NZ624019B2 - Method for inhibition of deubiquitinating activity - Google Patents

Method for inhibition of deubiquitinating activity Download PDF

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Publication number
NZ624019B2
NZ624019B2 NZ624019A NZ62401912A NZ624019B2 NZ 624019 B2 NZ624019 B2 NZ 624019B2 NZ 624019 A NZ624019 A NZ 624019A NZ 62401912 A NZ62401912 A NZ 62401912A NZ 624019 B2 NZ624019 B2 NZ 624019B2
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alkyl
compound
cancer
treatment
phenyl
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NZ624019A
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NZ624019A (en
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Rolf Larsson
Stig Linder
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Vivolux Ab
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Priority claimed from PCT/SE2012/000158 external-priority patent/WO2013058691A1/en
Publication of NZ624019A publication Critical patent/NZ624019A/en
Publication of NZ624019B2 publication Critical patent/NZ624019B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/02—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D223/06—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D223/08—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Abstract

Disclosed herein are compounds of the formula S-1 where the substituents are as defined herein. Examples of the compounds include (3E, 5E)?3,5?Bis(phenylmethylidene)azepan?4?one and (3E,5E)?5?[(4?chlorophenyl)methylidene]?3?[(4?nitrophenyl)methylidene]azepan?4?one. The compounds are capable of abrogating the deubiquitinating (DUB) activity of the 19S RP DUBs and are therefore suitable for treating cancer, in particular of cancer refractory to treatment by chemotherapy using compounds such as bortezomib. Also disclosed are corresponding methods of treatment and pharmaceutical compositions comprising the compounds. ating the deubiquitinating (DUB) activity of the 19S RP DUBs and are therefore suitable for treating cancer, in particular of cancer refractory to treatment by chemotherapy using compounds such as bortezomib. Also disclosed are corresponding methods of treatment and pharmaceutical compositions comprising the compounds.

Description

METHOD FOR TION OF DEUBIQUITINATING ACTIVITY FIELD OF THE INVENTION The invention generally s to a compound capable of abrogating the deubiquitinating (DUB) activity of the 19S RP DUBs. Also described is a method of treating cancer in a patient by inhibiting deubiquitinating ty. Also described is a method of ng a cancer in a patient who has proved resistant to treatment by at least one anti‐cancer ne. The invention also relates to a compound for use in the described method and to a pharmaceutical composition comprising the compound.
BACKGROUND OF THE INVENTION Tumor cells display enhanced sensitivity to disruptions in the ubiquitin‐proteasome system (UPS) making this an attractive target for the development of anti‐cancer therapies (1).
Ubiquitin‐tagged substrates are degraded by the 26S proteasome, a multi‐subunit complex comprising a proteolytic 20S core (20S CP) capped by 19S regulatory particles (19S RP) (2,3).
The 20S CP has evolved as an important target for anti‐cancer drug development, resulting in the approval of bortezomib (Velcade®) for treatment of myeloic leukemia (4).
O OH N NH B NH OH O CH3 CH3 omib The nd b‐AP15 (NSC687852) is known to induce p53‐independent and cathepsin‐D‐dependent apoptosis (5,6).
O O + + N N N O CH2 O O b‐AP15 (NSC687852) OBJECTS OF THE INVENTION It is an object of the invention to e a nd for use in a method of treating cancer in a patient by inhibiting deubiquitinating activity, in particular a cancer refractory to state‐of‐the‐art chemotherapy; and/or to e such a compound for treating cancer in a patient refractory to treatment with at least bortezomib or an agent sharing the mechanism of deubiquitinating activity inhibition of omib; and/or to provide a nd of the aforementioned kind, which has improved solubility at physiological pH in respect of onally equivalent compounds known in the art; and/or to provide a corresponding method; and/or to provide a pharmaceutical composition comprising the compound; and/or to at least provide the public with a useful choice.
Still further objects of the invention will become evident by ng the following summary of the invention, a number of preferred embodiments thereof illustrated in a drawing, and the appended claims.
SUMMARY OF THE INVENTION In one aspect, the invention provides a compound of the general structure S‐1 capable of abrogating the deubiquitinating (DUB) activity of the 19S RP DUBs wherein R1, R2 at double bond d1 and R3, R4 at double bond d2 can, independent of each other, have a configuration opposite to that of structure S1, X is CO or CS; R1 and R3 are, independent of each other, H or C1‐6 ‐alkyl; R2 and R4 are, independent of each other, H; C1‐6 ‐alkyl; C1‐5 CO; phenyl or 6‐membered heteroaryl optionally substituted by 1‐3 of: C1‐6 ‐alkyl,C1‐6 ‐alkoxy, CN, ‐COOC1‐6 ‐alkyl, COOH, NO2 , F, Cl, Br, I, CF3 , NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , CONR7R8, with the proviso that one or more of H in alkyl and alkoxy can be tuted by fluoro; R5 is any of H; C1‐6 ‐alkyl; C2‐6 ‐alkenyl; C1‐3 ‐alkoxy‐C2‐6 ‐alkyl‐; C1‐3 ‐alkoxy‐C2‐6 ‐alkenyl‐; aryl‐C0‐6 ‐alkyl‐; heteroaryl‐C0‐6 ‐alkyl‐; heterocyclyl‐C0‐6 ‐; cycloalkyl‐C0‐6‐alkyl‐; ‐C1‐6 ‐alkyl‐COOC1‐6‐alkyl; ‐C2‐6 ‐alkyl‐aryloxy; COR6; R6 is any of C1‐6 ‐alkyl; C2‐6 ‐alkenyl; C1‐6 ‐alkoxy; C1‐3 ‐alkoxy‐C1‐6 ‐alkyl‐; C1‐3 ‐alkoxy‐C1‐6 ‐alkenyl‐; aryl‐C0‐6 ‐alkyl‐; heteroaryl‐C0‐6 ‐alkyl‐; heterocyclyl‐C0‐6 ‐alkyl‐; cycloalkyl‐C0‐6‐alkyl‐; ‐C1‐6 ‐alkyl‐COOC1‐6‐alkyl; NH2 ; ‐NHC1‐6 ‐alkyl; ‐N(C1‐6 ‐alkyl)2 ; ‐C0‐6 ‐alkyl‐aryloxy; R7, R8 are, ndent of each other, H or C1 ‐C3 ‐alkyl.
In another aspect, the invention relates to the use of the compound of the invention in the manufacture of a medicament for treating cancer in a person tory to chemotherapy.
In another aspect, the invention relates to the use of a compound of the invention in the manufacture of a medicament for treating a cancer tumor refractory to treatment with bortezomib or an agent g the apoptosis generating activity of omib or any other anti‐cancer drug.
In another aspect, the invention provides a pharmaceutical composition sing the compound of the invention and a pharmaceutically acceptable carrier.
Certain statements that appear below are broader than what appears in the statements of the ion above. These ents are provided in the interests of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which defines the scope of the invention.
According to the present invention is disclosed a compound of the general structure S: capable of abrogating the deubiquitinating (DUB) activity of the 19S RP DUBs.
The nd of the invention is recognized as pertaining to a novel class of proteasome tors of which the known compound b‐AP15 is a representative.
In particular, according to the present invention, the compound of the invention inhibits the activity of two 19S RP DUBs, UCHL5 and USP14 while not affecting non‐proteasomal DUBs. More particularly, the compound of the invention has effect in the ent of a cancer tumor refractory to of‐the‐art chemotherapy due to over‐expression of the intrinsic apoptosis‐inhibitor Bcl‐2.
Most particularly, according to the present ion, the compound of the invention is effective in the treatment of a cancer tory to treatment with bortezomib or an agent g the mechanism of deubiquitinating inhibition of bortezomib. In another preferred embodiment, the compound is ive in the treatment of a cancer tory to any anti‐cancer drug known in the art.
In this application, "refractory to treatment" signifies that ent of a cancer with a single dose of an anti‐cancer medicine does not substantially reduce the growth rate of the cancer observed immediately prior to the treatment, such as reducing the growth rate per month by not more than 25 per cent or 10 per cent or even 5 percent or less. In particular, the method described is ent in treating a cancer in a patient which, after having received one or more, in particular two or three, standard doses of bortezomib or an agent sharing the apoptosis ting activity of bortezomib or any other anti‐cancer drug, exhibits a cancer growth rate per month reduced by not more than 25 per cent or 10 per cent or even 5 percent or less, such as any positive growth rate, in comparison with the cancer growth rate observed immediately prior to the single treatment or to the last of two or three or more treatments, respectively. An accepted measure of tumor growth is the change of volume of a sseminated cancer.
An example of a cancer amenable to treatment by the method described is multiple myeloma. Other examples of cancers amenable to treatment comprise lung cancer, prostate cancer, colon cancer, ovary cancer, pancreas cancer, breast cancer, neck & head In the nd of the general structure S‐1, R1, R2 at double bond d1 and R3, R4 at double bond d2 can, independent of each other, have a configuration opposite to that of formula S‐1, X is CO, CS, CH2 , CHC1‐6 ‐alkyl, NH or NC1‐6 ‐alkyl; R1 and R3 are, independent of each other, H or C1‐6 ‐alkyl; R2 and R4 are, ndent of each other, H; C1‐6 ‐alkyl; C1‐5 ‐alkylCO; phenyl or 6‐membered heteroaryl optionally substituted by 1‐3 of: C1‐6 ‐alkyl,C1‐6 ‐alkoxy, CN, ‐COOC1‐6 alkyl, COOH, NO2, F, Cl, Br, I, CF3 , NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , CONR7R8, with the proviso that one or more of H in alkyl and alkoxy can be substituted by fluoro; R5 is H; C1‐6 ‐alkyl; C2‐6 ‐alkenyl; C1‐3 ‐alkoxy‐C2‐6 ‐alkyl‐; C1‐3 ‐alkoxy‐C2‐6 ‐alkenyl‐; aryl‐C0‐6 ‐ alkyl‐; heteroaryl‐C0‐6 ‐alkyl‐; heterocyclyl‐C0‐6 ‐alkyl‐; cycloalkyl‐C0‐6 ‐alkyl‐; ‐C1‐6 ‐alkyl‐COOC1‐ 6 ‐alkyl; ‐C2‐6 ‐alkyl‐aryloxy; COR R6 is ed from: C1‐6 ‐alkyl; C2‐6 ‐alkenyl; C1‐6 ‐alkoxy; C1‐3 y‐C1‐6 ‐alkyl‐; C1‐3 ‐alkoxy‐C1‐6 ‐alkenyl‐; aryl‐C0‐6 ‐alkyl‐; heteroaryl‐C0‐6 ‐alkyl‐; heterocyclyl‐C0‐6 ‐alkyl‐; cycloalkyl‐C0‐6‐alkyl‐; ‐C1‐6 ‐alkyl‐COOC1‐6‐alkyl; NH2 ; 6 ‐alkyl; ‐N(C1‐6 ‐alkyl)2 ; ‐C0‐6 ‐alkyl‐aryloxy; R7, R8 are, independent of each other, H or C1‐3 ‐alkyl.
It is preferred for both of R1 and R3 to be or C1‐3 ‐alkyl.
