a) a glycolytic inhibitor (b) a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group; wherein (a) and (b) are different, for simultaneous, separate or sequential use in the treatment of cancer.
According to one aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a glycolytic inhibitor (b) a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group; wherein (a) and (b) are different, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
According to one aspect of the present invention, there is provided a composition as defined herein, for use in medicine.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament to prevent and/or inhibit tumour growth.
According to one aspect of the present invention, there is provided a composition as defined herein for preventing and/or inhibiting tumour growth.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament for use in the therapy of a condition or disease associated with one or more of steroid sulphatase (STS) activity; cell cycling; apoptosis; cell growth; glucose uptake by a tumour; tumour angiogenesis; microtubules formation; and apoptosis.
According to one aspect of the present invention, there is provided a composition as defined herein, for treatment of a condition or disease associated with one or more of steroid sulphatase (STS) activity; cell cycling; apoptosis; cell growth; glucose uptake by a tumour; tumour angiogenesis; microtubules formation; and apoptosis.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse levels of one or more of steroid sulphatase (STS) activity; cell cycling; apoptosis; cell growth; glucose uptake by a tumour; tumour angiogenesis; microtubules formation; and apoptosis.
According to one aspect of the present invention, there is provided a composition as defined herein for the treatment of a condition or a disease associated with adverse levels of one or more of steroid sulphatase (STS) activity; cell cycling; apoptosis; cell growth; glucose uptake by a tumour; tumour angiogenesis; microtubules formation; and apoptosis.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament for one or more of inhibiting steroid sulphatase (STS) activity; modulating cell cycling; modulating apoptosis; modulating cell growth; preventing and/or suppressing glucose uptake by a tumour; preventing and/or inhibiting tumour angiogenesis; disrupting microtubules; and inducing apoptosis.
According to one aspect of the present invention, there is provided a composition as defined herein for one or more of inhibiting steroid sulphatase (STS) activity; modulating cell cycling; modulating apoptosis; modulating cell growth; preventing and/or suppressing glucose uptake by a tumour; preventing and/or inhibiting tumour angiogenesis; disrupting microtubules; and inducing apoptosis.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament for modulating cell growth.
According to one aspect of the present invention, there is provided a composition as defined herein, for modulating cell growth.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament for treating a cancer.
According to one aspect of the present invention, there is provided a composition as defined herein, for the treatment of cancer.
The cancer may be any susceptible cancer. The cancer may be in the form of a solid tumour. The cancer may in one aspect be breast cancer, ovarian cancer, non-small lung cancer, endometrial cancer, haematological malignancy or prostate cancer. The cancer may in one aspect be breast cancer, ovarian cancer or prostate cancer.
According to one aspect of the present invention, there is provided use of a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group, in the manufacture of a medicament for rendering a tumour susceptible to action by a glycolytic inhibitor.
According to one aspect of the present invention, there is provided a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group for rendering a tumour susceptible to action by a glycolytic inhibitor.
According to one aspect of the present invention, there is provided use of a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group, in the manufacture of a medicament to intensify at least one of hypoxia and glycolysis in a tumour.
According to one aspect of the present invention, there is provided a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group to intensify at least one of hypoxia and glycolysis in a tumour.
According to one aspect of the present invention, there is provided use of a composition as defined herein, in the manufacture of a medicament for decreasing Adenosine 5′-triphosphate (ATP) levels in a tumour.
According to one aspect of the present invention, there is provided a composition as defined herein, for decreasing Adenosine 5′-triphosphate (ATP) levels in a tumour.
According to one aspect of the present invention, there is provided a method of treatment comprising administering to a subject in need of treatment a composition as defined herein.
According to one aspect of the present invention, there is provided a method of treatment comprising administering to a subject in need of treatment a composition as defined herein, in order to inhibit steroid sulphatase (STS) activity; modulate cell cycling; modulate apoptosis; modulate cell growth; prevent and/or suppress glucose uptake by a tumour; prevent and/or inhibit tumour angiogenesis; disrupt microtubules; and/or induce apoptosis.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
Some Advantages
One key advantage of the present invention is that the compositions of the present invention can prevent and/or inhibit tumour angiogenesis.
One key advantage of the present invention is that the compositions of the present invention can modulate cell cycling.
One key advantage of the present invention is that the compositions of the present invention can modulate apoptosis.
One key advantage of the present invention is that the compositions of the present invention can modulate cell growth.
One key advantage of the present invention is that the compositions of the present invention can prevent and/or suppress glucose uptake by a tumour.
One key advantage of the present invention is that the compositions of the present invention can disrupt microtubules.
One key advantage of the present invention is that the compositions of the present invention can induce apoptosis.
The present invention is based on the surprising finding that the compositions provide an effective treatment of cancer.
Another advantage of the compositions of the present invention is that they may be potent in vivo.
Some of the compositions of the present invention are also advantageous in that they may be orally active.
The compositions of the present invention may useful for the prevention and/or treatment of cancer, such as breast cancer, as well as (or in the alternative) non-malignant conditions, such as the prevention and/or treatment of inflammatory conditions—such as conditions associated with any one or more of: autoimmunity, including for example, rheumatoid arthritis, type I and II diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, endometriosis, ulcerative colitis and Crohn's disease, skin disorders e.g. acne, psoriasis and contact dermatitis; graft versus host disease; eczema; asthma and organ rejection following transplantation. In one aspect the compositions of the present invention may useful for the prevention and/or treatment of endometriosis. The compounds of the present invention are useful particularly when pharmaceuticals may need to be administered from an early age.
In one embodiment, the compounds of the present invention are useful for the treatment of breast cancer.
In one embodiment, the compounds of the present invention are useful for the treatment of prostate cancer.
In one embodiment, the compounds of the present invention are useful for the treatment of ovarian cancer.
Thus, some of the compounds of the present invention are also believed to have therapeutic uses other than for the treatment of endocrine-dependent cancers, such as the treatment of autoimmune diseases.
We have studied combinations of glycolytic inhibitor (such as 2-deoxy-D-glucose [2DG]) and compounds comprising a ring system substituted with at least one of a sulphamate group (such as STX140) in MCF-7 and LNCaP cells in vitro. A MCF-7 (ER+ve) breast xenograft model was used to examine the potency of the composition on the growth of solid tumours. We have found that the present composition disrupt the rapidly dividing aerobic cells and offer a method of targeting both the hypoxic and aerobic cells in tumours.
We have also studied combinations of glycolytic inhibitor (such as 2-deoxy-D-glucose [2DG]) and compounds comprising a ring system substituted with at least one of a sulphamate group (such as STX140) in MCF-7 and LNCaP cells in vivo. We have found that the present composition significantly reduced tumour growth. We have found that the present composition significantly reduced tumour growth. Moreover, use of the present composition may provide for lower doses to be used of compounds comprising a ring system substituted with at least one of a sulphamate group.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
Preferable Aspects
Glycolytic Inhibtor
As discussed herein, the present invention provides a composition comprising a glycolytic inhibitor. It will be understood that by glycolytic inhibitor it is meant an inhibitor of glycolytic ATP production.
In one preferred aspect the glycolytic inhibitor is a glucose analogue or a glucose conjugate. The terms glucose analogue and glucose conjugate are well understood by one skilled in the art. It will be understood that a glucose analogue mimics glucose while not being metabolised. It will be understood that a glucose conjugate includes materials such as glucose sulphate and glucuronide. In one aspect, the glucose conjugate may be an analogue of a glucose conjugate, such as a glucose sulphamate.
In one preferred aspect the glycolytic inhibitor is a compound of the formula
wherein each of R16, R17, R18 is independently selected from H, OH, OSO2NH2, OSO3H, SO3H, oxamate and halogen and wherein R19 is CH2OH.
In one preferred aspect the glycolytic inhibitor is a compound of the formula
wherein each of R16, R17, R18 is independently selected from H, OH, OSO2NH2, OSO3H, SO3H, oxamate, and halogen and wherein R19 is CH3.
Preferably R16 is H, R17 is OH, and R18 is OH.
Highly preferred glycolytic inhibitors for use in the present invention may be selected from 2-deoxy-D-glucose, 1,6-dichloro-1,6-dideoxy-2-deoxyglucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), 2-fluor-2-deoxy-D-glucose (2FG), 2-deoxy-D-galactose, and 3H-2-deoxyglucose.
A highly preferred glycolytic inhibitor for use in the present invention is 2-deoxy-D-glucose.
Compound
As described above the present invention provides a composition or product comprising a compound comprising a ring system substituted with at least one of a sulphamate group and an alkoxy group.
In one preferred aspect the compound comprises a ring system substituted with a sulphamate group and an alkoxy group.
In one preferred aspect the ring system is a steroidal ring system.
Preferably the compound comprises a steroidal ring system ring system substituted with a sulphamate group and an alkoxy group.
In one preferred aspect the compound is of Formula I
wherein R1 is selected from —OH and a sulphamate group ; wherein R2 is selected from —OH, a sulphamate group, ═O and—L—R3, wherein L is an optional linker group and R3 is selected from groups which are or which comprise one of (i) —SO2R5, wherein R5 is H, a hydrocarbyl group or a bond or group attached to the D ring, (ii) —NO2, (iii) —SOR6, wherein R6 is H or a hydrocarbyl group, (iv) —R7, wherein R7 is a halogen, (v) -alkyl, (vi) —C(═O)R8, wherein R8 is H or hydrocarbyl, (vii) —C≡CR9, wherein R9 is H or hydrocarbyl, (viii) —OC(═O)NR10R11, (wherein R10 and R11 are independently selected from H and hydrocarbyl,
(xiv) a nitrile group, (xv) an alcohol, (xvi) an ester, (xvii) an ether, (xviii) an amine and (xix) an alkene.
The compounds of the present invention may comprise other substituents. These other substituents may, for example, further increase the activity of the compounds of the present invention and/or increase stability (ex vivo and/or in vivo).
Steroidal Ring System
The compound of the present invention has a steroidal ring component—that is to say a cyclopentanophenanthrene skeleton, or bio-isosteres thereof.
As is well known in the art, a classical steroidal ring structure has the generic formula of:
In the above formula, the rings have been labelled and numbered in the conventional manner.