It is preferred for both of R2 and R4 to be H; C1‐6 ‐alkyl; C1‐5 CO; phenyl or 6‐membered heteroaryl optionally tuted by 1‐3 of: C1‐6 ‐alkyl,C1‐6 ‐alkoxy, CN, COOC1‐6 ‐alkyl, COOH, NO2 , F, Cl, Br, I, CF3 , NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , CONR7R8, with the proviso that one or more of H in alkyl and alkoxy can be tuted by fluoro, and wherein substitution of phenyl is preferably at one or more of positions 3, 4, 5.
It is particularly preferred for both of R2 and R4 to be phenyl substituted at one or more of positions 3, 4, 5 by 1‐3, preferably by 1 or 2, of: C1‐6 ‐alkyl, C1‐6 ‐alkoxy, CN, ‐COOC1‐6 alkyl, COOH, NO2 , F, Cl, Br, I, CF3 , NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , 8, with the proviso that one or more of H in alkyl and alkoxy can be substituted by fluoro. Most preferred are electron‐withdrawing substituents, in particular F, Cl, trifluoromethyl, NO2 , CN.
It is preferred for R5 to be selected from the group consisting of H, methyl, acetyl, COCH=CH2 , oxyethyl.
According to a preferred ment X = CO. According to another preferred embodiment R1 and R3 are both H. According to a third preferred embodiment, R2 and R4 are, independent of each other, phenyl or 6‐membered heteroaryl optionally substituted by 1‐3 of: C1‐6 ‐alkyl,C1‐6 ‐alkoxy, CN, ‐COOC1‐6 , COOH, NO2, F, Cl, Br, I, CF3 , NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , 8, phenyl being preferred and substitution of phenyl, if any, being preferred in one or more of ons 3, 4, 5.
According to a preferred ment R1, R2 at double bond d1 and R3, R4 at double bond d2 have the configuration of formula S‐1; X is CO, CS, CH2 , CHC1‐6 ‐alkyl, NH or NC1‐6‐alkyl; R1 and R3 are, independent of each other, H or C1‐6 ‐alkyl; R2 and R4 are, independent of each other, H; C1‐6 ‐alkyl; C1‐5 ‐alkylCO; phenyl or 6‐membered heteroaryl substituted with 1‐3 of: CN, NO2 , F, Cl, Br, I, NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , COC1‐6 ‐alkyl; R5 is H, C1‐6 ‐alkyl, C2‐6 ‐alkenyl, C1‐3 ‐alkoxy‐C1‐6 ‐alkyl, C1‐3 ‐alkoxy‐C1‐6 ‐alkenyl, aryl, heteroaryl, heterocyclyl, C1‐6 ‐alkyl‐heteroaryl, C1‐6 ‐alkyl‐heterocyclyl, C1‐6 ‐cycloalkyl, C1‐6 ‐alkyl‐aryl, CO‐C1‐6 ‐alkyl, CO‐vinyl, CO‐allyl, CO‐aryl, CO‐cycloalkyl. It is preferred, independent of each other, for X to be CO, for R2 and R4 to be substituted phenyl, for R5 to be selected from COR6, in particular from CO‐C1‐6 ‐alkyl, CO‐cycloalkyl, CO‐vinyl, CO‐allyl.
“Aryl” refers to a monocyclic or bicyclic hydrocarbon of from 6 to 10 carbon atoms comprising at least one aromatic ring. xy” refers to an aryl group bonded to an oxygen atom. “Heteroaryl” represents a clic ring system having 5 or 6 ring atoms, of which one or more are selected independently from , nitrogen, sulphur. "Alkyl" s straight or branched alkyl. “Alkenyl” s straight or branched alkenyl.
"Alkoxy" denotes straight or branched alkoxy. alkyl” refers to a saturated monocyclic hydrocarbon of from 3 to 7 carbon atoms.
Preferred compounds of the l structure S‐1 are disclosed in Tables 1 and 2.
Table 1. Preferred compounds X = CO, R1 = R3 = H, R5 is H or alkyl # R2 R4 R5 HCT116, MeJuSo‐UB, FMCA, IncuCyte lowest IC50 (μM) effective conc. (μM) 1516 phenyl phenyl H 1.2 1517 4‐methoxyphenyl 4‐methoxyphenyl H 1518 4‐chlorphenyl 4‐chlorophenyl H 1.6 1533 3‐acetylphenyl 3‐acetylphenyl H 1535 3‐nitrophenyl 3‐nitrophenyl H 5.9 1536 2‐nitrophenyl 2‐nitrophenyl H 6.9 1537 4‐nitrophenyl 4‐nitrophenyl H 4.1 1560 4‐nitrophenyl* H** H 1.0 1561 4‐fluorophenyl 4‐fluorophenyl H 1.0 1 1562 4‐fluoro‐3‐nitrophenyl 4‐fluoro‐3‐nitrophenyl H 0.5 0.25 1563 4‐nitrophenyl 4‐nitrophenyl methyl 1.5 0.5 1564 4‐fluorophenyl 4‐fluorophenyl methyl 0.9 16 1565 4‐fluoro‐3‐nitrophenyl 4‐fluoro‐3‐nitrophenyl methyl 1.5 0.5 1566 3‐nitrophenyl 3‐nitrophenyl methyl 2.3 0.5 1574 4‐fluorophenyl 4‐fluorophenyl propyl 2.8 0.5 1575 4‐nitrophenyl 4‐methoxyphenyl H 2.1 1 1576 4‐fluorophenyl 4‐methoxyphenyl H 1.6 8 1577 4‐fluorophenyl 4‐methoxyphenyl methyl 4.9 8 1582 4‐fluorophenyl 4‐chlorophenyl H 1.7 2 1583 4‐chlorophenyl 4‐nitrophenyl methyl 3.3 2 1584 4‐chlorophenyl 4‐nitrophenyl H 1.8 2 1585 rophenyl 4‐nitrophenyl H 1.5 2 1586 4‐chlorophenyl rophenyl H 1.1 0.5 1587 4‐fluorophenyl 4‐nitrophenyl methyl 3.0 1 1588 4‐nitrophenyl 4‐methoxyphenyl methyl 3.1 1 1589 4‐chlorophenyl 4‐fluorophenyl methyl 2.5 1 1590 4‐chlorophenyl 4‐methoxyphenyl methyl 2.0 1 1591 4‐nitrophenyl 4‐chlorophenyl H 0.9 0.5 1592 4‐chlorophenyl ophenyl H 12 4 1593 4‐nitrophenyl 4‐fluorophenyl methyl 2.9 1 1594 4‐nitrophenyl rophenyl methyl 2.6 1 1595 4‐fluorophenyl 4‐chlorophenyl methyl 2.3 1 1596 4‐nitrophenyl 4‐fluorophenyl H 2.6 2 1608 3‐chloro‐4‐fluoro‐ 3‐chloro‐4‐fluoro‐ methyl 1.8 1 phenyl phenyl 1609 4‐fluoro‐3‐trifluoro‐ 4‐fluoro‐3‐trifluoro‐ methyl 1.4 1 methyl‐phenyl methyl‐phenyl 1610 3,4‐difluorophenyl 3,4‐difluorophenyl methyl 1.7 1 1611 3‐fluoro‐5‐trifluoro‐ 3‐fluoro‐5‐trifluoro‐ H 1.1 0.5 methyl‐phenyl methyl‐phenyl 1612 3‐fluoro‐5‐trifluoro‐ 3‐fluoro‐5‐trifluoro‐ methyl 0.7 0.25 methyl‐phenyl methyl‐phenyl 1613 4‐nitrophenyl 4‐nitrophenyl H 25 32 1614 4‐nitrophenyl ophenyl methyl 7.4 8 1615 4‐chloro‐3‐trifluoro‐ 4‐chloro‐3‐trifluoro‐ methyl 0.9 0.5 phenyl methylphenyl 1616 3,4,5‐trifluoromethyl‐ 3,4,5‐trifluoromethyl‐ methyl 1.4 0.5 phenyl phenyl 1617 luoromethyl‐ 4‐trifluorometyl‐ methyl 1.6 1 phenyl phenyl 1618 3‐cyano‐4‐fluoro‐ 3‐cyano‐4‐fluoro‐ methyl 1.5 0.5 phenyl phenyl 1619 3‐carbonylamino‐ 3‐carbonylamino‐ H 30 32 phenyl phenyl 1620 3‐nitrophenyl 3‐nitrophenyl methyl >32 no effect 1621 4‐cyanophenyl 4‐cyanophenyl methyl 4.5 2 1622 4‐fluoro‐3‐trifluoro‐ 4‐fluoro‐3‐trifluoro‐ H 0.8 0.5 methyl‐phenyl methyl‐phenyl 1623 o‐4‐fluoro‐ 3‐cyano‐4‐fluoro‐ H 1.0 0.5 phenyl phenyl 1624 3‐fluoro‐4‐trifluoro‐ 3‐fluoro‐4‐trifluoro‐ methyl 1.8 1 methyl‐phenyl methyl‐phenyl 1625 4‐cyanophenyl 4‐cyanophenyl H 0.9 0.5 1626 3‐fluoro‐4‐trifluoro‐ 3‐fluoro‐4‐trifluoro‐ H 0.8 8 methyl‐phenyl ‐phenyl * or H or a mixture of H and 4‐nitrophenyl ** or 4‐nitrophenyl or a mixture of H and 4‐nitrophenyl Due to protonation of their amino group the solubility in aqueous media of azepanone compounds of which R5 is not acyl as well as of correspondingly substituted piperidin‐4‐ones increases with decreasing pH. However, according to an important aspect azepanone compounds of which R5 is not acyl (that is, not ‐COR6) have superior lity in aqueous media at physiological pH in comparison with correspondingly substituted din‐4‐ones. While the solubility of these azepanones and piperidine‐4‐ones increases in going from a high pH to a low pH, the increase starts at higher pH values for the azepanones than for the corresponding piperidin‐4‐ones. In this application "physiological pH" is a pH of from about 6 to about 8, in particular from 7.0 to 7.5.