In one aspect, the steroidal ring structure may contain any one or more of C, H, O, N, P, halogen (including Cl, Br and I), S and P.
At least one of the cyclic groups of the steroidal ring structure may be a heterocyclic group (a heterocycle) or a non-heterocyclic group.
At least one of the cyclic groups of the steroidal ring structure may be a saturated ring structure or an unsaturated ring structure (such as an aryl group).
Preferably, at least one of the cyclic groups of the steroidal ring structure is an aryl ring.
An example of a bio-isostere is when any one or more of rings A, B, C and D is a heterocyclic ring and/or when any one or more of rings A, B, C and D has been substituted and/or when any one or more of rings A, B, C and D has been modified; but wherein the bio-isostere has steroidal properties.
In this regard, the structure of a preferred steroidal ring structure can be presented as:
wherein each ring A′, B′, C′ and D′ independently represents a heterocyclic ring or a non-heterocyclic ring, which rings may be independently substituted or unsubstituted, saturated or unsaturated.
By way of example, any one or more of rings A′, B′, C′ and D′ may be independently substituted with suitable groups—such as an alkyl group, an allyl group, an hydroxy group, a halo group, a hydrocarbyl group, an oxyhydrocarbyl group etc.
The term “hydrocarbyl group” as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, a hydrocarbon group, an N-acyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
In one preferred embodiment of the present invention, the hydrocarbyl group is a hydrocarbon group.
Here the term “hydrocarbon” means any one of an alkyl group, an alkenyl group, an alkynyl group, an acyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
In one preferred embodiment of the present invention, the hydrocarbyl group is an oxyhydrocarbyl group.
The term “oxyhydrocarbyl group” as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
In one preferred embodiment of the present invention, the oxyhydrocarbyl group is an oxyhydrocarbon group.
Here the term “oxyhydrocarbon” means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably the oxyhydrocarbyl group is an alkoxy group. Preferably the oxyhydrocarbyl group is of the formula C1-6O (such as a C1-3O).
An example of D′ is a five or six membered non-heterocyclic ring having at least one substituent.
In one preferred embodiment, the ring D′ is substituted with an ethinyl group.
If any one of rings A′, B′, C′ and D′ is a heterocyclic ring, then preferably that heterocyclic ring comprises a combination of C atoms and at least one N atom and/or at least one O atom. Other heterocyclic atoms may be present in the ring.
Examples of suitable, preferred steroidal nuclei rings A′-D′ of the compounds of the present invention include rings A-D of oestrone and dehydroepiandrosterone.
Preferred steroidal nuclei rings A′-D′ of the compounds of the present invention include rings A-D of:
oestrones and substituted oestrones, viz:
oestrone
2-OH-oestrone
2-alkoxy-oestrone (such as C1-6 alkoxy-oestrone, such as 2-methoxy-oestrone)
4-OH-oestrone
6α-OH-oestrone
7α-OH-oestrone
16α-OH-oestrone
16β-OH-oestrone
oestradiols and substituted oestradiols, viz:
2-OH-17β-oestradiol
2-alkoxy-17β-oestradiol (such as C1-6 alkoxy-17β-oestradiol, such as 2-methoxy-17β-oestradiol)
4-OH-17β-oestradiol
6α-OH-17β-oestradiol
7α-OH-17β-oestradiol
2-OH-17α-oestradiol
2-alkoxy-17α-oestradiol (such as C1-6 alkoxy-17α-oestradiol, such as 2-methoxy-17α-oestradiol)
4-OH-17α-oestradiol
7α-OH-17α-oestradiol
16α-OH-17α-oestradiol
16α-OH-17α-oestradiol
16α-OH-17β-oestradiol
16β-OH-17α-oestradiol
16β-OH-17β-oestradiol
17α-oestradiol
17β-oestradiol
17α-ethinyl-17β-oestradiol
17β-ethinyl-17α-oestradiol oestriols and substituted oestriols, viz:
oestriol
2-OH-oestriol
2-alkoxy-oestriol (such as C1-6 alkoxy-oestriol, such as 2-methoxy-oestriol)
4-OH-oestriol
6α-OH-oestriol
7α-OH-oestriol
dehydroepiandrosterones and substituted dehydroepiandrosterones, viz:
dehydroepiandrosterones
6α-OH-dehydroepiandrosterone
7α-OH-dehydroepiandrosterone
16α-OH-dehydroepiandrosterone
16β-OH-dehydroepiandrosterone
In general terms the ring system A′B′C′D′ may contain a variety of non-interfering substituents. In particular, the ring system A′B′C′D′ may contain one or more hydroxy, alkyl especially lower (C1-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C1-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
In an alternative embodiment, the polycyclic compound may not contain or be based on a steroid nucleus. In this regard, the polycyclic compound may contain or be based on a non-steroidal ring system—such as diethylstilboestrol, stilboestrol, coumarins, and other ring systems. Other suitable non-steroidal compounds for use in or as the composition of the present invention may be found in U.S. Pat. No. 5,567,831.
R1 and R2
In one preferred aspect the compound is of Formula I
In one preferred aspect the compound is of Formula Ia
In one preferred aspect the compound is of Formula Ib
In one preferred aspect the compound is of Formula II
In one preferred aspect the compound is of Formula IIa
In one preferred aspect the compound is of Formula IIb
In one preferred aspect the compound is of Formula III
In one preferred aspect the compound is of Formula IIIa
In one preferred aspect the compound is of Formula IIIb
In one preferred aspect the compound is of Formula IVa or Formula IVb (preferably of Formula IVa)
In one preferred aspect the compound is of Formula IVc or Formula IVd (preferably of Formula IVc)
In one preferred aspect the compound is of Formula IVe or Formula IVf (preferably of Formula IVe)
In one preferred aspect the compound is of Formula Va or Formula Vb (preferably of Formula Va)
In one preferred aspect the compound is of Formula Vc or Formula Vd (preferably of Formula Vc)
R1
It will be appreciated by one skilled in the art that R1 is an optional group which may or may not be present. In one preferred aspect R1 is present. In this aspect R1 is a group selected from any one of —OH, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group or a sulphonamide group.
Sulphamate Group
In one aspect R1 is an optional sulphamate group.
The term “sulphamate” includes an ester of sulphamic acid, or an ester of an N-substituted derivative of sulphamic acid, or a salt thereof.
In one aspect R1 is a sulphamate group. In this aspect the compound of the present invention may be referred to as a sulphamate compound.
Preferably the sulphamate group of R1, is a sulphamate group of the formula
wherein R12 and R13 are independently selected from H or a hydrocarbyl group.
Preferably R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, acyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or aryl optionally contains one or more hetero atoms or groups (such as O, S and N).
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl, N-acyl, or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R12 and/or R13 is alkyl, the preferred values are those where R12 and R13 are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R5 and R6 are both methyl. When R12 and/or R13 is aryl, typical values are phenyl and tolyl (-PhCH3; o-, m- or p-). Where R5 and R6 represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R12 and R13 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. —OH— or —NH— to provide a 5-, 6- or 7-membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl, acyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the sulphatase inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl. A non-limiting example of a hydrocarbyl group is an acyl group.
In some embodiments, the sulphamate group may form a ring structure by being fused to (or associated with) one or more atoms in or on the steroidal ring system.
In some embodiments, there may be more than one sulphamate group. By way of example, there may be two sulphamates (i.e. bis-sulphamate compounds).
In some preferred embodiments, at least one of R12 and R13 is H.
In some preferred embodiments, each of R12 and R13 is H.
In some preferred embodiments R1 is a sulphamate group and the compound is suitable for use as an inhibitor of oestrone sulphatase (E.G. 3.1.6.2).
In some preferred embodiments if the sulphamate group on the sulphamate compound were to be replaced with a sulphate group to form a sulphate compound then the sulphate compound would be hydrolysable by a steroid sulphatase enzyme (E.C.3.1.6.2).
In some preferred embodiments if the sulphamate group on the sulphamate compound were to be replaced with a sulphate group to form a sulphate compound and incubated with a steroid sulphatase enzyme (E.C.3.1.6.2) at a pH 7.4 and 37° C. it would provide a Km value of less than 50 mM.
In some preferred embodiments if the sulphamate group on the sulphamate compound were to be replaced with a sulphate group to form a sulphate compound and incubated with a steroid sulphatase enzyme (E.C.3.1.6.2) at a pH 7.4 and 37° C. it would provide a Km value of less than 50 μM.
Other Substituents
The compound of the present invention may have substituents other than those of formula I. By way of example, these other substituents may be one or more of: one or more sulphamate group(s), one or more phosphonate group(s), one or more thiophosphonate group(s), one or more sulphonate group(s), one or more sulphonamide group(s), one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing group(s), one or more hydrocarbyl group(s)—such as an oxyhydrocarbyl group.
R2
R2 is selected from —OH, a sulphamate group, ═O and—L—R3, wherein L is an optional linker group and R3 is selected from groups which are or which comprise one of (i) —SO2R5, wherein R5 is H, a hydrocarbyl group or a bond or group attached to the D ring, (ii) —NO2, —SOR6, wherein R6 is H or a hydrocarbyl group, (iv) —R7, wherein R7 is a halogen, (v) -alkyl, (vi) —C(═O)R8, wherein R8 is H or hydrocarbyl, (vii) —C≡CR9, wherein R9 is H or hydrocarbyl, (viii) —OC(═O)NR10R11, wherein R10 and R11 are independently selected from H and hydrocarbyl,
(xiv) a nitrile group, (xv) an alcohol, (xvi) an ester, (xvii) an ether, (xviii) an amine and (xix) an alkene.
In some preferred embodiments R2 is of the formula —R3, in other words no group L is present.
In a preferred aspect R2 is selected from —OH, a sulphamate group.
In a highly preferred aspect R2 is a sulphamate group. Preferably R2 is a sulphamate group of the formula
wherein R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or aryl optionally contains one or more hetero atoms or groups (such as O, N and S).