Table 2. Preferred compounds X = CO, R1 = R3 = H, R5 = COR6 # R2 R4 R6 HCT116, MeJuSo‐UB, FMCA, IC50 lowest (μM) effective conc. (μM) 1505 4‐nitrophenyl 4‐nitrophenyl 2‐pyrrolidinyl 5.2 16 1507 4‐nitrophenyl 4‐nitrophenyl 2‐(1‐carboxyethyl‐ethyl) 4.4 8 1520 phenyl phenyl vinyl 1521 phenyl phenyl cyclobutyl 1525 4‐methoxyphenyl 4‐methoxyphenyl cyclobutyl 1526 oxyphenyl 4‐methoxyphenyl cyclopropyl 1527 4‐chlorophenyl 4‐chlorophenyl cyclobutyl 2 1546 4‐nitrophenyl 4‐nitrophenyl vinyl 1.2 2 1567 4‐nitrophenyl 4‐nitrophenyl methyl 0.6 0.5 1568 4‐fluorophenyl 4‐fluorophenyl vinyl 1.5 2 1569 4‐fluorophenyl 4‐fluorophenyl vinyl 2.0 4 1570 4‐fluoro‐3‐ 4‐fluoro‐3‐ vinyl 0.5 0.25 nitrophenyl nitrophenyl 1571 4‐fluoro‐3‐ 4‐fluoro‐3‐ methyl 0.9 0.25 nitrophenyl nitrophenyl 1572 3‐nitrophenyl 3‐nitrophenyl vinyl 2.5 0.5 1578 rophenyl 4‐methoxyphenyl methyl 7.0 8 1579 4‐fluorophenyl 4‐methoxyphenyl methyl 5.9 8 1580 ophenyl oxyphenyl vinyl 1.5 8 1581 4‐nitrophenyl 4‐methoxyphenyl methyl 7.2 8 1597 4‐nitrophenyl 4‐chlorophenyl methyl 1.3 1 1627 4‐trifluoromethyl‐ luoromethyl‐ methyl 0.7 1 phenyl phenyl 1628 3,4‐difluorophenyl 3,4‐ methyl 2.3 1 difluorophyenyl 1629 3,4,5‐ 3,4,5‐ methyl 0.6 1 trifluorophenyl trifluorophenyl 1630 4‐chloro‐3‐fluoro‐ 4‐chloro‐3‐fluoro‐ methyl 0.9 0.5 phenyl phenyl 1631 3‐chloro‐4‐fluoro‐ 3‐chloro‐4‐fluoro‐ methyl 1.0 32 phenyl phenyl 1633 rophenyl 4‐chlorophenyl 2‐acetoxyethyl 2.2 4 1635 4‐chlorophenyl 4‐chlorophenyl benzyl 1.4 2 1636 4‐chlorophenyl 4‐chlorophenyl 1‐(3‐phenyl‐2‐propenyl) 2.0 1 1637 4‐chlorophenyl 4‐chlorophenyl 3‐pyridyl 2.1 2 1638 4‐chlorophenyl 4‐chlorophenyl 2‐thiophenyl 2.0 2 1639 4‐chlorophenyl 4‐chlorophenyl 4‐hydroxy‐3‐ 1.2 1 ethoxybenzyl 1640 4‐chlorophenyl 4‐chlorophenyl methyl‐(2‐methoxy‐ 1.9 1 yl)phenyl 1641 4‐trifluoromethyl‐ 4‐trifluoromethyl‐ methyl‐3‐pyridyl 2.7 2 phenyl phenyl 1642 4‐trifluoromethyl‐ 4‐trifluoromethyl‐ 2‐oxo‐acetoxyethyl 1.2 1 phenyl phenyl 1643 4‐trifluoromethyl‐ 4‐trifluoromethyl‐ 3‐(3‐oxo‐propanoyloxy‐ 0.9 1 phenyl phenyl methyl) 1644 4‐trifluoromethyl‐ 4‐trifluoromethyl‐ 1‐oxo‐2‐(2‐pyridyl)ethyl 1.4 1 phenyl phenyl 1645 4‐chloro‐3‐fluoro‐ 4‐chloro‐3‐fluoro‐ 2‐oxo‐acetoxyethyl 2.5 2 phenyl phenyl 1646 4‐fluoro‐3‐nitro‐ 4‐fluoro‐3‐nitro‐ 2‐oxo‐acetoxymethyl 1.1 1 phenyl phenyl 1647 4‐fluoro‐3‐nitro‐ 4‐fluoro‐3‐nitro‐ 3‐(3‐oxo‐propanoyloxy‐ 0.7 0.25 phenyl phenyl methyl) 1648 4‐fluoro‐3‐nitro‐ 4‐fluoro‐3‐nitro‐ 1‐oxo‐2‐(2‐pyridyl)ethyl 0.8 0.5 phenyl phenyl 1649 3,4,5‐trifluoro‐ trifluoro‐ methyl‐(2‐methoxy‐ 0.9 0.5 phenyl phenyl carboxyl)phenyl 1650 trifluoro‐ 3,4,5‐trifluoro‐ methyl‐3‐pyridyl 1.9 1 phenyl phenyl 1651 3,4,5‐trifluoro‐ 3,4,5‐trifluoro‐ 2‐oxo‐acetoxyethyl 0.8 0.5 phenyl phenyl 1652 3,4,5‐trifluoro‐ 3,4,5‐trifluoro‐ 3‐(3‐oxo‐propanoyloxy‐ 0.6 0.5 phenyl phenyl methyl 1653 trifluoro‐ trifluoro‐ 1‐oxo‐2‐(2‐pyridyl)ethyl 0.71 1 phenyl phenyl 1654 4‐chloro‐3‐trifluoro 4‐chloro‐3‐fluoro‐ ‐(2‐methoxy‐ 1.2 1 methyl‐phenyl methyl‐phenyl carboxyl)phenyl 1655 4‐chloro‐3‐trifluoro 4‐chloro‐3‐trifluoro methyl‐3‐pyridyl 1.0 1 methyl‐phenyl methyl‐phenyl 1656 4‐chloro‐3‐trifluoro ro‐3‐trifluoro 2‐oxo‐acetoxyethyl 0.7 0.5 ‐phenyl methyl‐phenyl 1657 4‐chloro‐3‐trifluoro 4‐chloro‐3‐trifluoro acetyl 0.6 0.5 methyl‐phenyl methyl‐phenyl 1658 4‐chloro‐3‐trifluoro 4‐chloro‐3‐trifluoro 1‐oxo‐2‐(2‐pyridyl)ethyl 0.8 0.5 methyl‐phenyl methyl‐phenyl 1659 4‐trifluoromethyl‐ 4‐trifluoromethyl‐ 2‐acetoxyethyl 2.4 2 phenyl phenyl 1660 4‐chloro‐3‐fluoro‐ 4‐chloro‐3‐fluoro‐ 2‐acetoxyethyl 2.4 2 phenyl phenyl 1661 4‐chloro‐3‐fluoro‐ 4‐chloro‐3‐fluoro‐ methylcarboxyl 24 32 phenyl phenyl 1662 4‐fluoro‐3‐nitro‐ 4‐fluoro‐3‐nitro‐ 2‐acetoxyethyl 2.1 1 phenyl phenyl 1663 ro‐3‐nitro‐ 4‐fluoro‐3‐nitro‐ carboxyl 3.1 4 phenyl phenyl 1664 trifluoro‐ 3,4,5‐trifluoro‐ 2‐acetoxylethyl 2.8 4 phenyl phenyl 1665 3,4,5‐trifluoro‐ 3,4,5‐trifluoro‐ methylcarboxyl 5.0 8 phenyl phenyl 1666 4‐chloro‐3‐ 4‐chloro‐3‐ 2‐acetoxyethyl 1.3 1 trifluoromethyl‐ trifluoromethyl‐ phenyl phenyl The solubility in aqueous media of nds of the invention of which R5 is acyl (that is, ‐COR6) is substantially independent of pH.
Particularly preferred compounds are compounds nos. 1561, 1562, 1567, 1570, 1571, 1586, 1591, 1600, 1612, 1618, 1622, 1625, 1643, 1644, 1647, 1648, 1649, 1652, 1653, 1656, 1657, 1658, 1662. Most preferred compounds are nds nos. 1570, 1571, 1625, 1662.
Since the compound of the invention comprises a substituted 1,4‐pentene‐3‐one moiety it can exist in four cis/trans isomers EE, ZE, ES, ZZ. In defining the compound of the invention this isomerism is defined in the foregoing as "R1, R2 at double bond d1 and R3, R4 at double bond d2 can, independent of each other, have a configuration opposite to that of formula S1". The compound of the invention comprises any such isomer and any e of such isomers.
In synthesis the compound of the invention is obtained as a mixture of isomer but sometimes also in form of the isomer with the lowest solubility in the particular solvent, from which it precipitates or crystallizes. While pure isomers thus can be obtained under controlled conditions, the pharmacological effect of the compound is exhibited by all isomers. The reason for this is their equilibration in the presence of water or other ylic or sulfhydrylic solvent or agent, which is accelerated by acid and base catalysis. ingly, the term "compound of the invention" as used herein comprises a pure isomer of the aforementioned kind as well as a mixture of two or more such s. The rate of bration of the compound of the invention in aqueous body fluid is sufficient to provide for substantial equilibration within a single treatment period.
The nd of the invention comprises an azepane moiety, preferably an azepane‐4‐one moiety. According to an important aspect of the invention, the compound of the invention exhibits a cytotoxic activity superior to that of a structurally corresponding compound comprising a piperidine moiety, such as a 4‐piperidinone moiety.
According to another important aspect of the invention, the compound of the invention comprising an azepane moiety, in ular an azepan‐4‐one moiety exhibits a solubility in a liquid carrier suitable for administration to a patient, such as dimethyl sulfoxide, superior to that of a structurally corresponding nd comprising a piperidine moiety, such as a 4‐piperidinone moiety.
A "single treatment period" is the period of time elapsing between administration and consumption of the compound of the ion, that is, the point in time at which the concentration of the compound of the invention at a site of action, such as in a tumor, has been reduced by 90 % or 95 % or 99 % and more. In a ceutical composition, an isomer or a mixture of isomers of the compound of the invention is stabilized against isomerization by careful exclusion of moisture.
The method bed herein comprises administering to the patient in need a pharmacologically effective dose of the compound of the invention in a suitable pharmaceutical carrier, such as, for instance, dissolved or suspended in an s carrier or in a carrier comprising dimethyl ide or N,N‐dimethylacetamide. Administration can be by any suitable route, such as by intravenous, uscular, eritoneal or subcutaneous injection or infusion. Other methods of administration, in particular per os, are also contemplated, such as in form of tablets or hard or soft gelatin capsules.
The person skilled in the art knows how to determine a cologically effective dose.
Such a dose may be from 0.0001g/kg to 0.1 g/kg body weight, in particular from 0.001 g/kg to 0.01 g/kg kg body weight, consideration being given to whether the agent is administered systemically or locally. tent with DUB inhibition, treatment with the compound of the invention causes the accumulation of polyubiquitinated proteins of higher molecular weight in comparison with bortezomib treatment, and results in a stronger unfolded protein response. According to the invention, it has also been found that apoptosis induction by the compound of the invention s from that of bortezomib by being insensitive to disruption of the p53 tumor suppressor and insensitive to overexpression of the apoptosis inhibitor Bcl‐2.
According to the present invention treatment with the compound of the invention inhibits tumor progression in human and mouse tumor in vivo models of breast, lung, colon, head & neck carcinoma, and inhibits infiltration in an acute myeloid leukaemia (AML) model. In consequence, inhibiting the DUB activity of the 19S RP by the compound of the invention is disclosed to be a viable option for the treatment of cancer in humans and animals.
Thus, more specifically, is disclosed a method of treating in a person a cancer tumor refractory to state‐of‐the‐art chemotherapy sing administering, in a pharmaceutically acceptable carrier, a pharmacologically ive dose of the compound of the invention. The method described is particularly useful in the treatment of a patient having a tumor of which cells are refractory to treatment due to over‐expression of the intrinsic apoptosis‐inhibitor Bcl‐2.
According to a red aspect of the invention the 19S RP DUBs comprise UCHL5 and USP14. According to another preferred aspect of the invention the uitinating (DUB) activity of non‐proteasomal DUBs is not affected by the compound of the invention. The compound of the invention can be administered dissolved or suspended in a liquid carrier by any suitable route, such as by intravenous, intramuscular and subcutaneous administration. Alternatively or additionally, the compound of the invention can be administered perorally, such as in form of a tablet or capsule. A useful pharmacologically ive dose of the compound of the invention is from 0.0001g/kg to 0.1 g/kg body weight, in particular from 0.001 g/kg to 0.01 g/kg kg body , consideration being given to whether the compound is administered systemically or locally. The method may comprise selecting a person to be treated by ining the growth rate of the cancer prior to and upon stration of bortezomib or said active principle sharing the mechanism of deubiquitinating activitiy inhibition of bortezomib or said other anti‐cancer drug, a positive growth rate, in ular a growth rate of more than 5 % or more than % or more than 25 % per month constituting a selection marker.