When R12 and/or R13 is alkyl, the preferred values are those where R12 and R13 are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R12 and R13 are both methyl. When R12 and/or R13 is aryl, typical values are phenyl and tolyl (-PhCH3; o-, m- or p-). Where R12 and R13 represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R12 and R13 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. —O— or —NH— to provide a 5-, 6- or 7-membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
In one aspect R12 and R13 are each independently hydrocarbyl and H. In one preferred embodiment of the present invention R12 and R13 are each independently selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups,. C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl
In one aspect R12 and R13 are each independently selected from H and C1-10 alkyl. In one aspect R12 and R13 are each independently C1-10 alkyl. In one aspect R12 and R13 are each independently selected from H and C1-5 alkyl. In one aspect R12 and R13 are each independently C1-5 alkyl. In one aspect R12 and R13 are each independently selected from H and C1-3 alkyl. In one aspect R12 and R13 are each independently C1-3 alkyl. Preferably R12 and R13 are independently selected from —H and —CH3.
In some preferred embodiments, at least one of R12 and R13 is H.
In some preferred embodiments, each of R12 and R13 is H.
In some preferred aspects group R2 is in an a configuration. Preferably group R2 is in an a configuration on the 17 position of the D ring.
L
In some embodiments L is selected from a hydrocarbyl group, —NR14— and —O—, wherein R14 is H, a hydrocarbyl group or a bond.
Preferably L is selected from a hydrocarbon group, —NR14— and —O—.
In one aspect L is selected from an alkylene group (such as C1-10 alkylene, a C1-5 alkylene, a C1 or C2 alkylene), —NR14— and —O—.
In one aspect L is selected from a C1-10 alkylene group, —NR14— and —O—.
In one aspect L is selected from a C1 or C2 alkylene group, —NR14— and —O—.
Particularly preferred linkers are ═N—, —NH—, ═CH—, —CH2—, —CH2CH2— and ═CHCH2—, such as ═N—, —NH—, ═CH—, and —CH2—.
R3
As discussed above R3 is selected from (i) —SO2R5, wherein R5 is H, a hydrocarbyl group or a bond or group attached to the D ring, (ii) —NO2, (iii) —SORB, wherein R6 is H or a hydrocarbyl group, (iv) —R7, wherein R7 is a halogen, (v) -alkyl, (vi) —C(═O)R8, wherein R8 is H or hydrocarbyl, (vii) —C═CR9, wherein R9 is H or hydrocarbyl, (viii) —OC(═O)NR10R11, wherein R10 and R11 are independently selected from H and hydrocarbyl,
(xiv) a nitrile group, (xv) an alcohol, (xvi) an ester, (xvii) an ether, (xviii) an amine and (xix) an alkene.
R3 may be a cyclic group or an acyclic group.
When R3 is a cyclic group is may form a ring which is fused with the D ring of the steroid or which is not fused with the D ring of the steroid. When R3 forms a cyclic group which is fused with the D ring of the steroid, preferably R3 forms a ring joining adjacent members of the D ring, more preferably R3 forms a ring joining positions 16 and 17 of the D ring.
It will be appreciated by one skilled in the art that group R3 may be attached to optional L at any point on R3. Preferred points of attachment are shown when groups (ix) to (xiiii) are selected from optionally substituted groups of the formulae
—SO2R5
In one preferred aspect R3 is —SO2R5, wherein R5 is H, a hydrocarbyl group or a bond or group attached to the D ring
Preferably R5 is selected from H and hydrocarbyl. In one aspect R5 is hydrocarbyl. In one preferred embodiment of the present invention R5 is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R5 is selected from H and C1-10 alkyl. In one aspect R5 is C1-10 alkyl. In one aspect R5 is selected from H and C1-5 alkyl. In one aspect R5 is C1-5 alkyl. In one aspect R5 is selected from H and C1-3 alkyl. In one aspect R5 is C1-3 alkyl. Preferably R5 is —CH3.
Preferably R5 is —O—R15-D, wherein R15 is a linker and D is a member of the D ring. In a preferred aspect this provides a compound of the formula
R15 may be any suitable group. Particularly preferred are —O—CH2— and —N═CH—
In this aspect preferably R2 is —CH2—R3 or —NH—R3, for example in one preferred aspect R2 is —NH—SO2—CH3.
—NO2
In one preferred aspect wherein R3 is —NO2
In this aspect preferably R2 is —CH2—R3
—SOR6
In one preferred aspect R3 is —SOR6, wherein R6 is H or a hydrocarbyl group.
Preferably R6 is selected from H and hydrocarbyl. In one aspect R6 is hydrocarbyl. In one preferred embodiment of the present invention R6 is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R6 is selected from H and C1-C10 alkyl. In one aspect R6 is C1-C10 alkyl. In one aspect R6 is selected from H and C1-C5 alkyl. In one aspect R6 is C1-C5 alkyl. In one aspect R6 is —CH3. is selected from H and C1-3 alkyl. In one aspect R6 is C1-3 alkyl. Preferably R6 is —CH3.
In this aspect preferably R2 is —CH2—R3
R7
In one preferred aspect R3 is —R7, wherein R7 is a halogen
It will be appreciated that R7 may chlorine, fluorine, bromine or iodine. Preferably R7 is fluorine.
In this aspect preferably R2 is —CH2CH2—R3, namely —CH2CH2—R7.
In this aspect preferably R2 is —CH2CHX—R7 wherein X is a halogen. For example X may be F and R7 may be F such that R2 is —CH2CF2H.
In this aspect R2 may also be —CX2—R3, wherein each X is independently selected from halogens. For example each X may be F and R3 may be F such that R2 is CF3.
In this aspect R2 may be —CY2—R3 or —CY2CY2—R3, wherein each Y is independently selected from H and halogens. For example one or more Y may be F and R3 may be F. When only one Y is H and the remaining Y are H, R2 may be —CHY—R3 or —CH2CHY—R3, wherein Y is selected from H and halogens. For example Y may be F and R3 may be F.
-alkyl
In one preferred aspect R3 is -alkyl
In one preferred embodiment of the present invention R3 is selected from one of C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R3 is C1-10 alkyl. In one aspect R3 is C1-5 alkyl. In one aspect R3 is C1-3 alkyl. Preferably R3 is —CH3 or —CH2CH3.
In this aspect preferably R2 is R3.
In one preferred aspect when R3 is alkyl the compound is of Formula IVb
such as of Formula IVd
such as of Formula IVf
such as of Formula Vb
such as of Formula Vd
In one aspect the compound further comprises a further group denoted R2′ which is an alkyl group and preferably an alkyl group described under (v) herein. Thus in one preferred aspect the compound is selected from compounds of the formulae
wherein R2 and R2′ are independently selected from one of C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl. In a highly preferred aspect each of R2 and R2′ are —CH3.
—C(═O)R8
In one preferred aspect R3 is —C(═O)R8, wherein R8 is H or hydrocarbyl
Preferably R8 is selected from H and hydrocarbyl. In one aspect R8 is hydrocarbyl. In one preferred embodiment of the present invention R8 is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R8 is selected from H and C1-10 alkyl. In one aspect R8 is C1-10 alkyl. In one aspect R8 is selected from H and C1-5 alkyl. In one aspect R9 is C1-5 alkyl. In one aspect R8 is selected from H and C1-3 alkyl. In one aspect R8 is C1-3 alkyl. Preferably R8 is —CH3.
In this aspect preferably R2 is —CH2—R3 or R3, for example —C(═O)CH3.
—C≡CR9
In one preferred aspect R3 is —C≡CR9, wherein R9 is H or hydrocarbyl
Preferably R9 is selected from H and hydrocarbyl. In one aspect R9 is hydrocarbyl. In one preferred embodiment of the present invention R9 is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R9 is selected from H and C1-10 alkyl. In one aspect R9 is C1-10 alkyl. In one aspect R9 is selected from H and C1-5 alkyl. In one aspect R9 is C1-C5 alkyl. In one aspect R9 is selected from H and C1-3 alkyl. In one aspect R9 is C1-3 alkyl. Preferably R9 is —CH3.
In this aspect preferably R2 is —CH2—R3
—OC(═O)NR10R11
In one preferred aspect R3 is —OC(═O)NR10R11, wherein R10 and R11 are independently selected from H and hydrocarbyl
Preferably R10 and R11 are independently selected from H and hydrocarbyl. In one aspect R10 and R11 are independently selected from hydrocarbyl. In one preferred embodiment of the present invention R10 and R11 are independently selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R10 and R11 are independently selected from H and C1-10 alkyl. In one aspect R10 and R11 are independently selected from C1-10 alkyl. In one aspect R10 and R11 are independently selected from H and C1-5 alkyl. In one aspect R10 and R11 are independently selected from C1-5 alkyl. In one aspect R10 and R11 are independently selected from H and C1-3 alkyl. In one aspect R10 and R11 are independently selected from C1-3 alkyl. Preferably R10 and R11 are both H.
In this aspect preferably R2 is R3.
Cyclic Groups
In one preferred aspect R3 is
Preferably R3 is
In this aspect preferably R2 is selected from —CH2CH2—R3, ═N—R3 and —NH—R3
In one preferred aspect wherein R3 is
Preferably R3 is
Preferably R3 is
In this aspect preferably R2 is selected from ═CH—R3 and —CH2CH2—R3
In one preferred aspect wherein R3 is
Preferably R3 is
In this aspect preferably R2 is selected from ═CH—R3 and —CH2CH2—R3
In one preferred aspect R3 is
Preferably R3 is
Preferably R3 is selected from
In this aspect preferably R2 is selected from ═CH—R3 and —CH2CH2—R3
In one preferred aspect R3 is
Preferably R3 is
In this aspect preferably R2 is selected from ═CH—R3 and —CH2CH2—R3
In one preferred aspect R3 is selected from groups which are or which comprise one of a nitrile group, an alcohol, an ester, an ether, an amine and an alkene In some preferred aspects R3 is selected the groups nitrile, alcohol, ester, ether, amine and alkene. Preferably R3 is or comprises a nitrile group. Preferably R3 is a nitrile group.