The compound of the invention blocks cellular proteasome on, as confirmed by use of a reporter cell line, which expresses ubiquitin tagged to yellow fluorescent protein (UbG76V‐YFP) constitutively ed for proteasomal degradation (12). Immunoblotting and flow cytometry revealed a dose ent accumulation of the Ub‐YFP reporter (IC50=0.8 µM) suggesting an impairment of proteasome on. Since inhibition of proteasome on is characterized by defects in ubiquitin turnover (13) colon carcinoma HCT116 cells were d with the nd of the invention and the level of ubiqutin conjugation analyzed by immunoblotting. The treatment caused the rapid time dependent lation of polyubiquitinated proteins of a higher molecular weight in comparison with the 20S CP inhibitor bortezomib, suggesting that the compound of the invention inhibits an alternative branch of the UPS. The increase in polyubiquitin is associated with a strong toxic response characterized by induction of HSPA6 (Hsp70B'), HSPA1B and DNAJB1 (Hsp40).
The turnover of many cell cycle tory ns is controlled by the UPS including inhibitors of the cyclin‐dependent kinase p21Cip1, p27Kip1 and the tumor suppressor p53 (4).
Treatment with the compound of the invention increases their levels in a dose dependent manner without altering the levels of ornithine decarboxylase 1 (ODC1), an ubiquitin‐ independent proteasome substrate (8). The increase in cell cycle regulators was concomitant with growth arrest in the G2/M phase boundary and increased sub G1 DNA content. The cell cycle arrest observed is not ated with increased levels of DNA damage markers such as phosphorylated p53 (at Ser 15) (9) or H2AX (at Ser 139) (10), suggesting that b‐AP15 is not a genotoxic agent.
The increase in sub G1 DNA, e‐3 activation and cleavage of poly‐ADP ribose polymerase (PARP) and cytokeratin is associated with an overall decrease in cell viability at drug concentrations that induce the accumulation of polyubiquitin connecting UPS inhibition and sis. Apoptosis induction by bortezomib is sensitive to the status of the p53 tumor suppressor and over‐expression of the anti‐apoptotic Bcl‐2 oncoprotein (11, 12). By using isogenic clones of HCT116 colon cancer cells it was demonstrated that b‐AP15 induced apoptosis is insensitive to over‐expression of Bcl‐2 and disruption of the apoptotic regulators p52, BAX or PUMA. Measurement of cytotoxic activity shows that the compound of the invention is more toxic to the colon carcinoma cell line HTC‐116 than to immortalized l pigment epithelial cells (hTERT‐RPE1) and peripheral blood mononuclear cells . The compound of the invention exhibits a higher degree of cytotoxic activity s the HTC‐116 cells than towards normal cell types.
The observed reduction in cellular proteasome activity cannot be explained by tion of proteolytic activities of the ß subunits of the 20S CP. In vitro experiments using activity‐ specific substrates do not show inhibition in any of the proteolytic activities of the 20S CP or 26S proteasome, ociation of the 19S RP and 20S CP or inhibition of polyubiquitin g to the proteasome.
The compound of the invention comprises an α‐ß dienone entity with two sterically accessible ß carbons. A structurally similar pharmacophore has been earlier described to be comprised by a class of ubiquitin isopeptidase inhibitors (13). However, when cellular DUB activity was tested using ubiquitin 7‐amido‐4‐methylcoumarin (Ub‐AMC) on treated cells d with the compound of the invention, no reduction in Ub‐AMC ge was observed. This demonstrates that the compound of the invention is not a general DUB inhibitor. While not wishing to be bound by theory, the similarities in pharmacophore structure and the data showing that compound of the ion inhibits proteasome ty independent of the 20S CP indicate that the compound of the invention inhibits the proteasome by blocking the deubiquitinating activity of the 19S RP.
In vitro assays using Ub‐AMC and ed 19S RP or 26S proteasomes confirmed that the compound of the invention inhibits the deubiquitinating activity of both the 19S RP and 26S proteasome. Recombinant tin‐GFP is a substrate for 19S RP DUB ty (15).
Treatment of 19S RP with b‐AP15 efficiently inhibited the ge of Ub‐GFP and ubiquitinated HDM2. The type of ubiquitin bonds present in the polyubiquitin chain determines the fate of an ubiquitin‐modified substrate.
K48 linked polyubiquitin chains generally target conjoined proteins for degradation (14), whereas K63 linked chains are involved in non‐proteolytic roles including DNA repair (15) and mitotic chromosome segregation (16). Ubiquitin chain disassembly reactions ed that the compound of the invention ts 19S RP processing of both K48 and K63 linked ubiquitin tetramers. The inhibition of ubiquitin chain disassembly observed may account for the accumulation of high molecular weight tin conjugates in cells treated with the compound of the invention.
The deubiquitinating activity of the proteasome is attributed to the action of three DUBs, UCHL5, USP14 and POH1, all localized within the 19S RP (17‐19). Both UCHL5 and USP14 are sensitive to N‐ethylmaleimide (NEM), a general inhibitor of cysteine ses, whereas POH1 is itive to inhibition by NEM but sensitive to metal chelators such as N,N,N,N‐tetrakis‐(2‐pyridylmethyl)ethylenediamine (TPEN) (20). Inhibition experiments showed that residual DUB activity is present even after atment of 19S RP with NEM and the compound of the ion. This residual DUB activity was abolished upon co‐treatment of 19S RP with the compound of the invention and TPEN, suggesting that the compound of the invention ily inhibits one or both of the NEM sensitive cysteine DUBs. The ß‐carbons of the compound of the invention may serve as Michael acceptor moieties, resulting in covalent binding to cysteine residues in target proteins. In vitro assays , however, that the compound of the invention is a reversible inhibitor and that glutathione does not preclude the inhibitory activity of the compound.
To identify specifically which DUBs were inhibited by treatment with the compound of the invention, competitive labelling experiments were performed using hemagglutinin tagged ubiquitin ulphonone (HA‐UbVS), an active site directed probe that rsibly reacts with DUBs of the cysteine class (17). Incubation of 19S RP or 26S proteasomes with the compound of the invention abolished Ub‐VS labelling of two DUBs of molecular weights corresponding to UCHL5 and USP14. A similar result was obtained using UbVs on lysates d from drug‐treated cells. blot analysis showed a downward shift in molecular weight of both USP14 and UCHL5 due to loss of activity and decreased UbVs labelling. This is consistent with affinity‐purified proteasomes from the nd of the invention d cells ying reduced DUB activity confined to the proteasome and not evident in cell lysates. Additional in vitro assays showed minimal inhibition of the compound of the invention on recombinant non‐proteasomal cysteine DUBs, consistent with the notion that inhibition is not due to general cysteine vity.
The compound of the ion does substantially decrease and even stop tumor growth in vivo, as shown by its administration to mice bearing either a human tumor or mouse xenografts. When the compound of the invention is administered daily to SCID mice bearing FaDu head and neck carcinoma xenografts, significant inhibition of FaDu tumor growth is observed following daily treatment with the compound of the invention (treated/control tumor volume, T/C=0.4, p=<0.001). Tumor cell death was analyzed by measuring xenograft derived cytokeratin (CK18) in ation. Cytokeratin‐18 is a biomarker for apoptosis (21, 22); a significant increase in plasma levels of total human CK18 was observed (p=0.01).
Levels of caspase cleaved CK18 Asp396) increased moderately compared with total levels, suggesting that the compound of the invention has activity against tumor cells in vivo. The compound of the invention was also shown to inhibit tumor onset of HCT‐116Bcl2+ colon carcinoma xenografts in nude mice, as demonstrated by significant delay in tumor onset in comparison to vehicle treated controls. rly, the nd of the invention inhibits tumor growth in syngenic mice models using less frequent administration schedules.
Ubiquitin C‐terminal hydrolases (UCH) and ubiquitin specific proteases (USP) are major subgroups of the approximately one hundred DUBs encoded by the human genome (23).
The mechanism of specificity of the compound of the invention for UCHL5 and USP14 in the 19S RP may be related to unique conformations of these enzymes in the 19S RP or due to drug‐induced tions of the 19S RP structure. The present findings are consistent with s in the art indicating that loss of both UCHL5 and USP14, unlike loss of either one alone, leads to the accumulation of polyubiquitinated proteins and inhibition of cellular protein degradation (24).
The observation that DUB inhibition is ated with high molecular weight ubiquitin‐substrate xes seems to be of particular relevance. Strong expression of chaperone genes was observed in cells treated with the compound of the invention, indicating induction of a proteotoxic response. High‐molecular weight ubiquitin‐substrate complexes accumulating as a result of DUB inhibition by the compound of the invention seem to generate strong cytotoxicity.
The term ‘comprising’ as used in this specification and claims means ‘consisting at least in part of’. When interpreting statements in this specification and claims which includes the ‘comprising’, other features besides the features prefaced by this term in each statement can also be present. d terms such as ‘comprise’ and ‘comprised’ are to be interpreted in similar manner.
In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless ically stated otherwise, reference to such external documents is not to be construed as an ion that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
In the following the invention will be bed in greater detail by reference to red embodiments f rated by a drawing comprising a number of s.
DESCRIPTION OF THE FIGURES Figs. 1a to 1o are diagrams illustrating induction of dose‐dependent cytotoxicity after 72 hours of continuous compound exposure to the reporter cell line HCT‐116 by embodiments of the compound of the invention, as measured FMCA (Fluorometric Microculture Cytotoxicity Assay), as well as absence of such ion by structurally related compounds not comprised by the invention. Treated cells were compared to untreated controls (survival Figs. 2a to 2e are diagrams illustrating the superior solubility of compounds of the invention in an aqueous media at physiological pH; Figs. 3a to 3f are diagrams illustrating, by the method of Figs. 1a to 1o, the or cytotoxicity of azepanone compounds of the invention in relation to structurally corresponding piperidin‐4‐one compounds not comprised by the invention.
DESCRIPTION OF PREFERRED EMBODIMENTS Methods In vitro proteasome activity assays are performed in black 96‐well microtitier plates using human 20S proteasome (Boston Biochem) in reaction buffer (25 mM Hepes, 0.5 mM EDTA, 0.03 % SDS) with Suc‐LLVY‐AMC, Z‐LLE‐AMC or Boc‐LRRAMC used as substrates for some activity. De‐ubiquitinase activity assays are performed with human 19S RP (Boston Biochem) with ubiquitin‐AMC as substrate. For FaDu xenograft s a 100‐µl‐cell suspension containing 1x106 cells is injected subcutaneously into the flank of SCID. Upon 30 tumor take mice are randomized into control or treatment groups and administered with 5 mg kg‐1 compound of the invention or vehicle. In vivo levels of apoptosis and cell death are determined from the detection of caspase cleaved and total levels of ratin‐18 in plasma using M30 Apoptosense® and M65 ELISA®s assays (Peviva). The methods are described below in more detail.