In some preferred aspects R3 is selected from groups of the formula ═CH2, ═CH—CH3, ═C(CN)2, ═C(CH3)(CN), and —(R7a)n(CR14aR15a)pR8a, wherein n is 0 or or 1, p is an integer R7a is selected from ═CH—, —O— and NR13a; R8a is selected from —SO2—R9a, —C(O)OR17a, —OR10a, (CH2)q—X—R16a, —C≡N, —NR11aR12a—C≡CH and —CH═CH2; R9a is selected from H and hydrocarbyl, R10a is selected from H and hydrocarbyl; R11a and R12a are each independently selected from H and hydrocarbyl; R13a is selected from H and hydrocarbyl, R14a and R15a are each independently selected from H and hydrocarbyl, q is an integer, X is O or S, R16a is selected from H and hydrocarbyl and R17a is selected from H and hydrocarbyl.
In some preferred aspects R3 is a group of the formula —(R7a)n(CR14aR15a)pR8a, where n is 0 or 1, p is an integer, R7a is selected from ═CH—, —O— and NR13a; R8a is selected from —SO2—R9a, —C(O)OR17a, —OR10a, (CH2)q—X—R16a, —C≡N, —NR11aR12a—C≡CH and —CH═CH2; R9a is selected from H and hydrocarbyl, R10a is selected from H and hydrocarbyl; R11a and R12a are each independently selected from H and hydrocarbyl; R13 is selected from H and hydrocarbyl, R14a and R15a are each independently selected from H and hydrocarbyl, q is an integer, X is O or S, R16a is selected from H and hydrocarbyl and R17 is selected from H and hydrocarbyl.
In some preferred aspects R3 is a group of the formula —(CR14aR15a)pR8a, p is an integer; R8a is selected from —SO2-R9a, —C(O)OR17a, —OR10a, (CH2)q—X—R16a, —C≡N, —NR11aR12—C≡CH and —CH═CH2; R9a is selected from H and hydrocarbyl, R10a is selected from H and hydrocarbyl; R11a and R12a are each independently selected from H and hydrocarbyl, R14a and R15a are each independently selected from H and hydrocarbyl, q is an integer, X is O or S, R16a is selected from H and hydrocarbyl and R17a is selected from H and hydrocarbyl.
In some preferred aspects R3 is a group of the formula —(CH2)pR8a, p is an integer; R8a is selected from —SO2—R9a, —C(O)OR17a, —OR10a, (CH2)q—X—R16a, —C≡N, —NR11aR12a—C≡CH and —CH═CH2; R9a is selected from H and hydrocarbyl, R10a is selected from H and hydrocarbyl; R11a and R12a are each independently selected from H and hydrocarbyl, q is an integer, X is O or S, R16a is selected from H and hydrocarbyl and R17a is selected from H and hydrocarbyl.
In some preferred aspects R3 is a group of the formula —(R7a)nR8a, wherein n is 0 or 1, R7a (CH2)q—X—R16a, —C≡N, —NR11aR12a—C≡and —CH≡CH2; R9a is selected from H and hydrocarbyl, R10a is selected from H and hydrocarbyl; R11a and R12a are each independently selected from H and hydrocarbyl; R13a is selected from H and hydrocarbyl, q is an integer, X is O or S, R16a is selected from H and hydrocarbyl and R17a is selected from H and hydrocarbyl.
p may be any integer. p may be from 0 to 20. p may be from 0 to 10: Typically p is from 0 to 5. In one aspect p is 0, 1 or 2.
q may be any integer. q may be from 0 to 20. q may be from 0 to 10. Typically q is from 0 to 5. In one aspect q is 0, 1 or 2.
R8a is selected from —SO2—R9a, —C(O)OR17a, —OR10a, (CH2)q—X—R16a, —C≡N, —NR11aR12a—C≡CH and —CH═CH2. In one preferred aspect R8a is —SO2—R9a. In one preferred aspect R8a is —SO2—R9a wherein R9a is hydrocarbyl. Preferably in this aspect, R7a is —O—, n is 1 and p is 0 such that R3 is —O—SO2R9a.
R9a is selected from H and hydrocarbyl. In one aspect R9a is hydrocarbyl. In one preferred embodiment of the present invention R9a is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R9a is selected from H and C1-10 alkyl. In one aspect R9a is C1-10 alkyl. In one aspect R9a is selected from H and C1-5 alkyl. In one aspect R9a is C1-5 alkyl. In one aspect R9a is selected from H and C1-3 alkyl. In one aspect R9a is C1-3 alkyl. Preferably R9a is —CH2CH3.
In one aspect R9a is a substituted or unsubstituted amine. When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl, N-acyl, or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. In a preferred aspect, R9a is an unsubstituted amine, i.e. R9a is NH2.
R10a is selected from H and hydrocarbyl. In one preferred embodiment of the present invention R10a is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R10a is selected from H and C1-10 alkyl. In one aspect R10a is selected from H and C1-5 alkyl. In one aspect R10a is selected from H and C1-3 alkyl. In one aspect R10a is C1-3 alkyl. Preferably R10a is —H or —CH3.
As previously mentioned, R11a and R12a of NR11aR12a are each independently selected from H and hydrocarbyl. When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl, N-acyl, or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R11a and/or R12a is alkyl, the preferred values are those where R11a and R12a are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R11a and R12a are both methyl. When R11a and/or R12a is aryl, typical values are phenyl and tolyl (-PhCH3; o-, m- or p-). Where R11a and R12a represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R11a and R12a typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. —O— or —NH— to provide a 5-, 6- or 7-membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
In one aspect R11a and R12a are each independently hydrocarbyl. In one preferred embodiment of the present invention R11a and R12a are each independently selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl
In one aspect R11a and R12a are each independently selected from H and C1-10 alkyl. In one aspect R11a and R12a are each independently C1-10 alkyl. In one aspect R11a and R12a are each independently selected from H and C1-5 alkyl. In one aspect R11a and R12a are each independently C1-5 alkyl. In one aspect R11a and R12a are each independently selected from H and C1-3 alkyl. In one aspect R11a and R12a are each independently C1-3 alkyl. Preferably R1la and R12a are independently selected from —H and —CH3.
R13a is selected from H and hydrocarbyl. In one preferred embodiment of the present invention R13a is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R13a is selected from H and C1-10 alkyl. In one aspect R13a is selected from H and C1-5 alkyl. In one aspect R13a is selected from H and C1-3 alkyl. In one aspect R13a is C1-3 alkyl. Preferably R13a is —H.
R14a and R15a are each independently selected from H and hydrocarbyl. In one aspect R14a and R15a are each independently hydrocarbyl. In one preferred embodiment of the present invention R14a and R15a are each independently selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon, groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R14a and R15a are each independently selected from H and C1-10 alkyl. In one aspect R14a and R15a are each independently C1-10 alkyl. In one aspect R14a and R15a are each independently selected from H and C1-5 alkyl. In one aspect R14a and R15a are each independently C1-5 alkyl. In one aspect R14a and R15a are each independently selected from H and C1-3 alkyl. In one aspect R14a and R15a are each independently C1-3 alkyl.
Preferably R14a and R15a are independently selected from —H and —CH3.
X is selected from O or S. In one aspect X is S. In one aspect X is O.
R18a is selected from H and hydrocarbyl. In one preferred embodiment of the present invention R16a is selected from one of H, C1-C20 hydrocarbyl, C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C20 hydrocarbon, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C20 alkyl, C1C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one aspect R16a is selected from H and C1-10 alkyl. In one aspect R16a is selected from H and C1-5 alkyl. In one aspect R16a is selected from H and C1-3 alkyl. In one aspect R16a is C1-3 alkyl. Preferably R16a is —H.
In one highly preferred aspect R3a is a group selected from ═CHC(O)OEt, —CH2C(O)OEt, ═CHCH2OH, —CH2CH2OH, —CH2C≡N, ═CHC≡N, —NHCH2CH2N(Me)2, —OCH2CH2—OMe.
In one aspect R3a may be selected from the D substitutions shown below wherein each Q is independently selected from O, S, NH and CH2 and y is an integer from 3 to 8, preferably 5, 6, 7 or 8.
R4
As previously mentioned, the A ring of the steroidal ring system is optionally substituted with a group R4, wherein R4 is preferably selected from a hydrocarbyl group or an oxyhydrocarbyl group.
In one preferred embodiment of the present invention, the R4 is a oxyhydrocarbon group.
Here the term “oxyhydrocarbon” means, or R4 is, any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably the oxyhydrocarbyl group R4 is an alkoxy group. Preferably the oxyhydrocarbyl group R4 is of the formula C1-6O (such as a C1-3O). Preferably the oxyhydrocarbyl group R4 is of the formula —O(CH2)1-10CH3, —O(CH2)1-5CH3, —O(CH2)1-2CH3. In a highly preferred aspect R4 is methoxy.
Preferably the oxyhydrocarbyl group R4 is an ether group. Preferably the oxyhydrocarbyl group R4 is of the formula C1-6OC1-6 (such as a C1-3OC1-3). Preferably the oxyhydrocarbyl group R4 is of the formula —(CH2)1-10O(CH2)1-10CH3, —(CH2)1-5O(CH21-5CH3, —(CH2)1-2O(CH2)1-2CH3. In a highly preferred aspect R4 is —CH2OCH3.
In one preferred embodiment of the present invention, R4 is a hydrocarbon group. Preferably R4 is an alkyl group. Preferably the alkyl group is a C1-6 alkyl group (such as a C1-3 alkyl group). Preferably the hydrocarbyl group R4 is of the formula —(CH2)1-10CH3, —(CH2)1-5CH3, —(CH2)1-2CH3. In a highly preferred aspect R4 is ethyl.
In one preferred embodiment of the present invention R4 is selected from one of C1-C10 hydrocarbyl, C1-C5 hydrocarbyl, C1-C3 hydrocarbyl, hydrocarbon groups, C1-C10 hydrocarbon, C1-C5 hydrocarbon, C1-C3 hydrocarbon, alkyl groups, C1-C10 alkyl, C1-C5 alkyl, and C1-C3 alkyl.
In one preferred embodiment of the present invention, the R4 is a hydrocarbylsulphanyl group.
The term “hydrocarbylsulphanyl” means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur. That sulphur group may be optionally oxidised. Preferably the hydrocarbylsulphanyl is of the formula —S-hydrocarbyl wherein the hydrocarbyl is as described herein.
The term “hydrocarbylsulphanyl group” as used herein with respect to R4 means a group comprising at least C, H and S and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbylsulphanyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbylsulphanyl group may contain further hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, nitrogen.
In one preferred embodiment of the present invention, the R4 is a hydrocarbonsulphanyl group. The term “hydrocarbonsulphanyl group” as used herein with respect to R4 means a group consisting of C, H and S. Preferably the hydrocarbonsulphanyl is of the formula —S— hydrocarbon wherein the hydrocarbon is as described herein.
Preferably the hydrocarbonsulphanyl group R4 is of the formula C1-6S (such as a C1-3S). Preferably the oxyhydrocarbyl group R4 is of the formula —S(CH2)1-10CH3, —S(CH2)1-5CH3, —S(CH2)1-2CH3. In a highly preferred aspect R4 is —S—Me.
As previously mentioned, R4 is at position 2 or 4 of the A ring. Thus the compound may have the formula
wherein R1 and R2 are as specified herein, such as
Preferably R4 is at position 2 of the A ring.
In a further preferred aspect when the A ring is substituted with R1 and R4, R4 is ortho with respect to R1.
It will be appreciated by one skilled in the art that the proviso that R4 is at position 2 or 4 of the A ring, allows for R4 being at position 2 and 4 of the A ring, wherein each R4 is independently selected from the possibilities recited herein.
In a highly preferred aspect the compound is of Formula Va
wherein R1 is selected from —OH and a sulphamate group
wherein R2 is selected from —OH, a sulphamate group,
wherein the or each sulphamate group is of the formula
wherein R12 and R13 are independently selected from H and alkyl.
and wherein R4 is an alkoxy group.
In a highly preferred aspect the compound is selected from
In a highly preferred aspect the compound is
Distinct from Docetaxel, 2-Methoxyestradiol-3,17-O,O-bis-sulfamate (STX140) is a microtubule disruptor (Raobaikady 2005) which is orally bioavailable (Ireson 2004), not a substrate for p-glycoprotein (Suzuki, 2003) and can be dosed daily in vivo (Foster, submitted; Ireson 2004). The microtubule disruption leads to cell cycle arrest and subsequent apoptosis in both tumour and endothelial cells and inhibits in vitro angiogenesis (Newman et al., 2004) and in vivo angiogenesis (Foster, submitted; and Chander submitted).
STX140, was developed from the original steroid sulfatase (STS) inhibitor EMATE in order to overcome estrogenicity problems (Purohit et al, 1995-Biochemistry). The new class of A-ring modified anti-cancer compounds not only inhibited STS but were potent inhibitors of cell proliferation in vitro and in NMU induced tumours in vivo (Purohit et al, 2000, Int J Cancer).
The structurally related compound 2-MeOE2 is a well studied compound with known anti-cancer properties, and can inhibit cell proliferation and angiogenesis via a mechanism independent of hormone receptors (Fotsis et al., 1994). However, the clinical success of 2-MeOE2 has been limited as it has very poor oral bioavailability and is rapidly metabolised. In contrast to 2-MeOE2, STX140 avoids metabolism by 17βHSB due to its sulfate moiety at the C-17 position (Newman et al., 2006). STX140 has previously been shown to inhibit the proliferation of prostate LNCaP (androgen, AR+ve) cells (Day et al., 2003) and breast ER+ve MCF-7 cells (Suzuki et al., 2003b). STX140 inhibits doxorubicin and mitoxantrone resistant MCF-7 breast cancer proliferation (Suzuki et al., 2003a). The inhibition of in vivo angiogenesis by STX140 will increase hypoxia and make the tumour more reliant on glycolysis, thus sensitising the tumour to 2DG.
Composition
As described above according to one aspect of the present invention, there is provided a pharmaceutical composition comprising a composition as defined herein, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
In accordance with the present invention the composition of the present invention may comprise more than one biological response modifier.
The term biological response modifier (“BRM”) includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc.
BRMs may play a role in modulating the immune and inflammatory response in disorders. Examples of BRMs include: Tumour Necrosis Factor (TNF), granulocyte colony stimulating factor, erythropoietin, insulin-like growth factor (IGF), epidermal growth factor (EGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), interferons (IFNs), interleukins, tissue plasminogen activators, P-, E- or L-Selectins, ICAM-1, VCAM, Selectins, addressins etc.
Preferably, the biological response modifier is a cytokine.
A cytokine is a molecule—often a soluble protein—that allows immune cells to communicate with each other. These molecules exert their biological functions through specific receptors expressed on the surface of target cells. Binding of the receptors triggers the release of a cascade of biochemical signals which profoundly affect the behaviour of the cell bearing the receptor (Poole, S 1995 TibTech 13: 81-82). Many cytokines and their receptors have been identified at the molecular level (Paul and Sedar 1994, Cell 76: 241-251) and make suitable molecules of therapeutic value as well as therapeutic targets in their own right.
More details on cytokines can be found in Molecular Biology and Biotechnology (Pub. VCH, Ed. Meyers, 1995, pages 202, 203, 394, 390, 475, 790).
Examples of cytokines include: interleukins (IL)—such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8; IL-9, IL-10, IL-11, IL-12, IL-19; Tumour Necrosis Factor (TNF)—such as TNF-α; Interferon alpha, beta and gamma; TGF-β.
For the present invention, preferably the cytokine is tumour necrosis factor (TNF).
More preferably the cytokine is TNF-a.
TNF is a cytokine produced by macrophages and lymphocytes which mediates inflammatory and immunopathological responses. TNF has been implicated in the progression of diseases which include but are not limited to immunomodulation disorder, infection, cell proliferation, angiogenesis (neovascularisation), tumour metastasis, apoptosis, sepsis, and endotoxaemia.
The necrotising action of TNF in vivo mainly relates to capillary injury. TNF causes necrosis not only in tumour tissue but also in granulation tissue. It causes morphological changes in growth inhibition of and cytoxicity against cultured vascular endothelial cells (Haranka et al 1987 Ciba Found Symp 131: 140-153).
For the preferred aspect of the present invention, the TNF may be any type of TNF—such as TNF-α, TNF-β, including derivatives or mixtures thereof.
Teachings on TNF may be found in the art—such as WO-A-98/08870 and WO-A-98/13348.
The TNF can be prepared chemically or it can be extracted from sources. Preferably, the TNF is prepared by use of recombinant DNA techniques.
With this aspect of the present invention the compositions of the present invention are more potent in vivo than the compounds alone or TNF alone. Moreover, in some aspects the combination of compounds and TNF is more potent than one would expect from the potency of the compound alone i.e. this is a synergistic relationship between them.
In addition, the present invention contemplates the composition of the present invention further comprising an inducer of the biological response modifier—such as in vivo inducer of the biological response modifier.
In accordance with the present invention, the components of the composition can be added in admixture, simultaneously or sequentially. Furthermore, in accordance with the present invention it may be possible to form at least a part of the composition in situ (such as in vivo) by inducing the expression of—or increasing the expression of—one of the components. For example, it may be possible to induce the expression of—or increase the expression of—the biological response modifier, such as TNF. By way of example, it may be possible to induce the expression of—or increase the expression of—TNF by adding bacterial lipopolysaccharide (LPS) and muramyl dipeptide (MDP). In this regard, bacterial LPS and MDP in combination can stimulate TNF production from murine spleen cells in vitro and tumour regression in vivo (Fuks et al Biull Eksp Biol Med 1987 104: 497-499).
In the method of treatment, the subject is preferably a mammal, more preferably a human. For some applications, preferably the human is a woman.
The present invention also covers novel intermediates that are useful to prepare the compounds of the present invention. For example, the present invention covers novel alcohol precursors for the compounds. By way of further example, the present invention covers bis protected precursors for the compounds. Examples of each of these precursors are presented herein. The present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention.
Therapy
The compounds of the present invention may be used as therapeutic agents—i.e. In therapy applications.
The term “therapy” includes curative effects, alleviation effects, and prophylactic effects.
The therapy may be on humans or animals, preferably female animals.
Pharmaceutical Compositions
In one aspect, the present invention provides a pharmaceutical composition, which comprises a composition according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as—or in addition to—the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
Combination Pharmaceutical
The compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.
By way of example, the compounds of the present invention may be used in combination with other STS inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4-hydroxyandrostenedione (4-OHA)) and/or steroids—such as the naturally occurring neurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds. Examples of other STS inhibitors may be found in the above references. By way of example, STS inhibitors for use in the present invention include EMATE, and either or both of the 2-ethyl and 2-methoxy 17-deoxy compounds that are analogous to compound 5 presented herein.
In addition, or in the alternative, the compound of the present invention may be used in combination with a biological response modifier.
The term biological response modifier (“BRM”) includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc. For some applications, preferably, the biological response modifier is a cytokine. Examples of cytokines include: interleukins (IL)—such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-19; Tumour Necrosis Factor (TNF)—such as TNF-α; Interferon alpha, beta and gamma; TGF-β. For some applications, preferably the cytokine is tumour necrosis factor (TNF). For some applications, the TNF may be any type of TNF—such as TNF-α, TNF-β, including derivatives or mixtures thereof. More preferably the cytokine is TNF-α. Teachings on TNF may be found in the art—such as WO-A-98/08870 and WO-A-98/13348.
Administration
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
The compositions of the present invention may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
By way of further example, the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
Aside from the typical modes of delivery—indicated above—the term “administered” also includes delivery by techniques such as lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
The term “administered” includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
Thus, for pharmaceutical administration, the STS inhibitors of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
Cell Cycling
The compounds of the present invention may be useful in the method of treatment of a cell cycling disorder.
As discussed in “Molecular Cell Biology” 3rd Ed. Lodish at al. pages 177-181 different eukaryotic cells can grow and divide at quite different rates. Yeast cells, for example, can divide every 120 min., and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all; others, like the fibroblasts that assist in healing wounds, grow on demand but are otherwise quiescent.
Still, every eukaryotic cell that divides must be ready to donate equal genetic material to two daughter cells. DNA synthesis in eukaryotes does not occur throughout the cell division cycle but is restricted to a part of it before cell division.