Reagents. Reagents were obtained from the following sources: 20S proteasome (E‐360), 26S proteasome (E‐365), 19S proteasome (E‐366), VY‐AMC (S‐280), AMC (S‐230), Boc‐LRR‐AMC (S‐300), Ubiquitin‐AMC (U‐550), Tetra‐ubiquitin K63 (UC‐310), Tetra‐ubiquitin K48 (UC‐210), deconjugating enzyme set (KE10), HA‐Ubiquitin Vinyl Sulfone (U‐40212) (Boston Biochem); anti‐β‐actin (AC‐15), ODC‐1 1536) (Sigma Aldrich); anti‐LC‐3 (2775), APDH (2118), anti‐p44/42 MAPK (4695), anti‐Phospho‐p44/42 MAPK (9101)(Cell Signaling); N‐ethylmaleimide (34115) (EMD Chemicals); biquitin K48 (Apu2), anti‐Ubiquitin (MAB1510) (Millipore); anti‐p53 (DO1), anti‐UCHL5 (H‐110), Hdm2 (SMP14) (Santa Cruz); anti‐PARP (C2‐10), anti‐p27 (G173‐524), anti‐active Caspase 3 (C92‐605) (BD Biosciences); anti‐USP14 (A300‐919A) (Bethyl tories); anti‐HA (12CA5) (Roche). Bortezomib was obtained from the Department of Oncology, nska Hospital, Sweden.
Cell culture. MCF7 cells are maintained in % fetal calf serum. HCT‐116 p53 +/+, p53 ‐/‐, Bcl‐2 +/+, PUMA ‐/‐ and BAX ‐/‐ cells are maintained in McCoy´s 5A modified medium/10% fetal calf serum. The HCT‐116 p53 +/+, p53 ‐/‐, PUMA ‐/‐ and BAX ‐/‐ are generated as described (25). The HCT‐116 Bcl‐2 +/+ cell line was generated by transfecting parental HCT‐116 p53 +/+ cells with pCEP4 Bcl‐2 (Addgene d 16461) (26) and isolating high expression clones. FaDu and LLC3 cells are maintained in DMEM high glucose medium supplemented with 10% fetal calf serum, Na pyruvate, Hepes and non‐essential amino acids. 4T1.12B carcinoma cells are maintained in RPMI medium supplemented with % fetal calf serum. The proteasome reporter cell line MelJuSo Ub‐YFP was generated as described (12). Cells were maintained in Dulbecco’s Modified Eagle’s Medium/10 % fetal calf serum. The retinal lial cell line was generated as described (12). All cells are maintained in Dulbecco’s Modified Eagle Medium/10 % fetal calf serum. The retinal epithelial cell line was generated as described (28). All cells are maintained at 37 oC in 5 % CO2 .
Proteasome and DUB tion assays. In vitro proteasome activity assays using 20S CP (2nM) (Boston Biochem) are performed at 37 oC in 100‐µl reaction buffer (25 mM Hepes, 0.5 mM EDTA, 0.03 % SDS). Samples are incubated for 10 min with indicated compound followed by addition of 10 µM Suc‐LLVY‐AMC, Z‐LLE‐AMC or R‐AMC for the detection of chymotrypsin‐like, caspase‐like and trypsin‐like activity respectively. For DUB inhibition assays 19S RP (5 nM), 26S (5 nM) UCH‐L1 (5 nM), UCH‐L3 (0.3 nM), USP2CD (5 nM) USP7CD (5 nM) USP8CD (5 nM) and BAP1 (5 nM) are incubated with the compound of the invention followed by addition of ubiquitin‐AMC (1000 nM). scence is monitored using Wallac abel counter or Tecan Infinite M1000 ed with 360 nm excitation and 460 nm emission filters.
Substrate overlay assays. Native gel electrophoresis is med as described (29). In brief 4 µg of purified 26S proteasome n Biochem) is mixed with 10 or 50 µM of the compound of the invention and incubated at 37 oC for 10 min. Samples are resolved on 4% non‐denaturing PAGE. Gels are submerged in assay buffer (20 mM Tris‐HCL, 5 mM MgCl2 , 1 mM ATP, 0.1 mM VY‐AMC) and proteasomes are ized under UV illumination.
Ubiquitin‐cleavage assay. The recombinant Ub‐GFP plasmid pet19b Ub‐M‐GFP is generated as described (30). In brief recombinant Ub‐GFP is purified from BL21 E.coli cells by His affinity purification. For cleavage assays 19S RP (25 nM) is incubated with 10 mM NEM, 250 µM TPEN or 50 µM of the compound of the invention for 10 min followed by the addition of recombinant Ub‐GFP (200 nM). Ubiquitin chain disassembly reactions are performed essentially as above except K48‐ or K63‐linked ubiquitin tetramers (50 ng) are substituted for Ub‐GFP. The level of Ub‐GFP cleavage or ubiquitin disassembly is determined by immunoblotting with anti ubiquitin antibodies. The ubiquitinated Hdm2 substrate is generated according to the Boston Biochem protocol (K‐200). For the cleavage assay 19S RP (25 nM) is incubated with 50 μM of the compound of the invention or DMSO for 10 min followed by the addition of ubiquitinated Hdm2 substrate (100 nM). The cleavage of ubiquitinated Hdm2 substrate and ubiquitinated Hdm2 is determined by immunoblotting with anti‐Hdm2 antibodies.
Proteasome ion: HCT‐116 cells are d with bortezomib (100 nM) or the compound of the invention (1 μM) for 3 hours. After ation, the cells are lysed in 50 mM HEPES pH 7.4, 250 mM sucrose, 10 mM MgCl2 , 2 mM ATP, 1 mM DTT and 0.025 % digitonin. Samples are sonicated briefly and incubated for 15 min on ice. Proteasomes from these samples are isolated according to the manufacturer’s protocol.
UbVS labelling of DUBs. For labelling of DUBs in cell lysates sub confluent cells are harvested by trypsinization, washed three times with PBS, and centrifuged at 1500 RPM for 5 min. Cell pellets are lysed with buffer (50 mM HEPES pH 7.4, 250 mM e, 10 mM MgCl2 , 2 mM ATP, 1 mM DTT) on ice for 15 min. Debris is removed by centrifugation and 25 µg of protein is labelled with 1 µM HA‐UbVS for 30 min at 37 oC. Samples are resolved by SDS‐PAGE and analyzed by blotting with indicated antibodies.
Determination of cell apoptosis and viability. For determination of apoptosis parental HCT‐116 p53 +/+ cells are d with the increasing doses of the compound of the invention for 24 h. ent doses are based on the drug tration that resulted in l apoptosis over a 24 h period. HCT‐116 cells are seeded in 96‐well microtiter plates at 10,000 cells per well and incubated overnight. Cells are d with indicated drug for 24 h. At the end of the incubation period, NP40 is added to the tissue culture medium to 0.1 % and 25 μl of the content of each well was d using the M30‐Apoptosense® ELISA as previously described (31). Cell viability is determined by measuring acid phosphatase activity or using the FMCA method (32). For the acid phosphatase ty cells are seeded at 5000 cells per well in 96‐well culture plates and incubated for 12 h at 37 oC. Compounds are added to the cells in growth media and incubated for 72 h at 37 oC. Cells are washed with 200 μl warm PBS. 100 μl of para‐nitrophenyl phosphate (pNPP, 2mg/ml) in Na acetate buffer pH 5 (NaAc 0.1 M, 0.1% Triton‐X‐100) is added per well. Cells are incubated for 2 h after which reaction was stopped by addition of 1N NaOH. Absorbance is measured at 405 nm. The dose‐dependent cytotoxicity of a number of embodiments of the compound of the ion is illustrated in Figs. 1a‐1o.
For the FMCA assay cells are seeded in the drug‐prepared 384‐well plates using the pipetting robot Precision 2000 (Bio‐Tek Instruments Inc., Winooski, VT). The plates are incubated for 72 h and then transferred to an integrated HTS SAIGAN Core System consisting of an ORCA robot (Beckman Coulter) with CO2 incubator (Cytomat 2C, Kendro, Sollentuna, Sweden), dispenser module (Multidrop 384, Titertek, Huntsville, AL), washer module (ELx 405, k Instruments Inc), delidding station, plate hotels, barcode reader (Beckman Coulter), liquid handler (Biomek 2000, Beckman Coulter) and a multipurpose reader (FLUOstar Optima, BMG Labtech GmbH, urg, Germany) for automated FMCA. Survival index (SI) is defined as the scence of test wells in percentage of controls with blank values cted.
Cell‐cycle analysis. For determination of cell cycle HCT‐116 cells are treated with the compound of the invention or DMSO cells are harvested by trypsinisation, washed and fixed in 70% ice cold EtOH for 12 h. The cells are re‐suspended in staining solution containing propidium iodide (50 µg/ml) and RNAse A (0.5 µg/ml) in PBS. Samples are run on BD FACScalibur. The percentage of cells in each phase of the cell cycle is ined using ModFit software.
EXAMPLE 1. ary synthesis of red embodiments to the compound of the invention General information. All solvents used were of HPLC grade or better. When anhydrous conditions were required, an excess of 3 Å lar sieves were added to the t at least 24 h before use to ensure dryness. 1H NMR nuclear magnetic resonance (NMR) was recorded on a Bruker Advance DPX 400 spectrometer at 400.1 MHz. Low resolution electrospray ionization mass spectra were obtained using an Agilent mass spectrometer in positive ionization mode. Flash chromatography was performed on Merck silica gel 60 (230‐400 mesh). Analytical LCMS data were obtained with an Agilent mass spectrometer; Agilent 1100 system; A: ACE C8 column (50x3.0 mm, 5μM); nt: 10‐97 % acetonitrile in water/0.1 % TFA, in 3 min 1.0 mL/min, or B: xBridge C18 column (3.5 µM. 50x3.0 mm), nt 10 % to 97 % acetonitrile in 10 mM NH4 HCO3 (pH 10) in 3 min, 1 mL/min).