The relationship between eukaryotic DNA synthesis and cell division has been thoroughly analysed in cultures of mammalian cells that were all capable of growth and division. In contrast to bacteria, it was found, eukaryotic cells spend only a part of their time in DNA synthesis, and it is completed hours before cell division (mitosis). Thus a gap of time occurs after DNA synthesis and before cell division; another gap was found to occur after division and before the next round of DNA synthesis. This analysis led to the conclusion that the eukaryotic cell cycle consists of an M (mitotic) phase, a G1 phase (the first gap), the S (DNA synthesis) phase, a G2 phase (the second gap), and back to M. The phases between mitoses (G1, S, and G2) are known collectively as the interphase.
Many nondividing cells in tissues (for example, all quiescent fibroblasts) suspend the cycle after mitosis and just prior to DNA synthesis; such “resting” cells are said to have exited from the cell cycle and to be in the G0 state.
It is possible to identify cells when they are in one of the three interphase stages of the cell cycle, by using a fluorescence-activated cell sorter (FACS) to measure their relative DNA content: a cell that is in G1 (before DNA synthesis) has a defined amount x of DNA; during S (DNA replication), it has between x and 2x; and when in G2 (or M), it has 2x of DNA.
The stages of mitosis and cytokinesis in an animal cell are as follows
(a) Interphase. The G2 stage of interphase immediately precedes the beginning of mitosis. Chromosomal DNA has been replicated and bound to protein during the S phase, but chromosomes are not yet seen as distinct structures. The nucleolus is the only nuclear substructure that is visible under light microscope. In a diploid cell before DNA replication there are two morphologic chromosomes of each type, and the cell is said to be 2n. In G2, after DNA replication, the cell is 4n. There are four copies of each chromosomal DNA. Since the sister chromosomes have not yet separated from each other, they are called sister chromatids.
b) Early prophase. Centrioles, each with a newly formed daughter centriole, begin moving toward opposite poles of the cell; the chromosomes can be seen as long threads. The nuclear membrane begins to disaggregate into small vesicles.
(c) Middle and late prophase. Chromosome condensation is completed; each visible chromosome structure is composed of two chromatids held together at their centromeres. Each chromatid contains one of the two newly replicated daughter DNA molecules. The microtubular spindle begins to radiate from the regions just adjacent to the centrioles, which are moving closer to their poles. Some spindle fibres reach from pole to pole; most go to chromatids and attach at kinetochores.
(d) Metaphase. The chromosomes move toward the equator of the cell, where they become aligned in the equatorial plane. The sister chromatids have not yet separated.
(e) Anaphase. The two sister chromatids separate into independent chromosomes. Each contains a centromere that is linked by a spindle fibre to one pole, to which it moves. Thus one copy of each chromosome is donated to each daughter cell. Simultaneously, the cell elongates, as do the pole-to-pole spindles. Cytokinesis begins as the cleavage furrow starts to form.
(f) Telophase. New membranes form around the daughter nuclei; the chromosomes uncoil and become less distinct, the nucleolus becomes visible again, and the nuclear membrane forms around each daughter nucleus. Cytokinesis is nearly complete, and the spindle disappears as the microtubules and other fibres depolymerise. Throughout mitosis the “daughter” centriole at each pole grows until it is full-length. At telophase the duplication of each of the original centrioles is completed, and new daughter centrioles will be generated during the next interphase.
(g) Interphase. _Upon the completion of cytokinesis, the cell enters the G1 phase of the cell cycle and proceeds again around the cycle.
It will be appreciated that cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions.
Thus, the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers.
In addition, the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
For some applications, cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested. In one aspect cell cycling may be inhibited and/or prevented and/or arrested in the G2/M phase. In one aspect cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested.
By the term “irreversibly prevented and/or inhibited and/or arrested” it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. More particularly by the term “irreversibly prevented and/or inhibited and/or arrested” it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol I than control cells. Details on this protocol are presented below.
Thus, the present invention provides compounds which: cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ER−) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling; and/or cause regression of nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as a cell cycling agonist.
Procedure
Stage 1
MCF-7 breast cancer cells are seeded into multi-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows:
Control—no treatment
Compound of Interest (COI) 20 μM
Cells are grown for 6 days in growth medium containing the COI with changes of medium/COI every 3 days. At the end of this period cell numbers were counted using a Coulter cell counter.
Stage 2
After treatment of cells for a 6-day period with the COI cells are re-seeded at a density of 104 cells/well. No further treatments are added. Cells are allowed to continue to grow for a further 6 days in the presence of growth medium. At the end of this period cell numbers are again counted.
Cancer
As indicated, the compounds of the present invention may be useful in the treatment of a cell cycling disorder. A particular cell cycling disorder is cancer.
Cancer remains a major cause of mortality in most Western countries. Cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
Hence, the development of a pharmaceutical for anti-cancer treatment of hormone dependent and/or hormone independent tumours, yet lacking some or all of the side-effects associated with chemotherapy, would represent a major therapeutic advance.
It is known that oestrogens undergo a number of hydroxylation and conjugation reactions after their synthesis. Until recently it was thought that such reactions were part of a metabolic process that ultimately rendered oestrogens water soluble and enhanced their elimination from the body. It is now evident that some hydroxy metabolites (e.g. 2-hydroxy and 16alpha-hydroxy) and conjugates (e.g. oestrone sulphate, EIS) are important in determining some of the complex actions that oestrogens have in the body.
Workers have investigated the formation of 2- and 16-hydroxylated oestrogens in relation to conditions that alter the risk of breast cancer. There is now evidence that factors which increase 2-hydroxylase activity are associated with a reduced cancer risk, while those increasing 16alpha-hydroxylation may enhance the risk of breast cancer. Further interest in the biological role of estrogen metabolites has been stimulated by the growing body of evidence that 2-methoxyoestradiol is an endogenous metabolite with anti-mitotic properties. 2-MeOE2 is formed from 2-hydroxy estradiol (2—OHE2) by catechol estrogen methyl transferase, an enzyme that is widely distributed throughout the body.
Workers have shown that in vivo 2-MeOE2 inhibits the growth of tumours arising from the subcutaneous injection of Meth A sarcoma, B16 melanoma or MDA-MB-435 estrogen receptor negative (ER−) breast cancer cells. It also inhibits endothelial cell proliferation and migration, and in vitro angiogenesis. It was suggested that the ability of 2-MeOE2 to inhibit tumour growth in vivo may be due to its ability to inhibit tumour-induced angiogenesis rather than direct inhibition of the proliferation of tumour cells.
The mechanism by which 2-MeOE2 exerts its potent anti-mitogenic and anti-angiogenic effects is still being elucidated. There is evidence that at high concentrations it can inhibit microtubule polymerisation and act as a weak inhibitor of colchicine binding to tubulin. Recently, however, at concentrations that block mitosis, tubulin filaments in cells were not found to be depolymerised but to have an identical morphology to that seen after taxol treatment. It is possible, therefore, that like taxol, a drug that is used for breast and ovarian breast cancer therapy, 2-MeOE2 acts by stabilising microtubule dynamics.
While the identification of 2-MeOE2 as a new therapy for cancer represents an important advance, the bioavailability of orally administered oestrogens is poor. Furthermore, they can undergo extensive metabolism during their first pass through the liver. As part of a research programme to develop a steroid sulphatase inhibitor for breast cancer therapy, oestrone-3-O-sulphamate (EMATE) was identified as a potent active site-directed inhibitor. Unexpectedly, EMATE proved to possess potent oestrogenic properties with its oral uterotrophic activity in rats being a 100-times higher than that of estradiol. Its enhanced oestrogenicity is thought to result from its absorption by red blood cells (rbcs) which protects it from inactivation during its passage through the liver and which act as a reservoir for its slow release for a prolonged period of time. A number of A-ring modified analogues were synthesised and tested, including 2-methoxyoestrone-3-O-sulphamate. While this compound was equipotent with EMATE as a steroid sulphatase inhibitor, it was devoid of oestrogenicity.
We believe that the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer.
In addition or in the alternative the compound of the present invention may be useful in the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours.
Therapy Concerning Oestrogen
We believe that some of the compounds of the present invention may be useful in the control of oestrogen levels in the body—in particular in females. Thus, some of the compounds may be useful as providing a means of fertility control—such as an oral contraceptive tablet, pill, solution or lozenge. Alternatively, the compound could be in the form of an implant or as a patch.
Thus, the compounds of the present invention may be useful in treating hormonal conditions associated with oestrogen.
In addition or in the alternative the compound of the present invention may be useful in treating hormonal conditions in addition to those associated with oestrogen. Hence, the compound of the present invention may also be capable of affecting hormonal activity and may also be capable of affecting an immune response.
Neurodegenerative Diseases
We believe that some of the compounds of the present invention may be useful in the treatment of neurodegenerative diseases, and similar conditions.
By way of example, it is believed that STS inhibitors may be useful in the enhancing the memory function of patients suffering from illnesses such as amnesia, head. injuries, Alzheimer's disease, epileptic dementia, presenile dementia, post traumatic dementia, senile dementia, vascular dementia and post-stroke dementia or individuals otherwise seeking memory enhancement.
TH1
We believe that some of the compounds of the present invention may be useful in TH1 implications.
By way of example, it is believed that the presence of STS inhibitors within the macrophage or other antigen presenting cells may lead to a decreased ability of sensitised T cells to mount a TH1 (high IL-2, IFNγ low IL-4) response. The normal regulatory influence of other steroids such as glucocorticoids would therefore predominate.
Inflamatory Conditions
We believe that some of the compounds of the present invention may be useful in treating inflammatory conditions—such as conditions associated with any one or more of: autoimmunity, including for example, rheumatoid arthritis, type I and II diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn's disease, skin disorders e.g. psoriasis and contact dermatitis; graft versus host disease; eczema; asthma and organ rejection following transplantation.
By way of example, it is believed that STS inhibitors may prevent the normal physiological effect of DHEA or related steroids on immune and/or inflammatory responses.
The compounds of the present invention may be useful in the manufacture of a medicament for revealing an endogenous glucocorticoid-like effect.
Other Therapies
It is also to be understood that the compound/composition of the present invention may have other important medical implications.
For example, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890—viz:
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859. For ease of reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g. treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.
In addition, or in the alternative, the composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti- inflammatory activity; anti-immune activity, i.e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of the disorders listed selected from endometriosis, uterus fibromyoma, induction of mono-ovulation (in polycystic ovarian disease [PCOD] patients). induction of multiple follicullar development in (ART patients), preterm labor/cervical incompetency and recurrent abortion.