Names of al structures were determined using Marvin Scech 5.2.6, ChemAxon. (3E, 5E)‐3,5‐Bis(phenylmethylidene)azepan‐4‐one (# 1516) and (3E, 5E)‐3,5‐bis(4‐ methoxyphenylmethylidene)‐azepan‐4‐one (# 1517) Hexahydro‐4H‐azepin‐4‐one (0.45 g, 3.0 mmol), together with either benzaldehyde (0.70 g, 7.0 mmol), 4‐methoxybenzaldehyde (0.90 g, 7.0 mmol) or robenzaldehyde (0.92 g, 7.0 mmol) was dissolved in acetic acid (10 mL). Then sulfuric acid (conc. 1 mL) was added drop‐wise and the reactions were stirred for 24 hours at rt. Water (30 mL) was added and the precipitate filtered and dried in vacuo over night. No further purification was performed. Compound # 1516 was obtained with 99% purity determined by LCMS (System A) MS ESI+ m/z 290 . Compound # 1517 was also ed in 99% purity determined by LCMS (System A), MS ESI+ m/z 350 [M+H]+. Compound # 1518 was obtained in 91% purity; LCMS m A). MS ESI+ m/z 358 [M]+, 360 [M+2]+. (3E, 5E)‐3,5‐bis(phenylmethylidene)‐1‐(prop‐2‐enoyl)‐azepan‐4‐one (#1520) (3E, 5E)‐3,5‐Bix(phenylmethylidene)azepan‐4‐one (# 1516) (50.0 mg, 0.182 mmol) and acrylic acid (14.4 mg, 0.20 mmol), HBTU (58.4 mg, 0.182 mmol), triethylamine (36.7 mg, 0.364 mmol) were dissolved in DMF (2 mL) and stirred over night. Ethyl acetate and brine were added and the products were extracted. The combined organic layers were dried and evaporated. The crude product was diluted with methanol and ed by preparative HPLC. Compound # 1520 was obtained in 96% purity, + m/z 344 (2R)‐[(3E, 5E)‐3,5‐Bis(4‐nitrophenylmethylidene)‐4‐oxo‐1‐(pyrrolidin‐2‐yl‐carbonyl)‐azepan trifluoroacetate (# 1505) N‐Boc‐azepanone (100 mg, 0.47 mmol) and 4‐nitrobenzaldehyde (156 mg, 1.03 mmol) were dissolved in acetic acid (10 mL). Then sulfuric acid (conc. 1mL) was added dropwise and the reactions were stirred at room temperature for three days. Then more aldehyde and sulfuric acid were added and the reaction stirred another 24 hours, more acid was added twice 24 hours apart. The reaction was quenched by addition of water and the precipitated crude intermediates were ed off and washed with water. After drying the product in vacuo over night 2 x 35 mg (0.09 mmol) of the crude intermediate was weighed into two flasks and dissolved together with monoethyl ate (14.8 mg, 0.10 mmol) in DCM/DMF (2 mL, 4:1). Triethylamine (19.3 μL, 0.14 mmol) was added and the e stirred for 5 min before addition of HATU (38.6 mg, 0.10 mmol). After continuing stirring for 12 hours more triethylamine and HATU was added and the stirring continued for 4 hours. The solvents were evaporated and the residue purified by preparative HPLC. The residue was dissolved in dichloromethane/trifluroacetic acid (5 mL, 4:1), stirred for 40 min and concentrated again.
Compound # 1505 was obtained in 93% purity by LCMS (System A). MS ESI+ m/z 477 [M+H]+.
EXAMPLE 2. r exemplary syntheses of red ments of the compound of the invention ‐{[(3E, 5‐bis[(4‐nitrophenyl)methylidene]‐4‐oxoazepan‐1‐carbonyl}pyrrolidinium trifluoroacetate (compound # 1505). N‐boc Azepan‐4‐one (0.10 g, 0.47 mmol) and 4‐nitrobenzaldehyde (156 mg, 1.0 mmol) were dissolved in acetic acid (10 mL), conc. H2 SO4 (1 mL) was added drop‐wise and the reaction d at rt over the weekend. More aldehyde (156 mg) and H2 SO4 (1 mL) were added and stirring continued at rt over night. Another mL conc. H2 SO4 was added and reaction stirred over night again. Conc. H2 SO4 was added once more and the reaction stirred until complete (for two . Upon addition of water a brown precipitate was formed, filtered off, washed with water, and dried under vacuum to give 339.5 mg of brown solid Intermediate 1, which was used without further purification.
Intermediate 1 (35 mg, 0.09 mmol) and N‐boc proline (22 mg, 0.10 mmol) were dissolved in DCM/DMF (4:1, 2 mL). TEA (19 µL, 0.14 mmol) was added and the mixture stirred for 5 min, then HATU (38.6 mg, 0.10 mmol) was added and the reaction stirred at rt overnight. More TEA (19 µL, 0.14 mmol) and HATU (38.6 mg, 0.10 mmol) was added, and the reaction stirred for another 4 h. The reaction mixture was trated and then purified by preparative LC (40‐70 % ACN in 0.1% TFA) to give the product as a yellow solid. The solid was dissolved in DCM/TFA (4:1, 5 mL) and the solution d at rt for 40 min to remove the boc protective group. The TFA salt of the product was recovered as a yellow solid of 93% purity. LCMS A: Rt 1.94/1.99, m/z [M+H]+ 477.1, B: Rt 2.28. (3E,5E)‐1‐(4‐ethoxy‐4‐oxobutanoyl)‐3,5‐bis[(4‐nitrophenyl)methylidene]‐4‐oxoazepan‐1‐ium trifluoroacetate (compound # 1507). Intermediate 1 (35 mg, 0.09 mmol) and N‐boc proline (22 mg, 0.10 mmol) were dissolved in DCM/DMF (4:1, 2 mL). TEA (19 µL, 0.14 mmol) was added and the mixture stirred for 5 min, then HATU (38.6 mg, 0.10 mmol) was added and the reaction stirred at rt overnight. More TEA (19 µL, 0.14 mmol) and HATU (38.6 mg, 0.10 mmol) were added and the reaction stirred for another 4 h. The reaction mixture was concentrated and then purified on preparative LC (40‐70 % ACN in 0.1% TFA) to give the TFA salt of the product as a yellow solid of 95% purity. LCMS A: Rt 2.48/2.50 m/z [M+H]+ 508.1. B: Rt 2.48/2.52. (3E, 5‐bis[(4‐chlorophenyl)methylidene]azepan‐4‐one (compound # 1518).
Azepan‐4‐one hydrochloride (0.45 g, 3.0 mmol) and 4‐chlorobenzaldehyde (0.92 g, 6.6 mmol) were dissolved in acetic acid (10 mL), conc. H2 SO4 (1 mL) was added drop‐wise and the reaction stirred at rt for 24 h. After addition of water (30 mL) a precipitate was formed, filtered off, and dried in vacuum to give the product in 91% purity as a yellow solid. LCMS A: Rt 2.04 m/z [M]+ 358.1. (3E, 5E)‐3,5‐bis(phenylmethylidene)‐1‐(prop‐2‐enoyl)azepan‐4‐one (compound # 1520) .
Azepan‐4‐one hydrochloride (50 mg, 0.182 mmol), acrylic acid (14 µL, 0.20 mmol), TBTU (58 mg, 0.182 mmol) and TEA (37 mg, 0.364 mmol) were dissolved in DMF (2 mL) and stirred at rt overnight. Brine and ethyl acetate were added and the phases separated. The organic phase was dried and the solvents evaporated after filtration. The crude product was dissolved in acetic acid (2 mL) and H2 SO4 (0.2 mL). dehyde (50 µL) was added and the on stirred for 24 hours. Methanol and water were added to the mixture, which was purified by preparative LC. The title nd was ed in 96% purity as a yellow solid.
LCMS A: Rt 2.68 m/z [M+H]+ 344.1. (3E, 5E)‐3,5‐bis(phenylmethylidene)‐1‐cyclobutanecarbonylazepan‐4‐one (compound # 1521).
Azepan‐4‐one hydrochloride (50 mg, 0.182 mmol), cyclobutyric acid (14 µL, 0.20 mmol), TBTU (58 mg, 0.182 mmol) and TEA (37 mg, 0.364 mmol) were dissolved in DMF (2 mL) and stirred at rt overnight. Brine and ethyl acetate were added and the phases separated. The organic phase was dried and the solvents ated after filtration. The crude product was dissolved in acetic acid (2 mL) and H2 SO4 (0.2 mL). Benzaldehyde (50 µL) was added and the reaction stirred for 24 h. Methanol and water were added to the mixture, which was purified by preparative LC. The title compound was isolated in 96% purity as a yellow solid. LCMS A: Rt 2.68 m/z [M+H]+ 372.1. (3E,5E)‐1‐(2‐cyclopropylacetyl)‐3,5‐bis[(4‐methoxyphenyl)methylidene]azepan‐4‐one (compound 1526). Azepan‐4‐one hydrochloride (0.45 g, 3.0 mmol) and 4‐methoxybenz‐ aldehyde (0.90 g, 6.6 mmol) were dissolved in acetic acid (10 mL), conc. H2 SO4 (1 mL) was added drop‐wise, and the reaction stirred at rt for 24 h. Water (30 mL) was added. The precipitate was filtered off and dried in vacuum over night. The crude material (30 mg, 0.107 mmol), cyclopropylacetic acid (12 mg, 0.12 mmol), TBTU (41 mg, 0.13 mmol) and TEA (26 mg, 0.26 mmol) were dissolved in DMF (2 mL) and stirred at rt over night.
Methanol (1.5 mL) and water (0.5 mL) were added and the product was purified by preparative LC to yield the solid product in 95% purity. LCMS A: Rt 2.51 m/z [M+H]+ 432.2. (3E,5E)‐5‐[(3‐nitrophenyl)methylidene]‐3‐(phenylmethylidene)azepan‐4‐one (compound # 1560). N‐boc‐Azepan‐4‐one (0.10 g, 0.47 mmol) and 3‐nitrobenzaldehyde (156 mg, 1.0 mmol) were dissolved in acetic acid (5 mL), trated H2 SO4 (0.5 mL) was added drop‐ wise and the on stirred at rt for 4 days. Then more concentrated H2 SO4 (0.5 mL) and aldehyde (156 mg, 1.0 mmol) were added and ng continued at rt for three weeks. A mixture of the mono‐ and di‐condensation products was obtained. The mixture was purified by column chromatography (DCM/methanol) to give the intermediate amine Intermediate 2 as a brown oil (19 mg). Intermediate 2 was ved in acetic acid (1.5 mL) together with benzaldehyde.
Conc. H2 SO4 (0.05 mL) was added and the reaction stirred at rt overnight. Then more H2 SO4 was added and the stirring continued for a week. More aldehyde (156 mg, 1.0 mmol) and H2 SO4 was added and stirring continued for an additional 4 days. The reaction mixture was concentrated and purified by preparative LC to give the TFA‐salt of the product as yellow solid in 98% . LCMS System A: Rt 1.78 m/z [M+H]+ 335.1, System B: Rt 2.43/2.28. (3E,5E)‐1‐methyl‐3,5‐bis[(4‐nitrophenyl)methylidene]azepan‐4‐one (compound # 1563).
N‐methylazepan‐4‐one∙HCl (50 mg, 0.30 mmol) and 4‐nitrobenzaldehyde were dissolved in acetic acid (5 mL) and stirred for 10 min, then conc. H2 SO4 (50 µL) was added slowly and the mixture was stirred at rt overnight. More concentrated H2 SO4 (100 µL) was added and stirring was continued at rt for 6 h. Additional 500 µL of concentrated H2 SO4 was added and the reaction stirred overnight. A further 350 µL of conc. H2 SO4 was added and stirring continued for additional 5 h, during which period further H2 SO4 was added in two portions (500 µL and 250 µL). Then water ( 3 x reaction volume) was added and the mixture was stirred until rt was reached. The reaction mixture was extracted with ethyl acetate (3 x reaction volume). The phases were separated and the organic phase concentrated to yield a dark yellow s oil. The crude product was purified by preparative HPLC, (XBridge column; eluents 50 mM ammonium ate buffer at pH 10 and methanol) giving the title t as a yellow solid (26.3 mg). LCMS System A: Rt 1.87 m/z [M+H]+ 394.1, System B: Rt 2.57. (3E,5E)‐3,5‐bis[(4‐fluorophenyl)methylidene]‐1‐propylazepan‐4‐one (compound # 1574).