Compound Preparation
The compounds may be prepared by reacting an appropriate alcohol with a suitable chloride. By way of example, the sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable sulfamoyl chloride, of the formula R4R5NSO2Cl. Specific methods for preparing the compounds are provided in WO-A-02/16392, WO-A-2004/085459 and WO-A-2006/032885.
Typical conditions for carrying out the reaction are as follows.
Sodium hydride and a sulfamoyl chloride are added to a stirred solution of the alcohol in anhydrous dimethyl formamide at 0° C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgSO4. Filtration followed by solvent evaporation in vacuo and co-evaporated with toluene affords a crude residue which is further purified by flash chromatography.
Preferably, the alcohol is derivatised, as appropriate, prior to reaction with the sulfamoyl chloride. Where necessary, functional groups in the alcohol may be protected in known manner and the protecting group or groups removed at the end of the reaction.
Preferably, the sulphamate compounds are prepared according to the teachings of Page et al (1990 Tetrahedron 46; 2059-2068).
The present invention will now be described in further detail by way of example only with reference to the accompanying figures in which:—
The present invention will now be described only by way of example. However, it is to be understood that the examples also present preferred compounds of the present invention, as well as preferred routes for making same and useful intermediates in the preparation of same.
Materials and Methods
Compounds
2-MeOE2bisMATE (STX140) was synthesised at the Department of Pharmacy and
Pharmacology, University of Bath (Leese et al., 2006). The compound displayed spectroscopic and analytical data in accordance with structure. 2-DEOXY-D-GLUCOSE was obtained from Sigma (Irvine, England).
Cell Culture
MCF-7 and LNCaP cells were obtained from the American Tissue Culture Collection (ATCC). Cells were cultured in the following supplements, obtained from Sigma: RPMI 1640 with 10% foetal bovine serum, 1% L-glutamine, 1% MEM non-essential amino acids, 1% sodium bicarbonate solution, LNCaP cells also required 1% sodium pyruvate. Cells were maintained in an incubator at 37° C. under 5% CO2 and 95% air atmosphere.
Proliferation Assay
MCF-7 and LNCaP cells were seeded at 3000 cells/well or 5000 cells/well respectively, in 96 well plates. Compounds were added 4 hours after seeding of cells. Alamar blue 10 ml (Biosource, Nivelles, Belgium) was added and cell proliferation was measured 72 hrs later using a FluoStar Optima Plate reader.
Hypoxia Model
An Innova co-48 CO2 humidified incubator (Newbrunswick Scientific, St. Albans, U.K.) was used to study the effects of 2DG and STX140 on cells under hypoxic conditions. Oxygen was maintained at 1%, CO2 5% and temperature at 37° C.
Measurement of ATP
MCF-7 and LNCaP cells were seeded at 3000 cells/well or 5000 cells/well respectively, in 96 well plates. Compounds were added 4 hours following seeding of cells. The ATPlite assay procedure was carried out according to manufacturer's instructions (Perkin Elmer, Beaconsfield, U.K.) and ATP measured 72 hrs later using a FluoStar Optima Plate reader.
Photography
Cells were examined using a Zeiss inverted microscope fitted with a 20× and 40× Plan-Neofluor objective, and analysed through an Axiovision imaging system attached to the microscope.
Immunoblotting
Immunoblotting was performed as previously described (Newman et al., 2004). The following antibodies were used: Anti-PAMPK;1: 1000 dilution (Millipore, Chanders Ford, U.K.), Anti-PBCL-2; 1:100 dilution (Cell Signaling, MA, U.S.A.), Anti-PP70S6K; 1:1000 dilution (Cell Signaling), Anti-phoshpo-SAPK/JNK; 1:1000 dilution (Cell Signaling), Anti-p21; 1:500 dilution (BD Biosciences, Oxford, U.K.) or anti-beta-actin; 1:3000 dilution (Abcam, Cambridge, U.K.) primary antibody, the wash was then repeated and the secondary antibodies added which were either anti-rabbit; 1:5000 dilution (Cell Signaling), anti-mouse; 1:5000 dilution (Cell Signaling) or anti-goat; 1: 5000 dilution (Abcam). After thoroughly washing the membrane it was developed using ECF substrate (Amersham Biosciences, Little Chalfont, U.K.) and analysed using a STORM Optical Scanner. Whole cell lysate was loaded onto the immunoblot, a Bradford assay was used to assess the protein content of the nuclear extracts to ensure equal protein loading in each lane.
In Vivo Tumour Xenograft Model
MCF-7 Cell Tumours
Intact, ovariectomized, athymic, female MF-1 nude mice (nu-/nu-) were purchased from Harlan (Bicester, Oxon, UK) at 5 weeks of age (˜20-25 g in weight). All experiments were carried out under conditions that complied with institutional guidelines. Animals were kept in a 12-hour light/12-hour dark cycle and given food and water ad libitum. Monolayers of MCF-7 cells were removed by trypsinization, and the resultant cell suspension was centrifuged for 5 min. at 1,000×g and then resuspended in ice-cold matrigel (BD Biosciences). Five million MCF-7 cells were injected s.c. into the right flank of the animals. The tumours were allowed to grow for 2 weeks, when the tumours reached 100-150 mm3 in volume, mice were randomly divided into four treatment groups: 1) vehicle (10% tetrahydrofuran; 90% propylene glycol, 100 ml oral)+saline i.p. 200 ml; 2) vehicle+2DG (2 g/kg) i.p. 200 ml; 3) STX140 (5 mg/kg) 100 ml oral+saline i.p. 200 ml; 4) STX140 (5 mg/kg) 100 ml oral+2DG (2 g/kg) i.p. 200 ml. All treatments were administered daily for 4 weeks, 2DG was given i.p. (100 ml) and STX140 or vehicle was given orally (200 ml). Throughout the study, mice were weighed and tumour measurements were taken on a weekly basis. Tumour volumes were calculated using the formula: length×width2/2. At the end of the study, tumours were collected for immunohistochemical analysis.
LNCaP Cell Tumours
Athymic, male MF-1 nude mice (nu-/nu-) were purchased from Harlan (Bicester, Oxon,
UK) at 5 weeks of age (˜20-25 g in weight). All experiments were carried out under conditions that complied with institutional guidelines. Animals were kept in a 12-hour light/12-hour dark cycle and given food and water ad libitum. Monolayers of LNCaP cells were removed by trypsinization, and the resultant cell suspension was centrifuged for 5 min. at 1,000×g and then resuspended in ice-cold matrigel (BD Biosciences). Five million LNCaP cells were injected s.c. into the right flank of the animals. The tumours were allowed to grow for 3 weeks, when the tumours reached 100-150 mm3 in volume, mice were randomly divided into four treatment groups: 1) vehicle (10% tetrahydrofuran; 90% propylene glycol, 100 μl p.o.+saline i.p. 200 μl; 2) vehicle p.o.+2DG (2 g/kg) i.p.+200 μl; 3) STX140 (5 mg/kg) 100 μl p.o.+saline i.p. 200 μl; 4) STX140 (5 mg/kg) 100 μl p.o.+2DG (2 g/kg) i.p. 200 μl. All treatments were administered 5 days per week for 4 weeks, 2DG was given i.p. (200 μl) and STX140 or vehicle was given orally (100 μl). Throughout the study, mice were weighed and tumour measurements were taken on a weekly basis. Tumour volumes were calculated using the formula: length x width2/2.
Immunohistochemistry
von Willebrand's factor IHC were performed on paraffin embedded MDA-MB-231 tumor sections cut at 6 mm. After sectioning, rehydration and antigen retrieval steps, von Willebrand antibody (1:800, Abcam, Cambridge, United Kingdom) was applied to the section for 1 h at RT, followed by a goat polyclonal secondary antibody conjugated to FITC (30 min at RT). Sections were then mounted and viewed under a light or fluorescence microscope.
Statistics
All experiments were carried out in triplicate and data presented are representative of one of three such experiments, unless indicated. All errors shown are the mean±SE. Student's t test was used to assess the significance of the differences in cell proliferation for all experiments and ANOVA was used to look at the percentage change in ATP per live cell equivalent.
Results
Cell Proliferation Assays: Effect of 2DG Alone.
The ability of 2DG to inhibit the proliferation of LNCaP and MCF-7 cell lines was examined over a 5 day period under normoxic conditions (
Cell Proliferation Assays: Effect of 2DG and STX140, Alone and in Combination.
The growth inhibitory effects of 2DG and STX140, used alone and in combination were compared (
Under hypoxic conditions, 2DG alone reduces the proliferation of LNCaP cells by 75%, this inhibition is further decreased to 85% (p<0.05) with addition of STX140 (0.5μ) down to 87% (p<0.01) with addition of STX140 (1μ).
Effect of 2DG and STX140 on ATP Levels
The ATP levels of MCF-7 and LNCaP treated with 2DG and STX140 were studied (
Cell Morphology
Images show LNCaP and MCF-7 cells treated with STX140 (0.5 μM) and 2DG 8 mM) alone or in combination (
Effect of STX140 and 2DG In Vivo
Following inoculation, MCF-7 cells developed into stable growing tumours, with a “take rate” of 80%. Once tumours had reached approximately 100-150 mm3 in volume, compound dosing commenced for 28 days. At the end of dosing growth of the MCF-7 tumours (
Expression of von Willebrand's Factor
The endothelial specific marker, von Willebrand's factor, was used to assess vessel density in sections taken from MCF-7 xenografts taken from treated animals.
LNCaP Cell Tumours
Once tumours had reached approximately 100-150 mm3 in volume, compound dosing commenced for 5 days per week for 4 weeks. At the end of dosing growth of the LNCaP tumours was significantly inhibited by the combination of STX140 (5 mg/kg p.o.) and 2DG (2 g/kg i.p.) (p<0.001).