Azepan‐4‐one hydrochloride (0.25 g, 1.68 mmol) and 4‐fluorobenzaldehyde (0.416 g, 3.36 mmol) were dissolved in acetic acid (20 mL) and the solution stirred for 10 min, then conc.
H2 SO4 (200 µL) was slowly added and the solution was stirred at rt ght. More conc.
H2 SO4 (1 mL) was added and stirring continued at rt. Another mL of conc. H2 SO4 was added after 6 h, and the reaction stirred again overnight. The next day further 800 µL of conc. H2SO4 was added and stirring continued for a period of five days, during which two portions of H2 SO4 (1 mL and 0.5 mL) were added to the reaction mixture. Then water (3 x reaction volume) was added and the mixture stirred until rt was reached. The on mixture was extracted with ethyl acetate (10 x reaction volume). The c phase was concentrated by evaporation. Water was added to the residue. A precipitate was formed and filtered off. The solid was washed with water and dried in vacuum to give Intermediate 3 as a yellow solid. A n (15 mg, 0.05 mmol) thereof was dissolved in DCE‐Propanal (4 µL, 0.06 mmol) was added, and the e stirred for 15 min at rt. Then NaBH(OAc)3 (15.7 mg, 0.07 mmol) and acetic acid (2.6 µL, 0.05 mmol) were added and the reaction d at rt over night. The reaction was concentrated and the crude product purified by preparative LC giving the product (7.2 mg) in 90% purity. LCMS System A: Rt 2.02 m/z [M+H]+ 368.1, System B: Rt 3.21. )‐3‐[(4‐methoxyphenyl)methylidene]‐5‐[(4‐nitrophenyl)methylidene]azepan‐4‐one (compound # 1575). Azepan‐4‐one hydrochloride (0.25 g, 1.68 mmol) and 4‐nitrobenz‐ aldehyde (253 mg, 1.68 mmol) were ved in acetic acid (20 mL) and stirred for 10 min, then conc. H2 SO4 (1 mL) was slowly added and the mixture stirred at rt for 8 days. On days 1‐3 one n conc. H2 SO4 per day was added (0.5 mL, 0.75 mL, and 0.5 mL). Water (2 x reaction volume) was added and the mixture extracted with ethyl e (2 x reaction volume). The organic phase was concentrated by evaporation and dried to yield crude Intermediate 4. A portion of Intermediate 4 (100 mg, 0.41 mmol) was dissolved in acetic acid (6 mL) and stirred for 10 min, then concentrated H2 SO4 (0.6 mL) was added slowly and the reaction stirred at rt for 6 days. Upon addition of water the product precipitated as a yellow solid. The precipitate was filtered off, washed with water and dried in vacuum to give the title compound as a yellow solid in 98% purity. LCMS System A: Rt 1.82 m/z [M+H]+ 365.1, System B: Rt 2.41. 1H‐NMR (400 MHz, CDCl 3 ) [ppm] = 2.97‐2.99 (m, 2H), 3.41‐3.44 (m, 2H), 3.83 (bs, 3H), 4.28 (s, 2H), 7.06‐7.08 (d, 2H), 7.47 (s, 1H), 7.59‐7.62 (d, 2H), 7.76 (s, 1H), .80 (d, 2H), 8.27‐8.29 (d, 2H). (3E,5E)‐5‐[(4‐fluorophenyl)methylidene]‐3‐[(4‐methoxyphenyl)methylidene]‐1‐ methylazepan‐4‐one (compound # 1577). N‐methylazepan‐4‐one hydrochloride (75 mg, 0.46 mmol) and robenzaldehyde were dissolved in acetic acid (7 mL) and stirred for 10 min, then conc. H2 SO4 (350 µL) was added slowly and the mixture was stirred at rt for 8 days.
More conc. H2 SO4 was added during days 2‐4 (0.175 mL, 0.35 mL, 0.25 mL tively).
Water was added and the solution ted with ethyl acetate (twice the volume of reaction mixture). The organic phase was concentrated to give Intermediate 5. A portion of this intermediate (35 mg, 0.15 mmol) and 4‐methoxybenzaldehyde (17 µL, 0.15 mmol) were dissolved in acetic acid (2.5 mL) and stirred for 10 min, then conc. H2 SO4 (0.20 mL) was added slowly and the on stirred for five days. Water (2 x reation volume) was added and the reaction mixture extracted with ethyl acetate (2 x reaction volume). The organic layer was concentrated and water was added. A precipitate was formed and filtered off to give the title product (11.2 mg) in 91% purity as a yellow solid. LCMS System A: Rt 1.86 m/z [M+H]+ 352.1, System B: Rt 2.79. (3E,5E)‐1‐acetyl‐5‐[(4‐fluorophenyl)methylidene]‐3‐[(4‐ethoxyphenyl)methylidene]azepan‐4‐ one (compound # 1579). Azepan‐4‐one hydrochloride (0.25 g, 1.68 mmol) and 4‐fluoro‐ benzaldehyde (179 µL, 1.68 mmol) were dissolved in acetic acid (20 mL) and stirred for 10 min, then conc. H2 SO4 (1 mL) was slowly added and the mixture was stirred at rt for 8 days with on of conc. H2 SO4 during the first three days (0.5 mL, 0.75 mL and 0.5 mL respectively). Water (2 x reaction volume) was added and the mixture extracted with ethyl acetate (2 x mixture volume). The organic phase was concentrated and dried to give the crude Intermediate 6. A portion of this intermediate (100 mg, 0.46 mmol) was dissolved in acetic acid (6 mL) and stirred for 10 min, then concentrated H2 SO4 (0.6 mL) was added slowly and the on stirred at rt for 7 days. Water was added (1 x volume) and the e was neutralized with saturated aqueous NaHCO3 . The formed precipitate was filtered off, washed with water and dried in vacuum to yield Intermediate 7 (31.5 mg) as a yellow solid of 91% purity. LCMS System A: Rt 1.85 m/z [M+H]+ 338. Intermediate 7 (10 mg) was ved in DCM (1 mL) and TEA (5.0 µL, 0.04 mmol) was added. The mixture was stirred for 10 min, then acetyl chloride (2.3 µL, 0.03 mmol) was added and the reaction stirred at rt for 30 min. The reaction was washed with water, saturated aqueous NaHCO3 and brine. The organic phase was concentrated to give the title compound (6.4 mg) as a yellow solid of 90% purity. LCMS System A: Rt 2.35 m/z [M+H]+ 380.1, System B: Rt 2.37. 1H‐ NMR (400 MHz, CDCl3 ): [ppm] = 1.70, 1.90, 1.98 and 1.99 (4 x s, 3H, CH3 CO‐, signals from the two regioisomers and their acetate rotamers), 2.89‐3.01 (m, 2H), 3.68‐3.77 (m, 2H), 3.79, 3.79, 3.79, 3.08 (4 x s, 3H, ‐OMe, signals from the two regioisomers and their acetate rotamers), 4.65‐4.68 (m, 2H), 7.0‐7.04 and 7.098‐7.103 (2 x m, 2H), 7.22‐7.30 (m, 3H), 7.48‐7.62 (m, 5H). (3E,5E)‐5‐[(4‐chlorophenyl)methylidene]‐3‐[(4‐nitrophenyl)methylidene]azepan‐4‐one (compound # 1583). N‐methylazepan‐4‐one hydrochloride (75 mg, 0.46 mmol) and 4‐chlorobenzaldehyde (64 mg, 0.46 mmol) were dissolved in acetic acid (7 mL) and d for min, then conc. H2 SO4 (350 µL) was added slowly and the mixture was stirred at rt for 8 days. More conc. H2 SO4 was added during days 2‐4 (0.175 mL, 0.35 mL, 0.25 mL respectively). Water (2 x reaction volume) was added and the solution extracted with ethyl e (2 x reaction volume). The organic phase was concentrated to give Intermediate 8. A portion of the intermediate (35 mg, 0.14 mmol) and obenzaldehyde (69.5 mg, 0.46 mmol) were dissolved in acetic acid (2.5 mL) and stirred for 10 min, then conc. H2 SO4 (200 µL) was added slowly and the mixture was stirred at rt for 5 days. More conc. H2 SO4 (0.2 mL) was added, and stirring continued for 5 more days. Water (2 x on volume) was added and the solution extracted with ethyl acetate (2 x reaction volume). The organic phase was concentrated and the residue purified by preparative LC to give the title compound (1.8 mg) as a yellow solid of 94% purity. LCMS System A: Rt 1.98/2.04 m/z [M+H]+ 383.1, System B: Rt 2.82/2.98.
Abbreviations Boc tert‐butyloxycarbonyl ACN acetonitrile DCM romethane TFA trifluoroacetic acid DMF dimethylformamide TEA triethylamine Rt retention time TBTU O‐(benzotriazol‐1‐yl)‐N,N,N',N'‐tetramethyluronium tetrafluoroborate) rt room temperature LC liquid chromatography EDC l‐3‐[3‐dimethylaminopropyl]carbodiimide HATU 2‐(1H‐7‐Azabenzotriazol‐1‐yl)‐‐1,1,3,3‐tetramethyl uronium hexafluorophosphate; DCE 1,2‐Dichloroethane EXAMPLE 3. Pharmaceutical composition A (aqueous suspension). The compound of the invention (25 mg) is ved in 1 ml of dimethyl sulfoxide. The solution is added drop‐wise to 10 ml of vigorously stirred saline. The formed suspension, which can be stabilized by adding 1 % by weight of PVP, can be used for intramuscular, intravenous or subcutaneous administration.
EXAMPLE 4. Pharmaceutical composition B (tablet). Tablets for oral stration are produced by blending 2.0 g of the compound of the invention (powder, <10 mµ, 90 %) with microcrystalline cellulose (1.30 g), corn starch (0.50) g, silica (0.20) g, Mg stearate (0.12 mg).
The mixture is dry compressed to 400 mg tablets, which are sugar .
EXAMPLE 5. Pharmaceutical composition C (solution). The compound of the invention (10 mg) is dissolved in 0.5 ml of Cremophor EL (BASF Corp.) and absolute ethanol was added to 1.0 ml. The clear solution is filled into glass vials for injection.
EXAMPLE 6. Pharmaceutical composition D (solution). For intraperitoneal administration in animal studies an aqueous composition a stock on was prepared by dissolving the compound of the invention to a concentration of 2 mg/ml in phor EL/polyethylene glycol 400 1:1. (v/v) at room temperature or by heating to up to about 80 °C assisted by ultrasonication. A t of the stock solution was diluted 1:10 with 0.9 % saline and used immediately for IP injection.
EXAMPLE 7. ceutical composition E (solution). For intraperitoneal administration a % by weight Kolliphor HS15 stock solution was prepared by melting an entire container of Kolliphor HS15 (Sigma 42966) by warming to 60 °C and diluting with deionized water to % w/w. To nd # 1570 (18.0 mg) in a 10 mL sample tube was added 10.0 mL of the stock solution and the tube vortexed, treated with ultrasound at about 50 °C for about 2 h, and occasionally heated to about 83 °C. The clear solution ed was sterile filtered through a 0.2 µm cellulose syringe filter prior to ion. By the same procedure solutions of compounds # 1546 and # 1571 were ed; these nds were however not fully dissolved. The non‐dissolved residue was weighed, and the weight deducted from the starting weight of compound (18 mg). It was found that the prepared solutions (10 ml) contained 8.5 mg and 11.0 mg, respectively, of compounds # 1546 and # 1571.
EXAMPLE 8. Pharmaceutical composition F (solution). For intraperitoneal administration a stock on of 2‐hydroxypropyl‐β‐cyclodextrin (Aldrich 332593) was prepared by ving the cyclodextrin in deionized water to a concentration of 30% w/w. To compound # 1649 (15.0 mg) in a 10 mL sample tube was added 10.0 mL of the stock solution. The tube was vortexed, treated with ultrasound at about 50 °C for about 2 h, and occasionally heated to about 83 °C. The solution obtained was sterile filtered through a 0.2 µm cellulose syringe filter prior to injection. The weight of al compound # 1659 not dissolved was determined and used for ting the concentration of the filtered solution to 82.5 % of the attempted tration. By the same procedure a solution of compound # 1546 was prepared.
EXAMPLE 9. The nd of the invention induces proteasome inhibition. The reporter cell line MelJuSo Ub‐YFP, which is engineered to accumulate yellow fluorescent protein (YFP) upon proteasome inhibition (12), was used for compound evaluation. The accumulation of YFP was measured for 48 hours in an IncuCyte‐FLR system (Essen Bioscience, Essen, UK), which is an ted fluorescence microscope. Numbers of positive cells per field were used as a e of proteasome inhibition.
EXAMPLE 10. Determination of solubility of compounds of the invention in aqueous media.
In the ms of Figs.2a‐2e solubility is expressed as Log S (mmol/ml; software ACD/Labs Inc.) Solubility is determined in aqueous buffer at various pH values and predicted for pure water at 25 °C. The algorithm uses a set of >6,800 compounds as reference. The diagrams show that azepanones of the ion can have a substantially increased solubility, such as by a factor 2 or more, in aqueous media at physiological pH, such as at a pH from 6 to 8, in particular of from 7.0 to 7.5, in comparison with correspondingly substituted piperidin‐4‐ ones.
EXAMPLE 11. Azepanes/azepanones of the invention exhibit higher cytotoxicity than structurally corresponding piperidines/piperidin‐4‐ones Figs. 3b, 3d, 3f are ms illustrating the cytotoxicity of compounds of the invention nos. 1546, 1547, and 1570 with a 7‐membered ring moiety in comparison to structurally corresponding compounds not sed by the invention with a 6‐membered ring moiety. Their induction of dose‐dependent cytotoxicity was determined after 72 hours of continuous compound exposure to the reporter cell line HCT‐116. Treated cells were compared with untreated controls. Cytotoxicity is visualized as survival index (SI) over the range of about 90 % SI to about 0 % SI in dependence on compound concentration. It s from the Figures that the compounds of the invention are more cytotoxic that the reference nds since they are producing the same level of cytotoxicity at lower concentration.
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Claims (36)

What we claim is
1. A compound of the l structure S‐1 capable of abrogating the deubiquitinating (DUB) activity of the 19S RP DUBs wherein R1, R2 at double bond d1 and R3, R4 at double bond d2 can, independent of each other, have a uration opposite to that of structure S1, X is CO or CS; R1 and R3 are, independent of each other, H or C1‐6 ‐alkyl; R2 and R4 are, independent of each other, H; C1‐6 ‐alkyl; C1‐5 ‐alkylCO; phenyl or 6‐membered heteroaryl optionally substituted by 1‐3 of: C1‐6 ‐alkyl,C1‐6 y, CN, ‐COOC1‐6 , COOH, NO2 , F, Cl, Br, I, CF3 , NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 , CONR7R8, with the proviso that one or more of H in alkyl and alkoxy can be substituted by fluoro; R5 is any of H; C1‐6 ‐alkyl; C2‐6 ‐alkenyl; C1‐3 ‐alkoxy‐C2‐6 ‐alkyl‐; C1‐3 ‐alkoxy‐C2‐6 yl‐; aryl‐C0‐6 ‐alkyl‐; aryl‐C0‐6 ‐alkyl‐; heterocyclyl‐C0‐6 ‐alkyl‐; cycloalkyl‐C0‐6‐alkyl‐; ‐C1‐6 ‐alkyl‐COOC1‐6‐alkyl; ‐C2‐6 ‐alkyl‐aryloxy; COR6; R6 is any of C1‐6 ‐alkyl; C2‐6 ‐alkenyl; C1‐6 ‐alkoxy; C1‐3 ‐alkoxy‐C1‐6 ‐alkyl‐; C1‐3 ‐alkoxy‐C1‐6 ‐alkenyl‐; aryl‐C0‐6 ‐alkyl‐; heteroaryl‐C0‐6 ‐alkyl‐; heterocyclyl‐C0‐6 ‐alkyl‐; cycloalkyl‐C0‐6‐alkyl‐; ‐C1‐6 ‐alkyl‐COOC1‐6‐alkyl; NH2 ; ‐NHC1‐6 ‐alkyl; ‐N(C1‐6 ‐alkyl)2 ; ‐C0‐6 ‐alkyl‐aryloxy; R7, R8 are, independent of each other, H or C1 ‐C3 ‐alkyl.
2. The nd of claim 1 wherein X = CO.
3. The compound of claim 1 or claim 2, wherein R1 and R3 are both H.
4. The compound of any one of claims 1 to 3, wherein the substitution of phenyl is in one or more of positions 3, 4, 5.
5. The compound of any one of claims 1 to 4, wherein R5 is COR6, and R6 is selected from C1‐ 6‐alkyl, cycloalkyl, vinyl, allyl.
6. The compound of claim 5 wherein R6 is vinyl.
7. The compound of claim 5 wherein R6 is methyl.
8. The compound of claim 1, wherein R1, R2 at double bond d1 and R3, R4 at double bond d2 have the configuration of structure S‐1, X is CO or CS; R1 and R3 are, ndent of each other, H or C1‐6 ‐alkyl; R2 and R4 are, ndent of each other, H; C1‐6 ‐alkyl; C1‐5 ‐alkylCO; phenyl or ered heteroaryl substituted with 1‐3 of: CN, NO2 , F, Cl, Br, I, NH2 , NHC1‐6 ‐alkyl, N(C1‐6 ‐alkyl)2 ; R5 is H, C1‐6 ‐alkyl, C2‐6 ‐alkenyl, C1‐3 ‐alkoxy‐C2‐6 ‐alkyl, C1‐3 ‐alkoxy‐C2‐6 ‐alkenyl, aryl, heteroaryl, heterocyclyl, C1‐6 ‐alkyl‐heteroaryl, C1‐6 ‐alkyl‐heterocyclyl, C1‐6 ‐alkyl‐cycloalkyl, C1‐6 ‐alkyl‐aryl, CO‐C1‐6 ‐alkyl, CO‐vinyl, CO‐allyl, l, CO‐cycloalkyl.
9. The compound of claim 8, wherein X is CO.
10. The compound of claim 8 or claim 9, wherein R2 and R4 are substituted phenyl.
11. The compound of any one of claims 8 to 10, wherein R5 is selected from CO‐C1‐6 ‐alkyl, CO‐cycloalkyl, CO‐vinyl, CO‐allyl.
12. Use of the compound of any of claims 1 to 11 in the cture of a medicament for treating cancer in a person refractory to chemotherapy.
13. Use of a compound of any one of claims 1 to 11 in the manufacture of a medicament for treating a cancer tumor tory to ent with bortezomib or an agent sharing the apoptosis generating activity of bortezomib or any other anti‐cancer drug.
14. The use of claim 13, wherein cells of the tumor are tory to treatment due to over‐expression of the intrinsic apoptosis‐inhibitor Bcl‐2.
15. The use of claim 13 or claim 14, wherein 19S RP DUBs comprise UCHL5 and USP14.
16. The use of any one of claims 13 to 15, wherein the deubiquitinating (DUB) activity of non‐proteasomal DUBs is not affected.
17. The use of any one of claims 13 to 16, wherein the ment comprises the compound dissolved or suspended in a liquid carrier.
18. The use of any one of claims 13 to 17, wherein the medicament is for intravenous, intramuscular, intraperitoneal or subcutaneous ion or infusion.
19. The use of claim 13, wherein medicament is for peroral administration.
20. The use of claim 19, wherein the carrier is a tablet or capsule.
21. The use of any one of claims 13 to 20, wherein the medicament is administered to e a pharmacologically ive dose of the active agent.
22. The use of claim 21, wherein the pharmacologically effective dose of the active agent is from g/kg to 0.1 g/kg body weight, consideration being given to whether the agent is administered systemically or locally.
23. The use of claim 22, n the cologically effective dose of the active agent is from 0.001 g/kg to 0.01 g/kg kg body weight, consideration being given to whether the agent is administered systemically or locally.
24. The use of any one of claims 13 to 23, wherein "refractory to treatment" signifies that treatment of a cancer with a single dose of omib or an agent sharing the apoptosis generating activity of bortezomib or any other anti‐cancer drug does not substantially reduce the growth rate per month of the cancer observed immediately prior to the treatment.
25. The use of claim 24, wherein "refractory to treatment" signifies that treatment of a cancer with a single dose of bortezomib or an agent sharing the apoptosis generating activity of bortezomib or any other anti‐cancer drug does not reduce the growth rate per month of the cancer ed immediately prior to the treatment by more than 25 per cent.
26. The use of claim 25, wherein "refractory to treatment" signifies that treatment of a cancer with a single dose of bortezomib or an agent sharing the apoptosis generating activity of bortezomib or any other anti‐cancer drug does not reduce the growth rate per month of the cancer observed immediately prior to the treatment by more than 10 per cent.
27. The use of claim 26, wherein "refractory to treatment" signifies that treatment of a cancer with a single dose of bortezomib or an agent sharing the apoptosis ting activity of bortezomib or any other anti‐cancer drug does not reduce the growth rate per month of the cancer observed immediately prior to the treatment by more than 5 percent.
28. The use of any one of claims 13 to 27, wherein the cancer is selected from multiple myeloma, breast cancer, ovary , lung cancer, colon cancer, prostate cancer, as cancer.
29. The use of any one of claims 13 to 28, wherein the ment is to be administered by selecting a person to be treated by determining the growth rate of the cancer prior to and upon administration of bortezomib or said active principle sharing the mechanism of deubiquitinating activitiy inhibition of bortezomib or said other anti‐cancer drug, a positive growth rate, in particular a growth rate of more than 5 % or more than 10% or more than 25 % per month constituting a selection marker.
30. A ceutical composition comprising the nd of any of claims 1 to 11 and a pharmaceutically acceptable carrier.
31. The composition of claim 30, in the form of a tablet or capsule or other single‐dose preparation for peroral administration.
32. The composition of claim 30, in the form of a solution or suspension in a pharmaceutically able liquid carrier for injection or infusion.
33. The composition of claim 32, for intravenous, intramuscular, intraperitoeal or subcutaneous infusion or injection.
34. A compound as claimed in any one of claims 1 to 11, substantially as herein described with reference to any example thereof.
35. A use as claimed in any one of claims 12 to 29, substantially as herein described with reference to any example thereof.
36. A pharmaceutical composition as claimed in any one of claims 30 to 33, substantially as herein described with nce to any e thereof.
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