Discussion
The aim of this study was to improve inhibition of tumour cell proliferation using the combination of an alkoxy/sulphamate substituted ring system compound and a glycolytic inhibitor in vitro and in vivo. Tumour cells, even in the presence of oxygen, continue to rely on glycolysis rather than oxidative phosphorylation (Warburg, 1956). Several oncogenes have been implicated in the Warburg effect; the AKT oncogene is associated with enhanced glucose uptake and aerobic glycolysis, independent of HIF-1 (Elstrom et al., 2004). AKT assembles glucose transporters to the cell surface to enhance glucose uptake and activate hexokinase II (HK2) to phosphorylate and trap intracellular glucose. AKT is able to enhance glycolytic flux without affecting mitochondrial oxidative phosphorylation, contributing to the Warburg effect. The MYC oncogene activates virtually all glycolytic enzyme genes, including those encoding HK2, enolase, and lactate dehydrogenase A (LDHA) (Kim & Dang, 2005). The inner core of tumour cells are particularly resistant to many of the anti-cancer agents which target rapidly dividing cells (Liu et al., 2001).
Anaerobically metabolizing, slow growing tumour cells demonstrate another form of multidrug resistance (MDR) in addition to the MDR mechanisms previously identified, for example the ABC cassette family of transporter proteins, P-GP and MRP (Hendrikse, 2000; Kuwano et al., 1999) and topoisomerase (Fortune & Osheroff, 2000; Gupta et al., 2005).
STX140 causes caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL by activating caspases (Wood et al., 2004). STX140 has been shown to be 6 times more potent an inhibitor of breast cancer cells compared to 2-MeOE2 (Raobaikady et al., 2003) and can inhibit proliferation of HUVEC cells 60 fold more effectively than 2-MeOE2 and was 10-13 times more active as an inhibitor of vessel formation in a novel co-culture model system (Newman et al., 2004). STX140 was also effective in cells resistant to mitoxantrone or doxorubicin (Suzuki et al., 2003a) and was also a very active anti-tumour agent in vivo (Ireson et al., 2004).
STX140 also has good STS inhibitory properties in vitro and in vivo (Raobaikady et al., 2003) which is beneficial for the treatment of hormone dependent breast cancers. STX140 shows enhanced oral availability and improved pharmacokinetic properties (Ireson et al., 2004). Ho et al., showed that TNF-aand STX140 used together increased the potency of 2-substituted oestrogens as anti-angiogenic agents via synergistic induction of apoptosis in endothelial cells and had low cytotoxicity in fibroblasts (Ho et al., 2003). Fluorescent images of MDA-MB-231 cells treated with STX140 revealed disintegrated tubulin, condensed nuclei and microtubule damage. In addition, STX140 caused cell cycle arrest in MDA-MB-231 cells (Raobaikady et al., 2005). To summarise the anti-tumour affects of STX140 are caused by disruption of microtubules leading to cell cycle arrest and subsequent apoptosis. Furthermore, STX140 has potential in vitro and in vivo anti-angiogenic activity (Newman 2004, Chander 2007[submitted] and Foster et al., 2007 [submitted]).
The results from this study confirm that 2DG is a potent inhibitor of both LNCaP and MCF-7 cell proliferation, with increased potency under hypoxic conditions. Other groups have also showed 2DG to be very efficacious at inhibiting tumour cell growth (Lyon et al., 1988 and Kaplan et al., 1990).
In addition, Liu et al used two models to investigate the tumour cells dependency on glycolysis. The first represented osteosarcoma wild-type (wt) cells treated with agents which inhibit mitochondrial oxidative phosphorylation (Oxphos) by interacting with complexes I, III and V of the electron transport chain in different ways, inhibitors included
Rhodamine (Rho 123), rotenone, antimycin A and oligomycin. All of these Oxphos inhibitors were found to hypersensitize wild-type cells to 2DG. Cells treated with Rho 123 also became hypersensitive to oxamate, an analogue of pyruvate which blocks the step of glycolysis that converts pyruvate to lactic acid. The second model is r□ cells which have lost their mitochondrial DNA and therefore cannot undergo Oxphos. These cells were 10 and 4.9 times more sensitive to 2DG than oxamate, respectively, than wt cells. Overall, Liu et at showed that the glycolytic inhibitors oxamate and 2DG could be used to specifically target the slow-growing cells of a tumour and thereby increase the efficacy of current chemotherapeutic and irradiation protocols designed to kill rapidly dividing cells. They also hypothesised that glycolytic inhibitors could be particularly useful in combination with anti-angiogenic agents which should make tumours more anaerobic and therefore, reliant on glycoloysis (Liu et al., 2001).
Aft et al studied the effects of 2DG on breast cancer cells in vitro: 2DG treatment of breast cancer cells resulted in cessation of cell growth in a dose-dependent manner. Cell death was induced by 2DG caused by apoptosis, with induction of caspase 3 activity and cleavage of poly (ADP-ribose) polymerase. GLUT 1 transporter was elevated and glucose uptake elevated compared to non-2DG treated breast cancer cells. It was concluded that 2DG causes death in human breast cancer cells by activation of the apoptotic pathway, they accelerate their own demise by initially expressing high levels of glucose transporter protein, which allowed increased uptake of 2DG and subsequent induction of cell death. Aft et al revealed good evidence to support the targeting of glucose metabolism as a site for chemotherapeutic intervention by agents such as 2DG (Aft et al., 2002).
The results of this study revealed how 2DG depletes ATP in vitro. Xu et at showed how inhibition of glycolysis severely depleted ATP in cancer cells, especially those cells with mitochondrial defects and led to rapid dephosphorylation of the glycolysis-apoptosis interacting molecule BAD at Ser112, relocalization of BAX to mitochondria and massive cell death. 2DG was shown to utilise a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia (Xu et al., 2005).
Zhu et al showed in MCF-7 cells that 2DG reduced cell growth and intracellular ATP in a dose- and time-dependent manner. 2DG increased levels of phosphorylated AMPK and Sirt-1 and reduced phosphorylation of Akt. This study supported the hypothesis that dietary energy restriction (DER) inhibits carcinogenesis, partly by limiting glucose availability and that energy metabolism is a target for the development of energy restriction mimetic agents for chemoprevention (Zhu et al., 2005). Recently, Lampidis et at created 2-halogenated D-glucose analogs for improved activity, compared to 2DG. 2-fluor-2-deoxy-D-glucose (2FG) was more potent than 2DG in killing hypoxic tumour cells and would therefore could be more clinically effective when combined with standeard chemotherapeutic protocols (Lampidis et al., 2006).
The results of this study revealed how there is significant improvement in cell proliferation inhibition when a glycolytic inhibitor (such as 2DG) is combined with a alkoxy/sulphamate substituted ring system compound (such as STX140) in LNCaP cells. The combination of compounds was not more effective than either agent alone in MCF-7 cells in vitro. The in vitro proliferation assay used in these studies does not reflect the in vivo situation; there is no inner core of slow growing tumour cells that would be potentially recalcitrant to STX140 but sensitive to 2DG. The true benefit of combining agents with 2DG may only become apparent using in vivo tumour models.
The combination of 2DG and STX140 is the most effective at inhibiting tumour xenograft growth compared to either agent alone, in vivo. The dose of STX140 used in vivo was only 5 mg/kg, it would be possible to increase the efficiency of STX140 by increasing the dose to 20 mg/kg as used by Foster et al (Foster et al., 2006[submitted]).
Several groups have also showed beneficial effect when chemotherapeutic agents are combined with 2DG, with the hope of targeting the aerobic rapidly dividing and anaerobic, slowly dividing cells: Maschek et al utilised 2DG in combination with Adriamycin (ADR) or Paclitaxel in nude mouse xenograft models of human osteosarcoma and non-small cell lung cancer. Nude mice were implanted with osterosarcoma cells and divided into four groups: 1) untreated, 2) ADR alone, 3) 2DG alone, 4) ADR+2DG. Tumours were 50 mm3 or 300 mm3 in volume when treatment began. Starting with small or large tumours, the ADR+2DG combinations resulted in significantly slower tumour growth (and therefore longer survival) then the control, 2DG or ADR treatments (p<0.0001). Similar beneficial effects of combination treatment were found with 2DG and paclitaxel in MV522 non-small lung cancer xenograft model. Their results provided a rationale for initiating clinical trials using glycolytic inhibitors in combination with chemotherapeutic agents to increase their therapeutic effectiveness (Maschek et al., 2004). In 2005, Gupta et al showed how 2DG enhances the efficacy of etoposide in Ehrlich ascites tumour-bearing mice. Analysis of cells obtained from ascitic fluid as well as solid tumours during follow up-revealed etoposide induced cell death was mainly due to apoptosis, which was enhanced further by 2DG. In addition, 2DG did not increase the toxicity of etoposide at 60 mg/kg. Therefore, administration of 2DG can improve the local control of tumours without increasing normal tissue toxicity, so enhancing the therapeutic efficacy of topoisomerase inhibitor -based anti-cancer drugs like etoposide (Gupta et al., 2005).
The results of this study revealed how STX140 causes a decrease in von Willebrand's factor staining, thus supporting the anti-angiogenic potential of this compound observed in vitro (Newman et al., 2004). The combination of STX140 and 2DG was the most efficacious at inhibiting in vivo tumour growth, this supports the hypothesis that the present anti-angiogenic agents may potentiate the activity of 2DG due to an increase in hypoxia and greater reliance on glycolysis.
Raez et al have instigated a Phase 1 trial of 2DG and cocetaxel in patients with solid tumours and the drug combination was shown to be feasible and safe (Raez et al., 2005)
In conclusion, both STX140 and 2DG are very potent anti-cancer compounds in vitro and in vivo. There are clear advantages of using multi-targeting treatments, combining anti-angiogenic activity, microtubule disruption with 2DG to attack the whole of the tumour. The anti-angiogenic properties of STX140 results in less blood vessels, an increase in hypoxia and therefore more glycolysis, thus sensitizing the cell to 2DG. The microtubule disruption of STX140 will target rapidly dividing cells, inhibiting cell proliferation. Using multiple compounds reduces the likelihood of resistance so lower doses of microtubule disruptors can be used causing less off target effects. In the future it may be very beneficial to modify 2DG in order to make it more efficacious in cancer prevention.
All publications and patents mentioned in the above specification are herein incorporated by reference.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 0702446.6 | Feb 2007 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB08/00448 | 2/7/2008 | WO | 00 | 1/25/2010 |
